RESUMO
Acute pancreatitis (AP) is serious inflammatory disease of the pancreas. Accumulating evidence links diabetes with severity of AP, suggesting that endogenous insulin may be protective. We investigated this putative protective effect of insulin during cellular and in vivo models of AP in diabetic mice (Ins2Akita) and Pancreatic Acinar cell-specific Conditional Insulin Receptor Knock Out mice (PACIRKO). Caerulein and palmitoleic acid (POA)/ethanol-induced pancreatitis was more severe in both Ins2Akita and PACIRKO vs control mice, suggesting that endogenous insulin directly protects acinar cells in vivo. In isolated pancreatic acinar cells, insulin induced Akt-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) which upregulated glycolysis thereby preventing POA-induced ATP depletion, inhibition of the ATP-dependent plasma membrane Ca2+ ATPase (PMCA) and cytotoxic Ca2+ overload. These data provide the first mechanistic link between diabetes and severity of AP and suggest that phosphorylation of PFKFB2 may represent a potential therapeutic strategy for treatment of AP.
Assuntos
Células Acinares/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Glicólise/efeitos dos fármacos , Insulina/metabolismo , Insulina/farmacologia , Pancreatite/metabolismo , Substâncias Protetoras/farmacologia , Células Acinares/efeitos dos fármacos , Doença Aguda , Animais , ATPases Transportadoras de Cálcio/metabolismo , Ceruletídeo , Diabetes Mellitus Experimental/metabolismo , Ácidos Graxos Monoinsaturados , Masculino , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/patologiaRESUMO
BACKGROUND & AIMS: Chronic amino acid (AA) deficiency, as in kwashiorkor, reduces the size of the pancreas through an effect on mammalian target of rapamycin complex 1 (mTORC1). Because of the physiological importance of AAs and their role as a substrate, a stimulant of mTORC1, and protein synthesis, we studied the effect of acute protein and AA deficiency on the response to feeding. METHODS: ICR/CD-1 mice were fasted overnight and refed for 2 hours with 4 different isocaloric diets: control (20% Prot); Protein-free (0% Prot); control (AA-based diet), and a leucine-free (No Leu). Protein synthesis, polysomal profiling, and the activation of several protein translation factors were analyzed in pancreas samples. RESULTS: All diets stimulated the Protein Kinase-B (Akt)/mTORC1 pathway, increasing the phosphorylation of the kinase Akt, the ribosomal protein S6 (S6) and the formation of the eukaryotic initiation factor 4F (eIF4F) complex. Total protein synthesis and polysome formation were inhibited in the 0% Prot and No Leu groups to a similar extent, compared with the 20% Prot group. The 0% Prot diet partially reduced the Akt/mTORC1 pathway and the activity of the guanine nucleotide exchange factor eIF2B, without affecting eIF2α phosphorylation. The No Leu diet increased the phosphorylation of eIF2α and general control nonderepressible 2, and also inhibited eIF2B activity, without affecting mTORC1. Essential and nonessential AA levels in plasma and pancreas indicated a complex regulation of their cellular transport mechanisms and their specific effect on the synthesis of digestive enzymes. CONCLUSIONS: These studies show that dietary AAs are important regulators of postprandial digestive enzyme synthesis, and their deficiency could induce pancreatic insufficiency and malnutrition.
Assuntos
Insuficiência Pancreática Exócrina/etiologia , Leucina/deficiência , Pâncreas/patologia , Deficiência de Proteína/complicações , Animais , Dieta com Restrição de Proteínas/efeitos adversos , Modelos Animais de Doenças , Fator de Iniciação 2 em Eucariotos/metabolismo , Insuficiência Pancreática Exócrina/patologia , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Pâncreas/enzimologia , Fosforilação , Período Pós-Prandial , Biossíntese de Proteínas , Deficiência de Proteína/patologiaRESUMO
Endogenous CCK plays an important role in pancreatic regeneration after pancreatitis. We used primary culture of mouse pancreatic acinar cells to evaluate the effect of CCK on acinar cell morphology and gene expression and to determine signaling pathways required for proliferation of acinar cells in vitro. Over 4 days in culture, cells grew out from acini and formed patches of monolayer, which displayed a reduced expression of acinar cell markers including digestive enzymes and Mist1 and an increased expression of ductal and embryonic markers, including cytokeratin 7, ß-catenin, E-cadherin, pdx-1, and nestin. There was no appearance of stellate cell markers. CCK enhanced cellular spreading, DNA synthesis, and cyclin D1 expression. When signaling pathways were evaluated, CCK stimulation increased c-Jun expression, JNK and ERK activity, and AP-1 activation. Chemical inhibitors of JNK and ERK pathways, dominant-negative JNK and c-Jun, and c-Jun shRNA significantly inhibited CCK-induced DNA synthesis, CCK-induced AP-1 activation, and cyclin D1 expression. Furthermore, dominant-negative c-Jun reduced the increased expression of ß-catenin and the decreased expression of amylase during culture. These results show that MAPK/c-Jun/AP-1 pathway plays an important role in pancreatic acinar cell dedifferentiation and proliferation in culture. Monolayer culture can serve as a model to study acinar cell proliferation similar to regeneration after pancreatitis in vivo.
Assuntos
Células Acinares/metabolismo , Desdiferenciação Celular/fisiologia , Colecistocinina/farmacologia , Replicação do DNA/fisiologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Células Acinares/efeitos dos fármacos , Amilases/metabolismo , Animais , Caderinas/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina D1/metabolismo , Replicação do DNA/efeitos dos fármacos , Camundongos , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , beta Catenina/metabolismoRESUMO
Adaptive exocrine pancreatic growth is mediated primarily by dietary protein and the gastrointestinal hormone cholecystokinin (CCK). Feeding trypsin inhibitors such as camostat (FOY-305) is known to induce CCK release and stimulate pancreatic growth. However, camostat has also been reported to stimulate secretin release and, because secretin often potentiates the action of CCK, it could participate in the growth response. Our aim was to test the role of secretin in pancreatic development and adaptive growth through the use of C57BL/6 mice with genetic deletion of secretin or secretin receptor. The lack of secretin in the intestine or the secretin receptor in the pancreas was confirmed by RT-PCR. Other related components, such as vasoactive intestinal polypeptide (VIP) receptors (VPAC(1) and VPAC(2)), were not affected. Secretin increased cAMP levels in acini from wild-type (WT) mice but had no effect on acini from secretin receptor-deleted mice, whereas VIP and forskolin still induced a normal response. Secretin in vivo failed to induce fluid secretion in receptor-deficient mice. The pancreas of secretin or secretin receptor-deficient mice was of normal size and histology, indicating that secretin is not necessary for normal pancreatic differentiation or maintenance. When WT mice were fed 0.1% camostat in powdered chow, the pancreas doubled in size in 1 wk, accompanied by parallel increases in protein and DNA. Camostat-fed littermate secretin and secretin receptor-deficient mice had similar pancreatic mass to WT mice. These results indicate that secretin is not required for normal pancreatic development or adaptive growth mediated by CCK.
Assuntos
Pâncreas Exócrino/crescimento & desenvolvimento , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Secretina/metabolismo , Células Acinares/metabolismo , Animais , Colecistocinina/metabolismo , AMP Cíclico/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pâncreas Exócrino/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores dos Hormônios Gastrointestinais/genética , Secretina/genética , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
The endoplasmic reticulum (ER) is a multifunctional intracellular organelle responsible for the synthesis, processing and trafficking of a wide variety of proteins essential for cell growth and survival. Therefore, comprehensive characterization of the ER proteome is of great importance to the understanding of its functions and has been actively pursued in the past decade by scientists in the proteomics field. This review summarizes major proteomic studies published in the past decade that focused on the ER proteome. We evaluate the data sets obtained from two different organs, liver and pancreas each of which contains a primary cell type (hepatocyte and acinar cell) with specialized functions. We also discuss how the nature of the proteins uncovered is related to the methods of organelle purification, organelle purity and the techniques used for protein separation prior to MS. In addition, this review also puts emphasis on the biological insights gained from these studies regarding the molecular functions of the ER including protein synthesis and translocation, protein folding and quality control, ER-associated degradation and ER stress, ER export and membrane trafficking, calcium homeostasis and detoxification and drug metabolism.
Assuntos
Retículo Endoplasmático/química , Proteômica , Animais , Bovinos , Cricetinae , Bases de Dados de Proteínas , Cães , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Camundongos , Proteômica/métodos , RatosRESUMO
The rough endoplasmic reticulum (RER) is a central organelle for synthesizing and processing digestive enzymes and alteration of ER functions may participate in the pathogenesis of acute pancreatitis (AP). To comprehensively characterize the normal and diseased RER subproteome, this study quantitatively compared the protein compositions of pancreatic RER between normal and AP animals using isobaric tags (iTRAQ) and 2D LC-MALDI-MS/MS. A total of 469 unique proteins were revealed from four independent experiments using two different AP models. These proteins belong to a large number of functional categories including ribosomal proteins, translocon subunits, chaperones, secretory proteins, and glyco- and lipid-processing enzymes. A total of 37 RER proteins (25 unique in arginine-induced, 6 unique in caerulein-induced and 6 common in both models of AP) showed significant changes during AP including translational regulators and digestive enzymes, whereas only mild changes were found in some ER chaperones. The six proteins common to both AP models included a decrease in pancreatic triacylglycerol lipase precursor, Erp27, and prolyl 4-hydroxylase beta polypeptide as well as a dramatic increase in fibrinogen alpha, beta and gamma chains. These results suggest that the early stages of AP involve changes of multiple RER proteins that may affect the synthesis and processing of digestive enzymes.
Assuntos
Retículo Endoplasmático/metabolismo , Pâncreas/metabolismo , Pancreatite/metabolismo , Proteômica , Doença Aguda , Animais , Cromatografia Líquida , Imuno-Histoquímica , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
TGF-beta is an important regulator of growth and differentiation in the pancreas and has been implicated in pancreatic tumorigenesis. We have recently demonstrated that TGF-beta can activate protein kinase A (PKA) in mink lung epithelial cells (Zhang L, Duan C, Binkley C, Li G, Uhler M, Logsdon C, Simeone D. Mol Cell Biol 24: 2169-2180, 2004). In this study, we sought to determine whether TGF-beta activates PKA in pancreatic acinar cells, the mechanism by which PKA is activated, and PKA's role in TGF-beta-mediated growth regulatory responses. TGF-beta rapidly activated PKA in pancreatic acini while having no effect on intracellular cAMP levels. Coimmunoprecipitation experiments demonstrated a physical interaction between a Smad3/Smad4 complex and the regulatory subunits of PKA. TGF-beta also induced activation of the PKA-dependent transcription factor CREB. Both the specific PKA inhibitor H89 and PKI peptide significantly blocked TGF-beta's ability to activate PKA and CREB. TGF-beta-mediated growth inhibition and TGF-beta-induced p21 and SnoN expression in pancreatic acinar cells were blocked by H89 and PKI peptide. This study demonstrates that this novel cross talk between TGF-beta and PKA signaling pathways may play an important role in regulating TGF-beta signaling in the pancreas.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Pâncreas Exócrino/citologia , Pâncreas Exócrino/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas Exócrino/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.
Assuntos
Colecistocinina/sangue , Gabexato/análogos & derivados , Mitose/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Administração Oral , Animais , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/análise , Ésteres , Fatores de Iniciação em Eucariotos , Gabexato/administração & dosagem , Gabexato/farmacologia , Expressão Gênica/efeitos dos fármacos , Guanidinas , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Tamanho do Órgão/efeitos dos fármacos , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas/análise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas/metabolismo , Serina-Treonina Quinases TOR , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Endogenous CCK release induced by a synthetic trypsin inhibitor, camostat, stimulates pancreatic growth; however, the mechanisms mediating this growth are not well established. Early response genes often couple short-term signals with long-term responses. To study their participation in the pancreatic growth response, mice were fasted for 18 h and refed chow containing 0.1% camostat for 1-24 h. Expression of 18 early response genes were evaluated by quantitative PCR; mRNA for 17 of the 18 increased at 1, 2, 4, or 8 h. Protein expression for c-jun, c-fos, ATF-3, Egr-1, and JunB peaked at 2 h. Nuclear localization was confirmed by immunohistochemistry of c-fos, c-jun, and Egr-1. Refeeding regular chow induced only a small increase of c-jun and none in c-fos expression. JNKs and ERKs were activated 1 h after camostat feeding as was the phosphorylation of c-jun and ATF-2. AP-1 DNA binding evaluated by EMSA showed a significant increase 1-2 h after camostat feeding with participation of c-jun, c-fos, ATF-2, ATF-3, and JunB shown by supershift. The CCK antagonist IQM-95,333 blocked camostat feeding-induced c-jun and c-fos expression by 67 and 84%, respectively, and AP-1 DNA binding was also inhibited. In CCK-deficient mice, the maximal response of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively. These results indicate that camostat feeding induces a spectrum of early response gene expression and AP-1 DNA binding and that these effects are mainly CCK dependent.
Assuntos
Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Pâncreas/efeitos dos fármacos , Inibidores da Tripsina/farmacologia , Fator 2 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Carbamatos/farmacologia , Colecistocinina/deficiência , Colecistocinina/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Ésteres , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gabexato/análogos & derivados , Gabexato/farmacologia , Guanidinas , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Pirimidinonas/farmacologia , Receptores da Colecistocinina/antagonistas & inibidores , Fator de Transcrição AP-1/metabolismoRESUMO
Feeding stimulates pancreatic digestive enzyme synthesis at the translational level, and this is thought to be mediated by hormones and neurotransmitters. However, BCAAs, particularly leucine, stimulate protein synthesis in several tissues. We investigated whether BCAA stimulated the translational machinery in murine pancreas and whether their effects were independent of hormones. Rats and mice were administered (i.g. gavage) individual BCAA at 1.35 mg/g (body weight) and rat isolated pancreatic acini were incubated with BCAA under different conditions. Activation of translation initiation factors and total protein synthesis were analyzed. BCAA gavage stimulated the phosphorylation of the initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase (S6K), with leucine being the most effective. Leucine also increased the association of the initiation factors eIF4E and eIF4G, but did not affect the activity of the guanine nucleotide exchange factor eIF2B, nor total protein synthesis. BCAA acted independently of insulin signaling on isolated pancreatic acini from diabetic rats. The ability of leucine to promote phosphorylation of 4E-BP1 and S6K as well as enhance the assembly of the eIF4F complex was unimpaired in CCK-deficient mice. Finally, rapamycin (0.75 mg/kg) administered to rats 2 h before leucine gavage inhibited the phosphorylation of S6 and 4E-BP1 induced by leucine. We conclude that leucine may participate, as a signal as well as a substrate, in activating the translational machinery in pancreatic acinar cells independently of hormonal effects and that this action is through the mTOR pathway.
Assuntos
Aminoácidos de Cadeia Ramificada/farmacologia , Proteínas de Ligação a DNA/biossíntese , Leucina/farmacologia , Pâncreas Exócrino/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas/biossíntese , Fatores de Transcrição/biossíntese , Aminoácidos de Cadeia Ramificada/sangue , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Colecistocinina/deficiência , Colecistocinina/fisiologia , Proteínas de Ligação a DNA/metabolismo , Interações Medicamentosas , Imunossupressores/farmacologia , Insulina/sangue , Leucina/metabolismo , Leucina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Pâncreas Exócrino/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Fatores de Transcrição/metabolismoRESUMO
Endoplasmic reticulum (ER) stress mechanisms have been found to play critical roles in a number of diseases states, such as diabetes mellitus and Alzheimer disease, but whether they are involved in acute pancreatitis is unknown. Here we show for the first time that all major ER stress sensing and signaling mechanisms are present in exocrine acini and are activated early in the arginine model of experimental acute pancreatitis. Pancreatitis was induced in rats by intraperitoneal injection of 4.0 g/kg body wt arginine. Pancreatitis severity was assessed by analysis of serum amylase, pancreatic trypsin activity, water content, and histology. ER stress-related molecules PERK, eIF2alpha, ATF6, XBP-1, BiP, CHOP, and caspase-12 were analyzed. Arginine treatment induced rapid and severe pancreatitis, as indicated by increased serum amylase, pancreatic tissue edema, and acinar cell damage within 4 h. Arginine treatment also caused an early activation of ER stress, as indicated by phosphorylation of PERK and its downstream target eIF2alpha, ATF6 translocation into the nucleus (within 1 h), and upregulation of BiP (within 4 h). XBP-1 splicing and CHOP expression were observed within 8 h. After 24 h, increased activation of the ER stress-related proapoptotic molecule caspase-12 was observed along with an increase in caspase-3 activity and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL) staining in exocrine acini. These results indicate that ER stress is an important early acinar cell event that likely contributes to the development of acute pancreatitis in the arginine model.
Assuntos
Arginina , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/metabolismo , Pancreatite/patologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Pancreatite/induzido quimicamente , Ratos , Ratos WistarRESUMO
OBJECTIVE: We have recently reported that pancreatic growth driven by cholecystokinin released endogenously by feeding the synthetic trypsin inhibitor camostat requires the Ca-activated phosphatase calcineurin. In the present study, we evaluated a number of signal transduction pathways for their activation as part of the growth response and whether their activation was dependent on calcineurin. METHODS: Male ICR mice were fed with either chow or chow plus 1 mg/g of camostat. FK506 was administered at 3 mg/kg. After various times from 12 hours to 10 days, pancreatic samples were prepared and assayed for activity of various signal transduction pathway components. RESULTS: Camostat feeding increased the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and phosphorylation of the translation factor eukaryotic initiation factor 4E and activated the mammalian target of rapamycin pathway that leads to phosphorylation of the ribosomal protein S6 and of the eukaryotic initiation factor 4E binding protein but with different time courses. Treatment of mice with the calcineurin inhibitor FK506 totally blocked c-Jun NH2-terminal kinase activation, partially blocked the mammalian target of rapamycin pathway, and had no effect on extracellular signal-regulated kinase activation or the phosphorylation of eukaryotic initiation factor 4E. CONCLUSIONS: The pancreatic growth response is accompanied by activation of a number of signaling pathways regulating transcription and translation, some of which are dependent on and some independent of calcineurin.
Assuntos
Calcineurina/fisiologia , Pâncreas/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Animais , Ésteres , Fator de Iniciação 4E em Eucariotos/metabolismo , Gabexato/análogos & derivados , Gabexato/farmacologia , Guanidinas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fosforilação , Proteínas Quinases/fisiologia , Serina-Treonina Quinases TOR , Tacrolimo/farmacologiaRESUMO
While pancreatic protein synthesis and the initiation of translation are regulated by hormones and neurotransmiters, whether the elongation process is also regulated is unknown. Stimulatory doses of cholecystokinin (CCK) (100 pM), bombesin (10 nM), and carbachol (10 microM) increased elongation rates (measured as ribosomal half-transit time) in pancreatic acini in vitro. At the same time these secretagogues reduced elongation factor 2 (eEF2) phosphorylation, the main factor known to regulate elongation, and increased the phosphorylation of the eEF2 kinase. The mTOR inhibitor rapamycin reversed the dephosphorylation of eEF2 induced by CCK, as did treatment with the p38 MAPK inhibitor SB202190, the MEK inhibitor PD98059, and the phosphatase inhibitor calyculin A. Neither rapamycin, SB202190, PD98059 nor calyculin A had an effect on CCK mediated eEF2 kinase phosphorylation. Translation elongation in pancreatic acinar cells is likely regulated by eEF2 through the mTOR, p38, and MEK pathways, and modulated through PP2A.
Assuntos
Pâncreas/metabolismo , Fator 2 de Elongação de Peptídeos/química , Biossíntese de Proteínas , Animais , Bombesina/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Carbacol/farmacologia , Colecistocinina/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Quinase do Fator 2 de Elongação , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imidazóis/farmacologia , Toxinas Marinhas , Oxazóis/farmacologia , Fator 2 de Elongação de Peptídeos/metabolismo , Peptídeos/química , Fosforilação , Piridinas/farmacologia , Ratos , Transdução de Sinais , Sirolimo/farmacologia , Fatores de TempoRESUMO
To determine the mechanism of meal-regulated synthesis of pancreatic digestive enzymes, we studied the effect of fasting and refeeding on pancreatic protein synthesis, relative mRNA levels of digestive enzymes, and activation of the translational machinery. With the use of the flooding dose technique with L-[3H]phenylalanine, morning protein synthesis in the pancreas of Institute for Cancer Research mice fed ad libitum was 7.9 +/- 0.3 nmol phenylalanine.10 min(-1).mg protein(-1). Prior fasting for 18 h reduced total protein synthesis to 70 +/- 1.4% of this value. Refeeding for 2 h, during which the mice consumed 29% of their daily food intake, increased protein synthesis to 117.3 +/- 4.9% of the control level. Pancreatic mRNA levels of amylase, lipases, trypsins, chymotrypsin, elastases, as well as those for several housekeeping genes tested were not significantly changed after refeeding compared with fasted mice. By contrast, the major translational control pathway involving Akt, mTOR, and S6K was strongly regulated by fasting and refeeding. Fasting for 18 h decreased phosphorylation of ribosomal protein S6 to almost undetectable levels, and refeeding highly increased it. The most highly phosphorylated form of the eIF4E binding protein (4E-BP1) made up the 14.6% of total 4E-BP1 in normally fed animals, was only 2.8% after fasting, and was increased to 21.4% after refeeding. This was correlated with an increase in the formation of the eIF4E-eIF4G complex after refeeding. By contrast, feeding did not affect eIF2B activity. Thus food intake stimulates pancreatic protein synthesis and translational effectors without increasing digestive enzyme mRNA levels.
Assuntos
Ingestão de Alimentos/fisiologia , Pâncreas/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/biossíntese , Animais , Primers do DNA , Fator de Iniciação 4F em Eucariotos/biossíntese , Fator de Iniciação 4F em Eucariotos/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Pâncreas/enzimologia , Fosforilação , Testes de Precipitina , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores Acoplados a Proteínas G/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/biossíntese , Proteínas Quinases S6 Ribossômicas 70-kDa/genéticaRESUMO
CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca(2+)-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 +/- 11% of control. The immunosuppressant FK506 dose-dependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calcium-regulated heat-stable protein of 24 kDa. Another immunosuppressant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2alpha, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (eIF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the eIF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes.
Assuntos
Calcineurina/metabolismo , Pâncreas/fisiologia , Biossíntese de Proteínas/fisiologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/metabolismo , Colecistocinina/farmacologia , Ciclosporina/farmacologia , Quinase do Fator 2 de Elongação , Fator de Iniciação 2B em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos , Fator de Iniciação 4F em Eucariotos/metabolismo , Imunossupressores/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fator 2 de Elongação de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Ligação ao Cap de RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína S6 Ribossômica/metabolismo , Tacrolimo/farmacologiaRESUMO
Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2alpha. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 microg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2alpha phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2alpha phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.
Assuntos
Ceruletídeo , Fator de Iniciação 2B em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Pancreatite/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases , Doença Aguda , Animais , Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4E em Eucariotos/metabolismo , Camundongos , Pancreatite/induzido quimicamente , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Serina-Treonina Quinases TORRESUMO
Pancreatic secretagogues enhance acinar protein synthesis at physiological concentrations and inhibit protein synthesis at high concentrations. We investigated the potential role in this process of the eukaryotic translation initiation factor (eIF)2B. Cholecystokinin (CCK) at 10-100 pM did not significantly affect eIF2B activity, which averaged 35.4 nmol guanosine 5'-diphosphate exchanged per minute per milligram protein under control conditions; higher CCK concentrations reduced eIF2B activity to 38.2% of control. Carbamylcholine chloride (Carbachol, CCh), A-23187, and thapsigargin also inhibited eIF2B and protein synthesis, whereas bombesin and the CCK analog JMV-180 were without effect. Previous studies have shown that eIF2B can be negatively regulated by glycogen synthase kinase-3 (GSK-3). However, GSK-3 activity, as assessed by phosphorylation state, was inhibited at high concentrations of CCK, an effect that should have stimulated, rather than repressed, eIF2B activity. An alternative mechanism for regulating eIF2B is through phosphorylation of the alpha-subunit of eIF2, which converts it into an inhibitor of eIF2B. CCK, CCh, A-23187, and thapsigargin all enhanced eIF2alpha phosphorylation, suggesting that eIF2B activity is regulated by eIF2alpha phosphorylation under these conditions. Removal of Ca(2+) from the medium enhanced the inhibitory action of CCK on both protein synthesis and eIF2B activity as well as further increasing eIF2alpha phosphorylation. Although it is likely that other mechanisms account for the stimulation of acinar protein synthesis, these results suggest that the inhibition of acinar protein synthesis by CCK occurs as a result of depletion of Ca(2+) from the endoplasmic reticulum lumen leading to phosphorylation of eIF2alpha and inhibition of eIF2B.
Assuntos
Cálcio/metabolismo , Colecistocinina/farmacologia , Fator de Iniciação 2B em Eucariotos/metabolismo , Pâncreas/metabolismo , Biossíntese de Proteínas/fisiologia , Sincalida/análogos & derivados , Animais , Calcimicina/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Carbacol/farmacologia , Células Cultivadas , Colecistocinina/análogos & derivados , Agonistas Colinérgicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Ionóforos/farmacologia , Masculino , Pâncreas/citologia , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sincalida/farmacologia , Tapsigargina/farmacologiaRESUMO
Translational control of protein synthesis in the pancreas is important in regulating growth and the synthesis of digestive enzymes. Regulation of translation is primarily directed at the steps in initiation and involves reversible phosphorylation of initiation factors (eIFs) and ribosomal proteins. Major sites include the assembly of the eIF4F mRNA cap binding complex, the activity of guanine nucleotide exchange factor eIF2B, and the activity of ribosomal S6 kinase. All of these involve phosphorylation by different regulatory pathways. Stimulation of protein synthesis in acinar cells is primarily mediated by the phosphatidylinositol 3-kinase-mTOR pathway and involves both release of eIF4E (the limiting component of eIF4F) from its binding protein and phosphorylation of ribosomal S6 protein by S6K. eIF4E is itself phosphorylated by a distinct pathway. Inhibition of acinar protein synthesis can be mediated by inhibition of eIF2B following phosphorylation of eIF2alpha.