-
Interaction of Fat-stimulated Gastric InhibitoryPolypeptide on
Pancreatic Alpha and Beta Cell Function
C. A. VERDONK,R. A. RIZZA, R. L. NELSON, V. L. XV. Go, J. E.
GERICH, andF. J. SERVICE, Endocrine Research Unit, Gastroenterology
Research Unit,Departments of Medicine and Physiology, Mayo Medical
School andMayo Clinic, Rochester, Minnesota 55901
A B S T RA C T Gastric inhibitory polypeptide (GIP) isconsidered
to be the principal mediator of the entero-insular axis. A
glucose-insulin clamp technique wasused to study the effects of
differing blood glucoselevels on the insulinotropic and
glucagonotropicactions of fat-stimulated GIP in seven healthy
subjects,as well as the effect of physiologic hyperinsulinemiaon
GIP secretion. Blood glucose levels were clampedfor 4 h at 43+2
mg/dl (hypoglycemic clamp), 88+1mg/dl (euglycemic clamp), and 141+2
mg/dl (hypergly-cemic clamp) in the presence of a constant insulin
in-fusion (100 mU/kg per h).
Under hypoglycemic clamp conditions there was noincrease in
C-peptide nor glucagon after Lipomul in-gestion, despite an
increase of GIP of 51.7±8.7 ng/mlper 120 min. Under euglycemic
clamp conditions,small and inconsistent increases in C-peptide
andglucagon were observed after fat ingestion and a con-comitant
increase of GIP of 35.2±9.4 ng/ml per 120min. Under hyperglycemic
clamp conditions after fatingestion and a GIP increase of 24.0±5.7
ng/ml per 120min, C-peptide increased from 6.4±5 ng/ml to
11.0±1.1ng/ml (P < 0.01) but glucagon did not change.
Thesefindings confirm that in healthy man GIP exerts
itsinsulinotropic properties only under hyperglycemicconditions and
indicate that GIP is not glucagonotropic.
Under euglycemic clamp conditions (plasma glu-cose, 89±1 mg/dl)
and physiologic hyperinsulinemia(serum immunoreactive insulin,
137±3 ,uU/ml) GIPresponses to fat ingestion (39.7±9.8 ng/ml per 120
min)were not different from the GIP responses to fat inges-tion in
the absence of hyperinsulinemia (39.7±11.1ng/ml per 120 min).
Therefore, insulin under normogly-
This work was presented in part at the 39th Annual Meetingof the
American Diabetes Association, Los Angeles, Calif., 11June
1979.
Address reprint requests to Dr. Service at the Mayo
Clinic.Receivedfor publication 14 September 1979 and in revised
form 14 January 1980.
cemic conditions does not exert an inhibitory effecton
fat-stimulated GIP secretion. The higher GIP re-sponse to oral fat
in the hypoglycemic clamp, and thelower GIP response in the
hyperglycemic clamp com-pared to the response in the euglycemic
clamp suggestsan effect of glycemia itself on GIP secretion in
thepresence of hyperinstulinemia.
INTRODUCTION
Gastric inhibitory polypeptide (GIP)' is considered tobe the
gastrointestinal factor primarily responsible forthe greater plasma
insulin response to oral compared toparenteral nutrient
administration (1-3). Althoughingestion of carbohydrate, fat (4),
and some amino acids(5) results in increased levels of GIP,
increased levelsof insulin are observed only after oral
carbohydrate(4) and amino acid (5) and not after oral fat (4).
Theseobservations suggest that the insulin secretory
respon-siveness of the beta cell to GIP is influenced bysubstrate
or hormonal factors. There is in vitro evidencethat the
insulinogenic effect of GIP is glucose depend-ent (2, 6). Studies
in humans, however, have shownconflicting results. There is
evidence, using a glucoseclamp technique, that the insulinotropic
action of oralglucose-stimulated GIP (7) occurs only during
hyper-glycemia, but also evidence that amino acid-stimulatedGIP is
insulinotropic in the absence of hyperglycemia(5,8). The
involvement of a glucose-dependent mecha-nism for the
insulinotropic action of fat-stimulated GIPhas been reported from
nonsteady-state conditions (8-10), but has not been investigated
using a glucoseclamp.
Whether insulin released after nutrient ingestionalso regulates
the secretion of GIP as part of a negativefeedback system is
presently controversial. The re-duced GIP responses to oral fat
observed after an intra-
'Abbreviation uised in this )ai)er: GIP, gastric
inhibitorypolypeptide.
1119J. Clin. Invest. (© The American Society for Clinical
Investigation, Inc. 0021-9738/80/05/1119/07 $1.00Volume 65 May 1980
1119-1125
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Venous bolus of insulin or durinlg a coIncomitant infuisionof
gIlucose supported an inhibitorx action of insulinon the secretioni
of GIP (4, 8- 11). However, Andersenet al. (7), using a
glucose-insulin clacmp technique ateuglycem-nia and hyperglycemia,
fouind no feedbackinhib)ition of insulin on glucose-stimultlated
GIP secre-tioll.
The eurrent studies were undertaken to examinewhether the level
of glycemia moduilates the insulino-tropic effect of fat-stimulated
GIP, to determinewhether insulin inhibits the secretion of
fat-stimulatedGIP, anid to determine wvhether the level of
glycemiaitself miav inifluence the GIP responise to oral fat.
GIP,C-peptide, and glucagon responsies to fat ingestionNere
imieastured in healthy subjects while glycemiia was
miiaintainied by a glucose-insulin clamiip techni(lue in
thehypoglycemic, euglycemic, and hyperglycemic rangesdurinig a
concomitant inftusion of insulin at a rate suf-ficienit to achieve
physiologic hvperinssulinemnia.
METHODS
inormed conisenit was obtained fromil seveniioriiail
nonobesesul)ects (three males, four females) ages 36±5 y r (mean ±
SEM).All were within 10%of their ideal bodx weight and n1on1e had
afhimilv history of'diabetes mellitis.
Each subject was stu(die(l in the ov-ernight f:aste(d state at
eachglycemic clamiip level, and six of the seven subjects were
stud-iedl dIurinig saline infusion in the absence of'
glucose-insulillclamiip with and xvithout the ingestion of
Lipomutl. Each studywas sep)arated by 1-2 wk.
For each glucose clamp studv 18-gauge indwelling catheterswere
inserted into contralateral antecuibital veins, one forthe
conttinuous inf'usioni of crystalline insulini (pork U100, EliLilly
& Co., Indianapolis, Ind.), at the rate of 100 mU/kg per h1v
imleanls of a Harvard pump (Harvard Apparatus, millis,NMass.) and
one for the intermittent (every 20 min) wvithdrawalof' blood for
the determination of hormonies. Distal to theinisilini inf'usion
site in a separate f'orearmii vein, a double-lumiien caltheter was
inserted for continutiouis withdrawal of)lood(at a rate of 2 ml/h
for glucose analyN-sis by the Biostator.
The glucose clamp was achieved using the Biostator GCIIS(Life
Science Instruments, Elkhart, Ind.), wvhich permits con-tinuous
analysis and minuite-by-minute recording of plasmaglucose levels
(glucose-oxidase) as well as the infuision ofglucose according to
predetermined computer-containedalgorithlmls (12). Glucose (50
g/dl) was inf'used through theinsuilin infuision access site at
rates determined by theBiostator (mode 7:1). In each subject the
glucose clamp wasmaintainled at 45 mg/dl (hypoglycemic clamp), 140
mg/dl(hyperglycemic clamp), and at the basal overnight
fastingplasmiia gluicose level (euiglycemic clamp). The gltucose
in-fuision rate at the desired plasma gltucose level, determinedin
prelininary studies, w as 0.97+0.06 mg/kg per min,6.0(.1 miig/kg
per min, and 7.4±.1 mg/kg per mimi and the inverseof'the static
gain for glucose inf'usion was 18, 45, and 45 for thehypoglycemic,
euiglycemic, and hyperglycemiiic clampis, respec-tively. Becauise
the maximal infusion rate of gltucose that canbe infuised by the
Biostator is 1 g/min, additional glutcose forthe euglycemic and
hyperglycemic clamiips was given by avariable-speed infusion
Harvard puimp.
Each gltucose clamp study was conducted for a total of'240min.
The first 120 min w.ere devoted to obtaining stableplasma glulcose
and insulin conicentrations. Emutlsified cornoil
(Lipomul, Upjohn Co., Kalamazoo, Mich.), 67 g, was
adminiis-tered orally at 120 min At 20-min intervals duiring the
aclamps,glucose levels obtained by the Biostator w ere checked
againistthe reference method, YSI 23A glucose analyzer,
YellowSprings Instrumient Co., Yellow. Springs, Ohio. The
Bio-stator glutcose values for the hyperglycemic anld
euglycemicclamps were found to be consistently within ±O10% of'the
YSIreadings. The mediani percent difference between the twomethods
xwas 3.5%. For the hypoglycemic clamp the YSIglucose readinigs
wvere consistently greater than the Biostatorvalues with a mnedian
(lifference of 15%.
For the two stuidies wvithout the insulini-gluicose clampii),
withand without the Lipomutl ingestion, blood samiples were
oh-tained for 20-mimi intervals f'or glucose, insulin, and GIP
deter-miniationis for 140 mi.
Sertum samples were f'rozen for insuilini assay. Blood
samplesfor GIP and C-peptide were collectedl on ice in tubes
conttain-ing EDTA andl Trasvlol (500 kallikreini inhibitor
tuniits/Il;Sigmla Chemiiical Co., St. Louis, Io.) cenitrif'ugedl at
4°C afterwhich the plasma was frozen until assay. Blood for
glucagonlwas processed similarly except for the use of
benzamidine(0.1 M) instead of Trasvlol.
Hormonie assaq s. Plasmna GIP was measure(d bv the miiethodof'
Kuzio et al. (13). Purified GIP, obtained fromii Dr. J. C.Brown
(University of' British Columbia, \ancouver, BritishColtumbia,
Canca(la) was used as standardl and( tracer. Anlti-serumii R4817
wxas used at a final dilution of 1:100,000. Thisantiserumil detects
the tw ro molecular forms of imminuinoreactiveGIP (5,000 and 7,500
mol wt) present in postprandial blood.The limit of' detectioni,
intraassav and interassav coefficientof variation for the plasmiia
internal reference stanldardls were50 pg/ml, 7 anld 13%,
respectively. No cross-reactivity wasdetected with glucagon
(crystalline porcine glucagon, EliLilly & Co.), humiican
pancreatic polvpeptide (Eli Lillx & Co.),highly purified
cholecvstokinin, secretin, vasoactive intes-tinal peptide (all
gifts from Dr. XI. Mlutt, Stockholmii, Sw,veden),motilin (Dr. J. C.
Brow.n), and gastrin (Imperial ChemiiicalIndustries LTD, Londoni,
England) in concenitrations uip to10 ng/ml.
Insulini and( gluicagoni were measutred 1w the miietho(ds
ofHerbert et al. (14) and Faloona et al. (15), respectively.
Plasmaiit C-peptide wvas measured using the reagenits
andprocedure obtained from Calbiochem-Behri ng Corp., Amiieri-can
Hoechst Corp. (San Diego, Calif.). The characteristics ofthe rabbit
aintiserumiii against synthetic hulman C-peptide havebeen
descril)ed (16). 1211 synthetic tvrosvl C-peptide was tiusedas
tracer, a.nd synthetic human C-peptide as staindatrd.
Arialhjtic nmethods. Data in the text and figtures are givenias
mean±SE-M. The integrated plasma GIP response afteringestion of
Lipomntul was calculated from the area eircuim-scribed by the curve
(using the menan of the 100 and 120 mifor basal) in eaclh person
and expressed as ig/mIl per 120 min.The coefficient of variation of
the glucose claimps was caltcu-lated wvith the Biostator glucose
vallues at 5-mimi intervalsduiring the 60-240-mimi period of each
clamp.
Statistical evaluation was performed by m11eans of' the
two-tailed paired t tests. The rank sum test was ised to
compatrethe plasma GIP responses after Lipomutl ingestion tinder
thevarious claimp conditionis, since these responses were
nlotnormally distributed (17).
RESULTS
Hypoglycemic clamp. Fig. 1 shows the glucose,GIP, C-peptide,
insulin, and glucagon levels beforeand after Lipomul ingestion
during the hypoglycemicclamp. Glucose levels decreased
progressively and
1120 Verdonik, Rizza, Nelsoni, Go, Gerich, atnd Service
-
HORMONERESPONSESDURINGHYPOGLYCEMICCLAMPBEFOREANDAFTERLIPOMUL
(67g) INGESTION
INSULIN (O. IU/kq/h) +VARIABLE GLUCOSE
100 - GLUCOSE
i 80 LIPOMUL p.o.N\ ffO- t
6 40
20L
GASTRIC INHIBITORY ,POLYPEPTIDE
Soo.00 k
N400k-
200- INSULIN
N 500
-20 0 40 80 120 160 200 240
MEAN± SEM
n - 7
*P
-
HORMONERESPONSESDURINGEUGLYCEMICCLAMPBEFOREANDAFTERLIPOMUL (67g)
INGESTION
INSULIN (0.1U/kg/h) + MEANt SEMVARIABLE GLUCOSE na 7
GLUCOSE *P
-
EFFECTOF INSULIN ONGASTRIC INHIBITORYPOLYPEPTIDERESPONSESTO FAT
INGESTION
INSULIN 10.1 U/kg/h) +VARIABLE GLUCOSEor SALINE
LIPOMUL- 67 g p.o.GLUCOSE t
% 10090
E280180
0135
90_::
45
OL_960
720
480Q~
240
oL_
MEAN± SEMn-6
--o CONTROL*_ 'CLAMP'
* P
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at euglycemia. Although the GIP levels 120 min afterLipomul were
not returniing to base-line, they wereclose to a plateau
configuirationi in the last 60 min. Theobserved levels likely
represent maximal responses asthey are similar to those reported by
others after fatingestion (8, 9, 11). In addition, further sampling
be-yond 120 min was unlikely to have shown a differencein GIP
between control and euglycemic clamp studiesbecause differences
observed in GIP by others be-tween control aind glucose infusion
studies occuirredbefore 120 min after fat or galactose ingestion.
Theslight decreases in basal GIP during the 120-inin pre-Lipomul
period in the presence aind absence of hyper-insulinemia may
represent the effect of fasting on GIP.
The observation of a reduced GIP response after fatingestion
during hyperglycemnia compared to theresponse after the same
stimulus in the presence ofeuglycemia is consistent with previous
reports (8-10,25), but is open to an interpretation different from
anlinhibition of GIP by insulin alone. The increased GIPresponse to
oral fat during hypoglycemia coimpared toeuglycemia couipled with
the reduced GIP response inhyperglycemia (Fig. 3) in the presence
of similar serumninsulin levels (144+3 ,uU/ml for hypoglycemiia,
141+2,uU/ml for etuglycemia, and 183±5 ,uU/ml for hypergly-cemia)
stiggests that the glucose level itself in thepresence of
hyperinsulinemia affects the GIP responseto oral fat. Whether this
effect is on one or bothmolecuilar species remains to be
determined.
In summary, fat-stimulated GIP has insulinotropicactivity that
is glucose dependent, is not gluicagonotro-pic, is not inhibited by
physiologic hyperinsuilinemia ateuglycemia, but is influenced by
the ambient gluicoselevel in the presence of hyperinsulinemia.
ACKNOWLEDGMENTS
The excellent technical assistance of R. Westland, N. Reed,J.
King, K. Greene, B. Brick, I). Stenner, and D. Nash is grate-fully
acknowledged.
This investigation was supported in part by research
grantsAM-20973, AM 20411, AMX 5827, and RR 00036 from theNational
Institutes of Health, U. S. Public Health Service,and from the Mayo
Foundation.
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Interaction of Fat-stimulated GIP otl Alpha and Beta Cell
Fuinction 1125