Acta physiol. scand. 1972. 84. 29-33 From the Physiology Section, Johan Throne Holst’s Institute for Nutrition Research,

University of Oslo, Norway

Correlation between Hyperinsulinemia and Hyperphagia in Rats with Ventromedial Hypothalamic Lesions

BY

B. E. HUSTVEDT and A. Lov0

Received 16 June 1971

Abstract

HUSTVEDT, B. E. and A. Lover. Correlation between hyperinsulinemia and hyper- phagia in rats with uentromedial hypothalamic lesions. Acta physiol. scand. 1972. 84. 29-33.

Plasma levels of immunoreactive insulin ( IRI) and glucose were determined in unrestrained rats equipped with chronically implanted venous catheters. Electrolytic lesions were then placed bilaterally in the ventromedial hypothalamic area. Food intake was restricted in order to prevent hyperphagia. A 60-280 % increase in the IRI level with a concomitant small in- crease in blood glucose was demonstrated on the second day post-operatively. The animals were then given free access to food. The gain in body weight during the following 3 days of ad lib feeding and the post-operative increase in the IRI level of each animal was positively correlated.

Bilateral destruction of the ventromedial hypothalamic area (VMH of rats produces hyperphagia and extreme obesity (Hetherington and Ranson 1939). Elevated plasma levels of immmunoreactive insulin (IRI) have been reported in animals with such lesions (Hales and Kennedy 1964, Frohman and Bernadis 1968). The etiology of this hyperinsulinemia has not yet been established.

Firm knowledge of the plasma IRI and blood glucose changes during the first post-operative days is essential for a fruitful investigation of this problem. More- over, it is important that the nutritional state of lesioned animals remains virtually unchanged during the observation period as both over- and underfeeding will in- fluence plasma IRI and blood glucose levels. I n order to obtain blood samples under comparable and possibly basal conditions before and after the lesions were made it would seem advantageous to implant venous catheters permanently into a suitable vessel. Such an arrangement would be of particular value during the post-operative period since lesioned animals are aggressive and more difficult to handle than normal rats.

The investigation reported here was undertaken in order to study the earIy effects of VMH lesions on fasting plasma levels of insulin and glucose from unrestrained

29

30 B. E. HUSTVEDT A S D A. LOVO

animals under conditions during which hyperphagia was prevented. Furthermore, we have attempted to establish whether changes in these parameters might in some \yay be correlated to the body weight gain of the individual animals on a subse- quent ad lzb feeding schedule.

Material and Methods Male Wistar rats ( .4F/Han/hIo/Han) weighing 240-280 g were used. They were provided with a chronically implanted venous heart cannula according to Steffens (1969). The catheter (Silastic medical grade tubing, 0.020 in 1.D.: 0.037 in O.D.) was introduced into the external jugular vein under Xenibutal@ anesthesia (50 mg/ kg b.w.) . After catheterization the animals were kept in separate cages and fed ad lib. The animals recovered rapidly from the catheteri- xation. A modest loss of body weight took place during the first two days post-operztively. Then the food intake was normalized and the body weight increased again.

Stereotaxically guided lesions (David Kopf instruments hfodel 900) were produced by electrolysis 7 days after catheterization. An anodal d.c. current (1.5 m.4, 15 s) was passed through steel electrodes of even thickness (0.4 mm), insulated by varnish except for 0.5 mm a t the tip. The stereotasic coordinates used for destroying the ventromedial area of hypo- thalamus \\*ere 6.0 mm anterior to the inter-aural line, 0.6 mm lateral to the midline, and 1.0 min above the 1.entral floor of the skull.

The animals were offered 15 g of food daily for the first 5 days post-operatively which equals the average food intake of normal rats of the same size. After this initial period they were fed ad lib as before. The food intake \vas recorded daily, and the animals were weighed every 3 d a p . Only the body weight was recorded during the ad lib feeding period of the lesioned animals because it proved difficult to obtain reliable measurements of the food intake.

Blood samples isere obtained 5 days and 2 days before the VhIH destruction and again 2 days after the operation. The sampling was performed on unrestrained animals 8 h after withdrawal of food. S o handling of the animals took place during the last 2 h prior to sampling, and Heparin was not infused. Blood samples ( 2 ml) were drawn into heparinized syringes and immediately placed in ice-water. Duplicate aliquots of 25 ,ul each were secured for the glucose analysis. The rest of the blood \vas centrifuqed and plasma separated from the cells. T h e plasma was frozen and kept a t -25@ C for later analysis. Immediately after sampling the rats rcceiwd transfusions of heparinized fresh donor blood.

A?rnlyfical f i rorrdures

The blood glucose concentration was determined in 25 ,ul of whole blood. A commercial Flucose-oxidase reagent set was used (GLOX, Kabi). Plasma IRI was assayed in samples of 50 u1 in quadriplicate, according to Drskov (1967). A proinsulin-free preparation of crystal- line rat insulin (Batch R 169, Xovo) was used as standard. Plasma I R I concentrations were calculated by means of the computer program developed by Viilund et al. (1970) adapted for use on the CDC 3300 computer of the University of Oslo. The computer output consists of a table uf estimates with statistical confidence limits for each unknown, the variation between replicates of the unknowns and standards being taken into account.

Results The plasma IRI concentrations mere increased considerably 2 days after VMH de- struction compared to the level before the operation in all animals tested. This is demonstrated in Fig. 1. There was, however, a greater variation in IRI values after Irsion than before, 38-107 ,iiU/ml 2 days after lesion as compared to 18-37 pU/ml 2 da)s before lesion.

The IRI levels before VMH destruction also increased somewhat from the first to the second measurement. We believe that the concomitant improvement in the nutritional state of the animals as they regained their appetite after catheterization i, the most likely explanation for this observation. The average food intake on the

INSULIN I N HYPOTHALAMIC HYPERPHAGIA 31

0 5 DAYS BEFORE LESKXJ

2 DAYS BEFORE LESON

2 DAYS AFTER LESON

E 2 Z - v

L Fig. 1. Blood glucose and plasma immunoreactive insulin (IRI) N levels in rats before and after the 2 placement of ventromedial hypo- (3 0 thalamic lesions. The vertical bars in the IRI diagram represent the 8 95 % confidence interval. d

416 417 418 419 420 422

day of the first blood sampling was 8-10 g, whereas it had risen to 13-15 g 3 days later, when the second blood sample was taken. This corresponds closely to the average food intake of normal rats of this size. Thus the IRI level measured 2 days before VMH destruction is probably representative for normal rats of this age after 8 h of fasting.

The bIood glucose level 2 days before the lesions were produced was in the range of 75-80 mg/100 ml for all except one of the animals. As the concentration of blood glucose is markedly influenced by stress, the uniform level that was observed seems to confirm our impression that the blood sampling procedure was performed under comparable conditions in the different animals. All animals had a slightly increased blood glucose level 2 days post-operatively compared to the values measured 2 days before the operation.

I n Fig. 2 the individual increases in the fasting IRI level post-operatively is plotted against the subsequent gain in body weight of each animal during 3 days of ad lib feeding. The first day with free access to food is not included in this period in order to avoid the initial body weight increase due to the rapid filling of the digestive tract

32 B. E. HUSTVEDT AND A. LQVG

Fig. 2. The relation between the increase in plasma immunoreac- tive insulin (IRI) from 2 days before to 2 days after lesion, and the increase in body weight fol- lowing 3 days feeding ad lib. Each filled circle represents values from one animal. The linear re- gression line is stippled.

\+hich takes place when hyperphagic animals are fed ad lib. There is a reasonably good positive correlation between the 2 sets of values. The correlation coefficient equals 0.74 with a 95 76 confidence interval of 0.23-0.93. The correlation is signifi- cant. p < 0.02. All animals used in Fig. 2 exhibited varying degrees of hyper- phagia as their body weights increase considerably more than 1-2 g/day which is the ai'erage weight gain for normal rats of this age.

Discussion The present \tork shows that the plasma IRI level is considerably elevated already 2 day5 after VMH destruction in adult rats even when hyperphagia is prevented. Furthermorel this IRI elevation is found to be positively correlated to the subsequent gain in body weight of each animal after 3 days of ad lib feeding.

Hales and Kennedy (1964) obserr-ed increased fasting IRI concentrations in lesioned animals after 7 days of ad lib feeding but not as early as on the second post- operative day. They therefore attributed the hyperinsulinemia to the hyperphagia and obesity that accompanies the VMH lesions in adult rats. I t is well documented, indeed. that obesity leads to adaptive metabolic changes including an elevated IRI level (Karam, Grodsky and Forsham 1963). Hyperinsulinemia has also been reported i n voluntarily hyperphagic humans (Sims et al. 1968). However, Frohman et al. ( 1969 i found increased IRI levels on the 4th post-operative day in lesioned weanling rats which, they claim, develop obesity without hyperphagia. Recently, Han and Frohman ( 1970) reported hyperinsulinemia in hypophysectomized rats 5 days after VMH destruction as compared to hypophysectomized control animals. Both groups Ivcre tubefed equal amunots of food. They concluded, therefore, that hyperin- sulinemia may develop after VMH lesions are produced in the absence of both hyperphagia and pituitary hormones.

Our results extend the previous evidence for an early development of hyper- insulinemia in lesioned rats. Probably, the elevated IRI level is established even

INSULIN IN HYPOTHALAMIC HYPERPHAGIA 33 before the second post-operative day. In one single animal we have sampled blood 24 h post-operatively and found increased IRI concentration. Furthermore, we have confirmed that the hyperinsulinemia is not a result of adaptive metabolic changes following hyperphagia. The early onset of hyperinsulinemia after VMH destruction may indicate a direct nervous or humoral action on the endocrine pancreas causing increased insulin secretion. Alternatively, the operation may lead to other nervous or endocrine disturbances that result in insulin resistance of some tissue(s) . In that case, the elevated IRT level would be a secondary effect of such disturbances.

Normal rats given regular injections of longacting insulin exhibit increased food intake and accumulation of body fat (MacKay, Callaway and Barnes 1940). I t seems reasonable to expect that endogenous hyperinsulinemia will have the same effects. The first hypothesis is therefore an attractive one because it provides a simple ex- planation for the hyperphagia and obesity of lesioned animals. The correlation re- ported in this paper appears to support this hypothesis although it does not prove any causal relationship between the hyperinsulinemia and the subsequent hyperphagia.

If the primary effect of VMH lesions is increased insulin secretion, hypoglycemia in the fasting state post-operatively would be expected. This is not the case, how- ever. The blood glucose is in fact slightly elevated compared to the level 2 days be- fore the operation. In our opinion, this finding supports the alternative hypothesis of insulin resistance caused by VMH destruction. However, a firm conclusion as to the primary effect of such lesions cannot be drawn on basis of the presented results alone.

We thank Dr. Henrik Ege, Novo Industri A/S, for a gift of crystalline rat insulin and Miss Ase Turter, the Hormone Laboratory, Aker Hospital, Oslo, for valuable assistance with the insulin radioimmunoassay.

References FROHMAN, L. A. and L. L. BERNARDIS, Growth hormone and insulin levels in weanling rats

with ventromedial hypothalamic lesions. EndocrinoZogy 1968. 82. 1125-1 132. FROHMAN, L. A., L. L. BERNARDIS, J. D. SCHNATZ and L. SUREK, Plasma insulin and tri-

glyceride levels after hypothalamic Iesions in weanling rats. Amer. J. Physiol. 1969. 216.

HALES, C . N. and G. C. KENNEDY, Plasma glucose, non-esterified fatty acid and insulin con-

HAN, P. W. and L. A. FROHMAN, Hyperinsulinemia in tube-fed hypophysectomized rats bearing

HETHERINGTON, A. and S. W. RANSON, Experimental hypothalamico-hypophyseal obesity in the

KARAM, J. H., G . M. GRODSKY and P. H. FORSHAM, Excessive insulin response to glucose in

MACKAY, E. M., J. W. CALLAWAY and R. H. BARNES, Hyperalimentation in normal animals

URSKOV, H., Wick-chromatography for the immunoassay of insulin. Scand. 1. d in . Lab. Invest.

SIMS, E. A. H., R. F. GOLDMAN, C. M. GLUCK, E. S. HORTON, P. C. KELLEHER and D. W. ROWE, Experimental obesity in man. Trans. Ass. Amer. Phycns. 1968. 81. 153-170.

STEFFENS, A. B., A method for frequent sampling of blood and continuous infusion of fluids in the rat without disturbing the animal. Physial. Behav. 1969. 4. 833-836.

VOLUND, AA., L. G. HEDING, A. J. MOODY and E. PARRST, Computerized calculation and quality control of radioimmuno- or bioassays illustrated for the insulin assay. Diabetologia 1970. 6. 68.

1496-1501.

centrations in hypothalamic-hyperphagic rats. Biochem. ]. 1964. 90. 620-624.

hypothalamic lesions. Amer. J . Physiol. 1970. 219. 1632-1636.

rat. Proc. SOC. exp. B id . (N.Y.) 1939. 41. 465--466.

obese subjects as measured by immunochemical assay. Diabetes 1963. 12. 197-204.

produced by protamine insulin. J . Nutr . 1940. 20. 59-66.

1967. 20. 297-304.

3-723003. Acta physiol. scaad. Vol . 84: I