BIOCHIMICA ET BIOPHYSICA ACTA

BBA 269I 5

129

EFFECT OF LONGTERM SUCROSE FEEDING ON THE ACTIVITY OF SOME

ENZYMES REGULATING GLYCOLYSIS, LIPOGENESIS AND GLUCONEO-

GENESIS IN RAT LIVER AND ADIPOSE TISSUE

AHARON M. COHEN, SARA BRILLER AND ELEAZAR SHAFRIR Diabetic Unit, Isotope Laboratory for Endocrine Research and Department of Biochemistry, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem (Israel)

(Received March 27th, 1972)

SUMMARY

4- to 6-fold increases in the activity of liver pyruvate kinase (EC 2.7.1.4o), glucose-6-phosphate dehydrogenase (EC i.I.i.49), NADP-malate dehydrogenase (EC 1.1.1.4o ) and acetyl-CoA carboxylase (EC 6.4.I.2 ), relative to liver protein concentra- tion, were obtained in rats fed for 12 months on a 72% sucrose diet, as compared to rats kept on a standard laboratory chow. Feeding a 72 % starch diet resulted in only 2- to 3-fold increases in the activities of these enzymes. The activity of liver 6-phos- phogluconate dehydrogenase (EC 1.1.1.44 ) increased about 2-fold in the sucrose-fed rats and did not rise significantly on a starch diet. In adipose tissue, the activity of pentose shunt enzymes rose significantly only on the sucrose diet, whereas NADP- malate dehydrogenase rose 5-fold in sucrose and 2-fold in starch-fed rats. Fasting for 48 h resulted in a decrease in the activity of enzymes of glucose utilization and lipo- genesis in the liver and adipose tissue, which on all diets was of a similar proportion with respect to the activity prior to fasting. The activity of most enzymes was re- duced to 50-70% of the prefasting value; that of NADP-malate dehydrogenase de- creased more pronouncedly.

The activity of liver phosphoenolpyruvate carboxylase (EC 4.1.1 32) was slight- ly decreased on both starch and sucrose diets, whereas the activity of glucose-6-phos- phatase (EC 3.1.3.9) was significantly increased in the sucrose-fed rats, indicating adaptation to gluconeogenesis from the fructose component. Each of the enzymes of gluconeogenesis responded to 48 h fasting with a rise in activity, the extent of which was not apparently related to the previous diet.

The more extensive changes in enzyme activities on prolonged feeding of sucrose in comparison to starch are discussed with respect to the large share of ttle liver in the metabolism of carbohydrate intake on sucrose and to the role of the fruc- tose component of sucrose in producing a pattern of adaptation of liver enzymes con- ducive to lipogenesis and glucose intolerance.

Biochim. Biophys. Acta, 279 (1972) 129-138

13o A.M. COHEN ~!t al.

INTRODUCTION

There are several reports which demonstrate that sucrose feeding to rats results in impaired glucose tolerance ~, elevated serum lipid levels 2 and increased hepatic con- tent and synthesis of lipids a-7. A rise in the activity of some liver enzymes associated with glycolysis and lipogenesis has also been noted s-ll.

This study is part of an endeavor to define the pattern of enzyme adaptation developing as a particular response to long-term sucrose intake, which may lead to the intolerance to glucose and hyperlipidemia. The activities of several enzymes of regula- tory importance in the pathways of glycolysis, lipogenesis and gluconeogenesis were measured in the liver and adipose tissue, and compared to the corresponding activities in rats fed an isocaloric starch diet or a standard laboratory chow.

MATERIALS AND METHODS

Male albino rats of Hebrew University strain, weighing 6o to 7 ° g, were placed on diets ad libitum for a period of 12 months. Group I was fed a standard laboratory chow (a mixture of ground whole wheat, alfalfa and skimmed milk powder) containing by weight 21% protein, 6o~/0 high molecular weight carbohydrates, 5% fat and the rest, cellulose, inert material and salts. Group II was fed a synthetic diet containing by weight 18°/o casein, 5% butter, o/ 5/o salt mixture U.S.P. No. II and 72% corn- starch. Vitamins of the B group were added as a dry mix, and fat soluble vitamins were given twice weekly. Group I I I was given the same synthetic diet as Group II, except that sucrose was substituted for starch. Thus, the diets of Group II and I I I were isocaloric.

Group IV animals were taken from our inbred albino rats AMC. The parents of these rats were selected from a group which had been fed for 2 months the sucrose diet. A glucose tolerance test was made by giving intragastrically 35 ° mg glucose/Ioo g body weight to overnight fasted rats. Blood was taken in duplicate from the tail vein at o and 6o min, and the glucose determined by the Somogyi-Nelson methodtL The male and female with the highest rises in blood glucose at 6o min were mated. The offspring at the age of 21 to 28 days and body weight of 6o g were put on tile sucrose diet, and after 2 months a glucose tolerance test was performed. Offspring which had a glucose tolerance lower than the parents were separated, kept on sucrose as a special group and taken for enzymes assays after 7 months on the diet.

At the appropriate time the rats were anesthetized by ether in a large glass vessel through which oxygen was passing to prevent anoxia. The rats were then bled through the abdominal artery, and the serum was later separated and extracted with iso- propanol for triglyceride and cholesterol determinations t3. The liver and the epididy- mal adipose tissue were excised, washed in ice cold o.9% NaC1 solution, weighed and homogenized (25°,0, w/v) in a medium composed of 0.2 M sucrose, 20 mM triethanol- amine-HC1, pH 7.5, I mM dithioerythreitol and I mM disodium EDTA.

For the assay of glucose-6-phosphatase (EC 3.1.3.9) activity, according to the method of Hers~L a portion of the homogenate was centrifuged at 3ooo ×g for 15 rain and the supernatant fraction used. For the assay of other enzyme activities the super- natant fraction after centrifugation at IOOOOO >(g for 45 rain was used. The activity of glucose-6-phosphate- and 6-phosphogluconate dehydrogenases (EC 1.1.1.49 and

19iochim. Biophys. Acta, 279 (i972) i29- i38

E F F E C T OF SUCROSE F E E D I N G ON E N Z Y M E A C T I V I T Y 131

EC L I . I . 4 4 , respect ively) , N A D P - m a l a t e dehydrogenase ( "mala te enzyme" , EC L I . L 4 o ) , ace ty l -CoA carboxylase (EC 6.4.I .2 ), phosphoeno lpy ruva te ca rboxylase (EC 4 . L I 32) as well as the l iver p ro te in conten t was de t e rmined b y me thods used pre- viously15,1~. The ac t iv i ty of acetyl -CoA carboxylase was measured af ter ma x ima l pre- ac t iva t ion in the presence of ci t rate . P y r u v a t e kinase (EC 2.7.L4o) ac t i v i t y was as- sayed by the me thod of Bticher and Pfleiderer~L The measuremen t of ma x ima l ac t i v i t y of this enzyme was improved b y rais ing the A D P and phosphoeno lpy ruva te concen- t ra t ions to 1.5 mM and 2o mM, respect ively .

The ac t iv i ty of glucose-6-phosphatase , acetyl -CoA carboxylase and phospho- eno lpy ruva te carboxylase were measured at 37 °C. The ac t iv i ty of o ther enzymes was de te rmined at 3o °C. All enzyme act iv i t ies are expressed as nmoles of subs t ra te me tab - olized per mg soluble l iver protein, except glucose-6-phosphatase , the ac t i v i t y of which was re la ted to the prote in of the crude l iver homogenate .

RESULTS

General d a t a on the exper imen ta l an imal groups are summar ized in Table I. As specified in Mater ia ls and Methods, the s ta rch and sucrose diets offered to the ra ts were isocaloric, bu t the food consumpt ion var ied among the groups, which m a y have been responsible for the differences in body weight a t the t ime of enzyme assay. The average da i ly food in take , measured at r andom for one week dur ing the s ixth month of feeding was 15 _i I and 2o "- I g for the sucrose and s ta rch fed groups, respect ively. The s tarch-fed animals (Group II) weighed signif icant ly less (I9°/O) than the chow-fed ones (Group I). The mean ra t weight in sucrose-fed groups ( I I I and IV) was abou t 36% less than Group I, and 22% less than Group I I .

The to ta l l iver weight was not s ignif icant ly different between Groups I and II , bu t i t was somewhat lower in Group I I I . However , the amoun t of l iver re la t ive to body weight was s ignif icant ly increased in the two sucrose-fed groups, particularly., in Group IV compris ing the inbred animals.

Glucose tolerance was not affected b y long- term s tarch feeding, as seen from similar rises in b lood glucose level in Groups I and I I af ter a s t a n d a r d glucose load. The tolerance de te r io ra ted on sucrose feeding (Group I I I ) , pa r t i cu la r ly in the inbred animals (Group IV).

"FABLE I

GENERAL DATA ON THE EXPERIMENTAL ANIMAL GROUPS

Values given are the means ± S.E. for the number of animals indicated in parentheses for each group. The intragastric glucose load amounted to 35 ° mg/ioo g body wt.

Diet Group I (6) Group I I (9) Group H I (9) Group 11" (6) Laboratory chow Starch Sucrose Sucrose

Duration of diet (months) 8 I2 12 7 Bodywt (g) 54 ° ~- I5 448 ± I3" 353 ± 17" 341 ~ 29 Liver wt (g) I6.8 ~: 0. 7 15.4 _L O.5 I4.9 ± O-4 I5.4 ~ O.9 Liver wt/IOO g body wt 3.I ~ o.I 3.4 ± o.I 4.2 4~ o.2* 4.9 ~ o.6" Blood glucose (mg/ioo ml)

fasting 8I ~ 4 8o ~: 3 76 ± 5 lO5 ± I5 60 m i n a f t e r g l u c o s e l o a d lO9 ~2 4 lO6 ± 4 149 i 5 21o _@_ 16

* Differences from the laboratory chow-fed rats significant at the level of P < 0.02 at least.

Biochim. Biophys. Acta, 279 (1972) I29--138

132 A.M. COHEN e[ al.

TABLE II

L I V I ~ R E N Z Y M E A C T I V I T I E S R E L A T E D TO G L Y C O L Y S I S A N D L I P O G E N E S I S I N R A T S ON D I F F E R E N T D I E T S

Enzyme activity, in nmoles/min per mg protein, is given as the mean ± S.E. for the number of animals indicated for each group. The mean rise in activity is also given as the percentage difference from the laboratory chow fed group.

Enzyme Group I (6) Group I I (9) Group I I I (9) Group IV (6) Laboratory chow Starch Sucrose Sucrose Activity Activity % Rise Activity % Rise Activity % Rise

Pyruvatekinase 269 ~4- 15 567 zlz 71. 211 818 + 42* 304 981 _~ -~ 91"* 365 Glucose-6-phosphate

dehydrogenase 34 ± 2 85 ~ 13" 250 214 -J; I4"* 630 192 ~ 18"* 567 6-phosphogluconate

dehydrogenase 72 • 5 87 d_ 6 121 148 ~ 13"* 207 136 ~: 14"* 189 NADP-malate

dehydrogenase 26 ± 5 49 i 7* 188 132 ~. io** 5o8 128 ~ 14"* 492 Acetyl-CoA

carboxylase 13 ± 2 4 ° ~: 6* 3o8 72 ~ 4** 554 47 ~ 5** 362

• Difference from Group I statistically significant at a level of P < o.o5 at least. • * Difference from Group II statistically significant at a level of P < 0.o5 at least.

Serum lipids were moderate ly increased in the sucrose-fed rats of Group I I I in comparison to the starch-fed ones of Group II . Triglyceride levels rose from Io3 '-- 8 to I98 ~ 32 mg/ Ioo ml, and cholesterol levels from 8o c~ 5 to Io6 ~ I I mg/ Ioo ml.

The activities of several liver enzymes related to the pa thways of glycolysis and lipogenesis are listed in Table II. Starch feeding (Group I1), as compared to labora tory chow feeding, resulted in more than a 2-fold increase in the ac t iv i ty of pyruva te kinase and glucose-6-phosphate dehydrogenase, whereas only a slight rise was observed with regard to 6-phosphogluconate dehydrogenase activity. The ac t iv i ty of NADP-mala te dehydrogenase similarly rose 2-fold, whereas tha t of acetyl-CoA carboxylase as much

as 3-fold. Sucrose feeding (Group III) produced rises in enzymes of glycolysis and lipo-

genesis markedly higher than those seen with starch feeding. Glucose-6-phosphate dehydrogenase ac t iv i ty increased in particular, over 6-fold, and markedly exceeded tha t of 6-phosphogluconate dehydrogenase, in comparison to the reverse ratio in the ac t iv i ty of these two pentose shunt enzymes in rats kept on the s tandard chow. NADP-mala te dehydrogenase and acetyl-CoA carboxylase activities rose approxi- mate ly 5-fold on the sucrose diet.

Sucrose-fed inbred rats (Group IV) exhibited rises in the act ivi ty of most en- zymes quite similar to those of the sucrose-fed non- inbred animals. The lower act ivi ty of acetyl-CoA carboxylase in comparison to Group I I I ma y be due to the shorter sucrose feeding period in this group (7 versus I2 months).

Table I I I shows the effect of long-term feeding of starch and sucrose diets on NADPH-genera t ing enzymes of adipose tissue. The rises in act ivi ty of the pentose shun t enzymes on starch diet were not significant, bu t the act ivi ty of NADP-mala te dehydrogenase was doubled in comparison to the chow-fed rats. The rise in glucose-6- phosphate- and 6-phosphogluconate dehydrogenase activities on sucrose diet (Groups I I I and IV) were significant with respect to Group I, though small in magnitude. On the other hand, NADP-mala te dehydrogenase act ivi ty rose most pronouncedly on the sucrose diet. The rise was 5-fold in respect to chow feeding and 3-fold in respect to starch feeding.

Biochim. Biophys. Acta 279 (i97z) 129-i38

EFFECT OF SUCROSE FEEDING ON ENZYME ACTIVITY 133

T A B L E I I I

A D I P O S E T I S S U E E N Z Y M E A C T I V I T I E S R E L A T E D TO L I P O G E N E S I S I N R A T S O N D I F F E R E N T D I E T S

Enzyme activity, in nmoles /min per mg protein, is given as the mean 4- S.E. for the numbe r of animals indicated in parenthesis for each group. The mean rise in act ivi ty is also given as the percentage difference f rom the labora tory chow fed group.

Enzyme Group I (6) Group I I (9) Group I I I (9) Group I V (6) Laboratory chow Starch Sucrose Sucrose Activity Activity % Rise Activity % Rise Activity % Rise

Glucose-6-phosphate dehydrogenase 41.7 ± 4-7 55.4 2- 4-4 133 70.5 ~_ 6.o* 169 65.1 4- 4.9* 156

6-Phosphogluconate dehydrogenase 17. 7 ± 1. 3 19. 5 ± 2.0 i i o 25. 7 -4__ 2.7* 145 24. 7 ± 1.7" 14o

NADP-mala te dehydrogenase lO. 7 ~ 1.2 21.9 4- 4.1" 205 58.7 -~ 9.1" 548 53.3 ± 8.8* 498

* Difference from Group I statistically significant at a level of P < 0.0 5.

T A B L E IV

E F F E C T O F 4 8 h F A S T I N G O N L I V E R E N Z Y M E A C T I V I T Y OF DIET-ADAPTED RATS

Enzyme activity, in nmoles /min per mg protein, is given as the mean ± S.E. for the number of animals indicated in parentheses for each group. The mean act ivi ty after the 48 h fast is also expressed as the percentage of t ha t in the respective nonfas ted group.

Enzyme Group I : Laboratory chow Group I I : Starch Group I I I : Sucrose Fed (6) Fasted (8) % Fed (9) Fasted (9) % Fed (9) Fasted (9) %

Pyruva t e kinase 269 ± 15 189 4- 14" 7o.3 567 4- 7I 35 ° _k 28* 61. 7 818 32 42 529 ± 4 °* 64.7 Glucose-6-phosphate

dehydrogenase 34 ± 2 19 ~ 2* 55.9 85 ± 13 44 ± 5 51.7 214 4- 20 lO6 £ I I * 49.5 6-phosphogluconate

dehydrogenase 72 -i_ 5 49 4- 4* 68.0 87 J: 6 61 :~_ 8* 7o.1 148 i 13 i i o ± 14 74.4 NADP-mala te dehydrogenase 26 4- 5 I I ~z 2* 42.3 49 ± 6 16 ± 2 32.7 132 ± IO 63 ± 9 47.7 Acetyl-CoA

carboxylase 13 4- 2 8 4- I* 61.5 4 ° =E 6 26 ± 2* 65.0 72 4- 6 49 4- 8* 68.1

* Difference from the corresponding fed animals statist ically significant at a level of P < o.o 5 at least.

TABLE V

E F F E C T O F 4 8 h F A S T I N G O N A D I P O S E T I S S U E E N Z Y M E A C T I V I T Y OF D I E T - A D A P T E D R A T S

Enzyme activity, in nmoles /min per nlg protein, is given as the mean 4- S.E. for the n u m b e r of animals indicated in parentheses for each group. The mean act ivi ty after the 48 h fast is also expressed as the percentage of t ha t in the respective nonfas ted group.

Enzyme Group l : Laboratory chow Group I I : Starch Group I I I : Sucrose Fed (6) Fasted (8) % Fed (9) Fasted (9) % Fed (9) Fasted (9) %

Glucose-6- phospha te dehydrogenase 41-7 i 4.7 28.1 ~_ 3.2* 67. 5 55-4 ± 4.4 50.0 ± 6.0 90.0 70.5 =}- 6.0 49-5 ± 5 .0* 7 o.1

6-Phospho- gluconate dehydrogenase 17. 7 4- I 3 i i . i i 2.1" 62. 7 19. 5 ~- 2.0 15.8 4- 1.8 81.1 25.7 -E 2.7 17.8 ~ 2.1" 69.3

NADP-mala te dehydrogenase lO. 7 4- 1.2 5.6 -~ o.7" 52-3 21.9 4- 4.1 lO.5 .J: 1.9" 47.9 58.7 4- 9.1 22-5 i 2.3* 38.3

* Difference from the corresponding fed animals statist ically significant at a level of P < o.o5 at least.

Biochim. Biophys. Acta, 279 (1972) I29-138

134 A.M. COHEN e't al.

The prolonged main tenance of the d ie t a ry a da p t a t i on of l iver and adipose t issue enzymes m a y be seen from the effect of fast ing on thei r ac t iv i ty (Tables IV and V). In the chow-fed ra ts the ac t i v i t y of l iver p y r u v a t e kinase, g lucose-6-phosphate- and 6-phosphogluconate dehydrogenases , and acetyl-CoA carboxylase was reduced to abou t 2/3 of the value in nonfas ted ra ts (Table IV). The decrease in N A D P - m a l a t e dehydrogenase ac t iv i ty was more marked . In the s tarch-fed and sucrose-fed ra ts the ex ten t of reduct ion in enzyme act ivi t ies , due to fast ing was genera l ly s imilar to tha t seen in the chow-fed rats. Glucose-6-phosphate dehydrogenase ac t iv i ty fell somewhat more extensively , and again the decrease in N A D P - m a l a t e dehydrogenase ac t iv i ty was most prominent . The ex ten t of decrease in the ac t iv i ty of each enzyme was thus similar, p ropor t iona l to the prefas t ing value, and independen t of the previous d i e t a ry his tory. Thus, the enzyme ac t iv i ty in the l iver of sucrose-fed ra ts exceeded, af ter 48 h of fasting, those of the 48 h fas ted ra ts previous ly kept on starch, and even those of the chow-fed ra ts of Group I a t full a l imenta t ion . This is pa r t i cu la r ly evident in the case of acetyl -CoA carboxylase , poin t ing out the ample l ipogenic capac i ty acquired on the sucrose diet and pers is t ing in the fas ted s ta te .

In adipose tissue the decline in the ac t iv i ty of glucose-6-phosphate and 6-phos- phogluconate dehydrogenases in the chow-fed and sucrose-fed ra ts was s imilar in magn i tude in compar ison to the liver. On s tarch diet the decreases in the pentose- shunt enzyme act iv i t ies due to fast ing were insignificant, as were the increases due to s tarch feeding. A more m a r k e d decrease occurred in the ac t i v i t y of N A D P - m a l a t e dehydrogenase . This was especial ly evident on the sucrose diet to which this enzyme also responded most m a r k e d l y (Table I I I ) . In adipose tissue, as in the liver, the residual enzyme ac t i v i t y in the fast ing sucrose-fed ra ts was still higher than in the nonfas t ing chow-fed group.

Table VI presents the ac t iv i ty of two l iver enzymes concerned with gluconeo- genesis and thei r response to fasting. In the s tarch-fed rats, the ac t iv i ty of both g lucose-6-phosphatase and phosphoeno lpy ruva te carboxylase was sl ightly, though not significantly, lower than in the chow-fed ones. However , in the sucrose-fed group the ac t i v i t y of g lucose-6-phosphatase was or iginal ly elevated, whereas the ac t i v i t y of phosphoeno lpy ruva te carboxylase tended to be lower, in compar ison to the chow-fed group.

In response to 48 h fast the ac t i v i t y of g lucose-6-phosphatase rose s ignif icant ly in all groups. The rise in the sucrose-fed ra ts was significant and the value was the

TABLE VI

L I V E R E N Z Y M E A C T I V I T I E S R E L A T E D TO G L U C O N E O G E N E S I S I N

T H E I R R E S P O N S E TO 4 8 h F A S T

Enzyme activity, in nmoles/min per mg protein, is given as mean indicated in parentheses.

G~/oup Glucose- 6-phosphatase* *

Fed Fasted

R A T S ON D I F F E R E N T D I E T S A N D

S.E. for the number of animals

Phosphoenolpyruvate carboxylase Fed Fasled

I. Laboratory chow 129 i IO (6) I90 ~ 9 (8) I66 ~ I8 (6) 386 -a_ 3I (8) II. Starch lO4 ± 9 (9) i8i +~ 23 (9) 136 • 15 (9) 348 -L 22 (9) III. Sucrose I65 -r I2 ' (9) 225 ~ 2I (9) 132 ~ 21 (9) 390 ~ 28 (9)

* Difference from the corresponding chow-or starch-fed animals significant at P < o.o5. * * Activity expressed per mg protein in crude liver homogenate.

Biochim. Biophys. Acts, 279 (1972) 129-138

E F F E C T OF SUCROSE F E E D I N G ON ENZYME A C T I V I T Y 135

highest recorded. Percentage-wise it was somewhat lower (136%) than in the chow-fed and starch-fed groups (154 and 174%, respectively). Phosphoenolpyruvate carboxylase activity rose 2- to 3-fold in all groups as a result of fasting. The extent of the rise was not appreciably related to the previous diet.

DISCUSSION

The starch and sucrose diets offered to the experimental animals had a higher carbohydrate content than the standard laboratory chow; 72 as against 6o% (by weight). I t was therefore expected that long-term feeding of starch would produce notable increases in the activity of liver enzymes concerned with glucose utilization and lipogenesis. On the 72% sucrose diet these increases were substantially higher than on the starch diet, even though the amount of the diet consumed and the growth of the rats on sucrose was lower than on the laboratory chow or on starch diets.

Pyruvate kinase, one of the key enzymes of glycolysis, was reported to increase considerably in activity on sucrose-rich diets 8-11. In our long-term experiments pyru- vate kinase activity rose twice as much on the sucrose than on the starch diet. Since liver weight, relative to body weight, also increased on sucrose feeding, the total availability of pyruvate kinase as well as of other enzymes reported here was even greater, due to both specific induction and adaptive enlargement of the liver.

Liver pyruvate kinase activity is known to be susceptible to enhancement by feed-forward type cellular effectors such as fructose-I,6-diphosphate 18 and fructose-l- phosphate 19. In our experiments the activity of this enzyme was measured at maximal catalytic capacity conditions and it appears certain that the sucrose-induced incre- ment in activity was primarily due to an increase in the amount of enzyme protein. The decrease on fasting was relative to the prefasting activity attained on each diet, which also indicates that it was due to a decrease in the enzyme level rather than to activity modulation by effectors which, after 48 h of fasting, were most probably reduced to similar levels on all diets. These considerations apply to the sucrose-induced activity changes in other enzymes investigated here.

The remarkable rises in the activity of liver enzymes of NADPH production and especially the more than 5-fold rise in the activity of acetyl-CoA carboxylase, which is directly involved in fatty acid synthesis, emphasize the acquired capacity for en- hanced lipogenesis on the sucrose diet. This is well in agreement with the augmented incorporation of labeled precursors into lipids on sucrose diets seen by most investi- gators 2-7. Some investigators, who have similarly observed rises in the activity of en- zymes associated with lipogenesis, such as NADP-malate dehydrogenase and ATP citrate lyase (EC 4.I.3.8), did not obtain increases in the uptake of labeled acetate into lipids by liver slices of sucrose-fed rats 1°. Others observed a reduction in the incorpora- tion of labeled glucose into liver lipids both i n vi tro and in vivo2°, 21. In our hands tile sucrose diet consistently increased fatty acid and cholesterol synthesis from acetate in liver homogenates or in vivo 7 and increased the liver lipid content. Serum triglyceride and cholesterol levels were also increased.

In adipose tissue, as in the liver, the activity of NADPH-generating enzymes rose more markedly in the sucrose-fed than in the starch-fed rats. Thus the lipo- genesis-promoting effect of sucrose extends to adipose tissue as well, which is able to utilize directly both glucose and fructose ~2.

Biochim. Biophys. Acta, 279 (1972) 129-138

136 a.M. COHEN et al.

Certain differences were apparent in the pattern of behavior of enzymes of NADPH generation in the liver and adipose tissue. In the liver the original activity of glucose-6-phosphate dehydrogenase was about one half of that of 6-phosphogluco- nate dehydrogenase. As a result of the long-term sucrose diet the activity of the former rose to a much larger extent than that of the latter, so that 6-phosphogluconate de- hydrogenase now became rate limiting for entrance into the pentose shunt pathway of glucose metabolism. Conversely, as a result of the fast, the fall in the activity of glucose-6-phosphate dehydrogenase was more marked than that of 6-phosphogluco- nate dehydrogenase. Tile high susceptibility of liver glucose-6-phosphate dehydro- genase to nutritional regulation is thus evident, in contrast to 6-phosphogluconate dehydrogenase. This is also true in the case of NADP-malate dehydrogenase. The activity of this enzyme rose somewhat less impressively than that of glueose-6-phos- phate dehydrogenase as a result of sucrose diet, but declined more markedly on fasting.

In adipose tissue, on the other hand, the rise and fall in the activity of NADP- malate dehydrogenase on sucrose diet and fasting respectively, were more pronounced than the corresponding changes in the activity of glucose-6-phosphate dehydrogenase. These findings of more extensive adaptability of NADP-malate dehydrogenase in adipose tissue fit the conclusions of Katz et al. 2a, concerning the supply of NADPH during extensive lipogenesis in adipose tissue, which point out the increasing depen- dence on sources other than the pentose cycle. With regard to 6-phosphogluconate de- hydrogenase, the activity of which in adipose tissue is similar to, or lower than, that of glucose-6-phosphate dehydrogenase, no marked changes were observed, and the activity of the latter appeared rate limiting for entrance into the pentose shunt path- way throughout.

The response of hepatic enzymes of the gluconeogenic pathway in the sucrose- adapted animals is of interest. The basal activity of phosphoenolpyruvate carboxylase tended to decrease, but the extent of its rise after a 4 8 h fast was not appreciably affected. On the other hand, glucose-6-phosphatase activity in the ad l ibi tum sucrose- fed rats was substantially elevated. Bartley et al. n and Carrol ~4 also observed rises in glucose-6-phosphatase, as well as in fructose-I,6-diphosphatase activities on high sucrose diets.

The effect on glucose-6-phosphatase in sucrose-fed animals should be attributed to the fructose moiety, as the activity of this enzyme is known to rise on fructose diets ~5,26. Thus, fructose, as a glucogenic substrate, induced the activity of only one or two rate-limiting enzymes of gluconeogenesis, proximate to its entry into liver metabolism. As shown here on the sucrose diet, and elsewhere on fructose diets '-'6-28, this was coincident with the stimulation of glycolytic and lipogenic enzymes. In con- trast, starvation or diabetes are known to induce a rise in activity of glucose-6-phos- phatase and fructose-I,6-diphosphatase as well as of the two dicarboxylic acid shuttle enzymes pyruvate carboxylase and phosphoenolpyruvate carboxylase with coincident suppression of the activity of glycolytic and lipogenic enzymes. The selective induc- tion enzymes of a short segment in the pathway of gluconeogenesis with concurrent stimulation of the enzymes of glycolysis does not conform to the concept, suggested by Weber et al. ~9, that all four key enzymes of gluconeogenesis rise together as an ex- pression of one genome unit and that this occurs in conjunction with a reciprocal behavior of a group of key enzymes of glyeolysis.

The more marked increases in the activity of glycolytic and lipogenic enzymes

Biochim. Biophys. dcta, 279 (1972) I29 I38

EFFECT'OF SUCROSE FEEDING ON ENZYME ACTIVITY 13 7

in the liver of sucrose-fed as compared to starch-fed rats should be traced to the greater capacity of the liver to metabolize fructose and glucose. In the liver of rats on a regular laboratory chow fructose exceeds glucose in the rate of incorporation into pyruvate, C02 or fa t ty acids 3-fold, in the incorporation into :¢-glycerophosphate I9-fold 30 and the activity of liver fructokinase (EC 2.7.1.4) exceeds the combined activities of hexo- kinase (EC 2.7.1.1 ) and glucokinase (EC 2.7.1.2) by a factor of eleven al. In addition, there is also a difference in the sites of metabolism of these hexoses. The glucose ab- sorbed upon the hydrolysis of starch is available to all body tissues, the larger part being consumed by extrahepatic tissues. In the case of sucrose, most of the fructose moiety is absorbed intact in rats but, in contrast to glucose, the bulk of it must be metabolized by the liver, due to low extrahepatic fructose metabolisma2, 3a. Thus, the liver is exposed to a larger hexose load in the case of sucrose, with respect to starch, which is most likely to compel a greater induction of adaptive responses in the enzymes controlling hexose disposal.

The two-pronged metabolism of fructose in the liver, from the stage of fruc- tose-I-phosphate, may have a bearing on the reduced glucose tolerance. There is a rise in the flow of triose phosphate intermediates of glycolysislg,a°,al, a4 but, as demonstrated in short term fructose infusion experiments19, a~, there is a coincident flow of hexose phosphates in the converse direction of gluconeogenesis. Increased glucose-6-phos- phate production may provoke tile observed rises in glucose-6-phosphatase and glu- cose-6-phosphate dehydrogenase activities. The fact that glucose-6-phosphate ac- cumulation does not persist on more prolonged fructose administration at may be due to its breakdown by the induced glucose-6-phosphatase. At the same time the in- creased activity of glucose-6-phosphatase is not compatible with glucose phosphoryla- tion, and may interfere with glucose uptake.

On the other hand, the activity of liver glucokinase is not as high on fructose or sucrose as on glucose dietsal, 35,36 most probably in conjunction with the fact that the plasma levels of insulin, the inducer of this enzyme, are lower after fructose than after glucose or starch3S, ag. A metabolic setup seems then to develop on the sucrose regimen, in which the hepatic capacity to phosphorylate glucose and to channel glucose 6-phosphate to glycolysis is relatively low. Deficient hepatic glucose utilization was in fact demonstrated in fructose-fed rats 40. This may explain the observations that lipogenesis from glucose in contrast to other precursors is low in sucrose- adapted animals'-'t, °-~-, and a decrease in glucose tolerance occurs when a glucose load is administered.

ACKNOWLEDGEMENT

This work was supported by a Ford Foundation Grant No. DI I I /6 .

R E F E R E N C E S

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