p53 Mutant Human Glioma Cells Are Sensitive to UV-C-Induced Apoptosis Due to Impaired Cyclobutane Pyrimidine Dimer Removal

The p53 protein is a key regulator of cell responses to DNA damage, and it has been shown that It sensitizes glioma cells to the alkylating agent temozolomide by up-regulating the extrinsic apoptotic pathway, whereas it increases the resistance to chloroethylating agents, such as ACNU and BCNU, probably by enhancing the efficiency of DNA repair. However, because these agents induce a wide variety of distinct DNA lesions, the direct Importance of DNA repair is hard to access. Here, it is shown that the Induction of photoproducts by UV light (UV-C) significantly Induces apoptosis In a p53-mutated glioma background. This Is caused by a reduced level of photoproduct repair, resulting In the persistence of DNA lesions in p53-mutated glioma cells. UV-C-Induced apoptosis in p53 mutant glioma cells Is preceded by strong transcription and replication inhibition due to blockage by unrepaired photolesions. Moreover, the results Indicate that UV-C-induced apoptosis of p53 mutant glioma cells Is executed through the intrinsic apoptotic pathway, with Bcl-2 degradation and sustained Bax and Bak up-regulation. Collectively, the data Indicate that unrepaired DNA lesions Induce apoptosis In p53 mutant gliomas despite the resistance of these gliomas to temozolomide, suggesting that efficiency of treatment of p53 mutant gliomas might be higher with agents that Induce the formation of DNA lesions whose global genomic repair is dependent on p53. (Mol Cancer Res 2009;7(2):237-46)

Immobilization of primary cultures of insulin-releasing human pancreatic cells

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type 1 diabetes, however, it is severely limited by the shortage of organ donors. Ex vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 diabetes. It has recently been shown that, even in the absence of fibrotic over-growth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

Dyslipidaemia and insulin resistance in vertically HIV-infected children and adolescents

This cross-sectional study determined the influence of antiretroviral therapy (ART) on the lipid profile and insulin sensitivity of 119 perinatally HIV-infected Brazilian patients aged 6-19 years. Inadequate high-density lipoprotein cholesterol (HDL-c) concentrations were observed in 81.4% of patients. High concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDI.-c) and triglycerides (TG) were found in 33.9%, 9.7% and 35.6% of patients, respectively. There were statistically significant differences in mean concentrations of TC (P=0.004), HDL-c (P=0.015) and LDL-c (P=0.028) among children (< 10 years), early adolescents (10-14 years) and late adolescents (15-19 years). Children presented the highest mean concentrations of TC and LDL-c, and patients in late adolescence presented the lowest concentrations of HDL-c. Insulin sensitivity, assessed by the Homeostasis Model Assessment (HOMA) index, was diagnosed in 16.7% of patients, with a statistically higher proportion (P=0.034) of insulin-resistant children (33.3%) compared with adolescents (12.5%). There was a statistically significant association between TG concentrations and use of ART regimens containing protease inhibitors (PI) (P=0.0003). Children presented a higher prevalence of insulin resistance and dyslipidaemia compared with adolescents, suggesting that ART, especially Pls, may lead to metabolic complications. (C) 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Calcium intake and its relationship with adiposity and insulin resistance in post-pubertal adolescents

Background Dietary calcium intake has been described as being a negative contributor to adiposity. In adolescents, this relationship is not well established. The objectives of the present study were to compare the calcium intake of normal-weight and obese adolescents and to evaluate its relationship with adiposity and insulin resistance. Methods A cross-sectional analysis of 96 post-pubertal adolescents; 47 normal weight and 49 obese, mean age 16.6 (SD +/- 1.3) years. Body composition was assessed by dual-energy X-ray absorptiometry. Dietary intake was evaluated using a 3-day dietary record. The biochemical evaluation comprised the measurements of serum lipids, lipoproteins, glucose and insulin. Insulin resistance was calculated using the Homeostasis Model Assessment of Insulin resistance (HOMA-IR). Results The mean calcium intake, adjusted for energy, was lower in obese adolescents, 585.2 (+/- 249.9) mg, than in normal weight adolescents, 692.1 (+/- 199.5) mg. Only 4% of adolescents had an adequate intake of calcium. Calcium intake was inversely associated with body trunk fat, insulin and HOMA-IR in the obese group. The quartile analysis of calcium intake provided evidence that girls in the highest quartile had decreased adiposity and insulin resistance. Conclusions This study showed a negative relationship between calcium intake and body fat and insulin resistance, mainly in obese girls, and demonstrates the importance of an increased dietary calcium intake.

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.

High sodium intake enhances insulin-stimulated glucose uptake in rat epididymal adipose tissue

Objective: This study investigated the effect of different sodium content diets on rat adipose tissue carbohydrate metabolism and insulin sensitivity. Methods and Procedures: Male Wistar rats were fed on normal- (0.5% Na+; NS), high- (3.12% Na+; HS), or low-sodium (0.06% Na+; LS) diets for 3, 6, and 9 weeks after weaning. Blood pressure (BP) was measured using a computerized tail-cuff system. An intravenous insulin tolerance test (ivITT) was performed in fasted animals. At the end of each period, rats were killed and blood samples were collected for glucose and insulin determinations. The white adipose tissue (WAT) from abdominal and inguinal subcutaneous (SC) and periepididymal (PE) depots were weighed and processed for adipocyte isolation and measurement of in vitro rates of insulin-stimulated 2-deoxy-d-[H-3]-glucose uptake (2DGU) and conversion of -[U-C-14]-glucose into (CO2)-C-14. Results: After 6 weeks, HS diet significantly increased the BP, SC and PE WAT masses, PE adipocyte size, and plasma insulin concentration. The sodium dietary content did not influence the whole-body insulin sensitivity. A higher half-maximal effective insulin concentration (EC50) from the dose - response curve of 2DGU and an increase in the insulin-stimulated glucose oxidation rate were observed in the isolated PE adipocytes from HS rats. Discussion: The chronic salt overload enhanced the adipocyte insulin sensitivity for glucose uptake and the insulin-induced glucose metabolization, contributing to promote adipocyte hypertrophy and increase the mass of several adipose depots, particularly the PE fat pad.

Smad5 regulates Akt2 expression and insulin-induced glucose uptake in L6 myotubes

Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5. a transcription factor activated by BMP9, and Akt2. are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated ""in vivo"" and ""in vitro"" by dexamethasone Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and Increased in L6 myotubes compared to myoblasts The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes. (C) 2010 Elsevier Ireland Ltd. 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

Expression of Pancreatic Endocrine Markers by Mesenchymal Stem Cells From Human Umbilical Cord Vein

Background. Mesenchymal stem cells (MSCs) from human umbilical cord vein have great potential for use in cell therapy because of their ease of isolation, expansion, and differentiation, in addition to their relative acceptance from the ethical point of view. Obtaining the umbilical cord at birth does not present any risk to either mother or child. Objective. To isolate and promote in vitro expansion and differentiation of MSCs from human umbilical cord vein into cells with a pancreatic endocrine phenotype. Methods. Mesenchymal stem cells obtained from human umbilical cord vein via collagenase digestion were characterized at cytochemistry and fluorescent-activated cell sorting, and expanded in vitro. Differentiation of MSCs into an endocrine phenotype was induced using high-glucose (23 mmol/L) medium containing nicotinamide, exendin-4, and 2-mercaptoethanol. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was analyzed using immunofluorescence. Results. Cells isolated from the umbilical cord vein were MSCs as confirmed at cytochemistry and fluorescent-activated cell sorting. Expression of somatostatin, glucagon, and pancreatic and duodenal homeobox 1 by differentiated cells was demonstrated using immunofluorescence. Insulin was not expressed. Conclusions. The MSC differentiation protocol used in the present study induced expression of some endocrine markers. Insulin was not produced by these cells, probably because of incomplete induction of differentiation.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K`s regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3 beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis. (C) 2009 Published by Elsevier Inc.

Anti-inflammatory effect of atorvastatin ameliorates insulin resistance in monosodium glutamate-treated obese mice

Considering that inflammation contributes to obesity-induced insulin resistance and that statins have been reported to have other effects beyond cholesterol lowering, the present study aimed to it whether atorvastatin treatment has anti-inflammatory action in white adipose tissue of obese mice, consequently improving insulin sensitivity. Insulin sensitivity in vivo (by insulin tolerance test); metabolic-hormonal profile; plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and adiponectin; adipose tissue immunohistochemistry; glucose transporter (GLUT) 4; adiponectin; INF-alpha; IL-1 beta; and IL-6 gene expression; and I kappa B kinase (IKK)-alpha/beta activity were assessed in 23-week-old monosodium glutamate induced obese mice untreated or treated with atorvastatin for 4 weeks. Insulin-resistant obese mice had increased plasma triglyceride, insulin, TNF-alpha, and IL-6 plasma levels. Adipose tissue of obese animals showed increased macrophage infiltration, IKK-alpha (42%, P < .05) and IKK-beta (73%, P < .05) phosphorylation, and INF-alpha and IL-6 messenger RNA (mRNA) (similar to 15%, P < .05) levels, and decreased GLUT4 mRNA and protein (30%, P < .05) levels. Atorvastatin treatment lowered cholesterol, triglyceride, insulin, INF-alpha, and IL-6 plasma levels, and restored whole-body insulin sensitivity. In adipose tissue, atorvastatin decreased macrophage in and normalized IKK-alpha/beta phosphorylation; INF-alpha, IL-6, and GLUT4 mRNA; and GLUT4 protein to control levels. The present findings demonstrate that atorvastatin has anti-inflammatory effects on adipose tissue of obese mice, which may be important to its local and whole-body insulin-sensitization effects. (C) 2010 Published by Elsevier Inc.

The binding of synthetic triiodo L-thyronine analogs to human transthyretin: Molecular basis of cooperative and non-cooperative ligand recognition

Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. Several classes of small molecules can bind to TTR delaying its amyloid fibril formation, thus being promising drug candidates to treat TTR amyloidoses. In the present study, we characterized the interactions of the synthetic triiodo L-thyronine analogs and thyroid hormone nuclear receptor TR beta-selecfive agonists GC-1 and GC-24 with the wild type and V30M variant of human transthyretin (TTR). To achieve this aim, we conducted in vitro TTR acid-mediated aggregation and isothermal titration calorimetry experiments and determined the TTR:GC-1 and TTR:GC-24 crystal structures. Our data indicate that both GC-1 and GC-24 bind to TTR in a non-cooperative manner and are good inhibitors of TTR aggregation, with dissociation constants for both hormone binding sites (HBS) in the low micromolar range. Analysis of the crystal structures of TTRwt:GC-1(24) complexes and their comparison with the TTRwt X-ray structure bound to its natural ligand thyroxine (T4) suggests, at the molecular level, the basis for the cooperative process displayed by T4 and the non-cooperative process provoked by both GC-1 and GC-24 during binding to TTR. (C) 2010 Elsevier Inc. All rights reserved.

Skipping of exon 30 in C5 gene results in complete human C5 deficiency and demonstrates the importance of C5d and CUB domains for stability

The deficiency of complement C5 is rare and frequently associated with severe and recurrent infections, especially caused by Neisseria spp. We observed the absence of component C5 in the serum of 3 siblings from a Brazilian family with history of consanguinity. The patients had suffered from recurrent episodes of meningitis and other less severe infections. Sera from these patients were unable to mediate hemolytic activity either by the classical or alternative pathways and presented extremely low levels of C5 protein (13, 0.9 and 1.0 mu g/ml-normal range: 45-190 mu g/ml). Hemolytic activity could be restored by the addition of purified C5 to deficient serum. Sequencing of sibling C5 cDNA revealed a homozygous 153 bp deletion that corresponds precisely to exon 30. The parents carried the same deletion but only in one allele. Sequencing of the corresponding region in the genomic DNA revealed a C to C substitution within intron 30 and, most significantly, the substitution of GAG(4028) for GAA(4028) at the 3` end of exon 30 which is most likely responsible for skipping of exon 30. The resulting in-frame deletion in the C5 mRNA codes for a mutant C5 protein lacking residues 1289-1339. These residues map to the CUB and C5d domains of the C5 alpha chain. This deletion is expected to produce a non-functional and unstable C5 protein which is more susceptible to degradation. (C) 2009 Published by Elsevier Ltd.

Biodritin microencapsulated human islets of langerhans and their potential for type 1 diabetes mellitus therapy

Background. Microencapsulation of pancreatic islets with polymeric compounds constitutes an attractive alternative therapy for type 1 diabetes mellitus. The major limiting factor is the availability of a biocompatible and mechanically stable polymer. We investigated the potential of Biodritin, a novel polymer constituted of alginate and chondroitin sulfate, for islet microencapsulation. Methods. Biodritin microcapsules were obtained using an air jet droplet generator and gelated with barium or calcium chloride. Microencapsulated rat insulinoma RINm5F cells were tested for viability using the [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] [MTT] colorimetric assay. Microencapsulated rat pancreatic islets were coincubated with macrophages derived from mouse peritoneal liquid to assess the immunomodulatory potential of the microcapsules, using quantitative real time-PCR (qPCR). Biodritin biocompatibility was demonstrated by subcutaneous injection of empty microcapsules into immunocompetent Wistar rats. Insulin secretion by microencapsulated human pancreatic islets was evaluated using an electrochemoluminescent assay. Microencapsulated human islets transplanted into chemically induced diabetic mice were monitored for reversal of hyperglycemia. Results. The metabolic activity of microencapsulated RINm5F cells persisted for at least 15 days. Interleukin-1 beta expression by macrophages was observed during coculture with islets microencapsulated with Biodritin-CaCl2, but not with Biodritin-BaCl2. No statistical difference in glucose-stimulated insulin secretion was observed between nonencapsulated and microencapsulated islets. Upon microencapsulated islet transplantation, the blood glucose level of diabetic mice normalized; they remained euglycemic for at least 60 days, displaying normal oral glucose tolerance tests. Conclusion. This study demonstrated that Biodritin can be used for islet microencapsulation and reversal of diabetes; however, further investigations are required to assess its potential for long-term transplantation.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAD genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1 Delta) of this gene. The gat 1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gal is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans. (C) 2010 Elsevier Inc. All rights reserved.