Comp. Biochem, Physiol., 1974, Vol. 49B, pp. 393 to 406. Pergamon Press. Printed in Great Britain

THE I N VITRO METABOLISM OF PROPIONATE AND GLUCOSE BY THE RUMEN EPITHELIUM

T. E. C. WEEKES

Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland

(Received 8 November 1973)

Abstract--1. L-Lactate and pyruvate were formed when cattle rumen papillae were incubated in vitro with added propionate, valerate or glucose.

2. Butyrate inhibited the formation of pyruvate from propionate, while ammonium chloride inhibited L-lactate formation but stimulated pyruvate formation.

3. The presence of glucose together with propionate resulted in a syner- gistic increase in L-lactate and pyruvate formation.

4. The formation of L-lactate and pyruvate from added propionate and the activities of some enzymes involved in this conversion were similar using epithelium taken from four areas of the sheep tureen.

INTRODUCTION

THE RUMEN epithelium can metabolize propionate absorbed from the rumen with the formation of L-lactate (Pennington & Sutherland, 1956b) and pyruvate (Weekes, 1972); little is known, however, about the processes controlling pro- pionate metabolism by isolated but intact rumen epithelium. The control of propionate metabolism has been studied in more detail in sheep liver (Smith, 1971). In this tissue propionate utilization is limited by succinate dehydrogenase (E.C. 1.3.99.1) activity and multiple compartmentation of propionate and suc- cinate metabolism exists within the hepatic mitochondrion (Smith & Russell, 1967).

Propionate uptake by rumen epithelium incubated in v i tro was reduced in the presence of an equal concentration of butyrate, but was slightly stimulated by glucose (Pennington & Pfander, 1957). Pennington (1954) reported that 15 mM ammonium chloride partially inhibited propionate uptake by the rumen epithelium. The effects of these compounds on lactate and pyruvate formation by the rumen epithelium have not been reported. It was therefore decided to investigate the effects of other volatile fatty acids, intermediary metabolites and cofactors on lactate and pyruvate formation from added propionate by the rumen epithelium. A comparison of factors regulating propionate metabolism in the rumen epithelium and the liver may then help to explain the significance to the ruminant of pro- pionate metabolism by the rumen epithelium.

393

394 T . E . C . W~KES

MATERIALS AND M E T H O D S Chemicals and enzymes

Biochemica Test Combinations for glucose estimation using hexokinase (E.C. 2.7.1.1) were obtained from Boehringer Corp. (London) Ltd., London W5, England. L-a-Lecithin (Type II-S), n-valeric acid and a-oxoglutarate (monosodium salt) were obtained from Sigma (London) Chemical Co. Ltd., Kingston-upon-Thames, Surrey, England. All other chemicals and enzymes were obtained from the sources described by Weekes (1972).

Animals and sampling procedure Samples of ox (Bos taurus) rumen epithelium were collected from a local abattoir, the

animals used being similar to those described by Weekes (1974). Rumen epithelium was taken from the floor of the anterior ventral sac. Samples of sheep (Ovis arvies) rumen epithelium were obtained from breeding Scottish Blackface ewes slaughtered at the Rowett Institute. Details of these animals, their management and the slaughter procedure are given by Fell et al. (1972). These samples of rumen epithelium were also taken from the floor of the anterior ventral sac.

For the determination of enzyme activities and the extent of lactate and pyruvate forma- tion in different areas of the rumen wall, four non-breeding Scottish Blackface ewes were used. These animals were fed 1"0 kg/day of a lucerne-barley pelleted diet (Fell et al., 1972). Their rumens were removed immediately after slaughter and washed. Samples of rumen epithelium were taken from the following areas: (1) floor of the anterior ventral sac; (2) roof of the dorsal sac; (3) roof of the posterior dorsal blind sac; (4) floor of the ventral sac. These areas were chosen in order to compare samples of rumen epithelium bearing long and short papillae, and also to compare samples bearing different numbers of papillae per unit area.

All samples of rumen epithelium were transported to the laboratory in ice-cold Krebs- Ringer bicarbonate buffer, modified as described by Weekes (1971).

Incubation of rumen papillae For the comparison of different areas of the tureen wall, the whole rumen epithelium,

consisting of the squamous epithelium and the lamina propria, was cut into squares weighing about 25 rag, which were rinsed with the modified Krebs-Ringer bicarbonate buffer and blotted prior to incubation. All other incubations were performed using rumen papillae cut from the rumen wall. The procedures adopted for the in vitro incubation of rumen papillae with added substrates and for the estimation of lactate and pyruvate formation have been described previously (Weekes, 1972). Glucose was estimated by the method of Slein (1963) and propionate uptake during incubation by the method of Weekes (1973).

All incubation media were isotonic, additional substrates being substituted for NaCI present in the Krebs-Ringer bicarbonate buffer, and the pH of all solutions was adjusted to 7-1 unless otherwise stated. Duplicate flasks were used for each substrate addition and two flasks containing no additional substrates or cofactors (endogenous incubations) were included in each experiment. Two additional samples of papillae were placed in 20 ml ice-cold modified Krebs-Ringer bicarbonate buffer and homogenized without incubation before determination of metabolite concentrations (zero-time metabolite concentrations). A separate sample of papillae was used for determinations of dry matter content, performed in triplicate by drying overnight at 105°C.

Expression of results Results were expressed as/~mol metabolite formed/100 mg tissue dry matter per 2 hr.

Net metabolite formation was calculated by subtraction of metabolite levels in flasks without added substrate from metabolite levels in the experimental flasks. The metabolite levels found after endogenous incubations were similar to the zero-time metabolite con- centrations, suggesting that very little metabolite uptake or formation occurred in the absence

1N VITRO METABOLISM OF PROPIONATE AND GLUCOSE BY RUMEN EPITHELIUM 395

of added substrates. Lactate/pyruvate ratios refer to the metabolite concentrations present in flasks at the end of the 2-hr incubation, without correction for endogenous concentrations.

Enzyme assays Lactate dehydrogenase (E.C. 1.1.1.22), NADP-malate dehydrogenase (E.C. 1.1.1.40)

glutamate dehydrogenase (E.C. 1.4.1.3), aspartate aminotransferase (E.C. 2.6.1.1.) and alanine aminotransferase (E.C. 2.6.1.2) were assayed as described by Weekes (1972). ~-Hydroxybutyrate dehydrogenase (E.C. 1.1.1.30) was assayed in the debris-free homo- genate fraction (Weekes, 1972) prepared using a medium containing 0"20 M sucrose, 20 mM Tris-HCl buffer, pH 7"2, 1 mM EDTA and 1 mM GSH*. Enzyme activity was determined by a spectrophotometric assay based on that used by Koundakjian & Snoswell (1970). The final assay mixture contained 66"7 mM Tris-HCl buffer, pH 8"5, 1.8mM NAD +, 50mM nicotinamide, 1 mM NaCN, 1 mM CaCI2, 0"25 mg/ml L -rv- lecithin, 23"3 mM VL-fl-hydroxybutyrate and debris-free homogenate (0.50 ml), in a total volume of 3"00 ml. The reaction was started by the addition of DL-fl-hydroxybutyrate.

One unit of enzyme activity was defined as the amount of enzyme catalysing the forma- tion of 1/~nol reaction product or the utilization of 1/~mol of substrate per min at 25°C under the assay conditions employed.

RESULTS

General remarks

The effects of pH on metabolite formation by cattle tureen papillae are shown in Fig. 1. In the absence of added propionate, pyruvate concentrations were very low, accounting for the variable lactate/pyruvate ratio. In all subsequent studies incubations were carried out at pH 7.1, which allowed the greatest stimulation of metabolite formation by propionate, in comparison with endogenous incubations at the same pH.

The optimum propionate concentration for lactate formation by sheep and cattle rumen papillae varied between experiments within the range 15-25 raM, while pyruvate formation was greatest using 15 mM propionate. Lactate formation was inhibited when 45 or 50 mM propionate was used. In subsequent work 15 mM propionate was therefore used.

Hodson et al. (1967) reported that the uptake of propionate, butyrate and valerate by cattle rumen papillae, ketone body formation and mitoehondrial structure were adversely affected if the papillae were immersed in Krebs-Ringer bicarbonate buffer at 0°C for 40 min, compared with immersion in the same buffer at 39°C. Lactate formation from added propionate was not measured in their study. These experiments were therefore repeated using samples of cattle rurnen papillae immersed for 50 min either in ice-cold modified Krebs-Ringer bicarbonate buffer or in buffer maintained at 39°C. The preincubation temperature had no effect on subsequent net lactate formation or net propionate uptake by the papillae during incubations with 15 or 30 mM propionate.

* Abbreviations used are: ATP, adenosine triphosphate; EDTA, ethylenediamine- tetraacetate; GSH, reduced glutathione; Tris, 2-amino-2-hydroxymethylpropane-l,3- diol; VFA, volatile fatty acid.

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FIG. 1. Effect of incubation pH on (a) L-lactate formation and (b) pyruvate formation (O, O) and L-lactate/pyruvate ratio (A, A). Open symbols (O, zS) represent cattle rumen papillae incubated with no added substrate, closed symbols (0, A) incubations with 15 mM propionate. Experimental details are given in

the Materials and Methods section.

Net lactate and pyruvate formation in vitro from added propionate and enzyme activities in different areas of the rumen wall of sheep

With the exception of f l-hydroxybutyrate dehydrogenase activity, none of the properties measured showed any significant variation between the four areas of the rumen wall (Table 1), although there were significant differences between animals in net pyruvate ( P < 0.001) and net lactate ( P < 0.05) formation and in the epithelial dry matter content (P<0.05) . f l -Hydroxybutyrate dehydrogenase activity was significantly greater in areas 2 and 4 than in areas 1 and 3 (P<0.05) .

This broad uniformity in metabolic properties occurred despite wide differences in the appearance of the rumen epithelium. Samples taken from the floor of the anterior ventral sac (area 1) were the most heavily papillated, the papillae being large, rod shaped and darker coloured than those found in the other areas. The rumen epithelium taken from area 2, the roof of the dorsal sac, was light col- oured, bearing very few papillae, these being small and conical. Th e epithelium in area 3, the roof of the posterior dorsal blind sac, was similar to area 2, although the papillae were slightly larger and more numerous. Samples of epithelium taken from area 4, the floor of the ventral sac, were intermediate in colour between area 1 and areas 2 and 3. The individual papillae were similar in size and shape to those found in area l, but were less numerous.

I N VITRO METABOLISM OF PROPIONATE AND GLUCOSE BY RUMEN EPITHELIUM 397

TABLE a- -Ngr L-LACTATE AND PYRLrVATE FORMATION in vitro FROM ADDED PROPIONATE AND

ENZYME ACTIVITIES IN DIFFERENT AREAS OF THE RUMEN WALL OF SHEEP

Area of rumen wall

Property 1 2 3 4

Epithelial dry matter (%) Net L-lactate formation Net pyruvate formation

20"41 _+0"24 19"86+ 1"08 19"24+1"07 20"31 -+0"80 8"70-+ 1"21 6.78 __+ 0'78 7"78 __+ 1-30 7"63 -+ 0"87 1"56_+0"30 1"41_+0"10 1"52_+0"35 1"58_+0-32

Enzyme activities NADP-malate dehydrogenase Lactate dehydrogenase Glutamate dehydrogenase Aspartate aminotransferase Alanine aminotransferase

1"14__+0"29 1"08_+0"18 1"16+0-17 1"22-+0"17 68"3 -+ 12"2 58"6 -+ 8"8 57-5 + 9-3 56"2 -+ 6"0

0"93-+0"32 1-03+0"16 0"64__+0-10 0-95_+0"26 5"70 _+ 0"80 5"38 _+ 1 "22 5"25 __+ 1 "18 4"75 -+ 1 "29 0"93_+0"17 1-02__+0"20 0"68+0-19 0-80-+0"19

1 "06 __+ 0"16 0"71 -+ 0-08 1"00 _+ 0"21 fl-Hydroxybutyrate dehydrogenase 0"79 + 0"16

Area 1 = floor of anterior ventral sac; area 2 = roof of dorsal sac; area 3 = roof of posterior dorsal blind sac; area 4 = floor of ventral sac.

The net rates of L-lactate and pyruvate formation during incubation with 15 mM propionate are given as/~mol/100 mg dry matter per 2 hr and the levels of enzyme activity as U/g epithelium. The results are given as the mean_+ S.E.M. for four animals. Experimental details are given in Materials and Methods.

The effects of additional substrates and cofactors on L-lactate and pyruvate formation by cattle tureen papillae

(i) Volatile fat ty acids (VFAs). Cattle rumen papillae were incubated with 15 m M initial concentrations of acetate, propionate, butyrate, valerate and iso- butyrate, with and without the addition of 15 m M propionate (Table 2). In order to compare metabolite formation in the presence of VFAs with metabolite formation in the absence of added substrate, lactate and pyruvate formation was calculated by subtraction of the zero t ime concentrations f rom metabolite concentrations after 2-hr incubation. There was no significant lactate or pyruvate formation f rom acetate, butyrate or iso-butyrate and these VFAs had no effect on lactate formation f rom propionate. Pyruvate was taken up during incubation with butyrate (P<0 .05 ) and the presence of butyrate significantly reduced pyruvate formation f rom propionate and increased the lactate/pyruvate ratio in comparison with the incubation med ium containing 1 5 m M propionate ( P < 0 . 0 5 in both cases). Considerable amounts of lactate were formed during incubation with valerate and when valerate and propionate were present together there was an additive effect on lactate formation. Less pyruvate was formed when valerate rather than propionate was the substrate ( P < 0.001), but valerate did not significantly reduce pyruvate formation f rom propionate when the two VFAs were present together.

398 T . E . C . WEEKES

TABLE 2--L-LACTATE AND PYRUVATE FORMATION in vitro BY CATTLE RUMEN PAPILLAE INCUBATED WITH VFAs

Additional Lactate 15 mM Lactate Pyruvate - -

VFA propionate formation formation Pyruvate

Nil (endogenous) - - 0"27 0"02 49.8 Propionate - 5 -54 0"75 17"8

+ 4"91 0-56 22"9 Acetate - - 0"04 0"03 114.5

+ 6"06 0.51 20.8 Butyrate - 0"42 0"00 73 " 5

+ 7"10 0-05 84"9 Valerate - 5 "75 0"08 73 "2

+ 11-65 0"46 43.0 iso-Butyrate - 0'29 0"04 37"3

+ 4"09 0'36 32"5

S.E. of treatment differences 1 "68 0"19 23'5

All VFAs were used at an initial concentration of 15 mM, pH 7"1, with and without the addition of 15 mM propionate. Further experimental details a r e given in the Materials and Methods section.

The rates of L-lactate and pyruvate formation during incubation were cal- culated by subtraction of zero-time concentrations from metabolite concentrations after the 2-hr incubation and are expressed as/~mol/100 mg dry matter per 2 hr. Lactate/pyruvate ratios refer to the metabolite concentrations present in flasks at the end of the 2-hr incubation, without correction for zero-time concentrations. Values are means of three experiments, each using one animal, analysed as a 2 x 2 x 2 factorial design.

The effects of physiological concentrations of the three major VFAs on lactate and pyruvate formation in vitro were also investigated in a 2 × 2 × 2 factorial experiment, using papillae taken from four steers. The VFA concentrations used were 85 m M acetate, 2 4 m M propionate and 1 6 r a m butyrate, concentrations similar to those found in the rumen liquor of cattle fed on roughages (Bath & Rook, 1963). The results were similar to those reported in Table 2. Lactate and pyruvate were only formed in significant amounts in the presence of propionate, but butyrate significantly reduced pyruvate formation and increased the lactate/ pyruvate ratio when compared with incubations with propionate alone ( P < 0.001 in both cases). There were no significant interactions between acetate and pro- pionate or butyrate, or between acetate, propionate and butyrate together.

(ii) Succinate and ATP. Smith & Russell (1967) suggested that externally added succinate was metabolized in a different metabolic compartment by sheep liver mitochondria than was succinate derived from propionate. The stimulation of metabolism by A T P was less for added succinate than for propionate, but the addidon of 10 m M succinate increased the rate of metabolism of 5 m M pro- pionate. The interactions of propionate, succinate and A T P on net lactate and

IN VITRO METABOLISM OF PROPIONATE AND GLUCOSE BY RUMEN EPITHELIUM 399

pyruvate formation by cattle rumen papillae were therefore investigated in a 2 x 2 x 2 factorial experiment (Table 3). These results demonstrate that lactate and pyruvate were formed from added succinate, although succinate was a less effective substrate than an equal concentration of propionate.

TABLE 3--L-LACTATE AND PYRUVATE FORMATION in v i t ro BY CATTLE RUMI~ PAPILLAE

INCUBATED WITH PROPIONATE~ SUCCINATE AND A T P

Lactate L a c t a t e P y r u v a t e

A d d i t i o n f o r m a t i o n f o r m a t i o n P y r u v a t e

Nil (endogenous) 0"40 0"09 33"9 Propionate 6.19 1 "85 6"5 Succinate 2.79 0"47 15"4 ATP 0-70 0"10 34.7 Propionate + succinate 9.58 2"54 5.9 Propionate + ATP 7"25 1 "34 9"5 Succinate + ATP 4"87 0"49 21.2 Propionate + succinate + ATP 12.94 2" 33 8" 1 S.E. of treatment differences 0"92 0"17 3"9

The initial concentrations used were 15 mM propionate, 15 mM succinate and 5 mM ATP. Further experimental details are given in the Materials and Methods section. Lactate and pyruvate formation (/maol/100 mg dry matter per 2 hr) and lactate/pyruvate ratios were calculated as described in Table 2. The results are for four experiments, each using one animal, analysed as a 2 x 2 x 2 factorial design.

Lactate formation from succinate was significantly stimulated by ATP (P< 0.05) but the stimulation of lactate formation from propionate was not significant. Lactate formation from succinate and propionate together was approximately additive, but the combination of propionate, succinate and ATP tended to result in a more than additive lactate formation. Although this interaction was not statis- ticaUy significant, as its magnitude was variable, this trend was apparent with all four samples of papillae. Pyruvate formation from added propionate, but not from added succinate, was inhibited by ATP ( P < 0.05). There was a significant positive interaction between propionate and succinate in pyruvate formation ( P < 0.05).

The possibility that ATP was acting merely as a source of inorganic phosphate, facilitating the entry of succinate into mitochondria (Chappell & Robinson, 1968), was tested by repeating the incubations described in Table 3 in modified Krebs- Ringer bicarbonate buffer containing 20 m M phosphate, pH 7.1, using papillae from the same animals for both series of incubations. The addition of phosphate had no effect on lactate or pyruvate formation, confirming that propionate and succinate metabolism was not limited by phosphate.

(iii) Glucose. Incubation of cattle rumen papillae with 15 m M glucose resulted in L-lactate formation, together with small amounts of pyruvate relative to that

400 T . E . C . WEEKES

formed from propionate (Table 4). Preliminary experiments established this to be the opt imum glucose concentration for lactate and pyruvate formation. Th e formation of both lactate and pyruvate was greater than additive when propionate and glucose were present together (P < 0.001 in both cases). Th e inclusion of A T P in the incubation medium significantly reduced pyruvate formation from pro- pionate ( P < 0.001) and from glucose ( P < 0.05).

TABLE 4 - - L - L A C T A T E AND PYRUVATE FORMATION in vitro BY CATTLE RUMEN PAPILLAE

INCUBATED W I T H PROPIONATE, GLUCOSE AND A T P

Lactate Lactate Pyruvate

Addition formation formation Pyruvate

Nil (endogenous) - 0"68 0-03 40"6 Propionate 3 '75 1" 10 8-6 Glucose 3'70 0"20 34"2 ATP 0"44 0"11 41 '3 Propionate + glucose 12"06 1 "77 10" 1 Propionate + ATP 4"33 0"66 12"9 Glucose + ATP 1-46 0'04 48"3 Propionate + glucose + ATP 12'70 1-04 16"3 S.E. of treatment differences 1"20 0"12 6-5

The initial concentrations used were 15 mM propionate, 15 mM glucose and 5 mM ATP. Further experimental details are given in the Materials and Methods section. Lactate and pyruvate formation (/~mol/100 mg dry matter per 2 hr) and lactate/pyruvate ratios were calculated as described in Table 2. The results are for four experiments each using one animal, analysed as a 2 x 2 × 2 factorial design.

The uptake of glucose during the 2-hr incubation period was measured in two of the four experiments reported in Table 4. Glucose uptake in the absence of any other substrate averaged 22/zmol/100 mg dry matter per 2 hr, in the additional presence of 5 m M A T P it averaged 27 ~mol, in the presence of 15 m M propionate 25/zmol, and in the presence of propionate and A T P 32/zmol/100 mg dry matter per 2 hr. The initial glucose concentration in these incubations was 15 mM. Thus considerably more glucose was taken up than could be accounted for by lactate or pyruvate formation. Glucose was not synthesized by rumen papillae during incubation with additions of propionate, succinate, propionate + A T P or propionate + succinate + ATP. The zero-time free glucose concentration in the incubation medium was 20 + 5/~M (mean + S.E.M. for four experiments). Glucose concentrations decreased during incubation with the above substrates, averaging 15 + 6/~M (16) after a 2-hr incubation but this glucose uptake was not influenced by the substrate added.

(iv) Malonate. The formation of L-lactate and pyruvate from added pro- pionate was only partially inhibited by malonate (Fig. 2), in agreement with

I N VITRO M E T A B O L I S M OF P R O P I O N A T E A N D G L U C O S E B Y R U M E N E P I T H E L I U M 401

earlier studies (Pennington & Sutherland, 1965b). In contrast with the findings of Smith & Russell (1967), who worked with sheep liver mitochondria, the in- clusion of ATP did not reduce the malonate sensitivity of propionate metabolism to lactate and pyruvate. Indeed the sensitivity of pyruvate formation to malonate inhibition was increased in the presence of ATP, while L-lactate formation was significantly inhibited at a lower malonate concentration than was required in the absence of ATP (10 mM v. 15 raM).

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FIG. 2. Effect of malonate concentration of (a) L-lactate and (b) pyruvate forma- tion by cattle rumen papillae incubated in the absence of additional substrates (O), in the presence of 15 mM propionate (0) and in the presence of 15 mM propionate+5 mM ATP (A). Values for L-lactate and pyruvate formation were corrected for metabolite levels in control flasks containing no added sub- strate or inhibitor. Experimental details are given in the Materials and Methods

section.

(v) Ammonia. The addition of 10 mM ammonium chloride, a concentration within the range found in rumen liquor (Hungate, 1966), significantly reduced net lactate formation in comparison with incubations containing 15 mM pro- pionate alone (P<0.01) (Table 5). Net pyruvate formation was significantly increased by the inclusion of NH4C1 (P<0.05), these changes resulting in a significant reduction in the lactate/pyruvate ratio (P<0"01). These incubations were performed using sheep rttrnen papillae obtained from animals used in another study (Weekes, 1972), but similar results were obtained in one experiment using cattle rumen papillae.

402 T . E . C . WEEKeS

TABLE 5 - - T H E EFFECT OF 10 mM NH4C1 ON :NET LACTATE AND PYRUVATE FORMATION BY

SHEEP RUMEN PAPILLAE INCUBATED in vitro

Addition to incubation medium

10 mM NH4CI Property 15 mM propionate + 15 mM propionate

Net lactate formation 6"27 + 0"78 5'17 + 0'64* * Net pyruvate formation 0"88 + 0"18 1"01 + 0'19" Lactate/pyruvate 16-4 + 2"2 13'4 + 1 "5 * *

The net rates of L-lactate and pyruvate formation are expressed as/zmol/100 mg dry matter per 2 hr, as mean+ S.E.M. Further experimental details are given in the Materials and Methods section. The results are for papillae taken from ten breeding ewes fed on lucerne-barley cobs, analysed statistically using the paired comparison t-test. Statistical differences between values found using the two incubation media are indicated by * (P<0"05) and ** (P<0"01)

(vi) DL-fl-Hydroxybutyrate. T h e presence of 2 m M or 10 m M 3L-fl-hydroxy- butyrate did not result in any net formation of L-lactate or pyruvate in one experi- ment using cattle rumen papillae. Net lactate formation f rom propionate was not affected by the presence of DL-fl-hydroxybutyrate, but net pyruvate formation was slightly reduced.

DISCUSSION

This investigation has established some of the factors affecting L-lactate and pyruvate formation f rom added substrates by rumen papillae. In the intact rumen papilla at least three possible permeabil i ty barriers exist, namely, the outer layers of the s t ra tum corneum, the cell membranes of the metabolically active cells in the s t ra tum basale and the mitochondrial membranes of these cells. T h e present work was, however, carried out using rumen papillae incubated in vitro, since it was not possible to prepare either a viable suspension of epithelial cells (Weekes, 1974) or an undamaged mitochondrial suspension f rom rumen mucosa (Weekes, 1973).

T h e calculation of metabolite formation during incubation involved the sub- traction either of zero-t ime metabolite concentrations or of metabolite concentra- tions following endogenous incubations. Krebs et al. (1973) observed that this procedure may be open to question as it was not known what effect the addition of substrates may have on the endogenous rate of metabolism. T h e endogenous rates of lactate and pyruvate uptake or formation were, however, very small in the present experiments, so that the error resulting f rom this procedure is also likely to be small.

T h e net amounts of L-lactate and pyruvate formed in vitro and the enzymes concerned with the metabol ism of propionate and amino acids appeared to be uni formly distr ibuted between the four areas of the rumen wall that were sampled,

I N VITRO METABOLISM OF PROPIONATE AND GLUCOSE BY RUMEN EPITHELIUM 4 0 3

suggesting that incubations performed using papillae taken from the floor of the anterior ventral sac may be representative of the Tureen epithelium as a whole. The Tureen contents of animals fed on lucerne-barley cobs are, however, very homogenous (Weekes, 1972), so these results may not be typical for animals fed other rations. This uniformity may not extend to the enzymes involved in butyrate metabolism, since Baird et al. (1970)reported that fl-hydroxy-, ]~-methyl-glutaryl- CoA synthetase (E.C. 4.1.3.5) activity per unit weight was significantly greater using samples of ox rumen epithelium taken from the "ventral Tureen", com- pared with samples bearing few papillae taken from the "dorsal Tureen".

Weidemann & Krebs (1969) suggested that the reactions leading from pro- pionate to methylmalonyl-CoA may be in equilibrium in the rat kidney cortex. If this were so in the Tureen epithelium, the addition of phosphate would be ex- pected to inhibit propionate metabolism, while ATP would be stimulatory. The low activity of propionyl-CoA synthetase (E.C. 6.2.1.-) in the Tureen epithelium (Weekes, 1972) suggests that in this tissue the rate of propionyl-CoA formation may limit L-lactate and pyruvate formation, so that equilibrium conditions do not occur.

In agreement with the findings of Weigand et al. (1967), L-lactate was formed from valerate as well as propionate. The additive net lactate formation when propionate and valerate were present together suggests that their metabolic pathways were controlled by different rate-limiting reactions. Valerate metabolism results in the formation of propionyl-CoA and acetyl-CoA, further suggesting that propionate metabolism was controlled by the rate of formation of propionyl- CoA rather than by its subsequent metabolism or by competition for free CoA. Acetyl-CoA formed from valerate and butyrate could have stimulated propionate metabolism by combining with intramitochondrial oxaloacetate, deinhibiting succinate dehydrogenase, as occurs in sheep liver mitochondria (Smith, 1971). The failure of butyrate to stimulate significantly L-lactate formation suggests that this control mechanism is not important in the rumen epithelium. Indeed, Pennington & Pfander (1957) reported that 20 mM butyrate inhibited propionate uptake from a 20 mM solution by sheep rumen epithelium, in contrast with the results presented in Table 2. The presence of propionate slightly decreased ketone body formation from butyrate, while increasing butyrate uptake (Pennington & Pfander, 1957). Thus it is possible that butyrate was preferentially oxidized, allowing a greater proportion of the reduced propionate uptake to be metabolized to lactate and pyruvate. It should, however, be noted that Bush et al. (1970) reported that propionate did not inhibit ketogenesis from butyrate in the rumen epithelium.

The failure of acetate to influence lactate and pyruvate formation, even when an 85 mM concentration was used, agrees with the failure of acetate to influence propionate uptake (Pennington & Pfander, 1957). Acetate uptake was inhibited by propionate, and this effect, coupled with the low absolute capacity of the rumen epithelium to metabolize acetate (Cook e t a / . , 1969), probably explains the in- sensitivity, of propionate metabolism to acetate.

404 T . E . C . WEEKES

The inhibitory effect of ammonia on net lactate formation may result from the synthesis of glutamate, reducing the intramitochondrial ~-oxoglutarate concentra- tion, resulting in the utilization of malate formed from propionate as a four- carbon compound in the tricarboxylic acid cycle, rather than as a substrate for L-lactate formation.

The relatively small amounts of lactate and pyruvate formed from added suc- cinate suggests that succinate metabolism was limited by its poor permeability through the cell or mitochondrial membranes. Limited permeability to succinate has been reported by Hems et al. (1968) in rat liver slices and by Sherratt (1968) in rat and guinea-pig intestinal rings. Although propionate metabolism requires more ATP expenditure than does succinate metabolism, the latter was stimulated to a greater extent by the addition of ATP. Furthermore, in contrast with the findings of Smith & Russell (1967), working with sheep liver mitochondria, the inhibition of propionate metabolism by rnalonate was greater in the presence of ATP. Thus ATP may have stimulated the entry of both malonate and succinate into the metabolically active cells or their mitochondria, while propionate was able to penetrate freely into the mitochondria. The failure of ATP to stimulate consistently net lactate formation from propionate was probably not due to poor penetration of ATP into the metabolically active cells, since ATP consistently in- creased the lactate/pyruvate ratio compared with incubations with propionate alone.

The present studies confirmed the early work of Pennington & Sutherland (1956a), who were the first to demonstrate glycolysis in the tureen epithelium. When glucose and propionate were present together, a definite synergistic increase in net lactate and pyruvate formation was observed, but it is not clear whether these products were derived from glucose or propionate, or from both substrates. Pennington & Pfander (1957) reported that 11.1 mM glucose slightly increased the uptake of propionate by sheep tureen epithelium, while glucose uptake was slightly decreased by the presence of propionate. It is possible that glucose stimu- lated lactate and pyruvate formation from propionate, by inhibiting propionate oxidation, or propionyl-CoA synthetase may be allosterically activated by a gly- colytic intermediate, although no evidence for this is available.

Glucose uptake considerably exceeded lactate and pyruvate formation and, in contrast with the findings of Pennington & Pfander (1957), this uptake was slight- ly stimulated by the presence of propionate in the present study. A large part of this glucose was probably oxidized and propionate may have inhibited this oxidation, since propionyl-CoA inhibits pyruvate dehydrogenase (E.C. 1.2.4.1) (Bremner, 1969) and both propionyl-CoA and succinyl-CoA in hibitcitrate synthase (E.C. 4.1.3.7) (Smith & Williamson, 1971). If the glucose flux through the glycolytic pathway were unimpaired, this would result in the increased lactate and pyruvate formation.

A small incorporation of radioactive label from propionate into glucose was reported by Seto et al. (1971), using cattle tureen epithelium, in contrast with the lack of measurable net glucose synthesis in the present study. However, the very low activity of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase

1N V I T R O METABOLISM OF PROPIONATE AND GLUCOSE BY RUMEN EPITHELIUM 4 0 5

(E.C. 4.1.1.32) in the rumen epithelium (Young et al., 1969) would severely limit gluconeogenesis. Indirect evidence suggested that this enzyme was in fact involved in glyceride-glycerol synthesis rather than in gluconeogensis (Weekes, 1973).

A small extent of conversion of absorbed propionate to lactate and pyruvate in the rumen suggested by an indirect calculation (Weekes, 1972), and by direct measurements of propionate and L-lactate appearance in the portal blood of sheep given intraruminal infusions of VFAs (T. E. C. Weekes & A. J. F. Webster, unpublished work), indicate that this pathway is not important as a means of conserving three carbon units for gluconeogenesis in the liver. The findings of the present study suggest that propionate metabolism is controlled primarily by substrate availability and the rate of propionyl-CoA formation, and that lactate and pyruvate formation is relatively insensitive to the presence of other VFAs or to control at the level of succinate dehydrogenase. This would allow a relatively constant rate of lactate and pyruvate formation, in agreement with the suggestion made previously (Weekes, 1972) that this pathway functions to provide extra- mitochondrial N A D P H required for synthetic processes within the epithelial cell. I t is interesting that the presence of glucose could increase NADPH genera- tion in this pathway.

Acknowledgements--I wish to thank Mr. R. M. C. Crofts for help with the statistical treatment of the results. This work was carried out while I was in receipt of a Ministry of Agriculture, Fisheries and Food Postgraduate Studentship.

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Key Word Index--Bos taurus; Ovis aries; ruined epithelium; propionate metabolism; volatile fatty acids; L-lactate; pyruvate; glycolysis ; glucose metabolsim; malonate ; succinate.