Methylenetetrahydrofolate reductase (MTHFR) allele frequencies in Amerindians.

Neural tube defects (NTDs) have been associated with abnormalities of folate metabolism. Methylenetetrahydrofolate reductase (MTHFR) is the regulatory enzyme for the conversion of homocysteine to methionine. The C677T mutation in the MTHFR gene affects folate distribution, and homozygosity for the T allele may be associated with an increased risk of NTDs. A second mutation, an A1298C transversion in this same gene, is also associated with an increased risk for NTDs but only in conjunction with the 677T allele. A low incidence of NTDs has been observed in high-altitude populations; however, these studies did not provide information about the allele distribution of genes involved in folate metabolism. This investigation compares allele frequencies of the C677T and A1298C polymorphisms between Quechua people living at 3200-4200 m in the Peruvian Central Andes and an Aché group living at low altitude. Allele frequencies at both loci were not significantly different between the two populations. The absence of the 677T/677T genotypes and of the 677T/1298C arrangement in both groups may indicate a genetic contribution to reduced risk for NTDs; however, factors other than altitude are likely responsible for the low variant allele frequencies in these populations.

Selective pressure has not acted against hypercoagulability alleles in high-altitude Amerindians.

Elevated hematocrit increases blood oxygen carrying capacity in high-altitude populations, but blood viscosity and coaguability may increase concomitantly. Alleles of the beta-fibrinogen gene (FGB) associated with lower fibrinogen levels are more common in highland Amerindians (Quechua) than lowland Amerindians (Na-Dene). Although genetic drift could account for this, selection may have acted against transmission of hypercoagulability alleles at high altitude. To test this hypothesis, we compared allele frequencies between Quechua and more closely related lowlanders (Maya) at loci in the genes encoding beta-fibrinogen (FGB), factors V (F5), VII (F7) and XIII (F13), alpha2-integrin (ITGA2) and plasminogen activator inhibitor type 1 (PAI-1; SERPINE1). No significant differences in allele frequencies were found except 485arg in the gene encoding factor V, which was more common in the Quechua. These data do not support the hypothesis that selection has acted to eliminate alleles associated with hypercoagulability in Andean highlanders.

Intracellular convection, homeostasis and metabolic regulation.

Two views currently dominate experimental approaches to metabolic regulation. The first, let us call it Model 1, assumes that cells behave like a watery bag of enzymes. The alternative Model 2, however, assumes that 3-dimensional order and structure constrain metabolite behavior. A major problem in cell metabolism is determining why essentially all metabolite concentrations are remarkably stable (homeostatic) over large changes in pathway fluxes-for convenience, this is termed the [s] stability paradox. During large-scale transitions from maintenance metabolic rates to maximally activated work, contrasting demands of intracellular homeostasis versus metabolic regulation obviously arise. Data accumulated over the last 3-4 decades now make it clear that the demands of homeostasis prevail: during rest-work transitions, metabolites such as ATP and O(2) are notably and rigorously homeostatic; other intermediates usually do not vary by more than 0.5- to threefold over the resting condition. This impressive homeostasis is maintained despite changes in pathway fluxes that can exceed two orders of magnitude. Classical or Model 1 approaches to this problem can explain metabolite homeostasis, but the mechanisms for each metabolite, each enzyme locus, are necessarily specific. Thus Model 1 approaches basically do not provide a global explanation for the [s] stability paradox. Model 2 takes a different tack and assumes that an intracellular convection system acts as an over-riding 'assist' mechanism for facilitating enzyme-substrate encounter. Model 2 postulates that intracellular movement and convection are powered by macromolecular motors (unconventional myosins, dyneins, kinesin) running on actin or tubulin tracks. For fast and slow muscle fibers, microfilaments are concentrated near the periphery (where convection may be most important), but also extend throughout the actomyosin contractile apparatus both in horizontal and vertical dimensions. To this point in the development of the field, Model 1 and Model 2 approaches have operated as 'two solitudes', each considering the other incompatible with its own experimental modus operandi. In order to finally assemble a model that can sensibly explain a realistic working range of metabolic systems, opening of channels of communication between the above two very differing views of metabolic regulation would seem to be the requirement for the future.

Genetic polymorphisms in the Renin-Angiotensin system in high-altitude and low-altitude Native American populations.

Cardiovascular disease (CVD) is reportedly less common in high-altitude native populations than in lowlanders. To some extent, this is due to cultural and demographic factors; however, increased cardiovascular efficiency contributing to hypoxia adaptation may also be involved. Numerous genetic variants have been associated with cardiovascular health. If the decreased incidence of CVD in modern high-altitude populations reflects selective pressures having favoured the transmission of these alleles in their antecedents, it would be expected that these alleles would be more common in highlanders than in lowlanders. We tested this hypothesis by determining the allele frequencies of five polymorphic loci in genes encoding components of the renin-angiotensin system (RAS) that have alleles associated with hypertension and cardiovascular disease in a high-altitude native Andean population, Quechua from the Peruvian altiplano, and in a lowland Amerindian population, Maya from the Yucatan peninsula. The polymorphisms examined were 1) the insertion/deletion polymorphism in intron 16 of the angiotensin converting enzyme (ACE) gene; 2) the A/G2350 transition (ACE-8) in intron 17 of the ACE gene; 3) the A/C1166 transversion in the 3' untranslated region of the angiotensin II receptor (type 1) gene (AGTR1); 4) the G/AI9-83 transition in intron 8 of the renin gene (REN); and 5) the T/C704 (Met235Thr) transition mutation in angiotensinogen (AGT). There was no evidence for an over-representation of the RAS alleles associated with cardiovascular fitness in the high-altitude Amerindian population when compared to the lowland Amerindian population.

Going malignant: the hypoxia-cancer connection in the prostate.

The metabolic organization of both normal and malignant prostate cellular phenotypes involves some unusual and surprising features. In particular, both conditions exhibit ratios of NADH/NAD+ and NADPH/NADP+ characteristic of high oxidative states despite a chronic shortage of O2 in both conditions. In this paper, we observe that, in prostate cancer cells, the oxidizing power of the fatty acid synthesis (FAS) pathway is so large that redox is stabilized more favorably (more oxidized) than in normal prostate cells. This FAS-facilitated redox improvement occurs despite the fact that malignant cells are more O2 limited and therefore express more hypoxia inducible factor 1 (HIF1) and express hypoxia-regulated genes more robustly. This unusual metabolic situation clearly separates direct regulatory effects of redox balance from secondary effects of hypoxia per se. The physiological significance of the FAS pathway is thus the harnessing of its oxidizing power for improving redox balance despite conditions of more extreme hypoxia. Similar hypoxia defense strategies are found in animal species that are unusually tolerant to oxygen lack. Our hypothesis is that the metabolic organization in the "low zinc, low citrate" phenotype reflects an hypoxia-defense adaptation geared toward redox balance, with prostate cancer cells being relatively more oxidized, even if more hypoxic, than normal prostate cells. Recognition and understanding of these redox balancing and hypoxia defense functions may lead to new intervention strategies by developing new intracellular targets for prostate cancer therapy.

The lactate paradox in human high-altitude physiological performance.

For many years, physiologists have puzzled over the observation that, during maximum aerobic exercise, high-altitude natives generate lower-than-expected amounts of lactate; the higher the altitude, the lower the postexercise blood lactate peak. This paradoxical situation may be caused mainly by upregulated metabolic control contributions from cell ATP demand and ATP supply pathways.

Mechanism, origin, and evolution of anoxia tolerance in animals.

Organisms vary widely in their tolerance to conditions of limiting oxygen supply to their cells and tissues. A unifying framework of hypoxia tolerance is now available that is based on information from cell-level models from highly anoxia-tolerant species, such as the aquatic turtle, and from other more hypoxia-sensitive systems. The response of hypoxia-tolerant systems to oxygen lack occurs in two (defense and rescue) phases. The first lines of defense against hypoxia include a drastic, if balanced, suppression of ATP demand and supply pathways; this regulation allows ATP levels to remain constant, even while ATP turnover rates greatly decline. The ATP requirements of ion pumping are down-regulated by generalized 'channel' arrest in hepatocytes and by the arrest of specific ion channels in neurons. In hepatocytes, the ATP demands of protein synthesis are down-regulated on exposure to hypoxia by an immediate global blockade of the process (probably through translational arrest caused by complexing between polysomes and elongation factors). In hypoxia-sensitive cells, this translational arrest seems irreversible, but hypoxia-tolerant systems activate 'rescue' mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade of processes underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signal transduction pathway that leads to the specific activation of some genes (increased expression of several proteins) and to specific down-regulation of other genes (decreased expression of several other proteins). The functional roles of the oxygen-sensing and signal-transduction system include significant gene-based metabolic reprogramming - the rescue process - with maintained down-regulation of energy demand and supply pathways in metabolism throughout the hypoxic period. We consider that, through this recent work, it is becoming evident how normoxic-maintenance ATP turnover rates can be down-regulated by an order of magnitude or more - to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. Because the phylogenies of the turtles and of fishes are well known, we are now in an excellent position to assess conservative vs. adaptable features in the evolution of the above hypoxia-response physiology in these two specific animal lineages.

Genetic approaches to understanding human adaptation to altitude in the Andes.

Despite the initial discomfort often experienced by visitors to high altitude, humans have occupied the Andean altiplano for more than 10000 years, and millions of people, indigenous and otherwise, currently live on these plains, high in the mountains of South America, at altitudes exceeding 3000 m. While, to some extent, acclimatization can accommodate the one-third decrease in oxygen availability, having been born and raised at altitude appears to confer a substantial advantage in high-altitude performance compared with having been born and raised at sea level. A number of characteristics have been postulated to contribute to a high-altitude Andean phenotype; however, the relative contributions of developmental adaptation (within the individual) and genetic adaptation (within the population of which the individual is part) to the acquisition of this phenotype have yet to be resolved. A complex trait is influenced by multiple genetic and environmental factors and, in humans, it is inherently very difficult to determine what proportion of the trait is dictated by an individual's genetic heritage and what proportion develops in response to the environment in which the person is born and raised. Looking for changes in putative adaptations in vertically migrant populations, determining the heritability of putative adaptive traits and genetic association analyses have all been used to evaluate the relative contributions of nurture and nature to the Andean phenotype. As the evidence for a genetic contribution to high-altitude adaptation in humans has been the subject of several recent reviews, this article instead focuses on the methodology that has been employed to isolate the effects of 'nature' from those of 'nurture' on the acquisition of the high-altitude phenotype in Andean natives (Quechua and Aymara). The principles and assumptions underlying the various approaches, as well as some of the inherent strengths and weaknesses of each, are briefly discussed.

High-altitude acclimation increases the triacylglycerol/fatty acid cycle at rest and during exercise.

High-altitude acclimation alters lipid metabolism during exercise, but it is unknown whether this involves changes in rates of lipolysis or reesterification, which form the triacylglycerol/fatty acid (TAG/FA) cycle. We combined indirect calorimetry with [2-(3)H]glycerol and [1-(14)C]palmitate infusions to simultaneously measure total lipid oxidation, lipolysis, and rate of appearance (R(a)) of nonesterified fatty acids (NEFA) in high-altitude-acclimated (HA) rats exercising at 60% maximal O(2) uptake (VO(2 max)). During exercise, relative total lipid oxidation (%VO(2)) equaled sea-level control (SL) values; however, acclimation greatly stimulated lipolysis (+75%) but had no effect on R(a) NEFA. As a result, TAG/FA cycling increased (+119%), due solely to an increase in recycling (+144%) within adipocytes. There was no change in either group in these variables with the transition from rest to exercise. We conclude that, in HA, 1) acclimation is a potent stimulator of lipolysis; 2) rats do not modify TAG/FA cycling with the transition to exercise; and 3) in normoxia, HA and SL derive the same fraction of their total energy from lipids and carbohydrates.

Effects of forced diving on the spleen and hepatic sinus in northern elephant seal pups.

In phocid seals, an increase in hematocrit (Hct) accompanies diving and periods of apnea. The variability of phocid Hct suggests that the total red cell mass is not always in circulation, leading researchers to speculate on the means of blood volume partitioning. The histology and disproportionate size of the phocid spleen implicates it as the likely site for RBC storage. We used magnetic resonance imaging on Northern elephant seals to demonstrate a rapid contraction of the spleen and a simultaneous filling of the hepatic sinus during forced dives (P < 0.0001, R(2) = 0.97). The resulting images are clear evidence demonstrating a functional relationship between the spleen and hepatic sinus. The transfer of blood from the spleen to the sinus provides an explanation for the disparity between the timing of diving-induced splenic contraction ( approximately 1-3 min) and the occurrence of peak Hct (15-25 min). Facial immersion was accompanied by an immediate and profound splenic contraction, with no further significant decrease in splenic volume after min 2 (Tukey-Kramer HSD, P = 0.05). At the conclusion of the dive, the spleen had contracted to 16% of its predive volume (mean resting splenic volume = 3,141 ml +/- 68.01 ml; 3.54% of body mass). In the postdive period, the spleen required 18-22 min to achieve resting volume, indicating that this species may not have sufficient time to refill the spleen when routinely diving at sea, which is virtually continuous with interdive surface intervals between 1 and 3 min.

The evidence for hereditary factors contributing to high altitude adaptation in Andean natives: a review.

Humans have occupied the high plateaus and mountain valleys of the Andes and the Himalayas for thousands of years. Although sea level natives can, and often do, travel in these rarefied reaches, there is little doubt that natives born and raised in the "thin" air are better equipped to deal with the reduced availability of oxygen at altitude. What fraction of the hypoxia defense response of high altitude native populations is due to developmental adaptations acquired during growth and what fraction is due to a genetic component reflecting the effects of selective transmission of beneficial genetic variants through hundreds of generations of antecedents is as yet unresolved. This paper summarizes some of the studies that have been undertaken to address this issue in Andean indigenous populations, primarily with respect to those adaptations thought to be involved in the uptake, distribution and utilization of oxygen in children and adults. Specifically, it focuses on changes in chest morphology, pulmonary function, metabolism and hematology. Space constraints preclude extending this review to the large body of literature concerning prenatal and maternal adaptations although this critical stage in development has likely been subject to significant selective pressures. It is apparent that both nature and nurture influence the acquisition of a high altitude phenotype in humans and while there is some evidence for genetic adaptation in Andean highlanders, it is evident that these characteristics are expressed in concert with substantial environment-dependent developmental adjustments.

Physiological and biochemical correlates of brood size and energy expenditure in tree swallows.

Intra-population variation in many fitness-related traits (e.g. clutch size) is often attributed to variation in individual parental quality. One possible component of quality is the level at which each individual can expend energy while provisioning dependent young. We used breeding tree swallows (Tachycineta bicolor) to test whether adults with large, natural-sized broods and/or nestlings in good nutritional condition had relatively high daily energy expenditures (DEEs). Adults with high DEEs were predicted to have large internal organs and high metabolic capacities. We first measured the growth rate of nestlings in natural broods of five, six and seven over a 4-day period and then measured parental DEE using doubly labelled water. Adults were then dissected for analyses of body composition and to determine maximum enzyme activities in the pectoral muscle. Although the total mass gain of large broods was greater than that of small broods, parental DEE was independent of brood size. We hypothesize that adults matched their clutch size (and consequently, brood size) to their individual foraging efficiencies. When statistically controlling for the effects of brood size, in one of two years there was a positive correlation between DEE and brood mass. This suggests that among individuals rearing the same-sized broods there were reproductive benefits of a relatively high DEE. There was no correlation between either brood size or DEE and the mass of any internal organ or the metabolic capacity of the pectoral muscle.

An (1)H-MRS evaluation of the phosphocreatine/creatine pool (tCr) in human muscle.

The human gastrocnemius was examined with and without creatine supplementation under the conditions of rest, ischemic fatigue (IF), and recovery to perturb the pool sizes and equilibrium between phosphocreatine (PCr) and creatine (Cr). (1)H- and (31)P-magnetic resonance spectroscopy (MRS) were used to examine the total creatine (tCr) pool in each of the metabolic states. (31)P-MRS monitored the depletion of the PCr peak during IF to <5% of that at rest. (1)H-MRS focused on the tCr methyl peak at 3.02 ppm (dipolar coupled triplet), at which point it was expected that the triplet peak intensity would be similar both in IF and rest. Initial (1)H-MRS data showed the peak intensity during IF decreased, suggesting a change in tCr pool size. Subsequent studies of transverse relaxation time (T(2)) revealed that this decline was primarily due to a more rapid T(2) decay of the tCr peak in IF (T(2) approximately 40 ms) compared with at rest (T(2) approximately 162 ms). Because Cr is the major contributor to tCr in IF, it is possible that there is a pool of Cr displaying reduced mobility in vivo. Moreover, the residual dipolar coupled triplet observed at rest collapsed into a broad singlet during IF, suggestive of significant changes in the ordered environment experienced at rest for PCr compared with when it is converted to Cr during IF. In addition, these data suggest that in (1)H-MRS studies whose goals include quantitative estimates of tCr pool sizes, standardized metabolic conditions or careful T(2) evaluations will be required.

Effect of brood size manipulation on offspring physiology: an experiment with passerine birds.

The environment experienced during ontogeny has a significant impact on the physiological condition of offspring. This, in turn, forecasts survival probabilities and future reproductive potential. Despite the prominent role that the concept of condition plays in evolutionary studies, the physiological and biochemical characters that define it remain relatively unexplored. In this study, we quantified the impact of brood size manipulations on the physiology and biochemistry of nestling tree swallows (Tachycineta bicolor) shortly before they fledged. Over two breeding seasons, we either increased or decreased the number of individuals in a brood by a single nestling. Every 2-4 days, we determined the resting rate of oxygen consumption [V(O(2))] of individuals in each brood. Growth was followed until 16 days of age, at which time, to look for potential trade-offs in energy allocation, we measured total lipid mass, skeletal muscle and organ mass, indices of blood oxygen-carrying capacity and the activities of key metabolic enzymes in various tissues. Surprisingly, there was a minimal response of most characters to brood manipulation, suggesting that physiological and biochemical development is relatively invariant except perhaps under extreme conditions. Individuals reared in artificially enlarged broods, however, had a significantly lower body mass, body-size-adjusted [V(O(2))], gizzard mass and total lipid mass. These individuals also had decreased activity of cardiac 3-hydroxyacyl CoA dehydrogenase, suggesting a decreased capacity for oxidation of fatty acids. How these characters affect survival or the future adult phenotype remains unknown.

Pinniped diving response mechanism and evolution: a window on the paradigm of comparative biochemistry and physiology.

Starting even before the end of World War II, the discipline of comparative physiology and biochemistry experienced a period of unprecedented growth and development that pioneers in this field thought would never end. However, by the mid-1970s many of the major mechanistic problems in the field were pretty well understood in principle, and by the mid-1980s workers in the field widely recognized that the discipline was at the point of diminishing returns. One response to this was disillusionment, which turned out to be premature because the field was already absorbing molecular biology tools which has now caused a kind of renaissance in mechanistic physiology studies. The second major response to the sense of disillusionment led to a search for new approaches, and out of this endeavor the newly rejuvenated field of evolutionary physiology arose, and this research area too is now in a growth phase. These general patterns of growth and development in our discipline as a whole are particularly clearly evident in the field of aquatic mammals and birds. Between the 1930s and the 1970s, studies of diving physiology and biochemistry made great progress in mechanistically explaining the basic diving response of aquatic mammals and birds. Key components of the diving response (apnea, bradycardia, peripheral vasoconstriction, redistribution of cardiac output) were found in essentially all species analyzed and were generally taken to be biological adaptations. By the mid-1970s, this approach to unraveling the diving response had run 'out of steam' and was in conceptual stasis. The breakthrough which gave renewal to the field at this time was the development of microprocessor based monitoring of diving animals in their natural environments, which led to a flurry of studies mostly confirming the essential outlines of the diving response based upon laboratory studies and firmly placing it into a proper biological context, underlining its plasticity and species specificities. Now as we begin a new millenium, despite ever more detailed field monitoring of physiology, behavior and ecology, studies aimed at improving understanding of physiological mechanisms in diving are again approaching a point of diminishing returns. To avoid another conceptual stasis, what seems required are new initiatives which may arise from two differing approaches. The first is purely experimental, relying on magnetic resonance imaging (MRI) and spectroscopy (MRS) to expand the framework of the original 'diving response' concept. The second, evolutionary study of the diving response, is synthetic, linked to both field and laboratory studies. To date the evolution of the diving response has only been analyzed in pinnipeds and from these studies two kinds of patterns have emerged. (1) Some physiological and biochemical characters, required and used in diving animals, are highly conserved not only in pinnipeds but in all vertebrates; these traits are necessarily similar in all pinnipeds and include diving apnea, bradycardia, tissue specific hypoperfusion, and hypometabolism of hypoperfused tissues. (2) Another group of functionally linked characters are more malleable and include (i) spleen mass, (ii) blood volume, and (iii) hemoglobin (Hb) pool size. Increases in any of these traits (or in a morphological character, body size) improve diving capacity. Assuming that conserved physiological function means conserved sequences in specific genes and their products (and that evolving function requires changes in such sequences), it is possible to rationalize both the above trait categories in pinniped phylogeny. However, it is more difficult for molecular evolution theory to explain how complex regulatory systems like those involved in bradycardia and peripheral vasoconstriction remain the same through phylogenetic time than it is to explain physiological change driven by directional natural selection.

Evolution of human hypoxia tolerance physiology.

Analysis of human responses to hypobaric hypoxia in different lineages (lowlanders, Andean natives, Himalayan natives, and East Africans) indicates 'conservative' and 'adaptable' physiological characters involved in human responses to hypoxia. Conservative characters, arising by common descent, dominant and indeed define human physiology, but in five hypoxia response systems analyzed, we also found evidence for 'adaptable' characters at all levels of organization in all three high altitude lineages. Since Andeans and Himalayans have not shared common ancestry with East Africans for most of our species history, we suggest that their similar hypoxia physiology may represent the 'ancestral' condition for humans--an interpretation consistent with recent evidence indicating that our species evolved under 'colder, drier, and higher' conditions in East Africa where the phenotype would be simultaneously advantageous for endurance performance and for high altitude hypoxia. It is presumed that the phenotype was retained in low capacity form in highlanders and in higher capacity form in most lowland lineages (where it would be recognized by most physiologists as an endurance performance phenotype). Interestingly, it is easier for modern molecular evolution theory to account for the origin of 'adaptable' characters through positive selection than for conserved traits. Many conserved physiological systems are composed of so many gene products that it seems difficult to account for their unchanging state (for unchanging structure and function of hundreds of proteins linked in sequence to form the physiological system) by simple models of stabilizing selection.