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The alterations of glycogen phosphorylase in diabetic rats and the effects of vanadyl sulphate treatment Liu, Heyi 1992

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THE ALTERATIONS OF GLYCOGEN PHOSPHORYLASE IN DIABETIC RATSAND THE EFFECTS OF VANADYL SULPHATE TREATMENTbyHEYI LIUB.Sc., West China University of Medical Sciences, 1982M.Sc., West China University of Medical Sciences, 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCESinTHE FACULTY OF GRADUATE STUDIESFaculty of Pharmaceutical SciencesDivision of Pharmacology and ToxicologyWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1992©Heyi Liu, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives, It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.___________________Department of_____________________________The University of British ColumbiaVancouver, CanadaDateci __DE-6 (2/88)11ABSTRACTA supersensitivity to isoproterenol (ISO, 5x109 M)-induced phosphorylaseactivation in hearts and a decrease in phosphorylase activity in liver fromdiabetic rats have been previously reported. The nature of this supersensitivityhas been investigated in the present study. The effects of treatment with theinsulin-like agent vanadyl sulphate (VOSO4) on both diabetes-inducedalterations of phosphorylase in heart and liver were also investigated.No difference in the activity ratio (ratio=pH 6.2 activity/pH 8.2 activity) ofphosphorylase kinase was observed between diabetic and control hearts underbasal conditions. Similar to the profile of phosphorylase alterations in diabeticheart, the activation of phosphorylase kinase in response to ISO-stimulationwas significantly increased in diabetic hearts compared to control hearts,suggesting that the alteration of cardiac phosphorylase kinase in the diabeticcondition may be partially responsible for the supersensitivity of phosphorylaseactivation. The cyclic AMP-dependent protein kinase (PKA) cascade is one ofthe factors that are responsible for the regulation of phosphorylase kinase,which in turn activates phosphorylase by phosphorylation. However, a similarcorresponding alteration in ISO-induced elevation of cAMP levels was notobserved in diabetic hearts. On the contrary, the increase in cAMP levels indiabetic hearts was significantly lower than that in control rats. This observationstrongly challenges the possibility that the supersensitivity is due to an upregulation of the cAMP-PKA cascade.111In addition to PKA, phosphorylase kinase is also regulated by calcium. Whendiabetic hearts were perfused with the calcium channel blocker verapamil(5x108 M) prior to ISO stimulation, we demonstrated that the supersensitivity ofphosphorylase activation was abolished. Verapamil also prevented elevationof the cAMP level and activation of phosphorylase kinase by ISO in both controland diabetic hearts. Calcium overload and impaired calcium transport indiabetic heart have been previously reported. This, together with our resultssuggests that the supersensitivity may be due to diabetes-inducedabnormalities in calcium homeostasis in rat hearts. As calcium is also aregulator of phosphorylase kinase, the abnormalities of phosphorylase kinaseobserved in diabetic hearts are more likely due to altered calcium homeostasisin the hearts, rather than the up-regulation of the cAMP-PKA cascade.As an insulin-like agent, vanadyl sulphate has been shown to have euglycemiceffects and to improve cardiac performance of chronically diabetic animals. Ithas also been demonstrated that vanadium compounds have direct effects onthe enzymes of glycogen metabolism. In this study, the supersensitivity ofcardiac piosphorylase activation to ISO in diabetic rats was completelyabolished by treatment with VOSO4 given in drinking water for five weeks. Thetreatment also abolished the impairment in the ISO-induced cAMP elevation indiabetic heart.In diabetic livers, a partial restoration of the decreased hepatic phosphorylaseactivity was achieved by vanadyl sulphate treatment for five weeks, while acomplete restoration of hepatic phosphorylase activity was achieved after fivemonths treatment. The present study has thus provided further informationivtoward the understanding of the mechanism(s) involved in the supersensitivityof phosphorylase activation by catecholamines in diabetic rat heart. It has alsodemonstrated the beneficial effects of vanadyl sulphate treatment on theenzymes responsible for glycogen metabolism.VTABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTS vLIST OF FIGURES viiiLIST OF TABLES xiABBREVIATIONS xiiACKNOWLEDGMENTS xiiiDEDICATION xivINTRODUCTION 1I. Glycogen Phosphorylase and Its Regulation 4II. Phosphorylase Kinase and Its Regulation 11III. Vanadium and Its Insulin-Like Effects 18SUMMARY, RATIONALE AND PROPOSED EXPERIMENTS 20MATERIALS AND METHODS 21I. MATERIALS 21Chemicals and assay kits 21linstruments 22II. METHODS 221. Animal Models 222. Vanadyl Sulphate Treatment 223. Heart Perfusion, Drug Administrationand Sample Collection 234. Phosphorylase Assay 24vi5. Cardiac Phosphorylase Kinase Assay 286. Protein Determination 307. Cardiac cAMP Assay 328. Serum Glucose and Insulin Determination 329. Statistical Analyses 32RESULTS 33I. Validation of Assay Methods 331. Hepatic phosphorylase assay 332. Cardiac phosphorylase kinase assay 35II. General Features of Experimental Animals 381. Body weight of the animals 382. Serum insulin level 393. Serum glucose level 39Ill. Effects of ISO on Cardiac Phosphorylase,Phosphorylase Kinase and cAMPin Control and Diabetic Rats 461. Time course for ISO stimulationon cardiac phosphorylase 462. Supersensitivity of phosphorylaseactivation in response to ISO stimulation 503. Effects of ISO on cardiacphosphorylase kinase 504. Effects of ISO on cardiac cAMP 51IV. Effects of Verapamil Perfusion on CardiacPhosphorylase, Phosphorylase Kinase and cAMP 511. Effects of verapamil and ISO stimuTationon cardiac phosphorylase 522. Effects of verapamil and ISO stimulationon cardiac phosphoryTase kinase 52vii3. Effects of verapamil and ISOstimulation on cardiac cAMP 52V. Effects of Vanadyl Sulphate Treatment on CardiacPhosphorylase, Phosphorylase Kinase and cAMP 631. Effects of VS treatmenton cardiac phosphorylase 632. Effects of VS treatment oncardiac phosphorylase kinase 633. Effects of VS treatment on cAMP 64VI. Alteration of Hepatic Phosphorylase inDiabetes and the Effects of VS Treatment 641. Alterations of hepatic phosphorylase 642. Effects of Vanadyl Sulphate treatment 65DISCUSSION 73I. The Nature of the Supersensitivityof Phosphorylase Activation 73II. Reversal of Biochemical Alterations inDiabetic Rats by Vanadyl Sulphate Treatment 78A. Effects of VS treatment on generalfeatures of experimental animals 78B. Alteration of hepatic GP and theeffects of VS treatment 81C. Effects of VS treatment onalterations in cardiac GP, PPK and cAMP 85CONCLUSIONS 87REFERENCES 88viiiLIST OF FIGURESFigures Pages1. A schematic showing the interrelationshipsamong the cardinal forms of phosphorylase 72. The phosphorylase cascade showing how hormonalsignals activate phosphorylase through the twokinases and inactive glycogen synthase 93a. Suggested role for ATP/Mg2-bindingtotheosubunit 143b. Schematic diagram of the overall activityregulation of the y subunit 154. Synopsis of phosphorylase kinaseregulation in muscle cell 16-175. Standard curve for phosphorylase assayusing Na2HPO4as a standard 276. Standard curve for theLowry protein assay 317. Time course of the reaction rate forcardiac phosphorylase kinase assay 368. Effects of protein concentrationon the reaction rate of cardiacphosphorylase kinase assay 37ix9. Body weight of VS-treated andnon-treated control and diabetic rats 40-4 110. Serum insulin level of VS-treated andnon-treated control and diabetic rats 42-4311. Serum glucose levels of VS-treated andnon-treated control and diabetic rats 44-4512. Time course of cardiac phosphorylaseactivation by ISO in femalecontrol and diabetic rats 47-4813. Time course of cardiac phosphorylaseactivation by ISO in male rats 4914. Basal cardiac phosphorylase valuesand effects of ISO on phosphorylasein control and diabetic rats 53-5415. Basal cardiac phosphorylase kinasevalues and effects of ISO on phosphorylasekinase in control and diabetic rats 55-5616. Basal cardiac cAMP levels and effects of ISOon cAMP in control and diabetic rats 57-5817. Effects of verapamil (VP) perfusionon cardiac phosphorylase 59-6018. Effects of verapamil (VP) perfusionon cardiac phosphorylase kinase 61-6219. Effects of five-week vanadyl sulphatetreatment on cardiac phosphorylase 66-6720. Effects of five-week vanadyl sulphatetreatment on cardiac phosphorylase kinase 68-6921. Effects of diabetes and five-monthtreatment with vanadyl sulphate onhepatic phosphorylase 71-72xxiLIST OF TABLESTables Pages1. Biochemical and physical characteristicsof phosphorylase 52. Effects of caffeine concentration onhepatic phosphorylase assay 343. Diabetes-induced alterations inactivities of hepatic phosphorylase andthe effects of vanadyl sulphate treatment 70xiiABBREVIATIONSAC adenylate cyclase5’-AMP adenosine 5’-monophosphateATP adenosine triphosphateBSA bovine serum albuminC, CON controlcAMP cyclic adenosine 3’,5’-monophosphatecGMP cyclic guanosine1,5’-monophosphateEDTA ethylenediamine tetraacetate, disodium saltD, DIA diabeticG-l-P glucose-i-phosphateGP glycogen phosphorylaseGPa glycogen phosphorylase aGPb glycogen phosphorylase bISO isoproterenoln number of samplesp probabilityPKA cyclic AMP dependent protein kinasePP protein phosphatase/phosphorylase phosphatasePPK phosphorylase kinasePMSF phenylmethylsulfonyl fluoriderpm revolutions per minuteSTZ streptozotocinTCA tricholoroacetic acidSEM standard error of the meanV, VS vanadyl sulphateVP, Verp verapamilXIIIACKNOWLEDGMENTSI am very grateful to my supervisor Dr. John H. McNeill forgiving me the opportunity to work in his laboratory as a graduatestudent. I sincerely thank him for his knowledgeable guidance andpatience throughout my study.I would like to thank the member of my supervisory committee,Dr. Brownsey, Dr. Diamond, Dr. Macleod and Dr. Lyster for theirscientific input and advice.I would also like to thank our lab manager Mary Battell and Dr.Brian Rodrigues and Mrs. Sylvia Chan for all the help they offeredduring my experiments and thesis writing. I also thank Margaret Camand Zhen Yu for their friendship and help.I would like to express my gratitude to Dr. Margaret Prang, Dr.Maria Furstenwald, and Ms. Lynda Cowan for their moral support andconsistent encouragement.My thanks also go to all the nice colleagues and friends in ourlaboratory and the faculty for making my experience here enjoyable.DEDICATIONI wish to dedicate this thesis to my husband Yong-jiang Hei and my parentsRui Liu and Lan-fen Luo for their love and support.xiv1INTRODUCTIONDiabetes mellitus produces a number of secondary complications if notproperly controlled. Among the tissues affected are the eyes, kidneys, nerves,gastrointestinal tract, bladder, sex organs, blood vessels, various muscles, skin,liver and heart. As a result, diabetes is a major cause of death in NorthAmericans today (Pierce et a!., 1988).A variety of cardiovascular abnormalities have been shown to beassociated with diabetes in both humans and in experimental animal models inwhich diabetes is chemically-induced by streptozotocin (STZ) or alloxan(Kannel and McGee, 1979; Tahiliani and McNeill, 1986). While atherosclerosisand other forms of vascular pathology are prevalent in diabetic subjects andcould cause cardiac disease, a defect in cardiac performance has also beenobserved in the absence of major vascular alterations, suggesting an intrinsicabnormality of the cardiac muscle itself. This condition is defined as diabeticcardiomyopathy (Dhalla et a!., 1985; Fein & Sonnenblick, 1985; Zarich et a!.,1989; Fein, 1990; Fisher and Frier, 1990).Increasing evidence suggests that multiple factors are involved in theetiology of diabetic cardiomyopathy. Alterations in myocardial enzyme systemsand subcellular organelles such as sarcolemma, mitochondria, sarcoplasmicreticulum, as well as contractile proteins may all be contributing factors. Forinstance, the response of diabetic myocardium to catecholamines wasimpaired and was attributed to a decrease in the number of 13-adrenoceptors(Savarese & Berkowitz, 1979; Nishlo eta!., 1988); the Ca2-ATP se and theability of sarcoplasmic reticulum (SR) to transport Ca2 were reduced in the2diabetic heart (Lopaschuk et a!., 1982), and a defect in the Ca2 pump insarcolemma (SL) of the diabetic heart was also observed (Heyliger eta!., 1987).In addition, it has been repeatedly observed that the total activity of glycogenphosphorylase (GP), a key enzyme in the regulation of glucose metabolism andutilization, increases in cardiac tissue from diabetic animals and that asupersensitive GP activation occurs in response to catecholamine stimulation(Miller et aL, 1981, Miller eta!., 1983, Vadlamudi & McNeill, 1983, Miller et aL,1984), despite the fact that the number of B-receptors were reduced in diabetichearts (Savarese & Berkowitz, 1979; Nishio et aL, 1988).In addition to diabetic cardiomyopathy, liver disease is another severecomplication of diabetes. It has been shown that fatty liver and progressive liverdisease including cirrhosis are associated with diabetes. Cirrhosis has beenfound to be four times more common in diabetics than nondiabetics (Silvermanet aL, 1990). Moreover, diabetes is one of the four diseases together withhepatitis, cirrhosis, and drug allergy which showed a significant association withprimary liver cancer (Vecchia et a!., 1990). Additionally, significant alterations ofhepatic enzymes have been reported in diabetic subjects and it is believed thatthe liver undergoes unique biochemical changes in diabetes that could result inliver pathology (Miller, 1978, Silverman et aL, 1990). One of these enzymaticchanges in the livers of diabetic animals is the alteration of hepatic GP activity.However, in contrast to cardiac tissue, the activity of hepatic GP was reported tobe significantly decreased in diabetic rats (Khandelwal & Zebrowski, 1977,Ciudad eta!., 1988)As GP plays an important role in maintaining normal cardiac and hepaticfunctions, the alterations of its activity in both diabetic heart and liver may3represent one of the mechanisms for the development of cardiac abnormalitiesand hepatic pathology. Studies on the mechanisms responsible for thesupersensitivity of cardiac GP activation in diabetes are still controversial. Forexample, Miller and his colleagues tried to elucidate the role of phosphorylasekinase (PPK) in the supersensitivity of GP activation. One of their observationsshowed the activity of PPK was also significantly increased in the same diabeticrat hearts with the supersensitivity of GP activation and indicated that PPK wasinvolved, while another one showed that there was no change in PPK activity(Miller, 1981, 1984). It was also suggested that high levels of calcium orcalcium overload in the diabetic heart due to impaired calcium transport was acontributing factor. Again, there is no solid evidence to support this speculation(Vadlamudi & McNeill, 1983). Therefore, further studies are needed to clarifythe mechanisms for alteration of GP in diabetes.In addition to studies on the mechanisms responsible for thedevelopment of various complications in diabetes, the search for a better agentin the treatment of the disease has also been a subject of intensive research.Recently, the use of oral vanadium compounds in the treatment of chemically-induced diabetes has been suggested. Vanadium compounds have beenshown to produce an insulin-like effect in diabetic rats, causing a decrease inthe elevated blood glucose levels (Heyliger eta!., 1985; Ramanadham et aL,1989). It is yet to be determined, however, if the treatment can also correct thealterations of cardiac and hepatic GP in diabetic rats.This thesis will attempt to elucidate the mechanisms of GP alteration andthe effects of vanadium treatment on these changes. A brief description of the4biochemical properties of GP and its regulation, together with the pharmacologyof vanadium compounds is presented below.I. Glycogen Phosphorylàse and Its RegulationGP catalyzes the phosphorylytic cleavage of glycogen which releasesglucose-i-phosphate (G-1-P) and is thus a key enzyme in the regulation ofglycolysis. The physiologically active form of GP is a dimer of two identicalsubunits, each containing a single polypeptide chain (Mr=97,333) of 841 aminoacid residues to which pyridoxal phosphate (PLP), the cofactor, is attached by aSchiff base at lysine 679 (Jenkins et aL, 1981, Perutz, 1989). Under in vivoconditions, GP exists in two interconvertible forms; phosphorylases b and a(GPb and GPa). GPb, found in resting muscle, is inactive except in thepresence of AMP or certain analogues of 5’-AMP, and is inhibited by ATP orglucose-6-phosphate (G-6-P). GPa functions efficiently in the absence of AMP,although AMP activates it at low substrate concentrations and releases theenzyme from glucose inhibition (Sprang et a!., 1987). GP is an allosteric proteinand has two conformations: the T state and the R state, of which only the R statehas a high affinity for substrate and activators and therefore is catalyticallyactive (Table 1). GPb is predominantly in the T state, while GPa ispredominantly in the R state (Fletterick & Sprang, 1982).GP is controlled by both allosteric interactions and reversiblephosphorylation. Con and Con (1936) observed that the catalytic activity ofGPb could be triggered by AMP and this was the first example of enzymeregulation by an allosteric effector which is not a substrate. It was subsequentlyrecognized that as an allosteric protein, the equilibrium of the two5Table 1. Biochemical and Physical Characteristics of Glycogen Phosphorylasepropertymolecular weight (dimer)amino acids/monomerfunctional unitmetabolic interconversionco factorcharacteristics194 800841dimerserine 14-phosphatepyridoxal phosphateReaction Catalyzed+P1G-i-P + GN.,= 0.3Covalent ActivationPb kinase Pa(inactive) (active)phosphataseintersubunit AMP/ATP siteI site, near active siteactive site30.1-1Kd,mM1000.15Allosteric ActivationR is active conformerAMPPb(T) Pb(R)GlucoseL = 1/3000T is inactive conformerAMPPa(T)- —-Pa(R)GlucoseL = 1/3Kd,allosteric activators promote R conformation mMAMP intersubunit AMP/ATP site 0.002Glycogen storage site 1p Serine-14substrates P, G-1-P active siteF-1-P, UDPG inhibitors at active siteallosteric inhibitors promote T conformationADP, ATPpurinesglucoseAdapted from Fletterick and Sprang (1982)6conformational states of GP is modulated by small molecules, i.e. “allostericeffectors”. The effectors interact at specific sites on the protein and favor aparticular conformation. Positive allosteric effector signals such as AMP, G-1-P,inorganic phosphate (Pi) and glycogen promote the catalytically active R statewhile negative effector signals such as glucose, ATP and some unidentifiedligands are inhibitors, which bind to and stabilize the inactive T conformation(Figure 1) (Fletterick & Sprang, 1982, Perutz, 1989). Because GP can becontrolled by allosteric interaction, Con and Con originally assayed the differentGP activities in the presence and absence of AMP (Con & Con, 1940). GPaactivity can thus be assayed in the absence of AMP while the total GP activitycan be assayed in the presence of AMP. This principle has since been appliedto assays of GP in order to measure different activities of the enzyme.In addition to allosteric interaction, the activity of GP can also becontrolled by reversible phosphorylation. The inactive GPb is converted to GPa,the phosphorylated active form, by the addition of a phosphate at the hydroxylgroup of a senine-14 residue located near the N terminus of the molecule. PPKis the enzyme that catalyzes this reaction. On the other hand, active GPa isinactivated by the removal of the phosphate group to form GPb. This reaction iscatalyzed by one of protein phosphatases (PPs), phosphorylase phosphatase(Fletterick and Sprang, 1982; Perutz, 1989).Both PPK and PPs are very complex enzymes which are themselvesregulated by various nervous or hormonal signals. It is known that PPK can beregulated by both adrenergic stimulation and intracellular concentration ofCa2 (Pickett-Gies and Walsh, 1986). The PPs which are responsible forphosphorylase regulation, namely PP1 and PP2A (Ballou and Fisher, 1986)7T FORMKINASEPH OS P H ATAS ET FORMFigure 1. A chematic showing the interrelationship among thecardinal forms of phosphorylase. The striped species are the majorconforms in vivo. Phosphorylase b is activated covalently by the kinase or byAMP and substrates. Note that the phsophatase can only function on theinactive conformer of phosphorylase a.‘PURINES’GLUCOSEG, P1, G-1-PMPH FORMPHOSPHORYLASE bH FORMPHOSPHORYLASE aFletterik and Sprang (1982)8can be regulated by cAMP, Ca2 and insulin levels (Cohen, 1989, Cohen andCohen, 1989). These signals in turn regulate the degradation of glycogen. Therelationship between PPK, PP, GP and glycogen synthetase is schematicallyshown in Figure 2 (Fletterick & Sprang, 1982).It is clear from the above description that GP can be envisioned as atransducer that samples positive effector signals (nervous or hormonalstimulation, G-i-P, AMP, etc.) and negative effector signals (insulin-inducedsignals, glucose, ATP, etc.) and proceeds either to degrade glycogen for fuel, orto halt its degradation, allowing the cell to store glucose as glycogen for lateruse. The alteration in GP activity is thus a result of a change(s) in either positiveor negative signals. Although there are conflicting reports as to whether cardiacPPK is involved in the enhancement of the activity of GP in diabetic heart (Miller,1981, 1984), it certainly is a very important enzyme in the regulation of GPactivity and hence is worthy of further study. Extensive researches have alsobeen carried out on PPs and their regulation in recent years. A study onmolecular mechanism by which insulin regulates PPs (Dent et a!., 1990)showed that in mammalian skeletal muscle, insulin stimulates a protein kinasewhich activate type-i protein phosphatase (PP1), Since PP1 and PP2A are thetwo PPs responsible for phosphorylase regulation, it is possible that thesupersensitivity of GP activation in diabetic condition is the result of decreasedPP1 activity due to insulin deficiency. It is also possible that the abnormalities ofboth PPK and PPs resulted in the supersensitivity. While alterations in theactivity of PPs in the diabetic state and their significance in regulating GPactivity have been well recognized, they are not the focus of this thesis and willnot be discussed further.9hoirnonal signalscAMPprotein kinases7Thsynthetase b- P synthetase a phosphorytase kinase phosphorylase kinase\(active) (actwe)phosphatase phosphataseinhibitorinsulinglucocorticoidsglucose ) phosphorytase a - P phosphorylase - b(active)phosphataseFigure 2. Cascade for the hormonal activatation of phosphorylasethrough the two kinases and inactivation of glycogen synthase. Theinactivation (open arrows) is by the action of the phosphatase(s) that areregulated by phosphorylase and inhibitor protein(s). Glucose inactivatesphosphorylase a directly.synthetase aUDPGIG-1-P gtycogenphosphorylase aFletterick and Sprang (1982)10The assay method for cardiac GP is similar to other muscle GP assaysand has been well established ( Con and Con, 1940; McNeill, 1968). In thecase of hepatic GP, the same assay method cannot be applied exactly. In fact, itwas once considered that GP existed in rat liver in forms that were different fromthose of muscle. This was based on the observation that the dephosphorylatedform of hepatic GP could not be activated or assayed even in the presence ofAMP (Wosilait and Sutherland, 1956). However, this view has been challengedby other investigators. In 1975, observations made by different researchers(Agnes and Nuttall, 1975; Stalmans and Hers, 1975) indicated that thedephosphorylated form of GP in the liver was activated by AMP, i.e. the liver hasthe same a and b forms of GP as those found in other tissues. This point of viewhas now been widely accepted (Hers, 1976, Hems and Whitton, 1980). On theother hand, there are differences between muscle and hepatic GPb. Stalmansand Hers (1975) observed that hepatic GPb was not inactive under most assayconditions, especially for the enzymes from rats and mice. The activity of basalGPb also differed significantly in different species of animals. Additionally, itwas observed that the activity of rat or mouse liver GPb reached 25% of that ofthe GPa at pH 6.1 in the presence of 0.15 mM NaF (used for inhibitingphosphorylase phosphatase). When 1 mM AMP was also present, this valuerose to 50% (Stalmans and Hers, 1975, Hers, 1976). Thus, when the hepaticGPa of rats or mice is assayed, the interference due to the presence of GPbactivity has to be eliminated. The GPb activity was found to be stronglystimulated by fluoride and sulfate, but inhibited by caffeine and maleate (Hers,1976). Therefore the specific determination of GPa in the liver requires thepresence of caffeine in the assay system. A concentration of 0.5 mM caffeine11was used in earlier assays (Stalmans & Hers, 1975, Hue eta,’., 1975), while 1mM caffeine was applied in a later report (Blackmore et a!., 1985).II. Phosphorylase Kinase and Its RegulationIt has been well recognized that the conversion of GPb to GPa iscatalyzed by PPK (Krebs eta!., 1958). The reaction process and the GP-PPKcomplexes have been directly visualized and studied by scanning tunnellingand atomic force microscopy (Edstrom et aL, 1990):2 phosphorylase b +4 ATP phosphorylase a +4 ADPThe molecular weight of PPK is approximately 1.3x106 dalton and it isacomplex molecule composed of four types of subunits (cc4B’y.ö)with thespatial arrangement of a “butterfly” for the subunits (Figure 3). There are twoPPK isozymes, based on the difference in the size of the largest subunit, the asubunit. The two isozymes are designated as413y84 and x413y64(Pickett-Gies and Walsh, 1986, Edstrom eta!., 1990). Although they can coexistin the same muscle fiber, a particular type of muscle fiber may only contain aspecific form of the isozymes. For instance, the enzyme is predominant in red(slow-twitch) muscle such as cardiac PPK (Cooper et aL, 1980; Pickett-Gies andWalsh, 1985).The x and B subunits of PPK serve a regulatory function and theirphosphorylation by the cAMP-dependent protein kinase (PKA) results inactivation of the enzyme (Hayakawa et a!., 1973, Cohen, 1973). Theobservation that: (1) the isolated 7-subunit exhibits full catalytic activity that is12independent of Ca2 for the phosphorylation of both GP and PPK (Chan andGraves, 1982), and (2) that there is a substantial homology between thesequence of the Wy-subunit and that of the catalytic subunit of PKA (Reimann eta!., 1984, Harris et at, 1990) indicates that the y-component of the PPK is thecatalytic subunit. The primary structure of the 6 subunit is identical tocalmodulin, a calcium-binding protein. This subunit thus has the ability toactivate calmodulin-dependent enzymes (Cohen et at, 1978). Ca2+,binding to the 6-subunit results in the activation of PPK. This kind of activation isassociated with the phosphorylation and activation of GP, without covalentmodification of PPK and without an increase in cAMP or PKA (Walsh, 1986).The activation of PPK by Ca2 requires the binding of Ca2 to at leastthree of the tour Ca2 binding sites per 6-subunit, i.e. at least 12 mcI per mol ofthe holoenzyme (Burger et al., 1982, 1983). The corresponding physiologicalconcentration range is 2-25 jiM. The activation of PPK by Ca2 occursphysiologically. During nerve stimulation and muscle contraction, theintracellular concentration of Ca2+ rises from a resting level of 10-100 nM to 1-10 jiM. This is in the appropriate range to allosterically regulate PPK and thusprovides a physiological link whereby contractile activity is connected toenhanced glycogenolysis (Cohen, 1983; 1988). Ca2-induced PPK activationhas been well documented not only in skeletal muscle, but also in the heart(Ramachandran eta!., 1982) and the liver (Vandenheede eta!., 1979).Although sharing the common characteristics of calmodulin-dependentenzymes, PPK can be activated by Ca2 binded 6—subunit (calmodulin), theinteraction of 6—subunit with PPK is in a different manner from other calmodulindependent enzyemes: 6—subunit is tightly bind to PPK. It was reported that 6—13subunit was not resolved from PPK during the isolation of the enzyme, despitethe presence of EDTA or EGTA in the buffers (Cohen et a!., 1978; Shenolikar etaL, 1979). Nevertheless, the 6—subunit binds even more tightly in the presenceof Ca2+ and was not dissociated from PPK even in the presence of 8M urea(Picton eta!., 1981).Both phosphorylation of a and 13 subunits and Ca2 binding on PPK canactivate the enzyme. The mechanism of activation, however, is verycomplicated and yet to be completely understanded. It has been suggested(Heilmeyer, 1991) that there are ATP/Mg binding sites on both a and ‘ysubunitsand ADP/Mg binding sites on 13 subunit of PPK. As a prerequisite for expressionof activity of PPK, a binding of ATP/Mg and an exchange of ADP against ATP on‘ysubunit are necessary. The bindings of ATP/Mg and ADP/Mg on a and 13subunits can somehow trigger such an ATP/ADP exchange on y subunit andenable the enzyme to express its catalytic activity (Figure 5A, Heilmeyer, 1991).The phosphorylation of residues in a and 13 subunits and interactions ofCa2 binded 6 subunit (CaCm) with a, 13 and y subunits modulate the bindingsof ATP/Mg on a and y subunits and ADP/Mg bindings on 13 subunit. These ATP,ADP and CaCm bindings eventually affect and modulate the binding ofsubstrate (GPb ) on ‘y subunit and express the activity of PPK (Figure 5B,Heilmeyer, 1991).PPK is located at an interface between signalling and metabolicpathways. Therefore, it not only integrates signals from different signalpathways, but also receives information from metabolic pathways (Figure 6,Heilmeyer, 1991). Any hormonal or methabolic changes that can affected the14Ki +.ATP/Mg >1’(ATP/M9)a 1(1 +ATI3/Mg•TPADPKKADIYM9)7 KI(ATFYMg)7Phosphorylase a Phosphorylase bFigure 3a. Suggested role for ATP/Mg2-bindin to the x subunt.The arrow with 0 indicates the effect of ATP/Mg2+binding to c allowing anADP/ATP exchange on the subunit and consequently allowing an ADP/ATPexchange on the ‘y subunit and consequently the conversion of phosphorylase bto a. Ki, kinase.Heilmeyer (1991)15Calmodulin binding to the y, a and /3 subunits is indicatedby Ca Cm. The catalytic site on y is indicated by the substratebinding site. It binds ADP as well as ATP. The ATP/Mg2-bindinsite in a represents the site labelled by fluorescein isothiocyanate.The repetition of P indicates the multiphosphorylation loop on a.Binding of Mg2 to the 3 subunit is hypothetical. P26 and P11indicate phosphorylation sites in region A of the /3 subunit. P7 isthe phosphorylation of serine 700. ADP binding on the /3 subunit isproposed from the homology with the N-terminal region of a.Figure 3b. Schematic diagram of the overall activity regulation ofthe y subunit.6P700ADPHeilmeyer (1991)16Figure 4. Synopsis of phosphorylase kinase regulation in a musclecell. Incoming signals to phosphorylase kinase from signal pathways areindicated by large black arrows, incoming signals from metabolic pathways areindicated by dashed arrows. Metabolic pathways are indicated by large whitearrows. Interaction localizing phosphorylase kinase in specific cellularstructures are indicates by hatched arrows.Heilmeyer (1991)17/ Depolarisation p_Agonis7Plasma MembraneCa2 lp3 cAMPI IMe mbnes°gTroPonin/Phosphory1ase /Kinase \ /,/ —/LIPhosphorylaSe PhosphorylaseI GlycogenolysiSAMP GIc6P-——-- Glyco(ysis---- 02/COGlycolysis _zPlasma MembraneI I14eil[I I IADP1AMP ATPt%L/j IG fu coS 0 Lactate18levels of second messagers such as cAMP and calcium can trigger theactivation of PPK. Mentioned above that calcium release from nerve stimulationand mucle contraction resulted in the activation of PPK ( Walsh, 1986; Cohen1988) is the good example of this kind of regulation. The sum of this informationis then to determine the appropriate level of GPa.Inactive PPK isolated from resting skeletal muscle has minimal activity ata physiological pH (6.8-7.0) but considerable activity at pH values greater thanpH7.6 (Krebs et al., 1964). The phosphorylation of PPK causes a largeincrease in activity at physiological pH (6.8) but little activation above a pH of8.0 (Cohen, 1988). It is therefore convenient and accurate to monitor activationin tissue extracts by measuring an increase in the pH 6.8/8.2 activity ratio at asaturating concentration of Ca2+. The method for cardiac PPK assay has beendeveloped based on this pH profile of PPK activity (Sul at a!., 1983). The pHprofile of hepatic PPK is different from muscle tissue and its activity is notcompletely dependent on Ca2 (Cohen, 1988). The assay approach is thusdifferent from that of cardiac PPK.III. Vanadium and Its Insulin-Like EffectsVanadium is a widely distributed member of the first transition series.With an average content of 135 ppm, it is the twenty-first most abundant elementon the earth’s crust. Vanadium is a common constituent of living plants andanimals and its nutritional essentiality for normal growth has been suggested forsome species (Nechay, 1984). Vanadium deficiency in rats results in impairedfertility and reduced pup survival, whereas in chickens, there is a decreasedweight gain and feather growth, retarded skeletal development and an increase19in plasma triglyceride (Underwood, 1977). Human diploid fibroblasts alsorequire 0.25 ng/ml vanadium for optimal clonal growth (Carpenter, 1981).The chemistry of vanadium is very complicated. It has multiple stableoxidation states (from +2 to +5 differing by one unit of charge), hydrolyzes easilyand forms polymers frequently (Macura, 1980). The oxidation states ofbiological interest for vanadium are +3, +4, and +5. The predominant species ofvanadium in the body fluids is V03 (+5 oxidation state, also called vanadate).This form may enter certain cells by an anion transportation system (Nechay,1984). Two factors may account for many of the physiological effects ofvanadium: (1) mimicry of either phosphate ions or divalent cations, and (2)change from an anion V03 to a cation VO2 (+4 oxidation state, also calledvanadyl) through reduction by glutathione. Vanadate acts as an analogue ofphosphate in many biological processes. As for vanadyl, it is similar in size andbehaves like a simple divalent ions such as Ca2+, Mn2+, Zn2+, potentiallyhaving many biochemical and cellular sites of action (Nechay, 1984; Boyd andKustin, 1984).Apart from a variety of other biological effects (Macara, 1980, Jandhyalaand Hom, 1983, Nechay, 1984; Boyd and Kustin, 1984), vanadium given orallyto diabetic rats in the form of vanadate or vanadyl has been shown to haveinsulin-like effects on glucose metabolism and to correct diabetes-inducedchanges in cardiac performance (Heyliger et a!., 1985, Ramanadham et aL,1989). Additionally, in vitro studies have shown that vanadium does have directstimulatory or inhibitory effects on hepatic GP of cultured cells from differentspecies. It is thus highly possible that treatment with vanadium may correct thealteration in phosphorylase in both the diabetic heart and liver.20SUMMARY AND RATIONALE FOR THE PROPOSED EXPERIMENTSFrom the information presented, it is clear that the activity of GP isregulated by PPK and intracellular Ca2. The alteration of GP activity indiabetic heart and liver may thus be due to changes in PPK activity, and/orintracellular Ca2+ levels. Many investigators have reported that the myocardialcAMP-PKA cascade and intracellular calcium level are altered during diabetes(Ingebretsen eta!., 1981, Kawamura and Suzuki, 1990). We propose thatdiabetes-induced changes in PPK and/or calcium homeostasis are responsiblefor the supersensitivity of GP activation in diabetic rat hearts. We also proposethat vanadium treatment may correct the abnormalities of GP in both heart andliver of diabetic rats.The present study has been undertaken to address some of theseproblems. The following attempts were made to examine our hypothesesregarding the nature of diabetes-induced cardiac supersensitivity inphosphorylase activation: (1) to assay cardiac PPK activity in order to determineits involvement in the supersensitivity of cardiac GP activation; (2) to use thecalcium channel blocker verapamil in the heart experiments to examine ifcalcium is responsible for the supersensitivity of GP activation; (3) to assay thecardiac cAMP levels in all animals to examine the possible involvement ofcAMP-PKA cascade. Finally, cardiac GP activities , cardiac PPK activities,cardiac cAMP levels and hepatic GP activities in vanadyl sulphate-treatedanimals were measured to examine the effect of the treatment.21MATERIALS AND METHODSI. MATERIALSCHEMICALS AND ASSAY KITSBuffer chemicals like calcium chloride (CaCl.2H20), magnesium chloride(MgCI2), potassium chloride (KCI), sodium chloride (NaCI), sodium bicarbonate(NaHCO3), D-glucose, trichioroacetic acid (TCA) and ammonium molybdatewere obtained from BDH.The following chemicals, reagents and assay kits were obtained fromSigma: streptozotocin (STZ), phosphorylase b, protein assay kit, glucose-i-phosphate (G-1-P), bovine serum albumin fraction V (BSA), glycogen, Iisoproterenol, ethylenediaminetetraacetic acid (EDTA), caffeine, Tris 7-9,phenylm ethylsu Ifonyl fluoride (PMSF), glycerol phosphate, 2-mercaptoethanol,adenosine 5’-monophosphate (5’-AMP), magnesium acetate (Mg(Ac)2).1-Amino-2-naphthol-4-sulfonic acid (Fiske-Subbarow reagent) andvanadyl sulphate penta hydrate were purchased from Fisher Scientific.‘y32P..[ATP] and the assay kit for cAMP were obtained from Amersham.Insulin assay kit was obtained from lmmunocorp. Sodium fluoride was obtainedfrom Mallinckrodt Inc. Whatman ED-31 filter paper was used.22INSTRUMENTSBeckman J2-21 Centrifuge. Beckman J-6B Centrifuge. United TechnologiesParkard CrystalT.M Multi Detector RIA System. Parkard TRlCARBR 4530Liquid Scintillation Spectrometer. HP 8452A Diode Array Spectrophotometer.II. METHODS1. Animal ModelMale Wistar rats weighing 175-200 g were used predominantly in thisstudy. Diabetes was induced by a single tail vein injection of STZ (60 mg/kg)dissolved in 0.9% NaCl solution. Control rats received an injection of thevehicle. Rats:were anesthetized with halothane prior to injection. The vein wasdilated by immersing the tail in warm water. Three days after STZ injection,blood glucose levels were measured using a Glucometer Il (MilesLaboratories). Animals with glucose values greater than 15 mM wereconsidered diabetic.2. Vanadyl Sulphate TreatmentBoth treated and nontreated control and diabetic rats were maintainedwith free access to food and water for four to six weeks. Plain tap water wasgiven to non-treated groups, while vanadyl sulphate (VOSO4)in drinking water(1 mg/mI) was administered to treated groups of control and diabetic ratsbeginning two weeks after STZ injection. Treatment was continued for fiveweeks. Blood samples were collected at the time of sacrifice and the serum23was obtained by centrifugation. Body weight, serum glucose and insulin weremeasured. In addition, food and fluid intake were monitored in the vanadylsulphate treated control and diabetic rats.To observe the long term effects of vanadyl sulphate on hepatic GP, liversamples of control and diabetic rats that were either treated or not treated withvanadyl sulphate for five months were also studied.3. Heart Perfusion, Drug Administration and Sample CollectionThe animals were anesthetized with pentobarbital (60 mg/kg), the chestcavity opened and the heart removed. The Langendorff heart apparatus wasused for heart perfusion and drug administration in this study. The workingheart apparatus was once used to measure the time course and supersensitivityof ISO stimulation in control and diabetic hearts. For the Langendorif heart, thefilling pressure of perfusion was kept constant at 55 cm H20. The perfusate inthe two reservoirs was constantly bubbled with a mixture of 95%°2 and 5%CO2. Hearts were either perfused only with Chenoweth-Koelle (CK) buffer [i.e.solution with the following composition (mM): NaCI, 120; KCI, 5.6; CaCI2, 2.0;MgCI2, 2.1; NaHCO3,25; and glucose, 10] for 20 mm to measure basal GP,PPK and cAMP activities, or stimulated with 5X1 0 M ISO for another 30 sec tomeasure ISO-stimulated activities. For verapamil perfusion, the hearts were firstperfused with CK buffer containing 5X108 M verapamil (basal) andsubsequently stimulated with ISO. During this period, EDTA and ascorbic acid (both 10 mg/I) were added to the perfusion buffer to prevent the oxidation of ISO.After perfusion and drug administration, the hearts were freeze-clamped,powdered and stored at -80°C until assayed.24After removal of the heart, the liver was quickly removed and placed inliquid nitrogen. Liver samples were powdered and stored at -80°C untilassayed.4. Phosphorylase AssayBoth liver and heart GP activities were measured by determining therelease of inorganic phosphate from G-1-P using the method of Fiske andSubbarow. Inorganic phosphate is generated by a reversible enzymaticreaction catalyzed by phosphorylase:nGIucose1phosphate÷GIycogen() = nPO4+Glycogen(fl÷)The assay for cardiac GP was a modified method of McNeill and Brady(1966). About 50 mg of frozen powdered tissue was suspended in 2 ml of coldTris buffer (pH 6.8) containing 0.05 M Tris, 0.001 M EDTA and 0.01 M NaF. Thetissue was homogenized with a Polytron homogenizer at 4°C with a speedsetting of 6 for 10 seconds. The homogenate was then immediately centrifugedat 10,000 rpm for 10 mm at 0-4°C in Beckman J2-21 centrifuge. Thesupernatant fraction was used for the enzyme and protein assays. The reactionmixture contained 20 il supernatant and 0.5 ml reaction medium. The reactionmedium was made using Tris buffer (pH 6.8) and contained 0.48% glycogen,0.3% BSA and 1 iM G-1-P. In addition, the medium for the measurement oftotal phosphorylase activity (GPt) contained 0.001 M 5’-AMP. The reaction wascarried out at 37°C for 30 mm, except for the blanks which were kept at 4°C.The reaction was terminated by the addition of 1 ml 10% TCA and the mixture25was centrifuged at 3800 rpm for 30 mm at room temperature in a Beckman J-6BCentrifuge. The color development due to phosphate was carried out at roomtemperature with 0.8 ml of TCA mixture, 0.1 ml of Fiske & Subbarow reagentand 1.6 ml of shelf molybdate solution (containing H2S04 and ammoniummolybdate) for 30-45 mm. The concentration of phosphate was determinedfrom the absorbance at 660 nm in a HP-8452A Diode Array Spectrophotometer(Flowchart 1). Na2HPO4 (5 mM) was used to prepare standards with finalconcentrations ranging from 0.025 jiM to 0.3 iiM (Figure 5). The proteinconcentration of the supernatant fraction was determined by the Lowry methodusing a Sigma protein kit. Activities of both GPa and GPt are expressed innmoles of inorganic phosphate released per mm per mg protein. GPa was alsoexpressed as phosphorylase a ratio, i.e.Phosphorylase a Ratio (%) = (a Activity/Total Activity) x 100As described in the introduction, hepatic GPb of both rats and mice isactive under most assay conditions. Hepatic GP was assayed in this studyusing the same method as that for cardiac GP except that 1 mM caffeine waspresent in the reaction medium used for the determination of the GPa activity(i.e., the minus 5’-AMP medium fraction). This was done to block possibleinterference by active GPb. The final concentrations in the reaction mixturewere: 0.001 M EDTA, 0.01 M NaF, 0.48% Glycogen, 0.3% BSA, 1 jiM G-1-P, 1mM 5’-AMP and 1 mM caffeine.26FLOWCHART 1PHOSPHORYLASE ASSAYHeart or Liver Tissue (— 50 mg)+2 ml Tris Buffer4,Homogenization (Polytron 6, 4C, 10 sec)Centrifugation (10,000 rpm, <4C, 10 mm)Supernatantincubation with Reaction Medium (37’C, 30 mm)—AMP (Phosphorylase a) +AMP (Phosphorylase a+b)jrColor Reaction and Reading at 660 nm271.31G -‘e 877 1O88O----OIO5 0.216 6315Phosphate Concentration (jiM)Figure 5. Standard curve for phosphorylase assay using Na2HPO4as a standard. Samples were prepared for color reaction with FiskeSubbarrow reagent as described in Material and Method.285. Cardiac Phosphorylase Kinase AssayThe activity of cardiac PPK was determined by a method modified fromCohen and Walsh (1983). PPK catalyzes the conversion of GPb to GPa by thetransfer of the y—phosphate of ATP to a single specific serine residue on each ofthe two GPb subunits. If ATP is labeled with 32P at the y-position, the activity ofPPK can be assayed by measuring the incorporation of32P-radioactivity intoGPb from [y-32P]ATP:Phosphorylase b +[y-32P]ATP—e.-Phosphorylase a-32P + ADPApproximately 150 mg of heart tissue was suspended in 0.75 ml ofextraction buffer (pH 7.5) containing 30 mM Tris, 30 mM KCI, 5 mM EDTA, 100mM NaF and 1 mM PMSF. The tissue was homogenized with a Polytronhomogenizer at 4°C with the speed setting at 6 for 10 seconds and thehomogenate was then immediately centrifuged at 15,000 rpm for 15 mm at 0-4°C in a Beckman J2-21 Centrifuge. The supernatant fraction was diluted withan equal volume of dilution buffer (pH 6.8) containing 10 mM glycerolphosphate, 5 mM EDTA, 125 mM NaF and 45 mM 2-mercaptoethanol. Thissolution was the enzyme solution then used for the assay. The reactionmedium for the assay contained (i) assay buffer of either pH 6.8 or 8.6containing 125 mM Tris, 125 mM glycerol phosphate and 50 iiM CaCl2; and (ii)15 mg/mI GPb solution in 10 mM glycerol phosphate and 45 mM 2-mercaptoethanol (pH 6.8) in 1:1 ratio. A mixture of 40 il of the medium and 10jil of the enzyme solution was first preincubated at 30°C for 2 mm and the assaywas initiated by addition of 10 .il ATP solution containing 1 pCi [y-32P}ATP, 1829FLOWCHART 2PHOSPHORYLASE KINASE ASSAYHeart Tissue. (150 — 200 mg)+0,.75 m Extraction BufferHomogenization (Polytron 6, 4C, 10 sec)Centrifugation (15,000 rpm, <4’C, 15 minI‘I’Diluted Supernatant by Equal Volume of Dilution BufferPreincubation with Reaction Medium (30C, 2 minIpH 6..8 (Activation of the Enzyme) pH 8.2 (Enzyme Specific Activity)32Incubation with [i— PIATP at 30 C, 5 mm50 ii! on Whatman ET—31 Filter Paper. Washed with. TCA, Counted30mM ATP and 60 mM Mg(Ac)2. The assay was carried out at pH 6.8 and pH 8.2.The final concentrations in the reaction mixture were: 41.67 mM Tris, 41.67 mM(3-glycerol phosphate, 16.7 (IM CaCI2, 3 mM ATP, 10 mM magnesium acetate, 5mg/mi phosphorylase b, and 10 mg/mI glycogen.The reaction was terminated 5 mm later by spotting 50 jii of the mixtureon 1.5 cm2 Whatman ED-31 filter paper squares which were immediatelywashed with 10 % TCA for 30 mm. This was followed by several continuouswashes with 5% TCA and 95% ethanol. The filter paper was dried and countedin a Parkard TRICARBR4530 liquid scintillation counter (Flowchart 2). Theprotein content of the enzyme solution was determined using a Sigma proteinassay kit, and the activities expressed as nmol Pi transferred per mm per mgprotein. The activity at pH 8.2 is the enzyme specific activity. Activation of theenzyme can be expressed either as activity at pH 6.8, or by the ratio of activity atpH 6.8 to the activity at pH 8.2.6. Protein DeterminationThe concentration of protein in the enzyme solutions of bothphosphorylase and PhK assay was determined by a modified Lowry assaymethod using the Sigma protein assay kit. The analytical wavelength used was750 nm and the standard curve range was from 25 to 200 jig/mI (Figure 6). Foreach assay, one reagent blank and two buffer blanks were carried out at sametime.31t.224—CI-.--.Cu IUCrcS• 4oeo•800 -‘ 7o.oeaProtein Concentration (jig/mi)Figure 6. Standard curve for the Lowry protein assay. Bovine serumalbumin was used as a standard. The assay was performed using the Sigmaprotein assay kit.327. Cardiac cAMP assayCyclic AMP levels in the frozen heart tissues were determined by aradioimmunoassay kit supplied by Amersham. Briefly, about 20 mg of frozensamples were homogenized in 1 ml 6% TCA. After removal of TCA by washingthree times with ether, the ether was blown off with nitrogen gas andradioimmunoassys were performed on the ether free aqueous extracts. Resultsare expressed as picomoles of cAMP per mg wet tissue.8. Serum Glucose and Insulin DeterminationGlucose content of the serum was determined with the PeridochromRGlucose GOD-PAP assay kit. Serum insulin was measured by aradioimmunoassay using the Immunocorp insulin radioimmunoassay kit. Theassay was carried out following the standard procedures except that allquantities of either samples or reagents were reduced to half of that in theprotocol, i.e. half assay was used.9. Statistical AnalysisResults are presented as mean ± S.E.M. (standard error of mean).Statistical analysis was performed using two-way ANOVA followed by acomparison of group means with a Duncan’s test. When only two group meanswere compared, statistical analysis was performed using an unpaired Student’st-test. A probability of p<0.05 was taken as the level of statistical significance.33RESULTSI. Validation of Assay Methods1. Hepatic Phosphorylase AssayIn order to determine the concentration of caffeine necessary for theassay of hepatic phosphorylase, the hepatic phosphorylase assay was carriedout at three caffeine concentrations: 0.0, 0.5, and 1.0 mM using liver from controlrats (Table 2). The total phosphorylase activity was not affected by thepresence of caffeine. However, the a activity decreased with increase incaffeine concentration. Thus the phosphorylase a ratio also decreased. Theratio in the presence of 1 mM caffeine was significantly lower than the valueassayed without caffeine and was closer to the values reported in literature(Gilboe etah, 1972; Tan eta!., 1975).The possible effects of treatment with vanadyl sulphate on the assay ofhepatic phosphorylase was also examined (Table 2). No difference wasobserved between the values of non-treated and treated animals with regard toGPa ratio, a activity or the total activity at each assay with the variousconcentrations of caffeine, indicating that the treatment did not affect the assay.In addition, the concentrations of caffeine had similar effects on the a activityand the ratio in both treated and non-treated rats. Therefore, 1 mM caffeine wasthe concentration of choice in subsequent hepatic phosphorylase assays.Table2.EffectsofCaffeineConcentrationsonHepaticPhosphorylaseAssayPhosphorylaseActivityQimol Pi/mgprotein/mm)GPaRatio(-AMP/÷AMP)aActivityTotalActivity0.0mMCON83.8±2.30.29±0.0090.35±0.03CONV86.6±0.70.31±0.010.35±0.010.5mMCON79.5±1.60.28±0.020.35±0.02CONV83.0±0.90.29±0.010.35±0.02**1.0mMCON73.9±1.50.26±0.020.35±0.02CONV79.4±1.6*0.28±0.02*0.35±0.01Allvaluesaremean±SEMandrepresentthemeansofsixobservationsmadeoncontrol(CON)orcontrol-treated(CONV)animals.*denotesasignificantdecrease(p<0.05)intheactivityratioandtheaactivityincomparisonwiththevaluesobtainedintheabsenceofcaffeine.DatawereanalyzedbytwowayANOVAfollowedbyDuncan’smultiplecomparisontest.352. Cardiac Phosphorylase Kinase Assay.To determine the optimal assay conditions for cardiac phosphorylasekinase, different reaction time periods and different protein concentrations ofenzyme extract were tested. The reaction rate, defined as the total countsdivided by the reaction time, was used as a criterion. The assay was terminatedat different time points and the reaction rate at pH 6.8 and 8.2 was investigated(Figure 7). The reaction rate at pH 6.8 was linear over the period of 30 mm.However, the rate at pH 8.2 was only linear for the first 10 mm, then declinedwith increase in reaction time.To determine optimal concentration of enzyme extract (Figure 8), twodifferent enzyme extract solutions were used for the assay. The enzyme extractwas diluted with an equal volume of the dilution buffer for the diluted sample,while in the nondiluted sample, this step was excluded. The assay was carriedout at pH 6.8 and terminated at different time points. Although the reaction ratedid increase with the increase in the enzyme extract concentration and the ratevalue of the nondiluted samples was always higher than those of the dilutedsamples, the increase in the reaction rate was not constant over differentreaction time points. In addition, the increase of the reaction rate was notproportional to the increase in the concentration of the enzyme extract at mosttime points. Since only the reaction rate of diluted enzyme extract was constantand the rate of assay at pH 8.2 was only linear upto 10 mm, subsequent cardiacphosphorylase kinase assays were carried out for 5 mm using diluted enzymeextract.36EE0.0a,4—ci04—0a:Time (miniN pH 6.8A pH 8.2A-a-300 -240-180-120-60 -0- I - I I0 5 10 25 30 35Figure 7. Time Course of the Reaction Rate of CardiacPhosphorylase Kinase Assay. The assay was carried out at pH 6.8 and8.2 as described in Materials and Methods using cardiac tissue from the samecontrol rat. The assay was terminated at different time points. Data pointsrepresent the reaction rate at different reaction times.15 2037300250SEo.200C)0150Co1004-,C)5005 mm 10 mm 15mm 20mmTIME (mm)Figure 8. Effects of Protein Concentration on the Reaction Rate ofCardiac Phosphorylase Kinase. The assay was processed at pH 6.8 asdescribed in Materials and Methods with two different cardiac enzyme extractconcentrations. The enzyme extract of non-diluted samples was twice asconcentrated as compared to diluted samples. The assay was terminated atdifferent time points and each bar in the graph represents the reaction rate at aspecific time.25mm 30mm38II. General Features of Experimental AnimalsAs described in Materials and Methods, animals in the same body weightrange (170-200 g) were used for each set of experiments. The initial bodyweight, serum insulin and glucose levels were the same in rats used in differentexperiments. For all the experiments not involving vanadyl sulphate (VS)treatment, the rats were killed 6 weeks after diabetes was induced. The VStreated rats, however, were killed 8 weeks after diabetes induction. Bodyweight and serum samples were taken. Since the characteristics of non-treatedanimals were essentially the same, the general features of experimentalanimals were described as two major groups: treated and non-treated groups.The comparison was thus made between diabetic and control, or betweentreated and non-treated animals.1. Body Weight of the AnimalsFigure 9 presents body weights of rats used in experiments. There weretwo groups of animals: (1) 6-week non-treated diabetic and control rats and (2)8-week diabetic and control rats on vanadyl sulphate (VS)-treatment. In bothgroups, the body weight of diabetic animals was significantly lower than that ofthe controls. Although the rats in the VS-treated group were two weeks olderthan those in the 6-week group, the body weight of these animals was similar,indicating a slower body weight gain.392. Serum Insulin LevelSerum insulin levels of both VS-treated and non-treated control anddiabetic rats are presented in Figure 10. The insulin level in diabetic rats wassignificantly lower than that in the controls. Treatment with vanadyl sulphate didnot affect the insulin levels of treated diabetic rats. The insulin values of treatedcontrol rats were the same as that measured in the non-treated controls.3. Serum Glucose LevelIn the non-treated diabetic group, the serum glucose levels weresignificantly increased compared to controls. This increase in glucose levelswere largely eliminated by five week VS-treatment. The glucose levels oftreated diabetic rats (DIAV) were not different from controls (CON) (Figure 11).There was a variable response of diabetic rats to VS treatment: some becamecompletely euglycemic (41.7%), while the glucose level of others were onlypartially lowered(33.3%) or (25%) remained high.40Figure 9. Body Weight of VS-treated and Non-treated Control andDiabetic Rats. The body weights shown in the graph were recorded at thetime of sacrifice. The bars for the non-treated animals (CON & DIA) representthe mean of observations made with 36-37 rats used in different sets ofexperiments, while the bars for treated animals (CONV & DIAV) represent themeans of 12-14 animals involved in VS-treated study. An asterisk denotessignificant decrease (p<O.05) in body weight for diabetic animals in comparisonwith controls as analyzed by two way ANOVA followed by Duncan’s multiplecomparison test.41ECu0,I0Ui>-00U)60050040030020010006 WEEKS 8 WEEKSVS—TREATED42Figure 10. Serum Insulin Level of VS-treated and Non-treatedControl and Diabetic Rats. The serum samples were taken at the time ofsacrifice. Insulin levels were determined as described in Materials andMethods. The bars for the non-treated animals represent the mean ofobservations made with 35(CON)-37(DIA) rats used in the different sets ofexperiments, while the bars for treated animals represent the means of12(DIAV)-13(CONV) animals involved in VS-treated study. An asterisk denotessignificant decrease (p<O.05) in serum insulin level for diabetic animals ascompared to controls as analyzed by the Student-t test.SERUMINSULINLEVEL(pmol/I)-‘C)00000000IIIIC)I II__________HLJ44Figure 11. Serum Glucose Levels of VS-treated and Non-treatedControl and Diabetic Rats. The serum samples were taken at the time ofsacrifice. The glucose levels were determined as described in Materials andMethods. The bars for the non-treated animals represent the mean ofobservations made with 25(DIA)-27(CON) rats used in the different sets ofexperiments, while the bars for treated animals represent the means of12(DIAV)-13(CONV) animals involved in VS-treated study. An asterisk denotesa significant increase (p<O.05) in the serum glucose level for diabetic animals incomparison with their corresponding controls. The star denotes a significantdifference in comparison with non-treated controls as analyzed by two wayANOVA followed by Duncan’s multiple comparison test.E-jLULU-jUiCI)00-J(5a:Ui(I)453025201510506 WEEKS 8 WEEKSVS—TREATED46Ill. Effects of ISO on Cardiac Phosphorylase. Phosphorylase Kinase and cAMPin Control and Diabetic Rats.1. Time Course for ISO Stimulation on Cardiac Phosphorylase.The activation of phosphorylase in female rats was examined usingworking heart preparations. The phosphorylase activation following a bolusinjection of ISO (1 ml, 10-8 M ) is shown in Figure 12. The activation wasapparent at as early as 20 sec after the injection, peaked at 30 sec and thendeclined. After 60 sec, the phosphorylase a ratio returned to the basal level.The activation of phosphorylase in the diabetic rat heart displayed the sameactivation time course, but the magnitude of activation was much greater. Formale rats which were used in most of the experiments, a Langendorff heartperfusion preparation was used and ISO was present in the perfusate at aconcentration of 5x10 M. The time course of phosphorylase activation wasdifferent from that seen with the working heart method. As seen in Figure 13,phosphorylase was significantly activated 30 sec after ISO perfusion wasstarted; the activation was maintained and was even higher at 60 sec. TheLangendorff preparation was therefore used throughout the subsequentexperiments, and the perfusion with ISO was carried out for 30 sec.47Figure 12. Time Course of Cardiac Phosphorylase Activation byISO in Female Control and Diabetic Rats. The hearts were perfused inthe working heart apparatus for 20 mm and ISO was administered as a 1 mlbolus(1 8 M solution) into the inflow tubing cathetered into the left atria. Thehearts were subsequently freeze-clamped at different time points.Phosphorylase was assayed as described in Materials and Methods. The ratswere diabetic for six weeks. Data points represent means of observations madewith 5-7 rats. An asterisk denotes significant difference (p.<0.05) from controlsand was analyzed by the Student-t test.R DiabeticU Control.48**0(‘Ici)U)>100U)0070-60-50-40—30—20-100- I I I I I I0 10 20 30 40TIME (SEC)50 604960-50-04-,(a:40-CDC’,C3o0.U,o 20-01 O I I I I0 15 30 45 60TIME (SEC)Figure 13. Time Course of Cardiac Phosphorylase Activation byISO in Male Rats. The hearts were perfused in the Langendorif mode. ISOwas present in the perfusate at a concentration of 5x109 M. The hearts werefreeze-clamped at different time points and phosphorylase was assayed asdescribed in Materials and Methods. Data points represent means ofobservations made with 2-6 rats.S DiabeticD Control502. Supersensitivity of Phosphoiylase Activationin Response to ISO Stimulation.The basal activity of cardiac phosphorylase in male diabetic rats was thesame as that in control rats. However, the activation of phosphorylase by ISOwas much greater in the diabetic hearts. This was indicated by a higherphosphorylase a activity as well as a higher activation ratio (Figure 14). Itshould be noted that ISO stimulation did not alter the total phosphorylaseactivity, although this activity was significantly higher in the diabetic rat hearts(Figure 14, lower panel).3. Effects of ISO on Cardiac Phosphorylase KinaseThe phosphorylase kinase (PPK) profile obtained from the same cardiacsamples are presented in Figure 15. Although the PPK activity at pH 6.8 wasdifferent, the activation ratio (activity ratio of pH 6.8/8.2) of PPK was not differentbetween control and diabetic rat heart under basal conditions (lower panel ofFigure 15). However, when stimulated by ISO, the magnitude of PPK activationwas much greater in diabetic rats as shown by the higher activation ratio andactivity at pH 6.8. Similar to GP activation, activation of PPK in response to ISOstimulation was apparently also more sensitive in diabetic rats than in controls.On the other hand, the total activity of PPK at pH 8.2 was not changed by thediabetic state in either basal or the ISO-stimulated conditions, whereas the totalactivity of GP was significantly increased in the diabetic animals (Figure 14).514. Effects of ISO on cardiac cAMPThe basal level of cardiac cAMP was not different between control anddiabetic rats. When stimulated with ISO, cAMP levels were significantlyelevated only in hearts of the control rats. ISO stimulation of cAMP productionwas essentially negligible in the heart of diabetic rats (Figure 16, non-treatedgroup), in contrast to the situation for GP and PPK (Figure 14 and 15), where theactivation was significantly greater in the diabetic rats.IV. Effects of Verapamil Perfusion on Cardiac Phosphorylase. PhosphorylaseKinase and cAMP1. Effects of Verapamil and ISO Stimulation on Cardiac GP.Perfusion of rat hearts in the Langendorff mode with verapamil (5x108M) for 20 mm did not change the basal level of GP activity (Figure 17, lowerpanel). Subsequent perfusion with ISO significantly stimulated the GP activity.Interestingly, the activation was the same for both control and diabetic rats(Figure 17), i.e. the supersensitivity of GP activation by ISO in diabetic hearts(Figure 14) was abolished. Although the perfusion of verapamil abolisheddiabetes-induced supersensitivity of GP activation, it did not affect the increasein total activity of GP in diabetic hearts (Figure 14). Similar to the diabetic heartswithout verapamil perfusion, the total activity of GP in verapamil perfuseddiabetic hearts was still significantly higher than that of controls (Figure 17). Itwas also noted that the basal GPa activity was significantly higher in diabetic ratheart, which was not observed for hearts that were not perfused with verapamil.522. Effects of Verapamil and ISO Stimulation on Cardiac PPK.The basal activity ratio, pH 6.8 and 8.2 activities of PPK were not affectedby verapamil perfusion. Following the stimulation of ISO, however, theverapamil perfusion prevented the activation of cardiac PPK by ISO in bothcontrol and diabetic rats and thus abolished the increased sensitivity of PPKactivation by ISO in diabetic hearts (Figure 15 and 18) It should be also notedthat the total activity of PPK was again not different between control and diabeticrats and its not altered by the use of verapamil.3. Effects of Verapamil and ISO Stimulation on Cardiac cAMP.Cyclic AMP concentrations in cardiac tissues perfused with verapamilwere similar in control and diabetic rats under basal condition. Whencompared to non-treated control group, the basal levels of cAMP were notchanged by verapamil perfusion (Figure 16, VP-perfused group). The increasein cAMP levels after the stimulation with ISO were minimal in verapamilperfused hearts. Even in the control hearts, perfusion with verapamil prior to theapplication of ISO prevented the increase in cAMP levels observed in nonperfused controls (Figure 16, VP-perfused and non-treated groups). Therewere no difference observed in cAMP levels between control and diabetichearts in both basal and ISO stimulated conditions.53Figure 14. Basal Cardiac Phosphorylase Values and Effects of ISOon Phosphorylase in Control and Diabetic Rats. The hearts wereperfused in the Langendorff mode and were freeze-clamped either after 20 mmperfusion with CK buffer or after 20 mm perfusion with an additional 30 secperfusion with CK buffer containing ISO. ISO was present in the perfusate at aconcentration of 5x109 M. Phosphorylase was assayed as described inMaterials and Methods. The upper panel shows the activation ratio ofphosphorylase. The phosphorylase a activity and total activity are shown in thelower panel. Bars represent means of observations made with 6-8 rats. Thediabetic rats (DIA) were diabetic for six weeks and the controls (CON) were age-matched. An asterisk denotes a significant difference (p<0.05) in comparisonwith controls. A star denotes a significant difference in comparison with ISOstimulated controls (ISO-CON) by two way ANOVA followed by Duncan’smultiple comparison test.PhosphorylaseActvity(molP11mm/mgprotein)oPhosphorylaseaRatio(%1-0000000C’, 0 0 z C’)0t)C’) DD> 0 i-f i-f -1 0 C) i-f I-f.55Figure 15. Basal Cardiac Phosphorylase Kinase Values andEffects of ISO on Phosphorylase Kinase in Control and DiabeticRats. The phosphorylase kinase assay was carried out on the same cardiactissue that was used for the cardiac phosphorylase assay. The upper panelshows the activity ratio of phosphorylase kinase, whereas the lower panelshows the phosphorylase kinase activity at pH 6.8 and 8.2. Bars representmeans of observations made with 4-6 rats. An asterisk denotes a significantdifference (p.<O.05) in comparison to controls (CON). A star denotes asignificant difference in comparison with ISO-stimulated controls (ISO-CON) bytwo way ANOVA followed by Duncan’s multiple comparison test.C) C) t-t I 0 I-,PhosphorytaseKinaseActvity(molP1/mm/mgprotein)J)(npo0000o00000o000000 0 zIActivityRatio(pH68/8.2)0000000)(I) C) 0 z Cl)57Figure 16. Basal Cardiac CAMP and Effects of ISO on cAMP inControl and Diabetic Rats. The cAMP was assayed as described inMaterials and Methods. The non-treated group did not receive any treatment,the VANA-treated rats were treated with VS for five weeks, and VP-perfusedgroup were perfused with verapamil (5x108 M) for 20 mm before being freezeclamped for cAMP determination. Some of the hearts were stimulated with ISO(5x109 M) for 30 sec prior to being freeze-clamped. Bars represent means ofobservations made with 4-6 rats. An asterisk denotes a significant difference(p<0.05) when compared to control (CON) by two way ANOVA followed byDuncan’s multiple comparison test.CyclicAMPConcentration(fmol/mgTissue)(310)-ooooooo0III1III0I-,Z 0C)Z0I > z >z II*C,, C) 0 z C/)*-1 m > -I m -tJ-h m -Il C’, m—1*H—Lt59Figure 17. Effects of Verapamil (VP) Perfusion on CardiacPhosphorylase. The hearts from both control (CONVP) and diabetic (DIAVP)animals were perfused with verapamil (5x108 M) for 20 mm prior to freeze-clamping or prior to a 30 sec perfusion with ISO. Phosphorylase activity wasassayed as described in Materials and Methods. The upper panel shows theactivation ratio of phosphorylase; whereas the phosphorylase a activity andthe total activity are shown in the lower panel. Bars represent means ofobservations made with 5-6 rats. An asterisk denotes a significant difference incomparison with the control group as analyzed by one-way ANOVA followed byDuncan’s multiple comparison test.E C) 0 z -o I (1) 9 C) 0 z -U Cl) 9 -UPhosphorylaseActvity.(molPi/min/rrigprotein)a1’.) aa0PhosphorylaseaRatiof%)-1b)00000(SI 000> 0 .4 H 0 .4 C) .4 ‘4 1<61Figure 18. Effects of Verapamil (VP) Perfusion on CardiacPhosphorylase Kinase. The hearts from both control (CONVP) and diabeticgroup (DIAVP) rats were perfused with verapamil (5x108 M) for 20 mm prior tofreeze-clamping or prior to the 30 sec perfusion with ISO. PhosphorylaseKinase activity was assayed as described in Materials and Methods. The upperpanel shows the activity ratio of phosphorylase kinase. The activity at pH 6.8and 8.2 is shown in the lower panel. Bars represent means of observationsmade with 5-6 rats. An asterisk denotes a significant difference in comparisonwith the control group as analyzed by one-way ANOVA followed by theDuncan’s multiple comparison test.PhosphorylaseKinaseActvity(nmolP1/mm/mgprotein)000o00o00ActivityRatio(pH68/&2)ooppppZwiiC)II9) C) I-,0 oi0 z1’.)-o—1-4C’, o 00 00 0 a000-‘N)63V. Effects of Vanadyl Sulphate (VS) Treatment on Cardiac Phosphorylase.Phosphorylase Kinase and cAMP.1. Effects of VS treatment on Cardiac PhosphorylaseAfter treatment with oral VS (1OO mg/kg/day) for five weeks, the basalGPa activity and the GPa ratio were similar in control and diabetic rat hearts(Figure 19). VS treatment of control and diabetic rats did not alter basal GPaactivity and GPa ratio when compared to non-treated control and diabetic rats(Figure 14). More importantly, the supersensitivity of cardiac GP activation byISO that was observed in non-treated diabetic hearts (Figure 14) wascompletely eliminated with VS treatment. Additionally, the significantly highertotal activity of GP observed in non-treated diabetic heart was restored to thecontrol level after the treatment (Figure 19). Therefore, a complete normalprofile of phosphorylase was observed in diabetic hearts following chronic VStreatment.2. Effects of VS treatment on Cardiac Phosphorylase KinaseSimilar to the effects observed with GP, the significantly higher pH 6.8activity of cardiac PPK in diabetic rats (Figure. 15) was restored to control levelafter chronic treatment of the diabetic rats with VS. Likewise, the activation ofPPK by ISO perfusion was similar in both control and diabetic VS treated rathearts (Figure 20), i.e. the greater activation in both activity ratio and pH 6.8activity of PPK in diabetic non-treated hearts to ISO stimulation was alsoabolished by the treatment. It is worth noting that the total activity of PPK in the64VS-treated animals was significantly lower than that of the untreated rats forboth control and diabetic groups, although in both cases the total activity wasnot different between control and diabetic hearts (Figure 15, 20).3. Effects of VS-treatment on Cardiac cAMP.As seen in Figure 16, VS treatment did not change the basal levels ofcAMP in hearts of either control or diabetic rats when compared to non-treatedcontrols (Figure 16, non-treated groups). The treatment, however, did restorethe depression in ISO-induced increase in cAMP in the hearts of diabetic rats.Therefore, cardiac cAMP levels in both treated control and diabetic rats werenot different from non-treated controls in either basal or ISO stimulatedconditions (Figure 16, treated and non-treated groups).VI. Alteration of Hepatic Phosphorylase in Diabetes and the Effects of VanadylSulphate Treatment.1. Alterations of Hepatic PhosphorylaseThe phosphorylase activity in the livers of control and diabetic rats,treated or not treated with vanadyl sulphate (for five weeks or five months) wereexamined. Neither the diabetic state nor the vanadyl sulphate treatment alteredthe GPa ratio; the ratio did not change with age (Table 3). However, thediabetic state did change the a activity and the total activity of hepatic GP. Forexample, relative to their age-matched controls, diabetic rats from untreatedgroups revealed a significant decrease in both GPa activity and total activity.65Moreover, a trend was noted that the decrease became greater with theincrease in the duration of the diabetic state: both GPa and the total activity infive month untreated diabetic rats were less as compared to five week diabeticrats (Table 3).2. Effects of Vanadyl Sulphate Treatment.Diabetes-induced decrease in hepatic GPa and total GP activity wascorrected by treatment with vanadyl sulphate (Table 3 and Figure 21). Theeffects of the treatment were time dependent. While the treatment partiallyrestored both GPa and total activity of hepatic phosphorylase in five weekdiabetic rats, the five month treatment completely restored the phosphorylase aand total activity of diabetic rats. It should be noted that five month treatmentwith vanadyl sulphate slightly decreased the phosphorylase a and total activityin treated controls (Table 3, Figure 21); however, the shorter treatment of fiveweek group did not produce any change in the control rats. The response totreatment in the five week group was quite variable among the diabetic rats andranged from complete restoration to no effects. The effects of vanadyl sulphatewere quite consistent for rats treated for five months. Another interestingobservation was that the effect of vanadyl sulphate treatment on hepaticphosphorylase was independent of its euglycemic effects i.e. the rats withrestored hepatic phosphorylase activities did not necessarily have a normalizedserum glucose level, or vice versa.66Figure 19. Effects of Five Week Vanadyl Sulphate Treatment onCardiac Phosphorylase. Phosphorylase activity was determined in heartsfrom control (CONV) and diabetic (DIAV) rats which were treated with vanadylsulphate for five weeks. The hearts were perfused for 20 mm before freeze-clamping or before exposure to ISO for 30 sec. Phosphorylase was assayed asdescribed in Materials and Methods. The upper panel shows the activationratio of phosphorylase and the lower panel shows the phosphorylase a activityand total activity. Bars represent means of observations made with 6 rats. Anasterisk denotes a significant difference in comparison with the groups withoutISO perfusion as analyzed by one-way ANOVA followed Duncan’s multiplecomparison test.I Cl, C) 0 z Cl)PhosphorylaseActvity(moIPi/min/mgprotein)C) 0 zCw‘aD00PhosphorylaseaRatio(%) (71C)000000 H 0 0 ‘-4 ‘-468Figure 20. Effects of Five-Week Vanadyl Sulphate Treatment onCardiac Phosphorylase Kinase. Phosphorylase kinase activity wasdetermined in hearts from control (CONV) and diabetic (DIAV) rats which weretreated with vanadyl sulphate for five weeks. The hearts were perfused for 20mm before freeze-clamping or before exposure to ISO for 30 sec.Phosphorylase kinase was assayed as described in Materials and Methods.The upper panel shows the activity ratio of phosphorylase kinase. The lowerpanel shows the phosphorylase kinase activity at pH 6.8 and 8.2. Barsrepresent means of observations made with 5-6 rats. An asterisk denotes asignificant difference in comparison with the groups without ISO perfusion asanalyzed by one-way ANOVA followed by Duncan’s multiple comparison test.PhosphorylaseKinaseActvity(nmolP11mm/mgprotein)p0o0o0- 0 0 CL) 0 0 00ActivityRatio(pH6.8/8.2)00 3000 )C) 0 z Q Cl) 0 ) 0 z (1•) 0 rI a, > C) I > 0 p4 f-ION70Table 3. Diabetes-Induced Alteration in the Activity of HepaticPhosphorylase and the Effects of Vanadyl Sulphate TreatmentPhosphorylase Activity(mmol Pi/mg/min)Activity Ratioa Activity Total Activity6 wks CON 73.9±1.5 0.26±0.02 0.35±0.02* *DIA 68.4±2.9 0.14±0.006 0.20±0.015 wks CONV 79.4±0.8 0.28±0.01 0.35±0.02DIAV 78.6±1.6 0.19±0.02* 0.24±0.03*5 mths CON 77.6±1.9 0.29±0.02 0.38±0.02* *DIA 69.1±3.7 0.12±0.02 0.18±0.02CONV 77.4±0.8 0.26±0.01 0.34±0.02DIAV 73.8±1.6 0.22±0.01 0.30±0.02All values are mean± SEM of six animals. * denotes significant difference (p< 0.05) vscontrol vanadyl suphate-treated (CONV) by two-way ANOVA followed by Duncansmultiple comparison test.71Figure 21. Effects of Diabetes and Five-month Treatment withVanadyl Sulphate on Hepatic Phosphorylase. The hepaticphosphorylase activities were determined in livers from five month vanadylsulphate treated control and diabetic rats (CONV and DIAV) and untreated five-month diabetic (DIA) and control rats (CON). Phosphorylase a activity wasassayed in the presence of 1 mM caffeine as described in Materials andMethods. The upper panel shows the activation ratio and the lower panelshows the phosphorylase a and total activity. Bars represent means ofobservations made with 5-6 rats. An asterisk denotes significant difference(P<O.05) between DIA and all the other groups and a star significant differencebetween control and treated diabetic rats as analyzed by one-way ANOVAfollowed by Duncan’s multiple comparison test.C) 0 z C) 0 zPhosphorylaseActvity(rrioIP1/mm/mgprotein)0000aaaa0-r’U)0PhosphorylaseaRatio(%)00C) ‘4 ‘41 >C)C)00000-1 0 ‘4 C) ‘4 ‘473DISCUSSIONI. The Nature of the Supersensitivity of GP ActivationThe supersensitivity of GP activation by catecholamines in diabeticanimals has been well recognized. Miller et a!. (1981) showed that basalcardiac GP activity was not altered in acute alloxan-induced diabetes but theactivation of GP by epinephrine was significantly enhanced. Miller et al. alsodocumented a supersensitivity of GP activation by epinephrine inspontaneously diabetic Bio-Breeding/Worcester (BB/W) rats (Miller, 1983).Vadlamudi and McNeilI (1983) extended this study by showing asupersensitivity of GP activation by catecholamines in chronically diabetic rats.Additionally, they also reported a significantly greater total phosphorylaseactivity in the diabetic rat heart. Results obtained in the present investigationconfirmed that ISO (5x1O9M) caused a greater activation of GP in diabetichearts as compared to age-matched controls. While basal GPa activity was notdifferent between the diabetic and control rats, the total GP activity wassignificantly higher in the diabetic rats. Our observations are thus consistentwith previous reports. Supersensitivity of GP activation by catecholamines isalso found to be present in isolated cardiomyocytes from diabetic rats (Miller &Jaspers, 1991). Apparently, supersensitivity of cardiac GP activation is afundamental alteration that occurs in the myocyte and is directly related to thediabetic condition and insulin deficiency.As the activity of GP is regulated by PPK-induced phosphorylation andphosphorylase phosphatase (PP)-induced dephosphorylation, diabetes-relatedsupersensitivity of cardiac GP activation by ISO could be explained by either an74increase in the activity of PPK or a decrease in the activity of PP. It is alsopossible that the abnormality is due to the combined alterations of bothenzymes. Miller et al., (1981, 1983, 1984) observed that there were nostatistically significant differences between control and diabetic rat heartsperfused with or without epinephrine when the activity of PP was measured,which led to their conclusion that the diabetes-related defects in GP activationwas unlikely to be due to a PP deficiency (Miller and Jaspers 1991). However,others have reported a two fold decrease in the activity of PP in the liver extractsprepared from alloxan-diabetic rats (Foulkes et aI.,1984), which was largelyreversed by the administration of insulin (Dragland-Meserve et a!., 1985),indicating that the activity of PP can be regulated by insulin treatment (Cohn,1989). Hence, the elucidation of a role for the PP in the supersensitivity of GPactivation requires further studies and will not be discussed in the thesis.There have been few studies on the changes of PPK in diabetic rat heart.Miller et aL (1981) reported a two fold increase in the pH 6.8/8.2 ratio of PPK indiabetic rat heart in response to epinephrine. They also observed that the basalactivity ratio of PPK in diabetic rat heart was not different from that of thecontrols. Consistent with their report, we observed a significantly higher degreeof PPK activation in the diabetic rat hearts in response to ISO stimulation, whichcorrelated with the supersensitivity of GP activation. When stimulated with ISO,the increase in the activity of PPK at pH 6.8 and the activity ratio of pH 6.8/8.2were significantly greater in the diabetic than in the control rats. Theseobservations suggest that the increase in the sensitivity of ISO-inducedactivation of cardiac PPK may be involved in the occurrence of thesupersensitivity of GP activation. However, there have also been reports thatPPK was not changed in diabetic rat heart (Miller et a!., 1984). This is in direct75contrast to our experimental results as well as to the earlier report from thesame research group (Miller eta!., 1981). The explanation for the discrepancybetween this report and our observation is not clear, since only activity ratio wasused in the report and no details were presented on the absolute PPK activities.What are the mechanism(s) responsible for the diabetes-relatedalteration in cardiac PPK? In general, PPK is covalently regulated by the cAMP-dependent protein kinase (PKA) and also controlled by calcium (Heilmeyer,1991). Previous studies on the myocardial cAMP-PKA cascade system haveshown that : (1) Basal activity of cardiac adenylate cyclase (AC) in diabeticanimals was not different from the controls, while a significant depression wasobserved in the catecholamine stimulated activity of AC in diabetic rat hearts(Smith eta!., 1984, Michel eta!., 1985). (2) Basal cAMP, cGMP and PKA activitywere not altered by diabetes (Miller, 1981, 1984, Vadlamudi and McNeill,1983). In the present study, we also observed no difference in basal cAMPlevels between diabetic and control rats. On the other hand, reports on thechanges in catecholamine-stimulated levels of cAMP in diabetic hearts arecontroversial. Vadlamudi and McNeill (1983) documented similar changes incAMP levels in diabetic and control hearts after stimulation with ISO. However,Miller eta!. (1981, 1984) observed that hearts from diabetic rats accumulatedsignificantly less cAMP in response to epinephrine than did control hearts at allconcentrations tested. Ingebretsen eta!., (1981) also reported that ISO-inducedmyocardial changes in cAMP content and PKA activity ratios were depressed by50% in diabetic animals. Almira and Misbin (1989) reported similar findings.They demonstrated that basal cAMP levels were 2.5 times higher in diabeticcardiomyocytes than in control cells. Addition of M ISO caused only a 37%increase in the cAMP levels in diabetic myocytes as compared to a 250%76increase in the controls. Our results confirm that ISO-stimulated increase incAMP content from diabetic heart was markedly reduced as compared to theincrease in the controls. Hence, it appears that the catecholamine-stimulatedproduction of cAMP was defective in diabetic rat hearts. Since these changescould not cause greater activation of either PPK or GP, we suggest that thesupersensitivity of GP activation in diabetic hearts is not due to alterations in thecAMP-PKA cascade.Free intracellular calcium is another important regulator of PPK and GP.Diabetes-related abnormalities in cardiac calcium metabolism have beenreported previously. Three lines of evidence have been documented: (1)Isolated membrane studies have revealed that the activity of all the majorcalcium transporters are inhibited during diabetes (Heyliger eta!., 1987, Makinoet al., 1987); (2) Treatment of STZ-diabetic rats with the calcium antagonistverapamil decreased the severity of diabetic cardiomyopathy (Afzal eta!., 1988,1989); and (3) Supersensitivity of GP activation by epinephrine could beeffectively blocked by perfusion of diabetic rat hearts with a low calcium mediumor by using perfused hearts from adrenalectomized diabetic rats in which alower intracellular free calcium has been suggested (Miller et aL, 1984). Wealso investigated the role of intracellular Ca2+ in the mechanism of thesupersensitivity of GP activation by perfusion of rat hearts with verapamil (5x108 M) for 20 mm before ISO stimulation was applied. The perfusion did not affectthe basal or total activity of GP but abolished the supersensitivity of GPactivation in response to ISO stimulation. In the case of cardiac PPK, theperfusion caused an increase in the activities of PPK as compared to that of thenon-perfused hearts. On the other hand, the perfusion alone blocked thestimulatory effect of ISO on the enzyme activity, i.e., the activation of PPK in the77perfused hearts diminished after the perfusion. In agreement with ourobservations, Miller et a!. (1984) reported perfusion of diabetic hearts with asubphysiological concentration of calcium (0.83 mM), which produced a partialreverse of the diabetes-related supersensitivity of GP activation by epinephrine.Taken together, our experimental findings and the results of others suggest thatthe supersensitivity of GP activation by catecholamine in diabetic rat heart maybe partially related to an increase in the activation of PPK as a result of adefective calcium homeostasis.In summary, we demonstrated that the supersensitivity of GP activation indiabetic rat heart is correlated with an increase in the sensitivity of PPKactivation in response to ISO stimulation. The alterations in PPK activation,however, could not be explained by changes in one of the regulating systems ofPPK, the cAMP-PKA cascade, which is depressed instead of enhanced in thediabetic heart. Perfusion of diabetic rat heart with the calcium channel blockerverapamil successfully abolished the supersensitivity of GP activation as well asthe greater sensitivity of PPK activation by ISO stimulation. It is thus concludedthat defects in calcium metabolism plays a significant role in the mechanism ofthe supersensitivity of cardiac GP activation.78II. Reversal of Biochemical Alterations in Diabetic Rats by Vanadyl SulphateTreatment.The insulin-like effects of vanadium have long been recognized. Studieson using vanadium as a possible therapeutic agent for diabetes, however, wereinitiated by the report of Heyliger et at., (1985) who demonstrated that sodiumorthovanadate administered in drinking water was able to lower blood glucoselevels and improve cardiac performance of diabetic rats. Subsequent worksrevealed that the vanadyl form (V4j rather than the vanadate form (V5j ismore appropriate for chronic use in diabetic rats as it is equally effective but lesstoxic and more tolerable to diabetic animals (Ramanadham et al., 1989).Vanadyl sulphate (VS) was the compound used in the present study.A. Effects of VS Treatment on General Featuresof Experimental AnimalsAs a result of STZ treatment, body weights of diabetic rats weresignificantly lowered as compared to age-matched control animals. Diabeticrats also exhibited lower plasma insulin and higher levels of circulating glucose.VS-treatment did not show any significant effect on the weight-gain ofdiabetic animals. However, a slower weight-gain was noted in the VS-treatedcontrol rats. The treated rats were two weeks older than non-treated controlsbut the body weights were the same in the two groups. A decrease in foodintake and a significant reduction in body weight-gain has also been observedby other investigators in control rats receiving V03(Shechter et al., 1990).79Some studies have suggested that vanadium compounds can be easilytransported into brain, and similar to its action in peripheral tissues, it increasesglucose uptake and glucose metabolism in brain tissue. The increased glucosemetabolism within the central nerve system (CNS) may have then produced asignal to reduce eating, resulting in a reduction in body weight-gain. This effectof vanadate was less consistently observed in diabetic rats (Meyerovitch et a!.,1989). In the case of insulin, it is less likely that insulin will enter the CNS andexert direct effects on brain tissue. In fact, it has been shown that when insulinwas injected centrally into experimental animals, it rapidly lowered plasmaglucose levels by blocking glucose output from liver (Szabo and Szabo, 1983).The circulating level of insulin is lower in STZ diabetic rats than in thecontrols. VS treatment did not alter the serum insulin level in control animals,nor did it affect the significantly lowered insulin levels in diabetic rats. Whilethere is evidence that vanadium can induce or potentiate glucose stimulatedinsulin secretion from mouse or rat islet cells in vitro (Fagin et aL, 1987, Zhanget aL, 1991), it has been repeatedly reported that the normalization of elevatedblood glucose level in diabetic animals by vanadium treatment was notaccompanied by an increase in insulin secretion (Heyliger et aL, 1985;Ramanadham et a!., 1989). In fact, using both STZ-induced or spontaneouslydiabetic BB rats, Ramanadham and co-workers (1990) have shown that the VStreatment potentiated the in vivo glucose lowering effects of acute and chronicadministrations of insulin in STZ-diabetic rats and substituted for, or potentiated,the effects of chronic insulin therapy in spontaneously diabetic BB rats.Therefore, in addition to the insulin-mimetic effects of vanadium, beneficialeffects of vanadium in treated diabetic animals may be partially due to anenhanced in vivo sensitivity to insulin.80Five-weeks of VS treatment did not affect the serum glucose levels ofcontrol animals but lowered the serum glucose levels of diabetic rats. In thepresent study, almost half (42%) of the treated diabetic rats became euglycemic.Of the rest, thirty-three percent also had a lower serum glucose level, whereas21% of the animals remained hyperglycemic (glucose level remained the sameas non-treated diabetic rats, 16 to 20 mM or higher). The variable responses ofthe diabetic rats to vanadium treatment is believed to be due to differences inthe severity of the diabetic state in individual rats. Recently, it has been shownthat all diabetic rats treated with VS could be made euglycemic by individualdose-adjustment (Cam et a!., unpublished data). Additional observationsreported a sustained euglycemia and prevention of myocardial and metabolicabnormalities in diabetic rats following withdrawl from oral vanadyl treatment(Ramanadham eta!., 1989a, Pederson et aL, 1989). The euglycemic effect ofvanadium is thus profound and very persistent.The glucose lowering effects of vanadium are thought to be due to anincrease in glucose metabolism, namely an increase in glucose transport andoxidation. Alterations in glucose-transporter proteins and their mRNA levelshave been reported in liver, adipocytes and skeletal muscle of diabetic rats(Oka et a!., 1990; Strout et aL, 1990) and treatment with vanadium has beenreported to have effects on these alterations. For example, in skeletal muscle ofdiabetic rats, Strout et a!. (1990) demonstrated that diabetes led to a 70%decrease in the expression of insulin-responsive glucose transporters.Subsequent treatment of diabetic animals with vanadate resulted in renewedexpression of the transporter to 87% of control values. Furthermore, theirNorthern blot analysis on total skeletal muscle RNA from diabetic animals81revealed a 55% decline in the level of muscle glucose transporter mRNA, andvanadate treatment led to a 187% increase in the transporter mRNA overnormal levels. Therefore, one of the mechanisms by which vanadium affectsdiabetic hyperglycemia is to induce the expression of the insulin-responsiveglucose transporters at the pretranslational level. It should be noted that theinhibition of gluconeogenesis by vanadium compounds is another importantaspect which contributes to glucose-lowering effects.B. Alteration of Hepatic GP and Effects of VS treatmentHepatic abnormalities are associated with diabetes mellitus. A decreasein hepatic glycogen levels together with a decrease in GP activities have beenpreviously reported (Stearns and Benzo, 1981; Khandelwal eta!., 1977; Ciudadet al., 1988). While age-related decrease in glycogen content during diabeteshas been shown (Stearns and Benzo, 1981), no such studies have beenperformed on age-related changes in hepatic GP. In the present study, the totaland the a activity of hepatic GP were shown to be significantly decreased inrats which had been diabetic for six-weeks or five-months There was anapparent time dependence in the decrease of hepatic GP with the five-monthdiabetic rats having much lower hepatic GP activities than those in six-weekdiabetic rats. However, it should be noted that the phosphorylase a ratio, whichgenerally reflects the activation state of GP remained the same in these diabeticrats. The decrease in GPa activity may thus arise from a decrease in the totalhepatic GP, which could occur as a result of a compensatory mechanism ofdiabetic liver to adjust to the decrease in liver glycogen content during diabetes.82Previous studies have shown an improvement of hepatic GP activity withinsulin treatment (Khandelwal eta!., 1977). In view of the insulin-mimetic effectsof vanadium (Heyliger et al., 1985, Ramandham et a!., 1989), we treateddiabetic rats with VS for five weeks or five months and observed a significantimprovement of the hepatic GP activity in diabetic rat liver. While treatment withVS for five weeks only partially restored the hepatic GP activity, five-monthtreatment resulted in a complete recovery. Additionally, the large variation inthe response of diabetic rats to five-week VS treatment was greatly minimizedby extending the treatment to five months.Using sodium orthovanadate orally to treat diabetic rats for five weeks,Pugashenthi and Khandelwal (1990) reported a similar partial restoration ofhepatic GP: the total and the a activities of hepatic GP were restored to 70% ofcontrol values after the treatment. Our experiment not only demonstrated thatVS could produce a strong insulin-like effect on the hepatic GP activity but alsorevealed that these effects were time-dependent, i.e., a long-term application isrequired to achieve maximal effects of VS treatment. As the vanadyl form ofvanadium is less toxic than vanadate, it is advantageous to use VS, especially iflong term treatment is required.In our study, neither a five-week nor a five-month VS treatment were ableto activate hepatic GP (no change in the activity ratio), similar to the results offive-week sodium orthovanadate treatment by Pugashenthi and Khandelwal(1990). However, the decreased levels of upstream activators of GP such ashepatic PPK and PKA were restored to normal values with five-week vanadatetreatment (Pugashenthi and Khandelwal, 1990). The effects of these long-termtreatments in intact rats differs significantly from the acute effects of vanadate in83isolated liver cells. For example, incubation of hepatocytes from control ratswith 0.1-5 mM vanadate activated GP in a dose- and time-dependent manner(Bosch eta!., 1987). The activation was approximately 40% less in hepatocytesprepared from diabetic rats (Rodrigues-Gil et at., 1989). Apparently, theactivation of hepatic GP by vanadate wasCa2+dependent, since omission ofCa2 from the incubation media abolished the effect. Intracellular Ca2overload has been observed in isolated rat hepatocytes that were treated with0.25-1 mM vanadate (Richelmi et a!., 1989) and theCa2-transport system inrat adipocytes has been shown to be inhibited by 30 jiM vanadate or vanadyl(Delfert and McDonald, 1985). It is possible that high concentrations ofvanadium compounds alter intracellularCa2+homeostasis by directly acting onthe Ca2-transport system. Additionally, as a potent inhibitor of Na-KATPase (Nechay, 1984), vanadate or vanadyl at high concentrations couldinduce an increase in the intracellular concentration of Na+. This in turn couldlead to an increase in intracellular Ca2+ concentration via stimulation of theNa+Ca2+exchanger, a mechanism similar to that by which digitalis increasesintracellular Ca2+ concentration in cardiac cells. As PPK requires Ca2+ for itsactivity (Cohen, 1983 and 1988), it is likely that it may be involved in the effectsof vanadate on the activation of GP in hepatocytes. However, no directevidence is available to support this concept. It is also difficult to evaluate thephysiological relevance of these in vitro studies due to the concentration ofvanadate employed (up to 5 mM), which could never be reached by oraladministration of vanadate or VS.The mechanism by which VS restores GP activity in the liver has notbeen studied in detail. Our experimental findings do not provide directevidence for the elucidation of such a mechanism. Previous studies have84concentrated on studying changes in cardiac GP activity during diabetes. Forinstance, the total activity of cardiac GP in diabetic rats is significantly increasedas a result of increased synthesis of GP (Miller et al., 1989). This is in starkcontrast to hepatic GP activities which are decreased during diabetes. Thus, atissue-specific regulation of OP expression may be altered during diabetes.While no data is available, it is conceivable that the synthesis of hepatic GP maybe impaired during diabetes, leading to a decrease in the total hepatic GPactivity. The restoration of hepatic GP activities by VS treatment could be due tothe induction of general protein synthesis or a specific GP synthesis. The factthat we and others (Pugazhenthi and Khandelwal, 1990) did not observe achange in the protein concentration of liver extracts prepared from diabetic ratswould argue against a vanadate-induced increase in general protein synthesis.Furthermore, in a different model of animal diabetes such as db/db mice, the aand total activities of hepatic GP were significantly increased as compared tothe control mice, while vanadate-treatment caused a decrease in the GPactivities (Pugazhenthi et a!., 1991). This suggests that the alteration of OP isnot only tissue-specific but also species-specific, and that the effect of vanadateon hepatic GP depends upon the change of GP prior to the treatment. It wouldthus appear that the vanadate-induced restoration of hepatic OP to controllevels may be a non-specific phenomenon resulting from the euglycemic effectsof VS treatment. However, this is not consistent with our observation that somediabetic rats whose hepatic activities of GP were normalized were stillhyperglycemic while the hepatic GP activities of some euglycemic rats were stillunchanged. Thus, the recovery of hepatic GP appears to be independent of theeuglycemic effects of VS treatment and the exact mechanism(s) remains to bedetermined.85C. Effects of VS treatment on cardiac alterationsin GP, PPKand cAMP.As described in the introduction, various biochemical alterations havebeen documented in the cardiac tissues from diabetic animals. In most cases,many of these changes could be reversed by insulin treatment. Few studiesinvestigating the effects of VS treatment on these biochemical alterations havebeen carried out. Our laboratory pioneered the work on the beneficial effects ofvanadate treatment on decreased cardiac function in diabetic rats (Heyliger eta!., 1985). In the present study, in addition to the study of the changes inpathways leading to the activation of cardiac GP from chronically diabetic rats,we also explored the mechanism for these alterations and investigated theeffects of VS treatment. In direct contrast to findings in the liver, our resultsindicate that the total activity of cardiac GP was significantly increased indiabetes and that there was a supersensitivity of cardiac GP activation inresponse to ISO. Unlike the situation in the liver, five-week treatment with VShad no effect on the basal level or the ISO-stimulated levels of cAMP, GP orPPK in control rats, nor did it affect the basal levels of cAMP or activity ratio ofGP and PPK in the hearts from diabetic rats. The treatment did eliminate themore sensitive activation in both GP and PPK to ISO stimulation, decrease thehigher level of cardiac GP and normalize the ISO-stimulated cAMP production.A decrease in the synthesis of cardiac GP has been suggested to beresponsible for the decrease in total GP activity (Miller et a!., 1989). Thenormalization of total cardiac GP by VS treatment could therefore result from aninhibition of GP synthesis. This again indicates the tissue specific alteration ofGP in diabetes and tissue-specific response of VS-treatment. The effect of VS86treatment in abolishing the supersensitivity of cardiac GP activation could be adue to a direct effect of the treatment on cardiac GP or to an overall effect of thetreatment on the various components in the cascade leading to the activation ofGP, such as the level of cAMP and PPK. While the basal level of cAMP and theactivation of PPK (i.e. the activity ratio) were not altered by the treatment, theimpaired response of cAMP production and the sensitivity of PPK activation byISO were restored after VS treatment. Similar effects were observed inhepatocytes where the cAMP level was increased by vanadate treatment (VillarPalasi et aL, 1989). Since an increase in cAMP would generally lead toactivation of GP, the effect of the treatment on cAMP level apparently did notcontribute to abolishing the supersensitivity of GP activation, which is consistentwith our hypothesis that cAMP was not involved in the enhanced response ofGP activation to ISO. As discussed in the earlier sections, our results suggestthat the supersensitivity of cardiac GP activation to ISO occurs via an increasein intracellular Ca2. Hence, the effectiveness of VS-treatment to abolish thissupersensitivity suggests that the treatment might affect intracellular Ca2rather than directly acting on the GP per Se.87CONCLUSIONS•The mechanism(s) for supersensitivity of catecholamine-inducedcardiac GP activation in diabetic rats were investigated in this study.Our results suggest that alterations in cardiac calcium homeostasismay be partially responsible for the defects in GP activation, while thecAM P-PKA cascade may not be involved.• Vanadyl sulphate treatment was able to abolish the supersensitivityof cardiac GP activation in diabetic rats. The treatment also restoreddiabetes-induced changes in cardiac PPK and cAMP. These effectsmay contribute to the improvement of cardiac function in diabetic rathearts by the treatment.• Vanadyl sulphate treatment was able to correct the decrease in theactivity of hepatic GP, and the effect was time dependent. 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