Novel mechanism of chronic exposure of oleic acid-induced...
Transcript of Novel mechanism of chronic exposure of oleic acid-induced...
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Novel mechanism of chronic exposure of oleic acid-induced insulin
release impairment in rat pancreatic β-cells
Takanori Kudo, Jie Wu, Yoshiji Ogawa, Sechiko Suga, Noriyuki Hasegawa, Toshihiro
Suda, Hiroki Mizukami, Soroku Yagihashi, and Makoto Wakui
Third Department of Internal Medicine, Hirosaki University School of Medicine, 5
Zaifu-cho, Hirosaki 036-8562 (T.K., Y.O., N.H., T.S.)
Division of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and
Medical Center, Phoenix AZ 85013, USA (J.W.)
Department of Physiology, Hirosaki University School of Medicine, 5 Zaifu-cho,
Hirosaki 036-8562, Japan (S.S., M.W.)
Department of Pathology, Hirosaki University School of Medicine, 5 Zaifu-cho,
Hirosaki 036-8562, Japan (H.M., S.Y.)
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Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: Chronic oleic acid alters ATP sensitivity of KATP channel.
Please send all correspondence to: Jie Wu, M.D., Ph.D.
Director, Neural Physiology Laboratory
Neurology Research
Barrow Neurological Institute
St. Joseph’s Hospital and Medical Center
Phoenix, AZ 85013-4496
Tel: (602)-406-6376
Fax: (602)-406-7172
e-mail: [email protected]
Number of text pages: 37
Number of tables: 0
Number of figures: 6
Number of references: 40
Number of words in Abstract: 249
Number of words in Introduction: 607
Number of words in Discussion: 1,466
Nonstandard Abbreviations: OA, oleic acid; KATP channel, ATP-sensitive potassium
channel
Section Assignment: Endocrine and Diabetes
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Abstract
A sustained, high circulating level of free fatty acids (FFAs) is an important risk factor
for the development of insulin resistance, islet β-cell dysfunction and pathogenesis of
type 2 diabetes. Here, we report a novel mechanism of chronic exposure of oleic acid
(OA)-induced rat insulin release impairment. Following a four-day exposure to OA (0.1
mM), there was no significant difference in basal insulin release when comparing
OA-treated and untreated islets in the presence of 2.8 mM glucose, while glucose (16.7
mM)-stimulated insulin release increased 2-fold in control, but not in OA-treated, islets.
Perforated patch-clamp recordings showed that untreated β-cells exhibited a resting
potential of –62.1 ± 0.9 mV and were electrically silent, whereas OA-treated β-cells
showed more positive resting potentials and spontaneous action potential firing.
Cell-attached single channel recordings revealed spontaneous opening of ATP-sensitive
potassium (KATP) channels in control, but not in OA-treated, β-cells. Inside-out excised
patch recordings showed similar activity in both OA-treated and untreated β-cells in the
absence of ATP on the inside of the cellular membrane, while in the presence of ATP,
KATP channel activity was significantly reduced in OA-treated β-cells. Electron
microscopy demonstrated that chronic exposure to OA resulted in the accumulation of
triglycerides in β-cell cytoplasm and reduced both the number of insulin-containing
granules and insulin content. Collectively, chronic exposure to OA closed KATP
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channels by increasing the sensitivity of KATP channels to ATP, which in turn led to the
continuous excitation of β-cells, depletion of insulin storage and impairment of
glucose-stimulated insulin release.
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Introduction
Diabetes mellitus is a collection of diseases characterized by high levels of blood
glucose resulting from defects in insulin production, insulin action, or both. Type 2
diabetes may account for more than 90% of all diagnosed cases of diabetes. Onset
usually begins as insulin resistance develops—a disorder in which cells do not use
insulin properly and/or a gradual loss arises of the ability of pancreatic β-cells to
produce insulin as the need for insulin increases due to elevated circulating glucose
levels.
Patients afflicted with type 2 diabetes often are associated with having an elevated
body weight. A sustained, high circulating level of free fatty acids (FFAs) is thought to
cause an accumulation of triglycerides in tissues (Zhou et al., 1996; Man et al., 1997;
Shimabukuro et al., 1997), insulin resistance (Ferrannini et al., 1983; Lillioja et al.,
1985; Lee 1988) and islet β-cell dysfunction (Lee et al., 1994; Zhou and Grill, 1994;
Zhou et al., 1996; Bollheimer et al., 1998; Roche et al., 2000). Although it is still not
well-understood, several possible mechanisms have been postulated to interpret
FFA-induced insulin resistance. For instance, inhibition of both insulin-activated
carbohydrate oxidation (Bonadonna et al., 1989; Felley et al., 1989; Bevilacqua et al.,
1990) and glucose uptake appear to be involved in FFA-induced insulin resistance
(Boden et al., 1994). In addition, inhibition of glycogen synthesis by FFAs may also
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compete with insulin action, resulting in an increase of blood glucose concentration
(Boden et al., 1994). Also, FFAs may inhibit insulin synthesis (Lee et al., 1994;
Furukawa et al., 1999) and the glucose-fatty acid cycle may be responsible for the
impairment of insulin biosynthesis (Lee et al., 1994; Furukawa et al., 1999).
Furthermore, FFA-induced inhibition of insulin secretion may be mediated by a
decrease in glucose effectiveness on β-cells. Glucose-stimulated insulin secretion from
islet β-cells is triggered by an elevation of intracellular Ca2+ concentration ([Ca2+]i)
(Wollheim and Sharp, 1981), which occurs via the following steps (Ashcroft and
Ashcroft, 1992): 1) levels of intracellular ATP increase in the cell; 2) KATP channel
activity is inhibited by ATP, and 3) the opening of the Ca2+ entry pathway (via L-type
Ca2+ channel activation) leads to a subsequent increase in [Ca2+]i. Finally, with respect
to the effects of FFAs on the membrane properties of islet β-cells in vitro, acute
application of FFAs has been shown to induce activation of KATP channels and
hyperpolarization of β-cells (Larsson et al., 1996; Branstrom et al., 1997; Gribble et al.,
1998; Gravena et al., 2002; Branstrom et al., 2004), which then makes it more difficult
for β-cells to respond to glucose stimulation. Chronic exposure of β-cells to FFAs has
been shown to increase basal insulin release but decrease glucose-stimulated insulin
secretion (Elks, 1993; Zhou and Grill, 1994; Prentki and Corkey, 1996; Bjorklund et al.,
1997; Hosokawa et al., 1997; Bollheimer et al., 1998; McGarry and Dobbins, 1999;
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Zhang et al., 2005). However, the chronic effects of FFAs on β-cell membrane
properties, especially on KATP channel function, have not been studied.
The aim of the present investigation was, therefore, to examine the effects of
chronic exposure to OA on insulin release, β-cell electrophysiological properties, KATP
channel activity, insulin-containing granules and insulin content using multiple
experimental approaches. The presented results show that after exposure to OA for four
days, rat islet β-cells were excited rather than silent, and that an increased sensitivity of
KATP channels to ATP underlay the OA-induced β-cell excitation. The elucidation of
this novel mechanism—that chronic exposure to OA altered the sensitivity of KATP
channels to ATP—provides new insights into more fully understanding the cellular
mechanisms of insulin resistance associated with type 2 diabetes patients.
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Materials and Methods
This study was carried out in accordance with the Guidelines for Animal
Experimentation, Hirosaki University, Japan.
Islet isolation and OA loading. Adult, male Sprague-Dawley rats were used.
Islets were isolated from the pancreas by following the collagenase digestion method
(Suga et al., 1997) with some modifications. Briefly, rats were anesthetized with
pentobarbital (Dainabot, IL, USA) at 30 mg/kg body weight, and collagenase (type S,
Nitta Zelatin, Osaka, Japan), which was dissolved in Hank’s balanced salt solution
(HBSS, Sigma Chemical Co., St Louis, MO, USA) at 1 mg/mL, was injected into the
pancreatic duct through the bile duct. The distended pancreas was removed and
incubated for digestion at 37°C for 18 min. The digested pancreas was then washed with
HBSS, which included 2% bovine serum albumin (BSA), and filtered through stainless
mesh, and finally the islets were separated by the histopaque (specific gravity 1.077;
Sigma Chemical Co., St Louis, MO, USA) gradient method. Islets were incubated for 2
days with 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, NY, USA)
with 10% bovine serum, 10000 U/mL penicillin and 10 mg/mL streptomycin. Islets
were then separated into two groups. Islets in one group were transferred to culture
medium of the same composition as described above and incubated for 3 days. Islets in
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the other group were transferred to culture medium which contained 0.1 mM oleic acid
(Nacalai Tesque, Inc., Kyoto, Japan) and were also incubated for 3 days.
Isolation of islet β-cells. Following incubation for 3 days in culture medium with and
without OA, islets were further digested by disperse (1000 U/mL, Godo Shusei, Tokyo,
Japan) in order to be separated into single cells as previously described (Suga et al.,
1997). Separated cells that had been previously exposed to OA were then incubated for
an additional 24 h in the presence of 0.1 mM OA and used for electrophysiological
experiments. During the isolated cell preparations, the percentages of α-cells (8%),
β-cells (87%) and δ-cells (4%) were determined by immunohistochemical staining as
previously described (Suga et al., 2003).
For identification of β-cells in electrophysiological experiments, excitatory
responses to 16.7 mM glucose or 0.5 mM tolbutamide (Sigma Chemical Co., St Louis,
MO, USA ), tested at the end of experiments, were confirmed. Furthermore, the values
of percentages of cells showing the same results were used for judging whether β-cells
were included in those islets.
Histology of islet β-cells. Two dishes of islets, one incubated for 4 days with and the
other without OA, were prepared. For electron microscopy, a pellet of pancreatic islets
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from each group was fixed in ice-cold 2.5% glutaraldehyde in 0.05 M cacodylate buffer
(pH 7.4) overnight at 4°C and postfixed with 1% osmium tetroxide (Sigma Chemical
Co., St Louis, MO, USA) for 1 h at room temperature. After dehydration through an
ascending series of ethanol, the samples were embedded in epon. Ultrathin sections
from epon-embedded blocks were stained with uranyl acetate and lead citrate. They
were examined by a JEOL TEM-2000EX electron microscope (Nihon Densi, Tokyo,
Japan).
Measurement of total protein and insulin in islets. In order to measure the amount of
total protein in islets, two groups of islets, one incubated with and the other without OA
for 4 days, were prepared. In each dish, 200 islets were plated at the start of culture, and
the number of islets in the dish after 3 days of incubation was not different. The 200
islets were homogenized in 100 µL cold TE buffer (50 mM Tris, 1 mM EDTA, pH 7.4)
with 1 mM phenylmethyl-sulfonyl fluoride and 0.2 mM N-ethylmaleimide (Sigma
Chemical Co., St Louis, MO, USA). The protein concentration in each sample was
measured by a Bio-Rad protein assay (Bio-Rad, CA, USA). For measurement of the
amount of insulin in islets, islets were prepared in a similar way as described for
measurement of total protein. 20 islets from each group (i.e., one with and the other
without OA treatment) were hand-picked and homogenized in 100 µL cold acid ethanol.
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The homogenate was stored at 4°C overnight and then centrifuged at 2500 r.p.m for 30
min. The supernatant was collected and stored at –20°C until insulin assay experiments.
Samples were diluted 1:5000 in normal saline which contained 2% bovine serum
albumin. Insulin concentration was measured using a Rat Insulin ELISA (Mercodia,
Uppsala, Sweden).
Measurement of insulin secretion. In each experiment, 20 islets were placed on 8.0
mm polyethylene terephthalate membrane (FALCON, Cell Culture Insert, NJ, USA),
and then the membrane with islets was placed in HBSS which contained 2% BSA and
2.8 mM glucose for 1 h before measurement of insulin secretion. The medium was then
discarded and replaced with fresh medium having the same composition. During
exposure of the islets to the medium for 15 min, the medium was collected every 1 min
for insulin measurement (basal secretion). Islets were then exposed to a medium which
contained 16.7 mM glucose for 15 min and insulin secretion was also measured as
recently described. Then, the medium was replaced with one which contained the
original concentration of glucose (2.8 mM) and insulin secretion was again measured as
recently described. Each sample was then stored at –20°C until insulin assay
experiments were carried out as described above.
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Patch-clamp recordings. Isolated cells were kept in a 35-mm Petri dish and the dish
was placed on the stage of an inverted microscope (IMT-2, Olympus, Tokyo, Japan). A
patch-clamp amplifier (EPC-7, HEKA Electronik, Lambrecht, Germany) was used to
measure both membrane potentials and currents. The amphotericin B (Sigma Chemical
Co., St Louis, MO, USA)-perforation method was used to measure the membrane
potential of the cell. The resistance of the electrode, when filled with pipette solution,
ranged from 2 to 4 MΩ. During whole-cell current recordings, the membrane
capacitance of single cells ranged from 8 to 14 pF. Only experiments with series
resistances below 30 MΩ were accepted for data analysis. In the whole-cell
configuration, current-voltage relationships were obtained. In such experiments, the cell
membrane was held at -90 mV, and 10 mV-increasing step pulses having duration of
200 ms were applied. To record single channel currents, the cell-attached or inside-out
configuration was used. In the cell-attached configuration, the pipette potential with
reference to the bath solution potential was kept at 0 mV. In the inside-out configuration,
the potential difference through the membrane was kept at –60 mV, inside negative. The
open-time probability (Po) was calculated according to the equation Po = 1-Pc1/N, where
Pc is the total closed time probability and N is the total number of channels present. The
Pos of KATP channels are shown as a function of the concentration of adenosine
5’-triphosphate (ATP, Sigma Chemical Co., St Louis, MO, USA). The theoretical
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curves for ATP inhibition of KATP channel activity were obtained by using the Hill
equation (P/P(o)) = 1/1 + ([ATP]/ki)h, where P is the open-time probability with ATP
present at each concentration, P(o) is the open-time probability without ATP present,
[ATP] is the concentration of ATP, ki is the ATP concentration at which inhibition of
KATP channel activity is half-maximal, and h is the Hill coefficient. The mean values of
ki and h were used for obtaining the theoretical curves, which were taken from several
series of experiments in which various concentrations of ATP were examined in the
same patch. All electrophysiological experiments were carried out at room temperature
(22 ± 1° C).
Solutions. The extracellular solution for electrical recordings contained 135 mM NaCl,
5.6 mM KCl, 1.2 mM MgCl2, 1 mM CaCl2, 2.8 mM glucose and 10 mM HEPES. The
pH of the solution was 7.3. In experiments where the extracellular concentration of
glucose was elevated to 16.7 mM, the osmolarity of the control solution was adjusted by
adding sucrose. To record the membrane potential of the cell, the pipette solution
contained 135 mM KCl, 1.2 mM MgCl2, 2.8 mM glucose, 0.5 mM ethylene
glycol-bis(b-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 mM
N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) and 240 µg/mL
amphotericin B (Sigma Chemical Co., St Louis, MO, USA). The pH of this solution
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was 7.2. To record KATP channel activity in the cell-attached or inside-out configuration,
the ionic composition of the pipette solution was the same as that for membrane
potential recordings, but amphotericin B was omitted. For inside-out single channel
recordings, immediately following establishment of the inside-out configuration, the
bath solution was changed to a solution whose ionic composition was the same as the
pipette solution. During electrophysiological studies, cells in the experimental bath were
continuously exposed to a stream of extracellular solution throughout the experiments.
Statistics and data analysis. Data are expressed as mean ± S.E.M from several
experiments, and statistical significance was evaluated by using the two-tailed paired or
unpaired Student’s t-test. ANOVA analysis was also used. A value of p less than 0.05
was considered to be significant.
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RESULTS
Chronic exposure to OA induced impaired glucose-stimulated insulin secretion
from rat islets.
Initial experiments were performed to test insulin secretion after exposing β−cells to
OA (0.1 mM) for four days. Figure 1A shows the rates of insulin secretion from rat
islets. The rates of insulin secretion in a solution which contained 2.8 mM glucose were
19.4 ± 4.3 pg/islet/min (n = 6) in control (untreated) islets and 19.0 ± 2.4 pg/islet/min (n
= 6) in OA-treated islets (Figure 1B, left columns). There was no significant difference
when comparing basal insulin release between OA-treated and untreated islets (p >
0.05). When the glucose concentration was elevated to 16.7 mM (glucose stimulation),
control islets showed an elevation of insulin secretion (Fig. 1A, open symbols), while
OA-treated islets exhibited no clear elevation of insulin secretion due to high glucose
stimulation (Fig. 1A, black symbols). The average rates of insulin secretion during high
glucose stimulation were 39.2 ± 5.8 pg/islet/min (n = 6) in control islets and 24.6 ± 3.6
pg/islet/min (n = 6) in OA-treated islets (Fig. 1B, middle columns). The difference
when comparing high glucose-stimulated insulin secretion between control and
OA-treated islets is significant.
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Chronic exposure to OA affected isolated islet β-cell membrane potentials and
excitability.
In order to explore the possible cellular mechanisms of glucose-stimulated insulin
secretion impairment by chronic exposure to OA, we characterized the
electrophysiological properties of single β-cells isolated from rat islets treated and
untreated with OA. Under basal conditions (2.8 mM glucose in the external solution),
β-cells from control islets were mostly electrically silent (Fig. 2A), but β-cells from
OA-treated islets showed spontaneous action potential firing (Fig. 2B). The resting
membrane potentials of control β-cells at day 0 and day 4 were –62.1 ± 0.9 mV (n = 32)
and –64.3 ± 1.2 mV (n = 19), respectively, while in β-cells treated with OA at day 1, 2
and 4, it was –45.7 ± 3.1 mV (p > 0.05 vs. control cells at day 0, n = 14), –41.7 ± 3.3
mV (p > 0.05 vs. control cells at day 0, n = 10) and –36.4 ± 1.6 mV (p < 0.01 vs. control
cells at day 4, n = 18), respectively (Fig. 2C). These results suggest a time-dependent
depolarization of β-cells during chronic exposure to OA. An increase in the glucose
concentration to 16.7 mM depolarized membrane potential and elicited action potential
firing in control β-cells (Fig. 2A), but the glucose-stimulated effect was weaker in
OA-treated β-cells (Fig. 2B). These results indicate that unlike the previously observed
acute effects of FFAs, which resulted in β-cell hyperpolarization (Gribble et al., 1998),
chronic exposure to OA depolarized and excited rat β-cells.
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Current-voltage relationship curves of β-cells treated and untreated with OA.
In order to explore the ionic mechanism of depolarization of islet β-cells due to OA
treatment, β-cell membrane conductance was measured by obtaining current-voltage
relationships. As demonstrated in Fig. 3, the current-voltage relationship curves were
obtained by a series of step pulses from -90 to -30 mV at 10-mV increments, and the
results showed that the whole-cell current density (pA/pF) was clearly larger in control
β-cells compared to OA-treated β-cells (Fig. 3B), but the current density at around -90
mV was not changed, suggesting that a decrease in K+ conductance was likely
responsible for depolarization of OA-treated β-cells.
Effects of chronic exposure to OA on KATP channel activity in the cell-attached
recording configuration.
Why does chronic exposure to OA induce β-cell excitation? One possible explanation
is that chronic exposure to OA may close β-cell KATP channels, which would result in
β-cell depolarization and consequently excitation. To test this hypothesis, KATP channel
activity in both control and OA-treated β-cells was monitored using the cell-attached
single channel patch recording configuration. In control β-cells, spontaneous, inward
single channel currents were recorded at the pipette potential of 0 mV under basal
conditions (2.8 mM glucose in the bath; Fig. 4Aa, before tolbutamide application). The
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value of the open-time probability (Po) of the channel activity was 0.12 ± 0.2 (n = 12).
Application of the KATP channel blocker tolbutamide (0.5 mM) to the bath solution
completely inhibited channel activity (Fig. 4Ab, n = 5), suggesting that spontaneous
KATP channel activity was recorded. On the other hand, in β-cells isolated from islets
exposed to OA for four days, spontaneous KATP channel activity was rarely observed
(Fig. 4Ba), but application of the KATP channel opener diazoxide (0.1 mM) dramatically
opened KATP channels (Fig. 4Bb). These results clearly demonstrate that chronic
exposure to OA closed β-cell KATP channels.
Effects of chronic exposure to OA on the sensitivity of KATP channels to ATP in the
inside-out recording configuration.
In order to further examine the mechanisms by which chronic exposure to OA closed
β-cell KATP channels, the inside-out excised patch recording was employed.
Interestingly, in the absence of ATP on the inside of the cellular membrane, the values
of Po of KATP channel activity were 0.19 ± 0.02 (n = 8) in untreated β-cells (Figs. 5A, C)
and 0.20 ± 0.04 (p > 0.05, n=6) in OA-treated β-cells (Fig. 5B, C). However, when 10
µM ATP was applied to the inside of the membrane, KATP channel activity was
obviously reduced in OA-treated cells (Fig. 5B, C). In the presence of 10 µM ATP,
from six patches using OA-treated β-cells, the value of Po of KATP channel activity was
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0.02 ± 0.01, while from eight patches using control β-cells, the value of Po of KATP
channel activity was 0.12 ± 0.01. When comparing OA-treated and untreated β-cells in
the presence of 10 µM ATP, the values of Po are significantly different (p < 0.01, Fig.
5C, right columns). As shown in Fig. 5D, the sensitivity of KATP channels to different
concentrations of ATP was compared between OA-treated and untreated β-cells. Based
on estimations from the Hill equation, the values of ki and the Hill coefficient for ATP
inhibition of KATP channels were 12.5 ± 1.0 µM and 0.88 ± 0.04 in control β-cells (n =
6), but were 1.0 ± 0.4 µM (n = 5) and 0.92 ± 0.05 in OA-treated β-cells (n = 5). The ki
value was significantly lower in OA-treated β-cells (p < 0.01) but the values of the Hill
coefficient were not significantly different when comparing OA-treated and untreated
β-cells. These results indicate that chronic exposure to OA increased the sensitivity of
KATP channels to cytosolic ATP, which in turn resulted in the closure of KATP channels
when glucose was present at low concentrations (< 5.5 mM).
Effects of chronic exposure to OA on histological features and insulin content of
islet β-cells.
Data presented thus far demonstrate that in contrast to the acute effects of FFAs,
which lead to the opening of β-cell KATP channels, chronic exposure to OA closed β-cell
KATP channels. It is well known that the closure of β-cell KATP channels excites β-cells
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and triggers insulin release. To understand why chronic OA treatment excited β-cells,
but impaired glucose-stimulated insulin release (see Fig. 1), we compared histological
alterations of insulin-containing secretory granules and insulin content between
OA-treated and untreated islet β-cells using electron microscopy. As shown in Fig. 6,
insulin-containing secretory granules and Golgi apparata were well-developed in control
β-cells (A); however, in OA-treated β-cells, electron-dense inhomogeneous lipid
droplets were often deposited in the cytoplasm (B, dashed circle) and the total numbers
of insulin-containing granules were lower compared to control β-cells (A, B, arrows).
Furthermore, biochemical analysis demonstrated that total protein content was 44.9 ±
4.3 µg/islet in control islets (Fig. 6C, open column) and 48.7 ± 7.8 µg/islet in
OA-treated islets (Fig. 6C, black column, p > 0.05), suggesting that there was no
significant difference in total protein content when comparing OA-treated and untreated
islets. However, insulin content in OA-treated islets was significantly reduced compared
to control islets (Fig. 6D). The average insulin content was 19.5 ± 1.0 ng/islet in control
islets (n = 8) and 13.4 ± 0.7 ng/islet in OA-treated islets (p < 0.01, n = 8). These results
suggest that chronic exposure of islets to OA continuously excited β-cells, which
gradually depleted insulin content and led to an impairment of glucose-stimulated
insulin release.
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Discussion
The major and important discovery in the present study is that chronic exposure to
OA modulated rat pancreatic β-cell function in a different manner compared to acute
exposure to FFAs. In contrast to the acute effects resulting from exposure to FFAs,
which lead to the opening of β-cell KATP channels and consequently the silencing of
β-cells due to decreased sensitivity of KATP channels to ATP, chronic exposure (four
days) to OA closed β-cell KATP channels and excited β-cells by increasing the
sensitivity of KATP channels to ATP. After sustained β-cell excitation, both the total
numbers of intracellular insulin-containing granules and insulin content were decreased,
which caused an impairment of glucose-stimulated insulin release. Therefore, although
both acute and chronic exposure to FFAs results in a similar impairment of
glucose-induced insulin release, the underlying mechanism is different. Considering that
the pathogenesis of type 2 diabetes in patients having an elevated body weight is a
gradual process, this novel mechanism, which interprets how a sustained, high
circulating level of FFAs induces insulin release impairment and pancreatic β-cell
dysfunction, appears to closely resemble clinical conditions, and these new findings
help improve our understanding of the cellular mechanisms underlying insulin
resistance associated with type 2 diabetes.
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Chronic exposure to OA caused electrical excitation of islet β-cells when glucose
was present at low concentrations.
We found that chronic exposure to OA resulted in β-cell spontaneous action potential
firing even when glucose was present at low concentrations (Fig. 2B). Normally,
isolated islet β-cells are silent when the glucose concentration is below 5.5 mM (Fig.
2A) because in these conditions the membrane potential is below –50 mV. This resting
membrane potential is both established and maintained in part by KATP channel activity
(Suga et al., 2003), which is enough to prevent spontaneous action potential
development in low glucose (< 5.5 mM) conditions (Ashcroft and Rorsman, 1989).
When the glucose concentration is elevated, ATP production increases, which then
leads to the closure of KATP channels, β-cell depolarization and action potential firing.
Action potentials in islet β-cells allow Ca2+ entry through L-type Ca2+ channels
(Ashcroft and Rorsman, 1989), causing elevation of levels of [Ca2+]i, which triggers
exocytosis of insulin granules (Wollheim and Sharp, 1981). Therefore, it is clear that
the alteration of electrical properties of rat islet β-cells by chronic OA treatment plays
an important role in the development of OA-induced β-cell dysfunction and insulin
release impairment.
Accumulating lines of evidence indicate that acute or chronic exposure to FFAs
affects β-cell function and insulin release, hence FFAs are thought to be a critical risk
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factor in the development of insulin resistance associated with type 2 diabetes (Elks,
1993; Zhou and Grill, 1994; Larsson et al., 1996; Prentki and Corkey, 1996; Bjorklund
et al., 1997; Hosokawa et al., 1997; Bollheimer et al., 1998; Gribble et al., 1998;
McGarry and Dobbins, 1999; Gravena et al., 2002; Branstrom et al., 2004; Zhang et al.,
2005). Using both native pancreatic β-cells and heterologously expressed Kir6.2/SUR1
KATP channels, acute exposure of a recorded cell to FFAs strongly opened KATP
channels and hyperpolarized or silenced the tested cell under patch-clamp recording
configuration (Larsson et al., 1996; Gribble et al., 1998; Gravena et al., 2002;
Branstrom et al., 2004), and the underlying mechanisms of the FFA-induced KATP
channel opening included a reduction in the sensitivity of KATP channels to ATP
(Gribble et al., 1998). In experiments studying the effects of chronic exposure to FFAs,
it has been reported that a 1-2-day exposure to FFAs increased basal insulin release but
decreased glucose-stimulated insulin secretion (Elks, 1993; Zhou and Grill, 1994;
Prentki and Corkey, 1996; Bjorklund et al., 1997; Hosokawa et al., 1997; Bollheimer et
al., 1998; McGarry and Dobbins, 1999; Zhang et al., 2005). However, the membrane
properties of rat pancreatic β–cells and the electrophysiological alterations, especially
the functional changes of KATP channel activity, after chronic exposure to FFAs have
not been studied. The present investigation provides this missing evidence and proposes
that after chronic exposure (four days) to OA, an increased sensitivity of KATP channels
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to ATP may serve as an important mechanism, at least in part, to underlie chronic FFA
treatment-induced insulin release impairment.
Chronic exposure to OA increased the sensitivity of KATP channels to ATP.
As demonstrated in Fig. 5, KATP channel activity in the absence of ATP on the inside
of the cellular membrane was similar when OA-treated and untreated islet β-cells were
compared using inside-out patch recordings, suggesting that the basic function of KATP
channels was not altered by OA treatment. However, using the cell-attached
configuration, KATP channel activity in OA-treated β-cells mostly disappeared (Fig. 4).
This finding indicates that in OA-treated β-cells, KATP channels are functionally closed
by some intracellular mechanism. Since KATP channel activity in islet β-cells assists in
both establishing and maintaining the resting membrane potential (Ashcroft and
Rorsman, 1989), functional inhibition of KATP channel activity results in membrane
depolarization, which is responsible for the development of Ca2+-dependent action
potentials. Therefore, the functional closure of KATP channels under basal glucose
conditions (< 5.5 mM) explains why OA-treated β-cells showed spontaneous action
potential firing (Fig. 2).
KATP channel activity in islet β-cells is basically regulated by levels of intracellular
ATP and/or the ratio of ATP/ADP (Detimary et al., 1988). However, the sensitivity of
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KATP channels to ATP is also known to be modulated by other intracellular molecules.
For instance, intracellular Ca2+ was shown to increase the sensitivity of KATP channels
to ATP in rat islet β-cells (Nakano et al., 2002). Mg2+ has also been reported to inhibit
KATP channel activity by reducing intracellular concentrations of free ATP (Findley,
1987) and/or by increasing the affinity of KATP channels to ATP (Ashcroft and Kakei,
1989). Furthermore, the membrane phospholipid phosphatidylinositol-4,5-bis-phosphate
(PIP2) reduced the sensitivity of KATP channels to ATP (Baukrowitz et al., 1998; Huang
et al., 1998; Shyng and Nichols, 1998) by the mechanism of its minus charges
interfering with the efficacy of ATP binding to KATP channels (Fan and Makielski,
1997). In the present study, the ki value for ATP inhibition of KATP channel activity in
OA-treated β-cells was significantly lower when compared to untreated β-cells (Fig. 5),
indicating that chronic exposure of islet β-cells to OA increased the affinity of KATP
channels to ATP. At the present time, the precise mechanisms of how chronic exposure
to OA induced a change in the affinity of KATP channels to ATP are not clear. As
mentioned before, elevation of [Ca2+]i following chronic exposure to OA may be
involved in increasing the sensitivity of KATP channels to ATP (Nakano et al., 2002).
Chronic exposure to OA decreased both total numbers of insulin-containing
granules and insulin content.
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As illustrated in Fig. 6, total numbers of insulin-containing granules and insulin
content in islets were reduced following four-day exposure to OA, which is consistent
with previous findings (Zhou and Grill, 1994; Furukawa et al., 1999). However, the
rates of insulin secretion from both OA-treated and untreated islets in 2.8 mM glucose
were similar (Fig. 1), which is not consistent with previous studies that reported that
chronic exposure to FFAs elevated basal insulin release (Zhou and Grill, 1994; Zhou et
al., 1996; Bollheimer et al., 1998; McGarry and Dobbins, 1999; Zhang et al., 2005).
These different results can be explained by the present findings that β-cells isolated
from OA-treated islets were electrically excited even in low glucose (2.8 mM)
conditions (Fig. 2), and that over-activity of β-cells following long-term (four days)
exposure to OA seems to gradually deplete insulin storage. Under these conditions,
excitation of β-cells was sustained by a closure of KATP channels, and consequently
insulin was continuously secreted at a maximal, saturated level, but this saturated level
was obviously lower. This interpretation is supported by the following lines of
evidence: (1) in chronic OA-treated β-cells, both total numbers of insulin-containing
secretion granules and insulin content were clearly lower compared to untreated cells
(Fig. 6); (2) in chronic OA-treated β-cells, glucose (16.7 mM)-stimulated insulin release
was clearly impaired (Fig. 1); (3) the exposure time of β-cells to FFAs was longer in the
present study (four days) compared to previous studies (one or two days), and (4)
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FFA-induced hypersensitivity of islet β-cells to glucose has already been demonstrated
(Hosokawa et al., 1997; Liu et al., 1998).
Taken together, the present study provides a novel mechanism to interpret, at least in
part, chronic FFA-induced insulin release impairment. Unlike acute exposure to FFAs,
which results in the reduced sensitivity of KATP channels to ATP, chronic exposure to
OA increased the sensitivity of KATP channels to ATP, which led to the closure of KATP
channels when glucose was present at low concentrations (< 5.5 mM); therefore, β-cells
were continuously depolarized and excited, which in turn triggered long-term insulin
release under basal conditions, and ultimately led to gradual depletion of insulin storage
and impairment of glucose-stimulated insulin release.
Acknowledgements: Authors thank Kevin Ellsworth for his assistance in preparing the
manuscript.
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Figure legends
Fig. 1. Insulin secretion from islets treated and untreated with OA.
A, time course of insulin secretion from untreated (open circles) and OA-treated (filled
circles) islets. The glucose concentration was elevated from 2.8 mM to 16.7 mM. The
mean values from 6 experiments in each are shown. Vertical bars represent S.E.M. B,
the average rates of insulin secretion 30 min before, during and after stimulation with
glucose from 6 experiments each. *p < 0.05, **p < 0.01.
Fig. 2. Membrane potential recordings of islet β-cells.
A, untreated β-cell. The extracellular glucose concentration was 2.8 mM first, and then
elevated to 16.7 mM. B, OA-treated β-cell. Spontaneous action potential firing was seen
when the solution contained 2.8 mM glucose. Representative tracings from 12 and 20
experiments are shown in A and B, respectively. C, the values of β-cell resting
potentials were compared following different exposure times to OA. Each column
represents the average from ten to thirty-two cells, and the vertical bars represent ± S.E.
**p < 0.01.
Fig. 3. Current-voltage relationships in β-cells treated and untreated with OA.
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A, results from control (untreated) β-cells. Voltage pulses were applied to cells in the
presence of 1 µM nifedipine. Values are the means from 7 experiments. B, results from
OA-treated β-cells. Values are the means from 7 experiments.
Fig. 4. Single channel recordings of KATP channel activity in the cell-attached
recording configuration.
Aa, untreated β-cell. The pipette potential was 0 mV. Ab, tolbutamide (0.5 mM) was
applied to the bath solution. Ba, OA-treated β-cell. Bb, diazoxide (0.1 mM) was applied
to the bath solution. Representative traces from 5-12 experiments are shown. Dotted
lines indicate the level of channel closure.
Fig. 5. Sensitivity of KATP channels to ATP examined in the inside-out recording
configuration.
A, recordings from the membrane of an untreated β-cell. The potential difference
through the patch membrane was held at –60 mV, inside negative. The ATP
concentration in the bath was elevated from zero to 10 µM. B, recordings from the
membrane of an OA-treated β-cell. Dotted lines indicate the level of channel closure (A,
B). C, open-time probabilities (Po) of KATP channels with and without ATP examined in
membranes from untreated (open columns) and OA-treated (filled columns) β-cells.
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Mean values from 6 experiments with no ATP and 8 experiments with 10 µM ATP are
shown. Vertical bars represent S.E.M. D, concentration-response relationships for ATP
inhibition of KATP channel activity for untreated (open circles) and OA-treated (filled
circles) β-cells. KATP channel activity at each concentration of ATP is expressed relative
to that without ATP. Mean values from 5 experiments each are shown. Solid lines were
drawn based on the Hill equation. **p < 0.01.
Fig. 6. Electron micrographs of rat islet β-cells and amounts of total protein and
insulin in islets.
A, untreated β-cell. Insulin-containing secretory granules and Golgi apparata are
well-developed. B, OA-treated β-cell. Electron-dense inhomogeneous lipid droplets
were deposited in the cytoplasm. Total numbers of insulin granules were lower
compared to control cells. C, amount of total protein per islet, measured in untreated
and OA-treated islets. Mean values from 7 experiments each are shown. Vertical bars
represent S.E.M. The two values are not significantly different. D, amount of insulin per
islet, measured from untreated and OA-treated islets. Mean values from 8 experiments
each are shown. **p < 0.01.
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