The commonly occurring E23K and I337V Kir6.2 polymorphisms in the ATP-sensitive potassium (KATP) channel are more frequent in Caucasian type 2 diabetic populations. However, the underlying cellular mechanisms contributing to the pathogenesis of type 2 diabetes remain uncharacterized. Chronic elevation of plasma free fatty acids observed in obese and type 2 diabetic subjects leads to cytosolic accumulation of long-chain acyl CoAs (LC-CoAs) in pancreatic β-cells. We postulated that the documented stimulatory effects of LC-CoAs on KATP channels might be enhanced in polymorphic KATP channels. Patch-clamp experiments were performed on inside-out patches containing recombinant KATP channels (Kir6.2/SUR1) to record macroscopic currents. KATP channels containing Kir6.2 (E23K/I337V) showed significantly increased activity in response to physiological palmitoyl-CoA concentrations (100–1,000 nmol/l) compared with wild-type KATP channels. At physiological intracellular ATP concentrations (mmol/l), E23K/I337V polymorphic KATP channels demonstrated significantly enhanced activity in response to palmitoyl-CoA. The observed increase in KATP channel activity may result in multiple defects in glucose homeostasis, including impaired insulin and glucagon-like peptide-1 secretion and increased glucagon release. In summary, these results suggest that the E23K/I337V polymorphism may have a diabetogenic effect via increased KATP channel activity in response to endogenous levels of LC-CoAs in tissues involved in the maintenance of glucose homeostasis.
Type 2 diabetes is a multifactorial disease with both genetic and environmental components contributing to its development. Despite the investigation of polymorphic variations in genes encoding for key components in pathways controlling insulin secretion, their precise roles in the etiology of type 2 diabetes are not well understood.
Glucose homeostasis is maintained through the coordinated release of several hormones, including insulin, glucagon, and glucagon-like peptide-1 (GLP-1). A key component regulating the release of these hormones is the ATP-sensitive potassium (KATP) channel (1–3). Hormone secretion in the pancreatic β- and α-cell and in the intestinal L-cell is controlled through metabolic regulation of electrical activity, a process critically linked to glucose and fatty acid metabolism, which in turn regulates the activity of KATP channels that control membrane potential (1–4).
The KATP channel is a hetero-octameric protein complex comprised of the pore-forming inward-rectifier Kir6.2 subunit coupled to the high-affinity sulfonylurea receptor SUR1 subunit (5,6) in a stoichiometry of (Kir6.2)4/(SUR1)4. Mutations that reduce KATP channel activity can lead to the increased β-cell excitability and excessive insulin secretion that underlies the congenital disorder of persistent hyperinsulinemic hypoglycemia of infancy (7). In addition, transgenic animal models demonstrate that targeted overactivity of KATP channels severely impairs insulin secretion and leads to a severe neonatal diabetic phenotype (8). Therefore, enhanced β-cell KATP channel activity reduces the magnitude of the glucose-stimulated insulin secretory response in β-cells and can lead to the development of diabetes.
A recently discovered set of three commonly occurring single nucleotide polymorphisms (E23K, L270V, and I337V) in the Caucasian population has been found within the Kir6.2 gene (9,10). Both the E23K and I337V polymorphisms appear to be tightly linked, with >72% of those possessing E23K also possessing I337V (9,11). Recent clinical studies have indicated that homozygous polymorphic individuals are at an increased risk of developing type 2 diabetes compared with individuals with either heterozygous or wild-type KATP channels (9,11–13). Homozygous subjects also possess impaired glucose-stimulated insulin secretion (upon oral glucose challenge), and the prevalence of the Kir6.2 polymorphisms may be linked to an increase in BMI (14). In addition, recent evidence suggests that patients homozygous for the E23K polymorphism have a reduced glucagon secretion response during hyperglycemia (15) that could result from increased KATP channel activity (2). Taken together, the available evidence suggests that the homozygous polymorphic population may be especially susceptible to impairment of glucose homeostasis if they are overweight or obese, and/or that these polymorphisms may predispose patients to diabetes and excessive weight gain. However, the cellular mechanisms underlying these associations are still poorly understood.
At the cellular mechanistic level, it has been shown that the E23K polymorphism can decrease ATP sensitivity, rendering KATP channels slightly more active at higher ATP concentrations (16). In addition, it is known that plasma free fatty acid (FFAs) levels are elevated in obese (17) and diabetic (18) individuals with chronic elevation leading to an accumulation of long-chain acyl CoAs (LC-CoAs) in the cytosol, which can then directly activate β-cell KATP channels (19,20). Due to the cytosolic location of the polymorphisms in the Kir6.2 gene (Fig. 2D) and their possible effects on ATP and LC-CoA sensitivity, we hypothesized that binding of LC-CoAs may further potentiate polymorphic KATP channel activity relative to wild-type channels, thus reducing cellular excitability and contributing to altered nutrient-stimulated hormone release, such as reduced insulin secretion, that is observed in homozygous polymorphic individuals with type 2 diabetes (14).
Due to the coupling of the E23K and I337V polymorphisms in vivo, we have examined the sensitivity of the E23K/I337V double-polymorphic KATP channel to activation by the LC-CoA palmitoyl-CoA relative to wild-type (Kir6.2/SUR1) KATP channels in a recombinant expression system. Application of palmitoyl-CoA resulted in a dose-dependent increase in current of both wild-type and polymorphic KATP channels (Fig. 1). At palmitoyl-CoA concentrations of 100–1,000 nmol/l, polymorphic KATP channel current was significantly increased (approximately twofold) over wild-type at the same dose (Fig. 1C). For example, KATP channel current was increased by 389.69 ± 48.62% compared with 196.82 ± 20.30% in wild-type channels (P < 0.05) upon application of 100 nmol/l palmitoyl-CoA. With estimates of β-cell cytosolic free LC-CoA in the range of 0.5–1 μmol/l and binding affinities for various LC-CoA-utilizing enzymes in the nanomolar to micromolar range (21), our results show that in the presence of physiological concentrations of palmitoyl-CoA, polymorphic KATP channels are significantly more active than wild-type KATP channels.
To investigate the mechanisms responsible for the observed increase in sensitivity to LC-CoAs in the polymorphic KATP channel population, we utilized the Kir6.2ΔC26 truncation mutant to assess the contribution of the SUR1 subunit (Fig. 2D). The Kir6.2ΔC26 mutant can be expressed to yield functional ATP-sensitive potassium currents in the absence of the accessory SUR1 subunit (22). Although higher concentrations of palmitoyl-CoA were required to activate the Kir6.2ΔC26 channel, a similar significant increase in current magnitude in response to 500 nmol/l palmitoyl-CoA was observed between polymorphic (219.67 ± 37.27%) and wild-type (144.80 ± 17.20%) Kir6.2ΔC26 channels, as was noted in KATP channels comprised of both the Kir6.2 and SUR1 subunits (789.91 ± 89.30% in polymorphic KATP channels vs. 355.80 ± 52.56% in wild-type KATP channels) (Fig. 1C). These results, together with the observation that sulfonylurea sensitivity is unaffected in the presence of the E23K polymorphism (23), suggest that the principal mechanism by which the E23K/I337V polymorphism alters LC-CoA sensitivity lies primarily within the Kir6.2 subunit.
Increases in both open probability and single-channel amplitude could result in the observed increase in macroscopic polymorphic KATP channel activity in response to palmitoyl-CoA. To test which of these parameters is altered, we performed single-channel experiments on recombinant KATP channels expressed in tsA201 cells. Our results indicate that at 1 mmol/l ATP, application of 100 nmol/l palmitoyl-CoA to wild-type or polymorphic KATP channels does not significantly alter single-channel amplitude (Fig. 3). At a holding potential of −60 mV, wild-type channels had a measured conductance of 60.24 ± 0.97 pS versus polymorphic channels at 56.67 ± 2.70 pS (not significant [NS], n = 7), suggesting that the observed increase in KATP channel activity must occur through changes in open probability.
To gain insights into the physiological significance of these findings, we have examined ATP sensitivity in wild-type and polymorphic KATP channels in the absence and presence of palmitoyl-CoA. We found that 100 nmol/l palmitoyl-CoA decreased ATP sensitivity in both wild-type and polymorphic KATP channel populations (Fig. 4A and B), shifting the half-maximal inhibitory concentration (IC50) to the right 1.88- and 1.90-fold, respectively. However, our results indicate that ATP sensitivity is slightly increased at the respective IC50 concentrations versus wild-type channels in both the absence and presence of palmitoyl-CoA. This appears to be in contrast to the findings of Schwanstecher et al. (16), who noted a rightward shift in the ATP concentration-inhibition curve at all tested ATP concentrations. The study by Schwanstecher et al. noted an increased spontaneous open probability in KATP channels containing the single Kir6.2 E23K polymorphism in the absence of ATP. In the present study, we examined the single-channel properties of the E23K/I337V double polymorphism in tsA201 cells. In this recombinant expression system, we observed no significant difference between the spontaneous open probability of wild-type versus polymorphic KATP channels (wild-type 0.837 ± 0.08 vs. E23K/I337V 0.829 ± 0.04; NS, n = 7) as measured by noise analysis in the absence of ATP.
As physiological ATP concentrations in resting β-cells are estimated to be in the low millimolar range (24,25), we examined this portion of the concentration-response curve in greater detail (Fig. 4C and D). At 1 mmol/l ATP in the presence of 100 nmol/l palmitoyl-CoA, the polymorphic KATP channel population exhibited a significant increase in current (6.42 ± 1.21% of maximal KATP current vs. 3.61 ± 0.40% in wild-type). The difference in effect at low versus high ATP concentrations occurs due to a reduction in the Hill coefficient of ATP binding from 2.28 in wild-type to 1.58 in polymorphic KATP channels (Fig. 4F). After application of palmitoyl-CoA, the Hill coefficients were further reduced to 1.41 in the wild-type and 1.24 in the polymorphic KATP channel population. This reduction in Hill coefficient suggests that the ability of ATP to reach its binding site(s) may be impaired in the presence the E23K/I337V polymorphism. A further impairment of ATP binding after exposure to palmitoyl-CoA may occur due to competition at the ATP/LC-CoA binding site. A similar effect on ATP inhibition has been noted in cardiac sarcolemmal KATP channels in response to protein kinase C (26). In the presence of palmitoyl-CoA, the calculated crossover concentration was 100 μmol/l ATP, suggesting that at physiological [ATP]i the E23K/I337V polymorphism will render KATP channels more active than wild-type (Fig. 4D). From the ATP concentration-inhibition curves, it was calculated that the residual KATP channel activity at 1 mmol/l ATP was ∼40% greater in polymorphic KATP channels compared with wild-type KATP channels (Fig. 4E). Using a mathematical model of β-cell electrical activity, we found that increases in KATP channel conductance on the order of ∼25% are adequate to significantly reduce the bursting behavior characteristic of glucose-stimulated β-cells (27), suggesting that the observed increase in KATP channel activity in this study may be sufficient to alter insulin secretion. Taken together, the results and simulations indicate that physiological palmitoyl-CoA concentrations could lead to an increased basal polymorphic KATP channel activity resulting in dysfunctional glucose-stimulated hormone secretion.
Glucose homeostasis is properly maintained through a balanced secretion of hormones from multiple tissues. Interestingly, the KATP channel is involved in appropriate secretion of insulin (1), glucagon (2), and GLP-1 (3,28). Neuronal control of food intake and hormone secretion via glucose-sensing neurons in the hypothalamus represents an additional potentially impaired process in homozygous polymorphic individuals, as KATP channels are highly expressed in the central nervous system (29). It is therefore highly plausible that in addition to impaired insulin secretion from β-cells, the E23K/I337V polymorphism may result in inappropriate secretion of glucagon and GLP-1 and dysfunctional regulation of food intake.
In summary, we propose that increased polymorphic KATP channel activity in the presence of normal or elevated levels of intracellular acyl CoAs may result in decreased insulin and GLP-1 secretion coupled with increased glucagon release. Together, the inappropriate secretion of these hormones may contribute to the association of Kir6.2 polymorphisms with type 2 diabetes. Additional studies will be required to directly assess altered hormone secretion in cell lines expressing a homozygous population of polymorphic KATP channels. This study is the first to propose a mechanistic link between increased BMI and plasma FFA levels and the observed high prevalence of type 2 diabetes in the KATP channel homozygous polymorphic population.
RESEARCH DESIGN AND METHODS
Cell culture and transfection.
tsA201 cells (an SV40-transformed variant of the HEK293 human embryonic kidney cell line) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 25 mmol/l glucose, 2 mmol/l l-glutamine, 10% FCS, and 0.1% penicillin/streptomycin. Cells were kept at 37°C with 5% CO2. The KATP channel Kir6.2 subunit clone from mouse was generously provided by Dr. S. Seino (5). The SUR1 subunit clone from hamster was generously provided by Drs. L. Aguilar Bryan and J. Bryan (6). Clones were inserted into the mammalian expression vector pCDNA3. tsA201 cells were plated at 50–70% confluency on 35-mm culture dishes 4 h before transfection. Clones were then transfected into tsA201 cells using the calcium phosphate precipitation technique. Transfected cells were identified using fluorescence optics in combination with coexpression of the green fluorescent protein plasmid (pGreenLantern; Life Technologies, Gaithersburg, MD). Single-channel recordings were made from cells 24 h after transfection and macroscopic recordings 48–72 h after transfection. In contrast to cells expressing SUR1/Kir6.2, nontransfected cells did not exhibit any ATP-sensitive potassium current (data not shown).
The E23K and I337V mutations were introduced into the Kir6.2 gene using the protocol outlined in the QuikChange site-directed mutagenesis kit (Stratagene) and the generation of the mutations confirmed through sequence analysis. The ΔC26Kir6.2 truncation mutant was constructed as previously described (23).
The inside-out patch technique was used to measure macroscopic KATP channel currents in transfected tsA201 cells. Pipettes were back-filled with solution containing the following (in millimoles per liter): 134 KCl, 10 HEPES, 1.4 MgCl2, 1 EGTA, 6 KOH, and 10 glucose. The pH of the solution was adjusted to 7.4 with KOH. Macroscopic recordings were performed using patch pipettes pulled from borosilicate glass (G85150T; Warner Instrument, Hamden, CT) to yield pipettes with a resistance of 2–6 MΩ. Patch pipettes used for single-channel recordings were pulled from borosilicate glass (PG52151-4; World Precision Instruments, Sarasota, FL) to yield pipette resistances of 20–25 MΩ when filled with pipette solution. Once a GΩ seal was formed, the membrane patch was excised from the cell and positioned in the path of a multi-input perfusion pipette. 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 (20–22°C). An Axopatch 200B patch-clamp amplifier and Clampex 8.0 software (Axon Instruments, Foster City, CA) were used for data acquisition and analysis.
Single-channel amplitude measurements were analyzed using Fetchan software version 6.0 (Axon Instruments). For measurement of Po by noise analysis, the following equation was used: Po = 1 −(ς2/[i × I]), where ς2 is the variance measured in the absence of ATP minus the variance measured in between channel opening in the presence of 1 mmol/l ATP (i.e., baseline noise when all channels closed), i is the single channel amplitude, and I is the macroscopic mean current measured in the absence of ATP.
MgATP (Sigma, St. Louis, MO) was prepared from a 10-mmol/l stock and stored at −20°C until use. Palmitoyl CoA (Sigma) was dissolved in distilled water as 5-mmol/l stocks. Stock solutions were diluted in pipette solution to concentrations indicated in the text.
Recombinant macroscopic KATP currents were normalized to yield Irel, where Irel is the current under test conditions relative to the maximal control current observed and expressed as a percentage (i.e., Irel = Itest/Icontrol × 100). Statistical significance was assessed using the unpaired Student’s t test, with P values <0.05 considered significant. Data are expressed as the mean ± SE (NS, not significant).
Simulations of pancreatic β-cell electrical activity were generated using a mathematical model modified from that described by Bertram et al. (30). In this model, KATP channel activity as denoted by conductance is a variable and can thus be modified to reflect the experimentally observed changes in KATP channel activity.
This study was supported by funding from the Canadian Diabetes Association in honor of Violet D. Mulcahy and the Alberta Heritage Foundation for Medical Research (AHFMR). P.E.L. received salary support as an AHFMR Scholar and Canadian Institutes of Health Research New Investigator.
We thank Dr. Lynn Eisner for her expert technical assistance and Michael Wheeler (University of Toronto) for useful discussions.