Hormone-sensitive lipase (HSL) is known to mediate the hydrolysis not only of triacylglycerol stored in adipose tissue but also of cholesterol esters in the adrenals, ovaries, testes, and macrophages. To elucidate its precise role in the development of obesity and steroidogenesis, we generated HSL knockout mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutant HSL allele (HSL؊͞؊) were superficially normal except that the males were sterile because of oligospermia. HSL؊͞؊ mice did not have hypogonadism or adrenal insufficiency. Instead, the testes completely lacked neutral cholesterol ester hydrolase (NCEH) activities and contained increased amounts of cholesterol ester. Many epithelial cells in the seminiferous tubules were vacuolated. NCEH activities were completely absent from both brown adipose tissue (BAT) and white adipose tissue (WAT) in HSL؊͞؊ mice. Consistently, adipocytes were significantly enlarged in the BAT (5-fold) and, to a lesser extent in the WAT (2-fold), supporting the concept that the hydrolysis of triacylglycerol was, at least in part, impaired in HSL؊͞؊ mice. The BAT mass was increased by 1.65-fold, but the WAT mass remained unchanged. Discrepancy of the size differences between cell and tissue suggests the heterogeneity of adipocytes. Despite these morphological changes, HSL؊͞؊ mice were neither obese nor cold sensitive. Furthermore, WAT from HSL؊͞؊ mice retained 40% of triacylglycerol lipase activities compared with the wild-type WAT. In conclusion, HSL is required for spermatogenesis but is not the only enzyme that mediates the hydrolysis of triacylglycerol stored in adipocytes.

Methods GPI-LpL construct.A PCR-based strategy was used to ligate the DNA sequence encoding the last 37 amino acids of membrane decay accelerating factor (DAF) (9, 10) containing the GPI-anchoring sequence to a human LpL (hLpL) minigene (11) (see Figure 1a). This strategy required the elimination of the LpL termination codon

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...

C]TG, we observed that hearts also internalize intact core lipid. Inhibition of lipoprotein lipase (LPL) with tetrahydrolipstatin or dissociation of LPL from the heart with heparin reduced cardiac uptake of TG by 82 and 64%, respectively (P Ͻ 0.01). Palmitate uptake by the heart was not changed by either treatment. Uptake of TG was 88% less in hearts from LPL knockout mice that were rescued via LPL expression in the liver. Our data suggest that the heart is especially effective in removal of circulating TG and core lipids and that this is due to LPL hydrolysis and not its bridging function. fatty acids; lipoprotein lipase; triglycerides; lipid emulsion; very low-density lipoprotein; heart; myocyte FATTY ACIDS (FA) are an important fuel source for heart and skeletal muscle, providing over 70% of the energy needs for cardiac function (2,5,33). FA are delivered to cardiac myocytes in three ways. 1) FA are derived from the hydrolysis of triglyceride (TG) stored in adipose tissue via hormone-sensitive lipase and circulate complexed with albumin. 2) FA are produced from intracellular hydrolysis of TG in the core of internalized lipoproteins. 3) FA are also generated in the local capillary bed by lipoprotein lipase (LPL)-mediated hydrolysis of TG in circulating chylomicrons and very low-density lipoproteins (VLDL). Despite the fact that the molar concentration of FA in lipoprotein TG is an order of magnitude greater than that of albumin FA, it is widely believed that albumin-FA is the primary source of energy for the heart (21). However, cardiac muscle is the tissue with the greatest expression of LPL (9). Moreover, expression of LPL solely in the heart is adequate to maintain normal levels of plasma TG (19). Thus it is likely that hearts are continuously generating a large amount of FA from TG lipolysis.A number of early studies that measured FA delivery to muscle (11,14) were limited in their scope, measuring only the contribution of albumin-bound FA delivery to muscle without considering additional pathways. More recently, FA metabolism in isolated perfused working hearts has been studied and FA oxidation quantified (3,11,14,24,34). Comparison data on FA delivery to the heart under conditions that mimic those in vivo are limited. The contribution of lipoprotein-TG to heart energy production, especially in the postprandial period when the heart is bathed in dietary TG, is uncertain.This study had two objectives. The first was to compare the heart uptake of FA bound to albumin and FA derived from the hydrolysis of TG-rich lipoproteins with that of other tissues. Kinetic studies were performed in mice to assess two or more pathways of FA delivery concurrently. This allowed us to assess, in vivo, FA delivery to the heart in the context of whole body metabolism. Intralipid emulsion particles, which are similar in size and TG content to chylomicrons (17), and VLDL were utilized to determine lipoprotein particle FA delivery. In addition, palmitate complexed to bovine serum albumin (BSA) was used to assess free FA delivery ...

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes esterification of cellular cholesterol. To investigate the role of ACAT-1 in atherosclerosis, we have generated ACAT-1 null (ACAT-1؊/؊) mice. ACAT activities were present in the liver and intestine but were completely absent in adrenal, testes, ovaries, and peritoneal macrophages in our ACAT-1؊/؊ mice. The ACAT-1؊/؊ mice had decreased openings of the eyes because of atrophy of the meibomian glands, a modified form of sebaceous glands normally expressing high ACAT activities. This phenotype is similar to dry eye syndrome in humans. To determine the role of ACAT-1 in atherogenesis, we crossed the ACAT-1؊/؊ mice with mice lacking apolipoprotein (apo) E or the low density lipoprotein receptor (LDLR), hyperlipidemic models susceptible to atherosclerosis. High fat feeding resulted in extensive cutaneous xanthomatosis with loss of hair in both ACAT-1؊/؊:apo E؊/؊ and ACAT-1؊/؊:LDLR؊/؊ mice. Free cholesterol content was significantly increased in their skin. Aortic fatty streak lesion size as well as cholesteryl ester content were moderately reduced in both double mutant mice compared with their respective controls. These results indicate that the local inhibition of ACAT activity in tissue macrophages is protective against cholesteryl ester accumulation but causes cutaneous xanthomatosis in mice that lack apo E or LDLR.

Cholesterol ester (CE)-laden macrophage foam cells are the hallmark of atherosclerosis, and the hydrolysis of intracellular CE is one of the key steps in foam cell formation. Although hormone-sensitive lipase (LIPE) and cholesterol ester hydrolase (CEH), which is identical to carboxylsterase 1 (CES1, hCE1), were proposed to mediate the neutral CE hydrolase (nCEH) activity in macrophages, recent evidences have suggested the involvement of other enzymes. We have recently reported the identification of a candidate, neutral cholesterol ester hydrolase 1(Nceh1). Here we demonstrate that genetic ablation of Nceh1 promotes foam cell formation and the development of atherosclerosis in mice. We further demonstrate that Nceh1 and Lipe mediate a comparable degree of nCEH activity in macrophages and together account for most of the activity. Mice lacking both Nceh1 and Lipe aggravated atherosclerosis in an additive manner. Thus, Nceh1 is a promising target for the treatment of atherosclerosis.

Heparan sulfates, the carbohydrate chains of heparan sulfate proteoglycans, play an important role in basement membrane organization and endothelial barrier function. We explored whether endothelial cells secrete a heparan sulfate degrading heparanase under inflammatory conditions and what pathways were responsible for heparanase expression. Heparanase mRNA and protein by Western blot were induced when cultured endothelial cells were treated with cytokines, oxidized low-density lipoprotein (LDL) or fatty acids. Heparanase protein in the cell media was induced 2-10-fold when cells were treated with tumor necrosis factor alpha (TNFalpha) or interleukin 1beta (IL-1beta). Vascular endothelial growth factor (VEGF), in contrast, decreased heparanase secretion. Inhibitors to nuclear factor-kappaB (NFkappaB), PI3-kinase, MAP kinase, or c-jun kinase (JNK) did not affect TNFalpha-induced heparanase secretion. Interestingly, inhibition of caspase-8 completely abolished heparanase secretion induced by TNFalpha. Fatty acids also induced heparanase, and this required an Sp1 site in the heparanase promoter. Immunohistochemical analyses of cross sections of aorta showed intense staining for heparanase in the endothelium of apoE-null mice but not wild-type mice. Thus, heparanase is an inducible inflammatory gene product that may play an important role in vascular biology.

Fatty acids are the primary energy source for the heart. The heart acquires fatty acids associated with albumin or derived from lipoprotein lipase (LpL)-mediated hydrolysis of lipoprotein triglyceride (TG). We generated heart-specific LpL knock-out mice (hLpL0) to determine whether cardiac LpL modulates the actions of peroxisome proliferator-activated receptors and affects whole body lipid metabolism. Male hLpL0 mice had significantly elevated plasma TG levels and decreased clearance of postprandial lipids despite normal postheparin plasma LpL activity. Very large density lipoprotein-TG uptake was decreased by 72% in hLpL0 hearts. However, heart uptake of albumin-bound free fatty acids was not altered. Northern blot analysis revealed a decrease in the expression of peroxisome proliferatoractivated receptor ␣-response genes involved in fatty acid ␤-oxidation. Surprisingly, the expression of glucose transporters 1 and 4 and insulin receptor substrate 2 was increased and that of pyruvate dehydrogenase kinase 4 and insulin receptor substrate 1 was reduced. Basal glucose uptake was increased markedly in hLpL0 hearts. Thus, the loss of LpL in the heart leads to defective plasma metabolism of TG. Moreover, fatty acids derived from lipoprotein TG and not just albumin-associated fatty acids are important for cardiac lipid metabolism and gene regulation.The heart, unlike most skeletal muscles, is constantly undergoing contraction and relaxation, events that require a large amount of energy. Under normal conditions, the heart derives ϳ70% of its energy from the oxidation of long-chain fatty acids (FA) 1 and the remainder comes from glucose and lactate metabolism (1, 2). However, a number of conditions including fasting, aerobic exercise, and diabetes increase the contribution of FA to ATP production by the heart (3). FA are supplied to the heart from the hydrolysis of triglyceride (TG)-rich lipoproteins via lipoprotein lipase (LpL) (4) and via uptake of albuminbound FA derived from adipose TG stores. The relative importance of these two pathways for cardiac metabolism and regulation of FA-responsive genes is unknown. LpL controls FA uptake through the hydrolysis of TG in chylomicrons and very large density lipoproteins (VLDL) (5). Although most lipolysis of plasma TG is thought to occur in skeletal muscle and adipose, several lines of evidence suggest that cardiac muscle is an important site of regulation of plasma TG levels (4, 6). Cardiac muscle is the tissue with the greatest expression of LpL on a per gram basis (7). Moreover, mice expressing LpL only in the heart are able to maintain normal lipid levels (4, 6).We used the Cre-loxP recombination system to generate mice with a cardiac-specific ablation of the LpL gene to elucidate the role of cardiac LpL in heart and plasma lipoprotein metabolism and gene expression. This allowed us to investigate the role of LpL in the heart without directly altering LpL function and activity in skeletal muscle or adipose tissue. Our data show that the loss of LpL in the heart leads ...