Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada
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ABSTRACT |
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Obesity is a major risk factor for the development of type 2 diabetes. Studies suggest that increasing the amount of dietary energy derived from fat can lead to increased body weight. Coupled with reduced physical activity or energy expenditure, diets high in fat contribute to the development of the metabolic syndrome, obesity, and type 2 diabetes (1,2). Cardiovascular researchers have known for some time that the composition of dietary fat can influence ones risk for the development of atherosclerosis and coronary artery disease, with saturated long-chain and trans fatty acids having the largest detrimental effect (35). These observations parallel those found in studies linking fat composition to the frequency of type 2 diabetes and suggest a possible link between dietary fat composition and dysfunctional insulin secretion (6,7). The mechanisms by which dietary fat may alter insulin secretion are not fully characterized, in part due to the dual action of fatty acids on the process of insulin secretion. An acute free fatty acid (FFA) stimulus enhances glucose-stimulated insulin secretion (GSIS) (8). However, upon chronic FFA exposure, levels of acyl CoAs, the intracellular esters of FFAs, increase within ß-cells, contributing to decreased insulin output through several proposed mechanisms (9), including activation of ß-cell ATP-sensitive K+ channels (KATP channels).
The appropriate metabolic control of KATP channels is a critical component of normal GSIS. Under resting conditions, efflux of potassium through KATP channels maintains the ß-cell in a hyperpolarized inactive state. The metabolic signal initiated by glucose metabolism is linked to membrane excitability and ultimately the release of insulin through reduction of potassium efflux via closure of KATP channels.
Despite the large number of KATP channels estimated to populate the surface of an individual ß-cell, only 1% of the total KATP channel conductance is thought to be available even during resting periods (10). As such, only a small number of channels must be closed to initiate insulin secretion. Therefore, a relatively small increase in the plasma glucose concentration leading to subtle reductions in KATP channel activity can evoke a resting ß-cell to secrete insulin. Pharmacological agents that inhibit KATP channels (e.g., sulfonylureas) promote insulin secretion, whereas activators such as diazoxide limit insulin secretion. Endogenous KATP channel activators also exist, including MgADP and long-chain acyl CoAs. It has previously been shown by our group and others (1113) that acyl CoAs can potently activate KATP channels. Acyl CoAs are amphiphilic molecules comprised of a negatively charged acyl coenzyme A head group and a hydrophobic acyl chain. Acyl CoAs are the product of FFA esterification to the CoA moiety by acyl CoA synthetases, and thus specific dietary FFAs are directly esterified into acyl CoAs with the same acyl chain structure. Chronic upregulation of plasma FFA levels, as documented in both obese (14) and type 2 diabetic (15) individuals, can lead to accumulation of acyl CoAs in the cytosol of pancreatic ß-cells (9,16,17). Because the concentrations of these endogenous KATP channel activators are elevated in obesity and type 2 diabetes, it is important to understand the mechanisms by which they contribute to impaired GSIS.
The previously described E23K/I337V KATP channel polymorphisms have also been shown to alter channel activity and may increase the risk for development of type 2 diabetes in a large subset of the general Caucasian population (rev. in 18). Evidence suggests that these polymorphisms decrease ATP sensitivity (19) and increase KATP channel sensitivity to activation by palmitoyl-CoA (11), a common 16 carbonsaturated acyl CoA whose free-fat parent molecule is abundant in meat and dairy products.
To date, a comprehensive study on the effects of side-chain length, degree of saturation, and cis/trans double-bond conformation of various acyl CoAs has not been performed. Therefore, it was the aim of this study to investigate the KATP channelactivating properties of various common dietary acyl CoAs and to establish a molecular model to explain the process of acyl CoAmediated channel activation. Our results indicate that longer-chain saturated and trans fatty acids exert a larger stimulatory effect on the KATP channel and that the ability of any given acyl CoA to activate the channel is directly related to its hydrophobicity.
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RESEARCH DESIGN AND METHODS |
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Cell culture and transfection.
tsA201 cells (an SV40-transformed variant of the HEK293 human embryonic kidney cell line) were maintained in Dulbeccos modified Eagles medium supplemented with 25 mmol/l glucose, 2 mmol/l L-glutamine, 10% FCS, and 0.1% penicillin/streptomycin in a humidified incubator at 37°C with 5% CO2. Cells were passaged and plated at 5070% confluency on 35-mm culture dishes 4 h before transfection. Clones were then transfected into the tsA201 cells using the calcium phosphate precipitation technique. Transfected cells were identified using fluorescent optics in combination with coexpression of the green fluorescent protein plasmid (pGreenLantern; Life Technologies, Gaithersburg, MD). Macroscopic KATP channel recordings were then performed 4872 h after transfection.
Electrophysiology.
The inside-out patch-clamp technique was used to measure macroscopic KATP channel currents in transfected tsA201 cells. Patch pipettes were pulled from borosilicate glass (G85150T; Warner Instruments, Hamden, CT) to yield resistances between 2 and 6 mol/l when back filled with a buffer solution containing the following: 110 mmol/l KCl, 30 mmol/l KOH, 10 mmol/l EGTA, 5 mmol/l HEPES, and 1 mmol/l MgCl2. The pH of the solution was adjusted to 7.4 with KOH. Once a G
seal was formed, the membrane patch was excised from the cell and positioned in the path of a multi-input perfusion pipette. The cytosolic face of the membrane patch was held at a holding potential of 60 mV to elicit inward KATP channel currents, and the resulting upward current deflections were plotted using Origin graphing software (Microcal Software, Northampton, MA). Membrane patches were directly exposed to test solutions under symmetrical K+ conditions through this perfusion pipette (time to change solution at the tip of the recording pipette was <2 s.). All patch-clamp experiments were performed at room temperature (2022°C). An Axopatch 200B patch-clamp amplifier and Clampex 8.0 software (Axon Instruments, Foster City, CA) were used for data acquisition and analysis.
Experimental compounds.
MgATP (Sigma, Oakville, ON) was prepared as a 10-mmol/l stock and stored at 20°C until use. Long-chain acyl CoAs palmitoyl CoA (C16:0), cis- (9)-palmitoleoyl CoA (C16:1), stearoyl CoA (C18:0), cis-
(9)-oleoyl CoA (C18:1cis), trans-
(9)-elaidoyl CoA (C18:1trans), cis,cis-
(9,12)-linoleoyl CoA (C18:2), and all cis-
(5,8,11,14)-arachidonyl CoA were purchased from Sigma as Li+ salts and dissolved in ddH2O as 1-mmol/l stock solutions. Before use, stock solutions were sonicated for 5 min and diluted in pipette solution to concentrations indicated in the text.
Statistical analysis.
Recombinant macroscopic KATP channel currents were normalized and expressed as an increase in current relative to control (i.e., normalized KATP channel current = Itest/Icontrol), where Itest is the current elicited by the acyl CoA stimulus and Icontrol is the current elicited by 0.2 mmol/l MgATP. Statistical significance was assessed using the unpaired Students t test, with P < 0.05 considered statistically significant. Data are expressed as means ± SE.
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RESULTS |
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Acyl CoAs of differing side-chain composition may compete for a shared binding site on the KATP channel.
We performed a series of experiments testing the effects of simultaneous application of two acyl CoAs with different KATP channel stimulatory properties. Stearoyl CoA and linoleoyl CoA were used due to the drastically different levels of channel activation observed at 100 nmol/l (8.7 ± 0.7- vs. 2.9 ± 0.3-fold, respectively). While maintaining a constant total acyl CoA concentration at 100 nmol/l, increasing the relative ratio of linoleoyl CoA to stearoyl CoA from 1:1 to 2:1 resulted in a reduction of maximum normalized current to 5.3 ± 0.2-fold (Fig. 4A). A further increase in the linoleoyl CoAtostearoyl CoA ratio to 5:1 resulted in an additional decrease in KATP channel activation to 3.2 ± 0.6-fold, a level of activation not significantly different to that of linoleoyl CoA alone.
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In another experiment, the membrane patch was preexposed to 10 nmol/l stearoyl CoA to elicit significant KATP channel activation, and then the effects of either 100 or 500 nmol/l linoleoyl CoA were tested in the continued presence of stearoyl CoA. Under these conditions, linoleoyl CoA failed to reduce the stimulatory effect of stearoyl CoA (Fig. 4C). This suggests that, once bound, stearoyl CoA is unable to be competed off by saturating concentrations of linoleoyl CoA.
The hydrophobicity of the acyl chain predicts the efficacy of acyl CoAs on KATP channel activation.
We plotted the normalized KATP channel current in the presence of each tested acyl CoA against its partition coefficient between octanol and water (Fig. 5A). Partition coefficients (log P values) were obtained for FFAs with carbon chain lengths ranging from 8 to 20. A single exponential was fitted to the data obtained from this study and extrapolated to include log P values below that of palmitoleoyl CoA. Interestingly, as acyl chain length or degree of saturation increases, the log P value also increases, with stearoyl CoA having the longest and most saturated acyl tail as well as the largest log P value. Also plotted in Fig. 5 are results obtained from two previous studies (21,22). Data from the Branstrom et al. (22) study includes octanoyl CoA (C8:0), lauroyl CoA (C12:0), and myristoyl CoA (C14:0), while data from the Fox et al. (21) study add decanoyl CoA (C10:0) to the dataset. It has been previous shown that acyl CoAs with chain lengths <14 do not significantly activate KATP channels (12). This may result from their low partition coefficient and therefore a reduced ability to incorporate into hydrophobic environments, such as the lipid bilayer.
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Taking the above observations into consideration, we propose a model whereby the negatively charged CoA head group binds to a specific site(s) on the KATP channel, while the acyl tail partitions into the plasma membrane. Our observations indicate that saturated and long-chain acyl CoAs cause a large but slowed stimulatory response and do not readily unbind from the channel (Fig. 6A). In contrast, shorter-chain and unsaturated acyl CoAs, which have lower partition coefficients and are more mobile in hydrophobic environments, activate KATP channels but to a lesser extent, an effect that is readily reversible (Fig. 6B).
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DISCUSSION |
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Mechanisms of acyl CoAmediated KATP channel activation.
Lipid modulation is thought to play a major role in fine tuning KATP channel activity in vivo, given that under physiological nucleotide levels >99% of plasma membrane, KATP channels are closed (10). Acyl CoAs may represent one of the most important classes of lipid modulators, with a similar efficacy but greater potency and selectivity for KATP channels than the phosphoinositide phosphatidylinositol 4,5-bisphosphate (23,24). In pathological situations such as obesity (14) and type 2 diabetes (15), high circulating levels of FFAs lead to cytosolic accumulation of acyl CoAs (9), indicating that this class of ion channel modulators may play a contributory role in the mechanism by which obesity precipitates dysfunctional GSIS and increases susceptibility to type 2 diabetes.
In the current study, we have examined the effects of various acyl CoAs on KATP channel activity using physiological free concentrations 100 times below the critical micellar concentration (20). While all acyl CoAs tested in this study increased KATP channel activity, our results indicate the general rank order of efficacy was as follows: saturated CoAs, monounsaturated CoAs, and n-6 polyunsaturated acyl CoAs. We also found that longer-chain acyl CoAs activated the KATP channel to a greater extent than shorter-chain acyl CoAs. These data are consistent with other reports (12,22,25), indicating that increased acyl CoA side-chain length and saturation leads to increased KATP channel activity. However, based on results indicating that palmitoyl CoA and oleoyl CoA elicited similar KATP channel activation, previous studies have shown that this stimulatory effect is maximal at 16 carbons in side-chain length (22). Our data are in agreement with this observation, as these two acyl CoAs have a similar hydrophobicity (Fig. 5A). However, the fully saturated 18-carbon stearoyl CoA clearly elicited a greater response in both wild-type and E23K/I337V polymorphic KATP channels compared with palmitoyl CoA and oleoyl CoA.
Interestingly, the ability of a particular acyl CoA to activate the KATP channel may be directly related to its ability to partition into hydrophobic environments. This is the case not only with the acyl CoAs used in this study but also for acyl CoAs of shorter-chain length used in previous studies (21,22). Using the partition coefficient between water and octanol as an indication of hydrophobicity, we fit a single exponential curve to our KATP channel activation data that allows us to predict the effect of a given acyl CoA on the channel based on acyl chain hydrophobicity. At this point, it is unclear as to whether the relationship holds true for acyl CoAs of chain length >20. Further studies examining both natural and synthetic very longchain acyl CoAs will be required to probe the upper limits of our prediction.
An examination of the effect of trans fatty acids on the activity of KATP channels yielded interesting results. Although elaidoyl CoA and oleoyl CoA activated the KATP channel to a similar extent, our data show that KATP channels exposed to the trans 18-carbon monounsaturated elaidoyl CoA remained active for an extended period of time relative to the cis isoform oleoyl CoA once the acyl CoA stimulus was removed. This important observation reveals a possible mechanism by which trans monounsaturated fatty acids may be more detrimental to insulin secretion than the corresponding cis monounsaturated fatty acid. Prolonged channel activation could have a similar effect to that of an increased maximum response, such as occurs in the presence of saturated acyl CoAs in maintaining a hyperpolarized ß-cell membrane and reduced glucose responsiveness.
The specific interaction of long-chain acyl CoAs with the KATP channel is not currently well understood. A binding region for the CoA head group has been suggested to reside in the Kir6.2 subunit (26,27), but a precise mechanism detailing how binding of this head group and the associated acyl chain leads to stabilization of the open state is lacking. We have attempted to further our understanding of the nature of this interaction by investigating the importance of acyl CoA chain length and saturation. Our data show that acyl CoA efficacy and affinity is affected by the acyl chain structure (Fig. 3C and D). In addition, we found that 1) binding of polyunsaturated acyl CoAs could delay the activation efficacy of saturated acyl CoAs (Fig. 4A) and 2) persistent KATP channel activation induced by stearoyl CoA could not be diminished by a saturating concentration of the weakly activating linoleoyl CoA (Fig. 3C). Taken together, these data suggest that a shared binding site may be involved in the activation by various acyl CoAs and that this may be due to binding of the common CoA head group shared among each acyl CoA (see below). If the shared binding site(s) is initially unoccupied, then saturated and unsaturated acyl CoAs are equipotent in occupying the vacant site(s), as they share the identical CoA moiety (Fig. 4A). However, once bound, saturated acyl CoAs are unable to be competed off by unsaturated acyl CoAs, providing further evidence for the involvement of the acyl chain in the persistence and magnitude of activation. The slower response and persistent activation observed with saturated and trans monounsaturated acyl CoAs (Figs. 3C and D) may reflect a reduced lateral mobility of the saturated acyl chain within the membrane.
Additional support for this model comes from studies of acyl CoA incorporation into artificial membrane preparations. It has been previously shown that these amphiphilic molecules associate with membranes through insertion of the fatty acyl chain (20) and that this interaction becomes stronger with increased side-chain length (28). Cohen et al. (20) has also shown that lateral diffusion of acyl CoAs occurs in membranes leading to the formation of aggregates near areas of increased membrane curvature, which may include membrane-spanning proteins such as ion channels. Therefore, it is plausible that local acyl CoA concentrations may be higher in the proximity of KATP channels than in the surrounding membrane. In addition, incorporation of different acyl CoAs into the membrane may be constant, but the reduced lateral diffusion rate of saturated and trans acyl CoAs may increase the longevity of channel opening by maintaining the CoA head group in close proximity to its binding site on the KATP channel, as evidenced in Figs. 3B and D. Reduced lateral diffusion may also result in the increased time to activation and reduced washout of saturated and trans acyl CoAs as observed in this study.
Given the similarity in proposed crystal structure of the trans membrane domain of KcsA and KirBac1.1 and their sequence and structural homology with Kir6.2, we can speculate on potential interactions of acyl CoAs with Kir6.2 based on previous work performed on these related potassium channels (29). We have previously identified several positively charged residues in both the COOH-terminal and NH2-terminal domains of the cytosolic portion of the Kir6.2 subunit that are important for acyl CoA binding. In particular, residue R54 from one Kir6.2 subunit is thought to interact with R176 and R301 of the adjacent subunit to form a positively charged region to which the negatively charged CoA head group may bind (27). The exact placement of the head group in this region is currently unknown, but it appears that binding positions the fatty acyl chain to interact with the plasma membrane. Previous work by Williamson et al. (29) on the KcsA bacterial potassium channel indicates that fatty acids can bind to the -helices that contain the channel gate. Depending on the nature of this interaction, binding of fatty acids to these helices will likely result in alterations in their spatial arrangement and may lead to opening or closing of this gate. This fatty acid/
-helix interaction is highly dependent on the chain length. Binding occurs with a minimum acyl chain length of 10 carbons and becomes optimal at a tail length of 22 carbons (30). This matches very closely with our data showing an effect on KATP channel activity with acyl chains of
14 carbons (Fig. 5) (12,25) and is in agreement with our data indicating that the activation of KATP channels by acyl CoAs does not saturate with the 16-carbon palmitoyl CoA but is greater with the 18-carbon stearoyl CoA. Further studies to probe the binding of acyl chains to
-helices within the KATP channel are warranted to assess the similarity between fatty acid binding to KcsA and Kir6.2. The fact that the 10-carbon decanoyl CoA does not activate the ß-cell KATP channel may be as a result of the CoA binding site (including residues R54 and R176) lying below the plane of the membrane, necessitating a longer acyl chain to significantly partition into the lipid bilayer or through additional complexities of binding due to interactions with the SUR1 subunit. More detailed molecular modeling of CoA binding, including proper placement of the head group within this region, will be required before a firm mechanism for acyl CoAmediated KATP channel activation can be established.
Clinical relevance.
In both human and animal studies, high-fat diets routinely lead to insulin resistance, with most leading to dysfunctional GSIS (31,32,33,34). The observed inconsistency with regards to insulin secretion may result from two mechanisms. First, there is a biphasic effect of fatty acids on insulin secretion with an early stimulatory effect followed by a secondary inhibitory action that occurs with chronic fat exposure (35). Second, most studies have examined the effects of a mixture of fatty acids on insulin secretion, with the percentages of each fat modified to reflect the nature of the study (32). However, two recent large-scale prospective studies support our hypothesis that saturated and trans fatty acids are more detrimental to GSIS than unsaturated fatty acids. The study of Meyer et al. (7) of 36,000 women found that the incidence of type 2 diabetes was positively associated with higher intake of saturated animal fats and negatively associated with higher intake of unsaturated vegetable fats. These results are similar to those found in the Finnish and Dutch cohorts of the smaller Seven Countries Study (36). In addition, the Nurses Health Study examined
84,000 women, concluding that the incidence of type 2 diabetes was greater in those ingesting higher amounts of trans fatty acids and lower in those with a moderate increase in polyunsaturated fatty acid intake (6).
Summary.
In conclusion, we have shown that the direct exposure of KATP channels to long-chain acyl CoAs leads to increased channel activity in a manner dependent on both acyl chain length and degree of saturation. Saturated acyl CoAs are the most efficacious activators of KATP channels followed by monounsaturated and polyunsaturated acyl CoAs, respectively. The trans monounsaturated elaidoyl CoA activated KATP channels with similar efficacy to its cis monounsaturated counterpart (oleoyl CoA) but did not unbind from the channel as readily. This may result in a prolonged activation of KATP channels exposed to this and perhaps other as yet untested trans acyl CoAs, contributing to reduced ß-cell excitability and subsequent reductions in insulin secretion. Our results also indicate that different acyl CoAs can compete for a binding region on the KATP channel and that an advantage is given to those acyl CoAs with higher partitioning coefficients or reduced ability to unbind from or laterally move within the membrane. Finally, we have expanded on our previous findings (11) showing that E23K/I337V polymorphic KATP channels are more sensitive to activation by long-chain saturated and monounsaturated acyl CoAs compared with wild-type channels. Our data lend support to previous clinical studies that suggest replacement of saturated fat with either monounsaturated or polyunsaturated fat as a means to improving glucose tolerance in healthy (37) and glucose-intolerant (38) subjects and reducing the incidence of newly diagnosed type 2 diabetes (6) and has implications for dietary management of obese or type 2 diabetic individuals who are homozygous polymorphic for E23K.
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ACKNOWLEDGMENTS |
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Address correspondence and reprint requests to Peter E. Light, Department of Pharmacology, University of Alberta, 9-58 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada. E-mail: peter.light{at}ualberta.ca
Received for publication December 9, 2004 and accepted in revised form March 28, 2005
FFA, free fatty acid; GSIS, glucose-stimulated insulin secretion; KATP channel, ATP-sensitive K+ channel; SUR1, sulfonylurea receptor-1
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REFERENCES |
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