Dissociation of Bcl-2–Beclin1 Complex by Activated AMPK Enhances Cardiac Autophagy and Protects Against Cardiomyocyte Apoptosis in Diabetes

Diabetic cardiomyopathy is associated with suppression of cardiac autophagy, and activation of AMP-activated protein kinase (AMPK) restores cardiac autophagy and prevents cardiomyopathy in diabetic mice, albeit by an unknown mechanism. We hypothesized that AMPK-induced autophagy ameliorates diabetic cardiomyopathy by inhibiting cardiomyocyte apoptosis and examined the effects of AMPK on the interaction between Beclin1 and Bcl-2, a switch between autophagy and apoptosis, in diabetic mice and high glucose–treated H9c2 cardiac myoblast cells. Exposure of H9c2 cells to high glucose reduced AMPK activity, inhibited Jun NH₂-terminal kinase 1 (JNK1)–B-cell lymphoma 2 (Bcl-2) signaling, and promoted Beclin1 binding to Bcl-2. Conversely, activation of AMPK by metformin stimulated JNK1–Bcl-2 signaling and disrupted the Beclin1–Bcl-2 complex. Activation of AMPK, which normalized cardiac autophagy, attenuated high glucose–induced apoptosis in cultured H9c2 cells. This effect was attenuated by inhibition of autophagy. Finally, chronic administration of metformin in diabetic mice restored cardiac autophagy by activating JNK1–Bcl-2 pathways and dissociating Beclin1 and Bcl-2. The induction of autophagy protected against cardiac apoptosis and improved cardiac structure and function in diabetic mice. We concluded that dissociation of Bcl-2 from Beclin1 may be an important mechanism for preventing diabetic cardiomyopathy via AMPK activation that restores autophagy and protects against cardiac apoptosis.Diabetic cardiomyopathy, a clinical condition characterized by ventricular dysfunction, develops in many diabetic patients in the absence of coronary artery disease or hypertension (1,2). An increasing number of studies have demonstrated that hyperglycemia is central to the development of diabetic cardiomyopathy, which triggers a series of downstream signals that lead to cardiomyocyte apoptosis, chamber dilation, and cardiac dysfunction (3). In support of this view, diabetes-induced cardiac cell death has been observed in diabetic patients (3) and streptozotocin (STZ)-induced diabetic animals (4). The mechanisms of pathogenesis, however, remain elusive.Autophagy is a highly conserved process for bulk degradation and recycling of cytoplasmic components in lysosomes (5). In the heart, constitutive autophagy is a homeostatic mechanism for maintaining cardiac structure and function (6). However, excessive induction of autophagy may destroy the cytosol and organelles and release apoptosis-related factors, leading to cell death and cardiac dysfunction (7,8). Thus, autophagy appears to regulate both cell survival and cell death. Emerging evidence suggests that cross-talk occurs between autophagic and apoptotic pathways. For instance, the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) inhibits starvation-induced autophagy by binding to Beclin1, and this binding effectively sequesters Beclin1 away from the core kinase complex formed from Beclin1 and vacuolar sorting protein (VPS34), a class III phosphatidylinositol 3-kinase (PI3K), which is required for the induction of autophagy (9). Recently we demonstrated that in diabetic animals, suppression of autophagy is associated with an increase in cardiac apoptosis (10,11); however, whether the induction of autophagy serves as a protective response in the development of diabetic cardiomyopathy remains unknown.The AMP-activated protein kinase (AMPK) is a conserved cellular energy sensor that plays an important role in maintaining energy homeostasis (12). In addition, AMPK also regulates many other cellular processes, such as cell growth, protein synthesis (13,14), apoptosis (15,16), and autophagy (17,18). In the heart, AMPK is responsible for activation of glucose uptake and glycolysis during low-flow ischemia and plays an important role in limiting apoptotic activity associated with ischemia and reperfusion (19). Moreover, activation of AMPK by ischemia also stimulates autophagy and protects against ischemic injury (18). Mechanistically, AMPK appears to induce autophagy through phosphorylation and activation of ULK1 (the mammalian homolog of yeast autophagy-related gene 1 [Atg1]) (20,21); however, the molecular mechanism by which AMPK regulates the switch between autophagy and apoptosis in the development of diabetic cardiomyopathy remains to be established.In this study, we sought to determine whether autophagy plays a role in protection against cell death during the development of diabetic cardiomyopathy and to explore the mechanism by which activation of AMPK regulates the switch between autophagy and apoptosis in this disease. We found that activation of AMPK restores cardiac autophagy by disrupting the interaction between Beclin1 and Bcl-2 and protects against cardiac cell apoptosis, ultimately leading to improvement in cardiac structure and function in diabetic mice.     

Gene Deletion of the Kinin Receptor B1 Attenuates Cardiac Inflammation and Fibrosis During the Development of Experimental Diabetic Cardiomyopathy

OBJECTIVE

Diabetic cardiomyopathy is associated with increased mortality in patients with diabetes. The underlying pathology of this disease is still under discussion. We studied the role of the kinin B1 receptor on the development of experimental diabetic cardiomyopathy.

RESEARCH DESIGN AND METHODS

We utilized B1 receptor knockout mice and investigated cardiac inflammation, fibrosis, and oxidative stress after induction of streptozotocin (STZ)-induced diabetes. Furthermore, the left ventricular function was measured by pressure-volume loops after 8 weeks of diabetes.

RESULTS

B1 receptor knockout mice showed an attenuation of diabetic cardiomyopathy with improved systolic and diastolic function in comparison with diabetic control mice. This was associated with a decreased activation state of the mitogen-activated protein kinase p38, less oxidative stress, as well as normalized cardiac inflammation, shown by fewer invading cells and no increase in matrix metalloproteinase-9 as well as the chemokine CXCL-5. Furthermore, the profibrotic connective tissue growth factor was normalized, leading to a reduction in cardiac fibrosis despite severe hyperglycemia in mice lacking the B1 receptor.

CONCLUSIONS

These findings suggest that the B1 receptor is detrimental in diabetic cardiomyopathy in that it mediates inflammatory and fibrotic processes. These insights might have useful implications on future studies utilizing B1 receptor antagonists for treatment of human diabetic cardiomyopathy.Diabetic cardiomyopathy, as it occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality (1). Different pathophysiological stimuli are involved in its development and mediate tissue injury leading to left ventricular systolic and diastolic dysfunction. Accumulation of cardiac fibrosis with distinct changes in the regulation of the extracellular matrix (2,3), excessive generation of reactive oxygen species (4), and cardiac inflammation (5,6), characterized by increased levels of proinflammatory cytokines and transendothelial migration of immunocompetent cells, plays a role in the manifestation of diabetic cardiomyopathy. Experimental stimulation of the local tissue kallikrein-kinin system has been shown to be beneficial in different forms of cardiomyopathies (7–11). Most of these effects are attributed to the kinin B2 receptor (B2R), while the role of the kinin B1 receptor (B1R) in cardiac failure is still under discussion. In contrast to the B2R, which is constitutively expressed in the cardiac tissue, the B1R is expressed at very low levels under basal conditions. Nevertheless, it is highly inducible under pathological conditions by pathological mediators such as bacterial lipopolysaccharide (12), cytokines (13), and ischemia but also by hyperglycemia (14), as can be shown in different animal models of cardiomyopathy. Also, in endomyocardial biopsies of patients with end-stage heart failure, this upregulation could be demonstrated and correlated with increased expression of proinflammatory cytokines in those patients (15). Whether B1R upregulation is cardioprotective, parallel to that of the B2R (16,17), or is cardiotoxic (13,18,19) remains debated. To further clarify the role of the B1R in the pathogenesis of diabetic cardiomyopathy, we investigated the left ventricular function in an animal model of streptozotocin (STZ)-induced type 1 diabetes using B1R knockout mice. Furthermore, changes in the left ventricular remodeling, inflammation, and oxidative stress were analyzed.     

A Novel Mechanism by Which SDF-1β Protects Cardiac Cells From Palmitate-Induced Endoplasmic Reticulum Stress and Apoptosis via CXCR7 and AMPK/p38 MAPK-Mediated Interleukin-6 Generation

We studied the protective effect of stromal cell-derived factor-1β (SDF-1β) on cardiac cells from lipotoxicity in vitro and diabetes in vivo. Exposure of cardiac cells to palmitate increased apoptosis by activating NADPH oxidase (NOX)–associated nitrosative stress and endoplasmic reticulum (ER) stress, which was abolished by pretreatment with SDF-1β via upregulation of AMP-activated protein kinase (AMPK)–mediated p38 mitogen-activated protein kinase (MAPK) phosphorylation and interleukin-6 (IL-6) production. The SDF-1β cardiac protection could be abolished by inhibition of AMPK, p38 MAPK, or IL-6. Activation of AMPK or addition of recombinant IL-6 recaptured a similar cardiac protection. SDF-1β receptor C-X-C chemokine receptor type 4 (CXCR4) antagonist AMD3100 or CXCR4 small interfering RNA could not, but CXCR7 small interfering RNA completely abolished SDF-1β’s protection from palmitate-induced apoptosis and activation of AMPK and p38 MAPK. Administration of SDF-1β to diabetic rats, induced by feeding a high-fat diet, followed by a small dose of streptozotocin, could significantly reduce cardiac apoptosis and increase AMPK phosphorylation along with prevention of diabetes-induced cardiac oxidative damage, inflammation, hypertrophy, and remodeling. These results showed that SDF-1β protects against palmitate-induced cardiac apoptosis, which is mediated by NOX-activated nitrosative damage and ER stress, via CXCR7, to activate AMPK/p38 MAPK–mediated IL-6 generation. The cardiac protection by SDF-1β from diabetes-induced oxidative damage, cell death, and remodeling was also associated with AMPK activation.Intracellular accumulation of long-chain fatty acids in nonadipose tissues is associated with cellular dysfunction and cell death and may ultimately contribute to the pathogenesis of disease. For example, lipotoxic accumulation of long-chain fatty acids in the heart of the Zucker diabetic fatty rat leads to the development of pathogenic changes (1). Similarly, the pathogenic changes in the heart of diabetic patients are also associated with the increased cardiac triglyceride content and contributes to arrhythmia occurrence and reduced contractile function or sudden death (2). In cultured cardiac cells, palmitate induced cardiac cell death (3,4). Because palmitate and stearate, but not unsaturated fatty acids, are precursors for de novo ceramide synthesis, fatty acid–induced apoptosis was assumed to probably occur through ceramide; however, some studies did not support this notion (5,6). Chinese hamster ovary cells did not require de novo ceramide synthesis for palmitate-induced apoptosis, and palmitate supplementation rather overgenerated reactive oxygen species or reactive nitrogen species that initiate apoptosis (5). Other later studies also reported the importance of palmitate-induced oxidative and nitrosative damage in the induction of apoptotic cell death (3,7,8).Reportedly, palmitate induced endoplasmic reticulum (ER) stress and apoptosis in multiple tissues (9), and AMP-activated protein kinase (AMPK) activation inhibited palmitate-induced ER stress and apoptotic effects (9,10). Terai et al. (11) demonstrated the preventive effect of AMPK activation on hypoxia-induced ER stress and apoptosis in cardiac cells: hypoxia-induced C/EBP homologous protein (CHOP) expression and caspase 12 cleavage were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-β-d-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated AICAR’s cardioprotection (11). Another study showed the antiapoptotic effect of AMPK activation on tumor necrotic factor-α (TNF-α) (12). Furthermore, the AMPK antiapoptotic effect seemed associated with p38 mitogen-activated protein kinase (MAPK) and interleukin-6 (IL-6) (13,14). Therefore, AMPK activation is an attractive approach in the prevention and/or treatment of cardiac diseases. However, concerns have recently been raised about AICAR-mediated AMPK upregulation (15): 1) chemical AICAR unselectively stimulates AMPK phosphorylation in all cells when it is chronically administered in vivo, and 2) AICAR as an AMPK-specific activator will persistently upregulate AMPK, which is undesirable because it usually induces apoptosis (15–19).Chemokine stromal cell-derived factor (SDF-1), also known as chemokine (C-X-C motif) ligand 12 (CXCL12), regulates many essential biological processes, including cardiac and neuronal development, stem cell motility, neovascularization, angiogenesis, and tumorigenesis (20,21). Generally, SDF-1 mediates these disparate processes predominantly through CXC receptor 4 (CXCR4) and/or CXCR7 (22). Six variants of SDF-1, including SDF-1α, β, γ, δ, ε, and θ have been identified to date (23,24). SDF-1α and β are mostly involved in cardiovascular diseases (25–27). Previous studies predominantly showed the role of SDF-1α or β in directing stem cells into the damaged heart to repair tissue damage (21,28–30). Whether SDF-1 directly protects cardiac cells from lipotoxic effects has not yet been studied.Therefore the objectives of the current study are to determine:

• 1)whether SDF-1β protects cardiac cells from palmitate-induced nitrosative damage, ER stress, and apoptotic cell death;
• 2)whether any protective effect of SDF-1β against palmitate-induced cell death is mediated by activation of the AMPK-related protective pathway;
• 3)whether p38 MAPK and IL-6 are involved in the protective effect of SDF-1β from palmitate-induced cell death;
• 4)which subtype of SDF-1 receptors is required for SDF-1β’s protection from these palmitate-induced pathogenic effects; and
• 5)whether the antiapoptotic effect of SDF-1β on cardiac cells in vitro can be seen in the diabetic heart and whether the protective effect on cardiac cell death can lead to a prevention of cardiac pathogenic changes.

An understanding of these issues is very important for developing SDF-1β as a potential activator of AMPK to be used chronically in vivo. This peptide is an attractive candidate for clinical development because if it can activate AMPK, it will transiently activate AMPK only in cells expressing its receptor, unlike AICAR, which persistently and unselectively activates AMPK in all the cells in the body. Therefore, in vitro cultured cardiac H9C2 cells and also the primary cultures of neonatal cardiomyocytes, combined with pharmacological inhibitors and small interfering RNA (siRNA) approaches, were used. Diabetic rats were induced by being fed a high-fat diet (HFD) for 8 weeks, followed by a small dose of streptozotocin (STZ), as reported by others (31,32).     

C-Peptide Activates AMPKα and Prevents ROS-Mediated Mitochondrial Fission and Endothelial Apoptosis in Diabetes

Vasculopathy is a major complication of diabetes; however, molecular mechanisms mediating the development of vasculopathy and potential strategies for prevention have not been identified. We have previously reported that C-peptide prevents diabetic vasculopathy by inhibiting reactive oxygen species (ROS)-mediated endothelial apoptosis. To gain further insight into ROS-dependent mechanism of diabetic vasculopathy and its prevention, we studied high glucose–induced cytosolic and mitochondrial ROS production and its effect on altered mitochondrial dynamics and apoptosis. For the therapeutic strategy, we investigated the vasoprotective mechanism of C-peptide against hyperglycemia-induced endothelial damage through the AMP-activated protein kinase α (AMPKα) pathway using human umbilical vein endothelial cells and aorta of diabetic mice. High glucose (33 mmol/L) increased intracellular ROS through a mechanism involving interregulation between cytosolic and mitochondrial ROS generation. C-peptide (1 nmol/L) activation of AMPKα inhibited high glucose–induced ROS generation, mitochondrial fission, mitochondrial membrane potential collapse, and endothelial cell apoptosis. Additionally, the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and the antihyperglycemic drug metformin mimicked protective effects of C-peptide. C-peptide replacement therapy normalized hyperglycemia-induced AMPKα dephosphorylation, ROS generation, and mitochondrial disorganization in aorta of diabetic mice. These findings highlight a novel mechanism by which C-peptide activates AMPKα and protects against hyperglycemia-induced vasculopathy.C-peptide and insulin are cosecreted in equimolar amounts into the circulation from the pancreatic β-cells of Langerhans (1). C-peptide deficiency is a prominent attribute of type 1 diabetes (1). Deficiencies of C-peptide and insulin may also occur in the late stages of type 2 diabetes as a result of progressive loss of β-cells (2–4). Recent evidence demonstrates a beneficial role for C-peptide in diabetic neuropathy (1,5,6), nephropathy (1,6,7), and vascular dysfunction (1,5) and inflammation (1). C-peptide protects against diabetic vascular damage by promoting nitric oxide (NO) release (8) and suppressing nuclear factor-κB (9), which suppresses leukocyte-endothelium interactions (8,9). C-peptide may prevent atherosclerosis by inhibiting vascular smooth muscle proliferation and migration (10) and reducing venous neointima formation (11). However, the molecular mechanism by which C-peptide prevents diabetes complications is not understood well enough to permit its clinical implementation.Generation of reactive oxygen species (ROS) in response to high glucose is the leading cause of endothelial damage and diabetic vasculopathy (12). Protein kinase C (PKC)-dependent NADPH oxidase is considered a major cytosolic mediator of ROS generation in endothelial cells (13,14) that play a central role in hyperglycemia-induced endothelial cell apoptosis and vascular complications (15–17). Overproduction of intracellular ROS by mitochondria also occurs during the development of hyperglycemia-induced vascular complications (12,18,19). Altered mitochondrial dynamics due to mitochondrial fission were recently linked with endothelial dysfunction in diabetes (20,21). However, the mechanisms regulating production of cytosolic and mitochondrial ROS and their individual functions in regulating mitochondrial dynamics and apoptosis remain to be elucidated.AMP-activated protein kinase (AMPK) is an intracellular energy and stress sensor (22) and is an emerging target for preventing diabetes complications (23), as exhibited by the most common antihyperglycemic drugs, rosiglitazone (24) and metformin (25). AMPK prevents apoptosis of endothelial cells (26–28) by inhibiting ROS generation by NADPH oxidase (24,29) and mitochondria (30). Additionally, AMPK dephosphorylation is associated with diabetes (22,31,32). It has been reported that C-peptide inhibits high glucose–induced mitochondrial superoxide generation in renal microvascular endothelial cells (7). We recently demonstrated a key role for C-peptide in preventing high glucose–induced ROS generation and apoptosis of endothelial cells through inhibition of transglutaminase (17). However, the mechanism underlying C-peptide–mediated inhibition of intracellular ROS production and subsequent apoptosis remains unclear. Thus, we hypothesized that the potential protective role of C-peptide could be attributed to activation of AMPK, which results in reduced hyperglycemia-induced production of intracellular ROS and altered mitochondrial dynamics that suppress apoptosis of endothelial cells.In this study, we sought to elucidate the mechanism by which C-peptide protects against hyperglycemia-induced ROS production and subsequent endothelial cell damage. We examined the beneficial effect of C-peptide through AMPKα activation and subsequent protection against hyperglycemia-induced production of intracellular ROS, dysregulation of mitochondrial dynamics, mitochondrial membrane potential (∆Ψ_m) collapse, and apoptosis of endothelial cells. These studies were confirmed in vivo in mice with streptozotocin-induced diabetes using C-peptide supplement therapy delivered through osmotic pumps. Thus, our study implicates C-peptide replacement therapy as a potentially significant approach for preventing diabetes complications.     

Cardiac-Specific IGF-1 Receptor Transgenic Expression Protects Against Cardiac Fibrosis and Diastolic Dysfunction in a Mouse Model of Diabetic Cardiomyopathy

OBJECTIVE

Compelling epidemiological and clinical evidence has identified a specific cardiomyopathy in diabetes, characterized by early diastolic dysfunction and adverse structural remodeling. Activation of the insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) promotes physiological cardiac growth and enhances contractile function. The aim of the present study was to examine whether cardiac-specific overexpression of IGF-1R prevents diabetes-induced myocardial remodeling and dysfunction associated with a murine model of diabetes.

RESEARCH DESIGN AND METHODS

Type 1 diabetes was induced in 7-week-old male IGF-1R transgenic mice using streptozotocin and followed for 8 weeks. Diastolic and systolic function was assessed using Doppler and M-mode echocardiography, respectively, in addition to cardiac catheterization. Cardiac fibrosis and cardiomyocyte width, heart weight index, gene expression, Akt activity, and IGF-1R protein content were also assessed.

RESULTS

Nontransgenic (Ntg) diabetic mice had reduced initial (E)-to-second (A) blood flow velocity ratio (E:A ratio) and prolonged deceleration times on Doppler echocardiography compared with nondiabetic counterparts, indicative markers of diastolic dysfunction. Diabetes also increased cardiomyocyte width, collagen deposition, and prohypertrophic and profibrotic gene expression compared with Ntg nondiabetic littermates. Overexpression of the IGF-1R transgene markedly reduced collagen deposition, accompanied by a reduction in the incidence of diastolic dysfunction. Akt phosphorylation was elevated ∼15-fold in IGF-1R nondiabetic mice compared with Ntg, and this was maintained in a setting of diabetes.

CONCLUSIONS

The current study suggests that cardiac overexpression of IGF-1R prevented diabetes-induced cardiac fibrosis and diastolic dysfunction. Targeting IGF-1R–Akt signaling may represent a therapeutic target for the treatment of diabetic cardiac disease.Diabetes represents a major threat to human health, with global incidence projected to reach 300 million by 2025 (1,2). Cardiovascular complications including coronary heart disease and peripheral vascular disease are regarded as primary causes of morbidity and mortality in both type 1 and type 2 diabetes (3,4). In addition, clinical and experimental evidence supports the existence of a distinct diabetic cardiomyopathy, associated with adverse changes to the structure and function of cardiomyocytes, which can occur independent of macrovascular complications (5,6). Left ventricular (LV) diastolic impairments (in LV filling, relaxation, and/or diastolic distensibility) are evident early in disease progression (7–9), often followed by later onset of systolic dysfunction (e.g., reduced LV ejection fraction and fractional shortening) (10). Functional alterations in the diabetic heart occur concomitantly with development of the structural abnormalities cardiomyocyte hypertrophy and cardiac fibrosis (11–13). In both type 1 and 2 diabetes, this cardiomyopathy is a prognostic indicator, particularly for mortality (14). New approaches to rescue LV remodeling and dysfunction specifically in diabetic myocardium are thus highly desirable.Physiological heart growth or hypertrophy, which occurs during normal postnatal development and can be induced by exercise, is characterized by a normal cardiac structure and gene expression (15). In contrast, pathological hypertrophy is characterized by fibrosis, myofiber disarray, reduced cardiac output, and eventual heart failure (15). IGF-1, structurally and functionally related to insulin (16), plays a crucial role in stimulating physiological LV hypertrophy and conferring protection against cardiac dysfunction (17–19). The therapeutic potential of IGF-1 has thus been extensively examined in an array of cardiac pathologies, including heart failure and diabetes (18,19). In dilated cardiomyopathy, eccentric hypertrophy, and myocardial infarction (20–22), transgenic IGF-1 expression limited structural abnormalities such as myocyte necrosis and fibrosis (21). IGF-1 upregulation may also improve systolic function, via restoration of normal Ca²⁺ handling and increased cardiomyocyte contractility (17,20). Similar benefits have been proposed in diabetic myocardium (18,23), although in vivo cardiac functional studies have been limited. Furthermore, chronic IGF-1 may represent a flawed therapeutic approach for diabetes-induced LV dysfunction and remodeling, as both transgenic and pharmacological IGF-1 approaches significantly elevate systemic plasma IGF-1 concentrations and thus can have undesirable effects on nonmyocytes and other tissues. For instance, IGF-1 may induce fibroblast proliferation (24) and increase size of other organs including brain and kidney (17,25,26).The current study seeks to circumvent the problem of potential noncardiac IGF-1 effects by using a cardiomyocyte-specific IGF-1R transgenic (Tg) mouse (27), which develops physiological cardiac hypertrophy and enhanced systolic function, without histopathology (27). Despite compelling evidence for IGF-1 preservation of cardiac function, the role of cardiac IGF-1R specifically in protecting LV function and structure in the context of diabetes-induced cardiomyopathy in vivo has not been examined. Thus, the present study tested the hypothesis that cardiomyocyte-specific IGF-1R protects against diabetes-induced LV diastolic dysfunction and remodeling, using a streptozotocin (STZ)–induced mouse model of diabetes in vivo.     

Deficiency of Rac1 Blocks NADPH Oxidase Activation, Inhibits Endoplasmic Reticulum Stress, and Reduces Myocardial Remodeling in a Mouse Model of Type 1 Diabetes

OBJECTIVE

Our recent study demonstrated that Rac1 and NADPH oxidase activation contributes to cardiomyocyte apoptosis in short-term diabetes. This study was undertaken to investigate if disruption of Rac1 and inhibition of NADPH oxidase would prevent myocardial remodeling in chronic diabetes.

RESEARCH DESIGN AND METHODS

Diabetes was induced by injection of streptozotocin in mice with cardiomyocyte-specific Rac1 knockout and their wild-type littermates. In a separate experiment, wild-type diabetic mice were treated with vehicle or apocynin in drinking water. Myocardial hypertrophy, fibrosis, endoplasmic reticulum (ER) stress, inflammatory response, and myocardial function were investigated after 2 months of diabetes. Isolated adult rat cardiomyocytes were cultured and stimulated with high glucose.

RESULTS

In diabetic hearts, NADPH oxidase activation, its subunits'' expression, and reactive oxygen species production were inhibited by Rac1 knockout or apocynin treatment. Myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly increased in diabetic mice, which were accompanied by elevated expression of pro-fibrotic genes and hypertrophic genes. Deficiency of Rac1 or apocynin administration reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. These effects were associated with a normalization of ER stress markers'' expression and inflammatory response in diabetic hearts. In cultured cardiomyocytes, high glucose–induced ER stress was inhibited by blocking Rac1 or NADPH oxidase.

CONCLUSIONS

Rac1 via NADPH oxidase activation induces myocardial remodeling and dysfunction in diabetic mice. The role of Rac1 signaling may be associated with ER stress and inflammation. Thus, targeting inhibition of Rac1 and NADPH oxidase may be a therapeutic approach for diabetic cardiomyopathy.Diabetic cardiomyopathy has been defined as ventricular dysfunction that occurs in the absence of changes in blood pressure and coronary artery disease (1). Cardiac structural phenotypes of diabetic cardiomyopathy include cardiomyocyte apoptosis, cardiac hypertrophy, myocardial fibrosis, and interstitial inflammation (2,3), all of which significantly contribute to myocardial dysfunction. Three evident characteristic metabolic disturbances in diabetes, including hyperglycemia, hyperlipidemia, and hyperinsulinemia, are attributable to altered myocardial structure and function in diabetic cardiomyopathy (4). However, the signaling pathways associated with these metabolic triggers remain not fully understood in diabetic hearts.Several mechanisms involved in diabetic myocardial dysfunction have been suggested, which include increased oxidative stress, impaired calcium homeostasis, upregulation of the renin-angiotensin system, altered substrate metabolism, and mitochondrial dysfunction (3). These changes are closely related to reactive oxygen species (ROS) production. ROS is mainly produced by mitochondria and NADPH oxidase in cardiomyocytes. A cross-talk between mitochondria and NADPH oxidase has been suggested to sustain cellular ROS production under stresses (5–9). Selective inhibition of mitochondrial ROS has been shown to prevent diabetic cardiac changes in type 1 diabetic mice, confirming an important role of mitochondrial ROS (10). Our recent study has revealed that Rac1 via NADPH oxidase activation induces mitochondrial ROS production and plays an essential role in cardiomyocyte apoptosis and myocardial dysfunction in streptozotocin (STZ)-induced diabetes (8). Cell death by apoptosis is the predominant damage in diabetic cardiomyopathy (2). Cardiomyocyte death causes a loss of contractile tissue, which initiates a cardiac remodeling (11). Furthermore, Rac1/NADPH oxidase signaling has also been demonstrated to directly induce cardiac hypertrophy (12,13) and skin fibrosis (14,15). However, direct evidence is lacking as for the contribution of Rac1/NADPH oxidase to myocardial remodeling in the development of diabetic cardiomyopathy.In this study, we took advantage of the availability of mice with cardiomyocyte-specific Rac1 knockout to analyze the impact of Rac1 on NADPH oxidase activation, endoplasmic reticulum (ER) stress, hypertrophy, fibrosis, and inflammatory response in diabetic hearts. We further investigated the therapeutic effect of the NADPH oxidase inhibitor apocynin on diabetic cardiomyopathy in STZ-induced type 1 diabetic mice.     

AMP-Activated Protein Kinase�CDeficient Mice Are Resistant to the Metabolic Effects of Resveratrol

OBJECTIVE

Resveratrol, a natural polyphenolic compound that is found in grapes and red wine, increases metabolic rate, insulin sensitivity, mitochondrial biogenesis, and physical endurance and reduces fat accumulation in mice. Although it is thought that resveratrol targets Sirt1, this is controversial because resveratrol also activates 5′ AMP-activated protein kinase (AMPK), which also regulates insulin sensitivity and mitochondrial biogenesis. Here, we use mice deficient in AMPKα1 or -α2 to determine whether the metabolic effects of resveratrol are mediated by AMPK.

RESEARCH DESIGN AND METHODS

Mice deficient in the catalytic subunit of AMPK (α1 or α2) and wild-type mice were fed a high-fat diet or high-fat diet supplemented with resveratrol for 13 weeks. Body weight was recorded biweekly and metabolic parameters were measured. We also used mouse embryonic fibroblasts deficient in AMPK to study the role of AMPK in resveratrol-mediated effects in vitro.

RESULTS

Resveratrol increased the metabolic rate and reduced fat mass in wild-type mice but not in AMPKα1^−/− mice. In the absence of either AMPKα1 or -α2, resveratrol failed to increase insulin sensitivity, glucose tolerance, mitochondrial biogenesis, and physical endurance. Consistent with this, the expression of genes important for mitochondrial biogenesis was not induced by resveratrol in AMPK-deficient mice. In addition, resveratrol increased the NAD-to-NADH ratio in an AMPK-dependent manner, which may explain how resveratrol may activate Sirt1 indirectly.

CONCLUSIONS

We conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.Resveratrol is a natural polyphenolic compound found in grapes and red wine and has been shown to extend lifespan in many organisms, including yeast (1), flies (2), and worms (2–4). Resveratrol extended lifespan in mice on a high-fat diet (5) but not a regular diet (6). In mice with diet-induced obesity, resveratrol reduced fat accumulation and improved glucose tolerance and insulin sensitivity (5,7). In addition, resveratrol increases mitochondrial biogenesis and physical endurance. A resveratrol derivative with higher bioavailability is being tested in clinical trials for treating type 2 diabetes.Given its potential as a lead molecule for the development of drugs that treat metabolic disorders, it is critical to understand how resveratrol modulates metabolism. It is widely accepted that Sirt1, the founding member of the Sirtuin family (8) of NAD-dependent deacetylase, is the target of resveratrol (1,5,7). However, whether the putative Sirt1 activators such as resveratrol actually target Sirt1 in vivo is controversial because resveratrol increases Sirt1 activity in vitro only if the substrate is modified with a fluorescent tag (9,10). Resveratrol appears to increase the deacetylation rate by enhancing the affinity of Sirt1 for fluorescent-tagged peptides.Resveratrol also has a number of indirect effects (11), including stimulation of 5′ AMP-activated protein kinase (AMPK) (5,12,13). AMPK is a heterotrimeric protein consisting of an α-catalytic subunit and two regulatory subunits, β and γ (14). AMPK is a fuel-sensing kinase, which is activated by ATP-depleting conditions such as physical exercise, ischemia, and glucose deprivation. The catalytic subunit of AMPK has two isoforms, α1 and α2, which have different tissue expression patterns. Muscle expresses predominantly the α2-isoform (15), whereas fat and brain express predominantly the α1 isoform (16,17), and liver expresses both α1 and α2 isoforms (18). AMPKα1 and AMPKα2 knockout mice are viable, but AMPKα1/α2 double knockout causes embryonic lethality. Like resveratrol, activation of AMPK has been shown to reduce fat accumulation and increase glucose tolerance, insulin sensitivity, mitochondrial biogenesis, and physical endurance (19–23). Therefore, it is possible that the metabolic effects of resveratrol are mediated by AMPK. Supporting this possibility, resveratrol-mediated extension of lifespan in worms requires AMPK (24).Resveratrol may activate AMPK in several different ways. Resveratrol, as well as other polyphenols, can reduce ATP levels by inhibiting ATP synthase (25). Resveratrol can also activate AMPK without altering the AMP-to-ATP ratio. Dasgupta et al. (12) showed that, at lower doses, resveratrol can activate AMPK through a Sirt1-independent manner. Interestingly, Hou et al. (26) and Lan et al. (27) reported that the activity of liver kinase B (LKB)-1, one of the AMPK kinases that is important for AMPK activity, is activated by resveratrol in a Sirt1-dependent manner.     

Nutrient Stress Activates Inflammation and Reduces Glucose Metabolism by Suppressing AMP-Activated Protein Kinase in the Heart

OBJECTIVE

Heart failure is a major cause of mortality in diabetes and may be causally associated with altered metabolism. Recent reports indicate a role of inflammation in peripheral insulin resistance, but the impact of inflammation on cardiac metabolism is unknown. We investigated the effects of diet-induced obesity on cardiac inflammation and glucose metabolism in mice.

RESEARCH DESIGN AND METHODS

Male C57BL/6 mice were fed a high-fat diet (HFD) for 6 weeks, and heart samples were taken to measure insulin sensitivity, glucose metabolism, and inflammation. Heart samples were also examined following acute interleukin (IL)-6 or lipid infusion in C57BL/6 mice and in IL-6 knockout mice following an HFD.

RESULTS

Diet-induced obesity reduced cardiac glucose metabolism, GLUT, and AMP-activated protein kinase (AMPK) levels, and this was associated with increased levels of macrophages, toll-like receptor 4, suppressor of cytokine signaling 3 (SOCS3), and cytokines in heart. Acute physiological elevation of IL-6 suppressed glucose metabolism and caused insulin resistance by increasing SOCS3 and via SOCS3-mediated inhibition of insulin receptor substrate (IRS)-1 and possibly AMPK in heart. Diet-induced inflammation and defects in glucose metabolism were attenuated in IL-6 knockout mice, implicating the role of IL-6 in obesity-associated cardiac inflammation. Acute lipid infusion caused inflammation and raised local levels of macrophages, C-C motif chemokine receptor 2, SOCS3, and cytokines in heart. Lipid-induced cardiac inflammation suppressed AMPK, suggesting the role of lipid as a nutrient stress triggering inflammation.

CONCLUSIONS

Our findings that nutrient stress activates cardiac inflammation and that IL-6 suppresses myocardial glucose metabolism via inhibition of AMPK and IRS-1 underscore the important role of inflammation in the pathogenesis of diabetic heart.Type 2 diabetes is the most common metabolic disease in the world, affecting >250 million people, and cardiovascular disease is the leading cause of mortality in diabetes (1). Although the underlying mechanism by which diabetes increases cardiovascular events is unknown, perturbations in cardiac metabolism are among the earliest diabetes-induced alterations in the myocardium, preceding both functional and pathological changes, and may play a causative role in diabetic heart failure (2,3). Studies using isolated perfused-heart preparations, cultured cardiomyocytes, and positron emission tomography uniformly showed insulin resistance in human and animal models of diabetic heart (4,5). Diabetic heart is also characterized with elevated lipid oxidation with reciprocal reduction in glucose metabolism (6). Our recent study (7) found that chronic high-fat feeding impairs myocardial glucose metabolism, and this was associated with ventricular hypertrophy and cardiac dysfunction in obese mice. These findings highlight the importance of understanding the mechanism by which obesity and diabetes affect cardiac metabolism.Increasing evidence indicates the role of chronic inflammation and macrophage activation in insulin resistance (8,9). A cohort of recent studies (10–13) demonstrated increases in macrophage infiltration and cytokine expression in adipose tissue and their association with insulin resistance in obese humans and animal models. Tumor necrosis factor (TNF)-α is a proinflammatory cytokine that is secreted by macrophages and adipocytes and is shown to cause insulin resistance by inhibiting insulin signaling, AMP-activated protein kinase (AMPK), and the glucose transport system (14,15). Interleukin (IL)-6 is another proinflammatory cytokine that is elevated in obese diabetic subjects and is shown to cause insulin resistance by activating STAT3-suppressor of cytokine signaling 3 (SOCS3) expression and inhibiting the insulin signaling pathway in liver and adipose tissue (16–18). However, the role of IL-6 in insulin resistance remains debatable largely due to its differential effects on glucose metabolism in skeletal muscle, adipose tissue, and liver (19). Despite the wealth of information on the role of inflammation in peripheral insulin resistance, the impact of inflammation on cardiac metabolism has not been previously addressed. In this article, we demonstrate that diet-induced obesity increases macrophage and cytokine levels in heart. IL-6 reduces glucose metabolism by suppressing AMPK and insulin receptor substrate (IRS)-associated insulin signaling in heart, whereas IL-6–deficient mice are protected from diet-induced alterations in glucose metabolism. The fact that acute lipid infusion increases the inflammatory response and impairs myocardial glucose metabolism, similar to the effects of high-fat feeding, suggests the role of nutrient stress in the activation of toll-like receptor (TLR) 4 signaling and inflammation in heart. Since glucose is an important source of energy for a working heart, particularly during ischemia, our findings identify an important role of inflammation in the pathogenesis of diabetic heart failure.     

Mechanisms of Podocyte Injury in Diabetes: Role of Cytochrome P450 and NADPH Oxidases

OBJECTIVE

We investigated the role of cytochrome P450 of the 4A family (CYP4A), its metabolites, and NADPH oxidases both in reactive oxygen species (ROS) production and apoptosis of podocytes exposed to high glucose and in OVE26 mice, a model of type 1 diabetes.

RESEARCH DESIGN AND METHODS

Apoptosis, albuminuria, ROS generation, NADPH superoxide generation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo.

RESULTS

Exposure of mouse podocytes to high glucose resulted in apoptosis, with approximately one-third of the cells being apoptotic by 72 h. High-glucose treatment increased ROS generation and was associated with sequential upregulation of CYP4A and an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and Nox oxidases. This is consistent with the observation of delayed induction of NADPH oxidase activity by high glucose. The effects of high glucose on NADPH oxidase activity, Nox proteins and mRNA expression, and apoptosis were blocked by N-hydroxy-N′-(4-butyl-2-methylphenol) formamidine (HET0016), an inhibitor of CYP4A, and were mimicked by 20-HETE. CYP4A and Nox oxidase expression was upregulated in glomeruli of type 1 diabetic OVE26 mice. Treatment of OVE26 mice with HET0016 decreased NADPH oxidase activity and Nox1 and Nox4 protein expression and ameliorated apoptosis and albuminuria.

CONCLUSIONS

Generation of ROS by CYP4A monooxygenases, 20-HETE, and Nox oxidases is involved in podocyte apoptosis in vitro and in vivo. Inhibition of selected cytochrome P450 isoforms prevented podocyte apoptosis and reduced proteinuria in diabetes.Diabetic nephropathy in humans is characterized by increased urinary albumin excretion (microalbuminuria), which often progresses to proteinuria, one of the most important prognostic risk factors for kidney disease progression (1). Glomerular visceral epithelial cells, or podocytes, play a critical role in maintaining the structure and function of the glomerular filtration barrier. Careful morphometric analyses of renal biopsy in subjects with type 1 and type 2 diabetes (2–4) demonstrate that the density of podocytes is reduced not only in individuals with diabetic nephropathy, but also in patients with short duration of diabetes before the onset of microalbuminuria (4,5). Studies in experimental models of type 1 and type 2 diabetes have also documented that podocyte depletion represents one of the earliest cellular lesions affecting the diabetic kidney (6,7). Among various morphologic characteristics, the decreased number of podocytes in glomeruli is the strongest predictor of progression of diabetic nephropathy, where fewer cells predict more rapid progression (3,4). Although these observations identify podocyte depletion as one of the earliest cellular features of diabetic kidney disease, the mechanisms that underlie the loss of podocytes in diabetic nephropathy remain poorly understood.High glucose induces apoptosis (8), and there is evidence that podocyte apoptosis contributes to reduced podocyte number (9). High glucose, transforming growth factor-β (TGF-β), and angiotensin II (ANGII) induce apoptosis of cultured podocytes (9–12). ANGII appears to induce apoptosis in cultured rat glomerular epithelial cells at least partially via TGF-β because its apoptotic effect is attenuated by an anti–TGF-β antibody (12). There is also evidence that reactive oxygen species (ROS) contribute to podocyte apoptosis and depletion in cells exposed to high glucose and in experimental diabetic nephropathy (7). However, the sources of ROS and the kinetics of their generation have not been well characterized. We and others (13–15) have recently identified NADPH oxidases as major sources of ROS in kidney cortex and glomeruli of rats with type 1 diabetes. Six homologs of the cytochrome subunit of the phagocyte NADPH oxidase (Nox2/gp91^phox) have been cloned (16). At least three different Nox isoforms are expressed in the kidney cortex: Nox1, Nox2, and Nox4 (16). Cytochromes P450 (CYP450s) are significant sources of ROS in many tissues (17,18). CYP450 metabolizes arachidonic acid into hydroxyeicosatetraenoic acids (20-HETEs) and EETs (epoxyeicosatrienoic acids). 20-HETE, the ω-hydroxylation product of arachidonic acid, is one of the major CYP eicosanoids produced in the kidney cortex (19–21). The predominant CYP450 in the kidney cortex that synthesizes 20-HETE is cytochrome P450 of the 4A family (CYP4A) (19–21). 20-HETE has multiple and opposing functions depending on the site of production and target cells/tissues (19,22–24).In this study, we demonstrate that high glucose induces ROS production and apoptosis in cultured mouse podocytes through the upregulation of CYP4A with increased production of 20-HETE and upregulation of NADPH oxidases. Inhibition of 20-HETE production prevented podocyte apoptosis in vitro and decreased oxidative stress, podocyte apoptosis, and proteinuria in an in vivo model of type 1 diabetes.     

Rac1 Is Required for Cardiomyocyte Apoptosis During Hyperglycemia

OBJECTIVE

Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia.

RESEARCH DESIGN AND METHODS

Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose.

RESULTS

The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91^phox or p47^phox, or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function.

CONCLUSIONS

Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes.Diabetic cardiomyopathy has been defined as ventricular dysfunction that occurs in the absence of changes in blood pressure and coronary artery disease (1,2). Cell death by apoptosis is the predominant damage in diabetic cardiomyopathy (3,4). Diabetes increases cardiac apoptosis in animals and patients (3–7). Cardiomyocyte death causes a loss of contractile tissue, which initiates a cardiac remodeling (8). Loss of cardiomyocytes and hypertrophy of the remaining cells characterize the diabetic cardiomyopathy (9,10). Thus, suppression of cardiomyocyte apoptosis results in a significant prevention of the development of diabetic cardiomyopathy (4). However, the underlying mechanisms by which diabetes induce apoptosis remain not fully understood.All forms of diabetes are characterized by chronic hyperglycemia. Hyperglycemia induces reactive oxygen species (ROS) production in cardiomyocytes (6,11), which plays a crucial role in cardiomyocyte apoptosis in diabetes because the administration of antioxidant agents are able to rescue hyperglycemia-induced cardiomyocytes (4,6). The mechanisms activated by hyperglycemia, leading to myocardial oxidative stress and apoptosis, are not completely clarified.Although multiple sources of ROS have been demonstrated, NADPH oxidase is a critical determinant of the redox state of the myocardium (12–15). Higher myocardial NADPH oxidase activity has been detected in diabetes (16,17); more importantly, NADPH oxidase activity is markedly increased by high glucose levels (18). The NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b558, which contains gp91^phox and p22^phox, the cytosolic regulatory subunits p47^phox and p67^phox, and the small guanosine triphosphate-binding protein Rac. An important step for the assembly and function of this multicomponent NADPH oxidase complex is the heterodimerization of gp91^phox with p67^phox, which is mediated by Rac (19). Three isoforms of Rac (Rac1, Rac2, and Rac3) have been identified (20), and Rac1 is the predominant isoform expressed in cardiomyocytes (21). Thus, Rac1 activation may lead to myocardial oxidative stress and apoptosis during hyperglycemia. A recent study showed that Rac1 contributes to vascular injury in diabetes (22). However, no direct evidence is available on Rac1 and NADPH oxidase activation in cardiomyocyte apoptosis in diabetes.In this study, we generated cardiomyocyte-specific Rac1 knockout (Rac1-ko) mice; analyzed the impact of Rac1 on NADPH oxidase activation, mitochondrial ROS generation, and intracellular ROS production; and investigated the role of Rac1 and NADPH oxidase activation in cardiomyocyte apoptosis during hyperglycemia.     

High- and Moderate-Intensity Training Normalizes Ventricular Function and Mechanoenergetics in Mice With Diet-Induced Obesity

Although exercise reduces several cardiovascular risk factors associated with obesity/diabetes, the metabolic effects of exercise on the heart are not well-known. This study was designed to investigate whether high-intensity interval training (HIT) is superior to moderate-intensity training (MIT) in counteracting obesity-induced impairment of left ventricular (LV) mechanoenergetics and function. C57BL/6J mice with diet-induced obesity (DIO mice) displaying a cardiac phenotype with altered substrate utilization and impaired mechanoenergetics were subjected to a sedentary lifestyle or 8–10 weeks of isocaloric HIT or MIT. Although both modes of exercise equally improved aerobic capacity and reduced obesity, only HIT improved glucose tolerance. Hearts from sedentary DIO mice developed concentric LV remodeling with diastolic and systolic dysfunction, which was prevented by both HIT and MIT. Both modes of exercise also normalized LV mechanical efficiency and mechanoenergetics. These changes were associated with altered myocardial substrate utilization and improved mitochondrial capacity and efficiency, as well as reduced oxidative stress, fibrosis, and intracellular matrix metalloproteinase 2 content. As both modes of exercise equally ameliorated the development of diabetic cardiomyopathy by preventing LV remodeling and mechanoenergetic impairment, this study advocates the therapeutic potential of physical activity in obesity-related cardiac disorders.Obesity, sedentary lifestyle, and reduced aerobic capacity are known predictors of heart failure and a major challenge to the health care system of the Western society (1,2). In addition to increasing the risk of cardiovascular disease, obesity and diabetes have been associated with the development of a distinct cardiomyopathy with ventricular remodeling and the progression to cardiac dysfunction (3). Reduced mechanical efficiency is an important hallmark of obesity/diabetic cardiomyopathy (4,5), and recent experimental studies have demonstrated diabetes-related inefficiency to be due to impaired mechanoenergetics, where myocardial oxygen consumption (MVo₂) for nonmechanical processes is increased (6,7). Several obesity- or diabetes-induced changes, including increased fatty acid oxidation (4,8,9), impaired calcium handing (10,11), increased oxidative stress (12), and mitochondrial dysfunction (13), are factors that, most likely, contribute to increased MVo₂. Although exercise has been reported to induce mitochondrial and cellular adaptations that could potentially influence myocardial oxygen-consuming processes (such as increased antioxidant capacity, reduced oxidative stress, increased mitochondrial efficiency, and improved myocardial Ca²⁺ homeostasis [10,14,15]), no studies have assessed the consequences of exercise-induced adaptations in terms of left ventricular (LV) mechanical efficiency and mechanoenergetics properties in a model of obesity and insulin resistance.High-intensity exercise training induces a more pronounced increase in aerobic capacity and more evident cardiovascular adaptations compared with low- and moderate-intensity training (MIT) in healthy subjects (16–18). In a recent study by Tjønna et al. (19), high-intensity training (HIT) was also found to be superior to MIT in reducing cardiovascular risk factors in patients with metabolic syndrome. In addition, we found that high- but not MIT altered myocardial substrate utilization (decrease in fatty acid oxidation and increase in glucose oxidation) and increased mitochondrial respiratory capacity and LV mechanoenergetic properties in hearts from lean mice (18). Based on these findings, we hypothesized that exercise of high intensity would be superior to isocaloric moderate-intensity exercise in counteracting the unfavorable metabolic and functional changes that occur in the heart during obesity.     

Activation of AMP-Activated Protein Kinase Inhibits Oxidized LDL-Triggered Endoplasmic Reticulum Stress In Vivo

OBJECTIVE

The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL–triggered ER stress in vivo.

RESEARCH DESIGN AND METHODS

ER stress markers, sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) activity and oxidation, and AMPK activity were monitored in cultured bovine aortic endothelial cells (BAECs) exposed to HOG-LDL or in isolated aortae from mice fed an atherogenic diet.

RESULTS

Exposure of BAECs to clinically relevant concentrations of HOG-LDL induced prolonged ER stress and reduced SERCA activity but increased SERCA oxidation. Chronic administration of Tempol (a potent antioxidant) attenuated both SERCA oxidation and aberrant ER stress in mice fed a high-fat diet in vivo. Likewise, AMPK activation by pharmacological (5′-aminoimidazole-4-carboxymide-1-β-d-ribofuranoside, metformin, and statin) or genetic means (adenoviral overexpression of constitutively active AMPK mutants) significantly mitigated ER stress and SERCA oxidation and improved the endothelium-dependent relaxation in isolated mouse aortae. Finally, Tempol administration markedly attenuated impaired endothelium-dependent vasorelaxation, SERCA oxidation, ER stress, and atherosclerosis in ApoE^−/− and ApoE^−/−/AMPKα2^−/− fed a high-fat diet.

CONCLUSION

We conclude that HOG-LDL, via enhanced SERCA oxidation, causes aberrant ER stress, endothelial dysfunction, and atherosclerosis in vivo, all of which are inhibited by AMPK activation.LDL oxidation and glycation are known to promote atherosclerosis through several mechanisms that include promoting vascular proinflammatory responses, intracellular oxidative stress, and apoptosis associated with endothelial dysfunction (1,2). In addition, LDL oxidation is greatly enhanced by LDL glycation (3,4). For example, glycation of LDL slows the clearance of these particles from the circulation (5), increases their susceptibility to oxidative damage (6), enhances entrapment of extravasated particles in the vascular subintimal space, and increases chemotactic activity of monocytes (7). The presence of both glycated LDL and glycoxidized LDL in human atherosclerotic plaques has been confirmed by immunochemical methods both in vivo and in vitro (8–10). Increasing evidence suggests that glycation and oxidation of LDL induces apoptosis in arterial wall cells (11,12), and glycoxidized LDL triggers apoptosis in vascular smooth muscle cells (13,14). Overall, glycation of LDL promotes the formation of oxidized LDL, and this phenomenon contributes to accelerated atherosclerosis, an important pathologic corollary of diabetes.Endoplasmic reticulum (ER) stress has been linked to a wide range of human pathologies including diabetes (15–17), obesity (16,17), atherosclerosis (18), cancer, neurodegenerative disorders, and inflammatory conditions. ER stress may be triggered by high glucose, oxidative stress, Ca²⁺ overload, ischemia, and hypoxia. In addition, it causes the accumulation of unfolded and misfolded proteins, leading to an “unfolded protein response” (19). The normal ER is the principal site of protein synthesis, folding, and maturation. In unfolded protein response, unfolded or misfolded proteins are sent to the cytoplasm by a “retro-translocation mechanism” to be degraded by the ubiquitin proteasome system (20).AMP-activated protein kinase (AMPK), a sensor of cellular energy status, plays a critical role in controlling the cell''s energy balance and metabolism (21), and activation of AMPK is an important defensive response to stress (22). AMPK activation is neuroprotective (23), and also mediates at least some cardiovascular protective effects of drugs such as hydroxymethylglutaryl-CoA reductase inhibitors (e.g., the statins such as pravastatin and atorvastatin) and metformin (a biguanide that activates AMPK) (24,25). Activation of AMPK protects cardiomyocytes against hypoxic injury through attenuation of ER stress (26). However, whether AMPK alters oxidized LDL-induced ER stress in endothelial cells has not been investigated to date. In this study, we report that oxidized, glycated-LDL (HOG-LDL) via the oxidation and inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA), triggers ER stress in endothelial cells in vivo. In addition, we have uncovered evidence suggesting that AMPK activation attenuates ER stress by inhibiting SERCA oxidation caused by HOG-LDL.     

Hypothalamic AMP-Activated Protein Kinase Regulates Glucose Production

OBJECTIVE

The fuel sensor AMP-activated protein kinase (AMPK) in the hypothalamus regulates energy homeostasis by sensing nutritional and hormonal signals. However, the role of hypothalamic AMPK in glucose production regulation remains to be elucidated. We hypothesize that bidirectional changes in hypothalamic AMPK activity alter glucose production.

RESEARCH DESIGN AND METHODS

To introduce bidirectional changes in hypothalamic AMPK activity in vivo, we first knocked down hypothalamic AMPK activity in male Sprague-Dawley rats by either injecting an adenovirus expressing the dominant-negative form of AMPK (Ad-DN AMPKα2 [D¹⁵⁷A]) or infusing AMPK inhibitor compound C directly into the mediobasal hypothalamus. Next, we independently activated hypothalamic AMPK by delivering either an adenovirus expressing the constitutive active form of AMPK (Ad-CA AMPKα1³¹² [T172D]) or the AMPK activator AICAR. The pancreatic (basal insulin)-euglycemic clamp technique in combination with the tracer-dilution methodology was used to assess the impact of alternations in hypothalamic AMPK activity on changes in glucose kinetics in vivo.

RESULTS

Injection of Ad-DN AMPK into the hypothalamus knocked down hypothalamic AMPK activity and led to a significant suppression of glucose production with no changes in peripheral glucose uptake during the clamps. In parallel, hypothalamic infusion of AMPK inhibitor compound C lowered glucose production as well. Conversely, molecular and pharmacological activation of hypothalamic AMPK negated the ability of hypothalamic nutrients to lower glucose production.

CONCLUSIONS

These data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo.AMP-activated protein kinase (AMPK) is an evolutionarily conserved cellular energy sensor that regulates cellular metabolism (1). Consisting of a catalytic α subunit and two regulatory β and γ subunits, AMPK responds to an increase in intracellular AMP-to-ATP ratio and phosphorylates intracellular targets involved in cellular metabolism to promote ATP-generating processes and inhibit energy-consuming pathways. AMPK is expressed in a variety of tissues including the liver, skeletal muscles, adipose tissue, and the hypothalamus (1). AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC) (1), which prevents the conversion of acetyl-CoA to malonyl-CoA. A decrease in malonyl-CoA relieves the inhibition of carnitine palmitoyltransferase-1 (2) and favors the transfer of long-chain fatty acyl-CoA (LCFA-CoA) into the mitochondria for β-oxidation. Conversely, direct inhibition of AMPK increases malonyl-CoA and LCFA-CoA levels (3).Studies have emerged implicating that AMPK in the hypothalamus integrates nutritional and hormonal signals to regulate food intake (4–8). In particular, direct inhibition of hypothalamic AMPK lowers food intake (8), whereas selective activation of hypothalamic AMPK negates the ability of leptin to activate hypothalamic ACC, increase hypothalamic malonyl-CoA levels, and lower food intake (9). In light of the fact that the hypothalamus integrates nutritional and hormonal signals to not only regulate energy (10–12) but also glucose (13–17) homeostasis, and that accumulation of hypothalamic malonyl-CoA and LCFA-CoA levels lowers food intake as well as hepatic glucose production (18–20), a possibility arises that direct inhibition of hypothalamic AMPK activity could alter hepatic glucose production (Fig. 1A). This working hypothesis was first tested in the current study.Open in a separate windowFIG. 1.Molecular knockdown of hypothalamic AMPK by the dominant-negative form of AMPK (DN AMPK) is sufficient to lower glucose production. A: Schematic representation of the working hypothesis: Inhibition of hypothalamic AMPK activity by DN AMPK or compound C leads to the lowering of hepatic glucose production. B: Experimental procedure and clamp protocol. A bilateral MBH catheter was implanted on day 0. Adenovirus tagged with GFP (Ad-GFP) or adenovirus-expressing DN AMPK (Ad-DN AMPK) was injected into the MBH of a group of rats immediately after MBH catheter implantation. Venous and arterial cannulations were done on day 5, and the pancreatic clamp protocol was performed on day 8. In the Ad-GFP and Ad-DN AMPK–injected rats, no MBH infusions were given during the clamp experiments. In rats with no adenovirus injection, 5% DMSO control or compound C was infused into the MBH during the clamps. C: Hypothalamic AMPK activity was significantly diminished in animals injected with Ad-DN AMPK, compared with control animals with injection of Ad-GFP (*P < 0.001). Hypothalamic injection of Ad-DN AMPK led to an increase in glucose infusion rate (D) (*P < 0.01) and a decrease in glucose production (E) (*P < 0.001) compared with the GFP control. F: Suppression of glucose production during the clamp period (180–210 min) expressed as percentage reduction from basal steady state (60–90 min) (*P < 0.01 vs. GFP control). G: Glucose uptake was not significantly different from that of GFP control. Values are shown as means ± SEM. (A high-quality color representation of this figure is available in the online issue.)Second, hypothalamus glucose metabolism to lactate, and the subsequent conversion of lactate to pyruvate and acetyl-CoA, have been reported to lower hepatic glucose production (21). However, the downstream biochemical pathways that mediate the ability of hypothalamic glucose/lactate sensing to lower glucose production remain unclear, although it was hypothesized that the formation of malonyl-CoA via the enhanced flux of acetyl-CoA could be a necessary step (3,15). Given the well-established regulatory role of AMPK on the formation of malonyl-CoA from acetyl-CoA and that hypothalamic malonyl-CoA regulates glucose production (18), we next tested the possibility that direct activation of hypothalamic AMPK negates the ability of central nervous system glucose/lactate sensing to regulate glucose production.In summary, we tested the hypothesis that molecular and pharmacological changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to regulate glucose production in vivo.     

Lack of Type VIII Collagen in Mice Ameliorates Diabetic Nephropathy

OBJECTIVE

Key features of diabetic nephropathy include the accumulation of extracellular matrix proteins. In recent studies, increased expression of type VIII collagen in the glomeruli and tubulointerstitium of diabetic kidneys has been noted. The objectives of this study were to assess whether type VIII collagen affects the development of diabetic nephropathy and to determine type VIII collagen–dependent pathways in diabetic nephropathy in the mouse model of streptozotocin (STZ)-induced diabetes.

RESEARCH DESIGN AND METHODS

Diabetes was induced by STZ injections in collagen VIII–deficient or wild-type mice. Functional and histological analyses were performed 40 days after induction of diabetes. Type VIII collagen expression was assessed by Northern blots, immunohistochemistry, and real-time PCR. Proliferation of primary mesangial cells was measured by thymidine incorporation and direct cell counting. Expression of phosphorylated extracellular signal–regulated kinase (ERK1/2) and p27^Kip1 was assessed by Western blots. Finally, Col8a1 was stably overexpressed in mesangial cells.

RESULTS

Diabetic wild-type mice showed a strong renal induction of type VIII collagen. Diabetic Col8a1⁻/Col8a2⁻ animals revealed reduced mesangial expansion and cellularity and extracellular matrix expansion compared with the wild type. These were associated with less albuminuria. High-glucose medium as well as various cytokines induced Col8a1 in cultured mesangial cells. Col8a1⁻/Col8a2⁻ mesangial cells revealed decreased proliferation, less phosphorylation of Erk1/2, and increased p27^Kip1 expression. Overexpression of Col8a1 in mesangial cells induced proliferation.

CONCLUSIONS

Lack of type VIII collagen confers renoprotection in diabetic nephropathy. One possible mechanism is that type VIII collagen permits and/or fosters mesangial cell proliferation in early diabetic nephropathy.Diabetic nephropathy is the most common cause of end-stage renal failure leading to dialysis. Glomerular lesions are characterized by expansion of the mesangial matrix and thickening of peripheral glomerular basement membranes due to the synthesis and accumulation of extracellular matrix (ECM) (1,2). The degree of mesangial matrix expansion correlates with the progressive decline in the glomerular capillary surface area available for filtration and, hence, with the glomerular filtration rate (3). Early changes include a confined proliferation of mesangial cells followed by cell cycle arrest and hypertrophy (3–8). Several growth factors have been implicated in this process, among them transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF)-BB (4,9,10). During early stages, PDGF-BB potently increases proliferation and matrix synthesis of mesangial cells and induces the expression of TGF-β1 (4,5,11). Upregulation of the PDGF-BB pathway has been shown in kidneys from patients with diabetic nephropathy as well as in experimental models of diabetic nephropathy (12,13). Further, PDGF receptor antagonists attenuate diabetic nephropathy (4). Activation of the TGF-β1 loop leads to cell cycle arrest, induction of cyclin-dependent kinase inhibitors, and further ECM synthesis (3,14).Type VIII collagen, a nonfibrillar short-chain collagen, is a structural component of many extracellular matrices (15–17). Two highly homologous polypeptides, α1(VIII) and α2(VIII), form either homotrimeric or heterotrimeric molecules (18–20). Type VIII collagen is involved in cross-talk between cells and the surrounding matrix by modulating diverse cellular responses such as proliferation, adhesion, migration, chemotaxis, and metalloproteinase synthesis (21–23). It is highly expressed by vascular smooth muscle cells in response to PDGF-BB and is thought to be a key component of vascular remodeling (24–27). In healthy kidneys, expression of type VIII collagen has been demonstrated in glomerular arterioles, larger branches of renal arteries, and in rat glomeruli and mesangial cell in vitro (28,29). Increased mRNA as well as protein expression has been noted in glomeruli and the tubulointerstitium of biopsies of kidneys from patients with diabetic nephropathy (30,31). The functional role of collagen VIII, especially in the early phase of the disease, has not been investigated and remains obscure.To address the role of type VIII collagen in the pathogenesis of diabetic nephropathy, we applied the streptozotocin (STZ) model to mice with homozygous deletions of both collagen VIII genes and compared them with wild-type mice. The objectives of this study were to assess whether collagen VIII–dependent pathways are involved in the development of diabetic nephropathy and in various cellular and molecular processes associated with this disorder.     

Mammalian Target of Rapamycin Regulates Nox4-Mediated Podocyte Depletion in Diabetic Renal Injury

Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Pharmacological doses of the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduce albuminuria in diabetes. We explored the hypothesis that mTOR mediates podocyte injury in diabetes. High glucose (HG) induces apoptosis of podocytes, inhibits AMP-activated protein kinase (AMPK) activation, inactivates tuberin, and activates mTOR. HG also increases the levels of Nox4 and Nox1 and NADPH oxidase activity. Inhibition of mTOR by low-dose rapamycin decreases HG-induced Nox4 and Nox1, NADPH oxidase activity, and podocyte apoptosis. Inhibition of mTOR had no effect on AMPK or tuberin phosphorylation, indicating that mTOR is downstream of these signaling molecules. In isolated glomeruli of OVE26 mice, there is a similar decrease in the activation of AMPK and tuberin and activation of mTOR with increase in Nox4 and NADPH oxidase activity. Inhibition of mTOR by a small dose of rapamycin reduces podocyte apoptosis and attenuates glomerular injury and albuminuria. Our data provide evidence for a novel function of mTOR in Nox4-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. Thus, mTOR and/or NADPH oxidase inhibition may represent a therapeutic modality of diabetic kidney disease.There is increasing evidence that the mammalian target of rapamycin (mTOR) pathway is involved in the pathogenic manifestations of diabetic nephropathy. mTOR phosphorylation is enhanced in the kidney cortex of diabetic rats, and treatment of rats with rather large doses of the mTOR inhibitor rapamycin blocks diabetes-induced glomerular hypertrophy and albuminuria (1–3). The mechanism by which mTOR inhibition reduces albuminuria is unknown. Injury to glomerular epithelial cells or podocytes interferes with the integrity of the glomerular filtration barrier and contributes to albuminuria. In diabetic subjects and in diabetic animals, there is a decrease in podocyte number (4–8).mTOR, a highly conserved nutrient-responsive regulator of cell growth found in eukaryotes (9), is a serine/threonine protein kinase existing in two complexes, mTORC1 or mTORC2, consisting of distinct sets of protein-binding partners (10,11). mTORC1 is rapamycin sensitive and is thought to mediate many of its downstream effects through p70S6 kinase (p70S6K)/S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) (12). mTORC2 is largely rapamycin resistant and mediates phosphorylation of protein kinase B (PKB/Akt) at Ser⁴⁷³ (13). Although both complexes respond to hormones and growth factors, only mTORC1 is activated by nutrients and cellular energy status (12). mTOR activity is negatively regulated by the heterodimeric complex consisting of tuberin (TSC2) and hamartin (TSC1). Phosphorylation of tuberin serves as an integration point for a wide variety of environmental signals that regulate mTORC1 (11). Importantly, phosphorylation of tuberin by AMP-activated protein kinase (AMPK) maintains its tumor-suppressor activity and prevents the activation of mTORC1.We recently demonstrated that AMPK is inactivated in podocytes exposed to excess glucose and in an experimental model of type 1 diabetes (7,14). A functional link between AMPK and mTORC1 has been reported (15). AMPK is known to directly phosphorylate tuberin on conserved sites such as Thr¹²⁷¹ and Ser¹³⁸⁷, thereby maintaining the tuberin/hamartin complex active to prevent the activation of mTORC1 (16,17). We postulated that the activation of mTOR in type 1 diabetes reduces podocyte survival, providing a potential mechanism by which mTOR inhibition reduces albuminuria.In this study, we provide evidence that type 1 diabetes, or high glucose (HG)–induced podocyte apoptosis, is mediated by activation of the mTOR pathway through inactivation of AMPK/tuberin. We further demonstrate that in type 1 diabetes, the activation of mTOR enhances oxidative stress via upregulation of Nox4 and Nox1 expression and NADPH oxidase activity. Inhibition of the mTOR pathway by clinically relevant doses of rapamycin reverses the observed changes. Furthermore, we show that inhibition of mTOR decreases podocyte apoptosis, reduces glomerular basement membrane thickening (GBM) and foot process effacement, and attenuates mesangial expansion and albuminuria.