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Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose
151
Biochimica et Biophysica Acta, 540 (1978) 151--161
© Elsevier/North-Holland Biomedical Press
BBA 2 8 4 8 9 CYCLIC AMP-MEDIATED ACTIVATION OF HEPATIC GLYCOGENOLYSIS BY ]FRUCTOSE
THOMAS BRYAN MILLER, Jr. University of Massachusetts Medical School, Department of Biochemistry, Worcester, Mass. 01605 (U.S.A.)
(Received August 30th, 1977)
Summary Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continuous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3',5'-monophosphate (cyclic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results d e m o n s t r a t e that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.
Introduction Control of hepatic glycogen metabolism b y changes in circulating glucose levels has been well-documented, both in vivo and in vitro [1--5]. Sudden increases in circulating glucose in perfused rat liver resulted in activation of hepatic glycogen synthase through conversion of the D to I form [3--5]. In alloxan and streptozotocin diabetes, hepatic glycogen synthesis is no longer responsive to alterations in glucose concentrations due to a defect in the activation of glycogen synthase [5,6]. The lesion in glucose control of hepatic glycogen synthesis in diabetes m a y involve inadequate phosphorylation of glucose in the absence of glucokinase, a defect in the activation of glycogen synthase by synthase phosphatase, or a defect in the inactivation of synthase by synthase kinase. Whitton and Hems [7] have reported that in vivo treatment of rats with either fructose or glucose results in hepatic glycogen synthase
152
activation. Whereas several recent reports suggest a transient increase in hepatic glycogen phosphorylase with fructose [8--11], continued exposure to the sugar resulted in phosphorylase inactivation and/or inhibition [8~I0,!2,13]o While glucose itself may be the effector molecule responsible for synthase activatior~ the possibility remains that a metabolite of glucose may serve this funetion~ Therefore, it was decided to determine if fructose, the phosphorylation of which is independent of glucokinase, could serve as an activator of glycogen synthase, if fructose could be shown to activate synthase in normal liver, it should prove to be a useful tool in determining if deficient glucokinase was involved in lack of control of glycogen synthesis by glucose in diabetes° Further, such a study of the effects of fructose on the isolated perfused rat ]9~er should earify the direct action of fructose on the enzymes of glycogen metabolism. In this paper, we present evidence that fructose does not activate glycogen synthase, but rather, activates glycogen phosphorylase through a cyclic AMP mediated mechanism° Methods
and Materials
Male Sprague-Dawley rats weighing 100--150 g purchased from Charles River Breeding Laboratories were used after fasting for 18 h or being fed ad libitumo Isolated liver perfusion performed in situ was modified from the technique of Mortimore [14] as described by Exton and Park [15]. Livers were perfused at 37°C with medium containing Krebs-Henseleit bicarbonate buffer, pH 7.4~ 3% Fraction V bovine serum albumin and 25% washed bovine red blood cells a~ a constant flow of ? ml per minute° The perfusion medium was recirculated through the liver without added substrate for the first 30 rain of perfusion and then passed through the liver without recirculation for a further I0 rain with or without infusion of added substrate. Perfusions were terminated by freeze° clamping livers in Wollenberger clamps precooled in liquid nitrogen. The fro zen tissue was powdered and stored until analyses could be carried out as previously described [16]o Perfusate glucose was determined by the alkaline ferricyanide method using the Technicon Auto-Analyzer or by the glucose oxidase procedure. Hepatic glycogen synthase was extracted (I00 mg tissue/m]) with I00 mM KF, I0 mM EDTA, pH 7°8, and assayed on a 8000 ~g supernatant without added sulfate using the glueose-6-phosphate filter paper assay of Thomas et al° [17]o Tissue glycogen phosphorylase after being extracted (I00 mg tissue/5 ml) with 100 mM KF, 50 mM MES, 15 mM mercaptoethanol, 5 mM EDTA, pH 6.1, was centrifuged at 8000 x g and aliquots of the supernatant were assayed with and without added 5'-AMP using the filter paper technique described by Gilboe e~: al. [18]. Tubes without added AMP contained 0.5 mM caffeine as described by Stalmans and Hers [19]. Tissue protein kinase was extracted (100 mg tissue/5 ml) with 150 mM KF, 5 mM potassium phosphate, 2 mM EDTA, pH 6.8, by a 15 s homogenization on the Polytron at 2°C. After centrifugation at 12 000 xg for 15 rain, supernatant kinase activity was determined essentially as described by Corbin and geimann [20]. 20 ~l of the soluble'extract were added to tubes containing 50 #l of 17 mM potassium phosphate, pH 6.8, 50 pg of Type [la calf thymus histone, 0.033 mM 74abeled [32P]ATP (35 cpm/pmol), 6 mM mag-
153 nesium acetate and 2 ~M cyclic AMP. After I0 min at 30°C incubations were stopped by pipetting 50 t~ 1 of the reaction mixes onto 2 × 2 cm ET-31 filter paper sqaures, dropping them into a beaker containing 10% cold trichloroacetic acid and washing with stirring for 15 min. After 3 subsequent washes with 10% trichloroacetic acid for 15 min each at r o o m temperature, papers were washed successively with 95% ethanol and anhydrous ether, then dried and counted in toluene containing 0.5% PPO in a liquid scintillation counter. Protein kinase activity was expressed as pmol of 32p incorporated into histone per minute per mg protein and as percent protein kinase in the active form (cyclic AMP independent/total × 100). Phosphorylation of endogenous substrates was subtracted from total phosphorylation to calculate histone phosphorylation. Cyclic AMP was assayed as described by Gilman [21] after being extracted from tissue and purified as previously described [16]. Protein concentrations were determined b y the method of Lowry et al. [22] on trichloroacetic acid precipitates dissolved in NaOH. Tissue glucose 6-phosphate (Glc-6-P), fructose 6-phosphate (Fru-6-P), uridine diphosphoglucose (UDPGlc) and fructose 1-phosphate (Fru-l-P) were extracted and determined spectrophotometrically as described by Bergmeyer [23]. Fructose was purchased from Fisher Chemical Co. (Catalog No. L-95, Lot 741138). [~/-32p]ATP was prepared from carrier-free 32PO4 (New England Nuclear) as described by Schultz et al. [24]. Type IIa calf t h y m u s histone was purc]hased from Sigma Chemical Co. while Amberlite MB3 was a gift from R o h m and Haas of Philadelphia. Data are expressed as mean ± standard error of the mean and statistical significance determined by Student's t test. Numbers in parentheses indicate the number of livers per mean. Results
Perfusate glucose accumulation in livers from fed and fasted rats. In order to compare the acute effects of fructose with those of glucose, livers from fed and fasted rats were perfused in situ as described in Methods and Materials. Table I shows perfusate glucose accumulation at 0, 15 and 30 min of recirculation perfusion w i t h o u t added substrate, and for the final 10 rain of flow-through perfusion without added substrate, with 28 mM glucose or with 28 mM fructose. Accumulation of glucose in the absence of added substrate in perfusates from livers of fasted rats was 0, 6, 8 and 1 I mg/100 ml at 0, 15, 30 and 40 min of perfusion, respectively. Infusion of glucose at a rate of 35 mg/min for the final 10 min resulted in a flow-through concentration of glucose of 506 mg/100 ml while infusion of fructose at the same rate resulted in an effluent glucose concentration of 33 mg/100 ml. Similar results were obtained from livers of fed rats although glucose accumulation without added substrate was greater. This probably reflects the difference in glycogen levels and, therefore, glycogenolysis between livers from fasted and fed rats. The increased glucose accumulation when fructose was infused was at least partially due to fructose gluconeo= genesis. Effect of glucose and fructose on hepatic glycogen synthase. Table II shows
154 I
TABLE
PERFUSATE GLUCOSE WITH GLUCOSE AND
ACCUMULATION FRUCTOSE
IN LIVERS
FROM
FED
AND
FASTED
RATS
INFUSED
Livers were perfused with 100 ml of substrate-free perfusion medium in a reeircuiating system for 30 rain after which the medium was allowed to flow through the livers without recizculating for a further 10 rnin. Where indicated, glucose or fructose was infused to a final concentration of 500 m g[100 ml for the final 10 rain of perfusion. With control and glucose infusion, glucose was measured on perfusate samples by the alkaline ferdcyanide method. When fruciose was infused, perfusate glucose was determhled by glucose oxidase. Diet
Infusion
mg glucose/lO0 ml
15 rain
3 0 min
0
6 +- 1
8 ± 1
0
1 9 -+ 1
26 ± I
zero time Fasted
None Glucose Fructose None Glucose Fructose
Fed
40 rain 11 506 33 32 562 62
± 2 ± 18 ± 3 ± 2 ± 22 ± 4
the results obtained when hepatic glycogen synthase activities were determined on livers from the same series of peffusions described for Table t. In livers from fasted rats, glucose infusion for the final 10 rain promoted an increase in synthase activity from 42% I in the control up to 56% I in agreement with previous reports [3--5], The increase was due to a shift in synthase activity from the D to the I form although total activity was also increased. Fructose had no effect on glycogen synthase activity in livers from fasted rats. The effect of glucose was much greater in livers from fed rats where the Glc-6-P independent form of the enzyme was doubled by glucose infusion resulting in an increase from 23% I in the control up to 40% I. Here, fructose increased glycogen synthase activity although the effect was much less than that produced by glucose. Table II demonstrates that, while glucose is a p o t e n t activator of hepatic glycogen synthase, fructose is, at best, a very weak modulator of synthase activity under these conditions. TABLE n EFFECT
OF GLUCOSE
AND FRUCTOSE
ON GLYCOGEN
SYNTHASE
IN PERFUSED
RAT LIVERS
E x p e r i m e n t a l p r o c e d u r e s w e r e t h e s a m e as d e s c r i b e d i n T a b l e I. S y n t h a s e a c t i v i t y w a s e x p r e s s e d as n m o l o f g l u c o s e i n c o r p o r a t e d i n t o g l y c o g e n p e r m g p r o t e i n p e r 1 0 m i n o~ as % s y n t h a s e i n t h e a c t i v e (I) f o r m . Diet
Infusion
Glycogen synthase (nmol) --GlC-6-P
Control (12) Glucose (6) Fructose (6) Control (19) Glucose (8) Fructose (12)
Fa~ed
Fed
* ** *** T
P P P p
< < < <
0.01 compared to control. 0.05 compared to control. 0.001 compared to control. 0.025 compared to control.
11 16 14 9 18 12
± 0.8 + 1.6 -+ 1 . 3 + 0.8 -+ 2 . 2 ± 1.1
%1
÷Glc-6.P
* T * T
25 29 32 38 45 42
+ 1.3 + 1 . 8 ** ± 2.5 T -+ 1 . 9 + 2 . 7 ** ± 2.6
42 56 43 23 40 28
-+ 1 2 2 *** +- 2 -+ 1 -+ 4 * * * -+ 3 * *
155 TABLE
III
EFFECT LIVERS
OF
GLUCOSE
AND
FRUCTOSE
ON GLYCOGEN
PHOSPHORYLASE
IN PERFUSED
RAT
Data were obtained on a separate series of livers perfused 6 months after those described in Tables I and II while experimental procedures w e r e t h e s a m e a s d e s c r i b e d i n T a b l e I. P h o s p h o r y l a s e activity was e x p r e s s e d as n m o l o f g l u c o s e 1 - p h o s p h a t e i n c o r p o r a t e d i n t o g l y c o g e n p e r m g p r o t e i n p e r 1 0 r a i n . Diet
Infusion
Glycogen phosphorylase --AMP
Fasted
Control Glucose Fructose Control Glucose Fructose
Fed
* ** *** t
P P P p
< < < <
(9) (6) (6) (19) (S) (12)
(nmol) +AMP
262 + 31 151 + 27 * 7 0 1 -+ 1 2 0 * 364-+ 26 272 + 20 ** 968 + 29"**
670 + 47 668 + 56 8 8 1 -+ 1 3 7 1110+51 1 0 8 2 -+ 5 2 1295-+ 50t
0.01 compared to control. 0.0125 compared to control. 0.001 compared to control. 0,025 compared to control,
Effect of glucose and fructose on hepatic glycogen phosphorylase. Glycogen phosphorylase activity was determined next (Table III). Under these conditions, infusion of glucose into livers from either fed or fasted rats resulted in a significant decrease in phosphorylase activity determined in the absence b u t n o t the presence of 1 mM 5'-AMP. Fructose infusion resulted in a 2 to 3-fold increase in phosphorylase activity in livers from fasted and fed rats when assayed in t:he absence of 5'-AMP. Phosphorylase activity assayed in the presence of 5'-AMP was essentially unchanged b y fructose. Table III established that the effect o f acute administration of fructose to livers of fed or fasted rats was cle~cly different from that of glucose. Effect of glucose and fructose on hepatic protein kinase. Since the data in Tab][e III demonstrated activation of phosphorylase by fructose, it seemed TABLE EFFECT LIVERS
IV OF
GLUCOSE
AND
FRUCTOSE
ON PROTEIN
KINASE
ACTIVITY
IN PERFUSED
RAT
Experimental p r o c e d u r e s a n d t i s s u e s u s e d f o r a n a l y s e s w e r e t h e s a m e a s d e s c r i b e d i n T a b l e I. P r o t e i n k i n a s e a c t i v i t y w a s e x p r e s s e d a s p m o l o f 3 2 p i n c o r p o r a t e d i n t o h i s t o r i c p e r m g p r o t e i n p e r m i n o r as % protein kinase in the active form. Adenosine 3',5'-monophosphate is abbreviated as cyclic AMP. Diet
Infusion
Protein kinase ~cyclic
Fasted
Fed
Control Glucose Fructose Control Glucose Fructose
* P < 0.01 compared
(12) (6) (6) (19) (8) (12) to control.
32 34 59 38 41 70
-+ 2 -+ 3 + 8 * + 3 _+ 3 -+ 3 *
AMP
+ cyclic AMP
% Active
125 123 128 169 172 159
26 29 49 23 24 44
-+ 1 0 -+ 1 8 -+ 1 5 + 6 -+ 5 + 7
_+ 2 + 3 + 9 * -+ 1 + 1 + 4 *
156
TABLE
V
EFFECT LIVERS
OF
GLUCOSE
AND
FRUCTOSE
ON
INTRACELLULAR
CYCLIC
AMP
IN PERFUSED
RAT
E x p e r i m e n t a l p r o c e d u r e s w e r e t h e s a m e as d e s c r i b e d f o r T a b l e I w h i l e t h e t i s s u e s u s e d t o d e t e r m i n e c y c l i c A M P l e v e l s w e r e ~he s a m e as t h o s e u s e d i n T a b l e I I L
Diet
Infusion
Cyclic A M P / r a g t i s s u e (pmol)
Fasted
C o n t r o l (6) G l u c o s e (4) F r a c z o s e (6) Control (12) Glucose (12) Fructose (12)
Fed
P < 0.001 compared
1.14 0.97 2,87 0.89 (}.81 2.40
-+ 0 . 1 7 -+ (}.12 -* 0 . 4 3 ~ ~ (}.10 ± 0.09 +- 0 , 2 4 ~
~o c o n t r o l .
appropriate to examine protein kinase activity in these same livers (Table IV). Protein kinase activity was unaffected by infusion of glucose. Fructose infusion, on the other hand, resulted in increased activities of the protein kinase in the absence of exogenous cyclic AMP in livers from both fed and fasted rats. These data suggested that the fructose activation of glycogen phosphorylase could be explained by an increase in protein kinase activity° Further, it supported the idea that the glucose activation of glycogen synthase was apparently independent of cyclic AMP dependent protein kinase activity. Effect of glucose and fructose on hepatic cyclic AMP. Intraceltular cyclic AMP levels (Table V) were essentially unaltered by glucose. Fructose, on the other hand~ produced a very consistent change in tissue levels of the cyclic nucleotide. In livers from fed or fasted rats, fructose infusion resulted in a 2 to 3-fold increase in cyclic AMP. The effect of fructose to activate protein kinase can then be attributed to the increase in intracellular cyclic AMP effected by fructose. TABLE Vl EFFECT
OF
GLUCOSE
AND
FRUCTOSE
ON METABOLIC
INTERMEDIATES
IN P E R F U S E D
RAT
Experimental
procedures
w e r e t h e s a m e as d e s c r i b e d i n T a b l e I. D a t a o n f~ue$ose 1 - p h o s p h a t e l e v e l s w a s
LIVERS
obtained
f r o m the s a m e s e r i e s o f l i v e r s d e s c r i b e d i n T a b l e I I I . A l l o t h e r v a l u e s w e r e o b t a i n e d using t h e
t i s s u e s d e s c r i b e d i n T a b l e I. G l u c o s e 6 - p h o s p h a t e , f r u c t o s e 6 - p h o s p h a t e , u r i d i n e d i p h o s p h o g l u c o s e a n d f r u c t o s e 1 - p h o s p h a t e w e r e m e a s u r e d s p e c t r o p h o t o m e t r i c a l i y i n n e u t r a l i z e d p e r c h l o r a t e e x t r a c t s utilizing the e n z y m a t i c p r o c e d u r e s d e s c r i b e d in B e r y m e y e r [ 2 3 ] .
Diet
Fed
P < ** N o t *** P < ~" P < t'P Not
Infusion
Control (15) Glucose (8) Fructose (9) 0.001 compared
(mnol/g liver) Glc-6-P
Fru-6-P
UDPGlc
Fru-l-P
3 8 +- 3 125 z 12 * 92-+ 6 * * *
8 z 1 27 z 2 * 30-* 5t
183 z 14 2 1 2 ± 1 5 ** 165 ± 12"I~
3 6 3 x 33 4 1 1 z 3 9 ** 1 0 9 7 1 -* 1 3 7 *
to control.
significantly d i f f e r e n t f r o m c o n t r o l , 0.01 compared to control and glucose. 0 . 0 0 1 c o m p a r e d t o c o n t r o l b u t n o t significantly d i f f e r e n t f r o m g l u c o s e . significantly d i f f e r e n t f r o m c o n t r o l b u t P < 0 . 0 1 c o m p a r e d t o g l u c o s e .
157
Effects of glucose and fructose on hepatic glucose 6-phosphate, fructose 6-phosphate, uridine diphosphoglucose and fructose 1-phosphate. Since the initial intention was to utilize fructose as a tool to try to activate synthase through its conversion to metabolic intermediates, we also determined the levels of Glc-6-P, Fru-6-P, UDPGlc and Fru-l-P in the same tissues. Glucose infusion produced a 3-fold increase in Glc-6-P and although fructose increased Glc-6-P levels more than 2-fold, they were significantly lower than those promoted by glucose. Infusion of either glucose or fructose resulted in about the same 3-fold increase in Fru-6-P. The effects of glucose and fructose on UDPGlc levels were quite different. Glucose infusion tended to increase UDPGlc levels while fructose tended to decrease them. Although neither proved significantly different from the control, the effect of glucose was significantly different from that of fructose. While infusion of glucose resulted in no change in Fru1-P concentrations, fructose infusion produced a 30-fold increase. Therefore, the effects of glucose and fructose on these metabolic intermediates provided additional evidence that their actions were different.
TABLE VII EFFECTS
OF COLUMN
PURIFIED
FRUCTOSE
ON THE HEPATIC
GLYCOGEN
METABOLIC
COM-
PLEX IN FED RATS
E x p e r i m e n t a l p r o c e d u r e s w e r e t h e s a m e as d e s c r i b e d in T a b l e I a n d u n i t s are t h e s a m e as p r e v i o u s l y d e s c r i b e d . A l t h o u g h b o t h series o f livers w e r e p e r f u s e d e x a c t l y t h e s a m e , d a t a p r e s e n t e d f o r p h o s p h o r y l ase, k i n a s e , a n d c y c l i c A M P w a s o b t a i n e d o n a series r u n 6 m o n t h s a f t e r t h e series u s e d f o r s y n t h a s e a n d metabolite determinations. A solution of fructose was purified over a 1 X 2 0 c m A m b e r l i t e MB-3 column. E a c h v a l u e is t h e m e a n o f v a l u e s o b t a i n e d f r o m 6 livers. Assay
Synthase
Phosphorylase
Infusion
Control Fructose
Control Fructose
Activity --Glc-6-P
+Glc-6-P
%I
1 3 -+ 2 n m o l 13 + 2 nmol
37 + 4 nmol 37 + 3 nmol
34 ± 3 34 ± 4
--AMP
+AMP
324 ± 15 nmol 9 7 0 ± 76 n m o l *
1051 ± 35 nmol 1331 + 73 nmol *
---cyclic A M P
+cyclic AMP
% Active
212 + 12 pmol 226 ± 15 pmol
16 ± 1 46 + 12 **
Kinase
Control Fructose
3 6 -+ 4 p m o l 1 0 0 ± 2 8 p m o l **
Cyclic AMP
Control Fructose
1 . 0 6 -+ 0 . 1 0 p m o l / m g t i s s u e 2 . 4 8 + 0 . 3 7 p m o l / m g t i s s u e **
Metabolites
Control Fructose
* P < 0.001 compared to control. ** P < 0 . 0 1 c o m p a r e d t o c o n t r o l .
Glc-6-P
Fru-6-P
3 2 -+ 5 n m o l / g l i v e r 56 + 1 n m o l / g l i v e r
11 ± 2 n m o l / g liver 16 ± 4 n m o l / g l i v e r
UDPGlc , 1 2 5 ± 1 1 n m o l ] g liver 1 0 8 ± 1 3 n m o l / g liver
158 TABLE
VIII
EFFECT OF ACTIVITY
PERFUSION
TIME
AND
FRUCTOSE
CONCENTRATION
ON
PHOSPHORYLASE
E x p e r i m e n t a l p r o c e d u r e s w e r e t h e s a m e as d e s c r i b e d i n T a b l e I e x c e p t t h a t a f t e r t h e i n i t i a l 3 0 r a i n r e c i r c u o lation perfusion, flow-through perfusion was begun wiih or without constant fructose infusion and cont i n u e d f o r 5 o r 3 0 r a i n . P h o s p h o r y l a s e a c t i v i t i e s w e r e e x p r e s s e d as p r e v i o u s l y d e s c r i b e d . Perfusion time (min)
Fructose infused (raM)
Phosphorylase (nmol)
--AMP
+AMP
5 (6) 5 (6)
0 9
3 8 4 -+ 3 1 6 2 6 -+ 2 4 *
1 2 2 7 +- 7 2 1258 ± 54
30 (6) 30 (6)
0 28
4 2 0 -+ 2 9 815 ± 34 *
1 4 3 0 _+ 3 8 1 5 9 8 -+ 8 2 **
* P < 0.001 compared to control. ** P < 0 . 0 5 c o m p a x e d t o c o n t r o l .
Effects of column purification on the action of fructose. In o r d e r to determ i n e if t h e e f f e c t o f f r u c t o s e was d u e to an i m p u r i t y in t h e p r e p a r a t i o n , a s o l u t i o n o f f r u c t o s e was passed o v e r ! 20 cm m i x e d - b e d i o n - e x c h a n g e c o l u m n ( A m b e r l i t e M B 3 ) in o r d e r to r e m o v e a n y c h a r g e d c o n t a m i n a n t s . T h e c o l u m n p u r i f i e d f r u c t o s e was t h e n u s e d t o r e p e a t t h e e x p e r i m e n t in livers f r o m fed rats as s h o w n in Tables I - - V I . In T a b l e V I I , f r u c t o s e infusion resulted in p r o m o t i n g changes in t h e s a m e d i r e c t i o n s and w i t h t h e s a m e significance as s h o w n previously. Similar results to t h o s e s h o w n in T a b l e V I I w e r e o b t a i n e d w h e n a p r e p a r a t i o n o f f r u c t o s e p u r c h a s e d f r o m Sigma C h e m i c a l Co. was i n f u s e d u n d e r t h e s a m e c o n d i t i o n s (data n o t s h o w n ) . T h e r e f o r e , it a p p e a r s t h a t t h e effects o f f r u c t o s e to a c t i v a t e t h e g l y c o g e n p h o s p h o r y l a s e cascade m e d i a t e d t h r o u g h cyclic AMP a c t i v a t i o n o f h e p a t i c p r o t e i n kinase is d u e to a real e f f e c t o f t h e a c u t e a d m i n i s t r a t i o n o f f r u c t o s e r a t h e r t h a n a side e f f e c t o f s o m e c o n t a m i n a n t f o u n d in t h e p r e p a r a t i o n ° Effects of concentration and perfusion time on fructose activation of phosphorylase. Since glucose and f r u c t o s e e f f e c t s on t h e i n t e r c o n v e r s i o n o f glyc o g e n p h o s p h o r y l a s e m a y be t i m e a n d c o n c e n t r a t i o n d e p e n d e n t ° similar experim e n t s w e r e carried o u t o n livers f r o m fed rats at l o w e r f r u c t o s e c o n c e n t r a t i o n s and also at shorter and longer perfusion times (Table VIII}. Using the constant: infusion of fructose combined with the flow-through perfusion technique. perfusion of livers with only 9 mM fructose for 5 rain resulted in activation of phosphorylase. Likewise, perfusion of livers with 28 mM fructose for 30 rain resulted in the same phosphorylase activation. Although not shown, cyclic AMP concentrations and protein kinase activities were elevated to the same exten~ by fructose as shown in the previous tables. From these results, it appears that fructose at both low and high concentrations activates hepatic glycogen phosphoryiase as early as 5 rain and for as long as 30 min.
Discussion Fructose was chosen for these studies since it is a hexose which does not rely on hexokinase or glucokinase for its phosphorylation. Fructose is metabolized
159 primarily by the liver and is phosphorylated directly to fructose 1-phosphate b y a specific fructokinase. The Fru-l-P is then converted to dihydroxyacetone phosphate and glyceraldehyde, a reaction catalyzed by a liver isozyme of alsolase. Glyceraldehyde can then be phosphorylated by a kinase to glyceraldehyde 3-phosphate. Therefore, all 6 carbons can be used for the production of pyruvate or glucose as dictated by the metabolic state of the liver. Since fructose metabolism escapes the controls exerted at the hexokinase and phosphofructokinase steps, it has been suggested that fructose might be a good source of substrate for immediate energy needs in situations where glucose utilization is impaired. There are a number of reports suggesting that fructose might be useful in treatment of certain disease states. Several investigations, in vivo and in vitro, have led to the conclusion that fructose is more rapidly utilized than glucose [25--28]. It has also been suggested that fructose metabolism is unchanged by diabetes [29,30] and that fructose entry into the cell is independent of insulin [31]. Due to these findings, fructose infusion, in some cases, has been introduced for the treatment of diabetic ketoacidosis. However, more recent reports indicate that administration of fructose in vivo and infusion of fructose into the perfused rat liver result in tremendous increases in hepatic Fru-l-P at the expense of ATP and total adenosine phosphate depletion along with large decreases in inorganic phosphate [32--36]. These effects of fructose are inconsistent with normal liver function and metabolic regulation [34--36] and obviously fail to support the contention that fructose infusion might be beneficial. The present data reporting the effect of fructose to elevate intracellular hepatic cyclic AMP is also inconsistent with a beneficial effect of fructose in treating diabetic ketoacidosis or liver disease. The 3-fold increases in cyclic AMP in response to fructose were shown to activate phosphorylase and are also more than sufficient for activation of gluconeogenesis and lipolysis. Since both processes result in increased glucose production and/or fatty acid release, fructose administration to an already hyperglycemic patient could o n l y be detrimental rather than beneficial. Our observation that acute infusion of fructose into the perfused rat liver resulted in elevation of hepatic cyclic AMP does not explain h o w cyclic AMP metabolism is altered b y fructose. A recent report of the effects of hexose pho,~phates on adenyl cyclase activity from partially purified rat kidney plasma membranes by Fujino and Yasumasu [37] demonstrated that 10 -s M fructose 1-phosphate doubled the activity. This finding plus the effect of fructose administration to produce large increases in hepatic fructose 1-phosphate reported in this paper and b y others [32--36] can be used to hypothesize a mec:hanism for activation of phosphorylase by fructose. The infusion of fructose into the perfused liver resulting in increased fructose 1-phosphate could lead to activation of adenyl cyclase b y fructose 1-phosphate producing an elevation of cyclic AMP. The effect of cyclic AMP to activate phosphorylase through protein kinase and phosphorylase kinase activation has been well-documented. Van den Berghe et al. [12] and Thurston et al. [13] have reported that the administration of one large dose of fructose to mice produced a marked decrease in liver phosphorylase after 20 min. Our experiments were specifi-
160
cally designed to test the acute effects of fructose with alterations in glucose levels being kept to a mimimum. Fructose was infused at a constant rate and allowed to pass through the liver only one time so that the effect of fructose to promote glucose accumulation could be avoided. In previous reports ~8,12~13] fructose was administered in one large dose and no effort was made to prevent the rapid accumulation of glucose via fructose gluconeogenesis. Consequently, many of the effects of fructose observed at 20 and 30 rain might well be due to glucose accumulation. In support of this reasoning, Walli et al. [8] reported fructose activation of hepatic phosphorylase 5 rain after fructose administration whereas phosphorylase had become inactivated 25 rain later. Therefore~ the differences are probably due to the rapid production of glucose from fructose by the liver which then results in inactivation of phosphorylase. While a 20 rain period after administration of one large bolus of fructose might be sufficient for a rather complete conversion of fructose to glucose~ a five minute exposure or constant infusion would maintain fructose levels while glucose remained low. It seems possible that once glucose levels began to increase, a futile cycle might result with the fructose-mediated cyclic AMP increase rapidly activating phosphorylase and the glucose increase rapidly inactivating phasphorylase. Under these conditions, phosphorylase activity at any given moment might reflect the relative levels of fructose and glucose. Finally, the effect of high concentrations of fructose to increase hepatic intracellular cyclic AMP accumulation could serve to explain reported differences in the effect of glucagon or cyclic AMP to stimulate fructose gluconeogenesis. Early studies using the isolated perfused rat liver [38] demonstrated that glucagon had little effect on gluconeogenesis from 20 or 30 mM fructose or 40 mM dihydroxyacetone. It was concluded that an effect of glucagon on gluconeogenesis at the phosphofructokinase-fructose diphosphatase step was unlikely. More recent studies by Veneziale [39] using subsaturating concentrations of fructose and Blair et al. [40] using lower concentrations of dihydroxyacetone have demonstrated that glucagon can produce a large stimulation of gluconeogenesis under these conditions° It appears likely that the concentrations of fructose used in the earlier study [38] were sufficient to elevate cyclic AMP and therefore mask any further effect of glucagon which might be media ated through elevated cyclic AMP° In the later studies [39,40] where fructose concentrations were probably too low to increase cyclic AMP, the effect of glucagon became apparent.
Acknowledgements This research was supported by National Institutes of Health Grant AM18269. I wish to thank Mr. James M. Murphy and Mr. J. Vicalvi for their valuable technical assistance.
References I 2 3 4
K r e u t n e r , W. a n d G o l d b e r g , N.D. ( 1 9 6 7 ) Proc. Natl. A c a d . Sci. U.S. 58, 1 5 1 5 - - 1 5 1 9 De Wulf, H. a n d Hers, H . G . ( 1 9 6 7 ) Eur. J. B i o c h e m . 2, 5 0 - - 5 6 D u s c h i a z z o , H., Exton~ J . H . a n d P a r k , C.R. ( 1 9 7 0 ) P r o c . Natl, A c a d . ScL U.S. 65, 3 8 3 - - 3 8 7 G l i n s m a n n , W.~ P a u k , G. and H e m , E. ( 1 9 7 0 ) B i o c h e m . B i o p h y s . t~es. C o m m u n ; 39~ 7 7 4 - - 7 8 2
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Miller, T.B., Jr., Hazen, R. and Lamer, J. (1973) Biochem. Biophys. Res. Commun. 53, 4 6 6 - - 4 7 4 Whitton, P.D. and Hems, D.A. (1974) Biochem. Soc. Trans. 2, 917--920 Whitton, P.D. and Hems, D.A. (1975) Biochem. J. 150, 153--165 Walli, A.K., Birkmann, L. and Schimmassek, H. (1975) Biochem. Soc. Trans. 3, 1 0 3 9 - - 1 0 4 2 Jak ob, A. and Diem, S. (1974) Biochim. Biophys. Acta 362, 469--479 Jakob, A. (1976) Mol. Cell. Endocrinol. 6, 47--58 Geelen, M.J.H. (1977) Life Sci. 20, 1 0 2 7 - - 1 0 3 4 Van den Berghe, E., Hue, L. and Hers, H.G. (1973) Biochem. J. 134, 637--645 Thurston, J.H., Jones, E.M. and Hauhart, R.E. (1974) Diabetes, 23, 597---604 Mortimore, G.E. (1963) Am. J. Physiol. 204, 699--704 E x t o n , J.H. and Park, C.R. (1967) J. Biol. Chem. 242, 2622--2636 Miller, T.B., Jr. and Lamer, J. (1973) J. Biol. Chem. 248, 3483--3488 Thomas, J.A., Schlender, K.K. and Lamer, J. (1968) Anal. Biochem. 25, 486--499 Gilboe, D.P., Larson, K.L. and Nuttall, F.Q. (1972) Anal. Biochem. 47, 20--27 Staimans, W. and Hers, H.G. (1975) Eur. J. Biochem. 54, 341--350 Corbin, J.D. and Reimann, E.M. (1974) in Methods in Enzymology, pp. 287--290, Academic Press, New Yo rk Gilman, A.G. (1970) Proc. Natl. Acad. Sei. U.S. 6 7 , 3 0 5 - - 3 1 2 Lowry, O.H., Rosebrough, N.J. Farr, A:L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Bergmeyer, H.U. (1974) in Methods of E n z y m a t i c Analysis, pp. 1238--1242, 1308--1313 and 2225--2228, Academic Press, New Y o r k Schultz, G., Hardman, J.G., Schultz, K., Davis, J.W. and Sutherland, E.W. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1721--1725 Bergstrom, J. and Hultman, E. (1967) Acta Med. Scand. 182, 93--107 Kronert, E., Herzog, H. and Wolf, F. (1969) Med. Ernh'hrung. 10, 31--32 Miller, M., Craig, J.W., Drucker, W.R. and Woodward, H. (1956) J. Biol. (Yale) 2 9 , 3 3 5 Stuhlfauth, K. and Prosiegel, R. (1952) Klin. Wochenschr. 30, 206--290 Miller, M., Drucker, W.R., Owens, J.E., Craig, J.W. and Woodward, H. (1952) J. Clin. Invest. 31, 115--125 Chernick, S.S. and Chaikoff, I.L. (1951) J. Biol. Chem. 188, 389--396 Leuthardt, F. (1960) Schweizer. Med. Wochenschr. 9 0 , 4 5 5 - - 4 9 1 Maenpaa, P.H., Raivio, K.O. and Kekomaki, M.P. (1968) Science 161, 1253--1254 Butch, H.B., Max, Jr., P., Chyu, K. and Lowry, O.H. (1969) Biochem. Biophys. Res. Commun. 34, 619--626 Woods, H.F., Eggleston, L.V. and Krebs, H.A. (1970) Biochem. J. 119, 501--510 ]Bode, J.Ch., Bode, C., R u m p e l t , H.J. and Zelder, O. (1974) in Regulation of Hepatic Metabolism, pp. 267--281, Academic Press, New York Sestoft, L. (1974) in Regulation of Hepatic Metabolism, pp. 285--299, Academic Press, New Y ork Fujino, Y. and Yasumasu, I. (1975) Biochem. Biophys. Res. Commun. 65, 1067--1072 E x t o n , J.H. and Park, C.R. (1969) J. Biol. Chem. 244, 1424--1433 Veneziale, C.M. (1971) Biochemistry 18, 3443--3447 ]Blair, J.B., Cook, D.E. and Lardy, H.A. (1973) J. Biol. Chem. 248, 3601--3607
Biochimica et Biophysica Acta, 540 (1978) 151--161
© Elsevier/North-Holland Biomedical Press
BBA 2 8 4 8 9 CYCLIC AMP-MEDIATED ACTIVATION OF HEPATIC GLYCOGENOLYSIS BY ]FRUCTOSE
THOMAS BRYAN MILLER, Jr. University of Massachusetts Medical School, Department of Biochemistry, Worcester, Mass. 01605 (U.S.A.)
(Received August 30th, 1977)
Summary Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continuous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3',5'-monophosphate (cyclic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results d e m o n s t r a t e that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.
Introduction Control of hepatic glycogen metabolism b y changes in circulating glucose levels has been well-documented, both in vivo and in vitro [1--5]. Sudden increases in circulating glucose in perfused rat liver resulted in activation of hepatic glycogen synthase through conversion of the D to I form [3--5]. In alloxan and streptozotocin diabetes, hepatic glycogen synthesis is no longer responsive to alterations in glucose concentrations due to a defect in the activation of glycogen synthase [5,6]. The lesion in glucose control of hepatic glycogen synthesis in diabetes m a y involve inadequate phosphorylation of glucose in the absence of glucokinase, a defect in the activation of glycogen synthase by synthase phosphatase, or a defect in the inactivation of synthase by synthase kinase. Whitton and Hems [7] have reported that in vivo treatment of rats with either fructose or glucose results in hepatic glycogen synthase
152
activation. Whereas several recent reports suggest a transient increase in hepatic glycogen phosphorylase with fructose [8--11], continued exposure to the sugar resulted in phosphorylase inactivation and/or inhibition [8~I0,!2,13]o While glucose itself may be the effector molecule responsible for synthase activatior~ the possibility remains that a metabolite of glucose may serve this funetion~ Therefore, it was decided to determine if fructose, the phosphorylation of which is independent of glucokinase, could serve as an activator of glycogen synthase, if fructose could be shown to activate synthase in normal liver, it should prove to be a useful tool in determining if deficient glucokinase was involved in lack of control of glycogen synthesis by glucose in diabetes° Further, such a study of the effects of fructose on the isolated perfused rat ]9~er should earify the direct action of fructose on the enzymes of glycogen metabolism. In this paper, we present evidence that fructose does not activate glycogen synthase, but rather, activates glycogen phosphorylase through a cyclic AMP mediated mechanism° Methods
and Materials
Male Sprague-Dawley rats weighing 100--150 g purchased from Charles River Breeding Laboratories were used after fasting for 18 h or being fed ad libitumo Isolated liver perfusion performed in situ was modified from the technique of Mortimore [14] as described by Exton and Park [15]. Livers were perfused at 37°C with medium containing Krebs-Henseleit bicarbonate buffer, pH 7.4~ 3% Fraction V bovine serum albumin and 25% washed bovine red blood cells a~ a constant flow of ? ml per minute° The perfusion medium was recirculated through the liver without added substrate for the first 30 rain of perfusion and then passed through the liver without recirculation for a further I0 rain with or without infusion of added substrate. Perfusions were terminated by freeze° clamping livers in Wollenberger clamps precooled in liquid nitrogen. The fro zen tissue was powdered and stored until analyses could be carried out as previously described [16]o Perfusate glucose was determined by the alkaline ferricyanide method using the Technicon Auto-Analyzer or by the glucose oxidase procedure. Hepatic glycogen synthase was extracted (I00 mg tissue/m]) with I00 mM KF, I0 mM EDTA, pH 7°8, and assayed on a 8000 ~g supernatant without added sulfate using the glueose-6-phosphate filter paper assay of Thomas et al° [17]o Tissue glycogen phosphorylase after being extracted (I00 mg tissue/5 ml) with 100 mM KF, 50 mM MES, 15 mM mercaptoethanol, 5 mM EDTA, pH 6.1, was centrifuged at 8000 x g and aliquots of the supernatant were assayed with and without added 5'-AMP using the filter paper technique described by Gilboe e~: al. [18]. Tubes without added AMP contained 0.5 mM caffeine as described by Stalmans and Hers [19]. Tissue protein kinase was extracted (100 mg tissue/5 ml) with 150 mM KF, 5 mM potassium phosphate, 2 mM EDTA, pH 6.8, by a 15 s homogenization on the Polytron at 2°C. After centrifugation at 12 000 xg for 15 rain, supernatant kinase activity was determined essentially as described by Corbin and geimann [20]. 20 ~l of the soluble'extract were added to tubes containing 50 #l of 17 mM potassium phosphate, pH 6.8, 50 pg of Type [la calf thymus histone, 0.033 mM 74abeled [32P]ATP (35 cpm/pmol), 6 mM mag-
153 nesium acetate and 2 ~M cyclic AMP. After I0 min at 30°C incubations were stopped by pipetting 50 t~ 1 of the reaction mixes onto 2 × 2 cm ET-31 filter paper sqaures, dropping them into a beaker containing 10% cold trichloroacetic acid and washing with stirring for 15 min. After 3 subsequent washes with 10% trichloroacetic acid for 15 min each at r o o m temperature, papers were washed successively with 95% ethanol and anhydrous ether, then dried and counted in toluene containing 0.5% PPO in a liquid scintillation counter. Protein kinase activity was expressed as pmol of 32p incorporated into histone per minute per mg protein and as percent protein kinase in the active form (cyclic AMP independent/total × 100). Phosphorylation of endogenous substrates was subtracted from total phosphorylation to calculate histone phosphorylation. Cyclic AMP was assayed as described by Gilman [21] after being extracted from tissue and purified as previously described [16]. Protein concentrations were determined b y the method of Lowry et al. [22] on trichloroacetic acid precipitates dissolved in NaOH. Tissue glucose 6-phosphate (Glc-6-P), fructose 6-phosphate (Fru-6-P), uridine diphosphoglucose (UDPGlc) and fructose 1-phosphate (Fru-l-P) were extracted and determined spectrophotometrically as described by Bergmeyer [23]. Fructose was purchased from Fisher Chemical Co. (Catalog No. L-95, Lot 741138). [~/-32p]ATP was prepared from carrier-free 32PO4 (New England Nuclear) as described by Schultz et al. [24]. Type IIa calf t h y m u s histone was purc]hased from Sigma Chemical Co. while Amberlite MB3 was a gift from R o h m and Haas of Philadelphia. Data are expressed as mean ± standard error of the mean and statistical significance determined by Student's t test. Numbers in parentheses indicate the number of livers per mean. Results
Perfusate glucose accumulation in livers from fed and fasted rats. In order to compare the acute effects of fructose with those of glucose, livers from fed and fasted rats were perfused in situ as described in Methods and Materials. Table I shows perfusate glucose accumulation at 0, 15 and 30 min of recirculation perfusion w i t h o u t added substrate, and for the final 10 rain of flow-through perfusion without added substrate, with 28 mM glucose or with 28 mM fructose. Accumulation of glucose in the absence of added substrate in perfusates from livers of fasted rats was 0, 6, 8 and 1 I mg/100 ml at 0, 15, 30 and 40 min of perfusion, respectively. Infusion of glucose at a rate of 35 mg/min for the final 10 min resulted in a flow-through concentration of glucose of 506 mg/100 ml while infusion of fructose at the same rate resulted in an effluent glucose concentration of 33 mg/100 ml. Similar results were obtained from livers of fed rats although glucose accumulation without added substrate was greater. This probably reflects the difference in glycogen levels and, therefore, glycogenolysis between livers from fasted and fed rats. The increased glucose accumulation when fructose was infused was at least partially due to fructose gluconeo= genesis. Effect of glucose and fructose on hepatic glycogen synthase. Table II shows
154 I
TABLE
PERFUSATE GLUCOSE WITH GLUCOSE AND
ACCUMULATION FRUCTOSE
IN LIVERS
FROM
FED
AND
FASTED
RATS
INFUSED
Livers were perfused with 100 ml of substrate-free perfusion medium in a reeircuiating system for 30 rain after which the medium was allowed to flow through the livers without recizculating for a further 10 rnin. Where indicated, glucose or fructose was infused to a final concentration of 500 m g[100 ml for the final 10 rain of perfusion. With control and glucose infusion, glucose was measured on perfusate samples by the alkaline ferdcyanide method. When fruciose was infused, perfusate glucose was determhled by glucose oxidase. Diet
Infusion
mg glucose/lO0 ml
15 rain
3 0 min
0
6 +- 1
8 ± 1
0
1 9 -+ 1
26 ± I
zero time Fasted
None Glucose Fructose None Glucose Fructose
Fed
40 rain 11 506 33 32 562 62
± 2 ± 18 ± 3 ± 2 ± 22 ± 4
the results obtained when hepatic glycogen synthase activities were determined on livers from the same series of peffusions described for Table t. In livers from fasted rats, glucose infusion for the final 10 rain promoted an increase in synthase activity from 42% I in the control up to 56% I in agreement with previous reports [3--5], The increase was due to a shift in synthase activity from the D to the I form although total activity was also increased. Fructose had no effect on glycogen synthase activity in livers from fasted rats. The effect of glucose was much greater in livers from fed rats where the Glc-6-P independent form of the enzyme was doubled by glucose infusion resulting in an increase from 23% I in the control up to 40% I. Here, fructose increased glycogen synthase activity although the effect was much less than that produced by glucose. Table II demonstrates that, while glucose is a p o t e n t activator of hepatic glycogen synthase, fructose is, at best, a very weak modulator of synthase activity under these conditions. TABLE n EFFECT
OF GLUCOSE
AND FRUCTOSE
ON GLYCOGEN
SYNTHASE
IN PERFUSED
RAT LIVERS
E x p e r i m e n t a l p r o c e d u r e s w e r e t h e s a m e as d e s c r i b e d i n T a b l e I. S y n t h a s e a c t i v i t y w a s e x p r e s s e d as n m o l o f g l u c o s e i n c o r p o r a t e d i n t o g l y c o g e n p e r m g p r o t e i n p e r 1 0 m i n o~ as % s y n t h a s e i n t h e a c t i v e (I) f o r m . Diet
Infusion
Glycogen synthase (nmol) --GlC-6-P
Control (12) Glucose (6) Fructose (6) Control (19) Glucose (8) Fructose (12)
Fa~ed
Fed
* ** *** T
P P P p
< < < <
0.01 compared to control. 0.05 compared to control. 0.001 compared to control. 0.025 compared to control.
11 16 14 9 18 12
± 0.8 + 1.6 -+ 1 . 3 + 0.8 -+ 2 . 2 ± 1.1
%1
÷Glc-6.P
* T * T
25 29 32 38 45 42
+ 1.3 + 1 . 8 ** ± 2.5 T -+ 1 . 9 + 2 . 7 ** ± 2.6
42 56 43 23 40 28
-+ 1 2 2 *** +- 2 -+ 1 -+ 4 * * * -+ 3 * *
155 TABLE
III
EFFECT LIVERS
OF
GLUCOSE
AND
FRUCTOSE
ON GLYCOGEN
PHOSPHORYLASE
IN PERFUSED
RAT
Data were obtained on a separate series of livers perfused 6 months after those described in Tables I and II while experimental procedures w e r e t h e s a m e a s d e s c r i b e d i n T a b l e I. P h o s p h o r y l a s e activity was e x p r e s s e d as n m o l o f g l u c o s e 1 - p h o s p h a t e i n c o r p o r a t e d i n t o g l y c o g e n p e r m g p r o t e i n p e r 1 0 r a i n . Diet
Infusion
Glycogen phosphorylase --AMP
Fasted
Control Glucose Fructose Control Glucose Fructose
Fed
* ** *** t
P P P p
< < < <
(9) (6) (6) (19) (S) (12)
(nmol) +AMP
262 + 31 151 + 27 * 7 0 1 -+ 1 2 0 * 364-+ 26 272 + 20 ** 968 + 29"**
670 + 47 668 + 56 8 8 1 -+ 1 3 7 1110+51 1 0 8 2 -+ 5 2 1295-+ 50t
0.01 compared to control. 0.0125 compared to control. 0.001 compared to control. 0,025 compared to control,
Effect of glucose and fructose on hepatic glycogen phosphorylase. Glycogen phosphorylase activity was determined next (Table III). Under these conditions, infusion of glucose into livers from either fed or fasted rats resulted in a significant decrease in phosphorylase activity determined in the absence b u t n o t the presence of 1 mM 5'-AMP. Fructose infusion resulted in a 2 to 3-fold increase in phosphorylase activity in livers from fasted and fed rats when assayed in t:he absence of 5'-AMP. Phosphorylase activity assayed in the presence of 5'-AMP was essentially unchanged b y fructose. Table III established that the effect o f acute administration of fructose to livers of fed or fasted rats was cle~cly different from that of glucose. Effect of glucose and fructose on hepatic protein kinase. Since the data in Tab][e III demonstrated activation of phosphorylase by fructose, it seemed TABLE EFFECT LIVERS
IV OF
GLUCOSE
AND
FRUCTOSE
ON PROTEIN
KINASE
ACTIVITY
IN PERFUSED
RAT
Experimental p r o c e d u r e s a n d t i s s u e s u s e d f o r a n a l y s e s w e r e t h e s a m e a s d e s c r i b e d i n T a b l e I. P r o t e i n k i n a s e a c t i v i t y w a s e x p r e s s e d a s p m o l o f 3 2 p i n c o r p o r a t e d i n t o h i s t o r i c p e r m g p r o t e i n p e r m i n o r as % protein kinase in the active form. Adenosine 3',5'-monophosphate is abbreviated as cyclic AMP. Diet
Infusion
Protein kinase ~cyclic
Fasted
Fed
Control Glucose Fructose Control Glucose Fructose
* P < 0.01 compared
(12) (6) (6) (19) (8) (12) to control.
32 34 59 38 41 70
-+ 2 -+ 3 + 8 * + 3 _+ 3 -+ 3 *
AMP
+ cyclic AMP
% Active
125 123 128 169 172 159
26 29 49 23 24 44
-+ 1 0 -+ 1 8 -+ 1 5 + 6 -+ 5 + 7
_+ 2 + 3 + 9 * -+ 1 + 1 + 4 *
156
TABLE
V
EFFECT LIVERS
OF
GLUCOSE
AND
FRUCTOSE
ON
INTRACELLULAR
CYCLIC
AMP
IN PERFUSED
RAT
E x p e r i m e n t a l p r o c e d u r e s w e r e t h e s a m e as d e s c r i b e d f o r T a b l e I w h i l e t h e t i s s u e s u s e d t o d e t e r m i n e c y c l i c A M P l e v e l s w e r e ~he s a m e as t h o s e u s e d i n T a b l e I I L
Diet
Infusion
Cyclic A M P / r a g t i s s u e (pmol)
Fasted
C o n t r o l (6) G l u c o s e (4) F r a c z o s e (6) Control (12) Glucose (12) Fructose (12)
Fed
P < 0.001 compared
1.14 0.97 2,87 0.89 (}.81 2.40
-+ 0 . 1 7 -+ (}.12 -* 0 . 4 3 ~ ~ (}.10 ± 0.09 +- 0 , 2 4 ~
~o c o n t r o l .
appropriate to examine protein kinase activity in these same livers (Table IV). Protein kinase activity was unaffected by infusion of glucose. Fructose infusion, on the other hand, resulted in increased activities of the protein kinase in the absence of exogenous cyclic AMP in livers from both fed and fasted rats. These data suggested that the fructose activation of glycogen phosphorylase could be explained by an increase in protein kinase activity° Further, it supported the idea that the glucose activation of glycogen synthase was apparently independent of cyclic AMP dependent protein kinase activity. Effect of glucose and fructose on hepatic cyclic AMP. Intraceltular cyclic AMP levels (Table V) were essentially unaltered by glucose. Fructose, on the other hand~ produced a very consistent change in tissue levels of the cyclic nucleotide. In livers from fed or fasted rats, fructose infusion resulted in a 2 to 3-fold increase in cyclic AMP. The effect of fructose to activate protein kinase can then be attributed to the increase in intracellular cyclic AMP effected by fructose. TABLE Vl EFFECT
OF
GLUCOSE
AND
FRUCTOSE
ON METABOLIC
INTERMEDIATES
IN P E R F U S E D
RAT
Experimental
procedures
w e r e t h e s a m e as d e s c r i b e d i n T a b l e I. D a t a o n f~ue$ose 1 - p h o s p h a t e l e v e l s w a s
LIVERS
obtained
f r o m the s a m e s e r i e s o f l i v e r s d e s c r i b e d i n T a b l e I I I . A l l o t h e r v a l u e s w e r e o b t a i n e d using t h e
t i s s u e s d e s c r i b e d i n T a b l e I. G l u c o s e 6 - p h o s p h a t e , f r u c t o s e 6 - p h o s p h a t e , u r i d i n e d i p h o s p h o g l u c o s e a n d f r u c t o s e 1 - p h o s p h a t e w e r e m e a s u r e d s p e c t r o p h o t o m e t r i c a l i y i n n e u t r a l i z e d p e r c h l o r a t e e x t r a c t s utilizing the e n z y m a t i c p r o c e d u r e s d e s c r i b e d in B e r y m e y e r [ 2 3 ] .
Diet
Fed
P < ** N o t *** P < ~" P < t'P Not
Infusion
Control (15) Glucose (8) Fructose (9) 0.001 compared
(mnol/g liver) Glc-6-P
Fru-6-P
UDPGlc
Fru-l-P
3 8 +- 3 125 z 12 * 92-+ 6 * * *
8 z 1 27 z 2 * 30-* 5t
183 z 14 2 1 2 ± 1 5 ** 165 ± 12"I~
3 6 3 x 33 4 1 1 z 3 9 ** 1 0 9 7 1 -* 1 3 7 *
to control.
significantly d i f f e r e n t f r o m c o n t r o l , 0.01 compared to control and glucose. 0 . 0 0 1 c o m p a r e d t o c o n t r o l b u t n o t significantly d i f f e r e n t f r o m g l u c o s e . significantly d i f f e r e n t f r o m c o n t r o l b u t P < 0 . 0 1 c o m p a r e d t o g l u c o s e .
157
Effects of glucose and fructose on hepatic glucose 6-phosphate, fructose 6-phosphate, uridine diphosphoglucose and fructose 1-phosphate. Since the initial intention was to utilize fructose as a tool to try to activate synthase through its conversion to metabolic intermediates, we also determined the levels of Glc-6-P, Fru-6-P, UDPGlc and Fru-l-P in the same tissues. Glucose infusion produced a 3-fold increase in Glc-6-P and although fructose increased Glc-6-P levels more than 2-fold, they were significantly lower than those promoted by glucose. Infusion of either glucose or fructose resulted in about the same 3-fold increase in Fru-6-P. The effects of glucose and fructose on UDPGlc levels were quite different. Glucose infusion tended to increase UDPGlc levels while fructose tended to decrease them. Although neither proved significantly different from the control, the effect of glucose was significantly different from that of fructose. While infusion of glucose resulted in no change in Fru1-P concentrations, fructose infusion produced a 30-fold increase. Therefore, the effects of glucose and fructose on these metabolic intermediates provided additional evidence that their actions were different.
TABLE VII EFFECTS
OF COLUMN
PURIFIED
FRUCTOSE
ON THE HEPATIC
GLYCOGEN
METABOLIC
COM-
PLEX IN FED RATS
E x p e r i m e n t a l p r o c e d u r e s w e r e t h e s a m e as d e s c r i b e d in T a b l e I a n d u n i t s are t h e s a m e as p r e v i o u s l y d e s c r i b e d . A l t h o u g h b o t h series o f livers w e r e p e r f u s e d e x a c t l y t h e s a m e , d a t a p r e s e n t e d f o r p h o s p h o r y l ase, k i n a s e , a n d c y c l i c A M P w a s o b t a i n e d o n a series r u n 6 m o n t h s a f t e r t h e series u s e d f o r s y n t h a s e a n d metabolite determinations. A solution of fructose was purified over a 1 X 2 0 c m A m b e r l i t e MB-3 column. E a c h v a l u e is t h e m e a n o f v a l u e s o b t a i n e d f r o m 6 livers. Assay
Synthase
Phosphorylase
Infusion
Control Fructose
Control Fructose
Activity --Glc-6-P
+Glc-6-P
%I
1 3 -+ 2 n m o l 13 + 2 nmol
37 + 4 nmol 37 + 3 nmol
34 ± 3 34 ± 4
--AMP
+AMP
324 ± 15 nmol 9 7 0 ± 76 n m o l *
1051 ± 35 nmol 1331 + 73 nmol *
---cyclic A M P
+cyclic AMP
% Active
212 + 12 pmol 226 ± 15 pmol
16 ± 1 46 + 12 **
Kinase
Control Fructose
3 6 -+ 4 p m o l 1 0 0 ± 2 8 p m o l **
Cyclic AMP
Control Fructose
1 . 0 6 -+ 0 . 1 0 p m o l / m g t i s s u e 2 . 4 8 + 0 . 3 7 p m o l / m g t i s s u e **
Metabolites
Control Fructose
* P < 0.001 compared to control. ** P < 0 . 0 1 c o m p a r e d t o c o n t r o l .
Glc-6-P
Fru-6-P
3 2 -+ 5 n m o l / g l i v e r 56 + 1 n m o l / g l i v e r
11 ± 2 n m o l / g liver 16 ± 4 n m o l / g l i v e r
UDPGlc , 1 2 5 ± 1 1 n m o l ] g liver 1 0 8 ± 1 3 n m o l / g liver
158 TABLE
VIII
EFFECT OF ACTIVITY
PERFUSION
TIME
AND
FRUCTOSE
CONCENTRATION
ON
PHOSPHORYLASE
E x p e r i m e n t a l p r o c e d u r e s w e r e t h e s a m e as d e s c r i b e d i n T a b l e I e x c e p t t h a t a f t e r t h e i n i t i a l 3 0 r a i n r e c i r c u o lation perfusion, flow-through perfusion was begun wiih or without constant fructose infusion and cont i n u e d f o r 5 o r 3 0 r a i n . P h o s p h o r y l a s e a c t i v i t i e s w e r e e x p r e s s e d as p r e v i o u s l y d e s c r i b e d . Perfusion time (min)
Fructose infused (raM)
Phosphorylase (nmol)
--AMP
+AMP
5 (6) 5 (6)
0 9
3 8 4 -+ 3 1 6 2 6 -+ 2 4 *
1 2 2 7 +- 7 2 1258 ± 54
30 (6) 30 (6)
0 28
4 2 0 -+ 2 9 815 ± 34 *
1 4 3 0 _+ 3 8 1 5 9 8 -+ 8 2 **
* P < 0.001 compared to control. ** P < 0 . 0 5 c o m p a x e d t o c o n t r o l .
Effects of column purification on the action of fructose. In o r d e r to determ i n e if t h e e f f e c t o f f r u c t o s e was d u e to an i m p u r i t y in t h e p r e p a r a t i o n , a s o l u t i o n o f f r u c t o s e was passed o v e r ! 20 cm m i x e d - b e d i o n - e x c h a n g e c o l u m n ( A m b e r l i t e M B 3 ) in o r d e r to r e m o v e a n y c h a r g e d c o n t a m i n a n t s . T h e c o l u m n p u r i f i e d f r u c t o s e was t h e n u s e d t o r e p e a t t h e e x p e r i m e n t in livers f r o m fed rats as s h o w n in Tables I - - V I . In T a b l e V I I , f r u c t o s e infusion resulted in p r o m o t i n g changes in t h e s a m e d i r e c t i o n s and w i t h t h e s a m e significance as s h o w n previously. Similar results to t h o s e s h o w n in T a b l e V I I w e r e o b t a i n e d w h e n a p r e p a r a t i o n o f f r u c t o s e p u r c h a s e d f r o m Sigma C h e m i c a l Co. was i n f u s e d u n d e r t h e s a m e c o n d i t i o n s (data n o t s h o w n ) . T h e r e f o r e , it a p p e a r s t h a t t h e effects o f f r u c t o s e to a c t i v a t e t h e g l y c o g e n p h o s p h o r y l a s e cascade m e d i a t e d t h r o u g h cyclic AMP a c t i v a t i o n o f h e p a t i c p r o t e i n kinase is d u e to a real e f f e c t o f t h e a c u t e a d m i n i s t r a t i o n o f f r u c t o s e r a t h e r t h a n a side e f f e c t o f s o m e c o n t a m i n a n t f o u n d in t h e p r e p a r a t i o n ° Effects of concentration and perfusion time on fructose activation of phosphorylase. Since glucose and f r u c t o s e e f f e c t s on t h e i n t e r c o n v e r s i o n o f glyc o g e n p h o s p h o r y l a s e m a y be t i m e a n d c o n c e n t r a t i o n d e p e n d e n t ° similar experim e n t s w e r e carried o u t o n livers f r o m fed rats at l o w e r f r u c t o s e c o n c e n t r a t i o n s and also at shorter and longer perfusion times (Table VIII}. Using the constant: infusion of fructose combined with the flow-through perfusion technique. perfusion of livers with only 9 mM fructose for 5 rain resulted in activation of phosphorylase. Likewise, perfusion of livers with 28 mM fructose for 30 rain resulted in the same phosphorylase activation. Although not shown, cyclic AMP concentrations and protein kinase activities were elevated to the same exten~ by fructose as shown in the previous tables. From these results, it appears that fructose at both low and high concentrations activates hepatic glycogen phosphoryiase as early as 5 rain and for as long as 30 min.
Discussion Fructose was chosen for these studies since it is a hexose which does not rely on hexokinase or glucokinase for its phosphorylation. Fructose is metabolized
159 primarily by the liver and is phosphorylated directly to fructose 1-phosphate b y a specific fructokinase. The Fru-l-P is then converted to dihydroxyacetone phosphate and glyceraldehyde, a reaction catalyzed by a liver isozyme of alsolase. Glyceraldehyde can then be phosphorylated by a kinase to glyceraldehyde 3-phosphate. Therefore, all 6 carbons can be used for the production of pyruvate or glucose as dictated by the metabolic state of the liver. Since fructose metabolism escapes the controls exerted at the hexokinase and phosphofructokinase steps, it has been suggested that fructose might be a good source of substrate for immediate energy needs in situations where glucose utilization is impaired. There are a number of reports suggesting that fructose might be useful in treatment of certain disease states. Several investigations, in vivo and in vitro, have led to the conclusion that fructose is more rapidly utilized than glucose [25--28]. It has also been suggested that fructose metabolism is unchanged by diabetes [29,30] and that fructose entry into the cell is independent of insulin [31]. Due to these findings, fructose infusion, in some cases, has been introduced for the treatment of diabetic ketoacidosis. However, more recent reports indicate that administration of fructose in vivo and infusion of fructose into the perfused rat liver result in tremendous increases in hepatic Fru-l-P at the expense of ATP and total adenosine phosphate depletion along with large decreases in inorganic phosphate [32--36]. These effects of fructose are inconsistent with normal liver function and metabolic regulation [34--36] and obviously fail to support the contention that fructose infusion might be beneficial. The present data reporting the effect of fructose to elevate intracellular hepatic cyclic AMP is also inconsistent with a beneficial effect of fructose in treating diabetic ketoacidosis or liver disease. The 3-fold increases in cyclic AMP in response to fructose were shown to activate phosphorylase and are also more than sufficient for activation of gluconeogenesis and lipolysis. Since both processes result in increased glucose production and/or fatty acid release, fructose administration to an already hyperglycemic patient could o n l y be detrimental rather than beneficial. Our observation that acute infusion of fructose into the perfused rat liver resulted in elevation of hepatic cyclic AMP does not explain h o w cyclic AMP metabolism is altered b y fructose. A recent report of the effects of hexose pho,~phates on adenyl cyclase activity from partially purified rat kidney plasma membranes by Fujino and Yasumasu [37] demonstrated that 10 -s M fructose 1-phosphate doubled the activity. This finding plus the effect of fructose administration to produce large increases in hepatic fructose 1-phosphate reported in this paper and b y others [32--36] can be used to hypothesize a mec:hanism for activation of phosphorylase by fructose. The infusion of fructose into the perfused liver resulting in increased fructose 1-phosphate could lead to activation of adenyl cyclase b y fructose 1-phosphate producing an elevation of cyclic AMP. The effect of cyclic AMP to activate phosphorylase through protein kinase and phosphorylase kinase activation has been well-documented. Van den Berghe et al. [12] and Thurston et al. [13] have reported that the administration of one large dose of fructose to mice produced a marked decrease in liver phosphorylase after 20 min. Our experiments were specifi-
160
cally designed to test the acute effects of fructose with alterations in glucose levels being kept to a mimimum. Fructose was infused at a constant rate and allowed to pass through the liver only one time so that the effect of fructose to promote glucose accumulation could be avoided. In previous reports ~8,12~13] fructose was administered in one large dose and no effort was made to prevent the rapid accumulation of glucose via fructose gluconeogenesis. Consequently, many of the effects of fructose observed at 20 and 30 rain might well be due to glucose accumulation. In support of this reasoning, Walli et al. [8] reported fructose activation of hepatic phosphorylase 5 rain after fructose administration whereas phosphorylase had become inactivated 25 rain later. Therefore~ the differences are probably due to the rapid production of glucose from fructose by the liver which then results in inactivation of phosphorylase. While a 20 rain period after administration of one large bolus of fructose might be sufficient for a rather complete conversion of fructose to glucose~ a five minute exposure or constant infusion would maintain fructose levels while glucose remained low. It seems possible that once glucose levels began to increase, a futile cycle might result with the fructose-mediated cyclic AMP increase rapidly activating phosphorylase and the glucose increase rapidly inactivating phasphorylase. Under these conditions, phosphorylase activity at any given moment might reflect the relative levels of fructose and glucose. Finally, the effect of high concentrations of fructose to increase hepatic intracellular cyclic AMP accumulation could serve to explain reported differences in the effect of glucagon or cyclic AMP to stimulate fructose gluconeogenesis. Early studies using the isolated perfused rat liver [38] demonstrated that glucagon had little effect on gluconeogenesis from 20 or 30 mM fructose or 40 mM dihydroxyacetone. It was concluded that an effect of glucagon on gluconeogenesis at the phosphofructokinase-fructose diphosphatase step was unlikely. More recent studies by Veneziale [39] using subsaturating concentrations of fructose and Blair et al. [40] using lower concentrations of dihydroxyacetone have demonstrated that glucagon can produce a large stimulation of gluconeogenesis under these conditions° It appears likely that the concentrations of fructose used in the earlier study [38] were sufficient to elevate cyclic AMP and therefore mask any further effect of glucagon which might be media ated through elevated cyclic AMP° In the later studies [39,40] where fructose concentrations were probably too low to increase cyclic AMP, the effect of glucagon became apparent.
Acknowledgements This research was supported by National Institutes of Health Grant AM18269. I wish to thank Mr. James M. Murphy and Mr. J. Vicalvi for their valuable technical assistance.
References I 2 3 4
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