Regulation of metabolism during hibernation in brown bears (Ursus arctos): Involvement of cortisol, PGC-1α and AMPK in adipose tissue and skeletal muscle.

The purpose of this study was to investigate changes in expression of known cellular regulators of metabolism during hyperphagia (Sept) and hibernation (Jan) in skeletal muscle and adipose tissue of brown bears and determine whether signaling molecules and transcription factors known to respond to changes in cellular energy state are involved in the regulation of these metabolic adaptations. During hibernation, serum levels of cortisol, glycerol, and triglycerides were elevated, and protein expression and activation of AMPK in skeletal muscle and adipose tissue were reduced. mRNA expression of the co-activator PGC-1α was reduced in all tissues in hibernation whereas mRNA expression of the transcription factor PPAR-α was reduced in the vastus lateralis muscle and adipose tissue only. During hibernation, gene expression of ATGL and CD36 was not altered; however, HSL gene expression was reduced in adipose tissue. During hibernation gene expression of the lipogenic enzyme DGAT in all tissues and the expression of the FA oxidative enzyme LCAD in the vastus lateralis muscle were reduced. Gene and protein expression of the glucose transporter GLUT4 was decreased in adipose tissue in hibernation. Our data suggest that high cortisol levels are a key adaptation during hibernation and link cortisol to a reduced activation of the AMPK/PGC-1α/PPAR-α axis in the regulation of metabolism in skeletal muscle and adipose tissue. Moreover, our results indicate that during this phase of hibernation at a time when metabolic rate is significantly reduced metabolic adaptations in peripheral tissues seek to limit the detrimental effects of unduly large energy dissipation.

Amelioration of palmitate-induced metabolic dysfunction in L6 muscle cells expressing low levels of receptor-interacting protein 140.

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.

Genetic downregulation of receptor-interacting protein 140 uncovers the central role of Akt signalling in the regulation of fatty acid oxidation in skeletal muscle cells.

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. Our data show that, in skeletal muscle cells, RIP140 expression significantly impacts palmitate uptake and oxidation and that alterations in gene expression and Akt-atypical protein kinaseC-ζ signalling can partly explain these changes.

AMP-activated protein kinase α2 is an essential signal in the regulation of insulin-stimulated fatty acid uptake in control-fed and high-fat-fed mice.

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 Akt(Thr308) 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 pro-inflammatory markers.