Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses

Abstract Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

Vochysia tucanorum Mart. butanol fraction presents antitumoral activity in vivo and prevents the installation of cachexia in solid Ehrlich tumor model.

BACKGROUND: Cancer is a multifactorial disease caused by uncontrolled proliferation of cells. About 50-80% of cancer patients develop cachexia, a complex metabolic syndrome associated with an increase of mortality and morbidity. However, there are no effective therapies in medical clinic for cancer cachexia. Vochysia tucanorum Mart. is a common three of the Brazilian "Cerrado". The butanolic fraction of V. tucanorum (Fr-BuVt), very rich in triterpenes with various biological activities, might be interesting in being tested in cancer cachexia syndrome. Hence, the present study was undertaken to investigate the antitumoral activity of Fr-BuVt and its potential against cachexia development. METHODS: Ehrlich tumor was used as model of cancer cachexia. Ascitic Ehrlich tumor cells were collected, processed and inoculated subcutaneously in saline solution (1 × 107/100 µl; ≥95% viability) for the obtention of solid Ehrlich carcinoma. After inoculation, solid Ehrlich carcinoma-bearing mice were treated by 14 consecutive days by gavage with Fr-BuVt (200 mg/kg). Body weight and tumor volume were measure during the treatment period. Tumors were removed, weighed and properly processed to measure the content and phosphorylation levels of key-proteins involved to apoptotic and proliferation process by Western Blot. Muscles and adipose tissues were removed for weighed. Serum was collected to cytokines levels and energetic blood markers measurements. RESULTS: The treatment with the Fr-BuVt (200 mg/kg, 14 days) decreased the solid Ehrlich tumor volume and weight besides increased the expression of the pro-apoptotic proteins caspase-3 and BAX, but also decreased the expression of the proteins involved in proliferation NFκB, mTOR and ERK. In addition, our data shows that the administration of Fr-BuVt was able to prevent the installation of cancer cachexia in Ehrlich carcinoma-bearing mice, since prevented the loss of body weight, as well as the loss of muscle and adipose tissue. Moreover, an improvement in some blood parameters such as decrease in cytokines TNF-α and IL-6 levels is observed. CONCLUSIONS: The study revealed that Fr-BuVt has antitumoral activity and prevent installation of cancer cachexia in Ehrlich model. Therefore, Fr-BuVt may represent an alternative treatment for cancer cachexia.

High-fat diet obesity associated with insulin resistance increases cell proliferation, estrogen receptor, and PI3K proteins in rat ventral prostate.

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ERß) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ERß expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ERß and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Increased pancreatic islet mass is accompanied by activation of the insulin receptor substrate-2/serine-threonine kinase pathway and augmented cyclin D2 protein levels in insulin-resistant rats.

It is well known that glucocorticoids induce peripheral insulin resistance in rodents and humans. Here, we investigated the structural and ultrastructural modifications, as well as the proteins involved in beta-cell function and proliferation, in islets from insulin-resistant rats. Adult male Wistar rats were made insulin resistant by daily administration of dexamethasone (DEX; 1mg/kg, i.p.) for five consecutive days, whilst control (CTL) rats received saline alone. Structure analyses showed a marked hypertrophy of DEX islets with an increase of 1.7-fold in islet mass and of 1.6-fold in islet density compared with CTL islets (P < 0.05). Ultrastructural evaluation of islets revealed an increased amount of secreting organelles, such as endoplasmic reticulum and Golgi apparatus in DEX islets. Mitotic figures were observed in DEX islets at structural and ultrastructural levels. Beta-cell proliferation, evaluated at the immunohistochemical level using anti-PCNA (proliferating cell nuclear antigen), showed an increase in pancreatic beta-cell proliferation of 6.4-fold in DEX islets compared with CTL islets (P < 0.0001). Increases in insulin receptor substrate-2 (IRS-2), phosphorylated-serine-threonine kinase AKT (p-AKT), cyclin D(2) and a decrease in retinoblastoma protein (pRb) levels were observed in DEX islets compared with CTL islets (P < 0.05). Therefore, during the development of insulin resistance, the endocrine pancreas adapts itself increasing beta-cell mass and proliferation, resulting in an amelioration of the functions. The potential mechanisms that underlie these events involve the activation of the IRS-2/AKT pathway and activation of the cell cycle, mediated by cyclin D(2). These adaptations permit the maintenance of glycaemia at near-physiological ranges.

Efeitos da administração de dexametasona in vivo sobreglicemia, insulinemia e substratos circulantes sãodependentes do tempo de tratamento / Effects of dexamethasone administration in vivo on glycaemia, insulinaemia and circulating substrates are dependents of timeof treatment

Terapias a base de glicocorticóides estão freqüentemente associadas a alteração da sensibilidade à insulina. No presente trabalho avaliamos alguns parâmetros metabólicos como glicose, insulina, proteínas e colesterolplasmáticos em ratos tratados com dexametasona (DEX) (1mg/kg, peso corpóreo, ip.) por diferentes períodos de tempo (24h, 72h e 120h). Os ratos tratados com dexametasona apresentaram resistência periférica à insulina após 24h de administração da droga como indicam os valores de insulina plasmática de jejum (1,3 vs. 6,8 ng/ml para ratos controle [CTL] e DEX, respectivamente) e do índice HOMA. Resistência periférica à insulina adicional ocorre até o final do tratamento nos ratos DEX. A glicemia permanece moderadamente elevada até o período de 72h. Entretanto, observa-se marcante hiperglicemia após 120h (79 vs. 160 mg/dl para ratos CTL e DEX, respectivamente). Aumento significativo dos níveis de proteínas totais e albuminas plasmáticas ocorre a partir de 72h de tratamento e de colesterol total a partir de 120h. Glicogênio e gordura hepáticos aumentam de maneira tempo-dependente nos ratos DEX. Correlação negativa foi observada entre os valores de insulinemia de jejum e peso nos grupos tratados com dexametasona (r > 0,95). Portanto, administração de dexametasona, 1mg/kg, induz resistência periférica à insulina de maneira tempo-dependente a partir de 24h e aumento dos níveis circulantes de glicose e proteínas plasmáticos após 72h de tratamento.

Glucocorticoid therapies are often associated with insulin sensitivity alteration. In the present study we evaluated some metabolical parameters such as plasma glucose, insulin, protein and cholesterol levels in ratstreated with dexamethasone (DEX) (1mg/kg, body weight, ip.) in different periods (24h, 72h and 120h). Dexamethasonetreated rats show peripherical resistance after 24h of drug administration as indicated by the fasting plasma insulin values (1.3 vs.6.8 ng/ml for controls [CTL] and DEX rats, respectively) and by HOMA index. Additional peripheral insulin resistance occurred until the end of treatment in DEX rats. The glycaemia remained slightly elevated until 72h period. However, marked hyperglycaemia was observed after 120h (79 vs.160 mg/dl for CTL and DEX rats, respectively). Significantly increase of plasma albumin and total proteins levels occurred from 72h of treatment and total cholesterol from 120h. Hepatic glycogen and hepatic fat increased in a time-dependent manner in DEX rats. Negative correlation was observed between fasting insulin and body weight values in dexamethasone-treated groups (r > 0.95). Therefore, dexamethasone administration, 1mg/kg, induces insulin peripheral resistance in a time-dependent manner from 24h and increase of circulating plasma glucose and proteins levels after 72h of treatment.