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Endocrinology, doi:10.1210/en.2007-1138
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Endocrinology Vol. 149, No. 7 3679-3687
Copyright © 2008 by The Endocrine Society

Elevation in Intracellular Long-Chain Acyl-Coenzyme A Esters Lead to Reduced β-Cell Excitability via Activation of Adenosine 5'-Triphosphate-Sensitive Potassium Channels

Nicola J. Webster1, Gavin J. Searle1, Patrick P. L. Lam, Ya-Chi Huang, Michael J. Riedel, George Harb, Herbert Y. Gaisano, Andrew Holt and Peter E. Light

Department of Pharmacology (N.J.W., G.J.S., A.H., P.E.L.), University of Alberta, Edmonton, Alberta, Canada T6G 2H7; Departments of Medicine and Physiology (P.P.L.L., Y.-C.H., H.Y.G.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Cellular and Physiological Sciences (M.J.R.), University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3; and Surgical Medical Research Institute (G.H.), University of Alberta, Edmonton, Alberta, Canada T6G 2N8

Address all correspondence and requests for reprints to: Dr. Peter E. Light, Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: peter.light{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Closure of pancreatic β-cell ATP-sensitive potassium (KATP) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long-chain acyl-coenyzme A esters (acyl-CoAs) can potently activate KATP channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced β-cell excitability and insulin secretion observed in obesity and type 2 diabetes. However, reduced β-cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic β-cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse β-cells using virally delivered overexpression of long-chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on β-cell KATP channel activity and cell excitability was assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in KATP channel activity in AdACSL-1-infected β-cells cultured in medium supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected β-cells (with palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mM glucose. These results suggest a direct link between intracellular fatty ester accumulation and KATP channel activation, which may contribute to β-cell dysfunction in type 2 diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ATP-SENSITIVE POTASSIUM (KATP) channels play a key role in linking glucose metabolism to insulin secretion in the pancreatic β-cell. It has, therefore, been suggested that initial alterations in KATP channel activity, resulting in abnormal β-cell excitability, may be responsible for early β-cell dysfunction and impaired insulin secretion in type 2 diabetes (T2D) (1), preceding the loss of islet biomass and development of insulin resistance in the etiology of the disease. The recent rapid rise in T2D, however, has been mirrored by a significant increase in the prevalence of obesity, highlighting a major risk factor for the disease. This link has been strengthened by studies demonstrating that diets high in fat, coupled to reduced physical activity, significantly contribute to the development of obesity and T2D (2, 3), with diets rich in saturated long-chain fats having the most damaging effect (4, 5). The mechanisms linking dietary fat intake, obesity, and T2D, however, are not completely understood. There is, nevertheless, evidence indicating that the levels of plasma free fatty acids (FFAs) are elevated in both T2D and obesity (6, 7, 8) and may be implicated in β-cell insulin secretory dysfunction (8, 9, 10). Interestingly, growing evidence demonstrates that the intracellular esterified form of dietary FFAs, long-chain acyl-coenzyme A esters (acyl-CoAs), are potent modulators of KATP channels (11, 12, 13, 14).

Under resting conditions, the basal efflux of potassium ions through KATP channels maintains the pancreatic β-cell in a hyperpolarized inactive state. Closure of the channels in response to an elevated ATP/ADP ratio arising from glucose metabolism induces membrane depolarization, resulting in Ca2+ influx via voltage-gated Ca2+ channels and the concomitant release of insulin. Hence, KATP channels directly link pancreatic β-cell metabolism to changes in electrical activity and insulin secretion. This pivotal role of KATP channels in maintaining normal glucose homeostasis is demonstrated further both by the clinical efficacy of the sulfonylurea family of KATP channel inhibitors in the treatment of newly diagnosed T2D (15) (16) and by the observation that loss-of-function mutations in the genes encoding Kir6.2 and SUR1 (the β-cell KATP channel subunits) have been linked to the rare human disorder familial hyperinsulinemic hypoglycemia of infancy (17, 18). Conversely, monogenic activating mutations in the Kir6.2 and SUR1 genes severely impair insulin secretion and lead to a neonatal diabetic phenotype (19, 20, 21). Importantly, therapy with sulfonylureas is also an effective treatment in these patients (22). Thus, enhanced β-cell KATP channel activity, by whatever mechanism, will reduce the magnitude of glucose-stimulated insulin-secretion (GSIS) in β-cells and may, therefore, lead to the development of diabetes.

Acyl-CoAs represent one of the most important classes of endogenous KATP channel modulators (11, 12, 13, 23). In inside-out excised membrane patches, acyl-CoAs are potent activators of KATP channels, with the level of activation demonstrating both chain length and saturation dependence (14). Acyl-CoAs are formed in the cytosol by FFA esterification to the CoA moiety by acyl-CoA synthetase (ACSL). Chronic up-regulation of circulating plasma levels of FFAs, as observed in both the obese (6) and individuals with T2D (24), can lead to cytosolic accumulation of acyl-CoAs in pancreatic β-cells (10, 25). In T2D, increased glucose levels may compound this acyl-CoA accumulation by inhibition of fatty acid oxidation arising from increased glucose metabolism (9, 26). Therefore, elevated intracellular acyl-CoA concentrations in obesity and T2D are predicted to cause chronic activation of β-cell KATP channels, thereby hyperpolarizing the β-cell membrane and inhibiting insulin secretion. Indeed, it has been shown recently that elevation of intracellular acyl-CoAs can activate recombinant KATP channels in intact cells (27). However, a direct study of the effects of elevated endogenous acyl-CoAs on pancreatic β-cell KATP channel activity and whole-cell electrical activity has not been performed to date. Accordingly, we increased endogenous levels of dietary acyl-CoAs within physiologically intact primary mouse β-cells using overexpression of ACSL-1 and tested the effects of this manipulation on KATP channel activity and cellular excitability in response to glucose and sulfonylurea treatment. Results from our study strengthen the hypothesis that saturated fats can lead to β-cell dysfunction via chronic acyl-CoA modulation of β-cell KATP channels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Islet isolation and dissociation
Pancreatic islets from BALB/c mice were isolated by collagenase (Sigma Chemical Co., St. Louis, MO) digestion of the pancreas, purified by Ficoll density gradient, and then handpicked. Islets were dispersed into single cells 2 h after isolation by gentle agitation in a Ca2+-free buffer containing 0.0025% trypsin. Cells were plated onto poly-D-lysine-coated glass coverslips and maintained in culture for up to 4 d in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.2% NaHCO3 in a humidified incubator at 37 C/5% CO2. All animals were cared for according to the Canadian Council on Animal Care guidelines.

Cell culture and infection
At 24 h after dissociation, single-cell cultures were infected with an adenovirus containing an ACSL-1 clone driven under a cytomegalovirus promoter (AdACSL-1; a kind gift from Dr. J. R. B. Dyck, University of Alberta). Control cells received adenoviral delivery of a green fluorescent protein (GFP) clone (AdGFP) driven under the same promoter. Cells were exposed to 100 pfu/cell of either virus for 4 h, and then virus-containing medium was removed and cells cultured for another 48 h. For the final 24-h culture, medium was replaced with RPMI 1640 medium (supplemented as above) containing 0.3 mM FFAs. Palmitic acid, sodium oleate, and linoleic acid (Sigma) were dissolved by boiling in 63% ethanol with 0.003% NaHCO3. After evaporation of the ethanol during boiling, the dissolved fats were then diluted to the final concentration (0.3 mM) in RPMI 1640 medium (supplemented as above) containing an additional 3% albumin. In GFP-expressing control cells, culture medium was replaced with RPMI 1640 medium (supplemented as above) containing the additional BSA but no FFAs. INS1 cells were cultured in 75-cm2 flasks in a medium containing RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol. Infection and treatment with FFAs were as above for primary β-cells.

Electrophysiological measurements
The whole-cell perforated patch-clamp technique was used to measure basal KATP currents and membrane potential from single isolated β-cells infected with AdACSL-1 or AdGFP. Coverslips were placed in a recording chamber affixed to the stage of an inverted microscope (Zeiss Axiovert S100). Recordings were digitized at 2 kHz and filtered at 1 kHz using the Axopatch 200B patch-clamp amplifier and Clampex 8.0 (Molecular Devices Corp., Union City, CA). The chamber was continuously perfused with an external solution that contained (in mM) 140 NaCl, 1.5 CaCl2, 3.6 KCl, 2.0 NaHCO3, 0.5 NaH2PO4, 0.5 MgSO4, 5 HEPES, and 2.5 glucose (pH 7.4). Patch pipettes were pulled from borosilicate glass (GB150-86-15; Sutter Instrument Co., Novato, CA) to yield resistances from 1.7–2.0 M{Omega} when backfilled with buffer solution. Pipette tips were filled with a buffer solution containing (in mM) 76 K2SO4, 10 NaCl, 5 HEPES, 10 KCl, and 1 MgCl2 (pH 7.35) and then backfilled with the same internal solution containing 0.1 mg/ml amphotericin B. Once a gigaohm seal was formed, it took approximately 10–15 min to obtain satisfactory patch perforation. Experiments did not commence until series resistance was less than 25 M{Omega}. All experiments were performed at 30 C. No corrections were made for liquid junction potentials. β-Cells could be easily identified by size and distinguished from {alpha}-cells by their electrically quiescent state in perfusate containing 2.5 mM glucose and a membrane potential (VM) less than –70 mV.

Single-channel recordings were made from single β-cells and INS1 cells in the cell-attached configuration of the patch-clamp technique and were digitized at 5 kHz and filtered at 2 kHz. Bath perfusate for β-cells did not change. For INS1 cells, bath perfusate contained 1 mM glucose. Patch pipettes were pulled as above and back filled with solution containing (in mM) 110 KCl, 30 KOH, 1 MgCl, 5 HEPES, and 10 EGTA (pH 7.4). Cells were allowed to equilibrate in the bath perfusate for 10 min before recording commenced. Channel activity was recorded for 10 min in control perfusate before a 5-min application of 100 µM diazoxide.

Analysis of electrophysiological recordings
Whole-cell currents were normalized to percentage of maximum current obtained in each cell in the presence of 100 µM diazoxide. Single-channel events during the final 1 min before diazoxide application were analyzed using pClamp9 and the number of channels (N) in each patch estimated from the maximum superimposed channel events in diazoxide. Open probability (Popen) was calculated as NPopen/N.

Determination of acyl-CoA content
Large amounts of cellular lysates are not obtainable using primary β-cells; therefore, the model β-cell line INS1 was used for this part of the study. INS1 cells were grown to about 60% confluency in 75-cm2 flasks, infected with AdGFP/AdACSL-1 with or without 0.2 mM palmitiate/0.1 mM oleate or 0.3 mM linoleate as described above. The intracellular acyl-CoA content of INS1 cell lysates was determined using HPLC, as previously described (28). Values obtained for area under the curve were normalized to total protein content.

Western blotting
Islets were dispersed, as described above, and cells cultured in 35-mm petri dishes. Cell lysates were collected, snap-frozen in liquid N2, and stored at –80 C until use. For Western blot analysis, 40 µg protein was loaded onto 10% (wt/vol) polyacrylamide-SDS gels. After separation, proteins were transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA) and ACSL-1 detected by incubating membranes with an anti-ACSL-1 polyclonal antibody (1:2000 dilution for 1 h at room temperature; a kind gift from Dr. J. R. B. Dyck, University of Alberta).

Analysis of ATP/ADP ratios
Measurement of ATP and ADP levels was made using a luciferase-based assay adapted from Elmi et al. (29). Briefly, the protocol was modified as follows: cells were homogenized using a QIAshredder (QIAGEN, Valencia, CA), phosphoenolpyruvate and pyruvate kinase concentrations were increased to 250 µM and 1 µg/ml respectively, and luciferase and luciferin concentrations were increased to 8 nM and 34 µM, respectively.

Experimental compounds
Tolbutamide and diazoxide (Sigma) were dissolved in dimethylsulfoxide daily and diluted to the concentrations indicated in text before use. The final dimethylsulfoxide concentration of 0.1% did not affect KATP currents or membrane potential in β-cells.

Statistical significance
Statistical significance was assessed using the unpaired Student’s t test or ANOVA as required. P < 0.05 was considered significantly different. Data are expressed as means ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of ACSL-1 enhances basal KATP currents in primary β-cells
To elevate intracellular acyl-CoA levels, we used an adenoviral vector to overexpress ACSL-1 in primary β-cells. Overexpression of ACSL-1 was then confirmed by Western blot analysis using an ACSL-1-specific antibody (Fig. 1AGo). To facilitate elevation of acyl-CoAs levels, AdACSL-1-infected β-cells were cultured for the final 24 h in medium supplemented with 0.2 mM palmitate and 0.1 mM oleate. This experimental paradigm was designed to represent a chronic, rather than an acute, elevation in intracellular acyl-CoA levels. The perforated whole-cell configuration of the patch-clamp technique was used to compare basal ion channel activity with AdGFP-infected controls. Experiments were performed using a perfusate containing low glucose (2.5 mM) because this is a necessary experimental maneuver to detect whole-cell KATP currents in primary β-cells. Under these conditions, the ramp protocol outlined in Fig. 1Go triggered inward and outward currents in both groups of β-cells, with currents reversing close to predicted equilibrium potential for potassium (EK) (Fig. 1Go) and fully inhibited by 100 µM tolbutamide, a specific inhibitor of KATP channels(data not shown). However, in β-cells infected with AdACSL-1 cultured with palmitate/oleate (AdACSL-1+palmitate/oleate), basal currents were significantly larger than AdGFP-infected control currents. For example, at –50 mV, the mean basal current as a percentage of the maximum current obtained in the presence of 100 µM diazoxide was 18.5 ± 2.4% in control cells and 28 ± 2.9% in AdACSL-1+palmitate/oleate-infected cells. Current density values were 9.80 ± 2.06 pA/pF in control and 18.6 ± 2.62 pA/pF in AdACSL-1+palmitate/oleate (P = 0.02). Thus, in β-cells overexpressing ACSL-1 and treated with palmitate/oleate, the basal KATP currents observed in low glucose were significantly increased by over 51%, suggesting that more channels are in an open state or that channel conductance is increased. Infection with the AdACSL-1 virus alone (Fig. 1CiiGo) or treatment with 0.2 mM palmitate/0.1 mM oleate in the absence of virus (Fig. 1CiiiGo) did not significantly alter basal currents. To determine whether the observed changes in KATP channel activity were indirectly mediated via changes in the ATP/ADP ratio, we measured this ratio in all the experimental groups (Fig. 1BGo) and observed no significant differences between groups (P > 0.05).


Figure 1
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  		FIG. 1. Whole-cell KATP currents. A, Example Western blot of total ACSL-1 protein expression in dissociated islet cultures after infection with AdACSL-1 or AdGFP. B, ATP/ADP ratios in INS1 cells after infection with AdACSL-1 and/or addition of 0.2 mM palmitate/0.1 mM oleate to the culture medium for the final 24-h culture. C, Basal currents recorded from single β-cells: i, cells were infected with AdACSL-1 or AdGFP and 0.2 mM palmitate/0.1 mM oleate added to culture media of AdACSL-1-infected cells for the final 24-h culture, and currents were elicited by the ramp protocol shown below the figure; ii as i, except that medium was not supplemented with palmitate/oleate for the final 24 h; iii, as i, except cells were not infected with virus. Traces represent ensemble averages of current recordings from n = 7–9 cells. Grouped data are average currents recorded at –50 mV expressed as a percentage of maximal current recorded in the presence of 100 µM diazoxide. *, P < 0.05. There was no significant difference in the cell capacitance between groups; AdGFP = 5.4 ± 0.3 pF; AdACSL-1+palmitate/oleate = 5.35 ± 0.45 pF; P = 0.9.

 
To investigate the saturation and chain length dependence of the acyl-CoA effect, as previously reported by our group and others in recombinant KATP channels (14, 30), currents were also recorded from AdACSL-1-infected β-cells cultured in medium supplemented with the polyunsaturated FFA linoleate or the 10:0 medium-chain FFA decanoate in place of palmitate/oleate. Interestingly, currents were not enhanced after treatment with either FFA. Indeed, a decrease in current was observed in linoleate-treated cells. At –50 mV, mean basal currents expressed as a percentage of maximal current obtained in the presence of 100 µM diazoxide were 18.9 ± 2.3% in control cells and 10.6 ± 3.5% in AdACSL-1+linoleate-infected cells (n = 8; P < 0.05). Treatment with 0.3 mM linoleate (24 h) in the absence of virus did not significantly alter basal currents. KATP currents were also unaltered when AdACSL-1-infected β-cells were treated with decanoate (0.3 mM). Mean basal currents at –50 mV were 20.0 ± 2.9% of maximal current in control cells and 17.0 ± 2.6% of maximal current in AdACSL-1+decanoate-infected cells.

KATP channel open probability is increased in AdACSL-1-infected β-cells and INS1 cells
To investigate whether the increase in whole-cell K+ current in AdACSL-1+palmitate/oleate cells is due to an increase in Popen or an increase in single-channel unitary conductance of KATP channels, the cell-attached patch-clamp technique was used to measure single-channel activity and current amplitude in isolated β-cells (Fig. 2Go). In β-cells infected with AdACSL-1+palmitate/oleate, there was a significant 3-fold increase in KATP Popen (AdACSL-1+fat = 0.006 ± 0.0017, AdGFP = 0.002 ± 0.0009; P < 0.05, Fig. 2CGo) with no significant change in single-channel current amplitude (AdACSL-1+fat = 4.4 pA ± 0.16, AdGFP = 4.4 pA ± 0.08; Fig. 2Go, Aiii and Biii). We also verified that INS cells exhibit similar changes in Popen (KATP Popen AdACSL-1+fat = 0.0075 ± 0.0037, KATP Popen AdGFP = 0.00099 ± 0.00061; P < 0.05).


Figure 2
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  		FIG. 2. KATP single-channel activity and current amplitude. A and B, Example cell-attached single-channel recording from β-cells infected with AdGFP (A) or AdACSL-1 + 0.2 mM palmitate/0.1 mM oleate (B); i, last 2 min of recording in control perfusate (2.5 mM glucose) followed by application of 100 µM diazoxide for 5 min; ii, expansion of the section of trace marked by the open bar in i; iii; Gaussian fit to the all-levels current amplitude histogram generated by combining the all-levels current data from each cell in the data set. N is the number of events at a particular amplitude. Currents were recorded at room temperature. Pipettes contained solution that approximated intracellular K+ concentration (140 mM). Pipette potential was –15 mV, resulting in a VM of about –60 mV. C, Grouped data are Popen measured in the last 1 min of control solution before diazoxide application. n = 12 for AdGFP and n = 14 for AdACSL-1. *, P < 0.05. D, Grouped data (Popen) for cell-attached single-channel recording from INS1 cells infected with AdGFP or AdACSL-1 + 0.2 mM palmitate/0.1 mM oleate. n = 12 for AdGFP and n = 8 for AdACSL-1. *, P < 0.05.

 
HPLC analysis of cellular acyl-CoA levels
To confirm elevation of acyl-CoAs in AdACSL-1 infected β-cells and that palmitate/oleate and linoleate behave as similar substrates for ACSL-1, acyl-CoA content was measured in INS1 cells using HPLC. Treatment with either palmitate/oleate or linoleate alone did not appear to change the total acyl-CoA content relative to the GFP control group (percent change was –9.9 and 8.3%, respectively). Compared with GFP control, however, the total acyl-CoA content increased by 51% in the AdACSL-1 group, 103% in the AdACSL-1+palmtiate/oleate group, and 119% in the AdACSL-1+linoleate group (Fig. 3AGo). We also analyzed changes in specific acyl-CoA levels on selected HPLC samples as follows. The combination of ACSL-1 overexpression and treatment with palmitate/oleate significantly raised levels of palmitoyl-CoA (16:0) from 16.26 ± 6.39 to 34.0 ± 4.07 fmol/µg (Fig. 3Go, B and C). Levels of oleoyl-CoA (18:1) significantly increased from 9.83 ± 4.4 to 27.4 ± 7.1 fmol/µg in the same cells (data not shown). Treatment with linoleate in place of palmitate/oleate significantly raised levels of linoleoyl-CoA (18:2) from 2.92 ± 0.78 to 34.36 ± 6.53 fmol/µg (Fig. 3Go, B and C). Thus, treatment with palmitate/oleate and linoleate specifically increased levels of corresponding acyl-CoAs in AdACSL-1-infected β-cells.


Figure 3
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  		FIG. 3. HPLC analysis of cellular acyl-CoA levels. A, Grouped data showing percentage change in total acyl-CoAs in INS1 cells after infection with AdACSL-1 in the presence or absence of supplementary FFAs. FFAs (0.2 mM palmitate/0.1 mM oleate or 0.3 mM linoleate) were added to culture media for the final 24 h of culture. Data were obtained from runs on several different HPLC columns and thus are expressed as percent change relative to the control in each run on a particular column (AdGFP). B, Grouped data for levels of palmitoyl-CoA (16:0; open bars) and linoleoyl-CoA (18:2; hatched bars) from n = 3 experiments run on the same column. *, P < 0.05; **, P < 0.01. C, Representative HPLC traces showing specific long-chain acyl-CoA levels.

 
β-Cell excitability to glucose
A consequence of enhanced KATP channel activity in pancreatic β-cells would be a decrease in cell excitability to elevated glucose. Therefore, the electrical responsiveness of AdACSL-1+palmitate/oleate and AdGFP-infected β-cells to a glucose challenge was investigated (Fig. 4Go). After a change of glucose concentration from 2.5 to 10 mM, control cells exhibited membrane depolarization, followed by action potential generation (spiking) upon reaching a threshold voltage range from approximately –55 to –40 mV (Fig. 4AGoi). However, β-cells infected with AdACSL-1+palmitate/oleate were significantly less responsive to the same glucose challenge (Fig. 4AiiGo), both in the extent of membrane depolarization induced by the challenge (21.9 ± 1.7 mV positive shift in control cells and 15.2 ± 1.5 mV in AdACSL-1+fat-infected cells, Fig. 4BGo), and the frequency of subsequent spiking events (spike frequency in AdACSL-1+fat cells was about 60% less than controls, Fig. 4CGo). Mean initial resting VM in 2.5 mM glucose perfusate before commencement of the experiments was –76 ± 0.9 mV, similar to that previously reported for primary mouse β-cells when differences in Ek, predicted reversal potential (Erev) for KATP, and liquid junction potentials are accounted for (31, 32, 33). VM was not significantly different between the two groups, which might be expected given that at low glucose concentrations, VM is close to predicted Erev for KATP (when liquid junction potential is adjusted for), and thus small hyperpolarizations due to enhanced KATP activity will be more difficult to detect. However, mean VM recorded in perfusate containing 5 mM glucose was significantly more hyperpolarized in AdACSL-1+palmitate/oleate-infected β-cells compared with controls (AdGFP = –53.9 ± 2.1 mV and AdACSL-1+palmitate/oleate = –61.6 ± 2.1 mV; P < 0.05, Fig. 4DGo). Thus, at physiological glucose concentrations, acyl-CoA-mediated increases in KATP channel activity hold the β-cell membrane at more hyperpolarized potentials, resulting in a β-cell that will be less responsive to any given increase in glucose concentration.


Figure 4
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  		FIG. 4. Glucose-stimulated β-cell excitability. A, Representative traces showing β-cell membrane depolarization after a change of glucose concentration from 2.5 to 10 mM in AdGFP-infected β-cells (i) and AdACSL-1-infected cells (ii) (+0.2 mM palmitate/0.1 mM oleate). The 10 mM glucose was removed 2 min after the first spiking event. Expanded sections of trace taken from A (i and ii) represent the last 20 sec of recording in the presence of 10 mM glucose. B and C, Grouped data from n = 9 cells indicating mean depolarization in the presence of 10 mM glucose (B) and mean action potential spike frequency during the last 30 sec of recording in 10 mM glucose (C). D, Mean VM recorded from n = 5–7 β-cells in perfusate containing 5 mM glucose. *, P < 0.05.

 
β-Cell excitability to tolbutamide
Sulfonylureas are commonly used oral antidiabetic drugs that stimulate insulin secretion by closing KATP channels in pancreatic β-cells. Therefore, the electrical responsiveness of infected β-cells to clinically relevant concentrations of the sulfonylurea tolbutamide was also investigated (Fig. 5Go). In AdGFP-infected controls, perfusion with tolbutamide (0.3–300 µM) induced membrane depolarization in a dose-dependent manner. Once a threshold voltage range between approximately –55 and –40 mV had been achieved, cells exhibited characteristic spiking events. However, β-cells infected with AdACSL-1+palmitate/oleate were significantly less responsive to tolbutamide with regard to the size of depolarization induced by each effective concentration (P < 0.01 for 3–100 µM). For example, at 30 µM tolbutamide, there was only a 15 ± 1.6-mV positive shift in VM in cells infected with AdACSL-1+palmitate/oleate compared with a 24.1 ± 1.7 mV shift in controls. Consequently, mean spiking frequency in AdACSL-1+palmitate/oleate-treated cells at 30 µM tolbutamide was only 60% of that in AdGFP-infected cells. Furthermore, β-cell interspike VM during the depolarization plateau phase to tolbutamide was significantly more hyperpolarized in AdACSL-1+palmitate/oleate-infected β-cells compared with controls for the two concentrations of tolbutamide in the middle of the dose-response curve (P < 0.05; for 10 µM, control = –56.9 ± 2.4 mV and AdACSL-1+palmitate/oleate = –64 ± 1.8 mV, and for 30 µM, control = –50.8 ± 3.5 mV and AdACSL-1+palmitate/oleate = –59.5 ± 1.5 mV). Thus, in β-cells overexpressing ACSL-1 and treated with 0.2 mM palmitate/0.1 mM oleate, the resulting increase in KATP channel activity holds β-cell VM at more hyperpolarized potentials, and thus significantly higher doses of tolbutamide are required to induce changes in membrane excitability sufficient to trigger insulin secretion.


Figure 5
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  		FIG. 5. β-Cell excitability to tolbutamide. A, Representative traces showing β-cell membrane depolarization in response to the indicated concentrations of tolbutamide for AdGFP-infected β-cells (i) and AdACSL-1-infected cells (ii) (+0.2 mM palmitate/0.1 mM oleate). B and C, Grouped data from n = 7–11 cells indicating mean membrane depolarization in the presence of tolbutamide (B) and mean spike frequency during the last 30 sec of recording at each concentration (C). *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The pivotal role for KATP in controlling β-cell membrane potential has made the possible involvement of abnormal channel activity in precipitating altered β-cell excitability and T2D an area of increasing interest. In light of this, our and other recent studies have focused on characterizing potential endogenous activators of β-cell KATP, in particular saturated and unsaturated long-chain acyl-CoAs because levels of these lipids are elevated in T2D and obesity. Extending these studies to more physiologically relevant models, however, has proved to be problematic; long-term and/or high FFA treatment (i.e. to mimic the intracellular acyl-CoA accumulation occurring in individuals with high levels of circulating FFAs) leads to toxicity issues and/or changes in membrane fluidity of cultured β-cells, which makes it difficult to record changes in electrical activity. Furthermore, chronically elevating endogenous acyl-CoAs within intact primary cells via long-term/high FFA treatment also adds a layer of complexity to interpretation of the data, because it becomes difficult to identify a single lipid moiety responsible for mediating any observed effects. Adenoviral overexpression of ACSL-1, therefore, represents an elegant way to circumvent these problems (34) and has allowed us to specifically elevate common dietary acyl-CoAs within intact primary mouse β-cells and assess the effects of acyl-CoA accumulation on cell electrical activity and excitability.

Infection with AdACSL-1 followed by 24 h culture in the presence of palmitate and oleate, the most common saturated and monounsaturated FFAs in the Western diet, respectively, or linoleate, the primary {omega}-6 essential polyunsaturated FFA, significantly elevated levels of each corresponding acyl-CoA above that of basal. Patch-clamp analysis of KATP channel activity in these cells revealed a significant increase in KATP current in the AdACSL-1-infected β-cells cultured in the presence of palmitate but interestingly not linoleate. Indeed, the data suggest a slight protective effect of polyunsaturated FFAs on β-cell excitability, because a moderate decrease in KATP current was observed in AdACSL-1+linoleate-treated cells. Thus, we have confirmed our previous work on recombinant Kir6.2/SUR1 in excised membrane patches, which showed that acyl-CoAs enhance channel activity in a saturation-dependent manner (14). Furthermore, KATP current remained unaltered in AdACSL-1 infected cells cultured in the presence of the medium-chain FFA decanoate (10:0), also confirming the importance of chain length in acyl-CoA modulation of channel activity (14).

Our data are also consistent with other reports demonstrating direct KATP channel modulation by exogenously applied acyl-CoAs in recombinant expression systems and/or excised membrane patches (11, 12, 13) and are in agreement with two recent reports in which acute exogenous perfusion with nonesterified FFAs modulates KATP channel activity in Chinese hamster ovary cells (27) and β-cells (30). In these two latter studies, the authors argue that exogenously applied FFAs are rapidly transported into the cell and converted to acyl-CoAs, which in turn enhance KATP channel activity. We did not observe a significant change in KATP channel activity after treatment with exogenous palmitate/oleate in the absence of AdACSL-1 (Fig. 1Go). This is in agreement with a previous report showing no change in membrane potential after perfusion of mouse islets with palmitate (35). The discrepancy between our data and that of Branstrom et al. (30) and Shumilina et al. (27), however, most likely reflects the longer (24 h) treatment in our study. After 24 h culture in the presence of palmitate/oleate (or linoleate), we observed no elevation in acyl-CoA content, which supports our electrophysiology data. This presumably reflects the ability of primary β-cells to manage a moderate concentration of palmitate/oleate over this time period. Indeed, in the study by Branstrom et al. (30), channel activation in the presence of oleic acid was transient in nature, most likely representing an increase in channel activity in response to a transient rise in acyl-CoA levels occurring within 5 min of exposure to the oleic acid. We believe our AdACSL-1+palmitate/oleate experimental paradigm, however, represents a more chronic fat exposure, and in this treatment group, we observed a persistent activation of KATP channels. We propose that this activation may reflect direct and more permanent modulation of channel activity by an ACSL-1-driven chronic exposure to high levels of intracellular acyl-CoAs, as might be expected in obesity and T2D, where chronically elevated levels of circulating FFAs can lead to a cytosolic accumulation of acyl-CoAs (6, 10, 24, 25). Thus, chronic activation of KATP channels is likely mirrored in obesity and T2D and could account for altered β-cell excitability in these two disease states.

Interestingly, a modest increase in acyl-CoA content was observed in the AdACSL1 group not exposed to supplementary FFAs, yet no increase in KATP channel current was detected. A plausible explanation for this may involve the intrinsic intracellular acyl-CoA buffering capacity of the β-cell (e.g. acyl-CoA binding proteins), with the buffering capacity being exceeded in the AdACSL1+palmitate/oleate group but not by AdACSL-1 alone, resulting in elevation in free acyl-CoA levels and activation of KATP channels. Unfortunately, it was not possible to test this experimentally because the HPLC assay measures total (both free and buffered) acyl-CoA levels.

Importantly, our study also demonstrates that chronic cytosolic accumulation of acyl-CoAs translates directly to a decrease in β-cell excitability, as demonstrated by a more hyperpolarized membrane potential (Fig. 4Go) and decreased membrane depolarization and action potential spiking frequency in response to glucose and tolbutamide (Figs. 4Go and 5Go). The potential effects of this decrease in excitability are demonstrated by recent genetic studies linking monogenic mutations in Kir6.2 known to increase channel activity with a neonatal diabetic phenotype (21) as well as an association with T2D (36, 37, 38, 39, 40) and decreased insulin secretion (37, 40). The observation that the E23K polymorphism in KATP channels is associated with decreased insulin secretion in glucose-tolerant control subjects (37) is also highly supportive of a mechanism whereby altered KATP channel activity and decreased β-cell excitability is responsible for T2D. This proposition is strengthened further by a recent report showing rescue of the absent GSIS response in human T2D islets by the KATP channel inhibitor glibenclamide (41), consistent with the notion that β-cell KATP channel activity is increased in T2D islets. Our data therefore suggest that in obesity and T2D, chronic acyl-CoA accumulation would ultimately manifest as a reduction in GSIS and a decrease in sensitivity to sulfonylureas. Thus, we provide the first direct evidence for a mechanism through which exposure to high levels of circulating lipids may lead to the onset of T2D via reduced β-cell excitability. This hypothesis certainly appears to be supported by a study in which long-term (48 h) exposure of human pancreatic islets to high levels of palmitate/oleate resulted in reduced first-phase GSIS (42) and recent clinical data showing decreased sensitivity to sulfonylureas in patients with the Kir6.2 E23K polymorphism (43), which is known to increase channel sensitivity to acyl-CoAs (23, 44).

The effects of raising intracellular levels of acyl-CoAs on cell excitability in physiologically intact β-cells, however, is obviously multifaceted, and it is likely that many interlinked pathways may contribute to islet dysfunction after chronic exposure to elevated fat and intracellular acyl-CoA concentrations (10). Indeed, the effect of acyl-CoAs on GSIS can be both stimulatory and inhibitory, and thus the overall effect may depend on temporal fluctuations in acyl-CoA levels and the relative buffering by acyl-CoA binding proteins. FFAs also modulate protein expression in islets via regulation of transcription factors and nuclear receptors (45, 46, 47) and exert dual effects on β-cell excitability, dependent on the duration of exposure. Acute exposure to FFAs is known to stimulate insulin secretion (48, 49), whereas chronic exposure to FFAs attenuates glucose sensitivity (50, 51). The mechanisms underlying this biphasic response to FFAs likely reflect different signaling pathways. For example, the insulin-stimulatory effects of acute fat exposure are mediated by several signaling pathways including activation of the GPR40 orphan receptor (52) in the absence of changes in membrane potential (35). In contrast, chronic exposure to FFAs may act to reduce insulin secretion via multiple pathways such as saturation-dependent lipotoxicity (53). Additionally, our current data are consistent with a mechanism involving direct modulation of KATP channels by acyl-CoAs leading to reduced β-cell excitability.

A direct effect of acyl-CoAs on KATP channels is highly supported by our observation that the intracellular ATP/ADP ratio, perhaps the most important determinant of KATP channel activity, is not altered in any of the experimental groups (Fig. 1BGo). This confirms that enhanced KATP channel activity is not related to a decreased cytosolic ATP/ADP ratio that could arise as a consequence of altered fat metabolism. Incubation with elevated FFAs in the absence of ACSL-1 overexpression also did not significantly alter KATP channel activity (Fig. 1Go), further supporting the notion that the acyl-CoA molecules are the key regulators of KATP channel activity. Indeed, differential effects on KATP channel activity were observed when AdACSL-1-infected cells were cultured in medium supplemented with palmitate/oleate and linoleate, an observation that is strongly supported by our previous study (14) where we found that saturated and trans acyl-CoAs are more potent activators of KATP channels than are cis-, mono-, and polyunsaturated acyl-CoAs. The observation that KATP currents were unaltered in AdACSL-1-infected cells exposed to decanoate is also strongly supported by our and other previous studies that showed that the extent of KATP activation is highly dependent on chain length. Acyl chains of fewer than 14 carbons have very limited effects on KATP activity (14), yet presumably supplying cells with elevated decanoate for 24 h would bring about similar changes in metabolism as other fats used in this study. Thus, these latest results support the concept that dietary fat may be an important determinant of long-term changes in β-cell excitability via specific acyl-CoA regulation of KATP channel activity. Furthermore, we suggest that disrupting the delicate balance between free and buffered acyl-CoAs in β-cells may directly lead to alterations in β-cell excitability.

Summary
In summary, our data indicate a significant increase in KATP channel activity in intact primary mouse β-cells overexpressing ACSL-1. Consequently, cell excitability in response to glucose and tolbutamide is significantly attenuated in these cells. Our data, therefore, give credence for a novel excitability hypothesis, whereby altered β-cell electrical activity mediated, at least in part, through changes in the opening of KATP channels in response to elevated levels of acyl-CoAs, will contribute to whole islet dysfunction. We propose that in islets of obese individuals and individuals consuming diets rich in saturated fats, this resultant suppression of β-cell excitability by saturated acyl-CoAs may represent an early event that precedes β-cell death and subsequent reductions in β-cell mass via lipotoxicity (53). The recent report (54) showing that aging is associated with increased apoptosis and decreased proliferation of β-cells, leading to loss of β-cell mass, certainly supports our proposal that there are temporal aspects to the demise of the β-cell. If this is indeed the case, then initial secretory dysfunction arising from diet-induced alterations in β-cell excitability may be reversible and provide a window for therapeutic and/or dietary intervention to prevent the otherwise ultimate progression to T2D.


   Acknowledgments
 
We thank D. E. Dixon for the isolation of mouse islets and Dr. G. S. Korbutt for the use of his tissue culture facility. We also thank K. Strynadka, L. Jones, and C. St. Aubin for help with the acyl-CoA measurements.


   Footnotes
 
This work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR, MOP No. 67160). N.J.W. and M.J.R. were supported by Alberta Heritage Foundation for Medical Research (AHFMR) and Canadian Diabetes Association Fellowship and Studentship Awards respectively. P.P.L.L. was funded by a graduate doctoral studentship from the Canadian Digestive Health Foundation and Canadian Institute of Health Research. G.J.S. received salary support from the CIHR Strategic Training Initiative and support from AHFMR and Heart and Stroke Foundation of Canada Fellowships. P.E.L. received salary support as an AHFMR Senior Scholar.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 27, 2008

1 N.J.W. and G.J.S. contributed equally to this work. Back

Abbreviations: ACSL, Acyl-CoA synthetase-1; acyl-CoA, acyl-coenzyme A ester; FFA, free fatty acid; GFP, green fluorescent protein; GSIS, glucose-stimulated insulin-secretion; KATP, ATP-sensitive potassium; Popen, open probability; T2D, type 2 diabetes; Vm, membrane potential.

Received August 16, 2007.

Accepted for publication March 17, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ashcroft F, Rorsman P 2004 Type 2 diabetes mellitus: not quite exciting enough? Hum Mol Genet 13(Spec No 1):R21–R31
  2. Schrauwen P, Westerterp KR 2000 The role of high-fat diets and physical activity in the regulation of body weight. Br J Nutr 84:417–427[Medline]
  3. Astrup A 2001 The role of dietary fat in the prevention and treatment of obesity. Efficacy and safety of low-fat diets. Int J Obes Relat Metab Disord 25(Suppl 1):S46–S50
  4. Salmeron J, Hu FB, Manson JE, Stampfer MJ, Colditz GA, Rimm EB, Willett WC 2001 Dietary fat intake and risk of type 2 diabetes in women. Am J Clin Nutr 73:1019–1026[Abstract/Free Full Text]
  5. Meyer KA, Kushi LH, Jacobs Jr DR, Folsom AR 2001 Dietary fat and incidence of type 2 diabetes in older Iowa women. Diabetes Care 24:1528–1535[Abstract/Free Full Text]
  6. Golay A, Swislocki AL, Chen YD, Jaspan JB, Reaven GM 1986 Effect of obesity on ambient plasma glucose, free fatty acid, insulin, growth hormone, and glucagon concentrations. J Clin Endocrinol Metab 63:481–484[Abstract/Free Full Text]
  7. Golay A, Swislocki AL, Chen YD, Reaven GM 1987 Relationships between plasma-free fatty acid concentration, endogenous glucose production, and fasting hyperglycemia in normal and non-insulin-dependent diabetic individuals. Metabolism 36:692–696[CrossRef][Medline]
  8. Zraika S, Dunlop M, Proietto J, Andrikopoulos S 2002 Effects of free fatty acids on insulin secretion in obesity. Obes Rev 3:103–112[CrossRef][Medline]
  9. Yaney GC, Corkey BE 2003 Fatty acid metabolism and insulin secretion in pancreatic β-cells. Diabetologia 46:1297–1312[CrossRef][Medline]
  10. Corkey BE, Deeney JT, Yaney GC, Tornheim K, Prentki M 2000 The role of long-chain fatty acyl-CoA esters in β-cell signal transduction. J Nutr 130:299S–304S[Abstract/Free Full Text]
  11. Larsson O, Deeney JT, Branstrom R, Berggren PO, Corkey BE 1996 Activation of the ATP-sensitive K+ channel by long chain acyl-CoA. A role in modulation of pancreatic β-cell glucose sensitivity. J Biol Chem 271:10623–10626[Abstract/Free Full Text]
  12. Branstrom R, Leibiger IB, Leibiger B, Corkey BE, Berggren PO, Larsson O 1998 Long chain coenzyme A esters activate the pore-forming subunit (Kir6.2) of the ATP-regulated potassium channel. J Biol Chem 273:31395–31400[Abstract/Free Full Text]
  13. Gribble FM, Proks P, Corkey BE, Ashcroft FM 1998 Mechanism of cloned ATP-sensitive potassium channel activation by oleoyl-CoA. J Biol Chem 273:26383–26387[Abstract/Free Full Text]
  14. Riedel MJ, Light PE 2005 Saturated and cis/trans unsaturated acyl CoA esters differentially regulate wild-type and polymorphic β-cell ATP-sensitive K+ channels. Diabetes 54:2070–2079[Abstract/Free Full Text]
  15. Holman RR 2006 Long-term efficacy of sulfonylureas: a United Kingdom Prospective Diabetes Study perspective. Metabolism 55:S2–S5
  16. Proks P, Reimann F, Green N, Gribble F, Ashcroft F 2002 Sulfonylurea stimulation of insulin secretion. Diabetes 51(Suppl 3):S368–S376
  17. Aguilar-Bryan L, Bryan J 1999 Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev 20:101–135[Abstract/Free Full Text]
  18. Sharma N, Crane A, Gonzalez G, Bryan J, Aguilar-Bryan L 2000 Familial hyperinsulinism and pancreatic β-cell ATP-sensitive potassium channels. Kidney Int 57:803–808[CrossRef][Medline]
  19. Babenko AP, Polak M, Cave H, Busiah K, Czernichow P, Scharfmann R, Bryan J, Aguilar-Bryan L, Vaxillaire M, Froguel P 2006 Activating mutations in the ABCC8 gene in neonatal diabetes mellitus. N Engl J Med 355:456–466[Abstract/Free Full Text]
  20. Ashcroft FM, Rorsman P 2004 Molecular defects in insulin secretion in type-2 diabetes. Rev Endocr Metab Disord 5:135–142[CrossRef][Medline]
  21. Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, Slingerland AS, Howard N, Srinivasan S, Silva JM, Molnes J, Edghill EL, Frayling TM, Temple IK, Mackay D, Shield JP, Sumnik Z, van Rhijn A, Wales JK, Clark P, Gorman S, Aisenberg J, Ellard S, Njolstad PR, Ashcroft FM, Hattersley AT 2004 Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. N Engl J Med 350:1838–1849[Abstract/Free Full Text]
  22. Pearson ER, Flechtner I, Njolstad PR, Malecki MT, Flanagan SE, Larkin B, Ashcroft FM, Klimes I, Codner E, Iotova V, Slingerland AS, Shield J, Robert JJ, Holst JJ, Clark PM, Ellard S, Sovik O, Polak M, Hattersley AT 2006 Switching from insulin to oral sulfonylureas in patients with diabetes due to Kir6.2 mutations. N Engl J Med 355:467–477[Abstract/Free Full Text]
  23. Riedel MJ, Boora P, Steckley D, de Vries G, Light PE 2003 Kir6.2 polymorphisms sensitize beta-cell ATP-sensitive potassium channels to activation by acyl CoAs: a possible cellular mechanism for increased susceptibility to type 2 diabetes? Diabetes 52:2630–2635[Abstract/Free Full Text]
  24. Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD 1988 Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020–1024[Abstract]
  25. Corkey BE 1988 Analysis of acyl-coenzyme A esters in biological samples. Methods Enzymol 166:55–70[Medline]
  26. Prentki M, Corkey BE 1996 Are the β-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]
  27. Shumilina E, Klocker N, Korniychuk G, Rapedius M, Lang F, Baukrowitz T 2006 Cytoplasmic accumulation of long-chain coenzyme A esters activates KATP and inhibits Kir2.1 channels. J Physiol 575:433–442[Abstract/Free Full Text]
  28. Larson TR, Graham IA 2001 Technical advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues. Plant J 25:115–125[CrossRef][Medline]
  29. Elmi A, Idahl LA, Sehlin J 2000 Relationships between the Na+/K+ pump and ATP and ADP content in mouse pancreatic islets: effects of meglitinide and glibenclamide. Br J Pharmacol 131:1700–1706[CrossRef][Medline]
  30. Branstrom R, Aspinwall CA, Valimaki S, Ostensson CG, Tibell A, Eckhard M, Brandhorst H, Corkey BE, Berggren PO, Larsson O 2004 Long-chain CoA esters activate human pancreatic β-cell KATP channels: potential role in type 2 diabetes. Diabetologia 47:277–283[CrossRef][Medline]
  31. Manning Fox JE, Gyulkhandanyan AV, Satin LS, Wheeler MB 2006 Oscillatory membrane potential response to glucose in islet β-cells: a comparison of islet-cell electrical activity in mouse and rat. Endocrinology 147:4655–4663[CrossRef][Medline]
  32. Sakura H, Ashcroft SJ, Terauchi Y, Kadowaki T, Ashcroft FM 1998 Glucose modulation of ATP-sensitive K-currents in wild-type, homozygous and heterozygous glucokinase knock-out mice. Diabetologia 41:654–659[CrossRef][Medline]
  33. Zhang M, Goforth P, Bertram R, Sherman A, Satin L 2003 The Ca2+ dynamics of isolated mouse β-cells and islets: implications for mathematical models. Biophys J 84:2852–2870[Medline]
  34. Riedel MJ, Baczko I, Searle GJ, Webster N, Fercho M, Jones L, Lang J, Lytton J, Dyck JR, Light PE 2006 Metabolic regulation of sodium-calcium exchange by intracellular acyl CoAs. EMBO J 25:4605–4614[CrossRef][Medline]
  35. Warnotte C, Gilon P, Nenquin M, Henquin JC 1994 Mechanisms of the stimulation of insulin release by saturated fatty acids. A study of palmitate effects in mouse β-cells. Diabetes 43:703–711[Abstract]
  36. Gloyn AL, Weedon MN, Owen KR, Turner MJ, Knight BA, Hitman G, Walker M, Levy JC, Sampson M, Halford S, McCarthy MI, Hattersley AT, Frayling TM 2003 Large-scale association studies of variants in genes encoding the pancreatic β-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) confirm that the KCNJ11 E23K variant is associated with type 2 diabetes. Diabetes 52:568–572[Abstract/Free Full Text]
  37. Florez JC, Burtt N, de Bakker PI, Almgren P, Tuomi T, Holmkvist J, Gaudet D, Hudson TJ, Schaffner SF, Daly MJ, Hirschhorn JN, Groop L, Altshuler D 2004 Haplotype structure and genotype-phenotype correlations of the sulfonylurea receptor and the islet ATP-sensitive potassium channel gene region. Diabetes 53:1360–1368[Abstract/Free Full Text]
  38. Hansen SK, Nielsen EM, Ek J, Andersen G, Glumer C, Carstensen B, Mouritzen P, Drivsholm T, Borch-Johnsen K, Jorgensen T, Hansen T, Pedersen O 2005 Analysis of separate and combined effects of common variation in KCNJ11 and PPARG on risk of type 2 diabetes. J Clin Endocrinol Metab 90:3629–3637[Abstract/Free Full Text]
  39. Hani EH, Boutin P, Durand E, Inoue H, Permutt MA, Velho G, Froguel P 1998 Missense mutations in the pancreatic islet β-cell inwardly rectifying K+ channel gene (KIR6.2/BIR): a meta-analysis suggests a role in the polygenic basis of type II diabetes mellitus in Caucasians. Diabetologia 41:1511–1515[CrossRef][Medline]
  40. Nielsen EM, Hansen L, Carstensen B, Echwald SM, Drivsholm T, Glumer C, Thorsteinsson B, Borch-Johnsen K, Hansen T, Pedersen O 2003 The E23K variant of Kir6.2 associates with impaired post-OGTT serum insulin response and increased risk of type 2 diabetes. Diabetes 52:573–577[Abstract/Free Full Text]
  41. Del Guerra S, Lupi R, Marselli L, Masini M, Bugliani M, Sbrana S, Torri S, Pollera M, Boggi U, Mosca F, Del Prato S, Marchetti P 2005 Functional and molecular defects of pancreatic islets in human type 2 diabetes. Diabetes 54:727–735[Abstract/Free Full Text]
  42. Lupi R, Del Guerra S, Fierabracci V, Marselli L, Novelli M, Patane G, Boggi U, Mosca F, Piro S, Del Prato S, Marchetti P 2002 Lipotoxicity in human pancreatic islets and the protective effect of metformin. Diabetes 51(Suppl 1):S134–S137
  43. Sesti G, Laratta E, Cardellini M, Andreozzi F, Del Guerra S, Irace C, Gnasso A, Grupillo M, Lauro R, Hribal ML, Perticone F, Marchetti P 2006 The E23K variant of KCNJ11 encoding the pancreatic β-cell adenosine 5'-triphosphate-sensitive potassium channel subunit Kir6.2 is associated with an increased risk of secondary failure to sulfonylurea in patients with type 2 diabetes. J Clin Endocrinol Metab 91:2334–2339[Abstract/Free Full Text]
  44. Riedel MJ, Steckley DC, Light PE 2005 Current status of the E23K Kir6.2 polymorphism: implications for type-2 diabetes. Hum Genet 116:133–145[CrossRef][Medline]
  45. Lupi R, Bugliani M, Del Guerra S, Del Prato S, Marchetti P, Boggi U, Filipponi F, Mosca F 2006 Transcription factors of β-cell differentiation and maturation in isolated human islets: effects of high glucose, high free fatty acids and type 2 diabetes. Nutr Metab Cardiovasc Dis 16:e7–e8
  46. Prentki M, Joly E, El Assaad W, Roduit R 2002 Malonyl-CoA signaling, lipid partitioning, and glucolipotoxicity: role in β-cell adaptation and failure in the etiology of diabetes. Diabetes 51(Suppl 3):S405–S413
  47. Fatehi-Hassanabad Z, Chan CB 2005 Transcriptional regulation of lipid metabolism by fatty acids: a key determinant of pancreatic β-cell function. Nutr Metab (Lond) 2:1[CrossRef][Medline]
  48. Dobbins RL, Szczepaniak LS, Myhill J, Tamura Y, Uchino H, Giacca A, McGarry JD 2002 The composition of dietary fat directly influences glucose-stimulated insulin secretion in rats. Diabetes 51:1825–1833[Abstract/Free Full Text]
  49. Stein DT, Stevenson BE, Chester MW, Basit M, Daniels MB, Turley SD, McGarry JD 1997 The insulinotropic potency of fatty acids is influenced profoundly by their chain length and degree of saturation. J Clin Invest 100:398–403[Medline]
  50. Mason TM, Goh T, Tchipashvili V, Sandhu H, Gupta N, Lewis GF, Giacca A 1999 Prolonged elevation of plasma free fatty acids desensitizes the insulin secretory response to glucose in vivo in rats. Diabetes 48:524–530[Abstract]
  51. Sako Y, Grill VE 1990 A 48-hour lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and B cell oxidation through a process likely coupled to fatty acid oxidation. Endocrinology 127:1580–1589[Abstract/Free Full Text]
  52. Nolan CJ, Madiraju MS, Delghingaro-Augusto V, Peyot ML, Prentki M 2006 Fatty acid signaling in the β-cell and insulin secretion. Diabetes 55(Suppl 2):S16–S23
  53. Maedler K, Spinas GA, Dyntar D, Moritz W, Kaiser N, Donath MY 2001 Distinct effects of saturated and monounsaturated fatty acids on β-cell turnover and function. Diabetes 50:69–76[Abstract/Free Full Text]
  54. Maedler K, Schumann DM, Schulthess F, Oberholzer J, Bosco D, Berney T, Donath MY 2006 Aging correlates with decreased β-cell proliferative capacity and enhanced sensitivity to apoptosis: a potential role for Fas and pancreatic duodenal homeobox-1. Diabetes 55:2455–2462[Abstract/Free Full Text]




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