# Effects of Oral Glucose Load on Endothelial Function and  on Insulin and Glucose Fluctuations in Healthy Individuals

Authors: A. Major-Pedersen; N. Ihlemann; T. S. Hermann; B. Christiansen; H. Dominguez; B. Kveiborg; D. B. Nielsen; O. L. Svendsen; L. Køber; C. Torp-Pedersen
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Background/aims. Postprandial hyperglycemia, an independent risk factor for cardiovascular disease, is accompanied by endothelial dysfunction. We studied the effect of oral glucose load on insulin and glucose fluctuations, and on postprandial endothelial function in healthy individuals in order to better understand and cope with the postprandial state in insulin resistant individuals. Methods. We assessed post-oral glucose load endothelial function (flow mediated dilation), plasma insulin, and blood glucose in 9 healthy subjects.  Results. The largest increases in delta FMD values (fasting FMD value subtracted from postprandial FMD value) occurred at 3 hours after both glucose or placebo load, respectively: 4.80±1.41 (P = .009) and 2.34±1.47 (P = .15). Glucose and insulin
concentrations achieved maximum peaks at one hour post-glucose
load. Conclusion. Oral glucose load does not induce
endothelial dysfunction in healthy individuals with mean insulin
and glucose values of 5.6 mmol/L  and 27.2 mmol/L,
respectively, 2 hours after glucose load.


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## Body

## 1. Introduction

Postprandial hyperglycemia is an independent risk
factor for cardiovascular disease (CVD) in insulin-resistant individuals with
type 2 diabetes or with the prediabetic state, impaired glucose tolerance (IGT)
[1–10].
In line with these epidemiologic data, several
studies have shown impairment of endothelial function, one of the
earliest markers of atherosclerosis [11] after a meal or glucose
challenge, in individuals with diabetes or IGT [12–17].Endothelial function following acute hyperglycemia
has also been studied in healthy individuals. Some groups have shown  attenuation of endothelial function [18–26],
whereas others have shown no change [27–31]. A few studies have shown
acute hyperglycemia induced vasodilation in healthy individuals [32, 33], and only 2 studies (in
vitro/ex vivo studies) have specifically reported glucose induced enhancement
of the endothelial function [34, 35].These seemingly conflicting results could be
attributed to differences in methodology and subject characteristics: diverging
periods of post-glucose load observations, varying periods of exposition to
different concentrations of glucose, diverging criteria in subject selection,
enabling versus blocking insulin’s physiologic response, and finally
administrating oral glucose load versus intra-arterial or intravenous glucose
administration.Endothelial dysfunction is a predictor of
cardiovascular risk; consequently, postprandial endothelial dysfunction might
be a very early marker of atherosclerosis and, thus, an early potential target
for the prevention of cardiovascular complications in insulin resistance. In
order to better understand and cope with postprandial endothelial dysfunction and
accompanying postprandial hyperinsulinemia and hyperglycemia in insulin
resistant individuals, we find it important to study postprandial glucose and
insulin fluctuations and postprandial endothelial function in healthy,
insulin-sensitive people.


## 2. Material and Methods

### 2.1. Study Population

Ten non-smoking subjects, with no history of cardiovascular or other chronic
disease, with no signs of current acute disease, and no familiar disposition to
glucose intolerance, were included after screening a total of 13 individuals.
We assessed blood pressure, ECG, oral glucose tolerance test, calculations of body
mass index (BMI) and homeostasis model assessment (HOMA), and biochemical
parameters. Three subjects were excluded: two due to total cholesterol > 6
mmo/L and one due to history of anorexia nervosa. Participants' characteristics
are shown on Table1. All subjects gave written informed consent and the study
was approved by the ethics committee for Copenhagen and Frederiksberg counties.Table 1
Subject characteristics. Data are presented as mean value ±
SEM.
Variable(n = 10)Age (years)41.1±3.0Gender (F/M)4/6Bo dy mass
index (kg/m2)23.2±0.71Waist circumference (cm)80.7±2.9Glucose-fasting (mmol/l)4.12±0.12Glucose-2  hour post-glucose load (mmol/l)5.58±0.32P-fasting insulin (μU/ml)2.96±0.37HbA1c(%)5.06±0.2P-fasting
triglycerides (mmol/l)0.75±0.1P-CRP (mg/l)1.14±0.1HOMA (kg/m2)0.65±0.09


### 2.2. Study Design

Cross over study,n = 10. Subjects met on 2 separate days at 8:00
am after a 12-hour fast and after at least 18 hours abstinence from
exercise. On the “glucose day”, endothelial function was measured before a 75 g
oral glucose load (200 mL) and again at 1, 2, 3, and 4 hours after glucose
load. On the “placebo day”, the same procedure was followed, but the 75 g
glucose was substituted with placebo (200 mL tap water) as shown in Figure 1. The first FMD measurement of
the day commenced between 8:30 and 9:00 am, glucose/H20 was given between 9:00 and 9:30, and the last FMD measurement of
the day ended between 13:30 and 14:00.Figure 1
Flow chart showing the study sequence during a study day.
FMD1, FMD2, FMD3, and FMD4, respectively. Flow mediated dilation study
at 1, 2, 3, and 4 hours after glucose/placebo load. FMD0, flow mediated dilation study
before glucose/placebo load.We extrapolated our post-glucose load findings to
postprandial effects, since plasma glucose concentrations measured 2 hours after
the ingestion of 75 g glucose have been found to be closely related to those
measured after a standardized mixed meal [36].


### 2.3. Measurements of Endothelial Function

We used theflow mediated dilation
(FMD) method [37, 38]. 20 minutes after supine rest,
a pneumatic cuff was placed distal to the antecubital fossa, a 7.5 MHz linear
array transducer (Cypress/Siemens, Siemens Medical Solutions USA, Inc. Mountain
View, CA) was placed proximal to the antecubital fossa, and the brachial artery
was viewed in a 2-dimensional (B-mode) longitudinal plane. A baseline picture
was saved to ensure that the same brachial picture was found in the successive
studies. After acquiring a two-minute baseline image, the cuff was rapidly
inflated to 300 mmHg (VBM tourniquets, Braun Scandinavia) for 5 minutes,
followed by rapid cuff deflation. Images were registered during 4 minutes after
cuff deflation. Brachial diameters (at baseline and after cuff-deflation) were
measured simultaneously by automated software (VIA online flow mediated dilation
software, MD Medic) [39]. This software enabled us to
precisely localize the greatest EDV since dilation was depicted graphically (as
well as numerically).The following formula was used to calculate relative
change in FMD (expressed in %): ((Peak post cuff-deflation diameter − basline diameter)/baseline diameter)×100
[40].An intravenous cannula was inserted into a large
antecubital vein in the left arm for blood sampling.


### 2.4. Biochemical Measurements

Blood samples were drawn on both days before oral glucose load/placebo
administration and at 15, 30, 60, 120, 180, and 240 minutes after plasma was
separated after complete clot formation, subsequent to centrifugation, and
thereafter frozen at −80°C for posterior analysis of insulin (Immulite 2000, DPC
Scandinavia, Denmark).Whole venous blood glucose was measured at the
aforementioned times with HemoCue glucose (HemoCue AB, Angelholm, Sweden).


### 2.5. Data Analysis

Data are presented as mean ± SEM. Comparisons between time points on the same curve (pre-oral glucose load, 1hPG,
2hPG, 3hPG, and 4hPG) for FMD, insulin, and glucose were evaluated with paired studentt test for each curve separately. The level of statistical significance
used was P < .05. For comparison of FMD time response studies between
the 2 curves (post-glucose curve versus post-placebo curve), we used MIXED MODEL analysis (SAS statistical
software version 9.1, SAS Institute, Cary, NC, USA).Model assumptions of homogeneity of variance and
normal distribution of residuals were checked graphically. Subjects entered the
model as random effect as did the interaction between subject and time. Time
and experimental protocols
(glucose versus placebo) were entered as fixed effects.


## 2.1. Study Population

Ten non-smoking subjects, with no history of cardiovascular or other chronic
disease, with no signs of current acute disease, and no familiar disposition to
glucose intolerance, were included after screening a total of 13 individuals.
We assessed blood pressure, ECG, oral glucose tolerance test, calculations of body
mass index (BMI) and homeostasis model assessment (HOMA), and biochemical
parameters. Three subjects were excluded: two due to total cholesterol > 6
mmo/L and one due to history of anorexia nervosa. Participants' characteristics
are shown on Table1. All subjects gave written informed consent and the study
was approved by the ethics committee for Copenhagen and Frederiksberg counties.Table 1
Subject characteristics. Data are presented as mean value ±
SEM.
Variable(n = 10)Age (years)41.1±3.0Gender (F/M)4/6Bo dy mass
index (kg/m2)23.2±0.71Waist circumference (cm)80.7±2.9Glucose-fasting (mmol/l)4.12±0.12Glucose-2  hour post-glucose load (mmol/l)5.58±0.32P-fasting insulin (μU/ml)2.96±0.37HbA1c(%)5.06±0.2P-fasting
triglycerides (mmol/l)0.75±0.1P-CRP (mg/l)1.14±0.1HOMA (kg/m2)0.65±0.09


## 2.2. Study Design

Cross over study,n = 10. Subjects met on 2 separate days at 8:00
am after a 12-hour fast and after at least 18 hours abstinence from
exercise. On the “glucose day”, endothelial function was measured before a 75 g
oral glucose load (200 mL) and again at 1, 2, 3, and 4 hours after glucose
load. On the “placebo day”, the same procedure was followed, but the 75 g
glucose was substituted with placebo (200 mL tap water) as shown in Figure 1. The first FMD measurement of
the day commenced between 8:30 and 9:00 am, glucose/H20 was given between 9:00 and 9:30, and the last FMD measurement of
the day ended between 13:30 and 14:00.Figure 1
Flow chart showing the study sequence during a study day.
FMD1, FMD2, FMD3, and FMD4, respectively. Flow mediated dilation study
at 1, 2, 3, and 4 hours after glucose/placebo load. FMD0, flow mediated dilation study
before glucose/placebo load.We extrapolated our post-glucose load findings to
postprandial effects, since plasma glucose concentrations measured 2 hours after
the ingestion of 75 g glucose have been found to be closely related to those
measured after a standardized mixed meal [36].


## 2.3. Measurements of Endothelial Function

We used theflow mediated dilation
(FMD) method [37, 38]. 20 minutes after supine rest,
a pneumatic cuff was placed distal to the antecubital fossa, a 7.5 MHz linear
array transducer (Cypress/Siemens, Siemens Medical Solutions USA, Inc. Mountain
View, CA) was placed proximal to the antecubital fossa, and the brachial artery
was viewed in a 2-dimensional (B-mode) longitudinal plane. A baseline picture
was saved to ensure that the same brachial picture was found in the successive
studies. After acquiring a two-minute baseline image, the cuff was rapidly
inflated to 300 mmHg (VBM tourniquets, Braun Scandinavia) for 5 minutes,
followed by rapid cuff deflation. Images were registered during 4 minutes after
cuff deflation. Brachial diameters (at baseline and after cuff-deflation) were
measured simultaneously by automated software (VIA online flow mediated dilation
software, MD Medic) [39]. This software enabled us to
precisely localize the greatest EDV since dilation was depicted graphically (as
well as numerically).The following formula was used to calculate relative
change in FMD (expressed in %): ((Peak post cuff-deflation diameter − basline diameter)/baseline diameter)×100
[40].An intravenous cannula was inserted into a large
antecubital vein in the left arm for blood sampling.


## 2.4. Biochemical Measurements

Blood samples were drawn on both days before oral glucose load/placebo
administration and at 15, 30, 60, 120, 180, and 240 minutes after plasma was
separated after complete clot formation, subsequent to centrifugation, and
thereafter frozen at −80°C for posterior analysis of insulin (Immulite 2000, DPC
Scandinavia, Denmark).Whole venous blood glucose was measured at the
aforementioned times with HemoCue glucose (HemoCue AB, Angelholm, Sweden).


## 2.5. Data Analysis

Data are presented as mean ± SEM. Comparisons between time points on the same curve (pre-oral glucose load, 1hPG,
2hPG, 3hPG, and 4hPG) for FMD, insulin, and glucose were evaluated with paired studentt test for each curve separately. The level of statistical significance
used was P < .05. For comparison of FMD time response studies between
the 2 curves (post-glucose curve versus post-placebo curve), we used MIXED MODEL analysis (SAS statistical
software version 9.1, SAS Institute, Cary, NC, USA).Model assumptions of homogeneity of variance and
normal distribution of residuals were checked graphically. Subjects entered the
model as random effect as did the interaction between subject and time. Time
and experimental protocols
(glucose versus placebo) were entered as fixed effects.


## 3. Results

### 3.1. Endothelial Function

Delta FMD values (fasting FMD value subtracted from postprandial FMD value) ± SEM for
post-glucose load and post-placebo load showed increasing FMD delta values for
both days (see Table2). The largest
post-glucose load increase occurred at 3hPG (P = .009); compared to baseline, the increase was
already significant at 1hPG (P = 0.03). The largest post-placebo
increase also occurred at 3 hours post-placebo (P                                                                         = .15) but was not significant (see Table 2). When we compared the two curves employing mixed
models (post-glucose load curve versus post-placebo load curve), we found the
curves to be statistically different (P = .0007).Table 2
Effects of glucose load and placebo load on FMD, blood glucose and plasma insulin. Data
are presented as mean value ± SEM.
TimesDelta FMD
(%)Blood Glucose
(mmol/l)Plasma Insulin
(μU/l)Glucose
loadPlacebo
loadGlucose
loadPlacebo
loadGlucose loadPlacebo
loadPre-load004.12±0.124.46±0.172.96±0.374.24±0.671 h2.10±0.840.19±0.706.21±0.794.54±0.1534.42±4.993.30±0.552 h0.65±0.851.66±0.845.58±0.324.64±0.1927.24±3.032.89±0.483 h4.80±1.42.34±1.474.23±0.374.53±0.1712.67±2.543.44±0.824 h2.0±1.390.9±1.443.35±0.174.27±0.184.81±0.882.90±0.58


### 3.2. Metabolic Parameters

#### 3.2.1. Glucose

Post-glucose load mean blood
glucose concentration values showed a significant increase at both 1hPG (P =
.03) and 2hPG (P = .002). Conversely, a significant fall in blood sugar
was observed at 4hPG (P = .002, n = 6 since the study was interrupted
before the fourth postprandial hour in 3 subjects due to symptomatic
hypoglycemia). As expected, post-placebo load values showed no significant
change in blood glucose concentrations in the fasting state (see Table 2).


#### 3.2.2. Insulin

Mean insulin concentration post-glucose load values showed a marked increase at 1hPG                                                                        (P = .0006) and fell rapidly thereafter, achieving pre-glucose-like
values by 3hPG. Post-placebo load values fell across the 4 hour post-load
period, which was already apparent at one hour post placebo load (P = .0006)
(Table 2).


## 3.1. Endothelial Function

Delta FMD values (fasting FMD value subtracted from postprandial FMD value) ± SEM for
post-glucose load and post-placebo load showed increasing FMD delta values for
both days (see Table2). The largest
post-glucose load increase occurred at 3hPG (P = .009); compared to baseline, the increase was
already significant at 1hPG (P = 0.03). The largest post-placebo
increase also occurred at 3 hours post-placebo (P                                                                         = .15) but was not significant (see Table 2). When we compared the two curves employing mixed
models (post-glucose load curve versus post-placebo load curve), we found the
curves to be statistically different (P = .0007).Table 2
Effects of glucose load and placebo load on FMD, blood glucose and plasma insulin. Data
are presented as mean value ± SEM.
TimesDelta FMD
(%)Blood Glucose
(mmol/l)Plasma Insulin
(μU/l)Glucose
loadPlacebo
loadGlucose
loadPlacebo
loadGlucose loadPlacebo
loadPre-load004.12±0.124.46±0.172.96±0.374.24±0.671 h2.10±0.840.19±0.706.21±0.794.54±0.1534.42±4.993.30±0.552 h0.65±0.851.66±0.845.58±0.324.64±0.1927.24±3.032.89±0.483 h4.80±1.42.34±1.474.23±0.374.53±0.1712.67±2.543.44±0.824 h2.0±1.390.9±1.443.35±0.174.27±0.184.81±0.882.90±0.58


## 3.2. Metabolic Parameters

### 3.2.1. Glucose

Post-glucose load mean blood
glucose concentration values showed a significant increase at both 1hPG (P =
.03) and 2hPG (P = .002). Conversely, a significant fall in blood sugar
was observed at 4hPG (P = .002, n = 6 since the study was interrupted
before the fourth postprandial hour in 3 subjects due to symptomatic
hypoglycemia). As expected, post-placebo load values showed no significant
change in blood glucose concentrations in the fasting state (see Table 2).


### 3.2.2. Insulin

Mean insulin concentration post-glucose load values showed a marked increase at 1hPG                                                                        (P = .0006) and fell rapidly thereafter, achieving pre-glucose-like
values by 3hPG. Post-placebo load values fell across the 4 hour post-load
period, which was already apparent at one hour post placebo load (P = .0006)
(Table 2).


## 3.2.1. Glucose

Post-glucose load mean blood
glucose concentration values showed a significant increase at both 1hPG (P =
.03) and 2hPG (P = .002). Conversely, a significant fall in blood sugar
was observed at 4hPG (P = .002, n = 6 since the study was interrupted
before the fourth postprandial hour in 3 subjects due to symptomatic
hypoglycemia). As expected, post-placebo load values showed no significant
change in blood glucose concentrations in the fasting state (see Table 2).


## 3.2.2. Insulin

Mean insulin concentration post-glucose load values showed a marked increase at 1hPG                                                                        (P = .0006) and fell rapidly thereafter, achieving pre-glucose-like
values by 3hPG. Post-placebo load values fell across the 4 hour post-load
period, which was already apparent at one hour post placebo load (P = .0006)
(Table 2).


## 4. Discussion

Our principal finding was that oral glucose load did
not impair endothelial function in conduit arteries in our group of healthy,
insulin-sensitive individuals with mean 2hPG of
5.58 mmol/L. We found maximum glucose and insulin peak concentrations at
1hPG. Furthermore our findings imply that oral glucose load might enhance
endothelial function in these individuals.Many studies have examined
the effect of acute hyperglycemia on the healthy endothelium [18, 20, 22, 23, 30, 41]. These studies, however,
cannot be directly compared to ours since both the methodology and the purpose
of the studies diverge from our study. The aforementioned studies demonstrated
that hyperglycemia induced impaired endothelial function, in healthy
individuals, by either applying a local glucose/dextrose clamp or by
intra-arterial glucose infusion, hence obtaining elevated hyperglycemic values
(7.0 to 16.7 mmol/L). Yet, postprandial hormonal
mechanisms, such as those leading to the “incretin effect” [42], may not be triggered when
examined through the hyperglycemic clamp technique or local hyperglycemia. Some
of these studies [30, 41] had
also administered systemic ocreotide to suppress insulin secretion response to hyperglycemia. We
did not find it appropriate to inhibit insulin secretion in order to study the
physiologic endothelial post-glucose load response in healthy individuals since
we suspected that the post-oral-glucose load insulin response might be pivotal
in contributing to endothelial dependent vasodilation. Aside from keeping all
hormonal responses intact, we chose to investigate post-glucose load
endothelial function up to 4 hours after glucose load, because we were aware of
the possibility that the endothelial response might not necessarily occur
simultaneously with the increasing glucose and insulin concentrations. Our
study is further strengthened by the fact that “time to peak” (the time it
takes to achieve maximal dilation after cuff-deflation) varies individually, and
that, by using our automated edge-detecting software, we have been sure of
appreciating the largest dilation at each FMD measurement. Earlier studies
using the FMD method have been forced to find a spot more or less blindly,
between 40 and 90 seconds post cuff-deflation, for their  measurements [19, 25].As mentioned, differences in subject selection,
particularly regarding postprandial blood glucose concentrations, might too
have contributed to the apparently diverse findings reported in our study and
other studies. A continuous relationship between increasing fasting and
postprandial glucose concentrations and the risk of cardiovascular mortality in
healthy, normoglycemic subjects has been documented [9, 43, 44]. Furthermore, a strong
inverse relationship has been demonstrated between glucose levels, and
endothelial function and intima media thickness (IMT) across the normoglycemic
continuum; this was marked already at the 5.2 mmol/cut-off, and was even more
significant with fasting glucose above 5.7 mmol/L [45]. Interestingly,
a directly proportional risk gradient between CVD and postprandial glucose has been
evidenced at1hPG [9].In accordance with our selection criteria, our
subjects were metabolic healthier, with lower mean fasting and postprandial
glucose values than those in studies which employed a methodology similar to
ours and, yet, demonstrated impaired endothelial function [21, 25, 26].  To illustrate, Title’s study [19] reported impaired endothelial
function after oral glucose loading in 10 study subjects with a mean 1hPG of
7.9 mmol/L (as opposed to 6.2 mmol/L in our subjects). Thereby, the apparently
contradictory results between Title's study and ours are actually
complementary.A limitation to our study is that it does not shed
light on the cellular mechanisms responsible for the postprandial endothelial
response. Yet, several mechanistic studies support our findings. Taubert’s
group [34], for example, demonstrated
that insulin's NO vasodilatory effect was augmented in the presence of relative
hyperglycemia [34]. Indeed,
in our subjects, there was a short and modest postprandial plasma glucose
spike, followed by a larger postprandial hyperinsulinemic spike, which, in
conjunction, led to an enhancement of the endothelial function when compared to
baseline endothelial function.  Conversely, our group [46] has
previously proposed that persistent hyperglycemia inhibits PI3K-dependent signaling. Taubert's group
reported that glucose and insulin stimulated NO release was impaired in the presence of hyperglycemia lasting
beyond 2 hours, and that acute hyperglycemia did not increase NO release after
prolonged exposure to hyperglycemia, leading them to suggest that enhanced
superoxide generation in chronic hyperglycemia could be responsible for
accelerated NO degradation. These findings could explain why studies  where hyperglycemic clamps were employed,
and/or examined subjects with relatively high postprandial values, have not
found enhanced endothelial function [18–20, 23, 30, 47].The greatest increment in endothelial function in our
study occurred at 3 hours post-glucose load when glucose concentrations were
back to baseline levels yet with insulin concentrations still 6 times higher
than baseline insulin concentrations. Furthermore we observed a positive
post-glucose load delta value at the cessation of the study (4hPG) for FMD and
insulin concentrations. Interestingly, Cardillo et al. [48]  found maximal insulin dilator effect after 2
hours of insulin infusion, leading them to postulate that insulin has a
relatively slow-onset vasodilator effect. Accordingly, four hours of sustained
hyperinsulinemia in the healthy has been shown to induce endothelial dependent
vasodilation [49]. These observations could therefore explain our positive
findings and why research groups that have limited their postprandial
endothelial studies to 2 hours or less after a meal/glucose load [24] have not observed insulin
induced endothelium dependent vasodilation following a hyperglycemic spike.As aforementioned, post-glucose load glucose
concentrations correlate closely to post-standardized meal glucose
concentrations; yet we cannot directly compare our results with studies that
have used standardized meals since these include lipids. Admitting that a
standardized meal would have reflected a truer postprandial response, a
standardized meal would have obscured the action of glucose alone on the
endotheliuem, since it has been demonstrated that postprandial hyperlipidemia
induces postprandial endothelial dysfunction in the healthy endothelium  [50–52].


## 5. Conclusion

In short, we find that endothelial dysfunction is not
a normal, physiologic, post-glucose load response. Instead, this study finds
preserved post-glucose load endothelial function, thus implying that sugar derived from
meals does not promote endothelial dysfunction, providing that postprandial
insulin and glucose responses are preserved. It seems that the interaction of
glucose and insulin on the endothelial function is affected both by the
magnitude of glucose and insulin concentrations, and by the duration of
exposition to these.The immediate question arising from this study is
whether by targeting both postprandial glucose and insulin peaks in insulin
resistant individuals, so that both postprandial peaks resemble those of
insulin sensitive individuals, enhances postprandial endothelial function. There
is a pressing need for a greater therapeutic focus on the postprandial state,
accompanied by a redefinition of the “normal” postprandial state, marking the
true threshold for the onset of cardiovascular risk.



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*Source: 672021-2008-03-04.xml*