MODY2 in humans is characterized by mild but chronic hyperglycemia, with some MODY2 patients displaying peripheral insulin resistance, but diabetes-associated macro- and micro-vascular complications, overweight, and dyslipidemia are uncommon [22, 23]. Patients can usually manage their blood glucose levels by diet and exercise alone . The liver-specific gck knockout mice (gckw/–) appear normal at birth, with measurement of plasma insulin, triglyceride, cholesterol, ALT, and AST showing no difference compared with wild-type mice at up to 6 weeks of age . These parameter are similar to those observed in MODY2 patients. Fasting blood glucose levels in these mice increase with age and these mice spontaneously develop hyperglycemia after 6 weeks of age with impaired glucose tolerance and a decreased accumulation of hepatic glycogen . These mice however, do not exhibit signs of obesity with aging . No evidence of autoimmunity in the pancreatic islets is exhibited by these knockout mice . The gckw/– mouse, thus, may serve as a model of human MODY2 diabetes as it is associated with mild hyperglycemia and insulin resistance without hypoinsulinemia. These mice (gckw/– mice) were used to investigate the functional and structural changes in the myocardium that result from long-term (60 weeks) reduced liver gck expression (yielding mild hyperglycemia) followed by 4 weeks with or without treatment with insulin or rosiglitazone.
Diabetic cardiomyopathy describes diabetes-associated changes in myocardial structure and function. HOMA-IR is based on the dynamic interaction between glucose and insulin output and is a well-documented clinical representation of insulin resistance when compared to the gold standard reference, the euglycemic clamp . In several studies, an association between diabetic cardiomyopathy and HOMA-IR has been found. The HOMA index is an independent determinant of LV diastolic function . Insulin resistance has been correlated with increased left ventricular mass and risk of heart failure . In this study, we found that in gckw/– mice both serum glucose levels and insulin resistance were dramatically elevated compared with the control group (gckw/w mice). It has been demonstrated that the impairment of glucose homeostasis is able to affect the severity of heart disease.
MLC2 is part of the myosin complex, a hexameric complex of two heavy chains and four light chains, predominantly expressed in cardiac ventricle muscle . The regulatory light chains of the myosin complex, MLC2, can be phosphorylated leading to a conformational change, which consequently affects muscle contraction . Mutations in the human MLC2 gene have been associated with hypertrophic cardiomyopathy and lack of MLC2 in mice is embryonic lethal due to cardiac dysfunction that results in heart failure, a massive cardiac enlargement, wall thinning, and abnormalities in myofibril assembly . In this study, we found that in gckw/– mice the level of MLC2 protein expression was dramatically elevated compared with the control group (gckw/w mice). Koka et al.  revealed a coordinated down regulation of cytoskeletal contractile proteins such as myosin heavy-chainα (αMHC), and an up-regulation of MLC2 in diabetic mice, a result consistent with our results. The specific mechanism underlying these changes is unclear and we hypothesized that an increase in MLC2 levels may lead to myocardial hypertrophy by interfering with the assembly of myofibrils.
In several studies, an association between diabetic cardiomyopathy and cardiac hypertrophy, increased myocardial stiffness and reduced chamber compliance has been found . Clinical studies have demonstrated that diabetic cardiomyopathy is manifested with left ventricular hypertrophy associated with systolic/diastolic dysfunction and cardiac fibrosis in diabetic patients . In the present study, we observed cardiac hypertrophy in the gckw/– mouse, which was observed as an increase in the left ventricular wall thickness, a decrease of the left ventricular internal dimension, longer PR and QRS intervals, and increased myocardium MLC2 protein levels. Treatment with rosiglitazone results in PR and QRS intervals (Table 3), and MLC2 protein levels (Figure 3) being restored to wild-type levels, however, LVPW (Table 2) worsened after treatment with insulin or rosiglitazone.
A significant increase in collagen deposition around the intramural cardiacvessels and between myofibrils in heart biopsies from diabetic patients has been found . The PAS reaction is due to the accumulation of glycoprotein in the myocardium, which leads to interstitial fibrosis. Formation of glycoprotein has been suggested to be due to the increased formation of glucose-derived aldehydes, giving rise to so-called advanced glycation end products (AGEs) . The accumulation of glycoprotein increases myocardial stiffness by cross-linking. As seen in our study, reduced gck expression in the liver had an unfavorable effect on cardiac morphology and lead to the accumulation of collagen (Masson positive material) and glycoproteins (PAS positive material) in the myocardium (Figure 5). Glycoproteins increase intracellular oxidative stress, which contributes to additional cell damage. Oxidative stress is associated with mitochondrial damage and plays an important role in the development of diabetic cardiomyopathy .
Hyperglycemia-induced impairment of redox balance is considered to be a key trigger of diabetic complications, through the up-regulated generation of reactive oxygen species (ROS) , together with an impaired ability of the endogenous antioxidant defense system to remove them . The heart is particularly susceptible to oxidative damage, as it possesses lower levels of endogenous antioxidants in comparison to other organs. Cyba and Cybb are essential subunits of the NADPH oxidase, an enzyme considered to be a major source of O2˙- in cardiac cells catalyzing the one-electron reduction of oxygen using NADH or NADPH as the electron donor . Recent evidence suggests that NADPH oxidase derived superoxide is a key trigger of mitochondrial dysfunction and up regulated mitochondrial superoxide generation . SOD is one of the most important physiological antioxidants against free radicals and functions to prevent subsequent lipid peroxidation. Lipid peroxidation, which is a normal phenomenon that occurs continuously at lowlevels in every individual, is the most common consequence of oxidative stress. MDA is the end product of lipid peroxidation and is toxic to cells and cell membranes. Hyperglycemia contributes to the activation of free radicals and leads to tissue damage in different organs, such as endothelial dysfunction, hypertrophy and fibrosis . In the present study, we found that the MDA content and Cyba mRNA expression levels were significantly higher in gckw/– mice than in gckw/w control mice, which is concordant with the finding of significantly decreased SOD activity in the gckw/– mice. These results suggest that reduced liver gck expression may lead to a decreasein the antioxidant capacity of the diabetic myocardium, contributing significantly to oxidative stress and the resulting myocardial damage.
At the same time, our study showed that the mitochondrial volume density and number were increased in the gckw/– mice compared to gckw/w mice (Figure 6). After treatment with rosiglitazone or insulin, these parameters were restored to more normal values. Mitochondria are the center of fatty acid and glucose metabolism and thus are highly likely to be impacted by impaired metabolism associated with diabetes. Increased number and size of >mitochondria might be an adaptive response to hyperglycemia . It has recently been shown that insulin resistance drives the cardiac mitochondrial biogenesis regulatory program via PPARα, where activation was increased due to increased fat uptake and oxidation [40, 41]. The liver-specific glucokinase knockout mouse experienced long-term hyperglycemia, which results in decreased insulin receptor levels and ultimately leading to insulin resistance and attenuated glucose uptake . Insulin resistance drives the cardiac mitochondrial biogenesis regulatory program, which lead to increased mitochondrial volume density and number . Increased oxidative phosphorlyation and impairment in the electron transport chain contribute to increased ROS and superoxide (O2˙-) production . These changes cause morphological and functional abnormalities in cardiac tissue, thus suggesting that oxidative stress might be the unifying factor for the damaging effects of hyperglycemia .
The molecular signaling pathways implicated in myocardial damage in gckw/– mice have not been fully resolved. We demonstrate that the cardiac insulin receptor and Akt protein levels, key factors of the IR-PI3K-Akt pathway, were reduced in gckw/– mice, but were restored to wild-type levels by insulin or rosiglitazone treatment. The IR-PI3K-Akt pathway induces a hypertrophic response associated with cardioprotection , in which cardiac structure and function are preserved. Based on our observations, we postulate that prolonged exposure to increased levels of serum glucose depresses protectiveIR-PI3K-Akt signaling. Preservation of insulin receptor and Akt levels in the rosiglitazone-treated diabetic myocardium might thereby confer protection against pathological cardiac hypertrophy.
Metabolic disorders play major roles in the pathogenesis of diabetic cardiomyopathy. AMPK is a central regulator for glucose and fatty acid metabolism in mammalian cells, which acts as anenergy sensor, responding to a rise in AMP levels by increasing ATP generating pathways and reducing ATP-consuming pathways . AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits. Phosphorylation at Ser108 of theβ1 subunit seems to be required for the activation of AMPKenzyme. AMPK phosphorylation inhibits fatty acid and cholesterol synthesis and gluconeogenesis in the liver and stimulates fatty acid uptake and oxidation, glucose uptake, and mitochondrial biogenesis in skeletal muscle .
ACC is a cytosolic enzyme that catalyzes the carboxylation of cytosolic acetyl-CoA to form malonyl-CoA, which is the pivotal step of the fatty acid synthesis pathway . ACCβ is the major isoform in heart. We demonstrated that the levels of phosphorylated AMPK and ACC were dramatically lower in gckw/– mice, but only p-ACC was restored to wild-type levels with rosiglitazone treatment. It has previously been reported that AMPK regulates ACC phosphorylation . Phosphorylation by AMPK inhibits the enzymatic activity of ACC, and in turn malonyl-CoA levels . These results suggest that the fatty acid synthesis pathway may be enhanced in the myocardium of gckw/– mice, due to a reduction in ACC phosphorylation. The accumulation of fatty acid also causes a pathological ROS accumulation, which leads to damage in cardiomyocytes. It has been hypothesized that the dysregulation of the AMPK/ACC fuel-sensing and signaling network is a key factor in the development of insulin resistance and cardiac hypertrophy .