From the Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Boston, Massachusetts 02215
Received for publication, October 16, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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We have proposed that hyperglycemia-induced
dedifferentiation of Pancreatic The development of the endocrine pancreas and the maintenance of
In this study, we have extended our previous findings examining the
influence of the diabetic milieu on Animals--
Male Sprague-Dawley rats (Taconic Farms,
Germantown, NY), weighing 90-100 g were submitted to 85-95% Px or
sham Px surgery as previously described (13, 18). For partial
pancreatectomy, tissue removal was performed by gentle abrasion with
cotton applicators leaving the pancreas within 1-2 mm of the common
pancreatic bile duct and extending from the duct to the first part of
the duodenum. The proportion of the gastric lob removed was varied to
generate 85-95% Px rats that develop different degrees of
hyperglycemia (13). For sham surgery, the pancreatic tissue was only
lightly rubbed instead of being removed. Animals were weighed, and
blood was obtained in heparinized microcapillary tubes from snipped tails of fed rats (9-10 a.m.) weekly. Whole-blood glucose levels were
measured with a portable Medisense Precision QID® glucometer (Abbott
Laboratories, Bedford, MA). Rats were classified according to their
averaged blood glucose levels from 3 weeks after surgery; low
hyperglycemia (LPx) was assigned to Px rats with blood glucose levels
less than 150 mg/dl, and high hyperglycemia (HPx) was assigned above
150 mg/dl. To assess the effects of the duration of hyperglycemia on
islet gene expression, rats were anesthetized at 4 and 14 weeks after
surgery, and their islets were isolated with collagenase digestion of
the pancreatic remnant or sham pancreas. Islets were further separated
with a Histopaque density gradient (Histopaque-1077; Sigma) and
hand-picked under a stereomicroscope. Islets of similar size were used
for extraction of RNA. In several cases, it was necessary to pool
islets from two Px rats with similar glycemic levels to obtain an islet
yield that was sufficient for RNA extraction. Animals were kept under
conventional conditions with free access to water and standard pelleted
food. All animal procedures were approved by the Joslin Diabetes Center
Animal Care Committee.
RNA Extraction and Synthesis of cDNA--
Total RNA was
extracted from islets using Ultraspec RNA isolation reagent according
to the manufacturer's suggested protocols (Biotecx Laboratories,
Houston, TX) and quantified by spectrophotometry. RNA (500 ng) was
reverse-transcribed into cDNA in a final reaction solution of 25 µl containing the following: 1× Superscript first strand buffer (50 mM Tris-HCl, 75 mM KCl, and 3 mM
MgCl2) (Invitrogen), 40 units of Rnasin (Promega,
Madison, WI), 10 mM dithiothreitol, 1 mM dNTP,
50 ng of random hexamers, and 200 units of Superscript II Rnase
H Semiquantitative Radioactive Multiplex Polymerase Chain
Reaction--
PCRs were carried out in a volume of 25 µl consisting
of 10 ng of cDNA, 1× GeneAmp PCR Gold buffer (Applied Biosystems,
Foster City, CA), 1-2 mM MgCl2, 80-160
µM dNTP, 80-600 nmol of oligonucleotide primers (Sigma),
1.25 µCi of [ Plasma Insulin and Lipid Determination--
Plasma insulin was
measured by radioimmunoassay (Linco Research, St. Charles, MO). Plasma
lipids were measured from samples collected in
ETDA/diethyl-para-nitrophenyl phosphate (paraoxan)-coated tubes to avoid activation of lipoprotein lipase by heparin (19). Plasma
nonesterified fatty acids (NEFA) were measured by a colorimetric method (Wako Chemicals, Neuss, Germany). Plasma triglyceride were measured with a triglyceride assay kit (GPO Trinder; Sigma) using glycerol as a standard.
Phlorizin Treatment of Px Rats--
To reverse hyperglycemia, Px
rats were treated with phlorizin for the final 2 weeks of the 14-week
study period. Phlorizin was dissolved in 1,2-propanediol and injected
intraperitoneally twice a day (9 a.m. and 9 p.m.) at a dose of 0.8 g/kg/day. Sham animals received similar amounts of 1,2-propanediol as
phlorizin-treated Px rats.
Cell Size--
Glucose Clamp--
To assess the influence of short term
hyperglycemia on islet gene expression in vivo, catheterized
rats (~250 g) were infused for 4 days with glucose (500 g/liter
hydrated glucose, McGaw, Irvine, CA) or saline (4.5 g/liter) as
described (20, 21). The glucose infusion rate was regularly adjusted to
maintain the blood glucose level at ~200 mg/dl, with saline infusion
adjusted accordingly. At the end of the infusion period, islets were
isolated, and RNA was extracted for reverse transcription-PCR analysis. Experiments were performed on islet extracts from rats used in our
previous study (21).
Statistical Analysis--
All results are presented as
means ± S.E. Statistical analyses were performed using unpaired
Student's t test or one-way analysis of variance.
Changes in Fed Blood Glucose Levels and Body Weight after
Px--
Time course changes in blood glucose levels after Px are shown
in Fig. 1. As previously described
(13-15), slight variation in the proportion of pancreas removed
(~85-95%) resulted in rats with different degrees of hyperglycemia
after 4 weeks; blood glucose levels ranged from 114 to 370 mg/dl.
However, after 14 weeks, the range of blood glucose levels in Px rats
had clustered into two distinct groups with either low or high levels
of hyperglycemia (Fig. 1). Px rats were classified into groups
according to their averaged fed blood glucose levels; LPx below
150 mg/dl and HPx above 150 mg/dl. The time course changes in body
weight and fed blood glucose in LPx, HPx, and sham-Px rats are
illustrated in Fig. 2. As previously
described (13, 18), weight gain in Px rats was slightly decreased
during the first few days after surgery, resulting in significantly
lower body weights at 1 week postsurgery. Thereafter, Px rats gained
weight at the same rate as sham Px rats. After 8 weeks, LPx rats had
body weights not significantly different from age-matched sham rats,
whereas HPx rats had lower body weights throughout the study period.
Averaged post-3-week fed blood glucose levels ranged from 97 to 111 mg/dl in sham rats, from 108 to 141 mg/dl in LPx rats, and from 220 to
365 mg/dl in HPx rats. The blood glucose levels of HPx rats were
significantly increased by 1 week after surgery and remained
significantly higher compared with sham and LPx rats throughout the
study (p < 0.001). Blood glucose levels of LPx rats
were not different from sham at 1 week after surgery; however, the
averaged post-3-week blood glucose levels in LPx rats were
significantly higher than sham (p < 0.01).
Changes in Changes in Levels of Islet-associated Transcription Factor
mRNA--
After normalization of the specific gene to an internal
control gene (TBP, mRNA Levels of Islet Hormones--
Insulin and IAPP expression
were down-regulated after Px with a similar dependence on the degree
and duration of hyperglycemia. After 4 weeks, both insulin and IAPP
mRNA levels were unaltered in LPx rats but were reduced in HPx rats
(Table II). After 14 weeks, both genes were significantly reduced in
LPx rats and were further reduced in HPx rats (insulin reduced
by ~55%). In contrast, the mRNA levels for the mRNA Levels of Metabolic Enzymes--
Strikingly, the
down-regulation of several mRNA Levels of Ion Channels/Pumps--
The
expression of several ion channels important for the stimulation of
secretion were assessed in Px rats (Table II). Analyzed individually,
the mRNA levels of the pore-forming subunit of the ATP-sensitive
K+ channel Kir6.2, the mRNA Levels of Stress Genes--
Stress gene mRNA levels
in Changes in Plasma Insulin, NEFA, and Triglyceride Levels after
Px--
Changes in plasma insulin, NEFA, and triglyceride levels 4 and
14 weeks after surgery are shown in Fig.
3. At both time points, plasma insulin
levels (Fig. 3A) tended to be decreased in Px rats but were
only significantly reduced in HPx rats. Plasma NEFA (Fig. 3B) and triglyceride (Fig. 3C) levels were
unchanged at 4 weeks regardless of the level of hyperglycemia. At 14 weeks, NEFA and triglyceride levels were unchanged in LPx rats but
modestly increased in HPx rats.
Reversibility of Changes in Gene Expression after Px--
We
tested the reversibility of the changes in mRNA levels by using
phlorizin, an inhibitor of glucose reabsorption in the kidney.
Phlorizin blocks the Na+/glucose co-transporter in the
proximal tubules of the kidney, causing glucosuria and the
normalization of circulating glucose levels. We previously showed
(13-15) that expression levels of islet transcription factors,
hormones, metabolic enzymes, ion channels, and stress genes
were completely normalized in 4-week Px rats after treatment with
phlorizin. Here, we tested whether the altered islet gene expression in
14-week Px rats would be similarly normalized with 2-week phlorizin
treatment. Blood glucose levels in Px rats were completely normalized
during phlorizin treatment (Fig. 4).
Expression of the transcription factors, PDX-1 and Nkx6.1, shown
previously to be fully normalized with phlorizin treatment of 4-week Px
rats (13), were only partially reversed toward normal at 14 weeks,
remaining significantly reduced compared with sham levels (Fig.
5). Similarly, insulin and GLUT2
expression were partially restored after phlorizin treatment; GLUT2
mRNA levels were significantly decreased in phlorizin-treated Px
rats compared with sham. The up-regulated expression of c-Myc, LDH-A, HO-1, and glutathione peroxidase in Px rats tended to be partially reversed after phlorizin treatment (Fig. 5). However, LDH-A, HO-1, and
glutathione peroxidase mRNA levels were significantly increased in
phlorizin-treated Px rats compared with sham. In all cases shown in
Fig. 5, the vehicle had no effect on islet gene expression in
sham-treated rats. Similar results were observed for the other genes
altered in Table II (not shown).
Increased Islet Gene Expression after Glucose Clamp--
The influence of
short term hyperglycemia on islet gene expression was assessed using
in vivo glucose clamps. Gene expression levels after 4-day
glucose clamp (blood glucose maintained at ~200 mg/dl) relative to
saline-infused control rats are shown in Table
IV. In contrast to the effects of long
term hyperglycemia in Px rats (Table II), short term hyperglycemia
induced by glucose clamp caused relatively little change in islet gene
expression (Table IV). PDX-1 mRNA levels were unaltered after
glucose clamp, and BETA2/NeuroD and Nkx6.1 mRNA were modestly
reduced (Table IV). Insulin mRNA levels were unaltered after
glucose clamp, whereas IAPP mRNA levels were increased consistent
with previous findings in glucose-infused rats (20). Somatostatin
mRNA levels were unaltered after glucose clamp, whereas glucagon
mRNA was decreased, perhaps due the initial growth in In this study, we have extended our previous findings examining
the influence of the diabetic milieu on It is also of interest that this critical reduction of Loss of Changes in Gene Expression and the Loss of GIIS after Px--
The
loss of GIIS has been identified in not only type 2 and early type 1 diabetes but also in islet transplants (27-31). Islets from Px rats
display a similar defect in GIIS (16, 29). The down-regulation of
islet-associated transcription factors (PDX-1, BETA2/NeuroD, HNF1 Role of Hyperglycemia in the Loss of Deterioration of the
In conclusion, the findings of this study are consistent with our
hypothesis that a critical loss of -cells is a critical factor for the loss of
insulin secretory function in diabetes. Here we examined the effects of
the duration of hyperglycemia on gene expression in islets of partially
pancreatectomized (Px) rats. Islets were isolated, and mRNA was
extracted from rats 4 and 14 weeks after Px or sham Px surgery.
Px rats developed different degrees of hyperglycemia; low hyperglycemia
was assigned to Px rats with fed blood glucose levels less than 150 mg/dl, and high hyperglycemia was assigned above 150 mg/dl.
-Cell
hypertrophy was present at both 4 and 14 weeks. At the same time
points, high hyperglycemia rats showed a global alteration in gene
expression with decreased mRNA for insulin, IAPP, islet-associated
transcription factors (pancreatic and duodenal homeobox-1,
BETA2/NeuroD, Nkx6.1, and hepatocyte nuclear factor 1
),
-cell
metabolic enzymes (glucose transporter 2, glucokinase, mitochondrial
glycerol phosphate dehydrogenase, and pyruvate carboxylase), and ion
channels/pumps (Kir6.2, VDCC
, and sarcoplasmic reticulum
Ca2+-ATPase 3). Conversely, genes normally suppressed
in
-cells, such as lactate dehydrogenase-A, hexokinase I,
glucose-6-phosphatase, stress genes (heme oxygenase-1, A20, and Fas),
and the transcription factor c-Myc, were markedly increased. In
contrast, gene expression in low hyperglycemia rats was only minimally
changed at 4 weeks but significantly changed at 14 weeks, indicating
that even low levels of hyperglycemia induce
-cell dedifferentiation
over time. In addition, whereas 2 weeks of correction of hyperglycemia
completely reverses the changes in gene expression of Px rats at 4 weeks, the changes at 14 weeks were only partially reversed, indicating that the phenotype becomes resistant to reversal in the long term. In
conclusion, chronic hyperglycemia induces a progressive loss of
-cell phenotype with decreased expression of
-cell-associated genes and increased expression of normally suppressed genes, these changes being present with even minimal levels of hyperglycemia. Thus,
both the severity and duration of hyperglycemia appear to contribute to
the deterioration of the
-cell phenotype found in diabetes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells maintain specialized pathways of metabolism
that efficiently couple the secretion of insulin to circulating glucose
levels (1, 2). With the increased demand of insulin resistance and
obesity, an adaptation in
-cell mass and secretion can keep glucose
levels within a narrow range (3-5). The failure of
-cells to
adequately adapt to increased demand is fundamental to the pathogenesis
of all forms of diabetes. We have hypothesized that abnormal
-cell
function in diabetes is due to the loss of the unique expression
pattern of genes that optimize glucose-induced insulin secretion
(GIIS)1 and insulin synthesis
(5).
-cell differentiation is regulated by a network of transcription factors, which includes pancreatic and duodenal homeobox-1 (PDX-1), BETA2/NeuroD, Nkx6.1, and hepatocyte nuclear factors (HNFs). These factors regulate transcription of the insulin gene and genes involved in
-cell glucose sensing such as GLUT2 and glucokinase (6-12). Optimal
-cell function is dependent on expression of these genes and
the suppression of other genes, including lactate dehydrogenase-A (LDH-A), hexokinase I, and enzymes required for gluconeogenesis, that
can be predicted to interfere with optimal secretion.
-cell differentiation in the rat
partial pancreatectomy (Px) model of diabetes (13-15). Active
regeneration is seen during the first 7-10 days following surgery, but
by 14 days the morphology of the pancreas has stabilized, appearing
similar to what is seen at much later time points. We have found that
at the 4-week time point after surgery there is a loss of
-cell
differentiation associated with insulin secretory defects and
-cell
hypertrophy (13-17). Questions have been raised as to whether this
4-week time point provides a representative view of islet adaptation to
the diabetic milieu or whether the findings resulted from some
regeneration artifact. For these reasons, islet phenotype and
morphology were examined at both 4 and 14 weeks following partial pancreatectomy.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
reverse transcriptase (Invitrogen). Reverse
transcription reactions were incubated for 10 min at 25 °C, 60 min
at 42 °C, and 10 min at 95 °C. Resultant cDNA products were
diluted with 50 µl of H2O to a concentration
corresponding to 10 ng of starting RNA per 1.5 µl.
-32P]dCTP (3000 Ci/mmol; PerkinElmer
Life Sciences), and 2.5 units of AmpliTaq Gold DNA Polymerase (Applied
Biosystems, Foster City, CA). Table I
shows specific concentrations of MgCl2, dNTP, and oligonucleotide primers, along with multiplex PCR conditions for each
gene tested. Reactions were performed in a 9700 Thermocycler (Applied
Biosystems, Foster City, CA) in which samples underwent a 10-min
initial denaturing step, followed by the number of cycles indicated
(Table I), for durations of 1 min at 94 °C, 1 min at the annealing
temperature indicated in Table I, and 1 min at 72 °C. The final
extension step was 10 min at 72 °C. Amplimers were resolved by 6%
polyacrylamide gel electrophoresis in 1× Tris borate EDTA buffer. The
amount of [
-32P]dCTP incorporated into amplimers was
measured with a Storm 840 PhosphorImager and quantified with ImageQuant
software (Amersham Biosciences). The average intensity
of each product was expressed relative to the internal
control gene (ratio of specific product/control gene). These ratios
were then used to calculate the percentage of sham expression for each
Px animal in the same reverse transcription-PCR. We have previously
verified (13-15) that the multiplex PCR products for each set of
primers are linearly amplified. Control experiments were performed to
adjust the PCR conditions such that the number of cycles used was in
the exponential phase of amplification for all products and that each
PCR product in a multiplex reaction increased linearly with the amount
of starting material.
Sequences of oligonucleotide primers and PCR conditions
-Cell size was measured in sections of
pancreas from sham and Px rats stained with glucagon (rabbit
anti-bovine glucagon; gift of M. Appel, Worcester, MA) and
counterstained with hematoxylin as previously described (17). The
number of islet nuclei were counted (nuclei of cells staining for
glucagon were excluded), and the area of these cells was quantitated
using image analysis software (IP Lab Spectrum). Average
cross-sectional
-cell area was determined as the area of the cells
divided by the number of nuclei. An average of 725 ± 96 cells/animal were measured.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time course changes in fed whole blood
glucose levels in 85-95% Px rats.
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Fig. 2.
Time course changes in body weight and fed
blood glucose levels in Px rats classified according to their averaged
blood glucose levels post-Px. Values are means ± S.E. for
sham (open circles, n = 5), LPx
(closed circles, n = 6), and HPx
(closed squares, n = 11)
rats.
-Cell Phenotype--
The expression of
islet-associated transcription factors, islet hormones, specialized
-cell metabolic enzymes, and ion channels/pumps along with other
normally suppressed metabolic enzymes and stress genes were measured in
islets from sham, LPx, and HPx rats sacrificed 4 and 14 weeks after surgery.
-tubulin, cyclophilin, or 18 S rRNA),
mRNA levels in LPx and HPx islets were quantitated as a percentage
of sham (Table II). The expression levels
of several transcription factors (PDX-1; also known as IDX-1, IPF-1,
and STF-1),
-cell E-box trans-activator 2 (BETA2/NeuroD), Nkx6.1,
and hepatocyte nuclear factor 1
(HNF1
)) important for pancreas
and islet development and the maintenance of
-cell differentiation
were assessed in Px islets. After 4 weeks, the mRNA levels of
PDX-1, BETA2/NeuroD, Nkx6.1, and HNF1
were unaltered in islets from
LPx rats, whereas in HPx rats they were reduced by 50-60% compared
with sham (Table II). After 14 weeks, the mRNA levels for PDX-1,
Nkx6.1, and HNF1
were significantly reduced in LPx rats (30-40%)
and were further reduced in HPx rats (50-60%). Thus, expression
levels in LPx rats were unaltered at 4 weeks but significantly reduced
after 14 weeks. Therefore, the down-regulation of islet-associated
transcription factors after Px showed an association with increasing
blood glucose levels and the duration of hyperglycemia. On the other
hand, c-Myc is a transcription factor minimally expressed in
replicating cells, which is consistent with its low level expression in
adult islets (13, 22). After 4 weeks, c-Myc mRNA levels were
unaltered in LPx rats but were increased in HPx rats (Table II). After
14 weeks, c-Myc mRNA levels were significantly increased in LPx
rats and were increased further in HPx rats compared with sham control rats (p < 0.05 among groups). Therefore, the
up-regulation of c-Myc was associated with increasing blood glucose
levels and the duration of hyperglycemia.
Changes in islet gene mRNA levels in rats with different degrees of
hyperglycemia 4 and 14 weeks after Px
-cell hormone,
glucagon, and the
-cell hormone, somatostatin, were unchanged in Px
rats at both time points and thus showed no association with glycemia.
-cell-associated metabolic enzymes
(glucose transporter 2 (GLUT2), glucokinase, mitochondrial glycerol
phosphate dehydrogenase, and pyruvate carboxylase) after Px showed a
similar association with increasing blood glucose and the duration of
hyperglycemia. After 4 weeks, mRNA levels were unaltered in LPx
rats but were reduced by 40% in HPx rats (Table II). After 14 weeks,
the
-cell-associated metabolic enzymes were, individually, not
significantly changed in LPx rats. However, when the mRNA levels
for this group of genes (GLUT2, glucokinase, mitochondrial glycerol
phosphate dehydrogenase, and pyruvate carboxylase) were averaged for
each islet RNA preparation, the averaged mRNA levels in LPx rats
were significantly reduced compared with sham (25%, p < 0.05). They were further reduced (40-50%) in HPx rats at 14 weeks.
In contrast, several metabolic enzymes normally suppressed in
-cells
(hexokinase I, LDH-A, and the gluconeogenic enzyme, glucose-6-phosphatase) were markedly increased in HPx rats at 4 and 14 weeks (Table II). In LPx rats, hexokinase I, LDH-A, and glucose-6-phosphatase were unaltered at 4 weeks. However, at 14 weeks,
LDH-A mRNA levels were significantly increased, and hexokinase I
and glucose-6-phosphatase tended to be increased, although not significantly.
subunit of
voltage-dependent calcium channel, and sarcoplasmic
reticulum Ca2+-ATPase 3 were not significantly reduced in
LPx rats at 4 or 14 weeks. However, mRNA levels for this group of
ion channels (Kir6.2, the
subunit of voltage-dependent
calcium channel, and sarcoplasmic reticulum Ca2+-ATPase 3)
averaged for each experiment were significantly reduced in LPx rats at
14 weeks (25%, p < 0.05) but not at 4 weeks. In HPx
rats, the mRNA levels of Kir6.2, the
subunit of the
voltage-dependent calcium channel, and sarcoplasmic
reticulum Ca2+-ATPase 3 were each reduced to a similar
extent at both 4 and 14 weeks.
-cells are considerably lower than in the liver and other tissues
(23, 24). mRNA levels of the antioxidant enzyme heme oxygenase-1
(HO-1) were unchanged in LPx rats at 4 weeks but were significantly
increased at 14 weeks. Expression of both antioxidant genes, HO-1 and
glutathione peroxidase were markedly increased in islets of HPx rats at
4 and 14 weeks. mRNA levels of the antiapoptotic gene, A20, were unaltered in LPx rats but were increased in HPx rats at 4 weeks. At 14 weeks, A20 expression was increased to a similar extent in LPx and HPx
rats. Interestingly, expression of the proapoptosis cell surface
protein, Fas, was similarly altered; it was unchanged in LPx but
increased in HPx rats at 4 weeks and increased to similar levels in LPx
and HPx rats at 14 weeks.
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Fig. 3.
Measurement of plasma insulin, NEFA, and
triglyceride levels after Px. *, p < 0.05 versus sham.
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Fig. 4.
Time course changes in fed blood glucose
after phlorizin treatment of Px rats. At 12 weeks after Px,
phlorizin was administered daily for the next 2 weeks (0.8 g/kg/day).
Sham animals received a similar volume of vehicle (1,2-propanediol).
Values are means ± S.E. for sham vehicle-treated (open
circles, n = 4) and Px phlorizin-treated
(closed circles, n = 8)
rats.
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Fig. 5.
Reversibility of changes in islet gene
expression after Px. mRNA levels were compared by
semiquantitative multiplex reverse transcription-PCR in islet
preparations of sham (S, n = 5), sham
vehicle-treated (SP, n = 4), hyperglycemic
untreated Px (Px, n = 6), and
phlorizin-treated Px (PxP, n = 4) rats after
14 weeks. After normalization of the specific gene to an internal
control gene (TBP, -tubulin, cyclophilin, or 18 S rRNA), mRNA
levels were expressed as a percentage of sham. Values are means ± S.E. *, p < 0.05; **, p < 0.01; ***,
p < 0.001 versus sham for each gene. #,
p < 0.05; ##, p < 0.01 versus hyperglycemic untreated Px.
-Cell Size in Px Rats--
We previously found
hypertrophy of
-cells in 4-week hyperglycemic Px rats (13, 17). Here
we confirmed these findings (Table III)
and assessed the
-cell cross-sectional area 14 weeks after sham or
Px (Table III). Average
-cell cross-sectional area was increased by
~50% in 14-week Px rats. Thus,
-cell size was similarly increased
at 4 and 14 weeks after Px. It is noteworthy that of the three Px rats
of the 14-week group, all had increases of cell size, with two being
very hyperglycemic but one having a fed blood glucose level of only 121 mg/dl.
Effect of chronic hyperglycemia on -cell size 4 and 14 weeks after
Px
-cells,
leading to a reduction in the proportion of
-cells per islet. Gene
expression of representative
-cell metabolic enzymes, ion
channels/pumps and stress genes were also tested in glucose-clamped
rats; unchanged expression was found for GLUT2, Kir6.2, HO-1,
glutathione peroxidase, and A20 (Table IV). We previously found
increased c-Myc expression after glucose clamp (21). Note, as a caveat
to the comparison between the effects of hyperglycemia in the short
term after glucose clamp and the long term after Px, plasma insulin
levels were higher during glucose clamp (21), whereas they tend to be
lower after Px.
Effect of 4-day glucose clamp on islet gene expression
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell differentiation in the
rat 85-95% Px model of diabetes (13-15). Here we show that the
changes in
-cell gene expression in Px rats are associated with both
the severity and duration of hyperglycemia with the induction of
several normally suppressed genes and decreased expression of genes
that optimize
-cell function. The global disruption of gene
expression deteriorates over time, becoming resistant to reversal and
evident even with minimal hyperglycemia, thus demonstrating the
critical influence of hyperglycemia in the loss of
-cell
differentiation. This association between chronic hyperglycemia and
-cell dedifferentiation provides a molecular mechanism for the
-cell dysfunction found in diabetes.
-cell mass
results in two outcomes after 14 weeks. We noted a similar phenomenon
with transplanted encapsulated mouse islets several years ago, finding
that an identical number of islets either cured or did not cure
diabetic mouse recipients with there being no middle ground (25). In
the present Px study, a slight variation in the proportion of pancreas
removed may have been responsible for the rats initially having a full
range of blood glucose levels from low to middle and high values.
However, over time, blood glucose levels clustered into two distinct
groups. Some rats maintain nearly normal glucose levels that might be
equated with the state of impaired glucose tolerance in humans. The
other rats become highly hyperglycemic, with no rats maintaining
glucose levels in the middle range (140-250 mg/dl). Once
decompensation occurs, glucose levels may rise to high levels, probably
propelled by the combined forces of worsening glucotoxicity at the
-cell level and also by gluco- and lipotoxicity on insulin target
tissues leading to more severe insulin resistance. This decompensation phenomenon could explain why people with type 2 diabetes often present
with severe hyperglycemia and why patients with pre-type 1 diabetes
when stressed can develop severe hyperglycemia and then go into
remission once the stress recedes. Perhaps the LPx and HPx rats
represent different stages of diabetes development. We do not know if
the LPx rats, followed for a longer period of time, would display the
same changes in islet gene expression as HPx rats, which might lead to
decompensation with increasing hyperglycemia. It is equally possible
that the less severe phenotype would be stable and long lasting. Future
experiments will be necessary to address this question.
-Cell Differentiation in Px Rats--
This study and
others (13-15, 26) provide evidence that
-cell maladaptation to the
diabetic milieu is associated with a global disruption in
-cell gene
expression rather than with specific gene defects as occur with
maturity onset diabetes of the young 1-6 and mitochondrial diabetes.
Thus, under the influence of diabetes, otherwise normal
-cells show
progressively decreased expression of a panel of genes important for
glucose-stimulated insulin secretion and the regulation of
-cell
gene expression. Genes that are highly expressed in
-cells such as
insulin, GLUT2, glucokinase, mitochondrial glycerol phosphate
dehydrogenase, pyruvate carboxylase, potassium, and calcium channels
and several islet-associated transcription factors show a consistent
40-60% decrease in mRNA levels in Px rats. On the other hand,
concomitant up-regulation of several normally suppressed genes (LDH-A,
hexokinase I, glucose-6-phosphatase, stress genes, and the
transcription factor, c-Myc) appears to be an integral part of the
change in
-cell phenotype with diabetes. Similarly, in models of
diabetes derived by genetic modification such as the Zucker diabetic
fatty (ZDF) rat, progression to diabetes is associated with a similar
global alteration in
-cell gene expression (26).
,
and Nkx6.1) after Px could contribute to the altered expression of
genes essential for GIIS. None of the transcription factors were
completely shut off in Px rats, but recent studies implicate the
importance of modest reductions in the regulation of
-cell gene
expression (32, 33). Moreover, overexpression of PDX-1 can induce
insulin gene expression in nonislet tissue, which can improve glucose
homeostasis in diabetic mice (34). In Px rats, the down-regulation of
these and other potentially important islet transcription factors
(Pax6, PAN1, IB1, HNF3
, HNF4
1, HNF4
2/5) paralleled the
decreased expression of metabolic enzymes GLUT2, glucokinase,
mitochondrial glycerol phosphate dehydrogenase, and pyruvate
carboxylase. The expression of these genes favors the delivery of
glucose metabolites to mitochondria and the generation of metabolic
signals, such as ATP, that lead to an appropriate insulin secretory
response to an extracellular glucose stimulus (2, 5). Their decreased
expression coupled with up-regulation of normally suppressed metabolic
genes may be sufficient to interfere with this unique and possibly
fragile glucose recognition mechanism. In theory, the increased
expression of genes such as glucose-6-phosphatase and LDH-A could
up-regulate metabolic pathways diversionary to normal
-cell
metabolism and thus impair the efficiency of carbon flux through
glycolysis and the shuttling of molecules to mitochondria for oxidation
and ATP formation (35, 36). Normally, transported glucose is
efficiently metabolized by glycolysis and mitochondrial oxidation with
little if any carbon converted to lactate (37, 38).
-Cell Hypertrophy after Px--
Hypertrophy can be a
compensatory response to increased demand in terminally senescent
cells. Hypertrophy of
-cells has been found in Px rats (13, 17),
prediabetic ZDF rats with impaired glucose tolerance (4), and in
pregnancy (39). The up-regulation of c-Myc may be important in the
compensatory growth of
-cells, since this factor can lead to
hypertrophy in the absence of cell division (40). The development of
-cell hypertrophy is likely to be dependent upon the activation of
cell cycle inhibitors such as p21 and/or survival factors that prevent
c-Myc-induced proliferation and apoptosis (14, 41, 42).
-Cell
hypertrophy was evident 14 weeks after Px and thus appears unrelated to
the initial burst in regeneration of the endocrine pancreas that occurs
in the first 7-10 days after surgery (18). Rather, the findings
suggest that
-cell hypertrophy represents a stable response to the
diabetic environment that, although inadequate, may prevent more
serious metabolic decompensation.
-Cell Differentiation in Px
Rats--
Chronic hyperglycemia has been recognized as a leading cause
of
-cell dysfunction (5, 29, 31). In humans with diabetes, any
treatment that normalizes the plasma glucose profile leads to
improvements in insulin secretion (43, 44). Our findings suggest that
the severity of hyperglycemia plays a critical role in the progressive
loss of
-cell differentiation with diabetes. Circulating lipids were
not altered in Px rats at 4 weeks or in LPx rats at 14 weeks,
suggesting that they do not influence the changes in
-cell gene
expression in the Px model. Other studies have shown that fatty acids
can lead to alterations in
-cell gene expression (45-48), and the
changes in ZDF rats have been associated with elevated circulating
fatty acids (47, 49). However, a recent study in ZDF rats found
hyperglycemia, and not hyperlidemia, to be associated with the
decreased insulin gene expression in this model of diabetes (50).
Therefore,
-cell dysfunction in Px and ZDF rats may be similarly
mediated by chronic hyperglycemia. These findings do not rule out an
important role for intracellular lipid pathways in the
-cell
dysfunction of diabetes. Key deleterious changes could be caused by the
effects of high glucose concentrations working in concert with free
fatty acid substrate provided by normal levels of circulating
free fatty acid.
-Cell Phenotype with Time--
The
duration of exposure to hyperglycemia appears a critical factor in the
deterioration of the
-cell phenotype as indicated by the following:
1) short term hyperglycemia maintained by glucose clamp induced little
change in
-cell gene expression (Table IV), whereas long term
hyperglycemia after Px induced global alterations (Table II), 2)
changes in
-cell gene expression were fully reversible with
phlorizin treatment of Px rats at 4 weeks (13-15) but not at 14 weeks
(Fig. 5), and 3) low levels of hyperglycemia in Px rats induced
significant changes in gene expression after 14 weeks but not at 4 weeks (Table II). These data establish a relationship between the
duration of hyperglycemia and the deterioration of the
-cell
phenotype with diabetes. It is noteworthy that at 4 and 14 weeks, rats
with such similar minimal hyperglycemia have such striking differences
in
-cell phenotype. The
-cell hypertrophy of this later time
point is documented, although overall
-cell mass was not measured.
These results raise important questions about the apparent ability of
-cells with markedly altered phenotype to maintain enough insulin
output to keep glucose levels in the nearly normal range for long
periods of time. There may be important lessons about how
-cells can
adapt to prevent deterioration to frank diabetes. It is possible that
the
-cell changes seen in this rat model resemble those of humans
with the state of impaired glucose tolerance, a condition of mild
hyperglycemia that can persist for many years before deterioration to
the state of frank type 2 diabetes.
-cell differentiation contributes
to the
-cell dysfunction found in diabetes (5). However, despite a
marked loss in
-cell phenotype, insulin output can be well enough
maintained to keep glucose levels in a close to normal range.
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ACKNOWLEDGEMENTS |
---|
We thank the Joslin RIA Core for the insulin assay, and we thank the Animal Care Core.
![]() |
FOOTNOTES |
---|
* The Diabetes Endocrinology Research Center Core facilities were supported by National Institutes of Health Grant DK-36836. This work was also supported by National Institutes of Health Grants DK-35449 and 2U42 RR16606-02 and by the Diabetes Research and Wellness Foundation.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 postdoctoral fellowship from the Juvenile Diabetes
Foundation. Present address: Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney 2010, Australia.
§ To whom correspondence should be addressed: Islet Transplantation and Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2581; Fax: 617-732-2650; E-mail: Gordon.Weir@joslin.harvard.edu.
Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M210581200
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ABBREVIATIONS |
---|
The abbreviations used are: GIIS, glucose-induced insulin secretion; HNF, hepatocyte nuclear factor; LDH, lactate dehydrogenase; Px, partially pancreatectomized; LPx, low hyperglycemia; HPx, high hyperglycemia; NEFA, nonesterified fatty acid(s); PDX-1, pancreatic and duodenal homeobox-1; GLUT2, glucose transporter 2; ZDF, Zucker diabetic fatty; TBP, TATA-binding protein.
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