Inactivation of UCP1 and the Glycerol Phosphate Cycle Synergistically Increases Energy Expenditure to Resist Diet-induced Obesity
2008; Elsevier BV; Volume: 283; Issue: 41 Linguagem: Inglês
10.1074/jbc.m804268200
ISSN1083-351X
AutoresRea P. Anunciado‐Koza, Jozef Ukropec, Robert A. Koza, Leslie P. Kozak,
Tópico(s)Muscle metabolism and nutrition
ResumoOur current paradigm for obesity assumes that reduced thermogenic capacity increases susceptibility to obesity, whereas enhanced thermogenic capacity protects against obesity. Here we report that elimination of two major thermogenic pathways encoded by the mitochondrial uncoupling protein (Ucp1) and mitochondrial glycerol-3-phosphate dehydrogenase (Gdm) result in mice with increased resistance to diet-induced obesity when housed at 28 °C, provided prior adaptation occurred at 20 °C. Obesity resistant Gdm-/-·Ucp1-/- mice maintained at 28 °C have increased energy expenditure, in part through conversion of white to brown adipocytes in inguinal fat. Increased oxygen consumption in inguinal fat cell suspensions and the up-regulation of genes of mitochondrial function and fat metabolism indicated increased thermogenic activity, despite the absence of UCP1, whereas liver and skeletal muscle showed no changes in gene expression. Additionally, comparisons of energy expenditure in UCP1-deficient and wild type mice fed an obesogenic diet indicates that UCP1-based brown fat-based thermogenesis plays no role in so-called diet-induced thermogenesis. Accordingly, a new paradigm for obesity emerges in which the inactivation of major thermogenic pathways force the induction of alternative pathways that increase metabolic inefficiency. Our current paradigm for obesity assumes that reduced thermogenic capacity increases susceptibility to obesity, whereas enhanced thermogenic capacity protects against obesity. Here we report that elimination of two major thermogenic pathways encoded by the mitochondrial uncoupling protein (Ucp1) and mitochondrial glycerol-3-phosphate dehydrogenase (Gdm) result in mice with increased resistance to diet-induced obesity when housed at 28 °C, provided prior adaptation occurred at 20 °C. Obesity resistant Gdm-/-·Ucp1-/- mice maintained at 28 °C have increased energy expenditure, in part through conversion of white to brown adipocytes in inguinal fat. Increased oxygen consumption in inguinal fat cell suspensions and the up-regulation of genes of mitochondrial function and fat metabolism indicated increased thermogenic activity, despite the absence of UCP1, whereas liver and skeletal muscle showed no changes in gene expression. Additionally, comparisons of energy expenditure in UCP1-deficient and wild type mice fed an obesogenic diet indicates that UCP1-based brown fat-based thermogenesis plays no role in so-called diet-induced thermogenesis. Accordingly, a new paradigm for obesity emerges in which the inactivation of major thermogenic pathways force the induction of alternative pathways that increase metabolic inefficiency. Obesity is a disorder of energy homeostasis that results when energy intake exceeds energy expenditure. This simplistic relationship belies the complexity of the behavioral and physiological mechanisms underlying phenotypic variability among humans in both energy intake and energy expenditure (1Blundell J. Stubbs J. Bray G. Bouchard C. Handbook of Obesity. 2nd Ed. Marcel Dekker, Inc., New York2004: 427-460Google Scholar, 2Schutz Y. Jequier E. Bray G. Bouchard C. Handbook of Obesity. 2nd Ed. Marcel Dekker, Inc., New York2004: 615-629Google Scholar, 3Hill J. Saris W. Levine J. Bray G. Bouchard C. Handbook of Obesity. 2nd Ed. Marcel Dekker, Inc., New York2004: 631-653Google Scholar). A homeostatic model has been developed based on neuroendocrine polypeptides, secreted by peripheral tissues, that act on centers in the hypothalamus to reciprocally regulate feeding behavior and energy expenditure. Although it has been proposed that these neuropeptides act simultaneously to regulate energy intake and expenditure, the thermogenic targets in peripheral tissues are largely unknown (4Lowell B.B. Spiegelman B.M. Nature. 2000; 404: 652-660Crossref PubMed Scopus (1322) Google Scholar, 5Schwartz M.W. Woods S.C. Seeley R.J. Barsh G.S. Baskin D.G. Leibel R.L. Diabetes. 2003; 52: 232-238Crossref PubMed Scopus (310) Google Scholar, 6Levin B.E. Am. J. Physiol. 2007; 293: R988-R991Crossref PubMed Scopus (22) Google Scholar). Indeed, little attention has been given to centrally controlled thermogenic mechanisms that are independent of mitochondrial uncoupling proteins (UCPs), 3The abbreviations used are: UCP, uncoupling protein; DIO, diet-induced obesity; T3, triiodothyronine; T4, thyroxine; PPAR, peroxisome proliferator-activated receptor; WT, wild type; SERCA, sacroendoplasmic reticulum calcium ATPase; RER, respiratory exchange ratio. either UCP1, for which there is strong evidence for a role in rodent models of obesity (7Kozak L.P. Harper M.E. Annu. Rev. Nutr. 2000; 20: 339-363Crossref PubMed Scopus (94) Google Scholar), or the UCP homologues that continue to be proposed for mitochondrial uncoupling functions with little supporting evidence (8Krauss S. Zhang C.Y. Lowell B.B. Nat. Rev. Mol. Cell. Biol. 2005; 6: 248-261Crossref PubMed Scopus (570) Google Scholar, 9Brand M.D. Esteves T.C. Cell Metab. 2005; 2: 85-93Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Given the paucity of evidence for UCP1 activity in adult tissues, there is a pressing need to identify novel mechanisms for activating thermogenesis that can be applied to the obesity problem. The uncoupling of mitochondrial substrate oxidation from ATP synthesis by UCP1 in brown adipose tissue is widely recognized for its role in maintaining a normal body temperature during exposure to cold (10Ricquier D. Kader J.C. Biochem. Biophys. Res. Commun. 1976; 73: 577-583Crossref PubMed Scopus (194) Google Scholar, 11Foster D.O. Frydman M.L. Can. J. Physiol. Pharmacol. 1979; 57: 257-270Crossref PubMed Scopus (398) Google Scholar, 12Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1353) Google Scholar, 13Enerback S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1098) Google Scholar). There is also strong evidence that inducing UCP1-based thermogenesis through pharmacological or genetic manipulation reduces excessive adiposity (14Gambert S. Ricquier D. Curr. Opin. Clin. Nutr. Metab. Care. 2007; 10: 664-670Crossref PubMed Scopus (26) Google Scholar). However, these experimentally induced phenotypes may not be indicative of a normal physiological mechanism for the homeostatic maintenance of energy balance. Indeed, the response of UCP1-deficient mice and wild type mice to a high fat diet is strongly affected by ambient temperature. Mutant and wild type mice maintained near thermoneutrality have the same level of adiposity when fed a high fat diet (15Liu X. Rossmeisl M. McClaine J. Riachi M. Harper M.E. Kozak L.P. J. Clin. Investig. 2003; 111: 399-407Crossref PubMed Scopus (244) Google Scholar). However, as the ambient temperature declines, the resistance of UCP1-deficient mice to diet-induced obesity (DIO) suggests that alternative thermogenic mechanisms are activated/induced to generate sufficient heat to maintain body temperature. Unlike brown fat thermogenesis, these alternative biochemical and physiological mechanisms appear to be less efficient for the production and distribution of heat, requiring expenditure of more calories to maintain body temperature and thereby indirectly reducing adiposity. Inguinal fat depots in UCP1-deficient mice, acclimated to cold, acquire the morphology of brown adipocytes, with gene expression data indicating an enhanced capacity for β-oxidation of fatty acids in mitochondria, and altered patterns of protein expression for phospholambans, regulators of the SERCA activity, suggested that a thermogenic mechanisms based upon Ca2+ cycling may have been induced (16Ukropec J. Anunciado R.P. Ravussin Y. Hulver M.W. Kozak L.P. J. Biol. Chem. 2006; 281: 31894-31908Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). To enhance our ability to detect and evaluate alternative thermogenic mechanisms, we have achieved a further reduction in the endogenous thermogenic capacity of Ucp1 mutant mice by the targeted inactivation of the mitochondrial glycerol-3-phosphate dehydrogenase gene (Gdm) (17Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The glycerol-3-phosphate shuttle has been proposed as a source of metabolic inefficiency, because of the generation of only two instead of three ATPs/mol of NADH generated by glycolysis (18Lardy H. Shrago E. Annu. Rev. Biochem. 1990; 59: 689-710Crossref PubMed Scopus (23) Google Scholar). The protein levels of GDM and the cytoplasmic glycerol-3-phosphate dehydrogenase are much higher in interscapular brown adipose tissue than any other tissue (19Ratner P.L. Fisher M. Burkart D. Cook J.R. Kozak L.P. J. Biol. Chem. 1981; 256: 3576-3579Abstract Full Text PDF PubMed Google Scholar, 20Koza R.A. Kozak U.C. Brown L.J. Leiter E.H. MacDonald M.J. Kozak L.P. Arch. Biochem. Biophys. 1996; 336: 97-104Crossref PubMed Scopus (58) Google Scholar), suggesting that heat production from the glycerol phosphate shuttle is a parallel thermogenic pathway that could independently supplement UCP1 thermogenesis. Here we report that Gdm-/-·Ucp1-/- mice fed a high fat diet are more resistant to dietary obesity than mice lacking either UCP1 or GDM. The resistance of Gdm-/-·Ucp1-/- mice to DIO depends on a prior conditioning at a lower ambient temperature (20 °C). In addition, and most importantly, changes in the inducible thermogenic capacity that reduced DIO were sustained for a period of 10 weeks, even after switching to a higher ambient temperature (28 °C). We propose that in the absence of two recognized heat-generating systems, by being forced to utilize metabolically costly and less efficient alternative thermogenic mechanisms for maintaining body temperature, the Gdm-/-·Ucp1-/- mice become highly resistant to DIO and have increased insulin sensitivity. Animals and Study Design—Gdm-/-·Ucp1-/- mice on a C57BL/6J (B6) genetic background were generated as follows: Gdm-/-·Ucp1+/+ mice were crossed with Gdm+/+·Ucp1-/- to generate the F1 offspring (Gdm+/-·Ucp1+/-) that were intercrossed to generate the F2 mice. The double knock-out (Gdm-Ucp1-/-) mice were fully fertile and maintained by full-sib mating. Only male mice were studied. In the studies conducted, 10–12-week-old mice were single-housed, fed rodent chow (PicoLab Rodent Diet 20; LabDiet, Richmond, IN) ad libitum, and reared at 28 °C ambient temperature. All of the animal experiments were approved by the Pennington Biomedical Research Center Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines for care and use of laboratory animals. Acute Cold Exposure Experiment—Mice previously maintained at 28 °C were exposed to an ambient temperature of 4 °C, and body temperature was measured every 60 min for 3 h with a rectal probe (TH-8; Physitemp Instruments Inc., Clifton, NJ). Cold Adaptation Experiment—Mice maintained at 28 °C for at least 1 week, following a test for acute sensitivity to cold, were then subjected to a slow reduction (2 °C daily) of ambient temperature until the temperature reached 4 °C. The mice were kept at 4 °C for 24–48 h prior to sacrifice. High Fat Diet Experiments—Three studies were conducted in which the mice were fed a high fat diet (D12230; Research Diets Inc., New Brunswick, NJ). In Experiment 1, the mice fed a high fat diet were reared at 20 °C ambient temperature for 10 weeks and then at 28 °C for another 10 weeks. In Experiment 2, the mice fed a high fat diet were reared at 28 °C ambient temperature for 8 weeks. In Experiment 3, the mice fed a high fat diet were reared at 20 °C ambient temperature for 4 weeks and then at 28 °C for another 4 weeks. Phenotypes of Energy Balance—Body composition was analyzed by NMR (Bruker). Oxygen consumption, carbon dioxide production, and physical activity of individual mice were measured in 16-chamber Oxymax lab animal monitoring system, CLAMS (Columbus Instruments, Columbus, OH), housed in a temperature-controlled incubator. Quantitative Reverse Transcription-PCR—Total RNA was prepared from tissues homogenized in TRI reagent (Molecular Research Center Inc., Cincinnati, OH). Genomic DNA was removed from total RNA by treating with RNase-free DNase (Qiagen). RNA was further purified using the RNeasy kit (Qiagen) and protected from RNase degradation by treatment with SUPERase-In (Ambion, Austin, TX). The quality and quantity of the isolated RNA were assessed using a NanoDrop spectrometer (NanoDrop Technologies, Wilmington, DE). Quantitative reverse transcription-PCR was performed using total RNA with specific primers and probes designed using Primer-Express™, version 2.0.0 (Applied Biosystems, Foster City, CA). TaqMan probes were used for quantification of some target genes using the TaqMan one-step reverse transcriptase PCR mastermix (Applied Biosystems). Other target genes were quantified using SYBR® Green PCR mastermix (Applied Biosystems). All of the gene expression data were normalized to the level of cyclophilin B. Primer and probe sequences are available upon request. Metabolite and Hormone Determinations—Glucose tolerance and insulin tolerance tests were performed after overnight fasting using an intraperitoneal injection of 20% glucose solution (2 g/kg of body weight) or insulin (0.5 IU/kg of body weight; Humulin R, Eli Lilly and Company, Indianapolis, IN). Blood glucose levels were determined using a OneTouch Profile blood glucose meter (LifeScan Inc., Milpitas, CA). Blood triglyceride and ketone levels were measured using a Cardiochek PA Professional meter (HealthCheck Systems, Brooklyn, NY). Serum lactic acid levels were measured using a lactate assay kit (Trinity BioTech, Berkeley Heights, NJ). Tissue triglycerides were measured in chloroform/methanol extracts using the L-type TG H kit (Wako Chemicals, Richmond, VA). Total serum triiodothyronine (T3) and thyroxine (T4) levels were measured by enzyme immunoassay (Alpco Diagnostics, Salem, NH). Histology of Inguinal Fat—Inguinal fat pads were fixed in Bouin's solution (Sigma-Aldrich), and paraffin-embedded sections were stained with hematoxylin-eosin and examined using a Zeiss Axioskop 40 microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY). Tissue Oxygen Consumption—Oxygen consumption in inguinal adipose tissue was measured with a Clark-type polarographic oxygen sensor (Oxygraph, Hansatech Instruments, Norfolk, UK). Freshly dissected inguinal adipose tissue was finely minced in a freshly oxygenated (95% O2, 5% CO2) Krebs-Ringer phosphate buffer (pH 7.4) with 1% fatty acid-free bovine serum albumin. All measurements were performed with minced tissue in bovine serum albumin-free Krebs-Ringer bicarbonate buffer (pH 7.4). Basal respiration was monitored for 1–2 min, after which succinate (5 mm) was added, and respiration was measured for 5–7 min. Tissue oxygen consumption was normalized to DNA content, which was quantified using the fluorescent dye bisbenzimide (Hoechst 33258; Sigma) (21Labarca C. Paigen K. Anal. Biochem. 1980; 102: 344-352Crossref PubMed Scopus (4553) Google Scholar). Statistical Analysis—The data are expressed as the means ± S.E. Unpaired t test was used to compare differences between groups (Statview, version 5.0.1; SAS Institute Inc., Cary, NC). Analysis of variance with Bonferroni post hoc test was used when more than two groups were compared. Gdm-/-·Ucp1-/- Mice Increase Energy Expenditure during Cold Adaptation—To determine whether Gdm-/-·Ucp1-/- mice are able to survive acute cold exposure, wild type and Gdm-/-·Ucp1-/- mice maintained at 28 °C were placed at 4 °C. Wild type mice were fully able to defend their body temperature, whereas 100% of the Gdm-/-·Ucp1-/- became hypothermic, with their body temperature dropping to less than 30 °C within 1–3 h of cold exposure (supplemental Table S1). Cold-sensitive mutant mice were immediately returned to a 28 °C ambient temperature and allowed to recover. However, when the ambient temperature of these recovered Gdm-/-·Ucp1-/- mice was gradually reduced at a rate of 2 °C per day, 80–90% of the mice were able to tolerate an ambient temperature of 4 °C (supplemental Table S1). This ability to adapt to cold ambient temperatures resembles our previous findings with the Ucp1-/- mice. Gdm-/- mice are cold tolerant (17Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), unless they are made hypothyroid (22DosSantos R.A. Alfadda A. Eto K. Kadowaki T. Silva J.E. Endocrinology. 2003; 144: 5469-5479Crossref PubMed Scopus (47) Google Scholar), and the Gdm-/-·Ucp1-/- mice do not appear to have increased cold intolerance compared with Ucp1-/- mice. To determine the mechanism by which Gdm-/-·Ucp1-/- mice are able to survive cold adaptation, we measured energy expenditure using indirect calorimetry. At 28 °C, wild type mice had slightly higher oxygen consumption compared with Gdm-/-·Ucp1-/- mice (Fig. 1A); however, during acclimation to the cold environment, Gdm-/-·Ucp1-/- had higher VO2 than wild type mice (Fig. 1B). Because similar increases in VO2 consumption during cold adaptation were observed in our previous studies of cold-adapted Ucp1-/- mice and T3 or leptin-treated Lepob/ob·Ucp1-/- mice, an enhanced capacity to stimulate oxygen consumption over that normally found for wild type mice appears to be an essential requirement for adaptation to the cold. Gene expression analyses of tissues from wild type and Gdm-/-·Ucp1-/- mice showed that, similar to the adaptive response of Ucp1-/- mice (16Ukropec J. Anunciado R.P. Ravussin Y. Hulver M.W. Kozak L.P. J. Biol. Chem. 2006; 281: 31894-31908Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), adaptation of Gdm-/-·Ucp1-/- mice to the cold (4 °C) was accompanied by induction of genes associated with fat oxidation, mitochondrial biogenesis, mitochondria transporters, and transcription factors known to regulate these genes. This induction in gene expression was found in inguinal fat and red and white gastrocnemius skeletal muscle but not in liver (Table 1 and supplemental Tables S2–S5).TABLE 1Summary of gene expression in WT and Gdm–/–·Ucp1–/– mice during cold adaptation with chow diet and with high fat diet for 20 weeks (20 °C for 10 weeks and at 28 °C for 10 weeks) The values in bold type are significantly different at p < 0.05. The number of mice used was five/genotype in each study.GeneRatio of Gdm–/–·Ucp1–/– to WTCold adaptation to 4 °C with chow dietHigh fat diet at 20 °C for 10 weeks and at 28 °C for 10 weeksLiverGasInguinal fatLiverGasInguinal fatRedWhiteRedWhiteGlucose metabolismGlut21.00.8Glut41.61.42.31.01.12.7Pdk40.81.86.70.51.96.7Pepck0.42.310.41.22.50.83.636.8Lipid metabolismAcox0.71.91.91.20.70.91.31.7Acsl20.81.11.60.91.30.91.01.6Hsl2.21.91.01.90.61.22.1Cpt1a0.60.3Cpt1b1.41.26.40.70.99.3Scd10.81.71.61.30.40.70.92.1Mitochondrial functionSlc25a250.71.83.70.40.80.71.12.8Mt-co20.91.61.41.30.70.70.92.6Ion transportSlc20a21.21.41.70.91.02.3TranscriptionPgc1-α2.22.73.31.81.01.21.114PPARα0.82.93.13.21.31.11.510.8PPARδ1.01.71.81.30.41.11.01.8PPARγ21.41.40.50.31.31.92.5Srebp1a0.40.81.11.10.70.80.91.5Srebp1c1.20.81.20.51.21.11.22.3Thyroid metabolismT4-Dio20.70.76.30.81.330.3Calcium cyclingSerca2a1.31.01.21.30.61.01.51.7 Open table in a new tab Gdm-/-·Ucp1-/- Mice Are Protected from Diet-induced Obesity—A central tenet of thermogenesis and obesity has been that UCP1 is the major thermogenic mechanism underlying diet-induced thermogenesis (4Lowell B.B. Spiegelman B.M. Nature. 2000; 404: 652-660Crossref PubMed Scopus (1322) Google Scholar, 23Rothwell N.J. Stock M.J. Nature. 1979; 281: 31-35Crossref PubMed Scopus (1205) Google Scholar). Accordingly, energy expenditure was expected to be lower in UCP1-deficient mice than in wild type mice fed a high fat-high sucrose diet. In addition, fat oxidation may also be suppressed in the UCP1-deficient mice. We tested this hypothesis and found, that there was no difference in oxygen consumption between wild type and UCP1-deficient mice when they were fed either a low fat chow diet or an obesogenic diet (Fig. 2, A and D). Upon switching to the obesogenic diet, oxygen consumption increased equally in both Ucp1-/- and wild type mice (Fig. 2, A and D). Furthermore, the RER showed the expected drop from ∼0.92 to 0.78 when the fat composition of the diet was increased, and similar to the oxygen consumption data, the values are indistinguishable between Ucp1-/- and wild type mice (Fig. 2, B and D). Accordingly, the experiment showed highly significant effects of diet on both energy expenditure and substrate utilization, but no significant genotype effects (Fig. 2, B–D). Consistent with this energy expenditure data, Ucp1 mRNA levels in the interscapular brown fat of wild type mice fed chow or obesogenic diets at both 28 and 4 °C were not significantly different from each other; if anything the HF diet suppressed Ucp1 mRNA induction when mice were exposed to an ambient temperature of 4 °C for 1 week (Fig. 2E). This evidence does not support the idea that UCP1 is part of a mechanism for diet-induced thermogenesis in mice fed an obesogenic/cafeteria diet; rather, the increase in oxygen consumption when animals switch from a diet high in carbohydrates to one rich in lipids is likely a consequence in the reduction in P/O ratio when fat is metabolized compared with carbohydrate (24Brand M.D. Chien L.F. Ainscow E.K. Rolfe D.F. Porter R.K. Biochim. Biophys. Acta. 1994; 1187: 132-139Crossref PubMed Scopus (393) Google Scholar). If it is calorically more costly to maintain body temperature by UCP1-independent thermogenesis, then an additional deletion of a thermogenic pathway should further reduce the development of adiposity. Accordingly, we evaluated the progressive loss of thermogenic mechanisms on the effects of ambient temperature on the development of DIO in wild type, Ucp1-/-, Gdm-/-, and Gdm-/-·Ucp1-/- mice, rationalizing that the deletion of a second thermogenic mechanism would further increase the demand for alternative thermogenesis and increase resistance to DIO. Ten-week-old mutant and wild type mice were fed a high fat diet at an ambient temperature of 20 °C for 10 weeks, and then the ambient temperature was raised to 28 °C for another 10 weeks (Fig. 3). The rate of body weight gain at 20 °C was slower for all genotypes than at 28 °C and more attenuated for the Ucp -/-, Gdm-/-, and Gdm-/-·Ucp1-/- than wild type mice (Fig. 3A). The effects of ambient temperature were clearly evident from the variation in the deposition of fat mass (Fig. 3, B and D). The most striking effects of ambient temperature were observed on the severely attenuated body weight and fat mass gain in the Gdm-/-·Ucp1-/- mice when the ambient temperature was raised to 28 °C. Estimates of the rate of increase in fat mass of the four genotypes fed a high fat diet at ambient temperatures of 20 and 28 °C show that at 20 °C wild type mice have the largest rate of increase, whereas Ucp1-/-, Gdm-/-, and Gdm-/-·Ucp1-/- mice were not significantly different from each other (Fig. 3D). However, when the ambient temperature is increased to 28 °C, the rate of fat mass accumulation in wild type mice and Ucp1-/- mice increased at a similar rate, Gdm-/- was slightly slower, whereas the Gdm-/-·Ucp1-/- mice showed only a modest increase in rate from that observed at 20 °C (Fig. 3D). Two-way analysis of variance on the fat mass gain during weeks 11–17 at 28 °C showed that both effects of genotype (p < 0.0001) and time (p < 0.0001) were significant; the interaction between genotype and time was likewise significant (p = 0.0135). When the ambient temperature is increased to 28 °C, the differences in fat mass accumulation during weeks 11–17 in wild type mice (8.4 ± 0.49) and Ucp1-/- mice (8.7 ± 0.46) are similar; Gdm-/- (6.5 ± 0.44) was slightly decreased, whereas the Gdm-/-· Ucp1-/- mice (1.9 ± 0.49) showed a significant decrease in fat mass. Inspection of the data suggests that the UCP1 and GDM phenotypes are synergistic, and they function as parallel thermogenic systems. Elimination of both Gdm and Ucp1 genes resulted in much lower fat mass accumulation than what would be predicted by the additive effect of the single mutants. These data indicate that there is a synergistic effect of the single gene deletions in causing reduced fat mass accumulation in the Gdm-/-·Ucp1-/- mice. Fat-free mass is significantly reduced in GDM-deficient mice and must be considered in calculations of energy expenditure (Fig. 3C). The variation in food intake on the adiposity phenotypes was minor (Fig. 3E), possibly accounting for some of the difference in adiposity between wild type and Ucp1-/- mice but not for the much greater reduction in fat mass of Gdm-/- and Gdm-/-·Ucp1-/- mice. Higher Energy Expenditure in Gdm-/-·Ucp1-/- Mice— The striking resistance of Gdm-/-·Ucp1-/- mice to DIO at 28 °C suggested that a difference in energy expenditure might be present even at an ambient temperature near thermoneutrality. Energy expenditure, monitored by indirect calorimetry at temperature transition points during the study (weeks 11 and 12, when ambient temperature was increased from 20 to 28 °C, and weeks 20 and 21, when ambient temperature was again reduced from 28 to 20 °C) showed that the Gdm-/-·Ucp1-/- mice had higher VO2 than wild type mice during both light and dark periods at both ambient temperatures (Fig. 1, C and D, and Table 2). By comparing A and B in Fig. 1, one can deduce that at 28 °C energy expenditure in wild type and Gdm-/-·Ucp1-/- mice is similar, but as the ambient temperature is reduced during the gradual cold adaptation, energy expenditure in Gdm-/-·Ucp1-/- becomes higher (Fig. 1B). Accordingly, when mice were fed a high fat diet and maintained at 20 °C during the first 10 weeks, a higher energy expenditure in Gdm-/-·Ucp1-/- mice was expected (Fig. 1C and Table 2). The higher level of energy expenditure of Gdm-/-·Ucp1-/- mice persisted even after the ambient temperature was elevated to 28 °C when assayed during the 11th week (Fig. 1C and Table 2). Unexpectedly, this elevated energy expenditure in Gdm-/-·Ucp1-/- continued to the 21st week when the mice were again placed in the metabolic chamber, indicating that significantly higher energy expenditure was sustained in Gdm-/-·Ucp1-/- mice during a period of 10 weeks at 28 °C while they were fed a high fat diet (Fig. 1D and Table 2). This elevated energy expenditure coincided with the severe suppression of DIO in the Gdm-/-·Ucp1-/- mice at 28 °C (Fig. 3A). To assess the continued response of the mice to changes in ambient temperature, the ambient temperature was again reduced to 20 °C during the 21st week, and the expected ambient temperature-dependent increase in energy expenditure was observed both in the wild type and mutant mice (Fig. 1D). This enhanced capacity for energy expenditure was dependent on prior conditioning at 20 °C, because Gdm-/-·Ucp1-/- mice fed a high fat diet at 28 °C and not exposed to 20 °C had comparable levels of obesity as wild type mice (adiposity indexes of 0.45 ± 0.04 versus 0.42 ± 0.07; Fig. 4D) and indistinguishable rates of oxygen consumption (12 h of light, 2.6 ± 0.05 versus 2.5 ± 0.17 ml O2/g fat free mass (FFM)/h, p = 0.290; 12 h of dark, 3.2 ± 0.10 versus 3.0 ± 0.25 ml O2/gm fat free mass (FFM)/h; p = 0.273).TABLE 2Energy expenditure, RER, and physical activity in WT and Gdm–/–·Ucp1–/– mice fed a high fat diet and reared at 20 °C for 10 weeks and then at 28 °C for 10 weeks All of the values represent the means ± S.E. p values are the results of statistical analysis using a Student's t test.TemperatureLight conditionsWT (n = 6)Gdm–/–·Ucp1–/– (n = 7)°CVO2 (ml O2/g of FFM/h)11th week2012 h of light2.9 ± 0.093.3 ± 0.02ap < 0.052012 h of dark3.5 ± 0.133.9 ± 0.05ap < 0.052812 h of light1.8 ± 0.052.1 ± 0.01ap < 0.052812 h of dark2.4 ± 0.012.9 ± 0.04ap < 0.0521st week2812 h of light1.5 ± 0.041.8 ± 0.05ap < 0.052812 h of dark1.8 ± 0.042.3 ± 0.08ap < 0.052012 h of light2.3 ± 0.052.8 ± 0.09ap < 0.052012 h of dark2.7 ± 0.113.3 ± 0.11ap < 0.05RER11th week2012 h of light0.87 ± 0.0080.88 ± 0.0022012 h of dark0.86 ± 0.0070.89 ± 0.007ap < 0.052812 h of light0.93 ± 0.0070.89 ± 0.007ap < 0.052812 h of dark0.91 ± 0.0090.91 ± 0.00921st week2812 h of light0.85 ± 0.0110.86 ± 0.0082812 h of dark0.84 ± 0.0150.87 ± 0.0042012 h of light0.84 ± 0.0060.84 ± 0.0052012 h of dark0.82 ± 0.0010.84 ± 0.010Ambulatory physical activity (total counts)11th week2012 h of light3138 ± 10142624 ± 1052012 h of dark13129 ± 347810512 ± 8112812 h of light2626 ± 5002831 ± 2722812 h of dark10740 ± 279211259 ± 242921st week2812 h of light1757 ± 5801859 ± 3032812 h of dark6465 ± 16607561 ± 11352012 h of light2357 ± 6582685 ± 3822012 h of dark9843 ± 238511076 ± 1149a p < 0.05 Open table in a new tab Despite feeding a high fat diet, significant differences in RER were evident during the dark h at 20 °C and light hours at 28 °C (Table 2, 11th week). Gdm-/-·Ucp1-/- mice had higher RER during the dark hours at 20 °C and lower RER during the light hours at 28 °C (Table 2). Whether this 3.5% difference in RER is biologically significant is questionable. However, differences in RER between genotypes were not evident during the 21st week (Table 2). Ambulatory physical activity did not differ between genotypes during the 11th or 21st weeks (Table 2) and was much greater during the dark than light hours at both temperatures. Moreover, the mice were more active at 20 °C than at 28 °C at all measurement periods, but differences between g
Referência(s)