Low expression of Elovl6 may involve in fat loss in white adipose tissue of cancer-associated cachexia

Low expression of Elovl6 may involve in fat loss in white adipose tissue of cancer-associated cachexia | 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 Low expression of Elovl6 may involve in fat loss in white adipose tissue of cancer-associated cachexia Chenyang Jin, Shuangjie Wang, Xiangyu Sui, Qingyang Meng, Guohao Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3611425/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: Cancer-associated cachexia (CAC) arises from malignant tumors and leads to a debilitating wasting syndrome. In the pathophysiology of CAC, the depletion of fat performs a significant function. The mechanism of CAC-induced fat loss includes the enhancement of lipolysis, inhibition of lipogenesis, and browning of white adipose tissue (WAT), however, few lipid-metabolic enzymes have been reported to be involved in CAC. This study hypothesized that Elovl6, a critical enzyme for elongation of fatty acids, may involve in fat loss in CAC. Method: Transcriptome sequencing technology was employed to identify CAC-related genes in the WAT of a CAC rodent model. Then the expression level of Elovl6 and fatty acid composition were analyzed in a large clinical sample. Elovl6 was knocked down in 3T3-L1 mouse preadipocytes to compare with wild-type 3T3-L1 cells treated with tumor cell-conditioned medium. Result: In the WAT of patients with CAC, a significant decrease in the expression of Elovl6 was found, which correlates with the extent of body mass reduction in a linear relationship. Gas chromatographic analysis revealed an augmentation in palmitic acid (C16:0) and a reduction in linoleic acid (C18:2n6c) content in those tissue samples. Treating with tumor cell-conditioned medium, 3T3-L1 mouse preadipocytes showed a decrease of Elov16, and Elovl6-knockdown cells demonstrated reduced pre-adipocyte differentiation and lipogenesis. Likewise, the knockdown of Elovl6 in 3T3-L1 cells exhibited a significant rise in palmitic acid (C16:0) and a remarkable decrease in oleic acid (C18:1n9c) content. Conclusion: Overall, the expression of Elovl6 was diminished in the WAT of CAC patients. The decreased expression of Elovl6 might lead to fat loss in CAC by potentially altering the fatty acid composition in adipocytes. These findings suggest that Elovl6 may be used as a valuable biomarker for early diagnosis of CAC, and hold promise as a target for future therapies. Cancer-associated cachexia Elovl6 fat loss long-chain fatty acid Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION According to an international consensus published in 2011 [1], cancer-associated cachexia (CAC) is characterized as a wasting syndrome induced by cancer. It involves persistent skeletal muscle depletion and fat loss, which proves challenging to be fully reversed using standard nutritional interventions. CAC not only leads to a grim prognosis but also diminishes the quality of life in individuals with malignant tumors [2, 3]. However, our comprehension of the mechanisms behind CAC remains relatively limited. Previous laboratory and clinical studies have revealed the generation of pro-inflammatory cytokines during crosstalk among somatic cells, immune cells, and tumor cells. These cytokines may consequently influence skeletal muscle, adipose tissue, and the central nervous system, causing clinical symptoms such as decreased appetite, skeletal muscle atrophy, and fat loss [4]. Recently, the role of fat loss in the pathophysiology of CAC has been reported as an increasingly important issue. The latest study in CAC rodent models suggests that fat loss may occur before skeletal muscle atrophy and altered lipid metabolic processes may be a prerequisite for this process [5-7, 35, 36]. According to most studies, fat loss in CAC occurs through three primary mechanisms: enhancement of fat hydrolysis, inhibition of fat synthesis, and the transformation of white adipose tissue (WAT) [1, 8]. In this study, transcriptome sequencing of WAT has been carried out from tumor-bearing mice. A reduction in the expression level of elongase of very long-chain fatty acid (Elovl6) was then observed. Elovl6 was found abundantly in lipid-rich tissues like WAT and serves as the enzyme responsible for extending saturated fatty acids comprising 12-16 carbon atoms [14-16]. In rodent models, multiple studies indicated that Elovl6 has a critical role in regulating fatty acid metabolism. Notably, Elovl6 knockout mice have shown resistance to diet-induced insulin resistance [17, 18]. Under cold-acclimated conditions, Elovl6 knockout mice show reduced gene expression associated with the thermogenic process in brown adipose tissue (BAT), indicating impaired thermogenic capacity [19]. Furthermore, over the past few decades, numerous studies have highlighted the crucial role of long-chain fatty acids (LCFAs) in regulating adipocyte metabolism. LCFAs present within adipocytes can enhance adipose thermogenesis [9-11] and trigger inflammation in adipose tissue [12, 13]. These connections among LCFAs, Elovl6, and fatty acid metabolism point out that Elovl6 could be involved in CAC-induced fat loss. Thus, based on the existing literature mentioned above, it is reasonable to hypothesize that Elovl6 is involved in fat loss in CAC. Hence, this study aimed to investigate both the expression level and the function of Elovl6 in the process of fat loss in CAC. METHODS Patients and tissue samples This study comprised patients who received diagnoses at the General Surgery Department of Zhongshan Hospital, Fudan University, during the period spanning from July 2019 to June 2020. The criteria for inclusion were as follows: (1) Confirmation of gastric or colorectal cancer through a pathological assessment; (2) Underwent radical surgery at the hospital; (3) Availability of complete clinical data. The exclusion criteria were as follows: (1) Previous receipt of preoperative anticancer strategy (chemotherapy, targeted therapy, immunotherapy, or radiotherapy); (2) Presence of severe organ dysfunction (renal failure, liver insufficient, heart failure or chronic obstructive pulmonary disease), immunity impairment (autoimmune disorders, lymphoma, leukemia or acquired immune deficiency syndrome) or uncontrolled metabolic syndrome (hyperthyroidism or diabetes). All enrolled patients were briefed about the research and granted their consent to take part. Clinical data (age, gender, height, weight, weight loss, tumor site, and staging) for the enrolled patients were recorded. Nutrition-related laboratory parameters, including hemoglobin, albumin, pre-albumin, and total protein, were also recorded. According to the 2011 international consensus on CAC [1], patients were defined as CAC if they satisfied one or more of these conditions: (1) A decrease in body weight exceeding 5% during the preceding 6 months; (2) A weight reduction of over 2% coupled with a body mass index (BMI) below 20 kg/m 2 . Abdominal subcutaneous WAT was carefully separated and divided into approximately 500 mg pieces before being transferred to sample storage tubes. The specimens were then rapidly frozen using liquid nitrogen and preserved at -80℃ for subsequent examination. CAC rodent model Male C57/BL6 mice, weighing 18-20 grams and aged 6-7 weeks, were randomly assigned to two study groups: (1) healthy control group (CONT) and (2) tumor-bearing group (CAC). The mice were provided by the Shanghai Institutes for Biological Science. The CAC mice were subcutaneously injected with 5*10 6 Lewis lung carcinoma (LLC) cells dissolved in phosphate-buffered saline (PBS) near their ilium in the lower back. In contrast, the CONT group received PBS injections only. All mice were euthanized after 28 days following LLC cell or PBS injection. The samples of inguinal white adipose tissue (iWAT), epididymis white adipose tissue (eWAT), gastrocnemius, liver, and heart from each mouse were collected. The samples were promptly frozen in liquid nitrogen and preserved at -80℃ for subsequent examination. RNA sequencing Total RNA extraction followed the official guidelines provided by Invitrogen using the TRIzol method. Degradation and contamination of RNA were identified using agarose gel (1%) electrophoresis. The measurement of RNA concentration was determined by employing a NanoPhotometer spectrophotometer for OD260/280 and OD260/230. RNA integrity was assessed by an Agilent 2100 Bioanalyzer. Subsequently, mRNA was extracted and constructed into a cDNA library following official guidelines and technical documents from NEB (China). After assembly, the library underwent quantification with a Qubit 2.0 fluorometer and was then diluted to a concentration of 1.5 ng/μL using enzyme-free water. The insert fragments were detected by using an Agilent 2100 Bioanalyzer. The libraries were quantified using qRT-PCR, meanwhile ensuring an effective library concentration exceeding 2 nM. Sequencing of the library was performed by the Illumina Hiseq 4000 platform. Bioinformatic data processing The raw data in fastq format underwent initial processing and data cleaning with Perl scripts developed in-house. After that, the read number of each gene was counted and normalized. The reference genome and its corresponding index were assembled using Hisat2 v2.0.5 after being downloaded from the Ensembl database (Ensembl Mus musculus release-94). A differential expression analysis was performed between two sets of data utilizing the DESeq2 R package (version 1.16.1). Genes identified as differential expressions had an adjusted p-value (padj) below 0.05. The Gene Ontology (GO) database was used for enrichment analysis. Among the genes with differential expressions, GO terms that demonstrated a padj below 0.05 were deemed significantly enriched. Real-time PCR Total RNA from both animal and human tissue was extracted by using the TRIzol method. The reverse transcription of mRNAs was conducted by employing a Qiagen Quantinova kit. A real-time PCR reaction system was established with a Qiagen SYBR green PCR kit and was performed on a LightCycler 480 Platform. The list of PCR primers used was attached to supplemental materials. Western blotting RIPA lysis buffer was used for protein extraction from both tissue and cell samples, following which the proteins were quantified by the bicinchoninic acid method. Protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane before blocking with Tris-buffered saline supplemented with 5% skimmed milk powder for 1 hour. Following this, the membranes underwent an incubation at 4℃ overnight with the primary antibody and then were subjected to a 1-hour incubation with goat anti-rabbit IgG marked with horse radish peroxidase (HRP). The Tanon 5200 imaging system was used for identifying and analyzing stained bands. In this study, β-tubulin (CST, USA; 1:1000) and Elovl6 (Novus Biologicals, USA; 1:1000) were the primary antibodies used. FAME analysis A mixture of ether and petroleum ether was used to extract fatty acids. Fatty acid was esterified to form fatty acid methyl esters (FAMEs) before being injected into the gas chromatograph for flame ionization detection. The percentage of a particular fatty acid is determined by the ratio of the peak area of that fatty acid to the sum of the peak areas of all fatty acid components. Cell culture The 3T3-L1 mouse preadipocytes were cultivated in Dulbecco's modified eagle medium with high-glucose (HG-DMEM, Thermo Fisher), into which 10% neonatal calf serum (NCS) from AusgeneX and 1% penicillin-streptomycin solution (PSS) from ThermoFisher were added. Similarly, LLC and the C26 mouse colon cancer cell lines were cultured in HG-DMEM. These cultures received additional supplementation, including 10% fetal bovine serum (FBS) from AusgeneX and 1% PSS. The cells were kept in a microbiological incubator at 37°C with a 5% CO 2 environment. The Cell Bank at the Chinese Academy of Sciences generously provided all the cells. Differentiation of 3T3-L1 cells For the initial differentiation of 3T3-L1 cells, a differentiation-induced medium (DIM-I) was employed, consisting of DMEM with 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μg/mL insulin, 1% PSS, and 10% FBS. Subsequently, another differentiation-induced medium (DIM-II) was used to maintain the differentiation environment. DIM-II was composed of DMEM with 1 μg/mL insulin, 1% PSS, and 10% FBS. The 3T3-L1 cell differentiation process commenced two days after achieving 100% confluency (D0) by switching to the DIM-I medium. On day 2 (D2), the medium was switched to DIM-II, which was renewed every 2 days until further intervention. Preparation and use of CAC-mimic medium The preparation of a CAC-mimic medium has been described in our previous work [33]. Two days after passaging C26 cells (or 3T3-L1 cells), the medium supernatant was gathered and filtered. The CAC-mimic medium consisted of 66% C26 medium supernatant and 33% complete medium, replacing D4 in the CAC group to form a cachexic environment. A mixture of 66% 3T3-L1 medium supernatant and 33% complete medium served as the corresponding intervention for the negative control (NC) group. Interleukin-6 (IL-6) was added to the complete medium as a positive control at a final concentration of 100 ng/ml [34]. Cell transfection In 6-well plates, 3T3-L1 cells were cultured until they reached a confluency of 70-90% for transfection. Following a PBS rinse, 700 µl of reduced-serum medium (Opti-MEM) was added to each well. The transfection solution consisted of two mixtures. Liquid A contained 9 µl of ThermoFisher Lipofectamine 2000, while liquid B contained 75 pmol per well of siRNA for the Elovl6 KD group. In both cases, 150 µl of Opti-MEM was added. The negative control group also used the same volume but with only Opti-MEM. The procedure involved combining liquid A and B in equal volumes, incubating for five minutes, and then adding the mixture to the 6-well plate. After 8 hours of transfection, the medium was switched back to a complete medium. Following that, the cells were allowed to grow until they reached 100% confluency for differentiation, as previously described. Sangon Biotech in Shanghai (China) custom-designed and produced the siRNA used for Elovl6 knockdown. It featured a sense strand sequence of GCUCUUCGAACUGGUGCUUTT, and an antisense strand sequence of AAGCACCAGUUCGAAGAGCTT. Oil red O (ORO) staining ORO solid powder was dissolved in isopropanol to reach a concentration of 0.5% and then filtered with filter paper to obtain the ORO staining storage solution. It was stored in the dark at a temperature of 4°C. Upon being prepared for utilization, the storage solution was diluted 60% in water and filtered with filter paper to obtain the ORO staining working solution. The cells underwent two rounds of PBS rinsing, followed by fixation with 4% paraformaldehyde at room temperature (RT) for half an hour. Subsequently, they received another round of washing with diluted water. Following that, the cells were subjected to an ORO staining solution, allowing them to stain for 30 min at RT. Afterward, destaining was carried out by a single wash with absolute ethanol. After staining each well, 1 ml of isopropanol was introduced, and the absorbance was measured at 510 nanometers utilizing the TECAN spectrophotometer. Statistical analysis Statistical hypothesis testing and plotting were performed using Prism GraphPad 9.0.0. Normality testing was conducted using both the Anderson-Darling and the Shapiro-Wilk tests. Based on the normality of data, a Student's t-test or Kruskal-Wallis’s rank-sum test was used to evaluate overall mean differences between groups. The Chi-squared test was used to compare the proportion of categorical variables between groups. RESULT Transcriptional sequencing revealed low expression of Elov6 in the WAT of the CAC rodent model. First, a CAC rodent model was established by implanting LLC cells into mice and using subcutaneous injection of PBS as the control group (CONT). Following the implantation of LLC for 4 weeks, mice in the CAC group exhibited a notable reduction in tumor-free body weight and experienced fat and muscle atrophy in multiple locations (Fig. 1a and Fig. 1b), indicating the representativeness of this CAC rodent model. Next, eWATs were obtained from 6 mice for transcriptome sequencing, comprising 3 from the CAC and 3 from the CONT groups. In total, 31,447 genes were successfully identified. 715 genes showed significant differential expression in the WAT of CAC mice based on criteria for differential analysis (|log2(fold change) | greater than 1 and padj less than 0.05). Among these, 368 exhibited up-regulations, while 347 displayed down-regulations (Fig. 2a). Gene ontology (GO) database was used for GO annotation and enrichment analysis. A total of 328 GO terms were significantly different, including 25 GO terms related to fatty acid and lipid metabolism (Fig. 2c). Totally 24 genes were screened out that have differential expression at the same time, related to lipid metabolism (Fig. 2b). Functional annotation and classification of these genes were performed, and they were subsequently sorted by the significance of difference, as shown in Table 1 [14-24]. A large proportion of genes were found to be related to very long chain fatty acid (VLCFA) or LCFA metabolism pathway (Fig. 2d). Moreover, 2 out of the 3 genes with the most significant expression difference were related to the elongation process of LCFA carbon chains. Notably, the expression of Elovl6 was significantly downregulated in the WAT of CAC mice (log2FC = -2.27, padj = 0.0000897). This result was validated through qRT-PCR analysis of the WAT from CAC and CONT mice (Fig. 1c). The data indicated a significant downregulation of Elovl6 and its upstream regulatory gene steroid regulatory element-binding protein-1 (SREBP-1). Therefore, Elovl6 is the most potential gene for subsequent research. Table 1 10 differentially expressed genes related to LCFA/VLCFA metabolism in WAT of CAC mice Gene Log2(FC) padj Function Slc27a1 2.522 <0.0001 Mediating ATP-dependent LCFA transport into the cytosol [14] Elovl6 -2.273 0.0001 Catalyzing carbon chain lengthening of C12-C16 fatty acids [15,16] Hacd2 1.095 0.0009 Catalyzing the third step of LCFA carbon chain extension (dehydration) [17] Adtrp 2.445 0.0009 Hydrolyzing bioactive fatty acid esters of hydroxy fatty acid [18] Acot4 1.983 0.0014 Hydrolyzing lipoyl CoA to form free fatty acids and coenzyme A. Can also catalyze the hydrolysis of long-chain lipoyl CoA [19] Ehhadh 2.844 0.0049 One of the four enzymes of the peroxisomal β-oxidation pathway [20] Acadvl 1.448 0.0106 Catalyzing the initial stage of VLCFA mitochondrial fatty acid β-oxidation [21] Acadl 1.205 0.0114 Catalyzing the initial stage of LCFA mitochondrial fatty acid β-oxidation [22] Pnpla3 -2.988 0.0189 Mediating triglyceride hydrolysis in adipocytes [23] Irs2 1.951 0.0286 Regulating the biological effects of insulin and IGF1 [24] Low expression of Elovl6 in WAT of CAC patients could change fatty acid proportion and worsen weight loss. To validate the decreased expression of Elovl6 in WAT of CAC, 204 subjects were enrolled in our study, adhering to the aforementioned inclusion and exclusion standards. 13 patients among them were excluded due to metabolic diseases and preoperative neoadjuvant chemotherapy. In the end, the study encompassed 191 patients. Out of this group, 99 were CAC patients and 92 were weight-stable cancer patients (WS) served as the control group. The fundamental clinical information of CAC patients is presented in Table 2 for both groups. Except for weight loss and BMI, other factors such as gender, age, and tumor node metastasis classification (TMN) did not exhibit significant differences when comparing the CAC and WS groups, indicating comparable baseline matching between the two groups. Table 2 Clinical information of included CAC patients and weight-stable tumor patients Features CAC (n=99) WS(n=92) P Gender, Female (n, %) 36, 36.36% 40, 43.47% 0.315 Age (yr) 62.12 ± 11.90 62.10 ± 9.47 0.991 Height (cm) 163.04 ± 7.88 166.09 ± 7.85 0.008 Weight (kg) 55.88 ± 9.62 67.46 ± 9.45 <0.0001 BMI (kg/m2) 20.96 ± 2.86 24.39 ± 2.55 <0.0001 Weight loss(kg) 4.99 ± 3.32 0.38 ± 0.91 <0.0001 Stage, I - II (n, %) 75, 75.75% 62, 67.39% 0.199 Albumin (g/L) 39.75 ± 4.31 40.78 ± 4.24 0.421 Pre-albumin (mg/L) 189.90 ± 52.14 223.58 ± 50.67 0.022 Total protein (g/L) 64.38 ± 5.76 66.29 ± 4.91 0.014 LDL (mmol/L) 2.44 ± 0.88 2.45 ± 0.82 0.959 HDL-CH (mmol/L) 1.13 ± 0.41 1.13 ± 0.36 0.937 Total cholesterol(mmol/L) 3.91 ± 0.29 4.26 ± 0.93 0.852 Triglyceride(mmol/L) 1.17 ± 0.14 1.52 ± 0.73 0.018 RBC (10^12/L) 3.89 ± 0.62 4.21 ± 0.53 0.0002 WBC (10^9/L) 5.18 ± 1.72 5.66 ± 1.90 0.06 Hb (g/L) 115.65 ± 20.32 126.91 ± 19.41 0.0001 PLT (10^9/L) 220.56 ± 71.90 218.88 ± 74.53 0.874 HBA1C (%) 5.71 ± 1.42 5.90 ± 0.88 0.246 In both groups of patients, the qPCR results for genes associated with lipid metabolism in WAT demonstrated a notable reduction in Elovl6 and its upstream regulatory gene, SREBP-1. Aligning with the Western blot data presented in Figure 3c, this result confirms the consistency. The expression levels of genes related to lipolysis, which include hormone-sensitive lipase (HSL), peroxisome proliferator-activated receptor α (PPARα), and adipose triglyceride lipase (ATGL), were significantly upregulated in WAT of the CAC patients. In contrast, there were no notable differences in the expression of genes associated with adipogenesis, including enhancer-binding protein α (C/EBPα), C/EBPβ, PPARγ, and the lipogenic genes such as the mammalian target of rapamycin (mTOR) and fatty acid synthase (FASN) (Fig. 3a). Moreover, there was a significant downregulation in the expression of the gene Stearoyl-CoA desaturase-1 (SCD-1), which is implicated in the synthesis of polyunsaturated fatty acids. The result of Western blotting also showed a decrease in Elovl6 protein in CAC patients (Fig. 2e and Fig. 2f). Then, a correlation analysis of Elovl6 expression was performed with the degree of weight loss in CAC patients (Fig. 3b), suggesting a statistically significant linear relationship between the two variables. To identify alterations in fatty acid content in the WAT of CAC patients, fatty acids were isolated from tissue samples and converted into FAMEs that were suitable for GC analysis . It was found that the content of palmitic acid (C16:0) was significantly higher in the WAT of CAC patients. Correspondingly, the content of linoleic acid (C18:2n6c) was significantly lower (Fig. 3c). When comparing the relationship of overall C18 to C16 fatty acid composition between the two groups, it became evident that there was a notable reduction in the C18:C16 ratio within the WAT tissues of CAC patients (Fig. 3d). There were no significant variations observed in the relative contents of other fatty acids (Supplement Table S2). Elovl6 knockdown could inhibit lipogenesis and pre-adipocyte differentiation by altering fatty acid components in adipocytes in vitro To further explore the influence of Elovl6 on the morphological and functional aspects of adipocytes, a tumor cell conditioned medium was introduced to differentiate 3T3-L1 cells as an in vitro model of CAC. IL-6 added complete medium served as a positive control. Additionally, siRNA was used to suppress Elovl6 expression in 3T3-L1 preadipocytes, using empty lipofectamine as a control. Following the onset of differentiation in 3T3-L1 cells, the preadipocytes underwent gradual transformation into a full, round shape, with a concurrent rise in both the quantity and diameter of intracellular lipid droplets (Fig. 4a). In the 3T3-L1 cells, the qPCR results indicated a decreasing Elovl6 expression level during the differentiation process, while the upstream regulatory gene of Elovl6, SREBP-1, displayed a progressive rise in expression. Additionally, PPARγ, a gene related to differentiation, and ATGL, a major gene associated with lipolysis, were also gradually up-regulated (Fig. 4b). Upon achieving differentiation and maturation of 3T3-L1 cells, tumor cell conditioned medium was introduced to simulate a cachexic environment. In the CAC group, the adipocyte morphology underwent significant changes, including a reduction in both the quantity and volume of intracellular lipid droplets (Fig. 4a). qPCR assays indicated a decrease in the expression level of genes related to the differentiation process, such as C/EBPα, C/EBPβ, and PPARγ, as well as genes associated with lipid synthesis, including mTOR, FASN, and SCD-1. The expression level of Elovl6 and SREBP-1 was also notably reduced (Fig. 4c). The Western Blot analysis also showed a decrease in Elovl6 protein in the CAC group (Fig. 3d and Fig. 3e). After using siRNA to knock down Elovl6 expression in 3T3-L1 cells, a decrease was observed in the differentiation rate of 3T3-L1. The proportion of pre-adipocytes undergoing differentiation was reduced, and the volume of lipid droplets in the cytoplasm after differentiation was also reduced (Fig. 4f). qPCR analysis showed that silencing Elovl6 suppressed gene expression associated with fatty acid synthesis, hydrolysis, and adipogenesis (Fig. 4g), including SREBP-1, ATGL, PPARγ, and CEB/Pβ. Mature 3T3-L1 cells were collected from both Elovl6-KD and the control group, and their fatty acid composition was analyzed by GC. The findings indicated that the knockdown of Elovl6 in 3T3-L1 cells could cause alterations in fatty acid composition. This was evidenced by a notable elevation in the content of palmitic acid (C16:0) and a decrease in oleic acid (C18:1n9c) (Fig. 4h). These results suggested that low expression of Elovl6 may exaggerate tumor cell-induced adipocyte atrophy, probably through altering fatty acid component of adipocytes. DISCUSSION At the outset of this study, transcriptome sequencing on the WAT of the CAC rodent model was performed. A significant decrease in Elovl6 expression was observed in CAC. This result was verified by an in vivo study on rodent CAC model and clinical CAC samples. The reduced expression of Elovl6 was also correlated with the degree of weight loss in CAC patients, which further corroborates the relationship between Elovl6 and pathophysiological processes in CAC. The first question that comes to our mind is whether the low expression of Elovl6 in WAT of CAC could be a consequence of altered fatty acid metabolism in the CAC pathophysiological process. Currently, no studies have reported the expression and regulation of Elovl6 in CAC. In this study, expression of both Elovl6 and SREBP-1 was decreased in CAC patients, tumor-bearing mice, and 3T3-L1 cells undergoing a cachexic environment. Studies in other related fields have suggested that SREBP-1 can regulate Elovl6 expression. Moon and colleagues observed high expression of Elovl6 in transgenic mice overexpressing SREBP-1 [ 14 ]. Furthermore, synthetic agonists of LXRα, an upstream regulating gene of SREBP-1, induced high expression of Elovl6 and FASN in BAT adipocytes [ 20 ]. These reports revealed the positive regulatory effect of SREBP-1 on Elovl6. The SREBP family proteins are all inactivated precursor proteins anchored to the endoplasmic reticulum (ER) membrane. Upon formation of their structural domains, SREBPs can regulate both cholesterol and fatty acid metabolism [ 21 , 22 ]. According to relevant studies, the anabolic Akt-mTOR signaling pathway could upregulate SREBP-1 [ 23 , 24 ]. Thus, it is possible that the downregulation of Elovl6 expression in the WAT of CAC patients could be a phenomenon following the inhibition of the entire lipid anabolic process. It is worth noting that no alterations in mTOR mRNA expression were observed in either the CAC rodent model or clinical samples. In fact, in cancer-associated cachexia, decreased mTOR expression has been described exclusively in the skeletal muscle of CAC patients or mice, with no such reports in adipose tissue [ 25 – 27 ]. However, this study observed downregulation of SREBP-1, FASN, and SCD-1 expression in the WAT of CAC, indicating a downstream inhibition of the fatty acid synthesis pathway. In other studies, mRNA levels of genes related to adipose synthesis, including FAS, ACC, and SCD-1, also exhibited reductions in the adipose tissue of tumor-bearing mice [ 28 ]. These results suggested that the downregulation of SERBP-1 in WAT of CAC may be induced by a change in phosphorylation of mTOR and could trigger downstream inhibition of lipid synthesis [ 37 ]. Up to this point, it is reasonable to assume that Elovl6 is downregulated by the inhibition of the lipid synthesis process during CAC pathophysiology, possibly through the mTOR-SREBP axis. On the other hand, the low expression of Elovl6 may also contribute to the fat loss process. In this study, the knockdown of Elovl6 could suppress the differentiation of pre-adipocytes and alter lipid metabolism. Additionally, palmitic acid (C16:0) in WAT of CAC patients was increased, while linoleic acid (C18:2n6c) was decreased. After the knockdown of Elovl6 in 3T3-L1 cells, a similar change appeared in the fatty acid composition of the cells. A significant elevation in palmitic acid (C16:0) and a reduction in oleic acid (C18:1n9c) content was observed in CAC groups in vitro . This phenomenon not only matches the decreased expression of Elovl6 but also implies that Elovl6 might have a subsequent biological impact by altering the proportion of fatty acids in WAT. Indeed, the regulatory influence of fatty acids on lipid metabolism has been a long-standing research topic, particularly regarding polyunsaturated fatty acids (PUFAs). A study reported that feeding PUFA to mice inhibited FASN expression and related lipid anabolism [ 29 ]. Worgall et al found that oleic acid and PUFA could inhibit SREBP expression, ultimately leading to the inhibition of fatty acid synthesis [ 30 ]. In recent years, Ou et al further elaborated that PUFA could directly inhibit expression of LXR, an upstream regulatory gene of SREBP-1 [ 31 ]. These studies suggest that PUFA itself has an inhibitory effect on the lipid synthesis pathway. Since de novo synthesis of PUFA requires LCFA as a substrate, and Elovl6 is a crucial enzyme in LCFA synthesis within WAT, the decreased expression of Elovl6 in WAT of CAC patients may play a further regulatory function in the process of CAC fat loss by affecting the synthesis of PUFA. However, no such change of PUFA was observed either in the WAT of CAC patients or in Elovl6-KD 3T3-L1 cells. We think the primary reason for not acquiring positive results is the low amount of PUFA in WAT and 3T3-L1 cells. In contrast, monounsaturated FAs and saturated FAs could also affect pre-adipocyte differentiation and lipid metabolism process independently. Kobayashi et al. found that adding C18:1 and C20:1 fatty acid into the culture medium of 3T3-L1 could facilitate the differentiation process [ 32 ], which may support this hypothesis. Thus, additional evidence is still required to support this viewpoint. Comparisons with other studies and what does the current work add to the existing knowledge Currently, no studies have been conducted on Elovl6 and other fatty acid elongases in CAC. However, Tan et al. have found a significant reduction in the C18:C16 ratio and a decrease in the expression of SCD family genes in the WAT of Elovl6 KO mice, which indicates a suppression of the fatty acid desaturation process [ 14 ]. Interestingly, the WAT of CAC patients in this study exhibited a similar alteration. In this study, transcriptome sequencing was conducted on WAT in a CAC mouse model, and fatty acid GC analysis was performed on WAT in CAC patients. The resulting data provides valuable insights for future studies on CAC-induced fat loss. Additionally, the current study proposed the potential contribution of Elovl6 in the process of CAC-induced fat loss. This discovery not only presents a new biomarker for diagnosing and treating CAC in future clinical practice but also introduces a new theory for CAC research. Study strengths and limitations This study is closely connected to the clinical field. First, most WAT samples used in this study were obtained from CAC patients diagnosed by clinical doctors, which makes the result representative of real situations in the human body. Furthermore, both a decrease in Elovl6 expression and a change in the fatty acid component were discovered in CAC patients' fat tissue, indicating that Elovl6 could be a valuable biomarker for early identification of CAC. Finally, Elovl6 could potentially contribute to fat loss in CAC by altering the fatty acid composition in adipocytes in vitro , making it a promising target for future therapeutic interventions. A limitation of our research was that Elovl6 knockdown was performed in vitro , which may not reflect the actual conditions in the human body. Conclusion Overall, Elovl6 expression was found to be diminished in the WAT of CAC. Low expression of Elovl6 correlated with the severity of weight loss in CAC. In addition, reduced Elovl6 expression levels in WAT of CAC patients coincided with a reduction in C18 fatty acid content and an augmentation in C16 fatty acid content. Knockdown Elovl6 inhibited the differentiation of pre-adipocytes and downregulated the expression of genes associated with lipid synthesis. Elovl6-knockdown also induced a significant decrease in oleic acid (C18:1n9c) content and a notable rise in palmitic acid (C16:0) content, suggesting low expression of Elovl6 could suppress the CAC liposynthesis process possibly by altering the lipid content in adipocytes. The findings from this study indicate that Elovl6 has the potential to serve as a dependable biomarker for the early diagnosis of cancer-associated cachexia and may offer a promising target for future therapies. Abbreviations CAC cancer-associated cachexia WAT white adipose tissue Elovl6 elongase of very long-chain fatty acid 6 BAT brown adipose tissue LCFA long-chain fatty acid VLCFA very-long-chain fatty acid TG triglyceride HDL-C high-density lipoprotein cholesterol LDL low-density lipoprotein BMI body mass index LLC Lewis lung carcinoma PBS phosphate-buffered saline FAME fatty acid methyl ester GC gas chromatography NCS neonatal calf serum HG-DMEM Dulbecco's modified eagle medium with high-glucose PSS penicillin-streptomycin solution FBS fetal bovine serum IBMX 3-isobutyl-1-methyl-7H-xanthine ORO Oil red O GO Gene ontology SREBP-1 steroid regulatory element-binding protein-1 ATGL adipose triglyceride lipase HSL hormone sensitive lipase LPL lipoprotein lipase C/EBP enhancer-binding protein PPAR peroxisome proliferator-activated receptor FASN fatty acid synthase SCD-1 Stearoyl-CoA desaturase-1. Declarations Ethics approval and consent to participate This study was approved by ethics committee of Zhongshan Hospital of Fudan University (B2019-193R). All patients participating in this study had signed the informed consents. The animal experiments in this study were approved by the Animal Ethics Committee of Zhongshan Hospital of Fudan University (Shanghai, China). Consent for publication Not applicable. Availability of data and materials mRNA-seq dataset used in this study has been uploaded to the Gene Expression Omnibus database (GSE242812). Competing interests The authors declared that they have no competing interests. Funding This study received funding through grants from the Shanghai Natural Science Foundation ProjectQ7 (19ZR1409100) Authors’ contributions Qingyang Meng and Guohao Wu formulated and planned the experiments. Chenyang Jin and Shuangjie Wang gathered the data and conducted the biological experiments with assistance from Xiangyu Sui. Chenyang Jin analyzed and interpreted the data. Chenyang Jin and Shuangjie Wang were responsible for composing the paper. All authors subsequently reviewed and granted approval for the paper. Acknowledgements Not applicable. Footnote Chenyang Jin and Shuangjie Wang contribute equally to this work. References Fearon K, Strasser F, Anker SD, Bosaeus I, Bruera E, Fainsinger RL, et al. Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011;12(5):489–95. Martin L, Senesse P, Gioulbasanis I, Antoun S, Bozzetti F, Deans C, et al. Diagnostic Criteria for the Classification of Cancer-Associated Weight Loss. J Clin Oncol. 2015;33(1):90–U147. Daly L, Dolan R, Power D, Ni Bhuachalla E, Sim W, Fallon M et al. The relationship between the BMI-adjusted weight loss grading system and quality of life in patients with incurable cancer. J cachexia sarcopenia muscle. 2019. Baracos VE, Martin L, Korc M, Guttridge DC, Fearon KCH. Cancer-associated cachexia. Nat Reviews Disease Primers. 2018;4. Petruzzelli M, Schweiger M, Schreiber R, Campos-Olivas R, Tsoli M, Allen J, et al. A Switch from White to Brown Fat Increases Energy Expenditure in Cancer-Associated Cachexia. Cell Metabol. 2014;20(3):433–47. Meex RC, Hoy AJ, Mason RM, Martin SD, McGee SL, Bruce CR, et al. ATGL-mediated triglyceride turnover and the regulation of mitochondrial capacity in skeletal muscle. Am J Physiol Endocrinol metabolism. 2015;308(11):E960–70. Das SK, Eder S, Schauer S, Diwoky C, Temmel H, Guertl B, et al. Adipose triglyceride lipase contributes to cancer-associated cachexia. Science. 2011;333(6039):233–8. Ebadi M, Mazurak VC. Evidence and mechanisms of fat depletion in cancer. Nutrients. 2014;6(11):5280–97. Laiglesia LM, Lorente-Cebrián S, Prieto-Hontoria PL, Fernández-Galilea M, Ribeiro SMR, Sáinz N, et al. Eicosapentaenoic acid promotes mitochondrial biogenesis and beige-like features in subcutaneous adipocytes from overweight subjects. J Nutr Biochem. 2016;37:76–82. Shin S, Ajuwon K. Effects of Diets Differing in Composition of 18-C Fatty Acids on Adipo se Tissue Thermogenic Gene Expression in Mice Fed High-Fat Diets. Nutrients. 2018;10(2):256. Shin S, Ajuwon KM. Divergent Response of Murine and Porcine Adipocytes to Stimulation of Browning Genes by 18-Carbon Polyunsaturated Fatty Acids and Beta-Recep tor Agonists. Lipids. 2018;53(1):65–75. Oliver E, McGillicuddy FC, Harford KA, Reynolds CM, Phillips CM, Ferguson JF, et al. Docosahexaenoic acid attenuates macrophage-induced inflammation and im proves insulin sensitivity in adipocytes-specific differential effects between LC n-3 PUFA. J Nutr Biochem. 2012;23(9):1192–200. Alvheim AR, Torstensen BE, Lin YH, Lillefosse HH, Lock E-J, Madsen L, et al. Dietary Linoleic Acid Elevates the Endocannabinoids 2-AG and Anandamid e and Promotes Weight Gain in Mice Fed a Low Fat Diet. Lipids. 2013;49(1):59–69. Moon YA, Shah NA, Mohapatra S, Warrington JA, Horton JD. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins. J Biol Chem. 2001;276(48):45358–66. Matsuzaka T, Shimano H, Yahagi N, Yoshikawa T, Amemiya-Kudo M, Hasty AH, et al. Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs. J Lipid Res. 2002;43(6):911–20. Jakobsson A, Westerberg R, Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism. Prog Lipid Res. 2006;45(3):237–49. Morcillo S, Martin-Nunez GM, Rojo-Martinez G, Almaraz MC, Garcia-Escobar E, Mansego ML, et al. ELOVL6 genetic variation is related to insulin sensitivity: a new candidate gene in energy metabolism. PLoS ONE. 2011;6(6):e21198. Zhao H, Matsuzaka T, Nakano Y, Motomura K, Tang N, Yokoo T, et al. Elovl6 Deficiency Improves Glycemic Control in Diabetic db/db Mice by Expanding β-Cell Mass and Increasing Insulin Secretory Capacity. Diabetes. 2017;66(7):1833–46. Tan CY, Virtue S, Bidault G, Dale M, Hagen R, Griffin JL, et al. Brown Adipose Tissue Thermogenic Capacity Is Regulated by Elovl6. Cell Rep. 2015;13(10):2039–47. Jakobsson A, Jörgensen JA, Jacobsson A. Differential regulation of fatty acid elongation enzymes in brown adip ocytes implies a unique role for Elovl3 during increased fatty ac id oxidation. Am J Physiology-Endocrinology Metabolism. 2005;289(4):E517–E26. Shimano H. Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Prog Lipid Res. 2001;40(6):439–52. Bertolio R, Napoletano F, Mano M, Maurer-Stroh S, Fantuz M, Zannini A et al. Sterol regulatory element binding protein 1 couples mechanical cues an d lipid metabolism. Nat Commun. 2019;10(1). Porstmann T, Santos CR, Griffiths B, Cully M, Wu M, Leevers S, et al. SREBP Activity Is Regulated by mTORC1 and Contributes to Akt-Dependent Cell Growth. Cell Metabol. 2008;8(3):224–36. Laplante M, Sabatini DM. An Emerging Role of mTOR in Lipid Biosynthesis. Curr Biol. 2009;19(22):R1046–R52. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, et al. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol. 2001;3(11):1014–9. White JP, Baynes JW, Welle SL, Kostek MC, Matesic LE, Sato S, et al. The Regulation of Skeletal Muscle Protein Turnover during the Progress ion of Cancer Cachexia in the ApcMin/+ Mouse. PLoS ONE. 2011;6(9):e24650. White JP, Puppa MJ, Gao S, Sato S, Welle SL, Carson JA. Muscle mTORC1 suppression by IL-6 during cancer cachexia: a role for A MPK. Am J Physiology-Endocrinology Metabolism. 2013;304(10):E1042–E52. Bing C, Russell S, Becket E, Pope M, Tisdale MJ, Trayhurn P, et al. Adipose atrophy in cancer cachexia: morphologic and molecular analysis of adipose tissue in tumour-bearing mice. Br J Cancer. 2006;95(8):1028–37. Blake WL, Clarke SD. Suppression of Rat Hepatic Fatty Acid Synthase and S14 Gene Transcript ion by Dietary Polyunsaturated Fat. J Nutr. 1990;120(12):1727–9. Worgall TS, Sturley SL, Seo T, Osborne TF, Deckelbaum RJ. Polyunsaturated Fatty Acids Decrease Expression of Promoters with Ster ol Regulatory Elements by Decreasing Levels of Mature Sterol Regulator y Element-binding Protein. J Biol Chem. 1998;273(40):25537–40. Ou J, Tu H, Shan B, Luk A, DeBose-Boyd RA, Bashmakov Y et al. Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dep endent activation of the LXR. Proceedings of the National Academy of Sciences. 2001;98(11):6027-32. Kobayashi T, Fujimori K. Very long-chain-fatty acids enhance adipogenesis through coregulation of Elovl3 and PPARγ in 3T3-L1 cells. Am J Physiology-Endocrinology Metabolism. 2012;302(12):E1461–E71. Han J, Wang Y, Qiu Y, Sun D, Liu Y, Li Z, et al. Single-cell sequencing unveils key contributions of immune cell populations in cancer-associated adipose wasting. Cell Discov. 2022;8(1):122. Fasshauer M, Kralisch S, Klier M, Lossner U, Bluher M, Klein J, et al. Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocytes. Biochem Biophys Res Commun. 2003;301(4):1045–50. Sun D, Zuoyou D, Shen L, Yang F, Han J, Wu G. miR-410-3P inhibits adipocyte differentiation by targeting IRS-1 in cancer-associated cachexia patients. Lipids Health Dis. 2021;20. Han J, Shen L, Zhan Z, Liu Y, Zhang C, Guo R, et al. The long noncoding RNA MALAT1 modulates adipose loss in cancer-associated cachexia by suppressing adipogenesis through PPAR-γ. Nutr Metabolism. 2021;18(1):27. Liu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol. 2020;21(4):183–203. Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1.docx Supplementary Information Additional File 1: Table S1. List of qPCR primers; Table S2. Fatty acid profile of CAC and control patients Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Feb, 2024 Reviews received at journal 31 Jan, 2024 Reviews received at journal 30 Jan, 2024 Reviewers agreed at journal 25 Jan, 2024 Reviewers agreed at journal 25 Jan, 2024 Reviewers agreed at journal 11 Dec, 2023 Reviewers agreed at journal 23 Nov, 2023 Reviewers invited by journal 20 Nov, 2023 Editor assigned by journal 14 Nov, 2023 Submission checks completed at journal 14 Nov, 2023 First submitted to journal 14 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3611425","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":249472203,"identity":"917b0ada-0f46-443e-b456-1f1fc28bfd39","order_by":0,"name":"Chenyang Jin","email":"","orcid":"","institution":"Zhongshan Hospital of Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenyang","middleName":"","lastName":"Jin","suffix":""},{"id":249472205,"identity":"af5a1e46-1a7e-4d0c-a10b-79d643035a2d","order_by":1,"name":"Shuangjie Wang","email":"","orcid":"","institution":"Shanghai Institute for Biomedical and Pharmaceutical Technologies","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuangjie","middleName":"","lastName":"Wang","suffix":""},{"id":249472208,"identity":"a51f02c0-dfee-4dfa-991a-e4bc0aa5594b","order_by":2,"name":"Xiangyu Sui","email":"","orcid":"","institution":"Zhongshan Hospital of Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiangyu","middleName":"","lastName":"Sui","suffix":""},{"id":249472210,"identity":"f705f840-3519-4a55-8ccf-1f43f28aa1d3","order_by":3,"name":"Qingyang Meng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYBACAygtx8befIA0LcZ8PMcSSNOSOE8iR4FILezNxx7dKDuc3saQw8Dwo2IbEVp4jqUb55w7nNvGcPYAY8+Z20Rokcgxk85tA2ph7EtgZmwjRov8G7CWdDZmHgMitUjwgLUksLERrYUnLU0651y6YRsPW8JBovxi3374mHROmbW8/PzHBx/8qCBCCwSwNYOpA8SqB2mpI0HxKBgFo2AUjDgAALNVNvKZP1B2AAAAAElFTkSuQmCC","orcid":"","institution":"Zhongshan Hospital of Fudan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Qingyang","middleName":"","lastName":"Meng","suffix":""},{"id":249472211,"identity":"8501a712-2cc2-447f-8ca1-aa587dffde06","order_by":4,"name":"Guohao Wu","email":"","orcid":"","institution":"Zhongshan Hospital of Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guohao","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2023-11-14 16:59:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3611425/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3611425/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46567337,"identity":"187b6baf-b4d4-43a7-b7a9-85adf111f768","added_by":"auto","created_at":"2023-11-16 15:23:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":695024,"visible":true,"origin":"","legend":"\u003cp\u003eElovl6 expression was downregulated in CAC mice. \u003cstrong\u003ea\u003c/strong\u003e. Comparison of fat, muscle, liver, and heart weights between CAC (n=10) and control (n=10) mice. \u003cstrong\u003eb\u003c/strong\u003e. Comparison of body weight (BW) and tumor-free BW of CAC and control mice. \u003cstrong\u003ec. \u003c/strong\u003eqPCR analysis of mRNA relative expression amounts of genes associated with WAT hydrolysis, synthesis, and adipose neogenesis in CAC and control mice. The findings are presented in the form of mean ± SEM, with statistical significance indicated by *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3611425/v1/a5ad1050266c718e2de1bc9f.png"},{"id":46567340,"identity":"26457e19-36ee-4ffd-80b1-d280cd350892","added_by":"auto","created_at":"2023-11-16 15:23:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18649669,"visible":true,"origin":"","legend":"\u003cp\u003eScreening for differentially expressed genes in CAC and CONT mice. \u003cstrong\u003ea\u003c/strong\u003e. A Volcano plot illustrating the genes with differential expression in mRNA sequencing. \u003cstrong\u003eb\u003c/strong\u003e. Venny plot showing the overlap of differentially expressed genes, genes included in significant GO-terms (GO: Sig), and genes included in fatty-acid-related GO terms (GO: FA/lipid). \u003cstrong\u003ec\u003c/strong\u003e. 25 significant GO terms related to fatty acid metabolism. \u003cstrong\u003ed\u003c/strong\u003e. Chord diagram of 24 function-annotated genes.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3611425/v1/17eaf57f30a2bc26e17ebc9b.png"},{"id":46568012,"identity":"cf5149dc-e0db-4d56-88e9-8bbc4f1432f6","added_by":"auto","created_at":"2023-11-16 15:31:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1769199,"visible":true,"origin":"","legend":"\u003cp\u003eDown-regulation of Elovl6 in WAT of CAC patients may alter fatty acid proportion and exacerbate weight loss. \u003cstrong\u003ea\u003c/strong\u003e. qPCR analysis of genes associated with lipid metabolism in WAT of weight-stable tumor patients (WS) and CAC patients. \u003cstrong\u003eb. \u003c/strong\u003eCorrelation analysis between Elovl6 expression in WAT and degree of weight loss in CAC patients. \u003cstrong\u003ec.\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e. fatty acid analysis of WAT from the WS group and CAC group, showing the composition of different fatty acids (\u003cstrong\u003ec\u003c/strong\u003e.) and C18:C16 ratio (\u003cstrong\u003ed\u003c/strong\u003e.) between the two groups. \u003cstrong\u003ee\u003c/strong\u003e. and \u003cstrong\u003ef.\u003c/strong\u003eWestern blot analysis of Elovl6 in both the CAC and WS groups. The findings are presented in the form of mean ± SEM, with statistical significance indicated by *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3611425/v1/18495ec4ccddd7978b2996e7.png"},{"id":46567342,"identity":"d0c2aba9-3f8d-4a6d-9593-77f351653ec2","added_by":"auto","created_at":"2023-11-16 15:23:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21737166,"visible":true,"origin":"","legend":"\u003cp\u003eElovl6 knockdown inhibits pre-adipocyte differentiation and affects adipocyte fatty acid metabolism. \u003cstrong\u003ea. \u003c/strong\u003eMicrographs of 3T3-L1 cells before differentiation (D0), 6 days after differentiation(D6), and with conditioned medium added after differentiation (D6 CAC). IL-6 added group was used as a positive control (D6 IL-6). \u003cstrong\u003eb\u003c/strong\u003e. Changes in gene expression associated with lipid metabolism and adipogenesis were observed in 3T3-L1 cells at various time points following differentiation. \u003cstrong\u003ec\u003c/strong\u003e. Alterations in the relative expression of genes associated with lipid metabolism were observed in 3T3-L1 cells following differentiation when exposed to CAC conditioned medium (CAC) or a control (NC). \u003cstrong\u003ed.\u003c/strong\u003e and \u003cstrong\u003ee.\u003c/strong\u003eWestern blot analysis of Elovl6 in CAC, NC, and IL-6 groups. \u003cstrong\u003ef\u003c/strong\u003e. Micrographs of ORO staining in differentiated 3T3-L1 cells were captured after exposure to CAC CAC-conditioned medium, treated with Elovl6 si-RNA (Elovl6 KD) or control (NC). \u003cstrong\u003eg\u003c/strong\u003e. Changes in genes related to lipid metabolism of differentiated 3T3-L1 cells added CAC conditioned medium, treated with Elovl6 si-RNA (Elovl6 KD) or control (NC). \u003cstrong\u003eh.\u003c/strong\u003e Fatty acid analysis in differentiated 3T3-L1 cells, following treatment with Elovl6 si-RNA (Elovl6 KD) or the control (NC), was performed using gas chromatography. The findings are presented in the form of mean ± SEM, with statistical significance indicated by *\u003cem\u003e P\u003c/em\u003e \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3611425/v1/f0b04f8217582d371dfc87d4.png"},{"id":46567338,"identity":"acfe078e-84fb-49d4-be25-f3c27030aa59","added_by":"auto","created_at":"2023-11-16 15:23:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional File 1: \u003c/strong\u003eTable S1. List of qPCR primers; Table S2. Fatty acid profile of CAC and control patients\u003c/p\u003e","description":"","filename":"AdditionalFile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3611425/v1/9c84ceeafc18f81b605c820b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Low expression of Elovl6 may involve in fat loss in white adipose tissue of cancer-associated cachexia","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAccording to an international consensus published in 2011 [1], cancer-associated cachexia (CAC) is characterized as a wasting syndrome induced by cancer. It involves persistent skeletal muscle depletion and fat loss, which proves challenging to be fully reversed using standard nutritional interventions. CAC not only leads to a grim prognosis but also diminishes the quality of life in individuals with malignant tumors [2, 3]. However, our comprehension of the mechanisms behind CAC remains relatively limited. Previous laboratory and clinical studies have revealed the generation of pro-inflammatory cytokines during crosstalk among somatic cells, immune cells, and tumor cells. These cytokines may consequently influence skeletal muscle, adipose tissue, and the central nervous system, causing clinical symptoms such as decreased appetite, skeletal muscle atrophy, and fat loss [4]. Recently, the role of fat loss in the pathophysiology of CAC has been reported as an increasingly important issue. The latest study in CAC rodent models suggests that fat loss may occur before skeletal muscle atrophy and altered lipid metabolic processes may be a prerequisite for this process [5-7, 35, 36]. According to most studies, fat loss in CAC occurs through three primary mechanisms: enhancement of fat hydrolysis, inhibition of fat synthesis, and the transformation of white adipose tissue (WAT) [1, 8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, transcriptome sequencing of WAT has been carried out from tumor-bearing mice. A reduction in the expression level of elongase of very long-chain fatty acid (Elovl6) was then observed. Elovl6 was found abundantly in lipid-rich tissues like WAT and serves as the enzyme responsible for extending saturated fatty acids comprising 12-16 carbon atoms [14-16]. In rodent models, multiple studies indicated that Elovl6 has a critical role in regulating fatty acid metabolism. Notably, Elovl6 knockout mice have shown resistance to diet-induced insulin resistance [17, 18]. Under cold-acclimated conditions, Elovl6 knockout mice show reduced gene expression associated with the thermogenic process in brown adipose tissue (BAT), indicating impaired thermogenic capacity [19]. Furthermore, over the past few decades, numerous studies have highlighted the crucial role of long-chain fatty acids (LCFAs) in regulating adipocyte metabolism. LCFAs present within adipocytes can enhance adipose thermogenesis [9-11] and trigger inflammation in adipose tissue [12, 13]. These connections among LCFAs, Elovl6, and fatty acid metabolism point out that Elovl6 could be involved in CAC-induced fat loss.\u003c/p\u003e\n\u003cp\u003eThus, based on the existing literature mentioned above, it is reasonable to hypothesize that Elovl6 is involved in fat loss in CAC. Hence, this study aimed to investigate both the expression level and the function of Elovl6 in the process of fat loss in CAC.\u0026nbsp;\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003ePatients and tissue samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study comprised patients who received diagnoses at the General Surgery Department of Zhongshan Hospital, Fudan University, during the period spanning from July 2019 to June 2020. \u0026nbsp;The criteria for inclusion were as follows: (1) Confirmation of gastric or colorectal cancer through a pathological assessment; (2) Underwent radical surgery at the hospital; (3) Availability of complete clinical data. The exclusion criteria were as follows: (1) Previous receipt of preoperative anticancer strategy (chemotherapy, targeted therapy, immunotherapy, or radiotherapy); (2) Presence of severe organ dysfunction (renal failure, liver insufficient, heart failure or chronic obstructive pulmonary disease), immunity impairment (autoimmune disorders, lymphoma, leukemia or acquired immune deficiency syndrome) or uncontrolled metabolic syndrome (hyperthyroidism or diabetes). All enrolled patients were briefed about the research and granted their consent to take part.\u003c/p\u003e\n\u003cp\u003eClinical data (age, gender, height, weight, weight loss, tumor site, and staging) for the enrolled patients were recorded. Nutrition-related laboratory parameters, including hemoglobin, albumin, pre-albumin, and total protein, were also recorded.\u003c/p\u003e\n\u003cp\u003eAccording to the 2011 international consensus on CAC [1], patients were defined as CAC if they satisfied one or more of these conditions: (1) A decrease in body weight exceeding 5% during the preceding 6 months; (2) A weight reduction of over 2% coupled with a body mass index (BMI) below 20 kg/m\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAbdominal subcutaneous WAT was carefully separated and divided into approximately 500 mg pieces before being transferred to sample storage tubes. The specimens were then rapidly frozen using liquid nitrogen and preserved at -80℃ for subsequent examination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCAC rodent model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale C57/BL6 mice, weighing 18-20 grams and aged 6-7 weeks, were randomly assigned to two study groups: (1) healthy control group (CONT) and (2) tumor-bearing group (CAC). The mice were provided by the Shanghai Institutes for Biological Science. The CAC mice were subcutaneously injected with 5*10\u003csup\u003e6\u003c/sup\u003e Lewis lung carcinoma (LLC) cells dissolved in phosphate-buffered saline (PBS) near their ilium in the lower back. In contrast, the CONT group received PBS injections only. All mice were euthanized after 28 days following LLC cell or PBS injection. The samples of inguinal white adipose tissue (iWAT), epididymis white adipose tissue (eWAT), gastrocnemius, liver, and heart from each mouse were collected. The samples were promptly frozen in liquid nitrogen and preserved at -80℃\u0026nbsp;for subsequent examination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Total RNA extraction followed the official guidelines provided by Invitrogen using the TRIzol method. Degradation and contamination of RNA were identified using agarose gel (1%) electrophoresis. The measurement of RNA concentration was determined by employing a NanoPhotometer spectrophotometer for OD260/280 and OD260/230. RNA integrity was assessed by an Agilent 2100 Bioanalyzer. Subsequently, mRNA was extracted and constructed into a cDNA library following official guidelines and technical documents from NEB (China). After assembly, the library underwent quantification with a Qubit 2.0 fluorometer and was then diluted to a concentration of 1.5 ng/μL using enzyme-free water. The insert fragments were detected by using an Agilent 2100 Bioanalyzer. The libraries were quantified using qRT-PCR, meanwhile ensuring an effective library concentration exceeding 2 nM. Sequencing of the library was performed by the Illumina Hiseq 4000 platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatic data processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data in fastq format underwent initial processing and data cleaning with Perl scripts developed in-house. After that, the read number of each gene was counted and normalized. The reference genome and its corresponding index were assembled using Hisat2 v2.0.5 after being downloaded from the Ensembl database (Ensembl Mus musculus release-94). A differential expression analysis was performed between two sets of data utilizing the DESeq2 R package (version 1.16.1). Genes identified as differential expressions had an adjusted p-value (padj) below 0.05. The Gene Ontology (GO) database was used for enrichment analysis. Among the genes with differential expressions, GO terms that demonstrated a padj below 0.05 were deemed significantly enriched.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA from both animal and human tissue was extracted by using the TRIzol method. The reverse transcription of mRNAs was conducted by employing a Qiagen Quantinova kit. A real-time PCR reaction system was established with a Qiagen SYBR green PCR kit and was performed on a LightCycler 480 Platform. The list of PCR primers used was attached to supplemental materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRIPA lysis buffer was used for protein extraction from both tissue and cell samples, following which the proteins were quantified by the bicinchoninic acid method. Protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane before blocking with Tris-buffered saline supplemented with 5% skimmed milk powder for 1 hour. Following this, the membranes underwent an incubation at 4℃\u0026nbsp;overnight with the primary antibody and then were subjected to a 1-hour incubation with goat anti-rabbit IgG marked with horse radish peroxidase (HRP). The Tanon 5200 imaging system was used for identifying and analyzing stained bands. In this study,\u0026nbsp;β-tubulin (CST, USA; 1:1000) and Elovl6 (Novus Biologicals, USA; 1:1000) were the primary antibodies used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFAME analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of ether and petroleum ether was used to extract fatty acids. Fatty acid was esterified to form fatty acid methyl esters (FAMEs) before being injected into the gas chromatograph for flame ionization detection. The percentage of a particular fatty acid is determined by the ratio of the peak area of that fatty acid to the sum of the peak areas of all fatty acid components.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3T3-L1 mouse preadipocytes were cultivated in Dulbecco's modified eagle medium with high-glucose (HG-DMEM, Thermo Fisher), into which 10% neonatal calf serum (NCS) from AusgeneX and 1% penicillin-streptomycin solution (PSS) from ThermoFisher were added. Similarly, LLC and the C26 mouse colon cancer cell lines were cultured in HG-DMEM. These cultures received additional supplementation, including 10% fetal bovine serum (FBS) from AusgeneX and 1% PSS. The cells were kept in a microbiological incubator at 37°C with a 5% CO\u003csub\u003e2\u003c/sub\u003e environment. The Cell Bank at the Chinese Academy of Sciences generously provided all the cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferentiation of 3T3-L1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the initial differentiation of 3T3-L1 cells, a differentiation-induced medium (DIM-I) was employed, consisting of DMEM with 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μg/mL insulin, 1% PSS, and 10% FBS. Subsequently, another differentiation-induced medium (DIM-II) was used to maintain the differentiation environment. DIM-II was composed of DMEM with 1 μg/mL insulin, 1% PSS, and 10% FBS. The 3T3-L1 cell differentiation process commenced two days after achieving 100% confluency (D0) by switching to the DIM-I medium. On day 2 (D2), the medium was switched to DIM-II, which was renewed every 2 days until further intervention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation and use of CAC-mimic medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of a CAC-mimic medium has been described in our previous work [33]. Two days after passaging C26 cells (or 3T3-L1 cells), the medium supernatant was gathered and filtered. The CAC-mimic medium consisted of 66% C26 medium supernatant and 33% complete medium, replacing D4 in the CAC group to form a cachexic environment. A mixture of 66% 3T3-L1 medium supernatant and 33% complete medium served as the corresponding intervention for the negative control (NC) group. Interleukin-6 (IL-6) was added to the complete medium as a positive control at a final concentration of 100 ng/ml [34].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 6-well plates, 3T3-L1 cells were cultured until they reached a confluency of 70-90% for transfection. Following a PBS rinse, 700 µl of reduced-serum medium (Opti-MEM) was added to each well. The transfection solution consisted of two mixtures. Liquid A contained 9 µl of ThermoFisher Lipofectamine 2000, while liquid B contained 75 pmol per well of siRNA for the Elovl6 KD group. In both cases, 150 µl of Opti-MEM was added. The negative control group also used the same volume but with only Opti-MEM. The procedure involved combining liquid A and B in equal volumes, incubating for five minutes, and then adding the mixture to the 6-well plate. After 8 hours of transfection, the medium was switched back to a complete medium. Following that, the cells were allowed to grow until they reached 100% confluency for differentiation, as previously described. Sangon Biotech in Shanghai (China) custom-designed and produced the siRNA used for Elovl6 knockdown. It featured a sense strand sequence of GCUCUUCGAACUGGUGCUUTT, and an antisense strand sequence of AAGCACCAGUUCGAAGAGCTT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOil red O (ORO) staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eORO solid powder was dissolved in isopropanol to reach a concentration of 0.5% and then filtered with filter paper to obtain the ORO staining storage solution. It was stored in the dark at a temperature of 4°C. Upon being prepared for utilization, the storage solution was diluted 60% in water and filtered with filter paper to obtain the ORO staining working solution. The cells underwent two rounds of PBS rinsing, followed by fixation with 4% paraformaldehyde at room temperature (RT) for half an hour. Subsequently, they received another round of washing with diluted water. Following that, the cells were subjected to an ORO staining solution, allowing them to stain for 30 min at RT. Afterward, destaining was carried out by a single wash with absolute ethanol. After staining each well, 1 ml of isopropanol was introduced, and the absorbance was measured at 510 nanometers utilizing the TECAN spectrophotometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical hypothesis testing and plotting were performed using Prism GraphPad 9.0.0. Normality testing was conducted using both the Anderson-Darling and the Shapiro-Wilk tests. Based on the normality of data, a Student's t-test or Kruskal-Wallis’s rank-sum test was used to evaluate overall mean differences between groups. The Chi-squared test was used to compare the proportion of categorical variables between groups.\u003c/p\u003e"},{"header":"RESULT","content":"\u003cp\u003e\u003cstrong\u003eTranscriptional sequencing revealed low expression of Elov6 in the WAT of the CAC rodent model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, a CAC rodent model was established by implanting LLC cells into mice and using subcutaneous injection of PBS as the control group (CONT). Following the implantation of LLC for 4 weeks, mice in the CAC group exhibited a notable reduction in tumor-free body weight and experienced fat and muscle atrophy in multiple locations (Fig. 1a and Fig. 1b), indicating the representativeness of this CAC rodent model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, eWATs were obtained from 6 mice for transcriptome sequencing, comprising 3 from the CAC and 3 from the CONT groups. In total, 31,447 genes were successfully identified. 715 genes showed significant differential expression in the WAT of CAC mice based on criteria for differential analysis (|log2(fold change) | greater than 1 and padj less than 0.05). Among these, 368 exhibited up-regulations, while 347 displayed down-regulations (Fig. 2a). Gene ontology (GO) database was used for GO annotation and enrichment analysis. A total of 328 GO terms were significantly different, including 25 GO terms related to fatty acid and lipid metabolism (Fig. 2c). Totally 24 genes were screened out that have differential expression at the same time, related to lipid metabolism (Fig. 2b). Functional annotation and classification of these genes were performed, and they were subsequently sorted by the significance of difference, as shown in Table 1 [14-24]. A large proportion of genes were found to be related to very long chain fatty acid (VLCFA) or LCFA metabolism pathway (Fig. 2d). Moreover, 2 out of the 3 genes with the most significant expression difference were related to the elongation process of LCFA carbon chains. Notably, the expression of Elovl6 was significantly downregulated in the WAT of CAC mice (log2FC = -2.27, padj = 0.0000897). This result was validated through qRT-PCR analysis of the WAT from CAC and CONT mice (Fig. 1c). The data indicated a significant downregulation of Elovl6 and its upstream regulatory gene steroid regulatory element-binding protein-1 (SREBP-1). Therefore, Elovl6 is the most potential gene for subsequent research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003e10 differentially expressed genes related to LCFA/VLCFA metabolism in WAT of CAC mice\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLog2(FC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003epadj\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFunction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSlc27a1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMediating ATP-dependent LCFA transport into the cytosol [14]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eElovl6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-2.273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCatalyzing carbon chain lengthening of C12-C16 fatty acids [15,16]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHacd2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCatalyzing the third step of LCFA carbon chain extension (dehydration) [17]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdtrp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.445\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHydrolyzing bioactive fatty acid esters of hydroxy fatty acid [18]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcot4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.983\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHydrolyzing lipoyl CoA to form free fatty acids and coenzyme A. Can also catalyze the hydrolysis of long-chain lipoyl CoA [19]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEhhadh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.844\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOne of the four enzymes of the peroxisomal \u0026beta;-oxidation pathway [20]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcadvl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCatalyzing the initial stage of VLCFA mitochondrial fatty acid \u0026beta;-oxidation [21]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcadl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCatalyzing the initial stage of LCFA mitochondrial fatty acid \u0026beta;-oxidation [22]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePnpla3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-2.988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMediating triglyceride hydrolysis in adipocytes [23]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIrs2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.951\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRegulating the biological effects of insulin and IGF1 [24]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eLow expression of Elovl6 in WAT of CAC patients could change fatty acid proportion and worsen weight loss.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the decreased expression of Elovl6 in WAT of CAC, 204 subjects were enrolled in our study, adhering to the aforementioned inclusion and exclusion standards. 13 patients among them were excluded due to metabolic diseases and preoperative neoadjuvant chemotherapy. In the end, the study encompassed 191 patients. Out of this group, 99 were CAC patients and 92 were weight-stable cancer patients (WS) served as the control group. The fundamental clinical information of CAC patients is presented in Table 2 for both groups. Except for weight loss and BMI, other factors such as gender, age, and tumor node metastasis classification (TMN) did not exhibit significant differences when comparing the CAC and WS groups, indicating comparable baseline matching between the two groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eClinical information of included CAC patients and weight-stable tumor patients\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFeatures\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAC (n=99)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWS(n=92)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender, Female (n, %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e36, 36.36%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e40, 43.47%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.315\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (yr)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e62.12 \u0026plusmn; 11.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e62.10 \u0026plusmn; 9.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.991\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeight (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e163.04 \u0026plusmn; 7.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e166.09 \u0026plusmn; 7.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeight (kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e55.88 \u0026plusmn; 9.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e67.46 \u0026plusmn; 9.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBMI (kg/m2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20.96 \u0026plusmn; 2.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e24.39 \u0026plusmn; 2.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeight loss(kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.99 \u0026plusmn; 3.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.38 \u0026plusmn; 0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage, I - II (n, %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e75, 75.75%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e62, 67.39%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.199\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlbumin (g/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e39.75 \u0026plusmn; 4.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e40.78 \u0026plusmn; 4.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.421\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePre-albumin (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e189.90 \u0026plusmn; 52.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e223.58 \u0026plusmn; 50.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal protein (g/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e64.38 \u0026plusmn; 5.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e66.29 \u0026plusmn; 4.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLDL (mmol/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.44 \u0026plusmn; 0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.45 \u0026plusmn; 0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.959\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHDL-CH (mmol/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.13 \u0026plusmn; 0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.13 \u0026plusmn; 0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.937\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal cholesterol(mmol/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.91 \u0026plusmn; 0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.26 \u0026plusmn; 0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.852\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTriglyceride(mmol/L)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.17 \u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.52 \u0026plusmn; 0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRBC (10^12/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.89 \u0026plusmn; 0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.21 \u0026plusmn; 0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWBC (10^9/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.18 \u0026plusmn; 1.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.66 \u0026plusmn; 1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHb (g/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e115.65 \u0026plusmn; 20.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e126.91 \u0026plusmn; 19.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLT (10^9/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e220.56 \u0026plusmn; 71.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e218.88 \u0026plusmn; 74.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.874\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHBA1C (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.71 \u0026plusmn; 1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.90 \u0026plusmn; 0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.246\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIn both groups of patients, the qPCR results for genes associated with lipid metabolism in WAT demonstrated a notable reduction in Elovl6 and its upstream regulatory gene, SREBP-1. Aligning with the Western blot data presented in Figure 3c, this result confirms the consistency. The expression levels of genes related to lipolysis, which include hormone-sensitive lipase (HSL), peroxisome proliferator-activated receptor \u0026alpha; (PPAR\u0026alpha;), and adipose triglyceride lipase \u0026nbsp; \u0026nbsp; \u0026nbsp; (ATGL), were significantly upregulated in WAT of the CAC patients. In contrast, there were no notable differences in the expression of genes associated with adipogenesis, including enhancer-binding protein \u0026alpha; (C/EBP\u0026alpha;), C/EBP\u0026beta;, PPAR\u0026gamma;, and the lipogenic genes such as the mammalian target of rapamycin (mTOR) and fatty acid synthase (FASN) (Fig. 3a). Moreover, there was a significant downregulation in the expression of the gene Stearoyl-CoA desaturase-1 (SCD-1), which is implicated in the synthesis of polyunsaturated fatty acids. The result of Western blotting also showed a decrease in Elovl6 protein in CAC patients (Fig. 2e and Fig. 2f). Then, a correlation analysis of Elovl6 expression was performed with the degree of weight loss in CAC patients (Fig. 3b), suggesting a statistically significant linear relationship between the two variables.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTo identify alterations in fatty acid content in the WAT of CAC patients, fatty acids were isolated from tissue samples and converted into FAMEs that were suitable for GC analysis\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e It was found that the content of palmitic acid (C16:0) was significantly higher in the WAT of CAC patients. Correspondingly, the content of linoleic acid (C18:2n6c) was significantly lower (Fig. 3c). When comparing the relationship of overall C18 to C16 fatty acid composition between the two groups, it became evident that there was a notable reduction in the C18:C16 ratio within the WAT tissues of CAC patients (Fig. 3d). There were no significant variations observed in the relative contents of other fatty acids (Supplement Table S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElovl6 knockdown could inhibit lipogenesis and pre-adipocyte differentiation by altering fatty acid components in adipocytes \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further explore the influence of Elovl6 on the morphological and functional aspects of adipocytes, a tumor cell conditioned medium was introduced to differentiate 3T3-L1 cells as an in vitro model of CAC. IL-6 added complete medium served as a positive control. Additionally, siRNA was used to suppress Elovl6 expression in 3T3-L1 preadipocytes, using empty lipofectamine as a control. Following the onset of differentiation in 3T3-L1 cells, the preadipocytes underwent gradual transformation into a full, round shape, with a concurrent rise in both the quantity and diameter of intracellular lipid droplets (Fig. 4a). In the 3T3-L1 cells, the qPCR results indicated a decreasing Elovl6 expression level during the differentiation process, while the upstream regulatory gene of Elovl6, SREBP-1, displayed a progressive rise in expression. Additionally, PPAR\u0026gamma;, a gene related to differentiation, and ATGL, a major gene associated with lipolysis, were also gradually up-regulated (Fig. 4b). Upon achieving differentiation and maturation of 3T3-L1 cells, tumor cell conditioned medium was introduced to simulate a cachexic environment. In the CAC group, the adipocyte morphology underwent significant changes, including a reduction in both the quantity and volume of intracellular lipid droplets (Fig. 4a). qPCR assays indicated a decrease in the expression level of genes related to the differentiation process, such as C/EBP\u0026alpha;, C/EBP\u0026beta;, and PPAR\u0026gamma;, as well as genes associated with lipid synthesis, including mTOR, FASN, and SCD-1. The expression level of Elovl6 and SREBP-1 was also notably reduced (Fig. 4c). The Western Blot analysis also showed a decrease in Elovl6 protein in the CAC group (Fig. 3d and Fig. 3e).\u003c/p\u003e\n\u003cp\u003eAfter using siRNA to knock down Elovl6 expression in 3T3-L1 cells, a decrease was observed in the differentiation rate of 3T3-L1. The proportion of pre-adipocytes undergoing differentiation was reduced, and the volume of lipid droplets in the cytoplasm after differentiation was also reduced (Fig. 4f). qPCR analysis showed that silencing Elovl6 suppressed gene expression associated with fatty acid synthesis, hydrolysis, and adipogenesis (Fig. 4g), including SREBP-1, ATGL, PPAR\u0026gamma;, and CEB/P\u0026beta;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMature 3T3-L1 cells were collected from both Elovl6-KD and the control group, and their fatty acid composition was analyzed by GC. The findings indicated that the knockdown of Elovl6 in 3T3-L1 cells could cause alterations in fatty acid composition. This was evidenced by a notable elevation in the content of palmitic acid (C16:0) and a decrease in oleic acid (C18:1n9c) (Fig. 4h). These results suggested that low expression of Elovl6 may exaggerate tumor cell-induced adipocyte atrophy, probably through altering fatty acid component of adipocytes.\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAt the outset of this study, transcriptome sequencing on the WAT of the CAC rodent model was performed. A significant decrease in Elovl6 expression was observed in CAC. This result was verified by an \u003cem\u003ein vivo\u003c/em\u003e study on rodent CAC model and clinical CAC samples. The reduced expression of Elovl6 was also correlated with the degree of weight loss in CAC patients, which further corroborates the relationship between Elovl6 and pathophysiological processes in CAC.\u003c/p\u003e \u003cp\u003eThe first question that comes to our mind is whether the low expression of Elovl6 in WAT of CAC could be a consequence of altered fatty acid metabolism in the CAC pathophysiological process. Currently, no studies have reported the expression and regulation of Elovl6 in CAC. In this study, expression of both Elovl6 and SREBP-1 was decreased in CAC patients, tumor-bearing mice, and 3T3-L1 cells undergoing a cachexic environment. Studies in other related fields have suggested that SREBP-1 can regulate Elovl6 expression. Moon and colleagues observed high expression of Elovl6 in transgenic mice overexpressing SREBP-1 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, synthetic agonists of LXRα, an upstream regulating gene of SREBP-1, induced high expression of Elovl6 and FASN in BAT adipocytes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These reports revealed the positive regulatory effect of SREBP-1 on Elovl6. The SREBP family proteins are all inactivated precursor proteins anchored to the endoplasmic reticulum (ER) membrane. Upon formation of their structural domains, SREBPs can regulate both cholesterol and fatty acid metabolism [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. According to relevant studies, the anabolic Akt-mTOR signaling pathway could upregulate SREBP-1 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Thus, it is possible that the downregulation of Elovl6 expression in the WAT of CAC patients could be a phenomenon following the inhibition of the entire lipid anabolic process.\u003c/p\u003e \u003cp\u003eIt is worth noting that no alterations in mTOR mRNA expression were observed in either the CAC rodent model or clinical samples. In fact, in cancer-associated cachexia, decreased mTOR expression has been described exclusively in the skeletal muscle of CAC patients or mice, with no such reports in adipose tissue [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, this study observed downregulation of SREBP-1, FASN, and SCD-1 expression in the WAT of CAC, indicating a downstream inhibition of the fatty acid synthesis pathway. In other studies, mRNA levels of genes related to adipose synthesis, including FAS, ACC, and SCD-1, also exhibited reductions in the adipose tissue of tumor-bearing mice [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These results suggested that the downregulation of SERBP-1 in WAT of CAC may be induced by a change in phosphorylation of mTOR and could trigger downstream inhibition of lipid synthesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUp to this point, it is reasonable to assume that Elovl6 is downregulated by the inhibition of the lipid synthesis process during CAC pathophysiology, possibly through the mTOR-SREBP axis. On the other hand, the low expression of Elovl6 may also contribute to the fat loss process. In this study, the knockdown of Elovl6 could suppress the differentiation of pre-adipocytes and alter lipid metabolism. Additionally, palmitic acid (C16:0) in WAT of CAC patients was increased, while linoleic acid (C18:2n6c) was decreased. After the knockdown of Elovl6 in 3T3-L1 cells, a similar change appeared in the fatty acid composition of the cells. A significant elevation in palmitic acid (C16:0) and a reduction in oleic acid (C18:1n9c) content was observed in CAC groups \u003cem\u003ein vitro\u003c/em\u003e. This phenomenon not only matches the decreased expression of Elovl6 but also implies that Elovl6 might have a subsequent biological impact by altering the proportion of fatty acids in WAT. Indeed, the regulatory influence of fatty acids on lipid metabolism has been a long-standing research topic, particularly regarding polyunsaturated fatty acids (PUFAs). A study reported that feeding PUFA to mice inhibited FASN expression and related lipid anabolism [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Worgall et al found that oleic acid and PUFA could inhibit SREBP expression, ultimately leading to the inhibition of fatty acid synthesis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In recent years, Ou et al further elaborated that PUFA could directly inhibit expression of LXR, an upstream regulatory gene of SREBP-1 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These studies suggest that PUFA itself has an inhibitory effect on the lipid synthesis pathway. Since de novo synthesis of PUFA requires LCFA as a substrate, and Elovl6 is a crucial enzyme in LCFA synthesis within WAT, the decreased expression of Elovl6 in WAT of CAC patients may play a further regulatory function in the process of CAC fat loss by affecting the synthesis of PUFA. However, no such change of PUFA was observed either in the WAT of CAC patients or in Elovl6-KD 3T3-L1 cells. We think the primary reason for not acquiring positive results is the low amount of PUFA in WAT and 3T3-L1 cells. In contrast, monounsaturated FAs and saturated FAs could also affect pre-adipocyte differentiation and lipid metabolism process independently. Kobayashi et al. found that adding C18:1 and C20:1 fatty acid into the culture medium of 3T3-L1 could facilitate the differentiation process [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], which may support this hypothesis. Thus, additional evidence is still required to support this viewpoint.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eComparisons with other studies and what does the current work add to the existing knowledge\u003c/h2\u003e \u003cp\u003eCurrently, no studies have been conducted on Elovl6 and other fatty acid elongases in CAC. However, Tan et al. have found a significant reduction in the C18:C16 ratio and a decrease in the expression of SCD family genes in the WAT of Elovl6 KO mice, which indicates a suppression of the fatty acid desaturation process [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Interestingly, the WAT of CAC patients in this study exhibited a similar alteration. In this study, transcriptome sequencing was conducted on WAT in a CAC mouse model, and fatty acid GC analysis was performed on WAT in CAC patients. The resulting data provides valuable insights for future studies on CAC-induced fat loss. Additionally, the current study proposed the potential contribution of Elovl6 in the process of CAC-induced fat loss. This discovery not only presents a new biomarker for diagnosing and treating CAC in future clinical practice but also introduces a new theory for CAC research.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStudy strengths and limitations\u003c/h2\u003e \u003cp\u003eThis study is closely connected to the clinical field. First, most WAT samples used in this study were obtained from CAC patients diagnosed by clinical doctors, which makes the result representative of real situations in the human body. Furthermore, both a decrease in Elovl6 expression and a change in the fatty acid component were discovered in CAC patients' fat tissue, indicating that Elovl6 could be a valuable biomarker for early identification of CAC. Finally, Elovl6 could potentially contribute to fat loss in CAC by altering the fatty acid composition in adipocytes \u003cem\u003ein vitro\u003c/em\u003e, making it a promising target for future therapeutic interventions. A limitation of our research was that Elovl6 knockdown was performed \u003cem\u003ein vitro\u003c/em\u003e, which may not reflect the actual conditions in the human body.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, Elovl6 expression was found to be diminished in the WAT of CAC. Low expression of Elovl6 correlated with the severity of weight loss in CAC. In addition, reduced Elovl6 expression levels in WAT of CAC patients coincided with a reduction in C18 fatty acid content and an augmentation in C16 fatty acid content. Knockdown Elovl6 inhibited the differentiation of pre-adipocytes and downregulated the expression of genes associated with lipid synthesis. Elovl6-knockdown also induced a significant decrease in oleic acid (C18:1n9c) content and a notable rise in palmitic acid (C16:0) content, suggesting low expression of Elovl6 could suppress the CAC liposynthesis process possibly by altering the lipid content in adipocytes. The findings from this study indicate that Elovl6 has the potential to serve as a dependable biomarker for the early diagnosis of cancer-associated cachexia and may offer a promising target for future therapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecancer-associated cachexia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewhite adipose tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eElovl6\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelongase of very long-chain fatty acid 6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebrown adipose tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elong-chain fatty acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVLCFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003every-long-chain fatty acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etriglyceride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHDL-C\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-density lipoprotein cholesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elow-density lipoprotein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebody mass index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLewis lung carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFAME\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efatty acid methyl ester\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egas chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNCS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneonatal calf serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHG-DMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's modified eagle medium with high-glucose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epenicillin-streptomycin solution\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIBMX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3-isobutyl-1-methyl-7H-xanthine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eORO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOil red O\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSREBP-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esteroid regulatory element-binding protein-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATGL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadipose triglyceride lipase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehormone sensitive lipase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elipoprotein lipase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eC/EBP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenhancer-binding protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperoxisome proliferator-activated receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFASN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efatty acid synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSCD-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStearoyl-CoA desaturase-1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by ethics committee of Zhongshan Hospital of Fudan University (B2019-193R). All patients participating in this study had signed the informed consents. The animal experiments in this study were approved by the Animal Ethics Committee of Zhongshan Hospital of Fudan University (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emRNA-seq dataset used in this study has been uploaded to the Gene Expression Omnibus database (GSE242812).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received funding through grants from the Shanghai Natural Science Foundation ProjectQ7 (19ZR1409100)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQingyang Meng and Guohao Wu formulated and planned the experiments. Chenyang Jin and Shuangjie Wang gathered the data and conducted the biological experiments with assistance from Xiangyu Sui. Chenyang Jin analyzed and interpreted the data. Chenyang Jin and Shuangjie Wang were responsible for composing the paper. All authors subsequently reviewed and granted approval for the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnote\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChenyang Jin and Shuangjie Wang contribute equally to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eFearon K, Strasser F, Anker SD, Bosaeus I, Bruera E, Fainsinger RL, et al. Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011;12(5):489\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMartin L, Senesse P, Gioulbasanis I, Antoun S, Bozzetti F, Deans C, et al. Diagnostic Criteria for the Classification of Cancer-Associated Weight Loss. J Clin Oncol. 2015;33(1):90\u0026ndash;U147.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDaly L, Dolan R, Power D, Ni Bhuachalla E, Sim W, Fallon M et al. The relationship between the BMI-adjusted weight loss grading system and quality of life in patients with incurable cancer. J cachexia sarcopenia muscle. 2019.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBaracos VE, Martin L, Korc M, Guttridge DC, Fearon KCH. Cancer-associated cachexia. Nat Reviews Disease Primers. 2018;4.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePetruzzelli M, Schweiger M, Schreiber R, Campos-Olivas R, Tsoli M, Allen J, et al. A Switch from White to Brown Fat Increases Energy Expenditure in Cancer-Associated Cachexia. Cell Metabol. 2014;20(3):433\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMeex RC, Hoy AJ, Mason RM, Martin SD, McGee SL, Bruce CR, et al. ATGL-mediated triglyceride turnover and the regulation of mitochondrial capacity in skeletal muscle. Am J Physiol Endocrinol metabolism. 2015;308(11):E960\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDas SK, Eder S, Schauer S, Diwoky C, Temmel H, Guertl B, et al. Adipose triglyceride lipase contributes to cancer-associated cachexia. Science. 2011;333(6039):233\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eEbadi M, Mazurak VC. Evidence and mechanisms of fat depletion in cancer. Nutrients. 2014;6(11):5280\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLaiglesia LM, Lorente-Cebri\u0026aacute;n S, Prieto-Hontoria PL, Fern\u0026aacute;ndez-Galilea M, Ribeiro SMR, S\u0026aacute;inz N, et al. Eicosapentaenoic acid promotes mitochondrial biogenesis and beige-like features in subcutaneous adipocytes from overweight subjects. J Nutr Biochem. 2016;37:76\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eShin S, Ajuwon K. Effects of Diets Differing in Composition of 18-C Fatty Acids on Adipo se Tissue Thermogenic Gene Expression in Mice Fed High-Fat Diets. Nutrients. 2018;10(2):256.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eShin S, Ajuwon KM. Divergent Response of Murine and Porcine Adipocytes to Stimulation of Browning Genes by 18-Carbon Polyunsaturated Fatty Acids and Beta-Recep tor Agonists. Lipids. 2018;53(1):65\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOliver E, McGillicuddy FC, Harford KA, Reynolds CM, Phillips CM, Ferguson JF, et al. Docosahexaenoic acid attenuates macrophage-induced inflammation and im proves insulin sensitivity in adipocytes-specific differential effects between LC n-3 PUFA. J Nutr Biochem. 2012;23(9):1192\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAlvheim AR, Torstensen BE, Lin YH, Lillefosse HH, Lock E-J, Madsen L, et al. Dietary Linoleic Acid Elevates the Endocannabinoids 2-AG and Anandamid e and Promotes Weight Gain in Mice Fed a Low Fat Diet. Lipids. 2013;49(1):59\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMoon YA, Shah NA, Mohapatra S, Warrington JA, Horton JD. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins. J Biol Chem. 2001;276(48):45358\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMatsuzaka T, Shimano H, Yahagi N, Yoshikawa T, Amemiya-Kudo M, Hasty AH, et al. Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs. J Lipid Res. 2002;43(6):911\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJakobsson A, Westerberg R, Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism. Prog Lipid Res. 2006;45(3):237\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMorcillo S, Martin-Nunez GM, Rojo-Martinez G, Almaraz MC, Garcia-Escobar E, Mansego ML, et al. ELOVL6 genetic variation is related to insulin sensitivity: a new candidate gene in energy metabolism. PLoS ONE. 2011;6(6):e21198.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhao H, Matsuzaka T, Nakano Y, Motomura K, Tang N, Yokoo T, et al. Elovl6 Deficiency Improves Glycemic Control in Diabetic db/db Mice by Expanding \u0026beta;-Cell Mass and Increasing Insulin Secretory Capacity. Diabetes. 2017;66(7):1833\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTan CY, Virtue S, Bidault G, Dale M, Hagen R, Griffin JL, et al. Brown Adipose Tissue Thermogenic Capacity Is Regulated by Elovl6. Cell Rep. 2015;13(10):2039\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJakobsson A, J\u0026ouml;rgensen JA, Jacobsson A. Differential regulation of fatty acid elongation enzymes in brown adip ocytes implies a unique role for \u0026lt; i \u0026gt; Elovl3 during increased fatty ac id oxidation. Am J Physiology-Endocrinology Metabolism. 2005;289(4):E517\u0026ndash;E26.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eShimano H. Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Prog Lipid Res. 2001;40(6):439\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBertolio R, Napoletano F, Mano M, Maurer-Stroh S, Fantuz M, Zannini A et al. Sterol regulatory element binding protein 1 couples mechanical cues an d lipid metabolism. Nat Commun. 2019;10(1).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePorstmann T, Santos CR, Griffiths B, Cully M, Wu M, Leevers S, et al. SREBP Activity Is Regulated by mTORC1 and Contributes to Akt-Dependent Cell Growth. Cell Metabol. 2008;8(3):224\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLaplante M, Sabatini DM. An Emerging Role of mTOR in Lipid Biosynthesis. Curr Biol. 2009;19(22):R1046\u0026ndash;R52.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, et al. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol. 2001;3(11):1014\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWhite JP, Baynes JW, Welle SL, Kostek MC, Matesic LE, Sato S, et al. The Regulation of Skeletal Muscle Protein Turnover during the Progress ion of Cancer Cachexia in the ApcMin/+ Mouse. PLoS ONE. 2011;6(9):e24650.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWhite JP, Puppa MJ, Gao S, Sato S, Welle SL, Carson JA. Muscle mTORC1 suppression by IL-6 during cancer cachexia: a role for A MPK. Am J Physiology-Endocrinology Metabolism. 2013;304(10):E1042\u0026ndash;E52.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBing C, Russell S, Becket E, Pope M, Tisdale MJ, Trayhurn P, et al. Adipose atrophy in cancer cachexia: morphologic and molecular analysis of adipose tissue in tumour-bearing mice. Br J Cancer. 2006;95(8):1028\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBlake WL, Clarke SD. Suppression of Rat Hepatic Fatty Acid Synthase and S14 Gene Transcript ion by Dietary Polyunsaturated Fat. J Nutr. 1990;120(12):1727\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWorgall TS, Sturley SL, Seo T, Osborne TF, Deckelbaum RJ. Polyunsaturated Fatty Acids Decrease Expression of Promoters with Ster ol Regulatory Elements by Decreasing Levels of Mature Sterol Regulator y Element-binding Protein. J Biol Chem. 1998;273(40):25537\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOu J, Tu H, Shan B, Luk A, DeBose-Boyd RA, Bashmakov Y et al. Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dep endent activation of the LXR. Proceedings of the National Academy of Sciences. 2001;98(11):6027-32.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKobayashi T, Fujimori K. Very long-chain-fatty acids enhance adipogenesis through coregulation of Elovl3 and PPAR\u0026gamma; in 3T3-L1 cells. Am J Physiology-Endocrinology Metabolism. 2012;302(12):E1461\u0026ndash;E71.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHan J, Wang Y, Qiu Y, Sun D, Liu Y, Li Z, et al. Single-cell sequencing unveils key contributions of immune cell populations in cancer-associated adipose wasting. Cell Discov. 2022;8(1):122.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFasshauer M, Kralisch S, Klier M, Lossner U, Bluher M, Klein J, et al. Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocytes. Biochem Biophys Res Commun. 2003;301(4):1045\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSun D, Zuoyou D, Shen L, Yang F, Han J, Wu G. miR-410-3P inhibits adipocyte differentiation by targeting IRS-1 in cancer-associated cachexia patients. Lipids Health Dis. 2021;20.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHan J, Shen L, Zhan Z, Liu Y, Zhang C, Guo R, et al. The long noncoding RNA MALAT1 modulates adipose loss in cancer-associated cachexia by suppressing adipogenesis through PPAR-\u0026gamma;. Nutr Metabolism. 2021;18(1):27.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLiu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol. 2020;21(4):183\u0026ndash;203.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"lipids-in-health-and-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lhad","sideBox":"Learn more about [Lipids in Health and Disease](http://lipidworld.biomedcentral.com/)","snPcode":"12944","submissionUrl":"https://submission.nature.com/new-submission/12944/3","title":"Lipids in Health and Disease","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cancer-associated cachexia, Elovl6, fat loss, long-chain fatty acid","lastPublishedDoi":"10.21203/rs.3.rs-3611425/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3611425/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eCancer-associated cachexia (CAC) arises from malignant tumors and leads to a debilitating wasting syndrome. In the pathophysiology of CAC, the depletion of fat performs a significant function. The mechanism of CAC-induced fat loss includes the enhancement of lipolysis, inhibition of lipogenesis, and browning of white adipose tissue (WAT), however, few lipid-metabolic enzymes have been reported to be involved in CAC. This study hypothesized that Elovl6, a critical enzyme for elongation of fatty acids, may involve in fat loss in CAC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eTranscriptome sequencing technology was employed to identify CAC-related genes in the WAT of a CAC rodent model. Then the expression level of Elovl6 and fatty acid composition were analyzed in a large clinical sample. Elovl6 was knocked down in 3T3-L1 mouse preadipocytes to compare with wild-type 3T3-L1 cells treated with tumor cell-conditioned medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult: \u003c/strong\u003eIn the WAT of patients with CAC, a significant decrease in the expression of Elovl6 was found, which correlates with the extent of body mass reduction in a linear relationship. Gas chromatographic analysis revealed an augmentation in palmitic acid (C16:0) and a reduction in linoleic acid (C18:2n6c) content in those tissue samples. Treating with tumor cell-conditioned medium, 3T3-L1 mouse preadipocytes showed a decrease of Elov16, and Elovl6-knockdown cells demonstrated reduced pre-adipocyte differentiation and lipogenesis. Likewise, the knockdown of Elovl6 in 3T3-L1 cells exhibited a significant rise in palmitic acid (C16:0) and a remarkable decrease in oleic acid (C18:1n9c) content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOverall, the expression of Elovl6 was diminished in the WAT of CAC patients. The decreased expression of Elovl6 might lead to fat loss in CAC by potentially altering the fatty acid composition in adipocytes. These findings suggest that Elovl6 may be used as a valuable biomarker for early diagnosis of CAC, and hold promise as a target for future therapies.\u003c/p\u003e","manuscriptTitle":"Low expression of Elovl6 may involve in fat loss in white adipose tissue of cancer-associated cachexia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-16 15:23:04","doi":"10.21203/rs.3.rs-3611425/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-11T06:03:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-31T08:32:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-31T02:02:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a329eef4-e99b-4dac-8941-5a2854c03c74","date":"2024-01-26T00:42:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1fd83d03-592f-4680-8d4b-dae41e61385f","date":"2024-01-25T17:02:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f14d4767-1f7d-40c0-99d3-6a5a76222ecc","date":"2023-12-12T00:56:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"00c820b8-00c3-4ea9-9819-644f22c32e18_SNPRID","date":"2023-11-24T00:10:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-11-20T20:38:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-15T04:19:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-11-15T02:43:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Lipids in Health and Disease","date":"2023-11-14T16:54:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"lipids-in-health-and-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lhad","sideBox":"Learn more about [Lipids in Health and Disease](http://lipidworld.biomedcentral.com/)","snPcode":"12944","submissionUrl":"https://submission.nature.com/new-submission/12944/3","title":"Lipids in Health and Disease","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"066b3977-fe35-48c6-9421-a091488d8a92","owner":[],"postedDate":"November 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-04-29T12:36:40+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-16 15:23:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3611425","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3611425","identity":"rs-3611425","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}