Diabetes triggers a PARP1 mediated death pathway in the heart through participation of FoxO1.

Cardiomyocyte cell death is a major contributing factor for diabetic cardiomyopathy, and multiple mechanisms have been proposed for its development. We hypothesized that following diabetes, an increased nuclear presence of the Forkhead transcription factor, FoxO1, could turn on cardiac cell death through mediation of nitrosative stress. Streptozotocin (100 mg/kg) was used to induce irreversible hyperglycemia in Wistar rats, and heart tissues and blood samples extracted starting from 1 to 4 days. Diazoxide (100 mg/kg), which produced acute reversible hyperglycemia, were followed for up to 12 h. In both animal models of hyperglycemia, attenuation of survival signals was accompanied by increased nuclear FoxO1. This was accompanied by a simultaneous increase in iNOS expression and iNOS induced protein nitrosylation of GAPDH, increased GAPDH binding to Siah1 and facilitated nuclear translocation of the complex. Even though caspase-3 was cleaved during diabetes, its nitrosylation modification affected its ability to inactivate PARP. As a result, there was PARP activation followed by nuclear compartmentalization of AIF, and increased phosphatidyl serine externalization. Our data suggests a role for FoxO1 mediated iNOS induced S-nitrosylation of target proteins like GAPDH and caspase-3 in initiating cardiac cell death following hyperglycemia, and could explain the impact of glycemic control in preventing cardiovascular disease in patients with diabetes.

Severity of diabetes governs vascular lipoprotein lipase by affecting enzyme dimerization and disassembly.

OBJECTIVE: In diabetes, when glucose consumption is restricted, the heart adapts to use fatty acid (FA) exclusively. The majority of FA provided to the heart comes from the breakdown of circulating triglyceride (TG), a process catalyzed by lipoprotein lipase (LPL) located at the vascular lumen. The objective of the current study was to determine the mechanisms behind LPL processing and breakdown after moderate and severe diabetes. RESEARCH DESIGN AND METHODS: To induce acute hyperglycemia, diazoxide, a selective, ATP-sensitive K(+) channel opener was used. For chronic diabetes, streptozotocin, a ß-cell-specific toxin was administered at doses of 55 or 100 mg/kg to generate moderate and severe diabetes, respectively. Cardiac LPL processing into active dimers and breakdown at the vascular lumen was investigated. RESULTS: After acute hyperglycemia and moderate diabetes, more LPL is processed into an active dimeric form, which involves the endoplasmic reticulum chaperone calnexin. Severe diabetes results in increased conversion of LPL into inactive monomers at the vascular lumen, a process mediated by FA-induced expression of angiopoietin-like protein 4 (Angptl-4). CONCLUSIONS: In acute hyperglycemia and moderate diabetes, exaggerated LPL processing to dimeric, catalytically active enzyme increases coronary LPL, delivering more FA to the heart when glucose utilization is compromised. In severe chronic diabetes, to avoid lipid oversupply, FA-induced expression of Angptl-4 leads to conversion of LPL to inactive monomers at the coronary lumen to impede TG hydrolysis. Results from this study advance our understanding of how diabetes changes coronary LPL, which could contribute to cardiovascular complications seen with this disease.

Cardiac triglyceride accumulation following acute lipid excess occurs through activation of a FoxO1-iNOS-CD36 pathway.

Obesity due to nutrient excess leads to chronic pathologies including type 2 diabetes and cardiovascular disease. Related to nutrient excess, FoxO1 has a role in regulating fatty acid uptake and oxidation and triglyceride (TG) storage by mechanisms that are largely unresolved. We examined the mechanism behind palmitate (PA)-induced TG accumulation in cardiomyocytes. To mimic lipid excess, rat ventricular myocytes were incubated with albumin-bound PA (1 mM) or rats were administered Intralipid (20%). PA-treated cardiomyocytes showed a substantial increase in TG accumulation, accompanied by amplification of nuclear migration of phospho-p38 and FoxO1, iNOS induction, and translocation of CD36 to the plasma membrane. PA also increased Cdc42 protein and its tyrosine nitration, thereby rearranging the cytoskeleton and facilitating CD36 translocation. These effects were duplicated by TNF-α and reversed by the iNOS inhibitor 1400 W. PA increased the nuclear interaction between FoxO1 and NF-κB, reduced the nuclear presence of PGC-1α, and downregulated expression of oxidative phosphorylation proteins. In vivo a robust increase in cardiac TGs after Intralipid administration was also associated with augmentation of nuclear FoxO1 and iNOS expression. Impeding this FoxO1-iNOS-CD36 pathway could decrease cardiac lipid accumulation and oxidative/nitrosative stress and help ameliorate the cardiovascular complications associated with obesity and diabetes.

The increase in cardiac pyruvate dehydrogenase kinase-4 after short-term dexamethasone is controlled by an Akt-p38-forkhead box other factor-1 signaling axis.

Glucocorticoids increase pyruvate dehydrogenase kinase-4 (PDK4) mRNA and protein expression, which phosphorylates pyruvate dehydrogenase, thereby preventing the formed pyruvate from undergoing mitochondrial oxidation. This increase in PDK4 expression is mediated by the mandatory presence of Forkhead box other factors (FoxOs) in the nucleus. In the current study, we examined the importance of the nongenomic effects of dexamethasone (Dx) in determining the compartmentalization of FoxO and hence its transcriptional activity. Rat cardiomyocytes exposed to Dx produced a robust decrease in glucose oxidation. Measurement of FoxO compartmentalization demonstrated increase in nuclear but resultant decrease in cytosolic content of FoxO1 with no change in the total content. The increase in nuclear content of FoxO1 correlated to an increase in nuclear phospho-p38 MAPK together with a robust association between this transcription factor and kinase. Dx also promoted nuclear retention of FoxO1 through a decrease in phosphorylation of Akt, an effect mediated by heat shock proteins binding to Akt. Measurement of the nuclear and total expression of sirtuin-1 protein showed no change after Dx. Instead, Dx increased the association of sirtuin-1 with FoxO1, thereby causing a decrease in FoxO acetylation. Manipulation of FoxO1 through agents that interfere with its nuclear shuttling or acetylation were effective in reducing Dx-induced increase in PDK4 protein expression. Our data suggest that FoxO1 has a major PDK4-regulating function. In addition, given the recent suggestions that altering glucose use can set the stage for heart failure, manipulating FoxO could assist in devising new therapeutic strategies to optimize cardiac metabolism and prevent PDK4 induced cardiac complications.

Glucose-induced endothelial heparanase secretion requires cortical and stress actin reorganization.

AIMS: Heparanase, which specifically cleaves carbohydrate chains of heparan sulfate, has been implicated in the pathology of diabetes-associated complications. Using high glucose (HG) to replicate hyperglycaemia observed following diabetes, the present study was designed to determine the mechanism by which HG initiates endothelial heparanase secretion. METHOD AND RESULTS: To examine the effect of HG on endothelial heparanase, bovine coronary artery endothelial cells were incubated with 25 mM glucose. Strategies using different agonists and antagonists were used to determine the mechanism behind HG-induced heparanase secretion. In endothelial cells, heparanase colocalized with lysosomes predominately around the nucleus, and HG caused its dispersion towards the plasma membrane for subsequent secretion. ATP release, purinergic receptor activation, cortical actin disassembly, and stress actin formation were essential for this HG-induced heparanase secretion. With HG, phosphorylation of filamin likely contributed to the cortical actin disassembly, whereas Ca(2+)/calmodulin-dependent protein kinase II and p38 mitogen-activated protein kinase /heat shock protein 25 phosphorylation mediated stress actin formation. The endothelial secreted heparanase in response to HG demonstrated endoglucuronidase activity, cleaved heparan sulfate, and released attached proteins like lipoprotein lipase and basic fibroblast growth factor. CONCLUSION: Our results suggest that HG is a potent stimulator of endothelial heparanase secretion. These data may assist in devising new therapeutic strategies to prevent or delay the cardiovascular complications associated with diabetes.

Cleavage of protein kinase D after acute hypoinsulinemia prevents excessive lipoprotein lipase-mediated cardiac triglyceride accumulation.

OBJECTIVE: During hypoinsulinemia, when cardiac glucose utilization is impaired, the heart rapidly adapts to using more fatty acids. One means by which this is achieved is through lipoprotein lipase (LPL). We determined the mechanisms by which the heart regulates LPL after acute hypoinsulinemia. RESEARCH DESIGN AND METHODS: We used two different doses of streptozocin (55 [D-55] and 100 [D-100] mg/kg) to induce moderate and severe hypoinsulinemia, respectively, in rats. Isolated cardiomyocytes were also used for transfection or silencing of protein kinase D (PKD) and caspase-3. RESULTS: There was substantial increase in LPL in D-55 hearts, an effect that was absent in severely hypoinsulinemic D-100 animals. Measurement of PKD, a key element involved in increasing LPL, revealed that only D-100 hearts showed an increase in proteolysis of PKD, an effect that required activation of caspase-3 together with loss of 14-3-3zeta, a binding protein that protects enzymes against degradation. In vitro, phosphomimetic PKD colocalized with LPL in the trans-golgi. PKD, when mutated to prevent its cleavage by caspase-3 and silencing of caspase-3, was able to increase LPL activity. Using a caspase inhibitor (Z-DEVD) in D-100 animals, we effectively lowered caspase-3 activity, prevented PKD cleavage, and increased LPL vesicle formation and translocation to the vascular lumen. This increase in cardiac luminal LPL was associated with a striking accumulation of cardiac triglyceride in Z-DEVD-treated D-100 rats. CONCLUSIONS After severe hypoinsulinemia, activation of caspase-3 can restrict LPL translocation to the vascular lumen. When caspase-3 is inhibited, this compensatory response is lost, leading to lipid accumulation in the heart.

AMP-activated protein kinase confers protection against TNF-{alpha}-induced cardiac cell death.

AIMS: Although a substantial role for 5' adenosine monophosphate-activated protein kinase (AMPK) has been established in regulating cardiac metabolism, a less studied action of AMPK is its ability to prevent cardiac cell death. Using established AMPK activators like dexamethasone (DEX) or metformin (MET), the objective of the present study was to determine whether AMPK activation prevents tumour necrosis factor-alpha (TNF-alpha) induced apoptosis in adult rat ventricular cardiomyocytes. METHODS AND RESULTS: Cardiomyocytes were incubated with DEX, MET, or TNF-alpha for varying durations (0-12 h). TNF-alpha-induced cell damage was evaluated by measuring caspase-3 activity and Hoechst staining. Protein and gene estimation techniques were employed to determine the mechanisms mediating the effects of AMPK activators on TNF-alpha-induced cardiomyocyte apoptosis. Incubation of myocytes with TNF-alpha for 8 h has increased caspase-3 activation and apoptotic cell death, an effect that was abrogated by DEX and MET. The beneficial effect of DEX and MET was associated with stimulation of AMPK, which led to a rapid and sustained increase in Bad phosphorylation. This event reduced the interaction between Bad and Bcl-xL, limiting cytochrome c release and caspase-3 activation. Addition of Compound C to inhibit AMPK reduced Bad phosphorylation and prevented the beneficial effects of AMPK against TNF-alpha-induced cytotoxicity. CONCLUSION: Our data demonstrate that although DEX and MET are used as anti-inflammatory agents or insulin sensitizers, respectively, their common property to phosphorylate AMPK promotes cardiomyocyte cell survival through its regulation of Bad and the mitochondrial apoptotic mechanism.

Endothelial heparanase secretion after acute hypoinsulinemia is regulated by glucose and fatty acid.

Following diabetes, the heart increases its lipoprotein lipase (LPL) at the coronary lumen by transferring LPL from the cardiomyocyte to the endothelial lumen. We examined how hyperglycemia controls secretion of heparanase, the enzyme that cleaves myocyte heparan sulphate proteoglycan to initiate this movement. Diazoxide (DZ) was used to decrease serum insulin and generate hyperglycemia. A modified Langendorff technique was used to separate coronary from interstitial effluent, which were assayed for heparanase and LPL. Within 30 min of DZ, interstitial heparanase increased, an effect that closely mirrored an augmentation in interstitial LPL. Endothelial cells were incubated with palmitic acid (PA) or glucose, and heparanase secretion was determined. PA increased intracellular heparanase, with no effect on secretion of this enzyme. Unlike PA, glucose dose-dependently lowered endothelial intracellular heparanase, which was strongly associated with increased heparanase activity in the incubation medium. Preincubation with cytochalasin D or nocodazole prevented the high glucose-induced depletion of intracellular heparanase. Our data suggest that following hyperglycemia, translocation of LPL from the cardiomyocyte cell surface to the apical side of endothelial cells is dependent on the ability of the fatty acid to increase endothelial intracellular heparanase followed by rapid secretion of this enzyme by glucose, which requires an intact microtubule and actin cytoskeleton.

Cardiac glycogen accumulation after dexamethasone is regulated by AMPK.

Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (Dex)-treated animals, glycogen accumulation was enhanced. We examined the influence of 5'-AMP-activated protein kinase (AMPK) on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. After Dex, cardiac tissue had a limited contribution toward the development of whole body insulin resistance. Measurement of glucose transporter 4 (GLUT4) at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated with increased GLUT4 mRNA. Both total and phosphorylated AMPK increased after Dex. Immunoprecipitation of Akt substrate of 160 kDa (AS160) followed by Western blot analysis demonstrated no change in Akt phosphorylation at Ser(473) and Thr(308) in Dex-treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr(172), which correlated well with AS160 phosphorylation. In Dex-treated hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas glycogen synthase kinase-3-beta phosphorylation was augmented. Our data suggest that AMPK-mediated glucose entry combined with the activation of glycogen synthase and a reduction in glucose oxidation (Qi et al., Diabetes 53: 1790-1797, 2004) act together to promote glycogen storage. Should these effects persist chronically in the heart, they may explain the increased morbidity and mortality observed with long-term excesses in endogenous or exogenous glucocorticoids.

Protein kinase D is a key regulator of cardiomyocyte lipoprotein lipase secretion after diabetes.

The diabetic heart switches to exclusively using fatty acid (FA) for energy supply and does so by multiple mechanisms including hydrolysis of lipoproteins by lipoprotein lipase (LPL) positioned at the vascular lumen. We determined the mechanism that leads to an increase in LPL after diabetes. Diazoxide (DZ), an agent that decreases insulin secretion and causes hyperglycemia, induced a substantial increase in LPL activity at the vascular lumen. This increase in LPL paralleled a robust phosphorylation of Hsp25, decreasing its association with PKCdelta, allowing this protein kinase to phosphorylate and activate protein kinase D (PKD), an important kinase that regulates fission of vesicles from the golgi membrane. Rottlerin, a PKCdelta inhibitor, prevented PKD phosphorylation and the subsequent increase in LPL. Incubating control myocytes with high glucose and palmitic acid (Glu+PA) also increased the phosphorylation of Hsp25, PKCdelta, and PKD in a pattern similar to that seen with diabetes, in addition to augmenting LPL activity. In myocytes in which PKD was silenced or a mutant form of PKCdelta was expressed, high Glu+PA were incapable of increasing LPL. Moreover, silencing of cardiomyocyte Hsp25 allowed phorbol 12-myristate 13-acetate to elicit a significant phosphorylation of PKCdelta, an appreciable association between PKCdelta and PKD, and a vigorous activation of PKD. As these cells also demonstrated an additional increase in LPL, our data imply that after diabetes, PKD control of LPL requires dissociation of Hsp25 from PKCdelta, association between PKCdelta and PKD, and vesicle fission. Results from this study could help in restricting cardiac LPL translocation, leading to strategies that overcome contractile dysfunction after diabetes.

Acute dexamethasone-induced increase in cardiac lipoprotein lipase requires activation of both Akt and stress kinases.

Following dexamethasone (DEX), cardiac energy generation is mainly through utilization of fatty acids (FA), with DEX animals demonstrating an increase in coronary lipoprotein lipase (LPL), an enzyme that hydrolyzes lipoproteins to FA. We examined the mechanisms by which DEX augments cardiac LPL. DEX was injected in rats, and hearts were removed, or isolated cardiomyocytes were incubated with DEX (0-8 h), for measurement of LPL activity and Western blotting. Acute DEX induced whole body insulin resistance, likely an outcome of a decrease in insulin signaling in skeletal muscle, but not cardiac tissue. The increase in luminal LPL activity after DEX was preceded by rapid nongenomic alterations, which included phosphorylation of AMPK and p38 MAPK, that led to phosphorylation of heat shock protein (HSP)25 and actin cytoskeleton rearrangement, facilitating LPL translocation to the myocyte cell surface. Unlike its effects in vivo, although DEX activated AMPK and p38 MAPK in cardiomyocytes, there was no phosphorylation of HSP25, nor was there any evidence of F-actin polymerization or an augmentation of LPL activity up to 8 h after DEX. Combining DEX with insulin appreciably enhanced cardiomyocyte LPL activity, which closely mirrored a robust elevation in phosphorylation of HSP25 and F-actin polymerization. Silencing of p38 MAPK, inhibition of PI 3-kinase, or preincubation with cytochalasin D prevented the increases in LPL activity. Our data suggest that, following DEX, it is a novel, rapid, nongenomic phosphorylation of stress kinases that, together with insulin, facilitates LPL translocation to the myocyte cell surface.

Acute diabetes moderates trafficking of cardiac lipoprotein lipase through p38 mitogen-activated protein kinase-dependent actin cytoskeleton organization.

OBJECTIVE: Heart disease is a leading cause of death in diabetes and could occur because of excessive use of fatty acid for energy generation. Our objective was to determine the mechanisms by which AMP-activated protein kinase (AMPK) augments cardiac lipoprotein lipase (LPL), the enzyme that provides the heart with the majority of its fatty acid. RESEARCH DESIGN AND METHODS: We used diazoxide in rats to induce hyperglycemia or used 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and thrombin to directly stimulate AMPK and p38 mitogen-activated protein kinase (MAPK), respectively, in cardiomyocytes. RESULTS: There was a substantial increase in LPL at the coronary lumen following 4 h of diazoxide. In these diabetic animals, phosphorylation of AMPK, p38 MAPK, and heat shock protein (Hsp)25 produced actin cytoskeleton rearrangement to facilitate LPL translocation to the myocyte surface and, eventually, the vascular lumen. AICAR activated AMPK, p38 MAPK, and Hsp25 in a pattern similar to that seen with diabetes. AICAR also appreciably enhanced LPL, an effect reduced by preincubation with the p38 MAPK inhibitor SB202190 or by cytochalasin D, which inhibits actin polymerization. Thrombin activated p38 MAPK in the absence of AMPK phosphorylation. Comparable with diabetes, activation of p38 MAPK and, subsequently, Hsp25 phosphorylation and F-actin polymerization corresponded with an enhanced LPL activity. SB202190 and silencing of p38 MAPK also prevented these effects induced by thrombin and AICAR, respectively. CONCLUSIONS: We propose that AMPK recruitment of LPL to the cardiomyocyte surface (which embraces p38 MAPK activation and actin cytoskeleton polymerization) represents an immediate compensatory response by the heart to guarantee fatty acid supply when glucose utilization is compromised.

AMPK control of myocardial fatty acid metabolism fluctuates with the intensity of insulin-deficient diabetes.

Flexibility in substrate selection is essential for the heart to maintain production of energy and contractile function, and is managed through multiple mechanisms including PPAR-alpha and AMP-activated protein kinase (AMPK). Rats injected with 55 mg/kg STZ (D55) were kept for 4 days (acute diabetes; D55-A) prior to termination. Fatty acid (FA) oxidation increased in D55-A hearts, with no significant change in gene expression of PPAR-alpha, or its downstream targets. However, both AMPK and ACC phosphorylation were significantly higher in these hearts, effects that were reversed by insulin. Unexpectedly, when the duration of diabetes in D55 rats was extended to 6 weeks (chronic diabetes; D55-C), AMPK and ACC phosphorylation were comparable in control and D55-C hearts. In D55-C rat hearts, lack of AMPK activation was closely associated to an overload of plasma and cardiac lipids. To validate the relationship between lipids and cardiac AMPK activation, we either induced more severe diabetes (100 mg/kg STZ to provoke both hyperglycemia and hyperlipidemia acutely; D100-A) or infused intralipid (IL) to enlarge circulating lipids. There was no difference in cardiac AMPK and ACC phosphorylation in D100-A rats compared to control. Measurement of AMPK and ACC phosphorylation in control and D55-A hearts revealed that their phosphorylation was inhibited by acute intralipid infusion. Our data suggest that activation of AMPK is an adaptation that would ensure adequate cardiac energy production when glucose utilization is compromised. However, in severe diabetes, with the addition of augmented plasma and heart lipids, AMPK activation is prevented, and control of FA oxidation is likely through alternate mechanisms. Given that AMPK plays an important role in preventing cardiac ischemic/reperfusion damage, it is possible that in these diabetic hearts, the accelerated damage observed during exposure to ischemia/reperfusion could be a likely outcome of a compromised activation of AMPK.

Induction of mitochondrial nitrative damage and cardiac dysfunction by chronic provision of dietary omega-6 polyunsaturated fatty acids.

Increased awareness of obesity has led to a dietary shift toward "heart-friendly" vegetable oils containing omega-6 polyunsaturated fatty acid (omega-6 PUFA). In addition to its beneficial effects, omega-6 PUFA also exhibits proinflammatory and prooxidative properties. We hypothesized that chronic dietary omega-6 PUFA can induce free radical generation, predisposing the cardiac mitochondria to oxidative damage. Male Wistar rats were fed a diet supplemented with 20% w/w sunflower oil, rich in omega-6 PUFA (HP) or normal laboratory chow (LP) for 4 weeks. HP feeding augmented phospholipase A(2) activity and breakdown of cardiolipin, a mitochondrial phospholipid. HP hearts also demonstrated elevated inducible nitric oxide synthase expression, loss of Mn superoxide dismutase, and increased mitochondrial nitrotyrosine levels. In these hearts, oxidative damage to mitochondrial DNA (mDNA) was demonstrated by 8-hydroxyguanosine immunopositivity, overexpression of DNA repair enzymes, and a decrease in the mRNA expression of specific respiratory subunits encoded by the mDNA. Functionally, at higher workloads, HP hearts also demonstrated a greater decline in cardiac work than LP, suggesting a compromised mitochondrial reserve. Our study, for the first time, demonstrates that consumption of a high fat diet rich in omega-6 PUFA for only 4 weeks instigates mitochondrial nitrosative damage and causes cardiac dysfunction at high afterloads.

Acute intralipid infusion reduces cardiac luminal lipoprotein lipase but recruits additional enzyme from cardiomyocytes.

OBJECTIVE: Lipoprotein lipase (LPL) metabolizes the triglyceride (TG) core of lipoproteins. We evaluated whether circulating lipids can regulate LPL by influencing the transfer of enzyme from the myocyte to the endothelial lumen. METHODS: Acute intralipid (IL, 10% and 20%) infusion was performed in male Wistar rats. After 3 h, insulin resistance was assessed using a euglycemic hyperinsulinemic clamp. Cardiac LPL activity was determined by retrogradely perfusing the hearts with heparin. Immunogold electron microscopy visualized LPL, and heparanase was detected by immunofluorescence. Cardiac myocytes were also isolated, and heparin-releasable LPL activity was measured. RESULTS: IL infusion increased both plasma and cardiac lipids. Circulating basal plasma LPL activity increased for the duration of the infusion. Compared to control (CON) hearts, there was a substantial decrease in heparin-releasable LPL activity at the vascular lumen following 3 h of IL infusion, an effect unrelated to changes in gene and protein expression or whole-body insulin resistance. Although constant perfusion of CON hearts with heparin stripped off most of the luminal bound LPL, hearts from IL-infused animals continued to release excessive amounts of the enzyme, suggesting buildup of LPL within endothelial cells or at the endothelial basolateral surface. Immunogold labeling confirmed this observation and demonstrated robust anti-LPL staining at these sites, only in IL hearts. Perfusing hearts from IL-rats in vitro, in the absence of TG, allowed the accumulated enzyme pool to transfer to the coronary lumen. CONCLUSION: Our data suggest that acute amplification of lipids reduces cardiac luminal LPL but facilitates additional recruitment of cardiomyocyte enzyme. Should this mechanism occur globally, it could contribute towards management of hyperlipidemia.

Altered cardiac fatty acid composition and utilization following dexamethasone-induced insulin resistance.

Glucocorticoid therapy is often associated with impaired insulin sensitivity and cardiovascular disease. The present study was designed to evaluate cardiac fatty acid (FA) composition and metabolism following acute dexamethasone (Dex) treatment. Using the euglycemic hyperinsulinemic clamp, rats injected with Dex demonstrated a reduced glucose infusion rate. This whole body insulin resistance was also associated with a heart-specific increase in pyruvate dehydrogenase kinase 4 gene expression and a reduction in the rate of glucose oxidation. Dex treatment increased basal and postheparin plasma lipolytic activity. In the heart, palmitic and oleic acid levels were higher after 4 h of Dex and decreased to control (CON) levels within 8 h. Measurement of polyunsaturated FAs demonstrated a drop in linoleic and gamma-linolenic acid, with an increase in arachidonic acid (AA) after acute Dex injection. Tissue FA can be either oxidized or stored as triglyceride (TG). At 4 h, Dex augmented cardiac TG accumulation. However, this increase in tissue TG could not be maintained, such that at 8 h following Dex, TG declined to CON levels. AMP-activated protein kinase (AMPK) activation is known to promote FA oxidation through its control of acetyl-CoA carboxylase (ACC). Acute Dex promoted ACC phosphorylation, and increased cardiac palmitate oxidation, likely through its effects in increasing AMPK phosphorylation and total AMPK protein and gene expression. Whether these acute effects of Dex on FA oxidation, TG storage, and arachidonic acid accumulation can be translated into increased cardiovascular risk following chronic therapy has yet to be determined.

Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion.

Oxidative stress due to excessive reactive oxygen species (ROS) and depleted antioxidants such as glutathione (GSH) can give rise to apoptotic cell death in acutely diabetic hearts and lead to heart disease. At present, the source of these cardiac ROS or the subcellular site of cardiac GSH loss [i.e., cytosolic (cGSH) or mitochondrial (mGSH) GSH] has not been completely elucidated. With the use of rotenone (an inhibitor of the electron transport chain) to decrease the excessive ROS in acute streptozotocin (STZ)-induced diabetic rat heart, the mitochondrial origin of ROS was established. Furthermore, mitochondrial damage, as evidenced by loss of membrane potential, increases in oxidative stress, and reduction in mGSH was associated with increased apoptosis via increases in caspase-9 and -3 activities in acutely diabetic hearts. To validate the role of mGSH in regulating cardiac apoptosis, L-buthionine-sulfoximine (BSO; 10 mmol/kg ip), which blocks GSH synthesis, or diethyl maleate (DEM; 4 mmol/kg ip), which inactivates preformed GSH, was administered in diabetic rats for 4 days after STZ administration. Although both BSO and DEM lowered cGSH, they were ineffective in reducing mGSH or augmenting cardiomyocyte apoptosis. To circumvent the lack of mGSH depletion, BSO and DEM were coadministered in diabetic rats. In this setting, mGSH was undetectable and cardiac apoptosis was further aggravated compared with the untreated diabetic group. In a separate group, GSH supplementation induced a robust amplification of mGSH in diabetic rat hearts and prevented apoptosis. Our data suggest for the first time that mGSH is crucial for modulating the cell suicide program in short-term diabetic rat hearts.

Lysophosphatidic acid-mediated augmentation of cardiomyocyte lipoprotein lipase involves actin cytoskeleton reorganization.

The lipoprotein lipase (LPL)-augmenting property of lysophosphatidylcholine requires the formation of lysophosphatidic acid (LPA) (J Mol Cell Cardiol 37: 931-938, 2004). Given that the actin cytoskeleton has been implicated in regulating cardiomyocyte LPL, we examined whether LPL secretion after LPA involves actin cytoskeleton reassembly. Incubation of myocytes with LPA (1-100 nM) increased basal and heparin-releasable LPL (HR-LPL), an effect that was independent of shifts in LPL mRNA. The influence of LPA on myocyte LPL was reflected at the coronary lumen, with substantial increases of the enzyme at this location. Incubation of myocytes with cytochalasin D not only blocked LPA-induced augmentation of HR-LPL but also abrogated filamentous actin formation. These effects of LPA were likely receptor mediated. Exposure of myocytes to LPA facilitated significant membrane translocation of RhoA and its downstream effector Rho kinase I (ROCK I), and blocking this effect with Y-27632 appreciably reduced basal and HR-LPL activity. Incubation of adipose tissue with LPA also significantly enhanced basal and HR-LPL activity, suggesting that sarcomeric actin likely has a limited role in influencing the LPL secretory function of LPA in the myocyte. Comparable to LPA, hyperglycemia also caused significant membrane translocation of RhoA and ROCK I in hearts isolated from diazoxide-treated animals, effects that were abrogated using insulin. Overall, our data suggest that comparable to hyperglycemia, LPA-induced increases in cardiac LPL occurred via posttranscriptional mechanisms and processes that likely required RhoA activation and actin polymerization. Whether this increase in LPL augments triglyceride deposition in the heart leading to eventual impairment in contractile function is currently unknown.

beta-Agonist stimulation produces changes in cardiac AMPK and coronary lumen LPL only during increased workload.

Given the importance of lipoprotein lipase (LPL) in cardiac and vascular pathology, the objective of the present study was to investigate whether the beta-agonist isoproterenol (Iso) influences cardiac LPL. Incubation of quiescent cardiomyocytes with Iso for 60 min had no effect on basal, intracellular, or heparin-releasable (HR)-LPL activity. Similarly, Iso did not change HR-LPL in Langendorff isolated hearts that do not beat against an afterload. In the intact animal, LPL activity at the vascular lumen increased significantly in the Iso-treated group, together with a substantial increase in rate-pressure product. This LPL increase was likely via mechanisms regulated by activation of AMP-activated protein kinase (AMPK) and inactivation of acetyl-CoA carboxylase (ACC280). In glucose-perfused hearts, simply switching from Langendorff to the isolated working heart (that beats against an afterload) induced increases in AMPK and ACC280 phosphorylation and enhanced HR-LPL activity. Provision of insulin and albumin-bound palmitic acid to the working heart was able to reverse these effects. In these hearts, introduction of Iso to the buffer perfusate duplicated the effects seen when this beta-agonist was given in vivo. Our data suggest that Iso can influence HR-LPL only during conditions of increased workload, mechanical performance and excessive energy expenditure, and likely in an AMPK-dependent manner.

The metabolic "switch" AMPK regulates cardiac heparin-releasable lipoprotein lipase.

The "fuel gauge" AMP-activated protein kinase (AMPK) facilitates ATP production to meet energy demands during metabolic stress. Given the importance of lipoprotein lipase (LPL) in providing hearts with fatty acids (FA), the preferred substrate consumed by the heart, the objective of the present study was to investigate whether activation of AMPK influences LPL at its functionally relevant location, the coronary lumen. Hearts from overnight-fasted rats were first perfused with heparin to release LPL, and homogenates from these hearts were then used to measure total and phospho-AMPK-alpha by Western blotting. Manipulation of AMPK activity [with drugs like adenine 9-beta-D-arabinofuranoside (Ara-A) and insulin (that inhibit) or perhexiline and oligomycin (that stimulate)] and its influence on LPL was also determined. Fasting augmented the activity of both AMPK and luminal LPL on immediate removal of hearts, effects that still remained even after in vitro perfusion of hearts for 1 h. Inhibition of AMPK in fasted hearts using an inhibitor like Ara-A or through provision of insulin markedly lowered the enhanced luminal LPL activity. In contrast, AMPK activators, like perhexiline and oligomycin, produced a significant elevation in heparin-releasable LPL activity. Thus, with fasting or drugs that influence AMPK, a strong correlation between this metabolic switch and cardiac LPL activity was established. Our data suggest that, in addition to its direct role in promoting FA oxidation, AMPK-mediated recruitment of LPL to the coronary lumen could represent an immediate compensatory response by the heart to guarantee FA supply.