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First published online May 30, 2008
Journal of Experimental Biology 211, 1911-1918 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016519
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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

M. Trzcionka1,*, K. W. Withers2, M. Klingenspor1 and M. Jastroch1

1 Department of Animal Physiology, Faculty of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35043 Marburg, Germany
2 Centre for Systems Biology, University of Southern Queensland, Toowoomba, Queensland, Australia


Figure 1
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  		Fig. 1. Mass-specific resting metabolic rate (RMR; in ml O2 g–1 h–1) of fed and fasted cane toads acclimated to either 30°C (WA) or 10°C (CA), measured at their respective acclimation temperature. Values are means ± s.e.m., N=9 for warm acclimated and N=8 for cold acclimated toads. *P<0.05 (two-way ANOVA).

 

Figure 2
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  		Fig. 2. Full kinetic response of the proton leak rate to changes in membrane potential of liver and skeletal muscle mitochondria of fed WA cane toads. Liver mitochondria have a lower basal proton leak than skeletal muscle mitochondria. A rough extrapolation of the skeletal muscle and the liver curve would suggest that the lower liver proton leak is achieved by a decrease in the respiratory chain activity and not via a change in the proton leak kinetic function. Values are means ± s.e.m., N=9.

 

Figure 3
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  		Fig. 3. Effect of acclimation temperature on proton leak kinetics of isolated liver (A) and skeletal muscle mitochondria (B) of fed cane toads. Experiments were carried out using liver mitochondria of CA (open circles) and WA (filled circles) and skeletal muscle mitochondria of CA (open squares) and WA (filled squares) cane toads. A shift of the proton leak curve to the right indicates a lower proton conductance of CA liver mitochondria (A), while the acclimation temperature has no effect in skeletal muscle mitochondria (B). Values are means ± s.e.m. from eight (CA group) or nine (WA group) independent preparations. *P<0.05 (two-way ANOVA).

 

Figure 4
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  		Fig. 4. Effect of fasting on proton leak kinetics of isolated liver and skeletal muscle mitochondria of WA (A) and CA (B) cane toads. Fasting revealed two different mechanisms of decreasing the proton leak in liver mitochondria (fasted toads, grey circles; WA fed toads, filled circles; CA fed toads, open circles), dependent on acclimation temperature. In WA toads (A), fasting caused a shift of the proton leak curve to the right, with no change in state 4 respiration but increased state 4 potential, thus suggesting a decrease in proton conductance (similar to Fig. 3; P<0.05 comparing state 4 membrane potential). In contrast, fasting in CA cane toads (B) resulted not only in a significant decrease of membrane potential (P<0.05) but also in a strong tendency towards lower state 4 respiration rates, suggesting a decreased respiratory chain activity. In skeletal muscle mitochondria, fasting has no effect on proton leakage at either acclimation temperatures (fasted toads, grey circles; WA fed toads, filled squares;, CA fed toads, open squares). Values are means ± s.e.m., N=8 for CA toads and N=9 for WA toads.

 

Figure 5
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  		Fig. 5. Determination of the ANT content in isolated mitochondria by CAT titration. (A) CAT titre of respiration. State 3 respiration was titrated by successive additions of CAT to achieve state 4 respiration. ANT content was measured as the CAT titre where the steepest slope in the titration crosses the state 4 rate (broken lines). Results of a single representative determination are shown for liver mitochondria from fed cane toads acclimated to 30°C. (B,C) ANT contents of liver and skeletal muscle mitochondria of all experimental conditions measured by CAT titre (B, WA cane toads; C, CA cane toads). ANT content is about five times lower in liver mitochondria compared to skeletal muscle (P<0.05, two-way ANOVA.) but neither the temperature nor the nutritional status have an effect. Values are means ± s.e.m. from 4 independent preparations.*P<0.05 (two-way ANOVA).

 

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  		Fig. 6. Regulation of UCP2/3 gene expression in amphibians in response to cold and food deprivation. 5 µg total RNA were hybridized with homologous Xenopus laevis UCP2/3 probes. (A) Northern blot analysis showing tissue-specific expression of UCP2/3 in X. laevis. (B) UCP2/3 mRNA expression levels in liver and skeletal muscle of B. marinus in response to cold and fasting. In the liver, cold caused a significant upregulation of UCP2/3 mRNA expression levels while fasting had no effect. In skeletal muscle, no effect of acclimation temperature and fasting was found due to high individual differences. (C) The scatter plot overlaying the bar charts shows the individual values for B. marinus liver. Values are means of four animals per group; *P<0.05, two-way ANOVA.

 

Figure 7
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  		Fig. 7. Model summarizing mitochondrial bioenergetics in response to cold exposure and fasting. Effects on proton motive force (PMF, measured as membrane potential) and respiration (R, measured as state 4 respiration) in liver and skeletal muscle mitochondria. The boxes show the four conditions analyzed and include the state 4 membrane potentials (mV). Arrows indicate the changes in membrane potential and/or respiration in response to cold and/or fasting: black arrows, an increase; white arrows, a decrease; broken arrows, no change. In the liver, cold or fasting lead to an increase in membrane potential, while additional fasting in the cold causes an decrease in state 4 membrane potential and respiration rate. In skeletal muscle of cold-acclimated toads, fasting leads to a decrease in state 4 membrane potential.

 

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