P738The restoration effect of metformin on the beta-adrenergic signaling, amp-activated kinase and akt-kinase in the myocardium of diabetic rats
Authors: Bayrasheva V , Derkach K , Kornyushin O , Bondareva V , Galagudza M , Shpakov A 
Published: October 2019

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).