The regulation of Rasd1 expression by glucocorticoids and prolactin controls peripartum maternal insulin secretion.

The transition from gestation to lactation is characterized by a robust adaptation of maternal pancreatic ß-cells. Consistent with the loss of ß-cell mass, glucose-induced insulin secretion is down-regulated in the islets of early lactating dams. Extensive experimental evidence has demonstrated that the surge of prolactin is responsible for the morphofunctional remodeling of the maternal endocrine pancreas during pregnancy, but the precise molecular mechanisms by which this phenotype is rapidly reversed after delivery are not completely understood. This study investigated whether glucocorticoid-regulated expression of Rasd1/Dexras, a small inhibitory G protein, is involved in this physiological plasticity. Immunofluorescent staining demonstrated that Rasd1 is localized within pancreatic ß-cells. Rasd1 expression in insulin-secreting cells was increased by dexamethasone and decreased by prolactin. In vivo data confirmed that Rasd1 expression is decreased in islets from pregnant rats and increased in islets from lactating mothers. Knockdown of Rasd1 abolished the inhibitory effects of dexamethasone on insulin secretion and the protein kinase A, protein kinase C, and ERK1/2 pathways. Chromatin immunoprecipitation experiments revealed that glucocorticoid receptor (GR) and signal transducer and activator of transcription 5b (STAT5b) cooperatively mediate glucocorticoid-induced Rasd1 expression in islets. Prolactin inhibited the stimulatory effect of GR/STAT5b complex on Rasd1 transcription. Overall, our data indicate that the stimulation of Rasd1 expression by glucocorticoid at the end of pregnancy reverses the increased insulin secretion that occurs during pregnancy. Prolactin negatively regulates this pathway by inhibiting GR/STAT5b transcriptional activity on the Rasd1 gene.

UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression.

Endocrine pancreas from pregnant rats undergoes several adaptations that comprise increase in ß-cell number, mass and insulin secretion, and reduction of apoptosis. Lactogens are the main hormones that account for these changes. Maternal pancreas, however, returns to a nonpregnant state just after the delivery. The precise mechanism by which this reversal occurs is not settled but, in spite of high lactogen levels, a transient increase in apoptosis was already reported as early as the 3rd day of lactation (L3). Our results revealed that maternal islets displayed a transient increase in DNA fragmentation at L3, in parallel with decreased RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation (pAKT), a known prosurvival kinase. Wortmannin completely abolished the prosurvival action of prolactin (PRL) in cultured islets. Decreased pAKT in L3-islets correlated with increased Tribble 3 (TRB3) expression, a pseudokinase inhibitor of AKT. PERK and eIF2α phosphorylation transiently increased in islets from rats at the first day after delivery, followed by an increase in immunoglobulin heavy chain-binding protein (BiP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islets from L3 rats. Chromatin immunoprecipitation (ChIP) and Re-ChIP experiments further confirmed increased binding of the heterodimer ATF4/CHOP to the TRB3 promoter in L3 islets. Treatment with PBA, a chemical chaperone that inhibits UPR, restored pAKT levels and inhibited the increase in apoptosis found in L3. Moreover, PBA reduced CHOP and TRB3 levels in ß-cell from L3 rats. Altogether, our study collects compelling evidence that UPR underlies the physiological and transient increase in ß-cell apoptosis after delivery. The UPR is likely to counteract prosurvival actions of PRL by reducing pAKT through ATF4/CHOP-induced TRB3 expression.

Palmitate activates insulin signaling pathway in pancreatic rat islets.

OBJECTIVE: To investigate the action of palmitate on insulin receptor (IR) signaling pathway in rat pancreatic islets. The following proteins were studied: IR substrate-1 and -2 (IRS1 and IRS2), phosphatidylinositol 3-kinase, extracellular signal-regulated protein kinase-1 and -2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3). METHODS: Immunoblotting and immunoprecipitation assays were used to evaluate the phosphorylation states of IRS1 and IRS2 (tyrosine [Tyr]), ERK1/2 (threonine 202 [Thr202]/Tyr204), and STAT3 (serine [Ser727]). RESULTS: The exposure of rat pancreatic islets to 0.1-mmol/L palmitate for up to 30 minutes produced a significant increase of Tyr phosphorylation in IRS2 but not in IRS1. The association of phosphatidylinositol 3-kinase with IRS2 was also upregulated by palmitate. Exposure to 5.6-mmol/L glucose caused a gradual decrease in ERK1/2 (Thr202/Tyr204) and STAT3 (serine [Ser727]) phosphorylations after 30-minute incubation. The addition of palmitate (0.1 mmol/L), associated with 5.6-mmol/L glucose, abolished these latter effects of glucose after 15-minute incubation. CONCLUSIONS: Palmitate at physiological concentration associated with 5.6-mmol/L glucose activates IR signaling pathway in pancreatic beta cells.

Short-term modulation of extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase in pancreatic islets by glucose and palmitate: possible involvement of ceramide.

OBJECTIVES: The effect of glucose and palmitate on the phosphorylation of proteins associated with cell growth and survival (extracellular signal-regulated kinase 1/2 [ERK1/2] and stress-activated protein kinase/c-Jun NH2-terminal kinase [SAPK/JNK]) and on the expression of immediate early genes was investigated. METHODS: Groups of freshly isolated rat pancreatic islets were incubated in 10-mmol/L glucose with palmitate, LY294002, or fumonisin B1 for the measurement of the phosphorylation and the content of ERK1/2, JNK/SAPK, and v-akt murine thymoma viral oncongene (AKT) (serine 473) by immunoblotting. The expressions of the immediate early genes, c-fos and c-jun, were evaluated by reverse transcription-polymerase chain reaction. RESULTS: Glucose at 10 mmol/L induced ERK1/2 and AKT phosphorylations and decreased SAPK/JNK phosphorylation. Palmitate (0.1 mmol/L) abolished the glucose effect on ERK1/2, AKT, and SAPK/JNK phosphorylations. LY294002 caused a similar effect. The inhibitory effect of palmitate on glucose-induced ERK1/2 and AKT phosphorylation changes was not observed in the presence of fumonisin B1. Glucose increased c-fos and decreased c-jun expressions. Palmitate and LY294002 abolished these latter glucose effects. The presence of fumonisin B1 abolished the effect induced by palmitate on c-jun expression. CONCLUSIONS: Our results suggest that short-term changes of mitogen-activated protein kinase and AKT signaling pathways and c-fos and c-jun expressions caused by glucose are abolished by palmitate through phosphatidylinositol 3-kinase inhibition via ceramide synthesis.

Involvement of phosphatidylinositol-3 kinase/AKT/PKCzeta/lambda pathway in the effect of palmitate on glucose-induced insulin secretion.

OBJECTIVES: In the present study, a novel pathway by which palmitate potentiates glucose-induced insulin secretion by pancreatic beta cells was investigated. METHODS: Groups of freshly isolated islets were incubated in 10 mM glucose with palmitate, LY294002, wortmannin, and fumonisin B1 for measurement of insulin secretion by radioimmunoassay (RIA). Also, phosphorylation and content of AKT and PKC proteins were evaluated by immunoblotting. RESULTS: Glucose plus palmitate and glucose plus LY294002 or wortmannin (PI3K inhibitors) increased glucose-induced insulin secretion by isolated pancreatic islets. Glucose at 10 mM induced AKT and PKCzeta/lambda phosphorylation. Palmitate (0.1 mM) abolished glucose stimulation of AKT and PKCzeta/lambda phosphorylation possibly through PI3K inhibition because both LY294002 (50 microM) and wortmannin (100 nM) caused the same effect. The inhibitory effect of palmitate on glucose-induced AKT and PKCzeta/lambda phosphorylation and the stimulatory effect of palmitate on glucose-induced insulin secretion were not observed in the presence of fumonisin B1, an inhibitor of ceramide synthesis. CONCLUSIONS: These findings support the proposition that palmitate increases insulin release in the presence of 10 mM glucose by inhibiting PI3K activity through a mechanism that involves ceramide synthesis.

Retinal removal up-regulates cannabinoid CB(1) receptors in the chick optic tectum.

The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration and neuroprotection. The aim of this study was to evaluate the effects of unilateral retinal ablation on the expression of the cannabinoid receptor subtype 1 (CB(1)) at both protein and mRNA levels in the optic tectum of the adult chick brain. After different survival times postlesion (2-30 days), the chick brains were subjected to immunohistochemical, immunoblotting, and real-time PCR procedures to evaluate CB(1) expression. TUNEL and Fluoro-Jade B were used to verify the possible occurrence of cell death, and immunostaining for the microtubule-associated protein MAP-2 was performed to verify possible dendritic remodeling after lesions. No cell death could be observed in the deafferented tectum, at least up to 30 days postlesion, although Fluoro-Jade B could reveal degenerating axons and terminals. Retinal ablation seems to generate an increase of CB(1) protein in the optic tectum and other retinorecipient visual areas, which paralleled an increase in MAP-2 staining. On the other hand, CB(1) mRNA levels were not changed after retinal ablation. Our results reveal that CB(1) expression in visual structures of the adult chick brain may be negatively regulated by the retinal innervation. The increase of CB(1) receptor expression observed after retinal removal indicates that these receptors are not presynaptic in retinal axons projecting to the tectum and suggests a role of the cannabinoid system in plasticity processes ensuing after lesions.

Signal transducer and activator of transcription 3-regulated sarcoendoplasmic reticulum Ca2+-ATPase 2 expression by prolactin and glucocorticoids is involved in the adaptation of insulin secretory response during the peripartum period.

During pregnancy, the maternal endocrine pancreas undergoes, as a consequence of placental lactogens and prolactin (PRL) action, functional changes that are characterized by increased glucose-induced insulin secretion. After delivery, the maternal endocrine pancreas rapidly returns to non-pregnant state, which is mainly attributed to the increased serum levels of glucocorticoids (GCs). Although GCs are known to decrease insulin secretion and counteract PRL action, the mechanisms for these effects are poorly understood. We have previously demonstrated that signal transducer and activator of transcription 3 (STAT3) is increased in islets treated with PRL. In the present study, we show that STAT3 expression and serine phosphorylation are increased in pancreatic islets at the end of pregnancy (P19). STAT3 serine phosphorylation rapidly returned to basal levels 3 days after delivery (L3). The expression of the sarcoendoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2), a crucial protein involved in the regulation of calcium handling in beta-cells, was also increased in P19, returning to basal levels at L3. PRL increased SERCA2 and STAT3 expressions and STAT3 serine phosphorylation in RINm5F cells. The upregulation of SERCA2 by PRL was abolished after STAT3 knockdown. Moreover, PRL-induced STAT3 serine phosphorylation and SERCA2 expression were inhibited by dexamethasone (DEX). Insulin secretion from islets of P19 rats pre-incubated with thapsigargin and L3 rats showed a dramatic suppression of first phase of insulin release. The present results indicate that PRL regulates SERCA2 expression by a STAT3-dependent mechanism. PRL effect is counteracted by DEX and might contribute to the adaptation of maternal endocrine pancreas during the peripartum period.

ERK3 associates with MAP2 and is involved in glucose-induced insulin secretion.

The adaptation of pancreatic islets to pregnancy includes increased beta cell proliferation, expansion of islet mass, and increased insulin synthesis and secretion. Most of these adaptations are induced by prolactin (PRL). We have previously described that in vitro PRL treatment increases ERK3 expression in isolated rat pancreatic islets. This study shows that ERK3 is also upregulated during pregnancy. Islets from pregnant rats treated with antisense oligonucleotide targeted to the PRL receptor displayed a significant reduction in ERK3 expression. Immunohistochemical double-staining showed that ERK3 expression is restricted to pancreatic beta cells. Transfection with antisense oligonucleotide targeted to ERK3 abolished the insulin secretion stimulated by glucose in rat islets and by PMA in RINm5F cells. Therefore, we examined the participation of ERK3 in the activation of a cellular target involved in secretory events, the microtubule associated protein MAP2. PMA induced ERK3 phosphorylation that was companied by an increase in ERK3/MAP2 association and MAP2 phosphorylation. These observations provide evidence that ERK3 is involved in the regulation of stimulus-secretion coupling in pancreatic beta cells.

Characterization of the insulinotropic action of a phospholipase A2 isolated from Crotalus durissus collilineatus rattlesnake venom on rat pancreatic islets.

The ability of PLA2 and crotapotin, isolated from Crotalus durissus collilineatus rattlesnake venom, to stimulate insulin secretion from isolated rat islets was examined. PLA2 and crotapotin stimulated insulin secretion at 2.8 mmol/L glucose, whereas at a high glucose concentrations (16.7 mmol/L) only PLA2 stimulated secretion. Nifedipine (10 micromol/L) did not alter the ability of PLA2 to increase insulin secretion stimulated by a depolarizing concentration of K+ (30 mmol/L). PLA2 did not affect 14CO2 production but significantly increased the efflux of arachidonic acid from isolated islets. These results indicate that PLA2-stimulated secretion is not dependent on an additional influx of Ca2+ through L-type Ca(2+)-channels but rather is associated with arachidonic acid formation in pancreatic islets.