Mechanism of Activation of AMPK by Cordycepin.

Cordycepin (3'-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin monophosphate.

Osteoblast AMP-activated protein kinase regulates glucose metabolism and bone mass in adult mice.

Previous studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in glucose metabolism and bone mass regulation in adult mice. Here, we used the inducible Cre system to control the onset of Ampk disruption after birth by removing doxycycline supplementation. We conditionally inactivated Ampk in osterix (Osx)-expressing cells in 3-week-old Ampk-/- mice. After 6 months of Ampk inactivation, the Ampk-/- mice displayed lower serum osteocalcin levels as well as glucose intolerance and insulin resistance, as indicated by glucose tolerance and insulin tolerance tests, respectively, when compared with wild-type mice. After 18 months of Ampk inactivation, micro computed tomography showed significant reductions in trabecular bone volume and cortical bone thickness in the femur of Ampk-/- mice when compared with wild-type mice. Moreover, bone stiffness was significantly lower in Ampk-/- mice than in wild-type mice. This is the first study to show that osteoblast AMPK plays an important roles in glucose metabolism and in maintaining trabecular bone volume, cortical thickness, and bone strength in adult mice.

Ilexgenin A inhibits endoplasmic reticulum stress and ameliorates endothelial dysfunction via suppression of TXNIP/NLRP3 inflammasome activation in an AMPK dependent manner.

Ilexgenin A is a natural triterpenoid with beneficial effects on lipid disorders. This study aimed to investigate the effects of ilexgenin A on endothelial homeostasis and its mechanisms. Palmitate (PA) stimulation induced endoplasmic reticulum stress (ER stress) and subsequent thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation in endothelial cells, leading to endothelial dysfunction. Ilexgenin A enhanced LKB1-dependent AMPK activity and improved ER stress by suppression of ROS-associated TXNIP induction. However, these effects were blocked by knockdown of AMPKα, indicating AMPK is essential for its action in suppression of ER stress. Meanwhile, ilexgenin A inhibited NLRP3 inflammasome activation by down-regulation of NLRP3 and cleaved caspase-1 induction, and thereby reduced IL-1ß secretion. It also inhibited inflammation and apoptosis exposed to PA insult. Consistent with these results in endothelial cells, ilexgenin A attenuated ER stress and restored the loss of eNOS activity in vascular endothelium, and thereby improved endothelium-dependent vasodilation in rat aorta. A further analysis in high-fat fed mice showed that oral administration of ilexgenin A blocked ER stress/NLRP3 activation with reduced ROS generation and increased NO production in vascular endothelium, well confirming the beneficial effect of ilexgenin A on endothelial homeostasis in vivo. Taken together, these results show ER stress-associated TXNIP/NLRP3 inflammasome activation was responsible for endothelial dysfunction and ilexgenin A ameliorated endothelial dysfunction by suppressing ER-stress and TXNIP/NLRP3 inflammasome activation with a regulation of AMPK. This finding suggests that the application of ilexgenin A is useful in the management of cardiovascular diseases in obesity.

Screening of isoquinoline alkaloids for potent lipid metabolism modulation with Caenorhabditis elegans.

Metabolic syndrome and related disorders are increasingly prevalent in contemporary society, and thus pose the need for potent agents to control lipid accumulation in the body. This study indicates that Caenorhabditis elegans was effective in screening for potent lipid metabolism modulators with berberine as a model compound. Among the various isoquinoline alkaloids tested, sanguinarine, a benzophenanthridine alkaloid, was found to be the most potent. Sanguinarine, like berberine, reduced lipid accumulation through AMP-activated protein kinase activation. Analysis of AMPK (aak-1 and aak-2) RNAi worms revealed that effects were aak-2-dependent. Characterization of worms with knockdown nhr-49, a hormone nuclear receptor gene that functions as a key regulator of fat consumption, showed that both alkaloids were effective even in these markedly lipid-accumulating nhr-49 RNAi worms, suggesting that they predominantly affect lipid synthesis, rather than fatty acid ß-oxidation. The versatility of C. elegans for the purpose of lipid-modulating chemical screening and characterization of the underlying mechanisms is discussed.

AMP-activated protein kinase α2 subunit is required for the preservation of hepatic insulin sensitivity by n-3 polyunsaturated fatty acids.

OBJECTIVE: The induction of obesity, dyslipidemia, and insulin resistance by high-fat diet in rodents can be prevented by n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). We tested a hypothesis whether AMP-activated protein kinase (AMPK) has a role in the beneficial effects of n-3 LC-PUFAs. RESEARCH DESIGN AND METHODS: Mice with a whole-body deletion of the α2 catalytic subunit of AMPK (AMPKα2(-/-)) and their wild-type littermates were fed on either a low-fat chow, or a corn oil-based high-fat diet (cHF), or a cHF diet with 15% lipids replaced by n-3 LC-PUFA concentrate (cHF+F). RESULTS: Feeding a cHF diet induced obesity, dyslipidemia, hepatic steatosis, and whole-body insulin resistance in mice of both genotypes. Although cHF+F feeding increased hepatic AMPKα2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the cHF+F diet to a similar degree in both AMPKα2(-/-) and wild-type mice in ad libitum-fed state. However, preservation of hepatic insulin sensitivity by n-3 LC-PUFAs required functional AMPKα2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. Under hyperinsulinemic-euglycemic conditions, AMPKα2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 LC-PUFA treatment. CONCLUSIONS: Our results show that n-3 LC-PUFAs prevent hepatic insulin resistance in an AMPKα2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. AMPKα2 is also essential for hypolipidemic and antisteatotic effects of n-3 LC-PUFA under insulin-stimulated conditions.

Loss of AMP-activated protein kinase alpha2 subunit in mouse beta-cells impairs glucose-stimulated insulin secretion and inhibits their sensitivity to hypoglycaemia.

AMPK (AMP-activated protein kinase) signalling plays a key role in whole-body energy homoeostasis, although its precise role in pancreatic beta-cell function remains unclear. In the present study, we therefore investigated whether AMPK plays a critical function in beta-cell glucose sensing and is required for the maintenance of normal glucose homoeostasis. Mice lacking AMPK alpha2 in beta-cells and a population of hypothalamic neurons (RIPCre alpha2KO mice) and RIPCre alpha2KO mice lacking AMPK alpha1 (alpha1KORIPCre alpha2KO) globally were assessed for whole-body glucose homoeostasis and insulin secretion. Isolated pancreatic islets from these mice were assessed for glucose-stimulated insulin secretion and gene expression changes. Cultured beta-cells were examined electrophysiologically for their electrical responsiveness to hypoglycaemia. RIPCre alpha2KO mice exhibited glucose intolerance and impaired GSIS (glucose-stimulated insulin secretion) and this was exacerbated in alpha1KORIPCre alpha2KO mice. Reduced glucose concentrations failed to completely suppress insulin secretion in islets from RIPCre alpha2KO and alpha1KORIPCre alpha2KO mice, and conversely GSIS was impaired. Beta-cells lacking AMPK alpha2 or expressing a kinase-dead AMPK alpha2 failed to hyperpolarize in response to low glucose, although KATP (ATP-sensitive potassium) channel function was intact. We could detect no alteration of GLUT2 (glucose transporter 2), glucose uptake or glucokinase that could explain this glucose insensitivity. UCP2 (uncoupling protein 2) expression was reduced in RIPCre alpha2KO islets and the UCP2 inhibitor genipin suppressed low-glucose-mediated wild-type mouse beta-cell hyperpolarization, mimicking the effect of AMPK alpha2 loss. These results show that AMPK alpha2 activity is necessary to maintain normal pancreatic beta-cell glucose sensing, possibly by maintaining high beta-cell levels of UCP2.