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.

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.

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.

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.

Naringenin prevents obesity, hepatic steatosis, and glucose intolerance in male mice independent of fibroblast growth factor 21.

The molecular mechanisms and metabolic pathways whereby the citrus flavonoid, naringenin, reduces dyslipidemia and improves glucose tolerance were investigated in C57BL6/J wild-type mice and fibroblast growth factor 21 (FGF21) null (Fgf21(-/-)) mice. FGF21 regulates energy homeostasis and the metabolic adaptation to fasting. One avenue of this regulation is through induction of peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc1a), a regulator of hepatic fatty acid oxidation and ketogenesis. Because naringenin is a potent activator of hepatic FA oxidation, we hypothesized that induction of FGF21 might be an integral part of naringenin's mechanism of action. Furthermore, we predicted that FGF21 deficiency would potentiate high-fat diet (HFD)-induced metabolic dysregulation and compromise metabolic protection by naringenin. The absence of FGF21 exacerbated the response to a HFD. Interestingly, naringenin supplementation to the HFD robustly prevented obesity in both genotypes. Gene expression analysis suggested that naringenin was not primarily targeting fatty acid metabolism in white adipose tissue. Naringenin corrected hepatic triglyceride concentrations and normalized hepatic expression of Pgc1a, Cpt1a, and Srebf1c in both wild-type and Fgf21(-/-) mice. HFD-fed Fgf21(-/-) mice displayed greater muscle triglyceride deposition, hyperinsulinemia, and impaired glucose tolerance as compared with wild-type mice, confirming the role of FGF21 in insulin sensitivity; however, naringenin supplementation improved these metabolic parameters in both genotypes. We conclude that FGF21 deficiency exacerbates HFD-induced obesity, hepatic steatosis, and insulin resistance. Furthermore, FGF21 is not required for naringenin to protect mice from HFD-induced metabolic dysregulation. Collectively these studies support the concept that naringenin has potent lipid-lowering effects and may act as an insulin sensitizer in vivo.

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.