In obesity-related hypertension, activation of the renin-angiotensin system (RAS) has been reported despite marked fluid volume expansion. Adipose tissue expresses components of the RAS and is markedly expanded in obesity. This study evaluated changes in components of the adipose and systemic RAS in diet-induced obese hypertensive rats. RAS was quantified in adipose tissue and compared with primary sources for the circulating RAS. Male Sprague-Dawley rats were fed either a low-fat (LF; 11% kcal as fat) or moderately high-fat (32% kcal as fat) diet for 11 wk. After 8 wk, rats fed the moderately high-fat diet segregated into obesity-prone (OP) and obesity-resistant (OR) groups based on their body weight gain (body weight: OR, 566 +/- 10; OP, 702 +/- 20 g; P < 0.05). Mean arterial blood pressure was increased in OP rats (LF: 97 +/- 2; OR: 97 +/- 2; OP: 105 +/- 1 mmHg; P < 0.05). Quantification of mRNA expression by real-time PCR demonstrated a selective increase (2-fold) in angiotensinogen gene expression in retroperitoneal adipose tissue from OP vs. OR and LF rats. Similarly, plasma angiotensinogen concentration was increased in OP rats (LF: 390 +/- 48; OR: 355 +/- 24; OP: 530 +/- 22 ng/ml; P < 0.05). In contrast, other components of the RAS were not altered in OP rats. Marked increases in the plasma concentrations of angiotensin peptides were observed in OP rats (angiotensin II: LF: 95 +/- 31; OR: 59 +/- 20; OP: 295 +/- 118 pg/ml; P < 0.05). These results demonstrate increased activity of the adipose and systemic RAS in obesity-related hypertension.

Adipose tissue expresses components of the renin-angiotensin system (RAS). Angiotensin converting enzyme (ACE2), a new component of the RAS, catabolizes the vasoconstrictor peptide ANG II to form the vasodilator angiotensin 1-7 [ANG-(1-7)]. We examined whether adipocytes express ACE2 and its regulation by manipulation of the RAS and by high-fat (HF) feeding. ACE2 mRNA expression increased (threefold) during differentiation of 3T3-L1 adipocytes and was not regulated by manipulation of the RAS. Male C57BL/6 mice were fed low-(LF) or high-fat (HF) diets for 1 wk or 4 mo. At 1 wk of HF feeding, adipose expression of angiotensinogen (twofold) and ACE2 (threefold) increased, but systemic angiotensin peptide concentrations and blood pressure were not altered. At 4 mo of HF feeding, adipose mRNA expression of angiotensinogen (twofold) and ACE2 (threefold) continued to be elevated, and liver angiotensinogen expression increased (twofold). However, adipose tissue from HF mice did not exhibit elevated ACE2 protein or activity. Increased expression of ADAM17, a protease responsible for ACE2 shedding, coincided with reductions in ACE2 activity in 3T3-L1 adipocytes, and an ADAM17 inhibitor decreased media ACE2 activity. Moreover, ADAM17 mRNA expression was increased in adipose tissue from 4-mo HF-fed mice, and plasma ACE2 activity increased. However, HF mice exhibited marked increases in plasma angiotensin peptide concentrations (LF: 2,141 Ϯ 253; HF: 6,829 Ϯ 1,075 pg/ml) and elevated blood pressure. These results demonstrate that adipocytes express ACE2 that is dysregulated in HF-fed mice with elevated blood pressure compared with LF controls.

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 cardiomyocytespecific ␣-myosin heavy chain (MHC) promoter led to approximately a doubling of DGAT activity and TG content and reductions of ϳ35% 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.Triglyceride (TG) 2 is the major energy storage form in organs. The final step of TG synthesis, the conversion of diacylglycerol (DAG) to TG, is catalyzed by diacylglycerol acyltransferase (DGAT) enzymes. DGAT1 and DGAT2 are unrelated proteins that exhibit DGAT activity (1). DGAT1 belongs to a gene family that includes ACAT1 and ACAT2 (acyl-CoA:cholesterol acyltransferases 1 and 2) (1-3), whereas DGAT2 is a member of a larger gene family whose members include acylCoA:monoacylglycerol acyltransferase (3). Although both enzymes catalyze the same reaction in TG synthesis, they are functionally distinguished by their differences in regulation and substrate specificity (2, 4 -7). For example, DGAT1, but not DGAT2, will esterify other lipids such as retinol (2,8). DGAT1 is widely expressed in all tissues, with high expression in white adipose tissue, skeletal muscle, heart, and intestine (1); DGAT2 is primarily expressed in the liver (1, 2).Studies to understand the roles of DGAT1 and -2 have been performed using genetically modified mice. Investigators have studied whether DGATs regulate insulin actions and toxic effects of lipids on tissue. DGAT1 knock-out mice have reduced obesity on a high fat diet (6). Moreover, when these mice were crossed onto the agouti background they had increased insulin sensitivity (9). Transplantation of DGAT1-deficient adipose tissue into wild type (WT) mice decreased adiposity and increased insulin sensitivity (10). These experiments suggest that DGAT1 inhi...

Free fatty acids (FFAs) suppress appetite when injected into the hypothalamus. To examine whether lipoprotein lipase (LPL), a serine hydrolase that releases FFAs from circulating triglyceride (TG)-rich lipoproteins, might contribute to FFA-mediated signaling in the brain, we created neuron-specific LPL-deficient mice. Homozygous mutant (NEXLPL-/-) mice were hyperphagic and became obese by 16 weeks of age. These traits were accompanied by elevations in the hypothalamic orexigenic neuropeptides, AgRP and NPY, and were followed by reductions in metabolic rate. The uptake of TG-rich lipoprotein fatty acids was reduced in the hypothalamus of 3-month-old NEXLPL-/- mice. Moreover, deficiencies in essential fatty acids in the hypothalamus were evident by 3 months, with major deficiencies of long-chain n-3 fatty acids by 12 months. These results indicate that TG-rich lipoproteins are sensed in the brain by an LPL-dependent mechanism and provide lipid signals for the central regulation of body weight and energy balance.

Non-alcoholic 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, PKCε 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. In conclusion these data demonstrate that increase plasma APOC3 concentrations predispose mice to diet-induced NAFLD and hepatic insulin resistance.

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-␣ (myosin heavy chain [MHC]-PPAR␣ mice) exhibit phenotypic features of the diabetic heart, which are rescued by deletion of CD36, a fatty acid transporter, despite persistent activation of PPAR␣ 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-PPAR␣ mice with mice deficient for cardiac lipoprotein lipase (hsLpLko). MHC-PPAR␣/hsLpLko mice exhibit improved cardiac function and reduced myocardial triglyceride content compared with MHC-PPAR␣ mice. Surprisingly, in contrast to MHC-PPAR␣/CD36ko mice, the activity of the cardiac PPAR␣ gene regulatory pathway is normalized in MHC-PPAR␣/hsLpLko mice, suggesting that PPAR␣ ligand activity exists in the lipoprotein particle. Indeed, LpL mediated hydrolysis of very-low-density lipoprotein activated PPAR␣ 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. Key Words: cardiomyopathy Ⅲ lipids Ⅲ diabetes mellitus T ype 2 diabetes mellitus and its associated cardiovascular complications are a worldwide health threat. 1,2 Although patients with obesity-related diabetes mellitus have an increased incidence of heart failure after myocardial infarction, 3 they are also prone to develop heart failure in the absence of significant coronary artery disease. 4 These observations suggest that myocardial dysfunction in diabetes mellitus, metabolic syndrome, and obesity has distinct pathogenic features. Conclusions-MHC-PPAR␣ Clinical Perspective on p 435Multiple mechanisms have been proposed to drive diabetes mellitus-associated heart dysfunction, including glucose toxicity (advanced glycation end products), 5 microvascular disease, 6 mitochondrial dysfunction, 7,8 and lipid toxicity. 9,10 Evidence supports a role for lipid metabolic derangements in the development of cardiomyocyte dysfunction in the insulinresistant and diabetic heart 9,10 ; both lipid accumulation and excessive fatty acid (FA) oxidation (FAO) are postulated to cause cardiomyocyte toxicity. Human studies and animal models demonstrate that diabetes mellitus and obesity are associated with accumulation of myocyte fat. 11,12 Additionally, the insulin-resistant heart is unable to fully use glucose, forcing the organ to rely on FAs, leading to a vicious cycle of increased myocyte FA import, oxidation, and triglyceride accumulation, 10 signatures of a metabolic cardiomyopathy called lipotoxic cardiomyopathy. Reprogramming of the insulin-resistant heart toward FA use involves gene regulatory mechanisms. Peroxisome proliferator-activated...

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.

This article is available online at http://www.jlr.org Supplementary key words heart • triglyceride • diacylglycerol acyl transferase • ceramide • exercise • muscle hypertrophy • peroxisome proliferator-activated receptor Triglyceride (TG) is the major energy storage form in tissues. The fi nal step of TG synthesis, the conversion of diacylglycerol (DAG) to TG, is catalyzed by DAG acyltransferases (DGAT). Dgat1 belongs to a family of membranebound O-acyltranferase (MBOAT), which includes acyl CoA:cholesterol acyltransferase ( Acat ) 1 and Acat2 ( 1-3 ). Dgat1 is widely expressed in all tissues, with high expression in white adipose tissue, skeletal muscle, heart, and intestine ( 1, 2 ). Dgat2 is a member of the monoacylglycerol acyltransferase family and is primarily expressed in the liver and adipose tissue ( 2 ); DGAT2 is thought to be the primary source of DGAT activity in the liver ( 4 ).The effects of Dgat1 overexpression in tissues have suggested that conversion of DAG to TG can be a detoxifying process ( 5,6 ). Overexpression of Dgat1 in skeletal muscle increased tissue content of TG but improved insulin sensitivity ( 5 ). These data support the hypothesis that conversion of intermediary lipids to TG via DGAT1 is benefi cial. Abstract 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 specifi c 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. ; DK-79221 and AA-10914 (W.S.B.); HL-62583 (L.S.H.); and T32-07343 (R.K. and L.L) Abbreviations: ACO, acyl-CoA oxidase; ANF, atrial natriuretic factor; AOX, acylCoA oxidase; ATGL, adipose TG lipase; BNP, brain-type natriuretic peptide; CD36, cluster of differentiation 36; CPT, carnitine palmitoyl transferase; DAG, diacylglycerol; dual energy X-ray absorptiometry, DEXA; DGAT, diacylglycerol acyl transferase; DGATli, D...