Dexamethasone effects on liver pyruvate kinase

140

Biochimica et Biophysica Acta, 675 (1981) 140 142

Elsevier/North-HoUandBiomedicalPress

BBA Report BBA 21556

DEXAMETHASONE EFFECTS ON LIVER PYRUVATE KINASE MARK L. JOHNSON, JOEL B. HANSEN,JAMES C. DONOFRIO and CARLO M. VENEZIALE * Department of Cell Biology, Mayo Medical School, Rochester, MN 55901 [U.S.A.J

(Received November 3rd, 1980)

Key words: Pyruvate kinase; Dexamethasone;(Rabbit liver)

Dexamethasone in the medium perfusing isolated rabbit livers caused a fast-acting and reversible effect on liver pyruvate kinase. The effect was to lower the assayable V activity (units/g tissue) without changing the concentration (nmol/g enzyme protein). In effect, glucocorticoid lowered the specific activity (units/nmol of enzyme) by direct action on liver. The effect on liver pyruvate kinase is mediated by a relatively stable alteration; 30 rain after perfusate (with steroid) was replaced by perfusate (without steroid), the effect remained strongly evident.

The permissive action of glucocorticoids in modulating the effects of other gluconeogenic hormones is generally accepted [1 ]. Glucocorticoids also 'induce' the activity of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase [2], fructose bisphosphatase [3-5] and glucose-6-phosphatase [3,4,6]. Data supporting a steroid-mediated reciprocal inhibition of the glycolytic enzymes opposing the nonequilibrium gluconeogenic reactions have been lacking. However, we recently reported that the administration of triamcinolone to rabbits resulted in an inhibition of liver pyruvate kinase assayable activity and proved that this inhibition was due to a decrease in specific activity [7]. Because whole animals were used there was concern that extra-hepatic phenomena might have been responsible. In this paper we present liver perfusion data which establish that glucocorticolds in pharmacologic doses can act directly on rabbit liver to decrease the units/nmol of liver pyruvate kinase and do not change the concentration of enzyme protein. Liver pyruvate kinase activity and concentration: Rabbits weighing approx. 1200 g and fed a 50-60% * To whom reprint requests should be addressed.

carbohydrate diet ad lib. provided the livers. Samples of perfused livers were homogenized in 4 vol. of 50 mM sodium phosphate buffer, pH 7.5, containing 10% glycerol (v/v), 10 mM/3-mercaptoethanol, 1 mM EDTA, 2 mM MgSO4 and 2 mM fructose bisphosphate. In all studies the supernatant fraction of homogenates centrifuged at 200000 Xg for 15 min was used. The V activity assay was applied to a partially purified fraction of liver extract which was free of the muscle pyruvate kinase isoenzyme [7]. The radioimmunoassay for liver pyruvate kinase protein concentration has been described in detail [7]. Liver perfusion: The livers of anesthetized rabbits were cannulated and isolated for perfusion [8]. The perfusion buffer was a Krebs-Ringer's bicarbonate buffer containing 1% bovine serum albumin (radioimmunoassay grade, Insulin Free, Sigma Chemical Co., St. Louis, MO), 10 mM glucose, 1X final concentration of amino acids without L-glutamine (Gibco, Grand Island, N Y ) a n d 1000-fold-diluted vitamin mixture (solution B with ascorbic acid; Upjohn Co., Kalamazoo, MI). Red blood cells were not included. We used a continuously recirculating perfusion system. The apparatus consisted of a large perfusion reservoir (800 ml capacity) on which sat a

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141 porcelain crucible which contained the liver and was similar to the apparatus o f others [9]. Perfusate was pumped at a constant flow (1 ml/gm ~min -1) from the reservoir b o t t o m to the liver. The liver was allowed 5 - 1 0 min to equilibrate with recirculating perfusate after the initial in situ washout o f red blood cells. Following equilibration, a t = 0 liver sample of approx. 2 g was removed; one-half was frozen immediately in liquid N2 and accurately weighed; the other half, also weighed, was homogenized as described. Perfusate 'bleeding' from the wound was prevented by placing a ligature around the lobe just proximal to the cut. Samples were taken at the indicated intervals (see Tables). Dexamethasone (1.42" 10 -s M) was added to the perfusate immediately after the t = 0 sample. At 30 min a liver sample was taken. The liver was then perfused with 100 ml hormone-free buffer from a second reservoir. The main reservoir was emptied, washed with saline and refilled with 600 ml hormone-free perfusate. The liver was perfused from the main reservoir for the remainder of the experiment. Adenine nucleotide concentrations: The liver specimens (about 1 g) which had been frozen in liquid N2 were weighed and then homogenized in 4 vol. o f ice-cold 20% perchloric acid using a Polytron apparatus. The supernatant was neutralized and assayed for ATP, ADP and AMP according to the procedure of Adam [10]. We had previously shown a direct effect o f glucagon on liver pyruvate kinase activity using isolated rat hepatocytes [11] and subsequently established that for whole animals this was also due to a lowering o f the enzyme's specific activity [7]. Pyruvate kinase data from rabbit liver perfused with glucagon proved less than satisfactory; the data were not often reproducible for unexplained reasons. Therefore, we were deterred from perfusion experiments which included glucagon in the perfusate. The total amount o f adenine nucleotides was constant during the perfusion at 4.35 -+ 0.53 g m o l s / g wet weight (n = 24). ATP was 2.45 _+0.44 #mols/g wet weight, ADP was 1.21 _+0.25 and AMP was 0.70 -+ 0.25. Thus, a constant A T P : A D P : AMP ratio was also maintained. Table I shows the results of the control liver perfusions. The concentration (1.07 /~M), assayable activity (12.8 units/g) and calculated specific activity (12.0 units/nmol) were constant during

TABLE I PYRUVATE KINASE IN CONTROL PERFUSED LIVERS Values are shown as mean ± S.D. of three perfusions. Concentration was determined by radioimmunoassay and is reported as gM or gmol enzyme/1000 g wet weight tissue. Units/g represents the enzyme activity measured in the extracts. Units/nmol is the specific activity of liver pyruvate kinase calculated by dividing units/g by nmol/g. Time

Concentration (uM)

Units/g

Units/nmol

0

1.07 -+.08 1.04 -+.07 1.06 ±.08 1.02 ±.09 1.14 ±.07

12.6 ±1.2 12.3 ±1.2 12.6 ±1.1 12.4 ±0.7 14.1 ±0.6

11.8 -+1.9 11.8 -+0.6 11.9 ±0.2 12.3 -+l.6 12.4 ±0.4

30 min 60 min 120 min 5h

TABLE II PYRUVATE KINASE IN DEXAMETHASONE-PERFUSED LIVERS Data are presented as described in Table I (n = 3). Statistical analysis was performed by using Student's t-test. Dexamethasone was perfused for the 30 min interval following zero time liver sampling. Time

Concentration (~M)

Units/g

Units/nmol

0

1.09 -*.01 1.03 -+.04 1.03 +-0.4 1.22 -*.10 1.18 ±.05

12.1 ±1.2 9.9 ** ±1.0 8.9 *,** ±1.4 11.0 -+1.1 13.2 ±0.3

11.2 ±1.1 9.6 ** ±1.0 8.7 *,** -2_1.4 9.1 ±1.6 11.2 -+0.8

30 min 60 min 120 min 5h

* P < 0.05 compared to tde x = 0 ** P < 0.05 compared to tcontrol; corresponding time point from control perfusion

142 the 5 h of perfusion and agree closely with those reported for control intact rabbits [71. The addition of dexamethasone to the perfusate caused significant alterations in activity and specific activity, but did not significantly alter enzyme concentration (Table II). Two controls were used for statistical evaluation of the dexamethasone data. Comparisons were made to the t = 0 data from the dexamethasone perfusions and to the corresponding time point from the perfused control livers. As shown in Table II dexamethasone caused a very rapid decrease in activity and specific activity. The effect was maximal at 60 min but at 30 rain a decrease was already evident. The effect of dexamethasone was reversible: at 120 min, i.e., 90 min after hormone withdrawal, the enzyme activity was returning to normal. At 5 h, all values were identical to control. Our data clearly demonstrate that dexamethasone has a relatively fast-acting, direct effect on liver pyruvate kinase in the perfused liver. The magnitude of the decrease in specific activity was similar to the decrease observed in the rabbit 1 h post-injection [7]. Thus, even though a large initial concentration of steroid was perfused, the result was identical to that obtained in in vivo studies [7]. It has been suggested that glucocorticoids may regulate liver pyruvate kinase activity in vivo through allosteric inhibition by alanine which has been mobilized to the liver form peripheral tissue [12]. Our data argue against such a mechanism. The activity assay conditions we employ result in a significant dilution of enzyme and any associated inhibitor. Therefore, one may conclude

that the change in specific activity is probably due to a stable modification, perhaps covalent. A kinetic analysis of the enzyme in liver extract suggested both an increase in K m for phosphoenolpyruvate and a decrease in V. However, further verification must be obtained using purified enzyme isolated from liver of the glucocorticoid-treated rabbit.

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