Carla Beatriz Collares-Buzato

Modulation of gap and adherens junctional proteins in cultured neonatal pancreatic islets

Fetal and neonatal pancreatic islets have lower insulin secretory responses compared with adult islets. In culture conditions and after treatment with mammosomatotropic hormones, neonatal islets undergo maturation of the secretory machinery that might involve regulation of cell–cell contacts within the islet. This study is an investigation of the effect of prolonged culturing and in vitro treatment with prolactin on the expression of the gap junction–associated connexin 43 and the adherens junction–associated &bgr;-catenin in cultured neonatal rat islets. Pancreatic islets from neonatal Wistar rats were cultured for 24 hours or 7 days, and the treated group was exposed to 2 &mgr;g/mL prolactin daily for 7 days. Connexin 43 and &bgr;-catenin were barely detected at the cell–cell contacts in 24-hour-cultured islets, as revealed by immunocytochemical analysis. Nevertheless, both junctional proteins were well expressed at the junctional region in islet cells cultured for 7 days and showed even greater staining in islets after long-term prolactin treatment. In accordance with the morphologic data, neonatal islets cultured for 24 hours displayed a relatively low level of connexin 43, as determined by Western blot analysis. Culturing for 7 days or combined prolactin treatment induced a significant increase in connexin 43 expression; this was 40% greater in the prolactin-treated group than in the control group. Furthermore, an enhancement of the expression of &bgr;-catenin and translocation of this protein to the cell–cell contact site was also observed in neonatal islets cultured for 7 days compared with those cultured for 24 hours. In vitro prolactin treatment induced even greater expression of &bgr;-catenin in islet cells. A correlation was observed between the increased expression of these junctional proteins and an increase in insulin secretion in cultured neonatal islets. In conclusion, prolonged culturing and in vitro treatment with prolactin induce the modulation of gap and adherens junctional proteins in pancreatic islets, which may be an important event in the in vitro maturation process of neonatal islet cells.

Primary hypercholesterolaemia impairs glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice independently of high-fat diet and obesity.

We investigated whether primary hypercholesterolaemia per se affects glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice (LDLR(-/-)). Glucose plasma levels were increased and insulin decreased in LDLR(-/-) compared to the wild-type mice. LDLR(-/-) mice presented impaired glucose tolerance, but normal whole body insulin sensitivity. The dose-response curve of glucose-stimulated insulin secretion was shifted to the right in LDLR(-/-) islets. Significant reductions in insulin secretion in response to l-leucine or 2-ketoisocaproic acid were also observed in LDLR(-/-). Islet morphometric parameters, total insulin and DNA content were similar in both groups. Glucose uptake and oxidation were reduced in LDLR(-/-) islets. Removal of cholesterol from LDLR(-/-) islets corrected glucose-stimulated insulin secretion. These results indicate that enhanced membrane cholesterol content due to hypercholesterolaemia leads to a lower insulin secretion and glucose intolerance without affecting body insulin sensitivity. This represents an additional risk factor for diabetes and atherosclerosis in primary hypercholesterolaemia.

Mechanisms involved in the blood–brain barrier increased permeability induced by Phoneutria nigriventer spider venom in rats

We have recently demonstrated by electron microscopy, using lanthanum nitrate as an extracellular tracer, that the intravenous injection of Phoneutria nigriventer spider venom (PNV) induces blood-brain barrier (BBB) breakdown in rat hippocampus. One and nine days after PNV injection, tracer was found in pinocytic vesicles crossing the endothelium and in the interendothelial cleft, suggesting that BBB breakdown had occurred through enhanced transendothelial transport and/or tight-junction opening. In the present work, we investigated the mechanisms by which PNV (850 microg/kg, i.v.) increased the hippocampal microvascular permeability in rats 24 h after the endovenous administration. The expression and phosphorylation of some tight- and adherens junctions-associated proteins in hippocampal homogenate and hippocampal microvessel homogenate were assessed by Western blotting and immunoprecipitation. The microtubule-dependent transcellular transport was also evaluated by quantitative ultrastructural methods in pretreated rats with colchicine (0.5 mg/kg, i.p.), prior to PNV injection. Western blots showed no significant increase in the expression of the tight junction-associated proteins ZO-1 and occludin or in the adherens junction-associated beta-catenin after 24 h of PNV administration. Morphological study showed no alterations of the immunolabeling for occludin and ZO-1 in rat brain cryosection following PNV. In addition, no changes were observed in phosphotyrosine content of occludin and beta-catenin in PNV-treated rats compared with control animals. However, the disruption of microtubule-dependent transcellular transport by colchicine completely prevented (p<0.001) PNV-induced leakage of the BBB tracer. These findings indicate that the increased BBB permeability evoked by PNV in rats probably resulted from enhanced microtubule-dependent transendothelial vesicular transport, with no substantial involvement of the paracellular barrier in the time interval studied.

Influence of gender and time diet exposure on endocrine pancreas remodeling in response to high fat diet-induced metabolic disturbances in mice

In this study, we investigated a possible sexual dimorphism regarding metabolic response and structural and functional adaptations of the endocrine pancreas after exposure to a high-fat diet (HFd). On chow diet, male and female C57BL/6/JUnib mice showed similar metabolic and morphometric parameters, except that female islets displayed a relatively lower β-cell:non-β-cell ratio. After 30 days on HFd, both male and female mice showed increased weight gain, however only the males displayed glucose intolerance associated with high postprandial glycemia when compared to their controls. After 60 days on HFd, both genders became obese, hyperglycemic, hyperinsulinemic, insulin resistant and glucose intolerant, although the metabolic changes were more pronounced in males, while females displayed greater weight gain. In both genders, insulin resistance induced by HFd feeding was compensated by expansion of β-cell mass without changes in islet cytoarchitecture. Interestingly, we found a strong correlation between the degree of β-cell expansion and the levels of hyperglycemia in the fed state: male mice fed a 60d-HFd, showing higher glycemic levels also displayed a greater β-cell mass increase in comparison with female mice. Additionally, sexual dimorphism was also observed regarding the source of β-cell mass expansion following 60d-HFd: while in males, both hypertrophy and hyperplasia (revealed by morphometry and Ki67 immunoreaction) of β-cells were observed, female islets displayed only a significant increase in β-cell size. In conclusion, this study describes gender differences in metabolic response to high fat diet, paralleled by distinct compensatory morphometric changes in pancreatic islets.

Upregulation of the expression of tight and adherens junction-associated proteins during maturation of neonatal pancreatic islets in vitro.

Cell–cell contacts mediated by intercellular junctions are crucial for proper insulin secretion in the endocrine pancreas. The biochemical composition of the intercellular junctions in this organ and the role of junctional proteins in endocrine pancreatic dysfunctions are still unclear. In this study, we investigated the expression and cellular location of junctional and cytoskeletal proteins in cultured neonatal rat pancreatic islets. Neonatal B-cells had an impaired insulin secretion compared to adult cells. Cultured neonatal islets showed a time-dependent increase in the glucose-induced secretory response. The maturation of B-cells in vitro was accompanied by upregulation of the expression of some junctional proteins in islet cells. Neonatal islets cultured for only 24 h showed a low expression and a diffuse cytoplasmic location of the tight junctional proteins occludin and ZO-1 and of the adherens junctional proteins α- and β-catenins, as demonstrated by immunoblotting and immunocytochemistry. Culturing islets for up to 8 days significantly increased the cell expression of these junctional proteins but not of the cytoskeletal proteins vinculin and α-actinin. A translocation of ZO-1 and catenins to the cell–cell contact region, as well as a higher association of F-actin with the intercellular junction, were also observed in neonatal islets following prolonged culturing. ZO-1 and β-catenin were immunolocated in the endocrine pancreas of adult rats indicating that these junctional proteins are also expressed in this organ in situ. In conclusion, endocrine pancreatic cells express several junctional proteins that are upregulated following differentiation of the endocrine pancreas in vitro.

Cytoskeletal rearrangement and cell death induced by Bothrops alternatus snake venom in cultured Madin-Darby canine kidney cells

Bothrops snake venoms cause renal damage, with renal failure being the main cause of death in humans bitten by these snakes. In this work, we investigated the cytoskeletal rearrangement and cytotoxicity caused by Bothrops alternatus venom in cultured Madin-Darby canine kidney (MDCK) cells. Incubation with venom (10 and 100 microg/mL) significantly (p <0.05) decreased the cellular uptake of neutral red dye after 1 and 3 h. Venom (100 microg/mL) also markedly decreased the transepithelial electrical resistance (RT) across MDCK monolayers. Staining with rhodamine-conjugated phalloidin revealed disarray of the cytoskeleton that involved the stress fibers at the basal cell surface and focal adhesion-associated F-actin in the cell-matrix contact region. Feulgen staining showed a significant decrease in the number of cells undergoing mitosis and an increase in the frequency of altered nuclei. Scanning electron microscopy revealed a decrease in the number of microvilli and the presence of cells with a fusiform format. Flow cytometry with annexin V and propidium iodide showed that cell death occurred by necrosis, with little apoptosis, a conclusion supported by the lack of DNA fragmentation characteristic of apoptosis. Pretreating the cells with catalase significantly attenuated the venom-induced loss of viability, indicating a possible involvement of H2O2 in the cellular damage; less protection was observed with superoxide dismutase or N omega-nitro-L-arginine methyl ester. These results indicate that Bothrops alternatus venom is cytotoxic to cultured MDCK cells, possibly via the action of reactive oxygen species. This cytotoxicity could contribute to nephrotoxicity after envenoming by this species.

Histological and functional renal alterations caused by Bothrops alternatus snake venom: expression and activity of Na+/K+-ATPase.

BACKGROUND Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na+/K+-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na+/K+-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. METHODS Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na+/K+-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. RESULTS Venom caused lobulation of the capillary tufts, dilation of Bowmans capsular space, F-actin disruption in Bowmans capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na+/K+-ATPase α1 subunit were increased 6h post-venom, whereas Na+/K+-ATPase activity increased 6 h and 24 h post-venom. CONCLUSIONS Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na+/K+-ATPase expression and activity in the early phase of renal damage. GENERAL SIGNIFICANCE Enhanced Na+/K+-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.

Impaired compensatory beta-cell function and growth in response to high-fat diet in LDL receptor knockout mice

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta‐cells in C57BL/6 mice fed a high‐fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild‐type (WT) and LDLr−/− mice were assessed. HF diet‐fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta‐cell secretory response to glucose. Overall, LDLr−/− mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose‐stimulated insulin secretion. HF diet induced similarly in WT and LDLr−/− mice, a significant decrease in Cx36 beta‐cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta‐cell mass mainly due to beta‐cell hypertrophy/replication. Nevertheless, HF diet‐fed LDLr−/− mice showed no significant changes in beta‐cell mass, but lower islet–duct association (neogenesis) and higher beta‐cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr−/− mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta‐cell expansion.

Cell-to-cell contact dependence and junctional protein content are correlated with in vivo maturation of pancreatic beta cells.

In this study, we investigated the cellular distribution of junctional proteins and the dependence on cell-cell contacts of pancreatic beta cells during animal development. Fetus and newborn rat islets, which display a relatively poor insulin secretory response to glucose, present an immature morphology and cytoarchitecture when compared with young and adult islets that are responsive to glucose. At the perinatal stage, beta cells display a low junctional content of neural cell adhesion molecule (N-CAM), α- and β-catenins, ZO-1, and F-actin, while a differential distribution of N-CAM and Pan-cadherin was seen in beta cells and nonbeta cells only from young and adult islets. In the absence of intercellular contacts, the glucose-stimulated insulin secretion was completely blocked in adult beta cells, but after reaggregation they partially reestablished the secretory response to glucose. By contrast, neonatal beta cells were poorly responsive to sugar, regardless of whether they were arranged as intact islets or as isolated cells. Interestingly, after 10 days of culturing, neonatal beta cells, known to display increased junctional protein content in vitro, became responsive to glucose and concomitantly dependent on cell-cell contacts. Therefore, our data suggest that the developmental acquisition of an adult-like insulin secretory pattern is paralleled by a dependence on direct cell-cell interactions.

Increased expression of SNARE proteins and synaptotagmin IV in islets from pregnant rats and in vitro prolactin-treated neonatal islets

During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic beta-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.