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Laboratoire Physiopathologie Nutrition, CNRS ESA 7059, Université Paris, P7/D. Diderot, 75251 Paris Cedex 05, France
Address all correspondence and requests for reprints to: Prof. B. Portha, Laboratoire Physiopathology of Nutrition, CNRS ESA 7059, Université Paris, 7/D. Diderot, 2 place Jussieu, Tour 33, 75251 Paris Cedex 05, France. E-mail: portha{at}paris7.jussieu.fr
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Abstract |
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I/G) was severely decreased in M rats compared with
that in control (C) rats. When evaluated after administration of
acetylcholine, I was amplified to the same extent in M rats as in C
rats. After administration of the 
2A-adrenergic agonist
oxymetazoline, glucose-induced insulin release in M rats was not
significantly affected, whereas it was sharply decreased in C rats.
Finally, administration of yohimbine, an 2-adrenergic
antagonist, partially restored the lack of reactivity of the ß-cells
to glucose in the M rats, as I/G was amplified by 6-fold in the M
group and by 3.3-fold in the C group. We conclude that protein-energy
restriction starting early in life in rats brings about changes in the
overall activity of the autonomic nervous system that, in turn, are
responsible at least in part for the acquisition/maintenance of
decreased ß-cell reactivity to glucose in vivo. |
Introduction |
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In the present report we have quantitatively assessed in rats submitted
to protein-energy restriction early in life 1) the firing rates of the
vagus nerve and of the superior cervical ganglion to evaluate the
parasympathetic and sympathetic activities, respectively; and 2) the
in vivo glucose-induced insulin secretion under treatment
with the 2A-adrenoceptor agonist oxymetazoline, the
2-antagonist yohimbine, or the muscarinic agonist
acetylcholine (ACh).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Female Wistar rats bred in our colony were housed in a
temperature-controlled room with a 12-h light, 12-h dark cycle (lights
on at 0700 h). They were weaned 28 days after birth and from this
age were fed either standard (C rats) or protein-energy-restricted (M
rats) diets from 4 weeks of age. One member of each pair of littermates
was fed ad libitum (standard diet), with daily food intake
measured, and the intake of the other member of the pair (low protein
diet) was restricted to 65% of the ad libitum intake
(standard diet), with the food placed in the cage each evening (1 h
before the onset of the dark cycle). The choice of a moderate level
(35%) of food restriction was suggested by the study of Okitolonda
et al. (3), who reported that rats offered a low protein
diet after weaning spontaneously reduced their daily food intake to
65% of that of animals fed a normal isocaloric diet ad
libitum. Compulsory food restriction was necessitated by our
previous observation that Wistar rats given this low protein diet
ad libitum spontaneously maintained a normal food intake
(8).
The combination of low protein diet and 35% restriction was necessary to obtain, in our hands (5), growth arrest and impaired glucose-induced insulin secretion similar to those reported previously (3, 7).
Normal rats fed the standard diet ad libitum for 4 weeks were used as controls.
It is important to mention that no major alteration of the feeding pattern took place in the protein-energy-restricted group, as we verified that restricted rats had an excess of food available most of the time during the nocturnal feeding period and that the restricted rats never consumed in one short meal their daily food ration (in contrast to the findings usually reported in more severe food restriction protocols). As in the insulin secretion experiments performed at 1400 h, food was withdrawn in the two groups of rats in the morning of the study shortly after the onset of the light cycle, one may consider that the durations of subsequent fasting were comparable in the two groups.
After feeding the diets for 4 weeks, animals from each group underwent insulin secretion or nerve activity experiments.
Sympathetic and parasympathetic firing rate recordings
Firing rate measurements were performed at 1400 h.
Sympathetic activity was recorded at the level of the superior cervical
ganglion, and parasympathetic activity was recorded at the level of the
thoracic branch of the vagus nerve along the carotid artery as
previously described (17). Different rats were used for recording
either activity.
For sympathetic activity recording, the method was the following one. Briefly, rats were anesthetized (4 mg/100 g BW, ip, with pentobarbital; Sanofi, Libourne, France). The sympathetic nerve, which is close to the carotid artery, was dissected free of underlying tissues for a distance of about 1 cm until the superior cervical ganglion. The nerve was then covered with paraffin oil to prevent dehydration and carefully placed on a pair of recording silver wire electrodes (0.6 mm in diameter). Electrodes were connected to a high impedance probe, amplified by 104 with an alternative current amplifier (time constant, 0.2 sec) and filtered at low and high frequency cut-offs (1–80 kHz). The filtered, amplified signal was routed to an oscilloscope and a pen recorder and was stored on a magnetic tape for further analysis.
A very similar experimental design was used for parasympathetic activity recording. After exposure of the carotid artery, the thoracic branch of the vagus nerve was carefully separated from surrounding tissues. The follow-up of the procedure was identical to that described for sympathetic nerve activity recording.
In vivo glucose-induced insulin secretion tests
In untreated rats. Intravenous glucose tolerance tests
(ivGTT) were performed at 1400 h in 8-week-old M and C rats fasted
from 0900 h and under pentobarbital anesthesia (4 mg/100 g BW,
ip). A single injection of glucose (0.5 g glucose/kg BW) was
administered via a saphenous vein. Blood samples (200 µl) were
collected sequentially from the tail vein before (t0) and 5 (t5), 10
(t10), 20 (t20), and 30 (t30) min after the injection of glucose. They
were then centrifuged, and the plasma was separated. The plasma glucose
concentration was immediately determined on a 10-µl aliquot, and the
remaining plasma was kept at -20 C until RIA of insulin.
In rats treated with the 2-adrenergic
antagonist, yohimbine.To investigate whether the decreased
insulin response to glucose present in this model is related at least
in part to the increased adrenergic tone, we used an
2-adrenoceptor antagonist. As imidazolines and
derivatives are able to stimulate insulin release as well as increase
pancreatic vascular resistance (18), we selected a nonimidazoline
2-adrenoceptor antagonist, yohimbine. We used 10
µmol/kg BW yohimbine. This dose is in the range of that used in the
normal dog (19). Briefly, M and C rats were anesthetized, and after a
lag period of about 20 min, yohimbine hydrochloride (Sigma Chemical
Co., St. Louis, MO) was injected ip 5 min before the study of
glucose-induced insulin secretion.
In rats treated with the 2A-adrenergic
agonist, oxymetazoline. To determine the occupation of the
pancreatic 2-adrenoceptors in the M rats, we
studied the influence on glucose-induced insulin secretion of an
2A-adrenoceptor agonist, oxymetazoline, using the
protocol described previously by N’Guyen et al. (17).
Oxymetazoline was selected because it has been recently proposed that
in the pancreatic ß-cell, the 2A subtype, rather than
the 2B subtype, is specifically involved in the
inhibition of insulin release (20). To select the adequate
concentration, a dose-response study was performed in C rats. The
insulin secretion was studied in the presence of 0.34, 0.8, 3.4, 8.4,
or 16.8 nmol/kg BW oxymetazoline (ip injection), with a minimum of six
animals for each dose. It was significantly reduced by 33%, 51%,
80%, 92%, and 96%, respectively. We selected 3.4 nmol/kg BW
oxymetazoline to determine whether this dose (which reduced by 80% the
glucose-induced insulin response in C rats) is also effective in M
rats.
In rats treated with the cholinergic agonist, ACh. With the aim of testing whether the decreased parasympathetic tone had altered the response of the ß-cell to ACh, we attempted to reactivate the glucose-induced insulin release by ACh administered exogenously. Following the same general protocol, the first group of animals was injected ip with ACh chloride (Sigma Chemical Co.) at a dose of 9 nmol/kg BW 5 min before the study of glucose-induced insulin secretion. In a second group of animals, the ip injected dose was 27 nmol/kg BW.
Presentation of results and statistical analysis
Plasma glucose was determined using a glucose analyzer (Beckman,
Palo Alto, CA). Plasma immunoreactive insulin was estimated using
purified rat insulin as standard (Novo, Copenhagen, Denmark) and
porcine monoiodinated 125I-labeled insulin (5). Charcoal
was used to separate free from bound hormone (5). The method allows the
determination of 0.08 ng/ml with a coefficient of variation within and
between assays of 10%. The insulin and glucose responses during the
various ivGTT were calculated as the incremental plasma insulin values
integrated over the 30-min period following the glucose injection
(I; picomoles per min/liter-1) and the corresponding
incremental integrated plasma glucose values (G; nanomoles per
min/liter-1). The glucose disappearance rate was
calculated from the slope of the regression line obtained with the
logarithm of plasma glucose values between 5 and 30 min after glucose
injection. A minimum of seven animals was used for each series of
experiments.
Data are given as the mean ± SEM. Comparison between the groups was performed using Student’s test for unpaired data. For statistical evaluation of temporal variations, we used two-way ANOVA for repeated measures (between-group factor was treatment and within-group factor was time) followed by post-hoc testing (Fisher test) of individual means. For all analyses, P < 0.05 was considered significant.
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Results |
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). On the other hand, the
vagus nerve firing rate was largely lower in M rats compared with that
in C rats (C rats, 2.70 ± 0.35 spikes/sec; M rats, 0.60 ±
0.05 spikes/sec; n = 6; P < 0.001; Fig. 1).
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). Basal plasma insulin was 0.17 ±
0.06 nmol/liter for M rats and 0.94 ± 0.08 nmol/liter for C rats.
During the ivGTT, the peak plasma insulin concentration occurred at t5
in both groups, but the value was smaller (P < 0.01)
in the M rats (0.53 ± 0.09 nmol/liter) than in the C rats
(2.51 ± 0.16 nmol/liter; Fig. 2). The M rats
showed a very decreased I compared with that in C rats (993 ±
130 vs. 3910 ± 410 pmol/liter·min; P
< 0.001) as well as an increased G (25.8 ± 2.2 vs.
18.9 ± 1.2 mmol/liter·min; P < 0.001).
Consequently, the insulinogenic index (I/G) was significantly
decreased in M rats compared with C rats (36 ± 4 vs.
204 ± 20; P < 0.001; Tables 1 and 2).
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2-adrenergic
antagonist, yohimbine. In control rats, yohimbine
pretreatment amplified the glucose-induced insulin release compared
with that in untreated C rats (with a 2.6-fold increment of the I
value; P < 0.001). Their G was decreased by 24%,
and the resulting I/G was increased by 3.3-fold (Table 2
). As
indicated in Fig. 2 and when compared with that in untreated M rats,
yohimbine also increased in M rats the plasma insulin levels at t5
(P < 0.01), t10 (P < 0.01), t15
(P < 0.01), and t20 (P < 0.001), and
their I was increased by 3-fold. Their G was reduced by 52%, and
their I/G was 6-fold increased (Table 2).
In rats treated with the 2A-adrenergic
agonist, oxymetazoline. In control rats, oxymetazoline
pretreatment greatly decreased the glucose-induced insulin response
(Fig. 2 and Table 2). Their I was
decreased by 84% compared with that in untreated C rats
(P < 0.001), and their G was increased by 21%
(P < 0.05). Consequently, the insulinogenic index was
sharply decreased (by 84%, P < 0.001; Table 2). By
contrast, oxymetazoline exerted no significant effect in M rats on
their insulin release during ivGTT, and neither I nor G was
significantly modified compared with those in the untreated M group
(Table 2).
In rats treated with the cholinergic agonist, ACh. In the
first set of experiments, we used an ACh dosage of 9 nmol/kg BW. I
and G were not significantly affected by this dose in the
malnourished group or in the control group. Using ACh (27 nmol/kg BW),
we were able to increase glucose-induced insulin secretion in the C
rats, as attested by plasma insulin levels increased at t5
(P < 0.01), t10 (P < 0.01), t15
(P < 0.01), and t20 (P < 0.001; Fig. 3). Their I was increased by 2.4-fold compared with that in
untreated C rats, and their G was decreased by 24%. Accordingly,
their insulinogenic index was increased by 2.7-fold (Table 2). As
indicated in Fig. 3, 27 nmol/kg BW ACh in M rats also caused an
increase in plasma insulin levels at t5 (P < 0.01),
t10 (P < 0.01), t15 (P < 0.01), and
t20 (P < 0.01) compared with that in untreated M rats
(P < 0.001) with a resulting 1.8-fold increased I.
Their G was decreased by 27%, and their insulinogenic index was
2.7-fold increased.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has to be remembered that in the same rat model, the ß-cell mass was clearly decreased (11), and the in vivo insulin response to glucose was very poor (5), in agreement with previous reports in similar models (3, 7), a defect related at least in part to an intrinsic abnormality(ies) of the pancreatic ß-cells as shown in experiments using perfused pancreas (5, 10, 12). The impairment of glucose-induced insulin release was further confirmed using isolated islets (9, 21), and it has been partly attributed to a defect in the ability of glucose to increase Ca2+ uptake and/or to reduce Ca2+ efflux from the ß-cell (21).
However, to our knowledge, the importance of extrapancreatic factors in the modulation of the long term effect of protein-energy on pancreatic islet secretory function in vivo has never been taken into account. Among these factors, those of neural origin may play a crucial role. It is now well recognized that increased parasympathetic activity and/or decreased sympathetic activity prime the ß-cell secretory response to glucose stimulation (17, 22, 23). Accordingly, we hypothesized that prolonged protein-calorie restriction in young rats could lead to high sympathetic and/or low parasympathetic activities that would contribute to the low in vivo insulin secretory response to glucose stimulation.
As indicated by the firing rates recorded from the superior cervical ganglion and from the thoracic branch of the vagus nerve, both parasympathetic and sympathetic activities were greatly altered in malnourished rats tested in the postabsorptive state. Many studies have shown that pentobarbital decreases the activity of both parasympathetic and sympathetic activities (24, 25). However, in our experiments we do not think that the observed modifications of autonomic nervous activity are mainly related to pentobarbital action, as we did not find a parallel decrease in their activities (in fact, there was an increase in sympathetic activity and a decrease in parasympathetic activity).
Previous studies have shown that changes in basal plasma glucose levels may modify parasympathetic nerve activity in rodents. In rats it has been reported that iv glucose administration in the carotid artery increased the discharge rate in both the hepatic and pancreatic branches of the vagus nerve (26). A similar conclusion was reached using rats rendered chronically hyperglycemic (17). Moreover in Niijima’s experiments, the firing rate was closely related to the blood glucose level over a wide range of concentrations from 0.6–4.5 g/liter (26). Our findings of very low parasympathetic nerve activity in the presence of low blood glucose match these observations.
Concerning our observation of increased firing of the superior cervical ganglion, there have also been indications for an activation of the catecholaminergic system in adult rats submitted to perinatal long lasting undernutrition or young rats fed a low protein diet, as attested by acceleration of the norepinephrine turnover rate in brown adipose issue (27, 28) and white adipose tissue (29), increased turnover rate of brain dopamine and norepinephrine together with a higher tyrosine-hydroxylase activity (30), and increased serotonin turnover in the ventromedial hypothalamus (31). As the secretion of epinephrine and the release of norepinephrine in the rat mainly depend on sympathetic nervous system activation, our previous finding of elevated plasma catecholamine levels in malnourished rats (16) is probably attributable to their increased sympathetic activity. Concerning the control of sympathetic activity, it is recognized that it is acutely stimulated by carbohydrate feeding, especially glucose and fructose, in humans and rats (32, 33). Conversely, short term fasting suppresses sympathetic activity. Whether such an effect is mediated via diet-induced variations in plasma insulin and/or those in plasma glucose remains controversial. According to the experiments performed by Niijima in the rabbit (34), glucose itself suppresses sympathetic activity. Injection of a 10% glucose solution (but not mannose or saline) into the carotid artery or the general circulation decreases the discharge rate of the splanchnic nerve, whereas hypoglycemia increases it. Moreover N’Guyen et al. (17) reported a sharp decrease in the firing rates from the superior cervical ganglion in rats previously submitted to elevated and prolonged hyperglycemia, a finding consistent with the view of a suppressive effect of a chronic elevation of the blood glucose level on sympathetic nervous system activity. In addition, sympathetic nervous system activity is considerably reduced in streptozotocin-diabetic rats, which are hyperglycemic and hypoinsulinemic, whereas insulin injection into these rats along with the administration of glucose to maintain euglycemia stimulate sympathetic nerve activity. There are also in the literature some lines of evidence that glucose exerts its effect mainly at the level of CNS sites involved in the control of the autonomic nervous system output; Smythe and co-workers have shown that glucose modifies hypothalamic noradrenergic and serotoninergic neuronal activities (35, 36). An inverse relationship between the plasma glucose concentration and hypothalamic norepinephrine neuronal activity has been reported (37). As far as we can extrapolate from the conclusions drawn during chronic hyperglycemia/hyperinsulinemia, the increase in sympathetic activity and the decrease in parasympathetic activity in malnourished rats could therefore be viewed as merely consequences of hypoglycemia and not of hypoinsulinemia.
Although the roles of the individual counterregulatory hormones in hypoglycemia correction have been studied extensively, the mechanisms that link long term glucopenia with activation of the counterregulatory system are poorly understood. Both the CNS and extracerebral glucose sensors have been implied in the activation of counterregulatory hormone release during hypoglycemia (38, 39, 40, 41, 42), and it is conceivable that protein-calorie restriction early in life alters CNS control of counterregulatory hormone release. Of course, additional studies are warranted to test this hypothesis, but there are presently in the literature indications that protein deprivation significantly impairs a variety of neurotransmitter systems in the CNS, including ß-endorphin (43), norepinephrine (44), neuropeptide Y (45), and GRF (46).
Our data related to the insulin secretory response to glucose in
malnourished and control rats submitted to oxymetazoline, yohimbine, or
ACh treatments indicate that the increase in insulin release in
malnourished rats after yohimbine or ACh exposure is actually due to
the treatment itself and not to alterations in the glycemic profiles
after glucose loading; the respective G values in the control and
malnourished groups treated with yohimbine or ACh were, in fact,
decreased compared with corresponding G values in the untreated
control and malnourished groups. Clearly, these data strongly suggest
that the high sympathetic tone in the malnourished group contributes to
their low insulin secretion in vivo. The involvement of the
sympathetic nervous system in ß-cell hyporesponsiveness to glucose in
malnourished rats is evidenced by the data related to oxymetazoline and
yohimbine treatments. First, the greater enhancing effect of the
2-adrenoceptor antagonist yohimbine on the
glucose-induced insulin release in malnourished rats compared with that
in controls suggests that a higher endogenous
2-adrenergic tone does contribute to the impaired
ß-cell. Second, the inhibitory effect of oxymetazoline that was
detectable in the C rats was lacking in malnourished rats. Such a
pattern strongly argues for a functionally important role of high
adrenergic tone in the low ß-cell responsiveness to glucose in these
rats. By contrast, the evidence that the decrease in parasympathetic
activity is causally involved in the low insulin response to glucose is
weak, as ACh produced similar relative changes in both controls and M
rats.
In conclusion, whatever their precise cause, the alterations in the
autonomic nervous system identified in the present study indicate that
the impairment of the ß-cell secretory potential after
protein-calorie restriction coincides with and may be partly
attributable to an increase in sympathetic activity and a decrease in
parasympathetic activity. Further studies of such type of anomalies may
be relevant to the perturbation of islet function in
noninsulin-dependent diabetes (NIDDM) for at least two main reasons: 1)
it has been recently proposed that inadequate early nutrition greatly
increases susceptibility to the occurrence of NIDDM (47); and 2) it has
been suggested that catecholamines may play a role in the pathogenesis
of NIDDM, as serum catecholamine levels have been found to be elevated
in NIDDM patients compared with those in healthy subjects (48), and
enhanced ß-cell -adrenoreceptor activity has been frequently
reported in NIDDM patients compared with that in healthy subjects
(49, 50, 51, 52, 53, 54, 55). From a more general perspective, our data in the
protein-energy-restricted model reinforce the view of participation of
the autonomic nervous system in the in vivo ß-cell
reactivity in response to glucose.
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Acknowledgments |
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Footnotes |
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2 Recipient of a travel grant from Islet Research European Network
(concerted action of the European Union).
Received January 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Adrenergic blockade improves glucose-potentiated
insulin secretion in non-insulin-dependent diabetes mellitus. Diabetes 36:932–937[Abstract]
2-adrenergic blocker (DG-5128)
improves insulin secretion and in vivo glucose disposal in
NIDM patients. Diabetes 35:1085–1089[Abstract]
2-adrenoceptor antagonist on glucose tolerance in the
genetically obese mouse (C57BL/6J ob/ob). Metabolism 39:445–451[CrossRef][Medline]
2-adrenergic blocker
(MK-912) on pancreatic islet function in non-insulin-dependent diabetes
mellitus. Metabolism 40:1160–1167[CrossRef][Medline]
2-Adrenoceptor blockade does not enhance glucose-induced
insulin release in normal subjects or patients with
noninsulin-dependent diabetes. J Clin Endocrinol Metab 67:1054–1059This article has been cited by other articles:
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  P. Mitrani, M. Srinivasan, C. Dodds, and M. S. Patel Autonomic involvement in the permanent metabolic programming of hyperinsulinemia in the high-carbohydrate rat model Am J Physiol Endocrinol Metab, May 1, 2007; 292(5): E1364 - E1377. [Abstract] [Full Text] [PDF]
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P. Mitrani, M. Srinivasan, C. Dodds, and M. S. Patel Role of the autonomic nervous system in the development of hyperinsulinemia by high-carbohydrate formula feeding to neonatal rats Am J Physiol Endocrinol Metab, April 1, 2007; 292(4): E1069 - E1078. [Abstract] [Full Text] [PDF]
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 F. Ferreira, E. Filiputti, V. C. Arantes, L. F. Stoppiglia, E. P. Araujo, V. Delghingaro-Augusto, M. Q. Latorraca, M. H. Toyama, A. C. Boschero, and E. M. Carneiro Decreased Cholinergic Stimulation of Insulin Secretion by Islets from Rats Fed a Low Protein Diet Is Associated with Reduced Protein Kinase C{alpha} Expression J. Nutr., March 1, 2003; 133(3): 695 - 699. [Abstract] [Full Text] [PDF]
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A. Plagemann, T. Harder, A. Rake, K. Melchior, W. Rohde, and G. Dörner Hypothalamic Nuclei Are Malformed in Weanling Offspring of Low Protein Malnourished Rat Dams J. Nutr., October 1, 2000; 130(10): 2582 - 2589. [Abstract] [Full Text]
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)
and malnourished (
) rats recorded from vagus nerve and superior
cervical ganglion, respectively. Values are the mean ±
SEM of six rats each from the C and M groups. ***,
P < 0.001, malnourished vs.
controls.
and 
) and C
unrestricted (
and •) Wistar female rats. The animals were weaned
at 4 weeks of age and placed on either the restricted diet (35% food
restriction and low protein diet) or the control unrestricted diet.
Rats were tested after 4 weeks on their respective diets. The tests
were performed in intact rats (