Stimulation Of AMP-activated Kinase Research Articles

P738The restoration effect of metformin on the beta-adrenergic signaling, amp-activated kinase and akt-kinase in the myocardium of diabetic rats

Abstract Background The type 2 diabetic mellitus (DM2) induces the myocardial dysfunctions, the impairment of beta-adrenergic regulations and the alterations of energy homeostasis and apoptotic processes in the myocardium are the main causes of diabetic cardiomyopathy. Metformin (MF) is a first-line drug for management of DM2. However, MF effects on the adrenergic signaling, the cell metabolism and survival in the cardiomyocytes are still poorly understood. Purpose To study MF effects on the beta-adrenergic signaling in myocardial membranes and on the expression and activity of beta1- and beta3-adrenergic receptors (ARs), AMP-activated kinase (AMPK), Akt-kinase and the pro- (Bax) and anti-apoptotic (Bcl-2) proteins in the myocardium of diabetic rats. Methods In male rats, DM2 was induced by high-fat/high-carbohydrate diet (three months) and by a single treatment with low-dose streptozotocin. Diabetic rats were treated with MF at two doses, 100 and 200 mg/kg/day, during 28 days. The stimulating effects of beta-AR agonists on adenylyl cyclase (AC) activity in the cardiac membranes were studied by radioisotope method. The gene expression was estimated by qPCR, and the protein amount and their activity were studied by Western blotting. Four groups of animals, such as control (C, n=12), DM2 (D, n=12), and DM2 treated with MF at the doses 100 (D100, n=9) and 200 mg/kg/day (D200, n=9) were investigated. Results MF restores the stimulating AC effect of beta1-agonists, the beta1-AR gene expression and the beta1/beta3-ARs ratio, all reduced in DM2, and restoration effect of MF in Groups D100 and D200 was similar. Meanwhile, in Group D200, but not in Group D100, the AC effect of beta3-agonists and the expression of beta3-AR, both increased in DM2, returns to the control values. In diabetic myocardium, MF restores the AMPK and Akt-kinase activities, reduced in DM2, and it is illustrated by the increased phosphorylation of AMPK by Thr172 and Akt-kinase by Ser473 and Thr308. Despite the fact that MF at a dose of 200 mg/kg/day to a greater extent, as compared to a dose of 100 mg/kg/day, increases the “stimulating” Thr172-phosphorylation, but at the same time it also enhances the “inhibiting” Ser491-phosphorylation of AMPK, decreasing the resulting stimulation of AMPK. In Groups D100 and D200 the Bax/Bcl-2 ratio, significantly increased in DM2, is reduced. This data indicates the weakening of apoptosis in cardiomyocytes in DM2. Conclusion The four-week MF treatment improves the myocardial beta-adrenergic signaling, normalizing the expression and activity of beta1- and beta3-ARs, restores the activity of AMPK and Akt-kinase, the effector components of the signaling cascades responsible for cell survival and metabolism, and normalizes the expression of apoptotic factors. Thus, the cardioprotective MF effect is largely due to the improved myocardial signaling, including the beta1-AR, AMPK and Akt-kinase signaling pathways. Acknowledgement/Funding This work was supported by the Russian Science Foundation (No. 17-75-30052) and partially by the Government Assignment (AAAA-A18-118012290427-7).

AMP-activated protein kinase mediates effects of oxidative stress on embryo gene expression in a mouse model of diabetic embryopathy

Neural tube defects (NTDs) are a common malformation associated with diabetic embryopathy. Maternal hyperglycaemia-induced oxidative stress inhibits the expression of Pax3, a gene that is essential for neural tube closure, and increases the incidence of NTDs. Because oxidative stress can stimulate AMP-activated kinase (AMPK) activity, and AMPK can regulate gene transcription, we hypothesised that increased AMPK activity would mediate the adverse effects of maternal hyperglycaemia-induced oxidative stress on Pax3 expression and NTDs. Pregnant mice were made transiently hyperglycaemic by glucose injection, or hypoxic by housing in a hypoxic chamber, or were treated with antimycin A to induce oxidative stress, and AMPK activity in the embryos was assayed. The effects of stimulating AMPK activity with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on Pax3 expression and NTDs were determined. Vitamin E or glutathione ethyl ester was used to reduce oxidative stress, and compound C was used to inhibit AMPK activation. Murine embryonic stem cells were employed as an in vitro model to study the effects of oxidative stress on AMPK activity and the effects of AMPK stimulation on Pax3 expression. Maternal hyperglycaemia stimulated AMPK activity, and stimulation of AMPK with AICAR inhibited Pax3 expression (in vivo and in vitro) and increased NTDs (in vivo). Stimulation of AMPK by hyperglycaemia, hypoxia or antimycin A was inhibited by antioxidants. The AMPK inhibitor compound C blocked the effects of hyperglycaemia or AA on Pax3 expression and NTDs. Stimulation of AMPK in embryos during a diabetic pregnancy mediates the effects of hyperglycaemia-induced oxidative stress to disturb the expression of the critical Pax3 gene, thereby causing NTDs.

Berberine exerts anti-adipogenic activity through up-regulation of C/EBP inhibitors, CHOP and DEC2

Berberine exerts an anti-adipogenic activity that is associated with the down-regulation of C/EBPα and PPARγ. Stimulation of AMP-activated kinase (AMPK) caused by inhibition of mitochondrial respiration has been suggested to underlie such molecular regulation. In the present study, we show that berberine up-regulated the expression of two different sets of C/EBP inhibitors, CHOP and DEC2, while down-modulating C/EBPα, PPARγ and other adipogenic markers and effectors in differentiating 3T3-L1 preadipocytes and mature adipocytes. Data also suggested that the berberine-induced up-regulation of CHOP and DEC2 was attributable to selective activation of an unfolded protein response (UPR) and modified extracellular environment, respectively. As a result, the anti-adipogenic activity of berberine was diminished remarkably by adjusting the differentiation culture media and limitedly but consistently by knockdown of CHOP expression. Together, up-regulation of C/EBP inhibitors appears to underlie the berberine-induced repression of C/EBPα and PPARγ and, so, the inhibition of adipogenesis.

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2′,3′-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.

Epithelial Sodium Channel Inhibition by AMP-activated Protein Kinase in Oocytes and Polarized Renal Epithelial Cells

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.