Andrei I. Tarasov

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

OBJECTIVE Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS ZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

TCF7L2 regulates late events in insulin secretion from pancreatic islet β-cells.

OBJECTIVE Polymorphisms in the human TCF7L2 gene are associated with reduced insulin secretion and an increased risk of type 2 diabetes. However, the mechanisms by which TCF7L2 affect insulin secretion are still unclear. We define the effects of TCF7L2 expression level on mature β-cell function and suggest a potential mechanism for its actions. RESEARCH DESIGN AND METHODS TCF7L2 expression in rodent islets and β-cell lines was altered using RNAi or adenoviral transduction. β-Cell gene profiles were measured by quantitative real-time PCR and the effects on intracellular signaling and exocytosis by live cell imaging, electron microscopy, and patch clamp electrophysiology. RESULTS Reducing TCF7L2 expression levels by RNAi decreased glucose- but not KCl-induced insulin secretion. The glucose-induced increments in both ATP/ADP ratio and cytosolic free Ca2+ concentration ([Ca2+]i) were increased compared with controls. Overexpression of TCF7L2 exerted minor inhibitory effects on glucose-regulated changes in [Ca2+]i and insulin release. Gene expression profiling in TCF7L2-silenced cells revealed increased levels of mRNA encoding syntaxin 1A but decreased Munc18–1 and ZnT8 mRNA. Whereas the number of morphologically docked vesicles was unchanged by TCF7L2 suppression, secretory granule movement increased and capacitance changes decreased, indicative of defective vesicle fusion. CONCLUSION—TCF7L2 is involved in maintaining expression of β-cell genes regulating secretory granule fusion. Defective insulin exocytosis may thus underlie increased diabetes incidence in carriers of the at-risk TCF7L2 alleles.

Regulation of ATP production by mitochondrial Ca2

Stimulation of mitochondrial oxidative metabolism by Ca2+ is now generally recognised as important for the control of cellular ATP homeostasis. Here, we review the mechanisms through which Ca2+ regulates mitochondrial ATP synthesis. We focus on cardiac myocytes and pancreatic β-cells, where tight control of this process is likely to play an important role in the response to rapid changes in workload and to nutrient stimulation, respectively. We also describe a novel approach for imaging the Ca2+-dependent regulation of ATP levels dynamically in single cells.

The mitochondrial Ca2+ uniporter MCU is essential for glucose-induced ATP increases in pancreatic β-cells.

Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca2+ influx. As well as activating ATP-consuming processes, cytosolic Ca2+ increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca2+ transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca2+ uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca2+ and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca2+ uniporter, MCU, is required for depolarisation-induced mitochondrial Ca2+ increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na+-Ca2+ exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca2+ uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca2+ accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.

Role of KATP Channels in Glucose-Regulated Glucagon Secretion and Impaired Counterregulation in Type 2 Diabetes

Summary Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.

Insulin Gene Mutations Resulting in Early-Onset Diabetes: Marked Differences in Clinical Presentation, Metabolic Status, and Pathogenic Effect Through Endoplasmic Reticulum Retention

OBJECTIVE Heterozygous mutations in the human preproinsulin (INS) gene are a cause of nonsyndromic neonatal or early-infancy diabetes. Here, we sought to identify INS mutations associated with maturity-onset diabetes of the young (MODY) or nonautoimmune diabetes in mid-adult life, and to explore the molecular mechanisms involved. RESEARCH DESIGN AND METHODS The INS gene was sequenced in 16 French probands with unexplained MODY, 95 patients with nonautoimmune early-onset diabetes (diagnosed at <35 years) and 292 normoglycemic control subjects of French origin. Three identified insulin mutants were generated by site-directed mutagenesis of cDNA encoding a preproinsulin–green fluorescent protein (GFP) (C-peptide) chimera. Intracellular targeting was assessed in clonal β-cells by immunocytochemistry and proinsulin secretion, by radioimmunoassay. Spliced XBP1 and C/EBP homologous protein were quantitated by real-time PCR. RESULTS A novel coding mutation, L30M, potentially affecting insulin multimerization, was identified in five diabetic individuals (diabetes onset 17–36 years) in a single family. L30M preproinsulin-GFP fluorescence largely associated with the endoplasmic reticulum (ER) in MIN6 β-cells, and ER exit was inhibited by ∼50%. Two additional mutants, R55C (at the B/C junction) and R6H (in the signal peptide), were normally targeted to secretory granules, but nonetheless caused substantial ER stress. CONCLUSIONS We describe three INS mutations cosegregating with early-onset diabetes whose clinical presentation is compatible with MODY. These led to the production of (pre)proinsulin molecules with markedly different trafficking properties and effects on ER stress, demonstrating a range of molecular defects in the β-cell.

Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn2+, and that Zn2+ is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn2+ chelation and bound Zn2+ with high selectivity against Ca2+ and Mg2+. When added to cultured β cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn2+ concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled β cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn2+/insulin release occurred largely in small groups of adjacent β cells, with each forming a “secretory unit.” Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca2+ elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca2+ signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn2+-containing granules, ZIMIR may find applications in β-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.

Sulfonylurea improves CNS function in a case of intermediate DEND syndrome caused by a mutation in KCNJ11

Background A 12-week-old female presented with neonatal diabetes. Insulin therapy alleviated the diabetes, but the patient showed marked motor and mental developmental delay. The patient underwent genetic evaluation at the age of 6 years, prompted by reports that mutations in the KCNJ11 gene caused neonatal diabetes.Investigations Genomic sequencing of the ATP-sensitive potassium (KATP) channel gene KCNJ11 and in vitro functional analysis of the channel defect, and single-photon emission CT imaging before and after glibenclamide therapy.Diagnosis Genetic evaluation revealed a missense mutation (His46Leu) in KCNJ11, which encodes the Kir6.2 subunit of the KATP channel, conferring reduced ATP sensitivity. Functional studies demonstrated that the mutant channels were strongly inhibited by the sulfonylurea tolbutamide.Management Sulfonylurea (glibenclamide) treatment led to both improved glucose homeostasis and an increase in mental and motor function.

Ablation of AMP-activated protein kinase α1 and α2 from mouse pancreatic beta cells and RIP2.Cre neurons suppresses insulin release in vivo

Aims/hypothesisAMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain.MethodsThe catalytic α1 and α2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkα1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkα2 [also known as Prkaa2] alleles (Ampkα1−/−,α2fl/fl,), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca2+ were measured with standard techniques.ResultsTrigenic Ampkα1−/−,α2fl/fl expressing Cre recombinase and lacking both AMPKα subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca2+, while granule docking and insulin secretion were enhanced. Conversely, βAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion.Conclusions/interpretationInhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence insulin secretion in vivo.

Identification of the PIP2‐binding site on Kir6.2 by molecular modelling and functional analysis

ATP‐sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ fluxes across the plasma membrane. Channel closure is facilitated by ATP, which binds to the pore‐forming subunit (Kir6.2). Conversely, channel opening is potentiated by phosphoinositol bisphosphate (PIP2), which binds to Kir6.2 and reduces channel inhibition by ATP. Here, we use homology modelling and ligand docking to identify the PIP2‐binding site on Kir6.2. The model is consistent with a large amount of functional data and was further tested by mutagenesis. The fatty acyl tails of PIP2 lie within the membrane and the head group extends downwards to interact with residues in the N terminus (K39, N41, R54), transmembrane domains (K67) and C terminus (R176, R177, E179, R301) of Kir6.2. Our model suggests how PIP2 increases channel opening and decreases ATP binding and channel inhibition. It is likely to be applicable to the PIP2‐binding site of other Kir channels, as the residues identified are conserved and influence PIP2 sensitivity in other Kir channel family members.