Diva D. De León

Preexisting pancreatic acinar cells contribute to acinar cell, but not islet β cell, regeneration

It has been suggested that pancreatic acinar cells can serve as progenitors for pancreatic islets, a concept with substantial implications for therapeutic efforts to increase insulin-producing beta cell mass in patients with diabetes. We report what we believe to be the first in vivo lineage tracing approach to determine the plasticity potential of pancreatic acinar cells. We developed an acinar cell-specific inducible Cre recombinase transgenic mouse, which, when mated with a reporter strain and pulsed with tamoxifen, resulted in permanent and specific labeling of acinar cells and their progeny. During various time periods of observation and using several models to provoke injury, we failed to observe any chase of the labeled cells into the endocrine compartment, indicating that acinar cells do not normally transdifferentiate into islet beta cells in vivo in adult mice. In contrast, we observed a substantial role for replication of preexisting acinar cells in the regeneration of new acinar cells after partial pancreatectomy. These results indicate that mature acinar cells harbor a facultative acinar but not endocrine progenitor capacity.

Mechanisms of Disease: advances in diagnosis and treatment of hyperinsulinism in neonates

Hyperinsulinism is the single most common mechanism of hypoglycemia in neonates. Dysregulated insulin secretion is responsible for the transient and prolonged forms of neonatal hypoglycemia, and congenital genetic disorders of insulin regulation represent the most common of the permanent disorders of hypoglycemia. Mutations in at least five genes have been associated with congenital hyperinsulinism: they encode glucokinase, glutamate dehydrogenase, the mitochondrial enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase, and the two components (sulfonylurea receptor 1 and potassium inward rectifying channel, subfamily J, member 11) of the ATP-sensitive potassium channels (KATP channels). KATP hyperinsulinism is the most common and severe form of congenital hyperinsulinism. Infants suffering from KATP hyperinsulinism present shortly after birth with severe and persistent hypoglycemia, and the majority are unresponsive to medical therapy, thus requiring pancreatectomy. In up to 40–60% of the children with KATP hyperinsulinism, the defect is limited to a focal lesion in the pancreas. In these children, local resection results in cure with avoidance of the complications inherent to a near-total pancreatectomy. Hyperinsulinism can also be part of other disorders such as Beckwith–Wiedemann syndrome and congenital disorders of glycosylation. The diagnosis and management of children with congenital hyperinsulinism requires a multidisciplinary approach to achieve the goal of therapy: prevention of permanent brain damage due to recurrent hypoglycemia.

Recommendations from the Pediatric Endocrine Society for Evaluation and Management of Persistent Hypoglycemia in Neonates, Infants, and Children

Recommendations from the Pediatric Endocrine Society for Evaluation and Management of Persistent Hypoglycemia in Neonates, Infants, and Children Paul S. Thornton, MB, BCh, Charles A. Stanley, MD, Diva D. De Leon, MD, MSCE, Deborah Harris, PhD, Morey W. Haymond, MD, Khalid Hussain, MD, MPH, Lynne L. Levitsky, MD, Mohammad H. Murad, MD, MPH, Paul J. Rozance, MD, Rebecca A. Simmons, MD, Mark A. Sperling, MBBS, David A. Weinstein, MD, MMSc, Neil H. White, MD, and Joseph I. Wolfsdorf, MB, BCh

Calcineurin Signaling Regulates Human Islet β-Cell Survival

The calcium-regulated phosphatase calcineurin intersects with both calcium and cAMP-mediated signaling pathways in the pancreatic β-cell. Pharmacologic calcineurin inhibition, necessary to prevent rejection in the setting of organ transplantation, is associated with post-transplant β-cell failure. We sought to determine the effect of calcineurin inhibition on β-cell replication and survival in rodents and in isolated human islets. Further, we assessed whether the GLP-1 receptor agonist and cAMP stimulus, exendin-4 (Ex-4), could rescue β-cell replication and survival following calcineurin inhibition. Following treatment with the calcineurin inhibitor tacrolimus, human β-cell apoptosis was significantly increased. Although we detected no human β-cell replication, tacrolimus significantly decreased rodent β-cell replication. Ex-4 nearly normalized both human β-cell survival and rodent β-cell replication when co-administered with tacrolimus. We found that tacrolimus decreased Akt phosphorylation, suggesting that calcineurin could regulate replication and survival via the PI3K/Akt pathway. We identify insulin receptor substrate-2 (Irs2), a known cAMP-responsive element-binding protein target and upstream regulator of the PI3K/Akt pathway, as a novel calcineurin target in β-cells. Irs2 mRNA and protein are decreased by calcineurin inhibition in both rodent and human islets. The effect of calcineurin on Irs2 expression is mediated at least in part through the nuclear factor of activated T-cells (NFAT), as NFAT occupied the Irs2 promoter in a calcineurin-sensitive manner. Ex-4 restored Irs2 expression in tacrolimus-treated rodent and human islets nearly to baseline. These findings reveal calcineurin as a regulator of human β-cell survival in part through regulation of Irs2, with implications for the pathogenesis and treatment of diabetes following organ transplantation.

Novel Presentations of Congenital Hyperinsulinism due to Mutations in the MODY genes: HNF1A and HNF4A

CONTEXT Inactivating mutations in HNF1A and HNF4A cause the maturity-onset diabetes of youth (MODY)-3 and MODY1 forms of monogenic diabetes, respectively. Children carrying HNF4A (MODY1) mutations can present in early infancy with macrosomia and diazoxide-responsive hyperinsulinism. OBJECTIVE Our objective was to describe three novel cases of hyperinsulinism associated with MODY1 and MODY3 mutations. RESEARCH DESIGN AND METHODS Clinical data were obtained from chart review. Gene sequencing was performed on genomic DNA. RESULTS Case 1 was diagnosed at 20 months with persistent hyperinsulinemic hypoglycemia and was found to have a novel MODY3 HNF1A mutation, carried by her father who had diabetes. Case 2 was diagnosed with diazoxide-responsive hyperinsulinism at 3 months of age and had complete resolution of hyperinsulinism by 4 yr. She was found to have a novel MODY3 HNF1A missense mutation, also carried by her father. Case 3 presented as a newborn with diazoxide-responsive hyperinsulinism and later developed renal Fanconi syndrome, hypophosphatemic rickets, and hepatic glycogenosis. Although the latters features suggested Fanconi-Bickel syndrome, sequencing of the SLC2A2 gene was normal. The patient was found to have a known MODY1 mutation in HNF4A. In all cases, the hyperinsulinism improved with age. CONCLUSIONS The first two cases demonstrate that mutations in HNF1A (MODY3) can cause hyperinsulinism early in life and diabetes later, similar to the phenotype recently reported for HNF4A (MODY1) mutations. Case 3 indicates that the effects of HNF4A mutations in infancy may extend beyond pancreatic β-cells to produce a disorder similar to glucose transporter 2 deficiency involving both liver glycogen metabolism and renal tubular transport.

Re-Evaluating "Transitional Neonatal Hypoglycemia": Mechanism and Implications for Management

A Committee of the Pediatric Endocrine Society was recently asked by xxx to develop guidelines for evaluation and management of hypoglycemia in neonates, infants, and children. To aid in formulating recommendations for neonates, in this review, we analyzed available data on the brief period of hypoglycemia which commonly is observed in normal newborns during the transition from fetal to extrauterine life, hereafter referred to as transitional neonatal hypoglycemia in normal newborns. The goal was to better understand the mechanism underlying this phenomenon in order to formulate recommendations for recognizing neonates requiring diagnosis and treatment during the first days of life for disorders causing severe and persistent hypoglycemia. It has long been known that plasma glucose concentrations are lower in the first 1–3 days of life in normal newborn infants than at later ages. Not until the 1960s was it appreciated that hypoglycemia in neonates could sometimes be symptomatic and, as in older infants and children, cause seizures or permanent brain damage (1, 2). Although studies in laboratory animals have demonstrated postnatal developmental changes in specific enzymes involved in hepatic gluconeogenesis and ketogenesis (3, 4), it is unclear that such changes adequately explain transitional neonatal hypoglycemia in human newborns or if other mechanisms may be involved (5, 6). A National Institutes of Health conference outlined many of the “gaps in knowledge” about neonatal hypoglycemia and lamented the lack of a rational basis for defining hypoglycemia in neonates (7). For this re-evaluation of transitional neonatal hypoglycemia in normal newborns, we used the strategy routinely employed by pediatric endocrinologists for evaluation of hypoglycemia in older infants and children. This strategy, based on an examination of the major metabolic fuel and hormone responses to hypoglycemia, makes it possible to discover the mechanism of hypoglycemia and to make a specific diagnosis of the underlying cause (Figure; available at www.jpeds.com) (8). We reviewed published data in normal newborns on metabolic fuel and hormone responses during the period of transitional neonatal hypoglycemia. We focused on mean responses as being most likely representative of normal newborns, recognizing the possibility of heterogeneity, particularly with regard to peri-partum stresses and feeding practices. We found that transitional neonatal hypoglycemia most closely resembles known genetic forms of congenital hyperinsulinism, which cause a lowering of the plasma glucose threshold for suppression of insulin secretion. This conclusion is based on strong evidence supported by two or more independent reports and provides a novel perspective on both the diagnosis and management of hypoglycemia in the first several days after birth. Figure Hypoglycemia diagnosis based on plasma metabolic fuel responses. Measurement of major fuels (lactate as a gluconeogenic substrate, FFA from adipose tissue lipolysis, and beta-hydroxybutyrate as the major ketone from hepatic ketogenesis) at a time of hypoglycemia ...

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.

Infants with congenital hyperinsulinism owing to inactivating mutations in the KATP channel (KATPHI) who are unresponsive to medical therapy will require pancreatectomy to control the hypoglycemia. In preclinical studies, we showed that the GLP-1 receptor antagonist exendin-(9-39) suppresses insulin secretion and corrects fasting hypoglycemia in SUR-1−/− mice. The aim of this study was to examine the effects of exendin-(9-39) on fasting blood glucose in subjects with KATPHI. This was a randomized, open-label, two-period crossover pilot clinical study. Nine subjects with KATPHI received either exendin-(9-39) or vehicle on two different days. The primary outcome was blood glucose; secondary outcomes were insulin, glucagon, and GLP-1. In all subjects, mean nadir blood glucose and glucose area under the curve were significantly increased by exendin-(9-39). Insulin-to-glucose ratios were significantly lower during exendin-(9-39) infusion compared with vehicle. Fasting glucagon and intact GLP-1 were not affected by treatment. In addition, exendin-(9-39) significantly inhibited amino acid–stimulated insulin secretion in pancreatic islets isolated from neonates with KATPHI. Our findings have two important implications: 1) GLP-1 and its receptor play a role in the regulation of fasting glycemia in KATPHI; and 2) the GLP-1 receptor may be a therapeutic target for the treatment of children with KATPHI.

Neonatal Diabetes and Congenital Malabsorptive Diarrhea Attributable to a Novel Mutation in the Human Neurogenin-3 Gene Coding Sequence

OBJECTIVE The aim was to describe the clinical presentation and to characterize the genetic mutation present in a child with congenital malabsorptive diarrhea and neonatal diabetes. RESEARCH DESIGN AND METHODS Clinical data were obtained from chart review. Histopathological characterization of intestinal samples and neurogenin-3 (NEUROG3) sequencing were performed. Expression and function of the mutated NEUROG3 protein were assessed by Western blot analysis and luciferase reporter assay. RESULTS At birth, the proband was small for gestational age. She presented for evaluation with persistent diarrhea and a poor postnatal growth pattern. Although the pancreas was present, serum amylase and fecal elastase levels were decreased, and blood glucose levels were persistently elevated by 5 months of age. Immunostaining of a small intestine biopsy for chromogranin A demonstrated complete absence of neuroendocrine cells. Genetic analysis revealed a nonsense mutation (E123X) in the region encoding helix II of the NEUROG3 gene, leading to premature termination at amino acid 123. The mutated truncated NEUROG3 protein was identified by Western blot analysis. Reporter assays show decreased transactivation of the NEUROD1 promoter by mutant NEUROG3 protein as compared to wild type. CONCLUSIONS This report describes a newly identified nonsense mutation in human NEUROG3 that in the homozygous state is associated with neonatal diabetes and malabsorptive diarrhea.

Exendin-(9–39) Corrects Fasting Hypoglycemia in SUR-1–/– Mice by Lowering cAMP in Pancreatic β-Cells and Inhibiting Insulin Secretion

Congenital hyperinsulinism is a disorder of pancreatic β-cell function characterized by failure to suppress insulin secretion in the setting of hypoglycemia, resulting in brain damage or death if untreated. Loss-of-function mutations in the KATP channel (composed of two subunits: Kir6.2 and SUR-1) are responsible for the most common and severe form of congenital hyperinsulinism. Most patients are unresponsive to available medical therapy and require palliative pancreatectomy. Similar to the human condition, the SUR-1–/– mouse is hypoglycemic when fasted and hyperglycemic when glucose-loaded. We have previously reported that the glucagon-like peptide-1 receptor antagonist exendin-(9–39) raises fasting blood glucose in normal mice. Here we examine the effect of exendin-(9–39) on fasting blood glucose in SUR-1–/– mice. Mice were randomized to receive exendin-(9–39) or vehicle. Fasting blood glucose levels in SUR-1–/– mice treated with exendin-(9–39) were significantly higher than in vehicle-treated mice and not different from wild-type littermates. Exendin-(9–39) did not further worsen glucose tolerance and had no effect on body weight and insulin sensitivity. Isolated islet perifusion studies demonstrated that exendin-(9–39) blocked amino acid-stimulated insulin secretion, which is abnormally increased in SUR-1–/– islets. Furthermore, cAMP content in SUR-1–/– islets was reduced by exendin-(9–39) both basally and when stimulated by amino acids, whereas cytosolic calcium levels were not affected. These findings suggest that cAMP plays a key role in KATP-independent insulin secretion and that the GLP-1 receptor is constitutively active in SUR-1–/– β-cells. Our findings indicate that exendin-(9–39) normalizes fasting hypoglycemia in SUR-1–/– mice via a direct effect on insulin secretion, thereby raising exendin-(9–39) as a potential therapeutic agent for KATP hyperinsulinism.

A Mutation in the TMD0-L0 Region of Sulfonylurea Receptor-1 (L225P) Causes Permanent Neonatal Diabetes Mellitus (PNDM)

OBJECTIVE—We sought to examine the molecular mechanisms underlying permanenent neonatal diabetes mellitus (PNDM) in a patient with a heterozygous de novo L225P mutation in the L0 region of the sulfonylurea receptor (SUR)1, the regulatory subunit of the pancreatic ATP-sensitive K+ channel (KATP channel). RESEARCH DESIGN AND METHODS—The effects of L225P on the properties of recombinant KATP channels in transfected COS cells were assessed by patch-clamp experiments on excised membrane patches and by macroscopic Rb-flux experiments in intact cells. RESULTS—L225P-containing KATP channels were significantly more active in the intact cell than in wild-type channels. In excised membrane patches, L225P increased channel sensitivity to stimulatory Mg nucleotides without altering intrinsic gating or channel inhibition by ATP in the absence of Mg2+. The effects of L225P were abolished by SUR1 mutations that prevent nucleotide hydrolysis at the nucleotide binding folds. L225P did not alter channel inhibition by sulfonylurea drugs, and, consistent with this, the patient responded to treatment with oral sulfonylureas. CONCLUSIONS—L225P underlies KATP channel overactivity and PNDM by specifically increasing Mg-nucleotide stimulation of the channel, consistent with recent reports of mechanistically similar PNDM-causing mutations in SUR1. The mutation does not affect sulfonylurea sensitivity, and the patient is successfully treated with sulfonylureas.