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Agonism Increases the Capacity for Sympathetically Mediated Thermogenesis in Lean and ob/ob Mice
Laval Hospital Research Center and Department of Anatomy and Physiology (H.S., P.S., J.L., Y.D., D.R.), School of Medicine, Laval University, Québec, Canada G1K 7P4; and Department of Metabolic Disorders (J.P.B., G.C.), Merck Research Laboratories, Rahway, New Jersey 07065
Address all correspondence and requests for reprints to: Professor Denis Richard, Department of Anatomy and Physiology, School of Medicine, Laval University, Québec, Canada G1K 7P4. E-mail: denis.richard{at}phs.ulaval.ca.
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Abstract |
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modulates the expression of numerous genes involved in glucose and lipid homeostasis and plays a critical role in adipocyte differentiation. Expression of uncoupling protein (UCP)1, which is necessary for thermogenesis, is strongly stimulated by PPAR agonists but without an increase in energy expenditure. This study was designed to assess whether PPAR-induced UCP1 has any functional impact and, if so, whether it involves sympathetic activity. In a first phase, obese ob/ob C57BL/6J mice and lean controls were treated for 2 wk with the PPAR agonist [2-(2-[4-phenoxy-2-propylphenoxy]ethyl)indole-5-acetic acid] (COOH). COOH induced UCP1 expression in brown and white adipose tissues as well as that of other genes associated with substrate oxidation and thermogenesis. However, UCP1 induction did not increase energy expenditure, as assessed by indirect calorimetry and other energy balance measurements. In a second phase, mice received for an additional 2 wk a combination of COOH and the ß3-adrenergic receptor (ß3-AR) agonist CL-316243 to stimulate the adrenergic signaling pathway and assess whether COOH-induced UCP1 was physiologically functional. The ß3-AR agonist stimulated thermogenesis in lean and ob/ob mice, an effect that was much stronger in COOH-pretreated mice, which exhibited lower respiratory quotient, higher oxygen consumption, and marked weight and fat mass loss, compared with mice not pretreated with COOH. These results demonstrate that PPAR agonism increases the thermogenic potential of white and brown adipose depots in lean and obese mice. This enhanced capacity leads to increased thermogenesis under ß-adrenergic stimulation, suggesting that the sympathetic drive is blunted by PPAR agonism. |
Introduction |
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(PPAR) agonists are potent insulin sensitizers (1). Activation of PPAR results in remodeling of white adipose tissue (WAT) that is characterized by an increase in the number of smaller, more insulin-sensitive adipocytes that actively take up and efficiently retain lipids (2). PPAR activation also enhances the expression of genes that favor lipid uptake and retention and decreases the expression of genes that lower adipocyte lipid content (3, 4, 5). Furthermore, PPAR agonists stimulate adipocyte differentiation and apoptosis of large lipid-laden adipocytes (6). The generation of smaller adipocytes in rodent WAT by PPAR agonists is accompanied by a strong induction of uncoupling protein (UCP) 1 mRNA expression (7, 8, 9). This increase is the result of a positive transcriptional effect by PPAR resulting from its direct interaction with a functional peroxisome proliferator response element within the UCP1 promoter (10).
In rodents, brown adipose tissue (BAT) plays a central role in maintaining energy balance because BAT thermogenesis significantly increases energy expenditure (11). UCP1 is a critical positive regulator of thermogenesis in BAT. In fact, BAT UCP1 levels are relatively low in genetic models of obesity such as the ob/ob mouse, partly because of reduced sympathetic activity, its major modulator (12). The thermogenic activity of UCP1 in BAT is tightly regulated by the sympathetic nervous system via ß3-adrenergic receptors (ß3-AR) and is inducible by cold and hyperphagia (13, 14). A number of studies have shown that PPAR agonists induce UCP1 mRNA expression in adipose tissue of rodents (9, 15) whereas paradoxically increasing their food intake, body weight, and feeding efficiency (8, 16, 17). This suggests a lack of coupling between elevated UCP1 expression and actual thermogenic activity, conceivably because of a reduced sympathetic drive.
To address these issues, we investigated the effect of the PPAR agonist [2-(2-[4-phenoxy-2-propylphenoxy]ethyl)- indole-5-acetic acid] (COOH) (18) on UCP1 expression in lean and ob/ob mice as well as the metabolic impact of such induction through energy balance measurements. COOH bears a carboxylic acid pharmacophore in the place of the thiazolidinedione (TZD) moiety found in glitazones currently used as therapeutic agents in humans. As shown earlier (18), this non-TZD compound is a potent and highly selective PPAR full agonist with many features (except structure) similar to rosiglitazone. We analyzed PPAR agonist regulation of UCP1 expression as well as that of PPAR, PPAR
, PPAR coactivator 1 (PGC1), and muscle-type carnitine palmitoyltransferase 1 (mCPT1), all of which are important effectors of lipid metabolism and/or thermogenesis in adipose tissues (19, 20, 21). To extend the observations of previous studies examining PPAR regulation of UCP1 that have been limited to only assaying mRNA levels in whole tissues, we performed immunohistochemical studies to localize the protein within adipose tissue. Additionally, electron microscopy was carried out to determine the type of cells expressing UCP1. The impact of PPAR agonism on energy expenditure was analyzed by indirect calorimetry. We then cotreated lean and ob/ob mice with COOH and the ß3-AR agonist CL-316243 and assessed their thermogenic activity by measuring oxygen consumption (VO2) and weight loss. We also investigated the impact of this cotreatment on the expression of the catabolic and thermogenic genes analyzed for COOH treatment alone, as well as that of the ß3-AR itself, lipoprotein lipase (LPL), and the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). The ob/ob mouse allowed assessment of the effects of COOH in a model of impaired thermogenesis.
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Materials and Methods |
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agonist COOH (22) for 14 d at a dose of 30 mg/kg·d to induce UCP1 expression in adipose tissues. The PPAR ligand COOH was administered as a food admixture, and its concentration was adjusted to body weight and food intake twice a week. In the second phase following this period, all animals were implanted with an osmotic minipump (Alzet model 2001 or 2002, Alza, Palo Alto, CA) that delivered the ß3-AR agonist CL-316243 (CL, 1 mg/kg·d) (kindly provided by American Cyanamid, Pearl River, NY) to stimulate thermogenesis, or saline vehicle, as the control, for 14 d. Treatment with COOH was continued during CL administration. At the end of the COOH/CL phase, mice were killed and blood and tissue samples taken. The blood was immediately centrifuged (1500 x g, 15 min at 4 C), and the separated plasma was stored at –70 C until biochemical analysis was performed. Tissue samples were taken from inguinal WAT (IWAT), retroperitoneal WAT (RWAT), or intrascapular BAT and immediately fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.3). Data for indirect calorimetry, body composition, and immunohistochemistry of adipose tissue were collected from these mice. Supplementary lean and ob/ob mice (n = 5 per group) received COOH or the control diet for 2 wk to obtain tissue samples for electron microscopy and gene and blood analyses. Some supplementary lean mice (n = 6 per group) were killed after 3 d in the COOH/CL phase to collect tissue samples for gene analysis. Adipose tissue samples were snap frozen in liquid N2 and stored at –80 C for measurement of gene expression.
Indirect calorimetry
A subset of randomly selected mice (n = 4–5 per group) were individually monitored by indirect calorimetry to analyze VO2 and carbon dioxide production as well as the respiratory quotient (RQ) while treated with COOH alone or with the COOH/CL combination. Measurements were made continuously over 24 h each day (33 measurements for each mouse) in an open circuit system with an oxygen analyzer (Applied Electrochemistry, Pittsburgh, PA; S-3A1) and carbon dioxide analyzer (Applied Electrochemistry, CD-3A). Calorimetry data are average values for the second week of the COOH phase and daily average values for the first week of the COOH/CL phase.
Body composition
Dual x-ray absorptiometry (Piximus, LUNAR Corp., Madison, WI) was used to measure total fat and lean mass. This apparatus is a rapid (5-min image acquisition) and very precise small animal densitometer with an intraindividual variation of 2.2 and 0.86% for fat and lean tissue masses, respectively (23). Routine calibration was performed daily with a defined standard (phantom). Before measurement, mice were anesthetized with isoflurane and then placed on a specimen tray and put in the imaging area for analysis. After measurement a region-of-interest rectangle was moved and sized to cover the whole body, excluding the animal’s head. The PIXIMUS software automatically calculated the whole body fat and lean mass. Each mouse was measured three times, before the beginning of the COOH phase, at the end of the COOH phase, and at the end of the experiment. At the beginning of the experiment, lean mice had a body weight of 21.1 ± 0.3 g with 15.8 ± 0.3 g lean and 4.1 ± 0.2 g fat masses, respectively, whereas ob/ob mice weighed 45.1 ± 0.6 g with 19.1 ± 0.4 g lean and 25.7 ± 0.6 g fat masses, respectively.
Plasma determinations
Plasma glucose concentrations were measured with a glucose analyzer (Beckman Instruments, Carlsbad, CA). Plasma insulin was determined by RIA using a reagent kit from Linco Research (St. Charles, MO) with rat insulin standards. Plasma triglycerides were assayed by an enzymatic method using a reagent kit from Roche Diagnostics (Laval, Québec, Canada).
RNA isolation and analysis
Total RNA was prepared from frozen tissues using the Ultraspec RNA isolation kit (Biotecx, Houston, TX) and the RNAeasy 96 total RNA isolation kit (Qiagen, Valencia, CA) according to the manufacturers’ protocols. RNA concentration was estimated from absorbance at 260 nm. Expression levels of specific mRNAs listed below were quantified using quantitative fluorescent RT-PCR. RNA was first reverse transcribed using random hexamers in a protocol provided by the manufacturer (PE Applied Biosystems, Foster City, CA). Amplification of each target cDNA was then performed with TaqMan PCR reagent kits in the ABI Prism 7700 sequence detection system according to the protocols provided by the manufacturer (PE Applied Biosystems). The levels of mRNA were normalized to the amount of 18S rRNA (primers and probes from PE Applied Biosystems) detected in each sample. Results are expressed as target mRNA/18S mRNA for the two different experiments. The PCR was performed with the pairs of primers listed in Table 1
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Western blotting
Fifteen micrograms of whole protein extracts from BAT and IWAT were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. These membranes were then blocked for 1 h at room temperature in Tris-buffered saline (TBS) [50 mM Tris (pH 7.5), 150 mM NaCl] containing 0.04% Nonidet P-40, 0.02% Tween 20, and 5% nonfat milk followed by overnight incubation at 4 C with a sheep primary antibody against hamster UCP1. The membranes were then washed for 45 min followed by a 1-h incubation with antisheep IgG conjugated to horseradish peroxidase in TBS containing 1% nonfat milk. Finally, membranes were washed for 45 min in TBS and the immunoreactive bands detected by the enhanced chemiluminescence method.
Electron microscopy
Fragments from the adipose tissues of about 1 mm3 were fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.3) at 4 C for 24 h. The specimens were then washed in phosphate buffer (PB), postfixed in 1% (wt/vol) OsO4, dehydrated, and embedded in LR white resin (London Resin Co., Berkshire, UK) under UV light at 4 C. Ultrathin sections were collected on 200-mesh nickel grids and placed on droplets of 10% donkey serum in PB for 20 min at room temperature. After washing with PB, the grids were incubated with 25 µl of a sheep antihamster UCP1 antibody at a dilution of 1:25 in 1% (vol/vol) donkey serum and 1% (vol/vol) Tween 20 in 0.1 M PB (pH 7.3) overnight at 4 C in a humid chamber. The grids were washed three times and incubated with the second antibody, a donkey antisheep antibody, at a dilution of 1:20 in 1% donkey serum and 1% Tween 20 in 0.1 M PB (pH 7.3) for 2 h at room temperature. The secondary antibody was coupled to Immunogold (diameter of 15 nm) (BBI International, Agawam, MA). The grids were then washed in PB once, thoroughly cleaned with distilled water, and dried. For contrast, the grids were colored with uranyl acetate for 10 min and lead acetate for 6 min. Pictures were taken on an electron microscope at a magnification of x15,000 on a GEOL 1200EX transmission electron microscope (GEOL Ltd., Tokyo, Japan).
Statistical analysis
Data are presented as means ± SEMs. The main and interactive effects of the chronic treatment with COOH on the various dependent variables in the COOH phase were analyzed by ANOVA. In the COOH/CL phase of the experiment, ANOVA was used to analyze the effects of the four different treatment combinations [control (ctrl)/ctrl, ctrl/CL, COOH/ctrl, and COOH/CL] on the various dependent variables. Individual between-group differences were analyzed by Fisher’s post hoc least squares difference test. Differences were considered statistically significant at P < 0.05.
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, and COOH phase of Fig. 1). COOH had no significant effect on food intake but tended to slightly increase feeding efficiency (milligram weight gain per gram food ingested) in lean mice and did so significantly in ob/ob mice. Weight gain during the COOH phase was mostly due to a gain in lean mass in lean mice and a gain in both lean and fat masses in ob/ob obese mice (Table 2).
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). In the first days of CL treatment, there was a decrease in food intake in all CL-treated mice, compared with their control groups (not shown), which was not significant but could have partly contributed to weight loss. The latter was significantly greater in COOH/CL than in ctrl/CL mice, but COOH-treated mice nevertheless remained heavier than their untreated counterparts. Lean mice then tended to regain weight after a few days of CL treatment, which is in agreement with the well-known adaptation of metabolic activity to chronic treatment with ß3-AR agonists (24, 25). As shown in Table 2, at the end of the COOH/CL phase, CL had tended to reduce cumulative weight gain and feeding efficiency in mice not receiving COOH, and it had done so significantly in COOH-treated mice, whereas food intake remained unaffected. During the COOH/CL phase, fat mass gain tended to be reduced by CL in mice not treated with COOH and was so significantly in COOH-treated mice, whereas lean mass gain was similar in all groups. The ob/ob cohort (Fig. 1B) displayed the same response pattern as that observed in lean mice in terms of weight loss and progressive regain after CL treatment, except that in this cohort the rate of weight regain after a few days of CL treatment was more robust in mice not treated with COOH than in those receiving the PPAR agonist. Indeed, as shown in Table 2, cumulative weight gain was similar in ctrl/ctrl and ctrl/CL groups, whereas it was significantly lower in COOH/CL than in COOH/ctrl groups. Again as seen in lean mice, it is feeding efficiency rather than food intake that tended to parallel the changes in body weight gain. The COOH/CL mice displayed a massive decrease in fat mass gain, whereas their counterparts treated with COOH or CL alone gained fat. COOH, but not CL, increased lean mass gain in this ob/ob cohort.
Plasma glucose, insulin, and triglycerides
Plasma glucose and insulin levels at the end of the COOH phase confirmed the well-established insulin-sensitizing action of the PPAR agonist (Table 2) (26, 27). In lean mice, both glucose and insulin were decreased by COOH, whereas in ob/ob mice only glucose was significantly decreased by the PPAR agonist. COOH-treated ob/ob mice also displayed a large decrease in plasma triglycerides. During the COOH/CL phase, in both the lean and ob/ob cohorts, COOH and CL generally tended to decrease plasma glucose and insulin levels, their effects not being additive.
Energy expenditure and substrate oxidation
Indirect calorimetry performed on mice treated with COOH alone in the first phase of the experiment revealed that the PPAR agonist neither reduced RQ (Fig. 2, A and B) nor increased VO2 (Fig. 2, C and D) in lean and ob/ob mice and therefore did not induce thermogenic activity. In fact, COOH treatment significantly increased the RQ of ob/ob mice, with individual values over 1 at several time points, which is indicative of lipogenesis. The combined COOH/CL treatment, however, demonstrated that COOH had elevated the thermogenic capacity of lean and ob/ob mice because VO2 was significantly higher in COOH/CL mice than in those treated with CL alone. In addition, COOH/CL mice had significantly lower RQ values than ctrl/CL mice. In both phenotypes, the period characterized by low RQ values and high VO2 lasted for approximately 3 d, after which RQ and VO2 were restored to pre-CL values.
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). Immunohistochemistry demonstrated that UCP1 induction did not occur in all adipocytes but was restricted to cell clusters in which smaller adipocytes were found (Fig. 4). Analysis of the cellular localization of UCP1 in IWAT and RWAT revealed that the protein could be found in two different cell types (Fig. 3B). UCP1 was expressed in multilocular brown adipocytes rich in mitochondria and in some cells with the morphology of white adipocytes. The protein was present in some of the small mitochondria of these large monolocular adipocytes.
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). This reversal was especially apparent in ob/ob mice, which accrued a particularly large amount of BAT lipid in response to COOH treatment. Different patterns of UCP1 induction were observed in IWAT and RWAT, depending on treatment (Fig. 4). In lean mice, COOH induced UCP1 in distinct but numerous clusters in IWAT and in RWAT to a lesser extent. In ob/ob mice, COOH induced UCP1 expression in both IWAT and RWAT. CL treatment induced UCP1 in both WAT depots of lean mice but had little effect in ob/ob mice. Qualitatively, WAT of COOH/CL rats appeared to display the most numerous clusters of small brown adipocytes with high UCP1 expression.
Expression of genes related to substrate oxidation and thermogenesis
The expression of genes that code for proteins with key metabolic functions, including UCP1, was assessed at the end of the first phase (COOH alone) of the treatment. PPAR up-regulates catabolic lipid metabolism in the liver, BAT, and heart and skeletal muscle (19). mCPT1, which is up-regulated by activation of PPAR (28, 29), is the limiting step in the transport of long-chain fatty acids into mitochondria of muscle and WAT (21, 30). PGC1, a thermogenic regulator and coactivator of PPAR, is expressed only in tissues with a high capacity for ß-oxidation such as BAT and the heart (20). It is linked to UCP1 induction (31, 32), and both are implicated in increased energy dissipation through thermogenesis (33). In both lean and ob/ob mice, COOH led to an increase in UCP1 expression in IWAT and RWAT (Fig. 5A), in accordance with the protein data (Fig. 3), decreased PPAR mRNA expression (
30%, Fig. 5B), and markedly increased that of mCPT1, PPAR, and PGC1 (Fig. 5, C–E). Globally, COOH had a similar effect on the expression of all the genes examined in WAT of both lean and ob/ob mice, with the exception of UCP1, which was more robustly induced in ob/ob than in lean mice, perhaps because of its low levels of expression in control ob/ob mice. Levels of UCP2 and UCP3 were also analyzed, but COOH treatment had no effect on the expression of these UCP1 homologs (data not shown).
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). COOH and CL modulation of PGC1 was found to be similar to that of UCP1 (Fig. 6B). PPAR expression was significantly decreased by CL only in RWAT, in contrast with the down-regulation of the receptor by COOH in all three tissues. Expression of the ß3-AR was decreased by CL in RWAT and BAT. COOH had no impact on ß3-AR expression in BAT and IWAT but diminished the latter in RWAT (Fig. 6D). Therefore, suppression of thermogenic activity by COOH apparently cannot be attributed to a diminution in ß3-AR expression in BAT, as has been suggested by in vitro studies (34), but rather to a lack of sympathetic stimulation. PPAR expression was increased by CL in both WAT depots and by COOH in BAT (Fig. 6E). Surprisingly, the combined treatment with COOH and CL had no significant effect on PPAR expression in BAT. mCPT1 expression was increased by COOH in IWAT and RWAT (Fig. 6F). In BAT, COOH alone led to an increase in mCPT1 mRNA expression, which was completely abolished by the combined COOH/CL treatment, as was the case for PPAR. Quantitation was also made of 11ß-HSD1 expression, an enzyme that locally transforms inactive glucocorticoids into the potent glucocorticoid receptor agonists (corticosterone in rodents). COOH markedly decreased 11ß-HSD1 expression in adipose tissue as previously reported (18) (Fig. 6G). Interestingly, CL also decreased 11ß-HSD1 expression although not as efficiently as COOH; no additional effect was observed with dual treatment. LPL expression was decreased by COOH in RWAT and increased in IWAT and BAT (Fig. 6H) in accordance with previous findings in rats (35). CL led to a large increase in IWAT and BAT LPL mRNA but had no influence on RWAT LPL. Combined treatment with COOH and CL led to the largest induction of LPL in BAT.
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agonist on UCP1 expression and the potential metabolic impact of such induction. We report that the PPAR agonist COOH strongly increased UCP1 protein and other thermogenic genes in both WAT and BAT, without apparently enhancing energy expenditure. However, dual treatment with a PPAR and ß3-AR agonist led to increased thermogenesis and weight loss in lean and ob/ob obese mice relative to treatment with each agonist alone. Therefore, UCP1 induced by PPAR agonism is potentially functional and carries out its normal physiological role given the presence of an appropriate adrenergic stimulation. Additionally, the PPAR and ß3-AR agonist dual treatment had robust insulin-sensitizing and hypolipidemic effects without weight gain or fat accretion.
As shown with other PPAR agonists (7, 8, 9), COOH promoted brown adipocyte differentiation in BAT and sc (IWAT) and visceral (RWAT) WAT depots. COOH also induced UCP1 expression in BAT and WAT. The cell types expressing UCP1 in WAT depots in response to treatment with the PPAR agonist were identified as newly generated brown adipocytes and some morphologically white adipocytes. The strong induction of UCP1 in BAT and WAT by PPAR activation came without a concomitant increase in energy expenditure or thermogenesis, which is indicative of a lack of sympathetic activation, the major thermogenic inducer in rodents. In other conditions such as cold acclimatization (36), pharmacological ß3-adrenergic stimulation (37), or high-energy diet feeding (38), an elevation in BAT UCP1 content is associated with increased sympathetic signaling and thermogenesis. However, in this and other studies (9, 15) in which animals were treated with PPAR agonists, induction of UCP1 expression occurred in the context of increased body weight and higher feed efficiency in both lean and obese mice. The morphology of BAT in COOH-treated mice further underlines the inactive state of the tissue, with greater lipid accumulation in the form of large lipid droplets. Therefore, whereas activation of PPAR induces UCP1 expression, it apparently does not increase the activity of the UCP. This situation is not unique because it is reminiscent of cold-acclimated animals reintroduced to a warm environment, in which an abundant UCP1 is not activated by the sympathetic nervous system (39). The Syrian hamster exposed to a short photoperiod or fed a high-energy diet constitutes another instance in which the thermogenic machinery of BAT is augmented without a parallel increase in adrenergic activity (40, 41, 42).
The morphological transformation of adipose tissues brought about by COOH was accompanied by alterations in the expression of several genes other than UCP1 that are also involved in the regulation of lipid metabolism and energy homeostasis. COOH treatment resulted in the induction of genes associated with increased fatty acid oxidation (PPAR, mCPT1) and thermogenesis (PGC1) without any apparent increase in substrate oxidation. Analysis of oxygen consumption revealed that mice treated with COOH alone did not display decreased RQ or increased O2 consumption, and therefore did not increase fatty acid oxidation and thermogenesis. Individual RQ values above 1 in some COOH-treated ob/ob mice may indicate instead that these animals were actively engaged in lipogenesis, thereby explaining, at least partly, their gain in fat mass.
To establish the functionality of PPAR-induced UCP1 and determine whether a blunted sympathetic tone was the cause of the lack of increase in thermogenesis despite enhanced UCP1 expression, we introduced a dual treatment with COOH and a ß3-AR agonist after 2 wk of COOH treatment. We discovered that such dual-ligand treatment led to a higher thermogenic activity in lean and ob/ob mice. COOH/CL-treated mice, in comparison with those treated with CL alone, showed higher values for O2 consumption and lower RQ, both indicative of greater thermogenic performance. Elevated thermogenesis was accompanied by a more marked and prolonged weight loss, mainly due to fat mass loss. Indeed, in COOH/CL-treated mice, decreased thermogenesis and weight recovery did not occur as rapidly as in mice treated with CL alone. Analysis of adipose tissue depots by immunohistochemistry revealed synergistic effects of PPAR and ß3-AR agonists in enhancing UCP1 protein levels in IWAT and RWAT. In a single previous study, combined treatment with a PPAR agonist and norepinephrine was shown to have additive acute effects on UCP1 expression in adipocytes in vitro (43). However, to our knowledge, no physiological data on the synergistic effects of PPAR and ß3-AR agonists on thermogenesis and energy balance in vivo have been reported before the present study. It is noteworthy that the potentiation of the thermogenic response to ß3-adrenergic stimulation by prior PPAR activation was recently confirmed in COOH-treated mice exposed to cold; these animals maintained a higher body temperature than mice not treated with the PPAR agonist (Sell, H., and D. Richard, unpublished data).
COOH treatment led to the increased expression of genes involved in lipid use such as UCP1, mCPT1, and PPAR, and lipid accumulation such as LPL. Interestingly, CL treatment resulted in a gene expression pattern that closely resembled that of COOH. Indeed, CL increased PPAR, mCPT1, PGC1, and LPL expression, albeit to a somewhat lesser degree than did COOH. Both agonists shared the ability to reduce the expression of their own receptor but had no impact on the expression of the other receptor (except in RWAT, in which COOH reduced the expression of ß3-AR and CL reduced the expression of PPAR). In addition to the above effects of COOH and CL on the expression of proteins that constitute or directly modulate the activity of the thermogenic machinery, both agonists caused a significant down-regulation in the expression of 11ß-HSD1. Because this enzyme locally generates potent endogenous glucocorticoid receptor agonists, it can be considered antithermogenic due to the negative impact of glucocorticoids on energy dissipation (44, 45, 46). Despite such similar effects on gene expression by PPAR and ß3-AR agonists, the two transduction pathways are clearly distinct from each other at the physiological level in that ß3-AR activation does translate into increased thermogenesis, whereas PPAR activation per se does not. This establishes that increased UCP1 gene expression obtained by PPAR activation in a way not involving the ß3-AR/cAMP/protein kinase A/hormone-sensitive lipase pathway is not sufficient to increase thermogenesis (47). However, the present findings clearly demonstrate that the pattern of gene alteration resulting from PPAR activation, although without functional effect on energy expenditure alone, predisposes the adipocyte machinery to increase its response to a superimposed adrenergic challenge.
The reason for the absence of increased thermogenic activity despite the PPAR-mediated alterations in the expression of genes that would be expected to favor fatty acid oxidation and thermogenesis is not known. However, the findings with the dual PPAR-ß3 AR treatment points to an involvement of the sympathetic nervous system. In vivo, PPAR activation does not lead to an increase in sympathetic signaling and, as alluded to above, may even be associated with a reduction in sympathetic activity. This has not been demonstrated directly, but, given the major role of sympathetic activity in rodent thermogenesis, the increased PPAR-induced feed efficiency seen here and in other studies suggests such a reduction. In this regard, it is noteworthy that we have observed PPAR-induced modifications in neuropeptide expression patterns favoring orexigenic neuropeptides in the brain, which may be indicative of decreased sympathetic signaling for energy expenditure (Sell, H., and D. Richard, unpublished data). These findings suggest that PPAR agonism may directly or indirectly influence the central regulation of energy balance so as to promote energy intake and accumulation, and depress thermogenesis-related energy expenditure. A reduced sympathetic activity would decrease adipocyte lipolysis and fatty acid availability for oxidation, which could be compounded by the strong PPAR-mediated induction of fatty acid reesterification into triglycerides (48). By stimulating lipolysis, concomitant administration of a ß3-AR agonist could conceivably overcome the shortage of substrate availability, thereby allowing the full physiological activation of the primed thermogenic machinery. Potentiation of the thermogenic response to ß3- adrenergic stimulation by PPAR activation may therefore result from their partially additive effect on the expression of thermogenic (UCP1, PGC1) and antithermogenic (11ß-HSD1) proteins in the presence of adequate oxidative substrate availability brought about by adrenergic stimulation.
One major physiological action of PPAR agonists is their remodeling of adipose tissue. Numerous previous studies demonstrated that this remodeling is accompanied by in increase in UCP1 expression in BAT and WAT. The findings of this study corroborate these reports and further illustrate the impact of UCP1 induction on energy balance. Here it was shown that PPAR agonism alone leads to increased energy deposition, likely because of a lack of adequate sympathetic activity because ß3-AR agonism overcomes this situation and synergizes with PPAR agonism to increase thermogenic energy expenditure. This series of observations establishes the existence in vivo of cross-talk between two distinct signal transduction pathways in adipose tissue that profoundly impacts energy balance.
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Footnotes |
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Abbreviations: ß3-AR, ß3-Adrenergic receptor; BAT, brown adipose tissue; COOH, 4-phenoxy-2-propylphenoxy(ethyl)indole-5-acetic acid; ctrl, control; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; IWAT, inguinal WAT; KPBS, potassium PBS; LPL, lipoprotein lipase; mCPT1, muscle-type carnitine palmitoyltransferase 1; PB, phosphate buffer; PGC1, PPAR coactivator 1; PPAR, peroxisome proliferator-activated receptor; RQ, respiratory quotient; RWAT, retroperitoneal WAT; TBS, Tris-buffered saline; TZD, thiazolidinedione; UCP, uncoupling protein; VO2, oxygen consumption; WAT, white adipose tissue.
Received March 15, 2004.
Accepted for publication April 29, 2004.
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  M. Laplante, W. T. Festuccia, G. Soucy, P.-G. Blanchard, A. Renaud, J. P. Berger, G. Olivecrona, and Y. Deshaies Tissue-specific postprandial clearance is the major determinant of PPAR{gamma}-induced triglyceride lowering in the rat Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2009; 296(1): R57 - R66. [Abstract] [Full Text] [PDF]
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S. Sena, I. R. Rasmussen, A. R. Wende, A. P. McQueen, H. A. Theobald, N. Wilde, R. O. Pereira, S. E. Litwin, J. P. Berger, and E. D. Abel Cardiac Hypertrophy Caused by Peroxisome Proliferator- Activated Receptor-{gamma} Agonist Treatment Occurs Independently of Changes in Myocardial Insulin Signaling Endocrinology, December 1, 2007; 148(12): 6047 - 6053. [Abstract] [Full Text] [PDF]
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M. Laplante, W. T. Festuccia, G. Soucy, Y. Gelinas, J. Lalonde, and Y. Deshaies Involvement of adipose tissues in the early hypolipidemic action of PPAR{gamma} agonism in the rat Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2007; 292(4): R1408 - R1417. [Abstract] [Full Text] [PDF]
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S. L. Gray, E. Dalla Nora, E. C. Backlund, M. Manieri, S. Virtue, R. C. Noland, S. O'Rahilly, R. N. Cortright, S. Cinti, B. Cannon, et al. Decreased Brown Adipocyte Recruitment and Thermogenic Capacity in Mice with Impaired Peroxisome Proliferator-Activated Receptor (P465L PPAR{gamma}) Function Endocrinology, December 1, 2006; 147(12): 5708 - 5714. [Abstract] [Full Text] [PDF]
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E. Hondares, O. Mora, P. Yubero, M. R. de la Concepcion, R. Iglesias, M. Giralt, and F. Villarroya Thiazolidinediones and Rexinoids Induce Peroxisome Proliferator-Activated Receptor-Coactivator (PGC)-1{alpha} Gene Transcription: An Autoregulatory Loop Controls PGC-1{alpha} Expression in Adipocytes via Peroxisome Proliferator-Activated Receptor-{gamma} Coactivation Endocrinology, June 1, 2006; 147(6): 2829 - 2838. [Abstract] [Full Text] [PDF]
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, P < 0.05 comparing ctrl/CL mice with ctrl/ctrl mice and COOH/CL mice with COOH/ctrl mice (impact of CL treatment). *, P < 0.05 comparing COOH/ctrl treated mice with ctrl/ctrl mice and comparing COOH/CL mice with ctrl/CL mice (impact of COOH treatment).
