Diagnosis and treatment of hyperinsulinaemic hypoglycaemia and its implications for paediatric endocrinology

The most prevalent and serious genetic cause of congenital HH is due to defects in the genes encoding the K_ATP channel (also known as channelopathies) [33, 34]. The K_ATP channels are made up of two protein subunits; the sulphonlyurea receptor 1 (SUR1), which is encoded by the ATP Binding Cassette Subfamily C Member 8 (ABCC8) gene, and the inwardly rectifying potassium (Kir6.2), encoded by the Potassium Voltage-Gated Channel Subfamily J Member 11 (KCNJ11) gene. Both these genes are located on chrosome11p15.1. ABCC8 is 39 exons in length, whilst the KCNJ11 gene is only made up of 1 exon [35]. The K_ATP channel complex is composed of four outer SUR1 subunits and four inner (pore-making) Kir6.2 proteins. The SUR1 component regulates the activity of the Kir6.2 proteins, in addition to a role in initiating a response and sensitivity to sulphonylurea drugs and K_ATP channel opening drugs such as diazoxide [36, 37]. The inner Kir6.2 proteins allow influx of potassium ion concentrations across the membrane. Intracellular nucleotides such as ADP and ATP mediate the action of the K_ATP channel. A change in the ratio of ATP to ADP in the β-cell causes closure of the K_ATP channel and triggers depolarisation of the cell membrane and activates the voltage-gated calcium channels [38]. This in turn causes insulin release through exocytosis and thus promotes lowering of plasma glucose [39].

Recessive inactivating (loss-of-function) K_ATP channel gene mutations predominantly cause medically unresponsive diffuse congenital HH, while, mild -even adult-onset- HH have also been reported [5, 40‐42]. Autosomal dominant inherited mutations usually cause milder forms of congenital HH, whilst, medically unressponsive forms have also been reported [43, 44].

Another ten gene defects; GLUD1, HADH, GCK, SLC16A1, HNF1A, HNF4A, UCP2, HK1, PGM1and PMM2 have been proposed to cause congenital HH [19, 45]. Known as the metabolopathies due to mutations resulting in defects in the metabolic pathways that cause unregulated insulin release and associated hypoglycaemia.

During states of hypoglycaemia, alternative fuel sources are required. The Glutamate Dehydrogenase 1 (GLUD1) gene encodes for the mitochondrial enzyme; glutamate dehydrogenase (GDH) which is essential in glutamate metabolism and has an important role in generating amino acids as fuel substitutes in the absence of glucose [6]. GDH appears to be sensitive to the amino acid leucine and nucleotide phosphate potential [7]. Both of these regulate GDH activity and thus stimulate amino acid-mediated insulin release. The GLUD1 mutations are associated with recurrent fasting and postprandial HH and is also known as the hyperinsulinism/hyperammonaemia (HI/HA) syndrome because of features that include high serum ammonia (usually two-to-three-times the upper normal range) after consumption of a high protein meal. These infants usually are not lethargic/comatose as occurs in inborn errors of metabolism with hyperammonaemia, therefore, unless the serum ammonia level, the diagnosis of HI/HA syndrome may be missed. Urinary α-ketoglutarate is elevated in patients with HI/HA syndrome [46].

GLUD1 mutations account for the most prevalent metabolopathic cause of congenital HH. Studies to date have identified mutations in exons 6, 7, 11 and 12 in the 13 exon gene to cause congenital HH [47, 48]. Although GLUD1 activating mutations occur mainly as de novo, some have reported patients presenting with autosomal dominant forms also [48, 49]. Since K_ATP channels are unaffected by the mutations, patients are medically controlled by the channel activator; diazoxide and are maintained on a low protein diet. Patients carrying GLUD1 mutations have been reported to be more sensitive in developing epileptic seizures irrespective of the severity and frequency of hypoglycaemia episodes. This has been attributed to the shift in the glutamate metabolism leading to a decline in the inhibitory neurotransmitter γ-aminobutyric acid (GABA) [50].

Hydroxyacyl-CoA dehydrogenase (HADH) or short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) is another mitochondrial enzyme that oxidises fatty acids to produce ATP. Although this gene is expressed in the liver, kidneys, muscle and heart, it is most abundant in the pancreatic islets [51]. The HADH gene has 8 exonic regions and mutations inherited in an autosomal recessive manner appears to reduce the enzymes inhibitory action on GDH [52‐54]. Therefore, GLUD1 and HADH patients present similarly with protein sensitivity, although GLUD1 mutations also cause hyperammonemia. HADH gene mutations can lead either to severe neonatal congenital HH or to mild late-even adult-onset, protein–induced HH which is usually diazoxide responsive [55, 56]. Although the clinical and etiological relevance are not clear, patient with HADH mutations may have elevated plasma concentrations of 3-hydroxy-butyryl-carnitine and urinary 3-hydroxy-glutarate [54, 57].

The GCK gene is 12 exons long and encodes the enzyme; glucokinase (GCK). GCK can be found in the pancreatic β-cells, liver and brain [58]. The GCK protein, catalyses glucose to G6P as substrate for glycolytic pathway which generates ATP, therefore, is essential in the glucose-dependent insulin release. The GCK enzyme has high affinity to glucose, serving as a glucose-sensor in pancreatic β-cells. Dominant activating mutations in GCK cause alteration in the protein structure and functions from having a low affinity for glucose to having a high affinity and reduces the threshold for glucose-stimulated insulin release [59, 60]. Variable clinical presentations occur ranging from neonatal severe HH to mild hypoglycaemia or even adult-onset asymptomatic or severe congenital HH, majority of which respond to diazoxide whilst some require surgery [61‐65].

The Solute Carrier Family 16 Member 1 (SLC16A1) gene encodes for the monocarboxylate transporter (MCT1). This protein has a role in transporting monocarboxylates such as pyruvate or lactate into the Krebs cycle to produce ATP and promote insulin release independently of glucose [66]. However, the low expression of this gene in the normal pancreatic β-cell suggests a physiological response to ensure against HH. Autosomal dominant gain-of-function mutations in SLC16A1 cause the expression of MCT1 in β-cells. This in turn leads to glycolysis-generated pyruvate to continually enter the Krebs cycle and induces inappropriate insulin secretion in states of low plasma glucose during anaerobic exercise [67]. Congenital HH due to these mutations is also known as exercise-induced HH, since vigorous exercise stimulates hypoglycaemia. Diagnostically, SLC16A1 patients present with HH after a pyruvate load test [68]. Typically patients with exercise-induced HH develop hypoglycaemia following a bout of strenous exercise. Some patients may require therapy with diazoxide while others can be mananged with diet [8].

The hepatocyte nuclear factors 1A and 4A genes (HNF1A/HNF4A) encode for the HNF1-a and HNF4-a proteins, respectively. These are transcription factors for nuclear hormone receptors expressed in pancreatic β-cells and regulates glucose-dependent insulin secretion [69, 70]. Inactivating mutations in HNF1A and HNF4A causes congenital HH in children and then maturity-onset diabetes of youth (MODY type 3 in HNF1A and type 1 in HNF4A) [71‐73].

Hnf1a null mice have unregulated insulin release, but the mechanism for this remains yet to be determined [74]. The exact role of HNF4α in insulin regulation also is unknown since studies have found conflicting results using transgenic Hnf4a mouse models [69, 75]. Another potential explanation for this disease-causing mutation is the loss of peroxisome proliferators-activated receptor alpha (PPAR alpha; another nuclear factor). PPARα’s role appears to direct fatty acids to the β-oxidation pathway and thus lead to hyperinsulinism during glucose depletion [76]. Moreover, in support of this, the Hnf4 alpha null mice have a decrease in PPARα expression, whilst the Pparα knockout mice have been shown to develop fasting hypoglycaemia [75, 77].

Mutations in both HNF1A and HNF4A have been identified in macrosomic births and can range from mild transient to severe and permanent HH [5, 42, 71, 78]. Patients generally respond to diazoxide and HH resolves with age [5, 42, 79, 80]. Mutation in the HNF4A gene have been reported in cases with HH, increased level of glycogen in erythrocytes, elevated liver transaminases, and increased echogenicity on liver ultrasonography suggesting glycogenosis-like phenotype [81, 82]. Although, thought to be rare causes of congenital HH, mutations in HNF1A and HNF4A have been reported at higher incidences in some series, particularly in those have diazoxide-responsive HH [79, 83].

Uncoupling protein 2 (UCP2) is mitochondrial carrier protein and encodes for the UCP2 gene. Expressed in the pancreatic α- and β-cells, UCP2 inhibits glucose oxidation and activates glutamine oxidation by carrying four-carbon metabolites out of the mitochondria [84]. Inactivation of the UCP2 gene would therefore, promote glucose oxidation and thus cause HH. Initially, it appeared that UCP2 causing HH would resolve with age suggestive of a transient nature since the original cases were reported in patients of up to 6 years, however, new cases have emerged where HH continued beyond 10 years of age [85‐87]. In a recent study, UCP2 mutations were reported for the underlying molecular genetics of 2.4% in a cohort of 211 patients with diazoxide-responsive congenital HH [87]. These patients developed hypoketotic-hypoglycaemia after a fairly good fasting tolerance, while some developed symptomatic hypoglycaemia following an oral glucose load. Rapid decline in plasma glucose and inappropriate insulin secretion following glucose load suggested that mutations in UCP2 cause glucose-induced HH rather than fasting hypoglycaemia [87].

HK1 is located on chromosome 10 and encodes the enzyme; hexokinase 1 (HK1). HK1 is one of the key enzymes involved in regulating glucose homeostasis. Normally, HK1 expression is silenced in the pancreatic β-cells. However recently, a report identified a dominant gain-of-function mutation in the HK1 gene to cause HH in a family with “idiopathic hypoglycaemia of infancy” [16, 86].

Phosphoglucomutase 1 (PGM1) is involved in glycogen metabolism and a loss-of-function mutation in the PGM1 gene has been shown to be associated with hypoglycaemia [17]. Patients with these inactivating mutations have presented with fasting hyperketotic hypoglycaemia, as well as postprandial HH [18].

The phosphomannomutase 2 gene (PMM2) has recently been reported to cause HH as well as congenital polycystic kidney disease in 17 children from 11 unrelated families [19]. The group reported a promoter mutation (c.-167G > T) in the PMM2 gene in all affected patients. PMM2 encodes for an enzyme in glycosylation, deglycosylation in pancreatic β-cells has been shown altered insulin secretion.

At the time of hypoglycemia, once the critical sample has been obtained, if hypoglycemia is unresponsive to oral feeds, or the patient is unable to take an oral feed, a mini bolus of 2mls/kg 10% glucose should be administered intravenously over 1 min. To achieve normoglycemia and keep plasma glucose within the safe range, a continuous intravenous glucose infusion of 6–8 mg/kg/min should be commenced immediately. Patients with HH usually require a very high glucose infusion rate (GIR) to achieve and maintain normoglycaemia. Therefore, GIR might be adjusted according to consecutive plasma glucose measurements.

Glucagon induces glycogenolysis, gluconeogenesis, ketogenesis and lipolysis, and may be used in emergency situations where patients are unable to take oral feed and/or venous access is difficult to obtain [101, 102]. The recommended single dose is between 0.5–1 mg [103]. Glucagon, in high doses, may cause rebound hypoglycemia due to a paradoxical increase in insulin secretion [104]. Glucagon can also be administered as continuous intravenous/subcutaneous infusion at a rate of 5-10mcg/kg/h for short-term stabilization of plasma glucose as well as long-term non-surgical management of congenital HH, in combination with octreotide [105, 106].

In infants, frequent high calorie carbohydrate feeds may reduce the frequency and severity of hypoglycaemic episodes. As these patients usually have food aversion, a percutaneous gastrostomy (PEG) may help to feed frequently and provide an opportunity to administer enteral boluses of high calorie-carbohydrate solutions [107, 108]. Uncooked cornstarch may help to decrease the hypoglycaemic episodes, particularly to improve fasting tolerance during a prolonged overnight fast in children over the age of one year.

Diazoxide, the first-line drug for HH, binds to and opens the SUR1 subunit of the K_ATP channel [101, 102, 109, 110]. Diazoxide is usually effective in all forms of congenital HH, except severe, neonatal-onset, recessive, and focal forms caused by mutations in the ABCC8 and KCNJ11 [109]. Diazoxide needs an intact K_ATP channel activity to work properly. Therefore, diazoxide responsiveness has been the key point for the molecular genetics analysis, differential diagnosis and management strategies of HH. If a patient is unresponsive to the diazoxide, further genetic analysis for ABCC8/KCNJ11 and ¹⁸F–DOPA-PET/CT scan must be performed to differentiate histological subtypes, focal or diffuse disease. The initial dose for diazoxide is usually 5 mg/kg/day, in 3 divided doses and then gradually can be increased up to maximum dose of 15–20 mg/kg/day [111]. The dose should be adjusted to achieve appropriate fasting tolerance for age and normoglycemia under a normal feeding plan. Fluid retention, hypertrichosis and feeding problems are the most common side effects of diazoxide. Therefore, especially in the newborns, when trying to reduce the risk of fluid retention, a thiazide diuretic, chlorothiazide (7–10 mg/kg/day in 2 divided doses), should be administered with diazoxide. Hyperuricaemia, tachycardia and leukopenia, are other rare side effects [112] (Table 3). During follow up, if the diazoxide dose required to maintain normoglycaemia is lower than 5 mg/kg/day, consideration should be given to possibly withdrawing diazoxide in the hospital setting as some types of HH gradually resolve over time [113].

Octreotide, the second line drug of HH, is an 8 amino acid synthetic somatostatin analogue that inhibits insulin secretion by binding to somatostatin receptor 2 and 5 (SSTR2 and SSTR5) [114]. Activation of SSTR5 decreases the insulin gene promoter activity and inhibits calcium mobilization and acetylcholine activity [115]. Somatostatin also inhibits the K_ATP channel which results in reduced insulin secretion [102]. The initial dose of octreotide is 5 μg/kg/day given by subcutaneous injections at 6–8 h intervals. The recommended maximum dose is 30–35 μg/kg/ day. Long-term continuous, subcutaneous octreotide infusion with an insulin pump has also been reported as a feasible alternative to surgery for patients with monoallelic K_ATP-channel mutations [116]. The first response to octreotide administration is usually hyperglycaemia followed by a blunted effect within 24–48 h (tachyphylaxis), thereby, dose adjustment may be required to keep plasma glucose level at normoglycaemic levels [101, 117, 118]. Tachyphylaxis, necrotizing enterocolitis, anorexia, nausea, abdominal pain, diarrhoea, gall bladder pathologies, elevation of liver transaminases, pituitary hormone suppression, long QT syndrome and arrest in linear growth are the reported side effects in patients treated with octreotide [110, 119‐126] (Table 3). However, in recent studies evaluating the long-term effects of octreotide therapy in patients with congenital HH the effect of octreotide on linear growth have been found to be of no clinical relevance [110, 121].

Recently long-acting somatostatin analogs have been an effective option in the management of congenital HH. Octreotide long-acting release (LAR) is formulated with biodegradable microspheres [127]. This formulation provides the advantage of administration every 28 days. Lanreotide is also a synthetic octapeptide and it is recommended to inject every 28 days. LAR-octreotide and lanreotide have been successfully used in children with congenital HH [110, 128‐132]. The opportunity to administer LAR once in every 4 week not only increases treatment compliance but also increases quality of life (QoL) [128]. Therefore, increasing experiences with LAR may improve the treatment compliance and most favorable long-term outcome of patients with congenital HH.

Nifedipine, a calcium channel blocker, inhibits insulin secretion by inactivating the voltage-gated calcium channels [133]. Recommended dose is 0.25–2.5 mg/kg/day divided into 2–3 doses [102]. Hypotension is an uncommon side-effect [102] especially at doses above 0.5 mg/kg/day [134] (Table 3). Although, in many case reports nifedipine treatment has shown to have a favourable effect on HH, in a recent study exclusively investigating its use in HH due to mutations in the ABCC8 gene, HH has shown not responded nifedipine therapy [135]. It is, therefore, suggested that, mutations in the K_ATP channel genes might render the L-type calcium channel ineffective to therapy with nifedipine [135‐139].

Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor. mTOR is a serine and threonine protein kinase and regulates cellular growth by stimulating protein synthesis by increasing mRNA translation initiation and the capacity of the ribosomal protein machinery [140]. The mechanism of action for mTOR inhibitors in HH has not been fully elucidated. However it is reported that there is constitutive activation and overexpression of p-mTOR on the plasmalemmal aspect of the acinar cells, and activation on the plasmalemmal aspect of the ductal cells in the diffuse variant of congenital HH [141].

A number of autocrine growth factors like IGF-I, vascular endothelial growth factor (VEGF), and epidermal growth factor receptor (ErbB), as well as glucose, fatty acids, and amino acids activate mTOR pathway. Upregulation of mTOR leads increased insulin production in the pancreatic β-cells [142]. Conversely, inhibition of mTOR with rapamycin inhibits insulin secretion as well as β-cell growth [143]. Sirolimus can also induces β-cell apoptosis and promotes insulin resistance. Furthermore, mTORC1 inhibits PPARα-mediated expression of ketogenic genes thereby ketone body synthesis, therefore, down-regulation of the mTOR pathway may restores ketogenesis which inturn reduces the risk of brain injury in patients with HH [21]. Recently, it has been reported that sirolimus is effective and safe for the severe, diazoxide unresponsive diffuse congenital HH with no major side effects [144]. Following the first experience, several number of case reports indicating the successful use of sirolimus have been published [145‐149].

Although the optimal therapeutic blood levels for sirolimus in congenital HH patient have not been determined, ranges previously defined for the use of renal transplant patients, 5–15 ng/ml is advised [144]. The most reported adverse effects are stomatitis, increased risk of infection, immunosuppression, renal dysfunction, fatigue, pneumonitis and increased serum aminotransferase or lipid levels [150]. Since, sirolimus have severe side effects, particularly due to risk arising from immunesuppression, patients under sirolimus therapy, should be closely monitored for adverse effects. Despite limited clinical experience in CHI, mTOR inhibitors are thought to be a good option for selected patients who do not respond to diazoxide or octreotide. However, in a recent report evaluating the efficacy of sirolimus in 10 patients with diazoxide unresponsive congenital HH, mTOR inhibition has shown to achieve euglycemia, improve fasting tolerance, and reduced requirement for medical therapy in only three patients (30%) with certain side effects [151]. In addition, pancreatic tissue from two patients who did not respond to sirolimus showed no reduction in cell proliferation, further suggesting that mTOR signaling did not down-regulate proliferation in the pancreas of patients with congenital HH [151]. Nevertheless, as recommended the last option prior to surgery, trying sirolimus before performing surgical therapy may help avoiding from much more severe and lifelong complications of surgery. Even so, this does not eliminate the requirement for further insights into identifying the long-term adverse effects and efficacy of mTOR inhibitors.

The abnormal pancreatic β-cells are localised to a specific location in pancreas. Focal pancreatic lesions are generally 2-10 mm in size and appear as small regions of islet adenomatosis (nodular hyperplasia of islet-like cell clusters, including ductuloinsular complexes, Fig. 2d) [1]. Islet cells of particular lesion have large cytoplasm with dispersed abnormal nuclei of irregular and angular shape [168]. Focal disease is mostly sporadic and is associated with a paternally inherited K_ATP channel mutation and the loss of the corresponding maternal allele in the focal area [169]. This promotes β-cell proliferation by inducing the expression of insulin-like growth factor 2, inhibiting the tumor suppressor genes H19 and cyclin-dependent kinase inhibitor 1C (CDKN1C) [170]. The Fluorine-18 dihydroxyphenylalanine-positron emission tomography (¹⁸F–DOPA-PET) scanning can help to localize focal lesion. The principle of ¹⁸F–DOPA-PET scan is based on the tissue uptake of L-DOPA. Pancreatic islets are able to uptake L-DOPA and convert it to dopamine through DOPA decarboxylase. The uptake of the positron emitting tracer ¹⁸F–DOPA-PET is increased in β-cells with a high rate of insulin synthesis and secretion compared to unaffected areas (Fig. 2a and c). The sensitivity for detecting focal lesions varies between 88 and 94% with an accuracy of 100% [171]. Intraoperative and postoperative diagnosis of focal congenital HH is based on the presence of adenomatous hyperplasia of β-cells within the focal lesion [172].

The diffuse form accounts for 60–70% of all congenital HH cases, and affects all pancreatic β-cells. Morphology of the islets of Langerhans shows the presence of β-cells with abnormally large nuclei (Fig. 2b) [173]. Patients with diffuse congenital HH either have a homozygous recessive or a compound heterozygous mutation in K_ATP channel genes [174]. Patients are usually unresponsive to the medical therapy and require a near-total pancreatectomy (95–98% removal).

If the pancreatic histology neither fits the focal nor diffuse congenital HH category, this can be defined as atypical form. In these atypical forms some islets show signs of hyperplasia but others appear normal. A previous study reported that some patients with congenital HH have morphological mosaicism including coexistence of two types of islet: large islets with cytoplasm-rich cells and occasional enlarged nuclei and shrunken islets with -cells exhibiting little cytoplasm and small nuclei [170].

Detection of paternally inherited mutations in K_ATP channel genes and localization of lesion using imaging with ¹⁸F–DOPA-PET/CT scan provide possibility of completely cure of the disease by limited lesionectomy/partial pancreatectomy, with few or no surgical complications [41, 178, 179]. In proximal lesions in the head and neck of the pancreas, open resection of the lesion with a small rim of surrounding normal pancreatic tissue is carried out and pancreaticojejunostomy is performed to allow drainage of the distal pancreas. Laparascopic distal pancreatectomy is the surgical option for distal lesions (Fig. 3,) [9].

Patients with diffuse and atypical disease that are unresponsive to medical treatment usually require extensive surgery (subtotal- or near-total pancreatectomy). Although this might alleviate the recurrence risk of hypoglycaemia, this procedure caries a high risk of developing pancreatic exocrine insufficiency and diabetes which requires life-long pancreatic enzyme replacement and insulin therapy [9, 180‐183]. In near-total pancreatectomy, the tail, body, uncinate process and part of pancreatic head are resected, leaving a rim of pancreatic tissue surrounding the common bile duct and along the duodenum [9]. However, despite this radical pancreatectomy some children continue to have HH despite the removal of 95–98% of pancreatic tissue [181]. Diabetes can develop immediately after surgery or later on follow up [180]. These patients who undergo surgical resection should be monitored for glucose metabolism and diabetes [180‐183].