Electrical stimulation and lesions of the hypothalamus in alloxan diabetic rabbits

BRAIN RESEARCH

355

ELECTRICAL STIMULATION AND LESIONS OF THE HYPOTHALAMUS IN ALLOXAN DIABETIC RABBITS

NORIO KOKKA AND ROBERT GEORGE

Department of Pharmacology, The Center for the Health Sciences and Brain Research Institute, University of California, Los Angeles, Calif. (U.S.A.) (Received December 10th, 1965)

INTRODUCTION

It is well-known that injury or stimulation of the caudolateral part of the hypothalamus of normal animals may result in hyperglycemia1,2,23. This effect is presumed to be due ~0 an activation of the sympathetico-adrenal system since hypothalamic stimulatior, also increases epinephrine secretione,14,21,24. Conversely, lesions in the rostral areas of the hypothalamus produce hypoglycemia and hypersensitivity to insulin3,7,22,2s, an effect also prominently observed following hypophysectomy9. Thus far, studies are lacking in which the effect of hypothalamic stimulation on blood glucose or electrolytic lesions on the blood glucose response to insulin have been examined in diabetic animals. It seemed of interest to determine whether electrical stimulation of the hypothalamus could exacerbate the hyperglycemia and whether electrolytic lesions could increase sensitivity to insulin in alloxan diabetic rabbits. METHODS

Adult male albino rabbits, 2.5-4.0 kg body weight, were used. The animals were maintained at a constant room temperature of 21-22 ° under standardized conditions of lighting, fed a standard diet of Purina rabbit pellets # 5320, and allowed to drink tap water, ad lib. Diabetes mellitus was produced by intravenous administration of alloxan (100-150 mg/kg). Animals were screened for diabetes by examination of blood glucose levels 7-10 days following alloxan administration and were examined again after 2-3 weeks for persistence of diabetes. Electrical stimulation of the hypothalamus was performed in unanesthetized diabetic rabbits through bipolar electrodes implanted 10-14 days prior to the experiments. The electrodes consisted of stainless steel insect pins (Adams No. E-80 size) insulated to within 0.5-1.0 mm of the tips with varnish (Epoxylite insulator 6001-M) and fixed with their tips 1.0 mm apart; 2 pairs were implanted in most of the animals, Brain Research, 1 (1966) 355-362

356

N. KOKKA AND R. GEORGE

within 0.5 mm of the midline. Placement of stimulation and lesion electrodes was done stereotaxically, using the coordinates of Sawyer et al. 2e, under sodium pentobarbital (i.v. 35 mg/kg) and ethyl ether, via intubation, anesthesia. Stimulation was effected by means of a square-wave stimulator (Grass, Model S4-D); the parameters of stimulation were 50 c/s, 1.0-2.5 V, i msec duration and a peak current of 0.4 mA. A timer was connected to the stimulator to allow the stimulus to be applied during alternative quarter minutes for a period of 4 h. Electrolytic lesions were achieved by passing a constant current of 2 mA for 20 sec through a unipolar nichrome wire electrode insulated to within i.0 mm of the tip; the lesions were anodal. Two weeks were allowed for postoperative recovery before the animals were tested for a hypersensitivity to insulin. At the end of the experiments the animals were killed with sodium pentobarbital and the heads were perfused with 10O ml of 10 % formalin-saline through each carotid artery. The heads were then placed in 10 % formalin-saline for 5-7 days. After fixation the electrodes were removed first and then followed by removal of the brain. Electrode placements and lesions were identified by cutting frozen sections (80/~ in thickness) and staining according to the Nissl technique. Adrenal meduUectomy was done according to the technique of Evans for rats 12. All animals were injected intramuscularly with cortisone ac.~tate (5 mg/kg) and antibiotics for the first 3 postoperative days. The regenerated adrenals were examined microscopically for residual medullary tissue after serial sectioning and staining with hematoxylin and eosin. Blood samples (0.15-0.2 ml) were withdrawn from the marginal vein of an ear with heparinized syringes and analyzed for glucose by the anthrone method of Loewus z0. Prior to the experiments rabbits were lasted 18-20 h and two control blood samples were taken and analyzed for glucose. Crystalline zinc insulin (Squibb), diluted in physiological saline, was injected intravenously via a marginal ear vein. RESULTS

Effects of electrical stimulation Two pairs of electrodes were usually implanted in each alloxan diabetic rabbit with one pair placed in a rostral and the other in a more caudal region of the hypothalamus. Each of the rostral positions was stimulated first and those animals which showed an increase of 15 % or more in blood glucose within a 4-h stimulation peried were selected as having responded; this was based upon results observed in normal rabbits (unpublished observations) in which an increase of 15 % or more was found to be a significant rise (p value of < 0.01). The average blood glucose levels before and after stimulation of 9 animals can be seen in Table 1 (anterior hypGthalamic) where they are compared with the results observed after caudal stimulation in 8 of the same rabbits; the stimulation points are shown in Fig. 1. In these 8 animals a mean increase of 33 % was observed after stimulation of the anterior hypothalamus. Stimulation of the caudal sites did not produce a significant change, the mean increase in Brah, ~esearch, i (1966) 355-362

357

HYPOTHALAMIC STIMULATION AND LESIONS IN DIABETICS TABLE I

THE EFFECT OF HYPOTHALAMIC STIMULATION ON THE BLOOD GLUCOSE OF ALLOXAN DIABETIC RABBITS

Number of ~uuas

Anterior hypothalamus Posterior hypothalamus Supramammiilary and medialmammillary Supraoptic and preoptic

Blood glucose (mg/ lO0 ml)

.3

Initial

4-hour stimulation

Change

9 8

331 =t=45* 326 -4- 19

430 4- 39 338 =1=30

99 =E 13 12 =l=8

9 6

315 4- 30 323 ~ 12

318 ± 31 334 -4- 18

3 =l= 10 11 ~= 11

* Standard error.

b l o o d g l u c o s e b e i n g 3 % in these r a b b i t s . W h e n s t i m u l a t i o n o f t h e r o s t r a l r e g i o n s w a s r e p e a t e d in 6 a n i m a l s , t h e b l o o d g l u c o s e rose significantly f r o m a n initial level o f 289 -t- 44 r a g / 1 0 0 m l t o 421 :t: 65 m g / 1 0 0 ml. T h e e l e c t r o d e p l a c e m e n t s f o r these 9

~4PC SC

S? AQ

PONS

mm i i

Fig. 1. Diagrammatic sagittal view of the hypothalamus with stimulation points. Open circles indicate increased hyperglycemia, triangles no response. Abbr.: AC = Anterior commissure; AQ = Aqueduct of Sy[ :'ius; CC = Corpus callosum; DM = N. medialis dorsalis; HPC = Hippocampus; IPN = N. interpedun,=ularis; M = Massa intermedia; MM = N. mammillaris medialis; OCH = Optic chiasma; P = Anterior pituitary; PC = Posterior commissure; PV = N. paraventricularis; SC = Superior collicul-s; SP = Septum pellucidum; VM = N. ventromedialis. Brain Res6arch, 1 (1966) 355r~362

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N. KOKKA AND R. GEORGE

rabbits were situated in the anterior hypothalamic area and rostral border of the ventromedial nucleus. Stimulation of the preoptic, supraoptic, posterior hypothalamic and mammillary areas in 9 rabbits failed to produce any significant elevations in the blood glucose. The results and sumutation ...... " ooints ~ can be seen in Table I and Fig. !, respectively. Effect o f lesions on insulin sensitivity

!n these experiments, the insulin responses of ailoxan diabetic rabbits were determined before and after placement of lesions in hypothalamic areas which produced a blood glucose increase after electrical stimulation. To evaluate the effects of hypothalamic lesions on the insulin sensitivity of alloxan diabetic rabbits, the insulin response of each lesion bearing rabbit was compared with the mean insulin response of the controls receiving 0.025 U/kg of insulin. The maximum decrease in the blood glucose which occurred at any time during the 150-min experimental pe:'od was used to determine the insulin response. For the controls, the maximum blood glucose change was -12 :t: 2 9/0 after 0.025 U/kg of insulin and the standard deviation of the maximum decrease was + 5.1%. A blood glucose fall which exceeded that of the controls by 3 standard deviations (p < 0.01) was used as the criterion for determining a hypersensitivity to insulin in the lesioned animals. As shown in Table II, injection of 0.025 U/kg of insulin did not produce a significant change in the blood glucose level of the control group. After 0.50 U/kg of insulin, the blood glucose decreased markedly and was below the preinjection level after 150 min. Of 11 alloxan diabetic rabbits with anterior hypothalamic lesions (Table If),

TABLE II THE EFFECT OF HYPOTHALAMIC LESIONS ON THE INSULIN RESPONSE OF ALLOXAN DIABETIC RABBITS

Number Insulin Initial of ( U/kg) blood rabbits glucose (me~ 100 ml) Non-~s~ns: A. B.

7 6

Lesions: C. Anterior hypothalamus 4 D. Anterior hypothalamus 7 E. Posterior hypothalamus 4

Blood glucose change ( % of initial) after insulin (rain) 30

60

90

120

150

0.025 0.50

222~55" - 9 4 - 3 -44-2 - 8 4 - 2 -94-2 -44-3 308+66 -29 4- 11 -35 4-11 -35 4-8 -34 4-6-31 4-3

0.025

257 4- 69 -15d=5

-27 4- 7

0.025

243 4- 60 -12 4-4

-9 4 - 4

-7i3

-4-+-5 --4±5

0.025

382 4- 32

"4-3

-5!4

-3 4-4

* Standard error. Brain Research, 1 (1966) 355-362

-I 4-2

-33 ~ 8 - 3 6 + 8 - 3 6 +

I0

04-5

359

HYPOTHALAMIC STIMULATION AND LESIONS IN DIABETICS

Fig. 2. Photomicrograph of a coronal section from the brain of an insulin hypersensitive diabetic rabbit with anterior hypothalamic lesions. Magnification, 12 ×.

4 showed exaggerated responses to an insulin dose of 0.025 U/kg; the responses were comparable to those observed in animals without lesions receiving 20 times this dose (0.5 U/kg). The lesion site of one of the sensitive animals can be seen in Fig. 2. The remaining 7 rabbits with anterior lesions and 4 rabbits with lesions in the posterior hypothalamus showed the same responses to 0.025 U/kg of insulin as the controls.

Effect of insulin on adrenal meduilectomized rabbits Since anterior hypothalamic lesions produced a marked increase in the blood glucose lowering effect of insulin in the aUoxan diabetic rabbit, it seemed necessary to determine whether this effect was due to an alteration of sympathetico-adrenal activity. Rabbits were adrenal medullectomized and allowed to recover for 4-5 weeks; adrenal cortical regeneration was nearly maximal at the end of this period. Demedullated normal rabbits were injected with two test doses of insulin (0.10 and 0.50 U/kg i.v.) and their blood glucose levels were measured and compared over a 3.5-4 h period. It can be seen in Table Ill that adrenal medullectomy did not increase the normal response to either dose of insulin. DISCUSSION

The data indicate that the anterior hypothalamic area may influence the blood ~rciil!

I l~, l n ~,,~.j II~ 355-362 .RPsPa,a'C,~,, . . .

360

N. KOKKA AND R. GEORGE

sugar of alloxan diabetic rabbits. Electrical stimulation of this area exacerbates the diabetes by further elevation of the blood glucose whilst lesions, in 4 of 11 animals, produce a hypersensitivity to insulin. Hyperglycemia has also been seen in cats following hypothalamic stimulation, however, the effective areas were found to be in the lateral and posterior hypothalamic areasl,2a; this discrepancy may be due to a species difference. Although the mechanism(s) by which the hypothalamus influences carbohydrate metabolism has not been clearly elucidated, two major possibilities exist. One involves the activity of the sympathetico-adrenal system and the other the activity of the hypothalamo-hypophysial system. It is well-known that epinephrine produces hyperglycemia by its glycogenolytic action and that posterior hypothalamic stimulation in cats results in increased release of epinephrine from the adrenal medullae, ~4. Thus it is generally assumed that hypothalamic-induced hyperglycemia is mediated via activation of the sympathetico-adrenal system. The other possibility concerns the regulatory role of the hypothalamus on the rates of secretion of anterior pituitary 'diabetogenic' hormones (adrenocorticotropin and somatotropin). Hypothalamie control of adrenocorticotropin secretion has been well documentedte, x7 and recent TABLE llI INSULIN RESPONSES OF NORMAL AND ADRENAL DEMEDULLATED RABBITS

Insulin (U/kg)

Number of rabbits

Initial blood glucose (me~ 100 ml)

30

0 0.025

5 4

0.10 0.50

5 3

81 83 80 82

78 80 63 56

Blood ghtcose (mg/lO0 ml) after htsulin (min) 60

90

120

150

A. Normals:

B. Adrenal demedullated: 0

0.10 0.50

6

5 4

4444-

4* 6 4 2

85 4-3 86 4-3 82 4-7

4444-

5 4 2 7

83 .-!-4 64 4-5 75 4- 10

82 79 72 58

4- 6 d= 3 q- 3 4- 8

81 4-5 74 4-6 54 4-5

80 80 77 64

4444-

5 4 2 5

83 4-4 75 4-6 56.4=_8

81 78 76 69

4444-

4 2 2 5

88 4-6 75 4-5 64 4-11

82 80 78 77

4- 5 -4- 2 4- 3 4- 7

90 4-6 76 4-4 72 4- 11

* Standard error. reports have indicated that the hypothalamus similarly exerts an effect on somatotropin secretion as evidenced by the reduction of growth rates in young rats4,1s, 25, kittens 22 and puppies xl after placement of lesions and after pituitary transplantation in rats 19. More recently, the presence of a growth promoting factor in hypothalamic extracts has been reported~0,xs,27. Our results favor the latter view since adrenal medullectomy did not alter Brain Research, 1 (1966) 355-362

HYPOTHALAMIC STIMULATION AND LESIONS IN DIABETICS

361

sensitivity to insulin whilst anterior hypothalamic lesions produced a marked increase in response to insulin in 4 of 11 diabetic rabbits. It would appear that the increased hyperglycemia seen in alloxan diabetic rabbits is due to an accelerated release of one or more pituitary 'diabetogenic' hormones while the converse effect may occur after hypothalamic lesions. Further evidence to support this possibility comes from the following reports: (1) pituitary stalk section in the rabbit produces hypersensitivity to insulin which is comparable to that seen after hypophysectomy~L Similarly, stalk section in patients with diabetic retinopathy reduced their insulin requirement by at least 50 % (ref. 13); (2) hypothalamic lesions ameliorate or prevent diabetes of pancreatectomy in cats s, although this has not been confirmed in partially depancreatized diabetic rats 5. However, such lesions have been observed to ameliorate aUoxan diabetes in rats (unpublished data); (3) hypothalamic lesions increase insulin sensitivity of normal rats ~s and adrenal medullectomized rats (unpublished data); (4) chronic electrical stimulation of the hypothalamus produced permanent hyperglycemia in partially depancreatized normoglycemic catsZ3; (5) electrical stimulation of the anterior hypothalamus produced hyperglycemia in adrenal medullectomized rabbits (unpublished data). .

.

ACKNOWLEDGEMENTS

This work was supported by USPHS Grant NB-04499. The authors express their thanks to Joanne Finch and Larry Bidwell for technical assistance. SUMMARY

The effects of hypothalamic stimulation and lesions were studied in alloxan diabetic rabbits. Electrical stimulation of the anterior hypothalamic area increased the severity of diabetes, by further elevation of blood glucose, whereas electrolytic lesions in this area in some animals, produced a marked increase in the response to insulin. Stimulation of the preoptic, supraoptic, posterior and mammillary areas of the hypothalamus did not alter blood glucose levels. The response to insulin in diabetics with posterior hypothalamic lesions was not different from diabetics without lesions. Insulin sensitivity was compared in normal and adrenal demedullated rabbits and no difference was noted. The results indicate that the anterior hypothalamus in the rabbit plays a part in the regulation of blood glucose which is independent of the sympathetico-adrenal system; it more likely involves an alteration of one or more anterior pituitary secretions of 'diabetogenic' hormones. REFERENCES 1 ANAND, B. K., AND DUA, S., Blood sugar changes induced by electrical stimulation of the hypothalamus in cat. Indian J. reed. Res., 43 (1955) 123-127. 2 ANDERSON,E., McK. RIOCH,D., AND HAYMAKER,W., Disturbances in blood sugar regulation in animals subjected to transection of the brain stem. Acta neuroveg. (Wien), 5 (1952) 132-164. Brain Research, 1 (1966) 355-362

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3 BARRIS,R. W., AND INGRAM,W. R., The effect of experimental hypothalamic lesions upon blood sugar. Amer. J. PhyMol., 114 (1935) 555-561. 4 BERNARDIS,L. L., Box, B. M., AND STEVENSON,J. A. F., Growth following hypothalamic lesions in the weanling rat. Endocrinology, 72 (1963) 684-692. 5 BROBECK,J. R., TEPPERMAN,J., AND LONG, C. N. H., The effect of experimental obesity upon carbohydrate metabolism. Yale J. Biol. Med., 15 (1943) 893-904. 6 BRi.~CKE,F. V., KAINDL, F.~ AND MAYER, H., I~Iber die Veriinderung in der Zusammensetzung des Nebennierenmarkinkretes bei elektrischer Reizung des Hypothalamus. Arch. int. Pharmaco@n, 88 (1952)407-412. 7 D'AMOUR, M. C., AND KELLEn, A. D., Blood sugar studies following hypophysectomy and experimental lesions of hypothalamus. Proc. Soc. exp. Biol. (N. Y.), 30 (1933) 1175-1177. 8 DAVIS, L., CLEVELAND,O., AND INGRAM,W. R., Carbohydi'ate metabolism. The effect of hypothalamic lesions and stimulation of the autonomic nervous system. Arch. Neurol. Psychiat. (Chic.), 33 (1935) 592-613. 9 DE BODO,R. C., ANDALTZt;LER, N., Insulin hypersensitivity and physiological insulin antagonists. Physiol. Rev., 38 (1958) 389--445. 10 DEUBEN, R., AND MEITES,J., In vitro reinitiation of pituitary somatotropin release by an acid extract of hypothalamus. Proc. Soc. exp. Biol. (N. Y.), ! 18 (1965) 409-412. 11 ENDR(3CZI,E., KOVACS,S., AND SZALAY,GY., Einfluss von Hypothalamusl/isionen auf die Entwicklung des K6rpers und verschiedener Organe bei neugeborenen Tieren. Endokrinologie, 34 (1957) 168-175. 12 EVANS, G., The adrenal cortex and endogenous carbohydrate formation. Amer. J. Physiol., 114 (1936) 297-308. 13 FIELD,R. A., SCHEPENS,C. L., SWEET,W. H., AND APPELS,A., Effect of hypophyseal stalk section in advancing diabetic retinopathy. Diabetes, 11 (1962) 465-469. 14 FOLKOW,B., ANDV. EULER, U. S., Selective activation of noradrenaline and adrenaline producing cells in the cat's adrenal gland by hypothalamic stimulation. Circulation Res., 2 (1954) 191-195. ! 5 FRANZ, J., HASELBACH,C. H., AND LIBERT, O., Studies of the effect of hypothalamic extracts on somatotrophic pituitary function. Acta endocrinol., 41 (1962) 336-350. 16 GANONG,W. F., The central nervous system and the synthesis and release of adrenocorticotropic hormone. In A. V. NALBANDOV(Ed.), Advances in Neuroendocrinology, University of Illinois Press, Urbana, 1963, pp. 92-149. 17 HARRIS,G. W., Neural Controlofthe Pituitary Gland. Edward Arnold, London, 1955, pp. 103-131. 18 I-hNTON,G. G., AND STEVENSON,J. A. F., The effect of hypothalamic lesions on growth. Canad. J. Biochem., 40 (1962) 1239-1246. 19 HJALMARSON,A., AND AHREN, K., Studies on growth hormone secretion in rats with the hypophysis autotransplanted to the kidney capsule. Acta endocrinol., 49 (1965) 17-27. 20 LOEWUS, F. A., Improvement in anthrone method for determination of carbohydrates. Analyt. Chem., 24 (1952) 219. 21 MAGOUN,H. W., RANSON,S. W., ANDHETHERINGTON,A., The liberation of adrenin and sympathin induced by stimulation of the hypotha!amus. Amer. J. Physiol., 119 (1937) 615-622. 22 O'BRIEN, C. P., HAPPEL, L., AND BACH, L. M. N., Some hypothalamic effects on STH-influenced growth and insulin sensitivity in kittens. Fed_.Proc., 23 (1964) 205. 23 PATON,A., The hypothalamus and carbohydrate regulation. J. Endocrinol., 15 (1957) 33-39. 24 REDGATE,E. S., AND GELLHORN,E., Nature of sympathetico-adrenal discharge under conditions .of excitation of central autonomic structures. Amer. J. Physiol., 174 (1953) 475-480. 25 REICnL~, S., Growth and the hypothalamus. Endocrinology, 67 (1960) 760-773. 26 SAWYER,C. H., EVERETT, J. W., AND GREEn, J. D., The rabbit diencephalon in stereotaxic coordinates. J. comp. Neurol., 101 (1954)801-824. 27 SCHALLY,A. V., STEELMAN,S. L.,'A~D BOWERS,C. Y., Effect of hypothalamic extracts on release of growth hormone in vitro. Proc. Soc. exp. Biol. (N. Y.), ! 19 (1965) 208-212. 28 SPIRTOS,B. N., AND~HALMI,N. S., Increased insulin sensitivity in rats with hypothalamic lesions. Endocrinology, 65 (1959) 669-678. 29 WESTMAN, A., AND JACOnSOnN, D., Endokrinologische Untersuchungen an Kaninchen mit durchtrenntem Hypophysenstiel. Acta obstet, gynec, scand., 20 (1943) 392-433.

Braht Research, 1 (1966) 355-362