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Endocrinology 149(5):2121–2130
Copyright © 2008 by The Endocrine Society
doi: 10.1210/en.2007-1553
Peroxisome Proliferator-Activated Receptor-␥-Mediated
Positive Energy Balance in the Rat Is Associated with
Reduced Sympathetic Drive to Adipose Tissues and
Thyroid Status
William T. Festuccia, Serdar Oztezcan, Mathieu Laplante, Magalie Berthiaume, Chantal Michel,
Shinya Dohgu, Raphaël G. Denis, Marcia N. Brito, Nilton A. Brito, David S. Miller, William A. Banks,
Timothy J. Bartness, Denis Richard, and Yves Deshaies
Laval Hospital Research Center (W.T.F., M.L., M.B., C.M., R.G.D., D.R., Y.D.), Faculty of Medicine, Laval University,
Quebec, Canada G1V 4G5; Department of Biochemistry (S.O.), Medical Faculty of Istanbul, Istanbul University, Istanbul
34452, Turkey; Geriatrics Research (S.O., S.D., W.A.B.), Education, and Clinical Center, Veterans Affairs Medical Center,
St. Louis, St. Louis, Missouri 63106; Division of Geriatrics (W.A.B.), Department of Internal Medicine, St. Louis University
School of Medicine, St. Louis, Missouri 63104; Laboratory of Pharmacology and Chemistry (D.S.M.), National Institute of
Environmental Health Science, National Institutes of Health, Research Triangle Park, North Carolina 27709; and Biology
Department (M.N.B., N.A.B., T.J.B.), Georgia State University, Atlanta, Georgia 30302
Peroxisome proliferator-activated receptor-␥ (PPAR␥) activation up-regulates thermogenesis-related genes in rodent
white and brown adipose tissues (WAT and BAT) without increasing whole-body energy expenditure. We tested here
whether such dissociation is the result of a negative modulation of sympathetic activity to WAT and BAT and thyroid axis
components by PPAR␥ activation. Administration of the
PPAR␥ agonist rosiglitazone (15 mg/kg䡠d) for 7 d to male
Sprague Dawley rats increased food intake (10%), feed efficiency (31%), weight gain (45%), spontaneous motor activity
(60%), and BAT and WAT mass and reduced whole-body oxygen consumption. Consistent with an anabolic setting, rosiglitazone markedly reduced sympathetic activity to BAT and
WAT (>50%) and thyroid status as evidenced by reduced levels
of plasma thyroid hormones (T4 and T3) and mRNA levels of
BAT and liver T3-generating enzymes iodothyronine type 2
P
EROXISOME PROLIFERATOR-activated receptor (PPAR)-␥
is a ligand-activated nuclear receptor that is mainly expressed in white adipose tissue (WAT) and rodent brown adipose
tissue (BAT) where it controls the expression of several proteins
involved in lipid metabolism. Although the identity of functional
endogenous PPAR␥ ligands remains uncertain, the receptor is
specifically activated by thiazolidinediones, a class of synthetic
agonists that, because of their beneficial effect on whole-body inFirst Published Online January 24, 2008
Abbreviations: ARC, Arcuate region; BAT, brown adipose tissue;
CART, cocaine- and amphetamine-regulated transcript; CsA, cyclosporin A; D1 and D2, type 1 and type 2 iodothyronine deiodinase;
HBMEC, human brain microvascular endothelial cells; icv, intracerebroventricular; ING, inguinal; NE, norepinephrine; NETO, NE turnover;
P-gp, p-glycoprotein; PPAR, peroxisome proliferator-activated receptor;
pPVN, parvocellular subregion of the hypothalamic paraventricular
nucleus; RETRO, retroperitoneal; SMA, spontaneous locomotor activity;
TAG, triacylglycerol; THR, thyroid hormone receptor; UCP1, uncoupling protein 1; WAT, white adipose tissue.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
(ⴚ40%) and type 1 (ⴚ32%) deiodinases, respectively. Rosiglitazone also decreased mRNA levels of the thyroid hormone
receptor (THR) isoforms ␣1 (ⴚ34%) and ␤ (ⴚ66%) in BAT and
isoforms ␣1 (ⴚ20%) and ␣2 (ⴚ47%) in retroperitoneal WAT.
These metabolic effects were associated with a reduction in
mRNA levels of the pro-energy expenditure peptides CRH and
CART in specific hypothalamic nuclei. A direct central action
of rosiglitazone is, however, unlikely based on its low brain
uptake and lack of metabolic effects of intracerebroventricular administration. In conclusion, a reduction in BAT sympathetic activity and thyroid status appears to, at least partly,
explain the PPAR␥-induced reduction in energy expenditure
and the fact that up-regulation of thermogenic gene expression does not translate into functional stimulation of wholebody thermogenesis in vivo. (Endocrinology 149: 2121–2130,
2008)
sulin sensitivity, are widely used in the treatment of insulin-resistant states such as type 2 diabetes and metabolic syndrome (1, 2).
WAT and BAT, the major sites in rodents of energy storage
and nonshivering thermogenesis, respectively, are the main
targets of PPAR␥ activation. In these tissues, PPAR␥ activation is associated with an increase in the number of differentiated adipocytes with enhanced capacity to take up and
store lipids (3, 4); such an effect partially accounts for the
concomitant reduction in circulating nonesterified fatty acids
and triacylglycerol (TAG) levels. Paradoxically to enhanced
fat storage, PPAR␥ activation is also associated with increases not only in WAT and BAT mitochondrial biogenesis
and levels of the thermogenic uncoupling protein 1 (UCP1)
but also in those of key proteins involved in fatty acid oxidation and lipolysis (5– 8). In accordance with its positive
effects on the lipolytic, oxidative, and thermogenic machineries, in vitro PPAR␥ activation in fetal primary brown adipocytes, 3T3-L1 adipocytes, and primary white adipocytes
increases rates of lipolysis, fatty acid oxidation, O2 consumption, and uncoupled respiration (5–7).
Despite the potent catabolic profile found in vitro, in vivo
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Festuccia et al. • PPAR␥ Activation and Energy Balance
Endocrinology, May 2008, 149(5):2121–2130
administration of PPAR␥ agonists brings about opposite effects. Indeed, in the face of a robust increase in the expression
of lipolytic, oxidative, and thermogenic genes in WAT and
BAT, the net result of PPAR␥ activation in vivo is an increase
in energy storage rather than in energy expenditure. In fact,
administration of PPAR␥ agonists to rodents and humans
increases body weight and fat mass gains as well as food
intake and efficiency, all indicative of an anabolic condition
(9 –12). Notably, the high thermogenic, oxidative, and lipolytic potential (i.e. mRNA and protein content and concomitant increase in related processes when assessed in vitro)
induced by PPAR␥ activation are actuated at the functional
level in vivo only under pharmacological ␤-adrenergic stimulation (9, 13, 14). This strongly suggests that the PPAR␥induced up-regulation of BAT and WAT lipolytic, oxidative,
and thermogenic machineries are silenced by the in vivo
neural and hormonal milieu such that energy storage, rather
than expenditure, is favored.
Release of norepinephrine (NE) by the sympathetic nervous system innervating fat pads, along with the thyroid
hormone T3, constitute the major activators of WAT and BAT
lipolysis and thermogenesis (15, 16). In the present study, we
tested the hypothesis that the dissociation between gene
expression and biological processes found in vivo under
PPAR␥ activation is caused by a concomitant reduction in
sympathetic activity and thyroid status. To this end, we
assessed in rats the effects of the PPAR␥ agonist rosiglitazone
on WAT and BAT NE turnover rates (NETO), a measure of
sympathetic activity, and on the hypothalamic-pituitary-thyroid axis, including BAT and liver type 2 and 1 iodothyronine
deiodinase (D2 and D1, respectively) and thyroid hormone
receptor (THR) mRNA levels.
Because PPAR␥ is expressed at low levels in some hypothalamic regions (17), we further tested the hypothesis that
rosiglitazone exerts its effects on energy balance centrally.
This was addressed by assessing the effects of rosiglitazone
on the expression of hypothalamic neuropeptides involved
in the control of energy expenditure, the ability of rosiglitazone to cross the blood-brain barrier, and the impact of
central administration of rosiglitazone on energy balance.
Materials and Methods
Animals and treatment
Animal care and handling were performed in accordance with the
Canadian Guide for the Care and Use of Laboratory Animals. All experimental procedures received prior approval of the Laval University
animal care committee. Male Sprague Dawley rats (Charles River Laboratories, St. Constant, Quebec, Canada) initially weighing 175–200 g
were individually housed in stainless steel cages in a room kept at 23 ⫾
1 C with a 12-h light, 12-h dark cycle (lights on at 0800 h). After a 4-d
adaptation period, rats were matched by weight and divided into control
and rosiglitazone-treated groups that received a nonpurified powdered
rodent diet (Charles River Rodent Diet no. 5075; Woodstock, Ontario,
Canada; digestible energy content, 12.9 kJ/g) alone (control) or supplemented with the PPAR␥ agonist rosiglitazone (purchased as AVANDIA
at a local pharmacy) at a dose of 15 mg/kg䡠d for 7 d. This dose was
chosen based on preliminary studies that showed its effectiveness to
increase both visceral and sc adipose depot mass within a short period
of treatment (e.g. 7 d). Ground rosiglitazone was mixed with the powdered chow diet, and the desired dose was achieved by adjusting the
amount of drug to the average food consumption and body weight of
rats every other day.
Energy expenditure and spontaneous motor activity
O2 consumption, CO2 production, and respiratory quotient were
determined over 1 wk in an open-circuit system with an O2 (Applied
Electrochemistry, Naperville, IL; S-3A1) and a CO2 analyzer (Applied
Electrochemistry, CD-3A). Data are presented as ml/min䡠kg0.75 body
weight. Locomotor activity was measured with the AccuScan Digiscan
Activity Monitor (AccuScan Instruments, Columbus, OH), with the aid
of the VersaMax software (version 1.30; AccuScan Instruments). Rats
were placed individually in acrylic chambers (40 ⫻ 40 ⫻ 30 cm), and
motor activity was measured for 48 h after a 24-h adaptation period.
Cages contained an array of beams (every 2.5 cm) in all three dimensions
(16 in x-axis left-right, 16 in y-axis front-back, and 16 in z-axis vertical).
Motor activity was determined by breaks in photobeams in the horizontal plane and converted into spontaneous locomotor activity (SMA,
meters per day).
Adipocyte morphology by light microscopy, DNA, and
TAG content
Portions of retroperitoneal (RETRO) WAT and interscapular BAT
were fixed in 0.1 mm PBS (pH 7.4) containing 4% paraformaldehyde and
embedded in paraffin. Thin sections were mounted on glass slides and
dyed with hematoxylin/eosin. Digital images of tissue slices were captured and analyzed as previously described (4). Tissue DNA content was
determined using the DNeasy tissue kit (QIAGEN, Mississauga, Ontario, Canada) following manufacturer’s instructions. Tissue TAG content was measured after lipid extraction with chloroform-methanol (2:1)
with an enzymatic kit (Roche Diagnostics, Montreal, Canada).
NETO
NETO rate, a reliable index of sympathetic activity in a given tissue,
was estimated from the decline in tissue NE content after inhibition of
catecholamine synthesis with dl-␣-methyl-tyrosine ester (Sigma Chemical Co., St. Louis, MO). Rats were killed by anesthetic (ketamine/
xylazine) overdose before or 4 h after ip injection of dl-␣-methyl-tyrosine ester (350 mg/kg body weight), exactly as previously described
(18 –20). These time points are based on previous studies establishing the
linearity of the logarithmic decline in NE content in rat tissues after
inhibition of catecholamine synthesis (21, 22). The inguinal (ING) and
RETRO fat depots and interscapular BAT were rapidly removed,
weighed, frozen in liquid nitrogen, and stored at ⫺80 C for later determination of NE content. NETO experiments were repeated three
times with high reproducibility. Rates of NETO were calculated as the
product of the fractional turnover rate (k) and the endogenous NE
content at time 0 as previously described (23). Fractional turnover rate
(k) was calculated by the formula: k ⫽ (log [NE]0⫺ log [NE]4)/(0.434 ⫻
4), where [NE]0 and [NE]4 are the NE content at times 0 and 4 h,
respectively.
Tissue NE content
Tissue NE content was measured as previously described (22).
Briefly, tissues were homogenized in 0.2 n perchloric acid, 1 mm EDTA,
1% sodium metabisulfite, with dihydroxybenzylamide as an internal
standard, and centrifuged, and the supernatant destined for catecholamine quantification was extracted with alumina. Catecholamines were
eluted from alumina with the above homogenization solution without
internal standard and assayed by HPLC.
Intracerebroventricular (icv) rosiglitazone infusion
Cannulae were implanted into the third ventricle of isoflurane-anesthetized rats as previously described (24). Cannulae were connected
with silicon catheters to mini-osmotic pumps (model 2002; Alzet, Cupertino, CA) that delivered a solution of rosiglitazone maleate
(BRL49653; GlaxoSmithKline, Mississauga, Ontario, Canada) at doses of
50 and 500 ␮g/ml dissolved in sterile saline containing 12.5% dimethylsulfoxide (pH 7.4). Pumps implanted in the interscapular region delivered the solution at a rate of 0.5 ␮l/h for 7 d. Control rats were infused
with vehicle. Before and 2 d after surgery, rats were given analgesics
Festuccia et al. • PPAR␥ Activation and Energy Balance
(ketoprophene, 5 mg/kg, twice a day). Cannula positioning was evaluated microscopically a posteriori in all rats.
Hypothalamic dissection
Hypothalami were rapidly dissected using as landmarks the optic
chiasma rostrally and the mammillary bodies caudally and frozen in
liquid nitrogen for later RNA extraction and quantification of neuropeptide expression levels by PCR.
In situ hybridization for CRH and cocaine- and
amphetamine-related transcript (CART)
Hypothalamic mRNA levels of CRH and CART, two neuropeptides
involved in the central regulation of sympathetic activity, were measured by in situ hybridization essentially as previously described (25).
Briefly, hypothalamic sections were mounted onto poly-l-lysine-coated
slides, dehydrated in ethanol, fixed in paraformaldehyde, digested with
proteinase K (10 ␮g/ml), acetylated with 0.25% acetic anhydride, and
dehydrated in ethanol gradient. Sections were incubated overnight with
antisense 35S-labeled cRNA probe (107 cpm/ml) for CRH or CART at 60
C. Slides were rinsed with sodium chloride/sodium citrate solution,
digested with RNase-A, washed in descending concentrations of sodium
chloride/sodium citrate solution, and dehydrated in ethanol gradient.
Slides were defatted in toluene, dipped in NTB2 nuclear emulsion (Eastman Kodak, Rochester, NY), and exposed for 7 d before being developed. Slides were examined by dark-field microscopy using an Olympus
BX51 microscope (Olympus America, Melville, NY). Images were acquired with an Evolution QEi camera and analyzed with ImagePro plus
version 5.0.1.11 (MediaCybernetics, Silver Spring, MD). The system was
calibrated for each set of analyses to prevent saturation of the integrated
signal. Mean pixel densities were obtained by taking measurements
from both hemispheres of one to four brain sections and subtracting
background readings taken from areas immediately surrounding the
region analyzed.
RNA isolation and quantification
RNA was isolated from adipose depots and hypothalamus using
QIAzol and the RNeasy lipid tissue kit (QIAGEN). For cDNA synthesis,
expand reverse transcriptase (Invitrogen) was used following the manufacturer’s instructions, and cDNA was diluted in DNase-free water
(1:25) before quantification by real-time PCR. mRNA transcript levels
were measured in duplicate samples using a Rotor Gene 3000 system
(Montreal Biotech, Montreal, Quebec, Canada). The primers used for the
PCR are available upon request to the corresponding author. Chemical
detection of the PCR products was achieved with SYBR Green I (Molecular Probes, Willamette Valley, OR). At the end of each run, meltcurve analyses were performed, and a few samples representative of
each experimental group were run on agarose gel to ensure the specificity of the amplification. Results are expressed as the ratio between the
expression of the target gene and the housekeeping genes 36B4
(NM_022402) for adipose tissues and liver or GAPDH (NM_017008) for
hypothalamus, which were selected because no significant variation in
their expression was observed between control and rosiglitazonetreated rats.
Rosiglitazone transport through the blood-brain barrier
All in vivo experiments investigating rosiglitazone transport through
the blood-brain barrier were performed in male CD-1 mice (35– 40 g)
from our in-house colony (W.A.B., Veterans Affairs Medical Center, St.
Louis, MO). Mice were used because the methodology involves intracranial injection procedures that have been optimized for this species
and are not readily adaptable to the rat. At least in terms of blood-tobrain rosiglitazone transport, there appears to be no difference between
the two species based on the present findings in mice and previous
evidence in rats (26). In addition, to investigate the applicability of the
findings obtained with mice to other species, rosiglitazone transport in
and out of the brain was also investigated in human brain microvascular
endothelial cells, a human model of the blood-brain barrier (see below).
Endocrinology, May 2008, 149(5):2121–2130
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Blood-to-brain uptake
Mice had free access to food and water and were maintained on a 12-h
dark, 12-h light cycle in a room with controlled temperature (24 ⫾ 1 C)
and humidity (55 ⫾ 5%). Mice were anesthetized with an ip injection of
0.2 ml of a 40% urethane solution. The left jugular vein and the right
carotid artery were isolated (27). Approximately 1.2 ⫻ 106 cpm [3H]rosiglitazone (American Radiolabeled Chemical, St. Louis, MO) with or
without 1 ␮g unlabeled rosiglitazone was injected into the jugular vein
in 200 ␮l lactate-buffered Ringer’s solution containing 1% BSA. At 1, 2,
3, 4, 5, 7.5, 10, 20, and 30 min after injection, blood was freely collected
for about 15 sec from a cut in the carotid artery, and serum was isolated
by centrifugation. Mice were then immediately decapitated, blood was
collected, and the whole brain minus pineal and pituitary glands was
removed and weighed. Brain and serum samples were incubated with
tissue solubilizer (1 ml/200 mg tissue and 500 ␮l/50 ␮l serum) in a water
bath at 40 C for 24 h. Scintillation cocktail (10 ml) was added, and
samples were allowed to quench for 24 h in the dark before determination of radioactivity content.
The percentage of the iv dose injected per milliliter of serum was
calculated with equation 1: % Inj/ml ⫽ 100 ⫻ (cpm/ml serum)/ (cpm/
injection). The brain/serum ratio (microliters per gram) was obtained by
equation 2: brain/serum ratio ⫽ (cpm/brain)/[(cpm/␮l serum) ⫻ (brain
weight)]. The percentage of injected dose taken up per gram of brain (%
Inj/g) was calculated from equation 3: % Inj ⫽ (R ⫺ 10) (% S), where R
is brain-to-blood ratio expressed in microliters per gram calculated from
equation 2 above, 10 ␮l is taken as the volume of blood in 1 g of brain,
and % S is the % Inj/ml as calculated in equation 1 above.
Brain-to-blood transport
The icv injection method was used to study brain-to-blood transport
(28). Mice (n ⫽ 7–9 per group) were anesthetized with an ip injection of
urethane. After the scalp was removed, a hole was made through the
cranium 1.0 mm lateral and 0.5 mm posterior to the bregma with a
26-gauge needle. The entire needle except 2.5 mm of the tip was covered
with PE-10 tubing. This ensured that the tip of the needle penetrated the
skull and brain tissue forming the roof of the lateral ventricle but did not
penetrate the ventricular floor. One microliter of lactated Ringer’s solution with 1% BSA containing 5000 cpm of [3H]rosiglitazone with or
without unlabeled rosiglitazone (1.5 ␮g/mouse) or the P-glycoprotein
(P-gp) inhibitor SDZ PSC833 (100 ng/mouse) was injected into the
TABLE 1. Final body weight, cumulative food intake, feed
efficiency, O2 consumption (VO2), CO2 production (VCO2),
respiratory quotient (RQ), SMA, mass of WAT and BAT, and
serum levels of hormones and metabolites in rats treated or not
with rosiglitazone (RSG) for 7 d
Final body weight (g)
Body weight gain (g)
Food intake (g)
Feed efficiency (%)b
VO2 (ml/min䡠kg bw0.75)
VCO2 (ml/min䡠kg bw0.75)
RQ
SMA (m/d)
BAT (g)
ING WAT (g)
RETRO WAT (g)
Insulin (pM)
Glucose (mM)
NEFA (mM)
TAG (mM)
ACTH (␮M)
Leptin (ng/ml)
Adiponectin (␮g/ml)
Control
RSG
303 ⫾ 7
37 ⫾ 4
144 ⫾ 5
25 ⫾ 2
14.8 ⫾ 0.2
11.8 ⫾ 0.2
0.798 ⫾ 0.006
66 ⫾ 7
0.31 ⫾ 0.01
3.2 ⫾ 0.2
2.0 ⫾ 0.2
186 ⫾ 22
7.9 ⫾ 0.3
0.47 ⫾ 0.05
2.0 ⫾ 0.2
28 ⫾ 3
4.6 ⫾ 0.4
3.8 ⫾ 0.4
328 ⫾ 5a
54 ⫾ 4a
159 ⫾ 5a
33 ⫾ 2a
13.9 ⫾ 0.2a
11.1 ⫾ 0.2a
0.796 ⫾ 0.007
106 ⫾ 12a
0.67 ⫾ 0.02a
4.3 ⫾ 0.2a
2.7 ⫾ 0.1a
122 ⫾ 18a
7.8 ⫾ 0.1
0.20 ⫾ 0.07a
1.1 ⫾ 0.1a
16 ⫾ 1a
5.2 ⫾ 0.5
16.8 ⫾ 0.8a
Data are means ⫾ SEM of six to 12 rats. bw, Body weight; NEFA,
nonesterified fatty acids.
a
P ⬍ 0.05 vs. control.
b
Calculated as grams body weight gain per 100 g food ingested.
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Festuccia et al. • PPAR␥ Activation and Energy Balance
Endocrinology, May 2008, 149(5):2121–2130
lateral ventricle with a 1-␮l Hamilton syringe. The amount of rosiglitazone available for transport was determined in mice that were killed
with an overdose of urethane 10 –20 min before the icv injection. The
mice were decapitated and brains were harvested 10 min after the icv
injection. The whole brains were removed and incubated with tissue
solubilizer and scintillation cocktail as above and counted. The amount
of [3H]rosiglitazone transported was expressed as a percentage of the
counts available for transport (A): % transported ⫽ 100 ⫻ (A ⫺ 10-min
mean)/(A).
In vitro human blood-brain barrier model
Human brain microvascular endothelial cells (HBMECs) were purchased from Applied Cell Biology Research Institute (Kirkland, WA).
HBMECs were cultured in CS-C Complete Medium (Cell Systems Corp.,
Kirkland, WA) supplemented with 50 ␮g/ml gentamicin (Life Technologies, Inc., Invitrogen, Grand Island, NY) at 37 C with a humidified
atmosphere of 5% CO2/95% O2 air. HBMECs were treated with Passage
Reagent Group (Cell Systems) and then seeded on the inside of the
fibronectin/collagen IV-coated (0.1 and 0.5 mg/ml, respectively) polyester membrane (0.33 cm2, 0.4-␮m pore size) of a Transwell-Clear insert
(Costar, Corning, NY) (4 ⫻ 104 cells per well) placed in the well of a
24-well culture plate. Four to five days after seeding, HBMECs at passages 4 – 6 were used for the transport experiments.
3
[ H]Rosiglitazone transport
For the transport experiments, the medium was removed and HBMECs were washed with serum-free CS-C medium (Cell Systems). HBMECs were preincubated with or without PSC833 (10 ␮m) and cyclosporin A (CsA; 10 ␮m) (Sigma) for 30 min. To initiate the transport
experiments, [3H]rosiglitazone and [14C]sucrose (2 ␮Ci/ml, approximately 1.8 ⫻ 106 cpm/ml) with or without the P-gp inhibitors PSC833
(10 ␮m) and CsA (10 ␮m) were loaded on the luminal (100 ␮l) or
abluminal chamber (600 ␮l). The side opposite to that to which the
labeled materials were loaded is the collecting chamber. Samples were
removed from the collecting chamber at 5, 10, 20, and 60 min and
immediately replaced with an equal volume of fresh serum-free medium. The sampling volume from the luminal and abluminal chamber
was 50 and 300 ␮l, respectively. Samples were mixed with 6 ml scintillation cocktail (Bio-Safe II; Research Products International Corp.,
Mount Prospect, IL), and radioactivity was determined in a liquid scintillation counter. The permeability coefficient and clearance of [3H]rosiglitazone and [14C]sucrose was calculated as previously described (29).
Clearance was expressed as microliters of radioactive tracer diffusing
from the luminal to abluminal (influx) chamber or from the abluminal
to luminal (efflux) chamber and was calculated from the initial level of
radioactivity in the loading chamber and final level of radioactivity in
the collecting chamber: clearance (microliters) ⫽ [C]C ⫻ VC/[C]L, where
[C]L is the initial radioactivity in 1 ␮l of loading chamber (in cpm per
microliter), [C]C is the radioactivity in 1 ␮l of collecting chamber (in cpm
per microliter), and VC is the volume of collecting chamber (in microliters). During a 60-min period of the experiment, the clearance volume
increased linearly with time. The volume cleared was plotted vs. time,
and the slope was estimated by linear regression analysis. The slope of
clearance curves for the HBMEC monolayer plus Transwell membrane
was denoted by PSapp, where PS is the permeability ⫻ surface area
product (in microliters per minute). The slope of the clearance curve with
a Transwell membrane without HBMECs was denoted by PSmembrane.
The real PS value for the HBMEC monolayer (PSe) was calculated from
1/PSapp ⫽ 1/PSmembrane ⫹ 1/PSe. The PSe values were divided by the
surface area of the Transwell inserts (0.33 cm2) to generate the endothelial permeability coefficient (Pe, in centimeters per minute).
Serum determinations
Plasma glucose concentrations were measured by the glucose oxidase
method with the YSI 2300 STAT plus glucose analyzer. Plasma insulin,
leptin, adiponectin (Linco Research, St. Charles, MO), total and free T3
and T4 (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA),
TSH (Biotrak rat TSH; Amersham Biosciences, Oakville, Ontario, Canada) and ACTH (Somagen Diagnostic, Edmonton, Alberta, Canada)
were determined by RIA. Plasma TAG and nonesterified fatty acid levels
were measured by enzymatic methods (Roche Diagnostics, Montreal,
Quebec, Canada, and Wako Chemicals, Richmond, VA, respectively).
Statistical analysis
Results are expressed as means ⫾ sem. Simple effects of rosiglitazone
treatment were analyzed by Student’s unpaired t test. When appropriate, factorial ANOVA followed by either Newman-Keuls’ multiple
range test or Dunnett’s test or Tukey-Kramer’s was used for multiple
comparisons. P ⬍ 0.05 was taken as the threshold of significance.
Results
Final body weight and body weight gain were significantly increased (8 and 45%, respectively) by 7 d of rosiglitazone treatment (Table 1). The higher body weight of rosiglitazone-treated rats was associated with increases in food
intake (10%), feed efficiency (31%), and mass of brown and
white adipose depots (BAT, 116%; ING, 25%; RETRO, 25%).
In addition, rosiglitazone induced a small, but significant
decrease in the rates of O2 consumption and CO2 production
(⫺7%), despite markedly increasing SMA (60%). Confirming
its beneficial effects on insulin sensitivity and lipemia, rosiglitazone significantly reduced fasting plasma levels of insulin (⫺35%), nonesterified fatty acids (⫺60%), and TAG
(⫺45%) but did not alter fasting glycemia. Rosiglitazone also
significantly decreased plasma ACTH levels (⫺42%), left
leptin levels unchanged, and increased those of adiponectin
4-fold.
The effects of rosiglitazone on BAT and WAT NE content,
fractional turnover rates, and NETO are presented in Table
2. One week of rosiglitazone markedly reduced sympathetic
TABLE 2. Effect of rosiglitazone (RSG) treatment on NE content, fractional turnover rates (k), and NETO rates in rat BAT, RETRO, and
ING adipose depots
NE content (ng)a
BAT
Control
RSG
RETRO
Control
RSG
ING
Control
RSG
Data are means ⫾ SEM of 12 rats.
a
BAT, ng/tissue; WAT, ng/pad.
b
P ⬍ 0.05 vs. control.
k (%/h)
NETO (ng NE/tissue䡠h)
314 ⫾ 21
296 ⫾ 20
9.2 ⫾ 0.8
4.0 ⫾ 0.6b
29 ⫾ 3
12 ⫾ 2b
75 ⫾ 2
72 ⫾ 3
11.5 ⫾ 1.2
4.6 ⫾ 0.5b
8.6 ⫾ 1.0
3.3 ⫾ 0.3b
6.6 ⫾ 0.7
2.8 ⫾ 0.4b
8.2 ⫾ 1.6
3.9 ⫾ 0.5b
124 ⫾ 10
140 ⫾ 16.3
Festuccia et al. • PPAR␥ Activation and Energy Balance
activity to BAT and WAT as evidenced by the lower fractional turnover rates and NETO to interscapular BAT (⫺56
and ⫺58%, respectively), RETRO (⫺60 and ⫺62%), and ING
(⫺57 and ⫺53%) compared with untreated rats.
Because thyroid hormones exert an important role in the
amplification of sympathetically mediated activation of BAT
and WAT lipolysis and thermogenesis, we next investigated
the effects of rosiglitazone on the hypothalamic-pituitarythyroid axis and on the mRNA levels of the deiodinases and
THR isoforms. Rosiglitazone did not affect hypothalamic
TRH mRNA (Fig. 1A) or plasma TSH levels (Fig. 1B) but
significantly reduced plasma total and free T4 (⫺26 and
⫺32%, respectively; Fig. 1, C and D) and total T3 (⫺15%, Fig.
1E), whereas free T3 remained unaffected (Fig. 1F). In BAT,
rosiglitazone reduced the potential for peripheral conversion
of T4 to the biologically active T3 by decreasing BAT D2
(⫺40%, Fig. 2A). Rosiglitazone also significantly reduced
BAT THR␤ (⫺66%) and -␣1 (⫺34%) mRNA levels, without
affecting those of THR␣2. In the liver, where rosiglitazone
significantly reduced D1 mRNA levels (⫺32%, Fig. 2E), no
effect of the agonist was found on any of the three thyroid
hormone isoforms (Fig. 2, F–H). In RETRO, rosiglitazone
significantly reduced the mRNA levels of THR␣1 (⫺20%, Fig.
2J) and -␣2 (⫺47%, Fig. 2L), without affecting those of THR␤
(Fig. 2I).
The effects of rosiglitazone on BAT and WAT morphology
and UCP1 expression are depicted in Fig. 3. Noteworthy, the
rosiglitazone-induced increase in BAT UCP1 mRNA levels
(Fig. 3A) was associated with a marked increase in tissue
FIG. 1. Effect of a 7-d rosiglitazone (RSG) treatment on hypothalamic-pituitary-thyroid axis: hypothalamic TRH mRNA levels (A),
plasma TSH (B), total (C) and free T4 (D), and total (E) and free T3
(F). *, P ⬍ 0.05 vs. control rats.
Endocrinology, May 2008, 149(5):2121–2130
2125
TAG content (66%, Fig. 3B) and the percentage of unilocular
adipocytes (66%, Fig. 3, C and D). In RETRO, the rosiglitazone-induced increase in UCP1 mRNA levels (Fig. 3E) was
associated with an increase in DNA content (Fig. 3F) and a
reduction in adipocyte cell diameter (Fig. 3, G and H).
To investigate the central mechanisms by which PPAR␥
activation reduces sympathetic activity to WAT and BAT and
thyroid status, we assessed the effect of 1 wk of rosiglitazone
treatment on the hypothalamic mRNA levels of several neuropeptides involved in the control of energy homeostasis. As
depicted in Fig. 4A, rosiglitazone significantly reduced
whole hypothalamic mRNA levels of CART (⫺40%) and
CRH (⫺25%), without altering the mRNA levels of agoutirelated protein (AGRP), proopiomelanocortin (POMC),
melanocortin receptors 3 and 4 (MC3/4R), or neuropeptide
Y (NPY). By using in situ hybridization, we confirmed the
reductions in CART and CRH mRNA levels found in whole
hypothalamic extracts and localized them to specific hypothalamic nuclei. CART mRNA levels were significantly reduced in the arcuate region (ARC, Fig. 4B), but not in the
paraventricular, dorsomedial or lateral hypothalamus (data
not shown). CRH mRNA levels were more modestly, but
significantly reduced in the parvocellular subregion of the
hypothalamic paraventricular nucleus (pPVN, Fig. 4C).
To test the hypothesis that rosiglitazone affects sympathetic activity to BAT and WAT and energy balance by acting
directly in the brain, we first investigated the ability of rosiglitazone to cross the blood-brain barrier. Figure 5A shows
the relationship between the brain/serum ratio and time
after iv injection of [3H]rosiglitazone with or without unlabeled rosiglitazone. The brain/serum ratio exceeded the typical 8- to 12-␮l vascular space of the brain, indicating uptake
of [3H]rosiglitazone. There was no increase in the brain/
serum ratio over time, however, indicating that an equilibrium between brain and blood was rapidly reached. Unlabeled rosiglitazone paradoxically increased early brain
uptake. Figure 5B shows the percentage of the iv-injected
dose that was taken up per unit weight of brain (% Inj/g)
after correction for brain vascular space. The peak value for
[3H]rosiglitazone was 0.045% Inj/g and occurred 4 min after
injection. Coinjection of unlabeled rosiglitazone produced an
earlier and higher peak (0.134% Inj/g at 1 min) than labeled
rosiglitazone alone. This again indicated a paradoxical increase in blood-to-brain uptake of [3H]rosiglitazone when
coadministered with unlabeled rosiglitazone. Clearance of
[3H]rosiglitazone from blood was minimally affected by unlabeled rosiglitazone (Fig. 5B, inset). Because the presence of
a saturable brain-to-blood transporter is a classic explanation
for the paradoxical increase in tracer uptake in the presence
of unlabeled tracer observed here, the ability of the bloodbrain barrier to transport rosiglitazone out of the brain was
also assessed. Figure 5C shows that 77% of icv-injected
[3H]rosiglitazone was transported out of the brain within 10
min. Unlabeled rosiglitazone significantly inhibited transport. Figure 5D shows that the P-gp inhibitor PSC833 nearly
abolished brain-to-blood transport of [3H]rosiglitazone, thus
identifying P-gp as the brain-to-blood transporter of rosiglitazone. The presence of a brain-to-blood transport of rosiglitazone was also evaluated in a human endothelial model
of blood-brain barrier. Figure 5E shows the polarity of
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Endocrinology, May 2008, 149(5):2121–2130
Festuccia et al. • PPAR␥ Activation and Energy Balance
FIG. 2. Effect of a 7-day rosiglitazone (RSG)
treatment on BAT and liver mRNA levels of D2
and D1 (A and E, respectively) and on BAT, liver,
and RETRO mRNA levels of THR␤ (B, F, and I,
respectively), -␣1 (C, G, and J, respectively), and
-␣2 (D, H, and L, respectively). *, P ⬍ 0.05 vs.
control rats.
[3H]rosiglitazone transport across the HBMEC monolayer.
The permeability coefficient for brain-to-blood (efflux) transport of [3H]rosiglitazone was 2-fold higher than that for
blood-to-brain (influx). In contrast, [14C]sucrose, a paracellular permeability marker with a molecular weight similar to
that of rosiglitazone, did not display polarity of transport. As
shown in Fig. 5F, P-gp inhibitors reduced (17–20%) the permeability coefficient for [3H]rosiglitazone, confirming the
finding in mice that P-gp contributes to outflow of rosiglitazone from the brain.
Because a small amount of rosiglitazone did enter the
brain, its central action on energy balance was further investigated by centrally administering the agonist. As shown
in Table 3, 1 wk of icv infusion of rosiglitazone at two different doses did not affect body weight gain, food intake,
RETRO or BAT mass, or BAT mRNA levels of the sympathetically up-regulated genes FATP-1, D2, and UCP1.
Discussion
To address the question of why the PPAR␥-mediated upregulation of lipolytic, oxidative, and thermogenic machineries are not associated with a proportional increase in their
respective biological processes in vivo, we investigated the
impact of the thiazolidinedione rosiglitazone on the major
activators of BAT and WAT thermogenesis and lipolysis,
namely the sympathetic nervous system and thyroid hormones. Chronic rosiglitazone treatment markedly reduced
sympathetic activity to BAT and WAT, the thyroid status
(plasma thyroid hormones and tissue mRNA levels of THR
and deiodinases) and hypothalamic mRNA levels of the proenergy expenditure peptides CART and CRH. The study also
suggests that, although a small amount of rosiglitazone is
able to enter the brain, its effects on the sympathetic and
thyroid axes are not mediated by a direct action in the central
nervous system.
The study confirms that the well-established positive actions of rosiglitazone on insulin sensitivity and lipemia occur
despite increased body weight gain and adiposity (9, 10).
Although such positive energy balance can partially be ascribed to the increase in food intake, the marked increase in
feed efficiency shown here and previously by us (4, 8) and
others (9, 10) strongly suggests that in vivo PPAR␥ decreases
energy expenditure. Confirming this notion, rosiglitazone
induced a small (7%) but significant reduction in O2 consumption. In fact, this represents an underestimation of such
metabolic adaptation given the obligatory increase in energy
expenditure related to higher food intake and increased motor activity elicited by rosiglitazone.
One germane aspect of the anabolic state induced by in vivo
PPAR␥ activation is that it occurs simultaneously with an
up-regulation of lipolytic, oxidative, and thermogenic proteins in WAT and BAT, which are functional, as evidenced
in vitro in basal and stimulated conditions (5, 6) and in vivo
under chronic ␤3-adrenergic stimulation (13). Supporting the
hypothesis of a silencing of the thermogenic activity by the
in vivo neurohormonal conditions, rosiglitazone markedly
decreased sympathetic activity to BAT and WAT and plasma
thyroid hormone levels. That the latter occurred without
change in TSH or TRH suggests alternative modulation of
thyroid hormone secretion, the nature of which remains to be
determined. Importantly, rosiglitazone reduced mRNA levels of liver D1, a sensitive marker of peripheral thyroid status
in rodents (30), and BAT D2, which generates biologically
active T3 from T4. BAT D2 is essential for thermogenesis due
Festuccia et al. • PPAR␥ Activation and Energy Balance
Endocrinology, May 2008, 149(5):2121–2130
2127
FIG. 4. Effect of a 7-d rosiglitazone treatment on whole hypothalamic
neuropeptide relative mRNA levels and on arcuate CART and paraventricular CRH mRNA levels measured by quantitative PCR (A) and
by in situ hybridization (B and C). In A, control and RSG hypothalamic
neuropeptide mRNA levels were normalized for the housekeeping
GAPDH. Control values were transformed to 1 (horizontal line). In B
and C, OD values of the hybridization signal of CRH and CART mRNA
are presented together with representative photographs of the in situ
hybridization. *, P ⬍ 0.05 vs. control rats; n ⫽ 6.
FIG. 3. Effect of a 7-d rosiglitazone (RSG) treatment on BAT UCP1
mRNA levels (A), TAG content (B), and percentage of unilocular
adipocytes (C) and on RETRO WAT UCP1 mRNA levels (E), DNA
content (F), and adipocyte diameter (G). D and H are representative
photomicrographs of BAT and WAT sections, respectively, dyed with
hematoxylin/eosin. *, P ⬍ 0.05 vs. control rats.
to the important role of locally produced T3 in the amplification of BAT adrenergic signaling and in the direct activation of UCP1 expression (16, 31). Sympathetic regulation of
D2 is well established, and the reduction in its mRNA levels
seen here is more likely due to rosiglitazone-induced inhibition of BAT sympathetic activity than to a direct PPAR␥
action on its promoter (32). In addition to reducing BAT local
T3 production, rosiglitazone inhibited the expression of
THR␣1 and THR␤, which mediate the T3 effects on amplification of adrenergic signaling and on direct activation of
UCP1 expression, respectively (33). Similarly to BAT, rosiglitazone also reduced RETRO mRNA levels of THR␣1,
which mediates in WAT the stimulatory effects of circulating
T3 on adrenergic responsiveness and lipolysis (34). It can
therefore be suggested that the reduction in sympathetic
activity and thyroid status accounts for the dissociation between gene expression and biological processes observed in
vivo in rosiglitazone-treated rats.
The robust inhibitory effect of rosiglitazone on sympathetic activity and thyroid status has important functional
consequences on both BAT and WAT. Under PPAR␥ activation, BAT shifts from a thermogenic toward a lipid storage
function, as evidenced here by the marked increase in tissue
TAG content and number of unilocular brown adipocytes,
and by the strong stimulatory action of rosiglitazone on
uptake of circulating lipids by BAT (35). This shift in BAT
function occurs in the presence of high levels of UCP1, universally considered as a marker of BAT sympathetic activation and thermogenesis. This dissociation between BAT thermogenic capacity (up-regulation of thermogenic proteins)
and functional activity, however, is not without precedent. In
Syrian hamsters either exposed to a short photoperiod or fed
a high-energy diet (36, 37) and in cold-acclimated rats reintroduced to a warm environment (38), abundant UCP1 does
not result in increased thermogenesis because of the absence
of sympathetic activation. This is perhaps related to inadequate provision of lipolytic products (fatty acids) for thermogenesis in the absence of sympathetic activity and thyroid
hormones, as strongly suggested by defective cold adaptation in adipose triglyceride lipase-deficient mice, which display reduced lipolysis (39). The same concept can also be
applied to the PPAR␥ agonist-induced increase in the lipolytic machinery of WAT (8), which, likely because of reduced
sympathetic activity and thyroid status, and insulin sensitization, does not result in increased fatty acid release in vivo
(40). Furthermore, inhibition of sympathetic activity in WAT
2128
Endocrinology, May 2008, 149(5):2121–2130
FIG. 5. A, Murine brain/serum ratios of [3H]rosiglitazone (3H-RSG);
addition of unlabeled rosiglitazone (⫹RSG) paradoxically increased
brain uptake of [3H]rosiglitazone immediately after their iv injection.
B, Percentage of the iv injected dose of [3H]rosiglitazone taken up per
gram of brain; unlabeled RSG did not affect clearance from blood
(inset) but confirmed that it did increase brain uptake. C, In vivo
inhibition of brain-to-blood transport of [3H]rosiglitazone by unlabeled rosiglitazone; this confirms that rosiglitazone is effluxed from
brain by a saturable transporter. D, Inhibition of [3H]rosiglitazone
efflux by the P-gp inhibitor PSC833; this confirms that the efflux
system for rosiglitazone is P-gp. E, In vitro [3H]rosiglitazone transport
across HBMEC monolayer model of the blood-brain barrier; influx
and especially efflux of [3H]rosiglitazone exceeds that of the poorly
permeable control, sucrose. F, In vitro model efflux is inhibited by
P-gp inhibitors PSC833 and CsA. *, P ⬍ 0.05 vs. [3H]rosiglitazone (C,
D, and F) or influx (E); n ⫽ 3– 4.
might well be involved in the rosiglitazone-induced increase
in DNA content and reduction in adipocyte diameter observed here; both are indicative of cell proliferation, a process
that is strongly inhibited by the sympathetic drive to WAT
(41, 42).
To gain further insight into the mechanisms by which
PPAR␥ activation reduces sympathetic activity to rat adipose tissues, we screened the hypothalamus for changes
in the expression of several neuropeptides known to affect
whole-body energy balance. Rosiglitazone treatment was
associated with significant reductions in hypothalamic
CRH and CART mRNA levels that were further localized
to the pPVN and the ARC, respectively. Although hypothalamic CRH is related to changes in BAT sympathetic
outflow (43), our finding that CRH was mainly reduced in
neurons of the pPVN is not consistent with its participation in the rosiglitazone effects on sympathetic activity.
Festuccia et al. • PPAR␥ Activation and Energy Balance
These neurons indeed mainly control pituitary ACTH secretion (44), as confirmed here by lower plasma ACTH
levels, which further translate into lower adrenal weight
(45), an index of long-term hypothalamic-pituitary-adrenal axis activity, in rosiglitazone-treated rats compared
with controls. In contrast to parvocellular CRH, arcuate
CART-expressing neurons are anatomically linked to BAT
sympathetic nerves (46). Adenovirus-mediated CART
overexpression and peptide injection in the ARC increase
BAT sympathetic outflow (47), further supporting the possible involvement of arcuate CART neurons in the rosiglitazone-induced inhibition of the sympathetic drive to
BAT.
Because a variety of signals can impact hypothalamic neuropeptide expression, we tested the hypothesis of whether
rosiglitazone could cross the blood-brain barrier to act directly in the brain. We found that about 0.045% of an iv
injected dose of rosiglitazone was taken up per gram of brain.
However, the in vivo mouse studies showed a paradoxical
increase in the uptake of [3H]rosiglitazone when unlabeled
rosiglitazone was included in the injection, suggestive that a
brain-to-blood efflux system pumps rosiglitazone out of the
brain (48). We confirmed in both an in vivo mouse model and
in an in vitro human brain endothelial cell model using the
specific inhibitor PSC833 (49) that rosiglitazone is a substrate
for the brain-to-blood efflux system P-gp.
The modest uptake of rosiglitazone from blood and its
rapid, transporter-mediated efflux from the brain are not
supportive of a direct effect of the agonist on the central
nervous system. Although transport kinetics may conceivably be modified with long-term treatment, a lack of direct
central action is confirmed by the absence of change in food
intake and energy expenditure after 1 wk of icv infusion of
rosiglitazone. This suggests instead that rosiglitazone affects
food intake, sympathetic activity, locomotor activity, and
energy balance by eliciting a peripheral signal that then
reaches the brain. Although possible mediators are speculative at present, adiponectin, the plasma levels of which are
markedly increased by PPAR␥ activation as confirmed here,
constitutes an attractive candidate. Although still debated,
recent evidence suggests an involvement of adiponectin in
energy balance. Adiponectin overexpression results in
marked positive energy balance even on an obese ob/ob
background (50), whereas mice lacking adiponectin display
lower body weight gain than their wild-type counterparts in
response to a PPAR␥ agonist (51). Furthermore, similarly to
rosiglitazone, adiponectin has recently been shown to increase food intake, to reduce both energy expenditure (52)
and renal sympathetic activity (53), and to increase locomotion (54). Noteworthy, these central adiponectin effects seem
to at least partly take place in the ARC (52), in which CART
expression was decreased by rosiglitazone in the present
study. Whether adiponectin mediates the effects of rosiglitazone on the central regulation of energy balance awaits
direct demonstration.
In conclusion, this study presents strong evidence suggesting that the increased energy efficiency and energy storage associated with PPAR␥ activation in rodents are mediated, at least in part, by a down-regulation of sympathetic
activity to BAT and WAT and thyroid status, likely a con-
Festuccia et al. • PPAR␥ Activation and Energy Balance
Endocrinology, May 2008, 149(5):2121–2130
2129
TABLE 3. Effect of a 7-d icv vehicle or rosiglitazone (RSG) infusion on body weight gain, daily food intake, RETRO adipose tissue and
BAT mass, and BAT FATP-1, D2, and UCP1 mRNA levels
Body weight gain (g)
Food intake (g/d)
RETRO mass (g)
BAT mass (g)
BAT FATP-1/L27 mRNA
BAT D2/L27 mRNA
BAT UCP1/L27 mRNA
Data are means ⫾
SEM
Vehicle
RSG (50 ␮g/ml)
RSG (500 ␮g/ml)
54 ⫾ 1
25.0 ⫾ 0.8
1.37 ⫾ 0.04
0.23 ⫾ 0.03
2.2 ⫾ 0.3
2.0 ⫾ 0.3
4.0 ⫾ 0.6
56 ⫾ 2
25.8 ⫾ 1.9
1.35 ⫾ 0.12
0.24 ⫾ 0.02
2.5 ⫾ 0.3
1.9 ⫾ 0.1
4.0 ⫾ 0.4
52 ⫾ 0.3
25.4 ⫾ 0.8
1.32 ⫾ 0.02
0.25 ⫾ 0.02
2.2 ⫾ 0.3
2.3 ⫾ 0.2
4.0 ⫾ 0.6
of four rats.
sequence of an indirect (peripheral) action of PPAR␥ activation on central modulators of energy balance.
11.
Acknowledgments
We are very grateful for the invaluable professional assistance of
Mélanie Alain, Sébastien Poulin, Josée Lalonde, Yves Gélinas, Pierre
Samson, and Emily Kelso.
Received November 12, 2007. Accepted January 14, 2008.
Address all correspondence and requests for reprints to: Dr. Yves Deshaies,
Faculty of Medicine, Laval University, Laval Hospital Research Centre, Laval
Hospital–d’Youville Y3110, 2725 Chemin Sainte-Foy, Quebec, Canada G1V
4G5. E-mail: [email protected].
This work was supported by a grant from the Canadian Institutes of
Health Research (CIHR) to Y.D. and Veterans Affairs Merit Review and
R010513334 to W.A.B. W.T.F. and R.G.D. were recipients of a Postdoctoral Fellowship from the CIHR-funded Obesity Research Training Program led by the Laval Hospital Research Center. M.L. and M.B. held
studentships from the Natural Sciences and Engineering Research Council of Canada and the CIHR-funded Obesity Research Training Program,
respectively.
This manuscript is dedicated to the memory of Dr. Renato Hélios
Migliorini.
Disclosure Statement: The authors have nothing to disclose.
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