Measurements of substrate oxidation using (13)CO 2-breath testing reveals shifts in fuel mix during starvation

Most fasting animals are believed to sequentially switch from predominantly utilizing one metabolic substrate to another from carbohydrates, to lipids, then to proteins. The timing of these physiological transitions has been estimated using measures of substrate oxidation including changes in respiratory exchange ratios, blood metabolites, nitrogen excretion, or enzyme activities in tissues. Here, we demonstrate how (13)CO2-breath testing can be used to partition among the oxidation of distinct nutrient pools in the body (i.e., carbohydrates, lipids, and proteins) that have become artificially enriched in (13)C. Seventy-two Swiss Webster mice were raised to adulthood on diets supplemented with (13)C-1-L-leucine, (13)C-1-palmitic acid, (13)C-1-D-glucose, or no tracer. Mice were then fasted for 72 h during which [Formula: see text], [Formula: see text], δ(13)C of exhaled CO2, body temperature, body mass, and blood metabolites (i.e., glucose, ketone bodies, and triacylglycerols) were measured. The fasting mice exhibited reductions in body mass (29 %), body temperature (3.3 °C), minimum observed metabolic rates (24 %), and respiratory exchange ratio (0.18), as well as significant changes in blood metabolites; but these responses were not particularly indicative of changes in oxidative fuel mixture. Measurements of endogenous nutrient oxidation by way of (13)CO2-breath testing revealed a decrease in the rate of oxidation of carbohydrates from 61 to 10 % of the total energy expenditure during the first 6 h without food. This response was mirrored by a coincidental increase in rate of endogenous lipid oxidation from 18 to 64 %. A transient peak in carbohydrate oxidation occurred between 8 and 14 h, presumably during increased glycogen mobilization. A well-defined period of protein sparing between 8 and 12 h was observed where endogenous protein oxidation accounted for as little as 8 % of the total energy expenditure. Thereafter, protein oxidation continually increased accounting for as much as 24 % of the total energy expenditure by 72 h. This study demonstrates that (13)CO2-breath testing may provide a complementary approach to characterizing the timing and magnitude of sequential changes in substrate oxidation that occur during prolonged fasting and starvation.