Effect of chronic undernutrition on hepatocyte insulin receptors in rats

Effect of Chronic

Undernutrition

on Hepatocyte

Insulin

Receptors

in Rats

R. Harsha Rao, James M. Betschart, and Mohamed A. Virji The effect of moderate chronic undernutrition on insulin receptors was studied in male rats, pair-fed 60% of the daily food intake of ad libitum-fed littermates, for 8 weeks. Body weights of undernourished rats were consistently found to be 35% to 40% less than control littermates, with no period of growth arrest at any point in the 8-week study. The binding-displacement curves of labeled insulin to hepatocyte receptors in the two groups in the presence of unlabeled insulin were significantly different (P = .0258 after repeated measures ANOVA). Significantly lower binding was observed in hepatocytes from the undernourished group (P < .Ol) at all unlabeled insulin concentrations less than 20 nmol/L. In the absence of any unlabeled insulin, specific binding was reduced from 8.8% t 0.7%. (mean 2 SE) in controls, to 7.4% 5 0.3% in undernourished rats (P < .Ol). Half-maximal specific hormone binding to hepatocytes was achieved at a free insulin concentration of 362 nmol/L in the control group, compared with 447 nmol/L in the undernourished group, reflecting an increase of approximately 20%. The hypoglycemic response to intravenous insulin (0.1 U/kg body weight) was tested in a parallel experiment involving seven paired littermate rats, and found to be significantly impaired in the undernourished group (P = .0041 by repeated measures ANOVA). The nadir of glucose attained in the ad libitum-fed group was significantly lower than in the undernourished group (3.4 t 0.4 mmol/L v 4.1 2 0.3 mmol/L, P < .Ol), representing approximately a 20% reduction. The similarity in the degree of impairment in both insulin binding to its receptor and the hypoglycemic response to insulin, suggests that they may be related, and that reduced binding may contribute in part to the overall insulin resistance seen in malnourished states. It is also concluded that an acquired defect in the insulin receptor associated with malnutrition, may be responsible for the reduced insulin binding reported in cells of patients with malnutrition diabetes. Copyright

0 199 1 by W. B. Saunders Company

M

ALNUTRITION DIABETES, a major form of growth-onset diabetes seen in many developing countries, is characterized by p-cell failure, insulin deficiency, and a moderate to severe degree of insulin resistance, which can often result in insulin requirements that exceed 3 U/kg/d.‘,‘There is some preliminary evidence that malnutrition diabetes is associated with an impairment in the binding of insulin to the insulin receptor in red blood cells and lymphocytes, and that the defect is more severe than that seen in type 2 diabetes.3 Clinical states of malnutrition such as kwashiorkor and cancer-associated malnutrition are also characterized by insulin resistance despite insulinopenia.4,5 Fasting hypoglycemia is often seen in these subjects; however, this is not due to enhanced insulin sensitivity, but to an impaired ability to mobilize glycogen in response to glucagon.’ The pathophysiologic basis for insulin resistance in malnourished states has not been studied. Specifically, the effect of chronic malnutrition itself on the insulin receptor is not known. There is evidence to suspect that nutritional modulation of the insulin receptor could be an important part of the homeostatic response to chronic nutritional deprivation. For instance, short-term fasting and starvation are clearly associated with significant alterations in the insulin receptor in rat liver.6-’ However, studies in fasting and starvation cannot be extrapolated to chronic malnutrition, because the former are associated with significant weight loss, and the latter (except in its severest forms) is a relatively stable From the Departments of Medicine and Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA. Supported by grants from the Upjohn Company and from the Diabetes Research and Education Foundation. Address reprint requests to R. Harsha Rae, MD, Department of Medicine, Montefiore University Hospital, 3459 Fifth Ave, Pittsburgh, PA 15213. Copyright 0 1991 by W.B. Saunders Company 0026-0495/9114012-0011$03.00/0 1292

state characterized in adults by long-term weight maintenance, albeit at a less than ideal body weight, or in children by a lower than normal rate of weight gain with continued growth. The usual pattern of malnutrition seen in underdeveloped countries is of this variety (chronic and relatively moderate in severity), rather than the most extreme forms (kwashiokor/marasmus in children, and nutritional hypoproteinemia in adults).‘” This is also the pattern that is seen in most malnourished growth-onset diabetics in underdeveloped countries, w and in elderly, institutionalized diabetics in the United States.” It is in these situations that any effect of chronic malnutrition on the insulin receptor would assume significance. The study reported here was performed with the primary purpose of determining the effect of a chronic state of moderate malnutrition on insulin binding to its receptor in hepatocytes in rats, as a first step to identifying the mechanisms by which insulin resistance is mediated in clinical states associated with chronic malnutrition. As a corollary, it was hoped to be able to determine whether the reported impairment in insulin binding in malnourished diabetics could be ascribed to coexisting malnutrition. MATERIALS Experimental

AND METHODS

Animals

Four-week-old, male Sprague-Dawley rats were assigned in littermate pairs to either a control group fed ad libitum or an undernourished group that received 60% of the total food intake of the ad libitum-fed littermates, based on daily weighing of food for 8 weeks (n = 13 each). They were maintained on a 12-hour (6:00 AM to 6:00 PM) light/dark cycle. Daily food portions were provided for the undernourished rats after 5:OO PM each evening, in order to approximate feeding behavior in the two groups (ie, meal-driven in the undernourished rats, and at onset of darkness in ad libitum-fed animals). On the day before study, food was provided to the undernourished animals at 8:00 AM, based on the overnight intake of the control littermate, and food bins of the latter emptied as soon as the undernourished rats had consumed their portions Metabolism,

Vol40, No 12 (December), 1991: pp 1292-1297

1293

INSULIN RECEPTORS IN UNDERNUTRITION

(usually in 1 hour). Thus, the duration

of fasting was approximately

equal at 24 to 26 hours.

Diet Composition Tne diet had a physiological fuel value of 3.3 kcalig (gross energy value, 4.25 kcalig), containing 23% protein, 4.5% fat, and 5.8% crude fiber. All vitamins and minerals were provided in excess of the recommended daily allowances (RDA) for growing rats, to prelent disproportionate deficiency of any micronutrients. Specificall;,. the concentrations of nutrients known to play a role in glucose tolerance were as follows: potassium, 1.1% (RDA 0.18%); calc#um, 1% (RDA 0.5%); zinc, 70 parts per million (RDA 12 ppm); thiamine, 15 ppm (RDA 1.25 ppm); niacin, 95 ppm (RDA 15 ppn,); and pyridoxine, 60 ppm (RDA 7 ppm).

I/&in

Receptor Studies

On the day of study, rats were anesthetized with intraperitoneal penzobarbital (50 mgikg body weight) and hepatocytes were ibolated after hepatic perfusion via the portal vein with collagendsc, as described by Seglen.lJ A sample of portal venous blood was withdrawn before starting the perfusion. Insulin binding studies were carried out in Krebs-Ringer bicarbonate buffer (pH 7.5) at 3OY’ for 2.5 minutes, as described earlier.” The reaction was storped with the addition of ice-cold buffer, and bound and free hormone were separated by centrifuging intact cells in a microcentrifuge. Under these conditions, binding equilibrium is established witbin 10 minutes, and maintained for up to 80 minutes. Degradation of insulin (measured after precipitation with 5% ice-cold trichloracetic acid) averaged 6% (range, 3% to 8%). and was not different in the two groups. Receptor grade mono-iodoinsulin was purg:hased from New England Nuclear (Boston, MA).

Assessmenf of insulin Sensifivit), TOdetermine whether signiticant insulin resistance was a feature of ‘he animal model developed for study, the hypoglycemic response to insulin was studied in seven paired littermates in each group, after a 12-hour fast. The shorter fast was dictated by the need to prevent the development of relatively lower plasma glucose levels in the malnourished rats during a 24-hour fast. As noted, this is a consequence of an inability to mobilize glycogen, and not due to enhanced insulin sensitivity.” Nevertheless, the resultant differenc: in the starting baseline would lead to difficulty in interpreting the hypoglycemic response in the two groups. Rats were anesthetized with pentobarbital, given intraperitoneally, in a dose of 50 mg/ kg body weight, and repeated as necessary. The left femoral artery and femoral vein were cannulated with silastic catheters, which were Hushed periodically with heparinized saline to keep them patent. Animals were systemically anticoagulated with heparm :25 U/l00 g body weight, repeated every 90 minutes). Porcine insL,lin was Injected via the venous catheter in a dose of 0.1 U/kg body weight, and blood samples were drawn at 0. 5, 10, 15, 20, 25, 30, 10. 50. and 60 minutes for plasma glucose assay. All samples wer;: carefully withdrawn using a tuberculin syringe, in a volume of 0.5 mL/kg body weight, to equalize the effects of repetitive blood sarrpling on the hemodynamic and stress responses in animals of \*id:ly differing body weights.

Neuman-Keuls

tests,”

using the mean square

error for the interac-

tion between nutritional state and dose. After subtraction of nonspecific binding, nonlinear regression was performed by least squares using a pseud+Gauss-Newton iterative algorithm, with hormone bound to hepatocytes as the dependent variable, and the free insulin concentration as the independent variable to obtain the ED,,, for binding in each group (the concentration of insulin at which binding was half maximal), as a crude but quantifiable estimate of overall binding. Data from the insulin tolerance test were analyzed by repeated measures ANOVA to identify the effect of nutrition on the glucose response to insulin, and the nadir in the plasma glucose in each rat was compared with its paired littermate by the paired t test.

Laboratory Methods Plasma glucose was analyzed with a calorimetric glucose oxidase method, using o-dianisidine as chromogen.‘” Serum insulin was measured by radioimmunoassay,‘” using rat insulin as standard. Cellular DNA and protein content were measured by standard techniques.“‘,*’ RESULTS

Growth Rate Over

the

&week

study

period,

the

undernourished

ani-

mals (II = 13 in each group) grew continuously

at a rate that was approximately 60% of the rate of growth of the ad libitum-fed control littermates (Fig 1). At no time was there a period of cessation of growth in the undernourished group. The slopes of the respective linear regression lines drawn through the mean data points were 52 glwk in the control group and 29 g/wk in the undernourished group (P < .OOl, r = .99 in both groups). Thus, the rats in the undernourished group were clearly in a state of chronic mild to moderate undernutrition without any evidence of the growth arresting effects of acute severe malnutrition. In the context of the overall objective of the study, which was to study insulin receptors in a state of chronic malnutrition

(WEEKS)

i 300

ii 3

G

AGE

400

9

;

GAIN

1

500

3 E

WEIGHT

r i

200

iii lOOi

StaWical Analysrs Analysis of data was performed on a VAX 6410 computer, using the BMDP statistical analysis program.‘6 Binding data in the two groups were analyzed by repeated measures ANOVA, to identify the effect of the nutritional state on insulin binding at different cold hormone concentrations in six rats in each group. Mean binding at eacn concentration of insulin was compared in the two groups by

Fig 1. Weight gain in ad libitum-fed rats (0-O) and their chronically undernourished littermates (O----O), pair-fed a 60% food restricted diet (n = 13 in each group).

1294

RAO, BETSCHART, AND VIRJI

with continuing weight gain, the animal model clearly met the requirements. insulin Receptor Studies

Displacement curves of insulin binding in the presence of unlabeled hormone were significantly different (P = .0258) in hepatocytes from the two groups of rats, when analyzed by repeated measures ANOVA (Table 1). The specific binding of a trace concentration (34 pmol/L) of radioactive insulin to isolated hepatocytes in the absence of unlabeled insulin, was significantly lower in the malnourished group compared with the control animals (mean ? SE, 8.8% ? 0.7% v 7.4% f 0.3%, P < .Ol by Newman-Keuls test after ANOVA). Similarly, labeled insulin binding was significantly lower at all unlabeled hormone concentrations less than 20 nmol/L (P < .Ol). It was also significantly reduced at 40 nmol/L (P < .05). No differences were seen at higher insulin concentrations (100 to 1,000 nmol/L). Nonspecific binding was not different in the two groups. The ED,, for bound hormone (ie, the free hormone concentration at which total hormone bound to hepatocytes was half-maximal) was determined after curvilinear regression (pseudo-R’ from the regression = .9997 in each group). In the undernourished rats, the ED,, was 447 nmol/L, whereas it was 362 nmol/L in the ad libitum-fed controls, an increase of approximately 20%. Maximal insulin binding (the horizontal asymptote of the curvilinear regression) was similar in both groups (18.2 nmol/L/lOS hepatocytes in undernourished rats, and 17.5 nmol/L/lO’ hepatocytes in the controls). To confirm that these differences could not be attributed to changes in cell number or cell size in suspensions prepared from malnourished animals, protein and DNA content of the suspensions were also measured. These were

almost identical in the two groups: in cell suspensions from control and malnourished rats, respectively, protein concentrations were 14.98 2 1.76 and 14.82 t 1.8 pg/uL, and DNA concentrations were 95.2 5 3.4 and 89.0 2 6.1 ug/mL. Hormone Levels

Fasting portal venous plasma insulin levels were significantly lower in the undernourished animals (92 2 5 )LU/ mL) compared with control animals (144 ? 18 kU/mL, P < .05). Serum corticosterone was similar in both groups (306 ? 20 ng/mL u 303 t 30 ng/mL). Hypoglycemic Response to Insulin

Fasting plasma glucose was similar in both groups after the relatively brief period of fasting (7.3 + 0.5 mmol/L in controls, and 7.6 ? 0.5 mmol/L in the undernourished rats). Repeated measures ANOVA of data from the insulin tolerance test in the two groups showed that there was a significant effect of malnutrition on the pattern of the plasma glucose response to intravenously injected insulin (P = .0041) (Fig 2). At all time points before 50 minutes in the insulin tolerance test, mean plasma glucose levels were higher in the malnourished group than in the ad libitum-fed group. The nadir of glucose attained in the malnourished group was 4.1 +- 0.3 mmol/L, compared with the ad libitum-fed control littermates in which it was 3.4 2 0.4 mmol/L (P < .Ol). This represents a 20% impairment in hypoglycemic response to insulin in the malnourished animals. However, undernourished rats appeared to show a defective ability to recover from hypoglycemia, as evidenced by the fact that the 60-minute plasma glucose was significantly lower than in the controls (7.0 ? 0.3 v 8.6 * 0.9 mmol/L, P < .Ol). DISCUSSION

Table 1. Percentage Binding of Radiolabeled Insulin (34 pmol/L) Different Concentrations

of Unlabeled Insulin in Hepatocytes

Ad Libitum-Fed and Chronically Undernourished Pair-Fed a 60% Food-Restricted Insulin

Unlabeled Concentration

From Rats

Diet for 8 Weeks

Ad Libitum-Fed (n = 6)

(nmol/L)

Littermate

Undernourished* (n = 6)

0

12.15 + 0.79

10.91 2 0.29*

1 x IO_’

12.06 i 0.77

10.81 i 0.52%

2 x lo-’

11.69 2 0.73

10.81 + 0.332

1 x 100

11.20 k 0.72

10.38 + 0.39s

2 x 100

10.68 k 0.83

9.80 + 0.36$

4x

at

100

9.62 + 0.75

9.11 k 0.42$

1 x 10’

9.19 + 0.63

8.54 k 0.3OZ

2x

IO’

8.39 k 0.64

4x

10’

8.09 k 0.73

8.21 + 0.26 7.45 + 0.20t

1 x 102

6.77 ? 0.80

6.64 k 0.21

2 x 102

6.18 -t 0.54

6.19 + 0.29

1 x 103

4.83 2 0.27

4.89 f 0.17

1 x 105

3.36 + 0.12

3.54 2 0.23

NOTE. Values are mean 2 SEM. *Significance

of interaction

between

nutritional state and insulin

binding at different insulin concentrations,

p = 0.0253 by repeated

measures ANOVA. tP < .05, SP < .Ol versus ad libitum-fed group by Neuman-Keuls test after ANOVA.

The results of this study show that chronic undernutrition for 8 weeks in rats results in a 20% increase in the ED,, of insulin binding to its receptor, along with a 20% impairment in the maximal hypoglycemic response to insulin. The ED,, has been used here as a crude method of quantifying overall insulin binding to its receptor in each of the groups, in order to provide an estimate of the degree of impairment in the undernourished rats. This use specifically precludes any interpretation of the ED,, as a parameter with physiologic meaning (eg, with respect to affinity), because of the errors that are inherently associated with such a simplistic treatment.” The more traditional Scatchard analysis does permit the separation of overall binding defects (as reflected in the ED,,) into changes in receptor affinity or concentration at different sites, but the basic assumption in this analysis is that the binding should exclusively reflect hormone-receptor interactions. This precondition could not be satisfied in the present study, because postreceptor events could also have influenced the binding of insulin to its receptor at the temperature at which the assay was performed (30°C). Consequently, the affinity constants and receptor concentrations derived therefrom would have dubious physiologic relevance. For the purpose of this

1295

NSULIN RECEPTORS IN UNDERNUTRITION

1

3

0

10

20

MINUTES

+

Nadir (control)

30

40

50

60

AFTER INSULIN

Fig 2. Effect of malnutrition on the hypoglycemic response to intravenously injected insulin (0.1 U/kg), in littermate rats either fed ad libitum (0-O) or fed a 60% food-restricted diet (O----O). Responses are significantly different in the two groups (P = .0041 by repeated measures ANOVA). Nadir: lowest plasma glucose value attained in the test (n = 7 in each group).

study, the ED,,, suffices as a method of nonselectively quantifying the sum of all the factors at the receptor and postreceptor level (receptor affinity, concentration, internalization, degradation, etc) that could contribute to the reduced binding of insulin to cells.” The 20% increase in the ED,, in undernourished rats therefore reflects a reciprocal decrease in the overall ability of hepatocytes to bind insulin. The fact that the maximal binding capacity was the same in the two groups suggests that the increase in ED,,] was not a reflection of increased binding capacity in the undernourished group. The pattern in the undernourished rats, of insulin resistance with impaired insulin binding, resembles that seen with glucocorticoid administration.” However, the possibility can be excluded that increased glucocorticoid secretion from the stress of food restriction was responsible for the impaired receptor binding in undernourished rats, primarily because corticosterone levels were found to be similar in both groups. In addition, undernourished rats had substantially lower insulin levels, whereas corticosteroid administration is associated with hyperinsulinemia.

For these reasons, the decrease in insulin binding cannot be attributed to stress. Since the only difference between the two groups lay in the nutritional state, it must be concluded that the changes in the insulin receptor were mediated by long-term chronic malnutrition. Although short-term fasting and starvation can produce changes in the insulin receptor that affect both receptor affinity and tyrosine kinase activity,‘~” the difference between the two groups was observed after the same period of fasting, confirming that the observation was explicitly related to malnutrition itself. The pathophysiologic basis for the impairment in insulin receptor binding is not known. One possibility is that the generally disordered protein synthetic mechanism, characteristically seen in all tissues in severe malnutrition,‘” might also affect the synthesis of receptor proteins. Impaired cellular protein synthesis has been implicated in the permanent impairment in p-cell function seen in severely proteindeficient animals.15 Such gross alterations probably did not exist with the degree of nutritional deprivation used in the present study, because the undernourished rats continued to grow at a significant rate, and hepatocyte protein and DNA content were not affected. However, subtler abnormalities at the cellular level could be postulated as a potential mechanism by which insulin receptors might be affected, resulting in impaired binding. Given the moderate degree of nutritional deprivation, it is not certain that any impairment existed, or that impaired binding or insulin resistance were mediated in this fashion. It is nonetheless significant that the insulinopenia characteristic of more severe malnutrition was seen in these animals, suggesting that at least in the 6 ceil, a secretory or synthetic deficit was present. Equally noteworthy is the fact that. notwithstanding the moderate nutritional deprivation, the insulin resistance induced was significant enough to be demonstrable in an insulin tolerance test, which is a fairly insensitive measure of insulin resistance. This confirms a preliminary observation made over two decades ago by Heard and Turner in three dogs subjected to 30% to 50%~ protein restriction, that the fractional rate of glucose disappearance after insulin was reduced by .50%.” Parenthetically, they also observed that recovery from hypoglycemia is blunted in malnourished animals, as was shown in this study. Little is at present known of the possible cause-and-effect relationships that might exist in the paradoxical association of insulinopenia with insulin resistance and impaired insulin binding to its receptor in these animals. Malnutrition clearly has lasting effects on the functioning of beta cells that are not merely the result of short-term compensatory homeostatic mechanisms, such as those seen in fasting or starvation. Glucose intolerance is characteristically seen, with insulinopenia, impaired insulin secretory responses to all stimuli such as glucose, glucagon and amino acids,z.17 and fibrosis, and damage to the pancreas? This can lead to persistent impairment of glucose tolerance, which may be irreversible even with nutritional rehabilitation in both human,z”-‘z and experimental malnutrition.“2’ This proves that the changes are neither short-term nor compensatory, but permanent. Similarly, the insulin resistance and receptor defect occur contrary to expected compensatory rc-

1296

RAO, BETSCHART, AND VIRJI

sponses: sustained insulinopenia should normally result in increases in insulin sensitivity and insulin receptor affinity.34 This paradoxical association of insulin resistance and insulinopenia is also a feature of kwashiorkor4 and malnutrition diabetes.’ The similarity between these clinical states and the animal model of chronic malnutrition studied here may indicate the presence of a common mediating factor, the most obvious one being malnutrition itself. The finding that chronic malnutrition is associated with an acquired receptor defect, that coexists with the paradox outlined above, is one of some significance. It points to an unusual homeostatic response that may be characteristic of states associated with long-term chronic nutritional deprivation. For instance, in malnutrition diabetes too, the binding of radiolabeled insulin to erythrocytes and mononuclear cells is impaired by approximately 20% to 30% when even compared with obese type 2 diabetics.3 This degree of impairment is not dissimilar to that found in the present study, and it may well be attributable to malnutrition itself. The physiologic significance of a 20% reduction in insulin receptor binding is not clear, and such changes may play little or no part in mediating the insulin resistance of malnourished states. It is of interest that a similar degree of impairment (20%) in the hypoglycemic response to injected insulin was shown in the malnourished animals studied here. Although these similarities suggest a link, malnutrition equally induces other changes that impair insulin

action at a postreceptor level and affect intermediary metabolism,3s.3h and these may quite possibly be of much greater significance. Even if the impairment in receptor binding in chronic malnutrition does not contribute to insulin resistance, its association with insulinopenia separates chronic malnutrition from many other insulin resistant states, which are associated with hyperinsulinemia. Further exploration of the basis for these changes may help in understanding the homeostatic mechanisms that operate in states of chronic malnutrition. In conclusion, the results of this study indicate that chronic malnutrition can result in impaired binding of insulin to the insulin receptor, that might conceivably impair the hypoglycemic effect of insulin. They are similar in degree of severity to the changes reported in patients with malnutrition diabetes. In the context of the objectives of the study, it may therefore be concluded that (1) chronic malnutrition does affect the insulin receptor, and (2) it may mediate, in part, the impaired insulin binding seen in malnourished diabetics. ACKNOWLEDGMENT

The technical assistance of Mary Jo Dimasi and Vincent Ballarotto is gratefully acknowledged. The authors would also like to thank Janine Janosky, PhD, Department of Clinical Epidemiology and Preventive Medicine, University of Pittsburgh School of Medicine, for her advice and guidance in the statistical analyses.

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14. Seglen PO: Preparation of isolated rat liver cells. Methods Cell Biol 13:29-83,1976 15. Betschart JM, Virji MA, Perera MIR, et al: Alterations in hepatocyte insulin receptors in rats fed a choline-deficient diet. Cancer Res 46:4425-4430,1986 16. Jennrich R, Sampson P, Frane J: Analysis of variance and covariance with repeated measures, in Dixon WJ, Brown MB, Engelman L, et al (eds): BMDP Statistical Software Manual, vol 1. Berkeley, CA, University of California Press, 1988, pp 483-525 17. Winer BJ: Statistical Principles in Experimental Design (ed 2). New York, NY, McGraw-Hill, 1971, pp 514-603 18. Raabo E, Terkildsen TE: On the enzymatic determination of blood glucose. Stand J Clin Lab Invest 12:402-407,196O 19. Desbuquois B, Aurbach GD: Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocrinol Metab 33:732-738, 1971 20. Boer GJ: A simplified microassay of DNA and RNA using ethidium bromide. Anal Biochem 65:225-231, 1975 21. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 22. Rodbard D: Mathematics of hormone-receptor interaction. I. Basic principles. Adv Exp Med Biol36:289-326,1973 23. Kahn CR, Goldfine ID, Neville DM, et al: Alterations in insulin binding induced by changes in vivo in the levels of glucocorticoids and growth hormone. Endocrinology 10:1054-1066, 1978 24. Phillips LS: Nutrition, metabolism and growth, in Daughaday W (ed): Endocrine Control of Growth. New York, NY, Elsevier, 1981, pp 121-173 25. Swenne I, Grace CJ, Milner DG: Persistent impairment of insulin secretory response to glucose in adult rats after limited

INSULIN RECEPTORS IN UNDERNUTRITION

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