Simpson-Golabi-Behmel Syndrome Cells Research Articles

Endocrine disruption of adipose physiology: Screening in SGBS cells.

The increasing use of industrial chemicals has raised concerns regarding exposure to endocrine-disrupting chemicals (EDCs), which interfere with developmental, reproductive and metabolic processes. Of particular concern is their interaction with adipose tissue, a vital component of the endocrine system regulating metabolic and hormonal functions. The SGBS (Simpson Golabi Behmel Syndrome) cell line, a well-established human-relevant model for adipocyte research, closely mimics native adipocytes' properties. It responds to hormonal stimuli, undergoes adipogenesis and has been successfully used to study the impact of EDCs on adipose biology. In this study, we screened human exposure-relevant doses of various EDCs on the SGBS cell line to investigate their effects on viability, lipid accumulation and adipogenesis-related protein expression. Submicromolar doses were generally well tolerated; however, at higher doses, EDCs compromised cell viability, with cadmium chloride (CdCl2) showing the most pronounced effects. Intracellular lipid levels remained unaffected by EDCs, except for tributyltin (TBT), used as a positive control, which induced a significant increase. Analysis of adipogenesis-related protein expression revealed several effects, including downregulation of fatty acid-binding protein 4 (FABP4) by dibutyl phthalate, upregulation by CdCl2 and downregulation of perilipin 1 and FABP4 by perfluorooctanoic acid. Additionally, TBT induced dose-dependent upregulation of C/EBPα, perilipin 1 and FABP4 protein expression. These findings underscore the importance of employing appropriate models to study EDC-adipocyte interactions. Conclusions from this research could guide strategies to reduce the negative impacts of EDC exposure on adipose tissue.

Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity

BackgroundObesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity’s impact on EV secretion from human AT remains limited.MethodsWe investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography–mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry.ResultsEVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation.ConclusionsWe are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

Single-cell transcriptome dataset of human and mouse in vitro adipogenesis models

Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on these widely used in vitro models of human and mouse adipogenesis, and on cell-to-cell variability during this process.

Effects of allicin on human Simpson-Golabi-Behmel syndrome cells in mediating browning phenotype.

Obesity is a major health problem because it is associated with increased risk of cardiovascular disease, diabetes, hypertension, and some cancers. Strategies to prevent or reduce obesity focus mainly on the possible effects of natural compounds that can induce a phenotype of browning adipocytes capable of releasing energy in the form of heat. Allicin, a bioactive component of garlic with numerous pharmacological functions, is known to stimulate energy metabolism. In the present study, the effects of allicin on human Simpson-Golabi-Behmel Syndrome (SGBS) cells were investigated by quantifying the dynamics of lipid droplets (LDs) and mitochondria, as well as transcriptomic changes after six days of differentiation. Allicin significantly promoted the reduction in the surface area and size of LDs, leading to the formation of multilocular adipocytes, which was confirmed by the upregulation of genes related to lipolysis. The increase in the number and decrease in the mean aspect ratio of mitochondria in allicin-treated cells indicate a shift in mitochondrial dynamics toward fission. The structural results are confirmed by transcriptomic analysis showing a significant arrangement of gene expression associated with beige adipocytes, in particular increased expression of T-box transcription factor 1 (TBX1), uncoupling protein 1 (UCP1), PPARG coactivator 1 alpha (PPARGC1A), peroxisome proliferator-activated receptor alpha (PPARA), and OXPHOS-related genes. The most promising targets are nuclear genes such as retinoid X receptor alpha (RXRA), retinoid X receptor gamma (RXRG), nuclear receptor subfamily 1 group H member 3 (NR1H3), nuclear receptor subfamily 1 group H member 4 (NR1H4), PPARA, and oestrogen receptor 1 (ESR1). Transcriptomic data and the network pharmacology-based approach revealed that genes and potential targets of allicin are involved in ligand-activated transcription factor activity, intracellular receptor signalling, regulation of cold-induced thermogenesis, and positive regulation of lipid metabolism. The present study highlights the potential role of allicin in triggering browning in human SGBS cells by affecting the LD dynamics, mitochondrial morphology, and expression of brown marker genes. Understanding the potential targets through which allicin promotes this effect may reveal the underlying signalling pathways and support these findings.

Transcriptomic analysis of Simpson Golabi Behmel syndrome cells during differentiation exhibit BAT-like function

High-throughput RNA sequencing of human Simpson-Golabi-Behmel syndrome cells (SGBS) was performed during the time-course of adipogenic differentiation at day 4 (D04), 6 (D06), 8 (D08), and 10 (D10) to characterize transcriptomic changes and to identify key patterns involved in adipogenesis and browning. In the comparisons, 932 and 384 overlapping transcripts were consistently up- and down-regulated, respectively. Combining the results of protein-protein interaction network analysis MCODE and CytoHubba, 55 up-regulated hub genes from four clusters and 9 down-regulated genes were identified. The up-regulated hub genes were mainly enriched in brown adipocyte differentiation, extracellular matrix organization, and valine, leucine, and isoleucine degradation. The enrichment of downregulated hub genes was related to NRF2 signalling and glutathione metabolism, indicating that oxidative stress also plays a role. Analysis of overlapping down-regulated genes, targets of transcription factors, revealed enrichment in the IL-18 signalling pathway, which is involved in browning process and extracellular matrix organization via actomyosin mechanics and integrin-extracellular matrix interactions. Finally, the comparison transcriptomic analysis with the gene signature reported by BATLAS and PROFAT web-based tools showed an increased percentage of the brown phenotype, confirming that differentiated SGBS cells at D06, D08, and D10 gradually acquire BAT-like function.

Insulin Signaling Induces AKR1C3 to Promote Lipid Overload through FASN in SGBS Cells, as a Model of PCOS Adipocytes

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age, affecting 8‐13% of women. The characteristic androgen excess (AE) of PCOS is believed to promote comorbidities, such as obesity and type 2 diabetes. AE correlates with insulin resistance (IR) in these women and may be the driving factor for lipid overload. Aldo‐keto reductase family 1 member C3 (AKR1C3) is induced by insulin in PCOS adipocytes and may be central in driving the AE of PCOS. AKR1C3 predominantly catalyzes formation of androgens such as testosterone and 5α‐dihydrotestosterone in peripheral tissues, which are potent agonists for the androgen receptor (AR) and upregulate de novo lipogenesis. Fatty acid synthase (FASN) is induced by androgens and may be responsible for an altered lipid profile in mature adipocytes. Nuclear factor erythroid 2‐related factor 2 (NRF2) is a known transcription factor for AKR1C3 and is regulated by insulin. AKR1C3 overexpression in adipocytes by insulin activation of NRF2 could increase androgen receptor signaling and promote de novo lipogenesis. The resulting lipid overflow could exacerbate insulin resistance through a feedforward mechanism and potentiate the cardio‐metabolic risk seen in PCOS. We hypothesize that insulin signaling induces AKR1C3 to promote lipid overload through FASN in PCOS adipocytes. Using differentiated human Simpson‐Golabi‐Behmel Syndrome (SGBS) adipocytes as a model for PCOS adipocytes, we have confirmed that AKR1C3 is induced >600 fold by insulin. Using pharmacological inhibition, we found that the insulin induction of AKR1C3 and FASN is dependent on PI3K/AKT/mTOR measured by RT‐qPCR and immunoblot. Using NRF2 siRNA and ML‐385, a NRF2 inhibitor, we have determined that AKR1C3 and FASN induction are NRF2 dependent, indicating NRF2 may be one transcription factor that binds to the AKR1C3 promotor region in adipocytes. We also determined that FASN induction is both AKR1C3 and AR dependent using pharmacological inhibition/antagonism and siRNA, where its effect was measured by RT‐qPCR and immunoblot. Pharmacological inhibition with NRF2 or AKR1C3 inhibitors additionally downregulated de novo lipogenesis measured by Oil Red O staining, which is a phenotypic readout for FASN activity. These studies elucidate the signaling mechanism by which AKR1C3 and FASN are induced by insulin in adipocytes. We conclude that upregulation of AKR1C3 may promote lipid overload in PCOS adipocytes by induction of FASN, and that AKR1C3 is a possible therapeutic target to prevent the risk of cardio‐metabolic disease in PCOS women.

MicroRNA-27a-3p but Not -5p Is a Crucial Mediator of Human Adipogenesis.

MicroRNAs (miRNAs), a class of small, non-coding RNA molecules, play an important role in the posttranscriptional regulation of gene expression, thereby influencing important cellular functions. In adipocytes, miRNAs show import regulatory features and are described to influence differentiation as well as metabolic, endocrine, and inflammatory functions. We previously identified miR-27a being upregulated under inflammatory conditions in human adipocytes and aimed to elucidate its function in adipocyte biology. Both strands of miR-27a, miR-27a-3p and -5p, were downregulated during the adipogenic differentiation of Simpson–Golabi–Behmel syndrome (SGBS) cells, human multipotent adipose-derived stem cells (hMADS), and human primary adipose-derived stromal cells (hASCs). Using miRNA-mimic transfection, we observed that miR-27a-3p is a crucial regulator of adipogenesis, while miR-27a-5p did not alter the differentiation capacity in SGBS cells. In silico screening predicted lipoprotein lipase (LPL) and peroxisome proliferator activated receptor γ (PPARγ) as potential targets of miR-27a-3p. The downregulation of both genes was verified in vitro, and the interaction of miR-27-3p with target sites in the 3′ UTRs of both genes was confirmed via a miRNA-reporter-gene assay. Here, the knockdown of LPL did not interfere with adipogenic differentiation, while PPARγ knockdown decreased adipogenesis significantly, suggesting that miR-27-3p exerts its inhibitory effect on adipogenesis by repressing PPARγ. Taken together, we identified and validated a crucial role for miR-27a-3p in human adipogenesis played by targeting the essential adipogenic transcription factor PPARγ. Though we confirmed LPL as an additional target of miR-27a-3p, it does not appear to be involved in regulating human adipogenesis. Thereby, our findings call the conclusions drawn from previous studies, which identified LPL as a crucial regulator for murine and human adipogenesis, into question.

Elucidating the Role of Insulin‐induced AKR1C3 and the 11‐oxygenated Androgens in Promoting the Androgen Excess of Polycystic Ovary Syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age. The androgen excess (AE) of PCOS is believed to promote cardio-metabolic risk, but how this occurs is unknown. As a result, treatment of cardio-metabolic diseases in PCOS women is not entirely effective. AE correlates with insulin resistance (IR) in these women and may be driving lipotoxicity. Aldo-keto reductase family 1 member C3 (AKR1C3) is induced by insulin and may be central in driving the AE of PCOS. Our goal is to understand the role of insulin-induced AKR1C3 in promoting AE and lipotoxicity in PCOS adipocytes. AKR1C3 predominantly catalyzes the conversion of peripheral androgens such Δ4-androstene-3,17-dione (4AD) to testosterone (T) and 5α-androstan-3,17-dione (5AD) to 5α-dihydrotestosterone (DHT). T and DHT are potent agonists for the androgen receptor (AR), which upregulate de novo lipogenesis. Fatty acid synthase (FASN) is known to be induced by androgens and may be responsible for this altered lipid profile in mature adipocytes. AKR1C3 overexpression in adipocytes, in response to insulin, could increase AR signaling and increase de novo lipogenesis. This lipid overflow could exacerbate insulin resistance through a feedforward mechanism and promote the lipotoxic profile seen in PCOS. Additionally, the adrenal 11-oxygenated androgens may be an alternative source of AE and have been reported as the dominant androgens of PCOS. We hypothesize that insulin induction of AKR1C3 and the conversion of 11-oxygenated androgens into potent AR agonists in adipocytes may potentiate AE and lipotoxicity in PCOS. Using differentiated Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes as a model, we have shown that insulin increased AKR1C3 expression by >600 fold at the RNA level. Using pharmacological inhibition, we have preliminary data that shows insulin induction of AKR1C3 to utilize the PI3K/AKT/mTOR pathway as measured by RT-qPCR and immunoblot assays. Using a reverse-phase-HPLC discontinuous assay and post-reaction derivatization with dinitrophenylhydrazine, we determined the kcat and Km for the conversion of 11-ketoandrostene-3,17-dione (11K-4AD) to 11-ketotestosterone (11K-T) and 11-ketoandrostan-3,17-dione (11K-5AD) to 11-ketodihydrotestosterone (11K-DHT) using recombinant AKR1C3. We find that 11K-4AD and 11K-5AD are superior substrates for AKR1C3 when compared to 4AD and 5AD. Furthermore, we have quantified the formation of 11K-T in SGBS cells by stable-isotope-dilution (SID)-LC-MS/MS. This research will elucidate the mechanism of how AKR1C3 potentiates androgen excess and AR signaling to promote lipotoxicity in PCOS. Additionally, this work supports AKR1C3 as a potential therapeutic target in mitigating the androgen excess of PCOS.

In Depth Quantitative Proteomic and Transcriptomic Characterization of Human Adipocyte Differentiation using the SGBS Cell Line.

Most information on molecular processes accompanying and driving adipocyte differentiation are derived from rodent models. Here, a comprehensive analysis of combined transcriptomic and proteomic alterations during adipocyte differentiation in Simpson-Golabi-Behmel Syndrome (SGBS) cells is provided. The SGBS cells are a well-established and the most widely applied cell model to study human adipocyte differentiation and cell biology. However, the molecular alterations during human adipocyte differentiation in SGBS cells have not yet been described in a combined analysis of proteome and transcriptome. Here a global proteomic and transcriptomic data set comprising relative quantification of a total of 14372 mRNA transcripts and 2641 intracellular and secreted proteins is presented. 1153 proteins and 313 genes are determined as differentially expressed between preadipocytes and the fully differentiated cells including adiponectin, lipoprotein lipase, fatty acid binding protein 4, fatty acid synthase, stearoyl-CoA desaturase, and apolipoprotein E and many other proteins from the fatty acid synthesis, amino acid synthesis as well as glucose and lipid metabolic pathways. Preadipocyte markers, such as latexin, GATA6, and CXCL6, are found to be significantly downregulated at the protein and transcript level. This multi-omics data set provides a deep molecular profile of adipogenesis and will support future studies to understand adipocyte function.

A new human adipocyte model with PTEN haploinsufficiency

ABSTRACT Few human cell strains are suitable and readily available as in vitro adipocyte models. We used resected lipoma tissue from a patient with germline phosphatase and tensin homolog (PTEN) haploinsufficiency to establish a preadipocyte cell strain termed LipPD1 and aimed to characterize cellular functions and signalling pathway alterations in comparison to the established adipocyte model Simpson-Golabi-Behmel-Syndrome (SGBS) and to primary stromal-vascular fraction cells. We found that both cellular life span and the capacity for adipocyte differentiation as well as adipocyte-specific functions were preserved in LipPD1 and comparable to SGBS adipocytes. Basal and growth factor-stimulated activation of the PI3 K/AKT signalling pathway was increased in LipPD1 preadipocytes, corresponding to reduced PTEN levels in comparison to SGBS cells. Altogether, LipPD1 cells are a novel primary cell model with a defined genetic lesion suitable for the study of adipocyte biology.

Elevated UCP1 levels are sufficient to improve glucose uptake in human white adipocytes

Brown adipose tissue (BAT) has been considered beneficial for metabolic health by participating in the regulation of glucose homoeostasis. The browning factors that improve glucose uptake beyond normal levels are still unknown but glucose uptake is not affected in UCP1 knockout mice. Here, we demonstrate in human white adipocytes that basal/resting glucose uptake is improved by solely elevating UCP1 protein levels. Generating human white Simpson-Golabi-Behmel syndrome (SGBS) adipocytes with a stable knockout and overexpression of UCP1, we discovered that UCP1 overexpressing adipocytes significantly improve glucose uptake by 40%. Mechanistically, this is caused by higher glycolytic flux, seen as increased oxygen consumption, extracellular acidification and lactate secretion rates. The improvements in glucose handling are comparable to white-to-brown transitions, as judged by, for the first time, directly comparing in vitro differentiated mouse brown vs white adipocytes. Although no adipogenic, metabolic and mitochondrial gene expressions were significantly altered in SGBS cells, pharmacological inhibition of GLUT1 completely abrogated differences between UCP1+ and control cells, thereby uncovering GLUT1-mediated uptake as permissive gatekeeper. Collectively, our data demonstrate that elevating UCP1 levels is sufficient to improve human white adipocytes as a glucose sink without adverse cellular effects, thus not requiring the adrenergic controlled, complex network of browning which usually hampers translational efforts.

Differentiating SGBS adipocytes respond to PPAR\u03b3 stimulation, irisin and BMP7 by functional browning and beige characteristics

Brown and beige adipocytes are enriched in mitochondria with uncoupling protein-1 (UCP1) to generate heat instead of ATP contributing to healthy energy balance. There are few human cellular models to reveal regulatory networks in adipocyte browning and key targets for enhancing thermogenesis in obesity. The Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte line has been a useful tool to study human adipocyte biology. Here we report that SGBS cells, which are comparable to subcutaneous adipose-derived stem cells, carry an FTO risk allele. Upon sustained PPARγ stimulation or irisin (a myokine released in response to exercise) treatment, SGBS cells differentiated into beige adipocytes exhibiting multilocular lipid droplets, high UCP1 content with induction of typical browning genes (Cidea, Elovl3) and the beige marker Tbx1. The autocrine mediator BMP7 led to moderate browning with the upregulation of the classical brown marker Zic1 instead of Tbx1. Thermogenesis potential resulted from PPARγ stimulation, irisin and BMP7 can be activated in UCP1-dependent and the beige specific, creatine phosphate cycle mediated way. The beige phenotype, maintained under long-term (28 days) conditions, was partially reversed by withdrawal of PPARγ ligand. Thus, SGBS cells can serve as a cellular model for both white and sustainable beige adipocyte differentiation and function.

SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes

The 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-à-vis primary human subcutaneous adipocytes as a human white adipocyte model, guiding the selection of appropriate cell models in future metabolic research.

Collagen beta (1-O) galactosyltransferase 1 (GLT25D1) is required for the secretion of high molecular weight adiponectin and affects lipid accumulation.

Secretion of high molecular weight (HMW) adiponectin is dependent on post-translational modification (PTM) of conserved lysines in the collagenous domain. The present study aims to characterize the enzymes responsible for the PTM of conserved lysines which leads to HMW adiponectin secretion, and to define its significance in relation to obesity. Collagen beta (1-O) galactosyltransferase 1 (GLT25D1) was knocked down in HEK cells modified for the stable expression of adiponectin (adiponectin expressing human embryonic kidney cells, Adipo-HEK) as well as in Simpson Golabi-Behmel-Syndrome (SGBS) adipocytes. Knockdown of GLT25D1 caused a significant decrease in HMW adiponectin in Adipo-HEK cells with no change in total adiponectin. Knockdown in the SGBS cells caused an increase in lipid accumulation yet inhibited adipogenesis. Co-immunoprecipitation with adiponectin and mass spectrometry showed that adiponectin formed a protein complex with lysyl hydroxylase 3 (LH3) and GLT25D1. Transient overexpression of GLT25D1 showed that the intracellular retention of LH3 was dependent on GLT25D1. To determine whether changes in GLT25D1 were significant in obesity, mice were fed a standard chow or high-fat diet (HFD) for 5 weeks. GLT25D1 was significantly decreased in mice fed HFD which coincided with a decrease in HMW adiponectin. We conclude that GLT25D1 regulates HMW adiponectin secretion and lipid accumulation, consistent with changes in mice after high-fat feeding. These results suggest a novel function of GLT25D1 leading to decreased HMW adiponectin secretion in early obesity.

LipiD-QuanT: a novel method to quantify lipid accumulation in live cells

Lipid droplets (LDs) are the main storage organelles for triglycerides. Elucidation of lipid accumulation mechanisms and metabolism are essential to understand obesity and associated diseases. Adipogenesis has been well studied in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell lines. However, most techniques for measuring LD accumulation are either not quantitative or can be destructive to samples. Here, we describe a novel, label-free LD quantification technique (LipiD-QuanT) to monitor lipid dynamics based on automated image analysis of phase contrast microscopy images acquired during in vitro human adipogenesis. We have applied LipiD-QuanT to measure LD accumulation during differentiation of SGBS cells. We demonstrate that LipiD-QuanT is a robust, nondestructive, time- and cost-effective method compared with other triglyceride accumulation assays based on enzymatic digest or lipophilic staining. Further, we applied LipiD-QuanT to measure the effect of four potential pro- or antiobesogenic substances: DHA, rosiglitazone, elevated levels of D-glucose, and zinc oxide nanoparticles. Our results revealed that 2 µmol/l rosiglitazone treatment during adipogenesis reduced lipid production and caused a negative shift in LD diameter size distribution, but the other treatments showed no effect under the conditions used here.

Fibroblast growth factor-1 inhibits Wnt/β-catenin pathway during adipogenesis

To determine the time course and potential mechanism of fibroblast growth factor-1 (FGF-1) in the regulation of adipogenesis. We cultured human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes with recombinant FGF-1 and harvested cells at various stages prior to and during differentiation; at cell proliferation (D-3), confluence (D0), early (D3), middle (D7) and mature (D14) stages of differentiation. We determined lipid accumulation in mature adipocytes by morphological observation and quantitative measurement of oil red O staining. We also examined the expression of adipogenic genes and related markers involved in the Wnt/β-catenin pathway using quantitative Real-time PCR and Western blot. Compared to control SGBS cells, treatment with FGF-1 increased lipid accumulation; induced a sustained increase in the mRNA for peroxisome proliferater-activated receptor γ (PPARγ), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), adiponectin and glucose transporter type 4 (GLUT4); and promoted a sustained decrease in expression of markers of the Wnt/β-catenin pathway, β-catenin and transcription factor 4 (TCF4). The adipogenic effects of FGF-1 are apparent throughout the whole priming and differentiation period in human SGBS pre-adipocytes. Furthermore, our results suggest that FGF-1 promotes adipogenesis, at least in part, via a sustained decrease in activity of the Wnt/β-catenin pathway.

Angiotensin II directly impairs adipogenic differentiation of human preadipose cells.

Angiotensin II reduces adipogenic differentiation of preadipose cells present in the stroma-vascular fraction of human adipose tissue, which also includes several cell types. Because of the ability of non-adipose lineage cells in the stroma-vascular fraction to respond to angiotensin II, it is not possible to unequivocally ascribe the anti-adipogenic response to a direct effect of this hormone on preadipose cells. Therefore, we used the human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell strain to investigate the consequences of angiotensin II treatment on adipogenic differentiation under serum-free conditions, by assessing expression of typical adipocyte markers perilipin and fatty acid-binding protein 4 (FABP4), at the transcript and protein level. Reverse transcription-polymerase chain reaction showed that perilipin and FABP4 transcripts were, respectively, reduced to 0.33±0.07 (P<0.05) and 0.41±0.19-fold (P<0.05) in SGBS cells induced to adipogenic differentiation in the presence of angiotensin II. Western Blot analysis corroborated reduction of the corresponding proteins to 0.23±0.21 (P<0.01) and 0.46±0.30-fold (P<0.01) the respective controls without angiotensin II. Angiotensin II also impaired morphological changes associated with early adipogenesis. Hence, we demonstrated that angiotensin II is able to directly reduce adipogenic differentiation of SGBS preadipose cells.

MFAP5is related to obesity-associated adipose tissue and extracellular matrix remodeling and inflammation

Low-grade inflammation is involved in adipose tissue (AT) and extracellular matrix (ECM) remodeling and induces deposition of ECM proteins in AT. We have previously shown that MFAP5 (microfibrillar-associated protein 5) expression decreases in AT after weight loss. The aim of this study was to investigate MFAP5 localization in human AT and gene expression in adipocytes and the role of MFAP5 in adipocyte metabolism and inflammation. MFAP5 protein localization and gene expression were studied with immunohistochemistry and quantitative reverse transcriptase PCR (RT-qPCR) in human subcutaneous AT and cultured Simpson-Golabi-Behmel syndrome (SGBS) adipocytes, respectively. The effect of MFAP5 knock-down by siRNA on gene expression and insulin action was examined with RT-qPCR, western blot, and insulin-stimulated glucose uptake. The effect of different cytokines on MFAP5 gene and protein expression was investigated in cultured human SGBS preadipocytes. MFAP5 protein was highly expressed in AT, and gene expression decreased during adipocyte differentiation in SGBS cells. Treatment of preadipocytes with TNFα and TGFβ1 increased MFAP5 gene and protein expression. Furthermore, MFAP5 knock-down decreased the expression of genes involved in inflammation. Our results demonstrate that factors involving low-grade inflammation modulate MFAP5 expression and that the modified expression of MFAP5 may further regulate AT inflammation.

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.

Abstract 5303: Uptake and Efflux of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) in human adipocytes

Abstract Obesity has been linked to the development of numerous chronic diseases, including breast cancer. Although the association between obesity and breast cancer risk is clear in postmenopausal women, high body mass index (BMI) appears to protect against breast cancer in pre-menopausal women. The mechanistic basis of this “obesity paradox” is unknown. One hypothesis is that abundant adipose tissue in the mammary gland functions as a storage depot for lipid-soluble carcinogens of dietary origin, initially offering protection against exposures. Heterocyclic amines (HCAs) are pro-carcinogens that are formed from high-temperature cooked meats. Exposures to these compounds are ubiquitous in Western diets and 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP), the most mass-abundant HCA, is classified as a “probable human carcinogen.” To determine rates of influx of PhIP into adipocytes, differentiated Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes were treated with 5 µM plus 0.02 µCi 14C-PhIP for 0.5, 1, 2, 5, 10, 15, 30, 45, and 60 minutes, and media was removed at time points. Cells were re-suspended in scintillation fluid, and counted with a 1450 LSL &amp; Luminescence Count (Perkin Elmer). Influx was rapid and PhIP continued to accumulate in the cells between 15 and 60 minutes. To determine efflux, SGBS cells were exposed to 5 µM plus 0.02 µCi 14C-PhIP and allowed to equilibrate for 30 minutes. Media was removed and the cells were washed with PBS. Radioactivity retained in SGBS cells was counted at 0.5, 1, 5, 10, 30, and 60 minutes. SGBS cells retained 50% of the radioactivity for more than 10 minutes, compared to other studies where PhIP efflux from pancreatic acinii and hepatocytes occurs within the first 15 seconds. Our data demonstrates that human adipocyte efflux is significantly slower than those in other type of cells reported in the literature, suggesting that human adipocytes can sequester PhIP, thereby protecting the breast from dietary exposure to carcinogens. Citation Format: Aiwei Yao-Borengasser, Lora J. Rogers, Susan A. Kadlubar. Uptake and Efflux of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) in human adipocytes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5303. doi:10.1158/1538-7445.AM2014-5303