Paul L. Else

Docosahexaenoic acid (DHA) content of membranes determines molecular activity of the sodium pump: implications for disease states and metabolism

The omega-3 polyunsaturate, docosahexaenoic acid (DHA), plays a number of biologically important roles, particularly in the nervous system, where it is found in very high concentrations in cell membranes. In infants DHA is required for the growth and functional development of the brain, with a deficiency resulting in a variety of learning and cognitive disorders. During adulthood DHA maintains normal brain function and recent evidence suggests that reduced DHA intake in adults is linked with a number of neurological disorders including schizophrenia and depression. Here we report a high positive correlation between the molecular activity (ATP min−1) of individual Na+K+ATPase units and the content of DHA in the surrounding membrane bilayer. This represents a fundamental relationship underlying metabolic activity, but may also represent a link between reduced levels of DHA and neurological dysfunction, as up to 60% of energy consumption in the brain is linked to the Na+K+ATPase enzyme.

Proton conductance and fatty acyl composition of liver mitochondria correlates with body mass in birds.

The proton conductance of isolated liver mitochondria correlates significantly with body mass in mammals, but not in ectotherms. To establish whether the correlation in mammals is general for endotherms or mammal-specific, we measured proton conductance in mitochondria from birds, the other main group of endotherms, using birds varying in mass over a wide range (nearly 3000-fold), from 13 g zebra finches to 35 kg emus. Respiratory control ratios were higher in mitochondria from larger birds. Mitochondrial proton conductance in liver mitochondria from birds correlated strongly with body mass [respiration rate per mg of protein driving proton leak at 170 mV being 44.7 times (body mass in g)(-0.19)], thus suggesting a general relationship between body mass and proton conductance in endotherms. Mitochondria from larger birds had the same or perhaps greater surface area per mg of protein than mitochondria from smaller birds. Hence, the lower proton conductance was caused not by surface area changes but by some change in the properties of the inner membrane. Liver mitochondria from larger birds had phospholipid fatty acyl chains that were less polyunsaturated and more monounsaturated when compared with those from smaller birds. Phospholipid fatty acyl polyunsaturation correlated positively and monounsaturation correlated negatively with proton conductance. These correlations echo those seen in mammalian liver mitochondria, suggesting that they too are general for endotherms.

An allometric comparison of the mitochondria of mammalian and reptilian tissues: The implications for the evolution of endothermy

SummaryThe effects of body size and phylogeny on metabolic capacities were examined by comparing the mitochondrial capacities of 6 mammalian and 4 reptilian species representing 100-fold body weight ranges. The mammals examined included 3 eutherian, 2 marsupial and a monotreme species and the reptiles 2 saurian, 1 crocodilian and 1 testudine species. The tissues examined were liver, kidney, brain, heart, lung and skeletal muscle. Allometric equations were derived for tissue weights, mitochondrial volume densities, internal mitochondrial membrane surface area densities, tissue mitochondrial membrane surface areas both per gram and per total tissue and summated tissue mitochondrial membrane surface areas.For the mammals and reptiles studied a 100% increase in body size resulted in average increases of 68% in internal organ size and 107% in skeletal muscle mass. Similarly, total organ mitochondrial membrane surface areas increase in mammals and reptiles by an average 54% and for skeletal muscle by an average 96%. These values are similar to increases in standard (54 and 71%) and maximum (73 and 77%) organismal metabolism values found by other authors for mammals and reptiles respectively.Although the allometric exponents (or rates of change with increasing body size) of the mitochondrial parameters in mammals and reptiles are statistically the same, in general the total amount of mitochondrial membrane surface area in the mammalian tissues are four times greater than found in the reptilian tissues. These differences were not the result of any single ‘quantum’ factor but are the result of the mammals having relatively larger tissues with a greater proportion of their volume occupied by mitochondria and to a lesser extent increases in the internal mitochondrial membrane surface area densities. Mitochondrial volume density from this present study would appear to be the major factor involved in changing weight specific metabolism of tissues both as a result of changes in body size and in the evolution of endothermy in mammals from reptiles.

Polyunsaturated fatty acids, membrane function and metabolic diseases such as diabetes and obesity.

Lipids play an extraordinary range of roles in normal and deranged metabolism. In diabetes and obesity, lipids have often been seen just as impacting on the energy balance equation. New data are extending our understanding of how lipid subclasses influence carbohydrate and lipid metabolism at multiple control points: from the modulation of membrane proteins to the regulation of gene transcription.

Plasticity of oxidative metabolism in variable climates: molecular mechanisms

Converting food to chemical energy (ATP) that is usable by cells is a principal requirement to sustain life. The rate of ATP production has to be sufficient for housekeeping functions, such as protein synthesis and maintaining membrane potentials, as well as for growth and locomotion. Energy metabolism is temperature sensitive, and animals respond to environmental variability at different temporal levels, from within‐individual to evolutionary timescales. Here we review principal molecular mechanisms that underlie control of oxidative ATP production in response to climate variability. Nuclear transcription factors and coactivators control expression of mitochondrial proteins and abundance of mitochondria. Fatty acid and phospholipid concentrations of membranes influence the activity of membrane‐bound proteins as well as the passive leak of protons across the mitochondrial membrane. Passive proton leak as well as protein‐mediated proton leak across the inner mitochondrial membrane determine the efficacy of ATP production but are also instrumental in endothermic heat production and as a defense against reactive oxygen species. Both transcriptional mechanisms and membrane composition interact with environmental temperature and diet, and this interaction between diet and temperature in determining mitochondrial function links the two major environmental variables that are affected by changing climates. The limits to metabolic plasticity could be set by the production of reactive oxygen species leading to cellular damage, limits to substrate availability in mitochondria, and a disproportionally large increase in proton leak over ATP production.

The Evolution of Endothermy: Role for Membranes and Molecular Activity

On the basis of the comparative approach and three models of metabolism (endothermic and ectothermic vertebrates, body mass, and mammalian development), we suggest that a few common cellular processes, linked either directly or indirectly to membranes, consume the majority of energy used by most organisms; that membranes act as pacemakers of metabolism through changes in lipid composition, altering membrane characteristics and the working environment of membrane proteins—specifically, that changes in the membrane environment similarly affect the molecular activities (specific rates of activity) of membrane‐bound proteins; and that polyunsaturation of membranes increases whereas monounsaturation decreases the activity of membrane proteins. Experiments designed to test this theory using the sodium pump support this supposition. Potential mechanisms considered include fluidity, electrical fields, and related surface area requirements of lipids. In considering the evolution of endothermy in mammals, for example, if the first mammals were small, possibly nocturnal and active organisms, all these factors would favour increased polyunsaturation of membranes. Such changes (from monounsaturated to polyunsaturated membranes) would allow membranes to set the pace of metabolism in the evolution of endothermy.

Membrane fatty acid composition of rat skeletal muscle is most responsive to the balance of dietary n -3 and n -6 PUFA

The present study quantifies the relationships between diet fatty acid profile and fatty acid composition of rat skeletal muscle phospholipids. Young adult male Sprague-Dawley rats were fed, for 8 weeks, on one of twelve moderate-fat diets (25 % of total energy) differing only in fatty acid profile. SFA content ranged from 8-88 % of total fatty acids, MUFA 6-65 %, total PUFA 4-81 %, n-6 PUFA 3-70 % and n-3 PUFA 1-70 %. Diet PUFA included only essential fatty acids 18 : 2n-6 and 18 : 3n-3. The balance between n-3 and n-6 PUFA (PUFA balance) in the diet ranged from 1 : 99 to 86 : 14 % n-3 PUFA:n-6 PUFA. The slope of muscle phospholipid composition plotted against diet composition quantifies the response of muscle membrane composition to dietary fat (0, no response; 1, complete conformity with diet). The resulting slopes were 0.02 (SFA), 0.10 (PUFA), 0.11 (MUFA), 0.14 (n-3 PUFA) and 0.23 (n-6 PUFA). The response to PUFA balance was biphasic with a slope of 0.98 below 10 % diet PUFA balance and 0.16 above 10 %. Thus, low diet PUFA balance has greater influence on muscle composition than 18-carbon n-3 or n-6 PUFA individually. Equations provided may allow prediction of muscle composition for other diet studies. Diet PUFA balance dramatically affects muscle 20 : 4n-6 and 22 : 6n-3. This may have significant implications for some disease states in human subjects.

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease.

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimers disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.

Thermal acclimation and regulation of metabolism in a reptile (Crocodylus porosus): the importance of transcriptional mechanisms and membrane composition.

Energy metabolism is fundamental for animal fitness because it fuels locomotion, growth, and reproduction. Mitochondrial capacities often acclimate to compensate for negative thermodynamic effects. Our aim was to determine the importance of transcriptional regulation and membrane fatty acid composition in modulating oxidative capacities at body temperatures selected in a cold and a warm environment by a reptile (Crocodylus porosus). In the cool environment (mean selected Tb = 21°C), mRNA concentrations of the transcription factor peroxisome proliferator–activated receptor gamma (PPARγ) and its coactivator PPARγ coactivator 1 alpha (PGC‐1α), as well as of the cytochrome c oxidase (COX) subunits COX1 and COX5, were significantly higher in the liver but not in skeletal muscle compared with animals in the warm environment (mean selected Tb = 29°C). FOF1‐ATPase subunit α mRNA concentrations were significantly higher in both muscle and the liver in the cool animals. A positive relationship between PGC‐1α and PPARγ mRNA concentrations, with an indicator of mitochondrial density (16S rRNA) in muscle and COX and FOF1‐ATPase subunit α mRNA concentrations in liver, suggest that these proteins regulate quantity increases of mitochondria during acclimation. The percent saturated fatty acids in liver membranes of cool animals was significantly lower, and the n3 fatty acid content was significantly higher, compared with in warm animals. The n3 fatty acid content was positively related to COX enzyme activity in the liver, and there was a negative relationship between n7 fatty acid content and COX activity in muscle. Hence, metabolic acclimation is mediated by both transcriptional regulation and membrane fatty acid composition. The importance of PGC‐1α and PPARγ in a reptile indicate that the mechanisms that regulate metabolism are conserved among vertebrates.

An allometric comparison of microsomal membrane lipid composition and sodium pump molecular activity in the brain of mammals and birds

SUMMARY Previous research has shown that the lipid milieu surrounding membrane proteins may be an important factor in determining their activity. To investigate this we have examined sodium pump molecular activity and microsomal membrane lipid composition in the brain of five mammalian and eight avian species ranging in size from 30 g mice to 280 kg cattle and 13 g zebra finches to 35 kg emus, respectively. Sodium pump (Na+,K+-ATPase) activity was higher in the smaller species and showed a significant allometric decline with body mass in both the mammals (μmol Pi h-1 mg wet mass-1 = 6.2×mass-0.06) and birds (μmol Pi h-1 mg wet mass-1 = 5.4×mass-0.07). In small mammals, the elevated enzyme activity was related to allometric changes in both the concentration and the molecular activity (turnover rate) of sodium pumps, while in birds, no significant body-size-related variation was observed for either sodium pump concentration or molecular activity. Microsomal phospholipid fatty acid profile displayed little allometric variation in both the mammals and birds and was not correlated with molecular activity in either group. Brain phospholipids from both endothermic classes were dominated by the long chain n-3 polyunsaturate, docosahexaenoic acid [22:6 (n-3)], which accounted for an average of 28% and 34% of the total fatty acids in the mammals and birds respectively. Bird membranes also contained a relatively large percentage of 22:5 (n-6) as well as high levels of cholesterol. These results are discussed in relation to neurological function and the emerging field of membrane lipid rafts.