Exercise Increases Pancreatic β-cell Viability In A Model Of Type 1 Diabetes Through Il-6 Signaling.

Type 1 diabetes (T1D) is provoked by an autoimmune assault against pancreatic β cells. Exercise training enhances β-cell mass in T1D. Here, we investigated how exercise signals β cells in T1D condition. For this, we used several approaches. Wild-type and IL-6 knockout (KO) C57BL/6 mice were exercised. Afterward, islets from control and trained mice were exposed to inflammatory cytokines (IL-1β plus IFN-γ). Islets from control mice and β-cell lines (INS-1E and MIN6) were incubated with serum from control or trained mice or medium obtained from 5-aminoimidazole-4 carboxamide1-β-d-ribofuranoside (AICAR)-treated C2C12 skeletal muscle cells. Subsequently, islets and β cells were exposed to IL-1β plus IFN-γ. Proteins were assessed by immunoblotting, apoptosis was determined by DNA-binding dye propidium iodide fluorescence, and NO(•) was estimated by nitrite. Exercise reduced 25, 75, and 50% of the IL-1β plus IFN-γ-induced iNOS, nitrite, and cleaved caspase-3 content, respectively, in pancreatic islets. Serum from trained mice and medium from AICAR-treated C2C12 cells reduced β-cell death, induced by IL-1β plus IFN-γ treatment, in 15 and 38%, respectively. This effect was lost in samples treated with IL-6 inhibitor or with serum from exercised IL-6 KO mice. In conclusion, muscle contraction signals β-cell survival in T1D through IL-6.-Paula, F. M. M., Leite, N. C., Vanzela, E. C., Kurauti, M. A., Freitas-Dias, R., Carneiro, E. M., Boschero, A. C., and Zoppi, C. C. Exercise increases pancreatic β-cell viability in a model of type 1 diabetes through IL-6 signaling.

Enhanced Glucose-induced Intracellular Signaling Promotes Insulin Hypersecretion: Pancreatic Beta-cell Functional Adaptations In A Model Of Genetic Obesity And Prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

Chronic Continuous Exenatide Infusion Does Not Cause Pancreatic Inflammation And Ductal Hyperplasia In Non-human Primates.

In this study, we aimed to evaluate the effects of exenatide (EXE) treatment on exocrine pancreas of nonhuman primates. To this end, 52 baboons (Papio hamadryas) underwent partial pancreatectomy, followed by continuous infusion of EXE or saline (SAL) for 14 weeks. Histological analysis, immunohistochemistry, Computer Assisted Stereology Toolbox morphometry, and immunofluorescence staining were performed at baseline and after treatment. The EXE treatment did not induce pancreatitis, parenchymal or periductal inflammatory cell accumulation, ductal hyperplasia, or dysplastic lesions/pancreatic intraepithelial neoplasia. At study end, Ki-67-positive (proliferating) acinar cell number did not change, compared with baseline, in either group. Ki-67-positive ductal cells increased after EXE treatment (P = 0.04). However, the change in Ki-67-positive ductal cell number did not differ significantly between the EXE and SAL groups (P = 0.13). M-30-positive (apoptotic) acinar and ductal cell number did not change after SAL or EXE treatment. No changes in ductal density and volume were observed after EXE or SAL. Interestingly, by triple-immunofluorescence staining, we detected c-kit (a marker of cell transdifferentiation) positive ductal cells co-expressing insulin in ducts only in the EXE group at study end, suggesting that EXE may promote the differentiation of ductal cells toward a β-cell phenotype. In conclusion, 14 weeks of EXE treatment did not exert any negative effect on exocrine pancreas, by inducing either pancreatic inflammation or hyperplasia/dysplasia in nonhuman primates.

Microbial distribution in the root canal system after periapical lesion induction using different methods

The aim of this study was to evaluate the microbial distribution in the root canal system after periapical lesion induction in dogs' teeth using different methods. Fifty-two root canals were assigned to 4 groups (n=13). Groups I and II: root canals were exposed to the oral cavity for 180 days; groups III and IV: root canals were exposed for 7 days and then the coronal openings were sealed for 53 days. The root apices of groups I and III were perforated, while those of groups II and IV remained intact. After the experimental periods, the animals were euthanized and the anatomic pieces containing the roots were processed and stained with the Brown & Brenn method to assess the presence and distribution of microorganisms. The incidence of microorganisms at different sites of the roots and periapical lesions was analyzed statistically by the chi-square test at 5% significance level. All groups presented microorganisms in the entire root canal system. A larger number of microorganisms was observed on the root canal walls, apical delta and dentinal tubules (p<0.05), followed by cementum and cemental resorption areas. In spite of the different periods of exposure to the oral environment, the methods used for induction of periapical periodontitis yielded similar distribution of microorganisms in the root canal system.

Lesion of the subthalamic nucleus reverses motor deficits but not death of nigrostriatal dopaminergic neurons in a rat 6-hydroxydopamine-lesion model of Parkinson's disease

The objective of the present study was to determine whether lesion of the subthalamic nucleus (STN) promoted by N-methyl-D-aspartate (NMDA) would rescue nigrostriatal dopaminergic neurons after unilateral 6-hydroxydopamine (6-OHDA) injection into the medial forebrain bundle (MFB). Initially, 16 mg 6-OHDA (6-OHDA group) or vehicle (artificial cerebrospinal fluid - aCSF; Sham group) was infused into the right MFB of adult male Wistar rats. Fifteen days after surgery, the 6-OHDA and SHAM groups were randomly subdivided and received ipsilateral injection of either 60 mM NMDA or aCSF in the right STN. Additionally, a control group was not submitted to stereotaxic surgery. Five groups of rats were studied: 6-OHDA/NMDA, 6-OHDA/Sham, Sham/NMDA, Sham/Sham, and Control. Fourteen days after injection of 6-OHDA, rats were submitted to the rotational test induced by apomorphine (0.1 mg/kg, ip) and to the open-field test. The same tests were performed again 14 days after NMDA-induced lesion of the STN. The STN lesion reduced the contralateral turns induced by apomorphine and blocked the progression of motor impairment in the open-field test in 6-OHDA-treated rats. However, lesion of the STN did not prevent the reduction of striatal concentrations of dopamine and metabolites or the number of nigrostriatal dopaminergic neurons after 6-OHDA lesion. Therefore, STN lesion is able to reverse motor deficits after severe 6-OHDA-induced lesion of the nigrostriatal pathway, but does not protect or rescue dopaminergic neurons in the substantia nigra pars compacta.

Evaluation of simulated bone lesion in the head of the mandible by using multislice computed tomography

Conventional radiography has shown limitation in acquiring image of the ATM region, thus, computed tomography (CT) scanning has been the best option to the present date for diagnosis, surgical planning and treatment of bone lesions, owing to its specific properties. OBJECTIVE: The aim of the study was to evaluate images of simulated bone lesions at the head of the mandible by multislice CT. MATERIAL AND METHODS: Spherical lesions were made with dental spherical drills (sizes 1, 3, and 6) and were evaluated by using multislice CT (64 rows), by two observers in two different occasions, deploying two protocols: axial, coronal, and sagittal images, and parasagittal images for pole visualization (anterior, lateral, posterior, medial and superior). Acquired images were then compared with those lesions in the dry mandible (gold standard) to evaluate the specificity and sensibility of both protocols. Statistical methods included: Kappa statistics, validity test and chi-square test. Results demonstrated the advantage of associating axial, coronal, and sagittal slices with parasagittal slices for lesion detection at the head of the mandible. RESULTS: There was no statistically significant difference between the types of protocols regarding a particular localization of lesions at the poles. CONCLUSIONS: Protocols for the assessment of the head of the mandible were established to improve the visualization of alterations of each of the poles of the mandible's head. The anterior and posterior poles were better visualized in lateral-medial planes while lateral, medial and superior poles were better visualized in the anterior-posterior plane.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

Background: Toll-like receptor 4 (TLR4) is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS), and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic beta-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic beta-cells is still to be clearly established. We investigated whether TLR4 is present in beta-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis. Results: CD11b positive macrophages were practically absent from isolated human islets obtained from nondiabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to beta-cells. A significant loss of cell viability was observed in these beta-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in beta-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content. Conclusions: Taken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic beta-cells.

Expression of an activating mutation in the gene encoding the K(ATP) channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K(+) (K(ATP)) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of K(ATP) channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of K(ATP) channels, closed K(ATP) channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of K(ATP) channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Contuse lesion of the rat spinal cord of moderate intensity leads to a higher time-dependent secondary neurodegeneration than severe one - An open-window for experimental neuroprotective interventions

Secondary neurodegeneration takes place in the surrounding tissue of spinal cord trauma and modifies substantially the prognosis, considering the small diameter of its transversal axis. We analyzed neuronal and glial responses in rat spinal cord after different degree of contusion promoted by the NYU Impactor. Rats were submitted to vertebrae laminectomy and received moderate or severe contusions. Control animals were sham operated. After 7 and 30 days post surgery, stereological analysis of Nissl staining cellular profiles showed a time progression of the lesion volume after moderate injury, but not after severe injury. The number of neurons was not altered cranial to injury. However, same degree of diminution was seen in the caudal cord 30 days after both severe and moderate injuries. Microdensitometric image analysis demonstrated a microglial reaction in the white matter 30 days after a moderate contusion and showed a widespread astroglial reaction in the white and gray matters 7 days after both severities. Astroglial activation lasted close to lesion and in areas related to Wallerian degeneration. Data showed a more protracted secondary degeneration in rat spinal cord after mild contusion, which offered an opportunity for neuroprotective approaches. Temporal and regional glial responses corroborated to diverse glial cell function in lesioned spinal cord. (C) 2007 Elsevier Ltd. All rights reserved.

Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Biliary and pancreatic dysgenesis in mice harboring a mutation in Pkhd1

Autosomal recessive polycystic kidney disease is a hereditary fibrocystic disease that involves the kidneys and the biliary tract. Mutations in the PKHD1 gene are responsible for typical forms of autosomal recessive polycystic kidney disease. We have generated a mouse model with targeted mutation of Pkbd1 by disrupting exon 4, resulting in a mutant transcript with deletion of 66 codons and expression at similar to 30% of wild-type levels. Pkhd1(del4/d3l4) mice develop intrahepatic bile duct proliferation with progressive cyst formation and associated periportal fibrosis. In addition, these mice exhibit extrahepatic manifestations, including pancreatic cysts, splenomegaly, and common bile duct dilation. The kidneys are unaffected both histologically and functionally. Fibrocystin is expressed in the apical membranes and cilia of bile ducts and distal nephron segments but is absent from the proximal tubule. This pattern is unchanged in orthologous models of autosomal dominant polycystic kidney disease due to mutation in Pkd1 or Pkd2. Mutant fibrocystin in Pkhd1(del4/d3l4) mice also retains this expression pattern. The hypomorphic Pkhd1(del4/d3l4) mouse model provides evidence that reduced functional levels of fibrocystin are sufficient for cystogenesis and fibrosis in the liver and pancreas, but not the kidney, and supports the hypothesis of species-dependent differences in susceptibility of tissues to Pkbdl mutations.