An Ethanolic Extract of Artemisia dracunculus L. Enhances the Metabolic Benefits of Exercise in Diet-induced Obese Mice.

PURPOSE: The purpose of this study was to determine the effect of an ethanolic extract of Artemisia dracunculus L. (5011) combined with exercise on in vivo glucose and fat metabolism in diet-induced obese male mice. METHODS: After 8 wk of high-fat diet (HFD) feeding, 52 mice were randomly allocated to a voluntary wheel running group (HFD Ex), a 5011 + HFD sedentary group (5011 Sed), a 5011 + HFD Ex (5011 Ex), or an HFD sedentary group (HFD Sed) for 4 wk. Real-time energy expenditure and substrate utilization were measured by indirect calorimetry. A stable isotope glucose tolerance test was performed before and after the 4-wk wheel running period to determine changes in endogenous glucose production and glucose disposal. We also performed an analysis of genes and proteins associated with the early response to exercise and exercise adaptations in skeletal muscle and liver. RESULTS: When compared with HFD Ex mice, 5011 Ex mice had increased fat oxidation during speed- and distance-matched wheel running bouts. Both HFD Ex and 5011 Ex mice had reduced endogenous glucose during the glucose tolerance test, whereas only the 5011 Sed and the 5011 Ex mice had improved glucose disposal after the 4-wk experimental period when compared with HFD Sed and HFD Ex mice. 5011 Ex mice had increased Pgc1-α and Tfam expression in skeletal muscle when compared with HFD Ex mice, whereas Pdk4 expression was reduced in the liver of HFD Ex and 5011 Ex mice. CONCLUSIONS: Our study demonstrates that 5011, an ethanolic extract of A. dracunculus L., with a history of medicinal use, enhances the metabolic benefits of exercise to improve in vivo fat and glucose metabolism.

Difference in Housing Temperature-Induced Energy Expenditure Elicits Sex-Specific Diet-Induced Metabolic Adaptations in Mice.

OBJECTIVE: The aim of this study was to test whether increased energy expenditure (EE), independent of physical activity, reduces acute diet-induced weight gain through tighter coupling of energy intake to energy demand and enhanced metabolic adaptations. METHODS: Indirect calorimetry and quantitative magnetic resonance imaging were used to assess energy metabolism and body composition during 7-day high-fat/high-sucrose (HFHS) feeding in male and female mice housed at divergent temperatures (20°C vs. 30°C). RESULTS: As previously observed, 30°C housing resulted in lower total EE and energy intake compared with 20°C mice regardless of sex. Interestingly, housing temperature did not impact HFHS-induced weight gain in females, whereas 30°C male mice gained more weight than 20°C males. Energy intake coupling to EE during HFHS feeding was greater in 20°C versus 30°C housing, with females greater at both temperatures. Fat mass gain was greater in 30°C mice compared with 20°C mice, whereas females gained less fat mass than males. Strikingly, female 20°C mice gained considerably more fat-free mass than 30°C mice. Reduced fat mass gain was associated with greater metabolic flexibility to HFHS, whereas fat-free mass gain was associated with diet-induced adaptive thermogenesis. CONCLUSIONS: These data reveal that EE and sex interact to impact energy homeostasis and metabolic adaptation to acute HFHS feeding, altering weight gain and body composition change.

Validation of an equation for energy expenditure that does not require the respiratory quotient.

BACKGROUND: Energy expenditure (EE) calculated from respirometric indirect calorimetry is most accurate when based on oxygen consumption (VO2), carbon dioxide production (VCO2) and estimated protein metabolism (PM). EE has a substantial dependence of ~7% on the respiratory quotient (RQ, VCO2/VO2) and a lesser dependence on PM, yet many studies have instead estimated EE from VO2 only while PM has often been ignored, thus reducing accuracy. In 1949 Weir proposed a method to accurately calculate EE without using RQ, which also adjusts for estimated PM based on dietary composition. This RQ- method utilizes the calorimeter airflow rate (FR), the change in fractional O2 concentration (ΔFO2) and the dietary protein fraction. The RQ- method has not previously been empirically validated against the standard RQ+ method using both VO2 and RQ. Our aim was to do that. METHODS: VO2 and VCO2 were measured repeatedly in 8 mice fed a high protein diet (HPD) during exposure to different temperatures (n = 168 measurements of 24h gas exchange). The HPD-adjusted RQ+ equation was: EE [kcal/time] = VO2 [L/time]×(3.853+1.081RQ) while the corresponding RQ- equation was: EE = 4.934×FR×ΔFO2. Agreement was analyzed using the ratios of the RQ- to RQ+ methods along with regression and Bland-Altman agreement analyses. We also evaluated the standard equation using the dietary food quotient (FQ) of 0.91 as a proxy for RQ (FQ+ method). RESULTS: Ratio analysis revealed that the mean error of the RQ- method was only 0.11 ± 0.042% while the maximum error was only 0.21%. Error using the FQ+ method was 4 -and 10-fold greater, respectively. Bland-Altman analysis demonstrated that the RQ- method very slightly overestimates EE as RQ decreases. Theoretically, this error can be eliminated completely by imposing an incurrent fractional oxygen concentration at a value only slightly greater than the atmospheric level. CONCLUSIONS: The Weir 'RQ-free' method for calculating EE is a highly valid alternative to the 'gold standard' method that requires RQ. The RQ- approach permits reduced cost and complexity in studies focused on EE and provides a way to rescue EE measurement in studies compromised by faulty CO2 measurements. Practitioners of respirometry should consider adjusting EE calculations for estimated protein metabolism based on dietary composition.

Genetic variation for tolerance to high temperatures in a population of Drosophila melanogaster.

The range of thermal tolerance is one of the main factors influencing the geographic distribution of species. Climate change projections predict increases in average and extreme temperatures over the coming decades; hence, the ability of living beings to resist these changes will depend on physiological and adaptive responses. On an evolutionary scale, changes will occur as the result of selective pressures on individual heritable differences. In this work, we studied the genetic basis of tolerance to high temperatures in the fly Drosophila melanogaster and whether this species presents sufficient genetic variability to allow expansion of its upper thermo-tolerance limit. To do so, we used adult flies derived from a natural population belonging to the Drosophila Genetic Reference Panel, for which genomic sequencing data are available. We characterized the phenotypic variation of the upper thermal limit in 34 lines by measuring knockdown temperature (i.e., critical thermal maximum [CTmax]) by exposing flies to a ramp of increasing temperature (0.25°C/min). Fourteen percent of the variation in CTmax is explained by the genetic variation across lines, without a significant sexual dimorphism. Through a genomewide association study, 12 single nucleotide polymorphisms associated with the CTmax were identified. In most of these SNPs, the less frequent allele increased the upper thermal limit suggesting that this population harbors raw genetic variation capable of expanding its heat tolerance. This potential upper thermal tolerance increase has implications under the global warming scenario. Past climatic records show a very low incidence of days above CTmax (10 days over 25 years); however, future climate scenarios predict 243 days with extreme high temperature above CTmax from 2045 to 2070. Thus, in the context of the future climate warming, rising temperatures might drive the evolution of heat tolerance in this population by increasing the frequency of the alleles associated with higher CTmax.

Extreme physiological plasticity in a hibernating basoendothermic mammal, Tenrec ecaudatus.

Physiological plasticity allows organisms to respond to diverse conditions. However, can being too plastic actually be detrimental? Malagasy common tenrecs, Tenrec ecaudatus, have many plesiomorphic traits and may represent a basal placental mammal. We established a laboratory population of T. ecaudatus and found extreme plasticity in thermoregulation and metabolism, a novel hibernation form, variable annual timing, and remarkable growth and reproductive biology. For instance, tenrec body temperature (Tb) may approximate ambient temperature to as low as 12°C even when tenrecs are fully active. Conversely, tenrecs can hibernate with Tb of 28°C. During the active season, oxygen consumption may vary 25-fold with little or no change in Tb During the austral winter, tenrecs are consistently torpid but the depth of torpor may vary. A righting assay revealed that Tb contributes to but does not dictate activity status. Homeostatic processes are not always linked, e.g. a hibernating tenrec experienced a ∼34% decrease in heart rate while maintaining constant body temperature and oxygen consumption rates. Tenrec growth rates vary but young may grow ∼40-fold in the 5 weeks until weaning and may possess indeterminate growth as adults. Despite all of this profound plasticity, tenrecs are surprisingly intolerant of extremes in ambient temperature (<8 or >34°C). We contend that while plasticity may confer numerous energetic advantages in consistently moderate environments, environmental extremes may have limited the success and distribution of plastic basal mammals.

Reduced Nonexercise Activity Attenuates Negative Energy Balance in Mice Engaged in Voluntary Exercise.

Exercise alone is often ineffective for treating obesity despite the associated increase in metabolic requirements. Decreased nonexercise physical activity has been implicated in this resistance to weight loss, but the mechanisms responsible are unclear. We quantified the metabolic cost of nonexercise activity, or "off-wheel" activity (OWA), and voluntary wheel running (VWR) and examined whether changes in OWA during VWR altered energy balance in chow-fed C57BL/6J mice (n = 12). Energy expenditure (EE), energy intake, and behavior (VWR and OWA) were continuously monitored for 4 days with locked running wheels followed by 9 days with unlocked running wheels. Unlocking the running wheels increased EE as a function of VWR distance. The metabolic cost of exercise (kcal/m traveled) decreased with increasing VWR speed. Unlocking the wheel led to a negative energy balance but also decreased OWA, which was predicted to mitigate the expected change in energy balance by ∼45%. A novel behavioral circuit involved repeated bouts of VWR, and roaming was discovered and represented novel predictors of VWR behavior. The integrated analysis described here reveals that the weight loss effects of voluntary exercise can be countered by a reduction in nonexercise activity.

Hyperthermic overdrive: oxygen delivery does not limit thermal tolerance in Drosophila melanogaster.

The causes of thermal tolerance limits in animals are controversial. In many aquatic species, it is thought that the inability to deliver sufficient oxygen at high temperatures is more critical than impairment of molecular functions of the mitochondria. However, terrestrial insects utilize a tracheal system, and the concept of a mismatch between metabolic demand and circulatory performance might not apply to them. Using thermo-limit respirometry, it has been shown earlier in Drosophila melanogaster that CO(2) release rates at temperatures above the upper thermal limit (CT(max)) exceed the rate at CT(max). The nature of this post-CT(max), or "post-mortal" peak, is unknown. Either its source is increased aerobic mitochondrial respiration (hyperthermic overdrive), or an anaerobic process such as liberation of stored CO(2) from the hemolymph. The post-mortal peak of CO(2) release was found to be oxygen dependent. As the rate of CO(2) emission is a conservative indicator of rate of O(2) consumption, aerobic flux at the thermal limit is submaximal, which contradicts the theory that oxygen availability limits metabolic activity at high temperatures in insects. Consequently, the tracheal system should be capable of delivering sufficient oxygen for aerobic activity of the mitochondria at and above Ct(max).

Effects of temperature on responses to anoxia and oxygen reperfusion in Drosophila melanogaster.

Insects in general, and Drosophila in particular, are much more capable of surviving anoxia than vertebrates, and the mechanisms involved are of considerable biomedical and ecological interest. Temperature is likely to strongly affect both the rates of damage occurring in anoxia and the recovery processes in normoxia, but as yet there is no information on the effect of this crucial variable on recovery rates from anoxia in any animal. We studied the effects of temperature, and thus indirectly of metabolic flux rates, on survival and recovery times of individual male Drosophila melanogaster following anoxia and O(2) reperfusion. Individual flies were reared at 25°C and exposed to an anoxic period of 7.5, 25, 42.5 or 60 min at 20, 25 or 30°C. Before, during and after anoxic exposure the flies' metabolic rates (MRs), rates of water loss and activity indices were recorded. Temperature strongly affected the MR of the flies, with a Q(10) of 2.21. Temperature did not affect the slope of the relationship between time to recovery and duration of anoxic exposure, suggesting that thermal effects on damage and repair rates were similar. However, the intercept of that relationship was significantly lower (i.e. recovery was most rapid) at 25°C, which was the rearing temperature. When temperatures during exposure to anoxia and during recovery were switched, recovery times matched those predicted from a model in which the accumulation and clearance of metabolic end-products share a similar dependence on temperature.

A new approach for flow-through respirometry measurements in humans.

Indirect whole room calorimetry is commonly used in studies of human metabolism. These calorimeters can be configured as either push or pull systems. A major obstacle to accurately calculating gas exchange rates in a pull system is that the excurrent flow rate is increased above the incurrent flow rate, because the organism produces water vapor, which also dilutes the concentrations of respiratory gases in the excurrent sample. A common approach to this problem is to dry the excurrent gases prior to measurement, but if drying is incomplete, large errors in the calculated oxygen consumption will result. The other major potential source of error is fluctuations in the concentration of O(2) and CO(2) in the incurrent airstream. We describe a novel approach to measuring gas exchange using a pull-type whole room indirect calorimeter. Relative humidity and temperature of the incurrent and excurrent airstreams are measured continuously using high-precision, relative humidity and temperature sensors, permitting accurate measurement of water vapor pressure. The excurrent flow rates are then adjusted to eliminate the flow contribution from water vapor, and respiratory gas concentrations are adjusted to eliminate the effect of water vapor dilution. In addition, a novel switching approach is used that permits constant, uninterrupted measurement of the excurrent airstream while allowing frequent measurements of the incurrent airstream. To demonstrate the accuracy of this approach, we present the results of validation trials compared with our existing system and metabolic carts, as well as the results of standard propane combustion tests.

Critical oxygen partial pressures and maximal tracheal conductances for Drosophila melanogaster reared for multiple generations in hypoxia or hyperoxia.

In Drosophila melanogaster and other insects, increases in atmospheric oxygen partial pressure (aPO(2)) tend to increase adult body size and decrease tracheal diameters and tracheolar proliferation. If changes in tracheal morphology allow for functional compensation for aPO(2), we would predict that higher aPO(2) would be associated with higher critical PO(2) values (CritPO(2)) and lower maximal tracheal conductances (G(max)). We measured CritPO(2) and G(max) for adult and larval vinegar flies reared for 7-9 generations in 10, 21 or 40 kPa O(2). The CritPO(2), CO(2) emission rates and G(max) values were generally independent of the rearing PO(2) these flies had experienced, suggesting that minimal functional changes in tracheal capacities occurred in response to rearing PO(2). Larvae were able to continue activity during 20 min of anoxia. The lack of multigenerational rearing PO(2) effects on tracheal function suggests that the functional compensation at the whole-body level due to tracheal morphological changes in response to aPO(2) may be minimal; alternatively the benefits of such compensation may occur in specific tissues or during processes not assessed by these methods. In larvae, the CritPO(2) and the capacity for movement in anoxia suggest adaptations for life in hypoxic organic matter.

The hygric hypothesis does not hold water: abolition of discontinuous gas exchange cycles does not affect water loss in the ant Camponotus vicinus.

The discontinuous gas exchange cycle (DGC) of insects and other tracheate arthropods temporally decouples oxygen uptake and carbon dioxide emission and generates powerful concentration gradients for both gas species between the outside world and the tracheal system. Although the DGC is considered an adaptation to reduce respiratory water loss (RWL) - the "hygric hypothesis" - it is absent from many taxa, including xeric ones. The "chthonic hypothesis" states that the DGC originated as an adaptation to gas exchange in hypoxic and hypercapnic, i.e. underground, environments. If that is the case then the DGC is not the ancestral condition, and its expression is not necessarily a requirement for reducing RWL. Here we report a study of water loss rate in the ant Camponotus vicinus, measured while its DGC was slowly eliminated by gradual hypoxia (hypoxic ramp de-DGCing). Metabolic rate remained constant. The DGC ceased at a mean P(O2) of 8.4 kPa. RWL in the absence of DGCs was not affected until P(O2) declined below 3.9 kPa. Below that value, non-DGC spiracular regulation failed, accompanied by a large increase in RWL. Thus, the spiracular control strategy of the DGC is not required for low RWL, even in animals that normally express the DGC.

Breathe softly, beetle: continuous gas exchange, water loss and the role of the subelytral space in the tenebrionid beetle, Eleodes obscura.

Flightless, diurnal tenebrionid beetles are commonly found in deserts. They possess a curious morphological adaptation, the subelytral cavity (an air space beneath the fused elytra) the function of which is not completely understood. In the tenebrionid beetle Eleodes obscura, we measured abdominal movements within the subelytral cavity, and the activity of the pygidial cleft (which seals or unseals the subelytral cavity), simultaneously with total CO2 release rate and water loss rate. First, we found that E. obscura has the lowest cuticular permeability measured in flow-through respirometry in an insect (0.90 microg H2O cm(-2) Torr(-1) h(-1)). Second, it does not exhibit a discontinuous gas exchange cycle. Third, we describe the temporal coupling between gas exchange, water loss, subelytral space volume, and the capacity of the subelytral space to exchange gases with its surroundings as indicated by pygidial cleft state. Fourth, we suggest possible mechanisms that may reduce respiratory water loss rates in E. obscura. Finally, we suggest that E. obscura cannot exchange respiratory gases discontinuously because of a morphological constraint (small tracheal or spiracular conductance). This "conductance constraint hypothesis" may help to explain the otherwise puzzling phylogenetic patterns of continuous vs. discontinuous gas exchange observed in tracheate arthropods.

Oxygen reperfusion damage in an insect.

The deleterious effects of anoxia followed by reperfusion with oxygen in higher animals including mammals are well known. A convenient and genetically well characterized small-animal model that exhibits reproducible, quantifiable oxygen reperfusion damage is currently lacking. Here we describe the dynamics of whole-organism metabolic recovery from anoxia in an insect, Drosophila melanogaster, and report that damage caused by oxygen reperfusion can be quantified in a novel but straightforward way. We monitored CO(2) emission (an index of mitochondrial activity) and water vapor output (an index of neuromuscular control of the spiracles, which are valves between the outside air and the insect's tracheal system) during entry into, and recovery from, rapid-onset anoxia exposure with durations ranging from 7.5 to 120 minutes. Anoxia caused a brief peak of CO(2) output followed by knock-out. Mitochondrial respiration ceased and the spiracle constrictor muscles relaxed, but then re-contracted, presumably powered by anaerobic processes. Reperfusion to sustained normoxia caused a bimodal re-activation of mitochondrial respiration, and in the case of the spiracle constrictor muscles, slow inactivation followed by re-activation. After long anoxia durations, both the bimodality of mitochondrial reactivation and the recovery of spiracular control were impaired. Repeated reperfusion followed by episodes of anoxia depressed mitochondrial respiratory flux rates and damaged the integrity of the spiracular control system in a dose-dependent fashion. This is the first time that physiological evidence of oxygen reperfusion damage has been described in an insect or any invertebrate. We suggest that some of the traditional approaches of insect respiratory biology, such as quantifying respiratory water loss, may facilitate using D. melanogaster as a convenient, well-characterized experimental model for studying the underlying biology and mechanisms of ischemia and reperfusion damage and its possible mitigation.

Respiratory biology: they would be giants.

A recent study has shown that respiratory structures are disproportionately oversized in larger insects, and that oxygen supply to leg muscles may be physically constrained in the largest modern insects. High oxygen concentrations during the Carboniferous may have alleviated these physical constraints allowing the evolution of gigantic arthropods.

Respiratory biology: why insects evolved discontinuous gas exchange.

Many, but not all, insects breathe in a discontinuous gas-exchange cycle. A recent study has evaluated rival hypotheses for the evolution of this trait, concluding that the most likely is the one invoking minimization of respiratory water loss.

Discontinuous gas exchange in insects: a clarification of hypotheses and approaches.

Many adult and diapausing pupal insects exchange respiratory gases discontinuously in a three-phase discontinuous gas exchange cycle (DGC). We summarize the known biophysical characteristics of the DGC and describe current research on the role of convection and diffusion in the DGC, emphasizing control of respiratory water loss. We summarize the main theories for the evolutionary genesis (or, alternatively, nonadaptive genesis) of the DGC: reduction in respiratory water loss (the hygric hypothesis), optimizing gas exchange in hypoxic and hypercapnic environments (the chthonic hypothesis), the hybrid of these two (the chthonic-hygric hypothesis), reducing the toxic properties of oxygen (the oxidative damage hypothesis), the outcome of interactions between O(2) and CO(2) control set points (the emergent property hypothesis), and protection against parasitic invaders (the strolling arthropods hypothesis). We describe specific techniques that are being employed to measure respiratory water loss in the presence or absence of the DGC in an attempt to test the hygric hypothesis, such as the hyperoxic switch and H(2)O/CO(2) regression, and summarize specific areas of the field that are likely to be profitable directions for future research.

To DGC or not to DGC: oxygen guarding in the termite Zootermopsis nevadensis (Isoptera: Termopsidae).

The ability of some insects to engage in complex orchestrations of tracheal gas exchange has been well demonstrated, but its evolutionary origin remains obscure. According to a recently proposed hypothesis, insects may employ spiracular control of gas exchange to guard tissues against long-term oxidative damage by using the discontinuous gas-exchange cycle (DGC) to limit internal oxygen partial pressure (PO2). This manuscript describes a different approach to oxygen guarding in the lower termite Zootermopsis nevadensis. These insects do not display a DGC but respond to elevated oxygen concentrations by restricting spiracular area, resulting in a transient decline in CO2 emission. High internal CO2 concentrations are then maintained; restoring normoxia results in a transient reciprocal increase in CO2 emission caused by release of excess endotracheal CO2. These changes in spiracular area reflect active guarding of low internal O2 concentrations and demonstrate that regulation of endotracheal hypoxia takes physiological priority over prevention of CO2 build-up. This adaptation may reflect the need to protect oxygen-sensitive symbionts (or, gut bug guarding). Termites may eschew the DGC because periodic flushing of the tracheal system with air may harm the obligate anaerobes upon which the lower termites depend for survival on their native diet of chewed wood.

Respiratory physiology: strange cycles and the fruit-fly's tongue.

Drosophila in flight show an unexpected cyclicity in gas exchange even at constant metabolic flux rates--perhaps because regular proboscis extensions assist in providing oxygen to the fly's brain.

Respiratory and cuticular water loss in insects with continuous gas exchange: comparison across five ant species.

Respiratory water loss (RWL) in insects showing continuous emission of CO(2) is poorly studied because few methodologies can measure it. Comparisons of RWL between insects showing continuous and discontinuous gas exchange cycles (DGC) are therefore difficult. We used two recently developed methodologies (the hyperoxic switch and correlation between water-loss and CO(2) emission rates) to compare cuticular permeabilities and rates of RWL in five species of ants, the Argentine ant (Linepithema humile) and four common native ant competitors. Our results showed that RWL in groups of ants with moderate levels of activity and continuous gas exchange were similar across the two measurement methods, and were similar to published values on insects showing the DGC. Furthermore, ants exposed to anoxia increased their total water loss rates by 50-150%. These results suggest that spiracular control under continuous gas exchange can be as effective as the DGC in reducing RWL. Finally, the mesic-adapted Argentine ant showed significantly higher rates of water loss and cuticular permeability compared to four ant species native to dry environments. Physiological limitations may therefore be responsible for restricting the distribution of this invasive species in seasonally dry environments.