René Lafont

CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis.

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.

Lead phytotoxicity on wheat ( Triticum aestivum L. ) seed germination and seedlings growth

Lead (Pb) is an environmental pollutant extremely toxic to plants and other living organisms including humans. To assess Pb phytotoxicity, experiments focusing on germination of wheat seeds were germinated in a solution containing Pb (NO(3))(2) (0.05; 0.1; 0.5; 1g/L) during 6 days. Lead accumulation in seedlings was positively correlated with the external concentrations, and negatively correlated with morphological parameters of plant growth. Lead increased lipid peroxidation, enhanced soluble protein concentrations and induced a significant accumulation of proline in roots. Esterase activity was enhanced in the presence of lead, whereas α-amylase activity was significantly inhibited. Antioxidant enzymes activities, such as, ascorbate peroxidase, peroxidase, superoxide dismutase, catalase and glutathione S-transferase were generally significantly increased in the presence of lead in a dose-dependent manner. The present results thus provide a model system to screen for natural compounds able to counteract the deleterious effects of lead.

Identification and quantitative analysis of the phytoecdysteroids in Silene species (Caryophyllaceae) by high-performance liquid chromatography: Novel ecdysteroids from S. pseudotites

Many species in the genus Silene (Caryophyllaceae) have previously been shown to contain ecdysteroids and this genus is recognised as a good source of novel ecdysteroid analogues. We have used ecdysteroid-specific radioimmunoassays and the microplate-based Drosophila melanogaster B(II) cell bioassay for ecdysteroid agonist and antagonist activities to identify further phytoecdysteroid-containing species in this genus. The main ecdysteroid components from 10 Silene species (S. antirrhina, S. chlorifolia, S. cretica, S. disticha, S. echinata, S. italica, S. portensis, S. pseudotites, S. radicosa, S. regia) were isolated and identified, mainly by normal-phase and reversed-phase high-performance liquid chromatography. The amount of each ecdysteroid was determined by comparing chromatogram peak areas with those for reference 20-hydroxyecdysone (20E) on reversed-phase HPLC. 20E is the most abundant ecdysteroid in each of the Silene extracts. Polypodine B, 2-deoxy-20-hydroxyecdysone and ecdysone are also common ecdysteroids in these Silene species, but the proportions of these ecdysteroids vary between the Silene species. HPLC proved to be a quick and effective way to screen Silene species, determine ecdysteroid profiles and, hence, identify extracts containing novel analogues. An extract of the aerial parts of S. pseudotites was found to contain several new ecdysteroids. These have been isolated and identified spectroscopically (by NMR and mass spectrometry) as 2-deoxyecdysone 22beta-D-glucoside, 2-deoxy-20,26-dihydroxyecdysone and 2-deoxypolypodine B 3beta-D-glucoside. Additionally, (5alpha-H)-2-deoxyintegristerone A (5alpha-2H 91%, 5alpha-1H 9%) was isolated as an artefact. This study contributes to the understanding of ecdysteroid distribution in Silene species and provides further information on the chemotaxonomic significance of ecdysteroids in Silene species.

Quinoa Extract Enriched in 20‐Hydroxyecdysone Protects Mice From Diet‐Induced Obesity and Modulates Adipokines Expression

Besides their well‐known effect in the molting control in insects, ecdysteroids are steroid hormones that display potential pharmacologic and metabolic properties in mammals. The most common ecdysteroid, 20‐hydroxyecdysone (20E) is found in many plants such as quinoa. The aim of the present study was to investigate the ability of quinoa extract (Q) enriched in 20E supplementation to prevent the onset of diet‐induced obesity and to regulate the expression of adipocyte‐specific genes in mice. Mice were fed a standard low‐fat (LF) or a high‐fat (HF) diet with or without supplementation by 20E‐enriched Q or pure 20E for 3 weeks. Supplementation with Q reduced adipose tissue development in HF mice without modification of their body weight gain. This adipose tissue‐specific effect was mainly associated with a reduced adipocyte size and a decrease in the expression of several genes involved in lipid storage, including lipoprotein lipase and phosphoenolpyruvate carboxykinase. Furthermore, Q‐treated mice exhibited marked attenuation of mRNA levels of several inflammation markers (monocyte chemotactic protein‐1, CD68) and insulin resistance (osteopontin, plasminogen activator inhibitor‐1 (PAI‐1)) as compared to HF mice. Q supplementation also reversed the effects of HF‐induced downregulation of the uncoupling protein(s) (UCP(s)) mRNA levels in muscle. Similar results were obtained in mice fed a HF diet supplemented with similar amounts of pure 20E, suggesting that the latter accounted for most of the Q effects. Our study indicates that Q has an antiobesity activity in vivo and could be used as a nutritional supplement for the prevention and treatment of obesity and obesity‐associated disorders.

Quinoa extract enriched in 20-hydroxyecdysone affects energy homeostasis and intestinal fat absorption in mice fed a high-fat diet

In a previous study, we have demonstrated that a supplementation of a high-fat diet with a quinoa extract enriched in 20-hydroxyecdysone (QE) or pure 20-hydroxyecdysone (20E) could prevent the development of obesity. In line with the anti-obesity effect of QE, we used indirect calorimetry to examine the effect of dietary QE and 20E in high-fat fed mice on different components of energy metabolism. Mice were fed a high-fat (HF) diet with or without supplementation by QE or pure 20E for 3 weeks. As compared to mice maintained on a low-fat diet, HF feeding resulted in a marked physiological shift in energy homeostasis, associating a decrease in global energy expenditure (EE) and an increase in lipid utilization as assessed by the lower respiratory quotient (RQ). Supplementation with 20E increased energy expenditure while food intake and activity were not affected. Furthermore QE and 20E promoted a higher rate of glucose oxidation leading to an increased RQ value. In QE and 20E-treated HFD fed mice, there was an increase in fecal lipid excretion without any change in stool amount. Our study indicates that anti-obesity effect of QE can be explained by a global increase in energy expenditure, a shift in glucose metabolism towards oxidation to the detriment of lipogenesis and a decrease in dietary lipid absorption leading to reduced dietary lipid storage in adipose tissue.

The ecdysteroidome of Drosophila: influence of diet and development.

Ecdysteroids are the hormones regulating development, physiology and fertility in arthropods, which synthesize them exclusively from dietary sterols. But how dietary sterol diversity influences the ecdysteroid profile, how animals ensure the production of desired hormones and whether there are functional differences between different ecdysteroids produced in vivo remains unknown. This is because currently there is no analytical technology for unbiased, comprehensive and quantitative assessment of the full complement of endogenous ecdysteroids. We developed a new LC-MS/MS method to screen the entire chemical space of ecdysteroid-related structures and to quantify known and newly discovered hormones and their catabolites. We quantified the ecdysteroidome in Drosophila melanogaster and investigated how the ecdysteroid profile varies with diet and development. We show that Drosophila can produce four different classes of ecdysteroids, which are obligatorily derived from four types of dietary sterol precursors. Drosophila makes makisterone A from plant sterols and epi-makisterone A from ergosterol, the major yeast sterol. However, they prefer to selectively utilize scarce ergosterol precursors to make a novel hormone 24,28-dehydromakisterone A and trace cholesterol to synthesize 20-hydroxyecdysone. Interestingly, epi-makisterone A supports only larval development, whereas all other ecdysteroids allow full adult development. We suggest that evolutionary pressure against producing epi-C-24 ecdysteroids might explain selective utilization of ergosterol precursors and the puzzling preference for cholesterol. Summary: Quantifying the full complement of endogenous ecdysteroids in Drosophila provides insights into the metabolic pathways of ecdysteroid production, and how these vary with diet and development.

The metabolism of 20-hydroxyecdysone in mice: Relevance to pharmacological effects and gene switch applications of ecdysteroids

Ecdysteroids exert many pharmacological effects in mammals (including humans), most of which appear beneficial, but their mechanism of action is far from understood. Whether they act directly and/or after the formation of metabolites is still an open question. The need to investigate this question has gained extra impetus because of the recent development of ecdysteroid-based gene-therapy systems for mammals. In order to investigate the metabolic fate of ecdysteroids in mice, [1α,2α-(3)H]20-hydroxyecdysone was prepared and injected intraperitoneally to mice. Their excretory products (urine+faeces) were collected and the different tritiated metabolites were isolated and identified. The pattern of ecdysteroid metabolites is very complex, but no conjugates were found, in contrast to the classical fate of the (less polar) endogenous vertebrate steroid hormones. Primary reactions involve dehydroxylation at C-14 and side-chain cleavage between C-20 and C-22, thereby yielding 14-deoxy-20-hydroxyecdysone, poststerone and 14-deoxypoststerone. These metabolites then undergo several reactions of reduction involving, in particular, the 6-keto-group. A novel major metabolite has been identified as 2β,3β,6α,22R,25-pentahydroxy-5β-cholest-8(14)-ene. The formation of this and the other major metabolites is discussed in relation to the various effects of ecdysteroids already demonstrated on vertebrates.

Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry

Ecdysteroids are polyhydroxylated steroids that function as molting hormones in insects. 20-Hydroxyecdysone (a 27C-ecdysteroid) is classically considered as the major steroid hormone of Drosophilamelanogaster, but this insect also contains 28C-ecdysteroids. This arises from both the use of several dietary sterols as precursors for the synthesis of its steroid hormones, and its inability to dealkylate the 28C-phytosterols to produce cholesterol. The nature of Drosophila ecdysteroids has been re-investigated using both high-performance liquid chromatography coupled to enzyme immunoassay and a particularly sensitive nano-liquid chromatography-mass spectrometry methodology, while taking advantage of recently available ecdysteroid standards isolated from plants. In vitro incubations of the larval steroidogenic organ, the ring-gland, reveals the synthesis of ecdysone, 20-deoxy-makisterone A and a third less polar compound identified as the 24-epimer of the latter, while wandering larvae contain the three corresponding 20-hydroxylated ecdysteroids. This pattern results from the simultaneous use of higher plant sterols (from maize) and fungal sterols (from yeast). The physiological relevance of all these ecdysteroids, which display different affinities to the ecdysteroid receptors, is still a matter of debate.

16th International Ecdysone Workshop: July 10–14, 2006, Ghent University, Belgium

Chitin synthesis and degradation are recurrent, fundamental events of insect development. The final step of chitin anabolism is the polymerization of UDP-N-acetylglucosamine units which requires chitin synthase activity. We present here results on the cloning and characterization of the chitin synthase 1 (CfChS1) gene from the spruce budworm (SBW; Choristoneura fumiferana), an important North American forest pest insect. Using degenerate primers from conserved regions of other insect ChS, a CfChS1 fragment was isolated by PCR on a cDNA library made from freshly ecdysed, L6 SBW larvae. The full length of the CfChS1 cDNA was determined to be 5.3 kb by the sequencing of overlapping cDNA clones and 5-RACE experiments. The encoded enzyme is 1565 amino acid long and possesses the two catalytic domain signature sequences found in all insect ChS (EDR, position 858 to 860 and QRRW, position 895 to 898). Computer-assisted analysis also predicts the existence of 16 transmembrane helices in the amino acid sequence, implying that CfChS1 N-and C- termini share the same (extracellular) topological space. The expression of CfChS1 mRNA was closely associated with larval-larval and larval-pupal molts as well as with the formation of adult cuticle. In larvae, mRNA was generally absent during intermolt periods, but accumulated to high levels immediately after ecdysis, consistent with renewed chitin synthesis. Accumulation was also observed 24h before and after this event, and even up to 48h after ecdysis to the 6th larval instar. CfChS1 expression was restricted to the epidermis and did not accumulate in fat body or midgut tissues. Treatment of larvae with ecdysone and with the non-steroidal ecdysone agonist tebufenozide, repressed the transcription of CfChS1, within 6 to 12h of application. These results indicate that CfChS1 expression could be stimulated by the falling 20-hydroxyecdysone titers that characterize the late phase of the molting process. We are currently investigating the interplay between CfChS1 and other genes expressed during molting in the SBW.

Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo

The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.