( PDF ) Algumas características dos fígados de ratos com diabetes tipos 1 e 2 e suas respostas metabólicas à glutamina

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Algumas características dos fígados

de ratos com diabete tipos 1 e 2 e

suas respostas metabólicas à

glutamina

Denise Silva de Oliveira

Orientadora: Dra. Fumie Suzuki-

Kemmelmeier

Maringá

2006

Dissertação apresentada ao Programa de Pós

Graduação em Ciências Biológicas da

Universidade Estadual de Maringá, área de

concentração em Biologia Celular, para obtenção

do grau de Mestre

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Este é um trabalho de equipe, realizado no Laboratório de

Metabolismo Hepático da Universidade Estadual de Marin-

gá; os resultados estão descritos e discutidos nos artigos:

1) Denise Silva de Oliveira, Fumie Suzuki-Kemmelmeier,

Ciomar Aparecida Bersani Amado, Luci Tiemi Ide and

Adelar Bracht. Glycogen levels and energy status of the liver of

fasting rats with diabetes types 1 and 2. Brazilian Archives of

Biology and Technology.

2) Denise Silva de Oliveira, Fumie Suzuki-Kemmelmeier,

Ciomar Aparecida Bersani Amado, Luci Tiemi Ide and

Adelar Bracht. The metabolic responses to glutamine of livers

from rats with diabetes types 1 and 2. Archives of Physiology and

Biochemistry (submetido).

Ficha catalográfica  BCE/UEM

Oliveira, Denise Silva de

Algumas características dos fígados de ratos com

diabete tipos 1 e 2 e suas respostas metabólicas

à glutamina  Maringá: UEM, 2006.

37 p., tabelas, gráficos.

Diss. (mestrado)  UEM-DBQ, 2006.

Orientadora: Dr. Fumie Suzuki-Kemmelmeier

Diabete tipo 1;

2. Diabete tipo 2; 3. níveis

plasmáticos; 4. neoglicogênese; 5. ureogênese; 6.

amoniogênese; 7. consumo de oxigênio; 8. gluta-

mina.

I. Universidade Estadual de Maringá.

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RESUMO

NTRODUÇÃO E OBJETIVOS

 A utilização de glutamina é essencial para o

funcionamento normal de grande número de células e tecidos. Esta utilização

ocorre através da enzima glutaminase que, no fígado, predomina em hepató-

citos periportais. A glutaminase é considerada a enzima-chave para a utilização

da glutamina e sua atividade pode mudar sob certas condições patológicas. Em

ratos com diabete induzida pela estreptozotocina,

aumentos de até 12

vezes

foram relatados. Consistentemente, a utilização de glutamina por hepatócitos

isolados de ratos diabéticos também aumenta. Mas, um aumento máximo de

40% foi encontrado, valor modesto em comparação com o incremento na ativi-

dade da glutaminase. Nos experimentos com hepatócitos isolados o metabolis-

mo da glutamina foi medido durante 30 minutos. Porém, foi mostrado em ex-

perimentos com o fígado perfundido de ratos saudáveis, que a utilização de

glutamina possui uma fase de retardo de modo que mais de 30 minutos são

necessários para atingir condições de estado estacionário. Assim, medidas úni-

cas de metabolização de glutamina em hepatócitos isolados após 30 minutos de

incubação podem estar revelando um quadro incorreto sobre as diferenças

entre ratos controle e diabéticos se a dependência do tempo também tiver sido

alterada. Por esta razão, a influência do diabete sobre o metabolismo da gluta-

mina foi re-examinada no presente estudo. Fígados de ratos com diabete tipos

1 e 2 foram perfundidos com glutamina até atingir condições de estado esta-

cionário. Vários parâmetros, tais como neoglicogênese e ureogênese, foram

medidos com o objetivo de obter uma visão mais ampla e detalhada a respeito

das alterações causadas pelo diabete no metabolismo da glutamina. Além disto,

foram medidos vários parâmetros

ratos

com

diabete

tipos 1 e 2,

tais

como

glicogênio

hepático

a carga energética, de modo a caracterizar melhor o

modelo experimental a ser utilizado.

ÉTODOS



Ratos Wistar machos (220-250 g), alimentados com ração pa-

dronizada (Nuvilab



), foram utilizados. O diabete tipo 1 e o diabete tipo 2 foram

induzidos com estreptozotocina, utilizando procedimentos tradicionais. Os experi-

mentos de perfusão foram feitos após períodos de jejum de 24, 48 ou

72 horas.

fígado foi perfundido isoladamente no modo não-recirculante com tampão Krebs/

Henseleit-bicarbonato (pH 7,4), saturado com uma mistura de O

e CO

(95:5)

através de um oxigenador de membrana com aquecimento simultâneo a 36

C. A

concentração de oxigênio no perfusado efluente foi monitorada por polarografia. Os

seguintes metabólitos foram dosados no perfusado efluente através de métodos

enzimáticos: glicose, lactato, uréia, amônia, glutam

3) O consumo de oxigênio de fígados de ratos com diabete 1 durante

perfusão sem substratos foi sempre menor que aquele dos outros dois grupos.

4) A liberação de glicose de figados de ratos com diabete 1 em perfusão livre

de substratos foi muito alta no início da perfusão, sendo necessário um longo

período de pré-perfusão para diminuir os níveis basais. Valores iguais àqueles

de fígados de ratos controle e com diabete2 só foram obtidos em fígados de

ratos submetidos a jejum de 72 horas.

5) A resposta à infusão de glutamina (5 mM) foi mais rápida no fígado de

ratos com diabete1 em jejum de 24 horas para cinco das variáveis medidas

neste trabalho, que foram os aumentos na produção de glicose, lactato, amônia

e uréia e no consumo de oxigênio. Os tempos médios para atingir metade da

resposta máxima (t

) foram iguais a 32,66±2,26 minutos para a condição

controle e 17,55±2,58 minutos para a condição diabete1. Em geral, durante um

período inicial de 30 minutos após o início da infusão, os aumentos gerados pela

glutamina nestes parâmetros foram consideravelmente maiores nos fígados de

ratos com diabete1. Com o progresso da infusão de glutamina a produção de

todos os metabólitos tendeu a atingir estados estacionários iguais na condição

controle e na condição de diabete1. Ao menos no tocante à produção de glico-

se, o aumento do período de jejum para 48 e 72 horas não alterou a diferença

de comportamento entre o fígado de ratos controle e diabéticos.

6) O fígado de ratos com diabete2 respondeu à glutamina de modo muito

semelhante ao fígado de ratos controle.

7) Durante 90 minutos de perfusão livre de substrato, o fígado de ratos con-

trole produziu 9,60±0,31 µmol de glicose por grama de fígado, somente 0,36

µmol g

−1

resultantes de glicogenólise; sob as mesmas condições o fígado de

ratos com diabete1 produziu 19,14±2,29 µmol de glicose g

−1

, quantidade atri-

buível à glicogenólise, que foi de 19,08 µmol g

−1

. Esta glicogenólise no fígado de

ratos diabéticos foi reduzida a 11,48 µmol g

−1

pela infusão de glutamina (90 mi-

nutos). Na condição controle, uma síntese líquida de glicogênio de 1,0 µmol g

−1

ocorreu na presença de glutamina. Em termos globais, a nova síntese de glicose

(neoglicogênese) na presença de glutamina (90 minutos) foi similar nos fíga-

dos de ratos controle e diabéticos (32,7 e 29,3 µmol g

−1

, respectivamente).

ONCLUSÕES E

ISCUSSÃO



Os altos níveis de glicogênio hepático de ratos

com diabete1 exigem monitoramento da glicogenólise para uma correta ava-

liação neoglicogênese.

A resposta à glutamina do fígado de ratos com diabete2

não difere substancialmente daquela do fígado de ratos saudáveis (controle). O

fígado de ratos com diabete1, porém, responde diferentemente à glutamina. As

diferenças, no entanto, são transitórias e caracterizam-se principalmente por

uma aceleração dos fluxos metabólicos durante os estágios iniciais da infusão

(10-30 minutos). Com a aproximação do estado estacionário, as diferenças

tendem a desaparecer. A principal razão é que o metabolismo da glutamina na

condição controle continua a acelerar e não atinge o seu máximo antes dos 50

minutos de infusão. Marginalmente pode ocorrer também no fígado de ratos

com diabete1 uma desaceleração do metabolismo inicial da glutamina após 20

ou 30 minutos. Estes dados confirmam o metabolismo acelerado da glutamina

encontrado em hepatócitos isolados de ratos com diabete1 durante 30 minutos

de incubação. Eles sugerem, no entanto, que a velocidade da transformação

hepática da glutamina in vivo, onde predominam condições de estado estacio-

nário, é provavelmente similar em ratos saudáveis (controle) e diabéticos. Uma

transformação acelerada de glutamina em ratos com diabete1 estaria, portan-

to, restrita a episódios de transição entre diferentes estados estacionários.

ABSTRACT

NTRODUCTION AND AIMS



Glutamine utilization is essential for the normal

functioning of a great number of cells and tissues. Glutamine utilization occurs

via glutaminase which in the liver is more concentrated in periportal hepato-

cytes. Glutaminase is considered a key-enzyme for hepatic glutamine utilization

and its activity changes under certain pathological conditions. Increases up to

12-fold have been reported for streptozotocin diabetic rats. Consistently, gluta-

mine utilization by hepatocytes isolated from streptozotocin diabetic rats was

also increased. However, a maximal increase of 40% was observed, a modest

stimulation if one takes into account the enormous increase in glutaminase

activity. The experiments with isolated hepatocytes have been done by measur-

ing glutamine metabolism during 30 minutes. However, it has been shown in

experiments with the isolated perfused rat liver from healthy rats that the

utilization of glutamine is characterized by a lag phase so that much more than

30 minutes are required for attaining steady-state conditions. In this way,

single measurements of glutamine metabolism in isolated hepatocytes after 30

minutes incubation can reveal an untrue picture of the real differences between

healthy and diabetic rats if the time dependence is also changed. For this

reason, the influence of diabetes on glutamine metabolism was reexamined in

the present study. Livers from rats with diabetes types 1 and 2 were perfused

with glutamine until steady-state conditions were attained. Several parameters,

such as gluconeogenesis and ureagenesis were measured in order to obtain a

more ample and detailed vision about the changes caused by diabetes in gluta-

mine metabolism. Besides this, several parameters were measured in livers and

plasma of rats

with

diabetes

tipes 1 and 2,

such as the hepatic glycogen content,

the hepatic energy charge and the plasma levels of glucose, urea and ammonia,

in order to characterize more precisely the experimental model.

ETHODS



Male Wistar rats (220-250 g), fed with a laboratory diet (Nuvilab



were used. Both type 1 and type 2 diabetes were induced with streptozotocin, using

traditional procedures. The perfusion experiments were done after fasting periods

of 24, 48 or 72 hours. The isolated liver was perfused in the non-recirculating

mode. The perfusion fluid was the Krebs/Henseleit-bicarbonate buffer (pH 7.4), sa-

turated with a mixture of O

and CO

(95:5) by means of a membrane oxygenator

with simultaneous temperature adjustment (36

C). The oxygen concentration in the

effluent perfusate was monitored continuously by polarography. The following me-

tabolites were determined in the outflowing perfusate: glucose, lactate, urea,

ammonia, glutamate and alanine. Metabolic fluxes were calculated from the portal-

venous differences and the flow through the liver and referred to the wet weight of

the organ. Hepatic glycogen and adenine nucleotide levels were also determined as

well as the plasma concentrations of glucose, urea and ammonia.

ESULTS



1) The fasting glycemy obeyed the following decreasing

sequence: diabetes1 rats (7.55±0.47 mM) > diabetes2 rats (5.91± 0.25 mM) >

control rats (4.80±0.14 mM). The plasmatic levels of urea and ammonia in rats

with diabetes1 were superior to those of control rats and rats with diabetes2.

2) The hepatic glycogen levels of rats with diabetes1 after a 24-hours fast

were much higher that those of control rats and rats with diabetes2, but they

diminished when the fasting period was extended to 48 and 72 hours. The

opposite occurred with control rats and rats with diabetes2, so that the dif-

ference between the various groups vanished after a 72-hours fasting period.

3) Oxygen uptake of livers from rats with diabetes1 during substrate-free

perfusion was always smaller in comparison with that of the other two groups.

4) Glucose release in livers from rats with diabetes1 during substrate-free

perfusion was very high at the beginning of the perfusion and a long period of

pre-perfusion was necessary for dropping the basal rates. Even so, basal rates

similar to those of livers from control rats were obtained only after submitting

the rats with diabetes1 to a fasting period of 72 hours.

5) The response to glutamine infusion (5 mM) was faster in the liver of 24-

hours fasted rats with diabetes1 for five of the metabolic variables measured in

the present work; these variables were the increases in the productions of glu-

cose, lactate, ammonia and urea and the increase in oxygen uptake. The mean

times for reaching half-maximal response (t

) were equal to 32.66±2.26

minutes for the control and 17.55±2.58 minutes for the diabetes1 condition.

During an initial period of 30 minutes after starting infusion, the increases

caused by glutamine were considerably higher in the livers from rats with

diabetes1. As the glutamine infusion was continued, however, the production of

all metabolites tended to reach steady-state levels which were similar in the

control and diabetes1 conditions. At least with respect to glucose production,

extension of the fasting period to 48 or 72 hours, did not change the difference

between the behaviour of livers from control and diabetic rats.

6) The liver of rats with diabetes2 responded to glutamine in almost the same

way as the liver of control rats.

7) During a substrate-free perfusion period of 90 minutes the liver of control

rats produced 9.60±0.31 µmol glucose per gram liver, only 0.36 µmol g

−1

resulting from glycogenolysis; under the same conditions the liver of rats with

diabetes1 produced 19.14±2.29 µmol glucose g

−1

, an amount attributable to

glycogenolysis which was equal to 19.08 µmol g

−1

. This glycogen degradation in

the liver from diabetic rats was reduced to 11.48 µmol g

−1

by glutamine infusion

(90 minutes). In livers from control rats net glycogen synthesis of 1 µmol g

−1

occurred in the presence of glutamine. In global terms, new glucose synthesis

(gluconeogenesis) in the presence of glutamine (90 minutes) was the similar in

livers of control and diabetic rats (32.7 and 29.3 µmol g

−1

, respectively).

ONCLUSIONS AND

ISCUSSION



The high levels of hepatic glycogen in

fasted rats with diabetes1 require monitoring of glycogenolisis for a correct

evaluation of gluconeogenesis.

The response to glutamine of the liver from

rats with diabetes2, when under isolated perfusion, does not differ substantially

from that one of the liver from healthy rats (control). The liver of rats with

diabetes1, however, responds differently to glutamine, but the differences are

transient and characterized mainly by an acceleration of the metabolic fluxes

during the initial stages of glutamine infusion (10-30 minutes). When approach-

ing steady-state

conditions,

the differences tend to disappear.

The main reason

is that the glutamine metabolism in control livers continues to accelerate and

does not reach its maximum before 50 minutes infusion. To a smaller extent it

also occurs because the accelerated initial glutamine metabolism in diabetes1

livers is downregulated after 20 or 30 minutes. These data confirm the ac-

celerated glutamine metabolism found in hepatocytes isolated from rats with

diabetes1 during 30 minutes of incubation. They suggest, however, that the

velocity of hepatic transformation of glutamine in vivo, where steady-state con-

ditions predominate, is probably similar in both healthy (control) and diabetic

rats. An accelerated transformation of glutamine in rats with diabetes1 would

does be restricted to episodes of transition between two different steady-states.

Glycogen levels and energy status of the liver of fasting

rats with diabetes types 1 and 2

Denise Silva de Oliveira, Ciomar Aparecida Bersani Amado, Mirian Carvalho Martini,

Fumie Suzuki-Kemmelmeier and Adelar Bracht

Laboratory of Liver Metabolism; University of Maringá; e-mail: [email protected]; 87020900

Maringá (Brazil)

ABSTRACT

Glycogen levels and the energy status of livers from fasting rats with diabetes types 1 and 2 were

measured. The main purpose was to obtain basic information for planning investigations on gluco-

neogenesis from several substrates in fasting diabetic rats. Diabetes1 rats presented very high levels

of blood urea; blood ammonia was also increased. In diabetes2 rats the blood urea and ammonia

levels were within the normal range. After a 24 hours fast the hepatic glycogen levels of rats with

diabetes1 were 18.7 times higher than those of livers from normal rats. Rats with diabetes2 presented

2.6 more hepatic glycogen than control rats. In diabetes1 rats the glycogen levels decreased when the

fasting period was extended to 48 and 72 hours. The opposite occurred with control and diabetes2

rats so that after a 72 hours fast all groups presented nearly the same hepatic glycogen levels.

Consistently, glucose release by perfused livers from diabetes1 rats was considerably higher during at

least 60 minutes after initiating perfusion although the difference tended to diminish along with the

perfusion time. The hepatic ATP content of diabetes1 rats was similar to that of the control rats; in

diabetes2 rats the hepatic ATP content was increased. It can be concluded that regulation of glycogen

deposition and degradation in rats with diabetes1 differs markedly from that of rats with diabetes2

which, in turn, behave similarly to normal healthy rats.

Key words: Diabetes type 1; diabetes type 2; rats; hepatic glycogen; hepatic energy status.

INTRODUCTION

In spite of decades of experimental efforts there

are many aspects of the hepatic metabolism of

diabetic animals that have not yet be satis-

factorily clarified. Contradictory observations

are frequently found in the specialized literature.

For example, earlier experiments with isolated

hepatocytes of alloxan diabetic rats have lead to

the conclusion that hepatic gluconeogenesis

from alanine, pyruvate and fructose is con-

siderably increased in this preparation (Wagle et

al., 1975). Experiments with the isolated per-

fused rat liver, however, concluded that gluco-

neogenesis from lactate, glycerol and sorbitol is

not affected by alloxan and streptozotocin in-

duced diabetes, whereas gluconeogenesis from

alanine is absent in these animals (Ferraz et al.,

1997). Kleckner et al. (1987), however, had

found diminished rates of gluconeogenesis from

lactate in perfused livers from alloxan diabetic

rats. A common characteristic of all these

experiments is that livers from diabetic rats

present high rates of basal glucose release, i.e.,

before the introduction of the gluconeogenic

substrates. This could be due to glycogenolysis

or gluconeogenesis from endogenous substrates.

Actually, there are indications that the glycogen

levels of diabetic rats in the fasting state are

much higher than those in normal healthy rats

(Gannon and Nuttall, 1997), so that the elevated

basal rates of glucose release observed by

several authors could be the result of glyco-

genolysis. The present work represents an

attempt of investigating in detail how the hepatic

glycogen levels of diabetic rats behave during

various fasting periods. Such a study is im-

portant because it will produce basic information

which can be used for planning future

experiments in which gluconeogenesis from

several substrates in fasting diabetic rats will be

investigated. In order to widen the scope and

reach of the investigation both rats with diabetes

types 1 and 2 were analyzed. In addition, other

parameters were measured such as liver weight

and the hepatic energy status

MATERIAL AND METHODS

Materials. All enzymes and coenzymes used in

the enzymatic assays were purchased from

Sigma Chemical Co. (St Louis, USA). All

standard chemicals were from the best available

grade (98-99.8 % purity).

Animals and diabetes induction. Male Wistar

rats weighing 220–250 g fed with a standard

laboratory diet (Nuvilab



) were used in all

perfusion experiments. Type 1 diabetes (dia-

betes1) was induced by injecting adult rats with

streptozotocin. Streptozotocin was dissolved in

citrate buffer (10 mM; pH 4.5) and a single dosis

of 50 mg/kg was injected intraperitoneally. After

12 days blood glucose of fed rats was measured

and animals presenting glycemic levels equal or

above 16 mM were selected for the experiments.

Control rats were injected intraperitoneally with

citrate buffer (10 mM, pH 4.5).

Type 2 diabetes mellitus (diabetes2) was in-

duced as described by Portha et al. (1974). Male

newborn (2 days old) Wistar rats were injected

intraperitoneally with streptozotocin (150 mg/

kg) dissolved in citrate buffer (10 mM; pH 4.5).

Seven weeks later, diabetes was confirmed by

urine glucose levels, 24 hours urinary volume

and water intake. The protocol for these

experiments was approved by the Ethics Com-

mittee for Animal Experimentation of the Uni-

versity of Maringá.

Liver perfusion. Hemoglobin-free, non-recir-

culating perfusion was done according to the

technique described by Scholz and Bücher

(1965). For the surgical procedure, the rats were

anesthetized by intraperitoneal injection of

sodium thiopental (50 mg/kg).

After cannulation of the portal and cava veins

the liver was positioned in a plexiglass chamber.

The flow was maintained constant by a

peristaltic pump (Minipuls 3, Gilson, France)

and was adjusted between 30 and 35 ml min

−1

depending on the liver weight. The perfusion

fluid was Krebs/Henseleit-bicarbonate buffer

(pH 7.4), saturated with a mixture of oxygen and

carbon dioxide (95:5) by means of a membrane

oxygenator with simultaneous temperature ad-

justment at 36

C. The composition of the Krebs/

Henseleit-bicarbonate buffer is the following:

115 mM NaCl, 25 mM NaHCO

, 5.8 mM KCl,

1.2 mM Na

1.18 mM MgCl

, 1.2 mM

NaH

and 2.5 mM CaCl

. Glucose in the

outflowing perfusate was measured enzymat-

ically (Bergmeyer and Bernt, 1974).

Analytical. Glycogen was determined in freshly

isolated livers from anesthetized rats (sodium

thiopental, 50 mg/kg). Portions of approximately

2 g were freeze-clamped with liquid nitrogen.

This sample was homogenized and extracted

with 8 ml of 6% HClO

. The supernatant was

neutralized with 5 N K

and used for

enzymatic glycogen assay (Kepler and Decker,

1974).

For the measurement of the cellular adenine

nucleotide contents livers were freeze-clamped

in liquid nitrogen, homogenized and extracted

with perchloric acid. The neutralized extracts

were used for the enzymatic assay of ATP

(Lamprecht and Trautschold, 1974), ADP and

AMP (Jaworek et al., 1974).

Plasma concentrations of glucose, urea and

ammonia were assayed by collecting blood from

the vena cava. Blood was collected by means of

a syringe containing heparin after a surgical

incision into the abdominal cavity of

anaesthetized rats (sodium thiopental, 50

mg/kg). After sedimenting the blood cells in a

refrigerated centrifuge (10 minutes, 7000 rpm),

plasma was used for the enzymatic assays of

urea (Gutmann and Bergmeyer, 1974), ammonia

(Kearney and Kun, 1974) and glucose (Berg-

meyer and Bernt, 1974).

Treatment of data. Statistical analysis of the

data was done by means of the Statistica

program (Statsoft



, 1998). Mono- or multi-

variate variance analysis was applied according

to context with post-hoc testing; p < 0.05 was

adopted as a criterion of significance.

RESULTS AND DISCUSSION

Table 1 shows some characteristics of the

plasma of rats with diabetes types 1 and 2 in the

fasted state. After a fasting period of 24 hours

the plasma glucose concentrations of both rats

with diabetes1 and 2 were, respectively, 57.3%

and 23.1% above the normal values. These

values are smaller than those ones normally

found in fed diabetic rats (Portha et al., 1974;

Gannon and Nuttall, 1997). The ammonia and

urea concentrations of rats with diabetes2 were

normal, but rats with diabetes1 presented con-

siderably higher urea (+401.5%) and ammonia

(+90.9%) concentrations. These higher blood

urea and ammonia concentrations in rats with

diabetes1 are probably reflecting the higher rates

of aminoacid degradation (Squires et al., 1997)

which also increase the urinary urea excretion

(Kim et al., 2005). However, the increased urea

excretion is combined with a much higher

urinary volume and lower urinary urea

concentrations (Kim et al., 2005). The high

blood urea concentrations are, thus, the conse-

quence of both higher aminoacid catabolism and

impaired renal function. Investigations about the

urea cycle in the liver of diabetes1 rats are

highly desirable because this tissue is the main

source of urea. It is interesting to note that rats

with diabetes2 do not present such visible

alterations in nitrogen catabolism.

Figure 1 illustrates the changes in the hepatic

glycogen contents and the relative liver weights

as a function of the fasting time. Rats with

diabetes1 presented higher liver weights per

body weight (g liver per 100 gram body weight)

after a 24 hours fast. The difference vanished,

however, when the fasting period was increased.

For rats with diabetes2, the relative liver weight

was similar to that of the control rats, irrespec-

tive of the fasting period. The higher liver

weights of diabetes1 rats after a 24 hours fast

could be caused, partly at least, by their high

glycogen contents because of the pronounced

hygroscopic properties of this polysaccharide.

As shown in Figure 1 rats with type 1 diabetes

contained high levels of hepatic glycogen after a

24 hours fast, the diabetes1/control ratio equal to

18.7. It should be noted that the relatively high

levels of hepatic glycogen in 24 hours fasted

diabetes1 rats, 55.8 µmol g

−1

are actually much

lower than the maximal levels normally attained

by normal fed rats which may reach values as

high as 330 µmol g

−1

(Bazotte et al., 1990). Rats

with type 2 diabetes also presented higher he-

patic glycogen levels, namely 8.2 µmol g

−1

, but

Figure 1. Evolution of liver weight and glycogen

content as a function of the fasting time in control

rats and rats with diabetes1 and 2. Statistically

significant differences between diabetes1 and control

or diabetes2 and control are indicated by asterisks (p

< 0.05). Bars represent mean standard errors of a

minimum of 3 and a maximum of 17 determinations.

Figure 2. Rates of glucose production by rat livers

as a function of time after connecting the liver to

the perfusion apparatus. Livers were perfused with

Krebs/Henseleit-bicarbonate buffer (pH 7.4). Sam-

pling of the outflowing perfusate was initiated im-

mediately after connecting the liver to the perfusion

apparatus. Glucose in the outflowing perfusate was

measured enzymatically. Data are means ± mean

standard errors of 6 (control) and 12 (diabetes1) liver

perfusion experiments.

Perfusion time (minutes)

0.5

1.0

1.5

2.0

2.5

Glucose production (

mol min

−

)

 Diabetes1

 Control

Fasting time (hours)

Glycogen content (µmol glucosyl per gram liver)

,  Control

,



Diabetes 1

, Diabetes 2

Liver weight (g per 100 g body weight)

the diabetes2/control ratio was only 2.76.

Prolongation of the fasting period produced

decreases in the glycogen levels of rats

with dia-

betes1 and increases in the glycogen levels of

rats with diabetes2 as well as in control rats. At

72 hours all groups presented similar hepatic

glycogen concentrations. These observations

mean that no net glycogenesis took place in

diabetes1 rats during the period between 24

and 72 fast. Net glycogen synthesis, however,

must have occurred in control and diabetes2 rats

during the same period. In the absence of

exogenous sources this net glycogen accumula-

tion must have occurred at the expense of

gluconeogenesis, which in turn, depended ex-

clusively on endogenous sources.

Since diabetes1 rats present relatively high

glycogen levels, glucose release by perfused

livers of such animals should be relatively

pronounced if no exogenous gluconeogenic

substrates are added to the perfusion fluid. This

hypothesis was tested by perfusing livers form

both control and diabetes1 animals with

substrate-free perfusion fluid. Sampling of the

outflowing perfusate for glucose assay was

initiated immediately after connecting the liver

to the perfusion fluid. The results are shown in

Figure 2. Glucose release from control livers

was small and almost constant over the 60

minutes perfusion time. Livers from diabetes1

animals, however, showed initially very high

rates of glucose release, the diabetes1/control

ratio being equal to 15.6 at time zero. These high

rates diminished progressively during the sub-

sequent time and the diabetes1/control ratio was

reduced to 3.59 at 60 minutes perfusion time.

Table 1

Plasma concentrations of glucose, ammonia and urea in 24-hours fasted control and diabetic rats. Blood

was collected and analyzed enzymatically. Data are means ± SEM. Asteriscs are used to indicate values differing

statistically from the corresponding controls according to the Student-Newman-Keuls test applied after

monovariate variance analysis (p < 0.05).

Compound Control Diabetes1 Diabetes2

Glucose

4.80±0.14 (n=5) 7.55±0.47* (n=18) 5.91±0.25* (n=9)

Ammonia

0.11±0.01 (n=8) 0.21±0.03* (n=11) 0.14±0.01 (n=7)

Urea

4.83±0.26 (n=8) 24.22±1.68* (n=11) 4.74±0.42 (n=7)

Table 2

Hepatic contents of adenine nucleotides in 24-hours fasted control and diabetic rats. The liver freeze-

clamped in liquid nitrogen, extracted with cold perchloric acid and the adenine nucleotides were determined

enzymatically. Data are means ± SEM. Asteriscs are used to indicate values of the diabetes1 or diabetes2

conditions differing statistically from the corresponding controls according to the Student-Newman-Keuls test

applied after variance analysis (p < 0.05).

Animal

condition

ATP ADP AMP

Total

nucleotides

µmol (gram liver wet weight)

−1

Control (n=13)

1.42±0.17 1.16±0.05 0.35±0.05 2.93±0.11

Diabetes1 (n=2)

1.32±0.07 0.87±0.06* 0.23±0.04 2.42±0.18*

Diabetes2 (n=9)

2.16±0.08* 1.11±0.02 0.25±0.02 3.52±0.08*

This decrease represents most probably the

progressive diminution of the glycogen stores in

consequence of glycogenolysis.

Table 2 shows the tissue contents in adenine

nucleotides of livers from control, diabetes1 and

diabetes2 animals after a fasting period of 24

hours. Diabetes1 rats presented a small tendency

toward smaller ATP levels, but only the ADP

levels were statistically smaller than those found

in control livers. As a consequence, however,

the sum of total nucleotides (ATP+ADP+AMP)

was also somewhat smaller in diabetes1 rats.

Diabetes2 rats, on the other hand, presented

significantly higher ATP levels with no

significant changes in AMP and ADP levels. In

consequence, these rats also presented a higher

content in total adenine nucleotides. These

results do not reveal any significant ATP

depletion in diabetic rats after a 24 hours fast.

The reasons for the higher ATP levels in livers

from diabetes2 rats cannot be inferred from the

present data, but the phenomenon is clearly

worth of more specific investigations.

In conclusion, it is quite apparent that investiga-

tions concerning gluconeogenesis in livers from

diabetes1 rats must take into account the

relatively high glycogen levels which are

unavoidably associated to glycogenolysis in the

perfused liver, with high and declining rates of

glucose release. For distinguishing gluconeo-

genesis from glycogenolysis under these condi-

tions it will be indispensable to use radioactive

precursors which allow to measure newly

formed glucose in a specific manner. Alterna-

tively, gluconeogenesis can also be evaluated if

the glycogen levels are simultaneously

measured so that the appropriate corrections can

be done. Simple subtraction of the very high

basal rates after infusion of a gluconeogenic

substrate (Ferraz et al., 1997; Akimoto et al.,

2000) is a highly risky procedure which is likely

to lead to erroneous evaluations of the true

gluconeogenic activities.

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Bazotte, R.B. (2000), Rates of gluconeogenesis in

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Ferraz, M., Brunaldi, K., Oliveira, C.E. and Bazotte,

R.B. (1997), Hepatic glucose production from L-

alanine is absent in perfused liver of diabetic rats.

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(1987), Potential intercellular futile cycling of car-

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The metabolic responses to

glutamine of livers from rats

with diabetes types 1 and 2

Denise Silva de Oliveira, Ciomar Aparecida Bersani Amado, Luci Tiemi Ide,

Fumie Suzuki-Kemmelmeier and Adelar Bracht

Laboratory of Liver Metabolism, University of Maringá,

87020900 Maringá (Brazil)

Address for correspondence:

Adelar Bracht

Laboratory of Liver Metabolism

Department of Biochemistry

University of Maringá

87020900 Maringá, Brazil

E-mail: adeb[email protected].br

Abstract

Glutamine utilization by hepatocytes isolated from streptozotocin diabetic rats

has been reported to be accelerated during the first 30 minutes of incubation. In

the isolated perfused rat liver, however, the utilization of glutamine is characterized

by a lag phase so that much more than 30 minutes are required for attaining

steady-state conditions. For this reason, the influence of diabetes on glutamine

metabolism was reexamined in the present study. Livers from rats with diabetes

types 1 and 2 were perfused with glutamine until steady-state conditions were

attained for the productions of glucose, urea, ammonia and lactate and for oxygen

uptake. The responses of livers of rats with diabetes2 did not differ from those of

control rats. Liver of rats with diabetes1 responded differently to glutamine, but the

differences were transient and characterized mainly by an acceleration of the

metabolic fluxes during the initial stages of glutamine infusion (10-30 minutes).

When approaching steady-state

conditions,

the differences tended to disappear.

These results confirm the accelerated glutamine metabolism found in hepatocytes

isolated from rats with diabetes1 during 30 minutes of incubation. They suggest,

however, that the in vivo velocity of hepatic glutamine transformation, which occurs

mainly under steady-state conditions, is probably similar in both healthy and

diabetic rats. An accelerated transformation of glutamine in rats with diabetes1

would does be restricted to episodes of transition between two different steady-

states.

Key words: diabetes type 1; diabetes type 2; liver; glutamine metabolism;

gluconeogenesis; glycogenolysis.

Introduction

Many tissues and cells use glutamine, which is essential for their normal

functioning. These tissues and cells include kidney, intestine, liver, specific neurons

of the central nervous systems, cells of the auto-immune system and pancreatic β-

cells (Young and Ajami 2001). In the rat the turnover of glutamine is high (1300

µmol per hour per kg body weight) and the compound is the most prominent

carbon and nitrogen carrier between tissues (Squires and Brosnan 1983).

Glutamine is one of the most important aminoacids released from the skeletal

muscle in the catabolic state and the most important fuel for cells undergoing rapid

division such as those of the intestinal epitelium, lymphocytes and tumor cells

(Newsholme et al. 1985, Tildon and Zielke 1988, Souba 1991). Glutamine is also

the most important substrate for renal ammoniogenesis and it plays a prominent

role in the acid-base equilibrium (Brosnan et al. 1987).

Synthesis and hydrolysis of glutamine depend on the enzymes glutamine

synthetase and glutaminase, respectively. Studies in the perfused rat liver

demonstrated a high rate of glutamine utilization resulting in glucose synthesis and

urea production (Ross et al. 1967, Saheki and Katunuma 1975). Glutaminase

hydrolyses glutamine to glutamate and ammonia. There are two isozymes of

glutaminase usually described as the “kidney-type” and the “liver-type”. The latter

is restricted to the liver during both the neonatal and adult phases, whereas the

former is present in all glutamine utilizing tissues (Watford 1993). These isozymes

are products of two different genes, but the amino acid sequences are 73%

identical, indicating that they probably originated from a common ancestor

(Curthoys and Watford 1995, Chung-Bok et al. 1997). The high activity of both

glutaminase and glutamine synthetase in the liver allows to this organ to remove

from and to add glutamine to the circulation. In consequence, the liver plays an

important role in glutamine homeostasis (Watford et al. 2002).

The “liver-type” glutaminase is considered a key-enzyme for hepatic glutamine

utilization and its flux control coefficient has been reported to be 0.96 (Low et al.

1993). Its activity changes under certain pathological conditions such as diabetes,

for example (Watford et al. 1984, Labow et al. 2001). In streptozotocin diabetic

rats (diabetes1) increases up to 12-fold have been reported (Squires et al. 1997).

Consistently, increased glutamine utilization was observed in hepatocytes isolated

from streptozotocin diabetic rabbits and rats (Squires et al. 1997). It must be

pointed out that the experiments with isolated hepatocytes have been done by

measuring

accumulation in the presence of [

C]glutamine (Squires et al.

1997) or gluconeogenesis (Zaleski and Bryla 1978) during 30 minutes. It has been

shown in experiments with the isolated perfused rat liver from healthy rats,

however, that the utilization of glutamine is characterized by a long lag phase so

that usually much more than 30 minutes are required for attaining steady-state

conditions (Corbello-Pereira et al. 2004). In this way, single measurements of

glutamine metabolism in isolated hepatocytes after 30 minutes incubation could be

revealing an untrue picture of the real differences between healthy and diabetic rats

if the time dependence is also changed, as it as been demonstrated to occur in rats

bearing the Walker-256 tumor (Corbello-Pereira et al. 2004). For this reason, the

influence of diabetes on glutamine metabolism was reexamined in the present

study. Livers from rats with diabetes types 1 and 2 (diabetes1 and diabetes2) were

perfused with glutamine until steady-state conditions were attained. Several

parameters, such as gluconeogenesis, ureagenesis and glycogen contents were

measured in order to obtain a more ample and detailed vision about the changes

caused by diabetes in glutamine metabolism.

Material and methods

Materials

The liver perfusion apparatus was built in the workshops of the University of

Maringá. All enzymes and coenzymes used in the enzymatic assays were purchased

from Sigma Chemical Co. (St Louis, USA). All standard chemicals were from the

best available grade (98-99.8 % purity).

Animals and diabetes induction

Male Wistar rats weighing 220–250 g fed with a standard laboratory diet

(Nuvilab



) were used in all perfusion experiments. Type 1 diabetes (diabetes1) was

induced by injecting adult rats with streptozotocin. Streptozotocin was dissolved in

citrate buffer (10 mM; pH 4.5) and a single dosis of 50 mg/kg was injected

intraperitoneally. After 12 days blood glucose of fed rats was measured and animals

presenting glycemic levels equal or above 16 mM were selected for the

experiments. Control rats were injected intraperitoneally with citrate buffer (10

mM, pH 4.5).

Type 2 diabetes mellitus (diabetes2) was induced as described by Portha et al.

(1974). Male newborn (2 days old) Wistar rats were injected intraperitoneally with

streptozotocin (150 mg/kg) dissolved in citrate buffer (10 mM; pH 4.5). Seven

weeks later, diabetes was confirmed by urine glucose levels, 24 hours urinary

volume, and water intake. The protocol for these experiments was approved by the

Ethics Committee for Animal Experimentation of the University of Maringá.

Liver perfusion

For the surgical procedure, the rats were anesthetized by intraperitoneal

injection of sodium thiopental (50 mg/kg). Hemoglobin-free, non-recirculating

perfusion was done according to the technique described by Scholz and Bücher

(1965).

After cannulation of the portal and cava veins the liver was positioned in a

plexiglass chamber. The flow was maintained constant by a peristaltic pump

(Minipuls 3, Gilson, France) and was adjusted between 30 and 35 ml min

−1

depending on the liver weight. The perfusion fluid was Krebs/Henseleit-bicarbonate

buffer (pH 7.4), saturated with a mixture of oxygen and carbon dioxide (95:5) by

means of a membrane oxygenator with simultaneous temperature adjustment at

C. The composition of the Krebs/Henseleit-bicarbonate buffer is the following:

115 mM NaCl, 25 mM NaHCO

, 5.8 mM KCl, 1.2 mM Na

1.18 mM MgCl

, 1.2

mM NaH

and 2.5 mM CaCl

Analytical

Samples of the effluent perfusion fluid were collected according to the

experimental protocol and analysed for their metabolite contents. The following

compounds were assayed by means of standard enzymatic procedures: glucose

(Bergmeyer and Bernt 1974), urea (Gutmann and Bergmeyer 1974), ammonia

(Kearney and Kun 1974), lactate (Gutmann and Wahlefeld 1974), alanine

(Williamson 1974) and glutamate (Bernt and Bergmeyer 1974). The oxygen con-

centration in the outflowing perfusate was monitored continuously, employing a

teflon-shielded platinum electrode adequately positioned in a plexiglass chamber at

the exit of the perfusate (Clark 1956, Scholz and Bücher 1965). Metabolic rates

were calculated from input-output differences and the total flow rates and were

referred to the wet weight of the liver.

Glycogen was determined in isolated perfused livers. Portions of approximately 2

g were freeze-clamped with liquid nitrogen. This sample was homogenized and

extracted with 8 ml of 6% HClO

. The supernatant was neutralized with 5 N K

and used for enzymatic glycogen assay (Kepler and Decker 1974).

Treatment of data

The metabolic rates were expressed as µmol per minute per gram liver wet

weight (µmol min

−1

). Statistical analysis of the data was done by means of the

Statistica

program (Statsoft



, 1998). The results are mentioned in the tables as

the p values.

Results

Time courses of metabolite release and oxygen uptake

upon glutamine infusion

The metabolism of glutamine was investigated in livers of fasted rats in order to

minimize interference by endogenous glycogen. Due to the slow response of the

liver to glutamine, long infusion times are required in order to achieve steady state-

conditions at least for parameters such as glucose production, oxygen uptake and

urea production (Corbello-Pereira et al. 2004). Figure 1 shows the time courses of

the changes in the productions of glucose, lactate, ammonia, urea, alanine and

glutamate and in oxygen uptake upon 5 mM glutamine infusion. Control livers

presented the characteristic response delays already reported in a previous work

(Corbello-Pereira et al. 2004), except for glutamate production which increased

linearly during the whole infusion time. A quantitative evaluation of this delay is

provided by the time for half-maximal increase (t

), which was evaluated by

numerical interpolation. The mean values for oxygen uptake and the production of

glucose, lactate, ammonia and urea are listed in Table 1. The response of livers

from rats with diabetes2 was similar to that of control livers. The most important

difference was found in alanine production which was substantially higher in

diabetes2 livers. Actually, alanine production in these livers was still increasing

when the perfusion was interrupted at 100 minutes. Lactate production was also

higher in diabetes2 livers at the end of glutamine infusion, whereas the opposite

occurred with oxygen uptake. The kinetics of the changes in diabetes2 livers was

also similar to that of the control livers, an observation corroborated by the times

for half-maximal increase in Table 1. A significantly smaller t

value was found only

for urea production.

Basal oxygen uptake of diabetes1 livers (before glutamine infusion) was lower

(−22.5%) and remained lower during glutamine infusion (Figure 1B); the onset of

the response, however, was faster in diabetes1 livers, as indicated by the t

value

in Table 1. Excepting oxygen uptake, all other parameters in diabetes1 and control

livers tended to converge to similar values at the end of the glutamine infusion

period. Glucose, lactate, alanine, ammonia and urea productions and oxygen

uptake tended to stabilize at the end of the infusion, suggesting steady-state

conditions. Glutamate production, however, was still increasing after 90 minutes of

glutamine infusion. Pronounced differences between diabetes1 and control livers

were apparent during the first 30 minutes of glutamine infusion for glucose, lactate,

ammonia and urea production. This was caused mainly by the rapid initial increases

which followed glutamine infusion in diabetes1 livers. Excepting urea production,

this rapid increase was not sustained. The difference in the behaviour of diabetes1

and control livers can be evaluated more conveniently by comparing the t

values

in Table 1 and by analyzing the graphs in Figure 2 where the diabetes1/control

ratios are represented against the perfusion time. The t

’s

were always shorter in

diabetes1 livers (43.7% on average; Table 1). The more rapid onset of the meta-

bolic fluxes in diabetes1 livers appears as well defined peaks in the diabetes1/con-

trol ratio for lactate, ammonia and urea productions during the first 10-30 minutes

following the onset of glutamine infusion (Figure 2, panel B). For oxygen uptake,

the diabetes1/control ratio showed little variation, but it remained below unity

during the whole glutamine infusion period (Figure 2, panel A). For glucose produc-

tion the diabetes1/control ratio was high from the beginning, with a clear declining

tendency. This declining tendency was halted in the initial stages of glutamine

infusion, but unity was approached as the glutamine infusion was continued.

Interpretation of the response of glucose production in diabetes1 livers can be

difficulted by the high basal rates due to the high residual glycogen content

(Gannon and Nuttall 1997). In an attempt of reducing these basal rates, the fasting

periods were increased to 48- and 72-hours and glucose production was measured.

Figure 3 shows the results that were obtained. After a 48-hours fast (Figure 3A) the

basal rate of glucose production in diabetes1 livers was still higher in comparison

with control and diabetes2 livers. Introduction of glutamine produced a rapid

increase which stabilized at levels similar to those of control livers. The response of

diabetes2 livers was similar to the response of the control livers. After a 72-hours

fast (Figure 3B) the basal rates of glucose production were pratically the same for

diabetes and control livers. Here again the initial rapid increase of glucose

production in diabetes1 livers was present and the response was stable. Diabetes2

livers also tended to respond more rapidly, statistical significance, however, was

lacking. The diabetes1/control ratios, that were plotted against the perfusion time

in Figure 4, showed a clear peak during the 10-20 minutes following initiation of

glutamine infusion after a 72-hours fast, similar to that one found for lactate, urea

and ammonia productions after a fasting period of 24-hours.

Glycogenolysis, glycogenesis and gluconeogenesis during

glutamine infusion

The high glycogen levels of diabetes1 livers and the kinetics of glucose produc-

tion following glutamine infusion lead to the question about the relative contribution

of glycogenolysis and gluconeogenesis. In order to answer this question the

glycogen levels of diabetes1 and control livers (24-hours fasted) were measured at

10 and 100 minutes perfusion time, i.e., before and at the end of glutamine

infusion (Figure 1). These levels were compared with the total amounts of glucose

that were produced from 10 to 100 minutes perfusion time. Control experiments

were also done in which glucose release an oxygen uptake were measured without

substrate infusion. The results shown in Figure 5 reveal low, but steady rates of

glucose production in control livers during the whole perfusion time and higher, but

declining rates in diabetes1 livers. Oxygen uptake, which was higher in control

livers, showed almost no changes during the whole perfusion time. Table 2 shows

the measured glycogen contents after the pre-perfusion period (arbitrarily set as 10

minutes perfusion time in Figures 1 and 5) and the total glucose production

(∆glucose production) during substrate-free perfusion and perfusion in the presence

of glutamine. The latter was evaluated as the area under the curves between 10

and 100 minutes perfusion time. Derived parameters are gluconeogenesis and the

differences in glycogen content (∆glycogen content) between 10 and 100 minutes,

the negative sign meaning net glycogenolysis and the positive sign net

glycogenesis. Gluconeogenesis was estimated as the difference between the change

in glycogen content and the total glucose release. Control livers presented very low

glycogen levels after the pre-perfusion period which includes the time between

anesthesia and surgical preparation of the liver and stabilization of oxygen uptake.

In diabetes1 livers the glycogen levels were much higher than in the control livers,

but substantially lower than those found in vivo (Gannon and Nuttall 1997). This is

due to the very high rates of glucose release during the pre-perfusion period. In

absolute and relative terms net glycogen degradation was small during substrate-

free perfusion in control livers (−22.7%), but very high in diabetes1 livers

(−81.3%). In control livers the infusion of glutamine produced some net glycogen

synthesis (+63.2% above the 10 minutes levels) and a reduction in net

glycogenolysis in diabetes1 livers. In control livers, gluconeogenesis during

substrate-free perfusion was relatively high. In diabetes1 livers gluconeogenesis

was negligible, the total glycogenolysis pratically accounting for the total glucose

production during the whole substrate-free perfusion period. Total glucose release

during glutamine infusion was higher in diabetes1 livers (+28.5%), but this

difference was mainly due to glycogenolysis, because gluconeogenesis was similar

in both control and diabetes1 livers.

Discussion

The hypothesis raised in the introduction, that measurements of glutamine

metabolism in isolated hepatocytes after 30 minutes incubation could be revealing

an untrue picture of the real differences between healthy and diabetic rats (Zaleski

and Bryla 1978, Squires et al. 1997), was confirmed by the results obtained in the

present work. The changes in glutamine hepatic metabolism induced by diabetes1

are not compatible with a stable increase in glutamine transformation. Diabetes 1

only accelerates the initial response to the introduction of glutamine, the steady-

state rates, however, being similar in livers from control and diabetes1 rats. The

times required for attaining half-maximal rates, which were clearly shorter in the

diabetes1 condition, are a good quantitive index for the response acceleration. The

reason for the shorter half-maximal rate times in the diabetes1 condition was that

glutamine metabolism in control livers continued to accelerate and did not reach its

maximum before 50 minutes infusion. Additionally the accelerated initial glutamine

transformation into lactate and ammonia in diabetes1 livers was downregulated

after 20 or 30 minutes. These data suggest that the velocity of hepatic

transformation of glutamine in vivo, where steady-state conditions predominate, is

probably similar in both healthy (control) and diabetic rats. An accelerated

transformation of glutamine in rats with diabetes 1 would does be restricted to

episodes of transition between two different steady-states. It is appropriate to

emphasize that only diabetes1 promotes these alterations in glutamine metabolism.

Diabetes2 was pratically without action on the hepatic glutamine metabolism with

the exception of alanine production which was increased.

Concerning the shorter half-maximal times for glutamine metabolism, the action

of diabetes1 was similar to the influences of the Walker-256 tumor and of adjuvant-

induced arthritis. In both pathologies the metabolic responses to glutamine are

considerably accelerated (Yassuda-Filho 2002, Corbello-Pereira et al. 2004), with

similar reductions in the half-maximal times. The introduction of glucagon equally

reduces the half-maximal times in control livers, being almost without effect in the

arthritic condition (Yassuda-Filho 2002). At least in adjuvant-induced arthritic rats

the reduction in the half-maximal times is not caused by an acceleration of

glutamine transport into the hepatocyte (Yassuda-Filho 2002). In principle at least,

this possibility can also be excluded for diabetes1, because no significant changes

in Na

-dependent and Na

-independent glutamine transport across rat liver

sinusoidal membranes have been found (Low et al. 1992). If the changes in the

half-maximal times are not caused by glutamine transport into the hepatocyte,

their causes must be linked to glutaminase which catalyzes the initial and

irreversible step of glutamine metabolism. The flux control coefficient of gluta-

minase has been reported to be equal to 0.96 whereas values of 0.51 and −0.46

have been attributed, respectively, to the cell influx and efflux systems (Low et al.

1993). These values must be regarded with some caution because they have been

evaluated after 10 minutes incubation periods of labeled glutamine with isolated

hepatocytes and, in the perfused liver of normal rats at least, the glutamine

metabolism is small during the first 10 minutes. The observations that diabetes1

and adjuvant-induced arthritis do not affect hepatic glutamine transport, however,

are strong indications that glutaminase exerts a key-role in glutamine transforma-

tion. In this respect it must be emphasized that this enzyme is subject to complex

cellular control and that, most probably, knowledge of many of its regulatory

factors and properties is still fragmentary. The enzyme can be stimulated by

glucagon and by interaction with the mitochondrial membrane, for example (Lacey

et al. 1981, McGivan et al. 1985). This stimulation persists even after isolation of

mitochondria (Lacey et al. 1981), so that it probably also persists during liver

perfusion. The high levels of glucagon in diabetes1 (Del Prato et al. 1980, Widmaier

et al. 1991) are probably an important cause for the elevated levels of glutaminase

in this disease (Watford et al. 1984). This set of observations most likely explains

the initial higher rates of glutamine metabolism that were found in the diabetes1

condition in as much as no such phenomenon was found in the livers of rats with

diabetes2, which do not normally present higher glucagon levels (Giroix et al.

1984). It is also evident, however, that during the course of glutamine infusion

different events must have taken place in the liver of healthy/diabetes2 and

diabetes1 rats. The former presented a sigmoidal time dependence with an

accentuated increment in glutamine metabolism during the period of 20 to 40

minutes glutamine infusion, suggesting activation of glutaminase by means of a

glucagon-independent mechanism. In the diabetes1 condition, where the initial rate

of metabolism was high no further stimulation took place. Actually a tendency

toward diminution was even apparent, suggesting also a decrease in the

glutaminase activity. The factors or conditions involved in these opposing

phenomena cannot be inferred from the present data and depend, consequently, on

additional experimental work.

The contribution of glycogenolysis to glucose production in livers from fasted

control rats was minimal, as revealed by the glycogen levels at various times in the

presence and absence of glutamine. The small basal rates of glucose production in

these livers were, thus, the result of gluconeogenesis from endogenous substrates.

These substrates are most likely glycogenic amino acids. At least 12 aminoacids are

released by substrate-free perfused livers at a total rate of 0.5 µmol min

−1

(Zwiebel 1990). This rate of amino acid release is compatible with the basal glucose

production rate of up to 0.1 µmol min

−1

that is normally found in livers from 24

hours fasted control rats (Kleckner et al. 1987; Botini et al. 2005). Higher

concentrations of amino acids can even led to net glycogen accumulation, as it was

the case when glutamine was introduced. This is in contrast with the net

glycogenolytic activity that was found in both the absence and presence of

glutamine in livers from 24-hours fasted diabetes1 rats, a phenomenon that is

consistent with their high glycogen levels (Gannon and Nuttall 1997). Although

glycogenolysis and gluconeogenesis were both contributing to the net glucose

release in livers from diabetes1 rats, the initial burst that followed glutamine

infusion was most likely due to gluconeogenesis. This is corroborated by similar

bursts in other parameters strictly dependent on glutamine transformation (urea

and lactate production, for example) and also by the fact that the same burst was

also present in livers from 48- and 72-fasted diabetes1 rats whose basal rates of

glucose release were very close to those found in the control condition.

Consistently, at these fasting times the hepatic glycogen levels of control, diabetes1

and diabetes2 rats are pratically the same (Oliveira et al. 2006).

With respect to the gluconeogenic capacity of hepatocytes from diabetic rats

contradictory observations are frequently found in the specialized literature.

Experiments with isolated hepatocytes of alloxan diabetic rats have lead to the

conclusion that hepatic gluconeogenesis from alanine, pyruvate and fructose is con-

siderably increased in this preparation (Wagle et al. 1975). Experiments with the

isolated perfused rat liver, however, concluded that gluconeogenesis from lactate,

glycerol and sorbitol is not affected by alloxan and streptozotocin induced diabetes,

whereas gluconeogenesis from alanine is absent in these animals (Ferraz et al.

1997). Kleckner et al. (1987), on the other hand, have found diminished rates of

gluconeogenesis from lactate in perfused livers from alloxan diabetic rats. The high

basal rates of glycogenolysis due to the high glycogen levels and the way by which

the gluconeogenic activity was calculated from the total rates of glucose release

could have contributed to these different reports. It is quite apparent from the

results of the present investigation that measurement of gluconeogenesis in livers

from diabetes1 rats must take into account the relatively high glycogen levels which

Acknowledgements

This work was supported by grants from the Programa Nacional de Núcleos de

Excelência (PRONEX) and from the Conselho Nacional de Desenvolvimento Cientí-

fico e Tecnológico (CNPq).

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Table 1

Half-maximal rate times (t

) of glutamine action on several metabolic

fluxes. The t

values are expressed in terms of time after initiation of glutamine

infusion and were obtained by numerical interpolation (Stineman's interpolation)

within each individual glutamine response curve of the experimental series

illustrated by Figure 1. Data are means ± SEM and asteriscs are used to indicate

values of the diabetes1 or diabetes2 conditions differing statistically from the

corresponding controls according to the Student-Newman-Keuls test applied after

monovariate variance analysis (p < 0.05).

(minutes)

Metabolic fluxes

Control Diabetes1 Diabetes2

Glucose production 32.68±1.89

(n=11)

19.83±1.53*

(n=21)

29.99±1.30

(n=12)

Ammonia production 26.91±2.45

(n=6)

14.12±2.42*

(n=7)

23.47±0.78

(n=6)

Urea production 31.52±3.02

(n=6)

16.16±3.69*

(n=6)

23.80±0.95*

(n=6)

Lactate production 43.23±3.94

(n=6)

16.65±3.74*

(n=6)

34.90±2.23

(n=5)

Oxygen uptake 28.95±1.82

(n=11)

19.01±1.55*

(n=21)

27.38±1.46

(n=12)

Table 2

Glycogen contents, total glucose release (∆glucose release), changes in glycogen contents (∆glycogen content) and

gluconeogenesis of livers from 24-hours fasted rats during substrate-free perfusion and perfusion with 5 mM

glutamine. The experiments were done according to the protocols illustrated by Figures 1 (glutamine infusion) and 5 (substrate-

free perfusion). For glycogen determination the livers were freeze-clamped in liquid nitrogen at 10 or 100 minutes perfusion time.

Total glucose release (∆glucose release) was computed as the area under the corresponding curves in Figures 1A and 5A. Data are

means ± SEM and the superscripts of each pair of values refer to the various p values given in the bottom (Student's t test); p

values > 0.05 where omitted. Latin letters were used for comparing columns and greek letters when comparing lines.

Control Diabetes1

Condition

of the

perfusion

fluid

Perfusion time

(minutes)

Glycogen

content

∆Glucose

production

∆Glycogen

content

Gluconeo-

genesis

Glycogen

content

∆Glucose

production

∆Glycogen

content

Gluconeo-

genesis

µmol glucosyl units per gram liver wet weight

Pre-

perfusion

1.58±0.08

(n=6)

a,α

100

Perfusion time (minutes)

0.10

0.20

0.30

0.40

0.50

0.60

Glucose production (µmol min

−

)

A, glucose

‡

100

Perfusion time (minutes)

1.60

1.90

2.20

2.50

2.80

3.10

3.40

Oxygen uptake (µmol min

−

)

Figure 2. Time courses of the hepatic metabolite production changes in

diabetes1 relative to the control condition after a 24 hours fast. The

diabetes1/control ratios were calculated from the data presented in panels A to E in

Figure 1.

1.0

2.0

3.0

4.0

5.0

 Glucose production

 Oxygen uptake

Glutamine infusion

Diabetes1/control

100

Perfusion time (minutes)

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Diabetes1/control







Ammonia

production







Lactate

production

──

Urea production

Figure 3. Time courses of glucose production during glutamine infusion (5

mM) in livers from control and diabetic rats after a fasting periods of 48

and 72 hours. Glutamine infusion was initiated at 10 minutes perfusion time.

Legends: control,

; diabetes1, ; diabetes2, . Statistically signi-

ficant differences between control and diabetes1 are indicated by the symbol *.

10 20 30

50 60 70 80 90 100

Perfusion time (minutes)

0.10

0.20

0.30

0.40

0.50

0.60

0.70

Figure 4. Time courses of the hepatic glucose production changes in

diabetes1 relative to the control condition after different fasting periods.

The diabetes1/control ratios were calculated from the data presented in panels A of

Figure 1 and Figure 3.

10 20 30 40 50

80 90 100

Perfusion time (minutes)

1.0

2.0

3.0

4.0

5.0

Glucose production: diabetes1/control

Fasting period:

 24 hours

 48 hours

 72 hours

Glutamine infusion

Figure 5. Time courses of glucose production and oxygen uptake of livers

from 24 hours fasted rats under substrate-free perfusion. Legends: control,

; diabetes1, . Statistically significant differences between control and

diabetes1 are indicated by the symbols *.

20 30 40 50 60 70 80 90 100

Perfusion time (minutes)

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

Oxygen uptake (

mol min

−

)

, oxygen uptake

10 20 30 40

90 100

Perfusion time (minutes)

0.10

0.20

0.30

0.40

0.50

Glucose production (

mol min

−

)

, glucose production

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