Biochbrdcaa BiophydcaActa, !136(1992)2.L'~-230
223
© 1992ElsevierSciencePublishersB.V. All rights reserved0167-4889/92/$05.00
BBAMCR 13229
Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes M i c h a e l N. Berry, J o h n W. Phillips, R o l a n d B. G r e g o r y , A n t h o n y R. G r i v d l a n d Patricia G. W a l l a c e Departmentof MedicalBiochemistry,Schoolof Medicine,Hinders Unirersityof South Australia, Adelaide, Sot,thAustralia (Australia)
(ReceivedZ'~December1991) (Revisedmanuscriptreceived28 April 1992) Keywords: Isolatedhepalocyle;Malale-asparlateshuttle;Lactate metabolism;Reducing-equivalenttransfer. Energytran~uclion The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in Lsolatedhepatocytes of faste.'] rats. 6oth lactate removal and glucose synthesis were inh~ited by butylmalonate, aminooxyacetate or cycloserine ~nfirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butyimalonate was considerably reduced, yet the trans;er of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for bulylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulator/effect on uptake of sorbitol, a substrate whose oxidation principally involves the ~x-glycetophosphateshuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potenti',d (Zl~) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate ox!4ation was strongly inhibited when AV' fell from 130 to 105 mV whereas O 2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by A~. In the presence of added ammonium ions the removal of lactate was much less impaired by walinomycin,suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochonddal matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the bulyimalonate-sensitive, energy-dependent, malate-asparlate shuttle. Introduction "Ilze initial step of hepatic lactate metabolism generates NADH, that must be converted back to NAD + for further flux to proceed. This oxidation r~ay take place by means of an entirely cytoplasmic-coupled reaction associated with gluconeogenesis, in which the conversion of lactate to pyruvate is linked to reduction of 1,3-diphospboglycerate to glyceraldehyde 3-phosphate. There is also a mitochondriai route available for cytoplasmic NADH oxidation, which is indirect because the mitorbondrial inner membrane is almost impermeable to NADH [1-3]. This indirect pathway involves reducing-equivalent carder s)'stems, such as the malate-
Correspondenceto: M.N. Berry, Depatlmenl of Medkal Biochemistry, School oi Medicine, The Binders IJabe~ity ol" South Australia" G.P.O. Fox 2100)Adelaide,South Australia,Australia,5001.
aspartate [4,5] or a-glycerophosphate shuttles [6,7]. The aln,ost complete inhibition of lactate oxidation by the NADH dehydrogenase inhibitor, totenone [8], indicates that the transfer of reducing equivalents from cytoplasm to mitochondria must involve one or more NKO.dependent shuttles. "Early studies on hepatic gluconeogenesis from lactate assumed that the cytoplasmic couple catalysed by glyceraldehyde 3-phosphate and lactate dehydrogenases was the predominant mechanism for NADH oxidation [9]. in conjunction with this, it was postulated that the carbon skeletons for gluconeogenesis are provided by transamination of oxalacetate to aspartate that then leaves the mitochondria [10-12], a balance of nitrogen being maintained by an associated entry of glutamate and exit of a-ketoglutarate, These early studies also demonstrated, however, that hepetic gluconeogenesis from lactate is depressed not only by inhibitors of transamination [13,I4], but is al~ strongly
224 inh~ited both by butylmalonate [15,16] and by fluoromalate [17], agents that respectively inhibit malate transport and metabolism. Hence there is good evidence that malate transport into the mitochondfia takes place during hepatic lactate metabolism, the most likely mechanism being a malate-aspartate carrier-raediated exchange. In studies with isolated mitochondnal preparations LaNoue and co-workers [18-21] found that the entry of aspartate into mitochondria, in exchange for glutamate, is very slow and does not occur in energized mitochondria, thereby accounting for the unidirectional nature of the malate-aspartat¢ shuttle. They demonstrated that exchange in the opposite direction, i.e. entry of glutamate and eff!ux of aspartate, was stimulated by coupled respiration, but inhibited by uncouplers and valinomycin. Because effiux was not affected by nigericin [22,23] they concluded that transport in the physiological direction of aspartate efflux was promoted by the electrical potential gradient across the inner mitoehondrial membrane (at/'). Since it is now feasible to measure ag" in intact cells [24-26], we have examined the effects of mitochondrial energy"and redox states on lactate utilization by isolated heparacytes. In this paper we show that reducing equivalents generated in the cytoplasm from the oxidation of lactate can be transported to the mitochondria not only by an energy-dependent mechanism involving 1he malateaspartate shuttle but also, when ammonium ions are present, by an. energy-independent process that does not utilize this shuttle. Materials and Methods Collagenase and enzymes for metabolite determination were from Boehringer M.':anheim (Germany) as
was bovine serum albumin (fraction V) which was defatted by the method of Chen [27]. Palmitate (Sigma, USA) was neutralized, and dissolved in an ~;otonic sail solution containing 9% defatted bovine ser~Jmalbumin. Vaiinomycin, p-trifluoromethox)'phenylhydrazone (FCCP), hutylmalonate, aminooxyacetic acid and quinolinic acid were also obtained from Sigma (USA). Pcrfluorosuccinic acid was from Pfaltz and Bauer (CT., USA) and (S)-(-bcycloserine was from Aldrich (Wi, USA). All other chemicals were of the highest quality available. L[U-taC|lactate, [t4C]methyltriphenylphosphonium iodide (TPMP +) and the scintillation cocktail ACSll were all obtained from Amersham (Australia). Isolated liver cells from male Hooded Wistar rats (250-280 g body wtL starved for 24 hours to deplete liver glycogen, were prepared by a modification [28] of the method of Berry and Friend [29], in which calcium ions were included in the washing media. The cells (90-120 mg wet wt) were incubated at 3T'C in 2 ml of a balanced bicarbonate-buffered medium [30,31] containing aibumin, 2.25% (w/v), under a gas phase of 95% O,, 5% CO:. Lactate and pymvate were normally present in incubations at initial concentrations of 10 mM and 1 mM, respectively. This level of lactate ensured saturating concentrations were maintained throughout the experiment, whilst addition of pyruvate established a normal lactate/pymvate steady-state ratio from the commencement of the incubation period. Consumption of O2 was measured in the presence of CO: by a manometric method [32]. The water-insoluble inhibitors (va!inomycin and FCCP) were dissolved in acetone. The solution was added to the empty incubation vessels and allowed to evaporate to dryness. The inhibitors were then re-solubilized by the additior, of medium containing the bovine serum albumin and other incubation components. A~ the completitm of the
TABLE I Reducing equivalentsgeneratedfrom lactate oxidation
Hepatocytesfromfastedrats wer~incubatedfor 30 min as describedin Materialsand Methodswith the followingadditions:lactate, 10rnM; plmtvate, I raM: palmitate, 2 mM: butylmalonate.1OmM: aminooxyacelate,1O0/,M: cycloserine, 5 mM. The data are presentedas the mean± S.E. of at least 12separateexperiments.Reducingequivalentaccumulationin glucosewas determinedas 2x glucosewiththe estimateof the minimumquantityof reducingequivalentstransferredto the mitochondriaas (lactate -2xgluoosc). Addition
Lactate Lactate,palmitate Lactate,butylmalonate Lactate,palmitaze,butylmalonate Lactate,palmitate,aminooxyacetate Lactate,palmitate,cycloserine
Metabolicchanges(/~mol/30 rainper 100mgwetwt) reducing lactate glucose equivalents accumulatedin glucose - 4.53+_I].09 - 8.01± 0.12 - 2.46+ 0.15 - 4.56± 0.18 --2.464-_0.12 - 3.18__0.27
1.71± 0.06 4.05± C.05 0.93_-.I0.03 1.95+ 0.09 1.53.~.0.06 1.29± 0.04
3.90
minimumquard;y of reducing equivalents transferre~to mitoch~ndria 1.11 -0.09 0.60 0.66
3.06
-0.60
3.42 8.10 1.86 2.58
O.6O
225
NN
.~+~
i~ ~
~
• ~
~
~m
-
~.N N
N
-+~ --NN l l l i l | l
_~'~
~
.~.~
I.°
N~g~N~Nm
+r -i--¢
N ~
,
m~
.-i-i
.
+i
¢~.
°
=~-.-~+~'~ I
I
I
I
NN
~'~ F I I | I I I
•.~ ~
'
-~ ~ .
..
e~
N
l
~
,
II
N
N
N
~
t
~
,-.
~
,,Tr,,,
N
,-1
I
~
~
N
~
~ g g g g g g g
226 incubation period (30 rain), samples were deprote':nized with an equal volume of cold 1 M perchloric acid and neutralized. Metabolites were measured by standard enzymatic techniques [33] using a Cobas FARA automated analyser (Roche Diagnostics, BaseD, the data being transferred to a PDP 11/73 computer (D.E.C., USA) for subsequent processing. Where the mitochondrial inner membrane electrica| potential (A~) was measured, 0.4 ml samples of the incubation mixture were removed prior to acidification and processed as previously described [26]. For the measurement of CO 2 production, hepatocytes were incubated in the presence of either 0.2 gCi [U14C]lactate or [l-t4C]palmitate in vessels sealed with a serum cap from which hung a plastic well holding a filter paper wick. At the end of the incubation, 0.25 ml of 2-phenylethylamine was injected through the seal and the vessel acidified with 0.5 ml of 4 M perchloric acid. The vessel was kept on ice for a further 60 rain before the plastic well, containing the trapped CO 2, was transferred to 10 ml of ACS II scintillation fluid. ,,,o~,~u~"t~'~mitochondria a were prepared in 0.25 M sucrose, 5 mM N-[2-hydroxyet.hyl]piperazine-N'*[2e~hanesulphonic acid], pH 7A according to Ref. 34 and incubated as described by Strzelecki et al. [35]. Results and Discussion
IncolL'ement of malate transport in lactate oxidation Determination of the minimum quantity of reducing equivalents generated by lactate oxidation that pass to the mitocbondria can be achieved by a simple carbon balm,.ce study, based on the assumption that all the reducing equivalents, required for the reduction of the 1,3-diphosphoglycerate, are derived from lactate, whether or not palmitate is present (Table I). In the presence of palmitate, the conversion of lactate to glucose is stoichiometric so that by this mode of calculation, which assumes no reducing equivalents are transfei~ed from mitochondria to cytoplasm, the flow of reducing equivalents into the mitochondria is calculated as nil. However, in the presence of butylmalone.re, net lactate utilization and glucose production could be inhibited about 50%, whether or not lactate conversion to glucose was stimulated in the presence of palmitate. From this observation (Table l), it can be inferred that malate transport between cytoplasm and mitochondda plays a substantial role in lactate oxidation. We also noted that the presence of transaminase inhibitors such as aminooxyacetate or cycloserinc, resulted in a 60-70% inhibition of net lactate utilization and glucose synthesis (Table 1). This degree of inhibition by these agents is in keeping with conclusions that the bulk of the oxalacetate, formed by ~ruvate carboxylation within the mitochondria during gluconeogenesis from lactate, is transported to the
cytoplasm as aspartate. These results support previous work [13-17] suggesting that the malate-aspartate shuttle is the major system involved in transporting to the mitochondria reducing equivalents arising from hepatic lactate oxidation.
Effects of ammonium ions or polmitate on lactate oxidation The presence of ammmlinm ions stimulated both lactate oxidation and 0 2 consumption (Table II). It in inferred that some of the extra lactate consumed was oxidized in the Krebs Cycle, an inference supported by the observation that 14CO2 production from [U14C][actate was increased from 0.81 _+0.03 to 2.54 _+ 0.10 #mol/min per g wet wt (n = 4) in the presence of anunonium ions, while some lactate carbon accumulated in amino adds. Butylmalonate caused only a small reduction in lactate utilization, indicating that, in the presence of ammonium ions, the energy-dependent malate-aspartate shuttle, which can be expected to be strongly inhibited by hutylrn~! . . . . . ut'*., t[1¢ .......... t . a , l1~' UJ1 , m:~tkes a substantially lesser contn'bution to lactate oxidation than when ammonium ions are absent. The diminution in lactate utilization due to butylmalonate addition, both in the presence aad absence of ammonium ions, was accounted for by a corresponding decrease in glucose formation. The stimulatory actions of palmitate and ammonium ions on lactate utilization were not additive (Table II). Palmitate stimulated ammonium ion removal, presttmably by providing additional NADH for the reductive formation of glutamate, and brought about a substantial accumulation of lactate carbon in alanine, aspartate and glutamate, but reduced the rate of gluconeogenesis to a level below that of the control with lactate alone. Similar observations have been reported by Krebs et al. [36]. Since the accumulation of amino acids indicates that palmitate did not suppress the conversion of lactate to pyruvate or oxalacetate, the depression of gluconeogenesis in the presence of palmitate and ammonium ions was apparently the consequence of the diversion of carbon and reducing equivalents from glucose synthesis towards amino acid accumulation. Carbon balance measurements also indicate that palmitate overcame, to a large extent, the stimulator/ effect of ammonium ions on oxidation of lactate via th~ Krebs Cycle (decreasing i4CO2 formation from [U~4C]lactate from 2.54 + 0.10 to 0.91 + 0.04 itmol/min per g wet wt (n = 4)), reflecting the inhibitory effect of fatty acid on the conversion of pyruvate to acetyl CoA [37,38], Thus, the failure of ammonium ions and palmitale to bring about additive effects on lactate removal can be attributed to the abiJity of palmitate to diminish lactate oxidation in the Kxebs Cycle, and the depressive action of ammonium ions on gluconeogenesis from lactate in the presence of palmitate.
227 Urea formation was greatly enhanced by the addition of ammonium ions both in the absence and presence of palmitate, however, butylmalonate had no significant effect on this rate (Table !1). The relative insensitivity of lactate removal and urea formation to b,atylmalonate in the presence of ammonium ions suggests that the oxidation of cytoplasmic malate formed from the fumarate derived from arginosuccinate cleavage is not impaired by the inhibitor. Indeed, this is confirmed by the failure of malate to accumulate in the presence of botylmalonate under these circumstances (data not shown). Reducing equivalents produced in the cytosol in excess of the requirements for gluconeogenesis must re-enter the mitochondria for oxidation. An estimate of the reducing-equivalent transport [14] can be determined in a manner analogous to the calculation performed in Table I on the assumption that each tool of urea accumulating represents the generation in the cytosol of one mot of reducing equivalents as malate (Table II). Clearly under conditions where ureogenesis was stimulated there was an increased flux of reducing equivalents into the mitochondria, with butylmalonate having little to no effect on this flux. Hence, there ap~ars to be an alternative butylmalonate-insensitivemechanism for the disposal of the malate generated in the cytoplasm that operates during synthesis of urea from ammonium ions.
Energy ~
of malate-aspartote shuttle
If an energy-dependent shuttle is involvedin hepatic lactate metabolism, it can be anticipated that lactate removal will be depressed by agents that impair energy metabolism and reduce A~/'. Valinomycinis a particularly potent agent in this regard [24,26] and proved a more useful tool than the ,mcoupling agent, FCCP, which, at concentrations strongly inhibitory to lactate uptake brought about an unacceptably high increase in the number of cells taking up trypan blue. Valinomycin causes swelling of isolated mit~hondria [39], which can lead to difficulties in interpretation of results; but significant swellingdoes not occur in the mitochondria of isolated hepatocytes [26]. Accordingly, we titrated hepatocytes with valinomycinin the presence of lactate alone, or in combination with palmitate. When lactate was the only added substrate, low concentrations of valinomycinstimulated respiration and lactate utilization (Fig. la), the 14CO2production from [t4C]lactate increasing from 0.81 + 0.04 to 1.65+ 0.07 ttmol/min per g wet wt (n -4), indicating that the extra lactate was oxidized in the Krebs Cycle. Higher concentrations of the ionophore brought about a small decline in respiration and a substantial fall in lactate consumption (Fig. la). Even at 75 nM valinomycin,the cellular concentration of ATP was greater than 0.6/tmol/g wet wt, a level at which, in our experience, cellular integrity is maintained. Nevertheless, to exclude the
3
i
6
v
0
0
20
40
60
80
15
!00
b
,4
12
"
i' ~
6 t
3
20
40
60
80
100
Fig. 1. Effect of vatinomycin on the rates of o~8en consumption and lactate removal by hepatocytes. Isolated liver cells were prepared as d~seribed in Mate~als and Methods and incubated wi',h either:. (a) 10 mM lactate or, (b) 10 mM lactate plus 2 mM palmilate in the presence of a graded concentration range of valinomycin (0-100 nM). In both (a) and (b) the closed symbols (11, e) represent the rate of orygen consumption, #o and the open symbols (t3. o) the rate of lactate removal, ]I~1c- The data presented are for a single ext~dmeat and are representative of all five experimem5 performed.
possibility that the inhibition of lactate removal was due merely to cell damage or death induced by high concentrations of valinomycin, the effects of the ionophore on lactate metabolism in the presence of palmitate were examined (Fig. lb). Extremely high rates of respiration were achieved under these conditions, with A~P falling and 02 uptake increasing for each increment in valinomycinconcentration up to 30 nM and then remaining steady. The rate of lactate oxidation by hepatocytes rose gradually as Aq' decreased from 170 to 130 mV, and then fell sharply, maximuminhibition being observed at levelsof 105 mV (Fig. 2a), when the rate of cellular 0 2 consumptionwas almost twice the level in the absence of ionophore. Hence the inability of the cells to take up lactate was not attributable to damage leading to a non-specific failure of electron transport. A small inhibition of lactate uptake associated with a concomitant 20% stimulation of 0 2 consumption was also brought about by low concentrations of FCCP (data not shown). The observation that hepatocs'tes metabolized l~ruvate rapidly in the presence of the same concentration of valinomycin and level of d~' that inhibited lactate
228
a
i o
80
100 120 140 160 180
7
b
~6
~5 :~t. 4
i3 I
0
80
100 120 140 160 zeo A't{~)
Fig. 2. Relationship bern'curt the rates of lactate and pymvate remowal and the nfitochoadrial membrane potential (A~). Isolated he~too~es were incubated as descn'bed in Materials and Methods with the addition of either. (a) 10 raM lactate plus 2 mM palmitale or;, (b) 15 mM pymvate in the presence of a graded concentration gauge of valieomy~ (0-100 nM). The data presented arc from a single experiment and are rCl~CScntath'c of the three experiments performed.
utilization (Fig. 2b) suggests that the energy-dependent step of lactate removal is the initial oxidation to pyrurate. Moreover. addition of the artificial electron cartier methylene blue, which bypasses Site i ef the dectron transport chain, overcame the inh~itory effect of valinomycin on lactate removal (data not shown). These
studies confirm that the energy-dependence of the malate-aspartate shuttle, first demonstrated with isolated mitochondria by LaNoue and co-workers [18-22], can be observed with an intact cell preparation, and support their conclusions that operation of the shuRle is driven by A~. In the presence of ammonium ions the utilization of lactate w~s much less impaired by high concentrations of valinomycin (Table liD. Indeed, the transport of reducing equivalents into the mitochondria was only slighdy depressed under these conditions (Table ÁÁÁ). Thus, it appears that ammonium ions promoted an energy-independent oxidation of lactate, since they were able to stimulate its uptake at the same depressed lceels of cellular A_TP ~-nd d V associated with inh$ition of lactate oxidation by valinomycin in the absence of ammonium ions (data not shown). High concentrations of valinomycin inh~ited gluconeogenesis but did not depress amino acid accumulation, a process that also requires lactate o~dation to pymvate, Hence, lactate removal in the presence of ammoniur~ ions and valhzomycin must have involved the passage of reducing equivalents into the mitochondria. Similar findings were obtained when bepatocytes from h~pothyroid rats were employed (IL Gregory, unpublished observations), discounting the likelihood that zhe a-glycerophosphate shuttle is involved in this reducing-equivalent transfer since the a-glycerophosphate dehydrogeaase component of this shuttle has a very low activit3, in the hypothyroid state [40]. Moreover, the oxidation of sorbitol, a substrate which feeds reducing ~uivalents into the a-glycerophosphate shuttle [41] was not stimulated by ammonium ions (data not shown). Butyimalonate (Table II) inhibited lactate conversion to glucose but, like valinomycin, in the presence of ammonium ions caused minimal depression of amino acid formation from lactate. Taken together, these observations lead to the conclusion that, in the presence of ammonium
TABLE !!! Effect of t'a~inoatyc~ and ammordum ions on lactate r a e t ~
Hepaloc~es from fasted raLswere incubated for 30 min with.subs:rate additions as in Table IL Valinom3x-inseaspresent at 75 nM. Resultsare presentcd as the mean-+S~E, of at hast eight separate experiments. B/A =[~-lffdrm3~tyratel/[acetoacoate]. The minimum transfer of reducing equiwatentsfrom c~osol to mitochondria is estimated as descn'bedin Table It. Addition
Metabolicchanges(/zmol/30 rain per 100mg wet wl) lactate glucose urea alanine glutamate aspartate
B/A minimqm
traaslam of reducing equivalentsto mitochondria Lactatc,~imitatc Lactate.palmitate.va~cin Lactate, palmitate,ammonia Lactate, palmitate,ammonia, valinomy~n
-8.01_+0.12 4.05_+0.06 0.12_+0.01 0.164-0.01 0.06_+0.01 0 -1.26_+0.09 0.75_+0.05 0.14_+0.01 0.17+0.01 OJ]O 0 -7.17_+0.17 0.90_+0.02 1.35_+0.05 1.79_+0.06 0.46+0.02 3.26+_0.I0 6.70
1.22+-0.03 0.27+0,01 0.29+0.02
-6.45+_0.21 0.574-0.05 0.90+_0.04 2.79+_0.15 I.(M__.0.04 1.62-+0.09 6.21
0.19+0.01
229 ions, the transfer of lactate hydrogen to the mitochondria is accomplished by an NAD-linked mechanism that is clearly distinct from the butylmalonatesensitive, energy-dependent, malate-aspartate shuttle associated with gluconeogenesis from lactate in the absence of ammonium ions. To gain further insight into the nature of the transfer mechanism, Lsolated mitocbondria were incubated under similar conditions to those used by [aNoue and coworket,s [18-22] (for details of a representative experiment, see Table IV) except that ammonium ions (4.8 raM) were included. A time course was run, during which accamulation of intermediates '#as measured. Under control ~ndigio~s virtually no aspartate, glutamate or alanine was released into the incubation medium. However, when 0.8 pM FCCP was included, reducing ATP levels from 0.7 mM to 10 p.M, substantial amounts of the amino acids acc,raulated. When glutamate was added in place of FCCP considerable amounts of this amino acid were metabolised and aspartate and alanine were released. These data confirm the findings of LaNoue and coworkers [18-21] that, under energized conditions, aspartate exit from the mit~hondria does not occur without concomitant glutamate entry (even in the presence of ammonia ions). However, in the de-energized state both aspartate and gluta ~;;:te can pass ~hrough the inner mitochondrial membrane. These observations on isolated mitochondri,, :~ con. junction with the whole cell studies allow the formulation of a relatively simple scheme to account for the action of ammonium ions on lactate uptake (Fig. 3). It is based on the assumption that in rat liver there are only two major shuttles for the transfer of reducing equivalents into the mitoehondria, one utilising aglycerophosphate and the other, malate, as the hydrogen cartier. Our evidence suggests that the stimulation of lactate uptake by ammonium ions does not utilise the a-glycerophosphate shuttle, but neither does it involve the energy-dependent, butylmalonate-sensitive malate-aspartate shuttle associated with gluconeogenesis from lactate. Rather, we postulate that ammo-
I !='
1' a-I
223
© 1992ElsevierSciencePublishersB.V. All rights reserved0167-4889/92/$05.00
BBAMCR 13229
Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes M i c h a e l N. Berry, J o h n W. Phillips, R o l a n d B. G r e g o r y , A n t h o n y R. G r i v d l a n d Patricia G. W a l l a c e Departmentof MedicalBiochemistry,Schoolof Medicine,Hinders Unirersityof South Australia, Adelaide, Sot,thAustralia (Australia)
(ReceivedZ'~December1991) (Revisedmanuscriptreceived28 April 1992) Keywords: Isolatedhepalocyle;Malale-asparlateshuttle;Lactate metabolism;Reducing-equivalenttransfer. Energytran~uclion The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in Lsolatedhepatocytes of faste.'] rats. 6oth lactate removal and glucose synthesis were inh~ited by butylmalonate, aminooxyacetate or cycloserine ~nfirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butyimalonate was considerably reduced, yet the trans;er of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for bulylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulator/effect on uptake of sorbitol, a substrate whose oxidation principally involves the ~x-glycetophosphateshuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potenti',d (Zl~) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate ox!4ation was strongly inhibited when AV' fell from 130 to 105 mV whereas O 2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by A~. In the presence of added ammonium ions the removal of lactate was much less impaired by walinomycin,suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochonddal matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the bulyimalonate-sensitive, energy-dependent, malate-asparlate shuttle. Introduction "Ilze initial step of hepatic lactate metabolism generates NADH, that must be converted back to NAD + for further flux to proceed. This oxidation r~ay take place by means of an entirely cytoplasmic-coupled reaction associated with gluconeogenesis, in which the conversion of lactate to pyruvate is linked to reduction of 1,3-diphospboglycerate to glyceraldehyde 3-phosphate. There is also a mitochondriai route available for cytoplasmic NADH oxidation, which is indirect because the mitorbondrial inner membrane is almost impermeable to NADH [1-3]. This indirect pathway involves reducing-equivalent carder s)'stems, such as the malate-
Correspondenceto: M.N. Berry, Depatlmenl of Medkal Biochemistry, School oi Medicine, The Binders IJabe~ity ol" South Australia" G.P.O. Fox 2100)Adelaide,South Australia,Australia,5001.
aspartate [4,5] or a-glycerophosphate shuttles [6,7]. The aln,ost complete inhibition of lactate oxidation by the NADH dehydrogenase inhibitor, totenone [8], indicates that the transfer of reducing equivalents from cytoplasm to mitochondria must involve one or more NKO.dependent shuttles. "Early studies on hepatic gluconeogenesis from lactate assumed that the cytoplasmic couple catalysed by glyceraldehyde 3-phosphate and lactate dehydrogenases was the predominant mechanism for NADH oxidation [9]. in conjunction with this, it was postulated that the carbon skeletons for gluconeogenesis are provided by transamination of oxalacetate to aspartate that then leaves the mitochondria [10-12], a balance of nitrogen being maintained by an associated entry of glutamate and exit of a-ketoglutarate, These early studies also demonstrated, however, that hepetic gluconeogenesis from lactate is depressed not only by inhibitors of transamination [13,I4], but is al~ strongly
224 inh~ited both by butylmalonate [15,16] and by fluoromalate [17], agents that respectively inhibit malate transport and metabolism. Hence there is good evidence that malate transport into the mitochondfia takes place during hepatic lactate metabolism, the most likely mechanism being a malate-aspartate carrier-raediated exchange. In studies with isolated mitochondnal preparations LaNoue and co-workers [18-21] found that the entry of aspartate into mitochondria, in exchange for glutamate, is very slow and does not occur in energized mitochondria, thereby accounting for the unidirectional nature of the malate-aspartat¢ shuttle. They demonstrated that exchange in the opposite direction, i.e. entry of glutamate and eff!ux of aspartate, was stimulated by coupled respiration, but inhibited by uncouplers and valinomycin. Because effiux was not affected by nigericin [22,23] they concluded that transport in the physiological direction of aspartate efflux was promoted by the electrical potential gradient across the inner mitoehondrial membrane (at/'). Since it is now feasible to measure ag" in intact cells [24-26], we have examined the effects of mitochondrial energy"and redox states on lactate utilization by isolated heparacytes. In this paper we show that reducing equivalents generated in the cytoplasm from the oxidation of lactate can be transported to the mitochondria not only by an energy-dependent mechanism involving 1he malateaspartate shuttle but also, when ammonium ions are present, by an. energy-independent process that does not utilize this shuttle. Materials and Methods Collagenase and enzymes for metabolite determination were from Boehringer M.':anheim (Germany) as
was bovine serum albumin (fraction V) which was defatted by the method of Chen [27]. Palmitate (Sigma, USA) was neutralized, and dissolved in an ~;otonic sail solution containing 9% defatted bovine ser~Jmalbumin. Vaiinomycin, p-trifluoromethox)'phenylhydrazone (FCCP), hutylmalonate, aminooxyacetic acid and quinolinic acid were also obtained from Sigma (USA). Pcrfluorosuccinic acid was from Pfaltz and Bauer (CT., USA) and (S)-(-bcycloserine was from Aldrich (Wi, USA). All other chemicals were of the highest quality available. L[U-taC|lactate, [t4C]methyltriphenylphosphonium iodide (TPMP +) and the scintillation cocktail ACSll were all obtained from Amersham (Australia). Isolated liver cells from male Hooded Wistar rats (250-280 g body wtL starved for 24 hours to deplete liver glycogen, were prepared by a modification [28] of the method of Berry and Friend [29], in which calcium ions were included in the washing media. The cells (90-120 mg wet wt) were incubated at 3T'C in 2 ml of a balanced bicarbonate-buffered medium [30,31] containing aibumin, 2.25% (w/v), under a gas phase of 95% O,, 5% CO:. Lactate and pymvate were normally present in incubations at initial concentrations of 10 mM and 1 mM, respectively. This level of lactate ensured saturating concentrations were maintained throughout the experiment, whilst addition of pyruvate established a normal lactate/pymvate steady-state ratio from the commencement of the incubation period. Consumption of O2 was measured in the presence of CO: by a manometric method [32]. The water-insoluble inhibitors (va!inomycin and FCCP) were dissolved in acetone. The solution was added to the empty incubation vessels and allowed to evaporate to dryness. The inhibitors were then re-solubilized by the additior, of medium containing the bovine serum albumin and other incubation components. A~ the completitm of the
TABLE I Reducing equivalentsgeneratedfrom lactate oxidation
Hepatocytesfromfastedrats wer~incubatedfor 30 min as describedin Materialsand Methodswith the followingadditions:lactate, 10rnM; plmtvate, I raM: palmitate, 2 mM: butylmalonate.1OmM: aminooxyacelate,1O0/,M: cycloserine, 5 mM. The data are presentedas the mean± S.E. of at least 12separateexperiments.Reducingequivalentaccumulationin glucosewas determinedas 2x glucosewiththe estimateof the minimumquantityof reducingequivalentstransferredto the mitochondriaas (lactate -2xgluoosc). Addition
Lactate Lactate,palmitate Lactate,butylmalonate Lactate,palmitaze,butylmalonate Lactate,palmitate,aminooxyacetate Lactate,palmitate,cycloserine
Metabolicchanges(/~mol/30 rainper 100mgwetwt) reducing lactate glucose equivalents accumulatedin glucose - 4.53+_I].09 - 8.01± 0.12 - 2.46+ 0.15 - 4.56± 0.18 --2.464-_0.12 - 3.18__0.27
1.71± 0.06 4.05± C.05 0.93_-.I0.03 1.95+ 0.09 1.53.~.0.06 1.29± 0.04
3.90
minimumquard;y of reducing equivalents transferre~to mitoch~ndria 1.11 -0.09 0.60 0.66
3.06
-0.60
3.42 8.10 1.86 2.58
O.6O
225
NN
.~+~
i~ ~
~
• ~
~
~m
-
~.N N
N
-+~ --NN l l l i l | l
_~'~
~
.~.~
I.°
N~g~N~Nm
+r -i--¢
N ~
,
m~
.-i-i
.
+i
¢~.
°
=~-.-~+~'~ I
I
I
I
NN
~'~ F I I | I I I
•.~ ~
'
-~ ~ .
..
e~
N
l
~
,
II
N
N
N
~
t
~
,-.
~
,,Tr,,,
N
,-1
I
~
~
N
~
~ g g g g g g g
226 incubation period (30 rain), samples were deprote':nized with an equal volume of cold 1 M perchloric acid and neutralized. Metabolites were measured by standard enzymatic techniques [33] using a Cobas FARA automated analyser (Roche Diagnostics, BaseD, the data being transferred to a PDP 11/73 computer (D.E.C., USA) for subsequent processing. Where the mitochondrial inner membrane electrica| potential (A~) was measured, 0.4 ml samples of the incubation mixture were removed prior to acidification and processed as previously described [26]. For the measurement of CO 2 production, hepatocytes were incubated in the presence of either 0.2 gCi [U14C]lactate or [l-t4C]palmitate in vessels sealed with a serum cap from which hung a plastic well holding a filter paper wick. At the end of the incubation, 0.25 ml of 2-phenylethylamine was injected through the seal and the vessel acidified with 0.5 ml of 4 M perchloric acid. The vessel was kept on ice for a further 60 rain before the plastic well, containing the trapped CO 2, was transferred to 10 ml of ACS II scintillation fluid. ,,,o~,~u~"t~'~mitochondria a were prepared in 0.25 M sucrose, 5 mM N-[2-hydroxyet.hyl]piperazine-N'*[2e~hanesulphonic acid], pH 7A according to Ref. 34 and incubated as described by Strzelecki et al. [35]. Results and Discussion
IncolL'ement of malate transport in lactate oxidation Determination of the minimum quantity of reducing equivalents generated by lactate oxidation that pass to the mitocbondria can be achieved by a simple carbon balm,.ce study, based on the assumption that all the reducing equivalents, required for the reduction of the 1,3-diphosphoglycerate, are derived from lactate, whether or not palmitate is present (Table I). In the presence of palmitate, the conversion of lactate to glucose is stoichiometric so that by this mode of calculation, which assumes no reducing equivalents are transfei~ed from mitochondria to cytoplasm, the flow of reducing equivalents into the mitochondria is calculated as nil. However, in the presence of butylmalone.re, net lactate utilization and glucose production could be inhibited about 50%, whether or not lactate conversion to glucose was stimulated in the presence of palmitate. From this observation (Table l), it can be inferred that malate transport between cytoplasm and mitochondda plays a substantial role in lactate oxidation. We also noted that the presence of transaminase inhibitors such as aminooxyacetate or cycloserinc, resulted in a 60-70% inhibition of net lactate utilization and glucose synthesis (Table 1). This degree of inhibition by these agents is in keeping with conclusions that the bulk of the oxalacetate, formed by ~ruvate carboxylation within the mitochondria during gluconeogenesis from lactate, is transported to the
cytoplasm as aspartate. These results support previous work [13-17] suggesting that the malate-aspartate shuttle is the major system involved in transporting to the mitochondria reducing equivalents arising from hepatic lactate oxidation.
Effects of ammonium ions or polmitate on lactate oxidation The presence of ammmlinm ions stimulated both lactate oxidation and 0 2 consumption (Table II). It in inferred that some of the extra lactate consumed was oxidized in the Krebs Cycle, an inference supported by the observation that 14CO2 production from [U14C][actate was increased from 0.81 _+0.03 to 2.54 _+ 0.10 #mol/min per g wet wt (n = 4) in the presence of anunonium ions, while some lactate carbon accumulated in amino adds. Butylmalonate caused only a small reduction in lactate utilization, indicating that, in the presence of ammonium ions, the energy-dependent malate-aspartate shuttle, which can be expected to be strongly inhibited by hutylrn~! . . . . . ut'*., t[1¢ .......... t . a , l1~' UJ1 , m:~tkes a substantially lesser contn'bution to lactate oxidation than when ammonium ions are absent. The diminution in lactate utilization due to butylmalonate addition, both in the presence aad absence of ammonium ions, was accounted for by a corresponding decrease in glucose formation. The stimulatory actions of palmitate and ammonium ions on lactate utilization were not additive (Table II). Palmitate stimulated ammonium ion removal, presttmably by providing additional NADH for the reductive formation of glutamate, and brought about a substantial accumulation of lactate carbon in alanine, aspartate and glutamate, but reduced the rate of gluconeogenesis to a level below that of the control with lactate alone. Similar observations have been reported by Krebs et al. [36]. Since the accumulation of amino acids indicates that palmitate did not suppress the conversion of lactate to pyruvate or oxalacetate, the depression of gluconeogenesis in the presence of palmitate and ammonium ions was apparently the consequence of the diversion of carbon and reducing equivalents from glucose synthesis towards amino acid accumulation. Carbon balance measurements also indicate that palmitate overcame, to a large extent, the stimulator/ effect of ammonium ions on oxidation of lactate via th~ Krebs Cycle (decreasing i4CO2 formation from [U~4C]lactate from 2.54 + 0.10 to 0.91 + 0.04 itmol/min per g wet wt (n = 4)), reflecting the inhibitory effect of fatty acid on the conversion of pyruvate to acetyl CoA [37,38], Thus, the failure of ammonium ions and palmitale to bring about additive effects on lactate removal can be attributed to the abiJity of palmitate to diminish lactate oxidation in the Kxebs Cycle, and the depressive action of ammonium ions on gluconeogenesis from lactate in the presence of palmitate.
227 Urea formation was greatly enhanced by the addition of ammonium ions both in the absence and presence of palmitate, however, butylmalonate had no significant effect on this rate (Table !1). The relative insensitivity of lactate removal and urea formation to b,atylmalonate in the presence of ammonium ions suggests that the oxidation of cytoplasmic malate formed from the fumarate derived from arginosuccinate cleavage is not impaired by the inhibitor. Indeed, this is confirmed by the failure of malate to accumulate in the presence of botylmalonate under these circumstances (data not shown). Reducing equivalents produced in the cytosol in excess of the requirements for gluconeogenesis must re-enter the mitochondria for oxidation. An estimate of the reducing-equivalent transport [14] can be determined in a manner analogous to the calculation performed in Table I on the assumption that each tool of urea accumulating represents the generation in the cytosol of one mot of reducing equivalents as malate (Table II). Clearly under conditions where ureogenesis was stimulated there was an increased flux of reducing equivalents into the mitochondria, with butylmalonate having little to no effect on this flux. Hence, there ap~ars to be an alternative butylmalonate-insensitivemechanism for the disposal of the malate generated in the cytoplasm that operates during synthesis of urea from ammonium ions.
Energy ~
of malate-aspartote shuttle
If an energy-dependent shuttle is involvedin hepatic lactate metabolism, it can be anticipated that lactate removal will be depressed by agents that impair energy metabolism and reduce A~/'. Valinomycinis a particularly potent agent in this regard [24,26] and proved a more useful tool than the ,mcoupling agent, FCCP, which, at concentrations strongly inhibitory to lactate uptake brought about an unacceptably high increase in the number of cells taking up trypan blue. Valinomycin causes swelling of isolated mit~hondria [39], which can lead to difficulties in interpretation of results; but significant swellingdoes not occur in the mitochondria of isolated hepatocytes [26]. Accordingly, we titrated hepatocytes with valinomycinin the presence of lactate alone, or in combination with palmitate. When lactate was the only added substrate, low concentrations of valinomycinstimulated respiration and lactate utilization (Fig. la), the 14CO2production from [t4C]lactate increasing from 0.81 + 0.04 to 1.65+ 0.07 ttmol/min per g wet wt (n -4), indicating that the extra lactate was oxidized in the Krebs Cycle. Higher concentrations of the ionophore brought about a small decline in respiration and a substantial fall in lactate consumption (Fig. la). Even at 75 nM valinomycin,the cellular concentration of ATP was greater than 0.6/tmol/g wet wt, a level at which, in our experience, cellular integrity is maintained. Nevertheless, to exclude the
3
i
6
v
0
0
20
40
60
80
15
!00
b
,4
12
"
i' ~
6 t
3
20
40
60
80
100
Fig. 1. Effect of vatinomycin on the rates of o~8en consumption and lactate removal by hepatocytes. Isolated liver cells were prepared as d~seribed in Mate~als and Methods and incubated wi',h either:. (a) 10 mM lactate or, (b) 10 mM lactate plus 2 mM palmilate in the presence of a graded concentration range of valinomycin (0-100 nM). In both (a) and (b) the closed symbols (11, e) represent the rate of orygen consumption, #o and the open symbols (t3. o) the rate of lactate removal, ]I~1c- The data presented are for a single ext~dmeat and are representative of all five experimem5 performed.
possibility that the inhibition of lactate removal was due merely to cell damage or death induced by high concentrations of valinomycin, the effects of the ionophore on lactate metabolism in the presence of palmitate were examined (Fig. lb). Extremely high rates of respiration were achieved under these conditions, with A~P falling and 02 uptake increasing for each increment in valinomycinconcentration up to 30 nM and then remaining steady. The rate of lactate oxidation by hepatocytes rose gradually as Aq' decreased from 170 to 130 mV, and then fell sharply, maximuminhibition being observed at levelsof 105 mV (Fig. 2a), when the rate of cellular 0 2 consumptionwas almost twice the level in the absence of ionophore. Hence the inability of the cells to take up lactate was not attributable to damage leading to a non-specific failure of electron transport. A small inhibition of lactate uptake associated with a concomitant 20% stimulation of 0 2 consumption was also brought about by low concentrations of FCCP (data not shown). The observation that hepatocs'tes metabolized l~ruvate rapidly in the presence of the same concentration of valinomycin and level of d~' that inhibited lactate
228
a
i o
80
100 120 140 160 180
7
b
~6
~5 :~t. 4
i3 I
0
80
100 120 140 160 zeo A't{~)
Fig. 2. Relationship bern'curt the rates of lactate and pymvate remowal and the nfitochoadrial membrane potential (A~). Isolated he~too~es were incubated as descn'bed in Materials and Methods with the addition of either. (a) 10 raM lactate plus 2 mM palmitale or;, (b) 15 mM pymvate in the presence of a graded concentration gauge of valieomy~ (0-100 nM). The data presented arc from a single experiment and are rCl~CScntath'c of the three experiments performed.
utilization (Fig. 2b) suggests that the energy-dependent step of lactate removal is the initial oxidation to pyrurate. Moreover. addition of the artificial electron cartier methylene blue, which bypasses Site i ef the dectron transport chain, overcame the inh~itory effect of valinomycin on lactate removal (data not shown). These
studies confirm that the energy-dependence of the malate-aspartate shuttle, first demonstrated with isolated mitochondria by LaNoue and co-workers [18-22], can be observed with an intact cell preparation, and support their conclusions that operation of the shuRle is driven by A~. In the presence of ammonium ions the utilization of lactate w~s much less impaired by high concentrations of valinomycin (Table liD. Indeed, the transport of reducing equivalents into the mitochondria was only slighdy depressed under these conditions (Table ÁÁÁ). Thus, it appears that ammonium ions promoted an energy-independent oxidation of lactate, since they were able to stimulate its uptake at the same depressed lceels of cellular A_TP ~-nd d V associated with inh$ition of lactate oxidation by valinomycin in the absence of ammonium ions (data not shown). High concentrations of valinomycin inh~ited gluconeogenesis but did not depress amino acid accumulation, a process that also requires lactate o~dation to pymvate, Hence, lactate removal in the presence of ammoniur~ ions and valhzomycin must have involved the passage of reducing equivalents into the mitochondria. Similar findings were obtained when bepatocytes from h~pothyroid rats were employed (IL Gregory, unpublished observations), discounting the likelihood that zhe a-glycerophosphate shuttle is involved in this reducing-equivalent transfer since the a-glycerophosphate dehydrogeaase component of this shuttle has a very low activit3, in the hypothyroid state [40]. Moreover, the oxidation of sorbitol, a substrate which feeds reducing ~uivalents into the a-glycerophosphate shuttle [41] was not stimulated by ammonium ions (data not shown). Butyimalonate (Table II) inhibited lactate conversion to glucose but, like valinomycin, in the presence of ammonium ions caused minimal depression of amino acid formation from lactate. Taken together, these observations lead to the conclusion that, in the presence of ammonium
TABLE !!! Effect of t'a~inoatyc~ and ammordum ions on lactate r a e t ~
Hepaloc~es from fasted raLswere incubated for 30 min with.subs:rate additions as in Table IL Valinom3x-inseaspresent at 75 nM. Resultsare presentcd as the mean-+S~E, of at hast eight separate experiments. B/A =[~-lffdrm3~tyratel/[acetoacoate]. The minimum transfer of reducing equiwatentsfrom c~osol to mitochondria is estimated as descn'bedin Table It. Addition
Metabolicchanges(/zmol/30 rain per 100mg wet wl) lactate glucose urea alanine glutamate aspartate
B/A minimqm
traaslam of reducing equivalentsto mitochondria Lactatc,~imitatc Lactate.palmitate.va~cin Lactate, palmitate,ammonia Lactate, palmitate,ammonia, valinomy~n
-8.01_+0.12 4.05_+0.06 0.12_+0.01 0.164-0.01 0.06_+0.01 0 -1.26_+0.09 0.75_+0.05 0.14_+0.01 0.17+0.01 OJ]O 0 -7.17_+0.17 0.90_+0.02 1.35_+0.05 1.79_+0.06 0.46+0.02 3.26+_0.I0 6.70
1.22+-0.03 0.27+0,01 0.29+0.02
-6.45+_0.21 0.574-0.05 0.90+_0.04 2.79+_0.15 I.(M__.0.04 1.62-+0.09 6.21
0.19+0.01
229 ions, the transfer of lactate hydrogen to the mitochondria is accomplished by an NAD-linked mechanism that is clearly distinct from the butylmalonatesensitive, energy-dependent, malate-aspartate shuttle associated with gluconeogenesis from lactate in the absence of ammonium ions. To gain further insight into the nature of the transfer mechanism, Lsolated mitocbondria were incubated under similar conditions to those used by [aNoue and coworket,s [18-22] (for details of a representative experiment, see Table IV) except that ammonium ions (4.8 raM) were included. A time course was run, during which accamulation of intermediates '#as measured. Under control ~ndigio~s virtually no aspartate, glutamate or alanine was released into the incubation medium. However, when 0.8 pM FCCP was included, reducing ATP levels from 0.7 mM to 10 p.M, substantial amounts of the amino acids acc,raulated. When glutamate was added in place of FCCP considerable amounts of this amino acid were metabolised and aspartate and alanine were released. These data confirm the findings of LaNoue and coworkers [18-21] that, under energized conditions, aspartate exit from the mit~hondria does not occur without concomitant glutamate entry (even in the presence of ammonia ions). However, in the de-energized state both aspartate and gluta ~;;:te can pass ~hrough the inner mitochondrial membrane. These observations on isolated mitochondri,, :~ con. junction with the whole cell studies allow the formulation of a relatively simple scheme to account for the action of ammonium ions on lactate uptake (Fig. 3). It is based on the assumption that in rat liver there are only two major shuttles for the transfer of reducing equivalents into the mitoehondria, one utilising aglycerophosphate and the other, malate, as the hydrogen cartier. Our evidence suggests that the stimulation of lactate uptake by ammonium ions does not utilise the a-glycerophosphate shuttle, but neither does it involve the energy-dependent, butylmalonate-sensitive malate-aspartate shuttle associated with gluconeogenesis from lactate. Rather, we postulate that ammo-
I !='
1' a-I
