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.

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.