Stimulation by a-adrenergic agonists of Ca2+ fluxes, mitochondrial

Biochem. J. (1983) 212,555-565 555Printed in Great Britain

Stimulation by a-adrenergic agonists of Ca2+ fluxes, mitochondrialoxidation and gluconeogenesis in perfused rat liver

Wayne M. TAYLOR, Peter H. REINHART and Fyfe L. BYGRAVEDepartment ofBiochemistry, Faculty ofScience, Australian Natiornal University, Canberra, A.C.T. 2600,

A ustralia

(Received 29 November 1982/Accepted 24 February 1983)

Glucose output from perfused livers of 48 h-starved rats was stimulated by phenyl-ephrine (2pM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone orfructose were used as gluconeogenic precursors. Phenylephrine-induced increases inglucose output were immediately preceded by a transient efflux of Ca2+ and a sustainedincrease in oxygen uptake. Phenylephrine decreased the perfusate [lactatel/[pyruvatelratio when sorbitol or glycerol was present, but increased the ratio when alanine,dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in theperfusate [,8-hydroxybutyratel/[acetoacetateI ratio and increased total ketone-bodyoutput by 40-50% with all substrates. The oxidation of [1-14C]octanoate or2-oxo[1-14Clglutarate to 14CO2 was increased by up to 200% by phenylephrine. Allresponses to phenylephrine infusion were diminished after depletion of the hepatica-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol andglycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75%and 66% respectively. Data presented suggest that the mobilization of an intracellularpool of Ca2+ is involved in the activation of gluconeogenesis by a-adrenergic agonists inperfused rat liver. a-Adrenergic activation of gluconeogenesis is apparently accom-panied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. Anenhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrialcompartment may also be involved in the stimulation of glucose output from therelatively reduced substrates glycerol and sorbitol and may arise principally from anincreased flux through the malate-aspartate shuttle.

Ca2+ ions appear to be involved in the mechanismof action of a-adrenergic agonists in liver in both thefed and starved states (for reviews see: Exton, 1981;Williamson et al., 1981; Taylor et al., 1983).Although it is generally agreed that a-adrenergicagonists enhance glycogenolysis through a Ca2+-dependent activation of phosphorylase b kinase,such clearly defined regulatory site(s) have not beenelucidated for the stimulation of the gluconeogenicpathway by these agonists. Proposed sites ofaction of a-adrenergic agonists include pyruvatecarboxylase (Garrison & Borland, 1979; William-son et al., 1981), phosphoenolpyruvate carboxy-kinase (Merryfield & Lardy, 1982) and pyruvatekinase (Garrison et al., 1979, 1981). It has also beensuggested that these agonists may stimulate gluco-neogenesis from reduced substrates such as glycerol

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and sorbitol in hepatocytes, by promoting theexchange of reducing equivalents from the cyto-plasmic to the mitochondrial compartment (Kneeret al., 1979; Yip & Lardy, 1981). A Ca2+-dependentactivation of the mitochondrial glycerol phosphatedehydrogenase was purported to play a major role inthis exchange mechanism (Yip & Lardy, 1981).However, a-adrenergic agonists have also beenreported to stimulate gluconeogenesis by a mechan-ism apparently independent of Ca2+ (Kneer et al.,1979).

Previously we described a method for depletingthe a-adrenergic-sensitive Ca2+ pool in the perfusedrat liver (Reinhart et al., 1982a). In the present studythis technique is employed to examine further therole of Ca2+ in the stimulation of gluconeogenesis bya-adrenergic agonists in the perfused liver of starved

W. M. Taylor, P. H. Reinhart and F. L. Bygrave

rats. This study provides insights into the generalrole of Ca2+ and examines the possible role ofmitochondrial respiration and transfer of reducingequivalents across the mitochondrial inner mem-brane.

Experimental

Animals andperfusionsWistar-strain albino rats (200-280g body wt.)

were starved for 48 h before use. Rats wereanaesthetized with sodium pentobarbitone (50mg/kg), and the livers perfused with Krebs-Henseleit(1932) bicarbonate medium equilibrated wtih 02/CO2 (19:1) and modified to contain 1.3mm addedCaCl2 unless otherwise indicated. All other per-fusion details were as described previously (Rein-hart et al., 1982a).

Perfusate Ca2+ and oxygen determinationsPerfusate Ca2+ and oxygen concentrations were

continuously monitored wtih a Radiometer F2112Ca2+_selective electrode and Clark-type oxygenelctrode, respectively, as detailed by Reinhart et al.(1982a). Lag times for Ca2+ and oxygen responseswere determined as described previously (Reinhartetal., 1982b).

Other analyticalproceduresEffluent perfusate was assayed for glucose by the

glucose oxidase/peroxidase method, and lag timesfor glucose output response were measured with[3Hlinulin, both as described by Reinhart et al.(1982b). In some experiments, larger volumes ofperfusate (7ml) were collected in graduated centri-fuge tubes, centrifuged to remove contaminatingerythrocytes, and the supernatants (6 ml) mixed with0.2 ml of 2 M-HC1O4 containing 5 mM-Hepes [4-(2 - hydroxyethyl) - 1 - piperazine - ethanesulphonicacid]. Samples were left on ice for 10min and thenneutralized with 0.2 ml of 2 M-KOH, and theresulting KCl04 was removed by centrifugation at2000g for 5min. Portions were taken for assay ofpyruvate, lactate, acetoacetate and,-hydroxybuty-rate, essentially as described by Bergmeyer (1974).Where relatively large volumes of perfusate wererequired for these multiple assays, the valuesobtained necessarily represented the average at-tained over the time of sampling (7 ml corres-ponding to 10-15s sampling time), and correctionsfor response lag times were not made.

Production of 14C02 from labelled substrates wasestimated from perfusate samples (1 ml) taken bysyringe from either the inflow or outflow cannulaeclose to the liver. Perfusate samples were injectedinto sealed vials containing 2ml of 100mM-sodiumphosphate/citric acid buffer (pH 3.0), and fitted withcentre wells containing filter paper impregnated with

0.1 ml of Hyamine hydroxide. After 15 min, the filterpapers were transferred to scintillation vials con-taining 10ml of scintillant {0.6% (w/v) butyl-PBD[5-(biphenyl-4-yi)-2-(4-t-butylphenyl)- 1-oxa-3,4-di-azolel in 2-methoxyethanol/toluene (2:3, v/v) } andtheir radioactivity was determined in a Beckman LS300 scintillation counter. Samples of perfusionmedium taken before passage through the liver wereused for background correction and specific-radio-activity determination.

Chemicals and materialsPhenylephrine, noradrenaline hydrochloride,

adrenaline bitartrate, [Arg8lvasopressin, amino-oxy-acetate and the glucose assay kit (510-A) wereobtained from Sigma Chemical Co., St. Louis, MO,U.S.A. Ca2+-electrode membranes (F2002) andfilling solution (S43316) were obtained from Radio-meter, Copenhagen, Denmark. Lactate dehydro-genase and f-hydroxybutyrate dehydrogenase werefrom Boehringer, Mannheim, West Germany. Hy-amine hydroxide, [ 1-14Cloctanoate and 2-oxo[ 1-14C1-glutarate were supplied by New England Nuc-lear, Boston, MA, U.S.A. All other chemicals wereof analytical grade.

Results

Although the glycogen contents in livers ofstarved rats are reported to be low (Exton & Park,1967; Hems & Whitton, 1973) and thereforeunlikely to contribute significantly to glucose outputby the liver, it was important to establish this pointat the outset of the present experiments. Use wasmade of the finding that the transaminase inhibitoramino-oxyacetate, which blocks the malate-aspart-ate shuttle (Borst, 1963), inhibits gluconeogenesisfrom lactate (Arinze et al., 1973; Rognstad, 1980).

Data in Fig. 1 show that the presence of0.2 mM-amino-oxyacetate essentially abolished theincrease in glucose output induced by lactateinfusion in livers of 48h-starved rats. In addition,glucose output in the absence of added lactate wasvery low (cf. that observed in the perfused liver offed rats; Reinhart et al., 1982a,b), and was alsodecreased by approx. 40% by amino-oxyacetate.Moreover, phenylephrine-induced increases in glu-cose output in either the presence or the absence oflactate were largely prevented by the transaminaseinhibitor. The maximal rate of glucose outputinduced by phenylephrine in the absence of lactatewas only 5-10% of that observed in the fed rat (seeReinhart et al., 1982b), and substantial increases inglucose output were only induced by phenylephrinein the presence of lactate. In other control experi-ments amino-oxyacetate was shown to have littleeffect on basal oxygen consumption by the liver andto inhibit only slightly phenylephrine-induced in-

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Effect of a-agonists on gluconeogenesis

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Livers of 48 h-starved rats were perfused withKrebs-Henseleit buffer as described in the Experi-mental section in either the presence (0) or theabsence (0) of 0.2mM-amino-oxyacetate. Phenyl-ephrine () and L(+)-lactate ( ) were infusedfor the times indicated to give final concentrations of2pM and 2.5 mM respectively. Effluent samples werecollected, contaminating erythrocytes removed bycentrifugation, and samples of supernatant assayedfor glucose. Data shown are from a typicalexperiment of three experiments performed in thepresence of amino-oxyacetate and four experimentsin its absence.

creases in oxygen uptake and Ca2+ efflux(138 ± 21 Mm versus 106 + 18,UM, and 20.6 + 2.1 Mversus 19.8 ± 2.OpM respectively; ±S.E.M., n = 3).Finally, we showed that glycogenolysis is appar-ently insensitive to amino-oxyacetate, since phenyl-ephrine-induced glucose output from perfused liversof fed rats was only slightly inhibited by thetransaminase inhibitor (628 + 54pM versus562 + 4lUM, n = 4).We would thus conclude that the substrate- and

hormone-induced increase in glucose output ob-served in Fig. 1 and in the data reported below forthe livers of 48h-starved rats reflects that arisingfrom gluconeogenesis.Effect of phenylephrine on Ca2+ efflux, oxygenuptake and glucose output

Livers perfused with 2.5 mM-lactate were treatedwith a maximally effective concentration of phenyl-ephrine (2Mm) and showed rapid changes in Ca2+efflux, oxygen uptake and glucose output, as detailedin Fig. 2. Phenylephrine-induced increases in Ca2+efflux, oxygen uptake and glucose output were

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Livers of 48 h-starved rats were perfused as des-cribed in Fig. 1 in the presence of 2.5 mM-lactate for15 min, and then phenylephrine (2pM final concn.)was infused. Perfusate Ca2+ (a), oxygen uptake (b)and glucose output (c) were monitored as describedin the Experimental section. Increases in theseduring the initial 3 min of phenylephrine infusion areshown. Lag times for Ca2+, oxygen and glucoseresponses were determined separately for eachexperiment and data shown were corrected for these.Just before phenylephrine infusion, basal oxygenuptake was 382 + 38pM (n = 8), basal glucoseoutput was 84+ 9#M (n = 5) and perfusate Ca2+was 1.3 mM. Data shown for Ca2+ efflux and oxygenuptake are continuous traces from one of sixexperiments that gave similar results. Variationbetween experiments was small, with S.E.M. valuesless than 10% of mean. Glucose-output data are themeans + S.E.M. for five separate experiments.

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Fig. 3. Dependence on Ca2+ ofphenylephrine-induced increases in Ca2+ efflux, oxygen uptake and glucose outputLivers were initially perfused for 15 min as described in Fig. 2. Phenylephrine (2,UM) was then infused in five pulseseach of 4 min duration and at 4min intervals except the last, which was given 8 min after the fourth pulse. In someexperiments (hatched columns) the 1.3 mM-CaCl2 present in the perfusion medium (administered by infusion syringe)was removed 4min before the commencement of the phenylephrine infusions. In those experiments, 1.3 mM-CaCl2was re-administered for 2 min, 6 min before the fifth phenylephrine infusion. All other experiments were performedwith 1.3 mM-CaCl2 present throughout the entire perfusion (open columns). Perfusate Ca2+ (a), oxygen uptake (b)and glucose output (c) were determined as described in the Experimental section and the maximal increase induced byeach phenylephrine infusion is shown. Basal values for oxygen uptake and glucose output were 345 + 54pM and87 + 1 1pM respectively at a perfusate Ca2+ concentration of 1.3mM, and 341 + 5O,UM and 84 + 12puM respectively inexperiments where the CaCl2 infusion was terminated and perfusate Ca2+ decreased to approx. 6,UM just beforeinfusion of phenylephrine. These basal values remained essentially unchanged throughout the experimental period.Data shown are the means + S.E.M. for five separate experiments.

evident after 9.5 + 0.8, 10.5 + 1.1 and approx. 20srespectively. The increase in Ca2+ efflux was

transient, with a maximal increase of 21.7+2.3,UMbeing attained after 75s of phenylephrine infusion.Ca2+ efflux declined thereafter and reached basalvalues again by 2.5min (Fig. 2). In contrast withthe transient effect on Ca2+ efflux, phenylephrine-induced increases in oxygen uptake and glucoseoutput were maintained near maximal values untilthe a-agonist infusion was discontinued (results notshown). Near-maximal increases in oxygen uptakeand glucose output were achieved after 1.5 and 2 minof phenylephrine infusion respectively (Fig. 2). Theresponses in terms of Ca2+ efflux and oxygenconsumption to phenylephrine seen here are vir-tually identical with those seen with perfused liversfrom fed rats (Reinhart et al., 1982a,b).

The next set of experiments involved utilizingconditions that lead to the depletion of the a-

adrenergic-agonist-sensitive pool of Ca2+ in theperfused liver of the fed rat and were designed to test

the hypothesis that intracellular Ca2+ is essential fora-adrenergic-agonist-induced increases in gluconeo-genesis. The procedure involves perfusing the liverwithout added Ca2+ in the medium and to infusephenylephrine by repeated pulses (see Fig. 5 ofReinhart et al., 1982a). As shown in that paper, foursuch pulses, each of 90 s duration and given at 5 minintervals, were sufficient essentially to deplete theliver of the a-adrenergic-sensitive intracellular Ca2+pool.

Data in Fig. 3(a) show that when the livers of48 h-starved rats, perfused with buffer containing noadded Ca2+, were given four repeated pulses ofphenylephrine, the maximum rate of Ca2+ efflux wasdecreased from 20,UM to approx. 2pM, reflecting aloss of phenylephrine-sensitive intracellular Ca2 .

The response to the fifth pulse, given after a 2 minre-infusion of 1.3 mM-CaCl2 to the liver, was approx.70% of that which followed the first pulse. A paralleldiminution in oxygen uptake and gluconeogenicresponses was observed after the loss of intra-

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Table 1. Dependence on Ca2+ ofphenylephrine-induced increases in glucose outputLivers were initially perfused for 15 min as described in Fig. 2, except that the gluconeogenic substrates (each at2.5 mm final concn.) were present as shown. Phenylephrine was administered for 4 min, and after a further 4 min the1.3 mM-CaCl2 in the perfusion medium (administered by infusion syringe) was removed. The liver was then infusedwith five successive pulses of phenylephrine (2QM) each of 4min duration as described in Fig. 3. A 2min pulse of1.3 mM-CaCl2 was administered 6 min before the final phenylephrine infusion. Samples of perfusate were taken forglucose assay, and maximal increases in glucose output induced by phenylephrine were determined before CaCl2removal (+ 1.3 mM-Ca2+) during the fourth infusion of the a-agonist in buffer containing no added Ca2+ (Ca2+-depleted) and during the last infusion of phenylephrine, 4min after a 2 min re-administration of 1.3 mM-Ca2+(Ca2+ re-added). For each substrate the rate of basal glucose output was determined after the initial 15 min perfusionand was essentially unchanged throughout the period of perfusion. Data shown are means ± S.E.M. for at least fourseparate experiments.

Glucose output (#,M)

Phenylephrine-induced increase in output

SubstrateAlaninePyruvateLactateGlycerolDihydroxyacetoneSorbitolFructoseNone

Basal output33+541+683+771+6152+ 2478+ 11177 + 3414+4

+ 1.3 mM-Ca2+68 + 4.833 + 5.793 + 7.781+ 7.467 + 9.0114 + 1557+ 1116+ 2.1

Ca2+-depleted + 1.3 mM-Ca2+ re-added10+2.7 57+615+3.0 28+412+2.3 71 +922+6.8 53+823+8.0 59+ 1113±3.9 92+ 1317+5.0 38+83+0.8 7+2

cellular Ca2+ (Figs. 3b and 3c); these responses alsowere largely restored after a short re-infusion of1.3 mM-CaCl2. Fig. 3 also shows that in controlexperiments, where 1.3 mM-CaCl2 was continuouslypresent, responses to repeated pulses of phenyl-ephrine were only slightly diminished.

Stimulation of glucose output by phenylephrinewas also observed with a range of gluconeogenicsubstrates, as shown in Table 1. Increases in glucoseoutput ranged from 28% and 31% with fructose anddihydroxyacetone as substrate to 160% and 180%with sorbitol and alanine as substrate. Basal glucoseoutput was substantially higher with fructose ordihydroxyacetone than with the other substrates,and consequently the absolute increases in glucoseoutput induced by phenylephrine with fructose ordihydroxyacetone approached those observed withthe other substrates. With all substrates tested,increases in glucose output induced by phenyl-ephrine were substantially decreased when liverswere perfused under conditions which deplete thea-agonist-sensitive Ca2+ pool (Table 1). In theseCa2+-depletion experiments, increases in glucoseoutput in response to phenylephrine approachedmaximal values again when 1.3 mM-CaCl2 wasre-infused for 2min before phenylephrine treatment.Data similar to those shown for phenylephrine inTable 1 were also obtained with 50nM-adrenaline,50nM-noradrenaline and 2.5 munits of vasopres-sin/ml (results not shown). It should be noted that,although glucose output was still enhanced by

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adrenaline or noradrenaline at higher concentra-tions of hormone, oxygen consumption was inhibitedat catecholamine concentrations greater than 0.5pum.

Thus, and by analogy with information gainedearlier from experiments with fed rats (Reinhartet al., 1982a), the present experiments providestrong evidence that intracellular Ca2+ is required forphenylephrine-induced stimulation of gluconeogene-sis.

Effect ofphenylephrine on perfusate [lactate] /[pyru-vate] and [fJ-hydroxybutyratel/[acetoacetateI ratios

Since it has been suggested that a-adrenergicagonists may increase gluconeogenesis from rela-tively reduced substrates by enhancing the transferof reducing equivalents from the cytoplasmic to themitochondrial compartment (Kneer et al., 1979;Yip & Lardy, 1981), the effects of phenylephrine onperfusate [lactate]/[pyruvate] and [IJ-hydroxybuty-rate]/[acetoacetatel ratios, indicators of the cyto-plasmic and mitochondrial [NADH]/[NAD+] ratiosrespectively, were examined with a range of gluco-neogenic substrates.

Changes in [lactate]/[pyruvatel ratios were evi-dent 30-40s after phenylephrine infusion, to near-maximal values by 90s (Fig. 4). The [lactate]/[pyruvatel ratio was decreased by phenylephrinewhen the relatively reduced substrates glycerol orsorbitol were employed and the initial ratios wererelatively high (Fig. 4). With all other substratestested, phenylephrine induced an increase in the

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Samples of perfusate obtained as in Fig. 4 wereassayed for fl-hydroxybutyrate and acetoacetate asdescribed in the Experimental section. Substrateswere (O) glycerol, (A) sorbitol, (U) dihydroxy-acetone, (A) fructose and (0) alanine, each at a finalconcentration of 2.5 mm. Data shown are the meansof five separate experiments. For simplicity, S.E.M.values are not shown; however, they never exceeded0.2 and were generally less than 0.1.

Fig. 4. Effect of phenylephrine on the [lactatel/[pyru-vatel ratio

Livers were initially perfused for 15 min as des-cribed in Fig. 2, except that lactate was replaced bythe gluconeogenic substrates (0) glycerol, (A)sorbitol, (U) dihydroxyacetone, (A) fructose or (0)alanine, each at a final concentration of 2.5 mm.Phenylephrine (2pM) was then infused for 6 min.Perfusate lactate and pyruvate were assayed asdescribed in the Experimental section. Data shownare the means + S.E.M. for five separate experiments.

[lactatel/[pyruvatel ratio. Where fructose or di-hydroxyacetone was employed, the increase in the[lactatel/Ipyruvatel ratio was transient and hadreturned close to initial values by 6min of phenyl-ephrine administration.

[If-Hydroxybutyratel/[acetoacetatel ratios were

increased by phenylephrine irrespective of thesubstrate employed (Fig. 5). The ratio increasedwithin 20-30s of phenylephrine infusion, was nearmaximal by 90s and decreased slightly thereafter,but was still significantly above initial values at theend of the phenylephrine infusion (6min). Effectssimilar to those described for phenylephrine in Figs.4 and 5 were also obtained after perfusion withadrenaline, noradrenaline or vasopressin (resultsnot shown). However, effects of phenylephrineon ([lactate]/[pyruvatel and [fJ-hydroxybutyratel/[acetoacetatel ratios were not observed after liverswere perfused under conditions that deplete thea-agonist-sensitive Ca2+ pool as described by Rein-hart et al. (1982a) and used in Fig. 3 and Table 1(results not shown).

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Effect of agents that alter intracellular redox ratioson glucose outputThe transaminase inhibitor, amino-oxyacetate,

blocks the mitochondrial malate-aspartate shuttleand thereby diminishes the transfer of reducingequivalents between the cytoplasmic and mitochon-drial compartments. Data presented in Fig. 6 andTable 2 indicate that amino-oxyacetate (0.2 mM)inhibits basal glucose output from glycerol andsorbitol by 34% and 55% respectively. Moreover,phenylephrine-induced increases in glucose outputfrom glycerol or sorbitol were inhibited by this agent

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Fig. 6. Effect ofphenylephrine and amino-oxyacetate onglucose output

Livers were initially perfused for 15 min as des-cribed in Fig. 2, except that 2.5 mM-fructose (M) or2.5 mM-sorbitol (A) was used as gluconeogenicsubstrate. Phenylephrine (2pM, ) or amino-oxyacetate (0.2 mm,[I ) was then administered asindicated and perfusate samples were taken forglucose determination. Data shown are themeans + S.E.M. for four separate experiments.

by, 66% and 75% respectively. Amino-oxyacetate,however, had little effect on either basal or phenyl-ephrine-induced glucose output from fructose ordihydroxyacetone.

Phenazine methosulphate, an agent that rapidlyre-oxidizes reduced nicotinamide nucleotides, anddinitrophenol, an uncoupler of oxidative phos-phorylation, both increased glucose output fromglycerol and sorbitol. With glycerol as substrate,phenazine methosulphate (30,M) and dinitrophenol(25,UM) increased glucose output from 73 + 8 to1 16 + 13 and 134 + 18,UM (n = 4) respectively. Withsorbitol, however, dinitrophenol was much lesseffective, and variable responses were obtained(average increase of 15%), whereas phenazinemethosulphate was still an effective activator(112 + 10 versus 198 + 16,M, n = 4). Phenazinemethosulphate and dinitrophenol both inhibited glu-cose output from fructose or dihydroxyacetone andincreased total pyruvate and lactate outputs (resultsnot shown).

Effect of phenylephrine on ketogenesis and oxida-tion of[1-'4C]octanoate and 2-oxo[1-"4C]glutarateThe rate of oxidation of tracer amounts of

[1-14C]octanoate to '4CO2 has been used as ameasure of tricarboxylic acid-cycle flux in theperfused rat liver (Soboll et al., 1981). In the presentstudy, tricarboxylic acid-cycle flux was also moni-tored by following "4CO2 generation from 2-oxo-[1-14C]glutarate and gave results similar to thoseobtained with [ 1-14Cloctanoate. Relatively low ratesof "4CO2 production were observed in the absence ofgluconeogenic substrates when rates of ketone-bodyoutput were relatively high (see Table 3). Forexample, rates of octanoate oxidation were in-creased from a basal value of 0.38 + 0.09 ng-atom/min (n = 3) to 0.80+ 0.11 or 1.5 +0.20ng-atom/min (n = 4) on addition of sorbitol or fructoserespectively. Rates of ketogenesis were correspond-ingly decreased on addition of sorbitol or fructose(Table 3).

Table 2. Effect ofphenylephrine and amino-oxyacetate on glucose outputLivers were perfused as described in Fig. 6 with the gluconeogenic substrates shown, each at a final concentrationof 2.5 mm. Perfusate samples were taken just before and during infusion of phenylephrine or amino-oxyacetate(final concns. 2,UM and 0.2 mm respectively). Maximal changes in glucose output induced by these agents weredetermined as described in the Experimental section. Data shown are means + S.E.M. for four separate experiments.

Glucose output (pM)

SubstrateSorbitolGlycerolFructoseDihydroxyacetone

* P < 0.01 compared with no additions.

Additions ... None Phenylephrine107+ 10 254+ 16*70±9 146+ 19*

281 + 11 338 ± 14*196+14 251+18*

Amino-oxyacetate59 ± 10*46+7*

273 ± 9185 + 16

Amino-oxyacetate+ phenylephrine

97 + 1171 + 13

324 + 13253 + 20

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The effect of phenylephrine on [1-14C]octanoateoxidation is shown in Fig. 7(a). A rapid increase in

Table 3. Effect of phenylephrine on total ketone-bodyoutput

Perfusate samples were taken immediately beforeand 2min after infusion of phenylephrine (2,pM) asdescribed in Fig. 4. Ketone bodies were determinedas described in the Experimental section. Datashown are the means+ S.E.M. for five separateexperiments.

Total ketone-bodyoutput (#M)

Substrate Additions ... None PhenylephrineNone 103+5.8 111+6.1Alanine 29 ± 1.4 48 + 6.4*Glycerol 38 1.0 55 + 1.4*Dihydroxyacetone 39 ± 4.3 55 + 3.8*Fructose 34 + 2.5 51 + 2.2*Sorbitol 36 + 1.4 51 + 1.5*

* P < 0.01 compared with respective control.

octanoate oxidation was observed within 10- 15s ofphenylephrine infusion when either fructose orsorbitol was present. Maximal increases were ob-served between 30 and 60s of a-agonist infusion,and rates of oxidation declined thereafter. Somevariability in this latter portion of the phenyl-ephrine response was observed. In some experi-ments rates of oxidation were maintained at approx.70% of the maximal value, whereas in others the ratefell to approx. 10-20% above the basal value at thecompletion of the phenylephrine infusion. Essen-tially similar data were obtained when [1-14C1-octanoate was replaced with 2-oxo[1-_4Clglutarate(Fig. 7b). Effects of phenylephrine on oxidation of[1-14C]octanoate or 2-oxo[ 1-14C]glutarate wereinhibited by over 80% after livers were perfusedunder conditions which deplete the a-agonist-sensi-tive Ca2+ pool (results not shown).

Ketone-body output was measured during gluco-neogenesis from a range of substrates shown inTable 3. Phenylephrine induced a rapid increase inketogenesis, with maximal increases of 40-50%being observed within 2min of a-agonist infusion. In

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Fig. 7. Effect ofphenylephrine on [I-14C]octanoate or 2-oxoll-14C]glutarate oxidation to '4CO2Livers were perfused as described in Fig. 6, except that 2-oxol1-_4Clglutarate (53.6Ci/mol) or [1-_4Cloctanoate(25.1 Ci/mol) was continuously infused to give final concentrations of 0.20 and 0.55#m respectively. The substratesfructose (-) or sorbitol (A) were used at final concentrations of 2.5mm and phenylephrine () was infused at afinal concentration of 2,pM as indicated. Samples of perfusate were assayed for 14CO2 as described in theExperimental section. Data shown are from one experiment of four, each of which gave similar results.

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the absence of gluconeogenic substrates, ketone-body output was elevated and phenylephrine-induced increases were not significant under thoseconditions. Phenylephrine did not affect ketone-bodyoutput after livers had been perfused under condi-tions that deplete the a-agonist-sensitive Ca2+ pool(results not shown).

Discussion

Among the major points revealed in this study ofa-agonist-induced gluconeogenesis in the perfusedliver of starved rats are (a) the obligatory role ofCa2+ in the gluconeogenic response, (b) the varietyof metabolic events seemingly associated with thegluconeogenic response and which are also Ca2+-dependent, and (c) the rapidity with which theresponses are seen. Of particular interest in thisregard was the observation that ketogenesis andtricarboxylate-cycle activity are each increased bya-agonists concomitant with enhanced rates ofgluconeogenesis.An obligatory role for Ca2+ in a-agonist-induced

gluconeogenesis could be deduced from experimentsin which the a-agonist-sensitive intracellular pool ofCa2+ was manipulated by the administration ofphenylephrine through repeated pulses to the liverperfused with bicarbonate buffer containing noadded Ca2+ (Fig. 3, Table 1). In this way it could beshown that the smaller the a-agonist-induced Ca2+efflux (and presumably the greater the depletion ofthe a-agonist-sensitive Ca2+ pool), the smaller thegluconeogenic response. It was noteworthy that theCa2+-dependence of the a-agonist-induced increasein glucose output was observed with all gluco-neogenic substrates tested (Table 1 and Fig. 3).An additional feature of our experimental

approach (Reinhart et al., 1982a), hitherto appar-ently not undertaken in studies on gluconeogenesis,was the ability to measure rapid and continuouschanges in Ca2+ fluxes across the plasma membraneand in whole-tissue respiration; such respiration isattributable in large part to mitochondria (Reinhartet al., 1982b). In this way we were able to show thatsuch changes precede phenylephrine-induced in-creases in glucose output. Indeed the time-courseand magnitude of Ca2+ efflux induced by phenyl-ephrine in these perfusions with livers from starvedrats were virtually identical with the phenylephrine-induced Ca2+ efflux previously shown to precedeglycogenolysis in perfused livers of fed rats (Rein-hart et aL, 1982a). The important conclusion canthus be drawn that such changes in Ca2+ efflux areindependent of the nutritional status of the experi-mental animal. The findings also suggest that alteredCa2+ fluxes may be linked to the initial events in themechanism of action of a-adrenergic agonists in ratliver.

The important role of Ca2+ in the responses toa-adrenergic agonists in rat liver was furtherhighlighted by the findings that the changes inducedby phenylephrine in the redox state of the cyto-plasm (Fig. 4) and mitochondria (Fig. 5), as well asin ketogenesis (Table 3) and the oxidation of[1-14Cloctanoate or 2-oxo[1-'4C]glutarate (Fig. 7),were all considerably diminished after depletion ofthe a-agonist-sensitive Ca2+ pool. As with gluco-neogenesis (Fig. 3, Table 1), re-infusion of livers with1.3 mM-CaCl2 restored all of these responses.A requirement for Ca2+ in the stimulation of

gluconeogenesis by a-adrenergic agonists has beenreported previously and a number of possible sitesfor Ca2+ action have been suggested. Tolbert & Fain(1974) reported a Ca2+-dependent catecholamine-induced increase in glucose output from a range ofgluconeogenic substrates with rat hepatocytes. How-ever, in those studies, basal rates of gluconeogenesiswere markedly decreased by the omission of Ca2+.This is in contrast with the present study and earlierstudies with perfused liver (Friedmann & Ras-mussen, 1970; Sugano et al., 1980) or hepatocytes(Kneer et al., 1979), where Ca2+ omission did notcause appreciable changes in the basal rates ofgluconeogenesis. More recently, Garrison et al.(1979, 1981) have suggested that a cytoplasmicCa2+-dependent protein kinase may catalyse thephosphorylation and subsequent inhibition of pyru-vate kinase in response to catecholamines. Inaddition, an activation of phosphoenolpyruvatecarboxykinase, indirectly mediated by Ca2+, andinvolving the release of mitochondrial Fe2+, has beenproposed by Merryfield & Lardy (1982). It has alsobeen argued that Ca2+ may regulate pyruvatecarboxylase activity (Williamson et al., 1981).A Ca2+_sensitive exchange of reducing equiv-

alents between cytoplasmic and mitochondrial com-partments involving an increased mitochondrialglycerol phosphate dehydrogenase activity has beenproposed to account for the Ca2+-dependent in-crease in gluconeogenesis from reduced substratessuch as glycerol and sorbitol in hepatocytes pre-pared from 24h-starved rats (Kneer et al., 1979; Yip& Lardy, 1981). Concentrations of amino-oxy-acetate that block the malate-aspartate shuttlereportedly had no effect on noradrenaline-inducedglucose output from these relatively reduced sub-strates (Kneer et al., 1979).Some of the findings reported by Kneer et al.

(1979) and Yip & Lardy (1981) differ substantiallyfrom the data obtained with perfused livers asdocumented in the present study. The differentresults obtained may in part reflect differencesbetween the hepatocytes and perfused liver pre-parations used, as well as differences in the time ofa-agonist treatment and the techniques employed todeplete the relevant Ca2+ pool(s). In this regard, it is

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noteworthy that hepatocyte preparations have pre-viously been reported to become depleted of malateand aspartate, and consequently exhibit low mal-ate-aspartate-shuttle activities (Cederbaum et al.,1977). In addition, in relatively long-term experi-ments such as those of Kneer et al. (1979), inhibi-tion of reducing-equivalent transfer by amino-oxy-acetate may be by-passed as glycerol phosphateconcentrations rise and flux through the mito-chondrial glycerophosphate dehydrogenase in-creases (Williamson et al., 1971).

In our hands amino-oxyacetate significantly de-creased basal glucose output from sorbitol andglycerol and effectively blocked the phenylephrine-induced increases in glucose output observed withthese reduced substrates. Data in the present studythen indicate that a-adrenergic agonists may in-crease gluconeogenesis from glycerol and sorbitol bypromoting the removal of cytoplasmic reducingequivalents by the operation of the malate-aspart-ate shuttle, although some involvement of theglycerol phosphate-dihydroxyacetone phosphateshuttle cannot be ruled out. The mechanism where-by a-agonists may stimulate the malate-aspartateshuttle is unknown, but may be related either tochanges in total amounts of shuttle intermediates orto a change in the energization of the mitochondrialinner membrane, which may in turn affect theelectrogenic exchange of aspartate for glutamate.

Other data presented are consistent with thesuggestion that glucose output from glycerol andsorbitol may be enhanced by an increase in the rateof oxidation of cytoplasmic nicotinamide nucleo-tides. The electron acceptor phenazine methosul-phate, and to a lesser extent dinitrophenol, anuncoupler of oxidative phosphorylation, enhancedglucose output from both of these reduced sub-strates. In addition, phenylephrine-induced in-creases in glucose output with glycerol or sorbitolwere accompanied by a decrease in the [lactate]/[pyruvatel ratio and an increase in the [fJ-hydroxy-butyratel/[acetoacetatel ratio.

This study also shows an a-agonist-inducedelevation of the [I-hydroxybutyratel/[acetoacetateIratio (the mitochondiral redox ratio) with everysubstrate tested. Sugano et al. (1980) reported thatnoradrenaline increased the total cellular reducingequivalents in perfused liver of starved rats witheither pyruvate of lactate as substrate, and pre-sented some evidence for a mitochondrial site ofreducing-equivalent generation. In studies withhepatocytes where lactate was the substrate, Siess etal. (1978) demonstrated an increase in the mito-chondrial redox ratio and a lowered mitochondrial2-oxoglutarate content in response to catechol-amines. Our studies of the conversion of [1-14C]-octanoate or 2-oxo[1-_4C]glutarate into 14CO2 sug-gest that a-adrenergic agonists rapidly stimulate

tricarboxylic acid-cycle flux. We also observed a40-50% increase in total ketone-body output.Sugden et al. (1980) have reported the stimulation ofconversion of [1-14CIoleate into "4CO2 in rathepatocytes after administration of adrenaline orvasopressin. Data from the present study and that ofSugden et al. (1980) suggest that a-agonists mayincrease the mitochondrial redox ratio mainly as aresult of an increased rate of fl-oxidation of fattyacids and an increased flux through the tricar-boxylic acid cycle. A significant contribution ofcytoplasmic reducing-equivalent transfer to themitochondrial compartment may only occur whenthe initial cytoplasmic [NADHI/[NAD+] ratio isrelatively high, for example when glycerol or sorbitolis employed.

Although Ca2+ has been reported to stimulateboth isocitrate dehydrogenase and 2-oxoglutaratedehydrogenase (McCormack & Denton, 1981), anda -Ca2+-dependent inhibition of acetyl-CoA car-boxylase has been observed in hepatocytes treatedwith a-adrenergic agonists (Ly & Kim, 1981), it isnot known if these effects are involved in thestimulation of tricarboxylic acid-cycle flux andketogenesis described in the present study.We also report the Ca2+-dependent activation by

a-adrenergic agonists of glucose output from fruc-tose and dihydroxyacetone, substrates which enterthe gluconeogenic pathway at the triose phosphatelevel. Since these changes were observed in theabsence of significant changes in glycolysis(lactate +pyruvate output unchanged), a-adrenergicagonists may stimulate the gluconeogenic pathwayat other site(s) located between the triose phos-phates and glucose. A Ca2+-dependent inhibition offructose 6-phosphate 2-kinase, and lowered con-centration of the activator of phosphofructokinase,fructose 2,6-bisphosphate, have been described inhepatocytes after treatment with adrenaline(Richards & Uyeda, 1982). Thus a-agonists mayalso inhibit phosphofructokinase and decrease therate of cycling of fructose phosphates. However,such a proposed action may be limited in the starvedstate, as phosphofructokinase activity and thecycling of fructose phosphate are already extremelylow in starved-rat liver (Kummel, 1982; VanSchaftingen et al., 1980). Consequently, a futurestudy should examine the possibility that a-adren-ergic agonists may stimulate fructose 1 ,6-bis-phosphatase or glucose 6-phosphatase activities.

We are grateful to Mrs. J. Lindley for assistance insome of these experiments. This work was supported by agrant to F. L. B. from the National Health and MedicalResearch Council of Australia.

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Stimulation by a-adrenergic agonists of Ca2+ fluxes, mitochondrial