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Kir6.2 variant E23K increases ATP-sensitive K+ channel activity and is associated with impaired insulin release and enhanced insulin sensitivity in adults with normal glucose tolerance.

Villareal DT, Koster JC, Robertson H, Akrouh A, Miyake K, Bell GI, Patterson BW, Nichols CG, Polonsky KS - Diabetes (2009)

Bottom Line: Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity.The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT

Objective: The E23K variant in the Kir6.2 subunit of the ATP-sensitive K(+) channel (K(ATP) channel) is associated with increased risk of type 2 diabetes. The present study was undertaken to increase our understanding of the mechanisms responsible. To avoid confounding effects of hyperglycemia, insulin secretion and action were studied in subjects with the variant who had normal glucose tolerance.

Research design and methods: Nine subjects with the E23K genotype K/K and nine matched subjects with the E/E genotype underwent 5-h oral glucose tolerance tests (OGTTs), graded glucose infusion, and hyperinsulinemic-euglycemic clamp with stable-isotope-labeled tracer infusions to assess insulin secretion, action, and clearance. A total of 461 volunteers consecutively genotyped for the E23K variant also underwent OGTTs. Functional studies of the wild-type and E23K variant potassium channels were conducted.

Results: Insulin secretory responses to oral and intravenous glucose were reduced by approximately 40% in glucose-tolerant subjects homozygous for E23K. Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity. The hyperinsulinemic-euglycemic clamp revealed that hepatic insulin sensitivity is approximately 40% greater in subjects with the E23K variant, and these subjects demonstrate increased insulin sensitivity after oral glucose. The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.

Conclusions: The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion. Initially, insulin sensitivity is enhanced, thereby maintaining normal glucose tolerance. Presumably, over time, as insulin secretion falls further or insulin resistance develops, glucose levels rise resulting in type 2 diabetes.

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Related in: MedlinePlus

Reduced ATP and sulfonyurea sensitivity of mutant E23K channels. A: Representative currents (at −50 mV) from inside-out membrane patches from COS cells expressing KATP channel (Kir6.2 + SUR1): homomeric E23 channels (E/E), K23 channels (K/K), or heteromeric E23 and K23 channels (E/K). Patches were exposed to differing [ATP], and baseline current was determined by exposure to ATP (5 mmol/l). B: Steady-state dependence of membrane current on [ATP] (relative to current in zero ATP [Irel]) for E23- and K23-containing channels. K1/2 ATP = 7.5 μmol/l (E/E) and 16 μmol/l (K/K). Data points represent means ± SE (n = 24–28 patches). The fitted lines correspond to least-squares fits of a Hill equation (see research design and methods). **P < 0.01 vs. E/E channels by unpaired Student's t test (two tailed assuming equal variance). C: Representative currents recorded from inside-out membrane patches containing homomeric E/E or mutant K/K channels at −50 mV and in response to varying [tolbutamide]. Zero-channel current was determined by application of ATP (5 mmol/l). D: Steady-state dependence of current on [tolbutamide] (relative to current in zero tolbutamide [Irel]) for E/E (○) and K/K (●) variant channels (from records such as those shown in C). Data points represent the means ± SE (n = 6–19 patches). For all channels, the lines are fits of the product of two Hill components, each of the form (Irel = 1/([1 + {[Tolb]/K1/2}H]), with H fixed at 1.3 in each case (see research design and methods). The relative fraction and K1/2 values of each component were varied. The high-affinity component was 53 and 44% for wild-type and K/K channels, respectively. *P < 0.05 vs. wild-type KATP channel by unpaired Student's t test. The shaded box shows the reported range of serum tolbutamide concentrations from a cohort of 37 type 2 diabetic subjects receiving sulfonylurea therapy (49).
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Figure 3: Reduced ATP and sulfonyurea sensitivity of mutant E23K channels. A: Representative currents (at −50 mV) from inside-out membrane patches from COS cells expressing KATP channel (Kir6.2 + SUR1): homomeric E23 channels (E/E), K23 channels (K/K), or heteromeric E23 and K23 channels (E/K). Patches were exposed to differing [ATP], and baseline current was determined by exposure to ATP (5 mmol/l). B: Steady-state dependence of membrane current on [ATP] (relative to current in zero ATP [Irel]) for E23- and K23-containing channels. K1/2 ATP = 7.5 μmol/l (E/E) and 16 μmol/l (K/K). Data points represent means ± SE (n = 24–28 patches). The fitted lines correspond to least-squares fits of a Hill equation (see research design and methods). **P < 0.01 vs. E/E channels by unpaired Student's t test (two tailed assuming equal variance). C: Representative currents recorded from inside-out membrane patches containing homomeric E/E or mutant K/K channels at −50 mV and in response to varying [tolbutamide]. Zero-channel current was determined by application of ATP (5 mmol/l). D: Steady-state dependence of current on [tolbutamide] (relative to current in zero tolbutamide [Irel]) for E/E (○) and K/K (●) variant channels (from records such as those shown in C). Data points represent the means ± SE (n = 6–19 patches). For all channels, the lines are fits of the product of two Hill components, each of the form (Irel = 1/([1 + {[Tolb]/K1/2}H]), with H fixed at 1.3 in each case (see research design and methods). The relative fraction and K1/2 values of each component were varied. The high-affinity component was 53 and 44% for wild-type and K/K channels, respectively. *P < 0.05 vs. wild-type KATP channel by unpaired Student's t test. The shaded box shows the reported range of serum tolbutamide concentrations from a cohort of 37 type 2 diabetic subjects receiving sulfonylurea therapy (49).

Mentions: To examine the molecular basis for the differences in insulin secretion, we transiently expressed Kir6.2 with residue 23, being either glutamate (E23) or lysine (K23), together with the SUR1 subunit and measured ATP sensitivity in excised membrane patches (Fig. 3A). Homomeric K23 channels (K/K) exhibit a modest, yet significant, decrease in ATP inhibition compared with homomeric E23 (E/E) channels (K1/2,ATP = 16 μmol/l [n = 28 patches] and 7.5 μmol/l [n = 24 patches], respectively). A similar relative shift in ATP sensitivities was observed in the presence of physiological (2 mmol/l) Mg2+ for E/E and K/K channels (K1/2,ATP = 19 μmol/l [n = 19 patches] and 26 μmol/l [n = 17 patches], respectively). In contrast, the averaged KATP channel density was not different between cells expressing E/E or K/K channels (201 ± 59 and 128 ± 50 channels/patch, respectively; n = 11–19 patches). To recapitulate the heterozygous E23K genotype (E/K), cells were transfected with a 1:1 mixture of E/E and K/K cDNAs. Since four subunits generate the channels, five different ratios of subunits will be present in the resultant channels (1 of 16 channels will be homozygous E/E and homozygous K/K). The ensemble of expressed channels display intermediate ATP sensitivity (K1/2,ATP = 10.0 μmol/l [n = 10 patches] compared with homomeric K/K and E/E channels) (Fig. 3B). Our data are similar to those reported by Schwanstecher et al. (5) for recombinant K/K channels. For comparison, the dose-response curves are shown in Fig. 3B for two Kir6.2 mutations that impair insulin release and underlie NDM. The I182V mutation underlies a transient NDM, whereas the I296L mutation underlies a syndromic form of NDM (32,33). Importantly, the ATP sensitivities of mutant channels correlate with the severity of the disease (K1/2,ATP = 39 μmol/l for homomeric I182V [n = 17 patches] and 771 μmol/l for homomeric I296L channels [n = 5 patches]).


Kir6.2 variant E23K increases ATP-sensitive K+ channel activity and is associated with impaired insulin release and enhanced insulin sensitivity in adults with normal glucose tolerance.

Villareal DT, Koster JC, Robertson H, Akrouh A, Miyake K, Bell GI, Patterson BW, Nichols CG, Polonsky KS - Diabetes (2009)

Reduced ATP and sulfonyurea sensitivity of mutant E23K channels. A: Representative currents (at −50 mV) from inside-out membrane patches from COS cells expressing KATP channel (Kir6.2 + SUR1): homomeric E23 channels (E/E), K23 channels (K/K), or heteromeric E23 and K23 channels (E/K). Patches were exposed to differing [ATP], and baseline current was determined by exposure to ATP (5 mmol/l). B: Steady-state dependence of membrane current on [ATP] (relative to current in zero ATP [Irel]) for E23- and K23-containing channels. K1/2 ATP = 7.5 μmol/l (E/E) and 16 μmol/l (K/K). Data points represent means ± SE (n = 24–28 patches). The fitted lines correspond to least-squares fits of a Hill equation (see research design and methods). **P < 0.01 vs. E/E channels by unpaired Student's t test (two tailed assuming equal variance). C: Representative currents recorded from inside-out membrane patches containing homomeric E/E or mutant K/K channels at −50 mV and in response to varying [tolbutamide]. Zero-channel current was determined by application of ATP (5 mmol/l). D: Steady-state dependence of current on [tolbutamide] (relative to current in zero tolbutamide [Irel]) for E/E (○) and K/K (●) variant channels (from records such as those shown in C). Data points represent the means ± SE (n = 6–19 patches). For all channels, the lines are fits of the product of two Hill components, each of the form (Irel = 1/([1 + {[Tolb]/K1/2}H]), with H fixed at 1.3 in each case (see research design and methods). The relative fraction and K1/2 values of each component were varied. The high-affinity component was 53 and 44% for wild-type and K/K channels, respectively. *P < 0.05 vs. wild-type KATP channel by unpaired Student's t test. The shaded box shows the reported range of serum tolbutamide concentrations from a cohort of 37 type 2 diabetic subjects receiving sulfonylurea therapy (49).
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: Reduced ATP and sulfonyurea sensitivity of mutant E23K channels. A: Representative currents (at −50 mV) from inside-out membrane patches from COS cells expressing KATP channel (Kir6.2 + SUR1): homomeric E23 channels (E/E), K23 channels (K/K), or heteromeric E23 and K23 channels (E/K). Patches were exposed to differing [ATP], and baseline current was determined by exposure to ATP (5 mmol/l). B: Steady-state dependence of membrane current on [ATP] (relative to current in zero ATP [Irel]) for E23- and K23-containing channels. K1/2 ATP = 7.5 μmol/l (E/E) and 16 μmol/l (K/K). Data points represent means ± SE (n = 24–28 patches). The fitted lines correspond to least-squares fits of a Hill equation (see research design and methods). **P < 0.01 vs. E/E channels by unpaired Student's t test (two tailed assuming equal variance). C: Representative currents recorded from inside-out membrane patches containing homomeric E/E or mutant K/K channels at −50 mV and in response to varying [tolbutamide]. Zero-channel current was determined by application of ATP (5 mmol/l). D: Steady-state dependence of current on [tolbutamide] (relative to current in zero tolbutamide [Irel]) for E/E (○) and K/K (●) variant channels (from records such as those shown in C). Data points represent the means ± SE (n = 6–19 patches). For all channels, the lines are fits of the product of two Hill components, each of the form (Irel = 1/([1 + {[Tolb]/K1/2}H]), with H fixed at 1.3 in each case (see research design and methods). The relative fraction and K1/2 values of each component were varied. The high-affinity component was 53 and 44% for wild-type and K/K channels, respectively. *P < 0.05 vs. wild-type KATP channel by unpaired Student's t test. The shaded box shows the reported range of serum tolbutamide concentrations from a cohort of 37 type 2 diabetic subjects receiving sulfonylurea therapy (49).
Mentions: To examine the molecular basis for the differences in insulin secretion, we transiently expressed Kir6.2 with residue 23, being either glutamate (E23) or lysine (K23), together with the SUR1 subunit and measured ATP sensitivity in excised membrane patches (Fig. 3A). Homomeric K23 channels (K/K) exhibit a modest, yet significant, decrease in ATP inhibition compared with homomeric E23 (E/E) channels (K1/2,ATP = 16 μmol/l [n = 28 patches] and 7.5 μmol/l [n = 24 patches], respectively). A similar relative shift in ATP sensitivities was observed in the presence of physiological (2 mmol/l) Mg2+ for E/E and K/K channels (K1/2,ATP = 19 μmol/l [n = 19 patches] and 26 μmol/l [n = 17 patches], respectively). In contrast, the averaged KATP channel density was not different between cells expressing E/E or K/K channels (201 ± 59 and 128 ± 50 channels/patch, respectively; n = 11–19 patches). To recapitulate the heterozygous E23K genotype (E/K), cells were transfected with a 1:1 mixture of E/E and K/K cDNAs. Since four subunits generate the channels, five different ratios of subunits will be present in the resultant channels (1 of 16 channels will be homozygous E/E and homozygous K/K). The ensemble of expressed channels display intermediate ATP sensitivity (K1/2,ATP = 10.0 μmol/l [n = 10 patches] compared with homomeric K/K and E/E channels) (Fig. 3B). Our data are similar to those reported by Schwanstecher et al. (5) for recombinant K/K channels. For comparison, the dose-response curves are shown in Fig. 3B for two Kir6.2 mutations that impair insulin release and underlie NDM. The I182V mutation underlies a transient NDM, whereas the I296L mutation underlies a syndromic form of NDM (32,33). Importantly, the ATP sensitivities of mutant channels correlate with the severity of the disease (K1/2,ATP = 39 μmol/l for homomeric I182V [n = 17 patches] and 771 μmol/l for homomeric I296L channels [n = 5 patches]).

Bottom Line: Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity.The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT

Objective: The E23K variant in the Kir6.2 subunit of the ATP-sensitive K(+) channel (K(ATP) channel) is associated with increased risk of type 2 diabetes. The present study was undertaken to increase our understanding of the mechanisms responsible. To avoid confounding effects of hyperglycemia, insulin secretion and action were studied in subjects with the variant who had normal glucose tolerance.

Research design and methods: Nine subjects with the E23K genotype K/K and nine matched subjects with the E/E genotype underwent 5-h oral glucose tolerance tests (OGTTs), graded glucose infusion, and hyperinsulinemic-euglycemic clamp with stable-isotope-labeled tracer infusions to assess insulin secretion, action, and clearance. A total of 461 volunteers consecutively genotyped for the E23K variant also underwent OGTTs. Functional studies of the wild-type and E23K variant potassium channels were conducted.

Results: Insulin secretory responses to oral and intravenous glucose were reduced by approximately 40% in glucose-tolerant subjects homozygous for E23K. Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity. The hyperinsulinemic-euglycemic clamp revealed that hepatic insulin sensitivity is approximately 40% greater in subjects with the E23K variant, and these subjects demonstrate increased insulin sensitivity after oral glucose. The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.

Conclusions: The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion. Initially, insulin sensitivity is enhanced, thereby maintaining normal glucose tolerance. Presumably, over time, as insulin secretion falls further or insulin resistance develops, glucose levels rise resulting in type 2 diabetes.

Show MeSH
Related in: MedlinePlus