FFAR4 (GPR120) Signaling Is Not Required for Anti-Inflammatory and Insulin-Sensitizing Effects of Omega-3 Fatty Acids.

Free fatty acid receptor-4 (FFAR4), also known as GPR120, has been reported to mediate the beneficial effects of omega-3 polyunsaturated fatty acids (ω3-PUFAs) by inducing an anti-inflammatory immune response. Thus, activation of FFAR4 has been reported to ameliorate chronic low-grade inflammation and insulin resistance accompanying obesity. However, conflicting reports on the role of FFAR4 in mediating the effects of ω3-PUFAs are emerging, suggesting that FFAR4 may not be the sole effector. Hence analyses of the importance of this receptor in relation to other signaling pathways and prominent effects of ω3-PUFAs remain to be elucidated. In the present study, we used Ffar4 knockouts (KO) and heterozygous (HET) mice fed either low fat, low sucrose reference diet; high fat, high sucrose ω3-PUFA; or high fat, high sucrose ω6-PUFA diet for 36 weeks. We demonstrate that both KO and HET mice fed ω3-PUFAs were protected against obesity, hepatic triacylglycerol accumulation, and whole-body insulin resistance. Moreover, ω3-PUFA fed mice had increased circulating protein levels of the anti-inflammatory adipokine, adiponectin, decreased fasting insulin levels, and decreased mRNA expression of several proinflammatory molecules within visceral adipose tissue. In conclusion, we find that FFAR4 signaling is not required for the reported anti-inflammatory and insulin-sensitizing effects mediated by ω3-PUFAs.

Partial Disruption of Lipolysis Increases Postexercise Insulin Sensitivity in Skeletal Muscle Despite Accumulation of DAG.

Type 2 diabetes and skeletal muscle insulin resistance have been linked to accumulation of the intramyocellular lipid-intermediate diacylglycerol (DAG). However, recent animal and human studies have questioned such an association. Given that DAG appears in different stereoisomers and has different reactivity in vitro, we investigated whether the described function of DAGs as mediators of lipid-induced insulin resistance was dependent on the different DAG isomers. We measured insulin-stimulated glucose uptake in hormone-sensitive lipase (HSL) knockout (KO) mice after treadmill exercise to stimulate the accumulation of DAGs in skeletal muscle. We found that, despite an increased DAG content in muscle after exercise in HSL KO mice, the HSL KO mice showed a higher insulin-stimulated glucose uptake postexercise compared with wild-type mice. Further analysis of the chemical structure and cellular localization of DAG in skeletal muscle revealed that HSL KO mice accumulated sn-1,3 DAG and not sn-1,2 DAG. Accordingly, these results highlight the importance of taking the chemical structure and cellular localization of DAG into account when evaluating the role of DAG in lipid-induced insulin resistance in skeletal muscle and that the accumulation of sn-1,3 DAG originating from lipolysis does not inhibit insulin-stimulated glucose uptake.

Role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle.

During induction of the autophagosomal degradation process, LC3-I is lipidated to LC3-II and associates to the cargo isolation membrane allowing for autophagosome formation. Lipidation of LC3 results in an increased LC3-II/LC3-I ratio, and this ratio is an often used marker for autophagy in various tissues, including skeletal muscle. From cell studies AMPK has been proposed to be necessary and sufficient for LC3 lipidation. The aim of the present study was to investigate the role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle. We observed an increase in the LC3-II/LC3-I ratio in skeletal muscle of AMPKα2 kinase-dead (KD) (p<0.001) and wild type (WT) (p<0.05) mice after 12h of fasting, which was greater (p<0.05) in AMPKα2 KD mice than in WT. The fasting-induced increase in the LC3-II/LC3-I ratio in both genotypes coincided with an initial decrease (p<0.01) in plasma insulin concentration, a subsequent decrease in muscle mTORC1 signaling and increased (p<0.05) levels of the autophagy-promoting proteins, FoxO3a and ULK1. Furthermore, a higher (p<0.01) LC3-II/LC3-I ratio was observed in old compared to young mice. We were not able to detect any change in LC3 lipidation with either in vivo treadmill exercise or in situ contractions. Collectively, these findings suggest that AMPKα2 is not necessary for induction of LC3 lipidation with fasting and aging. Furthermore, LC3 lipidation is increased in muscle lacking functional AMPKα2 during fasting and aging. Moreover, LC3 lipidation seems not to be a universal response to muscle contraction in mice.

LKB1 regulates lipid oxidation during exercise independently of AMPK.

Lipid metabolism is important for health and insulin action, yet the fundamental process of regulating lipid metabolism during muscle contraction is incompletely understood. Here, we show that liver kinase B1 (LKB1) muscle-specific knockout (LKB1 MKO) mice display decreased fatty acid (FA) oxidation during treadmill exercise. LKB1 MKO mice also show decreased muscle SIK3 activity, increased histone deacetylase 4 expression, decreased NAD⁺ concentration and SIRT1 activity, and decreased expression of genes involved in FA oxidation. In AMP-activated protein kinase (AMPK)α2 KO mice, substrate use was similar to that in WT mice, which excluded that decreased FA oxidation in LKB1 MKO mice was due to decreased AMPKα2 activity. Additionally, LKB1 MKO muscle demonstrated decreased FA oxidation in vitro. A markedly decreased phosphorylation of TBC1D1, a proposed regulator of FA transport, and a low CoA content could contribute to the low FA oxidation in LKB1 MKO. LKB1 deficiency did not reduce muscle glucose uptake or oxidation during exercise in vivo, excluding a general impairment of substrate use during exercise in LKB1 MKO mice. Our findings demonstrate that LKB1 is a novel molecular regulator of major importance for FA oxidation but not glucose uptake in muscle during exercise.