Alcoholic Fermentation in Multicellular Organisms
Author(s): Aren van Waarde
Source: Physiological Zoology, Vol. 64, No. 4 (Jul. - Aug., 1991), pp. 895-920
Published by: The University of Chicago Press
Stable URL: http://www.jstor.org/stable/30157948
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895
InvitedPerspectivesin PhysiologicalZoology
inMulticellular
Fermentation
Alcoholic
Organisms
Aren
vanWaarde*
Departmentof Biology (AnimalPhysiology), GorlaeusLaboratories,
Leiden University,P.O. Box 9502, 2300RALeiden, The Netherlands
Accepted 11/27/90
Abstract
1. Ethanol is an end product of anaerobic metabolism in a surprisingly large variety of multicellular organisms. These include Angiosperms, Platyhelminthes, Aschelminthes, Acanthocephala, Arthropoda, and Vertebrata. 2. Ethanol formation
proceeds in two steps (via pyruvate decarboxylase and alcohol dehydrogenase).
3. Pyruvate decarboxylase of plants is located in the cytosol, whereas that of animals is intramitochondrial and probably part of the pyruvate dehydrogenase
complex. 4. Alcohol dehydrogenases offacultative anaerobes are NAD- rather
than NADP-dependent, with the enzyme in the parasitic worm Moliniformis dubius as the only known exception. 5. Regulation at the pyruvate branch point in
plants seems to involve three different mechanisms: (i) increases ofpyruvate decarboxylase and alcohol dehydrogenase activity due to increased gene expression, (ii) control ofpyruvate decarboxylase by the intracellular pH and the cytoplasmic NADH/NAD+ ratio, and (iii) inhibition of lactate dehydrogenase by ATP
at low pH values. There is no evidence for increased expression of the pyruvate
decarboxylase and alcohol dehydrogenase genes of animals during anoxia. Here,
pyruvate decarboxylase is controlled by the intracellularpH and the mitochondrialphosphorylation potential, whereas the ethanol/lactate production ratio is
dependent on the magnitude of the glycolytic flux. 6. The ethanol route has significant advantages (minimal acidosis, avoidance of osmotic problems, and endproduct inhibition of the glycolytic chain) but also has disadvantages (relatively
low energy yield, loss of carbobydrate carbon). This may explain why only a few
animals produce ethanol during anoxia, in contrast to plants, which generally
respond to reduced 02 availability by alcoholic fermentation.
Introduction
Sincethedaysof Pasteur,it hasbeen commonknowledgethatyeastsproduce
ethanol under anoxic conditions. However, few people are awareof the fact
thatmanyhigherorganismsfollowa similarstrategyforanoxicsurvival.
* Present
address:
Division
ofCardiology,
Thorax
ofGroningen,
P.O.Box
Center,
Academical
Hospital,
University
9700RB
TheNetherlands.
30.001,
Groningen,
1991.1 1991byTheUniversity
ofChicago.
Physiological
Zoology
64(4):895-920.
Allrights
reserved.
0031-935X/91/6404-9080$02.00
896 A.vanWaarde
The presentarticlegives an overviewof recent dataon ethanol production
during facultativeanaerobiosis.The subject matteris divided into four different topics: (i) Whichspecies excrete ethanol and what is the quantitative
importanceof ethanol as an anaerobicend product?(ii) By what mechanism
is ethanol produced (enzymatic reactions, subcellular distribution)? (iii)
How is metabolism at the pyruvatebranchpoint controlled? (Since at least
two enzymes, lactate dehydrogenase and pyruvatedecarboxylase,compete
for a common substrate,some form of control seems necessary.) (iv) What
are the advantagesand disadvantagesof the ethanol route? Consideration
of these may help to explain why alcoholic fermentation is ubiquitous in
plants but relativelyrareamong animals.
of theEthanol
Distribution
Pathway
Plants. Angiosperms may experience conditions of limited oxygen availability during all phases of their life cycle. Manyseeds have a testa which
is hardlypermeable to oxygen (Crawford1977, 1978; Cossins 1978). The
developing embryo is therefore subjected to a period of anoxia between
imbibition and the emergence of the radicle. This period is characterized
by relatively high activities of glycolytic enzymes and by accumulation of
ethanol. Leguminousplants like chick peas (Cicer arietinum;Aldasoroand
Nicolas 1980), green peas (Pisum sativum; Cossins et al. 1968; Koll6ffel
1968;Suzukiand Kyuwa1972;Leblova,Sinecka,andVanickova1974;Leblova
1978a, 1978b), beans (Phaseolus vulgaris;Doireau 1971; Leblova 1978b),
soybeans (Glycine max; Leblova1978b), or lentils (Lens esculenta; Leblova
1978a, 1978b), and grains like barley (Hordeum vulgare; Duffus 1968),
maize (Zea mays;Leblova1978b), and rye (Secale cereale; Leblova1978a)
produceethanol duringearlydevelopment.Afterthe testahas been ruptured,
oxygen can enter and the seedling switches to aerobic respiration.
Species that grow in marshlandsoften have to germinate under water.
Especiallyin a hot climate,dissolved oxygen concentrationsnearthe bottom
can be very low. Rice (Oryza sativa; Taylor 1942; Avadhaniet al. 1978;
Bertani,Brambilla,and Menegus 1980; Cobb and Kennedy 1987), wild rice
(Zizania aquatica; Campiranonand Koukkari1977), and barnyardgrass
(Echinochloa crus-galli; Rumpho and Kennedy 1981, 1983; Cobb and Ken-
nedy 1987) areplantsthatdo germinatein the absence of oxygen and emerge
from deep water.Ethanolis the main metabolicend productin these species
under anoxic conditions (Taylor 1942; Phillips 1947; App and Meiss 1958;
John and Greenway 1976; Campiranonand Koukkari1977; Avadhaniet al.
1978; Bertaniet al. 1980; Cobb and Kennedy 1987). During anaerobicgermination, shoots are formed but root development is suppressed. As soon
Alcoholic
Fermentation
inMulticellular
897
Organisms
as the shoot reaches an aerobic zone, oxygen is transporteddownwardand
root growth starts (Cobb and Kennedy 1987).
The meristematic zone of roots (i.e., the apex of the root that is very
activelygrowing) seems to experience a permanentfunctionalanaerobiosis.
Even under well-aerated conditions, the supply of oxygen to this tissue is
less than its oxygen demand (Crawford1978). Consequently, part of its
energy is produced by anaerobicpathways.Pea root tips contain high concentrations of ethanol even in an aerobic environment (Ramshorn 1957;
Betz 1958; Cossins 1978). Oxygen diffusion into ripening fruitsalso cannot
keep pace with oxygen demand.Hypoxicand hypercapnicconditionsprevail
in the deep tissues of apples, pears, and bananas,which may sometimes
lead to accumulationof ethanol (Zemlianukhinand Ivanov 1978).
The roots of manyplants endure environmentaloxygen deficiency during
flooding, which can be a seasonallyoccurringphenomenon. The watertable
tends to rise duringwinter,and in springit takestime to returnto its summer
level. At the beginning of the growthseason, the deeper-lying roots of trees
may thus be temporarilydeprived of oxygen (Crawford1978). Rhizomes
of marshplants, like the sea rush (Scirpus maritimus), bulrush (Schoenoplectus lacustris), reed-mace (Typha angustifolia), and reed sweet-grass
(Glyceria maxima) are adaptedto overwinteringin anaerobic mud (Crawford 1982; Monkand Brindle 1982). Ethanolis usually the majormetabolic
end productof rootsin flooded soils (Laing1940;Crawford1967;Wignarajah,
Greenway,and John 1976; Chirkova1978; Smith and ap Rees 1979b;Mendelssohn, McKee, and Patrick1981; Monk and Brindle 1982;Jackson and
Drew 1984), but there are exceptions to this rule. Roots of alders (Alnus
incana) produce glycerol, and those of birch trees (Betula pubescens)
malate upon flooding (Crawford1972). The anaerobic end products are
transportedupwardand excreted (ethanol evaporatesthrough branch lenticels and leaf stomata;Kenefick1962; Fulton and Erickson1964; Chirkova
1978;Hook 1984) or assimilatedinto macromolecules (Cossins and Beevers
1963; Cossins 1978; Crawford1978).
Animals. Those animal species in which ethanol production has been conclusively demonstrated are listed in table 1. It has been suggested that a
Chondrostean fish, the lake sturgeon (Acipenserfulvescens),
is also capable
of ethanol production (Singer, Mahadevappa,and Ballantyne 1990) since
the animals(1) show a considerableresistanceto anoxia, (2) do not develop
an oxygen debt, and (3) possess low levels of lactate dehydrogenase in
their tissues. However, until it has been proven thatalcohol dehydrogenase
is present and ethanol is excreted by anoxic sturgeons, the evidence must
be considered only circumstantial.
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andZebe
900 A.vanWaarde
Few people have searched for the presence of the ethanol pathway in
closely relatedspecies, although such informationmight be very interesting
from a phylogenetic point of view. Wissing (1986) has examined a large
number of fish species to see whether their myotomal muscles possess a
special alcohol dehydrogenase (geared toward ethanol formation). His
(negative) findings and those of other authorsare summarized in the Appendix. At present it is not known whether "nonproducers"thatare closely
relatedto ethanol-producingspecies lackthe gene coding for muscle alcohol
dehydrogenase (ADH) or fail to express this gene. The answerto this question may throw light on the origin of the ethanol route.
of Ethanol
Formation
TheMechanism
Microorganisms
Alcoholic fermentation is well characterized in microorganisms, where
ethanol is produced by two different pathways (Wilps and Sch6ttler 1980;
see fig. 1). Yeasts catabolize pyruvateto acetaldehyde by a cytosolic decarboxylase (PDC). Acetaldehyde is then converted to ethanol by alcohol
dehydrogenase. Redox balance is maintainedbecause the glyceraldehyde3-phosphate dehydrogenase (GAPDH) and ADH reactions are stoichiometrically coupled (Stryer1988). Bacteriaproduce ethanol from pyruvate
by the concertedactionsof pyruvatedehydrogenase(PDH), thiokinase(TK),
aldehyde dehydrogenase (AldDH), and ADH (Doelle 1975; Thauer,Jungermann, and Decker 1977). The metabolic scheme of figure 1 is an oversimplification,for acetyl-CoAcan also be transformedto acetate via acetylphosphate (Doelle 1975; Shoubridge 1980), though this does not affectthe
overall stoichiometry of the pathway.
Pyruvate Decarboxylation in Multicellular Organisms
The mechanism of ethanol formationin plants is very similarto thatof yeast
cells. Ethanol is formed by PDC and ADH, which are both soluble and
located in the cytoplasm (Davies, Grego, and Kenworthy1974; Laszloand
St.Lawrence1983). Mitochondriaare not requiredfor alcoholic fermentation
of glucose; thereforethe process can be studied in a high-speed supernatant
(Davies et al. 1974).
In contrast,conversion of pyruvateto acetaldehyde is intramitochondrial
in all ethanol-producing animals that have been examined (Wilps and
Schdttler 1980; Mouriket al. 1982; Barrettand Butterworth1984). Acetaldehyde diffuses to the cytoplasm and is there reduced to ethanol. The fer-
Alcoholic
Fermentation
inMulticellular
901
Organisms
Glucose
Pyruvate
NAD
coA
PDC
NADH-------PDH
p
Acetaldehyde
Acetyl-coA
NADH
NAD
Ethanol
TK
--------
ADP
ATP
coA
Acetate
AldDH
NADH-NAD
Acetaldehyde
NADH
ADH ADH
NAD
Ethanol
Fig. 1. Metabolic pathways for ethanol formation in yeasts (left) and bacteria (right). See the text for an explanation.
mentationprocess involves cooperationof cytosol and mitochondria,unlike
the situation in yeast.
In theory, mitochondrial decarboxylationof pyruvatecan take place in
one step or be a multistep process involving the concerted actions of PDH,
TK,and AldDH (see fig. 1). The latterroute was at firstfavored (Shoubridge
1980; Shoubridge and Hochachka 1981) because of an extra mole of ATP
obtainable at the thiokinase step and the following lines of evidence: (1)
injected 14C-acetateis rapidlyoxidized to 14C02 by normoxic goldfish, so a
thiokinase must be present; and (2) anoxic fish produce 14C-ethanolfrom
injected 14C-acetate.
Subsequently, it has become clear that the thiokinase route is relatively
unimportant.(a) In the step leading from acetyl-CoAto acetate, acetyl-CoA
hydrolase (EC 3.1.2.1) could well outcompete acetyl-CoAsynthetase (EC
902 A.vanWaarde
6.2.1.1, a thiokinase); in such an event, there would be no energetic advantage to ethanol formation.(b) Althoughacetyl CoA-synthetaseis kinetically
and thermodynamicallyreadilyreversible,aerobic operationof a thiokinase
in the direction of acetyl-CoAis no proof of its anaerobic operation in the
opposite direction. (c) Although"4C-ethanolcan be producedfrom injected
14C-acetateunder anoxic conditions, 14C-lactateis a much better precursor
(Shoubridge 1980; Shoubridge and Hochachka 1981). This suggests that
acetate is not an obligate intermediate in the pathway between pyruvate
and ethanol. (d) The enzyme aldehyde dehydrogenase is virtuallyabsent
in Carassiusmuscle (Nilsson 1988, 1990) and in mitochondriafrom Chironomus larvae (Wilps and Sch6ttler 1980). Acetate can therefore not be
hydrogenated in situ. (e) Permeabilized mitochondriaof Chironomusdo
not convert acetyl-CoAto ethanol (Wilps and Sch6ttler 1980). In a reconstituted system containing intact mitochondria,ADH, NADH,and pyruvate,
arsenite inhibits acetate formation, but ethanol production is unaffected
(Wilps and Sch6ttler 1980). This proves that acetate and ethanol are synthesized by differentpathways. (f) The reactions leading from acetate to
ethanol are thermodynamicallyuphill. Net acetate-to-ethanolconversion
thereforewould only be expected at very high levels of acetate or very low
levels of ethanol. Since acetate concentrationsin the muscle of anoxic goldfish are low and independent of oxygen availability(van den Thillart,Kesbeke, and van Waarde1976), acetate is probablynot the obligate precursor
of ethanol. (g) Barrettand Butterworth(1984) have partiallypurified the
mitochondrial pyruvatedecarboxylase of Panagrellus redivivus. Pyruvate
decarboxylaseactivityis localized in the mitochondrialmembranefraction,
but it can be solubilized by sonication or freeze/thawing and purified by
isoelectric precipitation at pH 5.5. Since PDC and PDH show the same
subcellular distributionand a constantactivityratio during purification,the
authors conclude that PDC is a single enzyme and probablya part of the
PDH complex.
For the reasons stated above, it appears that anaerobic decarboxylation
of pyruvateby animal mitochondriais a one-step reaction. However, elucidation of the precise biochemical mechanism of acetaldehyde formation
seems in order. It would be very interesting to isolate pyruvatedehydrogenase from red muscle of crucian carp or goldfish. Does the protein that
convertspyruvateto acetaldehyde copurifywith the PDH complex? Is PDH
of ethanol-producingfishdifferentfromthe enzyme in closely relatedspecies
that do not produce ethanol? In case such purificationmay prove difficult
or impossible, intactanimalsor isolated mitochondriacould be treatedwith
activators(dichloroacetate,lipoic acid) or inhibitors (arsenite) of pyruvate
dehydrogenaseto study their effects on pyruvatemetabolismunder aerobic
and anoxic conditions.
inMulticellular
Fermentation
903
Alcoholic
Organisms
If the pyruvatedecarboxylaseof ethanol-producinganimals is indeed part
of the pyruvatedehydrogenasecomplex, as proposedby Mouriket al. (1982)
and suggested by the experimental evidence of Barrettand Butterworth
(1984), acetaldehyde formationmay proceed by the mechanism illustrated
in figure 2. Reversible dissociation of bound enzymes underlies the metabolic depression thatoccurs in manyanimalsduringfacultativeanaerobiosis
E3-FAD
H
+,C-S
---. .
NADH,H- 6
NAD
,-- R-I-S'C-C--E1
CH3
E3-FADH2
H3C-C-COOH
S-
1
E2
-- S-S
5
COOH
E3FAD,'-'
H3C-C-OH
SH SH
CRC
C1-E1
0
CH3
H3C-C-ScoA
002
2
coASH
C2
H
S
H3C-C- OH
E2
R NC-S2
3
E
SH
CSOH
C-CH3
11
I1
CH3
H
H3C-CX-H
6C-S
,CCC--
E1
C H3
Fig. 2. Hypothetical mechanism of acetaldehyde formation by the mitochondrial pyruvate dehydrogenase complex. During normoxia, tight
coupling ofpyruvate decarboxylase (E,), dihydrolipoyltransacetylase
(E2), and dihydrolipoyldehydrogenase (E3) causes reactions 3-6 toproceed. Anoxia induces dissociation of the complex, enabling independent
operation ofpyruvate decarboxylase. Reactions 1 and 2 are now followed by reaction 7, and decarboxylation ofpyruvate leads to the release of acetaldehbyde.
904 A.vanWaarde
(Plaxtonand Storey 1986; Guderley,Jean, and Blouin 1989; Lazou,Michaelidis, and Beis 1989). Pyruvatedecarboxylase is located on the outside of
the pyruvatedehydrogenase complex (Lehninger 1970), and it may be released in ethanol-producing species during anoxia, so that pyruvatedecarboxylationbecomes independent of the operation of the electron transport chain.
Alcohol Dehydrogenase in Multicellular Organisms
According to Chirkova(1978), ADH from the roots of a flooding-resistant
plant (the willow Salix alba) is specially adapted for ethanol oxidation,
whereas ADHfroma flooding-intolerantplant (the poplar,Populuspetrowskiana) has ideal properties for acetaldehyde reduction. It is doubtful
whether these findings can be generalized. Purifiedenzymes from 13 different species with varyingtoleranceto flooding all show the greatestactivity
in the reducing direction (Davies et al. 1973; Brown,Marshall,and Munday
1976;John and Greenway 1976; Leblova 1978a).
Mostalcohol dehydrogenasesfromethanol-producingorganismsare NADrather than NADP-dependent.These include the enzyme from wild rice
(Campiranonand Koukkari1977), potato (Davies et al. 1973), barley(Duffus
1968), bean, lentil, pea, soybean, maize, cucumber, wheat, rice, rape, sunflower (Leblova 1978a), and goldfish (Mouriket al. 1982), but there exists
an exception to this rule. The parasiticworm Moliniformisdubius has no
alcohol:NAD oxidoreductase (EC 1.1.1.1) and large amounts of alcohol:
NADP oxidoreductase (EC 1.1.1.2, K6rting and Fairbairn1972). This exception is interesting since a stoichiometric coupling of NAD-dependent
glyceraldehyde-3-phosphatedehydrogenase(GAPDH)andADHis normally
required to maintainredox balance (Stryer1988).
Interorgan Lactate/EthanolShuttlesin FreshwaterFish
Cyprinidspossess two isozymes of alcohol dehydrogenase with different
catalyticproperties.Hepatopancreascontainsthe normalvertebrateisozyme
which has a high affinityfor ethanol (Mouriket al. 1982). This protein is
supposed to be involved in the oxidation of ethanol from the diet. The
lateral red and epaxial white muscles have a peculiar isozyme with low
affinityfor ethanol, which is involved in alcoholic fermentation (Mourik
et al. 1982). ADH activity in heart muscle and brain is below the limit of
detection; these tissues are only capable of lactate glycolysis (Mourik
et al. 1982).
Tracerexperiments (Shoubridge 1980;Shoubridgeand Hochachka1983)
and measurementsof lactategradientsover the sarcolemmaof the myotomal
inMulticellular
Fermentation
Alcoholic
905
Organisms
muscles (van Waarde,van den Thillart,and Dobbe 1982) have provided
evidence that lactate produced in peripheral tissues of anoxic goldfish is
taken up by the myotome, where it is decarboxylated.The ultimate fate of
blood lactate is excretion in the ambient water as alcohol and CO2. When
mitochondriaisolated from goldfish red muscle are incubatedwith lactate,
NAD, LDH,and ADH, lactate is quantitativelyconverted to ethanol (G. van
den Thillartand J. Warnaar,unpublished data;G. van den Thillartand M.
Verhagen,unpublished data).
Mitochondrialabundance in a tissue can be assessed by measurementof
the markerenzyme succinate oxidase. Van den Thillart (1977) found 1018 U/g in red muscle and 1.5-1.6 U/g in white muscle of goldfish. If we
assume PDCactivityis proportionalto mitochondrialabundance, the maximal rate of alcohol production by white fibers is only 10%-15%of that in
red muscle, but owing to its large mass, white muscle may still be a quantitativelyimportantethanol-producingorgan in the fish body.
A closer examination of Carassiusgills may produce interesting results.
Ekberg (1956) has observed a large anaerobic CO2production in isolated
gills of goldfishand cruciancarp,which is not due to a shiftof the bicarbonate
equilibriumby tissue acidificationand maysuggest the presence of pyruvate
decarboxylase.Localizationof PDCand ADH in the gill could be functional
since ethanol would be produced in the organ by which it is excreted.
of theEthanol
RoutebyEnzyme
Induction
Regulation
Plants.Angiospermsgenerallyshow an increase of ADHactivityin response
to flooding (Crawford1967; Francis,Devitt, and Steele 1974;Wignarajahet
al. 1976; Mendelssohn et al. 1981;Jenkin and ap Rees 1983; Rowlandand
Strommer1986) or oxygen deprivation(App and Meis 1958; Hagemanand
Flesher 1960; Kollikffel1968; Leblovaet al. 1969;Wignarajahand Greenway
1976;Wignarajahet al. 1976; Campiranonand Koukkari1977; Smithand ap
Rees 1979a; Bertani et al. 1980; Rumpho and Kennedy 1981; Monk and
Brandle 1982;Jenkin and ap Rees 1983; Hanson,Jacobsen, and Zwar1984;
Ricardet al. 1986;Johnson, Cobb, and Drew 1989). These changes roughly
correspond to increases in ethanol production. The rise of ADH activity
upon flooding or anoxic exposure is due to increased gene expression,
elevated levels of mRNA,and enhanced protein synthesis (John and Greenway 1976; Sachsand Freeling 1978; Ferl, Brennan,and Schwartz1980; Hanson et al. 1984; Ricardet al. 1986; Rowlandand Strommer1986). The ADH
isozymesthatare induced by oxygen deficiencyhavea 3-6-fold higheraffinity
for NADHthanthe normoxic isozyme, so their synthesis facilitatesalcoholic
fermentationof glucose (Mayneand Lea 1985). The inducer substance may
906 A.vanWaarde
either be ethanol (App and Meis 1958; Koll6ffel 1968) or acetaldehyde
(Hageman and Flesher 1960; Crawfordand McManmon1968; Koll6ffel
1968), depending on the species.
The capacity of plants for pyruvatedecarboxylationalso increases upon
flooding (Wignarajahet al. 1976) and exposure to anoxia (John and Greenway 1976; Wignarajahand Greenway 1976; Wignarajahet al. 1976; Laszlo
and St. Lawrence1983). The rise of enzyme activity is caused by de novo
synthesis of the PDCpolypeptide (John and Greenway1976; Laszloand St.
Lawrence1983). Absolute activities of PDCare 6-15 times lower than those
of ADH and closely approximatethe in vivo rates of alcoholic fermentation
(John and Greenway 1976; Wignarajahand Greenway 1976). The reaction
catalyzedby PDCis thereforeconsidered the rate-limitingstep in the ethanol
pathway.The presence of excess ADH is probablyessential to prevent accumulation of acetaldehyde, a highly reactive and toxic compound (Walia
and Lamon1989).
Although increases of ADH and PDC correspond to increases in ethanol
productionunder anoxic conditions, such a correlationis not observed during aerobicrecovery.Elevatedactivitiesof the two enzymes persistfor several
days, but ethanol production stops immediately upon reoxygenation (John
and Greenway1976). This indicatesthe ethanolpathwayis underfine control
by other mechanisms,such as the redox state,allostericeffectors,or covalent
modification.
Animals. ADH activityin the muscle of normoxic goldfish is high, and the
amount of enzyme is not altered by anoxic exposure (van Waardeet al.
1990a). In the nematodes Aphelenchusand Caenorbabditis,ADH activity
is likewise independentof oxygen availability(Cooperandvan Gundy1971).
Induction of ADH as observed in plants seems therefore to be absent in
these animal species.
PDCactivityin Moliniformisdubius is much (>16 times) lower than that
of ADH (Kirting and Fairbairn1972), indicating that the decarboxylaseis
limiting the rate of conversion of pyruvateto ethanol.
at thePyruvate
Branch
Point
Regulation
Plants. Davies and co-workers (Davies et al. 1974; Davies 1980) examined
the factors controlling lactate and ethanol production in cell-free extracts
prepared from pea (Pisum sativum) seeds and parsnip (Pastinaca sativa)
roots. They showed that PDC has an acid pH optimum, whereas LDHperforms optimally in the alkaline range. Nucleotides (ATP,ADP,AMP)do not
affect PDC, in contrastto LDH,which is inhibited by ATP,especially at low
pH values.At the onset of anoxic exposure, the NADH/NAD+ratioincreases,
inMulticellular
Alcoholic
Fermentation
907
Organisms
pHi is still >7, and LDHis not inhibited by ATP,so lactateaccumulatesand
the pH falls. The resulting acidosis activatesPDCand initiates competition
for pyruvate.The ratio between lactate production and ethanol formation
will be determined by (i) the pyruvateconcentration, (ii) the intracellular
pH, and (iii) the concentrationof ATP.The LDH/ADHactivityratio drops
when the pyruvateconcentrationincreases or the pH, falls. When the intracellular pH reaches 6.9, inhibition of LDHby ATPbecomes very important
and the production of lactate virtually stops. Ethanol then becomes the
majoranaerobicend product.John and Greenway(1976) reportedallosteric
activationof PDC by NADHand inhibition by NAD+.The immediate stop
of ethanol production upon reoxygenation is therefore probably due to
removal of NADH by the electron-transportchain. The disappearanceof
NADH causes inhibition of PDC and ADH, thus effectively blocking the
ethanol pathway.
Robertset al. (1984a) examined the ethanolpathwayin vivo by measuring
NADH(fluorescence), lactateand ethanol production('3C-nuclearmagnetic
resonance [NMR]spectroscopy), intracellularpH (31P-NMR
s1ectroscopy),
and ATP concentration (31P-NMRspectroscopy) in intact maize root tips
during hypoxia. The model of Davies et al. (1974) provides an accurate
descriptionof the in vivo situation.Hypoxiacauses a rapid(<2 min) increase
of NADH,followed by a transient(ca. 35 min) accumulationof lactate.After
35 min, pHi and lactate concentrations stabilize due to activation of the
ethanol route.These observationslead to the hypothesisthatH+ is the signal
triggering ethanol production, the pH drop being provided by lactate glycolysis. To test this hypothesis directly, the pHi of the root tips was manipulated prior to anoxic exposure by addition of various amounts of acetic
acid (0-5 mM) to the perfusion medium. The lag time between the onset
of hypoxia and the startof ethanol production can indeed by abolished by
lowering the cytoplasmicpH under a threshold value of 6.9. Viabilityof the
root tips and the eventual kinetics of ethanol production are not affected
by acid treatment,so acetic acid is not toxic at the concentrationsused in
this study.
Animals. Pyruvatedecarboxylationby intact mitochondria of Panagrellus
redivivusis inhibited by ATP(strong), NAD+(weak), and acetyl-CoA(moderate) but unaffected by ADP, AMP,cAMP,NADH, NADP+,and NADPH
(Barrettand Butterworth1984). Vanden ThillartandVerhagen(unpublished
data)haveobservedthatdecarboxylationof pyruvatein muscle mitochondria
of goldfish is modulated by the ATP/Piratio.When 10 mM Pi, 0.1 mM adenylylimidodiphosphate (AMPPNP,an ATPaseinhibitor), and no ATPare
present, PDC shows maximal activity. In the presence of 10 mM ATP,0.1
mMAMPPNP,and no Pi, the reaction rate is reduced to 15%of the maximal
908 A.vanWaarde
value. Changes in the pH of the incubation medium have little influence
on the rate by which anoxic mitochondria decarboxylatepyruvatein the
presence of 10 mM Pi, but a decline of the pH from 7.4 to 6.9 abolishes the
inhibition of PDCby ATP(10 mM). Mouriket al. (1982) have reportedthat
anaerobic pyruvatedecarboxylationin muscle mitochondriaof goldfish is
stimulated by ADP.
In all animal species that have been examined, lactate and alanine accumulateinitiallybut not duringthe laterstages of the anoxic period, when
ethanol becomes the majorend product (Cooper and van Gundy 1971;van
den Thillart et al. 1976; Wilps and Zebe 1976; van den Thillart and van
Waarde1990). Fish do not produce ethanol duringnormoxia (van den Thillart,van Berge Henegouwen, and Kesbeke 1983; van Waversveld,Addink,
and van den Thillart 1989) or physical exercise (Wissing and Zebe 1988)
but only duringenvironmentalhypoxia/anoxia (van den Thillartet al. 1983;
Wissing and Zebe 1988; van Waversveldet al. 1989). The ethanol/lactate
production ratioduringanoxia seems dependent on the glycolytic flux, relatively more lactate being produced when the flux increases (VanWaarde
et al. 1990a; see table 2).
These observationssuggest the following control at the pyruvatebranch
point. At the onset of anoxia, the NADH/NAD+ratio shifts to the more
TABLE2
Dependence of the lactate/ethanol production ratio in anoxic goldfish
on the glycolytic flux
Energydemand (CO2
..
production)a ............
Ethanolexcretion during
steady state" ............
Lactateformationduringsteady
state ...........
.......
Ethanol/lactateproduction
ratioc ..................
200C-acclimated
5SC-acclimated
282
83
289
68
302
27
.95
2.4
Note. Values expressed as Cgmol* 100 g-' h-1
1983. o
b Estimatedfrom the
in
decline
the epaxial white muscle (measuredby 31P-NMR
pH
spectroscopy),assuminga bufferingcapacityof 30 meq H+/pH unit and negligible leak-out
of protons duringanoxia (datafrom vanWaardeet al. 1990a).
c
Assuminga correlationbetween myotomaland whole-bodyratesof glycolysis.
a Datafrom van den Thillartet al.
Alcoholic
Fermentation
inMulticellular
909
Organisms
reducedstate,facilitatingglycolysisand causinga net drop in the intracellular
pH. Inorganic phosphate (Pi) accumulatesbecause anaerobic energy metabolism cannot maintain the free ADP concentration near the normoxic
level that is mainly determined by oxidative phosphorylation:
ATP-- ADP + Pi (cellular energy demand),
phosphocreatine + ADP * creatine + ATP (creatine kinase),
net: phosphocreatine -+ creatine + Pi (appearanceof Pi).
The declines of pHi and the ATP/ADP - Pi ratio activate PDC and initiate
competition for pyruvate.Ethanolconcentrationsare kept low by excretion,
whereaslactateaccumulatesin situ. The AGovalue for conversionof pyruvate
to ethanol and CO2is -8.8 kcal mol-', whereas that of the lactate dehy1
drogenase reaction is -6 kcal - mol-'. Ethanolformationis therefore thermodynamicallyadvantageousover lactateglycolysis, especially in the presence of elevated levels of lactate. However, the maximal rate of pyruvate
decarboxylationis relatively low since both the substrate (pyruvate) and
product (acetaldehyde) must be transported through the mitochondrial
membraneand the Vmax of PDCis at least an order of magnitude lower than
thatof LDH.The ratioof ethanol formationover lactateproduction is therefore critically dependent on the glycolytic flux. At very high flux (intense
exercise), virtuallyall carbohydratecarbon is converted to lactate. At low
fluxes, which occur during environmental anoxia, ethanol production becomes relatively important.
The differences between plants and animals as far as regulation at the
pyruvatebranchpoint is concerned are summarizedin table 3.
Functional
inAlcoholic
Trade-offs
Fermentation
Alcoholic fermentationof carbohydrateshas at least three significantadvantages over lactate glycolysis.
1. Assuming effective removal of CO2 (see below), ethanol formation
does not acidifythe cell, whereas coupling of Embden-Meyerhofglycolysis
with cell ATPasesgenerates two protons per mole of glucose consumed
(Hochachkaand Mommsen 1983; Partner1987). Ethanol-producingorganisms maythus preventthe acidosis thatis normallyassociatedwith anaerobic
metabolism. Everyalternativepathwayof glycogen degradation(to opines,
alanine, malate,succinate,propionate,or acetate) produces acid equivalents
(Pbrtner1987).
2. Ethanol is a neutral end product that can be transported by passive
diffusion and is easily excreted into the ambient water (Shoubridge and
910 A.vanWaarde
TABLE 3
Regulation at thepyruvate branchpoint
Plants
Animals
1. Hypoxia-inducedincreases
of the activities of PDC
and ADH due to increased
gene expression
2. Control of cytosolic PDC
by the NADH/NAD+ratio
and the intracellularpH
3. Inhibition of LDHby ATP
at low pH values
1. Appearanceof PDC during anoxia
(dissociation of the PDH complex)?
No changes in ADH gene expression
4. Ethanolpathwaytriggered
by a drop of the
intracellularpH
2. Controlof mitochondrialPDC by the
ATP/ADP - Pi ratio and the
intracellularpH
3. Thermodynamicadvantageof ethanol
formationover lactate production at
elevated levels of lactate (ethanol is
excreted into the ambient water in
contrastto lactate,which
accumulates in situ)
4. Ethanolpathwaytriggered by
declines of the ATP/ADP * Pi ratio
Hochachka 1980). The lactate anion on the contrary is highly charged
and usually accumulates in situ. Species that have to survive long periods
of anoxia and produce ethanol thus avoid the osmotic problems that normally arise from extensive glycogen degradation. Another well-known
solution of the osmotic problem is opine formation, that is, reductive
condensation of pyruvate with an amino acid, which occurs in many invertebrates.
3. Since ethanol concentrations are kept low by passive efflux, there is
no productinhibitionof the glycolyticchain.The period duringwhich anoxia
can be resisted is thus limited by the amount of available substrate (i.e.,
glycogen) ratherthan end-productaccumulation.
In vivo 31P-NMR
studies of plants (Roberts et al. 1984a, 1984b; Roberts,
Andrade, and Anderson 1985) and animals (van den Thillart et al. 1989;
van den Thillart and van Waarde 1990; van Waarde et al. 1990a, 1990b;
see fig. 3) have provided ample evidence to support the hypothesis that
the switch to alcohol production dampens metabolic acidosis and increases the period during which cells can resist anoxia. Mutant maize
roots that lack alcohol dehydrogenase do not regulate cytoplasmic pH
well and cannot maintain high ATPlevels in the absence of oxygen (Rob-
Alcoholic
Fermentation
inMulticellular
911
Organisms
erts et al. 1984a, 1984b). When the intracellular pH of hypoxic root tips
is experimentally lowered by exposure to various amounts of carbon
dioxide, survival is shortened in proportion to the pH decrease (Roberts
et al. 1984b). In anoxic goldfish, there is an initial drop of pHi due to
lactate glycolysis, but during the rest of the anoxic period, acid production
is suppressed (van den Thillart et al. 1989; van Waarde et al. 1990a,
1990b). The dampening of acidosis coincides with the onset of ethanol
production in the myotome (van den Thillart and van Waarde 1990; van
Waardeet al. 1990b). In a comparative study using different fish species,
anoxic survival correlated with the ability to stabilize the intracellular
pH (van Waardeet al. 1990a).
Althoughthe switch from lactateto ethanol as the anaerobicend product
may prolong anoxic survival,it should be emphasized that this metabolic
modificationhas its drawbacks,too.
1. Although identical to lactate glycolysis, the process has a relatively
low energy yield of 2 mol ATPper mole of glucose consumed, as opposed
to 4 mol ATPper mole of glucose in succinate production and 7 mol ATP
per glucose unit in propionate production (Pirtner 1987). This means that
relatively high glycolytic fluxes are required to meet a certain energy demand, and much glycogen has to be stored for long-term anoxic survival.
It is not surprisingthat hibernatingcruciancarp,which rely on the ethanol
pathway,have the highest glycogen stores known in any vertebrate. Liver
glycogen concentrations reach 30%of liver dryweight during the fall, and
the organ increases in size from 2% to 14%of total body weight (Holopainen and Hyvdirinen1985; Hyv1irinen,Holopainen, and Piironen 1985;
Piironen and Holopainen 1986). Glycogen stores in the myotome also
reach record highs of 3.5%on a dry-weightbasis (Hyvdirinenet al. 1985).
Anoxic crucian carp do not fall into torpor but remain conscious and capable of active avoidance of a net or of predators. Since the animals do
not feed during winter, huge glycogen reserves are presumably necessary
for glycolytic energy production. The requirementof large glycogen stores
is less stringent in invertebrates since these take up food under anoxic
stress (K6rting and Fairbairn1972; Wilps and Zebe 1976; Redecker and
Zebe 1988).
2. Because ethanol is excreted, the alcohol route is wasteful of carbon.
Ethanol-producinganimals that are exposed to long periods of anoxia will
need a good food supply between anoxic events to ensure growth and replenishment of liver glycogen. Cruciancarpinhabitsmall lakes and shallow
ponds with a high productionof invertebratesduring summer. Chironomus
larvaelikewise occur in shallow, eutrophic waterswhere the availabilityof
food is not a problem.
Carassius
mM
carassius
pH
15-7. 8
S1//
15
TH
I
p
10-7.4
15I
10-
I
+I
C
5-L-oA
S3
6
9
12
15
18
2
3
6
9
12
15
18
21
2
0 3 6 9 12 15 18 21 24 3 6 9 12 15 1821 24
lac 1.4A
4.0
eth 02
0.5
7.0 umol/g
1.6
7.5
PH
7.0
6.5
0
1
2
ANOXIA(h)
Fig. 3. Dampening of acidosis in freshwaterfish by the ethanol pathway.
Top, measurements performed on a free-swimming, cannulated crucian
carp (Carassius carassius) at 150C. Indicated are the concentrations of
Alcoholic
Fermentation
inMulticellular
913
Organisms
Carbonloss maybe one of the reasonswhy the ethanol route is ubiquitous
in the plant kingdom but relatively rare among animals. Plants have an
abundantcarbon supply in the form of atmospheric CO2,whereas animals
must feed to acquire substratesfor gluconeogenesis. However, a shortage
of essential nutrientsin plantswith anoxic roots may limit their capacityfor
CO2fixation.Animalsare exposed to "globalanoxia,"but in plants normally
only certainorgans (i.e., the roots) are deprived of oxygen. Plantsthus have
the opportunity to transportethanol upward and use it for carbohydrate
synthesis, while in animals the end product of fermentation is irreversibly lost.
3. Animals cannot allow ethanol or acetaldehyde to reach high concentrations since these compounds disturb the operation of the nervous
system. Only those species that can keep ethanol levels low by excretion
may opt for this survival strategy. The use of alcoholic fermentation also
requires efficient removal of metabolic carbon dioxide. If CO2accumulates in situ, the major advantage of ethanol production (dampening of
acidosis) disappearsbecause hydrationof CO2generates acid equivalents.
Animals that live in water are at an advantage here since both ethanol
excretion and elimination of CO2 are facilitated by the aquatic environment.
The ethanol route may furtherbe rareamong animals because its advantages become significantonly when high glycolytic fluxes have to be maintained for long periods of time (species that remain active, do not fall into
torpor, and are subjected to prolonged anaerobiosis). Conscious activity
duringweeks or monthsin an anoxic environmentrequiresmanyadaptations
on other levels than energy metabolism, such as recycling of neurotransmitters in the brain (Nilsson 1989a, 1989b). Relatively few animals may
have solved all problems of this situationand thus possess the ethanol pathway. Most species have opted for a stronger suppression of the metabolic
rate, which causes torpor, and an energetically more efficient form of anaerobic metabolism to ensure anoxic survival.
lactate (0) and ethanol (0) in the blood, theplasma pH (+), and the
amount of ethanol excreted in the environmental water (X). Bottom,
measurementsperformed on six restrained goldfish(Carassiusauratus) at
200 C by in vivo 31P-NMR
spectroscopy(pH) and enzymatic analysis of
tissue samples (lactate, ethanol). Indicated are the intracellularpH (0)
of white muscle (mean 1 SE), the lactate concentration in white muscle,
and the ethanol concentration in the blood. Data are from van den Thillart and Heisler (unpublished), van den Thillartand van Waarde 1990,
and van Waarde et al. 1990a.
914 A.vanWaarde
Appendix
TABLE
Al
Related species that do not produce ethanol
Species
Arthropoda(Insecta):
Culexpipiens ..................
Vertebrata(Pisces):
Abramus brama ................
Anguilla anguilla ..............
Barbus ticto ...................
Brachydanio rerio ..............
Colossomabrachyponum ........
Colossomamacropomum ........
Ctenopharyngodonidella ........
Cyprinuscarpio ................
Esox lucius ....................
Gasterosteusaculeatus ..........
Gobiogobio ....................
Hypopthalmichthysmolitrix ......
Ictalurus nebulosus .............
Leucaspius delineatus ...........
Leucaspius idus .................
Oreochromismossambicus .......
Percafluviatilis ................
Poecilia reticulata ..............
Rasborachrysotaenia ...........
Rutilus rutilus .................
Tinca tinca ...................
Xiphophorusmaculatus .........
Reference
Redeckerand Zebe 1988
Wissing 1986
van Waarde,van den
Thillart,and
Kesbeke 1983
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
van Waardeet al. 1990b
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
Wissing 1986
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