Mohammad I. Khan

Valproate Attenuates Accelerated Atherosclerosis in Hyperglycemic ApoE-Deficient Mice: Evidence in Support of a Role for Endoplasmic Reticulum Stress and Glycogen Synthase Kinase-3 in Lesion Development and Hepatic Steatosis

We have previously shown that glucosamine promotes endoplasmic reticulum (ER) stress in vascular cells leading to both inflammation and lipid accumulation--the hallmark features of atherosclerosis. Pretreatment with glycogen synthase kinase (GSK)-3 inhibitors protects cultured cells from ER stress-induced dysfunction. Here we evaluate the potential role of GSK-3 on the pro-atherogenic effects of hyperglycemia and ER stress. We show that GSK-3-deficient mouse embryonic fibroblasts do not accumulate unesterified cholesterol under conditions of ER stress. Furthermore, GSK-3 inhibitors, including valproate, attenuate ER stress-induced unesterified cholesterol accumulation in wild-type mouse embryonic fibroblasts. In vivo we show that hyperglycemic apoE-deficient mice have accelerated atherogenesis at the aortic root compared with normoglycemic control mice. Mice fed a diet supplemented with 625 mg/kg valproate have significantly reduced lesion volume relative to nonsupplemented controls. Valproate supplementation has no apparent effect on the plasma levels of either glucose or lipids or on the expression of diagnostic markers of ER stress in the lesion. Significant reductions were observed in total hepatic lipids (>50.4%) and hepatic GSK-3beta activity (>55.8%) in mice fed the valproate diet. In conclusion, dietary supplementation with low levels of valproate significantly attenuates atherogenesis in hyperglycemic apoE-deficient mice. The in vivo anti-atherogenic effects of valproate are consistent with its ability to inhibit GSK-3 and interfere with pro-atherogenic ER stress signaling pathways in vitro.

Hexosamine biosynthesis pathway flux promotes endoplasmic reticulum stress, lipid accumulation, and inflammatory gene expression in hepatic cells

There is increasing evidence that endoplasmic reticulum (ER) stress contributes to the development of atherosclerosis in diabetes mellitus. The purpose of this study was to determine the effects of increased hexosamine biosynthesis pathway (HBP) flux on ER stress levels and the complications of ER stress associated with diabetes and atherosclerosis in hepatic cells. Glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of the HBP, was overexpressed in HepG2 cells by use of an adenoviral expression system. The ER stress response and downstream effects, including activation of lipid and inflammatory pathways, were determined using real-time PCR, immunoblot analysis, and cell staining techniques. GFAT overexpression resulted in increased expression of ER stress markers, including Grp78, Grp94, calreticulin, and GADD153, relative to cells infected with an empty adenoviral vector. In addition, GFAT overexpression promoted lipid, but not cholesterol, accumulation in hepatic cells as well as inflammatory pathway activation. Treatment with 6-diazo-5-oxo-norleucine, a GFAT antagonist, blocked the effects of GFAT overexpression. Consistent with our in vitro data, hyperglycemic mice presented with elevated markers of hepatic ER stress, glucosamine and lipid accumulation. Together, these data suggest that HBP flux-induced ER stress plays a role in the development of hepatic steatosis and atherosclerosis under conditions of hyperglycemia.

Effect of Leptin on Vascular Calcification in Apolipoprotein E–Deficient Mice

Objective—The adipocytokine leptin has been proposed to increase cardiovascular risk in both obese and diabetic individuals. In the current study, therefore, we used apoE-deficient mice to examine the effects of leptin on both lesion size and calcification. Methods and Results—Mice were treated with once daily intraperitoneal injections of leptin (125 &mgr;g/mouse/d) for 2 months. The mice were then euthanized, and sections of the aortic root and thoracic aorta analyzed histomorphometrically. Measurements of lesion size and surface area occupied by atherosclerotic lesions did not reveal any differences between nontreated and leptin-treated animals. However, von Kossa staining of the aortic root demonstrated an 8.3±2.0-fold increase in lesion calcification as well as a 2.5±0.6-fold increase in valvular calcification in those animals treated with leptin. In addition, the percent total lesion area demonstrating ALP-positive staining was 5.4±2.1-fold greater in leptin-treated mice when compared to nontreated control mice. This increase in ALP staining was also accompanied by an increase in the expression of the osteoblast-specific markers, osteocalcin, and osteopontin. Conclusions—Based on these observations, we conclude that leptin may increase cardiovascular risk by promoting osteogenic differentiation and thus vascular calcification.

Endoplasmic Reticulum Stress and Glycogen Synthase Kinase-3β Activation in Apolipoprotein E–Deficient Mouse Models of Accelerated Atherosclerosis

Objective— The goal of this study was to examine the role of endoplasmic reticulum (ER) stress signaling and the contribution of glycogen synthase kinase (GSK)-3&bgr; activation in hyperglycemic, hyperhomocysteinemic, and high-fat–fed apolipoprotein E–deficient (apoE−/−) mouse models of accelerated atherosclerosis. Methods and Results— Female apoE−/− mice received multiple low-dose injections of streptozotocin (40 &mgr;g/kg) to induce hyperglycemia, methionine-supplemented drinking water (0.5% wt/vol) to induce hyperhomocysteinemia, or a high-fat (21% milk fat+0.2% cholesterol) diet to induce relative dyslipidemia. A subset of mice from each group was supplemented with sodium valproate (625 mg/kg), a compound with GSK3 inhibitory activity. At 15 and 24 weeks of age, markers of ER stress, lipid accumulation, GSK3&bgr; phosphorylation, and GSK3&bgr; activity were analyzed in liver and aorta. Atherosclerotic lesions were examined and quantified. Hyperglycemia, hyperhomocysteinemia, and high-fat diet significantly enhanced GSK3&bgr; activity and also increased hepatic steatosis and atherosclerotic lesion volume compared with controls. Valproate supplementation blocked GSK3&bgr; activation and attenuated the development of atherosclerosis and the accumulation of hepatic lipids in each of the models examined. The mechanism by which GSK3&bgr; activity is regulated in these models likely involves alterations in phosphorylation at serine 9 and tyrosine 216. Conclusion— These findings support the existence of a common mechanism of accelerated atherosclerosis involving ER stress signaling through activation of GSK3&bgr;. Furthermore, our results suggest that atherosclerosis can be attenuated by modulating GSK3&bgr; phosphorylation.

Glucosamine-supplementation promotes endoplasmic reticulum stress, hepatic steatosis and accelerated atherogenesis in apoE−/− mice

OBJECTIVES To determine the effects of glucosamine-supplementation on endoplasmic reticulum (ER) stress levels and atherogenesis, and to investigate the potential role of glucosamine in hyperglycemia-associated accelerated atherosclerosis. METHODS Five week old apolipoprotein E-deficient (apoE-/-) mice were provided with normal drinking water or water supplemented with 5% glucosamine (w/v) or 5% mannitol (w/v). To induce hyperglycemia, a separate group of apoE-/- mice received multiple low dose injections of streptozotocin (STZ). All mice were provided with a standard chow diet and were euthanized at 15 weeks of age. Hepatic and vascular ER stress levels and atherosclerotic lesion area at the aortic root were determined. RESULTS STZ-induced hyperglycemic and glucosamine-supplemented mice had significantly larger and more advanced atherosclerotic lesions than control mice. Indications of ER stress were increased in the livers and atherosclerotic lesions of hyperglycemic and glucosamine-supplemented mice but not in the controls. In glucosamine-supplemented mice accelerated atherosclerosis was independent of detectable changes in blood glucose concentration, glucose tolerance, plasma insulin, or plasma lipid levels. CONCLUSION Similar to hyperglycemia, glucosamine-supplementation promotes ER stress, hepatic steatosis and accelerated atherosclerosis. These findings support a model by which hyperglycemia promotes hepatic and vascular complications via a glucosamine intermediate.

Hyperglycaemia is associated with impaired vasa vasorum neovascularization and accelerated atherosclerosis in apolipoprotein-E deficient mice

OBJECTIVE A direct correlation between blood glucose levels and the microvascular complications of diabetes is well established. However, the effects of hyperglycaemia on the vasa vasorum, a microvascular network which surrounds and supplies the walls of large arteries, is not known. The objective of this study is to investigate the effects of hyperglycaemia on the vasa vasorum and to examine correlations between these effects and the development of atherosclerosis in a mouse model. METHODS The micro- and macrovascular effects of hyperglycaemia were examined in streptozotocin-injected apolipoprotein-E deficient (ApoE(-/-)) mice. Retina and aortic sinus were isolated from hyperglycaemic mice and normoglycaemic controls at 5-20 weeks of age. Retinal and vasa vasorum microvessel densities were quantified and correlated to atherosclerotic lesion development. The expression levels of pro-angiogenic factors including vascular endothelial growth factor (VEGF) and VEGF receptor 2 were examined. RESULTS In normoglycaemic ApoE(-/-) mice atherogenesis is associated with vasa vasorum expansion, which likely corresponds to the increasing blood supply demands of the thickening artery wall. In hyperglycaemic ApoE(-/-) mice there is no significant neovascularization of the vasa vasorum, despite the fact that lesions are significantly larger. This defect may result from a localized deficiency in VEGF. CONCLUSIONS These findings are the first evidence that hyperglycaemia alters the structure of the vasa vasorum. Such microvascular changes directly correlate, and may contribute to, the development and progression of atherosclerosis in hyperglycaemic ApoE-deficient mice.

Endurance interval training in obese mice reduces muscle inflammation and macrophage content independently of weight loss

Obesity is associated with chronic low‐grade inflammation that involves infiltration of macrophages into metabolic organs such as skeletal muscle. Exercise enhances skeletal muscle insulin sensitivity independently of weight loss; but its role in regulating muscle inflammation is not fully understood. We hypothesized that exercise training would inhibit skeletal muscle inflammation and alter macrophage infiltration into muscle independently of weight loss. Wild type C57BL/6 male mice were fed a chow diet or a high‐fat diet (HFD, 45% calories fat) for 6 weeks. Then, mice maintained on the HFD either remained sedentary (HFD Sed) or exercised (HFD Ex) on a treadmill for another 6 weeks. The exercise training protocol involved conducting intervals of 2 min in duration followed by 2 min of rest for 60 min thrice weekly. Chow‐fed control mice remained sedentary for the entire 12 weeks. Muscle cytokine and macrophage gene expression analysis were conducted using qRT‐PCR, and muscle macrophage content was also measured using immunohistochemistry. Muscle cytokine protein content was quantified using a cytokine array. The HFD increased adiposity and weight gain compared to chow‐fed controls. HFD Sed and HFD Ex mice had similar body mass as well as total and visceral adiposity. However, despite similar adiposity, exercise reduced inflammation and muscle macrophage infiltration. We conclude that Endurance exercise training modulates the immune‐metabolic crosstalk in obesity independently of weight loss, and may have potential benefits in reducing obesity‐related muscle inflammation.

Enteric glia promote functional recovery of CTM reflex after dorsal root transection.

&NA; Transected dorsal root axons of adult rats canbe induced to regenerate through the normally non‐permissive environment of the dorsal root entry zone (DREZ) into the spinal cord by implanting enteric glia (EG) into the DREZ. We have now examined whether the regenerating central axons make functional connections by studying the return of function of a behavioral response, the cutaneous trunci muscle (CTM) reflex. Implantation of EG into the spinal cord DREZ led to functional recovery of the CTM reflex in 82%, 72% and 70% of animals1, 2 and 3 months, respectively, after injury. In contrast, the CTMreflex did not recover in animals implanted with 3T3 or C6 glioma cells or with vehicle only.

Sex-Specific Differences in an ApoE(-/-):Ins2(+/Akita) Mouse Model of Accelerated Atherosclerosis.

Diabetic patients have a twofold to fourfold increased risk of cardiovascular disease. Despite a vast amount of research, the underlying mechanisms that predispose individuals with diabetes to the development of cardiovascular disease are unclear. To further our understanding of how diabetes promotes atherosclerosis, we have established, characterized, and manipulated a new model of hyperglycemia-induced atherosclerosis: the apolipoprotein E-deficient (ApoE(-/-)):Ins2(+/Akita) mouse. All mice were fed a standard chow diet. Male ApoE(-/-):Ins2(+/Akita) mice developed chronic hyperglycemia, which significantly accelerated atherosclerosis. Female ApoE(-/-):Ins2(+/Akita) mice presented hyperglycemia that normalized by 15 weeks of age. Despite the transient hyperglycemia, advanced atherosclerosis was observed at 15 weeks of age compared with ApoE(-/-) females. To better understand these differences, subsets of mice were castrated or ovariectomized at 5 weeks of age. Castrated ApoE(-/-):Ins2(+/Akita) mice showed a reduction in blood glucose levels that correlated with the amelioration of atherosclerosis. Interestingly, castrated normoglycemic ApoE(-/-) mice developed larger atherosclerotic lesions than sham-operated on controls. Ovariectomized ApoE(-/-):Ins2(+/Akita) mice presented chronic hyperglycemia, and atherosclerosis appeared to be advanced. We have characterized the distinctive sex-specific phenotypes exhibited by the ApoE(-/-):Ins2(+/Akita) mouse model and present evidence for the action of sex hormones on pancreatic β-cell function and the vasculature that affect the regulation of blood glucose levels and the development of atherosclerosis. This model will provide a test bed to further delineate these effects.

485 Endoplasmic Reticulum stress and Glycogen Synthase Kinase-3 beta activation in apolipoprotein e-deficient mouse models of accelerated atherosclerosis

Objective—The goal of this study was to examine the role of endoplasmic reticulum (ER) stress signaling and the contribution of glycogen synthase kinase (GSK)-3 activation in hyperglycemic, hyperhomocysteinemic, and high-fat–fed apolipoprotein E–deficient (apoE / ) mouse models of accelerated atherosclerosis. Methods and Results—Female apoE / mice received multiple low-dose injections of streptozotocin (40 g/kg) to induce hyperglycemia, methionine-supplemented drinking water (0.5% wt/vol) to induce hyperhomocysteinemia, or a high-fat (21% milk fat 0.2% cholesterol) diet to induce relative dyslipidemia. A subset of mice from each group was supplemented with sodium valproate (625 mg/kg), a compound with GSK3 inhibitory activity. At 15 and 24 weeks of age, markers of ER stress, lipid accumulation, GSK3 phosphorylation, and GSK3 activity were analyzed in liver and aorta. Atherosclerotic lesions were examined and quantified. Hyperglycemia, hyperhomocysteinemia, and high-fat diet significantly enhanced GSK3 activity and also increased hepatic steatosis and atherosclerotic lesion volume compared with controls. Valproate supplementation blocked GSK3 activation and attenuated the development of atherosclerosis and the accumulation of hepatic lipids in each of the models examined. The mechanism by which GSK3 activity is regulated in these models likely involves alterations in phosphorylation at serine 9 and tyrosine 216. Conclusion—These findings support the existence of a common mechanism of accelerated atherosclerosis involving ER stress signaling through activation of GSK3 . Furthermore, our results suggest that atherosclerosis can be attenuated by modulating GSK3 phosphorylation. (Arterioscler Thromb Vasc Biol. 2012;32:82-91.)