To the best of our knowledge, this is the first study to clearly illustrate that metformin, which is used worldwide as an antidiabetic medicine, protects against insulin resistance in cardiomyocytes induced by HUA and increases AMPK activation. Of course, we and others have previously shown that HUA induces insulin resistance in various cells, including cardiomyocytes, skeletal muscle cells and HepG2 cells [6–9]. Nevertheless, it has been unclear whether AMPK can regulate insulin resistance induced by HUA in cardiomyocytes in vitro and in vivo. Therefore, we used neonatal mouse primary cardiomyocytes and an acute hyperuricemia mouse model, which is regarded as similar to humans with primary hyperuricemia and can be utilized in translational research for human hyperuricemia.
Hyperuricemia is strongly associated with cardiovascular risk and a poor outcome in a variety of cardiovascular disease states, such as coronary artery disease, hypertension and heart failure [20–24]. However, the underlying mechanisms to explain this association have been nonexistent. In our previous study [6], we explored the impact of HUA on glucose uptake and insulin resistance in primary cardiomyocytes and found that HUA induces oxidative stress, which plays a crucial role in the progression of insulin resistance in cardiomyocytes. Moreover, recent studies have confirmed insulin resistance as a powerful independent predictor of mortality and morbidity in patients with heart failure [25–26]. These studies demonstrated that therapeutically targeting impaired insulin sensitivity may potentially be favorable for patients with chronic heart failure. To determine whether metformin protects against insulin resistance induced by HUA in cardiomyocytes, we exposed primary cardiomyocytes to HUA, pretreated them with metformin and then quantified the uptake of glucose with 2-NBDG, a fluorescent glucose analog, after insulin stimulation. We found that treatment with metformin may protect cardiomyocytes from HUA and inhibit insulin-induced glucose uptake in cardiomyocytes. These results suggested that HUA resulted in insulin resistance in cardiomyocytes, but this change was weakened by pretreatment with metformin.
Metformin is well known to activate AMPK, which is expressed in a variety of tissues and cells, including cardiomyocytes, and plays a pivotal role in the regulation of cellular energy metabolism under stress conditions [14]. Previous studies have shown that metformin reduces long-term and high insulin-induced insulin resistance in cardiomyocytes [27], and AMPK has been shown to protect against insulin resistance in skeletal muscle cells through restoration of GLUT4 translocation [28]. Consistent with the findings of these previous studies, we found that metformin could improve HUA-induced insulin resistance in primary cardiomyocytes. As expected, this change was blocked by compound C, an AMPK inhibitor, indicating that AMPK activation was responsible for the suppression of insulin resistance in cardiomyocytes. In addition, using an acute hyperuricemic mouse model, our study indicated that metformin improved the progression of insulin resistance induced by HUA, as shown by the glucose tolerance test (Fig. 5A) and insulin tolerance test (Fig. 5B).
Interestingly, another activator of AMPK, AICAR, had almost the same effects as metformin, suggesting that activation of AMPK phosphorylation promoted the observed protective effect of insulin resistance in cardiomyocytes. In fact, AICAR has been demonstrated to protect against myocardial ischemia-reperfusion injury after myocardial infarction in animals and humans [29–30]. However, what procedures following AMPK pathway activation are involved in cardioprotection?
The first possibility is the improvement of the insulin-activated Akt pathway, which is inhibited by HUA. A previous study demonstrated that phosphorylation of Akt regulates cell survival, growth and metabolism. Additionally, cardiomyocyte metabolism and growth are coordinated by the integration of intracellular and extracellular signals [31]. Furthermore, activation of Akt phosphorylation, a key protein kinase of insulin-induced glucose uptake, modulates glucose uptake stimulated by insulin in cardiomyocytes [6]. In the present study, we found that HUA could strongly inhibit Akt phosphorylation, translocation of GLUT4 and 2-NBDG glucose uptake induced by insulin in primary cardiomyocytes. Our findings suggest that activators of AMPK, such as metformin and AICAR, could prevent HUA-inhibited Akt phosphorylation, GLUT4 translocation and HUA-inhibited 2-NBDG glucose uptake in cardiomyocytes. Moreover, an acute hyperuricemia mouse model demonstrated inhibited Akt phosphorylation with insulin resistance and glucose intolerance.
Another possibility is the metabolic effects of AMPK phosphorylation activation. Metformin has been demonstrated to activate the phosphorylation of AMPK in cardiomyocytes and mouse cardiac tissues. In fact, a recent study suggested that short-term treatment with metformin protects against myocardial ischemia-reperfusion injury via the AMPK signaling pathway after myocardial infarction in mice [11]. Therefore, AMPK has been considered to have various cardioprotective effects in animals and humans. Improvement in AMPK production by metformin may have mitigated the progression of insulin resistance induced by HUA. Both AICAR and metformin are reported to enhance glucose uptake in skeletal muscle cells and heart muscle cells [9, 31]. Consistent with these reports, in the HUA plus insulin group, we found that pretreatment with metformin and AICAR nearly reverted glucose uptake to the level of the control group in cardiomyocytes. Therefore, the possibility exists that the AMPK-induced uptake of glucose triggers improvement in insulin resistance, which is induced by HUA, followed by the restitution of the metabolic switch.
The decreased Akt phosphorylation and translocation of GLUT4 in the HUA group were reversed in both the HUA plus AICAR group or the HUA plus metformin group, demonstrating that the activation of AMPK protects the insulin signaling pathway of Akt against inhibition by HUA in both the HUA plus AICAR group or the HUA plus metformin group.