Abstract
Metformin is a first-line drug for the management of type 2 diabetes. Recent studies suggested cardioprotective effects of metformin against ischemia/reperfusion injury. However, it remains elusive whether metformin provides direct protection against hypoxia/reoxygenation (H/R) injury in cardiomyocytes under normal or hyperglycemic conditions. This study in H9C2 rat cardiomyoblasts was designed to determine cell viability under H/R and high-glucose (HG, 33 mM) conditions and the effects of cotreatment with various concentrations of metformin (0, 1, 5, and 10 mM). We further elucidated molecular mechanisms underlying metformin-induced cytoprotection, especially the possible involvement of AMP-activated protein kinase (AMPK) and Jun NH(2)-terminal kinase (JNK). Results indicated that 5 mM metformin improved cell viability, mitochondrial integrity, and respiratory chain activity under HG and/or H/R (P < 0.05). The beneficial effects were associated with reduced levels of reactive oxygen species generation and proinflammatory cytokines (TNF-α, IL-1α, and IL-6) (P < 0.05). Metformin enhanced phosphorylation level of AMPK and suppressed HG + H/R induced JNK activation. Inhibitor of AMPK (compound C) or activator of JNK (anisomycin) abolished the cytoprotective effects of metformin. In conclusion, our study demonstrated for the first time that metformin possessed direct cytoprotective effects against HG and H/R injury in cardiac cells via signaling mechanisms involving activation of AMPK and concomitant inhibition of JNK.
Highlights
Diabetes mellitus is associated with a number of long-term complications, including nephropathy, retinopathy, stroke, and cardiovascular diseases, which lead to decreased quality of life and reduced life expectancy [1]
Introducing low concentrations (1 or 5 medium containing (mM)) of metformin into the cell culture medium significantly increased the H9C2 cells viability while a higher concentration of metformin (10 mM) aggravated the loss in cell viability induced by HG + H/R treatment (Figure 1), indicating dose-dependence for metformin-induced protective effects, and only a moderate concentration of metformin is cytoprotective against HG + H/R injury
Our results showed that the levels of P-AMPK were markedly lower and its inhibitory downstream target P-ACC were higher in HG or H/R groups than in control group (Figures 2(a) and 2(b)) and the cells in HG + H/R group had the lowest level of P-AMPK (Figure 2(a)) and the resultant highest level of P-ACC (Figure 2(b))
Summary
Diabetes mellitus is associated with a number of long-term complications, including nephropathy, retinopathy, stroke, and cardiovascular diseases, which lead to decreased quality of life and reduced life expectancy [1]. It is widely accepted that metformin leads to activation of AMP-activated protein kinase (AMPK) with increased levels of phosphorylated AMPK [13, 14], which has complex properties on cardiomyocyte functionality and ROS production [12]. In this context, we hypothesized that metformin played a direct protective role against I/R injury in diabetic hearts and we Journal of Diabetes Research tested this hypothesis in an in vitro study using H9C2 rat cardiomyoblasts exposed to H/R injury under a simulated hyperglycemic (HG) condition with or without coincubation with various concentrations of metformin.
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