James A. Svoboda

Insect steroid metabolism

Insects are unable to biosynthesize the steroid nucleus and generally require an exogenous source of sterols. Two salient areas of insect steroid metabolism are the dealkylation and conversion of dietary C28 and C29 plant sterols to cholesterol and other C27 sterols, and the biosynthesis and metabolism of the steroidal insect molting hormones. Certain azasteroids and nonsteroidal amines block this conversion of 24-alkyl sterols to cholesterol and/or disrupt molting and development in insects. These inhibitors have served in charting metabolic pathways for steroids in insects and are serving as models in developing selective pesticidal chemicals and chemotherapeutic agents for use against insects and other invertebrate pests and parasites. The mode of action of some of these inhibitors on molting and development has been investigated in vivo and in vitro. Certain of these inhibitors represent a new class of insect hormonal compounds with a novel mode of action—the disruption of molting hormone metabolism. Research on sterol metabolism in insects provides important information on the comparative biochemistry and physiological functions of steroids in living systems.

Metabolism of Steroids in Insects

Publisher Summary This chapter discusses metabolism of steroids in insects. Insects require a dietary or exogenous source of sterol for normal growth, metamorphosis, and reproduction, and the only known exceptions to this are those species in which a sterol source can be attributed to associated symbiotes. The hornworm utilizes the major sterols of its primary host plant, tobacco—campesterol, sitosterol, and stigmasterol—through their dealkylation and conversion to cholesterol. These conversions proceed through a series of oxidation-reductive steps, and a number of the metabolites and intermediates involved can be characterized. Cholesterol serves both as a structural component of cells and tissues and as a precursor for the ecdysones or molting hormones. While the sulfate and glucoside conjugates of sterols and ecdysteroids function, primarily, in the inactivation and excretion of steroids in insects, these conjugates can have other equally important roles related to the biosynthesis, metabolism, and transport of steroids, or the regulation of hormone titer.

The synthesis and the mass and nuclear magnetic resonance spectra of side chain isomers of cholesta-5,22-dien-3β-ol and cholesta-5,22,24-trien-3β-ol

Abstract The previously unknown 22- cis -cholesta-5,22-dien-3β-ol, 22- cis and 22- trans isomers of cholesta-5,22,24-trien-3β-ol, 20- iso -22- trans -cholesta-5,22,24-trien-3β-ol, 22,24- trans , trans -26-homocholesta-5,22,24-trien-3β-ol, and the known 22- trans -dehydrocholesterol were synthesized. Their infrared, mass and nuclear magnetic resonance spectra are presented. These compounds undergo fragmentation processes that are governed primarily by the site of the unsaturation and not by their stereochemistry. However, the C-methyl resonances in the nuclear magnetic resonance spectra are influenced by their chemical environment.

Desmosterol, an intermediate in dealkylation of β-sitosterol in the tobacco hornworm

The mechanism of dealkylation of phytosterols such as β-sitosterol to cholesterol in insects is an important and little understood biochemical transformation. This alteration of the sterol side chain provides a means for omnivorous and particularly phytophagous insects to obtain their essential cholesterol from foodstuffs that are either very low or completely lacking in cholesterol but which contain substantial quantities of plant sterols. Dealkylation has been reported or inferred for a number of insects and has definitely been demonstrated to occur in certain species through the use of semidefined diets, radiotracer labeled sterols, and/or gas-liquid chromatographic analysis (1,2,3). In these insects, only the final metabolite of this process, cholesterol or a cholestane derivative, has been identified, and nothing is known of the intermediate reactions. Recent research has greatly enhanced our understanding of the alkylation process that occurs during the biosynthesis of plant sterols (4,5,6). It would be of extreme interest from a comparative biochemical standpoint to determine whether the alkylation and dealkylation processes share certain common intermediates or proceed through common pathways. During a study in our laboratory on the rate of conversion of H3-β-sitosterol to cholesterol in the tobacco hornworm, Manducasexta (Johannson), an unknown H3-labeled sterol component was detected. The isolation and identification of this sterol as desmosterol (Δ5,24-cholestadien-3β-o1) and proof of its occurrence as an intermediate in the dealkylation process is the topic of this report.

Metabolic pathways of steroids in insects

This paper discusses sterol metabolism and utilization in insects with particular emphasis on the two known major metabolic pathways for steroids in insects: (1) The conversion of C28 and C29 plant sterols to cholesterol, including a comprehensive survey of the intermediates involved in the conversion of phytosterols to cholesterol, certain inhibitors of phytosterol metabolism, and the end products of sterol metabolism in insects. (2) The biosynthesis and metabolism of the steroid moulting hormones of insects (ecdysones), including the present state of our knowledge of the sterol precursors of the ecdysones, the ecdysteroid intermediates in their biosynthesis, and the inactivation of the ecdysteroids in insects. The biological and physiological significance of these metabolic pathways in insects is discussed, as well as their relationship to analogous pathways in plants and vertebrates.

Ecdysone metabolism: Ecdysone dehydrogenase-isomerase

An enzyme system that converts α-ecdysone to its hormonally less active 3α-epimer was detected only in the midgut of the tobacco hornworm,Manduca sexta (L.). This system appears to be specific for the ecdysones and may represent a metabolic control point for regulating molting hormone activity.

Makisterone A and 24-methylenecholesterol from the ovaries of the honey bee,Apis mellifera L

Makisterone A, a 28-carbon ecdysteroid (molting hormone) has been isolated from the ovaries of queen bees. Analysis by reversed-phase and silica high performance liquid chromatography (HPLC) in conjunction with a radioimmune assay (RIA) revealed about 11 ng of makisterone A present per gram of ovaries on a fresh weight basis. No C27 ecdysteroids were detected. The predominant neutral sterol present was 24-methylenecholesterol.

Azasteroids: Potent inhibitors of insect molting and metamorphosis

Based on previous structure-activity studies, four new azasteroids were synthesized and tested in several species of insects. These compounds, which are the most potent azasteroid inhibitors of insect growth and development tested to date, affect the hormone-regulated processes of molting and metamorphosis.

The inhibitive effects of azasterols on sterol metabolism and growth and development in insects with special reference to the tobacco hornworm

Several monoazasterols were found to be potent inhibitors of Δ24- and Δ22,24- reductase enzyme(s) in the tobacco hornworm,Manduca sexta (Johannson). Certain of these inhibitors also prevented normal larval development and pupation in the hornworm at dietary concentrations in the parts per million range. Comparative studies with several different insects indicated differences between the species with respect to the effects of the azasterols. The relationship of azasterol structure to the inhibitory effect(s) on sterol metabolism and larval development is discussed.

Sterol metabolism in the nematodeCaenorhabditis elegans

The metabolism of various dietary sterols and the effects of an azasteroid on sitosterol metabolism in the free-living nematodeCaenorhabditis elegans was investigated. The major unesterified sterols ofC. elegans in media supplemented with sitosterol, cholesterol or desmosterol included 7-dehydrocholesterol (66.5%, 40.5%, 31.2%, respectively), cholesterol (6.7%, 52.3%, 26.9%), lathosterol (4.4%, 3.6%, 1.7%) and 4α-methylcholest-8(14)-en-3β-ol (4.2%, 2.1%, 3.8%). Esterified sterols, representing less than 20% of the total sterols, were somewhat similar except for a significantly higher relative content of 4α-methylcholest-8(14)-en-3β-ol (23.3%, 23.4%, 10.6%). ThusC. elegans not only removes the substituent at C24 of dietary sitosterol but possesses the unusual ability to produce significant quantities of 4α-methylsterols. WhenC. elegans was propagated in medium supplemented with sitosterol plus 5 μg/ml of 25-azacoprostane hydrochloride, the azasteroid strongly interfered with reproduction and motility ofC. elegans and strongly inhibited the Δ24-sterol reductase enzyme system; excluding sitosterol, the major free sterols of azacoprostane-treatedC. elegans were cholesta-5, 7, 24-trien-3β-ol (47.9%), desmosterol (9.4%), fucosterol (2.1%) and cholesta-7,24-dien-3β-ol (2.0%). These 4 sterols are likely intermediates in the metabolism of sitosterol inC. elegans.