SV40-induced expression of calretinin protects mesothelial cells from asbestos cytotoxicity and may be a key factor contributing to mesothelioma pathogenesis.

The calcium-binding protein calretinin has emerged as a useful marker for the identification of mesotheliomas of the epithelioid and mixed types, but its putative role in tumor development has not been addressed previously. Although exposure to asbestos fibers is considered the main cause of mesothelioma, undoubtedly, not all mesothelioma patients have a history of asbestos exposure. The question as to whether the SV40 virus is involved as a possible co-factor is still highly debated. Here we show that increased expression of SV40 early gene products in the mesothelial cell line MeT-5A induces the expression of calretinin and that elevated calretinin levels strongly correlate with increased resistance to asbestos cytotoxicity. Calretinin alone mediates a significant part of this protective effect because cells stably transfected with calretinin cDNA were clearly more resistant to the toxic effects of crocidolite than mock-transfected control cells. Down-regulation of calretinin by antisense methods restored the sensitivity to asbestos toxicity to a large degree. The protective effect observed in clones with higher calretinin expression levels could be eliminated by phosphatidylinositol 3-kinase (PI3K) inhibitors, implying an important role for the PI3K/AKT signaling (survival) pathway in mediating the protective effect. Up-regulation of calretinin, resulting from either asbestos exposure or SV40 oncoproteins, may be a common denominator that leads to increased resistance to asbestos cytotoxicity and thereby contributes to mesothelioma carcinogenesis.

Dosage-dependent effects of Akt1/protein kinase Balpha (PKBalpha) and Akt3/PKBgamma on thymus, skin, and cardiovascular and nervous system development in mice.

Akt/protein kinase B (PKB) plays a critical role in the regulation of metabolism, transcription, cell migration, cell cycle progression, and cell survival. The existence of viable knockout mice for each of the three isoforms suggests functional redundancy. We generated mice with combined mutant alleles of Akt1 and Akt3 to study their effects on mouse development. Here we show that Akt1-/- Akt3+/- mice display multiple defects in the thymus, heart, and skin and die within several days after birth, while Akt1+/- Akt3-/- mice survive normally. Double knockout (Akt1-/-) Akt3-/-) causes embryonic lethality at around embryonic days 11 and 12, with more severe developmental defects in the cardiovascular and nervous systems. Increased apoptosis was found in the developing brain of double mutant embryos. These data indicate that the Akt1 gene is more essential than Akt3 for embryonic development and survival but that both are required for embryo development. Our results indicate isoform-specific and dosage-dependent effects of Akt on animal survival and development.

Insulin-secreting beta-cell dysfunction induced by human lipoproteins.

Diabetes is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent low density lipoprotein (LDL) and high density lipoprotein (HDL) particles in insulin-secreting beta-cells. Purified human very low density lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels and beta-cell proliferation and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of beta-cells involved caspase-3 cleavage and reduction in the levels of the c-Jun N-terminal kinase-interacting protein-1. In contrast, the proapoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of c-Jun N-terminal kinase. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of Akt/protein kinase B. In conclusion, human lipoproteins are critical regulators of beta-cell survival and may therefore contribute to the beta-cell dysfunction observed during the development of type 2 diabetes.

Insulin and IGF-1 enhance the expression of the neuronal monocarboxylate transporter MCT2 by translational activation via stimulation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin pathway.

MCT2 is the main neuronal monocarboxylate transporter essential for facilitating lactate and ketone body utilization as energy substrates. Our study reveals that treatment of cultured cortical neurons with insulin and IGF-1 led to a striking enhancement of MCT2 immunoreactivity in a time- and concentration-dependent manner. Surprisingly, neither insulin nor IGF-1 affected MCT2 mRNA expression, suggesting that regulation of MCT2 protein expression occurs at the translational rather than the transcriptional level. Investigation of the putative signalling pathways leading to translation activation revealed that insulin and IGF-1 induced p44- and p42 MAPK, Akt and mTOR phosphorylation. S6 ribosomal protein, a component of the translational machinery, was also strongly activated by insulin and IGF-1. Phosphorylation of p44- and p42 MAPK was blocked by the MEK inhibitor PD98058, while Akt phosphorylation was abolished by the PI3K inhibitor LY294002. Phosphorylation of mTOR and S6 was blocked by the mTOR inhibitor rapamycin. In parallel, it was observed that LY294002 and rapamycin almost completely blocked the effects of insulin and IGF-1 on MCT2 protein expression, whereas PD98059 and SB202190 (a p38K inhibitor) had no effect on insulin-induced MCT2 expression and only a slight effect on IGF-1-induced MCT2 expression. At the subcellular level, a significant increase in MCT2 protein expression within an intracellular pool was observed while no change at the cell surface was apparent. As insulin and IGF-1 are involved in synaptic plasticity, their effect on MCT2 protein expression via an activation of the PI3K-Akt-mTOR-S6K pathway might contribute to the preparation of neurons for enhanced use of nonglucose energy substrates following altered synaptic efficacy.

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.

The peroxisome proliferator-activated receptor (PPAR) β/δ agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3 (STAT3) activation and insulin resistance in human liver cells.

AIM/HYPOTHESIS: IL-6 induces insulin resistance by activating signal transducer and activator of transcription 3 (STAT3) and upregulating the transcription of its target gene SOCS3. Here we examined whether the peroxisome proliferator-activated receptor (PPAR)β/δ agonist GW501516 prevented activation of the IL-6-STAT3-suppressor of cytokine signalling 3 (SOCS3) pathway and insulin resistance in human hepatic HepG2 cells. METHODS: Studies were conducted with human HepG2 cells and livers from mice null for Pparβ/δ (also known as Ppard) and wild-type mice. RESULTS: GW501516 prevented IL-6-dependent reduction in insulin-stimulated v-akt murine thymoma viral oncogene homologue 1 (AKT) phosphorylation and in IRS-1 and IRS-2 protein levels. In addition, treatment with this drug abolished IL-6-induced STAT3 phosphorylation of Tyr⁷⁰⁵ and Ser⁷²⁷ and prevented the increase in SOCS3 caused by this cytokine. Moreover, GW501516 prevented IL-6-dependent induction of extracellular-related kinase 1/2 (ERK1/2), a serine-threonine protein kinase involved in serine STAT3 phosphorylation; the livers of Pparβ/δ-null mice showed increased Tyr⁷⁰⁵- and Ser⁷²⁷-STAT3 as well as phospho-ERK1/2 levels. Furthermore, drug treatment prevented the IL-6-dependent reduction in phosphorylated AMP-activated protein kinase (AMPK), a kinase reported to inhibit STAT3 phosphorylation on Tyr⁷⁰⁵. In agreement with the recovery in phospho-AMPK levels observed following GW501516 treatment, this drug increased the AMP/ATP ratio and decreased the ATP/ADP ratio. CONCLUSIONS/INTERPRETATION: Overall, our findings show that the PPARβ/δ activator GW501516 prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 phosphorylation and preventing the reduction in phospho-AMPK levels. These effects of GW501516 may contribute to the prevention of cytokine-induced insulin resistance in hepatic cells.

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Akt signalling through GSK-3beta, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy

Skeletal muscle size is tightly regulated by the synergy between anabolic and catabolic signalling pathways which, in humans, have not been well characterized. Akt has been suggested to play a pivotal role in the regulation of skeletal muscle hypertrophy and atrophy in rodents and cells. Here we measured the amount of phospho-Akt and several of its downstream anabolic targets (glycogen synthase kinase-3beta (GSK-3beta), mTOR, p70(s6k) and 4E-BP1) and catabolic targets (Foxo1, Foxo3, atrogin-1 and MuRF1). All measurements were performed in human quadriceps muscle biopsies taken after 8 weeks of both hypertrophy-stimulating resistance training and atrophy-stimulating de-training. Following resistance training a muscle hypertrophy ( approximately 10%) and an increase in phospho-Akt, phospho-GSK-3beta and phospho-mTOR protein content were observed. This was paralleled by a decrease in Foxo1 nuclear protein content. Following the de-training period a muscle atrophy (5%), relative to the post-training muscle size, a decrease in phospho-Akt and GSK-3beta and an increase in Foxo1 were observed. Atrogin-1 and MuRF1 increased after the hypertrophy and decreased after the atrophy phases. We demonstrate, for the first time in human skeletal muscle, that the regulation of Akt and its downstream signalling pathways GSK-3beta, mTOR and Foxo1 are associated with both the skeletal muscle hypertrophy and atrophy processes

Phosphorylation of phosphatidylinositol-3-kinase-protein kinase B and extracellular signal-regulated kinases 1/2 mediate reoxygenation-induced cardioprotection during hypoxia.

In vivo exposure to chronic hypoxia (CH) depresses myocardial performance and tolerance to ischemia, but daily reoxyenation during CH (CHR) confers cardioprotection. To elucidate the underlying mechanism, we tested the role of phosphatidylinositol-3-kinase-protein kinase B (Akt) and p42/p44 extracellular signal-regulated kinases (ERK1/2), which are known to be associated with protection against ischemia/reperfusion (I/R). Male Sprague-Dawley rats were maintained for two weeks under CH (10% O(2)) or CHR (as CH but with one-hour daily exposure to room air). Then, hearts were either frozen for biochemical analyses or Langendorff-perfused to determine performance (intraventricular balloon) and tolerance to 30-min global ischemia and 45-min reperfusion, assessed as recovery of performance after I/R and infarct size (tetrazolium staining). Additional hearts were perfused in the presence of 15 micromol/L LY-294002 (inhibitor of Akt), 10 micromol/L UO-126 (inhibitor of ERK1/2) or 10 micromol/L PD-98059 (less-specific inhibitor of ERK1/2) given 15 min before ischemia and throughout the first 20 min of reperfusion. Whereas total Akt and ERK1/2 were unaffected by CH and CHR in vivo, in CHR hearts the phosphorylation of both proteins was higher than in CH hearts. This was accompanied by better performance after I/R (heart rate x developed pressure), lower end-diastolic pressure and reduced infarct size. Whereas the treatment with LY-294002 decreased the phosphorylation of Akt only, the treatment with UO-126 decreased ERK1/2, and that with PD-98059 decreased both Akt and ERK1/2. In all cases, the cardioprotective effect led by CHR was lost. In conclusion, in vivo daily reoxygenation during CH enhances Akt and ERK1/2 signaling. This response was accompanied by a complex phenotype consisting in improved resistance to stress, better myocardial performance and lower infarct size after I/R. Selective inhibition of Akt and ERK1/2 phosphorylation abolishes the beneficial effects of the reoxygenation. Therefore, Akt and ERK1/2 have an important role to mediate cardioprotection by reoxygenation during CH in vivo.

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.

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

Bromati CR, Lellis-Santos C, Yamanaka TS, Nogueira TC, Leonelli M, Caperuto LC, Gorjao R, Leite AR, Anhe GF, Bordin S. UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression. Am J Physiol Regul Integr Comp Physiol 300: R92-R100, 2011. First published November 10, 2010; doi:10.1152/ajpregu.00169.2010.-Endocrine pancreas from pregnant rats undergoes several adaptations that comprise increase in beta-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 eIF2 alpha 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 beta-cell from L3 rats. Altogether, our study collects compelling evidence that UPR underlies the physiological and transient increase in beta-cell apoptosis after delivery. The UPR is likely to counteract prosurvival actions of PRL by reducing pAKT through ATF4/CHOP-induced TRB3 expression.

UPR-mediated TRIB3 expression correlates with reduced AKT phosphorylation and inability of interleukin 6 to overcome palmitate-induced apoptosis in RINm5F cells

Unfolded protein response (UPR)-mediated pancreatic beta-cell death has been described as a common mechanism by which palmitate (PA) and pro-inflammatory cytokines contribute to the development of diabetes. There are evidences that interleukin 6 (IL6) has a protective action against beta-cell death induced by proinflammatory cytokines; the effects of IL6 on PA-induced apoptosis have not been investigated yet. In the present study, we have demonstrated that PA selectively disrupts IL6-induced RAC-alpha serine/threonine-protein kinase (AKT) activation without interfering with signal transducer and activator of transcription 3 phosphorylation in RINm5F cells. The inability of IL6 to activate AKT in the presence of PA correlated with an inefficient protection against PA-induced apoptosis. In contrast to PA, IL6 efficiently reduced apoptosis induced by pro-inflammatory cytokines. In addition, we have demonstrated that IL6 is unable to overcome PA-stimulated UPR, as assessed by activating transcription factor 4 (ATF4) andC/EBP homologous protein (CHOP) expression, X-box binding protein-1 gene mRNA splicing, and pancreatic eukaryotic initiation factor-2 alpha kinase phosphorylation, whereas no significant induction of UPR by pro-inflammatory cytokines was detected. This unconditional stimulation of UPR and apoptosis by PA was accompanied by the stimulation of CHOP and tribble3 (TRIB3) expression, irrespective of the presence of IL6. These findings suggest that IL6 is unable to protect pancreatic beta-cells from PA-induced apoptosis because it does not repress UPR activation. In this way, CHOP and ATF4 might mediate PA-induced TRIB3 expression and, by extension, the suppression of IL6 activation of pro-survival kinase AKT. Journal of Endocrinology (2010) 206, 183-193

Low ethanol consumption induces enhancement of insulin sensitivity in liver of normal rats

Moderate amounts of alcohol intake have been reported to have a protective effect on the cardiovascular system and this may involve enhanced insulin sensitivity. We established an animal model of increased insulin sensitivity by low ethanol consumption and here we investigated metabolic parameters and molecular mechanisms potentially involved in this phenomenon. For that, Wistar rats have received drinking water either without (control) or with 3% ethanol for four weeks. The effect of ethanol intake on insulin sensitivity was analyzed by insulin resistance index (HOMA-IR), intravenous insulin tolerance test (IVITT) and lipid profile. The role of liver was investigated by the analysis of insulin signaling pathway, GLUT2 gene expression and tissue glycogen content. Rats consuming 3% ethanol showed lower values of HOMA-IR and plasma free fatty acids (FFA) levels and higher hepatic glycogen content and glucose disappearance constant during the IVITT. Neither the phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), nor its association with phosphatidylinositol-3-kinase (PI3-kinase), was affected by ethanol. However, ethanol consumption enhanced liver IRS-2 and protein kinase B (Akt) phosphorylation (3 times, P < 0.05), which can be involved in the 2-fold increased (P < 0.05) hepatic glycogen content. The GLUT2 protein content was unchanged. Our findings point out that liver plays a role in enhanced insulin sensitivity induced by low ethanol consumption. © 2005 Elsevier Inc. All rights reserved.

Oncogenic transformation induced by the Qin protein is correlated with transcriptional repression

The retroviral oncogene qin codes for a protein that belongs to the family of the winged helix transcription factors. The viral Qin protein, v-Qin, differs from its cellular counterpart, c-Qin, by functioning as a stronger transcriptional repressor and a more efficient inducer of tumors. This observation suggests that repression may be important in tumorigenesis. To test this possibility, chimeric proteins were constructed in which the Qin DNA-binding domain was fused to either a strong repressor domain (derived from the Drosophila Engrailed protein) or a strong activator domain (from the herpes simplex virus VP16 protein). The chimeric transcriptional repressor, Qin–Engrailed, transformed chicken embryo fibroblasts in culture and induced sarcomas in young chickens. The chimeric activator, Qin–VP16, failed to transform cells in vitro or in vivo and caused cellular resistance to oncogenic transformation by Qin. These data support the conclusion that the Qin protein induces oncogenic transformation by repressing the transcription of genes which function as negative growth regulators or tumor suppressors.

Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.