Responses of the perfused liver of neonatal type 2 diabetic rats to gluconeogenic and ammoniogenic substrates

doi:10.4236/health.2010.25071

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Vol.2, No.5, 477-483 (2010)

doi:10.4236/health.2010.25071

Health

Responses of the perfused liver of neonatal type 2

diabetic rats to gluconeogenic and ammoniogenic

substrates

Mirian Carvalho-Martini, Fumie Suzuki-Kemmelmeier, Denise Silva de Oliveira,

Jurandir Fernando Comar, Adelar Bracht*

Department of Biochemistry, University of Maringá, Maringá, Brazil; *Corresponding Author: [email protected]

Received 23 December 2009; revised 6 February 2010; accepted 8 February 2010.

ABSTRACT

The responses of livers from rats with type 2

diabetes to alanine (gluconeogenesis and am-

monia detoxification) and other gluconeogenic

substrates were investigated. The experimental

system was the isolated perfused rat liver.

Neonatal type 2 diabetes was induced with

streptozotocin. Ammoniogenesis from endoge-

nous substrates was 610% higher in livers from

diabetic rats when compared to the control

condition. Alanine (2.5 mM) ammoniogenesis

was 285% higher in livers of diabetic rats. Glu-

coneogenesis from the following substrates

was smaller in the liver of diabetic rats: Alanine

(43.5%), lactate (28.3%) and glycerol (30.5%).

Pyruvate gluconeogenesis was normal. The

high rate of ammoniogenesis explains the mod-

erate hyperammonemia of type 2 diabetic rats.

The enzymatic machinery of the gluconeo-

genic pathway of type 2 diabetic rats seems to

be adapted to low rates of glucose removal by

extrahepatic tissues. A significant contribution

of gluconeogenesis to the fasting hyperglycemia

can be expected only by short-term up-regulation

mechanisms.

Keywords: Type 2 Diabetes; Gluconeogenesis;

Ammoniogenesis; Alanine; Lactate

1. INTRODUCTION

Increased hepatic glucose production is characteristic of

type 1 diabetes mellitus [1,2]. Particularly in the fasted

state, elevated hepatic gluconeogenesis seems to be the

main cause for the hyperglycemic condition in type 1

diabetes [3]. If the hyperglycemic condition in type 2

diabetes is also at least partly dependent on enhanced

gluconeogenesis is not clear. There are reports claiming

that this dependence is more accentuated in severely

hyperglycemic patients and that it tends to diminish or

even vanish in moderately hyperglycemic patients [4].

There are also studies in which no enhanced gluconeo-

genesis was found in type 2 diabetic patients [5]. It is

generally believed that the enhanced gluconeogenesis is

caused by an increased efficiency of the gluconeogenic

pathway in combination with an augmented mobilization

of glucose precursors to the liver [6,7].

Although gluconeogenesis can be measured in vivo

using appropriate tracer techniques [4,8,9] it is of inter-

est to reproduce the increased hepatic gluconeogenesis

in isolated cell systems because this allows conclusions

about its mechanisms. In the isolated perfused rat liver

for example, the gluconeogenic activity reflects the en-

zymatic capacities. These, in turn, reflect the medium-

and long-term effects of the circulating hormones on the

expression of enzymes and other factors. For type 1 dia-

betes mellitus, increased gluconeogenesis in isolated

hepatocytes or the isolated perfused liver from a variety

of substrates has been found [10-15]. When alanine was

the substrate, enhanced gluconeogenesis was found in

the fasted state, combined with increased rates of urea

production and increased rates of alanine incorporation

into proteins [11]. These observations with alanine are

consistent with the increased rates of mobilization of this

amino acid in type 1 diabetes [6]. All these data reveal

that short-term regulation operates most probably as a

secondary mechanism for the enhanced gluconeogenesis

in type 1 diabetes, the medium and long-term expression

of key enzymes playing the decisive role [16].

Experiments in which gluconeogenesis was measured

in the liver of rats with type 2 diabetes have not been

done until now. They are of interest, however, because

they will provide the same information about the enzy-

matic machinery that is already available for type 1 dia-

betes. The hypothesis that can be formulated is that en-

This work was sponsored by the Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq) and Programa Nacional de Núcleos de

Excelência (PRONEX, Fundação Araucária-CNPq).

M. Carvalho-Martini et al. / HEALTH 2 (2010) 477-483

478

hanced gluconeogenesis should be detectable in the iso-

lated rat liver of type 2 diabetic rats in the same way as it

was detected in livers from type 1 diabetic animals. En-

hanced gluconeogenesis in the isolated organ would be

reflecting mainly medium- and long-term effects of the

circulating hormones on enzyme expression. With this

hypothesis in mind, in the present work experiments

were conducted with livers of type 2 diabetic rats. The

neonatal streptozotocin-induced rat model of type 2 dia-

betes mellitus was used. It has been praised as a good

model for type 2 diabetes in humans because it presents

several of its characteristics [17]. In addition to glu-

coneogenesis, nitrogen metabolism from alanine was

also measured. Evaluation of nitrogen metabolism is of

interest because, in addition to hyperglycemia, type 2

diabetic rats also present moderate hyperammonemia

[18]. For comparative purposes other gluconeogenic

precursors, such as lactate and pyruvate, were also in-

vestigated.

2. MATERIALS AND METHODS

The liver perfusion apparatus was built in the workshops

of the University of Maringá. Enzymes and coenzymes

used in the assay procedures and streptozotocin were

purchased from Sigma Chemical Co. (St. Louis, USA).

All other chemicals were from the best available grade.

Neonatal type 2 diabetes mellitus was induced as pre-

viously described [19,20]. Male newborn (2 days old)

Wistar rats were injected intraperitoneally with strepto-

zotocin (160 mg/kg) dissolved in citrate buffer. Control

rats were injected with citrate buffer. Seven weeks later,

diabetes was confirmed by blood glucose levels (8-10 mM),

glucose appearance in urine and 24 hours urinary vol-

ume (generally 500% above normal). After seven weeks

the mean weights of the control and diabetic rats were

220  2.8 and 203.2  5.2 g, respectively. All animal

experiments were done according to the universally ac-

cepted standards for animal experimentation.

Rats were fed ad libitum with a standard laboratory

diet (Purina), but food was withdrawn 24 hours prior to

the perfusion experiments. For the surgical procedure,

rats were anesthetized by intraperitoneal injection of

sodium pentobarbital (50 mg/kg). Hemoglobin-free,

non-recirculating perfusion was undertaken according to

the technique described elsewhere [21,22]. After cannu-

lation of the portal and cava veins the liver was posi-

tioned in a plexiglass chamber. The hepatic artery was

closed (monovascular perfusion) and the bile duct was

left open. The flow was maintained constant by a peri-

staltic pump (Minipuls 3, Gilson, France) and was ad-

justed to 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 mem-

brane oxygenator with simultaneous temperature adjust-

ment at 37oC. The composition of the Krebs-Henseleit-

bicarbonate buffer is: 115 mM NaCl, 25 mM NaHCO3,

5.8 mM KCl, 1.2 mM Na2SO4, 1.18 mM MgCl2, 1.2 mM

NaH2PO4 and 2.5 mM CaCl2. L-Alanine (2.5 mM), lac-

tate (2.5 mM), pyruvate (1 mM) or glycerol (2 mM) were

dissolved in the perfusion fluid. Samples of the effluent

perfusion fluid were collected at 4 minute intervals and

analyzed for their metabolite content. The following

compounds were assayed by means of standard enzy-

matic procedures [23]: lactate, pyruvate, glucose, urea,

ammonia, glutamine and glutamate. The oxygen con-

centration in the outflowing perfusate was monitored

polarographically employing a teflon-shielded platinum

electrode adequately positioned in a plexiglass chamber

at the exit of the perfusate [22].

Rats after a 24-hours’ fast were used because this

minimizes interference of endogenous glycogen [24]. As

shown by previous work the fasting glycogen levels of

control and type 2 diabetic rats are very low when com-

pared to the fed state [18].

Basal rates (absence of substrates) as well as incre-

ments caused by substrates (L-alanine, lactate, pyruvate

or glycerol) were evaluated. The latter were calculated

by subtracting the basal rates (before substrate infusion)

from the steady-state rates found at the end of the sub-

strate infusion period. The error parameters presented in

graphs and tables are standard errors of the means. Dif-

ferences between pairs of means were analyzed by

means of Student’s t test. The 5% level (p < 0.05) was

adopted as a criterion of significance.

3. RESULTS

Table 1 lists the basal rates of metabolite release of liv-

ers from 24-hours fasted rats perfused with substrate-

free medium. Under such conditions the livers are de-

pendent solely on endogenous sources. The rates were

referred to the wet liver weights which were not different

in control and diabetic rats, namely 3.31  0.09 and 3.53

 0.13 g per 100 g body weight, respectively. As re-

vealed by Table 1 most basal rates were low. This did not

occur with oxygen uptake and it should be noted that it

was 13.6% lower in the liver of diabetic rats. Ammonia

production, however, was 610% higher in the diabetic

condition. All other parameters were similar in both the

control and the diabetic condition.

Figure 1 illustrates the experimental protocol that was

employed in the present study as well as the time course

of the changes in three selected parameters. Sampling of

the effluent perfusate for metabolite determination was

always initiated after oxygen uptake stabilization (zero

time). Alanine infusion was initiated at 12 minutes in the

time scale of Figure 1. The time courses of the changes

M. Carvalho-Martini et al. / HEALTH 2 (2010) 477-483

479

Openly accessible at

0.1

0.2

0.3

0.4

Ammonia production

(µmol min

1

)

Ammonia diabetic

control

0.1

0.2

0.3

0.4

Glucose production (µmol min1 g1)

Glucose

control

diabetic

1.7

1.9

2.1

2.3

2.5

2.7

2.9

3.1

3.3

Oxygen uptake (µmol min

1

)

0 10 20 30 40 50

Perfusion time

(

minutes

)

Alanine infusion (2.5 mM)

Oxygen

control

diabetic

Figure 1. Time course of changes in oxygen uptake, glu-

cose production and ammonia production in livers from

fasted control and type 2 diabetic rats. Livers were per-

fused as described in Material and Methods. Alanine was

infused as indicated by the horizontal bar. Results obtained

with livers of control rats were represented by full symbols

and those of diabetic rats with empty symbols. Data are

means  SEM of 9 (control) and 5 (diabetic) liver perfu-

sion experiments.

in ammonia and glucose production and oxygen uptake

were represented. The basal values of ammonia produc-

tion, oxygen uptake and glucose release correspond to

those ones listed in Table 1. The introduction of alanine

caused increases in all parameters which tended to new

steady-states during the following 30 minutes. Differ-

ences between the control and diabetic conditions were

not only maintained but even accentuated because glu-

cose production from alanine was smaller in the diabetic

condition.

Figure 1 reveals that new steady-state levels were

reached in consequence of alanine infusion. The steady-

state rates and the increments caused by alanine infusion

were evaluated and listed in Table 2. In addition to the

three parameters that were represented in Figure 1, the

rates of urea, lactate, pyruvate, glutamate and glutamine

production were also shown in Table 2. Alanine infusion

caused increases in all parameters and not only in those

ones shown in Figure 1. The increment in ammonia

production caused by alanine was higher in the diabetic

condition. The increments in oxygen uptake, urea, glu-

cose and glutamine productions, however, were smaller

in the diabetic condition. Gluconeogenesis from alanine

was, thus, smaller in the liver of diabetic rats (43.5%).

The total nitrogen flux generated by alanine infusion can

be approximated by the sum of ammonia production + (2

 urea production) + (2  glutamine production) + glu-

tamate production, after subtracting the basal rates (be-

fore alanine infusion). In livers of control rats this cal-

culation yielded a total nitrogen flux of 1.18 µmol min1

g1; in the diabetic condition the corresponding value

was 0.88 µmol min1 g

1, the difference amounting to

25.6%.

Glucose production from lactate, pyruvate and glyc-

erol was investigated by using essentially the same ex-

perimental protocol illustrated for alanine in Figure 1.

The results are summarized in Tables 3 to 5. Table 3

shows that gluconeogenesis from 2.5 mM lactate was

28.3% smaller in livers from diabetic rats. The final

oxygen uptake was also considerably smaller in the dia-

betic condition. Pyruvate production from lactate tended

to be more pronounced in the diabetic condition, but

there was no statistical significance at the 5% level. For

pyruvate metabolism, on the other hand, no significant

differences were found in glucose production, oxygen

uptake and lactate production, as revealed by Table 4.

There was a strong tendency, however, for higher values

of glucose production in the diabetic condition (p =

0.061). Glucose production from glycerol however, was

30.5% smaller in the diabetic condition as revealed by

Table 5. Lactate production from glycerol, however, was

not statistically different. Pyruvate production from gly-

cerol was negligibly small.

4. DISCUSSIONS

The results reveal that the metabolism of livers from

neonatal type 2 diabetic rats presents a few differences

when compared to livers from normal and also type 1

diabetic rats. These differences encompass both ammo-

nia detoxification and carbohydrate metabolism. The

liver of type 1 diabetic rats has been reported to present

considerably higher rates of urea production [11]. This

makes a clear contrast with the liver of type 2 diabetic

rats where ureogenesis was found to be close to normal

(Tables 1 and 2). Consistently, the plasma urea level of

type 1 diabetic rats is very high, but normal in type 2

diabetic rats [2-18]. The perfused liver of type 2 diabetic

rats, on the other hand, presented higher rates of ammo-

nia production under both conditions examined in the

present work, i.e., during substrate-free perfusion and

during alanine infusion (Tables 1 and 2). In relative

terms the difference was more pronounced in the

M. Carvalho-Martini et al. / HEALTH 2 (2010) 477-483

480

Table 1. Basal rates of metabolites release and oxygen uptake in perfused livers from control and type 2 diabetic rats. Signifi-

cant differences are indicated by an asterisk (p < 0.05). The results come from experiments in which various substrates were

infused at 12 minutes perfusion time as illustrated by Figure 1.

Control Diabetic

Metabolic flux



mol min1 (g liver wet weight)1

Ammonia production 0.029  0.012 (n = 9) 0.206  0.028* (n = 5)

Urea production 0.140  0.016 (n = 9) 0.171  0.003 (n = 5)

Glutamine production 0.064  0.007 (n = 9) 0.062  0.006 (n = 5)

Glutamate production 0.034  003 (n = 9) 0.031  0.007 (n = 5)

Oxygen uptake 2.470  0.065 (n = 18) 2.135  0.063* (n = 17)

Glucose production 0.080  0.010 (n = 17) 0.064  0.006 (n = 16)

Lactate production 0.052  0.007 (n = 13) 0.051  0.014 (n = 11)

Pyruvate production 0.007  0.002 (n = 13) 0.009  0.006(n = 13)

Table 2. Metabolic fluxes in livers from control and type 2 diabetic rats caused by alanine infusion (2.5 mM). The data

were obtained from experiments in which alanine was infused during 30 minutes. Asterisks (*) and crosses (†) indicate

values in the diabetic condition that are statistically different from the corresponding control values according to Student’s

t test (p < 0.05).

Control (n = 9) Diabetic (n = 5)



mol min1 (g liver wet weight)1

Parameter

Rate in the presence of

alanine

Increment caused by

alanine

Rate in the presence of

alanine

Increment caused by

alanine

Ammonia production 0.081  0.025 0.052  0.016 0.312  0.041† 0.106  0.013*

Urea production 0.499  0.030 0.359  0.021 0.416  0.019 0.245  0.011*

Glutamine production 0.255  0.015 0.191  0.011 0.191  0.008† 0.129  0.005*

Glutamate production 0.060  0.005 0.026  0.002 0.053  0.010 0.022  0.004

Oxygen uptake 3.064  0.102 0.434  0.048 2.349  0.073† 0.258  0.026*

Glucose production 0.364  0.017 0.292  0.014 0.238  0.011† 0.165  0.013*

Lactate production 0.307  0.019 0.255  0.015 0.285  0.024 0.234  0.019

Pyruvate production 0.122  0.028 0.115  0.026 0.160  0.018 0.151  0.017

Table 3. Metabolic fluxes in livers from control and type 2 diabetic rats caused by lactate infusion (2.5 mM). The data

were obtained from experiments in which lactate was infused during 30 minutes. Asterisks (*) and crosses (†) indicate

values in the diabetic condition that are statistically different from the corresponding control values according to Student’s

t test (p < 0.05).

Control (n = 4) Diabetic (n = 5)



mol min1 (g liver wet weight)1

Parameter

Rate in the presence of

lactate

Increment caused by

lactate

Rate in the presence of

lactate

Increment caused by

lactate

Oxygen uptake 3.195  0.228 0.629  0.042 2.631  0.070† 0.531  0.029

Glucose production 0.998  0.119 0.922  0.118 0.737  0.021† 0.661  0.018*

Pyruvate production 0.200  0.101 0.182  0.100 0.334  0.097 0.324  0.101

M. Carvalho-Martini et al. / HEALTH 2 (2010) 477-483

481

Table 4. Metabolic fluxes in livers from control and type 2 diabetic rats caused by pyruvate infusion (1.0 mM). The data were

obtained from experiments in which pyruvate was infused during 20 minutes.

Control (n = 4) Diabetic (n = 3)



mol min1 (g liver wet weight)1

Parameter

Rate in the presence of

pyruvate

Increment caused by

pyruvate

Rate in the presence of

pyruvate

Increment caused by

pyruvate

Oxygen uptake 2.657  0.101 0.361  0.045 2.658  0.014 0.434  0.059

Glucose production 0.399  0.049 0.340  0.042 0.515  0.013 0.463  0.014

Lactate production 1.1430.063 1.103  0.059 1.232  0.047 1.158  0.022

Table 5. Metabolic fluxes in livers from control and type 2 diabetic rats caused by glycerol infusion (2.0 mM). The data

were obtained from experiments in which glycerol was infused during 20 minutes. Asterisks (*) and crosses (†) indicate

values in the diabetic condition that are statistically different from the corresponding control values according to Student’s

t test (p < 0.05).

Control (n = 4) Diabetic (n = 4)



mol min1 (g liver wet weight)1

Parameter

Rate in the presence of

glycerol

Increment caused by

glycerol

Rate in the presence of

glycerol

Increment caused by

glycerol

Oxygen uptake 2.696  0.126 0.173  0.066 2.077  0.078† 0.061  0.020

Glucose production 0.609  0.049 0.518  0.051 0.423  0.036† 0.362  0.035*

Lactate production 0.140  0.049 0.091  0.009 0.103  0.036 0.056  0.016

absence of alanine, a condition where ammonia comes

solely from endogenous catabolic reactions. Even the

second route of ammonia detoxification, namely gluta-

mine production [25], seems to be impaired in the liver

of type 2 diabetic rats, as can be judged from its lower

rates in the presence of alanine (Table 2). The higher

rates of ammonia production can be indicating that ni-

trogen catabolism in livers of type 2 diabetic rats sur-

passes the capacity of the urea cycle when this route

depends solely on endogenous substrates and on en-

dogenous substrates plus alanine. In principle this con-

clusion receives support from the higher plasma ammo-

nia levels in type 2 diabetic rats [18]. It must be re-

marked, however, that the difference in the plasma am-

monia levels between diabetic and control rats, as re-

ported previously [18], is relatively small (+ 28%) when

compared to the difference in ammoniogenesis (+ 285%

in the presence of alanine). Extrapolation to the in vivo

conditions is always subject to error, but it principle one

would expect a more severe hyperammonemia in type 2

diabetic rats. Possibly there is some in vivo mechanism

or mechanisms that avoid the development of severe

hyperammonemia. One of these mechanisms could be

higher rates of renal excretion. Alternatively, there could

be a more efficient transformation of ammonia into urea

and glutamine in vivo than that one found in the isolated

perfused liver due to the presence of factors capable of

stimulating ureogenesis.

With reference to the main hypothesis of the present

work, it is apparent from the results of the present study

that hepatic gluconeogenesis in neonatal type 2 diabetic

rats is lower than that in non-diabetic rats, at least with

the relevant precursors lactate, alanine and glycerol. This

can also be interpreted as meaning, in principle at least,

that the enzymatic machinery of the liver from neonatal

type 2 diabetic rats is not adapted to higher rates of glu-

coneogenesis but much more to the lower rates of glu-

cose uptake by peripheral tissues [26]. No significant

difference was found when pyruvate was the substrate.

There was a tendency toward higher rates of glucose

synthesis in the diabetic condition without statistical

significance, however, possibly due to the small number

of rats used in these experiments. However, pyruvate is

the only substrate used in the present work that induces,

in the once-through perfused liver at least, an oxidizing

state in the liver cells when it is present alone (i.e., very

low NADH/NAD ratios) [27]. This is a situation that

does not normally occur in vivo where the pyruvate

concentrations are normally very low and the lactate to

pyruvate ratios high [27]. These results with livers of

type 2 diabetic rats are in sharp contrast with those ob-

tained with the liver of type 1 diabetic rats, where in-

creased gluconeogenesis with virtually all substrates was

observed [10-15]. Although the rates of oxygen uptake

were always lower in the liver of diabetic rats even in

the presence of gluconeogenic substrates, it is unlikely

that ATP availability could be limiting glucose synthesis.

M. Carvalho-Martini et al. / HEALTH 2 (2010) 477-483

482

Openly accessible at

As shown previously, the ATP content of the hepatic

tissue of type 2 diabetic rats under the same conditions

as those used in the present work (24-hours fast) is even

higher than that of normal rats [18]. Furthermore, there

was a clear correlation between the increments in oxy-

gen uptake caused by lactate, alanine and pyruvate and

the gluconeogenic activity. This reinforces the general

notion that gluconeogenesis controls the extra oxygen

uptake and not the contrary.

It should be stressed with reference to the contribution

of gluconeogenesis to the fasting hyperglycemia in ani-

mals and patients with type 2 diabetes that conflicting

results have been reported. There are studies proposing a

significant, though relatively small, contribution of glu-

coneogenesis to the fasting hyperglycemia [4,6,8] while

others claim that the contribution is not significant [6,26].

Our results thus agree much more with those studies in

which no enhanced gluconeogenesis was found in type 2

diabetic patients.

Extrapolations of the observations of the present work

to the in vivo conditions or to different species (mice,

humans) must always be done carefully. Even so, it

should be stressed that in vivo lower rates of gluconeo-

genesis in humans or animals bearing type 2 diabetes

have never been reported. Consequently it seems worth

to discuss the possible factors that could lead to an at

least normal or slightly above normal in vivo gluconeo-

genesis in spite of impaired enzymatic machinery. If one

assumes that the enzymatic machinery is the result of

long- or even medium-term regulation, efficient mecha-

nisms of short-term up-regulation must be operative in

type 2 diabetes, at least in the rat. There are several pos-

sibilities to be considered: 1) different concentrations of

hormones able to stimulate gluconeogenesis; 2) higher

substrate concentrations in the diabetic condition; 3)

increased plasma concentrations of free fatty acid, which

are known to estimulate gluconeogenesis [24-28]. With

reference to the hormonal factors, it is known that glu-

cagon, in addition to its medium- and long-term effects

[16] can also promote short-term up-regulation of glu-

coneogenesis [29,30]. Insulin, in contrast, does not exert

short-term effects in the liver [10,11]. Elevated glucagon

levels have been found in type 2 diabetic humans [9-31]

and in neonatal streptozotocin type 2 diabetic rats at least

during certain stages after streptozotocin injection [17].

Consequently a short-term up-regulation by glucagon must

be considered as a real possibility. It must be mentioned,

however, that the stimulatory effect of glucagon on glu-

coneogenesis is relatively modest unless the cytosolic

NADH/NAD+ ratio is very high [29,30]. Concerning the

possible contribution of increased substrate concentra-

tions one cannot expect a significant contribution from

lactate. The latter is by far the most important glu-

coneogenic substrate, but its normal concentration in

blood (around 2 mM) is already saturating for glu-

coneogenesis [28,32] so that increments would not en-

hance glucose synthesis. Some positive effect favouring

gluconeogenesis in the diabetic state could be expected,

however, by a shift in the redox potential of the cytosolic

NAD-NADH couple towards a more oxidized state

(lower NADH/NAD+ ratios). This would have the con-

sequence of increasing the pyruvate concentration by

virtue of the near-equilibrium of the lactate dehydro-

genase reaction [27]. This positive effect is to be ex-

pected from the observation that when pyruvate was the

sole substrate, a condition which means a strong oxidiz-

ing state for the cytosolic NAD-NADH couple [27], the

difference between gluconeogenesis in the diabetic and

the normal state was practically abolished. In a specific

study with type 2 diabetic patients [31], plasma glycerol

was increased by a factor of 1.46 and glycerol glu-

coneogenesis was increased more than twofold. From

the glycerol concentration increase one would expect

maximally a 1.46-fold increase in gluconeogenesis from

this substrate. This disproportion, two-fold versus 1.46-

fold, can be regarded as an indication that another factor

or factors are contributing to the enhanced gluconeo-

genesis. One of these factors could be the more elevated

glucagon concentration, as already mentioned. However,

the more elevated fatty acid concentrations, which seem

to be a frequent phenomenon in type 2 diabetes [4,31],

could be equally contributing as stimulatory effectors.

In conclusion, the enzymatic machinery of the glu-

coneogenic pathway of neonatal type 2 diabetic rats

seems to be adapted to low rates of glucose removal by

extrahepatic tissues rather than to enhanced glucose

production. Gluconeogenesis at rates high enough to

contribute significantly to the fasting hyperglycemia can

be generated only by short-term up-regulation which

could, in principle, be produced by high glucagon, glyc-

erol and fatty acids concentrations.

5. ACKNOWLEDGEMENTS

The authors wish to thank Dr. Ciomar Bersani Amado for supplying

the diabetic rats.

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