Comp. Biochem. Physiol., 1978, VoL 59B, pp. 317 to 325. Pergamon Press. Printed in Great Britain

GLUCOSE PHOSPHORYLATION A N D DEPHOSPHORYLATION IN CHICKEN LIVER IRIS E. O'NEILL and DEREK R. LANGSLOW Veterinary Unit, Department of Biochemistry, Royal (Dick) School of Veterinary Studies, Summerhall, Edinburgh, EH9 IQH, Scotland (Received 22 Auoust 1977)

Abstract--1. Glucokinase was absent from chicken liver and only the low K,, hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The K,. of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efltux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function.

INTRODUCTION Glucose serves as the primary fuel for many animals, including chickens. Since some cell types are dependent on a continuous supply of glucose, the plasma glucose concentration must be carefully controlled under all physiological circumstances. The liver is the major regulator of the plasma glucose concentration, as it has the capacity to take up or produce glucose according to requirements. Chickens consume a diet consisting mostly of carbohydrate and protein and, during starvation, are able to maintain a plasma glucose concentration of around 12-14 mM for several days (Langslow & Hales, 1971; Hazelwood, 1976). Therefore, the chicken liver has the capacity to take up and utilise or to synthesise glucose readily, and has a cell membrane freely permeable to glucose (see review by Newsholme & Start, 1973). This flux is controlled principally by the activities of the glucose phosphorylating and glucose-6-phosphate dephosphorylating enzymes and by the intracellular glucose and glucose-6-phosphate concentrations. These parameters are in turn regulated nutritionally, hormonally and by the other intracellular processes in the liver. Both hexokinase and glucose-6-phosphatase catalyse reactions with large equilibrium constants and hence they operate as essentially non-equilibrium reactions. Four isozymes catalyse the phosphorylation of glucose in rat liver and these have been designated A, B, C and D, according to their chromatographic mobilities (Gonzalez et al., 1964, 1967; Katzen & Schimke, 1965). Isozymes A, B and C have low Km values for glucose and broad sugar specificities, while isozyme C is inhibited by high glucose concentrations. Isozyme D (glucokinase) has a high K~, for glucose and is virtually specific for this sugar (Vinuela et ai., 1963; Gonzalez et al., 1964, 1967; Salas et al., 1965; Parry & Walker, 1966). Glucokinase is adaptive in some species and responds to different diets, to starvation and to hormonal manipulation (see review by Weinhouse, 1976). 317

In most avian rivers, only two of the low Km hexokinases were reported to be present, and no glucokinase-type activity was detected except in homogenates of goose liver (Sols et al., 1964; Ureta et al., 1972, 1973). By contrast, other reports (Wallace & Newsholme, 1967; Pearce, 1970) have suggested that chicken liver does contain glucokinase-like activity and Pearoe (1970) has reported that alteration of the carbohydrate content of the diet could increase glucokinase activity in chickens. Hence, some confusion exists as to whether or not glucokinase is present in chicken liver. If glucokinase is absent from chicken liver, then a major control for glucose uptake by liver will be absent unless the chicken liver hexokinases have markedly different properties from those of rat liver. If the control of glucose efflux and influx is not mediated through the rate of glucose phosphorylation, then either the control of glucose-6-phosphatase or of glueose-6-phosphate concentration could be responsible for regulating the flux. Experimental evidence suggests that glucose-6-phosphatase is a multifunctional enzyme (see review by Nordlie, 1974). In addition to its phosphohydrolase activity, it can catalyse the phosphorylation of glucose from several phosphoryl donors, although the physiological significanoe of these activities is doubtful. The aim of the experiments described in this paper was to determine whether or not glucokinase activity was present in: chicken liver and to determine the roles played by glucose phosphorylating and glucose-6-phosphate dephosphorylating activities in controlling glucose flux into and out of chicken hepatocytes. MATERIALS AND METHODS

Animals

Chickens (aged 4-6 weeks) of the Thornber 606 strain were purchased from the Poultry Research Centre, Edinburgh. They were allowed access to food and water ad libitum except during starvation when water only was given.

318

IRIS E. O'NEILL and DEREK R. LANGSLOW Liver was homogenised either in 2 volumes Buffer A pH 7.5 (for hexokinase) or in i0 volumes of 80 mM citrate buffer pH 6.8 (for glucose-6-phosphatase). All centrifugations were at 0-2oc.

Centrifuge the homogenate at 600 g for i0 minutes Pellet / Rehomogenise the pellet in the appropriate buffer. Spin at 600g for 6 ::~inutes

/

Supernatant

',,

Residue ! (Nuclei, plasma membranes)

Supernatant 2

Combine the supernatants

I Spin at 23000g for 8 minutes

Eesidue 2 (Mitochondria, lysosomes, peroxisomes)

Residue 3 (Mierosomes)

Supcrnatarlt 3 Spin at 1050006 for 30 minutes

Supernatant 4 (Cell sap)

Each residue was resuspended in the appropriate buffer and rehomogenised to disperse the particles prior to assay.

Fig. 1. Preparation of subcellular fractions from chicken liver homogenates.

Preparation of liver homogenates Chickens were killed by decapitation and the livers removed, weighed and placed on ice. For hexokinase (E.C. 2.7.1.1), 50~o homogenates were prepared by hand-homogenisation in an ice-cold medium (pH 7.5) containing 154mM KC1, 4 m M MgSO4.7 H20, 4 m M EDTA disodium salt and 8 mM N-acetyl-L-cysteine (Buffer A; Dawson & Hales, 1969). For glucose-6-phosphatase (E.C. 3.1.3.9), 10-% homogenates were prepared in ice-cold 80mM sodium citrate (pH 6.8) using an all-glass handhomogeniser.

Glucose-6-phosphatase. Glucose-6-phophatase was assayed by measuring inorganic phosphate production in a medium containing final concentrations of 27mM sodium citrate (pH 6.8) and 13 mM glucose-6-phosphate. The reaction was started by adding freshly prepared homogenate or resuspended subcellular fractions. Incubations were at 37°C and reactions were terminated after 15 min by the addition of 1 ml of 10~ trichloracetic acid. The inorganic phosphate liberated was measured by the method of Fiske & Subbarow (1925). Reagent blanks with either giucose-6-phosphate or homogenate omitted were also determined.

Subcellular fractionation Hexokinase and glucose-6-phosphatase activities were measured in different subcellular fractions of liver prepared by a modification of the method of De Duve (1965). The details of the procedure are shown in Fig. 1.

Measurement of enzyme activities Hexokinase. Hexokinase was assayed by measuring giucose-6-phosphate production at 25°C according to the method of Vinuela et al. (1963). Final glucose concentrations of 0.83 and 83.3 mM were used. ATP was used to start the reaction, and blank rates without ATP were determined at both glucose concentrations. Hexokinase activity was routinely measured in high-speed supernatants prepared by centrifuging the initial homogenate at 105,000 g for 60 rain. These supernatants gave high blank rates of NADPH production, and hence they were dialysed against several changes (500 ml each time) of buffer A at room temperature for 4 h r before being assayed. This removed glucose from the supernatant and lowered the blank rate to zero. The total enzyme activity of hexokinase was the same before and after dialysis.

Measurement of protein, glycogen, glucose and free fatty acids Protein was estimated in homogenates and supernatants according to Lowry et al. (1951) using bovine serum albumin as standard. Glucose was measured in deproteinised solutions by a modification of the method of Hugget & Nixon (1957) in which 4-aminophenazone replaces o-dianisidine in the assay medium (Trinder, 1969). Glycogen was measured by the method of Walaas & Walaas (1950), and free fatty acids according to Laurell & Tibling (1967).

Treatment of experimental chickens Glucose loading. Glucose (2 g dissolved in 2 ml warm distilied water) was given orally to starved chickens by intubation. The frequency of administration and the total amount of glucose given during each experiment are described in the figures. Control birds were given 2 ml water by the same method. Glucose-rich diet. A diet containing 70% glucose, 15% casein, 8% gelatin, 0,3% cystine, 0.2% choline chloride, 0.1% tryptophan, 0.1% inositol, 6% mineral salts mixture

Glucose metabolism in chicken liver Table 1. The Michaelis constants of hexokinase for glucose, fructose and ATP Glucose I

Fructose

II

A

I

B

ATP

II

A

I

II

B

A

B

Km (mM) 0.072 0.080 0.071 5.41 4.32 2.62 1.37 1.24 0.66 Column I gives the values presented in this paper from liver supernatants, and column II gives values for the purified chicken liver hexokinases from Ureta et al. (1972). and 0.3% vitamin mixture was prepared (Pearce, 1970). The composition of the vitamin and mineral salts mixtures are described by Whitehead et al. (1976). Chickens, which had previously been- starved for 48 hr, were fed ad libitum for 4 days on the glucose-rich diet. A control group was also starved for 48 hr but refed the usual cereal-based diet (Chick Starter Crumbs supplied by West Cumberland Farmers Ltd).

Sources of chemicals and biochemicals ATP, ADP, AMP, NADP, glucose-6-phosphate and all the enzymes used were obtained from the Boehringer Corporation (London) Ltd., Ealing, London. Bovine serum albumin (Fraction V) was purchased from Armour Pharmaceutical Co., Eastbourne, Sussex, U.K. and all other chemicals were of Analar grade (or the best grade available) and were obtained from BDH Chemicals Ltd., Poole, Dorset, U.K. or Koch-Light Laboratories, Ltd., Coinbrook, Bucks, U.K. RESULTS

General properties of chicken liver hexokinase All hexokinase velocities were measured using freshly prepared, well-dialysed 105,000 g supernatants. The effects of increasing glucose or fructose concentrations were measured at 4 mM ATP and the Km for ATP was determined at 83.3 mM glucose (Table 1). The Michaelis constants of hexokinase for glucose, fructose and ATP were determined from LineweaverBurk double reciprocal plots. The Km for glucose was 72 pM and there was no evidence for an isozyme with a K , in the millimolar range. Furthermore, when

319

hexokinase activity was measured at several glucose concentrations from 0.017 to 83.3 raM, no inhibition of enzyme activity was found and the velocity-substrate curve had the simple hyperbolic form of the Michaelis-Menten equation. Concentrations of ATP up to 8.3 mM had no inhibitory effect on hexokinase activity measured at 83.3 ram glucose. Hexokinase activity was normally measured at low (L) 0.83 and high (H) 83.3 mM glucose. The ratio of velocities at high and low glucose concentrations was usually between 1.1 and 1.2, which is close to the theoretical ratio predicted by the Michaelis-Menten equation. Both AMP and ADP inhibited hexokinase activity competitively, with respect to ATE The inhibitor constants (K~) calculated from Dixon plots were 1.24 mM for ADP and 4.9 mM for AMP. N-acetyl-Dglucosamine is a competitive inhibitor of hexokinases. It exerts only moderate competitive inhibition, with respect to glucose, on rat hexokinase (Km/K i ratio = 0.02) but strong competitive inhibition of rat glucokinase (K~/K~ ratio = 15) (Vinuela et al., 1963). N-acetyl-o-glucosamine inhibited chicken liver hexokinases competitively with a K~ of 0.32 mM. The K~/K~ ratio was calculated to be 0.22.

General properties of chicken liver glucose-6-phosphatase The glucose-6-phosphatase activity in freshly prepared liver homogenates from fed and 24-hr starved chickens was measured over a range of glucose-6phosphate concentrations from 1.7 to 27 mM. The Michaelis constants were calculated from double reciprocal plots (Fi~ 2) and starvation was seen to lower the Km from 5.65 mM in fed chickens to 3.75 mM in starved chickens. Although the glucose concentration in the assay mixture did not affect the Km of glucose-6-phosphatase, glucose inhibited the activity of the enzyme measured with 13 mM glucose-6phosphate. An increase in glucose concentration beyond 3 mM caused a progressive inhibition of enzyme activity which was 50% at 17 mM. Glucose (24mM) inhibited the enzyme activity by 62% but higher glucose concentrations caused no further inhibition (Fig. 3).

20ed

I_ V

I I Km I

r.sfarved

IO-

~

I

Km H

05

I I 0

I/S

Fig. 2. The Michaelis constants of fiver glucose-6-phosphatase for glucose-6-phosphate in fed and 24-hr starved chickens. The mean K , for G6P for fed and 24-hr starved chickens was 5.65mM and 3.75 mM respectively. c'.a.P.

59/4e--o

320

IRlS E. O'NEILL and DEREK R. LANGSLOW I00 ~



(Davison & Langslow, 1975) and its distribution was not measured. All the glucose-6-phosphatase activity was particulate (Table 2). More than 60~o of the total activity was in Residue 1 and most of the remainder was in the microsomal fraction (Residue 3). Starvation did not alter the sub-cellular distribution of the enzyme.

\ \

Glucose phosphorylating activity in liver from chickens of different ages and in chicken brain and kidney C Q-

~

4O

t

I

I

I

I0

2O

30

4O

Glucose, mM

Fig. 3. Inhibition of glucose-6-phosphatase by increasing glucose concentrations. Values are the means of triplicate determinations on three different liver preparations.

No evidence for a high Km glucokinase-type activity was found in liver homogenates or supernatants from 4-6-week old chickens (Table 3). The ratio of hexokinase activity at 83.3 (H) to 0.83 m M (L) glucose concentration was always slightly greater than 1. Similar H/L ratios were found in liver homogenates and 105,000 g supernatants from 2-day old, 9-day old and 16-day old chickens. Supernatants (105,000 g) from rat liver consistently yielded H/L ratios of between 2.5 and 3, indicating the presence of glucokinase. The H/L ratios of hexokinase activity obtained from homogenates and 105,000 g supernatants of chicken brain and kidney were similar to those found in liver (Table 3). The increase in phosphorylating activity at the higher glucose concentration averaged 6 ~ in brain and 11~o in kidney.

Subcellular distribution of hexokinase and glucose-6phosphatase in livers from fed and 24-hr starved chickens

Effect of starvation and acute glucose loading on chicken liver hexokinase and glucose-6-phosphatase activities

The hexokinase activity of chicken liver is partly associated with the nuclear fraction (Residue 1) and partly with the 105,000g supernatant. There was almost no activity in the other sub-cellular fractions. Starvation did not alter the sub-cellular distribution of the enzyme (Table 2). Neither hexokinase nor glucose-6-phosphatase activity was found to be associated with glycogen in fed chickens since 86~o of the glycogen appeared in Residue 2. The glycogen content of liver from starved chickens is greatly reduced

Homogenates and supernatants were prepared from the livers of chickens which were either (a) fed or (b) starved for 24 hr or (c) starved for 24 hr and then given glucose orally (2 g at 1-hr intervals for 3 hrs) before being killed 1 hr after the final dose. The last more than doubled the plasma glucose concentration. Starvation for 24 hr caused a small but significant fall in the total hexokinase activity and glucose loading increased the activity towards fed values (Fig. 4).

~---Table 2. SubceUular distribution of hexokinase, glucose-6-phosphatase and glycogen ~ in livers from fed and 24-hr starved chickens Hexokinase 24-hr Fed starved Residue 1 Residue 2 Residue 3 Supernatant 4

42.8 3 2.2 52.1

+ 2.7 _ 0.1 + 0.1 4- 2.8

44.3 ___1.6 2.8 -4- 0.2 0.9 _ 0.1 52.1 4- 1.8

Glucose-6-Phosphatase 24-hr Fed starved 61.4 + 1 13.8 4- 0.5 21.8 4- 0.6 3 + 0.4

61.4 _+ 1 12.6 _ 0.8 22.3 + 0.7 3.7 4- 0.2

Glycogen Fed 8.7 86.1 8.7 0

All enzyme values are expressed as the percentage of total activity + S.E.M. of 4 observations. Glycogen values are expressed as the percentage of the homogenate total found in each fraction. Table 3. Total glucose phosphorylating activity in homogenates and supernatants from chicken brain, kidney and liver Homogenate Units/g Units/g tissue protein Glucose concn in assay Brain Kidney Liver

0.83 2.73 0.65 0.2

83.3 2.74 0.73 0.26

0.83 83.3 12.38 12.38 2.12 2.4 1.64 2.05

H/L ratio 1 1.13 1.25

Supernatant Units/g Units/g tissue protein 0.83 0.17 0.21 0.12

83.3 0.20 0.22 0.14

0.83 1.8 0.95 2.73

83.3 2.07 1.21 3.12

H/L ratio 1.13 1.09 1.14

Glucose metabolism in chicken liver A

E ~ ~Z

Hexokinase

Glucose - 6 - phosphalose

~ 2oo I-

321

I

=50

go o ~ e IoO o

÷

8"-

I I I I

C: Q)

I Fed

2 4 hour starved

Fed

glucose tO~

2 4 hour stor~

glucose I ~

Fig. 4. Changes in hexokinase and glucose-6-phosphatase activity with 24-hr starvation and starvation plus acute glucose loading. Values are means + S.E.M. of 7 observations.

300--

o, . 8 I •

8~o

J~ G} O.N 2 0 0 - -

J

0

I

2

I

4

]

6

I

]

8

,12

I0

Duroi'ion of si'arvoi'ion,

• i

I

I

24

hr

Fig. 5. Changes in glucose-6-phosphatase activity with duration of starvation. Values are means + S.E.M. of 4 observations. Neither starvation nor glucose loading altered the H/L ratio significantly. Glucose-6-phosphatase activity was more than doubled by 24-hr starvation, but refeeding glucose over 4 hr reduced the activity to fed values. The time course of the changes in glucose-6-phosphatase activity during starvation and glucose loading was studied. Fed chickens were starved from time zero and killed after 2, 4, 6, 8, 10 and 24 hr. A significant increase of 14% in the glucose-6-phosphatase activity was observed after 4 hr starvation. This increase was progressive with time until a plateau was reached after 10 hr starvation (Fig. 5). The changes in glucose-6-phosphatase activity following glucose loading of 24-hr starved chickens were studied by giving glucose to chickens orally (2 g) at time zero and killing groups after 0.5, 1, 2 and 4 hr. Those used after 2 and 4 hr were given a second dose of oral glucose after 1 hr to ensure that their plasma glucose concentration remained above 20 mM. A 20% decrease in glucose-6phosphatase activity was observed after 1 hr (Fig. 6). The enzyme's activity continued to fall gradually with time and was restored to fed values after 4 hr.

Effect of glucose-rich diet on hexokinase activity Pearce (1970) reported that a glucose-rich diet induced an increase in glucokinase-like activity in

chicken liver. A diet identical to Pearce's formulation (1970) was prepared and fed to chickens for 4 days. A control group was fed the normal cereal-based diet. Neither the plasma glucose nor free fatty acid concentrations of the chickens were altered by the diet (Table 4). There was no evidence for glucokinase activity in 105,000 g supernatants from these livers, and

~

100

75 o =.

i 5° 0

30 60 120 T i m e a f t e r glucose a d m i n i s t r a t i o n ,

240 min

Fig. 6. Changes in glucose-6-phosphatase activity with duration of glucose loading. Values are means + S.E.M. of 4 observations.

322

IRIS E. O'NEILL and DEREK R. LANGSLOW Table 4. The plasma glucose and FFA concentrations of chickens fed with control or glucose-rich diets Control diet Plasma glucose (mM) Plasma FFA ~M)

Glucose-rich diet

1

2

1

2

3

4

15 275

14.4 300

14.6 275

14.8 290

13.2 350

14.5 220

The values are for 6 individual chickens used on the same day. the H/L ratios were similar in both groups of chickens (Table 5). Phosphotransferase activity was measured both before and after dialysis but there was no high K , activity in either instance. The fall in total hexokinase activity following the glucose-rich diet was unexpected.

The plasma glucose and free fatty acid concentrations of these birds were the same as those fed a normal cereal-based diet. Hence the data presented here disagree with his findings. The Michaelis constants for chicken liver hexokinase activity in dialysed 105,000g supernatants agree well with those determined by Ureta et al. DISCUSSION (1972) on purified isozymes (Table 1). The slightly IS glucokinase present in chicken liver? greater Km for ATP might be due to some non-speciNo phosphorylating activity which could be attri- fic ATPase activity in the supernatants. The inhibitor buted to glucokinase was found in homogenates and constant (Ki) for ADP was similar to that reported 105,000g supernatants from chicken liver, and only for other hexokinases (Copley & Fromm, 1967). The low Km hexokinases could be detected. Ureta et al. inhibitor constant for AMP is about 5-times greater (1972) separated the hexokinase isozymes of chicken than the intracellular concentration of AMP, and is liver and also found no enzyme with the kinetic and thus unlikely to play any regulatory role in vivo. The chromatographic characteristics of glucokinase. Wal- absence of inhibition of chicken liver hexokinases by lace & Newsholme (1967) and Pearce (1970) have, high glucose concentration is in accordance with however, reported that about 20-25% of the glucose earlier studies (Ureta et al., 1972). N-acetyi-D-glucosamine, a competitive inhibitor of phosphorylating activity in chicken liver was due to glucokinase. Furthermore, when Pearce (1970) fed a hexokinases, exerts only moderate competitive inhibismall group of chickens a glucose-rich diet, the pro- tion, with respect to glucose, on rat hexokinase portion of phosphorylating activity ascribed to glu- (Km/Ki ratio = 0.02) but strong competitive inhibition cokinase increased from 25% to 50%. In both in- on rat glucokinase (Km/Ki ratio = 15) (Vinuela et al., stances, the amount of glucokinase activity was based 1963). Chicken liver hexokinase shows a competitive on the difference in phosphorylating activity between inhibition between these two types. The K~/K~ ratio 0.5 and 100 mM glucose, but they failed to take two of 0.22 is intermediate between the values calculated important factors into account. Firstly, the hexo- for rat liver phosphotransferases, and this provides kinases of chicken liver are not inhibited by high con- further evidence against the existence of glucokinase. The adaptive nature of rat liver glucokinase is well centrations of glucose, which is in contrast to rat liver hexokinase Type C (Dawson & Hales, 1969). established (Perez et al., 1964; Ureta et al., 1971). Secondly, no allowance was made for the expected There was no evidence from these studies that chicken increase in enzyme velocity (based on the Michaelis- liver hexokinases were adaptive. The ratio of phosMenten equation) when the substrate concentration phorylating activity observed at high and low glucose is raised from 0.5 to 100mM glucose. Using the concentrations was the same in fed, 24-hr starved and 48-hr starved chickens and in starved chickens refed Michaelis-Menten equation with glucose. This contrasts with the sharp fall in H/L ratio observed with rat liver phosphotransferases following starvation (Vinuela et al., 1963; O'Neill & where Vis the enzyme velocity with substrate concen- Langslow, unpublished observations). trations. Vma~is the maximum enzyme velocity and K , is the Michaelis constant, an increase in velocity Table 5. The total glucose phosphorylating activity in chicken liver alter a glucose-rich diet of about 15% would be expected when the substrate concentration is increased from 0.5 to 100 mM gluUnits/g Units/g H/L cose and 10% following an increase from 0.83 to liver protein ratio 83.3 mM glucose. The average 14% in activity found in this study and the 20% found by Pearce (1970) Glucose concn 0.83 83.3 0.83 83.3 -and Wallace & Newsholme (1967) are both close to in assay (raM) 3.64 1.26 the increases predicted by the Michaelis-Menten Control diet: 1 0.373 0.469 2.89 2 0.387 0.422 3.75 4.09 1.09 equation. As expected, neither chicken brain nor kidGlucose-rich ney showed any-glucokinase-type activity. diet: 1 0.229 0.251 2.17 2.38 1.10 There is, however, the observation by Pearce (1970) 2 0.193 0.253 2.07 2.71 1.31 that a glucose-rich diet increased glucokinase activity 3 0.277 0.286 2.68 2.77 1.03 in chicken liver, which remains to be explained. We 4 0.251 0.277 2.49 2.75 1.10 were unable to detect any change in the ratio of phosphorylating activity at high and low glucose concenThe values are from 6 individual chickens used on the trations after feeding a diet identical to that of Pearce. same day.

Glucose metabolism in chicken liver Subcellular distribution of hexokinase and glucose-6phosphatase

323

plasm to the lumen of the endoplasmic reticulum, and the other is the catalytic component, glucose-6-phosphatase, which is bound .to the lumenal surface of the membrane.

Manimalian hexokinases exist in both the soluble and particulate fractions of the cell, and the particulate activity is most often bound to mitochondria. The Changes in hexokinase and glucose-6-phosphatase ackinetic parameters of the bound and soluble forms tivity during starvation and glucose loading Starvation in chickens must reduce glucose inflow of the enzyme are different. Subcellular redistribution of the enzyme may, therefore, have a regulatory func- into, and increase glucose efflux from, liver if the tion in controlling glucose phosphorylation. The dis- plasma glucose concentration is to be maintained. tribution of hexokinase between the soluble and par- The small but consistent fall in hexokinase activity, ticulate fractions of the cell could be altered by chang- together with a doubling of glucose-6-phosphatase acing the concentrations of certain metab01ites. Both tivity, will alter the flux of glucose across the liver ATP and glucose-6-phosphate can solubilise rat brain cell membrane to produce net glucose efflux after hexokinase in rive while Mg 2+ and inorganic phos- 24-hr starvation. Acute glucose loading restores phate both inhibit solubilisation (Wilson, 1968; Hoch- enzyme activities to values found in the fed state. It man & Sacktor, 1973). In contrast to this, Purich & seems unlikely that any relationship between hepatic Fromm (1971) were unable to demonstrate any glycogen content and hexokinase activity exists in change in the solubilisation or binding of hexokinase chicken liver, since the distribution of glycogen was to rat brain mitochondria when metabolite concen- confined to the particulate fractions of the cell which contain little hexokinase activities, and also because trations were varied. We have shown that 24-hr starvation did not alter starvation, which reduces liver glycogen by 90% the ATP concentration, although the glueose-6-phos- (Davison & Langslow, 1975), did not alter the subcelphate concentration increased approximately 2-fold lular distribution of hexokinase. The doubling of chicken liver glucose-6-phospha(O'Neill & Langslow, unpublished observations). Such a small change is unlikely to influence the bind- tase activity after 24-hr starvation is similar to that ing of the enzyme to membrane portions of the cell. observed in rat liver homogenates (Arion & Nordlie, The distribution of hexokinase within the subcellular 1965). They observed a further increase in phosphofraction of chicken liver cells was not altered by either hydrolase activity after 48-hr starvation but only in 24- or 48-hr starvation, and a 50:50 distribution the presence of detergent. Gunderson & Nordlie between soluble and particulate enzyme always (1973) have shown that the enzyme, as it exists in the membrane of intact isolated avian hepatic nuclei, existed. The soluble to particulate ratio of chicken brain is almost totally active without any form of memhexokinase activity is quite different to that observed brane disruption. It shows marked contrast to the in chicken liver. Only 15% of brain hexokinase ac- activity in recovered fragments of endoplasmic reticutivity is found in 105,000 g supernatants. Knull et al. lure, which is latent until the membranes are dis(1973, 1974), working with 5-day old chicks, have solved. However, latency develops when the memreported a 50:50 distribution of hexokinase in soluble branes of these hepatic nuclei are disrupted, indicatand particulate fractions, and that a 20:80 ratio only ing a complex relationship between membrane strucexisted under energy-stress conditions. These differ- ture and enzymatic behaviour. The partial latency ences in ratio between our results and those of KnuU observed by Arion & Nordlie (1965) in liver homoet al. (1973, 1974) may he due to the different ages genates from 48-hr starved rats may be due to an of chickens used or to the stress of decapitation which artifact of the homogenization procedure, rather than might cause rapid changes in subcellular distribution. a true reflection of the activity in vivo. We found no About 60% of the phosphohydrolase activity of effect of several different detergents on the total gluchicken liver homogenates sedimented at 600 g, and cose-6-phosphatase activity (O'Neill & Langslow, unhence the total glucose-6-phosphatase activity could published observations) and thus the homogenization only be determined in homogenates. Wallace & procedure may be crucial. Newsholme (1967) measured glucose-6-phosphatase The increase in glucose-6-phosphatase activity durin 600 g supernatants from chicken liver and the ac- ing starvation probably involves some new enzyme tivities they reported are only 30% of those reported formation. Only a small part of the increase in here. enzyme activity will be accounted for by the fall in The biological advantage of the close association K,, observed on starvation. Arion & Nordlie (1965) of glucose-6-phosphatase with microsomes and showed that the treatment of rats for 3 days with nuclear membranes is not readily apparent. It could glucocorticoids increased liver glucose-6-phosphatase aid compartmentation of the glucose-6-phosphate and activity. However, this difference was not apparent thus enable the substrate for glucose-6-phosphatase if the homogenates were pretreated with deoxychoto be in a different part of the cell from those reac- late, and actinomycin D administration simultions which consume it, since the physiological situ- taneously with the glucocorticoids did not affect total ations which necessitate glucose-6-phosphate utilisa- glucose-6-phosphatase activity. They have suggested, tion by glycolysis and glycogen synthesis are different therefore, that the increase of glucose-6-phosphatase from those which necessitate glucose production. was due to the activation of a pre-existing enzyme. Arion et al. (1975) have proposed that there are two Such a situation after starvation in chickens seems components in the microsomal membrane. One is a unlikely, as detergents do not release enzyme activity. glucose-6-phosphate specific transport system which Glucose administration rapidly reduced glucose-6functions to move glucose-6-phosphate from the. cyto- phosphatase activity. The high plasma glucose con-

324

IRIS E. O'NEILL and DEREK R. LANGSLOW

centration, the fall in glucose-6-phosphate concentration and the increase in Km will all contribute to the rapid reduction of glucose-6-phosphatase activity in vivo. Other activities of glucose-6-phosphatase

Glucose-6-phosphatase is a multifunctionai enzyme (see review by Nordlie, 1974) and can use several phosphoryl donors to phosphorylate glucose. They are unlikely to be physiologically significant, however, as the pH optima of these activities varies between 5 and 6. One phosphoryl donor, carbamyl phosphate, is active around pH 7. However, there are good reasons for rejecting this activity as non-physiological. Firstly, the Km for carbamyl phosphate is 2.8 mM, while, in the cell, carbamyl phosphate is hardly detectable (at most, 0.1 #M; Raijman, 1974) and is found exclusively in the mitochondria, from which glucose-6-phosphatase is absent. Secondly, the activity requires very high glucose concentrations and is most apparent after treatment with detergents. It is therefore unlikely that carbamyl phosphate phosphotransferase will phosphorylate glucose in vivo. Conclusions

The evidence presented in this paper is overwhelmingly against the presence of glucokinase in chicken liver. The chicken liver hexokinases are typical low-K~, isozymes which are not inhibited by glucose and, provided cellular ATP concentrations are normal, will show maximum and essentially constant activity under different physiological conditions, since the plasma glucose concentration is generally above 10 mM. It therefore seems likely that glucose flux into and out of chicken hepatocytes is regulated by the activity of glucose-6-phosphatase and by the provision of glucose-6-phosphate. The assertion of Nordlie (1974) that regulation of this flux is not possible without the presence of another glucose phosphorylating activity in chicken liver is not upheld by the data presented in this paper. Hypothetical calculations, similar to those employed by Nordlie (1974), show that, while in the fed state hexokinase activity exceeds glucose-6-phosphatase activity above about 10--13mM, in the starved chicken glucose-6-phosphatase activity exceeds hexokinase activity at all glucose concentrations below 30 mM. Hence hexokinase and glucose-6-phosphatase activity alone can regulate glucose flux in and out of chicken hepatocytes.

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Glucose phosphorylation and dephosphorylation in chicken liver.

Comp. Biochem. Physiol., 1978, VoL 59B, pp. 317 to 325. Pergamon Press. Printed in Great Britain GLUCOSE PHOSPHORYLATION A N D DEPHOSPHORYLATION IN C...
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