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J Biol Chem, Vol. 274, Issue 51, 36281-36287, December 17, 1999


Mutation of the RIIbeta Subunit of Protein Kinase A Differentially Affects Lipolysis but Not Gene Induction in White Adipose Tissue*

Josep V. PlanasDagger §, David E. Cummings§, Rejean L. Idzerda, and G. Stanley McKnight||

From the Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7750

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Targeted disruption of the RIIbeta subunit of protein kinase A (PKA) produces lean mice that resist diet-induced obesity. In this report we examine the effects of the RIIbeta knockout on white adipose tissue physiology. Loss of RIIbeta is compensated by an increase in the RIalpha 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 beta 3-selective and nonspecific beta -adrenergic agonists to stimulate lipolysis is markedly compromised in mutant white adipose tissue. This defect was found in vitro and in vivo and does not result from reduced expression of beta -adrenergic receptor or hormone-sensitive lipase genes. In contrast, beta -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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein kinase A (PKA)1 transduces 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 (1). 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 (RIalpha , RIbeta , RIIalpha , and RIIbeta ) and two C subunit isoforms (Calpha and Cbeta ) are transcribed in mice (2). 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 (3).

We have generated null mutant mice lacking the RIIbeta isoform of PKA (4). Unlike some PKA subunit isoforms that are expressed ubiquitously, RIIbeta demonstrates very restricted tissue distribution. It is most abundant in white and brown adipose tissue and brain, with very limited expression elsewhere. RIIbeta 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 RIIbeta 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 beta -adrenergic agonists, ACTH, and glucagon, all signaling via cAMP to stimulate PKA, which reversibly phosphorylates three serine residues on HSL to activate the enzyme (5-7) and promote translocation to lipid droplets (8, 9). Lipolysis is inhibited by insulin, which stimulates a phosphodiesterase (PDE3B) that lowers cAMP levels (10). 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 RIIbeta 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mice-- We have previously described the generation of RIIbeta mutant mice (4, 11). 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.

cAMP-binding Capacity-- 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 mM EDTA, 3 mM EGTA, 120 mM NaCl, 4 mM dithiothreitol, 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 (12) 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.

PKA Activity-- 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 mM dithiothreitol, 5 mM magnesium acetate, 250 mM sucrose, 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 (13), 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.

In Vitro Lipolysis Assays-- 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 (N6-[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 (14). Glycerol release data were converted from µg/ml glycerol to picograms of glycerol released per cell by assuming 6 pg DNA/cell.

In Vivo Effects of Lipolytic Agents on Serum Glycerol and Free Fatty Acid Levels-- Isoproterenol (0.3 mg/kg), CL 316,243 (1.0 mg/kg), or normal saline was injected intraperitoneally into wild type or RIIbeta 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.

Plasma Levels of Metabolites and Hormones-- 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).

Tissue cAMP Assay-- 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).

mRNA Quantitation-- The steady-state amount of mRNA derived from specific genes was determined by solution hybridization as described previously (15). Briefly, total nucleic acid was isolated from individual tissues by proteinase K digestion and phenol/chloroform extraction (16). 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 (beta -ARs).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of RIIbeta Knockout on PKA Holoenzyme Properties in WAT-- We have previously published data showing that RIIbeta is the predominant R subunit in WAT and that its loss in RIIbeta knockouts is compensated for in this tissue by a 3-4-fold increase of the RIalpha isoform, arising by RIalpha protein stabilization (17). This is the only R subunit adjustment in mutant WAT, as neither RIbeta nor RIIalpha 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 (17).

In order to determine the cAMP affinities of PKA from wild type versus RIIbeta mutant WAT, the cAMP-binding capacity of tissue homogenates was measured. As shown in Fig. 1A, the RIalpha -containing mutant holoenzyme binds cAMP more avidly than does the RIIbeta -containing wild type holoenzyme, with Kd values of 170 and 400 nM, respectively. As a consequence, mutant PKA is more readily activated by cAMP than is wild type enzyme (Ka values of 80 and 220 nM, respectively, Fig. 1B). This increased cAMP sensitivity is reflected by a 4-fold elevation of basal PKA activity in mutant WAT (Fig. 1B, inset).


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  		Fig. 1.   Increased cAMP sensitivity of PKA in RIIbeta mutant WAT. A, cAMP-binding capacity of wild type and RIIbeta mutant WAT. 160 µg of protein from WAT homogenates was incubated under equilibrium conditions with varying concentrations of [3H]cAMP. Proteins were then precipitated with NH4SO4, trapped on glass microfiber filters, and assessed for radioactivity. Nonspecific binding to the filters was measured and subtracted from all samples as described under "Experimental Procedures." Results represent averages ± S.D. from three experiments, each comparing one pair of mice. B, PKA activity of wild type (WT) and RIIbeta mutant WAT. Kinase activity with Kemptide as a substrate was measured in WAT homogenates incubated with varying concentrations of cAMP (for the activity curve) or in the presence or absence of 5 µM cAMP (cA, inset). The small amount of kinase activity not inhibited by PKI was subtracted as background from all samples. Results in the PKA activity curve are means ± S.D. of triplicate samples from two mice; results in the inset represent three mice per group, each assayed in triplicate.

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. 1A, the maximal cAMP-binding capacity (and thus, total R subunit content) of RIIbeta 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. 1B). We have previously published Western blot analysis showing a corresponding 43% decrease in the amount of C subunit protein in mutant WAT (17).

Lipolysis in Mutant WAT-- It is well established that beta -adrenergic stimulation of WAT leads to increased intracellular cAMP, activation of PKA, and stimulation of hormone-sensitive lipase by direct PKA phosphorylation (5, 6). Accordingly, to determine the functional impact of RIIbeta deficiency on PKA-mediated signaling, the effects of various beta -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. 2A). However, mutant WAT shows a severely blunted capacity for lipolytic stimulation by isoproterenol, a non-selective agonist of beta 1-, beta 2-, and beta 3-adrenergic receptors. All of these receptor subtypes are expressed in murine WAT (18) and normally couple via a Galpha s-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. 2B).


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  		Fig. 2.   In vitro lipolysis assays of RIIbeta mutant and wild type WAT. A, glycerol release over time from freshly cultured epididymal WAT in the presence or absence of 20 µM isoproterenol (Iso). B, total glycerol release over 1 h (between minutes 15 and 75) from Fig. 1A. C and D, glycerol release over 1 h from cultured mutant and wild type WAT in the presence or absence of 20 µM isoproterenol, 10 µM CL 316,243 (Cl), 1 unit/ml adenosine deaminase (ADA), or 10 µM PIA, as indicated. Results are means ± S.D. from five experiments in A and B and three experiments in each of C and D. WT, wild type; KO, knockout.

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 Galpha i-coupled A1-receptors that lower cAMP levels. Significant and variable amounts of endogenous adenosine can be released from cultured adipocytes, confounding lipolysis assays (19). ADA circumvents this problem by eliminating endogenous adenosine, thereby decreasing inter-assay variability (20) but also enhancing lipolysis (21, 22). As expected, ADA increased basal and stimulated rates of lipolysis in both wild type and mutant WAT (Fig. 2C). In contrast, PIA decreased these rates for both groups (Fig. 2D), presumably by decreasing intracellular cAMP concentration (23). 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 beta 3-selective agonist, CL 316,243 (24), both of which were added at concentrations previously shown to stimulate lipolysis maximally (25).

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 beta -adrenergic receptor (beta -AR) subtypes, all of which are expressed in WAT. However, our results agree with prior reports indicating that beta 3-ARs predominate in regulating lipolysis. In murine WAT, beta 1-, beta 2-, and beta 3-AR mRNA transcripts are expressed in a 3:1:150 ratio, respectively (18), and it has been estimated that beta 3-ARs are responsible for at least 80% of the maximal isoproterenol-induced cAMP response (18, 26). Furthermore, it has been shown that various beta 3-selective agonists are equally potent as isoproterenol at activating PKA and lipolysis and that antagonism of beta 1- and beta 2-ARs has no effect on isoproterenol stimulation of either PKA or lipolysis (27).

Lipolysis was assayed in vivo by measuring serum glycerol levels in wild type and mutant mice injected with beta -adrenergic agents. In agreement with the in vitro findings, RIIbeta 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).


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  		Fig. 3.   Assessment of lipolysis in vivo from RIIbeta mutant and wild type mice. Serum glycerol was measured 20 min after intraperitoneal injection of normal saline, isoproterenol (0.3 mg/kg), or CL 316,243 (1.0 mg/kg). Results are means ± S.D. (n = 9/group for base line, n = 7/group for isoproterenol, n = 9/group for CL 316,243). WT, wild type; KO, knockout.

Expression Levels of HSL and beta -AR Genes-- To determine whether the perturbations in lipolysis seen in RIIbeta mutants result from alterations in the expression of either beta -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 beta -ARs and HSL could be affected by the state of feeding (28, 29), 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 beta -adrenergic receptor probes also detected no changes in beta 3-receptor mRNA levels, but levels of beta 1- and beta 2-receptor mRNAs were too low to be accurately quantitated by solution hybridization. Therefore, Northern blots probed with beta -AR-specific probes are shown in Fig. 4B using RNA from fed animals.


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  		Fig. 4.   Expression levels of HSL and beta -AR mRNAs in wild type and mutant WAT. A, HSL mRNA was measured by solution hybridization using riboprobes as described under "Experimental Procedures." Animals were either fasted 24 h or fasted and then refed for 6 h before harvesting WAT tissue for nucleic acid extraction. B, Northern blots were run on RNA isolated from total nucleic acid by LiCl precipitation to remove DNA. 15 µg of total RNA was run on each lane of a formaldehyde gel, and the transferred RNA was hybridized to DNA probes specific for each of the beta -AR genes following standard techniques (59). Exposure was overnight for beta 3 and 6 days for beta 2 and beta 1 mRNA. The beta 3 transcripts were very similar to the 2.1, 2.6, and 3.5-kilobase pair mRNAs described previously, and the beta 2 and beta 1 mRNAs were approximately 2.2 and 2.6 kilobase pairs, respectively (18).

PKA-dependent Gene Expression in RIIbeta Knockout WAT-- In order to determine the functional consequences of RIIbeta 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) (30, 31), lipoprotein lipase (LPL) (32), and the insulin-responsive glucose transporter GLUT4 (33). In contrast, PKA activation is associated with enhanced expression of PEPCK (30). All of these genes are regulated by other factors, especially insulin, and their levels of expression vary with the state of feeding (34, 35). 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 RIIbeta mutant WAT (Fig. 5), despite the RIIbeta -to-RIalpha 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.


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  		Fig. 5.   WAT gene expression. Solution hybridizations using total nucleic acid and riboprobes were used to quantitate the levels of the glucose transporter (GLUT4), lipoprotein lipase (LPL), phosphoenolpyruvate carboxykinase (PEPCK), acetyl-CoA-carboxylase (ACC), and leptin mRNA. Results are expressed as molecules per cell based on the specific activity of the probe and a value of 6 pg of DNA per cell. The mean ± S.D. is shown for five animals in each group. Statistically significant differences (p < 0.05) were determined by one-way analysis of variance, followed by the Fisher Protected Least Significant Difference test. Different letters over the error bars indicate significant differences. WT, wild type; KO, knockout.

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 simultaneously in 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.


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  		Fig. 6.   In vitro lipolysis and PEPCK gene expression in RIIbeta mutant and wild type WAT. Glycerol release was determined from cultured epididymal WAT in the presence or absence of 20 µM isoproterenol as in Fig. 2B. Cells were harvested after 6 h of incubation and subjected to solution hybridization to determine PEPCK mRNA levels. PEPCK results are means ± S.D. of triplicate samples.

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).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RIIbeta null mutant mice offer a model to study the specific functions of individual PKA isoforms. In both WAT and BAT, RIIbeta is normally the prevailing R subunit, expressed far more abundantly than any other isoform. Its loss is compensated for solely by an increase in RIalpha protein, producing a switch from a predominantly type II to type I holoenzyme (4, 17). By studying adipose tissue in RIIbeta 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 RIIbeta 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 RIalpha compensation is virtually complete in mutant BAT (4), there is a 30% loss of R subunits overall in mutant WAT, as assessed by total cAMP-binding capacity. However, there is also an approximately 50% loss of C subunits in this tissue, as judged by Western blot analysis and total PKA activity. Since an R:C ratio of at least 1:1 is maintained, the increased basal PKA activity seen in mutant WAT cannot be accounted for by a gross disregulation of C subunits due to an inadequate quantity of R subunits. Altered PKA activity also does not appear to be caused by any difference in overall cAMP concentration in mutant compared with wild type WAT (379 ± 177 versus 359 ± 64 nM, respectively). Instead, the increased basal PKA activity probably arises because the type I PKA in mutants has a greater affinity for cAMP and thus activates more easily than does the type II enzyme in wild types. In this regard, the PKA alterations seen in RIIbeta mutant WAT are analogous to those previously described in BAT (4). Both tissues show a 4-5-fold increase in basal PKA activity but a decrease in total cAMP-stimulated activity. The only differences between mutant WAT and BAT with respect to changes in PKA appear to be quantitative. Compared with BAT, WAT shows a greater loss of both total R subunits (30% decrease versus 10%) and C subunits (50% decrease versus 30%).

Loss of RIIbeta from mutant WAT markedly impairs PKA-mediated activation of a pre-formed enzyme (HSL), yet leaves PKA-regulated gene expression relatively unperturbed. The disruption of lipolytic stimulation is seen both in vitro and in vivo and occurs equally for signaling from the beta 3-receptor-specific agonist CL 316,243 and a nonspecific beta -agonist, isoproterenol. It is unlikely that the signaling defect in RIIbeta mutant WAT is caused by decreased expression of either beta -ARs or HSL, given that mRNA levels for these genes are unaffected. Conceivably, impairment of lipolytic hormonal response could be a consequence of the chronic stimulation of basal lipolysis seen in RIIbeta knockouts. However, adipocytes from transgenic mice deficient in the G-protein subunit, Gialpha 2, show a 3-fold increase in basal cAMP levels and an elevated basal rate of lipolysis but retain normal maximal response to beta -adrenergic agonists (36). Thus, continuous stimulation of basal lipolysis alone does not appear to limit lipolytic response to hormones. Another possibility is that maximal lipolytic stimulation in mutant WAT is rate-limited by the 50% loss of total C subunit protein in this tissue, whereas levels of C subunit are not rate-limiting for gene induction.

We favor the hypothesis that at least some of the elements mediating lipolytic stimulation (e.g. beta -ARs, adenylate cyclase, PKA, and HSL) may be co-localized within adipocytes to facilitate efficient signal transduction, and that lipolytic stimulation is impaired in RIIbeta mutants because RIIbeta participates specifically in the formation of this complex. The following observations from previous studies suggest the existence of a compartmentalized apparatus mediating lipolytic stimulation. (i) At any given intracellular cAMP concentration, the lipolytic response from catecholamines is greater than that from forskolin, a nonspecific adenylate cyclase activator (21, 22, 37). Hence, low concentrations of isoproterenol (~10 nM) can stimulate lipolysis without measurably altering overall cAMP levels, whereas low concentrations of forskolin (0.1-1.0 µM) increase intracellular cAMP levels without affecting lipolysis (37). (ii) The concentration of isoproteronol or beta 3-specific agonists required for half-maximal activation of adenylate cyclase activity in adipocyte membranes is ~80× greater than the concentration required to activate lipolysis (27, 38, 39). (iii) Recently a signaling complex including beta 2-AR, PKA, and phosphatases has been isolated that appears to be assembled by the scaffold protein, gravin (40). In summary, catecholamines stimulate lipolysis more potently than they increase overall intracellular cAMP, suggesting a preferential association between receptor, PKA, and perhaps its substrate, HSL.

In numerous cell types, co-localization of key components involved in PKA signaling is accomplished by protein kinase A anchoring proteins (AKAPs), multivalent binding proteins that serve as platforms for the assembly of signal transduction modules (41, 42). These targeting proteins bind simultaneously to specific sites on the amino terminus of R subunits and to discrete subcellular structures. By tethering PKA at precise intracellular sites, AKAPs ensure that the kinase is exposed to localized changes in cAMP adjacent to appropriate substrates, thus preventing cross-talk between functionally unrelated PKA signaling units within the same cell. Although AKAPs have not yet been described in WAT, they have been found in virtually all other tissues investigated. We have preliminary data demonstrating AKAPs expressed in WAT,2 and it seems reasonable to speculate that they may facilitate phosphorylation of HSL by PKA.

Since most of the mammalian AKAPs identified to date are strongly RII-specific, PKA-AKAP binding might be disrupted in RIIbeta mutant WAT due to the type II to type I isoform switch. This alteration could displace the PKA holoenzyme from local waves of cAMP generated by beta -adrenergic agonists and/or from HSL, which would explain the blunted lipolytic response to adrenergic stimuli seen in mutant WAT. Indeed, disruption of RII-AKAP binding has been shown to have diverse functional consequences in other model systems (43-45). Our findings that PKA-mediated gene expression remains relatively intact in RIIbeta mutant WAT suggest that high affinity anchoring is not required for this particular PKA function.

Leptin and to a lesser extent GLUT4 were the only gene products whose expression was markedly different in RIIbeta mutant compared with wild type WAT, comparing the refed state. Leptin is a long term anorexigenic factor, and in most settings its level mirrors the amount of body adiposity (46-49). We found the same strong positive correlation of leptin expression with feeding that has been reported previously (50-53). Because mRNA levels were near the lower limit of detection in fasted mice, it is difficult to determine whether the lack of a significant difference between mutants and wild types in this condition is meaningful. However, in fed mice there was nearly 5-fold less leptin mRNA in mutants than in wild types. It is possible that leptin expression is inhibited in mutants because of their abnormally high basal PKA activity, since leptin expression is reduced by agents that activate PKA (54-56). However, leptin levels in mutant mice may be low simply due to the reduced body fat.

The lean phenotype and resistance to diet-induced obesity observed in RIIbeta knockout mice could derive from biochemical defects characterized previously in BAT (4), changes in WAT metabolism described in this paper, or direct alterations in neuronal activity in regions of the brain that regulate feeding behavior or energy expenditure. The RIIbeta gene is highly expressed in each of these tissues, and gene knockout leads to significant changes in PKA activity in each tissue. In BAT, RIIbeta mutation causes an increase in both uncoupling protein 1 expression and basal metabolic rate. In RIIbeta mutant WAT we find an elevated basal rate of lipolysis when measured in vitro and a dramatic deficiency in the lipolytic response to beta -AR stimulation that is observed both in vitro and in vivo. Could these changes in BAT and WAT be sufficient to provoke a chronically lean phenotype?

It is unlikely that a stable lean phenotype could derive solely from peripheral changes in adipose tissue without some alteration in the neuronal systems regulating feeding behavior and energy expenditure. Loss of peripheral energy stores induced by interventions such as food restriction, exercise, or surgical excision of WAT are associated with reduced leptin levels that trigger centrally mediated increases in food intake and decreases in energy expenditure (57). Mutant WAT synthesizes substantially less leptin than does wild type, as evidenced by the decrease in leptin mRNA shown in Fig. 5 and a chronic 3-fold decrease in circulating leptin.3 The observation that RIIbeta mutants do not overeat in the setting of low fat stores and low leptin levels suggests that their central nervous system regulation of food intake is impaired. They appear to be hypersensitive to the inhibitory effects of leptin on feeding behavior and therefore do not increase food intake to the extent expected for chronically leptin-deficient animals. Possibly the mutation in PKA signaling alters the production of another factor from either WAT or BAT that modifies feeding behavior and prevents the mice from replenishing their fat stores by overeating.

The alternative possibility that the lean phenotype derives more directly from neuronal alterations in PKA is supported by three observations as follows: (i) the brain is the major integrator of signals that control both feeding and energy expenditure, and RIIbeta is highly expressed in the hypothalamus, the expected site of this integration; (ii) cAMP and PKA are strongly implicated as key intracellular mediators of several central regulators of energy balance, such as alpha -melanocyte-stimulating hormone, neuropeptide Y, and corticotropin releasing hormone; (iii) PKA alterations in the brain of RIIbeta knockout mice lead to defective motor learning (11) and resistance to the biochemical and behavioral effects of a D2-dopamine receptor antagonist, haloperidol (58), demonstrating that signaling is affected in neural tissue. Genetic approaches that allow the manipulation of PKA within specific tissues may help to resolve the relative contribution of neuronal and adipose tissues to the changes in body weight regulation seen in PKA mutant mice.

    ACKNOWLEDGEMENTS

We thank Bradford Lowell and Vedrana Susulic for their advice and reagents for lipolysis assays and Thong Su for superb technical assistance. We also thank S. Collins, D. S. Weigle, M. C. Schotz, D. K. Granner, K.-H. Kim, and M. D. Lane for their generous gifts of DNA probes.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM82375 (to G. S. M.) and by a grant from Pfizer Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avenue Diagonal 645, 08028 Barcelona, Spain.

§ Both authors contributed equally to this work.

Supported by National Institutes of Health Grant DK01964.

|| To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, Box 357750, Seattle, WA 98195-7750. Tel.: 206-616-4237; Fax: 206-616-4230; E-mail: mcknight@u.washington.edu.

2 A. Sikorski and G. S. McKnight, unpublished observations.

3 S. Schreyer, D. E. Cummings, G. S. McKnight, and R. LeBoeuf, unpublished data.

    ABBREVIATIONS

The abbreviations used are: PKA, protein kinase A; R, regulatory; C, catalytic; PKI, protein kinase inhibitor; BAT, brown adipose tissue; WAT, white adipose tissue; HSL, hormone-sensitive lipase; ACC, acetyl-CoA carboxylase; LPL, lipoprotein lipase; PEPCK, phosphoenolpyruvate carboxykinase; beta -AR, beta -adrenergic receptor; PIA, phenylisopropyladenosine; ADA, adenosine deaminase; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; AKAP, protein kinase A protein; PIA, N6-[R-(-)-1-methyl-2-phenyl]adenosine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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