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• W2061520929 abstract "Targeted disruption of the RIIβ subunit of protein kinase A (PKA) produces lean mice that resist diet-induced obesity. In this report we examine the effects of the RIIβ knockout on white adipose tissue physiology. Loss of RIIβ is compensated by an increase in the RIα isoform, generating an isoform switch from a type II to a type I PKA. Type I holoenzyme binds cAMP more avidly and is more easily activated than the type II enzyme. These alterations are associated with increases in both basal kinase activity and the basal rate of lipolysis, possibly contributing to the lean phenotype. However, the ability of both β3-selective and nonspecific β-adrenergic agonists to stimulate lipolysis is markedly compromised in mutant white adipose tissue. This defect was foundin vitro and in vivo and does not result from reduced expression of β-adrenergic receptor or hormone-sensitive lipase genes. In contrast, β-adrenergic stimulated gene transcription remains intact, and the expression of key genes involved in lipid metabolism is normal under both fasted and fed conditions. We suggest that the R subunit isoform switch disrupts the subcellular localization of PKA that is required for efficient transduction of signals that modulate lipolysis but not for those that mediate gene expression. Targeted disruption of the RIIβ subunit of protein kinase A (PKA) produces lean mice that resist diet-induced obesity. In this report we examine the effects of the RIIβ knockout on white adipose tissue physiology. Loss of RIIβ is compensated by an increase in the RIα isoform, generating an isoform switch from a type II to a type I PKA. Type I holoenzyme binds cAMP more avidly and is more easily activated than the type II enzyme. These alterations are associated with increases in both basal kinase activity and the basal rate of lipolysis, possibly contributing to the lean phenotype. However, the ability of both β3-selective and nonspecific β-adrenergic agonists to stimulate lipolysis is markedly compromised in mutant white adipose tissue. This defect was foundin vitro and in vivo and does not result from reduced expression of β-adrenergic receptor or hormone-sensitive lipase genes. In contrast, β-adrenergic stimulated gene transcription remains intact, and the expression of key genes involved in lipid metabolism is normal under both fasted and fed conditions. We suggest that the R subunit isoform switch disrupts the subcellular localization of PKA that is required for efficient transduction of signals that modulate lipolysis but not for those that mediate gene expression. protein kinase A regulatory catalytic protein kinase inhibitor brown adipose tissue white adipose tissue hormone-sensitive lipase acetyl-CoA carboxylase lipoprotein lipase phosphoenolpyruvate carboxykinase β-adrenergic receptor phenylisopropyladenosine adenosine deaminase 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride protein kinase A protein N 6-[R-(−)-1-methyl-2-phenyl]adenosine Protein kinase A (PKA)1transduces the cAMP-mediated signals from more than 30 different hormones and neurotransmitters, many of which may act simultaneously on a given cell to provoke discrete biological responses (1Krebs E.G. Curr. Top. Cell. Regul. 1972; 5: 99-133Crossref PubMed Scopus (356) Google Scholar). The properties of PKA that modulate its signaling specificity are poorly understood, although it has been speculated that regulatory (R) and catalytic (C) subunit isoform diversity confers at least some of this specificity by assembling into discrete holoenzyme complexes that differ in subcellular targeting and sensitivity to activation by cAMP. Four R subunit isoforms (RIα, RIβ, RIIα, and RIIβ) and two C subunit isoforms (Cα and Cβ) are transcribed in mice (2McKnight G.S. Curr. Opin. Cell Biol. 1991; 3: 213-217Crossref PubMed Scopus (153) Google Scholar). Each is encoded by a separate gene and expressed in a tissue-specific pattern. Although much is understood regarding the physical properties of individual PKA isoforms, relatively little is known about the biological roles of each isoform in vivo. Knockout mice lacking individual PKA subunit genes represent powerful tools to elucidate these functions (3McKnight G.S. Idzerda R.L. Kandel E.R. Brandon E.P. Zhuo M. Qi M. Bourtchouladze R. Huang Y. Burton K.A. Skalhegg B.S. Cummings D.E. Varshavsky L. Planas J.V. Motamed K. Gerhold K.A. Amieux P.S. Guthrie C.R. Millett K.M. Belyamani M. Su T. Hansson V. Levy F.O. Tasken K. Signal Transduction in Testicular Cells. Springer-Verlag, Heidelberg, Germany1996: 95-122Crossref Google Scholar). We have generated null mutant mice lacking the RIIβ isoform of PKA (4Cummings D.E. Brandon E.P. Planas J.V. Motamed K. Idzerda R.L. McKnight G.S. Nature. 1996; 382: 622-626Crossref PubMed Scopus (361) Google Scholar). Unlike some PKA subunit isoforms that are expressed ubiquitously, RIIβ demonstrates very restricted tissue distribution. It is most abundant in white and brown adipose tissue and brain, with very limited expression elsewhere. RIIβ knockout mice are lean on a standard diet and resist diet-induced obesity as well as some of its associated adverse consequences. On a standard diet they have a 50% reduction in adipose tissue mass throughout their bodies, despite normal food intake, lipid absorption, and adipocyte cellularity. These changes may arise at least in part from PKA perturbations in brown adipose tissue (BAT). PKA in mutant BAT is more sensitive to cAMP and shows increased basal enzyme activity, alterations that are associated with an induction of uncoupling protein 1. Elevated levels of this thermogenic molecule correlate with an increase in basal metabolic rate and body temperature and may contribute to the lean phenotype. Here we report studies of PKA-mediated functions in the white adipose tissue (WAT) of RIIβ knockout mice. In WAT, PKA integrates several different hormonal signals to regulate the lipolytic catabolism of stored triglycerides into fatty acids and glycerol by hormone-sensitive lipase (HSL). Lipolysis is increased by β-adrenergic agonists, ACTH, and glucagon, all signaling via cAMP to stimulate PKA, which reversibly phosphorylates three serine residues on HSL to activate the enzyme (5Stralfors P. Belfrage P. J. Biol. Chem. 1983; 258: 15146-15152Abstract Full Text PDF PubMed Google Scholar, 6Stralfors P. Bjorgell P. Belfrage P. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3317-3321Crossref PubMed Scopus (196) Google Scholar, 7Anthonsen M.W. Ronnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar) and promote translocation to lipid droplets (8Egan J.J. Greenberg A.S. Chang M.K. Wek S.A. Moos Jr., M.C. Londos C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8537-8541Crossref PubMed Scopus (343) Google Scholar, 9Hirsch A.H. Rosen O.M. J. Lipid Res. 1984; 25: 665-677Abstract Full Text PDF PubMed Google Scholar). Lipolysis is inhibited by insulin, which stimulates a phosphodiesterase (PDE3B) that lowers cAMP levels (10Degerman E. Landstrm T.R. Wijkander J. Holst L.S. Ahmad F. Belfrage P. Manganiello V. Methods. 1998; 14: 43-53Crossref PubMed Scopus (67) Google Scholar). PKA also regulates several lipogenic enzymes, generally inhibiting gene expression in opposition to insulin action. In addition, PKA mediates the induction of cAMP-response element-regulated genes in WAT, including the well studied PEPCK gene. We find that RIIβ mutant WAT has a blunted capacity for PKA-stimulated lipolysis, whereas PKA-mediated transcriptional regulation is relatively unaffected. Possible mechanisms underlying these changes are discussed. We have previously described the generation of RIIβ mutant mice (4Cummings D.E. Brandon E.P. Planas J.V. Motamed K. Idzerda R.L. McKnight G.S. Nature. 1996; 382: 622-626Crossref PubMed Scopus (361) Google Scholar, 11Brandon E.P. Logue S.F. Adams M.R. Qi M. Sullivan S.P. Matsumoto A.M. Dorsa D.M. Wehner J.M. McKnight G.S. Idzerda R.L. J. Neurosci. 1998; 18: 3639-3649Crossref PubMed Google Scholar). Wild type and mutant mice used in all experiments were age- and gender-matched and maintained on the same mixed C57BL/6 × 129Sv/J genetic background. For the experiments on the nutritional regulation of PKA-mediated gene expression, adult male mice were fasted for 24 h, after which half of the animals were sacrificed (fasted group) and the rest allowed to feed on standard mouse chow (Teklad Rodent Diet 8604) for 6 h (refed group) before being sacrificed. Epididymal fat pads were dissected, immediately frozen in liquid nitrogen, and stored at −70 °C. Male wild type and mutant mice were sacrificed by cervical dislocation, and epididymal fat pads were immediately removed, weighed, and frozen at −70 °C. WAT was homogenized (10% w/v) by Polytron treatment followed by sonication in a buffer containing 50 mm Hepes, pH 7.2, 5 mmEDTA, 3 mm EGTA, 120 mm NaCl, 4 mmdithiothreitol, 40 μg/ml leupeptin, 50 μg/ml aprotinin, 100 μg/ml soybean trypsin inhibitor, 3 mm AEBSF, and 10% glycerol. Homogenates were centrifuged at 25,000 × g for 15 min, and the internatants were harvested for cAMP binding assays. Protein concentration of these samples was determined by Bradford assay (Bio-Rad). cAMP binding capacity was measured by incubating 160 μg of WAT protein with varying concentrations of [3H]cAMP (1 nm to 5 μm) for 45 min at 37 °C in 220 μl of buffer containing 20 mm Tris, pH 7.0, 0.5 mm 3-isobutyl-1-methylxanthine, 1 mg/ml bovine serum albumin, 10 mm magnesium acetate, 5 mm NaF, 10 mm dithiothreitol, 200 μm ATP, 15 μg/ml leupeptin, 9 μg/ml aprotinin, 100 μg/ml soybean trypsin inhibitor, and 1.5 mm AEBSF. Proteins were precipitated with 3 ml of 80% NH4SO4 at 4 °C as described previously (12Doskeland S.O. Ogreid D. Methods Enzymol. 1988; 159: 147-150Crossref PubMed Scopus (39) Google Scholar) and then trapped by filtration though GF/F glass microfiber filters (Whatman). Rinsed filters were incubated for 10 min in 850 μl of 2% SDS to solubilize trapped proteins, and radioactivity of the filters and SDS solution was determined by liquid scintillation counting. Nonspecific binding was measured in replicate samples containing a 1,000-fold excess of unlabeled cAMP and was subtracted from total counts to determine specific binding. Epididymal fat pads were obtained and stored as described above. WAT samples were thawed into homogenization buffer (20 mm Tris, pH 7.0, 0.1 mm EDTA, 0.5 mm EGTA, 1% Triton X-100, 10 mmdithiothreitol, 5 mm magnesium acetate, 250 mmsucrose, 1 μg/ml leupeptin, 3 μg/ml aprotinin, 100 μg/ml soybean trypsin inhibitor, 0.5 mm AEBSF, 100 μm ATP), dispersed by Polytron treatment, and sonicated. Homogenates were centrifuged at 16,000 × g for 15 min, and the internatants were harvested and stored at −70 °C. Protein concentration of these samples was determined by Bradford assay (Bio-Rad). Protein kinase activity was measured using Kemptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as described (13Clegg C.H. Correll L.A. Cadd G.G. McKnight G.S. J. Biol. Chem. 1987; 262: 13111-13119Abstract Full Text PDF PubMed Google Scholar), in the presence or absence of varying amounts of exogenous cAMP (up to 5 μm). The small amount of kinase activity not inhibited by 5 μm PKI was subtracted as background to determine PKA-specific activity. Epididymal fat pads freshly removed from male mice were finely minced with ultra-thin razor blades for 2 min. Adipose tissue fragments were washed three times by gentle inversion followed by centrifugation at 20 × g for 1 min in a 37 °C solution containing 20 mm Hepes, pH 7.4, 120 mm NaCl, 1.3 mm CaCl2, 1.2 mm KH2PO4, 4.8 mm KCl, 0.6 mm MgSO4, and 1% bovine serum albumin. Washed tissue fragments were suspended in 1 ml of the above buffer (∼100 mg of tissue/ml) and incubated in a 37 °C water bath rotating at 40 rpm. Plastic vials were used for all steps involving live adipocytes. Lipolytic agents were added after 15 min, and 35-μl samples of the media were removed every 15 min for 2 h for glycerol determinations. Samples were variably supplemented with the following reagents: 20 μm isoproterenol, 10 μm CL 316,243 (Wyeth Ayerst Laboratories, Philadelphia), 1 unit/ml adenosine deaminase, and 10 μm PIA (N 6-[R-(−)-1-methyl-2-phenyl]adenosine). Glycerol was measured using a GPO-Trinder enzymatic assay (Sigma) with a standard curve generated from samples of known glycerol content. After the 37 °C incubation period, adipocytes were lysed in SET buffer (1% SDS, 5 mm EDTA, 10 mm Tris, pH 7.5) by Polytron treatment and sonication, centrifuged at 27,500 ×g for 20 min, and the internatants harvested for DNA quantitation using a fluorescent dye binding assay (14Labarca C. Paigen K. Anal. Biochem. 1980; 102: 344-352Crossref PubMed Scopus (4537) Google Scholar). Glycerol release data were converted from μg/ml glycerol to picograms of glycerol released per cell by assuming 6 pg DNA/cell. Isoproterenol (0.3 mg/kg), CL 316,243 (1.0 mg/kg), or normal saline was injected intraperitoneally into wild type or RIIβ mutant male mice; 20 min later ∼200 μl of blood was rapidly removed by retro-orbital bleeding. Plasma was isolated from whole blood to determine the content of glycerol or free fatty acids, as described below. Trinder-type enzymatic colorimetric assay systems were used to determine plasma levels of glycerol (Sigma), free fatty acids (Wako Chemicals, Inc., Richmond, VA), triglycerides (Roche Molecular Biochemicals), cholesterol (Roche Molecular Biochemicals), and glucose (Sigma). Plasma insulin was measured by radioimmunoassay (Linco Research, Inc., St. Charles, MO). Epididymal fat pads were homogenized (10% w/v) in cold 6% trichloroacetic acid by Polytron treatment. Homogenates were centrifuged at 13,000 × g for 15 min at 4 °C. Internatants were harvested and extracted five times with 5-fold excess volumes of water-saturated ether (to remove trichloroacetic acid). Extracted aqueous samples were dried and resuspended in 300 μl of NEN assay buffer. The cAMP concentration of these samples was determined by radioimmunoassay (NEN Life Science Products). The steady-state amount of mRNA derived from specific genes was determined by solution hybridization as described previously (15Uhler M.D. Carmichael D.F. Lee D.C. Chrivia J.C. Krebs E.G. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1300-1304Crossref PubMed Scopus (199) Google Scholar). Briefly, total nucleic acid was isolated from individual tissues by proteinase K digestion and phenol/chloroform extraction (16McKnight G.S. Lee D.C. Hemmaplardh D. Finch C.A. Palmiter R.D. J. Biol. Chem. 1980; 255: 144-147Abstract Full Text PDF PubMed Google Scholar). Samples were hybridized overnight with approximately 5000 cpm of [32P]CTP-labeled antisense RNA probe at 70 °C under paraffin oil. Free probe was then digested with RNase A and T1 for 1 h at 37 °C. Samples were precipitated with 10% trichloroacetic acid and collected on Whatman GF/C glass microfiber filters (Whatman) to trap hybridized probe. The amount of RNase-resistant probe was measured by liquid scintillation counting. Standard curves were generated from known amounts of appropriate sense strand RNA. The results were converted to molecules of mRNA per cell based on both the standard curves and the specific activity of the probe. cDNAs used as templates for the synthesis of RNA probes and standards were kindly provided by K.-H. Kim, Purdue University (ACC); M. D. Lane, The Johns Hopkins University (GLUT4); M. C. Schotz, UCLA (HSL and LPL); D. K. Granner, Vanderbilt University (PEPCK); D. S. Weigle, University of Washington (leptin); and S. Collins, Duke University (β-ARs). We have previously published data showing that RIIβ is the predominant R subunit in WAT and that its loss in RIIβ knockouts is compensated for in this tissue by a 3–4-fold increase of the RIα isoform, arising by RIα protein stabilization (17Amieux P.S. Cummings D.E. Motamed K. Brandon E.P. Wailes L.A. Le K. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1997; 272: 3993-3998Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). This is the only R subunit adjustment in mutant WAT, as neither RIβ nor RIIα is expressed at appreciable levels (data not shown). We have also demonstrated by high performance liquid chromatography/ion exchange chromatography that in WAT there is an isoform switch from a nearly pure RII-containing holoenzyme in wild types to an entirely RI-containing holoenzyme in mutants (17Amieux P.S. Cummings D.E. Motamed K. Brandon E.P. Wailes L.A. Le K. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1997; 272: 3993-3998Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). In order to determine the cAMP affinities of PKA from wild typeversus RIIβ mutant WAT, the cAMP-binding capacity of tissue homogenates was measured. As shown in Fig. 1 A, the RIα-containing mutant holoenzyme binds cAMP more avidly than does the RIIβ-containing wild type holoenzyme, with K d values of 170 and 400 nm, respectively. As a consequence, mutant PKA is more readily activated by cAMP than is wild type enzyme (K a values of 80 and 220 nm, respectively, Fig. 1 B). This increased cAMP sensitivity is reflected by a 4-fold elevation of basal PKA activity in mutant WAT (Fig. 1 B, inset). The overall complement of both R and C subunits is reduced in mutant WAT. The total number of R subunits (of any isoform) is reflected in the total cAMP-binding capacity at saturating cAMP concentrations. As shown in Fig. 1 A, the maximal cAMP-binding capacity (and thus, total R subunit content) of RIIβ mutant WAT is reduced by 30% compared with wild type. Similarly, the amount of C subunit in mutant WAT is decreased by 55%, as judged by the reduction in total cAMP-stimulated PKA activity (Fig. 1 B). We have previously published Western blot analysis showing a corresponding 43% decrease in the amount of C subunit protein in mutant WAT (17Amieux P.S. Cummings D.E. Motamed K. Brandon E.P. Wailes L.A. Le K. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1997; 272: 3993-3998Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). It is well established that β-adrenergic stimulation of WAT leads to increased intracellular cAMP, activation of PKA, and stimulation of hormone-sensitive lipase by direct PKA phosphorylation (5Stralfors P. Belfrage P. J. Biol. Chem. 1983; 258: 15146-15152Abstract Full Text PDF PubMed Google Scholar, 6Stralfors P. Bjorgell P. Belfrage P. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3317-3321Crossref PubMed Scopus (196) Google Scholar). Accordingly, to determine the functional impact of RIIβ deficiency on PKA-mediated signaling, the effects of various β-adrenergic agonists upon lipolysis were assessed. Lipolysis was assayed in vitro by glycerol release from cultured adipose tissue. The basal rate of lipolysis is mildly increased in mutant WAT, as might be predicted in view of the increased basal PKA activity (Fig. 2 A). However, mutant WAT shows a severely blunted capacity for lipolytic stimulation by isoproterenol, a non-selective agonist of β1-, β2-, and β3-adrenergic receptors. All of these receptor subtypes are expressed in murine WAT (18Collins S. Daniel K.W. Rohlfs E.M. Ramkumar V. Taylor I.L. Gettys T.W. Mol. Endocrinol. 1994; 8: 518-527PubMed Google Scholar) and normally couple via a Gαs-adenylate cyclase mechanism to activate PKA and lipolysis. Isoproterenol increased the rate of lipolysis more than 5-fold in wild types but only approximately 50% in mutants (Fig. 2 B). The in vitro lipolysis experiments were repeated in the presence of adenosine deaminase (ADA), with and without the A1-selective adenosine receptor agonist PIA. In differentiated adipocytes, adenosine and PIA both suppress lipolysis by interacting with Gαi-coupled A1-receptors that lower cAMP levels. Significant and variable amounts of endogenous adenosine can be released from cultured adipocytes, confounding lipolysis assays (19Schwabe U. Ebert R. Erbler H.C. Naunyn-Schmiedeberg's Arch. Pharmacol. 1973; 276: 133-148Crossref PubMed Scopus (132) Google Scholar). ADA circumvents this problem by eliminating endogenous adenosine, thereby decreasing inter-assay variability (20Honnor R.C. Dhillon G.S. Londos C. J. Biol. Chem. 1985; 260: 15122-15129Abstract Full Text PDF PubMed Google Scholar) but also enhancing lipolysis (21Allen D.O. Quesenberry J.T. J. Pharmacol. Exp. Ther. 1988; 244: 852-858PubMed Google Scholar, 22Allen D.O. Ahmed B. Naseer K. J. Pharmacol. Exp. Ther. 1986; 238: 659-664PubMed Google Scholar). As expected, ADA increased basal and stimulated rates of lipolysis in both wild type and mutant WAT (Fig. 2 C). In contrast, PIA decreased these rates for both groups (Fig. 2 D), presumably by decreasing intracellular cAMP concentration (23Fain J.N. Mol. Pharmaco.l. 1973; 9: 595-604PubMed Google Scholar). However, the essential findings described above persisted in all conditions. Mutant WAT showed a slightly higher basal rate of lipolysis but a severely blunted capacity for hormone-mediated lipolytic stimulation. This defect was found equally with isoproterenol and the β3-selective agonist, CL 316,243 (24Bloom 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), both of which were added at concentrations previously shown to stimulate lipolysis maximally (25Mantzoros C.S. Qu D. Frederich R.C. Susulic V.S. Lowell B.B. Maratos F.E. Flier J.S. Diabetes. 1996; 45: 909-914Crossref PubMed Scopus (310) Google Scholar). In both wild type and mutant WAT the maximal lipolytic responses were equal for isoproterenol and CL. This might seem surprising, given that isoproterenol stimulates lipolysis via all three β-adrenergic receptor (β-AR) subtypes, all of which are expressed in WAT. However, our results agree with prior reports indicating that β3-ARs predominate in regulating lipolysis. In murine WAT, β1-, β2-, and β3-AR mRNA transcripts are expressed in a 3:1:150 ratio, respectively (18Collins S. Daniel K.W. Rohlfs E.M. Ramkumar V. Taylor I.L. Gettys T.W. Mol. Endocrinol. 1994; 8: 518-527PubMed Google Scholar), and it has been estimated that β3-ARs are responsible for at least 80% of the maximal isoproterenol-induced cAMP response (18Collins S. Daniel K.W. Rohlfs E.M. Ramkumar V. Taylor I.L. Gettys T.W. Mol. Endocrinol. 1994; 8: 518-527PubMed Google Scholar, 26Susulic V.S. Frederich R.C. Lawitts J. Tozzo E. Kahn B.B. Harper M.-E. Himms H.J. Flier J.S. Lowell B.B. J. Biol. Chem. 1995; 270: 29483-29492Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). Furthermore, it has been shown that various β3-selective agonists are equally potent as isoproterenol at activating PKA and lipolysis and that antagonism of β1- and β2-ARs has no effect on isoproterenol stimulation of either PKA or lipolysis (27Murphy G.J. Kirkham D.M. Cawthorne M.A. Young P. Biochem. Pharmacol. 1993; 46: 575-581Crossref PubMed Scopus (39) Google Scholar). Lipolysis was assayed in vivo by measuring serum glycerol levels in wild type and mutant mice injected with β-adrenergic agents. In agreement with the in vitro findings, RIIβ knockout mice showed a blunted capacity for lipolytic stimulation by both isoproterenol and CL 316,243 (Fig. 3). Both agents increased serum glycerol levels by 130% in wild types but by less than 30% in mutants. In contrast with the in vitro results, basal levels of serum glycerol were not altered in mutant mice nor were serum levels of free fatty acids (data not shown). To determine whether the perturbations in lipolysis seen in RIIβ mutants result from alterations in the expression of either β-ARs or HSL, steady-state mRNA levels of these gene products were measured in WAT using solution hybridization and Northern blot. Because the expression of β-ARs and HSL could be affected by the state of feeding (28Sztalryd C. Kraemer F.B. Am. J. Physiol. 1994; 266: E179-E185Crossref PubMed Google Scholar, 29Sztalryd C. Kraemer F.B. Metabolism. 1995; 44: 1391-1396Abstract Full Text PDF PubMed Scopus (55) Google Scholar), experiments were performed both in 24-h fasted and fed mice. In order to synchronize the feeding status of the latter group, mice were first fasted for 24 h and then refed for 6 h before being sacrificed. As shown in Fig. 4, there were no significant differences between knockout and wild type WAT with regard to the expression of HSL regardless of nutritional status. Solution hybridizations with β-adrenergic receptor probes also detected no changes in β3-receptor mRNA levels, but levels of β1- and β2-receptor mRNAs were too low to be accurately quantitated by solution hybridization. Therefore, Northern blots probed with β-AR-specific probes are shown in Fig. 4 B using RNA from fed animals. In order to determine the functional consequences of RIIβ deficiency on PKA-mediated gene expression in WAT, steady-state levels of mRNA from PKA-regulated genes were examined by solution hybridization. It has been shown previously that several of the enzymes involved in lipogenesis are transcriptionally regulated by PKA. In WAT, PKA inhibits the expression of acetyl-CoA carboxylase (ACC) (30Foufelle F. Gouhot B. Perdereau D. Girard J. Ferre P. Eur. J. Biochem. 1994; 223: 893-900Crossref PubMed Scopus (28) Google Scholar, 31Kim K.H. López C.F. Bai D.H. Luo X. Pape M.E. FASEB J. 1989; 3: 2250-2256Crossref PubMed Scopus (171) Google Scholar), lipoprotein lipase (LPL) (32Antras J. Lasnier F. Pairault J. Mol. Cell. Endocrinol. 1991; 82: 183-190Crossref PubMed Scopus (26) Google Scholar), and the insulin-responsive glucose transporter GLUT4 (33Kaestner K.H. Flores R.J.R. McLenithan J.C. Janicot M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1933-1937Crossref PubMed Scopus (86) Google Scholar). In contrast, PKA activation is associated with enhanced expression of PEPCK (30Foufelle F. Gouhot B. Perdereau D. Girard J. Ferre P. Eur. J. Biochem. 1994; 223: 893-900Crossref PubMed Scopus (28) Google Scholar). All of these genes are regulated by other factors, especially insulin, and their levels of expression vary with the state of feeding (34Girard J. Perdereau D. Foufelle F. Prip B.C. Ferré P. FASEB J. 1994; 8: 36-42Crossref PubMed Scopus (224) Google Scholar, 35Goodridge A.G. Annu. Rev. Nutr. 1987; 7: 157-185Crossref PubMed Scopus (159) Google Scholar). Accordingly, experiments were performed using mice subjected to 24-h fasting and 6-h refeeding protocols as described above. Surprisingly, expression of these PKA-regulated genes was regulated normally in RIIβ mutant WAT (Fig. 5), despite the RIIβ-to-RIα isoform switch and consequent perturbations of PKA activity described above. As expected, expression of ACC and GLUT4 was low in fasted mice and increased with refeeding. However, there were no significant differences between wild type and mutant mice in the fasted state, and only a small but significant (p < 0.05) increase in GLUT4 mRNA comparing mutant with wild type in the refed group. LPL expression was completely unaffected by the knockout in either fasted or refed animals. PEPCK expression showed the anticipated induction with fasting but was expressed similarly in wild types and mutants, regardless of feeding status. To verify that PKA-mediated gene expression is unperturbed in mutant WAT even though PKA-mediated lipolytic stimulation is blunted, we examined PEPCK gene expression and lipolysis simultaneouslyin vitro. Lipolysis assays were performed on cultured adipose tissue as described above, with aliquots of media harvested every 15 min for 2 h to determine glycerol content. Incubations with or without isoproterenol continued for a total of 6 h, after which all cells were harvested and subjected to solution hybridization to measure PEPCK mRNA content. As shown in Fig. 6, there was a 2-fold increase in basal (unstimulated) lipolysis in mutant WAT compared with wild type, but isoproterenol stimulated lipolysis by about 6-fold in wild type samples, compared with only 1.4-fold in mutants. In contrast, PEPCK mRNA expression from these cells was induced approximately 5-fold in both mutant and wild type samples, and there was no change in basal PEPCK mRNA comparing mutant with wild type. Leptin expression was inhibited dramatically by fasting in both wild type and mutant WAT (Fig. 5). There was no significant difference between the two fasted groups, although mRNA levels were near the minimal level of detection in our assay. Leptin was induced by refeeding in both groups; however, the mean level in refed mutants was nearly five times less than that in wild types, and although there were large animal to animal variations, the difference was statistically significant (p < 0.05). RIIβ null mutant mice offer a model to study the specific functions of individual PKA isoforms. In both WAT and BAT, RIIβ is normally the prevailing R subunit, expressed far more abundantly than any other isoform. Its loss is compensated for solely by an increase in RIα protein, producing a switch from a predominantly type II to type I holoenzyme (4Cummings D.E. Brandon E.P. Planas J.V. Motamed K. Idzerda R.L. McKnight G.S. Nature. 1996; 382: 622-626Crossref PubMed Scopus (361) Google Scholar, 17Amieux P.S. Cummings D.E. Motamed K. Brandon E.P. Wailes L.A. Le K. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1997; 272: 3993-3998Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). By studying adipose tissue in RIIβ mutants we can identify those PKA signaling functions that require an RII-type holoenzyme versus those that can also be subserved by an RI-containing enzyme. Because PKA signaling anomalies in RIIβ mutant mice could theoretically arise from changes in the number of R or C subunits, rather than from the isoform switch, we quantified these proteins in WAT. Whereas RIα compensation is virtually complete in mutant BAT (4Cummings D.E. B" @default.
• W2061520929 created "2016-06-24" @default.
• W2061520929 creator A5003209103 @default.
• W2061520929 creator A5017044526 @default.
• W2061520929 creator A5056510569 @default.
• W2061520929 creator A5089583678 @default.
• W2061520929 date "1999-12-01" @default.
• W2061520929 modified "2023-10-18" @default.
• W2061520929 title "Mutation of the RIIβ Subunit of Protein Kinase A Differentially Affects Lipolysis but Not Gene Induction in White Adipose Tissue" @default.
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