Intrauterine protein restriction combined with early postnatal overfeeding was not associated with adult-onset obesity but produced glucose intolerance by pancreatic dysfunction.

0.19 MB·English

by  Coutinho, Grazielle Vitoria Ponti

Coutinhoetal.Nutrition&Metabolism2013,10:5
http://www.nutritionandmetabolism.com/content/10/1/5
RESEARCH Open Access
Intrauterine protein restriction combined with
early postnatal overfeeding was not associated
with adult-onset obesity but produced glucose
intolerance by pancreatic dysfunction
Grazielle Vitória Ponti Coutinho1, Felipe Rodrigues Coutinho1, Jaline Zandonato Faiad2, Marina Satie Taki1,
Silvia Regina de Lima Reis2, Letícia Martins Ignácio-Souza3, Adriene Alexandra Paiva2, Márcia Queiroz Latorraca3,
Maria Helena Gaíva Gomes-da-Silva3 and Maria Salete Ferreira Martins3*
Abstract
We investigated ifwhether intrauterine protein restriction incombination with overfeeding during lactation would
cause adult-onset obesity and metabolic disorders. After birth, litters from dams fed withcontrol (17%protein) and
low protein (6% protein) diets were adjusted to a size of four (CO and LO groups, respectively) or eight (CC and LC
groups, respectively) pups. All of theoffspring were fed a diet containing 12%protein from the time of weaning
until they were 90 dold. Compared to theCC and LC groups, the CO and LO groups had higher relative and
absolute food intakes, oxygen consumption and carbon dioxide production;lower brown adipose tissue weight
and lipid content and greater weight gain and absolute and relative white adiposetissue weight and absolute lipid
content. Compared with theCO and CCrats, theLC and LO rats exhibited higherrelative food intake, brown
adipose tissue weight and lipid content, reduced oxygen consumption, carbon dioxide production and
spontaneous activity, increased relative retroperitoneal adipose tissue weight and unaltered absolute white adipose
tissue weight and lipid content. The fastingserum glucose was similar among the groups. The area under the
glucose curve was higher inthe LOand CO rats than inthe LC and CCrats. The basal insulinemia and homeostasis
model assessment of insulin resistance (HOMA-IR) were lower in theLO group thanin theothergroups. The total
area under the insulin curve for theLO rats was similarto the CCrats, and both were lower than theCO and LC
rats. K was higherin the LO, LC and CO groups than in theCC group. Thus, intrauterine protein restriction
itt
followed by overfeeding during lactation did not induce obesity, but produced glucose intolerance by impairing
pancreatic function in adulthood.
Keywords: Intrauterine protein restriction, Postnatal overfeeding,Obesity,Visceral fat, Glucose tolerance
Introduction deprivation in pregnancy during the Dutch famine in the
Obesity is one of the most daunting health challenges of Second World War [3]. Data have shown that nutritional
this century. The obesity epidemic is a major health con- and hormonal status in early life permanently changes the
cern because obesity significantly increases the risk for a structure and function of body tissues and systems. In
variety of chronic metabolic diseases, such as type 2 dia- addition, overfeeding after birth may increase the risk of
betes, hypertension, hypercholesterolemia and heart dis- obesity and metabolic disorders. Early postnatal overnutri-
ease [1,2]. Epidemiologic studies show a higher incidence tion can be induced by litter size reduction, which is an
of obesity among men whose mothers experienced food appropriate experimental model to study short- and long-
termconsequencesofchildhoodobesity.Animalsraisedin
*Correspondence:[email protected] a small litter have been shown to develop hyperphagia,
3DepartamentodeAlimentoseNutrição,FaculdadedeNutrição,
hyperinsulinemia, hyperleptinemia and hypertension [4-6],
UniversidadeFederaldeMatoGrosso,Cuiabá,MatoGrosso,Brazil
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Coutinhoetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Coutinhoetal.Nutrition&Metabolism2013,10:5 Page2of9
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as well as higher total and visceral fat mass, lower HDL-C littersofmaleratsfromdamsfedwithCandLPdietswere
andcentralleptinresistanceinadultlife[7]. adjusted to a litter size of four (CO and LO groups, re-
Adipose tissue has been shown to play a crucial role in spectively) to induce early postnatal overfeeding or eight
metabolic disorders associated with obesity [8]. Research (CC and LC groups, respectively) to induce normal feed-
has demonstrated associations among the location of fat ing. After weaning (week 4), the offspring of all the litter
storage, insulin sensitivity and the concurrent impacts on groups were fed a diet containing 12% protein (mainten-
multiple other metabolic complications. Insulin resistance ance diet) and 3.74 kcal/g, until they were90 d old. All of
and metabolic syndrome are established consequences of thedietsusedinthepresentstudyaredescribedinTable1
expanding visceral fat mass [9], whereas the expansion of [15]. In the low protein and maintenance diets, ad-
subcutaneous fat is associated with improved glucose tol- justments were made to equalize the proportion of
erance and a lower risk of developing obesity related co- L-cystine in relation to the casein content offered in the
morbidities [10,11]. Moreover, recent studies have shown controldiet.Afterweaningtheanimalswerehousedfour
that higher gluteofemoral fat mass is associated with pro- per cage. The rats had free access to food and water, and
tectionagainstinsulinresistanceinobesemenandwomen they were kept under standard lighting conditions (12 h
[12,13]. The protective role of gluteofemoral body fat light–12hdarkcycle)at24°Cthroughouttheexperimen-
results from its distinct properties with regard to lipolysis talperiod.
andfattyaciduptake[14].
In the present study, we evaluated if whether intra- Experimentalprocedures
uterine proteinrestriction incombination with overfeed- The rats were weighed at birth, after weaning, and then
ing during lactation would cause adult-onset obesity and once a week throughout the experimental period.
thedevelopment ofmetabolic disorders. Recorded weekly, the weight and the naso-anal length
(NAL) of the rats were used to calculate the Lee index
Methods and materials [16]: Lee index= 3√weight body(g)/NAL (cm) × 1000
Animalsanddiets The food intake was recorded three times per week, and
The experimental procedures involving rats were per- the data were expressed using absolute and relative
formed in accordance with the guidelines of the Brazilian values. To assess the relative food intake, the total food
Society of Science in Laboratory Animals (SBCAL) and intake during the experimental period after weaning was
were approved by the ethics committee at the Federal normalizedper100gof bodyweightatage90d.Because
University of Mato Grosso (Process nº 23108.021972/10- it was not possible to evaluate all variables in the same
4). Male and virgin female Wistar rats (aged 85–90 d) animal, the number of individual experiments varied
were obtained from the university’s breeding colony. among the groups, but it was representative of at least
Mating was prompted by housing one male with four three different litters.
femalesovernight,andpregnancywasconfirmedbyexam- At 90 d of age, the animals were euthanized with CO
2
ining vaginal smears for the presence of sperm. Pregnant afterfasting12h.Bloodsampleswerecollected,andserum
females were randomly separated during pregnancy, they was obtained by centrifugation to determine the lipid pro-
were fed isoenergeticdietscontaining 17% proteinand 3.9 file (triglycerides, total cholesterol, LDL cholesterol and
kcal/g(control[C]);or6%proteinand3.8kcal/g(lowpro- HDL),bloodglucoseandinsulin.Serumaliquots(storedat
tein[LP]).Caseinservedastheproteinsource.Afterbirth, -80°C) of animals in the fed state were used to determine
Table1Compositionofthecontrolgrowth,low-proteinandcontrolmaintenancediets
Ingredientes(g/kg) Controlgrowth(17%protein) Low-protein(6%deprotein) Controlmaintenance(12%deprotein)
Maizestarch 397.0 480.0 439.42
Dextrinisedmaizestarch 130.5 159.0 146.47
Casein(84%deprotein) 202.0 71.5 132.0
Soyabeanoil 70.0 70.0 70.0
Fibre 50.0 50.0 50.0
Sucrose 100.0 121.0 111.6
AIN-93Gmineralmix* 35.0 35.0 35.0
AIN-93Gvitaminmix* 10.0 10.0 10.0
L-Cystine 3.0 1.0 2.2
Cholinebitartrate 2.5 2.5 2.5
AIN,AmericanInstituteofNutrition.
*See[15].
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the serum concentrations of albumin, total protein, free programmable MPT Reader DV 990BV6). Plasma or
fatty acid (FFA) and leptin. After a medium laparotomy, serum leptin was determined with a radioimmunoassay
the retroperitoneal white adipose tissue (RWAT) and epi- (RIA) kit (Linco Research, St. Louis, Missouri USA).
didymal white adipose tissue (EWAT) were removed, and Plasma or seruminsulinwasdeterminedwith an RIAkit
themassofthetissueswasmeasured.Thetissueswerefro- (Millipore, Massachusetts. USA). Serum triglycerides,
zeninliquidN andstoredat−80°Cforasubsequentana- total cholesterol (TC), high-density lipoprotein choles-
2
lysis of their lipid content. The interscapular brown terol (HDL), low-density lipoprotein cholesterol (LDL)
adiposetissue(IBAT)wasremoved,carefullycleanedtore- were analyzed by an enzymatic method using BT-3000
move adhering fat and muscle, weighed and stored simi- Plus analyzer (Wiener labs, Rosario, Argentina). The
larly to the white adipose tissues for a subsequent analysis CastelliindicesIandIIthatcorrelatewithatherogenicity
ofitslipidcontent. were obtained usingthefollowing formulas[19]:
Castelli index I ¼ TC=HDL ð1Þ
Oxygenconsumption/carbondioxideproductionand
respiratoryexchangecoefficientdetermination Castelli index II ¼ LDL=HDL ð2Þ
Oxygen consumption, carbon dioxide production and the
respiratory exchange coefficient (RQ) were measured in Serum glucose (in mg/dL), was determined using a
fed animals by a computer-controlled, open circuit calor- monitor with test strips to measure blood glucose (gluc-
imetersystem,theLE405GasAnalyzer(Panlab–Harvard ometer) (Accu-ChekW, Roche Diagnostics, Germany).
Apparatus, Holliston, MA, USA). Individual rats were FFA levels were determined by a spectrophotometric
placed in the metabolic chamber and allowed to acclimate method using kits by Wako Chemicals USA, Inc. The
for 4 hs prior to data collection. After acclimation the rats homeostasis model assessment of insulin resistance
weresinglyhousedinclearrespiratorychambersandroom (HOMA-IR) was used as the physiological index of insu-
air was circulated through the chambers at a flow rate of lin resistance. The HOMA-IR was calculated from the
0.8l/min.Theairflowwithineachchamberwasmonitored fasting glucose and the fasting insulin concentrations
by an Air Supply and Switching sensor (Panlab – Harvard usingthefollowing formula:
Apparatus). The gas sensors were calibrated before begin-
ning the experiments with primary gas standards contain- HOMA(cid:2)IR ¼ðfasting insulin ðpmo1=1ÞÞ
ing knownconcentrations ofO2 and CO2 (Air Liquid,São (cid:3)ðfasting glucose ðmmo1=1ÞÞ=22:5
Paulo,Brazil).Theanalyseswereperformedfora12hdark
periodineachchamber.Twohourswerediscountedtoac-
commodate the dark-to-light or light-to-dark transition Intraperitonealglucose-tolerancetest
periods. Therefore, each rat was evaluated for 10 h. The Aftera12hfast,glucose(200g/l)wasadministeredintra-
outdoor air reference values were sampled after every four peritoneally at a dose of 2 g/kg body weight. Blood sam-
measurements.Sampleairwassequentiallypassedthrough ples were obtained from the cut tip of the tail 0, 30, 60
O andCO sensorstodeterminetheO andCO content; and120minlatertodetermineserumglucoseandinsulin
2 2 2 2
from these data, measures of oxygen consumption (VO ) concentrations. The glucose and insulin responses during
2
and carbon dioxide production (VCO ) were estimated. the intraperitoneal glucose tolerance tests were calculated
2
TheVO andVCO were calculatedwithMetabolism2.2v by estimating the total area under the glucose curves,
2 2
software and expressed in mL.h-1.g-1, based on the With- usingthetrapezoidalmethod[20].
ersequation.TheRQwascalculatedasVCO /VO .Energy
2 2
expenditure(EE)(kilocaloriesperkilogramperh;heatpro- IntraperitonealInsulin-tolerancetest
duction)wascalculatedusingarearrangementoftheWeir A subcutaneous insulin tolerance test was performed on
equation((3.851+1.232×RQ)×VO2)[17,18]. the 83 day-old male rats, after a 12 h fast. The insulin
tolerance test consisted of a bolus injection of insulin
Evaluationofspontaneouslocomotoractivity (300 mU/kg body weight) beneath the dorsal skin of the
Spontaneouslocomotoractivitywasevaluatedduringa12h fasting rat. Blood samples were obtained from the cut
dark period. Information on motility was gathered using tip of the tail 0, 5, 10 and 15 min later to measure glu-
twohorizontalaxesand,acomputer-controlledlineardetec- cose levels. The rate constant for the elimination of
tion system from Harvard Instruments (Panlab, Holliston, serum glucose was calculated using the formula 0.693/
MA,USA). t1=2, where t1=2 is half-life. The serum glucose half-life
was calculated from the slope of a least-square analysis
Hormonesandbiochemicalparameters of the serum glucose concentrations from 0–15 min
The serum hormone levels were measured with an after the subcutaneous injection of insulin, when the
ELISA, using kits designed specifically for rats (GDV serumglucose concentrations declined linearly [21].
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Lipidcontent weight to the CO and CC groups, which had higherbody
The lipid contents of the IBAT, RWAT and EWAT weightsthantheLCrats.However,thebodyweightofthe
were determined by the gravimetric method following CO group was higher in relation to the CC group
chloroform–methanol (2:1) extraction, described by (P<0.01).Atthetimeofweaning,theLeeindexwaslower
Folchetal.[22]). in the CO and LO groups than in the CC and LC groups
(F =63.48,P<0.01). However, the Lee indices for the LC
1,20
Statisticalanalysis and LO groups were higher when compared to the CC
The results are expressed as the mean values with stand- and CO groups (F =243.00, P<0.001). The increases in
1,20
ard errors for the number of rats indicated. Unpaired t body weight from weaning to the end of the experiment
tests were used to analyze the size of the litters and the (28–90 d) and from birth to the end of the experiment
body weight of the newborn rats. Bartlett’s test for the (0–90 d) were significantly higher in the CO and LO
homogeneity of variances was initially used to determine groups than in the CC and LC groups (F =11.91,
1,27
whether the data complied with the assumptions neces- P<0.001andF =29.92,P<0.0001,respectively).Similarly,
1,27
sary for a parametric ANOVA. When necessary, the data the nutritional status was higher in the CO and LO
were log-transformed to correct for the variance in he- groups,comparedtotheCCandLCgroups(F =243.00;
1,20
terogeneity or non-normality [23]. A two-way ANOVA P<0.001).Attheendoftheexperimentalperiod,themean
(the effect of previous nutritional status and overfeeding) body weight and the mean Lee index of the rats from the
was used to compare the data from the CC, CO, LC and CO and LO groups were higher than the corresponding
LOgroups. When necessary, these analyses were comple- values for the rats from the CC and LC groups
mentedbytheleastsignificantdifferencetesttodetermine (F =22.08, P<0.001 and F =7.22,P<0.02,respectively).
1,20 1,20
the significance of the individual differences. P<0·05 indi- The absolute food intake and relative food intake were
cated statistical significance. All statistical analyses were higher in the CO and LO groups, compared to the CC
conductedusingtheStatisticsoftwarepackage(Stat-soft). and LC groups (F =8.59, P<0.01 and F =5.97, P<0.02,
1,27 1,27
respectively). The relative food intake in the LC and LO
Results groups was higher than in the CC and CO groups
Measurementsofanimalweight,increaseinbodyweight, (F =24.11,P<0.0001)(Table2).
1,27
Lee’sindexandfoodintake
ThelittersizeintheCandLPgroupsofpupswassimilar, Oxygenconsumption(VO ),carbondioxideproduction
2
but the body weight of the newborn LP rats was lower (VCO ),respiratoryquotient(RQ),energyexpenditure(EE)
2
than that of the C rats (data not shown). The increase in andspontaneousactivity
bodyweightfrombirthtoweaning(0–28d)wassimilarin Carbon dioxide production, oxygen consumption and en-
the LO and CO groups; in both groups, body weight was ergyexpenditurewerehigherintheratsfromtheCOand
higherthanintheLCandCCgroups.Thelowestincrease LO groups than in the rats from the CC and LC groups
inbodyweightwasobservedintheLCgroup(P<0.01).At (F =15.15, P<0.002; F =13.79, P<0.001; F =15.09,
1,12 1,12 1,12
the time of weaning, the LO group had a similar body P<0.01, respectively) and lower in the LC and LO groups
Table2Somaticprofileofratsmaintainedwiththecontrol(CCandLC)oroverfeedingdiets(COandLO)
Variables Groups
CC CO LC LO
n Mean SEM n Mean SEM n Mean SEM n Mean SEM
Incrementinbodyweight0-28d(g) 6 56.9b 1.1 6 69.7a 2.4 6 41.2c 1.8 6 65.7a 1.0
BWatweaning(g) 6 61.3b 1.5 6 74.4a 2.8 6 45.7c 12.4 6 68.9ab 1.8
Leeindexatweaning 6 108.5 0.8 6 102.5# 0.8 6 119.5* 0.8 6 114.0*# 0.6
Incrementinbodyweight28-90d(g) 6 317.4 4.8 6 359.4# 10.6 6 324.6 13.9 6 333.3# 6.1
Incrementinbodyweight0-90d(g) 6 374.4 4.8 6 429.1# 9.9 6 365.9 14.2 6 339.0# 6.2
FinalBW(g) 6 372.3 6.1 6 422.8# 8.2 6 371.5 13.9 6 413.7# 9.5
FinalLeeindex 6 284.1 2.8 6 293.6# 3.6 6 285.4 1.2 6 289.3# 1.6
Absolutefoodintake(g) 8 1030 16.6 8 1107# 0.1 7 1021 61.1 8 1125# 0.1
Relativefoodintake(g/100gBW) 8 253.6 7.1 8 272.8# 4.7 7 292.6* 12.6 8 312.47#* 5.6
BWbodyweight.
abcMeanvalueswithunlikesuperscriptlettersweresignificantlydifferent(P<0·05;two-wayANOVAfollowedbyleastsignificantdifference(LSD)test).
*Meanvaluesweresignificantlydifferentfromthoseofthecontrolrats(P<0·05;two-wayANOVA).
#Meanvaluesweresignificantlydifferentfromthoseofthenon-overfeedingrats(P<0·05;two-wayANOVA).
nNumbersofrats.
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than in the CC and CO groups (F =5.84, P<0.03; HDL-cholesterol, total cholesterol and Castelli I did not
1,12
F =6.10, P<0.02; F =5.90, P<0.05, respectively). The differ among the groups. However, the Castelli II values
1,12 1,12
respiratory quotient (RQ) did not differ among groups. werehigherin the CO group than in the LO, CC and LC
Spontaneousactivitywas lower intheLCand LOgroups, groups (P <0.05). The serum FFA concentrations were
compared to the CC and CO groups (F =9.86, P<0.01) lower in the CO and LO rats than in the CC and LC rats
1,12
(Table3). (F =4.89;P<0.05)(Table5).Theserumtotalproteinand
1,20
albuminconcentrationsdidnotdifferamonggroups(data
Measurementsofweightandlipidcontentofbrown notshown).
adiposetissueandretroperitonealwhiteadiposetissues
The weight and lipid content (g/total tissue) of the IBAT Metabolicandhormonalparameters
were significantly lower in the CO and LO groups than Basal serum glucose levels were similar among the
in the CC and LC groups (F =8.57, P<0.001 and groups. However, serum insulin levels and HOMA-IR
1,20
F =10.58,P<0.001,respectively)andhigherfortherats were higher in the CO and LC groups than in the CC
1,20
from the LC and LO groups, compared with the rats and LO groups. Additionally, in the LO group, these
from the CC and CO groups (F =5.09, P<0.02 and parameters were lower than in the CC group (P<0.05).
1,20
F =19.67, P<0.001, respectively). However, the lipid No differences were observed among the leptin concen-
1,20
content of the IBAT (g/100 g tissue) was higher in the trationlevelsofthegroups.Thetotalareaundertheglu-
LC and LO rats than in the CC and CO rats (F =5.04, cose curve for the CO and LO rats was higher than for
1,20
P<0.02). The absolute RWATand EWAT weights and as the CC and LC rats (F =13.25, P<0.01). The total area
1,21
wellasthelipidcontent(g/totaltissue)inthesedeposits, under the insulin curve was similar in the CO and LC
were higher in the CO and LO rats than in the CC and groups, and both were higher than the corresponding
LC rats (F =14.71, P<0.001; F =6.38; P<0.05 and values forthe LOandCCgroups(P<0.01)(Table 6).
1,20 1,20
F =5.89, P<0.02; F =6.09; P<0.02, respectively). The
1,26 1,27
relativeweightofEWAT(mg/gbodyweight) wassimilar Discussion
in the LO, LC and CO groups and those weights were In the present study, restricting protein in the diets of
higher in relation to the CC group (P<0.02). The relative rats during fetal life led to a low birth weight. Overfeed-
weight of RWAT (mg/g body weight) was significantly ing during lactation, induced by small litter size resulted
higher in the CO and LO groups than in the CC and LC in a higher body weight at weaning and in adulthood for
groups (F1,20=4.60, P<0.05) as well as in rats from the the offspring, regardless of the protein content of the
LC and LO groups compared with the rats from the CC mother’s diets during pregnancy. However, based on
and CO groups (F1,20=9.25, P<0.01). The lipid content in Lee’s index, the protein restriction during intrauterine
the RWAT (g/100 g tissue) and EWAT (g/100 g tissue) life resulted in obesity at weaning, independent of post-
didnotdifferamongthe groups(Table4). natal nutrition. It is well established that early overfeed-
ing induces a dramatic increase in body weight during
Biochemicalparameters thesucklingperiod [7,24,25].
The triglycerides levels were lower in the rats from the Postnataloverfeedingandnormalnutritionafterwean-
CO and LO groups, compared with the rats from the ing corrected the features typical of protein malnutrition
CC and LC groups (F =16.64; P<0.001), but triglycer- (hypoalbuminemia and low serum total protein), and
1,19
ides were higher in the LC and LO rats than in the CC resulted in a higher final body weight, Lee’s index, and
and CO rats (F =6.43; P<0.02). The LDL-cholesterol, visceral fat mass. An increased fat mass and adipocyte
1,19
Table3Oxygenconsumption(O ),carbondioxideproduction(CO ),respiratoryquotient(RQ)andspontaneous
2 2
activitywasmeasuredofratsmaintainedwiththecontrol(CCandLC)oroverfeedingdiets(COandLO)
Variables Groups
CC CO LC LO
n Mean SEM n Mean SEM n Mean SEM n Mean SEM
CO production(ml/mim/kg0,75) 6 56.9 1.3 6 63.2# 1.3 6 52.5* 1.5 6 59.4*# 1.5
2
O consumption(ml/mim/kg0,75) 6 67.9 1.5 6 75.6# 1.8 6 62.9* 1.2 6 70.5*# 2.0
2
RQ(VCO/VO) 6 0.84 0.1 6 0.84 0.1 6 0.83 0.1 6 0.84 0.1
2 2
Activity 6 6609 368 6 7223 515 6 5534* 563 6 4683* 407
*Meanvaluesweresignificantlydifferentfromthoseofthecontrolrats(P<0·05;two-wayANOVA).
#Meanvaluesweresignificantlydifferentfromthoseofthenon-overfeedingrats(P<0·05;two-wayANOVA).
nNumbersofrats.
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Table4Weightandlipidcontentinthewhiteandbrownadiposetissues(BAT)ofratsmaintainedwiththecontrol
(CCandLC)oroverfeedingdiets(COandLO)
Variables Groups
CC CO LC LO
n Mean SEM n Mean SEM n Mean SEM n Mean SEM
WeightIBAT(g) 6 0.468 0.1 6 0.391# 0.1 6 0.652* 0.1 6 0.501*# 0.1
LipidcontentIBAT(g/totaltissue) 6 0.20 0.1 6 0.16# 0.1 6 0.34* 0.1 6 0.23*# 0.1
LipidcontentIBAT(g/100gtissue) 6 48.7 3.2 6 50.1 3.4 6 61.3* 2.1 6 52.5* 4.4
WeightRWAT(g) 6 9.1 0.5 6 12.1# 0.8 6 10.1 0.4 6 12.5# 0.9
WeightRWAT(g/100gBW) 6 2.5 0.1 6 2.6# 0.3 6 2.7* 0.1 6 3.10*# 0.1
LipidcontentRWAT(g/totaltissue) 8 3.5 0.3 7 4.9# 0.4 7 3.7 0.4 8 4.1# 0.3
LipidcontentRWAT(g/100gtissue) 8 39.6 2.1 7 38.0 1.5 7 38.6 2.5 8 33.6 1.3
WeightEWAT(g) 6 6.6 0.5 6 8.7# 0.7 6 8.3 0.7 6 9.7# 0.8
WeightEWAT(g/100gBW) 6 1.8b 0.1 6 2.2a 0.1 6 2.4a 0.1 6 2.2a 0.2
LipidcontentEWAT(g/totaltissue) 8 2.8 0.1 8 3.3# 0.2 7 2.7 0.3 8 3.2# 0.2
LipidcontentEWAT(g/100gtissue) 8 35.7 0.8 8 36.4 0.6 6 34.3 1.2 7 34.3 0.7
BW,bodyweight;RWAT,retroperitonealwhiteadiposetissue;EWAT,epididymalwhiteadiposetissue.
abMeanvalueswithunlikesuperscriptlettersweresignificantlydifferent(P<0·05;two-wayANOVAfollowedbyleastsignificantdifference(LSD)test).
*Meanvaluesweresignificantlydifferentfromthoseofthecontrolrats(P<0·05;two-wayANOVA).
#Meanvaluesweresignificantlydifferentfromthoseofthenon-overfeedingrats(P<0·05;two-wayANOVA).
nNumbersofrats.
number has been observed in a similar animal model growth-restrictioninuterofollowedbypostnatalcatch-up
[26-28]. These animals were shown to exhibit enhanced growthcontributestoincreasedcentralfatmass[31].
11-β-hydroxysteroid dehydrogenase type 1 (11β- HSD1) Obesity is the result of an imbalance between energy
mRNA in adipose cells, an enzyme that converts corti- intake andenergyexpenditure; excessenergy isstored as
sone to its active form of cortisol [25]. It has been sug- fat whenever energy intake exceeds energy expenditure.
gestedthatexacerbationofadiposetissueinobeserodents Hyperphagia, among other factors, seems to have con-
andhumansoccurduetoelevatedglucocorticoidproduc- tributed to the excessive body weight gain that occurred
tion as a result of increased 11β- HSD1 [29,30]. We veri- in our overfed groups. Interestingly,anincreasedappetite
fied that prenatal protein restriction did not alter body was also observed in the prenatal protein restricted rats,
weightpersebutincreasedvisceralfatdeposits.However, when the food consumption data were normalized for
prenatalproteinrestrictioncombinedwithpostnatalover- bodyweight.Thisfindingcontradictstheobservationthat
feeding did not modify the body weight, Lee’s index, and in rodents, unlike in humans, the development of the
visceralpadweightandcontent.Therefore,ourresultsare hypothalamic appetite regulatory system occurs predom-
in disagreement with human studies that showed that inantly after birth [32]. Early postnatal overnutrition,
Table5Biochemicalparametersofratsmaintainedwiththecontrol(CCandLC)oroverfeedingdiets(COandLO)
Variables Groups
CC CO LC LO
n Mean SEM n Mean SEM n Mean SEM n Mean SEM
Triglycerides(mg/dl) 6 181.4 16.4 5 84.2# 7.8 6 190.9* 12.2 6 156.5*# 21.9
LDL-cholesterol(mg/dl) 6 26.6 4.2 6 33.0 3.4 6 30.8 4.7 6 30.5 2.3
HDL-cholesterol(mg/dl) 6 23.6 0.6 6 20.1 1.9 6 24.6 1.2 6 23.5 0.9
Totalcholesterol(mg/dl) 6 86.5 3.3 6 86.3 12.5 6 93.6 7.4 6 85.2 4.4
CastelliI 6 3.7 0.1 6 3.9 0.1 6 3.8 0.2 6 3.6 0.1
CastelliII 6 1.1b 0.2 6 2.1a 0.2 6 1.2b 0.2 6 1.3b 0.1
FFA(μmol/l) 6 0.99 0.1 6 0.72# 0.1 6 0.88 0.0 6 0.84# 0.0
FFA,freefattyacid.
abcMeanvalueswithunlikesuperscriptlettersweresignificantlydifferent(P<0·05;two-wayANOVAfollowedbyleastsignificantdifference(LSD)test).
*Meanvaluesweresignificantlydifferentfromthoseofthecontrolrats(P<0·05;two-wayANOVA).
#Meanvaluesweresignificantlydifferentfromthoseofthenon-overfeedingrats(P<0·05;two-wayANOVA).
nNumbersofrats.
Coutinhoetal.Nutrition&Metabolism2013,10:5 Page7of9
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Table6Fastingserumglucoseandinsulinconcentration,totalareaundertheglucose(AUG)andinsulin(AUI)curves,
glucosedisappearanceratio(K )obtainedfromtheintraperitonealinsulintolerancetestandHOMA-IRofrats
itt
maintainedwiththecontrol(CCandLC)oroverfeedingdiets(COandLO)
Variables Groups
CC CO LC LO
n Mean SEM n Mean SEM n Mean SEM n Mean SEM
Basalglucose(mmol/l) 7 7.0 0.1 7 7.1 0.1 3 6.8 0.0 6 7.0 0.1
Basalinsulin(pmol/l) 7 14.8b 0.8 7 35.3a 2.3 3 34.6a 1.3 6 3.3c 0.7
AUG(mmol/L.120min) 7 2065 31 4 2278# 95 7 2075 27 7 2183# 52
AUI(pmol/L.120min) 7 56b 4 4 200a 53 7 173a 44 7 78b 3
K (%/min) 8 0.28b 0.2 8 2.79a 0.6 7 3.05a 0.3 8 3.13a 0.4
itt
HOMA-IR 7 4.6b 0.3 7 11.1a 0.7 3 10.4a 0.4 6 1.0c 0.2
Leptin(μU/ml) 5 12.1 1.6 5 11.8 1.2 5 10.8 0.4 5 10.2 0.5
abcMeanvalueswithunlikesuperscriptlettersweresignificantlydifferent(P<0·05;two-wayANOVAfollowedbyleastsignificantdifference(LSD)test).
*#Meanvaluesweresignificantlydifferentfromthoseofthecontrolrats(P<0·05;two-wayANOVA).
nNumbersofrats.
induced by small litter size [6,7,24,25,27,33], causes per- Thus, we suggest that IBAT thermogenic activity was
sistent hyperphagia and alters the hypothalamic energy enhanced by the prenatal protein restriction and sup-
homeostasis mechanism in adulthood [2]. The hypothal- pressed (decreased) by overfeeding during lactation.
amusistheprimarycenterinthebrainthatregulatesfood Reduced responsiveness to sympathetic stimulation and
intake and body weight homeostasis [34]. It is subject to down-regulated adrenoreceptors in WAT, commonly
theinfluenceofseveralperipheralfactors,includingcircu- observed in obese rodents [41-43], could have contribu-
lating levels of leptin [35], that which are proportional to ted to the increase in lipid deposits in our overfed ani-
thetotalfatmass[36].Weverifiedthatthecirculatinglep- mals. Thiseffect is manifested by a decrease in fatty acid
tinlevelswerenotmodifiedbyearlyproteinrestrictionor mobilization [44], and our overfed animals exhibited
by overfeeding during lactation. Moreover, the serum lep- reduced FFA levels. FFA release from adipose tissue is
tin concentrations were not equivalent to the absolute and also down-regulated by fasting hyperinsulinemia [45]
relativeweightandlipidcontentoftheRWATandEWAT. and could, therefore, be another factor contributing to
These results were not surprising, at least for postnatal low FFA levels and reduced fatty acid mobilization, at
overfedrats,because,hyperleptinemiaoccursonlyatwean- leastintheCO group.
ing, when subcutaneous adipose tissue is increased [7] and Independent of prenatal nutrition, postnatal overfeed-
leptin is produced mainly by subcutaneous adipose tissue ing resulted in impaired glucose tolerance. Curiously,
[37,38]. It is noteworthy that the hyperphagia in rats exhi- the overfed groups (CO and LO) exhibited opposite
bitingunalteredserumleptinlevels,asobservedintheani- basal and stimulated serum insulin profiles and, varying
malsthatweresubmittedtointrauterineproteinrestriction hepatic insulin sensitivity, but similar peripheral insulin
orearlypostnataloverfeeding,suggestsofleptinresistance. sensitivity. These observations are based on the HOMA-
Spontaneous activity levels, O consumption and CO IR and K values, respectively. Insulin sensitivity esti-
2 2 itt
production were reduced in the animals submitted to mated by HOMA-IR predominantly expresses the ability
prenatal protein restriction that exhibited a lean profile. of basal insulin to suppress hepatic glucose production
In contrast, in the overfed rats that showed a phenotype in a fasting state, whereas K is more influenced by the
itt
of obesity, the activity levels did not change, but the O disposal of glucose after insulin application, reflecting
2
consumption and CO production increased. The higher peripheral insulin resistance [46]. Increase in the body
2
O consumption, CO production and energy expend- weight andLee’sindexwere not accompaniedbyperiph-
2 2
itureintheoverfedanimalscouldbeexplainedbyhigher eral insulin resistance, and the increase in visceral fat
body weight. One possible explanation for the different depots was not a determinant of liver insulin resistance.
phenotypes exhibited by our equally hyperphagic ani- It appears that hepatic insulin resistance was determined
mals is the different levels of facultative thermogenesis. by the serum insulin concentration, corroborating the
This hypothesis is reinforced by the diverse profile of observation that insulin sensitivity and insulin secretion
IBAT weight and lipid content in the overfed and pro- are reciprocal and inversely related [47]. Another ad-
tein restricted rats. It has been demonstrated that IBAT equate explanation for the unrelated visceral fat deposits
thermogenic activity is accompanied by a simultaneous and liver insulin resistance is the mild increase in vis-
elevation infatty acidsynthesisand IBATweight[39,40]. ceral adiposity observed in our overfed animals. The LC
Coutinhoetal.Nutrition&Metabolism2013,10:5 Page8of9
http://www.nutritionandmetabolism.com/content/10/1/5
group had relative RWATand EWAT weights and lipid Acknowledgements
contents similar to the LO group, however only the LC TheauthorsaregratefultoCelsoRobertoAfonsofortheexcellenttechnical
assistance.ThisworkwassupportedbytheBrazilianfoundationsCNPq
group exhibited hepatic insulin resistance. Remarkably,
(ConselhoNacionaldeDesenvolvimentoCientíficoeTecnológicogrant
whereas prenatal protein restriction and overfeeding number:620106/2008-5),CAPES(CoordenaçãodeAperfeiçoamentode
duringlactationresultedinhyperinsulinemiainthebasal PessoaldeNívelSuperior,grantnumber:PROCAD022/2007)andFAPEMAT
(FundaçãodeAmparoàPesquisadoEstadodeMatoGrosso,Grantnumber:
state and after glucose challenge, fetal protein restriction
449161/2009).ThisworkispartofadissertationpresentedbyGrazielle
combined with overfeeding during lactation, produced a VitóriaPontiCoutinhoasapartialrequirementfortheMaster’sdegreein
state of basal insulindeficiency.Although the insulin de- BiosciencesattheFacultyofNutrition,UniversidadeFederaldeMatoGrosso.
ficiency was counteracted by the increase in peripheral
Authordetails
and hepatic insulin sensitivity in the LO group, it was 1MestradoemBiociências,FaculdadedeNutrição,UniversidadeFederalde
not enough to warrant glucose homeostasis. The insulin MatoGrosso,Cuiabá,MatoGrosso,Brazil.2LaboratóriodeAvaliaçãoBiológica
deAlimentos,FaculdadedeNutrição,UniversidadeFederaldeMatoGrosso,
AUC after glucose challenge was similar in the LO and
Cuiabá,MatoGrosso,Brazil.3DepartamentodeAlimentoseNutrição,
CC groups. However, these results are not indicative of FaculdadedeNutrição,UniversidadeFederaldeMatoGrosso,Cuiabá,Mato
normal secretion, because the insulin AUC after GTT Grosso,Brazil.
provides an idea of the amount of insulin that acts on
Received:27September2012Accepted:19December2012
the tissues but cannot provide information on the dy- Published:10January2013
namic of the hormone in terms of secretion, extraction
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