Kerry Withers

The effects of fasting and cold exposure on metabolic rate and mitochondrial proton leak in liver and skeletal muscle of an amphibian, the cane toad Bufo marinus

SUMMARY Futile cycling of protons across the mitochondrial inner membrane contributes significantly to standard metabolic rate in a variety of ectothermic and endothermic animals, but adaptations of the mitochondrial bioenergetics to different environmental conditions have rarely been studied in ectotherms. Changes in ambient temperature and nutritional status have a great effect on the physiological demands of ectothermic amphibians and may require the adjustment of mitochondrial efficiency. In order to investigate the effect of temperature and nutritional status on the mitochondrial level, we exposed male cane toads to either 10°C or 30°C and fasted half of the animals in each group. Cold exposure resulted in a fourfold reduction of the resting metabolic rate whereas nutritional status had only minor effects. The mitochondrial adjustments to each condition were observed by comparing the proton leak kinetics of isolated liver and skeletal muscle mitochondria at 25°C. In response to cold exposure, liver mitochondria showed a decrease in proton conductance while skeletal muscle mitochondria were unchanged. Additional food deprivation had minor effects in skeletal muscle, but in liver we uncovered surprising differences in energy saving mechanisms between the acclimation temperatures: in warm-acclimated toads, fasting resulted in a decrease of the proton conductance whereas in cold-acclimated toads, the activity of the respiratory chain was reduced. To investigate the molecular mechanism underlying mitochondrial proton leakage, we determined the adenine-nucleotide transporter (ANT) content, which explained tissue-specific differences in the basal proton leak, but neither the ANT nor uncoupling protein (UCP) gene expression correlated with alterations of the proton leak in response to physiological stimuli.

Phylogenetic differences of mammalian basal metabolic rate are not explained by mitochondrial basal proton leak.

Metabolic rates of mammals presumably increased during the evolution of endothermy, but molecular and cellular mechanisms underlying basal metabolic rate (BMR) are still not understood. It has been established that mitochondrial basal proton leak contributes significantly to BMR. Comparative studies among a diversity of eutherian mammals showed that BMR correlates with body mass and proton leak. Here, we studied BMR and mitochondrial basal proton leak in liver of various marsupial species. Surprisingly, we found that the mitochondrial proton leak was greater in marsupials than in eutherians, although marsupials have lower BMRs. To verify our finding, we kept similar-sized individuals of a marsupial opossum (Monodelphis domestica) and a eutherian rodent (Mesocricetus auratus) species under identical conditions, and directly compared BMR and basal proton leak. We confirmed an approximately 40 per cent lower mass specific BMR in the opossum although its proton leak was significantly higher (approx. 60%). We demonstrate that the increase in BMR during eutherian evolution is not based on a general increase in the mitochondrial proton leak, although there is a similar allometric relationship of proton leak and BMR within mammalian groups. The difference in proton leak between endothermic groups may assist in elucidating distinct metabolic and habitat requirements that have evolved during mammalian divergence.

Determining the age of adult wild dogs (Canis lupus dingo, C. l. domesticus and their hybrids). I. Pulp cavity : tooth width ratios

In order to determine the age of adult wild dogs, we compared two methods (that of Thomson and Rose (TR method) and that of Knowlton and Whittemore (KW method)) of measuring and calculating pulp cavity:tooth width ratios on upper and lower canine teeth from 68 mixed-sex, known-age wild dogs of 9 months to 13 years of age reared at two localities. Although significant relationships (P = 0.0001) were found between age and pulp cavity ratios by both methods, the TR ratio calculation and measurement showed heteroscedasity in error variance whereas the KW ratios had a more stable error variance and were normally distributed. The KW method also found significant differences between pulp cavity ratios between teeth of the upper and lower jaws (P < 0.0001) and sex (P = 0.01) but not geographic origin (P = 0.1). Regressions and formulae for fitted curves are presented separately for male and female wild dogs. Males show greater variability in pulp cavity decrements with age than do females, suggesting a physiological difference between the sexes. We conclude that the KW method of using pulp cavity as a proportion of tooth width, measured 15 mm from the root tip and averaged over both upper canines, is the more accurate method of estimating the age of adult wild dogs.

Measuring the density of dingo teeth with machine vision

Collaboration between a mechatronics engineer and a biologist resulted in an unlikely application of machine vision. To deduce the density of the porous teeth, the volume had to be found. An expedient method was constructed for scanning the teeth before they had to be returned to their source and a simple method was derived for deducing their volume.

Mitochondrial Proton Conductance in Skeletal Muscle of a Cold‐Exposed Marsupial, Antechinus flavipes, Is Unlikely to Be Involved in Adaptive Nonshivering Thermogenesis but Displays Increased Sensitivity toward Carbon‐Centered Radicals

The organs and molecular mechanisms contributing to adaptive thermogenesis in marsupials are not known because some species apparently lack brown adipose tissue (BAT). The increased oxidative capacity and presence of uncoupling protein 3 (UCP3) in skeletal muscle led to speculations on whether uncoupled respiration sustains endothermy in the cold, as found for BAT. Here, we investigated the role of mitochondrial proton conductance in the small Australian marsupial Antechinus flavipes during cold exposure. Although there was a tendency toward higher oxidative capacity in skeletal muscle, indicating metabolic adjustments to the cold, we observed no change in basal proton conductance of isolated myotubular and liver mitochondria. In eutherians, 4‐hydroxynonenal (HNE) is an activator of mitochondrial uncoupling mediated by UCP3 and ANT (adenine nucleotide translocase). In the marsupial A. flavipes, proton conductance in myotubular mitochondria could be induced by HNE selectively in the cold‐acclimated group. Induced uncoupling activity could be attributed to the ANT as judged by inhibition with carboxyatractylate, while GDP, a putative inhibitor of rodent UCP3, had no detectable effects on marsupial UCP3. In contrast to previous expectations, basal proton conductance in the myotubular mitochondria of marsupials does not contribute to adaptive thermogenesis, as found for eutherian BAT. Increased sensitivity of proton conductance to HNE by the ANT suggests a greater requirement for mild uncoupling activity that may convey protection from lipid peroxidation and mitigate reactive oxygen species production during cold stress.

Torpor in the Carnivorous Marsupial Sminthopsis macroura: Effects of Food Quality and Quantity

The effects of food quality (dietary protein content) and food quantity (ad lib vs food-restriction) on torpor were determined in six stripe-faced dunnarts (Sminthopsis macroura) by measuring metabolic rate (MR) at Ta of 18°C. The aims of the study were to determine whether increased dietary protein content delays the commencement of torpor and reduces the duration of torpor and whether food restriction induces torpor earlier and for a longer period. There was no effect of dietary protein content on torpor, irrespective of whether S. macroura were food-restricted or whether food was available ad lib. Torpor in foodrestricted S. macroura occurred earlier, was deeper and lasted longer. Food-restricted S. macroura lost less weight during torpor. Food-restriction also reduced average daily metabolic rate.