Understanding One Half of the Sex Difference Equation: The Modulatory Effects of Testosterone on Diabetic Cardiomyopathy.

Diabetes is a prevalent disease, primarily characterized by high blood sugar (hyperglycemia). Significantly higher rates of myocardial dysfunction have been noted in individuals with diabetes, even in those without coronary artery disease or high blood pressure (hypertension). Numerous molecular mechanisms have been identified through which diabetes contributes to the pathology of diabetic cardiomyopathy, which presents as cardiac hypertrophy and fibrosis. At the cellular level, oxidative stress and inflammation in cardiomyocytes are triggered by hyperglycemia. Although males are generally more likely to develop cardiovascular disease than females, diabetic males are less likely to develop diabetic cardiomyopathy than are diabetic females. One reason for these differences may be the higher levels of serum testosterone in males compared with females. Although testosterone appears to protect against cardiomyocyte oxidative stress and exacerbate hypertrophy, its role in inflammation and fibrosis is much less clear. Additional preclinical and clinical studies will be required to delineate testosterone's effect on the diabetic heart.

The Effects of Hyperglycemia on Early Endothelial Activation and the Initiation of Atherosclerosis.

It is well established that patients with diabetes have an increased risk of developing atherosclerotic cardiovascular disease. The earliest detectable sign of atherosclerosis initiation is endothelial cell activation. Activated endothelial cells express adhesion proteins, P-selectin, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, which function to recruit monocytes to the subendothelial layer. This study examines the effect of hyperglycemia on endothelial cell activation and the initiation and progression of atherosclerosis. In vitro studies revealed that exposure of human aortic endothelial cells to elevated (30 mmol/L) glucose concentrations significantly increased the expression of P-selectin, E-selectin, and vascular cell adhesion molecule-1. In vivo studies showed that, before lesion development, 5-week-old hyperglycemic ApoE-/-Ins2+/akita mice had significantly increased expression of these adhesion proteins in the aortic sinus and increased macrophage infiltration, compared with normoglycemic ApoE-/- controls. At 25 weeks of age, ApoE-/-Ins2+/akita mice had significantly larger atherosclerotic plaques than ApoE-/- controls (0.022 ± 0.004 versus 0.007± 0.001 mm3; P < 0.05). Similar endothelial activation was observed in heterozygous ApoE+/-Ins2+/akita mice; however, detectable atherosclerotic lesions did not develop in the absence of dyslipidemia. Lowering blood glucose levels (by 55%) using a sodium-glucose cotransporter 2 inhibitor reduced endothelial activation. Together, these findings support a causative role for hyperglycemia in atherogenesis and highlight the importance of blood glucose regulation in preventing atherosclerotic cardiovascular disease.

Investigating the Effects of Sex Hormones on Macrophage Polarization.

Sex differences in the development and progression of cardiovascular disease are well established, but the effects of sex hormones on macrophage polarization and pro-atherogenic functions are not well described. We hypothesize that sex hormones directly modulate macrophage polarization, and thereby regulate the progression of atherosclerosis. Bone marrow-derived monocytes from adult male and female C57BL/6 mice were differentiated into macrophages using macrophage colony-stimulating factor (20 ng/mL) and pre-treated with either 17ß-estradiol (100 nM), testosterone (100 nM), or a vehicle control for 24 h. Macrophages were polarized into pro- or anti-inflammatory phenotypes and the effects of sex hormone supplementation on the gene expression of macrophage phenotypic markers were assessed using RT-qPCR. Inflammatory markers, including IL-1ß, were quantified using an addressable laser bead immunoassay. A transwell migration assay was used to determine changes in macrophage migration. Sex differences were observed in macrophage polarization, inflammatory responses, and migration. Pre-treatment with 17ß-estradiol significantly impaired the gene expression of inflammatory markers and the production of IL-1ß in inflammatory macrophages. In anti-inflammatory macrophages, 17ß-estradiol significantly upregulated the expression of anti-inflammatory markers and enhanced migration. Pre-treatment with testosterone enhanced anti-inflammatory mRNA expression and impaired the production of IL-1ß. Our observations suggest a protective role of 17ß-estradiol in atherogenesis that may contribute to the sexual dimorphisms in cardiovascular disease observed in human patients.

Investigating the Role of 17-Beta Estradiol in the Regulation of the Unfolded Protein Response (UPR) in Pancreatic Beta Cells.

Diabetes mellitus is clinically defined by chronic hyperglycemia. Sex differences in the presentation and outcome of diabetes exist with premenopausal women having a reduced risk of developing diabetes, relative to men, or women after menopause. Accumulating evidence shows a protective role of estrogens, specifically 17-beta estradiol, in the maintenance of pancreatic beta cell health; however, the mechanisms underlying this protection are still unknown. To elucidate these potential mechanisms, we used a pancreatic beta cell line (BTC6) and a mouse model of hyperglycemia-induced atherosclerosis, the ApoE-/-:Ins2+/Akita mouse, exhibiting sexual dimorphism in glucose regulation. In this study we hypothesize that 17-beta estradiol protects pancreatic beta cells by modulating the unfolded protein response (UPR) in response to endoplasmic reticulum (ER) stress. We observed that ovariectomized female and male ApoE-/-:Ins2+/Akita mice show significantly increased expression of apoptotic UPR markers. Sham operated female and ovariectomized female ApoE-/-:Ins2+/Akita mice supplemented with exogenous 17-beta estradiol increased the expression of adaptive UPR markers compared to non-supplemented ovariectomized female ApoE-/-:Ins2+/Akita mice. These findings were consistent to what was observed in cultured BTC6 cells, suggesting that 17-beta estradiol may protect pancreatic beta cells by repressing the apoptotic UPR and enhancing the adaptive UPR activation in response to pancreatic ER stress.

Investigating the protective effects of estrogen on ß-cell health and the progression of hyperglycemia-induced atherosclerosis.

Sex differences in the prevalence and development of diabetes and associated cardiometabolic complications are well established. The objective of this study was to analyze the effects of estrogen on the maintenance of ß-cell health/function and atherosclerosis progression, using a mouse model of hyperglycemia-induced atherosclerosis, the ApoE-/-:Ins2+/Akita mouse. ApoE-/-:Ins2+/Akita mice exhibit sexual dimorphism in the control of blood glucose levels. Male ApoE-/-:Ins2+/Akita mice are chronically hyperglycemic due to a significant reduction in pancreatic ß-cell mass. Female mice are only transiently hyperglycemic, maintain ß-cell mass, and blood glucose levels normalize at 35 ± 1 days of age. To determine the effects of estrogen on pancreatic ß-cell health and function, ovariectomies and estrogen supplementation experiments were performed, and pancreatic health and atherosclerosis were assessed at various time points. Ovariectomized ApoE-/-:Ins2+/Akita mice developed chronic hyperglycemia with significantly reduced ß-cell mass. To determine whether the observed effects on ovariectomized ApoE-/-:Ins2+/Akita mice were due to a lack of estrogens, slow-releasing estradiol pellets were inserted subcutaneously. Ovariectomized ApoE-/-:Ins2+/Akita mice treated with exogenous estradiol showed normalized blood glucose levels and maintained ß-cell mass. Exogenous estradiol significantly reduced atherosclerosis in both ovariectomized female and male ApoE-/-:Ins2+/Akita mice relative to controls. Together, these findings suggest that estradiol confers significant protection to pancreatic ß-cell health and can directly and indirectly slow the progression of atherosclerosis.NEW & NOTEWORTHY This study examines the effect(s) of estrogen on ß cell and cardiometabolic health/function in a novel mouse model of hyperglycemia-induced atherosclerosis (ApoE-/-:Ins2+/Akita). Using a combination of estrogen deprivation (ovariectomy) and supplementation strategies, we quantify effects on glucose homeostasis and atherogenesis. Our results clearly show a protective role for estrogen on pancreatic ß-cell health and function and glucose homeostasis. Furthermore, estrogen supplementation dramatically reduces atherosclerosis progression in both male and female mice.

The Role of Estrogen in Insulin Resistance: A Review of Clinical and Preclinical Data.

Insulin resistance results when peripheral tissues, including adipose, skeletal muscle, and liver, do not respond appropriately to insulin, causing the ineffective uptake of glucose. This represents a risk factor for the development of type 2 diabetes mellitus. Along with abdominal obesity, hypertension, high levels of triglycerides, and low levels of high-density lipoproteins, insulin resistance is a component of a condition known as the metabolic syndrome, which significantly increases the risk of developing cardiometabolic disorders. Accumulating evidence shows that biological sex has a major influence in the development of cardiometabolic disturbances, with females being more protected than males. This protection appears to be driven by female sex hormones (estrogens), as it tends to disappear with the onset of menopause but can be re-established with hormone replacement therapy. This review evaluates current knowledge on the protective role of estrogens in the relevant pathways associated with insulin resistance. The importance of increasing our understanding of sex as a biological variable in cardiometabolic research to promote the development of more effective preventative strategies is emphasized.

Investigating the Role of Endothelial Glycogen Synthase Kinase3α/ß in Atherogenesis in Low Density Lipoprotein Receptor Knockout Mice.

Risk factors for developing cardiovascular disease (CVD) are associated with inflammation and endothelial activation. Activated endothelial cells (ECs) express adhesion proteins that recruit monocytes to the subendothelial layer initiating plaque development. Understanding the mechanism(s) by which ECs increase adhesion protein expression will facilitate the development of therapies aimed at preventing CVD progression and mortality. Glycogen synthase kinase (GSK)3α/ß are constitutively active kinases which have been associated with many cellular pathways regulating cell viability and metabolism. While roles for myeloid GSK3α/ß in the development of atherosclerosis have been established, there is limited knowledge on the potential roles of endothelial GSK3α/ß. With the use of Cre recombinase technology, GSK3α/ß was knocked out of both ECs and macrophages (Tie2Cre GSK3α/ßfl/fl LDLR-/-). A bone marrow transplant was used to replenish GSK3α/ß in the myeloid lineage allowing the assessment of an endothelial-selective GSK3α/ß knockout (BMT Tie2Cre GSK3α/ßfl/fl LDLR-/-). In both models, adhesion protein expression, macrophage recruitment and plaque volume were reduced in GSK3α knockout mice. GSK3ß knockout had no significant effect. Results from this study are the first to suggest a pro-atherogenic role of endothelial GSK3α and support existing evidence for targeting GSK3α in the treatment of atherosclerotic CVD.

Deletion of Macrophage-Specific Glycogen Synthase Kinase (GSK)-3α Promotes Atherosclerotic Regression in Ldlr-/- Mice.

Recent evidence from our laboratory suggests that impeding ER stress-GSK3α/ß signaling attenuates the progression and development of atherosclerosis in mouse model systems. The objective of this study was to determine if the tissue-specific genetic ablation of GSK3α/ß could promote the regression of established atherosclerotic plaques. Five-week-old low-density lipoprotein receptor knockout (Ldlr-/-) mice were fed a high-fat diet for 16 weeks to promote atherosclerotic lesion formation. Mice were then injected with tamoxifen to induce macrophage-specific GSK3α/ß deletion, and switched to standard diet for 12 weeks. All mice were sacrificed at 33 weeks of age and atherosclerosis was quantified and characterized. Female mice with induced macrophage-specific GSK3α deficiency, but not GSK3ß deficiency, had reduced plaque volume (~25%) and necrosis (~40%) in the aortic sinus, compared to baseline mice. Atherosclerosis was also significantly reduced (~60%) in the descending aorta. Macrophage-specific GSK3α-deficient mice showed indications of increased plaque stability and reduced inflammation in plaques, as well as increased CCR7 and ABCA1 expression in lesional macrophages, consistent with regressive plaques. These results suggest that GSK3α ablation promotes atherosclerotic plaque regression and identify GSK3α as a potential target for the development of new therapies to treat existing atherosclerotic lesions in patients with cardiovascular disease.

Macrophage Function and the Role of GSK3.

Macrophages are present in nearly all vertebrate tissues, where they respond to a complex variety of regulatory signals to coordinate immune functions involved in tissue development, metabolism, homeostasis, and repair. Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed protein kinase that plays important roles in multiple pathways involved in cell metabolism. Dysregulation of GSK3 has been implicated in several prevalent metabolic disorders, and recent findings have highlighted the importance of GSK3 activity in the regulation of macrophages, especially with respect to the initiation of specific pathologies. This makes GSK3 a potential therapeutic target for the development of novel drugs to modulate immunometabolic responses. Here, we summarize recent findings that have contributed to our understanding of how GSK3 regulates macrophage function, and we discuss the role of GSK3 in the development of metabolic disorders and diseases.

Glycosphingolipids promote pro-atherogenic pathways in the pathogenesis of hyperglycemia-induced accelerated atherosclerosis.

INTRODUCTION: Three out of four people with diabetes will die of cardiovascular disease. However, the molecular mechanisms by which hyperglycemia promotes atherosclerosis, the major underlying cause of cardiovascular disease, are not clear. OBJECTIVES: Three distinct models of hyperglycemia-associated accelerated atherosclerosis were used to identify commonly altered metabolites and pathways associated with the disease. METHODS: Normoglycemic apolipoprotein-E-deficient mice served as atherosclerotic control. Hyperglycemia was induced by multiple low-dose streptozotocin injections, or by introducing a point-mutation in one copy of insulin-2 gene. Glucosamine-supplemented mice, which experience accelerated atherosclerosis to a similar extent as hyperglycemia-induced models without alterations in glucose or insulin levels, were also included in the analysis. Untargeted plasma metabolomics were used to investigate hyperglycemia-associated accelerated atherosclerosis in three disease models. The effect of specific significantly altered metabolites on pro-atherogenic processes was investigated in cultured human vascular cells. RESULTS: Hyperglycemic and glucosamine-supplemented mice showed distinct metabolomic profiles compared to controls. Meta-analysis of three disease models revealed 62 similarly altered metabolite features (FDR-adjusted p < 0.05). Identification of shared metabolites revealed alterations in glycerophospholipid and sphingolipid metabolism, and pro-atherogenic processes including inflammation and oxidative stress. Post-multivariate and pathway analyses indicated that the glycosphingolipid pathway is strongly associated with hyperglycemia-induced accelerated atherosclerosis in these atherogenic mouse models. Glycosphingolipids induced oxidative stress and inflammation in cultured human vascular cells. CONCLUSION: Glycosphingolipids are strongly associated with hyperglycemia-induced accelerated atherosclerosis in three distinct models. They also promote pro-atherogenic processes in cultured human cells. These results suggest glycosphingolipid pathway may be a potential therapeutic target to block or slow atherogenesis in diabetic patients.

The Role of Endoplasmic Reticulum Stress-Glycogen Synthase Kinase-3 Signaling in Atherogenesis.

Cardiovascular disease (CVD) is the number one cause of global mortality and atherosclerosis is the underlying cause of most CVD. However, the molecular mechanisms by which cardiovascular risk factors promote the development of atherosclerosis are not well understood. The development of new efficient therapies to directly block or slow disease progression will require a better understanding of these mechanisms. Accumulating evidence supports a role for endoplasmic reticulum (ER) stress in all stages of the developing atherosclerotic lesion however, it was not clear how ER stress may contribute to disease progression. Recent findings have shown that ER stress signaling through glycogen synthase kinase (GSK)-3α may significantly contribute to macrophage lipid accumulation, inflammatory cytokine production and M1macrophage polarization. In this review we summarize our knowledge of the potential role of ER stress-GSK3 signaling in the development and progression of atherosclerosis as well as the possible therapeutic implications of this pathway.

Glucosamine induces ER stress by disrupting lipid-linked oligosaccharide biosynthesis and N-linked protein glycosylation.

Glucosamine is an essential substrate for N-linked protein glycosylation. However, elevated levels of glucosamine can induce endoplasmic reticulum (ER) stress. Glucosamine-induced ER stress has been implicated in the development of diabetic complications, including atherosclerosis and hepatic steatosis. In this study, we investigate the potential relationship between the effects of glucosamine on lipid-linked oligosaccharide (LLO) biosynthesis, N-linked glycosylation, and ER homeostasis. Mouse embryonic fibroblasts (MEFs) were cultured in the presence of 0-5 mM glucosamine for up to 18 h, and LLO biosynthesis was monitored by fluorescence-assisted carbohydrate electrophoresis. ER stress was determined by quantification of unfolded protein response (UPR) gene expression. We found that exposure of MEFs to ≥1 mM glucosamine significantly impaired the biosynthesis of mature (Glc3Man9GlcNAc2) LLOs before the activation of the UPR, which resulted in the accumulation of an LLO intermediate (Man3GlcNAc2). The addition of 4-phenylbutyric acid (4-PBA), a chemical chaperone, was able to alleviate ER stress but did not rescue LLO biosynthesis. Other ER stress-inducing agents, including dithiothreitol and thapsigargin, had no effect on LLO levels. Together, these data suggest that elevated concentrations of glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation. We hypothesize that this pathway represents a causative link between hyperglycemia and the development of diabetic complications.

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 ß-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.

Deletion of Myeloid GSK3α Attenuates Atherosclerosis and Promotes an M2 Macrophage Phenotype.

OBJECTIVE: Glycogen synthase kinase (GSK)-3α/ß has been implicated in the pathogenesis of diabetes mellitus, cancer, Alzheimer, and atherosclerosis. The tissue- and homolog-specific functions of GSK3α and ß in atherosclerosis are unknown. This study examines the effect of hepatocyte or myeloid cell deletion of GSK3α or GSK3ß on atherosclerosis in low-density lipoprotein receptor (LDLR)(-/-) mice. APPROACH AND RESULTS: We ablated GSK3α or GSK3ß expression in hepatic or myeloid cells of LDLR(-/-) mice, and mice were fed a high-fat diet for 10 weeks. GSK3α or GSK3ß deficiency in hepatic or myeloid cells did not affect metabolic parameters, including plasma lipid levels. Hepatic deletion of GSK3α or GSK3ß did not affect the development of atherosclerosis or hepatic lipid content. Myeloid deletion of GSK3α, but not of GSK3ß, reduced atherosclerotic lesion volume and lesion complexity. Mice lacking GSK3α in myeloid cells had a less inflammatory and more anti-inflammatory plasma cytokine profile. Macrophages within atherosclerotic lesions of myeloid GSK3α-deficient mice, but not of GSK3ß-deficient mice, displayed reduced expression of markers associated with M1 macrophage polarization and enhanced expression of the M2 markers. Finally, bone marrow-derived macrophages were isolated and differentiated into classical M1 macrophages or alternative M2 macrophages in vitro. GSK3α deletion, but not GSK3ß deletion, attenuated the expression of genes associated with M1 polarization while promoting the expression of genes associated with M2 polarization by modulating STAT3 and STAT6 activation. CONCLUSIONS: Our findings suggest that deletion of myeloid GSK3α attenuates the progression of atherosclerosis by promoting an M2 macrophage phenotype.

Glycogen synthase kinase 3α deficiency attenuates atherosclerosis and hepatic steatosis in high fat diet-fed low density lipoprotein receptor-deficient mice.

Studies have implicated signaling through glycogen synthase kinase (GSK) 3α/ß in the activation of pro-atherogenic pathways and the accelerated development of atherosclerosis. By using a mouse model, we examined the role of GSK3α in the development and progression of accelerated atherosclerosis. We crossed Gsk3a/GSK3α-knockout mice with low-density lipoprotein receptor (Ldlr) knockout mice. Five-week-old Ldlr(-/-);Gsk3a(+/+), Ldlr(-/-);Gsk3a(+/-), and Ldlr(-/-);Gsk3a(-/-) mice were fed a chow diet or a high-fat diet for 10 weeks and then sacrificed. GSK3α deficiency had no detectible effect on any measured parameters in chow-fed mice. High-fat-diet fed Ldlr(-/-) mice that were deficient for GSK3α had significantly less hepatic lipid accumulation and smaller atherosclerotic lesions (60% smaller in Ldlr(-/-);Gsk3a(+/-) mice, 80% smaller in Ldlr(-/-);Gsk3a(-/-) mice; P < 0.05), compared with Ldlr(-/-);Gsk3a(+/+) controls. GSK3α deficiency was associated with a significant increase in plasma IL-10 concentration and IL-10 expression in isolated macrophages. A twofold to threefold enhancement in endoplasmic reticulum stress-induced IL-10 expression was observed in Thp-1-derived macrophages that were pretreated with the GSK3α/ß inhibitor CT99021. Together, these results suggest that GSK3α plays a pro-atherogenic role, possibly by mediating the effects of endoplasmic reticulum stress in the activation of pro-atherogenic pathways.

Protein kinase R-like endoplasmic reticulum kinase and glycogen synthase kinase-3α/ß regulate foam cell formation.

Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. This study investigated the potential role of glycogen synthase kinase (GSK)-3α/ß in proatherogenic ER stress signaling. Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin, or palmitate. Using small-molecule inhibitors of specific unfolded protein response (UPR) signaling pathways, we found that protein kinase R-like ER kinase (PERK), but not inositol requiring enzyme 1 or activating transcription factor 6, is required for the activation of GSK3α/ß by ER stress. GSK3α/ß inhibition or siRNA-directed knockdown attenuated ER stress-induced expression of distal components of the PERK pathway. Macrophage foam cells within atherosclerotic plaques and isolated macrophages from ApoE(-/-) mice fed a diet supplemented with the GSK3α/ß inhibitor valproate had reduced levels of C/EBP homologous protein (CHOP). GSK3α/ß inhibition blocked ER stress-induced lipid accumulation and the upregulation of genes associated with lipid metabolism. In primary mouse macrophages, PERK inhibition blocked ER stress-induced lipid accumulation, whereas constitutively active S9A-GSK3ß promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. These findings suggest that ER stress-PERK-GSK3α/ß signaling promotes proatherogenic macrophage lipid accumulation.

Detection and quantification of endoplasmic reticulum stress in living cells using the fluorescent compound, Thioflavin T.

The endoplasmic reticulum (ER) plays a central role in the co- and post-translational modification of many proteins. Disruption of these processes can lead to the accumulation of misfolded proteins in the endoplasmic reticulum - a condition known as endoplasmic reticulum stress. In recent years, the association of endoplasmic reticulum stress with a number of disease pathologies has increased interest in the study of this condition. Current methods to detect endoplasmic reticulum stress are indirect and retrospective. Here we describe a new method to detect and quantify endoplasmic reticulum stress in live cells using Thioflavin T (ThT), a small molecule that exhibits enhanced fluorescence when it binds to protein aggregates. We show that enhanced ThT-fluorescence correlates directly with established indicators of unfolded protein response activation. Furthermore, enhanced ThT-fluorescence can be detected in living cells within 20 min of application of an endoplasmic reticulum stress-inducing agent. ThT is capable of detecting endoplasmic reticulum stress induced by distinctly different conditions and compounds, in different cultured cell types as well as in mouse tissue samples. Pre-treatment with a potent endoplasmic reticulum stress-reducing agent, 4-phenylbutyric acid, mitigates the enhanced ThT signal. This new tool will be useful in future research investigating the role of protein misfolding in the development and/or progression of human diseases.

Oral glucosamine sulfate supplementation does not induce endoplasmic reticulum stress or activate the unfolded protein response in circulating leukocytes of human subjects.

Glucosamine sulfate is a dietary supplement that is marketed as a treatment for osteoarthritis. Recent evidence from animal and cell culture models have suggested that glucosamine treatment can promote the misfolding of proteins and the activation of the unfolded protein response (UPR). We investigated whether glucosamine sulfate supplementation activates the UPR in circulating leukocytes of human subjects. Cultured Thp1 human monocytes were exposed to increasing concentrations of glucosamine (0, 0.25, 1.0, 4.0 mmol · L(-1)) for 18 h. We observed a dose-dependent increase in intracellular glucosamine levels as well as the activation of UPR. To test the effect of glucosamine sulfate supplementation in humans, 14 healthy human subjects took 1500 mg · day(-1) glucosamine sulfate for 14 days. Metabolic parameters and blood samples were collected before and after supplementation. In humans, glucosamine sulfate supplementation did not alter metabolic parameters including lipid levels and glucose tolerance. Further, glucosamine sulfate supplementation did not affect intracellular glucosamine levels or activate the UPR in the leukocytes of human subjects. Our results indicate that in healthy human subjects, the recommended dose of glucosamine sulfate (1500 mg · day(-1)) for 14 days does not significantly alter intracellular glucosamine levels and does not activate the UPR in circulating leukocytes.

Endoplasmic reticulum stress and glycogen synthase kinase-3ß 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)-3ß 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 µg/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, GSK3ß phosphorylation, and GSK3ß activity were analyzed in liver and aorta. Atherosclerotic lesions were examined and quantified. Hyperglycemia, hyperhomocysteinemia, and high-fat diet significantly enhanced GSK3ß activity and also increased hepatic steatosis and atherosclerotic lesion volume compared with controls. Valproate supplementation blocked GSK3ß activation and attenuated the development of atherosclerosis and the accumulation of hepatic lipids in each of the models examined. The mechanism by which GSK3ß activity is regulated in these models likely involves alterations in phosphorylation at serine 9 and tyrosine 216. CONCLUSIONS: These findings support the existence of a common mechanism of accelerated atherosclerosis involving ER stress signaling through activation of GSK3ß. Furthermore, our results suggest that atherosclerosis can be attenuated by modulating GSK3ß phosphorylation.

Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3ß.

In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 µg/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3ß on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of ß-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3ß (Ad-GSK-3ß S9A) resulted in a >2-fold increase in GSK-3ß activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3ß activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.