Page Articles
1 of 49 in PresS. Am J Physiol Endocrinol Metab (October 30, 2007). doi:10.1152/ajpendo.00514.2007
Importance of Changes in Gastric Emptying for Postprandial Plasma
Glucose Fluxes in Healthy Humans
1
1
2
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H.J. Woerle , M. Albrecht , R. Linke , S. Zschau , C. Neumann3, M. Nicolaus1,
J. Gerich4, B. Göke1, J. Schirra1
1
Department of Internal Medicine II, Clinical Research Unit, Grosshadern
Ludwig-Maximilians-University,
Munich, Germany
2
Department of Nuclear Medicine, Grosshadern
Ludwig-Maximilians-University,
Munich, Germany
3
Outpatient Diabetes Center,
Leopoldstraße 32, 80802 Munich, Germany
4
Department of Medicine,
University of Rochester School of Medicine,
Rochester, New York, USA
Running title: Effects of pramlintide induced delay in gastric emptying on postprandial
glucose fluxes
Key Words: Postprandial, glucose, gastric emptying, pramlintide
Word count: 5189
Address correspondence to:
Hans-Juergen Woerle
Department of Internal Medicine II
Clinical Research Unit (CRU)
University of Munich, LMU, Grosshadern
Marchioninistr. 15
81377 Munich
Germany
Tel. +49-89-7095-0, -2282, -2271
eMail: [email protected]
1
Copyright © 2007 by the American Physiological Society.
Page 2 of 49
Abstract:
Regulation of postprandial (pp) plasma glucose excursions is complex. Insulin and
glucagon secretion are thought to play the predominant role. Nevertheless, only 50% of the
variation in pp plasma glucose excursions is explained by variations in plasma insulin and
glucagon, suggesting that other factors are involved. Theoretically, gastric emptying (GE)
should be another important factor. However the impact of GE on pp glucose fluxes has not
been studied. Accordingly, we examined the consequences of pramlintide- induced
retardation in GE on pp glycemia and glucose fluxes using a combination of a double isotope
approach and scintigraphically determined rates of gastric emptying. Fourteen healthy nondiabetic subjects were studied (8 men, 6 women, 40 3 years, bodyweight 78 4 kg, BMI 27.8
1.1kg/m2), 30 Hg pramlintide (PRAM) or placebo (PBO) was injected s.c. before subjects ate
a mixed meal, scrambled eggs and jello containing 50g glucose, ~25g fat and ~20g protein.
At 60 minutes 65±3 vs. 87±2 %, (p<0.001) of initial gastric contents remained in the
stomach (PBO vs. PRAM). Thereafter gastric emptying slopes paralleled one another until
240 min. The 50% retention times (T50) were lower when PBO was given (177±9 vs. 227±14
min, p<0.001). GE was greater from 240 min to the end of the experiment in the PRAM
experiment, so that only a small but still greater proportion of the meal remained in the
stomach at 330 min (5.3±1.8 vs. 11.4±3.0%, p<0.01). Reductions of GE by PRAM markedly
reduced pp plasma glucose peak and overall excursions (7.5±0.3 vs. 6.0±0.2 mmol/l, p<0.001
and 5.6±0.1 vs. 5.2±0.1 mmol/l, p<0.001, PBO vs. PRAM respectively) even though peak and
overall plasma insulin concentrations were both lower with PRAM (346±37 vs. 211±42
pmol/ml, p<0.01, and 164±13 vs.138±13 pmol/ml, p<0.01). No differences in pp plasma
glucagon concentrations were found (66 5 vs. 59 5 pg/ml, ns). The reduction in total glucose
appearance in plasma (13.1±0.6 vs. 11.0±0.5 Hmol*kg-1*min-1, p<0.001) was solely
attributable to reduction in meal-derived glucose appearance (10.2±0.5 vs. 7.0±0.4 Hmol*kg1
*min-1, p<0.001) since endogenous glucose appearance was greater with PRAM (2.9±0.2 vs.
4.0±0.2 Hmol*kg-1*min-1, p<0.001). Splanchnic glucose uptake was greater with PRAM
(26.5±1.6 vs. 32.5±2.1%, p=0.014).
Taken together, these data support the concept that GE is an important physiological
regulator of pp plasma glucose excursions and glucose fluxes in healthy humans.
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Introduction
Glucose fluxes have generally been attributed to an interaction between pancreatic
islet cell function (insulin and glucagon secretion) and hepatic and peripheral sensitivity to
insulin (1;2). Although this approach can explain to a large extent fasting glucose
homeostasis, it can explain only approximately 50% of the variation in postprandial glucose
excursions (20;22). The importance of postprandial hyperglycemia has recently been
emphasized because it precedes fasting hyperglycemia as a precursor of diabetes and has been
identified as an independent risk factor for cardiovascular disease (9).
Prominent among the other factors that may determine postprandial glucose
excursions, is glucose influx from the gut (12). This appears to be determined to a certain
extent by the rate of gastric emptying since previous studies suggest that as much as 40-50%
of variation in postprandial glucose excursions may be explained by differences in the rate of
gastric emptying (11).
The present studies were therefore undertaken to test the hypothesis that slowing of
gastric emptying would reduce postprandial entry of glucose into the systemic circulation and
thus lower postprandial plasma glucose excursions. For this purpose we administered a
physiological meal, delayed gastric emptying with the amylin analogue pramlintide, and
assessed the consequences on postprandial plasma glucose concentrations, endogenous
glucose production, exogenous (meal derived) glucose appearance, splanchnic sequestration
and peripheral plasma glucose disposal.
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METHODS
Subjects: Informed written consent was obtained from 14 healthy volunteers after the
Ludwig Maximilian University of Munich, Institutional Review Board had approved the
protocol. The subjects (8 men, 6 women 40 ± 3 years of age, body weight 78 ± 4 kg, BMI
27.8±1.1 kg/m2) had normal physical examinations, no gastrointestinal symptoms and normal
routine laboratory tests as well as normal glucose tolerance (assessed by oral glucose
tolerance testing according to World Health Organization criteria (24)). None of the subjects
had a family history of diabetes. For three days prior to the study, all subjects had been on a
weight maintaining diet containing at least 200 g carbohydrate and had abstained from alcohol
and exercise.
Protocol: Subjects restrained from food intake at least 10 hours prior to admission to
the clinical research centre between 7:00 and 7:30 am on the study day. A dorsal hand vein
was cannulated and temperature was maintained at 40°C with a thermoregulated lamp for
arterialized venous blood sampling (20).
At 8 AM, a primed (25 Hmol) continuous (~0.25 Hmol/kg/min) infusion of [1-13-C]
glucose was started via a forearm vein of the contralateral arm for measurements of plasma
glucose turnover. At least three hours were allowed for achievement of isotope equilibration.
Prior meal ingestion three baseline blood samples were collected for fasting glucose, insulin,
glucagon and [1-13-C] glucose enrichments, and subjects ingested a standardized meal
containing ~500 kcal within five minutes thereafter. The meal consisted of 3 scrambled eggs
and 100 ml jello containing 50 g of glucose. Three of the 50 g of glucose were 6,6dideuteroglucose for measurements of meal-derived glucose. Consumption of 200 ml
sparkling water was allowed throughout the 330 min postprandial period. With the meal 30
Hg of pramlintide or placebo (Amylin Pharmaceuticals, San Diego, USA) was injected into
the subcutaneous tissue of the lower abdominal wall. All subjects were studied on two
occasions separated by at least one day in randomized, single blinded order (pramlintide or
placebo). Results of the placebo experiments have partially been reported elsewhere under
different aspects. Subjects remained in a semi-supine position throughout the study period.
Over the initial 90 minutes of the postprandial period blood samples were taken at 15-minuteand at 30-minute-intervals thereafter upon the end of the experiments at 330 minutes
postprandial. The scrambled eggs and jelly were labeled with 99m Tc-Sn-colloid ~70 MBq
for measurements of gastric emptying by high-resolution scintigraphy (20 images/minute,
Orbiter, Siemens, Germany, Erlangen) starting immediately after completion of the meal for
the remainder of the experiment (14;15).
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Analytical Procedures: Gastric outlines on the maximum intensity image were defined
as the region of interest (ROI). Completion of gastric emptying was assumed at a reduction of
the initial activity to less than 5 %. Loss of activity was corrected for the radioactive half-life
of Tc99m (14). According to the procedure guideline of The Society of Nuclear Medicine (6)
a continuous framing rate of 30–60 seconds is recommended to accurately determine lag
phase and half-time of gastric emptying. Therefore, one the basis of our ‘high-resolution
scintigraphy' (20 images/minute) we calculated the time course of gastric emptying in 1
minute intervals. As recommended in the Procedure Guideline for Gastric Emptying and
Motility (6) we acquired the scintigraphic data in the left anterior oblique view with a singlehead camera in order to avoid a (mathematical) tissue attenuation correction. Since all
subjects were examined in a convenient semi-supine position movement artefacts were largely
bypassed. The cine display was used to confirm the stomach outline and to determine the
extent of patient motion so that the ROI was adjusted appropriately. In the very rare cases of a
significant subject motion the nuclear medicine postprocessing software package (Hermes,
Nuclear Diagnostics, Sweden) provides a software tool for motion correction.
Blood samples were collected for glucose concentrations, [1-13-C] glucose, [6,6-2H2]
glucose enrichments in oxalate-fluoride tubes, and for insulin and glucagon concentrations in
EDTA tubes containing a protease inhibitor. Samples were immediately placed in a 4°C ice
bath and plasma was separated within 30 min by centrifugation at 4°C. Plasma glucose
concentrations, [1-13-C] glucose enrichments, [6,6-2H2] glucose enrichments were measured
by previously described methods (4;18). Plasma insulin and glucagon concentrations were
determined by standard radioimmunoassays, as previously described (19). Coefficients of
variation (CV) for insulin and glucagon assays were 2.2 % and 4.0 % within assay,
respectively.
Calculations: Systemic release and uptake of glucose were calculated with steady state
equations prior to meal ingestion (23) and subsequently with non-steady state equations of
DeBodo et al (5) using a pool fraction of 0.65 and a volume of distribution of 200 ml/kg.
The rate of appearance of the oral glucose load in the systemic circulation was
calculated from [6,6-2H2] glucose data with the equation of Chiasson et al (3;13). Endogenous
glucose release was calculated as the difference between the overall rate of plasma glucose
appearance and the rate of appearance of exogenous glucose (13;16). Overall splanchnic
glucose disposal of the ingested glucose load was calculated as the difference between the
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amount of glucose ingested and the total appearance of the ingested glucose in the systemic
circulation during the 330 min postprandial period based on the proportion of the meal being
emptied as determined by measurements of gastric emptying at the end of the experiment.
Statistical analysis: Data are given as mean ± SEM unless otherwise specified using
Statistica statistical software (1998 edition, Statsoft Inc, Tulsa, Okla). Normality of the
distribution was assessed using the Kolmogorov-Smirnov test. For comparison of the two
groups and to compare baseline data with mean data after meal ingestion ANOVA for
repeated measurements followed by post-hoc comparison for paired groups was
performed. A p value less than 0.05 was considered statistically significant. Correlation
between variables was performed using Spearman’s regression analysis. Values for the
description of dynamic changes represent absolute values, unless specified otherwise.
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RESULTS
% Gastric Retention, Gastric Retention Slopes, Lag Periods, 50% Retention (T50) and %
Retention at 60- Minutes (Figure 1):
Gastric content decreased rapidly within 60 min to a mean value of 77±2 %, when
placebo was administered. At the end of the experiment only 6±2 % of the initial 99mTc
counts could be detected in the region of interest. In contrast, subcutaneous pramlintide
injection with the meal resulted in a significantly greater percentage of gastric content
remaining in the stomach at 60 minutes (92±1 %, p=0.0001 compared to placebo).
Pramlintide prolonged the lag period (25±8 vs. 74±11 min, p=0.0008), defined as the delay
between ingestion of food and a 10% reduction of isotope counts in the region of interest. A
significant overall effect of pramlintide on gastric emptying slopes could be detected.
Assessing retention slopes 0 – 60, 60 – 240, 240 – 330 minutes yielded significantly lower avalues for 0 – 60 minutes (a = -0.38±0.03 vs. -0.13±0.02, p<0.001), comparable slopes for 60
– 240 minutes (a = -0.31±0.02 vs. –0.27±0.02, p=0.08) and significantly greater a- values for
240 – 330 minutes (a = -0.17±0.03 vs. –0.33±0.04, p=0.004) in the pramlintide experiment.
Thus the significantly greater proportion of the meal remaining at the end of the experiment
(14±4 %, p=0.009) was largely due to the delay in gastric emptying within the first 60
minutes after meal ingestion which resulted in a significant delay of the 50% retention time
(T50) from 177±9 to 227±14 min (p=0.001). Within the first 60 minutes a total of 23±2 vs.
8±1 %, p=0.0001, of the meal was emptied by the stomach.
Plasma Glucose (Figure 2):
Plasma glucose concentrations prior to meal ingestion were comparable on both study
days (4.5±0.1 vs. 4.6±0.1 mmol/l, p=0.47, placebo vs. pramlintide, respectively). In the
placebo experiments plasma glucose concentrations rose to a maximum of 7.5±0.3 mmol/l at
60 min and returned to baseline levels at 180 min. In the pramlintide experiments peak
postprandial plasma glucose concentrations were significantly lower (6.0±0.2 mmol/l at 120
min compared to 7.5±0.3 mmol/l at 60 min, p=0.0002). Mean postprandial plasma glucose
concentrations were significantly reduced after pramlintide administration (5.6±0.1 vs.
5.2±0.1 mmol/l, p=0.004). Areas under the plasma glucose curves (AUC) over the entire
postprandial period were lower with pramlintide (354.2±31.1 vs. 207.0±25.9 mmol/l * min, p
= 0.003).
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Plasma Insulin and Glucagon (Figure 2):
Plasma insulin concentrations paralleled those of plasma glucose concentrations (see
above): Peak plasma insulin concentrations were significantly greater and earlier when
placebo was administered (346±37 pmol/ml at 45 min vs. 211±42 pmol/ml at 90 minutes,
both p=0.001) compared to pramlintide. Average plasma insulin concentrations were
significantly greater during the placebo experiment compared to pramlintide (188±15 vs.
134±13 pmol/ml, p=0.001).
Basal plasma glucagon concentrations were comparable on both study days
(59.7±13.4 vs. 58.1±5.8 pg/ml, p=0.68), decreased to a comparable extent (nadir 48.1±5.8 vs.
50.0±3.7 pg/ml at 90 min, p=0.70, placebo vs. pramlintide respectively) and overall were not
significantly different (65.6±4.7 vs. 59.3±5.0 pg/ml, p=0.074, placebo vs. respectively).
As a result of the above differences attributable primarily to differences in plasma
insulin concentrations, the plasma insulin-to-glucagon molar ratio during the initial 90
minutes after meal ingestion was significantly greater after placebo administration compared
to pramlintide (18.7± 1.5 vs. 9.0± 1.9, p=0.001) and significantly lower between 150 and 330
minutes averaging 5.0± 0.6 vs. 8.7± 1.1, p=0.001.
Rates of Total Plasma Glucose Appearance, Exogenous (Meal) Plasma Glucose Appearance
and Endogenous Plasma Glucose Appearance (Figure 4):
Rates of basal glucose turnover were comparable on both study days (9.0±0.3 vs. 9.4±0.5
Hmol*kg-1*min-1, p=0.11, placebo vs. pramlintide, respectively). Pramlintide reduced peak
(18.6±1.6 Hmol*kg-1*min-1 at 30 min vs. 12.7±1.0 Hmol*kg-1*min-1 at 120, p<0.01) and
overall rates of total glucose appearance in plasma (12.0±0.5 vs. 10.7±0.5 Hmol*kg-1*min-1,
p=0.005).
Endogenous glucose production decreased more promptly in the placebo experiment
reaching a nadir of 1.4±1.0 Hmol*kg-1*min-1 at 45 min compared to a nadir of 3.1±0.7
Hmol*kg-1*min-1 at 90 min in the pramlintide experiment. On average 3.5±0.2 vs. 4.4±0.2
Hmol*kg-1*min-1, p<0.001, of glucose were endogenously released in the placebo and
pramlintide experiments, respectively. This corresponded to a 60.6±1.5 vs. 55.0±1.0 %,
p=0.008, suppression of endogenous glucose production in the postprandial state.
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Rates of exogenous glucose appearance (meal-derived glucose) increased to a
maximum of 15.4±1.1 Hmol*kg-1*min-1 within 45 minutes after meal ingestion and declined
steadily thereafter when placebo was administered with the meal.
Pramlintide decreased peak (15.4±1.1 vs. 8.9±0.7 Hmol*kg-1*min-1, p<0.001) and overall
rates of exogenous glucose appearance (8.5±0.4 compared to 6.3±0.4 Hmol*kg-1*min-1,
p<0.0001). Exogenous glucose appearance accounted for 70.7±1.0 % of total glucose
appearance in the placebo experiment compared to 58.6±1.6% in the pramlintide experiment,
p<0.001. The differences in rates of total glucose appearance (1.3±0.4 Hmol*kg-1*min-1,
p=0.005, see above) could thus be solely explained by differences in exogenous glucose
appearance (2.2±0.3 Hmol*kg-1*min-1) since endogenous glucose appearance rates were
greater in the pramlintide experiment (0.9±0.2 Hmol*kg-1*min-1, p=0.001).
Splanchnic Glucose Uptake:
Splanchnic glucose uptake estimated from the calculated proportion of the emptied
gastric content, which did not appear in the systemic circulation, was significantly greater
when pramlintide was administered (26.5±1.6 vs. 32.5±2.1%, p=0.014) at the end of the
experiment.
Rates of Glucose Disappearance (Figure 5):
In placebo experiments rates of glucose disappearance were not significantly different
from baseline values during the initial 45 minutes after meal ingestion averaging 9.2±0.8
Hmol*kg-1*min-1, p=0.8, increased to maximum values of 17.1±1.5 Hmol*kg-1*min-1 at 90
minutes and decreased steadily thereafter. In the pramlintide experiment rates of
disappearance were not significantly different from baseline during the first 90 minutes
averaging 8.8±0.5 Hmol*kg-1*min-1, p=0.17, with peak values of 14.5±0.9 Hmol*kg-1*min-1 at
210 min which were significantly lower compared to the placebo experiment, p=0.001.
In the placebo experiment rates of plasma glucose appearance exceeded those of its
removal by 5.6±0.6 Hmol*kg-1*min-1 corresponding to a total excess of 332±35 Hmol*kg-1. In
the pramlintide experiment only 1.7±0.2 Hmol*kg-1*min-1 had not been removed from the
circulation corresponding to 201±20 Hmol*kg-1 of excessive glucose, both significantly lower
in the pramlintide experiments, p=0.001 and p=0.0001, respectively, explaining differences in
peak plasma glucose concentrations. Since rates of removal of excessive plasma glucose
exceeded its appearance by only -2.84±0.1 Hmol*kg-1*min-1 vs. -1.4±0.2 Hmol*kg-1*min-1,
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placebo vs. pramlintide, baseline plasma glucose values were not achieved until 120 minutes
after peak plasma glucose concentrations in both experiments, explaining differences in
overall plasma glucose excursions above baseline.
Regression analyses using rates of gastric emptying as the independent variable and
peak plasma glucose concentrations and rates of exogenous glucose appearance as dependent
variables:
A significant inverse correlation between rates of gastric retention at 45 minutes and
incremental peak plasma glucose concentrations (at 60 minutes in the placebo and 120
minutes in the pramlintide experiment) was found (R=0.702, p=0.001) (Figure 3a).
Furthermore, the differences in gastric retention between the placebo and the pramlintide
experiment correlated significantly with differences in incremental peak plasma glucose
concentrations at 60 and 120 minutes, respectively (r=0.474, p=0.001).
There was a significant correlation between overall rates of gastric emptying and rates
of exogenous glucose appearance (r=0.564, P=0.001). Furthermore, the differences in rates of
meal-derived glucose appearance between placebo and pramlintide followed to peak plasma
glucose concentrations (0-60 min and 0-120 min in the placebo and pramlintide experiments,
respectively) correlated significantly with the differences in rates of gastric emptying over the
prevailing time intervals (r=0.580, p=0.001) (Figure 3b).
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Discussion:
The present studies were undertaken to test the hypothesis that slowing of gastric
emptying would reduce postprandial entry of glucose into the systemic circulation and thus
lower postprandial plasma glucose excursions. The hormone amylin is co- secreted with
insulin from the pancreatic beta- cell (7). Amylin as well as its analogue pramlintide have
been shown to markedly delay gastric emptying (17). Accordingly, we used pramlintide to
delay gastric emptying and found that the delay of gastric emptying was associated with a
marked reduction of meal- derived glucose in plasma especially within the first 60 minutes
after meal ingestion and a significant reduction in postprandial plasma glucose excursions.
The reductions in postprandial plasma glucose excursions could be explained by both a
reduction in glucose appearance in plasma solely due to meal-derived glucose and a relatively
greater proportional uptake of glucose by peripheral tissues.
Pramlintide administration was associated with a significant overall effect on gastric
emptying. The delay in gastric emptying occurred primarily due to a ~20 % reduction in
gastric emptying within the first 60 minutes, whereas gastric emptying was found to be
comparable to placebo between 75 and 240 minutes and somewhat greater thereafter. This
delay in gastric emptying was accompanied by a significant reduction in peak and overall
postprandial plasma glucose concentrations. Rates of meal derived glucose appearance in the
systemic circulation are determined by rates of gastric emptying and by the amount of glucose
being sequestrated in the splanchnic bed (22). Previous studies in healthy humans found
approximately 25-30% of the glucose load being sequestrated in the splanchnic bed (13). In
our placebo experiment we found 26.7±1.6 % of glucose being sequestrated. Estimation of
splanchnic sequestration in the pramlintide experiment yielded a significant increase
(32.5±2.1%, p<0.05) in the proportion of the oral glucose load being sequestrated. However,
these estimates have to be interpreted with caution since calculations of sequestration depend
on the amounts of glucose being absorbed rather than emptied by the stomach. Differences in
glucose absorption and, i.e. greater rates in non-oxidative glycolysis cannot be excluded and
may abolish differences in calculated rates of glucose sequestration. On the other hand even if
splanchnic glucose sequestration had been comparable this would have occurred in the face of
significantly lower postprandial insulin to glucagon ratios which should have been
accompanied with lower splanchnic sequestration rather than comparable or even increased
rates. Thus it appears that modulation of gastric emptying, especially a reduction of the initial
emptying may have indeed improved splanchnic sequestration.
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Pramlintide has been reported to suppress postprandial glucagon concentrations (8).
However, we found no significant differences in plasma glucagon concentrations and, even
more importantly, greater rates of endogenous glucose production in the pramlintide
experiments presumably due to lower postprandial plasma insulin concentrations.
Plasma glucose concentrations increase until rates of plasma glucose removal equal its
appearance (21). Postprandially rates of plasma glucose appearance are largely determined by
meal- derived plasma glucose appearance responsible for the disequilibrium between rates of
glucose appearance and disappearance (22). Delay in gastric emptying was associated with a
reduction in the amount of meal derived glucose entering the circulation. This reduced the
disequilibrium between the rates of entry and exit of glucose from the circulation and thus
reduced postprandial peak plasma glucose excursions by ~ 40 %. The more glucose is rapidly
added to the plasma pool, the longer it will take to remove the additional amounts of glucose
(10). Accordingly, overall postprandial plasma glucose excursions are largely determined by
the initial efflux of glucose into the circulation. It is important to note that this occurred in the
face of significantly greater plasma insulin and comparable glucagon concentrations. Thus
modification of glucose efflux via the gut, especially deceleration of initial gastric emptying
had led to a relatively more sufficient disposal of glucose in the face of lower plasma insulin
concentrations.
Taken together, these studies highlight the importance of gastric emptying to
determine postprandial glucose fluctuations. The pramlintide induced initial delay in gastric
emptying was associated with a reduction in meal- derived glucose appearance in plasma.
Since endogenous glucose production was greater in the pramlintide experiment, reduction in
total glucose appearance was solely attributable to reductions in meal- derived glucose
appearance. A greater proportion of the meal derived glucose was sequestrated in the
splanchnic bed. Since the initial rapid efflux of glucose into the circulation largely determines
postprandial plasma glucose excursions, delay in gastric emptying significantly reduced
postprandial plasma glucose concentrations.
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Acknowledgements
We thank the laboratory staff of the Clinical Research Centre and are especially indebted to
Rita Schinkmann and Silke Herrmann for their superb help.
Grants
The present work was supported by the Deutsche Forschungsgemeinschaft Grant SchI527/5-2
to H.J. Woerle and J. Schirra, and an unrestricted Grant by Amylin Pharmaceuticals, San
Diego, USA, and the National Institute Diabetes and Digestive and Kidney Disease Grant
DK-20411 to J. Gerich
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Figure 1: % Gastric Retention, Gastric Retention Slopes, Lag Periods, 50% Retention (T50)
and % Retention at 60- Minutes
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Figure 2: Plasma Glucose, Insulin, Glucagon Concentrations, Insulin-Glucagon Molar Ratio
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Figure 3a: Gastric Retention at 45 Minutes vs. Incremental Peak Plasma Glucose
Concentrations (above)
Figure 3b: Differences in Rates of Meal-Derived Glucose Appearance Placebo and
Pramlintide vs. Differences in Rates of Gastric Emptying (below)
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Figure 4: Rates of Total Plasma Glucose Appearance, Exogenous (Meal) Plasma Glucose
Appearance and Endogenous Plasma Glucose Appearance
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Figure 5: Rates of Glucose Disappearance, Rates of excessive Glucose Appearance and
Removal
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15. Linke,R, Meier,M, Muenzing,W, Folwaczny,C, Schnell,O, Tatsch,K: Prokinetic
therapy: what can be measured by gastric scintigraphy? Nucl Med Commun 26:527-533,
2005
16. Mitrakou,A, Kelley,D, Mokan,M, Veneman,T, Pangburn,T, Reilly,J, Gerich,J: Role of
reduced suppression of glucose production and diminished early insulin release in
impaired glucose tolerance. N Engl J Med 326:22-29, 1992
17. Schmitz,O, Brock,B, Rungby,J: Amylin agonists: a novel approach in the treatment of
diabetes. Diabetes 53 Suppl 3:S233-S238, 2004
18. Stumvoll,M, Chintalapudi,U, Perriello,G, Welle,S, Gutierrez,O, Gerich,J: Uptake and
release of glucose by the human kidney: postabsorptive rates and responses to
epinephrine. J Clin Invest 96:2528-2533, 1995
19. Stumvoll,M, Perriello,G, Nurjhan,N, Bucci,A, Welle,S, Jansson,P-A, Dailey,G, Bier,D,
Jenssen,T, Gerich,J: Glutamine and alanine metabolism in NIDDM. Diabetes 45:863868, 1996
20. Woerle HJ, Szoke E, Meyer C, Dostou JM, Wittlin SD, Gosmanov NR, Welle SL,
Gerich JE: Mechanisms for abnormal postprandial metabolism in type 2 diabetes. Am J
Physiol Endocrinol Metab 290:E67-E77, 2006
21. Woerle, H. J. and Gerich, J. Normal glucose physiology. 2003. Encyclopedia Diabetes.
Ref Type: Generic
21
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22. Woerle,HJ, Meyer,C, Dostou,JM, Gosmanov,NR, Islam,N, Popa,E, Wittlin,SD,
Welle,SL, Gerich,JE: Pathways for glucose disposal after meal ingestion in humans. Am
J Physiol Endocrinol Metab 284:E716-E725, 2003
23. Wolfe R: Radioactive and stable isotope tracers in biomedicine: principles and practice
of kinetic analysis. New York, Wiley-Liss, 1992,
24. World Health Organization Expert Committee: Diabetes mellitus: a second report.
Geneva, Switzerland. Technical Report Series 646:1-80, 1980
22
Page 23 of 49
100
s.c. placebo with the meal
s.c. pramlintide with the meal
80
% gastric retention
n = 14
60
40
gastric retention
20
0
0 – 60
minutes
60 – 240
minutes
80
placebo
% gastric retention
100
240 - 330
minutes
60
40
20
a = -0.38 ± 0.03
0
a = -0.31 ± 0.02
p < 0.0001
p = 0.08
pramlintide
% gastric retention
a = -0.13 ± 0.02
100
a = -0.17 ± 0.03
a = -0.27 ± 0.02
p = 0.004
80
a = -0.33 ± 0.04
60
40
20
retention slopes (a)
0
60
120
180
minutes
250
s.c. placebo with the meal
200
s.c. pramlintide with the meal
150
n = 14
100
240
p = 0.001
300
p = 0.0001
100
80
60
p = 0.0008
40
50
20
0
0
lag period
[min]
50 % retention (T50)
[min]
retention 60 min
[%]
%
min
0
Page 24 of 49
9
400
insulin
glucose
300
7
200
6
100
5
4
0
placebo with the meal, n = 14
s.c. pramlintide with the meal, n = 14
30
100
insulin to glucagon
25
glucagon
80
molar ratio
pg / ml
20
60
15
10
40
5
20
0
-60
0
60
120
minutes
180
240
300
-60
0
60
120
minutes
180
240
300
pmol / ml
mmol / l
8
100
80
R= 0.70
p= 0.001
60
[ mg / dl ]
incremental peak plasma glucose concentrations
Page 25 of 49
40
20
0
50
60
70
80
90
100
12
10
[ µmol * kg-1 * min-1 ]
delta mean rates exogenous glucose appearance
gastric retention [ % ], t = 45 minutes
8
6
R= 0.58
p= 0.001
4
2
0
0
0,1
0,2
0,3
0,4
0,5
0,6
delta mean rates of gastric emptying [ % per min ]
0,7
Page 26 of 49
µmol / kg / min
20
total RA
15
10
5
0
s.c. placebo with the meal, n = 14
s.c. pramlintide with the meal, n = 14
µmol / kg / min
20
15
endogenous RA
10
5
0
µmol / kg / min
20
exogenous RA
15
10
5
0
-60
0
60
120
minutes
180
240
300
Page 27 of 49
dynamic changes of RA and RD
dynamic changes of RA and RD
with placebo, n = 14
total RA
with pramlintide, n = 14
RD
20
20
15
15
10
10
5
5
excessive rates of glucose
appearance and removal
excessive rates of glucose
appearance and removal
with placebo, n = 14
with pramlintide, n = 14
µmol / kg / min
10
RA - RD
µmol / kg / min
total RA
10
RA exceeding RD
5
RA exceeding RD
5
RD exceeding RA
RD exceeding RA
0
0
-5
-5
-60
0
60
120
minutes
180
240
300
-60
0
60
120
minutes
180
240
300
µmol / kg / min
µmol / kg / min
RD
Page 28 of 49
Importance of Changes in Gastric Emptying for Postprandial Plasma
Glucose Fluxes in Healthy Humans
1
1
2
3
H.J. Woerle , M. Albrecht , R. Linke , S. Zschau , C. Neumann3, M. Nicolaus1,
J. Gerich4, B. Göke1, J. Schirra1
1
Department of Internal Medicine II, Clinical Research Unit, Grosshadern
Ludwig-Maximilians-University,
Munich, Germany
2
Department of Nuclear Medicine, Grosshadern
Ludwig-Maximilians-University,
Munich, Germany
3
Outpatient Diabetes Center,
Leopoldstraße 32, 80802 Munich, Germany
4
Department of Medicine,
University of Rochester School of Medicine,
Rochester, New York, USA
Running title: Effects of pramlintide induced delay in gastric emptying on postprandial
glucose fluxes
Key Words: Postprandial, glucose, gastric emptying, pramlintide
Word count: 5189
Address correspondence to:
Hans-Juergen Woerle
Department of Internal Medicine II
Clinical Research Unit (CRU)
University of Munich, LMU, Grosshadern
Marchioninistr. 15
81377 Munich
Germany
Tel. +49-89-7095-0, -2282, -2271
eMail: [email protected]
1
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Abstract:
Regulation of postprandial (pp) plasma glucose excursions is complex. Insulin and
glucagon secretion are thought to play the predominant role. Nevertheless, only 50% of the
variation in pp plasma glucose excursions is explained by variations in plasma insulin and
glucagon, suggesting that other factors are involved. Theoretically, gastric emptying (GE)
should be another important factor. However the impact of GE on pp glucose fluxes has not
been studied. Accordingly, we examined the consequences of pramlintide- induced
retardation in GE on pp glycemia and glucose fluxes using a combination of a double isotope
approach and scintigraphically determined rates of gastric emptying. Fourteen healthy nondiabetic subjects were studied (8 men, 6 women, 40 3 years, bodyweight 78 4 kg, BMI 27.8
1.1kg/m2), 30 µg pramlintide (PRAM) or placebo (PBO) was injected s.c. before subjects ate
a mixed meal, scrambled eggs and jello containing 50g glucose, ~25g fat and ~20g protein.
At 60 minutes 65±3 vs. 87±2 %, (p<0.001) of initial gastric contents remained in the
stomach (PBO vs. PRAM). Thereafter gastric emptying slopes paralleled one another until
240 min. The 50% retention times (T50) were lower when PBO was given (177±9 vs. 227±14
min, p<0.001). GE was greater from 240 min to the end of the experiment in the PRAM
experiment, so that only a small but still greater proportion of the meal remained in the
stomach at 330 min (5.3±1.8 vs. 11.4±3.0%, p<0.01). Reductions of GE by PRAM markedly
reduced pp plasma glucose peak and overall excursions (7.5±0.3 vs. 6.0±0.2 mmol/l, p<0.001
and 5.6±0.1 vs. 5.2±0.1 mmol/l, p<0.001, PBO vs. PRAM respectively) even though peak and
overall plasma insulin concentrations were both lower with PRAM (346±37 vs. 211±42
pmol/ml, p<0.01, and 164±13 vs.138±13 pmol/ml, p<0.01). No differences in pp plasma
glucagon concentrations were found (66 5 vs. 59 5 pg/ml, ns). The reduction in total glucose
appearance in plasma (13.1±0.6 vs. 11.0±0.5 µmol*kg-1*min-1, p<0.001) was solely
attributable to reduction in meal-derived glucose appearance (10.2±0.5 vs. 7.0±0.4 µmol*kg1
*min-1, p<0.001) since endogenous glucose appearance was greater with PRAM (2.9±0.2 vs.
4.0±0.2 µmol*kg-1*min-1, p<0.001). Splanchnic glucose uptake was greater with PRAM
(26.5±1.6 vs. 32.5±2.1%, p=0.014).
Taken together, these data support the concept that GE is an important physiological
regulator of pp plasma glucose excursions and glucose fluxes in healthy humans.
2
Page 30 of 49
Introduction
Glucose fluxes have generally been attributed to an interaction between pancreatic
islet cell function (insulin and glucagon secretion) and hepatic and peripheral sensitivity to
insulin (1;2). Although this approach can explain to a large extent fasting glucose
homeostasis, it can explain only approximately 50% of the variation in postprandial glucose
excursions (20;22). The importance of postprandial hyperglycemia has recently been
emphasized because it precedes fasting hyperglycemia as a precursor of diabetes and has been
identified as an independent risk factor for cardiovascular disease (9).
Prominent among the other factors that may determine postprandial glucose
excursions, is glucose influx from the gut (12). This appears to be determined to a certain
extent by the rate of gastric emptying since previous studies suggest that as much as 40-50%
of variation in postprandial glucose excursions may be explained by differences in the rate of
gastric emptying (11).
The present studies were therefore undertaken to test the hypothesis that slowing of
gastric emptying would reduce postprandial entry of glucose into the systemic circulation and
thus lower postprandial plasma glucose excursions. For this purpose we administered a
physiological meal, delayed gastric emptying with the amylin analogue pramlintide, and
assessed the consequences on postprandial plasma glucose concentrations, endogenous
glucose production, exogenous (meal derived) glucose appearance, splanchnic sequestration
and peripheral plasma glucose disposal.
3
Page 31 of 49
METHODS
Subjects: Informed written consent was obtained from 14 healthy volunteers after the
Ludwig Maximilian University of Munich, Institutional Review Board had approved the
protocol. The subjects (8 men, 6 women 40 ± 3 years of age, body weight 78 ± 4 kg, BMI
27.8±1.1 kg/m2) had normal physical examinations, no gastrointestinal symptoms and normal
routine laboratory tests as well as normal glucose tolerance (assessed by oral glucose
tolerance testing according to World Health Organization criteria (24)). None of the subjects
had a family history of diabetes. For three days prior to the study, all subjects had been on a
weight maintaining diet containing at least 200 g carbohydrate and had abstained from alcohol
and exercise.
Protocol: Subjects restrained from food intake at least 10 hours prior to admission to
the clinical research centre between 7:00 and 7:30 am on the study day. A dorsal hand vein
was cannulated and temperature was maintained at 40°C with a thermoregulated lamp for
arterialized venous blood sampling (20).
At 8 AM, a primed (25 µmol) continuous (~0.25 µmol/kg/min) infusion of [1-13-C]
glucose was started via a forearm vein of the contralateral arm for measurements of plasma
glucose turnover. At least three hours were allowed for achievement of isotope equilibration.
Prior meal ingestion three baseline blood samples were collected for fasting glucose, insulin,
glucagon and [1-13-C] glucose enrichments, and subjects ingested a standardized meal
containing ~500 kcal within five minutes thereafter. The meal consisted of 3 scrambled eggs
and 100 ml jello containing 50 g of glucose. Three of the 50 g of glucose were 6,6dideuteroglucose for measurements of meal-derived glucose. Consumption of 200 ml
sparkling water was allowed throughout the 330 min postprandial period. With the meal 30
µg of pramlintide or placebo (Amylin Pharmaceuticals, San Diego, USA) was injected into
the subcutaneous tissue of the lower abdominal wall. All subjects were studied on two
occasions separated by at least one day in randomized, single blinded order (pramlintide or
placebo). Results of the placebo experiments have partially been reported elsewhere under
different aspects. Subjects remained in a semi-supine position throughout the study period.
Over the initial 90 minutes of the postprandial period blood samples were taken at 15-minuteand at 30-minute-intervals thereafter upon the end of the experiments at 330 minutes
postprandial. The scrambled eggs and jelly were labeled with 99m Tc-Sn-colloid ~70 MBq
for measurements of gastric emptying by high-resolution scintigraphy (20 images/minute,
Orbiter, Siemens, Germany, Erlangen) starting immediately after completion of the meal for
the remainder of the experiment (14;15).
4
Page 32 of 49
Analytical Procedures: Gastric outlines on the maximum intensity image were defined
as the region of interest (ROI). Completion of gastric emptying was assumed at a reduction of
the initial activity to less than 5 %. Loss of activity was corrected for the radioactive half-life
of Tc99m (14). According to the procedure guideline of The Society of Nuclear Medicine (6)
a continuous framing rate of 30–60 seconds is recommended to accurately determine lag
phase and half-time of gastric emptying. Therefore, one the basis of our ‘high-resolution
scintigraphy' (20 images/minute) we calculated the time course of gastric emptying in 1
minute intervals. As recommended in the Procedure Guideline for Gastric Emptying and
Motility (6) we acquired the scintigraphic data in the left anterior oblique view with a singlehead camera in order to avoid a (mathematical) tissue attenuation correction. Since all
subjects were examined in a convenient semi-supine position movement artefacts were largely
bypassed. The cine display was used to confirm the stomach outline and to determine the
extent of patient motion so that the ROI was adjusted appropriately. In the very rare cases of a
significant subject motion the nuclear medicine postprocessing software package (Hermes,
Nuclear Diagnostics, Sweden) provides a software tool for motion correction.
Blood samples were collected for glucose concentrations, [1-13-C] glucose, [6,6-2H2]
glucose enrichments in oxalate-fluoride tubes, and for insulin and glucagon concentrations in
EDTA tubes containing a protease inhibitor. Samples were immediately placed in a 4°C ice
bath and plasma was separated within 30 min by centrifugation at 4°C. Plasma glucose
concentrations, [1-13-C] glucose enrichments, [6,6-2H2] glucose enrichments were measured
by previously described methods (4;18). Plasma insulin and glucagon concentrations were
determined by standard radioimmunoassays, as previously described (19). Coefficients of
variation (CV) for insulin and glucagon assays were 2.2 % and 4.0 % within assay,
respectively.
Calculations: Systemic release and uptake of glucose were calculated with steady state
equations prior to meal ingestion (23) and subsequently with non-steady state equations of
DeBodo et al (5) using a pool fraction of 0.65 and a volume of distribution of 200 ml/kg.
The rate of appearance of the oral glucose load in the systemic circulation was
calculated from [6,6-2H2] glucose data with the equation of Chiasson et al (3;13). Endogenous
glucose release was calculated as the difference between the overall rate of plasma glucose
appearance and the rate of appearance of exogenous glucose (13;16). Overall splanchnic
glucose disposal of the ingested glucose load was calculated as the difference between the
5
Page 33 of 49
amount of glucose ingested and the total appearance of the ingested glucose in the systemic
circulation during the 330 min postprandial period based on the proportion of the meal being
emptied as determined by measurements of gastric emptying at the end of the experiment.
Statistical analysis: Data are given as mean ± SEM unless otherwise specified using
Statistica statistical software (1998 edition, Statsoft Inc, Tulsa, Okla). Normality of the
distribution was assessed using the Kolmogorov-Smirnov test. For comparison of the two
groups and to compare baseline data with mean data after meal ingestion ANOVA for
repeated measurements followed by post-hoc comparison for paired groups was
performed. A p value less than 0.05 was considered statistically significant. Correlation
between variables was performed using Spearman’s regression analysis. Values for the
description of dynamic changes represent absolute values, unless specified otherwise.
6
Page 34 of 49
RESULTS
% Gastric Retention, Gastric Retention Slopes, Lag Periods, 50% Retention (T50) and %
Retention at 60- Minutes (Figure 1):
Gastric content decreased rapidly within 60 min to a mean value of 77±2 %, when
placebo was administered. At the end of the experiment only 6±2 % of the initial 99mTc
counts could be detected in the region of interest. In contrast, subcutaneous pramlintide
injection with the meal resulted in a significantly greater percentage of gastric content
remaining in the stomach at 60 minutes (92±1 %, p=0.0001 compared to placebo).
Pramlintide prolonged the lag period (25±8 vs. 74±11 min, p=0.0008), defined as the delay
between ingestion of food and a 10% reduction of isotope counts in the region of interest. A
significant overall effect of pramlintide on gastric emptying slopes could be detected.
Assessing retention slopes 0 – 60, 60 – 240, 240 – 330 minutes yielded significantly lower avalues for 0 – 60 minutes (a = -0.38±0.03 vs. -0.13±0.02, p<0.001), comparable slopes for 60
– 240 minutes (a = -0.31±0.02 vs. –0.27±0.02, p=0.08) and significantly greater a- values for
240 – 330 minutes (a = -0.17±0.03 vs. –0.33±0.04, p=0.004) in the pramlintide experiment.
Thus the significantly greater proportion of the meal remaining at the end of the experiment
(14±4 %, p=0.009) was largely due to the delay in gastric emptying within the first 60
minutes after meal ingestion which resulted in a significant delay of the 50% retention time
(T50) from 177±9 to 227±14 min (p=0.001). Within the first 60 minutes a total of 23±2 vs.
8±1 %, p=0.0001, of the meal was emptied by the stomach.
Plasma Glucose (Figure 2):
Plasma glucose concentrations prior to meal ingestion were comparable on both study
days (4.5±0.1 vs. 4.6±0.1 mmol/l, p=0.47, placebo vs. pramlintide, respectively). In the
placebo experiments plasma glucose concentrations rose to a maximum of 7.5±0.3 mmol/l at
60 min and returned to baseline levels at 180 min. In the pramlintide experiments peak
postprandial plasma glucose concentrations were significantly lower (6.0±0.2 mmol/l at 120
min compared to 7.5±0.3 mmol/l at 60 min, p=0.0002). Mean postprandial plasma glucose
concentrations were significantly reduced after pramlintide administration (5.6±0.1 vs.
5.2±0.1 mmol/l, p=0.004). Areas under the plasma glucose curves (AUC) over the entire
postprandial period were lower with pramlintide (354.2±31.1 vs. 207.0±25.9 mmol/l * min, p
= 0.003).
7
Page 35 of 49
Plasma Insulin and Glucagon (Figure 2):
Plasma insulin concentrations paralleled those of plasma glucose concentrations (see
above): Peak plasma insulin concentrations were significantly greater and earlier when
placebo was administered (346±37 pmol/ml at 45 min vs. 211±42 pmol/ml at 90 minutes,
both p=0.001) compared to pramlintide. Average plasma insulin concentrations were
significantly greater during the placebo experiment compared to pramlintide (188±15 vs.
134±13 pmol/ml, p=0.001).
Basal plasma glucagon concentrations were comparable on both study days
(59.7±13.4 vs. 58.1±5.8 pg/ml, p=0.68), decreased to a comparable extent (nadir 48.1±5.8 vs.
50.0±3.7 pg/ml at 90 min, p=0.70, placebo vs. pramlintide respectively) and overall were not
significantly different (65.6±4.7 vs. 59.3±5.0 pg/ml, p=0.074, placebo vs. respectively).
As a result of the above differences attributable primarily to differences in plasma
insulin concentrations, the plasma insulin-to-glucagon molar ratio during the initial 90
minutes after meal ingestion was significantly greater after placebo administration compared
to pramlintide (18.7± 1.5 vs. 9.0± 1.9, p=0.001) and significantly lower between 150 and 330
minutes averaging 5.0± 0.6 vs. 8.7± 1.1, p=0.001.
Rates of Total Plasma Glucose Appearance, Exogenous (Meal) Plasma Glucose Appearance
and Endogenous Plasma Glucose Appearance (Figure 4):
Rates of basal glucose turnover were comparable on both study days (9.0±0.3 vs. 9.4±0.5
µmol*kg-1*min-1, p=0.11, placebo vs. pramlintide, respectively). Pramlintide reduced peak
(18.6±1.6 µmol*kg-1*min-1 at 30 min vs. 12.7±1.0 µmol*kg-1*min-1 at 120, p<0.01) and
overall rates of total glucose appearance in plasma (12.0±0.5 vs. 10.7±0.5 µmol*kg-1*min-1,
p=0.005).
Endogenous glucose production decreased more promptly in the placebo experiment
reaching a nadir of 1.4±1.0 µmol*kg-1*min-1 at 45 min compared to a nadir of 3.1±0.7
µmol*kg-1*min-1 at 90 min in the pramlintide experiment. On average 3.5±0.2 vs. 4.4±0.2
µmol*kg-1*min-1, p<0.001, of glucose were endogenously released in the placebo and
pramlintide experiments, respectively. This corresponded to a 60.6±1.5 vs. 55.0±1.0 %,
p=0.008, suppression of endogenous glucose production in the postprandial state.
8
Page 36 of 49
Rates of exogenous glucose appearance (meal-derived glucose) increased to a
maximum of 15.4±1.1 µmol*kg-1*min-1 within 45 minutes after meal ingestion and declined
steadily thereafter when placebo was administered with the meal.
Pramlintide decreased peak (15.4±1.1 vs. 8.9±0.7 µmol*kg-1*min-1, p<0.001) and overall
rates of exogenous glucose appearance (8.5±0.4 compared to 6.3±0.4 µmol*kg-1*min-1,
p<0.0001). Exogenous glucose appearance accounted for 70.7±1.0 % of total glucose
appearance in the placebo experiment compared to 58.6±1.6% in the pramlintide experiment,
p<0.001. The differences in rates of total glucose appearance (1.3±0.4 µmol*kg-1*min-1,
p=0.005, see above) could thus be solely explained by differences in exogenous glucose
appearance (2.2±0.3 µmol*kg-1*min-1) since endogenous glucose appearance rates were
greater in the pramlintide experiment (0.9±0.2 µmol*kg-1*min-1, p=0.001).
Splanchnic Glucose Uptake:
Splanchnic glucose uptake estimated from the calculated proportion of the emptied
gastric content, which did not appear in the systemic circulation, was significantly greater
when pramlintide was administered (26.5±1.6 vs. 32.5±2.1%, p=0.014) at the end of the
experiment.
Rates of Glucose Disappearance (Figure 5):
In placebo experiments rates of glucose disappearance were not significantly different
from baseline values during the initial 45 minutes after meal ingestion averaging 9.2±0.8
µmol*kg-1*min-1, p=0.8, increased to maximum values of 17.1±1.5 µmol*kg-1*min-1 at 90
minutes and decreased steadily thereafter. In the pramlintide experiment rates of
disappearance were not significantly different from baseline during the first 90 minutes
averaging 8.8±0.5 µmol*kg-1*min-1, p=0.17, with peak values of 14.5±0.9 µmol*kg-1*min-1 at
210 min which were significantly lower compared to the placebo experiment, p=0.001.
In the placebo experiment rates of plasma glucose appearance exceeded those of its
removal by 5.6±0.6 µmol*kg-1*min-1 corresponding to a total excess of 332±35 µmol*kg-1. In
the pramlintide experiment only 1.7±0.2 µmol*kg-1*min-1 had not been removed from the
circulation corresponding to 201±20 µmol*kg-1 of excessive glucose, both significantly lower
in the pramlintide experiments, p=0.001 and p=0.0001, respectively, explaining differences in
peak plasma glucose concentrations. Since rates of removal of excessive plasma glucose
exceeded its appearance by only -2.84±0.1 µmol*kg-1*min-1 vs. -1.4±0.2 µmol*kg-1*min-1,
9
Page 37 of 49
placebo vs. pramlintide, baseline plasma glucose values were not achieved until 120 minutes
after peak plasma glucose concentrations in both experiments, explaining differences in
overall plasma glucose excursions above baseline.
Regression analyses using rates of gastric emptying as the independent variable and
peak plasma glucose concentrations and rates of exogenous glucose appearance as dependent
variables:
A significant inverse correlation between rates of gastric retention at 45 minutes and
incremental peak plasma glucose concentrations (at 60 minutes in the placebo and 120
minutes in the pramlintide experiment) was found (R=0.702, p=0.001) (Figure 3a).
Furthermore, the differences in gastric retention between the placebo and the pramlintide
experiment correlated significantly with differences in incremental peak plasma glucose
concentrations at 60 and 120 minutes, respectively (r=0.474, p=0.001).
There was a significant correlation between overall rates of gastric emptying and rates
of exogenous glucose appearance (r=0.564, P=0.001). Furthermore, the differences in rates of
meal-derived glucose appearance between placebo and pramlintide followed to peak plasma
glucose concentrations (0-60 min and 0-120 min in the placebo and pramlintide experiments,
respectively) correlated significantly with the differences in rates of gastric emptying over the
prevailing time intervals (r=0.580, p=0.001) (Figure 3b).
10
Page 38 of 49
Discussion:
The present studies were undertaken to test the hypothesis that slowing of gastric
emptying would reduce postprandial entry of glucose into the systemic circulation and thus
lower postprandial plasma glucose excursions. The hormone amylin is co- secreted with
insulin from the pancreatic beta- cell (7). Amylin as well as its analogue pramlintide have
been shown to markedly delay gastric emptying (17). Accordingly, we used pramlintide to
delay gastric emptying and found that the delay of gastric emptying was associated with a
marked reduction of meal- derived glucose in plasma especially within the first 60 minutes
after meal ingestion and a significant reduction in postprandial plasma glucose excursions.
The reductions in postprandial plasma glucose excursions could be explained by both a
reduction in glucose appearance in plasma solely due to meal-derived glucose and a relatively
greater proportional uptake of glucose by peripheral tissues.
Pramlintide administration was associated with a significant overall effect on gastric
emptying. The delay in gastric emptying occurred primarily due to a ~20 % reduction in
gastric emptying within the first 60 minutes, whereas gastric emptying was found to be
comparable to placebo between 75 and 240 minutes and somewhat greater thereafter. This
delay in gastric emptying was accompanied by a significant reduction in peak and overall
postprandial plasma glucose concentrations. Rates of meal derived glucose appearance in the
systemic circulation are determined by rates of gastric emptying and by the amount of glucose
being sequestrated in the splanchnic bed (22). Previous studies in healthy humans found
approximately 25-30% of the glucose load being sequestrated in the splanchnic bed (13). In
our placebo experiment we found 26.7±1.6 % of glucose being sequestrated. Estimation of
splanchnic sequestration in the pramlintide experiment yielded a significant increase
(32.5±2.1%, p<0.05) in the proportion of the oral glucose load being sequestrated. However,
these estimates have to be interpreted with caution since calculations of sequestration depend
on the amounts of glucose being absorbed rather than emptied by the stomach. Differences in
glucose absorption and, i.e. greater rates in non-oxidative glycolysis cannot be excluded and
may abolish differences in calculated rates of glucose sequestration. On the other hand even if
splanchnic glucose sequestration had been comparable this would have occurred in the face of
significantly lower postprandial insulin to glucagon ratios which should have been
accompanied with lower splanchnic sequestration rather than comparable or even increased
rates. Thus it appears that modulation of gastric emptying, especially a reduction of the initial
emptying may have indeed improved splanchnic sequestration.
11
Page 39 of 49
Pramlintide has been reported to suppress postprandial glucagon concentrations (8).
However, we found no significant differences in plasma glucagon concentrations and, even
more importantly, greater rates of endogenous glucose production in the pramlintide
experiments presumably due to lower postprandial plasma insulin concentrations.
Plasma glucose concentrations increase until rates of plasma glucose removal equal its
appearance (21). Postprandially rates of plasma glucose appearance are largely determined by
meal- derived plasma glucose appearance responsible for the disequilibrium between rates of
glucose appearance and disappearance (22). Delay in gastric emptying was associated with a
reduction in the amount of meal derived glucose entering the circulation. This reduced the
disequilibrium between the rates of entry and exit of glucose from the circulation and thus
reduced postprandial peak plasma glucose excursions by ~ 40 %. The more glucose is rapidly
added to the plasma pool, the longer it will take to remove the additional amounts of glucose
(10). Accordingly, overall postprandial plasma glucose excursions are largely determined by
the initial efflux of glucose into the circulation. It is important to note that this occurred in the
face of significantly greater plasma insulin and comparable glucagon concentrations. Thus
modification of glucose efflux via the gut, especially deceleration of initial gastric emptying
had led to a relatively more sufficient disposal of glucose in the face of lower plasma insulin
concentrations.
Taken together, these studies highlight the importance of gastric emptying to
determine postprandial glucose fluctuations. The pramlintide induced initial delay in gastric
emptying was associated with a reduction in meal- derived glucose appearance in plasma.
Since endogenous glucose production was greater in the pramlintide experiment, reduction in
total glucose appearance was solely attributable to reductions in meal- derived glucose
appearance. A greater proportion of the meal derived glucose was sequestrated in the
splanchnic bed. Since the initial rapid efflux of glucose into the circulation largely determines
postprandial plasma glucose excursions, delay in gastric emptying significantly reduced
postprandial plasma glucose concentrations.
12
Page 40 of 49
Acknowledgements
We thank the laboratory staff of the Clinical Research Centre and are especially indebted to
Rita Schinkmann and Silke Herrmann for their superb help.
Grants
The present work was supported by the Deutsche Forschungsgemeinschaft Grant SchI527/5-2
to H.J. Woerle and J. Schirra, and an unrestricted Grant by Amylin Pharmaceuticals, San
Diego, USA, and the National Institute Diabetes and Digestive and Kidney Disease Grant
DK-20411 to J. Gerich
13
Page 41 of 49
Figure 1: % Gastric Retention, Gastric Retention Slopes, Lag Periods, 50% Retention (T50)
and % Retention at 60- Minutes
100
s.c. placebo with the meal
s.c. pramlintide with the meal
80
% gastric retention
n = 14
60
40
gastric retention
20
0
0 – 60
minutes
60 – 240
minutes
80
placebo
% gastric retention
100
240 - 330
minutes
60
40
20
a = -0.38 ± 0.03
0
a = -0.31 ± 0.02
p < 0.0001
p = 0.08
a = -0.13 ± 0.02
pramlintide
% gastric retention
100
a = -0.17 ± 0.03
a = -0.27 ± 0.02
p = 0.004
80
a = -0.33 ± 0.04
60
40
20
retention slopes (a)
0
60
120
180
minutes
250
s.c. placebo with the meal
200
s.c. pramlintide with the meal
150
n = 14
100
240
p = 0.001
300
p = 0.0001
100
80
60
p = 0.0008
%
min
0
40
50
20
0
0
lag period
[min]
50 % retention (T50)
[min]
retention 60 min
[%]
14
Page 42 of 49
Figure 2: Plasma Glucose, Insulin, Glucagon Concentrations, Insulin-Glucagon Molar Ratio
9
400
insulin
glucose
300
7
200
6
pmol / ml
mmol / l
8
100
5
4
0
placebo with the meal, n = 14
s.c. pramlintide with the meal, n = 14
30
100
insulin to glucagon
glucagon
80
25
molar ratio
pg / ml
20
60
15
10
40
5
20
0
-60
0
60
120
minutes
180
240
300
-60
0
60
120
180
240
300
minutes
15
Page 43 of 49
Figure 3a: Gastric Retention at 45 Minutes vs. Incremental Peak Plasma Glucose
Concentrations (above)
Figure 3b: Differences in Rates of Meal-Derived Glucose Appearance Placebo and
Pramlintide vs. Differences in Rates of Gastric Emptying (below)
80
R= 0.70
p= 0.001
[ mg / dl ]
incremental plasma glucose concentrations
100
60
40
20
0
50
60
70
80
90
100
12
10
[ µmol * kg-1 * min-1 ]
delta mean rates exogenous glucose appearance
gastric retention [ % ], t = 45 minutes
8
6
R= 0.58
p= 0.001
4
2
0
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
delta mean rates of gastric emptying [ % per min ]
16
Page 44 of 49
Figure 4: Rates of Total Plasma Glucose Appearance, Exogenous (Meal) Plasma Glucose
Appearance and Endogenous Plasma Glucose Appearance
µmol / kg / min
20
total RA
15
10
5
0
s.c. placebo with the meal, n = 14
s.c. pramlintide with the meal, n = 14
µmol / kg / min
20
15
endogenous RA
10
5
0
µmol / kg / min
20
exogenous RA
15
10
5
0
-60
0
60
120
180
240
300
minutes
17
Page 45 of 49
Figure 5: Rates of Glucose Disappearance, Rates of excessive Glucose Appearance and
Removal
dynamic changes of RA and RD
dynamic changes of RA and RD
with placebo, n = 14
total RA
with pramlintide, n = 14
RD
20
20
15
15
10
10
5
5
excessive rates of glucose
appearance and removal
excessive rates of glucose
appearance and removal
with placebo, n = 14
with pramlintide, n = 14
RA - RD
10
µmol / kg / min
µmol / kg / min
total RA
10
RA exceeding RD
5
RA exceeding RD
5
RD exceeding RA
RD exceeding RA
0
0
-5
-5
-60
0
60
120
minutes
180
240
300
-60
0
60
120
180
240
µmol / kg / min
µmol / kg / min
RD
300
minutes
18
Page 46 of 49
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