40
Biochimica et Biophysica Acta, 404 (1975) 40--48
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27714
L A C T A T E F O R M A T I O N BY R A T SMALL I N T E S T I N E IN V I T R O
HENRY J. L E E S E and J. RAMSEY BRONK Department of Biology, University of York, York Y01 5DD (U.K.)
(Received March 10th, 1975)
Summary The f o r m a t i o n of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate o u t p u t by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting a b o u t 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate o u t p u t up to 8 min. The time course o f lactate o u t p u t suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate form at i on by rings and muscle fr o m rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na* in the incubating medium were replaced by K ÷, lactate f o r m a t i o n by mucosal slices and rings was considerably reduced. Measurements of tissue lactate c o n t e n t before and during incubation revealed t h at a b o u t three-quarters of the lactate released by mucosal slices during the first 30 s o f incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant bot h in the presence and absence of glucose so t h a t the lactate released into the incubation medium is equivalent to the lactate f o r m e d by the slices. The role of the various tissue c o m p o n e n t s of the small intestine in lactate f o r m a t i o n is discussed in relation to sites of glucose entry.
Introduction Lactate f o rm a t i on by the small intestine has been studied extensively since the d e m o n s t r a t i o n of a rapid rate of glycolysis in this organ by Warburg et al. [ 1 ] . Much of the work on this topic has been carried out with mucosal scrapings or isolated cells incubated for periods of 1 h or longer. We have
41 recently shown [2] that the mucosa of rat small intestine is substantially depleted of endogenous adenine nucleotides in a matter of minutes, and in this condition the rate of tissue respiration is greatly diminished. Consequently, we have made a study of lactate formation by mucosal slices of rat jejunum incubated for short periods of 8 min o r less. The preparation that we used for most of this work consists solely of mucosal epithelium, uncontaminated by lamina propria or muscularis mucosa. However, for comparison we have also studied lactate production b y jejunal rings and smooth muscle in view of a recent report [3] on the contribution that the various parts of the gut wall make to the metabolism of the whole intestine. Methods Mucosal slices and rings of rat jejunum were prepared and incubated b y the method of Bronk and Parsons [4]. The composition of the incubation medium (a modified Krebs-Ringer bicarbonate) was as follows: 118 mM NaC1, 25 mM NaHCO3, 4.74 mM KC1, 1.19 mM MgSO4, 1.17 mM KH2PO4, 1.70 mM CaC12. Small pieces (approx. 5 mg dry wt) of the tissue remaining after removal of mucosal slices, were incubated as the 'muscle preparation'. Although this preparation consisted chiefly of muscle it contained all the tissues normally underlying the epithelial layer. In the experiments with mucosal slices, rings and muscle, 100-pl aliquots of the incubation medium were taken at intervals for the determination of lactate, b y the automated fluorimetric method of Leese and Bronk [5]. Stringent precautions were taken to avoid contaminating the samples with lactic acid from the surface of the fingers, since the slightest contact with the skin was sufficient to introduce a large error into the results. Samples were taken with an automatic 100-pl pipette (Oxford Laboratories) and added to Autoanalyser cups containing 2 ml distilled water (also pipetted automatically). The cups were then transferred to the analytical system with forceps. The lactate content of tissue samples were determined after centrifugation of the tissue from the medium as previously described [6].
Expression of results Lactate appearance in the medium bathing the mucosal slices and rings was expressed as pmol/g dry weight; the dry weight of the tissues was derived from the wet weight measured at the end of the experiment [6]. The muscle preparation was weighed on a torsion balance prior to incubation, and for this tissue, the lactate released into the medium was expressed as pmol/g dry weight, employing a dry weight/wet weight ratio of 0.20. Tissue lactate was also expressed as pmol/g dry weight. Results
Fig. 1 shows the increase in the lactate content of the incubation medium when mucosal slices were prepared and incubated in the absence of glucose (a) in normal Krebs-Ringer bicarbonate medium and (b) in a medium in which Na ÷ was completely replaced with K ÷. In both cases there was a rapid initial release
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of lactate which was of short duration and which was followed by a very slow rise in the lactate content of the medium. Although approximately twice as much lactate was always liberated in the sodium medium as compared with the potassium medium, there was considerable day to day variation in the absolute amounts. In subsequent experiments additions of glucose were made after a 30-s preincubation, rather than at zero time, since the initial lactate release was virtually complete by this time. The results obtained when 11.1 mM glucose was added to the mucosal slices after a 30-s preincubation are s h o w n in Fig. 2. The 30-s p o i n t represents the level of lactate in the medium prior to glucose 5C
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N a + ( e ) o r K + ( o ) as t h e p r e d o m i n a n t c a t i o n i n t h e i n c u b a t i o n m e d i u m . G l u c o s e w a s a d d e d t o a c o n c e n t r a tion of 11.1 mM after 30 s preincubation. T h e first p o i n t , i n e a c h c a s e , r e p r e s e n t s t h e l a c t a t e c o n t e n t o f t h e m e d i u m p r i o r t o g l u c o s e a d d i t i o n . V a l u e s a r e m e a n ± S . E . o4 at l e a s t f o u r d e t e r m i n a t i o n s .
43 addition. In the normal sodium-containing medium, glucose addition initiated a short period of rapid lactate release, lasting about 30 s, which was followed by a steady rise, greater in magnitude than in the corresponding period in the absence of glucose. The total a m o u n t of lactate formed following glucose addition (i.e. from 30 s to 8.5 min) was 24 pmol/g dry weight or about three times the a m o u n t formed in its absence 7.0 pmol/g dry weight. In the high potassium medium, over four times as much lactate was formed in the presence of glucose (9.7 pmol/g dry weight) than in its absence (2.3 pmol/g dry weight), but in each case the absolute amounts were less than those produced in the presence of sodium. The time course of lactate release indicates that there is an apparent negative Pasteur effect in isolated jejunal mucosa, since we have previously shown [2], that the QO2 of mucosal slices steadily diminishes under the conditions used for the experiments reported in Fig. 2 and this decrease in respiration would normally be expected to be associated with an increase in the rate of lactate formation as the contribution of mitochondrial respiration to the energy supply of this tissue decreases. Since ADP and AMP are thought to be important mediators of the Pasteur effect, the failure of lactate formation to increase as the oxygen uptake decreased could have been due to a shortage of phosphate acceptors. Experiments were therefore carried out to determine the rate of lactate formation following the addition of I mM ADP, since this rapidly restores the ADP and ATP content of mucosal slices [7]. For these experiments the preincubation period was extended from 30 s to 4 min to maximise the nucleotide depletion in the controls and the results are given in Fig. 3. When glucose was added alone, after a 4-min preincubation, the initial burst of lactate formation was smaller than that obtained when glucose was added after 30 s preincubation, and the total quantity of lactate formed in the 4--8-min 4O
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Fig. 3. Effect of glucose with or without A D P a d d e d after 4 m i n preincubation on lactate output b y mucosal slices of rat jejunum, incubated in a m e d i u m in which N a + was the predominant cation, e, glucose (11.1 m M ) without A D P ; m, glucose (11.1 m M ) with A D P (1 raM). The first point in each condition represents the lactate content of the m e d i u m prior to substrate addition, Values are m e a n +S.E. of at least four determinations.
44
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Fig. 4. E f f e c t o f g l u c o s e 6 - p h o s p h a t e a d d e d a f t e r 3 0 s o r 4 m i n p r e i n c u b a t i o n ( w i t h o r w i t h o u t A D P ) o n l a c t a t e o u t p u t b y m u c o s a l s l i c e s o f r a t j e j u n u m , i n c u b a t e d in a m e d i u m in w h i c h s o d i u m w a s t h e predominant cation, a glucose 6-phosphate (11.1 raM) added after 30 s preincubation; • glucose 6-phosp h a t e ( 1 1 . 1 r a M ) w i t h o u t A D P a d d e d a f t e r 4 m i n p r e i n c u b a t i o n ; ~., g l u c o s e 6 - p h o s p h a t e ( 1 1 . 1 m M ) w i t h A D P (1 m M ) a d d e d a f t e r 4 r a i n p r e i n c u b a t i o n . The first point in each condition represents the lactate c o n t e n t o f t h e m e d i u m p r i o r t o s u b s t r a t e a d d i t i o n . V a l u e s a r e m e a n • S.E. o f a t l e a s t f o u r d e t e r m i n a t i o n s .
period (8.6 p m o l / d r y wt) was only slightly greater than that formed in the equivalent 4--8-min period when glucose was added after 30 s (6.9 pmol/g dru wt). The addition of ADP failed to increase lactate formation in the presence of glucose, so that the depletion of this and the ot her adenine nucleotides cannot explain the lack of a Pasteur effect in mucosal tissue. A n o t h e r possible explanation for the failure of the slices to maintain lactate p r o d u c t i o n could be the loss of glycolytic intermediates from the cells. If this were true, glucose 6-phosphate might be expect ed to stimulate lactate f o rmatio n since hexokinase activity has been shown to be rate limiting for gly.colytic activity in cell-free systems. A series of incubations were carried out in which 11.1 mM glucose 6-phosphate was added after a preincubation period of either 30 s or 4 min; I mM ADP also added in some of the experiments in which there was a 4-min preincubation. However Fig. 4 shows that glucose 6-phosphate p r o d u c e d only a slight stimulation of lactate f o r m a t i o n by mucosal slices with or w i t h o u t ADP. These results suggest that our failure to observe a sustained rate of lactate f or m at i on in the slices was n o t due to a loss of glucose 6-phosphate since the data in Fig. 4 indicates that the epithelial cells remain largely impermeable to this intermediate. Although it seemed unlikely that 11.1 mM glucose was below the concentration required f or m a x i m u m glycolytic activity by the mucosat slices, lactate p r o d u c t i o n was measured at a series of glucose concent rat i ons in the range 0.5--56 mM. The results indicated that while the amounts of lactate f o r m e d with 5.55, 11.1, 16.5, 28 and 56 mM glucose were all about 25 pmol/g dry weight per 8 min, less lactate was f o r m e d with 0.5 mM glucose (10.7 pmol/g dry wt per 8 min). However, f r om these experiments it was obvious that 11.1 mM glucose was adequate to given maximal rates of lactate formation. Having described the pattern of lactate f o r m a t i o n by mucosal slices, it was of interest to com pa r e these results with those for rings of small intestine and
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Fig. 5. L a c t a t e o u t p u t b y m u c o s a l slices, rings, and m u s c l e of rat j e j u n u m , i n c u b a t e d in a m e d i u m in w h i c h Na + was the p r e d o m i n a n t c a t i o n , e , m u c o s a l slices; o, rings; A m u s c l e . G l u c o s e ( 1 1 . 1 raM) a d d e d after 3 0 s p r c i n c u b a t i o n . T h e first p o i n t in each c o n d i t i o n r e p r e s e n t s the l a c t a t e c o n t e n t of t h e m e d i u m prior t o s u b s t r a t e a d d i t i o n . V a l u e s are m e a n ±S.E. o f at least five d e t e r m i n a t i o n s .
strips of s m o o t h muscle from which the mucosa had been removed. The results of such a comparison are given in Fig. 5. Muscle and rings of whole wall are seen to behave differently from the mucosal slices in that they fail to produce an initial burst of lactate release after glucose addition. Instead these preparations show a progressive increase in the rate of lactate formation which was
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Fig. 6. L a c t a t e o u t p u t by rings o f rat j e j u n u m i n c u b a t e d u n d e r d i f f e r e n t ionic c o n d i t i o n s , o, s o d i u m - c o n taining m e d i u m ; e , p o t a s s i u m - c o n t a i n i n g m e d i u m . G l u c o s e ( 1 1 . 1 raM) a d d e d a f t e r 3 0 s p r e i n c u b a t i o n . V a l u e s are m e a n -+S.E. o f f o u r d e t e r m i n a t i o n s .
46 more pronounced in the rings than in the muscle. We have measured the oxygen uptake of rings (Leese and Bronk unpublished observations) and found that like mucosal slices, the QO2 declines as the incubation proceeds, so that the pattern of lactate formation by rings is consistent with a positive Pasteur effect. The substitution of K ~ for Na ~ in the incubating medium abolishes the active transport of glucose in rat jejunal rings [9]. It was of interest to know whether such a limitation of glucose entry would cause a parallel reduction in lactate production. The results of such experiments are shown in Fig. 6, which indicates that the high K ÷ medium reduced lactate formation to about onethird the level found in the sodium medium. Discussion We have studied lactate formation in mucosal slices, rings of whole wall and strips of the underlying smooth muscle of rat jejunum and found that the time course of lactate formation by each of these preparations was different, qualitatively and quantitatively from the other two. Our results illustrate the danger of assuming from in vitro studies that the total capacity of the intestine to produce a particular metabolite is the sum of the capacities of its individual parts, since the time course of lactate formation by rings of whole wall could not be predicted from the data obtained with mucosal and muscle layers separately. A similar conclusion was reached by Evans and Burdett [3] working with isolated epithelial cells of guinea pig small intestine. The observation that lactate was released from mucosal slices incubated w i t h o u t substrate in a short burst lasting about 30 s suggested that it may have been present in the tissue before the incubation began. Consequently we measured the lactate content of the mucosal tissue prior to incubation and found it to be 21 -+ 2 (6) pmol/g dry weight. This corresponded well with the initial a m o u n t of lactate released when the slices were prepared and incubated in the sodihm medium and suggested that most of this lactate was n o t produced by glycolysis during the first few seconds of incubation. After the initial release, we found that the lactate content of the tissue fell to 5 pmol/g dry weight, i.e. approx. 1 mM (assuming that 1 g dry weight tissue is associated with 5.25 ml tissue water) or about five times the concentration in the medium, and remained at this level irrespective of whether glucose was present in the medium or not t h r o u g h o u t the rest of the incubation period (4.4 -+ 0.2 (8) pmol/g dry weight at 8 min). This is in agreement with the data of Wilson [10] who showed that segments of intestine perfused according to the method of Fisher and Parsons [11], and everted sacs could sustain concentration gradients of lactate (serosal to mucosal) or up to nine times and 6--20 times, respectively. The values for tissue lactate content indicate that about three-quarters of the lactate released into the medium during the first 30 s was present in the tissue initially. After the first 30 s the tissue lactate c o n t e n t remained constant and therefore the lactate released to the medium provided a true messure of lactate production. It is significant that the burst of extra lactate formed on glucose addition after a 30-s preincubation represents net lactate synthesis since the lactate c o n t e n t of the tissue does not alter under these conditions. However, we must stress that this correspondence between lactate appearance and
47 lactate formation does not apply to the initial 30 s of incubation. It is interesting to note that substantially less lactate was released from slices prepared and incubated in high potassium medium (Fig. 1). It is likely that the preparation of mucosal slices in a Na ÷ medium results in Na ÷ uptake particularly in the cold, and that on incubation, an efflux Of Na ÷ and anions takes place across the epithelial cell wall to restore the original intracellular ionic concentrations. If this is the case, then the use of a medium high in potassium, would tend to prevent Na ÷ uptake so that there would be less Na ÷ efflux and loss of accompanying anions (such as lactate}. This explanation could account for the pattern of lactate release shown in Fig. 1. Lactate was formed by slices incubated in the presence of glucose in two stages (Fig. 2); a rapid phase (17 pmol/min per g dry wt) immediately following glucose addition and lasting approx. 1 min followed by a slow steady rate of production (1.8 pmol/min per g dry wt) lasting up to 8 min. This is the first time such a pattern of lactate formation has been reported for rat small intestine, and it seems likely that the present results were obtained because we used the mucosal slice preparation. We found no initial burst of lactate formation either with rings of whole wall or with slices of the underlying muscle tissue. However, the fact that we obtained an apparent Crabtree effect with the mucosal slices and not with rings is probably because glucose entered the mucosal cells via the latero-basal membrane in the slices. This view is supported by the fact that qualitatively the same pattern of lactate release occurred in both sodium and potassium media; both media support glucose uptake in slices but only the sodium medium supports glucose uptake in rings [9]. A number of groups have c o m m e n t e d on the apparent lack of a Pasteur effect in jejunal mucosa [12--18], but as far as we are aware, no-one has put forward the apparently simple explanation, that this phenomenon could be due to a reduction in glucose uptake during hypoxia or anaerobiosis [19], and that this limited the supply of substrate for glycolysis. This idea is amenable to testing, although the intracellular distribution of glucose during absorption is u n k n o w n and it is not clear whether all the glucose entering the epithelial cells is accessible to the glycolytic enzymes. The results in Fig. 5, show that there was a greater lactate formation by intact rings, than by mucosal slices and underlying muscle, and one possible explanation is to suppose that the muscle c o m p o n e n t of the rings is responsible for producing the bulk of the lactate, while the mucosal c o m p o n e n t serves as an active transport system, absorbing glucose from the medium and passing it to the muscle. When rings are incubated in a high potassium medium, the uptake of glucose by jejunal rings is drastically reduced [9], and the concomitant reduction in lactate formation under these conditions strongly suggests that glucose stemming from mucosal entry is the predominant substrate for glycolysis in this preparation.
Acknowledgements We thank Mrs L. Clift for excellent technical assistance and the Medical Research Council for the grant which made this research possible.
48
References ] 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
W a r b u r g , O., Posener, K. a n d Negelein, E. ( 1 9 2 4 ) B i o c h e m . Z. 152, 3 0 9 - - 3 4 4 B r o n k , J.R. a n d Leese, H.J. ( 1 9 7 3 ) J. Physiol. L o n d o n 235, 1 8 3 - - 1 9 6 Evans, E.M. a n d B u r d e t t , K. ( 1 9 7 3 ) G u t 14, 9 8 - - 1 0 3 B r o n k , J.R. a n d Parsons, D.S. ( 1 9 6 5 ) B i o c h i m . Biophys. A c t a 107, 3 9 7 4 0 4 Leese, H.J. a n d B r o n k , J.R. ( 1 9 7 2 ) Anal. B i o c h e m . 45, 2 1 1 - - 2 2 1 B r o n k , J.R. a n d Leese, H.J. ( 1 9 7 4 ) J. Physiol. L o n d o n 2 4 1 , 271 - 2 8 6 B r o n k , J.R. a n d Leese, H.J. ( 1 9 7 3 ) J. Physiol. L o n d o n 235, 1 8 3 - - 1 9 6 Srivastava, L.M. a n d Hiibscher, G. ( 1 9 6 6 ) B i o c h e m . J. 100, 4 5 8 4 6 6 B r o n k , J.R. a n d Leese, tt.J, ( 1 9 7 4 ) T r a n s p o r t at the cellular level (Sleigh, M. a n d J e n n i n g s , D,H., eds), p. 283, 2 8 t h Syrup. Soc. Exp. Biol. C a m b r i d g e U n i v e r s i t y Press Wilson, T.H. ( 1 9 5 4 ) B i o c h e m . J. 56, 5 2 1 - - 5 2 7 Fisher, R.B. a n d Parsons, D.S. ( 1 9 4 9 ) J. Physiol. L o n d o n 110, 2 8 1 - - 2 9 3 Dickens, F. a n d Weil-Malherbe, H. ( 1 9 4 1 ) B i o c h e m . J. 35, 7-~15 Wilson, T.H. a n d W i s e m a n , G. ( 1 9 5 4 ) J. Physiol. L o n d o n 123, 1 2 6 - - 1 3 0 S t e r n , B.K. a n d Reilly, R.W, ( 1 9 6 5 ) N a t u r e 205, 5 6 3 - - 5 6 5 L o h m a n n , V,, G r a e t z , H.O. a n d L a n g e n , P. ( 1 9 6 6 ) C u r r e n t A s p e c t s of B i o c h e m i c a l E n e r g e t i c s ( K a p l a n , N.O. a n d K e n n e d y , E.P., eds), pp. 1 1 1 - - 1 2 6 , A c a d e m i c Press, N e w Y o r k T e j w a n i , G.A. a n d R a m a i a h , A. ( 1 9 7 1 ) B i o c h e m . J. 125, 5 0 7 - - 5 1 4 L a m e r s , J.M.J. a n d H ~ l s m a n n , W.C. ( 1 9 7 2 ) B i o c h i m . Bioph~'s. A c t a 275, 4 9 1 - - 4 9 5 T e j w a n i , G.A., K a u r , J., A n a n t h a n a r a y a n a n , M. a n d Rarnaigh, A. ( 1 9 7 4 ) B i o c h i m . Biophys. A c t a 370, 120--129 Faust, R.A. ( 1 9 6 2 ) B i o c h i m . Biophys. A c t a 60, 6 0 4 - - 6 1 4
Biochimica et Biophysica Acta, 404 (1975) 40--48
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27714
L A C T A T E F O R M A T I O N BY R A T SMALL I N T E S T I N E IN V I T R O
HENRY J. L E E S E and J. RAMSEY BRONK Department of Biology, University of York, York Y01 5DD (U.K.)
(Received March 10th, 1975)
Summary The f o r m a t i o n of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate o u t p u t by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting a b o u t 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate o u t p u t up to 8 min. The time course o f lactate o u t p u t suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate form at i on by rings and muscle fr o m rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na* in the incubating medium were replaced by K ÷, lactate f o r m a t i o n by mucosal slices and rings was considerably reduced. Measurements of tissue lactate c o n t e n t before and during incubation revealed t h at a b o u t three-quarters of the lactate released by mucosal slices during the first 30 s o f incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant bot h in the presence and absence of glucose so t h a t the lactate released into the incubation medium is equivalent to the lactate f o r m e d by the slices. The role of the various tissue c o m p o n e n t s of the small intestine in lactate f o r m a t i o n is discussed in relation to sites of glucose entry.
Introduction Lactate f o rm a t i on by the small intestine has been studied extensively since the d e m o n s t r a t i o n of a rapid rate of glycolysis in this organ by Warburg et al. [ 1 ] . Much of the work on this topic has been carried out with mucosal scrapings or isolated cells incubated for periods of 1 h or longer. We have
41 recently shown [2] that the mucosa of rat small intestine is substantially depleted of endogenous adenine nucleotides in a matter of minutes, and in this condition the rate of tissue respiration is greatly diminished. Consequently, we have made a study of lactate formation by mucosal slices of rat jejunum incubated for short periods of 8 min o r less. The preparation that we used for most of this work consists solely of mucosal epithelium, uncontaminated by lamina propria or muscularis mucosa. However, for comparison we have also studied lactate production b y jejunal rings and smooth muscle in view of a recent report [3] on the contribution that the various parts of the gut wall make to the metabolism of the whole intestine. Methods Mucosal slices and rings of rat jejunum were prepared and incubated b y the method of Bronk and Parsons [4]. The composition of the incubation medium (a modified Krebs-Ringer bicarbonate) was as follows: 118 mM NaC1, 25 mM NaHCO3, 4.74 mM KC1, 1.19 mM MgSO4, 1.17 mM KH2PO4, 1.70 mM CaC12. Small pieces (approx. 5 mg dry wt) of the tissue remaining after removal of mucosal slices, were incubated as the 'muscle preparation'. Although this preparation consisted chiefly of muscle it contained all the tissues normally underlying the epithelial layer. In the experiments with mucosal slices, rings and muscle, 100-pl aliquots of the incubation medium were taken at intervals for the determination of lactate, b y the automated fluorimetric method of Leese and Bronk [5]. Stringent precautions were taken to avoid contaminating the samples with lactic acid from the surface of the fingers, since the slightest contact with the skin was sufficient to introduce a large error into the results. Samples were taken with an automatic 100-pl pipette (Oxford Laboratories) and added to Autoanalyser cups containing 2 ml distilled water (also pipetted automatically). The cups were then transferred to the analytical system with forceps. The lactate content of tissue samples were determined after centrifugation of the tissue from the medium as previously described [6].
Expression of results Lactate appearance in the medium bathing the mucosal slices and rings was expressed as pmol/g dry weight; the dry weight of the tissues was derived from the wet weight measured at the end of the experiment [6]. The muscle preparation was weighed on a torsion balance prior to incubation, and for this tissue, the lactate released into the medium was expressed as pmol/g dry weight, employing a dry weight/wet weight ratio of 0.20. Tissue lactate was also expressed as pmol/g dry weight. Results
Fig. 1 shows the increase in the lactate content of the incubation medium when mucosal slices were prepared and incubated in the absence of glucose (a) in normal Krebs-Ringer bicarbonate medium and (b) in a medium in which Na ÷ was completely replaced with K ÷. In both cases there was a rapid initial release
42 "D 4C
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F i g . 1. L a c t a t e o u t p u t b y m u c o s a l s l i c e s o f r a t j e j u n u m . S l i c e s w e r e i n c u b a t e d w i t h o u t s u b s t r a t e w i t h N a + ( e ) o r K + ( ) as t h e p r e d o m i n a n t c a t i o n i n t h e i n c u b a t i o n m e d i u m . V a l u e s a r e m e a n ~-S.E. o f at l e a s t f i v e determinations.
of lactate which was of short duration and which was followed by a very slow rise in the lactate content of the medium. Although approximately twice as much lactate was always liberated in the sodium medium as compared with the potassium medium, there was considerable day to day variation in the absolute amounts. In subsequent experiments additions of glucose were made after a 30-s preincubation, rather than at zero time, since the initial lactate release was virtually complete by this time. The results obtained when 11.1 mM glucose was added to the mucosal slices after a 30-s preincubation are s h o w n in Fig. 2. The 30-s p o i n t represents the level of lactate in the medium prior to glucose 5C
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F i g . 2. E f f e c t o f g l u c o s e o n l a c t a t e o u t p u t
by mucosal
slices of rat jejunum.
Slices were incubated
with
N a + ( e ) o r K + ( o ) as t h e p r e d o m i n a n t c a t i o n i n t h e i n c u b a t i o n m e d i u m . G l u c o s e w a s a d d e d t o a c o n c e n t r a tion of 11.1 mM after 30 s preincubation. T h e first p o i n t , i n e a c h c a s e , r e p r e s e n t s t h e l a c t a t e c o n t e n t o f t h e m e d i u m p r i o r t o g l u c o s e a d d i t i o n . V a l u e s a r e m e a n ± S . E . o4 at l e a s t f o u r d e t e r m i n a t i o n s .
43 addition. In the normal sodium-containing medium, glucose addition initiated a short period of rapid lactate release, lasting about 30 s, which was followed by a steady rise, greater in magnitude than in the corresponding period in the absence of glucose. The total a m o u n t of lactate formed following glucose addition (i.e. from 30 s to 8.5 min) was 24 pmol/g dry weight or about three times the a m o u n t formed in its absence 7.0 pmol/g dry weight. In the high potassium medium, over four times as much lactate was formed in the presence of glucose (9.7 pmol/g dry weight) than in its absence (2.3 pmol/g dry weight), but in each case the absolute amounts were less than those produced in the presence of sodium. The time course of lactate release indicates that there is an apparent negative Pasteur effect in isolated jejunal mucosa, since we have previously shown [2], that the QO2 of mucosal slices steadily diminishes under the conditions used for the experiments reported in Fig. 2 and this decrease in respiration would normally be expected to be associated with an increase in the rate of lactate formation as the contribution of mitochondrial respiration to the energy supply of this tissue decreases. Since ADP and AMP are thought to be important mediators of the Pasteur effect, the failure of lactate formation to increase as the oxygen uptake decreased could have been due to a shortage of phosphate acceptors. Experiments were therefore carried out to determine the rate of lactate formation following the addition of I mM ADP, since this rapidly restores the ADP and ATP content of mucosal slices [7]. For these experiments the preincubation period was extended from 30 s to 4 min to maximise the nucleotide depletion in the controls and the results are given in Fig. 3. When glucose was added alone, after a 4-min preincubation, the initial burst of lactate formation was smaller than that obtained when glucose was added after 30 s preincubation, and the total quantity of lactate formed in the 4--8-min 4O
g l u c o s e -+ A D P
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Fig. 3. Effect of glucose with or without A D P a d d e d after 4 m i n preincubation on lactate output b y mucosal slices of rat jejunum, incubated in a m e d i u m in which N a + was the predominant cation, e, glucose (11.1 m M ) without A D P ; m, glucose (11.1 m M ) with A D P (1 raM). The first point in each condition represents the lactate content of the m e d i u m prior to substrate addition, Values are m e a n +S.E. of at least four determinations.
44
40
glucose 6 - phosphate
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Fig. 4. E f f e c t o f g l u c o s e 6 - p h o s p h a t e a d d e d a f t e r 3 0 s o r 4 m i n p r e i n c u b a t i o n ( w i t h o r w i t h o u t A D P ) o n l a c t a t e o u t p u t b y m u c o s a l s l i c e s o f r a t j e j u n u m , i n c u b a t e d in a m e d i u m in w h i c h s o d i u m w a s t h e predominant cation, a glucose 6-phosphate (11.1 raM) added after 30 s preincubation; • glucose 6-phosp h a t e ( 1 1 . 1 r a M ) w i t h o u t A D P a d d e d a f t e r 4 m i n p r e i n c u b a t i o n ; ~., g l u c o s e 6 - p h o s p h a t e ( 1 1 . 1 m M ) w i t h A D P (1 m M ) a d d e d a f t e r 4 r a i n p r e i n c u b a t i o n . The first point in each condition represents the lactate c o n t e n t o f t h e m e d i u m p r i o r t o s u b s t r a t e a d d i t i o n . V a l u e s a r e m e a n • S.E. o f a t l e a s t f o u r d e t e r m i n a t i o n s .
period (8.6 p m o l / d r y wt) was only slightly greater than that formed in the equivalent 4--8-min period when glucose was added after 30 s (6.9 pmol/g dru wt). The addition of ADP failed to increase lactate formation in the presence of glucose, so that the depletion of this and the ot her adenine nucleotides cannot explain the lack of a Pasteur effect in mucosal tissue. A n o t h e r possible explanation for the failure of the slices to maintain lactate p r o d u c t i o n could be the loss of glycolytic intermediates from the cells. If this were true, glucose 6-phosphate might be expect ed to stimulate lactate f o rmatio n since hexokinase activity has been shown to be rate limiting for gly.colytic activity in cell-free systems. A series of incubations were carried out in which 11.1 mM glucose 6-phosphate was added after a preincubation period of either 30 s or 4 min; I mM ADP also added in some of the experiments in which there was a 4-min preincubation. However Fig. 4 shows that glucose 6-phosphate p r o d u c e d only a slight stimulation of lactate f o r m a t i o n by mucosal slices with or w i t h o u t ADP. These results suggest that our failure to observe a sustained rate of lactate f or m at i on in the slices was n o t due to a loss of glucose 6-phosphate since the data in Fig. 4 indicates that the epithelial cells remain largely impermeable to this intermediate. Although it seemed unlikely that 11.1 mM glucose was below the concentration required f or m a x i m u m glycolytic activity by the mucosat slices, lactate p r o d u c t i o n was measured at a series of glucose concent rat i ons in the range 0.5--56 mM. The results indicated that while the amounts of lactate f o r m e d with 5.55, 11.1, 16.5, 28 and 56 mM glucose were all about 25 pmol/g dry weight per 8 min, less lactate was f o r m e d with 0.5 mM glucose (10.7 pmol/g dry wt per 8 min). However, f r om these experiments it was obvious that 11.1 mM glucose was adequate to given maximal rates of lactate formation. Having described the pattern of lactate f o r m a t i o n by mucosal slices, it was of interest to com pa r e these results with those for rings of small intestine and
45
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Fig. 5. L a c t a t e o u t p u t b y m u c o s a l slices, rings, and m u s c l e of rat j e j u n u m , i n c u b a t e d in a m e d i u m in w h i c h Na + was the p r e d o m i n a n t c a t i o n , e , m u c o s a l slices; o, rings; A m u s c l e . G l u c o s e ( 1 1 . 1 raM) a d d e d after 3 0 s p r c i n c u b a t i o n . T h e first p o i n t in each c o n d i t i o n r e p r e s e n t s the l a c t a t e c o n t e n t of t h e m e d i u m prior t o s u b s t r a t e a d d i t i o n . V a l u e s are m e a n ±S.E. o f at least five d e t e r m i n a t i o n s .
strips of s m o o t h muscle from which the mucosa had been removed. The results of such a comparison are given in Fig. 5. Muscle and rings of whole wall are seen to behave differently from the mucosal slices in that they fail to produce an initial burst of lactate release after glucose addition. Instead these preparations show a progressive increase in the rate of lactate formation which was
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j f J ~ ~
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Fig. 6. L a c t a t e o u t p u t by rings o f rat j e j u n u m i n c u b a t e d u n d e r d i f f e r e n t ionic c o n d i t i o n s , o, s o d i u m - c o n taining m e d i u m ; e , p o t a s s i u m - c o n t a i n i n g m e d i u m . G l u c o s e ( 1 1 . 1 raM) a d d e d a f t e r 3 0 s p r e i n c u b a t i o n . V a l u e s are m e a n -+S.E. o f f o u r d e t e r m i n a t i o n s .
46 more pronounced in the rings than in the muscle. We have measured the oxygen uptake of rings (Leese and Bronk unpublished observations) and found that like mucosal slices, the QO2 declines as the incubation proceeds, so that the pattern of lactate formation by rings is consistent with a positive Pasteur effect. The substitution of K ~ for Na ~ in the incubating medium abolishes the active transport of glucose in rat jejunal rings [9]. It was of interest to know whether such a limitation of glucose entry would cause a parallel reduction in lactate production. The results of such experiments are shown in Fig. 6, which indicates that the high K ÷ medium reduced lactate formation to about onethird the level found in the sodium medium. Discussion We have studied lactate formation in mucosal slices, rings of whole wall and strips of the underlying smooth muscle of rat jejunum and found that the time course of lactate formation by each of these preparations was different, qualitatively and quantitatively from the other two. Our results illustrate the danger of assuming from in vitro studies that the total capacity of the intestine to produce a particular metabolite is the sum of the capacities of its individual parts, since the time course of lactate formation by rings of whole wall could not be predicted from the data obtained with mucosal and muscle layers separately. A similar conclusion was reached by Evans and Burdett [3] working with isolated epithelial cells of guinea pig small intestine. The observation that lactate was released from mucosal slices incubated w i t h o u t substrate in a short burst lasting about 30 s suggested that it may have been present in the tissue before the incubation began. Consequently we measured the lactate content of the mucosal tissue prior to incubation and found it to be 21 -+ 2 (6) pmol/g dry weight. This corresponded well with the initial a m o u n t of lactate released when the slices were prepared and incubated in the sodihm medium and suggested that most of this lactate was n o t produced by glycolysis during the first few seconds of incubation. After the initial release, we found that the lactate content of the tissue fell to 5 pmol/g dry weight, i.e. approx. 1 mM (assuming that 1 g dry weight tissue is associated with 5.25 ml tissue water) or about five times the concentration in the medium, and remained at this level irrespective of whether glucose was present in the medium or not t h r o u g h o u t the rest of the incubation period (4.4 -+ 0.2 (8) pmol/g dry weight at 8 min). This is in agreement with the data of Wilson [10] who showed that segments of intestine perfused according to the method of Fisher and Parsons [11], and everted sacs could sustain concentration gradients of lactate (serosal to mucosal) or up to nine times and 6--20 times, respectively. The values for tissue lactate content indicate that about three-quarters of the lactate released into the medium during the first 30 s was present in the tissue initially. After the first 30 s the tissue lactate c o n t e n t remained constant and therefore the lactate released to the medium provided a true messure of lactate production. It is significant that the burst of extra lactate formed on glucose addition after a 30-s preincubation represents net lactate synthesis since the lactate c o n t e n t of the tissue does not alter under these conditions. However, we must stress that this correspondence between lactate appearance and
47 lactate formation does not apply to the initial 30 s of incubation. It is interesting to note that substantially less lactate was released from slices prepared and incubated in high potassium medium (Fig. 1). It is likely that the preparation of mucosal slices in a Na ÷ medium results in Na ÷ uptake particularly in the cold, and that on incubation, an efflux Of Na ÷ and anions takes place across the epithelial cell wall to restore the original intracellular ionic concentrations. If this is the case, then the use of a medium high in potassium, would tend to prevent Na ÷ uptake so that there would be less Na ÷ efflux and loss of accompanying anions (such as lactate}. This explanation could account for the pattern of lactate release shown in Fig. 1. Lactate was formed by slices incubated in the presence of glucose in two stages (Fig. 2); a rapid phase (17 pmol/min per g dry wt) immediately following glucose addition and lasting approx. 1 min followed by a slow steady rate of production (1.8 pmol/min per g dry wt) lasting up to 8 min. This is the first time such a pattern of lactate formation has been reported for rat small intestine, and it seems likely that the present results were obtained because we used the mucosal slice preparation. We found no initial burst of lactate formation either with rings of whole wall or with slices of the underlying muscle tissue. However, the fact that we obtained an apparent Crabtree effect with the mucosal slices and not with rings is probably because glucose entered the mucosal cells via the latero-basal membrane in the slices. This view is supported by the fact that qualitatively the same pattern of lactate release occurred in both sodium and potassium media; both media support glucose uptake in slices but only the sodium medium supports glucose uptake in rings [9]. A number of groups have c o m m e n t e d on the apparent lack of a Pasteur effect in jejunal mucosa [12--18], but as far as we are aware, no-one has put forward the apparently simple explanation, that this phenomenon could be due to a reduction in glucose uptake during hypoxia or anaerobiosis [19], and that this limited the supply of substrate for glycolysis. This idea is amenable to testing, although the intracellular distribution of glucose during absorption is u n k n o w n and it is not clear whether all the glucose entering the epithelial cells is accessible to the glycolytic enzymes. The results in Fig. 5, show that there was a greater lactate formation by intact rings, than by mucosal slices and underlying muscle, and one possible explanation is to suppose that the muscle c o m p o n e n t of the rings is responsible for producing the bulk of the lactate, while the mucosal c o m p o n e n t serves as an active transport system, absorbing glucose from the medium and passing it to the muscle. When rings are incubated in a high potassium medium, the uptake of glucose by jejunal rings is drastically reduced [9], and the concomitant reduction in lactate formation under these conditions strongly suggests that glucose stemming from mucosal entry is the predominant substrate for glycolysis in this preparation.
Acknowledgements We thank Mrs L. Clift for excellent technical assistance and the Medical Research Council for the grant which made this research possible.
48
References ] 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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