1.PDX-1 Involved In Insulin Secretion Under Leucine Exposure 2.Presence Of Adenosine A2a Receptor In Thyroid Cells And Its Effects On Vascular Endothelial Growth Factor

Part 1: PDX-1 INVOLVED IN INSULIN SECRETION UNDER LEUCINE EXPOSUREBackground:Type 2 diabetes mellitus is a kind of heterogeneous syndrome of polygenic origin and involves both defective insulin secretion and peripheral insulin resistance.β-cell dysfunction is a sine qua non for the development of the disease.Some metabolic factors such as hyperglycemia and hyperlipidemia contribute to the progressive deterioration of glucose homeostasis characteristic of this disease.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.Pancreatic/duodenal homeobox-1(PDX-1),a major transactivator of tightly regulating insulin and glucose transporter-2(GLUT2) at the transcriptional level,is an early and critical event in facilitating development and maintaining the function of pancreaticβ-cells.Its activation leads to insulin transcription potentiation that subsequently results in insulin synthesis increase.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.Numerous studies in vitro and in vivo have demonstrated that chronic exposure to glucose or fatty acid is able to suppress 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.Objectives: Effect of leucine on GSIS in pancreaticβ-celts is quite controversial,and mechanism involved in the effect has not been elucidated yet.Consequently,we aimed to investigate effect of leucine on GSIS and its mechanism focusing on contribution of PDX-1,GCK and GLUT2.Methods:Rat insulinomaβ-cells(DN-PDX-1^# 28 and PDX-1^# 6) were cultured with or without 40 mM leucine or 500ng/ml doxycycline for 48h.Results:To further prove that the PDX-1 plays an important role in impaired insulin secretion and content induced by chronic leucine,DN-PDX-1#28 and PDX-1#6 cells were treated with or without 500 ng/ml doxycycline in the presence or absence of 40 mM leucine for 48 hrs.1.glucose-induced insulin secretion and intracellular insulin contentIn DN-PDX-1#28 cells,in comparison with control,leucine alone treatment and doxycycline alone treatment diminished high glucose-induced insulin secretion by 36%and 84%(P＜0.05),decreased intracellular insulin content by 23%and 62%(P＜0.05),respectively.In comparison with leucine treatment alone group,doxycycline plus leucine treatment decreased high glucose-induced insulin secretion by 67%(P＜ 0.05) and intracellular insulin content by 44%(P＜0.05).In PDX-1#6 cells,compared with control,leucine alone treatment diminished high glucose-induced insulin secretion by 26%(P＜0.05) and insulin content by 25%(P＜0.05),doxycycline alone treatment increased high glucose-induced insulin secretion by 20%(P＜0.05) and insulin content by 21%(P＜0.05).However,relative to leucine alone treatment,doxycycline plus leucine treatment significantly increased GSIS by 36%(P＜0.05) and insulin content by 21%(P＜0.05).2.The mRNA expression of PDX-1,GCK and GLUT2In DN-PDX-1#28 cells,in contrast to control,40 mM leucine significantly diminished the mRNA level of PDX-1 and its downstream targets,GCK and GLUT2 (P＜0.05),doxycycline alone treatment decreased the mRNA level of PDX-1,GCK and GLUT2(P＜0.05).In comparison with leucine treatment alone group, doxycycline plus leucine treatment decreased PDX-1,GCK and GLUT2 mRNA level (P＜0.05).In PDX-1#6 cells,compared with control,leucine alone treatment diminished PDX-1,GCK and GLUT2 mRNA level(P＜0.05),doxycycline alone treatment increased the mRNA level of PDX-1,GCK and GLUT2(P＜0.05).In comparison with leucine treatment alone group,doxycycline plus leucine treatment enhanced PDX-1,GCK and GLUT2 mRNA level(P＜0.05).Conclusions:The study indicated that chronic leucine might result in a decrease in PDX-1,in turn to depress GCK and GLUT2 resulting in decreased GSIS at high glucose and insulin content. Part 2: PRESENCE OF ADENOSINE A2A RECEPTOR IN THYROCYTES AND ITS EFFECT ON VASCULAR ENDOTHELIAL GROWTH FACTORBackground:During the thyroid disease,the actions of growth factors,especially vascular endothelial growth factor(VEGF) and insulin like growth factor-1(IGF-1),are attached great importance to.It is thought growth factors can regulate the growth, differentiation and function of the thyroid cells through endocrine,autocrine, paracrine ways,and take part in the process of thyroid disease.VEGF is one of the most important soluble angiogenic factors.It can increase the microvascular permeability and facilitate to the exudation of plasma fibrin which offers fibre network for the movement of many cells.It also can stimulate the proliferation of endothelial cells and make it easy for endothelial cells to move.All are favorable for vascular formation.Currently,at least five different mRNA species encoding VEGF have been characterized.These variants result from alternative splicing of the VEGF transcript and encode human isoforms containing VEGF protein of 121,145,165,189,and 206 amino acids.In the rat,a similar profile of VEGF splice variants has been described,each encoding one fewer amino acid than the human species.IGF-1 has the functions of accelerating proliferation,differention and metabolism of many cells,such as endothelial cells and smooth muscle cells. Furthermore,IGF-1 is also involved in angiogenesis via increasing VEGF gene transcript and protein secretion in thyroid carcinomas.Our previous research showed that in patients with untreated GD,there was a significant increase in serum VEGF and IGF-1 levels,and localized VEGF and IGF-1 expression in thyroid tissues was higher in those patients compared with healthy subjects.The autoantibody(immunoglobulin G,IgG) against thyrotropin(TSH) receptor(TSHR),named thyrotropin receptor antibody(TRAb),is believed to act via the TSHR and mimic the role of TSH to bring biological effect through signaling cascades in thyroid and thus alter thyroid growth and function.In clinic,there are abundant patients with no TRAb developing goiter and hyperthyroidial toxic symptoms.Therefore,it is very important to study the mechanism of thyroid angiogenic goiter formation to find effective therapeutic targets for this disease.Hyperthyroidism raises activity of the purine nucleotide cycle and thyroid hormones,especially T3,stimulate the synthesis and consumption of ATP simultaneously.Endogenous adenosine,ligand of adenosine A2a receptor(A2aR),is an intermediate product of adenosine nucleotide metabolism,which is released from metabolically active cells by facilitated diffusion or is generated extracellularly by degradation of released ATP.Adenosine receptors,members of the G protein-coupled receptor super-family,represent a potential therapeutic target for regulation of neovascularization.The adenosine A2aR,one of adenosine receptors,is found on cardiac myocytes,immune and inflammatory cells and,very abundantly,on some cells in the central nervous system.Previous studies have also implicated adenosine A2aR in the regulation of VEGF expression in various cell types.The interest in targeting adenosine receptor in the realm of thyroid diseases first arose based on the mouse transgenic models that developed thyroid diseases.Ledent et al.reported thyroid expression of an A2 adenosine receptor induced thyroid hyperplasia and hyperthyroidism.The over-expression of adenosine A2aR(Tg-A2aR model) in the transgenic mice developed severe hyper-functioning and a large goiter. In goitrous Tg-A2aR mouse thyroid tissue,microvessels were mostly located in hyperplastic zones,VEGF proteins were more strongly expressed than in controls, and IGF-1 mRNA was observed to be up-regulated.However,less is known regarding A2aR activation in thyroid under various relative physiological microenvironments.Objectives:1.The mRNA and protein expresson of A2aR in thyroid cells;2.The mRNA expresson of A2aR and TSHR influenced by A2aR agonist or TSH;3.The effect of A2aR,TSH or IGF-1 on mRNA and protein expression levels of VEGF,and the effect of A2aR on mRNA and protein expression levels of IGF-1.Methods:1.Cell culture:FRTL-5 thyroid cells(ATCC catalog number CRTL-1486) were grown in 6H media consisting of Coon's modified Ham's F12 medium supplemented with 5%fetal bovine serum and a mixture of six hormones:bovine thyrotropin, insulin,hydrocortisol,transferrin,glycyl-L-histidyl-L-lysine acetate,and somatostatin. Fresh medium was added to all cells every 2 or 3 days,and cell passaging was done every 7-10 days.After cells were grown to 90%confluence in 6H media and washed twice with prewarmed phosphate-buffered solution(PBS),the cells continued incubation with 4H media containing0.2%FBS,and no thyrotropin,no insulin.In the individual experiments,cells maintained in 4H media were treated for 48 hours with or without A2aR selective agonist CGS21680 or antagonist ZM241385 or TSH, IGF-1.In addition,neuron cells in primary cultured Wister rat forebrain were cultured as previously described and used as positive control in the PCR assays for A2aR described here.2.The mRNA levels of A2aR,TSHR and IGF-1 were measured using Real-Time PCR and Reverse-Transcription(RT)-PCR;RT-PCR was performed to detect VEGF isoforms mRNA levels.3.Immunofluorescence was used to observe distribution of A2aR protein.4.Immunoprecipitation(IP)-western blotting was used to semi-quantify A2aR and IGF-1 protein expression.5.ELISA was used to quantify intra-and extra-cellular VEGF protein contents.Results:1.A2aR expression in FRTL-5After PCR amplification,a distinct band of 140 bp for A2aR was observed in the 6H and 4H cells.The real-time PCR results showed that in contrast to the control,A2aR mRNA expressions were enhanced by 25.3%(P＜0.05) at 1.0μM CGS2168,while diminished by 34.6%(P＜0.05) at 10mU/ml TSH.Immunoprecipitation-Western blot results showed that the A2aR protein level was increased by its selective agonist CGS21680 by about 1.1-fold(P＞0.05). Immunoreactivity for the A2aR protein was clearly observed in the cell membrane.2.TSHR mRNA expression in FRTL-5Results of the RT-PCR and real-time PCR showed that TSHR mRNA expression was decreased by 8.4%(P＞0.05) at 1.0 mU/ml and by 30.5%(P＜0.05) at 10 mU/ml TSH,while increased by 1.19-fold(P＞0.05) at 0.1μM and by 1.35-fold(P＜0.05) at 1.0μM CGS21680 compared with those of control cells.3.VEGF gene transcription in FRTL-5We successfully detected four isoforms in non-stimulated FRTL-5 cells:VEGF₁₈₈, VEGF₁₆₄,VEGF₁₄₄ and VEGF₁₂₀,while the VEGF₂₀₄ isoform was not detected.Of them,the productions of both VEGF₁₂₀ and VEGF₁₆₄ isoforms appeared to be predominant.â‘ A2aR agonist CGS21680(1μM and 10μM) exposure predominantly enhanced the levels of VEGF₁₈₈ and VEGF₁₆₄ isoforms by approximate 2-fold(both P＜0.05,respectively),in contrast to the corresponding control.â‘¡When cells were treated with TSH(10 mU/ml),VEGF₁₆₄ and VEGF₁₈₈ were decreased by 32.6%(P＜0.05) and 35.2%(P＜0.05) compared to the control.â‘¢Isoforms of VEGF₁₆₄ and VEGF₁₂₀ were increased by 1.8-fold and 2.1-fold(P＜0.05,respectively) by 50ng/ml IGF-1.4.VEGF protein production in FRTL-5â‘ Correlation analysis of VEGF protein contents intracellular and extracellular:The extracellular VEGF content in each group was approximately 22.3-fold higher than the intracellular VEGF,and the Pearson correlation analyses between them showed that the "R" value was 0.978(P＜0.05).â‘¡Effect of A2aR on VEGF protein expression in FRTL-5:In comparison with the control,the media VEGF contents were increased by 23.3%(P＜0.05) at 1.0μM CGS21680 stimulation,while strongly inhibited by 31.6%(P＜0.05) with 10μM antagonist ZM241385 alone.In addition,in the presence of 1.0 or 10μM A2aR inhibitor ZM241385,1μM agonist CGS21680 induced effects could be reduced: the extracellular decreased by 16.2%(P=0.065)～24.2%(P=0.007), respectively,compared to 1μM agonist-treated FRTL-5 cells.â‘¢Effect of TSH and IGF-1 on VEGF release in FRTL-5:After TSH incubation, there was 16.9%(P＜0.05) at 10 mU/ml decrease in the media VEGF content in contrast to non-stimulated cells.While there was 39.0%(P＜0.05) increase of VEGF content in media by 50ng/ml IGF-1 compared with the control.5.IGF-1 expression in FRTL-5 cellsRT-PCR and real-time PCR showed that 10 mU/ml TSH(P＜0.05) diminished,while 1.0μM CGS21680(P＜0.05) pretreatment increased IGF-1 mRNA level.IP-Western blot results showed that agonist CGS21680(1.0μM) increased IGF-1 protein level by 1.6-fold(P＜0.05),but antagonist ZM241385(1.0μM) reduced it by 17.4%(P＜0.05).Conclusions:1.The mRNA and protein of adenosine A2aR is expressed in thyroid FRTL-5 cell line, especially this receptor protein is expressed in the cytomembrane.2.Transcription of A2aR,TSHR and VEGF isoforms(especially VEGF₁₆₄) was enhanced by CGS21680 and diminished by TSH simultaneously,which can be inferred that there maybe a receptor cross-talk between the two G protein-coupled receptors,such as norientation associativity.3.A2aR regulates mRNA and protein expression of VEGF and IGF-1.IGF-1 increases VEGF mRNA and protein expression in FRTL-5 cells,especially VEGF₁₆₄, the most common form of VEGF in process of angiogenesis.Consequently,we can infer that A2aR induced upregulation of VEGF in the thyroid is tightly associated with IGF-1.A2aR might be involved in thyroid angoigenic goiter visa augmentation of VEGF and IGF-1.