Glucose metabolism and beta -cell mass in adult offspring of rats protein and/or energy restricted during the last week of pregnancy

Eric Bertin, Marie-Nöelle Gangnerau, Danièle Bailbé, and Bernard Portha

Laboratoire de Physiopathologie de la Nutrition, Centre National de la Recherche Scientifique-ESA 7059, Université Paris 7/D. Diderot, 75251 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An association between low birth weight and later impaired glucose tolerance was recently demonstrated in several human populations. Although fetal malnutrition is probably involved, the biological bases of such a relationship are not yet clear, and animal studies on the matter are scarce. The present study was aimed to identify, in adult (8-wk) female offspring, the effects of reduced protein and/or energy intake strictly limited to the last week of pregnancy. Thus we have tested three protocols of gestational malnutrition: a low-protein isocaloric diet (5 instead of 15%), with pair feeding to the mothers receiving the control diet; a restricted diet (50% of the control diet); and a low-protein restricted diet (50% of low-protein diet). Only the low-protein diet protocols, independent of total energy intake, led to a lower birth weight. The adult offspring female rats in the three deprived groups exhibited no decrease in body weight and no major impairment in glucose tolerance, glucose utilization, or glucose production (basal state and hyperinsulinemic clamp studies). However, pancreatic insulin content and beta -cell mass were significantly decreased in the low-protein isocaloric diet group compared with the two energy-restricted groups. Such impairment of beta -cell mass development induced by protein deficiency limited to the last part of intrauterine life could represent a situation predisposing to impaired glucose tolerance.

fetal malnutrition; endocrine pancreas


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EPIDEMIOLOGICAL DATA have shown in different human populations that low birth weight, and especially thinness at birth, is associated with susceptibility to the development of impaired glucose tolerance or type 2 diabetes in adult life (10, 17, 19, 21, 24, 25). This association has been interpreted as long-term effects of nutritional factors that reduce fetal growth and impair the development of tissues regulating glucose metabolism (9, 22, 29). Some authors have suggested that it could be mediated through insulin resistance (17, 19, 24). However, reduced beta -cell function has also been described (4), and it is known that babies with intrauterine growth retardation have marked reductions in the size of the endocrine pancreas (31). Animal studies have also reported that fetal malnutrition is associated with persistently impaired pancreatic beta -cell function and development (5, 8). When a low-protein diet was used during the entire rat pregnancy, reduced proliferation rate, size, and insulin content of pancreatic islets were observed in fetuses at 21 days of pregnancy (5, 30). In fact, a 50% reduction in the mother's intake during the first 2 wk of gestation did not exert adverse effects on insulin secretion and action in 4-mo male offspring (28). When such food restriction was applied in the last week of the rat pregnancy, it did affect significantly the pancreatic insulin stores and the beta -cell mass in the fetuses or the offspring neonates (1, 8). These data suggest that the impact of fetal malnutrition on beta -cell mass development could be influenced by the type of malnutrition (energy and/or protein restriction) and/or the time course of malnutrition. To our knowledge, none of the studies so far published have undertaken to dissect the long-term impact of undernutrition 1) limited to the fetal stage of pancreas development, i.e., the 3rd wk of gestation (11, 27), or 2) designed to allow a separate analysis of the effect of protein deficiency per se from that of energy deficiency.

In the present study, we have therefore determined to what extent the glucose homeostasis, the insulin action and secretion, and the total pancreatic beta -cell mass are modified in adult female offspring of rats undernourished during the last trimester of their pregnancy. More specifically, we have analyzed on the adult progeny the impact of three different patterns of fetal malnutrition, strictly limited to the last week of pregnancy: energy restriction to 50% of the control diet, a low-protein diet with pair feeding to a control diet, or energy restriction to 50% of a control diet that was also a low-protein diet. Because undernourished mothers exhibit compensatory hyperphagia after the period of energy restriction (28), and to eliminate the possibility that their hyperphagia after delivery is responsible for the subsequent effects observed in their offspring, the pups were given as soon as delivered to foster mothers fed ad libitum with the standard diet.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Diets. The powdered semi-synthetic standard diet contained by weight (g/100 g) 68% starch, 4% cellulose, 5% lipid (corn oil), and 15% protein (casein), and by calories 72% carbohydrate, 12% lipid, and 15% protein. The powdered semi-synthetic low-protein diet contained by weight (g/100 g) 78% starch, 4% cellulose, 5% lipid (corn oil), and 5% protein (casein) and by calories 83% carbohydrate, 12% lipid, and 5% protein. Energy content per 100 g diet was the same (375 cal) in both diets. Both diets contained 2 g/100 g yeast, a salt mixture (3.5 g/100 g), and a vitamin mixture (2.2 g/100 g) as described in Picarel-Blanchot et al. (26).

Animals. Female Wistar rats bred in our colony were housed in a temperature-controlled room with a 12:12-h light-dark cycle (lights on 0700). Weighing 230-260 g, they were mated for one night (from 1700 to 900). On the next morning, the presence of sperm in the vaginal smear was confirmed, and this was taken as day 0.5 of pregnancy. After impregnation, females were transferred to individual plastic maternity cages. Pregnant females were fed standard diet ad libitum during the first 2 wk of pregnancy and then were assigned to one of the following four experimental conditions during the last week of pregnancy, from day 14.5 to delivery (day 22.5). Rats in the first group had their energy restricted to 50% of their pregnancy standard diet intake. Rats in the second group were energy restricted to 50% of their pregnancy intake but were fed a low-protein diet. Rats in the third group were pair fed to control rats and were fed the low-protein diet. Control rats (the fourth group) were given access to standard diet ad libitum throughout pregnancy and lactation. Subsequently in this article, offspring of mothers in the four groups will be referred to by their mother's diet group: standard diet restricted (CER), low-protein diet restricted (PER), low-protein pair fed (PR), and control (C), respectively. Offspring of mothers in the four groups were nursed by foster mothers fed with standard diet during their own pregnancy. Litters were culled at birth to 10 pups each. On day 28 after birth, all of the offspring were weaned on the standard diet. Females only were studied subsequently. Body weight measurements were taken weekly in pregnant mothers and every 2 wk in the female offspring from birth to the end of the experiment (at 8 wk of age). After feeding on the standard diet for 8 wk, randomly selected animals from each group underwent a glucose tolerance test or the measurement of in vivo insulin action with the glucose-insulin clamp technique. The pancreatic insulin content and the beta -cell mass were also measured in some animals in each group.

Plasma glucose and insulin were determined at 1000 on days 14.5 and 21.5 of pregnancy from blood samples withdrawn from the tail vein. It is important to mention that no major alteration of the feeding pattern took place in the restricted groups, because we have verified that rats had an excess of food available most of the time (e.g., for >= 11 h) during the nocturnal feeding period and that the restricted rats never consumed their daily food ration in one short meal. Because in plasma glucose and insulin measurements performed at 1000 food was withdrawn in the four groups of pregnant rats on the morning of the study shortly after light-cycle onset, one may therefore consider that the duration of subsequent fasting was comparable in the four groups.

In vivo glucose-induced insulin secretion tests. Intravenous glucose tolerance tests (IVGTT) were performed under pentobarbital sodium anesthesia (4 mg/100 g body wt ip) at 1400 in 8-wk-old CER, PR, PER, and C rats that had been fasted from 0900. A single injection of glucose (0.5 g glucose/kg body wt) was administered via a saphenous vein. Blood samples (200 µl) were collected sequentially from the tail vein before (time 0) and 5 (t5), 10 (t10), 20 (t20), and 30 (t30) min after the injection of glucose. They were then centrifuged, and the plasma was separated. Plasma glucose concentration was immediately determined on a 10-µl aliquot, and the remaining plasma was kept at -20°C until radioimmunoassayed for insulin.

Euglycemic-hyperinsulinemic clamp studies. Studies were performed at 1400 in rats fasted from 0900 according to a previously detailed procedure (3, 16). Rats were considered to be in the postabsorptive period, and the rate of glucose production was a measure of endogenous glucose production. Rats were anesthetized with pentobarbital. Body temperature was maintained at 37-38°C with heating lamps. One carotid artery was catheterized for blood sampling, and a tracheotomy was systematically performed to avoid respiratory problems during anesthesia.

Blood samples of 150 µl were collected 20 min after the end of the surgery for the determination of basal blood glucose and plasma insulin concentrations. Then insulin was infused at a constant rate in a saphenous vein, and blood glucose level was clamped at the level measured in the basal state by a variable infusion of glucose through the other saphenous vein with a Precidor pump (Infors, Basel, Switzerland). Insulin [a porcine monocomponent insulin (Actrapid), Novo, Copenhagen, Denmark] was dissolved in 0.9% NaCl containing 0.2% bovine serum albumin (Sigma). Infusion of exogenous glucose (7.5% solution) was started 5 min after insulin infusion. Then 25 µl of blood were sampled from the carotid artery every 5 min, and plasma glucose concentrations were determined within 60 s.

Steady-state plasma insulin levels were reached 30 min after start of the insulin infusion, and steady-state blood glucose levels were reached after 40-45 min. Blood samples (200 µl) were collected at 45, 50, and 55 min to determine blood glucose specific activity and plasma insulin concentrations. Coefficients of variation in plasma glucose and insulin concentrations during the clamp were 5 and 15%, respectively.

Endogenous glucose production. Endogenous glucose production in the basal state and during hyperinsulinemic clamp studies was assessed by a primed-continuous infusion of [3-3H]glucose (New England Nuclear, Dreiech, Germany). Labeled glucose was administered as an initial intravenous priming dose (4 µCi) followed immediately by a continuous intravenous infusion at a rate of 0.2 µCi/min. Steady-state glucose specific activity was established by 40 min in both the basal state and the clamp studies. The rate of glucose appearance (Ra) was then equal to the rate of glucose disappearance (Rd), and these two parameters were calculated by dividing the [3-3H]glucose infusion rate (dpm/min) by the steady-state value of glucose specific activity (dpm/g). In the basal state, the rate of endogenous glucose production is equal to Ra. In clamp studies, the rate of endogenous glucose production was calculated by subtracting the exogenous steady-state glucose infusion rate (SSGIR) from Ra. The rate of glucose utilization by the whole body mass (GUR) was calculated as GUR = Rd, and the glucose production rate (GPR) in the liver was calculated as GPR = Ra - SSGIR.

beta -Cell immunohistochemistry and morphometry. After excision, whole pancreases (three in each restricted and control group) were immediately weighed, fixed in aqueous Bouin's solution overnight, and embedded in paraplast. Each pancreas was subsequently sectioned (7 µm thick) throughout its length, and 10 sections taken at regular intervals (1 every 35 sections) were immunostained for insulin with a technique adapted from the peroxidase indirect labeling method, as previously described (23). The anti-insulin serum was purchased from ICN (65-104-1, ICN Pharmaceutical, Orsay, France); it was raised in the guinea pig against porcine insulin. Labeling was performed using a peroxidase-conjugated rabbit anti-guinea pig IgG (PO141, Dako, Trappes, France). The activity was revealed with a peroxidase substrate kit (Vector SG, Biosys-Vector, Compiègne, France). After staining, sections were mounted in Eukitt. Quantitative evaluation was performed using a computer-assisted image analysis based on an Olympus microscope connected via a color video camera to a Siemens PC computer and using the software Imagenia 2000 (Biocom, les Ulis, France). The area of the insulin-positive cells and the total area of the pancreatic cells were evaluated in each stained section. The beta -cell relative volume was obtained according to stereological methods by calculating the ratio of the area occupied by immunoreactive cells to that occupied by all pancreatic cells. The total beta -cell mass per pancreas was derived by multiplying the beta -cell relative volume by the total pancreatic weight.

Samples, analytic techniques, and calculations. Plasma glucose was determined with a glucose analyzer (Beckman, Palo Alto, CA). Blood samples for measuring glucose specific activity were deproteinized with Ba(OH)2-ZnSO4 and immediately centrifuged. An aliquot of the supernatant was used for determination of glucose by use of a glucose oxidase method. Another aliquot of the supernatant was evaporated to dryness at 60°C to remove tritiated water. The dry residue was dissolved in 0.1 ml of distilled water and counted with 3 ml ReadySolv-MP scintillation solution (Beckman). Immunoreactive insulin in the plasma and pancreases was estimated with purified rat insulin as standard (Novo, Copenhagen, Denmark) and with porcine monoiodinated 125I-labeled insulin (26). Charcoal was used to separate free from bound hormone. The method allows the determination of 2 µU/ml (0.08 ng/ml or 14 pmol/l) with a coefficient of variation within and between assays of 10%.

The insulin and glucose responses during the IVGTT were calculated as the incremental plasma insulin values integrated over the 30-min period after the glucose injection (Delta I, nmol · l-1 · min-1) and the corresponding incremental integrated plasma glucose values (Delta G, mmol · l-1 · min-1). Rd was calculated from the slope of the regression line obtained with the log-transformed plasma glucose values between 5 and 30 min after glucose administration and was expressed as percent per minute.

Statistical analysis. Results are given as means ± SE. Statistical analysis was performed using ANOVA (Fisher's test) for comparison of unpaired data between groups. A P value of 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Evolution of maternal body weight and plasma glucose and insulin during pregnancy. At day 14.5 of pregnancy, body weight and plasma glucose and insulin did not significantly differ among the four groups of mothers that gave birth to similar numbers of pups. In the three experimental groups of rats submitted to food restriction during the last week of pregnancy, the relative variation for body weight was quite different compared with the C group, with a lower weight gain in CER and PR groups, and even a decrease in body weight in the PER group (Table 1). One may note that in the PR group, the whole daily food supply was ingested.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Characteristics of pregnant rats on day 14.5 and during final, restricted week of gestation and of their offspring at birth

At day 21.5 of pregnancy, basal plasma glucose was similar in CER and PR groups, with a significant decrease compared with the C group. It was less decreased in the PER group, and the decrease did not reach significance. However, plasma insulin was more decreased in the PR than in the PER group (not significant), and even more than in the C group (P < 0.01), whereas an increase of insulin level was highlighted in the CER group. Thus the insulin-to-glucose ratio was increased only in the CER group and decreased (to a similar extent) in the PR and PER groups compared with the C group.

Characteristics of the offspring. As shown in Table 1, pups differed in their body weight value according to the diet changes in the last week of pregnancy. The pups from the PR and PER groups had a lower birth weight compared with the C group. The average number of pups per mother was similar in all groups studied. No relationship between offspring and mother body weights could be detected. Nursing the offspring of food-restricted mothers by non-food-restricted mothers immediately after birth corrected the decrease in birth weight within 2 wk. Thus final weights of dams at 8 wk of age did not significantly differ among the four groups.

The characteristics of the female offspring at the age of 8 wk are given in Table 2. Basal plasma glucose measured in the postabsorptive state was not significantly different among the four groups. However, plasma insulin level was found significantly lower in the CER group compared with the other groups.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Body weight, plasma glucose and insulin levels, and results of IVGTT in adult (8-wk) female rats born from mothers that received different protocols of food restriction during last week of pregnancy

Glucose tolerance and insulin secretory response to glucose in the offspring. In response to intravenous glucose load, the mean incremental glucose areas (Delta G) in the CER, PR, and PER rats were not significantly different from those in C rats (Fig. 1 and Table 2). However, a significant difference in Delta G was detected between CER and PR rats (P < 0.02). Rd was higher in CER than in PR and C rats (P < 0.02 and P < 0.01, respectively). Values of the mean incremental insulin areas (Delta I) were increased in PER rats and decreased in CER rats, whereas PR rats exhibited intermediary value and did not differ significantly from C rats. The Delta I-to-Delta G ratio was similar in PR and PER groups; it was significantly increased in PR and PER rats and significantly decreased in CER rats compared with C rats.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Glucose tolerance and plasma insulin response to glucose (0.5 g/kg iv) in adult (8-wk) female rats born from mothers that have been energy restricted (CER, black-triangle), protein restricted (PR, ), protein + calorie restricted (PER, triangle ), or control unrestricted (C, ) during the last week of pregnancy. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. C group; ddager  P < 0.01 vs. PR group; $ P < 0.001 vs. CER group.

In vivo insulin action in the offspring. The basal GPR per rat was significantly lower in the PR group than in the other groups (Table 3). This difference was still significant when the data were expressed per kilogram body weight. These data have to be interpreted in the presence of basal steady-state plasma insulin (SSPI) levels, which were not significantly different among the three restricted groups but were moderately and significantly elevated in the PR and PER rats, respectively, compared with the C levels. After a similar submaximal hyperinsulinemia, GPR was suppressed to the same extent in all groups. When the values were expressed per kilogram body weight, they were higher in PER than in PR rats (P < 0.05), whereas SSPI was lower, although not significantly, in PER than in PR rats. After submaximal hyperinsulinemia, GUR did not differ among groups. The same data could be observed whenever the results were expressed per kilogram body weight. However, one should note that higher and lower insulin infusion rates were needed in CER and PER groups, respectively, to obtain SSPI levels similar in these groups to those in the C group.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Glucose and insulin levels and glucose kinetics during a hyperinsulinemic-euglycemic clamp in adult (8-wk) Wistar female rats born from mothers with differing diets during pregnancy

Pancreatic insulin content and total beta -cell mass in the offspring. The 8-wk-old female PER rats exhibited a pancreas weight significantly higher than that of CER, PR, or C rats (P < 0.001), and the difference still persisted when the values were expressed per kilogram body weight. The insulin content per pancreas was significantly lower in the PR group and higher in the CER and PER groups compared with the C group. Analysis of pancreatic insulin content per gram of body weight showed the same tendency (Table 4).

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Pancreas weight, insulin content, and total pancreatic beta -cell mass in adult (8-wk) female rats born from mothers with differing diets during pregnancy

The relative beta -cell volume was significantly decreased in PR vs. C rats (and CER rats). The total pancreatic beta -cell mass was found higher in PER and CER rats than in PR rats (P < 0.05 and P < 0.02). However, total beta -cell mass values in CER and PER rats were found not significantly different from values in C rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our present data indicate that a low-energy and/or a low-protein diet in the third part of gestation does affect basal plasma glucose and insulin levels in pregnant rats, especially when expressed as relative variation during the restriction period [(day 21.5 - day 14.5)/day 14.5]. Compared with the C group, the relative variation of plasma glucose level was decreased in the CER and PR groups but not in the PER group, whereas the relative variation of the insulin level was increased in the CER group and decreased in the PR and PER groups. At day 21.5, the basal plasma insulin-to-glucose ratio was therefore increased only in the CER group and decreased to a similar extent in the PR and PER groups compared with the C group. This observation suggests that protein deprivation has a specific effect on the adaptive changes in insulin action and/or secretion in the last part of pregnancy. These data also suggest that hormonal compensatory changes occur in PER mother rats to maintain glycemia and nutrient flux despite the severity of the undernutrition attested by maternal weight stagnation. This is also illustrated by the lack of additional decrease in the birth weight of PER pups compared with that of PR pups, whereas the body weights of the mothers were significantly lower.

PR and PER, but not CER, pups had a significant decrease in their birth weight compared with C pups. This argues for a predominant effect of protein restriction over energy restriction on fetal growth retardation. One may retain as important for the interpretation of our data that the CER group exhibits mainly calorie deficiency, with limited associated protein malnutrition, whereas the PR group and the PER group were clearly protein deficient. Wade et al. (32), using diet compositions close to ours, indeed demonstrated that serum albumin level was not significantly affected by a reduction in food consumption as important as 50% after 6 wk (18% protein diet), whereas a 50% food restriction combined with 4% protein in the food significantly decreased serum concentration of albumin.

After birth, the pups of the four groups were submitted to a similar environment, and weight retardation was rapidly (<15 days) remedied in the protein-deprived groups; consequently no significant difference in body weight was detectable among groups at the age of 8 wk. The female offspring of mothers who had been malnourished according to different patterns in the third part of pregnancy acquired limited impairment of their glucose metabolism in adult life. Whatever the adult experimental group, there was no clear impairment of glucose tolerance compared with the C group after an intravenous charge of glucose. A similar conclusion was previously reported when a 65% reduction of daily energy intake was used during the same period of gestation (20). However, the Delta I-to-Delta G ratio was significantly decreased in CER rats and significantly increased to a similar extent in PR and PER rats compared with C rats. This decrease in the CER group could be partly related to the lower basal plasma insulin level observed in this group. Apart from long-term metabolic changes induced by malnutrition, the difference in the Delta I-to-Delta G ratio of PR and PER rats compared with C rats could also result from differential response to the anesthetic procedure. The basal plasma insulin-to-glucose ratio was enhanced by the anesthesia in the three experimental groups but not in the C group (with 1.86-, 1.97-, 1.57-, and 0.97-fold increases, respectively, in CER, PR, PER, and C rats), whereas no difference in its value could be detected between PR and PER rats vs. C rats in the awake state. Therefore, the aforementioned increase in the Delta I-to-Delta G ratio could be explained by a greater reactivity to anesthesia in the protein-restricted groups compared with the C group. As a matter of fact, enhanced reaction to stressful situations and increased basal plasma levels of epinephrine and norepinephrine have been reported in protein undernutrition rat models (15, 18).

In our study, insulin action was not dramatically altered whatever the model of malnutrition used. Comparison of the data related to GUR and GPR in the basal state in the restricted groups suggests that isolated protein restriction could contribute to a moderate but significant decrease in basal GPR and basal GUR. A previous study using protein restriction during gestation and lactation reported similar results in rats studied in the awake state (13). However, in our clamp studies under hyperinsulinemic and euglycemic conditions, analysis of GPR did not highlight any difference in the restricted groups compared with the C group, and no difference in GUR was detectable among the four groups. It is noticeable that a greater insulin infusion rate was needed during the clamp study in the CER group to get a SSPI value similar to that in the other groups. Therefore, an increased clearance of insulin would characterize the CER group. Our data show that the same SSPI as obtained during the clamp studies exerted the same efficiency on whole body GUR in the four groups and that restricted rats had no decrease in glucose utilization. Despite the possible enhanced reactivity of the rats to the anesthetized situation, we believe that extrapolating our data to the unanesthetized situation is nevertheless valid when the three restricted groups of rats are considered, because the three groups exhibited a similar relative change in their plasma insulin-to-glucose ratio from the awake to the anesthetized situation. A previous study based on 50% energy restriction during the entire pregnancy led to the same conclusion and did not show any effect on peripheral glucose utilization in adult female offspring (12).

The whole pancreas development was not dramatically changed despite an increase of the pancreas weight in PER rats (1.25-fold increase), with no impact on beta -cell mass or insulin content per milligram of pancreas. On the other hand, the CER rats got an increase, whereas PR rats got a decrease, in the insulin content expressed in microgram per milligram of pancreas or microgram per gram of body weight. In the absence of obvious change in insulin sensitivity, these data suggest that protein restriction exerts a deleterious effect on beta -cell development during a critical period such as the end of pregnancy. This was further confirmed by the decrease (28%) in beta -cell mass only in the PR group. The relatively higher insulin content found in the CER group without change in beta -cell mass could be explained by a lower beta -cell stimulation, as suggested by a lower basal insulinemia and a lower Delta I-to-Delta G ratio. Therefore, energy restriction (CER group) did not appear to induce negative effects on beta -cell development, at least under our experimental conditions. Previous reports, using more severe energy restriction (65% reduction of daily intake) in the same period, did not show any decrease of beta -cell mass or of pancreatic insulin content at 21 days of pregnancy and in adult life (1, 20). However, a significant and persistent decrease of beta -cell mass and of pancreatic insulin content was found in another study in which the same conditions and 50% energy restriction were used (8). In this work, only pups with the lowest weights were selected and kept for further analysis. This point could explain such discrepancy, because a vasculoplacental defect would have been potentially associated with the effect of maternal malnutrition on its own (7). Other studies have highlighted a persistent decrease in beta -cell mass in the adult offspring of protein-restricted mothers (5), with a lower replication rate of beta -cells and a lower islet vascularization in the fetuses (30) when protein malnutrition was maintained during the entire pregnancy. The decrease in beta -cell mass reported in the aforementioned reference (5) was very similar to what we found in the PR group. This reinforces the previous suggestion (27), that maternal malnutrition exerts long-term adverse effects on insulin secretion only when it is applied during the third part of gestation. Because amino acids are the main stimulus of insulin secretion and beta -cell growth during fetal life, this could explain why protein malnutrition appeared particularly deleterious during this period of development (6). It could imply a defect in differentiation (neogenesis) and/or replication of beta -cells, but the targets of these detrimental effects of malnutrition are presently unknown.

Finally, we would like to point out that some of the minor disturbances here discussed could be aggravated during aging or other physiopathological conditions leading to insulin action impairment (2, 14).

In conclusion, the present study is the first one to investigate in the rat the long-term effects of various fetal malnutrition protocols strictly limited to the third part of pregnancy, which corresponds to the crucial period for fetal rat pancreas development. Under these conditions, protein deficiency, but not energy restriction, induced persistent impairment of the development of the pancreas and especially of beta -cell mass (-28%). On the other hand, glucose utilization and production were only marginally affected in these models of malnutrition. These changes did not induce alterations of glucose tolerance in the adult under standard physiological conditions. However, their implication as diabetes-predisposing situations acting synergistically with other deleterious environmental and/or genetic conditions cannot be excluded.


    ACKNOWLEDGEMENTS

This work was partly supported by a grant from the Ministre de l'Education Nationale, de l'Enseignement Suprieur et de la Recherche (95-G-0103; programme interministériel "Aliment Demain").


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: E. Bertin, Laboratoire de Physiopathologie de la Nutrition, CNRS-ESA 7059, Université Paris 7/D. DIDEROT, Tour 33-43, 1er étage, 2 place Jussieu; 75251 Paris Cedex 05, France (E-mail: ebertin{at}chu_reims.fr).

Received 12 August 1998; accepted in final form 15 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alvarez, C., M. A. Martin, L. Goya, E. Bertin, B. Portha, and A. M. Pascual-Leone. Contrasted impact of maternal rat food restriction on the fetal endocrine pancreas. Endocrinology 138: 2267-2273, 1997[Abstract/Free Full Text].

2.   Berthelier, C., M. Kergoat, and B. Portha. Lack of deterioration of insulin action with aging in the GK rat: a contrasted adaptation as compared with nondiabetic rats. Metabolism 46: 890-896, 1997[Medline].

3.   Blondel, O., D. Bailbe, and B. Portha. Relation of insulin deficiency to impaired insulin action in NIDDM adult rats given streptozotocin as neonates. Diabetes 38: 610-617, 1989[Abstract].

4.   Cook, J. T. E., J. C. Levy, R. C. L. Page, J. A. G. Shaw, A. T. Hattersley, and R. C. Turner. Association of low birth weight with cell function in the adult first degree relatives of non-insulin dependent diabetic subjects. Br. Med. J. 306: 302-306, 1993[Medline].

5.   Dahri, S., A. Snoeck, B. Reusens-Billen, C. Remacle, and J. J. Hoet. Islet function in offspring of mothers on low-protein diet during gestation. Diabetes 40, Suppl. 2: 115-120, 1991[Medline].

6.   De Gasparo, M., G. R. Milner, P. D. Norris, and R. D. G. Milner. Effect of glucose and amino-acids on fetal rat pancreatic growth and insulin secretion in vitro. J. Endocrinol. 77: 241-248, 1978[Medline].

7.   De Prins, F. D., and F. A. Van Assche. Intra-uterine growth retardation and development of endocrine pancreas in the experimental rat. Biol. Neonate 41: 16-21, 1982[Medline].

8.   Garofano, A., P. Czernichow, and B. Brant. In utero undernutrition impairs rat beta-cell development. Diabetologia 40: 1231-1234, 1997[Medline].

9.   Gluckman, P., and J. Harding. The regulation of fetal growth. In: Human Growth, Basic and Clinical Aspects, edited by M. Hernandez, and J. Argente. Amsterdam: Elsevier, 1992, p. 253-286.

10.   Hales, C. N., D. J. P. Barker, P. M. S. Clark, L. J. Cox, C. Fall, C. Osmond, and P. D. Winter. Fetal and infant growth and impaired glucose tolerance at age 64. Br. Med. J. 303: 1019-1022, 1991[Medline].

11.   Hellerstrom, C., and I. Swenne. Functional maturation and proliferation of fetal pancreatic beta -cells. Diabetes 40, Suppl. 2: 89-93, 1991[Medline].

12.   Holemans, K., J. Verhaeghe, J. Dequeker, and F. A. Van Assche. Insulin sensitivity in adult female rats subjected to malnutrition during the perinatal period. J. Soc. Gynecol. Invest. 3: 71-77, 1996[Medline].

13.   Holness, M. J. Impact of early growth retardation on glucoregulatory control and insulin action in mature rats. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E946-E954, 1996[Abstract/Free Full Text].

14.   Holness, M. J., and M. C. Sugden. Suboptimal protein nutrition in early life later influences insulin action in pregnant rats. Diabetologia 39: 12-21, 1996[Medline].

15.   Keller, E. A., G. R. Cuadra, V. A. Molina, and O. A. Orsingher. Perinatal undernutrition affects brain modulatory capacity of beta -adrenergic receptors in adult rats. J. Nutr. 120: 305-308, 1990[Medline].

16.   Kergoat, M., and B. Portha. In vivo hepatic and peripheral insulin sensitivity in rats with non-insulin-dependent diabetes induced with streptozotocin. Assessment with the insulin-glucose clamp technique. Diabetes 34: 574-579, 1985[Abstract].

17.   Léger, J., C. Levy Marchal, J. Bloch, A. Pinet, D. Chevenne, D. Porquet, D. Collin, and P. Czernichow. Reduced final height and indications for insulin resistance in 20 year olds born small for gestational age: regional cohort study. Br. Med. J. 315: 341-347, 1997[Abstract/Free Full Text].

18.   Leon-Quinto, T., P. Adnot, and B. Portha. Alteration of the counterregulatory hormones in the conscious rat after protein-energy restriction. Diabetologia 40: 1028-1034, 1997[Medline].

19.   Lithell, H. O., P. M. McKeigue, L. Berglund, R. Mohsen, U.-B. Lithell, and D. A. Leon. Relation of size at birth to non-insulin dependent diabetes and insulin concentrations in men aged 50-60 years. Br. Med. J. 312: 406-410, 1996[Abstract/Free Full Text].

20.   Martin, M. A., C. Alvarez, L. Goya, B. Portha, and A. M. Pascual-Leone. Insulin secretion in adult rats that had experienced different underfeeding patterns during their development. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E634-E640, 1997[Abstract/Free Full Text].

21.   McCance, D. R., D. J. Pettitt, R. L. Hanson, L. T. H. Jacobsson, W. C. Knowler, and P. H. Bennett. Birth weight and non-insulin dependent diabetes: thrifty genotype, thrifty phenotype, or surviving small baby genotype? Br. Med. J. 308: 942-945, 1994[Abstract/Free Full Text].

22.   Metzger, B. E. Biphasic effects of maternal metabolism on fetal growth. Diabetes 40, Suppl. 2: 99-105, 1991[Medline].

23.   Michel, C., J. Chariot, M. Souchard, and C. Roze. Modifications of the endocrine pancreas in rats after ethionine destruction of acini. Cell. Mol. Biol. 28: 135-148, 1982[Medline].

24.   Phillips, D. I. W., D. J. P. Barker, C. N. Hales, S. Hirst, and C. Osmond. Thinness at birth and insulin resistance in adult life. Diabetologia 37: 150-154, 1994[Medline].

25.   Phipps, K., D. J. P. Barker, C. N. Hales, C. H. D. Fall, C. Osmond, and P. M. S. Clark. Fetal growth and impaired glucose tolerance in men and women. Diabetologia 36: 225-228, 1993[Medline].

26.   Picarel-Blanchot, F., C. Alvarez, D. Bailbé, A. M. Pascual-Leone, and B. Portha. Changes in insulin action and insulin secretion in the rat after dietary restriction early in life: influence of food restriction versus low-protein food restriction. Metabolism 44: 1519-1526, 1995[Medline].

27.   Portha, B. Development of the pancreatic beta -cells: growth pattern and functional maturation. In: Endocrine and Biochemical Development of the Fetus and Neonate, edited by J. M. Cuezva, A. M. Pascual-Leone, and M. S. Patel. New York: Plenum, 1990, p. 33-43.

28.   Portha, B., M. Kergoat, O. Blondel, and D. Bailbé. Underfeeding of rat mothers during the first two trimesters of gestation does not alter insulin action and insulin secretion in the progeny. Eur. J. Endocrinol. 133: 475-482, 1995[Medline].

29.   Poulsen, P., A. A. Vaag, K. O. Kyvik, D. Moller-Jensen, and H. Beck-Nielsen. Low birth weight is associated with NIDDM in discordant monozygotic and dizygotic twin pairs. Diabetologia 40: 439-446, 1997[Medline].

30.   Snoeck, A., C. Remacle, B. Reusens, and J. J. Hoet. Effect of a low-protein diet during pregnancy on the fetal rat endocrine pancreas. Biol. Neonate 5: 107-118, 1990.

31.   Van Assche, F. A., and L. Aerts. The fetal endocrine pancreas. Contrib. Gynecol. Obstet. 5: 44-57, 1979[Medline].

32.   Wade, S., F. Bleiberg-Daniel, B. Le Moullac, D. Iyakaremye, D. Biou, F. Gauthier, and D. Lemonnier. Value of serum transthyretin measurements in the assessment of marginal protein-energy malnutrition in rats. J. Nutr. 118: 1002-1010, 1988[Medline].


Am J Physiol Endocrinol Metab 277(1):E11-E17
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society