Insulin sensitivity and nutrient utilisation in skeletal muscle.

Obesity is a condition in which fat accumulation in adipose tissue is in excess to an extent that health may be impaired. Insulin resistance is integral to the pathophysiology of obesity-related metabolic complications. Central adiposity and skeletal muscle mass and function determine insulin sensitivity and metabolic risk. A high visceral fat-to-skeletal muscle mass-ratio contributes to an unfavourable metabolic profile. Epidemiological and experimental studies suggest that high dietary saturated fat intake is deleterious while polyunsaturated fatty acids (PUFAs), in particular n-3 PUFAs of marine origin, may be advantageous to metabolic health. The aim was to determine the effect of subcutaneous (SC) and visceral (IAB) fat, and long-chain saturated, n-3 and n-6 PUFAs, and the interactions between them, on insulin sensitivity and the pathways regulating energy metabolism in skeletal muscle. Thereby an adipose tissue-conditioned media-skeletal muscle myotube co-culture system was developed. Adipose tissue-conditioned medium (CM) was generated from SC and IAB fat biopsy of obese humans. Viability of the tissue explants was confirmed by the measurement of lactate dehydrogenase activity in the CM and nuclear DNA fragmentation of tissue explants. The concentrations of cytokines (leptin, adiponectin, interleukin (IL)-1β, IL 6, IL-8, tumor necrosis factor-α, resistin and plasminogen activator inhibitor-1) and long-chain fatty acids were determined in CM. CM from IAB but not SC fat reduced insulin-stimulated glucose uptake. The effect of IAB fat was predominantly mediated by IL-6 via the activation of a nuclear factor kappa B/mammalian target of rapamycin complex 1 (NFκB/mTORC1)-dependent pathway. Palmitic acid (PA; 16:0) reduced insulin-stimulated glucose uptake, an effect mediated by intramuscular accumulation of ceramide and the activation of NFκB and mTORC1. The effects of fatty acids were similar in the presence of CM from either fat depot, where the effect of PA was partially reversed by docosahexaenoic acid (DHA; 22:6n-3) and completely by linoleic acid (LA; 18:2n-6). The effect of each fatty acid in the presence or absence of CM from each fat depot on mRNA expression of key genes regulating muscle energy metabolism was determined. Protein phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)-α and acetyl-coenzyme A carboxylase (ACC)-β were also determined. PA increased SCD1 mRNA. DHA and LA increased AMPKα2 mRNA and AMPKα and ACCβ protein phosphorylation. Microarray analysis was used to determine the global gene expression changes in PAand DHA-treated L6 myotubes. DHA down-regulated lipogenic genes and upregulated genes which were involved in β-oxidation and mitochondrial function. When compared to PA, DHA down-regulated genes which were involved in lipid synthesis, endoplasmic reticulum metabolism and mitogen-activated protein kinase activity. Taken together, pro-inflammatory cytokines from IAB fat and PA induced insulin resistance in skeletal muscle and both were at least partly mediated by a NFκB/mTORC1-dependent pathway. In contrast, DHA and LA may improve insulin sensitivity by diverting fatty acids towards oxidation and subsequently reducing substrate availability for the formation of lipid metabolites including ceramide. A reduction in PA intake and substitution (rather than addition) of DHA and LA, together with a reduction in overall energy intake and increase in physical activity, is optimal for metabolic health.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201