The cholesterol oxidation product 7-ketocholesterol impairs pancreatic beta cell insulin secretion 

The cholesterol oxidation product 7-ketocholesterol impairs pancreatic beta cell insulin secretion | 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 The cholesterol oxidation product 7-ketocholesterol impairs pancreatic beta cell insulin secretion Wenjing Zhang, Ying Wu, Yuchen Zhao, Nan Wu, Jiahua Wu, Shuiya Sun, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4483308/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: The impairment of pancreatic beta cell function caused by glucolipotoxicity plays an important role in the pathogenesis of type 2 diabetes. Previous studies have shown that cholesterol can induce beta cell dysfunction. However, the effect of the cholesterol oxidation product 7-ketocholesterol in beta-cell function remains unclear. Methods: Cell proliferation, Glucose-stimulated insulin secretion (GSIS), perifusion, calcium imaging, total internal reflection fluorescence microscopy (TIRFM), reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, qPCR, and Western blotting were used to evaluate the effect and mechanism of 7-ketocholesterol on INS1 cells and islets. N-Acetyl-L-cysteine was used to rescue insulin secretion of beta-cells. GSIS, perifusion, calcium levels and exocytosis events verified that early-phase insulin secretion was impaired after 7-ketocholesterol treatment. Results: The results of CCK 8 and GSIS demonstrated that 25 μmol/L 7-ketocholesterol significantly decreased insulin secretion in the INS1 cells ( P < 0.05), as did 50 μmol/L 7-ketocholesterol in the primary islets ( P < 0.05). The islet perifusion analysis verified that the insulin secretion function was impaired with 7-ketocholesterol( P < 0.001). Calcium imaging showed that the intracellular calcium levels were decreased following 7-ketocholesterol treatment( P <0.001). TIRFM imaging inferred that 7-ketocholesterol could reduced insulin-secretory-granule exocytosis by decreased fusion events and increased kiss-and-run events to the membrane to attenuate insulin secretion ( P < 0.01). Further data showed that the level of Snap25 gene and protein expression related to insulin exocytosis was substantially downregulated. Further study showed that the reactive oxygen species (ROS) in INS1 cells was upregulated, and both the mitochondrial membrane potential (MMP) and level of adenosine triphosphate (ATP) was downregulated ( P < 0.05). The regulation of nuclear factor erythroid 2-related factor (NRF2) is an important transcription factor for oxidative stress, for which its nuclear translocation results in the subsequent activation of gene transcription of Gpx4 , Sod1 , Txnip , Nqo1 , and Ho1 in INS1 cells. In addition, 7-ketocholesterol-induced pancreatic beta cell dysfunction and oxidative stress was ameliorated by pretreatment with the antioxidant, N-Acetyl-L-cysteine. Conclusions: These findings suggested that 7-ketocholesterol impacted insulin exocytosis to decrease the insulin secretion of pancreatic beta cells involved in the oxidative stress. 7-ketocholesterol pancreatic beta cell perifusion insulin secretion exocytosis oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The failure and dysfunction of pancreatic beta cells represents the key pathogenesis associated with type 2 diabetes [ 1 ]. Evidence indicates that the cellular dysfunction induced by lipo- and gluco-lipotoxicity in pancreatic beta cells may contribute to the pathogenesis of type 2 diabetes [ 2 ]. However, the risk factor and underlying mechanisms of beta cell dysfunction have not been thoroughly elucidated to date. Previous studies have shown that free fatty acids (FFA) generate essential metabolic coupling factors in beta cells to amplify the signals required for insulin secretion, but become detrimental under conditions of fuel oversupply [ 3 – 6 ]. As a component of the cell membrane, cholesterol influences fluidity and synthesizes several important molecules to regulate cellular functions [ 3 ], and cholesterol metabolism disorders could inhibit glucose-stimulated insulin secretion (GSIS) by pancreatic beta cells [ 7 , 8 ]. Moreover, excess cholesterol can reduce glucose transporter activity to impact glucose uptake, reduce glucokinase activity, inhibit glycolysis, and block insulin exocytosis via increasing oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction [ 5 , 7 – 11 ]. Cholesterol is produced endogenously and can also be introduced through the diet. Furthermore, cholesterol is converted to other perhydrocyclopentanophenanthrene compounds via oxidation/reduction, and these intermediate products participate in the further metabolic conversion to eventually form bile acids, steroid hormones, and vitamin D3 [ 12 ]. Studies have reported that these cholesterol oxidation products (oxysterols) are more harmful than cholesterol for accelerating disease progression [ 12 – 15 ]. The primary auto-oxidated oxysterols were 7-ketocholesterol, 7b-hydroxysterol, and cholesterol-5a,6a-epoxide. Cholesterol oxidation can occur within different tissues, especially in the vessel wall, and comprises the main hydroxysterol in human atherosclerotic plaques [ 16 ]. As primarily oxide products, 7-ketocholesterol represents biologically active molecules and can have many side effects and is associated with many diseases (e.g., atherosclerosis and cardiovascular diseases, alzheimer’s disease, and age-related macular degeneration) [ 17 – 22 ]. The accumulation of 7-ketocholesterol in vascular endothelial causes endothelial injury via activation of the endoplasmic reticulum stress response and the up-regulation of vascular endothelial growth factor, interleukin (IL)-1, IL-6, and IL-8 expression [ 9 , 23 ]. In addition, 7-ketocholesterol could increase reactive oxygen species (ROS) and activate caspase8/caspase3 to trigger apoptosis in mesenchymal stem cells from patients with acute myeloid leukemia [ 19 ]. The study by Chang et al. demonstrated that a small amount of dietary 7-ketocholesterol contributed to accelerate hepatic steatosis and inflammation in obese mice [ 24 ]. A previous study reported that the level of 7-ketocholesterol was significantly higher in type 2 diabetes patients compared to non-diabetic healthy participants [ 25 ]. Therefore, 7-ketocholesterol could be an marker in type 2 diabetes patients [ 26 ]. However, the effects of 7-ketocholesterol on pancreatic beta cell function have not been directly reported. Pancreatic beta cells, as the most important component in islet tissues, are the only functional cells for insulin secretion in vivo. Insulin secretion could involved in the beta cell differentiation state, insulin production or processing, insulin-secretory-granule synthesis and translocation, as well as insulin release. Insulin synthesis is regulated by trans-activators including paired box gene 6 (PAX6), pancreatic and duodenal homeobox-1(PDX-1), MafA, and Neurogenic differentiation 1 (NeuroD1) at both the transcriptional and translational level [ 27 ]. And insulin secretion process begins with glucose uptake and metabolism to ATP that subsequently closes ATP-sensitive K + -channels, resulting in membrane depolarization, opening of voltage-dependent Ca 2+ channels, Ca 2+ influx and insulin granule exocytosis [ 28 , 29 ]. The exocytosis involved in the trafficking of insulin granules to the plasma membrane, the exocytotic fusion of the granules with the plasma membrane and eventually the retrieval of the secreted membranes by endocytosis [ 30 ]. The previous study showed the excess cholesterol could increase insulin granule size and reduced the docking and fusion of insulin granules at the plasma membrane [ 9 ]. Based on the above, we intend to answer the question of whether 7-ketocholesterol might affect the development of pancreatic beta cell dysfunction and elucidated the mechanism of beta cell dysfunction. According to our results, 7-ketocholesterol accelerated insulin secretion dysfunction, and reduced insulin exocytosis via oxidative stress. 2. Materials and Methods 2.1 Materials Collagenase V (C9263), 7-ketocholesterol (C2394), and Histopaque®-1077 were purchased from Sigma. 7-ketocholesterol was dissolved with anhydrous ethanol and diluted in culture medium as described previously [ 31 ]. The CCK-8 kit (40203ES60) and BCA protein assay (FD2001) kit were obtained from YEASEN and FUDE Biological Tech Co. Ltd., respectively. N-Acetyl-L-cysteine was purchased from Beyotime, Shanghai, China. RPMI high glucose medium, 10 mmol/L Hepes, and 1 mmol/L sodium-pyruvate were acquired from Thermofisher. C57BL/6 mice were purchased from GemPharmatech Co., Ltd. (Nanjing, CN). 2.2 Cell culture and islets dissection INS1 cells were cultured in RPMI 1640 medium with 10 mmol/L Hepes, 1 mmol/L sodium-pyruvate, 0.05 mol/L 2-mercaptoethanol, and 10% fetal bovine serum, and penicillin-streptomycin (100 µg/mL) in an atmosphere of 5% CO 2 at 37°C. The C57BL/6 mice were fasted overnight and anesthetized. The duodenum was ligated and the pancreas was perifused with 2−3 mL collagenase V at 1 mg/mL via the common bile duct and digested for 28 min at 37°C. The digestion was terminated by adding HBSS buffer containing 10% FBS. Followed by filtration and centrifugation, islets were isolated by density gradient centrifugation (Histopaque-1077, Sigma) and collected under a microscope. Finally, the islets were incubated in culture medium and treated with different concentrations of 7-ketocholesterol for 24h at 37℃. 2.3 7-ketocholesterol treatment and cell viability INS1 cells were treated with different concentrations of 7-ketocholesterol for 24 h at 37℃, cell viability was assessed using a CCK8 viability assay. The islets were handpicked into the plates and treated with different concentrations of 7-ketocholesterol for 24 h at 37℃, cell viability was assessed using a CCK8 viability assay. 2.4 GSIS Experiments were performed as described previously [ 7 ]. INS1 cells were seeded at a density of 5 × 10 5 /well in 1 mL medium in a 12-well plate overnight, after which 7-ketocholesterol was added to each well for 24 h. Subsequently, the cells were rinsed with PBS and starved with 0.01% BSA Kreb’s buffer for 30 min and changed to 2.8 mmol/L or 25 mmol/L glucose 0.01% BSA Kreb’s buffer for 1 h. The supernatant was collected and centrifuged for 1 min. The insulin content was measured using an insulin immunoassay kit (RT300, Ezaasy) and normalized to cell protein content. The islets were starved with 0.01% BSA Kreb’s buffer for 30 min, then handpicked into the 1.5 ml Eppendorf tube and stimulated by 2.8 mmol/L glucose in 0.1% BSA Kreb’s buffer for 1 h. After collecting supernatant, the islets were stimulated by 16.8 mmol/L glucose in 0.01% BSA Kreb’s buffer for 1 h in the same tube and the supernatant was collected again. The insulin content was detected with an insulin immunoassay kit (MS300, Ezaasy), and normalized to protein content of islets. The insulin secretion index was normalized to the average baseline insulin secretion in 2.8mmol/L glucose. 2.5 Islet perifusion The dissected islets were cultured overnight and incubated with 7-ketocholesterol, the islets were starved with 0.1% BSA Kreb’s buffer for 30 min and placed in a chamber at 37°C in a perifusion device [ 32 ]. After equilibrium, different fluid buffers (2.8 mmol/L glucose, 16.8 mmol/L glucose, 2.8 mmol/L glucose and 30mmol/L KCl in 0.1% BSA Kreb’s buffer) were perifused for 20 min, 30 min, 20min, and 20 min separately, and fractions were collected each minute. The insulin content was measured with an insulin immunoassay kit (MS300, Ezaasy) and normalized to islets protein content. The insulin levels were normalized to the average baseline insulin secretion in 2.8mmol/L glucose buffer according to the previous study[ 32 ]. The area under curve (AUC) was used to quantify the biphasic insulin secretion ( the phase I and phase II). 2.6 Calcium imaging Following treatment with 7-ketocholesterol, the isolated islets were incubated with 5 mmol/L fluo-4-AM (Invitrogen) in 2 mmol/L glucose-Kreb’s buffer for 45 min, and incubated with 2 mmol/L glucose-Kreb’s buffer for 15 min at 37°C after four washes. Afterwards, the culture dish was transferred on the stage of the laser confocal microscope (Zeiss, Germany) and 16.8 mmol/L glucose-Kreb’s buffer was added. Calcium imaging detected and image measurements were acquired at 10 s intervals and excitation at 488 nm/emission at 515 nm. The results were normalized to basal 2 mmol/L glucose. 2.7 ROS, MMP and ATP assay The analyses were performed using commercially available kits according to the manufacturer’s instructions. After removing the normal culture medium, 1 mL 2 µmol/L DCFH-DA was added in 1640 medium with no FBS at 37℃ for 20 min. The plates were then washed three times with 1640 medium with no FBS, and the cells were detected with fluorescence microscopy at an excitation of 488 nm and emission at 525 nm. After removing the 10% FBS 1640 medium and washing once with KRBH buffer, 1 mL 10% FBS 1640 medium and a 1 mL JC-1 staining working solution was added and stained at 37℃ for 20 min. The supernatant was discarded and washed twice with washing solution, 2 mL 10% FBS 1640 medium was added, and detected with fluorescence microscopy at an excitation of 488 nm and emission of 590 nm. The culture medium was removed and the cells were lysed and supernatant was centrifuged at 4℃ 12,000 × g for 5 min. The ATP concentration was detected using chemiluminometry in accordance with the instructions of the ATP assay kit (Beyotime, China). The ATP level was normalized by protein content. 2.8 Immunofluorescence Experiments were performed as previously described [ 7 ]. After treatment, the cell climbing sheets were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.3% Triton X-100 for 10 min. The cells were blocked in non-specific binding in 5% BSA for each section for 1 h at room temperature on each well and apply primary antibody NRF2 (bs-1074R, Bioss, 1:200) overnight at 4℃. Next, secondary anti-Rabbit immunofluorescence antibodies (A-11037, ThermoFisher Scientific) was applied and diluted at 1:200 in 5% BSA per well in a dark humidified chamber for 90 min at room temperature. DAPI was added for 5 min and a coverslip was added with VECTASHIELD mounting media. Images were obtained with a confocal microscope. 2.9 Total internal reflection fluorescence (TIRFM) The INS1 cells were seeded into MatTek dishes, and transient transfection was used with Lipofectamine 3000 (ThermoFisher Scientific, USA) and Vamp2-pHluorin [ 8 ]. The INS1 cells were treated with 7-ketocholesterol after 24 h. After starving the above cells in 2 mmol/L glucose KRBH (serum free, containing 0.1% BSA) for 2 h, the dishes were transferred to an incubator at 37 ℃ and imaged for 2 min to represent the baseline conditions. Glucose concentration of 25 mmol/L was added to the dish. TIRFM (Olympus IX81, Japan) was performed and controlled by Andor iQsoftware. Images were acquired at 100 ms intervals and at an excitation of 488 nm and emission of 509 nm. The images were analyzed using image analysis software. Fusion events were performed as described previously [ 33 ]. 2.10 qRT-PCR Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, USA) in accordance with the guidelines. Real-time quantitative PCR (RT-PCR) was performed in a 384-well plate (Roche, Switzerland) in Roche 480. Relative gene expression was calculated according to the comparative threshold cycle (2 −ΔΔCt ) method. The primer sequences are described in the supplementary materials (Table S1 ). 2.11 Western blot Proteins were extracted and underwent electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membranes were subsequently incubated with anti - PDX1 (5679, CST,1:1000), anti - GLUT2 (A12307, ABclonal, 1:1000), anti - SNAP25 (ab108990, Abcam, 1:1000), anti - KIF5B (ab167429, Abcam, 1:1000), anti - VAMP2 (ab181869, Abcam, 1:1000), anti - KEAP1 (A17061, ABclonal, 1:1000), anti - COX4 (4844, CST, 1:1000), anti - GAPDH (AC002, ABclonal, 1:5000), anti - β-Actin (ab227387, Abcam, 1:1000), anti - Lamin B1 (13435, CST, 1:1000) and anti - NRF2 (bs-1074R, Bioss,1:1000) overnight at 4 ℃. Then, secondary antibodies conjugated to horseradish peroxidase (anti-mouse, AS003; anti-rabbit AS014, ABclonal, 1:1000) were added for 1 h. The enhanced chemiluminescence method was used to detect the protein. 2.12 Statistical analysis The data from cell experiments were presented as the mean ± SEM, and the representative calcium imaging was selected and the results presented as the means ± SD. Differences between the two groups were analyzed with Student’s t -test, while one-way or two way analysis of variance (ANOVA) (GraphPad Prism 7) was used for multiple groups, and the data comparisons within the significant differences were analyzed using Tukey’s analysis. P < 0.05 was set as the threshold for statistical significance. 3. Results 3.1 The insulin secretion function in pancreatic beta cells is impaired following 7-ketocholesterol treatment 7-ketocholesterol is produced from cholesterol by automatic and/or enzymatic oxidation, and the addition of a functional ketone group at C7 [ 21 ]. Our previous study showed that the accumulation of cholesterol contributed to pancreatic beta cell dysfunction and apoptosis [ 34 ]. To investigate the cell viability of 7-ketocholesterol on pancreatic beta cells, different concentrations of 7-ketocholesterol were exposed to INS1 cells (0, 6.25, 12.5, 25, 50, 100, and 200 µmol/L) (Fig. 1 A) and islets (0, 25, 50, and 100 µmol/L) (Fig. 1 C) for 24 h separately. Furthermore, GSIS was conducted in INS1 cells and primary islets isolated from C57BL/6 mice exposed to different concentrations of 7-ketocholesterol (INS1 cells: 0, 12.5, 25, 50, and 100 µmol/L 7-ketocholesterol; islets: 0, 25, 50, and 100 µmol/L 7-ketocholesterol) (Fig. 1 B and 1 D). The results demonstrated that 25 µmol/L 7-ketocholesterol significantly decreased insulin secretion in the INS1 cells ( P < 0.05), as did 50 µmol/L 7-ketocholesterol in the primary islets ( P < 0.05) (Fig. 1 B and 1 D). Furthermore, the result of primary islets perifusion showed that insulin secretion was severely decreased in the islets after 50 µmol/L 7-ketocholesterol treatment, the AUC was reduced obviously at 7-ketocholesterol group( P < 0.001) (Fig. 1 E and 1 F). Since insulin secretion dysfunction is key risk factor in type 2 diabetes, these data show that beta cell activity was strongly impaired by 7-ketocholesterol during insulin secretion. 3.2 Gene and protein expression of insulin secretory granule fusion with the plasma membrane are affected by 7-ketocholesterol Insulin secretion could represent the level of the beta cell differentiation state, insulin production or processing, insulin-secretory-granule synthesis and translocation, as well as insulin release. qPCR and Western blot were used to detect the level of gene and protein expression related to insulin secretion, translocation, tracking, and beta cell differentiation. The results showed that the transcription regulators for beta cell development (e.g., Pdx1 , Neurod1 , Nkx6.1 , and Mafa ) which were important in beta-cell maturity state and in regulation of insulin secretion were not changed obviously after 7-ketocholesterol treatment (Fig. 2 B). GLUT2 is the principal beta cell glucose transporter and is essential for maintaining its function in insulin secretion, and Glut2 gene and protein expression was down-regulated (Fig. 2 C-D). The level of SNAP25 gene and protein expression were significantly decreased following treatment with 7-ketocholesterol (Fig. 2 A, C-D). SNAP25 plays an important role in tethering the insulin granules and promoting insulin secretory granule docking to effect the exocytosis [ 35 ]. The above studies revealed that insulin secretion was clearly impaired, and may be attributed to the exocytosis of insulin granules. 3.3 7-Ketocholesterol reduced insulin exocytosis and calcium influx Our study are consistent with the previous study that the insulin exocytosis was mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) proteins. To further estimate the changes in insulin granule exocytosis, TIRFM was used to further observe full and kiss-and‐run fusion events of insulin granule exocytosis in INS1 cells expressing Vamp2-pHluorin. The results showed that the fusion events in high glucose stimulation were obviously decreased following treatment with 7-ketocholesterol (p < 0.01, Fig. 2 E). Furthermore, the proportion of the kiss-and-run event was increased in the 7-ketocholesterol treatment group, which may further reduce exocytosis activity (p < 0.01, Fig. 2 F). The results unequivocally showed that the 25 µmol/L 7-ketocholesterol injury decreased the fusion and the exocytosis events to attenuate early insulin secretion. Primary exocytosis occurs involving insulin secretory granule docking to the plasma membrane, followed by priming and Ca 2+ -dependent release. Calcium is a critical intracellular stimulator involved in the release of insulin secretory granules. Thus, we performed functional calcium imaging to measure the levels of cytosolic calcium using laser confocal scanning microscopy with the [Ca 2+ ] indicator, Fluo-4AM probe. The results showed that 50 µmol/L 7-ketocholesterol could reduce the fluorescence intensity of intracellular calcium ions in the islets under high glucose stimulation (p < 0.001, Fig. 2 G). Thus, 7-ketocholesterol had access to decreased intracellular calcium ions and injury insulin exocytosis. 3.4 7-Ketocholesterol induced oxidative stress and mitochondrial dysfunction in INS1 cells A large number of studies have demonstrated that mitochondrial dysfunction and oxidative stress have played a paramount importance in beta cell function. Clearly, lipo-toxicity led to mitochondrial ROS production, and the MMP decreased. When the MMP decreased, ATP synthesis reaction of mitochondrial oxidative phosphorylation was inhibited, the insulin secretion was decreased. To explore whether oxidative stress is linked to the 7-ketocholesterol-mediated beta cell function injury, we detected ROS, MMP, and ATP levels in INS1 cells. After treatment with 25 µmol/L 7-ketocholesterol, intra-cellular ROS obviously increased ( P < 0.05), the MMP ( P < 0.05) and ATP level ( P < 0.05) were decreased (Fig. 3 A-C). After the emergence of oxidative stress, anti-oxidative pathway was activated in beta cells. The results showed that NRF2 was translocated from the cytosol into the nucleus. Once translocated into the nucleus, NRF2 can activate the expression of antioxidant response elements in their promoter, including antioxidant pathway genes and xenobiotic detoxification genes (e.g., NAD(P)H:quinone oxidoreductase 1 [Nqo1] and glutathione-S-transferases [Gsts]). Our results showed the gene or protein expression of Gpx4, Sod1, Txnip, Nqo1, Ho1 , and Cox4 were upregulated following treatment with 25 µmol/L 7-ketocholesterol (Fig. 3 D-G). 3.5 N-Acetyl-L-cysteine (NAC) ameliorated 7-ketocholesterol-induced oxidative stress To further investigate whether oxidative stress response pathway-dependent mechanisms are crucial for beta cell dysfunction, we assessed the effect of antioxidant NAC. After pretreatment with 100 µmol/L NAC, the level of ROS was decreased in INS1 cells( P < 0.01), the levels of MMP and ATP increased (Fig. 4 A-C). Subsequently, NAC cotreatment decreased 25 µmol/L 7-ketocholesterol-induced COX4 up-regulation ( P < 0.01, Fig. 4 D) and NRF2 translocation at nucleus ( P < 0.01, Fig. 4 F) to alleviate oxidative stress. 3.6 N-Acetyl-L-cysteine restored beta cell early insulin secretion function GSIS and islet perifusion focused on whether NAC could recover insulin secretion in 7-ketocholesterol-induced beta cell dysfunction. The function of insulin secretion was partially recovered in beta cells and could alleviate insulin secretion in both beta cells ( P < 0.05) and islets (Fig. 5 A-B). The perifusion in Fig. 5 C and 5 D showed that NAC could strengthen insulin secretion. The results demonstrated that NAC recovered the 7-ketocholesterol-induced calcium influx blocking in the islets under high-glucose stimulation ( P < 0.001) (Fig. 5 E). Furthermore, cotreatment with NAC obviously increased the fusion events ( P < 0.001) and decreased the proportion of kiss-and-run events ( P < 0.05) (Fig. 5 F-G). Further study revealed that GLUT2 and SNAP25 protein associated with insulin secretion and fusion were up-regulated following cotreatment with NAC ( P < 0.05) (Fig. 5 H). Previous studies reported that patients with diabetes had significantly higher serum 7-ketocholesterol levels and that 7-ketocholesterol could be an oxidative stress marker in patients with type 2 diabetes [ 25 , 26 ]. We demonstrated that 7-ketocholesterol affected insulin secretion of pancreatic beta cells involved in oxidative stress pathway activation and decreased exocytosis (Fig. 5 I). 4. Discussion Lipotoxicity in pancreatic beta cells may contribute to type 2 diabetes pathogenesis [ 2 , 36 ]. Cholesterol is a functional component of cell membranes, which maintains beta cell secretory granules and plasma membrane function and the fluidity of beta cells [ 37 ]. However, excessive cholesterol may directly impair beta cell function. Cholesterol can be oxidated into several oxysterols in vivo, and previous studies have shown that oxysterols were higher in the type 2 diabetes subjects [ 38 , 39 ]. As the predominant and most toxic oxidation product [ 13 , 15 ], 7-ketocholesterol can have various side effects and is associated with several diseases (atherosclerosis and cardiovascular diseases, alzheimer disease, age-related macular degeneration) [ 17 – 22 ]. A previous study [ 25 ] demonstrated that diabetic serum contained higher levels of 7-ketocholesterol. Our GSIS results confirmed that 7-ketocholesterol could lead to impaired insulin secretion. Pancreatic beta cell insulin secretion is a biphasic, where it consists of a rapid and transient first phase, followed by a slowly developing and sustained second phase to respond glucose stimulation [ 40 ]. In type 2 diabetes, the first phase may be completely absent and the second phase reduced. And Our study demonstrated that the first phase and second phase insulin secretion were decreased obviously after 7-ketocholesterol treatment. The results further confirmed the impact of oxysterols on insulin secretion. Insulin secretion is derived by glucose entry into the pancreatic beta cell by Glut2 and metabolism into ATP that subsequently shuts off the ATP-sensitive ­K + -channels, leading to membrane depolarization, voltage-dependent Ca 2+ channel opening, Ca 2+ influx, and insulin granule exocytosis [ 29 , 30 , 41 ]. Xu et al. used a TIRFM image of time-lapse visualization performed to observe the fusion processes in exocytosis. Excess cholesterol was found to reduce the number of glucose-stimulated fusion events, modulate the proportion of full fusion, and kiss-and-run fusion events [ 8 ]. However, there is currently no evidence to show the effect of 7-ketocholesterol on exocytosis. Our data shows that the full fusion events were reduced, and the proportion of kiss and run events was increased following 7-ketocholesterol treatment. These changes might lead to a reduction in insulin secretion. During the course of insulin release, the t-SNARE proteins, syntaxin 1 (STX1) and SNAP25 are localized in the plasma membrane, whereas the v-SNARE protein, VAMP2, is associated with insulin secretory granules [ 41 , 42 ]. Our data show the SNAP25 was down-regulated, whereas STX1 and VAMP2 were unchanged following 7-ketocholesterol treatment. Therefore, 7-ketocholesterol was inhibited insulin granule binding with binding partner, SNAP25 to decrease insulin granule fusion on the plasma membrane. Furthermore, the calcium acts as a stimulus for insulin secretion and also a signal to increase insulin synthesis[ 43 ]. Ca 2+ stimulates insulin secretion by regulating docking and initiating fusion of secretory granules with the plasma membrane, a process mediated by SNARE proteins. Ca 2+ entry is actually directed to the sites of exocytosis via the binding of the L-type Ca2 + channels to SNARE proteins[ 44 ]. Calcium imaging in our study showed that calcium influx was reduced under high glucose stimulation after 7-ketocholesterol treatment, the result is consisting with the downregulation SNAP25 and exocytosis. Glut2 was associated with glucose sensing and is necessary for transporting glucose into the cell during the initiation phase of GSIS[ 45 ]. FFAs lipotoxicity was damaged islet β-cells insulin secretion and inhibited the Glut2 expression[ 46 ]. The level of GLUT2 expression were reduced to further demonstrate that insulin secretion was dysfunctional after 7-ketocholesterol treatment. Characterized by increased ROS levels, oxidative stress is a notable factor in the pathogenesis of beta cell dysfunction in type 2 diabetes [ 47 ]. Excessive accumulation of saturated fatty acids can cause the generation of reactive oxygen species, resulting in oxidative stress, mitochondrial dysfunction, loss of mitochondrial membrane potential, impaired ATP production, and fracture and fragmentation of mitochondria, which ultimately leads to cell injuries [ 48 ]. Pancreatic beta cells in both rodents and humans are reportedly rich in mitochondria [ 49 ] and have low levels of classical antioxidant enzymes compared to other cell types, leading to vulnerable to mitochondria dysfunction and increasing oxidative damage[ 50 ]. Following pretreatment with 7-ketocholesterol, there was increased cellular ROS, decreased MMP and ATP levels, which indicated that 7-ketocholesterol enhanced cellular oxidative stress and damaged mitochondrial function. The evidence showed the NAC could reduced oxidative stress in many disease [ 51 ]. Our data showed the NAC was obviously eliminate cellular ROS and strength mitochondrial function; and the insulin secretion function recovered obviously. Through the regulation of cytoprotective gene expression, the KEAP1-NRF2 stress response pathway is the principal inducible defense against oxidative and electrophilic stresses. In response to stress, an intricate molecular mechanism facilitated by sensor cysteines within KEAP1 allows NRF2 to escape ubiquitination, accumulate within the cell, and translocate to the nucleus, where it can promote its antioxidant transcription program[ 52 ]. Our further study showed that antioxidant NRF2 was translocated from the cytoplasm into the nucleus after 7-ketocholesterol treatment, which then triggered the up-regulation of antioxidant genes to decrease oxidative stress. Maintaining redox homeostasis is important for cell function, while the antioxidation genes were up-regulated to alleviate injury, the antioxidant level could not prevent intracellular injury by 7-ketocholesterol, which accelerated beta cell damage in INS1 cells. Pancreatic beta cells undergo dynamic compensation and decompensation processes during the development of type 2 diabetes, in which metabolic stresses such as oxidative stress, endoplasmic reticulum stress and inflammatory signals are key regulators of beta cell dynamics [ 47 ]. The present study only focused on an oxidative stress pathway in insulin secretion and exocytosis, antioxidants can partly restore the insulin secretion. There must be other mechanisms that 7-ketocholesterol can affect insulin secretion. Thus, future studies may employ such other mechanisms of affecting insulin secretion to induce 7-ketocholesterol injury. Oxysterols are derived from cholesterol and provide a feedback mechanism of cholesterol biosynthesis to maintain cholesterol homeostasis. 7-ketocholesterol is one of the most important oxysterols and can be obtained either from food intake or from free-radical oxidation or the enzymatic oxidation of cholesterol in vivo [ 16 , 53 – 55 ]. Additionally, 7-ketocholesterol can be metabolized to 27-hydroxylated 7-ketocholesterol and aqueous products by cholesterol 27-hydroxylase (CYP27A1), reduced to 7β-hydroxycholesterol by hydroxysteroid dehydrogenase (HSD11B1) and/or esterified by sterol O-acyltransferase (SOAT). Following the inhibition of CYP27A1, increased flux was diverted to reduction and esterification. When esterification was inhibited, further reduction and increased metabolism followed [ 16 ]. 7-ketocholesterol was observed to undergo greater hepatic metabolism and excretion, and no increased accumulation was observed in the tissues in the Cyp27 −/− animals compared with the wild-type control mice [ 54 ]. Although 7-ketocholesterol has been shown to accumulate in human macrophage-foam cells and atherosclerotic lesions [ 55 ], dietary 7-ketocholesterol intake did not increase the levels of 7-ketocholesterol in the artery wall in mice. Moreover, the excessive dietary intake of 7-ketocholesterol does not affect cholesterol and glucose metabolism, which might be related to 7-ketocholesterol rebalance in vivo. The complexity of the generation and destination of 7-ketocholesterol makes it difficult to mimic a higher concentration of 7-ketocholesterol in vivo using a gene editing model or diet intervention model to accurately observe the adverse effects of 7-ketocholesterol in various organs. 5. Conclusions Our data demonstrated that 7-ketocholesterol levels significantly impaired insulin secretion function by affecting insulin exocytosis and the oxidative stress pathway. Abbreviations GSIS ：Glucose-stimulated insulin secretion TIRFM ：Total internal reflection fluorescence microscopy ROS : Reactive oxygen species MMP : Mitochondrial membrane potential ATP : Adenosine triphosphate NRF2 : Nuclear factor erythroid 2-related factor NAC : N-Acetyl-L-cysteine PAX6 : Paired box gene 6 PDX-1 : Pancreatic and duodenal homeobox-1 NeuroD1 : Neurogenic differentiation 1 AUC : Area under curve SNAREs : Soluble N-ethylmaleimide-sensitive factor attachment protein receptors Nqo1 : NAD(P)H:quinone oxidoreductase 1 Gsts : Glutathione-S-transferases STX1 : Syntaxin 1 7KC: 7-Ketocholesterol Declarations 7. Acknowledgements We are grateful to Professor Zhuoxian Meng (Zhejiang University) for his expert technical assistance in islet perfusion experiments. 8. Funding This work was supported by the National Natural Science Foundation of China (grant number 81870562, 61827825 and 41906095); the National Key Technology R&D Program of China (grant number 2009BAI80B02); and the Zhejiang Provincial Natural Science Foundation of China (grant number LZ22H070002 and LY22D060003). Author information Wenjing Zhang, Ying Wu and Yuchen Zhao contributed equally to this study and are co-first authors. Authors and Affiliations Department of Endocrinology and Metabolism, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China. Wenjing Zhang, Ying Wu, Yuchen Zhao, Nan Wu, Jiahua Wu, Shuiya Sun, Hong Wang, Sunyue He, Xihua Lin, Jiaqiang Zhou Department of Biomedical Engineering, Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou 310027, China Yingke Xu Contributions W. Z. wrote the main manuscript text, prepared figures 1-5 and provided funding acquisition; Y. W. and Y. Z. prepared figures 1-5. N. W, S. H and X. L. performed formal analysis, and H. W investigated the paper. S. S. provided the resources, J. W. and Y. X. provided data curation. J. Z. revised the manuscript and provided funding acquisition. All authors reviewed the manuscript. Corresponding author Jiaqiang Zhou Ethics declarations Ethical approval and consent to participate Ethical approval and consent to participate were no applicable. The mice were conducted at the Animal Research Center with approval of Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (ZCMU). Consent for publication No applicable Competing interest All data included in this study are available and the authors declare no conflict of interest. Availability of data and materials Data are available on request to the authors. References Hudish LI, Reusch JE, Sussel L. β Cell dysfunction during progression of metabolic syndrome to type 2 diabetes. J Clin Invest. 2019;129:4001–8. Lytrivi M, Castell AL, Poitout V, Cnop M. Recent Insights Into Mechanisms of β-Cell Lipo- and Glucolipotoxicity in Type 2 Diabetes. J Mol Biol. 2020;432:1514–34. Brunham LR, Kruit JK, Verchere CB, Hayden MR. Cholesterol in islet dysfunction and type 2 diabetes. J Clin Invest. 2008;118:403–8. Ye R, Onodera T. Lipotoxicity and β Cell Maintenance in Obesity and Type 2 Diabetes. 2019; 3:617–31. 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Empagliflozin-Enhanced Antioxidant Defense Attenuates Lipotoxicity and Protects Hepatocytes by Promoting FoxO3a- and Nrf2-Mediated Nuclear Translocation via the CAMKK2/AMPK Pathway. 2022; 11. Benito-Vicente A, Jebari-Benslaiman S, Galicia-Garcia U, Larrea-Sebal A, Uribe KB, Martin C. Molecular mechanisms of lipotoxicity-induced pancreatic β-cell dysfunction. Int Rev Cell Mol Biol. 2021;359:357–402. Baumel-Alterzon S, Scott DK. Regulation of Pdx1 by oxidative stress and Nrf2 in pancreatic beta-cells. Front Endocrinol (Lausanne). 2022;13:1011187. Raghu G, Berk M, Campochiaro PA, Jaeschke H, Marenzi G, Richeldi L, Wen FQ, Nicoletti F, Calverley PMA. The Multifaceted Therapeutic Role of N-Acetylcysteine (NAC) in Disorders Characterized by Oxidative Stress. Curr Neuropharmacol. 2021;19:1202–24. Baird L, Yamamoto M. The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway. Mol Cell Biol 2020; 40. Brown AJ, Jessup W. Oxysterols and atherosclerosis. Atherosclerosis. 1999;142:1–28. Lyons MA, Maeda N, Brown AJ. Paradoxical enhancement of hepatic metabolism of 7-ketocholesterol in sterol 27-hydroxylase-deficient mice. Biochim Biophys Acta. 2002;1581:119–26. Lyons MA, Brown AJ. 7-Ketocholesterol delivered to mice in chylomicron remnant-like particles is rapidly metabolised, excreted and does not accumulate in aorta. Biochim Biophys Acta. 2001;1530:209–18. Additional Declarations No competing interests reported. Supplementary Files supplementarymaterials.docx 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. 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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-4483308","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312183800,"identity":"48be637e-6392-4c1c-a23a-f6bc8fc6c167","order_by":0,"name":"Wenjing Zhang","email":"","orcid":"","institution":"Department of Endocrinology and Metabolism, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Zhang","suffix":""},{"id":312183801,"identity":"a9c9561d-e2e4-436a-8613-6216b2e73389","order_by":1,"name":"Ying 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Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jiaqiang","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-05-27 08:06:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4483308/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4483308/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58170230,"identity":"c3f33982-f5dc-481c-b282-4af7fd8c74ca","added_by":"auto","created_at":"2024-06-12 03:37:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":676805,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e7-Ketocholesterol (7KC) led to pancreatic beta cell dysfunction. \u003c/strong\u003eA) The cell viability of INS1 cells was evaluated after different concentrations of 7-ketocholesterol (0, 6.25, 12.5, 25, 50, 100, and 200 μmol/L) treatment for 24 h (n=3). B) GSIS was detected after 24 h treatment with different concentrations of 7-ketocholesterol (0, 12.5, 25, 50 and 100 μmol/L) in INS1 cells (n = 3). C) The cell viability of islets was evaluated with different concentrations of 7-ketocholesterol (0, 25, 50 and 100 μmol/L) treatment for 24 h (15 islets per well, n = 3). D) GSIS was detected after 24 h treatment with different concentrations of 7-ketocholesterol (0, 25, 50 and 100 μmol/L) in the islets (15 islets per well, n = 4). E) Islet perifusion showed that the insulin secretion was severely decreased following 50 μmol/L 7-ketocholesterol treatment (50 islets per perfusion, n = 3). \u0026nbsp;F) The AUC of insulin secretion in phase I and phase II (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"OnlineFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4483308/v1/7c205c948e734dfacfec9d6c.jpg"},{"id":58170228,"identity":"749516b5-2331-4daa-ab01-2a48b84926c9","added_by":"auto","created_at":"2024-06-12 03:37:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1154731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInsulin secretion and exocytosis were impaired after 7-ketocholesterol treatment in pancreatic beta cells.\u003c/strong\u003e A) The gene expression of insulin secretion in INS1 cells treated with 25 μmol/L 7-ketocholesterol for 24h (n = 3). B) The genes related to transcription regulators and functions of beta cell development in INS1 cells treated with 25 μmol/L 7-ketocholesterol for 24h (n = 3). C-D) Western blot for proteins associated with insulin synthesis and secretion in INS1 cells treated with 25 μmol/L 7-ketocholesterol for 24h (n = 3). E) The fusion events were obviously decreased following treatment with 25 μmol/L 7-ketocholesterol for 24h in INS1 cells (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, n = 5). F) The proportion of kiss-and-run events was increased in the INS1 cells treated for 24 h with 25 μmol/L 7-ketocholesterol (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, n = 5). G) 7-Ketocholesterol (50 μmol/L) reduced the fluorescence intensity of intracellular calcium ions in the islets under high glucose stimulation (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, n = 19).\u003c/p\u003e","description":"","filename":"OnlineFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4483308/v1/89e33006d7bb3b9030e6f673.jpg"},{"id":58170233,"identity":"e1659119-54ab-42fb-b808-a16b4deae934","added_by":"auto","created_at":"2024-06-12 03:37:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1279641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidative stress and mitochondrial function in pancreatic beta cells after 7-ketocholesterol treatment.\u003c/strong\u003e A) ATP levels were decreased in INS1 cells treated with 25 μmol/L 7-ketocholesterol for 24 h (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n = 3). B) The mitochondrial membrane potential was decreased after 24 h treatment with 25 μmol/L 7-ketocholesterol (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n = 3). C) Intracellular ROS was obviously increased after 24 h treatment with 25 μmol/L 7-ketocholesterol (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n = 3). D) Antioxidant gene expression was up-regulated following 24 h treatment with 25 μmol/L 7-ketocholesterol (n = 3). E) The gene expression associated with mitochondrial function was downregulated (n = 3). F) The western blot exhibiting NRF2 and KEAP1 expression remained unchanged, but COX4 was up-regulated (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n = 3). G) The nuclear import of NRF2 was observed by immunofluorescence after 24 h treatment with 25 μmol/L 7-ketocholesterol.\u003c/p\u003e","description":"","filename":"OnlineFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4483308/v1/65169da818ea5c2186c126ec.jpg"},{"id":58170229,"identity":"d8fe7652-1d34-4a4d-81c9-e173dd028b65","added_by":"auto","created_at":"2024-06-12 03:37:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1760642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial function recovered and oxidative stress decreased in pancreatic beta cells cotreated with NAC.\u003c/strong\u003e A) Intracellular ROS was reduced following NAC cotreatment (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, n=3). B) The mitochondrial membrane potential was increased after NAC was added (n = 3). C) ATP levels were increased following NAC cotreatment (n = 3). D) COX4 protein expression was recovered by NAC cotreatment (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n = 3). E-F) The nuclear import of NRF2 by immunofluorescence and western blot was observed after 7-ketocholesterol and NAC cotreatment (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n = 3).\u003c/p\u003e","description":"","filename":"OnlineFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4483308/v1/0455efe085d5cecf2f5a1dac.jpg"},{"id":58170231,"identity":"81607c16-49b0-4293-a3a8-11afa70faf3c","added_by":"auto","created_at":"2024-06-12 03:37:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1574053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe insulin secretion was recovered following cotreatment with NAC.\u003c/strong\u003e A and B) NAC recovered the glucose-stimulated insulin release in INS1 cells (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) and primary islets (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) (n = 3). C-D) NAC recovered insulin secretion by islet perifusion (n = 3). E) NAC recovered calcium influx in the islets under high glucose stimulation (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; n \u0026gt; 8). F, G) Fusion events were increased (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001) and the proportion of kiss-and-run events (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) were decreased after cotreat with NAC (n = 4). H) GLUT2 and SNAP25 protein expression were recovered by NAC in INS1 cells (GLUT2, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n=4; SNAP25, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n=3). I) Schematic diagram of the mechanisms of 7-ketocholesterol-induced insulin secretion dysfunction in pancreatic beta cells.\u003c/p\u003e","description":"","filename":"OnlineFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4483308/v1/2175dabb62bc562b2190abe3.jpg"},{"id":99317633,"identity":"32e5f34a-585d-4a92-b9e8-6faf12ba6e26","added_by":"auto","created_at":"2025-12-31 16:30:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7610958,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4483308/v1/b58c290c-87c7-43db-bdc9-a02a70d4b796.pdf"},{"id":58171359,"identity":"a475d4d9-d824-4740-baaa-7120b812ae19","added_by":"auto","created_at":"2024-06-12 03:45:40","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":18428,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4483308/v1/ac9d1eb0d9623182c95d23e9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The cholesterol oxidation product 7-ketocholesterol impairs pancreatic beta cell insulin secretion ","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe failure and dysfunction of pancreatic beta cells represents the key pathogenesis associated with type 2 diabetes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Evidence indicates that the cellular dysfunction induced by lipo- and gluco-lipotoxicity in pancreatic beta cells may contribute to the pathogenesis of type 2 diabetes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the risk factor and underlying mechanisms of beta cell dysfunction have not been thoroughly elucidated to date. Previous studies have shown that free fatty acids (FFA) generate essential metabolic coupling factors in beta cells to amplify the signals required for insulin secretion, but become detrimental under conditions of fuel oversupply [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As a component of the cell membrane, cholesterol influences fluidity and synthesizes several important molecules to regulate cellular functions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and cholesterol metabolism disorders could inhibit glucose-stimulated insulin secretion (GSIS) by pancreatic beta cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, excess cholesterol can reduce glucose transporter activity to impact glucose uptake, reduce glucokinase activity, inhibit glycolysis, and block insulin exocytosis via increasing oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Cholesterol is produced endogenously and can also be introduced through the diet. Furthermore, cholesterol is converted to other perhydrocyclopentanophenanthrene compounds via oxidation/reduction, and these intermediate products participate in the further metabolic conversion to eventually form bile acids, steroid hormones, and vitamin D3 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Studies have reported that these cholesterol oxidation products (oxysterols) are more harmful than cholesterol for accelerating disease progression [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The primary auto-oxidated oxysterols were 7-ketocholesterol, 7b-hydroxysterol, and cholesterol-5a,6a-epoxide. Cholesterol oxidation can occur within different tissues, especially in the vessel wall, and comprises the main hydroxysterol in human atherosclerotic plaques [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs primarily oxide products, 7-ketocholesterol represents biologically active molecules and can have many side effects and is associated with many diseases (e.g., atherosclerosis and cardiovascular diseases, alzheimer\u0026rsquo;s disease, and age-related macular degeneration) [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The accumulation of 7-ketocholesterol in vascular endothelial causes endothelial injury via activation of the endoplasmic reticulum stress response and the up-regulation of vascular endothelial growth factor, interleukin (IL)-1, IL-6, and IL-8 expression [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, 7-ketocholesterol could increase reactive oxygen species (ROS) and activate caspase8/caspase3 to trigger apoptosis in mesenchymal stem cells from patients with acute myeloid leukemia [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The study by Chang et al. demonstrated that a small amount of dietary 7-ketocholesterol contributed to accelerate hepatic steatosis and inflammation in obese mice [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A previous study reported that the level of 7-ketocholesterol was significantly higher in type 2 diabetes patients compared to non-diabetic healthy participants [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, 7-ketocholesterol could be an marker in type 2 diabetes patients [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the effects of 7-ketocholesterol on pancreatic beta cell function have not been directly reported.\u003c/p\u003e \u003cp\u003ePancreatic beta cells, as the most important component in islet tissues, are the only functional cells for insulin secretion in vivo. Insulin secretion could involved in the beta cell differentiation state, insulin production or processing, insulin-secretory-granule synthesis and translocation, as well as insulin release. Insulin synthesis is regulated by trans-activators including paired box gene 6 (PAX6), pancreatic and duodenal homeobox-1(PDX-1), MafA, and Neurogenic differentiation 1 (NeuroD1) at both the transcriptional and translational level [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. And insulin secretion process begins with glucose uptake and metabolism to ATP that subsequently closes ATP-sensitive K\u003csup\u003e+\u003c/sup\u003e-channels, resulting in membrane depolarization, opening of voltage-dependent Ca\u003csup\u003e2+\u003c/sup\u003e channels, Ca\u003csup\u003e2+\u003c/sup\u003e influx and insulin granule exocytosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The exocytosis involved in the trafficking of insulin granules to the plasma membrane, the exocytotic fusion of the granules with the plasma membrane and eventually the retrieval of the secreted membranes by endocytosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The previous study showed the excess cholesterol could increase insulin granule size and reduced the docking and fusion of insulin granules at the plasma membrane [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Based on the above, we intend to answer the question of whether 7-ketocholesterol might affect the development of pancreatic beta cell dysfunction and elucidated the mechanism of beta cell dysfunction. According to our results, 7-ketocholesterol accelerated insulin secretion dysfunction, and reduced insulin exocytosis via oxidative stress.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eCollagenase V (C9263), 7-ketocholesterol (C2394), and Histopaque\u0026reg;-1077 were purchased from Sigma. 7-ketocholesterol was dissolved with anhydrous ethanol and diluted in culture medium as described previously [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The CCK-8 kit (40203ES60) and BCA protein assay (FD2001) kit were obtained from YEASEN and FUDE Biological Tech Co. Ltd., respectively. N-Acetyl-L-cysteine was purchased from Beyotime, Shanghai, China. RPMI high glucose medium, 10 mmol/L Hepes, and 1 mmol/L sodium-pyruvate were acquired from Thermofisher. C57BL/6 mice were purchased from GemPharmatech Co., Ltd. (Nanjing, CN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture and islets dissection\u003c/h2\u003e \u003cp\u003eINS1 cells were cultured in RPMI 1640 medium with 10 mmol/L Hepes, 1 mmol/L sodium-pyruvate, 0.05 mol/L 2-mercaptoethanol, and 10% fetal bovine serum, and penicillin-streptomycin (100 \u0026micro;g/mL) in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe C57BL/6 mice were fasted overnight and anesthetized. The duodenum was ligated and the pancreas was perifused with 2\u0026minus;3 mL collagenase V at 1 mg/mL via the common bile duct and digested for 28 min at 37\u0026deg;C. The digestion was terminated by adding HBSS buffer containing 10% FBS. Followed by filtration and centrifugation, islets were isolated by density gradient centrifugation (Histopaque-1077, Sigma) and collected under a microscope. Finally, the islets were incubated in culture medium and treated with different concentrations of 7-ketocholesterol for 24h at 37℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 7-ketocholesterol treatment and cell viability\u003c/h2\u003e \u003cp\u003eINS1 cells were treated with different concentrations of 7-ketocholesterol for 24 h at 37℃, cell viability was assessed using a CCK8 viability assay.\u003c/p\u003e \u003cp\u003eThe islets were handpicked into the plates and treated with different concentrations of 7-ketocholesterol for 24 h at 37℃, cell viability was assessed using a CCK8 viability assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 GSIS\u003c/h2\u003e \u003cp\u003eExperiments were performed as described previously [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. INS1 cells were seeded at a density of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/well in 1 mL medium in a 12-well plate overnight, after which 7-ketocholesterol was added to each well for 24 h. Subsequently, the cells were rinsed with PBS and starved with 0.01% BSA Kreb\u0026rsquo;s buffer for 30 min and changed to 2.8 mmol/L or 25 mmol/L glucose 0.01% BSA Kreb\u0026rsquo;s buffer for 1 h. The supernatant was collected and centrifuged for 1 min. The insulin content was measured using an insulin immunoassay kit (RT300, Ezaasy) and normalized to cell protein content.\u003c/p\u003e \u003cp\u003eThe islets were starved with 0.01% BSA Kreb\u0026rsquo;s buffer for 30 min, then handpicked into the 1.5 ml Eppendorf tube and stimulated by 2.8 mmol/L glucose in 0.1% BSA Kreb\u0026rsquo;s buffer for 1 h. After collecting supernatant, the islets were stimulated by 16.8 mmol/L glucose in 0.01% BSA Kreb\u0026rsquo;s buffer for 1 h in the same tube and the supernatant was collected again. The insulin content was detected with an insulin immunoassay kit (MS300, Ezaasy), and normalized to protein content of islets. The insulin secretion index was normalized to the average baseline insulin secretion in 2.8mmol/L glucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Islet perifusion\u003c/h2\u003e \u003cp\u003eThe dissected islets were cultured overnight and incubated with 7-ketocholesterol, the islets were starved with 0.1% BSA Kreb\u0026rsquo;s buffer for 30 min and placed in a chamber at 37\u0026deg;C in a perifusion device [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. After equilibrium, different fluid buffers (2.8 mmol/L glucose, 16.8 mmol/L glucose, 2.8 mmol/L glucose and 30mmol/L KCl in 0.1% BSA Kreb\u0026rsquo;s buffer) were perifused for 20 min, 30 min, 20min, and 20 min separately, and fractions were collected each minute. The insulin content was measured with an insulin immunoassay kit (MS300, Ezaasy) and normalized to islets protein content. The insulin levels were normalized to the average baseline insulin secretion in 2.8mmol/L glucose buffer according to the previous study[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The area under curve (AUC) was used to quantify the biphasic insulin secretion ( the phase I and phase II).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Calcium imaging\u003c/h2\u003e \u003cp\u003eFollowing treatment with 7-ketocholesterol, the isolated islets were incubated with 5 mmol/L fluo-4-AM (Invitrogen) in 2 mmol/L glucose-Kreb\u0026rsquo;s buffer for 45 min, and incubated with 2 mmol/L glucose-Kreb\u0026rsquo;s buffer for 15 min at 37\u0026deg;C after four washes. Afterwards, the culture dish was transferred on the stage of the laser confocal microscope (Zeiss, Germany) and 16.8 mmol/L glucose-Kreb\u0026rsquo;s buffer was added. Calcium imaging detected and image measurements were acquired at 10 s intervals and excitation at 488 nm/emission at 515 nm. The results were normalized to basal 2 mmol/L glucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 ROS, MMP and ATP assay\u003c/h2\u003e \u003cp\u003e The analyses were performed using commercially available kits according to the manufacturer\u0026rsquo;s instructions. After removing the normal culture medium, 1 mL 2 \u0026micro;mol/L DCFH-DA was added in 1640 medium with no FBS at 37℃ for 20 min. The plates were then washed three times with 1640 medium with no FBS, and the cells were detected with fluorescence microscopy at an excitation of 488 nm and emission at 525 nm.\u003c/p\u003e \u003cp\u003eAfter removing the 10% FBS 1640 medium and washing once with KRBH buffer, 1 mL 10% FBS 1640 medium and a 1 mL JC-1 staining working solution was added and stained at 37℃ for 20 min. The supernatant was discarded and washed twice with washing solution, 2 mL 10% FBS 1640 medium was added, and detected with fluorescence microscopy at an excitation of 488 nm and emission of 590 nm.\u003c/p\u003e \u003cp\u003eThe culture medium was removed and the cells were lysed and supernatant was centrifuged at 4℃ 12,000 \u0026times; g for 5 min. The ATP concentration was detected using chemiluminometry in accordance with the instructions of the ATP assay kit (Beyotime, China). The ATP level was normalized by protein content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Immunofluorescence\u003c/h2\u003e \u003cp\u003eExperiments were performed as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. After treatment, the cell climbing sheets were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.3% Triton X-100 for 10 min. The cells were blocked in non-specific binding in 5% BSA for each section for 1 h at room temperature on each well and apply primary antibody NRF2 (bs-1074R, Bioss, 1:200) overnight at 4℃. Next, secondary anti-Rabbit immunofluorescence antibodies (A-11037, ThermoFisher Scientific) was applied and diluted at 1:200 in 5% BSA per well in a dark humidified chamber for 90 min at room temperature. DAPI was added for 5 min and a coverslip was added with VECTASHIELD mounting media. Images were obtained with a confocal microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Total internal reflection fluorescence (TIRFM)\u003c/h2\u003e \u003cp\u003eThe INS1 cells were seeded into MatTek dishes, and transient transfection was used with Lipofectamine 3000 (ThermoFisher Scientific, USA) and Vamp2-pHluorin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The INS1 cells were treated with 7-ketocholesterol after 24 h.\u003c/p\u003e \u003cp\u003eAfter starving the above cells in 2 mmol/L glucose KRBH (serum free, containing 0.1% BSA) for 2 h, the dishes were transferred to an incubator at 37 ℃ and imaged for 2 min to represent the baseline conditions. Glucose concentration of 25 mmol/L was added to the dish. TIRFM (Olympus IX81, Japan) was performed and controlled by Andor iQsoftware. Images were acquired at 100 ms intervals and at an excitation of 488 nm and emission of 509 nm.\u003c/p\u003e \u003cp\u003eThe images were analyzed using image analysis software. Fusion events were performed as described previously [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, USA) in accordance with the guidelines. Real-time quantitative PCR (RT-PCR) was performed in a 384-well plate (Roche, Switzerland) in Roche 480. Relative gene expression was calculated according to the comparative threshold cycle (2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e) method. The primer sequences are described in the supplementary materials (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Western blot\u003c/h2\u003e \u003cp\u003eProteins were extracted and underwent electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membranes were subsequently incubated with anti - PDX1 (5679, CST,1:1000), anti - GLUT2 (A12307, ABclonal, 1:1000), anti - SNAP25 (ab108990, Abcam, 1:1000), anti - KIF5B (ab167429, Abcam, 1:1000), anti - VAMP2 (ab181869, Abcam, 1:1000), anti - KEAP1 (A17061, ABclonal, 1:1000), anti - COX4 (4844, CST, 1:1000), anti - GAPDH (AC002, ABclonal, 1:5000), anti - β-Actin (ab227387, Abcam, 1:1000), anti - Lamin B1 (13435, CST, 1:1000) and anti - NRF2 (bs-1074R, Bioss,1:1000) overnight at 4 ℃. Then, secondary antibodies conjugated to horseradish peroxidase (anti-mouse, AS003; anti-rabbit AS014, ABclonal, 1:1000) were added for 1 h. The enhanced chemiluminescence method was used to detect the protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data from cell experiments were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and the representative calcium imaging was selected and the results presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Differences between the two groups were analyzed with Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, while one-way or two way analysis of variance (ANOVA) (GraphPad Prism 7) was used for multiple groups, and the data comparisons within the significant differences were analyzed using Tukey\u0026rsquo;s analysis. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was set as the threshold for statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The insulin secretion function in pancreatic beta cells is impaired following 7-ketocholesterol treatment\u003c/h2\u003e \u003cp\u003e7-ketocholesterol is produced from cholesterol by automatic and/or enzymatic oxidation, and the addition of a functional ketone group at C7 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Our previous study showed that the accumulation of cholesterol contributed to pancreatic beta cell dysfunction and apoptosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To investigate the cell viability of 7-ketocholesterol on pancreatic beta cells, different concentrations of 7-ketocholesterol were exposed to INS1 cells (0, 6.25, 12.5, 25, 50, 100, and 200 \u0026micro;mol/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and islets (0, 25, 50, and 100 \u0026micro;mol/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) for 24 h separately. Furthermore, GSIS was conducted in INS1 cells and primary islets isolated from C57BL/6 mice exposed to different concentrations of 7-ketocholesterol (INS1 cells: 0, 12.5, 25, 50, and 100 \u0026micro;mol/L 7-ketocholesterol; islets: 0, 25, 50, and 100 \u0026micro;mol/L 7-ketocholesterol) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The results demonstrated that 25 \u0026micro;mol/L 7-ketocholesterol significantly decreased insulin secretion in the INS1 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as did 50 \u0026micro;mol/L 7-ketocholesterol in the primary islets (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, the result of primary islets perifusion showed that insulin secretion was severely decreased in the islets after 50 \u0026micro;mol/L 7-ketocholesterol treatment, the AUC was reduced obviously at 7-ketocholesterol group(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Since insulin secretion dysfunction is key risk factor in type 2 diabetes, these data show that beta cell activity was strongly impaired by 7-ketocholesterol during insulin secretion.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Gene and protein expression of insulin secretory granule fusion with the plasma membrane are affected by 7-ketocholesterol\u003c/b\u003e \u003c/p\u003e \u003cp\u003eInsulin secretion could represent the level of the beta cell differentiation state, insulin production or processing, insulin-secretory-granule synthesis and translocation, as well as insulin release. qPCR and Western blot were used to detect the level of gene and protein expression related to insulin secretion, translocation, tracking, and beta cell differentiation. The results showed that the transcription regulators for beta cell development (e.g., \u003cem\u003ePdx1\u003c/em\u003e, \u003cem\u003eNeurod1\u003c/em\u003e, \u003cem\u003eNkx6.1\u003c/em\u003e, and \u003cem\u003eMafa\u003c/em\u003e) which were important in beta-cell maturity state and in regulation of insulin secretion were not changed obviously after 7-ketocholesterol treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). GLUT2 is the principal beta cell glucose transporter and is essential for maintaining its function in insulin secretion, and Glut2 gene and protein expression was down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). The level of SNAP25 gene and protein expression were significantly decreased following treatment with 7-ketocholesterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C-D). SNAP25 plays an important role in tethering the insulin granules and promoting insulin secretory granule docking to effect the exocytosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The above studies revealed that insulin secretion was clearly impaired, and may be attributed to the exocytosis of insulin granules.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 7-Ketocholesterol reduced insulin exocytosis and calcium influx\u003c/h2\u003e \u003cp\u003eOur study are consistent with the previous study that the insulin exocytosis was mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) proteins. To further estimate the changes in insulin granule exocytosis, TIRFM was used to further observe full and kiss-and‐run fusion events of insulin granule exocytosis in INS1 cells expressing Vamp2-pHluorin. The results showed that the fusion events in high glucose stimulation were obviously decreased following treatment with 7-ketocholesterol (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, the proportion of the kiss-and-run event was increased in the 7-ketocholesterol treatment group, which may further reduce exocytosis activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The results unequivocally showed that the 25 \u0026micro;mol/L 7-ketocholesterol injury decreased the fusion and the exocytosis events to attenuate early insulin secretion.\u003c/p\u003e \u003cp\u003ePrimary exocytosis occurs involving insulin secretory granule docking to the plasma membrane, followed by priming and Ca\u003csup\u003e2+\u003c/sup\u003e-dependent release. Calcium is a critical intracellular stimulator involved in the release of insulin secretory granules. Thus, we performed functional calcium imaging to measure the levels of cytosolic calcium using laser confocal scanning microscopy with the [Ca\u003csup\u003e2+\u003c/sup\u003e] indicator, Fluo-4AM probe. The results showed that 50 \u0026micro;mol/L 7-ketocholesterol could reduce the fluorescence intensity of intracellular calcium ions in the islets under high glucose stimulation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Thus, 7-ketocholesterol had access to decreased intracellular calcium ions and injury insulin exocytosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 7-Ketocholesterol induced oxidative stress and mitochondrial dysfunction in INS1 cells\u003c/h2\u003e \u003cp\u003eA large number of studies have demonstrated that mitochondrial dysfunction and oxidative stress have played a paramount importance in beta cell function. Clearly, lipo-toxicity led to mitochondrial ROS production, and the MMP decreased. When the MMP decreased, ATP synthesis reaction of mitochondrial oxidative phosphorylation was inhibited, the insulin secretion was decreased. To explore whether oxidative stress is linked to the 7-ketocholesterol-mediated beta cell function injury, we detected ROS, MMP, and ATP levels in INS1 cells. After treatment with 25 \u0026micro;mol/L 7-ketocholesterol, intra-cellular ROS obviously increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the MMP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and ATP level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003eAfter the emergence of oxidative stress, anti-oxidative pathway was activated in beta cells. The results showed that NRF2 was translocated from the cytosol into the nucleus. Once translocated into the nucleus, NRF2 can activate the expression of antioxidant response elements in their promoter, including antioxidant pathway genes and xenobiotic detoxification genes (e.g., NAD(P)H:quinone oxidoreductase 1 [Nqo1] and glutathione-S-transferases [Gsts]). Our results showed the gene or protein expression of \u003cem\u003eGpx4, Sod1, Txnip, Nqo1, Ho1\u003c/em\u003e, and \u003cem\u003eCox4\u003c/em\u003e were upregulated following treatment with 25 \u0026micro;mol/L 7-ketocholesterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 N-Acetyl-L-cysteine (NAC) ameliorated 7-ketocholesterol-induced oxidative stress\u003c/h2\u003e \u003cp\u003eTo further investigate whether oxidative stress response pathway-dependent mechanisms are crucial for beta cell dysfunction, we assessed the effect of antioxidant NAC. After pretreatment with 100 \u0026micro;mol/L NAC, the level of ROS was decreased in INS1 cells(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), the levels of MMP and ATP increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Subsequently, NAC cotreatment decreased 25 \u0026micro;mol/L 7-ketocholesterol-induced COX4 up-regulation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and NRF2 translocation at nucleus (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) to alleviate oxidative stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 N-Acetyl-L-cysteine restored beta cell early insulin secretion function\u003c/h2\u003e \u003cp\u003eGSIS and islet perifusion focused on whether NAC could recover insulin secretion in 7-ketocholesterol-induced beta cell dysfunction. The function of insulin secretion was partially recovered in beta cells and could alleviate insulin secretion in both beta cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and islets (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). The perifusion in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eD showed that NAC could strengthen insulin secretion. The results demonstrated that NAC recovered the 7-ketocholesterol-induced calcium influx blocking in the islets under high-glucose stimulation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, cotreatment with NAC obviously increased the fusion events (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and decreased the proportion of kiss-and-run events (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G). Further study revealed that GLUT2 and SNAP25 protein associated with insulin secretion and fusion were up-regulated following cotreatment with NAC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003ePrevious studies reported that patients with diabetes had significantly higher serum 7-ketocholesterol levels and that 7-ketocholesterol could be an oxidative stress marker in patients with type 2 diabetes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We demonstrated that 7-ketocholesterol affected insulin secretion of pancreatic beta cells involved in oxidative stress pathway activation and decreased exocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLipotoxicity in pancreatic beta cells may contribute to type 2 diabetes pathogenesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Cholesterol is a functional component of cell membranes, which maintains beta cell secretory granules and plasma membrane function and the fluidity of beta cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, excessive cholesterol may directly impair beta cell function. Cholesterol can be oxidated into several oxysterols in vivo, and previous studies have shown that oxysterols were higher in the type 2 diabetes subjects [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. As the predominant and most toxic oxidation product [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], 7-ketocholesterol can have various side effects and is associated with several diseases (atherosclerosis and cardiovascular diseases, alzheimer disease, age-related macular degeneration) [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A previous study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] demonstrated that diabetic serum contained higher levels of 7-ketocholesterol. Our GSIS results confirmed that 7-ketocholesterol could lead to impaired insulin secretion. Pancreatic beta cell insulin secretion is a biphasic, where it consists of a rapid and transient first phase, followed by a slowly developing and sustained second phase to respond glucose stimulation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In type 2 diabetes, the first phase may be completely absent and the second phase reduced. And Our study demonstrated that the first phase and second phase insulin secretion were decreased obviously after 7-ketocholesterol treatment. The results further confirmed the impact of oxysterols on insulin secretion.\u003c/p\u003e \u003cp\u003eInsulin secretion is derived by glucose entry into the pancreatic beta cell by Glut2 and metabolism into ATP that subsequently shuts off the ATP-sensitive \u0026shy;K\u003csup\u003e+\u003c/sup\u003e-channels, leading to membrane depolarization, voltage-dependent Ca\u003csup\u003e2+\u003c/sup\u003e channel opening, Ca\u003csup\u003e2+\u003c/sup\u003e influx, and insulin granule exocytosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Xu et al. used a TIRFM image of time-lapse visualization performed to observe the fusion processes in exocytosis. Excess cholesterol was found to reduce the number of glucose-stimulated fusion events, modulate the proportion of full fusion, and kiss-and-run fusion events [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, there is currently no evidence to show the effect of 7-ketocholesterol on exocytosis. Our data shows that the full fusion events were reduced, and the proportion of kiss and run events was increased following 7-ketocholesterol treatment. These changes might lead to a reduction in insulin secretion. During the course of insulin release, the t-SNARE proteins, syntaxin 1 (STX1) and SNAP25 are localized in the plasma membrane, whereas the v-SNARE protein, VAMP2, is associated with insulin secretory granules [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our data show the SNAP25 was down-regulated, whereas STX1 and VAMP2 were unchanged following 7-ketocholesterol treatment. Therefore, 7-ketocholesterol was inhibited insulin granule binding with binding partner, SNAP25 to decrease insulin granule fusion on the plasma membrane. Furthermore, the calcium acts as a stimulus for insulin secretion and also a signal to increase insulin synthesis[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Ca\u003csup\u003e2+\u003c/sup\u003e stimulates insulin secretion by regulating docking and initiating fusion of secretory granules with the plasma membrane, a process mediated by SNARE proteins. Ca\u003csup\u003e2+\u003c/sup\u003e entry is actually directed to the sites of exocytosis via the binding of the L-type Ca2\u0026thinsp;+\u0026thinsp;channels to SNARE proteins[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Calcium imaging in our study showed that calcium influx was reduced under high glucose stimulation after 7-ketocholesterol treatment, the result is consisting with the downregulation SNAP25 and exocytosis. Glut2 was associated with glucose sensing and is necessary for transporting glucose into the cell during the initiation phase of GSIS[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. FFAs lipotoxicity was damaged islet β-cells insulin secretion and inhibited the Glut2 expression[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The level of GLUT2 expression were reduced to further demonstrate that insulin secretion was dysfunctional after 7-ketocholesterol treatment.\u003c/p\u003e \u003cp\u003eCharacterized by increased ROS levels, oxidative stress is a notable factor in the pathogenesis of beta cell dysfunction in type 2 diabetes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Excessive accumulation of saturated fatty acids can cause the generation of reactive oxygen species, resulting in oxidative stress, mitochondrial dysfunction, loss of mitochondrial membrane potential, impaired ATP production, and fracture and fragmentation of mitochondria, which ultimately leads to cell injuries [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Pancreatic beta cells in both rodents and humans are reportedly rich in mitochondria [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and have low levels of classical antioxidant enzymes compared to other cell types, leading to vulnerable to mitochondria dysfunction and increasing oxidative damage[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Following pretreatment with 7-ketocholesterol, there was increased cellular ROS, decreased MMP and ATP levels, which indicated that 7-ketocholesterol enhanced cellular oxidative stress and damaged mitochondrial function. The evidence showed the NAC could reduced oxidative stress in many disease [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our data showed the NAC was obviously eliminate cellular ROS and strength mitochondrial function; and the insulin secretion function recovered obviously. Through the regulation of cytoprotective gene expression, the KEAP1-NRF2 stress response pathway is the principal inducible defense against oxidative and electrophilic stresses. In response to stress, an intricate molecular mechanism facilitated by sensor cysteines within KEAP1 allows NRF2 to escape ubiquitination, accumulate within the cell, and translocate to the nucleus, where it can promote its antioxidant transcription program[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Our further study showed that antioxidant NRF2 was translocated from the cytoplasm into the nucleus after 7-ketocholesterol treatment, which then triggered the up-regulation of antioxidant genes to decrease oxidative stress. Maintaining redox homeostasis is important for cell function, while the antioxidation genes were up-regulated to alleviate injury, the antioxidant level could not prevent intracellular injury by 7-ketocholesterol, which accelerated beta cell damage in INS1 cells.\u003c/p\u003e \u003cp\u003ePancreatic beta cells undergo dynamic compensation and decompensation processes during the development of type 2 diabetes, in which metabolic stresses such as oxidative stress, endoplasmic reticulum stress and inflammatory signals are key regulators of beta cell dynamics [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The present study only focused on an oxidative stress pathway in insulin secretion and exocytosis, antioxidants can partly restore the insulin secretion. There must be other mechanisms that 7-ketocholesterol can affect insulin secretion. Thus, future studies may employ such other mechanisms of affecting insulin secretion to induce 7-ketocholesterol injury.\u003c/p\u003e \u003cp\u003eOxysterols are derived from cholesterol and provide a feedback mechanism of cholesterol biosynthesis to maintain cholesterol homeostasis. 7-ketocholesterol is one of the most important oxysterols and can be obtained either from food intake or from free-radical oxidation or the enzymatic oxidation of cholesterol in vivo [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Additionally, 7-ketocholesterol can be metabolized to 27-hydroxylated 7-ketocholesterol and aqueous products by cholesterol 27-hydroxylase (CYP27A1), reduced to 7β-hydroxycholesterol by hydroxysteroid dehydrogenase (HSD11B1) and/or esterified by sterol O-acyltransferase (SOAT). Following the inhibition of CYP27A1, increased flux was diverted to reduction and esterification. When esterification was inhibited, further reduction and increased metabolism followed [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. 7-ketocholesterol was observed to undergo greater hepatic metabolism and excretion, and no increased accumulation was observed in the tissues in the Cyp27\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e animals compared with the wild-type control mice [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Although 7-ketocholesterol has been shown to accumulate in human macrophage-foam cells and atherosclerotic lesions [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], dietary 7-ketocholesterol intake did not increase the levels of 7-ketocholesterol in the artery wall in mice. Moreover, the excessive dietary intake of 7-ketocholesterol does not affect cholesterol and glucose metabolism, which might be related to 7-ketocholesterol rebalance in vivo. The complexity of the generation and destination of 7-ketocholesterol makes it difficult to mimic a higher concentration of 7-ketocholesterol in vivo using a gene editing model or diet intervention model to accurately observe the adverse effects of 7-ketocholesterol in various organs.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur data demonstrated that 7-ketocholesterol levels significantly impaired insulin secretion function by affecting insulin exocytosis and the oxidative stress pathway.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eGSIS\u003c/strong\u003e：Glucose-stimulated insulin secretion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTIRFM\u003c/strong\u003e：Total internal reflection fluorescence microscopy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS\u003c/strong\u003e: \u0026nbsp;Reactive oxygen species\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMMP\u003c/strong\u003e: Mitochondrial membrane potential\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATP\u003c/strong\u003e:\u0026nbsp;Adenosine triphosphate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNRF2\u003c/strong\u003e: \u0026nbsp;Nuclear factor erythroid 2-related factor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAC\u003c/strong\u003e: N-Acetyl-L-cysteine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePAX6\u003c/strong\u003e: Paired box gene 6\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePDX-1\u003c/strong\u003e: Pancreatic and duodenal homeobox-1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuroD1\u003c/strong\u003e: Neurogenic differentiation 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUC\u003c/strong\u003e: Area under curve\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSNAREs\u003c/strong\u003e: Soluble N-ethylmaleimide-sensitive factor attachment protein receptors\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNqo1\u003c/strong\u003e: NAD(P)H:quinone oxidoreductase 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGsts\u003c/strong\u003e: Glutathione-S-transferases\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTX1\u003c/strong\u003e: Syntaxin 1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;7KC:\u0026nbsp;\u003c/strong\u003e7-Ketocholesterol\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e7. Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Professor Zhuoxian Meng (Zhejiang University) for his expert technical assistance in islet perfusion experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant number 81870562, 61827825 and 41906095); the National Key Technology R\u0026amp;D Program of China (grant number 2009BAI80B02); and the Zhejiang Provincial Natural Science Foundation of China (grant number LZ22H070002 and LY22D060003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWenjing Zhang, Ying Wu and Yuchen Zhao contributed equally to this study and are co-first authors.\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Department of Endocrinology and Metabolism, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China.\u003c/p\u003e\n\u003cp\u003eWenjing Zhang, Ying Wu, Yuchen Zhao, Nan Wu, Jiahua Wu, Shuiya Sun, Hong Wang, Sunyue He, \u0026nbsp;Xihua Lin,\u0026nbsp;Jiaqiang Zhou\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDepartment of Biomedical Engineering, Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou 310027, China\u003c/p\u003e\n\u003cp\u003eYingke Xu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW. Z. wrote the main manuscript text, prepared figures 1-5 and \u0026nbsp;provided funding acquisition; Y. W. and Y. Z. prepared figures 1-5. N. W, S. H and X. L. performed formal analysis, and \u0026nbsp;H. W \u0026nbsp; investigated the paper. S. S. provided the resources, J. W. and Y. X. provided data curation. J. Z. revised the manuscript and provided funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiaqiang Zhou\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval and consent to participate were no\u0026nbsp;applicable. The mice were conducted at the Animal Research Center with approval of Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (ZCMU).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available\u0026nbsp;and the authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available on request to the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHudish LI, Reusch JE, Sussel L. β Cell dysfunction during progression of metabolic syndrome to type 2 diabetes. 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Biochim Biophys Acta. 2001;1530:209\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e\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":"7-ketocholesterol, pancreatic beta cell, perifusion, insulin secretion, exocytosis, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-4483308/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4483308/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u0026nbsp;\u003c/strong\u003eThe\u0026nbsp;impairment of pancreatic\u0026nbsp;beta cell function caused by\u0026nbsp;glucolipotoxicity plays\u0026nbsp;an\u0026nbsp;important\u0026nbsp;role\u0026nbsp;in\u0026nbsp;the\u0026nbsp;pathogenesis\u0026nbsp;of\u0026nbsp;type 2\u0026nbsp;diabetes. Previous studies have shown that cholesterol can induce beta cell dysfunction.\u0026nbsp;However, the effect of the cholesterol oxidation product\u0026nbsp;7-ketocholesterol in beta-cell function remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Cell proliferation, Glucose-stimulated insulin secretion (GSIS), perifusion, calcium imaging, total internal reflection fluorescence microscopy (TIRFM), reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, qPCR, and Western blotting were used to evaluate the effect and mechanism of 7-ketocholesterol on INS1 cells and islets. N-Acetyl-L-cysteine was used to rescue insulin secretion of beta-cells. GSIS, perifusion, calcium levels and exocytosis events verified that early-phase insulin secretion was impaired after 7-ketocholesterol treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The results of CCK 8 and GSIS demonstrated that 25 μmol/L 7-ketocholesterol significantly decreased insulin secretion in the INS1 cells (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), as did 50 μmol/L 7-ketocholesterol in the primary islets (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The islet perifusion analysis verified that the insulin secretion function was impaired with 7-ketocholesterol(\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). Calcium imaging showed that the intracellular calcium levels were decreased following 7-ketocholesterol treatment(\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u0026nbsp;TIRFM\u0026nbsp;imaging inferred that 7-ketocholesterol could reduced insulin-secretory-granule\u0026nbsp;exocytosis by decreased\u0026nbsp;fusion\u0026nbsp;events and increased kiss-and-run events\u0026nbsp;to the membrane to attenuate\u0026nbsp;insulin secretion (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01).\u0026nbsp;Further data showed that the level of Snap25 gene and protein expression related to insulin exocytosis was substantially downregulated.\u0026nbsp;Further study showed that the\u0026nbsp;reactive oxygen species (ROS)\u0026nbsp;in INS1 cells was upregulated, and both the mitochondrial membrane potential (MMP) and level of adenosine triphosphate (ATP) was downregulated\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The regulation of nuclear factor erythroid 2-related factor (NRF2) is an important transcription factor for oxidative stress, for which its nuclear translocation results in the subsequent activation of gene transcription of \u0026nbsp;\u003cem\u003eGpx4\u003c/em\u003e, \u003cem\u003eSod1\u003c/em\u003e, \u003cem\u003eTxnip\u003c/em\u003e, \u003cem\u003eNqo1\u003c/em\u003e, and \u003cem\u003eHo1\u003c/em\u003e in INS1 cells. In addition, 7-ketocholesterol-induced pancreatic beta cell dysfunction and oxidative stress was ameliorated by pretreatment with the antioxidant, N-Acetyl-L-cysteine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e These findings suggested that 7-ketocholesterol impacted insulin exocytosis to decrease the insulin secretion of pancreatic beta cells involved in the oxidative stress.\u003c/p\u003e","manuscriptTitle":"The cholesterol oxidation product 7-ketocholesterol impairs pancreatic beta cell insulin secretion ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-12 03:37:35","doi":"10.21203/rs.3.rs-4483308/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":"cfdb4628-2875-4a18-95d4-7f98ed51d97a","owner":[],"postedDate":"June 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T17:21:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-12 03:37:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4483308","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4483308","identity":"rs-4483308","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}