The Alternative Stimulatory G Protein α-Subunit XLαs Is a Critical Regulator of Energy and Glucose Metabolism and Sympathetic Nerve Activity in Adult Mice
2006; Elsevier BV; Volume: 281; Issue: 28 Linguagem: Inglês
10.1074/jbc.m511752200
ISSN1083-351X
AutoresTao Xie, Antonius Plagge, Oksana Gavrilova, Stephanie Pack, William Jou, Edwin W. Lai, Marga Frontera, Gavin Kelsey, Lee S. Weinstein,
Tópico(s)Regulation of Appetite and Obesity
ResumoThe complex imprinted Gnas locus encodes several gene products including Gsα, the ubiquitously expressed G protein α-subunit required for receptor-stimulated cAMP generation, and the neuroendocrine-specific Gsα isoform XLαs. XLαs is only expressed from the paternal allele, whereas Gsα is biallelically expressed in most tissues. XLαs knock-out mice (Gnasxlm+/p–) have poor suckling and perinatal lethality, implicating XLαs as critical for postnatal feeding. We have now examined the metabolic phenotype of adult Gnasxlm+/p– mice. Gnasxlm+/p– mice had reduced fat mass and lipid accumulation in adipose tissue, with increased food intake and metabolic rates. Gene expression profiling was consistent with increased lipid metabolism in adipose tissue. These changes likely result from increased sympathetic nervous system activity rather than adipose cell-autonomous effects, as we found that XLαs is not normally expressed in adult adipose tissue, and Gnasxlm+/p– mice had increased urinary norepinephrine levels but not increased metabolic responsiveness to a β3-adrenergic agonist. Gnasxlm+/p– mice were hypolipidemic and had increased glucose tolerance and insulin sensitivity. The similar metabolic profile observed in some prior paternal Gnas knock-out models results from XLαs deficiency (or deficiency of the related alternative truncated protein XLN1). XLαs (or XLN1) is a negative regulator of sympathetic nervous system activity in mice. The complex imprinted Gnas locus encodes several gene products including Gsα, the ubiquitously expressed G protein α-subunit required for receptor-stimulated cAMP generation, and the neuroendocrine-specific Gsα isoform XLαs. XLαs is only expressed from the paternal allele, whereas Gsα is biallelically expressed in most tissues. XLαs knock-out mice (Gnasxlm+/p–) have poor suckling and perinatal lethality, implicating XLαs as critical for postnatal feeding. We have now examined the metabolic phenotype of adult Gnasxlm+/p– mice. Gnasxlm+/p– mice had reduced fat mass and lipid accumulation in adipose tissue, with increased food intake and metabolic rates. Gene expression profiling was consistent with increased lipid metabolism in adipose tissue. These changes likely result from increased sympathetic nervous system activity rather than adipose cell-autonomous effects, as we found that XLαs is not normally expressed in adult adipose tissue, and Gnasxlm+/p– mice had increased urinary norepinephrine levels but not increased metabolic responsiveness to a β3-adrenergic agonist. Gnasxlm+/p– mice were hypolipidemic and had increased glucose tolerance and insulin sensitivity. The similar metabolic profile observed in some prior paternal Gnas knock-out models results from XLαs deficiency (or deficiency of the related alternative truncated protein XLN1). XLαs (or XLN1) is a negative regulator of sympathetic nervous system activity in mice. The incidence of both obesity and diabetes has markedly increased over the past two decades. A basic understanding of the regulation of energy and glucose metabolism is required for the identification of potential new therapeutic targets. One gene shown to be important for metabolic regulation is GNAS on chromosome 20q13 in humans (Gnas on chromosome 2 in mice) (1Weinstein L.S. Liu J. Sakamoto A. Xie T. Chen M. Endocrinology. 2004; 145: 5459-5464Crossref PubMed Scopus (247) Google Scholar, 2Weinstein L.S. Yu S. Warner D.R. Liu J. Endocr. Rev. 2001; 22: 675-705Crossref PubMed Scopus (393) Google Scholar). GNAS/Gnas encodes several protein products, including the ubiquitously expressed G protein α-subunit Gsα 2The abbreviations used are: Gsα, stimulatory G protein a subunit; BAT and WAT, brown and white adipose tissue, respectively; XLαs, extra-large Gsα isoform; XLN1, alternative transcript from Gnasxl promoter; NESP55, 55-kDa neuroendocrine-specific protein; FFA, free fatty acids; PPAR, peroxisomal proliferator activated receptor; Ab, antibody; RT, reverse transcription. 2The abbreviations used are: Gsα, stimulatory G protein a subunit; BAT and WAT, brown and white adipose tissue, respectively; XLαs, extra-large Gsα isoform; XLN1, alternative transcript from Gnasxl promoter; NESP55, 55-kDa neuroendocrine-specific protein; FFA, free fatty acids; PPAR, peroxisomal proliferator activated receptor; Ab, antibody; RT, reverse transcription. that couples receptors to cAMP generation, the alternative Gsα isoform XLαs, and the 55-kDa neuroendrocrine-specific secretory protein (NESP55). Heterozygous mutations resulting in partial Gsα deficiency lead to obesity in mice (3Chen M. Gavrilova O. Liu J. Xie T. Deng C. Nguyen A.T. Nackers L.M. Lorenzo J. Shen L. Weinstein L.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7386-7391Crossref PubMed Scopus (155) Google Scholar) and in patients with Albright hereditary osteodystrophy (1Weinstein L.S. Liu J. Sakamoto A. Xie T. Chen M. Endocrinology. 2004; 145: 5459-5464Crossref PubMed Scopus (247) Google Scholar, 2Weinstein L.S. Yu S. Warner D.R. Liu J. Endocr. Rev. 2001; 22: 675-705Crossref PubMed Scopus (393) Google Scholar). GNAS/Gnas is a complex imprinted gene whose major gene products are generated by alternative promoters and first exons that splice onto a common exon (exon 2) (1Weinstein L.S. Liu J. Sakamoto A. Xie T. Chen M. Endocrinology. 2004; 145: 5459-5464Crossref PubMed Scopus (247) Google Scholar, 2Weinstein L.S. Yu S. Warner D.R. Liu J. Endocr. Rev. 2001; 22: 675-705Crossref PubMed Scopus (393) Google Scholar) (Fig. 1). NESP55 and XLαs are expressed from oppositely imprinted Nesp and Gnasxl promoters; NESP55 is only expressed from the maternal allele, whereas XLαs is only expressed from the paternal allele. In contrast, the Gsα promoter is imprinted in a tissue-specific manner, with Gsα being biallelically expressed in most tissues but primarily expressed from the maternal allele in a small number of tissues. XLαs is structurally identical to Gsα except for a long amino-terminal extension encoded within its specific first exon (4Kehlenbach R.H. Matthey J. Huttner W.B. Nature. 1994; 372: 804-809Crossref PubMed Scopus (201) Google Scholar, 5Li T. Vu T.H. Zeng Z.L. Nguyen B.T. Hayward B.E. Bonthron D.T. Hu J.F. Hoffman A.R. Genomics. 2000; 69: 295-304Crossref PubMed Scopus (63) Google Scholar). In contrast to Gsα, which is ubiquitously expressed at high levels in virtually all tissues, XLαs has a more restricted tissue distribution with expression primarily limited to neuroendocrine tissues (4Kehlenbach R.H. Matthey J. Huttner W.B. Nature. 1994; 372: 804-809Crossref PubMed Scopus (201) Google Scholar, 6Pasolli H.A. Klemke M. Kehlenbach R.H. Wang Y. Huttner W.B. J. Biol. Chem. 2000; 275: 33622-33632Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). XLαs has also been shown to be expressed in adipose tissue in 4-day-old mice (7Plagge A. Gordon E. Dean W. Boiani R. Cinti S. Peters J. Kelsey G. Nat. Genet. 2004; 36: 818-826Crossref PubMed Scopus (213) Google Scholar). In vitro biochemical studies have shown that XLαs is capable of stimulating adenylyl cyclase (8Klemke M. Pasolli H.A. Kehlenbach R.H. Offermanns S. Schultz G. Huttner W.B. J. Biol. Chem. 2000; 275: 33633-33640Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 9Bastepe M. Gunes Y. Perez-Villamil B. Hunzelman J. Weinstein L.S. Jüppner H. Mol. Endocrinol. 2002; 16: 1912-1919Crossref PubMed Scopus (111) Google Scholar) and mediating receptor-stimulated cAMP production (9Bastepe M. Gunes Y. Perez-Villamil B. Hunzelman J. Weinstein L.S. Jüppner H. Mol. Endocrinol. 2002; 16: 1912-1919Crossref PubMed Scopus (111) Google Scholar), although its role in vivo is less clear. The XLαs promoter also generates a neural-specific truncated form of XLαs (XLN1) produced by splicing of exon 3 onto an alternative terminal exon (6Pasolli H.A. Klemke M. Kehlenbach R.H. Wang Y. Huttner W.B. J. Biol. Chem. 2000; 275: 33622-33632Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Whether the resultant truncated protein has any biological function or whether it can act as a dominant negative inhibitor of Gsα or XLαs signaling is unknown. NESP55 does not appear to be an important metabolic regulator in either humans (10Liu J. Litman D. Rosenberg M.J. Yu S. Biesecker L.G. Weinstein L.S. J. Clin. Investig. 2000; 106: 1167-1174Crossref PubMed Scopus (241) Google Scholar) or mice (11Plagge A. Isles A.R. Gordon E. Humby T. Dean W. Gritsch S. Fischer-Colbrie R. Wilkinson L.S. Kelsey G. Mol. Cell. Biol. 2005; 25: 3019-3026Crossref PubMed Scopus (115) Google Scholar). We originally generated a Gnas knock-out in which exon 2, an exon common to all transcripts, was disrupted (12Yu S. Yu D. Lee E. Eckhaus M. Lee R. Corria Z. Accili D. Westphal H. Weinstein L.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8715-8720Crossref PubMed Scopus (353) Google Scholar). Heterozygotes with mutation of the paternal allele (E2m+/p–), leading to complete XLαs deficiency and partial Gsα deficiency, failed to suckle, and most died soon after birth with hypoglycemia. 3S. Yu and L. S. Weinstein, unpublished observations. 3S. Yu and L. S. Weinstein, unpublished observations. Those that survived had markedly reduced adiposity, increased metabolic rate, and activity levels and greater than normal glucose tolerance and insulin sensitivity (13Yu S. Castle A. Chen M. Lee R. Takeda K. Weinstein L.S. J. Biol. Chem. 2001; 276: 19994-19998Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 14Yu S. Gavrilova O. Chen H. Lee R. Liu J. Pacak K. Parlow A.F. Quon M.J. Reitman M.L. Weinstein L.S. J. Clin. Investig. 2000; 105: 615-623Crossref PubMed Scopus (136) Google Scholar, 15Chen M. Haluzik M. Wolf N.J. Lorenzo J. Dietz K.R. Reitman M.L. Weinstein L.S. Endocrinology. 2004; 145: 4094-4102Crossref PubMed Scopus (43) Google Scholar). E2m+/p– mice had increased urinary norepinephrine excretion, suggesting that these metabolic effects may be the consequence of increased sympathetic nervous system activity (14Yu S. Gavrilova O. Chen H. Lee R. Liu J. Pacak K. Parlow A.F. Quon M.J. Reitman M.L. Weinstein L.S. J. Clin. Investig. 2000; 105: 615-623Crossref PubMed Scopus (136) Google Scholar). In contrast, E1m+/p– mice with paternal deletion of Gsα exon 1 leading only to partial Gsα deficiency had normal suckling and survival and developed obesity, glucose intolerance, and insulin resistance (3Chen M. Gavrilova O. Liu J. Xie T. Deng C. Nguyen A.T. Nackers L.M. Lorenzo J. Shen L. Weinstein L.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7386-7391Crossref PubMed Scopus (155) Google Scholar). Comparison of E2m+/p– and E1m+/p– mice indirectly implicates XLαs as an important regulator of postnatal feeding as well as energy and glucose metabolism in older animals and suggests that XLαs and Gsα may have opposite effects on metabolic regulation in the whole animal. We have recently generated mice with deficiency of only XLαs (Gnasxlm+/p–) and showed that these mice have poor suckling and perinatal survival (7Plagge A. Gordon E. Dean W. Boiani R. Cinti S. Peters J. Kelsey G. Nat. Genet. 2004; 36: 818-826Crossref PubMed Scopus (213) Google Scholar), similar to that observed in E2m+/p– mice (12Yu S. Yu D. Lee E. Eckhaus M. Lee R. Corria Z. Accili D. Westphal H. Weinstein L.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8715-8720Crossref PubMed Scopus (353) Google Scholar) and other mouse models with disruption or loss of the paternal Gnas allele (16Skinner J.A. Cattanach B.M. Peters J. Genomics. 2002; 80: 373-375Crossref PubMed Scopus (43) Google Scholar, 17Cattanach B.M. Peters J. Ball S. Rasberry C. Hum. Mol. Genet. 2000; 9: 2263-2273Crossref PubMed Scopus (45) Google Scholar, 18Cattanach B.M. Kirk M. Nature. 1985; 315: 496-498Crossref PubMed Scopus (507) Google Scholar), implicating XLαs as critical for postnatal feeding adaptation in mice. We have now examined the metabolic phenotype of adult Gnasxlm+/p– mice and show that these mice have markedly reduced adiposity, increased metabolic rate with increased lipid metabolism in adipose tissue, and increased glucose tolerance and insulin sensitivity. We provide evidence that these metabolic changes are due to increased sympathetic nervous system activity rather than adipocyte cell-autonomous effects. XLαs (or possibly XLN1) appears to be an important negative regulator of sympathetic nervous system activity in mice. We also show that XLαs expression in adipose tissue is developmentally regulated. Animals—Gnasxlm+/p– mice (7Plagge A. Gordon E. Dean W. Boiani R. Cinti S. Peters J. Kelsey G. Nat. Genet. 2004; 36: 818-826Crossref PubMed Scopus (213) Google Scholar) were repeatedly mated with CD1 wild-type mice (Charles River, Wilmington, MA) for >5 generations and were maintained on a standard pellet diet (NIH-07, 5% fat by weight) and 12 h:12 h light/dark cycle. Except when noted, all experiments were performed on 12–14-week old male mutant mice and wild-type littermates. Experiments were approved by the NIDDK Animal Care and Use Committee. Body Composition, Food Intake, Metabolic Rate, and Activity Measurements—Body composition was measured using the Minispec mq10 NMR analyzer (Bruker Optics Inc., Woodlands, TX) that was calibrated with corn oil for fat mass and rat muscle for lean mass. Food intake, metabolic rates (oxygen consumption rate by indirect calorimetry), and activity levels were determined as previously described (14Yu S. Gavrilova O. Chen H. Lee R. Liu J. Pacak K. Parlow A.F. Quon M.J. Reitman M.L. Weinstein L.S. J. Clin. Investig. 2000; 105: 615-623Crossref PubMed Scopus (136) Google Scholar). The effect of the β3-specific adrenergic agonist CL316243 (19Bloom J.D. Dutia M.D. Johnson B.D. Wissner A. Burns M.G. Largis E.E. Dolan J.A. Claus T.H. J. Med. Chem. 1992; 35: 3081-3084Crossref PubMed Scopus (278) Google Scholar) on metabolic rate was measured as follows. At 9 a.m. mice were placed into calorimetry chambers that were prewarmed to 30 °C. At 1 p.m. CL316243 at the indicated dose (from a 1 mg/ml stock in saline) or a saline vehicle control was injected intraperitoneally, and after a 1-h delay data were collected for 3 h. Food and water were available at all times. Microscopic Analysis—For standard histology, samples were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and cut and stained with hematoxylin and eosin. For mitochondrial staining, 10-μm cryostat sections were stained for succinate dehydrogenase by incubation in 130 mm Tris-HCl buffer (pH 7.4) containing 0.2 mm nitro blue tetrazolium and 60 mm sodium succinate (Sigma) at 37 °C for 30 min. The slides were then rinsed with deionized water, dehydrated, and washed with xylene before coverslip mounting. Hyperinsulinemic-Euglycemic Clamp Studies—Hyperinsulinemic-euglycemic clamps studies and in vivo glucose flux analysis were performed as previous described (15Chen M. Haluzik M. Wolf N.J. Lorenzo J. Dietz K.R. Reitman M.L. Weinstein L.S. Endocrinology. 2004; 145: 4094-4102Crossref PubMed Scopus (43) Google Scholar). Insulin levels on samples during the clamp studies were measured by radioimmunoassay kit (Linco Research, St. Charles, MO). Blood, Tissue, and Urine Chemistries—Blood was obtained by retroorbital bleed. Serum glucose, cholesterol, and triglyceride levels were measured with an auto-analyzer by the NIH Clinical Chemistry Laboratory. Serum insulin, leptin, glucagon, and adiponectin were measured by Linco Research assay services. For C-peptide/insulin ratios, both were measured by radioimmunoassay (Linco). Serum resistin was measured using an enzyme-linked immunosorbent assay kit (Linco). Serum corticosterone was measured by Ani Lytics, Inc., Gaithersburg, MD. Serum free fatty acids (FFA) were measured using a kit from Roche Diagnostics. Triglyceride and glycogen content of hindlimb muscles were measured as previously described (15Chen M. Haluzik M. Wolf N.J. Lorenzo J. Dietz K.R. Reitman M.L. Weinstein L.S. Endocrinology. 2004; 145: 4094-4102Crossref PubMed Scopus (43) Google Scholar). Urine was collected by bladder puncture, and catecholamines were measured by high performance liquid chromatography (20Eisenhofer G. Goldstein D.S. Stull R. Keiser H.R. Sunderland T. Murphy D.L. Kopin I.J. Clin. Chem. 1986; 32: 2030-2033Crossref PubMed Scopus (410) Google Scholar), and corrected by creatinine concentration within the same samples, which were measured by Ani-Lytics. Triglyceride Clearance Test—Clearance of triglycerides (12.5 μl of peanut oil/g of body weight delivered by gavage) from the circulation was measured in mice after a 4-h fast. Blood was taken before gavage and hourly for 6 h after gavage, and plasma triglycerides were measured (kit #TR22421, Thermo DMA, Waltham, MA). Glucose and Insulin Tolerance Tests—Glucose and insulin tolerance tests were performed in overnight-fasted mice after intraperitoneal injection of glucose (2 mg/g) or insulin (Humulin, 0.50 mIU/g). Blood glucose levels in tail vein bleeds were measured using a Glucometer Elite (Bayer, Elkhart, IN) immediately before and at the indicated times after injection. Portal Vein Insulin Injection and Immunoblot Analysis—Overnight-fasted mice were anesthetized with avertin (0.25 mg/g of body weight intraperitoneal). Insulin (100 μl of 15 μg/ml, Sigma) was injected via the portal vein. At 2 or 4 min after injection, interscapular brown adipose tissue (BAT), epididymal white adipose tissue (WAT), and hind leg muscles were dissected and immediately frozen. Tissues were then homogenized, and protein concentrations of protein extracts were measured as previously described (15Chen M. Haluzik M. Wolf N.J. Lorenzo J. Dietz K.R. Reitman M.L. Weinstein L.S. Endocrinology. 2004; 145: 4094-4102Crossref PubMed Scopus (43) Google Scholar). Immunoprecipitations were performed on tissue extracts (1 mg of protein) using an anti-Akt1 Ab (Upstate Biotechnology, Lake Placid, NY) as per manufacturer's instructions followed by immunoblot analysis using an anti-Akt1 phospho-Ser473 Ab (Upstate). Ab binding was determined by ECL (ECL kit, Amersham Biosciences). The same blots were stripped and rehybridized with the anti-Akt1 Ab. Quantitative Reverse Transcription-PCR—RNA was extracted from frozen tissues using TRIzol (Invitrogen) or an Mini RNeasy kit (Qiagen, Germantown, MD) and treated with DNase (DNA-free, Ambion, Austin, TX) to remove DNA contamination. Reverse transcription was performed using the SuperScript first-strand synthesis system (Invitrogen). Gene expression levels were measured by quantitative RT-PCR using a real time PCR machine (MxP3000, Stratagene, La Jolla, CA). PCR reactions (20 μl total volume) included cDNA, 100 nm primers, and 10 μl of SYBR Green MasterMix (Applied Biosystems, Foster City, CA). To get relative quantification, standard curves were simultaneously generated with serial dilutions of cDNA, and results were normalized to β-actin mRNA levels in each sample, which were determined simultaneously by the same method. Specificity of each RT-PCR product was indicated by its dissociation curve and the presence of a single band of expected size on acrylamide gel electrophoresis. Sequences for gene-specific primers are listed in the supplementary table. Semiquantitative RT-PCR—XLαs mRNA expression was examined in BAT and WAT by semiquantitative RT-PCR. After first-strand synthesis, duplex PCR was performed (32 cycles, 60 °C annealing temperature) using a pair of XLαs-specific primers (1 μm each) and β-actin-specific primers (100 nm each) (primer sequences listed in the supplemental table). Products were separated on agarose gels, and gels were stained with ethidium bromide. Statistical Analysis—Data are expressed as the mean ± S.E. Statistical significance between groups was determined using unpaired Student's t test (two-tailed) with differences considered significant at p < 0.05. Gnasxlm+/p– Mice Have Reduced Survival and Adiposity—In this study we examined the survival and adult metabolic phenotype of Gnasxlm+/p– mice, which were placed in a CD1 genetic background to compare these mice to other Gnas knock-out models that we have studied on the same genetic background. In most other inbred genetic backgrounds E2m+/p– mice have no survival beyond several days.3 In the CD1 background most Gnasxlm+/p– mice failed to suckle and died soon after birth, as was previously described in both these mice (7Plagge A. Gordon E. Dean W. Boiani R. Cinti S. Peters J. Kelsey G. Nat. Genet. 2004; 36: 818-826Crossref PubMed Scopus (213) Google Scholar) and E2m+/p– mice (12Yu S. Yu D. Lee E. Eckhaus M. Lee R. Corria Z. Accili D. Westphal H. Weinstein L.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8715-8720Crossref PubMed Scopus (353) Google Scholar). By 3 weeks of age, Gnasxlm+/p– mice had a survival rate that was ∼27% that expected by Mendelian inheritance (8 of 59 total offspring), and the effect appeared to be similar in males and females. This survival rate is similar to the survival rate that we previously reported for E2m+/p– mice in the CD1 background (23%) (12Yu S. Yu D. Lee E. Eckhaus M. Lee R. Corria Z. Accili D. Westphal H. Weinstein L.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8715-8720Crossref PubMed Scopus (353) Google Scholar). Growth curves (Fig. 2A) demonstrate that both male and female Gnasxlm+/p– mice grew very slowly through the first 30 days relative to their wild-type littermates, and the resulting weight differences were maintained during adulthood. Adult Gnasxlm+/p– mice weighed ∼45% less than wild-type littermates and had a small but significant reduction in nasoanal body length (Fig. 2B). Body composition analysis by NMR showed that Gnasxlm+/p– mice had a markedly reduced fat mass and increased lean mass relative to total body weight (Fig. 2C). Consistent with this, the relative weights of retroperitoneal and epididymal WAT pads and interscapular BAT pads were greatly reduced in Gnasxlm+/p– mice, whereas the relative weights of most other organs were unchanged (Fig. 2D). The relative weight of testes and lungs were slightly, although significantly increased in Gnasxlm+/p– mice as compared with controls. Histology of both BAT and WAT from Gnasxlm+/p– mice showed reduced lipid accumulation and size of adipocytes (Fig. 2E). BAT from Gnasxlm+/p– mice had markedly increased succinate dehydrogenase staining compared with controls (Fig. 2F), indicating that the mutant mice had greater mitochondrial content in BAT. Overall, our findings show that, similar to E2m+/p– mice (12Yu S. Yu D. Lee E. Eckhaus M. Lee R. Corria Z. Accili D. Westphal H. Weinstein L.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8715-8720Crossref PubMed Scopus (353) Google Scholar, 14Yu S. Gavrilova O. Chen H. Lee R. Liu J. Pacak K. Parlow A.F. Quon M.J. Reitman M.L. Weinstein L.S. J. Clin. Investig. 2000; 105: 615-623Crossref PubMed Scopus (136) Google Scholar), Gnasxlm+/p– mice have reduced preweaning survival, poor growth, and maintain a very lean phenotype in adulthood. Hypermetabolism in Gnasxlm+/p– Mice Is Associated with Increased Norepinephrine Levels—The markedly reduced adiposity of Gnasxlm+/p– mice indicates that they may have either reduced food intake or increased energy expenditure rates relative to controls. Both male and female Gnasxlm+/p– mice had significantly increased food intake when corrected for body mass (Fig. 3A), indicating that reduced adiposity is not due to hypophagia. Hyperphagia in Gnasxlm+/p– mice may be a physiologic response to low circulating leptin levels (Table 1), which is an expected consequence of their severely lean phenotype. Both resting and total energy expenditure (O2 consumption) rates were very significantly increased at both ambient temperature(24 °C)andthermoneutral temperature (30 °C) (Fig. 3B). There were no differences in the respiratory exchange ratio (ratio of CO2 produced to O2 consumed) (Fig. 3C) or activity levels (Fig. 3D) between wild-type and mutant mice. The metabolic changes are similar to those previously observed in E2m+/p– mice, although we found E2m+/p– mice to be hyperactive (14Yu S. Gavrilova O. Chen H. Lee R. Liu J. Pacak K. Parlow A.F. Quon M.J. Reitman M.L. Weinstein L.S. J. Clin. Investig. 2000; 105: 615-623Crossref PubMed Scopus (136) Google Scholar), whereas Gnasxlm+/p– mice were not. Sex and age differences between the mice examined in this and the prior E2m+/p– mouse study may account for these discrepant results.TABLE 1Serum chemistriesWild typeGnasxlm+/p-Glucose (mg/dl)180 ± 8152 ± 6ap < 0.01 versus wild-type littermates.Cholesterol (mg/dl)132 ± 782 ± 9ap < 0.01 versus wild-type littermates.Triglycerides (mg/dl)104 ± 1645 ± 18ap < 0.01 versus wild-type littermates.Insulin (ng/ml)4.14 ± 0.900.73 ± 0.11ap < 0.01 versus wild-type littermates.Glucagon (pm)101 ± 22145 ± 96Leptin (pm)692 ± 7939 ± 19ap < 0.01 versus wild-type littermates.Adiponectin (μg/ml)17.0 ± 1.111.8 ± 0.8ap < 0.01 versus wild-type littermates.Resistin (ng/ml)8.2 ± 1.210.1 ± 1.6Corticosterone (ng/ml)169 ± 58203 ± 48a p < 0.01 versus wild-type littermates. Open table in a new tab We next examined whether the hypermetabolic state could be explained by increased metabolic responsiveness to sympathetic stimulation by measuring the resting metabolic rate after administration of various doses of the β3-adrenergic agonist CL316243 at thermoneutral temperature (30 °C). Resting metabolic rate at thermoneutrality reflects the metabolic rate in the absence of adaptive thermogenesis with minimal endogenous stimulation of adipose tissue by the sympathetic nervous system (21Pennycuik P.R. Aust. J. Exp. Biol. Med. Sci. 1967; 45: 331-346Crossref PubMed Google Scholar, 22Zaror-Behrens G. Himms-Hagen J. Am. J. Physiol. 1983; 244: E361-E366PubMed Google Scholar). Because β3 adrenergic receptors are specifically expressed in adipose tissue and CL316246 has been shown to specifically activate adipose tissue (23Gavrilova O. Marscus-Samuels B. Reitman M.L. Diabetes. 2000; 49: 1910-1916Crossref PubMed Scopus (55) Google Scholar), increases in metabolic rate after CL316246 reflect the metabolic responsiveness of adipose tissue to catecholamine stimulation by β3-adrenergic receptors, a major receptor stimulated by sympathetic nerves in adipose tissue. Our results show that adipose tissue in Gnasxlm+/p– mice was not more sensitive to β3-adrenergic stimulation (Fig. 3E). In fact Gnasxlm+/p– mice tended to have an increased base-line activity (p = 0.079) and a somewhat reduced response to higher doses of the β3 agonist, suggesting that they may be subtly resistant to β3-adrenergic stimulation. There were no significant differences in the respiratory exchange ratio in response to the β3 agonist (Fig. 3F). These results are similar to what was previously observed before and after maximal β3 agonist stimulation in E2m+/p– mice (14Yu S. Gavrilova O. Chen H. Lee R. Liu J. Pacak K. Parlow A.F. Quon M.J. Reitman M.L. Weinstein L.S. J. Clin. Investig. 2000; 105: 615-623Crossref PubMed Scopus (136) Google Scholar). We also assessed the ability of the β3 agonist to acutely raise serum FFA levels, which is a measure of the acute lipolytic response in adipose tissue. In the fed state Gnasxlm+/p– mice tended to have elevated serum FFA levels compared with controls (p = 0.098) even though they had a reduced adipose mass (Fig. 4A). The increase in FFA levels measured 20 min after injection of CL316243 (100 μg/kg) was significantly lower in Gnasxlm+/p– mice than in controls. The ratio of unstimulated/stimulated FFA levels was significantly higher in Gnasxlm+/p– mice (0.33 ± 0.05 in Gnasxlm+/p– versus 0.21 ± 0.02 in control mice). These results are similar to the slightly reduced metabolic responsiveness to CL316243 observed in the mutant mice. In the fasted state (when insulin levels are low) Gnasxlm+/p– mice had 2-fold higher serum FFA levels than controls (Fig. 4B), indicative of a very high lipolytic rate in mutant mice. After administration of insulin (0.75 mIU/g of body weight), FFA levels fell to similar levels in Gnasxlm+/p– and control mice, indicating that the ability of insulin to suppress lipolysis is maintained in Gnasxlm+/p– mice. To assess overall sympathetic nerve activity, we measured urinary norepinephrine concentrations normalized to creatinine to correct for differences in urinary concentration and in lean mass. Similar to E2m+/p– mice (14Yu S. Gavrilova O. Chen H. Lee R. Liu J. Pacak K. Parlow A.F. Quon M.J. Reitman M.L. Weinstein L.S. J. Clin. Investig. 2000; 105: 615-623Crossref PubMed Scopus (136) Google Scholar), both male and female Gnasxlm+/p– mice had significantly increased urine norepinephrine (NE) levels (Fig. 3G). Urine epinephrine levels, which primarily reflect adrenomedullary activity, were also increased, although the difference was only significant in males. There was no significant difference in urine creatinine concentration between the two groups (data not shown). Overall, our results suggest that increased metabolic rate in adult Gnasxlm+/p– mice is primarily the result of increased sympathetic nervous system activity (and possibly adrenomedullary secretion) rather than an inherent difference in sensitivity to metabolic stimulation. Gnasxlm+/p– Mice Have Improved Glucose Tolerance and Insulin Sensitivity—Random serum glucose, insulin, cholesterol, and triglyceride levels were all significantly lower in Gnasxlm+/p– as compared with control mice (Table 1). Similar to E2m+/p– mice (15Chen M. Haluzik M. Wolf N.J. Lorenzo J. Dietz K.R. Reitman M.L. Weinstein L.S. Endocrinology. 2004; 145: 4094-4102Crossref PubMed Scopus (43) Google Scholar), Gnasxlm+/p– mice had an increased clearance rate of an oral triglyceride load and very low muscle triglyceride levels (Figs. 4, C and D), consistent with an increased rate of overall lipid metabolism. Glucose and insulin tolerance tests showed that Gnasxlm+/p– mice had increased glucose tolerance and insulin sensi
Referência(s)