Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

A low-protein diet leads to functional and structural pancreatic islet alterations, including islet hypotrophy. Insulin-signaling pathways are involved in several adaptive responses by pancreatic islets. We determined the levels of some insulin-signaling proteins related to pancreatic islet function and growth in malnourished rats. Adult male Wistar rats (N = 20 per group) were fed a 17 percent protein (normal-protein diet; NP) or 6 percent protein (low-protein diet; LP), for 8 weeks. At the end of this period, blood glucose and serum insulin and albumin levels were measured. The morphometric parameters of the endocrine pancreas and the content of some proteins in islet lysates were determined. The β-cell mass was significantly reduced (≅65 percent) in normoglycemic but hypoinsulinemic LP rats compared to NP rats. Associated with these alterations, a significant 30 percent reduction in insulin receptor substrate-1 and a 70 percent increase in insulin receptor substrate-2 protein content were observed in LP islets compared to NP islets. The phosphorylated serine-threonine protein kinase (pAkt)/Akt protein ratio was similar in LP and NP islets. The phosphorylated forkhead-O1 (pFoxO1)/FoxO1 protein ratio was decreased by 43 percent in LP islets compared to NP islets (P < 0.05). Finally, the ratio of phosphorylated-extracellular signal-related kinase 1/2 (pErk1/2) to total Erk1/2 protein levels was decreased by 71 percent in LP islets compared to NP islets (P < 0.05). Therefore, the reduced β-cell mass observed in LP rats is associated with the reduction of phosphorylation in mitogenic-related signals, FoxO1 and Erk proteins. The cause/effect basis of this association remains to be determined.

Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 µg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 ± 0.02 for CNTF-treated rats vs 0.53 ± 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18 percent (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87 percent, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91 percent over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.

Correlation of serum leptin and insulin levels of pregnant protein-restricted rats with predictive obesity variables

During pregnancy and protein restriction, changes in serum insulin and leptin levels, food intake and several metabolic parameters normally result in enhanced adiposity. We evaluated serum leptin and insulin levels and their correlations with some predictive obesity variables in Wistar rats (90 days), up to the 14th day of pregnancy: control non-pregnant (N = 5) and pregnant (N = 7) groups (control diet: 17 percent protein), and low-protein non-pregnant (N = 5) and pregnant (N = 6) groups (low-protein diet: 6 percent). Independent of the protein content of the diet, pregnancy increased total (F1,19 = 22.28, P < 0.001) and relative (F1,19 = 5.57, P < 0.03) food intake, the variation of weight (F1,19 = 49.79, P < 0.000) and final body weight (F1,19 = 19.52, P < 0.001), but glycemia (F1,19 = 9.02, P = 0.01) and the relative weight of gonadal adipose tissue (F1,19 = 17.11, P < 0.001) were decreased. Pregnancy (F1,19 = 18.13, P < 0.001) and low-protein diet (F1,19 = 20.35, P < 0.001) increased the absolute weight of brown adipose tissue. However, the relative weight of this tissue was increased only by protein restriction (F1,19 = 15.20, P < 0.001) and the relative lipid in carcass was decreased in low-protein groups (F1,19 = 4.34, P = 0.05). Serum insulin and leptin levels were similar among groups and did not correlate with food intake. However, there was a positive relationship between serum insulin levels and carcass fat depots in low-protein groups (r = 0.37, P < 0.05), while in pregnancy serum leptin correlated with weight of gonadal (r = 0.39, P < 0.02) and retroperitoneal (r = 0.41, P < 0.01) adipose tissues. Unexpectedly, protein restriction during 14 days of pregnancy did not alter the serum profile of adiposity signals and their effects on food intake and adiposity, probably due to the short term of exposure to low-protein diet.

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine

We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K + , 5 mM theophylline (Theo) and 200 µM carbamylcholine (Cch). No effect of glucose or high K + was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127 por cento above basal levels, respectively). High K + also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54 percent above basal levels, respectively. Similar results were obtained with Theo and Cch. A more marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179 percent above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396 percent above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP 3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K + that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells

Synergetic effect of glucose and prolactin on GLUT2 expression in cultured neonatal rat islets

We studied the synergistic effect of glucose and prolactin (PRL) on insulin secretion and GLUT2 expression in cultured neonatal rat islets. After 7 days in culture, basal insulin secretion (2.8 mM glucose) was similar in control and PRL-treated islets (1.84 ñ 0.06 per cent and 2.08 ñ 0.07 per cent of the islet insulin content, respectively). At 5.6 and 22 mM glucose, insulin secretion was significantly higher in PRL-treated than in control islets, achieving 1.38 ñ 0.15 per cent and 3.09 ñ 0.21 per cent of the islet insulin content in control and 2.43 ñ 0.16 per cent and 4.31 ñ 0.24 per cent of the islet insulin content in PRL-treated islets, respectively. The expression of the glucose transporter GLUT2 in B-cell membranes was dose-dependently increased by exposure of the islet to increasing glucose concentrations. This effect was potentiated in islets cultured for 7 days in the presence of 2 mug/ml PRL. At 5.6 and 10 mM glucose, the increase in GLUT2 expression in PRL-treated islets was 75 per cent and 150 per cent higher than that registered in the respective control. The data presented here indicate that insulin secretion, induced by different concentrations of glucose, correlates well with the expression of the B-cell-specific glucose transporter GLUT2 in pancreatic islets.

Inhibition of insulin release by jatrophone

In may countries, including Brazil, extracts of Jatrophona elliptica species are currently used for the treatment of several diseases. Recently it was shown that a purified compound from these plants inhibits contraction of smooth and cardiac muscle in the uM range, probably involving alterations membrane Ca2+ permeability and/or internal Ca2+ distribution. In the collagenase-isolated rat islets and in the absence of glucose, basal insulin secretion measured by radioimmunoassay averaged 122 ñ 13 uU/islet per 90 min (N=27). Jatrophone (1-100 uM/l) caused a dose-related inhibition of glucose-induced insulin release, over basal secretion, with an ID50 close to 8 uM/l. Complete inhibition of insulin release was obtained with 100 uM/l Jatrophone. However, at 100 uM/l (but not at 10 uM/l) concentration, Jatrophone also provoked a reduction in glucose metabolism by the islets which could explain, at least in part, the reduction in insulin secretion. After 120-min incubation,. the glucose metabolism, measured by the 14CO2 production, was reduced from 26.58 ñ 3.63 (N=42) to 7.48 ñ 1.36 (N=16) pmol/l per islet. In conclusion at lower concentrations (10 uM/l) Jatrophone could be a valuable tool for the study of the mechanism of insulin release induced either by glucose or other secretagogues

Effects of triiodothyronine on insulin release from cultured neonatal and adult rat islets

Insulin secretion from neonatal and adult rat islets maintained in culture for 7-9 days in the absence or in the presence of nMT3 was measured. In both neonatal and adult islets T3 treatment tends to inhibit insulin secretion only in the absence or in the presence of low glucose concentrations

Mecanismo da secrecao de insulina. / Mecanism of insulin secretion

Este trabalho apresenta uma breve revisao sobre o mecanismo da secrecao de insulina induzido pela glicose. A sequencia de eventos obedece as seguintes etapas: a) reconhecimento da glicose pelas celulas beta (provavelmente como consequencia de sua metabolizacao); b) alteracao do estado de oxido-reducao celular; c) alteracao da permeabilidade da membrana a varios ions, em especial ao K+ e Ca2+; d) aumento da concentracao intracelular da Ca2+; e) ativacao do sistema efetor responsavel pela extrusao dos granulos de insulina. Maior enfase sera dada as etapas iniciais desse processo

Validacao de um radiomunoensaio para dosagem de insulina murina. / Validation of a radioimmunoassay for dosage of murine insulin

Este artigo apresenta metodologia que torna possivel, em um radioimunoensaio, o uso de insulina bovina como refencial para avaliacao de insulina de rato. Como os radioimunoensaios utilizados para dosagem de diferentes substancias seguem o mesmo principio (inibicao competitiva), acredita-se que a metodologia apresentada possa ser utilizada tambem para avaliacao de outros hormonios, quando nao se dispuser do referencial adequado, de atividade biologica conhecida