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ENDOCRINE AND DIABETES
-Cells
Third Department of Internal Medicine (T.K., Y.O., N.H., T.S.) and Departments of Physiology (S.S., M.W.) and Pathology (H.M., S.Y.), Hirosaki University School of Medicine, Hirosaki, Japan; and Division of Neurology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona (J.W.)
Received April 5, 2006; accepted June 5, 2006.
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Abstract |
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-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 4-day exposure to 0.1 mM OA, there was no significant difference in basal insulin release when comparing OA-treated and untreated islets in the presence of 2.8 mM glucose, whereas 16.7 mM glucose-stimulated insulin release increased 2-fold in control, but not in OA-treated, islets. Perforated patch-clamp recordings showed that untreated -cells exhibited a resting potential of -62.1 ± 0.9 mV and were electrically silent, whereas OA-treated -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, -cells. Inside-out excised patch recordings showed similar activity in both OA-treated and untreated -cells in the absence of ATP on the inside of the cellular membrane, whereas in the presence of ATP, KATP channel activity was significantly reduced in OA-treated -cells. Electron microscopy demonstrated that chronic exposure to OA resulted in the accumulation of triglycerides in -cell cytoplasm and reduced both the number of insulin-containing granules and insulin content. Collectively, chronic exposure to OA closed KATP channels by increasing the sensitivity of KATP channels to ATP, which in turn led to the continuous excitation of -cells, depletion of insulin storage, and impairment of glucose-stimulated insulin release.
-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 et al., 1988), and islet -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 example, inhibition of both insulin-activated carbohydrate oxidation (Bonadonna et al., 1989; Felley et al., 1989; Bevilacqua et al., 1990) and glucose uptake seem to be involved in FFA-induced insulin resistance (Boden et al., 1994). In addition, inhibition of glycogen synthesis by FFAs may also compete with insulin action, resulting in an increase in blood glucose concentration (Boden et al., 1994). In addition, 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 -cells. Glucose-stimulated insulin secretion from islet -cells is triggered by an elevation of [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 -cells in vitro, acute application of FFAs has been shown to induce activation of KATP channels and hyperpolarization of -cells (Larsson et al., 1996; Branstrom et al., 1997, 2004; Gribble et al., 1998; Gravena et al., 2002), which then makes it more difficult for -cells to respond to glucose stimulation. Chronic exposure of -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; Zhang et al., 2005). However, the chronic effects of FFAs on -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, -cell electrophysiological properties, KATP channel activity, insulin-containing granules, and insulin content using multiple experimental approaches. The results show that after exposure to OA for 4 days, rat islet -cells were excited rather than silent and that an increased sensitivity of KATP channels to ATP underlies the OA-induced -cell excitation. The elucidation of this novel mechanism—that chronic exposure to OA altered the sensitivity of KATP channels to ATP—provides new insight into more fully understanding the cellular mechanisms of insulin resistance associated with type 2 diabetes patients.
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Materials and Methods |
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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. In brief, rats were anesthetized with pentobarbital (Dainabot, Abbott Park, IL) at 30 mg/kg body weight, and collagenase (type S; Nitta Zelatin, Osaka, Japan), which was dissolved in Hanks' balanced salt solution (HBSS; Sigma-Aldrich, St. Louis, MO) 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, filtered through stainless mesh, and finally the islets were separated by the Histopaque (specific gravity 1.077; Sigma-Aldrich) gradient method. Islets were incubated for 2 days with 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, NY) with 10% bovine serum, 10,000 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 the other group were transferred to culture medium, which contained 0.1 mM oleic acid (Nacalai Tesque, Kyoto, Japan) and were also incubated for 3 days.
Isolation of Islet -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) to be separated into single cells as described previously (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 
-cells (8%), -cells (87%), and 
-cells (4%) were determined by immunohistochemical staining as described previously (Suga et al., 2003).
For identification of -cells in electrophysiological experiments, excitatory responses to 16.7 mM glucose or 0.5 mM tolbutamide (Sigma-Aldrich), tested at the end of experiments, were confirmed. Furthermore, the values of percentages of cells showing the same results were used for judging whether -cells were included in those islets.
Histology of Islet -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 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-Aldrich) 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. To measure the amount of total protein in islets, two groups of islets, one group incubated with and the other group 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 of ice-cold TE buffer (50 mM Tris and 1 mM EDTA, pH 7.4) with 1 mM phenylmethylsulfonyl fluoride and 0.2 mM N-ethylmaleimide (Sigma-Aldrich). The protein concentration in each sample was measured by a Bio-Rad protein assay (Bio-Rad, Hercules, CA). For measurement of the amount of insulin in islets, islets were prepared in a similar way as described for measurement of total protein. Twenty islets from each group (i.e., one group with and the other group without OA treatment) were hand-picked and homogenized in 100 µl of ice-cold acid ethanol. The homogenate was stored at 4°C overnight and then centrifuged at 2500 rpm 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 kit (Mercodia, Uppsala, Sweden).
Measurement of Insulin Secretion. In each experiment, 20 islets were placed on 8.0-mm polyethylene terephthalate membrane (BD Falcon Cell Culture Inserts; BD Biosciences, Franklin Lakes, NJ), and then the membrane with islets was placed in HBSS, which contained 2% bovine serum albumin 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 that contained 16.7 mM glucose for 15 min, and insulin secretion was measured as described above (Suga et al., 2003). Then, the medium was replaced with medium that contained the original concentration of glucose (2.8 mM), and insulin secretion was again measured as described above. Each sample was then stored at -20°C until insulin assay experiments were carried out as described above.
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, Lambrecht/Pfalz, Germany) was used to measure both membrane potentials and currents. The amphotericin B (Sigma-Aldrich)-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 M
. During whole-cell current recordings, the membrane capacitance of single cells ranged from 8 to 14 pF. Only experiments with series resistances below 30 M 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, with the inside negative. The open-time probability (Po) was calculated according to the equation Po = 1 - P 1/Nc, where Pc is the total closed time probability, and N is the total number of channels present. The Po values of KATP channels are shown as a function of the concentration of ATP (Sigma-Aldrich). The theoretical 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).
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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. Analysis of variance was also used. A value of p less than 0.05 was considered to be significant.
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Results |
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-cells to 0.1 mM OA for 4 days. Figure 1A shows the rates of insulin secretion from rat islets. The rates of insulin secretion in a solution that 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 (Fig. 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), whereas 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.
Chronic Exposure to OA Affected Isolated Islet -Cell Membrane Potentials and Excitability. To explore the possible cellular mechanisms of glucose-stimulated insulin secretion impairment by chronic exposure to OA, we characterized the electrophysiological properties of single -cells isolated from rat islets treated and untreated with OA. Under basal conditions (2.8 mM glucose in the external solution), -cells from control islets were mostly electrically silent (Fig. 2A), but -cells from OA-treated islets showed spontaneous action potential firing (Fig. 2B). The resting membrane potentials of control -cells at day 0 and day 4 were -62.1 ± 0.9 mV (n = 32) and -64.3 ± 1.2 mV (n = 19), respectively, whereas in -cells treated with OA at day 1, 2, and 4, it was -45.7 ± 3.1 mV (p > 0.05 versus control cells at day 0; n = 14), -41.7 ± 3.3 mV (p > 0.05 versus control cells at day 0; n = 10), and -36.4 ± 1.6 mV (p < 0.01 versus control cells at day 4; n = 18), respectively (Fig. 2C). These results suggest a time-dependent depolarization of -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 -cells (Fig. 2A), but the glucose-stimulated effect was weaker in OA-treated -cells (Fig. 2B). These results indicate that unlike the previously observed acute effects of FFAs, which resulted in -cell hyperpolarization (Gribble et al., 1998), chronic exposure to OA depolarized and excited rat -cells.
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Current-Voltage Relationship Curves of -Cells Treated and Untreated with OA. To explore the ionic mechanism of depolarization of islet -cells due to OA treatment, -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 (current per membrane capacitance, picoampere/picofarad) was clearly larger in control -cells compared with OA-treated -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 -cells.
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-cell excitation? One possible explanation is that chronic exposure to OA may close -cell KATP channels, which would result in -cell depolarization and consequently excitation. To test this hypothesis, KATP channel activity in both control and OA-treated -cells was monitored using the cell-attached single-channel patch recording configuration. In control -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 value of 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. In contrast, in -cells isolated from islets exposed to OA for 4 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 -cell KATP channels.
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Effects of Chronic Exposure to OA on the Sensitivity of KATP Channels to ATP in the Inside-Out Recording Configuration. To further examine the mechanisms by which chronic exposure to OA closed -cell KATP channels, the inside-out excised patch recording was used. 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 -cells (Fig. 5, A and C) and 0.20 ± 0.04 (p > 0.05; n = 6) in OA-treated -cells (Fig. 5, B and 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. 5, B and C). In the presence of 10 µM ATP, from six patches using OA-treated -cells, the value of Po of KATP channel activity was 0.02 ± 0.01, whereas from eight patches using control -cells, the value of Po of KATP channel activity was 0.12 ± 0.01. When comparing OA-treated and untreated -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 -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 -cells (n = 6), but they were 1.0 ± 0.4 µM (n = 5) and 0.92 ± 0.05 in OA-treated -cells (n = 5). The ki value was significantly lower in OA-treated -cells (p < 0.01), but the values of the Hill coefficient were not significantly different when comparing OA-treated and untreated -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).
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Effects of Chronic Exposure to OA on Histological Features and Insulin Content of Islet -Cells. Data presented thus far demonstrate that in contrast to the acute effects of FFAs, which lead to the opening of -cell KATP channels, chronic exposure to OA closed -cell KATP channels. It is well known that the closure of -cell KATP channels excites -cells and triggers insulin release. To understand why chronic OA treatment excited -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 -cells using electron microscopy. As shown in Fig. 6, insulin-containing secretory granules and Golgi apparatuses were well developed in control -cells (A); however, in OA-treated -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 with control -cells (Fig. 6, A and 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 with 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 -cells, which gradually depleted insulin content and led to an impairment of glucose-stimulated insulin release.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-cell function in a different manner compared with acute exposure to FFAs. In contrast to the acute effects resulting from exposure to FFAs, which lead to the opening of -cell KATP channels and consequently the silencing of -cells due to decreased sensitivity of KATP channels to ATP, chronic exposure (4 days) to OA closed -cell KATP channels and excited -cells by increasing the sensitivity of KATP channels to ATP. After sustained -cell excitation, both the total number 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 -cell dysfunction, seems 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.
Chronic Exposure to OA Caused Electrical Excitation of Islet -Cells When Glucose Was Present at Low Concentrations. We found that chronic exposure to OA resulted in -cell spontaneous action potential firing even when glucose was present at low concentrations (Fig. 2B). Normally, isolated islet -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, -cell depolarization, and action potential firing. Action potentials in islet -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 -cells by chronic OA treatment plays an important role in the development of OA-induced -cell dysfunction and insulin release impairment.
Accumulating lines of evidence indicate that acute or chronic exposure to FFAs affects -cell function and insulin release; hence, FFAs are thought to be a critical risk 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 -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- to 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 -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 (4 days) to OA, an increased sensitivity of KATP channels 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 -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 -cells mostly disappeared (Fig. 4). This finding indicates that in OA-treated -cells, KATP channels are functionally closed by some intracellular mechanism. Since KATP channel activity in islet -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 -cells showed spontaneous action potential firing (Fig. 2).
KATP channel activity in islet -cells is basically regulated by levels of intracellular ATP and/or the ratio of ATP/ADP (Detimary et al., 1988). However, the sensitivity of KATP channels to ATP is also known to be modulated by other intracellular molecules. For example, intracellular Ca2+ was shown to increase the sensitivity of KATP channels to ATP in rat islet -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 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 negative 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 -cells was significantly lower compared with untreated -cells (Fig. 5), indicating that chronic exposure of islet -cells to OA increased the affinity of KATP channels to ATP. Currently, the precise mechanisms by which chronic exposure to OA induced a change in the affinity of KATP channels to ATP are not clear. As mentioned, 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. As illustrated in Fig. 6, total numbers of insulin-containing granules and insulin content in islets were reduced following 4-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 -cells isolated from OA-treated islets were electrically excited even in low glucose (2.8 mM) conditions (Fig. 2) and that overactivity of -cells following longterm (4-day) exposure to OA seems to gradually deplete insulin storage. Under these conditions, excitation of -cells was sustained by a closure of KATP channels; 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 -cells, both total numbers of insulin-containing secretion granules and insulin content were clearly lower compared with untreated cells (Fig. 6); 2) in chronic OA-treated -cells, 16.7 mM glucose-stimulated insulin release was clearly impaired (Fig. 1); 3) the exposure time of -cells to FFAs was longer in the present study (4 days) compared with previous studies (1 or 2 days); and 4) FFA-induced hypersensitivity of islet -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, -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.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: FFA, free fatty acid; KATP channel, ATP-sensitive potassium channel; HBSS, Hanks' balanced salt solution; OA, oleic acid.
Address correspondence to: Dr. Jie Wu, Neural Physiology Laboratory, Neurology Research, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013-4496. E-mail: jie.wu{at}chw.edu
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References |
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Ashcroft FM and Ashcroft SJH (1992) Mechanism of insulin secretion, in Insulin: Molecular Biology to Pathology (Ashcroft FM and Ashcroft SJH eds) pp 97-150, IRL, Oxford, UK.
Ashcroft FM and Kakei M (1989) ATP-sensitive K+ channels in rat pancreatic -cells: modulation by ATP and Mg2+ ions. J Physiol (Lond) 416: 349-367.
Ashcroft FM and Rorsman P (1989) Electrophysiology of the pancreatic -cell. Prog Biophys Mol Biol 54: 87-143.[CrossRef][Medline]
Baukrowitz T, Schulte U, Oliver D, Herlitze S, Krauter T, Tucker SJ, Ruppersberg JP, and Fakler B (1998) PIP2 and PIP as determinants for ATP inhibition of KATP channels. Science (Wash DC) 282: 1141-1144.
Bevilacqua S, Buzzigoli G, Bonadonna R, Brandi S, Oleggini M, Boni C, Geloni M, and Ferrannini E (1990) Operation of Randle's cycle in patients with NIDDM. Diabetes 39: 383-389.[Abstract]
Bjorklund A, Yaney G, McGarry JD, and Weir G (1997) Fatty acids and beta-cell function. Diabetologia 40: B21-B26.
Boden G, Chen X, Ruiz J, White JV, and Rossetti L (1994) Mechanism of fatty acid-induced inhibition of glucose uptake. J Clin Investig 93: 2438-2446.[Medline]
Bollheimer LC, Skelly RH, Chester MW, McGarry JD, and Rhodes CJ (1998) Chronic exposure to free fatty acid reduces pancreatic B-cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis translation. J Clin Investig 101: 1094-1101.[Medline]
Bonadonna RC, Zych K, Boni C, Ferrannini E, and DeFronzo RA (1989) Time dependence of the interaction between lipid and glucose in humans. Am J Physiol 57: E49-E56.[CrossRef]
Branstrom R, Aspinwall CA, Valimaki S, Ostensson CG, Tibell A, Eckhard M, Brandhorst H, Corkey BE, Berggren PO, and Larsson O (2004) Long-chain CoA esters activate human pancreatic beta-cell KATP channels: potential role in type 2 diabetes. Diabetologia 47: 277-283.[CrossRef][Medline]
Branstrom R, Corkey BE, Berggren PO, and Larsson O (1997) Evidence for a unique long chain acyl-CoA ester binding site on the ATP-regulated potassium channel in mouse pancreatic beta cells. J Biol Chem 272: 17390-17394.
Detimary P, Gilon P, and Henquin JC (1988) Interplay between cytoplasmic Ca2+ and the ATP/ADP ratio: a feedback control mechanism in mouse pancreatic islets. Biochem J 15: 269-274.
Elks ML (1993) Chronic perifusion of rat islets with palmitate suppresses glucose-stimulated insulin release. Endocrinology 133: 208-214.
Fan Z and Makielski JC (1997) Anion phospholipids activate ATP-sensitive potassium channels. J Biol Chem 272: 5388-5395.
Felley CP, Felly EM, van Melle GD, Frascarolo P, Jequier E, and Felber JP (1989) Impairment of glucose disposal by infusion of triglycerides in humans: role of glycemia. Am J Physiol 256: E747-E757.
Ferrannini E, Barrett EJ, Bevilacqua S, and De Fronzo RA (1983) Effect of fatty acids on glucose production and utilization in man. J Clin Investig 72: 1737-1747.[Medline]
Findley I (1987) The effects of magnesium upon adenosine triphosphate-sensitive potassium channels in rat insulin-secreting cell line. J Physiol (Lond) 391: 611-629.
Furukawa H, Carroll RJ, Swift HH, and Steiner DF (1999) Long-term elevation of free fatty acids leads to delayed processing of proinsulin and prohormone convertases 2 and 3 in the pancreatic beta-cell line MIN6. Diabetes 48: 1395-1401.[Abstract]
Gravena C, Mathias PC, and Ashcroft SJ (2002) Acute effects of fatty acids on insulin secretion from rat and human islets of Langerhans. J Endocrinol 173: 73-80.[Abstract]
Gribble FM, Proks P, Corkey BE, and Ashcroft FM (1998) Mechanism of cloned ATP-sensitive potassium channel activation by oleoyl-CoA. J Biol Chem 273: 26383-26387.
Hosokawa H, Corkey BE, and Leahy JL (1997) -Cell hypersensitivity to glucose following 24-h exposure of rat islets to fatty acids. Diabetologia 40: 392-397.[CrossRef][Medline]
Huang CL, Feng S, and Hilgemann DW (1998) Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by G
. Nature (Lond) 391: 803-806.[CrossRef][Medline]
Larsson O, Deeney JT, Branstrom R, Berggrent PO, and Corkey BE (1996) Activation of the ATP-sensitive K+ channel by long chain acyl-CoA. J Biol Chem 271: 10623-10626.
Lee HU, Lee HK, Koh CS, and Min HK (1988) Artificial induction of intravascular lypolysis by lipid-heparin infusions leads to insulin resistance in man. Diabetologia 31: 285-290.[Medline]
Lee Y, Hirose H, Ohneda M, Johnson JH, McGarry JD, and Unger RH (1994) -Cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-B-cell relationships. Proc Natl Acad Sci USA 91: 10878-10882.
Lillioja S, Bogardus C, Mott D, Kennedy LA, Knowler WC, and Howard BV (1985) Relationship between insulin-mediated glucose disposal and lipid metabolism in man. J Clin Investig 75: 1106-1115.[Medline]
Liu YQ, Tornheim K, and Leahy JL (1998) Fatty acid-induced beta cell hypersensitivity to glucose. Increased phosphofructokinase activity and lowered glucose-6-phosphate content. J Clin Investig 101: 1870-1875.[Medline]
Man ZW, Zhu M, and Shima K (1997) Impaired beta-cell function and deposition of fat droplets in the pancreas as a consequence of hypertriglyceridemia in OLET rat, a model of spontaneous NIDDM. Diabetes 46: 1718-1724.[Abstract]
McGarry JD and Dobbins RL (1999) Fatty acids, lipotoxicity and insulin secretion. Diabetologia 42: 128-138.[CrossRef][Medline]
Nakano K, Suga S, Takeo T, Ogawa Y, Suda T, Kanno T, and Wakui M (2002) Intracellular Ca2+ modulation of ATP-sensitive K+ channel activity in acetylcholine-induced activation of rat pancreatic -cells. Endocrinology 143: 569-576.
Prentki M and Corkey BE (1996) Are the beta-cell signaling molecules malonyl-CoA and cystolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM? Diabetes 45: 273-283.[Abstract]
Roche E, Maestre I, Martin F, Fuentes E, Casero J, Reig JA, and Soria B (2000) Nutrient toxicity in pancreatic beta-cell dysfunction. J Physiol Biochem 56: 119-128.[Medline]
Shimabukuro M, Koyama K, Chen G, Wang MY, Trieu F, Lee Y, Newgard CB, and Unger RH (1997) Direct antidiabetic effect of leptin through triglyceride depletion of tissues. Proc Natl Acad Sci USA 94: 4637-4641.
Shyng SL and Nichols CG (1998) Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science (Wash DC) 282: 1138-1141.
Suga S, Kanno T, Nakano K, Takeo T, Dobashi Y, and Wakui M (1997) GLP-I (7-36) amide augments Ba2+ current through L-type Ca2+ channel of rat pancreatic -cell in a cAMP-dependent manner. Diabetes 46: 1755-1760.[Abstract]
Suga S, Nakano K, Takeo K, Osanai T, Ogawa Y, Yagihashi S, Kanno T, and Wakui M (2003) Masked excitatory action of noradrenaline on rat islet -cells via activation of phospholipase C. Eur J Physiol 447: 337-344.[CrossRef][Medline]
Wollheim CB and Sharp GWG (1981) Regulation of insulin release by calcium. Physiol Rev 61: 914-973.
Zhang Y, Xiao M, Niu G, and Tan H (2005) Mechanisms of oleic acid deterioration in insulin secretion: role in the pathogenesis of type 2 diabetes. Life Sci 77: 2071-2081.[CrossRef][Medline]
Zhou YP and Grill VE (1994) Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Investig 93: 870-876.[Medline]
Zhou YP, Ling ZC, and Grill VE (1996) Inhibitory effects of fatty acids on glucose-regulated -cell function: association with increased islet triglyceride stores and altered effect of fatty acid oxidation on glucose metabolism. Metabolism 45: 981-986.[CrossRef][Medline]
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