Jenny E. Kanter

Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1

The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.

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.

Do Glucose and Lipids Exert Independent Effects on Atherosclerotic Lesion Initiation or Progression to Advanced Plaques

It is becoming increasingly clear that suboptimal blood glucose control results in adverse effects on large blood vessels, thereby accelerating atherosclerosis and cardiovascular disease, manifested as myocardial infarction, stroke, and peripheral vascular disease. Cardiovascular disease is accelerated by both type 1 and type 2 diabetes. In type 1 diabetes, hyperglycemia generally occurs in the absence of elevated blood lipid levels, whereas type 2 diabetes is frequently associated with dyslipidemia. In this review article, we discuss hyperglycemia versus hyperlipidemia as culprits in diabetes-accelerated atherosclerosis and cardiovascular disease, with emphasis on studies in mouse models and isolated vascular cells. Recent studies on LDL receptor–deficient mice that are hyperglycemic, but exhibit no marked dyslipidemia compared with nondiabetic controls, show that diabetes in the absence of diabetes-induced hyperlipidemia is associated with an accelerated formation of atherosclerotic lesions, similar to what is seen in fat-fed nondiabetic mice. These effects of diabetes are masked in severely dyslipidemic mice, suggesting that the effects of glucose and lipids on lesion initiation might be mediated by similar mechanisms. Recent evidence from isolated endothelial cells demonstrates that glucose and lipids can induce endothelial dysfunction through similar intracellular mechanisms. Analogous effects of glucose and lipids are also seen in macrophages. Furthermore, glucose exerts many of its cellular effects through lipid mediators. We propose that diabetes without associated dyslipidemia accelerates atherosclerosis by mechanisms that can also be activated by hyperlipidemia.

Type 1 diabetes promotes disruption of advanced atherosclerotic lesions in LDL receptor-deficient mice

Cardiovascular disease, largely because of disruption of atherosclerotic lesions, accounts for the majority of deaths in people with type 1 diabetes. Recent mouse models have provided insights into the accelerated atherosclerotic lesion initiation in diabetes, but it is unknown whether diabetes directly worsens more clinically relevant advanced lesions. We therefore used an LDL receptor-deficient mouse model, in which type 1 diabetes can be induced at will, to investigate the effects of diabetes on preexisting lesions. Advanced lesions were induced by feeding mice a high-fat diet for 16 weeks before induction of diabetes. Diabetes, independently of lesion size, increased intraplaque hemorrhage and plaque disruption in the brachiocephalic artery of mice fed low-fat or high-fat diets for an additional 14 weeks. Hyperglycemia was not sufficient to induce plaque disruption. Furthermore, diabetes resulted in increased accumulation of monocytic cells positive for S100A9, a proinflammatory biomarker for cardiovascular events, and for a macrophage marker protein, without increasing lesion macrophage content. S100A9 immunoreactivity correlated with intraplaque hemorrhage. Aggressive lowering primarily of triglyceride-rich lipoproteins prevented both plaque disruption and the increased S100A9 in diabetic atherosclerotic lesions. Conversely, oleate promoted macrophage differentiation into an S100A9-positive population in vitro, thereby mimicking the effects of diabetes. Thus, diabetes increases plaque disruption, independently of effects on plaque initiation, through a mechanism that requires triglyceride-rich lipoproteins and is associated with an increased accumulation of S100A9-positive monocytic cells. These findings indicate an important link between diabetes, plaque disruption, and the innate immune system.

Diabetes reduces the cholesterol exporter ABCA1 in mouse macrophages and kidneys

Accumulation of cholesterol in arterial macrophages may contribute to diabetes-accelerated atherosclerotic cardiovascular disease. The ATP-binding cassette transporter ABCA1 is a cardioprotective membrane protein that mediates cholesterol export from macrophages. Factors elevated in diabetes, such as reactive carbonyls and free fatty acids, destabilize ABCA1 protein in cultured macrophages, raising the possibility that impaired ABCA1 plays an atherogenic role in diabetes. We therefore examined the modulation of ABCA1 in two mouse models of diabetes. We isolated peritoneal macrophages, livers, kidneys, and brains from type 1 non-obese diabetic (NOD) mice and mice made diabetic by viral-induced autoimmune destruction of pancreatic β-cells, and we measured ABCA1 protein and mRNA levels and cholesterol contents. ABCA1 protein levels and cholesterol export activity were reduced by 40–44% (P < 0.01) in peritoneal macrophages and protein levels by 48% (P < 0.001) in kidneys in diabetic NOD mice compared with nondiabetic animals, even though ABCA1 mRNA levels were not significantly different. A similar selective reduction in ABCA1 protein was found in peritoneal macrophages (33%, P < 0.05) and kidneys (35%, P < 0.05) from the viral-induced diabetic mice. In liver and brain, however, diabetes had no effect or slightly increased ABCA1 protein and mRNA levels. The reduced ABCA1 in macrophages and kidneys was associated with increased cholesterol content. Impaired ABCA1-mediated cholesterol export could therefore contribute to the increased atherosclerosis and nephropathy associated with diabetes.

Rosiglitazone Inhibits Acyl-CoA Synthetase Activity and Fatty Acid Partitioning to Diacylglycerol and Triacylglycerol via a Peroxisome Proliferator–Activated Receptor-γ–Independent Mechanism in Human Arterial Smooth Muscle Cells and Macrophages

Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator–activated receptor (PPAR)-γ, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acyl-CoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-γ–independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-γ–independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis.

Inflammation and diabetes-accelerated atherosclerosis: myeloid cell mediators.

Monocytes and macrophages respond to and govern inflammation by producing a plethora of inflammatory modulators, including cytokines, chemokines, and arachidonic acid (C20:4)-derived lipid mediators. One of the most prevalent inflammatory diseases is cardiovascular disease, caused by atherosclerosis, and accelerated by diabetes. Recent research has demonstrated that monocytes/macrophages from diabetic mice and humans with type 1 diabetes show upregulation of the enzyme, acyl-CoA synthetase 1 (ACSL1), which promotes C20:4 metabolism, and that ACSL1 inhibition selectively protects these cells from the inflammatory and proatherosclerotic effects of diabetes, in mice. Increased understanding of the role of ACSL1 and other culprits in monocytes/macrophages in inflammation and diabetes-accelerated atherosclerosis offers hope for new treatment strategies to combat diabetic vascular disease.

Testing the Role of Myeloid Cell Glucose Flux in Inflammation and Atherosclerosis

Inflammatory activation of myeloid cells is accompanied by increased glycolysis, which is required for the surge in cytokine production. Although in vitro studies suggest that increased macrophage glucose metabolism is sufficient for cytokine induction, the proinflammatory effects of increased myeloid cell glucose flux in vivo and the impact on atherosclerosis, a major complication of diabetes, are unknown. We therefore tested the hypothesis that increased glucose uptake in myeloid cells stimulates cytokine production and atherosclerosis. Overexpression of the glucose transporter GLUT1 in myeloid cells caused increased glycolysis and flux through the pentose phosphate pathway but did not induce cytokines. Moreover, myeloid-cell-specific overexpression of GLUT1 in LDL receptor-deficient mice was ineffective in promoting atherosclerosis. Thus, increased glucose flux is insufficient for inflammatory myeloid cell activation and atherogenesis. If glucose promotes atherosclerosis by increasing cellular glucose flux, myeloid cells do not appear to be the key targets.

VASP Increases Hepatic Fatty Acid Oxidation by Activating AMPK in Mice

Activation of AMP-activated protein kinase (AMPK) signaling reduces hepatic steatosis and hepatic insulin resistance; however, its regulatory mechanisms are not fully understood. In this study, we sought to determine whether vasodilator-stimulated phosphoprotein (VASP) signaling improves lipid metabolism in the liver and, if so, whether VASP’s effects are mediated by AMPK. We show that disruption of VASP results in significant hepatic steatosis as a result of significant impairment of fatty acid oxidation, VLDL-triglyceride (TG) secretion, and AMPK signaling. Overexpression of VASP in hepatocytes increased AMPK phosphorylation and fatty acid oxidation and reduced hepatocyte TG accumulation; however, these responses were suppressed in the presence of an AMPK inhibitor. Restoration of AMPK phosphorylation by administration of 5-aminoimidazole-4-carboxamide riboside in Vasp−/− mice reduced hepatic steatosis and normalized fatty acid oxidation and VLDL-TG secretion. Activation of VASP by the phosphodiesterase-5 inhibitor, sildenafil, in db/db mice reduced hepatic steatosis and increased phosphorylated (p-)AMPK and p-acetyl CoA carboxylase. In Vasp−/− mice, however, sildendafil treatment did not increase p-AMPK or reduce hepatic TG content. These studies identify a role of VASP to enhance hepatic fatty acid oxidation by activating AMPK and to promote VLDL-TG secretion from the liver.

Acyl-CoA synthetase 1 is required for oleate and linoleate mediated inhibition of cholesterol efflux through ATP-binding cassette transporter A1 in macrophages.

Diabetes and insulin resistance increase the risk of cardiovascular disease caused by atherosclerosis through mechanisms that are poorly understood. Lipid-loaded macrophages are key contributors to all stages of atherosclerosis. We have recently shown that diabetes associated with increased plasma lipids reduces cholesterol efflux and levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in mouse macrophages, which likely contributes to macrophage lipid accumulation in diabetes. Furthermore, we and others have shown that unsaturated fatty acids reduce ABCA1-mediated cholesterol efflux, and that this effect is mediated by the acyl-CoA derivatives of the fatty acids. We therefore investigated whether acyl-CoA synthetase 1 (ACSL1), a key enzyme mediating acyl-CoA synthesis in macrophages, could directly influence ABCA1 levels and cholesterol efflux in these cells. Mouse macrophages deficient in ACSL1 exhibited reduced sensitivity to oleate- and linoleate-mediated ABCA1 degradation, which resulted in increased ABCA1 levels and increased apolipoprotein A-I-dependent cholesterol efflux in the presence of these fatty acids, as compared with wildtype mouse macrophages. Conversely, overexpression of ACSL1 resulted in reduced ABCA1 levels and reduced cholesterol efflux in the presence of unsaturated fatty acids. Thus, the reduced ABCA1 and cholesterol efflux in macrophages subjected to conditions of diabetes and elevated fatty load may, at least in part, be mediated by ACSL1. These observations raise the possibility that ABCA1 levels could be increased by inhibition of acyl-CoA synthetase activity in vivo. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).