Improved efficacy for ezetimibe and rosuvastatin by attenuating the induction of PCSK9.

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.

Biochemical characterization of cholesteryl ester transfer protein inhibitors.

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.

Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions.

BACKGROUND: Two macrophage ABC transporters, ABCA1 and ABCG1, have a major role in promoting cholesterol efflux from macrophages. Peritoneal macrophages deficient in ABCA1, ABCG1, or both show enhanced expression of inflammatory and chemokine genes. This study was undertaken to elucidate the mechanisms and consequences of enhanced inflammatory gene expression in ABC transporter-deficient macrophages. METHODS AND RESULTS: Basal and lipopolysaccharide-stimulated thioglycollate-elicited peritoneal macrophages showed increased inflammatory gene expression in the order Abca1(-/-)Abcg1(-/-)>Abcg1(-/-)>Abca1(-/-)>wild-type. The increased inflammatory gene expression was abolished in macrophages deficient in Toll-like receptor 4 (TLR4) or MyD88/TRIF. TLR4 cell surface concentration was increased in Abca1(-/-)Abcg1(-/-)>Abcg1(-/-)> Abca1(-/-)> wild-type macrophages. Treatment of transporter-deficient cells with cyclodextrin reduced and cholesterol-cyclodextrin loading increased inflammatory gene expression. Abca1(-/-)Abcg1(-) bone marrow-derived macrophages showed enhanced inflammatory gene responses to TLR2, TLR3, and TLR4 ligands. To assess in vivo relevance, we injected intraperitoneally thioglycollate in Abcg1(-/-) bone marrow-transplanted, Western diet-fed, Ldlr-deficient mice. This resulted in a profound inflammatory infiltrate in the adventitia and necrotic core region of atherosclerotic lesions, consisting primarily of neutrophils. CONCLUSIONS: The results suggest that high-density lipoprotein and apolipoprotein A-1 exert anti-inflammatory effects by promoting cholesterol efflux via ABCG1 and ABCA1 with consequent attenuation of signaling via Toll-like receptors. In response to a peripheral inflammatory stimulus, atherosclerotic lesions containing Abcg1(-/-) macrophages experience an inflammatory "echo," suggesting a possible mechanism of plaque destabilization in subjects with low high-density lipoprotein levels.

Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice.

HDLs protect against the development of atherosclerosis, but the underlying mechanisms are poorly understood. HDL and its apolipoproteins can promote cholesterol efflux from macrophage foam cells via the ATP-binding cassette transporters ABCA1 and ABCG1. Experiments addressing the individual roles of ABCA1 and ABCG1 in the development of atherosclerosis have produced mixed results, perhaps because of compensatory upregulation in the individual KO models. To clarify the role of transporter-mediated sterol efflux in this disease process, we transplanted BM from Abca1(-/-)Abcg1(-/-) mice into LDL receptor-deficient mice and administered a high-cholesterol diet. Compared with control and single-KO BM recipients, Abca1(-/-)Abcg1(-/-) BM recipients showed accelerated atherosclerosis and extensive infiltration of the myocardium and spleen with macrophage foam cells. In experiments with isolated macrophages, combined ABCA1 and ABCG1 deficiency resulted in impaired cholesterol efflux to HDL or apoA-1, profoundly decreased apoE secretion, and increased secretion of inflammatory cytokines and chemokines. In addition, these cells showed increased apoptosis when challenged with free cholesterol or oxidized LDL loading. These results suggest that the combined effects of ABCA1 and ABCG1 in mediating macrophage sterol efflux are central to the antiatherogenic properties of HDL.

Hepatic response to restoration of GLUT4 in skeletal muscle of GLUT4 null mice.

Expression of GLUT4 in fast-twitch skeletal muscle fibers of GLUT4 null mice (G4-MO) normalized glucose uptake in muscle and restored peripheral insulin sensitivity. GLUT4 null mice exhibit altered carbohydrate and lipid metabolism in liver and skeletal muscle. To test the hypothesis that increased glucose utilization by G4-MO muscle would normalize the changes seen in the GLUT4 null liver, serum metabolites and hepatic metabolism were compared in control, GLUT4 null, and G4-MO mice. The fed serum glucose and triglyceride levels of G4-MO mice were similar to those of control mice. In addition, the alternations in liver metabolism seen in GLUT4 nulls including increased GLUT2 expression and fatty acid synthesis accompanied by an increase in the oxidative arm of the pentose phosphate pathway were absent in G4-MO mice. The transgene used for GLUT4 restoration in muscle was specific for fast-twitch muscle fibers. The mitochondria hypertrophy/hyperplasia in all GLUT4 null skeletal muscles was absent in transgene-positive extensor digitorum longus muscle but present in transgene-negative soleus muscle of G4-MO mice. Results of this study suggest that the level of muscle GLUT4 expression influences mitochondrial biogenesis. These studies also demonstrate that the type and amount of substrate that muscle takes up and metabolizes, determined in part by GLUT4 expression levels, play a major role in directing hepatic carbohydrate and lipid metabolism.

Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo.

Macrophage ATP-binding cassette transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI), and ABCG1 have been shown to promote cholesterol efflux to extracellular acceptors in vitro and influence atherosclerosis in mice, but their roles in mediating reverse cholesterol transport (RCT) from macrophages in vivo are unknown. Using an assay of macrophage RCT in mice, we found that primary macrophages lacking ABCA1 had a significant reduction in macrophage RCT in vivo, demonstrating the importance of ABCA1 in promoting macrophage RCT, however substantial residual RCT exists in the absence of macrophage ABCA1. Using primary macrophages deficient in SR-BI expression, we found that macrophage SR-BI, which was shown to promote cholesterol efflux in vitro, does not contribute to macrophage RCT in vivo. To investigate whether macrophage ABCG1 is involved in macrophage RCT in vivo, we used ABCG1-overexpressing, -knockdown, and -knockout macrophages. We show that increased macrophage ABCG1 expression significantly promoted while knockdown or knockout of macrophage ABCG1 expression significantly reduced macrophage RCT in vivo. Finally, we show that there was a greater decrease in macrophage RCT from cells where both ABCA1 and ABCG1 expression were knocked down than from ABCG1-knockdown cells. These results demonstrate that ABCA1 and ABCG1, but not SR-BI, promote macrophage RCT in vivo and are additive in their effects.

Decreased atherosclerosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1-/- bone marrow.

OBJECTIVE: Recent studies indicate that the ATP-binding cassette transporter ABCG1 can promote cholesterol efflux from macrophages to high-density lipoprotein. This study was designed to assess the in vivo role of macrophage ABCG1 in atherosclerosis. METHODS AND RESULTS: Bone marrow from Abcg1-/- mice was transplanted into irradiated Ldlr-/- recipients, and atherosclerosis was evaluated by aortic root assay after 7 or 11 weeks of feeding on a Western diet. After 7 weeks, there was no difference in lesion area in mice receiving either wild-type or Abcg1-/- bone marrow, whereas after 11 weeks, lesion area was moderately but significantly reduced in Abcg1-/- recipients. ABCG1-deficient peritoneal macrophages showed induction of several liver X receptor target genes, such as Abca1 and Srebp1c, and a dramatic increase in apolipoprotein E (apoE) protein both in cell media and lysates, without parallel change in apoE mRNA. Abca1 knockdown prevented the increase in apoE secretion but had minimal effects on apoE accumulation in cell lysates of Abcg1-/- macrophages. Plasma apoE levels were markedly increased in recipients of Abcg1-/- bone marrow. CONCLUSIONS: These studies reveal an inverse relationship between Abcg1 expression and apoE accumulation and secretion in macrophages. The reduced atherosclerosis in recipients of Abcg1-deficient bone marrow may be explained by induction of Abca1 and an associated increase in macrophage apoE secretion.

Macrophage insulin receptor deficiency increases ER stress-induced apoptosis and necrotic core formation in advanced atherosclerotic lesions.

Insulin resistance in diabetes and metabolic syndrome is thought to increase susceptibility to atherosclerotic cardiovascular disease, but the underlying mechanisms are poorly understood. To evaluate the possibility that decreased insulin signaling in macrophage foam cells might worsen atherosclerosis, Ldlr(-/-) mice were transplanted with insulin receptor Insr(+/+) or Insr(-/-) bone marrow. Western diet-fed Insr(-/-) recipients developed larger, more complex lesions with increased necrotic cores and increased numbers of apoptotic cells. Insr(-/-) macrophages showed diminished Akt phosphorylation and an augmented ER stress response, leading to induction of scavenger receptor A and increased apoptosis when challenged with cholesterol loading or nutrient deprivation. These studies suggest that defective insulin signaling and reduced Akt activity impair the ability of macrophages to deal with ER stress-induced apoptosis within atherosclerotic plaques.

LXR-induced redistribution of ABCG1 to plasma membrane in macrophages enhances cholesterol mass efflux to HDL.

OBJECTIVE: This study examines the ABCG1-mediated cholesterol efflux and intracellular cholesterol transport by studying the ABCG1 localization and function in macrophages. METHODS AND RESULTS: HEK 293 cell overexpressing ABCG1, RNA interference, or macrophages from ABCG1 or ABCG4 knockout mice were used. ABCG1 but not ABCG4 had a major role in the increased cholesterol mass efflux produced by treatment of macrophages with LXR activators. In 293 cells, ABCG1 was found in the plasma membrane, Golgi, and recycling endosomes. In contrast, in basal macrophages, ABCG1 was predominantly intracellular, and redistributed to the plasma membrane after LXR activation. LXR activation increased macrophage cholesterol efflux to high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cyclodextrin in an ABCG1-dependent fashion. Suppression of ABCG1 expression increased cholesteryl ester formation and decreased SREBP2 target gene expression in macrophages, even in the absence of HDL acceptors. CONCLUSIONS: LXR activation induces redistribution of ABCG1 from intracellular sites to the plasma membrane and increases cholesterol mass efflux to HDL in an ABCG1-dependent fashion. ABCG1 acts in the macrophage plasma membrane to increase the availability of cholesterol to a variety of lipoprotein and nonlipoprotein acceptors while limiting the accumulation of cholesterol in the endoplasmic reticulum.

Use of GLUT-4 null mice to study skeletal muscle glucose uptake.

1. The present review focuses on the effects of varying levels of GLUT-4, the insulin-sensitive glucose transporter, on insulin sensitivity and whole body glucose homeostasis. 2. Three mouse models are discussed including myosin light chain (MLC)-GLUT-4 mice which overexpress GLUT-4 specifically in skeletal muscle, GLUT-4 null mice which express no GLUT-4 and the MLC-GLUT-4 null mice which express GLUT-4 only in skeletal muscle. Overexpressing GLUT-4 specifically in the skeletal muscle results in increased insulin sensitivity in the MLC-GLUT-4 mice. In contrast, the GLUT-4 null mice exhibit insulin intolerance accompanied by abnormalities in glucose and lipid metabolism. Restoring GLUT-4 expression in skeletal muscle in the MLC-GLUT-4 null mice results in normal glucose metabolism but continued abnormal lipid metabolism. 3. The results of experiments using these mouse models demonstrates that modifying the expression of GLUT-4 profoundly affects whole body insulin action and consequently glucose and lipid metabolism.

Localization of the GLUT8 glucose transporter in murine kidney and regulation in vivo in nondiabetic and diabetic conditions.

Kidney disease is a major complication of diabetes, and poor glycemic control is associated with the development of diabetic nephropathy. The precise mechanisms that lead to diabetic kidney disease still remain largely unknown and are under current investigation. Because glucose transporters in the kidney play an important role in the local maintenance of intracellular glucose and plasma glucose homeostasis, the tissue distribution and regulation of glucose transporter GLUT8, a new member of the glucose transporter family with important functions in cellular survival, were examined. To understand the normal regulation of GLUT8 expression in response to metabolic signals, fasting and feeding conditions were studied. Additionally, GLUT8 expression was studied using two different models of insulin resistance, GLUT4-/- and db/db mice. GLUT8 was localized to glomerular podocytes and tubular epithelial cells in the distal portion of the nephron. Expression of GLUT8 in the kidney was influenced by plasma glucose levels in vivo. Podocytes in kidneys of diabetic db/db mice express higher levels of GLUT8 compared with nondiabetic db/m mice. Because podocytes play an important role in glomerulosclerosis development and high levels of glucose have been shown to induce apoptotic cell death in various kidney cells, these data may provide further insight into the pathogenesis of glomerulosclerosis and diabetic nephropathy.

Altered hepatic and muscle substrate utilization provoked by GLUT4 ablation.

Studies were conducted to explore altered substrate utilization and metabolism in GLUT4 null mice. Liver fatty acid synthase mRNA and fatty acid synthesis rates were dramatically increased in GLUT4 null mice compared with control mice and were supported by increased rates of the pentose phosphate pathway oxidative phase and sterol regulatory binding protein mRNA expression. Increased GLUT2 protein content, glucokinase mRNA, and glucose-6-phosphate in GLUT4 null mice may provide substrate for the enhanced fatty acid synthesis. Increased fatty acid synthesis, however, did not lead to hepatic triglyceride accumulation in GLUT4 null mice because of increased hepatic triglyceride secretion rates. GLUT4 null mice rapidly cleared orally administered olive oil, had reduced serum triglyceride concentrations in the fed and the fasted state, and increased skeletal muscle lipoprotein lipase when compared with controls. Oleate oxidation rates were increased in GLUT4 null skeletal muscle in association with mitochondrial hyperplasia/hypertrophy. This study demonstrated that GLUT4 null mice had increased hepatic glucose uptake and conversion into triglyceride for subsequent use by muscle. The ability of GLUT4 null mice to alter hepatic carbohydrate and lipid metabolism to provide proper nutrients for peripheral tissues may explain (in part) their ability to resist diabetes when fed a normal diet.

Regulation of hepatic GLUT8 expression in normal and diabetic models.

GLUT8 is a novel glucose transporter protein that is widely distributed in tissues including liver, a central organ of regulation of glucose homeostasis. The purpose of the current study was to investigate expression and regulation of hepatic GLUT8 mRNA and protein. Therefore, Northern and immunoblot analysis, semiquantitative RT-PCR, and immunofluorescence microscopy were performed using mouse livers at different stages of embryonic and postnatal development and in type 1 (streprozotocin treated) and type 2 (GLUT4 heterozygous) diabetes. GLUT8 mRNA and protein expression in embryonic liver was differentially regulated depending on the prenatal and postnatal developmental stage of the mice. Immunofluorescence microscopy of liver from wild-type mice demonstrated the highest levels of GLUT8 protein in perivenous hepatocytes pointing to its role in regulation of glycolytic flux. In diabetic scenarios, GLUT8 mRNA levels were correlated with circulating insulin; specifically, GLUT8 mRNA decreased in a type 1 diabetes model and increased in a type 2 diabetes model, suggesting a regulatory role for insulin in GLUT8 mRNA expression. While up-regulation of GLUT8 protein occurred in both models of diabetes, only in streptozotocin diabetic livers was GLUT8 zonation altered. These data demonstrate that GLUT8 mRNA and protein are differentially regulated in liver in response to physiologic and pathologic (diabetes) milieu and suggests that GLUT8 is intimately linked to glucose homeostasis.

Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene.

Fetuin inhibits insulin-induced insulin receptor (IR) autophosphorylation and tyrosine kinase activity in vitro, in intact cells, and in vivo. The fetuin gene (AHSG) is located on human chromosome 3q27, recently identified as a susceptibility locus for type 2 diabetes and the metabolic syndrome. Here, we explore insulin signaling, glucose homeostasis, and the effect of a high-fat diet on weight gain, body fat composition, and glucose disposal in mice carrying two null alleles for the gene encoding fetuin, Ahsg (B6, 129-Ahsg(tm1Mbl)). Fetuin knockout (KO) mice demonstrate increased basal and insulin-stimulated phosphorylation of IR and the downstream signaling molecules mitogen-activated protein kinase (MAPK) and Akt in liver and skeletal muscle. Glucose and insulin tolerance tests in fetuin KO mice indicate significantly enhanced glucose clearance and insulin sensitivity. Fetuin KO mice subjected to euglycemic-hyperinsulinemic clamp show augmented sensitivity to insulin, evidenced by increased glucose infusion rate (P = 0.077) and significantly increased skeletal muscle glycogen content (P < 0.05). When fed a high-fat diet, fetuin KO mice are resistant to weight gain, demonstrate significantly decreased body fat, and remain insulin sensitive. These data suggest that fetuin may play a significant role in regulating postprandial glucose disposal, insulin sensitivity, weight gain, and fat accumulation and may be a novel therapeutic target in the treatment of type 2 diabetes, obesity, and other insulin-resistant conditions.

Cell type-specific expression and coregulation of murine resistin and resistin-like molecule-alpha in adipose tissue.

Adipocytes are the exclusive or predominant source of several secreted proteins that exert profound effects on systemic carbohydrate and lipid metabolism. Resistin, a 10-kDa adipose tissue specific secretory protein, has recently been implicated in exerting a negative effect on systemic insulin sensitivity. It is, however, not known how resistin mediates this insulin-desensitizing effect or what regulatory mechanisms control resistin expression. Resistin-like molecule-alpha (RELMalpha), a homolog of resistin originally identified by its upregulation in asthmatic lung, is another secreted protein expressed in adipose tissue. The regulation of RELMalpha in adipose tissue and its relationship to resistin expression has not been addressed so far. Here, we demonstrate that the expression of resistin and RELMalpha are similarly regulated in adipose tissue despite the fact that RELMalpha is exclusively expressed in the stromal vascular fraction of adipose tissue and not in adipocytes. Interestingly, this coregulation is limited to adipose tissue as the expression of RELMalpha in lung is independent of metabolic regulation. Additionally, we show that resistin and RELMalpha levels are not subject to regulation by proinflammatory stimuli. Finally, acute hyperglycemia leads to up-regulation of resistin and RELMalpha transcription in various adipose depots.