Epigenomic and Transcriptional Basis of Human Insulin Resistance

Development of insulin resistance is a key pathogenic component underlying metabolic syndrome and Type 2 diabetes (T2DM). Despite its importance, the molecular mechanisms underlying insulin resistance are poorly understood. Genome-wide association studies for T2DM and other metabolic traits have led to the identification of many candidate SNPs, but the majority of these SNPs are noncoding and determination of associated causal genes and/or specific tissue sites of action have been difficult. Adipocytes are critical regulators of mammalian metabolic homeostasis, with important effects on appetite, satiety, glucose and lipid homeostasis, energy expenditure, blood pressure, and immune function. Although insulin resistance (IR) in skeletal muscle, the major source of glucose disposal, is responsible for the bulk of the hyperglycemia observed in T2DM, muscle IR KO mice are generally healthy, IR also occurs in adipocytes, and inflammation within adipose tissue has been proposed as a primary mediator of IR, resulting in excess release of free fatty acids as well as alterations in adipokine release. Absence of adipose tissue, as observed in various lipodystrophies, or adipocyte-specific knockout of genes such as GLUT4 or insulin receptor, also alter systemic IR and T2DM risk. Transcriptional changes in adipocytes associated with metabolic disease. Despite the importance of adipocytes to metabolic disease, we have a poor understanding of how the adipocyte transcriptome changes in the disease state. Studies investigating transcriptional changes in whole adipose tissue have shown decreases in genes involved in adipogenesis, as well as alterations in inflammation, mitochondrial metabolism, lipid metabolism, detoxification, and insulin signaling. However, the vast majority of these studies use total adipose tissue samples, which does not allow for the exclusive evaluation of gene expression in mature adipocytes due to the substantial number of nonadipocyte cells (immune cells, fibroblasts, endothelial cells, pre-adipocytes, and mesenchymal cells) that also reside in adipose tissue. This is particularly relevant when studying metabolic disorders related to obesity-associated insulin resistance because these conditions are characterized by an increased influx of inflammatory cells into the adipose tissue. We performed RNA-seq on purified adipocytes collected from subcutaneous panniculus samples of 43 individuals in the first and 4th quartiles for HOMA-IR from a population of plastic surgery patients at our institution. Adipocytes were isolated via modified Rodbell protocol form 300g-2kg of starting material. 400ul of floated adipocyte material was used for RNA isolation. Total RNA from ~400 μl of thawed floated adipocytes was isolated in TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For RNA-seq library construction, mRNA was purified from 100 ng of total RNA by using a Ribo-Zero rRNA removal kit (Epicentre) to deplete ribosomal RNA and convert into double-stranded complementary DNA by using an NEBNext mRNA Second Strand Synthesis Module (E6111L). cDNA was subsequently tagmented and amplified for 12 cycles by using a Nextera XT DNA Library Preparation Kit (Illumina FC-131). Sequencing libraries were analyzed with Qubit and Agilent Bioanalyzer, pooled at a final loading concentration of 1.8 pM and sequenced on a NextSeq500. No technical or biological replicates were performed.