Low-grade inflammation in obesity is associated with accumulation of the macrophage-derived cytokine osteopontin (OPN) in adipose tissue and induction of local as well as systemic insulin resistance. Since glucose-dependent insulinotropic polypeptide (GIP) is a strong stimulator of adipogenesis and may play a role in the development of obesity, we explored whether GIP directly would stimulate OPN expression in adipose tissue and thereby induce insulin resistance. GIP stimulated OPN protein expression in a dose-dependent fashion in rat primary adipocytes. The level of OPN mRNA was higher in adipose tissue of obese individuals (0.13 ± 0.04 vs. 0.04 ± 0.01, P < 0.05) and correlated inversely with measures of insulin sensitivity (r = -0.24, P = 0.001). A common variant of the GIP receptor (GIPR) (rs10423928) gene was associated with a lower amount of the exon 9-containing isoform required for transmembrane activity. Carriers of the A allele with a reduced receptor function showed lower adipose tissue OPN mRNA levels and better insulin sensitivity. Together, these data suggest a role for GIP not only as an incretin hormone but also as a trigger of inflammation and insulin resistance in adipose tissue. Carriers of the GIPR rs10423928 A allele showed protective properties via reduced GIP effects. Identification of this unprecedented link between GIP and OPN in adipose tissue might open new avenues for therapeutic interventions.

Genome-wide association studies (GWASs) have identified thousands of genetic loci associated with cardiometabolic traits including type 2 diabetes (T2D), lipid levels, body fat distribution, and adiposity, although most causal genes remain unknown. We used subcutaneous adipose tissue RNA-seq data from 434 Finnish men from the METSIM study to identify 9,687 primary and 2,785 secondary cis-expression quantitative trait loci (eQTL; <1 Mb from TSS, FDR < 1%). Compared to primary eQTL signals, secondary eQTL signals were located further from transcription start sites, had smaller effect sizes, and were less enriched in adipose tissue regulatory elements compared to primary signals. Among 2,843 cardiometabolic GWAS signals, 262 colocalized by LD and conditional analysis with 318 transcripts as primary and conditionally distinct secondary cis-eQTLs, including some across ancestries. Of cardiometabolic traits examined for adipose tissue eQTL colocalizations, waist-hip ratio (WHR) and circulating lipid traits had the highest percentage of colocalized eQTLs (15% and 14%, respectively). Among alleles associated with increased cardiometabolic GWAS risk, approximately half (53%) were associated with decreased gene expression level. Mediation analyses of colocalized genes and cardiometabolic traits within the 434 individuals provided further evidence that gene expression influences variant-trait associations. These results identify hundreds of candidate genes that may act in adipose tissue to influence cardiometabolic traits.

Identifying the regulatory mechanisms of genome-wide association study (GWAS) loci affecting adipose tissue has been restricted due to limited characterization of adipose transcriptional regulatory elements. We profiled chromatin accessibility in three frozen human subcutaneous adipose tissue needle biopsies and preadipocytes and adipocytes from the Simpson Golabi-Behmel Syndrome (SGBS) cell strain using an assay for transposase-accessible chromatin (ATAC-seq). We identified 68,571 representative accessible chromatin regions (peaks) across adipose tissue samples (FDR < 5%). GWAS loci for eight cardiometabolic traits were enriched in these peaks (P < 0.005), with the strongest enrichment for waist-hip ratio. Of 110 recently described cardiometabolic GWAS loci colocalized with adipose tissue eQTLs, 59 loci had one or more variants overlapping an adipose tissue peak. Annotated variants at the SNX10 waist-hip ratio locus and the ATP2A1-SH2B1 body mass index locus showed allelic differences in regulatory assays. These adipose tissue accessible chromatin regions elucidate genetic variants that may alter adipose tissue function to impact cardiometabolic traits.

Alternate splicing events can create isoforms that alter gene function, and genetic variants associated with alternate gene isoforms may reveal molecular mechanisms of disease. We used subcutaneous adipose tissue of 426 Finnish men from the METSIM study and identified splice junction quantitative trait loci (sQTLs) for 6,077 splice junctions (FDR < 1%). In the same individuals, we detected expression QTLs (eQTLs) for 59,443 exons and 15,397 genes (FDR < 1%). We identified 595 genes with an sQTL and exon eQTL but no gene eQTL, which could indicate potential isoform differences. Of the significant sQTL signals, 2,114 (39.8%) included at least one proxy variant (linkage disequilibrium r2 > 0.8) located within an intron spanned by the splice junction. We identified 203 sQTLs that colocalized with 141 genome-wide association study (GWAS) signals for cardiometabolic traits, including 25 signals for lipid traits, 24 signals for body mass index (BMI), and 12 signals for waist-hip ratio adjusted for BMI. Among all 141 GWAS signals colocalized with an sQTL, we detected 26 that also colocalized with an exon eQTL for an exon skipped by the sQTL splice junction. At a GWAS signal for high-density lipoprotein cholesterol colocalized with an NR1H3 sQTL splice junction, we show that the alternative splice product encodes an NR1H3 transcription factor that lacks a DNA binding domain and fails to activate transcription. Together, these results detect splicing events and candidate mechanisms that may contribute to gene function at GWAS loci.

Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here, we demonstrate that the gut microbiota-initiated trimethylamine N-oxide (TMAO)-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme flavin-containing monooxygenase 3 (FMO3) conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue.

COVID-19 severity has varied widely, with demographic and cardio-metabolic factors increasing risk of severe reactions to SARS-CoV-2 infection, but the underlying mechanisms for this remain uncertain. We investigated phenotypic and genetic factors associated with subcutaneous adipose tissue expression of Angiotensin I Converting Enzyme 2 ( ACE2 ), which has been shown to act as a receptor for SARS-CoV-2 cellular entry. In a meta-analysis of three independent studies including up to 1,471 participants, lower adipose tissue ACE2 expression was associated with adverse cardio-metabolic health indices including type 2 diabetes (T2D) and obesity status, higher serum fasting insulin and BMI, and lower serum HDL levels (P<5.32x10 -4 ). ACE2 expression levels were also associated with estimated proportions of cell types in adipose tissue; lower ACE2 expression was associated with a lower proportion of microvascular endothelial cells (P=4.25x10 -4 ) and higher macrophage proportion (P=2.74x10 -5 ), suggesting a link to inflammation. Despite an estimated heritability of 32%, we did not identify any proximal or distal genetic variants (eQTLs) associated with adipose tissue ACE2 expression. Our results demonstrate that at-risk individuals have lower background ACE2 levels in this highly relevant tissue. Further studies will be required to establish how this may contribute to increased COVID-19 severity.

Complete characterization of the genetic effects on gene expression is needed to elucidate tissue biology and the etiology of complex traits. Here, we analyzed 2,344 subcutaneous adipose tissue samples and identified 34K conditionally distinct expression quantitative trait locus (eQTL) signals in 18K genes. Over half of eQTL genes exhibited at least two eQTL signals. Compared to primary signals, non-primary signals had lower effect sizes, lower minor allele frequencies, and less promoter enrichment; they corresponded to genes with higher heritability and higher tolerance for loss of function. Colocalization of eQTL with conditionally distinct genome-wide association study signals for 28 cardiometabolic traits identified 3,605 eQTL signals for 1,861 genes. Inclusion of non-primary eQTL signals increased colocalized signals by 46%. Among 30 genes with ≥2 pairs of colocalized signals, 21 showed a mediating gene dosage effect on the trait. Thus, expanded eQTL identification reveals more mechanisms underlying complex traits and improves understanding of the complexity of gene expression regulation.

Non-alcoholic fatty liver disease (NAFLD) is a fast-growing, underdiagnosed, epidemic. We hypothesise that obesity-related inflammation compromises adipose tissue functions, preventing efficient fat storage, and thus driving ectopic fat accumulation into the liver.

Reverse causality has made it difficult to establish the causal directions between obesity and prediabetes and obesity and insulin resistance. To disentangle whether obesity causally drives prediabetes and insulin resistance already in non-diabetic individuals, we utilized the UK Biobank and METSIM cohort to perform a Mendelian randomization (MR) analyses in the non-diabetic individuals. Our results suggest that both prediabetes and systemic insulin resistance are caused by obesity (p = 1.2×10-3 and p = 3.1×10-24). As obesity reflects the amount of body fat, we next studied how adipose tissue affects insulin resistance. We performed both bulk RNA-sequencing and single nucleus RNA sequencing on frozen human subcutaneous adipose biopsies to assess adipose cell-type heterogeneity and mitochondrial (MT) gene expression in insulin resistance. We discovered that the adipose MT gene expression and body fat percent are both independently associated with insulin resistance (p≤0.05 for each) when adjusting for the decomposed adipose cell-type proportions. Next, we showed that these 3 factors, adipose MT gene expression, body fat percent, and adipose cell types, explain a substantial amount (44.39%) of variance in insulin resistance and can be used to predict it (p≤2.64×10-5 in 3 independent human cohorts). In summary, we demonstrated that obesity is a strong determinant of both prediabetes and insulin resistance, and discovered that individuals' adipose cell-type composition, adipose MT gene expression, and body fat percent predict their insulin resistance, emphasizing the critical role of adipose tissue in systemic insulin resistance.

Fatty acid desaturase (FADS) variants associate with fatty acid (FA) and adipose tissue (AT) metabolism and inflammation. Thus, the role of FADS1 variants in the regulation of dietary linoleic acid (LA)-induced effects on AT inflammation was investigated.

Subcutaneous adipose tissue stores excess lipids and maintains energy balance. We performed expression quantitative trait locus (eQTL) analyses by using abdominal subcutaneous adipose tissue of 770 extensively phenotyped participants of the METSIM study. We identified cis-eQTLs for 12,400 genes at a 1% false-discovery rate. Among an approximately 680 known genome-wide association study (GWAS) loci for cardio-metabolic traits, we identified 140 coincident cis-eQTLs at 109 GWAS loci, including 93 eQTLs not previously described. At 49 of these 140 eQTLs, gene expression was nominally associated (p < 0.05) with levels of the GWAS trait. The size of our dataset enabled identification of five loci associated (p < 5 × 10-8) with at least five genes located >5 Mb away. These trans-eQTL signals confirmed and extended the previously reported KLF14-mediated network to 55 target genes, validated the CIITA regulation of class II MHC genes, and identified ZNF800 as a candidate master regulator. Finally, we observed similar expression-clinical trait correlations of genes associated with GWAS loci in both humans and a panel of genetically diverse mice. These results provide candidate genes for further investigation of their potential roles in adipose biology and in regulating cardio-metabolic traits.

Understanding differences in adipose gene expression between individuals with different levels of clinical traits may reveal the genes and mechanisms leading to cardiometabolic diseases. However, adipose is a heterogeneous tissue. To account for cell-type heterogeneity, we estimated cell-type proportions in 859 subcutaneous adipose tissue samples with bulk RNA sequencing (RNA-seq) using a reference single-nuclear RNA-seq data set. Cell-type proportions were associated with cardiometabolic traits; for example, higher macrophage and adipocyte proportions were associated with higher and lower BMI, respectively. We evaluated cell-type proportions and BMI as covariates in tests of association between >25,000 gene expression levels and 22 cardiometabolic traits. For >95% of genes, the optimal, or best-fit, models included BMI as a covariate, and for 79% of associations, the optimal models also included cell type. After adjusting for the optimal covariates, we identified 2,664 significant associations (P ≤ 2e-6) for 1,252 genes and 14 traits. Among genes proposed to affect cardiometabolic traits based on colocalized genome-wide association study and adipose expression quantitative trait locus signals, 25 showed a corresponding association between trait and gene expression levels. Overall, these results suggest the importance of modeling cell-type proportion when identifying gene expression associations with cardiometabolic traits.

High serum triglyceride (TG) levels is an established risk factor for coronary heart disease (CHD). Fat is stored in the form of TGs in human adipose tissue. We hypothesized that gene co-expression networks in human adipose tissue may be correlated with serum TG levels and help reveal novel genes involved in TG regulation.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms.

Exercise training is critical for the prevention and treatment of obesity, but its underlying mechanisms remain incompletely understood given the challenge of profiling heterogeneous effects across multiple tissues and cell types. Here, we address this challenge and opposing effects of exercise and high-fat diet (HFD)-induced obesity at single-cell resolution in subcutaneous and visceral white adipose tissue and skeletal muscle in mice with diet and exercise training interventions. We identify a prominent role of mesenchymal stem cells (MSCs) in obesity and exercise-induced tissue adaptation. Among the pathways regulated by exercise and HFD in MSCs across the three tissues, extracellular matrix remodeling and circadian rhythm are the most prominent. Inferred cell-cell interactions implicate within- and multi-tissue crosstalk centered around MSCs. Overall, our work reveals the intricacies and diversity of multi-tissue molecular responses to exercise and obesity and uncovers a previously underappreciated role of MSCs in tissue-specific and multi-tissue beneficial effects of exercise.

Sirtuin 1 (SIRT1) is implicated in the regulation of mitochondrial function, energy metabolism, and insulin sensitivity in rodents. No studies are available in humans to demonstrate that SIRT1 expression in insulin-sensitive tissues is associated with energy expenditure and insulin sensitivity.

Parkin, an E3 ubiquitin ligase, plays an essential role in mitochondrial quality control. However, the mechanisms by which Parkin connects mitochondrial homeostasis with cellular metabolism in adipose tissue remain unclear. Here, we demonstrate that Park2 gene (encodes Parkin) deletion specifically from adipose tissue protects mice against high-fat diet and aging-induced obesity. Despite a mild reduction in mitophagy, mitochondrial DNA content and mitochondrial function are increased in Park2 deficient white adipocytes. Moreover, Park2 gene deletion elevates mitochondrial biogenesis by increasing Pgc1α protein stability through mitochondrial superoxide-activated NAD(P)H quinone dehydrogenase 1 (Nqo1). Both in vitro and in vivo studies show that Nqo1 overexpression elevates Pgc1α protein level and mitochondrial DNA content and enhances mitochondrial activity in mouse and human adipocytes. Taken together, our findings indicate that Parkin regulates mitochondrial homeostasis by balancing mitophagy and Pgc1α-mediated mitochondrial biogenesis in white adipocytes, suggesting a potential therapeutic target in adipocytes to combat obesity and obesity-associated disorders.

Branched-chain amino acids (BCAAs) metabolite, 3-Hydroxyisobutyric acid (3-HIB) has been identified as a secreted mediator of endothelial cell fatty acid transport and insulin resistance (IR) using animal models. To identify if 3-HIB is a marker of human IR and future risk of developing Type 2 diabetes (T2D), we measured plasma levels of 3-HIB and associated metabolites in around 10,000 extensively phenotyped individuals. The levels of 3-HIB were increased in obesity but not robustly associated with degree of IR after adjusting for BMI. Nevertheless, also after adjusting for obesity and plasma BCAA, 3-HIB levels were associated with future risk of incident T2D. We also examined the effect of 3-HIB on fatty acid uptake in human cells and found that both HUVEC and human cardiac endothelial cells respond to 3-HIB whereas human adipose tissue-derived endothelial cells do not respond to 3-HIB. In conclusion, we found that increased plasma level of 3-HIB is a marker of future risk of T2D and 3-HIB may be important for the regulation of metabolic flexibility in heart and muscles.

Inter-tissue communication via secreted proteins has been established as a vital mechanism for proper physiologic homeostasis. Here, we report a bioinformatics framework using a mouse reference population, the Hybrid Mouse Diversity Panel (HMDP), which integrates global multi-tissue expression data and publicly available resources to identify and functionally annotate novel circuits of tissue-tissue communication. We validate this method by showing that we can identify known as well as novel endocrine factors responsible for communication between tissues. We further show the utility of this approach by identification and mechanistic characterization of two new endocrine factors. Adipose-derived Lipocalin-5 is shown to enhance skeletal muscle mitochondrial function, and liver-secreted Notum promotes browning of white adipose tissue, also known as "beiging." We demonstrate the general applicability of the method by providing in vivo evidence for three additional novel molecules mediating tissue-tissue interactions.

Individual risk of type 2 diabetes (T2D) is modified by perturbations to the mass, distribution and function of adipose tissue. To investigate the mechanisms underlying these associations, we explored the molecular, cellular and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression and modulate, in trans, the expression of 385 genes. We demonstrate, in human cellular studies, that reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and in mice, that adipose tissue-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that carriers of the KLF14 T2D risk allele shift body fat from gynoid stores to abdominal stores and display a marked increase in adipocyte cell size, and that these effects on fat distribution, and the T2D association, are female specific. The metabolic risk associated with variation at this imprinted locus depends on the sex both of the subject and of the parent from whom the risk allele derives.