High-Resolution Genomic Mapping Reveals Consistent Amplification of the Fibroblast Growth Factor Receptor Substrate 2 Gene in Well-Differentiated and Dedifferentiated Liposarcoma

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31 CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) ""single nucleotide polymorphism""/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at 1436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma. (C) 2011 Wiley-Liss, Inc.

High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets

Rafacho A, Cestari TM, Taboga SR, Boschero AC, Bosqueiro JR. High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets. Am J Physiol Endocrinol Metab 296: E681-E689, 2009. First published January 21, 2009; doi:10.1152/ajpendo.90931.2008.-Activation of insulin signaling and cell cycle intermediates is required for adult beta-cell proliferation. Here, we report a model to study beta-cell proliferation in living rats by administering three different doses of dexamethasone (0.1, 0.5, and 1.0 mg/kg ip, DEX 0.1, DEX 0.5, and DEX 1.0, respectively) for 5 days. Insulin sensitivity, insulin secretion, and histomorphometric data were investigated. Western blotting was used to analyze the levels of proteins related to the control of beta-cell growth. DEX 1.0 rats, which present moderate hyperglycemia and marked hyperinsulinemia, exhibited a 5.1-fold increase in beta-cell proliferation and an increase (17%) in beta-cell size, with significant increase in beta-cell mass, compared with control rats. The hyperinsulinemic but euglycemic DEX 0.5 rats also showed a significant 3.6-fold increase in beta-cell proliferation. However, DEX 0.1 rats, which exhibited the lowest degree of insulin resistance, compensate for insulin demand by improving only islet function. Activation of the insulin receptor substrate 2/phosphatidylinositol 3-kinase/serine-threoninekinase/ribosomalprotein S6 kinase pathway, as well as protein retinoblastoma in islets from DEX 1.0 and DEX 0.5, but not in DEX 0.1, rats was also observed. Therefore, increasing doses of dexamethasone induce three different degrees of insulin requirement in living rats, serving as a model to investigate compensatory beta-cell alterations. Augmented beta-cell mass involves beta-cell hyperplasia and, to a lower extent, beta-cell hypertrophy. We suggest that alterations in circulating insulin and, to a lesser extent, glucose levels could be the major stimuli for beta-cell proliferation in the dexamethasone-induced insulin resistance.

Exercise Intensity, Inflammatory Signaling, and Insulin Resistance in Obese Rats

DA SILVA, A. S. R., J. R. PAULI, E. R. ROPELLE, A. G. OLIVEIRA, D. E. CINTRA, C. T. DE SOUZA, L. A. VELLOSO, J. B. C. CARVALHEIRA, and M. J. A. SAAD. Exercise Intensity, Inflammatory Signaling, and Insulin Resistance in Obese Rats. Med. Sci. Sports Exerc., Vol. 42, No. 12, pp. 2180-2188, 2010. Purpose: To evaluate the effects of intensity of exercise on insulin resistance and the expression of inflammatory proteins in the skeletal muscle of diet-induced obese (DIO) rats after a single bout of exercise. Methods: In the first exercise protocol, the rats swam for two 3-h bouts, separated by a 45-min rest period (with 6 h in duration-DIO + EXE), and in the second protocol, the rats were exercised with 45 min of swimming at 70% of the maximal lactate steady state-MLSS (DIO + MLSS). Results: Our data demonstrated that both protocols of exercise increased insulin sensitivity and increased insulin-stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate 1 and serine phosphorylation of protein kinase B in the muscle of DIO rats by the same magnitude. In parallel, both exercise protocols also reduced protein tyrosine phosphatase 1B activity and insulin receptor substrate 1 serine phosphorylation, with concomitant reduction in c-jun N-terminal kinase and I kappa B kinase activities in the muscle of DIO rats in a similar fashion. Conclusions: Thus, our data demonstrate that either exercise protocols with low intensity and high volume or exercise with moderate intensity and low volume represents different strategies to restore insulin sensitivity with the same efficacy.

Low-intensity Ultrasound Increases FAK, ERK-1/2, and IRS-1 Expression of Intact Rat Bones in a Noncumulative Manner

Background Low-intensity pulsed ultrasound stimulation (LIPUS) reportedly increases osteogenesis in fracture models but fails in intact bone, suggesting LIPUS does not act on mechanotransduction and growth factor pathways of intact bone. Questions/Purposes We asked whether daily 20-minute LIPUS applied to intact tibias would act on bone proteins involved in mechanotransduction (focal adhesion kinase [FAK], and extracellular signal-regulated kinase-1/2 [ERK-1/2]), and growth factor signaling (insulin receptor substrate-1 [IRS-1]) pathways at 7, 14, and 21 days of treatment. Methods Immunoblotting was performed to detect FAK, ERK-1/2, and IRS-1 expression and activation from the stimulated intact tibias at 7, 14, and 21 days of daily 20-minute LIPUS. Results LIPUS increased FAK expression (at 7 days), ERK-1/2 (at 14 days), and IRS-1 (at 7 days), but expression decreased 7 days later, indicating a noncumulative effect of LIPUS. As only FAK expression was detected at 21 days, these observations suggest LIPUS influences nuclear reactions that may be modulated by a major cellular mechanism preferentially inhibiting IRS-1 expression and not FAK expression. Increased ERK-1/2 expression at 14 days suggests the differing mechanisms for promoting ERK-1/2, FAK, and IRS-1 syntheses. IRS-1 expression behaved similarly to FAK expression; therefore, LIPUS may modulate growth factor pathways. LIPUS increased sustained FAK and ERK-1/2 activation, but not IRS-1, suggesting sustained ERK-1/2 activation is not the result of mechanically induced growth factor activation. Conclusions LIPUS acts on mechanotransduction and growth factor pathways in intact bone in a noncumulative manner. Clinical relevance These data suggest LIPUS applied to intact bone acts on proteins involved in osteogenesis.

Biological effects of insulin on murine melanoma cells and fish erythrophoroma cells: A comparative study

Insulin is the hormone that plays an essential role in metabolism and mitosis of normal and tumor cells, exerting its pleiotropic effects through binding to specific membrane receptors and promoting the phosphorylation of tyrosine residues of the receptor itself and of other components of the signaling pathway. The aim of this study was to investigate the effects of insulin on melanogenesis and cell growth in three different cell lines: the goldfish GEM-81 erythrophoroma cells (undifferentiated and differentiated with 1.5% dimethylsulfoxide-DMSO), and the murine B16F10 and Cloudman S91 melanoma cells. Undifferentiated GEM-81 and B16F10 cells responded to insulin with a small increase of cell proliferation, whereas S91 cells responded with a decrease of growth. In the two mammalian cell lines, and in DMSO-differentiated GEM-81 cells, the hormone strongly inhibited melanogenesis, by decreasing tyrosinase activity. In undifferentiated GEM-81 cells, insulin had no effect on tyrosinase activity. An increase in the tyrosine phosphorylation status of pp 185 (insulin receptor substrate 1 and 2-IRS-1/2) phosphorylation degree was observed in S91 mouse melanoma and in differentiated GEM-81 erythrophoroma cells, suggesting that this specific protein was maintained during transformation process and participates in insulin signaling. Our results imply an ancient and diverse history of the insulin signaling system in vertebrate pigment cells. (C) 2008 Elsevier Inc. All rights reserved.

Vascular density and distribution of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) are significantly higher in patients with deeply infiltrating endometriosis affecting the rectum

Objective: To analyze vascular density and immunolocalization of angiogenic vascular endothelial growth factor (VEGF) and its receptor Flk-1 in the proliferative and secretory eutopic human endometrium. and in three different sites of endometriosis: the ovary, bladder, and rectum. Design: Prospective study. Setting: University hospital. Patient(s): Thirty women with endometriosis (10 ovarian, 1.0 bladder, 10 rectal) and 32 control women (10 proliferative endometrium, 10 secretory endometrium, 4 normal ovary, 4 normal bladder, 4 normal rectum). Intervention(s): Normal endometrial samples were obtained from women during laparoscopic ablation of subserous myoma, and biopsy specimens of endometriosis were obtained from patients undergoing surgery for the diagnosis and treatment of endometriosis. Normal tissues of ovary, bladder, and rectum were obtained from these organs beside the lesions of endometriosis. Main Outcome Measure(S): Blood vessels were quantified according to the number of von Willebrand factor-positive endothelial cells. The VEGF and Flk-1 distribution were evaluated semiquantitatively by immunohistochemical staining. Result(s): More blood vessels were found in cases of endometriosis, particularly rectal endometriosis, compared with the respective control samples and with the eutopic endometrium, and they were localized in endometrial stroma around the glands. The VEGF and Flk-1 expression levels were also higher in cases of endometriosis, especially rectal endometriosis. Conclusion(s): Vascularization and VEGF and Flk-1 expression are significantly higher in deeply infiltrating endometriosis affecting the rectum, reinforcing the hypothesis that antiangiogenesis therapy may constitute a new modality of treatment, especially in cases of deep endometriosis involving the rectum.

Augmentation of insulin secretion by leucine supplementation in malnourished rats: possible involvement of the phosphatidylinositol 3-phosphate kinase/mammalian target protein of rapamycin pathway

A regimen of low-protein diet induces a reduction of pancreatic islet function that is associated with development of metabolic disorders including diabetes and obesity afterward. In the present study, the influence of leucine supplementation on metabolic parameters, insulin secretion to glucose and to amino acids, as well as the levels of proteins that participate in the phosphatidylinositol 3-phosphate kinase (PI3K) pathway was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal protein diet (17%) without (NP) or with leucine supplementation (NPL) or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine was given in the drinking water during the last 4 weeks. As indicated by the intraperitoneal glucose tolerance test, LPL rats exhibited increased glucose tolerance as compared with NPL group. Both NPL and LPL rats had higher circulating insulin levels than controls. The LPL rats also showed increased insulin secretion by pancreatic islets in response to glucose or arginine compared with those observed in islets from LP animals. Glucose oxidation was significantly reduced in NPL, LP, and LPL isolated islets as compared with NP; but no alteration was observed for leucine and glutamate oxidation among the 4 groups. Western blotting analysis demonstrated increased PI3K and mammalian target protein of rapamycin protein contents in LPL compared with LP islets. A significant increase in insulin-induced insulin receptor substrate I associated PI3K activation was also observed in LPL compared with LP islets. These findings indicate that leucine supplementation can augment islet function in malnourished rats and that activation of the PI3K/maminalian target protein of rapamycin pathway may play a role in this process. (C) 2010 Elsevier Inc. All rights reserved.

Obesity induced by high-fat diet promotes insulin resistance in the ovary

Besides the effects on peripheral energy homeostasis, insulin also has an important role in ovarian function. Obesity has a negative effect on fertility, and may play a role in the development of the polycystic ovary syndrome in susceptible women. Since insulin resistance in the ovary could contribute to the impairment of reproductive function in obese women, we evaluated insulin signaling in the ovary of high-fat diet-induced obese rats. Female Wistar rats were submitted to a high-fat diet for 120 or 180 days, and the insulin signaling pathway in the ovary was evaluated by immunoprecipitation and immunoblotting. At the end of the diet period, we observed insulin resistance, hyperinsulinemia, an increase in progesterone serum levels, an extended estrus cycle, and altered ovarian morphology in obese female rats. Moreover, in female obese rats treated for 120 days with the high-fat diet, the increase in progesterone levels occurred together with enhancement of LH levels. The ovary from high-fat-fed female rats showed a reduction in the insulin receptor substrate/phosphatidylinositol 3-kinase/AKT intracellular pathway, associated with an increase in FOXO3a, IL1B, and TNF alpha protein expression. These changes in the insulin signaling pathway may have a role in the infertile state associated with obesity. Journal of Endocrinology (2010) 206, 65-74

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance. (C) 2011 Elsevier Inc. All rights reserved.

Endurance exercise training ameliorates insulin resistance and reticulum stress in adipose and hepatic tissue in obese rats

Obesity-induced endoplasmatic reticulum (ER) stress has been demonstrated to underlie the induction of obesity-induced JNK and NF-kappa B activation inflammatory responses, and generation of peripheral insulin resistance. On the other hand, exercise has been used as a crucial tool in obese and diabetic patients, and may reduce inflammatory pathway stimulation. However, the ability of exercise training to reverse endoplasmatic reticulum stress in adipose and hepatic tissue in obesity has not been investigated in the literature. Here, we demonstrate that exercise training ameliorates ER stress and insulin resistance in DIO-induced rats. Rats were fed with standard rodent chow (3,948 kcal kg(-1)) or high-fat diet (5,358 kcal kg(-1)) for 2 months. After that rats were submitted to swimming training (1 h per day, 5 days for week with 5% overload of the body weight for 8 weeks). Samples from epididymal fat and liver were obtained and western blot analysis was performed. Our results showed that swimming protocol reduces pro-inflammatory molecules (JNK, I kappa B and NF-kappa B) in adipose and hepatic tissues. In addition, exercise leads to reduction in ER stress, by reducing PERK and eIF2 alpha phosphorylation in these tissues. In parallel, an increase in insulin pathway signaling was observed, as confirmed by increases in IR, IRSs and Akt phosphorylation following exercise training in DIO rats. Thus, results suggest that exercise can reduce ER stress, improving insulin resistance in adipose and hepatic tissue.

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 A cells.

Potential Role of Growth Hormone in Impairment of Insulin Signaling in Skeletal Muscle, Adipose Tissue, and Liver of Rats Chronically Treated with Arginine

We have shown that rats chronically treated with Arginine (Arg), although normoglycemic, exhibit hyperinsulinemia and decreased blood glucose disappearance rate after an insulin challenge. Attempting to investigate the processes underlying these alterations, male Wistar rats were treated with Arg (35 mg/d), in drinking water, for 4 wk. Rats were then acutely stimulated with insulin, and the soleus and extensorum digitalis longus muscles, white adipose tissue (WAT), and liver were excised for total and/or phosphorylated insulin receptor (IR), IR substrate 1/2, Akt, Janus kinase 2, signal transducer and activator of transcription (STAT) 1/3/5, and p85 alpha/55 alpha determination. Muscles and WAT were also used for plasma membrane (PM) and microsome evaluation of glucose transporter (GLUT) 4 content. Pituitary GH mRNA, GH, and liver IGF-I mRNA expression were estimated. It was shown that Arg treatment: 1) did not affect phosphotyrosine-IR, whereas it decreased phosphotyrosine-IR substrate 1/2 and phosphoserine-Akt content in all tissues studied, indicating that insulin signaling is impaired at post-receptor level; 2) decreased PM GLUT4 content in both muscles and WAT; 3) increased the pituitary GH mRNA, GH, and liver IGF-I mRNA expression, the levels of phosphotyrosine-STAT5 in both muscles, phosphotyrosine-Janus kinase 2 in extensorum digitalis longus, phosphotyrosine-STAT3 in liver, and WAT as well as total p85 alpha in soleus, indicating that GH signaling is enhanced in these tissues; and 4) increased p55 alpha total content in muscles, WAT, and liver. The present findings provide the molecular mechanisms by which insulin resistance and, by extension, reduced GLUT4 content in PM of muscles and WAT take place after chronic administration of Arg, and further suggest a putative role for GH in its genesis, considering its diabetogenic effect. (Endocrinology 150: 2080-2086, 2009)

Therapeutic administration of KM+ lectin protects mice against Paracoccidioides brasiliensis infection via interleukin-12 production in a Toll-like receptor 2-dependent mechanism

KM+ is a mannose-binding lectin from Artocarpus integrifolia that induces interleukin (IL)-12 production by macrophages and protective T helper I immune response against Leishmania major infection. in this study, we performed experiments to evaluate the therapeutic activity of jackfruit KM+ (jfKM(+)) and its recombinant counterpart (rKM(+)) in experimental paracoccidioidomycosis. To this end, jfKM(+) or rKM(+) was administered to BALB/c mice 10 days after infection with Paracoccidiodes brasiliensis. Thirty days postinfection, lungs from the KM+-treated mice contained significantly fewer colony-forming units and little to no organized granulomas compared to the controls. In addition, lung homogenates from the KM+-treated mice presented higher levels of nitric oxide, IL-12, interferon-gamma, and tumor necrosis factor-a, whereas higher levels of IL-4 and IL-10 were detected in the control group. With mice deficient in IL-12, Toll-like receptor (TLR) 2, TLR4, or TLR adaptor molecule MyD88, we demonstrated that KM+ led to protection against P. brasiliensis infection through IL-12 production, which was dependent on TLR2. These results demonstrated a beneficial effect of KM+ on the severity of P. brasiliensis infection and may expand its potential use as a novel immunotherapeutic molecule.

The insulin signaling pathway in honey bee (Apis mellifera) caste development - differential expression of insulin-like peptides and insulin receptors in queen and worker larvae

The insulin/insulin-like signaling (IIS) pathway is an evolutionarily conserved module in the control of body size and correlated organ growth in metazoans. In the highly eusocial bees, the caste phenotypes differ not only in size and several structural features but also in individual fitness and life history. We investigated the developmental expression profiles of genes encoding the two insulin-like peptides (AmILP-1 and AmILP-2) and the two insulin receptors (AmInR-1 and AmInR-2) predicted in the honey bee genome. Quantitative PCR analysis for queen and worker larvae in critical stages of caste development showed that AmILP-2 is the predominantly transcribed ILP in both castes, with higher expression in workers than in queens. Expression of both InR genes sharply declined in fourth instar queen larvae, but showed little modulation in workers. On first sight, these findings are non-intuitive, considering the higher growth rates of queens, but they can be interpreted as possibly antagonistic crosstalk between the IIS module and juvenile hormone. Analyzing AmInR-1 and AmInR-2 expression in ovaries of queen and worker larvae revealed low transcript levels in queens and a sharp drop in AmInR-2 expression in fifth instar worker larvae, indicating relative independence in tissue-specific versus overall IIS pathway activity. (C) 2008 Elsevier Ltd. All rights reserved.

Assessment of the Expression of IR beta, IRS-1, IRS-2 and IGF-IR beta in a Rat Model of Intrauterine Growth Restriction

Objective: To investigate glomerular development and expression of insulin and insulin-like growth factor receptors in an experimental model of intrauterine growth restriction (IUGR). Material and Methods: We studied three groups of Sprague-Dawley fetuses: IUGR - restricted by ligation of the right uterine artery; C-IUGR - left horn controls, and EC - external controls (non-manipulated). Body and organs were weighed, and glomerular number and volume were analyzed. Expression of IR beta, IRS-1, IRS-2 and IGF-IR beta was analyzed in liver, intestine and kidneys by immunoblotting. Results: Organ/body weight ratios were similar. In IUGR, glomerular number and volume were increased compared to C-IUGR and EC (p < 0.001). In the IUGR liver, increases were found in IGF-IR beta compared to C-IUGR and EC; IR beta compared to EC, and IRS-2 compared to C-IUGR. However, decreases in IR beta were noted in IUGR compared to C-IUGR; IRS-1 compared to C-IUGR and EC, and IRS-2 compared to EC. In IUGR intestine, increases were detected in IR beta, IRS-1 and IGF-IR beta compared to C-IUGR and EC. In IUGR kidneys, increases were observed in IR beta and IGF-IR beta compared to C-IUGR and EC, and IRS-1 compared to EC. Decreased IRS-2 in the intestine and kidney were noticed in IUGR compared to C-IUGR and EC. Conclusion: IUGR fetuses had less glomeruli and alterations in insulin receptors, which may be associated with an increased risk of disease occurrence in adulthood. Copyright (C) 2010 S. Karger AG, Basel