Lack of control by glucose of ultradian insulin secretory oscillations in impaired glucose tolerance and in non insulin dependent diabetes mellitus

Lack of control by glucose of ultradian insulin

secretory oscillations in impaired glucose

tolerance and in non-insulin-dependent

diabetes mellitus.

N M O'Meara, … , E Van Cauter, K S Polonsky

J Clin Invest. 1993;92(1):262-271. https://doi.org/10.1172/JCI116560.

Normal subjects demonstrate the presence of ultradian oscillations (period 80-150 min) in insulin secretion rate (ISR) tightly coupled to glucose oscillations of similar period. These oscillations appear to be a function of the feedback loop linking glucose and insulin. The present study was undertaken to determine whether the control by glucose of the ultradian oscillations in insulin secretion is altered in impaired glucose tolerance IGT and in non-insulin-dependent diabetes mellitus (NIDDM). Patients with NIDDM (n = 7), IGT (n = 4), and matched nondiabetic controls (n = 5) were studied under three separate protocols that involved administration of glucose at either a constant rate of 6 mg/kg per min for 28 h or in one of two oscillatory patterns at the same overall mean rate. The amplitude of the

oscillations was 33% above and below the mean infusion rate, and their respective periods were 144 min (slow oscillatory infusion) or 96 min (rapid oscillatory infusion). Insulin, C-peptide, and glucose were sampled at 10-min intervals during the last 24 h of each study. ISRs were calculated by deconvolution of C-peptide levels. Analysis of the data showed that (a) the tight temporal coupling between glucose and ISR in the nondiabetic controls was impaired in the IGT and NIDDM groups as demonstrated by pulse analysis, cross-correlation analysis, and spectral analysis; (b) the absolute […]

Research Article

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Lack of Control

by Glucose

of Ultradian

Insulin

Secretory

Oscillations

in Impaired

Glucose Tolerance

and in Non-insulin-dependent

Diabetes

Mellitus

Niall M.O'Meara, Jeppe Sturis,Eve Van Cauter,andKennethS. Polonsky

TheDepartmentofMedicine, UniversityofChicago, Pritzker School ofMedicine, Chicago, Illinois60637

Introduction

Normal subjects demonstratethepresenceofultradian

oscilla-tions (period 80-150 min) in insulin secretion rate (ISR)

tightlycoupledtoglucose oscillations of similar period.These

oscillationsappear tobeafunctionof the feedback loop linking glucoseandinsulin.Thepresent study was undertaken to deter-mine whether the control by glucoseofthe ultradian oscilla-tions in insulin secretion is altered in impaired glucose

toler-ance IGT and in non-insulin-dependent diabetes mellitus

(NIDDM). Patients withNIDDM(a=7), IGT(a= 4),and

matched nondiabetic controls (a=5) werestudied under three separate protocols that involved administration ofglucose at eitheraconstant rateof 6mg/kgperminfor 28h orinoneof

two oscillatory patterns at the same overall mean rate. The

amplitude of the oscillations was 33% above and below the

meaninfusionrate, andtheir respective periodswere144min (slow oscillatory infusion)or96 min (rapidoscillatory

infu-sion).Insulin,C-peptide,andglucoseweresampledat10-min

intervals during the last 24hof each study. ISRswere calcu-lated bydeconvolutionof C-peptide levels. Analysis of the data

showed that(a)thetight temporal couplingbetweenglucose

andISR in thenondiabeticcontrolswasimpairedin theIGT

and NIDDMgroups asdemonstratedbypulse analysis,

cross-correlationanalysis,andspectral analysis; (b)theabsolute am-plitude of the ISR pulsesprogressivelydeclined with the

transi-tion fromobesitytoIGTtoNIDDM;and(c)theabsolute

am-plitude of the ISR oscillations failedtoincreaseappropriately

with increasing absolute amplitude of glucose oscillations in the

IGT and NIDDMsubjects comparedwith the controlgroup. In

conclusion,thepresentstudydemonstrates thatimportant

dy-namicpropertiesof the feedbacklooplinkinginsulinsecretion

andglucosearedisruptednotonlyinestablishedNIDDM but

also in conditions where glucose tolerance is only minimally

impaired. Furtherstudiesareneededtodetermine howearlyin thecourseof beta-celldysfunctionthis lack of controlby

glu-coseof the ultradianoscillationsininsulin secretionoccursand todefinemoreprecisely ifthis phenomenon playsa

pathoge-netic role in theonsetof

hyperglycemia

in

genetically

suscepti-ble individuals. (J. Clin. Invest. 1993.

92:262-271.)

Key

words:entrainment*feedbackloop*glucose*insulin

secretion-oscillations *impaired glucosetolerance *

non-insulin-depen-dent diabetes mellitus

AddressreprintrequeststoKenneth S.Polonsky, M.D.,Universityof

Chicago, DepartmentofMedicine, MC1027, 5841 SouthMaryland Avenue,Chicago,IL60637.

Receivedfor publication 9September1992andinrevisedform3

February1993.

During thepast10-15 years, many studies havedemonstrated

thatinsulinsecretion isacomplex oscillatoryprocess charac-terized by rapid oscillations with a periodicity of 8-15 min,

whicharesuperimposedonslower (ultradian) oscillationswith

a periodof 1.5-2 h (1-9). Both rapid and slow oscillations

have beenobserved under fasting conditions and during

con-stantglucose infusion.Theultradianoscillations have in

addi-tionbeen demonstrated duringconstant enteral nutrition and

following meals. The rapidpulsespersistin the isolated

per-fusedpancreas (10) and in isolated islets ( 11 )exposed to a constantglucoseconcentration, suggestingthat they occur inde-pendently of oscillations inplasma glucose and that they origi-nateduetointraislet mechanisms.Theultradianoscillationsin insulinsecretionare,by contrast, tightly coupled to glucose (6, 8,9). Studiesin normal weight subjects during constant glu-coseinfusion and following meals have demonstrated a high

degree of concomitance between these secretory oscillations

andoscillationsin glucose of comparable periodicity.In

addi-tion,theperiodicity ofthe insulin secretory oscillationscan be entrainedtotheperiod ofanoscillatoryglucoseinfusion (9). These findingsareentirely compatible withthe view that the

ultradianoscillations in insulin secretionare aproductofthe

insulin/glucose feedback mechanism(12, 13).

Wehavestudiedthetemporal interactionsbetweenglucose

and insulinsecretion by determiningtheextent towhich ultra-dianoscillationsininsulinsecretioncanbeentrained by exoge-nousglucose. Theconceptof entrainmentiscentraltothefield of nonlinear dynamics and has been used inmany different scientificfields, includingthestudy of biologicalsystems( 14). Briefly,ifanonlinear, self-oscillatingsystemis perturbed exoge-nously withaperiodic stimulus, differenttypesofoscillatory behaviors may emerge, one of whichis entrainment. When

entrainmentoccurs,theoscillationof thesystemreacts tothe exogenousstimulus by

adjusting

its

period

tothat ofthe

stimu-lus. The ability to entrain depends on a number of

factors,

includingtheperiod ofthestimuluscomparedwith the natural

periodofthesystem,the

amplitude

ofthestimulus,the

nonlin-earities ofthe system, and the

strength

ofthe

coupling

between theexogenousstimulus and thesystem.

In non-insulin-dependent diabetes mellitus

(NIDDM),'

the

oscillatorypatternof

3-cell

secretion is

disrupted.

The

per-sistent, regular rapidoscillationspresentin normal

subjects

are

replaced byirregular cyclesof shorter duration both insubjects

with established NIDDMandintheirfirst-degreerelatives( 15,

16).Theultradianoscillations inNIDDMarealsomore

irregu-lar.Inaddition, theyareof smaller

amplitude

and the propor-tionoftheseoscillationsthatareconcomitant with oscillations

1.Abbreviationsused in thispaper:BMI,bodymassindex; IGT,

im-paired glucose tolerance; ISR,insulin secretionrate;NIDDM,

non-in-sulin-dependentdiabetes mellitus.

Abstract

J. Clin. Invest.

©The AmericanSocietyforClinicalInvestigation,Inc. 0021-9738/93/07/262/10 $2.00

in glucose is much smaller ( 17, 18). These latter observations raise the possibility that specific dynamic properties ofthe feed-back loop linking glucose and insulin are disrupted in NIDDM. Such an alteration, if it could be confirmed, might be indicative of an important early (3-cell abnormality in suscepti-ble individualsevenbefore theonsetofoverthyperglycemia. The present study was therefore undertaken to determine ifthe abilitytoentrain 3-cell secretionwasreduced insubjects with established NIDDM and inpatients with early impaired glu-cosetolerance(IGT) who hadnotyet developedovert hypergly-cemia.

Methods

Subjects

Studies wereperformed inpatients withproven NIDDM(n = 7, 6 males, 1 female;2Caucasian,5Black),impairedglucose tolerance (n

=4, 2males, 2 females, 1 Caucasian,3 Black), and inagroupof obese

subjects(n=5,4males,1 female,3Caucasian, 1 Black, 1Hispanic)

who hadnormal glucose tolerance and no family history of diabetes. Thecriteriausedtodeterminewhetherglucose tolerancewasnormal

orimpairedin therespectivegroupswerethoseoftheNational Dia-betes DataGroup (19).Themeanage,weight,andbodymassindex (BMI) of the three groupsaredemonstrated in TableI. Noneofthe diabeticpatientswasreceivinginsulintherapyatthetime ofthestudy. Oralhypoglycemicagentswerediscontinuedatleast 2 wkbefore the

commencementof theexperiments.

Allstudieswereperformedin theClinical Research Center(CRC)

of theUniversityofChicago.Theprotocolwas_{approved by}the Institu-tionalReview Board,andallsubjectsgave written informedconsent.

Experimental protocol

Subjects were admittedto the CRC afteran overnightfast. Aftera

subjectwasadmitted,anintravenous catheterwasinserted into both

forearms,onefor glucose infusionandonefor bloodsampling. Begin-ningat0800hours, glucosewasinfusedintravenouslyforaperiod of28 h,duringwhich time thesubjectsremained in the recumbentposition. Eachstudy consisted ofaninitial 4-hperiod (0800-1200 hours)to

allowasteadystatetobeachievedfollowingthe transientglucoserise thatisassociated with the first few hoursofinitiatingaglucoseinfusion

(8).Thiswasfollowed byasubsequentperiod of24 h (1200-1200 hours)duringwhichtimesamplesweredrawnat10-min intervalsfor

glucose,insulin,andC-peptide.Lightsweredimmed between 2300 and 0700hours, toallow thesubjectstosleep. Potassium(40 meq)was givenorally every 12 h, andsubjectswereallowedfreeaccesstowater

butnotgivenanyfood forthedurationof theexperiment.

Eachsubjectwasstudied on at leasttwooccasions under the above protocol. On eachoccasion,glucosewasadministeredas a20% solu-tion viaacomputer-controlled pump(Flo-gard8000volumetric infu-sion pump; Travenol Laboratories, Deerfield,IL)followingdifferent patterns.Initially,eachsubjectreceivedaconstantglucoseinfusion ata rateof 6 mg/kg per min. The subsequent studies involved the adminis-trationofanoscillatoryglucose infusion with periods of 96 min and/or

144min, theseperiods having been shown to be 20% shorter and 20% longerrespectively than the endogenous period observed in normal

subjects duringconstantglucoseinfusion (9). The software controlling

theglucose infusion was custom written to allow the actual infusion

rate tobe recorded so that possible interruptions could be detected.

Eachoscillation was shaped as a sine wave and the amplitude was 33% aboveand below the meaninfusion rate. Subjects received identical volumesof glucose during each 28-h infusion period.

Analytical methods

Blood samples for insulin measurementswere allowed to clot at room

temperatureand theserumwasstoredat-20'C untilassayed. Samples for_C-peptideweredrawninto tubesat

40C

_containing500 Kallikrein

inhibitorunits/ml Trasylol and 1.2 mg/ml EDTA. The plasma was immediately separated and stored frozen until assayed. Serum insulin wasassayed by a double antibody technique (20). This assay has a lowerlimitofsensitivity of 20 pmol/liter, and the average intraassay coefficientofvariation is 8%. PlasmaC-peptide immunoreactivitywas

measured aspreviously described (21 ). The lower limit ofsensitivityof the assay is 0.02pmol/ml and theintraassaycoefficient of variation averages 6%. Plasma glucose was measured with a glucose analyzer (model23A; Yellow Springs Instrument Co., Yellow Springs, OH). Theintraassaycoefficient of variationofthis method is<3%.

Data

analysis

Therelative degree ofinsulin resistance was evaluated with the Homeo-stasis Model Assessment (HOMA) method (22),usingthe mean of 3-4 stressedfastingglucose and insulin levels obtained ondifferent occa-sionsfrom each subject.

SMOOTHING AND ESTIMATION OF INSULINSECRETION RATES

Theindividual glucose,insulin,andC-peptide profilesweresmoothed

usingathree-point movingaverageasinpreviousstudies ofoscillatory

insulin secretion ( 1, 6, 9). This procedure strongly dampens all

fluctua-tions shorter than 30 min, allowingabetter visualizationofslower

oscillationsat anexpenseofamodest reduction in theiramplitude.It also reduces measurement errorbyafactor of A. All further calcula-tionswereperformedonthe smoothedprofiles.Thesmoothed

C-pep-tide curve was used to derive insulin secretion rates by deconvolution (23). Thekinetic parameters used in the deconvolution program were derived aspreviouslyvalidated and described in detail (24).

PULSE ANALYSIS

Toidentify significant pulses, eachprofilewasanalyzedwith ULTRA,

acomputer program forpulsedetectionandquantification (25).The generalprincipleof thisalgorithmis the eliminationofallpeaksfor

which either the increment (difference between the peak and the

pre-ceding trough)orthe decrement(differencebetween thepeakand the nexttrough) does not exceed acertainthreshold related to measure-ment error.Thisproduces a so-called "clean"profile.Extensive simula-tion studies(25)haveindicated thatathreshold of twice theintraassay

coefficient ofvariation generally minimizes both false-positive and

false-negativeerrors.This thresholdwasthereforeusedtoidentify

sig-nificant pulsesofglucose. However, becausedeconvolution involves

anamplification ofmeasurementerror,amoreconservative threshold of three times theintraassay coefficient ofvariation ofC-peptidewas

usedtoquantify pulsesof insulin secretion. Inthis study, pulseanalysis wasperformedon theprofilessmoothed by thethree-point moving

averageso that the measurement errors werereduced by A. Thus

peaks of insulin secretionandglucosewereconsideredsignificantif

theirrespectiveincrements and decrements exceeded 10.39%(i.e.,3

X6%/ V3) and 3.46% (2 x 3%/

_f/),

respectively. Peaksthatdid not meet these threshold criteriawereeliminated fromthe datasetusingan iterativeprocess. For eachsignificantpulse, the absoluteamplitudewas

definedasthe difference between the level of thepeak and the level of thepreceding trough. Groupstatisticsonpulse amplitudeswerebased

onmedians,ratherthan means, becauseofthe non-Gaussiannatureof

pulse distribution.

Becauseglucose hasarelativelylonghalf-life,changes in

produc-tion and/orutilization maynotbereflectedassignificant peaksin the glucose concentration curve. We therefore identified all the

"shoulders"in the glucoseprofiles.Todothis, instantaneous deriva-tives of eachindividual clean glucoseprofilewereestimatedastheslope oftheglucosechanges during each sampling interval, and the shoulders were identified as slopes with an absolute value of zero (i.e.,

corre-spondingtotheeliminated peaks), preceded and followed by slopes of thesamesign.

ANALYSIS OF THE TEMPORAL ASSOCIATION BETWEEN

OSCILLATIONS OF GLUCOSEAND OSCILLATIONS INISR

Pulse-by-pulseanalysis of concomitance. Temporal associations

analysis oftheconcomitance. Significantpulsesofglucose and insulin secretion were consideredconcomitantiftheirpeakvalues occurred within 10 min of each other. The concomitance ratio of glucose pulses with ISR pulses was calculated as the number of concomitant glucose and ISRpulses divided bythe total numberof glucosepulses.

Con-versely, theconcomitanceratio ofISR pulseswithglucose pulses was calculated as the numberofconcomitant glucose and ISR pulses di-videdby the total numberofISR pulses.

Cross-correlation analysis. For each glucose, insulin, and ISR pro-fileabest-fitcurve wascalculatedusingarobustalgorithmproposed by Cleveland(26)with awindowof 15 datapoints.Eachprofilewasthen

divided by itscorresponding best-fit curve, thereby yielding a de-trendedprofile for whichdiurnalvariationswereremoved. For each

study, coefficients ofoverallcrogs-correlationwerethen calculated be-tweenpairs ofdetrendedprofiles(glucose vs.insulin,glucose vs. ISR, andinsulinvs. ISR) atlags of0, ±10, ±20, etc., up to±300 min.

Cross-correlation profiles from different studies were pooled using Fisher'szvalues(27). This procedureallowed themaximalcoefficient of cross-correlation foreachglucoseinfusion protocolandstudygroup tobeidentified along withthelagatwhich this occurred,thus provid-ingmeasuresofthe overallconcomitanceaswell as thecorresponding

lag between the ultradianoscillationsin glucose,insulin,and ISR.

Spectral analysis. To investigate the temporal nature ofthe profiles

obtainedduring glucoseinfusion, spectral analysiswasperformedon eachglucose,insulin, and ISR series. The methods aredescribedby

Jenkinsand Watts(28).To removecircadiantrends,eachserieswas detrendedwiththefirst differencefilter,and then spectralestimations

wereobtained usingaTukeywindowwith a widthof72 datapoints.

Eachindividualspectrum wasnormalizedassumingthe totalvariance of theseriestobe 100%. Thisallowed average values across the three groups to bestatistically compared.

STATISTICALANALYSIS

Results areexpressedasmean±SEM. Statistical tests wereperformed usingtheStatistical Analysis System (version6.04for personal com-puters; SASInstitute Inc., Cary, NC). Repeatedmeasuresanalysisof

variance was used toidentify overall differences between the three

studygroups and thestudiesusingthe threedifferent glucose infusion

protocols.Tukey's studentizedrange testwasusedfor post-hoc

compar-isons. Differenceswereconsideredtobesignificantif P<0.05. Results

Basallevels ofplasma glucose, serum insulin, andISR. The

clinical characteristicsand basallaboratoryvaluesof the study

groups are recorded in Table I. All three groups were well

matched for age,weight,and BMI.Fastingglucose and

glyco-hemoglobinconcentrationsweresignificantly higherin the dia-betic patients, whilethese valueswerenormal in the patients

TableI. Ages, Weight,BMI, BasalInsulin, C-Peptide, Glucose, andHbAI inthe ThreeStudy Groups

Control IGT NIDDM

(n= ()(n=4) (n=7)

Age(yr) 40.4±4.6 44.5±7.0 46.0±4.7

Wt(kg) 122±5.9 110±11 106±10

BMI

(kg/M2)

38.4±1.3 38.8±4.4 34.9±2.5 Insulin(pmol/liter) 172±16.6 128±4 132±2.8

C-peptide(nmol/liter) 1.06±0.13 0.84±0.08 0.87±0.13 Glucose(mmol/liter) 5.54±0.21 6.11±0.17 10.3±0.98*

Glycosylatedhemoglobin(%) 5.81±0.18 6.05±0.53

10.3±1.11

*_{P <0.00 1; t P<}

0.01

_{vs.controlsubjects.}

with IGT. Although basal glucose levels were slightly higher in

thosewith IGT compared with the nondiabeticcontrol

sub-jects, the differences were notsignificant. Thedifferences in the

fastinginsulin andC-peptideconcentrations were not

signifi-cant at the 5% level.Use oftheHomeostasis Model Assessment

(HOMA) method revealed nosignificant differences in degree

of insulin resistance between the three study groups (control,

8.0+1.4;

IGT,

5.2+0.7;

NIDDM,7.9±1.4;P >0.43).

Mean 24-h levels of plasma glucose, plasma C-peptide,

seruminsulin, andISR. Overall mean concentrations of

glu-cose,insulin, C-peptide,and the insulin secretory rates over the 24-h periodduring the constant glucoseinfusion studies are presented inTable II. In addition, the mean glucose levels and insulin secretory rates are shown forthe oscillatory studies. Insulin,C-peptidelevels, andinsulin secretion rates were

signif-icantly lower in the NIDDMpatientsthan in thenondiabetic

controls,and theglucose concentrationsintheNIDDM group

were significantly higher than in both the control and IGT

groups. The differences in glucose and insulin secretion rates between thepatientswith IGT and controls were not

signifi-cant. Theglucoselevels and insulinsecretionratesdidnot

de-pendonthe modeof glucose infusion. Fig. 1 depictsthemean

insulinsecretory rates andglucose levelsduringallstudies.The meanglucose levels were elevated andinsulin secretion rates

werereduced in thesubjects withNIDDMcompared to

con-trols, withvirtually no overlap between the two groups. Pa-tients with IGT tendedto havelowinsulin secretionrates in

relationtotheprevailing glucose concentration, although the

differences in secretion rate did not reach statistical

signifi-cance.

Number andamplitude ofoscillations. Representative pro-files ofglucose, insulin and ISRfromeachof the three study groupsduringthe constantandoscillatory infusionsare demon-strated inFigs.2-4. The number and absoluteamplitude ofthe

respectiveoscillations in eachexperimentalprotocol are shown in TableIIIandFig.5.The numberofglucose pulseswas

simi-TableII. Mean Values (±SEM)of Insulin, C-Peptide, Glucose, and InsulinSecretionduring Constant Glucose Infusion

andofGlucose and InsulinSecretionduring Oscillatory GlucoseInfusion

Control IGT NIDDM

Constantglucose (n=5) (n=4) (n=7)

Insulin(pmol/liter) 1174±145 591±101 397±99*

C-peptide(nmol/liter) 3.2±0.35 2.4±0.23 1.73±0.17*

Glucose(mmol/liter) 9.2±0.47 10.7±0.91 20.4±1.83t

Insulinsecretion

(pmol/m2permin) 426±48 310±26 229±23*

Oscillatoryglucose-144min (n=₅₎ (n=₃₎ (n=₆₎

Glucose(mmol/liter) 9.0±0.33 11.4±0.74 20.4±2.01 Insulin secretion

(pmol/m2permin) 416±31 345±19 242±26

Oscillatoryglucose-96min (n=5) (n=4) (n=5)

Glucose(mmol/liter) 9.4±0.52 10.3±0.54 19.1±1.91 Insulin secretion

(pmol/m2permin) 416±37 301±44 243±30

U

a ** _*

* * * * *

5 10 15 20

MEAN GLUCOSE LEVEL

25

30i

(mmol/L)

Figure1. Relationship betweenmeaninsulin

secretory

rates normal-izedby bodysurface

areaandmeanglucose

concentrationsduring

allglucoseinfusion studies. Eachmarking (control, +;IGT,.m,

NIDDM,*)

corre-spondstotheresultsof onestudy.

lar in the threegroupsexcept fora

significant

reduction

during

constant

glucose

infusion in the

subjects

with NTDDM

(P

<

_0.02).

The numberof

pulses

ofTSR didnotdiffer inanyof thethreegroupsinanyof the threeprotocols.

Two-way

analy-sis of variance for repeatedmeasures showed that the

ampli-tudeof

glucose

oscillationsincreased

during

the

oscillatory

in-fusion

studies,

the

changes

being

most marked with a slow

144-mmn

oscillatory period

(P<

_0.0001I).

Therewere no

signifi-cant differences in the

amplitude

of the

glucose

oscillations between the threegroups. However, differences in the

ampli-tude of the TSR oscillations were

highly significant

both be-tweenthegroups(P<

0.001I)

and betweenthe

glucose

infusion

protocols (P<

0.01I),

and thegroupdifferenceswererelatedto thedifferences in the infusion

protocols (P

<

0.002).

Tn the

controls, theincrease in

amplitude

of the

glucose

oscillations

brought

about

by

the

oscillatory glucose

infusion

protocols

was

thus

accompanied by

a

significant

increase in the

amplitude

of the oscillations in

TSR,

whereas in both the diabetic andTGT

groups,the

amplitude

ofTSRoscillations didnotincrease

signif-icantly

with the increase in

amplitude

of the

glucose

oscilla-tions

(Fig. 5).

Temporal

association between oscillations in

glucose

and ISR. Thetemporal association between oscillations in

glucose

andTSRwasevaluated

using

threeseparate

approaches:

pulse-4~-4,

by-pulse

analysis

of

concomitance,

cross-correlation

analysis,

and

spectral

analysis.

Each

approach

tothe

analysis

of the data

indicated a reduction in the normal

tight temporal coupling

between oscillations in glucose andTSR in the NIDDM and IGT

subjects although

the

significance

of the differences varied

depending

onthe

technique.

Pulse-by-pulse analysis

of

concomitance. The concomi-tancebetween individual pulses of

glucose

and TSR for each groupand

protocol

areshown inTable ITT. When the concomi-tance of

pulses

in TSRwith

glucose pulses

wasexamined with

repeated

measures two-way

analysis

of

variance,

therewas a

significant

difference between groups

(NIDDM

significantly

different from TGT andcontrol; P<

0.002)

and between

glu-coseinfusion

protocols

(P<

0.0002).

Similarly,

thepercentage

of

glucose pulses

thatwasconcomitant withanTSR

pulse

was

significantly

different between groups (NIDDM

significantly

different from control; P <

_0.03),

but not between

glucose

infusion

protocols.

Inorder to determine if these differences

wereconfirmed with other

analytical approaches,

cross-corre-lation and

spectral

analyses

were

applied.

Cross-correlation

analysis. Fig.

6 illustrates the coefficient of cross-correlation between

glucose

andTSRatdifferent

lags

for the threegroupsand the three

glucose

infusion

protocols.

Repeated

measurestwo-way

analysis

of variance showed that the maximalcoefficient of cross-correlation between

glucose

and TSR was different between the three groups

(TGT

and

NTDDM different fromcontrol;P<

0.0001I)

and between the three

glucose

infusion

protocols

(P <

0.002).

Similarly,

the maximalcoefficients of cross-correlation between

glucose

and

insulin weredifferent between thegroups

(TGT

andNTDDM

different fromcontrol; P<

0.001I)

and between the three

glu-coseinfusion

protocols

(P<

0.002).

Theresults forcorrelation

between TSR and insulin were

only

significant

between the

groups

(NTDDM

differentfromcontrol;P<

0.04).

Tn orderto

examine which of the three

glucose

infusion

protocols

most

clearly brought

outgroup

differences,

one-way

analysis

of

vari-ance was

performed

ondatafrom each

glucose

infusion

study

separately.

The 144-mmn

protocol

consistently

revealed the most

significant

differences. In addition to reduced overall

10 9

8-7 6

1600

14001-8200

200 W-\ \64

600

00 ~

4001 200~

1500

-1750-1 250

1000 1250

2500 2501J

0 240 480 720 960 1200 1440

TIME (min) 0 240 480TIME720(min)960 1200 1440

Figure2.Profiles of theglucoseinfusion rate, theglucoseandinsulinconcentrations,and theinsulinsecretoryratesinonerepresentativecontrol

subject.All three studiesperformedinthissubjectareshown:constantglucose infusion, left;slowoscillatory infusion, middle; rapid oscillatory infusion, right. Significant peaksareshownas arrowsin theglucoseand ISRprofiles,andshoulder(s)areshown in theglucose profileobtained

duringconstantglucoseinfusion.Afewinterruptionsoccurredthatwereallof short duration(<2min),andtheydidnothave any visible effectupontheglucoseprofiles.

700

-E-

600

-NN

E 500

-

NI-0

E ₄₀

(i) 300

-j~

200-+

+ .- a*

ui0200-zE

0-0-0

0 8

110

25001-a10001

500~

11

9

7]fJJVk\AJ

0 240 480 720 960 1200 1440

TIME (min)

400

- 200-0

300-°, o 200-1

2 \ 100]

zE _ E

o-151 W

-14]

8 ° 13]

, E 12

J

E

10

16-14 -12

-101

400 200

0

141

12

-10

-8-i

~~~~~~~~~~~~12001-.E10001 1000

Z

E ,51000

E

0.1

600 600J600

1250 1500

1500-I1000-10

01000

500 ... 500 ..,,.,,,, 500

0 240 480 720 960 1200 1440

TIME (min) 0 240 480TIME720(min)960 1200 1440

0 240 480 720 960 1200 1440 TIME (min)

Figure 3.Profiles ofthe glucoseinfusionrate, theglucose andinsulin concentrations,and theinsulinsecretoryratesin one representative IGT

patient.Allthreestudiesperformedinthispatientareshown:constantglucoseinfusion, left;slowoscillatory infusion,middle; rapid oscillatory

infusion,right.Afewinterruptionsoccurred that were allofshort duration (< 2 min), and they did not have any visible effect upon the glucose

profiles.

cross-correlation, theNIDDMgroup also demonstrated a

con-siderably increased lagat which the maximal coefficient was observed (see Fig. 6). Inotherwords, whiletheoverall

coeffi-cient ofcross-correlation between glucose and ISR was greatest

at alag ofzero min in the control and IGT groups for all

glucose infusion protocols, there was a lag in the NIDDM group which ranged from 15 to 35 min (peaks ofglucose occur-ring before peaksofISR) depending on the glucose infusion

protocol.Similarresults wereobtainedfor the cross-correlation betweenglucoseandinsulin.

Spectral analysis. Spectral analysisof theoscillatory

pro-files (Fig. 7)confirmedthe existenceofpeaks in the plasma glucosespectra in the three study groups at 96 and 144min, correspondingtotherespectiveperiods of the exogenous

infu-sion. Similar spectral peaksin insulin (not shown) and ISR

wereobserved in thenondiabeticcontrolsubjects.However the

Z 400 400

200. 200 \

0

0E

20 -W

191

8 -

18-, E

17-_E 16]

15

231 21 ]

19] 17]

15

A

\AA/V\/\

power of the insulin and ISR spectralpeaks was reduced in

boththe IGT andNIDDMgroups, this reductionbeingmost

marked in theNIDDMgroup. Duringthe96-minoscillatory

infusion the normalized powerofthe ISR spectrumfell from

29.1 in the controls to 13.6 in the subjects with IGT (P

<0.001 ) and 5.5 (P<0.001)in the subjectswith NIDDM.

Correspondingly,the ISR spectra basedonthe144-min oscilla-tory infusionsyielded respectivepowers of26.2, 5.9,and 3.0 in eachofthethree groups(P<0.001). Similarresultswere ob-tained for the insulin spectra.Noclear group differenceswere detected between the spectra from the constantglucose infu-sion protocol. Examinationof individualspectra also showed

that in a large

proportion

of the IGT (three of four) and

NIDDM (five of seven) patients,a discrepancy betweenthe dominantperiodof ISR andinsulincould be detected.In con-trast, noneofthe five nondiabetic controlsubjectsexhibited

400

/VV\/V\ 200

0

28-26 -24 -22

20-18

1200

1o00o 1 1100

E 1000

800 , 800 ₇₀₀

1000 1000

1500 ..5 0

0 240 480 720 960 120014 0 240 480 720 960 1200 1440 TIME (min) TIME(min)

0 240480 720 9601200l1440

TIME (min)

Figure 4. Profiles of the glucose infusion rate, theglucoseand insulinconcentrations, and the insulinsecretoryratesinone_{representative}

NIDDMpatient. All threestudiesperformedinthispatientareshown:constantglucoseinfusion, left;slowoscillatory infusion, middle; rapid

oscillatory infusion, right.Afewinterruptionsoccurred thatwereallof short duration(<2min),andtheydidnot_{have any}visibleeffectupon

Table III. Pulse Analysis ofthe Glucose and ISR Oscillations

Glucose ISR

% ofglucose % of ISR No.of Absolute pulses with No. of No. of Absolute pulses with Study Group pulses increment ISRpulses shoulders pulses increment glucosepulses

Constant glucose infusion Control(n=₅₎ 9.8±0.6 1.10±0.12 61.2±7.9 3.8±0.4 14.6±1.0 334±52 40.4±3.8 IGT (n=4) 8.3±0.9 1.18±0.30 61.3±9.5 3.5±1.5 13.5±1.0 260±68 39.1±9.9 NIDDM(n =7) 6.7±0.6 1.14±0.11 54.4±9.5 6.0±0.7 12.9±1.2 158±17 28.1±6.1

Oscillatory glucoseinfusion Control(n= 5) 10.2±0.2 3.43±0.26 70.5±4.5 - 11.0±0.5 566±91 66.4±2.8

(144min) IGT (n= 3) 10.0±0.0 3.04±0.23 63.3±16.7 14.3±0.9 248±30 46.0±13.2

NIDDM(n =6) 9.7±0.2 2.87±0.33 40.9±7.2 13.8±0.7 145±13 28.5±5.0

Oscillatoryglucoseinfusion Control(n= 5) 14.2±0.2 2.47±0.13 80.4±4.0 - 14.2±0.4 476±47 80.3±2.7

(96min) IGT (n=4) 14.3±0.3 2.07±0.20 61.3±9.5 13.0±1.2 292±76 66.1±4.5

NIDDM(n=5) 13.4±0.2 1.57±0.05 45.8±10.2 14.2±1.0 159±17 43.6±10.2

Results ofstatisticalanalyses are in the text.

anydiscrepancy betweeninsulinand ISR.Intheseindividuals,

the largest spectral peak was always observed at the

corre-sponding periodof the exogenousglucoseinfusion. Examples

of individual spectra illustrating these results are shown in Fig. 8.

changes to assume the period of exogenous pulsesofglucose duringexogenousoscillatory glucoseinfusions.Thus these

ul-tradian oscillations in insulin secretion were completely

en-trainable by exogenous glucose, implying that the inherent

Discussion

We havepreviously demonstrated thatin normal volunteers (9), ultradian oscillations of insulin secretionarepresent dur-ing constant intravenous glucose infusion and their period

CONTROL

E _0E

-~ ~ ~ ~ ~ ~ 5

IGT

N

C

.E

._

soo 500

200

100

CL

Figure 5. Relationships between * absoluteamplitudesin the three

different groups and the three

dif-ferent protocols.C,R,and S de-noteconstant,rapid oscillatory,

andslowoscillatory glucose

infu-sion, respectively. Inall groups, N administration ofoscillatory

glu-E cose infusion leads to an amplifi-* _{cation of the glucose oscillations} *, (lefithandbars),butonly in the o _{controlgroup isthis associated} with asignificant increase in the

0: absoluteamplitude of the ISR

oscillations(righthand bars).

CONSTANT INFUSION GLUCOSE LEADING ISR

-_CONTROL

---IGT

---NIDDM

RAPID OSCILLATORY INFUSION GLUCOSE LEADING ISR

CONTROL

---IGT ---NIDDM

)-.... .. .

-300 -200 -100 0 100 200 300 LAG (min)

SLOW OSCILLATORY INFUSION GLUCOSE LEADING ISR

.31.000

cr

0

Ir

C.) 0.00

U- 0.50 S2

7-o

-CONTROL ---IGT

----NIDDM

I-~~ ~ ~ ~ ~~ ~

-300-200 -100 0 1600 200 300 LAG (min)

Figure 6. Coefficient of

cross-correlation be-tweenglucoseand ISR inthe threestudy

groupsduringconstant

(top), rapid oscillatory (center),and slow

oscillatory (bottom) glucoseinfusion.

-E

E

0 n

nx E

E

E

E

0

0.

NIDDM

4- A

1- ~~~~~~~~~~~~-26

0

.p

CONSTANT INFUSION 96-MIN INFUSION 144-MIN INFUSION -J 0 F-z 0 0)

----GLUCOSE °70 _GLUCOSE ISR E 60- ISR

IE4 jX40 030 20. 20

---144192 240 288 0 48 96144192 240 28 ID(min) PERIOD(min)

Figure 7. Mean glucose(-- -)and ISR ( )spectraof the control subjects (top),IGTpatients (middle) and NIDDM patients (bot-tom). Error bars have been omitted for clarity. Left, middle, and right columnsshow the results from theconstant,rapid, and slow

oscilla-toryinfusionstudies, respectively.

properties of the insulin/glucose feedback loop playan

impor-tantrole inregulating and generating the oscillations. Thepresentstudywasdesignedtodetermine if specific dy-namic abnormalitiesare presentinthe feedback loop linking insulinsecretion and glucose in NIDDM duringconstantand oscillatory intravenous glucose infusion. Studies were per-formed in patients with overtNIDDM and matchedgroups

with impaired and normal glucose tolerance who received intra-venousglucose infusions eitherataconstantoroscillatoryrate

withaperiodicity of 96or 144min.In all threegroups, oscilla-tionsinglucose and ISRwereapparentduringconstantglucose infusion. In the groupwith normalglucose tolerance atight temporal couplingwasevident between the oscillations ofglu-coseand ISR. Incontrast,inpatients with IGT andNIDDM, therewasevidence ofuncoupling of this relationship between glucose and ISR andmany glucose independent oscillations wereapparent.Alterations in the ultradian oscillations in ISR have notpreviously been characterizedinsubjects with IGT. The abnormalrelationship between oscillations ofglucose and ISR in thisparticulargroup,which issimilartothat observed in theNIDDM group, suggests thatan uncouplingof the

tem-poral association between ultradian oscillationsinglucoseand

ISR isanearly manifestation of ,-cell dysfunctioninNIDDM. In thesestudies, evidence for lack of control by glucose of the ultradianoscillations ofISRwasdemonstratedusingthree different analytical approaches: (a) by analysis of concomi-tance of theindividual pulses ofglucose and ISR using the

programULTRA; (b) by cross-correlation analysis; and (c) by spectral analysis. Analysis of theconcomitance of the

individ-ualpulses demonstratedthateventhoughthe total numberof

70- 60-E

°

50-X

40-0 3c 30.

0~ 20-M 10

X)

C] z ID 70-.N E 60-° 50-X 40-LhJ 30 20- 10-LUJ en 0 I' I I I I II I I I I

48 96 144 19 PERIOD (m

Il

I I

48 96 144 19

PERIOD (mi

Figure 8. Individual

powerspectraof the datafromone

nondia-beticcontrolsubject (top),oneIGT patient

(center), andone 2 240 288 NIDDM patient

(bot-in) tom). Theuppertwo

panels correspondtothe

data showncenterof

---GLUCOSE Figs. 2 and3, while the

--INSULN _lower

panelisfrom a

ISR

diabetic subject

differ-entfrom theone

dis-played inFig. 4.Inthe controlsubject, glucose (-- ),insulin(---),

andISR ( ) all

ex-hibitadominant peak

2 240 288 at 144min(theperiod

in) of theglucoseinfusion).

Inthe IGT andNIDDM

patients, although

---GLUCOSE plasma glucose isclearly ---INSUUN entrainedat144min

ISR

and 96min,

respec-tively, this is neither the

casewith insulinnor

with ISR. Furthermore, thedominant period of insulin is different from the dominantperiod of ISRinboth the_IGT

i2 240 288

in) and NIDDMpatients.

glucose and ISR pulses in the 24-h profileswassimilar between thegroups, asignificant reduction in the proportion ofpulses inISR that wasconcomitant withpulses in glucose and vice versa wasobserved in NIDDM. Cross-correlation analysis re-vealed a reduction in the overall concomitance between the ultradian oscillations ofglucose, insulin, andISR, whichwas

significant for both the IGT and NIDDMgroups. The differ-ences weremostsignificant when the144-minoscillatory glu-coseinfusionprotocolwasused.In additiontoreduced coeffi-cients ofcross-correlation, this analytical approach also re-vealed that in the NIDDMgroupthelagatwhich the maximal correlationbetween oscillations ofglucoseand insulin and

be-tween oscillations ofglucoseandISRoccurredwasincreasedin theNIDDMgroup.Spectral analysisof the 24-hprofiles from theoscillatory glucoseinfusion studies confirmed the failure of

glucosetocontrol theperiodicityoftheISR oscillations.Inthe

control subjects, single coinciding peaks representingthe

pe-riod of infusionwereclearlyapparent inboth theglucoseand

ISR spectra. By contrast, in the subjects with NIDDM and

IGT, althoughaclearpeak representing the periodof infusion

was evident in theglucose spectra, the ISR profiles revealed eitherabroadspectrum withoutaclearlydominantperiodora dominantperioddifferent from theperiodofinfusion andwith

significantlyreducedpowerincomparisontothe controls.We have taken this dissociationbetween the dominantpeaksinthe spectra ofglucoseandISRtoreflectanunderlyingdefectinthe

insulin/glucose feedback mechanism. The results of the spec-tralanalysisgohand in hand with the results shown in Fig. 5, which illustrate thatamplification oftheglucose oscillations results inasimilar amplificationof the ISR oscillations in the

controlgroup,butnotin the IGT and NIDDMgroups.

Thedissociation between the glucose and ISR oscillations

duringtheoscillatoryinfusions in the IGTgroupin this study canthus be addedtothe list of abnormalities ind-cellfunction that have been observed inpatients studied in theearlystages ofNIDDM.Attenuated insulinresponsestooralglucosehave

also been described in subjects with IGT who later develop

NIDDM(29-31 ) and innormoglycemicco-twins of patients

withNIDDM(32),agroupwithaveryhighrisk ofdeveloping

NIDDM. O'Rahilly and co-workers (16) reported alterations in therapid oscillations in insulin in first-degreerelatives of

patients with NIDDM, and Gerich and co-workers (33) ob-serveddelayed attenuatedresponses tooralglucoseinpatients

with mild IGT. Therelationship between theseabnormalities

and thealteredultradian oscillationsin thepresentstudyisnot knownnorhas themostsensitivetestofearly d-cell

dysfunc-tion beendefined.Nevertheless the data from thecurrentstudy

taken inconjunction withthestudies quotedaboveillustrate that with the appropriate experimental approaches, intrinsic defects in the (3 cellmaybedemonstrable inpatientswith

im-paired glucose tolerance but normalglycosylatedhemoglobin

values.

Theprecisemechanismsresponsible for the loss of

entrain-mentin the IGT and NIDDMgroups are notknown. However

amodel of the interactions betweenglucose,insulinsecretion,

andaction recentlypublishedbyour group(12)mayprovide

aninitial frameworkwithwhichtoaddress thesequestions. In

this model,themaintenance oftheregularpattern of oscilla-torysecretionobserved in normalsubjectswasdependenton a numberoffactors, themostimportant ofthesebeingthe sim-plesigmoidal relationshipbetweenglucose and insulin

secre-tion,theexistence ofatime delay in the order of 25-50min betweentheincrease ininsulinsecretion and thesuppressionof

hepaticglucoseproduction,and thefact that insulinmust dif-fuse intoaremoteperipheralcompartmentbefore stimulating peripheralglucose utilization. Alterations in thedose-response

relationship between glucose and insulin secretion (34-38),

hepatic resistance to the action of insulin (39-43), or

pro-longedtime lags in the peripherydueto diminishedglucose

disposal (42)could allcontributetothealterations in the dy-namic interactions betweeninsulin secretionandglucose

ob-served in the IGTand NIDDMgroupsin this study. To

under-standfullythe lackof entrainment observed in thesegroups,

however, itmay be necessary to modifythe model toinclude

factors other than the insulin/glucose feedback mechanism. Thepresent study alsohighlightsthe systematic change in

(-cell

secretoryresponses asobesity progresses to IGT and ulti-mately NIDDM.In Table

TI,

themean 24-h insulin and

C-pep-tideconcentrationsas well as themean insulinsecretoryrates in the IGT group are intermediate between those observed in

theobeseandNIDDM groups. The data in Table III

demon-strate aprogressive decline in the absolute amplitude of the glucose and ISRpulses with thetransition from obesity to IGT toNIDDM.Thesimilarityin basal insulin and C-peptide levels betweentheNIDDMandIGT groups in Table I (both ofwhich were lower than the correspondingconcentrations in the obese

[nondiabetic] subjects)

raises the

_possibility

that

(3-cell

secre-toryfunction decreases beforethe

_development

ofsevere

hyper-glycemia in NIDDM. This is further illustrated in Fig. 1,

de-picting the relationship between mean glucose concentration

andmean ratesof insulin secretionthroughout the 24-h study where theinsulin secretoryresponsesinmanyof the IGT and NIDDMsubjectswere notdifferent despite the marked differ-encesinglucose levels. In thisregard, it is also ofinterest that all three methods usedtoanalyze the interaction between glucose

and insulin secretion demonstrated abnormalities in the

pa-tients with IGT thatwerequalitatively similar but lesssevere than the defects identified in the subjects with NIDDM. It is thereforepossible that theuncoupling of the ISR and glucose oscillations isakey factor in thedevelopmentofglucose intoler-anceinsusceptibleindividuals.

The lower association observed by both cross-correlation andspectralanalysisbetweenthepatternsof insulin and ISR oscillations in the IGT and NIDDMgroupsthan in the control groupisnotreadily explained in view of theequimolar secre-tion of insulin andC-peptidefrom the(-cell,which has been well documented(44-46).In contrast tonondiabetic subjects, NIDDM patients have elevated levels ofproinsulin and of proinsulin conversion intermediates (split proinsulins),

sug-gestingthat the(-cellinthis instance releases immature secre-torygranules into the circulation(47-52). Since conventional insulin radioimmunoassays cross-react with proinsulin, it is also possible that the dissociation observed between insulin

andISR in theNIDDMand IGTgroups canbeattributed in

part tooscillating proportions ofproinsulin secretion in

rela-tiontothesecretionofinsulin. Proinsulin concentrationswere

not,however, measured in theseexperiments.

Although(-cellentrainmentwaspreserved inobesity, the presentstudy raises thepossibilitythat obesesubjectsmayhave subtle abnormalities in (3-cell function. Previous studies in obeseindividualsquantitatinginsulin secretionratesand evalu-ating pulsatility following meals have demonstrated (3-cell se-cretory patterns to be similar to those observed in normal weight subjects(7,53). Inthe present study,detailed analysis

of the proportion ofISR pulses thatwereconcomitant with

pulses in glucoseyieldedconcomitance ratios of 66%and80% inthe96 min and 144 minoscillatory infusions, respectively. This contrasts with concomitance ratios exceeding 90% that werepreviously reportedforyounger, normalweight subjects

(9)studied under identicalexperimentalprotocols. The obese

subjectsin this studywerehoweverolder and presumablymore insulin resistant than the normalweight subjects studied

previ-ously. Additionalstudiesarecurrently underway todetermine

if thesedifferences could bedue to the different agesof the

subjects, and/or differences in weight and degree of insulin resistance. In light ofthe importance ofthe insulin/glucose feedback mechanism incontrollingtheoscillations in nondia-beticsubjects,thenotion that insulin resistancemay,together with intrinsic defectsin the( cell, contribute to alterations in ultradian oscillations isanintriguingpossibility thatshould be

systematicallyaddressed infuture studies.

Inconclusion,thepresent study demonstrates that the

insu-lin/glucose feedback mechanism, which plays an important

role intheregulation and generation oftheultradian oscilla-tions innondiabetic subjects, is disrupted inNIDDMand even

in conditionswhere glucosetolerance isonly minimally

im-paired. Theresultantuncoupling ofthe normaltight

dis-crepancybetweenthedominantperiodofthe spectra ofinsulin secretion and glucose. Similar changes are seen using cross-correlation analysis and pulse analysis. The dissociation be-tweentheoscillations becomesmoreseverewith the

progres-sion fromimpaired glucose tolerance to NIDDM. Whether the

abnormalsecretoryoscillationsplay apathogeneticroleinthe progression to overtdiabetesor whetherthey are merely reflec-tive of underlying defects in the : cell and peripheral tissues

remainstobedetermined.

Acknowledgments

Theauthors wishtothank the nursing staff of the ClinicalResearch

Center for expertcare ofthe subjects whoparticipated in the study. The authors also greatly appreciate the advice given by Theodore Karrison regarding statistical analyses and thetechnical contributions of Paul Rue and MarshaJackson.

Thiswork wassupported byNational Institutes of Health grants DK-31842, DK-13941, DK-20595, DK-26678, (Diabetes Research and Training Center, Clinical Nutrition Research Unit), and

RR-00055 (Clinical Research Center). Dr. O'Meara (390343) and Dr. Sturis (392574) wererecipients of Post Doctoral Fellowships from the Juvenile DiabetesFoundationInternational.

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