1 Department of Endocrinology and Metabolism M, Aarhus University Hospital, 8000 Aarhus, Denmark; 2 Guilford, Connecticut 06437; 3 Novo Nordisk, 2880 Bagsvaerd, Denmark; and 4 Department of Medicine and National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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Insulin is largely secreted as serial
secretory bursts superimposed on basal release, insulin secretion is
regulated through changes of pulse mass and frequency, and the insulin
release pattern affects insulin action. Coordinate insulin release is
preserved in the isolated perfused pancreas, suggesting intrapancreatic coordination. However, occurrence of glucose concentration oscillations may influence the process in vivo, as it does for ultradian
oscillations. To determine if rapid pulsatile insulin
release may be induced by minimal glucose infusions and to define the
necessary glucose quantity, we studied six healthy individuals during
brief repetitive glucose infusions of 6 and 2 mg · kg1 · min
1
for 1 min every10 min. The higher dose completely synchronized pulsatile insulin release at modest plasma glucose changes (~0.3 mM = ~5%), with large (~100%) amplitude insulin pulses at every single
glucose induction (n = 54) at a lag time of 2 min (P < 0.05), compared with small (10%) and rare (n = 3)
uninduced insulin excursions. The smaller glucose dose induced insulin
pulses at lower significance levels and with considerable breakthrough
insulin release. Periodicity shift from either 7- to 12-min or from 12- to 7-min intervals between consecutive glucose (6 mg · kg
1 · min
1)
infusions in six volunteers revealed rapid frequency changes. The
orderliness of insulin release as estimated by approximate entropy
(1.459 ± 0.009 vs. 1.549 ± 0.027, P = 0.016) was
significantly improved by glucose pulse induction (n = 6; 6 mg · kg
1 · min
1)
compared with unstimulated insulin profiles (n = 7). We
conclude that rapid in vivo oscillations in glucose may be an important regulator of pulsatile insulin secretion in humans and that the use of
an intermittent pulsed glucose induction to evoke defined and recurrent
insulin secretory signals may be a useful tool to unveil more subtle
defects in
-cell glucose sensitivity.
C-peptide; oscillations; fasting; -cell
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INTRODUCTION |
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INSULIN IS SECRETED in a distinctly pulsatile manner
(6), which gives rise to detectable high-frequency oscillations in insulin concentrations in the peripheral circulation. These
high-frequency events are caused by coordinated interislet secretory
activity, at an interval of 5-15 min per pulse (6, 11, 23). The
contribution of insulin secretory bursts to overall insulin secretion
was recently quantified in a canine model by direct sampling across the
pancreas (23) and in humans by employing high-frequency peripheral
blood sampling, a highly specific insulin assay, and validated
deconvolution analysis (25). In both species, the contribution of
pulsatile (to total) insulin secretion postabsorptively is at least
70-75%. Physiologically, the mechanisms regulating changes in
overall insulin secretion after islet-cell stimulation (22, 26) or inhibition (20, 24) primarily embody modulation of the pulsatile component of insulin secretion via changes in the mass and/or frequency
of insulin secretory bursts. It appears that the pulsatile secretory
pattern is important for overall -cell performance, in view of
impaired insulin pulsatility in type II diabetes (10) and
glucose-intolerant first degree relatives of type II diabetic patients
(16), and in view of defective release processing in glucose-tolerant
first degree relatives of type II diabetic patients (28). The
importance of pulsatile insulin release is further underscored by the
reported enhanced action of infused insulin on muscle (14), adipose
(27), and liver (9) tissues, when the hormone is delivered in a
pulsatile rather than constant manner. Accordingly, the specifically
pulsatile mode of insulin release has dual implications both for cell
biology and for insulin action(s) on target organs.
The mechanisms underlying the coordination of insulin release into short-lived and discrete secretory bursts have yet to be established. However, preserved pulsatility of insulin release from the isolated perfused pancreas (30) strongly indicates an intrapancreatic coordinating mechanism, likely an intrapancreatic neuronal pacemaker. This is supported by studies with nerve or ion-channel blockers showing a periodicity shift (e.g., after tetrodotoxin was administered; Ref. 29) and by experiments showing that intrahepatic transplanted (dispersed) islets establish a pulsatile release pattern simultaneously with (re)innervation (21). In contrast, studies examining in vivo vs. isolated perfused pancreas insulin pulsatility conclude that the in vivo pulsatile secretion patterns are more pronounced (5, 13). Therefore, further regulation through circulating substrates (or the central nervous system) may be important for in vivo pulsatile insulin secretion. Glucose concentrations are known to oscillate (6, 7, 11), and because this substrate is a major regulator of insulin release, a classical feedback regulation is plausible. Indeed, studies on the slower ultradian (approximately sesquihoral) insulin pulses show that this pattern of pulsatility may be entrained by glucose infusions (32). Furthermore, an in vitro study with a similar technique to entrain rapid insulin pulsatility revealed similar control of the insulin release process by glucose (31). We therefore performed the present study to examine the possible role of glucose oscillations on high frequency in vivo insulin pulsatility in the healthy human.
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METHODS |
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Subjects and Design
The protocol was approved by the Ethical Committee of Aarhus and was performed in accordance with the Helsinki Declaration. A total of 19 healthy adult volunteers with no family history of diabetes (mean age 25 ± 2 yr, 9 females and 10 males, and mean body mass index 23 ± 2 kg/m2) were studied. Each subject was assigned to one of three different protocols.Protocols
After an overnight fast, the volunteers were admitted to the Clinical Research Unit at the University Hospital of Aarhus. After catheter placement in an antecubital vein for infusion purposes and in a contralateral dorsal (heated) hand vein for sampling purposes, a 30-min resting period was allowed before the protocols were initiated.Protocol 1: dose response for induction of pulsatile secretion.
To test whether a periodic infusion of small amounts of glucose may
induce pulsatile insulin secretory bursts and to estimate the dose
required to do so, we studied each volunteer on two randomly assigned
occasions at glucose infusion rates of 6 or 2 mg · kg1 · min
1
recurrently every 10 min from 10 to 90 min (n = 6). Blood (1.5 ml) was collected at minute intervals from 1 to 90 min.
Protocol 2: effects of periodicity shifts.
The ability to determine the periodicity of pulsatile insulin secretion
was examined. In randomly assigned order, the subjects (n = 6)
were studied during glucose infusion rates of 6 mg · kg1 · min
1
with either 7- or 12-min intervals between infusions over a total study
time of 60 min, followed by an abrupt phase shift at time 60 min to the
other infusion interval (12 or 7 min, respectively) for the time window
of 60-120 min. Blood (1.5 ml) was collected at minute intervals
from 1-120 min.
Protocol 3: control study. Subjects (n = 7) were studied under basal conditions without any glucose infusion. Samples (1 ml) were collected every min for 75 min. Data in these subjects were compared with data obtained from subjects studied in protocol 1.
Sampling
Blood samples were centrifuged, and serum was pipetted and stored atAssays
Glucose. Plasma glucose concentrations were measured in duplicate by the glucose oxidation method (Beckman Instruments, Palo Alto, CA).
Insulin. Serum insulin concentrations were measured in triplicate by a two-site immunospecific insulin ELISA, as previously described (1). In brief, the assay uses two monoclonal murine antibodies (Novo Nordisk, Bagsvaerd, Denmark) specific for human insulin. The detection range of this insulin ELISA was 5-600 pM. At intermediate (150 pM), intermediate-high (200 pM), and high (350 pM) plasma insulin concentrations, the interassay variation coefficients (among triplicate) were 3.7, 4.0, and 4.5%, respectively. Corresponding intra-assay variations were 2.3, 2.1, and 2.0%. There is no cross-reactivity with proinsulin and split (32, 33)- and des(31, 32)-proinsulin; the antibodies cross-react 30 and 63% with split (65-66)-proinsulin and des(64, 65)-proinsulin, respectively, whereas C-peptide, insulin-like growth factor (IGF)-I, IGF-II, and glucagon do not cross-react (1).
C-peptide. C-peptide measurements were performed with a commercially available kit (K6218, DAKO Diagnostics, Cambridgeshire, UK). The assay is a two-site ELISA based on two monoclonal antibodies, employing the same principles referred to in Insulin. Each sample was assayed in duplicate, and the intra- and interassay (among triplicate) variation coefficients were 2.2 and 3.3%, respectively.
Data Analysis
Detection and quantification of pulsatile insulin secretion by
deconvolution analysis.
The serum insulin concentration time series was analyzed by a
deconvolution method for the purpose of detecting and quantifying insulin secretory bursts. Deconvolution was performed with a
multiparameter technique (35). In this analysis, the venous serum
insulin concentrations measured in samples collected at 1-min intervals
are assumed to result from five determinable and correlated parameters:
1) a finite number of discrete insulin secretory bursts
occurring at specific times and having 2) individual amplitudes
(maximal rate of secretion attained within a burst); 3) a
common half-duration (duration of an algebraically Gaussian secretory
pulse at half-maximal amplitude), which are superimposed on a
4) basal time-invariant insulin secretory rate; and 5)
a biexponential insulin disappearance model in the systemic circulation
consisting, as directly estimated earlier (25), of half-lives of 2.8 and 5.0 min and a fractional slow compartment of 0.28. Kinetic
parameters have previously been examined in healthy subjects, and in
the present study, the kinetic model was evaluated vs. observed data by
assuming variable insulin half-lives. The sum of squared residuals
(SSR, observed vs. best fit curve) was used to assess optimal insulin
kinetic parameters, and analysis of induced pulsatile secretion
resulted in mean (±SD) SSR values of 313 ± 30, 313 ± 32, 310 ± 35, 327 ± 35, and 345 ± 36 pM × min for insulin half-lives of 3, 4, 5, 7, and 10 min, respectively (P 0.01 for comparisons of
5 vs. 7 min and 5 vs. 10 min), thus resulting in the chosen second
half-life of 5 min, although this is shorter than reported by most
other groups. Employing half-lives of 3, 4, 5, 7, and 10 min did not affect the number of detected pulses during glucose pulse
induction studies. Assuming the foregoing nominal biexponential insulin
disappearance kinetics, we estimated the number, locations, amplitudes,
and half-duration of insulin secretory bursts, as well as a nonnegative
basal insulin secretory rate, for each data set by nonlinear
least-squares fitting of the multiparameter convolution integral for
each insulin time series. A modified Gauss-Newton quadratically
convergent iterative technique was employed with an inverse (sample
variance) weighting function. Parameters were estimated until their
values and the total fitted variance both varied by <1 part in
100,000. Asymmetric highly correlated variance spaces were calculated
for each parameter by the Monte Carlo support-plane procedure.
Secretory rates were expressed as mass units of insulin (pmol) released
per unit distribution volume (liters) per unit time (min). The mass of
hormone secreted per burst (time integral of the calculated secretory
burst) was thus computed as picomoles of insulin released per liter of
(systemic) insulin distribution volume. Because, for peripherally
deconvolved secretory rates, the calculated values represent
hepatic-vein insulin appearance, total insulin secretion was calculated
by use of C-peptide concentrations (34). When the deconvolution analysis was performed, basal secretion was adjusted to ensure that the
model allows 95% of troughs to fit. Likewise, the secretory burst
half-duration was adjusted to fit to individual observed insulin
secretory bursts, consisting of series of a total of four or more
consecutive data points building up to a peak and down to a trough. All
data analysis was performed blinded to treatment.
Quantification of irregularity. The regularity of serum insulin concentration time series was assessed by application of approximate entropy (ApEn), which is a model-independent and scale-invariant statistic (18, 19). ApEn assigns a single nonnegative number to a time series, in which larger absolute values correspond to greater apparent process randomness and smaller values correspond to more instance of recognizable patterns or consistent features in the data. Briefly, ApEn measures the logarithmic likelihood that runs of patterns that are close (within r) for m contiguous observations remain close (within the same tolerance width r) on next incremental comparisons; the precise mathematical definition is given in Ref. 17. For this study, we calculated ApEn values for all data sets, with m = 1 and r = 20% of the SD of the individual subject time series. Herein, as in Ref. 28, we applied ApEn with these input parameters to the first-differenced serum insulin concentration time series to minimize the effects of short-term nonstationarities in the data.
Auto- and cross-correlation analyses. The periodic nature of individual insulin and glucose profiles was assessed by autocorrelation analysis, and the relationship between the profiles of glucose and insulin was quantified by cross-correlation analysis. Visual inspection of the raw data revealed that slow trends dominated by a linear component were often observed. Because such trends can distort the subsequent correlation analyses, they were removed by subtracting from each profile its best-fit line calculated by linear regression analysis. All auto- and cross-correlation analyses were performed on series from which this procedure was first performed. In the autocorrelation analyses, the correlation coefficients between the time series and a copy of itself at lags of 0, 1, 2, 3, and up to 25 min were calculated. Similarly, in the cross-correlation analyses, the coefficients of cross correlation between the glucose and insulin series at lags of 0 (i.e., simultaneous values of glucose and insulin), ±1 (i.e., glucose leading insulin by 1 min or vice versa), ±2, ±3, up to ±25 min were calculated. The largest coefficient of cross correlation and the lag at which it occurred were assessed in each case. For both auto- and cross-correlation analyses, group statistical analysis of the correlation coefficients was performed after use of Fisher's z transformation (4).
Spectral analysis. As an alternative way of quantifying the degree of periodicity in the series, spectral analysis was performed. Each time series was detrended by using the first-difference filter. The spectral estimates were then calculated with the use of a Tukey window as described by Jenkins and Watts (8). The width of the window was chosen to be one-half of the length of the data series, yielding a good compromise between stability and fidelity.
Concentration changes. The mean changes in glucose and insulin concentrations resulting from the pulsatile glucose infusion were estimated by averaging the data for the period length across the observation period. Because the periodicity was defined by the infusion, we averaged data for the first minute, second minute, third minute, and so forth in each single period (i.e., mean of values for minutes 1, 11, 21, and 31, when glucose was infused every 10 min). The concentration changes occurring at basal (non-glucose-induced) conditions (protocol 4) were examined by cluster analysis (36). Cluster analysis defines significant (P < 0.05) up- and downstrokes and gives mean figures for the trough and peak concentrations as well as mean interpulse intervals. Cluster sizes of two for both peak and nadir and values of two times SD for both significant up- and downstrokes were used.
Statistics
The statistics dealing with data analysis are described in that section. All data in text and figures are given as means ± SE. Student's two-tailed paired and unpaired t-tests were used to evaluate statistical significance. ![]() |
RESULTS |
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Insulin and Glucose Concentrations
The glucose concentration changes that resulted from pulsatile glucose infusions are illustrated in Fig. 1A, which gives examples of time series of glucose concentrations during a total of 90 min, when glucose was infused every 10 min at rates of 2 and 6 mg · kg
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The changes in glucose concentrations were examined by autocorrelation
analysis, which showed that infusion of glucose at 6 mg · kg1 · min
1
every 10, 7, and 12 min resulted in significant (P < 0.05 in all 12 subjects and 18 data series) corresponding oscillations in the
glucose concentration. However, the infusion of glucose at 2 mg · kg
1 · min
1
did not elicit significant glucose concentration changes in some of the
subjects, although the pooled data revealed glucose concentration oscillations at the frequency induced. The infusion of glucose evoked
significant (P < 0.05) insulin oscillations at the
periodicity employed, at a rate of 6 mg · kg
1 · min
1
illustrated for pooled data in Fig.
2A and as examples in Fig. 2B. There was a significant cross correlation between glucose and insulin with 6 mg · kg
1 · min
1
glucose infusion for 1 min every 10 min, but not with 2 mg · kg
1 · min
1
every 10 min, as illustrated in Fig. 2C. The
change in glucose concentrations due to glucose induction at 6 mg · kg
1 · min
1
every 10 min was compared with glucose excursions during endogenous glucose production by comparing the variability in
first-differenced glucose concentrations given as percentage of total
glucose concentrations. During glucose induction, the glucose
concentration variability was similar (2.9 ± 0.4 vs. 2.6 ±0.4%,
induction vs. basal conditions, P > 0.1), indicating similar
glucose changes and hence that the induced glucose excursions are
similar to physiologically occurring glucose changes.
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Spectral analysis showed distinct peaks at the frequency induced in
every subject studied (n = 12; data series = 18) during infusion with 6 mg · kg
glucose1 · min
1
at either 7-, 10-, or 12-min intervals, with other peaks occurring as
smaller fractions of the dominant peak at the frequency induced (Fig.
2D).
In protocol 2 (period shift), the change in periodicity between
7 and 12 min per glucose pulse resulted in corresponding (entrained) changes in the pulsatile insulin secretory pattern (Fig.
3). This was confirmed by autocorrelation
analysis of each time series for each infusion paradigm (Fig.
2B) and by spectral analysis (Fig. 2D).
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Compared with the very regular insulin concentration oscillations
induced by periodic glucose infusions, the insulin concentration profiles during basal conditions exhibited greater variability, as
illustrated in Fig. 4.
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Insulin Secretion
The deconvolved insulin secretory profiles confirm that the observed pulsatile concentration profiles are due to highly regular insulin secretory bursts (Fig. 5). Only occasional noninduced insulin secretory bursts occurred, especially when a frequency of 10 min per pulse or less was chosen, as in protocol 1 where only 3 noninduced vs. 54 induced pulses occurred. Furthermore, the noninduced pulses were much smaller (~10% of induced pulses). Similarly, the ability to induce pulses was striking with 100% concordance (no pulses missing) during glucose infusion at a rate of 6 mg · kg
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Regularity Analysis
ApEn analysis of serum insulin concentration time series disclosed a significantly greater orderliness of the insulin release process during the glucose pulse induction (6 mg · kg ![]() |
DISCUSSION |
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In the present study, we have infused glucose in an intermittent
square-wave pattern to evaluate the possible role of glucose oscillations in directing rapid pulsatile insulin secretion in the
healthy human. We evaluated the effects of two different glucose doses
delivered at three different frequencies and explored the persistence
of the pulsatile pattern after abrupt withdrawal of the pulsed glucose
signal. We found that infusion of glucose at a rate of 6 mg · kg1 · min
1
every 7, 10, and 12 min (but not 2 mg · kg
1 · min
1
every 10 min) promotes pulsatile insulin secretion at an identical frequency with very few (or no) insulin secretory bursts that were not
glucose induced. Compared with basal (noninduced) pulsatile insulin
secretion, the induced pulses are larger, more clearly demarcated, and
occur with much greater regularity.
Whereas it is well known that insulin is secreted in a pulsatile manner (6), the regulatory mechanisms governing the common secretory activity among intrapancreatically dispersed islets are not established. The release of insulin from perifused islets is shown to be pulsatile, suggesting that ability to act as pacemaker resides within the islet (2, 3, 33), but it does not explain the common pulsatile release from dispersed islets. Intrapancreatic neuronal pacemaker activity provides a plausible mechanism, because the pulsatile insulin secretory pattern (albeit less regular) is preserved in the isolated perfused pancreas at constant glucose concentrations (30). Thus glucose oscillations do not seem to be the sole cause of insulin pulses (6, 7, 11). However, the presence of both glucose and insulin concentration oscillations would allow a possible role for a feedback mechanism. Studies on entrainment of ultradian (~90-min period) insulin oscillations have indicated such a role for glucose (15, 32). The present observations that punctuated glucose infusions at specific intervals may determine the periodicity of rapid endogenous pulsatile insulin secretion (every 7-12 min) suggest an additional time scale at which glucose can regulate the pulsatile secretion of insulin.
Although a plausible speculation is that endogenous circulating glucose
pulses govern the coordinate secretion of insulin from the islet
population, because well-defined exogenous glucose pulses further
improve the regularity of insulin release, it is equally plausible that
an intrapancreatic pacemaker responsible for controlling the timing of
insulin release is itself sensitive to glucose. In the latter
conjecture a similar improvement of regularity would occur. The present
observations are fully consistent with the hypothesis of an
intrapancreatic pacemaker that is sensitive to glucose changes. In this
model, a classical insulin-glucose feedback mechanism would operate to
temporally organize the pulsatile release pattern. Such a feedback loop
would involve insulin secretion in pulses, followed temporally by
tissue insulin actions, causing glucose concentration changes as
oscillations, and subsequent -cell/pacemaker responses to the
glucose concentration changes.
Whereas insulin secretory patterns are influenced by numerous other
factors, including neuronal factors, hormones, and metabolic substrates, the present demonstration of a rapid induction of an
obvious on/off secretory pattern consisting of insulin secretory bursts
of a 3- to 4-min duration followed by 6-7 min of no insulin release at all, when entrained by the rather modest changes in glucose
concentrations, in our view is highly indicative of glucose as the
dominant regulator. This notion certainly supports the hypothesis of
the -cell as a very sensitive fuel sensor (33). It may also serve to
link the cyclical metabolic processes observed in vitro (namely, of
cyclical glycolysis, causing cyclical generation of ATP, resulting in
intermittent depolarization and increases in intracellular calcium and
likely several other factors and steps) with the recurrent release of
insulin in bursts. This scenario would unfold from present in vivo
studies showing that cyclical increases in fuel supply are tightly
linked to cyclical insulin release at intervals as short as 7-12
min, which fall well within the normally observed range of rapid
spontaneous insulin pulsatility. Failure to entrain at this high
frequency would cast serious doubt on the glucose-feedback hypothesis.
Because virtually the entire -cell population in widely dispersed
islets seems to respond synchronously as a common organ, we have argued
that an intrapancreatic pacemaker responds to the experimentally
induced changes in glucose in vivo, possibly by sensing glucose through
cyclic glycolysis. This thesis is congruent with in vitro studies on
perifused islets and the isolated perfused pancreas of rats, where
small but rapid oscillations in ambient glucose concentrations
(amplitude of 10% of the mean glucose concentration) are able to
control the pulsatile pattern of insulin release (31). Our clinical
data substantially extend this in vitro concept to the normal human in
the fasting state.
In the present studies, we have used the term induction for the ability of modest increases in glucose concentrations to cause subsequent on/off pulsatile insulin secretory patterns. This term was chosen instead of the more familiar term entrainment (32), simply to discriminate between rapid oscillations studied herein and ultradian oscillations studied by entrainment.
The paradigm of a repetitive well-defined discrete physiological
(glucose) stimulus to cause very regular (in time and amplitude) and
dominant (on/off) insulin secretory responses, as evident in healthy
humans, may be a useful tool for evaluation of -cell glucose
sensitivity. Defects in one or more of the steps putatively involved in
glucose sensing-insulin secretion coupling are likely to be unmasked
with the subtle glucose stimulus to test
-cell glucose sensitivity.
Conversely, studies observing basal spontaneous insulin secretion
oscillations reflect multiple other linkages within the entire organism
in addition to postulated glucose-insulin feedback regulatory
mechanisms. For example, to the extent that an individual is insulin
resistant, spontaneous oscillations in insulin-induced glucose
concentration changes are likely damped, which impairment itself would
be reflected in secondarily disrupted pulsatile insulin secretion. In
contrast, introduction of discrete glucose pulses affords the
opportunity to test the pancreatic sensitivity to glucose more
directly, as well as dynamically. Under these conditions, an observed
impairment in insulin output can be linked more directly to pancreatic
glucose insensitivity (or lack of intrapancreatic coordination). Thus
we suggest that the rapid periodic glucose pulsatile infusion model may
add a new dimension to our understanding of normal and
pathophysiological altered glucose-regulated
-cell secretory
activity, in the presence or absence of other relevant secretagogues or
modulators of islet cell function.
A recent paper describes entrainment of rapid pulsatile insulin
secretion by infusion of 15 mg · kg1 · min
1
of glucose every 29 min, causing insulin periodicities of ~15 min per
pulse (12). In the present paper, a more rapid and more subtle glucose
stimulus model was tested, which resulted in concordance between
frequency of glucose and insulin oscillations. It may well be that
extending the periodicity too far causes release of insulin secretory
bursts between the induced secretory pulses.
In conclusion, the present experiments show that the modest
oscillations in peripheral glucose concentrations induced by periodic glucose infusions can control the pulsatile insulin release pattern. Induction occurs immediately and shows frequency control by period shifting. These observations may indicate application of such induction
models to further study in vivo -cell glucose sensitivity in health
and disease and to examine early
-cell dysfunction by experimentally
defined islet cell "pacing" across a spectrum of
pathophysiologically relevant glucose-infusion (pulse) frequencies.
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ACKNOWLEDGEMENTS |
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The expert technical assistance from Anette Mengel, Lene Trudsø, and Elsebeth Hornemann is highly appreciated.
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FOOTNOTES |
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The present work was supported by Novo Nordisk, The Danish Diabetes Foundation, and The University of Aarhus.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. Pørksen, Dept. M (Endocrinology and Metabolism), Aarhus Univ. Hospital, 8000 Aarhus C, Denmark (E-mail: porksen{at}dadlnet.DK).
Received 5 January 1999; accepted in final form 15 September 1999.
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