( PDF ) A restrição protéica durante a lactação altera o controle pelo sistema nervoso autônomo da secreção de insulina induzida pela glicose em ratos adultos

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FERNANDA TAÍS MECABÔ

A RESTRIÇÃO PROTÉICA DURANTE A LACTAÇÃO

ALTERA O CONTROLE PELO SISTEMA NERVOSO

AUTÔNOMO DA SECREÇÃO DE INSULINA INDUZIDA

PELA GLICOSE EM RATOS ADULTOS.

“Dissertação apresentada ao Curso de

mestrado em Ciências Biológicas

(áreas de concentração – Biologia Celular),

da Universidade Estadual de Maringá para

a obtenção do grau de Mestre em

Ciências Biológicas.”

Maringá - 2006

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“PROTEIN RESTRICTION DURING LACTATION

ALTERS THE AUTONOMIC NERVOUS SYSTEM

CONTROL ON GLUCOSE-INDUCED INSULIN

SECRETION IN ADULT RATS.”

Orientador: Prof. Dr. Paulo Cezar de Freitas Mathias

Orientanda: Fernanda Taís Mecabô

Maringá – 2006

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AGRADECIMENTOS:

Aos meus pais: Gentil e Rozenete. Minha vovozinha Irma e minha irmã Carol, pelo

apoio incondicional.

Ao meu amor: Antonio, pelo carinho, compreensão e paciência.

Aos colegas de laboratório: Dionizia, Sabrina, Ana Eliza, Fernanda, Diego,

Anderson, Rodrigo, Marcelo e Carioca, pela ajuda nas dificuldades e pelos

momentos de descontração.

Às técnicas: Maroly e Clarice pelo auxílio e amizade.

Ao Prof. Paulo por apostar na minha capacidade.

Aos professores da pós-graduação pelo empenho em transmitir o conhecimento

científico.

Resumo:

1. Objetivo geral: Avaliar a modulação promovida pelo sistema nervoso

autônomo (SNA) na secreção de insulina de ratos adultos que foram

submetidos à desnutrição protéica durante parte da lactação.

Bases teóricas: A desnutrição protéica é um problema importante nos

países em desenvolvimento. Quando mudanças no estado nutricional

ocorrem no início da vida, podem afetar o desenvolvimento dos

mecanismos de controle metabólico desse organismo. Evidências

epidemiológicas indicam que há uma relação entre desnutrição protéica e

um aumento na suscetibilidade a alterações na tolerância a glicose. Estudos

em modelos experimentais de desnutrição protéica perinatal documentam

alterações nas ilhotas pancreáticas. De maneira geral, ocorre uma

diminuição na magnitude da resposta secretória da célula β pancreática e

dessa forma, leva a uma permanente hipoinsulinemia. A glicose é

considerada a biomolécula mais importante na estimulação das células β

pancreáticas durante o processo de secreção de insulina. Todavia, o SNA

constituído pelo Sistema Nervoso Parassimpático (SNP) e Sistema Nervoso

Simpático (SNS) também exercem um importante papel na regulação da

secreção de insulina. O SNP, liberando o seu principal neurotransmissor, a

acetilcolina, através da ligação com receptores muscarínicos nas células β

pancreáticas , potencializa a secreção de insulina estimulada por glicose. O

SNS, liberando ou secretando catecolaminas (adrenalina e noradrenalina),

exerce um papel dual na secreção de insulina, sendo inibitório via

receptores α adrenérgico e estimulatório via receptores β adrenérgico.

Ilhotas pancreáticas de animais que sofreram desnutrição protéica

desenvolvem diversas alterações intrínsecas, como morfologia, expressão

de genes, atividade enzimática, etc. Entretanto, essas evidências não

excluem a possibilidade da participação do SNA como modulador da

secreção deficiente de insulina. Foi observado que a desnutrição protéica

depois do desmame provoca redução na atividade do SNP e aumento da

atividade do SNS. No entanto, é provável que as alterações sofridas por um

mamífero durante a lactação não sejam exatamente as mesmas que

aquelas sofridas pelo animal jovem, levando-se em conta que a lactação é

uma fase em que ocorre uma alta taxa de crescimento e desenvolvimento,

particularmente no que se refere ao Sistema Nervoso.

2. Objetivos específicos: Observar a secreção de insulina in vivo e in vitro,

estimulada por glicose e ainda a modulação exercida pelos agonistas e

antagonistas dos receptores muscarínicos (acetilcolina e atropina) e dos

receptores α adrenérgicos (yohimbina e oxymetazolina) e β adrenérgicos

(isoproterenol).

3. Procedimentos: Ratos Wistar cujas mães receberam nos 14 primeiros dias

da lactação uma dieta isocalórica contendo 4% de proteína (LP) ou uma

dieta contendo 23% de proteína (NP). Aos 21 dias eram desmamados e aos

81dias parte dos animais de ambos os grupos foi submetido ao teste de

tolerância à glicose intravenosa (ivGTT) associada ou não a aceticolina

(ach), atropina (atr), yohimbina (yoh) e oxymetazolina (oxy). O restante dos

animais foram sacrificados. Foi medido o comprimento e o peso corporal. O

índice de Lee foi calculado [peso corporal(g)

1/3

/comprimento naso-anal

(cm). Foram também isoladas e pesadas as gorduras periepididimais e

retroperitoneais para estimar a massa de gordura corporal e o cérebro para

aferir possíveis alterações. As ilhotas pancreáticas foram isoladas pelo

método da colagenase. Ilhotas do grupo NP e LP foram incubadas com

glicose na presença ou ausência de epinefrina (100nM) com propranolol

(100nm) ou com carbamilcolina (1mM) com e sem atr (10µM). Também

foram feitas incubações com iso (10µM) com e sem yoh (10µM).

4. Resultados: Ratos submetidos à desnutrição protéica durante a lactação

apresentaram o peso corporal reduzido em 23% e das gorduras

periepididimal e retroperitoneal em 30% e 49%, respectivamente, tudo

comparado aos NP. A insulinemia de jejum foi 41% menor nos LP sem,

contudo apresentar diferença significativa na glicemia de jejum. O ivGTT

mostrou que a desnutrição provocou intolerância à glicose e

hipoinsulinemia. Os animais LP apresentaram ∆I (calculado a partir da área

sob a curva - AUC) 31% menor que os NP(p<0.05). Ach afetou os LP

aumentando o ∆I (AUC) 64% e em apenas 18% nos NP. Atr teve efeito

somente nos NP reduzindo o ∆I (AUC) em 22% (p<0.05). Oxy reduziu o ∆I

(AUC) em 25% apenas nos NP. Por outro lado, yoh aumentou o ∆I(AUC)

em 65% nos LP e em somente 32% nos NP (p<0.05). Em ilhotas, a

carbamilcolina potencializou a secreção de insulina em 311% nos LP e em

594% nos NP (p<0.05). Epinefrina inibiu a secreção na mesma magnitude

nos dois grupos. Isso potencializou em 70% a secreção nos LP e apenas

35% nos NP (p<0.05).

5. Conclusões: A desnutrição protéica perinatal induz mudanças no

metabolismo que são mantidas na vida adulta. Dentre as alterações está o

baixo peso e a hipoinsulinemia. Ilhotas pancreáticas de animais LP

secretam menos insulina sob o estímulo da glicose tanto in vivo como in

vitro. A resposta secretória das ilhotas aos moduladores autonômicos

também está alterada. Dessa forma os resultados sugerem que a regulação

da secreção de insulina está comprometida, não só o controle pela glicose,

mas também os efeitos insulinotrópicos modulados pelo SNA em ratos

adultos que sofreram desnutrição protéica durante parte da lactação.

Summary:

6. Main Objective: To evaluate the modulation caused by the autonomic

nervous system (SNA) on the insulin secretion in adult rats submitted to

protein undernutrition during part of lactation.

Theoretical foundations: Protein undernutrition is an important problem in

developing countries. When changes in nutritional situation happen in the

beginning of life, they may affect the development of the organism’s

metabolic control mechanisms. Epidemiologic evidences indicate that there

is a relationship between protein undernutrition and increase in susceptibility

to alterations in glucose tolerance. Studies in perinatal protein undernutrition

experimental models show alterations in pancreatic islets. In a general way,

there is a decrease in the magnitude of the secretory response of the β

pancreatic cell, thus leading to permanent hypoinsulinemia. Glucose is

considered to be the most important biomolecule to the β pancreatic cells

stimulation, during the insulin secretion process. However, the SNA,

consisting of the Parasympathetic Nervous System (SNP) and the

Sympathetic Nervous System (SNS), also have an important role in the

regulation of insulin secretion. The SNP, releasing acetylcholine – its main

neurotransmitter – through the binding with muscarinic receptors in the

pancreatic β cells, enhances glucose-stimulated insulin secretion. SNS,

releasing or secreting catecholamines (adrenaline and noradrenaline), plays

a dual role in insulin secretion, being an inhibitor through α adrenergic

receptors and a stimulator through the β adrenergic receptors. Pancreatic

islets from animals who have suffered protein undernutrition develop several

intrinsic alterations, e.g. in morphology, genes expression, enzymatic

activity, etc. However, these evidences do not exclude the possibility of the

participation of the SNA as a modulator of the defective insulin secretion. It

has been observed that the protein undernutrition after weaning provokes a

decrease in SNP activity and increase in SNS activity. Nevertheless, it is

possible that the alterations suffered by a mammal during lactation are not

exactly the same as those ones suffered by the young animal, considering

that lactation is a period in which there is a high rate of growth and

development, especially regarding the central nervous system.

7. Specific Objectives: Observe glucose-induced insulin secretion in vivo and

in vitro, and also the modulation induced by agonists and antagonists of the

muscarinic receptors – acetylcholine and atropine, respectively – and of the

α adrenergic receptors yohimbine and oxymetazoline and one β adrenergic

agonist – isoproterenol in adults rats whose were fed with protein restriction

during part of lactation.

8. Procedures: Wistar rats whose mothers received an isocaloric diet

containing 4% protein (LP) or a 23% protein diet (NP), in the 14 days

following birth. They were weaned at the age of 21 days, and at the age of

81 days part of the animals from both groups was submitted to the

intravenous glucose tolerance test (ivGTT), associated or not to

acetylcholine (ach), atropine (atr), yohimbine (yoh) and oxymetazoline (oxy).

The rest of the animals were killed. Length and body weight were measured.

Lee index was calculated [body weight(g)

1/3

/nasoanal length (cm)].

Periepididymal and retroperitoneal fat was also isolated and weighed to

estimate body fat mass, as well as the brain, to assess possible alterations.

Pancreatic islets were isolated by the collagenase method. Islets from the

NP and LP groups were incubated with glucose in the presence and in the

absence of epinephrine (100nM) or with cholinergic agonist carbamylcholine

(1mM) with and without muscarinic antagonist atropine (10µM). Incubations

were made also with a β adrenergic agonist – isoproterenol (10µM), with

and without yohimbine (10µM).

9. Results: Rats submitted to protein restriction during lactation presented

their body weight reduced in 23% and their periepididymal and

retroperitoneal fat pads in 30 and 49%, respectively, all when compared to

NP rats. Fasting insulinemia was 41% lower in the LP rats though there

were no significant differences in the fasting glycemia. Protein undernutrition

provoked intolerance to glucose and hypoinsulinemia during ivGTT. LP

animals presented ∆I (calculated from the area under the curve) 31% lower

than NP rats (p<0.05). Ach affected LP rats, provoking a 64% increase in

their ∆I, while increasing only 18% in the NP animals. Atr affected only NP

animals, reducing ∆I (AUC) by 22% (p<0.05). Oxy reduced ∆I by 25% only in

NP rats. On the other hand, yoh increased ∆I by 65% in LP and only 32% in

NP (p<0.05). In islets, carbamylcholine increased insulin secretion by 311%

in the LP rats and 594% in the NP ones (p<0.05). Epinephrine inhibited

secretion in the same rate for both groups. Isoproterenol increased secretion

by 70% in LP and only 35% in NP (p<0.05).

10. Conclusions: Perinatal protein undernutrition induces changes in

metabolism which remain during adult life. Among the alterations is low

weight and hypoinsulinemia. Pancreatic islets from LP animals secrete less

insulin under glucose stimulus both in vivo and in vitro. Results suggest that

the regulation of insulin secretion is impaired, not only regarding glucose

control as well as the insulinotropic effects modulated by the SNA in adult

rats who suffered from protein underutrition during part of their lactation

period.

Protein restriction during lactation alters the autonomic nervous system

control on glucose-induced insulin secretion in adult rats.

CLARICE GRAVENA, ANA ELIZA ANDREAZZI, FERNANDA TAIS

MECABÔ, SABRINA GRASSIOLLI, VIVIANE M. SCANTAMBURLO &

PAULO C. F. MATHIAS

Department of Cell Biology and Genetics, State University of Maringá, 87020-900

Maringá, PR, Brazil.

Abstract

Involvement of autonomic nervous system (ANS) neurotransmitters on insulin secretion

in rats submitted to protein malnutrition during lactation was studied. During the first

2/3 of lactation, mothers received a 4% protein diet (LP). Control group received

normal diet (23% protein) (NP). After protein restriction, mothers received normal

diets. At 81 days rats were submitted to ivGTT. Plasma glucose and insulin

concentration (PIC) were measured. Glucose-induced insulin secretion (GIIS) was

tested in pancreatic islets. Fasting normoglycemia and hypoinsulinemia were observed

in LP rats. Glucose intolerance and low PIC in LP group were detected during ivGTT.

Acetylcholine or blockage of α-adrenoceptors induced high PIC increment in LP rats;

atropine or stimulation of α-adrenoceptors did not change PIC. Insulin secretion of LP

rat islets showed low glucose and carbachol responses. Epinephrine inhibited GIIS in

both islet groups. Hypoinsulinemia observed in lactation-malnourished rats might be

caused by alterations in GIIS regulation, including ANS modulation.

Keywords: Protein malnutrition, lactation, insulin secretion, autonomous nervous

system.

Correspondence: Paulo C. F. Mathias, Laboratory of Secretion Cell Biology, Dept. of

Cell Biology and Genetics, State University of Maringá, Av. Colombo 5790, CEP

87020-900, Maringá, PR, Brazil. Phone/Fax: 55 44 32 63 26 48. E-mail:

pmathias@uem.br

INTRODUCTION

Human perinatal malnutrition has been suggested as a risk to predisposing

metabolic and cardiovascular diseases later in life (Bhargava et al., 2004; Waterland &

Garza, 2002). Protein restriction during gestation and lactation in rodents induce

underweight, growth and development impairment in the offspring, and it is associated

with irreversible metabolic disturbances when the pups turn into adult life (Dollet et al.,

1988; Garofano et al., 1999; Langley-Evans, 1997; Rasschaert et al., 1995). It has been

recognized that central nervous system (CNS) is pivotal for metabolism and body

weight control, and the hypothalamus has been identified as the major area

concentrating many neurons implicated in the reception of signals from the periphery,

which induce efferent signals to correct excessive or insufficient food intake and energy

storage (Steffens et al., 1990). Protein malnutrition during gestation and lactation

resulted in underweight, hypoglycemia and hipoinsulinemia in the offspring at weaning,

and it was associated to structural and functional changes in the hypothalamus

(Plagemann et al., 2000a; Plagemann et al., 2000b).

Low plasma insulin concentration and glucose intolerance were observed in

adult rats which were protein-malnourished only during lactation (Barbosa et al., 1999;

Moura et al., 1996). Hypoinsulinemia was associated to an impairment of glucose-

induced insulin secretion observed in pancreatic islets isolated from protein-

malnourished suckling rats; however, the mechanisms which are injured by early

protein restriction are still unclear. A fluctuation in glucose concentrations is by far the

most important signal that stimulates pancreatic islets beta-cells to release insulin into

the blood stream. Glucose enters into beta-cells and immediately stimulates the cell’s

metabolism, leading to ionic events that induce a huge increment in intracellular

calcium. The cytosolic free calcium starts exocytosis of insulin granules (Meglasson et

al., 1989). Other nutrient secretagogues, such as amino acids and fatty acids also

induce insulin secretion; however, in spite of a cellular metabolic regulation, insulin

secretion is also under important neural control. The beta-cells’ membrane is equipped

with many receptors to neurotransmitters and neuropeptides. Autonomic nervous

system (ANS), through their neurotransmitters, modulates the nutrient-induced insulin

secretion. While parasympathetic branches potentiate, sympathetic ends in pancreatic

islets inhibit glucose-induced insulin secretion (Gilon & Henquin, 2001).

Lactation is considered a very important phase in development, including that of

the CNS (Lima et al., 1993). Protein restriction in this phase might cause functional

injuries to the brain, leading to altered neural regulation of glucose-induced insulin

secretion. Therefore, we investigated the effect of protein malnutrition during lactation

on insulin secretion modulated by ANS neurotransmitters in adult rats.

MATERIAL AND METHODS

Dietary manipulation of lactating rats

Female Wistar rats were fed a normal laboratory diet throughout pregnancy.

After delivery the rats were distributed into two groups and each lactating dam was kept

with six pups. Since gender differences in insulin levels and glucose tolerance have

been observed in some early poor protein feeding maneuvers (Lopes Da Costa et al.,

2004), only male offspring were used on the experiments. On the normal protein (NP)

group all dams received a normal-protein (23%) diet ad libitum during lactation,

although the protein reduced (4%) diet had the same amount of calories as the normal

one, as described previously (Barbosa et al., 1999). On the low protein (LP) group all

dams received a low-protein diet ad libitum during the first 14 days of lactation,

returning to normal diet for the remaining 1/3 of lactation. After 21 days the pups were

weaned and received normal diet for 60 days at which time they were used for tests, as

indicated by others (Barbosa et al. 1999). The State University of Maringá Ethical

Committee for Animal Experiments approved the described protocol.

Early Protein Effects on Adult Rats

Adult rats with 81-day-old, NP or LP groups were anaesthetized by an

intraperitoneal injection of pentobarbital sodium [5 mg/100 g body weight (BW)] and

killed by cervical dislocation. The Lee index was calculated from the ratio [BW

1/3

(g)/nasoanal length (cm)] × 1000, used as a predictor of obesity in MSG-rodents

(Bernardis and Patterson, 1968). The periepididymal and retroperitoneal fat pads were

removed, washed with saline solution and weighed. The fat mass of these tissues is used as

a simple reliable estimation of body fat in normal and obese rodents (Rogers and Webb,

1980). To obtain rat brains, before killing, animals were perfused transcardially with saline

solution; the brains were removed and weighed.

Glucose Tolerance Test

Under ketamine and xylasine anesthesia (55mg and 8mg/kg BW, respectively), a

silicone cannula was implanted into 81-days-old rats’ left jugular and attached to the

animals’ back. To avoid blood clotting, heparinized saline (50 IU heparin/ml) was

previously injected into the cannula.

Intravenous glucose tolerance tests (ivGTT) were performed at 8:00 h in 81-days-old

LP and NP rats after a 12h fast (19:00-07:00 h) and without any anesthesia rats were

injected with a glucose load (1g glucose/Kg BW) through the cannula. Blood samples

(300 µl) were collected, from the same cannula, sequentially before glucose load (t0)

and 5 (t5), 15 (t15), 30 (t30) and 60 (t60) min after the injection of glucose. They were

then centrifuged, and the plasma was separated and stored at –20

C for posterior

dosage of glucose concentration by the glucose oxidase method (Kit-Bio Diagnostic

Chemistry Industry®) and insulin by radioimmunoassay (RIA) (Moura et al., 1996).

The total increments of glycemia (∆G) and insulinemia (∆I) were calculated using the

area under glycemia and insulinemia curves for the 60min of ivGTT, subtracting fasting

values. Insulinogenic index was calculated by ∆I (pmol/l/60 min)/ ∆G (mmol/l/60 min).

To investigate whether the decreased insulin response to glucose presented in this

perinatal poor protein nutrition model is related, at least in part, to impairment of

parasympathetic activity, we attempted to reactivate the glucose-induced insulinemia

increase by acetylcholine (Ach) administered exogenously. We used 27 nmoles/kg BW

Ach injected intraperitonealy (ip) 5 min before the ivGTT in LP and NP rats.

To test parasympathetic activity through its muscarinic effect on beta cells we used 20

nmoles/kg BW of muscarinic antagonist atropine (Atr). The drug was ip injected 5 min

before the study of ivGTT in LP and NP rats. Adrenergic adrenoceptors activity was

also investigated, using α

- adrenoceptor antagonist yohimbine. We used 20 nmoles/kg

BW yohimbine hydrochloride that was ip injected 5 min before the glucose load in LP

and NP rats. We also studied the influence of an α

- adrenoceptor agonist,

oxymetazoline (16nmoles/kg BW), on glucose-induced insulinemia increase.

Oxymetazoline was selected because it has been proposed that in the pancreatic β-cell,

the α

adrenoceptor subtype is specifically involved in the inhibition of insulin release

(Ullrich & Wollheim, 1985). Oxymetazoline was ip injected 5min before glucose load

in rats.

To select the concentration of each drug used in the protocol of ivGTT a dose response

curve was performed, measuring plasma insulin concentrations (results not shown). The

doses chosen were obtained by values close to EC

, which reduces or increases in

about 20% the total glycemia increment induced by glucose load in NP rats.

Preparation of islets of Langerhans

Isolation of islets from rat pancreas was performed as previously described

(Boschero et al., 1995) with adaptations. Male adult Wistar rats, only used to islets

isolation, were deeply anesthetized with ketamine and xylasine (55 and 8 mg/kg BW,

respectively), and the abdominal wall was cut and open. A 10 ml Hank’s buffered saline

solution (HBSS) containing collagenase type V (0.7 mg/ml, Sigma Chemical CO., St.

Louis, MO) was injected into the common bile duct of the rat. The pancreas, swollen

with the collagenase solution, was quickly excised and incubated in a plastic culture

bottle for 15 min at 37 ºC. The suspension obtained was filtered with a 0.5mm metal

mesh and washed with HBSS, including 0.12% bovine serum albumin fraction V (BSA)

in 5 continuous washings. The islets were collected with the aid of a microscope.

Insulin secretion from islets stimulated by glucose and neurotransmitters

To each incubation protocol a pool of islets were used, from, at least, 6 rats of

each group. Groups of 4 islets placed on plastic coverslips were pre-incubated for 60

min in 1.0 ml of Krebs-Ringer bicarbonate-buffered solution containing 0.12% (wt/vol)

bovine albumin (fraction V), and glucose 5.6 mM, pH 7.4. The solution was

equilibrated against a mixture of CO

(5%) and O

(95%). After the adaptation to a low

glucose concentration solution, islets were submitted to incubations for further 60 min

in glucose 2.8; 5.6; 8.3; 11.1; 16.7 and 20.0 mM with Krebs-Ringer solutions. To test

the response to cholinergic stimulus, islets were incubated with 8.3 mM of glucose plus

1.0 mM of carbachol in the absence or in the presence of the muscarinic antagonist

atropine 10 µM. Isoproterenol 10 µM, a nonspecific β-adrenoceptor agonist was used

to incubate islets stimulated with 16.7 mM of glucose in the presence or in the absence

of a specific α

-adrenergic antagonist, yohimbine 10µM. Samples of incubation media

were taken and stored frozen until assay to measure secreted insulin by RIA.

Islet insulin content

Insulin content was extracted from 8 sets of 4 islets from each animal group.

Islets were incubated with 0.18 mmol/l HCl-ethanol (1:3, vol/vol) overnight at 4 °C

(Kaiser et al., 1991). The incubation was centrifuged at 10 000 rpm for 20 min and the

supernatants were used to measure the insulin content by RIA.

Data Analysis

Results were given as mean ± SEM. Data were submitted to Student t test or

variance analysis (ANOVA®). In the case of analyses with a significant F, the

differences between means were evaluated by Bonferroni t-test. P values less than 0.05

were considered statistically significant. The tests were performed using GraphPad

Prism version 3.02 for Windows (GraphPad Software®). Glucose concentration

secretion response curve was fitted with sigmoidal concentration response equation

(Insulin release = Min + (Max – Min)/(1+ 10

(log Ec50 – concentration) x Hill slope)

)), using the

same software package.

RESULTS

81 day old rats (LP) that were nursed by dams fed with a low protein diet had

lower body weight and nasoanal length than normal rats (NP) (Table I). LP animals also

presented lower fat storage as indicated by periepididymal and retroperitoneal fat pads.

However, the Lee index was not altered by this early malnutrition model, indicating that

animals were proportionally lean. Nevertheless, fasting plasma glycemia was not altered

by perinatal low protein diet, although the fasting insulinemia was significantly reduced

by 41% (Table I).

Both plasma glucose and insulin level responses to a glucose load (1g/kg BW, iv) were

affected by the low protein treatment (Figure 1). Plasma glucose was increased by 20%

at 5 min in LP rats, compared to the NP rats and remained greater throughout the

ivGTT. Conversely, plasma insulin, which had a fasting state 41% lower in LP animals

than in NP ones, had a further increase, although 46% less than NP animals 5 min after

the glucose load, returning to a state not different from NP at 60 min. The incremental

plasma glucose (∆G) was enhanced 32% in LP rats (505.78 ± 43.53 mmol/l/min)

compared to NP ones (343.17 ± 23.54 mmol/l/min), p<0.01. However, incremental

plasma insulin (∆I) decreased 31% in LP rats (69 182.00 ± 6 428.88 pmol/l/min)

compared to NP animals (100 446.36 ± 2 974.00 pmol/l/min), p<0.05. The

insulinogenic index (∆I/∆G) was significantly lower in LP (143.77 ± 20.49) than in NP

rats (320.86 ± 14.10), p<0.001.

In NP rats, plasma insulin concentration was affected by the exogenous Ach and

its muscarinic antagonist atropine. Ach induced a 18% increase in ∆I, while Atr reduced

it by 22%, when compared with the ∆I values obtained during the ivGTT without any

drug addition (p<0.05), as shown in “Figure 2”. In LP rats, the intraperitoneal injection

of Ach significantly enhanced by 63% the ∆I; however, Atr failed to alter ∆I (Figure 2,

open bars). The adrenergic antagonist yohimbine increased the ∆I by 32% and 65% in

NP and LP rats, respectively. The α

- agonist oxymetazoline reduced the ∆I by 25% in

NP rats but didn’t alter it in LP animals.

Islet insulin content was not different in both rat groups: 43.33 ± 3.95 ng/islet in

NP rats and 37.11 ± 2.70 ng/islet in LP ones (p = 0.2). The insulinotropic effect of

glucose on isolated islets of low protein rats was also evaluated, as shown in “Figure 3”.

Overall, LP islets secreted less insulin than NP islets; however, the calculated half-

maximal insulin release and the hill slope were not altered by the low protein treatment.

The effect of the cholinergic analogue carbachol (Cch) on islets was also measured.

“Figure 4” shows the results obtained with 8.3 mM of glucose. The insulinotropic effect

of Cch on glucose-stimulated insulin secretion in islets from NP rats was 6 fold when

compared to release stimulated by glucose alone (p<0.001); however, islets from LP

rats showed only a 3 fold increase (p<0.001). In results not shown, similar results were

obtained from concentrations of glucose (2.8 - 20.0 mM) in response to Cch 1.0 mM,

and the maximal response to Cch was obtained by 1.0 mM. Muscarinic antagonist

atropine at 10µM was able to inhibit the Cch insulinotropic effect by 80% and 50% in

islets from NP and LP rats, respectively. Atropine alone did not cause any change in

glucose-induced insulin release in islets from both animal groups (results not shown).

Stimulation of islets’ adrenoceptors is shown in “Figure 5”. Epinephrine, as expected,

inhibited the glucose-induced insulin release through the α-adrenergic receptor;

however, the magnitude of the response was the same (50%) in islets from both animal

groups. Isoproterenol is a non-specific β-adrenergic agonist and it potentiated (30%) the

insulin release stimulated by glucose in NP islets, whereas the insulinotropic effect was

greater (80%) in LP islets, p<0.05. The association of β-adrenergic stimulation with

isoproterenol and α-adrenergic blockage resulted, as expected, in the potentiation of the

isoproterenol effect; it was 30% and 23% in NP and LP islets respectively.

DISCUSSION

It has been shown that low protein treatment during gestation and lactation

caused increases of neural density in the hypothalamus of weaned rats, mostly in the

medial area, but low immunopositivity in neurons that express neuropeptide Y and

galanine has also been

detected (Plagemann et al., 2000b). These neurotransmitters have

been known as potent stimulators of food intake (Clark et al., 1984). Furthermore,

studies reported that electric stimulation of the hypothalamic ventromedial area (VMH)

induced hypophagia (Inoue et al., 1991); however, its lesion causes an increase in food

intake (Balkan et al., 1991). It has been observed that VMH is the origin of sympathetic

fibers. Animals with hypoinsulinemia that undergo protein restriction early in life,

including the lactation phase, show persistent underweight and reduction in food

ingestion, even after diet recovery (Barbosa et al., 1999; Marcelino et al., 2004).

Perinatal protein undernutrition induces changes in metabolism. The metabolic changes

are retained in adult life. It has been shown that metabolic imprinting caused by protein

restriction during perinatal life is associated with several degenerative diseases, diabetes

among them (Waterland & Garza, 1999). Our results indicate that those 81 day rats have

low body weight when compared to animals whose mothers were submitted to regular

protein diet during lactation. The LP rats showed low fat accumulation, without

changing the Lee index. In results not shown, LP animals have the same plasma protein

concentration as NP rats. This indicates that those adult rats, after the diet recovery, are

not malnourished; however, perinatal protein restriction caused enough permanent

metabolic changes to reduce the body weight, the length and fat tissue accumulation.

Metabolic imprinting caused by perinatal protein restriction is followed by alterations in

the neural and endocrine system (Young, 2002). We have previously found fasting

hypoinsulinemia and normoglycemia in LP rats (Moura et al., 1996). Increasing plasma

glucose concentration, during ivGTT, showed that LP rats have glucose intolerance and

low plasma insulin concentration. The insulinogenic index (∆I/∆G) of LP rats is low

when compared to NP rats. Those results suggest that this metabolic imprinting

impaired insulin secretion, but increased the tissue glucose uptake induced by insulin

action. We observed high tissue sensitivity to insulin in adult rats that were protein

malnourished during the lactation phase (Moura et al., 1997), even when protein

restriction manipulation was developed during gestation and lactation, tissues showed

increased sensitivity to insulin (Latorraca et al., 1998; Ozanne et al., 1996).

Many different factors contribute to regulate insulin secretion during oscillations

of plasma glucose concentration, among them the neural activity. The ANS, through

parasympathetic branch activity, enhances insulin secretion stimulated by glucose;

whereas sympathetic activity mostly inhibits it (Gilon & Henquin, 2001). It has been

shown that 60 day old rats who were protein malnourished for 4 weeks, presented low

parasympathetic activity and high sympathetic activity, as observed by electric activity

indicated by firing rates recorded from the thoracic branch of the vagus nerve and from

superior cervical ganglion, respectively (Leon-Quinto et al., 1998). This imbalance of

ANS was suggested as a cause of altered plasma insulin levels changes during ivGTT of

protein malnourished adult rats. Exogenous Ach treatment before glucose load induced

high blood insulin concentration in NP animals during ivGTT; although LP rats had

more significant blood insulin concentration increment. The increase of plasma insulin

provoked by exogenous Ach might be attributed to the occupation of the beta cells’

muscarinic receptors, leading to the potentiation of glucose-induced insulin secretion

(Gilon & Henquin, 2001). Results recorded from protein malnourished adult rats during

ivGTT with Ach pre treatment showed less effect on plasma insulin than on the control

rats (Leon-Quinto et al., 1998). Muscarinic antagonist atropine treatment caused, as

expected, a decrease in insulinemia as a result of the blocking of muscarinic receptors;

however, LP rats showed no significant changes in plasma insulin. The set of results

obtained by pretreatment with muscarinic agonist and antagonist suggest that LP rats

have low activation of pancreatic cholinergic ends. Alternatively, it could be suggest

that pancreatic beta cells from LP rats have high numbers of cholinergic receptors

and/or high receptor sensitivity; indeed, atropine treatment was not able to significantly

reduce the plasma insulin increment stimulated by the glucose load. This result can also

indicate that the dose of muscarinic antagonist used in this study, was not enough to

bind significant numbers of muscarinic receptors. One very important component of

insulin secretion control during feeding behavior is the increased vagal activity, which

releases Ach on the parasympathetic ends of pancreatic beta cells; while sympathetic

activity, also associated to feeding behavior, mostly inhibited glucose-induced insulin

secretion, by catecholamine action upon α

adrenergic receptors of pancreatic beta

cells (Peterhoff et al., 2003). Yohimbine, an α

-adrenergic antagonist, caused a

significant increase in plasma insulin in both animal groups, though the effect was

stronger on LP rats. This result indicates that α-adrenergic receptors of beta cells from

LP animals are more responsive than those of NP rats. Alternatively, the result suggests

that beta cells of LP rats have high number and/or high sensitivity of α-adrenergic

receptors. Alternatively, a low adrenergic activity could also leads parasympathetic

activity to stimulate the insulin secretion by Ach release in the pancreatic

parasympathetic terminals, increasing the plasma insulin more efficiently as shown by

LP rats. Oxymetazoline is a potent α

2A-

adrenergic agonist and causes inhibition of

insulin secretion (Ullrich & Wollheim, 1985). This adrenergic agonist, as expected,

reduced plasma insulin during ivGTT in NP rats; however, no changes were observed in

LP rats. This former observation could support the hypothesis that LP animals have

high number of α-adrenergic receptor, because the dose of oxymetazoline was not

enough to activate a great number of the receptors; however, it did with NP animals.

The results obtained with α-adrenergic agonists and antagonists can suggest that

sympathetic activity, as well as parasympathetic activity, is also impaired in LP rats.

In results not shown, ∆G changes caused by pretreatment of cholinergic and

adrenergic agents showed an inverse relationship with ∆I variations also caused by

drugs during the ivGTT.

Pancreatic islets isolated from LP rats showed a weak insulin secretory response

when stimulated by glucose, although their sensitivity was not changed. It is known that

insulin secretion is regulated by cellular metabolism stimulation, which is able to

provoke ionic changes in the membranes with depolarization and intracellular calcium

increase, through calcium uptake and intracellular calcium mobilization. The increased

intracellular calcium allowed beta cells to secrete insulin (Malaisse et al., 1978). It has

been shown that protein restriction during gestation and post natal life induces reduction

in cell metabolic activity as well as in calcium uptake when their islets are exposed to

high glucose concentration (Latorraca et al., 1999). Recently, it has been shown that rats

who were protein restricted during the 12 first days of lactation were not impaired on

their β- cell glucose stimulated metabolism (Barbosa et al., 2002), although the authors

observed that pancreatic islets from protein malnourished rats showed an impairment in

phosphoinositides metabolism when stimulated by glucose and carbachol. It could be

argued that the beta cells of perinatal protein restricted rats are not able to produce

enough insulin to the secretory demands. In fact, low protein offer during gestation and

lactation decreases total insulin content in pancreatic islets (Bertin et al., 1999; Petrik et

al., 1999); however it was showed that protein restriction only during lactation did not

significantly decrease insulin content of pancreatic islets (Barbosa et al., 2002). Indeed,

in our study islet insulin content of NP and LP was not different. Although for sure that

the metabolic imprinting promoted by perinatal protein restriction maneuver induces

changes in the glucose-induced insulin secretion in beta cells, it is very attractive to

consider that pancreatic islets from LP rats could have changed their secretory responses

to neurotransmitters. It has been shown that obese rodents induced by VMH lesion or by

neonatal monosodium glutamate treatment, present high parasympathetic activity

(Balbo et al., 2002; Campfield et al., 1983; Kiba, 2004; Lucinei Balbo et al., 2000). The

pancreatic islets isolated from the obese animals showed high and low insulin secretion

response to glucose and to cholinergic agonists, respectively; however, bilateral

subdiafragmatic vagotomy restored the secretory responses (Balbo et al., 2002;

Campfield et al., 1983; Kiba, 2004). The authors have concluded that the fasting

hyperinsulinemia observed in those obese animals is attributed to beta-cells’ high

responsivity to glucose; as well as the low secretory response to cholinergic agonists

was attributed to high parasympathetic activity, which could be caused by decrease of

the number and/or affinity of muscarinic receptors, or alternative changes in cellular

transduction.

Our results showed that the cholinergic secretory response was weak in islets

isolated from rats who were submitted to protein restriction during lactation; however,

we cannot conclude that there is a mechanism of down regulation of muscarinic

receptors. We have not any evidence of increase or decrease in parasympathetic activity

in LP rats.

Stimulation of α-adrenergic receptors of LP islets showed the same inhibition of

glucose-induced insulin secretion than NP islets, indicating that the reception of

adrenergic signals is intact in LP islets; however, β-adrenoceptor stimulation is more

responsive in LP than in NP islets. Whereas, the overall stimulation of adrenoceptors

inhibits the glucose-induced insulin release, as has been shown in well protein

nourished rats (Gilon & Henquin, 2001).

Recently, it was showed that mice with gene knockout that expressed muscarinic

receptor of M3R subfamily are underweight, hypophagic and hypoinsulinemic (Yamada

et al., 2001), as did adult rats who was protein malnourished during lactation. It has

been shown that the insulinotropic effect of cholinergic agonist in pancreatic islets is

majority displaying throughout M3R muscarinic receptors (Boschero et al., 1995;

Miguel et al., 2002). Pancreatic islets from M3R mice (-/-) showed less secretory

response to cholinergic agonist (Duttaroy et al., 2004). Whereas, we have any evidence,

it is possible that metabolic programming induced by early protein malnutrition, causes

reduction of M3R in pancreatic beta cells.

Taken together, our results indicate that lactation protein restriction leads to

underweight, glucose intolerance and hypoinsulinemia in adult rats. This is followed by

alterations on the control of insulin secretion induced by oscillations of glucose

concentration and by modulation of the autonomic nervous system.

Lactation is a very important phase to organism development, including Central

Nervous System. It has been observed that interventions that alter the childhood

nutrition, including lactation phase, induce changes on development of the infantile

(Sawaya et al., 2003) and this condition is a risk to develop chronic degenerative

diseases, such as obesity, hypertension and type 2 diabetes, mostly in developing

countries with nutritional transition (Fjeld et al., 1989; Popkin et al., 1996). Recently, it

was indicated that disturbance on insulin secretion control of stunted children, who were

early malnourished, is related to early protein restriction experimental model presented

in the current work (Martins & Sawaya, 2006). Epidemiologic studies combine with

experimental findings suggests that undernutrition in childhood has enormous impact on

health of this population. Further research is urgently demanded to reduce the morbidity

and mortality due to diseases originated by perinatal undernutrition.

Acknowlegdgments:

We gratefully acknowledge financial support from CNPq (Brazilian Council for

Scietific and Tecnological Development).

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Metab 13: 381-385.

NP LP

Body weight (g)

364.36 ± 3.60 278.95 ± 3.16 *

Body Lenght (cm)

23.36 ± 0.08 21.31 ± 0.10 *

Lee Index

300 ± 10.00 300 ± 10.00 NS

Brain weight (g/100g BW)

0.51 ± 0.01 0.55 ± 0.01 *

Epididymal fat (g/100g BW)

0.97 ± 0.04 0.68 ± 0.03 *

Retroperitoneal fat (g/100g BW)

0.81 ± 0.04 0.41 ± 0.03 *

Plasma glucose (mmol/l)

5700 ± 140 6340 ± 300 NS

Plasma insulin (pmol/l) 1480 ± 30 870 ± 30 *

Table I. Effect of hypoproteic diet during part of lactation on 81 days rats. Animals

were sacrificed with 14 h of fasting. The results present the mean±SEM of 28 rats from

each group.

p<0,001 (Student’s t test)

0 10 20 30 40 50 60

Time (min)

Plasma glucose (mmol/l)

0 10 20 30 40 50 60

2000

4000

6000

8000

10000

***

Tim e (m in)

Plasma insulim (pmol/l)

Figure 1. Changes in plasma glucose and insulin levels during ivGTT with glucose

load. The upper panel shows the plasma glucose and the low panel shows the plasma

insulin after ivGTT with glucose load 1g/Kg BW. The symbols present the mean of

changes in glycemia and insulinemia during ivGTT, provided by 6 rats from NP and LP

rats. The lines on top of the symbols show the SEM. *p<0.05. **p<0.01, ***p<0.001 in

comparison with the same time (Student’s t test).

100

200

***

Ach Atr Yoh

Oxy

ψψ

Ω

ΩΩ

Ω

ΩΩΩ

∆

I (%)

Figure 2. Changes in plasma insulin levels during ivGTT in rats pretreated with

antagonists and agonists of ANS. Bars represent the mean percentage of insulin

increment (area under curve) obtained on ivGTT with indicated drug. It used 6 different

rats to each drug treatment to NP or LP rats. Dot line represents the total insulin plasma

concentration from area under the curve calculated to all ivGTT (100%), which was

induced by glucose load on NP or LP rats, without any drug pretreatment. The lines on

top of bars indicate the SEM. The letters over the bars refer to differences (p<0.05)

among treatments: (a) acetylcholine, (b) atropine, (c) yohimbine, (d) oxymetazoline to

each group.

*p<0.05;**p<0.01;***p<0.001, comparing with NP rats.

Ω

p<0.05;

ΩΩ

p<0.01;

ΩΩΩ

p<0.001, NP comparing with results obtained without any drug.

p<0.01;

ΨΨ

p<0.001, LP comparing with results obtained without any drug. Data were

analyzed with ANOVA.

-2.75

-2.50

-2.25

-2.00

-1.75

-1.50

Glucose [log M]

***

insulin release (ng/islet/60min)

Figure 3. Dose response curve of glucose-induced insulin release in isolated

pancreatic islets . Symbols represent the mean of 20 different incubations batches of 4

islets incubated during 60 min to each glucose concentration of the curve. Lines on the

symbols represent the respective SEM. (*) p<0.05, (**)p<0.01, (***)p<0.001 when

compared with LP group (Student's t test).

NP LP

Glucose + + + + + +

Cch - + + - + +

Atr - - + - - +

insulin release(ng/islet/60min)

Figure 4. Effect of cholinergic activation on glucose-induced insulin secretion in

pancreatic islets. The bars represent the mean of insulin released from 10 diffrent

batches of islets isolated from NP or LP rats, incubated for 60 min, as indicated over

the bars. Islets were stimulated by 8.3 mM of glucose in the presence or in the absence

of carbachol (1mM) and/or atropine (10 µM). The lines on the top of bars represent

SEM. Letters over the bars represent significant differences (p<0,05) among results

from intra group by ANOVA. (a) glucose only, (b) glucose+Cch, (c) glucose+Cch+ Atr.

Glucose + + + + + + + +

Epi - + - - - + - -

Iso - - + + - - + +

Yoh - - - + - - - +

NP LP

bcd

acd

abd

abc

bcd

acd

abd

abc

insuilin release (ng/islet/60min)

Figure 5. Effect of adrenoceptors activation in pancreatic islets stimulated by

glucose 16.7 mM. The bars represent the mean of insulin released obtained from 10

batches of islets incubation with glucose in the presence or in the absence of adrenergic-

agonists and/or antagonist during 60 min. Line on the top of bars represent SEM.

Letters over the bars represent significant differences (p<0,05) among results from intra

group by ANOVA. (a) glucose 16,7mM only, (b) glucose+epinephrine 100nM, (c)

glucose+isoproterenol 10µM and (d) glucose+isoproterenol+yohimbine 10µM.

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