Extract
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Introduction
Type 2 diabetes is a chronic metabolic disease, which is
characterized by fasting hyperglycemia that worsens as the
disease progresses. Data from the UK Prospective
Diabetes Study (UK-PDS) have shown that an almost inevitable
progressive β-cell failure occurs despite the use of various
therapies aimed at ameliorating
hyperglycemia[1]. Several mechanisms may contribute to the progressive
β-cell failure in type 2 diabetes, including loss of
β-cell mass, β-cell exhaustion, and the cytotoxic effects of elevated glucose
and lipid levels. A growing body of evidence suggests that
islet amyloid deposits may play an important role in the loss
of β-cells and the progressive decline in insulin
secretion[2]. Westermark et
al[3] identified the major component of islet
amyloid as a 37 amino acid peptide and named it amylin or
islet amyloid polypeptide. In type 2 diabetes, this peptide
aggregates to form amyloid fibrils that are toxic to
β-cells[4]. The mechanism responsible for islet amyloid formation in
type 2 diabetes is still unclear, but it appears that an increase
in the secretion and expression of
amylin[5-6] can result in its onset.
Amylin is colocalized with insulin in the isle
β-cells and is cosecreted with insulin in response
to β-cell stimulation by both glucose and non-glucose secretagogues agents,
such as arginine[7]. Therefore, therapies that alter
endogenous insulin secretion are likely to cause parallel changes
in amylin secretion. In fact, previous studies have suggested
that sulfonylurea therapy increases the post-prandial amylin
concentration, but not so in insulin
therapy[8]. These changes in turn may influence the rate of the formation of
islet amyloids, which may be disadvantageous in the long
term.
Glucagon-like peptide 1 (GLP-1) is an incretin hormone
secreted from the intestinal L-cell in response to meal
ingestion. In the pancreas, GLP-1 stimulates meal-induced
insulin secretion in a glucose-dependent manner, dramatically
lowering post-prandial glucose
levels[9,10]. More recently, the direct effects on
β-cell growth and survival have been identified, with the GLP-1-stimulated proliferation and
differentiation of new β-cells leading to increased
β-cell mass[11-13]. Furthermore, GLP-1 delays gastric emptying. These
observations support GLP-1 as a novel candidate for the
treatment of type 2 diabetes. However, it is not clear whether
GLP-1 would elevate the amylin concentration, like
sulfonylurea, which would weaken its effectiveness on
diabetes therapy.
The Goto-Kakizaki (GK) rat has been used as an animal
model for type 2 diabetes. This animal is a non-obese,
spontaneously-diabetic rat produced by selective inbreeding of
Wistar rats with the highest glucose values during oral
glucose tolerance tests. In vitro investigations of the perfused
pancreas of GK rats as well as of diabetic animals
in vivo have revealed an impairment of the glucose-induced release
of insulin. Further, histological studies have shown an
irregular shape of some, but not all, islets in GK rats aged 3
months or older[14,15].
The purpose of this study was to examine the effect of the
GLP-1 derivative, recombined human GLP-1 [rhGLP-1 (7_36)],
on the fasting and post-prandial amylin concentrations and
islet amylin and insulin mRNA levels to determine whether
amylin secretion would be altered in a way that might affect
the action of insulinotropic drugs in GK rats.
Materials and methods
Rats Male spontaneously-diabetic GK rats (34 weeks
old) and non-diabetic Wistar rats were supplied by and bred
at Shanghai Slac Laboratory Animals. The rats were housed
under controlled conditions of constant temperature and
humidity.
Experimental protocol There were 4 groups in the
experiment. The Wistar rats were used as the normal control
group. The GK rats were divided randomly into 3 groups.
The first group, which was given saline solution, served as
the vehicle-treated group; the other two groups were given
rhGLP-1 (7_36), 56 µg·kg-1 for the GK/GL (given GLP-1 low
dose) group and 133 µg·kg-1 for the GK/GH (given GLP-1
high dose) group by subcutaneous injection ter in
die (tid) for 12 weeks, respectively (provided by Shanghai Huayi
Biolab, Shanghai, China).
Measurement of blood glucose In each rat group, the
body weight and fasting blood glucose (overnight fasting
for 18 h) were monitored at 0, 1, 3, 5, 7, and 11 weeks of the
treatment with rhGLP-1 (7_36). In the 11th week, the
post-prandial blood glucose level was measured at 30 and 60 min
after feeding by the Roche Glucotrend-2 glucometer (Lewes,
East Sussex, UK)[16].
Intraperitoneal glucose tolerance
test[17] and the measurement of plasma amylin
In the 12th week of treatment following overnight fasting for 18 h, the rats were subjected
to an intraperitoneal glucose tolerance test (IPGTT). A
glucose solution (20%, 1 g·kg_1 body weight) was injected
intraperitoneally, and blood samples were collected from the
ophthalmic vein at 0, 15, 30, 60, 120, and 180 min after the
glucose injection. The blood glucose levels were measured
using the Roche Glucotrend-2
glucometer[16]. The samples were centrifuged at 4 °C. The plasma samples were stored at
_20 °C until the assay. Plasma amylin was determined using
an ELISA kit (Uscn Life Science and Technology, Missouri
City, TX, USA) based on the standard
curve[18]. Directly after the glucose tolerance test, the rats were killed by
dislocation of the cervical vertebra, and the pancreatic tissues
were taken for further studies.
Histological examination The rat pancreata were fixed
in 10% neutral buffered formalin and embedded in paraffin.
Each pancreatic block was serially sectioned (6 μm) to avoid
any bias from regional changes in islet distribution and islet
cell composition; the sections were then mounted on slides.
Then 10 sections were randomly chosen at a fixed interval
throughout the block. Half the sections were stained with
hematoxylin-eosin (HE) for light microscopy.
Examination of islet number In total, 45 sections stained
with HE were chosen from each group. The number of islets
in each section was counted in 10 random, non-overlapping
fields under light microscopy (magnification ×250).
Immunohistochemistry For the immunohistochemical
demonstration of amylin, the streptavidin-biotin-peroxidase
complex (SABC) technique was
employed[19,20]. After routine deparaffinization, rehydration, and blocking of
endogenous peroxidase activity, the sections underwent antigen
retrieval. Subsequently, the sections were incubated with a
rabbit antiserum against rat amylin (AssayPro, Winfield, MO,
USA) antibody diluted at 1:100 in phosphate-buffered saline,
followed by incubation with biotinylated antirabbit
immunoglobulin G (IgG; Boster, Wuhan, China) and SABC reagent
incubation. Staining was visualized by incubation with
3,3´-diaminobenzidine-tetrahydrochloride (Boster, China). The
sections were counterstained with hematoxylin, mounted in
a neutral gum, and examined under light microscopy.
Fluorescent-quantitative PCR The total RNA of the
samples was extracted with Trizol (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer's instructions. Reverse
transcription (RT) of RNA was performed with a M-MLV
reverse transcriptase (Promega, Madison, WI, USA). The
primers used for the amplification of insulin were:
5'-CAAACAGCACCTTTGTGGTCC-3' (forward) and 5'
-TCCACAATGCCACGCTTCT-3' (reverse), and for amylin:
5'-AGCTGTTCTCCTCATCCTCTCG-3' (forward) and 5'-TGCCACATTCCTCTTCCCAT-3' (reverse). The RT reaction
was incubated at 37 °C for 1 h and inactivated at 90 °C for 10
min. Fluorescent-quantitative (FQ)-PCR was applied to
quantify amylin and insulin mRNA in GK rat pancreata.
The 25 µL FQ-PCR mixture consisted of 2 µL RT products,
2.5 µL of 10× PCR buffer, 2 µL of 25 mmol/L
MgCl2, 2.5 µL of 2 mmol/L dNTP mixture, 0.3 µL primer 1 (10 µmol/L), 0.3 µL
primer 2 (10 µmol/L), 0.5 µL 20× SYBR, and 0.3 µL DNA
Taq polymerase (5 U/µL). The PCR cycle was as follows: 94 °C
for 2 min, 40 cycles of 94 °C for 1 min, 60 °C for 1 min, and 95
°C for 15 s on a real-time PCR machine (Applied Biosystems
7000 Real Time PCR System, Foster City, CA 94404, USA).
For the melting curve analysis after the PCR amplification,
the fluorescence signal was measured at the end of the
elongation phase at 84 °C. The obtained mRNA level was
expressed relative to that of the GAPDH (internal control) PCR
product amplified from the same sample (sample PCR
product/GAPDH PCR product).
Statistical analysis Quantitative variables were
expressed as mean±SD. One-way ANOVA was used for the
analysis. If any significant change was found, post-hoc
comparisons were performed using Fisher's PLSD (Protected
least significant difference). The ratio of amylin/insulin mRNA
was assessed by the Mann-Whitney U-test. P<0.05 was
considered significant.
Results
Fasting and post-prandial blood glucose Both the
fasting and post-prandial blood glucose levels were significantly
higher in the untreated GK rats than the Wistar rats
(P<0.01; Table 1). The GK/GL and GK/GH rats treated with 56 and 133
µg·kg_1 rhGLP-1 (7_36), showed significantly lower blood
glucose levels at 30 and 60 min after feeding compared with
the untreated GK group (P<0.05; Table 1). There was no
significant difference in the fasting blood glucose level
(P>0.05).
Levels of blood glucose in IPGTT Before the glucose
administration, the fasting blood glucose levels were
significantly higher in the GK rats than in the Wistar rats
(P<0.01; Table 2 ). Following the intraperitoneal glucose injection in
the GK rats, the blood glucose level at 15 min had risen to a
greater extent than in the Wistar rats and continued to do so
for the remainder of the experiment. The GK/GL and GK/GH
rats treated with 56 and 133 µg·kg_1
rhGLP-1 (7_36) showed significantly lower blood glucose levels after glucose
loading compared with the untreated GK group
(P<0.05; Table 2). There was no significant difference at the fasting state
(P>0.05).
Amylin concentrations in plasma The
plasma amylin levels were lower in the untreated GK rats than in the Wistar
controls, both during basal conditions (P<0.05) and after the
glucose administration (P<0.01; Figure 1). The basal plasma
amylin levels in the GK/GL and GK/GH rats showed a
descending trend compared to those in the untreated GK rats
(P>0.05). In response to the intraperitoneal glucose
administration, the plasma amylin levels of the GK/GH rats
displayed a marked increase at 30 min after the injection
compared with the untreated GK rats (P<0.05; Figure 1), whereas
the increase in the GK/GL rats did not reach significance
(P=0.09). In the Wistar control rats, the levels had increased
significantly at 30 min, then remained at a similar level at 60
min (P<0.05; Figure 1).
Histology and number of islets In contrast to the
findings in the Wistar control rats (Figure 2A), the islets of the
GK rats usually had a very irregular shape, and the islets
sometimes had a broken appearance (Figure 2B). The
boundary between the islets and exocrine pancreas was irregular.
Some islet cells seemed degenerate and swollen. In many
sections, a few islets were also found that displayed a rounded,
clear-cut shape like the normal ones seen in the Wistar control
rats. The number of islets was markedly decreased in the GK
rats compared to the Wistar control rats (P<0.01;
Figure 3). No differences were found in the number of islets between
the GK/GL or GK/GH rats and untreated GK rats. However,
the GK/GL and GK/GH rats showed slight histological
amelioration (Figure 2C,2D).
Immunohistochemistry finding Immunostaining for
amylin showed conclusive positivity in many cells of the
pancreatic islets in the Wistar rats (Figure 4A). The exocrine
pancreata were completely negative for amylin. Few
scattered cells were immunopositive for amylin in the untreated
GK rats (Figure 4B). However, the GK/GL and GK/GH rats
showed more rich amylin-positive cells compared to the
untreated GK rats (Figure 4C,4D), although the
positively-stained cells were still less than those of the Wistar rats.
Amylin and insulin mRNA levels In the untreated GK
rats, the levels of amylin and insulin mRNA were
significantly reduced (P<0.01). However, there was a more
pronounced reduction in the levels of insulin mRNA than amylin
mRNA. The levels of insulin mRNA in the untreated GK rats
was 24% of that of the Wistar controls, whereas the levels of
amylin mRNA in untreated GK rats was 59% of that of the
Wistar controls. The GK/GL and GK/GH rats both showed
marked increases of amylin and insulin mRNA compared with
the untreated GK rats (Figure 5).
The amylin to insulin mRNA ratio of the untreated GK rats
was significantly higher than that of the Wistar rats
(P<0.05, 164.51%±43.86% vs 63.25%±13.76%). The ratio in the
GK/GL and GK/GH rats was decreased with the rhGLP-1 (7_36)
treatment compared to the untreated GK rats
(P<0.05; Figure 6).
Discussion
Type 2 diabetes is characterized by insulin resistance
and progressive β-cell dysfunction leading to insulin
deficiency[21]. The importance of aggressive glucose-lowering
therapy to prevent late diabetes complications in type 2
diabetes has been convincingly
established[22,23]. However, the UK-PDS also demonstrated that the antidiabetic treatment
used failed to maintain acceptable glycemic control in the
vast majority of patients, emphasizing the need for more
effective antidiabetic agents. Treatment failure was attributed
to progressive β-cell exhaustion in type 2 diabetes.
Recently, amylin has been suggested to be responsible for
β-cell failure during the progression of type 2
diabetes[1].
GLP-1 has been shown to acutely reduce plasma glucose
levels by increasing insulin release and synthesis, inhibiting
glucagon release, and decreasing gastric emptying and
appetite. Long-term beneficial effects have also been shown
in human and rodent models[24,25]. These findings indicate
that GLP-1 is potentially a very attractive agent for treating
type 2 diabetes. In the present study, we investigated the
effects of 12 weeks of treatment with rhGLP-1 (7_36) on blood
glucose and hormone profiles. Treatment with 56 and 133
µg·kg_1 RhGLP-1 (7_36) treatment markedly improved the
glucose tolerance and reduced the blood glucose level of GK
rats after glucose loading , whereas no difference was
observed in the fasting glucose level. The result was possibly
attributed to the short half-life of GLP-1 (7_36). GLP-1 (7_36)
was rapidly degraded by the ubiquitous enzyme dipeptidyl
peptidase IV[26]. This enzyme, which is present in the
bloodstream and on cell membranes, cleaves the GLP-1 (7_36)
peptide to yield the inactive GLP-1 (9_36) form. Therefore, the
subcutaneous injection tid treatment of the GLP-1 (7-36)
peptide is probably insufficient in ensuring the effective plasma
concentration of the GLP-1 (7_36) peptide, which results in
the loss of glycemic control and decreased glucose toxicity.
As we known, amyloid deposition, which is composed of
amylin, is a predominant factor resulting in β-cell failure in
type 2 diabetes[1]. In our study, few scattered cells were
immunopositive for amylin in the pancreatic islets of the
untreated GK rats compared to the Wistar rats. This finding
may indicate that the amylin content decreased in the islet
secretory cells of the GK rats. However, the GLP-1 (7_36)
treatment caused more abundant amylin-positive cells
compared to the untreated GK rats. That is, GLP-1 (7_36)
increased the amylin content. We then tested the plasma
amylin concentration after the glucose administration. The
result was consistent with the change of content. GLP-1
(7_36) elevated the levels of plasma amylin in response to the
intraperitoneal glucose administration. A limitation of our
study was that we only measured 3 time points after the
intraperitoneal glucose administration due to the low plasma
sample amount. Nevertheless, the parallel relationship
between the amylin content and amylin secretion was clear and
was consistent with previous
studies[27]. However, the increase in the
production and secretion of amylin may be an important contributor to amyloid fibril formation. That is,
GLP-1 (7_36) may promote the formation of amyloid deposition, but is this true?
To answer this question, we designed the protocol to
investigate the effect of GLP-1 (7_36) on the amylin mRNA
level. The present study, using FQ-PCR, examined the
changes in the amylin and insulin mRNA levels in GLP-1 (7_36)-treated
GK rats. In the untreated GK rats, the quantitative findings
revealed a marked reduction of the amylin and insulin mRNA
in the islets, whereas the level of amylin mRNA was less
reduced than that of insulin mRNA. Therefore, the ratio of
amylin to insulin mRNA was sharply increased in the
untreated GK rats compared to the Wistar rats, which was
almost 2-fold higher. It has been proposed that the
amylin/insulin ratio may be worth investigating rather than the
absolute amylin mRNA level. It is not clear whether the
increase in the amylin/insulin mRNA ratio in GK rats is a
primary feature or is secondary to hyperglycemia. Interestingly,
hyperglycemia results in the hypersecretion of amylin
relative to that of insulin as well as increasing the amylin /insulin
ratio in insulin-resistant rats. Furthermore, it was recently shown
that the level of amylin mRNA increased more than that of
insulin mRNA in insulinoma cell line, INS-1 cells, cultured in a
medium containing 100 μmol/L
isobutylmethylxanthine[28]. Thus it is not inconceivable that the expression of amylin may be
more sensitive to hyperglycemia and peripheral insulin
resistance than that of insulin under certain
conditions. An increased ratio of amylin/insulin expression seems to be a
marker for insulin cell dysfunction under certain conditions.
In our research, the treatment with GLP-1 (7_36)
significantly increased the amylin and insulin mRNA levels, but
markedly decreased the ratio of amylin/insulin mRNA in the
GK rats. This result further confirms that GLP-1 (7_36)
stimulated an augmentation of the secretion and expression of
amylin in islet cells, which was consistent with other findings.
An increase of insulin protein expression was also certified
in the GK rats treated with GLP-1 (7_36) by
immunohistochemistry[29]. Therefore, it is not clear whether the
augmentation resulted from the either the direct effect of GLP-1
(7_36) on stimulating amylin or the secondary effect of
GLP-1 (7_36) on stimulating insulin. Our other interest was in
exploring the influence of GLP-1 (7_36) treatment on the
decreased the ratio of amylin/insulin mRNA. The result
contributes notably to the clinical treatment of GLP-1 (7_36) in
type 2 diabetes, including pathophysiological changes.
Putative mechanisms promote amylin gene expression
separate from insulin gene expression. However, the effect of
GLP-1 (7_36) on the pathways and sites of amylin and
insulin gene expression regulation require further study.
Acknowledgments
We wish to thank Shanghai Slac Laboratory Animals for
the animal breeding.
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