- Email: [email protected]
Resistance of obese and non-obese, spontaneously hypertensive rats to alloxan-induced diabetes
Life Sciences, Vol. 33, pp. 1097-1103 Printed in the U.S.A.
Pergamon Press
RESISTANCE OF OBESE AND NON-OBESE, SPONTANEOUSLY HYPERTENSIVE RATS TO ALLOXAN-INDUCED DIABETES Bernard C. Uexler and John P. Mc Murtry May I n s t i t u t e for ~ledical Research of Jewish Hospital and Departments of Hedicine and Pathology, University of Cincinnati College of Hedicine, Cincinnati, Ohio 45229 (Received in final form July 6, 1983)
SUMMARY Male, 5 month old, massively obese, spontaneously hypertensive rats (Obese/SHR) were given I0 mg alloxan/lO0 g b.w., s . c . , to induce diabetes. Control Obese and non-obese/SHR were given saline. Insulin therapy was withheld. All of the animals were k i l l e d at 6 months of age. Alloxan caused a s l i g h t but s t a t i s t i c a l l y s i g n i f i c a n t increase in blood pressure, p i t u i t a r y and adrenal glandular hyperplasia, hyperlipidemia, hyperglycemia, and increased BUN levels. The giant sized i s l e t s of Langerhans in Obese/SHR showed only partial degranulation of the insulin-producing beta cells concomitant with residual but apparently adequate blood insulin levels, whereas the i s l e t s of non-obese/SHR exhibited v i r t u a l l y total beta cell degranulation and only trace amounts of blood insulin. The alloxanized, nonobese rats were severely emaciated; the alloxanized Obese/SHR maintained t h e i r obesity. Alloxan-treated, Obese and non-obese/SHR manifested gross and microscopic degenerative changes suggesting acceleration of the normal aging process. The genetically-programmed pathogenesis of diabetes, obesity, hypertension, and Cushingoid pathophysiology of Obese/SHR may be due to hyperadrenocorticism. The authors have been investigating a unique variant of the spontaneously hypertensive rat (SHR) (I) which develops intense hyperphagia and massive obesity concomitant with pronressively rising hiqh blood pressure. By the time these animals become 7 months old, they are hyperinsulinemic, hyperglycemic, hyperlipidemic, hypertensive, and massively obese, i . e . , Obese/SHR (2-5). The parents carryinq the qene(s) for the production of these Obese/SHR are lean. Only 25% of the offsprinq born to these parents become obese; 75% of the offsprinn are hypertensive but lean, i . e . , non-obese/SHR. Despite t h e i r outward appearance of normalcy, the non-obese/SHR also manifest hyperinsulinemia, hyperplastic i s l e t s of Langerhans, hyperglycemia, hyperlipidemia, f a t t y l i v e r , and copious retroperitoneal and periadrenal adipose tissue. All of these conditions are maximally developed in t h e i r Obese/SHR siblings. In addition, the Obese/SHR are hyperadrenocorticoid and manifest many of the degenerative changes seen in Cushingoid patients, e.g., hyperglycemia, adiposi t y , hypertension, thin skin, kidney stones, accelerated aging, etc. (2-5). Repeatedly bred, male and female rats also develop obesity, hyperglycemia, accelerated cardiovascular disease, and other Cushing's disease-like changes (6-8). When repeatedly bred rats are subjected to alloxan diabetes, they manifest dichotomous changes ranging from severe emaciation to obesity concomitant with a spectrum of vascular disease ranging from micro- to macroarteriosclerosis 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
1098
Alloxan Diabetes in Obese/SHR
Vol. 33, No. ii, 1983
(9-11). When young and old, normotensive Sprague-Dawley and hypertensive SHR are subjected to alloxan diabetes, they also manifest similar dichotomous changes (12,13). Therefore, we challenged Obese and non-obese/SHR with alloxan to determine how these animals with p r e - e x i s t i n g hyperglycemia, obesity and hypertension would respond. Methods The original breeder stock from which our colony of Obese/SHR were derived are called, "Corpulent-SHR" and were provided by Dr. Carl T. Hansen, N.I.H. At 5 weeks of age, the animals g e n e t i c a l l y programmed to become obese develop hyperphaqia and at 7 months of age they weigh 700 ± 30 g; non-obese/SHR siblings of equal age weigh 350 i I0 g. In this experiment, 5 month old, male, Obese/SHR and non-obese/SHR were starved for 24 hr and 80bese/SHR and 12 non-obese/SHR were given a sinqle sc i n j e c t i o n of I0 mq alloxan/lO0 g body weight; 7 Obese/ SHR and 12 non-obese/SHR were given an equal volume of saline ( d i l u e n t for alloxan). (A dose of alloxan above I0 mq was lethal in old Obese/SHR.) Insulin therapy was withheld and the animals were observed for 4 weeks. The animals were fed a regular commercial rat chow d i e t (Purina) which is r e l a t i v e l y low in f a t (4%) and tap water to drink ad libitum. The animals were housed in our a i r conditioned, humidity and light-contro~FTed-Animal Research Colony. Daily urinary glucose, ketones, and protein were estimated by Urostix (Ames). Weekly and f i n a l blood pressure (72 hrs p r i o r to autopsy) were measured using an i n d i r e c t t a i l cuff and microphonic manometer under l i g h t Seconal anesthesia. The animals were decapitated, blood samples were spun in a r e f r i g e r a t e d c e n t r i fuge and assayed for free f a t t y acids, t r i g l y c e r i d e s , total c h o l e s t e r o l , glucose, and blood urea nitrogen (BUN) using the automated techniques prescribed for the Auto Analyzer (Technicon Instruments). Plasma samples were assayed in duplicate at 2 d i l u t i o n s for i n s u l i n using a double-antibody procedure described by Morishige (14). The hearts and aortae of each animal were examined at autopsy for gross evidence of vascular disease. Pertinent organs from each rat were trimmed and weighed. Organs and tissues were fixed in formalin, embedded in p a r a f f i n , and sectioned at 3~m. Adjacent sections were stained with hematoxylin and eosin, Gomori's aldehyde fuchsin Ponceau for pancreatic beta c e l l s , the Hale stain for glycosaminoglycans, and the von Kossa stain for calcium. Sudan black B (frozen sections) were used to stain l i p i d s . S t a t i s t i c a l analysis of the results was performed using a one-way analysis of variance, chi-square t e s t , or Student's t test. Results General observations. None of the Obese/SHR or non-obese/SHR died during the 4 ~ course of this experiment. The progressively increasing adiposity chara c t e r i s t i c of Obese/SHR reached a plateau post-alloxan despite persistent hyperphagia. The non-obese/SHR manifested marked polydypsia and polyuria. Daily t e s t of the urine showed severe glucosuria i n i t i a l l y but only mild glucosuria and l i t t l e or no ketonuria or proteinuria in alloxanized animals during the last three weeks of the experiment. Blood ~ . Prior to the i n j e c t i o n of alloxan, the blood pressure of the ~/SHR was 160 * 2 mmHg vs 180 ± 2 mmHg for the non-obese/SHR but i t slowly increased in the alloxan-treated Obese and non-obese/SHR (Table I ) . Although the rise in blood pressure in the alloxan-treated animals was not of great magnitude, i t was s i g n i f i c a n t l y higher than the non-treated animals (P< 0.001) (Table I ) .
Vol. 33, No. ii, 1983
Alloxan Diabetes in Obese/SHR
1099
TABLE I Comparison of Blood Pressure, Organ and Body Weight of Obese Vs NonObese/SHR With and Without Alloxan-lnduced (4 Weeks) Diabetes Final b.w. 9
Pit
Ob + Allox 560*25
Heart
Kidney
Testis
mcj
Ob No Rx 575':1:28 9.5±0.7
(7)
Thymus Adrenal
540±64
B.P. mmH~
25.6±0.8
1546±21 1277.26
1424.28
158.2
12.3±1.0 b 483.21
24.8.0.3
1846.98 a 1584±66a 1673±37a 171±2a
II.4.0.7
186.13
20,1±0.6
1439±29 II06.17
13.9.1.1
123±II a 23.8.0.5 a II18±35 a 1627.52 a 1604~39 192.2 a
(8) non-ob 365,13 No Rx(12) non-ob + 281.8 a Allox (12)
Results are means * SE; a P~ 0.001. b p< 0.05
1644.26
182,2
(n) = number of samples.
Gravimetric data. Alloxan-treated Obese/SHR weighed only 15 gms less than t h e i r non-treated brothers (Table I ) , whereas alloxan-treated non-obese/SHR l o s t a considerable amount of body weight (Table I ) . C h a r a c t e r i s t i c a l l y , the p i t u i t a r y glands of the non-obese/SHR were heavier (P~O.O01) than t h e i r obese siblings. Alloxan treatment was associated with increased p i t u i t a r y gland weight in both Obese and non-obese/SHR (Table I ) . The thymi of Obese/SHR were massive in c o n t r a d i s t i n c t i o n to the involuted thymi found in non-obese/SHR (Table I ) . The adrenal glands of Obese/SHR were heavier than those of non-obese /SHR, t h e i r heart and kidney weights were considerably heavier than t h e i r nonobese but more hypertensive siblings (Table I ) . ~lyocardial and renal hypertrophy occurred in a l l cases except in non-obese/SHR in which hearts weighed s i g n i f i c a n t l y less than t h e i r controls (Table I ) . The testes of Obese/SHR (nontreated) weighed less than t h e i r alloxanized Obese and non-obese brothers. The gravimetric relationships described above obtained whether the organ weights were expressed on an absolute basis or as an organ weight:body weight X I00 ratio. Biochemistry. L i p i d s . Non-obese/SHR were hyperlipidemic, had a f a t t y l i v e r , and had extra perl'adrenal and retroperitoneal adipose tissue. All of these conditions were g r e a t l y exacerbated in the Obese/SHR. The i n j e c t i o n of alloxan caused a f u r t h e r increase in free f a t t y acids, t r i g l y c e r i d e s , and total cholesterol in both the Obese and non-obese/SHR (Table I I ) . The overall greatest increase in l i p i d levels post-alloxan occurred in non-obese/SHR. Glucose. Treatment with alloxan evoked a f u r t h e r increase in blood glucose l e v e l s , p a r t i c u l a r l y in the non-obese/SHR but the hyperglycemia was not severe. (Table I I ) . Blood urea nitrogen. The i n t e n s i f i c a t i o n of the p r e - e x i s t e n t d i a b e t e s - l i k e con-'o-n~Ttlo~'6-n--b~n caused a marked increase in B.U.N. levels (Table I I ) . I n s u l i n . [lost rodent models of obesity, including the Obese/SHR, are hyper~emic. Although the single i n j e c t i o n of alloxan caused a s t r i k i n g reduction in c i r c u l a t i n g i n s u l i n l e v e l s , there was some residual c i r c u l a t i n g i n s u l i n in Obese/SHR 4 weeks post-alloxan and only trace amounts in the nonobese/SHR (Fig. I ) .
ii00
Alloxan Diabetes in Obese/SHR
Vol. 33, No. ii, 1983
TABLE I I Comparison of L i p i d s , . G l u c o s e , and BLIN of Obese Vs Non-obese/SHR With and Without Alloxan-induced (4 Weeks) Diabetes Free f a t t y acid (meq/liter)
•
Ob No Rx (7)
396*47
Ob + A l l o x
456.31
non-ob
(8)
No Rx
(12)
non-ob + A l l o x (12)
Triqlyceride
Cholesterol
Glucose
BUN
mg% 610,16 II14,149 a
146,11
196.5
26,I
195,13 a
231±II a 47±2a
363*39
196,20
118,21
176,4
571,33 a
398,70 a
151,4 a
353±19 a 36,2 a
Results are means * SE; a p< 0.001.
25.I
(n) = number of samples.
100 ]=No
90
Rx
Obese [-~=AIIoxan 80
Rx
I ~ = N o RX N°rP'Obese~ ] = AIIoxan Rx
70
E 60
~
so
c
40
30
20
10
FIG. 1 Blood i n s u l i n l e v e l s of 6 month old male. Obese/SHR (n = 7) compared with Obese/SHR (n = 8) of the same age but given a l l o x a n ( i n s u l i n therapy withheld) when they were 5 months of age; male. non-obese s i b l i n g s of the Obese/SHR also k i l l e d at 6 months of age (12 were i n j e c t e d with s a l i n e and 12 were given a l l o x a n 4 weeks p r i o r to autopsy.) The height of the columns connote Mean * SE.
Vol. 33, No. ii, 1983
Alloxan Diabetes in Obese/SHR
ii01
Gross and microscopi c pathology. At autopsy, there were few discernible differences in the adiposity of the treated vs non-treated Obese/SHR. All of the Obese/SHR had excessive quantities of retroperitoneal and periadrenal adipose tissue and severe, f a t t y i n f i l t r a t i o n of the l i v e r . The alloxantreated Obese/SHR had l o s t considerable amounts of f u r , had greatly thinned skin, and v i r t u a l l y a l l of them displayed grossly v i s i b l e evidence of old and new myocardial necrosis. Alloxan-treated non-obese/SHE were emaciated, i . e . , total disappearance of a l l r e t r o p e r i t o n e a l , periadrenal, and mesenteric f a t . Both groups of non-obese/SHR displayed equally severe f a t t y l i v e r s . Like the alloxan-treated Obese/SHR, the alloxan-treated non-obese/SHR were allopoecic, t h e i r skin was abnormally t h i n , and they displayed an unusually high incidence (76%) of old and new confluent foci of myocardial necrosis and f i b r o s i s . Microscopic examination of the tissues of these animals confirmed the gross observations, e . g . , abnormally thin skin, hepatic l i p i d o s i s , severe myocardial damage, but no evidence of a r t e r i o - or a r t e r i o l o s c l e r o s i s in e i t h e r treated or non-treated animals. The p i t u i t a r y glands of both sub-strains of SHR manifested marked hyperplasia and basophilia. The alloxan-treated animals displayed frequent foci of c o l l o i d - c o n t a i n i n g cysts in the pars a n t e r i o r . The thymi of the Obese/SHR were massive and free of f a t t y i n f i l t r a t e . The zona glomerulosa of a l l of the Obese/SHR was t o t a l l y devoid of l i p i d , whereas this zone was replete with l i p i d in a l l of the non-obese/SHR. The giant sized i s l e t s of Langerhans in the alloxan-treated Obese/SHR were devoid of any pathology except that the beta c e l l s , a l b e i t considerably degranulated, reacted p o s i t i v e ly to the aldehyde fuchsin stain suggesting the presence of i n s u l i n . In contrast, the granules in the beta cells of the alloxan-treated non-obese/SHR had disappeared completely. The renal cortices of all of the animals were free of pathologic change except for the frequent presence of e o s i n o p h i l i c , proteinaceous c o l l o i d within the c o l l e c t i n g tubules, and a high, equal i n c i d ence of renal calculi in a l l groups. The testes were atrophic and aspermatic with d e f i n i t e exacerbation of this hypogonadotrophic condition in the alloxantreated animals. The morphologic composition of the microscopic lesions encountered in these 6 month old Obese and non-obese/SHR are identical to lesions in old SHR, the d e t a i l s of which have been published (2-5,13,15). Discussion These findings indicate that Obese/SHR and non-obese/SHR are comparatively r e s i s t a n t to the diabetogenic e f f e c t of alloxan. This was surprising since i n t a c t Obese/SHR c h a r a c t e r i s t i c a l l y manifest an unusually high m o r t a l i t y rate, are prone to succumb to r e l a t i v e l y innocuous stress (2-5), and because, in generaT, the pancreatic beta cells of obese rats are much more susceptible to diabetogenic agents than the beta c e l l s of lean rats (16). When a large dose of alloxan is given to most strains of r a t , about 50% of the animals succumb within the f i r s t 48-72 hrs of the induction of severe diabetes (9-12). The various sub-strains of SHR which we have injected with alloxan appear to be res i s t a n t to alloxan (12). We believe that the p r e - e x i s t e n t hyperglycemia and enlarged i s l e t s of Langerhans c h a r a c t e r i s t i c of SHR impart resistance to the beta c e l l - l y s i n g e f f e c t s of alloxan. Obese/SHR with giant-sized i s l e t s of Langerhans are the most r e s i s t a n t to alloxan. The mild glucosuria and v i r t u a l absence of ketonuria and proteinuria in Obese/SHR also a t t e s t to the resistance of Obese/SHR to the diabetogenic dose of alloxan. I t is of i n t e r e s t that the g e n e t i c a l l y programmed hyperphagia in the Obese/SHR persisted post-alloxan without weight gain. Hyperphagia is maintained by hyperinsulinemia and by elevated adrenal steroid levels (16,17). Since c i r c u l a t i n g i n s u l i n levels were low in these animals, i t is suggested that p e r s i s t e n t hyperphagia in the alloxan-treated Obese/SHR was due to t h e i r
1102
Alloxan Diabetes in Obese/SHR
Vol. 33, No. ii, 1983
c h a r a c t e r i s t i c hyperadrenocorticism and deranged hypothalamic function (2-5). The n e g l i g i b l e loss of body weight vs s i g n i f i c a n t loss of body weight and emaciation in alloxanized Obese vs non-obese/SHR attests to the greater r e s i s t ance of Obese/SHR to the diabetogenic action of alloxan. The emaciation and loss of body weight in the non-obese/SHR was metabolic and not due to aphagia since t h e i r food intake remained the same pre- and post-alloxan. The growing concensus is that lean rats lose more weight than t h e i r obese brothers because lean rats metabolize t h e i r adipose tissue under conditions of energy demand in order to preserve body protein, whereas obese rats u t i l i z e body protein while hoarding body f a t (18). I t is suggested that the n e g l i g i b l e loss of f a t and/ or body weight in the Obese/SHR vs s i g n i f i c a n t body weight loss in non-obese/ SHR was due to the greater residual levels of i n s u l i n in the Obese/SHR. The residual i n s u l i n levels may have been s u f f i c i e n t to i n h i b i t the release of glycerol and free f a t t y acids from the more copious adipose tissue mass of the Obese/SHR. The adipokinetic effectiveness of the residual amounts of i n s u l i n in the Obese/SHR was probably rendered more e f f e c t i v e since i t is well known that hepatic clearance of i n s u l i n is greatly impaired in obesity prolonging i t s systemic metabolism. The retarded breakdown of i n s u l i n reenforces the tendency toward hyperinsulinemia in obesity which enhances l i p i d storage, especially t r i g l y c e r i d e s , c h a r a c t e r i s t i c of obesity. The emaciation and higher glucose and l i p i d levels in the alloxan-treated non-obese/SHR attests to the fact that they did not have s u f f i c i e n t adipose tissue reserve and that t h e i r residual levels of i n s u l i n were too meager to be e f f e c t i v e metabolically. All of the alloxan-treated animals manifested increased blood pressure, increased incidence of myocardial necrosis, n e p h r o l i t h i a s i s , hypogonadism, hypothermia, alopoecia, thin skin, and poor wound healing. In humans and in animals, hyperadrenocorticism and diabetes are associated with abnormal connective tissue and ground substance metabolism, poor wound healing, and thinning of the skin. The skin of a l l o x a n - d i a b e t i c rats contains decreased quantities of glycosaminoglycans, e . g . , hyaluronic acid, dermatan s u l f a t e , e t c . , decreased collagen and e l a s t i n , all of which can be reversed by administration of i n s u l i n (19). The spectrum of degenerative changes induced by alloxandiabetes in these 5-6 month old Obese and non-obese/SHR are e s s e n t i a l l y i d e n t i cal to those lesions which appear spontaneously in old (2-3 yrs) SHR (15). This indicates that diabetes and hyperadrenocorticism in animals cause accelerated aging as observed in humans with advanced diabetes or Cushing's syndrome (20). Acknowledgements The authors are grateful for the dedication and technical expertise of D. Conatser, E. gominqo, G. Williamson, G. Heap and J. Wexler. The research was supported by grants from the National Heart, Lung and Blood Diseases I n s t i t u t e (HL-21418) and the National I n s t i t u t e on Aging (AG-585). References I. 2. 3. 4. 5. 6. 7. 8.
K. OKAMOTOand K. AOKI, Jap. Circ. J. 27 282-293 (1963). B.C. WEXLER, S.G. IAMS and J.P. MC MURI'ITY, Br. J. exp. Path. 61 195-207, (1980). B.C. WEXLER and J.P. MC MURTRY, Br. J. exp. Path. 62 146-157 (1981). B.C. WEXLER and J.P. MC MURTRY, Br. J. exp. Path. ~ 369-374 (1981). B.C. WEXLER and J.P. MC MURTRY, J. Gerontol 38 148--I'54 (1983). B.C. WEXLER, J. Atheroscler. Res. 4 57-80,(19--6~4). B.C. WEXLER, Acta endocr, Copenh. # 397-415 (1964). B.C. WEXLER and G.W. KITTINGER, J.-Atheroscler. Res. 5 317-329 (1965).
Vol. 33, No. ii, 1983
9. lO. If. 12. 13. 14. 15.
16. 17. 18. 19. 20.
Alloxan Diabetes in Obese/SHR
B.C. WEXLER, J. SAROFFand J.T. JUDD, Diabetes 19 311-323 (1970). B.C..WEXLER, Diabetes 19 324-336 (1970). m B.C. WEXLER, Atheroscle-r'osis 14 289-307 (1971). B.C. WEXLER, Exp. Gerontol. I~'-47-58 (1981). B.C. WEXLER, Age 3 93-99 (198--0-). W.K. MORISHIGE, C?A. UETAKE, F.C. GREENWOOD,J. AKAKA, Endocrinology lO0 1710-1718 (1977). ~. WEXLER, J.P. MC MURTRYand S.G. IAMS, J. Gerontol. 36 514-519 (1981). S. INOUE and G. BRAY, Horm. Metab. Res. I0 273-280 (1978). G.A. BRAY and D.A. YORK, Physiol. Rev. 5~'-719-809 (1979). M.N. GOODMAN,P.R. LARSEN, M.M. Y~APLAN,~.T. AOKI, V.R. YOUNGand N.B. RUDERMAN,Am. J. Physiol. 239 E277-E286 (1980). E.R. DALFRERESJr, B. P~ADHAKRISI~B~URTRY,M.S. CROUCH, J.S. BERENSON, Proc. Soc. exp. Biol. Med. 148 918-924 (1975). B.C. WEXLER, H~pothalamus Pi-{u-itary and Aging, A.V. Everitt and J.A. Burgess, edso, p. 333-361, Chas. C. Thomas, Springfield, I I I (1970).
1103
Pergamon Press
RESISTANCE OF OBESE AND NON-OBESE, SPONTANEOUSLY HYPERTENSIVE RATS TO ALLOXAN-INDUCED DIABETES Bernard C. Uexler and John P. Mc Murtry May I n s t i t u t e for ~ledical Research of Jewish Hospital and Departments of Hedicine and Pathology, University of Cincinnati College of Hedicine, Cincinnati, Ohio 45229 (Received in final form July 6, 1983)
SUMMARY Male, 5 month old, massively obese, spontaneously hypertensive rats (Obese/SHR) were given I0 mg alloxan/lO0 g b.w., s . c . , to induce diabetes. Control Obese and non-obese/SHR were given saline. Insulin therapy was withheld. All of the animals were k i l l e d at 6 months of age. Alloxan caused a s l i g h t but s t a t i s t i c a l l y s i g n i f i c a n t increase in blood pressure, p i t u i t a r y and adrenal glandular hyperplasia, hyperlipidemia, hyperglycemia, and increased BUN levels. The giant sized i s l e t s of Langerhans in Obese/SHR showed only partial degranulation of the insulin-producing beta cells concomitant with residual but apparently adequate blood insulin levels, whereas the i s l e t s of non-obese/SHR exhibited v i r t u a l l y total beta cell degranulation and only trace amounts of blood insulin. The alloxanized, nonobese rats were severely emaciated; the alloxanized Obese/SHR maintained t h e i r obesity. Alloxan-treated, Obese and non-obese/SHR manifested gross and microscopic degenerative changes suggesting acceleration of the normal aging process. The genetically-programmed pathogenesis of diabetes, obesity, hypertension, and Cushingoid pathophysiology of Obese/SHR may be due to hyperadrenocorticism. The authors have been investigating a unique variant of the spontaneously hypertensive rat (SHR) (I) which develops intense hyperphagia and massive obesity concomitant with pronressively rising hiqh blood pressure. By the time these animals become 7 months old, they are hyperinsulinemic, hyperglycemic, hyperlipidemic, hypertensive, and massively obese, i . e . , Obese/SHR (2-5). The parents carryinq the qene(s) for the production of these Obese/SHR are lean. Only 25% of the offsprinq born to these parents become obese; 75% of the offsprinn are hypertensive but lean, i . e . , non-obese/SHR. Despite t h e i r outward appearance of normalcy, the non-obese/SHR also manifest hyperinsulinemia, hyperplastic i s l e t s of Langerhans, hyperglycemia, hyperlipidemia, f a t t y l i v e r , and copious retroperitoneal and periadrenal adipose tissue. All of these conditions are maximally developed in t h e i r Obese/SHR siblings. In addition, the Obese/SHR are hyperadrenocorticoid and manifest many of the degenerative changes seen in Cushingoid patients, e.g., hyperglycemia, adiposi t y , hypertension, thin skin, kidney stones, accelerated aging, etc. (2-5). Repeatedly bred, male and female rats also develop obesity, hyperglycemia, accelerated cardiovascular disease, and other Cushing's disease-like changes (6-8). When repeatedly bred rats are subjected to alloxan diabetes, they manifest dichotomous changes ranging from severe emaciation to obesity concomitant with a spectrum of vascular disease ranging from micro- to macroarteriosclerosis 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
1098
Alloxan Diabetes in Obese/SHR
Vol. 33, No. ii, 1983
(9-11). When young and old, normotensive Sprague-Dawley and hypertensive SHR are subjected to alloxan diabetes, they also manifest similar dichotomous changes (12,13). Therefore, we challenged Obese and non-obese/SHR with alloxan to determine how these animals with p r e - e x i s t i n g hyperglycemia, obesity and hypertension would respond. Methods The original breeder stock from which our colony of Obese/SHR were derived are called, "Corpulent-SHR" and were provided by Dr. Carl T. Hansen, N.I.H. At 5 weeks of age, the animals g e n e t i c a l l y programmed to become obese develop hyperphaqia and at 7 months of age they weigh 700 ± 30 g; non-obese/SHR siblings of equal age weigh 350 i I0 g. In this experiment, 5 month old, male, Obese/SHR and non-obese/SHR were starved for 24 hr and 80bese/SHR and 12 non-obese/SHR were given a sinqle sc i n j e c t i o n of I0 mq alloxan/lO0 g body weight; 7 Obese/ SHR and 12 non-obese/SHR were given an equal volume of saline ( d i l u e n t for alloxan). (A dose of alloxan above I0 mq was lethal in old Obese/SHR.) Insulin therapy was withheld and the animals were observed for 4 weeks. The animals were fed a regular commercial rat chow d i e t (Purina) which is r e l a t i v e l y low in f a t (4%) and tap water to drink ad libitum. The animals were housed in our a i r conditioned, humidity and light-contro~FTed-Animal Research Colony. Daily urinary glucose, ketones, and protein were estimated by Urostix (Ames). Weekly and f i n a l blood pressure (72 hrs p r i o r to autopsy) were measured using an i n d i r e c t t a i l cuff and microphonic manometer under l i g h t Seconal anesthesia. The animals were decapitated, blood samples were spun in a r e f r i g e r a t e d c e n t r i fuge and assayed for free f a t t y acids, t r i g l y c e r i d e s , total c h o l e s t e r o l , glucose, and blood urea nitrogen (BUN) using the automated techniques prescribed for the Auto Analyzer (Technicon Instruments). Plasma samples were assayed in duplicate at 2 d i l u t i o n s for i n s u l i n using a double-antibody procedure described by Morishige (14). The hearts and aortae of each animal were examined at autopsy for gross evidence of vascular disease. Pertinent organs from each rat were trimmed and weighed. Organs and tissues were fixed in formalin, embedded in p a r a f f i n , and sectioned at 3~m. Adjacent sections were stained with hematoxylin and eosin, Gomori's aldehyde fuchsin Ponceau for pancreatic beta c e l l s , the Hale stain for glycosaminoglycans, and the von Kossa stain for calcium. Sudan black B (frozen sections) were used to stain l i p i d s . S t a t i s t i c a l analysis of the results was performed using a one-way analysis of variance, chi-square t e s t , or Student's t test. Results General observations. None of the Obese/SHR or non-obese/SHR died during the 4 ~ course of this experiment. The progressively increasing adiposity chara c t e r i s t i c of Obese/SHR reached a plateau post-alloxan despite persistent hyperphagia. The non-obese/SHR manifested marked polydypsia and polyuria. Daily t e s t of the urine showed severe glucosuria i n i t i a l l y but only mild glucosuria and l i t t l e or no ketonuria or proteinuria in alloxanized animals during the last three weeks of the experiment. Blood ~ . Prior to the i n j e c t i o n of alloxan, the blood pressure of the ~/SHR was 160 * 2 mmHg vs 180 ± 2 mmHg for the non-obese/SHR but i t slowly increased in the alloxan-treated Obese and non-obese/SHR (Table I ) . Although the rise in blood pressure in the alloxan-treated animals was not of great magnitude, i t was s i g n i f i c a n t l y higher than the non-treated animals (P< 0.001) (Table I ) .
Vol. 33, No. ii, 1983
Alloxan Diabetes in Obese/SHR
1099
TABLE I Comparison of Blood Pressure, Organ and Body Weight of Obese Vs NonObese/SHR With and Without Alloxan-lnduced (4 Weeks) Diabetes Final b.w. 9
Pit
Ob + Allox 560*25
Heart
Kidney
Testis
mcj
Ob No Rx 575':1:28 9.5±0.7
(7)
Thymus Adrenal
540±64
B.P. mmH~
25.6±0.8
1546±21 1277.26
1424.28
158.2
12.3±1.0 b 483.21
24.8.0.3
1846.98 a 1584±66a 1673±37a 171±2a
II.4.0.7
186.13
20,1±0.6
1439±29 II06.17
13.9.1.1
123±II a 23.8.0.5 a II18±35 a 1627.52 a 1604~39 192.2 a
(8) non-ob 365,13 No Rx(12) non-ob + 281.8 a Allox (12)
Results are means * SE; a P~ 0.001. b p< 0.05
1644.26
182,2
(n) = number of samples.
Gravimetric data. Alloxan-treated Obese/SHR weighed only 15 gms less than t h e i r non-treated brothers (Table I ) , whereas alloxan-treated non-obese/SHR l o s t a considerable amount of body weight (Table I ) . C h a r a c t e r i s t i c a l l y , the p i t u i t a r y glands of the non-obese/SHR were heavier (P~O.O01) than t h e i r obese siblings. Alloxan treatment was associated with increased p i t u i t a r y gland weight in both Obese and non-obese/SHR (Table I ) . The thymi of Obese/SHR were massive in c o n t r a d i s t i n c t i o n to the involuted thymi found in non-obese/SHR (Table I ) . The adrenal glands of Obese/SHR were heavier than those of non-obese /SHR, t h e i r heart and kidney weights were considerably heavier than t h e i r nonobese but more hypertensive siblings (Table I ) . ~lyocardial and renal hypertrophy occurred in a l l cases except in non-obese/SHR in which hearts weighed s i g n i f i c a n t l y less than t h e i r controls (Table I ) . The testes of Obese/SHR (nontreated) weighed less than t h e i r alloxanized Obese and non-obese brothers. The gravimetric relationships described above obtained whether the organ weights were expressed on an absolute basis or as an organ weight:body weight X I00 ratio. Biochemistry. L i p i d s . Non-obese/SHR were hyperlipidemic, had a f a t t y l i v e r , and had extra perl'adrenal and retroperitoneal adipose tissue. All of these conditions were g r e a t l y exacerbated in the Obese/SHR. The i n j e c t i o n of alloxan caused a f u r t h e r increase in free f a t t y acids, t r i g l y c e r i d e s , and total cholesterol in both the Obese and non-obese/SHR (Table I I ) . The overall greatest increase in l i p i d levels post-alloxan occurred in non-obese/SHR. Glucose. Treatment with alloxan evoked a f u r t h e r increase in blood glucose l e v e l s , p a r t i c u l a r l y in the non-obese/SHR but the hyperglycemia was not severe. (Table I I ) . Blood urea nitrogen. The i n t e n s i f i c a t i o n of the p r e - e x i s t e n t d i a b e t e s - l i k e con-'o-n~Ttlo~'6-n--b~n caused a marked increase in B.U.N. levels (Table I I ) . I n s u l i n . [lost rodent models of obesity, including the Obese/SHR, are hyper~emic. Although the single i n j e c t i o n of alloxan caused a s t r i k i n g reduction in c i r c u l a t i n g i n s u l i n l e v e l s , there was some residual c i r c u l a t i n g i n s u l i n in Obese/SHR 4 weeks post-alloxan and only trace amounts in the nonobese/SHR (Fig. I ) .
ii00
Alloxan Diabetes in Obese/SHR
Vol. 33, No. ii, 1983
TABLE I I Comparison of L i p i d s , . G l u c o s e , and BLIN of Obese Vs Non-obese/SHR With and Without Alloxan-induced (4 Weeks) Diabetes Free f a t t y acid (meq/liter)
•
Ob No Rx (7)
396*47
Ob + A l l o x
456.31
non-ob
(8)
No Rx
(12)
non-ob + A l l o x (12)
Triqlyceride
Cholesterol
Glucose
BUN
mg% 610,16 II14,149 a
146,11
196.5
26,I
195,13 a
231±II a 47±2a
363*39
196,20
118,21
176,4
571,33 a
398,70 a
151,4 a
353±19 a 36,2 a
Results are means * SE; a p< 0.001.
25.I
(n) = number of samples.
100 ]=No
90
Rx
Obese [-~=AIIoxan 80
Rx
I ~ = N o RX N°rP'Obese~ ] = AIIoxan Rx
70
E 60
~
so
c
40
30
20
10
FIG. 1 Blood i n s u l i n l e v e l s of 6 month old male. Obese/SHR (n = 7) compared with Obese/SHR (n = 8) of the same age but given a l l o x a n ( i n s u l i n therapy withheld) when they were 5 months of age; male. non-obese s i b l i n g s of the Obese/SHR also k i l l e d at 6 months of age (12 were i n j e c t e d with s a l i n e and 12 were given a l l o x a n 4 weeks p r i o r to autopsy.) The height of the columns connote Mean * SE.
Vol. 33, No. ii, 1983
Alloxan Diabetes in Obese/SHR
ii01
Gross and microscopi c pathology. At autopsy, there were few discernible differences in the adiposity of the treated vs non-treated Obese/SHR. All of the Obese/SHR had excessive quantities of retroperitoneal and periadrenal adipose tissue and severe, f a t t y i n f i l t r a t i o n of the l i v e r . The alloxantreated Obese/SHR had l o s t considerable amounts of f u r , had greatly thinned skin, and v i r t u a l l y a l l of them displayed grossly v i s i b l e evidence of old and new myocardial necrosis. Alloxan-treated non-obese/SHE were emaciated, i . e . , total disappearance of a l l r e t r o p e r i t o n e a l , periadrenal, and mesenteric f a t . Both groups of non-obese/SHR displayed equally severe f a t t y l i v e r s . Like the alloxan-treated Obese/SHR, the alloxan-treated non-obese/SHR were allopoecic, t h e i r skin was abnormally t h i n , and they displayed an unusually high incidence (76%) of old and new confluent foci of myocardial necrosis and f i b r o s i s . Microscopic examination of the tissues of these animals confirmed the gross observations, e . g . , abnormally thin skin, hepatic l i p i d o s i s , severe myocardial damage, but no evidence of a r t e r i o - or a r t e r i o l o s c l e r o s i s in e i t h e r treated or non-treated animals. The p i t u i t a r y glands of both sub-strains of SHR manifested marked hyperplasia and basophilia. The alloxan-treated animals displayed frequent foci of c o l l o i d - c o n t a i n i n g cysts in the pars a n t e r i o r . The thymi of the Obese/SHR were massive and free of f a t t y i n f i l t r a t e . The zona glomerulosa of a l l of the Obese/SHR was t o t a l l y devoid of l i p i d , whereas this zone was replete with l i p i d in a l l of the non-obese/SHR. The giant sized i s l e t s of Langerhans in the alloxan-treated Obese/SHR were devoid of any pathology except that the beta c e l l s , a l b e i t considerably degranulated, reacted p o s i t i v e ly to the aldehyde fuchsin stain suggesting the presence of i n s u l i n . In contrast, the granules in the beta cells of the alloxan-treated non-obese/SHR had disappeared completely. The renal cortices of all of the animals were free of pathologic change except for the frequent presence of e o s i n o p h i l i c , proteinaceous c o l l o i d within the c o l l e c t i n g tubules, and a high, equal i n c i d ence of renal calculi in a l l groups. The testes were atrophic and aspermatic with d e f i n i t e exacerbation of this hypogonadotrophic condition in the alloxantreated animals. The morphologic composition of the microscopic lesions encountered in these 6 month old Obese and non-obese/SHR are identical to lesions in old SHR, the d e t a i l s of which have been published (2-5,13,15). Discussion These findings indicate that Obese/SHR and non-obese/SHR are comparatively r e s i s t a n t to the diabetogenic e f f e c t of alloxan. This was surprising since i n t a c t Obese/SHR c h a r a c t e r i s t i c a l l y manifest an unusually high m o r t a l i t y rate, are prone to succumb to r e l a t i v e l y innocuous stress (2-5), and because, in generaT, the pancreatic beta cells of obese rats are much more susceptible to diabetogenic agents than the beta c e l l s of lean rats (16). When a large dose of alloxan is given to most strains of r a t , about 50% of the animals succumb within the f i r s t 48-72 hrs of the induction of severe diabetes (9-12). The various sub-strains of SHR which we have injected with alloxan appear to be res i s t a n t to alloxan (12). We believe that the p r e - e x i s t e n t hyperglycemia and enlarged i s l e t s of Langerhans c h a r a c t e r i s t i c of SHR impart resistance to the beta c e l l - l y s i n g e f f e c t s of alloxan. Obese/SHR with giant-sized i s l e t s of Langerhans are the most r e s i s t a n t to alloxan. The mild glucosuria and v i r t u a l absence of ketonuria and proteinuria in Obese/SHR also a t t e s t to the resistance of Obese/SHR to the diabetogenic dose of alloxan. I t is of i n t e r e s t that the g e n e t i c a l l y programmed hyperphagia in the Obese/SHR persisted post-alloxan without weight gain. Hyperphagia is maintained by hyperinsulinemia and by elevated adrenal steroid levels (16,17). Since c i r c u l a t i n g i n s u l i n levels were low in these animals, i t is suggested that p e r s i s t e n t hyperphagia in the alloxan-treated Obese/SHR was due to t h e i r
1102
Alloxan Diabetes in Obese/SHR
Vol. 33, No. ii, 1983
c h a r a c t e r i s t i c hyperadrenocorticism and deranged hypothalamic function (2-5). The n e g l i g i b l e loss of body weight vs s i g n i f i c a n t loss of body weight and emaciation in alloxanized Obese vs non-obese/SHR attests to the greater r e s i s t ance of Obese/SHR to the diabetogenic action of alloxan. The emaciation and loss of body weight in the non-obese/SHR was metabolic and not due to aphagia since t h e i r food intake remained the same pre- and post-alloxan. The growing concensus is that lean rats lose more weight than t h e i r obese brothers because lean rats metabolize t h e i r adipose tissue under conditions of energy demand in order to preserve body protein, whereas obese rats u t i l i z e body protein while hoarding body f a t (18). I t is suggested that the n e g l i g i b l e loss of f a t and/ or body weight in the Obese/SHR vs s i g n i f i c a n t body weight loss in non-obese/ SHR was due to the greater residual levels of i n s u l i n in the Obese/SHR. The residual i n s u l i n levels may have been s u f f i c i e n t to i n h i b i t the release of glycerol and free f a t t y acids from the more copious adipose tissue mass of the Obese/SHR. The adipokinetic effectiveness of the residual amounts of i n s u l i n in the Obese/SHR was probably rendered more e f f e c t i v e since i t is well known that hepatic clearance of i n s u l i n is greatly impaired in obesity prolonging i t s systemic metabolism. The retarded breakdown of i n s u l i n reenforces the tendency toward hyperinsulinemia in obesity which enhances l i p i d storage, especially t r i g l y c e r i d e s , c h a r a c t e r i s t i c of obesity. The emaciation and higher glucose and l i p i d levels in the alloxan-treated non-obese/SHR attests to the fact that they did not have s u f f i c i e n t adipose tissue reserve and that t h e i r residual levels of i n s u l i n were too meager to be e f f e c t i v e metabolically. All of the alloxan-treated animals manifested increased blood pressure, increased incidence of myocardial necrosis, n e p h r o l i t h i a s i s , hypogonadism, hypothermia, alopoecia, thin skin, and poor wound healing. In humans and in animals, hyperadrenocorticism and diabetes are associated with abnormal connective tissue and ground substance metabolism, poor wound healing, and thinning of the skin. The skin of a l l o x a n - d i a b e t i c rats contains decreased quantities of glycosaminoglycans, e . g . , hyaluronic acid, dermatan s u l f a t e , e t c . , decreased collagen and e l a s t i n , all of which can be reversed by administration of i n s u l i n (19). The spectrum of degenerative changes induced by alloxandiabetes in these 5-6 month old Obese and non-obese/SHR are e s s e n t i a l l y i d e n t i cal to those lesions which appear spontaneously in old (2-3 yrs) SHR (15). This indicates that diabetes and hyperadrenocorticism in animals cause accelerated aging as observed in humans with advanced diabetes or Cushing's syndrome (20). Acknowledgements The authors are grateful for the dedication and technical expertise of D. Conatser, E. gominqo, G. Williamson, G. Heap and J. Wexler. The research was supported by grants from the National Heart, Lung and Blood Diseases I n s t i t u t e (HL-21418) and the National I n s t i t u t e on Aging (AG-585). References I. 2. 3. 4. 5. 6. 7. 8.
K. OKAMOTOand K. AOKI, Jap. Circ. J. 27 282-293 (1963). B.C. WEXLER, S.G. IAMS and J.P. MC MURI'ITY, Br. J. exp. Path. 61 195-207, (1980). B.C. WEXLER and J.P. MC MURTRY, Br. J. exp. Path. 62 146-157 (1981). B.C. WEXLER and J.P. MC MURTRY, Br. J. exp. Path. ~ 369-374 (1981). B.C. WEXLER and J.P. MC MURTRY, J. Gerontol 38 148--I'54 (1983). B.C. WEXLER, J. Atheroscler. Res. 4 57-80,(19--6~4). B.C. WEXLER, Acta endocr, Copenh. # 397-415 (1964). B.C. WEXLER and G.W. KITTINGER, J.-Atheroscler. Res. 5 317-329 (1965).
Vol. 33, No. ii, 1983
9. lO. If. 12. 13. 14. 15.
16. 17. 18. 19. 20.
Alloxan Diabetes in Obese/SHR
B.C. WEXLER, J. SAROFFand J.T. JUDD, Diabetes 19 311-323 (1970). B.C..WEXLER, Diabetes 19 324-336 (1970). m B.C. WEXLER, Atheroscle-r'osis 14 289-307 (1971). B.C. WEXLER, Exp. Gerontol. I~'-47-58 (1981). B.C. WEXLER, Age 3 93-99 (198--0-). W.K. MORISHIGE, C?A. UETAKE, F.C. GREENWOOD,J. AKAKA, Endocrinology lO0 1710-1718 (1977). ~. WEXLER, J.P. MC MURTRYand S.G. IAMS, J. Gerontol. 36 514-519 (1981). S. INOUE and G. BRAY, Horm. Metab. Res. I0 273-280 (1978). G.A. BRAY and D.A. YORK, Physiol. Rev. 5~'-719-809 (1979). M.N. GOODMAN,P.R. LARSEN, M.M. Y~APLAN,~.T. AOKI, V.R. YOUNGand N.B. RUDERMAN,Am. J. Physiol. 239 E277-E286 (1980). E.R. DALFRERESJr, B. P~ADHAKRISI~B~URTRY,M.S. CROUCH, J.S. BERENSON, Proc. Soc. exp. Biol. Med. 148 918-924 (1975). B.C. WEXLER, H~pothalamus Pi-{u-itary and Aging, A.V. Everitt and J.A. Burgess, edso, p. 333-361, Chas. C. Thomas, Springfield, I I I (1970).
1103