Acta physiol. scand. 1974. 90. 757-763 From the Institute for Zoophysiloogy, and the Institute for Nutrition Research,

University of Oslo, Blindern, Norway

Insulin Sensitivity in Rats with Ventromedial Hypothalamic Lesions

BY

C. FAHLE HONGSLO. B. E. HUSTVEDT and A. L a w

Received 31 October 1973

Abstract

FAHLE HONGSLO, C., B. E. HUSTVEDT and A. L ~ v Q . Insulin sensitivity in rats with ventromedial hypothalamic lesions. Acta physiol. scand. 1974. 90. 757-763.

The insulin sensitivity of the diaphragm and parametrial adipose tissue in animals with ventro- medial hypothalamic (VMH) lesions and controls were assayed in viuo by studying the intra- peritoneal action of exogenous insulin. Lipogenesis and glycogen synthesis were stimulated to the same extent in lesioned and control animals. This implies that the co-existence of hyper- insulinemia and normoglycemia previously demonstrated in lesioned animals cannot be attri- buted to decreased insulin sensitivity. Increased gluconeogenesis in the lesioned animals is indicated by elevated levels of plasma urea. This may contribute to the maintainance of normoglycemia in the presence of hyperinsulinemia and normal insulin sensitivity. I t is sug- gested that the profound metabolic effects of VMH destruction is caused by a disturbance in the nervous control of gastro-intestinal functions. This may lead to alterations in the endocrine pancreatic secretion, and possibly also in liver metabolism.

Ventromedial hypothalamic lesions (VMH) in the adult rat give rise to hyperphagia and extensive accumulation of body fat (Hetherington and Ranson 1939). Animals with such lesions exhibit increased plasma insulin levels (Hales and Kennedy 1964). Hyperinsulinemia is present already 2 days post-operatively even when hyperphagia is prevented (Hustvedt and L0v0 1972). The described increase in plasma insulin concentration is therefore not the result of adaptive metabolic changes secondary to hyperphagia.

The presence of hyperinsulinemia in human obesity as well as in genetically obese laboratory animals is well known and has been regarded as secondary to a resistance by peripheral tissues to the insulin action. The question has therefore been raised as to wether the VMH lesions lead to nervous and/or endocrine disturbances that may result in increased insulin resistance in some tissue (s) . However, recent publica- tions dealing with these problems report responses to insulin in the normal range in diaphragm as well as in adipose tissue (Frohman et al. 1972, Han et al. 1972). Frohman et al. (1972) studied the action of endogenous and exogenous insulin in

757

758 C. FAHLE HONGSLO, B. E. HUSTVEDT A N D A. L 0 V D

the VMH lesioned weanling rat 3 weeks after surgery, while the work of Han et al. (1972) was performed on 200 g hypophysectomized rats 21 days post-operatively.

I t is known, however, that the metabolism of the VMH lesioned animal is changed already a few hours post-operatively so as to promote accumulation of body fat at the expense of other anabolic processes (Hustvedt and Lava 1973, Holm et al. in press). These animals will during the 3 first post-operative weeks accumulate significantly more lipids and less protein and water than their controls, even on a normalized food intake (Holm et al. in press). The magnitude of this in- creased lipid accumulation is in the order of 100 %. The obesity caused by VMH destruction is characterized by marked enlargement of the fat cells, with little or no change in cell number (Hirsch and Han 1969). This enlargement will probably alter the insulin sensitivity of the fat cells, because it is evidence for an inverse cor- relation between cell size and response to insulin in uitro by adipose tissue (Salans et al. 1968).

In order to evaluate the effect of VMH destruction ger se on insulin sensitivity, it therefore seems important to carry out such studies as early after the operation as possible. The present expriments were performed 2 days post-operatively on animale kept on a normalized food regimen after surgery.

Materials and methods Animals and animal care Female Wistar rats (AFIHanlMIZIIHan) weighing 160-180 g were used. The animals were kept in separate cages during the experimental period. The lighting of the animal room was automatically regulated to provide 12 h of darkness and 12 h of light, from 1600 to 0400 and 0400 to 1600 respectively. The rats were fed ad libitum before the operation. The food intake post-operatively was restricted to 12-13 g/day, the average food consumption of normal rats of this size. After hypothalamic surgery an automated food dispenser (Quarterman el al. 1970) supplied the daily portion of food uniformly and continuously. This was done in order to exclude adaptive metabolic effects due to mealfeeding and hyperphagia. The diet contained 15 % protein, 19 % fat and 66 % carbohydrate, made into a powdered mixture.

Surgical procedure Stereotaxically guided lesions (David Koph Instruments Model 900) were produced by electro- lysis under Nembutala anesthesia. An anodal current (1.5 mA, 15 s ) was passed through steel electrodes of even thickness (0.4 mm), thoroughly insulated by varnish except a t the tip. The stereotaxic coordinates used for destroying the ventromedial area of the hypothalamus were 6.0 mm anterior to the inter-aural line, 0.6 mm lateral to the midline, and 1.0 mm above the ventral floor of the skull. As other experiments have revealed no effect of sham-operation upon the metabolism 2 days post-operatively (L0ve and Hustvedt, in press), untreated animals were used as controls.

Histology The brain lesions of all animals surgically treated were histologically examined. All animals included in this study exhibited symmetrical bilateral lesions in the ventromedial hypothalamic area, and no interruption of the median eminence could be demonstrated.

Experimental procedure The insulin sensitivity of diaphragm and parametrial adipose tissue were assayed in vivo by studying the intraperitoneal action of exogenous insulin (Rafaelsen et al. 1965). The experi- ments were performed 2 days post-operatively after 16 h fasting, and were always started between 0900 and 1000. In each experiment 2 groups of animals ( 9 with VMH lesions and

INSULIN RESISTANCE I N HYPOTHALAMIC HYPERPHAGIA 759 8 untreated controls) were given 2.0 ,uCi D-glucose-14C ( U ) . (The Radiochemical Centre, Amersham), contained in 2.5 ml saline by i.p. injection. The solution contained 5 % bovine serum albumin (Sigma, fraction V) and 0.1 mg glucose/ml. Insulin was added to the same solution in desired concentration to give each rat 0.1, 1.0 and 10.0 mU respectively. Bovine crystalline insulin (Novo, lot. nr. 027667) was used. The in U Z U D incubation was discontinued by decapitation of the animals after 2 h. Blood was collected into heparinized tubes, immediate- ly placed on ice, and the cells were separated from the plasma by centrifugation. The hemi- diaphragms and the parametrial fat pads were dissected free and immediately frozen on an aluminium block cooled by ethanol and dry ice within 2 min after the decapitation.

Analytical procedures The plasma glucose concentration was determined in 50 ti1 of plasma. A commercial glucose oxidase reagent set was used (Gloxe, Kabi). The plasma urea concentration was assayed by using an enzymatic test kit supplied by Boehringer, Mannheim. The frozen hemi-diaphragms were weighed and hydrolyzed for 30 min by boiling with 30 c/o KOH. The glycogen deter- minations were always started immediately after the experiment. Glycogen was precipitated over night at 4" C in 66 % ethanol with 60 mM Na2SO4. After washing 2 times with 66 % ethanol the precipitate was dried and dissolved in 2 ml aq. dest. An aliquot of 1.8 ml was mixed with Insta-GelQ (Packard Instrument Comp., Inc.) and counted in a Packard Tri Carb. Liquid Scintillation Spectrometer. Counting efficiency was calculated by means of the channels ratio method.

The remaining 0.2 ml of solution was used for duplicate glycogen determinations. This was performed by an all enzymatic method according to Adolfsson 1972. The glycogen was hydrolyzed to glucose by amyloglucoxidase (Sigma, grade 11), and the liberated glucose was determined by the glucose oxidase method described. Rabbit liver glycogen (Sigma, Type 111) was used as standard. A separate standard curve was constructed during each run of the assay. The parametrical fat pads were weighed and homogenized in chloroform : methanol 2 : 1. The total lipids were isolated according to Folch et al. (1957), and transferred to counting vials and taken to dryness. The lipid material was weighed and 10 ml scintillation fluid (toluene containing 6.0 g PPO/l) was added. Differences between groups were compared by Student's t test.

Results Fig. 1 shows the specific I4C activity of parametrial lipids from animals with V M H lesions and their controls after in viuo incubation with D-glucose-"C ( U ) without exogenous inculin and with 0.1, 1.0 and 10.0 m u , respectively. This demonstrates a more than 100 96 increase in specific 14C activity of the extracted lipids from the V M H lesioned group compared to the controls when no insulin was administred. The difference is significant, p < 0.001. The lesioned animals exhibit a higher IIC

ADIPOSE TISSUE

O j - INSULIN, mU

Fig. 1. Specific 14C-activity of total extracted lipids (DPM/mg) from parametrial adipose tissue after i.p. injection of 2 pCi D-glu- cose-14C ( U ) together with 0, 0.1, 1.0 and 10.0 mU of insulin. The open columns give the activity of the control groups ( n = 8), while the hatched columns represent VMH lesioned animals (n = 9 ) . The standard de- viations of the means are given by the verti- cal bars.

C VMH

C. FAHLE HONGSLO, B. E. HUSTVEDT AND A. LBVQ

D I A P H R A G M

2405 F T

0 ,I 1.0 INSULIN.mU

Fig. 2

C VYH

10

100 - 60 -

20 -

30 -

10 - c VMH c vMH c VMH - 0,1 1 P

INSULIN, mU

Fig. 3 Fig. 2. Glycogen concentration (mg/g w.w), upper panel, and the 14C-activity in glycogen (DPM/mg w.w.), lower panel, in the diaphragm after i.p. injection of 2 pCi D-glucose-14C ( U ) together with 0, 0.1, 1.0 and 10.0 mU of insulin. The open columns give the activity of the control groups ( n = 8) , while the hatched columns represent VMH lesioned animals ( n = 9 ) . The standard deviations of the means are given by the vertical bars. Fig. 3. Plasma glucose concentration imgl100 ml) , upper panel, and plasma urea concentra- tion (mg/100 ml), lower panel, after i.p. injection of 2 pCi D-glucose-14C ( U ) together with 0, 0.1, 1.0 and 10.0 mU of insulin. The open columns give the activity of the control groups ( n = 8), while the hatched columns represent VMH lesioned animals ( n = 9) . The standard deviations of the means are given by the vertical bars.

incorportaion than their controls also after stimulation with 0.1 and 1.0 mU of insulin. However, the difference was not significant following administration of 10.0 mU insulin. The figure also shows that only the highest dose of insulin (10.0 m u ) stimulated the 14C incorporation in the control groups, while 1.0 mU gave rise to a significantly increased incorporation ( p < 0.025) among the lesioned animals.

The glycogen concentration in diphragms from VMH lesioned and control rats (mg glycogen/g wet weight) is shown in Fig. 2 , upper panel. This demonstrates the stimulating effect of exogenous administered insulin on glycogen synthesis. The figure also shows increased levels of glycogen in the lesioned animals compared to the controls for all groups when exception is made for the groups given 10.0 mU of insulin. The differences are significant, p < 0.05. Only the highest insulin dose (10.0 m u ) gave rise to any significant increase of the glycogen concentration. The

INSULIN RESISTANCE IN HYPOTHALAMIC HYPERPHAGIA 76 1

glycogen levels exhibit a 4 fold increase from 1 to 4 mg glycogen/g wet weight in lesioned animals as well as controls when compared to the unstimulated levels.

The I4C incorporation from D-g lu~ose -~~C ( U ) into glycogen in diaphragm is shown in Fig. 2, lower panel. A stimulating effect ( p < 0.001) of insulin on the I4C incorporation is manifest both in lesioned and control animals after injection of 1.0 m u . Following administration of 10.0 mU of insulin a 10 fold increase in 14C activity is observed. The response seems to be of the same magnitude both in VMH lesioned and control animals.

The mean plasma glucose concentrations of the different groups are shown in Fig. 3, upper panel. The values are within the normal range for fasting animals, 70-100 mg/100 ml. There are no significant differences between the mean glucose levels for animals given 10.0 mU of insulin and the groups given saline. I t may be pointed out that the mean plasma glucose concentrations in the groups of lesioned animals seem to be somewhat elevated compared to those of their controls. The dif- ference is significant for the groups given 0.1 and 1.0 mU insulin, p < 0.01.

The mean values for plasma urea concentration in the different groups are shown in the lower panel of Fig. 3. I t will be seen that the urea levels in the VMH lesioned are significantly higher than in their controls, p < 0.01.

Discussion

Increased lipogenesis is the most pronounced effect of the VMH lesion. In the present experiments this is clearly demonstrated by the increased I'C incorporation into total extracted parametrial lipids (Fig. 1 ) . Lipogensis is stimulated by exogenous insulin in both lesioned animals and their controls. However, only the highest in- sulin dose (10.0 m u ) had significant effect in the control group, whereas 1.0 mU caused an increased incorporation in the lesioned animals. The adipose tissue from these animals therefore semes to be more sensitive to the action of insulin than the controls. This is contrary to what is expected if decreased insulin sensitivity is the primary cause of the hyperinsulinemia. The glycogen concentration and the 14C incorporation into glycogen in diaphragm is increased in the lesioned animals. However, the glycogen synthesis is stimulated to the same degree in lesioned and control animals (Fig. 2 ) .

These findings imply that the co-existence of hyperinsulinemia and normo- glycemia demonstrated in these animals are not due to decreased insulin sensitivity. Our results thus support these presented by Frohman et al. (1972), and extend their conclusion so as to include adult VMH lesioned animals 2 days post-operatively. The conclusion is further supported by results from some recent experiments dealing with enzyme activities in muscle and liver after VMH surgery (Adolfsson et al., sub- mitted for publication). .

The study was carried out to test whether a decrease in the activities of enzymes of importance for glucose utilization were included among the early metabolic alteratinos following VMH destruction. No such decrease could be demonstrated

762 C. FAHLE HONGSLO, B. E. HUSTVEDT AND A. L 0 V 0

3 days post-operatively. This is in contrast to the finding of decreased activity of glycolytic enzymes in muscle tissue from the genetic obese-hyperglycemic mouse (obob), which is known to exhibit pronounced insulin resistance (Bray and York 1971, Adolfsson et al., submitted for publication).

We have recently pointed out that the early onset of hyperinsulinemia post- operatively indicates that VMH destruction may cause increased insulin secretion by a direct nervous or humoral action on the endocrine pancreas (Hustvedt and L0v0 1972). I t has been reported that basal gastric secretion is elevated in VMH lesioned animals (Ridley and Brooks 1965). Therefore, the possibility of a disturbance in the nervous control of gastro-intestinal functions post-operatively merits further discusion. This could modify insulin secretion directly by an altered activity in pancreatic nervous fibres, and indirectly by altering the output of gastro-intestinal hormones. The nervous participation in insulin secretion, as well as the insulinotropic effect of gastro-intestinal hormones, are well documented (Porte et al. 1973, Dupr6 1970).

I t seems difficult, however, to account for the normal blood glucose levels during fasting in presence of hyperinsulinemia and normal insulin sensitivity in terms of elevated insulin secretion only. In our opinion, the most likely reason for this find- ing is a greater output of glucose from the liver. The data presented give no direct evidence for changes in liver metabolism, but the elevated levels of plasma urea indicate increased gluconeogenesis. This is further supported by the increased amino acid catabolism of lesioned animals demonstrated by nitrogen balance studies (Holm et aE. in press).

These results raise the question of whether the secretion of glucagon, as well as that of insulin, may be altered in VMH lesioned animals. Increased release of glucagon would promote gluconeogenesis and accelerate the glucose release from glycogen. Possibly this could produce, in concert with the lipogenic effect of elevated insulin secretion, the accumulation of body fat at the expense of protein which characterizes the VMH syndrome. Current evidence suggests that stimuli affecting insulin release, as nervous stimulation and gastrointestinal hormones, also influence pancreatic glucagon secretion (Porte et al. 1972, Iversen 1971 ) .

However, there also remains the possibility that VMH destruction could produce changes in liver metabolism by alterations of the activity in hepatic nervous fibres. Recent studies have shown that enzymes implicated in hepatic glycogen and amino acid metabolism are under the influence of the autonomic nervous system (Shimazu and Fujimoto 1971, Black and Axelrod 1971).

In summary, we suggest that the profound metabolic effects of VMH destruction is caused by a disturbance in the nervous control of gastro-intestinal functions. This may lead to alterations in the endocrine pancreatic secretion, and possibly also in liver metabolism.

This study was supported by a research grant from Nordisk Insulinfond.

INSULIN RESISTANCE I N HYPOTHALAMIC HYPERPHAGIA

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