Owing to its critical role in the regulation of skeletal muscle metabolism, AMP-activated protein kinase (AMPK) remains a central focus of research for the treatment of insulin resistance. The purpose of the present study was to determine the role of AMPKα2 activity in the regulation of glucose uptake and fatty acid (FA) metabolism in insulin-resistant skeletal muscle. Male C57BL/6 mice were divided into groups fed a control diet (CD) or high-fat (60%) diet (HFD) for 6 weeks and were either wild-type (WT) or possessed an AMPKα2 dominant negative transgene (DN). After 6 weeks, hindlimbs of CD (n = 10) and HFD mice (n = 10) were perfused with or without 450 μU ml −1 insulin. Muscles of CD (n = 8) and HFD mice (n = 8) were used for measurement of basal protein expression. In CD mice, low AMPKα2 activity did not affect basal FA uptake (FAU), but it increased basal FA oxidation (FAO) by 28% and prevented the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased basal FAU by 147% (P < 0.05). In both WT and DN mice, HFD abolished the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased SIRT1 activity and decreased Protein Tyrosine Phosphatase 1B (PTP1B) expression and AktThr308 phosphorylation (P < 0.05). Adipose tissue protein expression of interleukin-6 and tumour necrosis factor α was increased by HFD in WT mice but not in DN mice (P < 0.05). Skeletal muscle interleukin-15 expression was decreased in both feeding conditions in the DN mice (P < 0.05). The data from this study suggest that in insulin-resistant conditions low AMPKα2 activity impacts the regulation of skeletal muscle FA metabolism via changes in SIRT1 activity, PTP1B expression and Akt phosphorylation and the expression of adipose tissue proinflammatory markers. A precursor and unfavourable indicator of type 2 diabetes mellitus is insulin resistance in skeletal muscle (DeFronzo et al. 1992;Båvenholm et al. 2003). While the mechanisms leading to the development of insulin resistance have not been completely identified, there is evidence that its progression is linked to alterations in fatty acid (FA) metabolism (Laybutt et al. 1999;Chavez & Summers, 2003;Aas et al. 2004 (Winder & Hardie, 1999;Ruderman & Prentki, 2004).AMPK has been studied extensively for its role as an energy sensor during states of low energy balance, as exemplified by muscle contraction or hypoxia (Hutber et al. 1997;Mu et al. 2001). Additionally, multiple studies have linked FA intake and high-fat diets to alterations in AMPK activity in liver and skeletal muscle (Suchankova Fillmore et al. 2010). A recent study has linked the deletion of AMPKα2 activity to an exacerbation of high-fat diet-induced insulin resistance as it pertains to glucose metabolism (Fujii et al. 2008). However, in another study, it was shown that reduced AMPK activity did not affect the development of obesity-induced lipid accumulation and insulin resistance ). As such, the role of low muscle AMPK activ...
New findings r What is the central question of this study?Receptor-interacting protein 140 (RIP140) is a known negative regulator of oxidative capacity. However, the effects of its downregulation on insulin-mediated fatty acid metabolism and of gene expression and Akt-atypical protein kinase C-ζ signalling in skeletal muscle remain incomplete. r What is the main finding and its importance?Our data indicate that downregulation of RIP140 significantly impacts both basal and insulin-mediated activation of Akt and atypical protein kinase C-ζ. This extends the assigned regulatory role of these signalling intermediates during insulin stimulation and suggests that Akt may also be an important regulator of fatty acid oxidation in basal conditions. The role of the nuclear co-repressor receptor-interacting protein 140 (RIP140) in metabolic regulation, gene and protein expression and insulin signalling in skeletal muscle cells remains to be delineated. To study this question, L6 myotubes were treated with or without an RNA interference oligonucleotide sequence to downregulate RIP140 expression and incubated with or without insulin (1 μm). Downregulation of RIP140 increased (P < 0.05) basal palmitate uptake (by 20%) and decreased (P < 0.05) basal palmitate oxidation (by 38%). In control small interfering RNA-treated cells, insulin increased (P < 0.05) glucose (by 31%) and palmitate uptake (by 20%) and decreased (P < 0.05) palmitate oxidation (by 35%). However, in RIP140 small interfering RNA-treated cells, insulin did not affect (P > 0.05) palmitate uptake and increased (P < 0.05) palmitate oxidation (by 79%). In insulin-mediated conditions, downregulation of RIP140 decreased (P < 0.05) Akt Ser473 and atypical protein kinase C-ζ Thr403/410 phosphorylation. As expected, downregulation of RIP140 was accompanied by an increase (P < 0.05) in cytochrome c oxidase subunit 4 isoform 1 and peroxisome proliferator-activated receptor receptor γ coactivator-1α mRNA content. Downregulation of RIP140 increased (P < 0.05) fatty acid transport protein 1 mRNA content and carnitine palmitoyltransferase 1b protein content and decreased (P < 0.05) medium chain acyl-CoA dehydrogenase mRNA content in basal conditions. In insulin-mediated conditions, downregulation of RIP140 increased (P < 0.05) carnitine palmitoyltransferase 1b, fatty acid transport protein 1 and fibroblast growth factor 21 mRNA content and decreased (P < 0.05) medium chain acyl-CoA dehydrogenase mRNA content and plasma membrane fatty acid translocase/cluster of differentiation 36 protein content.
We have shown that reduced expression of receptor-interacting protein 140 (RIP140) alters the regulation of fatty-acid (FA) oxidation in muscle. To determine whether a high level of FA availability alters the effects of RIP140 on metabolic regulation, L6 myotubes were transfected with or without RNA interference oligonucleotide sequences to reduce RIP140 expression, and then incubated with high levels of palmitic acid, with or without insulin. High levels of palmitate reduced basal (53%-58%) and insulin-treated (24%-44%) FA uptake and oxidation, and increased basal glucose uptake (88%). In cells incubated with high levels of palmitate, low RIP140 increased basal FA uptake and insulin-treated FA oxidation and glucose uptake, and decreased basal glucose uptake and insulin-treated FA uptake. Under basal conditions, low RIP140 increased the mRNA content of FAT/CD36 (159%) and COX4 (61%), as well as the protein content of Nur77 (68%), whereas the mRNA expression of FGF21 (50%) was decreased, as was the protein content of CPT1b (35%) and FGF21 (44%). Under insulin-treated conditions, low RIP140 expression increased the mRNA content of MCAD (84%) and Nur77 (84%), as well as the protein content of Nur77 (23%). Thus, a low level of RIP140 restores the rates of FA uptake in the basal state, in part via a reduction in upstream insulin signaling. Our data also indicate that the protein expression of Nur77 may be modulated by RIP140 when muscle cells are metabolically challenged by high levels of palmitate.
To elucidate the role of RIP140 in the regulation of fatty acid uptake (FAU) and oxidation (FAO) and glucose uptake (GU) in insulin‐resistant muscle cells, L6 myotubes were transfected with RNAi to genetically silence a control sequence (CS) or RIP140 (RIP). Cells were then incubated ± palmitic acid (FA: 400μM, 12h) to induce insulin resistance followed by incubation ± insulin (I: 1000 nM) before measurement of glucose and FA kinetics. In line with the presence of insulin resistance, FA treatment raised basal GU (122.2±5.2% vs 230.0±18.8%; P<0.05) and obliterated the I‐induced increase in GU (P<0.05). FA treatment decreased basal FAU (107.1±3.7% vs 45.4±10.5%; P<0.05) and FAO (176.8±19.6 vs 82.7±13.9%; P<0.05). I‐induced FAU and FAO was reduced by 24% (P<0.05) and 44% (P<0.05) with FA treatment. Silencing of RIP140 in FA‐treated cells did not affect basal GU or FAO but it was associated with higher FAU (45.4±10.5% vs 75.0±12.1%; P<0.05). With I stimulation, silencing of RIP140 in FA‐treated cells was associated with higher GU (29%; P<0.05), lower FAU (35%; P<0.05) and no change in FAO (P<0.05). Our data show that reduced RIP140 expression in FA‐treated muscle cells rescues insulin‐mediated GU and basal FAU but does not affect basal or I‐mediated FAO. Together, our results indicate that low RIP140 expression may partially restore insulin sensitivity when insulin resistance is induced by high FA treatment.USC Women in Science and Engineering, USC Integrative and Evolutionary Biology and USC Zumberge Research Innovation Fund
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