Impaired glucose-induced glucagon suppression after partial ...

J Clin Endocrin Metab. First published ahead of print June 2, 2009 as doi:10.1210/jc.2009-0826
Impaired glucose-induced glucagon suppression after partial
pancreatectomy
Henning Schrader 1* , Bjoern A. Menge 1* , Thomas G. K. Breuer 1 , Peter R. Ritter 1 ,
Waldemar Uhl 2 , Wolfgang E. Schmidt 1 , Jens J. Holst 3 , Juris J. Meier 1
1
: Department of Medicine I, St. Josef-Hospital, Ruhr-University Bochum, Germany
2
: Department of Surgery, St. Josef-Hospital, Ruhr-University Bochum, Germany
3
: Department of Biomedical Sciences, The Panum-Institute, University of Copenhagen,
Denmark
Short title: Glucagon levels after partial pancreatectomy
Word count: 3174
Precis: The glucose-induced suppression of glucagon secretion appears to be lost after
hemipancreatectomy in humans
Address for correspondence:
Juris J. Meier, M.D.
Assistant Professor
Department of Medicine I,
St. Josef-Hospital
Ruhr-University Bochum
Gudrunstr. 56
44791 Bochum
Germany
Tel.: (+49) 234-509-2711
Fax: (+49) 234-509-2713
E-Mail: juris.meier@rub.de
*: These authors contributed equally to the work
Disclosure statement: These studies were supported by grants from the Deutsche
Forschungsgemeinschaft (DFG grant-no. Me2096/5-1 to JJM) and the Ruhr-University of
Bochum (FoRUM grants to JJM). The authors declare no conflicts of interest.
Copyright (C) 2009 by The Endocrine Society

Abstract
Introduction: The glucose-induced decline in glucagon levels is often lost in patients with
type 2 diabetes. It is unclear whether this is due to an independent defect in alpha-cell
function or secondary to the impairment in insulin secretion. We examined whether a partial
pancreatectomy in humans would also impair post-challenge glucagon concentrations, and if
so, whether this could be attributed to the reduction in insulin levels.
Patients and methods: 36 patients with pancreatic tumours or chronic pancreatitis were
studied before and after ~50% pancreatectomy with a 240 min oral glucose challenge, and
the plasma concentrations of glucose, insulin, C-peptide, and glucagon were determined.
Results: Fasting and post-challenge insulin and C-peptide levels were significantly lower
after partial pancreatectomy (p < 0.0001). Likewise, fasting glucagon concentrations tended to
be lower after the intervention (p = 0.11). Oral glucose ingestion elicited a decline in
glucagon concentrations before surgery (p < 0.0001), but this was lost after partial
pancreatectomy (p < 0.01 vs. pre OP values). The loss of glucose-induced glucagon
suppression was found after both pancreatic head (p < 0.001) and tail (p < 0.05) resection.
The glucose-induced changes in glucagon levels were closely correlated to the respective
increments in insulin and C-peptide concentrations (p < 0.01).
Conclusions: The glucose-induced suppression in glucagon levels is lost after a 50% partial
pancreatectomy in humans. This suggest that impaired alpha-cell function in patients with
type 2 diabetes may also be secondary to reduced beta-cell mass. Alterations in glucagon
regulation should be considered as a potential side effect of partial pancreatectomies.
2

Introduction:
In health, glucose homoeostasis is tightly regulated by the interplay of insulin and glucagon
secretion from pancreatic islets (1). Thus, under conditions of prolonged fasting, glucagon
levels rise, enhancing hepatic glycogenolysis and gluconeogenesis, whereas oral or
intravenous glucose administration typically results in a decline in glucagon levels (2, 3).
Furthermore, a brisk increase in glucagon secretion in response to falling blood glucose
concentrations provides a safeguard against the development of hypoglycaemia (2, 3).
Alterations in glucagon secretion are frequently found in patients with type 2 diabetes (4).
Thus, an insufficient suppression of glucagon levels after oral glucose or meal ingestion is
thought to contribute significantly to the excessive hepatic glucose production in type 2
diabetic patients (2, 5-7). Such abnormalities in glucagon secretion have been reported not
only in patients with overt diabetes, but even in pre-diabetic subjects, such as individuals with
impaired glucose tolerance (IGT) (8). However, the aetiology of these defects in glucagon
secretion is less clear, and two different explanations have been expounded. 1) The lack of
glucose-induced suppression of glucagon levels in type 2 diabetes could represent a primary,
possibly genetically determined, defect in alpha-cell function, independent of other metabolic
abnormalities in such patients (9, 10). Such argumentation would imply that defects in
glucagon secretion predispose subjects to developing type 2 diabetes (11). Against this, we
and others have failed to detect any defects in alpha-cell function in individuals at high risk
for developing type 2 diabetes, such as first-degree relatives (12, 13). 2) Alternatively, the
lack of glucose-induced suppression of glucagon levels in patients with type 2 diabetes may
be secondary to the deficit in beta-cell mass and the overall reduction in insulin secretion in
such patients (14-16). In line with this hypothesis, a selective 50% reduction in beta-cell mass
induced by the beta cytotoxin, alloxan, led to the development of both fasting and
postprandial hyperglucagonaemia in Göttingen minipigs (17). Several experiments have
suggested a close interaction of alpha- and beta-cells at the individual islet level (14-16).
3

However, this does not exclude that systemic insulin concentrations would have an impact on
glucagon release as well. Therefore, in the present study, we examined the effects of a hemi-
pancreatectomy, leading to a ~50% reduction in both beta- and alpha-cell mass, on post-
challenge glucagon levels in humans. For these purposes, 36 patients undergoing pancreatic
surgery were studied before and after surgery. By these means, we addressed the questions:
(1) Is the glucose-induced suppression of glucagon levels impaired after hemi-pancreatectomy
in humans, and if so, (2) are the alterations in glucose-induced glucagon suppression related
to the respective changes in glycaemia or insulin secretion?
4

Materials and methods
Study design
A total of 36 patients undergoing pancreatic surgery for chronic pancreatitis, pancreatic
carcinoma, and benign pancreatic tumours were studied. All patients were examined before
and after pancreatic surgery with a 240 min oral glucose challenge, and the respective changes
in the plasma concentration profiles of glucose, insulin, C-peptide, and glucagon were
determined. Parts of this study related to the changes in glucose, insulin and C-peptide levels
after partial pancreatectomy have been communicated in a separate report (18). The study
protocol was approved by the ethics committee of the Ruhr-University Bochum (registration
number 2528). All patients provided written informed consent prior to study enrolment.
Patients:
A total of 36 patients (18 males, 18 females) undergoing pancreatic resections in the
Department of Surgery, St. Josef-Hospital, Ruhr-University Bochum, between the years 2004
and 2007 were included. One patient from the original cohort (18) was excluded, because
glucagon measurements were not available. The mean age of the patients was 59.4 ± 13.0
years, and the body mass index was 23.9 ± 4.2 kg/m 2 . Detailed patient characteristics have
previously been reported (18). 14 patients were undergoing surgery for chronic pancreatitis,
nine patients were treated for pancreatic adenocarcinoma, 12 patients underwent surgery for
the removal of benign pancreatic adenomas, and one patient was undergoing surgery for
because of a tumour of the papilla Vateri.
In 12 patients, distal pancreatectomies (pancreas tail resection) were performed, whereas
24 patients were treated with a proximal pancreatectomy (pancreas head resection). The latter
group comprised 17 patients undergoing pancreaticoduodenectomy with pylorus preservation,
6 patients undergoing duodenum-preserving pancreatic head resections according to Beger,
and one patient undergoing classic partial pancreaticoduodenectomy (Whipple’s operation).
5

The clinical diagnoses chronic pancreatitis, pancreatic carcinoma, pancreatic adenoma or
ampullary cancer were confirmed by an independent pathologist in all cases. All post-
operative experiments were conducted after full clinical recovery of the patients. The mean
time interval between the first oral glucose challenge and surgery was 8.6 ± 7.6 days, and the
second test was carried out 23.9 ± 27 days after surgery.
Experimental procedures
The experiments were performed as described (18) in the morning after an overnight fast with
subjects in a supine position throughout the experiments. Both ear lobes were made
hyperemic using Finalgon � (Nonivamid 4 mg/g, Nicoboxil 25 mg/g). The experiments were
started by the ingestion of the oral glucose load (75 g glucose in 300 ml) over 5 min, and
capillary and venous blood samples were drawn at t = -5, 0, 15, 30, 60, 90, 120, 150, 180,
210, and 240. Capillary blood samples (approximately 100 μl) were added to NaF (Microvette
CB 300; Sarstedt, Nümbrecht, Germany) for the immediate measurement of glucose. Venous
blood was drawn into chilled tubes containing EDTA and aprotinin (Trasylol � ; 20000
KIU/ml, 200 μl per 10 ml blood; Bayer AG, Leverkusen, Germany) and kept on ice. After
centrifugation at 4 °C, plasma for hormone analyses was kept frozen at -28 °C.
Measurements
Glucose was measured as described (18, 19) using a glucose oxidase method with a Glucose
Analyser 2 (Beckman Instruments, Munich, Germany).
Insulin was measured as described (18) using an insulin microparticle enzyme
immunoassay (MEIA), IMx Insulin, Abbott Laboratories, Wiesbaden, Germany. Cross-
reactivity with proinsulin was < 0.005%. The intra-assay coefficient of variation was 4 %.
C-peptide was measured as described (18) using an enzyme-linked immunoabsobent
6

assay (ELISA) from DAKOP Ltd., Cambrigshire, UK. Intra-assay coefficient of variation was
3.3 to 5.7 %, inter-assay variation was 4.6 to 5.7 %. Human insulin and C-peptide were used
as standards.
IR-glucagon was measured by a radioimmunoassay using antibody no. 4305 in ethanol-
extracted plasma, as described (20). The detection limit was < 1 pmol/l. Intra-assay
coefficient of variation was below 6.7 %. This assay specifically reacts with the free
unmodified C-terminus of the glucagon molecule. Therefore, it is theoretically possible that
other circulating forms glucagon than the 3500 dalton glucagon (especially proglucagon 1-61)
were detected as well. However, the quantitative contribution of these forms to the overall
glucagon immunoreactivity appears to be rather negligible (21).
Calculations:
The Matsuda index of insulin sensitivity and HOMA insulin resistance were calculated as
described (22, 23). The maximum glucose-induced suppression of glucagon levels was
calculated by expressing the nadir glucagon levels between t = 15 min and t = 240 min in
relation to the respective baseline levels.
Statistical Analysis
Subject characteristics are reported as mean ± SD, results are presented as mean ± SEM. Time
course evaluations were carried out by paired or unpaired analysis of variance (ANOVA), as
appropriate, using Statistica version 5.0 (Statsoft Europe, Hamburg, Germany). All other
parameters were compared by two-way ANOVA or Student’s t-test, as appropriate. A p-value
< 0.05 was taken to indicate significant differences. Regression analyses were carried out
using GraphPad Prism 4.
7

Results
Fasting glucose concentrations increased significantly after partial pancreatectomy, but the
immediate rise in post-challenge glucose excursions was lower following the intervention (p <
0.0001; Fig. 1A; (18)). This was accompanied by significant reduction in both fasting and
post-challenge insulin and C-peptide levels (p < 0.0001; Fig. 1B, D; (18)). There was also a
significant improvement in the Matsuda index of insulin sensitivity (6.8 ± 0.6 to 8.6 ± 0.6, p =
0.005), whereas HOMA IR was unchanged (1.7 ± 0.2 vs. 1.6 ± 0.1, p = 0.35).
Fasting glucagon levels tended to be lower after partial pancreatectomy (p = 0.11). Oral
glucose ingestion elicited a significant decline in glucagon concentrations in the experiments
carried out prior to surgery (p < 0.0001; Fig. 1C). Thus, the maximum suppression of
glucagon levels from baseline was -39 ± 3 % before and -22 ± 4 % after surgery (p = 0.0017).
Similarly, the difference between the mean glucagon concentrations after glucose ingestion
and baseline values (� glucagon) was markedly reduced after surgery (p < 0.01; Fig 2).
In order to determine, whether the impairment in glucose-induced glucagon suppression
after partial pancreatectomy was secondary to the development of hyperglycaemia or rather
due to the reduction in insulin secretion, the glucose-induced reductions in glucagon
concentrations were correlated to the respective glucose, insulin and C-peptide levels. There
was indeed a significant relationship between the increments in insulin and C-peptide levels
after the glucose load and both the mean changes in glucagon levels after the glucose load (�
glucagon) (Fig. 3). Interestingly, the associations of glucagon levels with insulin levels were
greater than with C-peptide concentrations. In contrast, neither the fasting glucose levels (r =
0.21, p = 0.081), nor the mean post-challenge glucose concentrations (r = 0.023, p = 0.85)
were related to the mean glucose-induced changes in glucagon levels (details not shown).
When the patients were grouped according to the respective sort of pancreatic resection,
the impairment in glucose-induced suppression of glucagon levels was apparent after both
pancreatic head (n = 24; p < 0.001; Fig. 4A) and tail (n = 12; 0 < 0.05; Fig. 4B) resection.
8

However, after pancreatic head resection fasting glucagon concentrations were almost
unchanged, whereas the post-operative glucagon concentrations were even higher compared
to pre-operative levels from t = 90 to 150 min after the glucose drink. In contrast, pancreatic
tail resection caused an ~45% reduction in fasting glucagon levels, and postoperative
glucagon levels remained lower than before surgery at all subsequent time points after the
glucose drink.
Moreover, the individual impact of the hemipancreatectomy on glucagon concentrations
seemed to differ to some extent between the different groups of patients studied. Thus, in
patients undergoing surgery for the removal of pancreatic adenomas or extrapancreatic
tumors, fasting glucagon levels were 31% lower after surgery, and the glucose-induced
decline in glucagon levels was no longer detectable (Fig. 5A). In patients undergoing surgery
for chronic pancreatitis, the overall concentrations of glucagon tended to be lower at all time
points compared to the other patient groups, but the impairment in glucose-induced glucagon
suppression was less apparent (Fig.5B). In patients treated for pancreatic carcinoma, fasting
glucagon levels were 23% lower after the operation, but post-challenge glucagon levels
tended to be even higher (Fig. 5C).
9

Discussion:
Impaired glucagon suppression after glucose administration is a characteristic hallmark of
type 2 diabetes (2, 4, 5). This has been held to be secondary to the loss of intra-islet insulin
secretion in such patients (4, 14, 16, 17, 24). The present studies were designed to address the
question, whether a 50% partial pancreatectomy would alter alpha-cell function in humans as
well. We report that the glucose-induced decline in glucagon concentrations was lost after
hemi-pancreatectomy. This impairment in alpha-cell function was closely related to the
changes in insulin secretion, but appeared to be independent of the increases in glycaemia.
Even though the maximum secretory capacity of the alpha-cells (e.g. in response to
arginine or hypoglycaemia) has not been tested directly in this study, there was a tendency
towards a reduction in fasting glucagon levels, consistent with the expected 50% reduction in
alpha-cell mass. This is in line with previous studies by Seaquist and Robertson
demonstrating an impaired glucagon response to i.v. arginine after hemi-pancreatectomy in
humans (25). However, although the maximum glucagon responses seem to be primarily
determined by the number of alpha-cells within the pancreas, the glucose-induced decline in
glucagon concentrations is largely controlled by other factors. In this regard, macronutrients,
and particularly glucose and amino acids, are important regulators (26, 27), but there is also
good evidence that endocrine hormones, such as glucagon-like peptide 1 (GLP-1) (28), gastric
inhibitory polypeptide (GIP) (12, 29), gastrin (30), and cholecystokinin (31), may play a role.
In addition to these well established nutrient and endocrine factors, a number of studies have
suggested a paracrine regulation of glucagon release (32). Thus, within the individual islets,
the beta-cells are typically located in the core region, whereas the alpha-cells are
predominantly located in the islet periphery. Since the blood flow within the islets is usually
centripetally directed, reaching the beta-cell rich core first, and then traversing into the mantle
region, the islet alpha-cells are being exposed to very high local insulin levels (33), and a
number of in vitro studies have provided evidence for an inhibitory effect of insulin on alpha-
10

cell secretion (34, 35). Consistent with such reasoning, previous studies in baboons and
minipigs have demonstrated a close inverse relationship between circulating insulin and
glucagon levels at a minute-by-minute basis (16, 17). Furthermore, selective destruction of
islet beta-cells in Göttingen minipigs has resulted in a loss of meal-induced glucagon
suppression and the development of overt hyperglycaemia (17). The present findings extend
these studies by showing that an inverse relationship between insulin and glucagon levels can
also be found at a systemic level, independent of the paracrine interaction of both hormones
within the individual islets. Thus, since the intra-islet relationship between alpha- and beta-
cells should not be affected by a hemi-pancreatectomy, the loss of glucose-induced
suppression of glucagon levels in the present experiments suggests that a general reduction in
systemic insulin levels may impair alpha-cell function as well. Of note, this effect was
apparent after both pancreatic head and tail resection. Such argumentation is also consistent
with previous studied by Robertson and colleagues showing elevated post-operative fasting
glucagon levels in healthy subjects who donated 50% of their pancreas for transplantation
(36). Taken together, these studies emphasize the importance of circulating insulin levels for
the maintenance of alpha-cell function and lend strong support to the postulate that the
hyperglucagonaemia in patients with type 2 diabetes develops as a consequence of the
reduction in beta-cell mass, rather than due to an independent, primary defect in alpha-cell
function. Such reasoning is also in line with the clinical finding that endogenous glucagon
levels often decline after exogenous insulin supplementation in patients with diabetes (37,
38). However, aside from these effects of systemic insulin, a direct paracrine interaction
between insulin and alpha-cells may still exist, as suggested by previous animal studies.
Arguably, the inverse relationship between beta- and alpha-cell secretion may also be
mediated by other secretory products of the beta-cell than just insulin. In this regard, a direct
inhibitory action of zinc on alpha-cell secretion has recently been postulated by some (39),
but not all groups (40). However, while such inhibitory actions of zinc may contribute to the
11

intra-islet inhibition of glucagon release, they are less likely to play a role for the impaired
glucose-induced suppression of glucagon secretion observed after hemipancreatectomy in this
study, where the paracrine regulation of hormone secretion within the individual islets was
not specifically altered.
Moreover, even though the observed relationships between insulin and glucagon levels
suggest a direct interaction between both islet hormones, other pancreatic hormones might
have contributed as well. In particular, somatostatin has been previously shown to act both as
a paracrine and endocrine regulator of alpha-cell secretion (41, 42). It is therefore possible
that the reduction in delta-cell mass and secretion induced by the hemipancreatectomy has
contributed to the observed impairment in glucose-induced glucagon suppression.
Alternatively, one might argue that the lack of glucagon suppression might have been
secondary to the partial removal of the duodenum leading to impaired glucose-induced GLP-1
secretion. Unfortunately, incretin levels were not determined in this study. However, since
alterations in glucagon secretion were found after both pancreaticoduodenectomy and after
pancreatic tail resection (where the duodenum is not affected at all), such explanation appears
less likely.
Although a tendency towards a loss of glucose-induced glucagon suppression was
detectable in all groups of patients studied, the reduction in fasting glucagon levels was more
pronounced in patients undergoing surgery for the removal of benign adenomas,
extrapancreatic tumours or pancreatic cancer than in patients with chronic pancreatitis. Most
likely this is due to the fact that in patients with chronic pancreatits the secretion of both
insulin and glucagon was already markedly abnormal prior to surgery, meaning that the
additional alterations induced by the hemipancreatectomy were less apparent than in the other
patient groups (43). Consistent with this, the pre-operative insulin levels and glucagon levels
were lowest in patients with chronic pancreatitis (18).
12

A number of previous studies in rats and mice have suggested that functional alterations in
islet secretion are only detectable after reductions in beta-cell mass in the range of ~80-90%
(44, 45), whereas less extensive pancreatic resections can be tolerated without measurable
disturbances in circulating islet hormone concentrations (46). These reports are contrasted by
larger animal studies, where alterations in both insulin and glucagon secretion occurred
already after an ~50% beta-cell loss (15, 17, 47). Furthermore, studies in individuals with
impaired fasting glucose, who typically already exhibit defects in insulin secretion (48), have
revealed an ~50% deficit in beta-cell mass even before the full onset of type 2 diabetes (49).
The present studies showing marked alterations in both insulin and glucagon secretion after
hemipancreatectomy are therefore consistent with these previous studies and re-emphasize the
importance of beta-cell mass for the maintenance of glucose homoeostasis.
There has been some debate regarding the distribution of islet alpha- and beta-cells within
the pancreas. Indeed, in different studies in humans and in animal models, the majority of
alpha- and beta-cells were found in the pancreatic tail, whereas Pancreatic Polypeptide
secreting cells were predominantly shown in the pancreatic head and the uncinate process (50-
54). In the present studies fasting glucagon concentrations were almost unchanged after
pancreatic head resection, but substantially lowered after removal of the pancreatic tail. This
may suggest a predominance of alpha-cells in the pancreatic tail compared to the head and
body regions. However, since the maximum glucagon responses have not been tested directly,
this study does not allow for valid conclusions in this regard.
The present and our previous study (18) have also shown significant improvements in
glycaemia within the first 90 min after the oral glucose load, which is surprising in light of the
concomitant reduction in insulin secretion. This may on the one hand be due to an
improvement insulin sensitivity induced by the removal of the abnormal pancreatic tissue. On
the other hand it seems possible that the transient reduction in glycaemia immediately after
glucose ingestion was caused by a delay in gastric emptying secondary to the surgical trauma.
13

In conclusion, the present study has shown that the glucose-induced suppression in
glucagon levels is lost after a 50% partial pancreatectomy in humans. The changes in post-
challenge glucagon levels are closely related to the impairment in insulin concentrations.
These findings suggest that the defects in alpha-cell function typically observed in patients
with type 2 diabetes are likely secondary to the reduction in beta-cell mass. Alterations in
glucagon regulation should be considered as potential side effects of partial pancreatectomies
in humans.
Acknowledgements: The excellent technical assistance of Birgit Baller, Yvonne Dabrowski,
Kirsten Mros, Heike Achner and Gudrun Müller is gratefully acknowledged.
These studies were supported by grants from the Deutsche Forschungsgemeinschaft (DFG
grant-no. Me2096/5-1 to JJM) and the Ruhr-University of Bochum (FoRUM grants to JJM).
14

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Figure legends:
Figure 1: Plasma concentrations of glucose (a), insulin (b), glucagon (c), and C-peptide (d) in
36 patients (18 males, 18 females) examined before (filled symbols) and after partial
pancreatectomy (open symbols). At t = 0 min, an oral glucose load (75g) was ingested. Data
are presented as means ± SEM. P-values Statistics were carried-out using paired repeated
measures ANOVA and denote A: Overall differences between the experiments, B: differences
over the time course and AB: differences between the experiments over the time course.
Asterisks indicate significant (p < 0.05) differences at individual time points versus control
subjects (one-way ANOVA).
Figure 2: Changes (�) in glucagon concentrations (calculated as mean glucagon
concentrations from t = 15 to t = 240 min minus basal glucagon levels) after the oral ingestion
of 75 g glucose in 36 patients (18 males, 18 females) examined before (filled symbols) and
after partial pancreatectomy (open symbols). Statistics were carried-out using paired t-tests.
Figure 3: Linear regression analysis between the changes (�) in glucagon concentrations
(calculated as mean glucagon concentrations from t = 15 to t = 240 min minus basal glucagon
levels) and the respective changes in insulin (A) and C-peptide concentrations (B) after the
oral ingestion of 75 g glucose in 36 patients (18 males, 18 females) examined before (filled
symbols) and after partial pancreatectomy (open symbols). Dashed lines indicate the
respective upper and lower 95 % confidence intervals. r = correlation coefficient.
Figure 4: Plasma glucagon concentrations in 24 patients undergoing pancreatic head
resection (A) and 12 patients undergoing pancreatic tail resection (B) examined before (filled
symbols) and after surgery (open symbols). At t = 0 min, an oral glucose load (75g) was
19

ingested. Data are presented as means ± SEM. P-values Statistics were carried-out using
paired repeated measures ANOVA and denote A: Overall differences between the
experiments, B: differences over the time course and AB: differences between the
experiments over the time course. Asterisks indicate significant (p < 0.05) differences at
individual time points versus control subjects (one-way ANOVA).
Figure 5: Plasma glucagon concentrations in 12 patients undergoing surgery for the removal
of benign pancreatic adenomas and one patient with a tumour of the papilla Vateri (A), 14
patients with chronic pancreatitis (B), and nine patients with pancreatic adenocarcinoma (C)
examined before (filled symbols) and after pancreatic surgery (open symbols). At t = 0 min,
an oral glucose load (75g) was ingested. Data are presented as means ± SEM. P-values
Statistics were carried-out using paired repeated measures ANOVA and denote A: Overall
differences between the experiments, B: differences over the time course and AB: differences
between the experiments over the time course.
20