ABSTRACT
@#The aim of this study is to investigate the effect of long non-coding RNA PLUTO on β cell function and its underlying mechanism. Islet cells of db/db, ob/ob and HFD mice were isolated and extracted by enzymatic method to detect the expression of PLUTO in islet cells of different obesity model mice. Glucolipid toxicity was used to stimulate islet primary cells and Min6 cells, and the expression of PLUTO was detected to elucidate the reasons of PLUTO down-regulation induced by obesity factors. RT-qPCR and ELISA were performed to analyze the function of PLUTO on β cells; Western blot and rescue experiments were carried out to elucidate the regulatory mechanism of β cells by PLUTO. The results showed that the expression levels of PLUTO were significantly decreased in obesity model mice compared with control mice; over-expression PLUTO in primary islets and Min6 cells could improve insulin biosynthesis and secretion; Western blot analysis showed that PLUTO promoted insulin secretion and synthesis by up-regulating the transcription and translation of Pdx1, which is the adjacent gene of PLUTO.
ABSTRACT
Aim To investigate the effects of Guaveno-ic acid (GA)on proliferation,insulin synthesis and secretion in INS-1 cells and their possible mechanism. Methods INS-1 βcells were cultured in vitro.Control group,medium group,model group,drug groups and positive group were set.INS-1 cells were treated with GA (0.3,1 ,3,1 0,30 nmol·L -1 )for 48 h.The cell proliferation was tested by MTT assay.Acid-alco-hol was used to extract the insulin in the cells and the amount of insulin synthesis of INS-1 cells was tested by RIA.5.6,1 6.7 mmol·L -1 glucose was used to chal-lenge INS-1 cells for 1 h to the insulin secretion model (BIS and GSIS)was tested,and the insulin secretion of INS-1 cells was tested via RIA.The mRNA expres-sion of insulin gene,PDX-1 and MafA was tested by q-PCR.Results Compared with medium group,GA could promote the proliferation of INS-1 cells signifi-cantly (P <0.01 )and promote the synthesis of insulin in INS-1 cells significantly (P <0.01 ).GA(0.3 ~30 nmol· L -1 )could promote the BIS,GSIS of INS-1 cells significantly (P <0.05 or P <0.01 ).GA (3,30 nmol·L -1 )could up-regulate the mRNA expression of insulin gene,PDX-1 ,MafA in INS-1 cells signifi-cantly (P <0.01 ).Conclusions GA could signifi-cantly improve the proliferation of INS-1 cells and pro-mote the insulin synthesis and secretion of INS-1 cells, which may be associated with up-regulation of insulin gene,PDX-1 ,MafA mRNA expression.
ABSTRACT
Objective To observe insulin synthesis and secretion in INS-1 under high glucose, and to clarify the effect of PTH1R. Methods After successful construction of recombinant PTH1R-siRNA vectors in INS-1 cell, insulin secretion and intracellular insulin content of control group, siPTH1R-Negative control group, PTHrP group, and siPTH1R group under 25 mmol/L glucose were measured by radioimmunoassay in INS-1 cell. Intracellular calcium were detected by Fluo-3/AM and the capability of glucose transport was calculated by assaying the uptake of [3H]-2-deoxy-D-glucose in cells.Results Compared with control group, and siPTH1R-NC group, PTHrP group showed increased capability of insulin secretion; PTHrP group had higher intracellular insulin levels than others; PTHrP group showed increased intracellular calcium; the uptake of [3H]-2-deoxy-D-glucose under high glucose after 48h of PTHrP group was increased(all P＜0.01). Conclusion There is a close relationship between PTH1R activation and insulin secretion and synthesis, PTH1R activation may be one of the protective mechanisms in maintaining function of β-cell under high glucose.