200th anniversary of lactate research in muscle

Copyright @ 200 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.8


200th Anniversary of Lactate Research in MuscleL. Bruce Gladden

Department of Kinesiology, Auburn University, Auburn, AL, United States

GLADDEN, L.B. 200th anniversary of lactate research in muscle. Exerc. Sport Sci. Rev., Vol. 36, No. 3, pp. 109Y115, 2008.This year, 2008, marks the bicentennial of research into lactate metabolism in muscle. Berzelius linked lactate accumulation to exercise in1807/1808 when he noted the presence of lactate in the muscles of ‘‘hunted stags.’’ Today, the exact mechanism of intramuscular lactateoxidation and the relationship of lactate dehydrogenase to mitochondria remain unresolved as animated debate surrounds the intracellularlactate shuttle. Key Words: intracellular lactate shuttle, lactate dehydrogenase, lactic dehydrogenase, isozymes, history

INTRODUCTION

Discovery of LactateLactate/lactic acid was discovered in sour milk by a

Swedish apothecary assistant, Carl Wilhelm Scheele(1742Y1786) in 1780 (2[pp6Y8]). Benninga (2[pp7Y8])provided a modern translation of Scheele’s isolation techni-que; it is quoted partially here: ‘‘Sour whey is evaporated toone eighth of its volume, the curd [precipitated milkproteins] is then removed by filtration. The filtrate issaturated with milk of lime [calcium hydroxide], filteredagain and diluted with three times its volume of water’’ (2).After a series of additional steps, Scheele concluded that(modern translation): ‘‘The lactic acid produced is as pure ascan be obtained by chemistry’’ (2). The new acid was named‘‘Mjolksyra,’’ meaning ‘‘acid of milk’’ (2[p8]). Lactate (Laj)occurs as the acid ingredient of sour dairy products,fermented fruits and vegetables, and sausages (2[p1]) and asa flavor modifier or enhancer in carbonated soft drinks. It hasalso been used in tanning leather, acid dyeing of wool, and asa pharmaceutical in the form of calcium lactate; its estershave been used as lacquer solvents, and small amounts havebeen used in plastics. Benninga’s book (2) details the historyof Laj and its large scale production for industrial purposesfrom a biotechnology perspective, offering a viewpoint thatis completely outside the mind-set of most biologicalscientists. Note that lactic acid is more than 99% dissociated

into Laj anions and protons (H+) at the physiological pHlevels found in muscle and blood; therefore, it will generallybe referred to as ‘‘Laj’’ in this review.

Scheele also discovered chlorine, manganese, arsenic acid,and silicon fluoride along with numerous other compounds(28[p461]). However, he is best recognized for his work onO2 in which he first demonstrated that air is composed oftwo components, only one of which will support combustion(28[p461]). Scheele discovered O2 as early as 1772, morethan a year before Joseph Priestley (1733Y1804). Unfortu-nately, his experiments (A Chemical Treatise on Air andFire) were not published until 1777, probably because ofprocrastination by his friend and advocate, Torbern Bergman(1735Y1784), who wrote an introduction to the book(28[pp49,461]). As a result, he shares credit for the discoveryof O2 with Priestley.

LACTATE AND EXERCISE

Although Scheele is remembered as the discoverer of Laj,it was Jons Jacob Berzelius (1779Y1848), another Swede,who indelibly joined Laj to the study of exercise when hereported the presence of Laj in the muscles of hunted stagsin either 1807 (19[p41]) or 1808 (20[pxxvii]). Berzeliusapparently convinced himself that the amount of Laj in amuscle was proportional to the amount of exercise that themuscle had performed (19[p41]). Mentioned less often is thefact that Berzelius also discovered pyruvic acid (28[p56]).Accordingly, it is fitting that we pause at the bicentennial ofresearch into lactate metabolism in muscle for a brief historicoverview and discussion of current controversies. Presently,there are at least three contentious areas in the physiological/biochemical study of Laj: 1) the role of Laj as either acausative or a preventive agent of muscle fatigue, 2) the roleof Laj in the acidosis of intense exercise, and 3) the

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PERSPECTIVES FOR PROGRESS

Address for correspondence: L. Bruce Gladden, Ph.D., FACSM, Department ofKinesiology, 2050 Memorial Coliseum, Auburn University, Auburn, AL 36849-5323(E-mail: [email protected]).

Accepted for publication: March 13, 2008Associate Editor: Stephen M. Roth, Ph.D., FACSM

0091-6331/3603/109Y115Exercise and Sport Sciences ReviewsCopyright * 2008 by the American College of Sports Medicine



intracellular pathway of Laj metabolism in muscle. Thisreview focuses on the third controversy. Laj metabolism inmuscle during and after exercise is of special importancebecause skeletal muscle is not only the most important sourceof Laj but is also the primary consumer of Laj. For example,during recovery from intense exercise, oxidation is the majorpathway for Laj removal, particularly in skeletal muscles.

LACTATE DEHYDROGENASE: DISCOVERYAND ISOZYMES

The discord surrounding intracellular Laj metabolism isnot only a story about Laj but also about the enzymethat catalyzes its formation, lactate dehydrogenase (LDH).Berzelius has a distant relationship to this side of the tale aswell because he was the first (in 1836) to propose the termcatalysis that he used to explain the action of compounds thatfacilitated chemical reactions without undergoing anychange themselves (19[pp30Y31]). A long period followedduring which scientists studied the ability of ‘‘ferments,’’tissues, or tissue extracts of various organisms to promotereactions. Along the way, the ferments were divided into twogroups: 1) those that were disorganized or soluble and 2)those that were organized or insoluble (i.e., they were madeup of intact microorganisms). In 1877, Willy Kuhne(1837Y1900) offered the name ‘‘enzymes’’ (Greek, ‘‘inyeast’’) for the unorganized ferments (28[pp300Y301]).Twenty years later, Eduard Buchner (1860Y1917) groundyeast cells with sand at a controlled temperature to derive anextract that was free of yeast cells but still able to fermentsucrose to ethanol, demonstrating conclusively that cellswere not essential for fermentation (28[pp87Y88]). Subse-quently in 1926, American James Batcheller Sumner(1877Y1955) first isolated the crystalline form of an enzyme,urease, from jack beans (28[p498Y499]).Perhaps not surprisingly, it was Otto Meyerhof (1884Y

1951) (28[p368]) in 1919, studying skeletal muscle, who firstdemonstrated the action of LDH. After perhaps hundreds ofinvestigations on the topic of LDH, pure LDH in crystallineform was isolated from the hearts of bullocks. Beginning inthe 1950s, several investigators provided evidence thatpreparations of LDH were heterogeneous. This work culmi-nated in 1959 when Markert and Møller (17), using thesuperior separation method of starch gel electrophoresiscombined with histochemical staining, clearly demonstratedfive different forms of the LDH enzyme. They (17) also firstproposed the term isozymes to designate the multiplemolecular forms of an enzyme. In 1962, Kaplan’s group (6)hypothesized the model of tetrameric organization of theLDH isozymes that still applies today: there are twomonomer subunits, H (heart) and M (muscle), which canbe combined in groups of four to yield five differentmolecular species; HHHH, HHHM, HHMM, HMMM,and MMMM. Modern nomenclature designates the mono-mers as A (muscle) and B (heart) yielding LDH1(B4), LDH2(B3A1), LDH3(B2A2), LDH4(B1A3), and LDH5(A4).Kaplan’s laboratory (6) went on to propose that the differentproperties of the isozymes were physiologically important inthe regulation of cellular metabolism. Specifically, it was

proposed that the sensitivity of the heart forms (H4, H3M1)to pyruvate inhibition directed pyruvate toward oxidation inan aerobic environment such as the heart, whereas therelative insensitivity of the muscle forms (H1M3, M4) topyruvate inhibition facilitated the anaerobic breakdown ofcarbohydrates to lactate in exercising muscles (6).

This scheme has been supported by circumstantial evi-dence. For example, muscle capacity for Laj utilization iscorrelated with both the oxidative capacity and the contentof the heart-type LDH. As reviewed by Van Hall (27), thereis also evidence that oxidative muscle fibers have approx-imately half of the total LDH activity of glycolytic fibers, andof that activity, a greater relative amount is in the heartforms (H4 and H3M1). Also, endurance training results in alower total LDH content and a decrease in the muscle forms(H1M3, M4) of LDH (27). However, Newsholme (21) hasquestioned the potential role of LDH isozyme profile in Laj

production versus utilization on several counts: 1) the LDHreaction is near-equilibrium, suggesting that pyruvate inhib-ition would have a negligible effect on flux through thereaction, 2) the differences between function of the isozymesmay be less in vivo than in vitro because of higher temperatureand other factors in the intracellular milieu, 3) the pyruvateinhibition may not apply at the high concentrations of theLDH enzyme found in vivo, and 4) the reported inhibitionmay be due to traces of the enol form of pyruvate that arelikely more prevalent in vitro than in vivo. Further, Van Hall(27) notes the following: 1) that the absolute amount of theheart forms of LDH remains fairly constant among fiber typesand with endurance training and 2) that the concentrationsof pyruvate and Laj required for LDH inhibition in vitro areseveral times greater than the highest concentrationsobserved in vivo. It is also possible that the binding of LDHto cellular proteins may change its kinetic parameters. Myconclusion is that the exact regulatory role of the LDHisozymes remains unknown.

LDH: CELLULAR LOCATION

Lactate dehydrogenase has long been considered a ‘‘soluble’’glycolytic enzyme found in the cytoplasm of cells, predom-inantly in the I band of striated muscle (18, 27). There is alsosignificant evidence that LDH binds reversibly to cytoskele-tal proteins including actin, tubulin, and troponin amongothers (see (18) for references). Furthermore, it seems clearthat an LDH fraction is localized to the sarcoplasmicreticulum (SR), particularly in glycolytic skeletal muscle(e.g., (1)). In agreement with the studies previously cited, itis commonly reported that glycolytic activity is foundexclusively in the supernatant fraction of tissue homoge-nates, a fraction that contains essentially no mitochondria.

The story of the location of LDH, and thereby ofintracellular Laj metabolism, became more provocativewith a 1971 publication of Baba and Sharma (1). They (1)cited several studies that reported LDH activity in themitochondrial fractions of tissues as well as numeroushistochemical studies that demonstrated LDH activity inmitochondria of various tissues. Their own results (1),obtained with a combination of histochemical and electron

110 Exercise and Sport Sciences Reviews www.acsm-essr.org



microscopy techniques in skeletal muscle, indicated thepresence of mainly heart-type isozymes in mitochondria andcytoplasm and primarily muscle-type isozymes in associationwith the SR. They conjectured that heart forms of LDHmight serve a role similar to that of glycerol phosphatedehydrogenase and malate dehydrogenase. However, theyconcluded that ‘‘Permeability of the mitochondria to lactatehas not been well-demonstrated, and the lactate shuttle re-mains a pure speculation’’ ((1); emphasis is mine).

Subsequently, via a variety of techniques, LDH was foundin association with mitochondria by Skilleter and Kun (25),Deimann et al. (8), Kline et al. (16), Brandt et al. (3), andChretien et al. (7) among others. In 1972, Skilleter and Kun(25) also reported the oxidation of Laj by isolated livermitochondria, although, perplexingly, the process seemed torequire energy. Szczesna-Kaczmarek (26) became the firstinvestigator to report direct mitochondrial oxidation of Laj

in skeletal muscle in 1990.

THE INTRACELLULAR LACTATE SHUTTLE

Finally, in 1999, Brooks et al. (5) fully elucidated anintracellular lactate shuttle that had only been inferred byBaba and Sharma (1). A central tenet of this intracellularshuttle was that Laj is an inevitable product of glycolysis,particularly during rapid glycolysis, because LDH has thehighest Vmax of any enzyme in the glycolytic pathway, and theKeq for pyruvate to Laj is far in the direction of Laj andnicotinamide adenine dinucleotide (NAD+) (4,5). Given thisinformation, Brooks et al. (4) questioned how it would bepossible for Laj to be converted back to pyruvate in thecytosol, thus permitting oxidation of Laj by well-perfusedtissues, a universally accepted phenomenon that provides thefoundation for the cell-to-cell lactate shuttle theory (9,10,27).One simple explanation would have been that LDH catalyzes anear-equilibrium reaction, allowing small changes in substrateandproduct concentrations to immediatelydirect the reaction inthe opposite direction. However, a different explanation wasproffered, and in recent years, the Brooks laboratory (4,5,11,13)has reported evidence of the following key components of anintracellular lactate shuttle in skeletal muscle: 1) direct uptakeand oxidation of Laj by isolated mitochondria without priorextramitochondrial conversion of Laj to pyruvate (Fig. 1), 2)presence of an intramitochondrial pool of LDH, and 3)presence of the Laj transporter, monocarboxylate transporter1 (MCT1), in mitochondria, presumably in the innermitochondrial membrane.

Operation of an intracellular lactate shuttle such asdescribed by the Brooks group (5) entails constant produc-tion of Laj in the cytosol, with the rate of productionincreasing with elevated glycolytic activity. Because of itshigher concentration relative to pyruvate, Laj would bethe primary monocarboxylate diffusing to mitochondria. Inthe original version of this shuttle, this Laj would then betransported across the inner mitochondrial membrane byMCT1. Once inside the mitochondrial matrix, mitochon-drial LDH would catalyze the conversion of Laj back topyruvate that would be oxidized through the PDH reactionto acetyl coenzyme A (CoA). The acetyl CoA would then

enter the tricarboxylic acid cycle. An important point is thatthis intracellular lactate shuttle would not only deliversubstrate in the form of Laj for conversion to pyruvate, itwould also deliver reducing equivalents (reduced form ofNAD+ (NADH)), thus supplanting or supplementing therole of the malate-aspartate and glycerol-phosphate shuttlesto varying degrees, depending on the rate of Laj formationand its rate of transport into mitochondria (9).

Is Brooks correct? Can mitochondria oxidize Laj directly?After Brooks’ proposal of the intracellular lactate shuttle,Rasmussen et al. (23) and Sahlin et al. (24) found noevidence that mitochondria can use Laj as a substratewithout prior conversion to pyruvate in the cytoplasm. Apreliminary report by Willis et al. (29) also found insignifi-cant activity of the proposed intracellular lactate shuttle inmitochondria isolated from rat skeletal muscle (Types I andIIb) and liver. Ponsot et al. (22) reported no sign of directmitochondrial Laj oxidation in skinned fibers from heartmuscle, glycolytic skeletal muscle, or oxidative skeletalmuscle. Finally, Yoshida et al. (30) from the Bonenlaboratory have also recently found minimal direct oxidationof Laj by either subsarcolemmal or intermyofibrillar mito-chondria from either red (oxidative) or white (glycolytic)skeletal muscle (Fig. 2).

A central point in this debate is the exact location of LDH.Although several investigators have reported that LDH isassociated with mitochondria (as previously cited), thesereports warrant further scrutiny. First, some researchers(23,24,30) note that the amount of LDH associated withmitochondria is quite small and conclude that the mito-chondria isolated by Brooks et al. (5) as well as others arecontaminated with cytoplasmic LDH. In fact, Chretien andcolleagues (7), who found LDH in mitochondria-enrichedpreparations of human skeletal muscle, considered the LDHto be a contaminant from the cytoplasm. On the contrary,Hashimoto and Brooks (11) point to the difference inmitochondrial isolation techniques and argue that others mostlikely lost LDH that was associated with mitochondria.

Figure 1. State 3 lactate and pyruvate oxidation by isolated rat skeletalmuscle mitochondria. Concentration of both substrates was set at10 mM, and 2.5 mM malate was included in the medium. Comparewith values reported by Yoshida et al. (30) in Figure 2 who contend thatthe results in this figure are likely due to contamination. Based on datafrom Table 1 of Brooks et al. (5).

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Clearly, it would be helpful if someone were to carefullycompare LDH activity and Laj oxidation in mitochondriaisolated by the methods of Brooks and coworkers as comparedwith mitochondria isolated by the techniques of otherinvestigators who do not find mitochondrial Laj oxidation.Another critical obstacle to the notion of Laj oxidation

within the mitochondrial matrix is the possible violation ofthe first law of thermodynamics as described by Sahlin et al.(24). Separate pathways for cytosolic pyruvate and NADHentry into the mitochondrial matrix provide for a largeoxidation/reduction potential gradient spanning the innermitochondrial membrane. Crucial for maintenance of thisgradient is the existence of a nonequilibrium step withinboth the glycerol phosphate and malate-aspartate shuttles.Neither empirical evidence nor theoretical conjecture hasbeen presented in support of a nonequilibrium step in theintracellular lactate shuttle hypothesis.

LDH IN THEMITOCHONDRIALINTERMEMBRANE SPACE?

In my opinion, the weight of the evidence is against thepresence of LDH in the mitochondrial matrix and, therefore,the oxidation of Laj within the mitochondrial matrix.

However, LDH may be present in the intermembrane spaceof mitochondria, perhaps attached (loosely?) to the innermitochondrial membrane. Baba and Sharma (1), as pre-viously noted, found LDH associated with mitochondria but,at the same time, questioned the permeability of mitochon-dria to lactate, leaving the intermembrane space as a likelylocation in the heart and skeletal muscle. Skilleter and Kun(25) undertook submitochondrial fractionation and arrivedat the conclusion that LDH in intact mitochondria ‘‘isprobably on the outer side of the inner membrane’’ in liver.Deimann et al. (8) used scanning transmission electronmicroscopy and found the reaction product for LDH ‘‘clearlyidentified in the intermembranous space of mitochondria’’ inrabbit glycolytic skeletal muscle. Using proteolytic disruptionof isolated liver mitochondria, Kline et al. (16) concludedthat LDH is ‘‘mainly in the outer membrane and periplasmicspace.’’ Brandt et al. (3) used digitonin fractionation ofmitochondria isolated from rat heart, kidney, liver, andlymphocytes; they reported that ‘‘the mitochondrial LDH islocated primarily in the periplasmic space.’’ Periplasmic spaceand intermembrane space are equivalent terms.

Recent observations of Hashimoto et al. (12) from theBrooks laboratory provided additional detail for the possi-bility of LDH in association with the inner mitochondrialmembrane. Using the techniques of confocal laser scanning

Figure 2. Lactate and pyruvate oxidation in intermyofibrillar (Panel A) and subsarcolemmal (Panel C) mitochondria from rat red and white muscle atdifferent concentrations (0.18, 1.8, and 10 mM). Lactate oxidation is also shown on a 30-fold more sensitive scale in both red and white muscleintermyofibrillar (Panel B) and subsarcolemmal (Panel D) mitochondria. Note negligible direct lactate oxidation in subsarcolemmal and intermyofibrillarmitochondria obtained from both red and white rat skeletal muscle. Compare with values reported by Brooks et al. (5) in Figure 1. Hashimoto and Brooks(11) contend that these results are likely due to loss of mitochondrial LDH in the isolation procedures. (Reprinted from Yoshida, Y., G.P. Holloway,V. Ljubicic, H. Hatta, L.L. Spriet, D.A. Hood, and A. Bonen. Negligible direct lactate oxidation in subsarcolemmal and intermyofibrillar mitochondriaobtained from red and white rat skeletal muscle. J. Physiol. 582:1317Y1335, 2007. Copyright * 2007 Blackwell Publishing. Used with permission.)




microscopy and immunoblotting after immunoprecipitationin L6 skeletal muscle cells, Hashimoto et al. (12) foundevidence suggesting that LDH, MCT1, the single-spantransmembrane glycoprotein CD147, and cytochrome oxi-dase are colocalized in the inner mitochondrial membrane,with the LDH enzyme apparently residing on the outersurface of the inner membrane. They (12) have called thisa lactate oxidation complex and have enumerated theirexperimental evidence supporting the existence of thiscomplex elsewhere (11). Potential location of LDH looselyattached to the outside of the inner mitochondrial mem-brane rekindles the possibility that such LDH might besusceptible to loss during the isolation of mitochondria frommuscle tissue. However, it seems unlikely that LDH(molecular weight of 134,000) can pass through an intact

outer mitochondrial membrane because it is impermeable tomolecules larger than 5000 daltons, whereas NAD+/NADHat a molecular weight of approximately 664 moves throughreadily. It also seems unlikely that LDH within themitochondrial intermembrane space would be susceptible todestruction by proteases used in mitochondrial isolation(trypsin, molecular weight ,23,000; nagarse, molecularweight ,27,000).

INTRACELLULAR LACTATE SHUTTLEREMAINS VIABLE

Despite the serious reservations previously outlined, itremains quite possible that an intracellular lactate shuttle

Figure 3. Illustration of the essential elements of possible intracellular (intramuscular) lactate shuttles, one with lactate dehydrogenase (LDH) in a lactateoxidation complex (12) on the inner mitochondrial membrane and one without LDH attached to the inner membrane or even in the intermembrane space.A high activity of cytosolic LDH is considered to guarantee lactate (Laj) formation in the cytoplasm under virtually all conditions but especially duringexercise. Although Laj could be formed throughout the cytosol, two particular locations are illustrated V one in association with the Na+/K+-ATPasepump in the sarcolemma; and the other, the Ca2+-ATPase in the SR membrane. The sarcolemma is illustrated by the thick double lines at the top of thecartoon, whereas the inner and outer mitochondrial membranes are dramatically enlarged to demonstrate possible Laj pathways. The dashed red andblack lines connecting Pyrj and Laj in the upper left quadrant indicate the near-equilibrium nature of the LDH reaction and the movement of Laj andPyrj mass from a site of net formation to a site of net removal. The gaps in the outer mitochondrial membrane illustrate that it is freely permeable to mostsmall molecules (but probably not permeable to LDH, trypsin, or nagarse; see text). Laj is shown in bold and red and larger than pyruvate (Pyrj) toindicate that Laj is typically present in much higher concentration than Pyrj. Whether Laj were converted back to Pyrj outside the intermembranespace, inside the space, or via a mitochondrial LDH, the resulting NADH + H+ would be shuttled across the inner mitochondrial membrane via the malate-aspartate and glycerol phosphate shuttles. Please note that illustration of these traditional shuttles on the inner mitochondrial membrane is not intendedto imply that their components are limited to this location; the reactions that are outside the inner membrane can occur throughout the cytosol. Pyrj

could be transported across the inner mitochondrial membrane by either a pyruvate carrier, (MPC, arguably more likely) or a monocarboxylate transporter(MCT1), both of which have been identified in the inner membrane. The MCT1/MPC in the lactate oxidation complex is not meant to imply thatHashimoto et al. (12) reported the presence of MPC there; their evidence supports MCT1. To avoid confusion, arrows indicating possible diffusion routesof NAD+ and NADH + H+ are not shown. COX indicates cytochrome oxidase; cLDH, cytosolic lactate dehydrogenase; CD147, single-span transmembraneglycoprotein; ETC II and III, electron transport chain complexes II and III; Gly, glycogen; Glu, glucose; Inner, inner mitochondrial membrane; Laj, lactate;MCT1, monocarboxylate transporter 1; mLDH, mitochondrial LDH; MPC, mitochondrial pyruvate carrier; NADH-dh, NADH dehydrogenase complex I;Outer, outer mitochondrial membrane; Pyrj, pyruvate. [Adapted from Hashimoto, T., R. Hussien, and G.A. Brooks. Colocalization of MCT1, CD147, andLDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex. Am. J. Physiol. Endocrinol. Metab.290:E1237YE1244, 2006. Copyright * 2006 The American Physiological Society. Used with permission.]




operates, albeit without intramatrix lactate metabolism(Fig. 3). It is reasonable to speculate that pyruvate andNADH concentrations are lowest adjacent to the innermitochondrial membrane where the pyruvate carrier and theNADH shuttles (malate-aspartate and glycerol phosphate)are moving pyruvate and NADH equivalents, respectively,into the mitochondria. In other words, actively oxidizingmitochondria would create ‘‘sinks’’ for the utilization ofpyruvate and NADH and, therefore, their uptake fromadjacent cytosolic locations. At the same time, sites ofcellular glycolysis would create driving concentrations ofLaj because the primary end product of glycolysis would beLaj due to the high activity of LDH as describedearlier. This situation would lead to the highest Laj

production and concentration at cytosolic locations remotefrom mitochondria. Then, because of the relatively higher[Laj] as compared with [pyruvatej], Laj would be theprimary species diffusing to areas near mitochondria. The[Laj] is typically approximately 10Y200 times greater than[pyruvatej] in skeletal muscle. Adjacent to mitochondria, orif Hashimoto et al. (12) are correct, in the intermembranespace, Laj and NAD+ would be converted back to pyruvateand NADH via LDH for uptake into the mitochondria. Sucha scheme, arguably analogous to the phosphocreatine shuttle,would be in accord with near equilibrium of the LDHreaction throughout the cytosol and would accommodateready Laj production with subsequent oxidation and less

transport of Laj out of the cell. I should note clearly thatspecific location of LDH, either in the intermembrane spaceor attached to the inner membrane, might not be arequirement for operation of an intracellular lactate shuttle.Finally, despite the evidence for the presence of MCT1 ina lactate oxidation complex (12), it is quite possible,perhaps probable, that pyruvate is transported across theinner mitochondrial membrane via the mitochondrialpyruvate carrier (MPC (14)) that has a very high affinityfor pyruvate.

COMPARTMENTATION OF METABOLISM

The model previously described is consistent with thecompartmentation of metabolism as described in severalstudies. James and colleagues (15) proposed that the Na+/K+-ATPase pump derives its energy heavily from glycolysis thatis closely associated with the pump, an idea that has recentlybeen supported in studies of mechanically skinned skeletalmuscle fibers. There is also considerable evidence for afunctional compartmentation of glycolysis with the SR.Figure 4 provides visual circumstantial evidence for glycolyticcompartmentation with the SR. Is it possible that Laj

derived from glycolysis associated with SR Ca2+ pumpingis shunted toward intermyofibrillar mitochondria, whereasLaj derived from glycolysis affiliated with Na+/K+-ATPase

Figure 4. Transmission electron microscopy (TEM) image of a human skeletal muscle fiber (original magnification �20,000; scale bar = 2 Km). The 60-nm-thick section has been cut precisely on the surface of the myofibril, enabling visualization of the triads situated in the I band on each side of the z line,with paired long mitochondria located transversely at the I band level wrapped around the contractile apparatus and in contact with the SR but clearlyseparated from the t tubules. Traces of longitudinal tubules of SR can be seen as strings from the terminal cisternae toward the fenestrated collar at thelevel of the M band. The abundant glycogen granules are visualized as black dots. Although some glycogen granules are obviously near mitochondria inthe micrograph, the density of granules surrounding the SR is suggestive of glycolytic compartmentation with the SR. Human muscle fibers were fixedwith 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 24 h then rinsed four times in 0.1 M sodium cacodylate buffer, postfixed with1% osmium tetroxide (OsO4) and 1.5% potassium ferrocyanide (K4Fe(CN)6) in 0.1 M sodium cacodylate buffer for 90 min, rinsed two times in 0.1 Msodium cacodylate buffer, dehydrated through a graded series of alcohol, infiltrated with graded mixtures of propylene oxide and Epon, and embedded in100% Epon. Ultra-thin sections (È60 nm) were cut using a Leica Supernova ultramicrotome and contrasted with uranyl acetate and lead citrate. Thesections were examined and photographed in a Philips EM 208 electron microscope and a Megaview III FW camera. Images were taken at �20,000magnification with a precalibrated transmission electron microscope. Figure courtesy of Niels Krtenblad and Joachim Nielsen, University of SouthernDenmark, Odense, Denmark. Not previously published.




activity is shunted toward subsarcolemmal mitochondria?Perhaps future studies will provide evidence in this regard.

CONCLUSIONS

In conclusion, research into muscle Laj metabolismremains a hotbed of activity 200 yr after the report ofelevated Laj in exercised muscle. A major source ofcontention surrounds the exact pathway(s) of intracellularLaj oxidation. The model of interest is the intracellularlactate shuttle. The key questions are as follows: 1) How canLaj be tracked from one intracellular location to another? 2)Where is LDH precisely located inside muscle cells? 3) Doesthe intracellular lactate shuttle model require fixed locationsof LDH to operate? Future studies may provide conclusiveanswers to these queries.

Acknowledgments

The author wishes to thank Matthew L. Goodwin, Andres Hernandez, andJames E. Harris for their criticism and discussion of the manuscript drafts.

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200th anniversary of lactate research in muscle

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