Victor Samokhvalov

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and beta-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial beta-oxidation. However, imbalance between fatty acid uptake and beta-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid beta-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid beta-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid beta-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.

Stimulation of glucose oxidation protects against acute myocardial infarction and reperfusion injury

AIMS During reperfusion of the ischaemic myocardium, fatty acid oxidation rates quickly recover, while glucose oxidation rates remain depressed. Direct stimulation of glucose oxidation via activation of pyruvate dehydrogenase (PDH), or secondary to an inhibition of malonyl CoA decarboxylase (MCD), improves cardiac functional recovery during reperfusion following ischaemia. However, the effects of such interventions on the evolution of myocardial infarction are unknown. The purpose of this study was to determine whether infarct size is decreased in response to increased glucose oxidation. METHODS AND RESULTS In vivo, direct stimulation of PDH in mice with the PDH kinase (PDHK) inhibitor, dichloroacetate, significantly decreased infarct size following temporary ligation of the left anterior descending coronary artery. These results were recapitulated in PDHK 4-deficient (PDHK4-/-) mice, which have enhanced myocardial PDH activity. These interventions also protected against ischaemia/reperfusion injury in the working heart, and dichloroacetate failed to protect in PDHK4-/- mice. In addition, there was a dramatic reduction in the infarct size in malonyl CoA decarboxylase-deficient (MCD-/-) mice, in which glucose oxidation rates are enhanced (secondary to an inhibition of fatty acid oxidation) relative to their wild-type littermates (10.8 ± 3.8 vs. 39.5 ± 4.7%). This cardioprotective effect in MCD-/- mice was associated with increased PDH activity in the ischaemic area at risk (1.89 ± 0.18 vs. 1.52 ± 0.05 μmol/g wet weight/min). CONCLUSION These findings demonstrate that stimulating glucose oxidation via targeting either PDH or MCD decreases the infarct size, validating the concept that optimizing myocardial metabolism is a novel therapy for ischaemic heart disease.

Direct and macrophage-mediated actions of fatty acids causing insulin resistance in muscle cells

Obesity is associated with insulin resistance and increased risk for developing type 2 diabetes. Enlarged adipocytes develop resistance to the anti-lipolytic action of insulin. Elevated levels of fatty acids in the plasma and interstitial fluids lead to whole-body insulin resistance by disrupting normal insulin-regulated glucose uptake and glycogen storage in skeletal muscle. A new understanding has been cultivated in the past 5 to 10 years that adipocytes and macrophages (resident or bone marrow-derived) in adipose tissue of obese animals and humans are activated in a pro-inflammatory capacity and secrete insulin resistance-inducing factors. However, only recently have fatty acids themselves been identified as agents that engage toll-like receptors of the innate immunity systems of macrophages, adipocytes and muscle cells to trigger pro-inflammatory responses. This review summarizes our observations that fatty acids evoke the release of pro-inflammatory factors from macrophages that consequently induce insulin resistance in muscle cells.

Palmitate- and lipopolysaccharide-activated macrophages evoke contrasting insulin responses in muscle cells

Factors secreted by macrophages contribute to whole body insulin resistance, acting in part on adipose tissue. Muscle is the major tissue for glucose disposal, but how macrophage-derived factors impact skeletal muscle glucose uptake is unknown, or whether the macrophage environment influences this response. We hypothesized that conditioned medium from macrophages pretreated with palmitate or LPS would directly affect insulin action and glucose uptake in muscle cells. L6-GLUT4myc myoblasts were exposed to conditioned medium from RAW 264.7 macrophages pretreated with palmitate or LPS. Conditioned medium from palmitate-treated RAW 264.7 macrophages inhibited myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation while activating JNK p38 MAPK, decreasing IkappaBalpha, and elevating inflammation markers. Surprisingly, and opposite to its effects on adipose cells, conditioned medium from LPS-treated macrophages stimulated myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation without affecting stress kinases or inflammation indexes. This medium had markedly elevated IL-10 levels, and IL-10, alone, potentiated insulin action in myoblasts and partly reversed the insulin resistance imparted by medium from palmitate-treated macrophages. IL-10 neutralizing antibodies blunted the positive influence of LPS macrophage-conditioned medium. We conclude that myoblasts and adipocytes respond differently to cytokines. Furthermore, depending on their environment, macrophages negatively or positively influence muscle cells. Macrophages exposed to palmitate produce a mixture of proinflammatory cytokines that reduce insulin action in muscle cells; conversely, LPS-activated macrophages increase insulin action, likely via IL-10. Macrophages may be an integral element in glucose homeostasis in vivo, relaying effects of circulating factors to skeletal muscle.

Epoxyeicosatrienoic acids protect cardiac cells during starvation by modulating an autophagic response

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid involved in regulating pathways promoting cellular protection. We have previously shown that EETs trigger a protective response limiting mitochondrial dysfunction and reducing cellular death. Considering it is unknown how EETs regulate cell death processes, the major focus of the current study was to investigate their role in the autophagic response of HL-1 cells and neonatal cardiomyocytes (NCMs) during starvation. We employed a dual-acting synthetic analog UA-8 (13-(3-propylureido)tridec-8-enoic acid), possessing both EET-mimetic and soluble epoxide hydrolase (sEH) inhibitory properties, or 14,15-EET as model EET molecules. We demonstrated that EETs significantly improved viability and recovery of starved cardiac cells, whereas they lowered cellular stress responses such as caspase-3 and proteasome activities. Furthermore, treatment with EETs resulted in preservation of mitochondrial functional activity in starved cells. The protective effects of EETs were abolished by autophagy-related gene 7 (Atg7) short hairpin RNA (shRNA) or pharmacological inhibition of autophagy. Mechanistic evidence demonstrated that sarcolemmal ATP-sensitive potassium channels (pmKATP) and enhanced activation of AMP-activated protein kinase (AMPK) played a crucial role in the EET-mediated effect. Our data suggest that the protective effects of EETs involve regulating the autophagic response, which results in a healthier pool of mitochondria in the starved cardiac cells, thereby representing a novel mechanism of promoting survival of cardiac cells. Thus, we provide new evidence highlighting a central role of the autophagic response in linking EETs with promoting cell survival during deep metabolic stress such as starvation.

Inhibition of malonyl-CoA decarboxylase reduces the inflammatory response associated with insulin resistance

We previously showed that genetic inactivation of malonyl-CoA decarboxylase (MCD), which regulates fatty acid oxidation, protects mice against high-fat diet-induced insulin resistance. Development of insulin resistance has been associated with activation of the inflammatory response. Therefore, we hypothesized that the protective effect of MCD inhibition might be caused by a favorable effect on the inflammatory response. We examined if pharmacological inhibition of MCD protects neonatal cardiomyocytes and peritoneal macrophages against inflammatory-induced metabolic perturbations. Cardiomyocytes and macrophages were treated with LPS to induce an inflammatory response, in the presence or absence of an MCD inhibitor (CBM-301106, 10 μM). Inhibition of MCD attenuated the LPS-induced inflammatory response in cardiomyocytes and macrophages. MCD inhibition also prevented LPS impairment of insulin-stimulated glucose uptake in cardiomyocytes and increased phosphorylation of Akt. Additionally, inhibition of MCD strongly diminished LPS-induced activation of palmitate oxidation. We also found that treatment with an MCD inhibitor prevented LPS-induced collapse of total cellular antioxidant capacity. Interestingly, treatment with LPS or an MCD inhibitor did not alter intracellular triacylglycerol content. Furthermore, inhibition of MCD prevented LPS-induced increases in the level of ceramide in cardiomyocytes and macrophages while also ameliorating LPS-initiated decreases in PPAR binding. This suggests that the anti-inflammatory effect of MCD inhibition is mediated via accumulation of long-chain acyl-CoA, which in turn stimulates PPAR binding. Our results also demonstrate that pharmacological inhibition of MCD is a novel and promising approach to treat insulin resistance and its associated metabolic complications.

Cytochrome P450-derived eicosanoids and heart function

ABSTRACT The cytochrome P450 monooxygenase system (CYP) is a multigene superfamily of enzymes, which are important in the metabolism of foreign and endogenous compounds. CYP isoforms metabolize a number of n‐3 and n‐6 polyunsaturated fatty acids (PUFA), including linoleic acid (18:2n6, LA), arachidonic acid (20:4n6, AA), ecosapentaenoic acid (20:5n3, EPA) and docosahexaenoic acid (22:6n3, DHA) into bioactive lipid mediators, termed eicosanoids. CYP‐derived eicosanoids have numerous effects toward physiological and pathophysiological events within the body, which depends on the type, quantity and timing of metabolites produced. Alterations in fatty acid composition and concentrations have been shown to have a role in cardiovascular disease (CVD). The functional role of CYP isozymes and CYP‐derived eicosanoids toward physiological and pathophysiological processes in the heart is a rapidly expanding field of research. Numerous studies have investigated the beneficial and detrimental effects of CYP epoxygenase derived metabolites of AA, epoxyeicosatrienoic acids (EET) and CYP &ohgr;‐hydroxylase products, hydroxyeicosatetraenoic acids (HETE), toward both cardiac and vascular function and disease. Emerging research is revealing the importance of other lipid mediators generated from CYP isozymes, such as epoxyeicosatetraenoic acids (EEQ) and epoxydocosapentaenoic acids (EDP), formed from the metabolism of EPA and DHA and metabolites of LA. Important determinants such as genetics, gender and age have a role in regulating the CYP‐derived eicosanoids produced from the metabolism n‐3 and n‐6 PUFA. Obtaining a better understanding of the complex role CYP‐derived eicosanoids have within the heart will provide valuable insight for both basic and clinical researchers investigation CVD.

A Disintegrin and Metalloprotease-17 Regulates Pressure Overload–Induced Myocardial Hypertrophy and Dysfunction Through Proteolytic Processing of Integrin β1

A disintegrin and metalloprotease-17 (ADAM17) belongs to a family of transmembrane enzymes, and it can mediate ectodomain shedding of several membrane-bound molecules. ADAM17 levels are elevated in patients with hypertrophic and dilated cardiomyopathy; however, its direct role in hypertrophic cardiomyopathy is unknown. Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17flox/flox/&agr;MHC-Cre; ADAM17f/f/Cre) and littermates with intact ADAM17 levels (ADAM17f/f) were subjected to cardiac pressure–overload by transverse aortic constriction. Cardiac function/architecture was assessed by echocardiography at 2 and 5 weeks post transverse aortic constriction. ADAM17 knockdown enhanced myocardial hypertrophy, fibrosis, more severe left ventricular dilation, and systolic dysfunction at 5 weeks post transverse aortic constriction. Pressure overload–induced upregulation of integrin &bgr;1 was much greater with ADAM17 knockdown, concomitant with the greater activation of the focal adhesion kinase pathway, suggesting that integrin &bgr;1 could be a substrate for ADAM17. ADAM17 knockdown did not alter other cardiomyocyte integrins, integrin &agr;5 or &agr;7, and HB-EGF (heparin-bound epidermal growth factor), another potential substrate for ADAM17, remained unaltered after pressure overload. ADAM17-mediated cleavage of integrin &bgr;1 was confirmed by an in vitro assay. Intriguingly, ADAM17 knockdown did not affect the myocardial hypertrophy induced by a subpressor dose of angiotensin II, which occurs independent from the integrin &bgr;1–mediated pathway. ADAM17-knockdown enhanced the hypertrophic response to cyclic mechanical stretching in neonatal rat cardiomyocytes. This study reports a novel cardioprotective function for ADAM17 in pressure overload cardiomyopathy, where loss of ADAM17 promotes hypertrophy by reducing the cleavage of cardiac integrin &bgr;1, a novel substrate for ADAM17. This function of ADAM17 is selective for pressure overload–induced myocardial hypertrophy and dysfunction, and not agonist-induced hypertrophy.

Effect of ischemia reperfusion injury and epoxyeicosatrienoic acids on caveolin expression in mouse myocardium.

Background: Caveolins (Cav) are structural proteins that insert into the plasma membrane to form caveolae that can bind molecules important in cardiac signal transduction and function. Cytochrome P450 epoxygenases can metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase reduces the protective effect. Aims: (1) To assess the effect of ischemia–reperfusion injury on expression and subcellular localization of caveolins. (2) To study the effect of EETs on caveolins. Methods: Hearts from soluble epoxide hydrolase null (KO) and littermate control (WT) mice were perfused in Langendorff mode and subjected to 20 minutes ischemia followed by 40 minutes reperfusion. Immunohistochemistry, immunoblot, and electron microscopy were performed to study localization of caveolins and changes in ultrastructure. Results: In WT heart, Cav-1 and Cav-3 were present in cardiomyocyte and capillary endothelial cell at baseline. After ischemia, Cav-1 but not Cav-3, disappeared from cardiomyocyte; moreover, caveolae were absent and mitochondrial cristae were damaged. Improved postischemic functional recovery observed in KO or WT hearts treated with 11,12-EET corresponded to higher Cav-1 expression and maintained caveolae structure. In addition, KO mice preserved the Cav-1 signaling after ischemia that lost in WT mice. Conclusions: Taken together, our data suggest that ischemia–reperfusion injury causes loss of Cav-1 and caveolins, and EETs-mediated cardioprotection involves preservation of Cav-1.

Cardiac hypertrophy in the newborn delays the maturation of fatty acid β-oxidation and compromises postischemic functional recovery

During the neonatal period, cardiac energy metabolism progresses from a fetal glycolytic profile towards one more dependent on mitochondrial oxidative metabolism. In this study, we identified the effects of cardiac hypertrophy on neonatal cardiac metabolic maturation and its impact on neonatal postischemic functional recovery. Seven-day-old rabbits were subjected to either a sham or a surgical procedure to induce a left-to-right shunt via an aortocaval fistula to cause RV volume-overload. At 3 wk of age, hearts were isolated from both groups and perfused as isolated, biventricular preparations to assess cardiac energy metabolism. Volume-overload resulted in cardiac hypertrophy (16% increase in cardiac mass, P < 0.05) without evidence of cardiac dysfunction in vivo or in vitro. Fatty acid oxidation rates were 60% lower (P < 0.05) in hypertrophied hearts than controls, whereas glycolysis increased 246% (P < 0.05). In contrast, glucose and lactate oxidation rates were unchanged. Overall ATP production rates were significantly lower in hypertrophied hearts, resulting in increased AMP-to-ATP ratios in both aerobic hearts and ischemia-reperfused hearts. The lowered energy generation of hypertrophied hearts depressed functional recovery from ischemia. Decreased fatty acid oxidation rates were accompanied by increased malonyl-CoA levels due to decreased malonyl-CoA decarboxylase activity/expression. Increased glycolysis in hypertrophied hearts was accompanied by a significant increase in hypoxia-inducible factor-1α expression, a key transcriptional regulator of glycolysis. Cardiac hypertrophy in the neonatal heart results in a reemergence of the fetal metabolic profile, which compromises ATP production in the rapidly maturing heart and impairs recovery of function following ischemia.