De León-Crutchlow Laboratory Research Overview

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The De León-Crutchlow lab's translational research program focuses on examining the molecular genetics and pathophysiology of disorders of insulin regulation, with an emphasis on identifying novel therapeutic targets and new therapies for these conditions through lab and patient-oriented research.

In many patients with congenital hyperinsulinism (HI) a causative genetic mutation is not initially identified. Therefore, a major focus the lab's work is pinpointing previously unidentified causative mutations of HI in these patients. This work is done in collaboration with Dr. Arupa Ganguly from the Department of Genetics at the University of Pennsylvania. Numerous approaches, such as whole exome and whole genome sequencing, are used to identify novel mutations when genetic testing indicates a negative mutation analysis in known genetic loci.

A second area of focus is the identification of mosaic mutations that are undetectable by standard mutation analysis in peripheral blood. In patients who undergo pancreatectomy, direct genetic sequencing is conducted on pancreatic tissue removed during surgery. In patients who do not undergo pancreatectomy, sequencing is conducted on cell free DNA obtained from blood samples. The lab employs modern sequencing methods to locate and identify novel mutations and remain on the cutting edge of emerging technology to identify these hard-to-find mutations.

Understanding the consequences of various mutations that cause hyperinsulinism is an important step to create better treatments for patients. The lab works to characterize both mutations identified in novel genes not previously associated with HI, but also new mutations that occur in known genetic loci. A major area of focus regarding novel genetic loci is studying the consequences of non-coding variants in HK1.

A second area of focus is the characterization of novel missense mutation in ABCC8 and KCNJ11. This is done in part through functional studies of mutant channels as part of an ongoing collaboration with Dr. Show-Ling Shyng from Oregon Health and Science University. In addition, novel mutations in ABCC8 and KCNJ11, as well as in other loci, are characterized by phenotyping carriers of these mutations, which involves oral glucose and protein tolerance tests, a 24-hour fast, and/or exercise tolerance testing.

Functional studies of islet cells isolated from surgically resected pancreas are also used to characterize novel mutations through studying insulin secretion in response to various stimuli. These studies help to elucidate how mutations that affect different steps of the insulin secretion pathway can have different functional outcomes in patients.

Understanding the underlying mechanisms of insulin secretion and discovery of novel modulators of insulin secretion are important focuses of the lab. Currently, the lab is evaluating the capacity of the voltage-gated potassium channel Kv7.1 and the calcium-activated chloride channel TMEM16A to regulate insulin secretion in pancreatic islets. This work is done in collaboration with Professor Toshi Hoshi from the Department of Physiology at the University of Pennsylvania. Both human and mouse islets are used to delineate the specific roles that these ion channels could play in the secretion of insulin. Perforated whole-cell patch-clamp measurements of membrane potential (Vm) and perifusion of isolated pancreatic islets are used to assess any changes in the kinetics of insulin secretion caused by changes in expression or activity of Kv7.1 or TMEM16A.

In another project, the lab explores the effect of hypoxia and the HIF1a pathway on the maturation, growth, and function of pancreatic beta cells. To assess the role of Hif1a pathway and hypoxia in the beta cell, the lab uses perifusion, plasma glucose, plasma insulin, and glucose tolerance tests of several mouse models.

The ultimate goal of the lab is turning discoveries into better and more effective treatments for patients. A clear example of our capabilities is the development of exendin-(9-39), a specific GLP-1 receptor antagonist, as a novel therapy for congenital hyperinsulinism. From the early proof-of-concept studies generated from studying the effects of this peptide on cytosolic cAMP and insulin secretion, to validating studies in a mouse model of congenital hyperinsulinism, and phase 2A clinical trials under an investigator-sponsored IND in adults, adolescents, children and infants with congenital hyperinsulinism, the lab team has demonstrated that exendin-(9-39) has the potential to become a much needed new therapy for this condition.

In addition to our own drug development programs, the De León-Crutchlow lab partners with biotech companies to evaluate the efficacy of new potential treatments to lower insulin secretion and raise fasting glucose in the Sur1-/- hyperinsulinism mouse model. A recent project demonstrated that glucagon like peptide 1 receptor (GLP-1R) targeted antibodies significantly decreased insulin secretion during perifusion in human hyperinsulinemic islets, as well as in both wild type and Sur1-/- mouse islets. The GLP-1R targeted antibodies also raised fasting plasma glucose levels and decreased the insulin to glucose ratio in mice.

Another project examined the effect of a selective nonpeptide somatostatin 5 (SST5) agonist on fasting hypoglycemia and insulin secretion. With treatment of the SSTR5 agonist, significant increase in fasting plasma glucose levels were observed in mice and a significant decrease in insulin secretion was observed in isolated pancreatic islets.

Yet another study in progress examines long-acting forms of glucagon on fasting plasma glucose levels in Sur1-/- mice. The lab will continue these efforts and our partnerships with pharmaceutical companies to test new treatments in animal models of hyperinsulinism and conduct clinical trials of these potential therapeutics with the goal of bringing new treatment options to the patients affected by congenital hyperinsulinism.

The De León-Crutchlow lab works to create novel models to open new avenues of research for congenital hyperinsulinism. These models range from cell lines, to mouse lines, and to a zebrafish model. A current cell model that is in development expresses a mutant insulin receptor. This new cell line will allow for the study of the function of mutant insulin receptors that have been identified in some patients and the effects of the mutations on insulin signaling.

The lab team also is in the process of developing novel mouse lines that can help study different modulators of insulin secretion and beta cell function. Some examples include a β cell specific KCNQ1 deletion mouse line and a β cell specific HIf1a deletion mouse line. These new mouse lines will allow for study of the specific consequences of various mutations on pancreatic β cell function and insulin secretion.

The lab team also in the process of characterizing a zebrafish line with an early stop codon in the ABCC8 gene with the ultimate goal of implementing the zebrafish as a model to validate novel genetic loci and test potential new therapies. One major advantage of the zebrafish model is the ability to analyze insulin secretion in vivo. Using expression of ins:GcAMP6s, which is a fluorescent genetically encoded Ca2+sensor in pancreatic β cells, we can visualize the elevations of intracellular [Ca2+] during insulin secretion under the control of the insulin promoter. New models will be created as the lab uncovers novel genetic etiologies causative of hyperinsulinism.

Hyperinsulinism: Genotyping, Phenotyping, Database and Repository (1989 – present)

For over three decades, the De León-Crutchlow lab has collected blood, saliva, pancreatic tissue, and stool samples from thousands of patients with hyperinsulinism. Our goal is to (1) determine the phenotypic and genetic underpinnings of hyperinsulinism, (2) identify novel genetic candidates causing insulin dysregulation, (3) build a comprehensive repository of samples representing different forms of congenital hyperinsulinism, and (4) allow for future, multidisciplinary analyses with scientists inside and outside of our Center.

Improving Understanding of Neuropathophysiologic Mechanisms of Disease in Hyperinsulinemic Hypoglycemia Through Advanced Neuroimaging Techniques (2019 – present)

This study seeks to build knowledge of the pathophysiologic mechanisms underlying neurodevelopmental consequences of hyperinsulinism. The primary objective of this study is to determine whether brain GluCEST percent contrast, as a measure of brain glutamate concentration, differs between individuals with subtypes of hyperinsulinism compared to those with T1DM, and healthy controls.

Efficacy of Vitamin E in Hyperinsulinism/Hyperammonemia (HI/HA) Syndrome (2019 – present)

Based on preclinical studies, the De León-Crutchlow lab team hypothesizes that vitamin E will inhibit GDH activity and may impact hyperinsulinemic hypoglycemia and hyperammonemia in subjects with HI/HA syndrome. The primary objective of this study is to determine an effective dose of vitamin E to reduce protein-induced hyperinsulinemia in pediatric and adult subjects with HI/HA.

Partnering with Industry (2008 – present)

Dr. De León-Crutchlow's clinical research program has established itself as one of the world’s premier hyperinsulinism research centers. The lab is continually partnering with pharmaceutical and industry sponsors to evaluate the safety, tolerability, and efficacy of investigational drugs and devices.