Genome-wide association study of diabetogenic adipose morphology in the GENetics of Adipocyte Lipolysis (GENiAL) Cohort

An increased adipocyte size relative to the size of fat depots, also denoted hypertrophic adipose morphology, is a strong risk factor for the future development of insulin resistance and type 2 diabetes. The regulation of adipose morphology is poorly understood. We set out to identify genetic loci associated with adipose morphology and functionally evaluate candidate genes for impact on adipocyte development. We performed a genome-wide association study (GWAS) in the unique GENetics of Adipocyte Lipolysis (GENiAL) cohort comprising 948 participants who have undergone abdominal subcutaneous adipose biopsy with a determination of average adipose volume and morphology. The GWAS identified 31 genetic loci displaying suggestive association with adipose morphology. Functional evaluation of candidate genes by small interfering RNAs (siRNA)-mediated knockdown in adipose-derived precursor cells identified six genes controlling adipocyte renewal and differentiation, and thus of potential importance for adipose hypertrophy. In conclusion, genetic and functional studies implicate a regulatory role for ATL2, ARHGEF10, CYP1B1, TMEM200A, C17orf51, and L3MBTL3 in adipose morphology by their impact on adipogenesis

Genome-wide association study of adipocyte lipolysis in the GENetics of adipocyte lipolysis (GENiAL) cohort.

OBJECTIVES:Lipolysis, hydrolysis of triglycerides to fatty acids in adipocytes, is tightly regulated, poorly understood, and, if perturbed, can lead to metabolic diseases including obesity and type 2 diabetes. The goal of this study was to identify the genetic regulators of lipolysis and elucidate their molecular mechanisms. METHODS:Adipocytes from abdominal subcutaneous adipose tissue biopsies were isolated and were incubated without (spontaneous lipolysis) or with a catecholamine (stimulated lipolysis) to analyze lipolysis. DNA was extracted and genome-wide genotyping and imputation conducted. After quality control, 939 samples with genetic and lipolysis data were available. Genome-wide association studies of spontaneous and stimulated lipolysis were conducted. Subsequent in vitro gene expression analyses were used to identify candidate genes and explore their regulation of adipose tissue biology. RESULTS:One locus on chromosome 19 demonstrated genome-wide significance with spontaneous lipolysis. 60 loci showed suggestive associations with spontaneous or stimulated lipolysis, of which many influenced both traits. In the chromosome 19 locus, only HIF3A was expressed in the adipocytes and displayed genotype-dependent gene expression. HIF3A knockdown in vitro increased lipolysis and the expression of key lipolysis-regulating genes. CONCLUSIONS:In conclusion, we identified a genetic regulator of spontaneous lipolysis and provided evidence of HIF3A as a novel key regulator of lipolysis in subcutaneous adipocytes as the mechanism through which the locus influences adipose tissue biology

Global transcriptome profiling identifies KLF15 and SLC25A10 as modifiers of adipocytes insulin sensitivity in obese women.

Although the mechanisms linking obesity to insulin resistance (IR) and type 2 diabetes (T2D) are not entirely understood, it is likely that alterations of adipose tissue function are involved. The aim of this study was to identify new genes controlling insulin sensitivity in adipocytes from obese women with either insulin resistant (OIR) or sensitive (OIS) adipocytes. Insulin sensitivity was first determined by measuring lipogenesis in isolated adipocytes from abdominal subcutaneous white adipose tissue (WAT) in a large observational study. Lipogenesis was measured under conditions where glucose transport was the rate limiting step and reflects in vivo insulin sensitivity. We then performed microarray-based transcriptome profiling on subcutaneous WAT specimen from a subgroup of 9 lean, 21 OIS and 18 obese OIR women. We could identify 432 genes that were differentially expressed between the OIR and OIS group (FDR ≤5%). These genes are enriched in pathways related to glucose and amino acid metabolism, cellular respiration, and insulin signaling, and include genes such as SLC2A4, AKT2, as well as genes coding for enzymes in the mitochondria respiratory chain. Two IR-associated genes, KLF15 encoding a transcription factor and SLC25A10 encoding a dicarboxylate carrier, were selected for functional evaluation in adipocytes differentiated in vitro. Knockdown of KLF15 and SLC25A10 using siRNA inhibited insulin-stimulated lipogenesis in adipocytes. Transcriptome profiling of siRNA-treated cells suggested that KLF15 might control insulin sensitivity by influencing expression of PPARG, PXMP2, AQP7, LPL and genes in the mitochondrial respiratory chain. Knockdown of SLC25A10 had only modest impact on the transcriptome, suggesting that it might directly influence insulin sensitivity in adipocytes independently of transcription due to its important role in fatty acid synthesis. In summary, this study identifies novel genes associated with insulin sensitivity in adipocytes in women independently of obesity. KFL15 and SLC25A10 are inhibitors of insulin-stimulated lipogenesis under conditions when glucose transport is the rate limiting step

NPC1 in human white adipose tissue and obesity

Abstract Background Genetic studies have implicated the NPC1 gene (Niemann Pick type C1) in susceptibility to obesity. Methods To assess the potential function of NPC1 in obesity, we determined its expression in abdominal white adipose tissue (WAT) in relation to obesity. NPC1 mRNA was measured by RT-qPCR in lean and obese individuals, paired samples of subcutaneous (sc) and omental (om) WAT, before and after weight loss, in isolated adipocytes and intact adipose pieces, and in primary adipocyte cultures during adipocyte differentiation. NPC1 protein was examined in isolated adipocytes. Results NPC1 mRNA was significantly increased in obese individuals in scWAT and omWAT and downregulated by weight loss. NPC1 mRNA was enriched in isolated fat cells of WAT, in scWAT versus omWAT but not modified during adipocyte differentiation. NPC1 protein mirrored expression of mRNA in lean and obese individuals. Conclusions NPC1 is highly expressed in human WAT adipocytes with increased levels in obese. These results suggest that NPC1 may play a role in adipocyte processes underlying obesity.</p

Genome-wide association study identifies genetic loci associated with fat cell number and overlap with genetic risk loci for type 2 diabetes

Interindividual differences in generation of new fat cells determine body fat and type 2 diabetes risk. We utilized the GENiAL cohort, which consists of participants who have undergone abdominal adipose biopsy, to perform a genome-wide association study (GWAS) of fat cell number (n=896). Candidate genes from the genetic study were knocked down by siRNA in human adipose derived stem cells. We report 318 SNPs and 17 genetic loci displaying suggestive (p&lt;1x10-5) association with fat cell number. Two loci pass threshold for GWAS-significance, on chromosome 2 (lead SNP rs149660479-G) and 7 (rs147389390-deletion). We filtered for fat cell number-associated SNPs (p&lt;1.00x10-5) using evidence of genotype-specific expression. Where this was observed we selected genes for follow-up investigation and hereby identified SPATS2L and KCTD18 as regulators of cell proliferation consistent with the genetic data. Furthermore, 30 reported type 2 diabetes-associated SNPs displayed nominal and consistent associations with fat cell number. Functional follow up of candidate genes identified RPL8, HSD17B12 and PEPD displaying effects on cell proliferation consistent with genetic association and gene expression findings. In conclusion findings presented herein identify SPATS2L, KCTD18, RPL8, HSD17B12, and PEPD of potential importance in controlling fat cell numbers (plasticity), the size of body fat and diabetes risk

Shared genetic loci for body fat storage and adipocyte lipolysis in humans

Total body fat and central fat distribution are heritable traits and well-established predictors of adverse metabolic outcomes. Lipolysis is the process responsible for the hydrolysis of triacylglycerols stored in adipocytes. To increase our understanding of the genetic regulation of body fat distribution and total body fat, we set out to determine if genetic variants associated with body mass index (BMI) or waist-hip-ratio adjusted for BMI (WHRadjBMI) in genome-wide association studies (GWAS) mediate their effect by influencing adipocyte lipolysis. We utilized data from the recent GWAS of spontaneous and isoprenaline-stimulated lipolysis in the unique GENetics of Adipocyte Lipolysis (GENiAL) cohort. GENiAL consists of 939 participants who have undergone abdominal subcutaneous adipose biopsy for the determination of spontaneous and isoprenaline-stimulated lipolysis in adipocytes. We report 11 BMI and 15 WHRadjBMI loci with SNPs displaying nominal association with lipolysis and allele-dependent gene expression in adipose tissue according to in silico analysis. Functional evaluation of candidate genes in these loci by small interfering RNAs (siRNA)-mediated knock-down in adipose-derived stem cells identified ZNF436 and NUP85 as intrinsic regulators of lipolysis consistent with the associations observed in the clinical cohorts. Furthermore, candidate genes in another BMI-locus (STX17) and two more WHRadjBMI loci (NID2, GGA3, GRB2) control lipolysis alone, or in conjunction with lipid storage, and may hereby be involved in genetic control of body fat. The findings expand our understanding of how genetic variants mediate their impact on the complex traits of fat storage and distribution

Selected pathways over-represented among differentially expressed genes between OIR and OIS women^a.

<p>Selected pathways over-represented among differentially expressed genes between OIR and OIS women<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178485#t002fn001" target="_blank">^a</a>.</p

Effects of <i>KLF15</i> and <i>SLC25A10</i> knockdownon lipogenesis <i>in vitro</i>.

<p><b>A.</b><i>KLF15</i> and <i>SLC25A10</i> were knocked down using 40nM of siRNA in SVF-derived human adipocytes differentiated <i>in vitro</i> and expression of the genes evaluated using real-time PCR. Results were analyzed using Students t-test and are presented as relative fold change ± SD vs. negative control. <b>B.</b> SVF-derived adipocytes differentiated <i>in vitro</i> were transfected with 40 nM of siRNA against <i>KLF15</i> and <i>SLC25A10</i> for 48 hours followed by evaluation of basal and insulin-stimulated lipogenesis. Relative insulin-stimulated lipogenesis was calculated against non-targeting siRNA NegC at insulin-stimulated state. Induction of lipogenesis by insulin for NegC was minimum 3-fold in all experiments. Results are based on three to five biological/independent experiments.*p<0.05, **p<0.01 and ***p<0.001.</p

Clinical characteristics.

<p>Clinical characteristics.</p