The insulin-like effect of growth hormone

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SHORT COMMUNICATIONS, PRELIMINARY NOTES

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T H E I N S U L I N - L I K E E F F E C T OF GROWTH HORMONE by J. H. O T T A W A Y

Department o[ Biochemistry, University College, London (England)

P i t u i t a r y g r o w t h h o r m o n e h a s b e e n r e p o r t e d b y OTTAWAY1 to s t i m u l a t e (the " i n s u l i n - l i k e " effect) a n d to depress t h e glucose u p t a k e of r a t d i a p h r a g m . KRAHL~ a n d PARK 3 h a v e been u n a b l e to s h o w a n y effect of g r o w t h h o r m o n e on d i a p h r a g m f r o m n o r m a l rats, a l t h o u g h KRAHL h a s r e p o r t e d a n " i n s u l i n - l i k e " effect on d i a p h r a g m f r o m h y p o p h y s e c t o m i s e d rats. T h e effect of g r o w t h h o r m o n e in vitro in a s t r a i g h t f o r w a r d i n c u b a t i o n of r a t d i a p h r a g m h a s been f u r t h e r i n v e s t i g a t e d . T h e i n h i b i t o r y effect of g r o w t h h o r m o n e at a c o n c e n t r a t i o n of IOO/~g/m was confirmed, b u t a t lower c o n c e n t r a t i o n s , especially a t 1-5 p g ml, t h e r e s u l t s were conflicting. Some series of e x p e r i m e n t s s h o w e d a n inhibition, a n d o t h e r s a n a c t i v a t i o n . I t w a s felt t h a t t h e g r o w t h h o r m o n e could indeed p r o d u c e t w o q u i t e different effects, b u t their i n d u c t i o n d e p e n d e d on unidentified c h a n g e s in e x p e r i m e n t a l conditions. T h e e l u c i d a t i o n of t h e s e c h a n g e s p r o v e d difficult b e c a u s e of t h e wide s c a t t e r of t h e c o n t r o l r a t e s of glucose u p t a k e , a n d a t e c h n i q u e to be described here h a s been developed in t h i s l a b o r a t o r y for m a k i n g t h e m u s c l e m e t a b o l i s e m o r e c o n s i s t e n t l y , T h e t e c h n i q u e described b y KRAHL h a s been f o u n d b y us to reduce v a r i a n c e of glucose u p t a k e , b u t also to r e n d e r t h e m u s c l e c o m p l e t e l y insensitive to g r o w t h h o r m o n e . A possible c a u s e of t h e s c a t t e r was t h a t d u r i n g p r e p a r a t i o n one half of t h e d i a p h r a g m r e m a i n e d in more or less a n a e r o b i c c o n d i t i o n s for a longer t i m e t h a n t h e other. I n t h e e x p e r i m e n t s below b o t h h e m i d i a p h r a g m s are t r e a t e d in a n identical way. T h e m e d i u m devised b y STADIE AND ZAPP4 w a s used, modified b y raising t h e p H to 7.2, to e n s u r e t h a t t h e g r o w t h h o r m o n e (I.E.P. 6.75 ) r e m a i n e d in solution. T h e whole d i a p h r a g m w a s excised f r o m a f a s t e d male albino rat, a n d w a s h e d for a m o m e n t in m e d i u m to r e m o v e a d h e r i n g blood. I t w a s t h e n placed in a Gooch crucible c o n t a i n i n g lO-15 m l of m e d i u m w i t h glucose; n i t r o g e n w a s p a s s e d t h r o u g h t h e sintered glass b o t t o m of t h e crucible, so t h a t t h e m u s c l e h a d t h e s a m e p O 2 in all t h e e x p e r i m e n t s . I t r e m a i n e d in t h i s solution for 2 m i n u t e s , d u r i n g w h i c h t i m e it w a s d i v i d e d into two along t h e c e n t r a l t e n d o n , w i t h o u t r e m o v i n g it from t h e liquid. T h e a d d i t i o n of ascorbic acid (0.0o 5 M) to t h e m e d i u m u s e d in t h i s p r e l i m i n a r y g a s s i n g was f o u n d to reduce t h e v a r i a t i o n in c o n t r o l u p t a k e s ; its u s e did n o t affect t h e t y p e of r e s u l t o b t a i n e d . E a c h h e m i d i a p h r a g m was t a k e n s i m u l t a n e o u s l y b y one operator, blotted, a n d divided into two, a n d t h e four pieces placed into four W a r b u r g flasks. T h e flasks were b e i n g g a s s e d in t h e b a t h (with o x y g e n ) 31~-4 m i n u t e s a f t e r excision. T h e rigid u s e of t h i s r o u t i n e led to t h e finding t h a t g r o w t h h o r m o n e s t i m u l a t e d t h e glucose u p t a k e of n o r m a l r a t d i a p h r a g m a t t h e c o n c e n t r a t i o n used (25 t,g m l in all t h e e x p e r i m e n t s r e p o r t e d here). A s t i m u l a t i o n w a s also o b s e r v e d at IO/,g/nil, b u t at 4 ° / ~ g / m l t h e h o r m o n e w a s a p p a r e n t l y w i t h o u t effect. I t w a s dissolved in a s m a l l q u a n t i t y of m e d i u m w i t h tile aid of a d r o p of N[io N a O H , a n d this solution d i l u t e d as required. T h e insulin-like a c t i v i t y d i s a p p e a r s q u i t e r a p i d l y from t h e solution a n d it is n e c e s s a r y to use it n o t m o r e t h a n 2o' after m a k i n g . T h e r e s u l t s were v e r y c o n s i s t e n t ; a s t i m u l a t i o n w a s f o u n d in 26 o u t of 29 pairs of o b s e r v a t i o n s (Table I). TABLE I E F F E C T O F GRO'*VTH I t O R M O N E ON G L U C O S E U P T A K E I N m g / g O F T I S S U E / h , O R N O R M A L R A T D I A P H R A * I M A F T E R 2 M I N U T E S I N A N A E R O B I C C O N D I T I O N S . G R O W T H H O R M O N E A D D E D ~ n vitro, C O N C E N T R A T J O N

25 # g / m l . GLUCOSE CONCENTRATION 2 m g nil I n c u b a t e d for 1-2 h o u r s Number of observations

Control 4- S,E.

3°

3.44 3- o.16 Difference

Growth hormone 4:. S.E.

4.06 ~ o.16 + 0.62 q_ o.13

t ~ 2.7o, p = o.oi

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(1953)

I t seems unlikely t h a t g r o w t h h o r m o n e p r e p a r e d b y the usual m e t h o d s itself contains insulin (PARK 5 et al.). There r e m a i n e d the possibility t h a t insulin was in some w a y liberated by growth h o r m o n e after the preliminary anaerobic t r e a t m e n t of the diaphragm, either in the muscle cells themselves or in the blood remaining in the blood vessels after excision. The e x p e r i m e n t s were repeated, using alloxan-diabetic rats. The r a t s were not used until at least IO d a y s after s u b c u t a n e o u s injection of 25 m g of alloxan per lOO g of rat, after the m e t h o d of KASS AND WAISBREIq% Table I I shows t h a t g r o w t h h o r m o n e had no effect on the glucose u p t a k e f r o m diabetic r a t diaphragm, i.e. t h a t from animals with a fasting blood sugar greater t h a n 15o mg %. A g r a p h showed t h a t the s t i m u l a t i o n induced b y g r o w t h h o r m o n e added in vitro varies inversely with the fasting blood s u g a r (correlation coefficient o.62), which is f u r t h e r evidence t h a t it is insulin which is directly responsible for the stimulation. TABLE II EFFECT

OF G R O W T H

HORMONE

ON G L U C O S E

UPTAKE

OF D I A B E T I C

RAT DIAPHRAGM.

ALL VALUES

IN

mg]g]h. CONDITIONS AS IN PREV1OUS TABLE Number q[ observations

Control t_ S.E.

Fasted rats <~ 150 r a g %

i1

3.5 ° ~ o.12

2. F a s t e d r a t s > 15o r a g %

28

Blood sugar at death

I.

3.

Difference

Growth Hormone ~ S.E.

3-59 • o-43 + 0.09 ~ 0.27

F a s t e d r a t s injected with insulin 60 r a g % 20

3 .68 ± o.23

(a) 3.57 • o.21

--o.I1

(a,c)

~ o.i6

4.59 ± 0.25

5.04 52 0.27 + 0.45 zc o.15

4. Fed r a t s ; m e a n b.s. 380 r a g % 380 m g % 12

2.23 ± o.I9

(b) 2.07 52 o.27

- - o . 1 6 ± o.15

5. Fed r a t s ; m e a n b.s. 43 ° r a g % , injected with insulin 255 r a g % I6 1.97 -4- o.13

(a)

2.28 ~ 0.o9 + o.31 4_ O.li

(b)

a N o t significantly greater t h a n zero b Significantly greater t h a n zero c Significantly different from response of n o r m a l rats (Table I)

I t was desirable to s h o w t h a t the s t i m u l a t i o n was increased w h e n the concentration of insulin in the tissue is raised above normal. Injection of insulin into n o r m a l animals would complicate i n t e r p r e t a t i o n because of the secretion of insulin a n t a g o n i s t s (SoMoGYlV). Injection of 1-2 units of insulin s u b c u t a n e o u s l y into fasting mildly diabetic r a t s 3o-45 m i n u t e s before d e a t h caused the insulin-like effect to rise to a significant level (Table I I , section 3) ; all r e s u t s from animals in which the blood sugar at d e a t h w a s greatly below n o r m a l have been excluded. M a n y of the animals w e n t into coma, and a n o t h e r e x p e r i m e n t was performed using unfasted diabetic rats. Injection of 0.2 unit of insulin 2 h o u r s before death b r o u g h t the blood sugar level down in m o s t cases to a b o u t ioo m g % . Some animals were given a s u p p l e m e n t a r y dose of a b o u t o.i unit a b o u t 3 ° m i n u t e s before death. Table II, section 5, shows t h a t this t r e a t m e n t effectively restored the "insulin-like" response to g r o w t h h o r m o n e . As a control, tissue was taken from uninjected diabetic animals from the same b a t c h and incubated at the same time. These e x p e r i m e n t s s h o w t h a t g r o w t h h o r m o n e can have, in certain conditions, an insulin-like effect on muscle f r o m n o r m a l rats, b u t n o t on muscle f r o m diabetic rats. Two explanations suggest themselves: t h a t insulin in some w a y " p o t e n t i a t e s " the tissue so t h a t it is able to r e s p o n d to the g r o w t h h o r m o n e ; or t h a t g r o w t h h o r m o n e is able to liberate insulin from a complex in which it is inactive. This h y p o t h e s i s is more fruitful especially if it is considered t h a t endogenous g r o w t h h o r m o n e m a y also liberate insulin in anaerobic conditions. This concept suggests t h a t the great variation in rate of glucose u p t a k e observed in d i a p h r a g m e x p e r i m e n t s is p a r t l y due to insulin being set free to an e x t e n t depending on the pO 2 of the muscle during preparation. Again, the fact t h a t KRAHL WaS unable to s h o w an insulin-like effect u p o n muscle from n o r m a l animals m a y have been because

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in his conditions the " l i b e r a t i o n " was conlplete before incubation of the tissue was b e g u n ; only with muscle from h y p o p h y s e c t o m i s e d animals, lacking a n y endogenous g r o w t h hormone, would it t h e n be possible to d e m o n s t r a t e an in vitro effect.

REFI~.RENCES 1 j. H. OTTAW:XY, Nature, 167 (195I) lO64. 2 M. E. KRAHL,Ann. N . Y . Acad. Sci., 54 (1951) 649. 3 C. R. PARK, Phosphorus Metabolism, Vol. II, 1953. J o h n s H o p k i n s Press, Publishers. 4 W. C. STADIE AND J. A. ZAPP, J. Biol. Chem., 17o (1947) 55. 5 C. R. PARK, D. A. BROWN, M. CORNBLATH, W. H. DAUGHADAY AND M. E. KRAHL, J. Biol. Chem., 197 (1952 ) 151. g . H. KASS AND B. A. WAISBREN, Proc. Soc. Exptl. Biol. Med., 60 (1945) 303 • 7 M. SOMOGYI, J. Biol. Chem., 174 (1948) 597. Received May 26th, 1953

FACTORS I N F L U E N C I N G T H E FORMATION OF C E R O I D IN T H E L I V E R S OF C H O L I N E - D E F I C I E N T RATS* I. D I E T A R Y FATS by W. G. B R U C E CASSELMAN**

Banting and Best Department o/Medical Research, The University o[ Toronto, Toronto, Ontario (Canada)

The golden yellow, lipid p i g m e n t which m a y occur in the cirrhotic livers of choline-deficient r a t s was n a m e d ceroid b y LILLIE and his associates 1. Studies on the in vitro p r e p a r a t i o n and histochemical properties of substances resembling it s u p p o r t the suggestion t h a t this material m i g h t be f o r m e d b y the a u t o x i d a t i o n of pathologically accumulated, u n s a t u r a t e d fats% T h r o u g h their influence u p o n tissue lipids, therefore, dietary fats m i g h t be expected to affect the formation of ceroid in the livers of choline-deficient rats. Cod liver oil favours thisS, 4,~, while replacing the lard in the diet b y b u t t e r decreases it and b y h y d r o g e n a t e d cottonseed oil p r e v e n t s it e. E v e n reduction in the a m o u n t of fat has an effectL This p r e l i m i n a r y note concerns a s y s t e m a t i c s t u d y of the influence of the degree of u n s a t u r a t i o n of the dietary fat u p o n the a m o u n t of ceroid formed in the livers of choline deficient rats. Seven similar g r o u p s of male, W i s t a r rats, each consisting of io animals initially weighing 58-65 g, were fed choline-deficient diets***. The g r o u p receiving the basal, fat-free diet was given m e t h y l linoleate, 5° mg per IOO g b o d y weight, orally twice weekly. The other 6 g r o u p s were fed diets in which io % of the sucrose in the basal m i x t u r e was replaced b y io % of a fat: h y d r o g e n a t e d cottonseed oils (Iodine N u m b e r = 12 or 36), cocoa b u t t e r (I. No. ~ 36), beef tallow (I. No. = 40), cottonseed oil (I. No. = i i i ) , or a m i x t u r e of 4 p a r t s cod liver oil (I. No. ~ 151) and 6 p a r t s corn oil (I. No. = 125). To reduce t h e incidence of fatal renal lesions, the diets were s u p p l e m e n t e d with choline chlorxde, " o/ for the first io days, and o.o 3 % for the n e x t 14 days, b u t n o t a n y thereafter. o.08/o I n d i v i d u a l food c o n s u m p t i o n s were determined daily, b o d y weights twice weekly. R a t s f r o m each group were sacrificed at r a n d o m b e t w e e n the 55th and i 2 6 t h days. Sections from 4 regions of each of their livers were graded for the a m o u n t of ceroid p r e s e n t (paraffin sections, Oil Red O method). * S u p p o r t e d in p a r t b y a g r a n t from the N a t i o n a l Research Council, Canada. Senior Medical Research Fellow, N a t i o n a l Research Council, Canada. *** Basal choline-deficient diet contained: extracted p e a n u t meal, 3 o % ; v i t a m i n a n d fat-free casein, 6 % ; powdered sucrose, 49.9o-49.98 % ; salt m i x t u r e 11, 3 %; v i t a m i n m i x t u r e n , i % ; choline chloride (see text), 0.00-o.08 % ; cod liver oil concentrate (2oo,ooo I.U. v i t a m i n A and 50,0o0 I.U. v i t a m i n D per g), o.oi %, and a-tocopherol acetate, o.oi %. W h e n fat was added, this replaced IO % of the sucrose. **