Faculty Bibliography

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- activation, and decreased insulin-stimulated Akt2 activity. Increased hepatic triglyceride content in the HFD-fed ApoC3Tg mice could be attributed to a approximately 70% increase in hepatic triglyceride uptake and approximately 50% reduction hepatic triglyceride secretion. CONCLUSION: These data demonstrate that increase plasma APOC3 concentrations predispose mice to diet-induced NAFLD and hepatic insulin resistance.

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.

Septic shock results from bacterial infection and is associated with multi-organ failure, high mortality, and cardiac dysfunction. Sepsis causes both myocardial inflammation and energy depletion. We hypothesized that reduced cardiac energy production is a primary cause of ventricular dysfunction in sepsis. The JNK pathway is activated in sepsis and has also been implicated in impaired fatty acid oxidation in several tissues. Therefore, we tested whether JNK activation inhibits cardiac fatty acid oxidation and whether blocking JNK would restore fatty acid oxidation during LPS treatment. LPS treatment of C57BL/6 mice and adenovirus-mediated activation of the JNK pathway in cardiomyocytes inhibited peroxisome proliferator-activated receptor alpha expression and fatty acid oxidation. Surprisingly, none of the adaptive responses that have been described in other types of heart failure, such as increased glucose utilization, reduced alphaMHC:betaMHC ratio or induction of certain microRNAs, occurred in LPS-treated mice. Treatment of C57BL/6 mice with a general JNK inhibitor (SP600125) increased fatty acid oxidation in mice and a cardiomyocyte-derived cell line. JNK inhibition also prevented LPS-mediated reduction in fatty acid oxidation and cardiac dysfunction. Inflammation was not alleviated in LPS-treated mice that received the JNK inhibitor. We conclude that activation of JNK signaling reduces fatty acid oxidation and prevents the peroxisome proliferator-activated receptor alpha down-regulation that occurs with LPS.

OBJECTIVE: There are several pathways that mediate the aberrant metabolism of glucose and that might induce greater vascular damage in the setting of diabetes. The polyol pathway mediated by aldose reductase (AR) has been postulated to be one such pathway. However, it has been reported that AR reduces toxic lipid aldehydes and, under some circumstances, might be antiatherogenic. METHODS AND RESULTS: Atherosclerosis development was quantified in 2 lines of transgenic mice expressing human AR (hAR) crossed on the apolipoprotein E knockout background. The transgenes were used to increase the normally low levels of this enzyme in wild-type mice. Both generalized hAR overexpression and hAR expression via the Tie 2 promoter increased lesion size in streptozotocin diabetic mice. In addition, pharmacological inhibition of AR reduced lesion size. CONCLUSIONS: Although in some settings AR expression might reduce levels of toxic aldehydes, transgenic expression of this enzyme within the artery wall leads to greater atherosclerosis

The purpose of this article is to review the basic and clinical science relating plasma triglycerides and cardiovascular disease. Although many aspects of the basic physiology of triglyceride production, its plasma transport, and its tissue uptake have been known for several decades, the relationship of plasma triglyceride levels to vascular disease is uncertain. Are triglyceride-rich lipoproteins, their influence on high-density lipoprotein and low-density lipoprotein, or the underlying diseases that lead to defects in triglyceride metabolism the culprit? Animal models have failed to confirm that anything other than early fatty lesions can be produced by triglyceride-rich lipoproteins. Metabolic products of triglyceride metabolism can be toxic to arterial cells; however, these studies are primarily in vitro. Correlative studies of fasting and postprandial triglycerides and genetic diseases implicate very-low-density lipoprotein and their remnants and chylomicron remnants in atherosclerosis development, but the concomitant alterations in other lipoproteins and other risk factors obscure any conclusions about direct relationships between disease and triglycerides. Genes that regulate triglyceride levels also correlate with vascular disease. Human intervention trials, however, have lacked an appropriately defined population and have produced outcomes without definitive conclusions. The time is more than ripe for new and creative approaches to understanding the relationship of triglycerides and heart disease.

OBJECTIVE: Patients with diabetes have increased cardiovascular risk. Atherosclerosis in these patients is often associated with increased plaque macrophages and dyslipidemia. We hypothesized that diabetic atherosclerosis involves processes that impair favorable effects of lipid reduction on plaque macrophages. RESEARCH DESIGN AND METHODS: Reversa mice are LDL receptor-deficient mice that develop atherosclerosis. Their elevated plasma LDL levels are lowered after conditional knockout of the gene encoding microsomal triglyceride transfer protein. We examined the morphologic and molecular changes in atherosclerotic plaques in control and streptozotocin-induced diabetic Reversa mice after LDL lowering. Bone marrow-derived macrophages were also used to study changes mediated by hyperglycemia. RESULTS: Reversa mice were fed a western diet for 16 weeks to develop plaques (baseline). Four weeks after lipid normalization, control (nondiabetic) mice had reduced plasma cholesterol (-77%), plaque cholesterol (-53%), and plaque cells positive for macrophage marker CD68+ (-73%), but increased plaque collagen (+116%) compared with baseline mice. Diabetic mice had similarly reduced plasma cholesterol, but collagen content increased by only 34% compared with baseline; compared with control mice, there were lower reductions in plaque cholesterol (-30%) and CD68+ cells (-41%). Diabetic (vs. control) plaque CD68+ cells also exhibited more oxidant stress and inflammatory gene expression and less polarization toward the anti-inflammatory M2 macrophage state. Many of the findings in vivo were recapitulated by hyperglycemia in mouse bone marrow-derived macrophages. CONCLUSIONS: Diabetes hindered plaque regression in atherosclerotic mice (based on CD68+ plaque content) and favorable changes in plaque macrophage characteristics after the reduction of elevated plasma LDL

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.

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 beta-adrenergic receptor (beta-AR) density. Pharmacological and genetic PKC inhibition prevented both palmitate-induced beta-AR insensitivity and the accompanying reduction in cell surface beta-ARs. Mice with excess lipid uptake due to either cardiac-specific overexpression of anchored lipoprotein lipase, PPARgamma, 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)alpha or PKCdelta. Thus, several lipids that are increased in the setting of lipotoxicity can produce abnormalities in beta-AR responsiveness. This can be attributed to PKC activation and reduced beta-AR levels.

Sphingolipids are membrane components and are involved in cell proliferation, apoptosis and metabolic regulation. In this study we investigated whether de novo sphingolipid biosynthesis in macrophages is regulated by inflammatory stimuli. Lipopolysaccharide (LPS) treatment upregulated Sptlc2, a subunit of serine palmitoyltransferase (SPT), mRNA and protein in Raw264.7 and mouse peritoneal macrophages, but Sptlc1, another subunit of SPT, was not altered. SPT activation by LPS elevated cellular levels of ceramides and sphingomyelin (SM). Pharmacological inhibition of nuclear factor kappa B (NFkappaB) prevented LPS-induced upregulation of Sptlc2 while transfection of p65 subunit of NFkappaB upregulated Sptlc2 and increased cellular ceramide levels. In contrast, MAP kinases were not involved in regulation of sphingolipid biosynthesis. Analysis of Sptlc2 promoter and chromatin immunoprecipitation (ChIP) assay showed that NFkappaB binding sites are located in Sptlc2 promoter region. Our results demonstrate that inflammatory stimuli activate de novo sphingolipid biosynthesis via NFkappaB and may play a critical role in lipid metabolism in macrophages.

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.