Understanding the pathways that maintain proper glucose homeostasis is a central focus of discovery efforts for treatments of Type 2 diabetes which now affects over 20 million Americans, with levels increasing at 6% a year (Stein et al. (2004) J. Clin. Endocrinol. Metab. 89:2522-2525). Proper glucose homeostasis requires a balance between glucose uptake by skeletal muscle and adipose tissue, and production by the liver. Type 2 diabetic patients lose this balance due to a reduction of glucose uptake during the fed state, as well as improper fasting gluconeogenesis by the liver. During the fasted state, glucagon secretion by the pancreas and resulting cAMP mediated signaling through CREB, as well as glucocorticoid release, result in gluconeogenic gene transcription. In the fed state this transcriptional program is suppressed by insulin signaling due to repression of the nuclear hormone receptor co-activator PGC-1α, which is necessary for CREB mediated transcription. Phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) are two rate-limiting enzymes for gluconeogenesis that are transcriptionally regulated by glucagon and insulin, and are widely used as markers for gluconeogenesis (Sutherland et al. (1996) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 351,:91-199).
Due to the important role of dysregulated gluconeogenesis in the pathology of Type 2 diabetes, further insight into the mechanisms of repression of these genes by insulin independent mechanisms could lead to treatments of insulin resistant individuals (Wu et al. (2005) Curr. Drug Targets Immune Endocr. Metabol. Disord. 5:51-59). Activation of AMPK (AMP activated kinase) is one insulin independent means of gluconeogenesis repression. AMPK has been termed a “master switch” of cellular energy status, being highly conserved from simple eukaryotes to humans. In mammalian systems it is activated in multiple organs during conditions that cause a low ATP/AMP ratio. These include exercise and starvation on the whole body level as well as many cellular stresses such as glucose deprivation, oxidative stress, ischemia, and exposure to metabolic poisons that inhibit ATP synthesis (Hardie et al. (2003) FEBS Lett. 546:113-120; Hardie (2004) J. Cell Sci. 117:5479-5487). When activated, AMPK switches on ATP generating processes and switches off those that consume ATP. In vivo and in vitro there is much evidence indicating that AMPK activation inhibits gluconeogenesis (Foretz et al. (1998) J. Biol. Chem. 273:14767-14771). Treatment of rat hepatoma cells or primary hepatocytes with the AMPK activator AICAR inhibits expression G6P and PEPCK (Lochhead et al. (2000) Diabetes 49:896-903). In vivo, activation of AMPK in the livers of fasted mice has been shown to reduce glucose production (Vincent et al. (1996) Diabetologia 39:1148-1155) and gluconeogenic gene expression (Foretz et al. (2005) Diabetes 54:1331-1339). Additionally, AMPK has been suggested to mediate the beneficial and detrimental effects of adiponectin and resistin, respectively, on hepatic glucose output. Recently this has been supported by the finding that genetic deletion of the AMPK alpha2 isoform in the mouse liver leads to glucose intolerance and hyperglycemia in the fasted state, which could be reversed by insulin. Yet, these animals were resistant to regulation of glucose production by the AMPK activators leptin and adiponectin (Andreelli et al. (2006) Endocrinology, en.2005-0898).
AMPK achieves its downstream effects by immediate direct phosphorylation of enzyme substrates as well as long-term effects on gene expression. For example, AMPK phosphorylates and inactivates acetyl CoA carboxylase, resulting in a suppression of the conversion of acetyl CoA to malonyl CoA. The lower levels of malonyl CoA allows entry of fatty acids into the mitochondria and their subsequent oxidation (Winder et al. (1997) J. Appl. Physiol. 82:219-225; Munday et al. (1988) Eur. J. Biochem. 175:331-338). Other direct targets that can be phosphorylated by AMPK include glycogen synthase, IRS-1, and HMG-CoA reductase (Jorgensen et al. (2004) Diabetes 53:3074-3081; Jakobsen et al. (2001) J. Biol. Chem. 276:46912-46916; Clarke et al. (1990) EMBO J. 9:2439-2446). AMPK's effects on transcription, and their role in mediating the physiological effects of AMPK activation are much less well understood, although it has been shown that AMPK activation decreases HNF4 expression levels leading to repression of its target genes including HNF-1alpha, GLUT2, and L-type pyruvate kinase (Hong et al. (2003) J. Biol. Chem. 278:27495-27501).