371
Lipid Composition and Mitochondrial Respiration in Warm- and Cold-Adapted Sea Bass Gianni Trigad*, Maudzio Pirini, Vittoda Ventrella, Alessandra Pagliarani, Fabiana Trombetti and Anna Rosa Borgatti Department of Biochemistry, Section of Veterinary Biochemistry, University of Bologna, Bologna, Italy The response to cold of liver and heart membrane lipid composition and mitochondrial respiration in reared sea bass (Dicentrarchus labrax L.) was investigated. Fish acclimation was followed during the natural seasonal cycle from A u g u s t to March. The data on the fatty acid composition of liver and heart polar lipids and on total lipids of liver mitochondria and microsomes did not indicate any increase in unsaturation in response to cold. The enzyme complexes of the liver and heart mitochondrial respiratory chain showed a repeated negative compensation for cold acclimation. The constancy of the break in the Arrhenius plot of liver cytochrome oxidase (EC 1.9.3.1.} was consistent with the lack of homeoviscous adaptation of membrane lipids. A thermoadaptive strategy based on the reduction of sea bass metabolic activity is suggested. Lipids 27, 371-377 (1992}.
Various physiological and biochemical mechanisms have evolved in poikilotherms to counter the effects of thermal variation on physical properties of cells and body fluids. A number of studies have been carried out to identify the temperature-activated mechanisms involved in maintaining homeostasis. Changes in the unsaturation of membrane lipids have generally been implicated in membrane viscotropic regulaton and thus in the modulation of membrane-bound enzyme activities {1-5}. However variations in the membrane cholesterol content (6) and in the ratio of phospholipid classes {3,7}, interactions between the polar heads of phospholipids (8) and/or lipids and proteins {8,9}, as well as direct temperature effects on pr~ tein chains (2), have been shown to contribute to homeoviscous adaptation. These studies have been mainly carried out on winter-active freshwater teleosts. In some poikilotherms, the slowing of life activities, similar to that characteristic for hibernating msmmals, was described as another adaptational strategy (2,10L The purpose of our study was to examine whether behavioral adaptation in the sea bass {Dicentrarchus labrax L.) is accompanied by changes in membrane lipid composition and/or by variations in the activity of membranebound enzymes, such as mitochondrial respiratory complexes. The choice of the sea bass was based on the following observations: below 10~ this species strongly reduces motility and stops eating (11); its marine origin implies reduced capability to synthesize polyunsaturated fatty acids (PUFA) (12). Our experiments were carried out
following the seasonal cycle to obtain more reliable results than one could obtain by artificial, season-independent cold-acclimation studies. Thus, our experimental design reproduced the natural conditions, fast included, by over~ wintering sea bass in Northern Italy (11).
MATERIALS AND METHODS Reagents. Solvents, acid-washed Chromosorb W 60-80 mesh and diethyleneglycolsuccinatewere purchased from Carlo Erba {Milan, Italy). Silver nitrate and Tris were obtained from Merck (Darmstadt, Germany}. Silicic acid, 100 mesh and 14% boron trifluoride in anhydrous methanol were purchased from Mallinkrodt {St. Louis, MO) and from BDH, Laboratory Chemical Division {Pools United Kingdom}, respectively. Fatty acid-free bovine serum albumin {BSA), sodium ethylenedismlnetetraacetate {Na2EDTA}, respiratory substrates, and inhibitors were from Sigma Chemical Co. (St. Louis, MOL Standards for fatty acid analyses were obtained from Sigma and from Applied Science Laboratories Ina {State College, PAL All chemicals were reagent grade Quartz-double distilled water was used for all solutions in the preparation of subcellular fractions and in the mitochondrial respiratory activity tests. Animals and diets. Adult sea bass {Dicentrarchus labrax L.) weighing about 100 g, reared in a commercial hatchery in Valle Campo-Comacchio (Ferrara, Italy),were used. Fish were fed a pelleted diet to which lipids had been added as oilmixture (cod liver oil/raw linseed oil/grape seed oil/corn oil, 2:1:1:1, by vol) amounting to 10% {13). Total dietary total lipid content was slightly higher {11.2%, w/w) due to the lipid content of other dietary components. The fatty acid pattern of the diet is shown in Table 1. Other details of diet composition were reported by Corti et al. (13).
Experimental desigr~ As shown in Figure 1 fish were kept at approximately 22~ from August until midOctober (warm-adapted or WA} and fed to satiation {2.5% of wet body weight/day). Following the seasonal lowering of environmental temperatur~ the water temperature gradually decreased to 10~ Food consumption gradually decreased with temperature and became undetectabl~ The daily ration was therefore gradually lowered to 0.5% of wet body weight and maintained until the end of the experiment. Fish sacrificed in December, after a month of acclimation at temperatures around 10-11~ to test the short-term cold adaptation to the threshold temperature of food uptake among sea bass (11), were defined as short~ *To whom correspondence should be addressed at the Department of Biochemistry, Section of Veterinary Biochemistry, University term cold-adapted (SCA). Fish were then kept at approxof Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy. imately 7 ~ until March to test the long-term adaptation Abbreviations: BSA, bovine serum albumin; EDTA, ethylenedi- at a temperature similar to that faced by reared sea bass Arn~netetraacetate; FA, fatty acids;GLC,gas-liquidchromatography; during winter in Northern Italy (7). This adaptation was LCA,lon~term cold-adapted;MUFA,monounsaturatedfatty acids; performed under two conditions as follows. Fish were ranPL, phospholipids;PUFA, polyunsaturatedfatty acids; SCA, shortterm cold-adapted; SFA, saturated fatty acids; TL, total lipids; domly divided into two groups. The first group was kept TMPD, N,N,N',N'-tetramethyl-p-phenylendiaminedihydrocldoride; in seawater tanks and diet was provided (Expt. 1). In Tris, Tris-(hydroxymethyl}smlnomethane;WA, warm-adapted. parallel, tlie second group was maintained at the same LIPIDS, Vol. 27, no. 5 (1992)
372
G. TRIGARI ET AL. TABLE 1 F a t t y Acid Composition of the Diet a
Fatty acid
wt/%
14:0 16:0 16:1 18:0 18:1 18:2n-6
1.5 10.4 1.7 3.4 33.4 31.7
18:3n-3 20:1n-9 20:4n-6 22:1n-9 20:5n-3
5.4 3.1 0.2 2.6 2.0
22:6n-3 Others b ZSFA ZMUFA IPUFA Zn-3 Y_n-6 I I • 100/ZSFA n-3/n-6
3.4 4.6 17.3 39.4 43.3 11.4 31.9 1.53 8.84 0.36
a Acronyms are listed in Materials and Methods. Values are means of at least 3 determinations. bSum of 12:0, 13:0, 15:0, iso-16:0, 17:0, iso-18:0, 19:1, 18:3n-6, 18:4n-3, 20:3n-6, 20:4n-3, 22:5n-3, 24:0 and 24:1.
30.
v
~2o SCA LCA
10
AW, ~,.pt. oc!, Noy., O%, .fan. Feb.. M,~r
..
3o s0
90 120 15o 180 21o 240 Days
FIG. 1. Temperature regimen during the experiment. Samplings are indicated by arrows.
temperature in synthetic-seawater tanks, but was not fed (Expt. 2). Since at this temperature sea bass usually do not eat (11), the two experiments were aimed at ascertaining whether any differences exist between fasting (Expt. 1) and starved (Expt. 2) sea bass. Data from b o t h experiments were not significantly different; therefore, the results from long-term cold-acclimated (LCA) fish are reported as averages of Expts. 1 and 2. The water temperature in the tanks was measured at the same hour each day during the entire experiment. Fish mortality, which was calculated as percentage of the initial number at the end of each acclimation period, was about 0.9% up to December and nil thereafter (Viviani, R., personal communication). Sampling procedures. Fish were randomly selected and killed at the end of each experimental period. Samples consisted of 80 WA, 40 SCA and 80 LCA fish (60 from LIPIDS, Vol. 27, no. 5 (1992)
Expt. 1 and 20 from Expt. 2). Liver and heart were removed immediately after death. Part of each tissue was stored in the dark under nitrogen at - 2 0 ~ for lipid analysis. Another portion was immediately used for the preparation of subcellular fractions:
Preparation of the mitochondrial and microsomal fractions. In each preparation, the liver and heart from 10-20 animals were repeatedly rinsed in ice-cold medium A (0.25 M sucrose, 5 mM sodium ethylenediaminetetraacetate (Na2EDTA), 16.5 mM Tris-HCL p H 7.4) and homogenized with an Ultraturrax in medium B (0.25 M sucrose, 1 mM Na~EDTA, 24 mM T~is-HCL 0.5 mg]mL fatty acidfree bovine serum albumin (BSA), p H 7.6). The mitochondrial fraction was obtained by differential centrifugation using the m e t h o d of Lyons and Raison (14) modified as follows. The homogenate was centrifuged at 750 • g for 10 min, and then the supernatant was filtered through a double layer of gauze (30 mesh) and centrifuged at 10,000 • g for 10 min. The mitochondrial pellet was resuspended in medium B and centrifuged at 13,000 • g for 10 min. Part of the sample was frozen in liquid nitrogen for lipid analysis, and another part was immediately used for the respiratory tests. The post-mitochondrial supern a t a n t from the liver was further centrifuged at 90,000 • g for 90 ~ The pellet was resuspended and twice washed by centrifuging at the same speed for 40 rain. The resulting microsomal fraction was stored in liquid nitrogen until u s e All steps were carried out at 0-4~ Protein in the various fractions was evaluated by the biuret method (15). The purity of microsomal and mitochondrial fractions was routinely t e s t e d by d e t e r m i n i n g (Na +, K+)-ATPase (EC 3.6.1.3) and cytochrome oxidase (EC 1.9.3.1) as enzymatic markers for plasma membrane and mitochondria, respectively. The negligible specific activity of (Na+, K+)ATPase in the mitochondrial fraction and of the cytochrome oxidase in microsomes indicated no appreciable mutual contamination of the fractions under study. Determination of mitochondrial respiratory activities. The respiratory activity of liver and heart mitochondria was evaluated polarographically with a Clark electrode using a Yellow Springs Instrument Model 53 oxygen monitor (Yellow Springs, OH). The three sites of oxidative phosphorylation were tested at 20~ by using as substrate~ respectively, glutamat~ succinate and ascorbate + N,N,N',N'-tetramethyl-p-phenylendiamine dihydrochloride (TMPD) under the conditions previously described {16}. The respiratory control ratio (RCR) and adenosine diphosphate (ADP):Oxygen (O) ratio were calculated as described by Estabrook (17). The Arrhenius plot of the liver cytochrome oxidase system was drawn by plotting the state 3 respiratory activity data using ascorbate + T M P D as substrate in the temperature range of 3-40~ as reported by Borgatti et as {18).The above assay was not carried out on heart because of insufficient mitochondrial material. Lipid analyses. Lipids were extracted according to Folch et as (19) from each liver mitochondrial and microsomal preparation containing 100-300 m g protein, from 1-5 g liver and heart pooled from 5 fish,and from 5 g of the diet. Total lipids (TL) were determined gravimetrically. Phospholipids (PL) were quantitatively evaluated by colorimetric determination of phosphorus (20) according to the method of Bartlett (21),as modified by Marinetti {22). Tissue lipidextracts were separated into neutral lipids and
373
TEMPERATURE ADAPTATION RESPONSES IN SEA BASS polar lipids according to Marks et aL (23). Activated silicic acid in the m a x i m a l ratio of i g per 50 m g P L was added to total lipids. After removal of neutral lipids by triplicate extraction with diethyl ether, polar lipids were obtained by triplicate extraction with methanol. A complete separation was obtained. The saponification of TL and polar lipids, the extraction of the f a t t y acids (FA), and the methylation using 14% B F a in methanol were carried out as previously described {24). Methyl esters of FA were analyzed by gas-liquid c h r o m a t o g r a p h y (GLC) and also b y c h r o m a t o g r a p h y on silicic acid columns i m p r e g n a t e d with 25% AgNO3 and b y catalytic hydrogenation. G L C was done on a Dani (Monza, Italy) 3600 gas chromatograph using a glass column (2 m • 4 re_m) filled with acidwashed Chromosorb W 60-80 m e s h and 20% (w/w) diethyleneglycolsuccinat~ The carrier gas was nitrogen. The GLC operating conditions and the m e t h o d s of identification of FA were reported previously (25). Methyl esters of FA separated b y GLC are given as weight percentages of total FA. The u n s a t u r a t i o n index (I) and the ratio I • 100/~ s a t u r a t e d f a t t y acids (SFA) were calculated according to Bloj et al. (26). Statistics. Results are presented as m e a n _+ s t a n d a r d error (8E). Statistical significance of differences between differently acclimated groups was determined by the Student's t-test. RESULTS AND DISCUSSION
Effect of cold-acclimation on lipid composition. Changes in lipid class composition have been reported as one of the possible responses to t e m p e r a t u r e lowering {27,28}. The content of total lipids (TL) and phospholipids (PL) of liver, heart and liver mitochondria and microsomes, shown in Table 2, indicates t h a t only in the liver T L and P L levels were changed by cold exposur~ The observed increase of liver T L in SCA p r o b a b l y reflects fat storage to face the winter. This would be consistent with previous studies which have shown t h a t eel accumulates triglycerides as energy source before fasting for the winter {28}. A T L increase was found b y Roche and P6r~s (27) in coldacclimated Dicentrarchus labrax fed during the entire experiment. However, other cold acclimation studies (29) t h a t were carried out under conditions of forced starvation and independent of season showed no T L variation. The P L increase in LCA is probably related to the consumption of previously stored neutral lipids. Other studies have indicated a widespread tendency of P L to increase after long-term cold~xposure (at least one month} {27,28}. No variations in T L and P L levels are apparent in heart and liver subceUular fractions consistent with the lack of fat storage function of this tissu~ The constancy of T L and PL levels in liver mitochondrial and microsomal fractions is also in line with literature reports on cold acclimated teleosts {6,9,30). The analysis of f a t t y acid composition was focused on liver and h e a r t polar lipids and on total lipids of liver mitochondrial and microsomal fractions. Liver polar lipids {Table 3) show only little variation related to cold acclimation, namely an increase of 20:5n-3 and thus of Zn-3, and a decrease of 18:1 resulting into a lowering of m o n o u n s a t u r a t e d f a t t y acids (MUFA). The decrease of M U F A is consistent with t h a t reported for t r o u t liver (31).
TABLE 2 Total Lipid and Phospholipid Content of Liver and Heart and Liver Subcellular Fractions a
Liver WA SCA LCA Heart WA SCA LCA
Liver Mitochondria WA SCA LCA Microsomes WA SCA LCA
nb
TLc
PL c
6 2 4
30.4 _+ 1.0 37.1 __ 0.5d 32.5 __- 2.9
2.5 --4"_0.1 2.2 +_ 0.1 3.2 + 0.1e
6 2 4
4.0 _ 0.8 4.0 _+ 0.5 3.6 _+ 0.8
1.8 +_ 0.1 1.6 +_ 0.1 1.8 +_ 0.2
TLf
pLf
6 2 4
19.6 + 1.8 18.4 +_ 1.2 17.1 _+ 1.8
13.4 +_ 0.9 13.2 _+ 2.4 10.9 +_ 0.5
6 2 4
42.2 _+ 4.6 40.2 + 4.4 51.9 _+ 2.1
23.3 +_ 3.9 20.5 +_ 1.7 28.9 +_ 1.2
a Acronyms are listed in Materials and Methods. bNumber of pools analyzed. c Expressed as wet wt percentages, mean _+ SE. dp ~
Lipid Composition and Mitochondrial Respiration in Warm- and Cold-Adapted Sea Bass Gianni Trigad*, Maudzio Pirini, Vittoda Ventrella, Alessandra Pagliarani, Fabiana Trombetti and Anna Rosa Borgatti Department of Biochemistry, Section of Veterinary Biochemistry, University of Bologna, Bologna, Italy The response to cold of liver and heart membrane lipid composition and mitochondrial respiration in reared sea bass (Dicentrarchus labrax L.) was investigated. Fish acclimation was followed during the natural seasonal cycle from A u g u s t to March. The data on the fatty acid composition of liver and heart polar lipids and on total lipids of liver mitochondria and microsomes did not indicate any increase in unsaturation in response to cold. The enzyme complexes of the liver and heart mitochondrial respiratory chain showed a repeated negative compensation for cold acclimation. The constancy of the break in the Arrhenius plot of liver cytochrome oxidase (EC 1.9.3.1.} was consistent with the lack of homeoviscous adaptation of membrane lipids. A thermoadaptive strategy based on the reduction of sea bass metabolic activity is suggested. Lipids 27, 371-377 (1992}.
Various physiological and biochemical mechanisms have evolved in poikilotherms to counter the effects of thermal variation on physical properties of cells and body fluids. A number of studies have been carried out to identify the temperature-activated mechanisms involved in maintaining homeostasis. Changes in the unsaturation of membrane lipids have generally been implicated in membrane viscotropic regulaton and thus in the modulation of membrane-bound enzyme activities {1-5}. However variations in the membrane cholesterol content (6) and in the ratio of phospholipid classes {3,7}, interactions between the polar heads of phospholipids (8) and/or lipids and proteins {8,9}, as well as direct temperature effects on pr~ tein chains (2), have been shown to contribute to homeoviscous adaptation. These studies have been mainly carried out on winter-active freshwater teleosts. In some poikilotherms, the slowing of life activities, similar to that characteristic for hibernating msmmals, was described as another adaptational strategy (2,10L The purpose of our study was to examine whether behavioral adaptation in the sea bass {Dicentrarchus labrax L.) is accompanied by changes in membrane lipid composition and/or by variations in the activity of membranebound enzymes, such as mitochondrial respiratory complexes. The choice of the sea bass was based on the following observations: below 10~ this species strongly reduces motility and stops eating (11); its marine origin implies reduced capability to synthesize polyunsaturated fatty acids (PUFA) (12). Our experiments were carried out
following the seasonal cycle to obtain more reliable results than one could obtain by artificial, season-independent cold-acclimation studies. Thus, our experimental design reproduced the natural conditions, fast included, by over~ wintering sea bass in Northern Italy (11).
MATERIALS AND METHODS Reagents. Solvents, acid-washed Chromosorb W 60-80 mesh and diethyleneglycolsuccinatewere purchased from Carlo Erba {Milan, Italy). Silver nitrate and Tris were obtained from Merck (Darmstadt, Germany}. Silicic acid, 100 mesh and 14% boron trifluoride in anhydrous methanol were purchased from Mallinkrodt {St. Louis, MO) and from BDH, Laboratory Chemical Division {Pools United Kingdom}, respectively. Fatty acid-free bovine serum albumin {BSA), sodium ethylenedismlnetetraacetate {Na2EDTA}, respiratory substrates, and inhibitors were from Sigma Chemical Co. (St. Louis, MOL Standards for fatty acid analyses were obtained from Sigma and from Applied Science Laboratories Ina {State College, PAL All chemicals were reagent grade Quartz-double distilled water was used for all solutions in the preparation of subcellular fractions and in the mitochondrial respiratory activity tests. Animals and diets. Adult sea bass {Dicentrarchus labrax L.) weighing about 100 g, reared in a commercial hatchery in Valle Campo-Comacchio (Ferrara, Italy),were used. Fish were fed a pelleted diet to which lipids had been added as oilmixture (cod liver oil/raw linseed oil/grape seed oil/corn oil, 2:1:1:1, by vol) amounting to 10% {13). Total dietary total lipid content was slightly higher {11.2%, w/w) due to the lipid content of other dietary components. The fatty acid pattern of the diet is shown in Table 1. Other details of diet composition were reported by Corti et al. (13).
Experimental desigr~ As shown in Figure 1 fish were kept at approximately 22~ from August until midOctober (warm-adapted or WA} and fed to satiation {2.5% of wet body weight/day). Following the seasonal lowering of environmental temperatur~ the water temperature gradually decreased to 10~ Food consumption gradually decreased with temperature and became undetectabl~ The daily ration was therefore gradually lowered to 0.5% of wet body weight and maintained until the end of the experiment. Fish sacrificed in December, after a month of acclimation at temperatures around 10-11~ to test the short-term cold adaptation to the threshold temperature of food uptake among sea bass (11), were defined as short~ *To whom correspondence should be addressed at the Department of Biochemistry, Section of Veterinary Biochemistry, University term cold-adapted (SCA). Fish were then kept at approxof Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy. imately 7 ~ until March to test the long-term adaptation Abbreviations: BSA, bovine serum albumin; EDTA, ethylenedi- at a temperature similar to that faced by reared sea bass Arn~netetraacetate; FA, fatty acids;GLC,gas-liquidchromatography; during winter in Northern Italy (7). This adaptation was LCA,lon~term cold-adapted;MUFA,monounsaturatedfatty acids; performed under two conditions as follows. Fish were ranPL, phospholipids;PUFA, polyunsaturatedfatty acids; SCA, shortterm cold-adapted; SFA, saturated fatty acids; TL, total lipids; domly divided into two groups. The first group was kept TMPD, N,N,N',N'-tetramethyl-p-phenylendiaminedihydrocldoride; in seawater tanks and diet was provided (Expt. 1). In Tris, Tris-(hydroxymethyl}smlnomethane;WA, warm-adapted. parallel, tlie second group was maintained at the same LIPIDS, Vol. 27, no. 5 (1992)
372
G. TRIGARI ET AL. TABLE 1 F a t t y Acid Composition of the Diet a
Fatty acid
wt/%
14:0 16:0 16:1 18:0 18:1 18:2n-6
1.5 10.4 1.7 3.4 33.4 31.7
18:3n-3 20:1n-9 20:4n-6 22:1n-9 20:5n-3
5.4 3.1 0.2 2.6 2.0
22:6n-3 Others b ZSFA ZMUFA IPUFA Zn-3 Y_n-6 I I • 100/ZSFA n-3/n-6
3.4 4.6 17.3 39.4 43.3 11.4 31.9 1.53 8.84 0.36
a Acronyms are listed in Materials and Methods. Values are means of at least 3 determinations. bSum of 12:0, 13:0, 15:0, iso-16:0, 17:0, iso-18:0, 19:1, 18:3n-6, 18:4n-3, 20:3n-6, 20:4n-3, 22:5n-3, 24:0 and 24:1.
30.
v
~2o SCA LCA
10
AW, ~,.pt. oc!, Noy., O%, .fan. Feb.. M,~r
..
3o s0
90 120 15o 180 21o 240 Days
FIG. 1. Temperature regimen during the experiment. Samplings are indicated by arrows.
temperature in synthetic-seawater tanks, but was not fed (Expt. 2). Since at this temperature sea bass usually do not eat (11), the two experiments were aimed at ascertaining whether any differences exist between fasting (Expt. 1) and starved (Expt. 2) sea bass. Data from b o t h experiments were not significantly different; therefore, the results from long-term cold-acclimated (LCA) fish are reported as averages of Expts. 1 and 2. The water temperature in the tanks was measured at the same hour each day during the entire experiment. Fish mortality, which was calculated as percentage of the initial number at the end of each acclimation period, was about 0.9% up to December and nil thereafter (Viviani, R., personal communication). Sampling procedures. Fish were randomly selected and killed at the end of each experimental period. Samples consisted of 80 WA, 40 SCA and 80 LCA fish (60 from LIPIDS, Vol. 27, no. 5 (1992)
Expt. 1 and 20 from Expt. 2). Liver and heart were removed immediately after death. Part of each tissue was stored in the dark under nitrogen at - 2 0 ~ for lipid analysis. Another portion was immediately used for the preparation of subcellular fractions:
Preparation of the mitochondrial and microsomal fractions. In each preparation, the liver and heart from 10-20 animals were repeatedly rinsed in ice-cold medium A (0.25 M sucrose, 5 mM sodium ethylenediaminetetraacetate (Na2EDTA), 16.5 mM Tris-HCL p H 7.4) and homogenized with an Ultraturrax in medium B (0.25 M sucrose, 1 mM Na~EDTA, 24 mM T~is-HCL 0.5 mg]mL fatty acidfree bovine serum albumin (BSA), p H 7.6). The mitochondrial fraction was obtained by differential centrifugation using the m e t h o d of Lyons and Raison (14) modified as follows. The homogenate was centrifuged at 750 • g for 10 min, and then the supernatant was filtered through a double layer of gauze (30 mesh) and centrifuged at 10,000 • g for 10 min. The mitochondrial pellet was resuspended in medium B and centrifuged at 13,000 • g for 10 min. Part of the sample was frozen in liquid nitrogen for lipid analysis, and another part was immediately used for the respiratory tests. The post-mitochondrial supern a t a n t from the liver was further centrifuged at 90,000 • g for 90 ~ The pellet was resuspended and twice washed by centrifuging at the same speed for 40 rain. The resulting microsomal fraction was stored in liquid nitrogen until u s e All steps were carried out at 0-4~ Protein in the various fractions was evaluated by the biuret method (15). The purity of microsomal and mitochondrial fractions was routinely t e s t e d by d e t e r m i n i n g (Na +, K+)-ATPase (EC 3.6.1.3) and cytochrome oxidase (EC 1.9.3.1) as enzymatic markers for plasma membrane and mitochondria, respectively. The negligible specific activity of (Na+, K+)ATPase in the mitochondrial fraction and of the cytochrome oxidase in microsomes indicated no appreciable mutual contamination of the fractions under study. Determination of mitochondrial respiratory activities. The respiratory activity of liver and heart mitochondria was evaluated polarographically with a Clark electrode using a Yellow Springs Instrument Model 53 oxygen monitor (Yellow Springs, OH). The three sites of oxidative phosphorylation were tested at 20~ by using as substrate~ respectively, glutamat~ succinate and ascorbate + N,N,N',N'-tetramethyl-p-phenylendiamine dihydrochloride (TMPD) under the conditions previously described {16}. The respiratory control ratio (RCR) and adenosine diphosphate (ADP):Oxygen (O) ratio were calculated as described by Estabrook (17). The Arrhenius plot of the liver cytochrome oxidase system was drawn by plotting the state 3 respiratory activity data using ascorbate + T M P D as substrate in the temperature range of 3-40~ as reported by Borgatti et as {18).The above assay was not carried out on heart because of insufficient mitochondrial material. Lipid analyses. Lipids were extracted according to Folch et as (19) from each liver mitochondrial and microsomal preparation containing 100-300 m g protein, from 1-5 g liver and heart pooled from 5 fish,and from 5 g of the diet. Total lipids (TL) were determined gravimetrically. Phospholipids (PL) were quantitatively evaluated by colorimetric determination of phosphorus (20) according to the method of Bartlett (21),as modified by Marinetti {22). Tissue lipidextracts were separated into neutral lipids and
373
TEMPERATURE ADAPTATION RESPONSES IN SEA BASS polar lipids according to Marks et aL (23). Activated silicic acid in the m a x i m a l ratio of i g per 50 m g P L was added to total lipids. After removal of neutral lipids by triplicate extraction with diethyl ether, polar lipids were obtained by triplicate extraction with methanol. A complete separation was obtained. The saponification of TL and polar lipids, the extraction of the f a t t y acids (FA), and the methylation using 14% B F a in methanol were carried out as previously described {24). Methyl esters of FA were analyzed by gas-liquid c h r o m a t o g r a p h y (GLC) and also b y c h r o m a t o g r a p h y on silicic acid columns i m p r e g n a t e d with 25% AgNO3 and b y catalytic hydrogenation. G L C was done on a Dani (Monza, Italy) 3600 gas chromatograph using a glass column (2 m • 4 re_m) filled with acidwashed Chromosorb W 60-80 m e s h and 20% (w/w) diethyleneglycolsuccinat~ The carrier gas was nitrogen. The GLC operating conditions and the m e t h o d s of identification of FA were reported previously (25). Methyl esters of FA separated b y GLC are given as weight percentages of total FA. The u n s a t u r a t i o n index (I) and the ratio I • 100/~ s a t u r a t e d f a t t y acids (SFA) were calculated according to Bloj et al. (26). Statistics. Results are presented as m e a n _+ s t a n d a r d error (8E). Statistical significance of differences between differently acclimated groups was determined by the Student's t-test. RESULTS AND DISCUSSION
Effect of cold-acclimation on lipid composition. Changes in lipid class composition have been reported as one of the possible responses to t e m p e r a t u r e lowering {27,28}. The content of total lipids (TL) and phospholipids (PL) of liver, heart and liver mitochondria and microsomes, shown in Table 2, indicates t h a t only in the liver T L and P L levels were changed by cold exposur~ The observed increase of liver T L in SCA p r o b a b l y reflects fat storage to face the winter. This would be consistent with previous studies which have shown t h a t eel accumulates triglycerides as energy source before fasting for the winter {28}. A T L increase was found b y Roche and P6r~s (27) in coldacclimated Dicentrarchus labrax fed during the entire experiment. However, other cold acclimation studies (29) t h a t were carried out under conditions of forced starvation and independent of season showed no T L variation. The P L increase in LCA is probably related to the consumption of previously stored neutral lipids. Other studies have indicated a widespread tendency of P L to increase after long-term cold~xposure (at least one month} {27,28}. No variations in T L and P L levels are apparent in heart and liver subceUular fractions consistent with the lack of fat storage function of this tissu~ The constancy of T L and PL levels in liver mitochondrial and microsomal fractions is also in line with literature reports on cold acclimated teleosts {6,9,30). The analysis of f a t t y acid composition was focused on liver and h e a r t polar lipids and on total lipids of liver mitochondrial and microsomal fractions. Liver polar lipids {Table 3) show only little variation related to cold acclimation, namely an increase of 20:5n-3 and thus of Zn-3, and a decrease of 18:1 resulting into a lowering of m o n o u n s a t u r a t e d f a t t y acids (MUFA). The decrease of M U F A is consistent with t h a t reported for t r o u t liver (31).
TABLE 2 Total Lipid and Phospholipid Content of Liver and Heart and Liver Subcellular Fractions a
Liver WA SCA LCA Heart WA SCA LCA
Liver Mitochondria WA SCA LCA Microsomes WA SCA LCA
nb
TLc
PL c
6 2 4
30.4 _+ 1.0 37.1 __ 0.5d 32.5 __- 2.9
2.5 --4"_0.1 2.2 +_ 0.1 3.2 + 0.1e
6 2 4
4.0 _ 0.8 4.0 _+ 0.5 3.6 _+ 0.8
1.8 +_ 0.1 1.6 +_ 0.1 1.8 +_ 0.2
TLf
pLf
6 2 4
19.6 + 1.8 18.4 +_ 1.2 17.1 _+ 1.8
13.4 +_ 0.9 13.2 _+ 2.4 10.9 +_ 0.5
6 2 4
42.2 _+ 4.6 40.2 + 4.4 51.9 _+ 2.1
23.3 +_ 3.9 20.5 +_ 1.7 28.9 +_ 1.2
a Acronyms are listed in Materials and Methods. bNumber of pools analyzed. c Expressed as wet wt percentages, mean _+ SE. dp ~
