The under-appreciated fats of life: the two types of polyunsaturated fats.

There are two types of polyunsaturated fatty acids (i.e. fats that contain multiple carbon-carbon double bonds) - omega-6 and omega-3. They are not interconvertible, and they contribute 'double-bonded carbons' to different depths in bilayer membranes, with different effects on membrane processes. This Commentary emphasises the importance of these fats for biological membrane function and examines their evolution and biochemistry. Omega-6 and omega-3 fatty acids are separately essential in the diet of animals, and they pass up the food chain largely from plants, with 'seeds' being a prevalent source of omega-6, and 'leaves' a prevalent source of omega-3. The dietary balance between these fatty acids has a strong influence on membrane composition. Although this aspect of diet has been little investigated outside of the biomedical field, emerging evidence shows it can alter important physiological capacities of animals (e.g. exercise endurance and adiposity), which has implications for activities such as avian migration and hibernation and torpor, as well as significant implications for human health. This Commentary will focus on the separate effects of omega-3 and omega-6 on membrane properties and will emphasise the importance of the balance between these two fatty acids in determining the function of biological membranes; I hope to convince the reader that fats should be considered first and foremost as the basic unit of biological membranes, and secondarily as a means of energy storage.

Honey bee caste lipidomics in relation to life-history stage and the long life of the queen.

Honey bees have evolved a system in which fertilised eggs transit through the same developmental stages but can become either workers or queens. This difference is determined by their diet through development. Whereas workers live for weeks (normally 2-6 weeks), queens can live for years. Unfertilised eggs also develop through the same stages but result in a short-lived male caste (drones). Workers and drones are fed pollen throughout their late larval and adult life stages, while queens are fed exclusively on royal jelly and do not eat pollen. Pollen has a high content of polyunsaturated fatty acids (PUFA) while royal jelly has a negligible amount of PUFA. To investigate the role of dietary PUFA lipids and their oxidation in the longevity difference of honey bees, membrane fatty acid composition of the three castes was characterised at six different life-history stages (larva, pupa, emergent and different adult stages) through mass spectrometry. All castes were found to share a similar membrane phospholipid composition during early larval development. However, at pupation, drones and workers increased their level of PUFA, whilst queens increased their level of monounsaturated fatty acids. After emergence, worker bees further increased their level of PUFA by 5-fold across most phospholipid classes. In contrast, the membrane phospholipids of adult queens remained highly monounsaturated throughout their adult life. We postulate that this diet-induced increase in membrane PUFA results in more oxidative damage and is potentially responsible for the much shorter lifespan of worker bees compared with long-lived queens.

Fatty acid composition of membrane bilayers: importance of diet polyunsaturated fat balance.

In one of the most extensive analyses to date we show that the balance of diet n-3 and n-6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under 'normal' conditions. Young adult male Sprague-Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8-88% of total FAs, monounsaturate (MUFA) 6-65%, total PUFA 4-81%, n-6 PUFA 3-70% and n-3 PUFA 1-70%. Diet PUFA included only essential FAs 18:2n-6 and 18:3n-3. Balance between n-3 and n-6 PUFA is defined as the PUFA balance (n-3 PUFA as % of total PUFA) and ranged 1-86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer-regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n-3 and n-6 PUFA had a biphasic influence on membrane composition. When n-3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications.

Regulation of membrane phospholipids during the adult life of worker honey bee.

Two female castes that are genetically identical are found in honey bees: workers and queens. Adult female honey bees differ in their morphology and behaviors, but the most intriguing difference between the castes is the difference in their longevity. Queens live for years while workers live generally for weeks. The mechanisms that mediate this extraordinary difference in lifespan remain mostly unknown. Both castes share similar developmental stages and are fed liquid food (i.e. a jelly) during development. However, after emergence, workers begin to feed on pollen while queens are fed the same larval food for their entire life. Pollen has a high content of polyunsaturated fatty acids (PUFA) while royal jelly has negligible amounts. The difference in food during adult life leads to drastic changes in membrane phospholipids of female honey bees, and those changes have been proposed as mechanisms that could explain the difference in lifespan. To provide further details on those mechanisms, we characterized the membrane phospholipids of adult workers at seven different ages covering all life-history stages. Our results suggest that the majority of changes in worker membranes occur in the first four days of adult life. Shortly after emergence, workers increase their level of total phospholipids by producing phospholipids that contained saturated (SFA) and monounsaturated fatty acids (MUFA). From the second day, workers start replacing fatty acid chains from those pre-synthesized molecules with PUFA acquired from pollen. After four days, worker membranes are set and appear to be maintained for the rest of adult life, suggesting that damaged PUFA are replaced effectively. Plasmalogen phospholipids increase continuously throughout worker adult life, suggesting that plasmalogen might help to reduce lipid peroxidation in worker membranes. We postulate that the diet-induced increase in PUFA in worker membranes makes them far more prone to lipid-based oxidative damage compared to queens.

The adult lifespan of the female honey bee (Apis mellifera): Metabolic rate, AGE pigment and the effect of dietary fatty acids.

Female honey bees can be queens or workers and although genetically identical, workers have an adult lifespan of weeks while queens can live for years. The mechanisms underlying this extraordinary difference remain unknown. This study examines three potential explanations of the queen-worker lifespan difference. Metabolic rates were similar in age-matched queens and workers and thus are not an explanation. The accumulation of fluorescent AGE pigment has been successfully used as a good measure of cellular senescence in many species. Unlike other animals, AGE pigment level reduced during adult life of queens and workers. This unusual finding suggests female honey bees can either modify, or remove from their body, AGE pigment. Another queen-worker difference is that, as adults, workers eat pollen but queens do not. Pollen is a source of polyunsaturated fatty acids. Its consumption explains the queen-worker difference in membrane fat composition of female adult honey bees which has previously been suggested as a cause of the lifespan difference. We were able to produce "queen-worker" membrane differences in workers by manipulation of diet that did not change worker lifespan and we can, thus, also rule out pollen consumption by workers as an explanation of the dramatic queen-worker lifespan difference.

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.

Experimental studies of blowfly (Calliphora stygia) longevity: A little dietary fat is beneficial but too much is detrimental.

This paper is one in a series of experimental studies on the effects of food composition on aging and longevity, using the golden-haired blowfly Calliphora stygia as the animal model. Here we examine how diet fat content affects blowfly life history traits such as longevity, reproduction, feeding rate, body mass, total fat content and membrane fatty acid composition. The highest median and maximum longevity was observed in blowflies fed on low fat diets, while high-fat diets caused more rapid death of the blowflies. A major result was that blowflies feeding on the lowest fat diet had the highest maximal lifespan demonstrating that low levels of diet fat enhanced blowfly lifespan. Diet also influenced gender-specific mortality rates; females lived longer on a high-fat diet, while males lived longer on a low fat diet. Furthermore, we provide data for and explain how blowfly feeding rates, egg production and male harassment affected blowfly longevity. Our results highlight the need for further studies to understand how dietary fats are metabolised and utilised in the golden-haired blowfly.

The exceptional longevity of an egg-laying mammal, the short-beaked echidna (Tachyglossus aculeatus) is associated with peroxidation-resistant membrane composition.

The echidna Tachyglossus aculeatus is a monotreme mammal from Australia that is exceptionally long-living. Its documented maximum lifespan of 50 years is 3.7 times that predicted from its body mass. Other exceptionally long-living mammals (naked mole-rats and humans) are known to have peroxidation-resistant membrane composition, raising the question about echidnas. Phospholipids were extracted from skeletal muscle, liver and liver mitochondria of echidnas and fatty acid composition measured. As with other exceptionally long-living mammals, membrane lipids of echidna tissues were found to have a lower content of polyunsaturates and a higher content of monounsaturates than predicted for their body size. The peroxidation index (=peroxidation susceptibility) calculated from this membrane composition was lower-than-expected for their body size, indicating that the cellular membranes of echidnas would be peroxidation-resistant. Additionally when the calculated peroxidation index was plotted against maximum lifespan, the echidna values conformed to the relationship for mammals in general. These findings support the membrane pacemaker theory of aging and emphasise the potential importance of membrane fatty acid composition in aging and in the determination of maximum longevity.

Phospholipid composition of the rat lens is independent of diet.

Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague-Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens.

The links between membrane composition, metabolic rate and lifespan.

The acyl composition of tissue phospholipids varies in a systematic manner among species. Phospholipids, and thus membrane bilayers, from the tissues of small mammal and bird species have a high content of docosahexaenoic acid (DHA) compared to large species. A similar difference exists between the tissues of endothermic mammals and ectothermic reptiles. High DHA content in phospholipids is associated with high metabolic activity and this observation has led to the development of the "membrane pacemaker" theory of metabolism. This proposes that highly polyunsaturated acyl chains impart physical properties to membrane bilayers that enhance and speed up the molecular activity of membrane proteins and consequently the metabolic activity of cells, tissues and the whole animal. The brain has highly polyunsaturated membranes irrespective of body size and possible reasons for this are discussed. Highly polyunsaturated acyl chains are very susceptible to peroxidative damage. It is suggested that these chemical properties of highly polyunsaturated membrane acyl chains have important implications for understanding aging and the determination of longevity.

Membrane phospholipid composition may contribute to exceptional longevity of the naked mole-rat (Heterocephalus glaber): a comparative study using shotgun lipidomics.

Phospholipids containing highly polyunsaturated fatty acids are particularly prone to peroxidation and membrane composition may therefore influence longevity. Phospholipid molecules, in particular those containing docosahexaenoic acid (DHA), from the skeletal muscle, heart, liver and liver mitochondria were identified and quantified using mass-spectrometry shotgun lipidomics in two similar-sized rodents that show an approximately 9-fold difference in maximum lifespan. The naked mole rat is the longest-living rodent known with a maximum lifespan of >28 years. Total phospholipid distribution is similar in tissues of both species; DHA is only found in phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS), and DHA is relatively more concentrated in PE than PC. Naked mole-rats have fewer molecular species of both PC and PE than do mice. DHA-containing phospholipids represent 27-57% of all phospholipids in mice but only 2-6% in naked mole-rats. Furthermore, while mice have small amounts of di-polyunsaturated PC and PE, these are lacking in naked mole-rats. Vinyl ether-linked phospholipids (plasmalogens) are higher in naked mole-rat tissues than in mice. The lower level of DHA-containing phospholipids suggests a lower susceptibility to peroxidative damage in membranes of naked mole-rats compared to mice. Whereas the high level of plasmalogens might enhance membrane antioxidant protection in naked mole-rats compared to mice. Both characteristics possibly contribute to the exceptional longevity of naked mole-rats and may indicate a special role for peroxisomes in this extended longevity.

Extended longevity of queen honey bees compared to workers is associated with peroxidation-resistant membranes.

In the honey bee (Apis mellifera), depending on what they are fed, female eggs become either workers or queens. Although queens and workers share a common genome, the maximum lifespan of queens is an order-of-magnitude longer than workers. The mechanistic basis of this longevity difference is unknown. In order to test if differences in membrane composition could be involved we have compared the fatty acid composition of phospholipids of queen and worker honey bees. The cell membranes of both young and old honey bee queens are highly monounsaturated with very low content of polyunsaturates. Newly emerged workers have a similar membrane fatty acid composition to queens but within the first week of hive life, they increase the polyunsaturate content and decrease the monounsaturate content of their membranes, probably as a result of pollen consumption. This means their membranes likely become more susceptible to lipid peroxidation in this first week of hive life. The results support the suggestion that membrane composition might be an important factor in the determination of maximum lifespan. Assuming the same slope of the relationship between membrane peroxidation index and maximum lifespan as previously observed for mammal and bird species, we propose that the 3-fold difference in peroxidation index of phospholipids of queens and workers is large enough to account for the order-of-magnitude difference in their longevity.

Membrane fatty acids as pacemakers of animal metabolism.

The recent discovery that the fatty acid composition of tissue phospholipids varies in a systematic manner among species has lead to the proposal that membrane fatty acid composition is an important determinant of the metabolic rate characteristic for each species. Endotherms (mammals and birds) have a basal metabolic rate (BMR) that is several times that of ectotherms and have more polyunsaturated membranes. In both birds and mammals, as species size increases there is a decrease in mass-specific BMR and a decrease in membrane polyunsaturation. Membrane-associated processes are significant components of BMR and important membrane proteins operate at much faster rates in species with high BMR than in those with low BMR. A series of "species-crossover" experiments show that the rate of this molecular activity is largely due to the nature of the membrane bilayer surrounding these membrane proteins such that polyunsaturated membranes are associated with fast membrane-associated processes. It is suggested that this influence is due to the physical properties that such polyunsaturated membranes possess. This has been called the membrane pacemaker theory of metabolism and provides a framework to understand factors such as the influence of diet on metabolism. It is noted that in the rat membrane fatty acid composition is a regulated parameter being more influenced by the balance between n-3 and n-6 polyunsaturates in the diet than it is by general diet content of saturated, monounsaturated and total polyunsaturated fats.

Extended longevity of wild-derived mice is associated with peroxidation-resistant membranes.

Two lines of mice, Idaho (Id) and Majuro (Ma), both derived from wild-trapped progenitors, have previously been shown to have extended lifespans in captivity when compared to a genetically heterogenous laboratory line of mice (DC). We have examined whether membrane fatty composition varies with lifespan within the species Mus musculus in a similar manner to that previously demonstrated between mammal species. Muscle and liver phospholipids from these long-living mice lines have a reduced amount of the highly polyunsaturated omega-3 docosahexaenoic acid compared to the DC mice, and consequently their membranes are less likely to peroxidative damage. The relationship between maximum longevity and membrane peroxidation index is similar for these mice lines as previously observed for mammals in general. It is suggested that peroxidation-resistant membranes may be an important component of extended longevity.

Oxidation-resistant membrane phospholipids can explain longevity differences among the longest-living rodents and similarly-sized mice.

Underlying causes of species differences in maximum life span (MLS) are unknown, although differential vulnerability of membrane phospholipids to peroxidation is implicated. Membrane composition and longevity correlate with body size; membranes of longer-living, larger mammals have less polyunsaturated fatty acid (PUFA). We determined membrane phospholipid composition of naked mole-rats (MLS > 28.3 years) and similar-sized mice (MLS = 3-4 years) by gas-liquid chromatography to assess if the approximately 9x MLS difference could be explained. Mole-rat membrane composition was unchanged with age. Both species had similar amounts of membrane total unsaturated fatty acids; however, mice had 9 times more docosahexaenoic acid (DHA). Because this n-3PUFA is most susceptible to lipid peroxidation, mole-rat membranes are substantially more resistant to oxidative stress than are mice membranes. Naked mole-rat peroxidation indices, calculated from muscle and liver mitochondrial membranes, concur with those predicted by MLS rather than by body size, suggesting that membrane phospholipid composition is an important determinant of longevity.

Calorie restriction in mice: effects on body composition, daily activity, metabolic rate, mitochondrial reactive oxygen species production, and membrane fatty acid composition.

Different levels of calorie restriction (CR) (125, 85, 50, or 40 kcal/wk for 1, 3, and 6 months) were examined in mice by using the paradigm of Weindruch and colleagues. Lean and total body mass increased on 125 and 85 kcal/wk, but there was negligible growth on low-energy intake. There was no CR-induced reduction in either daily activity or mass-specific metabolic rate. There was no CR-effect on in vitro reactive oxygen species production by liver or muscle mitochondria at 3 months, but after 6 months the effect was significantly reduced in liver mitochondria from 40 kcal/wk mice compared to 125 kcal/wk mice. Changes in the fatty acid composition of phospholipids from liver, kidneys, heart, brain, and skeletal muscle were observed following 1 month of CR.

Scaling of Na+,K+-ATPase molecular activity and membrane fatty acid composition in mammalian and avian hearts.

We have examined Na(+),K(+)-ATPase molecular activity and membrane fatty acid composition in the heart of six mammalian and eight avian species ranging in size from 30 g in mice to 280 kg in cattle and 13 g in zebra finches to 35 kg in emus, respectively. Na(+),K(+)-ATPase activity scaled negatively with body mass in both mammals and birds. In small mammals, the elevated enzyme activity was related to allometric changes in both the concentration and molecular activity (turnover rate) of Na(+),K(+)-ATPase enzymes, while in small birds, higher Na(+),K(+)-ATPase activity appeared to result primarily from an increased molecular activity of individual enzymes. The unsaturation index of cardiac phospholipids scaled negatively with body mass in both groups, while a significant allometric increase in monounsaturate content was observed in the larger mammals and birds. In particular, the relative content of the highly polyunsaturated docosahexaenoic acid (22:6n-3) displayed the greatest variation, scaling negatively with body mass and varying greater than 40-fold in both mammals and birds. Membrane fatty acid profile was correlated with Na(+),K(+)-ATPase molecular activity in both mammals and birds, suggesting a potential association between membrane lipid composition and the activity of membrane-bound enzymes in the hearts of endotherms.

The proton permeability of liposomes made from mitochondrial inner membrane phospholipids: no effect of fatty acid composition.

The proton permeability of the mitochondrial inner membrane has been shown to correlate with the fatty acid composition of its phospholipids. In this paper, we test the hypothesis that the proton permeability of the phospholipid bilayer portion of the membrane depends on phospholipid fatty acid composition. We measured the proton permeability of liposomes made from the mitochondrial inner membrane phospholipids of eight vertebrates, representing a ten-fold range of mitochondrial proton leak and a three fold range of unsaturation index. At a membrane potential (delta psi) of 160 mV at 37 degrees C, the liposomes all had the same proton leak rate, about 30 nmol protons min-1 mg-1 phospholipid. There was no correlation between liposome proton permeability and phospholipid fatty acid composition.

Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygen-consuming tissues of the rat.

Maintenance of an electrochemical proton gradient across the mitochondrial inner membrane against the significant proton permeability of the membrane accounts for 25-30% of resting oxygen consumption in hepatocytes. It has been proposed that proton leak could be a significant contributor to resting metabolic rate in mammals if it were present in other tissues. Mitochondria were isolated from the major oxygen-consuming tissues (liver, kidney, brain and skeletal muscle) of the rat. In each tissue, the mitochondria showed significant proton leak with the same characteristic non-linear dependence on membrane potential. Liver and kidney mitochondria showed similar membrane proton permeability per mg of mitochondrial protein; brain and muscle permeabilities were greater when expressed in this way. Differences in the kinetic response of the substrate oxidation and phosphorylating systems to membrane potential were observed. The substrate oxidation system was more active in kidney, brain and skeletal muscle mitochondria than in liver mitochondria per mg of mitochondrial protein. Liver and kidney phosphorylating systems were less active than brain and skeletal muscle per mg of mitochondrial protein. The control of oxidative phosphorylation was also assessed. The distribution of control in mitochondria isolated from the four tissue types was found to be similar.

Why are some mitochondria more powerful than others: insights from comparisons of muscle mitochondria from three terrestrial vertebrates.

We studied the molecular composition of muscle mitochondria to evaluate whether the contents of cytochromes or adenine nucleotide translocase (ANT) or phospholipid acyl compositions reflect differences in mitochondrial oxidative capacities. We isolated mitochondria from three vertebrates of similar size and preferred temperature, the rat (Rattus norvegicus), the cane toad (Bufo marinus) and the bearded dragon lizard (Pogona vitticeps). Mitochondrial oxidative capacities were higher in rats and cane toads than in bearded dragon, whether rates were expressed relative to protein, cytochromes or ANT. Inter-specific differences were least pronounced when rates were expressed relative to cytochrome A, a component of cytochrome C oxidase (CCO), or ANT. In mitochondria from rat and cane toad, cytochrome A was more abundant than C followed by B and then C(1), while in bearded dragon mitochondria, the cytochromes were present in roughly equal levels. Analysis of correlations between mitochondrial oxidative capacities and macromolecular components revealed that cytochrome A explained at least half of the intra- and inter-specific variability in substrate oxidation rates. ANT levels were an excellent correlate of state 3 rates while phospholipid contents were correlated with state 4 rates. As the % poly-unsaturation and the % 20:4n-6 in mitochondrial phospholipids were equivalent in toads and rats, and exceeded the levels in lizards, they may contribute to the inter-specific differences in oxidative capacities. We suggest that the numbers of CCO and ANT together with the poly-unsaturation of phospholipids explain the higher oxidative capacities in muscle mitochondria from rats and cane toads.