Rikke Kruse - Molecular mechanisms underlying mitochondrial dysfunction in skeletal muscle insulin resistance
Type 2 diabetes (T2D) and obesity are characterized by insulin resistance (IR) in skeletal muscle, liver, and adipose tissue, but the exact molecular mechanisms underlying IR are not fully understood. Mitochondria are dynamic organelles that continuously undergo fusion and fission to maintain a healthy mitochondrial population.
An altered morphology of mitochondria including reduced size and decreased expression of MFN2 in skeletal muscle in obesity and T2D have indicated a disordered mitochondrial dynamics in insulin resistant states. This finding is underlined by a positive correlation between MFN2 expression and insulin sensitivity. Interestingly, recent data indicate that both insulin and exercise regulate proteins governing mitochondrial dynamics but also proteins involved in the clearance of damaged mitochondria (mitophagy).
It has been estimated that approximately 35% of all mitochondrial proteins have at least one acetylation site. Interestingly, deacetylation of the fusion protein OPA1 at Lys926 and Lys931 increases its GTPase activity and thereby contributes to the maintenance of the mitochondrial morphology and respiration. Furthermore, mass spectrometry (MS)-based studies have shown that most metabolic enzymes contain at least one acetylated lysine residue. Protein acetylation is reflective of the cellular energy and nutritional status and lysine acetylation of mitochondrial proteins has been shown to correlate with insulin sensitivity. A recent paper identified increased abundance of lysine acetylation in skeletal muscle mitochondria from mice fed a high-fat diet for 12 weeks, however, the identity of these proteins were not investigated.
The major aims of the present project are 1) to determine whether the expression of regulators of mitochondrial dynamics is influenced by insulin in skeletal muscle of lean glucose-tolerant individuals and in patients with T2D and whether this is reflected in changes in the mitochondrial morphology and 2) to evaluate the effect of insulin on mitochondrial protein acetylation in human skeletal muscle and how this response as well as the basal acetylome in individuals with T2D..
The project will characterize the mRNA levels and protein abundance of mediators of mitochondrial dynamics in human skeletal muscle in lean, glucose-tolerant individuals and patients with T2D after overnight fasting and in the insulin-stimulated state of a euglycaemic-hyperinsulinemic clamp using RT-qPCR and western blotting. C2C12 myotubes with palmitate-induced IR will be used to assess the impact of IR on the mitochondrial morphology, which will be visualized using fluorescent microscopy. Moreover, this project will include a discovery-mode MS-based study that aims to characterize the acetylome of human skeletal muscle mitochondria from healthy, glucose-tolerant individuals and patients with T2D after overnight fasting and in the insulin-stimulated state of a euglycaemic-hyperinsulinemic clamp. We will take advantage of isobaric labeling to facilitate comparative analysis of post-translational modifications between individuals and in response to insulin. Finally, this project will use isobaric labeling in combination with a targeted quantitative proteomic approach to characterize the acetylation of the proteins regulating mitochondrial dynamics and to identify whether the acetylation status of these proteins are altered by insulin and/or palmitate-induced IR.