Palmitate increases L-type Ca²⁺ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

Abstract

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 mm, bound to fatty acid-free albumin) to intact islets exposed to 15 mm glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 mm) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 mm palmitate, an effect that was abolished in the presence of 10 μm isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.

Type-2 diabetes involves disturbances in both glucose and lipid metabolism. Diabetic subjects display an elevation of circulating free fatty acids (FFAs) leading to perturbation of insulin action in peripheral tissues but also to impaired glucose-stimulated insulin secretion (GSIS), the two salient features of the disease (for review, see Boden & Shulman, 2002). However, despite the unfavourable effects believed to be associated with the chronic exposure to elevated FFA levels associated with type-2 diabetes, several studies suggest that a lipid-derived signal is required for a normal secretory response. For instance, depletion of lipids from normal rat islets by chronic hyperleptinaemia abolishes GSIS (Koyama et al. 1997), a response that is re-instated by addition of FFAs. Moreover, a perfused pancreas preparation from fasted rats exhibits an abrogation of GSIS (Stein et al. 1996), which can be restored by adding FFA to the perfusate. Further support for the idea that a lipid signal may contribute to the physiological glucose responsiveness of pancreatic β-cells comes from the observation that short-term exposure of islets to FFAs potentiates GSIS (Malaisse & Malaisse-Lagae, 1968; Goberna et al. 1974; Campillo et al. 1979; Warnotte et al. 1994). The nature of this signal is as yet undetermined but among suggested candidates are malonyl-CoA and long-chain acyl-CoA (LC-CoA; Prentki & Corkey, 1996) as well as diacylglycerol (Corkey et al. 1989; Alcazar et al. 1997).

Previous experimental data suggest that a metabolite derived from exogenously added FFAs rather than the fatty acid itself exerts the potentiating effect (Vara & Tamarit-Rodriguez, 1986; Prentki & Corkey, 1996; Alcazar et al. 1997; Yaney et al. 2000; Thams & Capito, 2001). Lipid-derived intracellular second messengers have been proposed to stimulate the activity of various protein kinase C isoforms (Alcazar et al. 1997; Yaney et al. 2000; Thams & Capito, 2001) or to interact with ion channel activity (Branstrom et al. 1998; Gribble et al. 1998; Baukrowitz & Fakler, 2000). It has also been demonstrated that a stimulatory glucose concentration is a prerequisite for the ability of FFAs to potentiate insulin secretion (Goberna et al. 1974; Prentki et al. 1992), possibly indicating a ‘switch’ between glucose metabolism and β-oxidation of lipids. According to this hypothesis, stimulation of β-cells with glucose shifts fatty acid metabolism toward esterification and thus generation of cytosolic lipid-derived metabolites mediating the effects of FFAs on GSIS (Berne, 1975; Tamarit-Rodriguez et al. 1984). Finally, there is evidence that the effects of saturated fatty acids on insulin secretion are secondary to an increase in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i; Warnotte et al. 1994; Remizov et al. 2003) and that the concentration of free rather than bound FFA determines the potency on insulin release (Warnotte et al. 1994).

Insulin secretion from pancreatic β-cells can be stimulated by intracellular signals generated via metabolism of a variety of nutrients, whereof the most important is glucose. Glucose is taken up via glucose transporter 2 (Glut2) and its metabolism increases cytoplasmic ATP at the expense of ADP. This leads to closure of ATP-regulated potassium channels (K_ATP channels) and subsequent membrane depolarization. Voltage-gated Ca²⁺ channels are activated and the influx of extracellular Ca²⁺ leads to release of insulin-containing secretory granules (Ashcroft et al. 1994). A mouse pancreatic β-cell contains approximately 10 000 insulin granules that can be subdivided into different pools depending on their release competence (Eliasson et al. 1997; Olofsson et al. 2002). Granules belonging to the readily releasable pool (RRP) are immediately available for release. Once this pool has been emptied, the secretory capacity of the cell depends on replenishment of the RRP by granules residing in a reserve pool. The latter type of granules are not immediately available for release and must undergo a series of chemical modifications and perhaps even physical translocation within the cell to gain release competence (Eliasson et al. 1997; Olofsson et al. 2002).

In this study we have investigated the effect of palmitate, the most abundant saturated fatty acid in plasma, on pancreatic β-cell function in an attempt to determine the cellular site(s) of action. Effects on insulin release were correlated to changes of [Ca²⁺]_i, membrane whole-cell currents and measurements of cell capacitance. We report that palmitate potentiates GSIS via activation of voltage-sensitive L-type Ca²⁺ channels as well as by increasing the size of the RRP.

Methods

Islet and cell preparation

Female NMRI mice (Bomholtgaard, Ry, Denmark), fed a normal diet ad libitum, were killed by cervical dislocation and pancreatic islets were isolated by collagenase P digestion (Boehringer Mannheim, Bromma, Sweden). Care and use of animals was approved by the ethical committee of Lund University. The islets were cultured overnight in RPMI 1640 (SVA, Uppsala, Sweden) supplemented with 10% calf serum, 100 U ml⁻¹ penicillin and 10 μg ml⁻¹ streptomycin (all from Life Technologies, Täby, Sweden). Single β-cells were generated by shaking freshly isolated islets at low extracellular Ca²⁺ as previously described (Eliasson et al. 1997). Dispersed cells were plated on plastic Petri dishes (Nunc, Denmark) and maintained in culture for up to 2 days.

Preparation of fatty acid–BSA solutions

Palmitate was prepared in a solution bound to fatty acid-free bovine serum albumin (BSA; Boheringer Mannheim). Palmitate (Sigma) was dissolved in 95% ethanol and stoichiometric amounts of NaOH were added. The solution was dried using nitrogen gas; water was subsequently added and the solution heated to create a hot soap. The solution was stirred and BSA was added to a final concentration of 10% w/v. The pH was set to 7.4 (NaOH). The palmitate–BSA stock was aliquoted and stored at −20°C. Before use, the stock was diluted 1:10 in the appropriate extracellular solution to a total concentration of 0.5 or 1 mm palmitate. Using the stepwise equilibrium method described elsewhere (Spector et al. 1971), we estimate that 0.5 mm palmitate in the presence of 1% w/v BSA corresponds to 26 nm free fatty acid. Due to a high ratio between palmitate and BSA, it is difficult to accurately approximate the free concentration of palmitate when the total concentration was 1 mm but the free concentration is likely to be 10–100 μm (M. Sörhede-Winzell, personal communication, 2004). This great difference in unbound concentration for a moderate increase in total lipid concentration is due to multiple FFA binding sites on albumin with diverse affinity for the FFA (Richieri et al. 1993). All extracellular solutions used in the experiments were prepared 10% more concentrated to allow for addition of the palmitate stock; water was added to adjust the concentration under control conditions. BSA (1% w/v) was always included under control conditions.

Measurements of [Ca²⁺]_i

Changes in [Ca²⁺]_i were recorded by dual-wavelength microfluorimetry (Grynkiewicz et al. 1985). Most [Ca²⁺]_i measurements (Figs 1–3) were carried out in intact islets rather than single cells because the β-cell [Ca²⁺]_i oscillations undergo profound changes upon islet dissociation (Jonkers et al. 1999). However, one series was performed using isolated cells (Fig. 6). Briefly, intact islets or dispersed cells were loaded with 3 μm fura-2 in the presence of 0.007% w/v pluronic acid (Molecular Probes, Leiden, Netherlands) for 40 min at 37°C. During the experiments, the islet was held in place in the experimental chamber by a heat-polished glass pipette. Dispersed cells were plated on glass Petri dishes (MatTek Corporation, USA). When islets were utilized, the chamber was continuously superfused with a solution containing (mm): 140 NaCl, 3.6 KCl, 2 NaHCO₃, 0.5 NaH₂PO₄, 0.5 MgSO₄, 5 Hepes (pH 7.4 with NaOH), 2.6 CaCl₂ and glucose as indicated (solution EC-1). Measurements using dispersed cells were performed with a solution containing (mm): 138 NaCl, 5.6 KCl, 2.6 CaCl₂, 1.2 MgCl₂, 5 Hepes (pH 7.4 with NaOH) and glucose as specified (solution EC-2). The latter medium was used to allow direct comparison with the electrophysiological measurements, which are best performed in solution EC-2 as sealing is more difficult in the bicarbonate-containing solution. When stimulation involved elevation of the extracellular KCl concentration, the amount of NaCl was correspondingly reduced to maintain iso-osmolarity. The measurements were carried out using a microfluorimetry system (D104, PTI, Monmouth Junction, NJ, USA) and islet cells were studied in an optical plane at the lower surface of the islets. The fluorophore was excited alternately at 350 nm and 380 nm and emitted light was collected at 510 nm. The fluorescence (F) ratio, F₃₅₀/F₃₈₀ was determined at a ratio frequency of 10 Hz. The [Ca²⁺]_i was calculated using eqn (5) of Grynkiewicz et al.(1985) and a K_d of 224 nm. The maximum ratio (R_max) was achieved by addition of ∼100 μm ionomycin in the presence of 10 mm CaCl₂ at the end of each experiment. Background subtraction was performed after quenching of the fura-2 signal with 1 mm MnCl₂. The measurements were carried out at 32°C to allow comparison with the electrophysiological data.

Figure 1. The effect of palmitate on [Ca²⁺]_i in intact islets.

A, changes in [Ca²⁺]_i upon addition of 1 mm palmitate to an islet stimulated with 15 mm glucose. Note that palmitate in this islet, in addition to the sustained increase also generated a transient elevation in [Ca²⁺]_i. B, example of an experiment in which the effects of palmitate only resulted in a sustained elevation of [Ca²⁺]_i. Note reversibility of effect. The recordings in A and B are representative of recordings from 5 and 13 islets, respectively.

Figure 3. The effects of palmitate on [Ca²⁺]_i are not mediated by elevated intracellular cAMP levels.

Addition of 1 mm palmitate to an islet already exposed to 15 mm glucose and 10 μm forskolin increased the frequency of the oscillations as well as the average [Ca²⁺]_i. The recording is representative of 7 different islets.

Figure 6. Dual effects of palmitate on [Ca²⁺]_i in single β-cells.

Changes in [Ca²⁺]_i in response to stimulation with 75 mm K⁺ in isolated β-cells exposed to 0, 0.5 mm or 1 mm palmitate as indicated. The traces are representative for 7 recordings in each group.

Electrophysiology

Patch pipettes were prepared from borosilicate glass capillaries coated with Sylgard and heat-polished prior to use. The pipette resistance ranged between 3 and 5 MΩ when filled with pipette-filling solution and access resistance during the recordings was <50 MΩ or <10 MΩ in the perforated patch and standard whole-cell configurations, respectively. Only experiments with stable access resistance and small leak currents (<50 pA) were used. The seal resistance was typically >1 GΩ.

Whole-cell currents and exocytosis were recorded using an EPC-9 patch-clamp amplifier (HEKA Electronics, Lambrecht/Pfalz, Germany) and Pulse software (version 8.50). Most electrophysiological experiments were carried out in the perforated patch whole-cell configuration, in which the cell interior is unperturbed and cell metabolism maintained. Single dispersed cells were used and β-cells were functionally identified by inactivation of the Na⁺ current as described elsewhere (Gopel et al. 1999). Whole-cell currents were recorded in solution EC-2 supplemented with glucose as indicated. In some experiments (Figs 4, 5 and 7), Ca²⁺ currents were recorded using 2.6 mm Ba²⁺ as the charge carrier (Ca²⁺ omitted) to prevent Ca²⁺-mediated inactivation of the channels and to increase the magnitude of the current. In these experiments, solution EC-2 was also supplemented with 20 mm TEA (tetraethylammonium chloride) to block delayed rectifier K⁺ channels (NaCl was correspondingly reduced to maintain iso-osmolarity). The pipette-filling solution used in these experiments consisted of (mm): 76 Cs₂SO₄, 10 NaCl, 10 KCl, 1 MgCl₂, and 5 Hepes (pH 7.35 with CsOH). Recordings of K_ATP currents were performed in solution EC-2 and using an intracellular solution composed of (mm): 76 K₂SO₄, 10 NaCl, 10 KCl, 1 MgCl₂, and 5 Hepes (pH 7.35 with KOH).

Figure 4. A high concentration of palmitate inhibits β-cell Ca²⁺ currents.

A, current (I)—voltage (V) relationship for Ca²⁺ currents recorded before (grey trace) and 6 min after (black trace) addition of 1 mm palmitate. The curve superimposed on the data points was obtained by approximating the mean values to the equation:

where I is the peak current, G is the whole-cell conductance (control 3.1 ± 0.3 nS; palmitate 2.1 ± 0.2 nS), V_m is the voltage of the depolarizing pulse, V_r is the extrapolated reversal potential (control 53 ± 3 mV; palmitate 50 ± 3 mV), V_h is the membrane potential at which the activation is half-maximal (control −21 ± 2 mV; palmitate −25 ± 2 mV) and k is the slope coefficient (control 6.3 ± 1.2 mV; palmitate 6.3 ± 1.4 mV). Inset, example of Ca²⁺ current evoked by depolarizations to 0 mV before (grey trace) and 6 min after (black trace) addition of 1 mm palmitate. B, voltage-dependent activation of the β-cell Ca²⁺ current. The activation curve was derived by measuring the tail currents observed when returning to the holding potential after depolarizations to different voltages. The data points were approximated to Eq. 1. See text for details. Values in A and B are mean values ± s.e.m. for 7 and 6 experiments, respectively. *P < 0.05; **P < 0.01.

Figure 5. Stimulation of Ca²⁺ currents by a low concentration of palmitate.

A—B, same as in Fig. 4A but instead using 0.5 mm palmitate. Data in A and B are mean values ± s.e.m. of 8 and 9 experiments, respectively.

Figure 7. The stimulatory effects of palmitate are confined to L-type Ca²⁺ channels.

Ca²⁺ currents evoked by depolarizations to −10 mV under control conditions (left), after addition of 10 μm isradipine (middle) and 6 min after inclusion of palmitate in the continued presence of isradipine (right). The traces are representative of results obtained in 7 different cells.

The capacitance measurements (Figs 8 and 9) were carried out in solution EC-2 supplemented with 15 mm glucose, 20 mm TEA, 10 μm forskolin and 500 μm of the K_ATP channel opener diazoxide. The latter was included to prevent incessant glucose-stimulated release of granules from cells not yet voltage-clamped, which might have interfered with the measurements. The intracellular solution was the same as used for recordings of Ca²⁺ currents. The applied sine wave had a frequency of 500 Hz and a peak amplitude of 20 mV.

Figure 8. Palmitate stimulates exocytosis.

A, Ca²⁺ currents (bottom) and capacitance increases (middle) elicited by a train of four 500-ms depolarizations from −70 to 0 mV (top) before (grey traces) and after (black traces) inclusion of 0.5 mm palmitate in the extracellular solution. The experiment is representative of results obtained in 8 different β-cells. B, histogram of the average increase in cell capacitance (δC_m) plotted against the separate depolarizations as well as the total increase in cell capacitance at the end of the train. Data are mean values ± s.e.m. of eight experiments. *P < 0.05; **P < 0.01). C, relationship between cumulative integrated Ca²⁺ entry (ΣQ_Ca) and exocytosis (ΣδC_m) in β-cells under control condtions (grey squares) and 4 min after addition of palmitate (black circles). Data are mean values ± s.e.m. of 6 experiments. The lines superimposed on the data points were obtained by least-squares regression analysis.

Figure 9. Failure of palmitoyl-CoA to mimic effects of palmitate on exocytosis.

A, changes in cell capacitance upon infusion of an intracellular solution containing 0.22 μm free Ca²⁺ under control conditions and with the addition of 1 or 10 μm palmitoyl-CoA as indicated. B, histogram summarizing the average exocytotic speed (ΔC/Δt) achieved upon infusion of buffer solution alone as well as the same solution supplemented with 1 or 10 μm palmitoyl-CoA (p-CoA). Data in A and B are mean values ± s.e.m. of 7–15 experiments. C, histogram summarizing the average increase in cell capacitance (ΔC_m) in response to trains consisting of four 500-ms depolarizations (delivered at 1 Hz) to 0 mV with an intracellular solution supplemented with 0 μm (black bars) or 10 μm (other bars) palmitoyl-CoA. ΔC_m is plotted against the separate depolarizations as well as the total increase in cell capacitance at the end of the train. Data are mean values ± s.e.m. of 9 and 16 experiments.

The effects of palmitoyl-CoA (Sigma) on exocytosis were investigated using the standard whole-cell configuration and capacitance measurements (Fig. 9). These experiments were conducted in solution EC-2 supplemented with 5 mm glucose. The pipette solution consisted of (mm): 125 potassium glutamate, 10 KCl, 10 NaCl, 1 MgCl₂, 5 CaCl₂, 10 EGTA (free Ca²⁺ estimated to be 0.22 μm), 5 Hepes (pH 7.1 with KOH), 3 Mg-ATP and 0.1 cAMP. The intracellular solution was supplemented with 0, 1 or 10 μm palmitoyl-CoA dissolved in water.

The effects of palmitoyl-CoA on depolarization-evoked exocytosis were likewise evaluated using the standard whole-cell approach. These experiments were conducted using solution EC-2 supplemented with 20 mm TEA (equimolar replacement of NaCl) and 5 mm glucose. The pipette solution consisted of (mm): 125 CsOH, 125 glutamate, 10 CsCl, 10 NaCl, 1 MgCl₂, 5 Hepes (pH 7.15 with KOH), 3 Mg-ATP, 0.1 cAMP, 25 μm EGTA and 0 or 10 μm palmitoyl-CoA. Stimulation commenced after >3 min to allow complete wash-in of palmitoyl-CoA. The holding potential was held at −70 mV and the bath temperature was 32°C in all electrophysiological experiments.

Measurements of insulin secretion

Groups of 10–12 islets were pre-incubated at 37°C for 30 min in EC-1 supplemented with 1 mm glucose and 1 mg ml⁻¹ albumin (Sigma). The pre-incubation medium was removed and 1 ml of the experimental solution was added. After 60 min of incubation at 37°C, 0.3 ml solution was removed for analysis of insulin by radioimmunoassay (Heding, 1966). The incubation medium consisted of solution EC-1. Talbutamide, diazoxide, forskolin (all sigma) and isradipine (kind gift from Dr J. Striessing) were included as indicated (see figure legends).

Data analysis

Data are presented as mean values ± s.e.m. for the indicated number of experiments. The significance of difference between two means was evaluated using Student's t test, paired or unpaired as appropriate.

Results

Palmitate increases [Ca²⁺]_i by a K_ATP channel- and cAMP-independent mechanism

Figure 1A shows a recording of [Ca²⁺]_i in an islet exposed to 15 mm glucose. Introduction of the sugar resulted in a prominent initial increase followed by a series of [Ca²⁺]_i oscillations from a plateau level slightly elevated with respect to that observed in the presence of a non-stimulatory glucose concentration (5 mm). These oscillations reflect the glucose-induced bursting electrical activity in β-cells within intact islets (Nadal et al. 1999) that characteristically consists of a protracted initial period of electrical activity followed by shorter bursts of action potentials (Valdeolmillos et al. 1989). As depicted in Fig. 1A and B, addition of 1 mm palmitate to an islet already exposed to 15 mm glucose elicited a sustained increase in [Ca²⁺]_i. The average [Ca²⁺]_i (based on the calculated time average) increased by 29%, from 276 ± 12 nm in the presence of glucose alone to 356 ± 17 nm following addition of the FFA (P < 0.01).

In 5 out of 13 islets, inclusion of palmitate elicited, in addition to the sustained elevation, a transient increase which subsided to a new plateau slightly above that observed in the presence of glucose alone (Fig. 1A). In the remaining eight cells, [Ca²⁺]_i rose to the new plateau without the transient increase (Fig. 1B). The frequency of the oscillations (peak-to-peak) was 1.4 ± 0.3 min⁻¹ under control conditions and increased to 2.5 ± 0.7 min⁻¹ in the presence of 1 mm palmitate (P < 0.001). In islets exhibiting an irregular oscillatory pattern, the oscillations became more regular in the presence of palmitate (not shown). The effects of palmitate were fully reversible upon removal of the FFA (Fig. 1B).

We next investigated whether the effects of palmitate involved closure of K_ATP channels. Palmitate remained capable of increasing [Ca²⁺]_i in 6 out of 7 islets exposed to the K_ATP channel inhibitor tolbutamide (Fig. 2A) Addition of 400 μm tolbutamide generated an initial peak response after which [Ca²⁺]_i stabilized at a mean concentration of 382 ± 31 nm (n = 7). Subsequent inclusion of 1 mm palmitate in the extracellular medium transiently increased [Ca²⁺]_i to 468 ± 30 nm (n = 7; P < 0.01). We also examined the effects of palmitate in β-cells clamped at a depolarized membrane potential (∼−30 mV; Atwater et al. 1979) by a combination of 15 mm glucose, 500 μm diaozoxide and 30 mm KCl (Fig. 2B). The steady-state [Ca²⁺]_i in the presence of 500 μm diazoxide and 30 mm KCl averaged 474 ± 35 nm, and rose to 595 ± 47 nm (P < 0.001) after addition of 1 mm palmitate (n = 8). Again, [Ca²⁺]_i declined spontaneously towards the level noted before inclusion of the FFA. We attribute the spontaneous decline in [Ca²⁺]_i to Ca²⁺-mediated inactivation of the Ca²⁺ channels.

Figure 2. Palmitate modulates [Ca²⁺]_i independently of K_ATP channel closure.

A, effects of palmitate (1 mm) when added in the simultaneous presence of 400 μm tolbutamide and 15 mm glucose. B, effects of palmitate (1 mm) when added in the simultaneous presence of 15 mm glucose, 30 mm KCl and 500 μm diazoxide. Traces in A and B are representative of 6 and 8 experiments, respectively.

Since the effects of palmitate on the [Ca²⁺]_i resembled those previously obtained in membrane potential recordings from islets stimulated with cAMP-elevating agents like forskolin (increased burst duration, frequency and amplitude; Henquin et al. 1983; Ikeuchi & Cook, 1984; Eddlestone et al. 1985; Gromada et al. 1997) we tested whether the effects of palmitate were mediated via increased cAMP levels (Fig. 3). Islets exposed to 10 μm forskolin in the presence of 15 mm glucose oscillated regularly with a frequency of 2.4 ± 0.2 min⁻¹ and the mean [Ca²⁺]_i (calculated time average) amounted to 223 ± 15 nm. These values were not significantly different from that observed in the presence of 15 mm glucose alone. Inclusion of 1 mm palmitate in the extracellular forskolin-containing medium still increased the frequency of the oscillations to 3.5 ± 0.2 min⁻¹ (P < 0.05, an increase of 46%) and [Ca²⁺]_i to a stable 288 ± 19 nm (P = 0.01, an increase of 29%). Thus, it appears that palmitate and cAMP-increasing agents such as forskolin modulate β-cell electrical activity and [Ca²⁺]_i by distinct mechanisms.

We considered whether palmitate could increase [Ca²⁺]_i by mobilization from intracellular Ca²⁺ stores. However, in 4 out of 5 experiments, palmitate was still able to increase [Ca²⁺]_i in islets pre-treated with 5 μm of the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (not shown). In addition, when calcium was omitted from the extracellular solution (0.5 mm EGTA), palmitate was unable to increase [Ca²⁺]_i (n = 5; not shown). Collectively, these results suggest that the effects of palmitate are not mediated by mobilization of intracellular Ca²⁺ or via closure of K_ATP channels.

Dual effect of palmitate on whole-cell Ca²⁺ currents

We measured whole-cell calcium currents in single β-cells during depolarizations from a holding potential of −70 mV to membrane potentials between −50 and +60 mV (Figs 4 and 5). Seals were established in a Ca²⁺-containing solution that was thereafter replaced with a medium containing Ba²⁺. The solution was permitted to wash in until there was no change in the size of the current between two successive current (I)–voltage (V) protocols separated by a 2 min time interval (∼5 min after solution replacement). Contrary to our expectations, the peak current at 0 mV decreased by ∼35% 6 min after addition of 1 mm palmitate in 7 out of 11 experiments (Fig. 4A; P < 0.01). The decrease was of similar magnitude when measured 4 min after addition of palmitate (P = 0.01). In 4 out of 6 experiments where removal of palmitate was attempted, the inhibitory effect of the FFA on Ca²⁺ current amplitude was reversible. In the remaining four experiments, palmitate increased rather than decreased the Ca²⁺ current. The increase was 34 ± 7% and 24 ± 8% at −20 mV and −10 mV, respectively (P < 0.05). When all 11 experiments were analysed together, a mean decrease by 18 ± 9% was observed at 0 mV (P < 0.05).

The reduction of the peak Ca²⁺ current correlated with a shift in the gating of the Ca²⁺ channels determined by tail current analysis. The data points were approximated to the equation:

(1)

where I/I_max is the relationship between the relative current amplitude and the voltage of depolarization (V_m), V_h is the membrane potential at which I/I_max equals 0.5 and k_h is the steepness coefficient. The average current amplitude for depolarizations between +40 mV and +60 mV was taken to represent the maximum tail current (I_max). Values of V_h and k_h were −11 ± 2 mV and 14 ± 2 mV under control conditions and −17 ± 2 mV and 12 ± 1 mV in the presence of the FFA, respectively (Fig. 4B). The shift of ∼6 mV (P < 0.05) suggests that a smaller depolarization is required to activate the Ca²⁺ channels to the same extent in the presence of the FFA than under control conditions. The reduction in Ca²⁺ channel activity is thus balanced by the shift in Ca²⁺ channel activation and this may be the reason why no inhibitory effect of palmitate on Ca²⁺ current amplitude was detectable at voltages up to −20 mV (the peak of the β-cell action potential; Henquin & Meissner, 1984). Mouse β-cells are known to contain R- and P/Q-type Ca²⁺ channels in addition to the L-type Ca²⁺ channels (Schulla et al. 2003). We therefore attribute the apparent leftward shift in the β-cell Ca²⁺ current activation to inhibition of high-voltage-activated L-type Ca²⁺ channels, making the contribution of non-L-type Ca²⁺ channels larger. Since these channels activate at more negative voltages, this may account for the apparent leftward shift in the activation properties.

A stimulatory action of palmitate predominates at low concentrations of the FFA

Since 1mm palmitate induced an increase in [Ca²⁺]_i in intact islets but appeared to decrease Ca²⁺ currents in the majority of singleβ-cells and given the variable effects on Ca²⁺ current amplitude, we hypothesized that the efficient concentration of FFA within intact islets is perhaps lower than that acting on isolated cells, possibly reflecting diffusion barriers. The stimulatory effect of a low concentration of palmitate could thereby be superseded by an inhibiting action at high concentrations. This is suggested by analogy to the different effects reported when reconstituted L-type Ca²⁺ channels from porcine ventricular sarcolemma were exposed to high and low concentrations of palmitoyl-l-carnitine (Liu & Rosenberg, 1996). We therefore repeated our whole-cell calcium current measurements with 0.5 mm palmitate (free concentration 26 nm). As shown in Fig. 5A, this lower concentration of palmitate consistently increased peak calcium currents. Currents at −20 and −10 mV increased by ∼23% 6 min after addition of palmitate (n = 8; P < 0.05). A significant increase was also evident 4 min after inclusion of the FFA. The lower concentration of palmitate did not result in a shift in Ca²⁺ current gating (Fig. 5B).

To verify the existence of diffusion barriers within the intact pancreatic islet, we measured [Ca²⁺]_i in islets exposed to 15 mm glucose upon addition of 0.5 mm palmitate. Inclusion of this lower concentration of the FFA increased the [Ca²⁺]_i (calculated time average) in 6 out of 9 islets; the increase was 25 ± 4% and thus similar to results obtained with 1 mm palmitate (P < 0.01). However, in 3 out of 9 islets, addition of 0.5 mm palmitate did not affect [Ca²⁺]_i and when the data were added together there was no significant change in [Ca²⁺]_i. Subsequent addition of 1 mm palmitate generated an increase in [Ca²⁺]_i in all islets. The increase was 33 ± 7% compared to control conditions (15 mm glucose and 1% BSA) and thus equivalent to our previously obtained value with this palmitate concentration (P < 0.01; data not shown).

We next examined whether the dual effects of high and low concentrations of palmitate on Ca²⁺ current amplitude determined by electrophysiology, in dispersed cells could also be envisaged by a difference in [Ca²⁺]_i. We measured [Ca²⁺]_i in isolated β-cells exposed to 0, 0.5 or 1 mm palmitate in the presence of 15 mm glucose and 500 μm diazoxide (to suppress spontaneous electrical activity). Four minutes after addition of the FFA, the Ca²⁺ channels were activated by addition of 75 mm KCl to the extracellular medium. Figure 6 shows representative recordings in three cells exposed to the different palmitate concentrations. The [Ca²⁺]_i in the presence of 0, 0.5 or 1 mm palmitate before stimulation with high K⁺ did not differ in the three groups of cells (70 ± 12 nm, 60 ± 5 nm and 70 ± 6 nm, respectively). Under control conditions, the increase in [Ca²⁺]_i elicited by high K⁺ amounted to 760 ± 70 nm. The corresponding value in β-cells exposed to palmitate was 800 ± 50 nm (not statistically different from control) and 620 ± 40 nm (P < 0.05 versus 0.5 mm palmitate) in cells exposed to 0.5 and 1 mm palmitate, respectively. Thus, the inhibitory effect of high palmitate on the Ca²⁺ current documented by electrophysiology can also be detected by [Ca²⁺]_i measurements. These results support the idea that for the same nominal concentration, the effective FFA concentration acting on β-cells in intact islets is lower than that experienced by isolated cells.

Palmitate selectively modulates L-type Ca²⁺ channels

Pancreatic β-cells are equipped with several types of Ca²⁺ channels (Schulla et al. 2003). To determine whether the ability of palmitate to increase whole-cell Ca²⁺ currents resulted from interaction with any type of voltage-gated Ca²⁺ channel or was selective for a certain subtype, we explored the ability of 0.5 mm palmitate to augment Ca²⁺ currents in the presence of isradipine, a selective inhibitor of L-type Ca²⁺ channels (Fig. 7). Addition of 10 μm isradipine decreased the peak Ca²⁺ current at −10 mV by 64 ± 4%(n = 8; P < 0.01). This value is in reasonable agreement with previously reported effects of this dihydropyridine Ca²⁺ channel antagonists (Schulla et al. 2003). Importantly, palmitate was without effect under these conditions, thus indicating that the FFA selectively modulates L-type Ca²⁺ channels.

The effect of palmitate on insulin release

We next investigated the effect of palmitate on insulin release under the same conditions as those used for [Ca²⁺]_i measurements. As indicated in Table 1, 0.5 and 1 mm palmitate increased insulin secretion from isolated islets 1.6- to 2-fold compared to that observed in the presence of 15 mm glucose alone. In islets depolarized by 15 mm glucose and 400 μm tolbutamide, palmitate accelerated insulin release 1.7-fold. In the presence of 15 mm glucose, 500 μm diazoxide and 30 mm KCl, palmitate produced a 1.5-fold enhancement of insulin secretion. Finally, palmitate increased insulin release from islets stimulated by 15 mm glucose and 10 μm forskolin >1.6-fold. Collectively, these data reinforce the conclusion that the potentiating effect of palmitate on insulin release is independent of K_ATP channel closure or elevation of cAMP.

Table 1.

Effect of palmitate on insulin secretion from intact mouse islets

		Insulin secretion (ng islet−1 h−1)
Glucose (mm)	Substance tested (mm)	Control	Palmitate (1 mm)	Palmitate (0.5 mm)
1	—	0.14 ± 0.01	—	—
15	—	1.31 ± 0.12a	2.58 ± 0.12*	2.10 ± 0.15†*
15	Tolbutamide (0.4)	2.05 ± 0.10b	3.44 ± 0.19*c	—
15	Diazoxide (0.5), KCl (30)	2.40 ± 0.14b	3.67 ± 0.53*c	—
15	Forskolin (0.01)	2.15 ± 0.13b	3.56 ± 0.20*c	—

Data are mean values ± s.e.m. of 10–28 experiments.

^aP < 0.001 versus 1 mm glucose;

^*P < 0.001 versus palmitate-free control.

^†P < 0.05 versus 1 mm palmitate;

^bP < 0.001 versus 15 mm glucose alone.

^cP < 0.001 versus 15 mm glucose and 1 mm palmitate.

The effect of palmitate on exocytosis evoked by trains of depolarizations

To elucidate the cellular mechanism by which palmitate enhances insulin secretion, we applied high-resolution capacitance measurements of exocytosis to single β-cells (Fig. 8A). The perforated patch whole-cell technique was used to allow measurements in metabolically intact cells and exocytosis was elicited by trains consisting of four 500-ms depolarizing pulses to 0 mV. The pulses were applied under control conditions and 4 min after inclusion of 0.5 mm palmitate in the extracellular solution. Addition of the FFA augmented the total capacitance increase at the end of the train by ∼75% (Fig. 8B). Interestingly, the capacitance increase evoked by the first depolarization was augmented by ∼200% after addition of the palmitate. Exocytosis during the first depolarization is believed to largely represent the content of the readily releasable pool of granules (RRP; Gillis et al. 1996). The size of the RRP (in fF) can be estimated using the equation:

(2)

where S is the sum of the response to the first (ΔC₁) and the second (ΔC₂) pulse and R is the ratio ΔC₁/ΔC₂ (Gillis et al. 1996). Six cells exhibited clear depression in ΔC₂ under control conditions (R < 0.7) and were therefore included. We thus estimated that the RRP averaged 63 ± 15 fF under control conditions and increased to 148 ± 40 fF after addition of palmitate (P < 0.05). Thus, palmitate increased the size of the RRP >2.3-fold. As illustrated by the histogram in Fig. 8B, the stimulatory action of palmitate is restricted to the first depolarization and little enhancement is seen during the final three pulses. Control experiments were performed in which the BSA-containing control solution was maintained during the entire experiment. Protracted exposure of β-cells to BSA alone was without effect on exocytosis. The total increase in cell capacitance elicited by two successive four-pulse trains, both applied in the presence of BSA and with 4 min in between, averaged 86 ± 35 and 75 ± 19 fF (n = 5).

To investigate how much of the observed palmitate-induced enhancement of exocytosis could be attributed to the stimulation of Ca²⁺ influx (Renstrom et al. 1997), we determined the relationship between Ca²⁺ entry (Q_Ca) and exocytosis (C_m) during the four-pulse trains under control conditions and in the presence of palmitate. These data are summarized in Fig. 8C. It can be seen that the relationships are linear under both conditions. The slope of the curves was 1.8 ± 0.2 and 2.0 ± 0.9 fF pC⁻¹ under control conditions and after addition of palmitate, respectively. Thus, palmitate does not seem to increase the efficacy by which Ca²⁺ elicits exocytosis and the 20% stimulation of Ca²⁺ entry obtained in response to stimulation with palmitate can be expected to contribute only marginally to the enhancement of exocytosis/secretion. This conclusion is in agreement with the small effects on exocytosis obtained in response to variations of the Ca²⁺ current magnitude obtained by low concentrations of Co²⁺ or by increasing the extracellular Ca²⁺ concentration (Renstrom et al. 1996; Barg et al. 2001).

Effect of intracellular application of palmitoyl-CoA on exocytosis

We next studied the effects of the palmitate metabolite palmitoyl-CoA on exocytosis applying the standard whole-cell approach in conjunction with capacitance measurements (Fig. 9A). The standard whole-cell configuration allows intracellular application via the recording electrode. The rate of capacitance increase (ΔC/Δt) was measured in the steady-state when a linear capacitance increase was observed (30–80 s after establishment of the whole-cell configuration) to allow sufficient time for wash-in of palmitoyl-CoA. Exocytosis was unaffected by inclusion of palmitoyl-CoA (1 or 10 μm) in the pipette solution and ΔC/Δt values averaged ∼6 fF s⁻¹ both under control conditions and in the presence of either concentration of palmitoyl-CoA (Fig. 9B).

However, measurements of ΔC/Δt only report the effects of palmitoyl-CoA on the late stages of exocytosis while the possible effects on the RRP cannot be determined by this approach. Therefore, in another series of experiments exocytosis was elicited by trains consisting of four 500-ms depolarizing pulses from −70 to 0 mV. Inclusion of 10 μm palmitoyl-CoA had no significant effect on exocytosis (Fig. 9C). If anything, palmitoyl-CoA tended to reduce exocytosis. Stimulation commenced >3 min after establishment of the whole-cell configuration to ascertain complete wash-in of the lipid metabolite. The failure of palmitoyl-CoA to mimic the effects of palmitate suggests that another metabolite is responsible for the stimulatory action of the FFA on the RRP and exocytosis.

The effect of palmitate on whole-cell K_ATP currents

To ascertain that palmitate potentiates glucose-induced insulin secretion by a K_ATP channel-independent mechanism, we measured whole-cell K_ATP currents in single β-cells by ± 20 mV changes from the holding potential before and after addition of the FFA. The experiments were repeated in 0, 5 and 10 mm glucose and 0.5 or 1 mm palmitate was utilized. There was no significant difference in K_ATP conductance after addition of palmitate in the absence or presence of any of the glucose or palmitate concentrations. The K_ATP conductance in the absence of glucose averaged 1.4 ± 0.2 nS and 1.6 ± 0.2 nS before and 4 min after addition of 0.5 mm palmitate, respectively (n = 6). The corresponding values in the presence of 5 mm glucose were 0.47 ± 0.12 nS and 0.50 ± 0.12 nS and in 10 mm glucose 0.39 ± 0.08 nS and 0.41 ± 0.06 nS (n = 8 and 7). Values obtained 4 min after subsequent inclusion of 1 mm palmitate were 1.3 ± 0.2 nS, 0.61 ± 0.23 nS and 0.42 ± 0.10 nS in the presence of 0, 5 and 10 mm glucose, respectively. The failure of palmitate to detectably affect K_ATP channel activity is in agreement with an earlier report (Warnotte et al. 1994).

Discussion

The ability of FFAs to stimulate insulin secretion is well established but the cellular mechanisms are not well understood and effects on electrical activity (Warnotte et al. 1994), Ca²⁺ influx (Warnotte et al. 1994; Remizov et al. 2003) and the exocytotic machinery (Deeney et al. 2000) have been postulated. We have applied a combination of patch-clamp recordings of membrane currents, capacitance measurements of exocytosis, monitoring of the intracellular Ca²⁺ concentration and hormone release measurements. We demonstrate that acute stimulation with palmitate increases [Ca²⁺]_i via enhanced Ca²⁺ entry through L-type Ca²⁺ channels. We also show that palmitate augments exocytosis and stimulates insulin secretion by increasing the size of the readily releasable pool of granules (RRP). Here we discuss the functional consequences of these results and consider in turn the modulation of the Ca²⁺ current, the effects on exocytosis, the physiological implications and the possible relevance of our data to the understanding of type-2 diabetes.

Palmitate potentiates glucose-induced insulin secretion via a K_ATP channel-independent mechanism

The reports about effects of FFAs or LC-CoAs on K_ATP channels are numerous and variable. There are experiments demonstrating that lipid derived metabolites like phospholipids and LC-CoAs, when applied to the intracellular side of the plasma membrane, activate K_ATP channels (Branstrom et al. 1998; Gribble et al. 1998; Baukrowitz & Fakler, 2000; Branstrom et al. 2004). The latter effects may account for the deleterious effects on β-cell function exerted by long-term elevation of circulating FFAs. However, opening of the K_ATP channels (with resultant membrane repolarization) cannot explain the acute stimulatory action exerted by FFAs. Indeed, acute exogenous application of palmitate has no or little effect on K_ATP currents (Warnotte et al. 1994). We now confirm the former observation by showing that palmitate remains capable of increasing [Ca²⁺]_i and potentiating insulin secretion in islets stimulated with tolbutamide as well as in islets depolarized with high K⁺ in the presence of diazoxide. Furthermore, patch-clamp measurements revealed that palmitate did not affect whole-cell K_ATP channel conductance in single β-cells at any of the tested glucose concentrations (0, 5 and 10 mm). However, a recent study shows that extracellular application of oleic acid increases whole-cell K_ATP conductance in human β-cells (Branstrom et al. 2004) but no details were provided on how oleate was dissolved. Moreover, the increase in conductance correlated with the development of an inward current, a feature not expected for an increased whole-cell K_ATP conductance.

Palmitate increases [Ca²⁺]_i via activation of L-type Ca²⁺ channels

In agreement with previous data, pancreatic β-cells within intact islets respond to glucose with a first large increase in [Ca²⁺]_i followed by regular oscillations between a plateau level and discrete peaks (Valdeolmillos et al. 1989; Nadal et al. 1999). We now show that addition of palmitate leads to a sustained elevation of the [Ca²⁺]_i in islets already exposed to a stimulatory glucose concentration. In a subset (38%) of the islets, an additional transient increase could be observed. The findings that the effects of the FFA on [Ca²⁺]_i persisted after pre-treatment of the islets with the SERCA inhibitor thapsigargin, whereas they were abolished when extracellular Ca²⁺ is absent, reinforce the view that the increase in [Ca²⁺]_i does not depend on mobilization of Ca²⁺ from intracellular stores but principally reflects stimulated entry of extracellular Ca²⁺. This contrasts with a recent report showing that the increase in [Ca²⁺]_i elicited by palmitate in isolated β-cells exposed to 5 mm glucose in part results from mobilization of Ca²⁺ from intracellular stores (Remizov et al. 2003).

Observations similar to ours have previously been reported by others (Warnotte et al. 1994; Remizov et al. 2003) and it was suggested that palmitate exerts an effect on voltage-gated Ca²⁺ channels. However, the latter possibility has remained unpursued and we have therefore measured whole-cell currents through voltage-gated Ca²⁺ channels before and after addition of palmitate. We now demonstrate that 0.5 mm palmitate increases the peak calcium current by ∼23% at voltages between −20 mV and 0 mV. The stimulation of Ca²⁺ entry is attributable to increased activity of L-type Ca²⁺ channels and was abolished in the presence of the dihydropyridine blocker isradipine.

Palmitate stimulates Ca²⁺-dependent β-cell exocytosis

Measurements of cell membrane capacitance demonstrated that palmitate potentiates exocytosis elicited by a train of four 500-ms depolarizations by ∼75% (Fig. 8A and B). This is in good agreement with the 1.5- (50%; diazoxide + high extracellular K⁺) to 2-fold (100%; 15 mm glucose) enhancement of insulin secretion measured by radioimmunoassay. The observed FFA-induced stimulation of exocytosis/secretion depends on enhanced Ca²⁺ influx only to a minor extent and most of the effect (two-thirds) instead appears attributable to a more direct effect on the exocytotic process. Close inspection of the data reveals that whereas the response to the first depolarization was increased 3-fold, there was little stimulation during the latter part of the train (Fig. 8B). This finding suggests that palmitate principally acts by promoting release of granules that have already proceeded into the RRP, an effect that was not accompanied by a corresponding stimulation of granule mobilization (i.e. the supply of new granules for release) as indicated by the lack of effects of the lipid during the latter part of the train. Palmitate will therefore only be able to exert its stimulatory action provided new granules are continuously supplied for release (a process referred to as mobilization or priming) and the latter process may in fact be rate limiting for exocytosis in the long term.

No evidence that the acute effects of palmitate are mediated by generation of palmitoyl-CoA

It has previously been reported that palmitoyl-CoA accelerates insulin secretion from permeabilized HIT T-15 cells measured over 15 min but fails to produce a statistically significant sustained acceleration of exocytosis in NMRI β-cells measured as a stimulated rate of capacitance increase (Deeney et al. 2000). Surprisingly, the latter study revealed an early component of capacitance increase that was apparent very shortly upon establishment of the whole-cell configuration, before solution exchange between the pipette and cell interior has been completed. Here we confirm that palmitoyl-CoA fails to produce a sustained stimulation of the exocytotic rate in primary mouse β-cells (Fig. 9A). Furthermore, when exocytosis was stimulated by a train of depolarizations, inclusion of palmitoyl-CoA had no effect on either the capacitance increase in response to the first depolarizing pulse or the total capacitance increase at the end of the train. These data make it unlikely that palmitoyl-CoA represents the intracellular metabolite that mediates the effects on exocytosis and RRP filling

Physiological significance and possible relevance to diabetes

We report here that palmitate increases L-type Ca²⁺ currents when applied at a low concentration (0.5 mm). The normal FFA level in blood is around 0.5 mm, one-third of which is palmitate. Thus, in healthy individuals, the β-cell may be exposed to FFAs at concentrations stimulating Ca²⁺ channel activity. This raises the possibility that an intracellular lipid-derived signal contributes to the maintenance of the secretory capacity by promoting Ca²⁺ channel opening. We also demonstrate that a lipid signal is involved in potentiating exocytosis, independently of Ca²⁺ channel stimulation. Loss of a lipid-derived signalling molecule may contribute to the fact that islets depleted of lipids exhibit an impaired GSIS (Koyama et al. 1997).

The observation that a higher concentration of palmitate (1 mm) inhibits Ca²⁺ entry in β-cells may be of relevance for the understanding of the pathophysiology of type-2 diabetes. Type-2 diabetes and/or obesity is associated with a 2- to 3-fold elevation of the plasma FFA concentration (Lewis et al. 2002) and the FFA levels are thereby brought dangerously close to those where the inhibitory action of FFAs on Ca²⁺ channel activity predominate. This may potentially give rise to a vicious cycle where a slight inhibition of insulin secretion leads to progressive elevation of plasma FFA levels and further impairment of insulin secretion. We acknowledge that we have so far only investigated the acute effects of palmitate and studies of the long-term effects are necessary to explore this possibility.