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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Long-chain fatty acids acyl coenzyme A esters (LC-CoA) are obligate intermediates of fatty acid metabolism and have been shown to activate KATP channels but to inhibit most other Kir channels (e.g. Kir2.1) by direct channel binding. The activation of KATP channels by elevated levels of LC-CoA may be involved in the pathophysiology of type 2 diabetes, the hypothalamic sensing of circulating fatty acids and the regulation of cardiac KATP channels. However, LC-CoA are effectively buffered in the cytoplasm and it is currently not clear whether their free concentration can reach levels sufficient to affect Kir channels in vivo. Here, we report that extracellular oleic acid complexed with albumin at an unbound concentration of 81 ± 1 nm strongly activated KATP channels and inhibited Kir2.1 channels in Chinese hamster ovary (CHO) cells as well as endogenous Kir currents in human embryonic kidney (HEK293) cells. These effects were only seen in the presence of a high concentration of glucose (25 mm), a condition known to promote the accumulation of LC-CoA by inhibiting their mitochondrial uptake via carnitine-palmitoyl-transferase-1 (CPT1). Accordingly, pharmacological inhibition of CPT1 by etomoxir restored the effects of oleic acid under low glucose conditions. Finally, triacsin C, an inhibitor of the acyl-CoA synthetase, which is necessary for LC-CoA formation, abolished the effects of extracellular oleic acid on the various Kir channels. These results establish the direct regulation of Kir channels by the cytoplasmic accumulation of LC-CoA, which might be of physiological and pathophysiological relevance in a variety of tissues.

Inwardly rectifying potassium (Kir) channels serve diverse functions, ranging from the regulation of the resting membrane potential and the pacing of both cardiomyocytes and neurons to the regulation of insulin secretion and renal K+ transport (Nichols & Lopatin, 1997; Tarasov et al. 2004; Hebert et al. 2005). A characteristic feature of all Kir channels is their regulation by phosphoinositides (mostly PIP2) (Baukrowitz et al. 1998; Huang et al. 1998; Lopes et al. 2002). It is generally accepted that all Kir channels are only active when associated with phosphoinositides and therefore their activity is intimately coupled to the complex cellular metabolism of phosphoinositides (Xie et al. 1999; Baukrowitz & Fakler, 2000; Kobrinsky et al. 2000). Another class of anionic lipids that strongly affect Kir channels are long-chain fatty acids acyl coenzyme A esters (LC-CoA). Increased cytoplasmic LC-CoA levels have been postulated to activate KATP channels in pancreatic β-cells under hyperglycaemic conditions (Larsson et al. 1996; Branstrom et al. 2004) and KATP channels in the heart under ischaemic conditions (Liu et al. 2001), and to regulate neuronal KATP channels as part of a mechanism to sense the concentration of circulating fatty acids in the hypothalamus (Lam et al. 2005).

Both phosphoinositides and LC-CoA presumably bind to the same regulatory lipid-binding site in Kir channels that is composed of basic residues in the cytoplasmic N- and C-terminals (Schulze et al. 2003; Rapedius et al. 2005). However, the functional consequence of binding is quite distinct depending on the Kir channel subtype: whereas PIP2 and LC-CoA cause an increase in open probability of KATP channels and a concomitant decrease in sensitivity to ATP inhibition (Liu et al. 2001; Schulze et al. 2003), LC-CoA exert a marked inhibitory effect on all other tested Kir channels. This inhibition is thought to result from the displacement of the activator PIP2 from its binding site and, thus, LC-CoA can be regarded as competitive antagonists of PIP2 activation (Rapedius et al. 2005).

The physiological significance of the regulation of phosphoinositides has been demonstrated in studies showing that changes in phosphoinositide metabolism directly modulate Kir channels (Baukrowitz & Fakler, 2000; Kobrinsky et al. 2000; Cho et al. 2005) and is underlined by the observation that mutations in Kir channels that impair PIP2 interactions can lead to channelopathies such as Bartter's syndrome, Andersen's syndrome and congenital hyperinsulinism (Lopes et al. 2002; Lin et al. 2006). In contrast, although the effects of exogenous LC-CoA have been studied in detail, it is currently not known whether endogenously formed LC-CoA regulate Kir channel activity in cells. LC-CoA are obligate intermediates of fatty acid metabolism formed from long-chain fatty acids in the cytoplasm and shuttled into the mitochondria for β-oxidation. Conditions that promote the cytoplasmic accumulation of LC-CoA such as hyperglycaemia, ischaemia or hyperlipidaemia markedly increase cellular LC-CoA (Prentki et al. 1992; Larsson et al. 1996; Lam et al. 2005). However, the unbound (free) concentration of LC-CoA in the cytoplasm is effectively buffered by specific acyl-CoA binding proteins and currently unknown (Knudsen et al. 2002). Calculations suggest that the concentration of LC-CoA is below 10 nm under physiological conditions (Knudsen et al. 2002). In excised membrane patches, application of LC-CoA at concentrations between 100 and 1000 nm are necessary to modulate Kir channel activity (Liu et al. 2001; Branstrom et al. 2004; Rapedius et al. 2005). This raises the question of whether cytoplasmic levels of LC-CoA can rise sufficiently to affect Kir channels in vivo.

To gain further insight into the interplay of fatty acid metabolism and Kir channel activity, in the present study we explored the effects of oleic acid on three different types of Kir channels: recombinant KATP channels, Kir2.1 in Chinese hamster ovary (CHO) cells and native Kir channels in HEK293 cells. Our findings show that all Kir channels tested are modulated by long-chain fatty acid metabolism under conditions favouring the accumulation of LC-CoA in the cytoplasm.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Cell culture

Human embryonic kidney (HEK293) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 4.5 g l−1 glucose, 2 mm l-glutamine and 1% penicillin-streptomycin (Gibco, Invitrogen, Karlsruhe, Germany) at 37°C in 5% CO2. CHO cells were grown in DMEM supplemented with 10% fetal calf serum, 1 g l−1 glucose, 2 mm l-glutamine and 1% penicillin-streptomycin (Gibco Invitrogen) at 37°C in 5% CO2. At ∼80% confluence, CHO cells were transfected using the Polyfect reagent (Qiagen GmbH, Hilden, Germany) for the patch-clamp experiments or using Lipofectamin 2000 (Invitrogen, Karlsruhe, Germany) for immunocytochemistry, according to the manufacturer's directions.

Electrophysiology

Whole-cell patch-clamp experiments were performed at room temperature (21°C) in a 0.4-ml chamber with continuous superfusion (1 ml min−1). Borosilicate glass pipettes (tip resistance, 2–4 MΩ; GC 150 TF-10, Clark Medical Instruments, Pangbourne, UK) manufactured using a microprocessor-driven DMZ puller (Zeitz, Augsburg, Germany) were used in combination with an MS 314 (Marzhauser, Wetzlar, Germany) electrical micromanipulator. Currents were recorded in the fast whole-cell, voltage-clamp mode and low-pass filtered at 3 kHz by an EPC-9 amplifier (HEKA, Lambrecht, Germany). Data acquisition and analysis were performed by Pulse software. Whole-cell currents were elicited by 200-ms square-wave voltage pulses from –135 to +15 mV in 10-mV steps delivered each second from a holding potential of –20 mV. To enable rapid measurements of conductance changes during the experiment, voltage ramps were also employed. Typically, the command voltage was varied from –175 to +5 mV over a duration of 400 ms. The initial bath solution contained (mm): NaCl 150, MgCl2 1, CaCl2 2, N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (Hepes)/NaOH 10 and glucose 10 (pH 7.4). To study the acute effect of oleic acid on glucose-depleted cells, 10 mm mannitol was used instead of glucose; the concentration of other components were not modified. At the beginning of the experiment, the initial bath solution was changed to the solutions required to study Kir2.1, Kir6.2 or native Kir current in HEK293 cells (see below). The intracellular pipette solution contained contained (mm): potassium glutamate 130, KCl 10, MgCl2 1, EGTA 1, Hepes/KOH 10 and MgATP 1 (pH 7.2). Oleoyl-CoA (1 μm; Sigma) or palmitoyl-CoA lacking the 3′-ribose phosphate (palmitoyl-CoADephosho) (10 μm; JenaBioScience, Germany) were added to the pipette solution when indicated. Oleic acid (Sigma) was prepared as a stock solution of 164 mm in methanol. To study the long-term effects on Kir channels, oleic acid was added to DMEM growth medium containing either a high (25 mm) or low (5.5 mm) concentration of glucose. For control experiments, methanol (0.1%) was always included. For the acute experiments, oleoyl-CoA, etomoxir (HHAC Labor Dr Heusler GmbH, Stutensee, Germany) and triacsin C (Biomol) were added to the media, as required. To record Kir2.1 channels, the solution contained (mm): KCl 5, NaCl 135, MgCl2 1, CaCl2 2, Hepes/NaOH 10, glucose (high glucose) 25, mannitol (no glucose) 25 and albumin 0.023, pH 7.4. To record KATP channels, the solution contained (mm): KCl 30, NaCl 110, MgCl2 1, CaCl2 2, Hepes/NaOH 10, glucose (high glucose) 25, mannitol (no glucose) 25 and albumin 0.023, pH 7.4; To record endogenous Kir channels, the solution contained (mm): KCl 140, MgCl2 1, CaCl2 2, Hepes/NaOH 10, glucose (high glucose) 25, mannitol (no glucose) 25 and albumin 0.023; pH 7.4.

Original current tracings were depicted after low-pass filtering at 1 kHz. The pipette potential was corrected for the liquid-junction potential between the pipette and bath solution (Barry & Lynch, 1991). The results are reported as means ±s.e.m. of independent experiments (n), where n refers to the number of cells patch clamped. Statistical significance was evaluated using Student's two-tailed paired t test or one-way analysis of variance (ANOVA) as appropriate. P < 0.05 was considered significant.

Gene construction and immunocytochemistry

An N-terminal fusion construct of mKir2.1 (murine Kir2.1) with enhanced green fluorescent protein (EGFP) was designed by inserting the respective cDNA in-frame into the EGFP-C1 eukaryotic expression plasmid (BD Biosciences, Heidelberg, Germany). A haemagglutinin (HA) epitope was introduced into the extracellular domain of mKir2.1 at amino acid position 116 (Stockklausner et al. 2001).

To detect selectively the population of HA-tagged Kir2.1 channels expressed on the cell surface, anti-HA immunocytochemistry was performed in vivo without use of detergents. Antibody incubation was carried out in serum-free medium for 15 min at 37°C for the primary (monoclonal mouse anti-HA, 1 : 100, Santa Cruz Biotechnology, Heidelberg, Germany) and secondary antisera (anti-mouse IgG conjugated to cy-3, 1 : 1000, Dianova, Hamburg, Germany. After washing, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 15 min at 4°C. Cells were imaged with a confocal laser scanning microscope (LSM510, Zeiss) using the following laser and filter settings: EGFP, excitation 488 nm Ar/emission BP(band-pass)505–530 nm; cy-3, excitation 543 nm He/emission LP(long-pass)560 nm. Channel surface expression was quantified by fluorescence intensity measurements of anti-HA immunocytochemistry without use of detergents as described before (Stockklausner & Klocker, 2003). Intensity values of staining from two independent transfections were corrected for background staining and integrated over 10 areas of 0.2116 mm2 each (Scion Image 4.02). Surface fluorescence intensity values were related to the translation efficiencies as determined by EGIP fluorescence intensity. Data were analysed for statistically significant differences using the unpaired Student's t test. Data are given as mean ±s.e.m. in arbitrary units.

Determination of free fatty acid concentrations

Unbound oleic acid concentrations in the samples were measured by FFA Sciences (San Diego, CA, USA) using the acrylodated intestinal fatty acid-binding (ADIFAB) protein method developed by Richieri & Kleinfeld (1992). In brief, the concentration of unbound oleic acid was calculated using the ratio of fluorescence intensities of bound to unbound ADIFAB indicator at 505 and 432 nm, respectively. Measurements of acute effects (e.g. Figs 2 and 3) were made at room temperature whereas the concentration of oleic acid in the DMEM growth medium for the long-term effects (Fig. 4) was determined at 37°C as cells were incubated at this temperature.

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Figure 2. Oleoyl-CoA formedin vivoactivates KATP channels in CHO cells A, schematic diagram depicting cellular fatty acid metabolism. Long-chain fatty acids (LC-FA) taken up by cells are converted to long-chain fatty acids acyl coenzyme A esters (LC-CoA) by the LC-CoA synthetase and then conjugated to carnitine by the carnitine-palmitoyl-transferase-1 (CPT1) before they can be shuttled into the mitochondrial matrix for β-oxidation. The LC-CoA synthetase can be inhibited by triacsin C leading to low levels of LC-CoA. High levels of LC-CoA are expected (i) in the presence of a high glucose concentration that result in the excessive formation of malonyl-CoA inhibiting the CPT1 or (ii) during inhibition of the CPT1 by etomoxir. B, time course of KATP current activity at –135 mV under control conditions (□, n= 10), upon superfusion with oleic acid (81 nm unbound concentration) and 25 mm glucose in the absence (•, n= 10) or in the presence (⋄, n= 6) of 5 μm triacsin C. Current amplitude was normalized to the amplitude obtained immediately after the formation of the whole-cell configuration. C, time course of KATP current activity at –135 mV with oleic acid (81 nm unbound concentration) added to a glucose-free bath solution in the absence (□, n= 10) or in the presence (▴, n= 10) of 100 μm etomoxir. The current amplitude was normalized to the amplitude obtained immediately after the formation of the whole-cell configuration. D, mean KATP current density at –135 mV at the beginning (open bars) and after 10 min of whole-cell recording (filled bars) for the different experimental conditions as described in B and C+, with; –, without. ***P≤ 0.001, two-tailed paired t test. All data show means ±s.e.m.

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Figure 3. Oleoyl-CoA formed in vivo inhibits Kir2.1 currents in CHO cells and endogenous Kir currents in HEK293 cells A, time course of Kir2.1 current activity in CHO cells at -135 mV under control conditions (□, n= 6), upon superfusion with oleic acid (81 nm unbound concentration) and 25 mm glucose in the absence (•, n= 8) or in the presence (⋄, n= 7) of 5 μm triacsin C. Current amplitude was normalized to the amplitude obtained immediately after the formation of whole-cell configuration. B, time course of Kir2.1 current activity at –135 mV upon superfusion with oleic acid (81 nm unbound concentration) added to a glucose-free bath solution in the absence (□, n= 7), or in the presence (▴, n= 5) of 100 μm etomoxir. Current amplitude was normalized to the amplitude obtained immediately after the formation of the whole-cell configuration. C, mean current density at –135 mV measured at the beginning (open bars) and after 10 min of whole-cell recording (filled bars) for the different experimental conditions as described in A and B. +, with; –, without. **P≤ 0.01 and ***P≤ 0.001, two-tailed paired t tests. D, time course of endogenous Kir current activity in HEK293 cells at –135 mV in control conditions (□, n= 4), with oleic acid (81 nm unbound concentration) and 25 mm glucose in the absence (•, n= 8) or in the presence (⋄, n= 4) of 5 μm triacsin C in the bath. Current amplitude was normalized to the amplitude obtained immediately after the formation of the whole-cell configuration. E, time course of endogenous Kir current activity in HEK293 cells at –135 mV with oleic acid (81 nm unbound concentration) added to the glucose-free bath solution in the absence (□, n= 9), or in the presence (▴, n= 6) of 100 μm etomoxir. Current amplitude was normalized to the amplitude obtained immediately after the formation of the whole-cell configuration. F, mean endogenous Kir current amplitude in HEK293 cells at –135 mV at the beginning (open bars) and after 10 min of whole-cell recording (filled bars) for the different experimental conditions as described in D and E. +, with; –, without. *P≤ 0.05, two-tailed paired t test. All data show means ±s.e.m.

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Figure 4. Long-term inhibition of Kir currents by extracellular oleic acids A, time course of the endogenous Kir current activity in HEK293 cells upon incubation with a medium containing oleic acid at an unbound concentration of 18.4 nm (▴, n= 3−9) and under control conditions (□, n= 3–4). B, mean endogenous Kir current amplitude at –135 mV in the HEK293 cells incubated overnight with oleic acid (18.4 nm unbound concentration), glucose (25 mm glucose, ‘–’ represents 5.5 mm glucose), etomoxir (100 μm etomoxir) and triacsin C (5 μm triacsin C). +, with; –, without. *** and ###P≤ 0.001, ANOVA. ***Significantly different (P≤ 0.001, ANOVA) compared to addition of oleic acid to high glucose (25 mm)-containing growth medium; ###significantly different (P≤ 0.001, ANOVA) compared to addition of oleic acid together with etomoxir (100 μm) to low glucose (5.5 mm)-containing growth medium. +, with; –, without. C, mean current densitity in Kir2.1 expressing CHO cells at –135 mV incubated overnight at different conditions as detailed in B. * and #P≤ 0.05, ** and ##P≤ 0.01, ANOVA. * and **significantly different compared to addition of oleic acid to high glucose (25 mm)-containing growth medium; # and ##significantly different compared to addition of oleic acid together with etomoxir (100 μm) to low glucose (5.5 mm)-containing growth medium. +, with; –, without. All values show means ±s.e.m.

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Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Regulation of Kir channels by exogenous oleoyl-CoA in CHO and HEK293 cells

Oleoyl-CoA has opposite effects on KATP and Kir2.1 channels if applied to these channels in inside out patches (Rapedius et al. 2005). To study the effects of oleoyl-CoA on KATP (Kir6.2/SUR2A) and Kir2.1 channels in cells, the channels were expressed in CHO cells and oleoyl-CoA was infused to the cytoplasm via patch pipette in the whole-cell patch-clamp configuration. For KATP channels, 1 mm ATP was included in the pipette solution to prevent channel activation by the washout of ATP. Figure 1A shows that oleoyl-CoA (1 μm) strongly activated whole-cell KATP currents without reaching a steady state during the 10-min recording period. To determine whether the activation was due to a direct effect on the channel, the experiments were repeated with palmitoyl-CoA lacking the 3′ ribose phosphate on the CoA moiety (palmitoyl-CoADephospho), a substance that does not interact with Kir channels (Rapedius et al. 2005). Consistently, addition of 10 μm palmitoyl-CoADephospho did not significantly activate KATP currents, neither did control LC-CoA-free pipette solution (Fig. 1A). Furthermore, pipette application of oleoyl-CoA (1 μm) markedly reduced whole-cell Kir2.1 currents whereas palmitoyl-CoADephospho (10 μm) or a LC-CoA-free pipette solutions had no effect (Fig. 1B). These experiments show that infusion of LC-CoA into the cytoplam activated KATP but inhibited Kir2.1 channels in accordance with the results obtained in excised patches (Rapedius et al. 2005).

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Figure 1. Exogenous oleoyl-CoA activates KATP and inhibits Kir2.1 currents KATP (Kir6.2/SUR2A) and Kir2.1 channels were expressed in CHO cells. A, typical macroscopic current from a KATP-expressing CHO cell in response to a voltage ramp from –175 to +5 mV obtained directly after establishment of the whole-cell configuration (start) and 7 min after dialysing the cytosol with the pipette solution containing 1 μm oleoyl-CoA and 1 mm ATP (left panel). The right panel shows the time course of KATP current activity at –135 mV with 1 μm oleoyl-CoA (•, n= 5), without oleoyl-CoA (□,n= 5) and with 10 μm dephospho-palmitoyl-CoA (▴, n= 9) added to the pipette solution. Current amplitude was normalized to the amplitude obtained immediately after the formation of whole-cell configuration. B, typical macroscopic current from a Kir2.1-expressing CHO cell in response to a voltage ramp from –175 to +5 mV (left panel). The right panel shows the time course of Kir2.1 current activity at –135 mV with 1 μm oleoyl-CoA (•, n= 5), without oleoyl-CoA (□, n= 6) and with 10 μm dephospho-palmitoyl-CoA (▴, n= 4) added to the pipette solution. Current amplitude was normalized to the amplitude obtained immediately after the formation of whole-cell configuration. All data show means ±s.e.m.

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Regulation of Kir channels by LC-CoA formed in the cytoplasm

To address the question of whether LC-CoA produced in the cytoplasm can reach concentrations sufficient to affect the activity of Kir channels, CHO cells expressing KATP and Kir2.1 were exposed to a bath medium containing oleic acid (164 μm) complexed with albumin at a molar ratio of 7 : 1. The concentration of unbound oleic acids in the medium was 81 ± 1 nm as determined by the ADIFAB-fluorescence assay (see Methods). This represents a concentration about 10 times higher than the normal plasma concentration (about 7.5 nm) (Richieri & Kleinfeld, 1995), which has, however has been reported to occur under pathophysiological conditions (Kleinfeld et al. 1996; Kleinfeld & Okada, 2005). Superfusion with this medium had no effect on either KATP channels (Fig. 2C and D) or Kir2.1 channels (Fig. 3B and C). However, if the same experiment was repeated in the presence of 25 mm glucose, strong activation of KATP currents (Fig. 2B and D) and marked inhibition of Kir2.1 currents (51 ± 9%, Fig. 3A and C) reaching a steady state within about 8 min were observed. This outcome is not surprising because high glucose concentrations are known to promote the cytoplasmic accumulation of LC-CoA (Prentki et al. 1992, 2002) via excessive formation of malonyl-CoA which inhibits the enzyme carnitine-palmitoyl-transferase-1 (CPT1), which is necessary for the uptake of LC-CoA into mitochondria (Fig. 2A). Accordingly, in the presence of the CPT1 inhibitor etomoxir (100 μm), the effects of oleic acid on KATP and Kir2.1 currents were also observed in the absence of glucose (Figs 2C and D, and 3B and C). Etomoxir had no effect on KATP or Kir2.1 currents in the absence of oleic acid (data not shown). Finally, blockade of the LC-CoA formation by inhibition of the long-chain fatty acid acyl-CoA synthetase with triacsin C (10 μm) abolished the regulation of KATP and Kir2.1 currents by oleic acid in the presence of a high glucose concentration (Figs 2B and D, and 3A and C) indicating that cytoplasmic oleoyl-CoA is the regulatory factor.

We have recently shown that HEK293 cells express substantial endogenous Kir currents that are inhibited by exogenous oleoyl-CoA (Rapedius et al. 2005). Figure 3DF shows that these endogenous Kir currents were also regulated by oleoyl-CoA formed in the cytoplasm as (i) application of oleic acid (unbound concentration of 81 nm) resulted in a marked inhibition (59 ± 9%) of the Kir currents in the presence of high glucose (25 mm) but not in glucose-starved cell (Fig. 3DF), (ii) etomoxir restored the effect of oleic acid on glucose-starved cells (Fig. 3E and F) and (iii) incubation with triacsin C prevented the effects of superfusion with oleic acid (Fig. 3D and F).

Effects of ‘mildly’ elevated oleic acid levels on Kir channels

In the above experiments, cells were challenged with relatively high concentrations of unbound fatty acids (81 nm) inducing fast changes in Kir channel activity. To test the effect of lower concentrations, cells were incubated in a growth medium containing oleic acid at an unbound concentration of 18.4 ± 0.1 nm as determined by the ADIFAB-fluorescence assay. This condition did not affect endogenous Kir channels in HEK293 cells or Kir2.1 channels in CHO cells acutely; that is, during the time course of the whole cell-recordings (about 20 min, data not shown). However, it is intriguing that a decline of the endogenous Kir current was observed in HEK293 cells that became apparent at 5 h and was maximal at 7 h (Fig. 4A) after addition of oleic acid to the incubation medium, representing a 79% reduction of the Kir current compared to control (Fig. 4A and B). Similar results were obtained for recombinant Kir2.1 currents in CHO cells: 51% of the current was reduced after 8 h of incubation with oleic acid-containing medium (Fig. 4C). As with the acute effects, these long-term effects were only seen in the presence of a high glucose concentration (25 mm) but not in the presence of triacsin C (Fig. 4B and C). Furthermore, with a lower glucose concentration (5.5 mm), co-application of etomoxir (100 μm) restored the inhibitory effect of oleic acid on Kir currents in HEK293 and CHO cells (Fig. 4B and C). These results clearly indicated that the effects of oleic acid were mediated by the formation of oleoyl-CoA induced by extracellular oleic acid. However, the slow time course might point to an indirect mechanism such as a change in surface channel expression. To address the latter possibility, Kir2.1 surface expression was quantified by extracellular epitope tagging (see Methods). Relative surface expression of Kir2.1 channel protein was 0.78 ± 0.11 arbitrary units (n= 10) after incubation with oleic acid for 8 h, a value not significantly different from the control value (0.61 ± 0.05 arbitrary units; n= 10) measured without oleic acid (Fig. 5). Thus, the decline of the Kir currents does not result from a decreased membrane expression of Kir2.1 channels.

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Figure 5. Long-term oleic acid treatment does not affect surface expression of Kir2.1 Representative confocal images of opossum kidney (OK) cells expressing GFPKir2.1(EGFP-linked Kir2.1) with an extracellular haemagglutinin (HA) epitope after 8 h incubation with (OA) or without (CTRL) oleic acid (18.4 nm unbound concentration). Green fluorescence protein fluorescence (GFP) shows total protein expression, whereas α-HA immunocytochemistry (α-HA) without permeabilization of the cells selectively detects the surface population of channels (for details on quantification of surface expression see Methods).

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Effects of sustained exposure to oleic acid (6–20 h, 14 nm unbound oleic acid) on the KATP channels could not be resolved because of the low KATP channel activity in CHO cells under the recording conditions (i.e. with 1 mm ATP in the patch pipette, KATP channel activity was not detectable). Measurements in the absence of ATP (or lower ATP concentrations) proved to be difficult because the washout of cellular ATP activated KATP currents with a variable time course that often did not saturate during the time of recording. We conclude that long-term oleic acid exposure did not strongly activate KATP channels, in contrast to effects seen with the acute exposure; however, moderate changes might not have been resolved.

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

The aim of this study was to resolve the question of whether endogenously formed LC-CoA can regulate the activity of Kir channels in cells. To this end we measured the impact of fatty acid metabolism on KATP and Kir2.1 channels expressed in CHO cells. These channels respond to exogenously applied LC-CoA differently and, thus, would allow us to distinguish indirect effects from the direct regulation of channel activity by LC-CoA. We confirmed this bi-directional regulation of KATP and Kir2.1 channels under whole-cell patch clamp by infusion of 1 μm oleoyl-CoA into the cytoplasm via the patch pipette. This resulted in the activation of KATP but inhibition of Kir2.1 channels as expected. An ineffective LC-CoA derivative (palmitoyl-CoADephospho), or the absence of oleoyl-CoA in the pipette solution, had no effect. To test for the impact of cytoplasmically formed oleoyl-CoA on Kir channels, cells were superfused with a bath medium containing oleic acid and a high glucose concetration. This condition induced within minutes the activation of KATP channels and inhibition of Kir2.1 channels in CHO cells as well as inhibition of endogenous Kir channels in HEK293 cells. Several lines of evidence suggest that the effects of extracellular oleic acid were mediated by the cytoplasmic accumulation of oleoyl-CoA and its direct interaction with the Kir channels. Firstly, KATP, Kir2.1 and endogenous Kir channels are affected differently consistent with the direct effects of exogenous oleoyl-CoA on these channels. Secondly, the regulation by oleic acid was only seen under conditions that promote the cytoplasmic accumulation of LC-CoA (i.e. high glucose concentration) (Prentki et al. 1992, 2002), or inhibition of the mitochondrial uptake of LC-CoA by the CPT1 inhibitor etomoxir (Alam & Saggerson, 1998). Finally, triacsin C, which is a potent inhibitor of the long-chain fatty acid acyl-CoA synthetase, prevented the effects of oleic acid on the different Kir channels. Taken together these results establish that the cytoplasmic accumulation of LC-CoA represents a pathway to directly regulate the activity of Kir channels.

The experimental conditions to induce fast (acute) fatty acid effects simulated the pathophysiological situations of hyperglycaemia (25 mm glucose) and hyperlipidaemia (81 nm unbound oleic acid). The concentration of unbound free fatty acids in the human plasma is thought to range from 7 nm under physiological conditions to 100 nm in pathological conditions. However, higher concentrations may occur locally (Richieri & Kleinfeld, 1995; Kleinfeld et al. 1996; Kleinfeld & Okada, 2005). Although a lower concentration of unbound oleic acid (18.4 nm) had no acute effects (i.e. within 20 min) on Kir2.1, endogenous Kir and KATP currents, a pronounced inhibition developed within 7 h of the start of incubation with oleic acid with Kir2.1 and endogenous Kir channels. No marked effect was observed for KATP channels; however, the presence of high intracellular ATP may have obscured moderate changes in ATP sensitivity. The slow inhibition of Kir2.1 and endogenous Kir channels depended on the formation of oleoyl-CoA as it occurred only under conditions that promote LC-CoA accumulation (i.e. high glucose or addition of etomoxir) and was prevented by triacsin C. As LC-CoA might affect membrane trafficking (Faergeman & Knudsen, 1997), we tested whether the slow time course of inhibition might reflect a change in surface membrane expression. However, no significant change in membrane expression induced by oleic acid incubation was observed for Kir2.1 channels. Therefore, the formation and the accumulation of LC-CoA most probably requires extended time periods with lower concentration of LC fatty acids in the medium.

Our results regarding the acute regulation of KATP channels substantiate the hypothesis that long-chain fatty acids activate KATP channels in situations that promote LC-CoA accumulation as proposed previously for pancreatic β-cells (Larsson et al. 1996) and cardiac myocytes (Liu et al. 2001). This is also in good agreement with a recent study by Bränström et al. (2004) showing that high extracellular oleic acid (5 μm) can activate KATP channels in human pancreatic β-cells. However, our experiments also show that extracellular oleic acid failed to modulate KATP (and Kir channel) activity in CHO and HEK293 cells in the presence of low or normal glucose concentrations. This observation apparently contradicts the proposed role of KATP channels as a sensor for circulating fatty acids in neurons of the hypothalamus in the presence of a normal glucose concentration (Lam et al. 2005). The discrepancy might reflect differences in the SUR subtype (SUR1 in the hypothalamus, Lam et al. 2005), in the fatty acid metabolism (Kim et al. 2002) or fatty acid buffer capacity within hypothalamic neurons. Of note, inhibition of the fatty acid synthase in hypothalamic neurons by administration of C75 induces leptin- and insulin-like behavioural effects (e.g. reduction of food intake) that are thought to be mediated by an increase of malonyl-CoA (Schwartz & Porte, 2005). Possibly, the effect of C75 might be related to the activation of KATP channels by LC-CoA as high malonyl-CoA should promote the accumulation of LC-CoA and both leptin and insulin have been reported to activate KATP channels in the hypothalamus (Spanswick et al. 1997; Mirshamsi et al. 2004).

We have recently shown that, in addition to KATP channels, most Kir channels are affected by exogenous LC-CoA, and we report here the regulation of recombinant Kir2.1 and endogenous Kir channels by endogeneously formed LC-CoA. Given the widespread expression of Kir channels throughout the body and the general occurrence of LC-CoA in cells, the modulation of Kir channel activity by fatty acid metabolism might be of relevance in many tissues.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix
  • Alam N & Saggerson ED (1998). Malonyl-CoA and the regulation of fatty acid oxidation in soleus muscle. Biochem J 334, 233241.
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Appendix

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Acknowledgements

The authors gratefully acknowledge the technical help of E. Faber. We thank FFA Sciences (San Diego, CA, USA) for determining the unbound concentration of oleic acid. We are grateful to Dr S. Huber for helpful discussions and comments on the manuscript. This work was supported by a Deutsche Forschungsgemeinschaft grant to T.B. (Ba 1793/4-1). E.S. has been supported by a grant from the Alexander von Humboldt Foundation. A preliminary account of this work has been published in abstract form (Annual Meeting of the German Physiological Society 2005).