LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic ß cells.

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning ß cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and ß cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human ß cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in ß cells to maintain appropriate insulin release.

Revealing the tissue-level complexity of endogenous glucagon-like peptide-1 receptor expression and signaling.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used in the treatment of diabetes and obesity, yet visualizing the endogenous localization, organization and dynamics of a GPCR has so far remained out of reach. In the present study, we generate mice harboring an enzyme self-label genome-edited into the endogenous Glp1r locus. We also rationally design and test various fluorescent dyes, spanning cyan to far-red wavelengths, for labeling performance in tissue. By combining these technologies, we show that endogenous GLP1R can be specifically and sensitively detected in primary tissue using multiple colors. Longitudinal analysis of GLP1R dynamics reveals heterogeneous recruitment of neighboring cell subpopulations into signaling and trafficking, with differences observed between GLP1RA classes and dual agonists. At the nanoscopic level, GLP1Rs are found to possess higher organization, undergoing GLP1RA-dependent membrane diffusion. Together, these results show the utility of enzyme self-labels for visualization and interrogation of endogenous proteins, and provide insight into the biology of a class B GPCR in primary cells and tissue.

GC-Globulin/Vitamin D-Binding Protein Is Required for Pancreatic α-Cell Adaptation to Metabolic Stress.

GC-globulin (GC), or vitamin D-binding protein, is a multifunctional protein involved in the transport of circulating vitamin 25(OH)D and fatty acids, as well as actin scavenging. In the pancreatic islets, the gene encoding GC, GC/Gc, is highly localized to glucagon-secreting α-cells. Despite this, the role of GC in α-cell function is poorly understood. We previously showed that GC is essential for α-cell morphology, electrical activity, and glucagon secretion. We now show that loss of GC exacerbates α-cell failure during metabolic stress. High-fat diet-fed GC-/- mice have basal hyperglucagonemia, which is associated with decreased α-cell size, impaired glucagon secretion and Ca2+ fluxes, and changes in glucose-dependent F-actin remodelling. Impairments in glucagon secretion can be rescued using exogenous GC to replenish α-cell GC levels, increase glucagon granule area, and restore the F-actin cytoskeleton. Lastly, GC levels decrease in α-cells of donors with type 2 diabetes, which is associated with changes in α-cell mass, morphology, and glucagon expression. Together, these data demonstrate an important role for GC in α-cell adaptation to metabolic stress.

Proline synthesis through PYCR1 is required to support cancer cell proliferation and survival in oxygen-limiting conditions.

The demands of cancer cell proliferation alongside an inadequate angiogenic response lead to insufficient oxygen availability in the tumor microenvironment. Within the mitochondria, oxygen is the major electron acceptor for NADH, with the result that the reducing potential produced through tricarboxylic acid (TCA) cycle activity and mitochondrial respiration are functionally linked. As the oxidizing activity of the TCA cycle is required for efficient synthesis of anabolic precursors, tumoral hypoxia could lead to a cessation of proliferation without another means of correcting the redox imbalance. We show that in hypoxic conditions, mitochondrial pyrroline 5-carboxylate reductase 1 (PYCR1) activity is increased, oxidizing NADH with the synthesis of proline as a by-product. We further show that PYCR1 activity is required for the successful maintenance of hypoxic regions by permitting continued TCA cycle activity, and that its loss leads to significantly increased hypoxia in vivo and in 3D culture, resulting in widespread cell death.

Lack of ZnT8 protects pancreatic islets from hypoxia- and cytokine-induced cell death.

Pancreatic ß-cells depend on the well-balanced regulation of cytosolic zinc concentrations, providing sufficient zinc ions for the processing and storage of insulin, but avoiding toxic effects. The zinc transporter ZnT8, encoded by SLC30A8,is a key player regarding islet cell zinc homeostasis, and polymorphisms in this gene are associated with altered type 2 diabetes susceptibility in man. The objective of this study was to investigate the role of ZnT8 and zinc in situations of cellular stress as hypoxia or inflammation. Isolated islets of WT and global ZnT8-/- mice were exposed to hypoxia or cytokines and cell death was measured. To explore the role of changing intracellular Zn2+ concentrations, WT islets were exposed to different zinc concentrations using zinc chloride or the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). Hypoxia or cytokine (TNF-α, IFN-γ, IL1-ß) treatment induced islet cell death, but to a lesser extent in islets from ZnT8-/- mice, which were shown to have a reduced zinc content. Similarly, chelation of zinc with TPEN reduced cell death in WT islets treated with hypoxia or cytokines, whereas increased zinc concentrations aggravated the effects of these stressors. This study demonstrates a reduced rate of cell death in islets from ZnT8-/- mice as compared to WT islets when exposed to two distinct cellular stressors, hypoxia or cytotoxic cytokines. This protection from cell death is, in part, mediated by a reduced zinc content in islet cells of ZnT8-/- mice. These findings may be relevant for altered diabetes burden in carriers of risk SLC30A8 alleles in man.

Isoform-specific Roles of Prolyl Hydroxylases in the Regulation of Pancreatic ß-Cell Function.

Pancreatic ß-cells can secrete insulin via 2 pathways characterized as KATP channel -dependent and -independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP+ ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α. In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated ß-cell-specific knockout (KO) mice for all 3 isoforms of PHD (ß-PHD1 KO, ß-PHD2 KO, and ß-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the first phase of insulin secretion but enhanced the second phase of insulin secretion in isolated islets. None of the ß-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for ß-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both ß-PHD1 KO and ß-PHD3 KO had elevated ß-cell apoptosis and reduced ß-cell mass. Isolated islets from ß-PHD1 KO and ß-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP+ ratio. All 3 PHD isoforms are expressed in ß-cells, with PHD3 showing the most distinct expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, ß-PHD1 KO and ß-PHD3 KO mice had defective ß-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Prolyl-4-hydroxylase 3 maintains ß cell glucose metabolism during fatty acid excess in mice.

The α-ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is an HIF target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. Although PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about the effects of this highly conserved enzyme in insulin-secreting ß cells in vivo. Here, we show that the deletion of PHD3 specifically in ß cells (ßPHD3KO) was associated with impaired glucose homeostasis in mice fed a high-fat diet. In the early stages of dietary fat excess, ßPHD3KO islets energetically rewired, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in ßPHD3KO islets was associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes, and insulin secretion. Thus, PHD3 might be a pivotal component of the ß cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.

PDX1LOW MAFALOW ß-cells contribute to islet function and insulin release.

Transcriptionally mature and immature ß-cells co-exist within the adult islet. How such diversity contributes to insulin release remains poorly understood. Here we show that subtle differences in ß-cell maturity, defined using PDX1 and MAFA expression, contribute to islet operation. Functional mapping of rodent and human islets containing proportionally more PDX1HIGH and MAFAHIGH ß-cells reveals defects in metabolism, ionic fluxes and insulin secretion. At the transcriptomic level, the presence of increased numbers of PDX1HIGH and MAFAHIGH ß-cells leads to dysregulation of gene pathways involved in metabolic processes. Using a chemogenetic disruption strategy, differences in PDX1 and MAFA expression are shown to depend on islet Ca2+ signaling patterns. During metabolic stress, islet function can be restored by redressing the balance between PDX1 and MAFA levels across the ß-cell population. Thus, preserving heterogeneity in PDX1 and MAFA expression, and more widely in ß-cell maturity, might be important for the maintenance of islet function.

Vitamin-D-Binding Protein Contributes to the Maintenance of α Cell Function and Glucagon Secretion.

Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in ß cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.

Oncogenic IDH1 Mutations Promote Enhanced Proline Synthesis through PYCR1 to Support the Maintenance of Mitochondrial Redox Homeostasis.

Since the discovery of mutations in isocitrate dehydrogenase 1 (IDH1) in gliomas and other tumors, significant efforts have been made to gain a deeper understanding of the consequences of this oncogenic mutation. One aspect of the neomorphic function of the IDH1 R132H enzyme that has received less attention is the perturbation of cellular redox homeostasis. Here, we describe a biosynthetic pathway exhibited by cells expressing mutant IDH1. By virtue of a change in cellular redox homeostasis, IDH1-mutated cells synthesize excess glutamine-derived proline through enhanced activity of pyrroline 5-carboxylate reductase 1 (PYCR1), coupled to NADH oxidation. Enhanced proline biosynthesis partially uncouples the electron transport chain from tricarboxylic acid (TCA) cycle activity through the maintenance of a lower NADH/NAD+ ratio and subsequent reduction in oxygen consumption. Thus, we have uncovered a mechanism by which tumor cell survival may be promoted in conditions associated with perturbed redox homeostasis, as occurs in IDH1-mutated glioma.

A web-based versus paper questionnaire on alcohol and tobacco in adolescents.

OBJECTIVE: Our study was aimed at comparing health behavior data collected from a Web-based self-administered questionnaire (Web SAQ) versus a paper-and-pencil self-administered questionnaire and assessing the feasibility of the application. MATERIALS AND METHODS: One hundred and ninety (n = 190) pupils (ages 14-16 years) of senior high schools anonymously completed a questionnaire, with demographics and queries about lifestyle, alcohol, and tobacco use. For each class, the adolescents were randomly assigned to complete either the paper version of the questionnaire or the equivalent Web-based one, which used a customized platform developed for the purposes of this survey. RESULTS: Females who filled out the Web SAQ required significantly less time and completed a significantly higher percentage of its items. Although the majority of questions on tobacco and alcohol did not differ significantly across the two administration modes, there were gender-related differences in some sensitive information. Male adolescents on the Web SAQ accounted higher per hour drink consumption (r = 0.27, p = 0.015) and more numerous episodes of inebriety (r = 0.26, p = 0.010), whereas females seemed to state a younger age of alcohol onset (r = 0.33, p = 0.002). Females were more likely to report being monthly smokers on the Web SAQ (odds ratio = 0.37). Adolescents felt significantly less observed and females referred being more independent while compiling the Web SAQ. CONCLUSIONS: The findings of the study suggest that differences in reporting of some behavior of adolescents when using a Web SAQ do exist, despite the small-to-medium effect sizes. Exploiting the Web requires further investigation for extensive comprehension of the reasons for such differences.