From the Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Boston, Massachusetts 02215
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Differentiated pancreatic Pancreatic Recently, the study of the transcriptional regulation of the insulin
gene has led to the identification of several In this study, we tested the influence of chronic hyperglycemia on
pancreatic Partial Pancreatectomy (Px)--
Male Sprague-Dawley rats
weighing 90-100 g (Taconic Farms, Germantown, NY) were anesthetized
with sodium amobarbital (10% solution, 1 ml/kg,) and submitted to a
partial pancreatectomy or sham surgery as described previously (16).
For 90% pancreatectomy, most of the pancreatic tissue was removed by
gentle abrasion with cotton applicators, leaving intact the tissue
within 1-2 mm of the common bile duct and extending to the first loop
of the duodenum (pancreatic remnant). In some experiments, the
proportion of the gastric lobe removed was purposely varied to generate
85-95% partially pancreatectomized rats that stay normoglycemic or
develop mild to severe hyperglycemia after Px. Blood was obtained
weekly from the snipped tail of non-fasted rats (9-10 a.m.) in
heparinized microcapillary tubes, and whole blood glucose levels were
determined with a "One Touch II" glucometer (Lifescan, Milpitas,
CA). Four weeks after Px, the rats were anesthetized with sodium
amobarbital (10% solution, 1.1 ml/kg) and their islets isolated by
collagenase digestion of the pancreatic remnants or the entire sham
pancreas (11). The islets were then microdissected and handpicked under a stereomicroscope to ensure high purity of the islet preparation. The
islets were maintained on ice during the entire isolation except for 20 min of collagenase digestion. All animal procedures were approved by
the Animal Care Committee of the Joslin Diabetes Center.
RNA Extraction and Complementary DNA (cDNA)
Synthesis--
Most of the time, total RNA was extracted from islets
of individual rats following manufacturer's suggested protocols using Ultraspec (Biotecx Laboratories, Houston, TX). In a few cases, however,
it was necessary to pool islets from two Px rats with similar glycemia
to have enough RNA for further analysis. After quantification by
spectrophotometry, 500 ng of RNA was diluted to a final concentration
of 0.1 µg/µl and heated at 85 °C for 3 min. It was then
reverse-transcribed (RT) into cDNA in a 25-µl solution containing
50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM dNTPs, 50 ng of random hexamers, and 200 units of
Superscript II RNase H Primers--
Primer design was optimized for multiplex
polymerase chain reaction (PCR) with EugeneTM version 2.2 (Daniben Systems, Cincinnati, OH) using the following restrictions:
oligonucleotide length of 18-25 bases, GC content of 30-70%, melting
temperature close to 60 °C, product length within 150-650 bases,
and maximum 6-8 primer dimer formation allowed within multiplex
PCR.
Semiquantitative Radioactive Multiplex PCR--
Polymerization
reactions were performed in a Perkin-Elmer 9600 or 9700 Thermocycler in
a 50-µl reaction volume containing 3 µl of cDNA (20-ng RNA
equivalents), 80-160 µM cold dNTPs, 2.5 µCi of
[ Validation of Semiquantitative Multiplex RT-PCR--
To ensure
the validity of the measurement of mRNA levels by
semiquantitative-radioactive multiplex RT-PCR, control experiments were
performed using normal rat islet cDNA to show that the amount of
each amplimer obtained in a multiplex PCR was independent of the
presence of the other primers (cross-correlation analysis), excluding
the possibility of strong interferences between primers. The number of
cycles (1 min denaturation at 94 °C, 1 min at annealing temperature,
and 1 min extension at 72 °C) and the final reaction conditions
(MgCl2, cold dNTP and primer concentrations, cycle number,
and annealing temperature) were then adjusted to be in the exponential
phase of the amplification of each product (Fig. 1A). Finally, we verified that
the amount of each PCR product in a multiplex reaction increases
linearly with the amount of starting cDNA (from 2.5- to 80-ng RNA
equivalents), ensuring that changes in the ratio of PCR product to
control gene product truly reflect a change in mRNA abundance of
that gene relative to the control gene (Fig. 1B). Our
results (PCR amplification and quantitation) were highly reproducible,
as judged by multiple PCR determination from the same cDNA
preparation (coefficient of variation <7%).
Px Rat Classification--
In the first part of the study, rats
sacrificed after 4 weeks were classified into 4 groups according to
their averaged fed blood glucose levels 3 and 4 weeks post-Px. Low
hyperglycemic rats (LPx) were below 100 mg/dl, mildly hyperglycemic
rats (MPx) were within 100-150 mg/dl, highly hyperglycemic rats (HPx)
were within 150-250 mg/dl, and severely hyperglycemic rats (SPx) were above 250 mg/dl (range in mM are <5.6, 5.6-8.3, 8.3-13.9
and >13.9 respectively).
In the second part of the study, Px rats were classified according to
their averaged fed blood glucose levels 1 and 2 weeks post-Px in
moderately hyperglycemic (<160 mg/dl) and severely hyperglycemic
( Phlorizin Treatment--
Phlorizin was dissolved in
1,2-propanediol (0.2 g/ml) and injected intraperitoneally twice a day
at a dose of 0.8 g per kg body weight per day for 2 weeks (19).
Sham animals received similar amounts of 1,2-propanediol as
phlorizin-treated rats. To make sure that blood glucose levels were
normalized throughout the day, blood sampling was always performed just
before injection of the morning dose of phlorizin.
Plasma Insulin and NEFA Determination--
Plasma was prepared
from blood samples collected in EDTA/paraoxon
(diethyl-para-nitrophenyl phosphate)-coated tubes, to avoid heparin stimulation of triglyceride lipase and breakdown of circulating triglycerides to glycerol and NEFA (20). Plasma insulin levels were
determined by radioimmunoassay for rat insulin (Linco Research, St.
Charles, MO). Plasma NEFA levels were measured by a colorimetric method
(Wako Chemicals, Neuss, Germany).
Immunohistochemistry--
Remnant tissue from 4-week Px (treated
with or without phlorizin for the last 2 weeks) and remnant equivalent
from sham Px (at least 4 rats in each group) was fixed for 2 h at
room temperature in freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer and then processed for paraffin
embedding. For staining of transcription factors, paraffin sections
(5-7 µm) were deparaffinized and then microwaved in citrate buffer
(3 times, each for 5 min) for antigen retrieval. Sections from all
blocks were then rinsed in Tris saline, incubated for 10-20 min in
0.3% Triton X-100 (Fisher) with 1% lamb serum (Life Technologies,
Inc.), and following a second rinse, incubated in rabbit blocking serum
(Vector, Burlingame, CA). Sections were then incubated overnight at
4 °C with Nkx6.1 antibody (gift of Palle Serup, Hagedorn, Gentofe,
DK, dilution 1:2000). After washing, sections were incubated 1 h
at room temperature with donkey biotinylated anti-rabbit IgG (1:400,
Jackson ImmunoResearch) as the secondary antibody followed by
streptavidin-conjugated FITC (1:400, Jackson ImmunoResearch) for 1 h at room temperature. After rinsing extensively, slides were mounted
with DABCO glycerol anti-fading mounting media. Images of stained
sections were taken on a Zeiss LSM 410 microscope with a fluorescence
filter for FITC. The sections were then washed with 0.01 mM
HCl to remove the attached antibodies, rinsed with distilled water, and
incubated with 5% normal donkey serum (Jackson ImmunoResearch, West
Grove, PA) for 30 min at room temperature. The staining procedure was
repeated on the same sections with PDX-1 primary antibody overnight
(Hm-253, gift of Joel Habener, Boston, dilution 1:7500) and donkey
biotinylated anti-rabbit IgG (1:400, Jackson ImmunoResearch) as the
secondary antibody followed by streptavidin-conjugated Texas Red
(1:400, Jackson ImmunoResearch). Images of stained sections were taken with a fluorescence filter for Texas Red. Similar results were obtained
with sections not previously stained for Nkx6.1.
Control serum of appropriate species gave no cross-reactions. Sections
from Px and sham pancreas were stained and photographed in parallel and
at the same settings. Adobe Photoshop was used to make final figures.
For lactate dehydrogenase (LDH) immunolocalization, deparaffinized
non-microwaved sections were soaked in phosphate-buffered saline plus
1% lamb serum (Life Technologies, Inc.) and incubated overnight with a
rabbit anti-rat liver LDH antibody (final dilution 1:250, gift of Dr.
A. Völkl, Heidelberg, Germany) that recognizes the
A4- and A3B isoforms (21). Pancreatic
In another set of experiments, 4-week Px (treated or not with
phlorizin) and sham islets were isolated, pelleted, and fixed for
2 h at room temperature in freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer and then processed for plastic
embedding. After resin removal, the sections (1 µm) were soaked in
phosphate-buffered saline plus 1% lamb serum (Life Technologies, Inc.)
and stained for the endocrine non- Electron Microscopy and Measurement of Data Analysis--
Results are presented as means ± S.E.
for the indicated number of animals or islet preparations. Statistical
significance of differences between Px and sham groups was assessed by
one-way ANOVA followed by a test of Dunnett for comparison with sham or by a test of Newman-Keuls for multiple comparisons. For
immunohistochemistry data, representative islets from different type of
animals are presented.
Characteristics of Px Animals--
The evolution of body weight
and fed blood glucose levels after Px are illustrated in Fig.
2. During the first few days after surgery, body weight gain was slightly decreased in Px rats, resulting at 1 week in a statistically significantly lower body weight in all
groups of Px versus sham. Thereafter, Px animals gained
weight at the same rate as sham. In contrast, changes in blood glucose were very heterogeneous among Px animals, due to deliberate, but slight, variation in the amount of pancreas removed (~85-95% Px). Three and four weeks after Px, blood glucose ranged from 68 to 317 and
72 to 336 mg/dl, respectively, whereas sham blood glucose varied from
63 to 87 and 64 to 96 mg/dl at the same time points. These values were
determined on whole blood obtained in the morning from fed animals.
They are lower than the corresponding plasma glucose values determined
by the glucose oxidase method that range from 120 mg/dl in sham to
450-500 mg/dl in Px animals. Px rats were then classified according to
their averaged blood glucose at 3 and 4 weeks post-Px as low (LPx),
mildly (MPx), highly (HPx), and severely hyperglycemic (SPx) rats (see
"Experimental Procedures" and Tables
II and III
for classification criteria). The blood glucose of SPx rats was
significantly increased already by 1 week and remained significantly
higher than in any other group during the entire study (Fig. 2). MPx
and HPx blood glucose values were not different from sham at 1 week but
increased significantly thereafter. Although these two groups could not
be distinguished from each other at 2 weeks, they were significantly
different 3 and 4 weeks after Px. LPx blood glucose was slightly, but
not significantly, increased over sham at any time during this
study.
Changes in Islet Hormone mRNA Level--
The expression of 4 major islet hormones was evaluated 4 weeks after Px (Table II). As
previously reported (11), insulin mRNA level was reduced by 50% in
SPx islets. A similar reduction was also observed in HPx rats, but the
insulin mRNA level was completely normal in MPx and LPx islets
(Fig. 3). In contrast, islet amyloid
polypeptide (IAPP) mRNA levels were significantly increased in LPx
islets but gradually decreased from LPx to SPx which were significantly
reduced by 30% compared with sham (Table II). The mRNA levels of
non- mRNA Level of Genes Involved in Glucose Metabolism--
The
expression of several important determinants of Changes in Ion Channel/Pump mRNA Level--
The expression of
several ion channels important for glucose-induced insulin release was
also evaluated (Table II and Fig. 3, sets 1, 5, and
6). The mRNA levels of Kir6.2, the pore-forming subunit
of the ATP-sensitive K+ channels (K+-ATP
channels) (23), were significantly reduced by 40% in SPx and HPx
islets (Fig. 4, set 1) but remained essentially unaffected in LPx and MPx (Table II). In contrast, the mRNA levels of SUR1, the sulfonylurea receptor that constitutes the regulatory subunit of
the K+-ATP channels, were not significantly altered after
Px (Table II and Fig. 4, set 6). The mRNA levels of
VDCC Changes in Transcription Factor mRNA Level--
We next
evaluated the level of expression of transcription factors important
for Reversibility of Changes in Islet Gene Expression after
Px--
Because correlation between blood glucose levels and changes
in islet gene expression do not distinguish between cause and effect,
we tested whether these changes are due to chronic hyperglycemia using
phlorizin, an inhibitor of the Na+-glucose cotransporter in
the proximal kidney tubules (25). By promoting glucosuria, this drug
rapidly restored normoglycemia in severely hyperglycemic Px rats (Fig.
5). Phlorizin also slightly reduced body
weight gain in Px rats but to the same extent as the vehicle did in
sham (Fig. 5). As shown in Fig. 6
(left panels), the decrease in insulin, PDX-1, and Nkx6.1,
and the increase in LDH-A mRNA levels were already observed in
islets by 2 weeks after Px, with a similar dependence to the level of
hyperglycemia as observed after 4 weeks. Correction of hyperglycemia
for the next 2 weeks by phlorizin was followed by complete
normalization of islet gene expression in severely hyperglycemic Px
rats (Fig. 6, right panels, Fig.
7, not shown for other genes listed in
Tables II-III). Administration of vehicle alone had no effect on islet
gene expression or blood glucose levels in sham animals (Figs.
5-7).
Changes in Plasma NEFA and Insulin Levels after Px--
The
possibility that plasma NEFA or insulin levels could contribute to
changes in islet gene expression after Px was also investigated.
Although fed plasma insulin levels were significantly lower in severely
hyperglycemic Px rats versus shams, Px plasma NEFA levels
were not different from shams 2 and 4 weeks after Px (Fig.
8). After an overnight fast, plasma
insulin levels were decreased significantly compared with fed values,
with a large increase in plasma NEFA levels that was not different in
Px and sham animals. A significant reduction in plasma insulin levels was observed in Px rats treated with phlorizin but also in sham injected with vehicle alone. However, phlorizin treatment had no effect
on plasma NEFA levels (Fig. 8).
Immunohistochemical Analysis of Changes in Islet Gene Expression
after Px--
It has been shown that changes in insulin and GLUT2
mRNA levels after Px are accompanied by parallel changes in protein
levels in Pancreatic Reduced Transcription Factor Expression--
Expression of many
Changes in Gene Expression and Role of Hyperglycemia in Loss of
An inverse relationship between cell growth and their state of
differentiation has previously been reported in immortalized insulin
secreting cell lines (Philippe et al. (49) and Fleischer et al. (50)). Since both glucose and NEFA can induce
expression of early genes in insulin-secreting cell lines (Yamashita
et al. (51), Susini et al. (52), and Roche
et al. (53)), it is tempting to speculate that plasma NEFA
and hyperglycemia may be deleterious to
Even Px rats with only mild hyperglycemia have loss of
In conclusion, prolonged exposure to increasing levels of hyperglycemia
correlates with progressive loss of cells are unique in
their ability to secrete insulin in response to a rise in plasma
glucose. We have proposed that the unique constellation of genes they
express may be lost in diabetes due to the deleterious effect of
chronic hyperglycemia. To test this hypothesis, Sprague-Dawley rats
were submitted to a 85-95% pancreatectomy or sham pancreatectomy. One week later, the animals developed mild to severe chronic hyperglycemia that was stable for the next 3 weeks, without significant alteration of
plasma nonesterified fatty acid levels. Expression of many genes
important for glucose-induced insulin release decreased progressively
with increasing hyperglycemia, in parallel with a reduction of several
islet transcription factors involved in
cell development and
differentiation. In contrast, genes barely expressed in sham islets
(lactate dehydrogenase A and hexokinase I) were markedly increased, in
parallel with an increase in the transcription factor c-Myc, a
potent stimulator of cell growth. These abnormalities were accompanied
by
cell hypertrophy. Changes in gene expression were fully
developed 2 weeks after pancreatectomy. Correction of blood glucose by
phlorizin for the next 2 weeks normalized islet gene expression and
cell volume without affecting plasma nonesterified fatty acid levels,
strongly suggesting that hyperglycemia triggers these abnormalities. In
conclusion, chronic hyperglycemia leads to
cell hypertrophy and
loss of
cell differentiation that is correlated with changes in
c-Myc and other key transcription factors. A similar change in
cell differentiation could contribute to the profound derangement
of insulin secretion in human diabetes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells are highly specialized cells that secrete
insulin in response to a variety of stimuli, the most important being
glucose (1, 2). Their correct function is dependent on the expression
of a unique set of genes that allow
cells to respond to even small
increases in plasma glucose levels by releasing appropriate amounts of
insulin into the circulation (3). Type 2 diabetes is characterized by
the combination of insulin resistance and profound alteration in
glucose-stimulated insulin secretion (4). The latter can be ascribed,
at least in part, to the deleterious effect of even mild chronic
hyperglycemia and elevated plasma nonesterified fatty acids
(NEFA)1 on
pancreatic
cell function, a process often referred to as "gluco-lipotoxicity" (5, 6). In islets isolated from animal models
of diabetes, several defects have been identified at the level of gene
expression and/or enzymatic activity, but none by itself can entirely
account for the
cell defect characteristic of type 2 diabetes (5, 7, 8).
cell/islet transcription factors that are important for the development of the
endocrine pancreas, the tissue-specific expression of key
cell
genes and maintenance of
cell differentiation (9, 10). In islets of
diabetic animals and in long term culture of immortalized
insulin-secreting cells, chronic exposure to high glucose can result in
loss of insulin gene expression (11, 12). This loss is associated with
reduced expression and/or activity of the
cell transcription
factors PDX-1 and RIPE3b1 (11, 13, 14) and increased expression of the
liver-adipocyte transcription factor C/EBP
(14). Similar alterations
in gene expression have also been described in islets exposed in
vitro for 2 days to high glucose and NEFA (15). We have recently
hypothesized that these changes in islet gene expression could reflect
a loss of differentiation of
cells chronically exposed to the
diabetic milieu, leading to further deterioration of
cell function
and subsequent decreased secretory function (8).
cell differentiation, using a well characterized model
of chronic hyperglycemia in the rat, the 90% partial pancreatectomy (Px) (16). After an initial burst of pancreatic regeneration that lasts
no longer than ~10 days, these animals have a stable population of
mature islets exposed to chronic hyperglycemia from 1st week post-Px
(17). Px rats are thus considered a model of
cell adaptation to
increased secretory demand and exposure to the diabetic environment
(5). Our results show that graded levels of chronic hyperglycemia
in vivo leads to progressive loss of
cell
differentiation that can be reversed by normalization of blood glucose
levels with phlorizin, in the absence of changes in plasma NEFA. They
also suggest a link between stimulation of
cell growth and their
reduced state of differentiation in hyperglycemic animals.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
reverse transcriptase (Life
Technologies, Inc.). Reactions were incubated for 10 min at 25 °C,
1 h at 42 °C, and 10 min at 95 °C. The final cDNA
reaction products were then diluted with 50 µl of H2O to
a concentration corresponding to 20 ng of starting RNA (20-ng RNA
equivalents) per 3 µl and stored at
80 °C (18).
-32P]dCTP (3000 Ci/mmol), 2.5-30 pmol of appropriate
oligonucleotide primers, GeneAmp PCR buffer, and 5 units of AmpliTaq
Gold DNA polymerase (Perkin-Elmer). The oligonucleotide primers and
conditions used for multiplex PCR are indicated in Table
I. The thermal cycle profile used was a
10-min denaturing step at 94 °C to release DNA polymerase activity
(hot start PCR) followed by the number of cycles indicated in Table I
and a final extension step of 10 min at 72 °C. In each set, the gene
products of interest were amplified with an internal control gene
(cyclophilin,
-tubulin, the rat ribosomal phosphoprotein P0 (RRPP0)
or the TATA-binding protein (TBP) to correct for experimental
variations between samples (RNA quantification, starting cDNA, gel
loading, etc.). After removal of free [
-32P]dCTP by
gel filtration on Probequant G50 microcolumns (Amersham Pharmacia
Biotech), the amplimers were separated on a 6% polyacrylamide gel in
Tris borate EDTA buffer. The gel was dried, and the amount of
[
-32P]dCTP incorporated in each amplimer was measured
with a PhosphorImager and quantified with the ImageQuant software
(Molecular Dynamics, Sunnyvale, CA). The amount of each specific
product was then expressed relative to the internal control, giving a
ratio "specific product/control gene" for each sample. These ratios
were then expressed as a percent of the ratio in sham islet extracts
tested in the same RT-PCR. For each sample, a negative control (RT
reaction performed in the absence of RT enzyme) was performed to
exclude genomic DNA contamination of the cDNA.
Sequences of oligonucleotide primers and PCR conditions
View larger version (64K):
[in a new window]
Fig. 1.
Validation of multiplex PCR for amplification
of IAPP ( ), GLUT2 (
), and SERCA3 (
) using cyclophilin (
) as
internal control. A, 50-µl aliquots of PCR mix
(containing 1 mM MgCl2, 80 µM
dNTP, 200 nM of each primer, and cDNA corresponding to
20 ng of starting RNA) were run for increasing number of cycles; the
products were separated on a 6% polyacrylamide gel, and band
intensities were quantified using a PhosphorImager. B, the
number of 20 cycles (well within the logarithmic linear range of
amplification for all products) was then chosen for checking the
linearity of PCR amplification with increasing amounts of cDNA
(2.5- to 80-ng RNA equivalents). The relative band intensity of each
product to cyclophilin remained stable within this wide range of
starting cDNA. The light band between cyclophilin and SERCA3
products was observed when SERCA3 was amplified alone and is not due to
interactions between different pairs of primers in the multiplex
reaction.
160 mg/dl) rats. Severely hyperglycemic Px rats were then divided in
3 groups. The first one was sacrificed 2 weeks after Px, and the other
two were randomly assigned to phlorizin treatment or no treatment for
the next 2 weeks.
cells
mainly express the liver isoform 5 of LDH, a tetramer of LDH-A
(LDH-A4) (22). The sections were then incubated overnight
at 4 °C, washed with Tris buffer (pH 7.4), sequentially incubated
with goat anti-rabbit Ig and rabbit peroxidase anti-peroxidase
conjugate (Cappel Laboratories, Cochranville, PA), stained with
diaminobenzidene, and counterstained with hematoxylin. By using this
procedure on liver sections, we found the expected staining pattern of
LDH (21).
cells using a mixture of
antibodies as follows: rabbit anti-bovine glucagon (final dilution
1:3000, gift of Dr. M. Appel), rabbit anti-synthetic somatostatin
(final dilution 1:300, made in our own laboratory), and rabbit
anti-bovine pancreatic polypeptide (final dilution 1:3000, gift of Dr.
R. Chance, Lilly, Indianapolis). The sections were incubated with this
mixture of antibodies 40 h at 4 °C and processed as described
above for LDH staining. These sections were then used to evaluate
to non-
cell ratio in isolated islets.
Cell Size--
Islet
pellets were fixed in 2.5% glutaraldehyde in 0.1 M
phosphate buffer and processed for routine electron microscopy. Fifteen micrographs of each of two randomly picked islets per sample were taken
at a magnification of 3800 with a Philips 301 electron microscope.
cell cross-sectional area was determined on
cells with a visible nucleus (64-120 cells/animal) by planimetry using the IPLab Spectrum image analysis software (Scanalytics, Fairfax, VA).
cell volume was
estimated from these values using the formula for volume of sphere.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 2.
Changes in body weight and fed whole blood
glucose levels after Px. Whole blood glucose levels were
determined in the fed state (9-10 a.m.) with a portable glucometer. Px
rats were then classified according to their averaged blood glucose 3 and 4 weeks post-Px (see "Experimental Procedures" and Table II).
Body weight gain was significantly reduced only during the 1st week
after Px. Values are means ± S.E. for 11 sham ( ), 18 low
hyperglycemic (
), 10 mildly hyperglycemic (
), 4 highly
hyperglycemic (
), and 6 severely hyperglycemic (
) Px rats.
Significant differences between groups were determined by one-way ANOVA
followed by a test of Dunnett. *p < 0.01 and
**p < 0.001 versus controls.
Changes in pancreatic islet gene mRNA levels in rats with different
degree of hyperglycemia 4 weeks after Px
Changes in transcription factor mRNA levels in rats with different
degree of hyperglycemia 4 weeks after Px
cell hormones, glucagon and somatostatin, were not
significantly altered 4 weeks after Px. To verify that the decrease in
insulin mRNA level was not due to variable contamination with
exocrine tissue, amylase mRNA levels were measured in sham, MPx,
HPx, and SPx islets and were compared with exocrine tissue obtained at
the end of the islet isolation procedure. The exocrine contamination
was not different between sham, MPx, HPx, and SPx (3.31 ± 0.75 (n = 10), 3.15 ± 0.95 (n = 5),
4.05 ± 0.70 (n = 4), and 3.59 ± 0.78 (n = 6) % of total islet cDNA, respectively). Because of the possibility that amylase gene expression may be altered
in the diabetic state, these numbers must be regarded as only
approximations. Additionally, in sections of islets isolated from H/SPx
and sham immunostained for non-
cell hormones, the proportion of
non-
cells to
cells appeared similar in Px and sham islets (data
not shown). In our previous study, the
cell to non-
cell ratio
was equal or even slightly higher in SPx than in sham islets (11).
These observations exclude that an increase in exocrine contamination
or decrease in
cell to non-
cell ratio artifactually accounts
for the changes in
cell-specific gene expression observed in Px
islet RNA.
View larger version (56K):
[in a new window]
Fig. 3.
Comparison of insulin mRNA levels by
semiquantitative RT-PCR in islets of representative sham
(S), mildly hyperglycemic (MPx), and severely
hyperglycemic (SPx) rats (BG represents the averaged
blood glucose values 3 and 4 weeks post-Px). Insulin mRNA was
decreased by ~50% in SPx islets but remained unaffected in MPx
islets. Cyclophilin, used as internal control gene, did not change
between groups. Mean changes of insulin mRNA levels in Px
versus sham islets are indicated in Table II.
cell glucose
metabolism was also evaluated 4 weeks after Px (Table II). The mRNA
level of the glucose transporter 2 (GLUT2) was decreased to the same
extent as insulin mRNA in HPx and SPx islets. However, it was also
significantly decreased by 25% in MPx islets and moderately, although
not significantly, decreased in LPx islets (Table II). Similarly, a
progressive decrease with increasing blood glucose was observed for the
mitochondrial glycerol-phosphate dehydrogenase (mGPDH, the
rate-limiting enzyme of the glycerol-phosphate shuttle) and the
anaplerotic enzyme pyruvate carboxylase (Table II, Fig. 4, sets 5 and 6).
Glucokinase mRNA level was significantly reduced by 40% in SPx and
HPx islets (Table II and Fig. 4, set 5) but not in MPx and
LPx. Interestingly, lactate dehydrogenase-A (LDH-A) mRNA levels,
which in sham islets were only about 3% that found in liver (not
shown), were dramatically increased 2-3-fold in LPx and MPx islets and
up to 5-fold in HPx and SPx (Table II and Fig. 4, set 3). A
similar increase was also observed for hexokinase I (HK I) which was
expressed at low level in sham islets (Fig. 4, set 4).
View larger version (73K):
[in a new window]
Fig. 4.
Semiquantitative multiplex RT-PCR sets used
to compare the mRNA levels of several transcription factors,
glucose metabolism genes, and ion channels in sham and Px islets.
The internal control gene used in each particular set is highlighted in
italics. The results were obtained with islets from a
representative sham (S) and a severely hyperglycemic Px rat
(SPx) 4 weeks after Px (averaged 3- and 4-week blood glucose
levels were 76 and 311 mg/dl, respectively).
1D, the neuroendocrine isoform of the
subunit of
voltage-dependent Ca2+-channels, were markedly
decreased with increasing glycemia after Px (Table II and Fig. 4,
set 5). A similar reduction was observed for the mRNA
levels of the third isoform of the sarcoendoplasmic reticulum
Ca2+-ATPase (SERCA3) (Table II). In contrast, the
ubiquitously expressed isoform SERCA2B was only moderately reduced by
25% in SPx islets but remained totally unaffected in other Px groups
(Table II).
cell development and differentiation. PDX-1 mRNA level was
significantly reduced in HPx and SPx islets to 60% of sham (Fig. 4,
set 2) and moderately reduced in LPx and MPx islets (Table
II). A progressive reduction of similar, or even more severe, magnitude
was also observed for the mRNA levels of Nkx6.1, Pax6, and the
hepatocyte nuclear factors HNF1
, HNF4
, and HNF3
(Table II and
Fig. 4). In contrast, the mRNA levels of the basic helix loop helix
(bHLH) factor beta2 were only decreased by 35% in HPx and SPx and
remained completely normal in LPx and MPx islets. Both the islet-brain
1 transcription factor (IB1) that may play a role in the control of
GLUT2 expression (24) and the bHLH factor PAN1 showed a modest decrease
in all Px groups (Table II). In contrast, the ubiquitous factor SP1
remained unaffected after Px.
View larger version (24K):
[in a new window]
Fig. 5.
Effects of phlorizin treatment on body weight
and blood glucose levels after Px. Whole blood glucose levels were
determined in the fed state (9-10 a.m.) with a portable glucometer.
Only severely hyperglycemic Px rats were used (see "Experimental
Procedures" for classification criteria). Half of Px were treated
with phlorizin (0.8 g/kg body weight) for the next 2 weeks in two daily
IP injections, and half of sham were injected with comparable volume of
vehicle alone. Values are means ± S.E. for 6 sham ( ), 5 sham
injected with vehicle (
), 4 severely hyperglycemic (
), and 6 phlorizin-treated severely hyperglycemic (
) Px rats.
View larger version (25K):
[in a new window]
Fig. 6.
Time course and reversibility of changes in
islet gene expression after 90% partial pancreatectomy. After 2 weeks, Px rats were classified according to their averaged 1- and
2-week blood glucose levels in moderately hyperglycemic (M
<160 mg/dl) and severely hyperglycemic (Px 160 mg/dl). Severely
hyperglycemic rats were then divided in 3 groups. The first one was
sacrificed 2 weeks after Px, and the other two were randomly assigned
to phlorizin treatment (PxP) or no treatment (Px)
for the next 2 weeks. Half of sham (S) received vehicle
alone (SV). Values are means ± S.E. for the indicated
number of animals. *, p < 0.05; **, p < 0.01 versus sham (sacrificed on the same day), by test of
Dunnett after one-way ANOVA. ##, p < 0.01;
###, p < 0.001 versus
nontreated SPx rats by test of Newman-Keuls after one-way ANOVA.
View larger version (77K):
[in a new window]
Fig. 7.
Effect of 2-week normalization of blood
glucose by phlorizin treatment on changes in islet gene mRNA levels
after 90% Px. The different internal control genes used are
highlighted in italics. The results were obtained 4 weeks
after Px with islets from a representative sham (S), a sham
injected with vehicle alone (SV), two severely hyperglycemic
Px rats (Px), and two severely hyperglycemic Px rats treated
with phlorizin for the last 2 weeks (PxP). BG
shows the averaged blood glucose values 3 and 4 weeks post-Px on the
upper line, and the averaged blood glucose values 1 and 2 weeks post-Px (before phlorizin treatment) on the bottom
line.
View larger version (36K):
[in a new window]
Fig. 8.
Changes in plasma nonesterified fatty acid
and plasma insulin levels after Px. Only severely hyperglycemic
rats were used. Plasma was prepared from blood samples obtained on
EDTA-paraoxon-coated tubes at 2 and 4 weeks in the fed state and after
an overnight fast on day 25. Values are means ± S.E. for the
indicated number of sham (S), sham-injected with vehicle
(SV), severely hyperglycemic rats (Px), and
severely hyperglycemic rats treated with phlorizin
(PxP).
cells (11). To verify that LDH-A expression is increased in pancreatic
cells, we stained sections of pancreas obtained 4 weeks after Px for LDH-A4 (isoform 5), the main isoform
expressed in islets (22). In sham pancreas, most LDH-A4
immunoreactivity was observed in ductal epithelial cells, smooth muscle
of blood vessels, lymph nodes, and in endothelial cells, even those
within the islets (Fig. 9A).
However, 4 weeks after Px, LDH-A4 was significantly increased in the cytoplasm of endocrine cells throughout the islet (Fig. 9B), consistent with the increase in islet LDH-A
mRNA levels (Table II). These changes were totally reversed by 2 weeks of phlorizin treatment in Px rats (Fig. 9C). Sections
of pancreas obtained 4 weeks after Px were also stained for the
transcription factors PDX-1 and Nkx6.1. In control animals, PDX-1 and
Nkx6.1 were strongly expressed in the nuclei of most cells in the islet core (Fig. 9, D and G). Remarkably, PDX-1 and
Nkx6.1 staining was markedly decreased in islets of severely
hyperglycemic Px rats (Fig. 9, E and H), and they
were restored to sham levels after 2 weeks of phlorizin treatment (Fig.
9, F and I). Comparison of LDH-A4 and
Nkx6.1 staining revealed that the increase in LDH-A4 was
most pronounced in animals in which Nkx6.1 was most reduced (data not
shown).
View larger version (98K):
[in a new window]
Fig. 9.
Immunohistochemistry for LDH-A4
(immunoperoxidase, A-C), PDX-1 (Texas Red,
D-F), and Nkx6.1 (FITC, G-I) on
pancreatic sections from a representative 4-week sham (A, D,
and G), a severely hyperglycemic Px rat
(B, E, and H), and a severely
hyperglycemic Px rat treated for 2 weeks with phlorizin (C,
F, and I). Islets representative of
islet population observed in each group of rats have been selected for
illustration. The larger islets in B, E, and H
are representative of hypertrophic islets observed in severely
hyperglycemic animals. Magnification bars = 100 µm.
Cell Hypertrophy and Increased c-Myc mRNA Levels--
As
can be seen in Fig. 9, Px islets were markedly hyperplastic compared
with sham. Individual
cells were also hypertrophic. Four weeks
after Px, their cross-sectional area, determined by planimetry on
ultrastructural images, was increased by ~50% in severely
hyperglycemic rats, corresponding to an 85% increase in cell volume
(Table IV). In contrast, the
cell
volume of phlorizin-treated Px rats was not different from shams. The
proto-oncogene c-myc is a bHLH-leucine zipper transcription
factor that stimulates cell growth and LDH-A gene expression (26). Of
considerable note, c-Myc mRNA levels were increased in parallel to
LDH-A mRNA and blood glucose levels 2 and 4 weeks after Px and
returned to normal values after 2 weeks of blood glucose normalization
by phlorizin treatment (Fig. 6).
Effect of chronic hyperglycemia on cell size 4 weeks after Px
cell cross-sectional
area was determined by planimetry on ultrastructural images from two
islets randomly selected in each pellet. Values are means ± S.E.
for the number of cells (shown in parentheses) with a visible nucleus.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells are unique in their ability to secrete
insulin in response to a rise in plasma glucose, a function that, in
addition to insulin, requires adequate level of expression of a unique
set of genes. In the present study, we show that increasing levels of
chronic hyperglycemia trigger gradual loss of
cell differentiation
in a rat model of type 2 diabetes, as indicated by the following
observations. First, the expression of several genes important for
glucose-stimulated insulin secretion (glucose metabolism enzymes and
ion channels/pumps) was gradually decreased with increasing levels of
blood glucose. This reduction was already significant in the presence
of low to mild hyperglycemia (<150 mg/dl), even though insulin and
Kir6.2 mRNA levels were only affected in the presence of high to
severe hyperglycemia (>150 mg/dl). Second, expression of
ldh-A and hk I, two genes barely expressed in
normal islets, was markedly increased in Px islets. Third, the levels
of many, but not all, transcription factors important for
cell
development and regulation of gene expression (9, 10) decreased
progressively with increasing levels of hyperglycemia. That these
changes in islet gene expression correspond to a loss of
cell
differentiation is further supported by the observation that the
mRNA levels of the proto-oncogene transcription factor c-Myc
increase in parallel to blood glucose. An increase in c-Myc is
likely to stimulate cell growth and inhibit cell differentiation by
directly increasing the transcription of several target genes, one of
which is ldh-A (26), or by repressing the expression of
other genes (27, 28). Consistent with this hypothesis, we observed
cell hypertrophy in hyperglycemic Px rats.
cell transcription factors decreased gradually with increasing
levels of chronic hyperglycemia. HNF3
, HNF4
, HNF1
, Pax6,
PDX-1, and Nkx6.1 were most sensitive to chronic hyperglycemia, whereas
IB1 and the bHLH factors beta2 and PAN1 were little affected, and the
ubiquitous transcription factor SP1 remained totally unaffected.
Although expression of none of these factors was completely abolished,
their reduction could contribute with time to alteration of expression
of
cell genes involved in glucose-induced insulin release, as
suggested by the development of maturity-onset diabetes of the young in
subjects heterozygous for mutations of pdx-1,
hnf1
, and hnf4
(29-31). Thus, the
expression of some of the major members of the transcriptional network
responsible for
cell differentiation (9, 10) is altered in islets
from chronic hyperglycemic Px rats and could contribute to the major
changes observed in genes important for glucose-stimulated insulin secretion.
Cell Secretory
Function--
Glucose-stimulated insulin secretion is a highly
regulated process in pancreatic
cells that depends on correct
expression of insulin and the unique
cell metabolic and secretory
machinery (2, 3). The reduction of insulin gene expression that was seen only in highly and severely hyperglycemic Px rats probably contributes to the diminished insulin secretion of these rats (32).
However, the normal insulin expression in low and mildly hyperglycemic
rats indicates that alteration of insulin secretion in rats with
similar mild hyperglycemia (16, 33) cannot be ascribed to reduced
insulin gene expression but rather to altered expression/activity of
other genes important for glucose-stimulated insulin secretion. Among
them, the most sensitive to chronic hyperglycemia in Px islets were
GLUT2, glucokinase, mGPDH, pyruvate carboxylase, VDCC
1D,
and SERCA3. Their mRNA levels were all progressively reduced down
to 40-60% of sham values in the presence of increasing levels of
hyperglycemia, in parallel with the most sensitive transcription factors. In contrast, two genes weakly expressed in normal islets, hk I and ldh-A, increased up to 5-fold with
increasing glycemia. From our results, we estimate that the ratios
glucokinase/HK I and mGPDH/LDH are reduced to 25-50% of sham in LPx
and MPx islets and down to 10-20% in HPx and SPx islets. These global
alterations observed in Px islets could alter the preferential
stimulation of oxidative metabolism by glucose and thus adversely
influence glucose-stimulated insulin secretion (34-40). This
conclusion relies on the premise that the changes in mRNA levels
correspond to changes in protein levels and function; it is supported
by parallel changes in mRNA and protein levels of LDH-A, PDX-1, and
Nkx6.1. This concordance may not be verified for all genes,
e.g. glucokinase enzymatic activity versus
mRNA/protein levels (7, 41, 42). However, the increase in HK I
mRNA found in our Px rats is consistent with the increased HK
activity reported in diabetic animals which probably accounts for the
lower threshold for glucose-stimulated insulin secretion in Px islets
and in islets overexpressing HK I (41-43). The progressive decrease of
VDCC
1D and SERCA3 could also contribute to the
alteration of glucose-induced [Ca2+]i rise and
stimulation of insulin release in type 2 diabetes (7, 44, 45).
Cell Differentiation after
Px--
The causal role of hyperglycemia in increased c-myc
expression,
cell hypertrophy, and loss of
cell differentiation
in the Px model is strongly suggested by their complete normalization after 2 weeks treatment with phlorizin, a drug that normalized blood
glucose without increasing plasma insulin or changing plasma NEFA
levels. In fact, plasma insulin levels were even further decreased by
phlorizin treatment, either because of reduced stimulation of insulin
secretion by correction of hyperglycemia or possibly because of some
nonspecific effect of the vehicle, 1,2-propanediol. The lack of changes
in islet gene expression in shams injected with the vehicle, however,
argues against a nonspecific effect of the vehicle on islets. The
reversibility with phlorizin makes it also highly unlikely that the
loss of
cell differentiation after Px is due to the initial burst
of regeneration of islets after pancreatectomy (17). Phlorizin also
corrects insulin resistance in Px rats (46). Thus, it is possible that
an unknown factor changing in parallel to blood glucose and development
of insulin resistance after Px is the real signal leading to alteration
of
cell differentiation and secretory function, but this seems unlikely. On the other hand, plasma NEFA levels were not affected in Px
rats compared with sham, indicating that they do not play any role in
triggering loss of
cell differentiation after Px. However, we do
not exclude the possibility that the deleterious effect of
hyperglycemia on pancreatic
cell could involve alterations in
intracellular lipid metabolism and accumulation of triglycerides in the
cytoplasm of
cells, as seems to be the case in islets from Zucker
Diabetic Fatty rats (6, 47, 48).
cells through a common
mechanism, the stimulation of cell growth and increased
c-myc expression, along with other transcription factors
(14), leading to loss of
cell differentiation. Indeed, islet
hyperplasia is a common feature of many rodent models of type 2 diabetes, as are changes in expression of several genes investigated in
this study (7, 11, 14, 35, 44, 45, 54, 55). Furthermore, in
vitro experiments have shown that high glucose or elevated NEFA
can have similar effects on
cell gene expression and secretory
function (13, 15).
cell
differentiation with marked alterations in islet gene expression and
secretory function (33). Thus, it is possible that a similar mechanism
occurs during the progression of glucose intolerance to type 2 diabetes
in humans. Phlorizin treatment has been shown to correct
glucose-induced insulin secretion in Px rats when secretion is
expressed relative to the
cell mass remaining in the pancreas (32).
Thus, complete normalization of islet secretory function occurs in
parallel with correction of islet gene expression.
cell differentiation, as
indicated by altered expression of several key islet transcription factors and other islet genes important for normal glucose-stimulated insulin secretion. Similar alterations might be responsible for at
least part of the altered
cell secretory function of human diabetes. Our finding of increased c-Myc mRNA levels provides a
plausible link between stimulation of cell growth and loss of
cell
differentiation in hyperglycemic animals. However, this hypothesis must
be tested with genetic and more definitive approaches.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank G. Waeber and C. Bonny for giving us the sequence of IB1 before its publication. We also thank Monica Taneja and Chris J. Cahill for their expert technical help. The animal care and morphology core facilities were supported by Grant DK-36836 from the National Institutes of Health to the Joslin Diabetes Endocrinology Research Center.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK-35449 (to G. C. W.) and DK-44523 (to S. B.-W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the Belgian American Educational Foundation.
§ Recipient of a Career Development award from the American Diabetes Association.
¶ Current address: Diabetes and Metabolism, Dept. of Internal Medicine, University of Istanbul, Istanbul, Turkey.
Current address: Dept. of Endocrinology, University of
Catania, Garibaldi Hospital, Catania, Italy.
** To whom correspondence should be addressed: Islet Transplantation and Cell Biology, Joslin Diabetes Center, One Joslin Place, Rm. 535, Boston, MA 02215. Tel.: 617-732-2581; Fax: 617-732-2650; E-mail: weirg{at}joslab.harvard.edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NEFA, nonesterified fatty acids; RT, reverse-transcribed; PCR, polymerase chain reaction; Px, pancreatectomy; LPx, low hyperglycemic pancreatectomy; MPx, mildly hyperglycemic Px; HPx, highly hyperglycemic Px; SPx, severely hyperglycemic Px; IAPP, islet amyloid polypeptide; GLUT, glucose transporter; HK, hexokinase; mGPDH, mitochondrial glycerol-phosphate dehydrogenase; LDH, lactate dehydrogenase; K+-ATP channels, ATP-sensitive K+ channels; VDCC, voltage-dependent Ca2+ channels; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; HNF, hepatocyte nuclear factor; bHLH, basic helix loop helix; ANOVA, analysis of variance; FITC, fluorescein isothiocyanate; DABCO, 1,4-diazabicyclo[2.2.2]octane.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|