RESUMO
BACKGROUND: Mammalian target of rapamycin (mTOR) complexes are a key regulator of pancreatic beta cells mass and function. DEP-domain containing mTOR-interacting protein (DEPTOR) is a common part of mTOR complexes and whether DEPTOR loss in islet β cells affects insulin-secreting function has never been identified. OBJECTIVE: To assess the alternation of insulin secretion by silencing DEPTOR gene in pancreatic β cells NIT-1 and to explore the underlying mechanism. METHODS: Three siRNA sequences for silencing DEPTOR gene were designed and constructed, which were transfected with lipofectamine into NIT-1 cells. There were six groups: blank transfection group (NIT-1 cells plus Lipofectamin), negative control group (NC-FAM), positive control group (GAPDH), siRNA deptor 1 group (siRNA deptor385), siRNA deptor 2 group (siRNA deptor766), and siRNA deptor 3 group (siRNA deptor1275). The transfection efficiency was determined by fluorescence microscope. The relative expression level of DEPTOR mRNA was detected by quantitative-PCR. Insulin secretion in the cell conditioned medium was determined by insulin ELISA kit. The expression level of DEPTOR downstream key protein was detected by western blot assay. RESULTS AND CONCLUSION: Specific green fluorescence accumulated in a punctated pattern under fluorescence microscope, indicating that the effectiveness of transfection was eligible. Quantitative-PCR results showed two (siDEPTOR385 and siDEPTOR766) of the three siRNA sequences could significantly disrupt the expression of DEPTOR mRNA, which had significant difference with negative control group (P＜ 0.05). The ELISA results showed that the total amount of insulin secretion in the effective transfected groups was significantly increased (P＜ 0.05). Western blot assay results showed the grey levels of p-s6 and p-4EBP-1 proteins were significantly elevated, while p-AKT of those former was slightly decreased. These findings suggest that siRNA technology can effectively silence the DEPTOR gene in NIT-1 cells, which improves β-cell insulin secretion in a manner of mTORC1 activation.
RESUMO
<p><b>OBJECTIVE</b>To investigate the prevalence of chronic kidney disease (CKD) in subjects with different glucose metabolism status.</p><p><b>METHODS</b>Between January, 2015 and October, 2015, a total of 934 subjects without a previous diagnosis of diabetes visiting the Department of Endocrinology or Health Examination Center underwent oral glucose tolerance test (OGTT), which identified 266 subjects with normal glucose tolerance (NGT group), 243 pre-diabetic subjects, and 425 patients with diabetes mellitus group. The baseline characteristics and laboratory test data of the subjects were collected. The diagnosis of CKD was established for an eGFR <60 mL/min/1.73 m(2) or a ACR≥30 mg/g, and the prevalence of CKD were compared among the 3 groups. Logistic regression model was used to analyze the OR value of the risk factors of CKD.</p><p><b>RESULTS</b>The prevalences of CKD in NGT, pre-diabetic and diabetic groups were 10.2%, 26.3% and 32.5%, respectively. Pairwise comparisons showed that the prevalence of CKD was significantly higher in pre-diabetic group (P<0.001, OR=3.17, 95% CI 1.94-5.17) and diabetic group (P<0.001, OR=4.27, 95% CI 2.72-6.65) than in NGT group, and was comparable between the pre-diabetic and diabetic groups (P=0.115, OR=1.35, 95% CI 0.95-1.91). Logistic regression analysis, after adjustment for age, gender, blood pressure, hypertension, blood lipids and uric acid, showed that pre-diabetes (OR=2.03, P=0.044) and diabetes mellitus (OR=2.22, P=0.016) were independently associated with CKD.</p><p><b>CONCLUSION</b>Glucose metabolism status has a significant independent impact on the incidence of CKD, suggesting the importance of early detection of pre-diabetes and timely interventions in pre-diabetic subjects in prevention CKD.</p>