Kalyani G. Bharadwaj

DGAT1 Expression Increases Heart Triglyceride Content but Ameliorates Lipotoxicity

Intracellular lipid accumulation in the heart is associated with cardiomyopathy, yet the precise role of triglyceride (TG) remains unclear. With exercise, wild type hearts develop physiologic hypertrophy. This was associated with greater TG stores and a marked induction of the TG-synthesizing enzyme diacylglycerol (DAG) acyltransferase 1 (DGAT1). Transgenic overexpression of DGAT1 in the heart using the cardiomyocyte- specific α-myosin heavy chain (MHC) promoter led to approximately a doubling of DGAT activity and TG content and reductions of ∼35% in cardiac ceramide, 26% in DAG, and 20% in free fatty acid levels. Cardiac function assessed by echocardiography and cardiac catheterization was unaffected. These mice were then crossed with animals expressing long-chain acyl-CoA synthetase via the MHC promoter (MHC-ACS), which develop lipotoxic cardiomyopathy. MHC-DGAT1XMHC-ACS double transgenic male mice had improved heart function; fractional shortening increased by 74%, and diastolic function improved compared with MHC-ACS mice. The improvement of heart function correlated with a reduction in cardiac DAG and ceramide and reduced cardiomyocyte apoptosis but increased fatty acid oxidation. In addition, the survival of the mice was improved. Our study indicates that TG is not likely to be a toxic lipid species directly, but rather it is a feature of physiologic hypertrophy and may serve a cytoprotective role in lipid overload states. Moreover, induction of DGAT1 could be beneficial in the setting of excess heart accumulation of toxic lipids.

Rescue of Cardiomyopathy in Peroxisome Proliferator-Activated Receptor-α Transgenic Mice by Deletion of Lipoprotein Lipase Identifies Sources of Cardiac Lipids and Peroxisome Proliferator-Activated Receptor-α Activators

Background— Emerging evidence in obesity and diabetes mellitus demonstrates that excessive myocardial fatty acid uptake and oxidation contribute to cardiac dysfunction. Transgenic mice with cardiac-specific overexpression of the fatty acid–activated nuclear receptor peroxisome proliferator-activated receptor-α (myosin heavy chain [MHC]-PPARα mice) exhibit phenotypic features of the diabetic heart, which are rescued by deletion of CD36, a fatty acid transporter, despite persistent activation of PPARα gene targets involved in fatty acid oxidation. Methods and Results— To further define the source of fatty acid that leads to cardiomyopathy associated with lipid excess, we crossed MHC-PPARα mice with mice deficient for cardiac lipoprotein lipase (hsLpLko). MHC-PPARα/hsLpLko mice exhibit improved cardiac function and reduced myocardial triglyceride content compared with MHC-PPARα mice. Surprisingly, in contrast to MHC-PPARα/CD36ko mice, the activity of the cardiac PPARα gene regulatory pathway is normalized in MHC-PPARα/hsLpLko mice, suggesting that PPARα ligand activity exists in the lipoprotein particle. Indeed, LpL mediated hydrolysis of very-low-density lipoprotein activated PPARα in cardiac myocytes in culture. The rescue of cardiac function in both models was associated with improved mitochondrial ultrastructure and reactivation of transcriptional regulators of mitochondrial function. Conclusions— MHC-PPARα mouse hearts acquire excess lipoprotein-derived lipids. LpL deficiency rescues myocyte triglyceride accumulation, mitochondrial gene regulatory derangements, and contractile function in MHC-PPARα mice. Finally, LpL serves as a source of activating ligand for PPARα in the cardiomyocyte.

Apolipoprotein CIII overexpressing mice are predisposed to diet‐induced hepatic steatosis and hepatic insulin resistance

Nonalcoholic fatty liver disease (NAFLD) and insulin resistance have recently been found to be associated with increased plasma concentrations of apolipoprotein CIII (APOC3) in humans carrying single nucleotide polymorphisms within the insulin response element of the APOC3 gene. To examine whether increased expression of APOC3 would predispose mice to NAFLD and hepatic insulin resistance, human APOC3 overexpressing (ApoC3Tg) mice were metabolically phenotyped following either a regular chow or high‐fat diet (HFD). After HFD feeding, ApoC3Tg mice had increased hepatic triglyceride accumulation, which was associated with cellular ballooning and inflammatory changes. ApoC3Tg mice also manifested severe hepatic insulin resistance assessed by a hyperinsulinemic‐euglycemic clamp, which could mostly be attributed to increased hepatic diacylglycerol content, protein kinase C‐ϵ activation, and decreased insulin‐stimulated Akt2 activity. Increased hepatic triglyceride content in the HFD‐fed ApoC3Tg mice could be attributed to a ≈70% increase in hepatic triglyceride uptake and ≈50% reduction hepatic triglyceride secretion. Conclusion: These data demonstrate that increase plasma APOC3 concentrations predispose mice to diet‐induced NAFLD and hepatic insulin resistance. (HEPATOLOGY 2011;)

Chylomicron- and VLDL-derived Lipids Enter the Heart through Different Pathways IN VIVO EVIDENCE FOR RECEPTOR- AND NON-RECEPTOR-MEDIATED FATTY ACID UPTAKE

Lipids circulate in the blood in association with plasma lipoproteins and enter the tissues either after hydrolysis or as non-hydrolyzable lipid esters. We studied cardiac lipids, lipoprotein lipid uptake, and gene expression in heart-specific lipoprotein lipase (LpL) knock-out (hLpL0), CD36 knock-out (Cd36−/−), and double knock-out (hLpL0/Cd36−/−-DKO) mice. Loss of either LpL or CD36 led to a significant reduction in heart total fatty acyl-CoA (control, 99.5 ± 3.8; hLpL0, 36.2 ± 3.5; Cd36−/−, 57.7 ± 5.5 nmol/g, p < 0.05) and an additive effect was observed in the DKO (20.2 ± 1.4 nmol/g, p < 0.05). Myocardial VLDL-triglyceride (TG) uptake was reduced in the hLpL0 (31 ± 6%) and Cd36−/− (47 ± 4%) mice with an additive reduction in the DKO (64 ± 5%) compared with control. However, LpL but not CD36 deficiency decreased VLDL-cholesteryl ester uptake. Endogenously labeled mouse chylomicrons were produced by tamoxifen treatment of β-actin-MerCreMer/LpLflox/flox mice. Induced loss of LpL increased TG levels >10-fold and reduced HDL by >50%. After injection of these labeled chylomicrons in the different mice, chylomicron TG uptake was reduced by ∼70% and retinyl ester by ∼50% in hLpL0 hearts. Loss of CD36 did not alter either chylomicron TG or retinyl ester uptake. LpL loss did not affect uptake of remnant lipoproteins from ApoE knock-out mice. Our data are consistent with two pathways for fatty acid uptake; a CD36 process for VLDL-derived fatty acid and a non-CD36 process for chylomicron-derived fatty acid uptake. In addition, our data show that lipolysis is involved in uptake of core lipids from TG-rich lipoproteins.

DGAT1 deficiency decreases PPAR expression and does not lead to lipotoxicity in cardiac and skeletal muscle

Diacylglycerol (DAG) acyl transferase 1 (Dgat1) knockout (−/−) mice are resistant to high-fat-induced obesity and insulin resistance, but the reasons are unclear. Dgat1−/− mice had reduced mRNA levels of all three Ppar genes and genes involved in fatty acid oxidation in the myocardium of Dgat1−/− mice. Although DGAT1 converts DAG to triglyceride (TG), tissue levels of DAG were not increased in Dgat1−/− mice. Hearts of chow-diet Dgat1−/− mice were larger than those of wild-type (WT) mice, but cardiac function was normal. Skeletal muscles from Dgat1−/− mice were also larger. Muscle hypertrophy factors phospho-AKT and phospho-mTOR were increased in Dgat1−/− cardiac and skeletal muscle. In contrast to muscle, liver from Dgat1−/− mice had no reduction in mRNA levels of genes mediating fatty acid oxidation. Glucose uptake was increased in cardiac and skeletal muscle in Dgat1−/− mice. Treatment with an inhibitor specific for DGAT1 led to similarly striking reductions in mRNA levels of genes mediating fatty acid oxidation in cardiac and skeletal muscle. These changes were reproduced in cultured myocytes with the DGAT1 inhibitor, which also blocked the increase in mRNA levels of Ppar genes and their targets induced by palmitic acid. Thus, loss of DGAT1 activity in muscles decreases mRNA levels of genes involved in lipid uptake and oxidation.

Cardiomyocyte lipids impair β-adrenergic receptor function via PKC activation.

Normal hearts have increased contractility in response to catecholamines. Because several lipids activate PKCs, we hypothesized that excess cellular lipids would inhibit cardiomyocyte responsiveness to adrenergic stimuli. Cardiomyocytes treated with saturated free fatty acids, ceramide, and diacylglycerol had reduced cellular cAMP response to isoproterenol. This was associated with increased PKC activation and reduction of β-adrenergic receptor (β-AR) density. Pharmacological and genetic PKC inhibition prevented both palmitate-induced β-AR insensitivity and the accompanying reduction in cell surface β-ARs. Mice with excess lipid uptake due to either cardiac-specific overexpression of anchored lipoprotein lipase, PPARγ, or acyl-CoA synthetase-1 or high-fat diet showed reduced inotropic responsiveness to dobutamine. This was associated with activation of protein kinase C (PKC)α or PKCδ. Thus, several lipids that are increased in the setting of lipotoxicity can produce abnormalities in β-AR responsiveness. This can be attributed to PKC activation and reduced β-AR levels.

Cardiomyocyte Specific Deficiency of Serine Palmitoyltransferase Subunit 2 Reduces Ceramide but Leads to Cardiac Dysfunction

Background: The importance of de novo ceramide biosynthesis in maintaining cardiac function is unknown. Results: Deletion of serine palmitoyltransferase subunit Sptlc2 reduced cardiac ceramide and caused cardiac dysfunction associated with activation of ER stress. Conclusion: Reduced ceramide content by Sptlc2 deficiency does not protect against lipid toxicity associated with increased saturated acyl CoAs. Significance: Development of disease by lipotoxicity is caused by a number of changes in lipidome. The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.

Adipose-specific Lipoprotein Lipase Deficiency More Profoundly Affects Brown than White Fat Biology

Background: Lipoprotein lipase (LpL) is rate-limiting for plasma triglyceride lipolysis, but its importance in adipose development is uncertain. Results: Adipocyte LpL knock-out affected brown but not white fat composition. White fat was reduced when muscle LpL expression was increased. Conclusion: LpL distribution and adipose metabolism affect adipogenesis. Significance: All fat depots are not equally dependent on triglyceride uptake. Adipose fat storage is thought to require uptake of circulating triglyceride (TG)-derived fatty acids via lipoprotein lipase (LpL). To determine how LpL affects the biology of adipose tissue, we created adipose-specific LpL knock-out (ATLO) mice, and we compared them with whole body LpL knock-out mice rescued with muscle LpL expression (MCK/L0) and wild type (WT) mice. ATLO LpL mRNA and activity were reduced, respectively, 75 and 70% in gonadal adipose tissue (GAT), 90 and 80% in subcutaneous tissue, and 84 and 85% in brown adipose tissue (BAT). ATLO mice had increased plasma TG levels associated with reduced chylomicron TG uptake into BAT and lung. ATLO BAT, but not GAT, had altered TG composition. GAT from MCK/L0 was smaller and contained less polyunsaturated fatty acids in TG, although GAT from ATLO was normal unless LpL was overexpressed in muscle. High fat diet feeding led to less adipose in MCK/L0 mice but TG acyl composition in subcutaneous tissue and BAT reverted to that of WT. Therefore, adipocyte LpL in BAT modulates plasma lipoprotein clearance, and the greater metabolic activity of this depot makes its lipid composition more dependent on LpL-mediated uptake. Loss of adipose LpL reduces fat accumulation only if accompanied by greater LpL activity in muscle. These data support the role of LpL as the “gatekeeper” for tissue lipid distribution.

Cardiac metabolic compensation to hypertension requires lipoprotein lipase

Fatty acids (FAs) are acquired from free FA associated with albumin and lipoprotein triglyceride that is hydrolyzed by lipoprotein lipase (LpL). Hypertrophied hearts shift their substrate usage pattern to more glucose and less FA. However, FAs may still be an important source of energy in hypertrophied hearts. The aim of this study was to examine the importance of LpL-derived FAs in hypertensive hypertrophied hearts. We followed cardiac function and metabolic changes during 2 wk of angiotensin II (ANG II)-induced hypertension in control and heart-specific lipoprotein lipase knockout (hLpL0) mice. Glucose metabolism was increased in ANG II-treated control (control/ANG II) hearts, raising it to the same level as hLpL0 hearts. FA uptake-related genes, CD36 and FATP1, were reduced in control/ANG II hearts to levels found in hLpL0 hearts. ANG II did not alter these metabolic genes in hLpL0 mice. LpL activity was preserved, and mitochondrial FA oxidation-related genes were not altered in control/ANG II hearts. In control/ANG II hearts, triglyceride stores were consumed and reached the same levels as in hLpL0/ANG II hearts. Intracellular ATP content was reduced only in hLpL0/ANG II hearts. Both ANG II and deoxycorticosterone acetate-salt induced hypertension caused heart failure only in hLpL0 mice. Our data suggest that LpL activity is required for normal cardiac metabolic compensation to hypertensive stress.

Chronic ethanol consumption increases cardiomyocyte fatty acid uptake and decreases ventricular contractile function in C57BL/6J mice

Alcohol, a major cause of human cardiomyopathy, decreases cardiac contractility in both animals and man. However, key features of alcohol-related human heart disease are not consistently reproduced in animal models. Accordingly, we studied cardiac histology, contractile function, cardiomyocyte long chain fatty acid (LCFA) uptake, and gene expression in male C57BL/6J mice consuming 0, 10, 14, or 18% ethanol in drinking water for 3months. At sacrifice, all EtOH groups had mildly decreased body and increased heart weights, dose-dependent increases in cardiac triglycerides and a marked increase in cardiac fatty acid ethyl esters. [(3)H]-oleic acid uptake kinetics demonstrated increased facilitated cardiomyocyte LCFA uptake, associated with increased expression of genes encoding the LCFA transporters CD36 and Slc27a1 (FATP1) in EtOH-fed animals. Although SCD-1 expression was increased, lipidomic analysis did not indicate significantly increased de novo LCFA synthesis. By echocardiography, ejection fraction (EF) and the related fractional shortening (FS) of left ventricular diameter during systole were reduced and negatively correlated with cardiac triglycerides. Expression of myocardial PGC-1α and multiple downstream target genes in the oxidative phosphorylation pathway, including several in the electron transport and ATP synthase complexes of the inner mitochondrial membrane, were down-regulated. Cardiac ATP was correspondingly reduced. The data suggest that decreased expression of PGC-1α and its target genes result in decreased cardiac ATP levels, which may explain the decrease in myocardial contractile function caused by chronic EtOH intake. This model recapitulates important features of human alcoholic cardiomyopathy and illustrates a potentially important pathophysiologic link between cardiac lipid metabolism and function.