- Email: [email protected]
In vitro studies on the synthesis of intramuscular fat in the longissimus dorsh muscle of pigs
Livestock Production Science, 2 (1975) 59--68 © ~ s e v i e r Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
IN VITRO STUDIES ON THE SYNTHESIS OF INTRAMUSCULAR FAT IN THE LONGISSIMUS DORSI MUSCLE OF PIGS
KIRSTEN CHRLSTENSEN
Dcpartr~nt of Animal Physiology and Chemistry, Copenhagen (Des;mar.h) (Received October 10th, 1974)
ABSTRACT Christenzen, K., 1975. In vitro studies on the synthesis of intramuscular fat in the longimimus dorsi muscle of pigs. Livcst. Prod. 8¢L, 2: 59--68. The a m o u n t o f intramuscular fPt is one factor contributing to the palatability of meat. As a preliminary a t t e m p t to s t u d y the capability o f M. Iongissimus dorsi in pigs to syntF, esize intramuscular lipick, an in vitro technique was designed. Tissue slices were incubated with gluco~-U-C ~4 or acetate-l-C ~4 in the presence and absence o f non-radioactive g i u c o ~ and insulin. Following incubation, the quantities of C ~' -labelled total lipid=, l'atty acids, and individmd lipid ¢lmmes, produced from each substrata were determined. The results indicate t h a t intramuscular lipids can be synthesized in the muscle tissue, but at a slow rate. It Was found that the conversion o f giucote into total lipids, fatty acids, and trigiycerides was markedly stimulated by an increase o f the glucose concentration o f the medium from 2 to fi mM. I n ~ l l n had no effect on the m o u n t of 8[uco~ converted into total lipids. However, at both giucoze concentrations insulin significantly stimulated the utilization o f glucose for production o f glyceride-glycerol at the expemm of fatty a~id synthesis. Acetate was utilized t o a greater extent than glucose for synthesis o f fatty acids. ' r a e presence o f glucose and insulin had no effect on the utilization of acetate for production o f total lipids, fatty acids, and individual lipid clasass. The major part of the radioactivity of total lipids was incorporated into the triglycerides both when glueose-U-C '4 and acetate-l-C ~4 were used as substrates.
INTRODUCTION
The intensive selection for lean pigs has reduced the proportions of fat tissues in the bodies. From a meat quality point of view, however, it is of importance to keep a certain amount of fat in the meat, as fat contributes to the organoleptic quality of meat (Hornstein and Crowe, 1960). Therefore, attention has been paid to the content of intramuscular fat in pork. Little is known about the physiological factors regulating the synthem and deposition of intramuscular lipids in pigs. Christensen (1969, 1970) has obtained data on the in vivo synthesis of intramuscular lipids from glucose and acetate, which suggest that intramuscular fat under certain conditions may be synthesized and deposited independently of the other lipids in the organism. Lee and Kauffman (1971) concluded from their studies on lipo@mic enzyme
60 activities in pig intramuscular adipose tissue that marbling fat could be synthesized within the muscle tissue itself. In order to obtain information about the capacity of the longissimus dorsi muscle of pigs to synthesize lipids from different precursors, a series ¢~f in vitro studies were performed. The experiments to be reported here were designed to investigate the lipogenic capability of M. longissimus dorsi of pigs by measuring the incorporation of glucose.U-C ~4 and acetate I-C ~4 into total lipids,fatty acids, and individual lipid cla~s (tri-,di-,monoglycerides, cholesterol, cholesterol esters,nonesterified fatty acids, and phospholipids).
MATERIAL A N D M E T H O D S Four castrated male pigs of the Danish ]~mdrace breed were used as sources of skeletal muscle tissue. The animals weighed an average of 80 kg and had been fed a commercial diet semi ad libitum during the month before the experiment started. Approx. 2 h after the morning feed, the pigs were anaesthetized with 10 ml mebumal and 15 ml narcodorm. Skeletal muscle tissue from M. longissimus dorsi was then remov.~d from the region above the last rib of the left side of the body by means of a biopsy technique described by Staun and Christensen (1968). Local anaesthesia was omitted. The pigs were kept anaesthetized with halothane during the operation, which lasted 15--20 rain per pig. Tissue slices were prepared immediately with a razor blade longitudinal to the muscle fibers. 2 g were weighed out accurately and used for each incubation. All incubations were of 3 h duration and performed in 10 ml of Ca 2÷free Krebs-Ringer bicarbonate buffer, pH 7.4, under 95% O2 and 5% CO2 (Umbreit et al., 1957). The temperature ~as m ~aintained at 37°C by a thermostated water bath with a shaker operating at 70 strokes/min. Nonradioactive glucose and sodium acetate were added to the buffer as indicated in the tables of results. Uniformly labelled glucose (glucose-U-C14 ) and acetate-l-C L4 were used at a specifm activity of 1.3/J Ci/mg substrate. The insulin was ordinary insulin (pharmaceutical preparation, pH 3) containing 40 U/ml and purchased from NOVO Industry A/S, Copenhagen, Denmark. The insulin was diluted to the required concentrations with the buffer solution. The incubations were stopped by rinsing the tissues with cold 0.9% NaCI and transferring them immediately to homogenizer tubes with cold chloroform/methanol (2 : 1 v/v). In order to prevent contamination of the extractea lipids with radioactive precursor, non-radioactive glucose and acetate were added to the NaCl-solution. Total lipids were then extracted as dea^ribed by Folch et al. (1957). A phosphate solution (pH 6) was added to obtain a quantitative extraction of non-esterified fatty acids (NEFA) as outlined by Riis (1968). The amount of total lipids was determined gravimetrically. 5~-10 mg of total lipids were dissolved in 10 ml of a scintillation solution containing 0.5% 2, 5-diphenyloxazole (PPO) in toluene and left overnight in darkness to obtain a constant count rate.
61
Another 5--10 mg of total lipids were used for preparation of fatty acids. To~'~l iipids were saponified in 3% methanolic KOH by refluxing at 85°C 2or 60 rain. The KOH solutions were diluted with water and the nonsaponiflable lipid fraction was extracted with three 10 ml portions of diethyl ether and discarded. The aqueous phase was acidified wi.th HCI and the fatty acich were removed quantitatively by three successive 10 ml extractions with diethyl ether. The ether was evaporated and the fatty acids dissolved in 10 mi of a ~imil9r scintiUation solution to that used for total lipids. The rest of the total lipids (10--20 rag) was used for isolation of individual lipid classes by means of thin-layer chromatography. The lipids were dissolved in hexane and applied t~ 0.5 mm thick silica gel HR plates in a band by means of a chromatocharger ((~rnag). The plates were pre-developed overnight in chloroform/methanol (2 : 1 v/v), activated for 60 rain at 110°C, and stored in a desiccator before use. A two step development system was used for sepanation of the lipid fractions as described by Skipski et ah(1968} with the foll~,wing modifications. In the first step the solvent diisopropylether/ice acetic acid (96 : 4 v/v) was allowed to run 8 cm from the bottom of the plate. In the second step the solvent petrolether/diethyl ether/ice acetic acid (82 : 18 : I v/v/v) was run 18 cm from the b o t t o m of the plate. This system allows a clear separation of total lipids into the following fractions: Trigiycerides (TG); diglycerides (DG); monoglycerides (MG); nonestenfled fatty acids (NEFA); cholesterol (C); cholesterol esters (CE); and phospholipids (PL): The lipid-containing bands were made visible in iodine vapour. After disappearance of the colour, the appropriate region of the plate was sucked up using an aspiration device described by Goldrick and Hirsch (196~,~ The lipid classes were eluted quantitatively from the silica gel with several portions of diethyl ether. The recovery of this system was found to be 9 0 100%. After evaporation of the ~ther, the individual lipid classes were dissolved in a similar scintillation solution to that used for total lipids. All radioactivity measurements were performed on an automatic Beckman LS-100 liquid scintillationcounter. The efficiency of the counting was determined for each sample by means of an internal standard of palmitate-1-C ~4 dissolved in 100/zl toluene. RESULTS
Table I shows the effect of two different glucose concentrations on the utilization of glucose-U-C ~4 for the synthesis of total lipids and fatty acids when insulin is absent or present at a level of 10 mU/ml of buffer. The data demonstrate that glucose can be converted into lipids by pig Ionglssimus dorsi muscle in vitro. An increase of the glucose concentration of the medium from 2 to 5 mM doubled the utilization of glucose for the production of total }ipids. Addition of insulin to the medium had no effect on the conversion of glucose into total lipids. Table I also a ~ o ~ that isolated pig longlssimus dorsi muscle can synthesize fatty acids from gl,~co6e and that
62 TABLEI The effect of glucose and insulin on the incorporation of glucose-U-C'4 into total lipids and fatty acids by isolated pig longissimns dorsi muscle ~ Glucose concentraticn of buffer (mM)
Insulin 10 mU/ml
Total lipids
Fatty acids
Fatty acids
2 2 5
-+ --
6.8 ± 1.8 6 . 9 ± 1.4 13.9 ± 3.3
0.8 + 0.3 0 . 5 ± 0.1 1.7 ~ 0 . 6
12
5
+
12.I ± 1.7
0.7 ± 0.3
6
Total lipids
x 100
12 7
* Results are expressed as nanomoles of substrate converted to the product indicated per g tissue per 3 h. The values are the mean for four pigs + S.E.M. • e gluco~;e concentration present is important for the rate of synthesis. At b~,h glucose concentrations chosen only 12% of the synthesized lipids are fatty acids, whereas the addition o f insulin to the m e d i u m is seen t o reduce the proportion to 7%. The reason for this finding is presumably that insulin enhances the production of glyceride-glycerol at the expense o f the synthesi~ of fatty acids. The utilization o f glucose for synthesis o f glycerideglycerol may be calculated as the difference between t h e conversion rate o f giucoseU-C '4 into total llpids and, fatty acids + cholesterol + cholesterol esters. Such a calculation showed that the addition o f insulin t o the m e d i u m increased the relative conversion of glucose into glyceride-glycerol from 70 to 75% when the glucose concentration o f the m e d i u m was 2 raM, and from 71 to 76% at a glucose concentration o f 5 mM. As shown in Table II insulin has no effect on the incorporation of glucose-U-C 14 into individual lipid classes, whereas an increase of the glucose concentration of t h e m e d i u m from 2 to 5 mM stimulated the esterification o f m o n o - and diglycerides into trig~ycerides. From Table III, it is evident that acetate can be utilized for lipid synthesis b y longissimus dorsi tissue in vitro. Acetate was incorporated int~ fatty acids to a s o m e w h a t greater e x t e n t than was glucose on a molar basis. As illustrated in Table IV the major lipid c o m p o n e n t f o r m e d durivg the incubation was triglycerides. Only a b o u t 8% o f the radioactivity o f total hpids was found in cholesterol when acetate-l-C ~4 was used as a subs/rate compared to 15% when glucose-U-C 14 was used. Because o f great individual variations in the capacity of M. longissimus dorsi to utilize acetate for production of lipids in vitro, no significant effect of the addition of insulin, glucose, and glucose + insulin could be demonstrated in these studies.
DISCUSSION Intramuscular fat comprises b o t h adipose tissue deposited within the connective tissue strands of the muscle tissue and structural lipids constituting
-+ -+
2 2 5 5
71.8±1.5 71.8±0.4 75.8±1.7 75.1±1.5
**TG
2.3±0.4 2.3±0.2 1.5±0.2 1.1±0.1
DG
2.4±0.2 2.7~0.1 2.3±0.1 2.0~0.2
MG
2.7±0.1 2.7:0.5 1.7~0.2 1.9+0.3
NEFA
2.4±0.8 2.6±0.4 1.6±0.3 1.9±0.3
PL
16.5±1.5 15.3±1.7 15.2±0.9 15.9±1.5
C
1.6±0.5 2.4±0.8 1.5±0.1 1.7~0.2
CE
*Results are e x p r e ~ e d as percentage of the redioactivity o f total lipids found in individual lipid classes. The values are the mean for four pigs ± S.E.M. **TG = triglycerides; IX] = digiycer/des; MG ffi monoglycerides; N E F A - nonesterified fatty acids; PL = phospholipids; C = cholesterol; CE = cholesterolester.
Insulin 10 m U / m l
Glucose concentration o f buffer (raM)
The effect o f glucose Lind insulin on the incorporation o f giucose-U-C ~4 into different lipid classes by isolated pig long. hmimr,~ d ~ i muscle*
TABLE II
C~
64
TABLE
Ill
Tl~,eeffect of glucose and insulin on the incorporation of acetate-l-C 14 into total lipids and fatty acids by isolated pig longiasimus dorsi muscle* Addition**
Total lipids
F a t t y acids
F a t t y acids
× 100
Total lipids None Insulin Glucose Glucose + insulin
12.3 11.1 11.4 12.6
± ± i ±
6.4 4.7 6.2 6.9
3. ° 3.7 3.1 3.8
± + ± ±
1.9 2.3 2.0 2.5
26 33 27 30
* Results ere e x p r e s s e d as n a n o m o l e s o f s u b s t r a t e c o n v e r t e d t o t h e p r o d u c t i n d i e a t e d / g tissue per 3 h. T h e d a t a are t h e m e a n f o r f o u r pigs ± S.E.M. **All b u f f e r s c o n t a i n e d 2 m M a c e t a t e . I n s u l i n was a d d e d a t a level o f 1 0 m U / m l a n d glucose at 5 . m o l e a / m l .
important parts of the muscle membranes. Most of the in vitro investigatio_ns of muscle metabolism have utilizedspecialized muscles sucb as the diaphragm (Randle and Smith, 1958) and heart muscle (Beatty et al.,1972). Whole skeletal muscles (M~)orthy and Gould, 1969) or fiber groups of small muscles from laboratory animals have also been used (Beatty et al.,1966). In the bovine, which is relativelyrich in in~ramuscular fat,lipogenic in vitro studies have been performed with separated fat cellsby measuring the incorporation of C14-acetate int.()fatty acids (Chakrabarty and Romans, 1972). In the domestic pig, however, the individual skeletalmuscles are too big for isolation.The intramuscular adipose tissuestrands are also too sparse and thin for a rapid dissection. It was therefore decided to use thin slicescut longitudinally to the fibers of M. longissimus dorsi of the pig as a preliminary attempt to study the lipogenic capability of this tissue. The data presented here indicate that thin slicesof pig longissimus dorsi muscle may be used for studies of the lipogenic activity of this tissue.The rate of [ipogenesisexpressed as the conversion rate of either labelled glucose or acet~it~to total lipidsand fatty acids was found to be low. This finding was, h(,w:ver, exlmcted, as the amount of intramuscular fat was low (1.0-1.6%) antl had be :n accumulated during the growth period. The concentration of glucose in the medium was found to be highly important to the utilization of glucose-U-C ~4 for synthesis of total lipidsand fatty acids. Acetate was, however, the preferred precursor for the synthesis of fatty acids by this tissue. Since the diets normally fed to pigs m a y be composed of up to 8 0 % starch, glucose is the predominant substrate awii~ble for lipogenesis. However, acetic acid is produced in the digestive tract of pigs and appears in the blood stream (Friend et al., 1964). It is conceivable, therefore, that the longissimus dorsi muscle of pigs possesses an active acetyl CoA synthetase enzyme which could activate acetete and make it available for the synthesis of fatty acids.
80.2±2.9 79.722.6 84.722.7 82.221.1
None Ins~in ~ucose ~ucose+i~in
1.6±0.3 1.720.3 1.8±0.5 2.0±0.4
DG 1.7±~.5 1.8±0.6 1.9~0.8 1.7±0.5
MG 3.420.8 3.020.7 2.1±0.4 2.3~0.6
NEFA 3.3±0.4 2.220.5 1.9±0.3 1.720.5
PL 8.0~ 1.0 7.421.7 5.7±0.5 8.2±1.5
C
1.6±0.4 2.821.1 1.G±0.7 1.6±0.4
CE
*Results are expressed as percentage of the radioactivity of total lipids found in individual lipid classes. The values are the mvan for four pigs _+S.E.M. **All buffers contained 2 mM acetate. Insulin was added at a level of 10 mU/ml and glucose at 5 ~moles/ml. ***Abbreviations as in Table II.
***TG
Addition**
The effect of glucose and insulin on the incorporation of acetate-l-C '4 into different lipid claues by isolated pig longi~imus dor~i muscle*
TABLE IV
66 In vitro studies have shown that insulin stimulates glucose u p t a k e b y adipose tissue isolated from non-ruminants, including man, and that this causes an increased conversion o f glucose into fatty acids and glycerol (Leonards and Landau, 1960; Gries and Steinke, 1967). Goldrick et al. (1972) found that insulin stimulated the uptake o f glucose b y rat skeletal muscle in vitzo. The effect of insulin on the utilization of glucose for lipid synthesis in t h e domebtic pig is, however, n o t clearly demonstrated. While O ' H e a and Leveiile (1970) and Rosmos et al. (1971) reported that pig adipose tissue responded minimally to insulin addition in vitro, C1u~tensen and Goel (1972) found that adipose tissue isolated from Danish Landrace pigs responded significantly to insulin addition, b u t that b o t h the glucose and insulin concentrations of the medium affected the conversion rate o f glucose into lipids. In the present studies insulin was added at a level of 10 m U / m l buffer, which was found by the latter authors to stimulate maximally lipid synthesis at the glucose concentrations employed. Insulin had no effect on the conversion of glucoso-U-C ~4 into total lipids, b u t favoured the production of glycerideglycerol at the expense of fatty acid synthesis. Addition of insulin to the medium when acetate-l-C ~4 was used as a precursor was without effect both in the presence and absence of glucose. Further studies seem, however, to be needed in order to establish the effect of insulin in the present incubation system. The present results indicate that in vitro, pig longissimus dorsi muscle can ~'onvert glucose and acetate into different lipid components. The major part of the radioactivity o f total lipids was found in triglycerides, indicating that the esterifying capacity of this ti~que is quite high under the conditions employed. The results o f Tables II and IV also indicate that slices of pig longissimus dorsi muscle can convert glucose and acetate into cholesterol. This ability is Likely to predominate in the muscle tissue per so, as it has been observed that littlc synthesis of nonsaponifiable lipids takes place in the adipo:~e tissue of pigs (O'Hea and Leveille, 1968). Lee and Kauffman (1972) found that intramuscular adipose tissue possessed lipogenic enzyme activity. The data presented here show that intramuscular lipids can be synthesized in situ b u t at a low rate. REFERENCES Beatty, C.H., Petersop, R.D., Basinger, G.M. and Bocek, R.M., 1966. Major metabo;ie pathways for carbohydrate metabolism of voluntary skeletal muscle. Amer. J. Phydol. 210:404--410
Beatty, C.H., Young, M.IC, Dwyer, D. and Bocek, R.M., 1972. Glucose utilization of cardiac and skeletal muscle homogenates from fetal and adult Rhesus monkeys. Pediatr. Res., 6 : 8 1 3 - - 8 2 1
Chakrabarty, K. and Romans, J.R., 1972. Lipogenesis in the adipose celia of the bovine (Bos Taurus) as related to their intramuscular fat content. Comp. Biochem. Physiol., 41 B: 6 0 3 - - ~ 1 5
67 Christensen, K., 1969. Det intramuskulsere fed~ danneise og aflejring i relation til svine k6dets kvalitet. Thesis, Univ. Copenhagen, 96 pp. Christensen, IC, 1970. The rate of formation and depo6ition of intramuscular lipids in pigs as affected by various feeding factors. R. Vet. Agric. Univ. Yearbook, pp. 193--209. Copenhagen: K.V.L Christensen, K. and Goel, V.D., 1972. The influence of various glucose and insulin concentratio~ on in vitro lipid synthesis by adipose ti~ue isolated from adult pigs. Int. J. Biochem,, 3:591--597 Folch, J., Lees, M. and Stanley, G.H. Sloane, 1957. A simp'e method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226:497--509 Friend, D.W., Nicholson, J.W.G. and Cunningham, H.M., 1964. Volatile fatty acid and lactic acid content of pig blood. Can. J. Anim. Sci., 44:303--313 Goldrick, B. and Hirsch, J.J., 1963. A technique for quantitative recovery of lipids from chromatoplates. J. Lipid Res., 4:482--483 Goldrick, R.B., Hoffmann, C.C. and Reardon, M., 1972. Studies on lipid and carbohydrate metabolism in the rat. Aust. J. Exp. Biol. Med Sci., 50:289--308 Gries, A. and Steinke, J., 1967. Insulin and human adipose tissue in vitro: a brief review. Metabolism, 16:693---696 Hornstein, I. and Crowe, P.F., 1960. Flavor studies on beef and pork. Agric. Food Chem., 8:494--498 Lee, Y.B. and Kauffman, R.G., 1971. Lipogenic enzyme activities in intramuscular adipose ti~ue of the pig. J. Anita. Sci., 33:1144 (Abstract) Leonards, J.R. and Landau, B.P~, 1960. A study o~ '.~,, equiva!ence of metabolic patterz~ in rat tissue: insulin versus glucose concentrat~:L Arch. 3iochem. Biophys., 91 : 194--200 Moorthy, K.A. and Gould, M.K., 1969. Synthesi~ of gi~t,gen from glucose and lactate in isolated rat soleus muscle. Arch. Biochem. Biophys., 130:399--407 O'Hea, E.]C and Leveille, G.A., 1968. Lipid metabolism in isol.ted adipose ti~ue of the domestic pig. (Bus Domesticus). Comp. Biochem. Physiol., 26:1081--1089 O'Hea, E.IC and Leveille, G.A., 1970. Studies on the retponse of pig adipose ti~ue to insulin. Int. J. Biochem., 1:605--611 Randle, P.J. and Smith, G.H., 1958. Regulation of glucoee uptake by muscle. 1. The effects of insulin, anaerobinsis and cell poisons on the uptake of glucose and releue of potassium by isolated rat diaphragm. Biochem. J., 70:490--500 Riis, P.M., 1968. Methods for in vitro studies on the kit ~-tic~ of carbohydrate and lipid pools. R. Vet. Agric. Univ. Yearbook, pp. 12--34 Copenhagen: K.V.L. Rosmoe, D.R., Leveille, G..~ and Allee, G.L., 1971. In ~Jitro lipogenesis in adipoee tissue from alloxan-diabetic pigs. (Sus domesticus). Comp. Biochem. Physiol., 40:569--578 Skipski, V.P., Good, J.J., Barclay, M. and Reggio, R.B., 1968. Quantitative analy~;s of simple lipid classes by thin-layer chromatography. Biochim. Biophys. Acts, 152: 1 0 ~ 9 Staun, H. and Christeusen, K., 1968. Unders6gelse over biopsiers anvendelighed til bestemm~lse af k~dm~ngde og k6dkvalitet hos svin. Bil. Land6kon. Fors~gslab. efterih'sm6de, 152--159, Copenhagen Umbreit, W.W., Bun'is, R.H. and Stauffer, J.F., 1957. Manometric Techniques. Burgess Publishing Company, Minneapolis, Minn. 3rd edn, 149 pp. RESUMP.. Christensen, K., 1975. Etudes sur la synth~se de la graisse intramu~culaire au muscle longiuimns dora; du porc. Livest. Prod. ScL, 2 : 5 9 - 6 8 (en angiiis). II est reconnu que la pr/~ence de graimse dans le muscle am~liort, la saveur de la viande. Au cours d'~tades pr~liminaires sur la possibilit~ du muscle longissimus dorsi d'ol~rer In synthi~se des lipides, une technique Sl~ciale a ~t~ incubus en presence de glucose UC'" ou
68
d'ac~tate-l-C ~4 , avec ou sans apport de glucose VLsuelOU d'insuline. A la fin de I'incubation, il a ~t~ proc~d~ b la recherche des q u a n t i t ~ de lipides totaux marqu~ au C ~4, ainsi qu'au clas~ement des acides g r u libres et des lipides individuals provemmt de chaque sub* strat. Les r~sultats obtenus ont prouv~ que les lipides intramumeulaires peuvent @tre synth~tis~s lentement b l'int~rieur du muscle. II a ~t~ trouv~ an odtre que la transformation du glucose en lipides totaux, en acides g r u comme en triglyceridm s'est trouvt~e stimul~e par l'accroissement de la concentration du glucose darts le milieu nutritif, i des concentrations comprises entre 2 at 5 raM. La formation des lipides n'a p u 6t6 modifi6e en presence d'insuline. Cependant, I'action de l'imuline a entratnd, dams le c ~ d ' u u e plum grande concentration en glucose, une formation plum importante de glyc&rtdes-glye~rols au dttriment de celle des acides gras. Pour ees synthbsee, l'acetate marqu6 ,'est montr& plus efficace que le glucose. La pr~kence clans le milieu de glueme et d'insuline n'a pea eu de I'influence sttr la synthetic lipidique t partir de I'ae~tate. Quel que soit le milieu nutritif utilis~, la majeure partie de la radioactivitA a ~t& mise en ~vidence darts les triglycerides. ZUSAMMENFASSUNG Christensen, K., 1075. In vitro Studien tiber die Synthe6e dcr intramuskularen Lipide der Iongissimus dorsi Muskel beim Schwein. Livest. Prod. Sci., 2: 5g ~=f,8(in Englisch). Die Menge yon intramuskularem Fett hit einer der Faktoren, die zur Palabilitat des Fleisches beitragt. Um die Fllhigkeit der Synthese der intrs.'numkularen Lipide des M. longissimus dorsi beim Schweine studieren zu konnen, wurde ein in vitro System konstruiert. D u Muskelgewebe wurde in dUnne Scheiben geschnitten und mit GluL~se-U-C~4 oder Acetat-l-C ~4, mit und ohne inaktive G l u k o ~ u n d Insulin, inkubiert. Nach der Inkubation wurden die Mengen yon radioaktiv markierten Produkten (Gesamtfett, F e t t u u r e n und verschiedenen Lipidkomponenten) b ~ t i m m t . Die Ergebni~e erwtlhnen, dass intramuskulare Lipide in der langen Ruckenmumkel des Sehweines synthetisiert werden kOnnen; aber die Synthese geht langsam vor sich. Es wurde gefunden, deas eine ErhOhung der Kon~.entration an Glukose im Medium yon 2 bis 5 raM, die Einkorporierung yon Glukose in Gesamtfett, Fettsauren und Triglyceride m~rkant stimulierte. Insulin hatte keinen Effekt auf die Einkort-~orierung yon Giukose ins Gesamtfett. Dagegen stimulierte der Zusatz yon Insulin zum Medium den Verbraueh der Glukose fur die Produktion yon Glycerol auf Kosten der Syntheae yon Fetts~uren. Acetat wurde besser wie G l u k o ~ fur die Synthese yon F e t t u u r e n ausgenutzt. Der Zmmtz yon Insulin hatt~ keinen Effekt auf die Verwertung yon Acetst fur die Synthese yon Gesamtfett, F e t t u u r a n und verschiedenen Lipidkomponenten. D,~r gro~te Teil der Radioaktivit/lt der Gesamtlipide wurde in Triglycelide eingebaut, sowohl ob Glukose als auch Acetat als Substrate dienten.
IN VITRO STUDIES ON THE SYNTHESIS OF INTRAMUSCULAR FAT IN THE LONGISSIMUS DORSI MUSCLE OF PIGS
KIRSTEN CHRLSTENSEN
Dcpartr~nt of Animal Physiology and Chemistry, Copenhagen (Des;mar.h) (Received October 10th, 1974)
ABSTRACT Christenzen, K., 1975. In vitro studies on the synthesis of intramuscular fat in the longimimus dorsi muscle of pigs. Livcst. Prod. 8¢L, 2: 59--68. The a m o u n t o f intramuscular fPt is one factor contributing to the palatability of meat. As a preliminary a t t e m p t to s t u d y the capability o f M. Iongissimus dorsi in pigs to syntF, esize intramuscular lipick, an in vitro technique was designed. Tissue slices were incubated with gluco~-U-C ~4 or acetate-l-C ~4 in the presence and absence o f non-radioactive g i u c o ~ and insulin. Following incubation, the quantities of C ~' -labelled total lipid=, l'atty acids, and individmd lipid ¢lmmes, produced from each substrata were determined. The results indicate t h a t intramuscular lipids can be synthesized in the muscle tissue, but at a slow rate. It Was found that the conversion o f giucote into total lipids, fatty acids, and trigiycerides was markedly stimulated by an increase o f the glucose concentration o f the medium from 2 to fi mM. I n ~ l l n had no effect on the m o u n t of 8[uco~ converted into total lipids. However, at both giucoze concentrations insulin significantly stimulated the utilization o f glucose for production o f glyceride-glycerol at the expemm of fatty a~id synthesis. Acetate was utilized t o a greater extent than glucose for synthesis o f fatty acids. ' r a e presence o f glucose and insulin had no effect on the utilization of acetate for production o f total lipids, fatty acids, and individual lipid clasass. The major part of the radioactivity of total lipids was incorporated into the triglycerides both when glueose-U-C '4 and acetate-l-C ~4 were used as substrates.
INTRODUCTION
The intensive selection for lean pigs has reduced the proportions of fat tissues in the bodies. From a meat quality point of view, however, it is of importance to keep a certain amount of fat in the meat, as fat contributes to the organoleptic quality of meat (Hornstein and Crowe, 1960). Therefore, attention has been paid to the content of intramuscular fat in pork. Little is known about the physiological factors regulating the synthem and deposition of intramuscular lipids in pigs. Christensen (1969, 1970) has obtained data on the in vivo synthesis of intramuscular lipids from glucose and acetate, which suggest that intramuscular fat under certain conditions may be synthesized and deposited independently of the other lipids in the organism. Lee and Kauffman (1971) concluded from their studies on lipo@mic enzyme
60 activities in pig intramuscular adipose tissue that marbling fat could be synthesized within the muscle tissue itself. In order to obtain information about the capacity of the longissimus dorsi muscle of pigs to synthesize lipids from different precursors, a series ¢~f in vitro studies were performed. The experiments to be reported here were designed to investigate the lipogenic capability of M. longissimus dorsi of pigs by measuring the incorporation of glucose.U-C ~4 and acetate I-C ~4 into total lipids,fatty acids, and individual lipid cla~s (tri-,di-,monoglycerides, cholesterol, cholesterol esters,nonesterified fatty acids, and phospholipids).
MATERIAL A N D M E T H O D S Four castrated male pigs of the Danish ]~mdrace breed were used as sources of skeletal muscle tissue. The animals weighed an average of 80 kg and had been fed a commercial diet semi ad libitum during the month before the experiment started. Approx. 2 h after the morning feed, the pigs were anaesthetized with 10 ml mebumal and 15 ml narcodorm. Skeletal muscle tissue from M. longissimus dorsi was then remov.~d from the region above the last rib of the left side of the body by means of a biopsy technique described by Staun and Christensen (1968). Local anaesthesia was omitted. The pigs were kept anaesthetized with halothane during the operation, which lasted 15--20 rain per pig. Tissue slices were prepared immediately with a razor blade longitudinal to the muscle fibers. 2 g were weighed out accurately and used for each incubation. All incubations were of 3 h duration and performed in 10 ml of Ca 2÷free Krebs-Ringer bicarbonate buffer, pH 7.4, under 95% O2 and 5% CO2 (Umbreit et al., 1957). The temperature ~as m ~aintained at 37°C by a thermostated water bath with a shaker operating at 70 strokes/min. Nonradioactive glucose and sodium acetate were added to the buffer as indicated in the tables of results. Uniformly labelled glucose (glucose-U-C14 ) and acetate-l-C L4 were used at a specifm activity of 1.3/J Ci/mg substrate. The insulin was ordinary insulin (pharmaceutical preparation, pH 3) containing 40 U/ml and purchased from NOVO Industry A/S, Copenhagen, Denmark. The insulin was diluted to the required concentrations with the buffer solution. The incubations were stopped by rinsing the tissues with cold 0.9% NaCI and transferring them immediately to homogenizer tubes with cold chloroform/methanol (2 : 1 v/v). In order to prevent contamination of the extractea lipids with radioactive precursor, non-radioactive glucose and acetate were added to the NaCl-solution. Total lipids were then extracted as dea^ribed by Folch et al. (1957). A phosphate solution (pH 6) was added to obtain a quantitative extraction of non-esterified fatty acids (NEFA) as outlined by Riis (1968). The amount of total lipids was determined gravimetrically. 5~-10 mg of total lipids were dissolved in 10 ml of a scintillation solution containing 0.5% 2, 5-diphenyloxazole (PPO) in toluene and left overnight in darkness to obtain a constant count rate.
61
Another 5--10 mg of total lipids were used for preparation of fatty acids. To~'~l iipids were saponified in 3% methanolic KOH by refluxing at 85°C 2or 60 rain. The KOH solutions were diluted with water and the nonsaponiflable lipid fraction was extracted with three 10 ml portions of diethyl ether and discarded. The aqueous phase was acidified wi.th HCI and the fatty acich were removed quantitatively by three successive 10 ml extractions with diethyl ether. The ether was evaporated and the fatty acids dissolved in 10 mi of a ~imil9r scintiUation solution to that used for total lipids. The rest of the total lipids (10--20 rag) was used for isolation of individual lipid classes by means of thin-layer chromatography. The lipids were dissolved in hexane and applied t~ 0.5 mm thick silica gel HR plates in a band by means of a chromatocharger ((~rnag). The plates were pre-developed overnight in chloroform/methanol (2 : 1 v/v), activated for 60 rain at 110°C, and stored in a desiccator before use. A two step development system was used for sepanation of the lipid fractions as described by Skipski et ah(1968} with the foll~,wing modifications. In the first step the solvent diisopropylether/ice acetic acid (96 : 4 v/v) was allowed to run 8 cm from the bottom of the plate. In the second step the solvent petrolether/diethyl ether/ice acetic acid (82 : 18 : I v/v/v) was run 18 cm from the b o t t o m of the plate. This system allows a clear separation of total lipids into the following fractions: Trigiycerides (TG); diglycerides (DG); monoglycerides (MG); nonestenfled fatty acids (NEFA); cholesterol (C); cholesterol esters (CE); and phospholipids (PL): The lipid-containing bands were made visible in iodine vapour. After disappearance of the colour, the appropriate region of the plate was sucked up using an aspiration device described by Goldrick and Hirsch (196~,~ The lipid classes were eluted quantitatively from the silica gel with several portions of diethyl ether. The recovery of this system was found to be 9 0 100%. After evaporation of the ~ther, the individual lipid classes were dissolved in a similar scintillation solution to that used for total lipids. All radioactivity measurements were performed on an automatic Beckman LS-100 liquid scintillationcounter. The efficiency of the counting was determined for each sample by means of an internal standard of palmitate-1-C ~4 dissolved in 100/zl toluene. RESULTS
Table I shows the effect of two different glucose concentrations on the utilization of glucose-U-C ~4 for the synthesis of total lipids and fatty acids when insulin is absent or present at a level of 10 mU/ml of buffer. The data demonstrate that glucose can be converted into lipids by pig Ionglssimus dorsi muscle in vitro. An increase of the glucose concentration of the medium from 2 to 5 mM doubled the utilization of glucose for the production of total }ipids. Addition of insulin to the medium had no effect on the conversion of glucose into total lipids. Table I also a ~ o ~ that isolated pig longlssimus dorsi muscle can synthesize fatty acids from gl,~co6e and that
62 TABLEI The effect of glucose and insulin on the incorporation of glucose-U-C'4 into total lipids and fatty acids by isolated pig longissimns dorsi muscle ~ Glucose concentraticn of buffer (mM)
Insulin 10 mU/ml
Total lipids
Fatty acids
Fatty acids
2 2 5
-+ --
6.8 ± 1.8 6 . 9 ± 1.4 13.9 ± 3.3
0.8 + 0.3 0 . 5 ± 0.1 1.7 ~ 0 . 6
12
5
+
12.I ± 1.7
0.7 ± 0.3
6
Total lipids
x 100
12 7
* Results are expressed as nanomoles of substrate converted to the product indicated per g tissue per 3 h. The values are the mean for four pigs + S.E.M. • e gluco~;e concentration present is important for the rate of synthesis. At b~,h glucose concentrations chosen only 12% of the synthesized lipids are fatty acids, whereas the addition o f insulin to the m e d i u m is seen t o reduce the proportion to 7%. The reason for this finding is presumably that insulin enhances the production of glyceride-glycerol at the expense o f the synthesi~ of fatty acids. The utilization o f glucose for synthesis o f glycerideglycerol may be calculated as the difference between t h e conversion rate o f giucoseU-C '4 into total llpids and, fatty acids + cholesterol + cholesterol esters. Such a calculation showed that the addition o f insulin t o the m e d i u m increased the relative conversion of glucose into glyceride-glycerol from 70 to 75% when the glucose concentration o f the m e d i u m was 2 raM, and from 71 to 76% at a glucose concentration o f 5 mM. As shown in Table II insulin has no effect on the incorporation of glucose-U-C 14 into individual lipid classes, whereas an increase of the glucose concentration of t h e m e d i u m from 2 to 5 mM stimulated the esterification o f m o n o - and diglycerides into trig~ycerides. From Table III, it is evident that acetate can be utilized for lipid synthesis b y longissimus dorsi tissue in vitro. Acetate was incorporated int~ fatty acids to a s o m e w h a t greater e x t e n t than was glucose on a molar basis. As illustrated in Table IV the major lipid c o m p o n e n t f o r m e d durivg the incubation was triglycerides. Only a b o u t 8% o f the radioactivity o f total hpids was found in cholesterol when acetate-l-C ~4 was used as a subs/rate compared to 15% when glucose-U-C 14 was used. Because o f great individual variations in the capacity of M. longissimus dorsi to utilize acetate for production of lipids in vitro, no significant effect of the addition of insulin, glucose, and glucose + insulin could be demonstrated in these studies.
DISCUSSION Intramuscular fat comprises b o t h adipose tissue deposited within the connective tissue strands of the muscle tissue and structural lipids constituting
-+ -+
2 2 5 5
71.8±1.5 71.8±0.4 75.8±1.7 75.1±1.5
**TG
2.3±0.4 2.3±0.2 1.5±0.2 1.1±0.1
DG
2.4±0.2 2.7~0.1 2.3±0.1 2.0~0.2
MG
2.7±0.1 2.7:0.5 1.7~0.2 1.9+0.3
NEFA
2.4±0.8 2.6±0.4 1.6±0.3 1.9±0.3
PL
16.5±1.5 15.3±1.7 15.2±0.9 15.9±1.5
C
1.6±0.5 2.4±0.8 1.5±0.1 1.7~0.2
CE
*Results are e x p r e ~ e d as percentage of the redioactivity o f total lipids found in individual lipid classes. The values are the mean for four pigs ± S.E.M. **TG = triglycerides; IX] = digiycer/des; MG ffi monoglycerides; N E F A - nonesterified fatty acids; PL = phospholipids; C = cholesterol; CE = cholesterolester.
Insulin 10 m U / m l
Glucose concentration o f buffer (raM)
The effect o f glucose Lind insulin on the incorporation o f giucose-U-C ~4 into different lipid classes by isolated pig long. hmimr,~ d ~ i muscle*
TABLE II
C~
64
TABLE
Ill
Tl~,eeffect of glucose and insulin on the incorporation of acetate-l-C 14 into total lipids and fatty acids by isolated pig longiasimus dorsi muscle* Addition**
Total lipids
F a t t y acids
F a t t y acids
× 100
Total lipids None Insulin Glucose Glucose + insulin
12.3 11.1 11.4 12.6
± ± i ±
6.4 4.7 6.2 6.9
3. ° 3.7 3.1 3.8
± + ± ±
1.9 2.3 2.0 2.5
26 33 27 30
* Results ere e x p r e s s e d as n a n o m o l e s o f s u b s t r a t e c o n v e r t e d t o t h e p r o d u c t i n d i e a t e d / g tissue per 3 h. T h e d a t a are t h e m e a n f o r f o u r pigs ± S.E.M. **All b u f f e r s c o n t a i n e d 2 m M a c e t a t e . I n s u l i n was a d d e d a t a level o f 1 0 m U / m l a n d glucose at 5 . m o l e a / m l .
important parts of the muscle membranes. Most of the in vitro investigatio_ns of muscle metabolism have utilizedspecialized muscles sucb as the diaphragm (Randle and Smith, 1958) and heart muscle (Beatty et al.,1972). Whole skeletal muscles (M~)orthy and Gould, 1969) or fiber groups of small muscles from laboratory animals have also been used (Beatty et al.,1966). In the bovine, which is relativelyrich in in~ramuscular fat,lipogenic in vitro studies have been performed with separated fat cellsby measuring the incorporation of C14-acetate int.()fatty acids (Chakrabarty and Romans, 1972). In the domestic pig, however, the individual skeletalmuscles are too big for isolation.The intramuscular adipose tissuestrands are also too sparse and thin for a rapid dissection. It was therefore decided to use thin slicescut longitudinally to the fibers of M. longissimus dorsi of the pig as a preliminary attempt to study the lipogenic capability of this tissue. The data presented here indicate that thin slicesof pig longissimus dorsi muscle may be used for studies of the lipogenic activity of this tissue.The rate of [ipogenesisexpressed as the conversion rate of either labelled glucose or acet~it~to total lipidsand fatty acids was found to be low. This finding was, h(,w:ver, exlmcted, as the amount of intramuscular fat was low (1.0-1.6%) antl had be :n accumulated during the growth period. The concentration of glucose in the medium was found to be highly important to the utilization of glucose-U-C ~4 for synthesis of total lipidsand fatty acids. Acetate was, however, the preferred precursor for the synthesis of fatty acids by this tissue. Since the diets normally fed to pigs m a y be composed of up to 8 0 % starch, glucose is the predominant substrate awii~ble for lipogenesis. However, acetic acid is produced in the digestive tract of pigs and appears in the blood stream (Friend et al., 1964). It is conceivable, therefore, that the longissimus dorsi muscle of pigs possesses an active acetyl CoA synthetase enzyme which could activate acetete and make it available for the synthesis of fatty acids.
80.2±2.9 79.722.6 84.722.7 82.221.1
None Ins~in ~ucose ~ucose+i~in
1.6±0.3 1.720.3 1.8±0.5 2.0±0.4
DG 1.7±~.5 1.8±0.6 1.9~0.8 1.7±0.5
MG 3.420.8 3.020.7 2.1±0.4 2.3~0.6
NEFA 3.3±0.4 2.220.5 1.9±0.3 1.720.5
PL 8.0~ 1.0 7.421.7 5.7±0.5 8.2±1.5
C
1.6±0.4 2.821.1 1.G±0.7 1.6±0.4
CE
*Results are expressed as percentage of the radioactivity of total lipids found in individual lipid classes. The values are the mvan for four pigs _+S.E.M. **All buffers contained 2 mM acetate. Insulin was added at a level of 10 mU/ml and glucose at 5 ~moles/ml. ***Abbreviations as in Table II.
***TG
Addition**
The effect of glucose and insulin on the incorporation of acetate-l-C '4 into different lipid claues by isolated pig longi~imus dor~i muscle*
TABLE IV
66 In vitro studies have shown that insulin stimulates glucose u p t a k e b y adipose tissue isolated from non-ruminants, including man, and that this causes an increased conversion o f glucose into fatty acids and glycerol (Leonards and Landau, 1960; Gries and Steinke, 1967). Goldrick et al. (1972) found that insulin stimulated the uptake o f glucose b y rat skeletal muscle in vitzo. The effect of insulin on the utilization of glucose for lipid synthesis in t h e domebtic pig is, however, n o t clearly demonstrated. While O ' H e a and Leveiile (1970) and Rosmos et al. (1971) reported that pig adipose tissue responded minimally to insulin addition in vitro, C1u~tensen and Goel (1972) found that adipose tissue isolated from Danish Landrace pigs responded significantly to insulin addition, b u t that b o t h the glucose and insulin concentrations of the medium affected the conversion rate o f glucose into lipids. In the present studies insulin was added at a level of 10 m U / m l buffer, which was found by the latter authors to stimulate maximally lipid synthesis at the glucose concentrations employed. Insulin had no effect on the conversion of glucoso-U-C ~4 into total lipids, b u t favoured the production of glycerideglycerol at the expense of fatty acid synthesis. Addition of insulin to the medium when acetate-l-C ~4 was used as a precursor was without effect both in the presence and absence of glucose. Further studies seem, however, to be needed in order to establish the effect of insulin in the present incubation system. The present results indicate that in vitro, pig longissimus dorsi muscle can ~'onvert glucose and acetate into different lipid components. The major part of the radioactivity o f total lipids was found in triglycerides, indicating that the esterifying capacity of this ti~que is quite high under the conditions employed. The results o f Tables II and IV also indicate that slices of pig longissimus dorsi muscle can convert glucose and acetate into cholesterol. This ability is Likely to predominate in the muscle tissue per so, as it has been observed that littlc synthesis of nonsaponifiable lipids takes place in the adipo:~e tissue of pigs (O'Hea and Leveille, 1968). Lee and Kauffman (1972) found that intramuscular adipose tissue possessed lipogenic enzyme activity. The data presented here show that intramuscular lipids can be synthesized in situ b u t at a low rate. REFERENCES Beatty, C.H., Petersop, R.D., Basinger, G.M. and Bocek, R.M., 1966. Major metabo;ie pathways for carbohydrate metabolism of voluntary skeletal muscle. Amer. J. Phydol. 210:404--410
Beatty, C.H., Young, M.IC, Dwyer, D. and Bocek, R.M., 1972. Glucose utilization of cardiac and skeletal muscle homogenates from fetal and adult Rhesus monkeys. Pediatr. Res., 6 : 8 1 3 - - 8 2 1
Chakrabarty, K. and Romans, J.R., 1972. Lipogenesis in the adipose celia of the bovine (Bos Taurus) as related to their intramuscular fat content. Comp. Biochem. Physiol., 41 B: 6 0 3 - - ~ 1 5
67 Christensen, K., 1969. Det intramuskulsere fed~ danneise og aflejring i relation til svine k6dets kvalitet. Thesis, Univ. Copenhagen, 96 pp. Christensen, IC, 1970. The rate of formation and depo6ition of intramuscular lipids in pigs as affected by various feeding factors. R. Vet. Agric. Univ. Yearbook, pp. 193--209. Copenhagen: K.V.L Christensen, K. and Goel, V.D., 1972. The influence of various glucose and insulin concentratio~ on in vitro lipid synthesis by adipose ti~ue isolated from adult pigs. Int. J. Biochem,, 3:591--597 Folch, J., Lees, M. and Stanley, G.H. Sloane, 1957. A simp'e method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226:497--509 Friend, D.W., Nicholson, J.W.G. and Cunningham, H.M., 1964. Volatile fatty acid and lactic acid content of pig blood. Can. J. Anim. Sci., 44:303--313 Goldrick, B. and Hirsch, J.J., 1963. A technique for quantitative recovery of lipids from chromatoplates. J. Lipid Res., 4:482--483 Goldrick, R.B., Hoffmann, C.C. and Reardon, M., 1972. Studies on lipid and carbohydrate metabolism in the rat. Aust. J. Exp. Biol. Med Sci., 50:289--308 Gries, A. and Steinke, J., 1967. Insulin and human adipose tissue in vitro: a brief review. Metabolism, 16:693---696 Hornstein, I. and Crowe, P.F., 1960. Flavor studies on beef and pork. Agric. Food Chem., 8:494--498 Lee, Y.B. and Kauffman, R.G., 1971. Lipogenic enzyme activities in intramuscular adipose ti~ue of the pig. J. Anita. Sci., 33:1144 (Abstract) Leonards, J.R. and Landau, B.P~, 1960. A study o~ '.~,, equiva!ence of metabolic patterz~ in rat tissue: insulin versus glucose concentrat~:L Arch. 3iochem. Biophys., 91 : 194--200 Moorthy, K.A. and Gould, M.K., 1969. Synthesi~ of gi~t,gen from glucose and lactate in isolated rat soleus muscle. Arch. Biochem. Biophys., 130:399--407 O'Hea, E.]C and Leveille, G.A., 1968. Lipid metabolism in isol.ted adipose ti~ue of the domestic pig. (Bus Domesticus). Comp. Biochem. Physiol., 26:1081--1089 O'Hea, E.IC and Leveille, G.A., 1970. Studies on the retponse of pig adipose ti~ue to insulin. Int. J. Biochem., 1:605--611 Randle, P.J. and Smith, G.H., 1958. Regulation of glucoee uptake by muscle. 1. The effects of insulin, anaerobinsis and cell poisons on the uptake of glucose and releue of potassium by isolated rat diaphragm. Biochem. J., 70:490--500 Riis, P.M., 1968. Methods for in vitro studies on the kit ~-tic~ of carbohydrate and lipid pools. R. Vet. Agric. Univ. Yearbook, pp. 12--34 Copenhagen: K.V.L. Rosmoe, D.R., Leveille, G..~ and Allee, G.L., 1971. In ~Jitro lipogenesis in adipoee tissue from alloxan-diabetic pigs. (Sus domesticus). Comp. Biochem. Physiol., 40:569--578 Skipski, V.P., Good, J.J., Barclay, M. and Reggio, R.B., 1968. Quantitative analy~;s of simple lipid classes by thin-layer chromatography. Biochim. Biophys. Acts, 152: 1 0 ~ 9 Staun, H. and Christeusen, K., 1968. Unders6gelse over biopsiers anvendelighed til bestemm~lse af k~dm~ngde og k6dkvalitet hos svin. Bil. Land6kon. Fors~gslab. efterih'sm6de, 152--159, Copenhagen Umbreit, W.W., Bun'is, R.H. and Stauffer, J.F., 1957. Manometric Techniques. Burgess Publishing Company, Minneapolis, Minn. 3rd edn, 149 pp. RESUMP.. Christensen, K., 1975. Etudes sur la synth~se de la graisse intramu~culaire au muscle longiuimns dora; du porc. Livest. Prod. ScL, 2 : 5 9 - 6 8 (en angiiis). II est reconnu que la pr/~ence de graimse dans le muscle am~liort, la saveur de la viande. Au cours d'~tades pr~liminaires sur la possibilit~ du muscle longissimus dorsi d'ol~rer In synthi~se des lipides, une technique Sl~ciale a ~t~ incubus en presence de glucose UC'" ou
68
d'ac~tate-l-C ~4 , avec ou sans apport de glucose VLsuelOU d'insuline. A la fin de I'incubation, il a ~t~ proc~d~ b la recherche des q u a n t i t ~ de lipides totaux marqu~ au C ~4, ainsi qu'au clas~ement des acides g r u libres et des lipides individuals provemmt de chaque sub* strat. Les r~sultats obtenus ont prouv~ que les lipides intramumeulaires peuvent @tre synth~tis~s lentement b l'int~rieur du muscle. II a ~t~ trouv~ an odtre que la transformation du glucose en lipides totaux, en acides g r u comme en triglyceridm s'est trouvt~e stimul~e par l'accroissement de la concentration du glucose darts le milieu nutritif, i des concentrations comprises entre 2 at 5 raM. La formation des lipides n'a p u 6t6 modifi6e en presence d'insuline. Cependant, I'action de l'imuline a entratnd, dams le c ~ d ' u u e plum grande concentration en glucose, une formation plum importante de glyc&rtdes-glye~rols au dttriment de celle des acides gras. Pour ees synthbsee, l'acetate marqu6 ,'est montr& plus efficace que le glucose. La pr~kence clans le milieu de glueme et d'insuline n'a pea eu de I'influence sttr la synthetic lipidique t partir de I'ae~tate. Quel que soit le milieu nutritif utilis~, la majeure partie de la radioactivitA a ~t& mise en ~vidence darts les triglycerides. ZUSAMMENFASSUNG Christensen, K., 1075. In vitro Studien tiber die Synthe6e dcr intramuskularen Lipide der Iongissimus dorsi Muskel beim Schwein. Livest. Prod. Sci., 2: 5g ~=f,8(in Englisch). Die Menge yon intramuskularem Fett hit einer der Faktoren, die zur Palabilitat des Fleisches beitragt. Um die Fllhigkeit der Synthese der intrs.'numkularen Lipide des M. longissimus dorsi beim Schweine studieren zu konnen, wurde ein in vitro System konstruiert. D u Muskelgewebe wurde in dUnne Scheiben geschnitten und mit GluL~se-U-C~4 oder Acetat-l-C ~4, mit und ohne inaktive G l u k o ~ u n d Insulin, inkubiert. Nach der Inkubation wurden die Mengen yon radioaktiv markierten Produkten (Gesamtfett, F e t t u u r e n und verschiedenen Lipidkomponenten) b ~ t i m m t . Die Ergebni~e erwtlhnen, dass intramuskulare Lipide in der langen Ruckenmumkel des Sehweines synthetisiert werden kOnnen; aber die Synthese geht langsam vor sich. Es wurde gefunden, deas eine ErhOhung der Kon~.entration an Glukose im Medium yon 2 bis 5 raM, die Einkorporierung yon Glukose in Gesamtfett, Fettsauren und Triglyceride m~rkant stimulierte. Insulin hatte keinen Effekt auf die Einkort-~orierung yon Giukose ins Gesamtfett. Dagegen stimulierte der Zusatz yon Insulin zum Medium den Verbraueh der Glukose fur die Produktion yon Glycerol auf Kosten der Syntheae yon Fetts~uren. Acetat wurde besser wie G l u k o ~ fur die Synthese yon F e t t u u r e n ausgenutzt. Der Zmmtz yon Insulin hatt~ keinen Effekt auf die Verwertung yon Acetst fur die Synthese yon Gesamtfett, F e t t u u r a n und verschiedenen Lipidkomponenten. D,~r gro~te Teil der Radioaktivit/lt der Gesamtlipide wurde in Triglycelide eingebaut, sowohl ob Glukose als auch Acetat als Substrate dienten.