Transcriptional, epigenetic and lipidomic responses to metabolic interventions : implications for human obesity

Obesity is increasing worldwide in an epidemic manner and better prevention and treatments are urgently needed. Obesity is strongly associated with insulin resistance and predisposes for type 2 diabetes. A deeper understanding of the underlying mechanisms behind obesity-driven insulin resistance is needed to identify new potential treatment targets. Skeletal muscle is the main organ for glucose and fat metabolism. It is characterized by the ability to switch between glucose and fat as an energy source depending on the requirements and nutrient availability. This ability is referred to as metabolic flexibility. In the obese state, the skeletal muscle has a surplus of fatty acids and glucose, and this energy overload leads to stress and inflammation. This metabolic stress and inflammation, as well as the presence of excess fatty acids per se, impairs insulin signalling and metabolic flexibility in skeletal muscle. The overall aims of this thesis were to study the effects of energy alterations on gene expression, epigenetic marks, and lipid profile in blood and skeletal muscle to elucidate the underlying mechanisms leading to perturbations in skeletal muscle metabolism. The interplay between our genes and the environment to which we are exposed, can explain some of the obesity and insulin resistance epidemic. Different environmental factors may alter DNA methylation, which is an epigenetic modification that can alter the function of genes. In Paper I, we obtained peripheral blood from obese subjects before and after weight loss through a diet intervention and subsequent gastric bypass surgery. In the peripheral blood, we assessed the level of DNA promoter methylation on selected genes important for metabolism. Promoter DNA methylation of PGC1α was altered after diet-induced weight loss, while gastric bypass surgery, inducing a substantial weight loss, was associated with methylation changes of PDK4, TNF and IL6 promoters. In Paper II, we studied the effects of a short insulin exposure on skeletal muscle DNA methylation, and identified broad methylation changes in genes regulating metabolic pathways. One gene, previously not implicated in metabolic regulation, DAPK3, exhibited differential methylation status in response to insulin. DNA methylation of DAPK3 also differed between normal glucose tolerant and type 2 diabetic subjects, and changed in vivo in response to glucose ingestion. In Paper III we found that when skeletal muscle was exposed to AICAR, an activator of AMP-activated protein kinase, and a mimic of low cellular energy state, there was reduced expression of several cytokines, involved in metabolic inflammation. Using an unbiased bioinformatics approach, we identified the Sp1 transcription factor to be involved in the down-regulation of IL6 mRNA and the relatively unknown transcription factor Zbtb14 to alter LIF mRNA. In Paper IV, we again studied the effects of diet-induced weight loss in skeletal muscle of obese subjects. The three-week diet intervention reduced body weight and insulin levels. Gene expression of FATP1, MLYCD, PDK4, UCP3 and SCD1 was altered, all in favour to promote fatty acid oxidation in skeletal muscle. We also found an altered lipid profile, with specific lipid species associated with the improvement of skeletal muscle insulin sensitivity. In conclusion, we elucidate potential regulatory mechanisms of skeletal muscle metabolic health, which is of relevance in the overall understanding of the metabolic perturbations of obesity and insulin resistance. We show that DNA methylation is dynamic and a potential regulator of “immunometabolic” gene expression. We provide insight in how weight loss improves skeletal muscle metabolism and identify specific lipid species that may play a role in improving skeletal muscle insulin sensitivity. LIST OF SCIENTIFIC PAPERS Articles included in this thesis I. Kirchner H, Nylén C, Laber S, Barrès R, Yan J, Krook A, Zierath JR and Näslund E. Altered promoter methylation of PDK4, IL1B, IL6 and TNF after Roux-en Y gastric bypass. Surg Obes Relat Dis 2014; 10:671-678. II. Mudry JM, Lassiter DG, Nylén C, García-Calzón S, Näslund E, Krook A, Zierath JR. Insulin and glucose alter death-associated protein kinase 3 (DAPK3) DNA methylation in human skeletal muscle. Diabetes 2017; 66:651-662. III. Nylén C, Aoi W, Lassiter DG, Lundell, LS, Wallberg-Henriksson H, Näslund E, Pillon NJ and Krook A. AICAR-induced reduction of baseline IL-6 and LIF mRNA in skeletal muscle involves the transcription factor Sp1. Manuscript Submitted. IV. Nylén C, Lundell LS, Massart J, Zierath JR and Näslund E. Short-term low calorie diet remodels skeletal muscle lipid profile and metabolic gene expression in obsess adults. Manuscript Submitted. Articles not included in this thesis Al-Khalili L, Chibalin AV, Yu M, Sjödin B, Nylén C, Zierath JR, Krook A. MEF2 activation in differentiated primary human skeletal muscle cultures requires coordinated involvement of parallel pathways. Am J Physiol Cell Physiol 2004 Jun;286(6):C1410-6. Chibalin AV, Leng Y, Vieira E, Krook A, Björnholm M, Long YC, Kotova O, Zhong Z, Skane F, Steiler T, Nylén C, Wang J, Laakso M, Topham MK, Gilbert M, Wallberg-Henriksson H, Zierath JR. Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance. Cell 2008 Feb 8:132(3):37586. Lassiter DG, Nylén C, Sjögren R, Chibalin AV, Wallberg-Henriksson H, Näslund E, Krook A, Zierath JR. FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle. Diabetologia. In Press. CONTENTS