am j physiol endocrinol metab 2004 martín.pdf

doi: 10.1152/ajpendo.00242.2003286:E542-E550, 2004. First published 9 December 2003;Am J Physiol Endocrinol Metab

M. A. Martín, E. Fernández, A. M. Pascual-Leone, F. Escrivá and C. Alvarezrat endocrine pancreasmetabolism and insulin gene expression in fetal and adult Protein calorie restriction has opposite effects on glucose

You might find this additional info useful...

37 articles, 16 of which you can access for free at: This article citeshttp://ajpendo.physiology.org/content/286/4/E542.full#ref-list-1

3 other HighWire-hosted articles: This article has been cited by http://ajpendo.physiology.org/content/286/4/E542#cited-by

including high resolution figures, can be found at: Updated information and serviceshttp://ajpendo.physiology.org/content/286/4/E542.full

can be found at: MetabolismAmerican Journal of Physiology - Endocrinology and about Additional material and information

http://www.the-aps.org/publications/ajpendo

This information is current as of November 25, 2012.

Physiological Society. ISSN: 0193-1849, ESSN: 1522-1555. Visit our website at http://www.the-aps.org/. American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2004 by Americanendocrine and metabolic systems on any level of organization. It is published 12 times a year (monthly) by the

publishes results of original studies aboutAmerican Journal of Physiology - Endocrinology and Metabolism

by guest on Novem

ber 25, 2012http://ajpendo.physiology.org/

Dow

nloaded from

http://ajpendo.physiology.org/content/286/4/E542#cited-by

http://ajpendo.physiology.org/

Protein calorie restriction has opposite effects on glucose metabolismand insulin gene expression in fetal and adult rat endocrine pancreas

M. A. Martın,* E. Fernandez,* A. M. Pascual-Leone, F. Escriva, and C. AlvarezInstituto de Bioquımica, Centro Mixto: Consejo Superior de Investigaciones Cientıficas-UniversidadComplutense, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain

Submitted 29 May 2003; accepted in final form 1 December 2003

Martın, M. A., E. Fernandez, A. M. Pascual-Leone, F. Escriva,and C. Alvarez. Protein calorie restriction has opposite effects onglucose metabolism and insulin gene expression in fetal and adult ratendocrine pancreas. Am J Physiol Endocrinol Metab 286:E542–E550, 2004. First published December 9, 2003; 10.1152/ajpendo.00242.2003.—We previously demonstrated that fetuses fromundernourished pregnant rats exhibited increased �-cell mass andhyperinsulinemia, whereas keeping food restriction until adult agecaused reduced �-cell mass, hypoinsulinemia, and decreased insulinsecretion. Because these alterations can be related to insulin availabil-ity, we have now investigated early and long-term effects of proteincalorie food restriction on insulin mRNA levels as well as the possiblemechanisms that could modulate the endogenous insulin mRNAcontent. We used fetuses at 21.5 days of gestation proceeding fromfood-restricted rats during the last week of pregnancy and 70-day-oldrats undernourished from day 14 of gestation until adult age and withrespective controls. Insulin mRNA levels, glucose transporters, andtotal glycolysis and mitochondrial oxidative fluxes were evaluated.We additionally analyzed undernutrition effects on signals implicatedin glucose-mediated insulin gene expression, especially pancreaticduodenal homeobox-1 (PDX-1), stress-activated protein kinase-2(p38/SAPK2), and phosphatidylinositol 3-kinase. Undernourished fe-tuses showed increased insulin mRNA, oxidative glucose metabolism,and p38/SAPK2 levels, whereas undernutrition until adult age pro-voked a decrease in insulin gene expression, oxidative glucose me-tabolism, and PDX-1 levels. The results indicate that food restrictioncaused changes in insulin gene expression and content leading toalterations in glucose-stimulated insulin secretion. The molecularevents, increased p38/SAPK2 levels in fetuses and decreased PDX-1levels in adults, seem to be the responsible for the altered insulinmRNA expression. Moreover, because PDX-1 activation appears tobe regulated by glucose-derived metabolite(s), the altered glucoseoxidation caused by undernutrition could in some manner affectinsulin mRNA expression.

glucose metabolism; insulin messenger ribonucleic acid; pancre-atic duodenal homeobox-1 levels; insulin synthesis; insulin secre-tion

IT IS WIDELY KNOWN that dietary influences during early stages ofdevelopment present a risk factor for the onset of both perinataland later-life diseases. As indicated by the “thrifty phenotypehypothesis” (21), the endocrine pancreas may be particularlysusceptible to the effects of poor maternal nutrition, since fetaland postnatal periods are critical for �-cell development andmaturation of pancreatic function. In this way, it has beenshown that isocaloric low-protein diets throughout pregnancy

impair fetal endocrine pancreas development (35) and thesecretory response in pancreatic islets of fetal (12) and adult(33) animals. Similarly, maternal general food restriction inlate pregnancy also leads to alterations in �-cell development.Both decreases (5, 16) and increases (2) in pancreatic insulinstores and �-cell mass have been described in fetuses fromfood-restricted mothers, whereas in the neonatal period asignificant and persistent decrease in these parameters has beenfound at days 1 (16) and 4 (26) of postnatal life. This effectpersists until adulthood (26) and can provoke long-lastingconsequences in the endocrine pancreas under situations ofincreased insulin demand, such as aging (17) and pregnancy(6). Consequently, experiments with animal models have pro-vided enough evidence that nutritional intervention leads todifferent adaptations at cellular (16, 35), biochemical (4, 33,41), and molecular levels (3). The result indicates a persistentalteration of �-cell mass and several defects in insulin responsein food restricted animals.

Impaired insulin secretion in undernutrition has been relatedto alterations at different steps in the secretion mechanismitself (4, 33, 41). However, changes in �-cell mass and ininsulin release may also result from alterations in insulinbiosynthesis, as previously described in models of low-proteindiets (3, 35). Long-term regulation of insulin secretion is thencontrolled by the hormone availability and the �-cell ability toproduce it.

Insulin synthesis in �-cells is regulated by nutrients, amongwhich glucose is physiologically the most significant (40).However, mechanisms through which glucose regulates insulingene expression are not as well understood as those that controlinsulin secretion. Over short periods of time, glucose regulatesinsulin biosynthesis mainly by increasing the translation ofinsulin mRNA; but over longer periods, glucose increasesinsulin mRNA levels (13). An increase in glucose metabolismgenerates a sequence of secondary-stimulus coupling factors,which in turn activate downstream signaling elements influ-encing both insulin gene expression and insulin mRNA trans-lation (31). The homeodomain pancreatic duodenal ho-meobox-1 (PDX-1) is an important transcription factor in�-cells that seems to play an essential role in linking glucosemetabolism with insulin gene transcription (28). PDX-1 DNA-binding activity and insulin promoter activity are modulatedvia a signaling pathway involving stress-activated protein ki-nase-2 (p38/SAPK2) (27). Although p38/SAPK2 seems to beactivated in response to adverse stimuli and under stressconditions (29), it has also been shown that glucose metabo-

*These authors contributed equally to the article as first authors.Address for reprint requests and other correspondence: C. Alvarez, Depar-

tamento de Bioquımica y Biologıa Molecular, Facultad de Farmacia, Univer-sidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain (E-mail:[email protected]).

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

Am J Physiol Endocrinol Metab 286: E542–E550, 2004.First published December 9, 2003; 10.1152/ajpendo.00242.2003.

0193-1849/04 $5.00 Copyright © 2004 the American Physiological Society http://www.ajpendo.orgE542

by guest on Novem


Dow

nloaded from


lism is able to modulate the activity of p38/SAPK2 through acell-signaling pathway that implicates phosphatidylinositol3-kinase (PI 3-kinase) (42).

Using a rat model based on a protein calorie deficiency [65%reduction in daily food intake (14)], we previously establishedthat �-cell mass and insulin secretion increase in fetuses at 21.5days of gestation (2) and decrease in 70-day-old adult rats (26).However, the biochemical and molecular adaptations respon-sible for the alterations we found are currently unknown.Therefore, in this study, we have investigated the early andlong-term effects of protein calorie food restriction on insulinmRNA levels as well as the possible mechanisms that couldmodulate the endogenous insulin mRNA content. For thispurpose, glucose transporters (GLUT2 and GLUT1) as well astotal glycolysis and mitochondrial oxidative fluxes were eval-uated. Moreover, we also analyzed the undernutrition effectson signals implicated in glucose-mediated insulin gene expres-sion, especially PDX-1, p38/SAPK2, and PI 3-kinase.

MATERIAL AND METHODS

Animals and diets. Wistar rats bred in our laboratory under acontrolled temperature and artificial dark-light cycle (from 0700 to1900) were used throughout the study. Females were caged withmales, and mating was confirmed by the presence of spermatozoa ina vaginal smear. Animals were fed a standard laboratory diet (19 gprotein, 56 g carbohydrate, 3.5 g lipid, 4.5 g cellulose/100 g plus saltand vitamin mixtures). In both the fetal and adult periods, foodrestriction was established beginning on day 14 of pregnancy. In thefetal period, the pregnant undernourished group received 35% of thefood intake of a pregnant control group until 21.5 days of pregnancy.Then, mothers were put under intraperitoneal pentobarbital sodiumanesthesia (4 mg/100 g body wt), and fetal blood was obtained afteraxillary artery incision of fetuses while still connected to the maternalcirculation; serum was separated and stored frozen at �20°C untilanalyzed. Fetuses were individually weighed, and pancreases weredissected, weighed, and extracted for determination of insulin content(see Insulin content and insulin release from isolated pancreaticislets). In the adult period, rats were 65% food restricted during thelast week of gestation, and the food restriction was extended duringthe suckling and adult period until 70 days of life. At this time, the ratswere decapitated. Serum was separated and stored at �20°C untilanalyzed. The pancreas was dissected, weighed, and extracted fordetermination of its insulin content. Control rats in both periods weregiven access to food ad libitum. Water was available ad libitum to allgroups. Food intake of control and undernourished rats has beenpreviously reported (14).

All studies were conducted according to the principles and proce-dures outlined in the National Institutes of Health Guidelines for Careand Use of Experimental Animals.

Fetal islet culture and isolation. Islets from fetal rats (21.5 days ofgestation) were obtained according to the method of Hellerstrom et al.(23). Briefly, 20–24 pancreases were minced in sterile Hanks’ solu-tion. The fragments were transferred to a sterile vial containingHanks’ solution supplemented with 5–7 mg of collagenase (Boehr-inger Mannheim, Mannheim, Germany). The vial was shaken for 10min at 37°C, and the tissue digest was washed three times with Hanks’solution. The pellet was resuspended in culture medium and trans-ferred to 4–6 plastic dishes containing the same medium. The culturemedium consisted of RPMI 1640 (ICN, Nuclear Iberica, Madrid,Spain) with 200 mM L-glutamine, penicillin, and streptomycin and10% fetal bovine serum (ICN). The islets were maintained at 37°C inan atmosphere of 5% CO2 and the medium was renewed every 48 h.After 5–6 days in culture, the islets were gently detached from theplates, and clean islets were individually transferred under a dissectingmicroscope and immediately used for experiments. Reversal effects in

the islets after 8 days of culture can be disregarded, since all condi-tions were compared with control plates cultured for the same period.

Adult islet isolation. Islets of 70-day-old rats from each group wereisolated by the collagenase digestion procedure of Malaisse-Lagaeand Malaisse (25). Briefly, islets were isolated from the pancreases oftwo or three rats and subsequently separated from the remainingexocrine tissue by hand-picking under a dissecting microscope. Theislets were immediately used for experiments. Collagenase fromClostridium histolyticum was purchased from Boehringer Mannheim.Hanks’ solution saturated with O2-CO2 (95:5%) was used during theisolation procedure.

Insulin content and insulin release from isolated pancreatic islets.For the study of �-cell secretory function, islets in groups of sevenwere incubated for 90 min at 37°C in plastic microbeakers placed insealed glass vials and containing 1.0 ml of Krebs-Ringer bicarbonatemedium (24) equilibrated against a mixture of 95% O2-5% CO2 andsupplemented with 5.0 mg/ml BSA (fraction V; Sigma Chemical, St.Louis, MO) and one of the following: 2.8 or 16.7 mM glucose. At theend of the incubation period, aliquots of the medium were stored at�20°C until assayed for insulin by use of the RIA described below.

Groups of 20 freshly isolated islets were sonicated in acid-ethanol(1.5 ml 12 M HCl/100 ml ethanol) and stored at �20°C for determi-nation of insulin content, which was measured by RIA as describedbelow.

RNA isolation and Northern blot analysis. RNA was extracted frompancreas obtained from control and undernourished fetuses and adultrats by the guanidinium isothiocyanate-phenol-chloroform method(11). After quantification, total RNA (30 �g in fetuses and 40 �g inadults) was submitted to Northern blot analysis following the methodpreviously described (38). Insulin cDNA was kindly provided by S. J.Chan [Howard Hughes Medical Institute, Univ. of Chicago (10)] andused as probe. pRat ins1 contains a 399-bp EcoRI-HindIII fragmentencoding a preproinsulin I cDNA inserted into a pGEMHZ plasmid.Membranes were autoradiographed, and an 18S probe was used ascontrol for RNA loading. Relative densities of signals were deter-mined by densitometric scanning of the autoradiograms in a laserdensitometer (Molecular Dynamics, Sunnyvale, CA).

Western blot analyses for GLUTs. Groups of 300–500 islets percondition were frozen in liquid nitrogen and kept at �80°C untilanalyzed. From GLUT2 and GLUT1 determination the islets weresonicated in 3% SDS, 120 mM Tris�HCl, pH 6.8, 7.5 mM EDTA, 5%2-mercaptoethanol, 1.5 mM PMSF, and 15% glycerol. Equivalentamounts of protein were subjected to SDS-polyacrylamide gel elec-trophoresis on 10% polyacrylamide gels at 125 V. Proteins were thenelectrophoretically transferred to polyvinylidene difluoride (PVDF)filters (PVDF Protein Sequencing Membrane, Bio-Rad Laboratories,Madrid, Spain) at 100 V for 2 h. After transfer, the filters wereblocked with 5% (wt/vol) nonfat dry milk in phosphate-bufferedsaline for 2 h at room temperature. Antibodies against the GLUT2 andGLUT1 were purchased from Biogenesis (Dorset, UK) and were usedat dilutions of 1:1,000 and 1:5,000, respectively. The PVDF filterswere then washed four times for 10 min at 37°C with phosphate-buffered saline with 0.1% Tween 20 followed by a 1-h incubationwith goat anti-rabbit immunoglobulin G conjugated to horseradishperoxidase (Sigma BioSciences, St. Louis MO). The PVDF mem-branes were then washed as indicated above. Detection of antibody-antigen complexes was accomplished by the enhanced chemiluminis-cence method (BM Chemiluminiscence, Boehringer Mannheim). Op-tical densities of bands corresponding to specific glucose transporterswere determined by laser scanning densitometry (Molecular Dynam-ics). Immunoblots were performed under conditions of linearity ac-cording to the amount of protein loaded on the gel. The PVDF filterswere finally stained with Coomassie blue to confirm that in the sameWestern assay equal amounts of protein were analyzed as well as toensure the heterogenity of the protein composition pattern in thedifferent samples.

E543UNDERNUTRITION EFFECT ON RAT ENDOCRINE PANCREAS

AJP-Endocrinol Metab • VOL 286 • APRIL 2004 • www.ajpendo.org

by guest on Novem


Dow

nloaded from


Islet glucose metabolism. Glucose utilization was based on theformation of 3H2O from D-[5-3H]glucose, and oxidation of glucosewas measured as 14CO2 production from D-[6-14C]glucose. Themethod used to determine simultaneously the conversion of D-[5-3H]glucose into 3H2O and D-[6-14C]glucose into 14CO2 has beenreported elsewhere (24). For measurement of glucose utilization andoxidation, groups of 20 islets each were incubated in 40 �l ofbicarbonate-buffered medium (23) containing BSA (5 mg/ml),HEPES (10 mM), and the appropriate D-glucose concentration with atracer amount of both D-[5-3H]glucose and D-[6-14C]glucose (40�Ci/ml and 10 �Ci/ml, respectively; Amersham Pharmacia). Theincubation was at 37°C for 120 min. The islet metabolism was stoppedby the addition of 20 �l of a citrate-NaOH buffer (400 mM, pH 4.9)containing antimycin A (0.01 mM), rotenone (0.01 mM), and KCN(5.0 mM). The 14CO2 was trapped in 0.250-ml hyamine hydroxide(Packard) over a 60-min incubation period at 20°C. After a 20- to 22-hincubation at 20°C, the recovery of 3H2O formed by the islets wasdetermined by liquid scintillation counting (1209 Rackbeta; LKBWallac, Turku, Finland).

The values obtained concerning glucose use were corrected inaccordance with the recovery of 3H2O from known amounts of 3H2O,which in these experiments was 35 � 1.1%.

Determination of PDX-1, total p85, and p38/SAPK2. The contentof PDX-1 and the p85 subunit of PI 3-kinase and p38/SAPK2 wereanalyzed by Western blot. Protein extracts were obtained from isletssonicated in a homogenization buffer (10 �M leupeptin, 2 mMO-vanadate, 2 mM benzamidine, 10 �g/ml aprotinin, and 2 mMphenylmethylsulfonyl fluoride in 12.5 mM EGTA, 1.25 mM EDTA,and 0.25% Triton X-100, pH 7.6). Equal amounts of protein wereseparated on a 10% SDS-polyacrylamide gel. Proteins were thenelectrophoretically transferred to PVDF filters and probed with thedifferent antibodies: polyclonal anti-rat PDX-1 (from Chemicon, Te-mecula, CA), polyclonal anti-rat p85 subunit of PI 3-kinase (fromUpstate Biotechnology, Lake Placid, NY), and polyclonal anti-ratp38/SAPK2 (from Santa Cruz Biotechnology, Santa Cruz, CA). Therest of the Western blot procedure was as described for GLUTdeterminations, with the following antibody dilutions: anti-PDX-1,1:10,000; anti-p85, 1:2,000; and anti-p38, 1:1,000.

Other analytical procedures. The concentration of protein wasdetermined by the Bradford method (7) using a protein assay (Bio-Rad Laboratories) with gamma-globulin as standard. Immunoreactiveinsulin in serum samples and islet extracts was determined by RIAusing rat insulin as standard (INCSTAR, Stillwater, MN). Thismethod allows the determination of 2.0 ng/ml, with coefficients ofvariation within and between assays of 10%. Aliquots of 10 �lobtained from 30 �l of Ba(OH)2-ZnSO4 deproteinized blood wereused to determine glucose by a glucose oxidase method (BoeringerMannheim).

Statistics. Values are given as means � SE for the number of ratsstudied. A two-tailed t-test for independent observations was used forcomparisons of the two groups. For all other data comparisons, aone-way analysis of variance (ANOVA), followed by the protectedleast significant difference test, was used.

RESULTS

Characteristics of undernourished and control rats. Foodrestriction of pregnant rats during the 3rd wk of gestationprovoked a significant decrease in body and pancreas weight intheir fetuses compared with those in controls. No change inglycemia was found in undernourished fetuses, but a signifi-cant increase in plasma insulin was observed in this groupcompared with controls. In accord with this increase, the isletinsulin content was significantly higher in fetuses from under-nourished rats (Table 1).

Adult rats food restricted from day 14 of gestation until 70days of life showed a significant decrease in body and pancreasweight compared with controls. No significant difference wasfound in glycemia between the two groups, whereas insuline-mia and islet insulin content were significantly lower in un-dernourished than in control adult rats (Table 1).

Islet insulin secretion. In agreement with the higher insu-linemia and islet insulin content found in undernourishedfetuses, the output of insulin was significantly higher than incontrols, both in basal (2.8 mM) and in stimulating (16.7 mM)conditions. However, insulin release in response to an increasein glucose concentration from 2.8 to 16.7 mM was significantlylower in undernourished adult rats compared with the controlgroup (Table 2).

To compare the relative levels of insulin secretion, theinsulin secretion rate was normalized by the islet insulincontent. On this basis, fractional insulin secretion was similarin fetal and adult food-restricted and control rats, indicatingthat undernutrition affects insulin content but not the mecha-nism of secretory response (Table 2).

Steady-state insulin mRNA levels. In accord with islet insulincontent in undernourished fetuses, insulin mRNA levels wereelevated fourfold compared with the control group. Similarly,the reduction in islet insulin content in undernourished adultrats was accompanied by a reduction in insulin mRNA levelscompared with controls (Fig. 1).

GLUT2 and GLUT1 protein levels. Islet GLUT2 andGLUT1 protein content was measured by Western blot analysisof homogenates of islets isolated from control and undernour-ished fetal and adult rats (Fig. 2). The main peptide bandobserved had an approximate molecular mass of 45 kDa, whichis in close agreement with the estimated molecular mass of thetransporters. GLUTs consistently did not appear as a singleband in freshly isolated adult islets. This represents proteolyticdegradation of GLUTs occurring during the isolation proce-dure and could not be prevented by addition of proteaseinhibitors to the collagenase solution (37). For this reason, the

Table 1. Characteristics of fetuses at 21.5 days of gestation from control or undernourished mothersand control or undernourished 70-day-old rats

Fetuses Adults

C U C U

Body weight, g 5.04�0.05 4.17�0.09* 274.5�2.0 121.4�7.1*Pancreas weight, mg 20.01�0.43 17.51�0.53* 860.4�27.1 475.1�29.3*Glycemia, mg/100 ml 51.4�3.1 52.1�1.7 80.1�2.1 81.3�1.6Insulinemia, �U insulin/ml 171.3�10.2 218.1�9.7* 52.3�5.1 43.9�3.6*Islet insulin content, �U insulin/islet 1,541�93 3,144�117* 2,366�106 1,610�156*

Values are means � SE. Number of data, 10–12. *P � 0.05 relative to control rats. C, control; U, undernourished.

E544 UNDERNUTRITION EFFECT ON RAT ENDOCRINE PANCREAS


by guest on Novem


Dow

nloaded from


sum of all bands was considered for the densitometric analysis.This effect was not observed in the fetus, because islets wereobtained directly from culture 6 days after collagenase extrac-tion.

The densitometric analysis of data from five independentdeterminations (Fig. 2) revealed that the levels of GLUT2 andGLUT1 were similar in control and undernourished animals inboth adult and fetal stages.

Glucose metabolism in pancreatic islets. Figure 3 shows therate of glucose utilization and oxidation in islets of undernour-ished fetuses. The oxidation in the tricarboxylic acid (TCA)cycle of acetyl residues derived from exogenous D-glucose wasmeasured by the generation of 14CO2 from D-[6-14C]glucose,and glucose utilization was determined on the formation of

3H2O from D-[5-3H]glucose. At a low concentration of glucose(2.8 mM) the rate of glucose oxidation was not significantlydifferent in both fetal populations, but at a higher concentration(16.7 mM) it was much lower in the control fetuses than infetuses from undernourished mothers. At 16.7 mM, the rate ofglucose oxidation in fetuses from undernourished mothersincreased 2.5-fold, whereas in fetuses from control mothersthis increase was only 1.5-fold. In both fetal populations, therate of glucose utilization increased significantly at 16.7 mMglucose (�2.0-fold in undernourished fetal group and 1.9-foldin control fetuses). No major impairment of glycolytic flux wasfound in islets from undernourished fetuses compared withcontrol.

To investigate the relationship between glucose utilizationand oxidation, the production of 3H2O from D-[5-3H]glucosewas measured together with the generation of 14CO2 fromD-[6-14C]glucose. Fetuses from undernourished mothersshowed a preferential stimulation of glucose oxidation relativeto total glycolytic rate, as indicated by the increase in thepaired ratio found in this group when the glucose concentrationrose from 2.8 to 16.7 mM. However, this fact was not observedin the fetal control group, where the paired ratio remainedunchanged.

The rate of glucose utilization and oxidation in control andundernourished adult rats is shown in Fig. 4. Glucose oxidationwas much lower in islets from undernourished adult rats thanin controls at both low and high glucose concentrations. Inter-estingly, the increase of glucose concentration to 16.7 mMevoked a similar increase of the rate of glucose oxidation inboth adult populations (�4.6-fold in control and 4.7-fold inundernourished rats). In undernourished adult rats, the rate ofglucose utilization at the two concentrations of the hexose (2.8and 16.7 mM) was similar to that of controls. Raising themedium glucose concentration from 2.8 to 16.7 mM signifi-cantly increased the rate of glucose utilization in both adultpopulations, control and undernourished, 3.0- and 3.2-fold,respectively. Therefore, no major impairment of glycolytic fluxwas observed in islets from adult undernourished rats.

At 16.7 mM, the paired ratio between glucose oxidation andglucose utilization was much lower in islets from undernour-ished adult rats. The paired ratio at 16.7 mM was higher thanat 2.8 mM only in control adult populations. This fact indicatesthat a rise in glucose concentration (from 2.8 to 16.7 mM)stimulates the oxidation of acetyl residues in the TCA cyclerelative to total glycolytic rate in islets from control adults butnot in islets from undernourished adult rats.

Islet content of PDX-1, total p85, and p38/SAPK2. Thelevels of PDX-1, total p85, and p38/SAPK2 were evaluated in

Table 2. Insulin secretion and insulin secretion rate in islets from control and undernourished fetal and adult rats

Fetuses Adults

C U C U

Insulin release, �U insulin�islet�1 90 min�1

Glucose/2.8 mM 13.6�0.7 20.1�1.6* 19.2�0.2 18.4�0.8Glucose/16.7 mM 23.1�4.1† 55.2�3.9*† 673.2�24.3† 490.4�16.0*†

Insulin release, % islet insulin contentGlucose/2.8 mM 1.24�0.04 1.18�1.6 0.8�0.02 1.0�0.03Glucose/16.7 mM 2.85�0.07 3.13�0.12*† 27.4�1.3 29.0�1.4

Values are means � SE. Number of data, 12–14. *P � 0.05 relative to control rats. †P � 0.05 relative to glucose 2.8 mM within the same group.

Fig. 1. Representative Northern blot of pancreatic insulin mRNA as well as18S rRNA from control (C) and undernourished (U) fetal (A) and adult (B) rats.Thirty micrograms in fetuses and 40 �g in adults of total mRNA were loadedin each lane. Densitometric analysis of Northern blot data was corrected forloading with 18S rRNA. Results represent means � SE for 6 independentexperiments. FP � 0.05 relative to control rats.



by guest on Novem


Dow

nloaded from


islets from control and undernourished fetal and adult rats. Theresults showed that the islet levels of PDX-1 were similar incontrol and undernourished fetuses, whereas islet p38 contentwas increased by food restriction (1.9-fold over control value)in the fetal period (Fig. 5). In contrast, the amount of PDX-1 inthe islets from undernourished adult animals was threefolddecreased, and no diferences were found in the same period inthe levels of p38/SAPK2 (Fig. 6). Moreover, food restrictiondid not alter the islet content of total p85 regulatory subunitprotein of PI 3-kinase either in fetal or in adult period.

DISCUSSION

In a previous work (2), we demonstrated that a 65% proteincalorie food restriction during the 3rd wk of gestation provokedin pregnant rats impaired insulin secretion in vivo and glucoseintolerance at the end of gestation. Moreover, this disturbanceof maternal glucose homeostasis in this model led to anincrease in �-cell mass and hyperinsulinemia in the fetuses at21.5 days of gestation. However, from 4 days of life, concor-dant with the literature (16), we observed decreased �-cellmass and hypoinsulinemia, effects that persisted until adult age(26). Under these conditions, our experimental model showsdifferent responses depending on whether fetal or adult stagesare considered. Nevertheless, both periods have in common theexistence of alterations at the cellular level and defects ininsulin secretion as a consequence of the nutritional interven-tion. The present study shows how changes at the metabolicand molecular levels seem to be involved in alterations that wefound in insulin secretion in the two stages.

The hyperinsulinemia observed in undernourished fetusesappeared to be accompanied by increased islet insulin contentand enhanced glucose-dependent insulin secretion in vitro,whereas the adult hypoinsulinemia was accompanied by de-creased insulin content and insulin-secretory response in vitro.However, when the insulin secretion rate was normalized withthe insulin islet content, the fraction of insulin release in bothpopulations was similar to that in controls. These resultsindicate that �-cell secretory mechanisms seem not to bealtered in undernourished animals, and at the same time, theyappear to indicate a regulatory role of insulin content ininsulin-secretory response. Similarly, it has been reported thatincreases in insulin content lead to higher insulin secretion(43), whereas decreased insulin content and proinsulin biosyn-thesis can be related to decreases in glucose-induced insulinsecretion (44).

To determine whether the insulin content alterations that wefound could be caused by alterations in insulin mRNA levels,we assessed this parameter at steady state in pancreas ofundernourished and control animals, and, in agreement withthe above observations, we found it to be higher in undernour-ished fetuses and lower in undernourished adults comparedwith their respective controls. The increased mRNA levels inour undernourished fetuses may result from the increasedinsulin requirements as a consequence of postprandial hyper-glycemic episodes generated by the altered glucose homeosta-sis present in pregnant rats (2). An increase in insulin demandmodulated by insulin mRNA levels has been observed incorticosterone-induced insulin resistance and after pancreatec-

Fig. 2. GLUT2 and GLUT1 protein content in islets isolatedfrom C and U fetal (A) and adult (B) rats. Equal amounts ofprotein (30 �g for GLUT2 and 60 �g for GLUT1) weresubjected to electrophoresis. Blots correspond to a representa-tive determination. Bars show averaged results from 5 indepen-dent islet isolations. Data are expressed as means � SE.



by guest on Novem


Dow

nloaded from


tomy (20, 32). However, lower insulin mRNA levels seem tobe related to fasting periods and undernutrition (19, 39). It canbe deduced from our results that, in both fetal and adultanimals, undernutrition affects molecular mechanisms, leadingto opposite alterations in insulin mRNA levels and, therefore,in the insulin biosynthesis capacity of the �-cell.

The primary physiological stimulus of insulin transcriptionand synthesis is glucose (40). Because glucose regulates insu-lin production by signals coming from the glycolytic pathway(18), we evaluated the adaptive changes of glucose metabolismin islets of fetal and adult undernourished animals. Our aimwas to determine the extent to which the glucose metabolismcould be related to the alterations that we found in our under-nutrition model. The present results indicate that undernutritiondid not modify the levels of glucose transporters (GLUT2 orGLUT1) in fetal and adult islets. In addition, the total glyco-lytic flux measured in both populations revealed no majorperturbations in intracellular glucose concentration, which in-dicates no defects in the glucose-sensing mechanism. How-ever, we found impaired glucose oxidative metabolism in islet

cells. In accord with what we have shown related to insulinexpression and content, islets from undernourished fetusesexposed to a stimulating concentration of glucose (16.7 mM)significantly increased the oxidation of glucose-derived acetylresidues in the TCA cycle with respect to control values,whereas this pathway appeared severely reduced in islets fromundernourished adults. These results suggest that the alter-ations in insulin gene expression may be related, at least inpart, to defects in mitochondrial oxidation of glucose. To ourknowledge, this is the first time that a possible relation betweenglucose oxidation and insulin production has been reported inundernourished rats.

�-Cell mitochondrial metabolism has been proven to benecessary for glucose-induced insulin secretion, but in our fetaland adult undernourished islets, oxidative metabolism in theTCA cycle appeared respectively augmented or diminished,whereas the ratio of insulin secretion remained unaffected.Similar results have been found in other malnourished animalmodels. Impaired insulin secretion has been related to reducedactivity of glycerol phosphate shuttle (34), to defects in ATP-sensitive K� channel pathways (41), and to attenuated islet

Fig. 3. D-[6-14C]glucose oxidation (A), D-[5-3H]glucose utilization (B), andpaired ratio between glucose oxidation and utlization (C) in islets from fetusesproceeding from C and U mothers. Islets were incubated for 2 h at 37°C with2.8 and 16.7 mM glucose. Data are expressed as pmol produced�islet�1�h�1

and represent means � SE of 16–20 individual observations collected in aseries of 4 independent experiments. FP � 0.05 relative to C animals. ŒP �0.05 relative to 2.8 mM glucose within the same group.

Fig. 4. D-[6-14C]glucose oxidation (A), D-[5-3H]glucose utilization (B), andpaired ratio between glucose oxidation and utilization (C) in islets from C andU adult rats. Islets were incubated for 2 h at 37°C with 2.8 and 16.7 mMglucose. Data are expressed as pmol produced�islet�1�h�1 and representmeans � SE of 16–20 individual observations collected in a series of 4independent experiments. FP � 0.05 relative to C animals. ŒP � 0.05 relativeto 2.8 mM glucose within the same group.



by guest on Novem


Dow

nloaded from


phosphatidylinositol hydrolysis (4), whereas glucose oxidationwas not found affected. These results suggest, in accord withthose from others (15), a permissive role of mitochondrialglucose oxidation, so additional signals must be involved inglucose induced insulin secretion. In addition, our resultsconcerning alterations in the TCA cycle, leading to insulinchanges in synthesis but not in secretion, reveal that, althoughglucose-induced synthesis and secretion have some commonsteps in stimulus-coupling signaling, both processes must nec-essarily diverge at some point. It has been recently shown thatthe divergence steps seem to be at the level of anaplerosis,downstream of glycolysis and upstream of oxidative phosphor-ylation (1). The nature of the signals from glucose metabolismlinked to insulin biosynthesis and gene expression is notcurrently well known, but our results seem to place thesesignals at the TCA cycle level, according to a suggestion ofAlarcon et al. (1), who proposed that succinate and/or succinyl-

CoA could be the preferential anaplerotic specific signal forregulating glucose-induced insulin biosynthesis.

The coupling factors linking metabolism to insulin geneexpression are not yet completely understood. Insulin genetranscription is regulated by the interaction between severalregulatory elements and trans-acting factor (31), of whichPDX-1 is the main one in mediating the effect of glucose ininsulin gene expression (28). Although PDX-1 protein levels infetal undernourished islets were found similar to those incontrols, the levels of p38/SAPK2, the protein involved inPDX-1 activation (27), were markedly increased. SAPK2 is amember of an expanding family of mitogen-activated proteinkinases that is activated in response to adverse stimuli (29).The increased p38/SAPK2 levels suggest that food restrictionin our undernourished fetuses could lead to a stress-relatedresponse in the islets. Similar results have been found in isletsfrom neonatal rats subjected to a high-carbohydrate diet, whereincreased mRNA levels and SAPK2 activity were related to a

Fig. 5. Protein content of pancreatic duodenal homeobox-1 (PDX-1; A), totalp85 subunit of phosphatidylinositol (PI) 3-kinase (p85; B), and stress-activatedprotein kinase-2 (p38/SAPK2; C) in islets from C and U fetal rats. Resultsshow representative Western blot, and each lane contained 50 �g of proteinextracted from isolated islets. Bars correspond to densitometric quantificationof 6 independent determinations. Data are expressed as means � SE. FP �0.05 relative to control rats.

Fig. 6. Protein content of PDX-1 (A), total p85 subunit of PI 3-kinase (B), andp38/SAPK2 (C) in islets from C and U adult rats. Results show representativeWestern blot, and each lane contained 50 �g of protein extracted from isolatedislets. Bars correspond to densitometric quantification of 6 independent deter-minations. Data are expressed as means � SE. FP � 0.05 relative to controlrats.



by guest on Novem


Dow

nloaded from


stress-like situation provoked by dietary treatment (36).SAPK2 is also implicated in insulin gene transcription (30) andin �-cell proliferation (9). Moreover, it has been shown thatoverexpression of SAPK2 mimics the effects of glucose andresults in a hyperstimulation of the PDX-1 activation pathway(27). Therefore, it is reasonable to suggest that the increased�-cell mass (2) and insulin mRNA levels observed in under-nourished fetal pancreases may be mediated by increasedp38/SAPK2 protein levels in our model.

Besides stress, glucose metabolism is able to activate p38/SAPK2 via a pathway that involves PI 3-kinase (42). AlthoughPI 3-kinase levels were not found impaired, it cannot be ruledout that increased glucose oxidation in undernourished fetusescan cause higher activation of PI 3-kinase and, consequently,of p38/SAPK2. Even when it is not well known which mole-cule derived from glucose metabolism might activate PI 3-ki-nase, some studies emphasize again the importance of mito-chondrial metabolism by showing the pyruvate, glyceralde-hyde, and oxoisocaproate ability to stimulate PDX-1 DNA-binding and insulin promoter activity (42).

On the other hand, until 70 days of life, undernourishedanimals show a significant decrease in the levels of PDX-1.Recently, Arantes et al. (3) also found decreased levels ofPDX-1 protein in islets of rats submitted to a low-protein dietand suggest a link among diminished PDX-1 protein expres-sion and alteration in islet size and functionality. Accordingly,in animal models of type 2 diabetes, reduced PDX-1 activityhas been related to impaired glucose-stimulated insulin secre-tion (8), insulin gene expression, and insulin production (22). Itis possible that the loss of insulin mRNA found in adultundernourished animals can be partly attributable to a loss ofPDX-1. Similarly to the fetuses, the decrease found in glucoseoxidation can also be involved in a lower PDX-1 activation inundernourished adults.

In conclusion, the results of this work indicate that, accord-ing to our model, food restriction causes changes in the levelsof insulin gene expression and content, leading to alterations inglucose-stimulated insulin secretion. The molecular events, anincrease in p38SAPK2 levels in fetuses and a decrease inPDX-1 levels in adults, seem to be involved in the alteredinsulin mRNA expression. Moreover, because PDX-1 activa-tion appears to be regulated by glucose-derived metabolite(s),the altered glucose oxidation caused by undernutrition could insome manner affect insulin mRNA expression. The study ofmetabolic and molecular mechanisms involved in alterationscaused by undernutrition becomes essential to evaluate thefacts that can lead to adult diseases. In this context, our ratmodel provides an opportunity to assess early and long-termevents under physiological conditions.

ACKNOWLEDGMENTS

We thank Dr. Luis Goya for critical review of this manuscript and SusanaFajardo for technical assistance.

Parts of this work were presented at the 18th International DiabetesFederation Congress, Paris, August 24–29, 2003.

GRANTS

This work was supported in part by a grant from Universidad Complutensede Madrid, Spain (ref. no. PR48/01–9836) and by Direccion General deInvestigacion, Ministerio de Ciencia y Tecnologıa, Spain (ref. nos. BFI2001–2125 and BFI2002–200253).

REFERENCES

1. Alarcon C, Wicksteed B, Prentki M, Corkey BE, and Rhodes CJ.Succinate is a preferential metabolic stimulus-coupling signal for glucoseinduced proinsulin biosynthesis translation. Diabetes 51: 2496–2504,2002.

2. Alvarez C, Martın MA, Goya L, Bertin E, Portha B, and Pascual-Leone AM. Contrasted impact of maternal rat food restriction on the fetalendocrine pancreas. Endocrinology 138: 2267–2273, 1997.

3. Arantes VC, Teixeira VPA, Reis MAB, Latorraca MQ, Leite AR,Carneiro EM, Yamada AT, and Boschero AC. Expression of PDX-1 isreduced in pancreatic islets from pups of rat dams fed a low protein dietduring gestation and lactation. J Nutr 132: 3030–3035, 2002.

4. Barbosa FB, Capito K, Kofod H, and Thams P. Pancreatic islet insulinsecretion and metabolism in adult rats malnourished during neonatal life.Br J Nutr 87: 147–155, 2002.

5. Bertin E, Gangnerau fMN, Bellon G, Bailbe D, Arbelot de VacqueurA, and Portha B. Development of beta cell mass in fetuses of ratsdeprived of protein and/or energy in last trimester of pregnancy. Am JPhysiol Regul Integr Comp Physiol 283: R623–R630, 2002.

6. Blondeau B, Garofano A, Czernichow P, and Breant B. Age-dependentinability of the endocrine pancreas to adapt to pregnancy: a long-termconsequence of perinatall malnutrition in the rat. Endocrinology 140:4208–4213, 1999.

7. Bradford M. A rapid and sensitive method for the quantification ofmicrogram quanties of protein, utilizing the principle of protein-dyebinding. Anal Biochem 72: 248–254, 1976.

8. Brissova M, Shiota M, Nicholson WE, Gannon M, Knobel SM, PistonDW, Wright CVE, and Power AC. Reduction in pancreatic transcriptionfactor PDX-1 impairs glucose-stimulated insulin secretion. J Biol Chem277: 11225–11232, 2002.

9. Burns CJ, Squires PE, and Persaud SJ. Signaling through the p38 andp42/44 mitogen-activated families of protein kinases in pancreatic betacell proliferation. Biochem Biophys Res Commun 268: 541–546, 2000.

10. Chan SJ, Noyes BE, Agarwal KL, and Steiner DF. Construction andselection of recombinant plasmids containing full-length complementaryDNAs corresponding to rat insulins I and II. Proc Natl Acad Sci USA 76:5036–5040, 1979.

11. Chomczymski P and Sacchi N. Single step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction. Anal Bio-chem 162: 156–159, 1987.

12. Dahri S, Snoek A, Reusens-Billen B, Remacle C, and Hoet JJ. Isletfunction in offspring of mothers on low protein diet during gestation.Diabetes 40: 115–120, 1991.

13. Docherty K and Clark A. Nutrient regulation of insulin gene expresion.FASEB J 8: 20–27, 1994.

14. Escriva F, Rodriguez C, Cacho J, Alvarez C, Portha B, and Pascual-Leone AM. Glucose utilization and insulin action in adult rats submittedto prolonged food restriction. Am J Physiol Endocrinol Metab 263:E1–E7, 1992.

15. Eto K, Tsubamoto Y, Terauchi Y, Sugiyama T, Kishimoto T, Taka-hashi N, Yamauchi N, Kubota N, Murayama S, Aizawa T, AkanumaY, Aizawa S, Kasai H, Yazaki Y, and Kadowaki T. Role of NADHshuttle system in glucose-induced activation of mitochondrial metabolismand insulin secretion. Science 283: 981–985, 1999.

16. Garofano A, Czernichow P, and Breant B. Beta cell mass and prolif-eration following late fetal and early postnatal malnutrition in the rat.Diabetologia 41: 1114–1120, 1998.

17. Garofano A, Czernichow P, and Breant B. Effect of ageing on beta cellmass and function in rats malnourished during the perinatal period.Diabetologia 42: 711–718, 1999.

18. German MS. Glucose sensing in pancreatic islets beta cells: the key roleof glucokinase and the glycolytic intermediates. Proc Natl Acad Sci USA90: 1781–1785, 1993.

19. Giddings SJ, Chirgwin J, and Permutt MA. The effect of fasting andfeeding on preproinsulin messenger mRNA in rats. J Clin Invest 67:952–960, 1981.

20. Giddings SJ, Orland MJ, Weir GC, Bonner-Weir S, and Permutt MA.Impaired insulin biosynthetic capacity in a rat model for noninsulin-dependent diabetes: studies with dexamethasone. Diabetes 34: 235–240,1985.

21. Hales CN and Barker DJP. Type 2 (non-insulin-dependent) diabetesmellitus: the thrifty hypothesis. Diabetologia 35: 595–601, 1992.



by guest on Novem


Dow

nloaded from


22. Harmon SJ, Gleason CE, Tanaka Y, Oseid EA, Hunter-Berger KK,and Robertson RP. In vivo prevention of hyperglycemia also preventsglucotoxic effects on PDX-1 and insulin gene expression. Diabetes 48:1995–2000, 1999.

23. Hellerstrom C, Lewis NJ, Borg H, Johnson R, and Freinkel N.Methods for large scale isolation of pancreatic islets by tissue culture offetal rat pancreas. Diabetes 28: 769–776, 1979.

24. Malaisse WJ and Sener A. Hexose metabolism in pancreatic islets.Feedback control of D-glucose oxidation by functional events. BiochimBiophys Acta 971: 246–254, 1988.

25. Malaisse-Lagae F and Malaisse WJ. Insulin release by pancreatic islets.In: Methods in Diabetic Research (Part B), edited by Larner J and PohlSL. New York: Wiley, 1984, vol. 1, p. 147–152.

26. Martın MA, Alvarez C, Goya L, Portha B, and Pascual-Leone AM.Insulin secretion in adult rats that had experienced different underfeedingpatterns during their development. Am J Physiol Endocrinol Metab 272:E634–E640, 1997.

27. Mcfarlane WM, McKinnon CM, Felton-Edkins ZA, Cragg H, JamesRFL, and Docherty K. Glucose stimulates translocation of the homeodo-main transcription factor PDX-1 from the cytoplasm to the nucleus inpancreatic beta cells. J Biol Chem 274: 1011–1016, 1999.

28. Mcfarlane WM, Read ML, Gilligan M, Bujalska I, and Dockerty K.Glucose modulates the binding activity of the beta cell transcription factorIUF1 in a phosphorylation-dependent manner. Biochem J 303: 625–631,1994.

29. Mcfarlane WM, Smith SB, James RFL, Clifton AD, Doza YN, CohenP, and Docherty K. The p38/reactivating kinase mitogen-activated pro-tein kinase cascade mediates the activation of the transcription factorinsulin upstream factor I and insulin gene transcription by high glucose inpancreatic beta cells. J Biol Chem 272: 20936–20944, 1977.

30. McKinnon CM and Docherty K. Pancreatic duodenal homeobox-1,PDX-1, a major regulator of beta cell identity and function. Diabetologia44: 1203–1214, 2001.

31. Ohneda K, Ee H, and German M. Regulation of insulin gene transcrip-tion. Cell Dev Biol 11: 227–223, 2000.

32. Orland MJ, Chyn R, and Permutt MA. Modulation of proinsulinmessenger RNA after partial pancreactetomy in rats: relationship withglucose homeostasis. J Clin Invest 75: 2045–2055, 1985.

33. Rasschaert J, Reusens B, Dahri S, Sener A, Remacle C, Hoet J, andMalaisse WJ. Impaired activity of rat pancreatic islet mitochondrialglycerophosphate dehydrogenase in protein malnutrition. Endocrinology136: 2631–2634, 1995.

34. Sener A, Reusens B, Remacle Hoet JJ C, and Malaisse WJ. Nutrientmetabolism in pancreatic islets from protein malnourished rats. BiochemMol Med 59: 62–67, 1996.

35. Snoeck A, Remacle C, Reusens B, and Hoet J. Effect of a low proteindiet during pregnancy on the fetal rat endocrine pancreas. Biol Neonate 57:107–118, 1990.

36. Srinivasan M, Song F, Aalinkel R, and Patel MS. Molecular adaptationsin islets from neonatal rats reared artificially on a high carbohydrate milkformula. J Nutr Biochem 12: 575–584, 2001.

37. Thorens B, Charron MJ, and Lodish HF. Molecular physiology ofglucose transporters. Diabetes Care 13: 209–218, 1990.

38. Valverde AM, Navarro P, Teruel T, Conejo R, Benito M, and LorenzoM. Insulin and insulin-like growth factor I up-regulate GLUT-4 geneexpression in fetal brown adipocytes in a phosphoinositide 3-kinase-dependent manner. Biochem J 337: 397–405, 1999.

39. Vuguin P, Ma X, Yang X, Surane M, Liu B, and Barzilai N. Fooddeprivation limits insulin secretory capacity in postpuberal rats. PediatrRes 49: 468–473, 2001.

40. Welsh M, Scherberg N, Gilmore R, and Steiner DF. Translation controlof insulin biosynthesis: evidence for regulation of elongation, initiationand signal-recognition particle mediated translational arrest by glucose.Biochem J 235: 459–467, 1986.

41. Wilson MR and Hughes SJ. Impairerd glucose-stimulated insulin releasein islets from adult rats malnourished during foetal-neonatal life. Mol CellEndocrinol 142: 41–48, 1998.

42. Wu H, Macfarlane WM, Tadayyon M, Arch JRS, and James RFL.Insulin stimulates pancreatic-duodenal homeobox factor-1 (PDX1) DNA-binding activity and insulin promoter activity in pancreatic beta cells.Biochem J 344: 813–818, 1999.

43. Xu GC and Rothenberg PL. Insulin receptor signaling in the beta cellinfluences insulin gene expression and insulin content. Diabetes 47:1243–1252, 1998.

44. Zhou YP and Grill VE. Long-term exposure of rat pancreatic islets tofatty acids inhibits glucose induced insulin secretion and biosynthesisthrough a glucose fatty acid cycle. J Clin Invest 93: 870–876, 1994.



by guest on Novem


Dow

nloaded from


am j physiol endocrinol metab 2004 martín.pdf

Documents