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Studies of insulin sensitivity in vivo in weanling rats with hypothalamic obesity
Studies of Insulin Sensitivity In Vivo in Weanling Rats With Hypothalamic Obesity By Lawrence A. Frohman, Jack K. Goldman, and Lee L. Bernardis
Studies were performed in weanling male rats with bilateral lesions in the ventromedial hypothalamic nuclei (VMN) and sham-operated controls to evaluate (1) the endogenous insulin response to glucose, and (2) the sensitivity to exogenous insulin as manifested by both hypoglycemia and the utilization of simultaneously injected 14C-glucose. VMN rats exhibited basal hyperinsulinemia and an increased insulin secretory response to glucose.
VMN and control rats exhibited similar hypoglycemic responses to exogenous insulin. Utilization of intraperitoneally 14C-glucose by epididymal injected adipose tissue and diaphragm was similarly increased by insulin in the two groups of animals. The results exclude peripheral insulin resistance as the basis for hyperinsulinemia and support the belief that it is a direct consequence of the hypothalamic lesion.
T
HE PRESENCE OF HYPERINSULINEMIA in both human obesity and that of laboratory animals has generally been regarded to be secondary to a resistance by peripheral tissues to the action of insulin. The inverse correlation between the size of adipocytes and the response to insulin in vitro,’ the direct correlation between plasma insulin levels and body fat estimates,2 and the appropriate changes in both plasma insulin levels and tissue responsiveness to insulin with alterations in body weight’s3 have strengthened this concept. During the past several years, our investigations of the obesity observed in weanling rats after electrolytic destruction of the ventromedial hypothalamic nuclei have suggested that insulin sensitivity is normal or perhaps increased in these animals. After hypothalamic destruction, weanling rats exhibited normal food intake, weight gain, and plasma glucose levels, yet developed hyperinsulinemia and increased carcass fat;4 starvation resulted in mild hypoglycemia with at first, elevated and later, normal insulin levels;4.5 and glucose oxidation and conversion to lipid by adipose tissue in vitro was elevated in both the fed state and after food deprivation sufficient to eliminate hyperinsulinemia. 5 The lack of insulin resistance was also suggested by the enhanced disappearance of r4C-glucose from plasma after intravenous injection.e From fhe Departments of Medicine nnd Pathology, State University of New York at Buffalo, and the Veterans Administration Hospifal, Buffalo, N. Y. Received for publication April 28, 1972. Supported in part by USPHS Grants AM 11456 and 14418, and by Veterans Administration Clinical Invesfigaforship and Research Grant 01/3191-1 69-01. Lawrence A. Frohman, M.D.: Associate Professor of Medicine, Stale University of New York at Bufj%lo, Buffalo, N.Y. Jack K. Goldman, M.D.: Assistant Professor of Medicine, State University of New York at Buffalo, and Clinical Investigator, Buffalo Veterans Administration Hospifaf, Buffalo, N.Y. Lee L. Bernardis, Dr. Phil., Ph.D.: Research Associate Professor of Pathology, and Clinical Assisfanf Professor of Medicine, State University of New York at Buffalo, Buffalo, N. Y. Metabolism.
Vol. 21, No. 12 (December),
1972
1133
1134
FROHMAN, GOLDMAN, AND BERNARDIS
However, since the alterations in glucose oxidation and lipogenesis by adipose tissue in vitro have recently been demonstrated to occur after ventromedial hypothalamic destruction in streptozotocin-diabetic animals and are, therefore, in part, independent of the hyperinsulinemia,’ the direct evaluation of insulin responsiveness was deemed necessary to answer the question of hormonal sensitivity. This report describes the endogenous insulin responses to glucose and the effect of exogenous insulin on plasma glucose and on the metabolism of intraperitoneally injected 14C-glucose by epididymal adipose tissue, diaphragm and liver. MATERIALS
AND METHODS
Animafs and Operative Procedures Weanling male Sprague-Dawley rats were individually caged and allowed food (Teklad) and water ad lib. At 26-28 days of age, bilateral electrolytic lesions were placed in the ventromedial hypothalamic nuclei (VMN) with a stereotaxic instrument. The details of animal maintenance, lesion production and verification were identical to those previously described.4 Sham-operated animals served as controls. In all experiments, food intake and weight gain of VMN and control animals were similar.
Experimental
Procedures
Three weeks after lesion production, one Responses to Glucose. group of VMN and control animals were given glucose, 1.5 g/kg, intraperitoneally. Heparinized blood was collected from the tail vein prior to and at 30, 60, and 120 min after glucose. Plasma was separated at 4’ and immediately frozen. Food was removed from the cages 2 hr prior to the start of the experiment. Insulin Hypoglycemia. Three weeks after lesion production another group of animals was injected intraperitoneally with purified bovine insulin, 1.0 U/kg. Heparinized blood was collected from the tail vein prior to, and at 30 min after the injection of insulin. A third group of animals was similarly injected with 0.5 U/kg of insulin and blood collected at 0, 30, and 60 min. The same animals were retested 1 wk later using 0.25 U/kg of insulin. Animals were permitted free access to food until the time of insulin injection, at which time food was removed for the duration of the experiment. Two weeks after lesion production, a fourth Insulin Effects on I+%‘-glucose Utilization. group of animals was injected intraperitoneally with 2 &i of U-14C-glucose (New England Nuclear, S.A. 200 mC/mM) simultaneously with 0, 0.1 or 10 mU (0.6 or 60 mu/kg) of purified bovine insulin. Thirty minutes later, animals were decapitated. Trunk blood was collected in heparinized beakers and the plasma separated at 4’ and then frozen. Segments of liver and diaphragm were immediately frozen on dry ice and the epididymal fat pads placed in chloroform: methanol (2:l). In this experiment, food was removed from the animals 2 hr prior to the injection of insulin. Endogenous
Insulin
Analytical Procedures Plasma glucose was measured by a “micro” modification of the ferricyanide methods on an AutoAnalyzer. Plasma insulin was measured by a double antibody radioimmunoassay, the details of which have been previously described.4 The incorporation of 24C-glucose into glycogen, total lipids and fatty acids in adipose tissue, liver and was diaphragm was determined as previously described. 5.6 Tissue protein concentration measured by the method of Lowry et al.9 Data from animals whose lesions were asymmetric or impinged significantly on areas other than the ventromedial nucleus were excluded prior to statistical analysis. Differences between groups were compared by Students’ t test or by rank test when marked dissimilarities in variance were present.10
INSULIN
SENSITIVITY
1135
IN RATS
Insulin, pU/ ml plasma
80-1
T
60/ 40-1T
.A .. ..._ Control ( 121 ‘--c _________ __________Q
I I
Glucose,
I
I
mg/ 100 ml plasma
200-
______p
160.**’
Fig. 1. Plasma glucose and insulin responses to intraperitoneal glucose administration (1.5 g/kg) in VMN and control rats. Shown are the mean I? SE.
80 -1 I_
-1 30
I- _. ___..-I 60 Minutes
120
RESULTS Endogenous Insulin
Responses
to Glucose
After the intraperitoneal administration of glucose (1.5 g/kg) plasma glucose levels peaked at 30 min in both VMN and control rats (Fig. I). Although the glucose levels were consistently greater in control animals, the differences were not significant and the incremental responses were similar in the two groups. Plasma insulin levels were greater prior to, and at all times after glucose administration (p
Responses
to Exogenous Insulin
The plasma glucose response to 1.0 U/kg of insulin was similar in 10 VMN and 12 control animals (initial glucose: 130 + 8 mg/lOO ml vs. 140 -t 5; 30 min glucose: 63 1 5 vs. 69 + 8). Because the large dose of insulin might conceivably mask a slight degree of insulin resistance, smaller doses were next tested. Plasma glucose responses to 0.5 U/kg of insulin were also similar with the decline in glucose at 60 min being significant in both VMN (p
1136
FROHMAN, GOLDMAN, AND BERNARDIS
Plasma Glucose mg/1OOml
d Minutes
Fig. 2. Plasma glucose responses to intraperitoneat administration of 0.5 U/kg (left panel) and 0.25 U/kg (right panel) of insulin. Shown are the mean -t SE.
were observed in the hypoglycemic responses. In this experiment the decline in glucose at 60 min was significant in control (p
Utilization
Plasma glucose and insulin levels 30 min after the intraperitoneal administration of considerably smaller doses of insulin (0.1 and 10 mu/rat) are shown in Table 1. Whereas no change in plasma glucose was observed in response to insulin in control animals, small but significant decreases were noted in VMN rats injected with both 0.1 mU (p
0
Insulin 10 mU
Plasma glucose, mg/lOO ml VMN Control Plasma insulin, pU/ml VMN Control
96 f 99 f
2 2
92 f2 96 ?I 1
6622 96 k 1
53 -t 4 42 4 3
31 +5 13 ?I 1
3629 27 &I 3
INSULIN
SENSITIVITY
1137
IN RATS
AdiDose
Tissue 14C- Lipid DPWmg
14C- Fattv Acids
-I-
I 14C- Glycoqen DPWmg
20-
l15-
Fig. 3. Incorporation of radioactivity into adipose tissue total lipid, fatty acids, and glycogen expressed per mg tissue after the intraperitoneal injecwith or without tion of W-glucose insulin in VMN and control rats. Shown are the mean f SE. Number of animals in each group (from left to right) were 7, 6, 7, 6, 7, and 4.
1 ,_
VMN c
wO..lc Insulin, mU
and differences between VMN and control groups persisted. In animals given the 10 mU dose, however, there were marked increases in the incorporation of glucose radioactivity into total lipid, fatty acids and glycogen of both groups of animals. At this dose, the differences between VMN and control groups were eliminated. Adipose tissue protein was reduced in VMN as compared to control animals (8.9 * 0.4 pglmg vs. 13.8 * 0.7, p
1138 Diaphragm 3-
FROHMAN,GOLDMAN, AND BERNARDIS
W- Lipid DPWmg
0.l Insulin, mU
,-
10
Fig. 4. incorporation of radioactivity into diaphragm total lipid and glycogen expressed per mg tissue after the intraperitoneal injection of %-glucose with or without insulin in VMN and control rats. See legend to Fig. 3. Liver I%- Lipid DPM/mq
IT
2-
Fig. 5. Incorporation of radioactivity into liver total lipid, fatty acids, and glycogen expressed per mg tissue after the intraperitoneal injection of 14C-glucose with or without insulin in VMN and control rats. See legend to Fig. 3.
14C- Fatty Acids
DPM/mg
Insulin,
mU
INSULIN
SENSITIVITY
1139
IN RATS
The effects of insulin upon glucose utilization by the diaphragm are shown of 14C-glucose into diaphragm glycogen was similar in VMN and control animals in the absence of insulin, and the two groups of animals exhibited similar increases in glycogen incorporation in response to both the low and high dose of insulin. Glucose incorporation into diaphragm lipid was similar in saline treated VMN and control animals. The response to insulin by VMN animals was slightIy greater than controls at the low dose and significantly greater (p
in Fig. 4. Incorporation
DISCUSSION
The coexistence of hyperinsulinemia and normal glucose values in both human and animal obesity has generally been accepted as evidence for the existence of insulin resistance. With the demonstration of resistance to the action of insulin in animals with genetic obesity” and in both spontaneous2 and induced3 human obesity, the presence of elevated plasma insulin levels has been considered to be a response by the pancreas in an effort to overcome this resistance. Hyperinsulinemia has previously been reported in weanling rats with hypothalamic obesity,4 and the present results indicate that the insulin response to glucose was similarly increased. The insulin response, expressed as a percentage of basal insulin levels, tended to be similar to that in control animals and may, therefore, be a reflection of the islet hypertrophy which occurs after hypothalamic destruction.12 This pattern is, therefore, similar to that observed in other types of obesity. However, our results clearly and unequivocally demonstrate that the observed hyperinsulinemia is not associated with peripheral insulin resistance. The hypoglycemic effect of moderate doses of insulin was completely normal in nonhyperphagic weanling rats with VMN lesions. Similar results have also been reported in hyperphagic adult rats with hypothalamic lesions.r3 An increased sensitivity to the hypoglycemic effects of insulin is even suggested by the plasma glucose responses to the small doses of insulin administered in the experiment in which r4C-glucose utilization was examined. This experiment also provided possible indirect evidence of an effect of insulin on the liver which was not evident from analysis of the 14C-glucose results. After the intraperitoneal administration of both 0.1 and 10 mU of insulin, there were decreases in plasma insulin in both VMN and control
1140
FROHMAN,
GOLDMAN,
AND BERNARDIS
animals. The decreases were most striking after the administration of 0.1 mU of insulin, which was a quantity sufficient to increase plasma insulin levels only transiently, even if injected intravenously. The plasma insulin levels were slightly greater after the larger dose of insulin, but were still less than in the saline-injected animals. The decline in plasma glucose levels, therefore, although possibly attributable to the effects of the injected insulin on the diaphragm and the intraperitoneal adipose tissue, suggests as well a decrease in hepatic glucose production. The greater decline in glucose levels in VMN as compared to controI animals is probably due to the more rapid plasma glucose turnover in VMN rats.6 The present data does not permit one to determine conclusively the mechanism responsible for the decreased plasma insulin levels in the insulin treated rats. However, there exist at least two possibilities. First, a decrease in plasma glucose levels secondary to a decreased hepatic glucose output could result in a subsequent reduction in the release of insulin by the pancreas. Although this explanation is supported by the glucose changes in VMN rats, the minimal changes in control animals would appear to be inadequate to cause a decrease of 30 pU/ml in the level of plasma insulin. Secondly, the injected insulin could possibly have a direct effect on the suppression of insulin release, although Malaisse et al. have reported no diminution of insulin release by islets incubated in the presence of insulin.r4 Irrespective of the mechanism, the results again demonstrate the ability of VMN rats to reduce their elevated plasma insulin levels when subjected to decreasing glucose availability, as has been previously observed after prolonged fastinge5 Assessment of glucose utilization by epididymal adipose tissue, diaphragm, and liver in vivo was achieved by the intraperitoneal administration of 14Cglucose, using a technique previously reported by Rafaelson et aLI5 This procedure permitted the evaluation of both basal and insulin stimulated glucose utilization at doses of insulin, which had minimal effects on circulating glucose levels. Under basal conditions, glucose utilization was increased in VMN adipose tissue while it was normal in diaphragm. The insulin response was normal in both tissues. Of interest are some differences between the present results and those observed in vitro. We have previously reported that in vitro incorporation of 14C-glucose into adipose tissue glycogen by VMN rats was not increased, in contrast to the marked increase in lipogenesis5 Recent results have further indicated that glucose incorporation into adipose tissue glvcogen in vitro by VMN rats is actually resistant to the effects of insulin.le There is no obvious explanation for the resistance to this pathway of glucose metabolism, nor is there one for the difference between the in vitro results and those obtained in vivo both in basal and insulin stimulated conditions. However, the applicability of any in vitro observation is contingent upon its reflection of an in vivo phenomenon and the present studies, therefore, indicate that the “apparent” resistance of the glycogenetic pathway in vitro is due to a phenomenon not present in the intact animal. No increase in glucose incorporation into either lipids or glycogen was observed in the liver of either VMN or control animals in response to insulin,
INSULIN
SENSITIVITY
1141
IN RATS
To the contrary, there was a tendency to decreased incorporation in the presence of insulin. This phenomenon may well be a reflection of the marked increase in glucose utilization by adipose tissue and diaphragm resulting in decreased availability of r4C-glucose to the liver. However, evidence for hepatic responses to insulin already has been discussed with respect to changes in plasma glucose levels. There are several implications of the present results with respect to the hyperinsulinemia of VMN animals. First, whether or not a linear relationship exists between insulin and glucose responses, the increased “insulin/glucose ratios,” which result from elevated insulin levels with normal ghrcose levels, are not an indication of peripheral insulin resistance in VMN animals. It follows that there may also be other forms of obesity, either experimental or naturally occurring, in which this is also true. The present results should, therefore, serve as a cautionary note with respect to the interpretation of “insulin/glucose ratios” unless more specific evidence of insulin resistance is also available. The second implication of our results relates to the etiology of the hyperinsulinemia in VMN rats and supports our initial hypothesis of a primary central nervous system pathogenetic mechanism.* The hyperinsulinemia has been shown to the independent of hyperglycemia, food intake and weight gain4,r7 and occurs despite hypophysectomyl*‘lg and pair feeding.lg Furthermore, it occurs after the destruction of a locus (VMN) which when stimulated, causes the inhibition of insulin release .20 This locus is within the sympathetic zone of the hypothaIamus2r and its destruction could alter insuIin secretion by affecting the sympathetic tone in pathways which still remain to be defined. Alterations in adipose tissue metabolism of VMN rats resulting in increased lipogenesis have recently been shown to be in part, independent of the hyperinsulinemia’ and presumably are also due to alterations in neurogenic activity. The combination of hyperinsulinemia and altered adipose tissue metabolism could then explain the development of obesity. The present data exclude the presence of peripheral insulin resistance in these animals. The results do not specifically indicate whether the sensitivity of the liver to exogenous insulin is altered with respect to hepatic glucose production. However, they support our suggestion’ that basal hepatic glucose production must be increased in weanling VMN rats in order to maintain normoglycemia in the presence of increased rates of glucose removal from plasma and normal food intake. ACKNOWLEDGMENT The authors wish to thank Ismael Mills, Betty Stone, and Tom Vincent
Allende, Elizabeth for their excellent
Gabel, Marjorie Kodis, technical assistance.
Maureen
REFERENCES 1. Salans, L. B., KnittIe, J. L., and Hirsch, J.: The role of adipose cell size and adipose tissue insulin sensitivity in the carbohydrate intolerance of human obesity. J. Clin. Invest. 47253, 1968. 2. Bagdade, J. D., Bierman, E. L., and
Porte, D., Jr.: The significance of basal insulin levels in the evaluation of the insulin response to glucose in diabetic and non-diabetic subjects. J. Clin. Invest. 46: 1549, 1967. 3. Sims, E. A. H., and Horton, E. 5.:
1142
Endocrine and metabolic adaptation to obesity and starvation. Amer. J. Clin. Nutr. 21:1455, 1968. 4. Frohman, L. A., and Bernardis, L. L,: Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions. Endocrinology 82:1125, 1968. 5. -, Goldman, J. K., Schnatz, J. D., and Bernardis, L. L.: Hypothalamic obesity in the weanling rat: Effect of diet upon hormonal and metabolic alterations. Metabolism 20:501, 1971. L. L.: Metabol6. -, -, and Bernardis, ism of intravenously injected 14C-glucose in weanling rats with hypothalamic obesity. Metabolism 21:799, 1972. 7. Goldman, J. K., Schnatz, J. D., Bernardis, L. L., and Frohman, L. A.: Effects of ventromedial hypothalamic destruction in rats with preexisting streptozotocin induced diabetes. Metabolism 21:132, 1972. 8. Hoffman, W. S.: A rapid photoelectric method for the determination of glucose in blood and urine. J. Biol. Chem. 120:51, 1937. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, P. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265, 1951. IO. Snedecor, G. W., and Cochran, W. G.: Statistical Methods (ed. 6). Ames, Iowa, Iowa State University Press, 1967. II. Bray, G. A., and York, D. A.: Genetically transmitted obesity in rodents. Physiol. Rev. 51:598, 1971. 12. Han, P. J., Yu, Y. K., and Chow, S. L.: Enlarged pancreatic islets of tube-fed hypophysectomized rats bearing hypothalamic lesions. Amer. J. Physiol. 218:769, 1970. 13. Tucker, J., Tretheway, J. T., Stewart, C. A., Neville, R. W., and Hanley, T.: Insulin sensitivity and carbohydrate metabol-
FROHMAN,
GOLDMAN,
AND BERNARDIS
ism of rats with hypothalamic obesity. J. Endocr. 33 ~437. 1965. 14. Malaisse, W. J., Malaisse-Lagae, F., Lacy, P. E., and Wright, P. H.: Insulin secretion by isolated islets in presence of glucose, insulin and anti-insulin serum. Proc. Sot. Exp. Biol. Med. 124~497, 1967. 15. Rafaelson, 0. J., Lauris, V., and Renold, A. E.: Localized intraperitoneal action of insulin on rat diaphragm and epididymal adipose tissue in viuo. Diabetes 14:19, 1965. 16. Goldman, J. K., Bernardis, L. L., and Frohman, L. A.: Insulin responsiveness in uifro of diaphragm and adipose tissue from weanling rats with hypothalamic obesity. Hormone and Metabolic Research, in press. 17. Frohman, L. A., Bernardis, L. L., Schnaiz, J. D., and Burek, L.: Plasma insulin and triglyceride levels after hypothalamic lesions in weanling rats. Amer. J. Physiol. 216:1496, 1969. 18. Goldman, J. K., Schnatz, J. D., Bernardis, L. L., and Frohman, L. A.: Adipose tissue metabolism of weanling rats after destruction of ventromedial hypothalamic nuclei : Effect of hypophysectomy and growth hormone. Metabolism 19 :995, 1970. 19. Han, P. W., and Frohman, L. A.: Hyperinsulinemia in tube-fed hypophysectomized rats bearing hypothalamic lesions. Amer. J. Physiol. 219:1632, 1970. 20. Frohman, L, A., and Bernardis, L. L.: Effect of hypothalamic stimulation on plasma glucose, insulin, and glucagon levels. Amer. J. Physiol. 221:1596, 1971. 21. Ban, T.: The septo-preoptico-hypothalamic system and its autonomic function. In Tokizane, T., and Schade, J. P., (Eds.): Progress in Brain Research, Vol. 21A. Amsterdam, Elsevier, 1966, pp. l-43.
Studies were performed in weanling male rats with bilateral lesions in the ventromedial hypothalamic nuclei (VMN) and sham-operated controls to evaluate (1) the endogenous insulin response to glucose, and (2) the sensitivity to exogenous insulin as manifested by both hypoglycemia and the utilization of simultaneously injected 14C-glucose. VMN rats exhibited basal hyperinsulinemia and an increased insulin secretory response to glucose.
VMN and control rats exhibited similar hypoglycemic responses to exogenous insulin. Utilization of intraperitoneally 14C-glucose by epididymal injected adipose tissue and diaphragm was similarly increased by insulin in the two groups of animals. The results exclude peripheral insulin resistance as the basis for hyperinsulinemia and support the belief that it is a direct consequence of the hypothalamic lesion.
T
HE PRESENCE OF HYPERINSULINEMIA in both human obesity and that of laboratory animals has generally been regarded to be secondary to a resistance by peripheral tissues to the action of insulin. The inverse correlation between the size of adipocytes and the response to insulin in vitro,’ the direct correlation between plasma insulin levels and body fat estimates,2 and the appropriate changes in both plasma insulin levels and tissue responsiveness to insulin with alterations in body weight’s3 have strengthened this concept. During the past several years, our investigations of the obesity observed in weanling rats after electrolytic destruction of the ventromedial hypothalamic nuclei have suggested that insulin sensitivity is normal or perhaps increased in these animals. After hypothalamic destruction, weanling rats exhibited normal food intake, weight gain, and plasma glucose levels, yet developed hyperinsulinemia and increased carcass fat;4 starvation resulted in mild hypoglycemia with at first, elevated and later, normal insulin levels;4.5 and glucose oxidation and conversion to lipid by adipose tissue in vitro was elevated in both the fed state and after food deprivation sufficient to eliminate hyperinsulinemia. 5 The lack of insulin resistance was also suggested by the enhanced disappearance of r4C-glucose from plasma after intravenous injection.e From fhe Departments of Medicine nnd Pathology, State University of New York at Buffalo, and the Veterans Administration Hospifal, Buffalo, N. Y. Received for publication April 28, 1972. Supported in part by USPHS Grants AM 11456 and 14418, and by Veterans Administration Clinical Invesfigaforship and Research Grant 01/3191-1 69-01. Lawrence A. Frohman, M.D.: Associate Professor of Medicine, Stale University of New York at Bufj%lo, Buffalo, N.Y. Jack K. Goldman, M.D.: Assistant Professor of Medicine, State University of New York at Buffalo, and Clinical Investigator, Buffalo Veterans Administration Hospifaf, Buffalo, N.Y. Lee L. Bernardis, Dr. Phil., Ph.D.: Research Associate Professor of Pathology, and Clinical Assisfanf Professor of Medicine, State University of New York at Buffalo, Buffalo, N. Y. Metabolism.
Vol. 21, No. 12 (December),
1972
1133
1134
FROHMAN, GOLDMAN, AND BERNARDIS
However, since the alterations in glucose oxidation and lipogenesis by adipose tissue in vitro have recently been demonstrated to occur after ventromedial hypothalamic destruction in streptozotocin-diabetic animals and are, therefore, in part, independent of the hyperinsulinemia,’ the direct evaluation of insulin responsiveness was deemed necessary to answer the question of hormonal sensitivity. This report describes the endogenous insulin responses to glucose and the effect of exogenous insulin on plasma glucose and on the metabolism of intraperitoneally injected 14C-glucose by epididymal adipose tissue, diaphragm and liver. MATERIALS
AND METHODS
Animafs and Operative Procedures Weanling male Sprague-Dawley rats were individually caged and allowed food (Teklad) and water ad lib. At 26-28 days of age, bilateral electrolytic lesions were placed in the ventromedial hypothalamic nuclei (VMN) with a stereotaxic instrument. The details of animal maintenance, lesion production and verification were identical to those previously described.4 Sham-operated animals served as controls. In all experiments, food intake and weight gain of VMN and control animals were similar.
Experimental
Procedures
Three weeks after lesion production, one Responses to Glucose. group of VMN and control animals were given glucose, 1.5 g/kg, intraperitoneally. Heparinized blood was collected from the tail vein prior to and at 30, 60, and 120 min after glucose. Plasma was separated at 4’ and immediately frozen. Food was removed from the cages 2 hr prior to the start of the experiment. Insulin Hypoglycemia. Three weeks after lesion production another group of animals was injected intraperitoneally with purified bovine insulin, 1.0 U/kg. Heparinized blood was collected from the tail vein prior to, and at 30 min after the injection of insulin. A third group of animals was similarly injected with 0.5 U/kg of insulin and blood collected at 0, 30, and 60 min. The same animals were retested 1 wk later using 0.25 U/kg of insulin. Animals were permitted free access to food until the time of insulin injection, at which time food was removed for the duration of the experiment. Two weeks after lesion production, a fourth Insulin Effects on I+%‘-glucose Utilization. group of animals was injected intraperitoneally with 2 &i of U-14C-glucose (New England Nuclear, S.A. 200 mC/mM) simultaneously with 0, 0.1 or 10 mU (0.6 or 60 mu/kg) of purified bovine insulin. Thirty minutes later, animals were decapitated. Trunk blood was collected in heparinized beakers and the plasma separated at 4’ and then frozen. Segments of liver and diaphragm were immediately frozen on dry ice and the epididymal fat pads placed in chloroform: methanol (2:l). In this experiment, food was removed from the animals 2 hr prior to the injection of insulin. Endogenous
Insulin
Analytical Procedures Plasma glucose was measured by a “micro” modification of the ferricyanide methods on an AutoAnalyzer. Plasma insulin was measured by a double antibody radioimmunoassay, the details of which have been previously described.4 The incorporation of 24C-glucose into glycogen, total lipids and fatty acids in adipose tissue, liver and was diaphragm was determined as previously described. 5.6 Tissue protein concentration measured by the method of Lowry et al.9 Data from animals whose lesions were asymmetric or impinged significantly on areas other than the ventromedial nucleus were excluded prior to statistical analysis. Differences between groups were compared by Students’ t test or by rank test when marked dissimilarities in variance were present.10
INSULIN
SENSITIVITY
1135
IN RATS
Insulin, pU/ ml plasma
80-1
T
60/ 40-1T
.A .. ..._ Control ( 121 ‘--c _________ __________Q
I I
Glucose,
I
I
mg/ 100 ml plasma
200-
______p
160.**’
Fig. 1. Plasma glucose and insulin responses to intraperitoneal glucose administration (1.5 g/kg) in VMN and control rats. Shown are the mean I? SE.
80 -1 I_
-1 30
I- _. ___..-I 60 Minutes
120
RESULTS Endogenous Insulin
Responses
to Glucose
After the intraperitoneal administration of glucose (1.5 g/kg) plasma glucose levels peaked at 30 min in both VMN and control rats (Fig. I). Although the glucose levels were consistently greater in control animals, the differences were not significant and the incremental responses were similar in the two groups. Plasma insulin levels were greater prior to, and at all times after glucose administration (p
Responses
to Exogenous Insulin
The plasma glucose response to 1.0 U/kg of insulin was similar in 10 VMN and 12 control animals (initial glucose: 130 + 8 mg/lOO ml vs. 140 -t 5; 30 min glucose: 63 1 5 vs. 69 + 8). Because the large dose of insulin might conceivably mask a slight degree of insulin resistance, smaller doses were next tested. Plasma glucose responses to 0.5 U/kg of insulin were also similar with the decline in glucose at 60 min being significant in both VMN (p
1136
FROHMAN, GOLDMAN, AND BERNARDIS
Plasma Glucose mg/1OOml
d Minutes
Fig. 2. Plasma glucose responses to intraperitoneat administration of 0.5 U/kg (left panel) and 0.25 U/kg (right panel) of insulin. Shown are the mean -t SE.
were observed in the hypoglycemic responses. In this experiment the decline in glucose at 60 min was significant in control (p
Utilization
Plasma glucose and insulin levels 30 min after the intraperitoneal administration of considerably smaller doses of insulin (0.1 and 10 mu/rat) are shown in Table 1. Whereas no change in plasma glucose was observed in response to insulin in control animals, small but significant decreases were noted in VMN rats injected with both 0.1 mU (p
0
Insulin 10 mU
Plasma glucose, mg/lOO ml VMN Control Plasma insulin, pU/ml VMN Control
96 f 99 f
2 2
92 f2 96 ?I 1
6622 96 k 1
53 -t 4 42 4 3
31 +5 13 ?I 1
3629 27 &I 3
INSULIN
SENSITIVITY
1137
IN RATS
AdiDose
Tissue 14C- Lipid DPWmg
14C- Fattv Acids
-I-
I 14C- Glycoqen DPWmg
20-
l15-
Fig. 3. Incorporation of radioactivity into adipose tissue total lipid, fatty acids, and glycogen expressed per mg tissue after the intraperitoneal injecwith or without tion of W-glucose insulin in VMN and control rats. Shown are the mean f SE. Number of animals in each group (from left to right) were 7, 6, 7, 6, 7, and 4.
1 ,_
VMN c
wO..lc Insulin, mU
and differences between VMN and control groups persisted. In animals given the 10 mU dose, however, there were marked increases in the incorporation of glucose radioactivity into total lipid, fatty acids and glycogen of both groups of animals. At this dose, the differences between VMN and control groups were eliminated. Adipose tissue protein was reduced in VMN as compared to control animals (8.9 * 0.4 pglmg vs. 13.8 * 0.7, p
1138 Diaphragm 3-
FROHMAN,GOLDMAN, AND BERNARDIS
W- Lipid DPWmg
0.l Insulin, mU
,-
10
Fig. 4. incorporation of radioactivity into diaphragm total lipid and glycogen expressed per mg tissue after the intraperitoneal injection of %-glucose with or without insulin in VMN and control rats. See legend to Fig. 3. Liver I%- Lipid DPM/mq
IT
2-
Fig. 5. Incorporation of radioactivity into liver total lipid, fatty acids, and glycogen expressed per mg tissue after the intraperitoneal injection of 14C-glucose with or without insulin in VMN and control rats. See legend to Fig. 3.
14C- Fatty Acids
DPM/mg
Insulin,
mU
INSULIN
SENSITIVITY
1139
IN RATS
The effects of insulin upon glucose utilization by the diaphragm are shown of 14C-glucose into diaphragm glycogen was similar in VMN and control animals in the absence of insulin, and the two groups of animals exhibited similar increases in glycogen incorporation in response to both the low and high dose of insulin. Glucose incorporation into diaphragm lipid was similar in saline treated VMN and control animals. The response to insulin by VMN animals was slightIy greater than controls at the low dose and significantly greater (p
in Fig. 4. Incorporation
DISCUSSION
The coexistence of hyperinsulinemia and normal glucose values in both human and animal obesity has generally been accepted as evidence for the existence of insulin resistance. With the demonstration of resistance to the action of insulin in animals with genetic obesity” and in both spontaneous2 and induced3 human obesity, the presence of elevated plasma insulin levels has been considered to be a response by the pancreas in an effort to overcome this resistance. Hyperinsulinemia has previously been reported in weanling rats with hypothalamic obesity,4 and the present results indicate that the insulin response to glucose was similarly increased. The insulin response, expressed as a percentage of basal insulin levels, tended to be similar to that in control animals and may, therefore, be a reflection of the islet hypertrophy which occurs after hypothalamic destruction.12 This pattern is, therefore, similar to that observed in other types of obesity. However, our results clearly and unequivocally demonstrate that the observed hyperinsulinemia is not associated with peripheral insulin resistance. The hypoglycemic effect of moderate doses of insulin was completely normal in nonhyperphagic weanling rats with VMN lesions. Similar results have also been reported in hyperphagic adult rats with hypothalamic lesions.r3 An increased sensitivity to the hypoglycemic effects of insulin is even suggested by the plasma glucose responses to the small doses of insulin administered in the experiment in which r4C-glucose utilization was examined. This experiment also provided possible indirect evidence of an effect of insulin on the liver which was not evident from analysis of the 14C-glucose results. After the intraperitoneal administration of both 0.1 and 10 mU of insulin, there were decreases in plasma insulin in both VMN and control
1140
FROHMAN,
GOLDMAN,
AND BERNARDIS
animals. The decreases were most striking after the administration of 0.1 mU of insulin, which was a quantity sufficient to increase plasma insulin levels only transiently, even if injected intravenously. The plasma insulin levels were slightly greater after the larger dose of insulin, but were still less than in the saline-injected animals. The decline in plasma glucose levels, therefore, although possibly attributable to the effects of the injected insulin on the diaphragm and the intraperitoneal adipose tissue, suggests as well a decrease in hepatic glucose production. The greater decline in glucose levels in VMN as compared to controI animals is probably due to the more rapid plasma glucose turnover in VMN rats.6 The present data does not permit one to determine conclusively the mechanism responsible for the decreased plasma insulin levels in the insulin treated rats. However, there exist at least two possibilities. First, a decrease in plasma glucose levels secondary to a decreased hepatic glucose output could result in a subsequent reduction in the release of insulin by the pancreas. Although this explanation is supported by the glucose changes in VMN rats, the minimal changes in control animals would appear to be inadequate to cause a decrease of 30 pU/ml in the level of plasma insulin. Secondly, the injected insulin could possibly have a direct effect on the suppression of insulin release, although Malaisse et al. have reported no diminution of insulin release by islets incubated in the presence of insulin.r4 Irrespective of the mechanism, the results again demonstrate the ability of VMN rats to reduce their elevated plasma insulin levels when subjected to decreasing glucose availability, as has been previously observed after prolonged fastinge5 Assessment of glucose utilization by epididymal adipose tissue, diaphragm, and liver in vivo was achieved by the intraperitoneal administration of 14Cglucose, using a technique previously reported by Rafaelson et aLI5 This procedure permitted the evaluation of both basal and insulin stimulated glucose utilization at doses of insulin, which had minimal effects on circulating glucose levels. Under basal conditions, glucose utilization was increased in VMN adipose tissue while it was normal in diaphragm. The insulin response was normal in both tissues. Of interest are some differences between the present results and those observed in vitro. We have previously reported that in vitro incorporation of 14C-glucose into adipose tissue glycogen by VMN rats was not increased, in contrast to the marked increase in lipogenesis5 Recent results have further indicated that glucose incorporation into adipose tissue glvcogen in vitro by VMN rats is actually resistant to the effects of insulin.le There is no obvious explanation for the resistance to this pathway of glucose metabolism, nor is there one for the difference between the in vitro results and those obtained in vivo both in basal and insulin stimulated conditions. However, the applicability of any in vitro observation is contingent upon its reflection of an in vivo phenomenon and the present studies, therefore, indicate that the “apparent” resistance of the glycogenetic pathway in vitro is due to a phenomenon not present in the intact animal. No increase in glucose incorporation into either lipids or glycogen was observed in the liver of either VMN or control animals in response to insulin,
INSULIN
SENSITIVITY
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IN RATS
To the contrary, there was a tendency to decreased incorporation in the presence of insulin. This phenomenon may well be a reflection of the marked increase in glucose utilization by adipose tissue and diaphragm resulting in decreased availability of r4C-glucose to the liver. However, evidence for hepatic responses to insulin already has been discussed with respect to changes in plasma glucose levels. There are several implications of the present results with respect to the hyperinsulinemia of VMN animals. First, whether or not a linear relationship exists between insulin and glucose responses, the increased “insulin/glucose ratios,” which result from elevated insulin levels with normal ghrcose levels, are not an indication of peripheral insulin resistance in VMN animals. It follows that there may also be other forms of obesity, either experimental or naturally occurring, in which this is also true. The present results should, therefore, serve as a cautionary note with respect to the interpretation of “insulin/glucose ratios” unless more specific evidence of insulin resistance is also available. The second implication of our results relates to the etiology of the hyperinsulinemia in VMN rats and supports our initial hypothesis of a primary central nervous system pathogenetic mechanism.* The hyperinsulinemia has been shown to the independent of hyperglycemia, food intake and weight gain4,r7 and occurs despite hypophysectomyl*‘lg and pair feeding.lg Furthermore, it occurs after the destruction of a locus (VMN) which when stimulated, causes the inhibition of insulin release .20 This locus is within the sympathetic zone of the hypothaIamus2r and its destruction could alter insuIin secretion by affecting the sympathetic tone in pathways which still remain to be defined. Alterations in adipose tissue metabolism of VMN rats resulting in increased lipogenesis have recently been shown to be in part, independent of the hyperinsulinemia’ and presumably are also due to alterations in neurogenic activity. The combination of hyperinsulinemia and altered adipose tissue metabolism could then explain the development of obesity. The present data exclude the presence of peripheral insulin resistance in these animals. The results do not specifically indicate whether the sensitivity of the liver to exogenous insulin is altered with respect to hepatic glucose production. However, they support our suggestion’ that basal hepatic glucose production must be increased in weanling VMN rats in order to maintain normoglycemia in the presence of increased rates of glucose removal from plasma and normal food intake. ACKNOWLEDGMENT The authors wish to thank Ismael Mills, Betty Stone, and Tom Vincent
Allende, Elizabeth for their excellent
Gabel, Marjorie Kodis, technical assistance.
Maureen
REFERENCES 1. Salans, L. B., KnittIe, J. L., and Hirsch, J.: The role of adipose cell size and adipose tissue insulin sensitivity in the carbohydrate intolerance of human obesity. J. Clin. Invest. 47253, 1968. 2. Bagdade, J. D., Bierman, E. L., and
Porte, D., Jr.: The significance of basal insulin levels in the evaluation of the insulin response to glucose in diabetic and non-diabetic subjects. J. Clin. Invest. 46: 1549, 1967. 3. Sims, E. A. H., and Horton, E. 5.:
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Endocrine and metabolic adaptation to obesity and starvation. Amer. J. Clin. Nutr. 21:1455, 1968. 4. Frohman, L. A., and Bernardis, L. L,: Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions. Endocrinology 82:1125, 1968. 5. -, Goldman, J. K., Schnatz, J. D., and Bernardis, L. L.: Hypothalamic obesity in the weanling rat: Effect of diet upon hormonal and metabolic alterations. Metabolism 20:501, 1971. L. L.: Metabol6. -, -, and Bernardis, ism of intravenously injected 14C-glucose in weanling rats with hypothalamic obesity. Metabolism 21:799, 1972. 7. Goldman, J. K., Schnatz, J. D., Bernardis, L. L., and Frohman, L. A.: Effects of ventromedial hypothalamic destruction in rats with preexisting streptozotocin induced diabetes. Metabolism 21:132, 1972. 8. Hoffman, W. S.: A rapid photoelectric method for the determination of glucose in blood and urine. J. Biol. Chem. 120:51, 1937. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, P. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265, 1951. IO. Snedecor, G. W., and Cochran, W. G.: Statistical Methods (ed. 6). Ames, Iowa, Iowa State University Press, 1967. II. Bray, G. A., and York, D. A.: Genetically transmitted obesity in rodents. Physiol. Rev. 51:598, 1971. 12. Han, P. J., Yu, Y. K., and Chow, S. L.: Enlarged pancreatic islets of tube-fed hypophysectomized rats bearing hypothalamic lesions. Amer. J. Physiol. 218:769, 1970. 13. Tucker, J., Tretheway, J. T., Stewart, C. A., Neville, R. W., and Hanley, T.: Insulin sensitivity and carbohydrate metabol-
FROHMAN,
GOLDMAN,
AND BERNARDIS
ism of rats with hypothalamic obesity. J. Endocr. 33 ~437. 1965. 14. Malaisse, W. J., Malaisse-Lagae, F., Lacy, P. E., and Wright, P. H.: Insulin secretion by isolated islets in presence of glucose, insulin and anti-insulin serum. Proc. Sot. Exp. Biol. Med. 124~497, 1967. 15. Rafaelson, 0. J., Lauris, V., and Renold, A. E.: Localized intraperitoneal action of insulin on rat diaphragm and epididymal adipose tissue in viuo. Diabetes 14:19, 1965. 16. Goldman, J. K., Bernardis, L. L., and Frohman, L. A.: Insulin responsiveness in uifro of diaphragm and adipose tissue from weanling rats with hypothalamic obesity. Hormone and Metabolic Research, in press. 17. Frohman, L. A., Bernardis, L. L., Schnaiz, J. D., and Burek, L.: Plasma insulin and triglyceride levels after hypothalamic lesions in weanling rats. Amer. J. Physiol. 216:1496, 1969. 18. Goldman, J. K., Schnatz, J. D., Bernardis, L. L., and Frohman, L. A.: Adipose tissue metabolism of weanling rats after destruction of ventromedial hypothalamic nuclei : Effect of hypophysectomy and growth hormone. Metabolism 19 :995, 1970. 19. Han, P. W., and Frohman, L. A.: Hyperinsulinemia in tube-fed hypophysectomized rats bearing hypothalamic lesions. Amer. J. Physiol. 219:1632, 1970. 20. Frohman, L, A., and Bernardis, L. L.: Effect of hypothalamic stimulation on plasma glucose, insulin, and glucagon levels. Amer. J. Physiol. 221:1596, 1971. 21. Ban, T.: The septo-preoptico-hypothalamic system and its autonomic function. In Tokizane, T., and Schade, J. P., (Eds.): Progress in Brain Research, Vol. 21A. Amsterdam, Elsevier, 1966, pp. l-43.