Everardo M. Carneiro

Protein deficiency during pregnancy and lactation impairs glucose-induced insulin secretion but increases the sensitivity to insulin in weaned rats

We studied glucose homeostasis in rat pups from dams fed on a normal-protein (170 g/kg) (NP) diet or a diet containing 60 g protein/kg (LP) during fetal life and the suckling period. At birth, total serum protein, serum albumin and serum insulin levels were similar in both groups. However, body weight and serum glucose levels in LP rats were lower than those in NP rats. At the end of the suckling period (28 d of age), total serum protein, serum albumin and serum insulin were significantly lower and the liver glycogen and serum free fatty acid levels were significantly higher in LP rats compared with NP rats. Although the fasting serum glucose level was similar in both groups, the area under the blood glucose concentration curve after a glucose load was higher for NP rats (859 (SEM 58) mmol/l per 120 min for NP rats v. 607 (SEM 52) mmol/l per 120 min for LP rats; P < 0.005). The mean post-glucose increase in insulin was higher for NP rats (30 (SEM 4.7) nmol/l per 120 min for NP rats v. 17 (SEM 3.9) nmol/l per 120 min for LP rats; P < 0.05). The glucose disappearance rate for NP rats (0.7 (SEM 0.1) %/min) was lower than that for LP rats (1.6 (SEM 0.2) %/min; P < 0.001). Insulin secretion from isolated islets (1 h incubation) in response to 16.7 mmol glucose/l was augmented 14-fold in NP rats but only 2.6-fold in LP rats compared with the respective basal secretion (2.8 mmol/l; P < 0.001). These results indicate that in vivo as well as in vitro insulin secretion in pups from dams maintained on a LP diet is reduced. This defect may be counteracted by an increase in the sensitivity of target tissues to insulin.

Low protein diet impairs glucose-induced insulin secretion from and 45Ca uptake by pancreatic rat islets

Glucose-induced insulin secretion from and 45 Ca uptake by isolated pancreatic islets, derived from rats fed with normal (NPD) or low protein diel (LPD), were studied. Insulin secretion from both types of islets in response to increasing concentrations of glucose followed an S-shaped pattern. However, basal secretion observed at substimulatory concentrations of glucose (0–5.6 m m ), as well as maximal release, obtained at 16.7 m m or higher glucose concentrations were significantly reduced in islets from LPD. Furthermore, in LPD rat islets, the dose-response curve to glucose was clearly shifted to the right compared with NPD islets, with the half-maximal response occurring at 8.5 and 14.4 m m glucose for NPD and LPD islets, respectively. In islets from NPD rats, the 45 Ca content, after 5 or 90 min in the presence of 8.3 m m glucose, was higher than that observed for islets kept at 2.8 m m glucose and increased further at 16.7 m m glucose. After 5 min of incubation, the 45 Ca uptake by LPD islets in the presence of 8.3 m m glucose was slightly higher than basal values (2.8 m m glucose): however, no further increase in the 45 Ca uptake was noticed at 16.7 m m glucose. In LPD islets a significant increase in 45 Ca uptake over basal values was registered only at 16.7 m m glucose, after 90 min of incubation. These data indicate that the poor secretory response to glucose observed in islets from LPD rats may be related to a defect in the ability of glucose to increase Ca 2+ uptake and/or to reduce Ca 2+ efflux from β-cells.

Reduced insulin secretion in response to nutrients in islets from malnourished young rats is associated with a diminished calcium uptake.

Changes in (45)Ca uptake and insulin secretion in response to glucose, leucine, and arginine were measured in isolated islets derived from 4-week-old rats born of mothers maintained with normal protein (NP, 17%) or low protein (LP, 6%) diet during pregnancy and lactation. Glucose provoked a dose-dependent stimulation of insulin secretion in both groups of islets, with basal (2.8 mmol/L glucose) and maximal release (27.7 mmol/L glucose) significantly reduced in LP compared with NP islets. In the LP group the concentration-response curve to glucose was shifted to the right compared with the NP group, with the half-maximal response occurring at 16.9 and 13.3 mmol/L glucose, respectively. In LP islets, glucose-induced first and second phases of insulin secretions were drastically reduced. In addition, insulin response to individual amino acids, or in association with glucose, was also significantly reduced in the LP group compared with NP islets. Finally, in LP islets the (45)Ca uptake after 5 minutes or 90 minutes of incubation (which reflect mainly the entry and retention, respectively, of Ca(2+)), was lower than in NP islets. These data indicate that in malnourished rats both initial and sustained phases of insulin secretion in response to glucose were reduced. This poor secretory response to nutrients seems to be the consequence of an altered Ca(2+) handling by malnourished islet cells.

Amino acid sequence of TsTX-V, an α-toxin from Tityus serrulatus scorpion venom, and its effect on K+ permeability of β-cells from isolated rat islets of Langerhans

Highly purified Tityustoxin V (TsTX-V), an a-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of 86Rb+ release from depolarized rat pancreatic /gb-cells as a measure of K+ permeability changes, TsTX-V (5.6 μg/ml) was found to increase by 2.0–2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 μM veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects β-cell voltage-dependent K+ channels, thus increasing K+ permeability.

Descrição de dieta purificada para indução de quadro de desnutrição protéica em ratos

No presente trabalho e descrita a composicao de dieta hipoproteica (6% de proteina) purificada para inducao de quadro de desnutricao em roedores. A referida dieta foi padronizada em laboratorio a partir de modificacao da AIN-93 (documento do American Institute of Nutrition que estabelece os padroes nutricionais para roedores de laboratorio), visando a obtencao de animais desnutridos para estudar as alteracoes metabolicas decorrentes da desnutricao proteica associada a situacoes como exercicio fisico, gestacao e diabetes. A dieta em questao contem os seguintes componentes (g/ kg): amido de milho (480), caseina (71,5), dextrina de milho (159), sacarose (121), oleo de soja (70), microcelulose (50), mistura mineral AIN-93-G-MX (35), mistura de vitaminas AIN-93-G-VX, (10), L-cistina (1), cloridrato de colina (2,5). Ratos alimentados cronicamente com a dieta apresentaram sinais comumente presentes na desnutricao proteica humana e de animais de laboratorio: reducao do ganho de peso, hipoproteinemia, hipoalbuminemia, elevacao dos acidos graxos livres sericos e do glicogenio hepatico.