| Background:Recently,studies have attached great importance for the effect of leucine on glucose-stimulated insulin secretion(GSIS) and intracellular insulin content in pancreaticβ-cells.However,up to now,the results from different research groups have been quite controversial.Yang and his colleagues demonstrated that leucine was able to enhance GSIS in pancreaticβ-cells.However,Anello et al.reported that chronic leucine exposure impaired GSIS in a dose-dependent manner.Moreover,the mechanism of leucine affecting insulin secretion and content has not been elucidated yet.Consequently,we aimed to investigate the effects of leucine on insulin secretion and content,also to explore the mechanism involved in the effects in rat insulinomaβ-cells.AMP-activated protein kinase(AMPK) acts as a cellular energy regulator activated by increased intracellular AMP-to-ATP ratio.GSIS fromβ-cells is directly related with the generation of metabolic intermediates,therefore,AMPK is deemed as an attractive candidate for control of insulin secretion and content.Many studies have reported that high glucose or fatty acid could change insulin secretion by controlling AMPK activity in pancreaticβ-cells.However,fewer studies pay attention to AMPK activity changes under chronic leucine exposure.Du and his colleagues reported that leucine stimulated mammalian target of rapamycin(mTOR) signaling by inhibition of AMPK activity[11],which suggested a possible association between leucine and AMPK.Glucokinase(GCK),an enzyme phosphorylating glucose to glucose-6-phosphate, acts as a glucose sensor and regulates insulin secretion.GLUT2 is an important component for insulin secretion as well.Tiedge and Lenzen reported in their studies that the concordant regulation of GCK and GLUT2 genes might represent the basis regulation of GSIS.In 2006,Yang et al.firstly reported that leucine culture altered GCK expression in INS-1 cells,rat islets and human islets,moreover,glucokinase contributed tight control of insulin secretion.Though AMPK,GCK and GLUT2 were separately reported to be associated with insulin secretion,the relationship between them under leucine exposure remains unclear.Kim et al.demonstrated that AMPK could regulate GCK and GLUT2 expression at high glucose concentration.On the basis of these reports,we supposed that chronic leucine exposure might influence insulin secretion and content by altering AMPK,GCK and GLUT2 expression,and there might be a regulatory relationship between AMPK,GCK and GLUT2 at high leucine concentration.In addition,how AMPK regulating GCK or GLUT2 was worthy of being investigated.Numerous studies in vitro and in vivo have demonstrated that chronic exposure to glucose or fatty acid is able to suppress pancreatic/duodenal homeobox-1(PDX-1) expression,leading to decreased insulin secretion.The role of PDX-1 in pancreaticβ-cell insulin secretion derives from its effect on transactivating the expression of insulin and otherβ-cell-specific genes,such as GCK and GLUT2.This promoted us to speculate that PDX- 1 might be the bridge between AMPK and GCK or GLUT2.Objective:1.To observe the effect of elevated concentrations of leucine on pancreatic beta cells insulin secretion and insulin content.2.To observe the effect of high concentration of leucine on AMPK,PDX-1,GCK/GLUT2 mRNA and protein expression,to explore the possible mechanisms.3.To observe the correlation of AMPK and PDX-1,GCK/GLUT2 in pancreatic beta cells4.To observe whether AMPK regulates GCK/GLUT2 via PDX-15.To observe whether chronic leucine exposure affects insulin secretion and insulin content via(AMPK)-(PDX-1)-(GCK/GLUT2) pathway.6.To estimate the effects of chronic leucine exposure on insulin secretion,insulin content and the protein expression of PDX-1 as well as its downstream target, GLUT2 in rat INS-1 cells,also to investigate whether they are reversible or not after removal of high concentrations of leucine.Research Design and Methods:1.Cell culture and treatment:Rat insulinoma cell lines,INS-1 and RIN m5F cells (passage 20-40) were grown in monolayer culture in RPMI 1640 medium treated with either 0.5 mM AICAR or 10 m M compound C,or supplemented with or without elevated concentrations of leucine(10,20 or 40 mM),for 48 hrs.INS-1 stable cell lines,DN-PDX-1#28 and PDX-1#6 cells were cultured in RPMI 1640, were treated with either AICAR or Compound C for 48 hrs.2.Insulin secretion and insulin content assays:INS-1 cells were pre-cultured in 24-well plates in standard medium for 24 hrs,and then were treated with either AICAR or compound C,supplemented with or without elevated concentrations of leucine(10,20 or 40 mM) for 48 hrs.In addition,intracellular insulin contents were tested in INS-1 and RINm5F cells,respectively.The cells were treated the same as in the insulin secretion section described.Total protein content was determined as described above.Insulin content was normalized based on the respective cellular protein in each group.3.Cell counting kit-8(CCK-8):INS-1,RIN m5F,DN-PDX-1#28 and PDX-1#6 cells were seeded in 96-well plates at a density of 104 cells/well for 24 hrs.The cell viability was tested by CCK-8 method.4.AMPK,PDX-1,GCK,GLUT2 detection in INS-1,RIN m5F cells:with Real-time PCR method to test AMPK,PDX-1,GCK,GLUT2mRNA expression;with Western blotting to detect AMPK,PDX-1,GCK,GLUT2 protein expression. 5.AMPK,PDX-1,GCK,GLUT2 detection in DN-PDX-1#28 and PDX-1#6 cells: with Real-time PCR method to test AMPK,PDX-1,GCK,GLUT2mRNA expression; with Western blotting to detect AMPK,PDX-1,GCK,GLUT2 protein expression.Results:1.The effects of AICAR or compound C in INS-1 and RIN m5F cellsThe results showed that in contrast to control,AICAR treatment decreased GSIS at high glucose by 29%(P＜0.05) and reduced the intracellular insulin content by 30%(P＜0.05) in INS-1 cells.In RIN m5F cells,after 48 hrs of AICAR incubation, there is a 34%decrease in the intracellular content compared with control(P＜0.05). When AMPK activity was inhibited by compound C,the results were quite opposite. In comparison with the corresponding control,compound C treatment was able to enhance GSIS at high glucose by 17%in INS-1 cells(P＜0.05) and the intracellular insulincontent by 19%in INS-1 cells(P＜0.05) and 25%in RIN m5F(P＜0.05). However,neither AICAR nor compound C could affect the insulin secretion level at low glucose(3 mM) stimulation(P＞0.05) in INS-1 cells.In contrast to control,AICAR significantly strengthened the band of p-AMPK and weakened the bands of PDX-1 and its downstream targets,GCK and GLUT2 in INS-1(P＜0.05) and RIN m5F cells(P＜0.05).Compared to the corresponding control,compound C obviously decreased AMPK activity and showed enhanced bands of PDX-1,GCK and GLUT2 in INS-1(P＜0.05) and RIN m5F cells(P＜0.05). The results were confirmed by real-time PCR as well.Compared to the corresponding control,AICAR decreased PDX-1 mRNA levels by 38%and 31%,GCK mRNA levels by 21%and 57%,GLUT2 mRNA levels by 51%and 44%in INS-1 cells(P＜0.05) and RIN m5F cells(P＜0.05),respectively.In contrast to the corresponding control,compound C increased PDX-1 mRNA levels by 36%and 55%,GCK mRNA levels by 23%and 24%,GLUT2 mRNA levels by 30%and 40%in INS-1(P＜0.05) and RIN m5F cells(P＜0.05),respectively.2.The effects of AICAR or compound C in DN-PDX-1#28 cellsUnder non-induced condition(without 500 ng/ml doxycycline treatment),the PDX-1 protein expression showed a stained band and there was no significant difference between 24 and 48 or 72 hrs(P＞0.05).Under induced condition(with 500ng/ml doxycycline treatment),PDX-1 protein band became weaker with time extension;furthermore,the weakest bands occurred in cells treated with doxycycline for 72 hrs(P＜0.05).Consistent with this,GLUT2 and GCK expression had the similar diminishing consequence with PDX-1 described above.Under non-induced condition for 24,48 and 72 hrs,protein bands intensity of both GCK and GLUT2 presented no change(P＞0.05).Under induced condition for 24,48 and 72 hrs,there was parallel decrease in the protein expression of GCK and GLUT2(P＜0.05).The results suggested that PDX-1 performed its function well in the regulation of GCK and GLUT2.In order to confirm the function of PDX-1 in the process of AMPK regulating GCK and GLUT2,DN-PDX-1#28 cells were pretreated with or without 500 ng/ml doxycycline for 24 hrs prior to treatment with or without AICAR or compound C for a further 48 hrs.Radioimmunoassay,Western blotting and real-time PCR were performed for the detection.In comparison with control,doxycycline alone treatment obviously decreased high glucose-induced insulin secretion by 88%(P＜0.05),insulin content by 82%(P＜0.05),and compound C alone treatment significantly increased high glucose-induced insulin secretion by 17%(P＜0.05) and insulin content by 19%(P＜0.05).However,compared with doxycycline alone treatment,doxycycline plus compound C treatment could not significantly affect insulin secretion or content(P＞0.05).With regard to p-AMPK,PDX-1,GCK and GLUT2 protein expression changes,under non-induced condition,AICAR or Compound C had the same effects as those observed in INS-1 cells or RIN m5F cells. However,under induced condition,p-AMPK protein expression had the same result as that under non-induced condition;with regard to PDX-1,GCK or GLUT2 protein expression,neither AICAR nor Compound C could affect them(P＞0.05).The results were confirmed by real-time PCRas well.3.The effects of elevated concentrations of leucine in INS-1 and RIN m5F cellsOur results demonstrated that a 48-hr incubation with elevated concentrations of leucine led to a dose-dependent decrease of GSIS at high glucose in INS-1 cells and insulin content in both INS-1 and RIN m5F cells.In INS-1 cells,in comparison with control,40 mM leucine exposure significantly decreased high glucose-induced insulin secretion by 34%(P＜0.05)and diminished the intracellular insulin content by 24% (P＜0.05),respectively,and there was not apparent change in insulin secretion induced by low glucose stimulation in INS-1 cells(P＞0.05).There was no difference of insulin secretion at both low and high glucose stimulations between control and either 10 mM or 20 mM leucine treatment(P＞0.05),and the same results occurred in insulin content as well in INS-1 cells(P＞0.05).With regard to RIN m5F cells,the insulin content presented the same trend as in INS-1 cells,40 mM leucine diminished the intracellular insulin content by 24%(P＜0.05) in contrast to its control.Western blotting and real-time PCR were performed for p-AMPK,PDX-1,GCK and GLUT2 detection.In contrast to control,40 mM leucine exposure significantly enhanced p-AMPK protein expression in INS-1 cells(P＜0.05) and RIN m5F cells(P＜0.05).The protein levels of PDX-1 and its downstream targets,GCK and GLUT2 in 40 mM leucine-treated cells were much lower than those in cells that were cultured in leucine-absent media in INS-1 cells(P＜0.05) and RIN m5F cells(P＜0.05).Compared with control,neither 10 mM nor 20 mM leucine culture could statistically change the expression of those proteins described above in INS-1 cells(P＞0.05)and RIN m5F cells(P＞0.05).The results above were confirmed by real-time PCR as well.4.Leucine cytotoxicity detection on pancreatic cell linesThe results showed that in INS-1,RIN m5F,DN-PDX-1#28 and PDX-1#6 cells,compared with its corresponding control,the cell viability of 10 mM leucine was 99%(P＞0.05),97%(P＞0.05),98%(P＞0.05),98%(P＞0.05),the cell viability of 20 mM leucine was 98%(P＞0.05),97%(P＞0.05),98%(P＞0.05),99%(P＞0.05),the cell viability of 40 mM leucine was 97%(P＞0.05),97%(P＞0.05),96% (P＞0.05),95%(P＞0.05).The results testified that neither 10,nor 20 nor 40 mM leucine had cytotoxicity on pancreatic cell lines.5.Effects of AICAR or compound C in INS-1 and RIN m5F cells with chronic leueine treatmentIn INS-1 and RINm5F cells,either leucine alone,AICAR alone or compound C alone treatment induced the same results.Moreover,in comparison with leucine treatment,leucine plus AICAR co-treatment diminished high glucose-induced insulin secretion by 21%in INS-1 cells(P＜0.05)and intracellular insulin content by 23%in INS-1 cells(P＜0.05),and 26%in RIN m5F cells(P＜0.05).Furthermore,the reduced insulin secretion and content caused by chronic high leucine treatment were significantly recovered by leucine plus compound C co-treatment.In contrast to leucine treatment,leucine plus compound C co-treatment increased high glucose-induced insulin secretion by 33%in INS-1 cells(P＜0.05)and intracellular insulin content by 24%in INS-1 cells(P＜0.05) and 29%in RIN m5F cells(P＜0.05),respectively.Neither AICAR nor compound C produced significant difference at low glucose stimulation in comparison with control in INS-1 cells(P＞0.05).The weak bands representing protein expression of PDX-1,GCK and GLUT2, respectively,appeared in 40 mM leucine alone and AICAR alone groups,and the weakest band occurred in 40 mM leucine plus AICAR co-treatment group compared with control(P＜0.05).The reduced protein expression of PDX-1,GCK and GLUT2 induced by chronic high leucine was recovered almost close to normal in leucine plus compound C co-treatment group(P＜0.05).The results were confirmed by real-time PCR in both INS-1 and RIN m5F cells.6.Effects of increasing concentrations of leueine on insulin secretion,content andβ-cell protein expressionOur results showed that a 24-h incubation with increasing concentrations of leucine led to a decrease of high glucose-stimulated insulin secretion and insulin content,and the effect was significant at 40 mmol/l.In contrast to control,40 mmol/l leucine treatment decreased high glucose-stimulated insulin secretion by 11%(P= 0.026) and insulin content by 14%(P=0.008),but it could not affect the insulin secretion level at low glucose stimulation(P=0.01).In contrast to control,neither 10 or 20 mmol/l leucine could produce significant decrease of insulin secretion(P= 0.645 or P=0.250) and insulin content(P=0.870 or P=0.279).PDX-1 protein band became weaker and weaker with concentration increasing and the effect was significant at 40 mmol/l leucine treatment(Fig.2A,P=0.013).Similarly,GLUT2 showed the same trend as PDX-1 and the weakest band occurred in cells treated with 40 mmol/l leucine(Fig.2B,P=0.011).7.Effects of 24 h recovery in standard medium on insulin secretion,content andβ-cell protein expressionThe results indicated that in contrast to its corresponding control,40 mmol/l leucine treatment for 24 h decreased GSIS at high glucose by 11%(P=0.026) and insulin content by 14%(P=0.008),and 40 mmol/l leucine treatment for 48 h decreased GSIS at high glucose by 22%(P=0.003) and insulin content by 20%(P= 0.002).When removing leucine from media for an additional 24 h incubation,in comparison with those in cells that maintained in leucine treatment for 24 h or 48 h, the high glucose-induced insulin secretion was increased by 13%(P=0.032) and 27%(P=0.002),and insulin content was augmented by 10%(P=0.014) and 20% (P=0.003),respectively.The results showed that in contrast to control,leucine treatment for 24 h decreased PDX-1 and GLUT2 protein expression,and the effects were quite significant in cells treated with leucine for 48 h.The reduced PDX-1 and GLUT2 protein expression induced by leucine treatment for 24 h was almost recovered to normal in cells firstly treatment with leucine for 24 h and then was allowed for 24 h recovery in standard medium(P=0.013;P=0.015).In comparison with cells that maintained in leucine treatment for 48 h,the PDX-1 and GLUT2 protein bands in cells with 24 h recovery in standard medium was significantly strengthened(P=0.005;P=0.006).Conclusions:1.Under nomal condition,AMPK regulates GCK/GLUT2 via PDX-1.2.AMPK regulates GCK/GLUT2 via PDX-1 under high leucine exposure as well.3.Chronic leucine might result in an increase in AMPK and then a decrease in PDX-1,in turn to depress GCK and GLUT2 resulting in decreased GSIS at high glucose and insulin content.4.Chronic high concentrations of leucine exposure for 24 h is able to induce reversible impairment of high glucose-induced insulin secretion,insulin content and PDX-1,GLUT2 expression in INS-1 cells. |
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