The influence of hypothyroidism on the adrenergic stimulation of glycogenolysis in perfused rat liver

Biochimica et Biophvstca Acta, 798 (1984) 350 360

350

Elsevier BBA21735

T H E INFLUENCE O F H Y P O T H Y R O I D I S M ON T H E ADRENERGIC S T I M U L A T I O N OF G L Y C O G E N O L Y S I S IN P E R F U S E D RAT LIVER HUIBERT STORM *, CORNELIS VAN HARDEVELD and ANTON A.H. KASSENAAR

Department of Chemical Pathology, University Hospital, Rijnsburgerweg 10, 2333 AA Leiden (The Netherlands) (Received November 17th, 1983)

Key words: Hypothyroidism; Adrenergic stimulation; Glycogenolysis," (Rat liver)

The ability of noradrenaline (1 pM), phenylephrine (10 pM), and isoproterenol (1 #M) to stimulate glycogenolysis in euthyroid and hypothyroid pedused rat livers was investigated. It was found that hypothyroidism severely impaired a-receptor-mediated (noradrenaline, phenylephrine) glucose release. The initial Ca2+ efflux and K ÷ influx induced by these agonists in the euthyroid control group were almost totally absent in the hypothyroid group, while glycogen phosphorylase a activity in the hypothyroid rat livers was markedly lower than in the controls after infusing noradrenaline for 1 min. Diminished Ca2+ efflux (and possibly diminished K ÷ influx) is likely to play a role in the large impairment in the action of noradrenaline or phenylephrine on glycogenolysis in the pedused hypothyroid rat liver. After prolonged stimulation (15 min) with noradrenaline, however, the phosphorylase a activity in the hypothyroid and euthyroid groups did not differ significantly. This was accompanied by Ca2+ influx in the hypothyroid livers, probably facilitated by a fl-adrenergic effect of noradrenaline in this group. Hypothyroidism potentiated the effect of isoproterenoi on glycogenolysis. The glucose 6-phosphate content in the hypothyroid rat livers was markedly higher than in the euthyroid group after stimulation by noradrenaline or isoproterenol.

Introduction The requirement of thyroid hormones for normal free fatty acid mobilization in response to catecholamines has been convincingly demonstrated [1], but in the hypothyroid rat the catecholamine-induced mobilization of glucose might be deficient too [2]. Thus, a lack of substrates, i.e., glucose and free fatty acids, might be involved in the observed development of hypothermia in hypothyroid rats exposed to cold [3]. The effects of hypothyroidism on different stages of catecholamine-induced hepatic glyco-

* To whom reprint requests should be addressed. Abbreviation: EGTA, ethyleneglycol bis(fl-aminoethyl ether)N, N'-tetraacetic acid. 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

genolysis in the rat are not well understood and at some points conflicting. In the rat the effect of catecholamines on hepatic glucose release is mediated mainly by (x-adrenergic receptors [4]. It has been shown [5,6] that in hepatocytes of the hypothyroid rat activation of glycogen phosphorylase is shifted from an ~- to a t-receptor-mediated response, concomitant with a decreased [7] or unaltered [6] a-receptor response. In view of the stimulation of Ca2+-mediated metabolic processes by thyroid hormones in rat skeletal muscle, observed in our laboratory [8,9], it seemed of interest to investigate the effect of hypothyroidism on rat liver glycogenolysis induced by noradrenaline and phenylephrine, in which process Ca 2÷ is also intimately involved [10,11]. In addition, we examined the influence of hypothyroidism on the glycogenolytic effect of the fl-agonist iso-

351 proterenol. A preliminary report of a part of this work has been published [12]. Materials and Methods

Animals Male rats of a Wistar strain were used. Hypothyroidism was induced by one subcutaneous injection of 0.75 mCi Na131I given to animals which had been put on a low-iodine diet 1 week before. The experiments were performed 6 weeks after the injection, at which time the animals weighed about 250 g, Plasma L-thyroxin concentrations in the hypothyroid group were less than 10 nM, which is an 80% decrease compared to the euthyroid values (determined by radioimmunoassay, Clinical Assays, Travenol Laboratories, Inc., Cambridge, MA). Control rats were weight-matched (controls: 2.5-3-months-old, hypothyroid rats: 3.5-4months-old).

Perfusion The rats were anaesthetized with sodium pentobarbitone (60 m g / k g body weight) and the livers were perfused in situ in a non-recirculating system at a flow rate of 1 m l / m i n per g wet weight. The medium consisted of Krebs-Henseleit bicarbonate buffer [13], pH 7.4, supplemented with washed bovine erythrocytes (10-12% (w/v)), bovine serum albumin (3% (w/v)), glucose (10 mM), and pyruvate (0.15 mM). The Ca 2÷ and lactate concentrations were 1.3 and 1-2 mM, respectively. The medium was gassed with O2/CO 2 (19 : 1) and kept at 37°C. Phenylephrine, noradrenaline and isoproterenol were dissolved in saline and infused into the inflow line near to the liver. Phentolamine and propranolol were added to the perfusion reservoir.

Experimental design (I) In the first series of experiments, after perfusing the liver for 29 min (total perfusion time: t' = 29 min), an accessory lobe was cut out after a silk ligature had been tied around the base of the lobe, and freeze-clamped immediately between a pair of aluminium blocks that had been cooled in liquid N 2 [14]. In this lobe basal phosphorylase a and phosphorylase kinase activities and glucose 6-phosphate and glycogen content were de-

termined. The liver was allowed to recover during 16 min from the removal of the accessory lobe. Antagonists were added at t ' = 30 min and were used only in combination with noradrenaline. Infusion of noradrenaline or phenylephrine was started at t ' = 45 min (infusion time: t = 0 rain, which is indicated in the figures and tables) and lasted 15 min. At t ' = 60 min (t = 15 min in the figures and tables) the right main lobe was freezeclamped in order to determine agonist-induced phosphorylase a activity and glucose 6-phosphate content. Influent and basal effluent samples (1 ml) were taken at regular intervals, and after starting infusion of agonists, effluent samples were taken at the time points indicated in the figures. In these samples the concentrations of glucose, lactate, Ca 2÷, and K ÷ were determined. (II) In the second series of experiments, infusion of noradrenaline was started at t ' = 28 min (t = 0 ) and lasted 1 min, at which time point (t = 1) the right main lobe was freeze-clamped in order to determine agonist-induced phosphorylase a activity and glucose 6-phosphate content (as basal levels the values obtained in experimental series I were used). Influent and basal effluent samples were taken at regular intervals, and, after starting infusion of noradrenaline, effluent samples were taken every 4 s. In these samples the concentrations of Ca 2+ and K ÷ were determined. (III) In the last series of experiments infusion of isoproterenol was started at t ' = 28 min (t = 0 ) and lasted 15 rain, at which time point (t = 15) the right main lobe was freeze-clamped in order to determine agonist-induced phosphorylase a activity and glucose 6-phosphate content (as basal levels the values obtained in experimental series I were used). Influent and basal effluent samples were taken at regular intervals, and, after starting infusion of isoproterenol, effluent samples were taken at the time points indicated in the figures. In these samples the concentrations of glucose, lactate, Ca 2+, and K ÷ were measured. In all experimental series oxygen saturation was determined at regular intervals. In the figures and tables t = 0 refers to the time point at which infusion of agonists was started, and the effluent perfusate samples were corrected for the time-lag from the point of infusion to the point of sam-

352 piing. The total liver wet weight was determined at the end of each experiment (euthyroid: 9.9 + 0.2 g (n = 27); hypothyroid: 8.6 + 0.3 g (n = 28), P <

0.001). Analytical procedures Perfusate samples. Part of the perfusate was deproteinized with 3 vol. 0.6 M HC104 and the supernatant was used for the assay of glucose [15], and lactate [16]. In the remainder of the perfusate, from which erythrocytes were removed by centrifugation, Ca 2÷ was determined by flame photometry after dilution of samples and standards with 3.7 m M La203. K + was determined by flame photometry in undiluted erythrocyte-free perfusate. The oxygen saturation was measured with a Po2 electrode type E5047 (Radiometer, Copenhagen, Denmark). The rates of release or uptake of metabolites and ions, and 02 consumption were calculated from the differences in respective content of the influent and effluent medium multiplied by the flow rate. The basal oxygen consumption was not influenced by the antagonists used, and was about 16% lower ( P < 0.01) in the hypothyroid group (euthyroid: 2.00 + 0.08 (n = 12); hypothyroid: 1.69 _.+0.08 (n = 15) # m o l / m i n per g wet weight). Liver samples. Glycogen content was determined according to Keppler and Decker [17] and glucose 6-phosphate content as described by Lang and Michal [18]. To determine glycogen phosphorylase activity freeze-clamped liver was homogenized in 2 vol. ice-cold 100 m M glycylglycine, p H 7.4, containing 230 mM N a F and 3.75 mM EDTA. The homogenate was treated further as described by Stalmans and Hers [19]. Glycogen was purified twice before use. The phosphorylase reaction was stopped by the addition of 1 vol. (200/~1) ice-cold 5% (v/v) trichloroacetic acid, followed by the addition of 3.9 ml water. After centrifugation P~ was determined according to Fiske and SubbaRow [20] with the following modifications. To the supernatant were added 0.5 ml 2.5% (w/v) ammoniummolybdate, containing 0.02% CuSO 4 • 5H20, and 0.2 ml aminonaphtholsulfonic reagent [20]. At the final pH of this reaction mixture (pH 3) no significant acid hydrolysis of glucose 1-phosphate occurs, which otherwise leads to high blank

values. 1 unit of glycogen phosphorylase is the amount of enzyme which liberates 1 /tmol P~ per rain at 25°C. To determine phosphorylase kinase activity we used a modification of the methods described by Doorneweerd et al. [21] and Van de Werve et al. [22]. Freeze-clamped liver was homogenized in 2 vol. ice-cold 10 mM Tris, pH 7.4, 30 mM NaF, 250 mM sucrose and 9.8 mM EDTA. The homogenate was centrifuged at 50 000 × g for 30 min at 0°C. An appropriate dilution of the 50000 × g supernatant (10 #l), containing the enzyme activity, was pre-incubated for 1 min at 30°C with buffer (10 #1 250 mM fl-glycerophosphate, pH 7.4, 2.5 mM EGTA and 40 mM NaF), and rabbit skeletal muscle glycogen phosphorylase b (6 units dissolved in 20 /.tl 10 mM Tris, pH 7.4, 20 mM NaF). The reaction was started by addition of 10 btl of a mixture containing 25 mM ATP and 25 mM Mg (CH3COO)2, pH 7.4. After 12 rain at 30°C the reaction was stopped by addition of 1 ml ice-cold 10 mM ~-glycerophosphate, pH 6.8, containing 45 mM fl-mercaptoethanol, 20 mM NaF, and 10 mM EDTA. In this mixture phosphorylase a and total phosphorylase activity were measured in the direction of glycogen breakdown according to Arag6n et al. [23]. The phosphorylase kinase reaction was linear with time (measured up to 20 rain), substrate, and the amount of added enzyme. One unit of phosphorylase kinase is the amount of enzyme that converts 1 unit of rabbit skeletal muscle phosphorylase b into phosphorylase a per rain under the conditions of this assay. Glucose 6-phosphatase activity was measured as described by Baginski et al. [24] in homogenates of non-perfused fresh liver tissue. Protein was determined by the method of Lowry et al. [25] using crystalline bovine albumin as standard.

Chemicals L-Phenylephrine- HCI, L-isoproterenol-D-bitartrate, DL-propranolol" HCI, rabbit skeletal muscle glycogen phosphorylase b, ATP (disodium salt), and bovine serum albumin (fraction V) were obtained from Sigma Chemical Co. Phentolamine methanesulfonate was obtained from Ciba-Geigy, Na131I from Byk-Mallinckrodt, Petten, The Netherlands, and mollusc glycogen from Boeh-

353

ringer. All other chemicals were of the highest reagent grade available from the usual commercial sources.

stimulating glucose output was also severely decreased in the hypothyroid rat liver (Fig. 1D): the maximal response was reduced by 90% compared to euthyroid values (P < 0.001). In complete contrast to the observed effects of phenylephrine or noradrenaline on the stimulation of glucose release, addition of the fl-agonist isoproterenol resulted in a substantial mobilization of glucose in the hypothyroid rat livers, but not in the euthyroid group (P < 0.001 at the plateau, Fig. 1D).

Statistics All data are expressed as means + S.E. Student's t-test was used for the calculation of the significance of differences between means. Results

Glucose release The basal glycogen content of euthyroid and hypothyroid rat livers did not differ significantly (301 5:33 (n = 6) and 384 + 54 (n = 6) #mol glucose units/g wet weight, respectively). The effect of the mixed a-/3-agonist noradrenaline in stimulating glucose output was severely decreased in the hypothyroid rat liver (Fig. 1A): the maximal response was reduced by 65% compared to euthyroid values (P < 0.001). The noradrenaline-induced effects on glucose output were blocked effectively by the a-antagonist phentolamine in euthyroid livers (P < 0.01 at the plateau), while this antagonist blocked noradrenaline-induced glucose output significantly at t = 10 min in the hypothyroid group (P < 0.05, Fig. 1B). The/3-antagonist propranolol had no effect in both groups (Fig. 1C). The effect of the a-agonist phenylephrine in

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Glycogen phosphorylase and phosphorylase kinase The euthyroid and hypothyroid groups did not differ significantly with respect to the total glycogen phosphorylase and basal phosphorylase a activities (Table I). The data in Table I show furthermore that infusion of noradrenaline during 1 min induced a 3.4-fold increase of phosphorylase a over basal in the euthyroid (P < 0.001) and a 2.5-fold increase in the hypothyroid livers (P < 0.001). Phosphorylase in the euthyroid group was maximally activated by noradrenaline at this time point (95% of total phosphorylase activity). The amount of phosphorylase a in the hypothyroid group in the presence of noradrenaline was 47% lower than in the euthyroid group at t = 1 min (P < 0.005). After 15 min of stimulation with noradrenaline

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Fig. 1. Adrenergic stimulation of glucose release from perfused livers of fed euthyroid (open symbols) and hypothyroid (filled symbols) rats (Exps. I and III). A: noradrenaline 1 /~M; B: noradrenaline 1 ~tM+phentolamine 0.1 mM; C: noradrenaline 1 #M + propranolol 0.1 raM; D: phenylephrine 10/~M (circles) or isoproterenol I #M (triangles). Antagonists were added 15 min before administration of noradrenaline. Glucose output was expressed as rate of release above basal levels (d glucose; mean + S.E.). Number of observations in both groups (except where indicated otherwise) in A: 4-5; B: 4; C: 4; D: phenylephrine 5-6, isoproterenol 4. * Significantly different ( P < 0.05 or better) from euthyroid values (A,D). XSignificantly different (P < 0.05 or better) from the values with only noradrenaline present (B compared to A).

354

the phosphorylase a activity was increased 2.6-fold over basal in the euthyroid ( P < 0.005) and 2.9-fold over basal in the hypothyroid group ( P < 0.001). The changes in phosphorylase a activity between 1 and 15 rain with noradrenaline in both euthyroid and hypothyroid rats were not significant. However, the amount of phosphorylase a in the hypothyroid group now did not differ significantly from that in the controls. The phosphorylase a activity induced by noradrenaline at t = 15 min was decreased by phentolamine in the euthyroid and hypothyroid rats to about the same extent. The decrease in the euthyroid group, however, was border-line significant due to the rather high variability of the observations. The effects of propranolol on the activation of phosphorylase by noradrenaline in both euthyroid and hypothyroid rats at t = 15 min were not significant. Infusion of phenylephrine for 15 min produced a 2.6-fold increase of phosphorylase a over basal in the euthyroid livers ( P < 0.001), while there was no significant increase in the hypothyroid group (Table I). The amount of phosphorylase a in the hypothyroid animals in the presence of phenylephrine was 60% lower than in the euthyroid group at this time point ( P < 0.05). Thus, in the normal

animals, the final (15 min) phosphorylase a levels reached are the same, irrespective as to whether noradrenaline or phenylephrine was added. In the hypothyroid rat livers, however, the final value reached with noradrenaline was about 100% higher than with phenylephrine ( P < 0.05). In contrast to the higher degree of phosphorylase activation induced by phenylephrine in euthyroid compared to hypothyroid livers, infusing isoproterenol for 15 min showed a proportionally stronger stimulation in the hypothyroid group, which is corroborated by the observations on glucose release, although the amount of phosphorylase a did not differ significantly between the groups at the end of the infusion period (Table I). The activity of phosphorylase kinase, measured in the same liver lobes in which the basal phosphorylase activity was determined, was 59 + 5 (n = 6) and 65 + 3 (n = 4) U / g protein in euthyroid and hypothyroid livers, respectively. C a : + and K + f l u x e s

In order to gain support for the idea that the a-receptor-mediated effect of noradrenaline is diminished in the hypothyroid rat liver, we measured the al-receptor-mediated [10] Ca 2+ release. The

Table I A D R E N E R G I C A C T I V A T I O N OF G L Y C O G E N P H O S P H O R Y L A S E 1N P E R F U S E D E U T H Y R O I D A N D H Y P O T H Y R O I D R A T LIVERS Glycogen phosphorylase activity was determined in freeze-clamped liver lobes before and after adding the indicated agents. Data represent mean ± S.E. for the n u m b e r of observations indicated in parentheses. For the sake of clarity P-values better than 0.05 are not given in this table; exact P-values are given in the text. Total glycogen phosphorylase activity ( U / g protein) in euthyroid (58.8 _+4.2, n = 17) and hypothyroid (56.3 ± 2.4, n = 17) rat livers did not differ significantly. Additions

Time point (min)

Euthyroid

Hypothyroid

N o n e (basal) Noradrenaline (1 # M ) Noradrenaline (1 # M ) Noradrenaline (1/x M) + Phentolamine (0.1 m M ) Noradrenaline (1 # M ) + Propranolol (0.1 m M ) Phenylephrine (10/~ M) lsoproterenol (1/~M)

0 1 15

16.7_+ 1.8 (7) 56.1__+ 4.1 (4) ~ 44.2_+ 7.3 (5) a

11.9_+2.0(7) 29.8_+3.0(5) a'b 34.6_+4.0(5)"

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25.8_+3.2 (4) ~ 17.4+2.2 (5) b 20.6--+4.0 (4)

P < 0.05 vs. basal. b p < 0.05 vs. euthyroid. c p < 0.05 vs. noradrenaline 1/~M (15 rain).

Glycogen phosphorylase a ( U / g protein)

355 As was found with noradrenaline, the transient Ca 2+ release induced by the a-agonist phenylephrine was diminished in the hypothyroid group (Fig. 2B, P < 0.025). Furthermore, it can be seen in Fig. 2A and B that the secondary prolonged uptake of Ca 2+ in the hypothyroid rat livers was larger than in the controls. The /3-agonist isoproterenol did not stimulate Ca 2÷ efflux or influx in euthyroid and hypothyroid livers (Fig. 2C). As a-adrenergic activation also leads to changes in the K ÷ fluxes across the plasma membrane in rat liver [10,27], we measured the agonist-induced K ÷ fluxes. The transient K ÷ uptake, induced by noradrenaline, was much smaller in the hypothyroid livers (Fig. 2A, P < 0.01). Measurement of K ÷ in the 4-s samples, in which Ca 2÷ was determined, again showed the markedly decreased

transient Ca 2+ release, induced by noradrenaline in the euthyroid rat livers, was decreased in the hypothyroid group (Fig. 2A). As the interval between these samples was 1 min (first sample at 35 s), and the a]-receptor-mediated maximal rate of Ca 2÷ efflux in perfused rat liver takes place between 35 and 40 s after a-agonist administration [26], we studied the initial noradrenaline-induced Ca 2+ release more closely by measuring Ca 2÷ in effluent samples taken every 4 s. The data thus obtained show that the maximal Ca 2 + release (peak value between 30 and 40 s) was reduced by 75% ( P < 0.02), while the response in the hypothyroid livers was also slower in onset (Fig. 3A). The effect of noradrenaline on Ca 2+ release in the euthyroid rat livers was blocked effectively by phentolamine ( P <0.05), while propranolol did not influence Ca 2+ efflux (Table II).

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Fig. 3. Initial Ca 2+ release (A) and K + uptake (B) in perfused livers of fed euthyroid ( O O) and hypothyroid (O O) rats, induced by noradrenaline 1 #M (Exp. II). Ion fluxes were expressed over basal levels (ACa2+, AK ÷) mean _+ S.E. In both groups four observations, except where indicated otherwise. Significantly different from euthyroid values: * P < 0.05; ** P < 0.02; *** P < 0.01.

livers was blocked effectively by p h e n t o l a m i n e ( P < 0.005), while p r o p r a n o l o l did not influence K + u p t a k e (Table II). The transient K + influx i n d u c e d by phenyle p h r i n e was d i m i n i s h e d in the h y p o t h y r o i d group to an even greater extent than with n o r a d r e n a l i n e (Fig. 2B, P < 0.005). A n interesting p h e n o m e n o n was the transient u p t a k e of K ÷ i n d u c e d by i s o p r o t e r e n o l in the h y p o t h y r o i d group, while no K + influx was detect a b l e in the e u t h y r o i d rat livers (Fig. 2C). M o r e over, the transient i s o p r o t e r e n o l - i n d u c e d K ÷ influx in the h y p o t h y r o i d rat livers was a p p a r e n t l y n o t c o u p l e d to Ca 2+ efflux (Fig. 2C). A f t e r a d d i n g n o r a d r e n a l i n e , p h e n y l e p h r i n e , and isoproterenol, the s e c o n d a r y efflux of K + in the h y p o t h y r o i d g r o u p tended to be equal, smaller, a n d larger, respectively, c o m p a r e d to the e u t h y r o i d g r o u p (Fig. 2 A - C ) .

Glucose 6-phosphate

influx of K * in the h y p o t h y r o i d livers, though m a x i m a l values for K ÷ u p t a k e a p p a r e n t l y have n o t yet been r e a c h e d (Fig. 3B). T h e effect of n o r a d r e n a l i n e on K ÷ u p t a k e in the e u t h y r o i d rat

TABLE II THE EFFECT OF ANTAGONISTS ON PEAK VALUES OF NORADRENALINE-INDUCED ION FLUXES IN PERFUSED EUTHYROID RAT LIVERS Peak values (30-35 s after adding noradrenaline) obtained in Exp. I (Fig. 2A) were combined with those of Exp. lI (Fig. 3A, B). Data represent mean + S.E. for the number of observations indicated in parentheses. Peak values of ion fluxes in the hypothyroid group were too small to detect any effect of the blockers. Additions

C a 2+

release K ÷ uptake (#mol/min per g wet weight)

Noradrenaline (1 FM) Noradrenaline (1 #M) + Phentolamine (0.1 raM) Noradrenaline (1 #M)+ Propranolol (0.1 mM)

0.18:t:0.04 (9)

0.7_+0.1(8)

0.05_+0.02 (4) a

0.1 _+0.1 (3) b

0.13+0.12 (4)

0.5 5:0.1 (4)

a p < 0.05 vs. noradrenaline 1 #M. b p < 0.005 vs. noradrenaline 1 #M.

Infusion of n o r a d r e n a l i n e for 1 min or 15 min p r o d u c e d a m a r k e d rise of the glucose 6 - p h o s p h a t e c o n t e n t in e u t h y r o i d and h y p o t h y r o i d rat livers ( T a b l e III). The n o r a d r e n a l i n e - i n d u c e d glucose 6p h o s p h a t e level in the h y p o t h y r o i d group, however, was 6.9-fold higher at t = 1 min a n d 4.8-fold higher at t = 15 rain than in the e u t h y r o i d group ( P < 0.005 a n d P < 0.05, respectively). Phentolam i n e s u p p r e s s e d the glucose 6 - p h o s p h a t e levels i n d u c e d b y n o r a d r e n a l i n e at t = 15 min in b o t h groups: a decrease in the e u t h y r o i d rats of 45%, which was, however, not significant, a n d a 78% decrease in the h y p o t h y r o i d rats ( P < 0.025). Prop r a n o l o l d e c r e a s e d the glucose 6 - p h o s p h a t e content of the h y p o t h y r o i d livers to the same extent as p h e n t o l a m i n e at this time p o i n t ( P < 0.05), whereas this a n t a g o n i s t h a d no effect in the e u t h y r o i d group. T h e p h e n y l e p h r i n e - i n d u c e d glucose 6-phosp h a t e level in the e u t h y r o i d a n d h y p o t h y r o i d g r o u p s did not differ significantly. I s o p r o t e r e n o l infused for 15 min p r o d u c e d a m a r k e d l y increased glucose 6 - p h o s p h a t e level in the h y p o t h y r o i d group, which was 4.5-fold higher t h a n in the e u t h y r o i d rat livers ( P < 0.02). T h e highly elevated glucose 6 - p h o s p h a t e levels p r o d u c e d b y n o r a d r e n a l i n e or i s o p r o t e r e n o l in the h y p o t h y r o i d rat livers p r o m p t e d us to investigate the activity of glucose 6 - p h o s p h a t a s e in e u t h y r o i d

357 TABLE III GLUCOSE 6-PHOSPHATE CONTENT IN PERFUSED EUTHYROID AND HYPOTHYROID RAT LIVERS IN RESPONSE TO ADRENERGIC STIMULATION Glucose 6-phosphate was determined in freeze-clamped liver lobes before and after adding the indicated agents. Data represent mean ± S.E. for the number of observations indicated in parentheses. For the sake of clarity P-values better than 0.05 are not given in this table; exact P-values are given in the text. Additions

Time point (rain)

None (basal) Noradrenaline (1/*M) Noradrenaline (1 #M) Noradrenaline (1/~M)+ Phentolamine (0.1 raM) Noradrenaline (1/,M)+ Propranolol (0.1 raM) Phenylephrine (10 #M) Isoproterenol (1/*M)

Glucose 6-phosphate (nmol/g wet weight) Euthyroid

Hypothyroid

0 1 15

10± 2 (7) 37 + 26 (4) 58 ± 21 (5) ~

257 ± 33 (5) a.b 276 ± 61 (5) a.b

15

32 ± 4 (4) a

60 ± 25 (4) a,c

15 15 15

73 + 9 (4) ~ 93 + 27 (5) a 77 ± 12 (4) a

76 ± 25 (4) =,c 128 ± 45 (5) ~ 345 4- 53 (4) a.b

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(6)

1

a p < 0.05 vs. basal. b p < 0.05 vs. euthyroid. c p < 0.05 vs. noradrenaline 1 #M (15 rain).

a n d h y p o t h y r o i d rats. T h e livers o f N e m b u t a l anaesthetized animals were removed and imm e d i a t e l y h o m o g e n i z e d a n d p r o c e s s e d to det e r m i n e g l u c o s e - 6 - p h o s p h a t a s e activity. T h e activity of g l u c o s e - 6 - p h o s p h a t a s e w a s m a r k e d l y l o w e r in the h y p o t h y r o i d g r o u p : 49.4 _ 4.0 ( n = 4) v e r s u s 111,8 + 3.6 ( n = 4 ) / ~ m o l P i / m i n p e r g p r o t e i n for t h e e u t h y r o i d g r o u p ( P < 0.001).

Although the agonist-induced lactate release f r o m e u t h y r o i d a n d h y p o t h y r o i d rat livers d i d n o t d i f f e r s i g n i f i c a n t l y at e a c h t i m e p o i n t , the t o t a l a m o u n t of l a c t a t e r e l e a s e d in t h e e u t h y r o i d g r o u p d u r i n g the 1 5 - m i n i n t e r v a l was d e a r l y l a r g e r t h a n in t h e h y p o t h y r o i d g r o u p a f t e r a d m i n i s t r a t i o n o f p h e n y l e p h r i n e , w h i l e t h e reverse was true a f t e r

'1

Al

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I o~o ~

Lactate release

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, ........ 10

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5 10 15 0 5 10 15 Time (rain } Fig. 4. Lactate release from perfused livers of fed euthyroid (O O) and hypothyroid (e O) rats (Exps. I and Ill). A: noradrenaline 1 pM; B: phenylephrine 10 pM; C: isoproterenol 1 ~M. Lactate output was expressed as rate of release above basal levels (Alactate; means:t: S.E.). Number of observations in both groups in: A: 4-5; B: 5-6; C: 4.

358 infusing isoproterenol (Fig. 4A-C). In the hypothyroid group the phenylephrine-induced output was slower in onset, while the output induced by isoproterenol developed more rapidly than in the normal rats. Discussion The present results demonstrate that hypothyroidism severely impaired the a-receptor-mediated mobilization of glucose induced by phenylephrine and noradrenaline in perfused livers from fed male rats (Fig. 1A-D). Thus, as was already referred to in the introduction, an impaired mobilization of glucose might be involved in the development of hypothermia in hypothyroid rats exposed to cold. Furthermore, glucose output was markedly stimulated by the fl-agonist isoproterenol in the hypothyroid rats, whereas no release was observed in the controls (Fig. 1D). This finding is compatible with the potentiation of the fl-adrenergic stimulation of phosphorylase a formation in hypothyroidism reported by others [5,6]. The impairment of the short-term action (1 min infusion) of noradrenaline on phosphorylase activation (Table I) and of noradrenaline and phenylephrine on the initial Ca 2 + release (Figs. 2A and B, 3A) in the hypothyroid rat livers compared to the controls is in agreement with the results of the group of Kunos, who found that hypothyroidism impairs phosphorylase activation and 45 C a 2+ release in rat hepatocytes incubated during 3 rain with adrenaline [5] and phenylephrine [7], respectively. Yet, in another study [6], phosphorylase in euthyroid and hypothyroid rat hepatocytes was activated to the same extent by incubating with phenylephrine or adrenaline during 1 min. The reason for the discrepancy between these studies [5-7] is not clear, though differences in age, sex, and the method of inducing hypothyroidism might play a role [7]. Furthermore, the initial K + uptake, induced by noradrenaline or phenylephrine in the perfused rat liver preparations, which is reported to be an a-receptor-mediated effect [10,27], was almost totally absent in the hypothyroid animals (Figs. 2A and B, 3B, Table II). These findings could be explained by a decreased number of al-receptors,

while, however, effects of hypothyroidism on the coupling of receptor activation and release of intracellular Ca 2+ cannot be ruled out [7]. Alternatively, the total pool size of intracellular Ca 2~ could be decreased, since it was shown in one study that hypothyroidism caused a marked reduction of a slow turnover cellular pool of Ca 2+ in liver cells [28]. The impaired Ca 2+ release from intracellular pool(s) would lead to a decreased activation of phosphorylase (Table I) [7], and consequently to a lowered glucose output (Fig. 1A, D). The basal phosphorylase kinase data, which are in agreement with those found by Van de Werve et al. [22], show that the impaired phenylephrine- or noradrenaline-induced activation of phosphorylase in the hypothyroid livers is not likely to be ascribed to a decreased amount of phosphorylase kinase in this group. A decreased sensitivity of phosphorylase kinase to Ca 2 + in the hypothyroid group cannot be excluded, though all Ca 2 +-dependent enzymes studied till now in different thyroid states reveal unchanged Ca 2+ sensitivities [29,30]. In addition, it was observed in our laboratory that the sensitivity of rat skeletal muscle phosphorylase kinase to Ca 2+ was unchanged in hypothyroidism (Leijendekker, W.J. et al., unpublished data). The role played by K + in the control of glycogenolysis is unclear [4,27], though a-receptor-mediated Ca 2+ and K + fluxes seem to be conditionally linked (Fig. 2A, B) [10,27]. The net uptake of K + has been attributed to an activation of the Na +-K + pump [31]. The decreased K + influx after stimulation of the perfused hypothyroid rat liver with aoradrenaline or phenylephr!ne compared to euthyroid controls, observed in this study, might be explained by a decreased Na+-K + pump activity per se, as it is well-known that the thyroid state influences the activity of the Na+-K + pump [32,33]. However, in contrast to the effect of noradrenaline or phenylephrine, K + influx was stimulated by isoproterenol in the hypothyroid rat liver, but not in the controls (Fig. 2C). A more likely explanation is contained in the recent proposal that the activation of the Na +-K + pump by noradrenaline and other Ca2+-mobilizing agents is the result of a displacement of an inhibitory pool of Ca 2 + located on the internal face of the plasma membrane in the microenvironment of the pump

359 [34,35]. This theory is not incompatible with our findings of the largely decreased noradrenaline- or phenylephrine-induced ion fluxes in the hypothyroid rat liver. The reduced Ca 2+ efflux might reflect a decreased mobilization of Ca 2÷ from internal stores, i.e., mitochondria, endoplasmic reticulum, and plasma membrane, thus in the latter case leading to a reduction of the N a ÷ - K ÷ pump response in the hypothyroid rats. We observed that the hypothyroid rat livers accumulated Ca 2÷ during prolonged noradrenaline stimulation (Fig. 2A). Recently, the group of Exton [36] showed that both cAMP elevation and Ca 2÷ mobilization are required to induce net Ca 2÷ uptake in rat liver cells and perfused rat livers. This mechanism could explain our findings, since we observed a relatively larger fl-response in the hypothyroid group (Tables I and III; Fig. 1D), which is in agreement with other reports [5,6]. The proposed mechanism is supported by the observations that the secondary noradrenaline-induced accumulation of Ca 2 ÷ in the hypothyroid livers was largely suppressed by addition of either proprano1ol or phentolamine (results not shown), while infusion of phenylephrine or isoproterenol alone produced a smaller accumulation of Ca 2÷ than that seen during infusion of noradrenaline (cf. Fig. 2A-C). In conclusion, during prolonged stimulation of hypothyroid rat liver with noradrenaline a fl-receptor-stimulating component may facilitate cellular Ca 2÷ uptake. Since the hepatic fl-receptor is of the fl2-subtype [37], it is less responsive to noradrenaline than adrenaline [38]. Consequently, the use of adrenaline probably would enlarge the effects seen in this study. Although the enhanced Ca 2+ influx in the hypothyroid group during prolonged noradrenaline stimulation does not warrant any conclusion about changes in the cytosolic Ca 2+ concentration (see also Ref. 36), we observed a definitely higher activation of phosphorylase with noradrenaline as compared to phenylephrine (Table I), which in the latter case was coupled with a smaller C a 2+ influx. The observed similar blocking effect of phentolamine and propranolol on the glucose 6-phosphate accumulation after a 15-rain infusion of noradrenaline in the hypothyroid group also implicates that both ~- and fl-adrenergic effects of noradrenaline play a role in the stimulation of

glycogenolysis in these rats. The highly elevated glucose 6-phosphate content with noradrenaline and isoproterenol in the hypothyroid as compared to the euthyroid group is probably not caused by decreased glycolysis in the hypothyroid state, as the lactate output induced by these agonists was equal to or even larger than the control levels (Fig. 4A, C). A possible explanation for the accumulation of glucose 6-phosphate may be inferred from the observation that the maximal glucose-6-phosphatase activity in hypothyroid rat liver homogenates was more than 2-fold lower compared to the control value, which is in agreement with other reports on the influence of thyroid state on glucose-6-phosphatase activity [39-42]. On the other hand, isoproterenol induced a substantial glucose output in the hypothyroid rats, but not in the euthyroid group, thus implicating that glucose-6-phosphatase activity is not rate-limiting for glucose release in the hypothyroid group. Furthermore, the K m of the enzyme is about 2 mM [43], which is still more than 5-fold higher than the observed glucose 6-phosphate concentration. At present we have no explanation for the severely impaired glucose output in the hypothyroid group after prolonged noradrenaline stimulation, despite a phosphorylase activation comparable to the euthyroid level. Further studies on catecholamine regulation of glucose 6-phosphate metabolism in the hypothyroid state are needed to elucidate this question.

Acknowledgments We thank Mrs. E.T.J.M. Roeffen for expert technical assistance, Mrs. M.M. van WelijZwanenburg for excellent secretarial assistance and Mr. J. van Elk for the preparation of the figures.

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