Viswanathan Saraswathi

Defective Phagocytosis of Apoptotic Cells by Macrophages in Atherosclerotic Lesions of ob/ob Mice and Reversal by a Fish Oil Diet

Rationale: The complications of atherosclerosis are a major cause of death and disability in type 2 diabetes. Defective clearance of apoptotic cells by macrophages (efferocytosis) is thought to lead to increased necrotic core formation and inflammation in atherosclerotic lesions. Objective: To determine whether there is defective efferocytosis in a mouse model of obesity and atherosclerosis. Methods and Results: We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr−/− mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr−/− mice showed impaired efferocytosis, reflecting defective phosphatidylinositol 3-kinase activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr−/− macrophages showed an increased content of saturated fatty acids (FAs) and decreased &ohgr;-3 FAs (eicosapentaenoic acid and docosahexaenoic acid) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA/BSA complexes, whereas the defect in ob/ob macrophages was reversed by treatment with eicosapentaenoic acid/BSA or by feeding ob/ob mice a fish oil diet rich in &ohgr;-3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr−/− mice and this was reversed by a fish oil–rich diet. Conclusions: The findings suggest that in obesity and type 2 diabetes elevated levels of saturated FAs and/or decreased levels of &ohgr;-3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis and could be particularly important in type 2 diabetes and obesity.

PCB-induced oxidative stress in endothelial cells: modulation by nutrients

There is an increasing body of evidence suggesting that exposure to Superfund chemicals may have adverse consequences on many organ systems, as well as carcinogenic and atherogenic effects. This is particularly true for polyhalogenated aromatic hydrocarbons such as the polychlorinated biphenyls (PCBs). The vascular endothelium, which is constantly exposed to blood components including environmental contaminants, is extremely vulnerable to chemical insult as well as necrotic and apoptotic injury. Our recent studies suggest that certain PCBs, especially coplanar PCBs, can compromise normal functions of vascular endothelial cells by activating oxidative stress-sensitive signaling pathways and subsequent proinflammatory events critical in the pathology of atherosclerosis and cardiovascular disease. Our findings suggest that an increase in the level of cellular oxidative stress is a significant event in PCB-mediated endothelial cell dysfunction and that nutrients can modulate PCB-induced oxidative stress and endothelial toxicity. We have demonstrated that the dietary fat linoleic acid, the parent unsaturated fatty acid of the omega-6 family, can increase endothelial dysfunction induced by selected PCBs, probably by contributing to oxidative stress and as the result of the production of toxic metabolites called leukotoxins. The subsequent imbalance in the overall cellular oxidant/antioxidant status can activate oxidative stress- or redoxsensitive transcription factors, which in turn promote gene expression for inflammatory cytokines and adhesion molecules, intensifying the inflammatory response and endothelial cell dysfunction. Our data also suggest that antioxidant nutrients such as vitamin E can protect against endothelial cell damage mediated by PCBs or polyunsaturated dietary fats by interfering with oxidative stress-sensitive and proinflammatory signaling pathways. The concept that nutrition can modify or ameliorate the toxicity of Superfund chemicals is provocative and warrants further study as the implications for human health are significant. The information from such studies could be used to develop dietary recommendations and nutritional interventions for populations at high risk for exposure to PCBs, including communities living near Superfund sites and those exposed via occupation or diet.

The role of adipose tissue in mediating the beneficial effects of dietary fish oil

Fish oil improves several features of metabolic syndrome (MetS), such as dyslipidemia, insulin resistance and hepatic steatosis. Fish oil may mediate some of its beneficial effects by modulating the storage and/or secretory functions of adipose tissue (AT). The storage of triglycerides in AT is regulated by the availability of free fatty acids and the degree of lipolysis in AT. Fish oil has been shown to reduce lipolysis in several studies, indicating improved triglyceride storage. Importantly, AT secretes a variety of adipokines and fish oil feeding is associated with remarkable changes in the plasma levels of two key adipokines, adiponectin and leptin. Much attention has been focused on the contribution of adiponectin in fish oil-mediated improvements in MetS. However, emerging evidence also indicates a role of leptin in modulating the components of the MetS upon fish oil feeding. In addition to improving the storage and secretory functions of AT, fish oil, and the n-3 fatty acids found in fish oil, has been shown to reduce inflammation in AT. These effects may be in part a result of activation of peroxisome proliferator-activated receptor γ or inhibition of Toll-like receptor 4. Thus, there is compelling evidence that fish oil mediates its beneficial effects on MetS by improving AT storage and secretory functions and by reducing inflammation.

The role of lipolysis in mediating the proinflammatory effects of very low density lipoproteins in mouse peritoneal macrophages

Hypertriglyceridemia is an important risk factor for atherosclerosis, especially in obesity. Macrophages are one of the primary cell types involved in atherogenesis and are thought to contribute to lesion formation through both lipid accumulation and proinflammatory gene expression. In this study, we sought to determine the direct impact of triglyceride (TG)-rich VLDL-induced lipid accumulation on macrophage proinflammatory processes. Incubation of mouse peritoneal macrophages with 100 μg/ml VLDL for 6 h led to 2.8- and 3.7-fold increases in intracellular TGs and FFAs, respectively (P < 0.05). The inflammatory proteins tumor necrosis factor-α, interleukin-1β, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, matrix metalloproteinase 3 (MMP3), and macrophage inflammatory protein-1α (MIP-1α) were all upregulated by at least 2-fold (P < 0.05) in a dose-dependent manner in VLDL-treated macrophages. The increase in inflammatory gene expression coincided with the phosphorylation of the mitogen-activated protein kinase (MAPK) pathway members extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38 MAPK and was ameliorated by U0126, an inhibitor of ERK1/2. Inhibition of extracellular TG hydrolysis with tetrahydrolipstatin (Orlistat) resulted in the absence of intracellular TG and FFA accumulation and was accompanied by the amelioration of ERK1/2 phosphorylation and MIP-1α gene expression. These data indicate that VLDL hydrolysis, and the subsequent accumulation of intracellular FFAs and TGs, plays a substantive role in mediating the proinflammatory effects of VLDL. These data have important implications for the direct proatherogenic effects of VLDL on macrophage-driven atherosclerosis.

Brain insulin action augments hepatic glycogen synthesis without suppressing glucose production or gluconeogenesis in dogs

In rodents, acute brain insulin action reduces blood glucose levels by suppressing the expression of enzymes in the hepatic gluconeogenic pathway, thereby reducing gluconeogenesis and endogenous glucose production (EGP). Whether a similar mechanism is functional in large animals, including humans, is unknown. Here, we demonstrated that in canines, physiologic brain hyperinsulinemia brought about by infusion of insulin into the head arteries (during a pancreatic clamp to maintain basal hepatic insulin and glucagon levels) activated hypothalamic Akt, altered STAT3 signaling in the liver, and suppressed hepatic gluconeogenic gene expression without altering EGP or gluconeogenesis. Rather, brain hyperinsulinemia slowly caused a modest reduction in net hepatic glucose output (NHGO) that was attributable to increased net hepatic glucose uptake and glycogen synthesis. This was associated with decreased levels of glycogen synthase kinase 3β (GSK3β) protein and mRNA and with decreased glycogen synthase phosphorylation, changes that were blocked by hypothalamic PI3K inhibition. Therefore, we conclude that the canine brain senses physiologic elevations in plasma insulin, and that this in turn regulates genetic events in the liver. In the context of basal insulin and glucagon levels at the liver, this input augments hepatic glucose uptake and glycogen synthesis, reducing NHGO without altering EGP.

Linoleic acid-induced endothelial activation role of calcium and peroxynitrite signaling

Hypertriglyceridemia, an important risk factor of atherosclerosis, is associated with increased circulating free fatty acids. Research to date indicates that linoleic acid (LA), the major fatty acid in the American diet, may be atherogenic by activating vascular endothelial cells. However, the exact signaling mechanisms involved in LA-mediated proinflammatory events in endothelial cells still remain unclear. We previously reported increased superoxide formation after LA exposure in endothelial cells. The objective of the present investigation is to determine the role of calcium and peroxynitrite in mediating the proinflammatory effect of LA in vascular endothelial cells. LA exposure increased intracellular calcium, nitric oxide, and tetrahydrodiopterin levels as well as the expression of E-selectin. Inhibiting calcium signaling using 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid and heparin decreased the expression of E-selectin. Also, LA-mediated nuclear factor kappa B activation and E-selectin gene expression were suppressed by Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (a superoxide scavenger), NG-monomethyl-l-arginine (an endothelial nitric oxide synthase inhibitor), and 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) chloride (a peroxynitrite scavenger). LA exposure resulted in increased nitrotyrosine levels, as observed by Western blotting and immunofluorescence. Our data suggest that the proinflammatory effects of LA can be mediated through calcium and peroxynitrite signaling.

Dietary Fish Oil Exerts Hypolipidemic Effects in Lean and Insulin Sensitizing Effects in Obese LDLR−/− Mice

Obesity is often associated with dyslipidemia, insulin resistance, and hypertension. Together, these metabolic perturbations greatly increase the risk of developing cardiovascular disease and diabetes. Although fish oil is a well-established hypolipidemic agent, the mechanisms by which it mediates its lipid-lowering effects are not clear. In addition, it has not been established whether dietary fish oil has different effects in lean and obese mice. LDL receptor deficient (LDLR-/-) and leptin deficient mice on a LDLR-/- background (ob/ob;LDLR-/-) were fed a high fat diet (39% total fat) supplemented with 6% olive oil or fish oil for 6 wk. Fish oil supplementation resulted in lower concentrations of plasma total cholesterol (P < 0.01), triglycerides (P < 0.01), and free fatty acids (P < 0.001) in lean LDLR-/- mice, but not in ob/ob;LDLR-/- mice. In contrast, a fish oil diet did not modulate insulin sensitivity in lean LDLR-/- mice, but it improved insulin sensitivity in ob/ob;LDLR-/- mice (P < 0.05) compared with olive oil fed ob/ob;LDLR-/- mice. Interestingly, plasma adiponectin concentrations were significantly higher and hepatic steatosis was reduced in both mouse models upon fish oil feeding. Finally, fish oil fed LDLR-/- mice exhibited higher hepatic AMP activated protein kinase (AMPK) phosphorylation (P < 0.05), whereas AMPK phosphorylation was not elevated by fish oil feeding in ob/ob;LDLR-/- mice. Taken together, our data suggest that fish oil reduces hepatic steatosis in both lean and obese mice, has potent plasma lipid lowering effects in lean mice, and exerts insulin sensitizing effects in obese mice.

Inhibition of Long-Chain Acyl Coenzyme A Synthetases During Fatty Acid Loading Induces Lipotoxicity in Macrophages

Objectives—Obesity is often associated with hypertriglyceridemia and elevated free fatty acids (FFAs), which are independent risk factors for cardiovascular disease and diabetes. Although impairment of cholesterol homeostasis is known to induce toxicity in macrophages, the consequence of altered fatty acid homeostasis is not clear. Methods and Results—Long-chain acyl CoA synthetases (ACSLs) play a critical role in fatty acid homeostasis by channeling fatty acids to diverse metabolic pools. We treated mouse peritoneal macrophages (MPMs) with VLDL or FFAs in the presence of triacsin C, an inhibitor of the 3 ACSL isoforms present in macrophages. Treatment of macrophages with VLDL and triacsin C resulted in reduced TG accumulation but increased intracellular FFA levels, which induced lipotoxicity characterized by apoptosis. Treatment of MPMs with the saturated fatty acid stearic acid in the presence of triacsin C increased intracellular stearic acid and induced apoptosis. Stromal vascular cells collected from high-fat diet–fed mice displayed foam cell morphology and exhibited increased mRNA levels of macrophage markers and ACSL1. Importantly, all of these changes were associated with increased FFA level in AT. Conclusions—Inhibition of ACSLs during fatty acid loading results in apoptosis via accumulation of FFAs. Our data have implications in understanding the consequences of dysregulated fatty acid metabolism in macrophages.

Differential effects of eicosapentaenoic acid and docosahexaenoic acid in promoting the differentiation of 3T3-L1 preadipocytes

The objective of this study was to determine the effects of enrichment with n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the differentiation of 3T3-L1 preadipocytes. Enrichment with DHA but not EPA significantly increased the differentiation markers compared to control differentiated cells. DHA compared to EPA treatment led to a greater increase in adiponectin secretion and, conditioned media collected from DHA treated cells inhibited monocyte migration. Moreover, DHA treatment resulted in inhibition of pro-inflammatory signaling pathways. DHA treated cells predominantly accumulated DHA in phospholipids whereas EPA treatment led to accumulation of both EPA and its elongation product docosapentaenoic acid (DPA), an n-3 fatty acid. Of note, adding DPA to DHA inhibited DHA-induced differentiation. The differential effects of EPA and DHA on preadipocyte differentiation may be due, in part, to differences in their intracellular modification which could impact the type of n-3 fatty acids incorporated into the cells.

Impact of hematopoietic cyclooxygenase-1 deficiency on obesity-linked adipose tissue inflammation and metabolic disorders in mice

OBJECTIVE Adipose tissue (AT)-specific inflammation is considered to mediate the pathological consequences of obesity and macrophages are known to activate inflammatory pathways in obese AT. Because cyclooxygenases play a central role in regulating the inflammatory processes, we sought to determine the role of hematopoietic cyclooxygenase-1 (COX-1) in modulating AT inflammation in obesity. MATERIALS/METHODS Bone marrow transplantation was performed to delete COX-1 in hematopoietic cells. Briefly, female wild type (wt) mice were lethally irradiated and injected with bone marrow (BM) cells collected from wild type (COX-1+/+) or COX-1 knock-out (COX-1-/-) donor mice. The mice were fed a high fat diet for 16 weeks. RESULTS The mice that received COX-1-/- bone marrow (BM-COX-1-/-) exhibited a significant increase in fasting glucose, total cholesterol and triglycerides in the circulation compared to control (BM-COX-1+/+) mice. Markers of AT-inflammation were increased and were associated with increased leptin and decreased adiponectin in plasma. Hepatic inflammation was reduced with a concomitant reduction in TXB2 levels. The hepatic mRNA expression of genes involved in lipogenesis and lipid transport was increased while expression of genes involved in regulating hepatic glucose output was reduced in BM-COX-1-/- mice. Finally, renal inflammation and markers of renal glucose release were increased in BM-COX-1-/- mice. CONCLUSION Hematopoietic COX-1 deletion results in impairments in metabolic homeostasis which may be partly due to increased AT inflammation and dysregulated adipokine profile. An increase in renal glucose release and hepatic lipogenesis/lipid transport may also play a role, at least in part, in mediating hyperglycemia and dyslipidemia, respectively.