ORIGINAL ARTICLE Metabolic action of peroxisome proliferator

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International Journal of Obesity (2007) 31, 1660–1670 & 2007 Nature Publishing Group All rights reserved 0307-0565/07 $30.00 www.nature.com/ijo

ORIGINAL ARTICLE Metabolic action of peroxisome proliferator-activated receptor c agonism in rats with exogenous hypercorticosteronemia M Berthiaume1, M Laplante1, A Tchernof2 and Y Deshaies1 1 Faculty of Medicine, Laval Hospital Research Center, Laval University, Que´bec, Que´bec, Canada and 2Faculty of Medicine, CHUL Medical Research Center, Laval University, Que´bec, Que´bec, Canada

Objective: The beneficial metabolic actions of peroxisome proliferator-activated receptor g (PPARg) agonism are associated with modifications in adipose tissue metabolism that include a reduction in local glucocorticoid (GC) production by 11bhydroxysteroid dehydrogenase type 1 (11b-HSD1). This study aimed to assess the contribution of GC attenuation to PPARg agonism action on gene expression in visceral adipose tissue and global metabolic profile. Design: Rats were treated (2 weeks) with the PPARg agonist rosiglitazone (RSG, 10 mg/kg/day) with concomitant infusion of vehicle (cholesterol implant) or corticosterone (HiCORT, 75 mg/implant/week) to defeat PPARg-mediated GC attenuation. Measurements: mRNA levels of enzymes involved in lipid uptake (and lipoprotein lipase activity), storage, lipolysis, recycling, and oxidation in retroperitoneal white adipose tissue (RWAT). Serum glucose, insulin and lipids, and lipid content of oxidative tissues. Results: Whereas HiCORT did not alter RWAT mass, RSG increased the latter ( þ 33%) independently of the corticosterone status. Both HiCORT and RSG increased lipoprotein lipase activity, the mRNA levels of the de novo lipogenesis enzyme fatty acid synthase, and that of the fatty acid retention-promoting enzyme acyl-CoA synthase 1, albeit in a nonadditive fashion. Expression level of the lipolysis enzyme adipose triglyceride lipase was increased additively by HiCORT and RSG. PPARg agonism increased mRNA of the fatty acid recycling enzymes glycerol kinase and cytosolic phosphoenolpyruvate carboxykinase and those of the fatty acid oxidation enzymes muscle-type carnitine palmitoyltransferase 1 and acyl-CoA oxidase, whereas HiCORT remained without effect. HiCORT resulted in liver steatosis and hyperinsulinemia, which were abrogated by RSG, whereas the HiCORTinduced elevation in serum nonesterified fatty acid levels was only partially prevented. The hypotriglyceridemic action of RSG was maintained in HiCORT rats. Conclusion: The GC and PPARg pathways exert both congruent and opposite actions on specific aspects of adipose tissue metabolism. Both the modulation of adipose gene expression and the beneficial global metabolic actions of PPARg agonism are retained under imposed high ambient GC, and are therefore independent from PPARg effects on 11b-HSD1-mediated GC production. International Journal of Obesity (2007) 31, 1660–1670; doi:10.1038/sj.ijo.0803668; published online 19 June 2007 Keywords: glucocorticoids; 11b-hydroxysteroid dehydrogenase type 1; visceral adipose tissue; insulin sensitivity; lipemia; adipose tissue gene expression

Introduction Peroxisome proliferator-activated receptor g (PPARg) is a lipid-activated transcription factor expressed predominantly in adipose tissue, where the nuclear receptor modulates the

Correspondence: Dr Y Deshaies, Faculty of Medicine, Laval Hospital Research Center, Laval University, Laval Hospital – d’Youville Y3110, 2725 Ch SainteFoy, Que´bec, QC, Canada G1V 4G5. E-mail: [email protected] Received 23 January 2007; revised 7 April 2007; accepted 26 April 2007; published online 19 June 2007

expression of genes involved in glucose and lipid metabolism.1,2 Thiazolidinediones such as rosiglitazone and pioglitazone are synthetic PPARg agonists that are used clinically for treatment of type 2 diabetes.3 In addition to positive alterations in the adipokine profile that include increased expression of the insulin-sensitizing adiponectin,4,5 the beneficial effects of PPARg agonists on insulin sensitivity and on the lipid profile appear to be partly attributable to lipid retention in adipose tissue and consequent reduction in lipid exposure of nonadipose tissues.6–8 The lipid retention action of PPARg agonism is linked to its ability to induce the expression of genes involved in lipid

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1661 accumulation, including lipoprotein lipase (LPL),9 acyl-CoA synthase 1 (ACS1),10 diacylglycerol acyltransferase 1 (DGAT1),11 fatty acid synthase (FAS),12 and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C).13 PPARg agonism also affects local glucocorticoid (GC) metabolism in adipose tissue. Because elevated GC production, as seen for instance in Cushing’s syndrome, is causally associated with visceral obesity and deterioration of the metabolic profile,14,15 it has been suggested that some of the effects of PPARg agonism on adipose tissue remodeling and insulin sensitization may be attributable to its inhibitory effect on the expression of the enzyme 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1),16 particularly in visceral fat.9 The enzyme generates active GC (cortisol in humans and corticosterone in the rat) from inactive forms (cortisone and 11-dehydrocorticosterone), and it plays a central role in regulating intracellular GC concentration and action in adipose tissue and liver.17 Overexpression of 11b-HSD1 in adipose tissue results in a phenotype resembling the metabolic syndrome, whereas whole-body specific inhibition of 11b-HSD1 by genetic or pharmacologic means results in overall metabolic improvement.17 Because of the potential importance of local modulation of GC action in adipose tissue lipid metabolism, the present study aimed to assess whether defeating the PPARg-induced reduction in adipose tissue GC amplification via 11b-HSD1, achieved through introduction of an exogenous source of GC, would impact the beneficial global metabolic actions of PPARg agonism, namely insulin sensitization and reduction in lipemia. In addition, because the GC and PPARg pathways share several common targets in adipose lipid metabolism, the study also aimed at investigating their interactions on the expression of major genes of adipose tissue lipid uptake, storage, lipolysis, recycling, and oxidation. The retroperitoneal fat depot was selected for the latter aim because it is representative of visceral fat, the major adipose target of GC action.14,15

Methods Animals and treatments Thirty-two male Sprague–Dawley rats initially weighing 150– 175 g were purchased from Charles River Laboratories (St Constant, QC, Canada) and housed individually in stainlesssteel cages in a room kept at 23711C with a 12:12 h light– dark cycle (lights on at 0700 hours). Rats had free access to tap water and a stock diet (Charles River Rodent Diet #5075, Ralston Products, Woodstock, ON, Canada; digestible energy content:12.9 kJ/g). At 2 days after their arrival, the animals were randomly assigned to four groups of eight rats each according to a 2  2 factorial design. The factors were drug treatment, with two levels (Control, rosiglitazone (RSG)), and the corticosterone (CORT) status, with two levels (Vehicle, CORT implant, herein termed HiCORT). On days 1 and 8 (to maintain relatively constant levels of serum

CORT throughout the experiment), rats under isoflurane anesthesia were subcutaneously implanted in the interscapular region with pellets containing the vehicle (cholesterol) alone, or cholesterol and 75 mg of CORT. On day 11, blood samples were taken from the orbital sinus under isoflurane anesthesia for plasma CORT measurement. One control and one HiCORT group were given the ground stock diet (chow), whereas the other groups were given the same diet containing the PPARg agonist RSG (Avandia, purchased at a local pharmacy, 10 mg/kg/day) for the 2-week treatment period. The amount of RSG was adjusted twice weekly to the average food consumption of each group so as to provide the same amount per unit body weight to all groups. To ensure an identical terminal nutritional state in all groups, on the last day of treatment, food was removed at 0730 hours and rats were killed at 1330 hours. An initial study using identical experimental conditions was performed to establish the efficacy of the CORT implants. One control and one HiCORT group of eight rats each were studied, and at the end of the experimental period, CORT levels were measured in serum, retroperitoneal white adipose tissue (RWAT), and liver. The weight of the adrenal glands was used as an index of longterm activity of the hypothalamic–pituitary–adrenal axis, and the expression level of angiotensinogen, a GC target in adipose tissue, as an index of functional efficacy of exposure to high CORT levels.

Serum and tissue sampling Rats were killed by decapitation, trunk blood was collected and centrifuged (1500 g, 15 min at 41C), and serum was stored at 201C until later biochemical measurements. Retroperitoneal and inguinal white adipose tissue (IWAT), taken as representative of visceral and subcutaneous fat, respectively, brown adipose tissue (BAT, a major target of both GC and PPARg agonism), skeletal muscles (soleus and vastus lateralis), and samples of liver were excised and quickly weighed. Crude indices of global adiposity and lean mass were calculated as the sum of adipose depots and skeletal muscles, respectively. Tissue samples were immediately frozen in liquid nitrogen and stored at –801C for later processing.

Serum/tissue determinations Serum glucose concentrations were measured by the glucose oxidase method with the Beckman glucose analyzer. Insulin and CORT concentrations in serum, RWAT, and liver steroid extracts18 were determined by radioimmunoassay (RIA) using reagent kits from Linco Research (St Charles, MO, USA) with rat insulin and CORT as standards, respectively. Triglyceride (TG) concentrations in serum, liver, soleus, and adipose tissue lipid extracts19 were measured by an enzymatic method using a reagent kit from Roche Diagnostics (Montreal, QC, Canada) that allows correction for free glycerol. Serum nonesterified fatty acid (NEFA) levels were International Journal of Obesity

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1662 measured enzymatically using reagents from Wako Chemicals (Richmond, VA, USA).

Adipose tissue lipoprotein lipase activity Immediately upon tissue harvesting, samples (B50 mg) of retroperitoneal adipose tissue (RWAT) were homogenized, processed, and frozen exactly as described.20 For determination of total extractable LPL activity, thawed tissue homogenates were incubated at 281C with a substrate mixture containing [carboxyl-14C]triolein, and 14C-NEFA released by LPL were separated and counted. LPL activity is expressed as mUnits (1 mU ¼ 1mmol NEFA released/h at 281C) per fat depot. Adipose tissue total RNA isolation and analysis of mRNA levels of genes involved in adipose lipid metabolism Total RNA was prepared from RWAT samples using QIAzol and the RNeasy Lipid Tissue Kit (QIAGEN, Mississauga, ON, Canada). For cDNA synthesis, Expand reverse transcriptase (Roche Diagnostics, Laval, QC, Canada) was used following manufacturer’s instructions and cDNA was diluted in DNasefree water (1:25) before quantification by real-time PCR. The primers, designed using the Vector NTI program and synthesized by Invitrogen (Burlington, ON, Canada), are shown in Table 1, and were validated with a sample RWAT cDNA. mRNA transcript levels were measured using a Rotor Gene 3000 system (Montreal Biotech, Montreal, QC, Canada). Amplification and detection of target mRNA was performed with Platinum Taq polymerase and the intercalating dye Sybr-Green I. The genes studied were the following: Lipoprotein-triglyceride hydrolysis: LPL; NEFA synthesis/ esterification: FAS, ACS1, DGAT1; NEFA re-esterification (recycling): glycerol kinase (GyK), PEPCK-C; lipolysis: adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL); fatty acid oxidation: muscle-type carnitine palmitoyltransferase 1 (mCPT1), acyl-CoA oxidase (ACO). Because PPARg agonists and glucocorticoids modulate the expression of many genes, no suitable housekeeping gene not affected by

Table 1

treatments individually or in combination could be found. Levels of mRNA were determined using a standard curve generated with the plasmid corresponding to the target gene, and are expressed as number of copies per reaction. To control for sample loading, tissue samples were run in duplicate. Between-duplicate variation never exceeded 10%.

Statistical analysis Data are presented as means7s.e. Except for variables reported in Table 2 (Student’s unpaired t-test), data were analyzed by 2  2 factorial analysis of variance (ANOVA). The factors were the CORT status with two levels (Vehicle, HiCORT), and drug treatment with two levels (Control, RSG). Some variables were log transformed prior to analysis to ensure homogeneity of variance. Pairwise differences between individual group means were analyzed by Fisher’s protected least significant difference post hoc test. Differences were considered statistically significant at Po0.05. Statement of ethics We certify that all applicable institutional and government regulations concerning the ethical use of animals were followed during this research. The animals were cared for

Table 2 Effects of exogenous HiCORT on plasma and tissue CORT concentrations, adrenal weight, and RWAT mRNA levels of angiotensinogen

Corticosterone Serum (mM)a RWAT (ng/g tissue) Liver (ng/g tissue) Adrenal weight (mg) Angiotensinogen mRNA (cprc)

Vehicle

HiCORT

1.570.1 2874 40718 5673 144719

2.270.1b 58712b 214718b 2171b 223734b

Abbreviations: HiCORT, hypercorticosteronemia; RWAT, retroperitoneal white adipose tissue. Values are means7s.e.m. of 5–8 animals. aMeasured in trunk blood samples obtained at the end of the experiment. bDifferent from Vehicle, Po0.05 (Student’s unpaired t-test). ccpr, copies per reaction  10–3.

Primers used for the quantification of mRNA levels in retroperitoneal adipose tissue

Gene

50 Primer (50 –30 )

30 Primer (50 –30 )

Angiotensinogen PPARga 11b-HSD1 FAS ACS1 DGAT1 GyK PEPCK-C ATGL HSL mCPT1 ACO

AATCAACAGGTTTGTGCAGG GGTGAAACTCTGGGAGATCC AATGGGAGCCCATGTGGTATTG GAGTCCGAGTCTGTCTCCCGCTTGA GCCCCCATGCCTTGCAATTA TATTACTTCATCTTTGCTCC CCTGTCCATTGAAATGTGTCATCC TGGGTGATGACATTGCCTGG CACTTTAGCTCCAAGGATGA CCTGCTGACCATCAACCGAC CGGAAGCACACCAGGCAGTA TCAAGGAGAGTGCTACGGGT

GTGTCACGGAGAAGTTGTTC TCAGCAACCATTGGGTCAG GCACAGAGTGGATATCATCGTGG GCCGTGAGGTTGCTGTTGTCTGTAG GCCATTTGGCAGCCATTTTCC AAAGTAGGTGACAGACTCAG GCCATGAAGCCATGACAATTAGTG ACCTTGCCCTTATGCTCTGCAG TGGTTCAGTAGGCCATTCCT CCTCGATCTCCGTGATATTCCAGA GCAGCTTCAGGGTTTGTCGGAATA CGTCAGCTTGTTACTCAAAGGC

a

Abbreviations: ACO, acyl-CoA oxidase; ACS1, acyl-CoA synthase 1; ATGL, adipose triglyceride lipase; FAS, fatty acid synthase; DGAT1, diacylglycerol acyltransferase 1; GyK, glycerol kinase; HSL, hormone-sensitive lipase; mCPT1, muscle-type carnitine palmitoyl transferase 1; PEPCK-C, cytosolic phosphoenolpyruvate carboxykinase; PPARg, peroxisome proliferator-activated receptor g; 11b-HSD1, 11b-hydroxysteroid dehydrogenase type 1.

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1663 and handled in conformance with the Canadian Guide for the Care and Use of Laboratory Animals and the protocols were approved by Laval University animal protection committee.

Whereas HiCORT impacted body weight gain solely through a reduction in lean tissue mass, RSG increased fat mass alone, without any apparent interaction between the two treatments. Serum level of leptin, an important modulator of energy balance, was increased twofold in HiCORT rats compared with vehicle-implanted animals. RSG did not affect leptinemia in the latter, but largely prevented its elevation in HiCORT rats. Adipose tissue being a major site of action of both PPARg and GC, where the two systems can exert either similar or antagonistic actions, we next considered whether defeating, via exogenous CORT, the PPARg-mediated reduction in local GC activity would alter the effects of PPARg agonism on adipose morphology and the expression of major target genes. Visceral fat being the major site of GC action among fat depots, treatment effects were assessed mainly in RWAT. The two-week RSG treatment increased RWAT weight ( þ 33%, Figure 1a), DNA (2.3-fold, Figure 1b), and TG content ( þ 29%, Figure 1c) independently of the CORT status, whereas HiCORT was without effect on these variables. Because interactions exist between PPARg and GC signaling, we next examined treatment effects on PPARg and 11b-HSD1 gene expression in RWAT. HiCORT did not alter PPARg expression, whereas, as expected, RSG decreased the latter by approximately 35% in both vehicle- and CORTimplanted rats (Figure 1d). HiCORT nearly doubled 11bHSD1 expression, such increase being abolished by RSG, which potently diminished the expression of this gene regardless of the CORT status (Figure 1e). HiCORT also remained without effect on subcutaneous (inguinal) WAT and interscapular BAT weight, these tissues being in turn greatly enlarged by RSG ( þ 56% and 2.9-fold, respectively, Po0.001; data not shown). PPARg agonism and GC are liable to interact upon adipose lipid metabolism at several levels that include uptake, storage, lipolysis, and recycling, at least partly via modulation of the expression of genes encoding key enzymes of these pathways. To assess whether the local PPARg-mediated reduction in GC action (through downregulation of 11bHSD1) contributes to its effect on visceral adipose tissue lipid

Results The CORT implant successfully increased circulating CORT levels (1.5-fold) as well as those found in RWAT (twofold) and the liver (fivefold; Table 2). The efficacy of the implant was also confirmed by the reduced activity of the hypothalamic–pituitary–adrenal (HPA) axis, reflected in the much smaller adrenal gland weight. In a pilot experiment using conditions identical to those of the present study, portal blood CORT was shown to be highly correlated with that in trunk blood (r ¼ 0.92, P ¼ 0.0002, n ¼ 10), confirming that visceral WAT draining into the portal vein had effectively been exposed to high CORT levels. Higher expression levels in RWAT of the GC target gene angiotensinogen21 (1.5-fold) further confirmed the functionality of adipose exposure to exogenous high CORT. RSG did not significantly influence serum CORT concentrations, as determined by averaging two separate samples taken at day 11 and at the end of the treatment period, and slightly but significantly reduced (–11%, Po0.04) adrenal weight in vehicle-implanted animals (data not shown). Because both the HPA axis and PPARg agonism impact whole body energy balance and fat deposition, which in turn affect several of the target end points, treatment effects on food intake, body weight gain, food efficiency, and indexes of fat and lean mass were evaluated. HiCORT reduced body weight gain by half (Table 3). This resulted from the combination of a slight (–5%) but significant decrease in food intake and a frank reduction in food efficiency (–40%) compared with rats implanted with the vehicle pellet. Treatment with RSG did not affect body weight gain, food intake, and efficiency in vehicle-implanted rats, but partially prevented the reduction in body weight accretion in HiCORT rats, largely because of increased food intake.

Table 3

Cumulative food intake and morphometric variables in normal and HiCORT rats treated or not with RSG for 2 weeks Vehicle Control

Body weight gain (g) Food intake (g/14 days) Food efficiency (%) Fat mass indexc (g) Lean mass indexd (g) Serum leptin (ng/ml)

8072 33675 2471 3.270.1 1.3670.06 2.870.2

HiCORT RSG 8274 357712 2371 5.170.3* 1.2870.03 2.470.3

Control a

4077 31179a 1372a 3.270.4 1.1370.05a 5.871.4a

ANOVA RSG a,b

6177 36678b 1672a 5.570.4b 1.1470.04a 3.670.6b

C

D

CD

o0.0001 NS o0.0001 NS 0.0003 0.02

0.04 0.0002 NS o0.0001 NS NS

NS (0.06) NS NS NS NS

Abbreviations: ANOVA, analysis of variance; HiCORT, hypercorticosteronemia; RSG, rosiglitazone; NS, not significant; RWAT, retroperitoneal white adipose tissue Data are means7s.e.m. of eight animals. The ANOVA columns represent the level of significance of the global effects of the CORT status (C) with two levels (Vehicle and HiCORT) and Drug (D) with two levels (Control and RSG) and their interaction (C  D). aDifferent from Vehicle-implanted within same Drug treatment, Po0.05. b Different from Control within same CORT status; cCalculated as the sum of inguinal+retroperitoneal white+interscapular brown adipose tissues. dCalculated as the sum of the vastus lateralis+soleus muscles.

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Figure 1 Retroperitoneal adipose tissue weight (a), DNA (b), TG content (c), and mRNA levels of PPARg (d) and 11b-HSD1 (e) in control (Vehicle) and corticosterone-implanted (HiCORT) rats treated or not with RSG for 2 weeks. See footnote to Table 3 for significance of ANOVA table. Each bar represents the mean7s.e.m. of 7–8 animals. *Different from Vehicle within same CORT status; wdifferent from Vehicle within same Drug treatment, Po0.05.

metabolism, expression levels of major lipid genes, and LPL activity were quantified in RWAT of control and RSG-treated rats with or without exposure to exogenous CORT. With respect to pathways that contribute to fat accretion, RSG alone increased LPL activity (Figure 2a) without affecting its mRNA (not shown), and increased the mRNA levels of proteins involved in de novo lipid synthesis (FAS; 2.5-fold, Figure 2b) and esterification (ACS1, DGAT1; þ 24%, Figures 2c and d). HiCORT shared with RSG a tendency to increase LPL activity (P ¼ 0.06) and mRNA levels of FAS and ACS1. Notably, the combination of treatments did not result in additive effects, nor did the CORT status impact in any way the RSG effect on the mRNA levels of the genes examined. With regard to pathways contributing to fatty acid export, International Journal of Obesity

recycling, and oxidation, RSG alone increased mRNA levels of ATGL (1.9-fold, Figure 3a) but not that of HSL (Figure 3b), and greatly increased mRNAs of the re-esterification enzymes GyK (3.5-fold, Figure 3c) and PEPCK-C (2-fold, Figure 3d), as well as those of the oxidative enzymes mCPT1 (4.4-fold, Figure 3e) and ACO (4.1-fold, Figure 3f). Adipose triglyceride lipase was the only protein of these pathways of which the mRNA was altered by HiCORT, again in the same direction as RSG, both effects being additive. The CORT status did not impact the magnitude of the effect of RSG on mRNA levels of the FA esterification, lipolytic, recycling, and oxidation enzymes examined. Because GC and PPARg agonism exert diametrically opposed effects on insulin action and lipemia, we next

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Figure 2 Activity of LPL (a), and mRNA levels of FAS (b), ACS1 (c), and DGAT1 (d) in retroperitoneal adipose tissue of control (Vehicle) and corticosterone-implanted (HiCORT) rats treated or not with RSG for 2 weeks. See footnote to Table 3 for significance of ANOVA table. Each bar represents the mean7s.e.m. of 7–8 animals. *Different from Vehicle within same CORT status; wdifferent from Vehicle within same Drug treatment, Po0.05.

determined whether HiCORT would defeat the RSG-induced improvement in indices of insulin sensitivity and serum lipid levels. As assessed after a short-term (6-h) fast, HiCORT alone modestly but significantly decreased serum glucose (10%, Figure 4a) and increased insulinemia ( þ 29%, Figure 4b) relative to vehicle-implanted rats. RSG did not affect glycemia, but brought about an overall decrease in insulinemia (global drug effect, Po0.02), which was more pronounced in HiCORT (–29%, Po0.02) than in vehicleimplanted rats (18%, not significant). As expected in chowfed animals,22 HiCORT did not impact serum TG. Triglyceridemia was strongly reduced by RSG to half of untreated levels (Figure 4c). Serum NEFA were nearly doubled by HiCORT, and RSG exerted a significant overall reducing effect on NEFA, the pairwise difference between untreated and RSG-treated HiCORT groups nearing significance (P ¼ 0.06, Figure 4d). Quantification of TG in oxidative tissues indicated that HiCORT increased liver TG concentration ( þ 29%), whereas RSG greatly decreased the latter (58%), in fact totally preventing the steatotic effect of HiCORT (Figure 4e). The TG concentration of the soleus muscle was slightly but significantly elevated in HiCORT rats ( þ 12%), whereas RSG remained without effect regardless of the CORT status (Figure 4f).

Discussion This study aimed to assess the contribution of the PPARginduced reduction in GC action to its modulation of adipose

tissue gene expression and beneficial impact on the metabolic profile. To this end, the effects of chronic treatment with a PPARg agonist were assessed in rats exposed or not to high CORT. It was found that the rosiglitazone retained its ability to alter the expression of several key genes of adipose tissue lipid metabolism and to improve the metabolic profile, including reversal of many but not all of the deleterious consequences of exposure to high CORT, suggesting independence of its metabolic effects from GC modulation at the systemic and tissue levels. Because GC strongly impact energy balance, which in turn affects metabolic homeostasis, it was deemed relevant to investigate the interaction of the CORT status and PPARg agonism on determinants of whole body energetics. Glucocorticoids exert concentration-dependent effects on body energy, with low levels being anabolic and high levels being catabolic according to the dose-related recruitment of central and peripheral mineralocorticoid and glucocorticoid receptors.23 In the present study, the CORT levels achieved were sufficient to slightly decrease food intake, as opposed to low physiological levels, which are orexigenic,15,24 and to vastly reduce food efficiency, resulting in a 50% smaller body weight gain. Of note is the fact that the mass of all adipose tissue depots examined was preserved in HiCORT rats, establishing that reduced weight gain was linked to lean mass, a well-recognized catabolic effect of high GC.23 Remarkably, treatment with RSG abolished the HiCORTinduced decrease in food intake and prevented half of the reduction in weight gain, albeit exclusively through increased fat mass accretion. The orexigenic effect of PPARg agonism confirms earlier rodent studies.9,25,26 Therefore, PPARg agonism prevents the anorectic effect of hypercorticosteronemia, but not its catabolic action on lean mass. These data further establish the important notion that the combined metabolic effects of HiCORT and PPARg agonism discussed below were independent from altered energy intake, a major potential confounder. There are several pathways of adipose tissue lipid metabolism in which positive or negative interactions between the GC and PPARg systems are liable to occur. A first level of interaction is the expression level of the PPARg receptor itself, known to be upregulated by GC27 and downregulated by its own agonists,11,28 and that of 11b-HSD1, which is subject to feed-forward regulation by GC29 and inhibition by PPARg agonism.16 The present study confirms the downregulation of PPARg expression by its own agonists, and shows that chronic exposure to high levels of CORT did not affect PPARg expression or its agonist-mediated reduction, at least over the short time period studied here. It was also found that PPARg agonism potently decreased 11b-HSD1 expression, as previously reported,9,16,25 and that it completely overwhelmed the stimulatory effect of hyperCORT on the latter. Therefore, for any given level of systemic GC, PPARg agonism appears able to diminish 11b-HSD1-mediated local GC production in visceral adipose tissue, thereby limiting the impact of GC on lipid metabolism. International Journal of Obesity

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Figure 3 mRNA levels of ATGL (a), HSL (b), GyK (c), PEPCK (d), mCPT1 (e), and ACO (f) in retroperitoneal adipose tissue of control (Vehicle) and corticosteroneimplanted (HiCORT) rats treated or not with RSG for 2 weeks. See footnote to Table 3 for significance of ANOVA table. Each bar represents the mean7s.e.m. of 7–8 animals. *Different from Vehicle within same CORT status; wdifferent from Vehicle within same Drug treatment, Po0.05.

A second potential level of GC-PPARg interaction is adipose cell proliferation and fat accretion. As noted above, high GC levels such as those seen in Cushing’s syndrome promote visceral obesity.14,30 Based on studies in adrenalectomized animals, GC appear to impact fat cell size rather than cell number.25,31 In vitro, CORT limits adipocyte proliferation and favors differentiation instead.32 In the present study, it is noteworthy that high CORT levels resulted in the maintenance of body fat mass in the face of slightly reduced food intake and frank loss of lean mass, confirming the fat sparing effect of GC. With respect to PPARg agonism, its proliferative and fat accreting actions are well established.1,2,33 In rodent models, there exists a dose-dependent depot specificity of action of PPARg agonists on adipose cell proliferation and fat International Journal of Obesity

accretion, and low doses such as that used here increase both subcutaneous and visceral fat cellularity and mass.9,11,12,25 Because our previous study in adrenalectomized rats suggested that, in vivo, GC may facilitate PPARg-mediated proliferative and lipid storage capacity,25 it was deemed of interest to assess whether maintenance of high levels of CORT would impact the latter. The DNA and TG contents of RWAT indicate that the level of GC to which adipose tissue was exposed did not alter in any direction the robust positive action of RSG on proliferation and fat accretion. Therefore, whereas total absence of GC may partly restrain the PPARg-induced stimulation of adipose tissue development, the latter does not appear to be particularly sensitive to a relatively wide concentration range of GC.

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Figure 4 Serum glucose (a), insulin (b), triglycerides (TG, c), nonesterified fatty acids (NEFA, d), and liver (e) and soleus muscle (f) TG concentration in control (Vehicle) and corticosterone-implanted (HiCORT) rats treated or not with RSG for 2 weeks. See footnote to Table 3 for significance of ANOVA table. Each bar represents the mean7s.e.m. of 7–8 animals. *Different from Vehicle within same CORT status; wdifferent from Vehicle within same Drug treatment, Po0.05.

A third level at which GC and PPARg can interact is the direct or indirect modulation of specific pathways of adipose tissue lipid metabolism. Because GC cumulate stimulatory actions on both lipogenic and lipolytic processes,23 the net effect of GC on adipose tissue lipid metabolism is highly dependent upon many factors, including levels of GC themselves, interactions with other hormonal systems such as insulin,34 and regional specificity.29,35,36 Knowledge of the detailed mechanisms by which GC impact adipose tissue lipid metabolism remains fragmentary; however, a permissive action on the insulin-mediated stimulation of LPL,37,38 an inhibitory effect on the anti-lipolytic action of insulin,39 direct transcriptional activation of the fatty acid-synthesizing gene FAS,40 and inhibition of the expression of the fatty acid re-esterification gene PEPCK41 have been established. In

the case of PPARg, effects on adipose tissue lipid metabolism are manifold and include stimulation of lipid uptake, esterification, recycling, lipolysis, and oxidation,11,42–44 with an overall preponderance of lipid retention over fatty acid release pathways explaining fat accretion.11 Therefore, the GC and PPARg pathways are liable to interact either positively or negatively at several levels of adipose tissue metabolism. In the present study, high CORT levels and RSG treatment elicited three combinations of responses in adipose tissue. Firstly, HiCORT and RSG tended to share a stimulatory effect on genes/enzymes of lipid accretion, namely LPL activity, and expression levels of FAS and ACS1. Quantitatively, HiCORT and RSG induced nearly identical increases; however, their effects were not additive, which may be suggestive International Journal of Obesity

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1668 of common, saturable molecular mechanisms of action. Secondly, whereas HSL expression remained unaffected, both treatment conditions exerted a stimulatory effect on mRNA levels of the lipolytic enzyme ATGL, which in this case was additive. This confirms our previous finding that ATGL is a target of PPARg agonism,43 and reveals the as yet unrecognized positive modulation of ATGL expression in vivo by CORT. The GC-mediated induction of adipose tissue lipolysis was until now thought to be due to facilitation of catecholamine action45 and interference with that of insulin.39 The present findings strongly suggest that ATGL constitutes an additional level of modulation of lipolysis by GC, given that the enzyme has very recently been established as a major determinant of lipolytic rates in adipose tissue.46–48 Whether additivity of HiCORT and RSG is exerted via a common or distinct pathways and whether these are direct or indirect remain to be established. Thirdly, RSG exerted its expected stimulatory effect on the expression levels of fatty acid esterification (DGAT1), recycling (GyK, PEPCK), and oxidation genes (mCPT1, ACO),11,42,44 whereas those genes were unaffected by high CORT. It must be emphasized that such PPARg-mediated changes in gene expression patterns are translated into corresponding changes at the functional level.11 These pathways tend to reduce the output of lipolytic products and favor local storage or metabolism of fatty acids, and therefore constitute a key divergence between the GC and PPARg pathways. The lack of reduction in PEPCK expression under HiCORT conditions was unexpected;49,50 however, PEPCK expression is also modulated by the nutritional status,51 and the present conditions may not have been optimal to reveal an effect of CORT thereupon. Through modulation of adipose tissue metabolism and other mechanisms, PPARg agonism improves insulin sensitivity and lipemia, in direct contrast with the consequences of excess GC. We therefore wished to determine whether exposure to high CORT would dampen the PPARg-mediated metabolic improvement in indices of glucose and lipid metabolism. The findings demonstrate that this was not the case. Indeed, the insulin-sensitizing properties of RSG appeared to be fully maintained in the high CORT setting, as suggested by fasting insulinemia, a crude but reliable index of insulin sensitivity. Not unexpectedly, the RSG-induced insulin reduction was modest and nonsignificant in insulinsensitive, chow-fed control rats; however, RSG was able to negate the rise in insulin brought about by chronically high levels of CORT. With regard to lipid accumulation in oxidative tissues, as expected because of its well-established lipogenic52,53 and fatty acid oxidation-reducing action,54 the robust increase in local CORT concentration in the liver achieved under HiCORT conditions resulted in a marked increase in liver TG content. Treatment with RSG not only decreased liver TG in vehicle-implanted rats but also abrogated HiCORT-induced steatosis. PPARg agonists are thought to counteract liver lipid deposition mainly indirectly through increasing the secretion of the adipokine International Journal of Obesity

adiponectin, which strongly favors hepatic fatty acid oxidation.55,56 The present findings demonstrate that in normal rats such as the vehicle-implanted rats of the present study, any reduction in liver exposure to CORT that may conceivably occur in response to PPARg agonism is not the cause of the antisteatotic effect of the latter. Notably, HiCORT modestly promoted tissue TG accumulation also in skeletal muscle, an effect that RSG was unable to counteract. Despite the fact that PPARg agonism is thought to improve muscle insulin signaling partly through a reduction in exposure to circulating lipids,6–8 there is evidence that PPARg agonists actually tend to increase muscle TG content.57 This strongly suggests that the insulin-sensitizing properties of PPARg agonists in muscle involve changes in deleterious lipid intermediates rather than in inert TG. High ambient CORT robustly increased serum NEFA levels, an expected consequence of increased adipose tissue lipolysis58 and a probable contributor to CORT-induced insulin resistance.59,60 Treatment with RSG modestly decreased serum NEFA but was unable to fully counteract the effect of HiCORT. This may possibly relate to the additive effect of HiCORT and RSG on ATGL expression, which may not have been efficiently counteracted by recycling/oxidation pathways. It should also be noted that the NEFA-lowering action of PPARg agonism is dependent upon the nutritional status and is particularly marked in the postprandial, rather than the fasted state.6,9 In contrast, HiCORT did not alter fasting triglyceridemia, which was not unexpected in chow-fed rats because the impact of CORT on triglyceridemia is best revealed under high fat-feeding conditions.22 Maintenance of normal triglyceridemia in the present HiCORT conditions may have resulted from a balance between increased verylow-density lipoprotein (VLDL)-TG secretion61,62 and increased LPL-mediated clearance by adipose tissue. Treatment with RSG strongly decreased triglyceridemia regardless of the CORT status. Long-term PPARg agonism appears to lower circulating TG both by reducing liver VLDL-TG secretion and by stimulating clearance from plasma.6,63 The RSG-induced reduction in liver TG content, a key determinant of hepatic VLDL secretion,64 combined with high adipose tissue LPL activity, neither of which was affected by the CORT status, likely participated in maintenance of the hypotriglyceridemic effect of RSG in the high CORT setting. In summary, the glucocorticoid and PPARg pathways were shown here to exert both congruent (lipid accretion pathways) and opposite (lipid release) actions on specific aspects of adipose tissue metabolism, as suggested by expression levels or activity of enzymes involved in such processes and by serum lipid levels. In addition, the findings clearly demonstrate that PPARg agonism is able to retain most of its beneficial metabolic actions in the presence of high ambient GC imposed by infusion of exogenous CORT. Without underestimating the undeniable impact of GC on adipose tissue, liver, and circulating lipid metabolism, the present findings indicate that, in normal conditions, PPARg agonism impacts adipose tissue metabolism and improves

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1669 several aspects of glucose and lipid homeostasis independently from its modulation of systemic and local GC action.

16

Acknowledgements We acknowledge the invaluable professional assistance of Ms Jose´e Lalonde and Mr Yves Ge´linas. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) to YD. M Berthiaume was the recipient of a PhD Studentship award from CIHR-Laval University.

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