PPAR[gamma] isoforms differentially regulate metabolic networks to mediate mouse prostatic epithelial differentiation

Abstract

Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specific ablation of PPARγ in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARγ isoforms in PPARγ knockout prostate epithelial cells. Expression and activation of either PPARγ 1 or 2 reduced de novo lipogenesis and oxidative stress and mediated a switch from glucose to fatty acid oxidation through regulation of genes including Pdk4, Fabp4, Lpl, Acot1 and Cd36. Differential effects of PPARγ isoforms included decreased basal cell differentiation, Scd1 expression and triglyceride fatty acid desaturation and increased tumorigenicity by PPARγ1. In contrast, PPARγ2 expression significantly increased basal cell differentiation, Scd1 expression and AR expression and responsiveness. Finally, in confirmation of in vitro data, a PPARγ agonist versus high-fat diet (HFD) regimen in vivo confirmed that PPARγ agonization increased prostatic differentiation markers, whereas HFD downregulated PPARγ-regulated genes and decreased prostate differentiation. These data provide a rationale for pursuing a fundamental metabolic understanding of changes to glucose and fatty acid metabolism in benign and malignant prostatic diseases associated with systemic metabolic stress.

Citation: Cell Death and Disease (2012) 3, e361; doi:10.1038/cddis.2012.99

& 2012 Macmillan Publishers Limited All rights reserved 2041-4889/12 http://www.nature.com/CDDIS

Web End =www.nature.com/cddis

PPARc isoforms differentially regulate metabolic networks to mediate mouse prostatic epithelial differentiation

DW Strand1, M Jiang*,1, TA Murphy2, Y Yi3, KC Konvinse1, OE Franco1, Y Wang1, JD Young2,4 and SW Hayward*,1,5

Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specic ablation of PPARc in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARc isoforms in PPARc knockout prostate epithelial cells. Expression and activation of either PPARc 1 or 2 reduced de novo lipogenesis and oxidative stress and mediated a switch from glucose to fatty acid oxidation through regulation of genes including Pdk4, Fabp4, Lpl, Acot1 and Cd36. Differential effects of PPARc isoforms included decreased basal cell differentiation, Scd1 expression and triglyceride fatty acid desaturation and increased tumorigenicity by PPARc1. In contrast, PPARc2 expression signicantly increased basal cell differentiation, Scd1 expression and AR expression and responsiveness. Finally, in conrmation of in vitro data, a PPARc agonist versus high-fat diet (HFD) regimen in vivo conrmed that PPARc agonization increased prostatic differentiation markers, whereas HFD downregulated PPARc-regulated genes and decreased prostate differentiation. These data provide a rationale for pursuing a fundamental metabolic understanding of changes to glucose and fatty acid metabolism in benign and malignant prostatic diseases associated with systemic metabolic stress.

Cell Death and Disease (2012) 3, e361; doi:http://dx.doi.org/10.1038/cddis.2012.99

Web End =10.1038/cddis.2012.99 ; published online 9 August 2012

Subject Category: Cancer Metabolism

Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are age-related diseases associated with complications of metabolic syndrome (MetS).1 However, the molecular underpinnings of prostatic susceptibility to systemic metabolic dysfunction are poorly understood, in part because dietary and transgenic animal models display a limited recapitulation of human benign growth and stromal expansion or adenocarcinoma. Furthermore, unlike adipose, muscle and liver, understanding of the effects of systemic metabolic stressors on prostate growth and/or transformation are hampered by a limited understanding of the prostates normal nutritional metabolism.

Epidemiological links between BPH and diabetes have been recognized for many years2 and recent studies have demonstrated that the incidence and severity of BPH are correlated with obesity, atherosclerosis, diabetes mellitus, hyperinsulinemia, hyperglycemia and hypercholesterolemia.3,4,5,6,7 Although diabetes mellitus has a negative

correlation with the incidence of multiple cancers including prostate, diabetic patients exhibit increased mortality.8 Moreover, MetS as a set of comorbidities (obesity, insulin resistance, dyslipidemia and hypertension) is correlated with PCa incidence.9 Such associations have prompted the

1Department of Urologic Surgery, A-1302 MCN, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Engineering, and Vanderbilt University Medical Center, Nashville, TN 37232-2765, USA; 2Department of Chemical and Biomolecular Engineering, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Engineering, and Vanderbilt University Medical Center, Nashville, TN 37232-2765, USA; 3Department of Medicine and Institute for Integrative Genomics, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Engineering, and Vanderbilt University Medical Center, Nashville, TN 37232-2765, USA; 4Department of Molecular Physiology and Biophysics, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Engineering, and Vanderbilt University Medical Center, Nashville, TN 37232-2765, USA and 5Department of Cancer Biology, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Engineering, and Vanderbilt University Medical Center, Nashville, TN 37232-2765, USA*Corresponding authors: M Jiang, Department of Urologic Surgery, A-1302 MCN, Vanderbilt University Medical Center, Nashville, TN 37232-2765, USA. Tel: [phone]; Fax: [phone]; E-mail: mailto:[email protected]

Web End [email protected] or SW Hayward, Department of Urologic Surgery, A-1302 MCN, Vanderbilt University Medical Center, Nashville, TN 37232-2765. Tel: [phone]; Fax: [phone]; E-mail: mailto:[email protected]

Web End [email protected]

Received 09.5.12; revised 06.6.12; accepted 21.6.12; Edited by C Munoz-Pinedo

Keywords: prostate; differentiation; peroxisome proliferator-activated receptor gamma (g); PPARg; fatty acid metabolism; androgen receptorAbbreviations: acetyl-CoA, acetyl coenzyme A; AR, androgen receptor; ARE, androgen response element; BPH, benign prostatic hyperplasia; DHT, dihydrotestosterone; eWAT, epididymal white adipose tissue; HFD, high-fat diet; MetS, metabolic syndrome; mPrE-PPARg KO (mPrE-gKO), a PPARg knockout mouse prostate epithelial cell line spontaneously immortalized from an adult PBCre4 tg/0/PPARg ox/ox mouse prostate; mPrE-PPARgKO PPARg1 ( PPARg1 or g1), mPrE-gKO overexpressing mouse PPARg1 WT full-length cDNA; mPrE-PPARgKO PPARg2 ( PPARg2 or g2), mPrE-gKO overexpressing mouse

PPARg2 WT full-length cDNA; mPrE-PPARgKO empty vector ( EV), mPrE-gKO retrovirally transduced using a control empty vector; MUFAs, monounsaturated

fatty acids; KO, knockout; PCa, prostate cancer or prostate carcinoma; PIN, prostatic intraepithelial neoplasia; PPARg, peroxisome proliferator-activated receptor

gamma; ROS, reactive oxygen species; TCA, tricarboxylic acid; TZDs, thiazolidinediones; UGM, urogenital mesenchyme

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

2

investigation of metabolic genes and potential metabolic therapies in benign and malignant prostatic diseases.3,10

The peroxisome proliferator-activated receptors (PPARs) are a family of nuclear fatty acid receptors that regulate tissue-specic cellular metabolism and differentiation and have been widely sought after therapeutic targets for a number of obesity-related metabolic diseases owing to their ability to regulate glucose and fatty acid metabolism.11,12 A class of

PPARgamma (PPARg) agonists called thiazolidinediones (TZDs) are used in the treatment of insulin resistance and regulate a wide range of genes with tissue-specic effects.13

Historically, PPARg has been associated with pre-adipocyte expansion and differentiation,14 but other tissues also show a functional role for PPARg, including liver15 and

muscle.16 We showed previously that PPARg ablation in mouse prostate causes tumorigenesis and active auto-phagy,17,18 suggesting PPARg may provide a molecular link between systemic metabolism and prostate differentiation and growth.19 There are two isoforms of PPARg with the longer PPARg2 isoform using an alternate transcription start site containing a 30-amino acid N-terminal extension.

Our goal in this study was to garner a fundamental molecular and cellular understanding of the role of PPARg in mediating metabolic control of prostatic differentiation.

Because of the importance of individual PPARg isoforms in systemic metabolism and our previous work implicating

PPARg in prostate epithelial growth and differentiation, we chose to examine the potential roles of individual PPARg isoforms in mediating nutrient metabolism in the prostate, which has not been performed in any tissue. A prostatic epithelial cell line (mPrE-PPARgKO) restored with either

PPARg1 or PPARg2 isoform was used to determine how each isoform might contribute to prostatic metabolism, differentiation and disease. We show, using in vitro analysis, lipidomics and in vivo animal models that PPARg isoforms control overlapping and distinct metabolic programs in prostate epithelia that lead to functional changes in glucose and lipid metabolism and that these changes are coordinate with reduced lipogenesis, increased b-oxidation and markers of basal and luminal epithelial differentiation. Furthermore, we show in animals that prostate differentiation is oppositely affected after chronic treatment with a TZD versus high-fat diet (HFD) through disparate regulation of PPARg-and its downstream genes. These data suggest, as in other tissues, that PPARg agonization may directly or indirectly modulate the nutritional supply of glucose and lipids for prostate metabolism and differentiation.

Results

Restoration of PPARc2, but not PPARc1, reverses PPARcKO-induced mouse prostatic carcinogenesis. Alternative transcription start sites and splicing produce two PPARg isoforms, so only the longer PPARg2 isoform can be knocked out individually.20 In order to study the independent functions of PPARg1 and -g2 isoforms on prostate metabolism and differentiation, we developed a prostate epithelial cell line (mPrE-PPARgKO) with genetic knockout (KO) of both PPARg1 and -g2 isoforms from an adult PB-Cre4 tg/0/

PPARgox/ox double-transgenic male mouse.17 We then

restored mPrE-gKO cells with mouse PPARg1 cDNA ( PPARg1), PPARg2 cDNA ( PPARg2) or an empty vector

( EV) as control, respectively, to create an isogenic series

of cell lines for genetic and functional comparisons (see Materials and Methods).

In order to determine the effects of PPARg isoforms on tissue morphogenesis in vivo, mPrE-gKO and restored cell lines were each recombined with inductive 18-day fetal rat urogenital mesenchyme (UGM) and grafted into the kidney capsule for 2 months (Figures 1ac). Control mPrE-gKO EV

cells (empty vector-transfected) regenerated high-grade mouse prostatic intraepithelial neoplasia (HGPIN) (Jiang et al.17; Figure 1a) with predominantly Ck8/Ck18 luminal epithelial glands and few Ck14 basal cells (Figure 1d). Upon restoration with PPARg1, large areas of

Ck8/18 /Ck14 middle or highly differentiated adenocarcinoma were observed in PPARg1 tissue recombinants

(Figure 1b, black star, Figure 1e), but large uid-lled cysts were also formed (Figure 1b, white star). PPARg2 restoration resulted in the regeneration of Ck8/18/Ck14 acini that resembled developing prostate glands without evidence of tumor formation (Figures 1c and f). Furthermore, androgen receptor (AR) expression was demonstrated in regenerated tissues by immunohistochemical staining using EV or

PPARg2 cells, but not with PPARg1 cells (Figures 1gi).

These data indicate that restoration of PPARg2 isoform, but not PPARg1 isoform, reverses PPARg-decient mouse prostatic carcinogenesis through an increase in Ck14 basal cells.

PPARc isoforms 1 and 2 differentially regulate mouse prostate benign epithelial cell differentiation as well as luminal AR expression and function. In order to conrm the in vivo restoration of basal and luminal differentiation by PPARg2 expression shown in Figure 1, protein expression in mPrE-gKO EV, PPARg1 and PPARg2 cells was

examined by western blot, which revealed increases in both Ck14 and AR upon expression of PPARg2 (Figure 2a). To determine whether PPARg isoform expression increased the differentiation of basal cells in vitro, each mPrE-gKO cell line was double stained in culture for PPARg and Ck14 (Figure 2b(iiii)). Results demonstrated that although PPARg expression in PPARg1 cells was increased 14% versus EV cells, this resulted in an insignicant change in Ck14 expression (Figure 2b (ii versus iii)). Alternatively, and consistent with regeneration experiments in vivo (Figure 1f), PPARg2 restoration resulted in a 15% increase in PPARg cells as well as a 15% increase in Ck14 cells (Figure 2b(iii), quantied in Figure 2c). Interestingly, only 6

9% of the PPARg cells had overlapping Ck14 expression, suggesting a potential paracrine regulation of basal cell differentiation. To determine whether the increase in basal cells also resulted in increase luminal differentiation, cells were treated with dihydrotestosterone (DHT) and co-stained for AR and CK14 (Figure 2b(iv-vi)). In PPARg2-rescued cells, cells adjacent to PPARg cells were observed to have nuclear AR localization (Figure 2b(vi), arrowhead). In contrast, fewer cells were found with nuclear AR localization in EV or PPARg1 cells treated with DHT (Figure 2b

(iv versus v)). To conrm increased AR responsiveness, an androgen response element (ARE)-luciferase construct was

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

3

Figure 1 Restoration of PPARg2, but not PPARg1, reverses PPARg KO-induced mouse prostate carcinogenesis. mPrE-PPARgKO EV, PPARg1 or PPARg2 cell

lines were recombined with inductive rat UGM and grafted under the kidney capsule for 2 months (N 3 each). Histological analysis revealed regeneration of Ck14/Ck18/

AR HGPIN in mPrE-PPARg PPARgKO EV grafts (a, d and g). Restoration of PPARg1 resulted in a mixture of uid-lled cysts (black star) and CK14 /Ck18/AR

middle to highly differentiated adenocarcinoma (white star) (b, e and h). Restoration of PPARg2 resulted in regeneration of Ck14 /Ck18/AR benign acinus formation without any tumors (c, f and i)

transfected in each cell line, which demonstrated that

PPARg2 cells signicantly increased AR responsiveness 3-fold, while PPARg1 cells had no response and

mPrE-gKO EV cells had a 1.8-fold increase (Figure 2d).

The results showed restoration of PPARg2 rescues and drives mouse prostate benign epithelial cell differentiation associated with AR activation.

PPARc isoforms 1 and 2 regulate both overlapping and distinct metabolic networks. In order to determine the potential molecular disparity between PPARg1- and PPARg2-driven epithelial differentiation, microarrays were performed on mPrE-PPARgKO EV versus PPARg1 or PPARg2

cells. As outlined in Figure 3a, EV cells minus/plus

Rosiglitazone (Rosi) were examined to eliminate PPARg-independent effects of Rosi (Supplementary Figure 1a).

These independent effects were subtracted from results generated from comparison of EV versus PPARg1 and EV versus PPARg2 to identify PPARg-specic effects of

Rosi. Using INGENUITY software (Redwood City, CA, USA) and the signicance analysis of microarray (SAM) test for signicance, Figure 3b displays the top networks regulated by PPARg isoforms in prostate epithelial cells. Individual genes regulated by PPARg1 and PPARg2 (top 10 up and down-regulated genes shown in Supplementary Figure 1a)

included numerous focus molecules with functions related to amino acid, carbohydrate and lipid metabolism, drug

metabolism and cellular detoxication as well as inammation and immunity.

Restoration of PPARc1 or -c2 isoforms reduces lipogenesis/oxidative stress. Microarray data analysis of PPARg isoform-regulated genes showed a strong upregulation of genes involved in fatty acid metabolism, which has been shown to reduce de novo lipogenesis in some tissues;21 however, the inuence of fatty acid metabolism on prostate differentiation has not been examined. Accordingly, western blotting revealed that PPARg1 or PPARg2 expression resulted in a decrease in lipogenic pathways (Akt, mTOR, Fasn, Acc) and oxidative stress (Cox-2) (Figure 4a). Flow cytometry of dihydroethidium-stained cell lines conrmed a signicant reduction in reactive oxygen species (ROS) upon expression of PPARg isoforms (Figure 4b).

These data suggest that paracrine PPARg expression satises the endogenous lipid needs, thereby negating the need for de novo lipogenesis.

To conrm candidate genes identied by INGENUITY software analysis, qRT-PCR plates were custom-designed for analysis of PPARg-restored cell lines. Results showed that both PPARg isoforms regulated genes involved in metabolism (Table 1, Section I), including the modication (Elovl4, Scd1), transport (Cd36, Lpl, Fabp4) and b-oxidation (Acsf2, Lipa,

Acot1) of fatty acids. In addition, multiple genes involved in detoxication were upregulated (Table 1, Section II), notably

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

4

Figure 2 PPARg isoforms 1 and 2 differentially regulate prostate basal differentiation as well as luminal AR expression and function. (a) Western blot analysis of mPrE PPARgKO EV, PPARg1 or PPARg2 cells shows dual increase in Ck14 and AR in mPrE PPARgKO PPARg2 cells compared with EV or PPARg1 cells.

(b) ICC of PPARg (red) and Ck14 (green) in mPrE-PPARgKO EV (i), PPARg1 (ii) or PPARg2 (iii) cells in culture shows an increase in Ck14 cells in PPARg2, but

not PPARg1 cells. ICC for AR and Ck14 in mPrE-PPARgKO EV (iv), PPARg1 (v) or PPARg2 (vi) cells treated with DHT shows cytoplasmic AR in most mPrE

PPARgKO EV or PPARg1 cells, whereas PPARg2 cells displayed increased nuclear AR immunoreactivity in cells adjacent to Ck14 cells. (c) Quantitation of PPARg

immunoreactivity shows a signicant increase of 15% for PPARg-restored cells compared with EV cells, whereas only PPARg2-restored cells showed a signicant 15%

increase in Ck14 immunoreactivity compared with EV cells. (d) Androgen responsiveness was increased three-fold in PPARg2 cells compared with EV cells as

measured by ARE-luciferase (N 3). *P valueo0.05

including Cox-2, further conrming the decrease in ROS shown in Figure 4b. Finally, multiple markers of differentiation (Table 1, Section III) were upregulated, notably including regulation of basal cell (Trp63, Ck14) and luminal cell (Pbsn, AR, PTEN) markers by PPARg2, conrming the changes shown in Figure 1. These data suggest that increased fatty acid import results in a reduction in lipogenesis and oxidative stress.

PPARg isoforms differentially regulate glucose and fatty acid metabolism. One of the most interesting examples of isoform-specic changes in fatty acid modication genes was the differential regulation of Scd1, which was upregulated by PPARg2 but downregulated by PPARg1 (Table 1, Section I).

Scd1 is an ER-resident fatty acid desaturase strongly induced by dietary saturated fat and responsible for the production of monounsaturated fatty acids (MUFAs) from 12 to 19 carbon saturated fatty acids, and has been implicated in numerous metabolic diseases. MUFAs are the preferred substrates in the synthesis of major lipid classes including phospholipids, cholesterol esters, wax esters and triglycerides.22,23

When various lipid classes were analyzed (phospholipids, diglycerides, triglycerides and cholesterol esters) in PPARg1 and -g2-restored mPrE-gKO cells, we found that not only were total triglyceride stearic acid levels increased, but also that the

PPARg1-mediated Scd1 decrease resulted in a signicantly increased abundance of stearic acid and decreased

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

5

Figure 3 Microarray and network analyses of cell lines. (a) Schematic of microarray analysis. In order to distinguish PPARg isoform effects downstream of TZD treatment, PPARg KO cells (mPrE-PPARgKO EV) were treated with Rosiglitazone (Rosi) and these Rosi-independent results (representing 82 genes, see Supplementary Figure 1a)

were subtracted from the downstream regulation demonstrated in Rosi-treated PPARg1- (358 differentially regulated genes, see Supplementary Figure 1a) or PPARg2-(400 differentially regulated genes, see Supplementary Figure 1a) rescued cells. Further analysis showed that 230 genes were differentially regulated in PPARg1 versus

PPARg2-restored PPARg2-rescued cells. N 3 for each of the four samples. (b) Top networks differentially regulated by PPARg isoforms using INGENUITY

abundance of oleic acid (Figure 4c). As shown above, PPARg1 expression also failed to induce prostatic differentiation (Figures 1 and 2), indicating that an increased availability of MUFAs through PPARg2-mediated Scd1 expression may be benecial for normal prostate epithelial differentiation.

As for other PPARs,21 we found using qRT-PCR that PPARg1 and -g2 also regulated glucose metabolism genes, notably increasing Pdk4 expression (Table 1). Pdk4 phosphorylates and inactivates pyruvate dehydrogenase, resulting in the shunting of pyruvate toward lactate production rather than entry in the mitochondrial tricarboxylic acid (TCA) cycle. In tissues such as muscle, metabolic switching from glucose to fatty acid oxidation is mediated by increased Pdk4 expression.24 To determine whether increased Pdk4 expression resulted in altered glucose/lactate ux, we collected conditioned media from mPrE-PPARgKO and the isoform-restored cells over a 4-day period. Results demonstrated a decrease in glucose ux in PPARg-restored cells (Figure 4d), coordinate with the level of Pdk4 expression (Table 1), with

PPARg1 mediating the strongest upregulation of Pdk4 and decrease in glucose consumption. A signicant increase in

lactate production was observed in PPARg2-restored cells, indicating increased glycolytic metabolism. Furthermore, the increased glucose/lactate ratios observed in both PPARg-restored cells points to a clear shift away from glucose oxidation in the TCA cycle in favor of lactate production. Measurement of the IC50 of the glucose analog and oxidation inhibitor 2-deoxy-D-glucose (2DG) showed that mPrEPPARgKO EV cells were signicantly more sensitive to

inhibition of glucose metabolism than their PPARg-restored counterparts (Figure 4e), which are more likely to rely on fatty acid oxidation according to genes shown in Table 1.

These data demonstrate that although restoration of either PPARg isoform in PPARg KO HGPIN cells decreases de novo lipogenesis and oxidative stress, only PPARg2-regulated genes induce a metabolic switch for induction of a prostatic differentiation program, potentially through disparate regulation of Scd1.

TZD or HFD treatment drives opposing effects on mouse prostate metabolism and differentiation in vivo. PPARg has been hypothesized to provide a metabolic link between

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

6

Figure 4 PPARg isoforms equally decrease de novo lipogenesis and oxidative stress, but differentially modulate triglyceride saturation and glucose metabolism. (a) Ectopic expression of PPARg1 or PPARg2 in mPrE-PPARgKO cells resulted in decreased activation of lipogenic pathways including Akt, mTOR, Fasn and Acc. In addition,

Cox-2 levels were decreased indicative of lower levels of ROS. (b) Dihydroethidium staining followed by ow cytometry (N 3) conrmed a decrease in ROS. (c) Fatty acid

analysis of triglycerides by TLC/MS revealed increased levels and saturation of stearic acid (increased 18 : 0, decreased 18 : 1n9) in PPARg1-restored cells compared to EV

cells (N 3), consistent with the decreased expression of Scd1 (see Table 1). (d) Glucose/lactate ux analysis in mPrE-PPARgKO EV, PPARg1 or PPARg2 cells over

4 days (2 time points/day) demonstrated signicantly decreased glucose uptake in mPrE-PPARgKO PPARg1 cells and signicantly increased lactate secretion in

mPrE-PPARgKO PPARg2 cells compared to EV cells. (e) IC50 analysis of the glucose oxidation inhibitor 2DG suggested a reliance of mPrE-PPARgKO EV cells on

glucose in the absence of PPARg1 or PPARg2 cells, which highly regulate fatty acid transport and metabolism (see Table 1). *P value o0.05

obesity and tissue dysfunction.13 In order to determine whether TZDs or obesity affect PPARg-mediated prostatic differentiation in vivo, we fed male mice a Western diet or

Rosiglitazone chow for 6 months and examined their prostates for changes in morphology (Figure 5) and PPARg-regulated genes (Table 2). Signicant increases in overall animal weight were detected following HFD treatment only (Figure 5a). Notable increases in smooth muscle density were observed with TZD treatment (Figure 5d), with little epithelial hyperplasia. In addition, intra-muscular adipocytes were also highly enriched in TZD-treated animals (Figure 5d,

star), which made clean dissection for RNA analysis of prostate-specic genes extremely difcult. This is consistent with the deposition of intramuscular adipocytes in skeletal muscle of TZD-treated human subjects.25 Although PPARg2 is thought of as an adipose-specic gene, we were able to demonstrate using a PPARg2-specic antibody that it is also expressed sporadically in mouse prostate luminal epithelia (Figure 5f) as well as throughout the smooth muscle (Figure 5b).

As shown in Table 2, large increases in PPARg-regulated genes were observed in TZD-treated animals. Given the

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

7

Table 1 Comparison of gene expression from mPrE-PPARgKO EV, PPARg1 and PPARg2-restored cell lines by qRT-PCR

PPARc1 PPARc2Gene Function Cellular localization Fold induction P value Fold induction P value

I. Metabolic genesAcot1 Long chain fatty acid metabolism Cytoplasm 19.0 0.09 304.3 o0.05

Acsf2 Fatty acid oxidation Mitochondrion 69.1 o0.05 109.7 0.10 Adipor1 Fatty acid oxidation Plasma membrane 1.7 o0.05 2.7 o0.05

Cd68 Fatty acid transport Lysosome 2.1 0.27 11.8 o0.05 Cd36 Long chain fatty acid metabolism Cytoplasm, mitochondrion 3.2 0.09 32.1 o0.05

Dagla Lipolysis Plasma membrane 3.5 o0.05 5.5 0.08 Dgat2 Triglyceride synthesis Endoplasmic reticulum 1.4 0.10 3.3 0.08

Elovl4 Fatty acid elongation Endoplasmic reticulum 2.6 0.09 1.3 0.41

Fabp4 Fatty acid transport Cytoplasm, nucleus 72.7 o0.05 220.3 o0.05 Fbp2 Carbohydrate metabolism Cytoplasm 3.7 o0.05 2.1 0.11

Fetub Insulin responsiveness Extracellular 3.2 0.30 2.4 0.13

Gls2 Glutamine synthesis Mitochondrion 1.3 0.52 1.2 0.48

Glul Glutamine catabolism Mitochondrion 1.7 0.25 2.9 0.16 Lipa Lipolysis Lysosome 3.7 o0.05 6.1 o0.05

Lpl Lipolysis Plasma membrane 15.4 o0.05 121.1 o0.05 Lrp1 Fatty acid transport Plasma membrane 2.1 0.10 8.3 o0.05

Pdk4 Carbohydrate metabolism Mitochondrion 17.4 o0.05 5.1 o0.05 Pparg Nuclear receptor Nucleus 8.7 0.06 64.6 o0.05

Ppargc1a Pparg cofactor Nucleus 6.0 0.09 23.9 o0.05 Ppargc1b Pparg cofactor Nucleus 1.5 0.30 2.2 0.19

Scd1 Fatty acid desaturase Endoplasmic reticulum 2.2 0.07 2.6 o0.05

Txnip Carbohydrate metabolism Mitochondrion 1.1 0.64 7.9 0.08

II. Oxidative stress genesAldh1a1 Oxidative stress Cytoplasm 1.3 0.28 9.2 0.06 Aldh1a7 Oxidative stress Cytoplasm 2.0 0.12 8.7 o0.05

Aldh3a1 Oxidative stress Cytoplasm 4.8 0.19 11.8 o0.05 Aldh3b1 Oxidative stress Cytoplasm 3.7 0.16 9.7 o0.05

Aldh7a1 Oxidative stress Cytoplasm 4.9 o0.05 2.9 0.12 Aox3 Oxidative stress Cytoplasm 105.9 0.20 92.6 o0.05

Casp4 Cell death Endoplasmic reticulum 5.0 o0.05 21.6 o0.05 Cat Oxidative stress Mitochondrion 1.2 0.36 4.1 o0.05

Cyp17a1 Oxidative stress Mitochondrion 3.5 o0.05 4.5 0.09

Dhrs3 Oxidative stress Plasma membrane 13.8 0.23 31.9 o0.05

Gsta2 Oxidative stress Cytoplasm 3.9 0.07 18.7 0.24 Sirt5 Oxidative stress Mitochondrion 1.9 0.13 2.8 0.08 Sod3 Oxidative stress Extracellular 5.1 o0.05 8.1 0.11

III. Differentiation genesAr Steroid receptor Cytoplasm, nucleus 3.5 o0.05 10.8 o0.05

Krt14 Basal cell keratin Plasma membrane 1.2 0.60 4.1 0.14

Pbsn Prostate-specic differentiation Extracellular 1.7 0.41 2.7 0.19

Pten Lipid and protein phosphatase Plasma membrane 1.5 0.14 2.7 o0.05 Trp63 Basal cell marker Nucleus 3.3 o0.05 8.0 0.17

Acta2 Smooth muscle marker Structural 1.5 0.28 1.9 o0.05

increase in intraprostatic adipocytes after TZD treatment, it was unclear whether these changes were regulated in adipocytes or prostate. Therefore, immunohistochemistry was performed on metabolic proteins including Scd1, Lpl, Cd36, Fabp4 and Pdk4. Although ectopic PPARg expression was able to regulate some of the fatty acid metabolism genes in vitro (Table 1), immunoreactivity for Fabp4 (Supplementary Figure 1c), Lpl and Scd1 was low in mouse prostate and high in adjacent adipocytes, likely reecting the strong increases in RNA expression shown in Table 2, Section II. However, immunoreactivity for Cd36 (expressed in epithelia and stroma, Supplementary Figure 1b) and Pdk4 (expressed in epithelia, Figure 5ln) were strong, suggesting that these genes might be important in directly mediating the metabolism and differentiation of prostate, versus Fabp4 and Lpl, which may

indirectly mediate prostate differentiation through adjacent adipocyte fatty acid metabolism.

As PPARg-regulated genes were so drastically affected in TZD-treated animals, epididymal white adipose tissue (eWAT) gene expression was also examined by qRT-PCR under normal conditions or TZD treatment and was directly compared with that of prostate (Table 2, row 1 versus row 2). Results are compartmentalized into genes predominantly regulated by TZD in either eWAT (I) or prostate (II) or both (III). Contrary to common perceptions of both adipose and prostate, total PPARg levels (isoforms were not discriminated by qRT-PCR) were higher in prostate than eWAT under normal feeding conditions and AR levels were equivalently expressed (contamination by adipocytes in prostates of animals fed regular chow was minimal). However, under

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

8

Figure 5 Administration of TZD or HFD drives opposing effects on mouse prostate metabolism and differentiation in vivo. (a) Western diet-, but not TZD-fed animals signicantly increased total weights by 20 g (N 3 each). (b) PPARg2 is coexpressed in prostate smooth muscle. (cn) C57B male mice were fed control (a, f, i and l),

Rosiglitazone (b, g, j and m) or Western diet chow (e, h, k and n) for 6 months. PPARg2 levels were similar in prostate epithelia after TZD treatment (f versus g) but decreased in some acini of HFD-treated animals (h, star). Intramuscular adipocytes were increased in TZD-treated animals (g, star), which also resulted in increased AR expression increased in smooth muscle (j, arrow). AR expression was decreased in HFD-treated animals (* in k) similar to qRT-PCR analysis (see Table 2). PDK4 expression was similar in prostate tissue of TZD-treated animals (m, * indicates adipose) and decreased in HFD-treated animals (n) (see Table 2)

TZD treatment, AR (and probasin) levels increased in prostate and not eWAT. Notably, PPARg2 is allostatically induced in adipose under HFD,26 which could explain the lower levels of total PPARg in lean mouse eWAT versus prostate. Recent evidence of the molecular signature of various adipose depots could also explain the difference in PPARg-regulated genes in eWAT versus the intramuscular adipose of TZD-treated prostate,27 which may indicate the expansion of highly

metabolic brown adipose shown previously to be regulated by TZDs.28

Other notable differences between prostate and eWAT gene expression patterns included numerous oxidative stress genes (Cat, Gsta2, Sirt5, Nox1, Nfkb2) and the triglyceride-synthesizing enzyme Dgat2, which were highly enriched in adipose versus prostate. However, a number of fatty acid metabolism (Acsf2, Adipor1, Cd36, Lpl) and differentiation

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

9

Table 2 Comparison of gene expression from harvested tissues of control, TZD- or HFD-treated mice by qRT-PCR

Fold induction

Prostate (HFD)

I. TZD-regulated genes in adiposeDgat2 Triglyceride synthesis Endoplasmic reticulum 2459.2 4.4 53.1 8.3 Elovl4 Fatty acid elongation Endoplasmic reticulum 16.5 4.0 1.3 9.4

Fetub Insulin responsiveness Extracellular 223.3 5.7 96.8 124.6

Gsta2 Oxidative stress Cytoplasm 21.5 115.5 1.5 9.7

Ppargc1b Pparg cofactor Nucleus 6.3 1.9 2.0 5.1

Sirt5 Oxidative stress Cytoplasm 1492.8 1.4 1.9 11.5

Trp63 Prostate basal cell/adipocyte marker

Nucleus 153.9 2.1 36.3 6.2 Nox1 Oxidative stress Cytoplasm 508.9 2.7 1.1 1.3

Nfkb2 Oxidative stress, inammation Cytoplasm, nucleus 63.8 2.9 3.2 1.5II. TZD-regulated genes in prostateAcsf2 Fatty acid oxidation Mitochondrion 5.7 1.1 6.2 5.4

Acta2 Smooth muscle marker Structural 2.3 2.7 9.5 41.2

Adipor1 Fatty acid oxidation Plasma membrane 2.0 2.3 97.7 171.4

Ar Steroid receptor Cytoplasm, nucleus 1.4 2.1 33.8 81.1

Cd36 Long chain fatty acid metabolism Cytoplasm, mitochondrion 1.4 4.7 48.3 3

Cd68 Fatty acid transport Lysosome 3.7 2.8 473.5 31.3

Dagla Lipolysis Plasma membrane 2.9 1.0 7.8 77.4

Fabp4 Fatty acid transport Cytoplasm, nucleus 252.5 7.3 7669.7 169.6

Fbp2 Carbohydrate metabolism Cytoplasm 1.8 11.0 122.3 13.6

Glul Glutamine catabolism Mitochondrion 26.8 1.6 223.2 57.8

Krt14 Basal cell keratin Plasma membrane 1.8 2.5 5.2 5.1

Lpl Lipolysis Plasma membrane 4.1 7.8 1035.7 4.7

Pbsn Prostate-specic differentiation Extracellular 135.3 1.6 414.3 249.9

Pdk4 Carbohydrate metabolism Mitochondrion 2.3 14.7 7047.8 57.3

Tgm2 Wound healing Cytoplasm, nucleus 7.8 11.6 9939.9 123.4III. TZD-regulated genes in adipose and prostateAcot1 Long chain fatty acid metabolism Cytoplasm 3.0 36.5 38.9 254.1

Cat Oxidative stress Mitochondrion 250.3 5.4 68.0 9.4

Lipa Lipolysis Lysosome 9.8 2.1 4.7 1.5

Pparg Nuclear receptor Nucleus 11.3 9.7 76.1 3.7

Ppargc1a Pparg cofactor Nucleus 31.5 5.8 5.1 2.8

Pten Protein and lipid phosphatase Cytoplasm, nucleus 36.2 1.7 49.7 97.9

Scd1 Fatty acid desaturase Endoplasmic reticulum 21.9 13.2 128.0 4.3

Txnip Carbohydrate metabolism Plasma membrane, nucleus

1.0 14.7 60.0 2.4

Gene Function Cellular localization

Prostate versuse WAT ( TZD)

eWAT( TZD)

Prostate

( TZD)

(AR, probasin) genes were equally high in prostate, including the glucose oxidation inhibitor, Pdk4, which suggests that the differentiation induced by PPARg expression shown here may represent a metabolically regulated program of prostatic differentiation in vivo.

Given the regulation of prostate differentiation by PPARg2 in mouse, we also wanted to conrm the expression of

PPARg2 and some of its downstream-regulated genes in human prostate tissue. Figure 6a shows that PPARg2 is highly enriched in prostate smooth muscle and that Scd1 (upregulated by PPARg2 expression in vitro) is highly enriched in prostate basal cells (Figure 6b). Furthermore, CD36 was expressed in both epithelia and smooth muscle (Figure 6c), whereas PDK4 was expressed predominantly in epithelia (Figure 6d). The compartmentalization of cellular glucose and fatty acid metabolism (Figure 6e) suggests a model of stromalepithelial, as well as basalluminal interactions whose disruption by systemic metabolic disease may adversely affect the health and differentiation of prostate (Figure 6f). These results suggest that PPARg is a major metabolic regulator in the control of mouse and human prostate differentiation.

Discussion

A recent study of the global prevalence of glycemia and diabetes demonstrated an increase from 153 million affected individuals in 1980 to 347 million in 2008,29 which, according to epidemiological correlations, will likely have a major direct impact on prostate disease incidence. Upon maximal lipid storage capacity of white adipose tissue (WAT), peripheral tissues begin to store lipid in excess of their natural oxidative or storage capacity resulting in lipotoxicity, inammation and eventually insulin resistance.30 Recent evidence squarely positions prostatic diseases as sequelae of systemic metabolic dysfunction, including hyperinsulinemia, hyperglycemia and hypercholesterolemia31; however, the underlying etiologies of such susceptibilities remain unknown largely because of the absence of a molecular understanding of the basic metabolic machinery governing prostatic function.

Here, we demonstrate that expression of PPARg2 drives benign prostate epithelial cell differentiation. In mouse prostate PPARg2 was expressed in both smooth muscle and epithelium (Figures 5b and f), whereas in human prostate

PPARg2 expression seems to be restricted to smooth muscle

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

10

Figure 6 PPARg-regulated genes in human prostate tissues. (a) Double staining for PPARg2 and alpha smooth muscle actin (a-SMA) by immunouorescence revealed colocalization in a subset of smooth muscle. (b) SCD1 expression is enriched in prostate basal epithelia by immunohistochemical staining (IHC). (c) CD36 is expressed in smooth muscle and epithelia. (d) PDK4 is expressed predominantly in the epithelium. (e) Cellular model of metabolic genes and functions regulated by PPARg. (f) Tissue interaction model of the potential role of paracrine fatty acid metabolism in regulating prostate differentiation and diseases as comorbidities of diabetes and obesity

(Figure 6a). Stromalepithelial interactions have long been recognized to have a role in prostate differentiation,32 but the

underlying mechanisms remain elusive. These data suggest that paracrine fatty acid metabolism may drive epithelial differentiation, resulting in decreased glucose metabolism, oxidative stress and lipogenesis (Figure 4).

PPARg has been shown to regulate the balance between glucose and lipid oxidation in a tissue-specic manner.33

Here, we show that TZD treatment upregulated markers of prostatic differentiation in correlation with an increase in highly metabolic smooth muscle and intramuscular adipose (Figure 5, Table 2), which also correlated with the decreased glucose ux and lipogenesis shown in PPARg-restored cells in vitro (Figure 2).

The isoform-specic effects of PPARg have not been directly compared in any tissue. One of the most interesting

genes differentially regulated by PPARg isoforms in prostate was the fatty acid desaturase Scd1. Systemic deletion of Scd1 provides protection against obesity due to reduced fatty acid and triglyceride synthesis as well as increased oxidation.22,23

In contrast to other tissues, SCD1 expression is high in human prostate epithelia, but its expression and role in prostate cancer has been debated.34,35 Moreover, PPARg2 upregulated Scd1 expression and drove basal cell differentiation (Figure 2), and SCD1 was shown to be predominantly restricted to the basal cell compartment of normal human prostate (Figure 6b) whereas PPARg2 was predominantly expressed in smooth muscle (Figure 6a). As modeled in Figure 6f, these data led us to hypothesize that PPARg2-mediated import and oxidation of fatty acids may dictate prostate basal cell differentiation through increased SCD1.

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

11

Tissue-specic effects of PPARg agonization have demonstrated that the upregulation of liver fatty acid and sterol synthesis in HFD-fed rats could be reversed by PPARg agonists, whereas the same HFD stimulated PPARg downregulation and lipogenesis in muscle.13 Concordant with studies demonstrating positive effects of TZDs on HFD mouse prostates,36,37 we demonstrated here that chronic HFD treatment resulted in decreased androgen signaling and low-grade PIN, coordinate with decreased PPARg signaling (Figure 5, Table 2). These data suggest that an allostatic response to downregulate fatty acid import and metabolism may negatively affect prostate differentiation through metabolic switching.

Cells access and metabolize fatty acids through the activities of lipases (e.g., Lpl, Lipa), transporters (e.g., Fabp4, Cd36) and enzymes (acyl-coa synthetases, thioesterases), which supply the cell with acetyl coenzyme A (acetyl-CoA) for eventual entry into mitochondria for energy production. Alternatively, acetyl-CoA can be used as a building block for MUFA production (Scd1) and subsequently converted into triglycerides, cholesterol esters and phospholipids (Figure 6e). The therapeutic efcacy of targeting fatty acid metabolism (synthesis, modication, transport and oxidation) has had some success, but off-target effects have limited their broad usage.10 Similarly, more selective drugs targeting PPARg and Scd1 promising fewer side effects are being pursued.22,38 Future studies must be able to link changes in systemic metabolism to local metabolic changes in prostate, which mandates a deeper understanding of the fundamental metabolic infrastructure regulating prostatic differentiation and what allostatic changes may occur in response to systemic metabolic stress. The data presented here suggest that a microenvironment of PPARg2-mediated fatty acid metabolism by stroma or adipose may drive prostatic epithelial differentiation; however, under conditions of diabetes and obesity fatty acid supply may become saturated, leading to inammation and hyperplasia in prostate disease (Figure 6f).

Materials and MethodsGeneration of cell lines, microarray studies and qRT-PCR. mPrEPPARgKO cells were spontaneously immortalized from an adult PBCre4tg/0/

PPARgox/ox double-transgenic male mouse.17 The pQCXIP-empty vector, mouse PPARg1 or PPARg2 wild-type full-length cDNA (gifts from Drs. Y Eugene

Chen and Jifeng Zhang, University of Michigan Medical Center) were stably transfected into the mPrE-PPARggKO cells to generate mPrE

PPARggKO EV, mPrE-PPARgKO PPARg1 or mPrE-PPARgKO PPARg2

cell line, respectively.17Microarray and network analyses and qRT-PCR validation. RNA was isolated with Trizol (Ambion, Austin, TX, USA) and reverse transcribed by RNeasy columns (Qiagen, Valencia, CA, USA) followed by reverse transcription (Qiagen) for hybridization on mouse Genechip St. 1.0 microarrays using triplicate samples from each cell line treated with 5 mM Rosiglitazone (Rosi) (Cayman

Chemical, Ann Arbor, MI, USA) as well as mPrE-PPARgKO EV without Rosi.

PPARg-independent effects of Rosi generated in mPrE-PPARgKO EV cells

were subtracted from results comparing mPrE-PPARgKO EV versus PPARg-

restored cells. SAM was used to stringently select (FDRo0.05) statistically signicant genes. Both SAM and unsupervised hierarchical clustering analysis were carried out using TIGR MeV program. Their possible networks and canonic pathways were identied using INGENUITY software (https://apps.ingenuity.com

Web End =https://apps.ingenuity.com). Custom qRT-PCR plates manufactured by SABiosciences (Valencia, CA, USA) were designed to analyze selected PPARg-regulated genes in Rosi-treated mPrE-

PPARgKO EV, mPrE-PPARgKO PPARg1 and mPrE-PPARgKO PPARg2

cell lines in triplicate. Results were analyzed using SABiosciences qRT-PCR analysis software (SABiosciences.com) and represented fold changes based on

DDCt analysis.

Detection of ROS. Each cell line was grown to conuence, trypsinized, washed and incubated with 1 mM Dihydroethidium (Life Technologies, Carslbad,

CA, USA) for 15 min followed by three washes in PBS. Flow cytometry was then performed and results represent the average mean intensity (N 3). Statistical

analysis was performed by GraphPad Prism software (La Jolla, CA, USA) using Students unpaired t test.

Fatty acid prole (TLC/MS). Lipid class separation and fatty acid identication of cell lines were performed by the Vanderbilt Hormone Assay and Analytical Services Core (http://hormone.mc.vanderbilt.edu/

Web End =http://hormone.mc.vanderbilt.edu/). Briey, the Folch method of lipid extraction39 was followed by thin layer chromatography (TLC) was used to isolate triglycerides. Fatty acid analysis was performed by mass spectrometry (MS) with internal standards. Total triglycerides were normalized to protein concentration as determined by the Lowry method. Statistical analysis was performed on triplicate samples by GraphPad Prism software using one way ANOVA test.

Luciferase assay. Cells were grown to 70% conuency in 12-well culture plates and cotransfected with pRL-null (0.16 mg/well) and ARR2PB-Luciferase(1.44 mg/well) using 4 ml Lipofectamine 2000 in OptiMEM (Invitrogen, Grand Island, NY, USA). After 24 h transfection, media was replaced with 10% charcoal-stripped FBS in DMEM plus or minus 10 8 M DHT and incubated for 24 h. At this time, cells were lysed and dual luciferase activity measured using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) on a Turner Biosystems 20/20n luminometer (Promega). Results were statistically analyzed by Students unpaired t test (N 3) using GraphPad Prism.

Western blot. Western blotting was performed as described previously.17

Briey, 30 mg protein was loaded on 10% SDS acrylamide gels (Life Technologies) and transferred onto PVDF membranes (Millipore, Temecula, CA, USA). The following antibodies were used for detection: mTOR, phospho-mTOR, PTEN, ACC and AKT, phospho-AKT (S473) were from Cell Signaling (1 : 1000, Danvers, MA, USA), Cox-2 (1 : 500, Millipore), AR and PPARg (1 : 250, Santa Cruz

Biotechnology, Santa Cruz, CA, USA), FASN (1 : 1000, GeneTex, Irvine, CA, USA), Gapdh (1 : 1000, Abcam, Cambridge, MA, USA) and Ck14 (1 : 1000, Vector Labs, Burlingame, CA, USA). Blots were then incubated with secondary antibodies (1 : 1000, anti-mouse, anti-rabbit from GE Healthcare, Buckinghamshire, UK) for 45 min in 5% milk in TBST, washed and developed with Western Lightning Plus-ECL (Perkin Elmer, Waltham, MA, USA).

ICC/IHC. ICC/IHC was performed as described previously.17 Briey, cells were cultured on glass chamber slides (LabTek II, Naperville, IL, USA), washed with PBS, xed in 4% PFA for 15 min, blocked in 12% BSA for 1 h and incubated with primary antibodies (1 : 25 PPARg, Santa Cruz; 1 : 50 CK14, Vector Labs; 1 : 100

AR, Santa Cruz; 1 : 500 Ck8/18, Fitzgerald, Acton, MA, USA) in 12% BSA at 41C overnight followed by incubation with a uorescent secondary antibody (1 : 1000,

Molecular Probes, Eugene, OR, USA) for 45 min at 371C. Cells were counterstained and mounted with Dapi Vectashield (Vector Labs) and images were taken on a Zeiss Axioplan microscope. Tissues harvested from grafted (kidney capsule) or TZD- or HFD-treated mice (epididymal WAT, prostate) were xed in 10% formalin at 41C overnight, parafn embedded and then 5 mM sections cut for immunohistochemistry. Slides were deparafnized, rehydrated and endogenous peroxidases blocked with 2% H2O2 in methanol. Following citrate retrieval and blocking with 5% goat serum, slides were incubated at 41C overnight with the following antibodies: AR (1 : 200, Santa Cruz), PPARg2 (1 : 200, Abcam), smooth muscle actin (1 : 2000 Invitrogen), Ck14 (1 : 100), CK18 (1 : 500), PDK4 (1 : 500, ProteinTech, Chicago, IL, USA). Secondary antibodies for IHC were from Dako (Carpinteria, CA, USA) and used at 1 : 200 dilution. Secondary antibodies for IF (1 : 1000, Molecular Probes) were incubated for 45 min followed by mounting with DAPI Vectashield.

Tissue recombination. Tissue recombination was performed as described previously.17 Briey, 400 K of each mPrE-PPARgKO-related cell lines were recombined with 18-day fetal rat UGM and grafted in collagen into the kidney

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

12

capsule of male SCID mice for 2 months. Grafts were harvested and xed in formalin for morphologic and immunohistochemical analysis.

Glucose/lactate measurements. Extracellular uptake and excretion rates were determined in triplicate growth experiments. Eight separate 48-well tissue culture plates were seeded at a density of 20 K cells. One plate was sampled every 1014 h, whereby the conditioned medium was removed and frozen at

801C. The remaining cells on the plate were stained with crystal violet for assessment of cell number. Concentrations of medium glucose and lactate were determined using a YSI 2300 Stat Plus Glucose and Lactate Analyzer (YSI, Yellow Springs, OH, USA). Cell-specic rates of glucose consumption and lactate production were determined by regression analysis using the method of Glacken et al.40

Animal experiments. To examine the effects of TZD or HFD regimen on prostate gene expression in vivo, control, Rosiglitazone chow (0.188% Avandia) or Western diet chow (16% protein, 40% carbohydrate, 40% fat, 0.15% cholesterol) (Test Diets, Richmond, IN, USA) were fed to male C57B mice for 6 months at which time the animals were weighed (Students unpaired t test for statistical analysis) and tissues removed for formalin xation and storage at 801C for later

RNA extraction. Because of the extreme density of adipose directly surrounding the prostate of both TZD- and HFD-fed animals, care was taken to dissect away as much as possible. WAT was taken from the epididymal fat pad. RNA was extracted and cDNA synthesized for qRT-PCR on custom-designed plates (Qiagen) using an ABI 7900HT real-time PCR machine with a standard block according to the manufacturers instructions. Results represent triplicate experiments and fold changes were calculated as described above. P values were mostly insignicant, given the contamination by intramuscular adipocytes and were therefore not shown.

Conict of InterestThe authors declare no conict of interest.

Acknowledgements. All microarray experiments were performed in the Vanderbilt Genome Sciences Resource. The Vanderbilt Genome Sciences Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485), the Vanderbilt Digestive Disease Center (P30 DK58404) and the Vanderbilt Vision Center (P30 EY08126). This work was supported by a Department of Defense Prostate Cancer Training Award W81XWH-07-1-0479 to DWS, P20 DK090874 and 2R01 DK067049 to SWH, and R21 CA155964 to JDY. All fatty acid analyses were performed by the Vanderbilt Hormone Assay and Analytical Services Core. We thank Drs. Robert Matusik and David Degraff for helpful discussions and manuscript editing.

1. Hammarsten J, Peeker R. Urological aspects of the metabolic syndrome. Nat Rev Urol

2011; 8: 483494.

2. Bourke JB, Grifn JP. Diabetes mellitus in patients with benign prostatic hyperplasia. Br Med J 1968; 4: 492493.

3. Parsons JK, Carter HB, Partin AW, Windham BG, Metter EJ, Ferrucci L et al. Metabolic factors associated with benign prostatic hyperplasia. J Clin Endocrinol Metab 2006; 91: 25622568.

4. Berger AP, Bartsch G, Deibl M, Alber H, Pachinger O, Fritsche G et al. Atherosclerosis as a risk factor for benign prostatic hyperplasia. BJU Int 2006; 98: 10381042.

5. Burke JP, Rhodes T, Jacobson DJ, McGree ME, Roberts RO, Girman CJ et al. Association of anthropometric measures with the presence and progression of benign prostatic hyperplasia. Am J Epidemiol 2006; 164: 4146.

6. Michel MC, Mehlburger L, Schumacher H, Bressel HU, Goepel M. Effect of diabetes on lower urinary tract symptoms in patients with benign prostatic hyperplasia. J Urol 2000; 163: 17251729.

7. Freeman MR, Solomon KR. Cholesterol and benign prostate disease. Diff Res Biol Div 2011; 82: 244252.

8. Yeh HC, Platz EA, Wang NY, Visvanathan K, Helzlsouer KJ, Brancati FL. A prospective study of the associations between treated diabetes and cancer outcomes. Diab Care 2012; 35: 113118.

9. Grundmark B, Garmo H, Loda M, Busch C, Holmberg L, Zethelius B. The metabolic syndrome and the risk of prostate cancer under competing risks of death from other causes. Cancer Epidemiol Biomarkers Prev 2010; 19: 20882096.

10. Flavin R, Zadra G, Loda M. Metabolic alterations and targeted therapies in prostate cancer. J Pathol 2011; 223: 283294.

11. Lee CH, Olson P, Evans RM. Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 2003; 144: 22012207.

12. Cheatham WW. Peroxisome proliferator-activated receptor translational research and clinical experience. Am J Clin Nutr 2010; 91: 262S266S.

13. Hsiao G, Chapman J, Ofrecio JM, Wilkes J, Resnik JL, Thapar D et al. Multi-tissue, selective PPARgamma modulation of insulin sensitivity and metabolic pathways in obese rats. Am J Physiol Endocrinol Metab 2011; 300: E164E174.

14. He W, Barak Y, Hevener A, Olson P, Liao D, Le J et al. Adipose-specic peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle. Proc Natl Acad Sci USA 2003; 100: 1571215717.

15. Vidal-Puig A, Jimenez-Linan M, Lowell BB, Hamann A, Hu E, Spiegelman B et al. Regulation of PPAR gamma gene expression by nutrition and obesity in rodents. J Clin Invest 1996; 97: 25532561.

16. Norris AW, Chen L, Fisher SJ, Szanto I, Ristow M, Jozsi AC et al. Muscle-specic PPARgamma-decient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones. J Clin Invest 2003; 112: 608618.

17. Jiang M, Fernandez S, Jerome WG, He Y, Yu X, Cai H et al. Disruption of PPARgamma signaling results in mouse prostatic intraepithelial neoplasia involving active autophagy. Cell Death Differ 2010; 17: 469481.

18. Jiang M, Jerome WG, Hayward SW. Autophagy in nuclear receptor PPARgamma-decient mouse prostatic carcinogenesis. Autophagy 2010; 6: 175176.

19. Jiang M, Strand DW, Franco OE, Clark PE, Hayward SW. PPARgamma: A molecular link between systemic metabolic disease and benign prostate hyperplasia. Differentiation 2011; 82: 220236.

20. Fajas L, Auboeuf D, Raspe E, Schoonjans K, Lefebvre AM, Saladin R et al. The organization, promoter analysis, and expression of the human PPARgamma gene. J Biol Chem 1997; 272: 1877918789.

21. Clarke SD. Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome. J Nutr 2001; 131: 11291132.

22. Sampath H, Ntambi JM. The role of stearoyl-CoA desaturase in obesity, insulin resistance, and inammation. Ann N Y Acad Sci 2011; 1243: 4753.

23. Miyazaki M, Sampath H, Liu X, Flowers MT, Chu K, Dobrzyn A et al. Stearoyl-CoA desaturase-1 deciency attenuates obesity and insulin resistance in leptin-resistant obese mice. Biochem Biophys Res Commun 2009; 380: 818822.

24. Kelley DE. Skeletal muscle fat oxidation: timing and exibility are everything. J Clin Invest 2005; 115: 16991702.

25. Sears DD, Hsiao G, Hsiao A, Yu JG, Courtney CH, Ofrecio JM et al. Mechanisms of human insulin resistance and thiazolidinedione-mediated insulin sensitization. Proc Natl Acad Sci USA 2009; 106: 1874518750.

26. Medina-Gomez G, Gray SL, Yetukuri L, Shimomura K, Virtue S, Campbell M et al. PPAR gamma 2 prevents lipotoxicity by controlling adipose tissue expandability and peripheral lipid metabolism. PLoS Genet 2007; 3: e64.

27. Walden TB, Hansen IR, Timmons JA, Cannon B, Nedergaard J. Recruited vs. nonrecruited molecular signatures of brown, "brite," and white adipose tissues. Am J Physiol Endocrinol Metab 2012; 302: E19E31.

28. Kelly LJ, Vicario PP, Thompson GM, Candelore MR, Doebber TW, Ventre J et al. Peroxisome proliferator-activated receptors gamma and alpha mediate in vivo regulation of uncoupling protein (UCP-1, UCP-2, UCP-3) gene expression. Endocrinology 1998; 139: 49204927.

29. Danaei G, Finucane MM, Lu Y, Singh GM, Cowan MJ, Paciorek CJ et al. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2.7 million participants. Lancet 2011; 378(9785): 3140.

30. Chavez JA, Summers SA. Lipid oversupply, selective insulin resistance, and lipotoxicity: molecular mechanisms. Biochim Biophys Acta. 2010; 1801: 252265.

31. De Nunzio C, Aronson W, Freedland SJ, Giovannucci E, Parsons JK. The correlation between metabolic syndrome and prostatic diseases. Eur Urol 2012; 61: 560570.

32. Strand DW, Franco OE, Basanta D, Anderson AR, Hayward SW. Perspectives on tissue interactions in development and disease. Curr Mol Med 2010; 10: 95112.

33. Way JM, Harrington WW, Brown KK, Gottschalk WK, Sundseth SS, Manseld TA et al. Comprehensive messenger ribonucleic acid proling reveals that peroxisome proliferator-activated receptor gamma activation has coordinate effects on gene expression in multiple insulin-sensitive tissues. Endocrinology 2001; 142: 12691277.

34. Moore S, Knudsen B, True LD, Hawley S, Etzioni R, Wade C et al. Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma. Int J cancer 2005; 114: 563571.

35. Fritz V, Benfodda Z, Rodier G, Henriquet C, Iborra F, Avances C et al. Abrogation of de novo lipogenesis by stearoyl-CoA desaturase 1 inhibition interferes with oncogenic signaling and blocks prostate cancer progression in mice. Mol Cancer Ther 2010; 9: 17401754.

36. Vikram A, Jena G, Ramarao P. Pioglitazone attenuates prostatic enlargement in diet-induced insulin-resistant rats by altering lipid distribution and hyperinsulinaemia. Br J Pharmacol 2010; 161: 17081721.

37. Vikram A, Jena GB, Ramarao P. Increased cell proliferation and contractility of prostate in insulin resistant rats: linking hyperinsulinemia with benign prostate hyperplasia. Prostate 2010; 70: 7989.

38. Rikimaru K, Wakabayashi T, Abe H, Imoto H, Maekawa T, Ujikawa O et al. A new class of non-thiazolidinedione, non-carboxylic-acid-based higshly selective peroxisome

Cell Death and Disease

PPARg isoforms in prostate metabolism and differentiation DW Strand et al

13

proliferator-activated receptor (PPAR) gamma agonists: design and synthesis of benzylpyrazole acylsulfonamides. Bioorg Med Chem 2012; 20: 714733.39. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purication of total lipides from animal tissues. J Biol Chem 1957; 226: 497509.

40. Glacken MW, Adema E, Sinskey AJ. Mathematical descriptions of hybridoma culture kinetics: I. Initial metabolic rates. Biotechnol Bioeng 1988; 32: 491506.

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

Supplementary Information accompanies the paper on Cell Death and Disease website (http://www.nature.com/cddis

Web End =http://www.nature.com/cddis)

Cell Death and Disease