acid were from Fisher Scientific (Loughborough, UK). Methanol and acetonitrile (HPLC grade) were from VWR (Lutterworth, Leicestershire, UK).

Transcription

1 1 SUPPLEMENTAL MATERIAL 2 SUPPLEMENTARY METHODS Materials Unless stated otherwise, all solvents were from Rathburn Chemical Ltd. (Walkerburn, UK) and other chemicals from Sigma-Aldrich (Dorset, UK). HPLC gradient water and formic acid were from Fisher Scientific (Loughborough, UK). Methanol and acetonitrile (HPLC grade) were from VWR (Lutterworth, Leicestershire, UK) Quantitation of steroids and finasteride by LC-MS/MS Testosterone, DHT, 2,3,4-[ 13 C 3 ]-testosterone and 2,3,4-[ 13 C 3 ]-5α-DHT, were from Sigma- Aldrich (Dorset, UK). d9-finasteride was synthesised in-house (16) Samples were extracted via solid-phase extraction (Oasis HLB 30mg, Waters, Manchester, UK). Extraction cartridges were primed with methanol (1 ml) then water (1 ml). Samples (500 µl) were enriched with internal standard (1 ng); water (500 µl) was added. Samples were then mixed with a pipette and loaded onto primed extraction cartridges. After a wash step (50% methanol in water, 1 ml), analytes were eluted with methanol (1 ml). Eluates were dried under oxygen-free nitrogen (37 C) and reconstituted in mobile phase (100 µl). Injection volume was 10 µl LC-MS/MS analysis was performed on a Waters Acquity UPLC (Manchester, UK) with autosampler, coupled to an ABSciex QTRAP 5500 mass spectrometer (Warrington, UK), operated with Analyst Software version Analytes were separated at 35ºC on Kinetex C18 column (150 x 3 mm, 2.6 µm). Elution was achieved with a 9 minute linear gradient from 30:70 to 80:20 (methanol: water with 0.1% FA) at a flow rate of 250 µl/ min. Initial 1

2 hold was 1 minute with a linear gradient applied until 10 minutes, hold until 16 minutes, with a total run-time of 19 minutes. The mass spectrometer was operated in positive APCI mode, with curtain gas 25 psi, collision gas low, spray voltage 5 kv, nebuliser current 3.5 µa, source temperature 500 C, ion source gas 55 psi. Multiple reaction monitoring of the analyte quantifier and qualifier transitions were performed as summarised in Supplementary Table 1. Peak areas ratios of analyte/internal standard were calculated and calibration lines, with appropriate weighting factors applied and amounts in serum calculated by interpolation Quantitation of glucose, glycerol, d2-glucose and d5-glycerol by gas chromatography mass spectrometry (GC-MS) A standard curve was prepared in expected concentration ranges of glucose ( mm), glycerol ( µm), d2-glucose ( mm) and d5-glycerol ( µm) in plasma. Internal standards were 13 C 6 -glucose (Isotec; Dorset, UK), d2-glucose and d5-glycerol (Cambridge Isotope Laboratories, Inc., Andover, USA). An enrichment curve was prepared representing 0-12% enrichment for both glucose and glycerol, with d2-glucose and d5- glycerol respectively Acetonitrile (1 ml) was added to extraction wells (96-well Strata Impact protein precipitation plate, Phenomenex, Macclesfield, Cheshire, UK). Standards, enrichment standards and samples (200 µl) were added. Internal standard solution (25 µl) was added to standard curve and samples. Internal standard was not added to enrichment samples. After a 20 minute incubation (RT), vacuum was applied (0.3 bar) and extracts collected. Eluates were reduced to dryness under oxygen-free nitrogen (OFN; 37 C). Acetate derivatives were formed; to the dry residue pyridine: acetic anhydride (200 µl, 1:1, v/v) was added and this was allowed to stand (RT, 15 minutes). Derivatised samples were dried under OFN (37 C) 2

3 1 2 and reconstituted in 5% acetic anhydride in heptane. 1 µl of sample was injected onto the GC-MS system The gas chromatograph mass spectrometer (GC-MS) consisted of a Finnigan GC8000 TOP GC, a AS800 autosampler, a single quadropole Voyager mass spectrometer, operated via Xcalibur software version 1.2 (Finnigan, now Thermo Scientific, Manchester, UK). The analytical column was a HP-Innowax column (30 m x 0.32 mm x 0.25 µm; Agilent Technologies Ltd., Stockport, UK). The sample was injected (1 µl) by splitless injection, with injector temperature set to 260 C. The column oven temperature was 60 C at injection, programmed to reach 150 C at a rate of 30 C/minute, and then further programmed to reach 260 C at a rate of 10 C, with a final hold time of 3 minutes. Interface and source temperatures were 240 C and 175 C respectively. Helium gas was used as the mobile phase, at a flow rate of 2.5 ml/min. Mass spectra were recorded using negative ion chemical ionisation with methane as reagent gas at an electron energy of 70 ev. Selected ion monitoring was used with a total run-time of 18 minutes. Derivatives of glycerol, d5-glycerol and butanetriol were monitored from 0-10 minutes, and those of glucose, d2-glucose and 13 C 6 -glucose monitored from minutes. Monitored ions were: glycerol triacetate m/z 217, d5-glycerol triacetate m/z 222, butanetriol triacete m/z 231 (internal standard), glucose pentacetate m/z 287, d2-glucose pentacetate m/z 289, 13 C 6 -glucose pentacetate m/z 293 (internal standard). 21 3

4 SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1 Recruitment and participation rates Yes indicates an interest in participation, No indicates either an unfavourable response or no response. Two of the 4 participants who did not complete the study were assigned a study number following informed consent but did not participate further. Supplememtal Figure 2 Glucoseinfusion rates during euclyaemic hyperinsulinaemic clamp Graphs showing glucose infusion rates (steady state phases of the hyperinsulinaemic euglycaemic clamp; mins no insulin, mins low-dose insulin, mins high-dose insulin shown in grey). Open and closed symbols are data from subjects prior to or after receiving drug treatment respectively. N=14-16/ group. Data are mean± SEM. Supplementary Figure 3 Salivary cortisol concentrations Salivary cortisol levels through the day before (open shapes) and after (filled shapes) three months of dutasteride, finasteride or tamsulosin; absolute levels and variations through the day did not differ in response to treatment. Samples collected at waking, 30 minutes after waking ( 30 min ), 1200h, 1600h, and bedtime. Data points are mean; error bars indicate SEM. 4

5 Supplemental Table 1 Primers and probes for PCR Gene: Name Gene accession number Quantitation of transcript abundance by real-time qpcr ACACA: Acetyl-Coenzyme A carboxylase α isoform 1 NM_ PLIN2: Adipocyte differentiation-related protein (Perilipin 2) NM_ ADIPOQ: Adiponectin NM_ AR: Androgen receptor NM_ DGAT2: Diacylglycerol O-acyltransferase homolog 2 NM_ ESR1: Estrogen receptor α NM_ ESR2: Estrogen receptor β NM_ FASN: Fatty acid synthase NM_ GPD2: Glycerol-3-phosphate dehydrogenase 2 (mitochondrial) NM_ NR3C1: Glucocorticoid receptor NM_ ,NM_ ,NM_ ,NM_ ,NM_ LIPE: Hormone sensitive lipase NM_ Primer sequences Forward followed by reverse GATGTGGATGATGGGCTACA TGAGGCCTTGATCATTACTGG TCAGCTCCATTCTACTGTTCACC CCTGAATTTTCTGATTGGCACT GGTGAGAAGGGTGAGAAAGGA TTTCACCGATGTCTCCCTTAG GCCTTGCTCTCTAGCCTCAA GTCGTCCACGTGTAAGTTGC GAGGGGTCTGGGAGATGG TTGAGCCAGGTGACAGAGAA TTACTGACCAACCTGGCAGA ATCATGGAGGGTCAAATCCA TGGGTGATTGCCAAGAGC GTTTGAGAGGCCTTTTCTGC UPL Probe CAGGCACACACGATGGAC 11 CGGAGTGAATCTGGGTTGAT TCGCGCGTGAGGATCTAT AGTCCTAAAACAGTTGCAAGAGC TTCAAAAGAGCAGTGGAAGGA TTTCTTCGAATTTTATCGATGATG TGCTCGGAATCACAGACACT CAGGTCCATGTTGTGGATGA

6 LEP: Leptin NM_ LPL: Lipoprotein lipase NM_ HSD11B1: 11β-Hydroxysteroid dehydrogenase type 1 (11β HSD1) NM_ GPAM: Glycerol-3-phosphate acyltransferase 1 (mitochondrial) NM_ MCP1: Monocyte chemotactic protein 1 NM_ PPARG: Peroxisome proliferator-activated receptor γ (PPARγ) NM_ PPARGC1A: PPARγ coactivator 1α NM_ SRD5A1: 5α-Reductase type 1 (5αR1) NM_ SREBF1: Sterol regulatory element binding transcription factor 1 NM_ SREBF2: Sterol regulatory element-binding transcription factor 2 NM_ UCP 2: Uncoupling protein 2 NM_ PPIA: Peptidylprolyl isomerase A (cyclophilin A) NM_ TBP: TATA-binding protein NM_ TTGTCACCAGGATCAATGACA GTCCAAACCGGTGACTTTCT ATGTGGCCCGGTTTATCA CTGTATCCCAAGAGATGGACATT CAATGGAAGCATTGTTGTCG GGCAGCAACCATTGGATAAG GGAAAGTTTATCCAGTATGGCATT TGATATCTTCCTGGTCATCGTG TTCTGTGCCTGCTGCTCAT GGGGCATTGATTGCATCT TGACAGGAAAGACAACAGACAAAT GGGTGATGTGTTTGAACTTGATT TGAGAGGGCCAAGCAAAG ATAAATCACACGGCGCTCTT TTGGAGAAATCATGGAGTGGT ACTCTTCAAATTTCCGGAGGTA CGCTCCTCCATCAATGACA TGCGCAAGACAGCAGATTTA ATCTGGATCTCGCCAGAGG CCAGGCAGGTTTGTAGGTTG TGAAAGCCAACCTCATGACA GATGACAGTGGTGCAGAAGC ATGCTGGACCCAACACAAAT TCTTTCACTTTGCCAAACACC GAACATCATGGATCAGAACAACA ATAGGGATTCCGGGAGTCAT GAPDH: Glyceraldehyde-3-phosphate dehydrogenase AGCCACATCGCTCAGACAC

7 NM_ Tissue screening by reverse transcriptase-pcr SRD5A1: 5α-Reductase type 1 (5αR1) SRD5A2: 5α-Reductase type 1 (5αR2) GCCCAATACGACCAAATCC TGGCGCTTCTCTATGGACTT ACACAGCACCTGACACGAA GCCCTCTCCTCATAGTGCTG GCAAGCAGCATGTAACCTCA Assays used for real-time polymerase chain reaction analysis and primers used for isozyme distribution of 5αR1 and 5αR2. PCR = polymerase chain reaction

8 Supplemental Table 2 Conditions for LC-MS/MS assays for steroids and drugs in serum Precursor Product Declustering Collision Cell exit ion ions potential (V) energy (V) potential (V) m/z m/z Quan; qual Testosterone ; ; 31 14; 16 DHT Finasteride ; ; 41 14; 28 Dutasteride ; ; 55 48; C 3 -Testosterone ; ; 35 4; 8 13 C 3 -DHT d9-finasteride ; ; 41 34; 36 Using atmospheric pressure chemical ionisation; quan, quantifier ion; qual, qualifier ion; V, volts; DHT, dihydrotestosterone.

9 Supplemental Table 3 Effects of drug intervention on serum adipokines and cytokines Leptin pg/ml Adiponectin µg/ml Resistin ng/ml MCP-1 pg/ml IL-8 pg/ml Dutasteride Finasteride Tamsulosin P from ANOVA Before After Change Before After Change Before After Change (461) 3487 (504) 28 (281) (485) p=0.32 (1696) (1518) (928) (1899) (2052) 13.4 (0.5) 14.7 (0.6) 1.3 (1.0) 13.7 (0.7) 14.6 (0.5) 0.9 (0.7) 16.4 (0.2) 16.7 (0.4) 0.3 (0.4) p= (0.4) 24.0 (0.4) 0.4 (0.5) 23.3 (0.3) 23.6 (0.6) 0.2 (0.6) 27.0 (0.3) 29.9 (0.3) 2.9 (0.6) p= (17.5) 238 (20.2) 1 (17) 189 (17.4) 197 (21.5) 8 (14) 274 (29.4) 249 (28.6) -25 (15) p= (0.9) 5.6 (0.9) 0.0 (0.3) 6.0 (1.2) 6.3 (0.9) 0.3 (0.6) 7.3 (0.4) 6.2 (0.4) -1.1 (0.4) p=0.08 Data are mean (SEM). MCP-1, monocyte chemoattractant protein 1; IL-8, interleukin 8. Kruskal-Wallis test.

10 Supplemental Table 4 Effects of drug interventions on subcutaneous adipose mrna transcripts Dutasteride (n=13) Finasteride (n=14) Tamsulosin (n=12) P from ANOVA Before After Before After Before After AR (androgen receptor) 0.34 (0.05) 0.22 (0.04) 0.31 (0.03) 0.23 (0.03) 0.22 (0.03) 0.26 (0.05) p=0.045* SRD5A1 (5αR1) 0.28 (0.03) 0.29 (0.02) 0.28 (0.02) 0.24 (0.03) 0.30 (0.03) 0.31 (0.03) p=0.47 HSD11B1 (11βHSD1) 0.44 (0.08) 0.28 (0.07) 0.35 (0.06) 0.23 (0.03) 0.52 (0.16) 0.27 (0.09) p=0.58 NR3C1 (total GR) 0.35 (0.05) 0.34 (0.03) 0.34 (0.05) 0.31 (0.04) 0.26 (0.03) 0.27 (0.03) p=0.62 ESR1 (ERα) 0.32 (0.04) 0.26 (0.04) 0.40 (0.06) 0.32 (0.05) 0.32 (0.04) 0.32 (0.04) p=0.40 ESR2 (ERβ) 0.32 (0.04) 0.32 (0.06) 0.35 (0.04) 0.31 (0.04) 0.32 (0.04) 0.29 (0.04) p=0.86 ACACA (Acetyl CoA carboxylase) 0.20 (0.03) 0.25 (0.05) 0.27 (0.04) 0.24 (0.04) 0.21 (0.02) 0.26 (0.05) p=0.28 UCP2 (Uncoupling protein 2) 0.25 (0.05) 0.29 (0.08) 0.24 (0.03) 0.27 (0.04) 0.24 (0.04) 0.23 (0.02) p=0.65 FASN (Fatty acid synthase) 0.29 (0.04) 0.30 (0.06) 0.39 (0.07) 0.28 (0.06) 0.25 (0.05) 0.43 (0.14) p=0.17 LIPE (Hormone sensitive lipase) 0.36 (0.10) 0.24 (0.04) 0.44 (0.08) 0.34 (0.04) 0.33 (0.05) 0.32 (0.06) p=0.62 PLIN2 (Perilipin) 0.30 (0.04) 0.22 (0.04) 0.32 (0.04) 0.25 (0.03) 0.33 (0.05) 0.26 (0.03) p=0.97 PPARG (PPARγ) 0.46 (0.06) 0.40 (0.06) 0.49 (0.05) 0.46 (0.04) 0.45 (0.05) 0.48 (0.09) p=0.61 PPARGC1A (PPARγ, coactivator 1α) 0.43 (0.13) 0.19 (0.02) 0.38 (0.05) 0.34 (0.05) 0.26 (0.03) 0.25 (0.05) p=0.33

11 GPD2 (G3P dehydrogenase) 0.33 (0.03) 0.38 (0.04) 0.30 (0.03) 0.28 (0.02) 0.31 (0.03) 0.35 (0.03) p=0.36 LEP (Leptin) 0.19 (0.04) 0.17 (0.03) 0.19 (0.02) 0.12 (0.02) 0.18 (0.04) 0.17 (0.04) p=0.37 ADIPOQ (Adiponectin) 0.35 (0.03) 0.32 (0.06) 0.40 (0.04) 0.30 (0.04) 0.26 (0.03) 0.34 (0.06) p=0.10 MCP (0.06) 0.29 (0.04) 0.40 (0.07) 0.39 (0.08) 0.48 (0.07) 0.34 (0.06) p=0.47 DGAT (0.05) 0.29 (0.06) 0.31 (0.05) 0.24 (0.06) 0.18 (0.02) 0.32 (0.09) p=0.12 SREBF (0.04) 0.17 (0.06) 0.18 (0.03) 0.26 (0.08) 0.13 (0.02) 0.24 (0.06) p=0.28 SREBF (0.04) 0.44 (0.05) 0.44 (0.06) 0.40 (0.08) 0.36 (0.04) 0.44 (0.08) p=0.27 LPL (Lipoprotein lipase) 0.32 (0.04) 0.25 (0.04) 0.30 (0.03) 0.23 (0.03) 0.30 (0.03) 0.31 (0.05) p=0.39 GPAM (G3P acyl-transferase) 0.33 (0.06) 0.32 (0.06) 0.28 (0.04) 0.22 (0.03) 0.25 (0.04) 0.27 (0.07) p=0.62 Data are mean (SEM). Analysed by Kruskal-Wallis test. *p=0.02 dutasteride versus tamsulosin, and p=0.06 dutasteride versus finasteride.

12 Supplemental Figure Clinic patients reviewed 37 Primary care patients invited Healthy participant advertising 255 Information sheets requested or sent 95 Yes 160 No 58 Attend screening 44 Ineligible / Unable to participate 51 Consented 7 Not consented/ Ineligible 47 Completed study 4 Did not complete study 46 Included in final analysis

13 Supplemental Figure Dutasteride Prior After G;ucose Infusion Rate mg/min Finasteride Tamsulosin Prior After Prior After Duration of Tracer Infusions (mins)

14 Supplemental Figure 3