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Purified sarcolemmal membranes from mixed rat hindlimb muscle were solubilized with octylglucoside and the extract subjected to hydroxylapatite (HA) chromatography. Following protein elution with a sodium phosphate gradient and detergent removal by dialysis, the HA eluate was reconstituted into asolectin liposomes using a freeze-thaw procedure. Specific L-[14C]lactate transport activity eluting from the 0.2 M sodium phosphate fraction was 30-fold higher compared with native sarcolemmal vesicles (31.64 versus 1.06 nmol/min per mg). The reconstituted carrier exhibited Michaelis-Menten saturation kinetics with Km and Vmax. values of 46.2 +/- 6.6 mM and 498.7 +/- 17.2 nmol/15 s per mg respectively. L-Lactate transport activity was inhibited 57% by preincubation of proteoliposomes with 10 mM alpha-cyano-4-hydroxycinnamate, a known inhibitor of lactate transport. Analysis of the HA eluates by SDS/PAGE showed the presence of a 34 kDa band corresponding to lactate transport activity. Reconstitution of lactate transport activity eluting from the HA column, together with SDS/PAGE analysis suggests the presence of a 34 kDa polypeptide mediating sarcolemmal lactate exchange in rat skeletal muscle.

Hindlimb muscle sarcolemmal vesicles were purified from three age-matched groups of female Sprague-Dawley rats: sedentary control (CON; n = 10), sprint trained (ST; n = 8), and endurance trained (ET; n = 9). Membrane isolations from the three groups were not significantly different in protein yield or purification index. Blood lactate concentration was determined in resting CON rats and running ET and ST rats during the final week. Both the ST and ET groups were significantly higher in citrate synthase (vs. CON) in the soleus and mid-vastus lateralis. The time course of 1 mM L-(+)-lactate uptake in vesicles from the three groups showed no significant difference at any of the five time points tested under zero-trans conditions. Saturation kinetics were examined at nine lactate concentrations, and Lineweaver-Burk plots revealed no difference between groups in apparent Michaelis-Menten constant or maximal transport velocity. Vesicles from CON and ET rats were used to investigate cis inhibition of 0.1 mM L-(+)-lactate transport by four unlabeled monocarboxylates: L-(+)-lactate, D-(-)-lactate, pyruvate, and alpha-cyanohydroxycinnamate at 0.1, 1.0, and 10 mM. Under pH gradient-stimulated L-(+)-lactate transport conditions, cis inhibition was affected by neither D-(-)-lactate nor endurance training. We conclude that the lactate transporter has distinct cis-inhibitory specificity, is stereospecific, and is stimulated when confronted with parallel lactate and proton gradients but that spring and endurance training do not alter lactate transport rate or capacity under these conditions.

To evaluate the hypothesis that lactate supply is essential to maintain euglycemia during iron deficiency, female Sprague-Dawley rats were assigned to iron-sufficient (50 mg Fe2+/kg diet, +Fe), or iron-deficient (15 mg Fe2+/kg diet, -Fe) dietary groups and were injected with a specific beta 2-adrenergic inhibitor, ICI 118,551 (1.0 mg/kg body wt). Rats were studied at rest or after 30 min of running at 13.4 m/min 0% grade. Dietary iron deficiency decreased hemoglobin concentration 38%, but resting arterial concentrations of glucose ([Glc]), lactate ([La]), or alanine ([Ala]) were unaffected. Administration of ICI 118,551 (beta 2-blockade) decreased [La] and [Glc] 52 and 32% in resting -Fe rats, respectively. beta 2-Blockade attenuated the exercise-induced rise in [La] and decreased [Glc] 31% in exercising -Fe rats. [Ala] were unaffected by iron deficiency or exercise but decreased 24 and 18% because of beta 2-blockade in resting and exercising +Fe rats. Iron deficiency depleted resting liver glycogen concentration 45%, with no additional effect of exercise or beta 2-blockade. beta-Blockade decreased arterial insulin and increased arterial glucagon concentrations in resting -Fe and +Fe rats. During exercise glucagon concentration increased significantly more in -Fe than +Fe rats. Decreased arterial [La] with a corresponding decrease in arterial [Glc] in response to beta 2-blockade support the contention that lactate supply is critical to maintenance of euglycemia in -Fe rats at rest and during exercise.

To determine whether the working muscle is able to sustain ATP homeostasis during a hypoxic insult and the mechanisms associated with energy metabolic adaptations during the acclimatization process, seven male subjects [23 +/- 2 (SE) yr, 72.2 +/- 1.6 kg] were given a prolonged exercise challenge (45 min) at sea level (SL), within 4 h after ascent to an altitude of 4,300 m (acute hypoxia, AH), and after 3 wk of sustained residence at 4,300 m (chronic hypoxia, CH). The prolonged cycle test conducted at the same absolute intensity and representing 51 +/- 1% of SL maximal aerobic power (VO2 max) and between 64 +/- 2 (AH) and 66 +/- 1% (CH) at altitude was performed without a reduction in ATP concentration in the working vastus lateralis regardless of condition. Compared with rest, exercise performed during AH resulted in a greater increase (P < 0.05) in muscle lactate concentration (5.11 +/- 0.68 to 22.3 +/- 6.1 mmol/kg dry wt) than exercise performed either at SL (5.88 +/- 0.85 to 11.5 +/- 3.1) or CH (5.99 +/- 0.88 to 12.4 +/- 2.1). These differences in lactate concentration have been shown to reflect differences in arterial lactate concentration and glycolysis (Brooks et al. J. Appl. Physiol. 71: 333-341, 1991). The reduction in glycolysis at least between AH and CH appears to be accompanied by a tighter metabolic control. During CH, free ADP was lower and the ATP-to-free ADP ratio was increased (P < 0.05) compared with AH.(ABSTRACT TRUNCATED AT 250 WORDS)

There are several goals to this introductory paper in the symposium proceedings, "Current Concepts in Lactate Exchange." First, an attempt is made to set the historical context for the symposium and foreshadow how the paper of each participant contributes to our contemporary understanding of the field. As implied in the symposium title, an emphasis will be placed on the exchange of lactate for other metabolites and ions so that utilization can be temporally and spatially disassociated from formation. Thus, rather than a dead-end metabolite, which only accumulates during exercise, there appears to be great usefulness in the formation, exchange between cells, blood and organs, and utilization of lactic acid (lactate). Specific papers will deal with aspects of lactate release and uptake by skeletal muscle, hepatic lactate balance, the flux of dietary carbohydrate through various lactate pools in the synthesis of liver glycogen, lactate metabolism in the heart, properties of the sarcolemmal lactate transporter, and evolution of a model to predict lactate production from blood measurements. Second, in this review an attempt will be made to present and support a unifying hypothesis (the "lactate shuttle") in which the various aspects of lactate exchange may be integrated and understood. Emphasis will be placed on showing several corollaries between muscle and whole-body lactate metabolism. These are: temporal dependence on lactate uptake and release, the effects of beta-adrenergic stimulation on lactate formation and release, the effect of prior endurance training on lactate metabolism, the effect of lactate on glucose uptake and utilization, and the role of low oxygen tension (hypoxia) in loosening the control of glycolysis. The formation, exchange, and utilization of lactate represents a central means by which the coordination of intermediary metabolism in diverse tissues and different cells within tissues can be accomplished.

To evaluate the role played by gluconeogenesis in blood glucose homeostasis, female Sprague-Dawley rats were injected with mercaptopicolinic acid (MPA), a gluconeogenic inhibitor. Glucose kinetics were assessed by primed, continuous infusion of [U-14C]- and [6(-3)H]glucose via an indwelling jugular catheter at rest and during submaximal exercise at 13.4 m/min on level grade. Blood samples were taken from carotid catheters and analyzed for glucose and lactate concentrations and specific activities. Tissue glycogen samples were obtained from rats after exercise as well as from unexercised animals. When compared with the sham-injected animals, MPA-treated animals had 22% lower (5.92 +/- 0.36 vs. 7.62 +/- 0.21 mM) and 44% higher (1.90 +/- 0.11 vs. 1.32 +/- 0.09 mM) resting arterial glucose and lactate concentrations, respectively. Resting glucose appearance (Ra) rates were 20% lower in the MPA-treated animals (57.2 +/- 7.5 mumol.kg-1.min-1) than in the sham-injected animals (71.1 +/- 12.1 mumol.kg-1.min-1). During exercise, Ra increased to 174.7 +/- 32.8 mumol.kg-1.min-1 in sham-injected animals. In the MPA-treated animals, there was a 35% increase during the first 15 min of exercise, followed by a decrease to the resting values. MPA-treated animals had no measurable glucose recycling at rest or during exercise. Exercise decreased blood glucose concentration (35%) and increased blood lactate concentration (160%) in the MPA-treated animals. Exercising sham-injected animals had increased blood glucose (9.8%) but no change in blood lactate concentration. Moderate depletions in liver and skeletal muscle glycogen contents were observed after exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

Three weeks of dietary iron deficiency in weanling rats resulted in anemia (Hb, 3.9 vs. 14.2 g/dl in controls) and decreased oxidative capacities of skeletal muscle (as much as 90% below control values). Whole-animal maximal O2 consumption (VO2max), measured in a brief treadmill run of progressively increasing work load, was approximately 50% lower for iron-deficient rats than for controls, and maximal endurance capacity (time to exhaustion in a separate treadmill run at a constant, sub-Vo2max work load) was 90% lower for iron-deficient rats than for controls. Exchange transfusion, with packed erythrocytes or plasma, was used to adjust Hb to an intermediate concentration of approximately 9.5 g/dl in both iron-deficient and and control rats. This procedure corrected the Vo2max of iron-deficient rats to within 15% of control values, whereas endurance capacity showed no improvement. Our experimental dissociation of Vo2max and endurance capacity provides further evidence that Vo2max is not the sole determinant of endurance. We propose that defects in Vo2max during iron deficiency result primarily from diminished O2 delivery, whereas decreased endurance capacity reflects impaired muscle mitochondrial function.

In a comparison of traditional and theoretical exercise efficiency calculations male subjects were studied during steady-rate cycle ergometer exercises of "0," 200, 400, 600, and 800 kgm/min while pedaling at 40, 60, 80, and 100 rpm. Gross (no base-line correction), net (resting metabolism as base-line correction), work (unloading cycling as base-line correction), and delta (measurable work rate as base-line correction) efficiencies were computed. The result that gross (range 7.5-20.4%) and net (9.8-24.1%) efficiencies increased with increments in work rate was considered to be an artifact of calculation. A LINEAR OR SLIGHTLY EXPONENTIAL RELATIONSHIP BETWEEN CALORIC OUTPUT AND WORK RATE DICTATES EITHER CONSTANT OR DECREASING EFFICIENCY WITH INCREMENTS IN WORK. The delta efficiency (24.4-34.0%) definition produced this result. Due to the difficulty in obtaining 0 work equivalents, the work efficiency definition proved difficult to apply. All definitions yielded the result of decreasing efficiency with increments in speed. Since the theoretical-thermodynamic computation (assuming mitochondrial P/O = 3.0 and delta G = -11.0 kcal/mol for ATP) holds only for CHO, the traditional mode of computation (based upon VO2 and R) was judged to be superior since R less than 1.0. Assuming a constant phosphorylative-coupling efficiency of 60%, the mechanical contraction-coupling efficiency appears to vary between 41 and 57%.

Lactate metabolism was studied in six normal males using a primed continuous infusion of lactate tracer during continuous graded supine cycle ergometer exercise. Subjects exercised at 49, 98, 147, and 196 W for 6 min at each work load. Blood was sampled from the brachial artery, the iliac vein, and the brachial vein. Arteriovenous differences were determined for chemical lactate concentration and L-[1-14C]-lactate. Tracer-measured lactate extraction was determined from the decrease in lactate radioactivity per volume of blood perfusing the tissue bed. Net lactate release was determined from the change in lactate concentration across the tissue bed. Total lactate release was taken as the sum of tracer-measured lactate extraction and net (chemical) release. At rest the arms and legs showed tracer-measured lactate extraction, as determined from the isotope extraction, despite net chemical release. Exercise elicited an increase in both net lactate release and tracer-measured lactate extraction by the legs. For the legs the total lactate release (net lactate release + tracer-measured lactate extraction) was roughly equal to twice the net lactate release under all conditions. The tracer-measured lactate extraction by the exercising legs was positively correlated to arterial lactate concentration (r = 0.81, P less than 0.001) at the lower two power outputs. The arms showed net lactate extraction during exercise, which was correlated to the arterial concentration (r = 0.86). The results demonstrate that exercising skeletal muscle extracts a significant amount of lactate during net lactate release and that the working skeletal muscle appears to be a major site of blood lactate removal during exercise.

Patterns of postexercise glycogen repletion in heart, skeletal muscle, and liver in the absence of exogenously supplied substrates during the first 4 h of recovery were assessed. Female Wistar rats were run to exhaustion using continuous (1.0 mph, 15% grade) and intermittent (alternate 1-min intervals at 0.5 and 1.5 mph, 15% grade) exercise protocols. Rats at exhaustion were characterized by marked depletion of glycogen in heart (55%), skeletal muscle (94%), and liver (97%). Blood glucose levels at exhaustion (1.33 mumol/g) were only 37% of preexercise levels. There were no significant differences between continuous and intermittent exercise groups for any of the tissue glycogen or blood glucose values. Cardiac muscle was the only tissue capable of complete restoration of glycogen levels while relying exclusively upon endogenous substrates. Concentrations of endogenous substrates present at the end of exercise were insufficient to support restoration of blood glucose levels to preexercise values nor support glycogen repletion in skeletal muscle and liver during the initial 4-h food-restricted postexercise period. With subsequent feeding, skeletal muscle demonstrated a glycogen supercompensation effect at 24 h (181.1 and 191.8% of preexercise levels for continuous and intermittent exercise, respectively). Lactate concentration in all tissues at the point exhaustion (1.5--2.5 times resting levels) were only moderately elevated and returned to preexercise levels within 15 min. It was concluded that lactate removal after exercise contributed only minimally to the repletion of muscle glycogen.

We used endurance training and acute anemia to assess the interactions among maximal oxygen consumption (VO2max), muscle oxidative capacity, and exercise endurance in rats. Animals were evaluated under four conditions: untrained and endurance-trained with each group subdivided into anemic (animals with reduced hemoglobin concentrations) and control (animals with unchanged hemoglobin concentrations). Anemia was induced by isovolemic plasma exchange transfusion. Hemoglobin concentration and hematocrit were decreased by 38 and 41%, respectively. Whole body VO2max was decreased by 18% by anemia regardless of training condition. Anemia significantly reduced endurance by 78% in untrained rats but only 39% in trained animals. Endurance training resulted in a 10% increase in VO2max, a 75% increase in the distance run to exhaustion, and 35, 45, and 58% increases in skeletal muscle pyruvate-malate, alpha-ketoglutarate, and palmitylcarnitine oxidase activities, respectively. We conclude that endurance is related to the interactive effects of whole body VO2max and muscle oxidative capacities for the following reasons: 1) anemic untrained and trained animals had similar VO2max but trained rats had higher muscle oxidative capacities and greater endurance; 2) regardless of training status, the effect of acute anemia was to decrease VO2max and endurance; and 3) trained anemic rats had lower VO2max but had greater muscle oxidative capacity and greater endurance than untrained controls.

The concentration of lactate in the blood is the result of (1) those processes which produce lactate and contribute to its appearance in the blood and (2) those processes which catabolize lactate after its removal from the blood. Consequently, the concentration of lactate in the blood provides minimal information about the rate of lactate production in muscle. The accumulation of lactate beyond the lactate threshold [T(lact)] does provide an indication that the mechanisms of lactate removal fail to keep pace with lactate production. Lactate is produced in skeletal muscle as a direct result of increased metabolic rate and glycolytic carbon flow. Factors which influence lactate production in muscle include: the Vmax of lactic dehydrogenase (LDH), which is several times greater than the combined activities of enzymes which provide alternative pathways of pyruvate metabolism; the kM of LDH for pyruvate, which is sufficiently low to assure maximal stimulation of LDH in the conversion of pyruvate to lactate; and the K'eq of pyruvate to lactate conversion, which exceeds 1000. Recent studies on dog gracilis muscle in situ clearly indicate that lactate production occurs in contracting pure red muscle for reasons other than an O2 limitation on mitochondrial ATP production. In addition to failure of the essential assumption of the anaerobic threshold [T(an)] hypothesis that there exist limitations on O2 availability in muscles of healthy individuals during submaximal exercise, several groups of investigators have produced results which indicate that parameters associated with changes in pulmonary minute ventilation [i.e., the ventilatory threshold, T(vent)] do not always track changes in blood lactate concentration. Therefore, the T(an) hypothesis fails on the bases of theory and prediction. A series of kinetic tracer experiments to better understand lactate kinetics during exercise is proposed.

We determined the recovery time courses of muscle oxidases and liver enzymes after iron administration to iron-deficient rats. Female 21-day-old Sprague-Dawley rats were fed an iron-deficient (3 mg Fe/kg) or a control (50 mg Fe/kg) diet for 3 wk. The deficient rats were then injected with 50 mg Fe as iron dextran/kg body wt (Fe-T) or saline (Fe-) intraperitoneally. At 16, 40, 64, 112, and 180 h after injection, blood and tissue samples were taken to determine hemoglobin concentration (Hb), gastrocnemius glycolytic enzyme and oxidase activities, and liver amino acid catabolic enzyme activities. No changes were observed in any parameter across time in either the Fe- or control (Fe+) rats. In the Fe- rats, Hb, pyruvate + malate (P + M), 2-oxoglutarate (2-OG), and succinate oxidases (SO) were depressed to 33, 36, 44, and 7% of Fe+, respectively (P less than 0.05). At 16 h, Fe-T values were significantly elevated compared with Fe- rats but still only 40, 48, 55 and 10% of controls, respectively. Glutamate dehydrogenase (GDH) and alanine aminotransferase (AAT) of Fe- rats were 174 and 134% of control values (P less than 0.05). By the 180-h time point, Hb, P + M, 2-OG, and SO of Fe-T rats increased to 99, 84, 89, and 43% of Fe+ values, whereas GDH and AAT activities declined to 111 and 106% of controls. Glycolytic enzymes showed no systematic changes with iron deficiency or after iron administration.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of walking against vertical (gradient) and horizontal (trailing weight) forces was made during steady-rate exercise at 0.250, 500, and 750 kg-m/min with speeds of 3,0, 4.5, and 6.0 km/h. In all cases exponential relationships between energy expenditure (calculated from the steady-rate respiration) and increasing work rate and speed were observed which indicated that muscular efficiency during walking is inversely related to speed and work rate. "Work" (level, unloaded walking as the baseline correction), "delta" (measured work rate as the baseline correction), and "instantaneous" (derived from the equation describing the caloric cost of work) efficiencies were computed. All definitions yielded decreasing efficiencies with increasing work rates. At work rates above 250 kg-m/min the curves describing the relationship between energy expenditure and work rate were parallel for vertical and horizontal forces, indicating equivalent efficiencies in this range. Only the delta and instantaneous definitions accurately described these relationships for vertical and horizontal work. Determinations of combined work loads (gradient plus trailing weight) were made and the energy costs of both types of work found to be additive.

High-voltage electron microscopy at 1,500 kV was used to examine mitochondrial morphology in three skeletal muscles of the rat. The soleus, deep portion of the vastus lateralis, and superficial portion of the vastus lateralis muscles were examined to represent slow-twitch oxidative, fast-twitch oxidative, glycolytic, and fast-twitch glycolytic skeletal muscle fiber types, respectively. Muscle samples were removed from six female Wistar rats. The tissues were fixed using standard electron microscopic techniques and were sectioned transversely with respect to muscle fiber orientation to approximately 0.5-micron thickness. The sections were stained on grids with uranyl acetate and Reynolds' lead citrate. Results revealed a mitochondrial reticulum in all three skeletal muscle fiber types. Stereological analyses of the electron micrographs were performed to measure volume densities and surface-to-volume ratios of mitochondria in the muscle samples. Cross-sectional volume densities of mitochondria in the soleus (15.5 +/- 1%) and deep portion of the vastus lateralis (16.1 +/- 2%) were significantly greater (P less than 0.05) than in the superficial portion of the vastus lateralis (8.7 +/- 1%). Surface-to-volume ratios of mitochondria were not significantly different between fiber types. It was concluded that the mitochondria in mammalian limb skeletal muscle are a reticulum, or network.