Optimized protocol for shotgun label-free proteomic analysis of pancreatic islets.

Pancreatic islets are crucial in diabetes research. Consequently, this protocol aims at optimizing both the protein-extraction process and the proteomic analysis via shotgun methods for pancreatic islets. Six protocols were tested, combining three types of chemical extraction with two mechanical extraction methods. Furthermore, two protocols incorporated a surfactant to enhance enzymatic cleavage. The steps involved extraction and concentration of protein, protein quantification, reduction, alkylation, digestion, purification and desalination, sample concentration to ∼1 µl, and proteomic analysis using the mass spectrometer. The most effective protocol involves either a milder chemical extraction paired with a more intensive mechanical process, or a more robust chemical extraction paired with a gentle mechanical process, tailored to the sample's characteristics. Additionally, it was observed that the use of a surfactant proved ineffective for these types of samples. Protocol 5 was recently used with success to examine metabolic changes in pancreatic islets of non-obese diabetic mice exposed to low doses of fluoride ions (F-) and the primary pathways altered by the treatment.

Maternal protein restriction combined with postnatal sugar consumption alters liver proteomic profile and metabolic pathways in adult male offspring rats.

This study investigated the impact of maternal protein restriction (MPR) and early postnatal sugar consumption (SUG) on the liver health of adult male descendant rats. Male offspring of mothers fed a normal protein diet (NPD) or a low protein diet (LPD) were divided into four groups: Control (CTR), Sugar Control (CTR + SUG), LPD during gestation and lactation (GLLP), and LPD with sugar (GLLP + SUG). Sugar consumption (10% glucose diluted in water) began after weaning on day 21 (PND 21), and at 90 days (PND 90), rats were sacrificed for analysis. Sugar intake reduced food intake and increased water consumption in CTR + SUG and GLLP + SUG compared to CTR and GLLP. GLLP and GLLP + SUG groups showed lower body weight and total and retroperitoneal fat compared to CTR and CTR + SUG. CTR + SUG and GLLP + SUG groups exhibited hepatocyte vacuolization associated with increased hepatic glycogen content compared to CTR and GLLP. Hepatic catalase activity increased in GLLP compared to CTR. Proteomic analysis identified 223 differentially expressed proteins (DEPs) among experimental groups. While in the GLLP group, the DEPs enriched molecular pathways related to cellular stress, glycogen metabolic pathways were enriched in the GLLP + SUG and CTR + SUG groups. The association of sugar consumption amplifies the effects of MPR, deregulating molecular mechanisms related to metabolism and the antioxidant system.

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

OBJECTIVES: 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). 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.

Effects of low-level fluoride exposure on glucose homeostasis in female NOD mice.

Water fluoridation is an important public health measure for the control of dental caries. Recent animal studies have shown that low doses of fluoride (F) in the drinking water, similar to those found in public water supplies, increase insulin sensitivity and reduce blood glucose. In the present study we evaluated the effects of low-level F exposure through the drinking water on glucose homeostasis in female NOD mice. Seventy-two 6-week mice were randomly divided into 2 groups according to the concentration of F in the drinking water (0-control, or 10 mg/L) they received for 14 weeks. After the experimental period the blood was collected for analyses of plasma F, glucose and insulin. Liver and gastrocnemius muscle were collected for proteomic analysis. Plasma F concentrations were significantly higher in the F-treated than in the control group. Despite treatment with fluoridated water reduced plasma levels glucose by 20% compared to control, no significant differences were found between the groups for plasma glucose and insulin. In the muscle, treatment with fluoridated water increased the expression of proteins related to muscle contraction, while in the liver, there was an increase in expression of antioxidant proteins and in proteins related to carboxylic acid metabolic process. Remarkably, phosphoenolpyruvate carboxykinase (PEPCK) was found exclusively in the liver of control mice. The reduction in PEPCK, a positive regulator of gluconeogenesis, thus increasing glucose uptake, might be a probable mechanism to explain the anti-diabetic effects of low doses of F, which should be evaluated in further studies.

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.