Lipid rafts, caveolae and GPI-linked proteins.

Lipid rafts and caveolae are specialized membrane microdomains enriched in sphingolipids and cholesterol. They function in a variety of cellular processes including but not limited to endocytosis, transcytosis, signal transduction and receptor recycling. Here, we outline the similarities and differences between lipid rafts and caveolae as well as discuss important components and functions of each.

Caveolae: a regulatory platform for nutritional modulation of inflammatory diseases.

Dietary intervention strategies have proven to be an effective means of decreasing several risk factors associated with the development of atherosclerosis. Endothelial cell dysfunction influences vascular inflammation and is involved in promoting the earliest stages of lesion formation. Caveolae are lipid raft microdomains abundant within the plasma membrane of endothelial cells and are responsible for modulating receptor-mediated signal transduction, thus influencing endothelial activation. Caveolae have been implicated in the regulation of enzymes associated with several key signaling pathways capable of determining intracellular redox status. Diet and plasma-derived nutrients may modulate an inflammatory outcome by interacting with and altering caveolae-associated cellular signaling. For example, omega-3 fatty acids and several polyphenolics have been shown to improve endothelial cell function by decreasing the formation of ROS and increasing NO bioavailability, events associated with altered caveolae composition. Thus, nutritional modulation of caveolae-mediated signaling events may provide an opportunity to ameliorate inflammatory signaling pathways capable of promoting the formation of vascular diseases, including atherosclerosis.

The G(0)/G(1) switch gene 2 regulates adipose lipolysis through association with adipose triglyceride lipase.

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for triacylglycerol (TAG) hydrolysis in adipocytes. The precise mechanisms whereby ATGL is regulated remain uncertain. Here, we demonstrate that a protein encoded by G(0)/G(1) switch gene 2 (G0S2) is a selective regulator of ATGL. G0S2 is highly expressed in adipose tissue and differentiated adipocytes. When overexpressed in HeLa cells, G0S2 localizes to lipid droplets and prevents their degradation mediated by ATGL. Moreover, G0S2 specifically interacts with ATGL through the hydrophobic domain of G0S2 and the patatin-like domain of ATGL. More importantly, interaction with G0S2 inhibits ATGL TAG hydrolase activity. Knockdown of endogenous G0S2 accelerates basal and stimulated lipolysis in adipocytes, whereas overexpression of G0S2 diminishes the rate of lipolysis in both adipocytes and adipose tissue explants. Thus, G0S2 functions to attenuate ATGL action both in vitro and in vivo and by this mechanism regulates TAG hydrolysis.

Glucagon-like peptide 1 and pancreatic polypeptide responses to feeding in normal weight and overweight children.

BACKGROUND: Glucagon-like peptide 1 (GLP-1) and pancreatic polypeptide (PP) are intestinal hormones that are involved in the post-prandial satiety response. We sought to assess meal-related changes in these hormones in young children and determine whether differences exist between normal weight (NW) and overweight (OW) children. METHODS: Seven to 11-year-old healthy NW (n=20) and OW (n=12) volunteers were given a standardized breakfast and lunch following an overnight fast and had measurements of GLP-1 and PP over 9 hours. We characterized whether GLP-1 and PP changed from the pre-prandial to the post-prandial state and whether the serum levels corresponded to reported appetite. RESULTS: GLP-1 did not increase after eating, did not decline prior to the next meal, and did not correspond to satiety ratings in either group. PP increased post-prandially in OW children after both breakfast and lunch, but in the NW group PP only increased after breakfast. PP levels did not decline in either group as the next meal approached. CONCLUSIONS: In our study of school-age children, feeding had little effect on GLP-1 secretion and a variable effect on serum PP levels. Observed differences in the GLP-1 and PP responses between the NW and OW groups do not suggest there is an intrinsic abnormality in their secretion that causes weight gain.

Thiol oxidative stress induced by metabolic disorders amplifies macrophage chemotactic responses and accelerates atherogenesis and kidney injury in LDL receptor-deficient mice.

BACKGROUND: Strengthening the macrophage glutathione redox buffer reduces macrophage content and decreases the severity of atherosclerotic lesions in LDL receptor-deficient (LDLR(-/-)) mice, but the underlying mechanisms were not clear. This study examined the effect of metabolic stress on the thiol redox state, chemotactic activity in vivo, and the recruitment of macrophages into atherosclerotic lesions and kidneys of LDL-R(-/-) mice in response to mild, moderate, and severe metabolic stress. METHODS AND RESULTS: Reduced glutathione (GSH) and glutathione disulfide (GSSG) levels in peritoneal macrophages isolated from mildly, moderately, and severe metabolically-stressed LDL-R(-/-) mice were measured by HPLC, and the glutathione reduction potential (E(h)) was calculated. Macrophage E(h) correlated with the macrophage content in both atherosclerotic (r(2)=0.346, P=0.004) and renal lesions (r(2)=0.480, P=0.001) in these mice as well as the extent of both atherosclerosis (r(2)=0.414, P=0.001) and kidney injury (r(2)=0.480, P=0.001). Compared to LDL-R(-/-) mice exposed to mild metabolic stress, macrophage recruitment into MCP-1-loaded Matrigel plugs injected into LDL-R(-/-) mice increased 2.6-fold in moderately metabolically-stressed mice and 9.8-fold in severely metabolically-stressed mice. The macrophage E(h) was a strong predictor of macrophage chemotaxis (r(2)=0.554, P<0.001). CONCLUSIONS: Thiol oxidative stress enhances macrophage recruitment into vascular and renal lesions by increasing the responsiveness of macrophages to chemoattractants. This novel mechanism contributes at least in part to accelerated atherosclerosis and kidney injury associated with dyslipidemia and diabetes in mice.

Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord.

Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild-type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane-anchored cytoskeleton.

Salt inactivates endothelial nitric oxide synthase in endothelial cells.

There is a 1-4 mmol/L rise in plasma sodium concentrations in individuals with high salt intake and in patients with essential hypertension. In this study, we used 3 independent assays to determine whether such a small increase in sodium concentrations per se alters endothelial nitric oxide synthase (eNOS) function and contributes to hypertension. By directly measuring NOS activity in living bovine aortic endothelial cells, we demonstrated that a 5-mmol/L increase in salt concentration (from 137 to 142 mmol/L) caused a 25% decrease in NOS activity. Importantly, the decrease in NOS activity was in a salt concentration-dependent manner. The NOS activity was decreased by 25, 45, and 70%, with the increase of 5, 10, and 20 mmol/L of NaCl, respectively. Using Chinese hamster ovary cells stably expressing eNOS, we confirmed the inhibitory effects of salt on eNOS activity. The eNOS activity was unaffected in the presence of equal milliosmol of mannitol, which excludes an osmotic effect. Using an ex vivo aortic angiogenesis assay, we demonstrated that salt attenuated the nitric oxide (NO)-dependent proliferation of endothelial cells. By directly monitoring blood pressure changes in response to salt infusion, we found that in vivo infusion of salt induced an acute increase in blood pressure in a salt concentration-dependent manner. In conclusion, our findings demonstrated that eNOS is sensitive to changes in salt concentration. A 5-mmol/L rise in salt concentration, within the range observed in essential hypertension patients or in individuals with high salt intake, could significantly suppress eNOS activity. This salt-induced reduction in NO generation in endothelial cells may contribute to the development of hypertension.

Acid Sphingomyelinase Deficiency Prevents Diet-induced Hepatic Triacylglycerol Accumulation and Hyperglycemia in Mice.

Acid sphingomyelinase plays important roles in ceramide homeostasis, which has been proposed to be linked to insulin resistance. To test this association in vivo, acid sphingomyelinase deletion (asm(-/-)) was transferred to mice lacking the low density lipoprotein receptor (ldlr(-/-)), and then offsprings were placed on control or modified (enriched in saturated fat and cholesterol) diets for 10 weeks. The modified diet caused hypercholesterolemia in all genotypes; however, in contrast to asm(+/+)/ldlr(-/-), the acid sphingomyelinase-deficient littermates did not display hepatic triacylglyceride accumulation, although sphingomyelin and other sphingolipids were substantially elevated, and the liver was enlarged. asm(-/-)/ldlr(-/-) mice on a modified diet did not accumulate body fat and were protected against diet-induced hyperglycemia and insulin resistance. Experiments with hepatocytes revealed that acid sphingomyelinase regulates the partitioning of the major fatty acid in the modified diet, palmitate, into two competitive and inversely related pools, triacylglycerides and sphingolipids, apparently via modulation of serine palmitoyltransferase, a rate-limiting enzyme in de novo sphingolipid synthesis. These studies provide evidence that acid sphingomyelinase activity plays an essential role in the regulation of glucose metabolism by regulating the hepatic accumulation of triacylglycerides and sphingolipids during consumption of a diet rich in saturated fats.

Role of caveolin-1 in EGCG-mediated protection against linoleic-acid-induced endothelial cell activation.

Flavonoids can protect against inflammatory diseases such as atherosclerosis by decreasing vascular endothelial cell activation. Plasma microdomains called caveolae may be critical in regulating endothelial activation. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that flavonoids can down-regulate endothelial inflammatory parameters by modulating caveolae-regulated cell signaling. We focused on the role of caveolae and its major protein, caveolin-1, in mechanisms of linoleic-acid-induced endothelial cell activation and protection by the catechin epigallocatechin-3-gallate (EGCG). Exposure to linoleic acid for 6 h induced expression of both caveolin-1 and cyclooxygenase (COX)-2. Pretreatment with EGCG blocked fatty-acid-induced caveolin-1 and COX-2 expression in a time- and concentration-dependent manner. Similar results were observed with nuclear factor-kappa B DNA binding activity, which was also reduced by caveolin-1 silencing. Exposure to linoleic acid rapidly increased phosphorylation of several kinases, including p38 MAPK, extracellular signal regulated kinase 1/2 (ERK1/2) and amino kinase terminal (Akt), with maximal induction at about 10 min. Inhibitors of ERK1/2 and Akt down-regulated the linoleic-acid-induced increase in COX-2 protein, which also occurred after pretreatment with EGCG. Caveolin-1 silencing blocked linoleic-acid-induced phosphorylation of ERK1/2 and protein expression of COX-2, suggesting that specific MAPK signaling is caveolae dependent. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and protection by flavonoids such as EGCG.

Caveolin-1 regulates human immunodeficiency virus-1 Tat-induced alterations of tight junction protein expression via modulation of the Ras signaling.

The blood-brain barrier (BBB) is the critical structure for preventing human immunodeficiency virus (HIV) trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1-infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMECs). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMECs. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions on Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients.

Morphine causes rapid increases in glial activation and neuronal injury in the striatum of inducible HIV-1 Tat transgenic mice.

HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV-related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3-nitrotyrosine (3-NT) positive macrophages/microglia, was determined following a 2-day exposure to morphine (5 mg/kg/day via time-release, subcutaneous implant) and doxycycline in GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure. By contrast, combined Tat induction and morphine exposure, but neither manipulation alone, significantly increased the proportion of macrophages/microglia present in the striatum of transgenic mice, although morphine exposure was necessary to elevate 3-NT co-detection in Mac1-positive macrophages/microglia. Finally, Tat induction increased the percentage of neurons expressing active caspase-3, and this was even more significantly elevated by co-administration of morphine. In spite of elevations in caspase-3, neuronal TUNEL reactivity was unchanged in all groups, even after 10 days of Tat induction. Importantly, co-administration of naltrexone completely antagonized the effects of morphine. These findings indicate that morphine rapidly and significantly increases the activation of astrocytes and macrophages/microglia in the brains of inducible Tat transgenic mice, supporting the theory that early inflammatory changes in glia could underlie the development of HIVE in opiate-abusing AIDS patients.

Caveolae structure and function.

Studies on the structure and function of caveolae have revealed how this versatile subcellular organelle can influence numerous signalling pathways. This brief review will discuss a few of the key features of caveolae as it relates to signalling and disease processes.

Influence of salt on subcellular localization of nitric oxide synthase activity and expression in the renal inner medulla.

1. The aims of this study were: (i) to characterize the subcellular localization of nitric oxide synthase (NOS) 1 and NOS3 activity and expression within the cytosolic, plasma membrane and intracellular membrane subcellular fractions of the renal inner medulla of rats; and (ii) to determine whether NOS1 and NOS3 activity and expression in subcellular fractions of the renal inner medulla are regulated by dietary salt intake. Although the NOS system is important in maintaining Na(+) and water homeostasis, the identity of the NOS isoform that is sensitive to dietary Na(+) remains unclear. In addition, subcellular localization of both NOS1 and NOS3 has been shown to regulate enzymatic activity and influence the ability of NOS to produce nitric oxide (NO). 2. Renal inner medullae were dissected from male Sprague-Dawley rats and separated into cytosolic, plasma membrane and intracellular membrane fractions for measurement of NOS activity and western blot analysis. 3. On a normal-salt diet, NOS activity and NOS1 and NOS3 protein expression were present in all three subcellular fractions, although total NOS activity was enriched in the intracellular membrane fraction. In response to a high-salt diet, urinary nitrate/nitrite (NO(x)) increased. Despite an increase in NO(x) excretion, total NOS activity in the renal inner medullary homogenate was decreased. There were no detectable differences in NOS activity in the subcellular fractions. Expression of NOS1 protein was decreased in the cytoplasmic and plasma membrane fractions, although maintained in the intracellular membrane fraction, in response to high salt. Expression of NOS3 protein was unaffected by high salt. 4. In conclusion, we hypothesize that NOS1 localization in the intracellular membrane is important in increasing NO production to aid Na(+) and water homeostasis.

Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin.

In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

Gender as a regulator of atherosclerosis in murine models.

The risk of development and progression of atherosclerosis is different between males and females. Premenopausal women have a lower risk of developing atherosclerosis and cardiovascular disease than men. However, after the onset of menopause the protection associated with gender is lost and the risk of women developing atherosclerosis gradually approaches that of men. In an effort to treat the elevated risk of cardiovascular disease in postmenopausal women, hormone replacement therapy has been used. However, the results of the randomized trials of the Women's Health Initiative indicated that hormone replacement therapy may not be cardioprotective. The use of mouse models have aided in the understanding of atherosclerosis for many years. These models along with the gender effects attributed to sex hormones are being used to generate a more complete understanding of the development of atherosclerosis. Mice lacking one or both of the genes for estrogen receptors have highlighted the role of estrogen in atherosclerosis. In addition to estrogen, the effects of testosterone have been researched in many animal models and several mechanisms incorporating its role in cholesterol homeostasis have emerged. Our understanding of the pathways involved in gender effects on cardiovascular disease is incomplete, however, a plethora of animal models offer the opportunity to dissect the molecular mechanisms involved.

The role of caveolin-1 in PCB77-induced eNOS phosphorylation in human-derived endothelial cells.

Polychlorinated biphenyls (PCBs) may contribute to the pathology of atherosclerosis by activating inflammatory responses in vascular endothelial cells. Endothelial nitric oxide synthase (eNOS) is colocalized with caveolae and is a critical regulator of vascular homeostasis. PCBs may be proatherogenic by causing dysfunctional eNOS signaling. The objective of this study was to investigate the role of caveolin-1 in PCB-induced endothelial dysfunction with a focus on mechanisms associated with eNOS signaling. Cells derived from an immortalized human vascular endothelial cell line were treated with PCB77 to study nitrotyrosine formation through eNOS signaling. Phosphorylation studies of eNOS, caveolin-1, and kinases, such as Src, phosphatidylinositol 3-kinase (PI3K), and Akt, were conducted in cells containing either functional or small-interfering RNA-silenced caveolin-1 protein. We also investigated caveolin-1-regulated mechanisms associated with PCB-induced markers of peroxynitrite formation and DNA binding of NF-kappaB. Cellular exposure to PCB77 increased eNOS phosphorylation and nitric oxide production, as well as peroxynitrite levels. A subsequent PCB-induced increase in NF-kappaB DNA binding may have implications in oxidative stress-mediated inflammatory mechanisms. The activation of eNOS by PCB77 treatment was blocked by inhibitors of the Src/PI3K/Akt pathway. PCB77 also increased phosphorylation of caveolin-1, indicating caveolae-dependent endocytosis. Caveolin-1 silencing abolished both the PCB-stimulated Akt and eNOS phosphorylation, suggesting a regulatory role of caveolae in PCB-induced eNOS signaling. These findings suggest that PCB77 induces eNOS phosphorylation in endothelial cells through a Src/PI3K/Akt-dependent mechanism, events regulated by functional caveolin-1. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs.

Crystallographic studies of human MitoNEET.

MitoNEET was identified as an outer mitochondrial membrane protein that can potentially bind the anti-diabetes drug pioglitazone. The crystal structure of the cytoplasmic mitoNEET (residues 33-108) is determined in this study. The structure presents a novel protein fold and contains a [2Fe-2S] cluster-binding domain. The [2Fe-2S] cluster is coordinated to the protein by Cys-72, Cys-74, Cys-83, and His-87 residues. This coordination is also novel compared with the traditional [2Fe-2S] cluster coordinated by four cysteines or two cysteines and two histidines. The cytoplasmic mitoNEET forms homodimers in solution and in crystal. The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface. His-87 residue, which plays an important role in the coordination of the [2Fe-2S] cluster, is exposed to the solvent on the dimer surface. It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the [2Fe-2S] cluster.

How HIV protease inhibitors promote atherosclerotic lesion formation.

PURPOSE OF REVIEW: One of the aims of this review is to summarize recent clinical approaches used to determine the role of HIV protease inhibitors in the development of cardiovascular disease. Another aim is to discuss possible molecular mechanisms whereby HIV protease inhibitors may promote atherogenesis. RECENT FINDINGS: Several clinical studies have recently used ultrasonography to demonstrate increased intimal medial thickness and alterations in the structural characteristics of epi-aortic lesions in patients receiving HIV protease inhibitors. Molecular studies have indicated that several mechanisms are likely involved in mediating the effects of protease inhibitors. Possible mechanisms include inhibition of the proteasome, increased CD36 expression in macrophage, inhibition of lipoprotein lipase-mediated lipolysis, decreased adiponectin levels, and dysregulation of the NF-kappaB pathway. SUMMARY: The currently available data strongly suggest that HIV protease inhibitors negatively impact the cardiovascular system. As is often the case with complex diseases like atherosclerosis it appears that HIV protease inhibitors affect the cardiovascular system through several distinct mechanisms by affecting various components of the arterial wall directly or indirectly by influencing lipoprotein and glucose metabolism of the body.

Voluntary wheel running ameliorates vascular smooth muscle hyper-contractility in type 2 diabetic db/db mice.

Physical activity reduces cardiovascular disease related mortality in diabetic patients. However, it is unknown if the diabetic state reduces voluntary physical activity and, if so, if the voluntary physical activity at the reduced level is sufficient to improve cardiovascular risk factors. To address these two specific questions, we investigated voluntary wheel running performance in an obese and type 2 diabetic mouse model, the db/db mice. In addition, we determined the effects of running on body mass, blood glucose, insulin, plasma free fatty acids, cholesterol, and vascular smooth muscle hyper-contractility. Our results showed that daily running distance, time, and speed were significantly reduced in the db/db mice to about 23%, 32%, and 71%, respectively, of that in non-diabetic control mice. However, this low level of running was sufficient to induce a reduction in the vascular smooth muscle hyper-contractility, cholesterol, and some plasma free fatty acids, as well as to delay the decrease in blood insulin. These changes occurred in the absence of weight loss and a detectable decrease in blood glucose. Thus, the results of this study demonstrated that voluntary wheel running activity was dramatically reduced in db/db mice. However, the low levels of running were beneficial, in the absence of effects on obesity or blood glucose, with significant reductions in cardiovascular risk factors and potential delays in beta-cell dysfunction.

Increased expression of glutathione reductase in macrophages decreases atherosclerotic lesion formation in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: Thiol oxidative stress leads to macrophage dysfunction and cell injury, and has been implicated in the development of atherosclerotic lesions. We investigated if strengthening the glutathione-dependent antioxidant system in macrophages by overexpressing glutathione reductase (GR) decreases the severity of atherosclerosis. METHODS AND RESULTS: Bone marrow cells infected with retroviral vectors expressing either enhanced green fluorescent protein (EGFP) or an EGFP-fusion protein of cytosolic GR (GR(cyto)-EGFP) or mitochondrial GR (GR(mito)-EGFP) were transplanted into low-density lipoprotein receptor-deficient mice. Five weeks after bone marrow transplantation, animals were challenged with a Western diet for 10 weeks. No differences in either plasma cholesterol and triglyceride levels or peritoneal macrophage content were observed. However, mice reconstituted with either GR(cyto)-EGFP or GR(mito)-EGFP-expressing bone marrow had lesion areas (P<0.009) that were 32% smaller than recipients of EGFP-expressing bone marrow. In cultured macrophages, adenovirus-mediated overexpression of GR(cyto)-EGFP or GR(mito)-EGFP protected cells from mitochondrial hyperpolarization induced by oxidized low-density lipoprotein. CONCLUSION: This study provides direct evidence that the glutathione-dependent antioxidant system in macrophages plays a critical role in atherogenesis, and suggests that thiol oxidative stress-induced mitochondrial dysfunction contributes to macrophage injury in atherosclerotic lesions.