SUMMARY Diabetes is a major risk factor for atherosclerosis. Although atherosclerosis is initiated by deposition of cholesterol-rich lipoproteins in the artery wall, the entry of inflammatory leukocytes into lesions fuels disease progression and impairs resolution. We show that diabetic mice have increased numbers of circulating neutrophils and Ly6-Chi monocytes, reflecting hyperglycemia-induced proliferation and expansion of bone marrow myeloid progenitors and release of monocytes into the circulation. Increased neutrophil production of S100A8/A9, via an interaction with the receptor for advanced glycation end products on common myeloid progenitor cells, leads to enhanced myelopoiesis. Treatment of hyperglycemia reduces monocytosis, entry of monocytes into atherosclerotic lesions and promotes regression. In patients with type I diabetes plasma S100A8/A9 levels correlate with leukocyte counts and coronary artery disease. Thus, hyperglycemia drives myelopoiesis and thus promotes atherogenesis in diabetes.

Three forms of PPARs are expressed in the heart. In animal models, PPARγ agonist treatment improves lipotoxic cardiomyopathy; however, PPARγ agonist treatment of humans is associated with peripheral edema and increased heart failure. To directly assess effects of increased PPARγ on heart function, we created transgenic mice expressing PPARγ1 in the heart via the cardiac α-myosin heavy chain (α-MHC) promoter. PPARγ1-transgenic mice had increased cardiac expression of fatty acid oxidation genes and increased lipoprotein triglyceride (TG) uptake. Unlike in cardiac PPARα-transgenic mice, heart glucose transporter 4 (GLUT4) mRNA expression and glucose uptake were not decreased. PPARγ1-transgenic mice developed a dilated cardiomyopathy associated with increased lipid and glycogen stores, distorted architecture of the mitochondrial inner matrix, and disrupted cristae. Thus, while PPARγ agonists appear to have multiple beneficial effects, their direct actions on the myocardium have the potential to lead to deterioration in heart function.

OBJECTIVEPatients with diabetes have increased cardiovascular risk. Atherosclerosis in these patients is often associated with increased plaque macrophages and dyslipidemia. We hypothesized that diabetic atherosclerosis involves processes that impair favorable effects of lipid reduction on plaque macrophages.RESEARCH DESIGN AND METHODSReversa mice are LDL receptor–deficient mice that develop atherosclerosis. Their elevated plasma LDL levels are lowered after conditional knockout of the gene encoding microsomal triglyceride transfer protein. We examined the morphologic and molecular changes in atherosclerotic plaques in control and streptozotocin-induced diabetic Reversa mice after LDL lowering. Bone marrow–derived macrophages were also used to study changes mediated by hyperglycemia.RESULTSReversa mice were fed a western diet for 16 weeks to develop plaques (baseline). Four weeks after lipid normalization, control (nondiabetic) mice had reduced plasma cholesterol (−77%), plaque cholesterol (−53%), and plaque cells positive for macrophage marker CD68+ (−73%), but increased plaque collagen (+116%) compared with baseline mice. Diabetic mice had similarly reduced plasma cholesterol, but collagen content increased by only 34% compared with baseline; compared with control mice, there were lower reductions in plaque cholesterol (−30%) and CD68+ cells (−41%). Diabetic (vs. control) plaque CD68+ cells also exhibited more oxidant stress and inflammatory gene expression and less polarization toward the anti-inflammatory M2 macrophage state. Many of the findings in vivo were recapitulated by hyperglycemia in mouse bone marrow–derived macrophages.CONCLUSIONSDiabetes hindered plaque regression in atherosclerotic mice (based on CD68+ plaque content) and favorable changes in plaque macrophage characteristics after the reduction of elevated plasma LDL.

Levels of apolipoprotein B, the protein component of low-density lipoproteins, correlate with the risk of coronary heart disease. We examined whether genetic variation in apolipoprotein B is associated with myocardial infarction by studying apolipoprotein B-gene restriction-fragment-length polymorphisms in 84 patients with myocardial infarction and an equal number of matched controls. Southern blot analysis with apolipoprotein B-gene probes, performed after DNA was digested with the endonucleases XbaI and EcoRI, revealed alleles that we designated as X1, X2, and X3 and as R1 and R2, respectively. Similar studies with the endonuclease MspI revealed alleles of many different sizes (the difference was due to an insertion-deletion polymorphism), which we grouped as larger and smaller alleles and designated as ID1 and ID2, respectively. The frequencies of the X1, R1, and ID1 alleles were all significantly higher (P less than 0.01) in the cases than in the controls. None of the alleles, however, was significantly associated with variation in levels of low-density lipoprotein cholesterol or apolipoprotein B, and the functional importance of these alleles is therefore uncertain. Nonetheless, in addition to quantitative variation in apolipoprotein B levels in plasma, genetic variation at the apolipoprotein B locus may be a new and independent risk factor for myocardial infarction.

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Insulin-resistant apoB/BATless mice have hypertriglyceridemia because of increased assembly and secretion of very low density apolipoprotein B (apoB) and triglycerides compared with mice expressing only apoB (Siri, P., Candela, N., Ko, C., Zhang, Y., Eusufzai, S., Ginsberg, H. N., and Huang, L. S. (2001) J. Biol. Chem. 276, 46064 -46072). Despite increased very low density lipoprotein secretion, apoB/BATless mice have fatty livers. We found that hepatic mRNA levels of key lipogenic enzymes, acetyl-CoA carboxylase, fatty-acid synthase, and stearoyl-CoA desaturase-1 were increased in apoB/BATless mice compared with levels in apoB mice, suggesting increased lipogenesis in apoB/BATless mice. This was confirmed by determining incorporation of tritiated water into fatty acids. Neither the hepatic mRNA of the lipogenic transcription factor, SREBP-1c (sterol-response element-binding protein 1c), nor the nuclear levels of the mature form of SREBP-1 protein were elevated in apoB/BATless mice. By contrast, hepatic levels of peroxisomal proliferator-activated receptor 2 (PPAR␥2) mRNA and protein were specifically increased in apoB/BATless mice, as were hepatic mRNA levels of two targets of PPAR␥, CD36 and aP2. Treatment of apoB/BATless mice for 4 weeks with intraperitoneal injections of a PPAR␥ antisense oligonucleotide resulted in dramatic reductions of both PPAR␥1 and PPAR␥2 mRNA, PPAR␥2 protein, and mRNA levels of fatty-acid synthase and acetyl-CoA carboxylase. These changes were associated with decreased hepatic de novo lipogenesis and hepatic triglyceride concentrations. We conclude that hepatic steatosis in apoB/ BATless mice is associated with elevated rates of hepatic lipogenesis that are linked directly to increased hepatic expression of PPAR␥2. The mechanism whereby hepatic Ppar␥2 gene expression is increased and how PPAR␥2 stimulates lipogenesis is under investigation.We reported previously that apoB/BATless mice, a model generated by crossing mice expressing human apolipoprotein B (apoB) 3 (1) with mice lacking brown adipose tissue (BATless) (2), have hypertriglyceridemia and hypercholesterolemia because of a 2-3-fold increase in the secretion of very low density lipoprotein (VLDL) apoB and triglycerides (TG) relative to mice expressing apoB only (3). Similar levels of apoB mRNA in the livers of apoB and apoB/BATless mice indicated that the differences in apoB secretion resulted from differences in posttranscriptional regulation of VLDL assembly and secretion (4). There were no differences in hepatic levels of microsomal triglyceride transfer protein mRNA (3), a critical factor in the early co-and post-translational regulation of apoB-containing lipoprotein secretion (5). Low density lipoprotein receptor mRNA levels in liver were higher in apoB/BATless compared with apoB mice (3), indicating that increased apoB secretion in apoB/BATless mice did not result from reduced interactions of the low density lipoprotein receptor with nascent apoB lipoproteins (6).Associated with a rising prevalence of obesity and insuli...

Microsomal triglyceride (TG) transfer protein (MTP) is an endoplasmic reticulum lumenal protein consisting of a 97-kDa subunit and protein disulfide isomerase. It is believed that MTP delivers TG to nascent apoB molecules during the assembly of lipoprotein particles in the secretory pathway. Although in vitro studies have established the mechanism of TG transfer between donor and acceptor membranes, the mechanism of action of MTP in vivo remains unknown. The present studies were undertaken to examine whether or not the transfer of TG to nascent apoB in the endoplasmic reticulum involves the physical interaction between MTP and apoB. HepG2 cells were labeled with [3H]leucine, lysed in a nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP antiserum. We found that labeled apoB and protein disulfide isomerase were co-immunoprecipitated by this procedure. In addition, we were able to detect the 97-kDa subunit of MTP in these immunoprecipitates by immunoblot. The association of MTP and apoB, as assessed in pulse-labeled cells by co-immunoprecipitation, was transient; apoB was prominent on fluorgraphy at 10 min of chase but minimal thereafter. Oleic acid treatment, which protects apoB from rapid intracellular degradation by increasing TG availability, increased both the degree and the duration of association between MTP and apoB dramatically. Inhibition of TG synthesis by Triacsin D, on the other hand, significantly decreased the MTP-apoB binding. N-Acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor, which directly protects apoB from rapid intracellular degradation but does not affect TG synthesis, increased the interaction between MTP and apoB only slightly, although it did prolong it. Our results suggest that direct interaction between MTP and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells.

Insulin resistance is a common metabolic abnormality that is associated with an increased risk of both atherosclerosis and type 2 diabetes. The phenotype of insulin resistance includes a dyslipidemia characterized by an elevation of very low-density lipoprotein triglyceride, a reduction in high-density lipoprotein cholesterol, and the presence of small, triglyceride-enriched low-density lipoproteins. The underlying metabolic abnormality driving this dylipidemia is an increased assembly and secretion of very low-density lipoprotein particles, leading to an increased plasma level of triglyceride. Hypertriglyceridemia, in turn, results in a reduction in the high-density lipoprotein level and the generation of small, dense low-density lipoproteins; these events are mediated by cholesteryl ester transfer protein. In addition, hypertension, obesity, and a prothrombotic state are also integral components of the insulin resistance syndrome. In this review, we will provide a pathophysiologic basis, based on studies on humans and in tissue culture, for the dyslipidemia of insulin resistance. We will also review the effects of insulin resistance on the coagulation and fibrinolytic pathways. It is hoped that this review will allow health professionals better to evaluate and treat their patients with insulin resistance, thereby reducing the very much increased risk of atherosclerotic cardiovascular disease carried by these individuals.