Metformin is widely used to treat hyperglycemia in individuals with type 2 diabetes. Recently the LKB1/AMPactivated protein kinase (LKB1/AMPK) pathway was proposed to mediate the action of metformin on hepatic gluconeogenesis. However, the molecular mechanism by which this pathway operates had remained elusive. Surprisingly, here we have found that in mice lacking AMPK in the liver, blood glucose levels were comparable to those in wild-type mice, and the hypoglycemic effect of metformin was maintained. Hepatocytes lacking AMPK displayed normal glucose production and gluconeogenic gene expression compared with wild-type hepatocytes. In contrast, gluconeogenesis was upregulated in LKB1-deficient hepatocytes. Metformin decreased expression of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), while cytosolic phosphoenolpyruvate carboxykinase (Pepck) gene expression was unaffected in wild-type, AMPK-deficient, and LKB1-deficient hepatocytes. Surprisingly, metformin-induced inhibition of glucose production was amplified in both AMPK-and LKB1-deficient compared with wild-type hepatocytes. This inhibition correlated in a dose-dependent manner with a reduction in intracellular ATP content, which is crucial for glucose production. Moreover, metformin-induced inhibition of glucose production was preserved under forced expression of gluconeogenic genes through PPARγ coactivator 1α (PGC-1α) overexpression, indicating that metformin suppresses gluconeogenesis via a transcription-independent process. In conclusion, we demonstrate that metformin inhibits hepatic gluconeogenesis in an LKB1-and AMPK-independent manner via a decrease in hepatic energy state.

This review focuses on remarkable recent findings concerning the mechanism by which the LKB1 protein kinase that is mutated in Peutz-Jeghers cancer syndrome operates as a tumor suppressor. We discuss evidence that the cellular localization and activity of LKB1 is controlled through its interaction with a catalytically inactive protein resembling a protein kinase, termed STRAD, and an armadillo repeat-containing protein, named mouse protein 25 (MO25). The data suggest that LKB1 functions as a tumor suppressor by not only inhibiting proliferation, but also by exerting profound effects on cell polarity and, most unexpectedly, on the ability of a cell to detect and respond to low cellular energy levels. Genetic and biochemical findings indicate that LKB1 exerts its effects by phosphorylating and activating 14 protein kinases, all related to the AMP-activated protein kinase. The work described in this review shows how a study of an obscure cancer syndrome can uncover new and important regulatory pathways, relevant to the understanding of multiple human diseases.

Salicylate, a plant product, has been in medicinal use since ancient times. More recently it has been replaced by synthetic derivatives such as aspirin and salsalate, both rapidly broken down to salicylate in vivo. At concentrations reached in plasma following administration of salsalate, or aspirin at high doses, salicylate activates adenosine monophosphate-activated protein kinase (AMPK), a central regulator of cell growth and metabolism. Salicylate binds at the same site as the synthetic activator, A-769662, to cause allosteric activation and inhibition of dephosphorylation of the activating phosphorylation site, Thr172. In AMPK knockout mice, effects of salicylate to increase fat utilization and lower plasma fatty acids in vivo were lost. Our results suggest that AMPK activation could explain some beneficial effects of salsalate and aspirin in humans.The medicinal effects of willow bark have been known since the time of Hippocrates. The active component is salicylate, a hormone produced by plants in response to pathogen infection (1). For medicinal use it was largely replaced by aspirin (acetyl salicylate), which is rapidly broken down to salicylate in vivo (2, 3). Salicylate can also be administered as salsalate, which shows promise for treatment of insulin resistance and type 2 diabetes (4, 5). Aspirin and salicylate inhibit cyclo-oxygenases and hence prostanoid biosynthesis (6), as well as the protein kinase IKKβ in the NF-κB pathway (7). However, some effects of these drugs are still observed in mice deficient in these pathways (8).Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor conserved throughout eukaryotes. This heterotrimeric enzyme is composed of catalytic α Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts subunits and regulatory β and γ subunits (9, 10). Once activated in response to metabolic stress, AMPK phosphorylates targets that switch off adenosine triphosphate (ATP) consuming processes, while switching on catabolic pathways that generate ATP. AMPK is activated >100-fold by phosphorylation at Thr172 in the α subunit by the tumour suppressor protein kinase, LKB1, or the Ca 2+ -dependent kinase, CaMKKβ (9, 10). Binding of AMP or adenosine diphosphate (ADP) to the γ subunit triggers a conformational change that promotes phosphorylation and inhibits dephosphorylation (11-15), causing a switch to the active form. Binding of AMP (but not ADP) to a second site (15) causes further allosteric activation, leading to >1,000-fold activation overall (16). Most drugs or xenobiotics that activate AMPK work by inhibiting mitochondrial ATP synthesis and increasing the concentration of AMP and ADP (17). However, a synthetic activator, A-769662 (18), which also causes allosteric activation and inhibits Thr172 dephosphorylation, binds directly to AMPK at distinct site(s) (19-21).Salicylate, but not aspirin, activated AMPK when applied to HEK-293 cells, with its effects being significant at 1 mM and above ( Fig. 1A; it appears that the estera...

The inactivation of glycogen synthase kinase (GSK)3 has been proposed to play important roles in insulin and Wnt signalling. To define the role that inactivation of GSK3 plays, we generated homozygous knockin mice in which the protein kinase B phosphorylation sites on GSK3a (Ser21) and GSK3b (Ser9) were changed to Ala. The knockin mice were viable and were not diabetic. Using these mice we show that inactivation of GSK3b rather than GSK3a is the major route by which insulin activates muscle glycogen synthase. In contrast, we demonstrate that the activation of muscle glycogen synthase by contraction, the stimulation of muscle glucose uptake by insulin, or the activation of hepatic glycogen synthase by glucose do not require GSK3 phosphorylation on Ser21/ Ser9. GSK3 also becomes inhibited in the Wnt-signalling pathway, by a poorly defined mechanism. In GSK3a/ GSK3b homozygous knockin cells, Wnt3a induces normal inactivation of GSK3, as judged by the stabilisation of b-catenin and stimulation of Wnt-dependent transcription. These results establish the function of Ser21/Ser9 phosphorylation in several processes in which GSK3 inactivation has previously been implicated.

Recent studies indicate that the LKB1 tumour suppressor protein kinase is the major 'upstream' activator of the energy sensor AMP-activated protein kinase (AMPK). We have used mice in which LKB1 is expressed at only B10% of the normal levels in muscle and most other tissues, or that lack LKB1 entirely in skeletal muscle. Muscle expressing only 10% of the normal level of LKB1 had significantly reduced phosphorylation and activation of AMPKa2. In LKB1-lacking muscle, the basal activity of the AMPKa2 isoform was greatly reduced and was not increased by the AMP-mimetic agent, 5-aminoimidazole-4-carboxamide riboside (AICAR), by the antidiabetic drug phenformin, or by muscle contraction. Moreover, phosphorylation of acetyl CoA carboxylase-2, a downstream target of AMPK, was profoundly reduced. Glucose uptake stimulated by AICAR or muscle contraction, but not by insulin, was inhibited in the absence of LKB1. Contraction increased the AMP:ATP ratio to a greater extent in LKB1-deficient muscles than in LKB1-expressing muscles. These studies establish the importance of LKB1 in regulating AMPK activity and cellular energy levels in response to contraction and phenformin.

Adult skeletal muscle has the unique capacity to regenerate. Muscle regeneration is always associated with inflammation and notably macrophages (MPs), which play dual role. Soon after injury, inflammatory monocyte‐derived macrophages (M1 phenotype) stimulate myogenic cell proliferation. After phagocytosis of muscle debris, MPs switch their phenotype to acquire an anti‐inflammatory profile (M2) and stimulate myogenic cell differentiation and myofibre growth. Here, we explored the role of AMPK in the resolution of inflammation during muscle repair. AMPKα1 KO muscle shows both a delay and an impairment of post‐injury regeneration. These deficiencies are also observed in LysM‐CRE;AMPKfl/fl muscle, confirming the MP specificity of AMPK requirement. In vitro, AMPKα1 KO MPs hardly acquire a M2 profile upon cytokine stimulation. Their phagocytic activity is also altered. In vivo analysis of MP subpopulations (using the AMPKα1−/−;CX3CR1GFP/+ mouse) during muscle repair shows that the number of intramuscular MPs exhibiting the M2 phenotype is reduced in the AMPKα1 KO compared to the WT mouse. Accordingly, leukocytes from AMPKα1 KO muscle do not increase their expression of markers associated with the resolution of inflammation during muscle regeneration. These results strongly support that AMPKα1 is one key regulator of MP switch at time of resolution of inflammation and is essential for a proper muscle repair.

Supplemental Digital Content is available in the text.

Contraction induces marked metabolic changes in muscle, and the AMP-activated protein kinase (AMPK) is a good candidate to explain these effects. Recent work using a muscle-specific knockout of the upstream kinase, LKB1, has confirmed that the LKB1-->AMPK cascade is the signaling pathway responsible for many of these effects.