Altered hepatic glucose homeostasis in AnxA6-KO mice fed a high-fat diet

Annexin A6 (AnxA6) controls cholesterol and membrane transport in endo- and exocytosis,and modulates triglyceride accumulation and storage. In addition, AnxA6 acts as a scaffolding protein for negative regulators of growth factor receptors and their effector pathways in many different cell types. Here we investigated the role of AnxA6 in the regulation of whole body lipid metabolism and insulin-regulated glucose homeostasis. Therefore, wildtype (WT) and AnxA6-knockout (KO) mice were fed a high-fat diet (HFD) for 17 weeks. During the course of HFD feeding, AnxA6-KO mice gained less weight compared to controls, which correlated with reduced adiposity. Systemic triglyceride and cholesterol levels of HFD-fed control and AnxA6-KO mice were comparable, with slightly elevated high density lipoprotein (HDL) and reduced triglyceride-rich lipoprotein (TRL) levels in AnxA6-KO mice. AnxA6-KO mice displayed a trend towards improved insulin sensitivity in oral glucose and insulin tolerance tests (OGTT, ITT), which correlated with increased insulin-inducible phosphorylation of protein kinase B (Akt) and ribosomal protein S6 kinase (S6) in liver extracts. However,HFD-fed AnxA6-KO mice failed to downregulate hepatic gluconeogenesis, despite similar insulin levels and insulin signaling activity, as well as expression profiles of insulin-sensitive transcription factors to controls. In addition, increased glycogen storage in livers of HFDand chow-fed AnxA6-KO animals was observed. Together with an inability to reduce glucose production upon insulin exposure in AnxA6-depleted HuH7 hepatocytes, this implicates AnxA6 contributing to the fine-tuning of hepatic glucose metabolism with potential consequences for the systemic control of glucose in health and disease

Annexin A6 is critical to maintain glucose homeostasis and survival during liver regeneration in mice

Background and aims: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. Approach and results: Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. Conclusions: We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress.

Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis

The biliary epithelium gives rise to liver progenitor cells: RODRIGO-TORRES ET AL.

Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1β). However, HNF1β expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)-positive hepatocytes. In order to investigate the role of HNF1β- expressing cells we used a tamoxifen-inducible Hnf1βCreER/R26RYfp/LacZ mouse to lineage-trace Hnf1β+ biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1β+ cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1β+ cells to hepatocyte was detected. We next assessed the contribution of Hnf1β+ -derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl-1,4-dihydrocollidin (DDC)-diet and a choline-deficient ethionine-supplemented (CDE)-diet. The contribution of Hnf1β+ cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC-diet treatment, mice fed with a CDE-diet showed a small population of hepatocytes derived from Hnf1β+ cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome-wide expression profile of Hnf1β+ -derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways

Annexin A6 and late endosomal cholesterol modulate integrin recycling and cell migration.

Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVβ3 and α5β1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration

GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans.

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression. Circulating GDF15 levels manifest very modest changes in response to moderate caloric surpluses or deficits in mice or humans, differentiating it from classical intestinally derived satiety hormones and leptin. However, GDF15 levels do increase following sustained high-fat feeding or dietary amino acid imbalance in mice. We demonstrate that GDF15 expression is regulated by the integrated stress response and is induced in selected tissues in mice in these settings. Finally, we show that pharmacological GDF15 administration to mice can trigger conditioned taste aversion, suggesting that GDF15 may induce an aversive response to nutritional stress.This work and authors were funded by the NIHR Cambridge Biomedical Research Centre; NIHR Rare Disease Translational Research Collaboration; Medical Research Council [MC_UU_12012/2 and MRC_MC_UU_12012/3]; MRC Metabolic Diseases Unit [MRC_MC_UU_12012/5 and MRC_MC_UU_12012.1]; Wellcome Trust Strategic Award [100574/Z/12/Z and 100140]; Wellcome Trust [107064 , 095515/Z/11/Z , 098497/Z/12/Z, 106262/Z/14/Z and 106263/Z/14/Z]; British Heart Foundation [RG/12/13/29853]; Addenbrooke’s Charitable Trust / Evelyn Trust Cambridge Clinical Research Fellowship [16-69] US Department of Agriculture: 2010-34323-21052; EFSD project grant and a Royal College of Surgeons Research Fellowship, Diabetes UK Harry Keen intermediate clinical fellowship (17/0005712). European Research Council, Bernard Wolfe Health Neuroscience Endowment, Experimental Medicine Training Initiative/AstraZeneca and Medimmune

Estudio de la función de Annexina A6 en el hígado

[spa] Annexina A6 (AnxA6) es una proteína de unión a fosfolípidos de membrana de forma dependiente de calcio. Está implicada en múltiples procesos celulares como la homeostasis de colesterol y el tráfico intracelular. Partiendo de que AnxA6 es una de las proteínas más abundantes en el hígado, representando el 0.25% del total, el principal objetivo de esta tesis fue el estudio del papel de AnxA6 en este órgano a partir de ratones genoanulados para la proteína (AnxA6-/-). Teniendo en cuenta la fisiología y arquitectura comparable a los ratones WT de los hígados en ausencia de AnxA6, se procedió al estudio de AnxA6 durante el proceso de regeneración hepática tras la realización de una hepatectomía parcial (HP) de 2/3. Sorprendentemente, el 80% los ratones AnxA6-/- no sobrevivían más allá de las 72 horas post-HP. La regeneración hepática consiste en la activación de los hepatocitos por diversas citoquinas y factores de crecimiento que permiten la proliferación de las células hepáticas tras un daño hepático o una resección, como es el caso. En paralelo, el hígado y, el organismo en general, sufren una serie de adaptaciones metabólicas para mantener la homeostasis energética. El estudio del proceso de regeneración hepática demostró una menor expresión de las citoquinas TNFα e IL-6, encargadas de activar la transcripción de centenares de genes que podía estar compensada por una mayor activación del factor de crecimiento epidérmico (EGF). Además, se observó cierto retraso en la acumulación de lípidos en el hígado, necesarios para hacer frente a la elevada demanda energética derivada de la intensa proliferación. Tras la HP, los ratones experimentan una hipoglucemia muy elevada fruto de la resección del 60% de la masa hepática, con lo que se reducen los almacenes de glucógeno y los hepatocitos con capacidad gluconeogénica. A diferencia de los ratones WT, los AnxA6-/- tenían una drástica y rápida caída de los niveles de glucosa en sangre que no retornaban a valores normales, sugiriendo un coma hipoglucémico como causa de la baja supervivencia. Además, el análisis de la glucosa en sangre en los ratones AnxA6-/-, hizo evidente que en ausencia de AnxA6, y ante un estrés metabólico, los ratones eran incapaces de mantener la homeostasis de glucosa. Mediante el ayuno de los ratones AnxA6-/-, se determinó que el problema en la homeostasis de glucosa no era debido ni a una señalización defectuosa por parte de las hormonas insulina y glucagón, ni por una disfunción en el almacén y degradación de glucógeno. Estos resultados centraban a la gluconeogénesis (GNG) como la causa que provoca la hipoglucemia. La GNG es el proceso mediante el cual se produce glucosa a partir de sustratos no glucídicos como lactato, aminoácidos y glicerol. Así, se descartó un problema en la expresión de las enzimas clave de la GN; glucosa-6-fosfatasa, fosfoenolpiruvato carboxiquinasa y fructosa-1,6-bisfosfatasa. En cambio, se observó una inhibición en la producción de glucosa a partir de alanina tanto in vivo en ratones hipoglucémicos como en hepatocitos aislados. La alanina es el principal sustrato de la GNG tanto en condiciones de ayuno como durante la regeneración hepática. Está inhibición se determinó debida a una deficiencia en la captación de alanina, tal como se observó en hepatocitos aislados. El transportador de aminoácidos SNAT2, uno de los principales transportadores de alanina, entre otros aminoácidos neutros, se ha descrito esencial durante la regeneración hepática y el ayuno. En estas situaciones se sobreexpresa el transportador y se transloca a la membrana plasmática de los hepatocitos. Tras el ayuno, los hepatocitos deficientes en AnxA6 no presentan niveles de SNAT2 en la membrana sinusoidal indicando un impedimento en el tráfico del transportador. Por último, se ha determinado mediante la técnica de pull down la interacción entre AnxA6 y SNAT2. De manera que se propone un mecanismo mediante el cual AnxA6 interacciona con SNAT2 dirigiéndolo a la membrana plasmática. Con estos resultados, hemos determinado la implicación por primera vez de AnxA6 en el metabolismo de la glucosa a través de alanina. Así, sugerimos que en ausencia de AnxA6, el transportador SNAT2 no se transloca a la membrana plasmática, quedando retenido en algún compartimento celular. De esta forma, se inhibe la GNG a partir de alanina impidiendo la producción de glucosa y provocando una situación de hipoglucemia, que en el caso de la HP podría llevar a la muerte del animal.[eng] Annexin A6 (AnxA6) ) is a multifunctional calcium-dependent membrane binding protein involved in a plethora of different functions such as Ras-MAPK and PKCα signalling regulation, intracellular cholesterol homeostasis, actin-cytoskeleton and plasma membrane arrangement among others. Although AnxA6 is one of the major proteins in the liver (0.25% total protein), its function in the physiology of this organ remains unknown. After partial hepatectomy (PH) quiescent hepatocytes are triggered to progress through the cell cycle, showing a synchronous onset of DNA synthesis with a cellular response that involves cell activation and tissue remodelling. During this period of liver regeneration, the liver has to retain the major physiological tasks such as the synthesis and secretion of plasma proteins, lipid homeostasis and its metabolic function to ensure viability of the organism. The aim of the present study was to investigate the in vivo function of AnxA6 during liver regeneration using an AnxA6 knock-out (AnxA6-/-) mouse model. To this objective, 2/3 PH was performed in wild-type C57BL/6 control and AnxA6-/- mice. After PH, AnxA6-/- mice exhibit a dramatic reduction of survival rate (87.5% at 72h post-PHx). However, the exogenous administration of glucose before and during PH restored AnxA6-/- survival rate after PH, suggesting a link between AnxA6 and hepatic energetic metabolism. A comprehensive analysis of glucose metabolism experiments pointed to an impairment of liver gluconeogenesis in AnxA6-/- mice, also observed in starving mice. A biochemical approach allowed us to elucidate a role for AnxA6 in the intracellular trafficking of SNAT2, the main liver L-Alanine transporter, which is essential for gluconeogenic hepatic substrate uptake during liver regeneration and mice starvation. We conclude that AnxA6 is a new regulator of hepatic gluconeogenesis essential for the trafficking of the alanine transporter (SNAT2) into the hepatocyte sinusoidal plasma membrane and subsequent alanine uptake, the major gluconeogenic substrate during both liver regeneration and fasting

Annexin A6 regulates adipocyte lipid storage and adiponectin release

Lipid storage and adipokine secretion are critical features of adipocytes. Annexin A6 (AnxA6) is a lipid binding protein regulating secretory pathways and its role in adiponectin release was examined. The siRNA-mediated AnxA6 knock-down in 3T3-L1 preadipocytes impaired proliferation, and differentiation of AnxA6-depleted cells to mature adipocytes was associated with higher soluble adiponectin and increased triglyceride storage. The latter was partly attributed to reduced lipolysis. Accordingly, AnxA6 overexpression in 3T3-L1 adipocytes lowered cellular triglycerides and adiponectin secretion. Indeed, serum adiponectin was increased in AnxA6 deficient mice. Expression analysis identified AnxA6 protein to be more abundant in intra-abdominal compared to subcutaneous adipose tissues of mice and men. AnxA6 protein levels increased in white adipose tissues of obese mice and here, levels were highest in subcutaneous fat. AnxA6 protein in adipocytes was upregulated by oxidative stress which might trigger AnxA6 induction in adipose tissues and contribute to impaired fat storage and adiponectin release. (C) 2016 Elsevier Ireland Ltd. All rights reserved

Phenotypic characterization of Adig null mice suggests roles for adipogenin in the regulation of fat mass accrual and leptin secretion.

Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig-/-) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig-/- mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion