Sylvaine Cases

Triglyceride accumulation protects against fatty acid-induced lipotoxicity

Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic β-cell apoptosis and heart failure. Here, we demonstrate in cultured cells that the relative toxicity of two common dietary long chain fatty acids is related to channeling of these lipids to distinct cellular metabolic fates. Oleic acid supplementation leads to triglyceride accumulation and is well tolerated, whereas excess palmitic acid is poorly incorporated into triglyceride and causes apoptosis. Unsaturated fatty acids rescue palmitate-induced apoptosis by channeling palmitate into triglyceride pools and away from pathways leading to apoptosis. Moreover, in the setting of impaired triglyceride synthesis, oleate induces lipotoxicity. Our findings support a model of cellular lipid metabolism in which unsaturated fatty acids serve a protective function against lipotoxicity though promotion of triglyceride accumulation.

Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat

Triglycerides (or triacylglycerols) represent the major form of stored energy in eukaryotes. Triglyceride synthesis has been assumed to occur primarily through acyl CoA:diacylglycerol transferase (Dgat), a microsomal enzyme that catalyses the final and only committed step in the glycerol phosphate pathway. Therefore, Dgat has been considered necessary for adipose tissue formation and essential for survival. Here we show that Dgat-deficient (Dgat−/−) mice are viable and can still synthesize triglycerides. Moreover, these mice are lean and resistant to diet-induced obesity. The obesity resistance involves increased energy expenditure and increased activity. Dgat deficiency also alters triglyceride metabolism in other tissues, including the mammary gland, where lactation is defective in Dgat−/− females. Our findings indicate that multiple mechanisms exist for triglyceride synthesis and suggest that the selective inhibition of Dgat-mediated triglyceride synthesis may be useful for treating obesity.

Cloning of DGAT2, a Second Mammalian Diacylglycerol Acyltransferase, and Related Family Members

Studies involving the cloning and disruption of the gene for acyl-CoA:diacylglycerol acyltransferase (DGAT) have shown that alternative mechanisms exist for triglyceride synthesis. In this study, we cloned and characterized a second mammalian DGAT, DGAT2, which was identified by its homology to a DGAT in the fungus Mortierella rammaniana. DGAT2 is a member of a gene family that has no homology with DGAT1 and includes several mouse and human homologues that are candidates for additional DGAT genes. The expression of DGAT2 in insect cells stimulated triglyceride synthesis 6-fold in assays with cellular membranes, and DGAT2 activity was dependent on the presence of fatty acyl-CoA and diacylglycerol, indicating that this protein is a DGAT. Activity was not observed for acyl acceptors other than diacylglycerol. DGAT2 activity was inhibited by a high concentration (100 mm) of MgCl2 in anin vitro assay, a characteristic that distinguishes DGAT2 from DGAT1. DGAT2 is expressed in many tissues with high expression levels in the liver and white adipose tissue, suggesting that it may play a significant role in mammalian triglyceride metabolism.

ACAT-2, A Second Mammalian Acyl-CoA:Cholesterol Acyltransferase ITS CLONING, EXPRESSION, AND CHARACTERIZATION

The synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is an important component of cellular cholesterol homeostasis. Cholesterol ester formation also is hypothesized to be important in several physiologic processes, including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Mouse tissue expression studies and the disruption of the mouse ACAT gene (Acact) have indicated that more than one ACAT exists in mammals and specifically that another enzyme is important in mouse liver and intestine. We now describe a second mammalian ACAT enzyme, designated ACAT-2, that is 44% identical to the first cloned mouse ACAT (henceforth designated ACAT-1). Infection of H5 insect cells with an ACAT-2 recombinant baculovirus resulted in expression of a ∼46-kDa protein in cell membranes that was associated with high levels of cholesterol esterification activity. Both ACAT-1 and ACAT-2 also catalyzed the esterification of the 3β-hydroxyl group of a variety of oxysterols. Cholesterol esterification activities for ACAT-1 and ACAT-2 exhibited different IC50 values when assayed in the presence of several ACAT-specific inhibitors, demonstrating that ACAT inhibitors can selectively target specific forms of ACAT. ACAT-2 was expressed primarily in mouse liver and small intestine, supporting the hypothesis that ACAT-2 contributes to cholesterol esterification in these tissues. The mouse ACAT-2 gene (Acact2) maps to chromosome 15 in a region containing a quantitative trait locus influencing plasma cholesterol levels. The identification and cloning of ACAT-2 will facilitate molecular approaches to understanding the role of ACAT enzymes in mammalian biology.

Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase

Acyl-CoA:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of physiologically important lipids such as triacylglycerol and phospholipids. In the intestine, MGAT plays a major role in the absorption of dietary fat because resynthesis of triacylglycerol is required for the assembly of lipoproteins that transport absorbed fat to other tissues. MGAT activity has also been reported in mammalian liver and white adipose tissue. However, MGAT has never been purified to homogeneity from mammalian tissues, and its gene has not been cloned. We identified a gene that encodes an MGAT (MGAT1) in mice. This gene has sequence homology with members of a recently identified diacylglycerol acyltransferase gene family. Expression of the MGAT1 cDNA in insect cells markedly increased MGAT activity in cell membranes. In addition, MGAT activity was proportional to the level of MGAT1 protein expressed, and the amount of diacylglycerol produced depended on the concentration of either of its substrates, oleoyl-CoA or monooleoylglycerol. In mice, MGAT1 expression and MGAT activity were detected in the stomach, kidney, white and brown adipose tissue, and liver. However, MGAT1 was not expressed in the small intestine, implying the existence of a second MGAT gene. The identification of the MGAT1 gene should greatly facilitate research on the identification of the intestinal MGAT gene and on the function of MGAT enzymes in mammalian glycerolipid metabolism.

Triglyceride synthesis: insights from the cloning of diacylglycerol acyltransferase.

Although the biochemistry of triglyceride synthesis has been studied for decades, an understanding of the molecular processes involved has been lacking. The recent cloning of a gene encoding acyl coenzyme A : diacylglycerol acyltransferase, an enzyme that catalyses the final step in triglyceride synthesis, has opened this area to molecular investigation and has begun to provide new insights into triglyceride metabolism.

LPA3 receptor mediates chemotaxis of immature murine dendritic cells to unsaturated lysophosphatidic acid (LPA)

Increasing evidence supports roles for lipids in the biology of immune cells. In particular, bioactive lipids such as sphingosine‐1‐phosphate (S1P) bind to cognate G protein‐coupled receptors (GPCRs) and modulate leukocyte trafficking and homeostasis. Lysophosphatidic acid (LPA) represents a family of bioactive lipids, which differ in the length and degree of saturation of the fatty acyl chain. LPA is structurally related to S1P and exerts cellular effects by binding to five known GPCRs (LPA1–5). Its function in the immune system is less clear, although it was shown to induce chemotaxis of human dendritic cells (DCs) and activated T cells. In this study, we show that LPA can induce chemotaxis of immature but not mature mouse DCs and that only unsaturated and not saturated LPA species are efficient chemoattractants. However, both LPA species do not alter DC maturation or chemotaxis to other chemokines. The loss of DC migration capability correlated with the down‐regulation of expression of the receptors LPA3 and LPA5, and expression of LPA1, LPA2, and LPA4 did not change. A LPA3 antagonist reduced immature DC migration to LPA by 70%, suggesting that LPA3 mediates immature DC chemotaxis to unsaturated species of LPA. Furthermore, isolated, immature DCs from mice lacking LPA3 exhibited a 50% reduction in migration to LPA. In summary, our results indicate that immature mouse DCs migrate preferentially in response to unsaturated LPA and that LPA3 is important in this response.

Development of the mammary gland requires DGAT1 expression in stromal and epithelial tissues

Mammary gland development is a complex process that is dependent on interactions between the developing mammary epithelium and the surrounding stromal tissues. We show that mice lacking the triglyceride synthesis enzyme acyl CoA:diacylglycerol transferase 1 (DGAT1) have impaired mammary gland development, characterized by decreased epithelial proliferation and alveolar development, and reduced expression of markers of functional differentiation. Transplantation studies demonstrate that the impaired development results from a deficiency of DGAT1 in both the stromal and epithelial tissues. Our findings are the first to link defects in stromal lipid metabolism to impaired mammary gland development.

Adrenocortical Lipid Depletion Gene (ald) in AKR Mice Is Associated with an Acyl-CoA:Cholesterol Acyltransferase (ACAT) Mutation

ald, a recessive allele in AKR inbred mice, is responsible for complete adrenocortical lipid depletion in postpubertal males, which appears to be androgen dependent. Two recent observations (adrenocortical lipid depletion in acyl-CoA:cholesterol acyltransferase-deficient (Acact−/−) mice and the mapping of Acact to a region of chromosome 1 containing theald locus) prompted us to ask whether adrenocortical lipid depletion in AKR mice results from an Acact mutation. Refined genetic mapping of Acact and ald was consistent with colocalization of these loci. CrossingAcact−/− with AKR (ald/ald) mice yielded postpubertal male offspring characterized by adrenocortical lipid depletion, indicating that these loci are not complementational and are therefore allelic. Immunoblotting of preputial gland homogenates demonstrated that AKR mice had an ACAT protein with a lower molecular mass than other mouse strains. Analysis of Acact cDNA from AKR mice revealed a deletion of the first coding exon and two missense mutations. Despite these coding sequence differences, the ACAT protein from the ald allele catalyzed cholesterol esterification activity at levels similar to that of wild-type protein. We speculate that the adrenocortical lipid depletion resulting from theald mutation is caused by an altered susceptibility of the mutant protein to modifying factors, such as androgen production at puberty, in an as yet undetermined manner.