Metformin Inhibits Human Androgen Production by Regulating ...

Metformin Inhibits Human Androgen Production byRegulating Steroidogenic Enzymes HSD3B2 andCYP17A1 and Complex I Activity of the RespiratoryChainAndrea Hirsch, Dagmar Hahn, Petra Kempná, Gaby Hofer, Jean-Marc Nuoffer,Primus E. Mullis, and Christa E. Flück

Department of Pediatrics, Division of Pediatric Endocrinology, Diabetology and Metabolism (A.H., P.K.,G.H., P.E.M., C.E.F.), and Departments of Clinical Research (A.H., P.K., G.H., P.E.M., C.E.F.) and ClinicalChemistry (D.H., J.-M.N.), University Hospital Inselspital, University of Bern, 3010 Bern, Switzerland

Metformin is treatment of choice for the metabolic consequences seen in polycystic ovary syn-drome for its insulin-sensitizing and androgen-lowering properties. Yet, the mechanism of actionremains unclear. Two potential targets for metformin regulating steroid and glucose metabolismare AMP-activated protein kinase (AMPK) signaling and the complex I of the mitochondrial respi-ratory chain. Androgen biosynthesis requires steroid enzymes 17�-Hydroxylase/17,20 lyase(CYP17A1) and 3�-hydroxysteroid dehydrogenase type 2 (HSD3B2), which are overexpressed inovarian cells of polycystic ovary syndrome women. Therefore, we aimed to understand how met-formin modulates androgen production using NCI-H295R cells as an established model of steroid-ogenesis. Similar to in vivo situation, metformin inhibited androgen production in NCI cells bydecreasing HSD3B2 expression and CYP17A1 and HSD3B2 activities. The effect of metformin onandrogen production was dose dependent and subject to the presence of organic cation trans-porters, establishing an important role of organic cation transporters for metformin’s action.Metformin did not affect AMPK, ERK1/2, or atypical protein kinase C signaling. By contrast, met-formin inhibited complex I of the respiratory chain in mitochondria. Similar to metformin, directinhibition of complex I by rotenone also inhibited HSD3B2 activity. In conclusion, metformin in-hibits androgen production by mechanisms targeting HSD3B2 and CYP17-lyase. This regulationinvolves inhibition of mitochondrial complex I but appears to be independent of AMPK signaling.(Endocrinology 153: 4354–4366, 2012)

Exact regulation of human androgen biosynthesis re-mains obscure. But both steroidogenic enzymes

CYP17A1 and HSD3B2 certainly play an essential role forandrogen biosynthesis in health and disease (1). The mi-crosomal CYP17A1 enzyme possesses two distinct activ-ities, namely 17�-hydroxylase (OHase) and 17,20-lyase(lyase) (1). Human CYP17-OHase converts the substratespregnenolone (Preg) and progesterone (Prog) to 17�-hy-droxypregnenolone (17OHPreg) and 17�-hydroxypro-gesterone (17OHP) with equal efficiency (1). By contrast,

the CYP17-lyase predominantly converts 17OHPreg todehydroepiandrosterone (DHEA) through the so-called�5 pathway and has only little efficiency to convert17OHP to androstenedione (�4A) over the �4 pathway(2). Both CYP17 activities require electron supply fromthe flavoprotein P450 oxidoreductase (POR), andCYP17-lyase activity is additionally enhanced by proteinphosphorylation and cytochrome b5 (CYB5), which isthought to allosterically support the CYP17-POR inter-action (1, 3–5). CYP17-OHase activity is required for the

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2012 by The Endocrine Societydoi: 10.1210/en.2012-1145 Received February 7, 2012. Accepted June 18, 2012.First Published Online July 9, 2012

Abbreviations: �4A, Androstenedione; AICAR, 5-aminoimidazole-4-carboxamide; AMPK,AMP-activated protein kinase; AU, arbitrary units; C, complex; cpm, counts/min; CYB5,cytochrome b5; DHEA, dehydroepiandrosterone; FCCP, carbonyl cyanide p-trifluoro-me-thoxyphenyl-hydrazone; NADH, reduced nicotinamide adenine dinucleotide; OCT, organiccation transporter; OHase, 17�-hydroxylase; 17OHP, 17�-hydroxyprogesterone; 17OHPreg,17�-hydroxypregnenolone; PCOS, polycystic ovary syndrome; PKC, protein kinase C; PMAT,plasma membrane monoamine transporter; POR, P450 oxidoreductase; Preg, pregnenolone;Prog, progesterone; qRT-PCR, quantitative RT-PCR; TLC, thin-layer chromatography; TTBS,Tween 20 tris-buffered saline.

G L U C O C O R T I C O I D S - C R H - A C T H - A D R E N A L

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synthesis of glucocorticoids, whereas both CYP17-OHaseand lyase activities are essential for the production of sexsteroids. The microsomal HSD3B2 enzyme is also knownas �5-�4-isomerase because it converts �5 steroids (Preg,17OHPreg, and DHEA) to �4 steroids (Prog, 17OHP, and�4A). Therefore, HSD3B2 is crucial for the production ofmineralocorticoids, glucocorticoids, and sex steroids (6).

Polycystic ovary syndrome (PCOS) is the most commondisorder of hyperandrogenism and the leading cause offemale infertility (7, 8). Hyperandrogenism in PCOS pa-tients is of both adrenal and ovarian origin (9), may man-ifest in mid-childhood as premature exaggerated adre-narche (10, 11), and persists beyond menopause transition(12–14). In PCOS, androgen excess is caused by an in-creased expression of CYP17A1 and HSD3B2 in ovariantheca cells (15), but additional details of the pathomecha-nism remain unknown.

Metformin was first used as an effective insulin-sensi-tizing drug in PCOS patients with diabetes before it wasfound to ameliorate hyperandrogenism in PCOS (16–18).Although metformin has been in clinical use for years andis regarded as one of the first-line treatments for diabetesand anovulation in PCOS (19, 20), its exact mechanism ofaction remains elusive. Similarly, it is unclear why somePCOS patients seem resistant to metformin treatment withrespect to anovulation and hyperandrogenism. However,recent studies suggest that heterogeneity in the response tometformin may be partially explained by a variable func-tion of organic cation transporters (OCT), which areneeded to transport metformin into the cell (21, 22).

The search for the specific intracellular targets of met-formin is still ongoing. For the past 10 yr, AMP-activatedprotein kinase (AMPK) and complex I of the mitochon-drial respiratory chain have been proposed as potentialtargets of metformin’s action on glucose and steroid me-tabolism (23–29). Zhou et al. (23) showed that metforminactivates AMPK in primary hepatocytes. As a result, met-formin was thought to control glucose levels by inhibitionof glucose production in rat hepatocytes and by improvingglucose uptake into skeletal muscle cells. Thereafter, sev-eral research groups confirmed the action of metforminvia activated AMPK for several tissues including liver,muscle, and fat (28, 30, 31). Metformin has also beenreported to inhibit androgen production of human ovar-ian cells (32, 33), possibly through AMPK (27). By con-trast, recent studies by Foretz et al. (34) suggest an AMPK-independent effect of metformin on hepaticgluconeogenesis and propose a direct effect of metforminon the intracellular energy state. In line with that, alreadyin the year 2000, two research groups independently pro-posed that metformin has a direct (25) or indirect (24)

inhibitory effect on the complex I of the mitochondrialrespiratory chain in hepatocytes.

In previous work, our group investigated the role ofAMPK in steroidogenesis of human adrenal NCI-H295Rcells (35). We found that AMPK activation promotes an-drogen production by enhancing CYP17-lyase activity. Inthis study, we investigated several possible targets of ac-tion of metformin on steroid biosynthesis in NCI-H295Rcells hoping to find new insight into the basics of androgenproduction. Our main focus was on the effects of met-formin on AMPK signaling and on complex I of the mi-tochondrial respiratory chain. Overall, our studies con-firm that metformin acts independently of AMPK buttargets complex I of the respiratory chain for regulatingandrogen production.

Materials and Methods

Materials

Antibodies against phospho-AMPK� and AMPK�

(AMPK and ACC Antibody Sampler Kit 9957), MAPK/ERK (Phospho-ERK1/2 Pathway Sampler Kit 9911) andprotein kinase C (PKC) (Phospho-PKC Antibody SamplerKit 9921) were purchased from Cell Signaling Technology(Danvers, MA). �-Actin antibody was received fromSigma Chemical Co. (St. Louis, MO). Goat antirabbit andgoat antimouse horseradish peroxidase-conjugated anti-bodies were obtained from Santa Cruz Biotechnology(Santa Cruz, CA). Rabbit polyclonal antibody detectinghuman CYP17A1 was custom made by Genscript (Pisca-taway, NJ) (35), whereas chicken peptide antibodyagainst human HSD3B was custom made by Genetel Phar-maceutical Co. Ltd. (Guangdong, China). Radioactive-labeled [7(N)-3H]pregnenolone (14 Ci/mmol) and[1,2,6,7(N)-3H]DHEA (63 Ci/mmol) were procured fromPerkinElmer (Boston MA), and [1,2,6,7(N)-3H]17�-hy-droxypregnenolone (50 Ci/mmol) was purchased fromAmerican Radiolabeled Chemicals (St. Louis, MO).Trilostane was extracted in absolute ethanol from tabletscommercially available as Modrenal (Bioenvision, NewYork, NY). 5-Aminoimidazole-4-carboxamide (AICAR),rotenone, and all other chemicals for respirometric mea-surements were of standard analytical grade and were pur-chased from Sigma or Merck (Darmstadt, Germany).

Cell culture and treatmentHuman adrenal NCI-H295R cells were from the Amer-

ican Type Culture Collection (CRL-2128; Manassas, VA).Cells were cultured under standard conditions (growth

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medium) in DMEM/Ham’s F-12 medium containing L-glutamine and 15 mM HEPES (GIBCO, Life Technologies,Paisley, UK) supplemented with 5% NU-I serum (BectonDickinson, Basel, Switzerland), 0.1% selenium/insulin/transferrin, penicillin (100 U/ml; GIBCO), and strepto-mycin (100 �g/ml; GIBCO). The serum-free NCI-H295Rmedium contained DMEM/Ham’s F-12 medium, penicil-lin (100 U/ml; GIBCO), and streptomycin (100 �g/ml;GIBCO). For RNA and protein extraction experimentsand for steroid labeling experiments, cells were first grownin growth medium in six-well plates. After subculturingfor 24 h, the medium was replaced, and cells were treatedin serum-free medium for 48 h unless indicated differently.Metformin was dissolved in water at stock concentrationsof 1 M; the final concentration used for treatment was inthe range of 1–10 mM. AICAR (Sigma-Aldrich) was dis-solved in serum-free NCI-H295R medium at stock con-centrations of 10 mM; final concentration was 1 mM.

RNA isolation and semiquantitative RT-PCRTotal RNA from NCI-H295R cells and human adrenal

glands was isolated using a TRIzol method according tothe manufacturer’s instructions (Invitrogen, Life Technol-ogies, Paisley, UK). Similarly, total RNA was obtainedfrom human hepatocellular HepG2 (American Type Cul-ture Collection HB-8065) and monkey kidney COS1(CRL-1650) cells. RNA was reverse-transcribed to cDNAusing the Improm RNA transcriptase kit (Promega, Mad-ison, WI) with 0.5 �g random primer (Promega) per 1 �gof RNA at 42 C for 1 h.

For the semiquantitative RT-PCR, cDNA (resultingfrom 100 ng RNA) was amplified by Go-TAQ polymerase(Promega) and specific primers (sequences available uponrequest) in a final volume of 25 �l. PCR conditions wereas follows: 45 sec at 95 C, 45 sec at 50–60 C, and 45 secat 72 C for 30 or 40 cycles. PCR products were separatedby electrophoresis on gels containing 1.5% agarose, visu-alized by ethidium bromide staining, and detected on anAlpha Imager 3400 (Alpha Innotech, San Leandro, CA).

Real-time PCRThe transcripts of CYP17, HSD3B2, POR, and CYB5

were quantified by real-time PCR using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).In brief, PCR were performed in 96-well plates (Micro-Amp; Applied Biosystems) using cDNA prepared as de-scribed above. We used ABsolute QPCR SYBR Green Mix(ABgene; Thermo Fisher Scientific, Wohlen, Switzerland),1 �l (20 pmol/�l) specific primers (Microsynth, Balgach,Switzerland), and 50 ng cDNA in a total volume of 25 �l.Relative expression values were determined by the 2��Ct

method using 18S rRNA as the reference gene. Amplifi-

cation curves and the mean cycle threshold values werecalculated using 7500 Fast System SDS software (AppliedBiosystems), and the correction for the reference gene wascalculated as previously described (50). Final results arediagrammed as the percentage of expression of untreatedcells.

Steroid labelingSteroid metabolism was labeled by adding either

100,000 counts/min (cpm) [3H]Preg, [3H]DHEA, or[3H]17OHPreg for 90 min. Steroids were extracted frommedium as previously described (3) and separated on thin-layer chromatography (TLC) plates (Macherey-Nagel,Düren, Germany). For specific analysis of the CYP17 ac-tivities, cells were treated with 1 �M trilostane (a specificblocker of HSD3B) for 90 min before adding labeled ste-roids. The separated steroids were visualized on a FujiPhosphoImager FLA-7000 (Fujifilm, Dielsdorf, Ger-many), recognized using known standards and densito-metrically quantified using Multi Gauge software (Fujif-ilm). Specific steroid conversion was calculated aspercentage of radioactivity incorporated in a specific ste-roid hot spot when compared with the total radioactivityadded to the reaction.

Overexpression of OCTNCI-H295R cells were either transfected with

pEGFP-N3 empty vector or cDNA expressing vectorshOCT1 or hOCT2 (kindly provided by Giuliano Ciarim-boli and Eberhard Schlatter) (36) in six wells (1.5 � 106

cells per well; Falcon 3046; BD Biosciences, Basel, Swit-zerland) using Lipofectamine 2000 according to the man-ufacturer’s recommendations (Invitrogen). Transfectionmedium [DMEM containing L-glutamine and 15 mM

HEPES (GIBCO) supplemented with 10% NU-I serum(Becton Dickinson) and 0.1% selenium/insulin/transfer-rin (GIBCO)] was changed to normal growth medium af-ter 5 h, and cells were cultivated overnight. Thereafter,cells were treated with 0–10 mM metformin in serum-freemedium for 48 h, and steroid production was labeled byadding [3H]Preg (100,000 cpm/well) for 90 min beforeperforming TLC.

Protein extraction and Western blot analysisFor protein analyses, cells were treated as described

above, washed with ice-cold PBS, and harvested in 250 �llysis buffer [200 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1mM EDTA, 1% Triton X-100, and protease and/or phos-phatase inhibitors]. Lysates were passed three timesthrough a 25-gauge syringe and centrifuged at 13,000 �g for 10 min at 4 C, and supernatants were collected.Protein concentrations were measured using the DC Pro-

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tein Assay (Bio-Rad, Hercules, CA). For the Western blot,20 �g protein of total cell lysates was mixed with sodiumdodecyl sulfate loading buffer [62.5 mM Tris-HCl (pH6.8), 2% sodium dodecyl sulfate, 10% glycerol, 100 mM

dithiothreitol, and 0.01% bromophenol blue], heated for5 min at 95 C, separated on a 10% SDS-PAGE gel, andblotted on an Immobilon P transfer membrane (Millipore,Bedford, MA) using the semi-dry transfer method. Block-ing and staining with antibodies was performed accordingto manufacturer’s recommendations (Cell Signaling Tech-nology or Sigma-Aldrich, St. Louis, MO). For CYP17,membranes were blocked with 5% nonfat dry milk, andthe secondary antibody (1:5000) was dissolved in 1�Tween 20 tris-buffered saline (TTBS) with 5% BSA.HSD3B2 membranes were blocked with 10% nonfat drymilk, and the secondary antibody (1:10000) was dissolvedin 1� TTBS with 5% milk. Protein bands were visualizedby ECL substrate reagent (PerkinElmer Inc., Waltham,MA) and exposed on Hyperfilm MP (Amersham Pharma-cia Biotech, Dübendorf, Switzerland). When tested withdifferent antibodies, membranes were stripped with 0.2 M

NaOH for 30 min and washed with TTBS before restain-ing. �-Actin was used to control for equal loading.

Promoter activity experimentsTo assess the effect of metformin on promoter activi-

ties, NCI-H295R cells (150,000 cells per well in a 24-wellformat; Falcon 3047; BD Biosciences) were transfectedwith an empty vector (pGL3, �luc) or specific promoter-reporter constructs (�3.7CYP17, �1.05HSD3B2,�1.3b5, �325POR) using Lipofectamine 2000 (Invitro-gen). The transfection mixture per well contained 0.6 �gplasmid, 25 ng renilla luciferase reporter vector (pRL-TK;Promega, Dübendorf, Switzerland), 2 �l Lipofectamine2000, and 50 �l OptiMEM (GIBCO). Briefly, cells weretransfected in suspension for 5 h, and then cells werewashed and normal growth medium added overnight. Af-ter 24 h, cells were treated in serum-free medium with 10mM metformin for 48 h. Finally, cells were lysed and as-sayed for luciferase activity using the dual luciferase re-porter assay system and protocol (Promega).

Mitochondrial isolation and respiratory chainanalysis

Isolation of mitochondria and activity measurementsof citrate synthase (cs), reduced nicotinamide adenine di-nucleotide (NADH) coenzyme Q reductase complex (C)I,succinate dehydrogenase (CII), ubiquinol-cytochrome creductase (CIII), cytochrome c oxidase (CIV), and MgAT-Pase (CV) were determined spectrophotometrically as de-scribed (37). In brief, the assay buffer [25 mM potassiumphosphate (pH 7.2), 5 mM MgCl2, 2.5 mg/ml BSA] con-

sisted of 65 �M NADH, 65 �M ubiquinone, 2 mM KCN,and 4 �M antimycin A with or without 25 �M rotenone forcomplex I; 20 mM sodium succinate, 65 �M ubiquinone,50 �M 2,6-dichlorphenolindophenol, 2 mM KCN, 5 �M

antimycin A, and 3.75 �M rotenone with or without 1 mM

thenoyltrifluoroacetone for complex II; 50 �M oxidizedcytochrome c, 50 �M ubiquinol2, 2 mM KCN, and 5 �M

rotenone with or without 20 �M antimycin A for complexIII; and 15 �M reduced cytochrome c in 20 mM potassiumphosphate (pH 7.2) with or without 250 �M KCN forcomplex IV. The FoF1-ATP-ase (complex V) activity wasassayed by the method of Morava et al. (38) with slightmodifications. Each sample was run in the presence orabsence of oligomycin (12.6 �M), and the FoF1-ATP-aseactivity was expressed as the oligomycin-sensitive ATPaseactivity.

Respirometric measurementsWe used the OROBOROS oxygraph, a two-chamber

respirometer equipped with a Peltier thermostat and in-tegrated electromagnetic stirrers. The oxygen concentra-tion was recorded by the software DatLab (OROBOROSinstruments), and oxygen consumption rates were calcu-lated and expressed as specific oxygen consumption rates(picomoles O2 per second per 106 cells). Measurementswere performed using 0.5–1.5 � 106 cells/ml in 2 ml res-piration buffer Mir05 (39) at 37 C with continuous stir-ring. The following substrate-uncoupler-inhibitor titra-tion regime was applied: after measuring the routinerespiration (no additions), the cell membrane was perme-abilized with digitonin (10 �g/106 cells or 5 �g/106 cells).Complex I-dependent respiration was measured withmalate (2 mM) and pyruvate (5 mM) after addition of ADP(2 mM). Cytochrome c (10 �M) was added to test for theintegrity of the outer mitochondrial membrane. The max-imal coupled respiration of complexes I and II was stim-ulated by the addition of succinate (10 mM). Succinate-related pyruvate respiration was assessed as ratio ofpyruvate/malate respiration in relation to succinate respi-ration. The maximum capacity of the electron transportsystem was obtained by uncoupling with carbonyl cyanidep-trifluoro-methoxyphenyl-hydrazone (FCCP). Inhibi-tion with rotenone (0.5 �M) shows the maximal uncou-pled respiration via complex II. The residual oxygen con-sumption was estimated by inhibition of complex III withantimycin A (2.5 �M). This residual oxygen consumptionwas ascribed to nonmitochondrial sources and was there-fore subtracted from the initial oxygen uptake rates.

Proliferation assayThe CellTiter 96 AQueous nonradioactive cell prolif-

eration assay (Promega) was used to determine cell via-


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bility and proliferation of NCI-H295R cells treated withmetformin or rotenone. Briefly, cells were plated on 96-well plates at a density of 20,000 cells per well. The me-dium was changed to starvation medium, and cells wereallowed to grow for 24 h. Cells were then treated witheither 10 mM metformin or 200 nM rotenone. After 0, 6, 24,and 48 h, cell proliferation was assessed by adding 20 �lMTS/PMS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)/(phenazinemethosulfate)] to the culturemediumfor3hbefore reading theabsorbance at 490 nm (Sunrise microplate reader; Tecan, Sal-zburg, Austria). In this assay, the absorbance at 490 nm corre-lates directly to the number of viable cells.

Data analysisStatistical analyses were performed using the statistical

software Prism version 4 (GraphPad Software, Inc., SanDiego, CA). Statistical differences between mean valueswere evaluated by Student’s t test or one-way ANOVAfollowed by the Bonferroni posttest where appropriate.Quantitative data represent the mean of at least two in-

dependent experiments, and error bars are indicated as themean � SEM or � SD. Significance was set at P � 0.05, P �0.01, and P � 0.001.

Results

Metformin inhibits androgen production and theactivities of CYP17-lyase as well as HSD3B2 in NCI-H295R cells

Several clinical studies have demonstrated that met-formin treatment decreases elevated androgen concentra-tion in PCOS patients (16–18). An effect of metformin onsteroidogenesis has also been found in vitro. It decreasedsynthesis of Prog and estradiol in granulosa cells of thebovine ovaries (27). The steroidogenic pathways of thehuman adrenal cortex and the gonads employ the sameenzymes for the conversion of cholesterol to C19 steroids(precursors of androgens) (1). Also, human adrenal NCI-H295R cells are a well-established cell model for studyingsteroidogenesis (40, 41). Therefore, we investigated the

effect of metformin on androgen pro-duction in human adrenal NCI-H295Rcells in search of a cell model to studythe mechanism of action of metformin.We treated NCI-H295R cells with0–10 mM metformin for 48 h and testedfor changes in the steroid profile byTLC. Overall, we found a significantincrease in 17OHPreg and a decrease in�4A production in NCI-R cells aftertreatment with 10 mM metformin, sug-gesting an inhibitory effect of met-formin on CYP17-lyase and HSD3B2activities (Fig. 1, A and B). In addition,we were able to confirm that metformininhibits androgen production in ourNCI cell model.

CYP17 and HSD3B2 are essentialenzymes for the conversion of preg-nenolone to �4A. Thus, we tested theeffect of metformin on their activitiesspecifically. To assess the activities ofcombined CYP17-OHase and 17,20-lyase in metformin-treated cells, weblocked endogenous HSD3B2 activityby trilostane (a specific HSD3B2 inhib-itor) and labeled steroidogenesis with[3H]Preg (Fig. 1C). To assess the met-formin-mediated effect on 17,20-lyaseactivity alone, [3H]OHPreg was used assubstrate to label steroidogenesis in

FIG. 1. Effect of metformin on steroid production in NCI-H295R cells. Cells were treated with0–10 mM metformin (Met) for 48 h. Steroidogenesis was labeled using 100,000 cpm/35-mmwell of [3H]Preg, [3H]17OHPreg, or [3H]DHEA as substrates for 90 min. Steroids were thenextracted from the cell medium and resolved on TLC plates. To specifically characterize theCYP17-OHase and CYP17-lyase activities, HSD3B2 activity was blocked by 1 �M trilostane. A,Representative TLC showing the effect of metformin treatment on the steroidogenic profile ofNCI-H295R cells when using labeled Preg as substrate (overview); B, quantification of basaland metformin-affected steroidogenesis of NCI-H295R cells; C–E, representative TLC (upperpanel) and quantifications (lower panel) of specific CYP17-OHase (C), CYP17-lyase (D), andHSD3B2 (E) activities. Results are expressed as percentage of total radioactivity incorporated inspecific steroid products (B) or percentage of unstimulated cells (C–E) and represent the meanof three to four independent experiments. Error bars represent SD. *, P � 0.05; **, P � 0.01.11 DOC, 11-Deoxycortisol.


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trilostane-treated cells (Fig. 1D). Finally, the effect of met-formin on HSD3B2 activity was assessed by using its directsubstrate [3H]DHEA (Fig. 1E). As expected from steroidprofiling (Fig. 1, A and B), we found that 10 mM metformin

significantly inhibited the activities of CYP17-lyase as wellas HSD3B2 (Fig. 1, D and E). By contrast, OHase activityof CYP17 seemed to be unaffected by metformin (Fig. 1C).Metformin did not alter any of these enzyme activities

when tested directly in enzyme kineticassays using recombinant HSD3B2 andCYP17 proteins produced in yeast(data not shown) (42), indicating thatits action requires the cellular context.

Effect of metformin ontranscription and expression ofsteroidogenic genes

We used promoter reporter assays,quantitative real-time PCR, and West-ern blot analyses to assess the effect ofmetformin on transcription and geneexpression of CYP17A1, HSD3B2,CYB5, and POR. CYB5 and POR weretested because they are important co-regulators of CYP17 activities (1).

To test whether metformin modu-lates the activities of promoters of ste-roidogenic genes, we transfected NCI-H295R cells with specific promoterconstructs (�3.7CYP17, �1.05HSD3B2,�1.3CYB5, and �325POR) (40, 41) andtreated them with 10 mM metformin for48 h before measuring promoter activities.Metformindidnotchangepromoteractiv-ities of CYP17A1, HSD3B2, CYB5, orPOR,indicatingthatthereisnoeffectatthelevel of transcription (Fig. 2A).

To assess for an effect of metforminon gene expression and protein level,we treated NCIH295R cells with met-formin and extracted either total RNAfor quantitative RT-PCR (qRT-PCR)or prepared total protein extracts forWestern blot analyses. The qRT-PCRexperiments revealed an inhibition ofHSD3B2 expression after treatmentwith 10 mM metformin (Fig. 2B). Bycontrast, metformin did not change ex-pression of CYP17A1, CYB5, and POR(Fig. 2B). At the protein level, 10 mM

metformin did not significantly alterthe expression of CYP17 over 0–48 h(Fig. 2C). Qualitative analysis of theWestern blots for HSD3B2 showed anapparent increase 3–6 h after met-formin treatment followed by a de-

FIG. 2. How metformin affects the promoter activities and the expression of genes that arecrucial for androgen production. A, Effect on promoter activities of CYP17, HSD3B2, CYB5,and POR. NCI-H295R cells were transiently transfected with specific promoter luciferaseconstructs as indicated. After transfection, cells were treated with 10 mM metformin for 48 h,and promoter activation was assessed by dual luciferase assay readout (Promega) using TK-pRL as internal control. Data are expressed as percentage of untreated cells and represent themean � SEM of two independent experiments performed in duplicates. B, Gene expressionstudies. Total RNA was isolated from cells treated with 0–10 mM metformin for 48 h. Afterreverse transcription to cDNA, SYBR Green real-time PCR was performed to assess the geneexpression of CYP17, HSD3B2, CYB5, and POR. 18S rRNA served as internal control. Data areexpressed as percentage of expression of untreated cells and represent the mean � SD ofthree independent experiments. *, P � 0.05. C and D, Effect of metformin on proteinexpression of CYP17 and HSD3B2 by Western blot analyses of total cell extract from NCI-H295R cells treated with 10 mM metformin for 0–48 h using specific antibodies.Representative blots are depicted, whereas quantification of three independentexperiments � SD is given in numbers.


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crease after 48 h in line with qRT-PCR experiments (Fig.2C).

Human OCT influence the action of metformin onandrogen production in NCI-H295R cells

The OCT1–3 and the plasma membrane monoaminetransporter (PMAT) have been shown to play a role in thetherapeutic action of metformin (22, 43–46). Until now,it remained a conundrum why effective concentrations ofmetformin needed in cell experiments are vastly higher(usually �5 mM) than therapeutic plasma concentrationsin the clinical setting (�10 �M; range, 5–50 �M).

To study the role of metformin transporters in steroid-ogenic NCI-H295R cells, we assessed the expression pro-file of human OCT1–3 and human PMAT of NCI-H295Rcells as well as human adrenal tissues by semiquantitativeRT-PCR. We found that PMAT is readily expressed inNCI-H295R cells and human adrenal tissues (Fig. 3A). Incontrast, OCT1–3 seemed to be expressed at very lowlevels (40 cycles of PCR; Fig. 3A). In comparison, humanhepatocellular HepG2 cells expressed hOCT1 and -3 andPMAT already at 30 cycles of PCR (Fig. 3A). This tissue-specific expression of hOCT is in line with published lit-

erature (47) and entries in the Gene Expression Omnibus(GEO) database (www.ncbi.nlm.nih.gov/geo/). We there-fore hypothesized that overexpression of OCT in NCI-H295R cells may promote the androgen-lowering effect ofmetformin at lower concentrations. To test our hypothe-sis, cells were transfected with hOCT1, hOCT2, or theempty expression vector (pEGFP-N3) and treated with0–10 mM metformin, and the steroid profile was assessedby TLC. We found that, similar to untransfected cells (Fig1, A and B), empty vector-transfected cells showed a de-crease in �4A production only after treatment with 10 mM

metformin (Fig. 3B). However, when effectively overex-pressing hOCT1 or hOCT2 in NCI-H295R cells (see ex-pression profile in Fig. 3B), metformin was able to inhibit�4A production already at concentrations of less than 1mM. Also, hOCT2 seemed to be more effective thanhOCT1 in enhancing metformin’s action (Fig. 3B). More-over, to further characterize the interplay between OCTand metformin, we intended to block OCT by using quin-idine (kindly provided by Christian Lanz and Jürg Müller,Bern, Switzerland), expecting to abolish or decrease theeffect of metformin on steroidogenesis in NCI-H295Rcells (48). But quinidine was found to inhibit CYP17A1

activities in NCI-H295R cells directlyand is therefore not suitable for OCTtransporter studies in steroidogeniccells (data not shown).

Metformin does not act throughAMPK, MAPK/ERK1/2, or atypicalPKC to modulate androgenproduction in NCI-H295R cells

Several studies have demonstratedthat metformin enhances the phosphor-ylation/activation of AMPK (23, 27,30, 31). Therefore, the impact of met-formin on AMPK signaling in NCI-H295R cells was examined. We usedthe chemical AICAR as an establishedpositive control for AMPK phosphor-ylation/signaling. By contrast, com-pound C (an established inhibitor ofAMPK) was not used because previousstudies in our lab showed that it is adirect CYP17 inhibitor (35). Time-de-pendent analyses of the phosphoryla-tion status of AMPK in metformin-treated cells revealed that metformindid not change the phosphorylation ofAMPK (Fig. 4A). By contrast, in ourcontrol experiment, AICAR was foundto increase phosphorylation of AMPK

FIG. 3. Role of transporters for metformin’s effect on steroidogenesis in NCI-H295R cells. A,Gene expression profile of metformin transporters in NCI-H295R cells, human adrenal tissues,and HepG2 or COS1 cells. Semiquantitative RT-PCR analysis was performed for hOCT1–3 andPMAT mRNA expression. PCR (30 or 40 cycles) was performed on 100 ng of reverse-transcribed total RNA, and products were separated on 1.5% agarose gel and visualized withethidium bromide. Plasmids containing the cDNA of hOCT1 and hOCT2 as well as cDNA ofthe cell lines HepG2 and HT29 served as positive controls. 18S served as loading control. A,Representative RT-PCR result of three independent experiments is depicted. B, Enhancedeffect of metformin on steroidogenesis after overexpression of hOCT1/2. NCI-H295R cellswere transiently transfected with either hOCT1 or hOCT2 or the empty vector for control.Transfected cells were treated with 0–10 mM metformin for 48 h, and steroid production wasassessed using 80,000 cpm/35-mm well of [3H]Preg as substrate for 90 min. Steroids wereextracted from the cell medium and resolved on TLC plates. Quantification was performed onthree independent experiments. Results are expressed as fold of control regarding �4Aproduction. Error bars are �SD. *, P � 0.05; **, P � 0.01; ***, P � 0.001. In addition,overexpression of hOCT1/2 was assessed by performing RT-PCR on extracted RNA fromtransfected NCI cells (B, right panel).


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as expected (Fig. 4B). Because these results were not in linewith the reported findings for muscle or liver cells, weperformed exactly the same experiment in HepG2 cellsusing the NCI-H295R serum-free growth medium to ex-clude an experimental flaw. Similar to published results,treatment with 2 mM metformin increased phosphoryla-tion of AMPK within 3–24 h in these cells (data notshown), suggesting a cell-specific action of metformin.

To assess functional interplay of AMPK with met-formin on androgen production, we studied the steroidprofiles of NCI-H295R cells treated with 10 mM met-formin, 1 mM AICAR, or both after 48 h (Fig. 4C). AICARstimulation of AMPK increased CYP17A1-lyase activity,but did not change 17�-hydroxylase or HSD3B2 activity.Metformin alone decreased lyase activity, whereas met-

formin in combination with AICARabolished this effect, mirroring the ef-fect of AICAR alone (Fig. 4C). The ac-tivity of HSD3B2 was decreased bymetformin treatment only and re-mained unaffected by AICAR (Fig. 4C).Taken together, these data suggest anAMPK-independent mechanism of ac-tion of metformin on adrenal androgenproduction.

Previous studies have suggested thatmetformin involves the signaling path-ways of MAPK and atypical PKC for itsmetabolic effect on liver and muscle(29, 49). In addition, the MAPK path-way has been shown by us and others toregulate CYP17 and thus androgenproduction (50, 51). Therefore, we per-formed Western blot analyses to assessthe potential effect of 10 mM metforminon MAPK and atypical PKC signalingin NCI-H295R cells using specific an-tibodies. Metformin did not changeERK1/2 phosphorylation (Fig. 4D) oratypical PKC phosphorylation signifi-cantly in NCI-H295R cells (Fig. 4E),indicating that these signaling path-ways might not be crucial for met-formin’s action on androgens.

Metformin inhibits complex I ofthe mitochondrial respiratorychain

Previous studies suggested that themetabolic effect of metformin in hepa-tocytes is mediated by inhibiting com-plex I of the mitochondrial respiratorychain (24, 25). Therefore, we studied

the effect of metformin on the complexes of the mitochon-drial respiratory chain. NCI-H295R cells were treatedwith metformin for 48 h, and then mitochondria wereisolated and activities of the complexes were assessed us-ing specific spectrophotometric assays (37). Interestingly,metformin was found to inhibit complex I very specificallyin isolated NCI-H295R mitochondria, whereas assess-ment of activities for complexes II–V revealed no changesafter treatment with metformin (Fig. 5A). A direct inhibitoryeffect of metformin on complex I was also tested using iso-lated mitochondria of untreated NCI-H295R cells. Met-formin was observed to inhibit complex I activity in a dose-dependent manner (Fig. 5B). Yet 10 mM metformin was ableto inhibit complex I activity directly by only 18%, whereas

FIG. 4. Role of AMPK, MAPK (ERK1/2), and atypical PKC signaling in metformin’s inhibitoryeffect on androgen production. Western blot analyses were performed on total proteinpreparations from NCI-H295R cells treated with 10 mM metformin for 0–48 h (A, D and E) oronly 48 h (B). Specific antibodies recognizing signaling molecules as indicated were used; �-actin served as loading control. A and B, Effect of metformin on AMPK activity[phosphorylation (P)]. Stimulation with AICAR (an established AMPK activator) was used aspositive control when investigating NCI cells (B). C, Comparative effect of AICAR (AMPKactivator) and metformin on androgen-producing enzymes. NCI-H295R cells were treatedwith 10 mM metformin, and AMPK signaling was optionally stimulated with 1 mM AICAR for48 h. HSD3B2 activity was blocked by 1 �M trilostane to specifically assess the CYP17-OHaseand CYP17-lyase activities. Steroidogenesis was labeled with 100,000 cpm/35-mm well[3H]Preg, [3H]17OHPreg, or [3H]DHEA as substrates for 90 min. Steroids were extracted fromcell medium and resolved on TLC plates. Representative TLC (upper panel) and quantifications(lower panel) of CYP17-OHase/-lyase, CYP17-lyase and HSD3B2 activities are shown. D and E,Western blots showing the effect of metformin on MAPK (D) and atypical PKC signaling (E).A–E, Quantification was performed on three to four independent experiments. Quantitativeresults are expressed as a percentage of control; error bars are �SD. *, P � 0.05; **, P �0.01; ***, P � 0.001.


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treatment of NCI cells with the same concentration of met-formin led to an inhibition of 37%, indicating that met-formin exerts its effect both directly and indirectly.

In addition to the effect of metformin on the activity ofcomplex I in isolated mitochondria, the effect of met-formin on mitochondrial function was analyzed in moredetail by high-resolution respirometry (Fig. 5C). After incu-bation with metformin for 48 h, NCI-H295R cells presenteda reduced endogenous routine respiration. To further dissectthis respiratory defect, cells were permeabilized with digi-tonin followed by a substrate-uncoupler-inhibitor titrationregime. Metformin treatment resulted in a significant de-crease in respiration driven by substrates for complex I. Res-piration with the substrate for complex II and the total elec-tron transport capacity after FCCP titration showed nosignificant difference with and without metformin. More-over, NCI-H295R cells treated with metformin showed aremarkably reduced succinate-related pyruvate respirationof 0.85 � 0.06 to 0.53 � 0.09 (�38%; P � 0.056). Thisfinding is in line with the reduced complex I activity inducedby metformin. Taken together, metformin seems to specifi-cally decrease the oxygen flow induced by substrates forcomplex I.

Therefore, because the androgen-lowering effect ofmetformin seems to be mediated by specific inhibition ofcomplex I of the mitochondrial respiratory chain, directinhibitionof complex Ibyanyother specificmeans, shouldresult in a decrease in androgen production. To confirmthis, we treated our NCI-H295R cells with the specificcomplex I inhibitor rotenone (52) in a dose- and time-dependent manner and tested for changes in the steroidprofile. Treatment of NCI-H295R cells with 0–2000 nM

rotenone for 6, 24, or 48 h decreased �4A production after24 h, suggesting an effect at the level of CYP17A1 and/orHSD3B2 activities (data not shown). Specific testing forthese enzyme activities revealed that rotenone decreasedandrogen (�4A) production by inhibiting the activity ofHSD3B2 after 24 h in a dose-dependent fashion, similar tometformin (Fig. 6A). By contrast, unlike metformin, ro-tenone did not affect CYP17A1 activity (Fig. 6A). In ad-dition, to exclude direct effects of rotenone on the activ-ities of our enzymes of interest, we assessed the effect ofrotenone on the activities of CYP17A1 and HSD3B2 re-combinantly expressed in yeast microsomes (53). Variousconcentrations of rotenone had no direct effect on theenzyme activities of CYP17A1 and HSD3B2 (Fig. 6B).

In theory, inhibition of complex I results in a decreasein the oxidation ratio of NADH to NAD, which leads tofeedback inhibition of the pyruvate dehydrogenase en-zyme and thus enhanced production of mitochondrial py-ruvate (54). Pyruvate transported into the cytosol is thenmetabolized into lactate by cytosolic lactate dehydroge-nase. Thus, generally, an inhibition of complex I of theoxidative phosphorylation system (OXPHOS) system re-sults inan increased ratioof lactate topyruvate.Therefore,

FIG. 5. Effect of metformin on the complexes of the mitochondrialrespiratory chain. A, Cellular effect of metformin on specificcomplexes. NCI-H295R cells were cultured in serum-free medium (SM)with and without 10 mM metformin for 48 h. Mitochondria wereisolated, and the activities of the complexes were assessed byspectrophotometric assays. The specific activities of the respiratorychain complexes were obtained as milliunits per milligrammitochondrial protein and are expressed as ratios to citrate synthase(CS) activity serving as quality control of equal mitochondrial content.Complex I is NADH:ubiquinone oxidoreductase, complex II is succinatedehydrogenase, complex III is ubiquinol-cytochrome c reductase,complex IV is cytochrome c oxidase, and complex V is ATPase. Resultsare presented as mean of two to four independent experiments. Errorbars represent �SD. *, P � 0.05. B, Direct effect of metformin oncomplex I activity. NCI-H295R cells were cultured for 48 h, andmitochondria were isolated. Mitochondria complex I activity was thenmeasured in the presence or absence of 0.1–30 mM metformin. Dataare expressed as metformin concentration-dependent inhibition ofcomplex I activity (percent) and represent the mean and SD of threeindependent experiments. C, Respirometric measurements. NCI-H295Rcells were grown in serum-free medium (SM) with and without 10 mM

metformin for 48 h. Oxygen consumption was measured by high-resolution respirometry in respiration buffer, after the titration regimedescribed in the experimental section. Respiration rates weredetermined by subtracting the residual oxygen uptake rates measuredafter the addition of antimycin A (Ama). The addition of substrates,uncoupler, and inhibitors is indicated on the x-axis. Routine indicatesno addition (control). CytC, Cytochrome c; F, FCCP; PM, pyruvate andmalate; Rot, rotenone; S, succinate. Results are presented as the meanof two independent experiments � SEM done with cells permeabilizedwith 10 and 5 �g digitonin per 106 cells, respectively. *, P � 0.05.


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we determined the lactate to pyruvate ratio in the cell me-dium of NCI-R cells treated with 10 mM metformin for48 h or 200 nM rotenone for 24 h by gas chromatography/mass spectrometry in two independent experiments as de-scribed elsewhere (55). The lactate to pyruvate ratio ofuntreated cells was 11 � 0.04 arbitrary units (AU),whereas this ratio was markedly elevated approximately6-fold in metformin-treated cells (62.2 � 3.4 AU) andaround 4-fold in rotenone-treated cells (46.2 � 3.5 AU).

Metformin does not inhibit NCI-H295R cellproliferation in contrast to rotenone

Metformin has been shown to affect the cell cycle ofhuman prostate and breast cancer cells and is therefore apotential cancer drug (56). Because the NCI-H295R cellline originates from an adrenocortical carcinoma (57), wewere interested in the effect of metformin on cell viabilityand proliferation of NCI-H295R cells and whether an-drogen regulation runs by parallel mechanisms to cell cy-cle regulation. Furthermore, rotenone has been reportedto inhibit cell proliferation in HeLa (IC50 � 0.2 � 0.1 �M)and MCF-7 (IC50 � 0.4 � 0.1 �M) cells by disturbing thedynamics of microtubule assembly (58). Hence, we alsoanalyzed the effect of rotenone on the proliferation ofNCI-H295R cells. NCI-H295R cells were treated with 10

mM metformin or 200 nM rotenone for0–48 h in serum-free medium, and cellproliferation was assessed by a com-mercially available assay. Metformindid not affect the cell viability and pro-liferation of NCI-H295R cells (Fig.7A). We observed that rotenone tendedto inhibit the proliferation of NCI-H295R cells after 48 h; however, thistendency was not significant (P � 0.27)(Fig. 7B).

Discussion

In this study, we describe metformin’sregulatory inhibition of androgen bio-synthesis in a steroid cell model. Be-cause CYP17A1 and HSD3B2 are keyenzymes for androgen biosynthesis, wefocused the specific investigations onthose two steroid enzymes. Similar toobservations in clinical studies (16–18), metformin treatment reduces an-drogen production in NCI-H295Rcells. This seems to be caused by a de-crease in the activities of CYP17A1-lyase and HSD3B2 and by a decrease in

HSD3B2 but not CYP17A1 expression. Furthermore, weshow that the intracellular shift of metformin for its actionis transporter dependent. Finally, our studies reveal that inNCI-H295R cells, metformin does not act through sig-naling pathways including AMPK, MAPK, or atypicalPKC�/� signaling but that metformin specifically inhibitscomplex I of the respiratory chain in the mitochondrion.

Several clinical studies have shown that both the adre-nals and ovaries are the source of androgen excess inwomen suffering from PCOS (9) and that metformin treat-ment can ameliorate hyperandrogenism in these women(16–18). Ovarian theca cells originating from PCOSwomen are reported to have increased activities of CYP17and HSD3B2 (9, 15). In line with these findings, met-formin treatment decreases androgen production in ouradrenal NCI-H295R cell model by inhibiting the activitiesof CYP17-lyase and HSD3B2.

Recent studies have demonstrated that membranetransporters OCT play an essential role for the efficacy ofmetformin in clinical use (21, 22). Several nonsynony-mous single-nucleotide polymorphisms in the genes ofOCT1 and OCT2 have been found to decrease the capac-ity for transporting metformin intracellularly in vitro andhave therefore been suggested to cause failure to properly

FIG. 6. Effect of rotenone, a specific complex I inhibitor, on the activities of CYP17A1 andHSD3B2. NCI-R cells (A) and microsomes (B) were treated with 0–2000 nM rotenone, and thesteroid profile was assessed by TLC. A, Representative TLC and calculations of the activities ofCYP17A1 and HSD3B2 of the cell experiments. Results are presented as the mean of threeindependent experiments. Error bars represent � SD. *, P � 0.05; **, P � 0.01; ***, P �0.001. B, Representative TLC of the activities of CYP17A1 and HSD3B2 of the microsomeassays.


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reduce fasting glucose levels in some type 2 diabetic pa-tients under metformin therapy (59). We now show thatOCT1–3 are expressed at low levels in NCI-H295R cells,whereas HepG2 cells readily express hOCT1 and -3. Ac-cordingly, high concentrations of metformin (10 mM) areneeded to lower androgen production in NCI-H295Rcells, and overexpression of OCT1/2 results in an en-hanced effect of metformin at lower concentrations. Over-all, our in vitro studies show for the first time the impor-tant role of metformin transporters for its intracellularaction.

In the past, metformin has been thought to act throughdifferent signaling pathways. It has been described to ac-tivateAMPKvia liverkinaseB1 in several cells suchas liverand muscle cells as well as ovarian granulosa cells (26–28). However, whether AMPK is essential for metformin’saction remains controversial because AMPK-independenteffects of metformin have also been described (34, 60).Likewise, metformin has also been reported to act viaMAPK (ERK1/2) and atypical PKC signaling pathwaysthat may be associated with AMPK signaling (27, 29).Tosca et al. (27), for instance, have shown that metforminactivates AMPK and decreases phosphorylation ofMAPK3/MAPK1 (ERK1/2), resulting in reduced steroid-ogenesis of bovine ovarian cells. However, in contrast tothese results, human theca cells of PCOS woman seem tohave decreased activity of ERK1/2 with elevated andro-gens (61). On the other hand, in human granulosa cells,

metformin combined with insulin appears to activateERK1/2 and decrease mRNA expression as well as activityof CYP19 (P450 aromatase) for estrogen production (62).Our signaling studies suggest that metformin does not di-rectly target the AMPK, MAPK, or atypical PKC path-ways to regulate androgen production in NCI-H295Rcells. Possible explanations why our data are in contrast tothe investigations in ovary cells may include differences inthe cell model and experimental design or tissue-specificeffects of metformin.

In our opinion, the most important finding of this studyis that metformin inhibits complex I of the respiratorychain in mitochondria and that this leads to decreasedandrogen production. To confirm this, we show that di-rect inhibition of complex I by the specific inhibitor rote-none decreases androgen production by inhibitingHSD3B2 activity. In this respect, it is important to realizethat HSD3B2 activity depends on the NAD cofactor,where complex I maintains its pool through NADH oxi-dation. Thus, NAD is diminished in complex I-deficientcells. Interestingly, very recent studies have also suggestedcomplex I of the respiratory chain as the potential targetof metformin for its action on glucose metabolism (24, 25,34). Taken together, this may nicely explain the ideal pro-file of the drug metformin for the treatment of patientswith the hyperinsulinemic, hyperandrogenic form ofPCOS. Furthermore, rats treated with the complex I in-hibitor rotenone to mimic Parkinson’s disease have beenfound to have lower plasma testosterone levels than con-trol animals (63), confirming the important role of com-plex I of the respiratory chain for androgen production.Therefore, further investigations will aim at cofactors sup-porting complex I in its function to modulate androgenbiosynthesis. In addition, other NAD-dependent indirecteffects may be considered such as regulation of gene ex-pression through sirtuins. Sirtuins are histone deacetylasesthat require NAD as cofactor and are important playersin transcriptional regulation of metabolic genes and clockgenes (64). This issue will be the subject of our futurestudies. Whether these studies will elucidate how met-formin acts precisely to decrease HSD3B2 expression andCYP17A1-lyase activity remains to be seen. Alternatively,even other avenues of action need to be considered becauseinhibition of CYP17-lyase activity seems to be indepen-dent of complex I inhibition.

In summary, we demonstrate that metformin decreasesadrenal androgen production in NCI-H295R cells by tar-geting CYP17A1 and HSD3B2. Metformin inhibits com-plex I of the respiratory chain in mitochondria but seemsto exert no specific effect on signaling pathways includingAMPK, MAPK, and atypical PKC.

FIG. 7. Effect of metformin and rotenone on the proliferation of NCI-H295R cells. Cells were split into 96-well plates at a density of 20,000cells per well and cultured in serum-free medium in the presence orabsence of 10 mM metformin or 200 nM rotenone up to 48 h. Cellproliferation and viability were measured colorimetrically by aproliferation assay (Promega). Data are expressed as OD 490 nm thatcorresponds to the amount of viable cells. Results are given as mean �SEM of two independent experiments performed in duplicates. DMSO,Dimethylsulfoxide.


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Acknowledgments

We thank Giuliano Ciarimboli and Eberhard Schlatter (Mün-ster, Germany) for the hOCT1 and hOCT2 plasmids. We alsothank Christian Lanz and Jürg Müller (Bern, Switzerland) forquinidine and Merck/Serono AG (Geneva, Switzerland) for pro-viding metformin for our studies. Finally, we acknowledgeChantal Cripe-Mamie’s proofreading our manuscript.

Address all correspondence and requests for reprints to:Christa E. Flück, M.D., Pediatric Endocrinology and Diabetology,University Children’s Hospital Bern, Freiburgstrasse 15, Room G3812, 3010 Bern, Switzerland. E-mail: [email protected].

This work was supported by a grant from the Swiss NationalScience Foundation (31003A-130710 to C.E.F.) and an inde-pendent project grant by Merck/Serono, Switzerland (to C.E.F.).

Disclosure Summary: The authors A.H., D.H., P.K., G.H.,J.-M.N., and P.E.M. have nothing to disclose. C.E.F. received anindependent project grant from Merck/Serono, Switzerland.

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