Molecular dissection of pathways regulating glucose and lipid metabolism using siRNA

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. 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 β-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. 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. Insulin resistance has been linked to low-grade inflammation and increased circulatory inflammatory cytokines. We determined the role of inhibitor of nuclear factor-κβ kinase β (IKKβ) in TNF-α-induced insulin resistance in cultured human myotubes. Deletion of IKKβ fully protected against TNF-α-induced insulin resistance at the level of Akt and AS160 phosphorylation. Importantly, TNF-α-induced impairments in insulin action on glucose uptake and incorporation into glycogen were completely restored by siRNA-mediated gene silencing of IKKβ. 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-α and palmitate. siRNA-mediated SNARK silencing was without effect on basal or insulin-stimulated glucose and lipid metabolism, and failed to prevent TNF-αor palmitate-induced insulin resistance. Thus, SNARK does not directly influence skeletal muscle metabolism in human. 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. LIST OF PUBLICATIONS I. Rune, A. , Salehzadeh, F. , Szekeres, F. , Kuhn, I. , Osler, M.E. , and Al-Khalili, L. (2009). Evidence against a sexual dimorphism in glucose and fatty acid metabolism in skeletal muscle cultures from age-matched men and postmenopausal women. Acta Physiol (Oxf). In press. II. Bouzakri, K. , Zachrisson, A. , Al-Khalili, L. , Zhang, B.B. , Koistinen, H.A. , Krook, A. , and Zierath, J.R. (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, 89-96. III. Austin, R.L. , Rune, A. , Bouzakri, K. , Zierath, J.R. , and 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, 2066-2073. IV. Rune, A. , Osler, M.E. , Fritz, T. , and Zierath, J.R. (2009). Regulation of skeletal muscle sucrose, non-fermenting 1/AMP-activated protein kinaserelated kinase (SNARK) by metabolic stress and diabetes. Diabetologia. In press. LIST OF PUBLICATIONS NOT INCLUDED IN THIS THESIS I. Bouzakri, K. , Austin, R. , Rune, A. , Lassman, M.E. , Garcia-Roves, P.M. , Berger, J.P., Krook, A. , Chibalin, A.V. , Zhang, B.B. , and Zierath, J.R. (2008). Malonyl CoenzymeA decarboxylase regulates lipid and glucose metabolism in human skeletal muscle. Diabetes 57, 1508-1516. II. Barrès, R, Osler M.E. , Yan J., Rune A. , Fritz F. , Caidahl K. , Krook A. , and Zierath J.R. (2009). Non-CpG methylation of the PGC-1α promoter through DNMT3B controls mitochondrial density. Cell Metab. In press.