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Articles

A Protein-Restricted Diet during Pregnancy Alters in Vitro Insulin Secretion from Islets of Fetal Wistar Rats^{1 ,2}

Laboratory of Cell Biology Department of Biology, WHO Collaborating Center, Université Catholique de Louvain, 1348, Louvain-la-Neuve, Belgium

³To whom correspondence should be addressed. E-mail: remacle{at}bani.ucl.ac.be.

	ABSTRACT

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Previous studies indicate that insulin secretion from the fetuses of dams fed a low protein (LP) diet is reduced in response to leucine or arginine. The aim of this study was to locate the defect in the insulin secretion pathway induced by a LP diet during gestation. The effects of various secretagogues acting at different levels of the insulin secretion cascade were investigated in vitro in fetal islets from dams fed either a normal or a LP diet during pregnancy. Insulin content, insulin secretion and the cAMP content were then measured. Although insulin content of LP islets did not differ from that of control islets, insulin secretion from LP fetal islets was reduced when challenged by amino acids or cAMP enhancers. This reduction did not appear to be related solely to an altered islet cAMP content. An impairment of insulin secretion remained after stimulation of fetal LP islets with either metabolic or nonmetabolic secretagogues. The insulin secretion by LP islets was restored to normal, however, with barium or cytochalasin-B. These findings demonstrate that an in utero isocaloric LP diet impairs insulin secretion of the fetus. This alteration is located at the exocytosis step in the insulin secretion cascade and not in the insulin pool of the ß cell.

KEY WORDS: • protein malnutrition • glucose • amino acids • pregnancy • insulin • cAMP • rats

	INTRODUCTION

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The appropriate maternal metabolic environment is essential for the development of the fetus, including its endocrine pancreas. Indeed, when the former is disturbed such as in diabetic pregnancy, many changes occur in the structure and function of the fetal endocrine pancreas. Fetuses from mildly diabetic dams feature hypertrophy and hyperplasia of the islets (1) , leading to a higher insulin content (2) . The in vivo and in vitro proliferative capacity of these islets is increased (3 ,4) . Such islets are more sensitive to glucose stimulation than normal fetal islets (2 ,5) . In situations of severe maternal diabetes, fetal ß cells are degranulated (2) and their pancreatic insulin content is lower (4) . In response to glucose perfusion, these islets secrete less insulin (5) .

Major perturbation of the metabolic environment also occurs when the dams are fed a low protein (LP)⁴ diet during gestation. When an isocaloric LP diet containing 8% instead of 20% protein is given to dams from d 1 of pregnancy and maintained throughout gestation, the glycemia of dams and fetuses remains normal, whereas the amino acid profile in their serum is altered (6) . LP fetuses and newborns are smaller, and the development of their endocrine pancreas is impaired. Islet cell multiplication in vivo, islet size and pancreatic insulin content are decreased compared with pancreata of control fetuses. Moreover, these LP islets are less vascularized (7) . These islet alterations observed in vivo are associated with a decreased insulin release in response to leucine and arginine in vitro. In adulthood, insulin secretion remains lower in these rats even when weaned onto a normal diet (8) .

The importance of insulin for fetal growth throughout late gestation is well established (9) . In humans, specific fetal hypoinsulinemia is associated with intrauterine growth retardation (10) . For normal insulin-dependent fetal growth, insulin must then be produced by the fetal pancreatic ß cells in an appropriate quality and quantity, which is not the case in protein-restricted fetuses (10) .

Previous experiments could not attribute the deficiency in insulin release to a lower pool of insulin, a reduced secretory capacity or a lower sensitivity to secretagogues. This paper attempts to address this question by triggering different steps leading to insulin release. Therefore, metabolic (glucose, leucine, glutamine or -ketoisocaproate) or nonmetabolic (high extracellular potassium concentration, acetylcholine, tolbutamide, barium chloride, cytochalasin-B) secretagogues were used to analyze specific changes in intracellular pathways leading to insulin secretion. Cyclic AMP, which is a major second messenger in the amplification of the stimulus secretion coupling in the ß cell (11) , was also investigated.

The mechanistic basis of the secretory defect in LP islets is not known. The aim of the present study was to investigate the site of this defect. This was achieved using a range of metabolic and nonmetabolic secretagogues, which act at different steps of the insulin secretory pathway.

	MATERIALS AND METHODS

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Animals and diets.

Control fetuses (C) were from Wistar rats fed 20% protein diet during gestation; the LP fetuses were from dams fed an isocaloric, low protein diet (8%). Virgin female rats, 110 d old and weighing 200 g, belonging to a local stock bred at the Animal Center of the Université Catholique de Louvain, Belgium, were caged overnight with males and copulation was verified in the morning by detection of a vaginal plug. Midnight was considered as the time of mating on d 0 of gestation. Pregnant females were then housed singly under controlled conditions of light (14 h light, 10 h darkness), temperature (24°C), and humidity (60%). The rats were then given free access to their respective diets and to water. They were divided randomly into two groups starting from d 1 of gestation. After 21.5 d of gestation, dams and their fetuses were killed by decapitation.

Diets were purchased from Hope Farms (Woerden, The Netherlands) and their compositions are described elsewhere (12) . The diets were similar in fat content and were rendered isocaloric by the addition of carbohydrate to the LP diet. The amount of food consumed by C and LP dams was not determined in the current study because earlier work indicated no difference (7) .

The Institution’s guide for the care and use of laboratory animals was respected, and all procedures were performed with approval of the Animal Ethics Committees of the Université Catholique de Louvain, Belgium.

Culture of fetal islets.

RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, 11 mmol/L glucose and antibiotics (penicillin 0.2 x 10⁴ U/L, streptomycin 0.2 g/L) was used during the entire culture process, including the digestion of the pancreatic tissue.

The pancreata were removed aseptically from 21.5-d-old C and LP fetuses and placed in sterile cold medium. They were minced and transferred into a sterile tube containing 2 mL of medium with 5 mg collagenase (Boehringer, Mannheim, Germany). The tube was shaken by hand at 37°C for 7–8 min and the digestion was stopped by adding cold medium. The tissue digest was then washed twice with RPMI medium. Thereafter, the resulting cell pellets were suspended in 20 mL of medium and gently stirred at room temperature for 60 min. After centrifugation (180 x g for 10 min), the pellets were resuspended in medium (1 pancreas/2 mL RPMI 1640 medium). This suspension was distributed into 35-mm Petri dishes (2 mL/dish, Falcon 3001; Falcon Plastics, Los Angeles, CA). The culture dishes were incubated for up to 7 d at 37°C, in a humidified atmosphere of 5% CO₂ in air, according to a method derived from that of Hellerström et al. (13) and Mourmeaux et al. (14) . From d 2 of culture, the medium was replaced every 24 h and stored at -20°C for insulin assay.

Fetal islet cell proliferation.

On d 7 of culture, tritiated thymidine (Amersham, Rosendaal, The Netherlands; specific activity 814 GBq/mmol) was added to the Petri dishes (1 mCi/L). After 24 h, the dishes were washed with RPMI 1640 medium containing an excess of nonradioactive thymidine. The islets were fixed in Bouin Allen solution, embedded in paraffin and serially sectioned at a thickness of 7 µm. The slides were covered with L4-Ilford nuclear track emulsion, exposed for 18 d at 4°C and revealed with Kodak D 19 developer. The slides were then stained with hematoxylin-eosin, and the labeling index (LI) was calculated by dividing the number of labeled islet cell nuclei by the total number of islet cell nuclei. The LI was expressed as a percentage.

Incubation of cultured islets.

All of these experiments were performed with a Krebs-Ringer solution containing (mmol/L): NaCl, 120; KCl, 5; CaCl₂, 2; MgCl₂, 1; NaHCO₃, 22. It was supplemented with 5 g bovine serum albumin (BSA)/L (Fraction V, Calbiochem-Behring, San Diego, CA) and was gassed with 95% O₂/5% CO₂ to maintain a pH of 7.4. Glucose and test substances were added into the incubation medium without correction for osmolarity except when indicated.

In the first part of the study, batches of 10 free floating islets were picked up and incubated at 37°C in 1 mL of Krebs-Ringer medium containing various secretagogues: glucose alone (5.6 mmol/L); glucose (16.7 mmol/L) or glucose (5.6 mmol/L) together with leucine (10 mmol/L), arginine (10 mmol/L), theophylline (2 mmol/L), forskolin (1 µmol/L) or 5-tetradecanoyl-13-phorbol acetate (TPA; 25 nmol/L).

In the second part of the study, various secretagogues, which act at various steps of the insulin secretion pathway, were added to the incubation medium, which contained 16.7 mmol/L glucose. The secretagogues tested were tolbutamide (100 µmol/L), acetylcholine (100 µmol/L), leucine alone at 20 mmol/L or at 10 mmol/L together with glutamine (10 mmol/L) and -ketoisocaproate (KIC; 10 mmol/L). Cytochalasin-B (10 µmol/L) was added to 5.6 mol/L glucose (G5.6) or to glutamine and leucine (both at 10 mmol/L). Studies in which the extracellular concentration of K⁺ was raised to 50 mmol/L (KCl, 50 mmol/L) were also performed. In these experiments, the concentration of NaCl was decreased to maintain osmolarity. In further experiments, BaCl₂ (2.5 mmol/L) was used to substitute CaCl₂ in the incubation medium.

After 120 min, the incubation medium was removed and placed in a watch glass to verify that no islet had been taken; then the medium was frozen until the insulin assay was performed. To determine insulin and cAMP contents, islets were collected under microscopic observation and homogenized by sonification (30 s, 40 W) in 0.5 mL Krebs-Ringer solution. A sample of 100 µL from the homogenate extraction was added to 200 µL acid/ethanol [0.15 mol/L HCl in 75% (v/v) ethanol in water] to extract insulin.

To determine the cAMP content, 150 µL of the homogenate solutions was added to ice-cold ethanol to obtain a final concentration of 65% ethanol. The extracts were centrifuged at 2000 x g for 15 min at 4°C. Supernatants were transferred to fresh tubes and diluted with appropriate volumes of acetate buffer before analysis.

To eliminate variations due to differences in individual batches of islets, insulin secretion during incubation was expressed as a percentage of the islet insulin content at the start of the incubation, which is referred as fractional insulin release. The latter was obtained by adding the content measured at the end to the amount of released insulin.

Test substances.

Glucose, L-glutamine, L-leucine and L-arginine were purchased from Merck (Darmstadt, Germany), theophylline, forskolin, TPA, BaCl₂, acetylcholine chloride, cytochalasin-B and KIC from Sigma Chemical (St Louis, MO), and tolbutamide from ICN Biomedicals (Costa Mesa, CA).

Insulin and cAMP assays,

Insulin content and release were measured by RIA (Insik 5; Sorin, Italy). Immunoreactive insulin was estimated using purified rat insulin as a standard (Novo, Nordisk, Denmark), guinea-pig anti-human insulin antiserum and porcine monoionodinated [¹²⁵I]-insulin. Antibody against guinea-pig immunoglobulin (Ig)G was used for precipitation of the binding complex. To separate free from bound hormone, the samples were centrifuged at 1500 x g. This method allows the determination of 4 mU/L (27.5 pmol/L) with a CV within and between assay of 8.2%.

The cAMP content was analyzed by RIA (Biotra RPA 509; Amersham). Immunoreactive cAMP was estimated using cAMP standard, tracer adenosine 3',5'-cyclic phosphoric acid 2'-O-succinyl-3- [¹²⁵I] iodotyrosine methyl ester (0.058 MBq) and rabbit anti-succinyl cAMP antiserum. The precipitation of the labeled complex was carried out with a second antibody reagent (donkey anti-rabbit IgG antiserum). To separate the free from the bound cAMP, samples were centrifuged at 1500 x g. The assay allowed the determination of 1.0 pmol/L. The CV within and between assays was 6.1%.

Statistical analysis.

The data were analyzed by one- or two-way ANOVA including the Barlett test for homogeneity of variance, followed by Newman-Keuls test for individual differences between means as indicated. Results are expressed as mean ± SEM unless indicated otherwise. Differences with a P-value < 0.05 were considered significant.

	RESULTS

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Insulin secretion.

Batches of 10 neoformed fetal islets were incubated in Krebs-Ringer medium with different secretagogues. C and LP islets had insulin contents that did not differ [4.9 ± 0.2 pmol/islet (n = 164) and 4.7 ± 0.2 pmol/islet (n = 139), respectively]. In response to either 5.6 or 16.7 mmol/L glucose, the fractional insulin release was significantly lower in the LP group than in controls (P < 0.05) (Fig. 1 ). In the presence of 5.6 mmol/L glucose with 10 mmol/L leucine or arginine, fetal islets from both groups exhibited increased insulin secretion (P < 0.01), but it remained significantly lower in the LP islets than in the C group (P < 0.01) (Fig. 1) . In both groups, the inclusion of theophylline, a methylxanthine inhibitor of phosphodiesterase, with glucose and leucine in the incubation medium induced a significant potentiation of the insulin release ( P < 0.05). However, the secretion was lower in the LP group compared with the C group (P < 0.01) (Fig. 1) . Arginine added to glucose also induced a lower insulin release by LP islets compared with controls, both in the presence and the absence of theophylline (P < 0.01).

View larger version (19K): [in this window] [in a new window]   Figure 1. Fractional insulin release by cultured fetal islets from dams fed 20% (C) or 8% protein (LP) diets throughout gestation. Islets were incubated in Krebs-Ringer medium containing combinations of secretagogues, glucose (G5.6; 5.6 mmol/L), leucine (Leu; 10 mmol/L), arginine (Arg; 10 mmol/L) and theophylline (The, 2 mmol/L). Values are means ± SEM, n = 12–13. **P < 0.01, *P < 0.05 vs. C.

View larger version (15K): [in this window] [in a new window]   Figure 2. Fractional insulin release by cultured fetal islets from dams fed 20% (C) or 8% protein (LP) diets throughout gestation. Islets were incubated in Krebs-Ringer medium containing combinations of secretagogues, glucose (G5.6; 5.6 mmol/L ), theophylline (The, 2 mmol/L), forskolin (For, 1 µmol/L) and 5-tetradecanoyl-13-phorbol acetate (TPA; 25 nmol/L). Values are means ± SEM, n = 8–12. **P < 0.01 vs. C.

View larger version (16K): [in this window] [in a new window]   Figure 3. Fractional insulin release by cultured fetal islets from dams fed 20% (C) or 8% protein (LP) diets throughout gestation. Islets were incubated in Krebs-Ringer medium containing metabolic secretagogues, glucose at 5.6 mmol/L (G5.6) or at 16.7 mmol/L (G16.7), leucine (Leu; 10 mmol/L), -ketoisocaproate (KIC; 100 µmol/L) or leucine added to glutamine (Leu + Gln; 10 mmol/L). Values are means ± SEM, n = 9. **P < 0.01, *P < 0.05 vs. C

View larger version (16K): [in this window] [in a new window]   Figure 4. Fractional insulin release by cultured fetal islets from dams fed 20% (C) or 8% protein (LP) diets throughout gestation. Islets were incubated in Krebs-Ringer medium containing glucose and other nonmetabolic secretagogues, glucose at 5.6 mmol/L (G5.6) or at 16.7 mmol/L (G16.7), G5.6 added to acetylcholine (Ach; 100 µmol/L), or G16.7 added to tolbutamide (Tol 100 µmol/L), KCl (50 mmol/L) or BaCl2 (2.5 mmol/L). Values are means ± SEM, n = 9. **P < 0.01, *P < 0.05 vs. C.

View larger version (16K): [in this window] [in a new window]   Figure 5. Fractional insulin release by cultured fetal islets from dams fed 20% (C) or 8% protein (LP) diets throughout gestation. Islets were incubated in Krebs-Ringer medium containing cytochalazin-B (Cyt; 10 nmol/L) added to glucose at 16.7 mmol/L (G16.7) or to leucine and glutamine (Leu + Gln; 10 mmol/L). Values are means ± SEM, n = 9 observations. **P < 0.01, *P < 0.05 vs. C.

Islet cAMP content estimated per pmol of islet insulin was not different in low and high concentrations of glucose, in either the LP or the C group (Table 1 ). However, cAMP content was significantly lower in the LP group at both concentrations of glucose (5.6 or 16.7 mmol/L) (P < 0.01). This was also true in response to leucine, but not to arginine added to glucose. Moreover, in both groups, cAMP content was enhanced in the presence of pharmacologic agents such as theophylline, forskolin or TPA, and reached values that did not differ between the two groups, notwithstanding alterations in insulin secretion (Fig. 2) . The greatest effect on cAMP content was observed in the presence of forskolin.

As analyzed by the labeling index, the cell proliferation in 19.34 islets from 6 cultures was 11.7 ± 0.7% (n = 34) and 5.1 ± 0.7% (n = 19) in C and LP groups, respectively (P < 0.01).

	DISCUSSION

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This study was designed to investigate the cellular basis of the secretory defect present in islets from fetuses deprived of maternal protein.

The insulin content per islet was similar in the C and LP groups, suggesting that the reduced insulin release was not attributable to a decreased available pool of insulin but to alterations in the insulin secretion pathways. To test the metabolic disturbance in insulin secretion of fetal LP ß cells, secretory effects of glucose, glutamine, leucine and KIC were investigated. Glucose has to be metabolized in the cytosol as well as the mitochondria, whereas leucine and KIC require only mitochondrial metabolism to stimulate insulin release. In response to glucose, albeit a poor stimulus in the normal fetus (14 ,15) , fetal LP islets exhibited a lower insulin secretion than control islets. A similar reduction was observed when fetal LP islets were stimulated by leucine or KIC. In another study (16) , we found that insulin release stimulated by nutrient secretagogues was altered in the islets of adult rat offspring of dams fed the low protein diet during pregnancy and lactation and maintained on the LP diet throughout life. In parallel, the activity of the mitochondrial FAD-dependent glycerophosphate dehydrogenase was diminished in these islets, and coincided with an alteration in the oxidative metabolism of glucose. Moreover, L-leucine transamination to KIC was impaired in these rats (17) . These alterations, which are evident in adults, may also be present during fetal life and contribute to the deficient insulin secretion by the LP fetuses.

Because metabolic secretagogues were unable to restore to normal the lower insulin secretion from LP fetal islets, the secretory effect of arginine was then tested on C and LP islets. Arginine is weakly metabolized in islet cells (18) and acts on the ß cells differently from glucose and leucine; this amino acid is thought to depolarize the ß cell membrane because of its transport into the cell in a positively charged form (19) . It activates voltage-dependent Ca²⁺ channels, and Ca²⁺ influx ensues. Insulin secretion by fetal LP islets remained reduced in response to arginine. This indicates that there is a defect in the insulin secretory pathway, which is independent of metabolic processes.

Ca²⁺ influx into ß cells is a key step in the insulin secretory pathway. To investigate the involvement of Ca²⁺ in the altered insulin secretion of LP islets, KCl, tolbutamide, acetylcholine and Ba were used. KCl and tolbutamide induce insulin release by increasing the rate of entry of Ca²⁺ into the ß cell. Acetylcholine was added to increase cytosolic Ca²⁺ by releasing Ca²⁺ from intracellular stores in addition to the increase of Ca²⁺ influx through voltage-dependent Ca²⁺ channels (20) . In response to the two kinds of stimulation, insulin was secreted by the C as well as by the LP fetal islets, but the response of LP islets remained reduced compared with controls. In substituting Ca²⁺ with Ba²⁺, which permeates membranes through Ca²⁺ channels (21) and triggers directly exocytosis of insulin without any contribution of Ca²⁺ (22) , insulin was secreted by C and LP fetal islets and was not different in the two groups. This suggests that in fetal LP ß cells, defects in Ca²⁺ movements and/or activity of exocytotic enzymes including kinase proteins, which are Ca²⁺ dependent, are implicated in their reduced insulin secretion.

In this study, we detected a low cAMP content in the LP islets when stimulated with glucose and leucine. This might also explain in part their reduced insulin release. However, the level of cAMP in the LP islets in response to arginine, theophylline, forskolin and TPA was normal and was not associated with a recuperation of a normal insulin secretion. This implies that defects in the insulin secretion in the LP group were due to alterations in insulin secretory pathway at steps downstream from cAMP production.

Because none of the secretagogues tested were able to restore insulin secretion from LP fetal islets to normal, alterations in the exocytotic process were analyzed. Fetal islets were then stimulated by cytochalasin-B, which acts by reversibly changing the ß cell microfilamentous cell web involved in the last step of the insulin secretion cascade (23) . This fungal metabolite similarly increased insulin secretion in the two groups. These findings indicate that the readily releasable pool of insulin is not reduced in size and that a main alteration site of insulin secretion of the LP islets is also situated at the level of exocytotic process.

The persisting reduction of both the insulin secretion and replication of fetal ß cells in the LP group in vitro, even when they have been removed from the maternal environment and cultured for 1 wk in RPMI medium, shows that the protein-restricted diet of dams during pregnancy induces a lasting impairment of the sensitivity of the fetal ß cells to secretagogues as well as to mitogenic stimuli. The secretory and proliferative activities of the fetal ß cell are stimulated by amino acids whose effects are known at 14.5 d of gestation. Indeed, enrichment of the culture medium with essential amino acids results in a higher fetal ß cell replication, an increased insulin ß cell content as well as a higher basal and glucose-stimulated insulin release (24 ,25) . Because of decreased concentrations of essential and branched amino acids in the serum of LP fetuses and dams (6) , we suggest that the lower sensitivity to amino acids observed in vitro might be due to the low availability of amino acids in the fetomaternal unit. Low levels of taurine could also be involved because, when added into the culture medium, this amino acid enhances the insulin secretion by fetal islets in response to secretagogues (26) and restores the reduced insulin secretion by fetal LP islets when added during gestation to the drinking water of LP dams (27) .

In conclusion, our findings highlight the crucial role of a normal protein diet during pregnancy to ensure a normal insulin secretory responsiveness in the progeny. The alteration site of the insulin secretion by fetal islets from dams fed a low protein diet might consist of alterations in Ca²⁺ fluxes into ß cells or further events of the exocytotic process of insulin secretion.

	ACKNOWLEDGMENTS

 
We dedicate this investigation to the memory of J. J. Hoet. We thank J. C. Henquin and A. Sener for their precious advice in the accomplishment of this study. We also thank M. T. Ahn for skillful technical assistance.

	FOOTNOTES

 
¹ Presented in abstract form [Cherif, H., Reusens, B., Dahri, S., Remacle, C. & Hoet, J. J. (1997) Alterations in islets’ insulin secretion of fetus from pregnant rats fed an isocaloric low protein diet. Diabetologia 40 (suppl. 1) 520, A 134 (abs.)] and [Cherif, H., Reusens, B., Dahri, S., Remacle, C. & Hoet, J. J. (1997) Islets’ insulin secretion is altered in fetus from pregnant rats fed an isocaloric low protein diet. Diabetes 46 (suppl. 1) 1366, A 359 (abs.).]

² Supported by a grant from The Parthenon Trust, London, UK and a grant from the National Research Fund of Belgium.

⁴ Abbreviations used: C, control; Ig, immunoglobulin; KIC, -ketoisocaproate; LI, labeling index; LP, low protein diet; TPA, 5-tetradecanoyl-13-phorbol acetate.

Manuscript received October 25, 2000. Initial review completed November 29, 2000. Revision accepted February 16, 2001.

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