Determinants of glucose toxicity and its reversibility in the
pancreatic islet
-cell line, HIT-T15
Catherine E.
Gleason,
Michael
Gonzalez,
Jamie S.
Harmon, and
R. Paul
Robertson
Pacific Northwest Research Institute, and Departments of
Pharmacology and Medicine, University of Washington, Seattle,
Washington 98122
 |
ABSTRACT |
HIT-T15 cells, a
clonal
-cell line, were cultured and passaged weekly for 6 mo in
RPMI 1640 media containing various concentrations of glucose. Insulin
content decreased in the intermediate- and late-passage cells as a
continuous rather than a threshold glucose concentration effect. In a
second series of experiments, cells were grown in media containing
either 0.8 or 16.0 mM glucose from passages 76 through
105. Subcultures of passages 86, 92, and 99 that had been grown in media containing 16.0 mM
glucose were switched to media containing 0.8 mM glucose and also
carried forward to passage 105. Dramatic increases in
insulin content and secretion and insulin gene expression were observed
when the switches were made at passages 86 and 92 but not when the switch was made at passage 99. These
findings suggest that glucose toxicity of insulin-secreting cells is a
continuous rather than a threshold function of glucose concentration
and that the shorter the period of antecedent glucose toxicity, the
more likely that full recovery of cell function will occur.
insulin gene expression; PDX-1 binding
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INTRODUCTION |
THE RELATIONSHIP
BETWEEN fasting plasma glucose level and the magnitude of
glucose-induced insulin secretion is reciprocal (4),
suggesting that elevated glycemia might adversely affect
-cell
function. Adverse (glucotoxic) effects of glucose on cells have been
shown to occur when they are exposed chronically to supraphysiological
concentrations of this hexose. A molecular mechanism of action for
glucose toxicity has been identified in studies demonstrating decreased
insulin gene expression (3, 13, 14, 24, 25, 27, 28, 30, 33, 36,
37). However, these mechanistic studies have not addressed two
important functional issues. One is whether the adverse effects on
insulin-secreting cells of chronic exposure to high glucose
concentrations are glucose concentration related in a continuous manner
or whether there exists a threshold that must be reached before glucose
toxicity is expressed. The other is whether successful reversal of
glucose toxicity is related to the length of previous exposure to
supraphysiological glucose concentrations. The clinical relevance of
these questions stems from the need to understand the intensity with
which hyperglycemia should be treated in type 2 diabetes. It is often
suggested that the more quickly and more completely glycemia is
normalized through diet or drug treatment in diabetic patients, the
more likely residual
-cell function will be sufficient to help
maintain normoglycemia. Consequently, we designed experiments to answer
two questions. 1) Is induction of glucose toxicity of the
HIT-T15 cell over a defined period of time a continuous or a threshold
function of glucose concentration? 2) Is successful reversal
of glucose toxicity of the HIT-T15 cell inversely related to the
duration of time over which the antecedent glucose toxic state exists?
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RESEARCH MATERIALS AND METHODS |
HIT-T15 cell culture.
HIT-T15 cells were routinely grown in 5% CO2-95%
humidified air at 37°C, maintained in RPMI 1640 medium containing
10% fetal bovine serum, and passaged once weekly after
trypsin-EDTA detachment (41).
Insulin content and secretion.
Determination of intracellular insulin content and insulin secretion
during static incubations in 0.2 or 5.0 mM glucose was determined as
described previously (40).
Analysis of insulin mRNAs.
The abundance of insulin mRNAs in HIT-T15 cells was determined by
ribonuclease protection assay (RPA) using the Direct Protect RPA Kit
(Ambion, Austin, TX) as previously described (13).
Riboprobes were transcribed from template DNA using the MAXIscript In
Vitro Transcription Kit (Ambion). Insulin DNA template was generated by
inserting the 307-bp Pst I fragment of the human
preproinsulin genomic DNA (phins 214, ATCC) into the pSport 1 vector.
The insulin template was linearized with Kpn I and
transcribed with T7 RNA polymerase to generate a 330-bp RNA fragment (a
186-bp protected fragment).
-Actin was used for the control. The
-actin template DNA was transcribed with T7 RNA polymerase from a
linearized pTRIPLEscript plasmid containing a 250-bp mouse
-actin
gene fragment to generate a 304-bp RNA fragment (a 245-bp protected
fragment). The probes were gel purified on a 5% polyacrylamide-8 M
urea gel and eluted overnight at 37°C in elution buffer (0.5 M
NH4OAc, 1 mM EDTA, 0.1% SDS). Probes were precipitated
from the elution buffer with 10 µg of yeast tRNA and 1 ml of 100%
ethanol on ice for 15 min and resuspended in an appropriate volume of
RNase-free trypsin-EDTA buffer. The quantity of probe generated was
determined by spectrometry. Riboprobes (0.5 µg) were labeled with
Psoralen-biotin using the BrightStar Psoralen-Biotin Nonisotopic
Labeling Kit (Ambion).
HIT-T15 cells were subcultured in RPMI 1640 medium containing either
0.8 or 16 mM glucose in 100-mm2 dishes. The cells were
grown to confluence and subcultured in medium containing 11.1 mM
glucose for 48 h before harvesting. The cells were trypsinized,
transferred to 15-ml tubes, and washed with PBS. Cells were resuspended
in 1 ml lysis/denaturation solution and vortexed. Five microliters of
the appropriate probe were hybridized to 10 µl of cell lysate and
incubated overnight at 45°C. The unprotected mRNA fragments were
digested with 200 U of RNase T1 for 30 min at 37°C. The protected
fragments were precipitated with 0.5 ml of isopropanol and resuspended
in 10 µl of gel loading buffer. The fragments were separated on a 5%
polyacrylamide-8 M urea gel. RNA was transferred from the gel to a
positively charged nylon membrane (Ambion's BrightStar-Plus Membrane)
by electrotransfer in 1× Tris-borate-EDTA buffer and cross-linked by
ultraviolet illumination. The mRNA fragments were detected with
Ambion's Biodetect Nonisotopic Detection Kit, and the membrane was
exposed to X-ray film. Density of the insulin band was divided by the
density of the
-actin band within the same lane to correct for loading.
Analysis of transcription factor binding to the insulin promoter.
The binding of PDX-1 and RIPE-3b1-activator proteins to the insulin
promoter was analyzed by electrophoretic mobility shift assay (EMSA).
HIT-T15 cells were subcultured for 48 h in RPMI 1640 medium
containing 11.1 mM glucose. Nuclear extracts were prepared from these
cells as previously described (27). Oligonucleotide probes
consisting of the human insulin CT2 (
230 to
201) and rat insulin II
RIPE-3b1 (
126 to
101) fragments were annealed and end-labeled
with [32P]dCTP using the Klenow fragment of
Escherichia coli DNA polymerase I. The EMSA was performed as
previously described (27), with binding reactions
containing 10 or 20 µg of protein. Autoradiograms were analyzed and
quantitated by densitometry. Protein concentrations were determined by
bicinchoninic acid assay (Pierce, Rockford, IL).
Statistical analysis.
Statistical analysis was performed by Student's test (Fig.
1), by Pearson product-moment
correlations (Fig. 2), and by ANOVA with
Bonferroni/Dunnett 2 × 2 comparisons (Figs.
3-5).

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Fig. 1.
Insulin content of HIT-T15 cells cultured in RPMI 1640 media containing increasing concentrations of glucose. Passages are
grouped as early (p 76-79; n = 8),
intermediate (p 81-91; n = 11) and late (p 97-105; n = 8). Early
passage cells maintained insulin content regardless of the glucose
concentration in the media, whereas intermediate and late passage cells
maintained insulin content only when media with the lowest glucose
concentration was used (intermediate cells = 6,738 ± 324 vs.
3,975 ± 366, P < 0.001; late cells = 4,750 ± 322 vs. 2,172 ± 50. Values are means ± SE,
P = <0.0001; comparisons when using 0.8 vs. 12 mM
glucose in media).
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Fig. 2.
Data shown in Fig. 1 from the early, intermediate, and
late passage groups expressed as a percentage of the insulin content
data obtained when media contained 0.8 mM glucose. Regression analysis
using the best-fit equation of the line yielded highly statistically
significant correlations, suggesting continuous relationships between
declining insulin content and increasing glucose concentrations in the
media for the intermediate and late passage groups.
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Fig. 3.
Insulin content in HIT-T15 cells grown in 0.8 or 16 mM
glucose grown from passages 76 though 105. At
passages 86 (condition A), 92 (condition B), and 99 (condition C),
subcultures of cells previously grown in 16 mM glucose were introduced
into media containing 0.8 mM glucose and continued to passage
105. Cells grown in media containing 0.8 mM showed no significant
decrease in insulin content between passages 76 and
105. In contrast, cells grown in 16 mM glucose showed a
progressive decline in content (6,341 ± 63 vs. 93 ± 28, P < 0.0001). Cells whose media were changed at
passages 86 and 92 from 16 to 0.8 mM glucose
showed a dramatic increase in insulin content by passage 97 (condition A = 15,210 ± 531 vs. 4,434 ± 398; condition B = 15,555 ± 187 vs. 4,434 ± 398; both P < 0.0001 compared with cells grown in
media containing 0.8 mM glucose) that was sustained through
passage 105. In contrast, insulin content did not increase
dramatically when the switch in glucose concentration was made at
passage 99 (condition C = 1,809 ± 23 vs. 4,434 ± 398). All n = 4 for each data
point.
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Fig. 4.
Basal insulin secretion (IRI) during static incubations
in 0.2 mM glucose for 2 h from HIT-T15 cells grown in 0.8 or 16 mM
glucose from passages 76 through 105. At
passages 86, 92, and 99, subcultures of cells
previously grown in 16 mM glucose were introduced into media containing
0.8 mM glucose and continued to passage 105 as in Fig. 3.
Cells grown in media containing 0.8 mM glucose showed no significant
decrease in basal IRI between passages 76 and
105. In contrast, cells grown in 16 mM glucose showed a
progressive decline in basal IRI (132 ± 6 vs. 9 ± 2, P < 0.0001). Cells whose media were changed at
passages 86 and 92 from 16 to 0.8 mM glucose
showed a dramatic increase in basal insulin release by passage
97 (condition A = 206 ± 7 vs. 103 ± 3;
condition B = 201 ± 6 vs. 103 ± 3; both
P < 0.0001 compared with cells grown in media
containing 0.8 mM glucose) that was sustained through passage
105. In contrast, basal IRI did not increase dramatically when the
switch in glucose concentration was made at passage 99 (condition C = 37 ± 2 vs. 103 ± 3). All
n = 4 for each data point.
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Fig. 5.
Glucose-stimulated IRI during static incubations
containing 5.0 mM glucose for 2 h from HIT-T15 cells grown in 0.8 or 16 mM glucose grown from passages 76 through
105. At passages 86, 92, and 99, subcultures
of cells previously grown in 16 mM glucose were introduced into media
containing 0.8 mM glucose as in Fig. 3. Cells grown in media containing
0.8 mM glucose showed no significant decrease in glucose-stimulated IRI
between passages 76 and 105. In contrast, cells
grown in 16 mM glucose showed a progressive decline in
glucose-stimulated IRI (292 ± 35 vs. 8 ± 2, P < 0.0002). Cells whose media were changed at
passages 86 and 92 from 16 to 0.8 mM glucose
showed a dramatic increase in glucose-stimulated IRI by passages
91 and 97, respectively (condition A = 346 ± 25 vs. 187 ± 9; condition B = 490 ± 17 vs. 196 ± 5; both P < 0.0001 compared with cells grown in media containing 0.8 mM glucose). In
contrast, glucose-stimulated IRI did not increase dramatically when the
switch in glucose concentration was made at passage 99 (condition C = 43 ± 2 vs. 300 ± 12). All
n = 4 for each data point.
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RESULTS |
Glucose toxicity: a continuous or a threshold glucose
concentration-related event?
Experiments were conducted with HIT-T15 cells, beginning with
passage 76, that were carried forward for 30 wk, during
which the cells were split and passaged on a weekly basis until
passage 105 was reached. Cells were grown in RPMI 1640 containing one of five increasing (0.8, 1.6, 5.0, 8.0, 12.0 mM)
concentrations of glucose. For purposes of comparison, cell passages
were clustered into three groups: early passages (p
76-79); intermediate passages (p 81-91); and
late passages (p 97-105). Insulin content in the early
passages did not diminish, regardless of the glucose concentration in
the cell culture media (Fig. 1). Similarly, insulin content in the
intermediate and late passages did not decrease in cells cultured in
media containing 0.8 or 1.6 mM glucose. However, insulin content
decreased dramatically in the intermediate and late passage cells when
5.0, 8.0, or 12.0 mM glucose was used. Correlation analysis indicated a
continuous rather than a threshold glucose concentration effect on
insulin content in the intermediate and late passages (Fig. 2).
Duration of exposure to toxic concentrations of glucose as a
determinant of reversal of glucose toxicity.
To ascertain whether duration of antecedent exposure to
supraphysiological glucose concentrations is a determinant of the degree of reversibility of glucose toxicity, experiments were conducted
in which HIT-T15 cells were grown in RPMI 1640 media containing either
0.8 or 16.0 mM glucose. Cells were passaged weekly for 6 mo from
passages 76 through 105. Insulin content in the
later passages did not decrease in cells cultured in media containing
0.8 mM glucose, whereas a progressive decline in insulin content was
observed when cells were passaged in the presence of 16 mM glucose
(Fig. 3). Subcultures of passages 86, 92, and 99 that were being grown in media containing 16.0 mM glucose were switched
to media containing 0.8 mM glucose and carried forward to passage
105. A dramatic increase in insulin content was observed five
passages beyond the switch when the glucose concentrations were changed
at passages 86 and 92. However, this large
increase in insulin content was not observed when the switch to the
lower glucose concentration was made at passage 99 (Fig. 3).
Basal and glucose-stimulated insulin release from subcultures of the
cells cultured in either 0.8 mM or 16.0 mM glucose or those switched
from 16.0 to 0.8 mM glucose at various passages provided results
consistent with the insulin content measurements. Increases in basal
insulin release during static incubations in 0.2 mM glucose were
observed from the cells that were switched at passages 86 and 92; however, basal insulin secretion from passages switched at passage 99 did not change impressively (Fig.
4). Subcultures of the cells were also
exposed to stimulatory concentrations of glucose during static
incubations in buffer containing 5.0 mM glucose, a concentration that
is maximal for stimulation of insulin release from HIT-T15 cells.
Glucose-induced insulin responses were improved when cells were
switched from media containing 16.0 to 0.8 mM glucose at passages
86 and 92 (Fig. 5). In
contrast, no similar increase was observed when the cells were switched at passage 99. These data indicated that recovery of insulin
content, basal insulin secretion, and glucose-stimulated insulin
secretion are heavily influenced by the duration of exposure of the
cells to supraphysiological glucose concentrations in the media in
which they are grown.
To determine whether recovery of insulin content might be related to
recovery of insulin gene expression, levels of insulin mRNA were
determined in subcultures of cells that were grown in either 0.8 mM or
16.0 mM glucose and in cells that were switched from 16 mM glucose to
0.8 mM glucose at passages 86, 92, and
99. In two of two experiments, recovery of insulin mRNA was
detected in cells that were switched to media containing the lower
glucose concentration when the switches were made at passages 86 and 92, but not 99 (Fig.
6). EMSAs revealed recovery of PDX-1 and
RIPE-3b1-activator protein binding to the insulin promoter when the
switches were made at all three passages (Figs.
7 and 8).

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Fig. 6.
Ribonuclease protection assay (RPA) of insulin mRNA in
HIT-T15 cells. Photoimage of RPA for insulin mRNA. Cells had been grown
in media containing 16.0 mM glucose and then switched to 0.8 mM glucose
at passages 86 (A), 92 (B),
or 99 (C). Insulin/ -actin = 0.58, 0.00, 0.84, 0.26, and 0.02 for lanes 1-5,
respectively. Data representative of 2 separate experiments.
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Fig. 7.
Electrophoretic mobility shift assay for PDX-1 binding
and RIPE-3b1-activator binding to the insulin promoter region.
Experimental conditions were as indicated in legend for Fig. 6. Data
representative of 2 separate experiments.
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DISCUSSION |
These studies were designed to assess two hypotheses: first, that
the development of glucose toxicity in insulin-secreting cells over a
finite period of time is a continuous rather than a threshold function
of the glucose concentration in the environment of the
-cell;
second, that the magnitude of recovery from glucose toxicity is
inversely related to the length of antecedent exposure to
supraphysiological glucose concentrations. Consistent with our previous
observations (14, 27, 28, 30, 33, 37), we found no
evidence of diminished insulin content or defective insulin secretion
when cells were cultured in any of the glucose concentrations for
several weeks only. However, chronic exposure of the cells for up to 30 wk to glucose concentrations >1.6 mM resulted in progressive declines
in insulin content and insulin secretion. This deterioration appeared
to be a continuous function of increasing glucose concentrations rather
than being related to a threshold glucose concentration. For
interpretation of these findings, it is important to note that the
EC50 for glucose is ~1.0 mM and its maximal effect is
~2.0 mM for insulin secretion in HIT-T15 cells (40).
Pancreatic islets typically have an EC50 of 7.0-8.0 mM
glucose. To assess the second hypothesis, we chronically cultured
HIT-T15 cells in media containing either 0.8 or 16.0 mM glucose for 30 wk. In this paradigm, we intervened by switching subcultures of the
cells grown in the presence of 16.0 mM glucose to media containing 0.8 mM glucose at three intervals into the long-term culture. We observed
that the earlier the switch to the lower glucose concentration was
made, the greater the recovery of insulin content and secretion.
Unexpectedly, within five passages of switching to media containing the
lower glucose concentration, insulin content of the cells increased to
greater levels than the level observed in the control cells being
chronically grown in media containing 0.8 mM glucose. This was
associated with increased insulin gene expression and increases in
PDX-1 and RIPE-3b1-activator binding to the insulin promoter. The cells
switched at the latest passage failed to have such a strong recovery of
insulin content and insulin secretion. These findings indicate that an
early, finite window of time exists during which full recovery from
glucose toxicity is possible. The recovery of PDX-1 and
RIPE-3b1-activator binding, but not insulin mRNA in the passage
switched latest, may indicate that levels of other transcription
factors (24) not studied in these experiments had changed
and had not normalized sufficiently to allow full insulin gene expression.
Although it would have been more desirable to conduct these studies
using isolated islets, it is not possible to continuously culture
islets for 30 wk. In this situation, use of
-cell lines provides a
useful surrogate approach, even though one cannot assume that all our
findings are necessarily pertinent to authentic
-cells within
islets. Because we have previously demonstrated that no differences in
population doubling times exist when HIT-T15 (33) or
TC-6 (30) cells are grown in media containing 0.8 or
11.1 mM glucose, these adverse effects of supraphysiological glucose concentrations cannot be explained by accelerated aging. We have also
shown that other osmotically active moieties do not cause these
changes. To assess whether the adverse effect of elevated glucose
concentrations on hormone synthesis and secretion might be specific to
-cells, we performed control experiments using the glucagon
secreting cell line
TC-1/9 (32). In these experiments, chronic exposure to media containing supraphysiological glucose concentrations did not cause defective glucagon secretion or defective glucagon gene expression. Olson et al. (26) demonstrated
that supraphysiological glucose concentrations could decrease insulin gene expression in INS-1 cells, an insulin secreting cell line that
secretes insulin in response to physiological glucose concentrations. However, this glucose-induced decrease in insulin gene expression was
observed after only 48 h of exposure to elevated glucose
concentrations and therefore is more consistent with glucose
desensitization than with glucose toxicity. The associated diminished
binding of PDX-1 and RIPE-3b1-activator to the insulin promoter was
readily reversible when the cells were switched to media containing
physiological glucose concentrations.
In summary, these findings indicate that glucose toxicity of the
-cell is a continuous rather than a threshold function of glucose
concentration and that the shorter the period of antecedent glucose
toxicity, the greater the degree of recovery. Findings from experiments
such as these, in conjunction with many findings published by other
researchers (1-3, 5-12, 15-24, 29, 31, 34-36,
38, 39), suggest that abnormally elevated glucose concentrations in the
-cells' environment can cause a spectrum of changes. With short-term exposure to high glucose concentrations, decreases in
insulin secretion and insulin content can occur that are reversible upon return to normal glucose concentrations. The term glucose desensitization seems most apt to describe this sequence of events (16). On the other hand, a spectrum of pathophysiological
changes may occur with more prolonged exposure of the
-cell to
supraphysiological glucose concentrations. Using various experimental
models, many researchers have described adverse effects of glucose on
-cell function by the term "
-cell exhaustion." The
distinction between
-cell exhaustion and glucose toxicity is not
always clear. We favor the concept that the two may be in a
pathophysiological continuum (32). In this context,
-cell exhaustion might be earlier and more likely to be reversible,
whereas glucose toxicity is later and less likely to be reversible. In
this context, it seems likely that early, effective management by diet
and drugs of hyperglycemia in type 2 diabetes is an important aspect of preserving residual
-cell function. The same argument for meticulous glycemic control can be made after pancreas or islet transplantation.
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FOOTNOTES |
Address for reprints and other correspondence: R. Paul Robertson,
Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122 (E-mail: rpr{at}u.washington.edu).
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.
Received 9 March 2000; accepted in final form 22 June 2000.
 |
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