The role of mitochondrial glycerol-3-phosphate acyltransferase-1 in regulating lipid and glucose homeostasis in high-fat diet fed mice.

Glycerol-3-phosphate acyltransferase (GPAT) is involved in triacylglycerol (TAG) and phospholipid synthesis, catalyzing the first committed step. In order to further investigate the in vivo importance of the dominating mitochondrial variant, GPAT1, a novel GPAT1(-/-) mouse model was generated and studied. Female GPAT1(-/-) mice had reduced body weight-gain and adiposity when fed chow diet compared with littermate wild-type controls. Furthermore, GPAT1(-/-) females on chow diet showed decreased liver TAG content, plasma cholesterol and TAG levels and increased ex vivo liver fatty acid oxidation and plasma ketone bodies. However, these beneficial effects were abolished and the glucose tolerance tended to be impaired when GPAT1(-/-) females were fed a long-term high-fat diet (HFD). GPAT1-deficiency was not associated with altered whole body energy expenditure or respiratory exchange ratio. In addition, there were no changes in male GPAT1(-/-) mice fed either diet except for increased plasma ketone bodies on chow diet, indicating a gender-specific phenotype. Thus, GPAT1-deficiency does not protect against HFD-induced obesity, hepatic steatosis or whole body glucose intolerance.

Liver-directed overexpression of mitochondrial glycerol-3-phosphate acyltransferase results in hepatic steatosis, increased triacylglycerol secretion and reduced fatty acid oxidation.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first committed step in triacylglycerol (TAG) and phospholipid biosynthesis. GPAT activity has been identified in both ER and mitochondrial subcellular fractions. The ER activity dominates in most tissues except in liver, where the mitochondrial isoform (mtGPAT) can constitute up to 50% of the total activity. To study the in vivo effects of hepatic mtGPAT overexpression, mice were transduced with adenoviruses expressing either murine mtGPAT or a catalytically inactive variant of the enzyme. Overexpressing mtGPAT resulted in massive 12- and 7-fold accumulation of liver TAG and diacylglycerol, respectively but had no effect on phospholipid or cholesterol ester content. Histological analysis showed extensive lipid accumulation in hepatocytes. Furthermore, mtGPAT transduction markedly increased adipocyte differentiation-related protein and stearoyl-CoA desaturase-1 (SCD-1) in the liver. In line with increased SCD-1 expression, 18:1 and 16:1 in the hepatic TAG fraction increased. In addition, mtGPAT overexpression decreased ex vivo fatty acid oxidation, increased liver TAG secretion rate 2-fold, and increased plasma TAG and cholesterol levels. These results support the hypothesis that increased hepatic mtGPAT activity associated with obesity and insulin resistance contributes to increased TAG biosynthesis and inhibition of fatty acid oxidation, responses that would promote hepatic steatosis and dyslipidemia.

Glucose and fatty acid metabolism in McA-RH7777 hepatoma cells vs. rat primary hepatocytes: responsiveness to nutrient availability.

The overabundance of dietary fats and simple carbohydrates contributes significantly to obesity and metabolic disorders associated with obesity. The liver balances glucose and lipid distribution, and disruption of this balance plays a key role in these metabolic syndromes. We investigated (1) how hepatocytes balance glucose and fatty acid metabolism when one or both nutrients are supplied in abundance and (2) whether rat hepatoma cells (McA-RH7777) reflect nutrient partitioning in a similar manner as compared with primary hepatocytes. Increasing media palmitate concentration increased fatty acid uptake, triglyceride synthesis and beta-oxidation. However, hepatoma cells had a 2-fold higher fatty acid uptake and a 2-fold lower fatty acid oxidation as compared with primary hepatocytes. McA-RH7777 cells did not synthesize significant amounts of glycogen and preferentially metabolized the glucose into lipids or into oxidation. In primary hepatocytes, the glucose was mostly spared from oxidation and instead partitioned into both de novo glycogen and lipid synthesis. Overall, lipid production was rapidly induced in response to either glucose or fatty acid excess and this may be one of the earliest indicators of metabolic syndrome development associated with nutrient excess.

Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis.

Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Because both mtGPAT and carnitine palmitoyl transferase-1 are located on the outer mitochondrial membrane, we hypothesized that mtGPAT directs fatty acyl-CoA away from beta-oxidation and toward glycerolipid synthesis. Adenoviral-mediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme, which helps define the active site required for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have >2-fold increased enzyme activity. Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.

Mice deficient in apolipoprotein E but not LDL receptors are resistant to accelerated atherosclerosis associated with obesity.

The aims of this study were to determine whether mice induced to become obese also exhibited accelerated atherosclerosis, and to determine whether obesity itself or dyslipidemia associated with obesity enhanced atherosclerosis. Wild-type (C57BL/6) mice and mice deficient for the low density lipoprotein receptor (LDLR-/-) or apolipoprotein E (apoE-/-) were fed a low fat, rodent chow diet or a high fat, high sucrose (diabetogenic) diet to induce obesity. As compared with wild-type mice, diabetogenic diet-fed LDLR-/- mice became more obese and developed severe dyslipidemia. Consequently, atherosclerotic lesions were increased in the LDLR-/- mice 3.7-fold over chow fed values. ApoE-/- mice showed weight gain profiles similar to those observed for wild-type mice. However, no differences in plasma lipid levels, lipoprotein profiles or atherosclerotic lesion areas were observed between chow-fed and diabetogenic diet-fed apoE-/- mice. These data demonstrate that lipid storage and partitioning as mediated by the low density lipoproteins (LDL) receptor or apoE-/- have profound and opposing consequences for dyslipidemia and atherosclerosis susceptibility associated with obesity.

Pharmacological interference with intestinal bile acid transport reduces plasma cholesterol in LDL receptor/apoE deficiency.

Reduction of plasma cholesterol by statins is fundamental to prevent coronary heart disease. Such therapy is often sub-optimal, however, particularly in patients with reduced LDL receptors (familial hypercholesterolemia), and novel or adjuvant therapies are therefore warranted. Cholesterol elimination is profoundly influenced by the rate of its conversion to bile acids (BA), regulated by the enzyme Cyp7a1. Induced fecal loss of BA by resin treatment reduces plasma cholesterol, presumably through induction of hepatic LDL receptors (LDLR). We here describe the effect of PR835, a drug belonging to a new class of lipid-lowering agents that inhibit the Slc10a2 protein, the intestinal transporter responsible for active uptake of BA. Treatment reduced plasma cholesterol by 40% in mice devoid of both the LDLR and its ligand, apoE, while triglycerides and HDL cholesterol were unchanged. Cyp7a1 enzyme activity and mRNA were induced several-fold, and hepatic HMG CoA reductase mRNA increased, mirroring an induced synthesis of BA and cholesterol. The addition of a statin potentiated the effect, leading to reductions of plasma total and LDL cholesterol by 64% and 70%, respectively. These effects could not be attributed to induction of other known hepatic lipoprotein receptors and indicate the presence of new points of targeting in lipid-lowering therapy.

Loss of lymphotoxin-alpha but not tumor necrosis factor-alpha reduces atherosclerosis in mice.

Inflammatory processes are involved with all phases of atherosclerotic lesion growth. Tumor necrosis factor-alpha (TNFalpha) is an inflammatory cytokine that is thought to contribute to lesion development. Lymphotoxin-alpha (LTalpha) is also a proinflammatory cytokine with homology to TNFalpha. However, its presence or function in lesion development has not been investigated. To study the role of these molecules in atherosclerosis, the expression of these cytokines in atherosclerotic lesions was examined. The presence of both cytokines was observed within aortic sinus fatty streak lesions. To determine the function of these molecules in regulating lesion growth, mice deficient for TNFalpha or LTalpha were examined for induction of atherosclerosis. Surprisingly, loss of TNFalpha did not alter lesion development compared with wild-type mice. This brings doubt to the generally held concept that TNFalpha is a "proatherogenic cytokine." However, LTalpha deficiency resulted in a 62% reduction in lesion size. This demonstrates an unexpected role for LTalpha in promoting lesion growth. The presence of LTalpha was observed in aortic sinus lesions suggesting a direct role of LTalpha in modulating lesion growth. To determine which receptor mediated these responses, diet-induced atherosclerosis in mice deficient for each of the TNF receptors, termed p55 and p75, was examined. Results demonstrated that loss of p55 resulted in increased lesion development, but loss of p75 did not alter lesion size. The disparity in results between ligand- and receptor-deficient mice suggests there are undefined members of the TNF ligand and receptor signaling pathway involved with regulating atherogenesis.

LDL receptor but not apolipoprotein E deficiency increases diet-induced obesity and diabetes in mice.

The aim of this study was to determine whether phenotypes associated with type 2 diabetes are altered in dyslipidemic obese mice. C57BL/6 wild-type, low-density lipoprotein (LDL) receptor-deficient (LDLR-/-), and apolipoprotein E-deficient (apoE-/-) mice were fed a high-fat, high-carbohydrate diet (diabetogenic diet), and the development of obesity, diabetes, and hypertriglyceridemia was examined. Wild-type mice became obese and developed hyperglycemia, but not hypertriglyceridemia, in response to this diet. LDLR-/- mice fed the diabetogenic diet became more obese than wild-type mice and developed severe hypertriglyceridemia and hyperleptinemia. Surprisingly, glucose levels were only modestly higher and insulin levels and insulin-to-glucose ratios were not strikingly different from those of wild-type mice. In contrast, diabetogenic diet-fed apoE-/- mice were resistant to changes in glucose and lipid homeostasis despite becoming obese. These data suggest that modifications in lipoprotein profiles associated with loss of the LDL receptor or apoE function have profound and unique consequences on susceptibility to diet-induced obesity and type 2 diabetic phenotypes.