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The recognition of β cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain β cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic β cells. Inducible ablation of LDB1 in mature β cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted β cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of β cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative β cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of β cells and is a component of active enhancers in both murine and human islets.

Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis. AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.

The importance of glucokinase (GK) in the regulation of insulin secretion has been highlighted by the phenotypes of individuals with activating and inactivating mutations in the glucokinase gene (GCK). Here we report 10 individuals with congenital hyperinsulinism (HI) caused by eight unique activating mutations of GCK. Six are novel and located near previously identified activating mutations sites. The first recognized episode of hypoglycemia in these patients occurred between birth and 24 years, and the severity of the phenotype was also variable. Mutant enzymes were expressed and purified for enzyme kinetics in vitro. Mutant enzymes had low glucose half-saturation concentration values and an increased enzyme activity index compared with wild-type GK. We performed functional evaluation of islets from the pancreata of three children with GCK-HI who required pancreatectomy. Basal insulin secretion in perifused GCK-HI islets was normal, and the response to glyburide was preserved. However, the threshold for glucose-stimulated insulin secretion in perifused glucokinase hyperinsulinism (GCK-HI) islets was decreased, and glucagon secretion was greatly suppressed. Our evaluation of novel GCK disease-associated mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response. Article Highlights: Our evaluation of six novel and two previously published activating GCK mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response. These studies provide insights into the pathophysiology of GCK-hyperinsulinism and the dual role of glucokinase in β-cells and α-cells to regulate glucose homeostasis. [ABSTRACT FROM AUTHOR]

There is a significant unmet need for a safe and effective therapy for the treatment of children with congenital hyperinsulinism. We hypothesized that amplification of the glucagon signaling pathway could ameliorate hyperinsulinism associated hypoglycemia. In order to test this we evaluated the effects of loss of Prkar1a, a negative regulator of Protein Kinase A in the context of hyperinsulinemic conditions. With reduction of Prkar1a expression, we observed a significant upregulation of hepatic gluconeogenic genes. In wild type mice receiving a continuous infusion of insulin by mini-osmotic pump, we observed a 2-fold increase in the level of circulating ketones and a more than 40-fold increase in Kiss1 expression with reduction of Prkar1a. Loss of Prkar1a in the Sur1-/- mouse model of KATP hyperinsulinism significantly attenuated fasting induced hypoglycemia, decreased the insulin/glucose ratio, and also increased the hepatic expression of Kiss1 by more than 10-fold. Together these data demonstrate that amplification of the hepatic glucagon signaling pathway is able to rescue hypoglycemia caused by hyperinsulinism. [ABSTRACT FROM AUTHOR]

NLRP3 assembles an inflammasome complex that activates caspase-1 upon sensing various danger signals derived from pathogenic infection, tissue damage, and environmental toxins. How NLRP3 senses these various stimuli is still poorly understood, but mitochondria and mitochondrial reactive oxygen species have been proposed to play a critical role in NLRP3 activation. In this article, we provide evidence that the mitochondrial antiviral signaling protein MAVS associates with NLRP3 and facilitates its oligomerization leading to caspase-1 activation. In reconstituted 293T cells, full-length MAVS promoted NLRP3-dependent caspase-1 activation, whereas a C-terminal transmembrane domain-truncated mutant of MAVS (MAVS-ΔTM) did not. MAVS, but not MAVSΔTM, interacted with NLRP3 and triggered the oligomerization of NLRP3, suggesting that mitochondrial localization of MAVS and intact MAVS signaling are essential for activating the NLRP3 inflammasome. Supporting this, activation of MAVS signaling by Sendai virus infection promoted NLRP3-dependent caspase-1 activation, whereas knocking down MAVS expression clearly attenuated the activation of NLRP3 inflammasome by Sendai virus in THP-1 and mouse macrophages. Taken together, our results suggest that MAVS facilitates the recruitment of NLRP3 to the mitochondria and may enhance its oligomerization and activation by bringing it in close proximity to mitochondrial reactive oxygen species. [ABSTRACT FROM AUTHOR]

Inactivating mutations of β-cell KATP channels cause the most common and severe form of congenital hyperinsulinism (HI), a β-cell disorder that causes dysregulated insulin secretion and persistent hypoglycemia. Children with KATPHI are typically unresponsive to diazoxide, the only FDA-approved drug for HI. Octreotide, a sst2 peptide agonist that inhibits insulin secretion, is used as second line therapy, but poor efficacy and sst2-mediated side effects, such as glucagon suppression and necrotizing enterocolitis, limit its use in infants. Thus, most infants with KATPHI require pancreatectomy for intractable hypoglycemia. To address the unmet need for effective and safe therapies, we examined the ability of a selective sst5 nonpeptide agonist CRN02481 (Crinetics Pharmaceuticals) to suppress insulin secretion and prevent fasting hypoglycemia in the SUR1-/- mouse model of KATPHI. SUR1-/- mouse islets were treated with CRN02481 (100 nM) or vehicle and then stimulated with a physiologic amino acid mixture (4 mM). CRN02481 significantly decreased baseline (1.5 ± 0.2 vs. 2.4 ± 0.5 ng/uL, p≤0.01) and amino acid-stimulated (2.8 ± 1.5 vs. 4.4 ± 1.1 ng/uL, p≤0.05) insulin secretion. SUR1-/- mice were gavaged with CRN02481 (10 mg/kg/day) or PBS for 7 days (n=7/group) and fasting glucose, glucose tolerance and insulin tolerance tests were assessed. CRN02481 significantly increased fasting glucose and prevented fasting hypoglycemia (98 ± 18 vs. 44 ± 6 mg/dL, p<0.00001). During a glucose tolerance test CRN02481 increased post-glucose load glucose concentrations significantly by 60 and 90 minutes. CRN02481 significantly decreased the slope of decline of glucose concentrations after insulin administration (1 u/kg): -2.8 ± 2.3 vs. -5.7 ± 2.1, p=0.03). Collectively these data show that the sst5 agonist CRN02481 effectively suppresses insulin secretion and prevents fasting hypoglycemia in the KATPHI mouse model and is a potentially novel therapy for children with HI. Disclosure: C. Juliana: Research Support; Self; Crinetics. J. Chai: None. E.F. Rico-Bautista: Employee; Self; Crinetics Pharmaceuticals. Stock/Shareholder; Self; Crinetics Pharmaceuticals. S.F. Betz: Employee; Self; Crinetics Pharmaceuticals. Stock/Shareholder; Self; Crinetics Pharmaceuticals. D.D. De Leon: Consultant; Self; Crinetics, Novartis Pharmaceuticals Corporation, ProSciento, Soleno Therapeutics, Zealand Pharma A/S. Employee; Spouse/Partner; Merck & Co., Inc. Research Support; Self; Crinetics, Dexcom, Inc., Zealand Pharma A/S. Stock/Shareholder; Self; Merck & Co., Inc. Funding: Crinetics Pharmaceuticals (to D.D.D.) [ABSTRACT FROM AUTHOR]

Introduction: Congenital hyperinsulinism (HI) is the leading cause of persistent hypoglycemia in infants. Current models to study the most common and severe form of HI resulting from inactivating mutations in the ATP-sensitive potassium channel (K ATP ) are limited to primary islets from patients and the Sur1 mouse model. Zebrafish exhibit potential as a novel K -/- HI model since they express canonical insulin secretion pathway genes and those with identified causative HI mutations. Moreover, zebrafish larvae transparency provides a unique opportunity for in vivo visualization of pancreatic islets.ATP HI model since they express canonical insulin secretion pathway genes and those with identified causative HI mutations. Moreover, zebrafish larvae transparency provides a unique opportunity for in vivo visualization of pancreatic islets., Research Design and Methods: We evaluated zebrafish as a model for K ATP HI using a genetically encoded Ca 2+ sensor (ins:gCaMP6s) expressed under control of the insulin promoter in beta cells of an abcc8 -/- zebrafish line., Results: We observed significantly higher islet cytosolic Ca 2+ in vivo in abcc8 -/- compared with abcc8 +/+ zebrafish larvae. Additionally, abcc8 -/- larval zebrafish had significantly lower whole body glucose and higher whole body insulin levels compared with abcc8 +/+ controls. However, adult abcc8 -/- zebrafish do not show differences in plasma glucose, plasma insulin, or glucose tolerance when compared with abcc8 +/+ zebrafish., Conclusions: Our results identify that zebrafish larvae, but not adult fish, are a demonstrable novel model for advancement of HI research., Competing Interests: Competing interests: DDDL has received consulting fees from Zealand Pharma A/S, Crinetics Pharmaceuticals, Hanmi Pharmaceuticals, Eiger Pharma and Rhythm Pharmaceuticals. DDDL has received research funding from Zealand Pharma A/S, Rezolute, Crinetics Pharmaceuticals, Hanmi Pharmaceuticals, Twist Biosciences, Eiger Pharma, and Ultragenyx for studies not discussed in this manuscript., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)