Abstract
APPL1 is an adaptor protein that binds to both AKT and adiponectin receptors and is hypothesised to mediate the effects of adiponectin in activating downstream effectors such as AMP-activated protein kinase (AMPK). We aimed to establish whether APPL1 plays a physiological role in mediating glycogen accumulation and insulin sensitivity in muscle and the signalling pathways involved. In vivo electrotransfer of cDNA- and shRNA-expressing constructs was used to over-express or silence APPL1 for 1 week in single tibialis cranialis muscles of rats. Resulting changes in glucose and lipid metabolism and signalling pathway activation were investigated under basal conditions and in high-fat diet (HFD)- or chow-fed rats under hyperinsulinaemic–euglycaemic clamp conditions. APPL1 over-expression (OE) caused an increase in glycogen storage and insulin-stimulated glycogen synthesis in muscle, accompanied by a modest increase in glucose uptake. Glycogen synthesis during the clamp was reduced by HFD but normalised by APPL1 OE. These effects are likely explained by APPL1 OE-induced increase in basal and insulin-stimulated phosphorylation of IRS1, AKT, GSK3β and TBC1D4. On the contrary, APPL1 OE, such as HFD, reduced AMPK and acetyl-CoA carboxylase phosphorylation and PPARγ coactivator-1α and uncoupling protein 3 expression. Furthermore, APPL1 silencing caused complementary changes in glycogen storage and phosphorylation of AMPK and PI3-kinase pathway intermediates. Thus, APPL1 may provide a means for crosstalk between adiponectin and insulin signalling pathways, mediating the insulin-sensitising effects of adiponectin on muscle glucose disposal. These effects do not appear to require AMPK. Activation of signalling mediated via APPL1 may be beneficial in overcoming muscle insulin resistance.
Highlights
Resistance of skeletal muscle to the action of insulin is an essential pre-requisite for the development of type 2 diabetes and is frequently associated with obesity (Zimmet et al 2001)
The APPL2 antibody was from Abnova (Heidelberg, Germany), pY608-IRS1 antibody was from Biosource International (Camarillo, CA, USA), uncoupling protein (UCP) 3 antibody from Thermo Scientific (Rockford, IL, USA), total IRS1 and anti-peroxisome proliferator-activated receptor gamma coactivator (PGC) 1a from Calbiochem (Merck Chemicals), total GSK3a/b and pT642-TBC1D4 antibodies from Millipore (Billerica, MA, USA), GFP antibody from Molecular Probes (Leiden, The Netherlands), anti-FLAG and actin antibodies from Sigma, and all others from Cell Signaling Technology (Beverley, MA, USA)
In vivo electrotransfer (IVE) of EH114-GW-APPL1 was used to over-express APPL1 in the right Tibialis cranialis muscles (TCMs) of cohorts of chow-fed young adult rats, with EH114-EGFP administered to the left TCMs as paired control
Summary
Resistance of skeletal muscle to the action of insulin is an essential pre-requisite for the development of type 2 diabetes and is frequently associated with obesity (Zimmet et al 2001). Adiponectin circulates at a concentration of 5–10 mg/ml (Weyer et al 2001), orders of magnitude greater than the levels of other insulin-sensitising adipokines, such as leptin (Segal et al 1996). It is uncertain whether the relatively modest variations in the circulating level are of physiological significance or whether the primary regulation of adiponectin action occurs at the target tissue
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