Abstract
We have recently shown that in diabetic OVE26 mice (type I diabetes), the AMP-activated protein kinase (AMPK) is reduced along with cardiac dysfunction and decreased cardiac autophagy. Genetic inhibition of AMPK in cardiomyocytes attenuates cardiac autophagy, exacerbates cardiac dysfunction and increases mortality in diabetic mice. More importantly, we have found chronic AMPK activation with metformin, one of the most used antidiabetes drugs and a well-characterized AMPK activator, significantly enhances autophagic activity, preserves cardiac function and prevents most of the primary characteristics of diabetic cardiomyopathy in OVE26 mice, but not in dominant negative-AMPK diabetic mice. We conclude that AMPK activation protects cardiac structure and function by increasing cardiac autophagy in the diabetic heart.
Autophagy is essential in cell growth, development and homeostasis, where it maintains a balance between the synthesis, degradation and subsequent recycling of cellular components. It allows recycling of amino acids and removal of damaged organelles to eliminate oxidative stress and promote remodeling for survival. A low level of constitutive autophagy is cytoprotective by maintaining the quality of proteins and organelles. Constitutive autophagy in the heart under basal conditions is a homeostatic mechanism for maintaining cardiomyocyte size and global cardiac structure and function. Under normal or mildly stressed conditions, autophagy degrades and recycles cytoplasmic components, such as long-lived proteins and organelles, and selectively removes damaged mitochondria as a cytoprotective mechanism for limiting mitochondria-derived oxidative stress. In the heart, downregulation of autophagy would cause abnormal proteins and organelles to accumulate, leading to apoptosis and cardiac dysfunction, the pathologies seen in diseases such as myocardial hypertrophy, cardiomyopathy and ischemic heart disease.
Diabetic cardiomyopathy is characterized by ventricular dysfunction that develops in many diabetic patients in the absence of coronary artery disease or hypertension. Diabetic cardiomyopathy has become a major cause of diabetes-related morbidity and mortality. Despite the importance of this complication, the underlying mechanisms of diabetic cardiomyopathy are still incompletely understood. In the context of accumulation of polyubiquitinated proteins and aggregation of abnormal mitochondria in the heart, we determined the role of autophagy in the development of diabetic cardiomyopathy in OVE26 mice, an established type 1 diabetic mouse model. We have found that OVE26 mice develop marked cardiac dysfunction at 6 mo of age. These animals have very high blood glucose concentrations, reduced serum insulin values and elevated serum triglycerides, and they also exhibit cardiomyopathy characterized by clear morphological abnormalities and reduced cardiac contractility. Evidence for diabetes-induced suppression of autophagic activity was uncovered by protein gel blotting, which reveals a decrease in LC3-II levels in the hearts from OVE26 mice as compared with FVB mice. Autophagosomes can be frequently detected in hearts from FVB control mice, whereas they are rarely detected in OVE26 mouse hearts. Overall, these results suggest that basal levels of autophagy are important for protecting cardiomyocytes from hyperglycemic damage and that suppression of autophagy in diabetes contributes to the development of cardiomyopathy.
AMPK is a major regulator of cellular and whole-body energy homeostasis. It is activated in response to an increase in the intracellular AMP-to-ATP ratio during exercise, hypoxia, oxidative stress and glucose deprivation. Activation of AMPK requires the phosphorylation of its α subunit at Thr172 by two upstream kinases, Liver Kinase B1 (LKB1) and Ca2+/calmodulin-dependent kinase kinase (CaMKK)-β. Emerging evidence demonstrates that AMPK not only regulates cellular energy, but also other cellular processes such as cell growth, protein synthesis and autophagy. Interestingly, AMPK activity in diabetic OVE26 mice is significantly suppressed, suggesting that AMPK reduction might be related to a reduction of autophagy and cardiac dysfunction. To further test the role of AMPK on cardiac function and autophagy, we treated OVE26 mice and diabetic dominant negative AMPK transgenic mice with metformin and monitored cardiac autophagy and cardiac functions after 4 mo of treatment. Metformin is one of the most commonly prescribed antidiabetes drugs for the treatment of type 2 diabetes, and it also improves cardiac function and reduces the incidence of myocardial infarction in treated patients. Clinical and animal studies suggest that metformin provides cardiovascular protection independent of its hypoglycemic effects. More recent investigations have indicated that metformin prevents cardiac ischemia/reperfusion injury and heart failure through activation of AMPK. Indeed, chronic activation of AMPK by metformin restores cardiac autophagy in wild-type diabetic hearts, but this effect is abolished in mice deficient in AMPKα2, indicating that AMPK regulates cardiac autophagy in diabetic cardiomyopathy.
Autophagy is tightly controlled by the mammalian target of rapamycin (mTOR)-dependent signaling pathway, which phosphorylates and inhibits the ULK1 (the mammalian homolog of yeast Atg1) kinase complex and prevents autophagy induction. In mammals, compromised cellular energy production inhibits mTOR through activation of AMPK and, subsequently, phosphorylation of the tuberous sclerosis complex (TSC). Since mTOR negatively regulates autophagy, the AMPK-mTOR pathway has been considered an important mechanism in autophagy regulation in response to energy stress and glucose starvation. Consistent with this hypothesis, diabetic hearts exhibit activation of the TSC-mTOR signaling pathway, as reflected by decreased phosphorylation of raptor at both Ser722 and Ser792, and increased phosphorylation of mTOR at both Ser2448 and Thr2446, as well as its downstream effectors, 4 E binding protein 1 (4EBP1) and p70 ribosomal protein S6 kinase 1 (p70 S6K1). Activation of AMPK by metformin inhibits the TSC-mTOR pathway and restores cardiac autophagy in OVE26 mice. It is likely that diabetes activates TSC-mTOR signaling through inactivation of AMPK, which inhibits the ULK1 kinase complex, preventing the initiation of autophagy. Recently, several groups have reported that AMPK directly regulates autophagy by phosphorylating and activating ULK1. Whether or not AMPK inhibition in diabetes suppresses the phosphorylation of ULK1 in vivo warrants further investigation.
Acknowledgements
This study was supported by funding from the following: NIH (HL079584, HL080499, HL074399, HL089920 and HL096032 to M.Z. and 1P20RR024215-01 to Z.X. and M.Z.), the American Heart Association Scientist Development Grant (Z.X.), the Juvenile Diabetes Research Foundation (M.Z.), Oklahoma Center for the Advancement of Science and Technology (M.Z. and Z.X.), and the American Diabetes Association (M.Z.). Dr. M.H. Zou is a recipient of the National Established Investigator Award of the American Heart Association.
Punctum to: Xie Z, Lau K, Eby B, Lozano P, He C, Pennington B, Li H, Rathi S, Dong Y, Tian R, Kem D, Zou MH. Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice. Diabetes 2011; 6:1770 - 1778; PMID: 21562078; http://dx.doi.org/10.2337/db10-0351