| Type 2 diabetes is a variety of metabolism disorders characterized with insulin resistant and insulin secretion deficiency. It is a chronic disease seriously harm to human health. Given the high incidence of this disease. the current research focus is to find an effective therapeutic target.It has long been demonstrated that glucocorticoids contribute to the pathophysiology of metabolic disorder syndrome. including hypertension, obesity, and type 2 diabetes mellitus. Glucocorticoids work antagonistically to the action performed by insulin characterized by increased hepatic gluconeogenesis and decreased ability of insulin to inhibit glucose production. Glucocorticoid receptor (GR) is a steroid ligand activated transcription factor. However, the actions of glucocorticoids on target tissues, such as liver and adipose tissue, are not necessarily dependent on circulating glucocorticoid levels but rather on their prereceptor metabolism, which is regulated by 11β-hydroxysteroid dehydrogenase (11β-HSD1).11β-HSD1 is a NADPH-dependent enzyme that resides within the endoplasmic reticulum (ER) lumen and expresses highly in liver, adipose tissue, and skeletal muscle. This isozyme is a biodirectional enzyme which acts predominantly as a reductase in vivo, catalyzing the interconversion of the active hormone cortisol and inert cortisone. The physiological role of the enzyme is supposed to regulate local glucocorticoid levels in the target tissues, and then influence the key hepatic gluconeogenic enzymes including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Enhanced 11β-HSD1 activity results in production of excess tissue GCs and induction of local GR activation leads to hepatic gluconeogenesis and adipocyte differentiation, all of which are associated with the development of metabolic syndrome and visceral obesity. Because the ER membrane is practically impermeable to pyridine nucleotides. the effects of 11β-HSD1 are dependent on its reductase activity, which requires NADPH as the cofactor tomaintain its reductase activity. NADPH is regenerated by hexose-6-phosphate dehydrogenase (H6PDH). a microsomal enzyme located in the lumen of the ER and principally expressed in hepatocytes and adipocytes. sites of 11β-HSD1 and GR. In these target tissues. H6PDH utilizes glucose-6-phosphate (G6P) and NADP to produce NADPH. thus H6PDH is likely to be the crucial enzyme supplying NADPH for 11β-HSD1-induced amplification of tissue GR ligand cortisol/corticosterone production linked to the development of type 2 diabetes and obesity.We had successfully established a rat diabetic model induced by a combination of high fat diet and multiple low dose of STZ (30mg/kg. twice) injection, and observed the change of H6PDH,11β-HSD1, PEPCK and G6Pase in diabetic rats. The results showed that compared with control group, the expression and activity of H6PDH and 11β-HSD1 in livers were all increased in diabetic group, and the expression of PEPCK and G6Pase were also increased in diabetic group. Moreover, we examined the corticosterone levels of serum and liver in diabetic group. The results showed that the serum corticosterone in diabetic group was normal, while the liver corticosterone was significantly higher than control group, indicating that H6PDH and 11β-HSD1 increased abnormally in diabetes and caused tissue corticosterone level increased. The results in vivo showed that H6PDH and 11β-HSD1 were closely related to hepatic insulin resistance. To prove this point of view, we constructed siRNA plasmids of H6PDH and 11β-HSD1 respectively to inhibit the corresponding genes expression, then observed the interaction between the two enzymes. We also examined the change of gluconeogenesis and the insulin signaling pathway after inhibited H6PDH expression of corticosterone treated liver cells by siRNA. The results showed H6PDH siRNA and 11β-HSD1 siRNA can inhibit the protein expression and enzyme activity by each other. In CBRH-7919 the protein expression of PEPCK increased afther the treatment of 10-6 M corticosterone for 48 h resulting in hepatic insulin resistance, and the suppression on PEPCK by insulin reduced, also the expression of insulin signaling pathway proteins including IR-β, IRS-1, Akt, p-Akt reduced. After utilized H6PDH siRNA to inhibite the expression of H6PDH. we found the enhancement of PEPCK expression by corticosterone was weakened, and resumed the suppression on PEPCK by insulin. Furthermore, insulin resistance caused by corticosterone was declined, and the expression of IR-βand p-Akt was significantly increased after stimulated by insulin. It suggested that inhibition of H6PDH by siRNA can not only block the enhancement of gluconeogenesis stimulated by corticosterone. but also reduce insulin resistance stimulated by corticosterone. Therefore, the specific suppression of the two enzymes by siRNA technology could be considered as an effective target for the treatment of type 2 diabetes and metabolic syndrome.In vivo many hormones and growth factors regulate H6PDH and 11β-HSD1 including glucocorticoids. insulin and growth hormone. Blood glucose levels and insulin secretion is interaction. After eating blood glucose is elevation, promoting insulin secretion, lowering blood glucose, then it remained at normal levels. We had successfully established a rat diabetic model induced by a combination of high fat diet and multiple low dose of STZ injection. Here, we examined H6PDH and 11β-HSD1 expression from mRNA levels, protein levels and enzyme activity of diabetic and normal rats under fasting and non-fasting state. The results showed that compared with fasting state. H6PDH and 11β-HSD1 expression in livers were all increased under non-fasting state in both normal and diabetic rats, and the non-fasting diabetic group was the highest among the four experimental groups. The expression of GR. PEPCK. and G6Pase were also higher in diabetic group. The results of correlation analysis showed both H6PDH and 11β-HSD1 are correlated with blood glucose but no correlation with circulating insulin. Moreover, incubation of primary hepatocytes with increasing glucose (5,10,20,30 mM) caused dose-dependent increases in H6PDH. 11β-HSD1. GR. PEPCK and G6Pase expression. Also. G6P (1.2.5.5 mM) had a positive regulation on H6PDH and 11β-HSD1 in hepatocytes. In addition, primary hepatocytes treated with different doses of insulin in high glucose induced alteration of H6PDH and 11β-HSDl while in low glucose there was no significant effect. These findings suggest that glucose instead of insulin directly regulates H6PDH and 11β-IISD1. We examined the impact of glucose concentrations on interference effect by siRNA in CBRH-7919. The results showed H6PDH siRNA and 11β-HSD1 siRNA can selectively inhibit PEPCK in high glucose medium but in low glucose medium. Further we transfected the cells with H6PDH and 11β-HSD1 siRNA under low glucose, then replaced by high glucose medium. The results show that the protein expression of H6PDH.11β-HSD1 and PEPCK were increased in the normal cells and cells transfected with negative control plasmid after increasing the concentration of glucose in the medium, while all of them were reduced in the cells transfected with H6PDH and 11β-HSD1 siRNA. It suggested H6PDH and 11β-HSD1 siRNA can inhibit the expression of PEPCK only in high glucose, on the other hand, it can selectively inhibit PEPCK by the regulation of glucose concentration. These results have supplied a new method for the use of H6PDH or 11β-HSDl siRNA as a specific inhibitor in clinic.In summary, compared with control group, the expression of H6PDH.11β-HSD1. corticosterone. PEPCK and G6Pase in livers were all increased in diabetic group. H6PDH siRNA and 11β-HSD1 siRNA can inhibit each other in CBRH-7919. Inhibition of H6PDH by siRNA can not only block the enhancement of gluconeogenesis stimulated by corticosterone, but also reduce insulin resistance stimulated by corticosterone. Glucose instead of insulin directly regulates H6PDH and 11β-HSD1. H6PDH siRNA and 11β-HSD1 siRNA can selectively inhibit PEPCK by the regulation of glucose concentration.
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