Rat Leydig cell isolation
Rat Leydig cells contain the highest 11b-HSD1 activity in all cell types of rat tissues [12], and were used for rat 11b-HSD1 enzyme sources. Purified rat Leydig cells were obtained from 90day-old Sprague-Dawley rats by collagenase digestion of the testes followed by Percoll density centrifugation of the cell suspension, according to the previously described method [13]. Adult rat Leydig cells were harvested from the Percoll gradient at a band at 1.070 mg/ml. The purity of cell fractions was evaluated by histochemical staining for 3b-hydroxysteroid dehydrogenase activity with 0.4 mm etiocholanolone as the steroid substrate [14]. Enrichment of rat Leydig cells was typically more than 95%.Construction of expression human HSD11B1 plasmid and transfection
An expression plasmid was constructed to express human 11bHSD1 (HSD11B1) cDNA in pcDNA I expression vector from its original human HSD11B1 vector (pBluescriptSK+).[15]. The Escherichia coli transformants carrying an insert were selected by colony hybridization, and a clone with the insert in the correct orientation relative to the vector T7 promoter was identified by restriction mapping. All transfections were carried out on 80% confluent cultures in 12-well plates. Aliquots of 1 mg HSD11B1 pcDNA I were transfected into mammalian CHOP cells with the FuGENE Transfection Reagent (Roche) according to manufacturer’s protocol. Cells were allowed to grow for 24 hours in media containing 10% fetal bovine serum. Then media were removed and cells were harvested for 11b-HSD1 activity assay.Determination of half maximum inhibitory concentrations (IC50) and inhibitory mode
The IC50 was determined by adding different concentrations of each compound in the 11b-HSD1 or 11b-HSD2 reaction as described [17]. The mode of inhibition was assayed by adding various concentrations of steroid substrates in the presence of an inhibitor as described [18].
Animal treatment
Thirty male Sprague-Dawley rats (body weight 140?80 g) were randomly divided into three groups: vehicle control (normal diet), HFD and HFD plus 200 mg/kg curcumin, with 10 rats in each group. Rats were gavaged with vehicle (0.1% cellulose) in normal diet control and HFD groups, or with 200 mg/kg/day of curcumin (suspended in 0.1% cellulose) in the HFD plus curcumin group for two months. By the end of curcumin treatment, rats were euthanized. The body, liver, kidney and testis weights were recorded. Sera were collected after placing the bloods at room temperature for 25 min and centrifuged at 1500 g/min for 20 minutes. Sera were used for measurement of serum glucose, and lipid analysis.11b-HSD1 assay in intact rat Leydig cells and CHOP cells transfected with HSD11B1
11b-HSD1 reductase activity was performed in intact cells using endogenous cofactor NADPH as described previously [16]. In brief, each assay tube contained 25 nM substrate 11DHC (for rat) or cortisone (for human), spiked with 30,000 cpm their respective 3 H-11keto-steroid in the PBS buffer. 256103 cells 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. At the end of reaction, the reaction was stopped by adding 2 ml ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin layer plates in chloroform and methanol (90:10, v/v), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc.,
Serum glucose and lipid analysis
Serum glucose, total cholesterol, low density triglyceride (TG), lipoprotein (LDL), apolipoprotein A1 is (APOA1) and apolipoprotein B (APOB) were measured using a Hitachi 7600 biochemical analyzer (Hitachi, Japan) according to standard clinical protocol.
Statistics
Enzyme data were subjected to nonlinear analysis by GraphPad (Version 5, GraphPad Software Inc., San Diego, CA) for IC50. Lineweaver-Burk plot was used for the mode of inhibition. Data were subjected to analysis by one-way ANOVA followed by Tukey’s multiple comparisons testing to identify significant differences between groups when three were calculated. Differences were regarded as significant at P,0.05.Results Screening the selective inhibitors
Using microsomes from CHOP cells transfected with human HSD11B1 and adult rat testis as 11b-HSD1 sources, we screened many nutraceuticals, including curcumin, icariin and berberine, and found that only curcumin (compound 1) showed inhibitory effects against human and rat 11b-HSD1, with IC50 values of 10.6267.17 mM and 4.1860.24 mM, respectively. In intact CHOP cells transfected with human HSD11B1 and adult rat Leydig cells, curcumin showed inhibitory effects against human and rat 11b-HSD1, with IC50 values of 5.7862.22 mM and 2.2960.69 mM, respectively, indicating that curcumin was slightly potent when the enzyme was assayed in intact cells. We further used intact cells to screen curcumin derivatives (Fig. 2). Thiophenyl 1,4-pentadiene-3-one compounds 4 and 6 were among the most potent inhibitors (Table 1 and Fig. 3). Compound 4 [(1E,4E)1,5-bis(3-methylthiophen-2-yl) penta-1,4-dien-3-one] was 12.54 and 50.75 times more potent for the inhibition of human and rat 11b-HSD1 activity than curcumin, respectively (Table 1). Compound 6 [(1E,4E)-1,5-bis(thiophen-2-yl) penta-1,4-dien-3one] was 24.68 (human) and 31.44 (rat) times more potent than curcumin, respectively (Table 1). There are clear structure-activity responses for these compounds. Generally, the potencies of inhibiting 11b-HSD1 activity for cyclic pentadienone analogues were significantly reduced (Tables 1), indicating that the different structures in the central spacer may play a role in the effects of 11b-HSD1. For example, compound 9 [(1E,4E)-1,5-bis(3methylthiophen-2-yl) cyclopentanone] did not inhibit human
and rat 11b-HSD1 at 100 mM, and compound 16 [(1E,4E)-1,5bis(thiophen-2-yl) cyclohexanone] inhibited human 11b-HSD1 activity with reduced potency (IC50 = 3.57 mM) compared to the open chain pentadienone compound 6, IC50 = 93 nM). There was also species-dependent inhibition, human 11b-HSD1 was more sensitive to the inhibition by compound 8 and 11 than rat one (Table 1). Curcumin was slightly selective against both human and rat 11b-HSD2 with IC50 values of 14.56 and 11.92 mM, respectively (Table 1). However, compound 6, 8 and 11 were highly selective against human and rat 11b-HSD2 activity, since they did not inhibit human and rat 11b-HSD2 at all at 100 mM (Table 1). Compound 4 also inhibited human 11b-HSD2 activity with IC50 value of 19.58 mM.Mode of inhibition of curcumin derivatives
Using the compound 6 as an inhibitor to test the mode of inhibition of rat 11b-HSD1 activity, it was found that compound 6 inhibited rat 11b-HSD1 activity with a competitive mode (Fig. 4). This compound inhibited human liver 11b-HSD1 activity by the same mode (data not shown). Curcumin showed the similar mode to compound 6 as an inhibitor of 11b-HSD1 in both human and rat enzymes (data not shown).