Enhanced Lipolysis In Visceral And Subcutaneous Fat In Chronic Renal Failure

Background:White adipose tissue (WAT) plays an important role in regulating whole-body energy homeostasis.One of its major roles is to release fatty acids (FAs) under conditions of negative energy balance or prolonged exercise to provide energy for peripheral tissues.The molecular machinery involved in triacylglycerol (TAG) breakdown and FA release works in an orderly and regulated fashion, conferring to WAT the capacity to respond to various feeding conditions and to the energy demands of the body. The control of lipolysis is complex and involves multiple mechanisms.These include lipolytic (β-adrenergic agonists, ACTH, etc.) and anti-lipolytic(insulin, adenosine, etc.)hormones.Importantly, conditions that disrupted normal regulation of WAT lipolysis will result in lipotoxicity. Evidence from human studies and animal models suggests that lipid accumulation in the heart, skeletal muscle, pancreas, liver, and kidney play an important role in the pathogenesis of heart failure, obesity and diabetes.In addition, protein energy wasting, a state of decreased body stored protein and fat, has been observed and linked to the poor outcome in chronic renal failure.The mechanisms underlying the fat loss in CRF remains unclear.Accumulation of advanced oxidation protein products (AOPPs) has been found in subjects with metabolic syndrome as well as in patients with diabetes. AOPPs are the dityrosine-containing and cross-linking protein products formed during oxidative stress by reaction of plasma protein with chlorinated oxidants. Accumulation of AOPPs has been considered the marker of oxidant-mediated protein damage. Our recent studies have shown that chronic accumulation of AOPPs promotes inflammation in diabetic and non-diabetic kidney and worsen inflammation and oxidative stress in artery in a hyperlipidemic model. These data suggest the oxidized proteins, by themselves, may contribute to the persistent inflammation in the tissue. Furthermore, in vitro studies have shown AOPPs induce perturbation of various cells such as vascular endothelial cell, renal epithelia cells, and glomerular mesangial cell, suggesting AOPPs might be a pathological factor in cell function. Whether increased level of AOPPs, as seen in metabolic syndrome and type 2 diabetes, affects lipolysis of adipocytes remains unclearLipolysis in the WAT of humans and rodents is regulated in a step-wise fashion by adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and monoacylglycerol lipase (MAGL). The current literature suggests that in lean rodents ATGL and HSL are the major lipases for TAG and DAG, respectively, and account for～95% of lipase activity in murine WAT The current model is that ATGL initiates lipolysis by cleaving the first FA from TAG and then HSL and MAGL act on diacyglycerol (DAG) and monoacylglycerol, respectively, releasing two additional FAs and one glycerol molecule. Therefore, the orchestrated activation of ATGL, HSL, and MAGL seems to be required for complete lipolysis to occur in adipocytes. Binding of agonists to theβ-adrenergic receptors, coupled to adenylate cyclase via the stimulatory G protein, leads to an increase in cAMP and activation of protein kinase A (PKA). In rat HSL, PKA phosphorylates serine residues 563,659, and 660, leading to translocation of HSL to the lipid droplet and to great enhancement of lipolysis. Phosphorylation of perilipin A, a protein associated with the lipid droplet, by PKA has also been demonstrated to be necessary for activation of HSL and for catecholamine-induced lipolysis to occur. ATGL activity is also stimulated by catecholamines, but the molecular mechanism(s) underlying this effect is unknown. Although ATGL can be phosphorylated on serine residues 404 and 428 by a yet unidentified kinase, this does not seem to affect the activity of this lipase. There is compelling evidence that the protein comparative gene identification-58 (CGI-58) drastically enhances ATGL-mediated TAG hydrolysis without affecting HSL activity. Under basal conditions, CGI-58 is also localized to the lipid droplet in association with perilipin A. Upon hormonal stimulation, PKA phosphorylates perilipin A at serine residues 492 and 517, resulting in CGI-58 dissociation.Once dissociated, CGI-58 interacts with ATGL and potently activates this TAG lipase. In fact, addition of CGI-58 to ATGL containing extracts enhances TAG hydrolase activity by-20-fold.Lipolytic stimuli increase lipolysis by activating adenylyl cyclase and raising intracellular concentrations of cyclic AMP, with resultant activation of cyclic AMP-dependent protein kinase (PKA), which phosphorylates both perilipins and HSL. The phosphorylation of HSL is associated with an increase in hydrolytic activity of the enzyme and the translocation of HSLfrom the cytosol to the lipid droplet in some physiological settings. PKA has been shown to phosphorylate HSL at residues Ser 563, Ser 659, and Ser 660, all of which reside in a 150-amino acid stretch, termed the regulatory module.The regulatory module is found within the C-terminal domain of HSL, which also contains the catalytic triad.Although it is generally accepted that lipolysis is stimulated by PKA activation, as a consequence of GTP-binding protein (G protein)-coupled receptors acting through adenylyl cyclase and cyclic AMP, there is accumulating evidence to suggest that, in addition to PKA, G protein-coupled receptors and cyclic AMP can also activate mitogen-activated protein kinase (MAPK) pathways. it was proposed that ERK1/2 phophorylates HSL on Ser-600 and that this leads to increased activity of the enzyme.Loss of protein and fat stores, presenting as clinical wasting, is reported to have a prevalence of 30-60% and is an important risk factor for mortality in chronic kidney disease (CKD) patients.Resting energy expenditure is high in patients with cachexia from renal failure,moreover,fat re-distribution has been densitied in a model of CRF.The aim of the study is to investigate that weather abnormal lipolysis occurs in fat in a rat model of chronic renal failure and its related mechanisms.As yet, there is no research about whether AOPPs has an impact on lipolysis of adipocyte.Therefore,we test the possibility that induce lipolysis of adipocyte by AOPPs. In this study, we culture 3T3-L1 preadipocytes as the model in vitro to observe the effect of AOPP on lipolysis.MethodsThe first part:studies in vivo1.Animal model preparationsSD male rat were maintained on a 12/12-h light/dark cycle at 22℃and fed ad libitum a standard laboratory chow for a 1 week acclimation period.Subsequently, the animals were randomly assigned to two groups:sham (n=8), chronic renal failure(n=12), Surgical five-sixth nephrectomy (5/6Nx) and sham operations in rats were performed。Briefly, under pentobarbital anaesthesia, two-thirds of the left kidney was removed in the first stage of the procedure, and a week later the right kidney was totally excised (totally 5/6Nx). Control animals were sham-operated with only decapsulation of the kidney.2. Measurement of lipolysis in WAT fat pads ex vivo.Epididymal and inguinal,retroperitoneal fat pads were removed from sham-operated grop and 5/6Nx group, weighed and carefully minced thoroughly (20-30mg) using microscissors.fat pads were incubated at 37℃for some hours in the presence of Krebs-Ringer bicarbonate buffer containing fatty acid-free bovine serum albumin (2%BSA). After the incubation period, glycerol release into the medium was determined and the results were expressed as micromoles of glycerol released per 100 mg of adipose tissue per hour. The fat pads from the sham-operated rats served as negative controls for these experiments.3.Isolation and ceiling culture of adipocyte and identification of adipocyteIsolation of mature adipocytes from fat tissue was described above. Briefly, approximately 1 g of fat tissue was minced and digested in 0.75mg/ml collagenase solution (Collagenase typeⅠ) at 37℃for 1 h with gentle agitation.After filtration and centrifugation at 400g for 5 min and wash with phosphate-buffered saline (PBS), the floating top layer containing unilocular adipocytes was collected.cells (1×104-5×104) were placed in 25-cm2 culture flasks filled completely with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and were incubated at 37℃in 5% CO2. Cells floated up and adhered to the top inner ceiling surface of the flask. After 7 days, the medium was removed, and the flasks were inverted so that the cells were on the bottom. Application of oil red O staining to identify the mature adipocyte. Adipocytes were fixed with 3.7% paraformaldehyde for15-20 min., stained with 0.5% Oil Red O [w/v, dissolved in 60% isopropanol solution (diluted with water), filtered twice with filter paper], for 1h at room temperature. Then washed with PBS to remove unbound dye. Cells were visualized by light microscopy and photographed.4.basal and Isoproterenol-stimulated lipolysis in isolated rat adipocyteBriefly, the inguinal and epididymal, retroperitoneal fat pads were used as representative of subcutaneous (SC) and visceral (VC) adipose tissue. SC and VC fat pads were harvested and placed in weighing boats containing PBS (pH 7.4) at room temperature. Fat pads were weighed, and minced thoroughly (2-3 mm pieces in diameter) in collagenase solution (0.75mg/ml). This mixture was transferred to a 30 ml narrow-mouthed polypropylene bottle and incubated at 37℃with shaking at 220 rpm for 1 h. After digestion, the mixture was filtered through a 250-μm gauze mesh into a 50 ml conical polypropylene tube and was centrifuged for 5 minute at 400xg. The fat cells floated to the surface, and the stromal-vascular cells (capillary, endothelial, mast, macrophage, and epithelial cells) were sedimented.The stromal-vascular cells were removed by aspiration, and the fat cells were washed by suspending them in 10 ml of 200nM adenosine-replete KRB-2%BSA and centrifuging for 5 minute at 400 x g. This procedure was repeated three times. Stromal-vascular cells were absent, by histological examination, from the fat cell preparation after three washes. After centrifugation, resuspend 500ul of packed adipocytes in 1.5 mL of KRB-2%BSA for subsequent lipolysis assay. To assess the effects of isoproterenol on lipolysis, adipocytes were treated with 10umol/l isoproterenol for some hours. The medium was then collected and assayed for glycerol. The results were expressed as millimicromoles of glycerol released per ug of adipocyte protein per hour.5. Preadipocyte isolation and culture and identificationPreadipocytes were isolated by digesting freshly isolated adipose tissue with 0.75 mg/ml collagenase in Dulbecco's modified Eagle's medium (DMEM) for 1 h at 37℃in a shaking water bath. Stromal-vascular cells (containing preadipocytes) were spun down and washed twice with DMEM and filtered through a polypropylene mesh (pore size 250-um). After centrifugation, the cell pellet were resuspended in DMEM with 10% FBS and cultured in T25 flasks.2 days after reaching confluence (Day 0), the cell culture medium was changed to differentiation medium (high glucose DMEM supplemented with 10% FBS,0.5mmol/L isobutylmethylxanthine, 10μg/ml insulin,0.25μmol/L dexamethasone) for 48 h to induce differentiation. Then, the medium was replaced with high glucose DMEM with 10% FBS and 10μg/ml insulin. After Day 4, cells were maintained in DMEM plus 10% FBS with a media change every other day.Until on day 8-12, preadipocytes were differentiated into multiloculated mature adipocytes. Application of oil red O staining to identify the differentiation of preadipocytes. Adipocytes were fixed with 3.7% paraformaldehyde for15-20 min., stained with 0.5% Oil Red O [w/v, dissolved in 60% isopropanol solution (diluted with water), filtered twice with filter paper], for 1h at room temperature. Then washed with PBS to remove unbound dye. Cells were visualized by light microscopy and photographed. Isopropyl alcohol was added to dissolve the oil red O, the OD at 490 nm was determined by spectrophotometer. The results were expressed as TG accumulation(OD) per mg protein.6. Glycerol assayGlycerol content released in incubation medium served as an index of lipolysis and was determined at the absorption at 540 nm, by use of a colorimetric assay (GPO Trinder reaction) kit from sigma-aldrich. Lipolysis data were expressed as millimicromoles of glycerol per ug protein of adipocytes or mildigram adipose tissue per hour.7. Immunoblot AnalysisAdipose tissue was homogenized in ice-cold fractionation buffer (50 mM Tris-HCl, pH 7.4,1 mM EDTA,0.1 mM sodium orthovanadate [Na3VO4], 50 mM sodium fluoride [NaF],1μM leupeptin,1μM pepstatin and 20mM PMSF). cytosolic fractions were prepared following the procedures. The cell lysate was incubated on ice for 15 min and then centrifuged at 13, 000×g for 40 min at 4℃. The cytosolic fraction was localized below the layer of the fat cake. The cytosolic fraction was aspirated from below the solidified fat cake, and an aliquot was used to measure protein by the Bradford method.Samples were diluted 4:1 (v/v) with 5x Laemmli buffer, heated to 95℃and subjected to SDS-PAGE.Following SDS-PAGE the samples were transferred onto polyvinylidine difluoride membranesand and were blocked with 5% non-fat milk in TBS-T buffer (150 mM NaCl,20 mM Tris-HCl, pH 7.4,0.05% Tween-20), and incubated at 4℃for overnight with antibodies against rabbit anti-HSL antibody (1:5000), rabbit anti-HSLser563 antibody (1:1000), rabbit anti-HSL ser660 antibody (1:1000), rabbit anti-ATGL antibody (1:500), rabbit anti-CGI58 antibody (1:200), rabbit anti-P-PKA antibody (1:1000), rabbit anti-PKA antibody (1:1000), rabbit anti-P-ERK(1:2000) antibody, rabbit anti-ERK(1:2000) antibody, rabbit anti-P-JNK antibody (1:1000), rabbit anti-JNK antibody (1:1000), rabbit anti-P-P38 antibody (1:1000), rabbit anti-P38(1:1000) antibody,and mouse anti-β-actin antibody respectively. After being washed for 3×10 min in TBS-T buffer, the membranes were probed for 1 h with secondary antibodies conjugated to horseradish peroxidase. The blots were washed and then developed by use of an enhanced chemiluminescent detection method.8. StatisticsAll values are presented as mean±SEM. The significance of differences among mean values was determined by Independent-Samples T Test. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was<0.05.The second part:studies in vitro1. Preparation of AOPPsAOPP was prepared in vitro according to the method described by Witko-Sarsat and our laboratory. Briefly, endotoxin free MSA was incubated with HOCl at molar ratio 1:140 at room temperature for 30 min and then dialyzed against PBS to remove any free HOCl in the solution.AOPPs content was determined by measuring absorbance at 340 nm in acidic condition and was calibrated with Chloramines-T.2. Cell culture3T3-L1 preadipocytes were obtained from American Type Culture Collection (No.CL-173) and were cultured in high glucose Dulbecco's minimum essential medium (DMEM) with 10% fetal bovine serum (PAA,German),100 U/ml penicillin and 0.1 mg/ml streptomycin at 37℃in a 5% CO2 incubator.2 days after reaching confluence (Day 0), the cell culture medium was changed to differentiation medium (high glucose DMEM supplemented with 10% FBS, 0.5mmol/L isobutylmethylxanthine, 10μg/ml insulin,0.25μmol/L dexamethasone) for 48 h to induce differentiation. Then, the medium was replaced with high glucose DMEM with 10% FBS and 10μg/ml insulin. After Day 4, cells were maintained in DMEM plus 10% FBS with a media change every other day until experimental treatments were initiated.8-10 days after differentiation,3T3-L1 cells were incubated in DMEM overnight and treated with AOPP for the indicated concentration and time.3. Measurement of lipolysisDifferentiated 3T3-L1 preadipocytes were incubated with AOPPs in serum-free DMEM containing 2% BSA for different times and doses oor in the presence of 200 u g/ml AOPPs in combination with the specific MAPK inhibitors: SP600125,SB203580, H89 and PD98059 (all from Sigma) in free medium. All inhibitors were added 2 h before the initiation of stimulation to diminish intrinsic activity to a minimum. Glycerol content in the incubation medium was used as an index for lipolysis and was measured using a colorimetric assay (GPO-Trinder;Sigma). Results were corrected for cellular proteins, which were quantified using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) and were expressed as micromoles of glycerol per milligrams of protein.4. Detection of the protein expression of HSL,ATGL and CGI-58Cells were exposed to 200μg/ml unmodified MSA or 200μg/ml AOPPs for indicated time,0,6h,12 h and 24 h;or for indicated concentration,0,50,100,200μg/ml. After incubution,adipocytes were rinsed briefly with 1 ml of phosphate buffered saline (PBS). Proteins were extracted separated by 10% SDS-PAGE, and electrophoretically transferred to polyvinylidine difluoride membranes.Equivalent amounts of protein were loaded onto the gel for each treatment.Proteins were detected with enhanced chemiluminescence (ECL) system. Rabbit polyclonal anti-ATGL,HSL AND CGI-58 were used for Western blotting.5. Determination Phosphorylation activity of HSLCells were exposed to 200μg/ml unmodified MSA or 200μg/ml AOPPs for indicatedtime0,1h,3h,6h,12hand24h;or for indicated concentration,0,50,100,200μg/ml. Proteins were extracted,separated by 10% SDS-PAGE, and electrophoretically transferred to polyvinylidine difluoride membranes.Equivalent amounts of protein were loaded onto the gel for each treatment.Proteins were detected with enhanced chemiluminescence (ECL) system. Rabbit polyclonal anti-HSL ser563 and ser660 were used for Western blotting.6. Determination Phosphorylation activity of PKACells were exposed to 200μg/ml unmodified MSA or 200μg/ml AOPPs for indicated time,0,1 h,3 h 6h,12 h. Phosphorylation activity of PKA was detected by western blotting7. Determination Phosphorylation activity of MAPK familyCells were exposed to 200μg/ml unmodified MSA or 200μg/ml AOPPs for indicated time,0,1 h,3 h 6h,12 h. Phosphorylation activity of ERK,JNK and P38 were detected by western blotting.8. Statistics All values are mean±SEM. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was<0.05.ResultsThe first part:studies in vivo1. General dataData are summarized in Table 1. As expected, the CRF group exhibited a significant increase in serum creatinine and BUN concentration. In addition, the CRF animals showed a significantiy higher serum glycerol and a significantly lower body wt when compared with the sham-operated normal control group. Serum triglyceride concentration was markedly elevated in the CRF group as compared to the normal control animals.2.Lipolysis in VC and SC fat padsCompared to sham group, the CRF group exhibited a increased lipolysis in both SC(P<0.05) and VC(P<0.05) fat pads.3.Basal and hormonal regulation of lipolysis in isolated rat adipocyteCompared to sham group, the CRF group exhibited a increased lipolysis in VC(P<0.05) and SC(P<0.05) isolated rat adipocyte in both basal and hormonal-stimulated conditions.4.Differentiation of preadipocyteThe ratio of differentiation of preadipocyte in two groups have no change in SC(P>0.05) and VC(P>0.05) adipose tissue.5.HSL, MAPK and PKA phosphorylation and protein expression of HSL, ATGL, and CGI-58 in SC AND VC adipose tissue5.1 the phosphorylation of HSL on key serine residues and the total HSL protein expression in SC and VC adiposeIn order to assess the effects of chronic renal failure increased lipolysis in WAT, we examined phosphorylation of HSL on key serine residues as well as the protein content of HSL. Phosphorylation of HSL at serine 563 and 660 revealed that these variables were potently raised in the VC(P<0.05) and SC(P<0.05) adipose tissue of CRF Rats, despite no change in total HSL in the VC(P>0.05), but the content of HSL was markedly increased in SC(P<0.05)5.2 the expression of ATGL in SC and VC adipose tissueThe content of ATGL of sham-operated and CRF groups in VC adipose tissue have no change(P>0.05), but in SC adipose tissue the content of ATGL were significantly increased(P<0.05).5.3 the expression of CGI-58 in SC and VC adipose tissueIn VC(P<0.05) and SC(P<0.05) adipose tissue, the protein level of CGI-58 markedly increased in CRF rats relative to control animals.5.4 the binding of ATGL and CGI-58 protein in SC and VC adipose tissueIn order to assess the activity of ATGL, immunoprecipitation were applicated to evaluate the binding of ATGL with CGI-58. In VC(P<0.05) and SC(P<0.05) adipose tissue,the binding of ATGL with CGI-58 were significantly increased in CRF rats relative to control animals.5.5 the phosphorylation of PKA in SC and VC adipose tissueSince PKA phosphorylates and activates HSL, the phosphorylation and content of this kinase were determined. Phosphorylation of PKA was increased in both SC(P<0.05) and VC(P<0.05) adipose tissue of CRF Rats relative to control animals.5.6 the phosphorylation of MAPKs in SC and VC adipose tissueRecent studies indicated that ERK and JNK also regulated lipolysis, we also determined the phosphorylation and content of P44/42(P<0.05), JNK1/2(P<0.05), P38(P<0.05). Phosphorylation of MAPKs were significantly increased in the VC and SC adipose tissue of CRF Rats relative to control animals.Conclusion1.The lipolysis of fat is increased in chronic renal failure.2.In visceral adipose tissue,the activity of two key lipases,ATGL and HSL,were increased in CRF group compared to sham-oprated group, but the expression of total protein of ATGL and HSL did not change. In Subcutaneous adipose tissue,the activity and total protein expression of ATGL and HSL all increased in CRF rats relative to control animals.3.The activity of PKA and MAPKs,who can regulate the phosphorylation of lipase,were also increased in CRF rats.The second part:studies in vitro1. Characterization of AOPPThe protein concentration of AOPP and unmodified MSA were 9.2mg/ml and 10.5mg/ml, respectively. The real content of AOPP in the preparation of AOPP and unmodified MSA were 658.5μmol/l and 1.66μmol/l, respectively, which were 72.8nmol/mg protein and 0.16nmol/mg protein respectively after calibrated by protein concentration. The concentration of endotoxin in all preparations was lower than 0.25EU/ml.2. Effect of AOPP treatment on lipolysis in differentiated 3T3-L1 adipocytesAfter the stimulation of 3T3-L-1 with 200μg/ml AOPP for 0,6,12,24 hours,the glycerol release increase gradually with the prolonging of time(P<0.05). Moreover, the glycerol release expression augmented with the increase of AOPP doses(P<0.05). Unmodified MSA was without effect (P>0.05). These data indicated that AOPP stimulated lipolysis in 3T3-L1 adipocytes in a dose-and time-dependent manner.3. Effect of AOPP on expression of the related lipases to lipolysisHydrolysis of triacylglycerol in adipocytes is performed by catalysis of lipases and regulation of several important protein factors. In order to explore the effect of AOPP on expression of related lipases to lipolysis, we analyzed the protein expression of HSL, ATGL and CGI-58.AOPP treatment did not affect the expression of HSL,ATGL and CGI-58 protein.4. Effect of AOPP on phosphorylation of HSL To determine the relationship of the phosphorylation of HSL and AOPP-mediated lipolysis, the dose-dependent and time-dependent effects of AOPP on HSL activation was examined. After the stimulation of adipocytes with 200μg/ml AOPP or 200μg/ml MSA for0,1h,3h,6h,12h,24h,AOPPtreatment increased HSL phosphorylation,whereas it did not affect the expression of total HSL. The increase was detectable at 6h treatment, and was maximal at 12h treatment. To study the dose-dependent effect of AOPP on HSL activation,the adipocytes were stimulated with 0,50,100,200ug/ml AOPP, The increase was detectable at 50ug/ml treatment, and was maximal at 200ug/ml treatment.5. Effect of PKA pathway on AOPP-stimulated lipolysisTo verify whether the activation of cAMP-dependent PKA mediated AOPP-stimulated lipolysis in 3T3-L1 adipocytes, adipocytes were stimulated with 200μg/ml AOPP for 0, 1h,3h,6h,12h,AOPP treatment increased PKA phosphorylation,whereas it did not affect the expression of total PKA. The increase was maximal at 1 h treatment.6. Effect of ERK pathway on AOPP-stimulated lipolysisTo determine the relationship of the ERK pathway and AOPP-mediated lipolysis, adipocytes were stimulated with 200μg/ml AOPP for 0, 1h,3h,6h,12h,AOPP treatment increased ERK phosphorylation,whereas it did not affect the expression of total ERK. The increase was maximal at 12h treatment.7. Effect of JNK pathway on AOPP-stimulated lipolysisTo determine the relationship of the ERK pathway and AOPP-mediated lipolysis, adipocytes were stimulated with 200μg/ml AOPP for 0, 1h,3h,6h,12h,AOPP treatment increased JNK phosphorylation,whereas it did not affect the expression of total.JNK. The increase was maximal at 3h treatment.8. Effect of p38 pathway on AOPP-stimulated lipolysisTo determine the relationship of the p38 pathway and AOPP-mediated lipolysis, adipocytes were stimulated with 200μg/ml AOPP for 0, 1h,3h,6h,12h,AOPP treatment didn't increase P38 phosphorylation Conclusion:In the present study, we tested the hypothesis that AOPP induced lipolysis in adipocyte. Our results revealed that AOPP stimulated overproduction of glycerol in cultured 3T3-L1 cell, which was dependent on activation of PKA.