Eur. J . Biochem. 88, 79-85 (1978)
Inverse Effects of D-Galactosamine and Inorganic Phosphate on Glycogenolysis in Isolated Rat Hepatocytes Rita STERMANN, Shreepad R . WAGLE, and Karl DECKER Biochemisches Institut an der Medizinischen Fakultat der Albert-Ludwigs-Universitat. Freiburg i. Br (Received February 20, 1978)
D-Galactosamine (2 mM) lowered the intracellular Pi level of isolated rat hepatocytes within 20 min from 3.6 to 2.3 and the ATP content from 2.34 to 1.86 mmol per kg wet cells. At the same time, the rate of glycogenolysis decreased to 55% and of glucose output to 45%. Inorganic phosphate added to the incubation medium raised the intracellular Pi level in a dosedependent manner. At an extracellular Pi concentration of 20 mM, the intracellular Pi content rose within 20 min to 6.4 and the ATP content to 2.96 mmol per kg wet cells. This increase was accompanied by a two-fold stimulation of glycogenolysis and release of glucose. D-Galactosamine (2 mM) and potassium phosphate (20 mM) supplied simultaneously counteracted their individual effects. Since the activities of the enzymes involved in glucose formation from glycogen including the glucagon-stimulated adenyl cyclase activity were unaltered by D-galactosamine treatment, it is concluded that in isolated hepatocytes phosphorolysis of glycogen is the limiting step in its conversion to glucose, that the intracellular Pi level is a limiting factor of phosphorylase a activity, that the intracellular Pi content and the rate of glycogenolysis can be increased by raising the extracellular Pi concentration, and that the net influx from elevated medium Pi levels can effectively counteract the phosphate trapping capacity of galactosamine, but not that of 20 mM fructose. D-Galactosamine is metabolized in liver by the enzymes of the galactose pathway [l - 31 ;ATP-dependent D-galactosamine 1-phosphate formation is followed by uridylylation with UDP-glucose as donor resulting eventually in the accumulation of UDPhexosamines and -N-acetyl-hexosamines in the hepatocytes [4]. This uridylate trapping by the amino sugar was found to elicit a temporal deficiency of uridine phosphates and UDP-hexoses leading to irreversible liver injury [5]. As a result of UDP-glucose depletion in livers of D-galactosamine-treated animals, glycogen synthesis is arrested followed by an almost complete loss of glycogen 161. Studies on the effect of D-galactosamine on glycogen metabolism in hepatocytes in vitvo showed, on the other hand, that the amino sugar lowered the rate of glycogen breakdown, glucose output and lactate formation in hepatocytes [7]. ~ Ahhveviation. Cyclic AMP, adenosine 3’: 5’-monophosphate. Enzymes. Galactokinase or ATP: D-galactose l-phosphotransferase (EC 2.7.1.6) glucose-6-phosphatase or ~-glucose-6-phosphate phosphohydrolase (EC 3.1.3.9); ketohexokinase or ATP: o-fructose I-phosphotransferase (EC 2.7.1.3) ; phosphoglucomutase or cc-Dglucose-l,6-bisphosphate:a-D-glucose-1-phosphate phosphotransferase (EC 2.7.5.1); phosphorylase, liver glycogen phosphorylase or 1 , 4 - ~ - ~ - g l u c:a northophosphate cc-glycosyltransferase (EC 2.4.1.2).
The mechanism of this additional D-galactosamine effect on hepatic carbohydrate metabolism, however, could not be deduced from these experiments. The following feasible causes were considered. Firstly, that the amino sugar or one of its metabolites inhibits competitively an early step of glycogenolysis. Secondly, that galactosamine affects the interconversion of phosphorylase, e.g. by reducing the level of cyclic AMP or by a change of the phosphorylation state. Thirdly, that galactosamine induces a trapping of intracellular inorganic phosphate and thereby inhibits phosphorolysis of glycogen by depletion of a cosubstrate. A transient decrease of the inorganic phosphate content has been observed in liver after a fructose load [8] and treatment with 2-deoxy-D-galactose [9]. This communication will show that the decrease of the hepatic Pi content is responsible for the D-galactosamine-mediated inhibition of glycogenolysis.
~_ _
MATERIALS AND METHODS D-Galactosamine . HCl was obtained from C. Roth OHG (Karlsruhe), collagenase, grade I1 purity, from Worthington Biochemical Corp. (Freehold, N.J., U.S.A.), glycogen, coenzymes and enzymes from
Effects of Galactosamine and Pi on Glycogenolysis
80
Boehringer Mannheim GmbH (Mannheim). L-Lactic acid and oleate were purchased from Serva GmbH (Heidelberg), glucagon from Sigma Chemical CO. (St Louis, Mo., U.S.A.). a-~-(U-'~C]glucose l-phosphate was supplied by Amersham-Buchler GmbH (Braunschweig). Glassware brought in contact with hepatocytes was pretreated with siliclad (Clay Adams, Parsippany, N.J., U.S.A.). Female Wistar rats (Ivanovas, Kisslegg) weighing 165- 180 g were used. The animals had unlimited access to a standard diet (Altromin R) and water. Liver cells were prepared by the method of Berry and Friend [lo] without hyaluronidase and with slight modifications as reported previously [ll]. 1 ml of cell suspension (50- 60 mg of cells) in a 50-ml conical flask was supplemented with 2 ml of Umbreit/Ringer/carbonate (25 mM) buffer, pH 7.4 and the indicated additions. The vials were gassed with 95% 0 , / 5 % COZ and incubated at 37 "C in a water bath (90 oscillations x min-'). Reactions were discontinued by transfer of the vials into an ice bath, centrifugation at 3000 x g for 5 min at 4 "C, and deproteinization with perchloric acid (final concentration 0.1 M). Separation of liver cell homogenates into a cytosolic and a microsomal fraction was performed according to De Duve et al. [12]. Glucose [13], glycogen [14], glucose 1-phosphate and glucose 6-phosphate [15], ATP [16], ADP and AMP [17] and inorganic phosphate [18] were determined enzymatically. Cyclic AMP was assayed by the competitive protein-binding technique [I91 after purification of the samples by anion-exchange chromatography on Dowex AG 1 x 8 (formate) which had been equilibrated with 0.1 M formic acid. The intracellular content of inorganic phosphate was measured in the following way. After the incubation period, the cell suspension was cooled on ice and centrifuged rapidly at 4 "C for 5 min at 3000 x g . The pellet was washed once with 5 ml of ice-cold 10mM triethanolamine .HC1 buffer, pH 7.4, containing 250 mM sucrose and 0.2 mM sodium phosphate. The resulting pellet was frozen immediately in liquid nitrogen and stored at - 70 "C for 1 h. After thawing, the cells were disrupted by homogenization in 1 ml 0.5 M perchloric acid and centrifuged. The supernatant was neutralized by addition of potassium hydroxide and recentrifuged. Inorganic phosphate was determined in the supernatant. The washing of the cells with the triethanolamine/ sucrose/phosphate buffer did not alter the intracellular P, level (Table 1). However, more than 0.8 mM phosphate in the washing buffer yielded erroneously high values of intracellular P,. Phosphoglucomutase was assayed by the method of Lowry and Passonneau [20] and glucose-6-phosphatase according to Appelmans et al. [21]. The phosphorylase assay in the direction of glycogen synthesis was carried out at 37 "C according to Stalmans and
Table 1. Intracellular P , content of isolated rat hepatocytes after washing with hujjers containing sucrose and various Pi concentrations 50 mg of cells were incubated for 30 min in Umbreit/Ringer/carbonate buffer, pH 7.4, containing 0.4 mM Pi. For further details see text Pi concentration in washing buffer
Pi content
mM
mmol/kg wet wt
-
0.16 0.35 0.8 1.6 a
3.55 3.58 3.53 3.62 3.86 4.73
Cells not subjected to washing procedure.
Hers [22], whereby only the a form of the enzyme is measured. One unit of enzyme activity is defined as 1 pmol of ['4C]glucose 1-phosphate incorporated into glycogen per min. The phosphorylase a assay in the direction of glycogen breakdown was carried out by a modified [23] procedure of Maddaiah and Madsen [24]. Protein was determined according to Lowry et al. [25].
RESULTS AND DISCUSSION In the presence of D-galactosamine, glucose output by isolated hepatocytes was inhibited in a dosedependent manner [7]. This effect of the aminosugar appeared to be due to a decreased rate of glycogen breakdown. The inhibition of glycogenolysis and glucose production set in several min after D-galactosamine addition and remained constant for at least 60 min, indicating a rapid mechanism responsible for the D-galactosamine-specific effect on glycogenolysis. Several enzymes are involved in the formation of glucose from glycogen including the hormonally regulated interconvertible phosphorylase, phosphoglucomutase and glucose-6-phosphatase. The specific activities of these enzymes were assayed in extracts of hepatocytes preincubated with D-galactosamine and were found to be the same as those from control cells (Table 2); similar results were obtained with livers from rats treated with the amino sugar in vivo and when D-galactosamine was added to the enzyme assay itself. These findings are in line with previous determinations of metabolite levels in D-galactosaminetreated hepatocytes [7] which ruled out an impairment of the activities of phosphoglucomutase and glucose6-phosphatase as well as of the glycolytic pathway leading to lactate. The greatly reduced lactate levels found after D-galactosamine addition to a hepatocyte suspension [7] do not appear to be due to an enhanced
R. Stermann, S. R. Wagle, and K. Decker
81
Table 2. Activities oftlie enzynies involved in the conversion of glycogen to glucose, both in the presence and absence of l rn M u-galactosarnine Phosphorylase a and phosphoglucomutase were measured in the cytosolic, glucose-6-phosphatase in the rnicrosomal fraction of homogenized hepatocytes. Methods were: (A) cells homogenized by freezing (- 180 "C) and thawing; (B) cells homogenized by NZ cavilation [26] ( 5 atm, 20 min); (S) assayed by incorporation of [U-'4C]glucose 1-phosphate into glycogen at 37 "C; (D) assayed by glucose I-phosphate formation from added glycogen at 25 "C; for details see Methods ~
Enzyme
~
Method
Control
With galactosamine
u x g protein-' A; S B; S A; D
1772 174.7 69.0
158.9 167.9 65.8
Phosphoglucomutase
A
398
400
Glucose-6-phosphatase
A
85
81
Phosphorylase a
Table 3. Oxygen consumption by rat hepatocytes in the presence and absence of I rnM n-galactosamine About 50 mg of isolated cells were incubated in Umbreit/Ringer/ carbonate buffer, pH 7.4, in Warburg vessels; gas phase, air; 4 M KOH in center well. Additions were tipped in from the sidearm after thermal equilibration Additions
0 2
mM None Galactosamine
consumption
mmol x min-' x (kg wet wt)-' 2.32
1
2.0
Lactate Oleate
10 1
3.74
Galactosamine Lactate Oleate
1 10 1
3.38
oxidation of C3 compounds, since the 0 2 consumption both in the presence and in the absence of oleate and lactate is hardly affected by D-galactosamine treatment (Table 3). It follows that the decreased lactate production after addition of the amino sugar is the result of an impaired glycogenolysis rather than an inhibited glycolysis or enhanced oxidation. Phosphorylase a activity is influenced by many effectors; especially the level of cyclic AMP is thought to play an important role in the hormone-dependent regulation of hepatic phosphorylase activity [27]. A possible effect of D-galactosamine on the adenylate cyclase activity was investigated by measuring the glucagon-dependent response of the cyclic AMP concentration in hepatocytes in the presence and absence of 1 mM D-galactosamine (Fig. 1). The insensitivity
I
00
5
10
Time (min)
Fig. 1. Effect of 1 rnM D-gu/uctoSUmine on the glucagon-stimulated cyclic AMP,formation. Full symbols, o-galactosamine-treated cells; open symbols, control cells. (A, A) 10 nM glucagon added at zero time after 10 min preincubation; (0, 0 ) unstimulated cells
of the glucagon-elicited cyclic AMP burst to 1 mM D-galactosamine is in agreement with the corresponding glucagon-stimulated glucose production [7]: although the absolute value of glucose release into the incubation medium decreased in the presence of D-galactosaniine, its relative enhancement by glucagon remained about the same at all D-galactosamine concentrations tested. The relation of the active and the inactive form of pyruvate dehydrogenase, another interconvertible enzyme, was shown to depend on the phosphorylation state (ATP/ADP . Pi) and the energy charge, respectively 1281. Addition of D-galactosamine to hepatocytes elicited a transient decrease of the ATP content, the phosphorylation state and the energy charge (Table 4). Therefore, phosphorylase a activity was determined in extracts of D-galactosamine-pretreated hepatocytes both in the anabolic and catabolic direction (Table 2). Although the methods used for cell disruption were considered to preserve the content of active (phosphorylated) enzyme [29], no difference was found between D-galactosamine-pretreated and control hepatocytes. The intracellular level of inorganic phosphate can not only influence glycogenolysis by way of the phosphorylation state but also as a substrate of the glycogen phosphorylase reaction itself. The K, of Pi and purified liver phosphorylase a was shown [24] to depend on the glycogen concentration present in the assay and to vary between 7.3 mM (at 0.5 mM glycogen) and 1.09 mM (at 25.2 mM). By measurements of phosphorylase a activity in a crude cytosolic fraction of the hepatocytes a K , for Pi of 1.78 mM was obtained in the presence of 12 mM
Effects of Galactosamine and Pi on Glycogenolysis
82
Table 4. EfJect of D-galuctosamine and potassium phosphate on the phosphorylution state qf isolated rat hepatocytes 50 mg of cells were suspended in Utnbreit/Ringer/carbonate buffer, pH 7.4. After addition of the indicated compounds, the incubations were continued for 20 min. The cells were then processed and analyzed as described in Methods. The contents are given as mean values 5 S.D. ( n z 7) Additions
ATP
ADP
AMP
Pi
C ATP
+ADP + AMP
ATP/ ADP
Energy charge
Phosphorylation state
mmol/kg wet wt ~-
None
2.34i0.34
0.66k0.04
0.23k0.02
3.59t0.63
3.21 k 0 . 2 7
3.55k0.63
0.82+0.03
0.9Yk0.38
Phosphate,20mM
2.96k0.12
0.63+0.01
0.14k0.01
6.42k0.99
3.65It0.18
4.60i0.29
0.88k0.02
0.72f0.05
Galactosamine,2mM
1.86i0.39
0.94k0.09
0.30k0.03
2.33t0.83
3.02i0.28
1.92k0.36
0.75k0.04
0.82k0.15
Phosphate, 20 mM +- galactosamine, 2 mM
2.38+0.08
0.72k0.04
0.21+0.02
4.7Yf1.32
3.27k0.15
3,2750.22
0.83i0.03
0.68+0.05
75
1
I
50
25
-
Km,app = 1.78rnM
,'
-
1
0
1 2 l/[Pi](mM-')
3
4
0
10
20
30 40 [Pi] (mMi
50
60
Fig. 2. Dependence oJ the phosplioryluse a uciivity in the cytosolic fraction of heputocytes on Pi concentration. Enzyme activity was measured by glucose I-phosphate formation from added glycogen (see Methods). Y is expressed as nmol glycosyl groups released per min
Fig. 3. Dependence of glucose output on the eutrucellular concentration of inorganicphosphate. Full symbols, in the presence of 1 m M D-galactosamine; open symbols, control cells
glycogen and in the absence of 5'-AMP (Fig. 2). Since glycogen forms particles of varying molecular weight to which phosphorylase is tightly bound, it is not possible to evaluate the level of saturation of the enzyme in intact cells. Assuming conditions in isolated hepatocytes corres'ponding to a K , value of about 1.S mM, however, the intracellular Pi content of 3.6 mM (Table 4) would not be sufficient to saturate the phosphorylase a . It was considered possible that a reduction of the intracellular Pi level results in a decreased actual glycogenolytic activity of the enzyme. D-Galactosamine is rapidly converted to D-galactosamine 1-phosphate by ATP in the presence of galactokinase [l]and subsequently converted to UDP derivatives of the amino sugar. Their accumulation
within the hepatocyte not only leads to uridylate trapping [4] but also to a transient increase of the total organic phosphate. It was expected that this D-galactosamine-induced phosphate trapping provokes a deficit of intracellular inorganic phosphate whose duration depends on the rate of net influx of Pi from the extracellular space. The following experiments were designed to study the effect of D-galactosamine on the intracellular Pi content of isolated hepatocytes; also, the feasibility of an increased extracellular Pi concentration as a counterbalancing measure was tested. Uptake of Pi by Ehrlich ascites tumor cells [30] and increased phosphate contents as well as stimulation of the renal glucose output after severance of the blood supply [31]
R. Stermann, S. R. Wagle, and K. Decker I
I*
3
-r c
a,
3
. -
s p 0
c
ar
. 3
m 1 0
- 60 3e
-a-
-cn
-
-m
E
b
E
E
4 ,
3
%
c
2 "
B 80
3 40
P
x
a,
9
20
2
5 0
0
O
O
'
Time (min)
-
B 3 0 Time fmin)
4
0
D
c
3 80
c
a,
. 3
n,
1
- 60 a v)
-cne x
-mx
40
b a,
9
20
5
0
0
10
20 30 Time (min)
40
0
10
20 30 Time (rnin)
40
Fig. 4. Effects of'tlie extracellular concentrations of D-galactosamine and Pion glycogen hreakdown, gluco.ve ourpur and inrrucellular Pi content. Glycosyl groups released from glycogen; (0-) glucose output to the incubation medium; (A A) intracellular Pi content. (A) Control; (B) + 2 mM galactosamine; (C) + 20 m M P,; (D) + 20 mM Pi + 2 m M galactosamine (0-0)
had already been observed. After a reliable method was developed to determine the intracellular Pi content of cells incubated in excessive concentrations of extracellular Pi (Table l), it became possible to study the relation between the extracellular and intracellular levels of inorganic phosphate and the rate of glycogenolysis in isolated hepatocytes. The glucose output of D-galactosamine-pretreated hepatocytes could be restored by increasing the extracellular concentration of Pi (Fig.3). Surprisingly, the glucose release of untreated cells could also be enhanced by a factor of two. These results suggested that Pi penetrated the cell, that the activity of phosphorylase a in normal liver cells was limited by the intracellular Pi concentration, and that D-galactosamine reduced the Pi level
~~
of hepatocytes. The latter conclusion was confirmed by direct determination of the intracellular Pi content : 2 mM D-galactosamine in the medium decreased the Pi level within 20 min from 3.59 0.63 ( n = 7) to 2.23 '. 0.83 mmol (n = 7) per kg wet cells. Kinetic measurements of the influence of D-galactosamine and inorganic phosphate, separately and in combination, on intracellular Pi content, glycogen breakdown and glucose output underscored the role of phosphate (Fig.4). In the absence of D-galactosamine and at an extracellular Pi concentration of 0.4 mM, hepatocytes from fed rats whose glycogen content was 260 mmol glycosyl groups per kg wet cells degraded within 1 h 7 3 mmol glycosyl units of glycogen and released 42mmol of free glucose into the medium per kg of
Effects of Galactosamine and P, on Glycogenolysis
84 Table 5. Intracellular Pi contents of isolated rat hepatocytes ufter uddition of D-galactosamine,D-fructoseand potassium phosphate to the incubation medium 50 mg of cells were incubated in Umbreit/Ringer/carbonate buffer, pH 7.4. After 20 min of incubation, the cells were processed as described in Methods
120
-
--- 100.
r =0.833
e
.3
a,
Additions
P, content
-CAW 1
-
‘1
mM
mmol/kg wet wt
3.23
None
2
1.63
o-Fructose
20
1.23
Potassium phosphate
20
5.54
o-Galactosamine Potassium phosphate
2 20
3.36
o-Fructose Potassium phosphate
20 20
2.37
u-Galactosamine
--
a 3
+
3
40 0,
-rn 20
0
0
2
Intracllular
4
6
6 content (rnrnol/kq wet
a wt)
Fig. 5. Correlution between intrucellulur P, content und glucose output under dijferent conditions. (0)Control cells; (A) in the presence of 20 mM Pi; ( 0 ) in the presence of 2 mM u-galactosamine; (A) in the presence of P, and o-galactosamine
cells ; the intracellular Pi content remained constant at 3.5 mmol per kg wet cells (Fig.4A). Addition of 2 mM D-galactosamine to the medium induced a significant drop of the intracellular Pi accompanied by a reduced rate of glycogen breakdown and glucose output (Fig. 4B). This effect of D-glactosamine could be prevented by the simultaneous increase of the extracellular Pi concentration to 20 mM, resulting in an intracellular Pi content somewhat higher than that of control cells and a concomitantly increased rate of glycogen conversion to glucose (Fig. 4D). The effect of 20 mM extracellular Pi alone is depicted in Fig. 4 C showing enhanced intracellular Pi level, glycogenolysis and glucose output. Sulfate and potassium ions, usually the potassium salt of phosphate was used in these experiments, were found to be unable to stimulate glycogenolysis. Furthermore, the addition of 2 mM D-galactosamine elicited a transient decrease of the intracellular ATP content and concomitantly enhanced the concentrations of ADP and AMP (Table 4). As a result, the value of the energy charge dropped from 0.83 to 0.75. On the other hand, addition of 20mM inorganic phosphate to the medium raised the ATP level in hepatocytes. When D-galactosamine and inorganic phosphate were given simultaneously, their effects cancelled each other. An increase of the ATP content of yeast cells incubated in the presence of high Pi concentrations has been reported by Erecinska et al. [32]. An effect similar to that of 2 mM D-galactosamine could be observed when hepatocytes were incubated with 20 mM D-fructose (Table 5 ) ; the intracellular Pi level fell from 3.23 to 1.23 mmol per kg wet cells within 20 min ; the simultaneous addition of 20 mM phosphate, however, was not able to prevent this decrease completely. The quantitatively different re-
sponse to exogenous phosphate of the Pi decrease induced by fructose and that seen after D-galactosamine addition can be understood in view of the different rates of D-galactosamine phosphorylation by galactokinase [33] and of fructose 1-phosphate formation by ketohexokinase [34]. While the net influx of Pi from the medium containing 20 mM phosphate is able to compensate the phosphate trapping by D-galactosamine it appears to be insufficient to cope with the faster phosphorylation of D-fructose. The data of the D-galactosamine and Pi effect on glycogenolysis and glucose output can best be interpreted by a phosphatedependence of the phosphorylase a activity in the intact hepatocyte. Assuming a constancy of other regulatory factors during these experiments and a homogeneous distribution of Pi within the cell, the actual activity of phosphorylase a in the presence of the physiological Pi level is about 45% of its maximal capacity. The enhancement of glycogenolysis and glucose output by an increased Pi supply (Fig.5) is in line with this assumption. The skilful technical assistance of Ms J. Nowack and Ms A. Leonhardt is gratefully acknowledged. This work was supported by grants from the Deutsche Forschungsgenieinschaft, Bonn-Bad Godesberg, through Forschergruppe ‘Lehererkrunkungen’.
REFERENCES 1. Maley, F., Terantino, A . L., McGarrahan, J . F. & Del Giacco, R. (1968) Biochem. J . 107, 637-644. 2. Keppler, D. &Decker, K. (1969) Eur. d. Biochem. 10,219-225.
85
R. Stermann, S. R. Wagle, and K. Decker 3. Maley, F. (1970) Biochem. Biophys. Res. Commun. 39,371 - 378. 4. Keppler, D., Rudigier, J., Bischoff, E. & Decker, K. (1970) Eur. J . Biochem. 17,246-253. 5. Decker, K. & Keppler, D. (1974) Rev. Physiol. Biochem. Pharmacoi. 71, 77- 106. 6. Keppler, D., Lesch, R., Reutter, W. & Decker, K. (1968) Exp. Mol. Pathol. 9, 279-290. 7. Wagle, S. R., Stermann, R. & Decker, K. (1976) Biochem. Biophys. Res. Commun. 71, 622-628. 8. Maenpaa, P. H., Raivio, K. 0. d Kekomaki, M. P. (1968) Science (Wash. D.C.), 161, 1253-1254. 9. Starling, J. J. & Keppler, D. (1977) Eur. J . Biochem. 80, 373 - 379. 10. Berry, M. N. & Friend, D. S. (1969) J . Cell. B i d . 43, 506-520. 11. Hofmann, F. & Decker, K. (1975) Biochem. Soc. Trans. 3, 1084- 1086. 12. DeDuve, C., Pressmann, B. C., Cianetto, R., Wattiaux, R. & Appelmans, F. (1955) Biochem. J . 60, 604- 61 7. 13. Bergmeyer, H. U., Bernt, E., Schmidt, F. & Stork, H. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 1241- 1246, Verlag Chemie, Weinheim. 14. Keppler, D. & Decker, K. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 11711176, Verlag Chemie, Weinheim. 35. Bergmeyer, H. U. & Michal, G. (1974) in Methoden der Enzymutischen Analyse(Bergmeyer, H. U., ed.) 3rdedn, pp. 12791282, Verlag Chemie, Weinheim. 16. Lamprecht, W. & Trautschold, I. (1974) in Methoden dev Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 2151 2160, Verlag Chemie, Weinheim. 17. Jaworek, D., Gruber, W. & Bergmeyer, H. U . (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 2178-2181, Verlag Chemie, Weinheim.
18. Gawehn, K. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 2286-2289, Verlag Chemie, Weinheim. 19. Michal, G. & Wunderwald, P. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 21862194, Verlag Chemie, Weinheim. 20. Lowry, 0. H. & Passonneau, J. (1969) J . Biol. Chem. 244, 910-916. 21. Appelmans, F., Wattiaux, R. & DeDuve, C. (1955) Bioclzem. J . 59,438 - 445. 22. Stalmans, W. & Hers, H. G . (1975) Eur. J . Biochem. 54, 341 - 350. 23. Bergmeyer, H. U., Gawehn, K. & GraBI, M. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 539- 541, Verlag Chemie, Weinheim. 24. Maddaiah, V. T. & Madsen, N. B. (1966) J . B i d . Chem. 241, 3873-3881. 25. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 26. Ferber, E., Resch, K., Wallach, D. F. H. & Imm, W. (1972) Biochim. Biophys. Acta, 266, 494- 504. 27. Rail, T. W. d Sutherland, E. W. (1958) J . B i d . Chem. 232, 1065- 1076. 28. Siess, E. A. & Wieland, 0. H. (1976) Biochem. J . 156, 92 - 102. 29. Hue, L., Bontenips, F. & Hers, H. G . (1975) Biuchern. ,/. 152, 105 - 114. 30. Barankiewicz, J., Battell, M. L. &Henderson, J. F. (1977) Can. J . Biochem. 55, 834- 840. 31. Hems, D. A. & Brosnan, J. T. (1970) Biochem. J . 120,105- 111. 32. Erecinska, M., Stubbs, M., Miyata, Y., Ditre, C. M. & Wilson, D. F. (1977) Biochim. Biophys. Acta, 462, 20-35. 33. Walker, D. G.&Khan, H. H. (1968)Biochem. J . 108,169-175. 34. Sestoft, L. (1974) Biochim. Biophys. Acta, 343, 1 - 16.
R. Stermann and K. Decker*, Biochemisches Institut an der Medizinischen Fakultat der Albert-Ludwigs-Universitat, Hermann-Herder-Strasse 7, D-7800 Freiburg i. Br., Federal Republic of Germany S. R. Wagle, Department of Pharmacology, Indiana University School of Medicine. 1100 West Michigan Street, Indianapolis, Indiana, U.S.A. 46202
* To whom correspondence should be addressed.
Inverse Effects of D-Galactosamine and Inorganic Phosphate on Glycogenolysis in Isolated Rat Hepatocytes Rita STERMANN, Shreepad R . WAGLE, and Karl DECKER Biochemisches Institut an der Medizinischen Fakultat der Albert-Ludwigs-Universitat. Freiburg i. Br (Received February 20, 1978)
D-Galactosamine (2 mM) lowered the intracellular Pi level of isolated rat hepatocytes within 20 min from 3.6 to 2.3 and the ATP content from 2.34 to 1.86 mmol per kg wet cells. At the same time, the rate of glycogenolysis decreased to 55% and of glucose output to 45%. Inorganic phosphate added to the incubation medium raised the intracellular Pi level in a dosedependent manner. At an extracellular Pi concentration of 20 mM, the intracellular Pi content rose within 20 min to 6.4 and the ATP content to 2.96 mmol per kg wet cells. This increase was accompanied by a two-fold stimulation of glycogenolysis and release of glucose. D-Galactosamine (2 mM) and potassium phosphate (20 mM) supplied simultaneously counteracted their individual effects. Since the activities of the enzymes involved in glucose formation from glycogen including the glucagon-stimulated adenyl cyclase activity were unaltered by D-galactosamine treatment, it is concluded that in isolated hepatocytes phosphorolysis of glycogen is the limiting step in its conversion to glucose, that the intracellular Pi level is a limiting factor of phosphorylase a activity, that the intracellular Pi content and the rate of glycogenolysis can be increased by raising the extracellular Pi concentration, and that the net influx from elevated medium Pi levels can effectively counteract the phosphate trapping capacity of galactosamine, but not that of 20 mM fructose. D-Galactosamine is metabolized in liver by the enzymes of the galactose pathway [l - 31 ;ATP-dependent D-galactosamine 1-phosphate formation is followed by uridylylation with UDP-glucose as donor resulting eventually in the accumulation of UDPhexosamines and -N-acetyl-hexosamines in the hepatocytes [4]. This uridylate trapping by the amino sugar was found to elicit a temporal deficiency of uridine phosphates and UDP-hexoses leading to irreversible liver injury [5]. As a result of UDP-glucose depletion in livers of D-galactosamine-treated animals, glycogen synthesis is arrested followed by an almost complete loss of glycogen 161. Studies on the effect of D-galactosamine on glycogen metabolism in hepatocytes in vitvo showed, on the other hand, that the amino sugar lowered the rate of glycogen breakdown, glucose output and lactate formation in hepatocytes [7]. ~ Ahhveviation. Cyclic AMP, adenosine 3’: 5’-monophosphate. Enzymes. Galactokinase or ATP: D-galactose l-phosphotransferase (EC 2.7.1.6) glucose-6-phosphatase or ~-glucose-6-phosphate phosphohydrolase (EC 3.1.3.9); ketohexokinase or ATP: o-fructose I-phosphotransferase (EC 2.7.1.3) ; phosphoglucomutase or cc-Dglucose-l,6-bisphosphate:a-D-glucose-1-phosphate phosphotransferase (EC 2.7.5.1); phosphorylase, liver glycogen phosphorylase or 1 , 4 - ~ - ~ - g l u c:a northophosphate cc-glycosyltransferase (EC 2.4.1.2).
The mechanism of this additional D-galactosamine effect on hepatic carbohydrate metabolism, however, could not be deduced from these experiments. The following feasible causes were considered. Firstly, that the amino sugar or one of its metabolites inhibits competitively an early step of glycogenolysis. Secondly, that galactosamine affects the interconversion of phosphorylase, e.g. by reducing the level of cyclic AMP or by a change of the phosphorylation state. Thirdly, that galactosamine induces a trapping of intracellular inorganic phosphate and thereby inhibits phosphorolysis of glycogen by depletion of a cosubstrate. A transient decrease of the inorganic phosphate content has been observed in liver after a fructose load [8] and treatment with 2-deoxy-D-galactose [9]. This communication will show that the decrease of the hepatic Pi content is responsible for the D-galactosamine-mediated inhibition of glycogenolysis.
~_ _
MATERIALS AND METHODS D-Galactosamine . HCl was obtained from C. Roth OHG (Karlsruhe), collagenase, grade I1 purity, from Worthington Biochemical Corp. (Freehold, N.J., U.S.A.), glycogen, coenzymes and enzymes from
Effects of Galactosamine and Pi on Glycogenolysis
80
Boehringer Mannheim GmbH (Mannheim). L-Lactic acid and oleate were purchased from Serva GmbH (Heidelberg), glucagon from Sigma Chemical CO. (St Louis, Mo., U.S.A.). a-~-(U-'~C]glucose l-phosphate was supplied by Amersham-Buchler GmbH (Braunschweig). Glassware brought in contact with hepatocytes was pretreated with siliclad (Clay Adams, Parsippany, N.J., U.S.A.). Female Wistar rats (Ivanovas, Kisslegg) weighing 165- 180 g were used. The animals had unlimited access to a standard diet (Altromin R) and water. Liver cells were prepared by the method of Berry and Friend [lo] without hyaluronidase and with slight modifications as reported previously [ll]. 1 ml of cell suspension (50- 60 mg of cells) in a 50-ml conical flask was supplemented with 2 ml of Umbreit/Ringer/carbonate (25 mM) buffer, pH 7.4 and the indicated additions. The vials were gassed with 95% 0 , / 5 % COZ and incubated at 37 "C in a water bath (90 oscillations x min-'). Reactions were discontinued by transfer of the vials into an ice bath, centrifugation at 3000 x g for 5 min at 4 "C, and deproteinization with perchloric acid (final concentration 0.1 M). Separation of liver cell homogenates into a cytosolic and a microsomal fraction was performed according to De Duve et al. [12]. Glucose [13], glycogen [14], glucose 1-phosphate and glucose 6-phosphate [15], ATP [16], ADP and AMP [17] and inorganic phosphate [18] were determined enzymatically. Cyclic AMP was assayed by the competitive protein-binding technique [I91 after purification of the samples by anion-exchange chromatography on Dowex AG 1 x 8 (formate) which had been equilibrated with 0.1 M formic acid. The intracellular content of inorganic phosphate was measured in the following way. After the incubation period, the cell suspension was cooled on ice and centrifuged rapidly at 4 "C for 5 min at 3000 x g . The pellet was washed once with 5 ml of ice-cold 10mM triethanolamine .HC1 buffer, pH 7.4, containing 250 mM sucrose and 0.2 mM sodium phosphate. The resulting pellet was frozen immediately in liquid nitrogen and stored at - 70 "C for 1 h. After thawing, the cells were disrupted by homogenization in 1 ml 0.5 M perchloric acid and centrifuged. The supernatant was neutralized by addition of potassium hydroxide and recentrifuged. Inorganic phosphate was determined in the supernatant. The washing of the cells with the triethanolamine/ sucrose/phosphate buffer did not alter the intracellular P, level (Table 1). However, more than 0.8 mM phosphate in the washing buffer yielded erroneously high values of intracellular P,. Phosphoglucomutase was assayed by the method of Lowry and Passonneau [20] and glucose-6-phosphatase according to Appelmans et al. [21]. The phosphorylase assay in the direction of glycogen synthesis was carried out at 37 "C according to Stalmans and
Table 1. Intracellular P , content of isolated rat hepatocytes after washing with hujjers containing sucrose and various Pi concentrations 50 mg of cells were incubated for 30 min in Umbreit/Ringer/carbonate buffer, pH 7.4, containing 0.4 mM Pi. For further details see text Pi concentration in washing buffer
Pi content
mM
mmol/kg wet wt
-
0.16 0.35 0.8 1.6 a
3.55 3.58 3.53 3.62 3.86 4.73
Cells not subjected to washing procedure.
Hers [22], whereby only the a form of the enzyme is measured. One unit of enzyme activity is defined as 1 pmol of ['4C]glucose 1-phosphate incorporated into glycogen per min. The phosphorylase a assay in the direction of glycogen breakdown was carried out by a modified [23] procedure of Maddaiah and Madsen [24]. Protein was determined according to Lowry et al. [25].
RESULTS AND DISCUSSION In the presence of D-galactosamine, glucose output by isolated hepatocytes was inhibited in a dosedependent manner [7]. This effect of the aminosugar appeared to be due to a decreased rate of glycogen breakdown. The inhibition of glycogenolysis and glucose production set in several min after D-galactosamine addition and remained constant for at least 60 min, indicating a rapid mechanism responsible for the D-galactosamine-specific effect on glycogenolysis. Several enzymes are involved in the formation of glucose from glycogen including the hormonally regulated interconvertible phosphorylase, phosphoglucomutase and glucose-6-phosphatase. The specific activities of these enzymes were assayed in extracts of hepatocytes preincubated with D-galactosamine and were found to be the same as those from control cells (Table 2); similar results were obtained with livers from rats treated with the amino sugar in vivo and when D-galactosamine was added to the enzyme assay itself. These findings are in line with previous determinations of metabolite levels in D-galactosaminetreated hepatocytes [7] which ruled out an impairment of the activities of phosphoglucomutase and glucose6-phosphatase as well as of the glycolytic pathway leading to lactate. The greatly reduced lactate levels found after D-galactosamine addition to a hepatocyte suspension [7] do not appear to be due to an enhanced
R. Stermann, S. R. Wagle, and K. Decker
81
Table 2. Activities oftlie enzynies involved in the conversion of glycogen to glucose, both in the presence and absence of l rn M u-galactosarnine Phosphorylase a and phosphoglucomutase were measured in the cytosolic, glucose-6-phosphatase in the rnicrosomal fraction of homogenized hepatocytes. Methods were: (A) cells homogenized by freezing (- 180 "C) and thawing; (B) cells homogenized by NZ cavilation [26] ( 5 atm, 20 min); (S) assayed by incorporation of [U-'4C]glucose 1-phosphate into glycogen at 37 "C; (D) assayed by glucose I-phosphate formation from added glycogen at 25 "C; for details see Methods ~
Enzyme
~
Method
Control
With galactosamine
u x g protein-' A; S B; S A; D
1772 174.7 69.0
158.9 167.9 65.8
Phosphoglucomutase
A
398
400
Glucose-6-phosphatase
A
85
81
Phosphorylase a
Table 3. Oxygen consumption by rat hepatocytes in the presence and absence of I rnM n-galactosamine About 50 mg of isolated cells were incubated in Umbreit/Ringer/ carbonate buffer, pH 7.4, in Warburg vessels; gas phase, air; 4 M KOH in center well. Additions were tipped in from the sidearm after thermal equilibration Additions
0 2
mM None Galactosamine
consumption
mmol x min-' x (kg wet wt)-' 2.32
1
2.0
Lactate Oleate
10 1
3.74
Galactosamine Lactate Oleate
1 10 1
3.38
oxidation of C3 compounds, since the 0 2 consumption both in the presence and in the absence of oleate and lactate is hardly affected by D-galactosamine treatment (Table 3). It follows that the decreased lactate production after addition of the amino sugar is the result of an impaired glycogenolysis rather than an inhibited glycolysis or enhanced oxidation. Phosphorylase a activity is influenced by many effectors; especially the level of cyclic AMP is thought to play an important role in the hormone-dependent regulation of hepatic phosphorylase activity [27]. A possible effect of D-galactosamine on the adenylate cyclase activity was investigated by measuring the glucagon-dependent response of the cyclic AMP concentration in hepatocytes in the presence and absence of 1 mM D-galactosamine (Fig. 1). The insensitivity
I
00
5
10
Time (min)
Fig. 1. Effect of 1 rnM D-gu/uctoSUmine on the glucagon-stimulated cyclic AMP,formation. Full symbols, o-galactosamine-treated cells; open symbols, control cells. (A, A) 10 nM glucagon added at zero time after 10 min preincubation; (0, 0 ) unstimulated cells
of the glucagon-elicited cyclic AMP burst to 1 mM D-galactosamine is in agreement with the corresponding glucagon-stimulated glucose production [7]: although the absolute value of glucose release into the incubation medium decreased in the presence of D-galactosaniine, its relative enhancement by glucagon remained about the same at all D-galactosamine concentrations tested. The relation of the active and the inactive form of pyruvate dehydrogenase, another interconvertible enzyme, was shown to depend on the phosphorylation state (ATP/ADP . Pi) and the energy charge, respectively 1281. Addition of D-galactosamine to hepatocytes elicited a transient decrease of the ATP content, the phosphorylation state and the energy charge (Table 4). Therefore, phosphorylase a activity was determined in extracts of D-galactosamine-pretreated hepatocytes both in the anabolic and catabolic direction (Table 2). Although the methods used for cell disruption were considered to preserve the content of active (phosphorylated) enzyme [29], no difference was found between D-galactosamine-pretreated and control hepatocytes. The intracellular level of inorganic phosphate can not only influence glycogenolysis by way of the phosphorylation state but also as a substrate of the glycogen phosphorylase reaction itself. The K, of Pi and purified liver phosphorylase a was shown [24] to depend on the glycogen concentration present in the assay and to vary between 7.3 mM (at 0.5 mM glycogen) and 1.09 mM (at 25.2 mM). By measurements of phosphorylase a activity in a crude cytosolic fraction of the hepatocytes a K , for Pi of 1.78 mM was obtained in the presence of 12 mM
Effects of Galactosamine and Pi on Glycogenolysis
82
Table 4. EfJect of D-galuctosamine and potassium phosphate on the phosphorylution state qf isolated rat hepatocytes 50 mg of cells were suspended in Utnbreit/Ringer/carbonate buffer, pH 7.4. After addition of the indicated compounds, the incubations were continued for 20 min. The cells were then processed and analyzed as described in Methods. The contents are given as mean values 5 S.D. ( n z 7) Additions
ATP
ADP
AMP
Pi
C ATP
+ADP + AMP
ATP/ ADP
Energy charge
Phosphorylation state
mmol/kg wet wt ~-
None
2.34i0.34
0.66k0.04
0.23k0.02
3.59t0.63
3.21 k 0 . 2 7
3.55k0.63
0.82+0.03
0.9Yk0.38
Phosphate,20mM
2.96k0.12
0.63+0.01
0.14k0.01
6.42k0.99
3.65It0.18
4.60i0.29
0.88k0.02
0.72f0.05
Galactosamine,2mM
1.86i0.39
0.94k0.09
0.30k0.03
2.33t0.83
3.02i0.28
1.92k0.36
0.75k0.04
0.82k0.15
Phosphate, 20 mM +- galactosamine, 2 mM
2.38+0.08
0.72k0.04
0.21+0.02
4.7Yf1.32
3.27k0.15
3,2750.22
0.83i0.03
0.68+0.05
75
1
I
50
25
-
Km,app = 1.78rnM
,'
-
1
0
1 2 l/[Pi](mM-')
3
4
0
10
20
30 40 [Pi] (mMi
50
60
Fig. 2. Dependence oJ the phosplioryluse a uciivity in the cytosolic fraction of heputocytes on Pi concentration. Enzyme activity was measured by glucose I-phosphate formation from added glycogen (see Methods). Y is expressed as nmol glycosyl groups released per min
Fig. 3. Dependence of glucose output on the eutrucellular concentration of inorganicphosphate. Full symbols, in the presence of 1 m M D-galactosamine; open symbols, control cells
glycogen and in the absence of 5'-AMP (Fig. 2). Since glycogen forms particles of varying molecular weight to which phosphorylase is tightly bound, it is not possible to evaluate the level of saturation of the enzyme in intact cells. Assuming conditions in isolated hepatocytes corres'ponding to a K , value of about 1.S mM, however, the intracellular Pi content of 3.6 mM (Table 4) would not be sufficient to saturate the phosphorylase a . It was considered possible that a reduction of the intracellular Pi level results in a decreased actual glycogenolytic activity of the enzyme. D-Galactosamine is rapidly converted to D-galactosamine 1-phosphate by ATP in the presence of galactokinase [l]and subsequently converted to UDP derivatives of the amino sugar. Their accumulation
within the hepatocyte not only leads to uridylate trapping [4] but also to a transient increase of the total organic phosphate. It was expected that this D-galactosamine-induced phosphate trapping provokes a deficit of intracellular inorganic phosphate whose duration depends on the rate of net influx of Pi from the extracellular space. The following experiments were designed to study the effect of D-galactosamine on the intracellular Pi content of isolated hepatocytes; also, the feasibility of an increased extracellular Pi concentration as a counterbalancing measure was tested. Uptake of Pi by Ehrlich ascites tumor cells [30] and increased phosphate contents as well as stimulation of the renal glucose output after severance of the blood supply [31]
R. Stermann, S. R. Wagle, and K. Decker I
I*
3
-r c
a,
3
. -
s p 0
c
ar
. 3
m 1 0
- 60 3e
-a-
-cn
-
-m
E
b
E
E
4 ,
3
%
c
2 "
B 80
3 40
P
x
a,
9
20
2
5 0
0
O
O
'
Time (min)
-
B 3 0 Time fmin)
4
0
D
c
3 80
c
a,
. 3
n,
1
- 60 a v)
-cne x
-mx
40
b a,
9
20
5
0
0
10
20 30 Time (min)
40
0
10
20 30 Time (rnin)
40
Fig. 4. Effects of'tlie extracellular concentrations of D-galactosamine and Pion glycogen hreakdown, gluco.ve ourpur and inrrucellular Pi content. Glycosyl groups released from glycogen; (0-) glucose output to the incubation medium; (A A) intracellular Pi content. (A) Control; (B) + 2 mM galactosamine; (C) + 20 m M P,; (D) + 20 mM Pi + 2 m M galactosamine (0-0)
had already been observed. After a reliable method was developed to determine the intracellular Pi content of cells incubated in excessive concentrations of extracellular Pi (Table l), it became possible to study the relation between the extracellular and intracellular levels of inorganic phosphate and the rate of glycogenolysis in isolated hepatocytes. The glucose output of D-galactosamine-pretreated hepatocytes could be restored by increasing the extracellular concentration of Pi (Fig.3). Surprisingly, the glucose release of untreated cells could also be enhanced by a factor of two. These results suggested that Pi penetrated the cell, that the activity of phosphorylase a in normal liver cells was limited by the intracellular Pi concentration, and that D-galactosamine reduced the Pi level
~~
of hepatocytes. The latter conclusion was confirmed by direct determination of the intracellular Pi content : 2 mM D-galactosamine in the medium decreased the Pi level within 20 min from 3.59 0.63 ( n = 7) to 2.23 '. 0.83 mmol (n = 7) per kg wet cells. Kinetic measurements of the influence of D-galactosamine and inorganic phosphate, separately and in combination, on intracellular Pi content, glycogen breakdown and glucose output underscored the role of phosphate (Fig.4). In the absence of D-galactosamine and at an extracellular Pi concentration of 0.4 mM, hepatocytes from fed rats whose glycogen content was 260 mmol glycosyl groups per kg wet cells degraded within 1 h 7 3 mmol glycosyl units of glycogen and released 42mmol of free glucose into the medium per kg of
Effects of Galactosamine and P, on Glycogenolysis
84 Table 5. Intracellular Pi contents of isolated rat hepatocytes ufter uddition of D-galactosamine,D-fructoseand potassium phosphate to the incubation medium 50 mg of cells were incubated in Umbreit/Ringer/carbonate buffer, pH 7.4. After 20 min of incubation, the cells were processed as described in Methods
120
-
--- 100.
r =0.833
e
.3
a,
Additions
P, content
-CAW 1
-
‘1
mM
mmol/kg wet wt
3.23
None
2
1.63
o-Fructose
20
1.23
Potassium phosphate
20
5.54
o-Galactosamine Potassium phosphate
2 20
3.36
o-Fructose Potassium phosphate
20 20
2.37
u-Galactosamine
--
a 3
+
3
40 0,
-rn 20
0
0
2
Intracllular
4
6
6 content (rnrnol/kq wet
a wt)
Fig. 5. Correlution between intrucellulur P, content und glucose output under dijferent conditions. (0)Control cells; (A) in the presence of 20 mM Pi; ( 0 ) in the presence of 2 mM u-galactosamine; (A) in the presence of P, and o-galactosamine
cells ; the intracellular Pi content remained constant at 3.5 mmol per kg wet cells (Fig.4A). Addition of 2 mM D-galactosamine to the medium induced a significant drop of the intracellular Pi accompanied by a reduced rate of glycogen breakdown and glucose output (Fig. 4B). This effect of D-glactosamine could be prevented by the simultaneous increase of the extracellular Pi concentration to 20 mM, resulting in an intracellular Pi content somewhat higher than that of control cells and a concomitantly increased rate of glycogen conversion to glucose (Fig. 4D). The effect of 20 mM extracellular Pi alone is depicted in Fig. 4 C showing enhanced intracellular Pi level, glycogenolysis and glucose output. Sulfate and potassium ions, usually the potassium salt of phosphate was used in these experiments, were found to be unable to stimulate glycogenolysis. Furthermore, the addition of 2 mM D-galactosamine elicited a transient decrease of the intracellular ATP content and concomitantly enhanced the concentrations of ADP and AMP (Table 4). As a result, the value of the energy charge dropped from 0.83 to 0.75. On the other hand, addition of 20mM inorganic phosphate to the medium raised the ATP level in hepatocytes. When D-galactosamine and inorganic phosphate were given simultaneously, their effects cancelled each other. An increase of the ATP content of yeast cells incubated in the presence of high Pi concentrations has been reported by Erecinska et al. [32]. An effect similar to that of 2 mM D-galactosamine could be observed when hepatocytes were incubated with 20 mM D-fructose (Table 5 ) ; the intracellular Pi level fell from 3.23 to 1.23 mmol per kg wet cells within 20 min ; the simultaneous addition of 20 mM phosphate, however, was not able to prevent this decrease completely. The quantitatively different re-
sponse to exogenous phosphate of the Pi decrease induced by fructose and that seen after D-galactosamine addition can be understood in view of the different rates of D-galactosamine phosphorylation by galactokinase [33] and of fructose 1-phosphate formation by ketohexokinase [34]. While the net influx of Pi from the medium containing 20 mM phosphate is able to compensate the phosphate trapping by D-galactosamine it appears to be insufficient to cope with the faster phosphorylation of D-fructose. The data of the D-galactosamine and Pi effect on glycogenolysis and glucose output can best be interpreted by a phosphatedependence of the phosphorylase a activity in the intact hepatocyte. Assuming a constancy of other regulatory factors during these experiments and a homogeneous distribution of Pi within the cell, the actual activity of phosphorylase a in the presence of the physiological Pi level is about 45% of its maximal capacity. The enhancement of glycogenolysis and glucose output by an increased Pi supply (Fig.5) is in line with this assumption. The skilful technical assistance of Ms J. Nowack and Ms A. Leonhardt is gratefully acknowledged. This work was supported by grants from the Deutsche Forschungsgenieinschaft, Bonn-Bad Godesberg, through Forschergruppe ‘Lehererkrunkungen’.
REFERENCES 1. Maley, F., Terantino, A . L., McGarrahan, J . F. & Del Giacco, R. (1968) Biochem. J . 107, 637-644. 2. Keppler, D. &Decker, K. (1969) Eur. d. Biochem. 10,219-225.
85
R. Stermann, S. R. Wagle, and K. Decker 3. Maley, F. (1970) Biochem. Biophys. Res. Commun. 39,371 - 378. 4. Keppler, D., Rudigier, J., Bischoff, E. & Decker, K. (1970) Eur. J . Biochem. 17,246-253. 5. Decker, K. & Keppler, D. (1974) Rev. Physiol. Biochem. Pharmacoi. 71, 77- 106. 6. Keppler, D., Lesch, R., Reutter, W. & Decker, K. (1968) Exp. Mol. Pathol. 9, 279-290. 7. Wagle, S. R., Stermann, R. & Decker, K. (1976) Biochem. Biophys. Res. Commun. 71, 622-628. 8. Maenpaa, P. H., Raivio, K. 0. d Kekomaki, M. P. (1968) Science (Wash. D.C.), 161, 1253-1254. 9. Starling, J. J. & Keppler, D. (1977) Eur. J . Biochem. 80, 373 - 379. 10. Berry, M. N. & Friend, D. S. (1969) J . Cell. B i d . 43, 506-520. 11. Hofmann, F. & Decker, K. (1975) Biochem. Soc. Trans. 3, 1084- 1086. 12. DeDuve, C., Pressmann, B. C., Cianetto, R., Wattiaux, R. & Appelmans, F. (1955) Biochem. J . 60, 604- 61 7. 13. Bergmeyer, H. U., Bernt, E., Schmidt, F. & Stork, H. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 1241- 1246, Verlag Chemie, Weinheim. 14. Keppler, D. & Decker, K. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 11711176, Verlag Chemie, Weinheim. 35. Bergmeyer, H. U. & Michal, G. (1974) in Methoden der Enzymutischen Analyse(Bergmeyer, H. U., ed.) 3rdedn, pp. 12791282, Verlag Chemie, Weinheim. 16. Lamprecht, W. & Trautschold, I. (1974) in Methoden dev Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 2151 2160, Verlag Chemie, Weinheim. 17. Jaworek, D., Gruber, W. & Bergmeyer, H. U . (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 2178-2181, Verlag Chemie, Weinheim.
18. Gawehn, K. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 2286-2289, Verlag Chemie, Weinheim. 19. Michal, G. & Wunderwald, P. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 21862194, Verlag Chemie, Weinheim. 20. Lowry, 0. H. & Passonneau, J. (1969) J . Biol. Chem. 244, 910-916. 21. Appelmans, F., Wattiaux, R. & DeDuve, C. (1955) Bioclzem. J . 59,438 - 445. 22. Stalmans, W. & Hers, H. G . (1975) Eur. J . Biochem. 54, 341 - 350. 23. Bergmeyer, H. U., Gawehn, K. & GraBI, M. (1974) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.) 3rd edn, pp. 539- 541, Verlag Chemie, Weinheim. 24. Maddaiah, V. T. & Madsen, N. B. (1966) J . B i d . Chem. 241, 3873-3881. 25. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 26. Ferber, E., Resch, K., Wallach, D. F. H. & Imm, W. (1972) Biochim. Biophys. Acta, 266, 494- 504. 27. Rail, T. W. d Sutherland, E. W. (1958) J . B i d . Chem. 232, 1065- 1076. 28. Siess, E. A. & Wieland, 0. H. (1976) Biochem. J . 156, 92 - 102. 29. Hue, L., Bontenips, F. & Hers, H. G . (1975) Biuchern. ,/. 152, 105 - 114. 30. Barankiewicz, J., Battell, M. L. &Henderson, J. F. (1977) Can. J . Biochem. 55, 834- 840. 31. Hems, D. A. & Brosnan, J. T. (1970) Biochem. J . 120,105- 111. 32. Erecinska, M., Stubbs, M., Miyata, Y., Ditre, C. M. & Wilson, D. F. (1977) Biochim. Biophys. Acta, 462, 20-35. 33. Walker, D. G.&Khan, H. H. (1968)Biochem. J . 108,169-175. 34. Sestoft, L. (1974) Biochim. Biophys. Acta, 343, 1 - 16.
R. Stermann and K. Decker*, Biochemisches Institut an der Medizinischen Fakultat der Albert-Ludwigs-Universitat, Hermann-Herder-Strasse 7, D-7800 Freiburg i. Br., Federal Republic of Germany S. R. Wagle, Department of Pharmacology, Indiana University School of Medicine. 1100 West Michigan Street, Indianapolis, Indiana, U.S.A. 46202
* To whom correspondence should be addressed.
