A putative route to ecdysteroids: Metabolism of cholesterol in vitro by mildly disrupted prothoracic glands of Manduca sexta

Abstract

Mechanical disruption of day 7 fifth instar Manduca sexta larval prothoracic glands prior to in vitro incubation with [³H]cholesterol resulted in a dramatic 10-fold increase in its conversion to [³H]7-dehydrocholesterol (20–50%), when compared to intact gland incubations (2–5%). Both procedures resulted in a 0.5–2% conversion to [³H]ecdysteroids. Endogenous cholesterol levels also decreased by this same 20–50% during the incubation, suggesting that the added tracer [³H]cholesterol was equilibrated with the total endogenous cholesterol pool. Glands from earlier fifth instar larvae were capable of similar conversion of [³H]cholesterol to [³H]7-dehydrocholesterol but without concomitant conversion to [³H]ecdysteroids, while in day 7 glands, conversion to [³H]ecdysteroids was temporally correlated with both the in vitro secretory activity of intact glands and the endogenous hemolymph ecdysteroid titer. These data suggest that the rate-limiting step in ecdysteroid biosynthesis occurs after the synthesis of 7-dehydrocholesterol. In addition to 7-dehydrocholesterol and ecdysteroids, four intermediate polarity metabolites were detected following the incubation of disrupted prothoracic glands. One, M1, appears to be an immediate precursor of ecdysone and 3-dehydroecdysone. Another, M3, while not a precursor of the ecdysteroids, may be a degradation product of a proposed epoxide intermediate of 7-dehydrocholesterol. A hypothetical scheme for the biosynthesis of ecdysteroids from cholesterol is presented.