Carnosine in skeletal muscle: biological action and therapeutic implications

The worldwide prevalence of diabetes has risen to 8.5% among adults, which represents a staggering rise in prevalence from 4.7% in 1980. More than 90% of these individuals have type 2 diabetes (T2DM), a disease typically characterized by peripheral insulin resistance and underpinned by pancreatic β-cell dysfunction. Importantly, more than 80% of those with T2DM are either overweight or obese, which results in chronic elevated fatty acid and glucose concentrations in these individuals. The ensuing glucolipotoxic (GLT) environment drives much of the pathogenesis of T2DM (β-cell dysfunction and insulin resistance) and contributes to the development of mitochondrial stress, generation of reactive species, proinflammatory cytokines, and altered gene expression. There are currently a limited number of options to treat T2DM, and oral and injectable medications often become less effective over time. Thus, there is an urgent need to better understand the causes of diabetes and to identify new targets for the development of novel treatment strategies. Carnosine (β-alanyl-L-histidine) is an endogenously synthesised dipeptide that is widely and abundantly distributed in the skeletal muscles. Beneficial actions that have been credited to carnosine include, but are not limited to, intracellular buffering, metalion chelation, antioxidant, anti-glycating, and free-radical scavenging. This PhD project is focussed on investigating the biological actions and therapeutic potential of carnosine to combat T2DM, through targeted action to improve insulin resistance in skeletal muscle cells, and to augment insulin secretion from pancreatic β-cells. A diabetic model of glucolipotoxicity was generated by incubating pancreatic β-cells or myotubes in standard tissue culture media supplemented with 28mM glucose, 200μM palmitic acid, and 200μM oleic acid. Intracellular reactive species content was assayed using 2, 7-dichlorofluorescein diacetate dye (DCFDA), whereas 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) content, as well as insulin secretion, were assayed and quantified using respective ELISA assays. SDS-PAGE in conjunction with immunoblotting and semi-quantitative densitometry analysis was employed to determine protein expression. Glucose uptake was determined through 2-deoxy glucose-6-phosphate (2-DG6P) luminescence. Immunoprecipitation-mass spectrometry tandem techniques were utilised to study GLT-mediated protein adduction. Seahorse XF Cell Mito Stress Test kit was employed to preliminarily investigate the functional capacity of mitochondria in GLTexposed skeletal muscle cells. Using carnosine as a starting material and template, both synthetic and computational chemistry approaches were utilised to generate carnosine mimetics and putative carnosinase inhibitor molecules, respectively. Carnosine supplementation resulted in protection of cells against GLT-mediated generation of reactive species, and thereby enhanced glucose uptake into skeletal muscle and increased insulin secretion from pancreatic β-cells. Further investigation showed that carnosine prevented adduction or modification of between 65-90% of protein by 4-HNE or 3-NT in GLT-treated pancreatic islets and muscle cells. Analysis using Panther software showed that many of these proteins are involved in catalytic and binding activities, with the leading cellular function affected being metabolic processes. Importantly, and consistent with the aforementioned findings, addition of carnosine to GLT-treated cells significantly improved mitochondrial respiration in both mouse C2C12 muscle cells and a human skeletal muscle cell-line. By contrast, in human serum donated (with informed consent) by individuals who are either obese or type 2 diabetic, and are both diabetic and obese, several proteins associated with the immune system were detected to have formed adducts with both 3-nitrotyrosine and 4-hydroxynonenal. Screening of carnosine analogs identified 5 candidate drugs that were effective at scavenging reactive species whilst having no impact on cell viability. Subsequent in vivo experiments, carried out with collaborators, showed that one of these molecules reduced obesity in high-fat fed mice, whereas one was effective at improving glucose tolerance in these animals. This strategy offers potential therapeutic benefit to patients with obesity and diabetes. In summary, this body of work provides new insights into the biological actions and therapeutic implications of carnosine and associated analogues