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  • Metformin a biguanide derivative is


    Metformin, a biguanide derivative, is a commonly used drug in the treatment of type 2 diabetes, as it suppresses endogenous glucose output and increases peripheral insulin sensitivity [39], [40]. In recent years, attention has been concentrated on AMPK in the context of metformin's action [16], [41], but data on the activation of SIRT1 by metformin have also been published. Metformin has been reported to activate hepatic SIRT1 through AMPK-mediated induction of Nampt [19]. In the current study, we observed a stimulating effect of metformin on the enzymatic activity of SIRT1 in podocytes. We also demonstrated that HG concentrations do not alter metformin's ability to activate SIRT1 in podocytes. Moreover, metformin prevented hyperglycemia-induced reductions in SIRT1 Relebactam protein levels in these cells. In parallel, metformin increased AMPK phosphorylation in podocytes incubated in control or hyperglycemic medium. As a result of metformin action, HG-induced impairment of glucose uptake into podocytes was ameliorated. Therefore, despite AMPK activation, increasing SIRT1 protein levels and activity could be an important mechanism by which metformin overcomes insulin resistance in HG-exposed podocytes (Fig. 8). Accumulating data have shown that metformin acts mainly through AMPK, increasing the AMP/ADP and/or ADP/ATP ratios [16]. This effect may be reinforced by a decrease in the cytosolic NAD+/NADH ratio [42], [43]. It was reported that metformin decreased NAD+/NADH ratio in liver to inhibit glucose production via a novel direct target, mitochondrial glycerol-3-phosphate dehydrogenase [44]. Decreased AMPK activity and Nampt and SIRT1 expression have also been observed in the white adipose tissue of db/db mice, and metformin has been shown to increase AMPK activity and restore Nampt and SIRT1 levels [37]. Our results show that the NAD+/NADH ratio decreased in podocytes exposed to hyperglycemic conditions, which is in line with the decreased NAD+/NADH ratio observed in mesangial Relebactam grown in HG medium [45]. Metformin diminished the NAD+/NADH ratio in podocytes exposed to both SG and HG concentrations. These data are also supported by other studies demonstrating that metformin decreases the intracellular NAD+/NADH ratio [46]. Our data suggest that the reduced NAD+/NADH ratio in metformin-treated podocytes could result from SIRT1 activation and subsequent NAD+ consumption. The Nampt protein level was increased in podocytes treated with metformin, presumably to compensate for the NAD+ deficit. Our study demonstrated that the stimulatory effects of metformin on SIRT1 protein level and activity were not affected by transfection with SIRT1 siRNA. Metformin restored the decreased SIRT1 protein levels resulting from transfection and activated SIRT1 in cells transfected with scrambled or SIRT1 siRNA. SIRT1 protein expression did not change after downregulation of AMPK catalytic subunits AMPKα1 or AMPKα2. However, SIRT1 enzymatic activity was decreased in AMPKα1-depleted podocytes, suggesting that crosstalk between SIRT1 and AMPK prefers the AMPKα1 isoform. In contrast, SIRT1-AMPK crosstalk has not been shown to favor either AMPK isoform in skeletal muscle cells [47]. The same research group provided evidence of preferential activation of AMPKα1 by metformin, but metformin also significantly increased AMPK phosphorylation and the activities of both AMPKα1 and AMPKα2 in skeletal muscle cells, where AMPK activation is associated with increased rates of glucose uptake [48]. We found that the degree of AMPK phosphorylation is significantly reduced in podocytes with suppressed expression of SIRT1. Our results are in agreement with those obtained for retinal capillary endothelial cells with SIRT1 knocked down, which have reduced AMPK activation [49]. Our results suggest that metformin activates AMPK independent of SIRT1, because the increase in the degree of AMPK phosphorylation after metformin treatment was not affected by SIRT1 downregulation. A similar effect of metformin was observed in HepG2 cells [50]. We found that metformin-induced activation of AMPK in podocytes occurred regardless of the AMPKα1/AMPKα2 protein level. AMPKα1 and AMPKα2 have different expression patterns in various tissues, and they have been shown to control different metabolic actions [51]; thus, the cellular effects of AMPK activation may depend on whether the AMPKα1 or AMPKα2 isoform is stimulated. Notably, several metabolic actions of metformin have been demonstrated to occur independent of AMPK activation, such as the downregulation of glucose-6-phosphatase expression and inhibition of respiratory complex I, or are mediated by p38 MAPK- and PKC-dependent mechanisms [52], [53].