Elongation factor 1A1 regulates metabolic substrate preference in mammalian cells.

Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells.

The marine compound and elongation factor 1A1 inhibitor, didemnin B, provides benefit in western diet-induced non-alcoholic fatty liver disease.

Inhibition of eukaryotic elongation factor 1A1 (EEF1A1) with the marine compound didemnin B decreases lipotoxic HepG2 cell death in vitro and improves early stage non-alcoholic fatty liver disease (NAFLD) in young genetically obese mice. However, the effects of didemnin B on NAFLD in a model of long-term diet-induced obesity are not known. We investigated the effects of didemnin B on NAFLD severity and metabolic parameters in western diet-induced obese mice, and on the cell types that contribute to liver inflammation and fibrosis in vitro. Male 129S6 mice were fed either standard chow or western diet for 26 weeks, followed by intervention with didemnin B (50 µg/kg) or vehicle by intraperitoneal (i.p.) injection once every 3 days for 14 days. Didemnin B decreased liver and plasma triglycerides, improved oral glucose tolerance, and decreased NAFLD severity. Moreover, didemnin B moderately increased hepatic expression of genes involved in ER stress response (Perk, Chop), and fatty acid oxidation (Fgf21, Cpt1a). In vitro, didemnin B decreased THP-1 monocyte proliferation, disrupted THP-1 monocyte-macrophage differentiation, decreased THP-1 macrophage IL-1ß secretion, and decreased hepatic stellate cell (HSteC) proliferation and collagen secretion under both basal and lipotoxic (high fatty acid) conditions. Thus, didemnin B improves hepatic steatosis, glucose tolerance, and blood lipids in obesity, in association with moderate, possibly hormetic, upregulation of pathways involved in cell stress response and energy balance in the liver. Furthermore, it decreases the activity of the cell types implicated in liver inflammation and fibrosis in vitro. These findings highlight the therapeutic potential of partial protein synthesis inhibition in the treatment of NAFLD.

Two-Week Isocaloric Time-Restricted Feeding Decreases Liver Inflammation without Significant Weight Loss in Obese Mice with Non-Alcoholic Fatty Liver Disease.

Prolonged, isocaloric, time-restricted feeding (TRF) protocols can promote weight loss, improve metabolic dysregulation, and mitigate non-alcoholic fatty liver disease (NAFLD). In addition, 3-day, severe caloric restriction can improve liver metabolism and glucose homeostasis prior to significant weight loss. Thus, we hypothesized that short-term, isocaloric TRF would improve NAFLD and characteristics of metabolic syndrome in diet-induced obese male mice. After 26 weeks of ad libitum access to western diet, mice either continued feeding ad libitum or were provided with access to the same quantity of western diet for 8 h daily, over the course of two weeks. Remarkably, this short-term TRF protocol modestly decreased liver tissue inflammation in the absence of changes in body weight or epidydimal fat mass. There were no changes in hepatic lipid accumulation or other characteristics of NAFLD. We observed no changes in liver lipid metabolism-related gene expression, despite increased plasma free fatty acids and decreased plasma triglycerides in the TRF group. However, liver Grp78 and Txnip expression were decreased with TRF suggesting hepatic endoplasmic reticulum (ER) stress and activation of inflammatory pathways may have been diminished. We conclude that two-week, isocaloric TRF can potentially decrease liver inflammation, without significant weight loss or reductions in hepatic steatosis, in obese mice with NAFLD.

Bempedoic Acid Lowers Low-Density Lipoprotein Cholesterol and Attenuates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient (LDLR+/- and LDLR-/-) Yucatan Miniature Pigs.

OBJECTIVE: Bempedoic acid (BemA; ETC-1002) is a novel drug that targets hepatic ATP-citrate lyase to reduce cholesterol biosynthesis. In phase 2 studies, BemA lowers elevated low-density lipoprotein cholesterol (LDL-C) in hypercholesterolemic patients. In the present study, we tested the ability of BemA to decrease plasma cholesterol and LDL-C and attenuate atherosclerosis in a large animal model of familial hypercholesterolemia. APPROACH AND RESULTS: Gene targeting has been used to generate Yucatan miniature pigs heterozygous (LDLR+/-) or homozygous (LDLR-/-) for LDL receptor deficiency (ExeGen). LDLR+/- and LDLR-/- pigs were fed a high-fat, cholesterol-containing diet (34% kcal fat; 0.2% cholesterol) and orally administered placebo or BemA for 160 days. In LDLR+/- pigs, compared with placebo, BemA decreased plasma cholesterol and LDL-C up to 40% and 61%, respectively. In LDLR-/- pigs, in which plasma cholesterol and LDL-C were 5-fold higher than in LDLR+/- pigs, BemA decreased plasma cholesterol and LDL-C up to 27% and 29%, respectively. Plasma levels of triglycerides and high-density lipoprotein cholesterol, fasting glucose and insulin, and liver lipids were unaffected by treatment in either genotype. In the aorta of LDLR+/- pigs, BemA robustly attenuated en face raised lesion area (-58%) and left anterior descending coronary artery cross-sectional lesion area (-40%). In LDLR-/- pigs, in which lesions were substantially more advanced, BemA decreased aortic lesion area (-47%) and left anterior descending coronary artery lesion area (-48%). CONCLUSIONS: In a large animal model of LDLR deficiency and atherosclerosis, long-term treatment with BemA reduces LDL-C and attenuates the development of aortic and coronary atherosclerosis in both LDLR+/- and LDLR-/- miniature pigs.

Intervention with citrus flavonoids reverses obesity and improves metabolic syndrome and atherosclerosis in obese Ldlr-/- mice.

Obesity and its associated metabolic dysfunction and cardiovascular disease risk represent a leading cause of adult morbidity worldwide. Currently available pharmacological therapies for obesity have had limited success in reversing existing obesity and metabolic dysregulation. Previous prevention studies demonstrated that the citrus flavonoids, naringenin and nobiletin, protect against obesity and metabolic dysfunction in Ldlr-/- mice fed a high-fat cholesterol-containing (HFHC) diet. However, their effects in an intervention model are unknown. In this report, we show that, in Ldlr-/- mice with diet-induced obesity, citrus flavonoid supplementation to a HFHC diet reversed existing obesity and adipocyte size and number through enhanced energy expenditure and increased hepatic fatty acid oxidation. Caloric intake was unaffected and no evidence of white adipose tissue browning was observed. Reversal of adiposity was accompanied by improvements in hyperlipidemia, insulin sensitivity, hepatic steatosis, and a modest reduction in blood monocytes. Together, this resulted in atherosclerotic lesions that were unchanged in size, but characterized by reduced macrophage content, consistent with a more stable plaque phenotype. These studies further suggest potential therapeutic utility of citrus flavonoids, especially in the context of existing obesity, metabolic dysfunction, and cardiovascular disease.

Prevention of Diet-Induced Metabolic Dysregulation, Inflammation, and Atherosclerosis in Ldlr-/- Mice by Treatment With the ATP-Citrate Lyase Inhibitor Bempedoic Acid.

OBJECTIVE: Bempedoic acid (ETC-1002, 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel low-density lipoprotein cholesterol-lowering compound. In animals, bempedoic acid targets the liver where it inhibits cholesterol and fatty acid synthesis through inhibition of ATP-citrate lyase and through activation of AMP-activated protein kinase. In this study, we tested the hypothesis that bempedoic acid would prevent diet-induced metabolic dysregulation, inflammation, and atherosclerosis. APPROACH AND RESULTS: Ldlr-/- mice were fed a high-fat, high-cholesterol diet (42% kcal fat, 0.2% cholesterol) supplemented with bempedoic acid at 0, 3, 10 and 30 mg/kg body weight/day. Treatment for 12 weeks dose-dependently attenuated diet-induced hypercholesterolemia, hypertriglyceridemia, hyperglycemia, hyperinsulinemia, fatty liver and obesity. Compared to high-fat, high-cholesterol alone, the addition of bempedoic acid decreased plasma triglyceride (up to 64%) and cholesterol (up to 50%) concentrations, and improved glucose tolerance. Adiposity was significantly reduced with treatment. In liver, bempedoic acid prevented cholesterol and triglyceride accumulation, which was associated with increased fatty acid oxidation and reduced fatty acid synthesis. Hepatic gene expression analysis revealed that treatment significantly increased expression of genes involved in fatty acid oxidation while suppressing inflammatory gene expression. In full-length aorta, bempedoic acid markedly suppressed cholesteryl ester accumulation, attenuated the expression of proinflammatory M1 genes and attenuated the iNos/Arg1 ratio. Treatment robustly attenuated atherosclerotic lesion development in the aortic sinus by 44%, with beneficial changes in morphology, characteristic of earlier-stage lesions. CONCLUSIONS: Bempedoic acid effectively prevents plasma and tissue lipid elevations and attenuates the onset of inflammation, leading to the prevention of atherosclerotic lesion development in a mouse model of metabolic dysregulation.

Differential Lipotoxic Effects of Palmitate and Oleate in Activated Human Hepatic Stellate Cells and Epithelial Hepatoma Cells.

BACKGROUND/AIMS: Nonalcoholic fatty liver disease (NAFLD) progression to fibrosis, cirrhosis and hepatocellular carcinoma, alters the cellular composition of this organ. During late-stage NAFLD, fibrotic and possibly cancerous cells can proliferate and, like normal hepatocytes, are exposed to high concentrations of fatty acids from both surrounding tissue and circulating lipid sources. We hypothesized that primary human activated hepatic stellate cells and epithelial hepatoma (HepG2) cells respond differently to lipotoxic conditions, and investigated the mechanisms involved. METHODS: Primary activated hepatic stellate cells and HepG2 cells were exposed to pathophysiological concentrations of fatty acids and comparative studies of lipid metabolic and stress response pathways were performed. RESULTS: Both cell types remained proliferative during exposure to a combination of palmitate plus oleate reflective of the general saturated versus unsaturated fatty acid composition of western diets. However, exposure to either high palmitate or high oleate alone induced cytotoxicity in activated stellate cells, while only palmitate caused cytotoxicity in HepG2 cells. mRNA microarray and biochemical comparisons revealed that stellate cells stored markedly less fatty acids as neutral lipids, and had reduced capacity for beta-oxidation. Similar to previous observations in HepG2 cells, palmitate, but not oleate, induced ER stress and actin stress fiber formation in activated stellate cells. In contrast, oleate, but not palmitate, induced the inflammatory signal TXNIP, decreased cytoskeleton proteins, and decreased cell polarity preceding cell death in activated stellate cells. CONCLUSIONS: Palmitate-induced lipotoxicity was associated with ER stress pathways in both primary activated hepatic stellate cells and epithelial hepatoma cells, whereas high oleate caused lipotoxicity only in activated stellate cells, possibly through a distinct mechanism involving disruption of cytoskeleton components. This may have implications for optimal dietary fatty acid compositions during various stages of NAFLD.

Peroxisome proliferator-activated receptor δ agonist GW1516 attenuates diet-induced aortic inflammation, insulin resistance, and atherosclerosis in low-density lipoprotein receptor knockout mice.

OBJECTIVE: The peroxisome proliferator-activated receptor (PPAR) δ regulates systemic lipid homeostasis and inflammation. However, the ability of PPARδ agonists to improve the pathology of pre-established lesions and whether PPARδ activation is atheroprotective in the setting of insulin resistance have not been reported. Here, we examine whether intervention with a selective PPARδ agonist corrects metabolic dysregulation and attenuates aortic inflammation and atherosclerosis. APPROACH AND RESULTS: Low-density lipoprotein receptor knockout mice were fed a chow or a high-fat, high-cholesterol (HFHC) diet (42% fat, 0.2% cholesterol) for 4 weeks. For a further 8 weeks, the HFHC group was fed either HFHC or HFHC plus GW1516 (3 mg/kg per day). GW1516 significantly attenuated pre-established fasting hyperlipidemia, hyperglycemia, and hyperinsulinemia, as well as glucose and insulin intolerance. GW1516 intervention markedly reduced aortic sinus lesions and lesion macrophages, whereas smooth muscle α-actin was unchanged and collagen deposition enhanced. In aortae, GW1516 increased the expression of the PPARδ-specific gene Adfp but not PPARα- or γ-specific genes. GW1516 intervention decreased the expression of aortic proinflammatory M1 cytokines, increased the expression of the anti-inflammatory M2 cytokine Arg1, and attenuated the iNos/Arg1 ratio. Enhanced mitogen-activated protein kinase signaling, known to induce inflammatory cytokine expression in vitro, was enhanced in aortae of HFHC-fed mice. Furthermore, the HFHC diet impaired aortic insulin signaling through Akt and forkhead box O1, which was associated with elevated endoplasmic reticulum stress markers CCAAT-enhancer-binding protein homologous protein and 78kDa glucose regulated protein. GW1516 intervention normalized mitogen-activated protein kinase activation, insulin signaling, and endoplasmic reticulum stress. CONCLUSIONS: Intervention with a PPARδ agonist inhibits aortic inflammation and attenuates the progression of pre-established atherosclerosis.

PPARδ activation attenuates hepatic steatosis in Ldlr-/- mice by enhanced fat oxidation, reduced lipogenesis, and improved insulin sensitivity.

PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr(-/-) mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA ß-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKß1(-/-) hepatocytes. However, FA oxidation was only partially reduced in AMPKß1(-/-) hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKß1(-/-) hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.

Naringenin prevents cholesterol-induced systemic inflammation, metabolic dysregulation, and atherosclerosis in Ldlr⁻/⁻ mice.

Obesity-associated chronic inflammation contributes to metabolic dysfunction and propagates atherosclerosis. Recent evidence suggests that increased dietary cholesterol exacerbates inflammation in adipose tissue and liver, contributing to the proatherogenic milieu. The ability of the citrus flavonoid naringenin to prevent these cholesterol-induced perturbations is unknown. To assess the ability of naringenin to prevent the amplified inflammatory response and atherosclerosis induced by dietary cholesterol, male Ldlr⁻/⁻ mice were fed either a cholesterol-enriched high-fat or low-fat diet supplemented with 3% naringenin for 12 weeks. Naringenin, through induction of hepatic fatty acid (FA) oxidation and attenuation of FA synthesis, prevented hepatic steatosis, hepatic VLDL overproduction, and hyperlipidemia induced by both cholesterol-rich diets. Naringenin attenuated hepatic macrophage infiltration and inflammation stimulated by dietary cholesterol. Insulin resistance, adipose tissue expansion, and inflammation were alleviated by naringenin. Naringenin attenuated the cholesterol-induced formation of both foam cells and expression of inflammatory markers in peritoneal macrophages. Naringenin significantly decreased atherosclerosis and inhibited the formation of complex lesions, which was associated with normalized aortic lipids and a reversal of aortic inflammation. We demonstrate that in mice fed cholesterol-enriched diets, naringenin attenuates peripheral and systemic inflammation, leading to protection from atherosclerosis. These studies offer a therapeutically relevant alternative for the prevention of cholesterol-induced metabolic dysregulation.

Activation of peroxisome proliferator-activated receptor δ inhibits human macrophage foam cell formation and the inflammatory response induced by very low-density lipoprotein.

OBJECTIVE: Hypertriglyceridemia is an important risk factor for cardiovascular disease. Elevated plasma very low-density lipoprotein (VLDL) puts insulin-resistant patients at risk for atherosclerosis. VLDL readily induces macrophage lipid accumulation and inflammatory responses, for which targeted therapeutic strategies remain elusive. We examined the ability of VLDL to induce macrophage foam cells and the inflammatory response and sought to define the cell signaling cascades involved. We further examined the potential of peroxisome proliferator-activated receptor (PPAR) δ activation to attenuate both VLDL-stimulated lipid accumulation and cytokine expression. METHODS AND RESULTS: THP-1 macrophages exposed to VLDL displayed significant triglyceride accumulation, which was attenuated by PPARδ activation. PPARδ agonists stimulated a transcriptional program resulting in inhibition of lipoprotein lipase activity, activation of fatty acid uptake, and enhanced ß-oxidation. VLDL-treated macrophages significantly increased the expression of activator protein 1 associated cytokines interleukin-1ß, macrophage inflammatory protein 1α, and intercellular adhesion molecule-1. VLDL treatment significantly increased the phosphorylation of both extracellular signal-related kinase 1 and 2 and p38. VLDL reduced AKT phosphorylation as well as its downstream effector forkhead box protein O1, concomitant with increased nuclear forkhead box protein O1. Cells treated with PPARδ agonists were completely resistant to VLDL-induced expression of inflammatory cytokines, mediated by normalization of mitogen-activated protein kinase (MAPK)(erk) and AKT/forkhead box protein O1 signaling. CONCLUSIONS: The combined PPARδ-mediated reductions of lipid accumulation and inflammatory cytokine expression suggest a novel macrophage-targeted therapeutic option in treating atherosclerosis.

Naringenin decreases progression of atherosclerosis by improving dyslipidemia in high-fat-fed low-density lipoprotein receptor-null mice.

OBJECTIVE: Naringenin is a citrus flavonoid that potently inhibits the assembly and secretion of apolipoprotein B100-containing lipoproteins in cultured hepatocytes and improves the dyslipidemia and insulin resistance in a mouse model of the metabolic syndrome. In the present study, we used low-density lipoprotein receptor-null mice fed a high-fat diet (Western, TD96125) to test the hypothesis that naringenin prevents atherosclerosis. METHODS AND RESULTS: Three groups (chow, Western, and Western plus naringenin) were fed ad libitum for 6 months. The Western diet increased fasting plasma triglyceride (TG) (5-fold) and cholesterol (8-fold) levels compared with chow, whereas the addition of naringenin significantly decreased both lipids by 50%. The Western-fed mice developed extensive atherosclerosis in the aortic sinus because plaque area was increased by 10-fold compared with chow-fed animals. Quantitation of fat-soluble dye (Sudan IV)-stained aortas, prepared en face, revealed that Western-fed mice also had a 10-fold increase in plaque deposits throughout the arch and in the abdominal sections of the aorta, compared with chow. Atherosclerosis in both areas was significantly decreased by more than 70% in naringenin-treated mice. Consistent with quantitation of aortic lesions, the Western-fed mice had a significant 6-fold increase in cholesterol and a 4-fold increase in TG deposition in the aorta compared with chow-fed mice. Both were reduced more than 50% by naringenin. The Western diet induced extensive hepatic steatosis, with a 10-fold increase in both TG and cholesteryl ester mass compared with chow. The addition of naringenin decreased both liver TG and cholesteryl ester mass by 80%. The hyperinsulinemia and obesity that developed in Western-fed mice was normalized by naringenin to levels observed in chow-fed mice. CONCLUSIONS: These in vivo studies demonstrate that the citrus flavonoid naringenin ameliorates the dyslipidemia in Western-fed low-density lipoprotein receptor-null mice, leading to decreased atherosclerosis; and suggests a potential therapeutic strategy for the hyperlipidemia and increased risk of atherosclerosis associated with insulin resistance.

Naringenin Supplementation to a Chow Diet Enhances Energy Expenditure and Fatty Acid Oxidation, and Reduces Adiposity in Lean, Pair-Fed Ldlr-/- Mice.

SCOPE: Naringenin is a citrus-derived flavonoid that has potent lipid-lowering and insulin-sensitizing effects in obese mouse models of metabolic dysfunction. However, in these models, a significant effect of naringenin supplementation is the prevention of weight gain, which in itself can confer metabolic protection. Therefore, in the present study, the effect of naringenin supplementation in lean, chow-fed Ldlr-/- mice is investigated. METHODS AND RESULTS: In Ldlr-/- mice with isocaloric food consumption, treatment with naringenin for 8 weeks reduces body weight and adiposity compared to littermate controls pair-fed the chow diet alone. Furthermore, naringenin treatment reduces plasma lipids and enhances insulin sensitivity compared to chow-fed controls. Metabolic cage studies reveal that naringenin-treated mice have elevated energy expenditure with no change in ambulatory activity. Additionally, naringenin-treated mice have an increased respiratory exchange ratio and food consumption during the dark cycle. Treatment increases the expression of fatty acid oxidation genes in liver, and increased ß-hydroxybutyrate concentrations in plasma, indicating that one mechanism through which naringenin mediates metabolic improvement is enhanced hepatic fatty acid oxidation. CONCLUSIONS: These studies highlight the potential therapeutic utility of naringenin and suggest that this flavonoid maintains potent metabolic properties in the absence of obesity or a high-fat diet.

Naringenin enhances the regression of atherosclerosis induced by a chow diet in Ldlr-/- mice.

BACKGROUND AND AIMS: Naringenin is a citrus-derived flavonoid with lipid-lowering and insulin-sensitizing effects leading to athero-protection in Ldlr-/- mice fed a high-fat diet. However, the ability of naringenin to promote atherosclerosis regression is unknown. In the present study, we assessed the capacity of naringenin to enhance regression in Ldlr-/- mice with diet-induced intermediate atherosclerosis intervened with a chow diet. METHODS: Male Ldlr-/- mice were fed a high-fat, cholesterol-containing (HFHC) diet for 12 weeks to induce intermediate atherosclerosis and metabolic dysfunction. Subsequently, a group of these mice were sacrificed for baseline analyses and the remainder either 1) continued on the HFHC diet, 2) switched to a chow diet or 3) switched to chow diet supplemented with naringenin. RESULTS: After 12 weeks induction, intermediate lesions developed in the aortic sinus. Intervention with chow alone slowed lesion growth, while intervention with naringenin-supplemented chow completely halted lesion growth. Lesions were characterized by features of improved morphology. Compared to chow alone, naringenin reduced plaque macrophages and modestly increased smooth muscle cells. Investigating processes that contributed to improved plaque morphology, we showed naringenin further reduced plasma triglycerides and cholesterol compared to chow alone. Furthermore, elevated monocytosis and myelopoiesis were further corrected by intervention with naringenin compared to chow alone. Metabolically, naringenin enhanced the correction of insulin resistance, hepatic steatosis and obesity compared to chow alone, potentially contributing to enhanced regression. CONCLUSIONS: Naringenin supplementation to chow enhances atherosclerosis regression in male Ldlr-/- mice. These studies further underscore the potential therapeutic utility of naringenin.

Vitamin D intervention does not improve vascular regeneration in diet-induced obese male mice with peripheral ischemia.

Vitamin D appears to either promote or inhibit neovascularization in a disease context-dependent manner. The effects of vitamin D, alone or in combination with niacin, on endothelial cell (EC) angiogenic function and on revascularization in obese animals with peripheral ischemia are unknown. Here, we report that supplementation of high palmitate medium with vitamin D, niacin or both vitamins increased EC tube formation, which relies primarily on cell migration, and also maintained tube stability over time. Transcriptomic analyses revealed that both vitamins increased stress response and anti-inflammatory gene expression. However, vitamin D decreased cell cycle gene expression and inhibited proliferation, while niacin induced stable expression of miR-126-3p and -5p and maintained cell proliferation in high palmitate. To assess vascular regeneration, diet-induced obese mice received vitamin D, niacin or both vitamins following hind limb ischemic injury. Niacin, but not vitamin D or combined treatment, improved recovery of hind limb use. Histology of tibialis anterior sections revealed no improvements in revascularization, regeneration, inflammation or fibrosis with vitamin D or combined treatment. In summary, although both vitamin D and niacin increased angiogenic function of EC cultures in high fat, only niacin improved recovery of hind limb use following ischemic injury in obese mice. It is possible that inhibition of cell proliferation by vitamin D in high-fat conditions limits vascular regeneration and recovery from peripheral ischemia in obesity.

Common variants APOC3, APOA5, APOE and PON1 are associated with variation in plasma lipoprotein traits in Greenlanders.

OBJECTIVES: We undertook studies of the association between common genomic variations in APOC3, APOA5, APOE and PON1 genes and variation in biochemical phenotypes in a sample of Greenlanders. STUDY DESIGN: Genetic association study of quantitative lipoprotein traits. METHODS: In a sample of 1,310 adult Greenlanders, fasting plasma lipid, lipoprotein and apolipoprotein (apo) concentrations were assessed for association with known functional genomic variants of APOC3, APOA5, APOE and PON1. For significantly associated polymorphisms, between-genotype differences were examined in closer detail. RESULTS: We found that (1) the APOE restriction isotype was associated with variation in plasma total and LDL cholesterol and apo B (all p < .0001); (2) the APOC3 promoter genotype was associated with variation in plasma triglycerides, HDL cholesterol and apo A-I (all p < .002); (3) the APOA5 codon 19 genotype was associated with variation in plasma triglycerides (p = .027); and (4) the PON1 codon 192 genotype was associated with variation in total and LDL cholesterol and apo B (all p < .05). CONCLUSIONS: Taken together, our results suggest that common genetic variations in APOC3, APOA5, APOE and PON1 are associated with significant variation in intermediate traits in plasma lipoprotein metabolism in Greenlanders; the associations are similar to those observed for these variants in other populations.

Microarray data and pathway analyses for primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells.

As nonalcoholic fatty liver disease progresses to end-stage diseases, including fibrosis, cirrhosis and hepatocellular carcinoma, fibrotic activated hepatic stellate cells and cancerous epithelial cells can become abundant, changing the cellular composition of this organ. Despite potentially residing within the same diseased tissue, direct comparisons of global gene expression between activated hepatic stellate cells and hepatocellular carcinoma cells are lacking. Here we provide data collected using Affymetrix GeneChip microarrays to identify differential gene expression in cultured primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells. The dataset includes many genes involved in intermediary metabolism which were investigated in greater depth in our associated article (A.M. Hetherington, C.G. Sawyez, E. Zilberman, A.M. Stoianov, D.L. Robson, J.M. Hughes-Large, et al., 2016) [1]. Pathway analyses of known protein coding genes down-regulated or up-regulated by greater than 2.0-fold are also provided.

Enhanced synthesis of the oxysterol 24(S),25-epoxycholesterol in macrophages by inhibitors of 2,3-oxidosqualene:lanosterol cyclase: a novel mechanism for the attenuation of foam cell formation.

Oxysterols are key regulators of lipid metabolism and regulate gene expression by activating the liver X receptor (LXR). LXR plays a vital role in macrophage foam cell formation, a central event in atherosclerosis. It is known that addition of exogenous oxysterols to cultured macrophages activates LXR, leading to increased expression of ABCA1 and cholesterol efflux. In this study, we tested the novel hypothesis that stimulation of endogenous oxysterol synthesis would block foam cell formation induced by atherogenic lipoproteins. Macrophage synthesis of 24(S),25-epoxycholesterol, a potent LXR ligand, increased 60-fold by partial inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), a microsomal enzyme in both the cholesterol biosynthetic pathway and the alternative oxysterol synthetic pathway. When macrophages were challenged with human hypertriglyceridemic VLDL (HTG-VLDL), cellular cholesteryl ester accumulation increased 12-fold. This was reduced dramatically, by 65%, after preincubation with an OSC inhibitor (OSCi). The HTG-VLDL-induced accumulation of macrophage TG (70-fold) was unaffected by the OSCi or exogenous 24(S),25-epoxycholesterol, an effect associated with suppression of SREBP-1 processing. By contrast, TO901317, a synthetic LXR agonist, increased cellular TG significantly and markedly increased SREBP-1 processing. OSC inhibition decreased HTG-VLDL uptake through downregulation of LDL-receptor expression, despite substantial inhibition of cholesterol synthesis. Furthermore, OSC inhibition significantly upregulated ABCA1 and ABCG1 expression, which led to enhanced macrophage cholesterol efflux, an effect mediated through LXR activation. Therefore, increased macrophage synthesis of endogenous oxysterols represents a new mechanism for the dual regulation of LXR- and SREBP-responsive genes, an approach that inhibits foam cell formation without detrimental effect on TG synthesis.

Niacin promotes revascularization and recovery of limb function in diet-induced obese mice with peripheral ischemia.

Niacin can reduce vascular disease risk in individuals with metabolic syndrome, but in light of recent large randomized controlled trials outcomes, its biological actions and clinical utility remain controversial. Niacin can improve endothelial function, vascular inflammation, and vascular regeneration, independent of correcting dyslipidemia, in various lean rodent models of vascular injury. Here, we tested whether niacin could directly improve endothelial cell angiogenic function during combined exposure to excess fatty acids and hypoxia, and whether intervention with niacin during continued feeding of western diet could improve revascularization and functional recovery in obese, hyperlipidemic mice with peripheral ischemia. Treatment with niacin (10 µmol/L) increased human microvascular endothelial cell angiogenic function during exposure to high fatty acids and hypoxia (2% oxygen), as determined by tube formation on Matrigel. To assess revascularization in vivo, we used western diet-induced obese mice with unilateral hind limb femoral artery ligation and excision. Treatment for 14 days postinjury with once daily i.p. injections of a low dose of niacin (50 mg/kg) improved recovery of hind limb use, in association with enhanced revascularization and decreased inflammation of the tibialis anterior muscle. These effects were concomitant with decreased plasma triglycerides, but not increased plasma apoAI. Thus, niacin improves endothelial tube formation under lipotoxic and hypoxic conditions, and moreover, promotes revascularization and functional hind limb recovery following ischemic injury in diet-induced obese mice with hyperlipidemia. These data may have implications for niacin therapy in the treatment of peripheral ischemic vascular disease associated with metabolic syndrome.

Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease.