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.

Insulin resistance of skeletal muscle is a major defect in obesity and type 2 diabetes. Insulin resistance has been associated with a chronic subclinical inflammatory state in epidemiological studies and specifically with activation of the inhibitor B kinase (IBK)-nuclear factor-B (NF-B) pathway. However, it is unclear whether this pathway plays a role in mediating insulin resistance in muscle in vivo. We separately overexpressed the p65 subunit of NF-B and IBK␤ in single muscles of rats using in vivo electrotransfer and compared the effects after 1 wk vs. paired contralateral control muscles. A 64% increase in p65 protein (P Ͻ 0.001) was sufficient to cause muscle fiber atrophy but had no effect on glucose disposal or glycogen storage in muscle under hyperinsulinemic-euglycemic clamp conditions. Similarly, a 650% increase in IBK␤ expression (P Ͻ 0.001) caused a significant reduction in IB protein but also had no effect on clamp glucose disposal after lipid infusion. In fact, IBK␤ overexpression in particular caused increases in activating tyrosine phosphorylation of insulin receptor substrate-1 (24%; P ϭ 0.02) and serine phosphorylation of Akt (23%; P Ͻ 0.001), implying a moderate increase in flux through the insulin signaling cascade. Interestingly, p65 overexpression resulted in a negative feedback reduction of 36% in Toll-like receptor (TLR)-2 (P ϭ 0.03) but not TLR-4 mRNA. In conclusion, activation of the IBK␤-NF-B pathway in muscle does not seem to be an important local mediator of insulin resistance.inhibitor kinase-nuclear factor-B pathway; skeletal muscle; Tolllike receptors; in vivo electrotransfer TYPE 2 DIABETES AND OBESITY are intimately related and rapidly increasing human health problems worldwide.