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Desaturation and elongation of unsaturated fatty acids in hepatocytes from thermally acclimated rainbow trout
ARCHIVES OF BI~~~s~Y AND BIOPHYSICS Vol. 213, No. 1, January, pp. 5%66,1%X32
Desaturation
and Elongation of Unsaturated Fatty Acids in Hepatocytes from Thermally Acclimated Rainbow Trout’ PEGGY
A. SELLNER’
I)epartment Of &do@% Arizxnm
AND JEFFREY State
U%&mi&
R. HAZEL
Tempe,
Ariama
85.287
Received April 3,1981, and in revised form August 6,198l Hepatocytes isolated from 5- and 2O”C-acclimated rainbow trout (Salmo g&&zeri) were incubated with [1-‘*C]oleic, -linoleic, or -1inolenic acid to investigate the role of desaturation and elongation pathways in cold adaptation. Cells from both acclimation groups demonstrated a substrate preference in the order: 13~3 > l&2 > 13~1. When assayed at 5”C, cells from cold-acclimated trout were more efficient at producing derivatives of linolenic acid than cells from warm-acclimated trout. Cold acclimation resulted in degrees of compensation of desaturation and elongation rates that depended on the fatty acid family and specific reaction in the sequence. In the oleic acid family, reaction rates showed no compensation; partial compensation was observed in the linoleie acid family, and perfect (e.g., A6 desaturation) to overcompensation (e.g., A4 desaturation) in the linolenic acid family. Cells from cold-acclimated trout had higher rates of produetion of linolenicderived acids when assayed at 5 than 20°C. The proportion of the product of a given desaturation or elongation reaction that continued to the next reaction in the sequence was higher when assayed at 5 than 2O”C, increasing the probability of producing polyunsaturates. The results suggest that the substrate preference.of the desaturation and elongation enzymes, the compensation of reaction rates, and the possible “link” between certain reactions in the metabolic sequence may ensure the production of polyunsaturated fatty acids in cold-adapted trout.
One response of poikilotherms to changing environmental temperatures is the alteration of membrane lipid composition such that the degree of unsaturation of fatty acids is inversely related to the acclimation temperature (1). This has been termed a “homeoviscous adaptation” (2) and is presumed essential to preserving normal membrane function, The restructuring of lipid is not limited to membrane components, however, as changes in degree of unsaturation have been reported for neutral lipids (3) as well as phospholipids (4) in liver tissue of thermally acclimated rainbow trout. While the metabolic basis for increasing unsaturation is not entirely clear, there is
evidence that cold-adapted organisms have higher rates of production of unsaturated acids than warm-adapted organisms. This has been demonstrated for both procaryotes, which produce primarily monoenes, and eucaryotes, which generally increase unsaturation by incorporating polyunsaturates. For example, in E. co&, the ratio of oleie to palmitic acid produced by the fatty acid synthetase complex increased when assayed at 10°C compared to 40°C (5). Transfer of Bacillus megaterium cultures from 35 to 20°C caused a hyperinduction of synthesis of the A5-desaturase enzyme (6). Similar observations have been made with Tetrahymena, in which the activity of the palmitoyl CoA desaturase (A9) increased upon shifting the culture from 39.5 to 15°C (7). In the fish Pimebdus, A9-, A&, and A5-desaturase activities were higher in 15 than 30°C acclimated animals when assayed at the
’ Supported by NSF Grant PCM76-04313-AOl. 2 Present address: Department of Neurosurgical Research, Massachusetts General Hospital, Boston, Mass. 02114. 58
59
POLYUNSATURATEDFATTYACIDPRODUCTIONINTROUTHEPATOCYTES PARENT
FATTY
ACID
18:2 CHAIN ELONGATION 1 0:2 ~6 - DESATURATION . 18.3 ELONGATION
TO
20-C
ACID
22-C
ACID
I 20~3
Ah5- DESATURATION I 20~4 ELONGATION
TO
I 22~4
use of isolated hepatocyte preparations has several distinct advantages for studies of metabolism, including: (i) the availability of a homogeneous cell suspension from which many experimental aliquots can be apportioned; (ii) the diffusion of oxygen or substrates is not limiting, as is frequently the case with liver slices (13); and (iii) the study of regulatory interactions between reactions that take place in different subcellular compartments, but which compete for a common substrate, is possible. EXPERIMENTAL
A4-
DESATURATION
I 22:5
FIG. 1. Desaturation and elongation sequence. Linoleic acid (182, n - 6) and its derivatives are included as examples. C18, C20, and chain elongation reactions are named to distinguish between products and not to imply distinct enzymes. The phrase “dead end” denotes chain elongation of the substrate and incorporation without further metabolism of the acid.
same temperature (8). Thus, enhanced rates of desaturation may account for some of the fatty acid composition changes which accompany cold adaptation. In this study, the effects of thermal acclimation and assay temperature on fatty acid desaturation and elongation were determined in 5- and ZO”C-acclimated rainbow trout (Salmo gairdneri). Isolated hepatocytes were incubated with [l-‘4C]oleic, -linoleic, or -1inolenic acid, the parent acids of the three major families of polyunsaturated fatty acids (PUFA3) found in eucaryotes. These three fatty acids enter the desaturation and elongation sequence (Fig. 1) at the same step, i.e., A6 desaturation, which is known to be an important regulatory step in mammalian PUFA synthesis (9). Hepatocytes from rainbow trout have proved useful in studies of lipogenesis (10, 11) and gluconeogenesis (12). The 3Abbreviations used: PUFA, polyunsaturated fatty acids; BSA, bovine serum albumin; PPO, 2,5diphenyloxazole; POPOP, 1,4-bis[2(5-phenyloxazo1yl)jbenzene; PL, phospholipid; FFA, free fatty acid; NL, neutral lipid; GLC, gas-liquid chromatography.
PROCEDURES
Animals. Rainbow trout (Salmo gairdneri) were obtained from the Alchesay Fish Hatchery in Whiteriver, Arizona, and were maintained on pelleted trout food from Glencoe Mills, Minneapolis, Minnesota. The fatty acid composition of this diet has been reported elsewhere (4). Fish were acclimated to 5 or 20°C and a 12/12 photoperiod for at least 1 month prior to sacrifice. Preparation and incubation of hepatocgtaHepatocytes were prepared from 100 to 400-g animals by in situperfusion as described previously (11). In each experiment, hepatocytes obtained from two fish of the same acclimation group were pooled and taken up in 5-6 ml of Krebs-Ringer-imidazole buffer, pH 7.8 at 20°C containing 2% (w/v) bovine serum albumin (BSA) and 10 rnM glucose (Buffer A). Trypan blue was excluded by r95% of these freshly isolated cells. A small aliquot of the cell suspension was reserved for fatty acid analysis. One-milliliter portions of the cell suspension (6-10 mg protein) were incubated with [l-i%] oleic, -linoleic, or-linolenic acid (1.5 pCi in a total of ‘75 nmol, added in 5 ~1 ethanol) for 5 h at 20°C or 9 h at 5°C. Flasks were gassed continuously with Oz/COz (95/5) and effluent gas from each flask was bubbled through 1.5 ml of methylbenzethonium hydroxide to trap COz. At the end of the experiment, the contents of the COz trap were taken up in 10 ml of scintillation cocktail A (6 g PPO + 0.1 g POPOP per liter of toluene) and counted to estimate the extent of j3 oxidation. Incubations were terminated by diluting the cell suspensions with 3 ml of ice-cold Buffer A and immediately collecting the cells by centrifugation. Lipids were extracted from the cell pellet by the method of Bligh and Dyer (14). Ten percent of the total lipid extract was applied to a Silica Gel G plate which was developed in hexane/diethyl ether/formic acid (80/ 20/2) to separate phospholipid (PL), free fatty acid (FFA), and neutral lipid (NL). The regions of gel containing these lipids were scraped directly into scintillation vials and counted in 10 ml of cocktail B (6
60
SELLNER
g PPO + 0.1 g POPOP per liter of toluene/2-ethoxyethanol, 2/l) to determine total incorporation into lipid classes. The remaining 90% of the lipid extract, to be used for radio-gas-liquid ehromato~aphy, was separated by thin-layer chromatography in an identical manner, but the lipids were eluted from the gel with chlorform/methanol (Z/l). Fatty acid analyses. All lipid analyses were performed under nitrogen to prevent oxidation of the fatty acids. Lipid samples in chloroform/methanol were evaporated to dryness and saponified with 0.2 ml of 30% aqueous KOH in 5 ml of ethanol at 80°C for 2 h. Fatty acids were extracted with light petroleum ether following acidification with 1.5 ml of 6 N hydrochloric acid. Fatty acyl methyl esters were then prepared with 14% boron trifluoride in methanol (15), and separated by gas-liquid chromato~aphy on 10% EGSS-X on Gas-Chrom P using a HewlettPackard Model 534OA chromatograph equipped with an effluent splitter adjusted to a 91 split ratio. During each run, the column was heated from 130 to 205’C using a time-temperature program. Radioactive methyl esters were collected on 3-cm Estron filter rods and counted directly in 10 ml of scintillation cocktail A. In some cases, positions of the double bonds in a fatty acid were determined by eluting the methyl ester from the Estron filter rod with hexane and subjecting it to permanganate-periodate degradation according to von Rudloff (16). Electron rn~~os~~. Throughout the preparation of the samples for electron microscopy, isolated hepatoeytes were collected by gravitation rather than centrifugation to minimize cell damage. Small blocks of intact liver (taken before perfusion) and pellets of isolated hepatocytes taken before and after incubation (without added fatty acid) were fixed in Cazffree Krebs-Ringer-phosphate buffer (pH 7.4, containing 2% formaldehyde and 3% glutaraldehyde) for 1 h on ice. Samples were washed with buffer, postfixed on ice in buffered 1% 0~0~ for an additional hour, and washed again in buffer. Hepatocytes were encapsulated in 2% agar, and small blocks of suspended cells as well as blocks of liver were dehydrated in an ethanol series. All blocks were embedded in Epon/Araldite (l/l). Sections were cut with a glass knife on a Porter-Blum MT-l, stained with uranyl acetate and lead citrate, and viewed on a Philips EM 201 instrument. Analytical methods. Counts per minute were converted to disintegrations per minute using the external standard ratio and a quench calibration curve. Nanomoles were calculated (assuming no dilution of label by endogenous FFA) from the known specific activity of the substrate mixture, which was determined by counting a small aliquot. Lactate dehydrogenase assays to estimate cell viability were performed as described by Holbrook et ah (17). Protein was determined according to Lowry et al. (18) using
AND HAZEL BSA as a standard. Statistical analyses consisting of Student’s t test were performed according to Zar (19). ~~~~. [1-‘*C]Oleie (56 mCi/mmol), -1inoleic (60 mCi/mmol), and -1inolenic (60 mCi/mmol) acids were obtained from the Radiochemical Centre, Amersham, England. Nonradioactive oleic, linoleic, and linolenic acids were purchased from Sigma. Purities of the “cold” and “hot” fatty acids, determined by radioGLC analysis of methyl esters prepared from the supplied fatty acids, were: oleic, 99%, 97%; linoleic, 97%, 97%; and linolenic, 98%, 92%, respectively. Collagenase (Type IV, Chtridium histdyticum), hyaluronidase {Type I, bovine testes), bovine serum albumin (Fraction V), nicotinamide adenine dinueleotide (reduced form), methylbenzethonium hydroxide, PPO, and POPOP were also from Sigma. EGSS-X on Gas-Chrom P, Silica Gel G, and 14% boron trifluoride in methanol were from Applied Science Laboratories, and pyruvic acid was obtained from Eastman Chemicals. Estron filter rods were from the American Filtrona Company. RESULTS
Rates of LDH activity in freshly isolated cells were 1.34 k 0.26 and 1.33 f 0.30 gmol (min X mg)-’ from cold- and warm-acclimated fish, respectively. Hepatocyte viability (computed as the percentage of zerotime LDH activity) in both acclimation groups averaged 68% after 5 h at 20°C and 71-74s after 9 h at 5°C. Viability was also assessed by comparing the ultrastructure of isolated hepatocytes with intact liver (Fig. 2). Isolated cells (Fig. 2B) closely resembled in morphology cells of the intact liver (Fig. 2A). Surrounding the prominent nucleus were several mitochondria and large amounts of endoplasmic reticulum; discrete regions of glycogen granules were located toward the periphery of the cells. This pattern of organization remained unchanged after 5 h of incubation at 20X (Fig. 2C) or 9 h at 5°C (Fig. ZD), and there was no significant depletion of glycogen. The extent of ,kI oxidation, estimated from the amount of radioactivity recovered in the COZ trap, was always less than 3% of the total incorporated radioactivity. Absolute rates of l*C02 production (data not shown) in cells from warm-acclimated compared with cold-
POLYUNSATURATED
FATTY ACID PRODUCTION
IN TROUT HEPATOCYTES
61
FIG. 2. Ultrastructure of liver tissue and isolated hepatocytes from 5’C-acclimated trout. (A) Section of liver (x5700); (B) freshly isolated cell (X8660); (C) cells after incubation at 20°C for 5 h (x9100); (D) after incubation at 5’C for 9 h (x7400). n, nucleus; m, mitochondria; er, endoplasmic reticulum; g, glycogen; pm, plasma membrane.
acclimated fish were approximately 20 and 50% lower in the 20 and 5°C assays, respectively. Normalized values (Table I) indicated a tendency to degrade 182 to a greater extent than l&l or l&3, regardless of assay or acclimation temperature. The fatty acid composition of hepatocyte lipids is indicated in Table 11. Saturated and monounsaturated fatty acids were more abundant in ceils from warm-
acclimated trout. Polyunsaturated fatty acids of the linolenic acid (n - 3) family predominated over the linoleic acid (n - 6) family; the proportions of both families were greater in hepatocytes of coldacclimated fish. The total amount of exogenous fatty acid incorporated (nmol (g protein)-‘) averaged 30-40s of that supplied to the cells. Incorporation was nearly equivalent
SELLNER
AND HAZEL
TABLE
Temperature
(“C)
Acclimation
Substrate Assay
l&l (n-9)
20 5 20 5
1.00 0.19 f 0.04 1.00
20 5
in each experiment
when measured after 5 h at 20°C or 9 h at S’C, regardless of acclimation temperature or substrate (Fig. 3). The tendency for greater incorporation of substrate with increasing degree of unsaturation was only significant in cells from warm-acclimated fish assayed at 5°C (l&l < l&3, P < 0.05). Incorporation of isotope into each fatty acid was determined by radio-GLC analysis, and rates expressed as nmol (g protein X h)-l. Total incorporation of a given fatty acid was calculated by summing rates of incorporation into all three lipid classes (PL, FFA, NL). The rate of a given reaction (desaturation or elongation) was TABLE
II
FATTY ACID COMPOSITIONOF ISOLATED HEPATOCYTESFROM~PANDWGACCLIMATED RAINBOWTROUT Temperature
Saturated Monounsaturated n-9 Family n-6 Family n-3 Family 20:4 22.5 m5 22~6
(n-6) (n-6) (n-3) (n-3)
23.9 +. 1.99 21.9 + 1.88 15.4 + 1.08 34.5 f 1.56 4.4 + 0.43
1.1 f 0.37 3.4 k 0.89 24.3 k 0.75
183 (n-3)
1.72 + 0.60
0.73 f
0.43 -t 0.10 3.00 -t 0.96 1.21 2 0.27
0.23 It 0.04
to 29°C assay, l&l
0.28
1.12 k 0.18 0.96 2 0.35
= 1.00. Values represent
then calculated by summing total incorporation into the reaction product plus incorporation into subsequent fatty acids according to the scheme in Fig. 1. For example, in the linoleic acid family, the rate of A5 desaturation was approximated by summing rates of isotope incorporation into 20:4, 22:4, and 22:5, since A5 desaturation is required for the formation of the latter two acids. The results (Table III) include rates of incorporation of the unmetabolized substrate, and rates of incorporation of label into fatty acids synthesized de now and into fatty acids of other families. Radioactivity in the latter two groups presumably reflects the incorporation of labeled acetyl-CoA derived from ,f3oxidation of the substrate. & z .E 2 P
(“C)
5 25.8 + 1.16
182 (n-6)
1.02 + 0.23
ncnm from each CO, flask were normalized mean t SEM for four or five experiments.
Fatty acid/ family
I
8
Y-ACCLIMATED 18~1 18~2 18~3
20*-ACCLIMATED i8:3 18~1 182
20 28.7 36.2 24.3 14.6 27.0 3.5
2 + & zk
2.81 2.34 1.76 0.79
f 1.59 c!z 0.13
1.1 rl: 0.28 1.6 zk 0.18 20.8 + 1.06
AWe. Values represent weight percentage of a family of fatty acids or specific acids and are listed as the mean + SEM for six experiments.
FIG. 3. Total incorporation (nmol g protein-‘) of ‘*C-18:1,18:2, or 18:3 in hepatocytes from cold (5X)or warm (2O*C~-acclimated trout assayed at 5 and 20°C. Values are mean f SEM for four to six experiments.
63
POLYUNSATURATEDFATTYACIDPRODUCTIONINTROUTHEPATOCYTES TABLE
III
RATES OF PRODUCTION AND INCORPORATION OF UNSATURATED FAW FROM 5- AND 20°C-A~~~~~~~ TROUT 181 (n-9) Reaction
20°C
ACIDS INTO HEPATOCYTES
182 (n-6) 5°C
20°C
183 (n-3) 20°C
5°C
5°C
5°C Acclimated Substrate A6 Desaturation Cl8 Elongation A5 Desaturation C20 Elongation A4 Desaturation “Dead end” elongation De nuvo acids Other families
304 * 50 9.9 * 1.7 4.2 + 1.1
13.2 f 8.2 f 7.1 +
3.4 2.8 2.0
137 + 16 6.3 f 0.84” 2.5 IL 0.6Sb
8.6 k 3.1 k 4.9 f
1.2” 1.1 1.3
399 34.4 23.1 7.3 3.4 1.3
f + f k f +
29.1 + 43 + 18.6 f
89 5.5 4.3 1.1 0.48 0.26
146 20.2 15.3 4.8 1.6 0.77
6.1 22 5.0
11.4 17.1 10.5
f 24 f 2.4 f 2.1 f 0.34 k 0.13’ Y!z 0.12 k + f
2.2’ 6.9 1.7
257 50.1 33.2 18.4 9.6 3.7
+44 + 6.5 + 4.8 * 2.0 k 1.4 2 0.68
16.7 f 15.4 + 9.3 f
3.7 2.6 1.2
175 62.2 46.2 25.9 11.3 8.2
* 37 + 7.1 k 4.9 2 1.5 f 1.2 f 1.0”
10.0 + 12.2 * 7.5 +
0.92Q 3.5 1.2
20°C Acclimated Substrate A6 Desaturation Cl8 Elongation A5 Desaturation C20 Elongation A4 Desaturation “Dead end” elongation De nova acids Other families
283 + 79 10.8 k 1.0” 4.3 iz 0.18”
29.9 f 4.8 k 12.5 +
5.1’ 0.34 2.6
145 f 18 5.3 It 0.60 1.7 + 0.30
11.9 + 1.9 + 5.0 k
2.0 0.11 0.83
417 29.0 15.6 5.6 2.8 1.2
k 107 f 4.8 k 2.5 ” 0.45 f 0.34” f 0.30
38.9 k 6.2 2 14.7 f
8.0f 1.4 3.2
198 14.5 9.6 3.7 1.8 0.89 13.7 3.0 6.1
f 42 * 1.1 f 0.73 CL 0.45 + 0.34 f 0.28 * 2 +
2.1 0.62 0.72
415 64.0 49.2 20.3 9.1 4.9
+ 98 f 9.2 2 7.6 f 2.2 + 1.0 f 0.44d
38.4 k 19.7 + 18.7 5
9.5’ 5.8 3.9
192 42.2 28.7 14.0 4.2 2.6
+ 23 + 5.2 k 4.0 + 2.5 + 0.38 + 0.30
20.2 f 6.4 + 10.2 +
3.1 0.51 1.9
Note. Rates of incorporation (nmol (g protein. h)-‘) of radioactivity into substrate, derivatives, de nouo fatty acids, and acids of other families in hepatocytes assayed at 5 and 20°C. Values for production of derivatives were calculated as described under Experimental Procedures, and are listed as mean f SEM for three to four experiments (5°C acclimated) or four to five experiments (2O’C acclimated). Identical superscripts denote statistically different values: ‘-‘P < 0.05; 0 P < 0.10.
Since it has been reported that 18-carbon substrates which have been chain elongated can undergo A5 desaturation (20), it was necessary to confirm that the “dead end” elongation products were not desaturated, which would lead to an incorrect estimation of desaturation rates. For example, 20:3 (n - 6), assumed to be the Cl8 elongation product, 68, 11, 14, could actually be the A5, 11,14 isomer resulting from A5 desaturation of the “dead end” elongation product. Upon degradation of the corresponding acid in each family (20:2, n - 9; 20:3, n - 6; and 20:4, n 3), ~5% of the label was found in the C5 fragment. Other carboxyl-terminal fragments of 4 and 6 carbons were labeled to a minor extent (data not shown). Therefore, little modification of the “dead end”
elongation product was occurring in either acclimation group. In cells from both acclimation groups, rates of derivative production (desaturation and elongation) increased with increasing degree of unsaturation of the substrate (Table III); this trend was independent of assay temperature. Rates of derivative production at 20°C were higher than rates at 5°C (Q10= 1.3-1.8), except for metabolism of linolenic acid by cells from cold-acclimated fish. In this case, rates at 5°C were equal to or greater than rates at 20°C (Q10 < 1). Furthermore, Qlo values decreased with succeeding desaturation steps (A6, A5, A4 = 0.87, 0.80, 0.59, respectively), indicating an augmented production of the more polyunsaturated derivatives of linolenic acid at 5°C.
SELLNER
64
Rates of derivative production in cells from 5°C-acclimated trout at 5°C can be compared to those in cells from 20°C trout at 20°C to assess the steady-state levels of PUFA production in fully acclimated fish. Rates of derivative production from 181 were significantly (P < 0.05) lower in cold-acclimated fish. Rates of derivative production from 182 were consistently less in cold-acclimated fish, but only significantly so for the C20 elongation product. Rates of 183 derivative production in 5”C-acclimated trout were equal to or greater than rates in warm-acclimated fish, and the rate of A4 desaturation in cold-acclimated fish was significantly (P < 0.05) higher than in warm-acclimated fish. For a given assay temperature, rates of incorporation of ‘*C into de nouo synthesized fatty acids were greater in cells from cold-acclimated trout. Rates of formation of the “dead end” elongation product were significantly greater in cells from warmacclimated fish assayed at 20°C than coldacclimated fish assayed at 5°C. The percentage of incorporated radioactivity recovered in desaturation and elongation products is given in Table IV. An increase in utilization of the substrate with increasing degree of unsaturation is apparent; this trend was more pronounced in cells from cold- than warm-acclimated
TABLE
IV
PERCENTAGE OF INCO~ORATED RADIOA~IV~Y RECOVERED IN DESATURATION AND ELONGATION PRODUCTS Substrate Temperature Acclimation
20 5
(“C) Assay
l&l (n-9)
182 (n-6)
18:3 (n-3)
20 5 20 5
3.15 4.24 2.92 3.92
2.93 4.75 6.57 9.30
10.89 13.35 15.00 23.05
Note. Values represent the amount of radioactivity recovered in substrate derivatives divided by the total amount incorporated, and are the means of three to five experiments
AND HAZEL
animals at both assay temperatures. Production of derivatives was greater when assayed at 5°C than 20°C in all cases, though the differences were greater in cells from cold-acclimated trout. Cells from warm-acclimated fish appeared equally able to form derivatives from 181 and less able to metabolize 182 and 183 than cells from cold-acclimated fish. DISCUSSION
~epatocytes isolated from rainbow trout have been used in this laboratory to investigate lipogenesis (10, 11) and, in this study, PUFA metabolism. The results presented here indicate that even after several hours of incubation, cell viability is quite high (6874%) and overall morphology is preserved (Fig. 2). Although the micrographs presented here are all from tissues of cold-acclimated trout, previous studies with intact liver (21) have shown no ultrastructural differences between 5and 18”C-acclimated animals except for greater development of Golgi complexes in the former group. Qualitatively, there appears to be no depletion of glycogen stores during incubation, and no evidence of membrane damage (Fig. 2). Trout exhibit the same substrate preferences for desaturation and elongation reactions as mammals, i.e., rates of these reactions increase with increasing degree of unsaturation of the substrate (Table III). Maximal activities with acids of the n - 3 family may be of special significance in trout, as linolenic acid has been shown to be an essential fatty acid in this species (22). It is therefore not surprising that in hepatocyte lipids from cold- and warmacclimated trout, linoleie acid and its derivatives constitute only 15.4 and 14.6% by weight, respectively, while linolenic acid and derivatives comprise 34.5 and 27.0%, respectively (Table II). Both aeclimation groups have twice the amount of n - 3 acids as n - 6 acids, but cold acclimation results in higher proportions of linolenic acid derivatives. Production of n - 3 derivatives at 5°C is more efficient in cells from cold- than warm-acclimated trout (Table IV), as 23%
POLYUNSATURATEDFATTYACIDPRODUCTIONINTROUTHEPATOCYTES
of the incorporated radioactivity was recovered in desaturation and elongation products in the former group, and only 14% in the latter. This higher efficiency of production of linolenic acid derivatives, and the inverse sensitivity of these reactions to assay temperature in cells from cold-acclimated trout (Table III) may explain the accumulation of n - 3 acids in 5°C trout, and suggest that specialized mechanisms exist to augment the proportions of n - 3 derivatives in these animals. Levels of PUFA production in cells from 5°C trout compared with 20°C trout (Table III) show various degrees of compensation (23) when assayed at the respective acclimation temperatures. Rates of desaturation and elongation of l&l show almost no compensation, as rates in cells from 5”C-acclimated fish assayed at 5°C are significantly lower than those in cells from 20’Cacclimated fish at 20°C. Rates of derivative production from 18:2 in cold-acclimated trout are equal to or less than those in warm-acclimated trout, indicating partial compensation. With 18:3 as the substrate, rates in cold-acclimated fish are equal to or greater than rates in warmacclimated fish, demonstrating perfect compensation for most desaturation and elongation reactions, and overcompensation of the A4 desaturation step. These results are consistent with composition data, i.e., cold acclimation results in a significant decrease in n - 9 acids, little change in n - 6 acids, and a significant increase in n - 3 acids (Table II). The changes in desaturation and elongation rates can therefore account in part for the increased unsaturation of tissue lipids in 5°C trout. These trends in compensation are further reflected in the weight percentage of individual fatty acids (Table II). 20:4 and 22:5 (n - 6), the A5 and A4 desaturation products of l&2, respectively, are the major derivatives of linoleic acid found in trout. These acids show little difference in composition (Table II) or rates of production (Table III) between acclimation groups. In contrast, 20:5 and 22:6 (n - 3), the analogous derivatives of linolenic acid, show significant increases in composition with cold acclimation (Table II), consis-
65
tent with the perfect to overcompensation observed above. However, rates of production of these acids do not parallel the composition data, since much less 22:6 is produced than 20:5 (Table III). While the significantly higher rate of production of 22:6 in cold- than warm-acclimated fish (8.16 vs 4.92 nmol (g protein X h)-‘, Table III) can potentially account for the relative changes in levels of this acid which accompany cold acclimation, the rates seem insufficient to account for the high tissue levels characteristic of both acclimation groups. The efficiency of 22:6 production has been demonstrated in trout fed [14C]linolenic acid; 6 days after administration, nearly ‘70% of recovered radioactivity in tissue fatty acids was found in 22:6, while only 5% was found in 20:5 (24). Low mass levels of 20:5 and high levels of 22:6 could arise from selective long-term retention of the longer chain polyunsaturates, as has been observed in tissues of coho salmon (25). Another mechanism to ensure high production of polyunsaturates at a low temperature would be to increase the proportion of desaturation or elongation product that goes on to the next reaction in the sequence. For example, in forming derivatives of linoleic acid in cells of coldacclimated trout, 67% of the A&desaturation products continue to the Cl8 elongation step (23.1134.4, Table III) at 20°C; in the 5°C assay, the percentage is raised to ‘76%. In both acclimation groups, the proportion of the A5-desaturation products undergoing the C20 elongation step always decreases at the lower assay temperature, while the proportion of the C20 elongation products undergoing the A4 desaturation always increases, to as muoh as 73% (8.2/11.3, Table III) in cells from cold-acclimated trout metabolizing l&3. Certain of these desaturation and elongation reactions thus appear to be “linked” so that there is a greater likelihood that some products will be formed than others. These results may explain why 20:5 and 22:6 (n - 3), the A5- and A4desaturation products, are present in higher amounts and 22:5 (n - 3), the C20 elongation product, is present in lower
66
SELLNER
amounts in lipids from cold- than warmacclimated trout (Table II, Ref. (4)). Assessing the role of desaturase enzymes in mediating cold adaptation in trout involves at least three factors. First, the substrate preference of the desaturation and elongation enzymes is biased toward the production of derivatives from the most unsaturated fatty acids in both acclimation groups, regardless of assay temperature. Second, the degree of thermal compensation of rates is maximal with the n - 3 family, but depends on the specific reaction: A6 desaturation shows incomplete compensation in the n - 6 family but perfect compensation in the n - 3 family, and A4-desaturation rates illustrate overcompensation with 18:3 but not 18:2. Third, the possible “link” between reactions as affected by assay temperature also serves to produce certain polyunsaturated derivatives and not others. However, the compensation of desaturase activities, particularly for metabolizing linolenic acid and its derivatives by coldacclimated trout, appears to be an important mechanism for mediating and/or maintaining cold adaptation. Higher proportions of polyunsaturated fatty acids in cold-acclimated trout could also result from (i) selective incorporation into particular lipid classes (Sellner and Hazel, submitted for publication) or (ii) different turnover rates for molecular species of lipids. ACKNOWLEDGMENTS The cooperation of the U. S. Fish and Wildlife Service and the personnel of the Alchesay Trout Hatchery is greatly appreciated. REFERENCES 1. HAZEL, J. R., AND SEWER, P. A. (1980) in Animals and Environmental Fitness (Gilles, R., ed.), pp. 541-560, Pergamon Press, Oxford. 2. SINENSKY, M. (1974) Proc Nat. Ad Sci USA 71, 522-525.
AND
HAZEL
3. HAZEL, J. R. (1979) J. Exp. Zool 207,33-42. 4. HAZEL, J. R. (1979). Amer. J. Physiol 236, R91RlOl. 5. OKUYAMA, H., YAMADA, K., KAMEYAMA, Y., IKEZAWA, H., AKAMATSU, Y., AND NOJIMA, S. (1977) Biochemistry 16,2668-2673. 6. FUJII, D. K., AND FULCO, A. J. (1977) J. BioL Chem 252,3660-36’70. 7. NOZAWA, Y., AND KASAI, R. (1978) Biochim Bie phys Actu 529.54-66. 8. NINNO, R. E., DE TORRENGO, M. A. P., CASTUMA, J. C., AND BRENNER, R. R. (1974) B&him Bie phys. Acta 360,124-133. 9. BRENNER, R. R. (1977) in Function and Biosynthesis of Lipids (Bazan, N. G., Brenner, R. R., and Giusto, N. M., eds.), pp. 85-101, Plenum, New York. 10. HAZEL, J. R., AND SELLNER, P. A. (1979) J. Exp. zoo1 209,105-114. 11. HAZEL, J. R., AND PROSSER, C. L. (1979) J. Camp. Physiol 134,321-329. 12. WALTON, M. J., AND COWEY, C. B. (1979) Cump. Biochem Physid 62B. 75-79. 13. BERRY, M. N., AND FRIEND, D. S. (1969) J. Cell Biol. 43, 506-520. 14. BLIGH, E. G., AND DYER, W. J. (1959) Canad J. Bioch.em Physid 37,911-917. 15. MORRISON, W. R., AND SMITH, L. M. (1964) J. Lipid Res. 5,600~608. 16. VON RUDLOFF, E. (1956) Caned J. Chem 34,14131418. 17. HOLBROOK, J. J., LIWAS, A., STEINDEL, S. J., AND ROSSMAN, M. G. (1975) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 11, pp. 191-258, Academic Press, New York. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL., R. J. (1951) J. Biol Chmn 193, 265-275. 19. ZAR, J. H. (1974) Biostatistical Analysis, pp. 101107, Prentice-Hall, Englewood Cliffs, N. J. 20. BERNERT, J. T., Jr., AND SPRECHER, H. (1975) Biochim Biophys. Ada 398,354-363. 21. BERLIN, J. D., AND DEAN, J. M. (1967) J. Exp. Zool 164,117-132. 22. CASTELL, J. D., SINNHUBER, R. O., AND WALES, J. H. (1972) J. N&r. 102,77-86. 23. PROSSER, C. L. (1973) in Comparative Animal Physiology (Prosser, C. L., ed.), 3rd ed., pp. 362-428, Saunders, Philadelphia. 24. OWEN, J. M., ADRON, J. W., MIDDLETON, C., AND CoWEY, C. B. (1975) Lip& 10, 528-531. 25. PARKER, R. S., SELIVONCHICK, D. P., AND SINNHUBER, R. 0. (1980) Lipids 15.80-85.
Desaturation
and Elongation of Unsaturated Fatty Acids in Hepatocytes from Thermally Acclimated Rainbow Trout’ PEGGY
A. SELLNER’
I)epartment Of &do@% Arizxnm
AND JEFFREY State
U%&mi&
R. HAZEL
Tempe,
Ariama
85.287
Received April 3,1981, and in revised form August 6,198l Hepatocytes isolated from 5- and 2O”C-acclimated rainbow trout (Salmo g&&zeri) were incubated with [1-‘*C]oleic, -linoleic, or -1inolenic acid to investigate the role of desaturation and elongation pathways in cold adaptation. Cells from both acclimation groups demonstrated a substrate preference in the order: 13~3 > l&2 > 13~1. When assayed at 5”C, cells from cold-acclimated trout were more efficient at producing derivatives of linolenic acid than cells from warm-acclimated trout. Cold acclimation resulted in degrees of compensation of desaturation and elongation rates that depended on the fatty acid family and specific reaction in the sequence. In the oleic acid family, reaction rates showed no compensation; partial compensation was observed in the linoleie acid family, and perfect (e.g., A6 desaturation) to overcompensation (e.g., A4 desaturation) in the linolenic acid family. Cells from cold-acclimated trout had higher rates of produetion of linolenicderived acids when assayed at 5 than 20°C. The proportion of the product of a given desaturation or elongation reaction that continued to the next reaction in the sequence was higher when assayed at 5 than 2O”C, increasing the probability of producing polyunsaturates. The results suggest that the substrate preference.of the desaturation and elongation enzymes, the compensation of reaction rates, and the possible “link” between certain reactions in the metabolic sequence may ensure the production of polyunsaturated fatty acids in cold-adapted trout.
One response of poikilotherms to changing environmental temperatures is the alteration of membrane lipid composition such that the degree of unsaturation of fatty acids is inversely related to the acclimation temperature (1). This has been termed a “homeoviscous adaptation” (2) and is presumed essential to preserving normal membrane function, The restructuring of lipid is not limited to membrane components, however, as changes in degree of unsaturation have been reported for neutral lipids (3) as well as phospholipids (4) in liver tissue of thermally acclimated rainbow trout. While the metabolic basis for increasing unsaturation is not entirely clear, there is
evidence that cold-adapted organisms have higher rates of production of unsaturated acids than warm-adapted organisms. This has been demonstrated for both procaryotes, which produce primarily monoenes, and eucaryotes, which generally increase unsaturation by incorporating polyunsaturates. For example, in E. co&, the ratio of oleie to palmitic acid produced by the fatty acid synthetase complex increased when assayed at 10°C compared to 40°C (5). Transfer of Bacillus megaterium cultures from 35 to 20°C caused a hyperinduction of synthesis of the A5-desaturase enzyme (6). Similar observations have been made with Tetrahymena, in which the activity of the palmitoyl CoA desaturase (A9) increased upon shifting the culture from 39.5 to 15°C (7). In the fish Pimebdus, A9-, A&, and A5-desaturase activities were higher in 15 than 30°C acclimated animals when assayed at the
’ Supported by NSF Grant PCM76-04313-AOl. 2 Present address: Department of Neurosurgical Research, Massachusetts General Hospital, Boston, Mass. 02114. 58
59
POLYUNSATURATEDFATTYACIDPRODUCTIONINTROUTHEPATOCYTES PARENT
FATTY
ACID
18:2 CHAIN ELONGATION 1 0:2 ~6 - DESATURATION . 18.3 ELONGATION
TO
20-C
ACID
22-C
ACID
I 20~3
Ah5- DESATURATION I 20~4 ELONGATION
TO
I 22~4
use of isolated hepatocyte preparations has several distinct advantages for studies of metabolism, including: (i) the availability of a homogeneous cell suspension from which many experimental aliquots can be apportioned; (ii) the diffusion of oxygen or substrates is not limiting, as is frequently the case with liver slices (13); and (iii) the study of regulatory interactions between reactions that take place in different subcellular compartments, but which compete for a common substrate, is possible. EXPERIMENTAL
A4-
DESATURATION
I 22:5
FIG. 1. Desaturation and elongation sequence. Linoleic acid (182, n - 6) and its derivatives are included as examples. C18, C20, and chain elongation reactions are named to distinguish between products and not to imply distinct enzymes. The phrase “dead end” denotes chain elongation of the substrate and incorporation without further metabolism of the acid.
same temperature (8). Thus, enhanced rates of desaturation may account for some of the fatty acid composition changes which accompany cold adaptation. In this study, the effects of thermal acclimation and assay temperature on fatty acid desaturation and elongation were determined in 5- and ZO”C-acclimated rainbow trout (Salmo gairdneri). Isolated hepatocytes were incubated with [l-‘4C]oleic, -linoleic, or -1inolenic acid, the parent acids of the three major families of polyunsaturated fatty acids (PUFA3) found in eucaryotes. These three fatty acids enter the desaturation and elongation sequence (Fig. 1) at the same step, i.e., A6 desaturation, which is known to be an important regulatory step in mammalian PUFA synthesis (9). Hepatocytes from rainbow trout have proved useful in studies of lipogenesis (10, 11) and gluconeogenesis (12). The 3Abbreviations used: PUFA, polyunsaturated fatty acids; BSA, bovine serum albumin; PPO, 2,5diphenyloxazole; POPOP, 1,4-bis[2(5-phenyloxazo1yl)jbenzene; PL, phospholipid; FFA, free fatty acid; NL, neutral lipid; GLC, gas-liquid chromatography.
PROCEDURES
Animals. Rainbow trout (Salmo gairdneri) were obtained from the Alchesay Fish Hatchery in Whiteriver, Arizona, and were maintained on pelleted trout food from Glencoe Mills, Minneapolis, Minnesota. The fatty acid composition of this diet has been reported elsewhere (4). Fish were acclimated to 5 or 20°C and a 12/12 photoperiod for at least 1 month prior to sacrifice. Preparation and incubation of hepatocgtaHepatocytes were prepared from 100 to 400-g animals by in situperfusion as described previously (11). In each experiment, hepatocytes obtained from two fish of the same acclimation group were pooled and taken up in 5-6 ml of Krebs-Ringer-imidazole buffer, pH 7.8 at 20°C containing 2% (w/v) bovine serum albumin (BSA) and 10 rnM glucose (Buffer A). Trypan blue was excluded by r95% of these freshly isolated cells. A small aliquot of the cell suspension was reserved for fatty acid analysis. One-milliliter portions of the cell suspension (6-10 mg protein) were incubated with [l-i%] oleic, -linoleic, or-linolenic acid (1.5 pCi in a total of ‘75 nmol, added in 5 ~1 ethanol) for 5 h at 20°C or 9 h at 5°C. Flasks were gassed continuously with Oz/COz (95/5) and effluent gas from each flask was bubbled through 1.5 ml of methylbenzethonium hydroxide to trap COz. At the end of the experiment, the contents of the COz trap were taken up in 10 ml of scintillation cocktail A (6 g PPO + 0.1 g POPOP per liter of toluene) and counted to estimate the extent of j3 oxidation. Incubations were terminated by diluting the cell suspensions with 3 ml of ice-cold Buffer A and immediately collecting the cells by centrifugation. Lipids were extracted from the cell pellet by the method of Bligh and Dyer (14). Ten percent of the total lipid extract was applied to a Silica Gel G plate which was developed in hexane/diethyl ether/formic acid (80/ 20/2) to separate phospholipid (PL), free fatty acid (FFA), and neutral lipid (NL). The regions of gel containing these lipids were scraped directly into scintillation vials and counted in 10 ml of cocktail B (6
60
SELLNER
g PPO + 0.1 g POPOP per liter of toluene/2-ethoxyethanol, 2/l) to determine total incorporation into lipid classes. The remaining 90% of the lipid extract, to be used for radio-gas-liquid ehromato~aphy, was separated by thin-layer chromatography in an identical manner, but the lipids were eluted from the gel with chlorform/methanol (Z/l). Fatty acid analyses. All lipid analyses were performed under nitrogen to prevent oxidation of the fatty acids. Lipid samples in chloroform/methanol were evaporated to dryness and saponified with 0.2 ml of 30% aqueous KOH in 5 ml of ethanol at 80°C for 2 h. Fatty acids were extracted with light petroleum ether following acidification with 1.5 ml of 6 N hydrochloric acid. Fatty acyl methyl esters were then prepared with 14% boron trifluoride in methanol (15), and separated by gas-liquid chromato~aphy on 10% EGSS-X on Gas-Chrom P using a HewlettPackard Model 534OA chromatograph equipped with an effluent splitter adjusted to a 91 split ratio. During each run, the column was heated from 130 to 205’C using a time-temperature program. Radioactive methyl esters were collected on 3-cm Estron filter rods and counted directly in 10 ml of scintillation cocktail A. In some cases, positions of the double bonds in a fatty acid were determined by eluting the methyl ester from the Estron filter rod with hexane and subjecting it to permanganate-periodate degradation according to von Rudloff (16). Electron rn~~os~~. Throughout the preparation of the samples for electron microscopy, isolated hepatoeytes were collected by gravitation rather than centrifugation to minimize cell damage. Small blocks of intact liver (taken before perfusion) and pellets of isolated hepatocytes taken before and after incubation (without added fatty acid) were fixed in Cazffree Krebs-Ringer-phosphate buffer (pH 7.4, containing 2% formaldehyde and 3% glutaraldehyde) for 1 h on ice. Samples were washed with buffer, postfixed on ice in buffered 1% 0~0~ for an additional hour, and washed again in buffer. Hepatocytes were encapsulated in 2% agar, and small blocks of suspended cells as well as blocks of liver were dehydrated in an ethanol series. All blocks were embedded in Epon/Araldite (l/l). Sections were cut with a glass knife on a Porter-Blum MT-l, stained with uranyl acetate and lead citrate, and viewed on a Philips EM 201 instrument. Analytical methods. Counts per minute were converted to disintegrations per minute using the external standard ratio and a quench calibration curve. Nanomoles were calculated (assuming no dilution of label by endogenous FFA) from the known specific activity of the substrate mixture, which was determined by counting a small aliquot. Lactate dehydrogenase assays to estimate cell viability were performed as described by Holbrook et ah (17). Protein was determined according to Lowry et al. (18) using
AND HAZEL BSA as a standard. Statistical analyses consisting of Student’s t test were performed according to Zar (19). ~~~~. [1-‘*C]Oleie (56 mCi/mmol), -1inoleic (60 mCi/mmol), and -1inolenic (60 mCi/mmol) acids were obtained from the Radiochemical Centre, Amersham, England. Nonradioactive oleic, linoleic, and linolenic acids were purchased from Sigma. Purities of the “cold” and “hot” fatty acids, determined by radioGLC analysis of methyl esters prepared from the supplied fatty acids, were: oleic, 99%, 97%; linoleic, 97%, 97%; and linolenic, 98%, 92%, respectively. Collagenase (Type IV, Chtridium histdyticum), hyaluronidase {Type I, bovine testes), bovine serum albumin (Fraction V), nicotinamide adenine dinueleotide (reduced form), methylbenzethonium hydroxide, PPO, and POPOP were also from Sigma. EGSS-X on Gas-Chrom P, Silica Gel G, and 14% boron trifluoride in methanol were from Applied Science Laboratories, and pyruvic acid was obtained from Eastman Chemicals. Estron filter rods were from the American Filtrona Company. RESULTS
Rates of LDH activity in freshly isolated cells were 1.34 k 0.26 and 1.33 f 0.30 gmol (min X mg)-’ from cold- and warm-acclimated fish, respectively. Hepatocyte viability (computed as the percentage of zerotime LDH activity) in both acclimation groups averaged 68% after 5 h at 20°C and 71-74s after 9 h at 5°C. Viability was also assessed by comparing the ultrastructure of isolated hepatocytes with intact liver (Fig. 2). Isolated cells (Fig. 2B) closely resembled in morphology cells of the intact liver (Fig. 2A). Surrounding the prominent nucleus were several mitochondria and large amounts of endoplasmic reticulum; discrete regions of glycogen granules were located toward the periphery of the cells. This pattern of organization remained unchanged after 5 h of incubation at 20X (Fig. 2C) or 9 h at 5°C (Fig. ZD), and there was no significant depletion of glycogen. The extent of ,kI oxidation, estimated from the amount of radioactivity recovered in the COZ trap, was always less than 3% of the total incorporated radioactivity. Absolute rates of l*C02 production (data not shown) in cells from warm-acclimated compared with cold-
POLYUNSATURATED
FATTY ACID PRODUCTION
IN TROUT HEPATOCYTES
61
FIG. 2. Ultrastructure of liver tissue and isolated hepatocytes from 5’C-acclimated trout. (A) Section of liver (x5700); (B) freshly isolated cell (X8660); (C) cells after incubation at 20°C for 5 h (x9100); (D) after incubation at 5’C for 9 h (x7400). n, nucleus; m, mitochondria; er, endoplasmic reticulum; g, glycogen; pm, plasma membrane.
acclimated fish were approximately 20 and 50% lower in the 20 and 5°C assays, respectively. Normalized values (Table I) indicated a tendency to degrade 182 to a greater extent than l&l or l&3, regardless of assay or acclimation temperature. The fatty acid composition of hepatocyte lipids is indicated in Table 11. Saturated and monounsaturated fatty acids were more abundant in ceils from warm-
acclimated trout. Polyunsaturated fatty acids of the linolenic acid (n - 3) family predominated over the linoleic acid (n - 6) family; the proportions of both families were greater in hepatocytes of coldacclimated fish. The total amount of exogenous fatty acid incorporated (nmol (g protein)-‘) averaged 30-40s of that supplied to the cells. Incorporation was nearly equivalent
SELLNER
AND HAZEL
TABLE
Temperature
(“C)
Acclimation
Substrate Assay
l&l (n-9)
20 5 20 5
1.00 0.19 f 0.04 1.00
20 5
in each experiment
when measured after 5 h at 20°C or 9 h at S’C, regardless of acclimation temperature or substrate (Fig. 3). The tendency for greater incorporation of substrate with increasing degree of unsaturation was only significant in cells from warm-acclimated fish assayed at 5°C (l&l < l&3, P < 0.05). Incorporation of isotope into each fatty acid was determined by radio-GLC analysis, and rates expressed as nmol (g protein X h)-l. Total incorporation of a given fatty acid was calculated by summing rates of incorporation into all three lipid classes (PL, FFA, NL). The rate of a given reaction (desaturation or elongation) was TABLE
II
FATTY ACID COMPOSITIONOF ISOLATED HEPATOCYTESFROM~PANDWGACCLIMATED RAINBOWTROUT Temperature
Saturated Monounsaturated n-9 Family n-6 Family n-3 Family 20:4 22.5 m5 22~6
(n-6) (n-6) (n-3) (n-3)
23.9 +. 1.99 21.9 + 1.88 15.4 + 1.08 34.5 f 1.56 4.4 + 0.43
1.1 f 0.37 3.4 k 0.89 24.3 k 0.75
183 (n-3)
1.72 + 0.60
0.73 f
0.43 -t 0.10 3.00 -t 0.96 1.21 2 0.27
0.23 It 0.04
to 29°C assay, l&l
0.28
1.12 k 0.18 0.96 2 0.35
= 1.00. Values represent
then calculated by summing total incorporation into the reaction product plus incorporation into subsequent fatty acids according to the scheme in Fig. 1. For example, in the linoleic acid family, the rate of A5 desaturation was approximated by summing rates of isotope incorporation into 20:4, 22:4, and 22:5, since A5 desaturation is required for the formation of the latter two acids. The results (Table III) include rates of incorporation of the unmetabolized substrate, and rates of incorporation of label into fatty acids synthesized de now and into fatty acids of other families. Radioactivity in the latter two groups presumably reflects the incorporation of labeled acetyl-CoA derived from ,f3oxidation of the substrate. & z .E 2 P
(“C)
5 25.8 + 1.16
182 (n-6)
1.02 + 0.23
ncnm from each CO, flask were normalized mean t SEM for four or five experiments.
Fatty acid/ family
I
8
Y-ACCLIMATED 18~1 18~2 18~3
20*-ACCLIMATED i8:3 18~1 182
20 28.7 36.2 24.3 14.6 27.0 3.5
2 + & zk
2.81 2.34 1.76 0.79
f 1.59 c!z 0.13
1.1 rl: 0.28 1.6 zk 0.18 20.8 + 1.06
AWe. Values represent weight percentage of a family of fatty acids or specific acids and are listed as the mean + SEM for six experiments.
FIG. 3. Total incorporation (nmol g protein-‘) of ‘*C-18:1,18:2, or 18:3 in hepatocytes from cold (5X)or warm (2O*C~-acclimated trout assayed at 5 and 20°C. Values are mean f SEM for four to six experiments.
63
POLYUNSATURATEDFATTYACIDPRODUCTIONINTROUTHEPATOCYTES TABLE
III
RATES OF PRODUCTION AND INCORPORATION OF UNSATURATED FAW FROM 5- AND 20°C-A~~~~~~~ TROUT 181 (n-9) Reaction
20°C
ACIDS INTO HEPATOCYTES
182 (n-6) 5°C
20°C
183 (n-3) 20°C
5°C
5°C
5°C Acclimated Substrate A6 Desaturation Cl8 Elongation A5 Desaturation C20 Elongation A4 Desaturation “Dead end” elongation De nuvo acids Other families
304 * 50 9.9 * 1.7 4.2 + 1.1
13.2 f 8.2 f 7.1 +
3.4 2.8 2.0
137 + 16 6.3 f 0.84” 2.5 IL 0.6Sb
8.6 k 3.1 k 4.9 f
1.2” 1.1 1.3
399 34.4 23.1 7.3 3.4 1.3
f + f k f +
29.1 + 43 + 18.6 f
89 5.5 4.3 1.1 0.48 0.26
146 20.2 15.3 4.8 1.6 0.77
6.1 22 5.0
11.4 17.1 10.5
f 24 f 2.4 f 2.1 f 0.34 k 0.13’ Y!z 0.12 k + f
2.2’ 6.9 1.7
257 50.1 33.2 18.4 9.6 3.7
+44 + 6.5 + 4.8 * 2.0 k 1.4 2 0.68
16.7 f 15.4 + 9.3 f
3.7 2.6 1.2
175 62.2 46.2 25.9 11.3 8.2
* 37 + 7.1 k 4.9 2 1.5 f 1.2 f 1.0”
10.0 + 12.2 * 7.5 +
0.92Q 3.5 1.2
20°C Acclimated Substrate A6 Desaturation Cl8 Elongation A5 Desaturation C20 Elongation A4 Desaturation “Dead end” elongation De nova acids Other families
283 + 79 10.8 k 1.0” 4.3 iz 0.18”
29.9 f 4.8 k 12.5 +
5.1’ 0.34 2.6
145 f 18 5.3 It 0.60 1.7 + 0.30
11.9 + 1.9 + 5.0 k
2.0 0.11 0.83
417 29.0 15.6 5.6 2.8 1.2
k 107 f 4.8 k 2.5 ” 0.45 f 0.34” f 0.30
38.9 k 6.2 2 14.7 f
8.0f 1.4 3.2
198 14.5 9.6 3.7 1.8 0.89 13.7 3.0 6.1
f 42 * 1.1 f 0.73 CL 0.45 + 0.34 f 0.28 * 2 +
2.1 0.62 0.72
415 64.0 49.2 20.3 9.1 4.9
+ 98 f 9.2 2 7.6 f 2.2 + 1.0 f 0.44d
38.4 k 19.7 + 18.7 5
9.5’ 5.8 3.9
192 42.2 28.7 14.0 4.2 2.6
+ 23 + 5.2 k 4.0 + 2.5 + 0.38 + 0.30
20.2 f 6.4 + 10.2 +
3.1 0.51 1.9
Note. Rates of incorporation (nmol (g protein. h)-‘) of radioactivity into substrate, derivatives, de nouo fatty acids, and acids of other families in hepatocytes assayed at 5 and 20°C. Values for production of derivatives were calculated as described under Experimental Procedures, and are listed as mean f SEM for three to four experiments (5°C acclimated) or four to five experiments (2O’C acclimated). Identical superscripts denote statistically different values: ‘-‘P < 0.05; 0 P < 0.10.
Since it has been reported that 18-carbon substrates which have been chain elongated can undergo A5 desaturation (20), it was necessary to confirm that the “dead end” elongation products were not desaturated, which would lead to an incorrect estimation of desaturation rates. For example, 20:3 (n - 6), assumed to be the Cl8 elongation product, 68, 11, 14, could actually be the A5, 11,14 isomer resulting from A5 desaturation of the “dead end” elongation product. Upon degradation of the corresponding acid in each family (20:2, n - 9; 20:3, n - 6; and 20:4, n 3), ~5% of the label was found in the C5 fragment. Other carboxyl-terminal fragments of 4 and 6 carbons were labeled to a minor extent (data not shown). Therefore, little modification of the “dead end”
elongation product was occurring in either acclimation group. In cells from both acclimation groups, rates of derivative production (desaturation and elongation) increased with increasing degree of unsaturation of the substrate (Table III); this trend was independent of assay temperature. Rates of derivative production at 20°C were higher than rates at 5°C (Q10= 1.3-1.8), except for metabolism of linolenic acid by cells from cold-acclimated fish. In this case, rates at 5°C were equal to or greater than rates at 20°C (Q10 < 1). Furthermore, Qlo values decreased with succeeding desaturation steps (A6, A5, A4 = 0.87, 0.80, 0.59, respectively), indicating an augmented production of the more polyunsaturated derivatives of linolenic acid at 5°C.
SELLNER
64
Rates of derivative production in cells from 5°C-acclimated trout at 5°C can be compared to those in cells from 20°C trout at 20°C to assess the steady-state levels of PUFA production in fully acclimated fish. Rates of derivative production from 181 were significantly (P < 0.05) lower in cold-acclimated fish. Rates of derivative production from 182 were consistently less in cold-acclimated fish, but only significantly so for the C20 elongation product. Rates of 183 derivative production in 5”C-acclimated trout were equal to or greater than rates in warm-acclimated fish, and the rate of A4 desaturation in cold-acclimated fish was significantly (P < 0.05) higher than in warm-acclimated fish. For a given assay temperature, rates of incorporation of ‘*C into de nouo synthesized fatty acids were greater in cells from cold-acclimated trout. Rates of formation of the “dead end” elongation product were significantly greater in cells from warmacclimated fish assayed at 20°C than coldacclimated fish assayed at 5°C. The percentage of incorporated radioactivity recovered in desaturation and elongation products is given in Table IV. An increase in utilization of the substrate with increasing degree of unsaturation is apparent; this trend was more pronounced in cells from cold- than warm-acclimated
TABLE
IV
PERCENTAGE OF INCO~ORATED RADIOA~IV~Y RECOVERED IN DESATURATION AND ELONGATION PRODUCTS Substrate Temperature Acclimation
20 5
(“C) Assay
l&l (n-9)
182 (n-6)
18:3 (n-3)
20 5 20 5
3.15 4.24 2.92 3.92
2.93 4.75 6.57 9.30
10.89 13.35 15.00 23.05
Note. Values represent the amount of radioactivity recovered in substrate derivatives divided by the total amount incorporated, and are the means of three to five experiments
AND HAZEL
animals at both assay temperatures. Production of derivatives was greater when assayed at 5°C than 20°C in all cases, though the differences were greater in cells from cold-acclimated trout. Cells from warm-acclimated fish appeared equally able to form derivatives from 181 and less able to metabolize 182 and 183 than cells from cold-acclimated fish. DISCUSSION
~epatocytes isolated from rainbow trout have been used in this laboratory to investigate lipogenesis (10, 11) and, in this study, PUFA metabolism. The results presented here indicate that even after several hours of incubation, cell viability is quite high (6874%) and overall morphology is preserved (Fig. 2). Although the micrographs presented here are all from tissues of cold-acclimated trout, previous studies with intact liver (21) have shown no ultrastructural differences between 5and 18”C-acclimated animals except for greater development of Golgi complexes in the former group. Qualitatively, there appears to be no depletion of glycogen stores during incubation, and no evidence of membrane damage (Fig. 2). Trout exhibit the same substrate preferences for desaturation and elongation reactions as mammals, i.e., rates of these reactions increase with increasing degree of unsaturation of the substrate (Table III). Maximal activities with acids of the n - 3 family may be of special significance in trout, as linolenic acid has been shown to be an essential fatty acid in this species (22). It is therefore not surprising that in hepatocyte lipids from cold- and warmacclimated trout, linoleie acid and its derivatives constitute only 15.4 and 14.6% by weight, respectively, while linolenic acid and derivatives comprise 34.5 and 27.0%, respectively (Table II). Both aeclimation groups have twice the amount of n - 3 acids as n - 6 acids, but cold acclimation results in higher proportions of linolenic acid derivatives. Production of n - 3 derivatives at 5°C is more efficient in cells from cold- than warm-acclimated trout (Table IV), as 23%
POLYUNSATURATEDFATTYACIDPRODUCTIONINTROUTHEPATOCYTES
of the incorporated radioactivity was recovered in desaturation and elongation products in the former group, and only 14% in the latter. This higher efficiency of production of linolenic acid derivatives, and the inverse sensitivity of these reactions to assay temperature in cells from cold-acclimated trout (Table III) may explain the accumulation of n - 3 acids in 5°C trout, and suggest that specialized mechanisms exist to augment the proportions of n - 3 derivatives in these animals. Levels of PUFA production in cells from 5°C trout compared with 20°C trout (Table III) show various degrees of compensation (23) when assayed at the respective acclimation temperatures. Rates of desaturation and elongation of l&l show almost no compensation, as rates in cells from 5”C-acclimated fish assayed at 5°C are significantly lower than those in cells from 20’Cacclimated fish at 20°C. Rates of derivative production from 18:2 in cold-acclimated trout are equal to or less than those in warm-acclimated trout, indicating partial compensation. With 18:3 as the substrate, rates in cold-acclimated fish are equal to or greater than rates in warmacclimated fish, demonstrating perfect compensation for most desaturation and elongation reactions, and overcompensation of the A4 desaturation step. These results are consistent with composition data, i.e., cold acclimation results in a significant decrease in n - 9 acids, little change in n - 6 acids, and a significant increase in n - 3 acids (Table II). The changes in desaturation and elongation rates can therefore account in part for the increased unsaturation of tissue lipids in 5°C trout. These trends in compensation are further reflected in the weight percentage of individual fatty acids (Table II). 20:4 and 22:5 (n - 6), the A5 and A4 desaturation products of l&2, respectively, are the major derivatives of linoleic acid found in trout. These acids show little difference in composition (Table II) or rates of production (Table III) between acclimation groups. In contrast, 20:5 and 22:6 (n - 3), the analogous derivatives of linolenic acid, show significant increases in composition with cold acclimation (Table II), consis-
65
tent with the perfect to overcompensation observed above. However, rates of production of these acids do not parallel the composition data, since much less 22:6 is produced than 20:5 (Table III). While the significantly higher rate of production of 22:6 in cold- than warm-acclimated fish (8.16 vs 4.92 nmol (g protein X h)-‘, Table III) can potentially account for the relative changes in levels of this acid which accompany cold acclimation, the rates seem insufficient to account for the high tissue levels characteristic of both acclimation groups. The efficiency of 22:6 production has been demonstrated in trout fed [14C]linolenic acid; 6 days after administration, nearly ‘70% of recovered radioactivity in tissue fatty acids was found in 22:6, while only 5% was found in 20:5 (24). Low mass levels of 20:5 and high levels of 22:6 could arise from selective long-term retention of the longer chain polyunsaturates, as has been observed in tissues of coho salmon (25). Another mechanism to ensure high production of polyunsaturates at a low temperature would be to increase the proportion of desaturation or elongation product that goes on to the next reaction in the sequence. For example, in forming derivatives of linoleic acid in cells of coldacclimated trout, 67% of the A&desaturation products continue to the Cl8 elongation step (23.1134.4, Table III) at 20°C; in the 5°C assay, the percentage is raised to ‘76%. In both acclimation groups, the proportion of the A5-desaturation products undergoing the C20 elongation step always decreases at the lower assay temperature, while the proportion of the C20 elongation products undergoing the A4 desaturation always increases, to as muoh as 73% (8.2/11.3, Table III) in cells from cold-acclimated trout metabolizing l&3. Certain of these desaturation and elongation reactions thus appear to be “linked” so that there is a greater likelihood that some products will be formed than others. These results may explain why 20:5 and 22:6 (n - 3), the A5- and A4desaturation products, are present in higher amounts and 22:5 (n - 3), the C20 elongation product, is present in lower
66
SELLNER
amounts in lipids from cold- than warmacclimated trout (Table II, Ref. (4)). Assessing the role of desaturase enzymes in mediating cold adaptation in trout involves at least three factors. First, the substrate preference of the desaturation and elongation enzymes is biased toward the production of derivatives from the most unsaturated fatty acids in both acclimation groups, regardless of assay temperature. Second, the degree of thermal compensation of rates is maximal with the n - 3 family, but depends on the specific reaction: A6 desaturation shows incomplete compensation in the n - 6 family but perfect compensation in the n - 3 family, and A4-desaturation rates illustrate overcompensation with 18:3 but not 18:2. Third, the possible “link” between reactions as affected by assay temperature also serves to produce certain polyunsaturated derivatives and not others. However, the compensation of desaturase activities, particularly for metabolizing linolenic acid and its derivatives by coldacclimated trout, appears to be an important mechanism for mediating and/or maintaining cold adaptation. Higher proportions of polyunsaturated fatty acids in cold-acclimated trout could also result from (i) selective incorporation into particular lipid classes (Sellner and Hazel, submitted for publication) or (ii) different turnover rates for molecular species of lipids. ACKNOWLEDGMENTS The cooperation of the U. S. Fish and Wildlife Service and the personnel of the Alchesay Trout Hatchery is greatly appreciated. REFERENCES 1. HAZEL, J. R., AND SEWER, P. A. (1980) in Animals and Environmental Fitness (Gilles, R., ed.), pp. 541-560, Pergamon Press, Oxford. 2. SINENSKY, M. (1974) Proc Nat. Ad Sci USA 71, 522-525.
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3. HAZEL, J. R. (1979) J. Exp. Zool 207,33-42. 4. HAZEL, J. R. (1979). Amer. J. Physiol 236, R91RlOl. 5. OKUYAMA, H., YAMADA, K., KAMEYAMA, Y., IKEZAWA, H., AKAMATSU, Y., AND NOJIMA, S. (1977) Biochemistry 16,2668-2673. 6. FUJII, D. K., AND FULCO, A. J. (1977) J. BioL Chem 252,3660-36’70. 7. NOZAWA, Y., AND KASAI, R. (1978) Biochim Bie phys Actu 529.54-66. 8. NINNO, R. E., DE TORRENGO, M. A. P., CASTUMA, J. C., AND BRENNER, R. R. (1974) B&him Bie phys. Acta 360,124-133. 9. BRENNER, R. R. (1977) in Function and Biosynthesis of Lipids (Bazan, N. G., Brenner, R. R., and Giusto, N. M., eds.), pp. 85-101, Plenum, New York. 10. HAZEL, J. R., AND SELLNER, P. A. (1979) J. Exp. zoo1 209,105-114. 11. HAZEL, J. R., AND PROSSER, C. L. (1979) J. Camp. Physiol 134,321-329. 12. WALTON, M. J., AND COWEY, C. B. (1979) Cump. Biochem Physid 62B. 75-79. 13. BERRY, M. N., AND FRIEND, D. S. (1969) J. Cell Biol. 43, 506-520. 14. BLIGH, E. G., AND DYER, W. J. (1959) Canad J. Bioch.em Physid 37,911-917. 15. MORRISON, W. R., AND SMITH, L. M. (1964) J. Lipid Res. 5,600~608. 16. VON RUDLOFF, E. (1956) Caned J. Chem 34,14131418. 17. HOLBROOK, J. J., LIWAS, A., STEINDEL, S. J., AND ROSSMAN, M. G. (1975) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 11, pp. 191-258, Academic Press, New York. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL., R. J. (1951) J. Biol Chmn 193, 265-275. 19. ZAR, J. H. (1974) Biostatistical Analysis, pp. 101107, Prentice-Hall, Englewood Cliffs, N. J. 20. BERNERT, J. T., Jr., AND SPRECHER, H. (1975) Biochim Biophys. Ada 398,354-363. 21. BERLIN, J. D., AND DEAN, J. M. (1967) J. Exp. Zool 164,117-132. 22. CASTELL, J. D., SINNHUBER, R. O., AND WALES, J. H. (1972) J. N&r. 102,77-86. 23. PROSSER, C. L. (1973) in Comparative Animal Physiology (Prosser, C. L., ed.), 3rd ed., pp. 362-428, Saunders, Philadelphia. 24. OWEN, J. M., ADRON, J. W., MIDDLETON, C., AND CoWEY, C. B. (1975) Lip& 10, 528-531. 25. PARKER, R. S., SELIVONCHICK, D. P., AND SINNHUBER, R. 0. (1980) Lipids 15.80-85.