Reducing-equivalent transfer to the mitochondria during gluconeogenesis and ureogenesis in hepatocytes from rats of different thyroid status

Biochimica el BiaF-JO~dcaActa. 1137(1992)34-38

34

© 1992ElsevierS~nce ~ r s

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Reducing-equivalent transfer to the mitochondria during gluconeogenesis and ureogenesis in hepatocytes from rats of different thyroid stares R o l a n d B. G r e g o r y , J o h n W . Phillips a n d M i c h a e l N . B e r r y Depmmm~ of Med~al BiochoniuO; Schoolof Me :~c, The I:End~.~rsU n h ' - ~ of South A ~ r a l ~ Adelaide tAu~ralia)

(Received I0 January 1992) (Revisedmanuscriptrvcci~122 April 1992)

Keyv.ords: Reducing-equivalenttransfer,Ureogenesi~Hypat~Toid: Hyperl~Tokh(Iso~tedrat hepatocyte) Isolated hepatoc~les from hypothyroid, eutbyroid and hyperthyroid rats have been employed to investigate the relative imlmrtance of reducing-equivalent shuttles for the transfer of hydrogen between c3"toplasmand mitochondria during simultaueous ureogenesis and gluconeogenesis. In cells from :'_~thyroid anhnals, a 58% d e p r e ~ n of glucose formation and 68% reduction in ureogene~is were induced by n-bu~lmalonate, an inh~itor of the matte shuttle. A more reduced state of the cytoplasmic compatln~nt and a substantial fall in the concentrations of p~avate, aspanate, alanine and glutamate accompanied this inhibition. Preincubation of cells with n-butylmalonate yielded greater inh~hou effects than observed in the absence of preincubation. The inhibito~ effects on gluconeogenesis and ureogenesis were less in cells from eutl~Toid rats and were ve~ much reduced in the case of glucos~esynthesis and absent in the case of ureogenesis, in cells from hyperthyroid rats. It is inferred that both the malate-aspmlate and a-glycerophnsphate shuttles may function in the wansfer of reducing equivalents from cytoplasm to mitochondria during ureogenesis in hepatocytes. The major inlu'bition by n-butylmalonate of glucose and urea synthesis in hepatocytes from bypothyrcid rats is due to the diminished activity of the a-glycerophosphate shuttle in these cells. Moreover, it follows that the NADH arising from the cytoplasmicmaline debydrogenase-eatabjsedreaction is accessible to both the malate-aspartate shuttle and the a-glycerophosphate shuttle.

Introductien Urea synthesis from ammonium ions and CO 2 occurs in the liver by means of a pathway that is compartmented between the mitochondrial and cytosolic spaces [1]. In the cytoplasm, malate is a by-product of urea synthesis, and the reducing equivalents derived from malate, are tran~erred to the mitochondria for oxidation [2,3], a process that can be accomplished by malate carriers such as the dicarboxTlate carrier, malate/ citrate carder, or malate/a-ketoglutarale carder [3,4].

Correspondenceto: R.B. Gregory,Departmentof Medical Biochemistry. School of MedicL'te. The Hinders Universityof South Anstrafia, G.P.O. Box 2100, Adelaide, South Australia,5001, Australia~ Abbreviations: Jo, rate of respiration; J ~ , late of gluconec~ genesis; J ~ . rate of lactate removal; J , ~ rate of ureogenesis~ l~or,~, rate of renmvalof ammoniumions; G/D, [a-glycerophosphate]/[dihydrm~cetonephosphate]ratio; L/P [lactate]/[pyravate] ratio.

The net activity of hydrogen shuttles is influenced by thyroid status, since the activity of the a-glycerophosphate shuttle is increased in the hyperthyroid state and decreased in the hypothyroid state [5-7]. On the other hand, ureogenic capacity is stimulated in the hypothyroid state [8,9], suggesting that the a-glycero* phosphate shuttle is not important for the reducingequivalent transfer from cytoplasm to mitochondria associated with ureogenesis. To determine the relative importance of malate and a-glycerophosphate shuttles in hydrogen transfer during nrcogenesis, we have examined the roles played by the different hydrogen shuttles dur:mg the synthesis of urea in hepatooytes derived from 24 h-fasted hypothyroid, euthyroid and byperthyroid rats, and have used n-butylmalonate to inhibit malate transfer across the mitochondrial membrane [4,10] during incubation of cells in the presence of lactate, NH4C! and omithine. Our results imply that in euthyroid and hyperthyroid animals, the hepatic a-glycerophosphate shuttle can play a major role in the transfer to the mitochondria of

35 cytoplasmic reducing equivalents from malate, produced during urcogenesis. Materials and MetheOs

Materials Collagenase, carbonic anbydrase and enzymes necessary for the assay of motabolites were obtained from Boehringer Mannheim (Germany), as was bovine serum albumin (fraction V), which was defatted by the method of Chen [11]. n-Butylmalonic acid came from Aldrich (USA), and 3~',5-triiodo-L-thyrouine (T 3) and palmitic acid were purchased from Sigma (USA). Palmitic acid was neutralized and dissolved in 0.15 M NaCi containing 9% (w/v) bovine serum albumin. Other chemicals were of the highest quality commercially available.

Methods Hyperthyroidism was induced in male Hooded Wistar rats (280-320 g) by four daily subcutaneous injections in the inguinal region, of 170 ng T 3 / g body mass, m~d the rats were used for experimentation on the fifth day after commencement of administration of T 3. The T 3 solution was prepared by dissclving 1 mg T 3 in 50 #1 0.1 M NaOH and diluting this solution to 2 ml with 0.9% (w/v) NaCI. Hypothyroid rats were obtained by thyroid-parathyroidectomy of young rats when the animals had a body mass of 130-160 g. They were then kept for at least 6 weeks, after which their body mass was approx. 73% of the mass of control animals and plasma concentrations of T 3 and free T 4 (thyroxine) were < 0.5 nM and < 10pM, respectively. In the postoperative weeks, the animals" drinking water was supplemented with 0.2% (w/v) calcium lactate. Hepatocytes from 24 h-fasted eutbyroid, hypothyroid or hyperthyroid male Hooded Yftstar rats of similar age were prepared as described in Ref. 12. Incubations of cells (approx. 100 mg wet wt) were performed for 30 min at 37~C, as described earlier [13] and 0 2 consumption in the presence of CO 2 was measured manometrically [14]. Respiration rates, which were recorded over the period 15 to 30 min, were virtually linear. Reactions were terminated by the addition to the incubation mixture of an equal volume of ice-cold 1 M perchloric acid. The denatured protein was removed by centrifugation, the superuatants neutralized with 2 M KOH and the resulting precipitate of potassium perchlorate removed by centrifugation. Metabolite a s s a ~ were performed on the neutralized porchloric acid extracts of the incubatmns, according to standard methods [15], by means of a COBAS F A R A automated analyzer (Roche Diagnnsti~, B~le) and the data transferred to a PDPI1/73 computer (DEC, USA) for processing. P~tc~ oi" respiration, substrate removal or product formation, are given a s / t m o i / m i n per g wet wt cells.

Results Preincubation of cells with n-butylmalonate enhances its inhibttory effect It was observed that when n-butylmalouata was added to cells incubated in the presence of 10 mM lactate, 15 mM NH4Ci and 2 mM oruithine, inh~ition of glucose synthesis became apparent only after 15-20 rain and was less than that observe~ when cells were pre--:ncubated fo~ 15 .~.~nwith the inh~itor. For example, glucose production was decreased by 61% when cells from a hypothyroid rat were preincubated with n-butylmalonate and subsequently incubated v.~th lactate: NH4C! and ornithine for 30 rain, but was reduced only 24% if non-preincubated cells were used. The degree of inhibition of ureogenesis was also enhanced by preincubation of the cells with n-butylmalonate, viz., 72% compared with 45% for no preincobation. The increase in potency of the inh~itor upon preincubation, which may represent either a slow uptake of inhibitor by the cells, or possibly a slow intracellular conversion to another (inhibitory) compound, has not been further investigated. However, as a result of these observations, cells used in this investigation have been pre-incubated for 15 rain with or without 10 m M nbutylmalonate (unless stated otherwise) before addition of substrates. Under the conditions used, the rates of removal of lactate and ammonium ions and the rates of formation of urea and glucose, were linear over the incubation period. Levels of pyruvate varied in a manner that depended on thyroid status. The presence of n-lmtylmalonate decreased the magnitude of the initial accumulation of pyruvate and resulted in the near depletion of pyruvate by 30 rain, in cells from hyputhyroid rats (Fig. 1). The concentrations of other measured metabolites were approximately steady-state after 30 rain (not shown).

Effect of n-butylmalonate in hepatocytes from rats of different thyroid status Table i shows the effects of both thyroid status and n-butylmalonate on the rates of respiration, ginconeogenesis, ureogenesis and of lactate and ammonium ion removal, in the presence or absence of butylmalonate, 3o, - / ~ and J l ~ t e were lowest in cells from hypothyroid rats and highest in hepatocytes from hyperthyroid animals. In the absence of n-butylmalonate, Ju,~ was highest in cells from hypothyroid rats. The addition of n-butylmalonate caused a large degree of inhibition (50-70%) of Jeuco~ and 31actate in cells from euthyroid and hypothyroid animals. In hepatocytes from hyperthyroid rats, ./o~o~ and ./la~t~ were inhibited only 26% and 19%, respectively, by the addition of n-butylmalonate. These differences in inhibition between hypothyroid and hyperthyroid states were even

TABLE I TIoToid status and the effecI of n-bu~,lmalonate on respiration and rates of urea and g~ucme formation Hepatoojtes from rats of d/fferant thyroid status were preincubated for 15 m/n at 3"F'Cin the absence or presence of 10 mM n-butylmaionate. and tl~n incutJated for a further 30 rain at 37=Cin the pre~enceof ndded 10 n ~ lactate, t5 mM NH4C! and 2 mM omithine. Other details are in Mat¢,'i~ and Methods. Mean values are shown with S.E. and the number of experiments (n). n-Butylmatonate n ~ / d

9 9 7 7 8 8

+ + +

Eathym~ Hype~

~raol/min per g wet wt 7o 7~=~:

7~

1 ~

I'~.wate !ttmol/30 min)

L/P

7===

4.57+__0.15 2.62+-0.18 5.83_+0.12 3.t)7-+0-a-6 7.39-+0.14 6.78+0.14

2.63+-0.13 0.84__+0.11 2.28+__0.12 1.40_+0.15 2.13-+0.08 2.26__+0.09

--2.~_+0.12 -0.71+0.12 -4.03+0.|1 --I.97__+0.22 -5.42_+0.09 --4.38_+0.13

-5.07_+0.23 --1.76-+0.20 -4.79±0.30 -2.85_+0-21 --4.70+0.16 --4.72+__0.17

0.63+__0.05 0.054-__0.01 0.42_+0.04 0.17__+0.04 0.15_+0.01 0.20-+0.01

22.1+ I.8 693 -+227 14.5-+ 0.7 159 ± 56 15-2__+ 1.1 28.3-+ 0.8

0.31+__0.02 0.14+__0.03 0.84_+0.03 0__30+0.08 !.24-+0.04 0.92+0.03

~o. ,¢

0 0

5

zo 15 20 25 T:Lme ( min )

30

Fig. !. The effect of n-butylmaionate on pyruvate levels in hepatocytes from rats of different th3"xbid status. Cells, preincubated for 15

rain with or without 10 mM n-butylmaionate,were then incubated in the presence of t0 mM !.~tate. 15 mM NH4CI and 2 mM oraithine. The figure shows the quantity of pyra~'ate accumulated at different times during the 30 rain incubation. The 0.ata show representagive plots of up to 5 separate ex~riments in each case. Open symbols, n-butylmaionateabsent; filled symbols,n-butylmalonatepresent, liypothyroid ( o , e), euthyroid ( 123,• ) and h~perthyroid t r.,, • ).

m o r e m a r k e d /It relation to u r e a synthesis. Thus, •/ ~ c , ~ a n d 7~,~ w e r e inhibited 6 5 - 6 8 % by n-butylm a l o n a t e a d d e d to cells from hypothyroid rats, 3 9 - 4 1 % in ceils from euthyroid rats but w e r e not affected by the addition o f n-butylmalonate to hepatocytes from h ~ e r t b y r o i d animals. A similar t r e n d was evident for the inh~ition o f 70 (Table !). A large increase in the ratio ( L / P ) , reflecting a severe depression o f pyruvate accumulation (Table I, Fig. 1), was also associated with the inhibition by n-butylmalouate in hepatocytes from hypothyroid rats. In cells from euthyroid rats, the L / P ratio a n d the pyruvate level w e r e similarly affected, but to a lesser extent, whilst relatively m i n o r changes occ u r r e d in the ratio, L / P , a n d in the pyravate level, in response to the addition o f n-butylmalonate to cells from ~ p e r t b y r o / d rats. T h e addition o f n-butylmalonate brought about increases in the total a-ketoglutarate (Table I!), o f the o r d e r o f 3-fold in cells from hypothyroid rats a n d 5-fold in hepatocytes from euthyroid animals, b~J~ only 2-fold in cells from hyperthyroid rats. In cells from hypothyroid rats, a 2-fold increase occurred in the m a l a t e level (Table !1). T h e substantial lowering (by up to 80%) by n-bu13,1malonate of the tot~: a m o u n t s o f aspartate, alanine plus O u t a m a t e

TABLE 11 Thyroid status o..d the effect of n.burybnalonate m intermediates formed during gluconeogenesis and ureogene~ Conditions were as descrihed in the legend to Table I. Shown are the total amounts (/zmoDin the incubation vessels,of a-ketoglutarate (aKG), malate, alanine (Ala), aspartate (Asp) and glutamate (Glu) after a 30*min incubation. B/A is the ratio [3-hydrox3'butyrate]/[acetoacetate].The number of experiments is given by n unless otherwise indicated ~-. parenthesis. Status Hypothyroid Eutbyroid Hyperthyroid

n-Butylmaionate n 9 + 9 7 + 7 8 + 8

aKG 0.03_+0.01(6) 0.09+_0.01 0.05±i~.9! 0.24±0.04 0.03-+0.002 0.06_+0.01

Malate 0.02_+0.003(5) 0.04-+0.004 0.06±0.02 0.09.+.0.01 0.06+0.01 0.07-+0.01

Asp Ala 0.09_+0.01(7) 0_68+0.06 0.04 (2) 0.11+0.02(8) 0.42-+0.11 !.21_+0.15 0.09_+0.02.(4) 0-22+_0.07(5) 0.70_+0.08 1.40_+9.09 0 3 6 - + 0 . 0 4 0.84±0.07

Glu 0.12+_0.01 0.05_+0.01 0.40_+0.04 0.22_+0.03 0.55_+0.02 0.63_+0.03

B/A 0.12_+0.01 0.06-+0.01(6) 0.17+0.02 0.09_+0.01(6) 0.13-+0.02 0.16_+0.02

measured at the end of 30 min incubation in cells from euthyroid and bypothyroid rats, was less evident in hepatocytos derived from hyperthyroid animals, since in the latter case, although a 40% to 50% decrease occurred in the amounts of aspartate and alanine measured, the amount of glutamate was not decreased (Table il). The ratio of [3-hydroxybutyrate]/[acetoacetate], an indicator of the mitochondrial N A D H / N A D + redox state, was low in the presence of NH~CI and was lowered further still when n-butyimalonate was added, except in cells fi'om hyperthyroid rats (Table ll). Qualitatively similar changes to those observed in the L / P ratio (Table 1) were observed in the ratio G / D . Thus, for hepatoeytes incubated in the presence of lactate, ornithine and NH :Cl, and in the absence of n-butylmalonate, the G / D values were 4.9 + 0.4 (n = 5), 2.9 +_0.6 (n = 7) and Z6 _+0.7 (n = 7) in cells from hypothyroid, euthyroid or hyperthyroid rats, respectively, but when 10 mM n-botylmalonate was included, the values of G / D were 10.8 -4-_1.0 (n = 6), 4.6 + 0.9 (n = 6) and 2A + 0.2 (n = 3), respectively. The n-butylmalonate-induced depletion of the pyrurate level in cells from hypothyroid rats could not be prevented by the inclusion of 20 mM or 40 mM glucose as a source of glycolytically-derived pyruvate (data not shown). O n the other hand, the inclusion of 10 mM alanine in the incubations, which generated pyruvate by transamination, prevented to a large degree, the inhibitory effects of n-butylmalonate in hepatocytes from hypothyroid rats. Thus, rates of gluconeogcnesis and urcogenesis in ceils from thes~ animals were 0.31 + 0.04 (n = 4) and 1.84 +_0.02 (n = 2), respectively, when added alanine was also present. This compares with control values of 0.29 and 1.91 for ]e.~c,,~ and Jura respectively, obtained in two experiments in the presence of added lactate, NH4CI, ornithine and alanine but the absence of n-butylmalon~te. Discussion To study the transfer of reducing equivalents from cytoplasm to mitochondria during gluconeogenesis and ureogenesis, isolated hepatocytos from fasted rats were incubated with lactate, ornithine and NH4CI. Lactate acts as a precursor for glucose and also provides the carbon skeleton for a~artate, a substrate in the reaction-forming argininosuccinate [16]. Cleavage of this compound gives rise to arginine and fumarate in the cytoplasm. Subsequently, arglnine is hydrolysed to yield urea and ornithine, whilst fumarate is converted to malate. Hence, the synthesis of urea delivers reducing equivalents in the form of malate to the cytoplasm [3]. In theory, gluconeogenesis from lactate does not require reducing equivalent transfer between mitoehondria and cytoplasm, since a coupling of glyceralde-

hyde-3-phosphate dehydrogenase and lactate debydlogenase might be expected to maintain a steady-state cytoplasmic redox state. However, under certain conditions, such as those employed in this study, J l ~ t ~ ~reatly exceeds Jgt. . . . with the result that cytosolie reducing equivalents become excessive to the requirements of gluconeogenesis [17]. A sustained rate of urea production is thus dependent on the mitochondrial oxidation of these reducing equivalents. In this study, n-butylmalonate, an inhibitor of malate transfer across the mitochondrial membrane [10], was employed to decrease the rates of processes dependent on such malate transfer. When isolated hepatocytes were incubated in the presence of lactate, ammonium ions and ornithine, Ju,=a was greatest in hepatocytes from hypothyroid rats, in agreement with the results of Marti et al. [8] and our eatlicr work [9]. The degree of inhibition of urea synthesis by n-butTlmalonate was influenced by the thyroid status of the rat from which the hepatocytes were derived, varying from almost 70% inhibition (hypotbyroid) to no inhibition (hyperthyroid). These result~ can be interpreted in terms of the activities of the hydrogen-shuttles that are available for the transfer of reducing equivalents from the cytoplasm to the mitochondria. The a-glycerophosphate shuttle has a very low activity in the hypothyroid state and a markedly elevated activity in the hyperthyroid state [5], reflecting the induction by thyroid hormone of mitochondrial a-glycerophosphate dehydrogenase (EC 1.1.99.5) [5,18]. Thus, in hepatocytes from euthyroid rats, and more particularly, in cells from hyperthyroid rats, it is most likely that a coupling of cytoplasmic malate dehydrogenase (EC 1.1.1.37) and the a-glycerophosphate shuttle enables the reducing equivalents in malate, formed during urea synthesis, to be transferred to the mitochondria when the other mechanism for accomplishing this, the malate-aspartate shuttle, is substantially inhibited by n-butylmalonate. In cells from hypothyroid rats, the low level of a-glycerophosphate shuttle actkity does not allow the shuttle to compensate for the loss of malate-aspartate shuttle activity in the presence of n-butylmalonate. The net result is an accumulation of reducing equivalents in the cytoplasm, as shown by increases in L / P and G / D , a depletion of the pyruvate concentration, and inhibition of urea and glucose syntheses. The lowering of the pyruvate concentration to very low levels by the action of n-butylmalonate in cells from hypothyroid rats could be expected to lower the rate of formation of oxaloacetate by pyravate carboxTIase (EC 6.4.L1), in turn resulting in a decreased rate of formation of intramitochondrial aspartate from oxaloacetate and glutamate. The concentrations of aspartare, glutamate and alanine were lowered by the action of n-butytmalonate, with aspartate in particular, falling

38 t o almost n o n - d e t e c t a b l e levels. T h e increased a - k e t o glutarate/glutamate ratio when n-butylmalonate was a d d e d , p r o b a b l y reflects the oxidised state o f the mitoc h o n d r i a , as r e p r e s e n t e d b y t h e low value o f the r a t i o [ 3 - h y d r o x F b u t y r a t e ] / [ a c e t o a c e t a t e ] . T h e s e effects have b e e n n o t e d in cells f r o m e u t h y r o i d r a t s [3]. W e infer t h a t provision o f l ~ , u v a t e , e i t h e r directly [17], o r i~directly via alanine, largely p r e v e n t e d t h e i n h ~ i t o r y effects o f n*butylmalonate o n u r e a synthesis, by restoring levels o f o x a l o a c e t a t e in t h e m i t o c h o n d r i a [3]. A d d e d glucose w a s a p p a r e n t l y not able t o furnish sufficient p y r u v a t e in t h e p r e s e n c e o f n - b u t y l m a l o n a t e in cells f r o m h y p o t h y r o i d rats. In e a r l i e r w o r k , c o m p a r t m e n t a t i o n o f cyto.12.~mic r e d u c i n g equivalents into several N A D H pools w a s s u g g e s t e d [19], these pools b e i n g accessible to different reducing-equivalent shuttles [20]. O n the o t h e r h a n d , evidence exists f o r a single N A D H pool in t h e cytop l a s m [21]. T h e results o f t h e p r e s e n t study imply that, u n d e r t h e conditions e m p l o y e d , cytoplasmic r e d u c i n g equivalents f r o m malate, arising d u r i n g areogenesis, have access t o e i t h e r the a - g l y c e r o p h o s p h a t e shuttle o r to m a l a t e - m e d i a t e d shuttles f o r t r a n s f e r to the mitoc h o n d r i a , indicating t h a t these r e d u c i n g equivalents c a n flow to t h e m i t o c h o n d r i a b y m o r e t h a n o n e hydrog e n shuttle. B e c a u s e o f this, t h e a - g l y c e r o p h o s p h a t e shuttle h a s t h e capacity t o play a n i m p o r t a n t role in u r e o g e n e s l s in t h e h y p e r t h y r o i d state. Agknewledgmeats T h i s w o r k w a s s u p p o r t e d , in part, by a g r a n t flora t h e N a t i o n a l H e a l t h a n d Medical R e s e a r c h Council o f Anstrafia. W e t h a n k Eija K o r p e l a i n e n , L e o n i e Blight, Elizabeth W'dliams, R o b e r t N a p o l i a n d J u d y - A n n e Kelly f o r skilled techn/cal assistance, the staff o f the r a d i o - i m m u n o a s s a y l a b o r a t o r y a t H i n d e r s Medical C e n t r e f o r the m e a s u r e m e n t s o f thyroid h o r m o n e s in r a t p l a s m a , a n d J u l i e - A n n e B u r t o n a n d D i a n a Tanevski f o r assistance in t y p i n g the m a n u s c r i p t .

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