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• W2003157316 abstract "Peroxisome proliferator-activated receptor γ (PPARγ) is an essential regulator of adipocyte differentiation, maintenance, and survival. Deregulations of its functions are associated with metabolic diseases. We show here that deletion of one PPARγ allele not only affected lipid storage but, more surprisingly, also the expression of genes involved in glucose uptake and utilization, the pentose phosphate pathway, fatty acid synthesis, lipolysis, and glycerol export as well as in IR/IGF-1 signaling. These deregulations led to reduced circulating adiponectin levels and an energy crisis in the WAT, reflected in a decrease to nearly half of its intracellular ATP content. In addition, there was a decrease in the metabolic rate and physical activity of the PPARγ+/- mice, which was abolished by thiazolidinedione treatment, thereby linking regulation of the metabolic rate and physical activity to PPARγ. It is likely that the PPARγ+/- phenotype was due to the observed WAT dysfunction, since the gene expression profiles associated with metabolic pathways were not affected either in the liver or the skeletal muscle. These findings highlight novel roles of PPARγ in the adipose tissue and underscore the multifaceted action of this receptor in the functional fine tuning of a tissue that is crucial for maintaining the organism in good health. Peroxisome proliferator-activated receptor γ (PPARγ) is an essential regulator of adipocyte differentiation, maintenance, and survival. Deregulations of its functions are associated with metabolic diseases. We show here that deletion of one PPARγ allele not only affected lipid storage but, more surprisingly, also the expression of genes involved in glucose uptake and utilization, the pentose phosphate pathway, fatty acid synthesis, lipolysis, and glycerol export as well as in IR/IGF-1 signaling. These deregulations led to reduced circulating adiponectin levels and an energy crisis in the WAT, reflected in a decrease to nearly half of its intracellular ATP content. In addition, there was a decrease in the metabolic rate and physical activity of the PPARγ+/- mice, which was abolished by thiazolidinedione treatment, thereby linking regulation of the metabolic rate and physical activity to PPARγ. It is likely that the PPARγ+/- phenotype was due to the observed WAT dysfunction, since the gene expression profiles associated with metabolic pathways were not affected either in the liver or the skeletal muscle. These findings highlight novel roles of PPARγ in the adipose tissue and underscore the multifaceted action of this receptor in the functional fine tuning of a tissue that is crucial for maintaining the organism in good health. White adipose tissue (WAT) 2The abbreviations used are: WAT, white adipose tissue; FFA, free fatty acid; HFD, high fat diet; IGF-1, insulin growth factor 1; WT, wild type; PPARγ, peroxisome proliferator-activated receptor γ; RER, respiratory exchange ratio; SD, standard diet; TG, triglyceride(s); qRT-PCR, quantitative real-time PCR. 2The abbreviations used are: WAT, white adipose tissue; FFA, free fatty acid; HFD, high fat diet; IGF-1, insulin growth factor 1; WT, wild type; PPARγ, peroxisome proliferator-activated receptor γ; RER, respiratory exchange ratio; SD, standard diet; TG, triglyceride(s); qRT-PCR, quantitative real-time PCR. plays a dual role in regulating energy homeostasis (1Cinti S. Prostaglandins Leukot. Essent. Fatty Acids. 2005; 73: 9-15Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). First, it is a tissue that responds to nutrient intake by storing excess energy in the form of triglycerides (TG) and to metabolic demands associated with fasting or exercise by releasing the stored TG as free fatty acids (FFAs) and glycerol (2Mandrup S. Lane M.D. J. Biol. Chem. 1997; 272: 5367-5370Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Second, WAT is an endocrine organ in addition to its energy reserve functions. In fact, it integrates metabolic signals and secretes molecules, called adipokines, which in turn impact on multiple target organs, such as the liver, muscle, or brain. Therefore, it contributes significantly to the control of whole body energy homeostasis (3Juge-Aubry C.E. Somm E. Giusti V. Pernin A. Chicheportiche R. Verdumo C. Rohner-Jeanrenaud F. Burger D. Dayer J.M. Meier C.A. Diabetes. 2003; 52: 1104-1110Crossref PubMed Scopus (243) Google Scholar, 4Fruhbeck G. Gomez-Ambrosi J. Muruzabal F.J. Burrell M.A. Am. J. Physiol. 2001; 280: E827-E847Crossref PubMed Google Scholar, 5Fried S.K. Bunkin D.A. Greenberg A.S. J. Clin. Endocrinol. Metab. 1998; 83: 847-850Crossref PubMed Scopus (1410) Google Scholar).Deregulation of WAT functions in obesity or lipodystrophy is often linked to metabolic disorders, such as dyslipidemia, atherosclerosis, hypertension, insulin resistance, glucose intolerance, and prothrombotic and proinflammatory states (6Simha V. Garg A. Curr. Opin. Lipidol. 2006; 17: 162-169Crossref PubMed Scopus (92) Google Scholar, 7Jan V. Cervera P. Maachi M. Baudrimont M. Kim M. Vidal H. Girard P.M. Levan P. Rozenbaum W. Lombes A. Capeau J. Bastard J.P. Antivir. Ther. 2004; 9: 555-564PubMed Google Scholar, 8Domingo P. Matias-Guiu X. Pujol R.M. Francia E. Lagarda E. Sambeat M.A. Vazquez G. Aids. 1999; 13: 2261-2267Crossref PubMed Scopus (207) Google Scholar, 9Koutnikova H. Cock T.A. Watanabe M. Houten S.M. Champy M.F. Dierich A. Auwerx J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14457-14462Crossref PubMed Scopus (159) Google Scholar, 10Chao L. Marcus-Samuels B. Mason M.M. Moitra J. Vinson C. Arioglu E. Gavrilova O. Reitman M.L. J. Clin. Invest. 2000; 106: 1221-1228Crossref PubMed Scopus (335) Google Scholar). Thus, WAT functional integrity is required for the balanced body metabolism of a healthy organism.PPARγ (NR1C3) is highly expressed in the WAT, where it plays an important role in adipogenesis and in lipid metabolism (11Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2707) Google Scholar, 12Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3091) Google Scholar, 13Chawla A. Schwarz E.J. Dimaculangan D.D. Lazar M.A. Endocrinology. 1994; 135: 798-800Crossref PubMed Scopus (617) Google Scholar). Suppression of PPARγ expression in preadipocytes impairs their differentiation (14Rosen E.D. Sarraf P. Troy A.E. Bradwin G. Moore K. Milstone D.S. Spiegelman B.M. Mortensen R.M. Mol. Cell. 1999; 4: 611-617Abstract Full Text Full Text PDF PubMed Scopus (1626) Google Scholar, 15Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar). Furthermore, specific deletion of PPARγ in mature adipocytes causes their death, accompanied by macrophage infiltration in the affected WAT (16Imai T. Takakuwa R. Marchand S. Dentz E. Bornert J.M. Messaddeq N. Wendling O. Mark M. Desvergne B. Wahli W. Chambon P. Metzger D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4543-4547Crossref PubMed Scopus (306) Google Scholar). In humans, heterozygous PPARγ mutations are responsible for partial lipodystrophy, severe insulin resistance, steatosis, and hypertension (17Agarwal A.K. Garg A. J. Clin. Endocrinol. Metab. 2002; 87: 408-411Crossref PubMed Scopus (245) Google Scholar, 18Agostini M. Gurnell M. Savage D.B. Wood E.M. Smith A.G. Rajanayagam O. Garnes K.T. Levinson S.H. Xu H.E. Schwabe J.W. Willson T.M. O'Rahilly S. Chatterjee V.K. Endocrinology. 2004; 145: 1527-1538Crossref PubMed Scopus (54) Google Scholar, 19Clement K. Hercberg S. Passinge B. Galan P. Varroud-Vial M. Shuldiner A.R. Beamer B.A. Charpentier G. Guy-Grand B. Froguel P. Vaisse C. Int. J. Obes. Relat. Metab. Disord. 2000; 24: 391-393Crossref PubMed Scopus (128) Google Scholar, 20Hegele R.A. Anderson C.M. Wang J. Jones D.C. Cao H. Genome Res. 2000; 10: 652-658Crossref PubMed Scopus (90) Google Scholar, 21Hegele R.A. Cao H. Frankowski C. Mathews S.T. Leff T. Diabetes. 2002; 51: 3586-3590Crossref PubMed Scopus (225) Google Scholar). The activation of PPARγ improves insulin sensitivity in both humans and mice. Agonists of PPARγ, such as the thiazolidinedione Pioglitazone, are used clinically and are effective in reducing hyperglycemia, hyperinsulinemia, and hyperlipidemia in patients suffering from type 2 diabetes (22Miyazaki Y. Mahankali A. Matsuda M. Mahankali S. Hardies J. Cusi K. Mandarino L.J. DeFronzo R.A. J. Clin. Endocrinol. Metab. 2002; 87: 2784-2791Crossref PubMed Scopus (532) Google Scholar, 23Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3443) Google Scholar, 24Boden G. Homko C. Mozzoli M. Showe L.C. Nichols C. Cheung P. Diabetes. 2005; 54: 880-885Crossref PubMed Scopus (97) Google Scholar). Together, these facts underline the functions of PPARγ in adipocyte differentiation and survival and underscore its role in WAT integrity and whole body homeostasis.Although the homozygous deletion of PPARγ in a mouse model was shown to be embryonic lethal, the survival of PPARγ-/- mice by inactivation of PPARγ in all tissues except the trophoblasts was successful. These animals suffered from lipodystrophy, insulin resistance, and hypotension (25Duan S.Z. Ivashchenko C.Y. Whitesall S.E. D'Alecy L.G. Duquaine D.C. Brosius F.C. Gonzalez F.J. Vinson C. Pierre M.A. Milstone D.S. Mortensen R.M. J. Clin. Invest. 2007; 117: 812-822Crossref PubMed Scopus (138) Google Scholar). However, deletion of only one PPARγ allele had some intriguing effects (15Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar, 26Miles P.D. Barak Y. He W. Evans R.M. Olefsky J.M. J. Clin. Invest. 2000; 105: 287-292Crossref PubMed Scopus (373) Google Scholar). In fact, PPARγ+/- mice were resistant to obesity induced by a high fat diet (HFD) and, under these conditions, remained more sensitive to insulin then their WT counterparts (27Yamauchi T. Kamon J. Waki H. Murakami K. Motojima K. Komeda K. Ide T. Kubota N. Terauchi Y. Tobe K. Miki H. Tsuchida A. Akanuma Y. Nagai R. Kimura S. Kadowaki T. J. Biol. Chem. 2001; 276: 41245-41254Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar). Decreased PPARγ activity under HFD conditions had a positive outcome on the development of obesity and diabetes. Based on these observations, a novel approach in type 2 diabetes therapy would include the use of PPARγ antagonists, potentially with fewer side effects compared with the present day synthetic agonists (thiazolidinediones).Taking advantage of our PPARγ+/- mouse model, we aimed at understanding how deletion of one allele of PPARγ, which significantly reduces the activity of the receptor via a gene dosage effect, would affect WAT function and whole body metabolism, when the mice are fed with a standard diet (SD), a condition which does not exacerbate the lipid storage function of the WAT. The results reported herein show that deletion of one PPARγ allele affects specifically the expression of genes associated with metabolic pathways in the WAT. In addition to genes involved in lipid storage, genes involved in glycolysis, de novo fatty acid synthesis, and lipolysis were also down-regulated in the PPARγ heterozygous mice, creating a strong energy deficit in these animals. These defects in WAT functions correlated with a lowering of the metabolic rate of the whole body and were accompanied by a reduction in physical activity. These results cast doubt on a potential long term use of PPARγ antagonists for the treatment of type 2 diabetes.EXPERIMENTAL PROCEDURESIn Vivo Animal Study—WT and PPARγ+/- male mice, of a mixed background Sv129/C56BL/6, were maintained at 23 °C on a 12-h light-dark cycle. The animals studied were between 10 and 12 weeks of age. They had free access to water and to an SD, except during fasting, when they had free access to water only, food being withdrawn for 24 h. In some experiments, 5–6-week-old animals were fed with an SD containing 0.004% of Pioglitazone (w/w) for 5 weeks. The Pioglitazone treatment protocol was adapted from Ref. 15Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar, a study that involved PPARγ+/- animals too. Pioglitazone was kindly provided by Takeda Chemical Industries (Switzerland). The standard food pellets containing the Pioglitazone as well as the control pellets were produced by Provimi-Kliba (Switzerland). For analysis, the animals were killed in the morning between 9 and 11 a.m. by cervical dislocation, and tissues were rapidly frozen in liquid nitrogen. The animal experimentation protocols were approved by the Commission de Surveillance de l'Expérimention Animale of the Canton de Vaud (Switzerland).RNA Preparation—The RNA from epidydymal WAT, gastrocnemius skeletal muscle, and liver was extracted from the frozen tissues using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. The RNA for microarray analyses was further purified using Qiagen RNeasy columns (Qiagen). The RNA quality was assessed by capillary electrophoresis on a 2100 Bioanalyzer (Agilent Technologies).Microarray Experiment and Data Processing—To minimize interindividual variation due to the mixed background of the mouse strain, each PPARγ+/- animal had a WT counterpart coming from the same litter.Three independent sets of total RNA samples (three WT and three PPARγ+/- animals) from epidydymal WAT and gastrocnemius skeletal muscle were isolated. cRNA was synthesized from 5 μg of total RNA, according to Ref. 71. After purification using a Qiagen RNeasy column, aliquots of 20 μg of cRNA were fragmented. Each fragmented cRNA (15 μg) was then hybridized to an Affymetrix “Mouse Genome 430 2.0 Array” Gene-Chip microarray. Hybridization, washing, and scanning were according to Affymetrix instructions.Data from the scanned chips were analyzed using the Affymetrix MAS 5.0 software (28Hubbell E. Liu W.M. Mei R. Bioinformatics. 2002; 18: 1585-1592Crossref PubMed Scopus (494) Google Scholar, 29Liu W.M. Mei R. Di X. Ryder T.B. Hubbell E. Dee S. Webster T.A. Harrington C.A. Ho M.H. Baid J. Smeekens S.P. Bioinformatics. 2002; 18: 1593-1599Crossref PubMed Scopus (358) Google Scholar). To identify differentially expressed transcripts, pairwise comparison analyses were carried out. Each experimental sample was compared with each reference sample, resulting in nine pairwise comparisons. Transcripts were considered to be differentially expressed if their levels changed in the same direction in seven of nine comparisons. Further data filtering and analyses were performed with the Genespring (Agilent) and the Ingenuity Pathway Analysis 4.0 software.Quantitative RT-PCR—Single-stranded cDNA templates for quantitative real time (qRT)-PCR analysis were synthesized using Superscript II reverse transcriptase and random priming, starting from the same RNAs used for the microarray analysis, and from additional independent experiments as described above. Amplicons were designed using the Primer Express software (Applied Biosystems), and their sequences were checked by BLAST against the mouse genome to ensure that they were specific for the gene being assayed. The efficiency of each primer pair was tested in a cDNA dilution series. The list of primers is available on demand.Real time PCR was carried out in optical 384-well plates and labeled by using the SYBR green master mix (Applied Biosystems), and the fluorescence was quantified with a 7900HT SDS system (Applied Biosystems). The relative expression level of target genes was normalized according to geNorm, using β-actin, tubulin α2, and hypoxanthine guanine phosphoribosyl-transferase 1 as references to determine the normalization factor (30Vandesompele J. De Preter K. Pattyn F. Poppe B. Van Roy N. De Paepe A. Speleman F. Genome Biol. 2002; 3research0034.1-research0034.11Crossref Google Scholar). Fold changes were calculated from the ratio of means of the normalized quantities and their statistical significance was determined by a paired Student's t test.ATP Level Measurements—Frozen WAT homogenate was transferred into a plastic tube containing 6% HClO4. Following centrifugation, the supernatant was recovered and neutralized with 5.5 m KOH. The ATP concentration was measured with an ATP determination kit, a time-stable assay from Biaffin GmbH&Co KG (Germany). The kit allows quantitative determination of small amounts of ATP by a bioluminescence assay involving the oxidation of the firefly luciferase depending on the ATP present in the extracts. The ATP concentration was derived according to the manufacturer's instructions.Glycerol Level Measurements—The glycerol content was measured with a glycerol measuring kit (Randox). Briefly, the glycerol present in the samples was converted into a colored product measured at a wavelength of 520 nm. The glycerol concentration was then determined according to the manufacturer's instructions.Metabolic Measurements—Metabolic cage studies were conducted in a comprehensive laboratory animal monitoring System (8-chamber CLAMS system; Columbus Instruments, Columbus, OH). The mice were adapted to powdered food for 24 h before they were introduced into the metabolic cages, where a 48-h acclimation preceded the 24-h recording time. Information was collected on the metabolic activity, food intake, water drinking, and physical activity.Blood was collected from the orbital sinus between 9:00 and 11:00 a.m., using heparinized microcapillary tubes and immediately centrifuged. The serum fraction was frozen immediately. Depending on the experiment, the animals were either normally fed or fasted for 24 h.The plasma concentrations of TG, free fatty acids (FFAs), glycerol, and ketone bodies were measured at the Mouse Clinic Institute (ICS; Strasbourg, France) on a Olympus AU-400 automated laboratory work station (Olympus-SA France) using commercial reagents (Olympus Diagnostica GmbH, Lismeehan, Ireland).The plasma leptin and adiponectin concentrations were measured using the mouse leptin enzyme-linked immunosorbent assay kit and the mouse adiponectin enzyme-linked immunosorbent assay (Linco Reserach).The plasma glucose levels were measured with an Accu-Chek Sensor glucometer (Roche Applied Science), and the plasma insulin concentrations were measured with an Ultra mouse insulin enzyme-linked immunosorbent assay kit (Mercodia SA).RESULTSDecreased Metabolic Rate in PPARγ+/-Mice—In PPARγ+/- animals, PPARγ mRNA and protein (PPARγ1 and PPARγ2) levels were reduced by half compared with those of WT mice (31Rieusset J. Seydoux J. Anghel S.I. Escher P. Michalik L. Soon Tan N. Metzger D. Chambon P. Wahli W. Desvergne B. Mol. Endocrinol. 2004; 18: 2363-2377Crossref PubMed Scopus (30) Google Scholar). This prompted us to explore the impact of this reduced PPARγ expression on whole body metabolism in the absence of any excess energy challenge, as is usually done with HFD feeding in assessing the role of PPARγ in lipid storage. Instead, the PPARγ+/- mice were fed with an SD. Metabolic parameters of the PPARγ+/- mice and their WT littermates were determined using metabolic cages. As expected, both mutated and WT animals consumed more O2 and produced more CO2 during the dark cycle, when they are generally more active (Fig. 1A, left). Although the PPARγ+/- mice had a similar weight (Table S1) and ate an equal amount of food (data not shown), they consumed less oxygen and produced less CO2 during both the light and dark cycles when compared with their WT counterparts, a difference reflected in a decrease of 14% in the metabolic rate (heat production) of PPARγ+/- animals (Fig. 1A). This effect was clearly PPARγ-dependent, since a 5-week treatment with SD containing the PPARγ agonist Pioglitazone, at 0.004%, alleviated the metabolic rate difference between the two genotypes (Fig. 1A, right). Moreover, there was a trend, not statistically significant, for increased O2 consumption, CO2 production, and a higher metabolic rate in Pioglitazone-treated PPARγ+/- mice, whereas such a tendency was not observed in WT animals.FIGURE 1Metabolic rate, fuel consumption, and total physical activity in PPARγ+/- and control mice.A, oxygen (O2) consumption, carbon dioxide (CO2) production, and metabolic rate in the PPARγ+/- and control mice. O2 and CO2 were measured by indirect calorimetry and expressed as average VO2 and VCO2/kg of body weight/h during a 24-h monitoring session (light/dark). Metabolic rate (heat) is calculated from the oxygen production and the respiratory exchange ratio (RER) and is expressed as average kcal/h/kg of body weight during a 24-h monitoring session (light/dark). B, the fuel consumption (RER) is the ratio of CO2 produced to the amount of O2 consumed and serves as a guide of the fuel type consumption (carbohydrate (RER = 1.0) or fat (RER = 0.7)). C, the total physical activity was measured as the horizontal and rearing movements during the 24-h monitoring period (total of light and dark movements). The activity is expressed as the average number of times a mouse crosses both the x and y axes at least twice. n = 12 (CTL experiments); n = 5 (Pioglitazone experiments). Values are expressed as mean ± S.E.; *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Deletion of One PPARγ Allele Does Not Affect the Carbohydrate to Lipid Ratio in Metabolic Fuel Utilization—To determine whether the decrease in the metabolic rate of PPARγ+/- animals was associated with alterations in the use of carbohydrates versus lipids as fuel molecules, we calculated the respiratory exchange ratio (RER). The RER (equal to VO2/VCO2) indicates whether lipids (RER = 0.7) or carbohydrates (RER = 1.0) are being oxidized to produce energy. Both genotypes consumed carbohydrates as the main energy source (Fig. 1B, left). This result disagrees with the notion of a metabolic compensation through increased fat oxidation in PPARγ+/- mice (9Koutnikova H. Cock T.A. Watanabe M. Houten S.M. Champy M.F. Dierich A. Auwerx J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14457-14462Crossref PubMed Scopus (159) Google Scholar). Moreover, the Pioglitazone treatment had no significant effect on the choice of fuel type (Fig. 1B, right).Decreased Physical Activity in PPARγ+/-Mice—Since a decreased metabolic rate in the mutated animals may correlate with a change in behavior, we measured their physical activity (horizontal and rearing movements). Interestingly, the PPARγ+/- mice presented a 23% decrease in total activity (Fig. 1C, left). This phenotype correlated with a strong decrease in the plasma adiponectin concentration in PPARγ+/- mice, whereas the leptin level remained unchanged (Figs. 2A and S1). This observation is in agreement with the reduced spontaneous motor activity of transgenic mice overexpressing an antisense adiponectin RNA, resulting in decreased circulating adiponectin levels (32Saito K. Arata S. Hosono T. Sano Y. Takahashi K. Choi-Miura N.H. Nakano Y. Tobe T. Tomita M. Biochim. Biophys. Acta. 2006; 1761: 709-716Crossref PubMed Scopus (30) Google Scholar). The Pioglitazone treatment corrected this decrease in physical activity, suggesting an implication of PPARγ (Fig. 1C, right). In brief, reduced PPARγ levels decreased the metabolic rate and the physical activity of mice without changing their fuel preference.FIGURE 2Plasma profile of PPARγ+/- and control mice.A, plasma adiponectin concentrations; B, plasma insulin concentrations; C, plasma glucose concentrations; D, FFA plasma levels; E, plasma glycerol concentrations; F, plasma ketone body concentrations; G, plasma TG concentrations. A, fed WT, n = 11; fed PPARγ+/-, n = 12. B–G, fed WT, n = 7; fasted WT, n = 11; fed PPARγ+/-, n = 6; fasted PPARγ+/-, n = 6. Values are expressed as mean ± S.E.; *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PPARγ+/-and WT Mice Have Similar Plasma Insulin and Glucose Profiles—Since a decreased metabolic rate might impact on glucose and lipid homeostasis, we analyzed the plasma profile of the WT and PPARγ+/- mice. The plasma insulin concentration was normal in unchallenged animals and was decreased after a 24-h fast as expected, but no difference was observed between WT and mutant mice (Fig. 2B). Moreover, the glycemia was also normal in PPARγ+/- mice, which however had a significantly attenuated response to fasting (Fig. 2C). In fact, the fasting glycemia was higher in the PPARγ+/- mice compared with that of the WT animals. Fasting for 24 h decreased the glucose level by 45% in WT mice, whereas it was decreased by only 30% in the PPARγ+/- animals. Thus, after fasting, PPARγ+/- mice presented a less pronounced hypoglycemia.The Plasma Lipid Profile of PPARγ+/-Mice Reveals an Alteration in Lipolytic Activity—In fed conditions, the plasma FFA concentrations were normal, and no deregulation was observed in PPARγ+/- mice (Fig. 2D). WT animals responded normally to fasting by liberating FFAs from the WAT into the circulation, thus increasing the plasma FFA concentration. Remarkably, no significant increase was observed in the PPARγ+/- mice, suggesting a deregulation of the lipolytic activity of the PPARγ+/- WAT. This defect was confirmed by measuring the circulating glycerol concentration. As for the FFAs, there was no difference in the fed glycerol concentration between WT and PPARγ+/- mice (Fig. 2E). However, the PPARγ+/- mice responded less well to fasting, since they increased their plasma glycerol concentration by only 32%, compared with the 62% monitored in WT mice. Given that the fasting glycerol and FFA concentrations are indicators of the lipolytic activity in the WAT, we concluded that PPARγ+/- mice might have a decreased lipolytic activity. This alteration should also be detectable in the WAT itself, in which the total glycerol (glycerol + glycerol-3-P) originates from glycolysis, glyceroneogenesis, and lipolysis. There was a 23% decrease in total glycerol content of the PPARγ+/- WAT, suggesting that at least one of the three above functions or all of them were impaired (Fig. 3).FIGURE 3Glycerol content in white adipose tissue of fed PPARγ+/- and WT mice. WT, n = 11; PPARγ+/-, n = 12. Values are expressed as mean ± S.E.; *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Reduced circulating FFA levels should have consequences for ketone body synthesis in the liver, which depends on FFA availability. We measured the ketone body concentrations after fasting in both WT and PPARγ+/- mice (Fig. 2F). PPARγ+/- mice were less efficient in producing ketone bodies, since their plasma concentration of this peripheral organ fuel was 33% lower than in WT animals. Thus, this decreased supply in ketone bodies might reflect the reduced availability of FFAs in PPARγ+/- mice. The TG concentrations were increased by 38% after fasting in WT mice, which reflects the recycling to TG-very low density lipoprotein by the liver of a portion of the FFA liberated by the WAT during fasting (Fig. 2G). In agreement with the observations reported above, the plasma TG concentration was not increased in PPARγ+/- mice, in contrast to that measured in WT animals. The reason why fed PPARγ+/- animals also presented reduced ketone body levels remains to be elucidated (Fig. 2F).Based on the results described so far, we hypothesized that the PPARγ+/- mice decreased their metabolic rate and their physical activity to adapt to a diminished energy supply. Three organs, the liver, skeletal muscle, and WAT, are primarily involved in energy supply and consumption. Deletion of one PPARγ allele might have affected the expression pattern of PPARγ target genes. This possibility was tested by assessing the expression of genes involved in metabolic pathways of the three key organs mentioned above by microarray analysis (WAT and skeletal muscle) and/or qRT-PCR (WAT and liver). The expression of genes not represented in the microarray was analyzed by qRT-PCR.The Expression of Metabolic Genes Is Not Affected in the Liver of PPARγ+/-Mice—The liver is one of the major organs responsible for whole body energy balance. PPARγ is expressed at low levels in the liver under normal conditions but is increased in steatosis induced by HFD or other pathophysiological conditions (11Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2707) Google Scholar, 12Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3091) Google Scholar, 13Chawla A. Schwarz E.J" @default.
• W2003157316 created "2016-06-24" @default.
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• W2003157316 date "2007-10-01" @default.
• W2003157316 modified "2023-09-27" @default.
• W2003157316 title "Adipose Tissue Integrity as a Prerequisite for Systemic Energy Balance" @default.
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