Diabetes is a progressive metabolic diseases characterized by elevated plasma glucose levels and about 3.2 million Korean people aged over 30 years or older had diabetes based on the data in 2010. Chronic hyperglycemia of diabetes induces diverse complications including diabetic nephropathy, retinop...
Diabetes is a progressive metabolic diseases characterized by elevated plasma glucose levels and about 3.2 million Korean people aged over 30 years or older had diabetes based on the data in 2010. Chronic hyperglycemia of diabetes induces diverse complications including diabetic nephropathy, retinopathy, neuropathy, cardiovascular disorders, erectile dysfunction, and liver diseases, as result of damages of different cells. Therefore, diabetes patients need to be achieved glycemic control for reducing the risk of diabetic complication, however, only about half of patients with type 2 diabetes success on their glycemic control. That means a thorough understanding about intracellular mechanism under diabetes is needed to manage expertly. Diabetic nephropathy is the most frequent complication, in which, one of the vulnerable and damaged cells by hyperglycemia is mesangial cells. Emerging evidences show that cellular signalings such as inflammation, apoptosis and fibrosis, are activated in mesangial cells, which could lead dysfunction of kidney ultimately. Moreover, renal lipotoxicity also has been implicated in kidney abnormalities in obese type 2 diabetic individuals by both over-accumulation of circulating lipid and abnormal lipid synthesis. Increased expression of lipogenesis regulators, such as liver X receptor alpha (LXRα), sterol regulatory element binding protein (SREBP) and carbohydrate response element binding protein (ChREBP), and accumulation of lipid droplets in glomerulus have been reported. Furthermore, diabetes also may be a cause of liver diseases from non-alcoholic fatty liver to hepatocellular carcinoma. Particularly, type 2 diabetes are more often related with obesity and the abdorminal adipose depots release free fatty acids which are transported to the liver, which could lead chronic liver disease in obese patients. Because hyperglycemia induces detrimental effects on diverse cellular signaling of different organs as mentioned above, some proteins reported altered expressions or post-translational modifications by high glucose level can be candidates for developing therapeutic agents. Hence, for more obvious understanding of the proteins regulated by high glucose level, this study is focused on three molecules, ChREBP, thioredoxin interacting protein (TXNIP) and glycogen synthase kinase-3β (GSK-3β). 1) The regulation mechanism and function of ChREBP which is known as a glucose sensor, were investigated in kidney mesangial cells under high glucose. 2) The signaling mechanism of TXNIP known to be increased significantly by high glucose, was explored both in hepatocytes under high fatty acid circumstance and in the livers of high fat diet mice. 3) The function of GSK-3β reported to be related in diverse cellular signaling was inspected in kidney mesangial cells under high glucose. Results were as followings: Treatment with 25 mM glucose (high glucose, HG) increased cellular O-GlcNAc and O-GlcNAcylated ChREBP in mesangial cells compared to normal 5.5 mM glucose. PUGNAc, a drug that increases O-GlcNAc, augmented the expression of ChREBP targets, whereas DON, a drug that decreases O-GlcNAc and O-GlcNAcase overexpression, mitigated the increase with HG. O-GlcNAc augmented the protein stability, transcriptional activity and the nuclear translocation of ChREBP. HG treatment also induced lipid accumulation in mesangial cells as determined by Oil Red O staining. In addition, HG triggered expression of hypoxia-inducible factor 1-alpha, vascular endothelial growth factor and extracellular matrix components related to nephrosclerosis. The ChREBP mutant, W130A, did not exhibit HG-induced lipid accumulation and fibrotic proteins, suggesting the Trp-130 residue in the MCR3 domain is important in the development of glomerulosclerosis. In conclusion, HG increased the O-GlcNAcylated ChREBP level, which resulted in lipid accumulation and upregulation of fibrotic proteins in mesangial cells. Palmitic acid (PA) increased TXNIP, and inflammatory and lipogenic proteins in both AML12 and H4IIE cells. It also increased the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), which mediates the expression of lipogenic markers and lipid accumulation. In addition, PA increased protein arginine methylation transferase-1 (PRMT1) and PRMT1 siRNA abolished the increase in lipogenic markers with PGC-1α. Furthermore, TXNIP interacted with PRMT1 in PA-treated hepatocytes. In vivo, levels of lipogenic proteins, inflammatory molecules, PGC-1α, and PRMT1 were increased in the livers of HFD mice compared with those fed a chow diet, and were ameliorated in HFD TXNIP-deficient mice. HG treatment increased p-Ser9 GSK3β level in nucleus, which is generally known as an inactive form, while decreased it in cytosol in mesangial cells. The elevated p-Ser9 GSK3β mediated the endoplasmic reticulum (ER) stress and apoptosis induced by HG, and augmented Ser9 phosphorylation by LiCl exacerbated the apoptosis, whereas ectopic expression of S9A mutant or kinase dead mutant blocked reversely, suggesting the importance of Ser9 phosphorylation and kinase activity independent on Ser9 phosphorylation. Furthermore, nuclear p-Ser9 GSK3β was more phosphorylated on Tyr 216 residue under HG condition. Nuclear factor κB (NFκB), one of the GSK3β target, was activated by p-Ser9 GSK3β under HG condition suggesting the possibility that p-Ser9 GSK3β exert the detrimental effects through NFκB. Finally, diabetic patients, streptozotocin-induced diabetic rats and db/db mice kidney showed increased nuclear p-Ser9 GSK3β. As conclusions, 1) HG-induced O-GlcNAcylated ChREBP leads lipogenesis and fibrosis in mesangial cells, in which Trp-130 residue in the MCR3 domain is important. 2) TXNIP mediates hepatic lipogenesis via PRMT1 and PGC-1α regulation and inflammation in vitro and in vivo, implying that targeting of TXNIP and PRMT1 could be a potential therapeutic approach for the treatment of NAFLD. 3) p-Ser9 GSK3β has function significantly including kinase activity and HG induces apoptosis by p-Ser9 GSK3β possibly through NFκB transcriptional activity in mesangial cells. Therefore, the present results suggest that precise regulation of these proteins is the important therapeutic targets for the diabetic complications.
Diabetes is a progressive metabolic diseases characterized by elevated plasma glucose levels and about 3.2 million Korean people aged over 30 years or older had diabetes based on the data in 2010. Chronic hyperglycemia of diabetes induces diverse complications including diabetic nephropathy, retinopathy, neuropathy, cardiovascular disorders, erectile dysfunction, and liver diseases, as result of damages of different cells. Therefore, diabetes patients need to be achieved glycemic control for reducing the risk of diabetic complication, however, only about half of patients with type 2 diabetes success on their glycemic control. That means a thorough understanding about intracellular mechanism under diabetes is needed to manage expertly. Diabetic nephropathy is the most frequent complication, in which, one of the vulnerable and damaged cells by hyperglycemia is mesangial cells. Emerging evidences show that cellular signalings such as inflammation, apoptosis and fibrosis, are activated in mesangial cells, which could lead dysfunction of kidney ultimately. Moreover, renal lipotoxicity also has been implicated in kidney abnormalities in obese type 2 diabetic individuals by both over-accumulation of circulating lipid and abnormal lipid synthesis. Increased expression of lipogenesis regulators, such as liver X receptor alpha (LXRα), sterol regulatory element binding protein (SREBP) and carbohydrate response element binding protein (ChREBP), and accumulation of lipid droplets in glomerulus have been reported. Furthermore, diabetes also may be a cause of liver diseases from non-alcoholic fatty liver to hepatocellular carcinoma. Particularly, type 2 diabetes are more often related with obesity and the abdorminal adipose depots release free fatty acids which are transported to the liver, which could lead chronic liver disease in obese patients. Because hyperglycemia induces detrimental effects on diverse cellular signaling of different organs as mentioned above, some proteins reported altered expressions or post-translational modifications by high glucose level can be candidates for developing therapeutic agents. Hence, for more obvious understanding of the proteins regulated by high glucose level, this study is focused on three molecules, ChREBP, thioredoxin interacting protein (TXNIP) and glycogen synthase kinase-3β (GSK-3β). 1) The regulation mechanism and function of ChREBP which is known as a glucose sensor, were investigated in kidney mesangial cells under high glucose. 2) The signaling mechanism of TXNIP known to be increased significantly by high glucose, was explored both in hepatocytes under high fatty acid circumstance and in the livers of high fat diet mice. 3) The function of GSK-3β reported to be related in diverse cellular signaling was inspected in kidney mesangial cells under high glucose. Results were as followings: Treatment with 25 mM glucose (high glucose, HG) increased cellular O-GlcNAc and O-GlcNAcylated ChREBP in mesangial cells compared to normal 5.5 mM glucose. PUGNAc, a drug that increases O-GlcNAc, augmented the expression of ChREBP targets, whereas DON, a drug that decreases O-GlcNAc and O-GlcNAcase overexpression, mitigated the increase with HG. O-GlcNAc augmented the protein stability, transcriptional activity and the nuclear translocation of ChREBP. HG treatment also induced lipid accumulation in mesangial cells as determined by Oil Red O staining. In addition, HG triggered expression of hypoxia-inducible factor 1-alpha, vascular endothelial growth factor and extracellular matrix components related to nephrosclerosis. The ChREBP mutant, W130A, did not exhibit HG-induced lipid accumulation and fibrotic proteins, suggesting the Trp-130 residue in the MCR3 domain is important in the development of glomerulosclerosis. In conclusion, HG increased the O-GlcNAcylated ChREBP level, which resulted in lipid accumulation and upregulation of fibrotic proteins in mesangial cells. Palmitic acid (PA) increased TXNIP, and inflammatory and lipogenic proteins in both AML12 and H4IIE cells. It also increased the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), which mediates the expression of lipogenic markers and lipid accumulation. In addition, PA increased protein arginine methylation transferase-1 (PRMT1) and PRMT1 siRNA abolished the increase in lipogenic markers with PGC-1α. Furthermore, TXNIP interacted with PRMT1 in PA-treated hepatocytes. In vivo, levels of lipogenic proteins, inflammatory molecules, PGC-1α, and PRMT1 were increased in the livers of HFD mice compared with those fed a chow diet, and were ameliorated in HFD TXNIP-deficient mice. HG treatment increased p-Ser9 GSK3β level in nucleus, which is generally known as an inactive form, while decreased it in cytosol in mesangial cells. The elevated p-Ser9 GSK3β mediated the endoplasmic reticulum (ER) stress and apoptosis induced by HG, and augmented Ser9 phosphorylation by LiCl exacerbated the apoptosis, whereas ectopic expression of S9A mutant or kinase dead mutant blocked reversely, suggesting the importance of Ser9 phosphorylation and kinase activity independent on Ser9 phosphorylation. Furthermore, nuclear p-Ser9 GSK3β was more phosphorylated on Tyr 216 residue under HG condition. Nuclear factor κB (NFκB), one of the GSK3β target, was activated by p-Ser9 GSK3β under HG condition suggesting the possibility that p-Ser9 GSK3β exert the detrimental effects through NFκB. Finally, diabetic patients, streptozotocin-induced diabetic rats and db/db mice kidney showed increased nuclear p-Ser9 GSK3β. As conclusions, 1) HG-induced O-GlcNAcylated ChREBP leads lipogenesis and fibrosis in mesangial cells, in which Trp-130 residue in the MCR3 domain is important. 2) TXNIP mediates hepatic lipogenesis via PRMT1 and PGC-1α regulation and inflammation in vitro and in vivo, implying that targeting of TXNIP and PRMT1 could be a potential therapeutic approach for the treatment of NAFLD. 3) p-Ser9 GSK3β has function significantly including kinase activity and HG induces apoptosis by p-Ser9 GSK3β possibly through NFκB transcriptional activity in mesangial cells. Therefore, the present results suggest that precise regulation of these proteins is the important therapeutic targets for the diabetic complications.
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