Eur. J. Biochem. 102, 117-124 (1979)

The Stimulation of the Mitochondria1 Uncoupler-Dependent ATPase in Isolated Hepatocytes by Catecholamines and Glucagon and Its Relationship to Gluconeogenesis Michael A. TITHERADGE, Janet L. STRINGER, and Robert C. HAYNES, Jr Department of Pharmacology, University of Virginia School of Medicine (Received May 31, 1979)

Mitochondria prepared from isolated hepatocytes incubated with glucagon, epinephrine and norepinephrine, exhibited stimulated rates of uncoupler-dependent ATPase activity, although no effect of the hormones was apparent on the basal ATPase activity in the absence of 2,4-dinitrophenol. The hormonal stimulation of the ATPase activity behaved in a dose-dependent manner with increasing concentrations of the effectors. Glucagon exhibited an effect on the ATPase only after an initial lag period during which time there was an elevation in the intracellular level of adenosine 3’,5’-monophosphate (cyclic AMP), and incubation of the cells with cyclic AMP itself resulted in a stimulation of the ATPase. The effect of the catecholamines was not mediated through cyclic AMP, but through an a-receptor mechanism. Norepinephrine stimulation of the mitochondrial ATPase exhibited a shorter lag period than that demonstrated for glucagon, strengthening the postulate that the glucagon and catecholamine effects are mediated by independent mechanisms. The hormonal stimulation of the uncoupler-dependent ATPase activity is correlated with the stimulation of gluconeogenesis produced by glucagon and catecholamines both in terms of dose of hormone required and time of onset of effect. It is proposed that the stimulation of the ATPase leads, in the intact cell, to an increased rate of formation of ATP. This contributes to the control of gluconeogenesis from three-carbon acids at the pyruvate carboxylation reaction through increases in the mitochondrial ATP/ADP ratios, and it helps provide the increased ATP for cytosolic reactions that is demanded by an acceleration of gluconeogenesis.

The rate of hepatic gluconeogenesis from three carbon precursors is known to be under the acute control of both glucagon and catecholamines [l - 51. We have previously shown that one site of action of these hormones on gluconeogenesis lies at the level of the hepatic mitochondria by stimulation of the pyruvate carboxylation step [6- 101. Recent studies have expanded the known effects of glucagon on hepatic mitochondria. It appears now that this hormone stimulates the mitochondria to utilize various substrates at an accelerated rate and by this means produce an enhanced energized state of the mitochondria [ l l - 161. Not only is there a stimulation of the electron transport chain as evidenced by the increased rate of uncoupled respiration, there is also a stimulation of the other major component of the ATP-forming process, the ATPase [17]. Yamazaki Enzymes. Pyruvate carboxylase (EC 6.4.1.1); mitochondrial ATPase or ATP phosphohydrolase (EC 3.6.1.3).

[17,18] has suggested that this stimulation of the ATP-forming reactions in the mitochondrion would allow ATP-dependent processes such as the pyruvate carboxylase and carbamyl phosphate synthetase reactions to proceed at a higher rate, with the concomitant stimulation of gluconeogenesis and ureogenesis. Before the hormonal activation of the ATPase can be seriously considered to have a significant relationship to the acute hormonal stimulation of gluconeogenesis it must satisfy certain criteria. The stimulation must be found with the same hormones and at the same dose of hormones that affect gluconeogenesis. It must occur at an appropriate time in relation to the events that follow the administration of hormones. If it is produced by the action of the catecholamine hormones, it must be mediated by an a-adrenergic mechanism. Furthermore, since glucagon and the catecholamines stimulate gluconeogenesis in isolated perfused livers and in hepatocyte preparations, the hormones should also stimulate the

118

Glucagon and Catecholamines Stimulate Hepatic Mitochondrial ATPdse

ATPase under these conditions where extrahepatic effects of the hormones are not present. In this report we have tested the ATPase response and find that it does satisfy these criteria. Using these data as a foundation, we discuss possible roles the activation of ATPase may fulfil in stimulation of gluconeogenesis.

MATERIALS AND METHODS Isolation and Incubation of'Hepatocytes Male Wistar rats weighing 200-300 g were fasted for 18hand anesthetized with 10 - 20mg sodium pentobarbital injected intraperitoneally. Liver cells were isolated essentially by the method of Berry and Friend [19] as described previously [7]. In most experiments, 10-ml aliquots of the cell suspension in Krebs/Ringer bicarbonate buffer, pH 7.4 (protein concentration approximately 25 mgiml), were incubated in 125 ml conical flasks, after being gassed with 95% 02/5% C 0 2 , at 37°C with a shaking rate of 60-70 cycles min- '.The general experimentaldesign was to incubate the cells with 15 mM lactate, 5 mM pyruvate for a period of 30 min, remove samples for the assay of glucose, and then add the hormones, cyclic nucleotide or vehicle. After a further 10 rnin of incubation in the presence of the effector, samples were again removed for the assay of glucose or cyclic AMP and the mitochondria prepared as below. In experiments where norepinephrine was used to stimulate the cells, 20 pM L-propranolol was generally added to the incubation medium 5 min prior to the addition of the norepinephrine to eliminate any change in activity as a result of a rise in intracellular cyclic AMP. Hormones, antagonists, and cyclic AMP were added to the cells as small volumes of 100-fold concentrated solutions. Glucagon was added in 0.01 M NaOH containing 1 mg/ml albumin; L-propranolol and dihydroergotamine were added in 0.01 M HCl. Controls received additions of the appropriate vehicle. Isolation of Mitochondria The isolation of mitochondria from the cells was carried out essentially as described previously [7]. After removing samples for glucose and cyclic AMP assay, the cells were added to 25 ml ice-cold buffer containing 0.3 M sucrose, 5 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonicacid, and 1 mM EDTA, adjusted to pH 7.4, and centrifuged at 50 x g for 2 min at 0-4°C. This was repeated once to remove traces of salts, hormones, cyclic nucleotides and substrates, and the cells resuspended in 1.5- 2.5 ml of the same buffer. The cells were then homogenized in a Dounce homogenizer and the homogenate centrifuged at 500 x g for 10 min. The supernatant was then

transferred and centrifuged at 9000 x g for 10 min and the resultant pellet resuspended to a final concentration of approximately 15 mg/ml. Adenine Nucleotide Distribution Thedistributionsofadenine nucleotides between the cytosol and mitochondria were estimated essentially according to the method of Zuurendonk and Tager [20]. After incubation of the cells in Krebs/Ringer buffer containing 15 mM lactate, 5 mM pyruvate for 30 min, hormone was added and the incubation continued for an additional 10 min. A 200-pl sample of the cell suspension was then rapidly mixed with 2 ml of ice-cold medium containing 0.25 M sucrose, 20 mM morpholinopropane sulphonate buffer, pH 7.0, 3 mM EDTA, 0.12 m M atractyloside and 4.0 mg/ml digitonin. After 30 s of incubation the suspension was centrifuged for 20 s at 3000 x g at 4 "C, and the supernatant and pellets acidified to 0.3 M perchloric acid. This procedure resulted in the appearance of 95 % of the total lactate dehydrogenase of the cell suspension in the supernatant and 98 % of the glutamate dehydrogenase in the pellet or mitochondria1 fraction. The total adenine nucleotides of intact cells were measured after rapid deproteinization of a cell suspension with 0.3 M perchloric acid, followed by neutralization of the deproteinized extract with potassium bicarbonate. Assays Mitochondrial pyruvate carboxylation was measured as described previously [6]. Mitochondrial ATPase activity was measured by the release of inorganic phosphate at 30 "C over a period of 8 min in the absence and presence of 0.18 mM 2,4-dinitrophenol as described by Weiner and Lardy [21]. The protein was always equalized to 0.5 mg/ml in the assay. Glucose was assayed on a Technicon AutoAnalyzer by means of the glucose oxidase method using a single vial glucose reagent kit from Boehringer [22]. Adenosine 3' :5'-monophosphate (cyclic AMP) was assayed by radioimmunoassay using the automated radioimmunoassay procedure of Brooker et al. [23]. Protein was determined by the method of Lowry et al. [24] using crystalline bovine serum albumin as standard. Adenine nucleotides were determined using the fluorometric assays described by Lowry et al. [25]. Muter ials Type IV collagenase was obtained from Worthington Biochemicals; crystallized bovine serum albumin from the Pentex Division of Miles Laboratories ; atractyloside, cyclic AMP, glucagon, epinephrine bitartrate, norepinephrine bitartrate, dihydroergotamine tartrate, sodium pyruvate, ATP,

M . A. Titheradge, J. L. Stringer, and R. C. Haynes, Jr

119

2,4-dinitrophenol from Sigma; glucose oxidase from Boehringer ; L-propranolol from Ayerst ; L-lactate from Schwarz-Mann; digitonin from Calbiochem ;and NaH14C03 from Amersham Searle.

cluded in Table 1 demonstrating that the stimulation of gluconeogenesis and carboxylation of pyruvate took place in these particular experiments as well as the stimulation of the ATPase. The rates of both pyruvate carboxylation and ATPase activity in mitochondria prepared from cells are approximately 50% of those rates found in mitochondria prepared from the intact animal. This has previously been reported by Garrison and Haynes [7] with respect to pyruvate carboxylation. No satisfactory explanation for this discrepancy has been found up until this time.

RESULTS Stimulation qf the Mitochondrial ATPase in Hepatocytes by Glucagon, Epinephrine, Norepinephrine and Cyclic A M P Yamazaki et al. [17] have reported that glucagon treatment of intact rats stimulated the uncoupleractivated ATPase as assayed in hepatic mitochondria subsequent to the hormone treatment. Table 1 shows the effect of incubating hepatocytes for 10 min with maximally effective concentrations of glucagon, cyclic AMP, epinephrine, and norepinephrine on the mitochondrial ATPase. The data indicate that epinephrine, norepinephrine, and cyclic AMP, in addition to glucagon, stimulate the mitochondrial uncouplerdependent ATPase activity when added directly to the cells, thus eliminating the requirement for extrahepatic factors which could regulate the ATPase. Epinephrine and norepinephrine appeared to be more effective than glucagon in stimulating both the ATPase and pyruvate carboxylation (P < 0.01). No hormone effects were apparent on the basal ATPase activities in the absence of uncoupler. The stimulation of the ATPase thus resembles that of gluconeogenesis [7, lo], pyruvate carboxylation and decarboxylation [7, 101 and the stimulation of oxidation of various substrates [16] in that they are produced by these hormones acting on isolated hepatocytes or the isolated perfused liver. Reference data are in-

Mediation of the Catecholamine Response by an a-Adrenergic Mechanism During recent years it has been discovered that the catecholamine hormones act on rat liver predominantly by an a-type mechanism rather than through the agency of cyclic AMP. Actions in liver mediated by the a-receptor include the stimulation of glycogenolysis, gluconeogenesis, and pyruvate carboxylation [ 10, 26- 321. The experiments in Table 2 were performed to determine if the ATPase response to epinephrine was also a-mediated. For this study epinephrine was chosen because it has both CI and 0 actions, each of which can be selectively eliminated by specific blocking agents. Adrenergic blocking agents are generally considered to be more specific than adrenergic agonists and for this reason were used instead of ‘typical’ cx or agonists that, in fact, may have some degree of both a and characteristics. Hepatocytes were incubated for 25 min with 15 mM lactate, 5 mM pyruvate, treated with the a-antagonist, dihydroergotamine (20 pM), or the a-antagonist, L-propranolol (20 pM); 5 min later 1 pM epinephrine or vehicle was added and the

Table 1. Ejfect of glucagon, epinephrine and norepinephrine on rates o j miiochondriul A TPuse uctivity, rate of pyruvate curho.~yluiion und glucose production by heputocytes Isolated hepatocytes were incubated with 15 mM lactate, 5 mM pyruvate for 30 min, sampled for glucose assay, then treated with hormone and incubated for an additional 10 min. The cell suspension was again sampled for glucose assay and mitochondria prepared. Preparation of mitochondria and assays of glucose, pyruvate carboxylation and ATPase activity were carried out as described in Materials and Methods. ATPase activity was assayed in the absence and presence of 0.18 mM 2,4-dinitrophenol. Results are means f S.E. for eight different cell preparations Group

Treatment of cells

Mitochondrial ATPase activity ~~~

basal

~

~~~~~

+ 2,4-dinitrophenol

Mitochondria1 pyruvate carboxylation

nmol min-’ (mg protein)-’ .~

-

1.

2.

a

Control 10 pM glucagon 10 pM epinephrine 10 pM norepinephrine

15.8 1.3 15.6 f 1.8 17.4 f 1.6 14.0 f 1.6

40.8 f 4.1 57.8 f 5.0” 78.8 j~7.8” 77.4 7.4“

Control 10 pM glucagon 1 m M cyclic A M P

14.4 f 0.8 16.1 f 0.8 15.2 f 0.7

64.4 k 7.4 85.8 f 7.9“ 110.7 f 9.6“

P < 0.001

*

Cellular glucosc production

pmol min-’ (g dry wt)-’

+

f 0.6” f 0.6” f 0.9”

0.43 4.56 6.84 -t 0.43” 6.41 f 0.28” 6.55 k 0.27a

6.6 f 0.9 12.6 2 1.0” 15.5 f 1.8”

4.98 f 0.33 6.98 k 0.49” 6.41 f 0.46”

4.0 7.0 8.1 9.3

f 0.5

Glucagon and Catecholamines Stimulate Hepatic Mitochondria1 ATPase

120

Table 2. The ejyects of the a and fl antagonists (dihydroergotamine and L-proprunololj on the epinephrine-induced stimulation o j ATPase. The effects of these agents on gluconeogenesis, cyclic A M P production and mitochondrial pyruvate carboxylation Isolated liver cells were incubated with 15 mM lactate, 5 mM pyruvate for 25 min, and then the antagonists (20 pM) or vehicle was added. Epinephrine (1 pM) was added at 30 rnin and the incubation continued for an additional 10 min. Samples were removed for glucose assay at 30 and 40 min and cyclic A M P samples at 31, 35, and 40 min. Mitochondria were prepared and assayed as described in Materials and Methods, as were cyclic A M P and glucose samples. Uncoupler-dependent ATPase activity was assayed in the presence of 0.18 mM 2,4-dinitrophenol. Results are expressed as means f S.E. for eight separate preparations Antagonist

Epine- UncouplerCyclic A M P at phrine dependent ATPase (1 pM) activity 1 min 5 min ~~~

HCI (0.01 M) 1.-Propranolol (20 pM) Dihydroergotamine (20 pM) HCI (0.01 M) 1.-Propranolol (20 pM) Dihydroergotamine(20 pM) A

P < 0.05.

P < 0.01.

-

+ + +

~

~~

nmol min-' (mg protein)-'

pmol (mg protein)-'

44.8 f 3.8 41.0 f 4.5 44.0 k 3.8 77.9 f 5.4b 69.2 k 4.7b 46.7 k 3.5

3.0f0.3 2.8f0.3 2.9 f 0.2 4.7 f 0.4b 3.4 ? 0.2 4.6 ? 0.4h

3.1f0.3 2.8k0.3 2.5 f 0.2 4.3 & 0.8" 3.2 k 0.4 3.7 2 0.4"

Pyruvate carboxylation

Cellular glucose production

nmol min-' (mg protein)-'

pmol min-' (g dry wt)-'

4.3f0.8 3.9f0.7 4.5 f 0.6 9.3 f 0.9' 7.8 k 0.7b 4.4 f 0.6

4.95 0.21 4.93 f 0.17 4.80 f 0.16 6.49 f 0.30' 6.06 k 0.28" 5.45 k 0.23

10 min

2.5k0.2 2.4k0.1 2.4 f 0.2 3.2 f 0.3" 2.7 f 0.2 3.2 f 0.3"

*

' < 0.001

incubation continued for a further 10 min. It can be seen that the effect of epinephrine on the ATPase was blocked by a concentration of dihydroergotamine that did not affect the rise in cyclic AMP at any of the times examined. On the other hand, L-propranolol did not affect the stimulation of the ATPase by epinephrine, while it did inhibit the rise in cyclic AMP. The addition of either dihydroergotamine or L-propranolol had no effect of any of the functions measured in the absence of epinephrine. This pattern of response indicates epinephrine stimulates the ATPase by an a-receptor mechanism as is the case with the stimulation of gluconeogenesis and pyruvate carboxylation [lo]. Data on these latter functions in our experiments are included as an additional validation of the action of the blocking agents.

Dose Response Relationships

Glucagon concn (M)

Fig. 1. EJfect (4increasing concentrations o/ glucagon on mitochnndrial uncoupler-dependent ATPase activity ( A ) . Correlation with mitochondria1 pyruvate carboxylation ( B ) und glucose output by the hepatocytes ( C ) . Cells were incubated with 1.5 mM lactate, 5 m M pyruvate for 30 min and treated with glucagon to give the indicated concentration. After a further 10 min of incubation, mitochondria were prepared and assayed as described under Materials and Methods. Glucose samples were removed from the incubation medium after 30 and 40 min. Points are means f S.E. for six experiments

The previous study of Yamazaki et al. [I71 describing the stimulation of the uncoupler-dependent ATPase by glucagon used large doses of the hormone to ensure adequate treatment of intact rats. To rule out the possibility that the ATPase response was an artefact of high dosage, unrelated to physiological effects of the hormone, responses to varying doses of glucagon were determined in the hepatocyte system. As this paper also reports an effect of catecholamine hormones on the ATPase, similar titrations of norepinephrine, in the presence of L-propranolol, were also carried out. In Fig. 1 and 2 the response of hepatocytes to various concentrations of glucagon and norepinephrine are displayed in terms of ATPase, pyruvate carboxylation, and gluconeogenesis. In Fig. 1, it can be seen that the concentration of glucagon required to reach half-maximal stimulation was ap-

M. A. Titheradge, J. L. Stringer, and R. C. Haynes, JI

121

proximately 5 nM, with a maximal stimulation being achieved at 1 pM. Fig.2 shows that the effective concentrations of norepinephrine occur over a much narrower range than that of glucagon, with a half maximal stimulation being achieved at approximately 0.7 pM and maximal at 10 pM. The data indicate that the stimulation of the ATPase is sensitive to varying physiological concentrations of hormones, and that there is an excellent correlation between the stimulation of the ATPase with the stimulation of pyruvate carboxylation of gluconeogenesis at all doses of hormones tested.

Time Course of ATPase Response Relative to Other Mitochondria1 and Cellular Responses to Hormones

Norepinephrine conc (pM)

Fig. 2. Ej7eec.t of increasing concentrations of norepinephrine on the mitochondria1 uncoupler-dependent ATPase activity ( A ) . Correlation with mitochondria1 pyruvate carboxylation ( B ) and glucose output by the hepatocytes (C). Cells were incubated with 15 mM lactate, 5 mM pyruvate for 25 min and then 20 pM L-propranolol was added. At 30 min norepinephrine was added to give the indicated concentration and at 40 rnin the reaction terminated, mitochondria prepared and assayed as described under Materials and Methods. Points are means f S.E. for six experiments

28

It has been established that the stimulation of carboxylation of pyruvate in mitochondria occurs within 4 min after addition of glucagon and 2 rnin after addition of epinephrine to hepatocytes. At these early times there is also a detectable increase in the rate of glucose synthesis [7,10]. If the stimulation of the uncoupler-dependent ATPase is to be considered of potential significance in the control of these processes, it is necessary that it take place at or before their initiation. Fig.3 and 4 show the time course of the activation of the ATPase by 10 pM glucagon and 10 pM norepinephrine in the presence of 20 pM L-propranolol, respectively. The data points are the mean values for three experiments in each case. The data indicate that the activation of the ATPase is indeed an early event following addition of hormones to hepatocytes. After stimulation by glucagon, the activation of the ATPase is preceded only by the rise

32 36 40 Time of incubation (min)

44

Fig. 3. Time course of the stimulation of the mitochondria1 uncoupler-dependent ATPuse by glucagon (0,a) and correlation with the increase in gbcose synthesis (0,w), cyclic A M P levels (A, A), andmitochondrialpyruvatecarboxylation ( 0 , r).Control levels are shown as open symbols. Isolated hepatocytes were incubated in eight flasks containing 15 mM lactate, 5 mM pyruvate for 30 min, then 10 pM glucagon was added t o four flasks and vehicle t o the other four flasks. A further flasks was incubated for 28 min t o give a control value before the addition of hormone. Flasks were then removed at 2 , 5 , 1 0 and 15 min after the addition of glucagon or vehicle, samples removed for glucose and cyclic AMP assay and mitochondria prepared. Assays were carried out as described under Materials and Methods. Each point is the mean of three experimental values

Glucagon and Catecholamines Stimulate Hepatic Mitochondria1 ATPase

122

I

- 10.0

$5

40

-.-

a,

1

c - 8.0 '5 g

- 6.0

35 m

a

3

I

I

a,

30

5

?

2 a

- 4.0 T.-

-

u,

0

- 2.0

25

28

32

40 36 Time of incubation (min)

44

-0

Fig. 4. Time course of the stimulalioii o/' the 11iitoc17oi7clricrluticou~)/er-de~~et~~lciiI A TPase by norepinephrine (0, 0 ) .and correlation with the increase in glucose synthesis (0, m), cyclic A M P levels ( A , A) and mitochondria1 pyruvate carhoxylatioij (v, v). Methods were as described under Fig. 3 except that after 25 min 20 pM ~.-propranololwas added to each Hask. Control levels are shown as open symbols. Each point is the mean of three experimental values

Table 3. Effects of norepinephrine und glucugon on the distribution of adenine nucleotides within isolated hepatocytes Isolated liver cells were incubated for 25 rnin in Krebs/Ringer bicarbonate buffer containing 15 mM lactate, 5 mM pyruvate before addition of L-propranolol or vehicle. After 30 rnin glucagon or norepinephrine was added to a final concentration of 10 pM, and the incubation continued for an additional 10 min. The distribution of nucleotides was estimated by the digitonin fractionation procedure described in Materials and Methods. Results are means & S.E. for five different cell preparations ~

Treatment of cells

Nucleotides in intact cells _ ~ _

-

~

__ ATP

~~~

ATP ATP

ADP

Nucleotides in mitochondria1 fraction

Nucleotides in cytosol

-

ATP

ADP

ADP

~

~

ATP ATP

ADP

ADP

ADP

-~

Control Norepinephrine (10 pM) + 1.-propranolol (20 Glucagon (10 pM)

!-w

a

P < 0.01.

nmol (mg cell dry wt)-'

nmol (mg cell dry wt)-'

nmol (mg cell dry wt)-' 10.30 k 0.60 2.36 f 0.08

4.4

8.80 f 0.56

1.42 f 0.05

6.2

1.77 f 0.15

9.30 i 0.42 2.23 i 0.29 9.88 k 0.37 2.17 k 0.24

4.4 4.8

7.19 i 0.43" 1.27 k 0.13 7.77 0.43" 1.29 f 0.09

5.9 6.1

2.31 f 0.17b 0.73 + 0.06 2.19 k 0.16b 0.72 f 0.03

+

0.82

0.06

2.2

3.2" 3.0"

P < 0.001

in cyclic AMP. No effects are apparent on either the ATPase, pyruvate carboxylation or gluconeogenesis during the first 2 min of incubation, however, after 5 min all three functions are significantly elevated. After stimulation with norepinephrine plus L-propranolol, there is a marked stimulation of ATPase activity within 2 min which was maximal at 10 min. The increased ATPase activity is seen as early as the increase in rate of pyruvate carboxylation. The data thus demonstrate that there is an effect on the ATPase similar to that reported earlier for pyruvate carboxylation and gluconeogenesis, that is that the stimulation by catecholamines is significantly more rapid than the

stimulation by glucagon and proceeds without any appreciable time lag [lo].

Effect of Catecholamines on the Adenine Nucleotide Distribution in Isolated Hepatocytes Previous data of Siess et al. [34] and Bryla et al. [15] have indicated that glucagon treatment of isolated hepatocytes results in an increased intramitochondrial concentration of ATP and an enhanced ATP/ADP ratio, suggesting that the stimulation of the mitochondrial ATPase by glucagon is also manifest when

M. A . Titheradge, J. L. Stringer, and R. C . Haynes. J r

the enzyme is operating in the direction of ATP synthesis and that the effect is not an artefact of the lengthy preparation of the mitochondria. We have therefore examined the adenine nucleotide distribution in isolated cells using the digitonin fractionation procedure of Zuurendonk and Tager [20] after treatment of the cells with norepinephrine plus L-propranolol. The results are shown in Table 3. Despite the fact that there is an enhanced rate of gluconeogenesis with consequent increased ATP utilization, the ATP/ADP ratio in the whole cells is not significantly altered. Norepinephrine treatment results in an elevation of the mitochondrial ATP content with a concomitant rise in the mitochondrial ATP/ADP ratio. There is a significant decrease in the cytosolic ATP content, although the change in the cytosolic ATP/ADP is not significant in these experiments. This effect of catecholamines has also recently been reported by Siess et al. [35]. The effects of glucagon in this system are included in Table 3 to serve as a positive control and validate the observed a-adrenergic stimulation by norepinephrine.

DISCUSSION Hormonal stimulation of the mitochondrial ATPase may be a significant component in the control of gluconeogenesis inasmuch as the response is appropriate in terms of hormone selectivity, dose sensitivity, promptness of reaction, the a-adrenergic nature of catecholamine stimulation, and occurrence in isolated hepatocytes. While definitive proof of its role is not yet available, it fits well with what is now known about the nature of the acute mitochondrial responses to hormones. Most, if not all, data available are consistent with the concept that has evolved from the work of Yamazaki and others that hormonal stimulation of the liver results in an accelerated rate of mitochondrial energization as evidenced by increased rates of oxidative phosphorylation [ l l - 13,15,16], an elevated proton gradient and membrane potential [12,13], and enhanced performance of various energy-dependent functions such as citrulline formation [15,18], reverse electron flow, energy-dependent transhydrogenase, and 8-anilino-1-naphthalenesulfonic acid fluorescence [36]. The stimulation of the uncoupler-dependent ATPase suggests that hormones facilitate the utilization of mitochondrial energy, presumably conserved as the transmembrane protonmotive force, to synthesize ATP. Two consequences of such an effect are obvious. First, the more rapid formation of ATP would tend to increase the ATP/ADP ratio within the mitochondria, thereby contributing to the activation of pyruvate carboxylase [37- 391 as well as providing increased ATP as a substrate for the enzyme. It has been de-

123

monstrated that mitochondria isolated from hormonetreated rats are able to maintain an elevated level of ATP in vitro even though the treated mitochondria were utilizing ATP faster for carboxylation of pyruvate than were control mitochondria [6,13]. It has also been shown, together with an elevated ATP/ADP ratio, in isolated hepatocytes for both glucagon [15, 341 (and present work) and now catecholamines [35]. This effect is likely to be of major importance in the stimulation of gluconeogenesis from pyruvate, and it presumably also acts in unison with the increased level of acetyl-CoA [6,34], decreased level of glutamate [34] and increased intramitochondrial pH [12] to stimulate pyruvate carboxylation. The second consequence of an increase in formation of ATP is the potential for a stimulated rate of transfer by exchange to the cytosol, where high-energy phosphate bonds are needed (as GTP) for the phosphoenolpyruvate carboxykinase reaction and (as ATP) for the formation of 1,3-bisphosphoglyceric acid in the 3-phosphoglyceric acid kinase reaction. Analysis of the cytosol of hepatocytes stimulated by glucagon [34] (and present work) and catecholamines [35] (and present work) revealed a decline in ATP content. This indicates that the increased demand for ATP by the acceleration of gluconeogenesis is such that, at least momentarily, it exceeds the ability of the mitochondria to supply ATP to the cytosol; therefore supply and demand come into balance at a decreased concentration of ATP. Nevertheless there has to be an ongoing stimulated rate of formation and delivery of ATP to the cytosol to accommodate the increased needs of the gluconeogenic reactions in the new steady state that results from hormonal stimulation. The hormonal stimulation of gluconeogenesis from glutamine would not be subject to control, as far as is known, by the ATP/ADP ratio in mitochondria. The studies of Siess and Wieland [16,40] suggest this stimulation is likely to be the consequence of an increase in oxidation of 2-oxoglutarate to malate via a portion of the tricarboxylic acid cycle. A stimulation of oxidation of this acid has been demonstrated after glucagon treatment [ l l , 161. This may result from the general increased rate of substrate oxidations seen after hormone treatment [ l l - 13,15,16] that in turn may be the consequence of a specific activation of one or more of the mitochondrial enzymes such as reported by Siess and Wieland [16] for succinate dehydrogenase. The experiments reported here strengthen the concept of identity or near identity of action of several gluconeogenic hormones at the mitochondrial level. Thus glucagon, the catecholamines, and the glucocorticoids have been shown to stimulate pyruvate carboxylation, pyruvate decarboxylation, and the oxidation of various substrates [6,12,40]. To this list is now added the stimulation of uncoupler-activated ATPase by both glucagon and catecholamines.

124

M . A. Titheradge, J . L. Stringer, and R. C. Haynes, J r : Glucagon and Catecholamines Stimulate Hepatic Mitochondria1 ATPase

We gratefully acknowledge the very capable aid of Eleanor Cassada in the preparation of this manuscript and Ralph A. Picking for able technical assistance during the course of this work. We should like to thank D r J. C. Garrison for helpful discussion and criticisms. The work was supported by National Institutes of Health Grant AM-14347. Cyclic A M P measurements were performed by the University of Virginia Diabetes Center, National Institutes of Health Grant AM-22125.

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M. A. Titheradge, Department of Biochemistry, School of Biological Sciences, University of Sussex, Biology Building, Falmer, Brighton, Sussex, Great Britain, BN1 9QG J. L. Stringer and R . C. Haynes, Jr*, Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A. 22908

* T o whom correspondence should be addressed

The stimulation of the mitochondrial uncoupler-dependent ATPase in isolated hepatocytes by catecholamines and glucagon and its relationship to gluconeogenesis.

Eur. J. Biochem. 102, 117-124 (1979) The Stimulation of the Mitochondria1 Uncoupler-Dependent ATPase in Isolated Hepatocytes by Catecholamines and Gl...
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