CRISPR/Cas9 ADCY7 Knockout Stimulates the Insulin Secretion Pathway Leading to Excessive Insulin Secretion.

Aim: Despite the enormous efforts to understand Congenital hyperinsulinism (CHI), up to 50% of the patients are genetically unexplained. We aimed to functionally characterize a novel candidate gene in CHI. Patient: A 4-month-old boy presented severe hyperinsulinemic hypoglycemia. A routine CHI genetic panel was negative. Methods: A trio-based whole-exome sequencing (WES) was performed. Gene knockout in the RIN-m cell line was established by CRISPR/Cas9. Gene expression was performed using real-time PCR. Results: Hyperinsulinemic hypoglycemia with diffuse beta-cell involvement was demonstrated in the patient, who was diazoxide-responsive. By WES, compound heterozygous variants were identified in the adenylyl cyclase 7, ADCY7 gene p.(Asp439Glu) and p.(Gly1045Arg). ADCY7 is calcium-sensitive, expressed in beta-cells and converts ATP to cAMP. The variants located in the cytoplasmic domains C1 and C2 in a highly conserved and functional amino acid region. RIN-m(-/-Adcy7) cells showed a significant increase in insulin secretion reaching 54% at low, and 49% at high glucose concentrations, compared to wild-type. In genetic expression analysis Adcy7 loss of function led to a 34.1-fold to 362.8-fold increase in mRNA levels of the insulin regulator genes Ins1 and Ins2 (p ≤ 0.0002), as well as increased glucose uptake and sensing indicated by higher mRNA levels of Scl2a2 and Gck via upregulation of Pdx1, and Foxa2 leading to the activation of the glucose stimulated-insulin secretion (GSIS) pathway. Conclusion: This study identified a novel candidate gene, ADCY7, to cause CHI via activation of the GSIS pathway.

Functional evaluation of 16 SCHAD missense variants: Only amino acid substitutions causing congenital hyperinsulinism of infancy lead to loss-of-function phenotypes in vitro.

Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), encoded by the HADH gene, is a ubiquitously expressed mitochondrial enzyme involved in fatty acid oxidation. This protein also plays a role in insulin secretion as recessive HADH mutations cause congenital hyperinsulinism of infancy (CHI) via loss of an inhibitory interaction with glutamate dehydrogenase (GDH). Here, we present a functional evaluation of 16 SCHAD missense variants identified either in CHI patients or by high-throughput sequencing projects in various populations. To avoid interactions with endogenously produced SCHAD protein, we assessed protein stability, subcellular localization, and GDH interaction in a SCHAD knockout HEK293 cell line constructed by CRISPR-Cas9 methodology. We also established methods for efficient SCHAD expression and purification in E. coli, and tested enzymatic activity of the variants. Our analyses showed that rare variants of unknown significance identified in populations generally had similar properties as normal SCHAD. However, the CHI-associated variants p.Gly34Arg, p.Ile184Phe, p.Pro258Leu, and p.Gly303Ser were unstable with low protein levels detectable when expressed in HEK293 cells. Moreover, CHI variants p.Lys136Glu, p.His170Arg, and p.Met188Val presented normal protein levels but displayed clearly impaired enzymatic activity in vitro, and their interaction with GDH appeared reduced. Our results suggest that pathogenic missense variants of SCHAD either make the protein target of a post-translational quality control system or can impair the function of SCHAD without influencing its steady-state protein level. We did not find any evidence that rare SCHAD missense variants observed only in the general population and not in CHI patients are functionally affected.

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism.

Human glucokinase (GCK) acts as the body's primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and ß-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a ß-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the ß-type variant lacks a burst phase. Comparative hydrogen-deuterium exchange mass spectrometry of unliganded enzymes demonstrates that a disordered active site loop, which folds upon binding of glucose, is protected from exchange in the α-type variant. The α-type variant also displays an increased level of exchange within a ß-strand located near the enzyme's hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, ß-type activation causes no substantial difference in global or local exchange relative to that of unliganded, wild-type GCK. Together, these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas ß-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK's unique kinetic cooperativity.

The Hypoglycemic Phenotype Is Islet Cell-Autonomous in Short-Chain Hydroxyacyl-CoA Dehydrogenase-Deficient Mice.

Congenital hyperinsulinism of infancy (CHI) can be caused by inactivating mutations in the gene encoding short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), a ubiquitously expressed enzyme involved in fatty acid oxidation. The hypersecretion of insulin may be explained by a loss of interaction between SCHAD and glutamate dehydrogenase in the pancreatic ß-cells. However, there is also a general accumulation of metabolites specific for the enzymatic defect in affected individuals. It remains to be explored whether hypoglycemia in SCHAD CHI can be uncoupled from the systemic effect on fatty acid oxidation. We therefore transplanted islets from global SCHAD knockout (SCHADKO) mice into mice with streptozotocin-induced diabetes. After transplantation, SCHADKO islet recipients exhibited significantly lower random and fasting blood glucose compared with mice transplanted with normal islets or nondiabetic, nontransplanted controls. Furthermore, intraperitoneal glucose tolerance was improved in animals receiving SCHADKO islets compared with those receiving normal islets. Graft ß-cell proliferation and apoptosis rates were similar in the two transplantation groups. We conclude that hypoglycemia in SCHAD-CHI is islet cell-autonomous.

Dipeptidyl peptidase-4 expression in pancreatic tissue from patients with congenital hyperinsulinism.

Congenital hyperinsulinism (CHI) is caused by unregulated insulin release and leads to hyperinsulinaemic-hypoglycaemia (HH). Glucagon like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), peptide YY (PYY) and the enzyme; dipeptidyl peptidase-4 (DPP-4) all regulate appetite and glucose homeostasis. These proteins have been identified as possible contributors to HH but the mechanism remains poorly understood. We aimed to look at the expression pattern of pancreatic DPP-4 in children with focal and diffuse CHI (FCHI and DCHI, respectively). Using immunohistochemistry; we determined DPP-4 expression patterns in the pancreas of CHI patients. DPP-4 was found to be expressed in the pancreatic ß, α and δ-cells in and around the focal area. However, it was predominantly co-localised with ß-cells in the paediatric tissue samples. Additionally, proliferating ß-cells expressed DPP-4 in DCHI, which was absent in the FCHI pancreas. Insulin was found to be present in the exocrine acini and duct cells of the DCHI pancreas suggestive of exocrine to endocrine transdifferentiation. Furthermore, 6 medically-unresponsive DCHI pancreatic samples showed an up-regulation of total pancreatic DPP-4 expression. In conclusion; the expression studies have shown DPP-4 to be altered in HH, however, further work is required to understand the underlying role for this enzyme.

Type 2 diabetes and congenital hyperinsulinism cause DNA double-strand breaks and p53 activity in ß cells.

ß cell failure in type 2 diabetes (T2D) is associated with hyperglycemia, but the mechanisms are not fully understood. Congenital hyperinsulinism caused by glucokinase mutations (GCK-CHI) is associated with ß cell replication and apoptosis. Here, we show that genetic activation of ß cell glucokinase, initially triggering replication, causes apoptosis associated with DNA double-strand breaks and activation of the tumor suppressor p53. ATP-sensitive potassium channels (KATP channels) and calcineurin mediate this toxic effect. Toxicity of long-term glucokinase overactivity was confirmed by finding late-onset diabetes in older members of a GCK-CHI family. Glucagon-like peptide-1 (GLP-1) mimetic treatment or p53 deletion rescues ß cells from glucokinase-induced death, but only GLP-1 analog rescues ß cell function. DNA damage and p53 activity in T2D suggest shared mechanisms of ß cell failure in hyperglycemia and CHI. Our results reveal membrane depolarization via KATP channels, calcineurin signaling, DNA breaks, and p53 as determinants of ß cell glucotoxicity and suggest pharmacological approaches to enhance ß cell survival in diabetes.

Order-disorder transitions govern kinetic cooperativity and allostery of monomeric human glucokinase.

Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity-onset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder-order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder-order cycle of the small domain as a "time-delay loop," which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.

[Mutation analysis of the GLUD1 gene in patients with glutamate dehydrogenase congenital hyperinsulinism].

OBJECTIVE: To investigate the glutamate dehydrogenase 1 (GLUD1) gene mutation of three patients diagnosed as glutamate dehydrogenase congenital hyperinsulinism (GDH-HI). METHODS: Three patients diagnosed as GDH-HI and their parents were involved in the study. PCR-DNA direct sequencing was used to analyze the exons 6,7,10,11 and 12 of the GLUD1 gene. RESULTS: In the first case, an R269H heterozygous mutation was found in the GLUD1 gene, with autosomal dominant inheritance. In the second case, there was a de novo S445L heterozygous mutation of the GLUD1 gene. No mutation was detected in the third case. CONCLUSION: In Chinese, R269H, S445L heterozygous mutation of the GLUD1 gene can lead to GDH-HI. Genetic analysis is necessary in making genetic diagnosis of congenital hyperinsulinsm.

Regulation of glutamate metabolism and insulin secretion by glutamate dehydrogenase in hypoglycemic children.

In addition to its extracellular roles as a neurotransmitter/sensory molecule, glutamate serves important intracellular signaling functions via its metabolism through glutamate dehydrogenase (GDH). GDH is a mitochondrial matrix enzyme that catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate in a limited number of tissues in humans, including the liver, the kidney, the brain, and the pancreatic islets. GDH activity is subject to complex regulation by negative (GTP, palmitoyl-coenzyme A) and positive (ADP, leucine) allosteric effectors. This complex regulation allows GDH activity to be modulated by changes in energy state and amino acid availability. The importance of GDH regulation has been highlighted by the discovery of a novel hypoglycemic disorder in children, the hyperinsulinism-hyperammonemia syndrome, which is caused by dominantly expressed, activating mutations of the enzyme that impair its inhibition by GTP. Affected children present in infancy with hypoglycemic seizures after brief periods of fasting or the ingestion of a high-protein meal. Patients have characteristic persistent 3- to 5-fold elevations of blood ammonia concentrations but do not display the usual neurologic symptoms of hyperammonemia. The mutant GDH enzyme shows impaired responses to GTP inhibition. Isolated islets from mice that express the mutant GDH in pancreatic beta cells show an increased rate of glutaminolysis, increased insulin release in response to glutamine, and increased sensitivity to leucine-stimulated insulin secretion. The novel hyperinsulinism-hyperammonemia syndrome indicates that GDH-catalyzed glutamate metabolism plays important roles in 3 tissues: in beta cells, the regulation of amino acid-stimulated insulin secretion; in hepatocytes, the modulation of amino acid catabolism and ammoniagenesis; and in brain neurons, the maintenance of glutamate neurotransmitter concentrations.

Activating mutations in the human glucokinase gene revealed by genetic selection.

We describe the discovery of 11 new activating mutations in the human glk gene associated with the disease persistent hyperinsulinemic hypoglycemia of infancy (PHHI). Three of the newly identified substitutions colocalize to a region of the glucokinase polypeptide where a synthetic allosteric activator binds. Of these substitutions, I211F is the most active variant identified to date, with a k(cat)/K(0.5,glucose) value (6.6 x 10(4) M(-1) s(-1)) that is 12-fold higher than that of wild-type glucokinase. The stimulatory mutations described herein represent surreptitious genetic determinants of PHHI. They also identify novel features of the glucokinase scaffold that could be targeted during the development of diabetes therapeutics.

Molecular physiology of mammalian glucokinase.

The glucokinase (GCK) gene was one of the first candidate genes to be identified as a human "diabetes gene". Subsequently, important advances were made in understanding the impact of GCK in the regulation of glucose metabolism. Structure elucidation by crystallography provided insight into the kinetic properties of GCK. Protein interaction partners of GCK were discovered. Gene expression studies revealed new facets of the tissue distribution of GCK, including in the brain, and its regulation by insulin in the liver. Metabolic control analysis coupled to gene overexpression and knockout experiments highlighted the unique impact of GCK as a regulator of glucose metabolism. Human GCK mutants were studied biochemically to understand disease mechanisms. Drug development programs identified small molecule activators of GCK as potential antidiabetics. These advances are summarized here, with the aim of offering an integrated view of the role of GCK in the molecular physiology and medicine of glucose homeostasis.