| Summary: | AMP-activated protein kinase (AMPK) has been proposed to be a therapeutic target for patients with type 2 diabetes and the metabolic syndrome. In skeletal muscle AMPK stimulates glucose uptake and fatty acid oxidation, whereas in liver it inhibits fatty acid and cholesterol synthesis. The Rab GTPase activating proteins Akt substrate of 160 kDa (AS160) and tre-2/USP6, BUB2, cdc16 domain family member 1 (TBC1C1) have been identified as potential targets of both protein kinase B (PKB, also known as Akt) and AMPK which mediate glucose transporter 4 (GLUT4) translocation to the plasma membrane in response to insulin and 5-aminoimidazole-4-carboxamide riboside (AICAR) respectively in muscle. Previous work in our laboratory has demonstrated that AICAR modestly stimulates basal glucose transport, yet inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes, which is in contrast to the effect of AICAR in skeletal muscle. Currently the role of AMPK in adipocytes remains poorly characterised despite the importance of fat tissue in energy homeostasis. To address this, the molecular mechanism of AMPK activation by known stimuli, the acute effect of various AMPK activators on glucose transport, the effect of AMPK inhibition and knockdown on AICAR mediated inhibition of insulin-stimulated glucose transport and the effect of acute AICAR treatment on PKB substrate phosphorylation in 3T3-L1 adipocytes was investigated. In addition the effect of sustained AMPK activation on glucose transport and insulin signaling in 3T3-L1 adipocytes, and the effect of sustained AMPK activation on insulin signaling in human adipose tissue was also investigated. The AMPK activators; sorbitol, metformin, rosiglitazone, arsenite, azide, hydrogen peroxide and isoproterenol were all shown to stimulate AMPK activity in the presence of the Ca2+/Calmodulin dependent protein kinase kinase (CaMKK) inhibitor STO-609, suggesting that these activators activate AMPK via a CaMKK-independent pathway in 3T3-L1 adipocytes. However, A23187-stimulated AMPK activity was abrogated in the presence of STO-609. Isoproterenol, sodium azide and rosiglitazone, were all shown to cause an increase in the ADP/ATP ratio in 3T3-L1 adipocytes compared to control as assessed by high performance liquid chromatography, suggesting that they stimulate AMPK activity in an LKB1-dependent manner. These results suggest a possible role for CaMKK as an upstream AMPK kinase in 3T3-L1 adipocytes, in addition to LKB1. There may also exist other upstream AMPK kinases in 3T3-L1 adipocytes that are both nucleotide and calcium independent since sorbitol, metformin, arsenite, hydrogen peroxide and leptin were found to activate AMPK independently of CaMKK and also showed no significant effect on adenine nucleotide ratios. Sorbitol, rosiglitazone, AICAR, isoproterenol and A769662 all significantly inhibited insulin-stimulated glucose transport. Furthermore, in the presence of the AMPK inhibitor, Compound C, the inhibitory effect of AICAR on insulin-stimulated glucose transport was no longer apparent. However, AICAR still displayed a tendency to inhibit insulin-stimulated glucose transport in 3T3-L1 adipocytes infected with adenoviruses expressing a dominant negative AMPK mutant.The effect of AICAR on basal and insulin-stimulated AS160/TBC1D1 phosphorylation at phospho-Akt substrate (PAS) sites, was assessed. AICAR did not alter AS160/TBC1D1 phosphorylation compared to basal levels, nor perturb insulin-stimulated AS160/TBC1D1 phosphorylation at PAS sites. In addition, AICAR did not appear to alter the phosphorylation of any other proteins at PAS sites. Prolonged AMPK activation by AICAR in 3T3-L1 adipocytes also significantly inhibited insulin-stimulated glucose transport and was not associated with altered PKB protein expression or insulin-stimulated PKB Ser473 phosphorylation. In addition, chronic AMPK activation by metformin in adipose tissue of type 2 diabetic subjects was not associated with altered expression of three key insulin signalling molecules; PKB, the phosphoinositide 3-kinase (PI3K) p85 subunit and insulin receptor substrate 1 (IRS-1). Overall these results suggest a prominent role for LKB1 as an AMPK kinase and a potential role for CaMKK as an AMPK kinase in adipocytes. This study also suggests that both acute and prolonged AMPK activation in adipocytes inhibits insulin-stimulated glucose uptake, however the precise mechanism of inhibition has yet to be elucidated.
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