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Glycerokinase and desaturase activity in pig, chicken and sheep intestinal epithelium
Comp. Biochem. Physiol.,
1969, Vol. 31, pp. 47 to 54. Pergamon Press. Printed in Great Britain
GLYCEROKINASE AND DESATURASE ACTIVITY IN PIG, CHICKEN AND SHEEP INTESTINAL EPITHELIUM R. B I C K E R S T A F F E and E. F. A N N I S O N Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, (Received
17 February 1969)
A b s t r a c t - - 1 . A comparative study was made of glycerokinase and desaturase
activity in the intestinal epithelium of pigs, chickens and sheep. 2. Glycerokinase, assayed by the incorporation of (1-t*C) glycerol into glycerol-3-phosphate, was found to be located in the particle-free supematant of all three species examined. Substantial conversion of glycerol to glycerol-3phosphate occurred but only a low incorporation into glycerides was observed even in the presence of microsomes. Glycerol and glycerol-3-phosphate were separated by cellulose thin-layer chromatography which gave clean separations and efficient recoveries. 3. Desaturation of (1-14C) stearic acid was only observed in sheep mucosal tissue. The desaturase enzyme was located in the microsomal fraction. INTRODUCTION FOR MANYyears it was believed that glycerol liberated during the digestion of fats was not utilized for glyceride synthesis in intestinal epithelium. Subsequent work, however, has shown that the intestinal epithelium of rats (Saunders & Dawson, 1962), hamster (Clark & Hfibscher, 1962) and man (Holt, 1964) incorporates free glycerol into glyceride-glycerol and that the enzyme, glycerokinase or ATP:glycerol phosphotransferase (2.7.1.30) responsible for the phosphorylation of glycerol is located in the cytoplasm of intestinal epithelial cells (Haessler & Isselbacher, 1963). In the present studies, glycerokinase activity was measured in epithelial tissue of pigs, chicken and sheep which have not been previously examined. The intestinal lumen of ruminants contains very little, if any, monoglycerides so that availability of monoglycerides is severely limited for the synthesis of triglycerides in the intestinal cell. The conversion of absorbed glycerol to glycerol-3-phosphate for the synthesis of triglycerides is therefore of prime importance to ruminant epithelial tissue. Glycerokinase activity was measured by the extent of incorporation of radioactive glycerol into either glycerol-3-phosphate or total lipid. A new and improved technique was used for the separation of glycerol and glycerol-3phosphate and was applied to all the subcellular fractions from the intestinal epithelial tissue of pigs, chickens and sheep. Desaturation of fatty acids has been reported to occur in liver (Elovson, 1965), adipose tissue (Gellhorn & Benjamin, 1965) and mammary tissue (Lauryssens e t al., 47
48
R. BICKERSTAFI~AND E. F. ANNI$ON
1961). R e c e n t w o r k b y B o u c r o t & C l e m e n t (1965) d e m o n s t r a t e d d e s a t u r a s e a c t i v i t y in r a t i n t e s t i n a l e p i t h e l i a l tissue, so it was of i n t e r e s t to c o m p a r e d e s a t u r a s e a c t i v i t y in n o n - r u m i n a n t ( p i g a n d c h i c k e n ) a n d r u m i n a n t ( s h e e p ) i n t e s t i n a l e p i t h e l i u m , p a r t i c u l a r l y s i n c e a c h a r a c t e r i s t i c f e a t u r e o f r u m i n a n t s is t h e h y d r o g e n a t i o n o f u n s a t u r a t e d f a t t y a c i d s in t h e r u m e n . MATERIALS AND METHODS
Experimental animals Newly weaned pigs, Sus scrofa (gilts, aged 3-4 weeks, fed creep feed Sowlac-21), sheep, Ovis aries (rams, aged 2-3 years, fed hay and oats) and chickens, Gallus domesticus (hens, aged 12-18 months, fed a complete commercial layer's ration) were killed and a length of small intestine removed as previously described (Bickerstaffe & Annison, 1969).
Preparation of homogenates and subcellular fractions from intestinal mucosa T h e general procedure of Hiibscher et al. (1965) was used for the preparation of tissue homogenates from all three species. Homogenization was in 0-25 M sucrose-5 m M E D T A 3 m M Tris buffer, p H 7"4. Subcellular fractions of debris/brush border/nuclei, mitochondria and microsomes were obtained by centrifugation for 10 rain at 1500 g, 15 rain at 12,000g and 60 min at 105,000g respectively. T h e final supernatant constituted the particle-free supernatant (PFS). T h e purity of the fractions was examined as described earlier (Bickerstaffe & Annison, 1969).
Assay of glycerokinase Glycerokinase activity was determined by the extent of incorporation of radioactive glycerol into either glycerol-3-phosphate or total lipid. T h e assay system (final volume 3 ml) contained: 250 ktmole potassium phosphate buffer (pH 7"4), 20 ktmole A T P , 0"1 k~mole CoA, 20/,mole MgClz, 2-10/~g (1-14C) glycerol and 0"6-3/~g palmitic acid bound to albumin so as not to exceed the fatty acid-albumin ratio of 2 : 1 (Laurell, 1957). T h e reaction was started by adding the enzyme preparation (5-10 mg protein). Incubations were carried out at 37°C for 40-60 min after which the reaction was stopped by the addition of 7"5 ml methanol followed by 6"5 ml of water and 13"0 ml chloroform. T h e upper aqueous phase was re-extracted with chloroform as described previously (Bickerstaffe & Annison, 1969) and the lipid extract (final volume 20 ml) counted directly. Alternatively, an aliquot (10 ml) was separated into lipid fractions by thin-layer chromatography on silica gel-G (thickness 0"4 ram) with the solvent system toluene-diethyl ether-ethanol-acetic acid (250 : 240 : 10 : 1, v/v). T h e various lipid fractions were counted in toluene (15 ml) containing 0"4% of 2.5diphenyloxazole (PPO) in a series 4000 Packard Tri-Carb liquid scintillation spectrometer. T h e aqueous residue was made up to 20 ml with water and assayed for glycerol-3phosphate by transferring an aliquot (0"5 ml) to a thin-layer plate (20 x 20 cm) coated with cellulose (Whatman CC41, thickness 0"4 ram) ; bands corresponding to glycerol and glycerol3-phosphate were located against authentic reference compounds chromatographed on an adjacent point of the thin-layer plate and detected by spraying with an aqueous solution of Na~CO3 (2%, w/v) and KMnO4 (1%, w/v) for glycerol (R! 0"75) and an aqueous solution of 60% perchloric (5% v/v), 1 N HC1 (10%, v/v) and 4% (w/v) ammonium molybdate (25%, v/v) for glycerol-3-phosphate (R! 0"25). T h e bands of cellulose corresponding to the positions of glycerol and glycerol-3-phosphate were scraped off and counted directly as a suspension in 15 ml toluene containing 3"8% Cab-O-Sil and 0.4% PPO. Alternatively, the cellulose bands were eluted with water and samples (1 ml) counted in toluene : Triton X 100 (2 : 1, v/v; 15 ml) containing 0-4% PPO. T h e limits of detection were 50-500/zg glycerol or glycerol-3-phosphate per thin-layer plate and recoveries were always greater than 92 per cent but less than 100 per cent.
GLYCEROKINASE AND DESATURASE ACTIVITY IN INTESTINAL EPITHELIUM
49
Assay of desaturase activity Desaturase activity was determined by the extent of conversion of labelled saturated fatty acid to the corresponding monoenoic acid. The incubation system (final volume 3 ml) contained: 250/zmole potassium phosphate buffer (pH 7"4), 20/~mole ATP, 0"1/Lmole CoA, 20/~mole MgC12, 270/zmole (1-14C) stearic acid (250 m/zc) bound to albumin 1.8/~mole NADH and 0-4 ~tmole NADPH. After the addition of the enzyme preparation (5-10 mg) the medium was gassed with an oxygen-carbon dioxide (95 : 5~o) mixture and incubations were continued for 60 min. The reaction was stopped by the addition of methanol (7"5 ml) and the lipids, including free fatty acids, were extracted from the incubation medium as described above and transmethylated by refluxing at 80°C with a mixture of methanolbenzene-concentrated sulphuric acid (20:10 : 1, v/v) for 90 rain. The methyl esters were extracted into petroleum ether, dried over anhydrous sodium sulphate and transferred to a gas radio chromatograph as described by Hitchcock & James (1964). The esters were separated on a 130-cm straight column (i.d. 4 mm) containing 100-200 mesh Celite supporting 10% Apiezon grease at 185°C. Alternatively, the methyl esters were separated into saturated and mono-unsaturated ester bands by argentation thin-layer chromatography with AgNOs (10%) impregnated silica gel-G and double development with the solvent system petroleum ether-diethyl ether (95 : 5, v/v). The ester bands were located with dichlorofluorescein and the appropriate areas were eluted with ether and counted in toluene (15 ml) containing 0"4~o PPO. The desaturase activity was expressed as the percentage of radioactivity appearing in the monoenoic acid. Protein determination The method of Gomal et al. (1949) was used. Materials Palmitic acid, stearic acid, ATP, CoA, NADH and NADPH were supplied by Sigma Chemical Company. (1-1aC) stearic acid and (1-1aC) glycerol were purchased from the Radiochemical Centre, Amersham, Bucks. Radioactive glycerol-3-phosphate was prepared from (1-14C) glycerol by using commercial glycerokinase (Boehringer Company) and the incubation conditions described by Kuhn (1967). The reaction was stopped by the addition of 0"5 ml trichloracetic acid (10~o, w/v) and glycerol-3-phosphate and glycerol were separated by thin-layer chromatography and isolated as described above. The yield of (1-a4C) glycerol-3-phosphate was 70 per cent.
RESULTS Previous investigations of glycerokinase activity have relied on column ionexchange chromatography(Smith & Hiibscher, 1966) and paper chromatography (DeFreitas & Depocas, 1963) to separate glycerol and g]ycerol-3-phosphate. In our hands, the use of columnion-exchangechromatographywas found satisfactory for separating glycerol and glycerol-3-phosphate (Table 1), but incomplete recovery of glycerol-3-phosphate was always observed (Table 1). Good separations and quantitative recoveries of these substrates were achieved by cellulose thin-layer chromatography and the application of the technique to subcellular fractions from pig, sheep and chicken intestinal epithelial tissues, which had been incubated with (1-I4C) glycerol, gave the results in Table 2. All tissue fractions showed some glycerokinase activity but most of the enzyme was located in the P F S (Table 2). Although substantial conversion of glycerol to glycerol-3-phosphate occurred in the incubations of total homogenate and microsomes plus PFS, examination of the
R. BICKERSTAFFEAND E. F. ANNISON
50
TABLE 1--SEPARATIONOF (laC) LABELLEDGLYCEROL(10'4 m/~c, 11"1 mg) AND GLYCEROL-3PHOSPHATE (18"7 rn/~c, 12"4 mg) ON AN ION-EXCHANGERESIN (AMBERLITE-IR-4B FORMATE FORM) COLUMN(10 x 1 cm)
Fraction 1 2 3 4 5 6 7
Solvent
Radioactivity (m/~c)
10 ml water 10 ml water 10 ml water 10 ml I M NH~OH 10 ml 1 M NH4OH 10 ml 1 M NH4OH 10 ml 1 M NH4OH
8-5 2-3 0"05 5"1 5.6 0"9 0"03
Material eluted Glycerol Glycerol Glycerol Glycerol-3-phosphate Glycerol-3-phosphate Glycerol-3-phosphate Glycerol-3-phosphate
Recovery of radioactivity (%) 104
62
TABLE 2--INcoRPORATION OF
(1-t4C) GLYCEROL INTO GLYCEROL-3-PHOSPHATE WHEN INCUBATED WITHSUBCELLULAR FRACTIONS FROM PIG, SHEEP AND CHICKEN INTESTINALEPITHELIAL TISSUE
Species Fraction Homogenate Mitochondria Mierosomes Microsomes + P F S Particle-free supernatant
Pig
Chicken
Sheep
1.46 0.29 0"63 0"71 1 "83
1.37 0"78 1.20 1"32 2-22
0.84 0"19 0"42 0'47 1 "20
(PFS) Results are in terms of glycerokinase specific activity (/~g glycerol incorporated/hr per mg protein). Incubation conditions were as described in the text. TABLE 3--INCORPORATION OF (I-14C) GLYCEROL INTO TOTALLIPIDS WHEN INCUBATED WITH SUBCELLULAR FRACTIONS OF PIG, SHEEP AND CHICKEN INTESTINALEPITHELIALTISSUE
Species Fraction Homogenate Mitochondria Microsomes Microsomes + PFS Particle-free supernatant
Pig
Chicken
Sheep
120 63 152 327 * 15
80 60 180 240 * 40
66 35 80 175 * 34
(PFS) Results are in terms of enzyme specific activity (m/~g glycerol incorporated into total lipid/hr per mg protein). Incubation conditions were as described in the text. * Results calculated on basis of microsomal protein.
GLYCEROKINASE AND DESATURASE ACTIVITY IN INTESTINAL EPITHELIUM
51
lipids showed that the incorporation of glycerol into lipids was low even though the incubation system had all the necessary enzymes and coenzymes for triglyceride synthesis (Table 3). T h e total lipid was examined by thin-layer chromatography and in all species examined (1-t4C) glycerol was distributed between the phospholipid and triglyceride fractions when incubated with the total homogenate, microsomes and microsomes plus P F S (Table 4). If it is assumed that the activity of intact mucosal T A B L E 4 - ' - I N c o R P O R A T I O N OF RADIOACTIVITY INTO PHOSPHOLIPIDS (PL), MONOGLYCERIDES (MG), FREE FATTY ACIDS (FFA), DIGLYCERIDES (DO) AND TRIGLYCERIDES (TG) AFTER
INCUBATIONOF (1-14C) GLYCEROLWITHTOTALHOMOOENATE(4--8 mg), MICROSOMES(1--2 rag) AND MICROSOMESPLUS PARTICLE-FREESUPEm'~ATANT(PFS, 2-3 mg) FROM PIG, SHEEP AND CHICKEN MUCOSAL TISSUE
Radioactivity in lipid fraction (% of the total for the tissue fraction) Tissue fraction Species Pig
Sheep
Chicken
Lipid fraction
Homogenate
Microsomes
Microsomes + PFS
PL MG FFA DG TG PL MG FFA DG TG PL MG FFA DG TG
6"6 0"7 -7"6 85"1 16"4 2.0 -5"0 76'3 4.0 0"6 -6"7 88'6
26"3 --3"5 70"2 31 "3 4"1 -7.5 55'5 33-3 0'6 -4"0 56'6
4'9 --8"1 86"7 4"5 --10-0 84"7 1 "8 1"8 -8"8 87.7
For further details, see the text. tissue is directly proportional to that of the homogenate, then a rough estimate can be made of the capacity of the epithelial tissue of the three species to phosphorylate glycerol. Although the synthetic capacity/g of sheep tissue is less than that of the pig and chicken (Table 5), when account is taken of the more widespread distribution of triglyceride synthetase along the sheep intestine (Bickerstaffe & Annison, 1969), sheep tissue has a greater capacity than pig or chicken tissue to phosphorylate glycerol, and to utilize the product for glyceride synthesis. T h e subcellular fractions from pig, sheep and chicken intestinal epithelial tissue were also examined for their capacity to desaturate (1-t4C) stearic acid. No
52
R. BICKERSTAFFE AND
E. F. ~ N I S O N
T A B L E 5 ~ R E L A T I V E RATES OF PHOSPHORYLATION OF ( 1 - 1 4 C ) GLYCEROL BY PIG, SHEEP AND CHICKEN INTESTINAL E P I T H E L I U M EXPRESSED IN TERMS OF NET W E I G H T ( A ) AND LENGTH OF INTESTINES ( B )
Phosphorylation of glycerol (/zmoles) Species
A (g net wt/hr)
Pig Sheep Chicken
3"2 1.8 3"0
B (30 cm intestine/hr) 28"8 18"0 24"0
desaturation was obtained in any of the subcellular fractions from pig and chicken, but sheep subcellular fractions showed desaturase activity (Table 6). The enzymes necessary for the activation and desaturation of stearic acid were located in the microsomal fraction of sheep mucosal tissue. The microsomal particles were also incubated with (1-14C) palmitic acid but no desaturation was obtained with this acid. TABLE 6--INTRACELLULARDISTRIBUTION OF DESATURASE ACTIVITY IN PIG, SHEEP AND CHICKEN INTESTINAL EPITHELIAL TISSUE
Species Fraction
Pig
Chicken
Sheep
Homogenate Mitochondria Microcomes Microsomes + PFS Particle-free supernatant (PFS)
0 0 0 0 0
0 0 0 0 0
4'5 5"1 15"2 15"9 0
The results are expressed as percentage desaturation of (1-14C) stearic acid/5 mg protein. Incubation conditions were as described in the text. DISCUSSION Glycerokinase has been found in kidney (Kalckar, 1939), liver (Wieland & Suyter, 1957), m a m m a r y tissue (McBride & Korn, 1964) and intestinal mucosa (Bublitz & Kennedy, 1954), but demonstration of this enzyme has frequently been confounded by the existence of an active glycerophosphatase in the enzyme preparations. T h e present work demonstrates that glycerokinase occurs in the cytoplasm of pig, sheep and chicken intestinal epithelial cells, and is in accord with similar results obtained in rat and hamster intestinal mucosal tissue (Haessler & Isselbacher, 1963). In all three species, glycerol-3-phosphate from glycerol is incorporated into lipids but only to a slight extent. Examination of the lipids after incubation of (1-14C) glycerol with microsomal particles showed that the radioactivity was
GLYCEROKINASE AND DESATURASE ACTIVITY IN" INTESTINAL E P I T H E L I U M
53
distributed between the phospholipids and triglycerides but on addition of the particle-free supernatant a shift towards triglycerides occurred. A higher enzyme specific activity was also noted under these conditions. It therefore seems that the PFS is providing both glycerokinase and the soluble enzyme phosphatidate hydrolase. Nevertheless, even in the presence of the PFS, the net incorporation of glycerol-3-phosphate into glycerides was low. This observation probably reflects the level of glycerol-3-phosphate available for glyceride synthesis since the optimum level of 6 m M (Bickerstaffe & Annison, 1969) was not reached in the present incubations. Obviously, the availability of glycerokinase is not inhibiting glyceride synthesis in any of the species studied. The significance of glycerokinase activity in vivo was not assessed in the present experiment, but in certain non-ruminants glycerol-3-phosphate contributes to less than 25 per cent of the synthesized triglycerides (Kayden et al., 1967), and the monoglyceride pathway predominates. In ruminants, however, glycerol-3-phosphate is probably the main precursor of triglycerides since only small amounts of monoglycerides are absorbed from the small intestine. Although glucose probably provides most of the glycerol-3phosphate in sheep mucosal tissue the above results indicate that glycerokinase may provide an alternative source of glycerol-3-phosphate. In fact, the glycerokinase level is substantially higher than the non-ruminant values but in vivo experiments are needed to assess the quantitative importance of the enzyme to the animal. Glycerol can also arise from the hydrolysis of monoglycerides by an intracellular monoglyceride lipase in the intestinal cell. Such a lipase has been demonstrated in intestinal mucosal tissue, and it is possible that monoglycerides are incorporated into higher glycerides after hydrolysis and reincorporation of the products by the glycerol-3phosphate pathway. Dawson (1967) speculates that the presence of an intramucosal lipase, glycerokinase, and the intracellular concentration of free fatty acid and monoglyceride may control which glyceride pathway is utilized in the intestinal cell. Desaturation of fatty acids has been demonstrated under both in vitro and in vivo conditions in several mammalian tissues but only recently in rat intestinal epithelial tissue (Boucrot & Clement, 1965). The present results indicate the absence of desaturation in pig and chicken epithelial tissue, but stearic acid was desaturated to oleic acid by sheep mucosal tissue. A possible explanation for the negative results with non-ruminant tissue might be the deacylation of stearyl CoA. The ready activation of (9,10-8H) palmitate as shown by its incorporation into lipids, however, clearly demonstrates that acylation must occur under the incubation conditions used. T h e desaturation of stearic acid by sheep intestinal epithelium partially reverses the extensive ruminal hydrogenation of dietary unsaturated fatty acids, but the physiological significance of this desaturation is not yet known. REFERENCES BICKERSTAFFER. & ANNISON E. F. (1969) Triglyceride synthesis by the small intestinal epithelium of the pig, sheep and chicken. Biochern.d7. 111, 419-429.
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R. BICKERSTAFFEAND E. F. ANNISON
BLIGH E. G. & DYER W. J. (1959) A rapid method of total lipid extraction and purification. Can. `7. biochem. Physiol. 37, 911-917. BOUCROT P. & CLEMENT J. (1965) Metabolic conversion of palmitic and stearic acids during their digestion and absorption. Arch. Sci. Physiol. 19, 181-196. BUBLITZ C. & KENNEDY E. P. (1954) Glycerokinase in mucosa of the small intestine of the rat. `7. biol. Chem. 211, 951-961. CLARKB.&HOBsCHER G. (1962) Synthesis ofphosphatides in isolated mitochondria. Nature, Load. 195, 599-600. DAWSON A. M. (1967) Absorption of fats. Br. Med. Bull. 23, 247-251. DEFREITAS A. S. W. & DEPOCASF. (1963) Quantitative isolation and assayof neutral glyceride glycerol and phosphoglyceride glycerol. Can. `7. Biochem. 42, 195-202. ELOVSON J. (1965) Conversions of palmitic and stearic acid in the intact rat. Biochim. biophys. Acta 106, 291-303. GELLHORN A. & BENJAMIN W. (1965) Lipid biosynthesis in adipose tissue during ageing and in diabetes. Ann. N. Y. Acad. Sci. 131,344--356. GORNALL A. G., BARDAWILLC. J. & DAVID M. M. (1949) Determination of serum proteins by means of the biuret reaction. `7. biol. Chem. 73, 751-766. HAESSLER H. A. & ISSELBACHER K. J. (1963) T h e metabolism of glycerol by intestinal mucosa. Biochim. biophys. Acta 73, 427--436. HITCHCOCK C. H. S. & JAMESA. T. (1964) Oxidation of unsaturated fatty acids by leaf tissue. `7. Lipid Res. 5, 593-599. HOLT P. R. (1964) Otilisation of 14C-glycerol for intestinal glyceride esterification : studies in patients with chyluria. `7. din. Invest. 43, 349-356. HOBSCHER G., WEST G. R. & BRINDLEY D. N. (1965) Studies on the fractionation of mucosal homogenates from the small intestines. Biochem.`7. 97, 629-642. KALCKARH. (1939) T h e nature of phosphoric esters formed in kidney extracts. Biochem.J. 33, 631-641. KAYDEN H. J., SENIOR J. R. & MATTSON F. H. (1967) T h e monoglyceride pathway of fat absorption in man. jT. din. Invest. 46, 1695-1703. KUHN N. J. (1967) Esterification of glycerol-3-phosphate in lactating guinea pig mammary gland. Biochem.`7. 105, 213-223. LAURELL S. (1957) Turnover rate of unesterified fatty acids in human plasma. Actaphysiol. scand. 41,158-167. LAURY$SENSM., VERBEKE R. & PEETERS G. (1961) Metabolism of 1-14C stearate in the isolated cow udder..7. LipidRes. 2, 383-388. McBRIDE O. W. & KORN E. D. (1964) Presence of glycerokinase in guinea pig mammary gland and the incorporation of glycerol into glycerides. `7. Lipid Res. 5, 442-447. SAUNDERSD. R. & DAWSONA. M. (1962) Studies on the metabolism of glycerol by the small intestines in vitro and in vivo. Biochem.`7. 82, 477-483. SMITH M. E. & HOBSCHER G. (1966) T h e biosynthesis of glycerides by mitochondria from rat liver. Biochem.`7. 101,308-316. WIELAND O. & SUYTER M. (1957) Glycerokinase: its isolation and properties. Biochem. Z. 329, 320-331.
Key Word Index--Glycerokinase; intestine; desaturation of fatty acids ; digestion ; microsomal desaturase; pig; chicken; sheep.
1969, Vol. 31, pp. 47 to 54. Pergamon Press. Printed in Great Britain
GLYCEROKINASE AND DESATURASE ACTIVITY IN PIG, CHICKEN AND SHEEP INTESTINAL EPITHELIUM R. B I C K E R S T A F F E and E. F. A N N I S O N Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, (Received
17 February 1969)
A b s t r a c t - - 1 . A comparative study was made of glycerokinase and desaturase
activity in the intestinal epithelium of pigs, chickens and sheep. 2. Glycerokinase, assayed by the incorporation of (1-t*C) glycerol into glycerol-3-phosphate, was found to be located in the particle-free supematant of all three species examined. Substantial conversion of glycerol to glycerol-3phosphate occurred but only a low incorporation into glycerides was observed even in the presence of microsomes. Glycerol and glycerol-3-phosphate were separated by cellulose thin-layer chromatography which gave clean separations and efficient recoveries. 3. Desaturation of (1-14C) stearic acid was only observed in sheep mucosal tissue. The desaturase enzyme was located in the microsomal fraction. INTRODUCTION FOR MANYyears it was believed that glycerol liberated during the digestion of fats was not utilized for glyceride synthesis in intestinal epithelium. Subsequent work, however, has shown that the intestinal epithelium of rats (Saunders & Dawson, 1962), hamster (Clark & Hfibscher, 1962) and man (Holt, 1964) incorporates free glycerol into glyceride-glycerol and that the enzyme, glycerokinase or ATP:glycerol phosphotransferase (2.7.1.30) responsible for the phosphorylation of glycerol is located in the cytoplasm of intestinal epithelial cells (Haessler & Isselbacher, 1963). In the present studies, glycerokinase activity was measured in epithelial tissue of pigs, chicken and sheep which have not been previously examined. The intestinal lumen of ruminants contains very little, if any, monoglycerides so that availability of monoglycerides is severely limited for the synthesis of triglycerides in the intestinal cell. The conversion of absorbed glycerol to glycerol-3-phosphate for the synthesis of triglycerides is therefore of prime importance to ruminant epithelial tissue. Glycerokinase activity was measured by the extent of incorporation of radioactive glycerol into either glycerol-3-phosphate or total lipid. A new and improved technique was used for the separation of glycerol and glycerol-3phosphate and was applied to all the subcellular fractions from the intestinal epithelial tissue of pigs, chickens and sheep. Desaturation of fatty acids has been reported to occur in liver (Elovson, 1965), adipose tissue (Gellhorn & Benjamin, 1965) and mammary tissue (Lauryssens e t al., 47
48
R. BICKERSTAFI~AND E. F. ANNI$ON
1961). R e c e n t w o r k b y B o u c r o t & C l e m e n t (1965) d e m o n s t r a t e d d e s a t u r a s e a c t i v i t y in r a t i n t e s t i n a l e p i t h e l i a l tissue, so it was of i n t e r e s t to c o m p a r e d e s a t u r a s e a c t i v i t y in n o n - r u m i n a n t ( p i g a n d c h i c k e n ) a n d r u m i n a n t ( s h e e p ) i n t e s t i n a l e p i t h e l i u m , p a r t i c u l a r l y s i n c e a c h a r a c t e r i s t i c f e a t u r e o f r u m i n a n t s is t h e h y d r o g e n a t i o n o f u n s a t u r a t e d f a t t y a c i d s in t h e r u m e n . MATERIALS AND METHODS
Experimental animals Newly weaned pigs, Sus scrofa (gilts, aged 3-4 weeks, fed creep feed Sowlac-21), sheep, Ovis aries (rams, aged 2-3 years, fed hay and oats) and chickens, Gallus domesticus (hens, aged 12-18 months, fed a complete commercial layer's ration) were killed and a length of small intestine removed as previously described (Bickerstaffe & Annison, 1969).
Preparation of homogenates and subcellular fractions from intestinal mucosa T h e general procedure of Hiibscher et al. (1965) was used for the preparation of tissue homogenates from all three species. Homogenization was in 0-25 M sucrose-5 m M E D T A 3 m M Tris buffer, p H 7"4. Subcellular fractions of debris/brush border/nuclei, mitochondria and microsomes were obtained by centrifugation for 10 rain at 1500 g, 15 rain at 12,000g and 60 min at 105,000g respectively. T h e final supernatant constituted the particle-free supernatant (PFS). T h e purity of the fractions was examined as described earlier (Bickerstaffe & Annison, 1969).
Assay of glycerokinase Glycerokinase activity was determined by the extent of incorporation of radioactive glycerol into either glycerol-3-phosphate or total lipid. T h e assay system (final volume 3 ml) contained: 250 ktmole potassium phosphate buffer (pH 7"4), 20 ktmole A T P , 0"1 k~mole CoA, 20/,mole MgClz, 2-10/~g (1-14C) glycerol and 0"6-3/~g palmitic acid bound to albumin so as not to exceed the fatty acid-albumin ratio of 2 : 1 (Laurell, 1957). T h e reaction was started by adding the enzyme preparation (5-10 mg protein). Incubations were carried out at 37°C for 40-60 min after which the reaction was stopped by the addition of 7"5 ml methanol followed by 6"5 ml of water and 13"0 ml chloroform. T h e upper aqueous phase was re-extracted with chloroform as described previously (Bickerstaffe & Annison, 1969) and the lipid extract (final volume 20 ml) counted directly. Alternatively, an aliquot (10 ml) was separated into lipid fractions by thin-layer chromatography on silica gel-G (thickness 0"4 ram) with the solvent system toluene-diethyl ether-ethanol-acetic acid (250 : 240 : 10 : 1, v/v). T h e various lipid fractions were counted in toluene (15 ml) containing 0"4% of 2.5diphenyloxazole (PPO) in a series 4000 Packard Tri-Carb liquid scintillation spectrometer. T h e aqueous residue was made up to 20 ml with water and assayed for glycerol-3phosphate by transferring an aliquot (0"5 ml) to a thin-layer plate (20 x 20 cm) coated with cellulose (Whatman CC41, thickness 0"4 ram) ; bands corresponding to glycerol and glycerol3-phosphate were located against authentic reference compounds chromatographed on an adjacent point of the thin-layer plate and detected by spraying with an aqueous solution of Na~CO3 (2%, w/v) and KMnO4 (1%, w/v) for glycerol (R! 0"75) and an aqueous solution of 60% perchloric (5% v/v), 1 N HC1 (10%, v/v) and 4% (w/v) ammonium molybdate (25%, v/v) for glycerol-3-phosphate (R! 0"25). T h e bands of cellulose corresponding to the positions of glycerol and glycerol-3-phosphate were scraped off and counted directly as a suspension in 15 ml toluene containing 3"8% Cab-O-Sil and 0.4% PPO. Alternatively, the cellulose bands were eluted with water and samples (1 ml) counted in toluene : Triton X 100 (2 : 1, v/v; 15 ml) containing 0-4% PPO. T h e limits of detection were 50-500/zg glycerol or glycerol-3-phosphate per thin-layer plate and recoveries were always greater than 92 per cent but less than 100 per cent.
GLYCEROKINASE AND DESATURASE ACTIVITY IN INTESTINAL EPITHELIUM
49
Assay of desaturase activity Desaturase activity was determined by the extent of conversion of labelled saturated fatty acid to the corresponding monoenoic acid. The incubation system (final volume 3 ml) contained: 250/zmole potassium phosphate buffer (pH 7"4), 20/~mole ATP, 0"1/Lmole CoA, 20/~mole MgC12, 270/zmole (1-14C) stearic acid (250 m/zc) bound to albumin 1.8/~mole NADH and 0-4 ~tmole NADPH. After the addition of the enzyme preparation (5-10 mg) the medium was gassed with an oxygen-carbon dioxide (95 : 5~o) mixture and incubations were continued for 60 min. The reaction was stopped by the addition of methanol (7"5 ml) and the lipids, including free fatty acids, were extracted from the incubation medium as described above and transmethylated by refluxing at 80°C with a mixture of methanolbenzene-concentrated sulphuric acid (20:10 : 1, v/v) for 90 rain. The methyl esters were extracted into petroleum ether, dried over anhydrous sodium sulphate and transferred to a gas radio chromatograph as described by Hitchcock & James (1964). The esters were separated on a 130-cm straight column (i.d. 4 mm) containing 100-200 mesh Celite supporting 10% Apiezon grease at 185°C. Alternatively, the methyl esters were separated into saturated and mono-unsaturated ester bands by argentation thin-layer chromatography with AgNOs (10%) impregnated silica gel-G and double development with the solvent system petroleum ether-diethyl ether (95 : 5, v/v). The ester bands were located with dichlorofluorescein and the appropriate areas were eluted with ether and counted in toluene (15 ml) containing 0"4~o PPO. The desaturase activity was expressed as the percentage of radioactivity appearing in the monoenoic acid. Protein determination The method of Gomal et al. (1949) was used. Materials Palmitic acid, stearic acid, ATP, CoA, NADH and NADPH were supplied by Sigma Chemical Company. (1-1aC) stearic acid and (1-1aC) glycerol were purchased from the Radiochemical Centre, Amersham, Bucks. Radioactive glycerol-3-phosphate was prepared from (1-14C) glycerol by using commercial glycerokinase (Boehringer Company) and the incubation conditions described by Kuhn (1967). The reaction was stopped by the addition of 0"5 ml trichloracetic acid (10~o, w/v) and glycerol-3-phosphate and glycerol were separated by thin-layer chromatography and isolated as described above. The yield of (1-a4C) glycerol-3-phosphate was 70 per cent.
RESULTS Previous investigations of glycerokinase activity have relied on column ionexchange chromatography(Smith & Hiibscher, 1966) and paper chromatography (DeFreitas & Depocas, 1963) to separate glycerol and g]ycerol-3-phosphate. In our hands, the use of columnion-exchangechromatographywas found satisfactory for separating glycerol and glycerol-3-phosphate (Table 1), but incomplete recovery of glycerol-3-phosphate was always observed (Table 1). Good separations and quantitative recoveries of these substrates were achieved by cellulose thin-layer chromatography and the application of the technique to subcellular fractions from pig, sheep and chicken intestinal epithelial tissues, which had been incubated with (1-I4C) glycerol, gave the results in Table 2. All tissue fractions showed some glycerokinase activity but most of the enzyme was located in the P F S (Table 2). Although substantial conversion of glycerol to glycerol-3-phosphate occurred in the incubations of total homogenate and microsomes plus PFS, examination of the
R. BICKERSTAFFEAND E. F. ANNISON
50
TABLE 1--SEPARATIONOF (laC) LABELLEDGLYCEROL(10'4 m/~c, 11"1 mg) AND GLYCEROL-3PHOSPHATE (18"7 rn/~c, 12"4 mg) ON AN ION-EXCHANGERESIN (AMBERLITE-IR-4B FORMATE FORM) COLUMN(10 x 1 cm)
Fraction 1 2 3 4 5 6 7
Solvent
Radioactivity (m/~c)
10 ml water 10 ml water 10 ml water 10 ml I M NH~OH 10 ml 1 M NH4OH 10 ml 1 M NH4OH 10 ml 1 M NH4OH
8-5 2-3 0"05 5"1 5.6 0"9 0"03
Material eluted Glycerol Glycerol Glycerol Glycerol-3-phosphate Glycerol-3-phosphate Glycerol-3-phosphate Glycerol-3-phosphate
Recovery of radioactivity (%) 104
62
TABLE 2--INcoRPORATION OF
(1-t4C) GLYCEROL INTO GLYCEROL-3-PHOSPHATE WHEN INCUBATED WITHSUBCELLULAR FRACTIONS FROM PIG, SHEEP AND CHICKEN INTESTINALEPITHELIAL TISSUE
Species Fraction Homogenate Mitochondria Mierosomes Microsomes + P F S Particle-free supernatant
Pig
Chicken
Sheep
1.46 0.29 0"63 0"71 1 "83
1.37 0"78 1.20 1"32 2-22
0.84 0"19 0"42 0'47 1 "20
(PFS) Results are in terms of glycerokinase specific activity (/~g glycerol incorporated/hr per mg protein). Incubation conditions were as described in the text. TABLE 3--INCORPORATION OF (I-14C) GLYCEROL INTO TOTALLIPIDS WHEN INCUBATED WITH SUBCELLULAR FRACTIONS OF PIG, SHEEP AND CHICKEN INTESTINALEPITHELIALTISSUE
Species Fraction Homogenate Mitochondria Microsomes Microsomes + PFS Particle-free supernatant
Pig
Chicken
Sheep
120 63 152 327 * 15
80 60 180 240 * 40
66 35 80 175 * 34
(PFS) Results are in terms of enzyme specific activity (m/~g glycerol incorporated into total lipid/hr per mg protein). Incubation conditions were as described in the text. * Results calculated on basis of microsomal protein.
GLYCEROKINASE AND DESATURASE ACTIVITY IN INTESTINAL EPITHELIUM
51
lipids showed that the incorporation of glycerol into lipids was low even though the incubation system had all the necessary enzymes and coenzymes for triglyceride synthesis (Table 3). T h e total lipid was examined by thin-layer chromatography and in all species examined (1-t4C) glycerol was distributed between the phospholipid and triglyceride fractions when incubated with the total homogenate, microsomes and microsomes plus P F S (Table 4). If it is assumed that the activity of intact mucosal T A B L E 4 - ' - I N c o R P O R A T I O N OF RADIOACTIVITY INTO PHOSPHOLIPIDS (PL), MONOGLYCERIDES (MG), FREE FATTY ACIDS (FFA), DIGLYCERIDES (DO) AND TRIGLYCERIDES (TG) AFTER
INCUBATIONOF (1-14C) GLYCEROLWITHTOTALHOMOOENATE(4--8 mg), MICROSOMES(1--2 rag) AND MICROSOMESPLUS PARTICLE-FREESUPEm'~ATANT(PFS, 2-3 mg) FROM PIG, SHEEP AND CHICKEN MUCOSAL TISSUE
Radioactivity in lipid fraction (% of the total for the tissue fraction) Tissue fraction Species Pig
Sheep
Chicken
Lipid fraction
Homogenate
Microsomes
Microsomes + PFS
PL MG FFA DG TG PL MG FFA DG TG PL MG FFA DG TG
6"6 0"7 -7"6 85"1 16"4 2.0 -5"0 76'3 4.0 0"6 -6"7 88'6
26"3 --3"5 70"2 31 "3 4"1 -7.5 55'5 33-3 0'6 -4"0 56'6
4'9 --8"1 86"7 4"5 --10-0 84"7 1 "8 1"8 -8"8 87.7
For further details, see the text. tissue is directly proportional to that of the homogenate, then a rough estimate can be made of the capacity of the epithelial tissue of the three species to phosphorylate glycerol. Although the synthetic capacity/g of sheep tissue is less than that of the pig and chicken (Table 5), when account is taken of the more widespread distribution of triglyceride synthetase along the sheep intestine (Bickerstaffe & Annison, 1969), sheep tissue has a greater capacity than pig or chicken tissue to phosphorylate glycerol, and to utilize the product for glyceride synthesis. T h e subcellular fractions from pig, sheep and chicken intestinal epithelial tissue were also examined for their capacity to desaturate (1-t4C) stearic acid. No
52
R. BICKERSTAFFE AND
E. F. ~ N I S O N
T A B L E 5 ~ R E L A T I V E RATES OF PHOSPHORYLATION OF ( 1 - 1 4 C ) GLYCEROL BY PIG, SHEEP AND CHICKEN INTESTINAL E P I T H E L I U M EXPRESSED IN TERMS OF NET W E I G H T ( A ) AND LENGTH OF INTESTINES ( B )
Phosphorylation of glycerol (/zmoles) Species
A (g net wt/hr)
Pig Sheep Chicken
3"2 1.8 3"0
B (30 cm intestine/hr) 28"8 18"0 24"0
desaturation was obtained in any of the subcellular fractions from pig and chicken, but sheep subcellular fractions showed desaturase activity (Table 6). The enzymes necessary for the activation and desaturation of stearic acid were located in the microsomal fraction of sheep mucosal tissue. The microsomal particles were also incubated with (1-14C) palmitic acid but no desaturation was obtained with this acid. TABLE 6--INTRACELLULARDISTRIBUTION OF DESATURASE ACTIVITY IN PIG, SHEEP AND CHICKEN INTESTINAL EPITHELIAL TISSUE
Species Fraction
Pig
Chicken
Sheep
Homogenate Mitochondria Microcomes Microsomes + PFS Particle-free supernatant (PFS)
0 0 0 0 0
0 0 0 0 0
4'5 5"1 15"2 15"9 0
The results are expressed as percentage desaturation of (1-14C) stearic acid/5 mg protein. Incubation conditions were as described in the text. DISCUSSION Glycerokinase has been found in kidney (Kalckar, 1939), liver (Wieland & Suyter, 1957), m a m m a r y tissue (McBride & Korn, 1964) and intestinal mucosa (Bublitz & Kennedy, 1954), but demonstration of this enzyme has frequently been confounded by the existence of an active glycerophosphatase in the enzyme preparations. T h e present work demonstrates that glycerokinase occurs in the cytoplasm of pig, sheep and chicken intestinal epithelial cells, and is in accord with similar results obtained in rat and hamster intestinal mucosal tissue (Haessler & Isselbacher, 1963). In all three species, glycerol-3-phosphate from glycerol is incorporated into lipids but only to a slight extent. Examination of the lipids after incubation of (1-14C) glycerol with microsomal particles showed that the radioactivity was
GLYCEROKINASE AND DESATURASE ACTIVITY IN" INTESTINAL E P I T H E L I U M
53
distributed between the phospholipids and triglycerides but on addition of the particle-free supernatant a shift towards triglycerides occurred. A higher enzyme specific activity was also noted under these conditions. It therefore seems that the PFS is providing both glycerokinase and the soluble enzyme phosphatidate hydrolase. Nevertheless, even in the presence of the PFS, the net incorporation of glycerol-3-phosphate into glycerides was low. This observation probably reflects the level of glycerol-3-phosphate available for glyceride synthesis since the optimum level of 6 m M (Bickerstaffe & Annison, 1969) was not reached in the present incubations. Obviously, the availability of glycerokinase is not inhibiting glyceride synthesis in any of the species studied. The significance of glycerokinase activity in vivo was not assessed in the present experiment, but in certain non-ruminants glycerol-3-phosphate contributes to less than 25 per cent of the synthesized triglycerides (Kayden et al., 1967), and the monoglyceride pathway predominates. In ruminants, however, glycerol-3-phosphate is probably the main precursor of triglycerides since only small amounts of monoglycerides are absorbed from the small intestine. Although glucose probably provides most of the glycerol-3phosphate in sheep mucosal tissue the above results indicate that glycerokinase may provide an alternative source of glycerol-3-phosphate. In fact, the glycerokinase level is substantially higher than the non-ruminant values but in vivo experiments are needed to assess the quantitative importance of the enzyme to the animal. Glycerol can also arise from the hydrolysis of monoglycerides by an intracellular monoglyceride lipase in the intestinal cell. Such a lipase has been demonstrated in intestinal mucosal tissue, and it is possible that monoglycerides are incorporated into higher glycerides after hydrolysis and reincorporation of the products by the glycerol-3phosphate pathway. Dawson (1967) speculates that the presence of an intramucosal lipase, glycerokinase, and the intracellular concentration of free fatty acid and monoglyceride may control which glyceride pathway is utilized in the intestinal cell. Desaturation of fatty acids has been demonstrated under both in vitro and in vivo conditions in several mammalian tissues but only recently in rat intestinal epithelial tissue (Boucrot & Clement, 1965). The present results indicate the absence of desaturation in pig and chicken epithelial tissue, but stearic acid was desaturated to oleic acid by sheep mucosal tissue. A possible explanation for the negative results with non-ruminant tissue might be the deacylation of stearyl CoA. The ready activation of (9,10-8H) palmitate as shown by its incorporation into lipids, however, clearly demonstrates that acylation must occur under the incubation conditions used. T h e desaturation of stearic acid by sheep intestinal epithelium partially reverses the extensive ruminal hydrogenation of dietary unsaturated fatty acids, but the physiological significance of this desaturation is not yet known. REFERENCES BICKERSTAFFER. & ANNISON E. F. (1969) Triglyceride synthesis by the small intestinal epithelium of the pig, sheep and chicken. Biochern.d7. 111, 419-429.
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R. BICKERSTAFFEAND E. F. ANNISON
BLIGH E. G. & DYER W. J. (1959) A rapid method of total lipid extraction and purification. Can. `7. biochem. Physiol. 37, 911-917. BOUCROT P. & CLEMENT J. (1965) Metabolic conversion of palmitic and stearic acids during their digestion and absorption. Arch. Sci. Physiol. 19, 181-196. BUBLITZ C. & KENNEDY E. P. (1954) Glycerokinase in mucosa of the small intestine of the rat. `7. biol. Chem. 211, 951-961. CLARKB.&HOBsCHER G. (1962) Synthesis ofphosphatides in isolated mitochondria. Nature, Load. 195, 599-600. DAWSON A. M. (1967) Absorption of fats. Br. Med. Bull. 23, 247-251. DEFREITAS A. S. W. & DEPOCASF. (1963) Quantitative isolation and assayof neutral glyceride glycerol and phosphoglyceride glycerol. Can. `7. Biochem. 42, 195-202. ELOVSON J. (1965) Conversions of palmitic and stearic acid in the intact rat. Biochim. biophys. Acta 106, 291-303. GELLHORN A. & BENJAMIN W. (1965) Lipid biosynthesis in adipose tissue during ageing and in diabetes. Ann. N. Y. Acad. Sci. 131,344--356. GORNALL A. G., BARDAWILLC. J. & DAVID M. M. (1949) Determination of serum proteins by means of the biuret reaction. `7. biol. Chem. 73, 751-766. HAESSLER H. A. & ISSELBACHER K. J. (1963) T h e metabolism of glycerol by intestinal mucosa. Biochim. biophys. Acta 73, 427--436. HITCHCOCK C. H. S. & JAMESA. T. (1964) Oxidation of unsaturated fatty acids by leaf tissue. `7. Lipid Res. 5, 593-599. HOLT P. R. (1964) Otilisation of 14C-glycerol for intestinal glyceride esterification : studies in patients with chyluria. `7. din. Invest. 43, 349-356. HOBSCHER G., WEST G. R. & BRINDLEY D. N. (1965) Studies on the fractionation of mucosal homogenates from the small intestines. Biochem.`7. 97, 629-642. KALCKARH. (1939) T h e nature of phosphoric esters formed in kidney extracts. Biochem.J. 33, 631-641. KAYDEN H. J., SENIOR J. R. & MATTSON F. H. (1967) T h e monoglyceride pathway of fat absorption in man. jT. din. Invest. 46, 1695-1703. KUHN N. J. (1967) Esterification of glycerol-3-phosphate in lactating guinea pig mammary gland. Biochem.`7. 105, 213-223. LAURELL S. (1957) Turnover rate of unesterified fatty acids in human plasma. Actaphysiol. scand. 41,158-167. LAURY$SENSM., VERBEKE R. & PEETERS G. (1961) Metabolism of 1-14C stearate in the isolated cow udder..7. LipidRes. 2, 383-388. McBRIDE O. W. & KORN E. D. (1964) Presence of glycerokinase in guinea pig mammary gland and the incorporation of glycerol into glycerides. `7. Lipid Res. 5, 442-447. SAUNDERSD. R. & DAWSONA. M. (1962) Studies on the metabolism of glycerol by the small intestines in vitro and in vivo. Biochem.`7. 82, 477-483. SMITH M. E. & HOBSCHER G. (1966) T h e biosynthesis of glycerides by mitochondria from rat liver. Biochem.`7. 101,308-316. WIELAND O. & SUYTER M. (1957) Glycerokinase: its isolation and properties. Biochem. Z. 329, 320-331.
Key Word Index--Glycerokinase; intestine; desaturation of fatty acids ; digestion ; microsomal desaturase; pig; chicken; sheep.