Mutations in SLC2A2 gene reveal hGLUT2 function in pancreatic ß cell development.

The structure-function relationships of sugar transporter-receptor hGLUT2 coded by SLC2A2 and their impact on insulin secretion and ß cell differentiation were investigated through the detailed characterization of a panel of mutations along the protein. We studied naturally occurring SLC2A2 variants or mutants: two single-nucleotide polymorphisms and four proposed inactivating mutations associated to Fanconi-Bickel syndrome. We also engineered mutations based on sequence alignment and conserved amino acids in selected domains. The single-nucleotide polymorphisms P68L and T110I did not impact on sugar transport as assayed in Xenopus oocytes. All the Fanconi-Bickel syndrome-associated mutations invalidated glucose transport by hGLUT2 either through absence of protein at the plasma membrane (G20D and S242R) or through loss of transport capacity despite membrane targeting (P417L and W444R), pointing out crucial amino acids for hGLUT2 transport function. In contrast, engineered mutants were located at the plasma membrane and able to transport sugar, albeit with modified kinetic parameters. Notably, these mutations resulted in gain of function. G20S and L368P mutations increased insulin secretion in the absence of glucose. In addition, these mutants increased insulin-positive cell differentiation when expressed in cultured rat embryonic pancreas. F295Y mutation induced ß cell differentiation even in the absence of glucose, suggesting that mutated GLUT2, as a sugar receptor, triggers a signaling pathway independently of glucose transport and metabolism. Our results describe the first gain of function mutations for hGLUT2, revealing the importance of its receptor versus transporter function in pancreatic ß cell development and insulin secretion.

Metabolic Stress Impairs Pericyte Response to Optogenetic Stimulation in Pancreatic Islets.

Pancreatic islets are highly vascularized micro-organs ensuring whole body glucose homeostasis. Islet vascular cells play an integral part in sustaining adequate insulin release by beta cells. In particular, recent studies have demonstrated that islet pericytes regulate local blood flow velocity and are required for maintenance of beta cell maturity and function. In addition, increased metabolic demand accompanying obesity alters islet pericyte morphology. Here, we sought to explore the effects of metabolic stress on islet pericyte functional response to stimulation in a mouse model of type 2 diabetes, directly in the pancreas in vivo . We found that high fat diet induced islet pericyte hypertrophy without alterations in basal local blood flow. However, optogenetic stimulation of pericyte activity revealed impaired islet vascular responses, despite increased expression of genes encoding proteins directly or indirectly involved in cell contraction. These findings suggest that metabolic stress impinges upon islet pericyte function, which may contribute to beta cell failure during T2D.

Detection of extracellular glucose by GLUT2 contributes to hypothalamic control of food intake.

The sugar transporter GLUT2, present in several tissues of the gut-brain axis, has been reported to be involved in the control of food intake. GLUT2 is a sugar transporter sustaining energy production in the cell, but it can also function as a receptor for extracellular glucose. A glucose-signaling pathway is indeed triggered, independently of glucose metabolism, through its large cytoplasmic loop domain. However, the contribution of the receptor function over the transporter function of GLUT2 in the control of food intake remains to be determined. Thus, we generated transgenic mice that express a GLUT2-loop domain, blocking the detection of glucose but leaving GLUT2-dependent glucose transport unaffected. Inhibiting GLUT2-mediated glucose detection augmented daily food intake by a mechanism that increased the meal size but not the number of meals. Peripheral hormones (ghrelin, insulin, leptin) were unaffected, leading to a focus on central aspects of feeding behavior. We found defects in c-Fos activation by glucose in the arcuate nucleus and changes in the amounts of TRH and orexin neuropeptide mRNA, which are relevant to poorly controlled meal size. Our data provide evidence that glucose detection by GLUT2 contributes to the control of food intake by the hypothalamus. The sugar transporter receptor, i.e., "transceptor" GLUT2, may constitute a drug target to treat eating disorders and associated metabolic diseases, particularly by modulating its receptor function without affecting vital sugar provision by its transporter function.

Structure-Activity Relationship Studies of Retro-1 Analogues against Shiga Toxin.

High-throughput screening has shown that Retro-1 inhibits ricin and Shiga toxins by diminishing their intracellular trafficking via the retrograde route, from early endosomes to the Golgi apparatus. To improve the activity of Retro-1, a structure-activity relationship (SAR) study was undertaken and yielded an analogue with a roughly 70-fold better half-maximal effective concentration (EC50) against Shiga toxin cytotoxicity measured in a cell protein synthesis assay.

Integrin ß1 Optimizes Diabetogenic T Cell Migration and Function in the Pancreas.

T cell search behavior is dictated by their need to encounter their specific antigen to eliminate target cells. However, mechanisms controlling effector T cell motility are highly tissue-dependent. Specifically, how diabetogenic T cells encounter their target beta cells in dispersed islets throughout the pancreas (PA) during autoimmune diabetes remains unclear. Using intra-vital 2-photon microscopy in a mouse model of diabetes, we found that CXCR3 chemokine downregulated CD8+ T cell motility specifically within islets, promoting effector cell confinement to their target sites. By contrast, T cell velocity and directionality in the exocrine tissue were enhanced along blood vessels and extracellular matrix fibers. This guided migration implicated integrin-dependent interactions, since integrin blockade impaired exocrine T cell motility. In addition, integrin ß1 blockade decreased CD4+ T cell effector phenotype specifically in the PA. Thus, we unveil an important role for integrins in the PA during autoimmune diabetes that may have important implications for the design of new therapies.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

Metabolism Regulates Exposure of Pancreatic Islets to Circulating Molecules In Vivo.

Pancreatic ß-cells modulate insulin secretion through rapid sensing of blood glucose and integration of gut-derived signals. Increased insulin demand during pregnancy and obesity alters islet function and mass and leads to gestational diabetes mellitus and type 2 diabetes in predisposed individuals. However, it is unclear how blood-borne factors dynamically access the islets of Langerhans. Thus, understanding the changes in circulating molecule distribution that accompany compensatory ß-cell expansion may be key to developing novel antidiabetic therapies. Here, using two-photon microscopy in vivo in mice, we demonstrate that islets are almost instantly exposed to peaks of circulating molecules, which rapidly pervade the tissue before clearance. In addition, both gestation and short-term high-fat-diet feeding decrease molecule extravasation and uptake rates in vivo in islets, independently of ß-cell expansion or islet blood flow velocity. Together, these data support a role for islet vascular permeability in shaping ß-cell adaptive responses to metabolic demand by modulating the access and sensing of circulating molecules.

Retro-1 Analogues Differentially Affect Oligonucleotide Delivery and Toxin Trafficking.

Retro-1 is a small molecule that displays two important biological activities: First, it blocks the actions of certain toxins by altering their intracellular trafficking. Second, it enhances the activity of oligonucleotides by releasing them from entrapment in endosomes. This raises the question of whether the two actions involve the same cellular target. Herein we report the effects of several Retro-1 analogues on both toxins and oligonucleotides. We found analogues that affect toxins but not oligonucleotides and vice-versa, while Retro-1 is the only compound that affects both. This indicates that the molecular target(s) involved in the two processes are distinct.

Synthesis, Chiral Separation, Absolute Configuration Assignment, and Biological Activity of Enantiomers of Retro-1 as Potent Inhibitors of Shiga Toxin.

The Shiga toxin (Stx) family is composed of related protein toxins produced by the bacteria Shigella dysenteriae and certain pathogenic strains of E. coli. No effective therapies for Stx intoxication have been developed yet. However, inhibitors that act on the intracellular trafficking of these toxins may provide new options for the development of therapeutic strategies. This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of Retro-1, a compound active against Stx and other such protein toxins. Retro-1 works by inhibiting retrograde transport of these toxins inside cells. In vitro experiments proved that the configuration of the stereocenter at position 5 is not crucial for the activity of this compound. X-ray diffraction data revealed (S)-Retro-1 to be slightly more active than (R)-Retro-1.

(S)-N-Methyldihydroquinazolinones are the Active Enantiomers of Retro-2 Derived Compounds against Toxins.

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of a new compound, named Retro-2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM, respectively.

N-methyldihydroquinazolinone derivatives of Retro-2 with enhanced efficacy against Shiga toxin.

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.