RESUMEN
AIMS/HYPOTHESIS: Diabetes mellitus type 2 is characterised by hyperglucagonaemia, resulting in hepatic glucose production and hyperglycaemia. Considering that insulin inhibits glucagon secretion and gene transcription, hyperglucagonaemia in the face of hyperinsulinaemia in diabetes mellitus type 2 suggests that there is insulin resistance also at the glucagon-producing pancreatic islet alpha cells. However, the molecular mechanism of alpha cell insulin resistance is unknown. Therefore, the effect of molecules implicated in conferring insulin resistance in some other tissues was investigated on insulin-induced inhibition of glucagon gene transcription in alpha cells. METHODS: Reporter gene assays and biochemical techniques were used in the glucagon-producing hamster pancreatic islet alpha cell line InR1-G9. RESULTS: From among 16 agents tested, chronic insulin treatment was found to abolish insulin-induced inhibition of glucagon gene transcription. Overproduction of constitutively active protein kinase B (PKB) still inhibited glucagon gene transcription after chronic insulin treatment; together with a markedly reduced insulin-induced phosphorylation and, thus, activation of PKB, this indicates that targets upstream of PKB within the insulin signalling pathway are affected. Indeed, chronic insulin treatment markedly reduced IRS-1 phosphorylation, insulin receptor (IR) autophosphorylation and IR content. Cycloheximide and in vivo labelling experiments attributed IR downregulation to enhanced degradation. CONCLUSIONS/INTERPRETATION: These results show that an extended exposure of alpha cells to insulin induces IR downregulation and loss of insulin-induced inhibition of glucagon gene transcription. They suggest that hyperinsulinaemia, through IR downregulation, may confer insulin resistance to pancreatic islet alpha cells in diabetes mellitus type 2.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Células Secretoras de Glucagón/fisiología , Glucagón/genética , Insulina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Cricetinae , Regulación hacia Abajo , Activación Enzimática , Células Secretoras de Glucagón/efectos de los fármacos , Insulina/fisiología , Proteínas Sustrato del Receptor de Insulina , Interleucina-1beta/farmacología , Ratones , Ratones Endogámicos , Fosforilación , Plásmidos , TransfecciónRESUMEN
AIMS/HYPOTHESIS: Inappropriate insulin secretion and biosynthesis are hallmarks of beta cell dysfunction and contribute to the progression from a prediabetic state to overt diabetes mellitus. During the prediabetic state, beta cells are exposed to elevated levels of proinflammatory cytokines. In the present study the effect of these cytokines and mitogen-activated protein kinase kinase kinase 1 (MEKK1), which is known to be activated by these cytokines, on human insulin gene (INS) transcription was investigated. METHODS: Biochemical methods and reporter gene assays were used in a beta cell line and in primary pancreatic islets from transgenic mice. RESULTS: IL-1beta and MEKK1 specifically inhibited basal and membrane depolarisation and cAMP-induced INS transcription in the beta cell line. Also, in primary islets of reporter gene mice, IL-1beta reduced glucose-stimulated INS transcription. A 5'- and 3'-deletion and internal mutation analysis revealed the rat insulin promoter element 3b (RIPE3b) to be a decisive MEKK1-responsive element of the INS. RIPE3b conferred strong transcriptional activity to a heterologous promoter, and this activity was markedly inhibited by MEKK1 and IL-1beta. RIPE3b is also known to recruit the transcription factor MafA. We found here that MafA transcription activity is markedly inhibited by MEKK1 and IL-1beta. CONCLUSIONS/INTERPRETATION: These data suggest that IL-1beta through MEKK1 inhibits INS transcription and does so, at least in part, by decreasing MafA transcriptional activity at the RIPE3b control element. Since inappropriate insulin biosynthesis contributes to beta cell dysfunction, inhibition of MEKK1 might decelerate or prevent progression from a prediabetic state to diabetes mellitus.
Asunto(s)
Células Secretoras de Insulina/efectos de los fármacos , Insulina/genética , Interleucina-1beta/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Factores de Transcripción Maf/genética , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Interferón gamma/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Transcripción Genética/efectos de los fármacos , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
A pancreatic islet cell-specific enhancer sequence (PISCES) shared by the rat insulin-I, glucagon, and somatostatin genes binds the paired domain-containing transcription factor Pax6 and confers strong transcriptional activity in pancreatic islet cell lines. It was found recently that Pax6 plays a major role in islet development. In the present study, transgenic mice were used to investigate PISCES-mediated transcription in normal adult islets in vivo. In several independent mouse lines expressing a PISCES-luciferase reporter transgene, the PISCES motif directed gene expression in the adult eye, cerebellum, and discrete brain areas, consistent with the tissue distribution of Pax6. These tissues contain two Pax6 isoforms caused by alternative splicing, only one of which was found to bind the PISCES motif in electrophoretic mobility shift assays. No reporter gene expression was detected in adult pancreatic islets or in any other peripheral organ tested. RT-PCR analysis confirmed that Pax6 mRNA is present in adult islets. These results demonstrate that the PISCES motif is sufficient to direct highly tissue-specific gene expression in whole animals. The lack of PISCES-mediated transcription in adult islets indicates that the Pax6 protein(s) expressed in adult pancreatic islets function differently from the ones in the eye and cerebellum.