Impact of skeletal muscle lipid droplet dynamics on oxidative metabolism and insulin sensitivity – DYNAMISE

Obesity and type 2 diabetes mellitus (T2DM) have been associated with ectopic fat deposition in non-adipose tissues such as skeletal muscle which plays an important role in the etiology of insulin resistance. Ectopic lipids mainly accumulate as triacylglycerol (TAG) stored in lipid droplets (LDs) in skeletal muscle. An inverse association between intramyocellular TAG (IMTG) content and peripheral glucose disposal has been repeatedly reported in sedentary subjects. It is now well established that IMTG is associated with elevated levels of lipotoxic intermediates such as diacylglycerols and ceramides that inhibit insulin signaling and interfere with glucose metabolism. However, how IMTG mediates lipotoxicity in obese sedentary subjects is poorly understood so far. LDs are dynamic organelles resulting from the balance between storage and breakdown of TAG by lipases. We could recently show that elevated adipose triglyceride lipase expression/activity in skeletal muscle contributes to lipotoxicity and insulin resistance. A causal relationship was shown in vitro in human myotubes. Our preliminary data also suggest that LDs hydrolysis may regulate the availability of fatty acids for mitochondrial oxidation and of lipid ligands for activation of the nuclear receptor peroxisome proliferator-activating receptor-beta (PPARbeta). We therefore hypothesize that skeletal muscle LDs dynamics is an important determinant of lipotoxicity, insulin sensitivity, and oxidative metabolism.
This project aims to investigate the role of skeletal muscle LDs dynamic in obesity-related insulin resistance and T2DM. The central aim of this proposal is to investigate the metabolic consequences of changes in skeletal muscle LDs dynamics on lipid and glucose homeostasis in vitro in a human skeletal muscle cell culture model, and in vivo in transgenic mice models. In this proposal, we will evaluate the metabolic role of key proteins of LDs dynamics (PLIN5, G0S2 and CGI-58) in the regulation of lipid, glucose metabolism and insulin sensitivity, 1) in vitro in human primary myotubes through overexpression and knockdown studies, 2) in vivo by investigating muscle-specific PLIN5 knockout mice (Plin5 skm-/-), and by crossing mice overexpressing hCGI-58 specifically in skeletal muscle (MCK-hcgi58) with muscle-specific PPARbeta knockout mice (pparbeta skm-/-).
In vitro studies will be carried out in human primary myotubes derived from vastus lateralis biopsies of lean healthy subjects. Overexpression and knockdown studies will be performed using adenovirus gene delivery. Lipid intermediates will be measured by lipidomic, insulin signaling by western blot and metabolic studies will be performed using radiolabeled tracers to determine insulin sensitivity, glucose and lipid metabolism.
In vivo, wild-type and transgenic mice will be investigated under normal chow and high fat diets in sedentary condition and after a 4-week chronic exercise challenge on treadmill. Insulin sensitivity will be determined by insulin tolerance test and hyperinsulinemic euglycemic clamps. Whole-body energy metabolism will be assessed over 24h in metabolic chambers. Lipid intermediates, insulin signaling and mitochondrial fat oxidative capacity will be measured in skeletal muscle ex vivo.
The role of LDs dynamics in the regulation of lipotoxicity, insulin sensitivity and oxidative metabolism in skeletal muscle is underappreciated and has been poorly investigated so far. This project will provide important insights into the pathophysiology of obesity-related insulin resistance and T2DM, and may provide novel targets for prevention.