EFFECT

OF

INSULIN

CONSUMPTION RAT

AND

AND

GLUCOSE

GLYCOGEN

ON T H E

SYNTHESIS

GLUCOSE BY T H E

DIAPHRAGM V. V. A b r a m o v a a n d V. P. F e d o t o v

UDC 615.357.379+615.31:547.455.623].015.42:612.217.1.015.32

Insulin activated both the uptake of glucose-C 14 f r o m the m e d i u m and its incorporation into glycogen by the incubated diaphragm: the p e r c e n t a g e of glucose-C 14 incorporated into glycogen i n c r e a s e d with an i n c r e a s e in the dose of insulin. An i n c r e a s e in the concentration of gluc o s e in the medium also caused this m o r e rapid a s s i m i l a t i o n by the m u s c l e tissue. However, significant stimulation of glycogen synthesis was o b s e r v e d only in the p r e s e n c e of a high (unphysiological) glucose concentration in the incubation fluid; the p e r c e n t a g e of glucose-C 14 inc o r p o r a t e d into glycogen r e m a i n e d s m a l l and constant. It is postulated that the glucose-C 14 found in the case of s u b s t r a t e control is the r e s u l t of exchange of the glucose r e s i d u e s of g l y cogen with i n t r a c e l l u l a r glucose. KEY WORDS: insulin; glycogen synthesis; glucose utilization; r a t diaphragm.

The effec't of insulin on c a r b o h y d r a t e m e t a b o l i s m in m u s c l e tissue is m a n i f e s t e d p r i m a r i l y as an inc r e a s e in the a s s i m i l a t i o n of s u g a r s by the tissue and in the rate of glycogen formation [9, 10]. An i n c r e a s e in the concentration of exogenous glucose also leads to an i n c r e a s e in its absorption by m u s c l e tissue, but the effect of this factor on glycogen formation has not been adequately studied [4, 6]. The purpose of this investigation was to make a c o m p a r a t i v e a n a l y s i s of the effect of insulin (hormonal control) and glucose (substrate control) on the a s s i m i l a t i o n of glucose and glycogen s y n t h e s i s by the r a t diaphragm. EXPERIMENTAL F e m a l e W i s t a r r a t s weighing 120-150 g were used. and deprived of food for 18-24 h before s a c r i f i c e .

METHOD The r a t s w e r e kept under o r d i n a r y conditions

P r e p a r a t i o n s of the diaphragm w e r e obtained by the method of Kipnis and Cori [3]. K r e b s - R i n g e r bicarbonate buffer solution, s a t u r a t e d with carbogen gas m i x t u r e (4.2% CO 2 and 95.8% O2) at 37~ the pH of which was adjusted to 7.4 by means of 1.3 % NaHCO 3, was used to isolate and incubate the d i a p h r a g m s . The isolated d i a p h r a g m s were washed for 5-7 min in cold buffer, divided into h a l f - d i a p h r a g m s , each of which was placed in a s e p a r a t e 50-ml flask containing 2 ml of incubation medium, and incubated for 90 min at 37~ In the t e s t s with insulin the medium contained glucose in a concentration of 250 mg% and uniformly labeled glucose-C 14 (0.2 gCi/ml). In the t e s t s with different glucose concentrations in the medium the quantity Of glucose-C 14 was changed so that the specific radioactivity r e m a i n e d constant in all the samples. Glycogen was isolated f r o m the d i a p h r a g m by alcoholic precipitation and hydrolyzed in 1 N H2SO 4 [11]. The total quantity of glucose in the glycogen digest was d e t e r m i n e d by the glucose oxidase method [1]. The absorption of glucose by the d i a p h r a g m was d e t e r m i n e d f r o m the difference between the concentrations of L a b o r a t o r y of Biological Standardization of H o r m o n e s , Institute of E x p e r i m e n t a l Endocrinology and Hormone C h e m i s t r y , Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the A c a d e m y of Medical Sciences of the USSR N. A. Yudaev.) T r a n s l a t e d f r o m Byulleten' ~ k s p e r i m e n t a l ' n o i Biologii i Meditsiny, Vol. 78, No. 7, pp. 47-50, July, 1974. Original a r t i c l e submitted June 18, 1973. 9 1974 Consultants Bureau, a division of Plenum Publishing Corporation, 227 West 17th Street, New York, N. Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission of the publisher. A copy of this article is available from the publisher for $15.00.

764

2 500

2 500

A

2 000

2088 ....t1500

! 508

/

7 0OO : . . . . . . . . . .

6oa O.z

l OOO

i

i

7

ZO

./

600

)

)'80

700

t

i

i

i

200 300 400 500

Fig. 1. Effect of insulin (A) and glucose (B) on uptake of glucose by the rat diaphragm. Mean values (M ~=m) of 6-8 experiments. A b s c i s s a , A: insulin c o n centration (in m i l l i u n i t s / m l on a l o g a r i t h mic scale), in B: glucose concentration in medium (in mg % on a logarithmic scale); ordinate for both graphs: uptake of glucose ( p u l s e s / m i n / m g wet weight of tissue). Broken line m a r k s basal level of glucose uptake (in s a m p l e s without h o r mone).

TABLE 1. Effect of Various C o n c e n t r a tions of Insulin and Glucose in the Medium on Glycogen Synthesis by the Rat Diaphragm* Composition of incubation

reed ium

[Quantityofglu- t Quantit~ of glucose 9 in .g1y c o~ ell [ e o s e - C 4 m glvtdlgest 0n vgz ] eogen ~tiges$(fn I00 mg wet wt. proses/rain/rag of tissue) wet wt. of -

t2ssue)

Glucose 250 mg %1 Insulin{in nYi11"iuni~s/ml) ] 1

10 Glucose (in mg ~ i00 200 300 400 500

470-+38,2? 618---+38,4?

179--+ 17,6

116--+13,1 328--+23,8 ~ 466-+ 27,4 I"

162~+21,0 195• i5,2 173--+22,4 238+--25,2

52m3,6 66--+2,25 115-----12: :~ 134--_+11 ~: 179-----2,14 $

288• i8,5 **

glucose-C 14 in the medium before and a f t e r incubation. Radioactivity was m e a s u r e d by means of a Packard liquid s p e c t r o m e t e r , using B r a y ' s scintillation fluid. The results were analyzed by Student's method with a level of significance P < 0.05, EXPERIMENTAL

RESULTS

AND DISCUSSION

The absorption of glucose-C14 by the diaphragm m u s cle i n c r e a s e d proportionally to the logarithm of the insulin concentration in the incubation medium within the dose range of 0.1-10 m i l l i u n i t s / m l (Fig. 1A). The g r e a t e s t absorption took place with insulin in a concentration of 10 m i l l i u n i t s / m l and remained virtually unchanged with a f u r t h e r i n c r e a s e in its concentration. In the different exp e r i m e n t s this maximal absorption amounted to 160-200 % of the absorption in the control samples (v#ithout insulin). With an i n c r e a s e in the concentration of exogenous glucose (from 100 to 500 mg%) its uptake by the diaphragm also increased (Fig. 1B). However, the magnitude and c h a r a c t e r of the two controlling factors - hormonal and substrate - differed, evidently reflecting differences in the m e c h a n i s m of their effect on glucose utilization by the m u s cle. Quantitative estimation of glucose in the glycogen digest c a r r i e d out on the same samples (Table 1) showed that insulin stimulates glycogen formation; both the total quantity of glucose (endogenous and exogenous) in the glycogen digest and the quantity of glucose-C 14 incorporated into glycogen (exogenous glucose) were increased. Insulin evidently does not change the relative proportions of m e t a bolites of endogenous and exogenous glucose incorporated into glycogen. This o c c u r s for two r e a s o n s : first, with a high concentration of exogenous glucose (250 mg %) the t r a n s p o r t p r o c e s s e s do not limit the velocity of glycogen synthesis from exogenous glucose; second, glycogen f o r m a tion from both endogenous and exogenous s o u r c e s under these conditions depends on glycogen synthesis activity. Insulin is known to stimulate the activity- of this enzyme, as was f i r s t shown on homogenates of the diaphragm incubated with insulin [7, 8].

By c o n t r a s t with insulin, an increase in the glucose concentration in the incubation medium did not give rise to a proportional i n c r e a s e in glycogen formation. The total quantity of glucose in the glycogen digest was increased only in the presence of the highest glucose concentration (500 rag%) in the medium. The incorporation of g l u c o s e Cl4 into glycogen, it is interesting to note, was proportional to and i n c r e a s e d significantly with an i n c r e a s e in the concentration of exogenous glucose. This fact can be explained on the assumption that the change in the quantity of glue o s e - C 14 in the glycogen digest reflects an i n c r e a s e not only in the rate of glycogen synthesis but also in the rate of the subsequent exchange of the glucose residues in the glycogen with the intraeellular glucose. It has been shown that a m y l o - l , 6 - g l u c o s i d a s e , an enzyme catalyzing the h y d r o l y s i s of glycogen at the b r a n c h ing points of the polysaccharide chain, can i n c r e a s e the incorporation of glueose-C 14 into glycogen either through the partial r e v e r s a l of h y d r o l y s i s in the p r e s e n c e of a high concentration of glucose-C 14 o r through an exchange reaction taking place between glucose-C14 and the glucose in the s u b s t r a t e - e n z y m e complex [2, 5]. *Here and in Table 2, mean values (M =~m) from 6-8 e x p e r i m e n t s are given. P < 0.001 c o m p a r e d with values in the absence of insulin. Sand ** denote P < 0.01 and P < 0.05, r e spectively, c o m p a r e d with values for a glucose concentration of 100 rag%.

765

TABLE 2. Effect of Various Concentrations of Insulin and Glucose on the Conversion of A s s i m ilated Glucose into Glycogen Quant. ofglucose- ~ 4 as-

Quant.of glucc~e-C14ingly-

Composition simi'lated(in

cogendiggs~

of incubation ] ~g/g wet wt. ] (in pmsesrmint medium ]of fiss:) ,m~ wet wt. [ oftissue) I

Ratio II/I (in %)

It

Glucose 250 mg % Insulin(in milliunits/m1) 0 I

1O Glucose

(inl~0g%) 200 300 400 500

9,1• 12,2• 14.3•

0,79• 2,42• 3,8•

9 20 27

6,1~0,13 9,9-----0,18

0,42-----0,01 0,56-+-0,012 0,98• 1,15+0,1 1,53•

6.7 5.8 8,4 7,9 7.1

11,7-+'0,06

14,5• 21,4•

In the p r e s e n t e x p e r i m e n t s i n c r e a s e d glucose concentrations in the medium led to m a r k e d stimulation of glucose utilization by the tissue, with a consequent i n c r e a s e in the i n t r a c e l l u l a r content of glucose-C 14 and also, as a possible r e s u l t of this, an i n c r e a s e in the incorporation of glucose-C 14 into the glycogen f r a c tion under the influence of a m y l o - l , 6 - g l u c o s i d a s e . Calculated values of the quantity of glucose-C 14 utilized under the influence of insulin and glucose and the r e l a t i v e proportion of the a s s i m i l a t e d glucose metabolized into glycogen under these conditions a r e given in Table 2. The i n c r e a s e in the relative proportion of a s s i m i l a t e d glucose i n c o r p o r a t e d into glycogen takes place only under the influence of specific hormonal control, but in the c a s e of s u b s t r a t e control, d e s pite m a r k e d stimulation of the a s s i m i l a t i o n of exogenous glucose by the tissue, the relative p r o p o r t i o n of this glucose i n c o r p o r a t e d into glycogen r e m a i n s s m a l l and virtually unchanged w h a t e v e r the glucose c o n c e n tration used. LITERATURE 1~

2. 3,

4. 5. 6. 7. 8. 9. 10. 11.

766

CITED

V. K. Gorodetskii, in: Modern Methods in B i o c h e m i s t r y [in Russian], Vol. 1, Moscow (1964), p. 311. H. G. H e r s , W. Verhue, and M. Mathieu, in: W. J. Whetan (editor), Control of Glycogen Metabolism, London (1964), p. 151. C. Kipnis and K. Cori, J. Biol. Chem., 224, 681 (1957). T. Kuzuya, E. S a m o l s , and R. H. Williams, J. Biol. Chem., 240, 2277 (1965). J. L a m e r and L. H. Schliselfeld, Biochim. Biophys. Acta, 20, 53 (1956). J. L a r n e r , C. V i l l a r - P a l a s i , and D. J. Richman, Ann. New York Acad. Sci., 822, 345 (1959). C. V i l l a r - P a l a s i and J. L a r n e r , Biochim. Biophys. Acta, 3_~9, 171 (1960). C. V i l l a r - P a l a s i and J. L a m e r , Arch. Biochem., 94., 351 (1961). C. V i l l a r - P a l a s i and J. L a r n e r , Vitamins and H o r m o n e s , 26, 65 (1968). C. V i l l a r - P a l a s i and J. L a r n e r , Ann. Rev. Biochem., 39, 733 (1970). O. Walaas and E. J. Walaas, J. Biol. Chem., 187,769 (1950).