《Different concentrations of palmitic acid on the INS- cell function and apoptosis.doc》由会员分享，可在线阅读，更多相关《Different concentrations of palmitic acid on the INS- cell function and apoptosis.doc（12页珍藏版）》请在三一文库上搜索。
1、 Different concentrations of palmitic acid on the INS-1 cell function and apoptosis Of: Guo Yaju, MO Zhaohui, Chen Branch, Xie Xiaoyun Abstract Objective To investigate the concentrations of palmitic acid at different times on the INS-1 cell function and apoptosis. Methods to 0,0.1,0.2,0.3,0.4 mmol
2、/ L palmitic acid and pancreatic INS-1 cells 6h, 24h, using MTT, flow cytometry and RT-PCR, INS-1 cells in islet cell viability, apoptosis and insulin gene expression and insulin secretion test. The results of islet INS-1 cells in the palmitic acid in 6h culture, with the palmitic acid concentration
3、, the basal insulin secretion increased (P <0.05), apoptosis was no significant change, and intervention by 24h, palmitic acid, with palmitic acid concentration, insulin INS-1 cells reduced basal insulin secretion, apoptosis was significantly increased, decreased insulin gene expression. Conclusi
4、on Long-term high-fat prejudice activity of INS-1 cell function, promoting -cell apoptosis. Keywords: palmitic acid; islet INS-1 cells; lipotoxicity Obesity is an important type 2 diabetes risk factors, both associated with marked hyperinsulinemia, insulin resistance and Type 2 diabetes, abnormal li
5、pid metabolism disorder of glucose metabolism in one of the reasons was lipid metabolism, so fat the more toxic and more attention. abnormal lipid metabolism has been suggested now start earlier than the increase of blood sugar may be the initial factor for diabetes, it is proposed to diabetes to “l
6、ipid” disease, but also suggested that hyperglycemia is the occurrence of toxic lipid preconditions, in the absence of high blood sugar, lipid toxicity is difficult to occur. Therefore, this study observed at different concentrations of palmitic acid and at different times of the INS-1 pancreatic is
7、let cell function and apoptosis. 1 Materials and methods 1.1 Materials and reagents islet INS-1 cell line (Wuhan cell collection centers); palmitic acid (sigma company), insulin radioimmunoassay kit (Beijing Chemclin biotechnology company), RT-PCR reagents required (Huamei Company ), the cell cultur
8、e medium (Bibico sugar), fetal calf serum (Evergreen companies). 1.2 Experimental Methods 1.2.1 Cell culture and experimental group rats were insulinoma INS-1 cells containing 10% FBS in RPMI1640 complete medium (10% fetal bovine serum, 11.2mmol / L glucose, 10mmol / L Hepes, 1mmol / L pyruvate sodi
9、um, 50moL / L -mercaptoethanol, 100/ml penicillin and streptomycin 100ug/ml) in culture medium was changed 1 day, 7 days passage 1, cells in logarithmic growth phase for experiments. Check the growth of good cells in INS-1, inoculated in 6 well plates, cultured cell fusion 60% to 70% in the control
10、group (control) plus the complete medium without palmitic acid 2ml, 0.1mmol experimental groups were added with / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L palmitic acid in the complete medium 2ml, each hole located 4 replicates, 37 C, 5% CO2 under the conditions of incubation. 1.2.2 Determination of
11、 insulin in each group were incubated for 6h, 24h supernatants were collected, -20 C preserved, with the insulin content was measured by radioimmunoassay. 1.2.3 MTT cell viability in each well 5000 cells were seeded into 96-well plates, adherent cells after 2 days to be added 5mg/ml PBS configuratio
12、n of MTT 20l, incubated for 4h, termination of culture, supernatant discarded smoking, add 150l DMSO, shaker shock 10min, enzyme-linked immunosorbent determination of detector wavelength on the absorbance of 570nm. 1.2.4 Detection of apoptosis would interfere with the completion of the cell into sin
13、gle cell suspension, 1000r/min centrifugal 5min, supernatant; pre-cooled at 4 , 70% cold ethanol, fixed, hanging from the cell, tight seal with the sealing film , 4 preservation; adjusting the cell concentration of 106 cells / ml, take 1ml cell suspension, washed 3 times with PBS, cells resuspended
14、in 1ml PI dye liquor, 37 30min incubation flow analysis can be carried out; PI dye final concentration of 50g/ml, RNase A final concentration of 20g/ml; flow cytometry MCYCLE software analysis system calculates the normal, apoptosis and the percentage of dead cell populations. 1.2.5 RT-PCR detection
15、 of insulin mRNA expression in INS-1 cells, respectively, after including 0mmol / L, 0.1mmol / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L palmitic acid in the complete medium cultured 24h, washed 2 times with PBS after the other with the Trizol reagent to extract total RNA, 2l taking the total RNA, wi
16、th Oligo (dT) 18primer primer AMW in 25l reverse transcription reaction using reverse transcription first strand cDNA synthesis, INS-1 cells according to cDNA sequence of insulin binding to GADPH as internal reference; insulin primer sequences were: upstream 5-CCAGGCTTTTGTCAAACAGCA-3 , downstream 5-
17、ACGGGACTTGGGTGTGTATAGAA-3; GADPH primer sequence was 5-TGGGTGTGAACCACGAGAA-3 , downstream primer was 5-GGCATGGACTGTGGTCATGA-3 ; reaction conditions: 94 for denaturing 3min; 94 3 s, 54 40s, 72 40s, 30 cycles; 72 and then extended 5min. 1.3 Statistical analysis All data were repeated three times to x
18、+-s, said the results of application of SPSS 10.0 software processing, the number in each group after the test of homogeneity of variance among groups using single factor analysis of variance. 2 Results 2.1 with different concentrations of palmitic acid on the basis of INS-1 cells in dynamic changes
19、 of insulin secretion of INS-1 cells at 0.1mmol / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L of palmitic acid in the complete culture INS-1 cell culture-based 6h, basal insulin secretion compared with the control group increased (P <0.05), no difference among the groups; culture 24h after the 0.1mm
20、ol / L, 0.2mmol / L, 0.3mmol / L basal insulin secretion gradually decreased, 0.4mmol / L PA, than 0.2,0.3 mmol / L PA, increase (see Table 1), and statistically significant (P <0.01). Table 1 with different concentrations of palmitic acid at different times of the INS- 1 cells, basal insulin sec
21、retion Note: Compared with the control group, * P <0.05, compared with 0.1PA group P <0.01,0.4 PA 0.2,0.3 PA group than in the group and # P <0.01 2.2 after the intervention of different concentrations of PA intervention MTT test 6h, palmitic acid, 0.1mmol / l MTT value of the low concentra
22、tion of the experimental group and control group no significant difference P> 0.05, while higher concentration (0.2mmol/LPA, 0.3mmol/LPA, 0.4 mmol / LPA) MTT values compared with the control group was significantly lower (P <0.05), while higher concentration see no significant difference betwe
23、en groups, but by the intervention 24h, different concentrations of palmitic acid group than the control group OD value significantly decreased (P <0.05), and with the palmitic acid concentration was regressive. Table 2, after the intervention of different concentrations of PA OD values Note: Com
24、pared with the control group * P value <0.05, compared between two groups P <0.05, statistically significance 2.3 Flow cytometry palmitic acid on the INS-1 cell apoptosis compared with the control group, INS-1 cells in different concentrations of palmitic acid in cultured 6h, no significant ch
25、anges of apoptosis, and 24h, with training with increasing concentrations of palmitic acid, apoptosis rate increased. Table 3 at different times with different concentrations of PA INS-1 cells when the rate (%) (x +-s) Note: Compared with the control group, * P value < 0.05, compared among groups
26、 P <0.01, statistically significant 2.4 palmitic acid on the INS-1 cell insulin gene expression in islets of palmitic acid intervention in INS-1 cells after 6h, compared with the control group, the concentration of insulin gene mRNA expression was not significantly change, and after 24h of insuli
27、n palmitic acid intervention gene mRNA expression was significantly decreased. Note: The figure 1,2,3,4,5 represent the concentration of palmitic acid 0mmol / L, 0.1mmol / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L , A group of representatives of PA intervention 6h, B 24h group intervention on behalf
28、of PA Figure 1, INS-1 palmitic acid on the impact of insulin mRNA Links to free paper download http:/ 3 Discussion INS-1 used in this study cells from the rat insulinoma cell line, can stabilize the secretion of insulin, insulin -cells can be used to study the ideal cell, in order to further clarify
29、 the free fatty acid on lipid INS-1 cell toxicity, the Experimental observation of different concentrations of palmitic acid at different times on the INS-1 islet cell function and apoptosis. The results showed that in a short time (6h), the free fatty acids can stimulate islet INS-1 cells secrete i
30、nsulin in a dose dependent nature, which may be short of free fatty acids can stimulate islet cell exocytosis-related; 6h, palmitic acid intervention no significant changes in insulin gene expression, further evidence of short-term free fatty acids stimulate insulin secretion by cells to achieve exo
31、cytosis . At the same flow cytometry no significant change in apoptosis rate, but decreased cell viability MTT shows, and 24h, the role of free fatty acids, INS-1 cell apoptosis was significantly increased, with increasing concentrations of palmitic acid, cells apoptosis rate increased. while insuli
32、n secretion decreased, but was observed in this experiment 0.4mmol / L PA increased when the basal insulin secretion may be related to palmitic acid concentration, INS-1 cells more, the occurrence of self- solution, insulin release into the culture medium on. and studies have shown 1, exposure to 0.
33、4mol / L palmitic acid observed in 12h, the cell autolysis, this study supports the conclusions of this study, phosphate-mTOR is inhibition of autolysis of a classical signal pathway activation in INS-1 cells after PA treatment, the observed decreased P-mTOR; free fatty acid oxidation in cells is us
34、ually non-toxic 9, but the long chain of coenzyme derivatives such as esters or triglycerides, lysophosphatidylcholine, they can promote nerve sheath lipid -cell apoptosis 2; ceramide have been proposed for free fatty acids may be mediated by -cell apoptosis in the media 3 6; endoplasmic reticulum s
35、tress and oxidative stress is mediated by free fatty acids a key factor in cell apoptosis, has tips and cell autolysis of the 7,9. PA role in pancreatic islet cells for 24h, INS-1 cells reduced basal insulin secretion and insulin gene from the results of RT-PCR analysis, a long time because of the r
36、ole of palmitic acid in INS-1 cells, the insulin gene transcription and translation decrease, studies have reported the role of free fatty acids in the short and long INS-1 cells induced changes in cell function is completely different mechanisms and through the signaling pathway induced. PA role in
37、 pancreatic islet cells for 24h, INS-1 cells reduced basal insulin secretion and insulin gene from the results of RT-PCR analysis, a long time because of the role of palmitic acid in INS-1 cells, the insulin gene transcription and translation decrease, studies have reported the role of free fatty ac
38、ids in the short and long INS-1 cells induced changes in cell function is completely different mechanisms and through the signaling pathway induced. The results of this study short and long observation of free fatty acids on the INS- different functions of a cell mechanisms need further study, but a
39、 long high free fatty acid can promote apoptosis of islet cells, islet cells can occur even autolysis. References 1 Sung-E Choi, Sung-Mi Lee, Youn-Jung Lee, et al.Protective role of autophagy in palmitate-induced INS-1 beta cell death. Endocrinology, 2008,150 (1) :126-134. 2 Assimacopoulos-Jeannet F
40、. Fat storage in pancreas and in insulin-sensitive tissues in pathogenesis of type 2 diabetes. International Joural of Obesity ,2004,25:53-57. 3 Lupi R, Dotta F, Marselli L, et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence th
41、at beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl- 2 regulated. Diabetes, 2002,51:1437-1442. 4 Maedler K, Spinas GA, Dyntar D, et al.Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function. Diabetes ,2001,50:69-76. 5 Shi
42、mabukuro M, Zhou YT, Levi M, et al.Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci USA, 1998, 95:2498-2502. 6 Okuyama R, Fujiwara T, Ohsumi J. High glucose potentiates palmitate-induced NO-Mediated cytotoxicity through generation of superoxide in clona
43、l beta-cell HIT-T15. FEBS Lett ,2003,545:219-223. 7 Hansen M. Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and Ca2 +. Cell death and differentiation ,2007,14:1576-1582. 8 Scherz-Shouval R, Elazar Z. ROS, mitochondria and the regulation of autophagy. Trends Cell Biol ,2007,17:422-427. 9 Vincent Poitout, Derek Hagman. Regulation of the Insulin Gene by Glucose and Fatty Acids. J Nutr, 2006,36 (4): 873-876. Links to free paper download http:/12