Joshua W. Knowles, Ph.D. - US grants

PROJECT SUMMARY/ABSTRACT Dramatic increases in insulin resistance (IR) prevalence are expected in the U.S. and throughout the world in coming years. Since IR is an important risk factor for cardiovascular disease, discovery of more efficient ways of preventing and treating this condition would have a huge public health impact. Over the past decades, development of new drugs aimed at preventing cardiometabolic disease has slowed down substantially, but recent advances in human genetics offer new exciting opportunities for drug development. Genome-wide associations studies (GWAS) have discovered >150 loci associated with IR and closely related traits over the past decade; but for the vast majority of these, the causal gene has not been definitely identified and the mechanisms leading to IR are unknown. We have performed colocalization analyses to prioritize 50 plausible candidate genes from 164 GWAS loci associated with IR-related traits, and we now aim to establish and characterize genes causally associated with IR using a rigorous series of experiments combining CRISPR-based gene perturbation with single-cell RNA sequencing and detailed phenotyping in human adipocytes, skeletal myocytes and mouse models. In Aim 1, we will perform CROP-seq ? CRISPR-based transcriptional interference (CRISPRi) followed by single- cell RNA-seq (scRNA-seq) ? in human adipocytes to characterize differentially expressed genes and pathways after knockdown of 50 genes selected based on colocalization analyses. In Aim 2, we will evaluate metabolic phenotypes, such as glucose uptake, lipolysis, insulin signaling, adipogenesis, mitochondrial function, fatty acid oxidation, and metabolite profiles in human adipocytes and skeletal myocytes after CRISPRi knockdown of 25 genes, guided by expression profiles from aim 1. In Aim 3, we will create and breed knockout mouse models for three IR-related genes, and then compare wildtype and knockout mice with regards to fat distribution, glucose and insulin tolerance, energy expenditure, physical activity, food intake, lipid profiles, kidney and liver panels, cellular transcriptome, and histopathology of different tissues in mice on chow and after high-fat feeding. By combining a range of innovative methods including high-throughput gene perturbations followed by single cell transcriptomics, in vitro and in vivo experiments to characterize loci established using human genetics, we expect to establish causal genes and mechanisms of action for novel genes involved in development of IR. This is a first important step towards development of new drugs to address the huge and increasing unmet need posed by IR. Our proposal integrates a range of innovative approaches in different model systems providing a translational framework that is likely to lead to new important insights into insulin resistance, type 2 diabetes and cardiovascular disease which could have a huge public health impact.

PROJECT SUMMARY/ABSTRACT Decreased insulin sensitivity (insulin resistance, IR) is a fundamental abnormality in patients with type 2 diabetes (T2D), and a major risk factor for cardiovascular disease (CVD). We led a genome wide association study (GWAS) for direct measures of IR and identified a novel IR gene, N-acetyl transferase 2 (NAT2). Non- synonymous coding variants in NAT2 were associated with increased IR independently of body mass index as well as IR-related traits. Knockdown and overexpression of the mouse ortholog Nat1 led to changes in glucose homeostasis in adipocytes and myoblasts. Nat1 deficient mice (Nat1 KO) had decreased insulin sensitivity and elevations in fasting blood glucose, insulin and triglycerides. Nat1 is highly co-regulated with key mitochondrial genes and RNA-interference mediated silencing of Nat1 leads 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. Nat1 KO mice have a decrease in basal metabolic rate and exercise capacity without altered thermogenesis versus Nat1 wild type (Nat1 WT) mice. Nat1 KO mice also have changes in plasma metabolites and lipids, such as decreased levels of acylcarnitines, and indirect calorimetry data shows decreased utilization of fats for energy, suggesting that Nat1 deficiency is associated with an impaired fatty acid oxidation (FAO). New data indicate that supernatant from Nat1 deficient liver cells results in IR in adipocytes. Our overall hypothesis is that Nat1 binds to and regulates key mediators of mitochondrial function and energy balance in the liver ultimately leading to IR. Using our unique resources including a liver specific knockout mouse (Nat1 LKO), we will test this hypothesis and elucidate the mechanisms of insulin resistance caused by Nat1 deficiency. Nat1 is known to acetylate certain drugs and carcinogens but the endogenous substrate/s are unknown. Studies in Aim 1 will identify Nat1 protein-protein interactions and Nat1 acetylation substrates that regulate energy balance and metabolism. Our hypothesis is that Nat1 binds key regulators of mitochondrial function. In Aim 2 we will define the specific mitochondrial defects in Nat1 deficiency. Our hypothesis is that Nat1 deficiency causes impaired FAO and that this can be rescued by augmenting ?-oxidation. In Aim 3 we will define mediators of local and systemic effects of Nat1 deficiency. Nat1 is highly expressed in the liver with more modest expression in insulin-sensitive tissues. We believe that hepatic Nat1 mediates whole body insulin sensitivity specifically through signaling intermediates that act through effects on adipose and skeletal muscle. We will confirm this through detailed phenotyping, including euglycemic clamp, of liver specific Nat1 KO. We will also identify secreted factors that impair insulin sensitivity in Nat1 deficiency building on our co-culture data from Nat1 deficient liver cells and adipocytes. These aims will define the pathophysiological role of the novel IR gene Nat1, thereby increasing our understanding of IR, which is a necessary step towards new therapies.

PROJECT SUMMARY/ABSTRACT Insulin resistance is a physiological state in which normal levels of insulin fail to regulate blood glucose levels, and even in the absence of type 2 diabetes, there is strong evidence that insulin resistance dramatically increases risk for atherosclerosis and overt cardiovascular disease. In the past few years, we have identified 13 susceptibility loci for insulin resistance, but the causal gene and mechanisms are unknown for all but three of these loci, and the role of the ten remaining loci for development of insulin resistance has not been studied systematically. This represents a gold mine for in-depth physiological and mechanistic studies as increased understanding of the links between obesity, insulin resistance and cardiovascular disease may lead to new approaches to prevention and treatment that could have a huge public health impact. To establish and characterize genes associated with insulin resistance, we plan experiments in large human cohorts with functional follow-up using zebrafish and cell-based models. We will characterize suggested insulin resistance loci using detailed phenotypic information from large population-based samples (total N=13,811) assessed with dynamic measures of glucose and insulin metabolism, metabolomic, transcriptomic, epigenomic and proteomic profiling together with in silico data on gene regulation and transcription from public resources. Next, we will take 55 candidate genes forward to our pipeline for efficient characterization in zebrafish using high-throughput visualization techniques and biochemical measurements. We use CRISPR-Cas9 techniques to knockout the orthologous 55 genes from the 10 loci that are uncharacterized to date, and study the effect of perturbing these genes on insulin resistance. Finally, we will prioritize five candidate genes for mechanistic studies using gene knockdown in adipocytes and hepatocytes to study glucose, insulin and lipid metabolism, gene expression and metabolic pathways. By performing detailed follow-up analyses of loci hypothesized to be involved in insulin resistance, we expect to establish causal genes and mechanisms of action for several of these loci. The in-depth characterization using in vivo and in vitro models will provide further evidence towards causality and the mechanisms of action, as well as a first evaluation of which could be viable drug targets. Our approach of integrating comprehensive characterization in humans with experiments in functional model systems provides a translational framework, which by design is more likely to yield findings relevant for human biology and medicine. Importantly, we have access to unique study materials, state-of-the art methodology, and have a strong track record of successful collaborations in this field. Our work is anticipated to benefit the scientific community, to lead to new important insights into insulin resistance, cardiovascular disease and type 2 diabetes.