Skeletal muscle-specific overproduction of constitutively activated c-Jun N-terminal kinase (JNK) induces insulin resistance in mice

Abstract

Aims/hypothesis Although skeletal muscle insulin resistance has been associated with activation of c-Jun N-terminal kinase (JNK), whether increased JNK activity causes insulin resistance in this organ is not clear. In this study we examined the metabolic consequences of isolated JNK phosphorylation in muscle tissue. Methods Plasmids containing genes encoding a wild-type JNK1 (WT-JNK) or a JNK1/JNKK2 fusion protein (rendering JNK constitutively active; CA-Jnk) were electroporated into one tibialis anterior (TA) muscle of C57Bl/6 mice, with the contralateral TA injected with an empty vector (CON) to serve as a within-animal control. Results Overproduction of WT-JNK resulted in a modest (~25%) increase in phosphorylation (Thr183/Tyr185) of JNK, but no differences were observed in Ser307 phosphorylation of insulin receptor substrate 1 (IRS-1) or total IRS-1 protein, nor in insulin-stimulated glucose clearance into the TA muscle when comparing WT-JNK with CON. By contrast, overexpression of CA-Jnk, which markedly increased the phosphorylation of CA-JNK, also increased serine phosphorylation of IRS-1, markedly decreased total IRS-1 protein, and decreased insulin-stimulated phosphorylation of the insulin receptor (Tyr1361) and phosphorylation of Akt at (Ser473 and Thr308) compared with CON. Moreover, overexpression of CA-Jnk decreased insulin-stimulated glucose clearance into the TA muscle compared with CON and these effects were observed without changes in intramuscular lipid species. Conclusions/interpretation Constitutive activation of JNK in skeletal muscle impairs insulin signalling at the level of IRS-1 and Akt, a process which results in the disruption of normal glucose clearance into the muscle.

Fatty acid metabolism, energy expenditure and insulin resistance in muscle

“…Ceramide has been reported to cause insulin resistance by impairing insulin signalling at the level of Akt (SchmitzPeiffer et al 1999, Bruce et al 2006, Holland et al 2007). In addition, ceramide is a potent activator of inflammatory molecules, including c-Jun N-terminal kinase (JNK; Westwick et al 1995) and nuclear factor kB/inducer of k kinase (IKK) (Wang et al 1999), which have been reported to be associated with the development of muscle insulin resistance (Itani et al 2002, Sriwijitkamol et al 2006, Henstridge et al 2012. However, while inflammation has been proposed as a critical factor causing insulin resistance, studies carried out by our group and other groups suggest that inflammation is not involved in the initiation of lipid-induced insulin resistance, but may be more important in the exacerbation and maintenance of insulin resistance once obesity is established .…”

Genetic manipulation of cardiac Hsp72 levels does not alter substrate metabolism but reveals insights into high-fat feeding-induced cardiac insulin resistance

“…Lipid content was determined in the cardiac tissue using the methods previously described (Henstridge et al 2012;Matthews et al 2010;Weir et al 2013). Briefly, samples (20-30 mg wet weight) were homogenized in 100 μL PBS buffer, pH 7.47.…”

“…Heart samples were lysed and protein concentration was determined and resolved by SDS-PAGE as previously described (Henstridge et al 2012). Immunoblotting was performed using the following primary antibodies: Hsp72 , H sp90 (Enz o Life Scien ces (fo rmerly Stressgen), PA, USA), and GAPDH (Cell Signaling, Danvers, USA).…”

Molecular characterisation of the adiponectin receptors, AdipoR1 and AdipoR2

“…This likely reflects a combination of the low level of expression of the receptors and intrinsic limitations of the approach 209 . Thus, we employed an alternate approach to address the physiological significance by characterizing the effect of overexpression of WT and canonical palmitoylation defective receptor constructs in mouse skeletal muscle 212,215 . Overexpression of the WT receptors for two weeks resulted in a constitutive increase in phosphorylation of AMPK, AKT and ERK.…”

“…To investigate the requirement for adiponectin receptor palmitoylation in a more physiological setting we employed in vivo electrotransfer (IVE) 212,215 to characterize the effects of overexpression of WT or canonical palmitoylation defective constructs, namely R1 C124A & R2 C135A…”