Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

AIMS: Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. METHODS: The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. RESULTS: M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. CONCLUSIONS: Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes.

3-Nitropropionic acid modifies neurotrophin mRNA expression in the mouse striatum: 18S-rRNA is a reliable control gene for studies of the striatum.

OBJECTIVE: The aim of the present study was to determine the changes in the mRNA levels of neurotrophins and their receptors in the striatal tissue of mice treated with 3-nitropropionic acid (3-NP). METHODS: At 1 and 48 h after the last drug administration, the mRNA expression of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 as well as their receptors p75, TrkA, TrkB and TrkC, was evaluated using semi-quantitative (semi-Q) and real-time RT-PCR. ß-actin mRNA and ribosomal 18S (18S rRNA) were tested as internal controls. RESULTS: 3-NP treatment did not affect mRNA expression of all neurotrophins and their respective receptors equally. Also, differences in neurotrophin and receptor mRNA expression were observed between semi-Q and real-time RT-PCR. Real-time RT-PCR was more accurate in evaluating the mRNA expression of the neurotrophins than semi-Q, and 18S rRNA was more reliable than ß-actin as an internal control. CONCLUSION: Neurotrophins and their receptors expression is differentially affected by neuronal damage produced by inhibition of mitochondrial respiration with 3-NP treatment in low, sub-chronic doses in vivo.

Immunization with Cry1Ac from Bacillus thuringiensis increases intestinal IgG response and induces the expression of FcRn in the intestinal epithelium of adult mice.

We have shown that Cry1Ac protoxin from Bacillus thuringiensis is a potent mucosal and systemic immunogen with adjuvant properties. Interestingly, we have observed that Cry1Ac preferentially induces high specific IgG responses in intestinal fluid when it is intraperitoneally administered to mice; therefore, in the present study, we used this protocol, as a model to address the influence of systemic immunization on the induction of the intestinal IgG response. The data shown indicate that upon intraperitoneal immunization with Cry1Ac, significant intestinal specific IgG cell responses were produced in the lamina propria, accompanied by an increased frequency of intestinal IgG+ lymphocytes and epithelial cells containing IgG. Considering that FcRn is the receptor responsible for the transport of IgG in neonatal intestinal epithelia, but it is developmentally downregulated in the rodent intestine, we analysed whether upon intestinal IgG induction, FcRn mRNA expression was induced in intestinal epithelial cells, of adult mice. Whereas in intestinal epithelia of unimmunized adult mice FcRn mRNA was not detected, in Cry1Ac immunized mice it was expressed, although the level was lower in comparison with that found in neonatal epithelia. Then using flow cytometry and immunofluorescence we confirmed that the expression of the protein FcRn was induced in the intestines of adult immunized mice especially in the large intestine. Finally, we found that Cry1Ac also increased FcRn expression in isolated intestinal epithelial cells stimulated in vitro. The outcomes suggest that the expression of FcRn in intestinal epithelium might be reactivated upon immunization, and possibly facilitate IgG transport.

Clinical evolution of diabetic rats after transplant of electrofused pancreatic islet cells and dermic cells.

This work was designed to study an alternative treatment of diabetes mellitus by using a transplant of hybrid cells obtained by the electrofusion of pancreatic islet cells from a healthy donor with dermic cells obtained from a recipient. The hybrid cells kept the capacity of insulin production, its regulation, and the natural control of glycemia, as well as the factors of histocompatibility to avoid the rejection. Four groups of four rats each were established: Group 1. Healthy animals (healthy control), Group 2. Diabetized non-treated animals (diabetic control), Group 3. Transplant recipient rats with extraction of dermic cells which were mixed with pancreatic insular cells from a healthy donor (transplant without fusion), and Group 4. Transplant recipient rats, with extraction of dermic cells which were electrofused with pancreatic insular cells from a healthy donor (transplant with fusion). For the Group 4, the cells were combined and they were submitted to dielectrophoresis conditions with an alternating current pulse of 15 s of 10 V RMS of 0.5 MHz. The fusion was made with a direct current pulse of 1 ms of 300 V. Clinical signs were registered (weight, diuresis, food and water intake), and several biochemical parameters in blood which included basal glycemia, uric acid, cholesterol, triglycerides, glutamate oxalacetate transaminase, glutamate pyruvate transaminase, urea, creatinine, insulin, glycated hemoglobin were registered. Additionally, ketone bodies and glucose were also measured in urine. All determinations were made at 30, 60, and 90 days. Animals of Group 1 maintained its parameters within the normal ranges. Rats of Group 2 presented alterations corresponding to a diabetic state in almost all the parameters measured, none of the animals showed a tendency to improve spontaneously, two of the rats died at 66 and 72 days. The Group 3 showed a clinical profile similar to the diabetic control group without improvement, only one rat died at day 33, while in the rats transplanted with fusion (Group 4) an improvement was observed on some parameters including body weight, water intake and glycemia. Although insulin concentration was under the normal range, it was higher than in the Group 3. None rat died. These results indicate that it is possible to improve the diabetic profile by the transplant of dermic cells from a diabetic animal fused with insular cells from a healthy donor in the recipient animal.