Joshua E. Basford

Adipocyte LDL receptor–related protein–1 expression modulates postprandial lipid transport and glucose homeostasis in mice

Diet-induced obesity and its serious consequences such as diabetes, cardiovascular disease, and cancer are rapidly becoming a major global health threat. Therefore, understanding the cellular and molecular mechanisms by which dietary fat causes obesity and diabetes is of paramount importance in order to identify preventive and therapeutic strategies. Increased dietary fat intake results in high plasma levels of triglyceride-rich lipoproteins (TGRL). Tissue uptake of TGRL has been shown to promote glucose intolerance. We generated mice with an adipocyte-specific inactivation of the multifunctional receptor LDL receptor-related protein-1 (LRP1) to determine its role in mediating the effects of TGRL on diet-induced obesity and diabetes. Knockout mice displayed delayed postprandial lipid clearance, reduced body weight, smaller fat stores, lipid-depleted brown adipocytes, improved glucose tolerance, and elevated energy expenditure due to enhanced muscle thermogenesis. We further demonstrated that inactivation of adipocyte LRP1 resulted in resistance to dietary fat-induced obesity and glucose intolerance. These findings identify LRP1 as a critical regulator of adipocyte energy homeostasis, where functional disruption leads to reduced lipid transport, increased insulin sensitivity, and muscular energy expenditure.

Melanocortin signaling in the CNS directly regulates circulating cholesterol

Cholesterol circulates in the blood in association with triglycerides and other lipids, and elevated blood low-density lipoprotein cholesterol carries a risk for metabolic and cardiovascular disorders, whereas high-density lipoprotein (HDL) cholesterol in the blood is thought to be beneficial. Circulating cholesterol is the balance among dietary cholesterol absorption, hepatic synthesis and secretion, and the metabolism of lipoproteins by various tissues. We found that the CNS is also an important regulator of cholesterol in rodents. Inhibiting the brains melanocortin system by pharmacological, genetic or endocrine mechanisms increased circulating HDL cholesterol by reducing its uptake by the liver independent of food intake or body weight. Our data suggest that a neural circuit in the brain is directly involved in the control of cholesterol metabolism by the liver.

Defective Lipid Delivery Modulates Glucose Tolerance and Metabolic Response to Diet in Apolipoprotein E–Deficient Mice

OBJECTIVE—Apolipoprotein E (ApoE) regulates plasma lipid levels via modulation of lipolysis and serving as ligand for receptor-mediated clearance of triglyceride (TG)-rich lipoproteins. This study tested the impact of modulating lipid delivery to tissues on insulin responsiveness and diet-induced obesity. RESEARCH DESIGN AND METHODS—ApoE+/+ and apoE−/− mice were placed on high-fat–high-sucrose diabetogenic diet or control diet for 24 weeks. Plasma TG clearance, glucose tolerance, and tissue uptake of dietary fat and glucose were assessed. RESULTS—Plasma TG clearance and lipid uptake by adipose tissue were impaired, whereas glucose tolerance was improved in control diet–fed apoE−/− mice compared with apoE+/+ mice after an oral lipid load. Fat mass was reduced in apoE−/− mice compared with apoE+/+ mice under both dietary conditions. The apoE−/− mice exhibited lower body weight and insulin levels than apoE+/+ mice when fed the diabetogenic diet. Glucose tolerance and uptake by muscle and brown adipose tissue (BAT) was also improved in mice lacking apoE when fed the diabetogenic diet. Indirect calorimetry studies detected no difference in energy expenditure and respiratory quotient between apoE+/+ and apoE−/− mice on control diet. Energy expenditure and uncoupling protein-1 expression in BAT were slightly but not significantly increased in apoE−/− mice on diabetogenic diet. CONCLUSIONS—These results demonstrated that decreased lipid delivery to insulin-sensitive tissues improves insulin sensitivity and ameliorates diet-induced obesity.

Ginsenoside Rb1 reduces fatty liver by activating AMP-activated protein kinase in obese rats

Ginsenoside Rb1 (Rb1), a natural compound extracted from ginseng, exerts anti-obesity activity and improves insulin sensitivity in high-fat diet (HFD)-induced obese rats. The objective of the current study was to evaluate the protective effect of Rb1 on fatty liver in HFD-induced obese rats and to elucidate underlying mechanisms. After chronic intraperitoneal administration, Rb1 (10 mg/kg) significantly ameliorated hepatic fat accumulation in HFD-induced obese rats, as demonstrated by reduced liver weight, hepatic triglyceride content, and histological evaluation of liver sections by hematoxylin and eosin and Oil Red O staining. Using primary cultured rat hepatic cells, we found that the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase 1 (CPT1), a key enzyme in fatty acid β-oxidation, were significantly elevated in Rb1-treated hepatocytes compared with those of vehicle-treated cells. HPLC analysis revealed that Rb1 increased the cellular AMP/ATP ratio, which is associated with elevated activation of hepatic AMP-activated protein kinase (AMPK) and phosphorylated acetyl-CoA carboxylase. Consistent with the activation of AMPK, Rb1 stimulated the expression of genes encoding fatty acid oxidative enzymes and proteins, and suppressed the expression of genes encoding enzymes or proteins that function in lipogenesis, assessed by quantitative PCR. We conclude that Rb1 has a potent ability to reduce hepatic fat accumulation and might be useful as a therapeutic agent for fatty liver disorder.

Myeloid-Specific Krüppel-Like Factor 2 Inactivation Increases Macrophage and Neutrophil Adhesion and Promotes Atherosclerosis

Rationale: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. Methods and Results: Cell-specific knockout mice were generated by Cre/loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout (myeKlf2−/−) mice were similar to myeKlf2+/+ macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2+/+ macrophages, myeKlf2−/− macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2−/− mice also adhered more robustly to endothelial cells, and fewer myeKlf2−/− neutrophils survived in culture over a 24-hour period in comparison with myeKlf2+/+ neutrophils. When myeKlf2−/− mice were mated to Ldlr−/− mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2−/−Ldlr−/− mice compared with myeKlf2+/+Ldlr−/− littermates. The increased atherosclerosis in myeKlf2−/−Ldlr−/− mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2+/+Ldlr−/− mice. Conclusions: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.

Lack of Association Between Adiponectin Levels and Atherosclerosis in Mice

Objective—Adiponectin is an adipocyte-derived, secreted protein that is implicated in protection against a cluster of related metabolic disorders. Mice lacking adiponectin display impaired hepatic insulin sensitivity and respond only partially to peroxisome proliferator-activated receptor &ggr; agonists. Adiponectin has been associated with antiinflammatory and antiatherogenic properties; however, the direct involvement of adiponectin on the atherogenic process has not been studied. Methods and Results—We crossed adiponectin knockout mice (Adn−/−) or mice with chronically elevated adiponectin levels (AdnTg) into the low-density lipoprotein receptor–null (Ldlr−/−) and the apoliprotein E–null (Apoe−/−) mouse models. Adiponectin levels did not correlate with a suppression of the atherogenic process. Plaque volume in the aortic root, cholesterol accumulation in the aorta, and plaque morphology under various dietary conditions were not affected by circulating adiponectin levels. In light of the strong associations reported for adiponectin with cardiovascular disease in humans, the lack of a phenotype in gain- and loss-of-function studies in mice suggests a lack of causation for adiponectin in inhibiting the buildup of atherosclerotic lesions. Conclusion—These data indicate that the actions of adiponectin on the cardiovascular system are complex and multifaceted, with a minimal direct impact on atherosclerotic plaque formation in preclinical rodent models.

Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of monocyte chemoattractant protein-1.

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed &agr;-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Apolipoprotein E4 Impairs Macrophage Efferocytosis and Potentiates Apoptosis by Accelerating Endoplasmic Reticulum Stress

Background: Apolipoprotein E4 (apoE4) is associated with inflammatory metabolic diseases. Results: Human APOE4 gene replacement mice displayed elevated tissue inflammation. APOE4 macrophages showed impaired efferocytosis, increased apoptosis, and endoplasmic reticulum stress. Conclusion: ApoE4 structural abnormalities induce ER stress to promote inflammation. Significance: Reducing ER stress and/or apoE4 structure correctors may reduce inflammatory metabolic disease risk in human apoE4 subjects. Apolipoprotein (apo) E4 is a major genetic risk factor for a wide spectrum of inflammatory metabolic diseases, including atherosclerosis, diabetes, and Alzheimer disease. This study compared diet-induced adipose tissue inflammation as well as functional properties of macrophages isolated from human APOE3 and APOE4 mice to identify the mechanism responsible for the association between apoE4 and inflammatory metabolic diseases. The initial study confirmed previous reports that APOE4 gene replacement mice were less sensitive than APOE3 mice to diet-induced body weight gain but exhibited hyperinsulinemia, and their adipose tissues were similarly inflamed as those in APOE3 mice. Peritoneal macrophages isolated from APOE4 mice were defective in efferocytosis compared with APOE3 macrophages. Increased cell death was also observed in APOE4 macrophages when stimulated with LPS or oxidized LDL. Western blot analysis of cell lysates revealed that APOE4 macrophages displayed elevated JNK phosphorylation indicative of cell stress even under basal culturing conditions. Significantly higher cell stress due mainly to potentiation of endoplasmic reticulum (ER) stress signaling was also observed in APOE4 macrophages after LPS and oxidized LDL activation. The defect in efferocytosis and elevated apoptosis sensitivity of APOE4 macrophages was ameliorated by treatment with the ER chaperone tauroursodeoxycholic acid. Taken together, these results showed that apoE4 expression causes macrophage dysfunction and promotes apoptosis via ER stress induction. The reduction of ER stress in macrophages may be a viable option to reduce inflammation and inflammation-related metabolic disorders associated with the apoE4 polymorphism.

HDAC9 Knockout Mice Are Protected From Adipose Tissue Dysfunction and Systemic Metabolic Disease During High-Fat Feeding

During chronic caloric excess, adipose tissue expands primarily by enlargement of individual adipocytes, which become stressed with lipid overloading, thereby contributing to obesity-related disease. Although adipose tissue contains numerous preadipocytes, differentiation into functionally competent adipocytes is insufficient to accommodate the chronic caloric excess and prevent adipocyte overloading. We report for the first time that a chronic high-fat diet (HFD) impairs adipogenic differentiation, leading to accumulation of inefficiently differentiated adipocytes with blunted expression of adipogenic differentiation-specific genes. Preadipocytes from these mice likewise exhibit impaired adipogenic differentiation, and this phenotype persists during in vitro cell culture. HFD-induced impaired adipogenic differentiation is associated with elevated expression of histone deacetylase 9 (HDAC9), an endogenous negative regulator of adipogenic differentiation. Genetic ablation of HDAC9 improves adipogenic differentiation and systemic metabolic state during an HFD, resulting in diminished weight gain, improved glucose tolerance and insulin sensitivity, and reduced hepatosteatosis. Moreover, compared with wild-type mice, HDAC9 knockout mice exhibit upregulated expression of beige adipocyte marker genes, particularly during an HFD, in association with increased energy expenditure and adaptive thermogenesis. These results suggest that targeting HDAC9 may be an effective strategy for combating obesity-related metabolic disease.

Transplanted Perivascular Adipose Tissue Accelerates Injury-Induced Neointimal Hyperplasia

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed &agr;-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.