Nat1 Deficiency Is Associated with Mitochondrial Dysfunction and Exercise Intolerance in Mice

SummaryWe recently identified human N-acetyltransferase 2 (NAT2) as an insulin resistance (IR) gene. Here, we examine the cellular mechanism linking NAT2 to IR and find that Nat1 (mouse ortholog of NAT2) is co-regulated with key mitochondrial genes. RNAi-mediated silencing of Nat1 led to mitochondrial dysfunction characterized by increased intracellular reactive oxygen species and mitochondrial fragmentation as well as decreased mitochondrial membrane potential, biogenesis, mass, cellular respiration, and ATP generation. These effects were consistent in 3T3-L1 adipocytes, C2C12 myoblasts, and in tissues from Nat1-deficient mice, including white adipose tissue, heart, and skeletal muscle. Nat1-deficient mice had changes in plasma metabolites and lipids consistent with a decreased ability to utilize fats for energy and a decrease in basal metabolic rate and exercise capacity without altered thermogenesis. Collectively, our results suggest that Nat1 deficiency results in mitochondrial dysfunction, which may constitute a mechanistic link between this gene and IR

Predictive network modeling in human induced pluripotent stem cells identifies key driver genes for insulin responsiveness.

Insulin resistance (IR) precedes the development of type 2 diabetes (T2D) and increases cardiovascular disease risk. Although genome wide association studies (GWAS) have uncovered new loci associated with T2D, their contribution to explain the mechanisms leading to decreased insulin sensitivity has been very limited. Thus, new approaches are necessary to explore the genetic architecture of insulin resistance. To that end, we generated an iPSC library across the spectrum of insulin sensitivity in humans. RNA-seq based analysis of 310 induced pluripotent stem cell (iPSC) clones derived from 100 individuals allowed us to identify differentially expressed genes between insulin resistant and sensitive iPSC lines. Analysis of the co-expression architecture uncovered several insulin sensitivity-relevant gene sub-networks, and predictive network modeling identified a set of key driver genes that regulate these co-expression modules. Functional validation in human adipocytes and skeletal muscle cells (SKMCs) confirmed the relevance of the key driver candidate genes for insulin responsiveness

Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene

Journal ArticleDecreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol- stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity

Interactions of physical activity, muscular fitness, adiposity, and genetic risk for NAFLD

Genetic predisposition and unhealthy lifestyle are risk factors for nonalcoholic fatty liver disease (NAFLD). We investigated whether the genetic risk of NAFLD is modified by physical activity, muscular fitness, and/or adiposity. In up to 242,524 UK Biobank participants without excessive alcohol intake or known liver disease, we examined cross‐sectional interactions and joint associations of physical activity, muscular fitness, body mass index (BMI), and a genetic risk score (GRS) with alanine aminotransferase (ALT) levels and the proxy definition for suspected NAFLD of ALT levels > 30 U/L in women and >40 U/L in men. Genetic predisposition to NAFLD was quantified using a GRS consisting of 68 loci known to be associated with chronically elevated ALT. Physical activity was assessed using accelerometry, and muscular fitness was estimated by measuring handgrip strength. We found that increased physical activity and grip strength modestly attenuate genetic predisposition to elevation in ALT levels, whereas higher BMI markedly amplifies it (all p values < 0.001). Among those with normal weight and high level of physical activity, the odds of suspected NAFLD were 1.6‐fold higher in those with high versus low genetic risk (reference group). In those with high genetic risk, the odds of suspected NAFLD were 12‐fold higher in obese participants with low physical activity versus those with normal weight and high physical activity (odds ratio for NAFLD = 19.2 and 1.6, respectively, vs. reference group). Conclusion: In individuals with high genetic predisposition for NAFLD, maintaining a normal body weight and increased physical activity may reduce the risk of NAFLD

<i>TCF21</i> and the environmental sensor aryl-hydrocarbon receptor cooperate to activate a pro-inflammatory gene expression program in coronary artery smooth muscle cells

<div><p>Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor <i>TCF21</i> as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of <i>TCF21</i> expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of <i>AHR</i>, its heterodimerization partner <i>ARNT</i>, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including <i>IL1A</i>, <i>MMP1</i>, and <i>CYP1A1</i>. TCF21 was shown to bind in <i>AHR</i>, <i>ARNT</i> and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD.</p></div

Schematic representation of AHR-TCF21 interactions that may modulate the effect of environmental stimuli on the progression of inflammation and atherosclerotic plaque formation.

<p>Environmental toxins, including dioxin and tobacco, as well as endogenous activators such as ox-LDL activate the AHR pathway, leading to increased inflammatory burden in the plaque. TCF21 can further increase the burden by increasing AHR expression as well as interacting with AHR at its downstream genes.</p

Endogenous regulation of the AHR pathway in SMC, and in vivo expression of <i>AHR</i> in atherosclerotic vascular disease.

<p>(a) <i>IL1A</i> mRNA levels are increased by dioxin and ox-LDL, and co-treatment with αNF, an AHR pathway antagonist, reverses these effects. (b) <i>AHR</i> mRNA levels are elevated in ligated carotid arteries with ruptured plaques compared to stable plaques. (c) Laser capture microdissection shows increased <i>AHR</i> mRNA levels to be located in the plaque region of the atherosclerotic aortic root. (d) <i>AHR</i> mRNA levels are elevated in atherosclerotic human carotid arteries compared to normal arteries. (e) In vivo protein expression patterns in human carotid artery plaque tissues. AHR-TCF21 unique interactors from the BIOGRID protein-protein interaction database display clustering patterns in mass-spectrometry and high resolution isoelectric focusing proteomic data from human carotid artery plaques. Proteomic datasets were constructed from patients with asymptomatic (10) and symptomatic (10) carotid stenoses. Mass-spec and high resolution isoelectric focusing yielded 8–9000 recovered proteins. Hierarchical clustering revealed AHR to be located in a cluster of genes that include immune related genes, transcription factors, and cell cycle regulated genes, such as IRAK4, SP1, and XPO1, respectively.</p

TCF21 binds in the <i>CYP1A1</i> locus, and interacts with AHR to promote transcription.

<p>(a) UCSC Genome Browser view of TCF21 binding upstream of the <i>CYP1A1</i> gene in a region of open chromatin as defined by ATAC-seq with a high predicted affinity for AHR-ARNT binding. (b) TCF21 binding to <i>CYP1A1</i> upstream region is demonstrated with ChIP-qPCR. (c) Transfection studies with a composite TCF21-AHR binding site reporter plasmid shows additive effects on transcription with combined <i>TCF21</i> expression plasmid and treatment of the cells with dioxin. <i>TCF21</i>-DRE-pLuc is a luciferase construct with alternating TCF21 and AHR binding motifs subcloned proximal to the promoter. DRE, dioxin response element. (d) Deletion of the TCF21 binding motif in the reporter plasmid abrogates the effects of <i>TCF21</i> over-expression, verifying that TCF21 has a direct transcriptional effect in these experiments. (*p<0.05; **p<0.005).</p

AHR expression in HCASMC is mediated by an eQTL that modulates <i>TCF21</i> binding.

<p>(a) The downstream region of the <i>AHR</i> gene on chr7 (located on >20kb distance from the transcription end site) contains a strong open chromatin region and enhancer in HCASMC marked by ATAC-Seq, H3K27ac tracks and binding of AP1 transcription factors, JUN and JUND, as well as TCF21. Enhanced view shows that SNP rs10265174 directly overlaps the ATAC-Seq open chromatin, H3K27ac enhancer mark, JUN and JUND ChIP-Seq peaks, and is within a broad TCF21 ChIP-Seq peak. (b) The LocusZoom plot shows <i>AHR</i> gene eQTL distribution in HCASMC across 1Mb (16.9–17.9Mb) on chr7 encompassing <i>AHR</i> gene, with SNP rs10265174 being the top eQTL in the locus. (c) rs10265174 alters the position weight matrix scores for AP1 and TCF4 transcription factors (data from Haploreg). (d) TCF21 ChIP-qPCR shows enrichment at the <i>AHR</i> upstream genomic region compared to IgG control (p<0.05).</p