]:)abetologa 10, 459--463 (1974) 9 by Srnger-Verlag 1974 Effectveness of the Intestnal Polyetdes, IRP, GIP, VIP and Motln on Insuln Release n the Rat D.S. Turner, LaurMne Etherdge, V. Marks, J.C. Brown and V. Mutt Receved: February 11, 1974, and n revsed form: June 4, 1974 Summary. Intestnal nsuln releasng olyetde (IRP) and Gastrc nhbtory olyetde (GIP) have a smlar effect on ntravenous glucose tolerance n the rat. Both augment the nsuln resonse to ntravenous glucose and ncrease the rate of glucose dsaearance. VIP and motln have no dscernble effect. Plasma nsuln doseresonse curves to IRP and GIP are smlar; both etdes stmulate nsuln release n the resence of small Mood glucose ncrements. A drect comarson of the nsuln releasng otency of IRP and GIP s not ossble as the former s not yet avalable n ure form. Key words: Intestnal hormones; nsuln release; ntestnal nsuln releasng olyetde (IRP); gastrc nhbtory etde (GIP); motlha; vasoactve ntestnal olyetde (VIP); glucagom A fracton from orcne duodenojejunal mucosa, ntestnal nsuln releasng olyetde (IRP), has been reared [1] and the suggeston made that t s resonsble for the greater lasma nsuln resonse to oral as comared to ntravenous glucose. The ossblty that the effects observed could be due to contamnaton of IRP by secretn, ancreozymn/cholecystoknn (CCK) or ntestnal glucagon-lke mmunoreactvty (GLI) has been nvestgated and consdered unlkely [2] on the evdence avalable. Three addtonm ntestnal olyetdes, vasoactve ntestnal olyetde (VIP) [3], gastrc nhbtory olyetde (GIP) [4] and motln [5] have recently been urfed. All aear to be hormonally actve and GIP has been shown to stmulate nsuln release [6]. It was consdered necessary, therefore, to nvestgate each of them to assess ther ablty to exlan the observed effects of IRP on nsuln secreton and ther ossble nvolvement n the ntestnal regulaton of nsuln release. Studes smlar to those revously reorted wth IRP [1, 2] were erformed usng ure or artally ure rearatons of GIP, VIP and motln. In addton further studes were made of the condtons affectng IRP and GIP medated nsuln release n rats n vvo. Materals and Methods Intestnal nsuln releasng olyetde (IRP) was reared by the method of Turner etal. [2]. A artally ure GIP rearaton, contanng aroxmately 40% GIP, and ure GIP were reared by the method of Broml etal. [4]. Vasoactve ntestnal olyetde was reared accordng to Sad and Mutt [3] and motln by the method of Brown et al. [5]. Insuln-free ork glucagon was gven by Dr. Mary Root, Llly Research Laboratores, Indanaols, U.S.A. All solutons for njecton contaned gelatn, 0.05~o, as a carrer roten. Overnght fasted twelve week old male Wstar rats weghng between 260 and 280 g were used for all exerments. Rats were anaesthetsed by the ntraertoneal njecton of sodum entobarbtone, 40 mg/kg. Intravenous njectons and blood samlng were carred out as revously descrbed [2]. Blood glucose was measured by a glucose oxdase rocedure [7] and lasma nsuln by a double antbody radommunoassay [8], usng human nsuln as standard. Radommunoassay for GIP was erformed on the IRP rearaton used here by J. C.B. and by Dr. S.R. Bloom of the Mddlesex Hostal, London. J.C.B. found an aarent GIP content of 1.2%, but the dluton sloe of the cross reactvty was markedly dfferent from that of GIP standards and other samles havng excellent nsuln releasng actvty showed no detectable GIP content. Dr. S.R. Bloom found a content of 400 ng/mg. It s thus dffcult to be certan of the nature or quantty of GIP mmunoreactvty resent n the IgP rearaton. Results 1. The Effect of Partally Pure GIP on Intravenous Glucose Tolerance n the Rat The effect of artally ure GIP, njected ntravenously at a dose of 5 ~g/kg, on the blood glucose and lasma nsuln resonse to ntravenous glucose s shown n Fg.. The glucose dsaearance rate n rats that receved glucose lus GIP was faster than that n the rats recevng glucose alone and ther mean blood glucose levels were sgnfcantly lower at 30 ran (< 0.02) and 60 ran (<0.05) after glucose njecton. Mean lasma nsuln levels n rats gven GIP n addton to glucose were sgnfcantly hgher at 5 ran (< 0.01) and 10 mn (/)<0.05) than n anmals gven glucose alone.
460 D.S. Turner et al. : Intestnal Polyetdes, II%P, GIP, VII ) and Motln 2. Dose Resonse to Pure GIP The curve obtaned by lottng "glucose deend. ent" nsuln releasng actvty -- exressed as a ercentage of control values -- aganst the dose of GIP s shown n Fg. 2. Plasma nsuln levels are gven n Table 1. The data reresent mean lasma nsuln levels observed n grous of eght rats, fve mnutes after the ntravenous njecton of glucose lus GIP, and n the fgure are exressed as a ercentage of the 5 mn nsuln level n control anmals recevng glucose alone. Sgnfcant augmentaton of nsuln release (< 0.01) was roduced by 0.125 Fg/kg GIP. A dose of 1.0 Fg/kg GIP had a maxmal nsuln releasng effect. At a hgher dose, 2.0 Fg/kg there was a suggeston of a reducton n effect. A lot of the mean 5 ran lasma nsuln levels aganst log GIP dose s shown n Fg. 3 and demonstrates a log-lnear relatonsh over much of the observed range. Log dose resonse to GIP Effect of GIPon IvGrT n the rat o 250- -~ 15o o O "a E 100 50' G[ucose,uJ.J/m[ 150 ""t 100 '"~...~ Insutn o',b 3'o 6'olo' 3'0 6o Mnutes ost glucose ---- Glucose ---~-- Glucose-GIP Fg. l. The effect of GIP on ntravenous glucose tolerance n the rat. Mean S.E.M. of blood glucose and lasma nsuln levels n grous of 8 rats after the ntravenous njecton of 5.0 Fg/kg 40% GIP n glucose (0.625 g/kg) over 1 89 mn. Control anmals receved glucose alone, 0.625 g/kg. @ glucose alone, -----A... glucose lus GIP Dose resonse to IRP, GI~ VIP~ glucagon end mottn ~5 - c La 150 - to0 Fg. 3. Log suln levels 0.0625 0.125 0.25 ' 0.50 1.0 2.0/Lg/kg GIP dose resonse to GIP. Mean 5 mn lasma nas a ercentage of values for glucose alone, lotted aganst log dose of GIP Data obtaned wth IRP and glucagon are ncluded for comarson. Crude IRP at a dose of 62.5 ~g/kg was suffcent to stmulate sgnfcantly nsuln release, whle 0.5 ~g/kg glucagon was requred to roduce a sgnfcant stmulaton. 3. The Effect of Motln and VIP on Insuln Release The lack of effect uon glucose medated nsuln release of ether VIP or motln at varous dose levels s shown n Fg. 2 and Table 1. In consequence no further studes were made of these two etdes. r ' o 9.."... """. ~ " 815o " to ~e--............ ~ 100 0.25 0.5 1.0 2.0 ~g/kg GIP, VIPand motln mg/kg IRP Fg. 2. The dose resonse to IRP, GIP, VIP, glueagon and motln. Mean 5 ran lasma nsuln levels exressed as a ercentage of those n anmals whch receved 0.625 g/kg glucose.v. alone over ]~ ran. The doses of etde ndcated were dssolved n the.v. glucose njecton.... []... It~P, A GIP, A" VIP,... 0------- Motln, O Gluca,gon 4. The Effect of Glucose Dosage on the Insulnaemc Resonse to IRP a) A constant dose of IEP (0.5 mg/kg) was admnstered ntravenously, smultaneously wth dfferng quanttes of glucose. Blood glucose and lasma nsuln levels were measured 5 mn after the njecton. The results are shown n Fg. 4. The njecton of IRP lus glucose, 78 mg/kg, resulted n a 13 rag/100 ml ncrement n the mean 5 ran blood gncose level and was assocated wth a sgnfcant ncrease n lasma nsuln level comared to anmals gven II~P n salne. The mean rse n lasma nsuln was equal to that ordnarly assocated wth a rse n blood glucose of about 150 rag/ 100 ml roduced by ntravenous glucose alone. Wth ncreasng amounts of glucose added to the II~P a dose resonse curve for nsuln release aganst blood glucose concentraton was observed. Ths moved towards a lateau at blood glucose levels above rag/100 ml. b) On ths occason the resonse to dfferen~ amounts of ntravenous glucose n the resence or absence of II~P was nvestgated. The results are
D.S. Turner et al. : Intestnal Polyetdes, IRP, GIP, VIP and Motfln 461 shown n Fg. 5. Fve mnute lasma nsuln levels were sgnfcantly hgher at ~ll glucose dose levels n anmals gve IBP lus glucose than n those gven glucose alone. IRP was not assocated wth any sgnfcant dfference n 5 ran blood glucose levels at any glucose dose excet the lowest. glucose alone. There were no dfferences n 5 ran blood glucose levels between the two grous. b) Because of dfferences between IRP and GIP treated anmals n the 5 mn blood glucose resonse to the lowest doses of glucose n exerments 4b and 5 a, further exerments were carred out to test the re- Table 1. Mean 4- S.E.M. 5 ran lasma nsuln levels n rats recevng the dose of etde ndcated together wth glucose 0.625 g/#g, admnstered.v. over 11,/2 ran No. of Dose of Polyetde Polyetde anmals er grou Zero 0.0625 Fg/kg a 0.125 ~g/kga 0.25 ~g/kga 0.50 ~g/kg a 1.0 Fg/kg a 2.0 ~g/kg a IBP 8 145+13.06 2534-27.39 2724-18.51 3264-20.21 3344-24.37 3714-25.02 3904-14.40 for <0.005 < 0.00t < 0.001 < 0.001 < 0.001 < 0.001 dfference from control GIP 8 1014-4.35 1264-17.85 > 0.20 1704-20.20 < 0.01 1774-16.93 < 0.005 1924-24.37 < 0.01 2354-25.93 < 0.O01 161 Motln to 8 924-5.95 -- 1034-13.76 > 0.50 1174-11.35 1144-10.39 101:]:16.17 > 0.60 964-6.92 > 0.70 VIP 6 1424-34.80 -- -- 1794-17.58 >0.50 -- 1624-25.93 >0.70 223~:31.03 >0.10 Glucagon 8 2084-15.94 -- 2684-24.06 2304-30.22 > 0.90 3524-34.63 < 0.005 4004-29.42 < 0.001 6024-45.87 < 0.001 a For IRP read mg/kg ~, z, oo 300 Dose resonse to glucose of rats recevng IRP Insuln ffu/ml Dose resonse to gtucosen rats recevng IRP Glucose mg/ml 5 ~ Insuln level 5' Glucose level f2. s.e -5 100 b0 100 I I I I 100 150 250 Brood glucose revels after njecton Fg. 4. Dose resonse to glucose of rats recevng II~P, 0.5 mg/kg. Mean 4- S.E.M. 5 ran lasma nsuln levels followng.v. njecton of glucose lus IRP over 11/2 ran, lotted aganst mean blood glucose levels at 5 ran n grous of eght rats recevng glucose doses: @ zero, O 78 mg/kg, A 156 mg/kg, A 312 mg/kg, [] 625 m~/kg, [] 1230 mg/kg 5. Glucose Dose and the Insulnaemc Resonse to GIP a) The results of exerments smlar to 4b, but n whch ure GIP, 0.5 Fg/kg was substtuted for IRP, are shown n Fg. 6. The 5 mn lasma nsuln levels were sgnfcantly hgher, at all glucose dosages, n anmals recevng GIP lus glucose than n those gven o I 0 78 155 312 625 0 78 156 312 625 mg/kg Gtucosenjected Fg. 5. Dose resonse of rats to glucose, and to glucose lus 0.5 mg/kg IRP. Mean 4- S.E.M. of 5 mn lasma nsuln levels n grous of eght rats and mean 4- S.E.M. 5 ran blood glucose levels followng ntravenous njecton over 11~ mn. Clear columns show results wth glucose alone and hatched columns those wth glucose lus IRP roducbflty of the aarent reducton of blood glucose levels n IRP treated anmals gven small doses of glucose, comared wth anmals gven glucose wthout IRP. In these exerments control anmals were gven glucose (78 mg/kg body weght) and exermental anmals receved a smlar amount of glucose lus ether IRP (0.5 mg/kg) or GIP (0.5 vg/kg). The results are shown n Fg. 7. There was no dfference n the
462 D.S. Turner at al. : Intestnal Polyetdes, I1%P, GIP, VIP and Yfotln 5 mn blood glucose level between any of the three grous and I~F and GIP evoked smlarly augmented 5 ran lasma nsuln resonses. Dose resonse to glucose n rats recevng GIP Insuln Glucose u/ml mg/100ml ~' 400 5' Insuln lever 5' Glucose level 3OO 2OO 0 78 156 312 625 0 78 156 312 625 Glucose dose mg/kg Fg. 6. Dose resouse of rats to glucose, and to glucose lus 0.5 Fg/kg GIP. Mean 4. S.E.M. of 5 ran lasma, nsuln levels n grous of eght rats and mean 4- S.E.M. 5 ran blood glucose levels followng ntravenous njecton over 1 89 ran. Clear columns show results wth glucose alone and hatched columns those wth glucose lus I1%P Effect of IRP and GIP on resonse to.v. glucose, 78 mg/kg -g loo E Glucose /~u/ml 5O Insuln 5O Fg. 7. The effect of I1%P and GIP on the resonse to ntravenous glucose, 78 mg/kg. Mean 4. S.E.M. 5 ran lasma nsuln and blood glucose levels n grou.s of eght rats followng.v. rejecton over 1 I/z mm Dscusson The effect of artally urfed GIP on the lasma nsuln resonse to ntravenous glucose, n the rat, resembled that revously descrbed for Il%P [2]. Moreover, the shae of the dose resonse curve to the two rearatons was smlar; both substances roduced log-lnear dose-resonse curve. Both II~P and GIP augmented nsuln release wth blood glucose ncrements whch were too small to roduce sgnfcant rses n lasma nsuln levels when roduced by ntravenous glucose alone. Ths s remnscent of the st- uaton often observed durng oral glucose tolerance tests n whch a large rse n lasma nsuln s assocated wth small ncrements n blood glucose concentraton. Both GIP and II~P would seem to qualfy, therefore, for consderaton as the ntestnal medator of the augmented nsulnaemc resonse to enterc glucose admnstraton. It s clear that nether VIP nor motln need be consdered further, snce, unlke II~P and GIP, nether olyetde had a sgnfcant effect on glucose stmulated nsuln release. A drect comarson of the nsuln releasng otency of IEP and GIP was not ossble due to the known, but unquantfed, murty of the IRP rearaton currently avalable. The comaratve data for glucagon used under the same condtons suggests that on a weght for weght bass GIP has an nsuln releasng otency aroxmately four tmes that of glucagon, or n molar terms there s an aroxmately sx fold dfference n otency. Our exerments revealed no essental dfference between the nsuln releasng effect of IEP and GIP. The queston arses, therefore, as to whether the actve comonent of the II~P rearaton s GIP or an as yet uncharactersed olyetde, and secondly whether GIP s normally nvolved n the nsuln secretory resonse to oral glucose n man. A onter to the ossble non-dentty of IRP and GIP comes from the observaton that extracts of duodenum, a source of GIP, do not exhbt sgnfcant nsuln releasng actvty when reared by methods used to roduce IRP from the jejunum [9, 10]. The fnal answers to both questons must awat further exerments, and ultmately the urfcaton and chemcal charactersaton of the actve comonent of IRP. It s lkely that GIP s closely related to, but not necessarly dentcal wth, II~P, and that, as wth other gastrontestnal olyetdes there s some sharng or overlang of structure and bologcal actvtes. Acknowledgements. The authors wsh to thank the Brtsh Dabetc Assocaton and the Wellcome Foundaton Lmted for ther generous fnancal assstance. References 1. Turner, D.S., Marks, V.: Enhancement of glucosestmulated nsuln release by an ntestnal olyetde n rats. Lancet 1972 I, 1095-- 1097 2. Turner, D.S., Shabaan, A., Etherdge, Laurane, Marks, V. : The effect of an ntestnal oly-etde fracton on nsuln release n the rat n vtro and ~ vvo. Endocrnology 93, 1325-- 1328 (1973) 3. Sad, S.I., Mutt, V. : Isolaton from orcne-ntestnal wall of a vasoactve octaeosaetde related to seeretn and to glueagon. Euro. J. Bochem. 28, 199--204 (1972) 4. Bro~:a, J.C., Mutt, V., Pederson, 1%. A. : Further urfcaton of a olyetde demonstratng enterogastrone actvty. J. Physol. (Lond.) 209, 57--64 (1970) 5. Brown, J.C., Cook, M.A., Dryburgh, J.l~.: Motln, a gastrc motor actvty-stmulatng olyetde; fnal
D.S. Turner st al. : Intestnal Polyetdes, IRP, GIP, VIP and Motln 463 urfcaton, amno acd comoston, and c-termnal resdues. Gastroenterology 62, 401--404 (973) 6. I)ur@, J., Ross, S.A., Watson, D., Broom, J.C.: Stmulaton of nsuln secreton by Gastrc Inhbtory Polyetde n man. J. c]n. Endocr. 37, 826--828 (1973) 7. Huggett, A.St.G., xon, D.: ~se of glucose oxdase, eroxdase, and o-dansdne n determnaton of blood and urnary glucose. Lancet 1957 II, 368--370 8. Samols, E., Bflkus, D.A.: A comarson of nsuln radommunoassays. 1)roe. Soc. ex. Bol. (~q.y.) 115, 79--84 (1964) 9. Moody, A.J., Markussen, J., Schach-Fres, A., Sundby, F., Malasse, W. J., Malasse-Lagae, F. : The nsuln releasng actvtes of extracts of ork ntestne. Dabetologa 6, 135--140 (1970) 0. Turner, D.S. : Unublshed observatons I)r. D.S. Turner Det. of Bochemstry Unv. of Surrey Guldford, Surrey GU2 5XI-I England