A Novel Pathway of Insulin Sensitivity in Chromogranin A Null Mice: A CRUCIAL ROLE FOR PANCREASTATIN IN GLUCOSE HOMEOSTASIS*

Chromogranin A (CHGA/Chga), a proprotein, widely distributed in endocrine and neuroendocrine tissues (not expressed in muscle, liver, and adipose tissues), generates at least four bioactive peptides. One of those peptides, pancreastatin (PST), has
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  ANovelPathwayofInsulinSensitivityinChromograninANullMice  ACRUCIALROLEFORPANCREASTATININGLUCOSEHOMEOSTASIS * Receivedforpublication,May13,2009,andinrevisedform,July23,2009  Published,JBCPapersinPress,August25,2009,DOI10.1074/jbc.M109.020636 JiaurR.Gayen ‡ ,MaziyarSaberi ‡ ,SimonSchenk  ‡ ,NilimaBiswas ‡ ,SuchetaM.Vaingankar ‡ ,WaiW.Cheung § ,SoniaM.Najjar ¶ ,DanielT.O’Connor ‡  ** ,GautamBandyopadhyay ‡ ,andSushilK.Mahata ‡ ** 1 FromtheDepartmentsof  ‡ Medicine, § Pediatrics,and   MolecularGenetics,UniversityofCalifornia,SanDiegoand  ** Veterans AffairsSanDiegoHealthcareSystem,LaJolla,California92093-0838andthe ¶ CenterforDiabetesandEndocrineResearchand DepartmentofPhysiologyandPharmacology,UniversityofToledoCollegeofMedicine,Toledo, Ohio 43614-5804 ChromograninA(CHGA/Chga),aproprotein,widelydistrib-uted in endocrine and neuroendocrine tissues (not expressedin muscle, liver, and adipose tissues), generates at least fourbioactive peptides. One of those peptides, pancreastatin(PST), has been reported to interfere with insulin action. WegeneratedaChgaknock-out(KO)mousebythetargeteddele-tion of the  Chga  gene in neuroendocrine tissues. KO micedisplayed hypertension, higher plasma catecholamine, andadipokine levels and lower IL-6 and lipid levels compared with wild type mice. Liver glycogen content was elevated, butthe nitric oxide (NO) level was diminished. Glucose, insulin,and pyruvate tolerance tests and hyperinsulinemic-euglyce-mic clamp studies established increased insulin sensitivity inliverbutdecreasedglucosedisposalinmuscle.Despitehighercatecholamine and ketone body levels and muscle insulinresistance, KO mice maintained euglycemia due to increasedliver insulin sensitivity. Suppressed mRNA abundance of phosphoenolpyruvate carboxykinase and glucose-6-phos-phatase(G6Pase)inKOmicefurthersupportthisconclusion.PST administration in KO mice stimulated phosphoenol-pyruvate carboxykinase and G6Pase mRNA abundance andraised the blood glucose level. In liver cells transfected withG6Pase promoter, PST caused transcriptional activation in aprotein kinase C (PKC)- and NO synthase-dependent man-ner. Thus, PST action may be mediated by suppressing IRS1/2-phosphatidylinositol 3-kinase-Akt-FOXO-1 signaling andinsulin-induced maturation of SREBP1c by PKC and a highlevel of NO. The combined effects of conventional PKC andendothelialNOsynthaseactivationbyPSTcansuppressinsu-lin signaling. The rise in blood PST level with age and indiabetes suggests that PST is a negative regulator of insulinsensitivity and glucose homeostasis. Chromogranin A (CHGA/Chga), 2 the index member of thechromogranin/secretogranin protein family (1, 2), is a propro-tein that gives rise to biologically active peptides such as thedysglycemic hormone pancreastatin (PST; human CHGA-(250–301)) (3), the vascular smooth muscle vasodilator vasostatin (human CHGA-(1–76)) (4), and a catecholaminerelease inhibitory peptide, catestatin (human CHGA-(352–372); bovine Chga-(344–364)) (5). Several studies, including  invivo  analyses of experimental animals (6, 7), showed that PSTinhibits insulin release in response to glucose (3), reduces glu-cose uptake in adipocytes and hepatocytes (8), and triggers gly-cogenolysis (6). Genetic analysis in humans supports a role forPSTindecreasingglucoseuptakeby   50%(9)whileincreasingthe spillover of free fatty acids but not amino acids. Moreoverthe PST level is elevated in patients with Type 2 diabetes mel-litus (9–11). Taken together, the data suggest that PST is animportant player in metabolism.TofurtherdelineatetheroleofPSTinmetabolism,wetestedwhether removal of PST, a negative regulator of insulin action,stabilizes glucose levels in knock-out (KO) mice and protectsagainst metabolic disorders. To this end, we characterizedmore extensively the phenotype of the global  Chga  KO mouse(12), which we had found to be hypertensive (resulting fromelevation in catecholamine release) and hyperadrenergic (12).We herein report that by comparison with wild type (WT), KOmiceareeuglycemicdespitelowplasmainsulinlevels.Contrary to what is found in hypertension (13), hypertensive KO miceexhibited a high plasma adiponectin level. EXPERIMENTALPROCEDURES  Animals —Young adult (3 months old) and adult (6 monthsold) WT (31  1 g) and KO (37  1 g) mice with mixed genetic *  Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealthGrants R01 DA011311 (to S. K. M.), DK60702 (to D. T. O.), P01 HL58120 andU01 HL69758 (to S. K. M. and D. T. O.), and DK54254 (to S. M. N.). This work was also supported by grants from the Department of Veterans Affairs (toS. K. M. and D. T. O.) and United States Department of Agriculture GrantUSDA 38903-02315 (to S. M. N.). 1  To whom correspondence should be addressed: Dept. of Medicine (0838),UniversityofCalifornia,SanDiegoSchoolofMedicineandVeteransAffairsSan Diego Healthcare System, 9500 Gilman Dr., La Jolla, CA 92093-0838. Tel.: 858-552-8585 (ext. 2637) or 858-534-0639; Fax: 858-642-6425; 2  The abbreviations used are: CHGA/Chga, human/mouse chromogranin A; CHGA  / Chga , human/mouse chromogranin A gene; Akt, serine/threoninekinase; Fasn ,fatty-acidsynthasegene;FOXO-1,theforkheadtranscriptionfactor;  Gpat  , glycerol-3-phosphate acyltransferase gene; G6Pase, glucose-6-phosphatase; G6pase ,glucose-6-phosphatasegene;KO,knock-out;GTT,glucosetolerancetest;IRS,insulinreceptorsubstrate;ITT,insulintolerancetest; NEFA, non-esterified fatty acid;  Pepck1 , phosphoenolpyruvate car-boxykinase-1 gene;  Ppar    , peroxisome proliferator-activated receptor    gene; PKC, protein kinase C; cPKC, conventional PKC; PST, pancreastatin;SREBP1c, sterol-regulatory element-binding protein 1c;  Srebp1c  , sterol-regulatory element-binding protein 1c gene;  Ucp2 , uncoupling protein 2gene; WT, wild type; NO, nitric oxide; GDR, glucose disposal rate; PI, phos-phatidylinositol; cPI, cPKC peptide inhibitor; ANOVA, analysis of variance;NOS, nitric-oxide synthase; eNOS, endothelial NOS.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 42, pp. 28498–28509, October 16, 2009Printed in the U.S.A. 28498  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 284•NUMBER 42• OCTOBER 16, 2009   a t   unk n owni  n s  t  i   t   u t  i   on , on O c  t   o b  er 1  5  ,2  0  0  9 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   background (129svJ  C57BL/6) were used in this study. BothWT and KO mice were generated from the srcinal foundercarrying mixed genotype (50% 128svJ, 50% C57BL/6) and weremaintained by brother/sister mating. Animals were kept in a12-h dark/light cycle and fed standard chow   ad libitum . TheInstitutionalAnimalCareandUtilizationCommitteeapprovedall procedures. Equal numbers of male and female mice werefasted overnight (12 h) for each experiment except for theclamp study where only male mice were used.  Dual Energy X-ray Absorptiometry Scan —Following anes-thesia with isoflurane, overnight fasted mice underwent dualenergy x-ray absorptiometry scanning to measure total tissueand fat mass and lean body mass using a PixiMus mouse densi-tometer (GE Healthcare).  MeasurementofBloodGlucose,Insulin,C-peptide,Glucagon, Adipokine, Lipid, Cytokine, and PST Levels in Fasted Mice —Blood glucose was measured using microcuvettes (HemoCue,Lake Forest, CA) and a Glucose 201 analyzer (HemoCue). Amouse insulin enzyme-linked immunosorbent assay kit (Milli-pore, Billerica, MA) was used to determine plasma insulin.C-peptide was measured using an enzyme-linked immunosor-bent assay kit (Shibayagi Co. Ltd.). Glucagon was measuredusing an enzyme-linked immunosorbent assay kit (WakoChemicals). A radioimmunoassay kit (Peninsula Laboratories,San Carlos, CA) was used to measure plasma PST. Plasma trig-lyceride and non-esterified fatty acid (NEFA) levels wereassayed by triacylglycerol and NEFA C test kits, respectively,from Wako Diagnostics (Richmond, VA). Enzyme-linkedimmunosorbent assay kits were used to determine plasma lev-els of leptin and adiponectin (Millipore) and IL-6 (Pierce).  BloodKetone(   -Ketone)Assay —Theblood(tailsnip)ketonelevel(mmol/liter)wasmeasuredusingthePrecisionXtraBloodKetone Monitoring System (Abbott Laboratories) according tomanufacturer’s instructions.  Determination of Liver Glycogen and NO Content  —Liver tis-sue(25–100mg)wasdissolvedinhotKOH(30%)beforeglyco-gen was precipitated in 3 ml of 95% ethanol (at 4 °C overnight)and centrifuged at 5000 rpm for 12 min. The pellet was redis-solved in 5 ml of water, and 1 ml was mixed with 3 ml of anthrone reagent (0.2% anthrone (Sigma) in concentratedH 2 SO 4 )(14)todeterminetheconcentrationat620nmbycom-parison with standard glycogen (Sigma). A nitrate/nitrite col-orimetric assay kit (Cayman Chemical Co.) was used to deter-mine liver NO content. Glucose, Insulin, and Pyruvate Tolerance Tests —Dextrose (2g/kg intraperitoneally; glucose tolerance test (GTT)), humaninsulin (0.40 IU/kg intraperitoneally; Novolin, Novo NordiskInc.;insulintolerancetest(ITT)),orpyruvate(2g/kgintraperi-toneally; pyruvate tolerance test) were injected into fasting (12h) mice before determining the tail vein glucose level at 0–150min following injection.  Hyperinsulinemic-Euglycemic Clamp —Briefly mice wereanesthetized (80 mg/kg ketamine, 0.5 mg/kg acepromazine,and 16 mg/kg xylazine) via intramuscular injection. Twomicrourethane catheters (0.012-  m inner diameter; Dow CorningSilastic)wereinsertedinthejugularvein,andthecan-nulas were externalized to the midscapula region and securedwithin tubing. After 5 days of recovery, mice were fasted (3 h)and placed in a Lucite restrainer (Braintree Scientific, Brain-tree,MA).After90min,anequilibratingtracersolutionof  D -[3- 3 H]glucose (5   Ci/h; PerkinElmer Life Sciences) was infused via the jugular cannula for 90 min. At the end of the equilibra-tionperiod( t   0min;fastedfor6h),bloodwascollected(15  lin duplicate) and deproteinized to assess tracer specific activity and basal hepatic glucose production. In addition,  75   l of whole blood was collected by cutting the tail vein for the meas-urementoffastingplasmainsulinandfreefattyacidconcentra-tions. After blood collection, mice were infused with insulin(12.0 milliunits/kg/min; Humulin   R, Eli Lilly and Co.) and D -[3- 3 H]glucose (5   Ci/h; PerkinElmer Life Sciences). Bloodglucose was assessed every 10 min, and a glucose solution (50%dextrose; Hospira, Inc.) was infused at a variable rate to main-tain blood glucose at  120  5 mg/dl. The clamp was termi-nated when steady state conditions were maintained for  30min (  130 min). At this time, blood was sampled (15   l induplicate) 10 min apart to assess glucose disposal rate (GDR)andinsulin-stimulatedhepaticglucoseproductionandtoverify that steady state conditions for specific activity of the tracerwere achieved. An additional 75   l of whole blood was alsocollectedattheendoftheclamptomeasureplasmainsulinandfree fatty acids concentrations during the clamp. Glucose Production in Primary Hepatocytes —Mice wereinfused with collagenase (Blendzyme, Roche Applied Science)through the inferior vena cava, and hepatocytes were isolatedby the method of Smedsrød and Pertoft (15). Percoll-purifiedhepatocyteswereculturedinWilliam’smediumE(Invitrogen),2m M Glutamax,10n M dexamethasone,5%fetalcalfserum,andantibiotics. Dexamethasone was withdrawn 6 h later, and cul-tures were exposed initially to PST (20 n M ) for 20 h and then toglucose-free, serum-free Dulbecco’s modified Eagle’s mediumcontaining PST for an additional 4 h. Dulbecco’s modifiedEagle’s medium was then replaced by phosphate-bufferedsaline containing 20 m M  HEPES, 0.2% bovine serum albumin,16 m M  lactate, and 4 m M  pyruvate with or without insulin (20n M ) and incubated for another 4 h. At the end, glucose levels inthe media were assayed by a kit (Cayman Chemicals, AnnArbor, MI), and cellular protein levels were determined by theBradford assay (Bio-Rad).  Primary Adipocytes and L6 Muscle Cell Cultures —Adipo-cytes were isolated from mouse visceral adipose tissue follow-ingaproceduredescribedbyKarnieli etal. (16).L6musclecellswere obtained from the laboratory of Dr. A. Klip (Hospital forSickChildren,Toronto,Canada).L6myoblastcellsweregrownin   -minimum Eagle’s medium supplemented with 10% fetalbovine serum and then switched to 2% fetal bovine serum fordifferentiation into myotubes. After 8 days in differentiationmedium, myotubes were used for glucose uptake studies. Glu-cose uptake in muscle and adipocytes cells was carried out inHEPES-salt buffer with or without 20 n M  insulin treatment for30 min followed by incubation (6 min) with 2-[ 3 H]deoxyglu-cose (0.1 m M  final concentration). After final incubation, cul-tureswerewashedwithphosphate-bufferedsaline,countedforradioactivity, and normalized against protein.  Immunoblotting and Signal Transduction Analyses —A groupof eight KO mice were injected with PST (20   g/g of body weight intraperitoneally twice daily) for 7 days. On the 8th day, PancreastatinandInsulinSensitivity  OCTOBER 16, 2009• VOLUME 284•NUMBER 42  JOURNAL OF BIOLOGICAL CHEMISTRY   28499   a t   unk n owni  n s  t  i   t   u t  i   on , on O c  t   o b  er 1  5  ,2  0  0  9 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   WT,KO,andKO  PSTmice( n  4ineachgroup)werefastedfor 12 h and injected either with vehicle or with insulin (0.4milliunits/g of body weight intraperitoneally). Mice were sacri-ficed after 20 min of treatment to collect tissues, which weresnap frozen in liquid nitrogen. Tissues from vehicle-treatedmice were considered as basal. Frozen tissues were homoge-nized in liquid nitrogen and lysed in a lysis buffer containingphosphatase and protease inhibitors as described elsewhere(17). Antibodies were purchased from Cell Signaling Technol-ogy (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz,CA). The chemiluminescence kit from Pierce was used fordetection of signals.  Real Time PCR —RNA was extracted using an RNA purifica-tion kit (RNeasy Plus, Qiagen, Valencia, CA) according to themanufacturer’sspecifications.AfterDNasedigestion,100ngof RNA was transcribed into cDNA in a 20-  l reaction using aHigh Capacity cDNA Archive kit, analyzed, and amplified.PCR was performed in a 25-  l reaction containing 5   l of cDNA (one-fifth diluted), 2  SYBR Green PCR Master Mix,and a 400 n M  concentration of each primer. Cycle threshold(Ct) values were used to calculate the amount of amplifiedPCR product relative to glyceraldehyde-3-phosphate dehy-drogenase and 18 S rRNA.  PI 3-Kinase Assay —Primary hepatocytes were preincubatedwith 10   M  myristoylated conventional PKC (cPKC) peptideinhibitor (cPI; myr-RFARKGALRQKNV; Promega) for 1 h fol-lowed by incubation with PST (100 n M ) for 30 min and thenwith insulin (20 n M ) for 15 min. Hepatocytes were chilled,washed with cold phosphate-buffered saline, and then lysed inradioimmuneprecipitationassaybuffercontaining1%NonidetP-40 and protease and phosphatase inhibitors (all from Sigma)as described by Backer  et al.  (18). An aliquot of 500   g of totalcellproteinextractusedforeachreactionwasimmunoprecipi-tated in lysis buffer with 4   g of anti-insulin receptor substrate(IRS) 1/2 antibody (Upstate Biotechnology Inc). Immunopre-cipitates were washed and analyzed for PI 3-kinase activity asdescribed by Backer  et al.  (18). cPKC Assay —We purchased peptide inhibitors PKC    pseu-dosubstrate (sc-3097), PKC    pseudosubstrate (sc-3098), PKC  peptide inhibitor (sc-3095), and PKC   peptide inhibitor(C2–4) (sc-3094) from Santa Cruz Biotechnology and non-peptideinhibitorsLY333531(IC 50 forPKC  IandPKC  II,5–6n M ), rottlerin (IC 50  for PKC  , 4 n M ), and Go¨6976 (IC 50  forPKC   and PKC  , 3–6 n M ) from Calbiochem. Primary hepato-cytes and HepG2 cells were incubated with 10   M  peptideinhibitors, 10   M  cPI, and 10 n M  non-peptide inhibitors for 1 hfollowedbyincubationwithPST100n M for15min.Aftertreat-ment, cells were chilled, washed, and lysed as described above.The kinase reaction mixture (120   l) contained 20 m M  Tris-HCl, pH 7.5, 10 m M  MgCl 2 , 0.5 m M  CaCl 2 , 25   M  phosphati-dylserine, 6   M  [   - 32 P]ATP (5000–8000 cpm/pmol), and 25  M  peptide substrate derived from neurogranin (Santa CruzBiotechnology). In some assays, PMA (1   M ) or 1:2 diolein(10   M ) was used as diacylglycerol. Reactions were initiatedby the addition of [   - 32 P]ATP, proceeded for 10 min at 30 °Cwith linear kinetics, and were terminated by spotting onphosphocellulose papers. Papers were washed six times with5% acetic acid. The radioactivities on the papers were ana-lyzedbycounting.Allassayswereperformedinsixreplicatesand expressed as the mean value  S.E. Statistics —Data are expressed as the mean  S.E. Curve fit-ting was accomplished in the program Kaleidagraph (Synergy Software,Reading,PA).Multiplecomparisonsweremadeusingeither one-way ANOVA followed by Bonferroni’s post hoc testor two-way ANOVA. Statistical significance was concluded at  p  0.05. RESULTS  Body Composition and Plasma Lipid and Adipokine Profile —Thegaininbodyweight(Fig.1  A )andtotalfatmasswere20and25%, respectively (Fig. 1  B ) in 6-month-old KO mice. Afteradjusting for increased body weight, the gain in fat mass was25% of the gain in body weight. Total abdominal fat was alsoincreased(by   48%)predominantlyattheepididymalandsub-epidermal regions and surrounding the adrenal gland (Fig. 1,  C  and  D ). Relative to WT, plasma triglyceride, NEFA, and IL-6levels were lower (Fig. 1,  E–G  ), whereas those of adiponectinand leptin (adjusted to fat mass) were higher in KO mice (Fig.1 G  ). Because IL-6 was shown to be elevated in patients withType 2 diabetes mellitus (19) and adiponectin was shown toimproveinsulinsensitivity(20,21),thesefindingscorrelatewellwith the data on hypertensive KO mice (decreased IL-6 andincreasedadiponectinlevels),suggestingincreasedinsulinsen-sitivity. The increase in fat mass may result from elevated lipiduptake and storage.  Insulin Metabolism and Action —Basal insulin levels weremarkedly (79%) lower in KO than WT mice (Fig. 2  A ). This isattributed to enhanced insulin clearance (as suggested by the  6-foldincreaseinthesteadystateC-peptide/insulinratio;Fig.2  B ) but not reduced insulin secretion (as suggested by intactplasmaC-peptidelevels;Fig.2  A ).Therapidinitialriseininsulinlevels during the first 7 min postglucose administration (Fig.2 C  ) suggests that the insulin secretion rate in response toglucose may instead be twice as high in KO mice lacking PST versus  WT (0.107  versus  0.057 ng/ml/min in WT;  p  0.05)(Fig. 2 C  ). Although actual assessment of insulin secretion by intravenous GTT was not carried out, a number of previouspublications, either in perfused pancreas or in isolated betacells, have demonstrated that PST inhibits the first phase of glucose-induced insulin secretion primarily by inhibiting aG-protein-coupled rise in calcium (22–25). Therefore, micewith PST deficiency might have enhanced insulin secretionduring GTT.Despitelowerbasalinsulinlevels(Fig.2  A ),fastingbloodglu-coselevelswerenormalinKOascomparedwithWTmice(Fig.2  A ).Moreoverinsulin(Fig.2 C  ;areaunderthecurve,71.0  6.6 versus 34.4  9.5ng/min/dlinWT;  p  0.008)andglucose(Fig.2  D ; area under the curve, 5462.5  582.3  versus  7918.8  532mg/min/dlinWT;  p  0.009)excursionsinresponsetoglucosechallenge during an intraperitoneal GTT were significantly improved. This suggests that KO mice are insulin-sensitive.InanITTin3-month-oldmice,hypoglycemiawascompara-ble between WT (51%) and KO (50%) mice (Fig. 2  E  ), whichreturned to the basal level 120 min after insulin injection. In6-month-old WT mice, hypoglycemia during ITT was lesser(by 11%), and the recovery from hypoglycemia was faster (90 PancreastatinandInsulinSensitivity  28500  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 284•NUMBER 42• OCTOBER 16, 2009   a t   unk n owni  n s  t  i   t   u t  i   on , on O c  t   o b  er 1  5  ,2  0  0  9 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   min)ascomparedwith3-month-oldmice(Fig.2  F  ).Incontrast,in 6-month-old KO mice, although hypoglycemia was compa-rable between 3- and 6-month-old mice, the blood glucose didnot return to the control level even after 150 min of insulininjection (Fig. 2  F  ). This could be attributed to elevated glucosedisposal and/or reduced gluconeogenesis in KO mice.In response to glucose challenge, the plasma glucagon leveldroppedtoasimilarextentinWT(by14.8%)andKO(by17.6%)mice (Fig. 2  F  ). This rules out a potential differential regulationof insulin action by glucagon in KO and WT mice.  Insulin Action in Vivo: Hyperinsulinemic-Euglycemic Clamp —FastingbloodglucosewasslightlylowerinWT versus KOmice FIGURE 1.  Body composition, adipose tissue, and plasma adipocytokines.  6-month-old male mice were fasted for 12 h before being subjected to a dualenergyx-rayabsorptiometryscantomeasuretotaltissuemass(  A )andtotalfatmass( B ).Aphotographoftheintra-abdominalcavityinWT( C  )andKO( D )micereveals fat deposition in the epididymal and subepidermal regions.  E  , plasma triglyceride in 12-h fasted mice.  F  , plasma NEFA in 12-h fasted mice.  G , plasmaadipokineandcytokinelevelsin12-hfastedmice.Experimentswereperformedon n  6pergroup.  A , B , E  ,and G ,valuesareexpressedasmean  S.E.(  p  0.05 versus  WT). PancreastatinandInsulinSensitivity  OCTOBER 16, 2009• VOLUME 284•NUMBER 42  JOURNAL OF BIOLOGICAL CHEMISTRY   28501   a t   unk n owni  n s  t  i   t   u t  i   on , on O c  t   o b  er 1  5  ,2  0  0  9 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   (Fig.3  A ).Duringtheclamp,asteady statebloodglucoseconcentrationof   120 mg/dl was reached at   70min and was maintained until theclamp was completed at 130 min.Blood glucose was maintained at asteady state level. Fig. 3  B  demon-strates that the specific activity of the tracer was in steady state at theend of the clamp, although the spe-cificactivitywassignificantlyhigherin KO  versus  WT at the end of theclamp period (  p  0.05). There wasno difference in specific activity during the basal infusion period.The overall glucose infusion raterequired to maintain euglycemiaduringtheclampwascomparableinWTandKOmice(Fig.3 C  ),suggest-ingthatwholebodyinsulinsensitiv-itywascomparablebetweengroups.However, the total glucose disposalrate during the clamp (GDR total )was 35% lower in KO  versus WT mice (Fig. 3  D ). In line withthis, the insulin-stimulated GDR(which is calculated as GDR clamp  GDR basal ), which primarily repre-sents glucose disposal by skeletalmuscle, was reduced by    68% inKO compared with WT mice (Fig.3  E  ;  p  0.006). This finding is sur-prising given the positive effect of adiponectin on glucose transport inmuscle (26, 27) and L6 muscle cells(Fig. 3 G  ). Nonetheless these dataindicate that,  in vivo , KO micedevelopinsulinresistanceinskeletalmuscle.In contrast to the insulin resist-ance in skeletal muscle, hepaticinsulin sensitivity was significantly higher in KO mice compared withWT mice as evidenced by a greatersuppression (by 32.2%) of hepaticglucose production during theclamp (Fig. 3  F  ;  p  0.0001). Fastinghepatic glucose production/GDRwas not different between groups(WT  versus  KO, 20  2  versus  24  3 mg/kg/min;  p  value not signifi-cant). The improved hepatic insulinsensitivity could, at least in part, beduetohigheradiponectinlevels(28,29).Moreovertheimprovedhepaticinsulin sensitivity likely compen-sates for the muscle insulin resist-ancefoundintheKOmiceandthus FIGURE 2. Metabolicparametersinfasting(12h)mice.  A , fasting blood glucose, plasma insulin and C-pep-tide levels.  B , C-peptide/insulin molar ratio at steady state. Plasma insulin levels ( C  ) and blood glucose duringGTT ( D ) in fasting mice are shown (performed on  n  8 and analyzed by two-way ANOVA). Fasting bloodglucose in 3-month-old mice ( E  ), fasting blood glucose in 6-month-old mice ( F  ), and fasting plasma glucagonduring ITT ( G ) are shown (performed on  n  8 and analyzed by two-way ANOVA). Values are expressed asmean  S.E.   , WT  versus  KO. *,  p  0.05; **,  p  0.01; ***,  p  0.001 as compared with the basal level. PancreastatinandInsulinSensitivity  28502  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 284•NUMBER 42• OCTOBER 16, 2009   a t   unk n owni  n s  t  i   t   u t  i   on , on O c  t   o b  er 1  5  ,2  0  0  9 www. j   b  c . or  gD  ownl   o a d  e d f  r  om 
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