Molecular dissection of pathways regulating glucose and lipid metabolism using siRNA

<p>Type 2 diabetes is a complex chronic disease that is associated with skeletal muscle insulin resistance. The aim of this thesis is to investigate pathways regulating glucose and lipid metabolism in human skeletal muscle.</p><p>Whole body metabolism differs between men and women. Epidemiological studies show gender-dependent variations in risk factors for type 2 diabetes. Therefore, we tested the hypothesis that intrinsic sex differences contribute to skeletal muscle metabolism and gene expression. Sex-dependent differences in expression of genes important for glucose and lipid metabolism were observed in skeletal muscle biopsies from age-matched male and female subjects. When the same genes were analyzed in cultured myotubes from the same subjects no sex-dependent differences were found. Furthermore, basal and insulin-stimulated glucose metabolism, as well as lipid uptake and beta-oxidation, were similar in myotubes between the sexes. Thus, we provide evidence against sexual dimorphism in metabolism in cultured myotubes. Differences seen between males and females in vivo are likely a consequence of systemic factors.</p><p>The complexity of the insulin signaling pathway is magnified by the existence of multiple isoforms of key signaling molecules. Insulin receptor substrate (IRS) 1 and 2 are the first post-receptor signaling molecules in the insulin signaling cascade, while Akt is a downstream kinase important for metabolic responses and cell survival. siRNA-mediated gene silencing was used to determine the relative contribution of IRS and Akt isoforms to glucose and lipid metabolism in human myotubes. Our results provide evidence that IRS-1 and Akt2 are required for cell growth and differentiation, as well as insulin-stimulated glucose uptake and incorporation into glycogen, whereas IRS-2 and Akt1 are dispensable. In contrast, IRS-2 was necessary for insulin action on lipid uptake and metabolism. Thus, we provide evidence for isoform-specific regulation in the insulin signaling cascade governing glucose and lipid metabolism.</p><p>Insulin resistance has been linked to low-grade inflammation and increased circulatory inflammatory cytokines. We determined the role of inhibitor of nuclear factor-κbeta kinase beta (IKKbeta) in TNF-alpha-induced insulin resistance in cultured human myotubes. Deletion of IKKbeta fully protected against TNF-alpha-induced insulin resistance at the level of Akt and AS160 phosphorylation. Importantly, TNF-alpha-induced impairments in insulin action on glucose uptake and incorporation into glycogen were completely restored by siRNA-mediated gene silencing of IKKbeta.</p><p>The AMPK-related kinase SNF (sucrose, non-fermenting) 1/AMPK-related kinase (SNARK) has been linked to obesity and type 2 diabetes. We show that SNARK expression is unaltered in skeletal muscle from type 2 diabetic patients. Conversely, SNARK expression is increased in obese humans, and in cultured myotubes exposed to metabolic stressors including TNF-alpha and palmitate. siRNA-mediated SNARK silencing was without effect on basal or insulin-stimulated glucose and lipid metabolism, and failed to prevent TNF-alpha- or palmitate-induced insulin resistance. Thus, SNARK does not directly influence skeletal muscle metabolism in human.</p><p>In conclusion, siRNA has been used to validate several molecular mechanisms governing insulin-dependent control of glucose and lipid metabolism in skeletal muscle from normal glucose tolerant and type 2 diabetic men and women. Isoform-specific gene targeting of insulin signaling proteins has potential to prevent or correct insulin resistance in skeletal muscle.</p><h3>List of scientific papers</h3><p>I. Rune A, Salehzadeh F, Szekeres F, Kühn I, Osler ME, Al-Khalili L (2009). Evidence against a sexual dimorphism in glucose and fatty acid metabolism in skeletal muscle cultures from age-matched men and post-menopausal women. Acta Physiol (Oxf). Jun 6: Epub ahead of print <br><a href="https://pubmed.ncbi.nlm.nih.gov/19508405">https://pubmed.ncbi.nlm.nih.gov/19508405</a><br><br></p><p>II. Bouzakri K, Zachrisson A, Al-Khalili L, Zhang BB, Koistinen HA, Krook A, Zierath JR (2006). siRNA-based gene silencing reveals specialized roles of IRS-1/Akt2 and IRS-2/Akt1 in glucose and lipid metabolism in human skeletal muscle. Cell Metab. 4(1): 89-96 <br><a href="https://pubmed.ncbi.nlm.nih.gov/16814735">https://pubmed.ncbi.nlm.nih.gov/16814735</a><br><br></p><p>III. Austin RL, Rune A, Bouzakri K, Zierath JR, Krook A (2008). siRNA-mediated reduction of inhibitor of nuclear factor-kappaB kinase prevents tumor necrosis factor-alpha-induced insulin resistance in human skeletal muscle. Diabetes. 57(8): 2066-73. Epub 2008 Apr 28 <br><a href="https://pubmed.ncbi.nlm.nih.gov/18443205">https://pubmed.ncbi.nlm.nih.gov/18443205</a><br><br></p><p>IV. Rune A, Osler ME, Fritz T, Zierath JR (2009). Regulation of skeletal muscle sucrose, non-fermenting 1/AMP-activated protein kinase-related kinase (SNARK) by metabolic stress and diabetes. Diabetologia. Aug 4: Epub ahead of print <br><a href="https://pubmed.ncbi.nlm.nih.gov/19652946">https://pubmed.ncbi.nlm.nih.gov/19652946</a><br><br></p>