返回顶部显示 81 - 90 件，共 108 件产品CAT.#SizePriceGST A3-3，重组人 GS630.1 mg$460.00GST A3-3，重组大鼠 GS330.1 mg$460.00GST A4-4，重组人 GS640.1 mg$460.00GST Alpha 检测试剂盒GS41Kit$655.0 0消费税M1 -1 免疫前对照GS67c0.1 mL$110.00GST M1-1，重组人GS650.1 mg$460.00GST M2-2，重组人GS660.1 mg$460.00GST M4-4，重组人GS740.1 mg$460.00GST M5-5，重组人GS590.1 mg$460.00GST P1-1 Preimmune ControlGS72c1.0 mL$110.00PaginationFirst page« FirstPrevious page‹ Previous…Page3Page4Page5Page6Page7Page8Current page9Page10Page11Next pageNext ›Last pageLast »返回顶部显示 71 - 80 件，共 108 件产品CAT.#SizePriceCypExpress 2E1，10.0 克 CE2E1.1010.0 克 3,760.00 克 CypExpress 2E1，250 毫克 CE2E1.250250 毫克 225.00 克 CypExpress 3A4，1.0 克 CE3A4。 11.0 克$500.00CypExpress 3A4, 10 克CE3A4.1010.0 克$3,760.00CypExpress 3A4, 250 mgCE3A4.250250 mg$225.00GST A1-1 免疫前对照GS62c1.0 mL$110.00GST A1-1，重组牛GS850.1 mg$460.00GST A1-1，重组人GS600.1 mg$460.00GST A2-2，重组人GS610.1 mg$460。 00消费税A2 -2, Recombinant RatGS310.1 mg$460.00分页第一页«第一上一页‹上一页…第3页4页5页6页7当前页8页9页10页11下一页下一页›最后一页最后»返回顶部显示 61 - 70 件，共 108 件产品CAT.#SizePriceCypExpress 2C19, 1.0 GramCE2C19.11.0 Gram$500.00CypExpress 2C19, 10 GramsCE2C19.1010.0 Grams$3,760.00CypExpress 2C19, 250 mgCE2C 19.250250 毫克$225.00CypExpress 2C9, 1.0 克CE2C9.11.0 克$500.00CypExpress 2C9, 10克CE2C9.1010.0 克$3,760.00CypExpress 2C9, 250 毫克CE2C9.250250 毫克$225.00CypExpress 2D6, 1.0 克$500.00CypExpress 2D6, 10 克CE2D6.1010.0 克ms$3,760.00CypExpress 2D6, 250 mgCE2D6.250250 mg$225.00CypExpress 2E1, 1.0 GramCE2E1.11.0 Gram$500.00分页首页« FirstPrevious page‹ 上一页…Page3Page4Page5Page6Current page7Page8Page9Page10Page11Next pageNext ›Last pageLast »返回顶部显示 51 - 60 件，共 108 件产品CAT.#SizePriceCypExpress 19A1, 250 mgCE19A1.250mg250 mg$210.00CypExpress 1A1, 1.0 GramCE1A1.11.0 Gram$500.00CypExpress 1A1, 10.0 GramsCE1A1.10 10.0 克$3,760.00CypExpress 1A1, 250 毫克CE1A1.250250 毫克$225.00CypExpress 1A2, 1.0 GramCE1A2.11.0 克$500.00CypExpress 1A2, 10 克CE1A2.1010.0 克$3,760.00CypExpress 1A2, 250 毫克CE1A2.250250 毫克$225.00CypExpress 2B6, 1 克CE2B6.11.0 克$500 .00CypExpress 2B6, 10 克CE2B6.1010.0 克$3,760.00CypExpress 2B6, 250 毫克CE2B6.250250 毫克$225.00分页首页« FirstPrevious page‹ Previous…Page2Page3Page4Page5Current page6Page7Page8Page9Page10…Next pageNext ›Last pageLast »返回顶部显示 41 - 50 件，共 108 件产品CAT.#SizePrice CYPerior CYP2CE，CPR 修饰，1.0 nmolCY34.1nmol1.0 nmol$275.00CYPerior CYP2CE，CPR 修饰，10.0 nmolCY34.10nmol10.0nmol 2,410.00CYPerior C YP2CE 与 hCPR，1.0 nmolCY33.1nmol1.0 nmol$275.00 CYPerior CYP2CE 与 hCPR，10.0 nmolCY33.10nmol10.0nmol$2,410.00CYPerior CYP3_N1A 与 CPR 修饰，1.0 nmolCY48.1nmol1.0 nmol$275.00CYPerior CYP3_N1A 与 CPR 修饰，10.0 nmolCY48 .10nmol10.0 nmol$2,410.00CYPerior CYP3_N1A 与 hCPR，1.0 nmolCY47.1nmol1。 0 nmol$275.00CYPerior CYP3_N1A with hCPR, 10.0 nmolCY47.10nmol10.0nmol$2,410.00CypExpress 19A1, 1 GramCE19A1.1g1.0 Gram$465.00CypExpress 19A1, 10 克CE19A1.101 0.0 克$3,760.00分页第一页«第一上一页‹上一页1页2页3页4当前页5页6页7页8页9…下一页下一页›最后一页最后»返回顶部显示 31 - 40 件，共 108 件产品CAT.#SizePriceCYPerior CYP1A1_Mam，CPR 修饰，1.0 nmolCY08.1nmol1.0 nmol$290.00CYPerior CYP1A1_Mam，CPR 修饰，10.0 nmolCY08.10nmol10.0nmol$2,605.0具有 CPR 修饰的 0CYPerior CYP2ABGS，1.0 nmolCY38.1nmol1.0 nmol $275.00CYPerior CYP2ABGS 加 CPR 修饰，10.0 nmolCY38.10nmol10.0nmol$2,410.00CYPerior CYP2ABGS 加 hCPR，1.0 nmolCY37.1nmol1.0 nmol$275.00CYPerior CYP2ABGS 加 hCPR，10.0 nmol CY37.10nmol10.0nmol$2,410.00CYPerior CYP2ABGSFTCEH，CPR 修饰，1.0 nmolCY40.1nmol1。 0 nmol$275.00CYPerior CYP2ABGSFTCEH 进行 CPR 修饰，10.0 nmolCY40.10nmol10.0nmol$2,410.00CYPerior CYP2ABGSFTCEH 进行 hCPR，1.0 nmolCY39.1nmol1.0 nmol$275.00CYPerior CYP2ABGSFTCE H with hCPR, 10.0 nmolCY39.10nmol10.0nmol$2,410.00PaginationFirst page« FirstPrevious page‹ PreviousPage1Page2Page3Current page4Page5Page6Page7Page8Page9...下一页下一页 ›最后一页最后 »返回顶部显示 21 - 30 件，共 108 件产品CAT.#SizePriceAnti-Rat CYP450 1A1，MonoclonalPM100.05 mg$290.00Anti-Rat CYP450 2B1，MonoclonalPM250.1 mL$315.00Anti-Rat CYP450 2B1/2B2，MonoclonalPM201.0 mL$290.00抗大鼠 CYP450 2E1 , MonoclonalPM320.1 mL$360.00Anti-Rat CYP450 3A1, MonoclonalPM400.1 mL$360.00Anti-Rat GST A (Ya) Goat PolyclonalGS090.1 mL$380.00Anti-Rat GST M1 (Yb1), Goat PolyclonalGS230.1 mL$380.00CYP450 1A1/1A2免疫前山羊血清PR31c1.0 mL$110.00CYP450 2B4 免疫前山羊血清PR30c1.0 mL$110.00CYP450 山羊免疫前IgGPH17c0.5 mL$110.00分页第一页« 第一上一页‹ 上一页1页2当前页3页4页5页6页7页8页9…下一页下一页›最后一页最后» SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes AbstractThe Simpson Golabi Behmel Syndrome (SGBS) pre-adipocyte cell strain is widely considered to be a representative in vitro model of human white pre-adipocytes. A recent study suggested that SGBS adipocytes exhibit an unexpected transient brown phenotype. Here, we comprehensively examined key differences between SGBS adipocytes and primary human white subcutaneous (PHWSC) adipocytes. RNA-Seq analysis revealed that extracellular matrix (ECM)-receptor interaction and metabolic pathways were the top two KEGG pathways significantly enriched in SGBS adipocytes, which included positively enriched mitochondrial respiration and oxidation pathways. Compared to PHWSC adipocytes, SGBS adipocytes showed not only greater induction of adipogenic gene expression during differentiation but also increased levels of UCP1 mRNA and protein expression. Functionally, SGBS adipocytes displayed higher ISO-induced basal leak respiration and overall oxygen consumption rate, along with increased triglyceride accumulation and insulin-stimulated glucose uptake. In conclusion, we confirmed that SGBS adipocytes, which are considered of white adipose tissue origin can shift towards a brown/beige adipocyte phenotype. These differences indicate SGBS cells may help to identify mechanisms leading to browning, and inform our understanding for the use of SGBS vis-脿-vis primary human subcutaneous adipocytes as a human white adipocyte model, guiding the selection of appropriate cell models in future metabolic research. IntroductionA human preadipocyte cell strain derived from the stromal vascular fraction of subcutaneous adipose tissue of a male infant with Simpson-Golabi-Behmel syndrome (SGBS) was first described in 20011. SGBS is a rare overgrowth syndrome characterized by a broad spectrum of clinical manifestations including multiple congenital abnormalities, facial and cardiac abnormalities, defective lung segmentation, Wilms tumor, macrocephaly, and organomegaly2,3,4,5. While it could be lethal in affected males, with up to 50% neonatal mortality rate, the syndrome shows a milder form in female carriers6,7,8. Several affected individuals鈥?body weights have been reported to be in the 97th percentile6, 7, 9 but it is not known whether body temperature is elevated in patients with SGBS syndrome. Wabitsch et al. previously reported that SGBS cells responded to catecholamine exposure with an increase in glucose uptake upon insulin stimulation and effective inhibition of cathecolamine-stimulated lipolysis1. Genomic rearrangements and point mutations involving the glypican (GPC)-3, GPC-4 and oral-facial-digital syndrome (OFD1) genes have been shown to be associated with SGBS. These genes are involved in the regulation of cell division and growth5, 10, 11.Neither transformed nor immortalized, the SGBS preadipocyte cell strain is capable of retaining their potential for adipose differentiation up to 50 generations1. This cell strain thus serves as an almost unlimited source of human preadipocytes and a widely used tool to study human adipocyte biology12,13,14,15,16,17.During the differentiation process, SGBS cells developed a gene expression pattern similar to that found in differentiating primary human preadipocytes with a characteristic increase in fat cell-specific mRNAs encoding lipoprotein lipase (LPL), glycerol-3-phosphate dehydrogenase (GPDH), GLUT4, leptin and others1. Subsequent studies performed in SGBS adipocytes and primary human subcutaneous adipocytes isolated from lipoaspirates showed a similar trend in the induction of FABP4 and PPAR纬 mRNA and FABP4 secretion during differentiation18. Another study characterizing SGBS adipocytes and primary human omental adipocytes derived from obese and non-obese individuals concurred that the cell lines are similar in terms of the morphology, induction of adipocyte-specific gene expression and GPDH activity13. However, inherent differences in metabolic profiles of SGBS adipocyte and primary human adipocytes derived from adult subcutaneous depot remain largely unclear. More recently SGBS adipocytes were reported to display a transient feature of brown adipocytes at day 14 and eventual differentiation and maturation to white adipocyte phenotype by day 28 of differentiation19. UCP1 expression appeared to be an intrinsic property of SGBS adipocytes as it remained high at day 14 when differentiated in a rosiglizatone free medium. Moreover, when exposed to cold at 30鈥壜癈, both UCP1 expression and mitotracker co-staining were elevated, implying a cold stimulus driven inducible brown phenotype19. Given that SGBS is often used as a representative model of primary human white SC adipocytes13, 18, 20,21,22,23, the validation is required to identify and characterize any potential differences in their metabolic phenotype. We conducted a comparative study and found that while both these cell types have overlapping similarities, they also exhibit distinct metabolic signatures that may explain differences in their capacities for adipocyte differentiation and maturation, lipid metabolism and thermogenic activity. Understanding these differences could open opportunities to understand the browning of adipose tissue and also further our knowledge of the strength and limitations of each cell line and provide guidance on the choice of cellular models in future research on human fat cell development and metabolism.ResultsFully differentiated SGBS adipocytes display a transcriptomic profile different to primary human white SC adipocytes (PHWSC)Approximately 15000 genes were read by RNA-Seq, of which 1667 genes were upregulated and 2050 genes were downregulated in SGBS adipocytes relative to PHWSC (Fig.聽1a and supplementary Table聽S2 and supplementary Table聽S3; log FC鈥?gt;鈥壜扁€?.6; FDR鈥?lt;鈥?.05).Figure 1Global transcriptome profiling of SGBS and PHWSC adipocytes (a) Volcano plots displaying a number of differentially expressed genes in SGBS adipocytes compared to PHWSC adipocytes. Red dots denote statistically significant genes. Genes with FDR鈥?lt;鈥?.05 and log (FC)鈥?gt;鈥?.60 were included. (b) Heat map clustering top 1000 significantly upregulated (red) or downregulated (blue) genes between SGBS and PHWSC adipocytes. For PHWSC adipocytes in RNA-Seq, experiments were performed in cells derived from 4 different donors (SC_1 to SC_4, n鈥?鈥?). For SGBS adipocytes, experiments were performed in duplicates (SGBS_1 and SGBS_2) from the same SGBS adipocyte.Full size imageNext we performed unsupervised clustering on the top 1,000 genes with the most variable RNA-seq counts across all experiments. The results were visualized using a heat map and clusters were analyzed for enriched gene ontologies (GO). Genes downregulated in SGBS were associated with the membrane (GO_cellular component), extra-cellular matrix (GO_cellular component) and cell adhesion (GO_biological function) gene ontologies. Conversely, genes upregulated in SBGS adipocytes were associated with lipid metabolism (GO_biological function) and mitochondria (GO_cellular component) gene ontologies (Fig.聽1b). When we generated an alternate heat map using all differentially expressed genes with a FDR鈥?lt;鈥?.05 (Supplementary Fig.聽S1), it was reassuring to see that the enriched gene ontologies were in perfect concordance. We next analysed all differentially expressed genes, both up and downregulated combined, in the context of KEGG pathways. Here, 20 KEGG pathways were enriched in SGBS when compared to PHWSC adipocytes and the top 2 significantly enhanced pathways were ECM-receptor interaction (FDR鈥?鈥?.18E-06) and metabolic pathways (FDR鈥?鈥?.07E-05) (Table聽1). Genes significantly up or downregulated within the metabolic pathways in SGBS adipocytes were further analysed for KEGG pathway enrichment and an interaction map was built using Cytoscape (Supplementary Table聽S4 and Supplementary Fig.聽S2). The interaction map highlighted significant positive enrichment (red coloured nodes) in oxidative phosphorylation, TCA cycle, fatty acid elongation, butanoate metabolism and steroid biosynthesis pathways in SGBS adipocytes when compared to PHWSC adipocytes (Supplementary Fig.聽S2 and Supplementary Table聽S4).Table 1 List of KEGG pathways enriched in SGBS adipocytes when compared to PHWSC adipocytes.Full size tableNext, we examined whether RNA-Seq analyses could reveal further insights into the lineage of SGBS adipocytes, referencing a comprehensive list of adipocyte lineage markers reported to date as a base (Table聽2). SGBS adipocytes display expression profiles consistent generally with WAT lineage, although upregulation of several markers relating to brown/beige lineage was also seen. Of interest, the UCP1 gene, a bona fide marker of brown fat, along with genes PDK4, PREX1, SIRT1, PLIN5, EAR2, SHOX2, SLC27A1, were markedly upregulated (FDR鈥?鈥?.037) in SGBS adipocytes, when compared to PHWSC adipocytes.Table 2 List of adipocyte and adipose tissue lineage markers analysed in SGBS adipocytes compared to PHWSC adipocytes.Full size tableSGBS and PHWSC adipocytes exhibit distinct gene expression patterns during differentiationWe next analysed when these cellular models might diverge. Adipogenesis was initiated in SGBS and PHWSC adipocytes and RNA from both cell types was harvested on days 0, 4, 8, and 12 for gene expression studies. Nine genes-of-interest relating to adipogenesis, lipid storage and metabolism, and browning were chosen for real-time PCR validation. With increasing days of differentiation, both SGBS and PHWSC preadipocytes showed upregulation of genes involved in adipocyte differentiation but the magnitude of induction of PPAR纬, CEBP伪, adiponectin and leptin was 10鈥?5 folds higher in SGBS compared to PHWSC adipocytes (Fig.聽2a) suggesting they had facilitated differentiation. For instance, using PHWSC adipocytes at day 0 as a reference, the fold change for adiponectin was about 14 times lower in PHWSC adipocytes when compared to SGBS at D12. Lipid storage genes FABP4 and FITM2 also showed a significant upregulation over the course of adipocyte differentiation (Fig.聽2b). FITM2 (fat storage-inducing transmembrane proteins 2) is involved in triglyceride partitioning and its gene expression levels has been previously reported to increase with differentiation in 3T3-L1 mouse adipocytes24, 25. PGC1伪, a transcriptional co-activator of UCP1 expression and a key gene in cellular energy metabolism26, 27, was significantly higher in PHWSC adipocytes when compared to SGBS (P鈥?lt;鈥?.05). Interestingly, the expression of browning marker UCP1 was almost non-existent in PHWSC adipocytes with approximately 1200 fold higher expression in SGBS adipocytes (Fig.聽2c). The discordance between UCP1 and PGC1伪 expression may be due to alternative transcriptional activators of UCP1 mRNA in SGBS adipocytes27. Gene expression data assessed using RNA-Seq and qRT-PCR methods showed good correlation (r鈥?鈥?.8289, P鈥?lt;鈥?.0002, supplementary Fig.聽S3).Figure 2Expression profiling of differentiating SGBS and PHWSC adipocytes, for genes involved in (a) adipogenesis (b) lipid storage (c) browning. Days of differentiation are indicated as D0, D4, D8, D12 respectively. For experiments involving PHWSC adipocytes, experiments were performed in duplicates from cells derived from 3 different donors. For SGBS adipocytes, experiments were performed in duplicates for 3 independent experiments from the same SGBS adipocyte. Data represented as mean鈥壜扁€塖D and was normalized to SC control D0 samples. Two-way ANOVA was used to test for statistical significance. *P鈥?lt;鈥?.05, **P鈥?lt;鈥?.01, ***P鈥?lt;鈥?.001, ****P鈥?lt;鈥?.0001.Full size imageSGBS adipocytes display some features consistent with a UCP1-driven thermogenic machineryConsistent with the gene expression data, Fig.聽3a and supplementary Fig.聽S4 show that UCP1 protein expression was significantly higher in mature SGBS adipocytes as compared to PHWSC adipocytes. UCP1 protein expression persisted in SGBS adipocytes even after depletion of T3, rosiglitazone or both from the differentiation media (Fig.聽3a). Several key adipogenic markers were also examined for their protein expression levels. While PPAR纬 and FABP4 appeared to be similar between the two cell types, the protein expression levels of adiponectin and FITM2 were found to be significantly higher in SGBS adipocytes (Fig.聽3b). In order to assess whether upregulation of UCP1 gene and protein expression had functional significance in SGBS adipocytes, we measured mitochondrial DNA content and mitochondrial function in both adipocyte cell types. As shown in Fig.聽3c, metabolic signatures of SGBS were clearly different from PHWSC adipocytes. Basal respiration, maximal respiration, and spare respiratory capacity were significantly higher in SGBS when compared to PHWSC adipocytes (P鈥?lt;鈥?.05). At a glance (Fig.聽3c), the leak respiration, which represents uncoupling of oxidative phosphorylation, was significantly higher in SGBS adipocytes (P鈥?lt;鈥?.05). To test if the observation was UCP1-dependent, we measured OCR and leak respiration in adipocytes treated with isoproterenol (ISO) in the presence of increasing dose of fatty acid free (FAF)-BSA to sequester free fatty acids released during lipolysis (Fig.聽3d). ISO treatment led to a sharp rise in OCR and as expected, introduction of FAF-BSA diminished the effect of ISO in a dose dependent manner. ISO-induced leak respiration, which is considered to be a better representation of UCP1 activity was nearly two fold higher than basal leak respiration in absence of FAF-BSA. The presence of 0.5% FAF-BSA significantly reduced ISO-induced leak respiration when compared to no-BSA or 0.25% FAF-BSA conditions. There was significantly higher ratio of mitochondrial DNA to nuclear DNA content (P鈥?鈥?.0079) in SGBS adipocytes (FC鈥?鈥?.54鈥壜扁€?.73) when compared to PHWSC adipocytes (Fig.聽3e). When the mitochondrial respiration data was normalized to mitochondrial DNA content, the differences between SGBS and PHWSC adipocytes were no longer significant. Hence, the differences in mitochondrial respiration could be contributed by a combination of factors, including differences in mitochondrial DNA content, mitochondrial activity and the thermogenic activity in SGBS adipocytes, which are partially but not fully mediated by UCP1.Figure 3UCP1 protein expression and mitochondrial respiration in SGBS adipocytes display characteristics suggestive of browning capacity. (a) Top panel: UCP1 protein expression in differentiated SGBS and PHWSC adipocytes. Bottom panel: UCP1 protein expression in SGBS adipocytes differentiated in medium depleted with either T3 or rosiglitazone or both. (b) Protein expressions of adipogenic and lipid metabolism markers including PPAR纬, adiponectin, FITM2 and FABP4 in differentiated SGBS and PHWSC adipocytes. (c) Mitochondrial respiratory function or oxygen consumption rate (OCR) measured by Seahorse mitochondrial flux analyser. Bar graphs denote functional readouts of mitochondrial oxidative capacity. (d) Representative time course of OCR of ISO-treated SGBS adipocytes in the presence of either 0%, 0.25% or 0.5% FAF BSA, as well as UCP1-mediated basal leak respiration in SGBS adipocytes. (e) Relative mitochondrial DNA content in SGBS and PHWSC adipocytes. UCP1 protein blots were cropped for a more concise presentation and the respective full-length blots are available in supplementary Fig.聽S4. For the experiments involving PHWSC adipocytes, experiments were performed in duplicates from cells derived from 3 different donors. For SGBS adipocytes, experiments were performed in duplicates during 3 independent experiments from the same SGBS adipocyte. Data are expressed as means鈥壜扁€塖D. *P鈥?lt;鈥?.05.Full size imageHigher lipid accumulation and greater insulin stimulated glucose uptake in SGBS adipocytesIt is known that BAT activation plays a role in regulating glucose and lipid metabolism28. Intracellular lipid droplet accumulation was assessed using the hydrophilic stain Nile red (AdipoRed), which fluoresces in the presence of hydrophobic triglycerides. SGBS adipocytes were observed to have accumulated 4 times more triglycerides than PHWSC adipocytes at D12 post differentiation (P鈥?lt;鈥?.0001) (Fig.聽4a). Finally, we performed insulin-stimulated radioactive glucose uptake assay to assess any potential differences in insulin action between the two cell types. SGBS adipocytes showed significantly higher (P鈥?lt;鈥?.0079) relative glucose uptake (1.87鈥壜扁€?.2) in response to insulin stimulation compared to PHWSC adipocytes (1.17鈥壜扁€?.08) (Fig.聽4b). It is generally observed that mouse adipocytes, such as 3T3-L1 adipocytes, are more responsive to insulin stimulation, which would make it a more suitable model for studying glucose uptake29,30,31, when compared to both SGBS and PHWSC adipocytes.Figure 4SGBS adipocytes have higher lipid accumulation and insulin stimulated glucose uptake. (a) Total lipid accumulation in SGBS and PHWSC adipocytes. Lipid accumulation was quantified as florescence after incubation of adipocytes with Nile red dye. (b) Relative insulin-stimulated glucose uptake assay in SGBS versus PHWSC adipocytes. Glucose uptake was measured using radiolabelled de-oxy glucose. For the experiments involving PHWSC adipocytes, experiments were performed in duplicates from cells derived from 3 different donors. For SGBS adipocytes, experiments were performed in duplicates during 3 independent experiments from the same SGBS adipocyte. Data represented as Means鈥壜扁€塖D. **P鈥?lt;鈥?.01, ****P鈥?lt;鈥?.0001.Full size imageDiscussionThe present study demonstrated that mature SGBS adipocytes and primary white human subcutaneous adipocytes derived from obese, non-diabetic individuals display characteristics that are metabolically distinct. Of note, the SGBS is an overgrowth disorder and although not all causes of SGBS have been identified, one cause of SGBS is a mutation of the glypican-3 gene (GPC3) on the X chromosome locus q26.1. Moreover, GPC3 is known to regulate Hedgehog signalling during development of tissues, including mesenchyme and progenitors of adipose tissue. However, a GPC3 mutation was not found in the seminal paper that described the characteristics of the SGBS cell line1. The precise mutation in the SGBS adipocytes widely used as a human derived in vitro model for adipocyte development and metabolism has not yet been characterised. However, transcriptomic analysis and the clustering of differentially expressed genes revealed several biological pathways enriched in SGBS adipocytes compared to the PHWSC adipocytes. The two most significant paths relate to extracellular matrix (ECM) and metabolic pathways.ECM plays a critical role in maintaining the structural integrity and communication between adipocytes. ECM-receptor interactions are associated with pro-inflammatory changes and dysregulation of adipocyte metabolism; excessive accumulation of ECM is known to correlate positively with BMI levels32, 33 and obesity-related inflammation contributing to a adipose tissue dysfunction and reduced adipose tissue expandability21. Our results showed 32 genes involved in ECM-receptor interactions being significantly up-regulated in PHWSC adipocytes when compared to SGBS adipocytes. One confounding factor, however, could be the obese status of the subjects in this study. This could be potentially addressed by other studies that involve adipocytes derived from lean subjects in the future.The expression of genes involved in metabolic pathways and mitochondrial respiration differed greatly between SGBS and primary adipocytes derived from obese adult individuals. Differences in protein expression levels of adipogenic markers such as adiponectin and FITM2 provided further evidence that the overall adipocyte regulation were clearly distinct between the two cell types. Interestingly, previous studies have shown that adiponectin could modulate UCP1 expression in rodent BAT and reducing visceral fat mass, preventing adipocyte hypertrophy, accelerating overall energy expenditure34 and also enhance cold-induced browning of SC adipose tissue by promoting M2 macrophage polarization35, suggesting that adiponectin may have a role in regulating UCP1 expression in SGBS adipocytes. 220 genes implicated in metabolism were significantly upregulated and 94 genes were significantly downregulated in SGBS as compared to PHWSC. The two metabolic genes that differed the most in their expression were ALDH3B2 and CYP19A1. ALDH3B2 is involved in the removal of aldehydes generated under conditions of oxidative stress within lipid droplets36, and it was found to be ~350 fold higher in SGBS adipocytes. CYP19A1, also known as aromatase, is responsible for the aromatization of A ring of C19 steroids. Typically, it is expressed at very low levels and hardly detectable in fully differentiated, lipid-laden adipocytes37, 38. In agreement with this, our data showed its expression to be more than 300 fold lower in SGBS adipocytes when compared to PHWSC adipocytes. Even though McInnes et al. reported that the aromatase expression in SGBS mimics that of isolated primary human adipocytes14, the changes in expression levels were not compared in parallel with primary human adipocytes and the origin of primary human adipocytes used was unclear. Further studies will be needed to verify the differences in observations and whether there is any functional significance in the regulation of genes mentioned above.Our results showed that probably the most compelling difference between SGBS adipocytes and PHWSC lies in UCP1 mRNA and protein expression as well as its higher mitochondrial oxygen consumption profile. It is tempting to speculate that both UCP1-dependent and UCP1-independent leak respiration were involved in the energy metabolism of SGBS adipocytes. UCP1-independent leak respiration could be due to the inherently high mitochondrial content39, elevated oxygen consumption rate, alternatively increase fatty acid flux40, and even UCP1 being inserted incorrectly into the membrane, making it constantly leaky41. An alternative UCP1-independent futile thermogenic pathway, analogous to the futile creatine cycle described by Kazak et al.42, might also be involved in maintaining metabolic homeostasis in SGBS adipocytes. Future work to address these missing links would be warranted. The gene expression patterns in Table聽2 confirmed that the more consistent patterns of SGBS involved white adipose tissue lineage markers, supported by upregulation of genes typically expressed in WAT, yet capable of displaying its thermogenic properties at the same time.The fact that SGBS cells are derived from the white adipose tissue of a 3-month-old infant could also be a key contributing factor to the differences observed1. It is known that the function and composition of adipose tissue vary substantially between an early age and in adulthood. In infancy, it performs an active role in heat generation and principally comprises brown adipose tissue (BAT). By adulthood, it performs a more passive role in preventing heat loss and the composition shifts in favour of WAT. But even WAT from neonates is significantly different to that from adults. It is capable of responding to 尾-adrenergic stimulus and showed enhanced oxygen consumption rate when compared to adult adipocytes43. In addition, adipocytes derived from neonatal interscapular region expressed detectable levels of UCP1 mRNAs and showed molecular signatures of developmentally-programmed BAT (e.g. high expression of ZIC1)44. It would be interesting for future studies to fully characterise WAT from a healthy infant, and compare these characteristics to the SGBS cells.Given their high differentiation capacity up to 50 passages1, 20, SGBS cells remain as a valuable in-vitro test model for studies related to human adipocyte metabolism. However, given the differences observed, we would caution against solely using SGBS to model primary human adult subcutaneous adipocytes. Understanding similarities and differences between the different adipocyte models can assist in the selection of appropriate models in future studies that examine human fat cell development and metabolism.MethodsCell cultureAll subjects recruited for this study were patients from the National University Health System (NUHS, Singapore) who underwent bariatric surgery and had a body mass index (BMI) of鈥夆墺鈥?5鈥塳g/m2. All participants provided informed consent and ethics approval was obtained from the National Healthcare Group Domain Specific Review Board (reference number 2014/00396, Singapore). All procedures were carried out in accordance with the approved guidelines and regulations. Fresh adipose tissue specimens were obtained laparoscopically from the subcutaneous (SC) abdominal region during the surgery, without any additional surgical procedures. Primary human SC preadipocytes derived from 4 different individuals were prepared by collagenase digestion using a previously established protocol45. Once the cells displayed fibroblastic morphology, adipogenesis was initiated using commercial preadipocyte growth media and differentiation cocktail (Lonza, MD, USA). SC adipocytes were harvested on Day 12 and are henceforth referred to as the PHWSC adipocytes. Human SGBS adipocytes were kindly donated by Professor David Silver from DUKE-NUS, Singapore. SGBS cell growth and differentiation were performed using the standard protocol1, 20. The optimal media recommended for differentiating SGBS adipocytes differ from the media generally used for PHWSC adipocytes. To confirm that the differences observed between the two adipocytes types were not attributable to the composition of the respective growth and differentiation cocktail, Lonza media was used to differentiate SGBS adipocytes on a separate occasion. Characteristics for SGBS adipocytes were found to be consistent using either type of media (data not shown).RNA-Seq experiment and analysisTotal RNA was extracted from SGBS adipocytes (in duplicates) and PHWSC adipocytes (n鈥?鈥? derived from 3 females and 1 male with an average age of 39.5鈥壜扁€?5.9 years old) using Qiagen RNeasy plus kit (Qiagen Inc, CA, USA). Poly-A mRNA was then enriched with oligodT beads (Life Technologies) from approximately 5鈥壜礸 of total RNA. 100 ng of poly-A mRNA recovered was used to construct multiplexed strand-specific RNA-Seq libraries as per manufacturer鈥檚 instructions (NEXTflexTM Rapid Directional RNA-Seq Kit (dUTP-Based) v2). Individual library quality was assessed with an Agilent 2100 Bioanalyzer and quantified with a QuBit 2.0 fluorometer before pooling for sequencing on a HiSeq 2000 (1鈥壝椻€?01鈥塨p read). The pooled libraries were quantified using the KAPA quantification kit (KAPA Biosystems) prior to cluster formation. 20鈥?6 million reads were mapped in all samples with a rate of 95%. Fastq-formatted reads were processed with Trimmomatic46 to remove adapter sequences and trim low quality bases (LEADING: 3 TRAILING: 3 SLIDINGWINDOW: 4:15 MINLEN: 36). Reads were aligned to the human genome (hg19) using Tophat version 2 (settings鈥搉o-coverage-search鈥搇ibrary-type鈥?鈥塮r-firststrand). Feature read counts were generated using htseq-count (Python package HTSeq47 default union-counting mode, strand鈥?鈥塺everse). Differential Expression analysis was performed using the edgeR package in both 鈥榗lassic鈥?and generalized linear model (glm) modes to contrast the SGBS cell strain and primary human subcutaneous adipocytes (PHWSC adipocytes) derived from obese subjects. Genes with at least 1 count per million reads were included for edgeR analysis. Unsupervised clustering was performed using the heatmap.3 function from the GMD R package48. The raw htseq-counts were first normalized using variance stabilizing transformation from the DESeq2 package49 before cluster analysis. iPathwayGuide (Advaita bioinformatics) and DAVID Bioinformatics, web-based analysis tools were further employed to identify the top biological pathways and metabolic pathways respectively. KEGG pathway interaction map of metabolism related genes was built in Cytoscape version 3.3 using CluePedia and ClueGo plugin50, 51.Real-time PCR and mitochondrial DNA contentTotal RNA was isolated using Qiagen RNeasy plus kit and quantified using nano-drop 2000 spectrophotometer (Thermo Scientific, IL, USA). cDNA was synthesized from 800鈥塶g of RNA using ABI high capacity cDNA synthesis kit (Applied Biosystems, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) using Qiagen QuantiFast SYBR Green PCR Kit was employed to quantify relative gene expression in samples. Primers involved in various pathways of interest such as adipogenesis (PPAR纬, CEBP伪, Adiponectin, Leptin), lipid storage (FABP4, FITM2) and browning (UCP1, PGC1伪, DIO2) were prioritized in this study1, 13, 19, 20, 25, 52,53,54,55. NCBI primer blast and ABI primer express 3.0 were used to generate primer sequences (Supplementary Table聽S1).Differences in mitochondrial DNA content between SGBS and PHWSC adipocytes was determined by real-time PCR using TaqMan probes specific for mitochondrial DNA (mtDNA) and nuclear DNA (nDNA or 18鈥塻 rRNA) as previously described (Supplementary Table聽S1)56. Taqman probes were labelled at the 5鈥?end with fluorescent reporter FAM and the 3鈥?ends were labelled with a quencher TAMRA. mtDNA content was measured by the ratio of relative expressions of mtDNA to that of nDNA.Western blottingThe cellular lysate was obtained using RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors. Briefly, 30鈥壜礸 of protein from cell culture lysates was separated using 10% acrylamide gel, transferred to nitrocellulose membrane, blocked with 5% milk (0.1% Tween 20), and incubated overnight at 4鈥壜癈 separately with UCP1 (Thermo Scientific), PPAR纬 (Santa Cruz, TX, USA), adiponectin (Abcam, MA, USA), FITM2 (antibody made in-house, kindly provided by Dr. David Silver, DUKE-NUS, Singapore), FABP4 (Santa Cruz, generously given by Dr. Sun Lei, DUKE-NUS, Singapore), and GAPDH (Cell Signalling) antibodies. Following overnight incubation, blots were incubated with HRP-conjugated secondary antibody for 2鈥塰 and exposed to x-ray films using chemiluminescent detection method (EMD Millipore, MA, USA).Mitochondrial respiration analysisSeahorse XFe24 analyzer (Seahorse Biosciences, MA, USA) was used to measure mitochondrial respiration in adipocytes. SGBS and PHWSC adipocytes were seeded on XFe24 tissue culture plates at 25,000 cells/well and were differentiated as described above. On day 12, maintenance media was replaced by pH 7.4 XF assay medium (Seahorse Biosciences), containing 1鈥塵M pyruvate and 25鈥塵M glucose and adipocytes were kept for incubation at 37鈥壜癈 ambient CO2 free incubator for 45鈥塵in; The concentration of dissolved oxygen above the cell monolayer was measured by the XFe24 analyzer solid state sensor probe and the change in dissolved oxygen concentration is known as the oxygen consumption rate (OCR). Baseline OCR (OCR Baseline) was measured 3 times for 4鈥塵in each separated by a 2鈥塵in mix and a 2鈥塵in wait. Following the measurement of basal respiration, ATPase inhibitor oligomycin (2鈥壜礛) (Sigma鈥揂ldrich, Oakville, ON, Canada) was injected to measure proton leak (OCR oligomycin). A mitochondrial inner membrane uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (1.2鈥壜礛) (Sigma鈥揂ldrich), was subsequently introduced to drive maximal respiration (OCR FCCP). Finally, a mixture of rotenone (1鈥壩糓) and antimycin A (1鈥壩糓) (OCR R/A) was added to block respiratory chain complexes I and III and thus check for non-mitochondrial respiration. Parameters of mitochondrial bioenergetics were expressed as a percentage of the basal OCR. For further evaluation of UCP1-dependent leak respiration, cells were replaced with assay medium in the presence or absence of 0.25% or 0.5% BSA to sequester free fatty acids and block intracellular fatty acid-mediated proton leakage. 1鈥壜礛 isoproterenol (ISO) were added post-oligomycin injection to drive 尾-adrenergic stimulation of lipolysis39, 57. Oxygen consumption rates were normalized to protein concentration. ISO-induced leak respiration rates were deduced from means of 4 highest values and represented as fold increase of basal leak respiration.Lipid accumulation and radioactive glucose uptake assayDifferentiated adipocytes were washed with PBS and stained with AdipoRed (Lonza) according to the manufacturer鈥檚 protocol. After 30鈥塵in, fluorescence readings representing triglyceride accumulation were measured with excitation at 485鈥塶m and emission at 572鈥塶m. Insulin-stimulated glucose uptake assay was performed as previously described with some modifications58. Differentiated adipocytes were first incubated in serum-free media supplemented with 0.2% BSA for 6鈥塰 prior to the assay. Subsequently, they were washed to completely remove glucose and were incubated in PBS with 0.2% BSA for 30鈥塵in at 37鈥壜癈. Cells were subjected to insulin stimulation (100鈥塶M) for 15鈥塵in and the assay was initiated by the addition of [3H] 2-deoxyglucose (5鈥壜礐i/mL) and deoxyglucose (5鈥塵M). At the end of 20鈥塵in incubation, the activity was terminated by washing the cells three times with ice-cold PBS. Cells were then lysed using RIPA buffer and incubated on ice for 20鈥塵in. Finally, cell-associated radioactivity was determined using a liquid scintillation counter (Perkin Elmer).StatisticsFor the RNA-Seq studies, PHWSC adipocytes derived from 4 independent donors were being used (n鈥?鈥?); while for all other experiments, adipocytes from 3 independent donors were used (n鈥?鈥?), in duplicates. 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The authors thank Professor David Silver and Assistant Professor Sun Lei from DUKE-NUS medical school, Singapore for providing the FIT2 and FABP4 antibodies, respectively.聽A.V. is funded by Medical Research Council (MRC), the Metabolic Diseases Unit of the MRC, and the British Heart foundation.锘?/p>Author informationAuthor notesChia Rou Yeo and Madhur Agrawal contributed equally to this work.AffiliationsDepartment of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, 117599, Singapore, SingaporeChia Rou Yeo,聽Madhur Agrawal,聽Chin Meng Khoo,聽Vanna Chhay,聽M. Shabeer Yassin,聽E. Shyong Tai聽 聽Sue-Anne TohMolecular Engineering Laboratory, Biomedical Sciences Institutes, A*Star, 138668, Singapore, SingaporeShawn HoonDepartment of Surgery, National University Hospital, 119074, Singapore, SingaporeAsim ShabbirMetabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke鈥檚 Hospital, University of Cambridge, Cambridge, CB2 0QQ, UKManu Kunaal Shrivastava聽 聽Antonio Vidal-PuigFood Science and Technology Program, Department of Chemistry, National University of Singapore, Singapore, 117542, SingaporeShiqi HuangDepartment of Medicine, National University Health System, 119228, Singapore, SingaporeChin Meng Khoo,聽E. Shyong Tai聽 聽Sue-Anne TohWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UKAntonio Vidal-PuigAuthorsChia Rou YeoView author publicationsYou can also search for this author in PubMed聽Google ScholarMadhur AgrawalView author publicationsYou can also search for this author in PubMed聽Google ScholarShawn HoonView author publicationsYou can also search for this author in PubMed聽Google ScholarAsim ShabbirView author publicationsYou can also search for this author in PubMed聽Google ScholarManu Kunaal ShrivastavaView author publicationsYou can also search for this author in PubMed聽Google ScholarShiqi HuangView author publicationsYou can also search for this author in PubMed聽Google ScholarChin Meng KhooView author publicationsYou can also search for this author in PubMed聽Google ScholarVanna ChhayView author publicationsYou can also search for this author in PubMed聽Google ScholarM. Shabeer YassinView author publicationsYou can also search for this author in PubMed聽Google ScholarE. Shyong TaiView author publicationsYou can also search for this author in PubMed聽Google ScholarAntonio Vidal-PuigView author publicationsYou can also search for this author in PubMed聽Google ScholarSue-Anne TohView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsC.R.Y. and M.A. conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing and revising; S.H. RNA-seq data analysis and interpretation, manuscript writing; A.S. subject recruitment for bariatric surgery and adipose tissue obtainment; M.K.S. collection and assembly of data, manuscript revising; C.M.K. data analysis and interpretation; S.H., V.C. and M.S.Y. collection and assembly of data; E.S.T. and A.V. data analysis and manuscript revising; S.A.T. financial support, conception and design, data analysis and interpretation, manuscript writing and revising, final approval of manuscript. All authors reviewed the manuscript.Corresponding authorCorrespondence to Sue-Anne Toh.Ethics declarations Competing Interests The authors declare that they have no competing interests. Additional informationPublisher\'s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Electronic supplementary material Supplementary informationRights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article鈥檚 Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article鈥檚 Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articleYeo, C.R., Agrawal, M., Hoon, S. et al. SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes. Sci Rep 7, 4031 (2017). https://doi.org/10.1038/s41598-017-04369-2Download citationReceived: 01 December 2016Accepted: 19 May 2017Published: 22 June 2017DOI: https://doi.org/10.1038/s41598-017-04369-2 Giulia Tini, Vijayalakshmi Varma, Rosario Lombardo, Greg T. Nolen, Gregory Lefebvre, Patrick Descombes, Sylviane M茅tairon, Corrado Priami, Jim Kaput Marie-Pier Scott-Boyer Genes Nutrition (2020) Claudia G. C谩rdenas-Le贸n, Ang茅lica Montoya-Contreras, Kristina M盲emets-Allas, Viljar Jaks Luis A. Salazar-Olivo In Vitro Cellular Developmental Biology - Animal (2020) 脕gnes Klus贸czki, Zolt谩n Ver茅b, Attila V谩mos, Pamela Fischer-Posovszky, Martin Wabitsch, Zsolt Bacso, L谩szl贸 F茅s眉s Endre Krist贸f Scientific Reports (2019) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Reply to: 鈥楤rowning capabilities of human primary adipose-derived stromal cells compared to SGBS cells鈥?/a> Chia Rou YeoMadhur AgrawalSue-Anne Toh Matters Arising Open Access 15 Jun 2020 Advertisement Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.

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