Temperature-adaptive lipid responses

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LIPID COMPOSITIONAL CORRELATES OF TEMPERATURE-ADAPTIVE. INTERSPECIFIC DIFFERENCES IN MEMBRANE PHYSICAL STRUCTURE. JAMES A.
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The Journal of Experimental Biology 203, 2105–2115 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JEB2742

LIPID COMPOSITIONAL CORRELATES OF TEMPERATURE-ADAPTIVE INTERSPECIFIC DIFFERENCES IN MEMBRANE PHYSICAL STRUCTURE JAMES A. LOGUE1, ART L. DE VRIES2, ELFRIEDA FODOR1 AND ANDREW R. COSSINS1,* Biology Research Division, School of Biological Sciences, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK and 2Department of Molecular and Integrative Physiology, University of Illinois, Urbana, IL 61801, USA 1Integrative

*Author for correspondence (e-mail: [email protected])

Accepted 26 April; published on WWW 22 June 2000 Summary constant proportion in all species. Exactly opposite effects Teleost species from cold environments possess more were evident for phosphatidylethanolamine (PtdEth). disordered brain synaptic membranes than species from Thus, the compositional adaptation for PtdCho occurred warm habitats, thereby providing equivalent physical largely by exchange of polyunsaturated and monostructures at their respective habitat temperatures. We unsaturated fatty acid in the sn-2 position, whilst for have related this adaptive interspecific biophysical PtdEth it involved exchanges between saturates and monoresponse to the fatty acid composition of brain membranes unsaturates at the sn-1 position. This difference may be from 17 teleost species obtained from Antarctic, temperate related to the different molecular shapes of the two and semi-tropical waters, as well as from rat and turkey phosphoglycerides and the need to maintain the balance as representative homeotherms. Cold-adaptive increases between bilayer-stabilising and -destabilising tendencies. in membrane disorder (determined by fluorescence This comparative study provides a more comprehensive anisotropy with diphenylhexatriene as probe) were view of the compositional adjustments that accompany and correlated with large and linear increases in the proportion perhaps account for temperature-adaptive interspecific of unsaturated fatty acids, from 35 to 60 % in differences in membrane physical structure. phosphatidylcholine (PtdCho) and from 55 to 85 % in phosphatidylethanolamine (PtdEth). For PtdCho, the coldadaptive increase in unsaturation was associated almost entirely with increased proportions (from 7 to 40 %) Key words: temperature adaptation, brain, membrane, fluorescence anisotropy, DPH, phosphatidylcholine, phosphatidylethanolamine, of polyunsaturated fatty acids (PUFAs), with monofatty acid unsaturation, molecular species. unsaturates (MUFAs) providing an approximately

Introduction Certainly the most consistent biochemical response of poikilothermic organisms to environmental cooling is an increase in fatty acid unsaturation of both membrane and depot lipids (Cossins, 1994; Hazel and Williams, 1990). This response has been observed over two distinctive time scales; first, when organisms are conditioned over days and weeks to the cold or warm and, second, when comparing species inhabiting extreme cold and warm thermal environments. The first is a clear short-term phenotypic response to altered temperature thought to be linked to seasonally adjusted thermal resistance (‘resistance acclimation’) of the whole organism. The second occurs over evolutionary time and again has been linked with adaptation of thermal resistance limits and physiological performance to specific thermal habitats. This compositional response is generally interpreted as causing a disordering of the bilayer to offset the cold-induced ordering. It is thus compensatory in conserving a particular membrane physical condition in the face of thermally induced

disturbance, as reflected in the widely used term homeoviscous adaptation (Cossins, 1994). Although there is some uncertainty as to the precise nature of the conserved membrane condition (Cossins, 1994; Hazel, 1995), biophysical techniques, such as fluorescence polarisation spectroscopy, provide a useful semiquantitative estimate of the extent to which biophysical structure is conserved, a parameter termed homeoviscous efficacy (HE) (Cossins and Raynard, 1987). Studies on membranes isolated from a range of tissues of thermally acclimated fish have shown HEs ranging from zero (i.e. no response in muscle sarcoplasmic reticulum; Cossins et al., 1978) to 100 %, when biophysical properties are conserved at each of the different acclimation temperatures (Crockett and Hazel, 1995; Lee and Cossins, 1990). Most preparations possessed HEs of 30–50 % (Cossins and Raynard, 1987). With regard to interspecific comparisons, Cossins and Prosser (1977) examined brain synaptosomal membranes from an Arctic, a temperate and a warm-adapted desert spring

2106 J. A. LOGUE AND OTHERS species of teleost fish and demonstrated 100 % HE for particular species pairs. This was extended to purified brain synaptic membranes by Behan-Martin et al. (1993), who showed 70–100 % HE, depending on the spectroscopic probe employed, for a somewhat wider range of species including, for the first time, an Antarctic teleost, Notothenia neglecta. This response was considerably greater than that observed in corresponding membranes in thermally acclimated goldfish suggesting that, in this respect at least, the evolutionary adaptation of a species greatly exceeds the potential for adaptation of any individual. The compositional basis of interspecific adaptation has been linked to membrane unsaturation (Cossins and Prosser, 1977; Farkas and Roy, 1989), although little exact detail is given beyond this general statement. Here, we provide a detailed and more precise understanding of how lipid compositional adjustments underpin the conservation of membrane physical structure over the full range of temperatures experienced by vertebrate animals. Brain synaptic preparations have been isolated from 17 teleost species obtained from Antarctic, temperate and tropical habitats and also from a representative species of a bird and a mammal. We correlate differences between species in membrane biophysical structure with differences in lipid fatty acid composition and demonstrate positionally specific but opposing changes in the two major phosphoglyceride classes. Materials and methods Animals Table 1 lists the species used in this work. Where possible, fish were maintained at the temperature specified for at least 21 days. In Liverpool, the tilapia, striped bass and rainbow trout were fed to satiety once daily with standard trout pellets (Trouw, Longridge, Preston, UK). Gourami were fed once daily with tropical fish food (Tetramin, Tetra Werke, Melle, Germany). Wild-caught fish from Antarctic and Florida were held for a few days, during which time they were unfed. Specimens of the Antarctic Pagothenia borchgrevinki were acclimated at 4 °C for 17 days, during which time they were unfed. Black cod were caught in New Zealand and transported by air freight to the US Antarctic Research Program base at McMurdo Sound. Isolation of brain synaptic membranes The method was modified from that described previously (Behan-Martin et al., 1993; Cossins and Prosser, 1982). The lysed synaptosomal pellet was resuspended by gentle homogenisation in 2.5 ml of 10 mmol l−1 imidazole, pH 7.4, and layered on a discontinuous sucrose gradient consisting of equal volumes of 0.9 and 1.0 mol l−1 sucrose in 10 mmol l−1 imidazole, pH 7.4. After centrifugation at 100 000 g for 120 min, the material occupying the interface was removed, diluted 10-fold with lysing medium and pelleted at 100 000 g for 60 min. This pellet was resuspended by homogenisation (all-glass homogeniser) in a small volume of lysing medium

and either used immediately or stored frozen at −80 °C. Preliminary experiments demonstrated that storage over several months has no effect upon the values or temperaturedependence of 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy. Previous studies have shown that these membrane fractions are of synaptosomal origin, as judged from marker enzyme assays, and have negligible mitochondrial contamination (Behan-Martin et al., 1993). Lipid extraction, phospholipid fractionation and fatty acid analysis Extraction of a total lipid fraction, separation of the major phospholipid headgroup classes and transmethylation were performed as described previously (Lee and Cossins, 1990). Fatty acid methyl esters were separated on a gas–liquid chromatograph (Series 610, ATI Unicam, Cambridge, UK) equipped with a fused silica, free fatty acid phase capillary column (30 m×0.25 mm, J&W Scientific, PhaseSep, Queensferry, Clwyd, UK). Methyl esters were identified by comparing peak retention times with authentic standards whose identity had been confirmed by mass spectrometry. Fatty acid compositions were calculated on a percentage by mass basis directly from the chromatographic output using data-analysis software from ATI Unicam. Molecular species analysis by high-performance liquid chromatography Phospholipid molecular species composition was determined following a published procedure (Takamura and Kito, 1991). High-performance liquid chromatography (HPLC) was performed on a PU4100 liquid chromatograph equipped with a PU4110 ultraviolet/visible detector (ATI Unicam, Cambridge, UK) using a reverse-phase octadecylsilyl (ODS) column (Supercosil, 5 µm, 4.6 mm i.d. × 25 cm, Supelco, Suplechem UK Ltd) and an acetonitrile:2-propanol (80:20, v/v, HPLC grade) solvent system at a flow rate of 1 ml min−1. The detection wavelength was 254 nm. Peaks were identified by reference to retention times of authentic standards and published data (Bell and Dick, 1991; Takamura and Kito, 1991). Fluorescence anisotropy Membrane physical properties were determined by measurements of fluorescence anisotropy using 1,6-diphenyl1,3,5-hexatriene (DPH) as probe. We used the T-format polarisation fluorometer described previously (Cossins and Macdonald, 1986) or a PC1 spectrofluorometer (ISS Inc, Urbana, Illinois, USA), both giving identical results. Results Fig. 1A compares the physical structure of brain synaptic membranes obtained from teleost, avian and mammalian species. Physical structure was determined by measurements of DPH anisotropy. At any given temperature, membranes from Antarctic species displayed lower anisotropies, and by

Temperature-adaptive lipid responses 2107 Table 1. List of species Order (Family) Perciformes (Nototheniidae) Perciformes (Nototheniidae) Perciformes (Nototheniidae) Perciformes (Zoarcidae) Perciformes (Nototheniidae) Perciformes (Nototheniidae) Salmoniformes (Salmonidae) Perciformes (Moronidae) Perciformes (Haemulidae) Perciformes (Haemulidae) Perciformes (Lutjanidae) Perciformes (Lutjanidae) Perciformes (Sparidae) Perciformes (Sciaenidae) Batrachoidiformes (Batrachoididae) Perciformes (Cichlidae) Perciformes (Belontiidae) − −

Trivial name

Species

Source

−1 and 4

Bald notothen

Pagothenia borchgrevinki

Emerald rock cod

Trematomus bernachii

Antarctic cod

Dissostichus mawsoni

Eelpout

Lycodichthys dearborni

Rock perch

Notothenia neglecta

New Zealand black cod Rainbow trout

Notothenia angustata Oncorhynchus mykiss

Wild-caught, Christchurch, New Zealand Farmed, UK

Striped bass

Morone saxatilis

Farmed, UK

17

White grunt

Haemulon plumieri

Wild-caught, Miami, Florida

24

Blue grunt

Haemulon sciurus

Wild-caught, Miami, Florida

24

Grey snapper

Lutjanus griseus

Wild-caught, Miami, Florida

24

School master

Lutjanus apodus

Wild-caught, Miami, Florida

24

Pinfish

Lagodon rhomboides

Wild-caught, Miami, Florida

24

Red drum

Sciaenops ocellatus

Wild-caught, Miami, Florida

24

Toadfish

Opsanus beta

Wild-caught, Miami, Florida

24

Tilapia

Oreochromis mossambicus

Farmed, UK

25

Pearl gourami

Trichogaster leeri

Aquarium trade, UK

28

Rat Turkey

Rattus rattus Meleagris gallopavo

Laboratory stock Farmed, UK

37 41

inference greater disorder, than those from temperate species over the full range of measurement temperatures. Temperate species possessed more disordered membranes than semitropical species and these in turn showed greater disorder than the membranes of the representative mammal and bird. Thus, a clear correlation exists between synaptic membrane order and species adaptation temperature. Measurement of anisotropy at each of the respective adaptation temperatures allows a direct comparison among species (Fig. 1B). With the exception of the Antarctic species and black cod, values for all the fish, avian and mammalian species were nearly identical, although the value for the pearl gourami was somewhat lower. This indicates that for these species the differences in anisotropy shown in Fig. 1A were sufficient to offset completely the direct

Wild-caught, McMurdo Sound, Antarctica Wild-caught, McMurdo Sound, Antarctica Wild-caught, McMurdo Sound, Antarctica Wild-caught, McMurdo Sound, Antarctica Wild-caught, South Georgia

Adaptation/ acclimation temperature (°C)

−1 −1 −1 0 4 8

effects of temperature upon anisotropy. The Antarctic species showed somewhat higher anisotropies indicating that they were not sufficiently disordered to provide the same anisotropy as all the other species. The fatty acid composition of the two major phospholipid headgroup classes, phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth), were determined in replicate membrane preparations for each species. The major fatty acids were 16:0, 18:0, 18:1n-9, 20:4n-6, 22:4n-6 and 22:6n-3. Phosphatidylethanolamine also contained appreciable quantities of alkenyl chains, predominantly alk16:0 and alk18:0 together with alk18:1n-7 and alk18:1n-9 as minor components. Fig. 2 shows that the proportion of unsaturated fatty acids for each phosphoglyceride class was inversely and

2108 J. A. LOGUE AND OTHERS Turkey

0.34

A

Rat Toadfish (24°C) Tilapia (25°C)

0.28

DPH anisotropy

Blue grunt (24°C)

0.22

0.16 Striped bass (17°C) Trout (8°C) Notothenia (0°C) Pagothenia (