Scholarly article on topic 'Increased Plasma Incretin Concentrations Identifies a Subset of Patients with Persistent Congenital Hyperinsulinism without KATP Channel Gene Defects'

Increased Plasma Incretin Concentrations Identifies a Subset of Patients with Persistent Congenital Hyperinsulinism without KATP Channel Gene Defects Academic research paper on "Clinical medicine"

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The Journal of Pediatrics
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Abstract of research paper on Clinical medicine, author of scientific article — Yanqin Shi, Hima B. Avatapalle, Mars S. Skae, Raja Padidela, Melanie Newbould, et al.

Congenital hyperinsulinism causes profound hypoglycemia, which may persist or resolve spontaneously. Among 13 children with congenital hyperinsulinism, elevated incretin hormone concentrations were detected in 2 with atypical, persistent disease. We suggest that incretin biomarkers may identify these patients, and that elevated hormone levels may contribute to their pathophysiology.

Academic research paper on topic "Increased Plasma Incretin Concentrations Identifies a Subset of Patients with Persistent Congenital Hyperinsulinism without KATP Channel Gene Defects"

The Journal of Pediatrics •



Increased Plasma Incretin Concentrations Identifies a Subset of Patients with Persistent Congenital Hyperinsulinism without

KATP Channel Gene Defects

Yanqin Shi, PhD1, Hima B. Avatapalle, MBChB2, Mars S. Skae, MBChB, MPhil2, Raja Padidela, MD2, Melanie Newbould, FRCPath3, Lindsey Rigby, BSc, RGN, RSCN2, Sarah E. Flanagan, PhD4, Sian Ellard, PhD, FRCPath4, Jacques Rahier, MD, PhD5, Peter E. Clayton, MBChB, MD2,6, Mark J. Dunne, PhD1, Indraneel Banerjee, MD2,

and Karen E. Cosgrove, PhD1

Congenital hyperinsulinism causes profound hypoglycemia, which may persist or resolve spontaneously. Among 13 children with congenital hyperinsulinism, elevated incretin hormone concentrations were detected in 2 with atypical, persistent disease. We suggest that incretin biomarkers may identify these patients, and that elevated hormone levels may contribute to their pathophysiology. (J Pediatr 2015;166:191-4).

Congenital hyperinsulinism (CHI), characterized by inappropriate release of insulin from pancreatic b cells, is associated with brain injury due to hypoglycemia.1-3 The severity of hyperinsulinism varies and may be transient or persistent. More than 70% of all patients with CHI currently have no identified genetic basis.4-6 The most common causes of persistent CHI are mutations in the ABCC8 and KCNJ11 genes, which cause focal (CHI-F) or diffuse (CHI-D) histological variants of CHI.1,2 A recently described histopathological variant of CHI, termed atypical CHI (CHI-A), accounts for 10% of patients undergoing pancreatectomy for treatment of CHI.7 Diagnosing CHI-A is difficult because no associated mutations have been reported, affected patients are variably responsive to medications, and imaging with fluorine-18-labeled L-dihydroxy-phenylalanine positron emission tomography and computed tomography, which differentiates between CHI-F and CHI-D, is unable to identify CHI-A.7

The incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP-1) are secreted from enteroendocrine cells and exert a substantial regulatory influence on insulin secretion.8 The aim of the present study was to compare fasting and postprandial plasma GLP-1 and GIP concentrations among patients with persistent forms of CHI (CHI-F, CHI-D, and CHI-A) and those patients with transient disease. Our results suggest a positive association between an elevated GLP-1 (7-36):GIP and CHI-A, which should be investigated as a potential diagnostic biomarker for CHI-A.


Thirteen patients with CHI were recruited with local Ethical Board approval and parental consent at a UK National

Referral Centre for CHI. The Table summarizes the patients' clinical profiles, with the classification of CHI

based on established diagnostic criteria.


CHI Congenital hyperinsulinism

CHI-A Atypical congenital hyperinsulinism

CHI-D Diffuse congenital hyperinsulinism

CHI-F Focal congenital hyperinsulinism

GIP Glucose-dependent insulinotropic peptide

GLP-1 Glucagon-like peptide 1

analysis of the exons and intron/exon boundaries of the ABCC8 and KCNJ11 genes was performed on all patients using genomic DNA extracted from peripheral blood leukocytes. ABCC8 analysis included screening for the recently reported deep intronic cryptic splicing mutation.10 No further testing was performed in the patients with transient CHI. In the patients with atypical disease, Sanger sequencing of the exons and intron/exon boundaries of the HADH, GCK, and HNF4A genes was performed. HNF4A analysis included the coding exons 1d-10 and the P2 pancreatic promoter, and HADH analysis included screening for the deep intronic splicing mutation.10

For analysis of plasma peptides, patients fasted for 4 hours before blood sample collection and more bloods samples were collected 20 minutes (30 minutes for patients subjected to an oral glucose tolerance test) after the start of feeding. Formula feeds (Table), obtained from Nutricia (Trowbridge, United Kingdom), were given at a rate of 20 mL/kg body weight. Two patients (2 and 13) were subjected to standard oral glucose tolerance test.

All samples were centrifuged at 1300 x g for 10 minutes at 4°C, after which the plasma was removed and stored at

From the 1Faculty of Life Sciences, University of Manchester; Departments of 2Pediatric Endocrinology and 3Pediatric Histopathology, Central Manchester University Hospitals nhs Foundation Trust, Manchester, United Kingdom; institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, United Kingdom; 5Department of Pathology, Cliniques Universitaires Saint Luc, Brussels, Belgium; and 6Manchester Academic Health Science Centre, Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom

Funded by a National Institute for Health Research Manchester Biomedical Research Centre award (R00388 [to K.C. who was supported by a Research Councils UK Academic Fellowship]) and the National Institute for Health Research UK (NIHR CRF 20121115 [to M.D., I.B., K.C.]). The authors declare no conflicts of interest.

Portions of this study were presented as a poster at the European Society of Pediatric Endocrinologists' meeting, September 20-23, 2012 in Leipzig, Germany.

0022-3476/Copyright © 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (

Table. Clinical characteristics of the CHI patient cohort

Age at

sample Birth

Classification Age at collection, weight, Gestation, Genetic Medical Surgical

Patient of CHI diagnosis mo kg wk cause treatment treatment Outcome Feed

1 Transient 6 d 1 2.08 38 Unknown Diazoxide No Normoglycemic Breast milk

2 Transient 1y 190 3.45 40 + 2 Unknown Diazoxide No Normoglycemic OGTT

3 Transient 4d 16 2.86 40 Unknown Diazoxide No Normoglycemic Nutrini Peptisorb

4 Transient 2d 24 3.5 40 Unknown Diazoxide No Normoglycemic Polycal

5 Transient 1d 1 2.49 40 Unknown Diazoxide No Normoglycemic Cow & Gate/


6 Diffuse 2d 27 4.4 40 ABCC8 Octreotide No Continuous Nutrini Peptisorb

Comp hetero therapy

7 Diffuse 3d 26 3.25 40 ABCC8 Diazoxide No Continuous Nutrini Peptisorb

Maternal therapy

8 Diffuse 4d 51 3.4 42 KCNJ11 Diazoxide/ No Continuous Nutrini Peptisorb

Paternal octreotide therapy

9 Diffuse 6d 33 4.73 40 ABCC8 Diazoxide/ Near-total Cured Whole milk

Homozygous octreotide pancreatectomy

10 Focal 3 mo 49 3.62 40 ABCC8 Diazoxide Subtotal Cured Whole milk

Paternal pancreatectomy

11 Focal 7d 48 4.9 38 + 5 ABCC8 Diazoxide/ Subtotal Cured Infantrini

Paternal octreotide pancreatectomy

12 Atypical 2 y, 7 mo 36 2.72 34 Unknown Diazoxide/ Near-total Cured Nutrini Peptisorb

octreotide pancreatectomy

13 Atypical 21 mo 37 3.4 40 Unknown Diazoxide No Continuous OGTT


Comp hetero, compound heterozygous; OGTT, oral glucose tolerance test.

All patients were treated for hypoglycemia and classified as transient, diffuse, focal, or atypical based on clinical characteristics, genotyping, fluorine-18 labeled L-dihydroxyphenylalanine positron emission tomography and computed tomography diagnosis or pancreatic histology after surgery. Four patients with persistent disease (patients 9-12) underwent surgery to alleviate hyperinsulinism; samples forthis study were obtained after recoveryfrom surgery. Patients 1-5 and 8-11 were sampled in the absence of drug treatment. Fortransient patients, disease resolution occurred byfollow-up visits at age 8 months (patient 1), 5 years (patient 2), 9 months (patient 3), 15 months (patient 4), and 5 months (patient 5). Four patients with persistent disease are currently receiving medical interventions (patients 6, 7, 8, and 13) and were sampled during ongoing treatment.

-80°C before analysis. Plasma GLP-1 (7-36) and total GIP concentrations were assessed in triplicate by enzyme-linked immunosorbent assay (ALPCO, Salem, New Hampshire and EMD Millipore, Billerica, Massachusetts, respectively). Incretin hormone concentrations and ratios were analyzed for differences between all patient groups. Statistical significance was determined by 1-way ANOVA and the Tukey post hoc test where appropriate, with a P value <.05 considered significant. All data are presented as mean ± SEM.


All patients were screened for mutations in ABCC8/KCNJ11 (Table). Two patients (12 and 13) had late-onset presentation of persistent CHI, with no characteristic triggers for hypoglycemic events, and both were genotype-negative for defects in ABCC8, KCNJ11, HADH, GCK, and HNF4A. After decline and eventual failure of responses to medical therapy, patient 12 required a 95% pancreatectomy. Examination of the resected pancreas revealed a heterogeneous pattern of pancreatic histopathology and abnormal expression of hexokinase 1, suggestive of CHI-A based on published criteria.7,11

In all patients, postprandial incretin concentrations increased above basal levels, with no differences between the patients with CHI-F and those with CHI-D when analyzed separately (Figure). The average basal GLP-1 (7-36) and GIP concentrations were not significantly different across the patient groups (transient CHI vs persistent CHI-F/CHI-D

and CHI-A). On average, postprandial GLP-1 (7-36) concentrations increased to 3.9 ± 0.9 pmol/L in the patients with transient CHI (n = 4) and to 4.3 ± 1.0 pmol/L in those with CHI-F/CHI-D (n = 6), and was markedly higher in the patients with CHI-A, measured at 82.2 pmol/L in patient 12 and 16.5 pmol/L in patient 13 (P < .05; Figure, A). These 2 patients with atypical disease also exhibited greater fold changes in postprandial GIP concentrations, increasing by 14-fold, compared with 8-fold in patients with transient CHI and 7-fold in those with CHI-F/CHI-D (Figure, B).

The postprandial GLP-1 (7-36):GIP was similar in patients with transient CHI and those with CHI-F/CHI-D (0.1 ± 0.03 [n = 4,patients 1-4] vs0.13 ±0.03 [n = 6, patients 6-11]),but was markedly elevated in patients with CHI-A (0.52 ± 0.06, P < .001; n = 2, patients 12 and 13) (Figure, C).


Incretin hormones have potent regulatory effects on hormone secretion, and inappropriately increased GLP-1 or GIP secretion after gastric surgery has been linked to hyper-insulinemic hypoglycemia.12,13 In the present study, 2 patients with CHI-A had strikingly higher postprandial GLP-1 (7-36):GIP compared with patients with CHI-F, CHI-D, or transient CHI (Figure).

In patients with CHI-A, the genetic basis of disease is unknown and may vary, although heterogeneous expression of hexokinase 1 in b cells is associated with inappropriate insulin release in atypical patients requiring pancreatectomy.11

January 2015


Figure. A and B, Basal and postprandial ("stimulated") concentrations of GLP-1 (7-36) and GIP in patients with CHI. C, GLP-1 (7-36):GIP in the 3 patient groups. The integrated incretin ratio values showed no differences in the transient and CHI-F/CHI-D patient groups, but was significantly larger in patients with CHI-A (P < .001).

We found a similar profile of hexokinase 1 expression in b cells in the tissue of patient 12, but could not evaluate this in patient 13, who was stable on medical therapy. Interestingly, studies on rat insulinoma cell lines have demonstrated up-regulation of hexokinase 1 gene expression in response to GLP-1 and GIP in vitro, suggesting a possible link between incretin pathology and CHI-A.14'15

Despite variable incretin secretion stimuli in the patients in this study and ongoing medical treatment that may have minimized observed differences, both patients with CHI-A had a markedly elevated postprandial GLP-1 (7-36):GIP compared with the other patient groups. Our preliminary findings suggest an alternative cause of hyper-insulinemic hypoglycemia independent of KATP channel-driven mechanisms that may affect b cells directly or may increase insulin secretion via enhanced incretin action. We speculate that both of our patients with CHI-A possibly would have benefited from the GLP-1 receptor antagonist-based therapy described by Calabria et al,16 which in 1 patient might have obviated the need for near-total pancreatectomy.

In summary, CHI-A currently represents a subgroup of patients with persistent and medically unresponsive hypoglycemia that cannot be detected by imaging techniques and for which there is no genetic or endocrine biomarker. Based on our observations, we suggest that investigation of plasma incretin profiles in patients with CHI may identify this group of patients to be followed up in multicenter studies. ■

We acknowledge the invaluable support from the Northern Congenital Hyperinsulinism Service team.

Submitted for publication Jun 27, 2014; last revision received Jul 30, 2014; accepted Sep 9, 2014.

Reprint requests: Dr Karen E. Cosgrove, PhD, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. E-mail:


1. De Leon DD, Stanley CA. Mechanisms of disease: advances in diagnosis and treatment of hyperinsulinism in neonates. Nat Clin Pract Endocrinol Metab 2007;3:57-68.

2. Mohamed Z, Arya VB, Hussain K. Hyperinsulinaemic hypoglycaemia: genetic mechanisms, diagnosis and management. J Clin Res Pediatr Endocrinol 2012;4:169-81.

3. Avatapalle HB, Banerjee I, Shah S, Pryce M, Nicholson J, Rigby L, et al. Abnormal neurodevelopmental outcomes are common in children with transient congenital hyperinsulinism. Front Endocrinol 2013;4:60.

4. Banerjee I, Skae MS, Flanagan SE, Rigby L, Patel L, Didi M, et al. The contribution of rapid KATP channel gene mutation analysis to the clinical management of children with congenital hyperinsulinism. Eur J Endo-crinol 2011;164:733-40.

5. Kapoor RR, Flanagan SE, Arya VB, Shield JP, Ellard S, Hussain K. Clinical and molecular characterisation of 300 patients with congenital hy-perinsulinism. Eur J Endocrinol 2013;168:557-64.

6. Snider KE, Becker S, Boyajian L, Shyng SL, MacMullen C, Hughes N, et al. Genotype and phenotype correlations in 417 children with congenital hyperinsulinism. J Clin Endocrinol Metab 2013;98:E355-63.

7. Sempoux C, Capito C, Bellanne-Chantelot C, Verkarre V, de Lonlay P, Aigrain Y, et al. Morphological mosaicism of the pancreatic islets: a novel anatomopathological form of persistent hyperinsulinemic hypoglycemia of infancy. J Clin Endocrinol Metab 2011;96:3785-93.

8. Campbell JE, Drucker DJ. Pharmacology, physiology, and mechanisms of incretin hormone action. Cell Metab 2013;17:819-37.

9. Banerjee I, Avatapalle HB, Padidela R, Stevens A, Cosgrove KE, Clayton PE, et al. Integrating genetic and imaging investigations into the clinical management of congenital hyperinsulinism. Clin Endocrinol 2013;78:803-13.

10. Flanagan SE, Xie W, Caswell R, Damhuis A, Vianey-Saban C, Akcay T, et al. Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation. Am J Hum Genet 2013;92:131-6.

Increased Plasma Incretin Concentrations Identifies a Subset of Patients with Persistent Congenital Hyperinsulinism 193 without KATP Channel Gene Defects

11. Henquin JC, Sempoux C, Marchandise J, Godecharles S, Guiot Y, Nenquin M, et al. Congenital hyperinsulinism caused by hexokinase I expression or glucokinase-activating mutation in a subset of b-cells. Diabetes 2012;62:1689-96.

12. Palladino AA, Sayed S, Levitt Katz LE, Gallagher PR, De Leon DD. Increased glucagon-like peptide-1 secretion and postprandial hypoglyce-mia in children after Nissen fundoplication. J Clin Endocrinol Metab 2009;94:39-44.

13. Myint KS, Greenfield JR, Farooqi IS, Henning E, Holst JJ, Finer N. Prolonged successful therapy for hyperinsulinaemic hypoglycaemia after gastric bypass: the pathophysiological role of GLP1 and its response to a somatostatin analogue. Eur J Endocrinol 2012;166:951-5.

14. Wang Y, Egan JM, Raygada M, Nadiv O, Roth J, Montrose-Rafizadeh C. Glucagon-like peptide-1 affects gene transcription and messenger ribonucleic acid stability of components of the insulin secretory system in RIN 1046-38 cells. Endocrinology 1995;136:4910-7.

15. Wang Y, Montrose-Rafizadeh C, Adams L, Raygada M, Nadiv O, Egan JM. GIP regulates glucose transporters, hexokinases, and glucose-induced insulin secretion in RIN 1046-38 cells. Mol Cell Endo-crinol 1996;116:81-7.

16. Calabria AC, Li C, Gallagher PR, Stanley CA, De Leon DD. GLP-1 receptor antagonist exendin-(9-39) elevates fasting blood glucose levels in congenital hyperinsulinism owing to inactivating mutations in the ATP-sensitive K+ channel. Diabetes 2012;61:2585-91.