Interaction between poly(A)-binding protein PABPC4 and nuclear receptor corepressor NCoR1 modulates a metabolic stress response.

Mitochondria are organelles known primarily for generating ATP via the oxidative phosphorylation process. Environmental signals are sensed by whole organisms or cells and markedly affect this process, leading to alterations in gene transcription and, consequently, changes in mitochondrial function and biogenesis. The expression of mitochondrial genes is finely regulated by nuclear transcription factors, including nuclear receptors and their coregulators. Among the best-known coregulators is the nuclear receptor corepressor 1 (NCoR1). Muscle-specific knockout of NCoR1 in mice induces an oxidative phenotype, improving glucose and fatty acid metabolism. However, the mechanism by which NCoR1 is regulated remains elusive. In this work, we identified the poly(A)-binding protein 4 (PABPC4) as a new NCoR1 interactor. Unexpectedly, we found that silencing of PABPC4 induced an oxidative phenotype in both C2C12 and MEF cells, as indicated by increased oxygen consumption, mitochondria content, and reduced lactate production. Mechanistically, we demonstrated that PABPC4 silencing increased the ubiquitination and consequent degradation of NCoR1, leading to the derepression of PPAR-regulated genes. As a consequence, cells with PABPC4 silencing had a greater capacity to metabolize lipids, reduced intracellular lipid droplets, and reduced cell death. Interestingly, in conditions known to induce mitochondrial function and biogenesis, both mRNA expression and PABPC4 protein content were markedly reduced. Our study, therefore, suggests that the lowering of PABPC4 expression may represent an adaptive event required to induce mitochondrial activity in response to metabolic stress in skeletal muscle cells. As such, the NCoR1-PABPC4 interface might be a new road to the treatment of metabolic diseases.

Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice.

AIMS/HYPOTHESIS: Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR(-/-)) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca(2+) handling in these islets. METHODS: Isolated islets from both LDLR(-/-) and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca(2+) level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0-10mmol/l) of methyl-beta-cyclodextrin (MßCD). RESULTS: The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR(-/-) than in WT islets, paralleled by an impairment of Ca(2+) handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR(-/-) compared with WT islets. Removal of excess cholesterol from LDLR(-/-) islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca(2+) handling was also normalized in cholesterol-depleted LDLR(-/-) islets. Cholesterol removal from WT islets by 0.1 and 1.0mmol/l MßCD impaired both GSIS and Ca(2+) handling. In addition, at 10mmol/l MßCD WT islet showed a loss of membrane integrity and higher DNA fragmentation. CONCLUSION: Abnormally high (LDLR(-/-) islets) or low cholesterol content (WT islets treated with MßCD) alters both GSIS and Ca(2+) handling. Normalization of cholesterol improves Ca(2+) handling and insulin secretion in LDLR(-/-) islets.

Ciliary neurotrophic factor (CNTF) protects non-obese Swiss mice against type 2 diabetes by increasing beta cell mass and reducing insulin clearance.

AIMS/HYPOTHESIS: Ciliary neurotrophic factor (CNTF) improves metabolic variables of obese animals with characteristics of type 2 diabetes, mainly by reducing insulin resistance. We evaluated whether CNTF was able to improve other metabolic variables in mouse models of type 2 diabetes, such as beta cell mass and insulin clearance, and whether CNTF has any effect on non-obese mice with characteristics of type 2 diabetes. METHODS: Neonatal mice were treated with 0.1 mg/kg CNTF or citrate buffer via intraperitoneal injections, before injection of 250 mg/kg alloxan. HEPG2 cells were cultured for 3 days in the presence of citrate buffer, 1 nmol/l CNTF or 50 mmol/l alloxan or a combination of CNTF and alloxan. Twenty-one days after treatment, we determined body weight, epididymal fat weight, blood glucose, plasma insulin, NEFA, glucose tolerance, insulin resistance, insulin clearance and beta cell mass. Finally, we assessed insulin receptor and protein kinase B phosphorylation in peripheral organs, as well as insulin-degrading enzyme (IDE) protein production and alternative splicing in the liver and HEPG2 cells. RESULTS: CNTF improved insulin sensitivity and beta cell mass, while reducing glucose-stimulated insulin secretion and insulin clearance in Swiss mice, improving glucose handling in a non-obese type 2 diabetes model. This effect was associated with lower IDE production and activity in liver cells. All these effects were observed even at 21 days after CNTF treatment. CONCLUSIONS/INTERPRETATION: CNTF protection against type 2 diabetes is partially independent of the anti-obesity actions of CNTF, requiring a reduction in insulin clearance and increased beta cell mass, besides increased insulin sensitivity. Furthermore, knowledge of the long-term effects of CNTF expands its pharmacological relevance.

Pregnancy restores insulin secretion from pancreatic islets in cafeteria diet-induced obese rats.

Insulin resistance during pregnancy is counteracted by enhanced insulin secretion. This condition is aggravated by obesity, which increases the risk of gestational diabetes. Therefore, pancreatic islet functionality was investigated in control nonpregnant (C) and pregnant (CP), and cafeteria diet-fed nonpregnant (Caf), and pregnant (CafP) obese rats. Isolated islets were used for measurements of insulin secretion (RIA), NAD(P)H production (MTS), glucose oxidation ((14)CO(2) production), intracellular Ca(2+) levels (fura-2 AM), and gene expression (real-time PCR). Impaired glucose tolerance was clearly established in Caf and CafP rats at the 14th wk on a diet. Insulin secretion induced by direct depolarizing agents such as KCl and tolbutamide and increasing concentrations of glucose was significantly reduced in Caf, compared with C islets. This reduction was not observed in islets from CP and CafP rats. Accordingly, the glucose oxidation and production of reduced equivalents were increased in CafP islets. The glucose-induced Ca(2+) increase was significantly lower in Caf and higher in CafP, compared with all other groups. CP and CafP islets demonstrated an increased Ca(2+) oscillation frequency, compared with both C and Caf islets, and the amplitude of oscillations was augmented in CafP, compared with Caf islets. In addition, Ca(v)alpha1.2 and SERCA2a mRNA levels were reduced in Caf islets. Ca(v)alpha1.2, but not SERCA2a, mRNA was normalized in CafP islets. In conclusion, cafeteria diet-induced obesity impairs insulin secretion. This alteration is related to the impairment of Ca(2+) handling in pancreatic islets, in especial Ca(2+) influx, a defect that is reversed during pregnancy allowing normalization of insulin secretion.

Blood amino acids concentration during insulin induced hypoglycemia in rats: the role of alanine and glutamine in glucose recovery.

Our purpose was to determine the blood amino acid concentration during insulin induced hypoglycemia (IIH) and examine if the administration of alanine or glutamine could help glycemia recovery in fasted rats. IIH was obtained by an intraperitoneal injection of regular insulin (1.0 U/kg). The blood levels of the majority of amino acids, including alanine and glutamine were decreased (P < 0.05) during IIH and this change correlates well with the duration than the intensity of hypoglycemia. On the other hand, the oral and intraperitoneal administration of alanine (100 mg/kg) or glutamine (100 mg/kg) accelerates glucose recovery. This effect was partly at least consequence of the increased capacity of the livers from IIH group to produce glucose from alanine and glutamine. It was concluded that the blood amino acids availability during IIH, particularly alanine and glutamine, play a pivotal role in recovery from hypoglycemia.

Day-restricted feeding during pregnancy and lactation programs glucose intolerance and impaired insulin secretion in male rat offspring.

AIM: The maternal environment during pregnancy and lactation plays a determining role in programming energy metabolism in offspring. Among a myriad of maternal factors, disruptions in the light/dark cycle during pregnancy can program glucose intolerance in offspring. Out-of-phase feeding has recently been reported to influence metabolism in adult humans and rodents; however, it is not known whether this environmental factor impacts offspring metabolism when applied during pregnancy and lactation. This study aims to determine whether maternal day-restricted feeding (DF) influences energy metabolism in offspring. METHODS: Pregnant and lactating Wistar rats were subjected to ad libitum (AL) or DF during pregnancy and lactation. The offspring born to the AL and DF dams were intra- and interfostered, which resulted in 4 group types. RESULTS: The male offspring born to and breastfed by the DF dams (DF/DF off) were glucose intolerant, but without parallel insulin resistance as adults. Experiments with isolated pancreatic islets demonstrated that the male DF/DF off rats had reduced insulin secretion with no parallel disruption in calcium handling. However, this reduction in insulin secretion was accompanied by increased miRNA-29a and miRNA34a expression and decreased syntaxin 1a protein levels. CONCLUSION: We conclude that out-of-phase feeding during pregnancy and lactation can lead to glucose intolerance in male offspring, which is caused by a disruption in insulin secretion capacity. This metabolic programming is possibly caused by mechanisms dependent on miRNA modulation of syntaxin 1a.

Biochemical characterization of two crotamine isoforms isolated by a single step RP-HPLC from Crotalus durissus terrificus (South American rattlesnake) venom and their action on insulin secretion by pancreatic islets.

Crotamine, a neurotoxin present in the venom of the South American rattlesnake Crotalus durrisus terrificus exists as several polymorphic variants, as demonstrated by recombinant DNA technology (Smith and Schmidt, Toxicon 28 (1990) 575-585). We have isolated native crotamine by chromatography on Sephadex G75, and have purified two crotamine isoforms (F2 and F3) by a single step of RP-HPLC. Native crotamine and RP-HPLC fractions F2 and F3 produced skeletal muscle spasms and spastic paralysis in mice. At low glucose concentrations (2.8-5.6 mmol/l), none of the crotamines altered the insulin secretion by rat isolated islets. In the presence of 16.7 mmol glucose/l, F2 (5 microg/ml), but not F3, increased insulin secretion two-fold, whereas native crotamine (1.5, 5 and 16.5 microg/ml) potentiated the secretion dose-dependently. The increase in insulin secretion induced by F2 fraction (5 microg/ml) was similar to that obtained with 16.5 microg of native crotamine/ml. These results indicate that the mode of action of the F2 and F3 isoforms in beta-cells is different from that in muscle cells. This difference may be related to the binding affinity of each isoform for the Na(+) channels located in the beta-cell membrane. Crotamine isoforms may be valuable tools for studying the involvement of Na(+) channels in the mechanism of insulin secretion.

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

Highly purified Tityustoxin V (TsTX-V), an alpha-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 beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 micrograms/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 microM 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 beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.

Biological and structural characterization of a new PLA2 from the Crotalus durissus collilineatus venom.

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A2 (PLA2) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA2s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA2s such as from the Bothrops snake venom. Also, this PLA2 induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and p-BPB showed a similar reduction. The p-BPB did not reduce significantly the myotoxic activity induced by the PLA2, but the anhydrous acetic acid treatment and the pre-incubation of PLA2 with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiforae (Gram-negative), which was drastically reduced by incubation of this PLA2 with p-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or beta-wing region may be involved in the binding of this PLA2 to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.

Determination of the amino acid sequence of a new phospholipase A(2) (MIDCA1) isolated from Micrurus dumerilii carinicauda venom.

A new Phospholipase A(2) (PLA(2)) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA(2) showed a low enzymatic activity when compared with other PLA(2)s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA(2)s, such as Naja naja, showed significant differences in the beta-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA(2), supporting the view that other regions of the protein are involved in the biological effects.

Vagotomy ameliorates islet morphofunction and body metabolic homeostasis in MSG-obese rats.

The parasympathetic nervous system is important for ß-cell secretion and mass regulation. Here, we characterized involvement of the vagus nerve in pancreatic ß-cell morphofunctional regulation and body nutrient homeostasis in 90-day-old monosodium glutamate (MSG)-obese rats. Male newborn Wistar rats received MSG (4 g/kg body weight) or saline [control (CTL) group] during the first 5 days of life. At 30 days of age, both groups of rats were submitted to sham-surgery (CTL and MSG groups) or subdiaphragmatic vagotomy (Cvag and Mvag groups). The 90-day-old MSG rats presented obesity, hyperinsulinemia, insulin resistance, and hypertriglyceridemia. Their pancreatic islets hypersecreted insulin in response to glucose but did not increase insulin release upon carbachol (Cch) stimulus, despite a higher intracellular Ca(2+) mobilization. Furthermore, while the pancreas weight was 34% lower in MSG rats, no alteration in islet and ß-cell mass was observed. However, in the MSG pancreas, increases of 51% and 55% were observed in the total islet and ß-cell area/pancreas section, respectively. Also, the ß-cell number per ß-cell area was 19% higher in MSG rat pancreas than in CTL pancreas. Vagotomy prevented obesity, reducing 25% of body fat stores and ameliorated glucose homeostasis in Mvag rats. Mvag islets demonstrated partially reduced insulin secretion in response to 11.1 mM glucose and presented normalization of Cch-induced Ca(2+) mobilization and insulin release. All morphometric parameters were similar among Mvag and CTL rat pancreases. Therefore, the higher insulin release in MSG rats was associated with greater ß-cell/islet numbers and not due to hypertrophy. Vagotomy improved whole body nutrient homeostasis and endocrine pancreatic morphofunction in Mvag rats.

Prolactin induces maturation of glucose sensing mechanisms in cultured neonatal rat islets.

The effects of PRL treatment on insulin content and secretion, and 86Rb and 45Ca fluxes from neonatal rat islets maintained in culture for 7-9 days were studied. PRL treatment enhanced islet insulin content by 40% and enhanced early insulin secretion evoked by 16.7 mM glucose. Insulin release stimulated by oxotremorine-M, a muscarinic agonist, in the presence of glucose (8.3 or 16.7 mM) was unchanged by PRL treatment. However, PRL treatment potentiated phorbol 12,13-dibutyrate-stimulated insulin secretion in the presence of the above glucose concentrations. PRL treatment potentiated the reduction in 86Rb efflux induced by glucose or tolbutamide and enhanced the increase in 86Rb efflux evoked by diazoxide. PRL treatment slightly potentiated the increment in 45Ca uptake induced by high concentrations of K+, but failed to affect the increment evoked by 16.7 mM glucose. Since glucose-induced 45Ca uptake was not affected by PRL, we suggest that the enhancement in first phase insulin secretion evoked by glucose in the PRL-treated islets occurs at a step in the secretory process that may involve protein kinase-C. These data further support observations that PRL treatment increases islet sensitivity to glucose.

Prolactin treatment increases GLUT2 but not the G protein subunit content in cell membranes from cultured neonatal rat islets.

Neonatal rat islets exhibit a reduced secretory response to glucose, compared to adult rat islets. The maturation of the secretory response is stimulated by prolactin (PRL). We show here by immunoblot analysis that PRL increases the beta-cell/liver glucose transporter GLUT2 in membrane fractions from cultured neonatal rat islets. This increase (+86%) may explain, at least in part, the development of a mature glucose response. G proteins modulate insulin secretion from pancreatic beta-cells. We show here by immunoblot analysis that, in contrast to the effect on GLUT2, PRL treatment does not modify the G protein subunits alpha i2, alpha i3, alpha o, alpha s, alpha q and beta 35 and beta 36, in cultured neonatal islets.

Glucose- and insulin-induced phosphorylation of the insulin receptor and its primary substrates IRS-1 and IRS-2 in rat pancreatic islets.

The presence of tyrosine-phosphorylated proteins was studied in cultured rat pancreatic islets. Immunoblotting performed with total extracts of islets cultured in the presence of 1.8 or 5.6 mM glucose revealed at least three distinct tyrosine-phosphorylated bands (25 kDa, 95 kDa and 165-185 kDa). After 12 h incubation in medium containing 1.8 mM glucose, a pulse exposition to 11 or 22 mM glucose or to 10(-7) M insulin led to a substantial increase in the phosphorylation of all three bands, with no appearance of novel bands. Immunoprecipitation with specific antibodies demonstrated that the signal detected at 95 kDa corresponds to the beta subunit of the insulin receptor (IR) while the band at 165-185 kDa corresponds to the early substrates of the insulin receptor, IRS-1 and IRS-2. Immunoprecipitation with IRS-1 or IRS-2 antisera detected their association with the lipid metabolizing enzyme phosphatidylinositol 3-kinase (PI 3-kinase). Thus, this is the first demonstration that elements involved in the insulin-signalling pathway of traditional target tissues are also present in pancreatic islets and are potentially involved in auto- and paracrine-signalling in this organ.

Restoration of insulin secretion in pancreatic islets of protein-deficient rats by reduced expression of insulin receptor substrate (IRS)-1 and IRS-2.

Autocrine and paracrine insulin signaling may participate in the fine control of insulin secretion. In the present study, tissue distribution and protein amounts of the insulin receptor and its major substrates, insulin receptor substrate (IRS)-1 and IRS-2, were evaluated in a model of impaired glucose-induced insulin secretion, the protein-deficient rat. Immunoblot and RT-PCR studies showed that the insulin receptor and IRS-2 expression are increased, whilst IRS-1 protein and mRNA contents are decreased in pancreatic islets of protein-deficient rats. Immunohistochemical studies revealed that the insulin receptor and IRS-1 and -2 are present in the great majority of islet cells; however, the greatest staining was localized at the periphery, suggesting a co-localization with non-insulin-secreting cells. Exogenous insulin stimulation of isolated islets promoted higher insulin receptor and IRS-1 and -2 tyrosine phosphorylation in islets from protein-deficient rats, as compared with controls. Moreover, insulin-induced IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activity are increased in islets of protein-deficient rats. The reduction of IRS-1 and IRS-2 protein expression in islets isolated from protein-deficient rats by the use of antisense IRS-1 or IRS-2 phosphorthioate-modified oligonucleotides partially restored glucose-induced insulin secretion. Thus, the impairment of insulin cell signaling through members of the IRS family of proteins in isolated rat pancreatic islets improves glucose-induced insulin secretion. The present data reinforced the role of insulin paracrine and autocrine signaling in the control of its own secretion.

Tetracaine stimulates extracellular Ca2+-independent insulin release.

The effect of the local anesthetic, tetracaine, on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i and insulin secretion in pancreatic B-cells was studied. At a physiological level of [Ca2+]o, tetracaine (0.1-5 mM) dose-dependently inhibited insulin secretion induced by 22 mM glucose. Paradoxically, at the same glucose concentration but in the absence of external Ca2+, tetracaine dose-dependently increased insulin secretion. At a low glucose level (2.8 mM) tetracaine failed to affect secretion, either in the presence or absence of external Ca2+. At high (22 mM) or low (2.8 mM) glucose, [Ca2+]i was increased by tetracaine in a dose-dependent manner. Tetracaine (2 mM) also increased the 45Ca efflux from isolated islets. This effect was of the same magnitude at both low and high glucose concentrations, and was independent of the presence of extracellular Ca2+. Finally, tetracaine increased 45Ca efflux from islets perifused in the presence of thapsigargin. In conclusion, our data indicate that tetracaine releases Ca2+ from a thapsigargin-insensitive store in pancreatic B-cells. Under suitable experimental conditions, insulin release can be elicited by a [Ca2+]o-independent pathway. The existence of a ryanodine-like Ca2+ channel in pancreatic B-cells is proposed.

Differences in K+ permeability between cultured adult and neonatal rat islets of Langerhans in response to glucose, tolbutamide, diazoxide, and theophylline.

The effects of glucose, tolbutamide, and diazoxide on K+ permeability in neonatal and adult rat pancreatic islets, maintained in culture 1 week, were investigated by measuring the 86Rb outflow rate from prelabeled islets. In the absence of glucose, the 86Rb efflux was significantly lower in neonatal than adult islets. Raising the glucose concentration to 2.8, 5.6, 8.3, and 11.1 mM produced a marked reduction in the 86Rb efflux in adult islets but only a minor reduction in neonatal islets. The effect of tolbutamide to reduce, and diazoxide to increase, the 86Rb efflux was also less in neonatal islets. These results are discussed with respect to previously reported differences in insulin secretion from neonatal and adult islets in culture.

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.