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Fatty acid synthesis in camel (Camelus dromedarius) hump and sheep (Ovis aries) tail fat
Comp. Biochera. Physiol. Vol. 68B, pp. 551 to 554
0305-0491/81/040551-04502.00/0
© Pergamon Press Ltd 1981. Printed in Great Britain
FATTY ACID SYNTHESIS IN CAMEL (CAMELUS DROMEDARIUS) H U M P AND SHEEP (OVIS ARIES) TAIL FAT B. EMMANUEL Department of Animal Biology, School of Veterinary Medicine, University of Shiraz, Shiraz, Iran (Received 17 July 1980)
Abstract--1. Fatty acid synthesis was studied in camel hump and sheep tail fat, utilizing tissue slices in the presence of labelled compounds including acetate, propionate, pyruvate, butyrate, 3-hydroxybutyrate, glucose and palmitate. In addition, the oxidation rates of these substances to CO2 were also measured. 2. In both species, oxidation rates in a decreasing order were: pyruvate, acetate, glucose, butyrate, 3-hydroxybutyrate, propionate and palmitate. In general, tail fat was more active than hump in this respect. 3. In both species, acetate was incorporated into fatty acids at a greater rate than glucose. Tail fat utilized more 3-hydroxybutyrate than hump; the reverse case was true for butyrate.
INTRODUCTION
In man (Shrago et al., 1969), and avian species (O'Hea & Leveille, 1969) fatty acid synthesis mainly occurs in the liver. In other species including rat, mouse and rabbit (Leveille, 1967; Jansen et al., 1967; Leung et al., 1974) lipogenesis takes place in both adipose and liver tissues. In contrast, in ruminants adipose tissue is the major site for fatty acid synthesis. Of the total fatty acid production in sheep, 5 and 92~ occurred in liver and adipose tissue, respectively (Ingle et al., 1972b). Other studies confirm this observation (Hanson & Ballard, 1967, 1968; Hood et al., 1972). The tail fat and hump are the major fat stores in fat-tailed sheep, and camel, and they comprise about 12 and 20~ of the total body weight, respectively (McFarlene, 1964; Khachadurian et al., 1966). Previous work in this laboratory (Emmanuel & Nahapetian, 1980) had shown that the major fatty acids in the hump of camel (Camelus dromedarius) were palmitic, stearic, oleic and myristic acids with small quantities of odd-numbered, palmitoleic, linoleic and linolenic acids, In true ruminants (sheep, cow and goat), butyrate which is the end product of carbohydrate fermentation in the rumen (Hungate et al., 1961) is converted to ketone bodies in the rumen epithelium (Pennington, 1952; Annison et al., 1957; Hird & Weidemann, 1964; Emmanuel, 1980). The resulting 3-hydroxybutyrate and some butyrate which escapes metabolism in the rumen epithelium are then incorporated into body lipids (Kronfeld, 1970). On the other hand, butyrate is not oxidized in the rumen epithelium of camel (Chandrasena et aL, 1979; Emmanuel, 1980), and plasma concentrations of 3-hydroxybutyrate are very low (Chandrasena et al., 1979). Therefore, differences would be expected between the camel and sheep with respect to fatty acid synthesis from butyrate and 3-hydroxybutyrate. The present work was aimed to compare the fatty acid synthesis pattern in camel hump and sheep tail 551
fat, utilizing lipogenic precursors including acetate, propionate, butyrate, 3-hydroxybutyrate, pyruvate, glucose and palmitate. In addition, the oxidation of these substances to CO2 was also measured. MATERIALSAND METHODS Chemicals
Radioactive substrates including I-l-t4CJacetic acid, sodium salt (60.1 mCi/mmol); I-2-14C]acetic acid, sodium salt (59mCi/mmol); [l-t4C]propionic acid, sodium salt (57 mCi/mmol); n-[1-t4C]butyric acid, sodium salt (24 mCi/mmol); DL-3-hydroxy[3-1'~C]butyric acid, sodium salt (20mCi/mmol); I-l-14C]pyruvic acid, sodium salt (13,34 mCi/mmol); o-[U.14C]glucose (248 mCi/mmol), and [1-14C]palmitic acid (57.9mCi/mmol) were purchased from Radiochemical Centre, Amersham, England. Casein hydrolysate (extra soluble), bovine albumin and DL-methionine were products of General Chemicals, Laboratory Park, Chagrin Falls, OH 44022. L-tryptophan and hyamine-hydroxide from BDH Chemical Ltd., Poole, England. Bovine insulin (24.3 I.U./mg) from Sigma Chemical Co., St. Louis, MO 63118. Sampling and incubation Hump and tail fat were obtained from animals immedi~,tely after slaughter, kept at 39°C in physiological saline and transferred to the laboratory within 30 rain. Tissue slices (0.5 mm thick were cut by means of a tissue slicer (Arthor H. Thomas Co., Philadelphia). The incubation was carried out in Erlenmeyer flasks as described previously (Emmanuel, 1980). The incubation medium contained 200 mg tissue, 3 ml of Krebs-Ringer bicarbonate (Ca-free) buffer, pH 7.4 (Laser, 1961), containing per 100ml, 50rag bovine albumin, 30 mg casein hydrolysate, 1 mg tryptophan and ling methionine; 0.5I.U. bovine insulin; 15 #mols glucose; 0.5#Ci of radioactive substrate and 75 gmol of unlabelled substances (with the exception of palmitate and glucose which were 1.5 and 30 #mol, respectively) in a total volume of 3.2 ml. Insulin was reported to enhance fatty acid synthesis from acetate and glucose in ruminants adipose tissue (Skarda &Bartos, 1969; Yang & Baldwin, 1973; Baldwin et al., 1973). Glucose provides
552
B. EM~A~UF.~ Table 1. Percent fatty acid composition of camel hump and sheep tail fat Fatty acid
Hump
Tail fat
14:0 15:0 16:0 17:0 18:0 16:1 18:1 18:2 18:3 Unknown
7.07 1.08 30.42 1.68 24.60 1.69 28.10 2.04 1.55 2.45
4.84 2.10 28.57 2.60 7.81 3.46 39.71 2.60 .... 8.31
Symbols used are: 14:0 for a normal saturated fatty acid with 14 carbon atoms; 16:1 for a normal monoenoic acid with 16 carbon atoms. NADPH and ~-glycerol phosphate through hexose monophosphate shunt and Embden-Meyerhof pathway, respectively which are needed for lipogenesis. Glucose stimulated fatty acid synthesis from acetate, lactate and pyruvate (Yang & Baldwin, 1973; Whitehurst et al,, 19781. The flasks were then alternately evacuated and refilled with oxygen gas for 30 sec, using hypodermic needles inserted through the stopper. The reaction mixture was incubated in a shaking water bath at 37°C for 2 hr. In each experiment, two control samples containing all compounds, but tissue were used. Collection of C02 At the end of incubation time, the flasks were cooled in ice-water, and 0.5 ml of hyamine-hydroxide were injected into the central glass well and 0.5 ml of I N-H2SO,, were added to the incubation medium to terminate the reaction and to release CO2. The reaction mixture was shaken for an extra 2hr at room temperature. The released 1'*CO2 was collected and counted as described previously (Emmanuel, 1980). Extraction and identifit ation of fatty acids To remove labelled sabstrates which were not incorporated into fatty acids. The above acidified tissue was washed with 200 ml of physiological saline, utilizing a glass filter holder for vacuum with Teflon coated screen (Karl Kolb GmbH & Co., KG, D-6079 Buchschlag-Frankfurt, Germany). The tissue was then transferred to a screw capped test tube, and 10 ml of 0.5 N methanolic sodium hydroxide
were added, and the sample was kept oternight tit 95 ( ' The resulting fatty acids were extracted with n-hexane as described by Kaneda (19661. The acidic extracts were evaporated under N 2 gas, and the residue was transferred with 0.5 ml of n-hexane to a scintillation vial and counted as described (Emmanuel, 1980}. Fatty acids were identified as described (Emmanuel & Nahapetian, 108(tt.
RESULTS AND DIS(I SSION Fatty acid composition of camel hump and sheep tail fat are tabulated in Table 1. Major fatty acids found were palmitic, stearic, oleic and myristic acids. Small quantities of odd-numbered fatty acids (pentadecanoate and heptadecanoate), and palmitoleic and linoleic were also present. The main difference in the composition of fatty acids between the hump and tail fat was in the proportion of stearic and oleic acids. Of the total fatty acids in hump, 24.6 and 28.1°~; were stearic and oleic, respectively; whereas tail fat contained lower stearic acid (7.810/0), and higher oleic acid (39.71%). In addition tail fat lacked linolenic acid. These results support previous observations made on camel hump and sheep tail fat (Pathak & Trivedi, i958; Khachadurian et al., 1966: Mirgani, 19771. Data on the oxidation of various substances to CO2 are presented in Table 2. The extent of oxidation of pyruvate was markedly higher than that of other substrates in both species. Similarly, Prior & Jacobson (1979) found high conversion rate of pyruvate to CO2 in bovine adipose tissue, In their studies, the rate of oxidation of pyruvate labelled in carboxyl group was about 7 times higher than that of pyruvate labelled in carbonyl group, suggesting that C O : release mainly takes place at the conversion of pyrurate to acetyl-CoA. Acetate labelled in carboxyl group produced almost twice as much CO2 as acetate labelled in methyl group (Table 2). Lindsay & Ford (1964), using [1-~4C]acetate and [2-~4C]acetate in sheep concluded that the oxidation rate of carbon 1 was 1.58 times that of carbon 2. Oxidation rates of acetate are closely related to observations made on bovine adipose tissue (Hanson & Ballard, 1967; Yang & Baldwin, 1973). Whitehurst et al. (19781 reported higher values (3.41 pmol/2 hr per g tissuet. Oxidation rate of acetate was higher than glucose in both tissues tested, supporting previous reports on sheep and cow
Table 2. Conversion of acetate, propionate, pyruvate, butyrate, 3-hydroxybutyrate, glucose, and palmitate to CO z and fatty acids in camel hump and sheep tail fat Substrate [1-~4C]Acetate [2-14C]Acetate [1-14C]Propionate [l-14C]Pyruvate n-[ l-~4C]Butyrate [3-14C]BHB * o-[U-14C]Glucose [l-igC]Palmitate
Tail fat CO2 0.920(0.1941 0.580 (0.075) 0.043(0.0121 11.290(2.235) 0.200 (0.067) 0.055 (0.023) 0.310 (0.034) 0.013 (0.003)
Hump COz 1.103 (0.1731 0.500(0.1141 0.032(0.007t 3.520(0.546) 0.110 (0.035) 0.062(0.0131 0.200 (0.045) 0.007(0.003)
Tail fat FAt
Hump FA+
1.8 (0.23) 3.3(0.44t 0.2(0.05) 0.7(0.09) 2.4 (0,34) 1.4 (0.211 0.7 (0,141 1.7 (0.29)
1.4 (0,25t 1.3(0.311 0.1 (0.04) 0.4t0.011 4.2 (0.69) 0.6 (0.131 0.5 (0.181 1.5 (0.311
Values are expressed as #mols substrate converted/2 hr per g wet wt tissue. * Refers to DL-3-hydroxy [3-1'~C]butyrate. ? Refers to fatty acid. Values in brackets are _+SD (n = 7-10 observationst.
553
Lipogenesis in camel hump and sheep tail fat (Hanson & Ballard, 1967; Yang & Baldwin, 1973; Whitehurst et al., 1978). The rate of glucose oxidation in both tissues was within the same range found in adipose tissue of ruminant species (Hanson & Ballard, 1969; Pothoven & Beitz, 1973; Yang & Baldwin, 1973). The oxidation rate of butyrate in tail fat was 2 times higher than in hump. Previous studies (Emmanuel, 1980) had shown that the rumen epithelium and liver of sheep oxidized much more butyrate to CO2 than that of camel. Little 3-hydroxybutyrate, propioHate and palmitate were oxidized in both species. The results on fatty acid synthesis are shown in Table 2. The incorporation rate of acetate into fatty acids was higher than that of glucose. These observations fully support other reports in ruminants (Balmain et al., 1954; Hanson & BaUard, 1967, 1968; Bauman et al., 1970; Hood et al., 1972; Ingle et al., 1972a; Baldwin et al., 1973; Yang & Baldwin, 1973; Liepa et al., 1978). In contrast to ruminants, in monogastrics, glucose is the principal substance for lipogenesis (Balmain et al., 1954; Linzell et al., 1969; Bauman et al., 1970). Incorporation of glucose into fatty acids entails its conversion to pyruvate through glycolysis, and decarboxylation of pyruvate to acetyl-CoA in the mitochondria. Acetyl-CoA translocation to the cytoplasm involves citrate. This compound is cleaved to acetyl-CoA and oxaloacetate in the cytoplasm by ATP-citrate lyase (EC 4.1.3.7). Oxaloacetate can not diffuse back to the mitochondria, therefore it is converted to malate utilizing NAD-malate dehydrogenase (EC 1.1.1.37), and malate is decarboxylated to pyruvate via NADP-malate dehydrogenase (EC 1.1.1,40). The resulting pyruvate is transferred to mitochondria and carboxylated by pyruvate carboxylase (EC 6.4.1.1) thereby regenerating mitochondrial oxaloacetate (Ballard et al., 1969; Bauman & Davis, 1975). The activities of ATP-citrate lyase (Bauman et al., 1970, 1973; Gumma et al., 1973), NADP-malate dehydrogenase (Hanson & BaUard, 1968; Bauman et al., 1970, 1973; Gumma et al., 1973; Mellenberger et al., 1973), and pyruvate carboxylas¢ (Hanson & hallard, 1967) are very low in adult ruminant tissues. On the other hand, the activity of acetyl-CoA synthetase (EC 6.2.1.1) in ruminant adipose tissue is higher than in monogastrics (Hanson & Ballard, 1967). In addition, in ruminants dietary carbohydrates are converted to volatile fatty acids (acetate, propionate and butyrate) in the rumen and very little (5-10~o) (Bensadoun et al., 1962) glucose escapes fermentation, resulting in lower plasma glucose concentration in ruminant species. In view of the point raised, the relatively less significant role of glucose in fatty acid synthesis in ruminants can be easily understood. The compound 3-hydroxybutyrate was incorporated into fatty acids to a greater extent in tail fat than in hump; the reverse case was true for butyrate. In contrast to true ruminants (cow, sheep and goat), negligible quantities of butyrate are oxidized to ketone bodies and CO2 in camel rumen epithelium (Chandrasena et al., 1979; Emnaanuel, 1980), despite the fact that the rumen function in the camel yields the same products at rates and in proportions comparable to those in true ruminants (Hungate et al., 1959; Williams, 1963). Therefore, utilization of butyrate in lipogenesis can explain in part the metabolic fate of this substance in camel. Since DL-3-hydroxybutyrate was used in the
present studies, and the o ( - ) , and L(+) isomers are metabolized at different rates (Lehninger & GreviUe, 1953; Webber & Edmond, 1977), the results on this substrate might have been different if each isomer was utilized separately. Propionate was incorporated into fatty acids of hump and tail fat at rates of 0.10 and 0.20 #mol/2 hr per g tissue, respectively. This compound is utilized for the synthesis of odd-numbered fatty acids. Of the total fatty acids, 2.76 and 4.70~o are odd-numbered (pentadecanoate and heptadecanoate) in hump and tail fat, respectively (Table 1); therefore the observed results are expected. Palmitate was incorporated at rates of 1.5 and 1.7/~mol/2 hr per g tissue in hump and tail fat, respectively. In general, tail fat was more active than hump in oxidizing substrates tested to CO2 and in incorporating them into fatty acids; butyrate was utilized at a higher rate for fatty acid synthesis in hump. Acknowledgements--The author wishes to thank Mr T. Vaseghee for his excellent technical assistance. This research was supported by a grant from Shiraz University Research Council.
REFERENCES
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LEHNINGER A. L. & Gm:VtLLE G. D. (1953) The enzymic oxidation of d- and I-fl-hydroxybutyrate. Biochim. hiophys. Acta 12, 188 202. LEUNG T. T.. BAUMAN D. E., SCOTT R. A. & GROOM W. J. (1974) Fatty acid synthesis in the rabbit (Abstr.). Fedn Proc. Fedn Am. Sots exp. Biol, 33, 663. LEVEILLEG, A, (1967) In vivo fatty acid synthesis in adipose tissue and liver of meal-fed rats. Proc. Soc. exp. Biol. Med. 125, 85. LINt)SAY D. B. & FORD E. J. H. (1964) Acetate utilization and turnover of citric acid cycle components in pregnant sheep. Biochem. J. 90, 24-30. LIEPA G. U,, BEITZ D. C. & LIND~g J. R. (1978) Fatty acid synthesis in ruminanting and nonruminanting goats. J. Nutr. 108, 1733 1739. LINZELL J. L., MEPHAM T. B.. ANNISON E. F. & WEST C. E. (1969) Mammary metabolism in lactating cows: Arteriovenous differences of milk precursors and the mammary metabolism of [14C]glucose and I-'4C]acetate. Br. J. Nutr. 23, 319-332. MCFARLENE W, V. (1964} Handbook of Physiology, Adaptation to the Environment (Edited by DILl. D. B,) pp. 509 539. Waverly Press, Baltimore. MELLENBERGER R. W., BAUMAN D. E. & NELSON D. R. (1973) Metabolic adaptations during lactogenesis: Fatty acid and lactose synthesis in cow mammary tissue. Biochem. J. 136, 741 748. MmGANI T. (1977) Fatty acid composition of hump triglycerides of the camel Camelus dromedarius. Comp. Biochem. Physiol. 58B, 211 2t3. O'HEA E. K. & LEVEILLE G. A. (1969) Lipid biosynthesis and transport in the domestic chick (Gallus domesticus}. Comp. Biochem. Physiol. 30, 149-159. PATHAK S. P. & TRIVEDI B. N. (1958) The component glycerides of camel (Camelus dromedarius) depot fats. J. Sci. Fd Agriv. 533-535. PENNINGTON R. J. [1952) The metabolism of shortchain fatty acids in the sheep. 1. Fatty acid utilization and ketone body production by tureen epithelium and other tissues. Biochem. J. 51. 251 258. POTHOVEN M. A. & BEITZ D. C. (1973) Effect of adipose tissue site, animal weight, and long-term fasting on lipogenesis in the bovine. J. Nutr. 103, 468-475. PmOR R. L. & JACOBSONJ. J. (1979) Lactate, pyruvate and acetate interactions during in vitro lipogenesis in bovine adipose tissue. J. Anim. Sci. 49, 1410-1416. SHRAGO E., SPENNETTAY., & GORDON E. (•969) Fatty acid synthesis in human adipose tissue. J. hiol. Chem. 244, 2761-2766.
0305-0491/81/040551-04502.00/0
© Pergamon Press Ltd 1981. Printed in Great Britain
FATTY ACID SYNTHESIS IN CAMEL (CAMELUS DROMEDARIUS) H U M P AND SHEEP (OVIS ARIES) TAIL FAT B. EMMANUEL Department of Animal Biology, School of Veterinary Medicine, University of Shiraz, Shiraz, Iran (Received 17 July 1980)
Abstract--1. Fatty acid synthesis was studied in camel hump and sheep tail fat, utilizing tissue slices in the presence of labelled compounds including acetate, propionate, pyruvate, butyrate, 3-hydroxybutyrate, glucose and palmitate. In addition, the oxidation rates of these substances to CO2 were also measured. 2. In both species, oxidation rates in a decreasing order were: pyruvate, acetate, glucose, butyrate, 3-hydroxybutyrate, propionate and palmitate. In general, tail fat was more active than hump in this respect. 3. In both species, acetate was incorporated into fatty acids at a greater rate than glucose. Tail fat utilized more 3-hydroxybutyrate than hump; the reverse case was true for butyrate.
INTRODUCTION
In man (Shrago et al., 1969), and avian species (O'Hea & Leveille, 1969) fatty acid synthesis mainly occurs in the liver. In other species including rat, mouse and rabbit (Leveille, 1967; Jansen et al., 1967; Leung et al., 1974) lipogenesis takes place in both adipose and liver tissues. In contrast, in ruminants adipose tissue is the major site for fatty acid synthesis. Of the total fatty acid production in sheep, 5 and 92~ occurred in liver and adipose tissue, respectively (Ingle et al., 1972b). Other studies confirm this observation (Hanson & Ballard, 1967, 1968; Hood et al., 1972). The tail fat and hump are the major fat stores in fat-tailed sheep, and camel, and they comprise about 12 and 20~ of the total body weight, respectively (McFarlene, 1964; Khachadurian et al., 1966). Previous work in this laboratory (Emmanuel & Nahapetian, 1980) had shown that the major fatty acids in the hump of camel (Camelus dromedarius) were palmitic, stearic, oleic and myristic acids with small quantities of odd-numbered, palmitoleic, linoleic and linolenic acids, In true ruminants (sheep, cow and goat), butyrate which is the end product of carbohydrate fermentation in the rumen (Hungate et al., 1961) is converted to ketone bodies in the rumen epithelium (Pennington, 1952; Annison et al., 1957; Hird & Weidemann, 1964; Emmanuel, 1980). The resulting 3-hydroxybutyrate and some butyrate which escapes metabolism in the rumen epithelium are then incorporated into body lipids (Kronfeld, 1970). On the other hand, butyrate is not oxidized in the rumen epithelium of camel (Chandrasena et aL, 1979; Emmanuel, 1980), and plasma concentrations of 3-hydroxybutyrate are very low (Chandrasena et al., 1979). Therefore, differences would be expected between the camel and sheep with respect to fatty acid synthesis from butyrate and 3-hydroxybutyrate. The present work was aimed to compare the fatty acid synthesis pattern in camel hump and sheep tail 551
fat, utilizing lipogenic precursors including acetate, propionate, butyrate, 3-hydroxybutyrate, pyruvate, glucose and palmitate. In addition, the oxidation of these substances to CO2 was also measured. MATERIALSAND METHODS Chemicals
Radioactive substrates including I-l-t4CJacetic acid, sodium salt (60.1 mCi/mmol); I-2-14C]acetic acid, sodium salt (59mCi/mmol); [l-t4C]propionic acid, sodium salt (57 mCi/mmol); n-[1-t4C]butyric acid, sodium salt (24 mCi/mmol); DL-3-hydroxy[3-1'~C]butyric acid, sodium salt (20mCi/mmol); I-l-14C]pyruvic acid, sodium salt (13,34 mCi/mmol); o-[U.14C]glucose (248 mCi/mmol), and [1-14C]palmitic acid (57.9mCi/mmol) were purchased from Radiochemical Centre, Amersham, England. Casein hydrolysate (extra soluble), bovine albumin and DL-methionine were products of General Chemicals, Laboratory Park, Chagrin Falls, OH 44022. L-tryptophan and hyamine-hydroxide from BDH Chemical Ltd., Poole, England. Bovine insulin (24.3 I.U./mg) from Sigma Chemical Co., St. Louis, MO 63118. Sampling and incubation Hump and tail fat were obtained from animals immedi~,tely after slaughter, kept at 39°C in physiological saline and transferred to the laboratory within 30 rain. Tissue slices (0.5 mm thick were cut by means of a tissue slicer (Arthor H. Thomas Co., Philadelphia). The incubation was carried out in Erlenmeyer flasks as described previously (Emmanuel, 1980). The incubation medium contained 200 mg tissue, 3 ml of Krebs-Ringer bicarbonate (Ca-free) buffer, pH 7.4 (Laser, 1961), containing per 100ml, 50rag bovine albumin, 30 mg casein hydrolysate, 1 mg tryptophan and ling methionine; 0.5I.U. bovine insulin; 15 #mols glucose; 0.5#Ci of radioactive substrate and 75 gmol of unlabelled substances (with the exception of palmitate and glucose which were 1.5 and 30 #mol, respectively) in a total volume of 3.2 ml. Insulin was reported to enhance fatty acid synthesis from acetate and glucose in ruminants adipose tissue (Skarda &Bartos, 1969; Yang & Baldwin, 1973; Baldwin et al., 1973). Glucose provides
552
B. EM~A~UF.~ Table 1. Percent fatty acid composition of camel hump and sheep tail fat Fatty acid
Hump
Tail fat
14:0 15:0 16:0 17:0 18:0 16:1 18:1 18:2 18:3 Unknown
7.07 1.08 30.42 1.68 24.60 1.69 28.10 2.04 1.55 2.45
4.84 2.10 28.57 2.60 7.81 3.46 39.71 2.60 .... 8.31
Symbols used are: 14:0 for a normal saturated fatty acid with 14 carbon atoms; 16:1 for a normal monoenoic acid with 16 carbon atoms. NADPH and ~-glycerol phosphate through hexose monophosphate shunt and Embden-Meyerhof pathway, respectively which are needed for lipogenesis. Glucose stimulated fatty acid synthesis from acetate, lactate and pyruvate (Yang & Baldwin, 1973; Whitehurst et al,, 19781. The flasks were then alternately evacuated and refilled with oxygen gas for 30 sec, using hypodermic needles inserted through the stopper. The reaction mixture was incubated in a shaking water bath at 37°C for 2 hr. In each experiment, two control samples containing all compounds, but tissue were used. Collection of C02 At the end of incubation time, the flasks were cooled in ice-water, and 0.5 ml of hyamine-hydroxide were injected into the central glass well and 0.5 ml of I N-H2SO,, were added to the incubation medium to terminate the reaction and to release CO2. The reaction mixture was shaken for an extra 2hr at room temperature. The released 1'*CO2 was collected and counted as described previously (Emmanuel, 1980). Extraction and identifit ation of fatty acids To remove labelled sabstrates which were not incorporated into fatty acids. The above acidified tissue was washed with 200 ml of physiological saline, utilizing a glass filter holder for vacuum with Teflon coated screen (Karl Kolb GmbH & Co., KG, D-6079 Buchschlag-Frankfurt, Germany). The tissue was then transferred to a screw capped test tube, and 10 ml of 0.5 N methanolic sodium hydroxide
were added, and the sample was kept oternight tit 95 ( ' The resulting fatty acids were extracted with n-hexane as described by Kaneda (19661. The acidic extracts were evaporated under N 2 gas, and the residue was transferred with 0.5 ml of n-hexane to a scintillation vial and counted as described (Emmanuel, 1980}. Fatty acids were identified as described (Emmanuel & Nahapetian, 108(tt.
RESULTS AND DIS(I SSION Fatty acid composition of camel hump and sheep tail fat are tabulated in Table 1. Major fatty acids found were palmitic, stearic, oleic and myristic acids. Small quantities of odd-numbered fatty acids (pentadecanoate and heptadecanoate), and palmitoleic and linoleic were also present. The main difference in the composition of fatty acids between the hump and tail fat was in the proportion of stearic and oleic acids. Of the total fatty acids in hump, 24.6 and 28.1°~; were stearic and oleic, respectively; whereas tail fat contained lower stearic acid (7.810/0), and higher oleic acid (39.71%). In addition tail fat lacked linolenic acid. These results support previous observations made on camel hump and sheep tail fat (Pathak & Trivedi, i958; Khachadurian et al., 1966: Mirgani, 19771. Data on the oxidation of various substances to CO2 are presented in Table 2. The extent of oxidation of pyruvate was markedly higher than that of other substrates in both species. Similarly, Prior & Jacobson (1979) found high conversion rate of pyruvate to CO2 in bovine adipose tissue, In their studies, the rate of oxidation of pyruvate labelled in carboxyl group was about 7 times higher than that of pyruvate labelled in carbonyl group, suggesting that C O : release mainly takes place at the conversion of pyrurate to acetyl-CoA. Acetate labelled in carboxyl group produced almost twice as much CO2 as acetate labelled in methyl group (Table 2). Lindsay & Ford (1964), using [1-~4C]acetate and [2-~4C]acetate in sheep concluded that the oxidation rate of carbon 1 was 1.58 times that of carbon 2. Oxidation rates of acetate are closely related to observations made on bovine adipose tissue (Hanson & Ballard, 1967; Yang & Baldwin, 1973). Whitehurst et al. (19781 reported higher values (3.41 pmol/2 hr per g tissuet. Oxidation rate of acetate was higher than glucose in both tissues tested, supporting previous reports on sheep and cow
Table 2. Conversion of acetate, propionate, pyruvate, butyrate, 3-hydroxybutyrate, glucose, and palmitate to CO z and fatty acids in camel hump and sheep tail fat Substrate [1-~4C]Acetate [2-14C]Acetate [1-14C]Propionate [l-14C]Pyruvate n-[ l-~4C]Butyrate [3-14C]BHB * o-[U-14C]Glucose [l-igC]Palmitate
Tail fat CO2 0.920(0.1941 0.580 (0.075) 0.043(0.0121 11.290(2.235) 0.200 (0.067) 0.055 (0.023) 0.310 (0.034) 0.013 (0.003)
Hump COz 1.103 (0.1731 0.500(0.1141 0.032(0.007t 3.520(0.546) 0.110 (0.035) 0.062(0.0131 0.200 (0.045) 0.007(0.003)
Tail fat FAt
Hump FA+
1.8 (0.23) 3.3(0.44t 0.2(0.05) 0.7(0.09) 2.4 (0,34) 1.4 (0.211 0.7 (0,141 1.7 (0.29)
1.4 (0,25t 1.3(0.311 0.1 (0.04) 0.4t0.011 4.2 (0.69) 0.6 (0.131 0.5 (0.181 1.5 (0.311
Values are expressed as #mols substrate converted/2 hr per g wet wt tissue. * Refers to DL-3-hydroxy [3-1'~C]butyrate. ? Refers to fatty acid. Values in brackets are _+SD (n = 7-10 observationst.
553
Lipogenesis in camel hump and sheep tail fat (Hanson & Ballard, 1967; Yang & Baldwin, 1973; Whitehurst et al., 1978). The rate of glucose oxidation in both tissues was within the same range found in adipose tissue of ruminant species (Hanson & Ballard, 1969; Pothoven & Beitz, 1973; Yang & Baldwin, 1973). The oxidation rate of butyrate in tail fat was 2 times higher than in hump. Previous studies (Emmanuel, 1980) had shown that the rumen epithelium and liver of sheep oxidized much more butyrate to CO2 than that of camel. Little 3-hydroxybutyrate, propioHate and palmitate were oxidized in both species. The results on fatty acid synthesis are shown in Table 2. The incorporation rate of acetate into fatty acids was higher than that of glucose. These observations fully support other reports in ruminants (Balmain et al., 1954; Hanson & BaUard, 1967, 1968; Bauman et al., 1970; Hood et al., 1972; Ingle et al., 1972a; Baldwin et al., 1973; Yang & Baldwin, 1973; Liepa et al., 1978). In contrast to ruminants, in monogastrics, glucose is the principal substance for lipogenesis (Balmain et al., 1954; Linzell et al., 1969; Bauman et al., 1970). Incorporation of glucose into fatty acids entails its conversion to pyruvate through glycolysis, and decarboxylation of pyruvate to acetyl-CoA in the mitochondria. Acetyl-CoA translocation to the cytoplasm involves citrate. This compound is cleaved to acetyl-CoA and oxaloacetate in the cytoplasm by ATP-citrate lyase (EC 4.1.3.7). Oxaloacetate can not diffuse back to the mitochondria, therefore it is converted to malate utilizing NAD-malate dehydrogenase (EC 1.1.1.37), and malate is decarboxylated to pyruvate via NADP-malate dehydrogenase (EC 1.1.1,40). The resulting pyruvate is transferred to mitochondria and carboxylated by pyruvate carboxylase (EC 6.4.1.1) thereby regenerating mitochondrial oxaloacetate (Ballard et al., 1969; Bauman & Davis, 1975). The activities of ATP-citrate lyase (Bauman et al., 1970, 1973; Gumma et al., 1973), NADP-malate dehydrogenase (Hanson & BaUard, 1968; Bauman et al., 1970, 1973; Gumma et al., 1973; Mellenberger et al., 1973), and pyruvate carboxylas¢ (Hanson & hallard, 1967) are very low in adult ruminant tissues. On the other hand, the activity of acetyl-CoA synthetase (EC 6.2.1.1) in ruminant adipose tissue is higher than in monogastrics (Hanson & Ballard, 1967). In addition, in ruminants dietary carbohydrates are converted to volatile fatty acids (acetate, propionate and butyrate) in the rumen and very little (5-10~o) (Bensadoun et al., 1962) glucose escapes fermentation, resulting in lower plasma glucose concentration in ruminant species. In view of the point raised, the relatively less significant role of glucose in fatty acid synthesis in ruminants can be easily understood. The compound 3-hydroxybutyrate was incorporated into fatty acids to a greater extent in tail fat than in hump; the reverse case was true for butyrate. In contrast to true ruminants (cow, sheep and goat), negligible quantities of butyrate are oxidized to ketone bodies and CO2 in camel rumen epithelium (Chandrasena et al., 1979; Emnaanuel, 1980), despite the fact that the rumen function in the camel yields the same products at rates and in proportions comparable to those in true ruminants (Hungate et al., 1959; Williams, 1963). Therefore, utilization of butyrate in lipogenesis can explain in part the metabolic fate of this substance in camel. Since DL-3-hydroxybutyrate was used in the
present studies, and the o ( - ) , and L(+) isomers are metabolized at different rates (Lehninger & GreviUe, 1953; Webber & Edmond, 1977), the results on this substrate might have been different if each isomer was utilized separately. Propionate was incorporated into fatty acids of hump and tail fat at rates of 0.10 and 0.20 #mol/2 hr per g tissue, respectively. This compound is utilized for the synthesis of odd-numbered fatty acids. Of the total fatty acids, 2.76 and 4.70~o are odd-numbered (pentadecanoate and heptadecanoate) in hump and tail fat, respectively (Table 1); therefore the observed results are expected. Palmitate was incorporated at rates of 1.5 and 1.7/~mol/2 hr per g tissue in hump and tail fat, respectively. In general, tail fat was more active than hump in oxidizing substrates tested to CO2 and in incorporating them into fatty acids; butyrate was utilized at a higher rate for fatty acid synthesis in hump. Acknowledgements--The author wishes to thank Mr T. Vaseghee for his excellent technical assistance. This research was supported by a grant from Shiraz University Research Council.
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