Publish Ahead of Print, published online October 27, 2010

Congenital hyperinsulinism and hypersensitivity in homozygous and heterozygous carriers of Kir6.2 (KCNJ11) mutation V290M mutation: KATP channel inactivation mechanism and clinical management

*Karen J. Loechner2, *Alejandro Akrouh1, Harley T. Kurata1, Carlo Dionisi-Vici3, Arianna Maiorana3, Milena Pizzoferro4, Vittoria Rufini5, Jean de Ville de Goyet6, Carlo Colombo7, Fabrizio Barbetti7, 8, Joseph C. Koster1, and Colin G. Nichols1

*K. J. L. and A. A. contributed equally

(1) Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110; (2) Department of Pediatrics, 3341 MBRB, UNC School of Medicine, Chapel Hill, NC 27599; (3) Unit of Metabolic Diseases, Department of Pediatrics, Bambino Gesù Children’s Hospital, Rome, Italy; (4) Unit of Nuclear Medicine, Department of Radiology, Bambino Gesù Children's Hospital, Rome, Italy; (5) Department of Nuclear Medicine, Catholic University of the Sacred Heart, Rome, Italy; (6) Dept. of Surgery, Bambino Gesù Children’s Hospital, Rome, Italy; (7) Laboratory of Monogenic Diabetes, Bambino Gesù Children's Hospital IRCCS, Rome, Italy; (8) Dept. of Internal Medicine, University of Tor Vergata, and Laboratory of Monogenic Diabetes, Bambino Gesù Children's Hospital IRCCS, Rome.

Running title: Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Address correspondence and reprint requests to: Colin G. Nichols [email protected] or Fabrizio Barbetti [email protected]

Submitted 22 May 2010 and accepted 15 October 2010.

This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org.

Copyright American Diabetes Association, Inc., 2010 Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

+ Objective: The ATP-sensitive K -channel (KATP) controls secretion from the islet. Gain- or loss-of-function mutations in channel subunits underlie human neonatal diabetes mellitus (NDM) and congenital hyperinsulinism (HI), respectively. In this study we sought to identify the mechanistic basis of KATP-induced HI in two probands, and characterize the clinical course.

Research Design and Methods: We analyzed HI in two probands and characterized the course of clinical treatment in each, as well as properties of mutant KATP channels expressed in COSm6 cells using Rb efflux and patch-clamp methods.

Results: We identified mutation V290M in the pore-forming Kir6.2 subunit in each proband. In vitro expression in COSm6 cells supports the mutation resulting in an inactivating phenotype, which leads to significantly reduced activity in intact cells when expressed homomerically, and to a lesser extent when expressed heteromerically with WT subunits. In one heterozygous proband, fluoro-DOPA scan revealed a causal focal lesion, indicating uniparental disomy with loss of heterozygosity. In a second family, the proband, homozygous for the mutation, was diagnosed with severe -unresponsive hypersinsulinism at 2 weeks of age. The patient continues to be treated successfully with and amlodipine. The parents and a male sibling are heterozygous carriers without overt clinical HI. Interestingly, both the mother and the sibling exhibit evidence of abnormally enhanced glucose tolerance.

Conclusions: V290M results in inactivating KATP channels that underlies HI. Homozygous individuals may be managed medically, without pancreatectomy. Heterozygous carriers also show evidence of enhanced glucose sensitivity, consistent with incomplete loss of KATP channel activity.

+ he ATP-sensitive K -channel (KATP) Importantly, the V290M mutation is present regulates insulin secretion from the in the homozygous state in one of the HI- T pancreatic β-cell by coupling changes affected probands, and is heterozygous in the in metabolism to changes in electrical unaffected parents and one sibling. Oral activity. KATP overactivity suppresses insulin glucose tolerance tests on the heterozygous release and causes neonatal diabetes mellitus mother and sibling suggest hyper-responsivity (NDM) (1;2), whereas KATP underactivity in both individuals. causes hypersecretion and congenital hyperinsulinemia (HI) (3;4;5). MATERIALS AND METHODS HI mutations can cause aberrant channel Genetics and Molecular Biology. Genomic synthesis or trafficking, or altered channel DNA was isolated from whole using gating (5;6). Mature KATP channels are hetero- the DNeasy Tissue Isolation kit (Qiagen, octomers of four pore-forming Kir6.2 subunits Valencia, CA, USA). KCNJ11 was amplified (KCNJ11) and four sulfonylurea receptor by PCR and directly sequenced. The subunits (ABCC8) (7;8;9). We report a novel identified V290M mutation was engineered Kir6.2 mutation (V290M), identified in two into mouse Kir6.2 cDNA in pCMV6B using unrelated HI probands. V290M reduces the Quikchange site-directed mutagenesis kit channel activity by causing an inactivating (Stratagene, La Jolla, CA), and confirmed by phenotype, explaining the HI outcome. direct sequencing.

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Clinical studies. Oral Glucose Tolerance basis to evaluate the safety and efficacy of the Testing (OGTT) D-glucose was given orally two treatment regimens. and blood drawn via peripheral IV at baseline Fluoro-DOPA analysis of . 18F-L- and then at hourly intervals. Samples were DOPA PET-CT study (4 MBq/Kg of 18F-L- assayed for serum glucose, insulin, and DOPA administered intravenously 45 minutes proinsulin at the Mayo Clinical Laboratory before acquisition) was performed using a (Rochester, MN). For the mother, the glucose hybrid machine (Gemini GXL, Philips Medical load (Glucola) was 75g. For the sibling child, Systems). PET scan was performed under the load was 1g/kg, and the duration of the general anesthesia and glucose infusion to OGTT was truncated to 3 hours due to age. Of maintain normoglycemia, after 6h-fasting, note, behavioral changes (e.g., hunger, without stopping medications. lethargy) that often followed a meal were Expression of KATP channels in COSm6 cells reported for the sibling. Given that . COSm6 cells were cultured in Dulbecco’s heterozygous KATP channel mutations have Modified Eagle Medium plus 10 mM glucose been identified in patients with HI (6), the (DMEM-HG), supplemented with fetal calf mother requested testing for her and her son. serum (FCS, 10%). Cells were transfected This case report was submitted to the IRB at with cDNA using FuGENE 6 Transfection UNC and declared “exempt”. Reagent (Roche Diagnostics, Indianapolis, Continuous Glucose Montoring System IN), and then plated on sterile glass coverslips (CGMS) Due to parental wishes to decrease the overnight prior to patch-clamp experiments. frequency of Octeotide injections, medical Electrophysiological methods. Patch-clamp therapy was adjusted while monitoring under experiments were performed at room CGMS as an off-label use. A sensor was placed temperature on COSm6 cells that fluoresced on three separate occasions for Proband #1 green under UV illumination, 3-5 days post- (CareLink(TM), Medtronic MiniMed, Inc) transfection. Membrane patches were voltage- after application of topical anesthetic. clamped using an Axopatch 1-D amplifier Medtronic (Caremark) provided training to the (Axon Instruments, Union City, CA). All parents on use and how to mark events such as currents were measured at a membrane medication and meals, as well as to corroborate potential of -50mV. Data were collected using hypo- (sensor set at <80) or hyperglycemic the pClamp8.2 software suite (Axon (>200) events detected by external blood Instruments, Union City, CA) and Microsoft glucose meter. Reference ranges were chosen Excel. Bath and pipette control solutions for the alarm settings to avoid (KINT) contained (mM): 150 KCl, 10 HEPES, and minimize glycosuria, as well as avoid and 1 EGTA (pH 7.4). Where indicated, ATP excessive fingersticks for the child. Sensors was added to the bathing solution as were placed for a maximum of 5 days and dipotassium salts. Tolbutamide was dissolved corresponded to periods when treated with (1) in KINT from a 100 mM stock solution in 100 Octreotide alone, or (2) Octreotide + mM KOH. Amlodipine. Of note, the child was tapered off Macroscopic 86Rb+ efflux assays. COSm6 cells amlodipine while on CGMS for a period of 2 in 12-well plates were incubated for 24 hr in weeks. After monitoring with Octreotide alone, culture medium containing 86RbCl (1 μCi/mL) amlodipine (0.1mg/kg divided twice daily) was 2 days after transfection. Cells were washed re-introduced for 5 days prior to CGMS testing. twice with Ringer’s solution (Basal) (in mM: Baseline glucose levels and excursions (high 118 NaCl, 2.5 CaCl2, 1.2 KH2PO4, 4.7 KCl, 25 and low) were documented on a continuous NaHCO3, 1.2 MgSO4, 10 HEPES; pH 7.4) with or without metabolic inhibition (MI)(1 mM 2-

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

deoxy-D-glucose and 2.5 μg/mL oligomycin). H At selected time points, solution was removed Irel = 100/(1 + ( [ATP] / k1/2 ) ) and replaced with fresh solution; after (Eq. 3) completion of the assay, cells were lysed with 1% SDS and removed. Collected samples were where Irel is the current relative to that in the assayed in a scintillation solution. Raw data is absence of ATP, [ATP] is the ATP 86 + shown as percent Rb efflux relative to total concentration, k1/2 is the half-maximal counts. inhibitory ATP concentration, H is the Hill 86 + The rate constant of KATP-specific Rb coefficient which was fixed at 1.3. efflux (k2) was obtained by fitting a single- Immunoblotting. 48 hours post transfection, exponential equation: cells were washed twice with cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 Relative flux = 1- exp [ - (k1 + k2) * t ] mM KH2PO4) and then incubated at 4ºC, in (Eq. 1) 300 µL of lysis buffer (150 mM NaCl, 20 mM HEPES, 10 mM EDTA, 1% NP-40, one where apparent rate constant for nonspecific “Complete Mini” protease inhibitor [Roche efflux (k1) was obtained from untransfected Diagnostics, Indianapolis, IN] per 10 mL at cells. Glibenclamide was added to Ringer’s pH 7). Lysates were centrifuged for 5 minutes solution plus metabolic inhibition from a 1 at 13,000 rpm in 4ºC and then transferred to mM stock solution in DMSO. Results are clean microcentrifuge tubes, resolved with presented as mean ± s.e.m. (standard error of SDS-PAGE (7.5% acrylamide) and the mean). Statistical tests and p-values are transferred to PVDF membranes pre-soaked noted in figure legends where appropriate. in methanol. Filters were blocked overnight in Estimation of Po,zero using Noise Analysis. TBS-T buffer (200 mM NaCl, 20 mM Tris- Mean Po,zero was estimated from stationary HCl, 0.1% Tween, pH 7.4) plus 5% nonfat fluctuation analysis of macroscopic currents dry milk at 4ºC. Filters were incubated and in isolated membrane patches (10;11). Short rocked for 1 hour in a 1:1000 dilution of anti- (<1 s) recordings of currents were analyzed in SUR1 antibody (affinity-purified from rabbit) zero [ATP] and in 5 mM [ATP] (for in TBS-T plus 5% milk, washed three times estimation of ATP-independent noise). for 5 minutes each in TBS-T, then bathed in Currents were filtered at 1 kHz and digitized 1:1000 dilution of secondary antibody (goat, at 3 kHz with 12-bit resolution. Mean patch anti-rabbit IgG, horseradish peroxidase current (I), and variance (σ) in the absence of linked, from Pierce) in TBS-T plus 5% milk. ATP were obtained by subtraction of mean Filters were washed an additional 3x in TBS- current and variance in 5 mM ATP (i.e. T for 5 minutes each before appliance of an assuming all channels fully closed). Single enhanced chemiluminescence system for channel current (i) was assumed to be -3.75 detection of horseradish peroxidase pA at –50 mV, corresponding to WT single (SuperSignal West Pico Chemiluminescent channel conductance of 75 pS (12). Po,zero was Substrate) and subsequent exposure to then estimated from the following equation: autoradiography film (Midwest Scientific, St. Louis, MO). 2 Po,zero = 1-[σ /(i * I)]. (Eq. 2) RESULTS Quantitative analysis of ATP inhibition. The Genetic pedigree of HI in two separate ATP dose-response was quantified by fitting families with V290M mutation. Fig. 1 shows the raw data with a Hill equation: available pedigrees for two families in which

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

the neonatal probands were clinically Variable clinical presentation. Shortly after diagnosed with HI. Proband 1 (female, birth, proband 1 developed cyanosis, blood gestation 38 weeks, birth weight (BW) 3.5 kg, glucose <20 mg/dL, and was transferred to 75-90th centile (13)) was referred by outside neonatal intensive care at an outside hospital hospital (OSH) at 2 weeks for evaluation of (OSH). The presence of a heart murmur on persistent hypoglycemia (blood glucose <30 DOL #2 prompted an echocardiogram mg/dl), despite initiation of dextrose- (ECHO) that detected a small patent ductus containing intravenous fluids and diazoxide arteriosus which resolved spontaneously. (15 mg/kg/d). Genotyping (Athena Despite frequent breast-feeding, supplemental Diagnostics) identified homozygous V290M and intravenous feeds, hypoglycemic episodes mutation in KCNJ11 in the proband, and (<30 mg/dL) continued. Diazoxide treatment heterozygous V290M in each of the was initiated at 15mg/kg/day on DOL #10. unaffected mother (gestation 40 weeks, BW Laboratory assessment prior to initiation of 3.9 kg, 75-90th centile), father (gestation 40 diazoxide revealed insulin levels (56, 70 weeks, BW 4.5 kg, 95-97th centile), and male pmol/l) on two occasions when blood glucose sibling (gestation 35 weeks, BW 3.0 kg, 75- <40 mg/dL. Pituitary testing at the time of 90th centile), but not in an unaffected male hypoglycemia showed intact sibling (gestation 36 weeks, BW 2.7 kg, 25- counterregulatory responses (not shown). In 50th centile). Additional sequencing revealed family 2, both the proband and the father no coding mutations in GCK, GLUD1 or carry the V290M mutation in KCNJ11, yet ABCC8 genes in the proband. The ancestors only the proband suffers from HI. Lack of of the mother and father came from the same mutations in other candidate genes suggested small town in Germany, suggesting the same paternal uniparental disomy with loss of founder mutations. There is a family history heterozygosity of the maternal allele, of in two maternal great aunts characteristic of focal HI (14). Fluoro-DOPA and a paternal great-grandmother, but no scanning (Fig. 1B), provided clear evidence family history of frank hypoglycemia. of a focal lesion in the body of the pancreas. Proband 2 was a female born at 38 weeks Extemporary histological examination gestation (BW 3.4 kg, 75-90th centile) to an performed during surgery revealed focal Italian family, and had the first episode of adenomatous hyperplasia of islet cells, hypoglycemia at day of life (DOL) #2 (blood prompting complete excision of the lesion, glucose 29 mg/dl, treated with glucose effectively curing the patient and providing infusion). She was referred at 8 weeks to further corroborative evidence of focal Bambino Gesù Pediatric Hospital for diffuse hyperinsulinism. cyanosis and tremor; plasma glucose was 40 Clinical course and treatment experience in mg/dl with simultaneously elevated insulin of Proband 1. At DOL #16, proband 1 was 215 pMol/l and low free fatty acids (220 transferred to UNC Children’s Hospital. mmol/l). Diazoxide therapy (15 mg/kg/d) was Blood glucose was <40 mg/dL and the patient started, then tapered to 4 mg/kg/d. was still receiving diazoxide. In addition, Genotyping identified heterozygous V290M repeat insulin level was 42 pMol/l with blood mutation in KCNJ11, in both the proband and glucose of 38 mg/dl. She was started on the apparently unaffected father (not available subcutaneous Octreotide (4 μg/kg/day given for further testing). Additional sequencing of in divided doses every 8 hours). Due to proband DNA revealed no coding mutations persistent hypoglycemia and concomitant in ABCC8, GCK or HNF4α genes. tachyphlyaxis commonly found with Octreotide (15), dosing was increased steadily

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

to 12 μg/kg/day (divided doses every 6 level than with Octreotide alone and the there hours). Diazoxide was tapered off, with were no episodes of blood glucose levels <60 fasting blood glucose maintained >60 mg/dL. mg/dL in the presence of Octreotide + On DOL #21, ECHO revealed systolic Amlodipine. Subsequent introduction of murmur, mild/moderate left ventricular bedtime cornstarch further improved control hypertrophy (LVH), and mild without dangerous excursions but was not for age (blood pressure (BP) 80-90 mm Hg tolerated due to typical side effects of bloating (systolic)/30-40 mm Hg (diastolic)). and abdominal discomfort, and was Consequently, amlodipine (0.1 mg/kg/d given discontinued. in divided dosing twice daily orally) was Effect of V290M mutation on KATP channels initiated to treat both LVH and associated expressed in COSm6 cells. To examine hypertension. Repeat ECHO at DOL #46 was mutation effects on KATP channel activity, we normal. measured 86Rb+ efflux across the plasma CGMS Evaluation. The patient is currently 6 membrane of COSm6 cells co-transfected years old with normal HbA1c (5.6%), IGF-1 with SUR1, and WT Kir6.2, Kir6.2[V290M], (123 ng/mL) and IGF-BP3 (4.3 μg/mL), and or 1:1 mixture of these subunits. Efflux serum insulin (119 pMol/l) at ambient glucose curves were fit with the two-pathway model of 78 mg/dl. Growth is steady between the 25- (see Methods), untransfected cells providing 50%ile on Octreotide (now 8 μg/kg/day the efflux rate (k1) for the non-KATP pathway. divided 4 times daily). Historically, she was Homomeric Kir6.2[V290M] (M/M) channels 86 allowed to outgrow her amlodipine. Due to show considerably reduced Rb efflux rates, desire to decrease the number of frequency of with k2 reduced by ~80% under metabolically injections, re-introduction of amlodipine was inhibited or diazoxide stimulated conditions evaluated systematically using Continuous (Figure 2). Cells expressing heteromeric Glucose Monitoring (CGMS, Medtronic WT+V290M (V/M) channels show CareLink®, see Methods). intermediate efflux rates (k2, reduced by Given long-standing debate as to usefulness ~50%) (Figure 2B). These data confirm that of calcium channel antagonists in HI the V290M mutation results in reduced KATP treatment (16;17), CGMS also permitted channel activity in intact cells, and predicts ascertaining response to amlodipine, which that insulin hyper-secretion will be seen in may inhibit insulin release through direct vivo for heterozygous carriers, and more inhibition of β-cell calcium channels (18), severely so in the homozygous state. and/or via inhibition of these channels Inactivating phenotype of V290M channels. through decreases in cyclic AMP (19). Lysates of COSm6 cells expressing Additive effects of amlodipine plus recombinant channels were assayed by octreotide, would therefore be expected to immunoblotting (Fig. 3A). Anti-SUR1 blots help to minimize peak/trough effects of each show a doublet at ~170kDa, corresponding to individual agent. As shown in Fig. 1C, SUR1 protein, with a similar fraction of Octreotide alone maintained average blood complex-glycosylated SUR1 to WT, glucose levels in the desired range, but indicating that the lower activity observed in marked excursions were still present, with V290M channels is not due to reduced episodes >300 mg/dl. There was only 1 channel density at the surface of the plasma episode of blood glucose <60 mg/dl in >3000 membrane. sensor readings (see Figure 1B). Following The activity of WT and mutant KATP channels introduction of Amlodipine, baseline mean was further examined in inside-out membrane blood glucose rose and stabilized at a higher patch-clamp experiments (Figs. 3-5). Upon

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

membrane excision, WT channels typically even 50% reduction is sufficient to cause open to a steady state level, with open enhanced glucose tolerance and probability of 0.3-0.5 (20). In marked hypersecretion of insulin (23;24). Given that contrast, V290M channels open, but then heteromeric V290M/WT channels exhibit exhibit a rapid decay in macroscopic current, ~50% reduction of KATP conductance (Figure typically resulting in much smaller steady 2), and the anecdotal evidence of state currents than is observed in WT hypersensitivity to glucose exhibited by the channels. Following patch excision, V290M proband’s brother, we pursued oral glucose channels inactivated with time constant of 2.0 tolerance (OGTT) testing in the mother ± 0.5 secs (n=5), while WT channels showed (Table 1) and the brother (Table 2), both of essentially no inactivation following excision whom are heterozygous for the V290M (Fig. 3B). Steady-state ATP sensitivity of mutation. Blood glucose in normal adults will V290M channels was similar to WT (Fig. be below 200mg/dL at 1 hour and below 4A,C), but current density was considerably 140mg/dL at 2 hours and in normal children lower than WT (Fig. 4B), reflecting the was reported to be 103+ 21mg/dl at 1 hour dramatic inactivation that occurs. (25). Even though neither mother nor brother Physiologically, the major determinant of display overt HI under fed conditions, glucose channel activation is stimulation by Mg- was close to fasting levels in both at 1 hour, nucleotides and, as shown in Fig. 4D, and even lower at 2 hours, suggestive of a MgADP activation is intact in V290M “supranormal” response. Proinsulin levels channels, although inactivation follows the rose during the OGTT, and although within MgADP activation, reducing steady-state reported ranges for normal subjects (26), currents in MgADP. proinsulin:insulin ratios at 1 and 2 hours are PIP2 rescues channels from the inactivated above normal range for young individuals state. Following patch excision, exogenously (26). applied phosphatidylinositol bisphosphate (PIP2) incorporates into the membrane inner DISCUSSION leaflet, and typically increases activity of WT Molecular basis of hyperinsulinism associated channels by 2-3 fold, as a result of increased with Kir6.2[V290M] mutation. We describe open state stability (21;22) (Fig. 5). Following two unrelated families with the Kir6.2[V290M] inactivation to steady state levels, application mutation. By recombinant expression of wild of PIP2 to V290M patches caused much type or V290M mutant Kir6.2 subunits, we greater relative increase in current (~50-fold) show that the mutation results in partial loss of to 1902.58 ± 560.63 pA (n=8), similar to the KATP channel activity in the heterozygous case, WT patch current on excision. During PIP2 and more severe loss in the homozygous case exposure, inactivation became progressively (Fig. 2). The loss of function results from slower and less complete in response to induction of an inactivation phenomenon (Fig. application and removal of ATP (Fig. 5B). 3B), similar to that resulting from HI- During this process, the rate and extent of associated mutations at Kir6.2 residue R301 inactivation both decreased in correlation with (27). These mutations resulted in both loss of the estimated open probability (Po) (Figure functional membrane channels, as well as an 6A) as expected for an inactivation process inactivating phenotype. The V290M mutation occuring from the closed state (see appears to reduce channel activity solely Discussion). through induction of inactivation, since surface OGTT evaluation in heterozygous carriers in expression appears normal (Fig. 3A). Structural family 1. In mouse models of reduced KATP, analysis suggests that V290 participates in the

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

generation of an inter-subunit salt bridge postprandial hypoglycemia, and may warrant involving R301 and E290 (Fig. 3C). We caution during meals and/or modification of suggest that V290M and R301 mutations meal carbohydrate composition for such induce the same inactivating phenotype by individuals. There are few published studies destabilizing this salt bridge, causing the focusing on heterozygous carriers of HI channel to enter a long-lived inactivated state mutations. Huopio et al. (32) reported that after closure in the absence of ATP. The carriers of the SUR1 V187D mutation had observed relationship between K1/2,ATP and Po normal glucose tolerance and insulin is an emergent property of gating schemes in secretory capacity. However, V187D reduces which ATP preferentially stabilizes the closed KATP channel activity by affecting trafficking state of the channel (28), and loss of to the surface membrane, and this is readily inactivation paralleling increase in open state reversible, even simply by exposure to stability with PIP2 is then predicted by the sulfonylureas (33), and it is unclear whether assumption that inactivation occurs from the the presence of even a single wild-type unliganded closed state (Figure 6B). subunit might be sufficient for normal Variable presentation of hyperinsulinism trafficking in the heterozygous case, such that associated with Kir6.2[V290M] mutation. In there is minimal effect on KATP density in family 1, HI in the neonatal period is clearly heterozygous carriers. Otherwise, for associated with the homozygous condition. In recessive loss of function HI mutations that the proband of family 2, the mutation presents do cause reduction in channel density in the in a focal lesion with loss of heterozygosity. heterozygous case, it seems that glucose KATP channel activity reduction is graded hyper-responsivity might be a feature, from the heteromeric to the homomeric although clearly this requires further study in expression, with severe (>80%) loss in the bigger cohorts. homomeric case; this is consistent with the There are now several reports of patients with clinical HI phenotype of these two probands. hyperinsulinism due to loss-of-function (LOF) In the heterozygous case, reduction of channel KATP mutations ‘crossing-over’ to diabetes in activity is ~50%. Previous extensive studies later life, including heterozygous carriers of of animal models of genetic suppression of SUR1 mutations (34;35). Again, this is KATP activity predict that such a reduction is predicted from mouse models of loss of KATP sufficient to cause glucose hyper-responsivity, activity (36;37), which become glucose- with left-shifted glucose-dependence of intolerant as adults, and diabetic on high fat insulin secretion, but unaltered basal secretion diets. It is notable that the proinsulin:insulin (29;30;31). The relevance of this to humans levels in the Het carriers were in the normal has remained unclear, but seems a reasonable range (0.1-0.2) in the fasted state, and fell explanation for the apparent glucose hyper- appropriately in the first hour of the OGTT, but responsivity of the two carrier relatives in then rose to relatively higher levels at 2 and 3 family 2. In this regard, it is notable that the hours than has been reported for normal young affected probands in each family and the three subjects, and reminiscent of the elevated ratios unaffected heterozygous carriers in family 1 seen in Type 2 diabetics (26;38). Conceivably, were at the upper range of normal this apparently elevated proinsulin:insulin ratio birthweights, and large relative to the reflects β-cell ‘overwork’ and might be a unaffected sibling. The anecdotal observation harbinger of a tendency to ‘crossover’ in later of the mother in family 1 that the 4 -year old life. heterozygous carrier child can become hungry Treatment options for KATP-dependent HI. and/or lethargic after eating might reflect Standard treatment options for HI are

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

essentially limited to diazoxide (activating support this management to date for this KATP and suppress insulin secretion), or child. Moreover, glucose levels normalize and octreotide (long-acting somatostatin analog medical therapy can be stopped at some point suppressing glucose actions on the beta-cell), for many HI children (42;44;45;46), whereas glucagon (counter-regulator to insulin), or patients who undergo major resection of the subtotal pancreatectomy (17;39;40;41). L- pancreas will tend to develop diabetes at or type (DHP-sensitive, CaV1) channel blockers around puberty, another consideration when are mechanistically an attractive option to assessing the pros and cons of surgical versus directly modulate insulin secretion. Previous medical management. results have been mixed (16;42), but some patients have achieved stable blood sugar CONCLUSIONS levels on nifedipine monotherapy. In the We report hyperinsulinism due to a KATP present case, improvement in both LVH and mutation that results in an inactivating hypertension in addition to hypoglycemia are channel, in two different families. A shown in a neonate on both amlodipine and homozygous affected child is well controlled octreotide. The contribution of amlodipine to with medical therapy without surgery. blood glucose was assessed more directly Clinically unaffected heterozygous carriers using CGMS when the proband became of show signs of hypersecretion, and we suggest school-age. The main finding was a rise in that these may represent an unappreciated basal glucose levels with no values below 70 cohort with subclinical features. mg/dL while on the same dose of octreotide in the presence of amlodipine. Given its Author Contributions: K.J.L., A.A., J.C.K. mechanism of Cav1 channel inhibition, this researched data, wrote manuscript; H.T.K., C. finding is not surprising and supports the D-V., A.M., M.P., V.R., J.deV.deG., C.C. results of Aynsley Green et al. (43). In the researched data; F.B., C.G.N. wrote present case, amlodipine also permitted manuscript. extension of octreotide dosing from every 6- to every 8-hours, thereby saving one injection ACKNOWLEDGEMENTS daily (a significant improvement in quality of This work was supported by National life). Institutes of Health Grant DK69445 (to A key clinical management question for HI is CGN). FB is a member of the Early Onset whether to pursue medical or surgical therapy. Diabetes Study Group of the Italian Society of Long-term medical therapy, as in this case, Pediatric and Diabetology presents a less expensive option than subtotal (SIEDP). We would like to thank Medtronic pancreatectomy, the procedure that would be for both clinical and technical support during indicated by the mutation and mode of the use of CGMS monitoring for the second inheritance. In addition, excellent glucose subject. Humanstudies for the second patient control, normal growth and development and and family were carried out under IRB the variable risk of postsurgical diabetes, all “exempt” category at UNC (KJL).

REFERENCES 1. Koster JC, Marshall BA, Ensor N, Corbett JA, Nichols CG: Targeted overactivity of K(ATP) channels induces profound neonatal diabetes. Cell 100:645-654, 2000 2. Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, Slingerland AS, Howard N, Srinivasan S, Silva JM, Molnes J, Edghill EL, Frayling TM, Temple IK, Mackay D, Shield JP,

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Sumnik Z, van Rhijn A, Wales JK, Clark P, Gorman S, Aisenberg J, Ellard S, Njolstad PR, Ashcroft FM, Hattersley AT: Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. New England Journal of Medicine 350:1838-1849, 2004 3. Thomas PM, Cote GJ, Wohllk N, Haddad B, Mathew PM, Rabl W, Aguilar-Bryan L, Gagel RF, Bryan J: Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy [see comments]. Science 268:426-429, 1995 4. Nichols CG, Shyng SL, Nestorowicz A, Glaser B, Clement JP, Gonzalez G, Aguilarbryan L, Permutt MA, Bryan J: Adenosine Diphosphate As an Intracellular Regulator Of Insulin Secretion. Science 272:1785-1787, 1996 5. Huopio H, Shyng SL, Otonkoski T, Nichols CG: K(ATP) channels and insulin secretion disorders. Am J Physiol Endocrinol Metab 283:E207-216., 2002 6. Flanagan SE, Clauin S, Bellanne-Chantelot C, de Lonlay P, Harries LW, Gloyn AL, Ellard S: Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism. Hum Mutat 30:170-180, 2009 7. Shyng S, Nichols CG: Octameric stoichiometry of the KATP channel complex. J Gen Physiol 110:655-664, 1997 8. Inagaki N, Gonoi T, Seino S: Subunit stoichiometry of the pancreatic beta-cell ATP-sensitive K+ channel. FEBS Lett 409:232-236, 1997 9. Clement JP, Kunjilwar K, Gonzalez G, Schwanstecher M, Panten U, Aguilar-Bryan L, Bryan J: Association and stoichiometry of K(ATP) channel subunits. Neuron 18:827-838, 1997 10. Neher E, Stevens CF: Conductance fluctuations and ionic pores in membranes. Ann. Rev. Biophys. Bioeng. 6:345-381, 1977 11. Sigworth FJ: The variance of sodium current fluctuations at the node of Ranvier. J. Physiol. 307:97-129, 1980 12. Shyng S, Ferrigni T, Nichols CG: Control of rectification and gating of cloned KATP channels by the Kir6.2 subunit. Journal of General Physiology 110:141-153, 1997 13. Bonellie S, Chalmers J, Gray R, Greer I, Jarvis S, Williams C: Centile charts for birthweight for gestational age for Scottish singleton births. BMC Pregnancy Childbirth 8:5, 2008 14. Glaser B, Ryan F, Donath M, Landau H, Stanley CA, Baker L, Barton DE, Thornton PS: Hyperinsulinism caused by paternal-specific inheritance of a recessive mutation in the sulfonylurea-receptor gene. Diabetes 48:1652-1657, 1999 15. Hofland LJ, van der Hoek J, Feelders R, van der Lely AJ, de Herder W, Lamberts SW: Pre- clinical and clinical experiences with novel somatostatin ligands: advantages, disadvantages and new prospects. J Endocrinol Invest 28:36-42, 2005 16. Muller D, Zimmering M, Roehr CC: Should nifedipine be used to counter low blood sugar levels in children with persistent hyperinsulinaemic hypoglycaemia? Arch Dis Child 89:83-85, 2004 17. Hussain K, Aynsley-Green A: Management of hyperinsulinism in infancy and childhood. Ann Med 32:544-551, 2000 18. Malaisse WJ, Boschero AC: Calcium antagonists and islet function. XI. Effect of nifedipine. Horm Res 8:203-209, 1977 19. Hansen JB, Arkhammar PO, Bodvarsdottir TB, Wahl P: Inhibition of insulin secretion as a new drug target in the treatment of metabolic disorders. Current Medicinal Chemistry 11:1595- 1615, 2004

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20. Nichols CG: KATP channels as molecular sensors of cellular metabolism. Nature 440:470- 476, 2006 21. Shyng SL, Nichols CG: Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science 282:1138-1141., 1998 22. Enkvetchakul D, Nichols CG: Gating mechanism of KATP channels: function fits form. Journal of General Physiology 122:471-480, 2003 23. Koster JC, Remedi MS, Flagg TP, Johnson JD, Markova KP, Marshall BA, Nichols CG: Hyperinsulinism induced by targeted suppression of beta cell KATP channels. Proceedings of the National Academy of Sciences of the United States of America 99:16992-16997, 2002 24. Remedi MS, Rocheleau JV, Tong A, Patton BL, McDaniel ML, Piston DW, Koster JC, Nichols CG: Hyperinsulinism in mice with heterozygous loss of K(ATP) channels. Diabetologia 49:2368-2378, 2006 25. Knopf CF, Cresto JC, Dujovne IL, Ramos O, de Majo SF: Oral glucose tolerance test in 100 normal children. Acta Diabetol Lat 14:95-103, 1977 26. Fritsche A, Madaus A, Stefan N, Tschritter O, Maerker E, Teigeler A, Haring H, Stumvoll M: Relationships among age, proinsulin conversion, and beta-cell function in nondiabetic humans. Diabetes 51 Suppl 1:S234-239, 2002 27. Lin YW, Bushman JD, Yan FF, Haidar S, MacMullen C, Ganguly A, Stanley CA, Shyng SL: Destabilization of ATP-sensitive potassium channel activity by novel KCNJ11 mutations identified in congenital hyperinsulinism. J Biol Chem 283:9146-9156, 2008 28. Enkvetchakul D, Loussouarn G, Makhina E, Shyng SL, Nichols CG: The kinetic and physical basis of K(ATP) channel gating: toward a unified molecular understanding. Biophys J 78:2334-2348, 2000 29. Koster JC, Remedi MS, Flagg TP, Johnson JD, Markova KP, Marshall BA, Nichols CG: Hyperinsulinism induced by targeted suppression of beta cell KATP channels. Proc Natl Acad Sci U S A 99:16992-16997., 2002 30. Remedi MS, Rocheleau JV, Tong A, Patton BL, McDaniel ML, Piston DW, Koster JC, Nichols CG: Hyperinsulinism in mice with heterozygous loss of K(ATP) channels. Diabetologia 49:2368-2378, 2006 31. Nichols CG, Koster JC, Remedi MS: beta-cell hyperexcitability: from hyperinsulinism to diabetes. Diabetes, Obesity & Metabolism 9 Suppl 2:81-88, 2007 32. Huopio H, Vauhkonen I, Komulainen J, Niskanen L, Otonkoski T, Laakso M: Carriers of an inactivating beta-cell ATP-sensitive K(+) channel mutation have normal glucose tolerance and insulin sensitivity and appropriate insulin secretion. Diabetes Care 25:101-106., 2002 33. Yan F, Lin CW, Weisiger E, Cartier EA, Taschenberger G, Shyng SL: Sulfonylureas correct trafficking defects of ATP-sensitive potassium channels caused by mutations in the sulfonylurea receptor. Journal of Biological Chemistry 279:11096-11105, 2004 34. Huopio H, Reimann F, Ashfield R, Komulainen J, Lenko HL, Rahier J, Vauhkonen I, Kere J, Laakso M, Ashcroft F, Otonkoski T: Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1. J Clin Invest 106:897-906., 2000 35. Grimberg A, Ferry RJ, Jr., Kelly A, Koo-McCoy S, Polonsky K, Glaser B, Permutt MA, Aguilar-Bryan L, Stafford D, Thornton PS, Baker L, Stanley CA: Dysregulation of insulin secretion in children with congenital hyperinsulinism due to sulfonylurea receptor mutations. Diabetes 50:322-328., 2001 36. Remedi MS, Koster JC, Markova K, Seino S, Miki T, Patton BL, McDaniel ML, Nichols CG: Diet-induced glucose intolerance in mice with decreased beta-cell ATP-sensitive K+

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channels. Diabetes 53:3159-3167, 2004 37. Nichols CG, Koster JC, Remedi MS: beta-cell hyperexcitability: from hyperinsulinism to diabetes. Diabetes Obes Metab 9 Suppl 2:81-88, 2007 38. Tura A, Pacini G, Kautzky-Willer A, Ludvik B, Prager R, Thomaseth K: Basal and dynamic proinsulin-insulin relationship to assess beta-cell function during OGTT in metabolic disorders. Am J Physiol Endocrinol Metab 285:E155-162, 2003 39. Mazor-Aronovitch K, Landau H, Gillis D: Surgical versus non-surgical treatment of congenital hyperinsulinism. Pediatr Endocrinol Rev 6:424-430, 2009 40. Aynsley-Green A, Hussain K, Hall J, Saudubray JM, Nihoul-Fekete C, De Lonlay-Debeney P, Brunelle F, Otonkoski T, Thornton P, Lindley KJ: Practical management of hyperinsulinism in infancy. Arch Dis Child Fetal Neonatal Ed 82:F98-F107, 2000 41. Stanley CA: Hypoglycemia in the neonate. Pediatr Endocrinol Rev 4 Suppl 1:76-81, 2006 42. Hussain K, Aynsley-Green A, Stanley CA: Medications used in the treatment of hypoglycemia due to congenital hyperinsulinism of infancy (HI). Pediatr Endocrinol Rev 2 Suppl 1:163-167, 2004 43. Lindley KJ, Dunne MJ, Kane C, Shepherd RM, Squires PE, James RF, Johnson PR, Eckhardt S, Wakeling E, Dattani M, Milla PJ, Aynsley-Green A: Ionic control of beta cell function in nesidioblastosis. A possible therapeutic role for calcium channel blockade. Arch Dis Child 74:373-378, 1996 44. Dacou-Voutetakis C, Psychou F, Maniati-Christidis M: Persistent hyperinsulinemic hypoglycemia of infancy: long-term results. J Pediatr Endocrinol Metab 11 Suppl 1:131-141, 1998 45. Hussain K, Aynsley-Green A: Management of hyperinsulinism in infancy and childhood. Annals of Medicine 32:544-551, 2000 46. Arnoux JB, de Lonlay P, Ribeiro MJ, Hussain K, Blankenstein O, Mohnike K, Valayannopoulos V, Robert JJ, Rahier J, Sempoux C, Bellanne C, Verkarre V, Aigrain Y, Jaubert F, Brunelle F, Nihoul-Fekete C: Congenital hyperinsulinism. Early Hum Dev 86:287-294 47. Antcliff JF, Haider S, Proks P, Sansom MS, Ashcroft FM: Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit. EMBO Journal 24:229-239, 2005 48. Enkvetchakul D, Loussouarn G, Makhina E, Nichols CG: ATP interaction with the open state of the K(ATP) channel. Biophysical Journal 80:719-728, 2001 49. Enkvetchakul D, Nichols CG: Gating mechanism of KATP channels: function fits form. Journal of General Physiology 122:471-480, 2003

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Table 1. OGTT in Heterozygous Mother Time serum glucose insulin proinsulin proinsulin/ (mg/dL) (pmol/L) (pmol/L) insulin ratio Baseline 82 40.6 3.4 0.083744 t=1 hour 75 350 17 0.048571 t=2 hour 74 133 13 0.097744 t=3 hour 63 14 6.7 0.478571 t=4 hour 72 9.1 4.7 0.516484 t=5 hour 73 6.3 3.1 0.492063 .

Table 2. OGTT in Heterozygous Brother Time serum glucose insulin proinsulin proinsulin/ (mg/dL) (pmol/L) pmol/L insulin ratio Baseline 77 35 4.2 0.12 t=1 hour 90 280 15 0.053571 t=2 hour 79 54.6 11 0.201465 t=3 hour 66 5.7 18.2 0.313187

FIGURE LEGENDS Fig 1. V290M pedigrees and clinical responses to therapy (A) Arrow indicates the V290M proband in each case; black, grey and empty symbols indicate genetically homozygous, heterozygous, and unaffected individuals, respectively. (B) Results Fluoro-DOPA scan of proband 2 indicates focal lesion (yellow arrowheads) in the body of the pancreas. (C) Mean daily glucose levels and standard deviation, for final period on octreotide alone (8 mcg/kg/day divided every 6 hours), following switch to octreotide 8 mcg/kg/day divided every 6 hours plus amlodipine (0.1mg/kg/day divided every 12 hours). Gray area represents target range for alarm for CGMS (70-140 mg/dL glucose). Fig. 2. Decreased KATP activity in homomeric V290M channels and intermediate activity with heteromeric WT and V290M channels (A) Representative 86Rb+ efflux shown as cumulative counts relative to total counts as a function of time, under basal conditions, in the presence of metabolic inhibition, or the presence of KATP channel opener diazoxide. (B) Data were fit to a single exponential and rate constants were obtained from untransfected cells, k1, for non- KATP channel 86Rb+ leak and from transfected cells, k1, for KATP channel-mediated 86Rb+ current. Graphs show compiled data (means +/- SEM) from a 4-6 experiments. Figure 3. Molecular basis of loss of KATP activity (A) Western blots of FLAG-tagged SUR1 (fSUR1) from COSm6 cells expressing fSUR1 alone, or co-expressing fSUR1 with either WT Kir6.2 or mutant Kir6.2[V290M]. The mature (cell-surface) complex-glycosylated and immature, core-glycosylated fSUR1 bands are indicated by upper and lower arrows, respectively. In the absence of Kir6.2, fSUR1 is exclusively core glycosylated. (B) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT or mutant V290M KATP channels at -50mV membrane potential (+50mV pipette). Patches were excised from the cell (arrow) into zero ATP solution, then subsequently exposed to 5 mM ATP.

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

(C) Structural model of Kir6.2 tetramer (47) indicates location of V290. R301 and E292 of adjacent Kir6.2 subunits form a salt bridge that is essential for channel function. V290 lies within 7 angstroms of R301 and mutations at this site may destabilize the intersubunit interface. Figure 4. Mutant V290M channels have unaltered ATP-sensitivity and intact MgADP activation (A) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT or mutant V290M KATP channels at -50mV membrane potential (+50mV pipette). Patches were exposed to different concentrations of ATP as indicated. (B) Mean steady state patch current following isolation (+ s.e.m.). (C) Steady-state dependence of membrane current on ATP relative to current in the absence of ATP. Solid line represents mutant V290M channels while dashed line represents WT channels, fitted with the Hill equation by least-squares method. (D) Representative currents recorded by inside-out excised patch-clamp technique from COSm6 cells expressing WT or mutant V290M KATP channels at -50mV membrane potential (+50mV pipette). Patches were exposed to different concentrations of ATP, and ADP (in the presence of 0.5. mM free Mg2+) as indicated. Figure 5. Inactivation is PIP2-sensitive (A,B) Current recordings of COSm6 patches expressing WT (A) and mutant V290M (B) channels. In WT channels, the current approximately doubles following addition of PIP2. However, in V290M channels, steady-state channel activity increases several-fold, accompanied by a marked reduction in inactivation, following exposures to PIP2, a channel open-state stabilizer. Figure 6. Kinetic mechanism of inactivation (A) Fraction of inactivating current (following ATP removal), versus steady state current (relative to maximal attained following repeat PIP2 exposure – relative open probability) at selected timepoints following exposures to PIP2 in V290M channels. Individual patches are indicated by different symbols, large symbol indicates starting values prior to first exposure to PIP2. (B) A 3-state model of channel activity (with an unliganded open and closed state, and with ATP binding to the closed state) is adequate to explain multiple featiures of KATP gating (28;48;49). A fourth, inactivated, state (CIN) that is coupled to the unliganded closed state can explain both the phenomenon of inactivation, and the finding that PIP2 (which acts to shift the KCO equilibrium towards the open state) both reduces and slows inactivation.

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Figure 1

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Figure 2

Figure 3

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Figure 4

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Figure 5

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Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11

Figure 6

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