Insulin-dependent GLUT4 is a risk factor for cancer in the prostate

Insulin-dependent GLUT4 is a risk factor for cancer in the prostate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Insulin-dependent GLUT4 is a risk factor for cancer in the prostate Pedro Gonzalez-Menendez, Alba Moran-Alvarez, Juan C. Mayo, Rafael Cernuda-Cernuda, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2619954/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Diabetic men are less likely to suffer prostate cancer, and insulin signalling through insulin receptors has been long considered. However, the role of insulin-dependent glucose transporters has yet to be elucidated. The unique metabolic properties of prostate cancer are attributed to the central role of androgens. Androgen-sensitive tumour cells have higher mitochondrial activity, while castration-resistant cells exhibit aerobic glycolysis. In addition, to glycolysis, one of the hallmarks of cancer metabolism is increased glucose uptake. However, the prostate's oncogenic value of glucose transporters (GLUTs) needs to be better characterized. This research aims to discover the relevance of insulin-dependent glucose transporters to cancer progression and their importance in the protective role of diabetes in prostate cancer. Methods: Androgen-sensitive LNCaP and androgen-insensitive PC-3 cells were used in vitro . Castration-resistant LNCaP-R cells and cells overexpressing GLUT1 or GLUT4 were established from LNCaP cell line. In addition, TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice and prostatic samples from patients were employed. Results: We found that androgens stimulate insulin-independent glucose transporters, while androgen independence is associated with GLUT4 overexpression. The ectopic overexpression of GLUT4 promotes the characteristics of a castration-resistant phenotype. Metabolomics confirmed that hormone-resistant prostate cancer cells show an oxidative metabolism with a clear enrichment in amino acid metabolism. Diabetic TRAMP mice showed total tumour regression, while insulin administration restored proliferation and recovered GLUT4 levels. The levels of GLUT4 increase along with tumour progression in TRAMP mice, and it is reduced by castration and streptozotocin-induced diabetes. Finally, the levels of GLUT4 accumulation in tumour tissues compared to normal epithelial in patients' samples showed a clear co-location with nuclear AR. Conclusion: Here it is confirmed the relevance of insulin-mediated glucose uptake through GLUT4 with prostate cancer progression and its relation to the reduced occurrence of prostate cancer in diabetic men. Prostate cancer glucose transporters insulin diabetes GLUT4 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background First described by Otto Warburg more than 90 years ago, cancer cells usually exhibit an intrinsic metabolic switch that is considered one of the hallmarks of cancer[ 1 ]. Tumour cells typically show increased nutrient uptake compared to normal cells, particularly glucose and glutamine, to facilitate their growth[ 2 ]. The higher glucose uptake feeds the so-called ‘aerobic glycolysis’ observed in these cells, leading to higher lactate production and release. Consequently, aerobic glycolysis equips tumour cells with the necessary macromolecular precursors for cell growth without compromising energy production[ 3 ]. Thus, targeting glucose metabolism is one of oncology’s most promising treatment options[ 4 ]. This higher glucose uptake rate observed in cancer compared to normal cells can be explained by changes in protein levels or the membrane location of glucose transporters. The GLUT (glucose transporter)/ SLC2A family is related to the transcriptional activation of several oncogenes[ 5 ]. Even though such an increase in glucose uptake has been mainly associated with GLUT1 overexpression, it may also involve other members of the GLUT family of transporters, including the structurally related insulin-dependent GLUT4[ 6 ]. Why carcinogenesis becomes insulin-sensitive in some tissues is one of the unanswered questions in oncology. Prostate metabolism is highly specialized to produce citrate in the prostatic fluid by inhibiting the isomerization of citrate to isocitrate. Such inhibition compromises the tricarboxylic acid (TCA) cycle, meaning glycolysis is favoured in the normal prostate epithelium. Zinc accumulates in the prostate epithelia and inhibits the TCA cycle enzyme aconitase (ACO2) that prevents citrate oxidation to isothiocyanate. This characteristic metabolism suggests that prostate cancer also displays a unique metabolism. Oxidative phosphorylation (OXPHOS) is stimulated in the first stages of carcinogenesis[ 7 ]. Although like many other tumours, in the later stages, prostate cancer (PCa) tissue is considered glycolytic[ 8 ]. Given this unique metabolic profile, glucose metabolism was not considered to be as important as glutamine or lipid metabolism in PCa and has thus received less research attention than in other cancers[ 9 ]. Androgens stimulate citrate production and secretion and near all the metabolic machinery that characterize the gland. In prostate tumors, androgens regulate zinc and aspartate transporters[ 10 ]. Metabolic shift between glycolysis and oxidative phosphorylation in prostate tumors also relies on AR function during oncogenic transformation. Androgen stimulation increases glucose uptake[ 11 ] but also, it drives lipogenesis and oxidative phosphorylation [ 12 – 14 ]. In addition, the transition to a castration-resistant phenotype (CRPC) is accompanied by changes in glucose consumption and biomass production. Clinical imaging using the radiolabelled glucose analogue 18F-fluorodeoxyglucose (FDG) is not suitable to detect local prostate cancer, whereas neuroendocrine prostate cancer is thought glycolytic and detectable on FDG imaging. Which confirms the metabolic shift during transition to androgen independence[ 15 ]. During the last decade, the number of studies showing the importance of glucose metabolism in PCa has increased dramatically and several inhibitors of glucose metabolism are in early phase clinical trials, however further investigation is still needed[ 15 ]. Molecularly, the mitochondrial pyruvate carrier protein has been shown to be transcriptionally regulated by AR signalling, increasing the glucose flux towards the mitochondria[ 16 ] and some reports indicate that androgens regulate the expression of GLUT1 in PCa cells[ 17 , 18 ]. GLUT1 production has been observed in normal prostate glands as well as in the most aggressive tumours[ 19 ] but less is known about other glucose transporters. The reduce prevalence of prostate cancer in diabetic patients suggest a role of insulin glucose transporters in prostate cancer still unknown. Recently, our own group reported the expression and production of insulin dependent GLUT4 glucose facilitative transporter in PCa cells[ 20 , 21 ], however there are no data regarding its regulation or role in tumour progression yet. Its expression could be somehow linked to the inverse relationship between diabetes and PCa progression[ 22 ], but the molecular mechanisms of which are unknown. Thus, the main aim of this work was to decipher the role of GLUT4 during PCa progression and determine its involvement in the inverse relationship between diabetes and prostate cancer. Methods Cell culture, transfection, and treatments Human androgen-sensitive LNCaP cells were purchased from the European Collection of Cell Cultures (catalogue no. 89110211) and cultured in RPMI 1640 medium (Lonza, catalogue no. 12-719F) supplemented with 10% FBS, 2 mM l-glutamine, 15 mM HEPES and a 1% antibiotic–antimycotic cocktail. PC-3 cells, a human androgen-insensitive cell line, were purchased from American Type Culture Collection (catalogue no. CRL-1435) and grown in DMEM/F12 medium (Lonza, catalogue no. BE12-702F) supplemented with 10% FBS, 2 mM l -glutamine and a 1% antibiotic–antimycotic cocktail. Both cell lines were authenticated by short tandem repeat profiling. Cell lines were grown at 37°C in a humidified 5% CO 2 environment. GLUT1- or GLUT4-overexpressing LNCaP cells were obtained by using FuGENE®HD (Promega, catalogue no. E2311) following the manufacturer’s instructions. Plasmids pcDNA3.2/v5-DEST hGLUT1 (Addgene plasmid no. 18085) and pcDNA3.2/v5-DEST hGLUT4 (Addgene plasmid no. 18087) were supplied by Wolf Frommer (Heinrich Heine University Düsseldorf) [ 23 ]. Cells were selected by incubation with 300 µg/mL G418 (Merck, catalogue no. A1720). To inhibit the Pentose Phosphate Pathway (PPP), cells were treated with 10 µM of 6-aminonicotinamide (6AN, Merck, catalogue no. A68203) for 48 hours. Androgen stimulation was performed by growing androgen-sensitive cells in androgen-deprived medium supplemented with dextran–charcoal FBS (FBS chst ). Then, 5 nM of dihydrotestosterone (DHT) (Merck, catalogue no. A8380) was used to stimulate ARs. 20 µM of the antiandrogen bicalutamide (CDX, Merck, catalogue no. B9061) was employed in the presence of DHT. Insulin (Ins, Merck, catalogue no. I9278) was added at 1 µM. For all treatments, cells were left to attach for 48 hours. The glucose concentration was always set at 2 g/L (11 mM). Vehicle alone was added to the control group. Animal procedure Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice (C57BL/6-Tg(TRAMP)8247Ng/J) were used (The Jackson Laboratory)[ 24 ]. Prostates were classified by a pathologist as well, moderately, or poorly differentiated tumours. Mice were maintained under controlled environmental conditions (12:12 light:dark) with ad libitum access to food and drinking water. Animals were sacrificed in a CO 2 chamber and the genitourinary tract was immediately dissected and frozen in liquid nitrogen or fixed in 10% phosphate-buffered formalin. Castration was done by radical bilateral orchiectomy after anaesthesia with 100 mg/kg ketamine (Imalgene; Merial) and analgesia with 20 mg/kg xylazine (Rompun; Bayer). For androgen replacement in castrated mice, 5 days after surgery testosterone was injected s.c. (2.5 mg/kg body weight). Animals were sacrificed at the indicated times. For diabetes induction, 50 mg/kg streptozotocin (STZ, Merck) was administered i.p. daily for 5 days. After two weeks, 0.6 U insulin (Humulin 70:30, Lilly) was injected s.c. daily. Insulin was always administered in the morning. Fasting blood glucose levels were measured with levels above 250 mg/dL for at least three consecutive days indicating diabetes. Mice exhibiting levels over 500 mg/dL were discarded. Patient samples Paraffin-embedded tissue samples from 132 patients were collected and their medical records, including data related to relapses, metastases, perineural invasion and PSA levels, were provided (Table 1 ). Samples were classified by Gleason score by the hospital pathologist. Frozen non-pathologic, hyperplastic and tumour tissues were collected from 20 patients. Patients underwent surgery at Hospital Universitario Central de Asturias, Hospital Valle del Nalón (Asturias) or Hospital Universitario Marqués de Valdecilla (Cantabria). Table 1 Clinical characteristics of diabetic patients Patient Age T1DM 1 Age at diagnosis PSA 2 Gleason Tumour Grade Glycosylated Hb (A1c) % Glucose 3 1 61 Yes 15 15 - - 7.1 154 2 61 Yes 39 39 6 (3 + 3) 3 8.5 158 3 59 Yes 12 12 7 (3 + 4) 2 6.9 157 4 69 Yes 3 3 7 (3 + 4) 2 9.6 298 1 T1DM, Type 1 Diabetes Mellitus 2 Levels of PSA, in ng/ml 3 Fasting glucose levels, in mg/dl PAGE and immunoblotting Protein extracts from culture cells were obtained, separated and electrotransferred as previously described[ 25 ]. For tissues, a section of 50 mg representing all the prostatic lobes were cut and embedded in 1 mL of chilled RIPA lysis buffer. Samples were homogenized employing an ULTRA-TURRAX® homogenizer, and same procedure to culture cells was followed. Antibodies (Supplementary Table 1) were visualized by binding horseradish peroxidase-conjugated secondary antibodies and detected with chemiluminescence substrate. Densitometry values were obtained using ImageJ and Image Studio software. To study N-glycosylation of GLUT4 protein, muscle and prostate tissue from TRAMP mice were homogenized in the protein extraction buffer described by Haga Y. et al [ 26 ]. Tissue lysates were incubated in ice for 30 minutes, then they were centrifugated for 15 min at 15000xg and supernatant were collected. Supernatants were concentrated using Amicon Ultra-0.5 centrifugal filter devices (Amicon Ultra 10K device. 100000 BNWL, Millipore) following manufacturer instructions. Concentrated proteins were diluted in 1:4 in DEPC treated Water and stored upon usage. Protein extracts were quantified using Bradford reagent (Merck). To remove glycosylation residues, 20 µg of protein were treated with PNGaseF reagent ( New England Biolabs ) under denaturing conditions following manufacturer's instructions. Treated extracts were loaded into an SDS-page (7.5% acrylamide) and electrophoresis and western-blot were performed like the other experiments. Immunocytochemistry and Immunohistochemistry studies Cells were fixed in phosphate-buffered 2% paraformaldehyde (H 7.4). For IHC, 5-µm thick paraffin-embedded tissue sections were deparaffinised and hydrated in a graded series of EtOH solutions. Antigen retrieval was carried out by microwaving. After blocking with 3% goat serum in TBS (Tris-HCl 20 mM pH 7.4, 150 mM NaCl), samples were incubated for 20 min at room temperature with 0.15% Tween-20 for cytoplasmic permeabilization or 0.1% Triton X-100 for nuclear permeabilization. Immunofluorescence was observed under a Leica TCS SP8 confocal microscope. The antibodies and dilution factors used in immunocytochemistry and immunohistochemistry studies are listed in Supplementary Table 1. AR and GLUT-4 immunostaining in human biopsies Double immunolabeling with a monoclonal mouse anti-AR and a rabbit polyclonal anti-GLUT4 antiserum was carried out in two groups of prostate biopsies from diabetic and non-diabetic human patients (n = 4 for each group). Briefly, the sections were immersed in EDTA pH 9.0 at 95°C for 20 minutes for antigen retrieval and subsequently incubated in the anti-AR antiserum at 4 ºC overnight (1:5000 dilution). The AR immunolabeling was detected using a standard procedure with the Vectastain ABC kit (Vector) and diaminobenzidine (DAB) as chromogen. In previous experiments, this method had proved to be more effective than IF when labelling with this particular antiserum. Due to the amplification of the signal obtained with the ABC method, a highly diluted primary antibody provided a clear signal, with little background staining. Immunofluorescence was then carried out to detect GLUT4 on these same sections. The samples were incubated with the anti-GLUT4 antiserum (1:250 dilution) at 4 ºC overnight and, subsequently, in an AlexaFluor 488 goat-anti-rabbit antiserum to visualize the immunolabeled regions. DAPI was used as a nuclear counterstaining. In order to obtain merged pictures with the double immunostaining, the sections were photographed with a Zeiss Axiocam 712 monochrome camera mounted on a Nikon Eclipse 80i microscope under both bright field and fluorescence conditions. The bright field pictures corresponding to AR-immunolabeling were then inverted, turned red and merged with those of GLUT-4 immunostaining and DAPI counterstaining with Adobe Photoshop CS8. To measure the GLUT-4 immunostaining intensity of each biopsy, 9 pictures were randomly taken within the section using the 20x objective. The average intensity of immunostaining (in a range of 0-255 values in a grayscale) was measured with the ImageJ software. In a parallel experiment, samples of prostatectomy from 15 patients diagnosed with different Gleason scores (which ranged between 3 + 3 and 4 + 5) were immunostained for GLUT-4 following a protocol like the one detailed above, omitting the initial AR immunostaining. Tumor regions with different Gleason grades (ranging between 3 and 5) were present in all the samples, which also contained healthy (non-tumour) glands. A minimum of 5 pictures from each (including non-tumour regions) were taken using the 20x objective and the same photographic conditions. To obtain an estimate of the average immunostaining intensity within the prostatic gland epithelia, the stromal areas were manually removed in all photographs with Adobe Photoshop CS8. Next, GLUT-4 immunostaining was measured using the ImageJ software. Flow cytometry The cell cycle was studied by staining with 50 µg/ml propidium iodide (PI) in cells fixed with 70% ethanol. Surface GLUT1 levels were monitored as previously reported[ 27 ]. Data analysis was performed using FlowJo (version 10.6.2) and Kaluza (version 2.1) software. Real-time PCR RNA was isolated using TriReagent® (Merck) and cDNA was synthesised using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). qRT-PCR was performed using the Light Cycler 480 SYBR Green I Master Kit (ThermoFisher Scientific). The specific primers are listed in Supplementary Table 1. Metabolic assays Glucose uptake was measured as described previously[ 20 , 28 ] , . For standardisation, number of cells were previously estimated. ATP/AMP determination was achieved by HPLC. Nucleotide extraction and measurement were performed as previously described[ 25 ]. Protein concentration was quantified by Bradford Assay for standardisation. Glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase enzyme activities were measured as previously described[ 29 ] and employed to evaluate the activity of PPP. Stable isotope labelling experiments Cells were grown in culture media as mentioned above but supplemented with 2 g/L U- 13 C 6 -glucose (Merck) for 24 hours. Extraction of the intracellular metabolites with methanol and ultrapure water, derivatisation and GC-MS measurement of metabolite-specific isotopic distributions were carried out as previously reported[ 30 ]. Heatmap and clustering analysis were performed using the heatmap3 add-in package with R project open-source software (version 3.4)[ 31 ]. Enrichment and pathway analysis were done using MetaboAnalyst (version 4.0)[ 32 ]. Extracellular lactate levels Cells were seeded in 24-well plates and harvested by scraping when reached 70% confluence. After centrifugation to eliminate cell debris, the cell culture medium was collected and deproteinised with 4 M perchloric acid and neutralised with 2 M KOH. The pH was adjusted to 7.4. The lactate determination assay was performed following the manufacturer’s instructions (Bioquochem S.L.). PSA levels PSA was measured by ELISA following the manufacturer’s instructions (Human Diagnostics). Total protein concentration was quantified by Bradford Assay for standardisation. Fasting blood glucose and glucose tolerance test Fasting blood glucose was measured using a One Touch Ultra Easy™ glucometer (Johnson & Johnson). Blood was taken from the tail vein in mice fasted overnight. To measure glucose tolerance, mice received an intraperitoneal injection of glucose (2 mg/g body weight) and blood glucose levels were determined after 15 min, 30 min, 1 hour and 2 hours. The AUC was then calculated by the linear trapezoidal method. Blood insulin levels Insulin levels were measured in the plasma of overnight-fasted mice. Blood was extracted post-mortem, collected in EDTA-treated tubes and immediately centrifuged at 3000 × g for 10 min at RT. Samples were frozen at -80°C until analysis. Insulin levels were measured by ELISA following the manufacturer’s instructions (Millipore, catalogue no. EZRMI-13K). Blood testosterone levels Blood was extracted post-mortem, collected in EDTA-treated tubes, and immediately centrifuged at 3000 × g for 10 min. Samples were frozen at -80°C until analysis. Testosterone levels were measured by ELISA following the manufacturer’s instructions (Cayman). Statistics Data are represented as individual values, or the mean and error bars represent the standard error of the mean (SEM). Outliers were identified using the Rout test, while normality was studied using the Shapiro–Wilk or Kolmogorov–Smirnov tests depending on the number of samples. If the samples followed a normal distribution, the significance was calculated using the two-tailed unpaired Student’s t-test for comparison of two groups while one-way ANOVA followed by Fisher’s LSD test was used for comparison of more than two groups. For samples that did not present a normal distribution, we used the non-parametric Kruskal–Wallis test followed by Benjamini, Krieger and Yekutieli’s two-stage linear step-up procedure. The tests were performed using Prism GraphPad (version 8). The significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 Results Androgens regulate insulin independent GLUT transporters in androgen dependent LNCaP cells Androgens regulate glucose metabolism in prostate cancer cells through the modulation of glycolic proteins. However, the role of insulin dependent glucose transporters in prostate cancer progression is still under unknown. To confirm androgen control over energy metabolism in the prostate we studied the effect of DHT treatment on glucose uptake and glucose transporters production androgen sensitive LNCaP cells. Thus, to verify DHT biological activity, first we confirmed DHT incubation significantly increased cell proliferation as expected (Fig. 1 a). In addition, we found that DHT increased the uptake of glucose (Fig. 1 b), it reduced the production and release of lactate (Fig. 1 c) and diminished Pentose Phosphate Pathway (PPP) activity (Fig. 1 d). Incubation with the antiandrogen bicalutamide (CDX) restored the levels to those of the control cells, indicating the specificity of androgen signalling in these effects. In addition, DHT increased the production of GLUT1 and GLUT3 both insulin independent glucose transporters. However, it does not affect GLUT4, and it reduced the levels of GLUT12 both insulin dependent glucose transporters. Effects also restored by CDX pre-incubation (Fig. 1 e). GLUT1 activity in cells is primarily regulated through translocation of GLUT1 to the plasma membrane. Since GLUT1 appeared to be the main target of androgens in LNCaP cells, we studied the membrane exposure of the transporter after the addition of androgens. We found that androgens removal by charcoal-stripped FBS incubation slightly decreased the presence of GLUT1 in the membrane ( Fig. 1 f, left pannel) as shown by flow cytometry immunostaining. However, membrane GLUT1 levels were not restored after DHT treatment nor influenced by CDX treatment ( Fig. 1 f, right pannel) . Master metabolic regulators, AMPK or AKT, were also found to be regulated by androgens (Fig. 1 g). The stimulation of LNCaP cells with DHT increased the level of pAMPK Thr172 by six-fold and significantly decreased the phosphorylation of pAKT Ser473 . Again, CDX recovered their levels showing the specificity of androgens. This increment in AMPK activity might support the increment of glucose uptake since it participates in their translocation to the cell membrane. To confirm the effects of androgen signalling on glucose transporters, we employed hormone-sensitive and castration-resistant LNCaP cells (LNCaP-R). LNCaP-R were previously established by permanent growth without androgens[ 33 ]. First, we noticed that GLUT1 was significantly lower in the castration-resistant LNCaP-R corroborating its dependence of androgens but GLUT3, GLUT4 and GLUT12 were higher in LNCaP-R than in the LNCaP parental cells (Fig. 1 h). Overexpression of GLUT4 favours an androgen-independent phenotype Glucose uptake is the first step in the metabolic shifting of cancer cells but the effect of glucose transporters on prostate cancer phenotype is scarcely known. We investigated the effects of GLUT1 and GLUT4 overexpression, an insulin independent and dependent glucose transporter, on LNCaP cells. First, overexpression was confirmed by western blotting ( Supplementary Fig. 1a ). As expected, the overexpression of both transporters increased glucose uptake in hormone-sensitive LNCaP (Fig. 2 a), although GLUT1 overexpressing cells grow faster than LNCaP Mock or LNCaP GLUT4 ( Supplementary Fig. 1b ). Interestingly, GLUT1 and GLUT4 overexpressing cells exhibited opposite tendency of lactate production. LNCaP cells overexpressing GLUT1 showed a significant reduction of lactate release, while overexpression of GLUT4 significantly enhanced its into the culture media (Fig. 2 b). There were no significant differences in PPP activity in both overexpressing clones and the parental mock-transfected LNCaP cells ( Supplementary Fig. 1c ). However, GLUT1-overexpressing cells were more sensitive than parental cells to glucose-6-phosphate dehydrogenase (G6PDH) inhibition, the first enzyme in the PPP pathway. (Fig. 2 c) To investigate the role of glucose transporters on AR location and activity, western blot was performed after GLUT1 or GLUT4 overexpression. Though total protein levels did not change, nuclear AR was significantly lower in LNCaP GLUT4 cells (Fig. 2 d) but prostate-specific antigen (PSA) release was significantly increased in LNCaP GLUT4 cells versus parental LNCaP or LNCaP GLUT1 (Fig. 2 e). A decrease in nuclear AR with an increased release of PSA production might be an indicator of the androgen independence of GLUT4-overexpressing prostate cancer cells. This fact was confirmed by investigating the response of LNCaP GLUT4 cells to DHT in terms of cell proliferation. DHT treatment increased the proliferation of LNCaP Mock cells as expected, and this increase was prevented by co-incubation with CDX. However, LNCaP GLUT4 cells did not show any response to DHT (Fig. 2 f). Likewise, the number of cells in the S-phase of the cell cycle was increased in LNCaP Mock studied by flow cytometry while it was significantly decreased in LNCaP GLUT4 cells after treatment with DHT (Fig. 2 g). All these results point that overexpression of GLUT4 encourages an androgen independence. Hormone resistant cells have an increase in glucose uptake and a glycolytic metabolism Prostate metabolism is highly specialised to produce citrate in prostatic fluid, which provides the gland with unique metabolic properties. The normal prostate epithelium favours glycolysis over oxidative phosphorylation under aerobic conditions, given the physiological truncation of the TCA cycle. Metabolic reprogramming in prostate tumours leads to a reduction of glycolysis and enhanced mitochondrial oxidative phosphorylation, which are believed to be regulated by androgen signalling. However, progression to a hormone independent phenotype increase glycolytic metabolism in the tumour prostate. First, we study differences in mitochondrial metabolism between hormone sensitive LNCaP cells and hormone independent PC-3 cells. As shown in Fig. 3 a, hormone-resistant PC-3 cells showed higher levels of glucose uptake, a reduce production of ATP (Fig. 3 b) and a lower activity of PPP when compared to LNCaP cells (Fig. 3 c) than androgen-sensitive LNCaP cells which implies a glycolytic phenotype. To determine whether androgen signalling influence the TCA cycle in prostate cancer cells, we investigated glucose flux by GC-MS-based 13 C metabolic analysis. Cells were incubated with 13 C 6 -labelled glucose for 24 hours and the incorporation of 13 C from the 13-labelled glucose in the metabolites was determined by measuring their isotopologue distribution by GC-MS. Mass isotopologue distributions were employed to calculate the molar fraction of 13 C incorporated. Thus, m0, m1 and m2 refer to the incorporation of zero, one and two 13 C atoms in the metabolite, respectively. Lactate, glutamate and TCA pathways allowed us to analyse metabolic flux by calculating the different molar fractions of 13 C incorporated in each metabolite. The absence of AR in PC-3 reduced the molar fraction of M0 in lactate (30% in comparison with LNCaP cells) and significantly increased the incorporation of 13 C into citrate. The incorporation of 13 C in succinate was much faster in LNCaP than in PC-3 (Fig. 3 d). Although the differences between LNCaP and PC-3 might be due to different metabolic players, such as TP53, our results suggest that AR signalling has a clear metabolic influence on the TCA cycle and favours glycolysis in prostate cancer cells. Enrichment and pathway analyses were performed using MetaboAnalyst 4.0 to compare LNCaP vs. PC-3. The pathway enrichment analysis showed that androgen dependency promoted tryptophan metabolism, gluconeogenesis and Warburg rewiring, as well as increasing the cells’ reductive power by increasing glutathione metabolism (Fig. 3 e). The alanine, arginine and proline, glutathione and glutamate metabolism pathways also showed significant differences in both comparisons. The TCA cycle was also shown to be affected by AR presence, confirming that androgen-sensitive and insensitive cells have different energy phenotypes (Fig. 3 f). Facilitative insulin-independent and -dependent GLUT levels increase with PCa progression The role of insulin-independent and -dependent glucose transporters in cancer progression was studied using TRAMP mouse prostate tissues. Tumours were classified by a pathologist into three categories: well (WD), moderately (MD) or poorly differentiated (PD) and tissues were collected in 24- and 32-week-old animals. The androgen-dependent prostate secretory protein of 94 amino acids ( Psp94 ) gene was chosen as a marker of hormone dependence of tumours since rodents have no counterpart of PSA. As shown in Supplementary Fig. 2a , PD tumours exhibited an androgen-insensitive phenotype since they did not express Psp94 . The levels of insulin-independent GLUT1 and GLUT3 transporters increased along with tumour progression, as previously suggested by others[ 15 ]. But interestingly, it was found that also insulin-dependent transporter GLUT4 increased in prostate tumour tissues with progression, showing a significant increment in PD tumours. In the case of GLUT12, also considered to be an insulin-responsive glucose transporter, no significant differences were found (Fig. 4 a). Immunostaining confirmed that insulin-responsive GLUT4 was found in epithelial prostate tissue the highest levels found within the most aggressive areas of the tumours, and they were increased in PD when compared with WT (Fig. 4 b,c). GLUT4 validation was performed by immunostaining of muscle and adipose tissue ( Supplementary Fig. 2b ). GLUT4 staining concurs with conventional prostate markers BCL-2, AR and Hypoxia-Inducible Factor (HIF)1α, which are all increased in androgen-resistant tumours ( Supplementary Fig. 2c ). While production of GLUT1 and GLUT4 is increased during PCa progression, the expression of Scl2a1 and Scl2a4 was significantly reduced in tumour tissues compared to normal tissues (Fig. 4 d, e), while there were no differences in Scl2a3 or Scl2a12 (data not shown). To confirm the effect of androgens in vivo , TRAMP mice were surgically castrated for 5 days and then treated daily with testosterone for a further 5 days. The effect of castration was confirmed by genitourinary (GU) weight/ Body weight (BW) ( Supplementary Fig. 3a ). Castration significantly reduced GU weight, while testosterone recovered it. GLUT1 levels did not change shortly after castration but they are significantly reduced after testosterone injection ( Fig. 5 a ) . Regarding GLUT4, a double band was detected. Significant differences were found in the abundance of upper and lower band. GLUT4 upper band was significantly reduced or even absence after castration (Fig. 5 a, b ) . Double band of GLUT4 might be related with the glycosylation of the transporter (upper band). The glycosylation of GLUT4 is related with a higher activation in the transport of glucose[ 34 ]. Intriguingly, the glycosylated form of GLUT4 disappeared and protein levels significantly decreased after castration in TRAMP mice and the upper band was again detected in presence of testosterone (Fig. 5 a-c). The treatment of TRAMP prostate protein samples with the amidase PNGase F, which cleaves at N-acetylglucosamine (GlcNAc) and asparagine residues of high mannose oligosaccharides, confirmed the changes in electrophoresis mobility claimed as a demonstration of glycosylation of the transporter. Muscle protein samples, a positive control of GLUT4 glycosylation was employed as positive controls ( Fig. 5 d ) . Surprisingly, the transcription of Scl2a1 was upregulated after testosterone administration, perhaps as a consequence of protein reduction ( Supplementary Fig. 3b ) while Scl2a4 did not change significantly pointing a functional modification by castration more than a transcriptional regulation ( Supplementary Fig. 3c ). Castration did not alter the levels of pAMPK Thr172 , though a significant increase was found after testosterone treatment vs CON. On the other hand, pAKT Ser473 was augmented after acute castration, but its levels were not restored by testosterone injection in TRAMP mice (Fig. 5 e). TRAMP mice were also long-term castrated by surgery at 12 weeks of age and further sacrificed when they were 24 or 32 weeks old. The effect of castration was confirmed by the significant reduction of GU weight/BW (Supplementary Fig. 3d) . GLUT1 protein levels were not significantly changed after permanent castration of TRAMP mice, although the disappearance of the upper band of GLUT4 was further confirmed ( Supplementary Fig. 3e) . TRAMP mice have higher levels of insulin and diabetes slows prostate cancer progression in TRAMP mice Hyperinsulinemia has been associated with aggressive prostate cancer and with the accelerated growth of LNCaP cell xenografts[ 35 ]. Our previous results suggested that the insulin-dependent GLUT4 transporter might be involved in PCa progression and it might be responsible, at least in part, for the deleterious effects of insulin in the prostate since insulin activates AKT and promotes GLUT4 translocation. First, we confirmed that LNCaP and PC-3 were sensitive to insulin. As shown in Fig. 6 a, LNCaP and PC-3 cells increased the uptake of glucose in response to insulin. Then, we studied the levels of insulin in wild-type and TRAMP mice at 24 weeks of age. TRAMP mice showed significantly higher levels of insulin than WT mice (Fig. 6 b) and, interestingly, castration reduced blood insulin levels, while testosterone injection recovered it (Fig. 6 c). However, there were no differences in the blood glucose levels in 16-week-old WT and TRAMP mice (Fig. 6 d). Fasting blood glucose tolerance was the lowest in TRAMP mice, as shown in Fig. 6 e. The area under the curve (AUC), which correlates with the ability to eliminate glucose from blood, was also lower in TRAMP mice than in WT and castrated mice. Altogether, these results indicate that TRAMP mice show higher blood insulin levels and better glucose tolerance during the first stages of the disease. The relative risk of developing prostate cancer is reduced in men with diabetes. To evaluate the impact of diabetes on PCa progression in TRAMP mice, Type 1 Diabetes Mellitus (T1DM) was induced by streptozotocin (STZ) treatment for 5 days. All treated mice showed over 250 mg/dL glucose after 1 week of the treatment, which was the criterion used to consider them as diabetic ( Supplementary Fig. 4a ). Histological analysis of the pancreas confirmed the loss of eosinophilic, granule-containing insulin-producing beta cells within the islets of Langerhans ( Supplementary Fig. 4b ). Insulin injection restored glucose blood levels to normal within 2 hours but this effect only lasted 24 hours ( Supplementary Fig. 4c ). For this reason, diabetic TRAMP mice were treated daily with insulin, started two weeks after the first injection with STZ. Relevant anatomical changes were found during necropsy in the prostate glands of TRAMP (CON), diabetic TRAMP (STZ) and diabetic TRAMP plus insulin (STZ + Ins) mice, as shown in Fig. 7 a. Diabetes reduced the size and histopathology of the prostate glands in TRAMP mice. The prostates of STZ-induced diabetic TRAMP mice did not show any relevant pathology. Shortly after insulin treatment, some of the animals developed a PIN (4/5), showed an area of PD tumour (1/5) or exhibited scattered areas of neuroendocrine carcinoma (1/5). The body and genitourinary tract were weighed at 24 weeks of age. Bodyweight was reduced in diabetic mice, likely due to white adipose tissue (WAT) depletion, and a significant reduction in the GU/BW ratio was found in STZ mice compared to CON. However, insulin treatment did not recover GU weight (Fig. 7 b). Even though the levels of circulating testosterone were slightly reduced after diabetes induction, the differences were not significant (Fig. 7 c). Lowering of tumour progression by diabetes was confirmed by western blot of the proliferating cell nuclear antigen (PCNA) (Fig. 7 d). STZ reduced PCNA production while insulin recovered its levels. Besides, the pro-apoptotic protein BAX was significantly increased in diabetic compared to control mice, while insulin recovered its levels. No differences were found in other tumour markers, such as TP53, P21/CDKN1 or BCL-2. To evaluate the role of androgens in the protective effect of diabetes on PCa, we studied AR levels and location. STZ did not induce changes in total AR levels, though after insulin stimulation it seems to be located in some nuclei ( Supplementary Fig. 4d ). However, Psp94 mRNA levels did not show any significant difference (data not shown). It is well known that insulin promotes activation of the PI3K/AKT pathway, either by direct interaction with its receptor or by Insulin-like Growth Factor (IGF) signalling, and this pathway is promoted in the most aggressive tumours. Levels of IGF1 Receptor (IGF1R)β and IGF Binding Protein (IGFBP)3 were analysed by western blot. There was no difference in IGF1Rβ between groups, and IGFBP3 in the prostate was somewhat reduced by insulin treatment in diabetic mice ( Supplementary Fig. 4e ). Instead, AKT phosphorylation was significantly reduced in diabetic mice, while insulin restored its levels (Fig. 7 e). To assess the implication of GLUT transporters in the protection of STZ-induced diabetic TRAMP mice against PCa progression, we studied the mRNA expression and protein production of GLUT4 transporters (data not shown). Induction of diabetes in the prostate reduced Slc2a4 expression (Fig. 7 f). Interestingly, although there was no difference in total GLUT4 protein levels between groups, diabetes led to the disappearance of the upper band found in the GLUT4 signal and insulin consistently restored it (Fig. 7 g). These results might indicate that GLUT4 is a facilitative glucose transporter involved in the protective role of diabetes in PCa in TRAMP mice. GLUT4 is increased in prostate cancer tissues To translate our results to patient samples, we studied the production and location of GLUT4 in prostate tumours. Samples were classified as non-tumour or hyperplasic and according to their Gleason score in the tumour tissues. Tumours were classified as Gleason score < 7 or ≥ 7 according to pathologists. The samples with Gleason scores ≥ 7 were characterised by higher PSA and BCL2 levels ( Supplementary Fig. 5a ) and decreased levels of GLUT1 protein during the first stages of tumour growth ( Supplementary Fig. 5b ). However, insulin-responsive GLUT4 increased with tumour progression, showing the double band that indicate its activate status. (Fig. 8 a). In addition, GLUT4 tissue distribution was examined. Samples of prostatectomy from 15 patients diagnosed with different Gleason scores (ranged between 3 + 3 and 4 + 5) were immunoassayed for GLUT4. GLUT4 immunolabeling intensity was significantly higher in Gleason grade 3 and 4 regions than in healthy (non-tumour) control areas (Fig. 8 b, c). A decrease in GLUT4 intensity was detected in Gleason grade 5 (less differentiated tumour) regions, which did not show significant differences when compared to control ones. Such difference in protein location was evident when these tumour regions were located next to healthy glands (Fig. 8 d). From a database of all patients treated for prostate cancer in the Urology service at the Central University Hospital of Asturias, between January 2016 and November 2018, out of 807 patients, only 4 patients had been previously diagnosed with type 1 diabetes mellitus, all of them diagnosed in adulthood. GLUT levels were also confirmed in patient samples (Table 1 ). Double immunolabeling with anti-AR and anti-GLUT4 antiserum was carried out in prostate biopsies from diabetic and non-diabetic human patients (n = 4 for each group). All the nuclei of the prostate epithelia, both from diabetic and non-diabetic patients, were stained and the signal was robust in all samples. No differences were observed between both groups. DAB-immunolabeled nuclei acquired little or no DAPI staining (Fig. 9 a,b) From 407 patients of prostate cancer treated at “Hospital Valle del Nalon” Asturias and “Hospital Universitario Marqués de Valdecilla” roughly 10% of the patients included in the study were diabetic. Most of the patients were diagnosed with Type 2 Diabetes Mellitus (T2DM) and only 1 suffered Type 1 Diabetes (T1DM) (Table 2 ). Most of the patients were treated with metformin alone or in combination with insulin or stimulators of insulin production. From those, relapses and perineural invasion did not show any difference between diabetic and non-diabetic patients (Table 3 ). Table 2 Distribution of diabetic prostate cancer patients and treatments Diabetics N (%) 1 Treatment N (%) 2 Type 1 1 (0.25) Insulin 1 (100) Type 2 41 (10.07) Diet 4 (9.75) Metformin 19 (46.34) Metformin + Insulin 3 (7.31) Stimulator of Insulin production 3 6 (14.63) Metformin + Insulin + SIP 3 (7.31) Stimulator + Metformin 3 5 (12.19) 1 N = 407. Percentage from the total of Prostate Cancer patients. 2 The % is referred to the number of patients with T1DM or T2DM, respectively. 3 Different stimulators of insulin production were employed: Repaglinide, Daonil and Glimepirin. Table 3 Relapses and perineural invasions in prostate cancer patients Distribution of patients (%) Non-diabetic (N = 90) Diabetic (N = 42) Relapses 31 (34.44) 9 (21.43) Perineural invasion 1 37 (48.68) 15 (48.38) 1 Information about perineural invasion was not facilitated for all patients Altogether, this data confirms that diabetes plays a protective role in prostate cancer and our results indicate that insulin dependent facilitative transporters of glucose, such as GLUT4, is a relevant protein implicated, while until know it was completely ignored. Discussion PCa is unique from the metabolic point of view. The prostate is specialised for the production and secretion of citrate in prostatic fluid. Androgens stimulate citrate production and regulate all metabolic pathways in the prostate, including glucose metabolism[ 36 ]. Here, we try to understand the role of glucose transporters sensitive to insulin in the progression of prostate cancer and their role on the lower incidence prostate cancer in individuals suffering diabetes. In this manuscript, the role of androgen signalling in glucose metabolism is confirmed since glucose uptake is increased after androgen treatment of LNCaP cells while lactate production and PPP activity are decreased. We showed in our previous work that GLUT1 levels are regulated in response to glucose deprivation in androgen sensitive PCa cells promoting survival and antioxidant pathways with no effects on androgen independent prostate cancer [ 25 ]. Here we show that androgens stimulate GLUT1 production, although the location on the cell membrane is not changed, suggesting that its overexpression in another organelle must be considered to explain its role on the survival of prostate cancer cells. Interestingly, androgens stimulate the production of insulin independent glucose transporters GLUT1 and GLUT3 as previously reported. AMPK activity that it is promoted by androgens, and it activates GLUT1, plays a role in mitochondrial metabolism of glucose in androgen-sensitive cells[ 11 ]. AMPK is also a regulator of GLUT1 synthesis and trafficking [ 37 ]. Thus, GLUT1 stimulation by androgens in vitro can be triggered via AMPK activation, as it is suggested by our results. Interesting, on the contrary DHT does not seem to influence insulin dependent transporters and even more, hormone-resistant LNCaP cells showed increase levels of insulin-sensitive GLUT4 and GLUT12. Though tumour cells often increase glucose uptake the physiologic role of glucose transporters in cancer has been scarcely study. Mostly GLUT1 and GLUT3 have gained attention as relevant factors in the accelerated metabolism of cancer cells[ 38 ] Overexpression of GLUT4 in androgen dependent prostate cancer cells promotes glycolysis and lactate production and more interestingly, it stimulates PSA production in an androgen independent manner. Metabolomics also confirmed the dependence of prostate cancer metabolism of androgens. Although differences between LNCaP and PC-3 cannot be attributed only to AR, since they also differ in TP53, which plays a principal role in glycolysis[ 39 , 40 ]. Although glucose uptake is higher in androgen-resistant cells, the levels of labelled lactate and, thus, glycolytic activity, are enhanced in androgen-insensitive cells which indicates the transition from glycolytic to oxidative in hormone resistant phenotypes previously described[ 36 ]. In addition to a decrease in aerobic glycolysis, as indicate m + 3 lactate molar fraction, some metabolites of the TCA, like succinate, incorporated lower levels of 13 C in PC-3 than LNCaP cells according to previous results[ 41 ]. A possible explanation for these results is that AR signalling reduces the utilisation of glucose through pyruvate production through the upregulation of PPP. Previous studies have confirmed that AR promotes PPP from glucose[ 42 ]. Moreover, pathway analysis showed that the alanine, aspartate, and glutamate metabolism pathway is one of the most affected. This pathway is intricately linked with glycine, serine, and threonine metabolism, which was also found to be significantly impacted[ 43 ]. It was recently revealed that serine, that can be produced from the glycolytic metabolite glycerate-3P, and one-carbon metabolism pathways are linked with neuroendocrine PCa[ 44 ]. In fact, both pathways should also be considered for androgenic regulation. Both GLUT1 and GLUT4 are highly produced by poorly differentiated tumours, which suggests their potential as biomarkers in advanced stages of cancer. In any case, only the levels of GLUT4 are reduced by surgical castration and restored by testosterone in TRAMP mice. We observed N-glycosylation of GLUT4 in TRAMP mice tumours that disappeared after castration and could be causally related to GLUT4 stability and trafficking, as proposed by others[ 45 ]. GLUT4 is regulated in vivo by the absence of androgens, while it seems to be androgen-independent in vitro . This can be explained by the close connection between testosterone and insulin production. Insulin reduces the production of sex hormone-binding globulin, increasing active testosterone levels[ 46 ]. Inversely, testosterone also promotes insulin production in pancreatic beta cells via non-genomic actions of AR[ 47 ]. Insulin has been proposed as a risk factor for PCa patients because of its mitogenic and antiapoptotic activity[ 48 ]. However, its role in tumour progression is still poorly understood and mechanistically limited. TRAMP mice show higher insulin levels, and in agreement, castrated TRAMP mice show lower blood insulin levels. Interestingly, hormone deprivation therapy in humans leads to insulin resistance[ 49 , 50 ], which is associated with both the effect found in androgen deprivation therapy and the role of insulin in PCa progression. Perhaps one of the most interesting data in this regard is that the risk of cancer incidence associated with diabetes is decrease in the prostate, being the molecular scenario still discussed. We demonstrated experimentally that T1D protects TRAMP mice of prostate cancer. Diabetic mice induced by STZ treatment reversed completely prostate carcer phenotype. We found that the levels of PCNA, a proliferation marker in PCa[ 51 ], was reduced and the pro-apoptotic protein BAX was increased in STZ-treated mice, and that insulin reverted this effect. Insulin stimulates IGF secretion, which is related to PCa aggressiveness[ 52 ], and IGFBP has been proposed as a predictive marker in advanced PCa, like PSA[ 53 ]. However, in TRAMP mice, IGF-1R or IGFBP3 levels do not change after STZ-induced diabetes or insulin treatment. One aspect to consider is that T1DM reduces visceral obesity. Periprostatic fat influences cancer progression through the secretion of adipokines, proinflammatory cytokines[ 54 ]. Moreover, fatty acids are important nutrients in the prostate[ 55 ]. In diabetic TRAMP mice GLUT4 glycosylation is highly reduced. The activation of AKT might be related to the increased production of GLUT4 in tumoral tissue. However, AKT activity is inhibited in diabetic TRAMP mice. The importance of GLUT4 in cancer has been proposed in oral squamous cell carcinoma patients. Here, GLUT4 was significantly associated with a poor overall survival and recurrence-free survival. In addition, the ectopic overexpression of GLUT4 in cell lines caused a significant increase in migration in vitro and in vivo, whereas GLUT4 silencing reversed the cancer phenotype[ 56 ]. Also, it has been shown that GLUT4 plays a major role in basal glucose uptake in breast cancer cells[ 57 ], and specific knockdown of GLUT4 in breast cancer cell lines impairs glucose uptake and reduces lactate production and more recently, the involvement of ALKBH5-mediated m 6 A RNA demethylation in control of GLUT4 expression and the sensitization of HER2-targeted therapy in breast cancer.[ 58 ] Conclusions In conclusion, diabetes and cancer are two aged-associated pathologies more prevalent each year in western countries. Targeting dysregulated glucose metabolism has focused attention on the therapy of cancer. However, challenging both is by itself a new difficulty in clinics. We experimentally demonstrated in this work that T1D induced by STZ injection avoids prostate cancer progression in the TRAMP mice. It seems insulin might promote PCa progression by incrementing GLUT4 independently of androgen signalling. Our results prove for the first time, the relevance of insulin-dependent glucose transporters in the progress of prostate tumours in vivo. They also suggest their value as therapeutic targets, particularly in the advanced stages of the tumour, characterised by increased glucose uptake and a glycolytic phenotype. Abbreviations 2DG: Deoxyglucose; 6AN: 6-aminonicotinamide; 6GPD: 6-phosphogluconate dehydrogenase; ACO2: Aconitase; AR: Androgen Receptor; AUC: Area Under the Curve; BD: Body Weight; CAS: Castrated; CDX: Bicalutamide; CON: Control; CRPC: Castration-Resistant Prostate Cancer; DAB: Diaminobenzidine; DHT: Dihydrotestosterone; FBS: Fetal Bovine Serum; FBS CHST : Charcoal-stripped FBS; FDG: Fluorodeoxyglucose; G6PDH: Glucose-6-phosphate dehydrogenase; GlcNAc: N-acetylglucosamine; GLUT: Glucose Transporter; GU: Genitourinary tract; HDAC: Histone deacetylase; HIF: Hypoxia-Inducible Factor; IF: Immunofluorescence; IGF: Insulin-like Growth Factor; IGF1R: IGF1 Receptor; IGFBP: Insulin-like Growth Factor Binding Protein; IHC: Immunocytochemistry; Ins: Insulin; MD: Moderately-differentiated tumor; OXPHOS: Oxidative phosphorylation; PCa: Prostate Cancer; PCNA: Proliferating Cell Nuclear Antigen; PD: Poorly-differentiated tumor; PNGase: N-glycosidase; PPP: Pentose Phosphate Pathway; PSA: Prostate Specific Antigen; SEM: Standard Medium Error; STZ: Streptozotocin; T1DM: Type 1 Diabetes Mellitus; T2DM: Type 2 Diabetes Mellitus; TCA: Tricarboxylic Acid Cycle; TES: Testosterone; TRAMP: Transgenic Adenocarcinoma of Mouse Prostate; WD: Well-differentiated tumor Declarations Ethics approval and consent to participate This work was carried out in accordance with the World Medical Association Declaration of Helsinki for the ethical principles for Medical Research involving human subjects. All patients were informed, and consent was obtained prior to sample collection and the protocol was approved by “Comite de Etica de la Investigacion del Principado de Asturias”. Experiments and procedures with mice were conducted in accordance with European Directive 2012/63/UE and were approved by the Ethical Committee Board for Animal Experiments at the University of Oviedo. Consent for publication All authors consent to publication Availability of the data and material Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rosa M Sainz ( [email protected] ). Competing interests The authors declare no competing interests. Funding This work was supported by the “Agencia Estatal de Investigacion” (MINECO-BFU2016-79139-R, and MCI-20-PID2019-111418RB-I00) and co-funded by the European Regional Development Fund (FEDER) and by the “Gobierno del Principado de Asturias, Programa de Grupos de Investigacion 2018-2020” (IDI/2018/000239). The EU is acknowledged for the provision of FEDER funds for the purchase of the GC-MS/MS instrument. Authors' contributions P.G.M., J.C.M. and R.M.S. designed the study. P.G.M. performed the experiments. A.M.A study GLUT4 production and activation. R.C.C. performed confocal microscopy studies and morphologic analysis. A.A.A. assisted with animal experimentation. D.H., P.R.G. and J.I.G.A. performed mass spectrometry analysis and metabolomics. R.A.A. assisted with flow cytometry. M.A.M., M.D.E. collected and oversaw prostate cancer patients’ and C.L. gather and supervised diabetic patients. P.G.M, J.C.M., D.H, P.R., J.I.G.A. and R.M.S interpreted and discussed the data. P.G.M and R.M.S conceived, analysed the data and wrote the manuscript with assistance from the other authors. All authors critically reviewed the manuscript. Acknowledgements P.G.M. thanks the finantial support of “Ministerio de Universidades de España, Subvenciones para la Recualificacion del Sistema Universitario Español María Zambrano (MU-21-UP2021-030). A.M.A was funded by “Asociacion Española Contra el Cancer” (SV-19-AECC-FPI-2). A.A.A. was supported by the University of Oviedo “Ayudas predoctorales para la realización de tesis doctorales modalidad A” (PAPI-18-PF-06). C.L is recipient from a Margarita Salas Post-doctoral grant from the University of Barcelona. We thank Sandrina Kinet and Naomi Taylor for their helpful assistance in the labelling and detection of GLUT1. We thank Marta Alonso-Guervos and Ana Salas Bustamante for their technical support with the confocal microscopy and flow cytometry assays. We thank Scientific and Technical Services (SCTs) from the University of Oviedo for their technical support, including biological, biotechnical, and biomedical testing, optical microscopy and image processing, mass spectrometry, and the animal facility. We also thank the Molecular Histopathology in Animal Models of Cancer of the IUOPA for the processing and analysis of prostate samples. Materials & correspondence Rosa M. Sainz, PhD. Department of Morphology and Cell Biology, University Institute of Oncology of Asturias (IUOPA). School of Medicine, C/Julian Claveria 6, 33006 Oviedo, SPAIN. Phone # 34 985103610, e-mail: [email protected] Pedro Gonzalez-Menendez, PhD. Department of Morphology and Cell Biology, University Institute of Oncology of Asturias (IUOPA). School of Medicine, C/Julian Claveria 6, 33006 Oviedo, SPAIN. Phone # 34 985103610, e-mail: [email protected] References Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov [Internet]. Cancer Discov; 2022 [cited 2023 Jan 11];12:31–46. Available from: https://pubmed.ncbi.nlm.nih.gov/35022204/ Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science (1979). 2009;324:1029–33. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The Biology of Cancer: Metabolic Reprogramming Fuels Cell Growth and Proliferation. Cell Metab. 2008;7:11–20. Vander Heiden MG, DeBerardinis RJ. Understanding the Intersections between Metabolism and Cancer Biology. Cell. Cell Press; 2017. p. 657–69. Pavlova NN, Zhu J, Thompson CB. The hallmarks of cancer metabolism: Still emerging. Cell Metab [Internet]. Cell Metab; 2022 [cited 2023 Jan 11];34:355–77. Available from: https://pubmed.ncbi.nlm.nih.gov/35123658/ Barron CC, Bilan PJ, Tsakiridis T, Tsiani E. Facilitative glucose transporters: Implications for cancer detection, prognosis and treatment. Metabolism. 2016;65:124–39. Singh KK, Desouki MM, Franklin RB, Costello LC. Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues. Mol Cancer. BioMed Central; 2006;5:14. Barfeld SJ, Itkonen HM, Urbanucci A, Mills IG. Androgen-regulated metabolism and biosynthesis in prostate cancer. Endocrine Related Cancer. 2014;21:T57–66. Vaz C V., Alves MG, Marques R, Moreira PI, Oliveira PF, Maia CJ, et al. Androgen-responsive and nonresponsive prostate cancer cells present a distinct glycolytic metabolism profile. Int J Biochem Cell Biol. 2012;44:2077–84. Ahmad F, Cherukuri MK, Choyke PL. Metabolic reprogramming in prostate cancer. Br J Cancer. 2021. Tennakoon JB, Shi Y, Han JJ, Tsouko E, White MA, Burns AR, et al. Androgens regulate prostate cancer cell growth via an AMPK-PGC-1α-mediated metabolic switch. Oncogene. 2014;33:5251–61. Gonthier K, Poluri RTK, Audet-Walsh É. Functional genomic studies reveal the androgen receptor as a master regulator of cellular energy metabolism in prostate cancer. Journal of Steroid Biochemistry and Molecular Biology. Elsevier Ltd; 2019. p. 105367. Higgins LH, Withers HG, Garbens A, Love HD, Magnoni L, Hayward SW, et al. Hypoxia and the metabolic phenotype of prostate cancer cells. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2009;1787:1433–43. Massie CE, Lynch A, Ramos-Montoya A, Boren J, Stark R, Fazli L, et al. The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis. EMBO J. 2011;30:2719–33. Gonzalez-Menendez P, Hevia D, Mayo JC, Sainz RM. The dark side of glucose transporters in prostate cancer: Are they a new feature to characterize carcinomas? Int J Cancer. 2017; Bader DA, Hartig SM, Putluri V, Foley C, Hamilton MP, Smith EA, et al. Mitochondrial pyruvate import is a metabolic vulnerability in androgen receptor-driven prostate cancer. Nat Metab. Nature Research; 2019;1:70–85. Vaz C V., Marques R, Alves MG, Oliveira PF, Cavaco JE, Maia CJ, et al. Androgens enhance the glycolytic metabolism and lactate export in prostate cancer cells by modulating the expression of GLUT1, GLUT3, PFK, LDH and MCT4 genes. J Cancer Res Clin Oncol. 2016;142:5–16. Wang J, Xu W, Wang B, Lin G, Wei Y, Abudurexiti M, et al. GLUT1 is an AR target contributing to tumor growth and glycolysis in castration-resistant and enzalutamide-resistant prostate cancers. Cancer Lett [Internet]. Cancer Lett; 2020 [cited 2022 Dec 20];485:45–55. Available from: https://pubmed.ncbi.nlm.nih.gov/32428663/ Reinicke K, Sotomayor P, Cisterna P, Delgado C, Nualart F, Godoy A. Cellular distribution of Glut-1 and Glut-5 in benign and malignant human prostate tissue. J Cell Biochem. 2012;113:553–62. Gonzalez-Menendez P, Hevia D, Rodriguez-Garcia A, Mayo JC, Sainz RM. Regulation of GLUT transporters by flavonoids in androgen-sensitive and-insensitive prostate cancer cells. Endocrinology. Endocrine Society; 2014;155:3238–50. Raina K, Ravichandran K, Rajamanickam S, Huber KM, Serkova NJ, Agarwal R. Inositol Hexaphosphate Inhibits Tumor Growth, Vascularity, and Metabolism in TRAMP Mice: A Multiparametric Magnetic Resonance Study. Cancer Prevention Research. 2013;6:40–50. Grossmann M, Wittert G. Androgens, diabetes and prostate cancer. Endocr Relat Cancer. 2012;19:F47-62. Takanaga H, Frommer WB. Facilitative plasma membrane transporters function during ER transit. The FASEB Journal. 2010;24:2849–58. Greenberg NM, DeMayo F, Finegold MJ, Medina D, Tilley WD, Aspinall JO, et al. Prostate cancer in a transgenic mouse. Proc Natl Acad Sci U S A. 1995;92:3439–43. Gonzalez-Menendez P, Hevia D, Alonso-Arias R, Alvarez-Artime A, Rodriguez-Garcia A, Kinet S, et al. GLUT1 protects prostate cancer cells from glucose deprivation-induced oxidative stress. Redox Biol. 2018;17:112–27. Haga Y, Ishii K, Suzuki T. N-glycosylation is critical for the stability and intracellular trafficking of glucose transporter GLUT4. J Biol Chem [Internet]. J Biol Chem; 2011 [cited 2022 Dec 20];286:31320–7. Available from: https://pubmed.ncbi.nlm.nih.gov/21757715/ Oburoglu L, Tardito S, Fritz V, de Barros SC, Merida P, Craveiro M, et al. Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification. Cell Stem Cell. 2014;15:169–84. Montel-Hagen A, Kinet S, Manel N, Mongellaz C, Prohaska R, Battini JL, et al. Erythrocyte Glut1 triggers dehydroascorbic acid uptake in mammals unable to synthesize vitamin C. Cell [Internet]. Cell; 2008 [cited 2022 Dec 21];132:1039–48. Available from: https://pubmed.ncbi.nlm.nih.gov/18358815/ Tian WN, Braunstein LD, Apse K, Pang J, Rose M, Tian X, et al. Importance of glucose-6-phosphate dehydrogenase activity in cell death. Am J Physiol. 1999;276:C1121-31. Fernández-Fernández M, Rodríguez-González P, Hevia Sánchez D, González-Menéndez P, Sainz Menéndez RM, García Alonso JI. Accurate and sensitive determination of molar fractions of 13 C-Labelled intracellular metabolites in cell cultures grown in the presence of isotopically-labelled glucose. Anal Chim Acta. 2017; Zhao S, Guo Y, Sheng Q, Shyr Y. Advanced heat map and clustering analysis using heatmap3. Biomed Res Int. Hindawi Publishing Corporation; 2014;2014:986048. Chong J, Wishart DS, Xia J. Using MetaboAnalyst 4.0 for Comprehensive and Integrative Metabolomics Data Analysis. Curr Protoc Bioinformatics. John Wiley and Sons Inc.; 2019;68. Sainz RM, Reiter RJ, Tan DX, Roldan F, Natarajan M, Quiros I, et al. Critical role of glutathione in melatonin enhancement of tumor necrosis factor and ionizing radiation-induced apoptosis in prostate cancer cells in vitro. J Pineal Res. 2008;45:258–70. Haga Y, Ishii K, Suzuki T. N-Glycosylation Is Critical for the Stability and Intracellular Trafficking of Glucose Transporter GLUT4. Journal of Biological Chemistry. 2011;286:31320–7. Venkateswaran V, Haddad AQ, Fleshner NE, Fan R, Sugar LM, Nam R, et al. Association of Diet-Induced Hyperinsulinemia With Accelerated Growth of Prostate Cancer (LNCaP) Xenografts. JNCI Journal of the National Cancer Institute. 2007;99:1793–800. Bader DA, McGuire SE. Tumour metabolism and its unique properties in prostate adenocarcinoma. Nat Rev Urol. Nature Research; 2020;17:214–31. Wu N, Zheng B, Shaywitz A, Dagon Y, Tower C, Bellinger G, et al. AMPK-Dependent Degradation of TXNIP upon Energy Stress Leads to Enhanced Glucose Uptake via GLUT1. Mol Cell. 2013;49:1167–75. Ancey PB, Contat C, Meylan E. Glucose transporters in cancer – from tumor cells to the tumor microenvironment. FEBS Journal. Blackwell Publishing Ltd; 2018;285:2926–43. Alimirah F, Panchanathan R, Chen J, Zhang X, Ho S-M, Choubey D. Expression of androgen receptor is negatively regulated by p53. Neoplasia. Neoplasia Press; 2007;9:1152–9. Kruiswijk F, Labuschagne CF, Vousden KH. p53 in survival, death and metabolic health: a lifeguard with a licence to kill. Nat Rev Mol Cell Biol. Nature Research; 2015;16:393–405. Quiros-Gonzalez I, Gonzalez-Menendez P, Mayo JC, Hevia D, Artime-Naveda F, Fernandez-Vega S, et al. Androgen-Dependent Prostate Cancer Cells Reprogram Their Metabolic Signature upon GLUT1 Upregulation by Manganese Superoxide Dismutase. Antioxidants (Basel) [Internet]. MDPI; 2022 [cited 2022 Dec 23];11. Available from: http://www.ncbi.nlm.nih.gov/pubmed/35204196 Tsouko E, Khan AS, White MA, Han JJ, Shi Y, Merchant FA, et al. Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth. Oncogenesis. Nature Publishing Group; 2014;3:e103. Mattaini KR, Sullivan MR, Vander Heiden MG. The importance of serine metabolism in cancer. Journal of Cell Biology. Rockefeller University Press; 2016. p. 249–57. Reina-Campos M, Linares JF, Duran A, Cordes T, L’Hermitte A, Badur MG, et al. Increased Serine and One-Carbon Pathway Metabolism by PKCλ/ι Deficiency Promotes Neuroendocrine Prostate Cancer. Cancer Cell. Cell Press; 2019;35:385-400.e9. Liu X, Choi RY, Jawad SM, Arnold JT. Androgen-induced PSA expression requires not only activation of AR but also endogenous IGF-I or IGF-I/PI3K/Akt signaling in human prostate cancer epithelial cells. Prostate. 2011;71:766–77. Pasquali R, Casimirri F, De Iasio R, Mesini P, Boschi S, Chierici R, et al. Insulin regulates testosterone and sex hormone-binding globulin concentrations in adult normal weight and obese men. J Clin Endocrinol Metab. 1995;80:654–8. Navarro G, Xu W, Jacobson DA, Wicksteed B, Allard C, Zhang G, et al. Extranuclear Actions of the Androgen Receptor Enhance Glucose-Stimulated Insulin Secretion in the Male. Cell Metab. 2016;23:837–51. Hsing AW, Chua S, Gao Y-T, Gentzschein E, Chang L, Deng J, et al. Prostate Cancer Risk and Serum Levels of Insulin and Leptin: a Population-Based Study. JNCI Journal of the National Cancer Institute. Oxford University Press; 2001;93:783–9. Hamilton EJ, Gianatti E, Strauss BJ, Wentworth J, Lim-Joon D, Bolton D, et al. Increase in visceral and subcutaneous abdominal fat in men with prostate cancer treated with androgen deprivation therapy. Clin Endocrinol (Oxf). 2011;74:377–83. Rao PM, Kelly DM, Jones TH. Testosterone and insulin resistance in the metabolic syndrome and T2DM in men. Nat Rev Endocrinol. Nature Publishing Group; 2013;9:479–93. Zhong W, Peng J, He H, Wu D, Han Z, Bi X, et al. Ki-67 and PCNA expression in prostate cancer and benign prostatic hyperplasia. Clin Invest Med. 2008;31:E8–15. De Nunzio C, Aronson W, Freedland SJ, Giovannucci E, Parsons JK. The Correlation Between Metabolic Syndrome and Prostatic Diseases. Eur Urol. 2012;61:560–70. Terracciano D, Bruzzese D, Ferro M, Mazzarella C, Di Lorenzo G, Altieri V, et al. Preoperative insulin-like growth factor-binding protein-3 (IGFBP-3) blood level predicts gleason sum upgrading. Prostate. 2012;72:100–7. Laurent V, Guérard A, Mazerolles C, Le Gonidec S, Toulet A, Nieto L, et al. Periprostatic adipocytes act as a driving force for prostate cancer progression in obesity. Nat Commun. 2016;7:10230. Watt MJ, Clark AK, Selth LA, Haynes VR, Lister N, Rebello R, et al. Suppressing fatty acid uptake has therapeutic effects in preclinical models of prostate cancer. Sci Transl Med. American Association for the Advancement of Science; 2019;11. Chang YC, Chi LH, Chang WM, Su CY, Lin YF, Chen CL, et al. Glucose transporter 4 promotes head and neck squamous cell carcinoma metastasis through the TRIM24-DDX58 axis. J Hematol Oncol [Internet]. BioMed Central Ltd.; 2017 [cited 2022 Dec 26];10:1–12. Available from: https://jhoonline.biomedcentral.com/articles/10.1186/s13045-016-0372-0 Garrido P, Osorio FG, Morán J, Cabello E, Alonso A, Freije JMP, et al. Loss of GLUT4 induces metabolic reprogramming and impairs viability of breast cancer cells. J Cell Physiol [Internet]. J Cell Physiol; 2015 [cited 2022 Dec 23];230:191–8. Available from: https://pubmed.ncbi.nlm.nih.gov/24931902/ Liu H, Lyu H, Jiang G, Chen D, Ruan S, Liu S, et al. ALKBH5-Mediated m6A Demethylation of GLUT4 mRNA Promotes Glycolysis and Resistance to HER2-Targeted Therapy in Breast Cancer. Cancer Res [Internet]. American Association for Cancer Research (AACR); 2022 [cited 2022 Dec 26];82:3974–86. Available from: https://aacrjournals.org/cancerres/article/82/21/3974/709951/ALKBH5-Mediated-m6A-Demethylation-of-GLUT4-mRNA Supplementary Files SupplementaryMaterialGonzalezMenendezPetal2023.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2619954","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":179126643,"identity":"790c9448-e915-4ef2-ba8d-8067f3836d1a","order_by":0,"name":"Pedro Gonzalez-Menendez","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pedro","middleName":"","lastName":"Gonzalez-Menendez","suffix":""},{"id":179126644,"identity":"45ac6268-3789-4db1-aabe-6f83dfe482ef","order_by":1,"name":"Alba Moran-Alvarez","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alba","middleName":"","lastName":"Moran-Alvarez","suffix":""},{"id":179126645,"identity":"8ef1a4a0-103c-4cc5-b3a9-c3b8d86a7d61","order_by":2,"name":"Juan C. Mayo","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juan","middleName":"C.","lastName":"Mayo","suffix":""},{"id":179126646,"identity":"a6d20d73-8326-42ab-b70e-c691893bdb4b","order_by":3,"name":"Rafael Cernuda-Cernuda","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Cernuda-Cernuda","suffix":""},{"id":179126647,"identity":"eb3f7ba4-877d-4c94-8c5b-fb581bac3325","order_by":4,"name":"Alejandro Alvarez-Artime","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Alvarez-Artime","suffix":""},{"id":179126648,"identity":"285fca3a-7b84-4f26-ad6d-caaf938e6764","order_by":5,"name":"David Hevia","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Hevia","suffix":""},{"id":179126649,"identity":"a32c5d9f-d296-495c-948a-d95da1162fed","order_by":6,"name":"Pablo Rodriguez-Gonzalez","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Rodriguez-Gonzalez","suffix":""},{"id":179126650,"identity":"8d7a712e-1f66-4c6b-a7ee-a359d85023c7","order_by":7,"name":"Jose I. Garcia-Alonso","email":"","orcid":"","institution":"Universidad de Oviedo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jose","middleName":"I.","lastName":"Garcia-Alonso","suffix":""},{"id":179126651,"identity":"a4ec5b0d-22a9-4f56-8f61-cf84a7adfbf3","order_by":8,"name":"Carmen Lambert","email":"","orcid":"","institution":"Hospital Universitario Central de Asturias","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Lambert","suffix":""},{"id":179126652,"identity":"4a7dec2c-503f-4974-a153-7c06ee116e0c","order_by":9,"name":"Elias Delgado","email":"","orcid":"","institution":"Hospital Universitario Central de Asturias","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elias","middleName":"","lastName":"Delgado","suffix":""},{"id":179126653,"identity":"c641d46c-ff43-459c-b0d3-476dd470e7cc","order_by":10,"name":"Rebeca Alonso-Arias","email":"","orcid":"","institution":"Hospital Universitario Central de Asturias","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rebeca","middleName":"","lastName":"Alonso-Arias","suffix":""},{"id":179126654,"identity":"d1247654-fa69-4df0-ab17-52cdc964c510","order_by":11,"name":"Miguel Alvarez-Mugica","email":"","orcid":"","institution":"Hospital Valle del Nalon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Alvarez-Mugica","suffix":""},{"id":179126655,"identity":"f50a6a27-5fea-4fc7-b3b8-087acdf26aba","order_by":12,"name":"Mario Dominguez-Esteban","email":"","orcid":"","institution":"Hospital Universitario Marques de Valdecilla","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Dominguez-Esteban","suffix":""},{"id":179126656,"identity":"a3b32af6-ca2e-487f-8989-a5bc4bcc37c4","order_by":13,"name":"Rosa M. Sainz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIie3OoQ6DMBCA4cMM0wRbkmV9hSpmSHiV1jCDXpCoIrHbW2xmaqJJBaYPwIIBw8wegLmxbFNbCriJfubM/bkDsKy/5GSv6Q6TAaAZCZKTkw/MJi6SXIn2fg4jb38LmiZVS3DzxphQzXOKu5jv6mRNmVYIkKbmBLjAVCoGdRJgLmIEOBl5rGgFZlJF5KLfCbmaH4NquCKlcg4VeibhcAVGHqta4Wcy5kcdbzHTIVqgxJyQYtP5vQyjValOfp/iyHPLkce+LGbuW5ZlWT88AONlQ0nwzDRsAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3048-5582","institution":"Universidad de Oviedo","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Rosa","middleName":"M.","lastName":"Sainz","suffix":""}],"badges":[],"createdAt":"2023-02-23 10:06:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2619954/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2619954/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":33643744,"identity":"b67bdce0-ed4c-41ed-9806-14cf8a1295ea","added_by":"auto","created_at":"2023-03-01 18:54:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1872242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAndrogens regulate the metabolism of glucose in the prostate\u003c/strong\u003e. (a) Androgens stimulate cell growth of LNCaP cells. Cells were seeded in 6-well plates. 5nM DHT was added to the culture and cells counted after 48h. 20 µM bicalutamide (CDX) was employed as an AR antagonist. (b) Glucose uptake was assessed by incubation with 2-deoxy-[1-\u003csup\u003e3\u003c/sup\u003eH]-glucose for 10 min at RT in LNCaP cells grown without androgens (CON) or in the presence of 5 nM DHT and in the presence of DHT+CDX. Uptake is presented as relative values and data are shown as individual values and mean ± SEM (n = 3). (c) Lactate levels in the extracellular medium were determined by fluorometry. Individual values and mean ± SEM are shown (n = 3). (d) PPP activity (G6PDH and 6GPD activity) was examined by enzymatic assay. Results are shown as individual values and average ± SEM (n = 3). (e) GLUT levels were analysed by western blot under the same conditions and a representative western blot is shown. Results are shown as individual values and mean ± SEM (n = 5). Protein bands were quantified and normalised to β-actin. (f) Surface GLUT1 levels were determined using an eGFP-tagged HTLV receptor-binding domain fusion protein. A representative histogram of three experiments is shown of LNCaP cells cultured in complete media (CTRL) and in androgen-depleted medium (FBS\u003csub\u003echst\u003c/sub\u003e), and in FBS\u003csub\u003echst\u003c/sub\u003e\u003csup\u003e \u003c/sup\u003emedia supplemented with DHT, or with DHT and bicalutamide (DHT + CDX). The grey histogram represents the negative control. g) The levels of pAMPK\u003csup\u003eThr172\u003c/sup\u003e and pAKT\u003csup\u003eSer473\u003c/sup\u003e were monitored by western blot. Results are presented as the ratio of phosphorylated to total protein ± SEM (n = 3). (h) GLUT levels were examined by western blot in LNCaP and castration-resistant LNCaP-R cells. Protein bands were quantified and normalised to ACTB. A representative experiment is shown. Protein levels are presented as mean ± SEM (n = 3). An arbitrary value of 1.0 was assigned to LNCaP. * p\u0026lt;0.05 * p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig1..png","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/c9060df7d7534541014afecb.png"},{"id":33643742,"identity":"046b81c4-d669-424f-b603-6bb9aa93f0c4","added_by":"auto","created_at":"2023-03-01 18:54:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":963877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLUT1 and GLUT4 overexpression changes the metabolic phenotype of prostate cancer cells\u003c/strong\u003e. (a) Glucose uptake was monitored by fluorometry in LNCaP\u003csup\u003eGLUT1\u003c/sup\u003e, LNCaP\u003csup\u003eGLUT4\u003c/sup\u003e and cells with an empty vector as control (LNCaP\u003csup\u003eMock\u003c/sup\u003e). An arbitrary value of 1.0 was assigned to one replicate value of LNCaP\u003csup\u003eMock\u003c/sup\u003e. Results are presented as mean ± SEM (LNCaP\u003csup\u003eMock\u003c/sup\u003e n=4; LNCaP\u003csup\u003eGLUT1\u003c/sup\u003e and LNCaP\u003csup\u003eGLUT4\u003c/sup\u003e n=8 each). (b) Extracellular lactate concentrations are presented as mean ± SEM (n=3). Arbitrary 1.0 was given to one of the replicates of LNCaP\u003csup\u003eMock\u003c/sup\u003e. (c) PPP activity was measured after 24 h treatment. Mean ± SEM are presented (n=3). (d) Nuclear and cytoplasmic AR levels were determined by western blot. β-actin (ACTB) was used as the cytosolic standard and HDAC2 was used as the nuclear standard. One representative experiment is shown. Mean of the 3 independent experiments is shown (n=3). A relative value of 1.0 was given to LNCaP\u003csup\u003eMock\u003c/sup\u003e. (e) Released PSA was measured by ELISA. Mean in ng PSA/µg protein ± SEM (n=3) are shown. (f) LNCaP\u003csup\u003eMock\u003c/sup\u003e and LNCaP\u003csup\u003eGLUT4 \u003c/sup\u003e5 × 10\u003csup\u003e4\u003c/sup\u003e cells were incubated during 48 hours in absence of androgens (CON) and incubated with 5 nM DHT or with DHT plus 20 µM CDX (initial concentration of). Cells were counted after that and mean ± SEM are presented (n=3). (g) Percentage of cells in the S-phase was analysed after 24 hours of treatment. Data is shown as mean ± SEM (n=3). * p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig2..png","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/9e9dc4c85907bb911dffcbb9.png"},{"id":33643747,"identity":"b05e75e6-fec1-4809-87b2-af751cdb82e7","added_by":"auto","created_at":"2023-03-01 18:54:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1856356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAR signalling favours glycolysis in prostate cancer cells. \u003c/strong\u003e(a) Glucose uptake was monitored by incubating LNCaP and PC-3 cells with 2-deoxy-[1-\u003csup\u003e3\u003c/sup\u003eH]-glucose for 10 min at RT. Uptake for each cell line is shown as mean ± standard error of the mean (SEM) (n=3). An arbitrary value of 1.0 was assigned to LNCaP cells. (b) ATP and AMP levels in LNCaP and PC-3 cells were measured by HPLC. Data were standardised to protein concentration and ATP/AMP ratio is represented. Mean ± SEM are shown (n=3). (c) PPP activity (G6PDH and 6GPD activity) was determined by enzymatic assay. An arbitrary value of 1.0 was assigned to LNCaP cells. Results are presented as mean ± SEM (n=3). (d) Cells were cultured for 24 hours with d-glucose-\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e.\u003c/sub\u003e A schematic representation of the TCA cycle is presented showing the main metabolites and their fraction that is isotopically labelled. Blue circles represent the number of potential labelled carbons in each compound. Graphs show the \u003csup\u003e13\u003c/sup\u003eC-labelled molar fraction of each compound. Enrichment among LNCaP and PC-3 cells are graphed. “m” corresponds to non-labelled metabolite and “m + n” indicates labelled at “n” carbons. Results are shown as mean ± SEM of 3 independent experiments. (e) Pathway impacts were analysed using MetaboAnalyst software. The pathway impact is plotted on the X-axis while log(p-value) is shown on the Y-axis. A colour scale from red to white is used where red indicates further significance while white corresponds to a p-value of 1. * p\u0026lt;0.05; **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig3..png","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/8f8a3c3175e51a3575f23706.png"},{"id":33643746,"identity":"2689d526-2d72-44a6-b65a-83cb6e819393","added_by":"auto","created_at":"2023-03-01 18:54:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10894628,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in GLUT levels along with prostate cancer progression. (a) GLUT levels were determined by western blot in prostate tissues from 24- and 32-week-old TRAMP mice. A representative experiment is shown (left panel). Results are shown as individual values and mean ± SEM (n=3). An arbitrary value of 1.0 was given to WT mice. (b) GLUT4 production was determined and located by immunohistochemistry in prostate tissues from 32-week-old TRAMP mice. Nuclei were counterstained with DAPI. A micrograph of each group is shown (c) GLUT-4 immunostaining intensity was measure, and 10 pictures were randomly taken within each section using the 20x objective. The average intensity of immunostaining (in a range of 0-255 values in a grayscale) was measured with the ImageJ software. (d) qPCR analysis of \u003cem\u003eScl2a1\u003c/em\u003e and (e) of \u003cem\u003eScl2a4 \u003c/em\u003ewas performed in prostate samples from 24- and 32-week-old TRAMP mice. Data is shown as individual values and mean±SEM * p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig4..png","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/187eb6510108c0ef886ccf84.png"},{"id":33643748,"identity":"8a005246-5028-4827-a83f-d95001aa4be8","added_by":"auto","created_at":"2023-03-01 18:54:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2820184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCastration regulates in GLUT4 levels in TRAMP mice\u003c/strong\u003e. (a) The levels of GLUT1 and GLUT4 were measured in 24-week-old TRAMP mice, surgically castrated TRAMP mice (CAS), and surgically castrated TRAMP mice treated with 2.5 mg/Kg/day testosterone daily for 5 days after castration (CAS + TEST). A representative western blot is shown. (b) Individual values of GLUT1 are presented (n=5), and (c) Individual values of GLUT4 upper and lower bands are presented (n=5). (e) pAMPK\u003csup\u003eThr172\u003c/sup\u003e and pAKT\u003csup\u003eSer473\u003c/sup\u003e were determined in the mouse prostate. The phosphorylated/total protein ratio was calculated. A representative western blot is shown. Individual values and mean ± SEM are represented (n=5). * p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig5..png","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/e489b570af0d1a1dbe98d2af.png"},{"id":33643745,"identity":"4b39818b-7860-468a-ad8d-8334dae12a1b","added_by":"auto","created_at":"2023-03-01 18:54:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":605466,"visible":true,"origin":"","legend":"\u003cp\u003eCirculating insulin levels are higher in TRAMP mice. (a) 2-deoxyglucose (2DG) uptake was monitored by fluorometry in LNCaP and PC-3 cells treated with 1 µM of insulin 1 hour before the assay. Results are standardised to protein concentration and presented as relative individual values and mean ± SEM. An arbitrary value of 1.0 was given to one LNCaP and one PC-3 control sample. (b) Insulin levels (ng/mL) were determined by ELISA in plasma samples from 24-week-old C57BL/6 (WT) and TRAMP mice. Results are presented as individual values and mean ± SEM (WT n=11; TRAMP n=12). (c) Circulating insulin levels were assessed in plasma collected from control 24-week-old TRAMP mice (CON), 10 days after surgical castration (CAS), and in castrated mice after administration of 2.5 mg/kg/day testosterone (CAS + TES) for 5 days. Results are shown as individual values and mean ± SEM (n = 5). (d) Fasting glucose blood levels (mg/dL) were measured in 24 weeks old WT and TRAMP mice. Results are shown as individual measurements and mean ± SEM (WT n = 5, TRAMP n = 8). (e) Glucose tolerance tests were performed in 16-week-old WT, TRAMP, and surgically castrated TRAMP mice (surgery at 12 weeks old). 2 mg glucose/g body weight was injected i.p. and glucose was measured at 0, 15-, 30-, 60- and 120-min. Results (mg/dL) are presented as mean ± SEM (n = 4). The area under the curve (AUC) of the glucose tolerance test was calculated. An arbitrary value of 1.0 was given to one replicate from WT mice. Individual results and mean are shown ± SEM (n = 4). * p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig6..png","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/1a4e8a71822a7723cd7e1cc4.png"},{"id":33643750,"identity":"64d2e596-d2e1-49a3-981d-d3bc2dfc7406","added_by":"auto","created_at":"2023-03-01 18:54:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6373673,"visible":true,"origin":"","legend":"\u003cp\u003eSTZ-induced T1DM reduces prostate cancer progression. (a) Type I diabetes mellitus was induced by treating 16-week-old TRAMP mice with 50 mg streptozotocin/kg bodyweight for 5 days (STZ). Diabetic animals were given 0.6 U of Humulin 70:30 for 6 weeks until sacrificed at 24 weeks old (STZ + Ins). Images of the urogenital tracts were taken after the sacrifice (above). Haematoxylin–eosin staining was performed in fixed tissues (below). Original magnification 200× and 400×. A representative picture is shown for each experimental group. (b) Genitourinary/body weight ratios were calculated after sacrifice. Results are shown as individual values and mean ± SEM (n=6). (c) Circulating testosterone levels were measured in plasma by ELISA. Results are reported in pg/m. Individual values and mean ± SEM (n=6) are shown. (d) PCNA, Bcl2, BAX, TP53 and p21 protein levels in prostatic tissue were determined by western blot. ACTB was employed as the internal standard and three samples per group are shown. An arbitrary value of 1.0 was assigned to one of the TRAMP results and amounts of PCNA and BAX protein are presented as individual data and mean ± SEM (n=6). (e) pAKT\u003csup\u003eSer476\u003c/sup\u003e levels were analysed in prostatic tissue by western blot and three representative samples per condition are shown. pAKT\u003csup\u003e Ser476\u003c/sup\u003e/AKT ratio was calculated and normalised to ACTB. Results are shown as individual measurement and mean ± SEM (n = 6). (f) GLUT4 levels were determined by western plot in prostatic tissues. Three representative samples per group are shown. ACTB was used as the internal control. Protein amounts for the upper and lower bands were calculated independently and the results are shown as individual data and mean ± SEM (n=6). One control was arbitrarily assigned a value of 1.0. * p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig7..png","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/2ac39fa2b118b0fbb9b685bc.png"},{"id":33643751,"identity":"85bf7979-a144-4c48-ab59-6ba9db0a29be","added_by":"auto","created_at":"2023-03-01 18:54:46","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":7717406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInsulin dependent glucose transporters increase in tumors with higher Gleason score.\u003c/strong\u003e GLUT4 production in the prostates of patients with prostate cancer determined by western blot. Samples were classified as Non-tumour (Norm), hyperplasic (Hyp), and tumour samples were classified according to their Gleason score (\u0026lt;7 and ≥7). Protein bands were quantified and normalised to ACTB. Data are shown as mean ± SEM (n = 5). (b) Representative micrographs of GLUT4 immunolabeling (shown in green) in several regions of human prostatectomy samples. Tumor regions with Gleason’s grades 3 (GS3) and 4 (GS4) and Gleason’s grade 5 (GS5). DAPI labeling (in blue) was merged with GLUT-4 immunostaining in the lower panels. Calibration bar (for all panels): 50 µm. (c) Sections were photographed with a Zeiss Axiocam 712 monochrome camera mounted on a Nikon Eclipse 80i microscope and 9 pictures were randomly taken within the section using the 20x objective, Quantification was performed by using ImageJ software. * p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig8..jpg","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/e46a146dffdd430f63a72259.jpg"},{"id":33644367,"identity":"f9456f84-5b98-4861-8ba5-18251ebd4554","added_by":"auto","created_at":"2023-03-01 19:02:46","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4493,"visible":true,"origin":"","legend":"\u003cp\u003eAR immunohistochemistry in non-diabetic and diabetic prostate tumors. (a) Representative micrographs of prostatic biopsies from non-diabetic (GS6) and non-diabetic (B) subjects. The sections were immunolabeled with anti-GLUT4 (green) antisera. (b) anti-AR (red) and anti-GLUT-4 (green) antisera. A few non-AR-immunolabeled nuclei are shown in blue (DAPI staining). Calibration bars: 50 µm.\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/da372a0eae0e4725476310f5.png"},{"id":33804904,"identity":"1218e1b3-d467-42cb-ba25-58f3b585cf5d","added_by":"auto","created_at":"2023-03-05 21:18:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3154604,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/e723bb82-d866-4414-8250-a0333f7563f0.pdf"},{"id":33644368,"identity":"6b2fadd5-606a-4468-97e8-9e714995ff43","added_by":"auto","created_at":"2023-03-01 19:02:46","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":1190453,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialGonzalezMenendezPetal2023.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2619954/v1/7a971067c46a77fd1ed667af.pdf"}],"financialInterests":"","formattedTitle":"Insulin-dependent GLUT4 is a risk factor for cancer in the prostate","fulltext":[{"header":"Background","content":"\u003cp\u003eFirst described by Otto Warburg more than 90 years ago, cancer cells usually exhibit an intrinsic metabolic switch that is considered one of the hallmarks of cancer[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Tumour cells typically show increased nutrient uptake compared to normal cells, particularly glucose and glutamine, to facilitate their growth[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The higher glucose uptake feeds the so-called \u0026lsquo;aerobic glycolysis\u0026rsquo; observed in these cells, leading to higher lactate production and release. Consequently, aerobic glycolysis equips tumour cells with the necessary macromolecular precursors for cell growth without compromising energy production[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, targeting glucose metabolism is one of oncology\u0026rsquo;s most promising treatment options[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This higher glucose uptake rate observed in cancer compared to normal cells can be explained by changes in protein levels or the membrane location of glucose transporters. The GLUT (glucose transporter)/\u003cem\u003eSLC2A\u003c/em\u003e family is related to the transcriptional activation of several oncogenes[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Even though such an increase in glucose uptake has been mainly associated with GLUT1 overexpression, it may also involve other members of the GLUT family of transporters, including the structurally related insulin-dependent GLUT4[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Why carcinogenesis becomes insulin-sensitive in some tissues is one of the unanswered questions in oncology.\u003c/p\u003e \u003cp\u003eProstate metabolism is highly specialized to produce citrate in the prostatic fluid by inhibiting the isomerization of citrate to isocitrate. Such inhibition compromises the tricarboxylic acid (TCA) cycle, meaning glycolysis is favoured in the normal prostate epithelium. Zinc accumulates in the prostate epithelia and inhibits the TCA cycle enzyme aconitase (ACO2) that prevents citrate oxidation to isothiocyanate. This characteristic metabolism suggests that prostate cancer also displays a unique metabolism. Oxidative phosphorylation (OXPHOS) is stimulated in the first stages of carcinogenesis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although like many other tumours, in the later stages, prostate cancer (PCa) tissue is considered glycolytic[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Given this unique metabolic profile, glucose metabolism was not considered to be as important as glutamine or lipid metabolism in PCa and has thus received less research attention than in other cancers[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Androgens stimulate citrate production and secretion and near all the metabolic machinery that characterize the gland. In prostate tumors, androgens regulate zinc and aspartate transporters[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Metabolic shift between glycolysis and oxidative phosphorylation in prostate tumors also relies on AR function during oncogenic transformation. Androgen stimulation increases glucose uptake[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] but also, it drives lipogenesis and oxidative phosphorylation [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, the transition to a castration-resistant phenotype (CRPC) is accompanied by changes in glucose consumption and biomass production. Clinical imaging using the radiolabelled glucose analogue 18F-fluorodeoxyglucose (FDG) is not suitable to detect local prostate cancer, whereas neuroendocrine prostate cancer is thought glycolytic and detectable on FDG imaging. Which confirms the metabolic shift during transition to androgen independence[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. During the last decade, the number of studies showing the importance of glucose metabolism in PCa has increased dramatically and several inhibitors of glucose metabolism are in early phase clinical trials, however further investigation is still needed[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMolecularly, the mitochondrial pyruvate carrier protein has been shown to be transcriptionally regulated by AR signalling, increasing the glucose flux towards the mitochondria[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and some reports indicate that androgens regulate the expression of GLUT1 in PCa cells[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. GLUT1 production has been observed in normal prostate glands as well as in the most aggressive tumours[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] but less is known about other glucose transporters. The reduce prevalence of prostate cancer in diabetic patients suggest a role of insulin glucose transporters in prostate cancer still unknown. Recently, our own group reported the expression and production of insulin dependent GLUT4 glucose facilitative transporter in PCa cells[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], however there are no data regarding its regulation or role in tumour progression yet. Its expression could be somehow linked to the inverse relationship between diabetes and PCa progression[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], but the molecular mechanisms of which are unknown.\u003c/p\u003e \u003cp\u003eThus, the main aim of this work was to decipher the role of GLUT4 during PCa progression and determine its involvement in the inverse relationship between diabetes and prostate cancer.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eCell culture, transfection, and treatments\u003c/h2\u003e\n\u003cp\u003eHuman androgen-sensitive LNCaP cells were purchased from the European Collection of Cell Cultures (catalogue no. 89110211) and cultured in RPMI 1640 medium (Lonza, catalogue no. 12-719F) supplemented with 10% FBS, 2 mM l-glutamine, 15 mM HEPES and a 1% antibiotic\u0026ndash;antimycotic cocktail. PC-3 cells, a human androgen-insensitive cell line, were purchased from American Type Culture Collection (catalogue no. CRL-1435) and grown in DMEM/F12 medium (Lonza, catalogue no. BE12-702F) supplemented with 10% FBS, 2 mM l -glutamine and a 1% antibiotic\u0026ndash;antimycotic cocktail. Both cell lines were authenticated by short tandem repeat profiling. Cell lines were grown at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e environment.\u003c/p\u003e\n\u003cp\u003eGLUT1- or GLUT4-overexpressing LNCaP cells were obtained by using FuGENE\u0026reg;HD (Promega, catalogue no. E2311) following the manufacturer\u0026rsquo;s instructions. Plasmids pcDNA3.2/v5-DEST hGLUT1 (Addgene plasmid no. 18085) and pcDNA3.2/v5-DEST hGLUT4 (Addgene plasmid no. 18087) were supplied by Wolf Frommer (Heinrich Heine University D\u0026uuml;sseldorf) [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cells were selected by incubation with 300 \u0026micro;g/mL G418 (Merck, catalogue no. A1720).\u003c/p\u003e\n\u003cp\u003eTo inhibit the Pentose Phosphate Pathway (PPP), cells were treated with 10 \u0026micro;M of 6-aminonicotinamide (6AN, Merck, catalogue no. A68203) for 48 hours. Androgen stimulation was performed by growing androgen-sensitive cells in androgen-deprived medium supplemented with dextran\u0026ndash;charcoal FBS (FBS\u003csub\u003echst\u003c/sub\u003e). Then, 5 nM of dihydrotestosterone (DHT) (Merck, catalogue no. A8380) was used to stimulate ARs. 20 \u0026micro;M of the antiandrogen bicalutamide (CDX, Merck, catalogue no. B9061) was employed in the presence of DHT. Insulin (Ins, Merck, catalogue no. I9278) was added at 1 \u0026micro;M. For all treatments, cells were left to attach for 48 hours. The glucose concentration was always set at 2 g/L (11 mM). Vehicle alone was added to the control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eAnimal procedure\u003c/h2\u003e\n\u003cp\u003eTransgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice (C57BL/6-Tg(TRAMP)8247Ng/J) were used (The Jackson Laboratory)[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Prostates were classified by a pathologist as well, moderately, or poorly differentiated tumours. Mice were maintained under controlled environmental conditions (12:12 light:dark) with ad libitum access to food and drinking water. Animals were sacrificed in a CO\u003csub\u003e2\u003c/sub\u003e chamber and the genitourinary tract was immediately dissected and frozen in liquid nitrogen or fixed in 10% phosphate-buffered formalin.\u003c/p\u003e\n\u003cp\u003eCastration was done by radical bilateral orchiectomy after anaesthesia with 100 mg/kg ketamine (Imalgene; Merial) and analgesia with 20 mg/kg xylazine (Rompun; Bayer). For androgen replacement in castrated mice, 5 days after surgery testosterone was injected s.c. (2.5 mg/kg body weight). Animals were sacrificed at the indicated times.\u003c/p\u003e\n\u003cp\u003eFor diabetes induction, 50 mg/kg streptozotocin (STZ, Merck) was administered i.p. daily for 5 days. After two weeks, 0.6 U insulin (Humulin 70:30, Lilly) was injected s.c. daily. Insulin was always administered in the morning. Fasting blood glucose levels were measured with levels above 250 mg/dL for at least three consecutive days indicating diabetes. Mice exhibiting levels over 500 mg/dL were discarded.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003ePatient samples\u003c/h2\u003e\n\u003cp\u003eParaffin-embedded tissue samples from 132 patients were collected and their medical records, including data related to relapses, metastases, perineural invasion and PSA levels, were provided (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Samples were classified by Gleason score by the hospital pathologist. Frozen non-pathologic, hyperplastic and tumour tissues were collected from 20 patients. Patients underwent surgery at Hospital Universitario Central de Asturias, Hospital Valle del Nal\u0026oacute;n (Asturias) or Hospital Universitario Marqu\u0026eacute;s de Valdecilla (Cantabria).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eClinical characteristics of diabetic patients\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePatient\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAge\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eT1DM\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAge at diagnosis\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePSA\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGleason\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTumour Grade\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGlycosylated Hb (A1c) %\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGlucose\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e154\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (3\u0026thinsp;+\u0026thinsp;3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e158\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e59\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (3\u0026thinsp;+\u0026thinsp;4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e157\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (3\u0026thinsp;+\u0026thinsp;4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e298\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"9\"\u003e\u003csup\u003e1\u003c/sup\u003e T1DM, Type 1 Diabetes Mellitus\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"9\"\u003e\u003csup\u003e2\u003c/sup\u003e Levels of PSA, in ng/ml\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"9\"\u003e\u003csup\u003e3\u003c/sup\u003e Fasting glucose levels, in mg/dl\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003ePAGE and immunoblotting\u003c/h2\u003e\n\u003cp\u003eProtein extracts from culture cells were obtained, separated and electrotransferred as previously described[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. For tissues, a section of 50 mg representing all the prostatic lobes were cut and embedded in 1 mL of chilled RIPA lysis buffer. Samples were homogenized employing an ULTRA-TURRAX\u0026reg; homogenizer, and same procedure to culture cells was followed. Antibodies \u003cstrong\u003e(Supplementary Table\u0026nbsp;1)\u003c/strong\u003e were visualized by binding horseradish peroxidase-conjugated secondary antibodies and detected with chemiluminescence substrate. Densitometry values were obtained using ImageJ and Image Studio software.\u003c/p\u003e\n\u003cp\u003eTo study N-glycosylation of GLUT4 protein, muscle and prostate tissue from TRAMP mice were homogenized in the protein extraction buffer described by Haga Y. et al [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. Tissue lysates were incubated in ice for 30 minutes, then they were centrifugated for 15 min at 15000xg and supernatant were collected. Supernatants were concentrated using Amicon Ultra-0.5 centrifugal filter devices (Amicon Ultra 10K device. 100000 BNWL, Millipore) following manufacturer instructions. Concentrated proteins were diluted in 1:4 in DEPC treated Water and stored upon usage. Protein extracts were quantified using Bradford reagent (Merck). To remove glycosylation residues, 20 \u0026micro;g of protein were treated with PNGaseF reagent (\u003cem\u003eNew England Biolabs\u003c/em\u003e) under denaturing conditions following manufacturer's instructions. Treated extracts were loaded into an SDS-page (7.5% acrylamide) and electrophoresis and western-blot were performed like the other experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunocytochemistry and Immunohistochemistry studies\u003c/h2\u003e\n\u003cp\u003eCells were fixed in phosphate-buffered 2% paraformaldehyde (H 7.4). For IHC, 5-\u0026micro;m thick paraffin-embedded tissue sections were deparaffinised and hydrated in a graded series of EtOH solutions. Antigen retrieval was carried out by microwaving. After blocking with 3% goat serum in TBS (Tris-HCl 20 mM pH 7.4, 150 mM NaCl), samples were incubated for 20 min at room temperature with 0.15% Tween-20 for cytoplasmic permeabilization or 0.1% Triton X-100 for nuclear permeabilization. Immunofluorescence was observed under a Leica TCS SP8 confocal microscope. The antibodies and dilution factors used in immunocytochemistry and immunohistochemistry studies are listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;1.\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eAR and GLUT-4 immunostaining in human biopsies\u003c/h2\u003e\n\u003cp\u003eDouble immunolabeling with a monoclonal mouse anti-AR and a rabbit polyclonal anti-GLUT4 antiserum was carried out in two groups of prostate biopsies from diabetic and non-diabetic human patients (n\u0026thinsp;=\u0026thinsp;4 for each group). Briefly, the sections were immersed in EDTA pH 9.0 at 95\u0026deg;C for 20 minutes for antigen retrieval and subsequently incubated in the anti-AR antiserum at 4 \u0026ordm;C overnight (1:5000 dilution). The AR immunolabeling was detected using a standard procedure with the Vectastain ABC kit (Vector) and diaminobenzidine (DAB) as chromogen. In previous experiments, this method had proved to be more effective than IF when labelling with this particular antiserum. Due to the amplification of the signal obtained with the ABC method, a highly diluted primary antibody provided a clear signal, with little background staining. Immunofluorescence was then carried out to detect GLUT4 on these same sections. The samples were incubated with the anti-GLUT4 antiserum (1:250 dilution) at 4 \u0026ordm;C overnight and, subsequently, in an AlexaFluor 488 goat-anti-rabbit antiserum to visualize the immunolabeled regions. DAPI was used as a nuclear counterstaining. In order to obtain merged pictures with the double immunostaining, the sections were photographed with a Zeiss Axiocam 712 monochrome camera mounted on a Nikon Eclipse 80i microscope under both bright field and fluorescence conditions. The bright field pictures corresponding to AR-immunolabeling were then inverted, turned red and merged with those of GLUT-4 immunostaining and DAPI counterstaining with Adobe Photoshop CS8. To measure the GLUT-4 immunostaining intensity of each biopsy, 9 pictures were randomly taken within the section using the 20x objective. The average intensity of immunostaining (in a range of 0-255 values in a grayscale) was measured with the ImageJ software.\u003c/p\u003e\n\u003cp\u003eIn a parallel experiment, samples of prostatectomy from 15 patients diagnosed with different Gleason scores (which ranged between 3\u0026thinsp;+\u0026thinsp;3 and 4\u0026thinsp;+\u0026thinsp;5) were immunostained for GLUT-4 following a protocol like the one detailed above, omitting the initial AR immunostaining.\u003c/p\u003e\n\u003cp\u003eTumor regions with different Gleason grades (ranging between 3 and 5) were present in all the samples, which also contained healthy (non-tumour) glands. A minimum of 5 pictures from each (including non-tumour regions) were taken using the 20x objective and the same photographic conditions. To obtain an estimate of the average immunostaining intensity within the prostatic gland epithelia, the stromal areas were manually removed in all photographs with Adobe Photoshop CS8. Next, GLUT-4 immunostaining was measured using the ImageJ software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eFlow cytometry\u003c/h2\u003e\n\u003cp\u003eThe cell cycle was studied by staining with 50 \u0026micro;g/ml propidium iodide (PI) in cells fixed with 70% ethanol. Surface GLUT1 levels were monitored as previously reported[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Data analysis was performed using FlowJo (version 10.6.2) and Kaluza (version 2.1) software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eReal-time PCR\u003c/h2\u003e\n\u003cp\u003eRNA was isolated using TriReagent\u0026reg; (Merck) and cDNA was synthesised using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). qRT-PCR was performed using the Light Cycler 480 SYBR Green I Master Kit (ThermoFisher Scientific). The specific primers are listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;1.\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eMetabolic assays\u003c/h2\u003e\n\u003cp\u003eGlucose uptake was measured as described previously[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e. For standardisation, number of cells were previously estimated. ATP/AMP determination was achieved by HPLC. Nucleotide extraction and measurement were performed as previously described[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Protein concentration was quantified by Bradford Assay for standardisation. Glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase enzyme activities were measured as previously described[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] and employed to evaluate the activity of PPP.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eStable isotope labelling experiments\u003c/h2\u003e\n\u003cp\u003eCells were grown in culture media as mentioned above but supplemented with 2 g/L U-\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e-glucose (Merck) for 24 hours. Extraction of the intracellular metabolites with methanol and ultrapure water, derivatisation and GC-MS measurement of metabolite-specific isotopic distributions were carried out as previously reported[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Heatmap and clustering analysis were performed using the heatmap3 add-in package with R project open-source software (version 3.4)[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Enrichment and pathway analysis were done using MetaboAnalyst (version 4.0)[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eExtracellular lactate levels\u003c/h2\u003e\n\u003cp\u003eCells were seeded in 24-well plates and harvested by scraping when reached 70% confluence. After centrifugation to eliminate cell debris, the cell culture medium was collected and deproteinised with 4 M perchloric acid and neutralised with 2 M KOH. The pH was adjusted to 7.4. The lactate determination assay was performed following the manufacturer\u0026rsquo;s instructions (Bioquochem S.L.).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003ePSA levels\u003c/h2\u003e\n\u003cp\u003ePSA was measured by ELISA following the manufacturer\u0026rsquo;s instructions (Human Diagnostics). Total protein concentration was quantified by Bradford Assay for standardisation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eFasting blood glucose and glucose tolerance test\u003c/h2\u003e\n\u003cp\u003eFasting blood glucose was measured using a One Touch Ultra Easy\u0026trade; glucometer (Johnson \u0026amp; Johnson). Blood was taken from the tail vein in mice fasted overnight.\u003c/p\u003e\n\u003cp\u003eTo measure glucose tolerance, mice received an intraperitoneal injection of glucose (2 mg/g body weight) and blood glucose levels were determined after 15 min, 30 min, 1 hour and 2 hours. The AUC was then calculated by the linear trapezoidal method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eBlood insulin levels\u003c/h2\u003e\n\u003cp\u003eInsulin levels were measured in the plasma of overnight-fasted mice. Blood was extracted post-mortem, collected in EDTA-treated tubes and immediately centrifuged at 3000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at RT. Samples were frozen at -80\u0026deg;C until analysis. Insulin levels were measured by ELISA following the manufacturer\u0026rsquo;s instructions (Millipore, catalogue no. EZRMI-13K).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eBlood testosterone levels\u003c/h2\u003e\n\u003cp\u003eBlood was extracted post-mortem, collected in EDTA-treated tubes, and immediately centrifuged at 3000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min. Samples were frozen at -80\u0026deg;C until analysis. Testosterone levels were measured by ELISA following the manufacturer\u0026rsquo;s instructions (Cayman).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistics\u003c/h2\u003e\n\u003cp\u003eData are represented as individual values, or the mean and error bars represent the standard error of the mean (SEM). Outliers were identified using the Rout test, while normality was studied using the Shapiro\u0026ndash;Wilk or Kolmogorov\u0026ndash;Smirnov tests depending on the number of samples. If the samples followed a normal distribution, the significance was calculated using the two-tailed unpaired Student\u0026rsquo;s t-test for comparison of two groups while one-way ANOVA followed by Fisher\u0026rsquo;s LSD test was used for comparison of more than two groups. For samples that did not present a normal distribution, we used the non-parametric Kruskal\u0026ndash;Wallis test followed by Benjamini, Krieger and Yekutieli\u0026rsquo;s two-stage linear step-up procedure. The tests were performed using Prism GraphPad (version 8). The significance is indicated as * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eAndrogens regulate insulin independent GLUT transporters in androgen dependent LNCaP cells\u003c/h2\u003e\n\u003cp\u003eAndrogens regulate glucose metabolism in prostate cancer cells through the modulation of glycolic proteins. However, the role of insulin dependent glucose transporters in prostate cancer progression is still under unknown. To confirm androgen control over energy metabolism in the prostate we studied the effect of DHT treatment on glucose uptake and glucose transporters production androgen sensitive LNCaP cells. Thus, to verify DHT biological activity, first we confirmed DHT incubation significantly increased cell proliferation as expected (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). In addition, we found that DHT increased the uptake of glucose (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb), it reduced the production and release of lactate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec) and diminished Pentose Phosphate Pathway (PPP) activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). Incubation with the antiandrogen bicalutamide (CDX) restored the levels to those of the control cells, indicating the specificity of androgen signalling in these effects.\u003c/p\u003e\n\u003cp\u003eIn addition, DHT increased the production of GLUT1 and GLUT3 both insulin independent glucose transporters. However, it does not affect GLUT4, and it reduced the levels of GLUT12 both insulin dependent glucose transporters. Effects also restored by CDX pre-incubation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee). GLUT1 activity in cells is primarily regulated through translocation of GLUT1 to the plasma membrane. Since GLUT1 appeared to be the main target of androgens in LNCaP cells, we studied the membrane exposure of the transporter after the addition of androgens. We found that androgens removal by charcoal-stripped FBS incubation slightly decreased the presence of GLUT1 in the membrane \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef, \u003cstrong\u003eleft pannel)\u003c/strong\u003e as shown by flow cytometry immunostaining. However, membrane GLUT1 levels were not restored after DHT treatment nor influenced by CDX treatment \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef, \u003cstrong\u003eright pannel)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eMaster metabolic regulators, AMPK or AKT, were also found to be regulated by androgens (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg). The stimulation of LNCaP cells with DHT increased the level of pAMPK\u003csup\u003eThr172\u003c/sup\u003e by six-fold and significantly decreased the phosphorylation of pAKT\u003csup\u003eSer473\u003c/sup\u003e. Again, CDX recovered their levels showing the specificity of androgens. This increment in AMPK activity might support the increment of glucose uptake since it participates in their translocation to the cell membrane.\u003c/p\u003e\n\u003cp\u003eTo confirm the effects of androgen signalling on glucose transporters, we employed hormone-sensitive and castration-resistant LNCaP cells (LNCaP-R). LNCaP-R were previously established by permanent growth without androgens[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. First, we noticed that GLUT1 was significantly lower in the castration-resistant LNCaP-R corroborating its dependence of androgens but GLUT3, GLUT4 and GLUT12 were higher in LNCaP-R than in the LNCaP parental cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eOverexpression of GLUT4 favours an androgen-independent phenotype\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eGlucose uptake is the first step in the metabolic shifting of cancer cells but the effect of glucose transporters on prostate cancer phenotype is scarcely known. We investigated the effects of GLUT1 and GLUT4 overexpression, an insulin independent and dependent glucose transporter, on LNCaP cells. First, overexpression was confirmed by western blotting (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;1a\u003c/strong\u003e). As expected, the overexpression of both transporters increased glucose uptake in hormone-sensitive LNCaP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea), although GLUT1 overexpressing cells grow faster than LNCaP\u003csup\u003eMock\u003c/sup\u003e or LNCaP\u003csup\u003eGLUT4\u003c/sup\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;1b\u003c/strong\u003e). Interestingly, GLUT1 and GLUT4 overexpressing cells exhibited opposite tendency of lactate production. LNCaP cells overexpressing GLUT1 showed a significant reduction of lactate release, while overexpression of GLUT4 significantly enhanced its into the culture media (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). There were no significant differences in PPP activity in both overexpressing clones and the parental mock-transfected LNCaP cells (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;1c\u003c/strong\u003e). However, GLUT1-overexpressing cells were more sensitive than parental cells to glucose-6-phosphate dehydrogenase (G6PDH) inhibition, the first enzyme in the PPP pathway. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec)\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eTo investigate the role of glucose transporters on AR location and activity, western blot was performed after GLUT1 or GLUT4 overexpression. Though total protein levels did not change, nuclear AR was significantly lower in LNCaP\u003csup\u003eGLUT4\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed) but prostate-specific antigen (PSA) release was significantly increased in LNCaP\u003csup\u003eGLUT4\u003c/sup\u003e cells versus parental LNCaP or LNCaP\u003csup\u003eGLUT1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). A decrease in nuclear AR with an increased release of PSA production might be an indicator of the androgen independence of GLUT4-overexpressing prostate cancer cells. This fact was confirmed by investigating the response of LNCaP\u003csup\u003eGLUT4\u003c/sup\u003e cells to DHT in terms of cell proliferation. DHT treatment increased the proliferation of LNCaP\u003csup\u003eMock\u003c/sup\u003e cells as expected, and this increase was prevented by co-incubation with CDX. However, LNCaP\u003csup\u003eGLUT4\u003c/sup\u003e cells did not show any response to DHT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef). Likewise, the number of cells in the S-phase of the cell cycle was increased in LNCaP\u003csup\u003eMock\u003c/sup\u003e studied by flow cytometry while it was significantly decreased in LNCaP\u003csup\u003eGLUT4\u003c/sup\u003e cells after treatment with DHT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg). All these results point that overexpression of GLUT4 encourages an androgen independence.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eHormone resistant cells have an increase in glucose uptake and a glycolytic metabolism\u003c/h2\u003e\n\u003cp\u003eProstate metabolism is highly specialised to produce citrate in prostatic fluid, which provides the gland with unique metabolic properties. The normal prostate epithelium favours glycolysis over oxidative phosphorylation under aerobic conditions, given the physiological truncation of the TCA cycle. Metabolic reprogramming in prostate tumours leads to a reduction of glycolysis and enhanced mitochondrial oxidative phosphorylation, which are believed to be regulated by androgen signalling. However, progression to a hormone independent phenotype increase glycolytic metabolism in the tumour prostate.\u003c/p\u003e\n\u003cp\u003eFirst, we study differences in mitochondrial metabolism between hormone sensitive LNCaP cells and hormone independent PC-3 cells. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, hormone-resistant PC-3 cells showed higher levels of glucose uptake, a reduce production of ATP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb) and a lower activity of PPP when compared to LNCaP cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec) than androgen-sensitive LNCaP cells which implies a glycolytic phenotype. To determine whether androgen signalling influence the TCA cycle in prostate cancer cells, we investigated glucose flux by GC-MS-based \u003csup\u003e13\u003c/sup\u003eC metabolic analysis. Cells were incubated with \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e-labelled glucose for 24 hours and the incorporation of \u003csup\u003e13\u003c/sup\u003eC from the 13-labelled glucose in the metabolites was determined by measuring their isotopologue distribution by GC-MS. Mass isotopologue distributions were employed to calculate the molar fraction of \u003csup\u003e13\u003c/sup\u003eC incorporated. Thus, m0, m1 and m2 refer to the incorporation of zero, one and two \u003csup\u003e13\u003c/sup\u003eC atoms in the metabolite, respectively. Lactate, glutamate and TCA pathways allowed us to analyse metabolic flux by calculating the different molar fractions of \u003csup\u003e13\u003c/sup\u003eC incorporated in each metabolite. The absence of AR in PC-3 reduced the molar fraction of M0 in lactate (30% in comparison with LNCaP cells) and significantly increased the incorporation of \u003csup\u003e13\u003c/sup\u003eC into citrate. The incorporation of \u003csup\u003e13\u003c/sup\u003eC in succinate was much faster in LNCaP than in PC-3 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). Although the differences between LNCaP and PC-3 might be due to different metabolic players, such as TP53, our results suggest that AR signalling has a clear metabolic influence on the TCA cycle and favours glycolysis in prostate cancer cells.\u003c/p\u003e\n\u003cp\u003eEnrichment and pathway analyses were performed using MetaboAnalyst 4.0 to compare LNCaP vs. PC-3. The pathway enrichment analysis showed that androgen dependency promoted tryptophan metabolism, gluconeogenesis and Warburg rewiring, as well as increasing the cells\u0026rsquo; reductive power by increasing glutathione metabolism (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee). The alanine, arginine and proline, glutathione and glutamate metabolism pathways also showed significant differences in both comparisons. The TCA cycle was also shown to be affected by AR presence, confirming that androgen-sensitive and insensitive cells have different energy phenotypes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n\u003ch2\u003eFacilitative insulin-independent and -dependent GLUT levels increase with PCa progression\u003c/h2\u003e\n\u003cp\u003eThe role of insulin-independent and -dependent glucose transporters in cancer progression was studied using TRAMP mouse prostate tissues. Tumours were classified by a pathologist into three categories: well (WD), moderately (MD) or poorly differentiated (PD) and tissues were collected in 24- and 32-week-old animals. The androgen-dependent prostate secretory protein of 94 amino acids (\u003cem\u003ePsp94\u003c/em\u003e) gene was chosen as a marker of hormone dependence of tumours since rodents have no counterpart of PSA. As shown in \u003cstrong\u003eSupplementary Fig.\u0026nbsp;2a\u003c/strong\u003e, PD tumours exhibited an androgen-insensitive phenotype since they did not express \u003cem\u003ePsp94\u003c/em\u003e. The levels of insulin-independent GLUT1 and GLUT3 transporters increased along with tumour progression, as previously suggested by others[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. But interestingly, it was found that also insulin-dependent transporter GLUT4 increased in prostate tumour tissues with progression, showing a significant increment in PD tumours. In the case of GLUT12, also considered to be an insulin-responsive glucose transporter, no significant differences were found (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Immunostaining confirmed that insulin-responsive GLUT4 was found in epithelial prostate tissue the highest levels found within the most aggressive areas of the tumours, and they were increased in PD when compared with WT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb,c). GLUT4 validation was performed by immunostaining of muscle and adipose tissue (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2b\u003c/strong\u003e). GLUT4 staining concurs with conventional prostate markers BCL-2, AR and Hypoxia-Inducible Factor (HIF)1\u0026alpha;, which are all increased in androgen-resistant tumours (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWhile production of GLUT1 and GLUT4 is increased during PCa progression, the expression of \u003cem\u003eScl2a1\u003c/em\u003e and \u003cem\u003eScl2a4\u003c/em\u003e was significantly reduced in tumour tissues compared to normal tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, e), while there were no differences in \u003cem\u003eScl2a3\u003c/em\u003e or \u003cem\u003eScl2a12\u003c/em\u003e (data not shown).\u003c/p\u003e\n\u003cp\u003eTo confirm the effect of androgens \u003cem\u003ein vivo\u003c/em\u003e, TRAMP mice were surgically castrated for 5 days and then treated daily with testosterone for a further 5 days. The effect of castration was confirmed by genitourinary (GU) weight/ Body weight (BW) (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3a\u003c/strong\u003e). Castration significantly reduced GU weight, while testosterone recovered it. GLUT1 levels did not change shortly after castration but they are significantly reduced after testosterone injection \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cstrong\u003e)\u003c/strong\u003e. Regarding GLUT4, a double band was detected. Significant differences were found in the abundance of upper and lower band. GLUT4 upper band was significantly reduced or even absence after castration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b\u003cstrong\u003e)\u003c/strong\u003e. Double band of GLUT4 might be related with the glycosylation of the transporter (upper band). The glycosylation of GLUT4 is related with a higher activation in the transport of glucose[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Intriguingly, the glycosylated form of GLUT4 disappeared and protein levels significantly decreased after castration in TRAMP mice and the upper band was again detected in presence of testosterone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). The treatment of TRAMP prostate protein samples with the amidase PNGase F, which cleaves at N-acetylglucosamine (GlcNAc) and asparagine residues of high mannose oligosaccharides, confirmed the changes in electrophoresis mobility claimed as a demonstration of glycosylation of the transporter. Muscle protein samples, a positive control of GLUT4 glycosylation was employed as positive controls \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed\u003cstrong\u003e)\u003c/strong\u003e. Surprisingly, the transcription of \u003cem\u003eScl2a1\u003c/em\u003e was upregulated after testosterone administration, perhaps as a consequence of protein reduction (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3b\u003c/strong\u003e) while \u003cem\u003eScl2a4\u003c/em\u003e did not change significantly pointing a functional modification by castration more than a transcriptional regulation (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3c\u003c/strong\u003e). Castration did not alter the levels of pAMPK\u003csup\u003eThr172\u003c/sup\u003e, though a significant increase was found after testosterone treatment vs CON. On the other hand, pAKT\u003csup\u003eSer473\u003c/sup\u003e was augmented after acute castration, but its levels were not restored by testosterone injection in TRAMP mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e\n\u003cp\u003eTRAMP mice were also long-term castrated by surgery at 12 weeks of age and further sacrificed when they were 24 or 32 weeks old. The effect of castration was confirmed by the significant reduction of GU weight/BW \u003cstrong\u003e(Supplementary Fig.\u0026nbsp;3d)\u003c/strong\u003e. GLUT1 protein levels were not significantly changed after permanent castration of TRAMP mice, although the disappearance of the upper band of GLUT4 was further confirmed (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3e)\u003c/strong\u003e.\u003c/p\u003e\n\u003ch2\u003eTRAMP mice have higher levels of insulin and diabetes slows prostate cancer progression in TRAMP mice\u003c/h2\u003e\n\u003cp\u003eHyperinsulinemia has been associated with aggressive prostate cancer and with the accelerated growth of LNCaP cell xenografts[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our previous results suggested that the insulin-dependent GLUT4 transporter might be involved in PCa progression and it might be responsible, at least in part, for the deleterious effects of insulin in the prostate since insulin activates AKT and promotes GLUT4 translocation.\u003c/p\u003e\n\u003cp\u003eFirst, we confirmed that LNCaP and PC-3 were sensitive to insulin. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, LNCaP and PC-3 cells increased the uptake of glucose in response to insulin. Then, we studied the levels of insulin in wild-type and TRAMP mice at 24 weeks of age. TRAMP mice showed significantly higher levels of insulin than WT mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb) and, interestingly, castration reduced blood insulin levels, while testosterone injection recovered it (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). However, there were no differences in the blood glucose levels in 16-week-old WT and TRAMP mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). Fasting blood glucose tolerance was the lowest in TRAMP mice, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee. The area under the curve (AUC), which correlates with the ability to eliminate glucose from blood, was also lower in TRAMP mice than in WT and castrated mice. Altogether, these results indicate that TRAMP mice show higher blood insulin levels and better glucose tolerance during the first stages of the disease.\u003c/p\u003e\n\u003cp\u003eThe relative risk of developing prostate cancer is reduced in men with diabetes. To evaluate the impact of diabetes on PCa progression in TRAMP mice, Type 1 Diabetes Mellitus (T1DM) was induced by streptozotocin (STZ) treatment for 5 days. All treated mice showed over 250 mg/dL glucose after 1 week of the treatment, which was the criterion used to consider them as diabetic (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4a\u003c/strong\u003e). Histological analysis of the pancreas confirmed the loss of eosinophilic, granule-containing insulin-producing beta cells within the islets of Langerhans (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4b\u003c/strong\u003e). Insulin injection restored glucose blood levels to normal within 2 hours but this effect only lasted 24 hours (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4c\u003c/strong\u003e). For this reason, diabetic TRAMP mice were treated daily with insulin, started two weeks after the first injection with STZ.\u003c/p\u003e\n\u003cp\u003eRelevant anatomical changes were found during necropsy in the prostate glands of TRAMP (CON), diabetic TRAMP (STZ) and diabetic TRAMP plus insulin (STZ\u0026thinsp;+\u0026thinsp;Ins) mice, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea. Diabetes reduced the size and histopathology of the prostate glands in TRAMP mice. The prostates of STZ-induced diabetic TRAMP mice did not show any relevant pathology. Shortly after insulin treatment, some of the animals developed a PIN (4/5), showed an area of PD tumour (1/5) or exhibited scattered areas of neuroendocrine carcinoma (1/5). The body and genitourinary tract were weighed at 24 weeks of age. Bodyweight was reduced in diabetic mice, likely due to white adipose tissue (WAT) depletion, and a significant reduction in the GU/BW ratio was found in STZ mice compared to CON. However, insulin treatment did not recover GU weight (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb). Even though the levels of circulating testosterone were slightly reduced after diabetes induction, the differences were not significant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eLowering of tumour progression by diabetes was confirmed by western blot of the proliferating cell nuclear antigen (PCNA) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed). STZ reduced PCNA production while insulin recovered its levels. Besides, the pro-apoptotic protein BAX was significantly increased in diabetic compared to control mice, while insulin recovered its levels. No differences were found in other tumour markers, such as TP53, P21/CDKN1 or BCL-2.\u003c/p\u003e\n\u003cp\u003eTo evaluate the role of androgens in the protective effect of diabetes on PCa, we studied AR levels and location. STZ did not induce changes in total AR levels, though after insulin stimulation it seems to be located in some nuclei (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4d\u003c/strong\u003e). However, \u003cem\u003ePsp94\u003c/em\u003e mRNA levels did not show any significant difference (data not shown).\u003c/p\u003e\n\u003cp\u003eIt is well known that insulin promotes activation of the PI3K/AKT pathway, either by direct interaction with its receptor or by Insulin-like Growth Factor (IGF) signalling, and this pathway is promoted in the most aggressive tumours. Levels of IGF1 Receptor (IGF1R)\u0026beta; and IGF Binding Protein (IGFBP)3 were analysed by western blot. There was no difference in IGF1R\u0026beta; between groups, and IGFBP3 in the prostate was somewhat reduced by insulin treatment in diabetic mice (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4e\u003c/strong\u003e). Instead, AKT phosphorylation was significantly reduced in diabetic mice, while insulin restored its levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee).\u003c/p\u003e\n\u003cp\u003eTo assess the implication of GLUT transporters in the protection of STZ-induced diabetic TRAMP mice against PCa progression, we studied the mRNA expression and protein production of GLUT4 transporters (data not shown). Induction of diabetes in the prostate reduced \u003cem\u003eSlc2a4\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef). Interestingly, although there was no difference in total GLUT4 protein levels between groups, diabetes led to the disappearance of the upper band found in the GLUT4 signal and insulin consistently restored it (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eg). These results might indicate that GLUT4 is a facilitative glucose transporter involved in the protective role of diabetes in PCa in TRAMP mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003eGLUT4 is increased in prostate cancer tissues\u003c/h2\u003e\n\u003cp\u003eTo translate our results to patient samples, we studied the production and location of GLUT4 in prostate tumours. Samples were classified as non-tumour or hyperplasic and according to their Gleason score in the tumour tissues. Tumours were classified as Gleason score\u0026thinsp;\u0026lt;\u0026thinsp;7 or \u0026ge;\u0026thinsp;7 according to pathologists. The samples with Gleason scores\u0026thinsp;\u0026ge;\u0026thinsp;7 were characterised by higher PSA and BCL2 levels (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;5a\u003c/strong\u003e) and decreased levels of GLUT1 protein during the first stages of tumour growth (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;5b\u003c/strong\u003e). However, insulin-responsive GLUT4 increased with tumour progression, showing the double band that indicate its activate status. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea). In addition, GLUT4 tissue distribution was examined. Samples of prostatectomy from 15 patients diagnosed with different Gleason scores (ranged between 3\u0026thinsp;+\u0026thinsp;3 and 4\u0026thinsp;+\u0026thinsp;5) were immunoassayed for GLUT4. GLUT4 immunolabeling intensity was significantly higher in Gleason grade 3 and 4 regions than in healthy (non-tumour) control areas (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb, c). A decrease in GLUT4 intensity was detected in Gleason grade 5 (less differentiated tumour) regions, which did not show significant differences when compared to control ones. Such difference in protein location was evident when these tumour regions were located next to healthy glands (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eFrom a database of all patients treated for prostate cancer in the Urology service at the Central University Hospital of Asturias, between January 2016 and November 2018, out of 807 patients, only 4 patients had been previously diagnosed with type 1 diabetes mellitus, all of them diagnosed in adulthood. GLUT levels were also confirmed in patient samples (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Double immunolabeling with anti-AR and anti-GLUT4 antiserum was carried out in prostate biopsies from diabetic and non-diabetic human patients (n\u0026thinsp;=\u0026thinsp;4 for each group). All the nuclei of the prostate epithelia, both from diabetic and non-diabetic patients, were stained and the signal was robust in all samples. No differences were observed between both groups. DAB-immunolabeled nuclei acquired little or no DAPI staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ea,b)\u003c/p\u003e\n\u003cp\u003eFrom 407 patients of prostate cancer treated at \u0026ldquo;Hospital Valle del Nalon\u0026rdquo; Asturias and \u0026ldquo;Hospital Universitario Marqu\u0026eacute;s de Valdecilla\u0026rdquo; roughly 10% of the patients included in the study were diabetic. Most of the patients were diagnosed with Type 2 Diabetes Mellitus (T2DM) and only 1 suffered Type 1 Diabetes (T1DM) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Most of the patients were treated with metformin alone or in combination with insulin or stimulators of insulin production. From those, relapses and perineural invasion did not show any difference between diabetic and non-diabetic patients (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eDistribution of diabetic prostate cancer patients and treatments\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDiabetics\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eN (%)\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTreatment\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eN (%)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eType 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1 (0.25)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInsulin\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 (100)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"6\" align=\"left\"\u003e\n\u003cp\u003eType 2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"6\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e41 (10.07)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDiet\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4 (9.75)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMetformin\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19 (46.34)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMetformin\u0026thinsp;+\u0026thinsp;Insulin\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 (7.31)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStimulator of Insulin production\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (14.63)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMetformin\u0026thinsp;+\u0026thinsp;Insulin\u0026thinsp;+\u0026thinsp;SIP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 (7.31)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStimulator\u0026thinsp;+\u0026thinsp;Metformin\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (12.19)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003csup\u003e1\u003c/sup\u003e N\u0026thinsp;=\u0026thinsp;407. Percentage from the total of Prostate Cancer patients.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003csup\u003e2\u003c/sup\u003e The % is referred to the number of patients with T1DM or T2DM, respectively.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003csup\u003e3\u003c/sup\u003e Different stimulators of insulin production were employed: Repaglinide, Daonil and Glimepirin.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eRelapses and perineural invasions in prostate cancer patients\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eDistribution of patients (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eNon-diabetic (N\u0026thinsp;=\u0026thinsp;90)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDiabetic (N\u0026thinsp;=\u0026thinsp;42)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRelapses\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31 (34.44)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (21.43)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePerineural invasion\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37 (48.68)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15 (48.38)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003e\u003csup\u003e1\u003c/sup\u003e Information about perineural invasion was not facilitated for all patients\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eAltogether, this data confirms that diabetes plays a protective role in prostate cancer and our results indicate that insulin dependent facilitative transporters of glucose, such as GLUT4, is a relevant protein implicated, while until know it was completely ignored.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePCa is unique from the metabolic point of view. The prostate is specialised for the production and secretion of citrate in prostatic fluid. Androgens stimulate citrate production and regulate all metabolic pathways in the prostate, including glucose metabolism[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here, we try to understand the role of glucose transporters sensitive to insulin in the progression of prostate cancer and their role on the lower incidence prostate cancer in individuals suffering diabetes. In this manuscript, the role of androgen signalling in glucose metabolism is confirmed since glucose uptake is increased after androgen treatment of LNCaP cells while lactate production and PPP activity are decreased.\u003c/p\u003e \u003cp\u003eWe showed in our previous work that GLUT1 levels are regulated in response to glucose deprivation in androgen sensitive PCa cells promoting survival and antioxidant pathways with no effects on androgen independent prostate cancer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Here we show that androgens stimulate GLUT1 production, although the location on the cell membrane is not changed, suggesting that its overexpression in another organelle must be considered to explain its role on the survival of prostate cancer cells. Interestingly, androgens stimulate the production of insulin independent glucose transporters GLUT1 and GLUT3 as previously reported. AMPK activity that it is promoted by androgens, and it activates GLUT1, plays a role in mitochondrial metabolism of glucose in androgen-sensitive cells[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. AMPK is also a regulator of GLUT1 synthesis and trafficking [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, GLUT1 stimulation by androgens \u003cem\u003ein vitro\u003c/em\u003e can be triggered via AMPK activation, as it is suggested by our results. Interesting, on the contrary DHT does not seem to influence insulin dependent transporters and even more, hormone-resistant LNCaP cells showed increase levels of insulin-sensitive GLUT4 and GLUT12. Though tumour cells often increase glucose uptake the physiologic role of glucose transporters in cancer has been scarcely study. Mostly GLUT1 and GLUT3 have gained attention as relevant factors in the accelerated metabolism of cancer cells[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eOverexpression of GLUT4 in androgen dependent prostate cancer cells promotes glycolysis and lactate production and more interestingly, it stimulates PSA production in an androgen independent manner.\u003c/p\u003e \u003cp\u003eMetabolomics also confirmed the dependence of prostate cancer metabolism of androgens. Although differences between LNCaP and PC-3 cannot be attributed only to AR, since they also differ in TP53, which plays a principal role in glycolysis[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough glucose uptake is higher in androgen-resistant cells, the levels of labelled lactate and, thus, glycolytic activity, are enhanced in androgen-insensitive cells which indicates the transition from glycolytic to oxidative in hormone resistant phenotypes previously described[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In addition to a decrease in aerobic glycolysis, as indicate m\u0026thinsp;+\u0026thinsp;3 lactate molar fraction, some metabolites of the TCA, like succinate, incorporated lower levels of \u003csup\u003e13\u003c/sup\u003eC in PC-3 than LNCaP cells according to previous results[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A possible explanation for these results is that AR signalling reduces the utilisation of glucose through pyruvate production through the upregulation of PPP. Previous studies have confirmed that AR promotes PPP from glucose[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, pathway analysis showed that the alanine, aspartate, and glutamate metabolism pathway is one of the most affected. This pathway is intricately linked with glycine, serine, and threonine metabolism, which was also found to be significantly impacted[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. It was recently revealed that serine, that can be produced from the glycolytic metabolite glycerate-3P, and one-carbon metabolism pathways are linked with neuroendocrine PCa[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In fact, both pathways should also be considered for androgenic regulation.\u003c/p\u003e \u003cp\u003eBoth GLUT1 and GLUT4 are highly produced by poorly differentiated tumours, which suggests their potential as biomarkers in advanced stages of cancer. In any case, only the levels of GLUT4 are reduced by surgical castration and restored by testosterone in TRAMP mice. We observed N-glycosylation of GLUT4 in TRAMP mice tumours that disappeared after castration and could be causally related to GLUT4 stability and trafficking, as proposed by others[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. GLUT4 is regulated \u003cem\u003ein vivo\u003c/em\u003e by the absence of androgens, while it seems to be androgen-independent \u003cem\u003ein vitro\u003c/em\u003e. This can be explained by the close connection between testosterone and insulin production. Insulin reduces the production of sex hormone-binding globulin, increasing active testosterone levels[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Inversely, testosterone also promotes insulin production in pancreatic beta cells via non-genomic actions of AR[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInsulin has been proposed as a risk factor for PCa patients because of its mitogenic and antiapoptotic activity[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, its role in tumour progression is still poorly understood and mechanistically limited. TRAMP mice show higher insulin levels, and in agreement, castrated TRAMP mice show lower blood insulin levels. Interestingly, hormone deprivation therapy in humans leads to insulin resistance[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], which is associated with both the effect found in androgen deprivation therapy and the role of insulin in PCa progression. Perhaps one of the most interesting data in this regard is that the risk of cancer incidence associated with diabetes is decrease in the prostate, being the molecular scenario still discussed. We demonstrated experimentally that T1D protects TRAMP mice of prostate cancer. Diabetic mice induced by STZ treatment reversed completely prostate carcer phenotype. We found that the levels of PCNA, a proliferation marker in PCa[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], was reduced and the pro-apoptotic protein BAX was increased in STZ-treated mice, and that insulin reverted this effect. Insulin stimulates IGF secretion, which is related to PCa aggressiveness[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], and IGFBP has been proposed as a predictive marker in advanced PCa, like PSA[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, in TRAMP mice, IGF-1R or IGFBP3 levels do not change after STZ-induced diabetes or insulin treatment.\u003c/p\u003e \u003cp\u003eOne aspect to consider is that T1DM reduces visceral obesity. Periprostatic fat influences cancer progression through the secretion of adipokines, proinflammatory cytokines[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Moreover, fatty acids are important nutrients in the prostate[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In diabetic TRAMP mice GLUT4 glycosylation is highly reduced. The activation of AKT might be related to the increased production of GLUT4 in tumoral tissue. However, AKT activity is inhibited in diabetic TRAMP mice.\u003c/p\u003e \u003cp\u003eThe importance of GLUT4 in cancer has been proposed in oral squamous cell carcinoma patients. Here, GLUT4 was significantly associated with a poor overall survival and recurrence-free survival. In addition, the ectopic overexpression of GLUT4 in cell lines caused a significant increase in migration in vitro and in vivo, whereas GLUT4 silencing reversed the cancer phenotype[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Also, it has been shown that GLUT4 plays a major role in basal glucose uptake in breast cancer cells[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and specific knockdown of GLUT4 in breast cancer cell lines impairs glucose uptake and reduces lactate production and more recently, the involvement of ALKBH5-mediated m\u003csup\u003e6\u003c/sup\u003eA RNA demethylation in control of GLUT4 expression and the sensitization of HER2-targeted therapy in breast cancer.[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, diabetes and cancer are two aged-associated pathologies more prevalent each year in western countries. Targeting dysregulated glucose metabolism has focused attention on the therapy of cancer. However, challenging both is by itself a new difficulty in clinics. We experimentally demonstrated in this work that T1D induced by STZ injection avoids prostate cancer progression in the TRAMP mice. It seems insulin might promote PCa progression by incrementing GLUT4 independently of androgen signalling. Our results prove for the first time, the relevance of insulin-dependent glucose transporters in the progress of prostate tumours in vivo. They also suggest their value as therapeutic targets, particularly in the advanced stages of the tumour, characterised by increased glucose uptake and a glycolytic phenotype.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e2DG: Deoxyglucose; 6AN: 6-aminonicotinamide; 6GPD: 6-phosphogluconate dehydrogenase; ACO2: Aconitase; AR: Androgen Receptor; AUC: Area Under the Curve; BD: Body Weight; CAS: Castrated; CDX: Bicalutamide; CON: Control; CRPC: Castration-Resistant Prostate Cancer; DAB: Diaminobenzidine; DHT: Dihydrotestosterone; FBS: Fetal Bovine Serum; FBS\u003csub\u003eCHST\u003c/sub\u003e: Charcoal-stripped FBS; FDG: Fluorodeoxyglucose; G6PDH: Glucose-6-phosphate dehydrogenase; GlcNAc: N-acetylglucosamine; GLUT: Glucose Transporter; GU: Genitourinary tract; HDAC: Histone deacetylase; HIF: Hypoxia-Inducible Factor; IF: Immunofluorescence; IGF: Insulin-like Growth Factor; IGF1R: IGF1 Receptor; IGFBP: Insulin-like Growth Factor Binding Protein; IHC: Immunocytochemistry; Ins: Insulin; MD: Moderately-differentiated tumor; OXPHOS: Oxidative phosphorylation; PCa: Prostate Cancer; PCNA: Proliferating Cell Nuclear Antigen; PD: Poorly-differentiated tumor; PNGase: N-glycosidase; PPP: Pentose Phosphate Pathway; PSA: Prostate Specific Antigen; SEM: Standard Medium Error; STZ: Streptozotocin; T1DM: Type 1 Diabetes Mellitus; T2DM: Type 2 Diabetes Mellitus; TCA: Tricarboxylic Acid Cycle; TES: Testosterone; TRAMP: Transgenic Adenocarcinoma of Mouse Prostate; WD: Well-differentiated tumor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was carried out in accordance with the World Medical Association Declaration of Helsinki for the ethical principles for Medical Research involving human subjects. All patients were informed, and consent was obtained prior to sample collection and the protocol was approved by \u0026ldquo;Comite de Etica de la Investigacion del Principado de Asturias\u0026rdquo;. Experiments and procedures with mice were conducted in accordance with European Directive 2012/63/UE and were approved by the Ethical Committee Board for Animal Experiments at the University of Oviedo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to publication\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of the data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rosa M Sainz ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the \u0026ldquo;Agencia Estatal de Investigacion\u0026rdquo; (MINECO-BFU2016-79139-R, and MCI-20-PID2019-111418RB-I00) and co-funded by the European Regional Development Fund (FEDER) and by the \u0026ldquo;Gobierno del Principado de Asturias, Programa de Grupos de Investigacion 2018-2020\u0026rdquo; (IDI/2018/000239). The EU is acknowledged for the provision of FEDER funds for the purchase of the GC-MS/MS instrument.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.G.M., J.C.M. and R.M.S. designed the study. P.G.M. performed the experiments. A.M.A study GLUT4 production and activation. R.C.C. performed confocal microscopy studies and morphologic analysis. A.A.A. assisted with animal experimentation. D.H., P.R.G. and J.I.G.A. performed mass spectrometry analysis and metabolomics. R.A.A. assisted with flow cytometry. M.A.M., M.D.E. collected and oversaw prostate cancer patients\u0026rsquo; and C.L. gather and supervised diabetic patients. P.G.M, J.C.M., D.H, P.R., J.I.G.A. and R.M.S interpreted and discussed the data. P.G.M and R.M.S conceived, analysed the data and wrote the manuscript with assistance from the other authors. All authors critically reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.G.M. thanks the finantial support of \u0026ldquo;Ministerio de Universidades de Espa\u0026ntilde;a, Subvenciones para la Recualificacion del Sistema Universitario Espa\u0026ntilde;ol Mar\u0026iacute;a Zambrano (MU-21-UP2021-030). A.M.A was funded by \u0026ldquo;Asociacion Espa\u0026ntilde;ola Contra el Cancer\u0026rdquo; (SV-19-AECC-FPI-2). A.A.A. was supported by the University of Oviedo \u0026ldquo;Ayudas predoctorales para la realizaci\u0026oacute;n de tesis doctorales modalidad A\u0026rdquo; (PAPI-18-PF-06). C.L is recipient from a Margarita Salas Post-doctoral grant from the University of Barcelona. We thank Sandrina Kinet and Naomi Taylor for their helpful assistance in the labelling and detection of GLUT1. We thank Marta Alonso-Guervos and Ana Salas Bustamante for their technical support with the confocal microscopy and flow cytometry assays. We thank Scientific and Technical Services (SCTs) from the University of Oviedo for their technical support, including biological, biotechnical, and biomedical testing, optical microscopy and image processing, mass spectrometry, and the animal facility. We also thank the Molecular Histopathology in Animal Models of Cancer of the IUOPA for the processing and analysis of prostate samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials \u0026amp; correspondence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRosa M. Sainz, PhD. Department of Morphology and Cell Biology, University Institute of Oncology of Asturias (IUOPA). School of Medicine, C/Julian Claveria 6, 33006 Oviedo, SPAIN. Phone # 34 985103610, e-mail: [email protected]\u003c/p\u003e\n\u003cp\u003ePedro Gonzalez-Menendez, PhD. Department of Morphology and Cell Biology, University Institute of Oncology of Asturias (IUOPA). School of Medicine, C/Julian Claveria 6, 33006 Oviedo, SPAIN. Phone # 34 985103610, e-mail: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov [Internet]. Cancer Discov; 2022 [cited 2023 Jan 11];12:31\u0026ndash;46. Available from: https://pubmed.ncbi.nlm.nih.gov/35022204/\u003c/li\u003e\n\u003cli\u003eVander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science (1979). 2009;324:1029\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eDeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The Biology of Cancer: Metabolic Reprogramming Fuels Cell Growth and Proliferation. Cell Metab. 2008;7:11\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eVander Heiden MG, DeBerardinis RJ. Understanding the Intersections between Metabolism and Cancer Biology. Cell. Cell Press; 2017. p. 657\u0026ndash;69. \u003c/li\u003e\n\u003cli\u003ePavlova NN, Zhu J, Thompson CB. The hallmarks of cancer metabolism: Still emerging. Cell Metab [Internet]. Cell Metab; 2022 [cited 2023 Jan 11];34:355\u0026ndash;77. Available from: https://pubmed.ncbi.nlm.nih.gov/35123658/\u003c/li\u003e\n\u003cli\u003eBarron CC, Bilan PJ, Tsakiridis T, Tsiani E. Facilitative glucose transporters: Implications for cancer detection, prognosis and treatment. Metabolism. 2016;65:124\u0026ndash;39. \u003c/li\u003e\n\u003cli\u003eSingh KK, Desouki MM, Franklin RB, Costello LC. Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues. Mol Cancer. BioMed Central; 2006;5:14. \u003c/li\u003e\n\u003cli\u003eBarfeld SJ, Itkonen HM, Urbanucci A, Mills IG. Androgen-regulated metabolism and biosynthesis in prostate cancer. Endocrine Related Cancer. 2014;21:T57\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eVaz C V., Alves MG, Marques R, Moreira PI, Oliveira PF, Maia CJ, et al. Androgen-responsive and nonresponsive prostate cancer cells present a distinct glycolytic metabolism profile. Int J Biochem Cell Biol. 2012;44:2077\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eAhmad F, Cherukuri MK, Choyke PL. Metabolic reprogramming in prostate cancer. Br J Cancer. 2021. \u003c/li\u003e\n\u003cli\u003eTennakoon JB, Shi Y, Han JJ, Tsouko E, White MA, Burns AR, et al. Androgens regulate prostate cancer cell growth via an AMPK-PGC-1\u0026alpha;-mediated metabolic switch. Oncogene. 2014;33:5251\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eGonthier K, Poluri RTK, Audet-Walsh \u0026Eacute;. Functional genomic studies reveal the androgen receptor as a master regulator of cellular energy metabolism in prostate cancer. Journal of Steroid Biochemistry and Molecular Biology. Elsevier Ltd; 2019. p. 105367. \u003c/li\u003e\n\u003cli\u003eHiggins LH, Withers HG, Garbens A, Love HD, Magnoni L, Hayward SW, et al. Hypoxia and the metabolic phenotype of prostate cancer cells. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2009;1787:1433\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eMassie CE, Lynch A, Ramos-Montoya A, Boren J, Stark R, Fazli L, et al. The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis. EMBO J. 2011;30:2719\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eGonzalez-Menendez P, Hevia D, Mayo JC, Sainz RM. The dark side of glucose transporters in prostate cancer: Are they a new feature to characterize carcinomas? Int J Cancer. 2017; \u003c/li\u003e\n\u003cli\u003eBader DA, Hartig SM, Putluri V, Foley C, Hamilton MP, Smith EA, et al. Mitochondrial pyruvate import is a metabolic vulnerability in androgen receptor-driven prostate cancer. Nat Metab. Nature Research; 2019;1:70\u0026ndash;85. \u003c/li\u003e\n\u003cli\u003eVaz C V., Marques R, Alves MG, Oliveira PF, Cavaco JE, Maia CJ, et al. Androgens enhance the glycolytic metabolism and lactate export in prostate cancer cells by modulating the expression of GLUT1, GLUT3, PFK, LDH and MCT4 genes. J Cancer Res Clin Oncol. 2016;142:5\u0026ndash;16. \u003c/li\u003e\n\u003cli\u003eWang J, Xu W, Wang B, Lin G, Wei Y, Abudurexiti M, et al. GLUT1 is an AR target contributing to tumor growth and glycolysis in castration-resistant and enzalutamide-resistant prostate cancers. Cancer Lett [Internet]. Cancer Lett; 2020 [cited 2022 Dec 20];485:45\u0026ndash;55. Available from: https://pubmed.ncbi.nlm.nih.gov/32428663/\u003c/li\u003e\n\u003cli\u003eReinicke K, Sotomayor P, Cisterna P, Delgado C, Nualart F, Godoy A. Cellular distribution of Glut-1 and Glut-5 in benign and malignant human prostate tissue. J Cell Biochem. 2012;113:553\u0026ndash;62. \u003c/li\u003e\n\u003cli\u003eGonzalez-Menendez P, Hevia D, Rodriguez-Garcia A, Mayo JC, Sainz RM. Regulation of GLUT transporters by flavonoids in androgen-sensitive and-insensitive prostate cancer cells. Endocrinology. Endocrine Society; 2014;155:3238\u0026ndash;50. \u003c/li\u003e\n\u003cli\u003eRaina K, Ravichandran K, Rajamanickam S, Huber KM, Serkova NJ, Agarwal R. Inositol Hexaphosphate Inhibits Tumor Growth, Vascularity, and Metabolism in TRAMP Mice: A Multiparametric Magnetic Resonance Study. Cancer Prevention Research. 2013;6:40\u0026ndash;50. \u003c/li\u003e\n\u003cli\u003eGrossmann M, Wittert G. Androgens, diabetes and prostate cancer. Endocr Relat Cancer. 2012;19:F47-62. \u003c/li\u003e\n\u003cli\u003eTakanaga H, Frommer WB. Facilitative plasma membrane transporters function during ER transit. The FASEB Journal. 2010;24:2849\u0026ndash;58. \u003c/li\u003e\n\u003cli\u003eGreenberg NM, DeMayo F, Finegold MJ, Medina D, Tilley WD, Aspinall JO, et al. Prostate cancer in a transgenic mouse. Proc Natl Acad Sci U S A. 1995;92:3439\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eGonzalez-Menendez P, Hevia D, Alonso-Arias R, Alvarez-Artime A, Rodriguez-Garcia A, Kinet S, et al. GLUT1 protects prostate cancer cells from glucose deprivation-induced oxidative stress. Redox Biol. 2018;17:112\u0026ndash;27. \u003c/li\u003e\n\u003cli\u003eHaga Y, Ishii K, Suzuki T. N-glycosylation is critical for the stability and intracellular trafficking of glucose transporter GLUT4. J Biol Chem [Internet]. J Biol Chem; 2011 [cited 2022 Dec 20];286:31320\u0026ndash;7. Available from: https://pubmed.ncbi.nlm.nih.gov/21757715/\u003c/li\u003e\n\u003cli\u003eOburoglu L, Tardito S, Fritz V, de Barros SC, Merida P, Craveiro M, et al. Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification. Cell Stem Cell. 2014;15:169\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eMontel-Hagen A, Kinet S, Manel N, Mongellaz C, Prohaska R, Battini JL, et al. Erythrocyte Glut1 triggers dehydroascorbic acid uptake in mammals unable to synthesize vitamin C. Cell [Internet]. Cell; 2008 [cited 2022 Dec 21];132:1039\u0026ndash;48. Available from: https://pubmed.ncbi.nlm.nih.gov/18358815/\u003c/li\u003e\n\u003cli\u003eTian WN, Braunstein LD, Apse K, Pang J, Rose M, Tian X, et al. Importance of glucose-6-phosphate dehydrogenase activity in cell death. Am J Physiol. 1999;276:C1121-31. \u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-Fern\u0026aacute;ndez M, Rodr\u0026iacute;guez-Gonz\u0026aacute;lez P, Hevia S\u0026aacute;nchez D, Gonz\u0026aacute;lez-Men\u0026eacute;ndez P, Sainz Men\u0026eacute;ndez RM, Garc\u0026iacute;a Alonso JI. Accurate and sensitive determination of molar fractions of 13 C-Labelled intracellular metabolites in cell cultures grown in the presence of isotopically-labelled glucose. Anal Chim Acta. 2017; \u003c/li\u003e\n\u003cli\u003eZhao S, Guo Y, Sheng Q, Shyr Y. Advanced heat map and clustering analysis using heatmap3. Biomed Res Int. Hindawi Publishing Corporation; 2014;2014:986048. \u003c/li\u003e\n\u003cli\u003eChong J, Wishart DS, Xia J. Using MetaboAnalyst 4.0 for Comprehensive and Integrative Metabolomics Data Analysis. Curr Protoc Bioinformatics. John Wiley and Sons Inc.; 2019;68. \u003c/li\u003e\n\u003cli\u003eSainz RM, Reiter RJ, Tan DX, Roldan F, Natarajan M, Quiros I, et al. Critical role of glutathione in melatonin enhancement of tumor necrosis factor and ionizing radiation-induced apoptosis in prostate cancer cells in vitro. J Pineal Res. 2008;45:258\u0026ndash;70. \u003c/li\u003e\n\u003cli\u003eHaga Y, Ishii K, Suzuki T. N-Glycosylation Is Critical for the Stability and Intracellular Trafficking of Glucose Transporter GLUT4. Journal of Biological Chemistry. 2011;286:31320\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eVenkateswaran V, Haddad AQ, Fleshner NE, Fan R, Sugar LM, Nam R, et al. Association of Diet-Induced Hyperinsulinemia With Accelerated Growth of Prostate Cancer (LNCaP) Xenografts. JNCI Journal of the National Cancer Institute. 2007;99:1793\u0026ndash;800. \u003c/li\u003e\n\u003cli\u003eBader DA, McGuire SE. Tumour metabolism and its unique properties in prostate adenocarcinoma. Nat Rev Urol. Nature Research; 2020;17:214\u0026ndash;31. \u003c/li\u003e\n\u003cli\u003eWu N, Zheng B, Shaywitz A, Dagon Y, Tower C, Bellinger G, et al. AMPK-Dependent Degradation of TXNIP upon Energy Stress Leads to Enhanced Glucose Uptake via GLUT1. Mol Cell. 2013;49:1167\u0026ndash;75. \u003c/li\u003e\n\u003cli\u003eAncey PB, Contat C, Meylan E. Glucose transporters in cancer \u0026ndash; from tumor cells to the tumor microenvironment. FEBS Journal. Blackwell Publishing Ltd; 2018;285:2926\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eAlimirah F, Panchanathan R, Chen J, Zhang X, Ho S-M, Choubey D. Expression of androgen receptor is negatively regulated by p53. Neoplasia. Neoplasia Press; 2007;9:1152\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eKruiswijk F, Labuschagne CF, Vousden KH. p53 in survival, death and metabolic health: a lifeguard with a licence to kill. Nat Rev Mol Cell Biol. Nature Research; 2015;16:393\u0026ndash;405. \u003c/li\u003e\n\u003cli\u003eQuiros-Gonzalez I, Gonzalez-Menendez P, Mayo JC, Hevia D, Artime-Naveda F, Fernandez-Vega S, et al. Androgen-Dependent Prostate Cancer Cells Reprogram Their Metabolic Signature upon GLUT1 Upregulation by Manganese Superoxide Dismutase. Antioxidants (Basel) [Internet]. MDPI; 2022 [cited 2022 Dec 23];11. Available from: http://www.ncbi.nlm.nih.gov/pubmed/35204196\u003c/li\u003e\n\u003cli\u003eTsouko E, Khan AS, White MA, Han JJ, Shi Y, Merchant FA, et al. Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth. Oncogenesis. Nature Publishing Group; 2014;3:e103. \u003c/li\u003e\n\u003cli\u003eMattaini KR, Sullivan MR, Vander Heiden MG. The importance of serine metabolism in cancer. Journal of Cell Biology. Rockefeller University Press; 2016. p. 249\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eReina-Campos M, Linares JF, Duran A, Cordes T, L\u0026rsquo;Hermitte A, Badur MG, et al. Increased Serine and One-Carbon Pathway Metabolism by PKC\u0026lambda;/\u0026iota; Deficiency Promotes Neuroendocrine Prostate Cancer. Cancer Cell. Cell Press; 2019;35:385-400.e9. \u003c/li\u003e\n\u003cli\u003eLiu X, Choi RY, Jawad SM, Arnold JT. Androgen-induced PSA expression requires not only activation of AR but also endogenous IGF-I or IGF-I/PI3K/Akt signaling in human prostate cancer epithelial cells. Prostate. 2011;71:766\u0026ndash;77. \u003c/li\u003e\n\u003cli\u003ePasquali R, Casimirri F, De Iasio R, Mesini P, Boschi S, Chierici R, et al. Insulin regulates testosterone and sex hormone-binding globulin concentrations in adult normal weight and obese men. J Clin Endocrinol Metab. 1995;80:654\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eNavarro G, Xu W, Jacobson DA, Wicksteed B, Allard C, Zhang G, et al. Extranuclear Actions of the Androgen Receptor Enhance Glucose-Stimulated Insulin Secretion in the Male. Cell Metab. 2016;23:837\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eHsing AW, Chua S, Gao Y-T, Gentzschein E, Chang L, Deng J, et al. Prostate Cancer Risk and Serum Levels of Insulin and Leptin: a Population-Based Study. JNCI Journal of the National Cancer Institute. Oxford University Press; 2001;93:783\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eHamilton EJ, Gianatti E, Strauss BJ, Wentworth J, Lim-Joon D, Bolton D, et al. Increase in visceral and subcutaneous abdominal fat in men with prostate cancer treated with androgen deprivation therapy. Clin Endocrinol (Oxf). 2011;74:377\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eRao PM, Kelly DM, Jones TH. Testosterone and insulin resistance in the metabolic syndrome and T2DM in men. Nat Rev Endocrinol. Nature Publishing Group; 2013;9:479\u0026ndash;93. \u003c/li\u003e\n\u003cli\u003eZhong W, Peng J, He H, Wu D, Han Z, Bi X, et al. Ki-67 and PCNA expression in prostate cancer and benign prostatic hyperplasia. Clin Invest Med. 2008;31:E8\u0026ndash;15. \u003c/li\u003e\n\u003cli\u003eDe Nunzio C, Aronson W, Freedland SJ, Giovannucci E, Parsons JK. The Correlation Between Metabolic Syndrome and Prostatic Diseases. Eur Urol. 2012;61:560\u0026ndash;70. \u003c/li\u003e\n\u003cli\u003eTerracciano D, Bruzzese D, Ferro M, Mazzarella C, Di Lorenzo G, Altieri V, et al. Preoperative insulin-like growth factor-binding protein-3 (IGFBP-3) blood level predicts gleason sum upgrading. Prostate. 2012;72:100\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eLaurent V, Gu\u0026eacute;rard A, Mazerolles C, Le Gonidec S, Toulet A, Nieto L, et al. Periprostatic adipocytes act as a driving force for prostate cancer progression in obesity. Nat Commun. 2016;7:10230. \u003c/li\u003e\n\u003cli\u003eWatt MJ, Clark AK, Selth LA, Haynes VR, Lister N, Rebello R, et al. Suppressing fatty acid uptake has therapeutic effects in preclinical models of prostate cancer. Sci Transl Med. American Association for the Advancement of Science; 2019;11. \u003c/li\u003e\n\u003cli\u003eChang YC, Chi LH, Chang WM, Su CY, Lin YF, Chen CL, et al. Glucose transporter 4 promotes head and neck squamous cell carcinoma metastasis through the TRIM24-DDX58 axis. J Hematol Oncol [Internet]. BioMed Central Ltd.; 2017 [cited 2022 Dec 26];10:1\u0026ndash;12. Available from: https://jhoonline.biomedcentral.com/articles/10.1186/s13045-016-0372-0\u003c/li\u003e\n\u003cli\u003eGarrido P, Osorio FG, Mor\u0026aacute;n J, Cabello E, Alonso A, Freije JMP, et al. Loss of GLUT4 induces metabolic reprogramming and impairs viability of breast cancer cells. J Cell Physiol [Internet]. J Cell Physiol; 2015 [cited 2022 Dec 23];230:191\u0026ndash;8. Available from: https://pubmed.ncbi.nlm.nih.gov/24931902/\u003c/li\u003e\n\u003cli\u003eLiu H, Lyu H, Jiang G, Chen D, Ruan S, Liu S, et al. ALKBH5-Mediated m6A Demethylation of GLUT4 mRNA Promotes Glycolysis and Resistance to HER2-Targeted Therapy in Breast Cancer. Cancer Res [Internet]. American Association for Cancer Research (AACR); 2022 [cited 2022 Dec 26];82:3974\u0026ndash;86. Available from: https://aacrjournals.org/cancerres/article/82/21/3974/709951/ALKBH5-Mediated-m6A-Demethylation-of-GLUT4-mRNA\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Prostate cancer, glucose transporters, insulin, diabetes, GLUT4","lastPublishedDoi":"10.21203/rs.3.rs-2619954/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2619954/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Diabetic men are less likely to suffer prostate cancer, and insulin signalling through insulin receptors has been long considered. However, the role of insulin-dependent glucose transporters has yet to be elucidated. The unique metabolic properties of prostate cancer are attributed to the central role of androgens. Androgen-sensitive tumour cells have higher mitochondrial activity, while castration-resistant cells exhibit aerobic glycolysis. In addition, to glycolysis, one of the hallmarks of cancer metabolism is increased glucose uptake. However, the prostate's oncogenic value of glucose transporters (GLUTs) needs to be better characterized. This research aims to discover the relevance of insulin-dependent glucose transporters to cancer progression and their importance in the protective role of diabetes in prostate cancer.\u003c/p\u003e\n\u003cp\u003eMethods: Androgen-sensitive LNCaP and androgen-insensitive PC-3 cells were used \u003cem\u003ein vitro\u003c/em\u003e. Castration-resistant LNCaP-R cells and cells overexpressing GLUT1 or GLUT4 were established from LNCaP cell line. In addition, TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice and prostatic samples from patients were employed.\u003c/p\u003e\n\u003cp\u003eResults: We found that androgens stimulate insulin-independent glucose transporters, while androgen independence is associated with GLUT4 overexpression. The ectopic overexpression of GLUT4 promotes the characteristics of a castration-resistant phenotype. Metabolomics confirmed that hormone-resistant prostate cancer cells show an oxidative metabolism with a clear enrichment in amino acid metabolism. Diabetic TRAMP mice showed total tumour regression, while insulin administration restored proliferation and recovered GLUT4 levels. The levels of GLUT4 increase along with tumour progression in TRAMP mice, and it is reduced by castration and streptozotocin-induced diabetes. Finally, the levels of GLUT4 accumulation in tumour tissues compared to normal epithelial in patients' samples showed a clear co-location with nuclear AR.\u003c/p\u003e\n\u003cp\u003eConclusion: Here it is confirmed the relevance of insulin-mediated glucose uptake through GLUT4 with prostate cancer progression and its relation to the reduced occurrence of prostate cancer in diabetic men.\u003c/p\u003e","manuscriptTitle":"Insulin-dependent GLUT4 is a risk factor for cancer in the prostate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-01 18:54:40","doi":"10.21203/rs.3.rs-2619954/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0aff7add-56bb-4ed0-a597-2098ccf7f260","owner":[],"postedDate":"March 1st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-29T00:19:33+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-01 18:54:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2619954","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2619954","identity":"rs-2619954","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}