Jeffrey R. Hazel

The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment

It is clear from the literature reviewed that modifications in membrane lipid composition play a major role in the adaptation of diverse organisms to specific environments and physiological circumstances. Acyl chain and molecular species restructuring in phospholipids are the most ubiquitous adaptations to environmental insult, being implicated in membrane adjustments to temperature, pressure, water activity, pH and salinity. In contrast, other adaptations (e.g. modulation of anionic phospholipids (salinity adaptation), trehalose content (dehydration) and the PC/PE ratio (temperature acclimation] appear to be more context specific. Although the volume of correlative data relating membrane composition to environmental state is impressive, several questions must be explicitly addressed in future research if a mechanistic understanding of the role of lipids in fine tuning membrane function is to be achieved. These include: (1) Adaptation thresholds--How much environmental variation is required before an acclimatory response is initiated, and is the extent of membrane perturbation induced by such minimally effective stimuli similar for different stress vectors? Interspecific comparisons of the Na+/K(+)-ATPase of fish collected at different depths indicate that species must be separated in depth by a distance corresponding to a pressure difference of 20 MPa before pressure adaptation is evident. Assuming a dT/dP value of 0.23 (Table 1), a 20 MPa change in pressure corresponds to ca. a 5 degrees C change in temperature, which agrees well with the minimal temperature change required to elicit changes in the lipid composition of plasma membranes in kidney tissue of thermally-acclimating trout. A pressure of 20 MPa also corresponds approximately to the maximum depth from which deep sea animals survive being brought to the surface. Collectively, these observations suggest that the minimally effective stimuli for both temperature and pressure adaptation are similar. Comparable data are not available for other environmental variables. (2) Signal transduction--What signals are being sensed and how are they transduced into an adaptational response? In some cases, it is clear that the enzymes of lipid metabolism respond directly (either by a variation in catalytic rate or substrate preference) to variations in the physical environment in an apparently adaptive manner (e.g. refer Sections VI.A.1 and VI.B.2). It seems unlikely, however, that such direct effects can explain the totality of the adaptive capacity of organisms, especially given the evidence for the induction of desaturase synthesis in cold adaptation (refer to Section VI.A.2).(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation of biological membranes to temperature: molecular species compositions of phosphatidylcholine and phosphatidylethanolamine in mitochondrial and microsomal membranes of liver from thermally-acclimated rainbow trout

SummaryMolecular species profiles were determined for both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of mitochondrial and microsomal membrane fractions from liver tissue of thermally-acclimated rainbow trout,Salmo gairdneri. The predominant molecular species of PC were 16:0/22:6, 16:0/18:1, 16:0/20:3 and 16:0/22:5, whereas predominant molecular species of PE were 18:1/20:4, 14:0/16:0, 18:0/22:6 and 18:1/22:6. PE possessed short chain saturates (primarily 14:0/16:0) and monoenes (primarily 14:0/16:1) not present in PC and larger proportions of polyunsaturated (18:0/22:6, 18:0/22:5 and 18:1/22:6. and diunsaturated molecular species than PC. Differences between membrane fractions were most evident in warm (20°C)-acclimated trout. Mitochondria contained higher proportions of long-chain, polyunsaturated molecular species of PE, but less of the corresponding species of PC than other membrane fractions. Rankings based on unsaturation index were accordingly: mitochondria ≥heavy microsomes>light microsomes for PE, but heavy microsomes>light microsomes>-mitochondria for PC. Mitochondria were notable for high proportions of diunsaturated molecular species of both phosphatides. Growth at cold temperatures (5°C) was generally associated with a replacement of shorter chain mono- and dienoic molecular species (16:0/18:1, 16:1/18:1, 14:0/16:2 and 18:1/18:1 in the case of PC and 14:0/16:1, 14:0/16:2 and 16:1/18:1 for PE), and occasionally saturates, with long-chain, polyunsaturated molecular species (for PC, C36–38: 16:0/22:6, 16:1/22:6, 16:0/20:3 and 16:0/20:5; for PE, C38–40: 18:1/20:4, 16:1/22:6, 18:0/20:5, 18:2/20:4, 18:0/22:5 and 18:0/22:6). However, compositions of mitochondrial PE and PC from heavy microsomes were not significantly influenced by acclimation temperature. The role of phospholipase A2, in addition to other metabolic processes, in mediating these changes is discussed.

Desaturation and elongation of unsaturated fatty acids in hepatocytes from thermally acclimated rainbow trout

Abstract Hepatocytes isolated from 5- and 20 °C-acclimated rainbow trout ( Salmo gairdneri ) were incubated with [1- 14 C]oleic, -linoleic, or -linolenic 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: 18:3 > 18:2 > 18:1. 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 linoleic acid family, and perfect (e.g., Δ6 desaturation) to overcompensation (e.g., Δ4 desaturation) in the linolenic acid family. Cells from cold-acclimated trout had higher rates of production of linolenic-derived 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 20 °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 poly-unsaturated fatty acids in cold-adapted trout.

The effects of assay temperature upon the pH optima of enzymes from poikilotherms: A test of the imidazole alphastat hypothesis

SummaryThe effects of assay temperature and pH upon the activities of acetyl CoA carboxylase, fatty acid synthetase, NADH- and succinate-cytochrome c reductases, and NADH-ferricyanide reductase were examined in tissues of rainbow trout. In all cases pH optima shifted to alkaline pH values as the assay temperature was reduced. For trout liver acetyl CoA carboxylase the pH optimum increased from 6.8 at 25°C to 7.36 at 5°C and was characterized by a ΔpHopt/ΔT of −0.0272; similarly, the pH optimum for trout liver fatty acid synthetase increased from 6.29 to 7.15 over the same temperature range and was characterized by a ΔpHopt/ΔT of −0.043. Additional values of ΔpHopt/ΔT were: −0.044 for both NADH- and succinate-cytochrome c reductases, and −0.0325 to −0.040 for NADH-ferricyanide reductase. The observed values for ΔpHopt/ΔT did not differ significantly from the ΔpH/ΔT reported for arterial blood of rainbow trout (Randall and Cameron, 1973). The parallel temperature dependence of enzyme pH optima and both the extracellular and intracellular pH is interpreted as supporting the imidazole alphastat hypothesis of acid base regulation in poikilotherms. Temperature-induced changes in blood pH and acid-base status appear to defend against changes in net protein charge and enzyme function.

Changes in desaturase activity and the fatty acid composition of microsomal membranes from liver tissue of thermally-acclimating rainbow trout

SummaryRainbow trout (Salmo gairdneri) were acclimated to either 5 or 20°C, and then transferred to the opposite temperature, and changes in the fatty acid composition of liver microsomal membranes and the activities of the hepatic Δ9, Δ6, and Δ5 desaturases were measured at intervals of up to one month post-transfer. Inital changes (days 0–3) in fatty acid composition were: (1) an increase in the proportion of saturates and a decrease in the proportion of polyunsaturates during warm acclimation, and (2) a decrease in the proportion of saturates during cold acclimation. The activity of the Δ6 desaturase approximately doubled immediately following the changes in temperature, but alterations in Δ9 and Δ5 desaturase activities required at least 3 days to occur. The results indicate that desaturase enzymes do not play a major role in the initial adaptation of membrane fatty acid composition to changes in temperature. However, the desaturase enzymes may be involved in the later stages (3–28 days) of the acclimatory process. The proportion of monoenes was well correlated with Δ9 desaturase activity during both transfers, and appeared to be adjusted as required to offset changes in the proportion of polyunsaturates.

Thermal adaptation in biological membranes : functional significance of changes in phospholipid molecular species composition

Quantities of 1-palmitoyl 2-docosahexaenoyl phosphatidylcholine (16∶0/22∶6-PC or PDPC) increase from 24 to 40 weight percent as a consequence of cold acclimation in mitochondrial membranes of rainbow trout liver (J. Comp. Physiol. 156, 665–674, 1986). The present study was undertaken to assess the impact of such a large change in the proportions of a single molecular species on the fluidity, lateral packing (as sensed by phospholipase A2), and permeability of biological membranes. These properties were examined in multilamellar liposomes prepared from binary mixtures of dipalmitoyl phosphatidylcholine (DPPC) and PDPC in proportions increasing from 10 to 40 mole% PDPC. Glucose permeability was positively correlated with both assay temperature and PDPC content. The temperature dependence of Na+ permeability declined steadily as the mole fraction of PDPC increased; consequently, sodium permeability was positively correlated with PDPC content at 5°C, but inversely correlated at 20°C. Phospholipase A2 activity was independent of both assay temperature and vesicle composition. Vesicles of all compositions displayed a single transition in the temperature dependence of 1,6 diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization, which shifted to lower temperature and broadened as proportions of PDPC increased. At temperatures belowthe transition, fluidity was positively correlated with the mole fraction of PDPC, but interfacial and deeper regions of the bilayer were affected differently by variations in PDPC content. Nonelectrolyte permeability was the only index of membrane structure or function to be significantly correlated with the fluidity of the bilayer interior. The tendencies of PDPC to both fluidize the membrane and to reduce the temperature sensitivity of electrolyte permeation may promote the adaptation of membrane function to low temperature.

Thermal adaptation in biological membranes: interacting effects of temperature and pH

SummaryThe interacting effects of pH and temperature on membrane fluidity were studied in plasma membranes isolated from liver of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20°C. Fluidity was determined as a function of temperature under conditions of both constant (in potassium phosphate buffer) and variable pH (in imidazole buffer, consistent with imidazole alphastat regulation) from the fluorescence anisotropy of two probes: 1,6-diphenyl-1,3,5-hexatriene, which intercalates into the bilayer interior, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene which is anchored at the membrane/water interface. The temperature dependence of the anisotropy parameter for 1,6-diphenyl-1,3,5-hexatriene in plasma membranes of 20°C-acclimated trout was greater when determined in phosphate (ΔAP per °C=-0.047) than in imidazole buffer (ΔAP per °C=-0.022); similar, but less significant, trends were noted with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene. In contrast, the temperature dependence of fluidity (ΔAP/°C in the range-0.0222 to-0.027) did not vary with buffer composition in membranes of 5°C-acclimated trout. In phosphate buffer, anisotropy parameter values for 1,6-diphenyl-1,3,5-hexatriene were significantly lower in 5°C-than 20°C-acclimated trout, indicating a less restricted probe environment following cold acclimation and nearly perfect compensation (∼91%) of fluidity. Temperature-dependent patterns of acid-base regulation were estimated to account for 11–40% of the fluidization evident in membranes of 5°C-trout, but a period of cold acclimation was required for complete fluidity compensation. In contrast, no homeoviscous adaptation was evident in imidazole buffer, indicating that membrane fluidity is sensitive to buffer composition. Accordingly, vesicles of bovine brain phosphatidylcholine, suspensions of triolein, and plasma membranes of 5°C-acclimated trout were consistently more fluid in imidazole than phosphate buffer. Membranes of 5°C-acclimated trout were enriched in molecular species of phosphatidylcholine containing 22:6n3 (at the expense of species containing 18:1n9 and 18:2n6) compared to membranes of 20°C-trout; consequently, the unsaturation index was significantly higher (3.29 versus 2.73) in trout maintained at 5 as opposed to 20°C. It is concluded that: 1) the chemical composition of the internal milieu can significantly influence the physical properties of membrane lipids; 2) temperature-dependent patterns of intracellular pH regulation may partially offset the ordering effect of low temperature on membrane fluidity in 20°C-acclimated trout transferred to 5°C, but not in 5°C-acclimated trout transferred to warmer temperatures; 3) the majority of the thermal compensation of plasma membrane fluidity resulting from a period of temperature acclimation most likely reflects differences in membrane composition between acclimation groups; 4) imidazole apparently interacts with trout hepatocyte plasma membranes in a unique way.

Incorporation of 1-14C-acetate into fatty acids and sterols by isolated hepatocytes of thermally acclimated rainbow trout (Salmo gairdneri)

SummaryRates of 1-14C-acetate incorporation into specific fatty acids and sterol fractions were determined at assay temperatures of 5°C and 20°C in hepatocytes isolated from warm (20°C)- and cold (5°C)- acclimated rainbow trout (Salmo gairdneri). Rates of sterol lipogenesis were 2.5- to 3-fold higher in hepatocytes from cold-acclimated trout. Rates of acetate oxidation and of total fatty acid lipogenesis did not differ significantly between acclimation groups. Fatty acid compositions did not change significantly during the experiment (9–12 h), but hepatocytes from cold-acclimated trout possessed significantly higher levels of polyunsaturates and unsaturates of the linolenic acid (n-3) family, and significantly lower levels of monounsaturates than did hepatocytes from warm-acclimated animals. Hepatocytes from cold-acclimated trout channeled a larger percentage of their total acetate incorporation into unsaturated fatty acids at 5°C than at 20°C due primarily to increased recovery of acetate in polyunsaturates and monoenes at 5°C. In contrast, hepatocytes from warm-acclimated trout channeled a slightly smaller percentage of their total acetate incorporation into unsaturates at 5°C than at 20°C. Hepatocytes from warm-acclimated trout incorporated significantly more 1-14C-acetate into the unsaturated fatty acid fraction (due primarily to incorporation into the diene fraction and less importantly all other classes of unsaturates) and significantly less into the saturated fatty acid fraction than hepatocytes from cold-acclimated trout when assayed at 20°C; similar but less dramatic differences were observed at 5°C. Consequently, unsaturated/saturated ratios for acetate incorporation ranked: warm-acclimated at 20°C≃warm-acclimated at 5°C≧cold-acclimated at 5°C>cold-acclimated at 20°C. These results suggest that regulation of the relative rates of unsaturated and saturated fatty acid synthesis is involved in lipid restructuringduring adaptation from one temperature regime to another, but that other mechanisms must be invoked to explain the maintenance of observed steady state differences between the fatty acid compositions of warm- and cold-acclimated trout.

Rapid changes in the phospholipid composition of gill membranes during thermal acclimation of the rainbow trout,Salmo gairdneri

SummaryThe phospholipid composition of gill tissue was determined in rainbow trout (Salmo gairdneri) undergoing thermal acclimation between 5°C and 20°C for a period of up to 28 days. Proportions of phosphatidylethanolamine (PE) and cardiolipin (CL) increased during cold acclimation and decreased during warm acclimation; proportions of phosphatidylcholine (PC) changed in the opposite direction (i.e., decreased during cold acclimation). In contrast, levels of phosphatidylserine,-inositol, and sphingomyelin did not vary significantly. Thermal modulation of headgroup composition occurred rapidly as reflected by changes in the ratio of PC-to-PE, which rose significantly from 2.40±0.09 to 2.92±0.09 within 72 h of transfer from 5 to 20°C; adaptation to 5°C was equally rapid. Proportions of PE changed more rapidly than those of PC during cold adaptation, whereas the opposite was true during warm acclimation. Both the time course and the direction of the observed changes in phospholipid composition suggest that such adjustments may contribute to the homeoviscous regulation of membrane properties, particularly during the initial stages of thermal adaptation.

Determination of the phospholipid composition of trout gill by latroscan TLC/FID: Effect of thermal acclimation

The phospholipid composition of gill tissue from thermally acclimated rainbow trout,Salmo gairdneri, was determined by Iatroscan analysis following an initial development of the chromarods in a non-polar solvent to remove neutral lipids. Standard curves for all phospholipids, although linear through most of the concentration range tested (1–40 μg), extrapolated to negative intercepts on the ordinate, indicating a decline in sensitivity at low phospholipid levels. In addition, the concentration dependence of the Iatroscan response varied by nearly 6-fold among phospholipids. Of the major phospholipids, only lysophosphatidylcholine could not be quantitated accurately because of the presence of an interfering peak. Quantitation by Iatroscan yielded results which, in general, agreed well (within 5%) with results obtained by an independent phosphate analysis. Only in the case of phosphatidylinositol (PI) did the two analytical methods differ significantly; proportions of PI were 55% higher when determined by Iatroscan as opposed to phosphate analysis. Gill tissue from 5 C-acclimated trout possessed higher proportions of phosphatidylethanolamine than tissue from 20 C-acclimated trout. The Iatroscan provided a rapid and reliable means of quantitating the proportions of all the major phospholipids of trout gill, although there are some limitations to the general applicability of the technique.