Introduction
Glucocorticoids (GCs) have a wide range of physiological and pharmacological roles in mammalian functions [1]. Excessive GCs under conditions such as stress and Cushing’s syndrome cause a spectrum of clinical features, including metabolic syndrome [2]. GCs increase glucose output in the liver, induce fat accumulation, dampen glucose-dependent insulin sensitivity in the adipose tissue, thus increasing the risks of metabolic syndrome [3]. Intracellular levels of GCs (cortisol in the human or corticosterone, CORT, in the rat) are regulated by 11b-hydroxysteroid dehydrogenase (11bHSD), which has two known isoforms: an NADP+/NADPH dependent 11b-HSD1 oxidoreductase that behaves a primary reductase in the liver and fat tissues (Fig. 1) and an NAD+ dependent 11b-HSD2 [4,5]. 11b-HSD2 acts a unidirectional oxidase to prevent cortisol from stimulating the mineralocorticoid receptor in kidney and colon, and the mutation of human 11b-HSD2 gene (HSD11B2) causes severe hypertension and hypokalemia [5]. Recently, 11b-HSD1 inhibition has gained attention as a potentially effective method for treating metabolic syndrome, including type 2 diabetes [6]. 11b-HSD1 knockout mice are resistant to diet-induced obesity and glucose intolerance, and 11bHSD1 over-expression in the fat tissue causes metabolic syndrome [3,7]. We screened many nutraceuticals to study whether they had inhibitory effects on 11b-HSD1 with selectivity against 11bHSD2. One of chemicals is curcumin (Fig. 2, compound 1). In this study, we investigated the therapeutic efficacy of curcumin for high-fat-diet (HFD)-induced metabolic syndrome in a rat model, and we also screened 12 curcumin analogues (Fig. 2) to test whether these compounds specifically inhibit 11b-HSD1 activity. It is important that 11b-HSD1 inhibitors should not significantly inhibit 11b-HSD2 in order to avoid undesirable sodium retention, hypokalemia, and hypertension.
Figure 1. Interconversion of cortisol and cortisone by two 11bhydroxysteroid dehydrogenase (11b-HSD) isoforms. 11b-HSD1 catalyzes the conversion of cortisone into cortisol in the liver or fat tissues, and 11b-HSD2 catalyzes the conversion of cortisol into cortisone in kidney or colon tissues.and 3H-cortisone were prepared from labeled 3H-CORT or 3Hcortisol as described earlier [8]. Cold CORT, 11DHC, cortisol and cortisone were purchased from Steraloids (Newport, RI). Curcumin, icariin and berberine was purchased from Sigma-Aldrich Company (St Louis, MO, USA). The library of nutraceuticals was obtained from Wenzhou Medical College (Wenzhou, China). Human liver and kidney microsomes were purchased from Gentest (Woburn, MA). Male Sprague-Dawley rats (body weight 140?80 g) were purchased from Wenzhou Medical College Animal Center (Wenzhou, China) for HFD-induced metabolic syndrome treatment experiment. Male Sprague-Dawley rats (90 days old) were purchased from Charles River Laboratories (Wilmington, MA) for the isolation of Leydig cells for 11b-HSD1 enzyme inhibition assay. Both animal protocols were approved by the Institutional Animal Care and Use Committee of the Rockefeller University and Wenzhou Medical College.Materials and Methods Chemical and animals
[1,2,6,7-3H] Corticosterone (3H-CORT) and [1,2,6,7-3H] cortisol (3H-cortisol) were purchased from Dupont-New England Nuclear (Boston, MA).
Chemistry
Curcumin analogues were synthesized by coupling the appropriate aldehyde with acetone, or cyclopentanone or cyclohexanone in an alkaline medium, respectively as described [9,10].Figure 2. Structures of curcumin and its pentadienone analogues. Ar = aryl group; n = carbon numbers, where n = 0, designating open chain pentadienone compound, and n = 2, 3 designating cyclopentadienone compound. spectroscopy were performed. The reaction was carried out with a rate 2:1 of substituted aldehydes and ketones. For example, the reaction of 5-bromofuran-2-aldehyde with three respective ketones in alkaline medium was performed to generate these compounds as previously described [9,10].Washington, DC) as described previously [11]. The percentage conversion of 11DHC to CORT or cortisone to cortisol was calculated by dividing the radioactive counts identified as 11-OHsteroids by the total counts.11b-HSD1 assay in rat and human microsomes Preparation of microsomes
Rats were euthanized by CO2, and livers, testes and kidneys were collected. Rat liver, testis and kidney microsomes were prepared as described previously [11]. In brief, rat liver and kidney were homogenized in 0.01 mM PBS buffer containing 0.25 M sucrose, and nuclei and large cell debris were removed by centrifugation at 15006 g for 10 min. The post-nuclear supernatants were centrifuged twice at 105,0006g, the resultant microsomal pellets were resuspended. Protein contents were measured by Bio-Rad Dye Reagent Concentrate (Cat.#500-0006). Microsomes were used for measurement of 11b-HSD1 and 11b-HSD2 activities. The rat testis and liver or human liver microsomes were used for 11b-HSD1 assay. 11b-HSD1 activity was performed in the microsome according to a previously described method [17]. In brief, the assay tubes contained 25 nM substrate 11DHC (for rat) or cortisone (for human), spiked with 30,000 cpm their respective 3H-11keto-steroid, 0.2 mM NADPH and 5 mM glucose-6-phosphaare in the PBS buffer. 2 mg microsomes were added to each tube to initiate the reaction and the reaction mixture was incubated for up to 2 hrs, during which the reaction is within the linear range. The rest procedure was similar to 11b-HSD1 assay in intact cells.
11b-HSD2 assay in rat and human kidney microsomes
The rat and human kidney microsomes were used for 11bHSD2 sources. 11b-HSD2 activity was performed in the microsome according to a previously described method [17]. In brief, the assay tubes contained 25 nM substrate CORT (for rat) or cortisol (for human), spiked with 30,000 cpm their respective 3 H-11b-hydroxyl steroid, 0.2 mM NAD+ and 0.1 mM DTT in the PBS buffer. 8 (rat) or 20 (human) mg kidney microsomes were added to each tube to initiate the reaction and the reaction mixture was incubated for up to 3 hrs, during which the reaction is within the linear range. The rest procedure was similar to 11bHSD1 assay. The percentage conversion of CORT to 11DHC or cortisol to cortisone was calculated by dividing the radioactive counts identified as 11keto-steroids by the total counts.
