Retraction Note to: L-glutamine supplementation induces insulin resistance in adipose tissue and improves insulin signalling in liver and muscle of rats with diet-induced obesity.

In light of forensic evidence indicating duplication and/or manipulation of western blot images the Editor-in-Chief is retracting the article cited above.

Double-stranded RNA-activated protein kinase is a key modulator of insulin sensitivity in physiological conditions and in obesity in mice.

The molecular integration of nutrient- and pathogen-sensing pathways has become of great interest in understanding the mechanisms of insulin resistance in obesity. The double-stranded RNA-dependent protein kinase (PKR) is one candidate molecule that may provide cross talk between inflammatory and metabolic signaling. The present study was performed to determine, first, the role of PKR in modulating insulin action and glucose metabolism in physiological situations, and second, the role of PKR in insulin resistance in obese mice. We used Pkr(-/-) and Pkr(+/+) mice to investigate the role of PKR in modulating insulin sensitivity, glucose metabolism, and insulin signaling in liver, muscle, and adipose tissue in response to a high-fat diet. Our data show that in lean Pkr(-/-) mice, there is an improvement in insulin sensitivity, and in glucose tolerance, and a reduction in fasting blood glucose, probably related to a decrease in protein phosphatase 2A activity and a parallel increase in insulin-induced thymoma viral oncogene-1 (Akt) phosphorylation. PKR is activated in tissues of obese mice and can induce insulin resistance by directly binding to and inducing insulin receptor substrate (IRS)-1 serine307 phosphorylation or indirectly through modulation of c-Jun N-terminal kinase and inhibitor of κB kinase ß. Pkr(-/-) mice were protected from high-fat diet-induced insulin resistance and glucose intolerance and showed improved insulin signaling associated with a reduction in c-Jun N-terminal kinase and inhibitor of κB kinase ß phosphorylation in insulin-sensitive tissues. PKR may have a role in insulin sensitivity under normal physiological conditions, probably by modulating protein phosphatase 2A activity and serine-threonine kinase phosphorylation, and certainly, this kinase may represent a central mechanism for the integration of pathogen response and innate immunity with insulin action and metabolic pathways that are critical in obesity.

Modulation of gut microbiota by antibiotics improves insulin signalling in high-fat fed mice.

AIMS/HYPOTHESIS: A high-fat dietary intake induces obesity and subclinical inflammation, which play important roles in insulin resistance. Recent studies have suggested that increased concentrations of circulating lipopolysaccharide (LPS), promoted by changes in intestinal permeability, may have a pivotal role in insulin resistance. Thus, we investigated the effect of gut microbiota modulation on insulin resistance and macrophage infiltration. METHODS: Swiss mice were submitted to a high-fat diet with antibiotics or pair-feeding for 8 weeks. Metagenome analyses were performed on DNA samples from mouse faeces. Blood was collected to determine levels of glucose, insulin, LPS, cytokines and acetate. Liver, muscle and adipose tissue proteins were analysed by western blotting. In addition, liver and adipose tissue were analysed, blinded, using histology and immunohistochemistry. RESULTS: Antibiotic treatment greatly modified the gut microbiota, reducing levels of Bacteroidetes and Firmicutes, overall bacterial count and circulating LPS levels. This modulation reduced levels of fasting glucose, insulin, TNF-α and IL-6; reduced activation of toll-like receptor 4 (TLR4), c-Jun N-terminal kinase (JNK), inhibitor of κ light polypeptide gene enhancer in B cells, kinase ß (IKKß) and phosphorylated IRS-1 Ser307; and consequently improved glucose tolerance and insulin tolerance and action in metabolically active tissues. In addition, there was an increase in portal levels of circulating acetate, which probably contributed to an increase in 5'-AMP-activated protein kinase (AMPK) phosphorylation in mice. We observed a striking reduction in crown-like structures (CLS) and F4/80(+) macrophage cells in the adipose tissue of antibiotic-treated mice. CONCLUSIONS/INTERPRETATION: These results suggest that modulation of gut microbiota in obesity can improve insulin signalling and glucose tolerance by reducing circulating LPS levels and inflammatory signalling. Modulation also appears to increase levels of circulating acetate, which activates AMPK and finally leads to reduced macrophage infiltration.

Cytotoxic drugs for patients with breast cancer in the era of targeted treatment: back to the future?

BACKGROUND: Despite current trend of targeted therapy development, cytotoxic agents are a mainstay of treatment of patients with breast cancer. We reviewed recent advances in cytotoxic therapy for patients with metastatic breast cancer (MBC). MATERIALS AND METHODS: Medline searches were conducted for English language studies using the term 'MBC' and 'cytotoxic drugs'. The data search was restricted to the period 2000-2011. RESULTS: Several novel cytotoxic compounds, all microtubule inhibitors, have been approved for clinical use in MBC: (i) nab-paclitaxel, reported to improve tumour response and decrease hypersensitivity reactions in comparison with other taxanes; (ii) ixabepilone, shown to have clinical benefit in taxane- and anthracycline-resistant disease and (iii) eribulin, shown to improve overall survival in heavily pre-treated patients, when compared with best available standard treatment. Agents, such as larotaxel, vinflunine, trabectidin and formulations, including cationic liposomal paclitaxel or paclitaxel poliglumex, are currently under evaluation in phase II/III trials. CONCLUSION: Toxicity and chemotherapy resistance are still major limitations in the treatment of patients with MBC. Further research into new cytotoxic compounds is needed in order to maximise benefit, whilst minimising toxicity.

Multidisciplinary approach in the treatment of patients with small cell bladder carcinoma.

Small cell carcinoma of the urinary bladder (SCCUB) is considered to be a tumor with a neuroendocrine phenotype characterised by aggressive behaviour and poor prognosis. Small cell carcinoma of the urinary bladder comprises 0.35 to 1% of all bladder cancers and is frequently observed in combination with other histological subtypes of carcinoma. Clinical presentation is characterized by advanced stage at diagnosis and rapidly progressive disease. In daily clinical practice there is no gold standard for the management of patients affected by this disease. Treatment of patients with limited disease combines neoadjuvant platinum-based chemotherapy followed by specific local treatment of the primary tumour. Cystectomy or radiotherapy should be proposed on an individual basis. In the metastatic setting, prognosis remains poor with a potential benefit from chemotherapy containing platinum compounds. Treatment of small cell carcinoma of the urinary bladder is based on evidence obtained from case reports and retrospective analyses. Due to low disease frequency there is a lack of randomized trials to provide guidance as to optimal therapy. Thus, systemic and local approaches are extrapolated from the literature available for the treatment of small cell carcinomas at other (non-urological) sites. We provide an overview of the currently available literature with it's main focus on the treatment of either locally advanced or metastatic small cell carcinoma of the urinary bladder.

Aspirin attenuates insulin resistance in muscle of diet-induced obese rats by inhibiting inducible nitric oxide synthase production and S-nitrosylation of IRbeta/IRS-1 and Akt.

AIM/HYPOTHESIS: High-dose aspirin treatment improves fasting and postprandial hyperglycaemia in patients with type 2 diabetes, as well as in animal models of insulin resistance associated with obesity and sepsis. In this study, we investigated the effects of aspirin treatment on inducible nitric oxide synthase (iNOS)-mediated insulin resistance and on S-nitrosylation of insulin receptor (IR)-beta, IRS-1 and protein kinase B (Akt) in the muscle of diet-induced obese rats and also in iNos (also known as Nos2)-/- mice on high fat diet. METHODS: Aspirin (120 mg kg-1 day-1 for 2 days) or iNOS inhibitor (L-NIL; 80 mg/kg body weight) were administered to diet-induced obese rats or mice and iNOS production and insulin signalling were investigated. S-nitrosylation of IRbeta/IRS-1 and Akt was investigated using the biotin switch method. RESULTS: iNOS protein levels increased in the muscle of diet-induced obese rats, associated with an increase in S-nitrosylation of IRbeta, IRS-1 and Akt. These alterations were reversed by aspirin treatment, in parallel with an improvement in insulin signalling and sensitivity, as measured by insulin tolerance test and glucose clamp. Conversely, while aspirin reversed the increased phosphorylation of IkappaB kinase beta and c-Jun amino-terminal kinase, as well as IRS-1 serine phosphorylation in diet-induced obese rats and iNos -/- mice on high-fat diet, these alterations were not associated with the improvement of insulin action induced by this drug. CONCLUSIONS/INTERPRETATION: Our data demonstrate that aspirin treatment not only reduces iNOS protein levels, but also S-nitrosylation of IRbeta, IRS-1 and Akt. These changes are associated with improved insulin resistance and signalling, suggesting a novel mechanism of insulin sensitisation evoked by aspirin treatment.

L-glutamine supplementation induces insulin resistance in adipose tissue and improves insulin signalling in liver and muscle of rats with diet-induced obesity.

AIMS/HYPOTHESIS: Diet-induced obesity (DIO) is associated with insulin resistance in liver and muscle, but not in adipose tissue. Mice with fat-specific disruption of the gene encoding the insulin receptor are protected against DIO and glucose intolerance. In cell culture, glutamine induces insulin resistance in adipocytes, but has no effect in muscle cells. We investigated whether supplementation of a high-fat diet with glutamine induces insulin resistance in adipose tissue in the rat, improving insulin sensitivity in the whole animal. MATERIALS AND METHODS: Male Wistar rats received standard rodent chow or a high-fat diet (HF) or an HF supplemented with alanine or glutamine (HFGln) for 2 months. Light microscopy and morphometry, oxygen consumption, hyperinsulinaemic-euglycaemic clamp and immunoprecipitation/immunoblotting were performed. RESULTS: HFGln rats showed reductions in adipose mass and adipocyte size, a decrease in the activity of the insulin-induced IRS-phosphatidylinositol 3-kinase (PI3-K)-protein kinase B-forkhead transcription factor box 01 pathway in adipose tissue, and an increase in adiponectin levels. These results were associated with increases in insulin-stimulated glucose uptake in skeletal muscle and insulin-induced suppression of hepatic glucose output, and were accompanied by an increase in the activity of the insulin-induced IRS-PI3-K-Akt pathway in these tissues. In parallel, there were decreases in TNFalpha and IL-6 levels and reductions in c-jun N-terminal kinase (JNK), IkappaB kinase subunit beta (IKKbeta) and mammalian target of rapamycin (mTOR) activity in the liver, muscle and adipose tissue. There was also an increase in oxygen consumption and a decrease in the respiratory exchange rate in HFGln rats. CONCLUSIONS/INTERPRETATION: Glutamine supplementation induces insulin resistance in adipose tissue, and this is accompanied by an increase in the activity of the hexosamine pathway. It also reduces adipose mass, consequently attenuating insulin resistance and activation of JNK and IKKbeta, while improving insulin signalling in liver and muscle.

Circulating ghrelin concentrations are lowered by intracerebroventricular insulin.

AIMS/HYPOTHESIS: Ghrelin is a peptide that is mainly produced by the stomach and stimulates food intake, adiposity and weight gain. Previous studies have documented that plasma levels of ghrelin are reduced by insulin, but the mechanisms that mediate this effect are unclear. METHODS: To determine whether phosphatidylinositol 3-kinase (PI(3)K) and/or mitogen-activated protein kinase (MAPK) are involved in this insulin action, we tested the intracerebroventricular (i.c.v.) effect of specific inhibitors of PI(3)K (LY294002 and wortmannin) and MAPK (PD98059 and UO126) on the insulin-mediated reduction of ghrelin levels in rats. RESULTS: Intracerebroventricular treatment with insulin reduced ghrelin levels. Inhibition of PI(3)K specifically blocked the insulin-induced reduction in ghrelin concentration, whereas inhibition of MAPK had no effect on insulin-mediated actions. Moreover, pretreatment with i.c.v. PI(3)K inhibitors blocked the reduction of ghrelin levels after OGTT-induced hyperglycaemia and hyperinsulinaemia. CONCLUSIONS/INTERPRETATION: These data demonstrate that changes in insulin action in the central nervous system regulate circulating ghrelin levels and that PI(3)K is a specific mediator of this action.

Regulation of insulin signalling by hyperinsulinaemia: role of IRS-1/2 serine phosphorylation and the mTOR/p70 S6K pathway.

AIM/HYPOTHESIS: Several epidemiological studies have suggested an association between chronic hyperinsulinaemia and insulin resistance. However, the causality of this relationship remains uncertain. METHODS: We performed chronic hyperinsulinaemic-euglycaemic clamps and delineated, by western blotting, an IR/IRSs/phosphatidylinositol 3-kinase(PI[3]K)/Akt pathway in insulin-responsive tissues of hyperinsulinaemic rats. IRS-1/2 serine phosphorylation, IR/protein tyrosine phosphatase 1B (PTP1B) association, and mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70 S6K) activity were also evaluated in the liver, skeletal muscle and white adipose tissue of hyperinsulinaemic animals. RESULTS: We found that chronic hyperinsulinaemic rats have insulin resistance and reduced levels of glycogen content in liver and muscle. In addition, we demonstrated an impairment of the insulin-induced IR/IRSs/PI3K/Akt pathway in liver and muscle of chronic hyperinsulinaemic rats that parallels increases in IRS1/2 serine phosphorylation, IR/PTP1B association and mTOR activity. Despite a higher association of IR/PTP1B, there was an increase in white adipose tissue of chronic hyperinsulinaemic rats in IRS-1/2 protein levels, tyrosine phosphorylation and IRSs/PI3K association, which led to an increase in basal Akt serine phosphorylation. No increases in IRS-1/2 serine phosphorylation and mTOR activity were observed in white adipose tissue. Rapamycin reversed the insulin resistance and the changes induced by hyperinsulinaemia in the three tissues studied. CONCLUSIONS/INTERPRETATION: Our data provide evidence that chronic hyperinsulinaemia itself, imposed on normal rats, appears to have a dual effect, stimulating insulin signalling in white adipose tissue, whilst decreasing it in liver and muscle. The underlying mechanism of these differential effects may be related to the ability of hyperinsulinaemia to increase mTOR/p70 S6K pathway activity and IRS-1/2 serine phosphorylation in a tissue-specific fashion. In addition, we demonstrated that inhibition of the mTOR pathway with rapamycin can prevent insulin resistance caused by chronic hyperinsulinaemia in liver and muscle. These findings support the hypothesis that defective and tissue-selective insulin action contributes to the insulin resistance observed in hyperinsulinaemic states.

Selective impairment of insulin signalling in the hypothalamus of obese Zucker rats.

AIM/HYPOTHESIS: By acting in the brain, insulin suppresses food intake. However, little is known with regard to insulin signalling in the hypothalamus in insulin-resistant states. METHODS: Western blotting, immunohistochemistry and polymerase chain reaction assays were combined to compare in vivo hypothalamic insulin signalling through the PI3-kinase and MAP kinase pathways between lean and obese Zucker rats. RESULTS: Intracerebroventricular insulin infusion reduced food intake in lean rats to a greater extent than that observed in obese rats, and pre-treatment with PI3-kinase inhibitors prevented insulin-induced anorexia. The relative abundance of IRS-2 was considerably higher than that of IRS-1 in hypothalamus of both lean and obese rats. Insulin-stimulated phosphorylation of IR, IRS-1/2, the associations of PI 3-kinase to IRS-1/2 and phosphorylation of Akt in hypothalamus were decreased in obese rats compared to lean rats. These effects seem to be mediated by increased phosphoserine content of IR, IRS-1/2 and decreased protein levels of IRS-1/2 in obese rats. In contrast, insulin stimulated the phosphorylation of MAP kinase equally in lean and obese rats. CONCLUSION/INTERPRETATION: This study provides direct measurements of insulin signalling in hypothalamus, and documents selective resistance to insulin signalling in hypothalamus of Zucker rats. These findings provide support for the hypothesis that insulin could have anti-obesity actions mediated by the PI3-kinase pathway, and that impaired insulin signalling in hypothalamus could play a role in the development of obesity in this animal model of insulin-resistance.

Peroxisome proliferator-activated receptor gamma coactivator-1-dependent uncoupling protein-2 expression in pancreatic islets of rats: a novel pathway for neural control of insulin secretion.

AIMS/HYPOTHESIS: Sympathetic inputs inhibit insulin secretion through alpha2-adrenergic receptors coupled with Gi protein. High adrenergic tonus generated by exposure of homeothermic animals to cold reduces insulin secretion. In this study we evaluate the participation of UCP-2 in cold-induced regulation of insulin secretion. METHODS: Static insulin secretion studies, western blotting and immunohistochemistry were used in this investigation. RESULTS: Exposure of rats to cold during 8 days promoted 60% ( n=15, p<0.05) reduction of basal serum insulin levels concentration accompanied by reduction of the area under insulin curve during i.p. GTT (50%, n=15, p<0.05). Isolated islets from cold-exposed rats secreted 57% ( n=6, p<0.05) less insulin following a glucose challenge. Previous sympathectomy, partially prevented the effect of cold exposure upon insulin secretion. Islets isolated from cold-exposed rats expressed 51% ( n=6, p<0.5) more UCP-2 than islets from control rats, while the inhibition of UCP-2 expression by antisense oligonucleotide treatment partially restored insulin secretion of islets obtained from cold-exposed rats. Cold exposure also induced an increase of 69% ( n=6, p<0.05) in PGC-1 protein content in pancreatic islets. Inhibition of islet PGC-1 expression by antisense oligonucleotide abrogated cold-induced UCP-2 expression and partially restored insulin secretion in islets exposed to cold. CONCLUSION/INTERPRETATION: Our data indicate that sympathetic tonus generated by exposure of rats to cold induces the expression of PGC-1, which participates in the control of UCP-2 expression in pancreatic islets. Increased UCP-2 expression under these conditions could reduce the beta-cell ATP/ADP ratio and negatively regulate insulin secretion.

Prolactin-signal transduction in neonatal rat pancreatic islets and interaction with the insulin-signaling pathway.

During pregnancy, pancreatic islets undergo structural and functional changes in response to an increased demand for insulin. Different hormones, especially placental lactogens, mediate these adaptive changes. Prolactin (PRL) mainly exerts its biological effects by activation of the JAK2/STAT5 pathway. PRL also stimulates some biological effects via activation of IRS-1, IRS-2, PI 3-kinase, and MAPK in different cell lines. Since IRS-2 is important for the maintenance of pancreatic islet cell mass, we investigated whether PRL affects insulin-signaling pathways in neonatal rat islets. PRL significantly potentiated glucose-induced insulin secretion in islets cultured for 7 days. This effect was blocked by the specific PI 3-kinase inhibitor wortmannin. To determine possible effects of PRL on insulin-signaling pathways, fresh islets were incubated with or without the hormone for 5 or 15 min. Immunoprecipitation and immunoblotting with specific antibodies showed that PRL induced a dose-dependent IRS-1 and IRS-2 phosphorylation compared to control islets. PRL-induced increase in IRS-1/-2 phosphorylation was accompanied by an increase in the association with and activation of PI 3-kinase. PRL-induced IRS-2 phosphorylation and its association with PI 3-kinase did not add to the effect of insulin. PRL also induced JAK2, SHC, ERK1 and ERK2 phosphorylation in neonatal islets, demonstrating that PRL can activate MAPK. These data indicate that PRL can stimulate the IRSs/PI 3-kinase and SHC/ERK pathways in islets from neonatal rats.

Insulin signalling pathways in aorta and muscle from two animal models of insulin resistance--the obese middle-aged and the spontaneously hypertensive rats.

AIMS/HYPOTHESIS: The aim of this study was to investigate insulin signalling pathways directly in vivo in skeletal muscle and thoracic aorta from obese middle-aged (12-month-old) rats, which have insulin resistance but not cardiovascular disease, and from spontaneously hypertensive rats (SHR), an experimental model of insulin resistance and cardiovascular disease. METHODS: We have used in vivo insulin infusion, followed by tissue extraction, immunoprecipitation and immunoblotting. RESULTS: Obese middle-aged rats and the SHR showed marked insulin resistance, which parallels the reduced effects of this hormone in the insulin signalling cascade in muscle. In aortae from obese middle-aged rats, the PI 3-kinase/Akt pathway is preserved, leading to a normal activation of endothelial nitric oxide synthase. In SHR this pathway is severely blunted, with reductions in eNOS protein concentration and activation. Both animals, however, showed higher concentrations and higher tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoforms in aortae. CONCLUSIONS/INTERPRETATION: Alterations in the IRS/PI 3-K/Akt pathway in muscle of 12-month-old rats and SHR could be involved in the insulin resistance of these animals. The preservation of this pathway in aorta of 12-month-old rats, apart from increases in MAP kinase protein concentration and activation, could be a factor that contributes to explaining the absence of cardiovascular disease in this animal model. However, in aortae of SHR, the reduced insulin signalling through IRS/PI 3-kinase/Akt/eNOS pathway could contribute to the endothelial dysfunction of this animal.

Tissue-specific regulation of early steps in insulin action in septic rats.

Sepsis is known to induce insulin resistance, but the exact molecular mechanism involved is unknown. In the present study we have examined the levels and phosphorylation state of the insulin receptor and of insulin receptor substrate 1 (IRS-1), as well as the association between IRS-1 and phosphatidylinositol 3-kinase (PI 3-kinase) in the liver and muscle of septic rats by immunoprecipitation and immunoblotting with anti-insulin receptor, anti-IRS-1, anti-PI 3-kinase and anti-phosphotyrosine antibodies. There were no changes in the insulin receptor concentration and phosphorylation levels in the liver and muscle of septic rats. IRS-1 protein levels were decreased by 40+/-3% (p < 0.01) in muscle but not in liver of septic rats. In samples previously immunoprecipitated with anti-IRS-1 antibody and blotted with antiphosphotyrosine antibody, the insulin-stimulated IRS-1 phosphorylation levels in the muscle of septic rats decreased by 38+/-5% (p < 0.01) and insulin-stimulated IRS-1 association with PI 3-kinase decreased by 44+/-7% in muscle (p < 0.01) but no changes were seen in liver. These data suggest that there is a tissue-specific regulation of early steps of insulin signal transduction in septic rats, and the changes observed in muscle may have a role in the insulin resistance of these animals.

Insulin modulates leptin-induced STAT3 activation in rat hypothalamus.

Insulin and leptin have overlapping effects in the control of energy homeostasis, but the molecular basis of this synergism is unknown. Insulin signals through a receptor tyrosine kinase that phosphorylates and activates the docking proteins IRSs (insulin receptor substrates), whereas the leptin receptor and its associated protein tyrosine kinase JAK2 (Janus kinase 2) mediate phosphorylation and activation of the transcription factor STAT3 (signal transducer and activator of transcription). Here, we present evidence for the integration of leptin and insulin signals in the hypothalamus. Insulin induced JAK2 tyrosine phosphorylation, leptin receptor phosphorylation which, in the presence of leptin, augmented the interaction between STAT3 and this receptor. Insulin also increased the leptin-induced phosphorylation of STAT3 and its activation. These results indicate that insulin modulates the leptin signal transduction pathway, and may provide a molecular basis for the coordinated effects of insulin and leptin in feeding behavior and weight control.