Supplementary MaterialsSupplementary Data. is usually a critical target of LKB1/AMPK signals in the regulation of gluconeogenesis. Finally, we show that metformin, one of the most widely prescribed type 2 diabetes therapeutics, requires LKB1 in the liver to lower blood glucose levels. Introduction The adenosine monophosphateCactivated protein kinase (AMPK) is usually a conserved regulator of the cellular response to low energy, and it is activated when intracellular adenosine triphosphate (ATP) concentrations decrease and AMP concentrations increase in response to nutrient deprivation and pathological stresses (1). In budding yeast, the AMPK homolog Snf1 is usually activated in response to glucose limitation. In mammals, AMPK has a DNAJC15 crucial role in many metabolic processes, including glucose uptake and fatty acid oxidation in muscle mass, fatty acid synthesis and gluconeogenesis in the liver, and the regulation of food intake in the hypothalamus (1-4). AMPK exists as a heterotrimer, composed of the catalytic kinase subunit and two associated regulatory subunits, and (1). Upon energy stress, AMP directly binds to tandem repeats of crystathionine- synthase (CBS) domains in the AMPK subunit, causing a conformation switch that exposes the activation loop in the subunit, allowing it to be phosphorylated SNS-032 inhibition by an upstream kinase (1). The sequence flanking the crucial activation loop threonine (Thr172 in human AMPK) is usually conserved across species, and its phosphorylation is absolutely required for AMPK activation. Three papers (5-7) recently reported that this kinase LKB1 is usually biochemically sufficient to activate AMPK in vitro and is genetically required for AMPK activation by energy stress in a number of mammalian cell lines. Because of this potent connection to AMPK, we began to consider the possibility that LKB1 might normally function as a central regulator of organismal metabolism. In the liver, AMPK is usually regulated in response to adipokines such as adiponectin and resistin, which serve to stimulate and inhibit AMPK activation, respectively (8, 9). Exercise and several current diabetes therapeutics activate AMPK in muscle mass and in liver and are SNS-032 inhibition thought to therapeutically take action in part through stimulation of this pathway in those tissues (10-16). However, Ca2+ calmodulin-dependent protein kinase kinase (CAMKK) also activates AMPK (14-16). CAMKK phosphorylates and activates AMPK in response to calcium, whereas LKB1 appears to be responsible for regulating AMPK under energy stress conditions that involve the accumulation of intracellular AMP (17-20). Moreover, in budding yeast, you will find three AMPK kinases (AMPKKs) that are functionally redundant, and all three contribute to metabolic regulation (21-24). Therefore, it was unclear whether LKB1, CAMKK, or another AMPKK might regulate AMPK activity in crucial metabolic tissues in mammals. We genetically deleted LKB1 in adult mouse liver and examined its role in AMPK activation and the effect of the loss of this pathway on glucose homeostasis. We also examined the therapeutic response to metformin, which is a drug widely used to lower blood glucose concentrations in diabetes patients. Finally, we have defined a signaling pathway by which LKB1 regulates a specific CREB (cAMP response elementCbinding protein) coactivator that serves as a rate-limiting switch controlling gluconeogenesis in the liver. LKB1 deletion in liver results in loss of AMPK activation We generated cohorts of mice that were either wild-type for LKB1 or were homozygous for any conditional floxed allele of LKB1 (25) by breeding 0.01 at all time points. (B) Glucose-tolerance SNS-032 inhibition test (GTT) on mice of indicated genotypes 2 weeks after adenoviral Cre injection. (C) Insulin-tolerance test (ITT) on mice of indicated genotypes 2 weeks after adenoviral Cre injection. No significant difference was observed. Data represents the mean + SEM for six mice of each genotype. Average T0 glucose levels for the wild-type mice = 180 mg/dl; average T0 glucose levels for the L/L mice = 355 mg/dl. The increase in blood glucose in mice lacking liver LKB1 was accompanied over time by compensatory increases in blood insulin levels, as expected for mice with normal SNS-032 inhibition pancreatic function (fig. S3). Despite these changes in blood glucose and insulin profiles, mice lacking LKB1 in the liver did not demonstrate increased body weight compared with their control littermates, even when placed on a high-fat diet for 2 months (fig. S4). LKB1 loss results in increased gluconeogenic and lipogenic gene expression The observed hyperglycemia in the mice lacking hepatic LKB1 may result from an failure SNS-032 inhibition to appropriately turn off gluconeogenesis. To.