Metabolic Downregulation and Inhibition of Carbohydrate Catabolism during Diapause in Embryos ofArtemia franciscana

Diapause embryos were collected from ovigerous females ofArtemia franciscanaat the Great Salt Lake, Utah, and were synchronized to within 4 h of release. Respiration rate for these freshly released embryos across a subsequent 26-d time course showed a rapid decrease during the first several days followed thereafter by a much slower decline. The overall metabolic depression was estimated to be greater than 99%. However, proton conductance of mitochondria isolated from diapause and postdiapause embryos was identical. Because proton leak is apparently not downregulated during diapause, mitochondrial membrane potential is likely compromised because of the very low metabolic rate observed for diapause embryos. Given that trehalose is the primary fuel used by these embryos, we measured metabolic intermediates along the catabolic pathway from trehalose to acetyl-CoA for both diapause and postdiapause (active) embryos in order to identify sites of metabolic inhibition. Comparison of product-to-substrate ratios for sequential enzymatic steps revealed inhibition during diapause at trehalase, hexokinase, pyruvate kinase, and pyruvate dehydrogenase. Measurements of ATP, ADP, and AMP allowed calculations of substantial decreases in ATP∶ADP ratio and in adenylate energy charge during diapause. The phosphorylation of site 1 for pyruvate dehydrogenase (PDH) subunit E1α was higher in diapause embryos than in postdiapause embryos, which is consistent with PDH inhibition during diapause. Taken together, our findings indicate that restricted substrate availability to mitochondria for oxidative phosphorylation contributes to downregulating metabolic rate during diapause.