Biphasic Patterns of Peripheral Insulin

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1 Biphasic Patterns f Peripheral Insulin and Glucse Levels After Lunch in Nrmal Subjects DAVID C ROBBINS, MD, JONATHAN JASPAN, MD, FRCP, BARBARA VASQUEZ, PhD, AND EVE VAN CAUTER, PhD The dynamic relatinship f glucse cncentratins and insulin secretin during the pstabsrptive state is cmplex and has been assciated with a variety f cyclic rhythms. T study the pattern f insulin and glucse respnse immediately after a mixed meal, we cllected bld every 15 min frm 0730 t 1645 h frm eight nrmal resting men (age 24.9 ±2.1 yr). They tk identically cnstituted mixed meals at 0800 and 45 h. Cncentratins f glucse and insulin were measured in samples taken thrughut the study, whereas levels f C-peptide, glucagn, and a-nh 2 were determined in samples taken after 30 h nly. Cmputer-assisted analysis was used t identify significant increments and declines in cncentratins and t quantify the cincidence f peaks f glucse, C-peptide, glucagn, and a-nh 2 with peaks f insulin. Cefficients f crrelatin between data pints were calculated fr each individual. The patterns f bld insulin and glucse after breakfast and lunch were different. After breakfast, a single simultaneus peak in insulin and glucse ccurred 60 min after starting the meal. In cntrast, the pattern after lunch in seven f the eight subjects was clearly biphasic. There were secndary, significant cincident peaks in serum insulin, glucse, and C-peptide ccurring h after the meal was served. The secndary peak appeared unrelated t the late absrptin f prtein because it was nt assciated with cnsistent changes in serum a-nh 2 cncentratin. Erratic variatins characterized the pstlunch pattern f glucagn levels, excluding a rle fr this cunterregulatry hrmne in the cntrl f the biphasic insulin and glucse respnse. Of the measured substances, nly glucse, insulin, and C-peptide demnstrated significant crss-crrelatins. These data indicate that in this well-defined grup f healthy vlunteers, the respnse f plasma glucse, serum insulin, and C-peptide t lunch is biphasic. Diabetes Care 10:293-99, 1987 nrmal subjects. Relatively late and large secndary cin- The dynamics f insulin secretin and glucse hmestasis during basal and pststimulatry cnditins are cmplex and incmpletely understd, Variatins in peripheral insulin cncentratins are nt slely dependent n the actin f varius pancreatic (3- cell secretaggues (1-7). During the basal state, it has been shwn in humans (3-5,7), mnkeys (2,6), and dgs (5) that insulin levels fluctuate rhythmically with a perid f 9 13 min. Cmplex patterns f scillatins in serum insulin and plasma glucse cncentratins have als been fund after the ingestin f ral glucse (8). Kraegenetal. (9), fr example, measured insulin and glucse levels in bld samples taken every 10 min during a 50-g ral glucse tlerance test in 12 cident peaks in insulin and glucse ccurred amng all sub- jects. In a preliminary study designed t establish reference val- ues fr serum insulin and plasma glucse levels amng am- bulatry nrmal subjects cnsuming standard mixed meals, we bserved secndary peaks in these cmpunds, especially after lunch (10). In this study we have statistically evaluated these secndary peaks t determine whether they crrelate with changes in levels f related substrates r f glucagn. Our data demnstrate that a secndary, synchrnus incre- ment in serum insulin and plasma glucse levels, unrelated t direct nutrient absrptin, is a cnsistent and nrmal respnse after the mixed nn meal. DIABETES CARE, VOL. 10 NO. 3, MAY-JUNE

2 BIPHASIC PATTERNS OF INSULIN AND GLUCOSE/D. C. ROBBINS AND ASSOCIATES MATERIALS AND METHODS Subjects. Eight nrmal-weight men, aged 24.9 ± 2.1 yr (mean ± SD) were studied (). They were free f acute r chrnic disease and had taken n medicatin fr 3 wk befre the study. Inclusin criteria included willingness t eat a standard American Diabetes Assciatin diet; absence f abnrmalities n rutine hemgram, urinalysis, r liver and kidney functin tests; and n first-degree relative with a diagnsis f diabetes mellitus. Subjects were instructed t ingest a balanced diet that included 300 g/day carbhydrate fr at least 3 days befre entry. The mean ± SD weight f the grup as a percentage f the ideal bdy weight was ± 2.0% (). Subjects were admitted t the Clinical Research Center (CRC) after btaining infrmed cnsent. They were asked t refrain frm vigrus exercise fr 72 h befre entry. Fd was limited t what was served t them at the CRC. The cmpsitin f the meals was accrding t the recmmendatins f the ADA (60% CHO, 20% fat, 20% prtein, g/day crude dietary fiber), and calries fr each subject were based n 1.2 times the basal energy requirement (12). Meals were served at 0800, 45, and 1645 h, and there was a snack at 2000 h. The distributin f calries amng the meals and snack was 25, 20, 40, and 15%, respectively. The subjects finished the entire meal within 30 min after it was served. N testing was perfrmed n study day 1 (admissin). On the 2nd day, a 75-g ral glucse tlerance test was perfrmed after an vernight fast. After sampling and 3 h after the ral glucse, the subject was given the rutine breakfast, and the meal plan was resumed as described abve. Plasma glucse cncentratins befre and during the test were nrmal fr each subject accrding t the criteria f the Natinal Diabetes Study Grup (13). N further testing tk place n study day 3, which was intended t ensure unifrm dietary preparatin and regulatin f activity fr the test perid (day 4). On day 4 at 0630 h, an intravenus line was placed in a frearm vein t allw frequent bld withdrawal. The line was kept patent by the slw infusin f nrmal saline. The diet plan n this day nly was changed t ensure that the subjects received identically cnstituted meals fr breakfast and lunch. Thus, the nly difference between the meals was the time f ingestin. The meals, served at 0800 and 45 h, cnsisted f 240 g f lw-fat milk, 82 g raw range sectins (withut skin), 60 g grund beef (after cking), 50 g whle wheat bread, 10 g crn il margarine, 100 g tmat, 10 g lettuce, 67 g apple slices (with skin), and 69 g banana slices. The subjects were bserved during meal times t ensure that the entire meal was eaten within min after serving. The test perid lasted until 1645 h. Subjects were in the recumbent r sitting psitin in bed, and activities were limited t reading, watching televisin, r quiet cnversatin thrughut the test perid. Bld was withdrawn every 15 min thrughut the study. Analytical methds. Plasma glucse was analyzed by the autmated glucse xidase methd. Serum insulin (14) and plasma C-peptide (15) were measured by radiimmunassay. Plasma glucagn was measured by radiimmunassay with the 30K antiserum f Unger et al. (16). Plasma a-nh 2 cncentratins were measured phtmetrically (17). The intraassay cefficients f variatin (s) fr glucse, insulin, C-peptide, glucagn, and a-nh 2 were 2, 4.5, 4, 4.9, and 5%, respectively. The crrespnding interassay s were 3, 7, 9.2, 9, and 8%, respectively. Statistical analyses. Fr each f the measured plasma cnstituents and fr each individual, the mean, SD, and f all data pints were calculated. Cmputer-assisted analysis f the data was used t identify significant peaks by previusly described methds (18-24). The significance f the peaks was determined in terms f relative increments and declines. An increase r decrease in cncentratin was cnsidered significant if it exceeded, in relative terms, twice the intraassay A peak was cnsidered significant if bth its increment and subsequent decline were significant. Every peak was characterized by the time f ccurrence and the height f its maximum. Cincidence f an insulin peak with a peak f anther variable was defined as ccurrence f the tw peaks at the same sampling time ±15 min. TABLE 1 Means, SDs, and cefficients f variatin (s) Insulin (n = 38) Glucse (n = = 38) C-peptide (n = 22) a-nh; (n = 22) Glucagn (n = 22) Subject (M-U/ml) (mg/dl) (pm/dl) (mg/dl) Mean ±SD (pg/ml) Grup mean 32.2 ± ± ± ± ± ± ± ± ± ± 102 ± ± ± ± ± ±9 101 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±5 45 dt dt dt dt 7 42 dt 9 72 dt it dt ±6 294 DIABETES CARE, VOL. 10 NO. 3, MAY-JUNE 1987

3 BIPHASIC PATTERNS OF INSULIN AND GLUCOSE/D. C. ROBBINS AND ASSOCIATES Cefficients f crrelatin between data pints were calculated fr all pssible pairs f measured bld cnstituents and fr every individual. Fr each prfile, the area under the pstmeal glucse and insulin curve was estimated as the sum f glucse and insulin levels during the 3-h perid beginning at either 0800 r 45 h. All statistical tests were tw tailed. The maximum prbability level fr significance was.05. Grup results were expressed as means ± SD. The methds f pulse identificatin and statistical evaluatin used in this study have been cmpared with ther published methds f pulse identificatin (25). A detailed descriptin f the pulse analysis methd is prvided by Van Cauter (26). Amng the salient features f the pulse analysis methdlgy used are the fllwing. 1) It is very sensitive because it uses a small threshld (2 s rather than 3 s used in several ther methds). 2) It des nt prduce an unacceptably high rate f false psitives because the criteria f significance f a peak are applied t bth the ascending and declining limb. 3) There is n limit t the number f data pints that can be included in an ascending r declining limb s that slwly rising r falling levels are adequately evaluated. RESULTS Table 1 lists the mean, SD, and fr each subject and prfile f measured hrmne r substrate. The s were highest fr insulin, reflecting sharp peaks ver relatively lw basal levels. The variability f C-peptide was cnsiderably less than that f insulin (39 ± 7 vs. 70 ± 9%, respectively, P <.01). The cncentratins f glucse, glucagn, and a- NH 2 were mre stable, with average s f ^15%. The prfiles f plasma glucse and serum insulin in the entire grup f subjects are shwn in Fig. 1. The principal peaks immediately after the tw meals were clearly defined and ccurred within 1.45 h after starting the meal. During the mrning hurs, ne r mre additinal glucse and insulin peaks, which were asynchrnus and f cnsiderably lesser magnitude, were seen. After lunch, secndary peaks f larger magnitude than thse fund during the mrning hurs were bserved between 1300 and 1430 h. In the eight subjects, there were 41 significant glucse peaks and 40 significant insulin peaks. Thirty-tw f these were cincident (i.e., a cncmitance rati f 78% fr glucse vs. insulin lo lo r IO i i IO O eu i * IO IO IO CO IO IO IO IO in IO t IO IO c O IO IO 9 CO nr TIME OF DAY (hurs) FIG. 1. Prfiles f plasma glucse and serum insulin in each f 8 subjects. Subject numbers crrespnd t thse in Table 1. Meal times are represented by shaded areas. Asterisks indicate significant peaks as defined in text. Nte differing scales. In subject 3, asterisk befre lunch dentes significance f each f 3 pints befre lunch as designated by curved line. Biphasic respnse t lunch is evident in all but 1 subject (n. 2). DIABETES CARE, VOL. 10 NO. 3, MAY-JUNE

4 BIPHAS1C PATTERNS OF INSULIN AND GLUCOSE/D. C. ROBBINS AND ASSOCIATES i i I I I i i i i i i i i i i i i i n i i i i i O O 1 1ZU 90 TT B D " Mil n 00 CO CO Ul O) 00 CD CO Time f Day (Hurs) Ol FIG. 2. Grup mean (± SD) acrss individuals fr plasma glucse (A), serum insulin (B), plasma C-peptide (C), glucagn (D), and serum a-nh 2 (E)» Meal times are represented by shaded areas. and f 80% fr insulin vs. glucse). Of the 9 glucse peaks that did nt cincide with an insulin peak, 4 were cincident with an insulin "shulder," and 1 was cincident with a nnsignificant insulin peak. Thus, a crrespndence between a glucse peak and a cincident peak r shulder f insulin was bserved in 90% f the cases. Similarly, f the 8 insulin peaks that did nt cincide with a glucse peak, 5 were cincident with a glucse shulder, suggesting a crrespndence between insulin and glucse changes in 37 f 40 (92.5%) cases. Amng the 32 cincident and significant glucse and insulin peaks, the maxima cincided exactly in 21 f 32 (66%) cases, the glucse maximum preceded the insulin maximum by 15 min in 7 f 32 (22%) cases, and the insulin maximum preceded the glucse maximum in 4 f 32 (12%) cases. Thus, we were unable t shw a lag r lead relatinship between insulin and glucse changes, which appear t be cincident. The mean prfiles acrss individuals f plasma glucse, serum insulin, plasma C-peptide, plasma glucagn, and plasma ct-nh 2 cncentratins are shwn in Fig. 2. In all subjects the plasma glucse cncentratins shwed a detectable increment in the first sample drawn after starting each meal (Fig. 2A), with the highest values ccurring 60 min after the beginning f breakfast. The glucse respnse t lunch (mean peak value 133 ± 13 mg/dl) was nt significantly different frm that after breakfast (mean peak value 137 ± 20 mg/dl, P >.10). The areas under the pstmeal curves were als similar (mean area ± 99.5 after lunch vs ± 1.3 after breakfast, P >.10), whereas the pattern f changes differed between the tw meals. After lunch, in additin t the peak ccurring 60 min after the meal, anther peak ccurred between 1330 and 1400 h, i.e., 296 DIABETES CARE, VOL. 10 NO. 3, MAY-JUNE 1987

5 BIPHASIC PATTERNS OF INSULIN AND GLUCOSE/D. C. ROBBINS AND ASSOCIATES h after the meal was served. All but ne subject exhibited this biphasic respnse t lunch. In cntrast, after breakfast, glucse levels after the majr pstmeal elevatin were scattered thrughut the perid f 0945 t 30 h and did nt result in a cnsistent biphasic respnse. Figure 2B shws the crrespnding average pattern in serum insulin cncentratins. The similarity with the pattern f glucse levels was evident. All subjects had a majr pstmeal peak after breakfast and after lunch. After lunch, a secndary significant insulin rise f smaller magnitude was bserved 1-2 h after the majr pstmeal peak in all subjects. In cntrast, after breakfast, secndary peaks were fund in nly six subjects and ccurred spradically in the 0945-t- 30 h perid withut apparent interindividual synchrnizatin and withut cnsistent assciatin with a cincident glucse peak. In cntrast t glucse, the initial insulin respnse after lunch was significantly less than after the identical meal taken at breakfast (mean peak value 60.5 ± 19.5 after lunch vs. 82 ± 26 jxu/ml after breakfast, P <.01). Similarly, the integrated respnse ver the 3-h perid after meals, as estimated by the area under the curve, was als larger after breakfast (514.8 ± 108.3) than after lunch (397.0 ± 63.5, P <.01). The calculatin f the ratis f insulin peak t glucse peak after breakfast (0.68 ± 0.20) and after lunch (0.45 ± 0.15) further indicated a difference in the respnse f insulin t a given glucse lad at different times f the day (P <.01). The pattern f C-peptide levels paralleled thse f insulin and glucse (Fig. 2C), suggesting that the biphasic respnse f insulin t lunch was due t increased p-cell secretin rather than t altered clearance. The pssibility that the secndary rise in plasma glucse might have ccurred in respnse t the effects f an insulin cunterregulatry hrmne was als investigated. Measurements f plasma glucagn, perfrmed n samples drawn between 30 and 1645 h, shwed randm, erratic variatins f plasma glucagn in all subjects thrughut the afternn. N cnsistent pstmeal pattern emerged (Fig. 2D), excluding a rle fr glucagn in the cntrl f the biphasic pstlunch glucse and insulin respnses. The secndary rise in pstlunch glucse pattern culd als have been related t delayed absrptin f a prtin f the meal such as prtein. We therefre measured the cncentratin f a-nh 2 during the perid f 30 t 1645 h. All subjects had a significant rise in the cncentratin f a-nh 2 with a peak between 1215 and 1230 h, clearly attributable t nutrient absrptin. The rest f the prfile reflected randm fluctuatins in cncentratin, thereby excluding the pssibility that a delayed absrptin f a cmpnent f the meal accunted fr the secndary glucse peak. On average, the fluctuatins f glucse, insulin, and C- peptide were highly crrelated. Thus, the cefficients f crrelatin between insulin and glucse, C-peptide and glucse, and insulin and C-peptide were.78 ±.18,.79 ±.08, and.88 ±.05, respectively (all significant at P <.001). In cntrast, glucagn and a-nh 2 did nt crrelate with glucse, insulin, r C-peptide. DISCUSSION Our bservatins shw that changes in insulin and glucse after a mixed meal are cmplex. After lunch, in additin t the expected immediate pstmeal peak, secndary statistically significant cincident peaks in insulin and glucse cncentratins ccurred in all but ne f the subjects. In cntrast with the incnsistent insulin and glucse elevatins bserved after the early pstbreakfast peak, the secndary pstlunch peaks were f greater magnitude and ccurred predictably h after the meal was served. These biphasic pstlunch patterns may reflect a phenmenn similar t that detected by Kraegen et al. (9) during a 50-g ral glucse tlerance test administered t previusly fasting nrmal subjects. In this earlier study, secndary and even tertiary cincident peaks in bld insulin and glucse levels appeared during a similar bservatin perid. The aim f this study was t dcument and analyze mvements in metablic hrmne and substrate cncentratins under carefully cntrlled cnditins in prperly prepared nrmal subjects, a prerequisite t further experimentatin designed t further evaluate and analyze these phenmena. Sme f ur data readily rule ut certain pssible explanatins fr the secndary pstprandial peaks in insulin and glucse. Fr example, the fact that a secndary peak in C-peptide cncentratin was cincident with that f insulin implicated increased (3-cell secretin rather than decreased peripheral insulin clearance (27). Analysis f glucagn prfiles did nt suggest that glucse and insulin fluctuatins were caused by r related t glucagn changes. Furthermre, the secndary insulin peaks did nt seem t be related t asynchrnus absrptin and appearance f glucse and a-nh 2, bth f which are f$-cell secretaggues. Althugh ur data indicate that biphasic r multiphasic patterns in insulin and glucse cncentratins shuld be cnsidered a nrmal respnse t lunch and are nt restricted t the respnse t ral glucse, further investigatin is required t determine whether such patterns als ccur after meals ther than lunch. Between 1.5 and 2.5 h after meals were served, there were significant and cincident peaks in insulin and glucse in ne f eight subjects after breakfast but in seven subjects after lunch. Thus, it shuld be nted that patterns f insulin and glucse qualitatively cmpatible with the hypthesis f a biphasic respnse t breakfast were ccasinally bserved in ur study. Such patterns resulted in mean insulin and glucse curves shwing asymmetric early pstbreakfast peaks. These early peaks, cmpared with thse after lunch, had a brader declining rather than ascending prtin. Hwever, when secndary pstbreakfast peaks were bserved, their timing was incnsistent, and their magnitudes were ften near r belw ur criteria fr significance. Nevertheless, the pssibility that a biphasic respnse after breakfast similar t that bserved after lunch but f lesser magnitude may have ccurred cannt be excluded. It must, hwever, be emphasized that we studied a rela- D1ABETES CARE, VOL. 10 NO. 3, MAY-JUNE

6 BIPHAS1C PATTERNS OF INSULIN AND GLUCOSE/D. C. ROBBINS AND ASSOCIATES tively hmgeneus grup f yung vlunteers. Further studies are necessary t evaluate the extent t which this biphasic respnse is a nrmal phenmenn in ther grups, e.g., wmen and bese but nrmglycemic subjects, and whether disturbances f this pattern are present in abnrmal states, particularly diabetes. The fact that a relatively straightfrward bservatin such as the biphasic respnse t lunch described here has nt been previusly reprted is prbably due t inadequate sampling frequencies and lack f detailed data analysis. Fr example, in sme reprts describing nrmal insulin and glucse levels thrughut the day, bld sampling was perfrmed at hurly r half-hurly intervals. Because the secndary glucse and insulin pstlunch peaks ccurred within ~15 min, such data were prbably t widely spaced t bserve them. Furthermre, the practice f averaging data frm a grup f subjects withut prir analysis f each individual prfile may bscure secndary peaks because their times f ccurrence vary slightly amng subjects. This may have been the case in the study by Rizza et al. (28), wh shwed a relatively large shulder rather than a distinct secndary peak after lunch. Thus, apprpriate methds must be applied t differentiate significant peaks frm methdlgic variatin and randm, nncincident changes in hrmne and substrate cncentratins (22). On the ther hand, althugh the mrning and nn meals given in this study were identical, there were ther differences between the meals that may have effected the subsequent respnse. First, the time elapsed since the previus meal was 12 h fr breakfast but nly 4 h fr lunch. Thus, the shrter perid f fasting befre lunch may be accmpanied by, fr example, relatively increased hepatic and muscle glycgen stres. It is pssible that the glycgen-replete liver either takes up less r prduces mre glucse after lunch than after breakfast and thereby cntributes t r causes the fluxes in bld glucse. Secnd, there are imprtant differences in the hrmnal milieu between early and late mrning hurs (29). Fr example, the cncentratins f several insulin cunterregulatry hrmnes, e.g., crtisl, are greater in the early mrning, which may cause r cntribute t relative insulin resistance (30-32). Despite ur inability t demnstrate asynchrnus absrptin f carbhydrate and prtein, it is entirely pssible that at least glucse is absrbed frm the gut in a biphasic r multiphasic manner. This imprtant pssibility shuld be differentiated frm changes in peripheral glucse disappearance r hepatic glucse prductin. Cmplex kinetic studies emplying the ingestin and intravenus infusin f labeled glucse tracer will be necessary t distinguish amng these intriguing pssibilities. Finally, because this investigatin was nt cntinued beynd 1645 h, we cannt dismiss the pssibilities that there may be additinal peaks after meals, that this phenmenn might be present after the evening meal, and that meals might prvke a dampened scillatry insulin and glucse respnse pattern. ACKNOWLEDGMENTS: We gratefully acknwledge the skilled assistance f Nancy Perrine and Nreen Bssi in the preparatin f the manuscript and the technical assistance f Patricia Mead. The studies were perfrmed at the Clinical Research Center at the University f Vermnt (RR-109). This study was supprted in part by NIH Grants AM , AM-25913, and AM and by an Actins Cncertees grant frm the Belgian Ministry f Scientific Plicy. Frm the Department f Medicine, Metablic Unit, University f Vermnt, Burlingtn, Vermnt, and the Natinal Institutes f Health, Phenix, Arizna (D.C.R., B. 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