AMERICAN
JOURNAL
OF PHYSIOLOGY
229, No. 2, August 1975.
Vol.
Printed
in U.S.A.
Hyporesponsiveness to glucoprivation postnatal period in the rat IRIT GIL-AD, GIULIANA Department of Pharmacology,
GIL-AD,
UDESCHINI, University of Milan,
DANIELA COCCHI, 20129 Milan, Italy
GIULIANA UDESCHINX, DANXELA Coccm, AND bfti LLER. Hyporesponsiveness to glucopriua tiun during postnatal period in the rat. Am. J. Physiol. 229(2) : 512-517. 1975.Z-Deoxy-D-glucose (Z-DG) , a glucose analogue, blocks glycolysis and induces intracellular glucoprivation. In the adult rat intraIRIT,
EUGE NXO II.
peritoneal administration of 2-DG or its injection into the lateral ventricle (NT) of the brain induces hyperglycemia which is divorced from a rise in plasma insulin (IRI). Tn the present study, responsiveness to Z-DG-induced glucoprivation, after central or intraperitoneal injection of the drug, was studied in rats of 7, 14, 21, and 28 days of age and compared to that of the adult rat (50 days old). In 7-, 14-, 21-, and Z&-day-old rats, the overall blood glucose (BG) response to IVT-injected 2-DG was equivalent to 4,3.3, 17, and 33 %, respectively, of the BG response present in the adult rat. Following intraperitoneal injection of Z-DG, the BG response evoked in the same age groups corresponded to 27, 31, 41, and 93 %, respectively, of the adult response. Base-line plasma IRX levels were significantly lower in pups than in adults and increased progressively with age, but no difference was present in IRI levels between 2-DG-treated and control pups whether the 2-DG was given via the lateral ventricle or intraperitoneally. These results demonstrate the presence in the infant rat of clear-cut hyporesponsiveness to Z-DG-induced glucoprivation. The different response pattern between experiments involving central and peripheral 2-DG administration supports the existence of separate peripheral glucoreceptors for 2-DG and their earlier ontogenic activation. Since in the infant mammal glucose is of minor relevance as an energy substrate, an interrelationship appears to be present between requirements for fuel(s) and homeostatic response to fuel deprivation. 2-DG glucoprivation; hyperglycemia; glucoreceptors; peripheral glucoreceptors;
insulin secretion; infant rat
AND
EUGENIO
E. MULLER
of Z-DG (26). R ecently it has been reported that in the rat, increased feeding, as a homeostatic response to 2-DGinduced cellular glucopenia, appears quite late during ontogeny, i.e., after weaning (18). The work reported here was designed to study the ontogeny of the hyperglycemic and blood insulin-lowering effect of 2-DG-induced glucoprivation after both central and peripheral administration of the drug. Since the evoked hyperglycemia and hypoinsulinemia is the more relevant emergency mechanism in the reflex response to glucoprivation in the adult rat (26), the study of these autonomic-endocrine responses (5, 12, 17) in the neonatal rat, the metabolic needs of which are quite dissimilar from those of the adult (16, 35), appeared to be of interest. MATERIALS
AND
METHODS
Animals Pregnant albino Wistar rats were obtained through the courtesy of Zambon S.p.A. Milano, in the 3rd wk of gestation. Pregnant rats were fed a standard laboratory diet and given tap water. Standard vivarium conditions included a room temperature of 22 =t 2’C and 14 h/day artificial light (0600-2000). After birth the newborn were allowed to suckle at will and remained with their dams until the start of the experiments. All litters were reduced to a standard size of 8-10. Pups of 7 and 14 days of age of both sexes were used, while only females were used from the other age groups (21, 28, and 50 days). Rats were weaned at 21 days and kept in housing conditions similar to the ones previously described.
central
Experimental
of both central and peripheral glucoreceptors sensitive to blood glucose in the measure that they can utilize it has been proposed, and their contribution to the short-term control of hunger has been emphasized (20, 28). Recently the function of these glucose-sensitive elements in the body regulatory adjustments to glucoprivation has been investigated with the aid of a glucose-analogue, Z-deoxy-Dglucose (2-DG) (23-25). A mong the many reflexive responses engendered by 2-DG-induced glucoprivation there is pronounced hyperphagia (33) and a striking hyperglycemia divorced from a rise in plasma immunoreactive insulin (IRI) (12). Previous work done in this laboratory has indicated that different populations of glucoreceptors in the brain mediate the hyperphagic or hyperglycemic, insulin-lowering effects THE
during
EXISTENCE
Procedures
Central 2-DG experiments. In 7- and 14-day-old pups, 2DG (Nutritional Biochemicals Corporation or BDH) was administered by a direct intraventricular (IVT) injection into the right lateral ventricle of the brain (27). In the other age groups the drug was given through a small polyethylene cannula (PE- 10) implanted into the right lateral ventricle 2-4 days before the experiment (I). The dose of Z-DG used was directly related to the actual mean brain weight of each group, and was determined on the basis of a dose of 4 mg per brain given to 50 day-old rats (see RESULTS). Control rats received an equivalent volume of an isosmolar NaCl solution. Adequate blood collection in the newborn (7 and 14 days old) necessitated exsanguination; hence each time period is represented by different animals. In the other 512
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ONTOGENY
OF 2-DG HYPERGLYCEMIC
RESPONSE
age groups the same animal was used for a different time period. Blood was collected in heparinized polyethylene tubes cooled in ice, 30, 60, and 90 min following injection, either from the decapitated trunks in 7- and 14-day-old pups, or from the orbital vessels (31) in the other age groups. An aliquot of blood was removed for blood glucose (BG) determination by the glucose oxidase method (Biochemical Test Combination, Boehringer Manheim Corp.). The remainder was centrifuged at 4°C and plasma was then separated and frozen until it was assayed in duplicate for immunoreactive insulin (IRI) by a double-antibody radioimmunoassay (11). Crystalline pork insulin (lot PJ 5589) and guinea pig pork-insulin antiserum were gifts of Dr. L. A. Frohman, Chicago. Pork insulin was iodinated with 1251 (Sorin, Saluggia, Italy) by the method of Greenwood et al. (14). The specific activity obtained was on the order of 1251-labeled pork insulin was purified be160-200 mCi/mg. fore being used by cellulose-absorption chromatography to remove the immunologically inactive components. For the insulin standard, crystalline pork insulin, wi-t-h a biological potency 25 U/mg was used. Separation of bound from free hormone was accomplished by precipitation of the antibodybound hormone with rabbit anti-guinea pig y-globulins. In this system the recovery of added insulin to rat plasma is about 90 %. The sensitivity of the assay, with an initial specific activity of 200 mCi/mg, is 0.2 pU, corresponding to 2.0 pu/ml. To minimize interassay variability, when possible, samples were run in the same assay and insulin and blood glucose levels were expressed as absolute values; otherwise, they were expressed as increments or decrements (A values) of base-line values (see RESULTS). intraPeripheral 2-DG experiments. 2-DG was administered peritoneally (ip) at the dose of 400 mg/kg to all age groups. Control animals received an equivalent volume of an isosmolar NaCl solution. Blood was collected and assayed for BG and IRI as described above. Glucose administration. D-Glucose was administered at the dose of 1 g/kg ip in all age groups. Blood was collected 5, 10, and 15 min after injection, and BG and IRI levels were determined as described above. 2-DG determination. In one experiment (see RESULTS) plasma was analyzed for 2-DG concentration by the method of Cramer and Neville (7). Statistical analyses were performed by the Student’s t test, and a probability p of SO.05 was taken as the level of significant difference. In some experiments the overall rise in blood glucose was calculated by triangulation. Areas under the mean blood glucose curves obtained at each age period were expressed in relation to the areas obtained in 50-day-old rats (see RESULTS). RESULTS
Effect of Central 2-DG Administratim
on Blood Glucose
The effect of IVT administration of 2-DG in rats of different ages is shown in Fig. I. In 7- and 14-day-old pups, 2-DG, at doses of 1 mg and 1.6 mg/lO ~1, respectively, did not evoke any significant hyperglycemic response. The overall BG response was equivalent to 4 and 3.3 %, respectively,
513 7 DAYS -
14 DAYS
21 DAYS
1. Effect of 2-DG given intraventricularly (IVT) on blood levels. Z-DG was given at doses of I, 1.6 mg/lO ~1 and 2.0, 2.2, and 4 mg/20 ~1, in 7, 14-, 21-, 2%, and 50-day-old rats, respectively. Control rats received IVT isosmolar solutions of NaCl in same voluses. Each point is mean =t SE of 6-8 determinations. Asterisks in this and Figs. 2-6 indicate a significant (P < 0.05) difference between experimental and control groups. FIG. @UCOS~
of the BG response present in 50-day-old rats. In 2 1-day-old rats, Z-DG, at the dose of 2 mg/lO ~1, induced a modest hyperglycemic effect (mean ABG levels at 30,60, and 90 min were 18, 28, and 26 mg/lOO ml), and the overall response was only 17 % thatt of the adult BG response. The hyperglycemic effect of 2-DG given centrally (2.2 mg/ZO ~1) in 28day-old rats was higher than that present in 21 -day-old animals (mean ABG levels were 40, 48, and 37 mg/ 100 ml), but the overall BG response was only 33 % that of the adult (Z-DG, 4 mg/ZO pl), in which mean ABG levels were 120, 148, and 125 mg/lOO ml, respectively. EJect of Peripheral
Z-DG Administration
on Blood Glucose
The effect of intraperitoneal injection of Z-DG (400 mg/ kg) on BG is shown in Fig. 2. It is evident that a hyperglycemic response was present from 7 days of age (mean ABG levels at 30, 60, and 90 min were 40, 38, and 42 mg/lOOml, respectively, on 7-day-old rats). When evaluated in comparison with the overall BG response present in 50day-old rats, the response in newborn animals was 27 %. There was a progressive increase in the BG response to 2-DG with advancing age. At 14 days of age the overall BG response was 31 % of the adult response; at 21 days of age, 41% (mean ABG levels were 60, 40, 36, and 62, 64, and 60 mg/lOO ml at 14 and 2 1 days of age, respectively). A striking increase in the BG response was present at 28 days of age (mean ABG levels were 92, 168, and 174 mg/lOO ml, and the overall response was 93 % of the adult response in which mean ABG levels were 98, I 76, and 186 mg/lOO ml.
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UDESCHINI,
GIL-AD,
1 31
1 II
L BP
50 DAYS 29
290
27
270
I
21
/
I
210
170
*f
in Plasma
Since Z-DG reacts slowly with glucose Oxidase, the plasma glucose measurements in the peripheral Z-DG experiments might be a mixture of glucose and Z-DG. Direct measurements of the sugar following administration of a dose of 400 mg/kg in 14-day-old rats showed, at 30 min, levels corresponding to 10 mg/lOO ml and at 60 and 90 min, levels of less than 5 mg/ 100 ml.
230
lB0
Intraperitoneal injection of glucose induced a rise in BG at 5, 10, and 15 min in all age groups (Fig. 5). In 7-day-old rats the BG rise did not evoke an appreciable rise in plasma insulin (mean AIR1 levels were 2.0, 1.5, and 8.0 pU/ml 5, IO, and 15 min after glucose administration). In 14-day-old rats mean AIRI levels were 3.0, 14.0, and Il.0 pU/ml, respectively, In 21- and 28-day-old rats the insulin response to exogenous glucose was higher than in the preceding age groups but lower than in adult rats (mean AIR1 levels were 14.0, 35.0, 23.0, and 16.0, 30.0, and 24.0 pU/ml, respectively). In adults mean AIR1 levels were 24.0, 57.0 and 68.0 pU/ml, respectively (Fig. 6). Z-DG Determination
25B 23
AND MtiLLER
E$ect of ExogenousGlucoseonBlood GlucoseandPlasmaInsulin
21 DAYS
14 DAYS
COCCHX,
I’
190 110 90
I4
70 r
30 t
--.-~
r-
TIME ( MIN )
r’o
levels.
]1 14 DAYS
1 21 DAYS
SALINE
SALINE
lo-
11
on blood glucose in all age groups. Control rats received intraperitoneally an isosmolar solution of NaCI. Each point is mean I#I SE of 6-8 determinations. FIG.
7 DAYS
1
$0
Effect of Z-DG given intraperitoneally 2-DG was given at dose of 400 mg/kg 2.
sm
50 DAYS
28 DAYS
f
251
25-1
T 2-06
After neither central nor peripheral Z-DG administration did any infants die or even show behavioral signs of acute Z-DG glucoprivation. These signs were present only when Z-DG was given to adults. Insulin Response to Central or Peripheral
I
3’0
L
9’0
11
Z-DG Administration
Despite the presence of a hyperglycemic response in all age groups in the peripheral Z-DG experiments and in Zl-, 28-, and 50-day-old rats in the central experiments involving IVT administration of the sugar, no significant increase in plasma IRI levels was observed at any time (Figs. 3 and 4).
8’0
of 2-DG given IVT levels. Doses and animals
(IRI)
7 DAYS
I
to
t
on plasma immunoreactive inare same as reported in Fig. 1.
21DAYS
14 DAYS
SALINE
I
Base-Line
Blood Glucose and insulin
I
9’0
TIME I MIH 1
FKG. 3. Effect
sulin
3’0
1
Levels
There was a clear-cut increase in basal BG levels between 7 and 14 days of age; in the following age groups no further BG increments were present (Figs. 1 and 2). Mean (& SE) basal BG values in 7-, 14-, 21-, 28- and 50-day-old rats were 71 &4,99&7,93&3,83&5,and92&4mg/lOOml,respectively (pooled determinations of experiments involving both IVT and intraperitoneal Z-DG administration.) Basal plasma insulin levels progressively increased with age. Mean IRI levels were 2.5 & 0.5, 6.0 h 1.0, 7.5 -J=2.0, 13 k 1.0, and 19.5 =t 2.0 PI-?/ml in 7-, 14-, Zl-, and 50-day-old rats, respectively (pooled determinations of experiments involving both IVT and intraperitoneal Z-DG administration).
28 DAYS
= CA
z
I
1
$0
# FIG. 4. Effect
61 DAYS
s’o
$0
J
I
I $0
TIME
I MIN I
If0
9’0
1
of Z-DG given intraperitoneally on plasma Dose and animals are same as reported in Fig. 2.
IRI
levels.
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ONTOGENY
OF
J 01
8
HYPERGLYCEMIC
515
RESPONSE
OLUCOIE
1 BLUCOSE
S 4
Z-IX
*E--U /
#’ 3
50 DAYS
1
* f
BlUCQ8E
I
T
10
6
11
TM
(MIMJ
I
15
1
5. Effect of intraperitoneal injection of glucose (1 g/kg) on blood glucose levels in rats of all age groups. Values are expressed as change (A) from base-line levels. Each point is mean =t SE of 6-8 determinations. FIG.
5
21 OAYS
14 DAYS
7 DAYS
I
0/ 6
-4
0 1 10
*;f 1 15
SAL.
-(o 50 DAY8 BLUCOSE
GLUCOSE
TIME
6. Effect of glucose on plasma are same as reported in Fig. 5. FIG.
I MIM 1
IRI
levels.
Dose
and
animals
In the infant rat a clear-cut hyporesponsiveness to the state of glucoprivation induced by Z-DG was present after central or peripheral administration of the sugar. Z-DG was given centrally at doses which were directly related to the weight of the brain at different ages. In this way the doses that were instilled were in excess of those that would have been calculated on the basis of the ratio of 1 to 20 between the central and peripheral routes, which is valid in the adult rat (23). Nevertheless, central response to Z-DG was greatly
reduced and even more impaired than that elicited by the systemic injection. No hyperglycemia effect was elicitable in 7- and I4-day-old rats, and in 21- and 28-day-old rats the action of the sugar was still unequivocally lower than in adults. Peripheral Z-DG administration, however, induced a modest hyperglycemia from 7 days of age; this response increased progressively with age, and at 28 days of age was already comparable to the adult response. Hyperglycemia following systemic injection of 2-DG has been previously observed by Houpt and Epstein (18) in 3-day-old rats. When administered peripherally, 2-DG evokes neurological signs, viz., drowsiness, stupor, and ataxia, which imply that activation of both central and peripheral glucoreceptors occurred in animals (33). Although workers agree that the production of cerebral glucoprivation by 2-DG is the critical event, the possibility that peripheral glucoprivation is of equal or greater importance was not excluded. The present data, which clearly showed in the young rat a different response pattern when 2-DG was given IVT than when it was injected intraperitoneally, support the existence of separate peripheral glucoreceptors for 2-DG and their earlier ontogenit activation. In this context, proof has been furnished recently by Novin et al. (28) for the existence of vagally mediated peripheral glucoreceptors important in initiation of 2-DG-induced feeding behavior. The possibility was considered that in experiments involving its peripheral administration Z-DG itself might have interferred in the blood glucose measurements, but this is made unlikely by the rapid metabolization of the drug after its systemic injection ((33) and this study). The hyperglycemia and the blood-insulin lowering effect which follow central or systemic 2-DG administration are, in animals as well as in human beings, reflexive autonomic responses due mainly to a discharge of catecholamines from the adrenal medulla (5, 17). The effect of epinephrine is to inhibit insulin secretion in the presence of hyperglycemia and to antagonize the action of insulin on glucose uptake by muscle and adipose tissue, thereby increasing glucose availability to the brain. Starting in the neonatal period, the adrenal medulla is capable of increasing the secretion of epinephrine under the influence of various stimuli (32). However, its secretory capacity in the neonate is lower than in the mature organism (6). It could be hypothesized that reduced adrenomedullary secretion was responsible for the poor blood glucose rise present in experiments involving IVT or intraperitoneal injection. However, in the experiments witd IVT Z-DG, a reduced hyperglycemic response was present also in 28-day-old rats, at an age when the secretory capacity of the adrenal medulla is fully developed (9). Moreover, in this study, catecholamine secretion was always sufficient to suppress the hyperglycemia-induced insulin rise. Immaturity of the glucose-sensitive elements which monitor the state of glucoprivation (20), or their inability to ” sense” glucose as a metabolic fuel, appear more plausible explanations for the sluggish response to glucoprivation noticed in the young rat. The second hypothesis, i.e., lack of detection of glucose as a metabolic substrate, appears to be more likely if the peculiar metabolic requirements of the infant period are considered. Before birth the fetal rat receives its supply of nutrients in the form of glucose and amino
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516
GIL-AD,
acids via the placenta (15). After birth there is a dramatic change in the nutrient supply, and the newborn animal is now on a natural high-fat, low-glucose diet (16), so that until weaning oxidation of lipids serves as the main supply of energy (16). Proteins contribute little to the metabolism of the newborn since only 4 % of the basal calories come from protein catabolism, whereas in the adult this value reaches 17 % (2 I). Predominance of lipid metabolism is reflected in the young mammal by the presence of high plasma concentrations of nonesterified fatty acids, glycerol, and ketone bodies, and by a low respiratory quotient (8, 19, 30) Neonatal brain is not exceptional in its nutritional requirements and depends heavily on fuels other than glucose to support energy metabolism. Glucose utilization on the 1st day of life is, in fact, less than 1/l&h that of the adult, and remains constant for 12 days, when it rises to about sth that of the adult (22). On the other hand, enzymes for ketone body utilization are present in higher concentration in the brain of suckling rats than they are in the brain of adults (29). Since it has been shown, by the use of various substrates of the glycolytic pathway, that brain glucoreceptors do not monitor the availability of intracellular free glucose, but rather the rate of oxidative metabolism (lo), it is not unlikely that the almost complete insensitivity to glucose deficiency in the postnatal period may be due to the minor relevance of this substrate as metabolic fuel. The lack of behavioral signs to acute Z-DG glucoprivation noticed in the present study is another indication that glucose is not a dominant fuel for the infant brain. The relatively lower activities of the enzymes concerned in ketone body utilization in peripheral tissues (29) might explain the higher sensitivity to Z-DG glucoprivation present in the studies involving peripheral administration of this sugar. Experiments in which glucose was administered peripherally were aimed to evaluate properly the effectiveness of Z-DG in inhibiting the insulin secretion due to the blood glucose rise. Plasma IRI levels increased progressively with age from very low base-line values in the immediate postnatal period; similarly, there was a progressive increase in the in-
UDESCHINI,
COCCHI,
AND
MijLLER
sulin response to exogenous glucose with advancing age. These results confirm similar findings in both animal (2, 4) and man (3, 34). In 7- and 14-day-old rats lack of the insulin response to Z-DG-evoked hyperglycemia (upon peripheral administration) cannot, by any means, be ascribed to the inhibitory action of Z-DG. At these ages, in fact, exogenous glucose was also unable to induce a consistent insulin response. At successive ages, however (21 and 28 days), inhibition of the insulin rise in response to endogenous hyperglycemia has to be attributed to the inhibitory effect of 2-DG. This is mediated by the adrenals and catecholamine secretion in the experiments involving central Z-DG administration (12) and in addition by direct inhibition of the beta cells in the experiments in which the sugar was given peripherally (13). The fact that, despite the presence of a diminished hyperglycemic response to glucoprivation, the blood-insulin-lowering effect of Z-DG is fully operative stresses the relevance of the adrenergic or direct inhibition of insulin secretion by the pancreas in the metabolic adjustment to glucose deprivation. Such a mechanism, which allows an increased availability of glucose for the brain, is of relatively minor importance in the immediate postnatal life (l-15 days) when glucose utilization in the brain is poor, but undoubtedly plays a significance role later on, when a rise in the rate of glucose utilization occurs. In summary, the hyporesponsiveness to 2-DG glucoprivation of the newborn rat does not extend only to the control of food intake (18), but apparently involves also the autonomic-endocrine response to hyperglycemia. Collectively, our data suggest that the magnitude of the reflexive response to a lack of a metabolizable substrate is related to the quantitative importance of the latter as a fuel. assistance
The fully
skilful technical acknowledged. This investigation
74.00140.04 Send
supported
of the Consiglio reprint
requests
of Pharmacology, Milan, Italy. Received
was
for
to Dr.
Lavinia
Frizzi
is grate-
in
Nazionale
University publication
of Miss
Eugenio
of Milan,
25 September
part by Contract Ricerche, Italy. E. Miiller, 2nd Chair
CT
delle
Via
Vanvitelli
32,
Dept. 20129
1974.
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ONTOGENY 16.
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19. 20. 21.
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RESPONSE
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postnatal
17.
OF Z-DG HYPERGLYCEMIC P., AND 0. KOLDOVSKY. development. Oxford
517 26. MUELLER, E. E., A. PECILE, D. COCCHI, AND V. R. OLGXATI. Hyperglycemic or feeding response to glucoprivation and hypothalamic glucoreceptors. Am. J. Physiol. 226: 1100-1109, 1974. 27. NOBLE, E. P., R. J* WURTMAN, AND R. J* AXIZLROD. A simple and rapid method for injecting H3 norepinephrine into the lateral ventricle of the rat brain. Life Sci. 6 : 281-291, 1967, 28. NOVIN, D., D. A. VANDER WEELE, AND M. REZEK. Infusion of 2-deoxy-D-glucose into the hepatic system causes eating: evidence for peripheral glucoreceptors. Science 18 1 : 858-860, 1973. 29. PAGE, M. A., H. A. KREBS, AND D. H. WILLIAMSON. Activities of enzyme of ketone bodies utilization in brain and other tissues of suckling rats. Biochem. J. 12 1 : 49-53, 1971. 30. PERSSON, B., AND J. GENTZ. The pattern of blood lipids, glycerol and ketone bodies during the neonatal period, infancy and childhood. Acta Paediat. 55: 353-362, 1966. 31. RERUP, C., AND I. LUNDQUIST. Blood glucose levels in mice. I. Evaluation of a new technique of multiple serial samplings. Acta Erzdocrinol. 52 : 357-367, 1966. 32. ROFFI, J., J, L. FROGER, AND M. F. DEBRAY. Depletion de 1’adrknaIine surr&alienne chez le nouveau-n& de Rat et de Lapin. Co??+ Rend. Acad. Sci. 277 : 595-597, 1973. 33. SMITH, G. P., AND A. N. EPSTEIN. Increased feeding in response to decreased glucose utilization in the rat and monkey. Am. J. Physiol. 217 : 1083-1087, 1969. AND D. O’BRIEN. Plasma insulin 34. STIMMLER, L., Jm V. BRAZIE, levels in the newborn infants of normal and diabetic mothers. Lancet. 1: 137-138, 1964.
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JOURNAL
OF PHYSIOLOGY
229, No. 2, August 1975.
Vol.
Printed
in U.S.A.
Hyporesponsiveness to glucoprivation postnatal period in the rat IRIT GIL-AD, GIULIANA Department of Pharmacology,
GIL-AD,
UDESCHINI, University of Milan,
DANIELA COCCHI, 20129 Milan, Italy
GIULIANA UDESCHINX, DANXELA Coccm, AND bfti LLER. Hyporesponsiveness to glucopriua tiun during postnatal period in the rat. Am. J. Physiol. 229(2) : 512-517. 1975.Z-Deoxy-D-glucose (Z-DG) , a glucose analogue, blocks glycolysis and induces intracellular glucoprivation. In the adult rat intraIRIT,
EUGE NXO II.
peritoneal administration of 2-DG or its injection into the lateral ventricle (NT) of the brain induces hyperglycemia which is divorced from a rise in plasma insulin (IRI). Tn the present study, responsiveness to Z-DG-induced glucoprivation, after central or intraperitoneal injection of the drug, was studied in rats of 7, 14, 21, and 28 days of age and compared to that of the adult rat (50 days old). In 7-, 14-, 21-, and Z&-day-old rats, the overall blood glucose (BG) response to IVT-injected 2-DG was equivalent to 4,3.3, 17, and 33 %, respectively, of the BG response present in the adult rat. Following intraperitoneal injection of Z-DG, the BG response evoked in the same age groups corresponded to 27, 31, 41, and 93 %, respectively, of the adult response. Base-line plasma IRX levels were significantly lower in pups than in adults and increased progressively with age, but no difference was present in IRI levels between 2-DG-treated and control pups whether the 2-DG was given via the lateral ventricle or intraperitoneally. These results demonstrate the presence in the infant rat of clear-cut hyporesponsiveness to Z-DG-induced glucoprivation. The different response pattern between experiments involving central and peripheral 2-DG administration supports the existence of separate peripheral glucoreceptors for 2-DG and their earlier ontogenic activation. Since in the infant mammal glucose is of minor relevance as an energy substrate, an interrelationship appears to be present between requirements for fuel(s) and homeostatic response to fuel deprivation. 2-DG glucoprivation; hyperglycemia; glucoreceptors; peripheral glucoreceptors;
insulin secretion; infant rat
AND
EUGENIO
E. MULLER
of Z-DG (26). R ecently it has been reported that in the rat, increased feeding, as a homeostatic response to 2-DGinduced cellular glucopenia, appears quite late during ontogeny, i.e., after weaning (18). The work reported here was designed to study the ontogeny of the hyperglycemic and blood insulin-lowering effect of 2-DG-induced glucoprivation after both central and peripheral administration of the drug. Since the evoked hyperglycemia and hypoinsulinemia is the more relevant emergency mechanism in the reflex response to glucoprivation in the adult rat (26), the study of these autonomic-endocrine responses (5, 12, 17) in the neonatal rat, the metabolic needs of which are quite dissimilar from those of the adult (16, 35), appeared to be of interest. MATERIALS
AND
METHODS
Animals Pregnant albino Wistar rats were obtained through the courtesy of Zambon S.p.A. Milano, in the 3rd wk of gestation. Pregnant rats were fed a standard laboratory diet and given tap water. Standard vivarium conditions included a room temperature of 22 =t 2’C and 14 h/day artificial light (0600-2000). After birth the newborn were allowed to suckle at will and remained with their dams until the start of the experiments. All litters were reduced to a standard size of 8-10. Pups of 7 and 14 days of age of both sexes were used, while only females were used from the other age groups (21, 28, and 50 days). Rats were weaned at 21 days and kept in housing conditions similar to the ones previously described.
central
Experimental
of both central and peripheral glucoreceptors sensitive to blood glucose in the measure that they can utilize it has been proposed, and their contribution to the short-term control of hunger has been emphasized (20, 28). Recently the function of these glucose-sensitive elements in the body regulatory adjustments to glucoprivation has been investigated with the aid of a glucose-analogue, Z-deoxy-Dglucose (2-DG) (23-25). A mong the many reflexive responses engendered by 2-DG-induced glucoprivation there is pronounced hyperphagia (33) and a striking hyperglycemia divorced from a rise in plasma immunoreactive insulin (IRI) (12). Previous work done in this laboratory has indicated that different populations of glucoreceptors in the brain mediate the hyperphagic or hyperglycemic, insulin-lowering effects THE
during
EXISTENCE
Procedures
Central 2-DG experiments. In 7- and 14-day-old pups, 2DG (Nutritional Biochemicals Corporation or BDH) was administered by a direct intraventricular (IVT) injection into the right lateral ventricle of the brain (27). In the other age groups the drug was given through a small polyethylene cannula (PE- 10) implanted into the right lateral ventricle 2-4 days before the experiment (I). The dose of Z-DG used was directly related to the actual mean brain weight of each group, and was determined on the basis of a dose of 4 mg per brain given to 50 day-old rats (see RESULTS). Control rats received an equivalent volume of an isosmolar NaCl solution. Adequate blood collection in the newborn (7 and 14 days old) necessitated exsanguination; hence each time period is represented by different animals. In the other 512
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ONTOGENY
OF 2-DG HYPERGLYCEMIC
RESPONSE
age groups the same animal was used for a different time period. Blood was collected in heparinized polyethylene tubes cooled in ice, 30, 60, and 90 min following injection, either from the decapitated trunks in 7- and 14-day-old pups, or from the orbital vessels (31) in the other age groups. An aliquot of blood was removed for blood glucose (BG) determination by the glucose oxidase method (Biochemical Test Combination, Boehringer Manheim Corp.). The remainder was centrifuged at 4°C and plasma was then separated and frozen until it was assayed in duplicate for immunoreactive insulin (IRI) by a double-antibody radioimmunoassay (11). Crystalline pork insulin (lot PJ 5589) and guinea pig pork-insulin antiserum were gifts of Dr. L. A. Frohman, Chicago. Pork insulin was iodinated with 1251 (Sorin, Saluggia, Italy) by the method of Greenwood et al. (14). The specific activity obtained was on the order of 1251-labeled pork insulin was purified be160-200 mCi/mg. fore being used by cellulose-absorption chromatography to remove the immunologically inactive components. For the insulin standard, crystalline pork insulin, wi-t-h a biological potency 25 U/mg was used. Separation of bound from free hormone was accomplished by precipitation of the antibodybound hormone with rabbit anti-guinea pig y-globulins. In this system the recovery of added insulin to rat plasma is about 90 %. The sensitivity of the assay, with an initial specific activity of 200 mCi/mg, is 0.2 pU, corresponding to 2.0 pu/ml. To minimize interassay variability, when possible, samples were run in the same assay and insulin and blood glucose levels were expressed as absolute values; otherwise, they were expressed as increments or decrements (A values) of base-line values (see RESULTS). intraPeripheral 2-DG experiments. 2-DG was administered peritoneally (ip) at the dose of 400 mg/kg to all age groups. Control animals received an equivalent volume of an isosmolar NaCl solution. Blood was collected and assayed for BG and IRI as described above. Glucose administration. D-Glucose was administered at the dose of 1 g/kg ip in all age groups. Blood was collected 5, 10, and 15 min after injection, and BG and IRI levels were determined as described above. 2-DG determination. In one experiment (see RESULTS) plasma was analyzed for 2-DG concentration by the method of Cramer and Neville (7). Statistical analyses were performed by the Student’s t test, and a probability p of SO.05 was taken as the level of significant difference. In some experiments the overall rise in blood glucose was calculated by triangulation. Areas under the mean blood glucose curves obtained at each age period were expressed in relation to the areas obtained in 50-day-old rats (see RESULTS). RESULTS
Effect of Central 2-DG Administratim
on Blood Glucose
The effect of IVT administration of 2-DG in rats of different ages is shown in Fig. I. In 7- and 14-day-old pups, 2-DG, at doses of 1 mg and 1.6 mg/lO ~1, respectively, did not evoke any significant hyperglycemic response. The overall BG response was equivalent to 4 and 3.3 %, respectively,
513 7 DAYS -
14 DAYS
21 DAYS
1. Effect of 2-DG given intraventricularly (IVT) on blood levels. Z-DG was given at doses of I, 1.6 mg/lO ~1 and 2.0, 2.2, and 4 mg/20 ~1, in 7, 14-, 21-, 2%, and 50-day-old rats, respectively. Control rats received IVT isosmolar solutions of NaCl in same voluses. Each point is mean =t SE of 6-8 determinations. Asterisks in this and Figs. 2-6 indicate a significant (P < 0.05) difference between experimental and control groups. FIG. @UCOS~
of the BG response present in 50-day-old rats. In 2 1-day-old rats, Z-DG, at the dose of 2 mg/lO ~1, induced a modest hyperglycemic effect (mean ABG levels at 30,60, and 90 min were 18, 28, and 26 mg/lOO ml), and the overall response was only 17 % thatt of the adult BG response. The hyperglycemic effect of 2-DG given centrally (2.2 mg/ZO ~1) in 28day-old rats was higher than that present in 21 -day-old animals (mean ABG levels were 40, 48, and 37 mg/ 100 ml), but the overall BG response was only 33 % that of the adult (Z-DG, 4 mg/ZO pl), in which mean ABG levels were 120, 148, and 125 mg/lOO ml, respectively. EJect of Peripheral
Z-DG Administration
on Blood Glucose
The effect of intraperitoneal injection of Z-DG (400 mg/ kg) on BG is shown in Fig. 2. It is evident that a hyperglycemic response was present from 7 days of age (mean ABG levels at 30, 60, and 90 min were 40, 38, and 42 mg/lOOml, respectively, on 7-day-old rats). When evaluated in comparison with the overall BG response present in 50day-old rats, the response in newborn animals was 27 %. There was a progressive increase in the BG response to 2-DG with advancing age. At 14 days of age the overall BG response was 31 % of the adult response; at 21 days of age, 41% (mean ABG levels were 60, 40, 36, and 62, 64, and 60 mg/lOO ml at 14 and 2 1 days of age, respectively). A striking increase in the BG response was present at 28 days of age (mean ABG levels were 92, 168, and 174 mg/lOO ml, and the overall response was 93 % of the adult response in which mean ABG levels were 98, I 76, and 186 mg/lOO ml.
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UDESCHINI,
GIL-AD,
1 31
1 II
L BP
50 DAYS 29
290
27
270
I
21
/
I
210
170
*f
in Plasma
Since Z-DG reacts slowly with glucose Oxidase, the plasma glucose measurements in the peripheral Z-DG experiments might be a mixture of glucose and Z-DG. Direct measurements of the sugar following administration of a dose of 400 mg/kg in 14-day-old rats showed, at 30 min, levels corresponding to 10 mg/lOO ml and at 60 and 90 min, levels of less than 5 mg/ 100 ml.
230
lB0
Intraperitoneal injection of glucose induced a rise in BG at 5, 10, and 15 min in all age groups (Fig. 5). In 7-day-old rats the BG rise did not evoke an appreciable rise in plasma insulin (mean AIR1 levels were 2.0, 1.5, and 8.0 pU/ml 5, IO, and 15 min after glucose administration). In 14-day-old rats mean AIRI levels were 3.0, 14.0, and Il.0 pU/ml, respectively, In 21- and 28-day-old rats the insulin response to exogenous glucose was higher than in the preceding age groups but lower than in adult rats (mean AIR1 levels were 14.0, 35.0, 23.0, and 16.0, 30.0, and 24.0 pU/ml, respectively). In adults mean AIR1 levels were 24.0, 57.0 and 68.0 pU/ml, respectively (Fig. 6). Z-DG Determination
25B 23
AND MtiLLER
E$ect of ExogenousGlucoseonBlood GlucoseandPlasmaInsulin
21 DAYS
14 DAYS
COCCHX,
I’
190 110 90
I4
70 r
30 t
--.-~
r-
TIME ( MIN )
r’o
levels.
]1 14 DAYS
1 21 DAYS
SALINE
SALINE
lo-
11
on blood glucose in all age groups. Control rats received intraperitoneally an isosmolar solution of NaCI. Each point is mean I#I SE of 6-8 determinations. FIG.
7 DAYS
1
$0
Effect of Z-DG given intraperitoneally 2-DG was given at dose of 400 mg/kg 2.
sm
50 DAYS
28 DAYS
f
251
25-1
T 2-06
After neither central nor peripheral Z-DG administration did any infants die or even show behavioral signs of acute Z-DG glucoprivation. These signs were present only when Z-DG was given to adults. Insulin Response to Central or Peripheral
I
3’0
L
9’0
11
Z-DG Administration
Despite the presence of a hyperglycemic response in all age groups in the peripheral Z-DG experiments and in Zl-, 28-, and 50-day-old rats in the central experiments involving IVT administration of the sugar, no significant increase in plasma IRI levels was observed at any time (Figs. 3 and 4).
8’0
of 2-DG given IVT levels. Doses and animals
(IRI)
7 DAYS
I
to
t
on plasma immunoreactive inare same as reported in Fig. 1.
21DAYS
14 DAYS
SALINE
I
Base-Line
Blood Glucose and insulin
I
9’0
TIME I MIH 1
FKG. 3. Effect
sulin
3’0
1
Levels
There was a clear-cut increase in basal BG levels between 7 and 14 days of age; in the following age groups no further BG increments were present (Figs. 1 and 2). Mean (& SE) basal BG values in 7-, 14-, 21-, 28- and 50-day-old rats were 71 &4,99&7,93&3,83&5,and92&4mg/lOOml,respectively (pooled determinations of experiments involving both IVT and intraperitoneal Z-DG administration.) Basal plasma insulin levels progressively increased with age. Mean IRI levels were 2.5 & 0.5, 6.0 h 1.0, 7.5 -J=2.0, 13 k 1.0, and 19.5 =t 2.0 PI-?/ml in 7-, 14-, Zl-, and 50-day-old rats, respectively (pooled determinations of experiments involving both IVT and intraperitoneal Z-DG administration).
28 DAYS
= CA
z
I
1
$0
# FIG. 4. Effect
61 DAYS
s’o
$0
J
I
I $0
TIME
I MIN I
If0
9’0
1
of Z-DG given intraperitoneally on plasma Dose and animals are same as reported in Fig. 2.
IRI
levels.
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ONTOGENY
OF
J 01
8
HYPERGLYCEMIC
515
RESPONSE
OLUCOIE
1 BLUCOSE
S 4
Z-IX
*E--U /
#’ 3
50 DAYS
1
* f
BlUCQ8E
I
T
10
6
11
TM
(MIMJ
I
15
1
5. Effect of intraperitoneal injection of glucose (1 g/kg) on blood glucose levels in rats of all age groups. Values are expressed as change (A) from base-line levels. Each point is mean =t SE of 6-8 determinations. FIG.
5
21 OAYS
14 DAYS
7 DAYS
I
0/ 6
-4
0 1 10
*;f 1 15
SAL.
-(o 50 DAY8 BLUCOSE
GLUCOSE
TIME
6. Effect of glucose on plasma are same as reported in Fig. 5. FIG.
I MIM 1
IRI
levels.
Dose
and
animals
In the infant rat a clear-cut hyporesponsiveness to the state of glucoprivation induced by Z-DG was present after central or peripheral administration of the sugar. Z-DG was given centrally at doses which were directly related to the weight of the brain at different ages. In this way the doses that were instilled were in excess of those that would have been calculated on the basis of the ratio of 1 to 20 between the central and peripheral routes, which is valid in the adult rat (23). Nevertheless, central response to Z-DG was greatly
reduced and even more impaired than that elicited by the systemic injection. No hyperglycemia effect was elicitable in 7- and I4-day-old rats, and in 21- and 28-day-old rats the action of the sugar was still unequivocally lower than in adults. Peripheral Z-DG administration, however, induced a modest hyperglycemia from 7 days of age; this response increased progressively with age, and at 28 days of age was already comparable to the adult response. Hyperglycemia following systemic injection of 2-DG has been previously observed by Houpt and Epstein (18) in 3-day-old rats. When administered peripherally, 2-DG evokes neurological signs, viz., drowsiness, stupor, and ataxia, which imply that activation of both central and peripheral glucoreceptors occurred in animals (33). Although workers agree that the production of cerebral glucoprivation by 2-DG is the critical event, the possibility that peripheral glucoprivation is of equal or greater importance was not excluded. The present data, which clearly showed in the young rat a different response pattern when 2-DG was given IVT than when it was injected intraperitoneally, support the existence of separate peripheral glucoreceptors for 2-DG and their earlier ontogenit activation. In this context, proof has been furnished recently by Novin et al. (28) for the existence of vagally mediated peripheral glucoreceptors important in initiation of 2-DG-induced feeding behavior. The possibility was considered that in experiments involving its peripheral administration Z-DG itself might have interferred in the blood glucose measurements, but this is made unlikely by the rapid metabolization of the drug after its systemic injection ((33) and this study). The hyperglycemia and the blood-insulin lowering effect which follow central or systemic 2-DG administration are, in animals as well as in human beings, reflexive autonomic responses due mainly to a discharge of catecholamines from the adrenal medulla (5, 17). The effect of epinephrine is to inhibit insulin secretion in the presence of hyperglycemia and to antagonize the action of insulin on glucose uptake by muscle and adipose tissue, thereby increasing glucose availability to the brain. Starting in the neonatal period, the adrenal medulla is capable of increasing the secretion of epinephrine under the influence of various stimuli (32). However, its secretory capacity in the neonate is lower than in the mature organism (6). It could be hypothesized that reduced adrenomedullary secretion was responsible for the poor blood glucose rise present in experiments involving IVT or intraperitoneal injection. However, in the experiments witd IVT Z-DG, a reduced hyperglycemic response was present also in 28-day-old rats, at an age when the secretory capacity of the adrenal medulla is fully developed (9). Moreover, in this study, catecholamine secretion was always sufficient to suppress the hyperglycemia-induced insulin rise. Immaturity of the glucose-sensitive elements which monitor the state of glucoprivation (20), or their inability to ” sense” glucose as a metabolic fuel, appear more plausible explanations for the sluggish response to glucoprivation noticed in the young rat. The second hypothesis, i.e., lack of detection of glucose as a metabolic substrate, appears to be more likely if the peculiar metabolic requirements of the infant period are considered. Before birth the fetal rat receives its supply of nutrients in the form of glucose and amino
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 16, 2019.
516
GIL-AD,
acids via the placenta (15). After birth there is a dramatic change in the nutrient supply, and the newborn animal is now on a natural high-fat, low-glucose diet (16), so that until weaning oxidation of lipids serves as the main supply of energy (16). Proteins contribute little to the metabolism of the newborn since only 4 % of the basal calories come from protein catabolism, whereas in the adult this value reaches 17 % (2 I). Predominance of lipid metabolism is reflected in the young mammal by the presence of high plasma concentrations of nonesterified fatty acids, glycerol, and ketone bodies, and by a low respiratory quotient (8, 19, 30) Neonatal brain is not exceptional in its nutritional requirements and depends heavily on fuels other than glucose to support energy metabolism. Glucose utilization on the 1st day of life is, in fact, less than 1/l&h that of the adult, and remains constant for 12 days, when it rises to about sth that of the adult (22). On the other hand, enzymes for ketone body utilization are present in higher concentration in the brain of suckling rats than they are in the brain of adults (29). Since it has been shown, by the use of various substrates of the glycolytic pathway, that brain glucoreceptors do not monitor the availability of intracellular free glucose, but rather the rate of oxidative metabolism (lo), it is not unlikely that the almost complete insensitivity to glucose deficiency in the postnatal period may be due to the minor relevance of this substrate as metabolic fuel. The lack of behavioral signs to acute Z-DG glucoprivation noticed in the present study is another indication that glucose is not a dominant fuel for the infant brain. The relatively lower activities of the enzymes concerned in ketone body utilization in peripheral tissues (29) might explain the higher sensitivity to Z-DG glucoprivation present in the studies involving peripheral administration of this sugar. Experiments in which glucose was administered peripherally were aimed to evaluate properly the effectiveness of Z-DG in inhibiting the insulin secretion due to the blood glucose rise. Plasma IRI levels increased progressively with age from very low base-line values in the immediate postnatal period; similarly, there was a progressive increase in the in-
UDESCHINI,
COCCHI,
AND
MijLLER
sulin response to exogenous glucose with advancing age. These results confirm similar findings in both animal (2, 4) and man (3, 34). In 7- and 14-day-old rats lack of the insulin response to Z-DG-evoked hyperglycemia (upon peripheral administration) cannot, by any means, be ascribed to the inhibitory action of Z-DG. At these ages, in fact, exogenous glucose was also unable to induce a consistent insulin response. At successive ages, however (21 and 28 days), inhibition of the insulin rise in response to endogenous hyperglycemia has to be attributed to the inhibitory effect of 2-DG. This is mediated by the adrenals and catecholamine secretion in the experiments involving central Z-DG administration (12) and in addition by direct inhibition of the beta cells in the experiments in which the sugar was given peripherally (13). The fact that, despite the presence of a diminished hyperglycemic response to glucoprivation, the blood-insulin-lowering effect of Z-DG is fully operative stresses the relevance of the adrenergic or direct inhibition of insulin secretion by the pancreas in the metabolic adjustment to glucose deprivation. Such a mechanism, which allows an increased availability of glucose for the brain, is of relatively minor importance in the immediate postnatal life (l-15 days) when glucose utilization in the brain is poor, but undoubtedly plays a significance role later on, when a rise in the rate of glucose utilization occurs. In summary, the hyporesponsiveness to 2-DG glucoprivation of the newborn rat does not extend only to the control of food intake (18), but apparently involves also the autonomic-endocrine response to hyperglycemia. Collectively, our data suggest that the magnitude of the reflexive response to a lack of a metabolizable substrate is related to the quantitative importance of the latter as a fuel. assistance
The fully
skilful technical acknowledged. This investigation
74.00140.04 Send
supported
of the Consiglio reprint
requests
of Pharmacology, Milan, Italy. Received
was
for
to Dr.
Lavinia
Frizzi
is grate-
in
Nazionale
University publication
of Miss
Eugenio
of Milan,
25 September
part by Contract Ricerche, Italy. E. Miiller, 2nd Chair
CT
delle
Via
Vanvitelli
32,
Dept. 20129
1974.
REFERENCES 1. ALTAFFER, F. B., F. D. BALBIAN VERSTER, S. HALL, C. LONG, AND P. D’ENCARNACAO. A simple and inexpensive cannula technique for chemical stimulation of the brain. Physiol. Behav. 5 : 119-121, 2. ASPLUND,
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E., E. M~NTOJA, AND C. LOPEZ QUKJADA. Relationship between insulin concentrations in plasma and pancreas of foetal and weaning rats. J. Endocrinol. 48 : 553-56 1, 1970. 5. BROWN, J., AND H. L. BACHRACH. Effects of 2-deoxy-n-glucose on blood glucose levels in the rat. Proc. Sot. Exftl. Biol. Med. 100 : 641-643, 1959. COMLINE, K. S., AND M. SILVER. Catecholamine secretion by the adrenal medulla of the foetal and newborn foal. J. Physiol., London 216: 659-682, 1971. 7. CRAMER, F. B., AND G. A. NEVILLE. A calorimetric method for estimating %deoxy-D-glucose. J. Franklin lizst. 254 : 379-381, 1953. 8. CROSS, K. W., 1. P. TGZRD, AND D. A. I-I. TRYUIALL. The gaseous.
the
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1957. R~~TSXNEN. Adrenaline and noradrenaline medulla during post-natal development of the rat. Endocrinology 60 : 753-760, 1959. FXORENTINI, A., AND E. E. MILLER. Sensitivity of central chemoreceptors controlling blood glucose and body temperature during glucose deprivation. J. Physiol., London. In press. FROHMAN, L. A., AND L. L. BERNARDIS. Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions. Endocrinology 82 : 1125-l 132, 1968. FROHMAN, L. A., E. E:, MUELLER, AND D, COCCHI. Central nervous system mediated inhibition of insulin secretion due to 2-deoxy-Dglucose. Harm. Metab. Res. 5: 21-26, 1973. GOLDSMITH, S. J., R. S. YALOW, AND S. A. BERSON. Effects of 2-deoxy-D-glucose on insulin secretory responses to intravenous glucose, glucagon, tolbutamide and arginine in man. Diabetes 19 : 453-457, 1970. GREENWOOD, F. C., Wm M. HUNTER, AND J. S. GLOVER. The preparation of 13Wabelled human growth hormone of high specific radioactivity. Biochem, J. 89: 114-123, 1963. HAGERMAN, D. D., AND C. A. VILLEE. Transport functions of the placenta. Phvsiol. Rev. 40 : 313-330. 1960.
9. ER~~NK~,
1970. K.
The effect of glucose on the insulin secretion in newborn rats. In: The Structure and Metabolism of the Pancreatic 1&q edited by S. Falkmer, S. B. Hellman, and I. B. Tgjedal. New York: Pergamon, 1970, pa 477484. 3. BAIRD, J+ D., AND J. W. FARJ~UHAR. Insulin-secreting capacity in newborn infants of normal and diabetic women. Lancet 1: 71-74,
foetal
of
metabolism 0,
AND L.
in the adrenal
10.
11.
12.
13.
6.
14.
15.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 16, 2019.
ONTOGENY 16.
18.
19. 20. 21.
22*
23.
24.
25,
RESPONSE
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