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International audience; Functional properties of the paracellular pathway depend critically on the set of claudins (CLDN) expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX) syndrome caused by genetic variations in the CLDN10 gene and familial hypomagnesemia with hypercalciuria and nephrocalcinosis caused by genetic variations in the CLDN16 or CLDN19 genes. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, CLDN16, and CLDN19 in both paraffin-embedded and frozen kidney sections from the adult human, mouse, and rat using immunohistochemistry and immunofluorescence, respectively. Here, CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junctions in human and rodent kidneys. Weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in the cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidneys. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of the TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junctions almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidneys.NEW & NOTEWORTHY This study used immunohistochemistry and immunofluorescence to investigate the distribution of claudin 10, 16, and 19 in the human, mouse, and rat kidney. The findings showed distinct junctional pathways in both human and rodent kidneys, supporting the existence of different permeation profiles in all species investigated.

Objectives Apoptosis-Inducing Factor (AIF) is a protein involved in mitochondrial electron transport chain assembly/stability and programmed cell death. The relevant role of this protein is underlined because mutations altering mitochondrial AIF properties result in acute pediatric mitochondriopathies and tumor metastasis. By generating an original AIF-deficient mouse strain, this study attempted to analyze, in a single paradigm, the cellular and developmental metabolic consequences of AIF loss and the subsequent oxidative phosphorylation (OXPHOS) dysfunction. Methods We developed a novel AIF-deficient mouse strain and assessed, using molecular and cell biology approaches, the cellular, embryonic, and adult mice phenotypic alterations. Additionally, we conducted ex vivo assays with primary and immortalized AIF knockout mouse embryonic fibroblasts (MEFs) to establish the cell death characteristics and the metabolic adaptive responses provoked by the mitochondrial electron transport chain (ETC) breakdown. Results AIF deficiency destabilized mitochondrial ETC and provoked supercomplex disorganization, mitochondrial transmembrane potential loss, and high generation of mitochondrial reactive oxygen species (ROS). AIF-/Y MEFs counterbalanced these OXPHOS alterations by mitochondrial network reorganization and a metabolic reprogramming toward anaerobic glycolysis illustrated by the AMPK phosphorylation at Thr172, the overexpression of the glucose transporter GLUT-4, the subsequent enhancement of glucose uptake, and the anaerobic lactate generation. A late phenotype was characterized by the activation of P53/P21-mediated senescence. Notably, approximately 2% of AIF-/Y MEFs diminished both mitochondrial mass and ROS levels and spontaneously proliferated. These cycling AIF-/Y MEFs were resistant to caspase-independent cell death inducers. The AIF-deficient mouse strain was embryonic lethal between E11.5 and E13.5 with energy loss, proliferation arrest, and increased apoptotic levels. Contrary to AIF-/Y MEFs, the AIF KO embryos were unable to reprogram their metabolism toward anaerobic glycolysis. Heterozygous AIF+/- females displayed progressive bone marrow, thymus, and spleen cellular loss. In addition, approximately 10% of AIF+/- females developed perinatal hydrocephaly characterized by brain development impairment, meningeal fibrosis, and medullar hemorrhages; those mice died 5 weeks after birth. AIF+/- with hydrocephaly exhibited loss of ciliated epithelium in the ependymal layer. This phenotype was triggered by the ROS excess. Accordingly, it was possible to diminish the occurrence of hydrocephalus AIF+/- females by supplying dams and newborns with an antioxidant in drinking water. Conclusions In a single knockout model and at 3 different levels (cell, embryo, and adult mice) we demonstrated that by controlling the mitochondrial OXPHOS/metabolism, AIF is a key factor regulating cell differentiation and fate. Additionally, by providing new insights into the pathological consequences of mitochondrial OXPHOS dysfunction, our new findings pave the way for novel pharmacological strategies., Highlights • Mitochondrial AIF loss triggers OXPHOS/metabolism impairment. • AIF-/Y MEFs reprogram their metabolism by enabling an AMPK/GLUT-4 glycolytic pathway. • OXPHOS dysfunction associated with AIF loss generates a P53/P21 senescent phenotype. • The lack of metabolic plasticity provokes embryonic lethality in the AIF KO animals. • AIF+/- females develop immune cell exhaustion and perinatal hydrocephaly.

Mitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF− /Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF− /Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid β-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid β-oxidation (e.g., palmitic acid assimilation), the AIF− /Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF− /Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics.

Intestine contributes to energy homeostasis through the absorption, metabolism, and transfer of nutrients to the organism. We demonstrated previously that hepatocyte nuclear receptor-4α (HNF-4α) controls intestinal epithelium homeostasis and intestinal absorption of dietary lipids. HNF-4γ, the other HNF-4 form highly expressed in intestine, is much less studied. In HNF-4γ knockout mice, we detect an exaggerated insulin peak and improvement in glucose tolerance during oral but not intraperitoneal glucose tolerance tests, highlighting the involvement of intestine. Moreover, the enteroendocrine L-type cell lineage is modified, as assessed by the increased expression of transcription factors Isl1, Foxa1/2, and Hnf4a, leading to an increase of both GLP-1–positive cell number and basal and stimulated GLP-1 plasma levels potentiating the glucose-stimulated insulin secretion. Using the GLP-1 antagonist exendin (9-39), we demonstrate a direct effect of GLP-1 on improved glucose tolerance. GLP-1 exerts a trophic effect on pancreatic β-cells, and we report an increase of the β-cell fraction correlated with an augmented number of proliferative islet cells and with resistance to streptozotocin-induced diabetes. In conclusion, the loss of HNF-4γ improves glucose homeostasis through a modulation of the enteroendocrine cell lineage.

OBJECTIVE In healthy rodents, intestinal sugar absorption in response to sugar-rich meals and insulin is regulated by GLUT2 in enterocyte plasma membranes. Loss of insulin action maintains apical GLUT2 location. In human enterocytes, apical GLUT2 location has not been reported but may be revealed under conditions of insulin resistance. RESEARCH DESIGN AND METHODS Subcellular location of GLUT2 in jejunal enterocytes was analyzed by confocal and electron microscopy imaging and Western blot in 62 well-phenotyped morbidly obese subjects and 7 lean human subjects. GLUT2 locations were assayed in ob/ob and ob/+ mice receiving oral metformin or in high-fat low-carbohydrate diet–fed C57Bl/6 mice. Glucose absorption and secretion were respectively estimated by oral glucose tolerance test and secretion of [U-14C]-3-O-methyl glucose into lumen. RESULTS In human enterocytes, GLUT2 was consistently located in basolateral membranes. Apical GLUT2 location was absent in lean subjects but was observed in 76% of obese subjects and correlated with insulin resistance and glycemia. In addition, intracellular accumulation of GLUT2 with early endosome antigen 1 (EEA1) was associated with reduced MGAT4a activity (glycosylation) in 39% of obese subjects on a low-carbohydrate/high-fat diet. Mice on a low-carbohydrate/high-fat diet for 12 months also exhibited endosomal GLUT2 accumulation and reduced glucose absorption. In ob/ob mice, metformin promoted apical GLUT2 and improved glucose homeostasis. Apical GLUT2 in fasting hyperglycemic ob/ob mice tripled glucose release into intestinal lumen. CONCLUSIONS In morbidly obese insulin-resistant subjects, GLUT2 was accumulated in apical and/or endosomal membranes of enterocytes. Functionally, apical GLUT2 favored and endosomal GLUT2 reduced glucose transepithelial exchanges. Thus, altered GLUT2 locations in enterocytes are a sign of intestinal adaptations to human metabolic pathology.

SummaryIn obesity, insulin resistance is linked to inflammation in several tissues. Although the gut is a very large lymphoid tissue, inflammation in the absorptive small intestine, the jejunum, where insulin regulates lipid and sugar absorption is unknown. We analyzed jejunal samples of 185 obese subjects stratified in three metabolic groups: without comorbidity, suffering from obesity-related comorbidity, and diabetic, versus 33 lean controls. Obesity increased both mucosa surface due to lower cell apoptosis and innate and adaptive immune cell populations. The preferential CD8αβ T cell location in epithelium over lamina propria appears a hallmark of obesity. Cytokine secretion by T cells from obese, but not lean, subjects blunted insulin signaling in enterocytes relevant to apical GLUT2 mislocation. Statistical links between T cell densities and BMI, NAFLD, or lipid metabolism suggest tissue crosstalk. Obesity triggers T-cell-mediated inflammation and enterocyte insulin resistance in the jejunum with potential broader systemic implications.

Acute liver failure (ALF) is a rare syndrome with a difficult clinical management and a high mortality rate. During ALF, several molecular pathways governing oxidative stress and apoptosis are activated to induce massive tissue injury and suppress cell proliferation. There are few anti-ALF drug candidates, among which is the C-type lectin Reg3a, or human hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which displayed promising properties for tissue regeneration and protection against cellular stress in transgenic mice. We report on substantial preclinical and clinical advances in the development of a recombinant (rc) full-length human HIP/PAP protein as an anti-ALF drug. The curative effects and mechanisms of action of rcHIP/PAP were investigated in murine Fas-induced ALF. Primary hepatocytes were cultured with cytotoxic doses of tumor necrosis factor a/actinomycin-D, transforming growth factor b, agonistic Fas antibody or hydrogen peroxide, and various concentrations of rcHIP/PAP. Cell viability, proliferation index, apoptosis, and oxidation were monitored. We found that rcHIP/PAP significantly improved survival in Fas-intoxicated mice in a dose-dependent and time-dependent manner, with optimum effects when it was injected at advanced stages of ALF. Primary hepatocytes were efficiently protected against multiple cell death signals by rcHIP/PAP. This survival benefit was linked to a depletion of oxidized biomolecules in injured liver cells due to a strong reactive oxygen species scavenging activity of rcHIP/PAP. Clinically, an escalating dose phase 1 trial demonstrated a good tolerability and pharmacokinetic profile of rcHIP/PAP in healthy subjects. Conclusion: The rcHIP/PAP protein exhibited significant curative properties against ALF in mice. It is a free-radical scavenger that targets a broad spectrum of death effectors and favors liver regeneration. The good safety profile of rcHIP/PAP during a phase 1 trial encourages evaluation of its efficacy in patients with ALF. (HEPATOLOGY 2010;00:000-000)

Introduction Le Glucagon-Like Peptide 1 (GLP-1) est une hormone peptidique secretee par les cellules L enteroendocrines qui amplifie la secretion d’insuline en reponse au glucose. Les taux circulants de GLP-1 sont habituellement diminues en cas d’obesite. Nous avons etudie le fonctionnement des cellules L enteroendocrines chez le sujet obese morbide et chez la souris nourrie avec un regime hyperlipidique. Patients et methodes Les cellules L de 75 echantillons de jejunum de sujets obeses morbides obtenus lors de chirurgie bariatrique (Roux-en-Y), ont ete quantifies par immunofluorescence. Leur nombre a ete correle aux differents parametres metaboliques des sujets, avant et apres chirurgie. Des souris C57BL6/J ont recu, pendant 8 semaines, une alimentation standard ou hyperlipidique (4 % et 34 % en lipides, respectivement). Les cellules enteroendocrines ont ete quantifiees par cytometrie en flux, les taux plasmatiques d’hormones ont ete determines par Multiplex et l’expression de facteurs de transcription a ete mesuree par Q-PCR. Resultats Parmi les sujets obeses, ceux ayant une alimentation favorisant les lipides au detriment des glucides, ont un nombre plus eleve de cellules L. En revanche, aucun lien n’a pu etre etabli entre le nombre de cellules L et les taux plasmatiques de GLP-1. Chez la souris, des 2 semaines de regime hyperlipidique, le nombre de cellules L est plus eleve, a la fois dans l’intestin proximal et distal. La secretion de GLP-1 en reponse au glucose est egalement augmentee ainsi que l’expression de facteurs de transcription impliques dans la differenciation enteroendocrine en aval de la Neurogenine 3. Conclusion Chez l’homme et la souris une alimentation riche en lipides conduit a une augmentation du nombre de cellules L. En revanche, les consequences sur la secretion de GLP-1 different, conduisant a s’interroger sur l’importance de la duree de l’obesite dans la mise en place des adaptations metaboliques.