Page 1 of 35 Diabetes
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1 KCNQ1 Long QT syndrome patients have hyperinsulinemia and symptomatic
3 Running title: Hyperinsulinemia and hypoglycemia in KCNQ1 LQTS
4
5 Signe S. Torekov, PhD1,2; Eva Iepsen, MD1,2; Michael Christiansen, MD3, Allan Linneberg,
6 PhD4; Oluf Pedersen, DMSci1,2,5; Jens J. Holst, DMSci1,2; Jørgen K. Kanters, PhD1,6; Torben
7 Hansen, PhD2,7
8 1Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of
9 Copenhagen, 2200 Copenhagen, Denmark, 2Novo Nordisk Foundation Center for Basic Metabolic
10 Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen.
11 3Department of Clinical Biochemistry, SSI, 2300 Copenhagen, 4Research Centre for Prevention and
12 Health, Glostrup University Hospital, 2600 Glostrup, 5Faculty of Health Sciences, University of
13 Aarhus, 8000 Aarhus, Denmark. Denmark. 6Gentofte, Aalborg & Herlev University Hospitals, 2950
14 Hellerup, Denmark, 7Faculty of Health Sciences, University of Southern Denmark, 5230 Odense,
15 Denmark.
16
17 Corresponding author
18 Signe S. Torekov, Assistant Professor, PhD
19 Department of Biomedical Sciences and Novo Nordisk Foundation Center for Basic Metabolic
20 Research, Faculty of Health and Medical Sciences, University of Copenhagen,
21 Blegdamsvej 3B, DK-2200 Copenhagen N, Denmark, TEL +45 353 27509,
23
24 Word count: 3,857 words, 6 figures, 2 tables and Supplementary appendix (2 tables and 1 figure)
Diabetes Publish Ahead of Print, published online December 18, 2013 Diabetes Page 2 of 35
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1 Patients with loss of function mutations in KCNQ1 have KCNQ1 long QT syndrome (LQTS).
2 KCNQ1 encodes a voltage-gated K+ channel located in both cardiomyocytes and pancreatic beta
3 cells. Inhibition of KCNQ1 in beta cells increases insulin secretion. Therefore KCNQ1 LQTS
4 patients may exhibit increased insulin secretion.
5 Fourteen patients, from six families, diagnosed with KCNQ1 LQTS were individually matched to
6 two randomly chosen BMI-age-gender-matched control participants and underwent oral glucose
7 tolerance test, hypoglycemia questionnaire and continuous glucose monitoring.
8 KCNQ1 mutation carriers showed increased insulin release (AUC:45.6±6.3vs.26.0±2.8 min*nmol/l
9 insulin), and beta cell glucose sensitivity and had lower levels of plasma glucose and serum
10 potassium upon oral glucose stimulation and increased hypoglycemic symptoms. Prolonged oral
11 glucose tolerance test in four available patients and matched controls revealed hypoglycemia in
12 carriers after 210 min. (range:1.4–3.6vs.4.1–5.3 mmol/l glucose), and 24-hour glucose profiles
13 showed that the patients spent 77±18 min. per 24-hours in hypoglycemic states (<3.9 mmol/l
14 glucose) with 36±10 min. <2.8 mmol/l glucose vs. 0 min (<3.9 mmol/l glucose) for the control
15 participants.
16 The phenotype of patients with KCNQ1 LQTS, caused by mutations in KCNQ1, includes, besides
17 long QT, hyperinsulinemia, clinically relevant symptomatic reactive hypoglycemia and low
18 potassium following an oral glucose challenge, suggesting that KCNQ1 mutations may explain
19 some cases of “essential” reactive hypoglycemia.
20 Page 3 of 35 Diabetes
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1 The blood glucose level increases after a meal intake, leading to formation of ATP in the beta-cell,
2 closure of the ATP-dependent potassium channel and thereby a reduction of the ATP-sensitive
3 potassium current, depolarization of the beta-cell and increased insulin secretion. Kv7.1 is another
4 potassium channel causing a voltage-gated repolarization current. Kv7.1 is encoded by KCNQ1 and
5 expressed both in beta cells(1) and cardiomyocytes(2). Functional mutations in KCNQ1 lead to
6 KCNQ1 Long QT syndrome (LQTS) with a predicted population prevalence of 1:2000, a disease
7 characterized by a delayed cardiac repolarization, prolonged QT interval on the ECG, syncope,
8 malignant arrhythmias and sudden death(3), but the clinical and physiological significance of
9 functional KCNQ1 mutations in beta cells is unknown. Interestingly, carriers of frequent intronic
10 single nucleotide polymorphisms (SNP)s (rs2237892, rs2237895, rs2237897) in KCNQ1, have
11 increased susceptibility for type 2 diabetes(4;5) and impaired beta cell function(6;7). Recent studies
12 show that inhibition of Kv7.1 in beta cells increases exocytosis and insulin secretion(1), due to
13 delayed repolarization(8) and KCNQ1 knock-down with siRNA similarly increases insulin
14 exocytosis and secretion(1). Over-expression of KCNQ1 in cultured MIN6 cells decreases both
15 glucose, pyruvate and tolbutamide induced insulin secretion (9) and the intronic SNP risk allele of
16 KCNQ1 rs2237895 decreases exocytosis and insulin secretion (1), suggesting that the risk alleles of
17 the intronic KCNQ1 SNPs are gain of function polymorphisms and increase the susceptibility of
18 Type 2 diabetes due to increased KCNQ1 expression and thereby decreased insulin exocytosis and
19 secretion(1;9). Consequently, we hypothesize that LQTS patients with loss of function mutations of
20 KCNQ1, may exhibit increased insulin secretion due to delayed repolarization of the beta-cell
21 causing increased exocytosis. In this study we show that KCNQ1 LQTS patients have postprandial
22 hyperinsulinemia, reactive hypoglycemia and experience symptoms of hypoglycemia.
23 Diabetes Page 4 of 35
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1 Research Design and Methods
2 Study participants
3 Fourteen patients, from six nuclear families, diagnosed with KCNQ1 LQTS(10) were recruited from
4 the outpatient clinics at the cardiology departments at Gentofte and Aalborg hospitals, Denmark.
5 Each patient was matched with respect to BMI, age and gender with two randomly chosen control
6 subjects from the Inter99 (11;12) or the Health(13) studies. A computer algorithm was applied to
7 randomly select matching control subjects, to be invited to participate in the study, solely based on
8 their best match with gender, BMI and age. Six out of fourteen patients were in standard long QT
9 therapy with beta-blocking agents and six out of fourteen patients had experienced syncope (of
10 these six, three were taking beta-blocking agents). Before the examinations all subjects were fasting
11 overnight (including not taking any medication), and were free of any medication in the morning of
12 examination. The characteristics of the two study groups are shown in table 1. Informed written
13 consent was obtained from all individuals before participation. The study was approved by the
14 Ethical Committee of Copenhagen County (H-4-2010-036) and was in accordance with the
15 principles of the Helsinki Declaration.
16 Genetics
17 The long QT syndrome patients were originally screened for mutations in genes known to cause
18 long QT syndrome: KCNQ1, KCNH2, KCNE1, KCNE2, SCN5A, using published single strand
19 conformation polymorphism (SSCP)-techniques followed by bi-directional sequencing of aberrant
20 conformers, as previously described(14), and were found to be heterozygous carriers of the
21 missense mutations p.H363N(15) (n = 4), p.R366W(16) (n = 4), the frameshift/insertion mutation
22 p.R401Pfs 62*(2) (n = 2), and the nonsense mutation p.Q530X(17) (n = 4). The protein reference
23 sequence used was NP_000209.2.
24 Oral glucose tolerance test, electrocardiogram and hypoglycemia questionnaire Page 5 of 35 Diabetes
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1 After an overnight fast, blood samples for measurements of plasma glucose, serum insulin, serum
2 C-peptide, serum proinsulin, serum potassium, plasma GLP-1, plasma GIP and plasma glucagon
3 were taken prior to a standard 75 g oral glucose tolerance test (OGTT). Blood sampling was
4 repeated every 15 minutes until 180 minutes after start of the OGTT. Before each blood sampling
5 ECG recordings were made with a MAC1600 ECG machine (GE Healthcare, Milwaukee, WI). QT
6 intervals were corrected by heart rate with Bazett’s correction method.
7 An adapted standard hypoglycemia questionnaire (18)
8 (www.hypoglycemia.asn.au/2012/hypoglycemia-questionnaire;
9 www.lisasaslove.com/uploads/8/4/4/9/8449235/hypoglycemia_questionnaire.pdf)
10 (see supplementary table 1) was filled at home by all participants except one patient (F, 56 years)
11 who died in between the physical examinations and answering the questionnaire. The patient
12 smoked, lost consciousness, and set herself on fire and later died at the hospital, due to her burns.
13 Whether the unconsciousness was due to self limiting arrhythmias, hypoglycemia or other causes is
14 unknown.
15 Prolonged OGTT and continuous blood glucose measurements
16 Follow-up studies were made in four available patients and four matched control individuals (Table
17 1) who underwent a prolonged OGTT (6 hours) and continuous glucose measurements with Ipro2
18 (Medtronic, Watford, UK) for 3 up to 7 days. The group of four out of 14 patients represented those
19 that agreed to participate in additional studies for up to 7 days duration. The OGTT´s were carried
20 out as described above except that they were continued until 6 hours after glucose ingestion. The
21 continuous glucose measurements were conducted according to the manufacturer´s manual (Ipro2,
22 Medtronic, Watford, UK). IPro2 uses a retrospective algorithm to convert sensor signal to glucose
23 values based on self-monitored capillary blood glucose readings. Therefore, all participants
24 received a glucose meter (Contour; Bayer Diabetes Care, Lyngby, Denmark) to ensure uniform Diabetes Page 6 of 35
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1 measurements for conversion of sensor signals. Hypoglycemic time, normoglycemic time, and
2 hyperglycemic time were calculated from the 24-h glucose profiles and defined as time spent with a
3 blood glucose <3.9 (and <2.8), between 3.9 and 7.8, and >7.8 mmol/L, respectively (Table 2). None
4 of the four patients for the follow-up studies were taking beta-blocking agents.
5 Biochemical and anthropometric measures
6 BMI was calculated as weight (kg) / height (m)2. The percentage of fat was measured with
7 bioimpedance analyzer, Biodynamics BIA 310e (Biodynamics, Seattle, Washington, USA). Plasma
8 glucose was measured by a glucose oxidase method (Granutest, Merck, Darmstadt, Germany) with
9 a detection limit of 0.11 mmol/l and intra- and inter-assay coefficients of variation of <1%.
10 Radioimmunological determinations of fully processed glucagon and total plasma glucagon-like-
11 peptide (GLP) -1 and gastric inhibitory peptide (GIP) were performed as described(19-21). The
12 analytical detection limit was 1 pmol/l and intra- and inter-assay coefficients of variation were <6%
13 and <15%, respectively. Serum insulin (excluding des (31, 32) split products and intact proinsulin)
14 was measured using the AutoDELFIA insulin kit (Perkin-Elmer, Wallac, Turku, Finland). Serum C-
15 peptide concentrations were measured by a time-resolved fluoroimmunoassay (Auto-DELFIA C-
16 peptide kit; Perkin-Elmer/Wallac, Turku, Finland). Total intra- and inter-assay coefficients of
17 variation were <3% and 4%, respectively. The analytical detection limit was 3 pmol/l. Proinsulin
18 was measured by ELISA using Sunrise Touchscreen photometer (Tecan Austria GmbH, Salzburg,
19 Austria). Total intra- and inter-assay coefficients of variation were <4% and 7%, respectively. The
20 analytical detection limit was 0·3 pmol/l. Serum potassium was measured using Vitros 5600 (Otho
21 Clinical Diagnostics, Cedex, France). Total intra- and inter-assay coefficients of variation were
22 <1%. The analytical detection limit was 1 mmol/l.
23 Sample size Page 7 of 35 Diabetes
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1 OGTT: The aim of the study was to test differences in insulin secretion among 14 KCNQ1 LQTS
2 patients and 28 matched controls. With a mean difference in the maximal insulin response at 60 min
3 of 212 pmol/l insulin, we will be able to reject the null hypothesis that this response difference is
4 zero with a probability (power) > 0.8.
5 Prolonged OGTT: The aim of the study was to test differences in glucose level among 4 KCNQ1
6 LQTS patients and 4 matched controls. With a mean difference in the minimum glucose level at
7 210 min of 1.5 mmol/l glucose, we will be able to reject the null hypothesis that this response
8 difference is zero with a probability (power) > 0.8. Sample size calculation was carried out with PS
9 Power and sample size calculation 3.0.34 (22;23).
10 Data calculations
11 Area under the curve (AUC) was calculated using GradPad Prism 5. Insulinogenic index (serum
12 insulin at 30-min (pmol/l) - fasting serum insulin (pmol/l)) / (plasma glucose at 30-min (mmol/l) -
13 fasting plasma glucose (mmol/l)) was calculated as a measure of beta-cell response to an oral
14 glucose load(24). Whole-body insulin sensitivity was estimated from oral glucose tolerance data by
15 applying the Matsuda insulin sensitivity index (10,000/√((fasting plasma glucose × fasting serum
16 insulin) × (mean plasma glucose × mean serum insulin during OGTT))(25;26). Fasting homeostasis
17 model assessment of insulin resistance (HOMA-IR) index was calculated as (fasting plasma glucose
18 (mmol/l) × fasting serum insulin (pmol/l)) / 22.5). Prehepatic insulin secretion rates (ISR) for each
19 individual were calculated using a two-compartment model of C-peptide kinetics and population-
20 based C-peptide kinetic parameters allowing calculations of values adjusted for clinical status, age,
21 weight and height, and sex using the ISEC software(27;28). The individually calculated ISR values,
22 for the time points 0 - maximum plasma glucose levels, were plotted against the individual plasma
23 glucose concentrations to establish the dose response relationship for each individual. The slopes of Diabetes Page 8 of 35
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1 these approximately linear relationships were regarded as measures of beta cell responsiveness to
2 glucose(29).
3 Statistics
4 Statistical analysis was carried out with a mixed model ANOVA with repeated measurements (SAS
5 version 9.2 PROC MIXED, Cary, NC) contrasting patient results versus control subjects in
6 matching pairs (SAS version 9.2 PROC MIXED, Cary, NC) and tested with Scheffes post-Hoc test
7 for multiple comparison. Values of insulin were logarithmically transformed before analysis. The
8 data are shown as mean ± SEM. P values are given for the overall ANOVA with asterisks in figures
9 indicating significantly different time points. The total hypoglycemia frequency score and total
10 hypoglycemia severity score of each participant were calculated from the sum of the point scores
11 given for each question answer (Figure 4, supplementary table 1 and 2). The differences in scores
12 between patients and control participants were tested with Students t-test. Regression analyses
13 between the QT interval and glucose, insulin or potassium levels were made using SAS version 9.2
14 PROC MIXED, Cary, NC A p-value < 0.05 was considered significant.
15
16
17 Results
18 The KCNQ1 LQTS patients had lower plasma glucose levels compared to matched control
19 participants three hours after glucose ingestion (4.4 ± 0.3 vs. 5.4 ± 0.5 mmol/l glucose, P = 0.03,
20 Figure 1) and had significantly increased insulin responses to oral glucose stimulation (AUC: 45.6
21 ± 6.3 vs. 26.0 ± 2.8 min*nmol /l insulin), P < 10-5 as well as significantly increased serum levels of
22 C-peptide and pro-insulin and higher ISR, P < 10-5 (Figure 2). Beta cell sensitivity to glucose,
23 evaluated as the slope of the relation between ISR and glucose was significantly greater in mutation
24 carriers (2.8 ± 0.31 vs. 1.9 ± 0.18, p < 10-5, Figure 3) and so was the insulinogenic index (41.5 ± 6.6 Page 9 of 35 Diabetes
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1 vs. 24.3 ± 3.7, P < 10-3). Hypoglycemia frequency and severity score was significantly higher
2 among patients compared to control participants, P < 10-3 (Figure 4 and Supplementary table 1).
3 There was no difference in HOMA-IR between the groups (10.0 ± 1.0 vs. 9.5 ± 1.3, P > 0.05) while
4 the Matsuda index was significantly lower in the patients (11.0 ± 1.0 vs. 16.7 ± 1.5, P < 10-3).
5 Serum potassium levels were significantly lower during the OGTT in mutation carriers compared to
6 control participants, P < 10-4. There were no significant differences between the groups in
7 circulating levels of glucagon, GLP-1 and GIP, P > 0.05 (Figure 5).
8 During the OGTT at the time corresponding to insulin peak levels, the patients significantly
9 increased their QT interval by 10 ± 2 ms, as compared to 5 ± 3 ms in control subjects, P < 10-3.
10 There was no significant correlation between the QT interval and circulating glucose, insulin or
11 potassium levels during the 3- hour OGTT (P > 0.05).
12 Follow-up studies, including a prolonged oral glucose tolerance test for 6 hours, undertaken in four
13 available patients with matching control individuals, showed that all four patients were
14 hypoglycemic (plasma glucose < 3.9 mmol/l glucose) 3.5 - 5 hours after glucose ingestion in
15 contrast to control subjects who remained normoglycemic (plasma glucose nadir: 1.4, 2.5, 3.3 and
16 3.6 mmol/l vs. nadir: 4.1, 4.3, 4.7 and 5.0 mmol/l), P < 10-4 (Figure 6). Also serum potassium levels
17 were lower 3-6 hours after oral glucose load among patients, P < 10-3 (Figure 6). The same
18 participants underwent continuous glucose measurements for 3 up to 7 days. Twenty-four hour
19 glucose profiles showed that the KCNQ1 LQTS patients spent 77 ± 18 min. per 24 hours in
20 hypoglycemic states (< 3.9 mmol/l glucose) with 36 ± 10 min. of this period spent at < 2.8 mmol/l
21 glucose vs. 0 min (< 3.9 mmol/l glucose) for the control participants, P < 0.05 (Table 2). The
22 hypoglycemic episodes occurred 3-5 hours after meals. The patient subgroup reported symptoms
23 like extreme cravings for sweets, irritability, weakness, dizziness, shakiness, depression or mood
24 swings and anxiety or nervousness as well as night awakening and severe night sweats correlating Diabetes Page 10 of 35
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1 to the exact time points of hypoglycemia, whereas the matching subgroup of control participants did
2 not report any of the above mentioned symptoms during the period of continuous glucose
3 monitoring.
4 There were no differences in baseline observations (Table 1) or metabolic responses between the
5 group of patients that participated in the follow-up studies and the whole patient group (P > 0.7).
6 There were no differences in baseline observations or metabolic responses between the group of
7 patients taking beta-blocking agents (6/14 patients) or patients who had experienced syncope (6/14
8 patients) and the group that did not (P > 0.8). Furthermore, after exclusion of the group of patients
9 taking beta-blocking agents and their corresponding control participants from the analyses, a similar
10 difference remained between patients and control participants with regards to glucose, insulin and
11 potassium response (P < 0.05, Supplementary figure 1).
12
13 Discussion
14 We here report a novel extracardial phenotype in KCNQ1 LQTS patients: postprandial
15 hyperinsulinemic reactive hypoglycemia with clinically relevant symptoms of hypoglycemia. Until
16 now, although KCNQ1 is widely expressed, the only extracardial symptom reported in LQTS
17 patients is sensorineural deafness in patients homozygous for KCNQ1 loss of function mutations.
18 The lower postprandial glucose levels were recorded three hours after glucose ingestion indicating a
19 delayed glycemic reaction to hyperinsulinemia, similar to what has been observed in genetically
20 determined hyperinsulinaemia due to insulin receptor mutations (30). Therefore, we performed a
21 prolonged oral glucose tolerance test for 6 hours in four patients that were available for extended
22 examination. Indeed, we observed that the patients became markedly hypoglycemic three and a half
23 hours after glucose ingestion in contrast to the matched control participants. Consistent with this
24 observation, twenty-four hour glucose profiles showed that the KCNQ1 LQTS patients had Page 11 of 35 Diabetes
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1 symptomatic hypoglycemic episodes in their own living environment, three to five hours after meal
2 intake. Furthermore, the low circulating glucose levels observed in KCNQ1 LQTS patients hours
3 after an oral glucose load are in agreement with observations of low glucose levels in KCNQ1 KO
4 mice(31).
5 The patients also had lower serum potassium levels. This is presumably due to insulin activating the
6 sodium-potassium ATPase to move potassium from the extracellular to the intracellular
7 compartment(32). Other mechanisms leading to low potassium levels might include fecal loss of
8 potassium as reported for KCNQ1 KO mice(33).
9 Patients with KCNQ1 LQTS are characterized by rare episodes of syncope, ventricular
10 tachyarrhythmia and cardiac arrest, which hitherto have been ascribed to their long QT interval.
11 However, we now show that these patients besides long QT interval also suffer from
12 hyperinsulinemic reactive hypoglycemia along with low potassium levels and symptoms of
13 hypoglycemia. Hypoglycemia may cause sympathetic activation which is associated with increased
14 propensity for arrhythmias and sudden death (34;35) in KCNQ1 LQTS patients. The insulin-
15 induced hypokalemia will also decrease the repolarisation reserve, by a reduction of the rapid
16 delayed rectifier current IKr(36), thus further increasing the risk of cardiac arrhythmia and cardiac
17 arrest(34). Furthermore, in a population-based study of 2570 elderly people without diabetes it was
18 demonstrated that hyperinsulinemia was associated with significantly increased QT-interval and
19 increased risk of sudden death(37). In addition, a recent study revealed that QT prolongation and
20 hypokalemia were common in diabetes patients with severe hypoglycemia, which increased their
21 risk of fatal arrhythmia and death(38). The combination of hyperinsulinemia, symptomatic
22 postprandial hypoglycemia and low serum potassium levels might thus further increase the
23 propensity for cardiac events in KCNQ1 LQTS patients. Diabetes Page 12 of 35
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1 None of the LQTS patients had previously been diagnosed with reactive hypoglycemia. The clinical
2 symptoms characterizing this patient group, e.g. syncope, have previously been solely ascribed to
3 their arrhythmia. However, e.g. syncope could also be a sign of hypoglycemia and our study
4 suggests that the KCNQ1 LQTS patients also have symptoms related to hypoglycemia.
5 The higher sensitivity of the beta cell to increments in plasma glucose which was a feature of the
6 mutation carriers is in agreement with the observations found in beta cell studies when blocking
7 KCNQ1(1;8). Thus, our findings confirm that blocking KCNQ1 increases insulin release in vivo
8 presumably due to prolonged depolarization of the beta-cell causing calcium influx and insulin
9 exocytocis. This observation also correlate with the genome wide association study observations
10 that intronic SNPs in KCNQ1 are associated with type 2 diabetes(4;5) and reduced serum insulin
11 levels(6;7), which could be secondary to increased expression of the channel and thereby decreased
12 insulin exocytosis(1). Taken together these findings underline that the voltage-gated K+ channel
13 encoded by KCNQ1 is a key player in insulin secretion.
14 Pancreatic β-cells express a variety of K+ channels regulated by voltage (Kv channels) and/or by the
15 intracellular Ca2+ concentration (KCa channels). Inhibition of Kv channels with TEA+ extends
16 action potential duration. Consequently, blockade of Kv channels is a potent tool to augment insulin
17 release(39;40). In the absence of KCNQ1 channels the repolarization is delayed, leaving it to the
18 other K+ channels to repolarize the beta-cell.
19 One question in the questionnaire included timing of the hypoglycemia event, whereas the other
20 questions do not distinguish between post-prandial or fasting symptoms. However, as activation of
21 Kv channels requires membrane depolarization, targeting Kv channels affect insulin secretion only
22 in the presence of elevated glucose concentrations or other depolarizing stimuli (39;40). This is in
23 agreement with our finding that insulin secretion is only abnormally increased post-prandially and
24 not during fasting when glucose levels are low. Page 13 of 35 Diabetes
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1 The mutation carriers exhibited moderately decreased insulin sensitivity during the first hours after
2 glucose stimulation, but not in the fasted state. This postprandial insulin resistance might be a
3 protective mechanism against severe postprandial hyperinsulinemic hypoglycemia. The observation
4 is reminiscent of the syndrome of autosomal-dominant hyperinsulinemic hypoglycemia linked to a
5 mutation in the human insulin receptor gene where hyperinsulinemia co-exists with a moderate
6 insulin resistance(30).
7 Our findings suggest that functional KCNQ1 mutations underlie some cases of “essential”
8 postprandial hypoglycemia. This syndrome is characterized by appearance of reactive
9 hypoglycemia occurring up till four hours after food intake without conspicuous cause. Thus, ECG
10 monitoring and genetic testing should be considered when other causes of reactive hypoglycemia
11 (e.g. gastrointestinal surgery or medications) have been excluded.
12 The glucagon levels were not significantly different between patients and control participants at the
13 time of hypoglycemia, although acute hypoglycemia under normal physiological circumstances will
14 increase glucagon levels. This lack of appropriate glucagon response may be due to the counter-
15 regulatory response impairment observed when recurrent hypoglycemia occurs (41), as we indeed
16 observed happened in the patients during the continuous glucose monitoring. Furthermore, a
17 mutated KCNQ1 channel in pancreatic delta cells may increase somatostatin secretion, in the same
18 manner as insulin is increased, and thereby inhibit glucagon. Both conditions may contribute to
19 worsen the hypoglycemia. Also, a relatively elevated insulin secretion from pancreatic beta-cells of
20 KCNQ1 patients may have suppressed their alpha-cell glucagon response. As compared to
21 controls, the relatively lower glucagon levels in KCNQ1 mutation patients during both OGTTs are
22 consistent with a paracrine effect of beta-cell hypersecretion suppressing alpha-cell glucagon
+ 23 release (42). In addition, studies have shown that lack of another K channel, namely the KATP
24 channel causes beta-cell depolarization and insulin secretion. This is in contrast to what happens in Diabetes Page 14 of 35
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1 the alpha cell, where the depolarization caused by lack of the KATP channel, is associated with
2 reduced rather than increased electrical activity and thereby low glucagon secretion(43). Because
3 alpha-cells possess a different complement of voltage-gated ion channels involved in action
4 potential generation than beta cells (43), it is likewise plausible that lack of the KCNQ1 channel
5 causes insulin secretion in beta cells, but inhibits glucagon secretion from alpha cells.
6 A previous study of carriers of frequent intronic SNPs in KCNQ1 indicated that impairment of
7 incretin secretion might be involved in the reduced beta-cell function among SNP carriers(44). In
8 our study of carriers of functional KCNQ1 mutations, we do not find any difference in circulating
9 incretin levels between patients and control individuals. This is in agreement with recent studies of
10 L-cells(45) and recent human genetic studies(46), indicating that KCNQ1 does not have a major
11 influence on incretin secretion.
12 Six out of 14 patients were in standard long QT type 1 treatment with beta-blocking agents.
13 However, all subjects were fasting overnight and free of any medication for at least 24 hours before
14 the morning of examination. Furthermore, since there was no difference in patient baseline
15 characteristics or metabolic responses among the group of patients taking beta-blocking agents and
16 the group that did not, and since studies have shown that beta-adrenergic blockade has little effect
17 on glucose regulation(47), the beta-blockers are unlikely to explain the metabolic differences
18 between patients and control individuals. In addition, excluding the group of patients taking beta-
19 blocking agents and their corresponding control participants from the analyses did not affect the
20 highly significant difference between patients and control participants with regards to all measured
21 metabolic responses.
22 We did not test other stimuli than glucose or study patients with other known LQTS causing gene
23 alterations both of which would be of interest for future studies. Page 15 of 35 Diabetes
15
1 In conclusion, besides prolonged QT interval, patients with KCNQ1 LQTS were characterized by
2 hyperinsulinemia upon an oral glucose load, postprandial hypoglycemia, symptoms of clinical
3 hypoglycemia and lower potassium levels, all of which increase risk of cardiac events. We confirm
4 that the voltage-gated K+ channel encoded by KCNQ1 is involved in insulin secretion and suggest
5 that KCNQ1 mutations may explain some cases of “essential” reactive hypoglycemia.
6
7 Acknowledgements
8 Author contributions
9 SS Torekov 1-5; E Iepsen 2,4.5, M Christiansen 1,4,5; A Linneberg 1,4,5; O Pedersen 1,2,4,5; JJ
10 Holst 1-5; JK. Kanters 1,2,4,5; Torben Hansen 1,2,4,5 were responsible for (1) conception and
11 design, (2) analysis and interpretation of data, (3) drafting the article, (4) revising it critically for
12 important intellectual content; and (5) final approval of the version to be published.
13 Statements of assistance
14 We are most grateful to the study participants. From the Novo Nordisk Foundation of Basic
15 metabolic Research, University of Copenhagen, we thank A. Forman, T. Lorentzen, M. Modest and
16 G. Klavsen for technical assistance and A. Nielsen for data management. From the Department of
17 Biomedical Sciences, University of Copenhagen, we thank S. Pilgaard and L. Albæk for technical
18 assistance.
19 Guarantors
20 SS Torekov, O Pedersen, JJ Holst, JK. Kanter and Torben Hansen
21 Funding
22 The research was supported by grants from the Danish Research Council, the Danish Strategic
23 Research Foundation, the Fraenkel Foundation, the Novo Nordisk Foundation, the Lundbeck
24 Foundation Centre of Applied Medical Genomics for Personalized Disease Prediction, Prevention Diabetes Page 16 of 35
16
1 and Care, the University of Copenhagen, and the University Investment Capital (UNIK): Food,
2 Fitness & Pharma for Health and Disease from the Danish Ministry of Science, Technology and
3 Innovation.
4 Conflict of interests
5 No conflicts of interest relevant for the present studies.
6 Disclosure
7 The study was presented at EADS, Barcelona, September 2013
8
9
10
11
12 Page 17 of 35 Diabetes
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1 Table 1 Baseline characteristics of study participants
Characteristic KCNQ1 Patientsubgroup Control group Controlsubgroup Ppatient Psubgroup
Long QT patients for follow up for follow up vs. control vs whole
group
N (m/w) 14 (5/9) 4 (2/2) 28 (10/18) 4 (2/2)
Age (years) 44 ± 3 46 ± 7 46 ± 2 47 ± 7 1 0.9
BMI (kg/m2) 28.5 ± 1.7 30.2 ± 1.4 29.0 ± 1.1 30.5 ± 1·0 1 0.7
Fat % 31.1 ± 1.6 32.4 ± 1.6 31.4 ± 1.3 31.7 ± 1.7 0.9 0.8
QTB-interval (ms) 481 ± 8 480 ± 9 425 ± 4 423 ± 7 <0.0001 0.9
2
3 Diabetes Page 18 of 35
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1 Table 2 Twenty-four hour glucose profiles during continuous glucose measurements for 3 up
2 to 7 days
Blood glucose level KCNQ1 Long QT patients Control participants
mmol/l Minutes ± SEM per 24 hours Minutes ± SEM per 24 hours
(Percentage ± SEM per 24 hours) (Percentage ± SEM per 24 hours)
>7.8 30 ± 10 (2.1 ± 0.7%) 59 ± 18 (4.1 ± 1.4%)
3.9-7.8 1,333 ± 199 (92.6 ± 14.1%) 1,381 ± 251 (95.9 ± 17.4%)
<3.9* 77 ± 19 (5.3 ± 1.3%) 0
<2.8* 36 ± 10 (2.5 ± 0.7%) 0
3
4 Mean minutes ± SEM and mean percentage ± SEM per 24 hours spent in different glucose states
5 during continuous glucose measurements for 3 up to 7 days in four patients and four matched
6 control participants. *P<0.05 when analyzed patients vs. control participants for each blood glucose
7 level state
8 Page 19 of 35 Diabetes
19
1 Figure text
2 Figure 1
3 Plasma glucose levels during an oral glucose tolerance test in 14 patients with KCNQ1 long QT
4 syndrome due to functional mutations in KCNQ1 (closed circles) and 28 randomly chosen BMI-
5 gender and age-matched control participants (open squares) (Mean±SEM), P = 0.03.
6
7 Figure 2
8 Serum insulin (upper left panel), C-peptide (upper right panel), pro-insulin (lower left panel) levels
9 and insulin secretion rate (ISR) (lower right panel) during an oral glucose tolerance test in 14
10 patients with KCNQ1 long QT syndrome due to functional mutations in KCNQ1 (closed circles)
11 and 28 randomly chosen BMI-, gender-, and age-matched control participants (open squares)
12 (Mean±SEM), P < 10-5.
13
14 Figure 3
15 Illustration of the beta cell responsiveness to glucose. The calculated insulin secretion rates (ISR)
16 values were plotted against plasma glucose to establish the dose response relationship for each
17 individual. The slopes of these approximately linear relations were regarded as measures of beta
18 cell responsiveness to glucose. For illustrative purposes the mean values of glucose at time 0, 15, 30
19 and 45 were plotted on the X-axis, however all data analysis and statistics were calculated for each
20 individual. The slope was significantly greater among KCNQ1 mutation carriers (closed circles) vs.
21 control individuals (open squares) (2.8 ± 0.3 vs. 1.9 ± 0.1, P < 10-5).
22
23
24 Diabetes Page 20 of 35
20
1 Figure 4
2 Hypoglycemia questionnaire score (see supplementary table 1) of 13 patients with KCNQ1 long QT
3 syndrome due to functional mutations in KCNQ1 (black bars) and 26 randomly chosen BMI-gender
4 and age-matched control participants (white bars). The total hypoglycemia frequency score and
5 total hypoglycemia severity score of each participant were calculated from the sum of the point
6 scores given for each question answer (supplementary table 1 and 2). The differences in scores
7 between patients and control participants were tested with Students t-test. (Mean±SEM), Pfrequency <
-3 -4 8 10 , Pseverity < 10
9
10 Figure 5
11 Serum potassium (upper left panel), plasma glucagon (upper right panel), plasma GIP (lower left
12 panel) and plasma GLP-1 (lower right panel) levels, during an oral glucose tolerance test in 14
13 patients with KCNQ1 long QT syndrome due to functional mutations in KCNQ1 (closed circles)
14 and 28 randomly chosen BMI-gender and age-matched control participants (open squares). Ppotassium
-4 15 < 10 and Pglucagon, PGLP-1 and PGIP > 0.05.
16
17 Figure 6
18 Plasma glucose (upper left panel), serum potassium (upper right panel), plasma glucagon (lower left
19 panel) and serum insulin (lower right panel) levels during an extended glucose tolerance test for six
20 hours in 4 patients with KCNQ1 long QT syndrome with functional mutations in KCNQ1 (closed
21 circles) and 4 randomly chosen BMI-gender and age-matched control participants (open squares)
-4 -3 22 (Mean±SEM), Pglucose < 10 and Ppotassium and Pinsulin <10 , Pglucagon> 0.05.
23
24 Page 21 of 35 Diabetes
21
1 Figure 1
9
8
7
* 6 Plasma glucose [mmol/l] Plasma 5
4 0 50 100 150 200 Time [min] 2 Diabetes Page 22 of 35
22
1 Figure 2
2
600 * * * 4000 * * * ** *
400 3000 * *
* 2000
200 Serum insulin [pmol/l] Seruminsulin
Serum C-peptide [pmol/l] SerumC-peptide 1000
0 0 0 50 100 150 200 0 50 100 150 200 Time [min] Time [min] 100 * * 80 * * * 15 * * * * * 60 ] -1 * 10 *min 40 -1
5 Serum proinsulin [pmol/l] Serum proinsulin 20 [pmol*kg Insulin secretionrate Insulin
0 0 0 50 100 150 200 0 50 100 150 200 Time [min] Time [min] 3
4 Page 23 of 35 Diabetes
23
1 Figure 3
2 ] -1 *min -1 15
10
5
0 4 5 6 7 8 9 Mean plasma glucose [mmol/l] Insulin secretion [pmol*kg rateInsulin 3 Diabetes Page 24 of 35
24
1 Figure 4
** 30 ***
20
10
Hypoglycemia score Hypoglycemia 0 Frequency Severity
2 Page 25 of 35 Diabetes
25
1 Figure 5
4.5
* 10
4.0
5 Serum potasium [mmol/l] Serum potasium Plasma glucagonPlasma [pmol/l]
3.5
0 0 50 100 150 200 0 50 100 150 200 25 Time [min] Time [min] 60
20
40 15
10 GIP [pmol/l] GIP 20 Plasma GLP-1 [pmol/l] GLP-1 Plasma 5
0 0 0 50 100 150 200 0 50 100 150 200 Time [min] Time [min] 2
3 Diabetes Page 26 of 35
26
1 Figure 6
* 10 5.0 * 8 * * 4.5 * * * 6 * 4.0 4
3.5 2 Plasma glucose[mmol/l] Plasma Serum potasium [mmol/l] Serum potasium
0 3.0 0 100 200 300 400 0 100 200 300 400 Time [min] Time [min]
800 * 20
600 15
400 10 Insulin [pmol/l] Insulin
5 200 Plasma glucagon [pmol/l] glucagon Plasma
0 0 0 100 200 300 400 0 100 200 300 400 Time [min] Time [min] 2 3 Page 27 of 35 Diabetes
27
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22 5 Unoki H, Takahashi A, Kawaguchi T, Hara K, Horikoshi M, Andersen G, Ng DP, Holmkvist J, 23 Borch-Johnsen K, Jorgensen T, Sandbaek A, Lauritzen T, Hansen T, Nurbaya S, Tsunoda T, Kubo M, 24 Babazono T, Hirose H, Hayashi M, Iwamoto Y, Kashiwagi A, Kaku K, Kawamori R, Tai ES, Pedersen O, 25 Kamatani N, Kadowaki T, Kikkawa R, Nakamura Y, Maeda S: SNPs in KCNQ1 are associated with 26 susceptibility to type 2 diabetes in East Asian and European populations. Nat Genet 2008;40:1098- 27 1102.
28 6 Holmkvist J, Banasik K, Andersen G, Unoki H, Jensen TS, Pisinger C, Borch-Johnsen K, 29 Sandbaek A, Lauritzen T, Brunak S, Maeda S, Hansen T, Pedersen O: The type 2 diabetes associated 30 minor allele of rs2237895 KCNQ1 associates with reduced insulin release following an oral glucose 31 load. PLoS ONE 2009;4:e5872.
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35 8 Ullrich S, Su J, Ranta F, Wittekindt OH, Ris F, Rosler M, Gerlach U, Heitzmann D, Warth R, 36 Lang F: Effects of I(Ks) channel inhibitors in insulin-secreting INS-1 cells. Pflugers Arch 37 2005;451:428-436.
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11 13 Thuesen BH, Cerqueira C, Aadahl M, Ebstrup JF, Toft U, Thyssen JP, Fenger RV, Hersoug LG, 12 Elberling J, Pedersen O, Hansen T, Johansen JD, Jorgensen T, Linneberg A: Cohort Profile: The 13 Health2006 cohort, Research Centre for Prevention and Health. Int J Epidemiol 2013.
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18 15 Kanters JK, Haarmark C, Vedel-Larsen E, Andersen MP, Graff C, Struijk JJ, Thomsen PE, 19 Christiansen M, Jensen HK, Toft E: T(peak)T(end) interval in long QT syndrome. J Electrocardiol 20 2008;41:603-608.
21 16 Larsen LA, Andersen PS, Kanters JK, Jacobsen JR, Vuust J, Christiansen M: A single strand 22 conformation polymorphism/heteroduplex (SSCP/HD) method for detection of mutations in 15 23 exons of the KVLQT1 gene, associated with long QT syndrome. Clin Chim Acta 1999;280:113-125.
24 17 Huang L, Bitner-Glindzicz M, Tranebjaerg L, Tinker A: A spectrum of functional effects for 25 disease causing mutations in the Jervell and Lange-Nielsen syndrome. Cardiovasc Res 2001;51:670- 26 680.
27 18 Hepburn DA, MacLeod KM, Pell AC, Scougal IJ, Frier BM: Frequency and symptoms of 28 hypoglycaemia experienced by patients with type 2 diabetes treated with insulin. Diabet Med 29 1993;10:231-237.
30 19 Holst JJ: Molecular heterogeneity of glucagon in normal subjects and in patients with 31 glucagon-producing tumours. Diabetologia 1983;24:359-365.
32 20 Orskov C, Rabenhoj L, Wettergren A, Kofod H, Holst JJ: Tissue and plasma concentrations of 33 amidated and glycine-extended glucagon-like peptide I in humans. Diabetes 1994;43:535-539.
34 21 Lindgren O, Carr RD, Deacon CF, Holst JJ, Pacini G, Mari A, Ahren B: Incretin hormone and 35 insulin responses to oral versus intravenous lipid administration in humans. J Clin Endocrinol Metab 36 2011;96:2519-2524.
37 22 Dupont WD, Plummer WD, Jr.: Power and sample size calculations for studies involving linear 38 regression. Control Clin Trials 1998;19:589-601. Page 29 of 35 Diabetes
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1 23 Dupont WD, Plummer WD, Jr.: Power and sample size calculations. A review and computer 2 program. Control Clin Trials 1990;11:116-128.
3 24 Torekov SS, Larsen LH, Glumer C, Borch-Johnsen K, Jorgensen T, Holst JJ, Madsen OD, Hansen 4 T, Pedersen O: Evidence of an Association Between the Arg72 Allele of the Peptide YY and 5 Increased Risk of Type 2 Diabetes. Diabetes 2005;54:2261-2265.
6 25 Matsuda M, DeFronzo RA: Insulin sensitivity indices obtained from oral glucose tolerance 7 testing: comparison with the euglycemic insulin clamp. Diabetes Care 1999;22:1462-1470.
8 26 Lucio M, Fekete A, Weigert C, Wagele B, Zhao X, Chen J, Fritsche A, Haring HU, Schleicher ED, 9 Xu G, Schmitt-Kopplin P, Lehmann R: Insulin sensitivity is reflected by characteristic metabolic 10 fingerprints--a Fourier transform mass spectrometric non-targeted metabolomics approach. PLoS 11 ONE 2010;5:e13317.
12 27 Hovorka R, Koukkou E, Southerden D, Powrie JK, Young MA: Measuring pre-hepatic insulin 13 secretion using a population model of C-peptide kinetics: accuracy and required sampling schedule. 14 Diabetologia 1998;41:548-554.
15 28 Hovorka R, Soons PA, Young MA: ISEC: a program to calculate insulin secretion. Comput 16 Methods Programs Biomed 1996;50:253-264.
17 29 Kjems LL, Holst JJ, Volund A, Madsbad S: The influence of GLP-1 on glucose-stimulated insulin 18 secretion: effects on beta-cell sensitivity in type 2 and nondiabetic subjects. Diabetes 2003;52:380- 19 386.
20 30 Hojlund K, Hansen T, Lajer M, Henriksen JE, Levin K, Lindholm J, Pedersen O, Beck-Nielsen H: 21 A novel syndrome of autosomal-dominant hyperinsulinemic hypoglycemia linked to a mutation in 22 the human insulin receptor gene. Diabetes 2004;53:1592-1598.
23 31 Boini KM, Graf D, Hennige AM, Koka S, Kempe DS, Wang K, Ackermann TF, Foller M, Vallon V, 24 Pfeifer K, Schleicher E, Ullrich S, Haring HU, Haussinger D, Lang F: Enhanced insulin sensitivity of 25 gene-targeted mice lacking functional KCNQ1. Am J Physiol Regul Integr Comp Physiol 26 2009;296:R1695-R1701.
27 32 Clausen T: Hormonal and pharmacological modification of plasma potassium homeostasis. 28 Fundam Clin Pharmacol 2010;24:595-605.
29 33 Vallon V, Grahammer F, Volkl H, Sandu CD, Richter K, Rexhepaj R, Gerlach U, Rong Q, Pfeifer 30 K, Lang F: KCNQ1-dependent transport in renal and gastrointestinal epithelia. Proc Natl Acad Sci U S 31 A 2005;102:17864-17869.
32 34 Robinson RT, Harris ND, Ireland RH, Lee S, Newman C, Heller SR: Mechanisms of abnormal 33 cardiac repolarization during insulin-induced hypoglycemia. Diabetes 2003;52:1469-1474.
34 35 Reno CM, Daphna-Iken D, Chen YS, Vanderweele J, Jethi K, Fisher SJ: Severe hypoglycemia- 35 induced lethal cardiac arrhythmias are mediated by sympathoadrenal activation. Diabetes 36 2013;62:3570-3581. Diabetes Page 30 of 35
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1 36 Guo J, Massaeli H, Xu J, Jia Z, Wigle JT, Mesaeli N, Zhang S: Extracellular K+ concentration 2 controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines. J 3 Clin Invest 2009;119:2745-2757.
4 37 van NC, Sturkenboom MC, Straus SM, Hofman A, Kors JA, Witteman JC, Stricker BH: Serum 5 glucose and insulin are associated with QTc and RR intervals in nondiabetic elderly. Eur J Endocrinol 6 2010;162:241-248.
7 38 Tsujimoto T, Yamamoto-Honda R, Kajio H, Kishimoto M, Noto H, Hachiya R, Kimura A, Kakei 8 M, Noda M: Vital Signs, QT Prolongation, and Newly Diagnosed Cardiovascular Disease during 9 Severe Hypoglycemia in Type 1 and Type 2 Diabetic Patients. Diabetes Care 2013.
10 39 MacDonald PE, Wheeler MB: Voltage-dependent K(+) channels in pancreatic beta cells: role, 11 regulation and potential as therapeutic targets. Diabetologia 2003;46:1046-1062.
12 40 Drews G, Krippeit-Drews P, Dufer M: Electrophysiology of islet cells. Adv Exp Med Biol 13 2010;654:115-163.
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19 43 Gromada J, Ma X, Hoy M, Bokvist K, Salehi A, Berggren PO, Rorsman P: ATP-sensitive K+ 20 channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type 21 and SUR1-/- mouse alpha-cells. Diabetes 2004;53 Suppl 3:S181-S189.
22 44 Mussig K, Staiger H, Machicao F, Kirchhoff K, Guthoff M, Schafer SA, Kantartzis K, Silbernagel 23 G, Stefan N, Holst JJ, Gallwitz B, Haring HU, Fritsche A: Association of type 2 diabetes candidate 24 polymorphisms in KCNQ1 with incretin and insulin secretion. Diabetes 2009;58:1715-1720.
25 45 Rogers GJ, Tolhurst G, Ramzan A, Habib AM, Parker HE, Gribble FM, Reimann F: Electrical 26 activity-triggered glucagon-like peptide-1 secretion from primary murine L-cells. J Physiol 27 2011;589:1081-1093.
28 46 Smushkin G, Sathananthan M, Sathananthan A, Dalla MC, Micheletto F, Zinsmeister AR, 29 Cobelli C, Vella A: Diabetes-associated common genetic variation and its association with GLP-1 30 concentrations and response to exogenous GLP-1. Diabetes 2012;61:1082-1089.
31 47 Toft-Nielsen M, Hvidberg A, Hilsted J, Dige-Petersen H, Holst JJ: No effect of beta-adrenergic 32 blockade on hypoglycaemic effect of glucagon-like peptide-1 (GLP-1) in normal subjects. Diabet 33 Med 1996;13:544-548. 34 35 Page 31 of 35 Diabetes Supplementary appendix KCNQ1 Long QT syndrome patients have hyperinsulinemia and symptomatic hypoglycemia
Contents Contents ...... 1 Supplementary table 1 Hypoglycemia questionnaire...... 2 Supplementary table 2. Hypoglycaemia scores for each specific question ...... 3 Supplementary figure 1 ...... 4
1
Diabetes Page 32 of 35 Supplementary appendix KCNQ1 Long QT syndrome patients have hyperinsulinemia and symptomatic hypoglycemia
Supplementary table 1 Hypoglycemia questionnaire. % KCNQ1 patients / % Control participant# Frequency† Severity£
Never Rarely Occasionally Usually Always Mild Moderate Severe (0) (1) (2) (3) (4) (1) (2) (3) 1.Cravings for sweets 0/8 8/12 62/50 8/23 23/8 46/31 15/54 38/8 2.Irritability if a meal 15/58 23/23 23/12 8/8 31/0 31/31 31/12 23/0 is missed* 3.Tired or weak if a 8/46 23/19 31/23 23/8 15/0 31/35 31/15 31/4 meal is missed* 4.Often hungry* 0/8 31/19 46/15 23/0 0/0 31/46 54/46 15/0 5.Dizziness when 8/50 38/35 38/15 8/0 8/0 62/42 15/4 15/0 standing suddenly* 6.Generally dizzy* 31/81 38/15 8/4 15/0 8/0 38/19 31/0 0/0 7.Tired or 38/65 31/15 23/15 8/4 0/0 31/27 23/8 8/0 uncomfortable some hours after a meal* 8.Trouble 15/35 77/38 0/27 8/0 0/0 62/46 15/19 8/0 concentrating 9.Heart palpitations 23/62 62/19 8/19 8/0 0/0 62/31 15/8 0/0 10.Occasional 46/62 46/31 8/8 0/0 0/0 38/35 8/4 8/0 shakiness 11.Occasional blurry 15/62 54/23 8/15 23/0 0/0 46/27 23/12 15/0 vision* 12.Depression or 46/46 38/31 0/23 8/0 8/0 38/38 8/15 8/0 mood swings 13.Frequent anxiety or 54/73 38/23 0/4 8/0 0/0 38/23 8/4 0/0 nervousness 14.Aggression* 31/69 54/27 0/4 0/0 15/0 46/27 0/4 23/0 15.Night awakening* 8/31 23/12 38/31 23/27 8/0 38/46 38/19 15/4 16.Night sweats* 15/38 8/27 38/12 38/15 0/8 38/31 31/31 15/0 17.Frequent sweating* 8/42 38/12 31/27 23/19 0/0 38/23 31/31 23/4 #Numbers in boxes indicate the percentage of 13 KCNQ1 LQTS patients and 26 control participants that gave the particular answer with percentage of patients written before / and the percentage of control participants written after /. †Frequency point score: Never: 0 point; Rarely: 1 point; Occasionally: 2 points; Usually: 3 points; Always: 4 points. The total frequency score of each participant was calculated from the sum of the point scores given for each question. £Severity point score: Mild: 1 point; Moderate: 2 points; Severe: 3 points. The total severity score of each participant was calculated from the sum of the point scores given for each question. *P<0.05. For each specific question, the difference in scores between patients and control participants was tested with Student’s t-test (see supplementary table 2) The total mean frequency and severity point score of patients and control participants, calculated from the sum of the point score given for each question, are shown in Figure 4.
2
Page 33 of 35 Diabetes Supplementary appendix KCNQ1 Long QT syndrome patients have hyperinsulinemia and symptomatic hypoglycemia
Supplementary table 2. Hypoglycaemia scores for each specific question
Frequency Severity
Patients Control Patients Control
1.Cravings for sweets 2.5 (0.3) 2.1 (0.2) 1.9 (0.3) 1.6 (0.1)
2.Irritability if a meal is missed* 2.2 (0.4) 0.7 (0.2) 1.6 (0.3) 0.6 (0.1)
3.Tired or weak if a meal is missed* 2.1 (0.3) 1.1 (0.2) 1.9 (0.3) 0.8 (0.2)
4.Often hungry* 2.0 (0.2) 1.8 (0.1) 1.8 (0.2) 1.4 (0.1)
5.Dizziness when standing suddenly* 1.7 (0.3) 0.7 (0.1) 1.4 (0.3) 0.6 (0.1)
6.Generally dizzy* 1.3 (0.4) 0.2 (0.1) 1.0 (0.2) 0.2 (0.1)
7.Tired or uncomfortable some hours after 1.0 (0.3) 0.6 (0.2) 1.0 (0.3) 0.4 (0.1) a meal* 8.Trouble concentrating 1.0 (0.2) 0.9 (0.2) 1.2 (0.2) 0.8 (0.1)
9.Heart palpitations 1.0 (0.3) 0.6 (0.2) 0.9 (0.2) 0.5 (0.1)
10.Occasional shakiness 0.6 (0.2) 0.5 (0.1) 0.8 (0.2) 0.4 (0.1)
11.Occasional blurry vision* 1.4 (0.3) 0.5 (0.1) 1.4 (0.3) 0.5 (0.1)
12.Depression or mood swings 0.9 (0.4) 0.8 (0.2) 0.8 (0.3) 0.7 (0.1)
13.Frequent anxiety or nervousness 0.6 (0.3) 0.3 (0.1) 0.5 (0.2) 0.3 (0.1)
14.Aggression* 1.1 (0.4) 0.4 (0.1) 1.2 (0.3) 0.4 (0.1)
15.Night awakening* 2.0 (0.3) 1.6 (0.1) 1.6 (0.2) 1.0 (0.1)
16.Night sweats* 2.0 (0.3) 1.3 (0.2) 1.5 (0.3) 0.9 (0.1)
17.Frequent sweating* 1.7 (0.1) 1.2 (0.1) 1.7 (0.3) 1.0 (0.2)
*P<0.05. For each specific question the difference in scores between patients and control participants were tested with Students t-test (mean ±SEM) The total mean frequency and severity point score of patients and control participants, calculated from the sum of the point score given for each question, are shown in Figure 4.
3
Diabetes Page 34 of 35 Supplementary appendix KCNQ1 Long QT syndrome patients have hyperinsulinemia and symptomatic hypoglycemia
Supplementary figure 1
Plasma glucose, serum insulin and serum potassium levels during an oral glucose tolerance test in 8 treatment naïve patients with KCNQ1 long QT syndrome due to functional mutations in KCNQ1
(closed circles) and 16 randomly chosen BMI-gender and age-matched control participants (open squares) (Mean±SEM), *P < 0.05.
4
Page 35 of 35 Diabetes Supplementary appendix KCNQ1 Long QT syndrome patients have hyperinsulinemia and symptomatic hypoglycemia
Supplementary figure 1
10
*
8 *
*
6
Plasmaglucose [mmol/l] 4
0 50 100 150 200 Time [min]
800
* * 600 * * * * 400
200 Serum insulin [pmol/l] Serum insulin
0 0 50 100 150 200 Time [min]
4.6
4.4
4.2 *
4.0
3.8 Serumpotasium [mmol/l]
3.6 0 50 100 150 200 Time [min]
5