TURNOVER OF TRIACYLGLYCEROLS IN RAT ADIPOSE TISSUE MAINTAINED IN CULTURE* W. W. CHKISTII:. M. L. HUNTIX and R. C. VISNON The Hannah Research Institute. Ayr. U.K.
Abstract--l. Rat adipose tissue was pulse-labelled with [l-“‘Cl fatty acids and was then maintained in tissue culture for up to 7 days. 2. After 1 day in culture. the proportion of the label recovered in the triacylglycerols tended lo decrease slowly while that in the unesterified fatty acids increased but a high level of esterifying activity remained in the tissue at the end of the culture period. 3. By measuring the rate of disappearance of particular molecular species of the labelled triacylglycerols. the half-life of triacylglycerols in the tissue was found to be 22-25 days. 4. The rate of desaturation of [I-“Clstearic acid was maintained in culture.
Son Ltd (Rayleigh. U.K.). Food (diet 418, Oxoid Ltd.) and water were supplied ud tihirutzr. Rats were killed by cerebral dislocation. samples of parametrial adipose tissue were removed immediately and placed in sterile isotonic saline at 37 C.
In adipose tissue. the triacylglycerols are continually turning over ia. hydrolysis and resynthesis is occurring (reviewed by Steinberg. 1964; Steinberg & Vaughan. 1965; Shapiro. 1977). Attempts have been made to determine the turnover rate of the fatty acid componcnts in adipose tissue (reviewed by Stein & Stein. 1965) but it is never clear whether the rate-limiting step is lipolysis of the triacylglycerols, oxidation of the fatty acids or export from the tissue as considerable reesterification may occur. The basal rate of lipolysis as measured by giycerol release has been used to determine triacylglycerol turnover and may be a better indicator but fipolysis is markedly influenced by hormonal factors including those induced by stress so there may be doubts as to the validity of extrapolating the results of comparatively short term experiments. In alddition. such factors as the nutritional state of the animal. the rate of growth of the tissue and interactions with other organs in the whole animal could markedly influence the nature and interpretation of the results. This study represents an attempt to determine the rate of triacylgly~erol turnover in rat adipose tissue maintained in culture by measuring the rate of turnover of a specific triacylglyceroi that is not resynthesized. The values obtained should indicate the mean rate of lipolysis over the period. MATERIALS
AND
Adipose tissue slices weighing 3- 4 mg were cut free-hand and transferred in approximately 80 mg aliquots to 25 ml Erlcnmcyer flasks containing medium 199 (2.5ml) with Earle’s salts containing t.-glutamine. hcpes (25 mM). insulin (10 lig;ml) and streptomycin sulphatc (100pgiml). hereafter rcfcrred to as medium 199 culture medium, together with bovine serum albumin (~mg/ml). To this was added a solution (0.2 ml) of the [i-‘4C]labclled fatty acid (2 jtmol. 2.5 CcCi)in 0.1 M KOH containing albumin (40 mg/ml). the flasks were sealed with Suba-seai stoppers and were incubated at 37 C for 4 hr. At the end of this period. the ti?suc slices were removed from the flasks and washed in fresh medium 199 culture medium to remove any of the residual labelled substrate adhering physically to the tissue. Tissue slices (approx 20mg). labellcd as above. in medium 199 culture medium (2Sml) were maintained in culture in 25ml Erlenmeyer flasks sealed with Suba-seal stoppers at 33 C as described by Vernon (1978). fn addttion. unlabelled tissue was maintained in culture in a similar way for the duration of the experiment (generally 7 days) before being incubated with a labelled Fatty acid as described above.
METHODS
Mtrrc~riuls
Insulin (24 units/mg). bovine serum albumin (fraction V. essentially fatty acid free), penicillin-G (I650 units/mg) and streptomycin sulphate were obtained from Sigma London Chemical Co. (Poole. U.K.). medium 199 with Earle’s salts and containing t.-glutamine and 25 mM Hepes was supplied by Gibco: Bio-Cult Ltd. (Paisley. U.K.) and [ i-14C]palmitic. stearic and oleic acids were from the Radiochemical Centre. (Amersham, U.K.). The bovine serum albumin was dialysed before use (Hanson & Ballard. 1968). Female Wist,ar rats were obtained
from A. Tuck and
* HRI Publication No. 1048. 517
At appropriate time intervals. the slices were removed from the medium and the lipids were extracted: aliquots of the medium itself were treated similarly. Procedures for the extraction of lipids. separation of lipid classes for liquid scintillation counting and for preparative isolation of the triacylglycerols were described elsewhere (Christie rt ctl.. 1976). Triacylglycerols were fractionated according to degree of unsaturation by preparative TLC on thin layers of silica gel G (0.5 mm thick) containing silver nitrate (IO?;, w w): hexane: diethyl ether (80:20, v/v) was the developing solvent. Up to 5 mg of triacylglycerols were separated on a 10 x 20 cm plate, Bands were visualised under u.v.-light after spraying with 2’,7’-dichlorofluorescein solution (O.Ol”,, (w/v) in methanol) and were identified by reference to authentic standards chromatographed alongside. Fractions
518
W. W.
CHKISTII..M. L. HUNTI:H and
were recovered from the adsorbent by solvent extraction using the procedure of Akesson (1969) for liquid scintillation counting. [l-‘4CJstearic acid and [I-‘4C]oleic acid formed from it in the tissue were separated by silver nitrate chromatography in the form of the methyl ester derivatives by a procedure described elsewhere (Christic, 1974).
RESULTS
As a preliminary to determining the metabolic fate of lipid classes in rat adipose tissue maintained in culture, adipose tissue slices were pulse-labelled by incubating with a labelled fatty acid. generally [ I-‘4C]palmitic acid though [ I-‘4C]stearic and oleic acids were used in certain experiments. in the medium. After 4 hr when a high proportion of the substrate was incorporated into lipids (Christie & Vernon, 1975). the slices were removed from the incubation medium and were washed before being placed in the culture medium. some of the properties of which were described earlier (Vernon. 1978). The compositions of the labelled-lipids in the cultured tissue were determined at intervals for up to 7 days in total. In addition. unlabelled tissue was maintained in culture for the full duration of the experiment then was incubated with the. labelled fatty acid to determine the residual esterifying activity of the tissue. The results obtained when palmitic acid was used are listed in Table I. The total amount of radioactivity recovered from the tissue at the end of the experiment was not significantly different from that incorporated at the beginning so the results are expressed in terms of the relative proportion of labelled fatty acid in each lipid class present. At the end of the pulse-labelling period (day 0). more than 80”,, of the labelled substrate was incorporated into triacylglycerols and much of the remainder was into diacylglycerols; trace amounts only of labelled-unesterified fatty acids were present. After 1 day in culture. most of the diacylglycerols in the tissue had been converted to triacylglycerols. presumably by esterification with fatty acids endogenous to the tissue, for example those synthesized dr ~oro. The diacylglycerols formed at day 0 were entirely of the SIP I. 2-configuration so were presumably intermediates in the biosynthetic pathway to triacylglycerols rather than say products of lipolysis (Henderson (‘r (I/., 1979). Throughout the remainder of the period that the tissue was maintained in culture. the proportion of unesterified fatty acid present increased significantly and there was a concomitant decrease Table I.
Proportional
distribution (“,,) of [“C]16:0 to time. Values represent
R. G.
VI.WNON
in the proportion of the triacylglycerols. Results virtually indistinguishable from these were obtained in a single experiment with [I-‘“Cloleic acid and in two with [I-“Clstearic acid as the labelled substrate. The unlabelled tissue maintained in culture for 7 days was capable of esterifying [1-‘4C]palmitic acid at a rate 39 + 6.3:, of that on day 0 but this must be considered as a minimum value because of the high levels of unesterified fatty acids present in the tissue at this time which would dilute the substrate so that its precise concentration was unknown. Somewhat higher values (58 and 65”,, respectively) were obtained in the limited number of experiments with [I-“C]oleic and -stearic acids as substrates. Triacylglycerols were again the main lipid class formed but in this instance. a higher proportion of unesterified fatty acid remained within the tissue. No change in the apparent residual esterifying activity was observed when the tissue was transferred to fresh medium 199 culture medium after 4 days in culture. The lipids in the medium in which the tissue was cultured were also extracted and analysed. Trace amounts only (
Abstract--l. Rat adipose tissue was pulse-labelled with [l-“‘Cl fatty acids and was then maintained in tissue culture for up to 7 days. 2. After 1 day in culture. the proportion of the label recovered in the triacylglycerols tended lo decrease slowly while that in the unesterified fatty acids increased but a high level of esterifying activity remained in the tissue at the end of the culture period. 3. By measuring the rate of disappearance of particular molecular species of the labelled triacylglycerols. the half-life of triacylglycerols in the tissue was found to be 22-25 days. 4. The rate of desaturation of [I-“Clstearic acid was maintained in culture.
Son Ltd (Rayleigh. U.K.). Food (diet 418, Oxoid Ltd.) and water were supplied ud tihirutzr. Rats were killed by cerebral dislocation. samples of parametrial adipose tissue were removed immediately and placed in sterile isotonic saline at 37 C.
In adipose tissue. the triacylglycerols are continually turning over ia. hydrolysis and resynthesis is occurring (reviewed by Steinberg. 1964; Steinberg & Vaughan. 1965; Shapiro. 1977). Attempts have been made to determine the turnover rate of the fatty acid componcnts in adipose tissue (reviewed by Stein & Stein. 1965) but it is never clear whether the rate-limiting step is lipolysis of the triacylglycerols, oxidation of the fatty acids or export from the tissue as considerable reesterification may occur. The basal rate of lipolysis as measured by giycerol release has been used to determine triacylglycerol turnover and may be a better indicator but fipolysis is markedly influenced by hormonal factors including those induced by stress so there may be doubts as to the validity of extrapolating the results of comparatively short term experiments. In alddition. such factors as the nutritional state of the animal. the rate of growth of the tissue and interactions with other organs in the whole animal could markedly influence the nature and interpretation of the results. This study represents an attempt to determine the rate of triacylgly~erol turnover in rat adipose tissue maintained in culture by measuring the rate of turnover of a specific triacylglyceroi that is not resynthesized. The values obtained should indicate the mean rate of lipolysis over the period. MATERIALS
AND
Adipose tissue slices weighing 3- 4 mg were cut free-hand and transferred in approximately 80 mg aliquots to 25 ml Erlcnmcyer flasks containing medium 199 (2.5ml) with Earle’s salts containing t.-glutamine. hcpes (25 mM). insulin (10 lig;ml) and streptomycin sulphatc (100pgiml). hereafter rcfcrred to as medium 199 culture medium, together with bovine serum albumin (~mg/ml). To this was added a solution (0.2 ml) of the [i-‘4C]labclled fatty acid (2 jtmol. 2.5 CcCi)in 0.1 M KOH containing albumin (40 mg/ml). the flasks were sealed with Suba-seai stoppers and were incubated at 37 C for 4 hr. At the end of this period. the ti?suc slices were removed from the flasks and washed in fresh medium 199 culture medium to remove any of the residual labelled substrate adhering physically to the tissue. Tissue slices (approx 20mg). labellcd as above. in medium 199 culture medium (2Sml) were maintained in culture in 25ml Erlenmeyer flasks sealed with Suba-seal stoppers at 33 C as described by Vernon (1978). fn addttion. unlabelled tissue was maintained in culture in a similar way for the duration of the experiment (generally 7 days) before being incubated with a labelled Fatty acid as described above.
METHODS
Mtrrc~riuls
Insulin (24 units/mg). bovine serum albumin (fraction V. essentially fatty acid free), penicillin-G (I650 units/mg) and streptomycin sulphate were obtained from Sigma London Chemical Co. (Poole. U.K.). medium 199 with Earle’s salts and containing t.-glutamine and 25 mM Hepes was supplied by Gibco: Bio-Cult Ltd. (Paisley. U.K.) and [ i-14C]palmitic. stearic and oleic acids were from the Radiochemical Centre. (Amersham, U.K.). The bovine serum albumin was dialysed before use (Hanson & Ballard. 1968). Female Wist,ar rats were obtained
from A. Tuck and
* HRI Publication No. 1048. 517
At appropriate time intervals. the slices were removed from the medium and the lipids were extracted: aliquots of the medium itself were treated similarly. Procedures for the extraction of lipids. separation of lipid classes for liquid scintillation counting and for preparative isolation of the triacylglycerols were described elsewhere (Christie rt ctl.. 1976). Triacylglycerols were fractionated according to degree of unsaturation by preparative TLC on thin layers of silica gel G (0.5 mm thick) containing silver nitrate (IO?;, w w): hexane: diethyl ether (80:20, v/v) was the developing solvent. Up to 5 mg of triacylglycerols were separated on a 10 x 20 cm plate, Bands were visualised under u.v.-light after spraying with 2’,7’-dichlorofluorescein solution (O.Ol”,, (w/v) in methanol) and were identified by reference to authentic standards chromatographed alongside. Fractions
518
W. W.
CHKISTII..M. L. HUNTI:H and
were recovered from the adsorbent by solvent extraction using the procedure of Akesson (1969) for liquid scintillation counting. [l-‘4CJstearic acid and [I-‘4C]oleic acid formed from it in the tissue were separated by silver nitrate chromatography in the form of the methyl ester derivatives by a procedure described elsewhere (Christic, 1974).
RESULTS
As a preliminary to determining the metabolic fate of lipid classes in rat adipose tissue maintained in culture, adipose tissue slices were pulse-labelled by incubating with a labelled fatty acid. generally [ I-‘4C]palmitic acid though [ I-‘4C]stearic and oleic acids were used in certain experiments. in the medium. After 4 hr when a high proportion of the substrate was incorporated into lipids (Christie & Vernon, 1975). the slices were removed from the incubation medium and were washed before being placed in the culture medium. some of the properties of which were described earlier (Vernon. 1978). The compositions of the labelled-lipids in the cultured tissue were determined at intervals for up to 7 days in total. In addition. unlabelled tissue was maintained in culture for the full duration of the experiment then was incubated with the. labelled fatty acid to determine the residual esterifying activity of the tissue. The results obtained when palmitic acid was used are listed in Table I. The total amount of radioactivity recovered from the tissue at the end of the experiment was not significantly different from that incorporated at the beginning so the results are expressed in terms of the relative proportion of labelled fatty acid in each lipid class present. At the end of the pulse-labelling period (day 0). more than 80”,, of the labelled substrate was incorporated into triacylglycerols and much of the remainder was into diacylglycerols; trace amounts only of labelled-unesterified fatty acids were present. After 1 day in culture. most of the diacylglycerols in the tissue had been converted to triacylglycerols. presumably by esterification with fatty acids endogenous to the tissue, for example those synthesized dr ~oro. The diacylglycerols formed at day 0 were entirely of the SIP I. 2-configuration so were presumably intermediates in the biosynthetic pathway to triacylglycerols rather than say products of lipolysis (Henderson (‘r (I/., 1979). Throughout the remainder of the period that the tissue was maintained in culture. the proportion of unesterified fatty acid present increased significantly and there was a concomitant decrease Table I.
Proportional
distribution (“,,) of [“C]16:0 to time. Values represent
R. G.
VI.WNON
in the proportion of the triacylglycerols. Results virtually indistinguishable from these were obtained in a single experiment with [I-‘“Cloleic acid and in two with [I-“Clstearic acid as the labelled substrate. The unlabelled tissue maintained in culture for 7 days was capable of esterifying [1-‘4C]palmitic acid at a rate 39 + 6.3:, of that on day 0 but this must be considered as a minimum value because of the high levels of unesterified fatty acids present in the tissue at this time which would dilute the substrate so that its precise concentration was unknown. Somewhat higher values (58 and 65”,, respectively) were obtained in the limited number of experiments with [I-“C]oleic and -stearic acids as substrates. Triacylglycerols were again the main lipid class formed but in this instance. a higher proportion of unesterified fatty acid remained within the tissue. No change in the apparent residual esterifying activity was observed when the tissue was transferred to fresh medium 199 culture medium after 4 days in culture. The lipids in the medium in which the tissue was cultured were also extracted and analysed. Trace amounts only (
