The metabolism of the small intestine: Glycolysis in the mucosa of the small intestine of the sheep

Comp. Biochem. PhysloL, 1971, VoL 38B,pp. 5 to 20. Pergamon Press. Printed in Great Britain

THE METABOLISM OF T H E SMALL I N T E S T I N E : GLYCOLYSIS IN THE MUCOSA OF T H E SMALL I N T E S T I N E OF THE SHEEP K. W. J. WAHLE1, * D. G. A R M S T R O N G a and H. S. A. S H E R R A T T 2 1Department of Agricultural Biochemistry, University of Newcastle upon Tyne; and ~Department of Pharmacology, Medical School, University of Newcastle upon Tyne, NE1 7RU, U.K. (Received 11 June 1970)

Abstract--1. Between 78-119 per cent of the glycolytic activity of homogenares of the mucosa from the jejunum of sheep was recovered in the particle-free supernatant fraction. 2. High rates of L-lactate formation were obtained with glucose 6-phosphate as substrate; these were about two to three times those with fructose or glucose. Some L-glycerol3-phosphate was formed from all three substrates. 3. There was an increase in the specific activities of supernatant fractions with the age of the sheep over the period of 194 days investigated. 4. It was not possible, in two experiments, to demonstrate any effects of different diets on the glycolyticactivity of the jejunum. INTRODUCTION THE METABOLISMof adult ruminants differs from that of most monogastric animals since ingested carbohydrate is mainly converted to acetic, propionic and butyric acids in the rumen by microbial fermentation. These fermentation products are absorbed through the rumen wall and may provide a major part of the total energy requirements of the sheep (Bergman et al., 1965; Seeley e t a / . , 1959). The glucose requirements of ruminants fed conventional diets are met by gluconeogenesis from propionic acid and amino acids (Armstrong, 1965). In ruminants fed hay diets little a-linked glucose polymer (starch) enters the small intestine, although when high grain diets are fed the amounts are increased significantly (MacRae & Armstrong, 1969): this is particularly true for diets containing ground maize (see Armstrong & Beever, 1959). If such carbohydrate is hydrolyzed to monosaccharide in the small intestine and absorbed it would make an appreciable contribution to the glucose requirements of these animals (Armstrong & Beever, 1969). By contrast, in monogastric animals dietary carbohydrates are completely absorbed as monosaccharides from the upper part of the small intestine (Crane, 1960). It might be noted that digestion and absorption of carbohydrates in the pre-ruminant stage of the ovine and bovine resembles that of monogastric species (Jarrett & Potter, 1952; McCandless & Dye, 1959). * Present address: Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB.

K. W. J. WAHLE,D. G. ARMSTRONG,AND H. S. A. SHERRATT In connection with a study in the Department of Agricultural Biochemistry, University of Newcastle upon Tyne, of various aspects of carbohydrate digestion in the sheep, it was felt desirable to obtain information concerning glycolysis in the mucosa of the small intestine of this species. It was known that mucosa from the small intestine of the rat forms large amounts of lactate from added glucose in vitro whereas that from the guinea pig produces much less (Wilson 1956; Clark & Sherratt, 1967; Sherratt, 1968). However, cell-free preparations from both species readily form lactate from glucose, fructose or glucose 6-phosphate (Srivastava & Hiibscher, 1969; Clark & Sherratt, 1967). After starvation for as little as twelve hours the glycolytic activity of rat small-intestinal mucosa decreases and is restored on refeeding after only two hours. This effect is mainly due to changes in the activity of hexokinase (E.C. 2.7.1.1) (Srivastava & Htibscher, 1966a; Srivastava et al., 1968) and is induced by oral but not by intravenous administration of glucose (Shakespeare et al., 1969). T h e activities of some other glycolytic enzymes in rat jejunum (Stifel et al., 1968) and in human jejunum (Rosensweig et al., 1968) have also been reported to be influenced by the nature of the diet. T h a t glycolytic activity in the small intestine is not necessarily dependent on carbohydrate digestion therein is shown by this study of such activity in the sheep. A preliminary account of some of this work has already appeared (Wahle & Sherratt, 1967). MATERIALS AND METHODS Animals Small intestines were removed without delay from sheep that had been stunned and bled at a local abattoir. They were washed through with cold 0'14 M NaC1 to remove digesta and, unless otherwise stated, surrounded by crushed ice. The time between slaughter and arrival of the samples at the laboratory was usually about one hour. The sex and approximate age and diet of the sheep were noted when possible. Most animals were castrate Suffolk Halfbred crosses with occasionally some of the Clun and Cheviot breeds. Their ages at slaughter usually varied from 3-12 months depending on the time of year, since all were born in spring. Their diet, determined by inspection of their rnmen contents, also varied with the time of year. In general, hay and high levels of concentrates were fed in winter and grass was eaten during spring and summer. Chemicals and enzymes Glucose 6-phosphate (sodium salt), galactose 1-phosphate (sodium salt), galactose 6phosphate (sodium salt), mannose 1-phosphate (potassium salt), ribose 5-phosphate (sodium salt), fructose 1, 6-diphosphate (sodium salt), DL-glycerol 3-phosphate (sodium salt), D-3phosphoglycerate (sodium salt), glycogen, adenosine, guanosine, uridine, cytidine, ATP, ADP, NAD + and yeast hexokinase (E.C. 2.7.1.1) (150 units/rag of protein), lactate dehydrogenase (E.C. 1.1.1.27) (400 units/rag of protein) and L-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) (50 units/mg of protein) were obtained from Sigma Chemical Co., London, S.W.6. Glucose 1-phosphate (sodium salt), rabbit muscle lactate dehydrogenase (360 units/rag of protein) and yeast hexokinase (140 units/rag of protein) were purchased from Boehringer Corp., London, W.5. Fructose 6-phosphate (barium salt) and all other carbohydrates used were obtained from British Drug Houses, Poole, Dorset. Other chemicals used were of A.R. grade where possible.

METABOLISM OF THE SMALL INTESTINE

Determination of L-lactate and L-glycerol 3-phosphate These were determined enzymically (Hohorst, Kreutz & Biicher, 1959). Determination of protein Protein was measured as described by Miller (1959). Preparation of the particle-free supernatant fraction from small intestinal mucosa Mucosa was separated from whole intestine by scraping with a microscope slide as described by Porteus & Clark (1965) and unless otherwise stated fresh mucosa was used. The separated mucosa was homogenized in 0"3 M marmitol, 2 mM EDTA, pH 7"4, at 0°C as described by Clark & Sherratt (1967). The PFS* fraction was obtained by centrifugation of the homogenate at 45,000 rev/min (150,000 g av.) in the 50 rotor of a Spinco Model L ultracentrifuge for 45 min at 2°C. Determination of the glycolytic activity of the particle-free supernatant fraction This was measured by determining the amount of L-lactate produced from various substrates. In some experiments the L-glycerol 3-phosphate production was also measured. The reaction system for glycolysis contained KHCOs (16 mM), (NH4)~HPO4 (20 raM), nicotinamide (16"5 raM), ATP (2 mM), NAD + (1 mM) and MgC12 (12 raM) and either glucose (25 mM), fructose (25 mM) or glucose 6-phosphate (10 mM) and PFS fraction (containing ca. 2 mg of protein) in a total volume of 1"0 ml, pH 7"4, at 40°C in centrifuge tubes. The reaction was started by the addition of the supernatant fraction, and after an appropriate time, it was stopped by the addition of 1"0 ml of 0"3 N HC104. After removal of denatured protein by centrifugation in a bench centrifuge the protein-free supernatant was used for assay of lactate and L-glycerol 3-phosphate. Unless otherwise stated, at least three tubes were used for each determination of glycolytic activity, usually with incubation times of 10, 20 and 30 min respectively, and the activity was calculated from the initial rate of lactate or L-glycerol 3-phosphate formation, since these were not generally linear with time (Fig. 1). EXPERIMENTAL AND RESULTS

General remarks T h e P F S fraction from the mucosa of the small intestine readily formed lactate from glucose 6-phosphate using the conditions given by Clark & Sherratt (1967). A preliminary survey using these conditions indicated little difference in the specific glycolytic activity along the whole length of the small intestine (Wahle & Sherratt, 1967). Owing to the length of the small intestine of the adult sheep, (20-25 m), all experiments were done using the segment of the jejunum between 0.5-0.75 m posterior to the opening of the bile and pancreatic duct. T h e conditions subsequently used for the assay of lactate formation from 10 m M glucose 6-phosphate by PFS fractions from sheep small intestinal mucosa have been given in the Materials and Methods section; the p H and the concentrations of A T P and Mg 2+ were chosen from a consideration of the results shown in Fig. 2 and the assays were conducted at 40°C since 39.5°C is the normal body temperature of the sheep. T h e initial rates of lactate production were proportional to the concentration of added protein in the range used, 1-4 mg/ml per incubation. An A T P concentration of 2 m M was maximal for lactate production, although *

Abbreviation: PFS, particle-free supematant.

K . W . J . WAHLE,D. G. ARMSTRONGAND H. S. A. SHERRATT

8

1,600

E 1,200

~.~_ ,== o ~ ~

800

~E o ~u

400

J _1

V 0

I

I

I

I0

20

30

Time,

min

FIc. 1. Time course of glycolysis from 10raM glucose 6-phosphate by the P F S fraction from sheep jejunal mucosa at different temperatures. L-Lactate formed at 30°C (1), 35°C (O) and 40°C ( 1 ) : L-glycerol 3-phosphate formed at 30°C ([]), 35°C (C)) and 40°C (A). Experimental details are given in the Materials and Methods section. -(a)

® ~

-(c)

-(b)

j~80--

~.~

60-

c E

40

t ~ o

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°

rrro

0

4

8

ATP, mM

0

I0

M g 2+,

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6.0

I 7 '0

,,

T 8.0

f 9'0

pH

FIG. 2. Variation in rate of L-lactate production ( 0 ) and in L-glycerol 3-phosphate production (©) by P F S fractions from sheep jejunal mucosa using 10raM glucose 6-phosphate as substrate: (a) with A T P concentration (using 16 m M Mg 2+ and p H 7"0), (b) with M g 2+ concentration (using 2 m M A T P and p H 7"0) and (c) with p H (using 2 m M A T P and 12 m M Mg2+). Different P F S fractions were used in each experiment. Experimental details are given in the Materials and Methods section.

METABOLISM OF THE SMALL INTESTINE

L-glycerol 3-phosphate formation increased slowly with increasing A T P concentration (Fig. 2a). Lactate production was maximal at 12 m M Mg z+, although Lglycerol 3-phosphate formation decreased slowly with increasing Mg ~+ concentration (Fig. 2b). There was a broad pH optimum between pH 7.4 and 8-5 for lactate production, although L-glycerol 3-phosphate production increased steadily with increasing pH (Fig. 2c). Srivastava & Hiibscher (1966a) used 6 m M A T P and 16 m M Mg ~+ at pH 7.6 and 30°C to assay lactate formation by PFS fractions. Plots of the rates of lactate formation against the concentrations of NAD +, glucose 6-phosphate, fructose and glucose gave typical hyperbolic curves and these gave no indication of the presence of a low affinity glucokinase (E.C. 2.7.1.2) (see Lea & Walker, 1965) and none was detected by direct spectrophotometric assay (K. W. J. Wahle, unpublished work). Nicotinamide had no effect on either lactate or L-glycerol 3phosphate production but it was included in the glycolytic assay system as a precaution against enzymic destruction of NAD+. Mean values for glycolytic activity in the PFS fraction expressed as a percentage of that in the homogenate were 78 + 9.9 (mean + S.E.M., 16 comparisons) with 25 m M glucose as substrate and 119+12.6 (mean _+ S.E.M., 8 comparisons) with 10 m M glucose 6-phosphate as substrate. This distribution of glycolytic activity agrees with results obtained in other species (Srivastava & Hiibscher, 1966a; Clark & Sherratt, 1967).

Rates of L-lactate and L-glycerol 3-phosphate production by particle-free supernatant fractions from sheep jejunal mucosa Considerably more lactate was formed from glucose 6-phosphate than from glucose (Table 1) suggesting that hexokinase is a rate-limiting step in glycolysis; this has also been found for most other species investigated (Srivastava & Hiibscher, TABLE 1--FORMATION OF L-LACTATE AND L-GLYCEROL 3-PHOSPHATE IN PARTICLE-FREE SUPERNATANT FRACTIONS FROM THE JEJUNAL MUCOSA OF THE SHEEP Rate of L-lactate formation

Rate of L-glycerol 3 - p h o s p h a t e formation

None G l u c o s e (25 r a M ) G l u c o s e 6 - p h o s p h a t e (10 m M ) G l u c o s e (25 r a M ) a n d hexokinase

4.62 + 0-19 (18) 17"5 + 0"76 (13) 60.2 + 3"73 (38) 86"3, 96"0

-3"4 + 0"8 (4) 17"0 + 1"9 (11) 9.3, 4"9

(1 unit) Fructose (25 raM)

39"3 + 2-4 (4)

4.2 _+0"6 (4)

Additions

T h e rates of L-lactate a n d L-glycerol 3 - p h o s p h a t e f o r m a t i o n are g i v e n as n m o l e s / r a i n p e r nag of p r o t e i n as m e a n s + S . E . M . w i t h t h e n u m b e r of p r e p a r a t i o n s i n p a r e n thesis or as i n d i v i d u a l values w h e r e appropriate. T h e s e data i n c l u d e results q u o t e d in Fig. 3 a n d t h e c o n t r o l e x p e r i m e n t s in T a b l e 4. E x p e r i m e n t a l details are given in t h e M a t e r i a l s a n d M e t h o d s section.

10

K. W. J. WAHLE,D. G. ARMSTRONGANDH. S. A. SHEmRATT

1966a; Clark & Sherratt, 1967). The addition of crystalline yeast hexokinase increased the rate of glycolysis with glucose as substrate to an even greater extent than when glucose 6-phosphate was used (Table 1). There was an optimum concentration of 1-0 unit/ml of added hexokinase, more being inhibitory, a finding similar to that obtained with mucosal preparations from monogastric species (Srivastava & Htibscher, 1966a; Clark & Sherratt, 1967). Lactate was also formed from fructose at rates between those from glucose and glucose 6-phosphate (Table 1), suggesting the presence of ketohexokinase (E.C. 2.7.1.3), fructose 1-phosphate aldolase (E.C. 4.1.2.7) and glyceraldehyde kinase (E.C. 2.7.1.18) as reported in some monogastric species (Heinz & Lamprecht, 1968). Some L-glycerol 3-phosphate was also formed from glucose, fructose and glucose 6-phosphate (Table 1). A strict comparison of the rates of lactate production from various substrates by PFS fractions shown for the sheep in Table 1 with those obtained from the small intestinal mucosa of monogastric animals (cat, rat, guinea pig, rabbit and pigeon) is not possible since there were differences in the assay conditions (Srivastava & Hiibscher, 1966a; Clark & Sherratt, 1967). Nevertheless, the values for a given substrate are of similar magnitude and further, the differences in rates for different substrates are in the same order. The specific glycolytic activities of jejunal mucosal PFS fractions depended on the time of year when the animals were killed. This is illustrated in Fig. 3 o

=o ~

t25

EcL

o~

~,oo

Regression e q u a t i o n .

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Y= 2 4 . 7 3 + 0 . 3 4 3 X S i g n i f i c a n t at the l % l e v e l

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~25

~o tD

I 50 September I st, 1967

f I00 Time,

I 150 days

f 200 l March 18th, 1968

FIG. 3, Rates of L-lactate formation from 10 mM glucose 6-phosphate prepared from untreated jejunal mucosa of sheep killed at different times of the year. The animals, born in the spring of 1967, were between 6-12 months old when slaughtered. The regression line (O--O) and equation for the variation of rate of lactate production/ mg of protein in the PFS fraction with the time of year calculated from these results are shown on the Figure. Some of these results are included in the data quoted in Table 1. Experimental details are given in the Materials and Methods section.

METABOLISM OF THE SMALL INTESTINE

11

showing the regression of the increase of specific glycolytic activity with time between September 1967 and March 1968 which is significant at the 1 per cent level. The opportunity arose in June 1967 to measure glycolysis in supernatant fractions from the small-intestinal mucosa of two early-weaned calves (ruminating) that had been fed experimentally a high grain diet. The rates of formation of Llactate from glucose 6-phosphate (Table 2) were similar to those obtained with sheep (Table 1) while those from glucose and for the formation of L-glycerol 3phosphate from glucose 6-phosphate, glucose and fructose were greater (Table 2). TABLE 2--FORMATION OF L-LACTATE AND L-GLYCEROL 3-PHOSPHATE IN PARTICLE-FREE SUPERNATANT FRACTIONS FROM THE SMALL INTESTINAL MUCOSA OF THE BOVINE

Additions Glucose (25 mM) Glucose 6-phosphate (10 mM) Fructose (25 mM)

Rate of L-lactate formation

Rate of L-glycerol 3 - p h o s p h a t e formation

31-3, 35"4 70"2, 77-9 48"2, 56"1

8-0, 9"7 36"3, 39"5 12-2, 16"1

T h e rates o f L-lactate a n d L-glycerol 3 - p h o s p h a t e f o r m a t i o n are given as m o l e s / m i n p e r m g of p r o t e i n a n d are q u o t e d for two preparations. E x p e r i m e n t a l details are given in the Materials a n d M e t h o d s section.

L-lactate production from various substrates by particle-free supernatant fractions from sheep jejunal mucosa In view of the large variety of carbohydrate residues that enter the smallintestine in unfermented plant material and in microbial cells, it was desirable to test various carbohydrates as substrates for lactate production. However, only a few compounds at concentrations of 10 and 30 mM increased lactate production above the endogenous rate (Table 3). No formation of lactate was detected from sucrose, arabinose, ribose, xylose, rhamnose, DL-glyceraldehyde, galactose 1-phosphate, galactose 6-phosphate, DLglycerol 3-phosphate, cytidine or uridine. The only monosaccharide utilized, other than glucose or fructose, was mannose (Table 3) which is presumably phosphorylated by hexokinase to mannose 6-phosphate and which is then converted to fructose 6-phosphate by phosphomannose isomerase (E.C. 5.3.1.8). Lactate was formed from glucose 1-phosphate and from glycogen indicating the presence of phosphoglucomutase (E.C. 2.7.5.1) and glycogen phosphorylase (E.C. 3.1.3.17), and from maltose because of the presence of maltase (E.C. 3.2.1.20) (Hembrey et al., 1967). Lactate production was also observed from the glycolytic intermediates fructose 6-phosphate, fructose 1,6-diphosphate and 3-D-phosphoglycerate (the last two also assayed using 2 mM ADP replacing ATP). The high rate of formation of lactate from ribose 5-phosphate is because of the high activity of pentose phospfiate cycle enzymes in sheep small-intestinal mucosa (Bell &

12

K . W . J . WAHLE,D. G. ARMSTRONGAND H. S. A. SHERRATT

Sherratt, 1967) since some intermediates and enzymes are c o m m o n to both this cycle and to glycolysis (Srivastava & Hiibscher, 1966a, b; Clark & Sherratt, 1967). TABLE 3--FORMATION OF L-LACTATEFROM VARIOUS SUBSTRATES IN PARTICLE-FREE SUPERNATANT F~CT,ONS FROM THE JEJUNAL MUCOSA OF THE SHEEP

Lactate formed Substrate Glucose 6-phosphate Glycogen* Mannose Maltose Glucose 1-phosphate Ribose 5-phosphate Mannose 1-phosphate Fructose 6-phosphate Fructose 6-phosphate+ADP Fructose 1,6-diphosphate Fructose 1,6-diphosphate+ADP 3-Phosphoglycerate 3-Phosphoglycerate + ADP Adenosine Guanosine

Concentration 10mM

Concentration 30mM

1360 + 132 (8) 140_+ 12 (3) 134+_ 21 (3) 82, 83 928 _+ 82 (3) 953, 1110 155, 163 550 + 36 (4) 676, 713 449 + 57 (3) 519, 531 68, 117 122, 162 364 276

-278 + 71 139+ 35 131, 163 1170 + 450 686, 898 138, 166 489 + 29 447, 615 207 + 27 182, 312 23 95 194 294

(3) (3) (3) (6) (3)

*Substrate concentrations 10 and 30 mM with respect to total glucose residues. The total amount of L-lactate formed during 20 rain incubation with the substrate, after subtraction of the endogenous lactate formed in the absence of substrate using the same supernatant fraction, is expressed as nmoles +_S.E.M. with the number of experiments in parentheses where applicable, or as individual values, since the formation of lactate was not necessarily linear. Glucose 6-phosphate was included in each experiment as a standard. Other experimental details are given in the Materials and Methods section, except that where indicated ATP was replaced by an equimolar amount of ADP in the assay system. Rates of endogenous lactate formation were in the range quoted in Table 1. Lactate production from the purine nucleosides, adenosine and guanosine is p r e s u m a b l y due to their splitting to free purines and ribose 1-phosphate and the conversion of ribose 1-phosphate to ribose 5-phosphate. Significant quantities of nucleotides derived f r o m the enzymic breakdown of ribonucleic acids of r u m e n micro-organisms reach the small intestine (Barnard, 1969).

Glycolysis in preparations from stored jejunal mucosa T h e effect of storage on the glycolytic activity of mucosal preparations was investigated in an attempt to overcome the restrictions imposed by the erratic availability of fresh material. I t was found that when particle-free supernatant preparations were stored at - 1 5 ° C or at - 1 9 6 ° C for up to 3 days there was a loss of between 5-15 per cent of glycolytic activity with glucose 6-phosphate as

~TABOL~S~OF T~E SMALL~NT~STINE

13

substrate. Because of these losses PFS fractions were prepared from intestinal mucosa that had been stored in various ways. When mucosa was stored at 4°C or at - 15°C for 24 hr there was a 20-40 per cent loss of activity of the subsequently prepared PFS fractions. Treatment of mucosa with liquid nitrogen at -196°C followed immediately by thawing sometimes, though not always, produced an increase in the specific glycolytic activity of the PFS fraction (Wahle & Sherratt, 1967). With glucose as substrate there was a mean increase of activity compared with untreated intestinal mucosa of 115.4 _+14.9 per cent (S.E.M.) in eight experiments and with glucose 6-phosphate as substrate 126.4 + 10.8 per cent (S.E.M.) in thirteen experiments. The results of a further storage experiment are detailed in Table 4 in which the specific glycolytic activities of the PFS fractions determined after storage of the mucosa are expressed as percentages of the control values (no storage). When mucosa was stored in liquid nitrogen values were 90 per cent after 24 hr, 88 per cent after 48 hr and 81 per cent after 72 hr (Table 4). When it was stored at - 15°C after freezing in liquid nitrogen the values were 76 per cent after 24 hr, 76.5 per cent after 48 hr and when stored on ice there was no loss of activity for 8 hr. The rate-limiting enzymes for glycolysis in preparations from intestinal mucosa of the rat are hexokinase and phosphofructokinase (E.C. 2.7.1.11) (Srivastava & Hfibscher, 1966a; Clark & Sherratt, 1967). Much of the hexokinase in mucosal homogenates is associated with the mitochondrial fraction and this bound enzyme does not apparently contribute to glycolysis in these preparations (Srivastava et al., 1968) and this is also found with sheep jejunal mitochondrial fractions (K. W. J. Wahle, unpublished work). Wilson (1968) has shown that hexokinase is released from brain mitochondria by treatment with liquid oxygen. Phosphofructokinase is also partly associated with mitochondria from some other tissues (Mansour et al., 1966). Therefore, increases in specific glycolytic activity of PFS fractions sometimes observed after a brief treatment of mucosa with liquid nitrogen may be explained by the release of bound rate-limiting enzymes.

A comparison of the glycolytic activity of jejunal mucosa of sheep fed on a high carbohydrate diet or on a fresh grass diet With the kind co-operation of Dr. E. R. Orskov of the Rowett Research Institute, Aberdeen, samples of small-intestinal mucosa were obtained in August, 1968 from four sheep that had been fed entirely on concentrate high carbohydrate rations (barley) (Orskov, 1969). These samples were compared with similarly treated samples of intestine from four sheep that had been fed fresh grass and slaughtered in the local abattoir, matched as closely as possible for age. All animals were Suffolk Half bred cross castrates about six months old and they were killed by stunning and bleeding. The samples of mucosa from Aberdeen were transported to Newcastle in liquid nitrogen and assayed within twenty-four hours. Samples from grass fed sheep killed locally were stored in liquid nitrogen for the same time. There were no significant differences in the specific glycolytic activities for the PFS fractions prepared from the two groups (Table 5).

3.

M u c o s a frozen in l i q u i d n i t r o g e n a n d s u b s e q u e n t l y s t o r e d at

2.

L e n g t h of storage (hr) % of control

% of c o n t r o l

L e n g t h of storage (hr)

% of c o n t r o l

L e n g t h of storage (hr)

5 100"3 + 6"8 (3)

76"2 + 4"7 (4)

24

89"5 + 0"7 (3)

24

8 100"8 + 10"7 (4)

76-1 + 6-7 (3)

48

88"0 + 9-0 (4)

48

12 94-5 + 9"6 (3)

75-0, 76-2

72

81.1 + 6"4 (3)

72

26 54"0

81"7

96

Specific glycolytic activity of t h e particle-free s u p e r n a t a n t fraction

Small intestines (jejunal region) were b r o u g h t to t h e l a b o r a t o r y o n ice. M u c o s a was frozen in plastic t u b e s p u t i n l i q u i d n i t r o g e n or in t h e deep-freeze, a n d t h a w e d in h o t w a t e r (60°C). S u p e r n a t a n t fractions were p r e p a r e d f r o m mucosa, b o t h before a n d after storage, a n d t h e glycolytic activity was m e a s u r e d as t h e rate of L-lactate f o r m a t i o n f r o m glucose 6 - p h o s p h a t e . T h e glycolytic activities of fractions f r o m s t o r e d m u c o s a were e x p r e s s e d as a p e r c e n t a g e of t h o s e f r o m u n t r e a t e d m u c o s a (control) f r o m t h e same i n t e s t i n e ( m e a n s + S . E . M . w i t h t h e n u m b e r of s a m p l e s in p a r e n t h e s e s w h e r e a p p r o p r i a t e ) . T h e glycolytic activities f r o m u n t r e a t e d m u c o s a were i n t h e r a n g e q u o t e d in T a b l e 1. O t h e r e x p e r i m e n t a l details are given in t h e Materials a n d M e t h o d s section.

I n t e s t i n e stored o n ice (0°C)

( - 15oc)

M u c o s a frozen a n d s t o r e d in liquid n i t r o g e n ( - 196°C)

1.

T r e a t m e n t of m u c o s a

TABLE 4-----EFFECT OF STORAGE OF JEJUNAL MUCOSA UNDER VARIOUS CONDITIONS ON THE SPECIFIC GLYCOLYTIC ACTIVITY OF THE PARTICLEFREE SUPERNATANT FRACTION

(13

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O

"

15

METABOLISM OF THE SMALL INTESTINE

TABLE 5 - - A COMPARISON OF GLYCOLYTIC ACTIVITY IN THE PARTICLE-FREE SUPERNATANT FRACTIONS OF THE JEJUNAL MUCOSA FROM SHEEP FED ON HIGH CONCENTRATE CARBOHYDRATE RATIONS OR ON GRASS

Substrate Glucose (25 mM) Glucose 6-phosphate (10 mM)

L-Lactate formed (nmoles/min per mg of protein) High carbohydrate diet Grass diet 22"9 _+2"3 (4) 65"8 + 4"3 (4)

28"6 + 0-7 (4) 56-0 + 2"7 (4)

E x p e r i m e n t a l d e t a i l s a r e g i v e n i n t h e t e x t . T h e r e s u l t s a r e g i v e n as m e a n s + S.E.M. with the number of animals in parentheses.

A comparison of the glycolytic activity of jejunal mucosa obtained from sheep fed on pelleted grass or on chopped grass and killed by stunning and bleeding or by anaesthesia with nembutal before killing Badawy (1964) reported that the trauma and bleeding involved in the normal method of slaughter of sheep induced a marked sloughing of intestinal mucosa and it was considered desirable to examine the effect of this on the glyeolytic activity of the mucosa subsequently obtained. The opportunity was also taken to examine the effect of physical form of the diet. For this investigation samples of mucosa from fourteen to sixteen weeks old, parasite-free sheep fed rigidly controlled diets for an investigation of carcass yield (Thomson et al., 1969) were obtained in November 1967 through the kind co-operation of the Director and staff of the Grassland Research Institute, Hurley. Six of the sheep were killed by stunning and bleeding: of these three had been fed on a pelleted dried grass diet and three on the same dried grass fed chopped. Eight sheep were anaesthetized by intravenous sodium nembutal and the intestines were removed before slaughter; of these four had been fed on pelleted grass and four on the chopped grass. On the chopped grass diet there were no significant differences in glycolytic activity due to the method of slaughter with either glucose or glucose 6-phosphate as substrate (Table 6). The same was true with glucose as a substrate for the mueosa taken from the animals on the pelleted grass diet. However, with glucose 6-phosphate the activities of mucosa from the animals that had been stunned and bled were about 20 per cent lower (P = 0.05) (Table 6). Thus it would appear that under certain conditions the trauma of bleeding might adversely affect some of the values obtained for glycolytic activity in PFS fractions of intestinal mueosa. However, bearing in mind the high cost of using the nembutal treatment (with no commercial value of the carcass) and the overall results observed in the experiment it is considered that for this purpose the use of material from conventionally slaughtered animals is still justified. This experiment and that described in the previous paragraph gave no indication that diets differing in physical state, or in chemical composition, had any large effect on glycolytic activity in the jejunal mueosa.

TABLE 6 - - A

14.0 + 4.25 (3) 41.3 + 1.9 (3)

11.2 + 1.02 (3) 48.5 + 4"90 (3)

Pelleted grass diet Bleeding Nembutal

13-6 + 0.87 (3) 53.0 + 2.65 (3)

11.1 + 0-82 (4) 51.1 + 3.26 (4)

Chopped grass diet Bleeding Nembutal

Experimental details are given in the text. T h e results are given as means + S.E.M. with the number of animals in parentheses.

Substrate Glucose (20 mM) Glucose 6-phosphate (10 m M)

Method of killing

L-Lactate formed (nmoles/min per mg of protein)

A N D B L E E D I N G OR A N A E S T H E T I Z E D B Y N E M B U T A L B E F O R E K I L L I N G

COMPARISON OF G L Y C O L Y T I C A C T I V I T Y IN T H E PARTICLE-FREE S U P E R N A T A N T FRACTIONS OF T H E JEJUNAL MUCOSA FROM SHEEP

FED O N P E L L E T E D OR O N C H O P P E D GRASS A N D K I L L E D B Y S T U N N I N G

~q

).

oo

~o

Z

O

METABOLISM OF THE SMALL INTESTINE

17

DISCUSSION Glycolysis in sub-cellularprcparations from thc smaU-intcstinal mucosa of thc shccp appcars to bc similarto that found with preparationsof this tissuefrom monogastric animals (Srivastava & Hfisbchcr, 1966a; Clark & Shcrratt, 1967) although carbohydratc digestion and metabolism of adult ruminants differsfrom that of other species (Armstrong, 1965). The specificglycolyticactivitieswith glucose, fructoseor glucose 6-phosphate as substratc arc as high as thosc of subcellularpreparationsfrom monogastric animals. Slicesof sheep intestinalmucosa form only moderate amounts of lactatefrom glucose (K. W. J. Wahlc, T. E. C. Wcckcs & H. S. A. Shcrratt,unpublished work) thus rcscmbling slicesof mucosa from guinea pig rather than from rat intestine(Shcrratt, 1968). There is little variation in specificglycolyticactivitiesof supernatant fractions prcparcd from mucosa from differentregions along the Icngth of the small intestineof the sheep (Wahle & Sherratt, 1967; K. W. J. Wahlc, T. E. C. Wcekes & H. S. A. Shcrratt, unpublished work). The rolesof glycolysisin intestinalmucosa arc not clcar,indeed glycolysismay bc a sidc issue in thc absorption of monosaccharides (Crane, 1960). With sheep on dietswhcrc littlcglucose is absorbed from the lumen of the small intestinethc main substratefor glycolysisin the jejunum is presumably glucose supplied by the blood (even though systemic blood glucosc concentrations may often bc only 2 m M or Icss). Glycolysismay provide energy for absorptionof solutesand water (Barry et al., 1960; Gilman & Koclle, 1961) and the L-glycerol 3-phosphatc necessary for synthesisof triglyccridesfrom absorbed dietaryfattyacids (Bickerstaffe& Annison, 1969). Formation of lactatcfrom glucose does not necessarily mcan loss of the limitcd supply of carbohydrate availableto sheep since this may bc recovered by gluconcogenesis. The activitiesof some glycolyticenzymes in the small-intcstinalrnucosa of rats arc partly dctcrmincd by thc prcscncc of glucose in the lumen (Stifelet al., 1967; Srivastavaet al., 1969). It is clear from the present study that significant glycolyticactivityisfound in the mucosa of sheep fed rationswhich ensure virtually no ~-linkcd glucose polymer cntcring the small intestine(i.e.those animals fed dricd grasses (Table 6)). What is uncertain is whether the activitiesof such enzymes may sometimes be increasedin animals fed rations(high in ground maize) which resultin apprcciablcamounts of thcsc polymcrs cntcringthe small intcstine. It may also bc noted that in contrastto the intcrmittentflow of digcsta into the small intestineoccurring in monogastric animals, that into the small intestineof ruminants is continuous. An apparcnt relationship between the specific glycolytic activity of jejunal preparations from the sheep and season was found (Fig. 3). The significanceof this relationshipis not clcar from the present studics. It may rcflcctthe age of the animals (allwere born in the spring of 1967) or alternativelyreflectchange from summcr grazing to winter feeding. Yet again,there may be a seasonalfluctuation in specific glycolytic activity not induced by age or dict. Srivastava & Hfibschcr (1968) found that the specificglycolyticactivityof rat small-intestinal

18

K. W. J. WAHLE, D. G. ARMSTRONGAND H. S. A. SHERRATT

mucosal homogenates varied during the first 77 days of life with the highest activity at about 28 days. Further the lactate dehydrogenase activity of bull serum is higher in winter than in summer (Roussel & Stallcup, 1967). SUMMARY 1. L-Lactate formation by the particle-free supernatant fraction of sheep jejunal mucosa was investigated. 2. Conditions for L-lactate formation from glucose 6-phosphate were determined. 3. Between 78-119 p e r cent of the glycolytic activity of jejunal homogenates was recovered in the supernatant fraction. 4. High rates of L-lactate formation were obtained with glucose 6-phosphate as substrate; these were about two to three times the rates with fructose or glucose. 5. T h e phosphorylation of glucose was a rate-limiting step in glycolysis since the addition of yeast hexokinase increased the rates with glucose as substrate to those found with glucose 6-phosphate. 6. Some L-glycerol 3-phosphate was also formed from glucose, fructose and glucose 6-phosphate. 7. Sheep jejunal supernatant fractions formed some L-lactate from mannose, maltose, glycogen, ribose 5-phosphate, adenosine and guanosine. No lactate was formed from several other monosaccharides tested. 8. T h e r e was an increase in the specific activities of supernatant fractions with the age of the sheep over the period of 194 days investigated. 9. It was not possible, in two experiments, to demonstrate any effects of different diets on the glycolytic activity of the jejunum. Acknowledgements--We wish to thank the staff of the Manners Slaughter House, Ponteland, Northumberland, for their help in providing the small intestines used in this study, and C. R. Strong, Esq., B.Sc., for help with some of the experiments. Unilever Ltd. generously provided financial support and a maintenance grant for K. W. J. W.

REFERENCES ARMSTRONG D. G. (1965) Carbohydrate metabolism in ruminants and energy supply. In Physiology of Digestion in The Rumen (Edited by DOUGHERTYR. W.), pp. 272-288. Butterworths, London. ARMSTRONGD. G. & BEEVERD. E. (1969) Part-abomasal digestion of carbohydrate in the adult animal. Proc. Nutr. Soc. 28, 121-131. BADAWA¥A. M. (1964) Changes in the protein and non-protein nitrogen in the digesta of sheep, In The Role of the Gastro-intestinal Tract in Protein metabolism (Edited by MUNROEH. N.), pp. 175-185. BlackweU Scientific, Oxford. BARNARD E. A. (1969) Biological function of pancreatic ribonuclease. Nature, Lond. 221, 340-344. BARRYB. A., MATTHEWSJ. & SMYTHD. H. (1961) Transfer of glucose and fluid by different parts of the small intestine of the rat. ft. Physiol. 157, 279-288. BELLJ. W. & S~mRRATTH. S. A. (1967) The reactons of the pentose phosphate cycle in the particle-free supernatant fraction from the mucosa of the small intestine. Comp. Biochem. Physiol. 20, 319-322.

METABOLISMOF THE SMALLINTESTINE

19

BERGMAN E. G., REID R. S., MURRAY M. G., BROCKMANJ. M. • WHITELAW F. O. (1965) Interconversions and production of volatile fatty acids in the sheep rumen. Biochem..7. 97, 53-58. BIC~aSTAFF R. & ANNISON E. F. (1969) Triglyceride synthesis by the small-intestinal epithelium of the pig, sheep and chicken. Biochem.ft. 111,419--429. CLARK B. & SHF_a~RaTrH. S. A. (1967) Glycolysis and oxidations in preparations from smallintestinal mucosa of four species. Comp. Biochem. Physiol. 20, 223-243. Ca~-E R. K. (1960) Intestinal absorption of sugars. Physiol. Rev. 40, 789-825. GILMAN A. & KOELLE E. S. (1960) Ion transport in the gut. Circulation 21, 948-954. HEMBaY F. G., BELL M. C. & HALL R. F. (1967) Intestinal carbohydrase activity and carbohydrate utilization in mature sheep, ft. Nutr. 93, 175-185. HEXNZ F. & LAMPrmCHT W. (1968) Enzyme des Fructosestoffwechsels: Enzymeactivit~iten in der Diinndarmmukosa verschiedener Laboratoriumstiere. Comp. Biochern. Physiol. 27, 319-327. HOHOaST H. J., KPa~VTZ F. H. & B0CHEa T. (1959) ~ b e r Metabolitgehalte und MetabolitKonzentrationen in der Leber der Ratte. Biochem. Z. 332, 18--46. JAaaETT I. G. & POTTER B. J. (1952) Carbohydrate metabolism in the young lamb. Aust.ft. exp. Biol. Med. 30, 207-212. LEA M. A. & WALKER D. G. (1965) Factors affecting hepatic glycolysis and some changes that occur during development. Biochem.ft. 94, 655-665. McCANDLES E. L. & DYE S. A. (1950) Physiological changes in intermediary metabolism of various species of ruminants incident to functional development of tureen. Am. ft. Physiol. 162, 434--446. M A c R ~ J. C. & ARMSTaONC D. G. (1969) Studies on intestinal digestion in the sheep. 2. Digestion of some carbohydrate constituents in hay, cereal and hay-cereal rations. Br.ft. Nutr. 28, 377-387. MANSOUa T. E., WAKID N. & St'ROUSE H. M. (1966) Studies on heart phosphofructokinase. Purification, crystallization, and properties of sheep heart phosphofructokinase, ft. Biol. Chem. 241, 1512-1521. MILLER G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. ORSKOV E. R. (1969) Ann. Report of Studies in Animal Nutrition and Allied Sciences, p. 55. Rowett Research Institute, Bucksbum, Aberdeen, Scotland. POaTEUS J. & CLARK B. (1965) The isolation and characterization of subcellular components of the epithelial cells of rabbit small intestine. Biochem.ft. 96, 159-171. ROSENSWEIC N. S., STIFEL F. B., HERM~'~ R. G. & ZAKIM D. (1968) T h e dietary regulation of the glycolytic e n z y m e s - - I I . Adaptive changes in human jejunum. Biochim. biophys. ~icta 170, 228-234. ROUSSEL J. D. & STALLCUP O. T. (1967) Influence of age and season on lactic dehydrogenase activity in blood serum of bulls. Am.ft. vet. Res. 28, 721-723. SEELV.Y R. C., AaMSTaONC D. G. & MAcRAE J. C. (1969) Feed carbohydrates--the contribution of the end products of their digestion to energy supply in the ruminant. In Energy Metabolism of Farm Animals (Edited by BLAXTEa K. L., KIP.LANOWSKIJ. and THOaBEK G.), pp. 93-100. Oriel Press, Newcastle upon Tyne. SHA~St°EAI~ P., SRIVASTAVAL. M. & HOBSCHEa G. (1969) Glucose metabolism in the mucosa of the small intestine. T h e effect of glucose on hexokinase activity. Biochem. ft. 111, 6367. SHERI~,TT H. S. P~. (1968) The metabolism of the small intestine. Oxygen uptake and L-lactate production along the length of the small intestine of the rat and the guinea pig. Comp. Biochem. Physiol. 24, 745-761. SRIVASTAVAL. M. & HOBSCHER G. (1966a) Glucose metabolism in the mucosa of the small intestine. Glycolysis in subcellular preparations from the cat and rat. Biochem. ft. 100, 458-466.

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K. W. J. WAHLE,D. G. ARMSTRONGAND H. S. A. SHERRATT

SRIVASTAVAL. M. & HI~BSCHER G. (1966b) Glucose metabolism in the mucosa of the small intestine. Enzymes of the pentose phosphate pathway. Biochem.d% 101,48-55. SRIVASTAVAL. M. ~ HOBSCHER G. (1968) T h e effect of age on glycolytic and hexokinase activities in the mucosa of rat smallintestine. Biochem.ff. 110, 607-608. SRIVASTAVAL. M., SHAKESPEAREP. & HIJBSCHER G. (1968) Glucose metabolism in the mucosa of the small intestine. A study of hexokinase activity. Biochem.ff. 109, 35-42. STIFEL F. B., ROSENSWEIGN. S., ZAKIM D. & HEaMAN R. H. (1968) Dietary regulation of glycolytic enzymes--I. Adaptive changes in rat jejunum. Biochim. biophys. Acta 170, 221-227. TrtOMSON D. J., AaMSTaONG D. G. & PRESCOTT J. H. D. (1969) Influence of physical structure on the utilization of dried grass and silage. Proc. 3rd General Meeting of the European Grassland Federation Braunschweig, pp. 253-258. WAHLE K. W. J. & SHERRATT S . 8. A. (1967) Glycolysis in the particle-free supernatant fraction f r o m t h e mucosa ofthe smallintestine. Biochem.ff. 104, 53P. WILSON J. E. (1968) Brain hexokinase. A proposed relation between soluble particulate distribution and activity in vivo. ft. biol. Chem. 243, 3640-3647. WILSON T. H. (1956) T h e role of lactic acid production in glucose absorption from the intestine, ft. biol. Chem. 222, 751-763.

Key Word lndex--Glycolysis; L-lactate; L-glycerol 3-phosphate; glucose; sheep small intestine; jejunal mucosa; carbohydrate digestion; diet; age; hexokinase; subcellular localization of glycolysis.