Genetic inactivation of zinc transporter SLC39A5 improves liver function and hyperglycemia in obesogenic settings

Background & Aims: Recent studies have revealed a role for zinc in insulin secretion and systemic glucose homeostasis. Randomized placebo-controlled zinc supplementation trials have demonstrated improved glycemic traits in patients with type II diabetes (T2D). Moreover, carriers of rare loss-of-function variants in the zinc efflux transporter SLC30A8 have been reported to reduce T2D risk. Despite this accumulated evidence, mechanistic understanding of how zinc influences systemic glucose homeostasis and consequently T2D risk remains unclear. Methods: To further explore the relationship between zinc and metabolic traits, we searched the exome database of the Regeneron Genetics Center-Geisinger Health System DiscovEHR cohort for genes that regulate zinc levels and associate with changes in metabolic traits. We then explored our main finding using in vitro and in vivo models. Results: We identified rare loss-of-function (LOF) variants (MAF<1%) in Solute Carrier Family 39, Member 5 (SLC39A5) associated with increased circulating zinc (p=4.9x10-4). Trans-ancestry meta-analysis across four studies exhibited nominal association of SLC39A5 LOF variants with decreased T2D risk (OR 0.82, 95%CI 0.68-0.99, p=3.7x10-2). To explore the mechanistic aspects of these associations, we generated mice lacking Slc39a5. Slc39a5-/- mice display improved liver function and reduced hyperglycemia when challenged with congenital or diet-induced obesity. These improvements result from elevated hepatic zinc levels and concomitant activation of hepatic AMPK and AKT signaling, in part due to zinc mediated inhibition of hepatic protein phosphatase activity. Furthermore, under conditions of diet-induced non-alcoholic steatohepatitis (NASH), Slc39a5-/- mice display significantly attenuated fibrosis and inflammation. Conclusions: Taken together, these results suggest SLC39A5 as a potential therapeutic target for non-alcoholic fatty liver disease (NAFLD) and consequent metabolic derangements including T2D.

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LncRNA NEAT1/microRNA-129-5p/SOCS2 axis regulates liver fibrosis in alcoholic steatohepatitis

Journal of Translational Medicine ◽

10.1186/s12967-020-02577-5 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Junfeng Ye ◽

Yuanqiang Lin ◽

Ying Yu ◽

Di Sun

Keyword(s):

Liver Fibrosis ◽

Liver Function ◽

Inflammatory Factors ◽

Cytokine Signaling ◽

Alcoholic Fatty Liver ◽

Alcoholic Steatohepatitis ◽

Inflammation Reaction

Abstract Background Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to play an essential role in non-alcoholic fatty liver disease. However, the role of NEAT1 in regulation of alcoholic steatohepatitis (ASH) remains largely unknown. This study aims to explore the role of NEAT1 in ASH by mediating microRNA-129-5p (miR-129-5p) targeting suppressor of cytokine signaling 2 (SOCS2). Methods NEAT1, miR-129-5p and SOCS2 expression in serum of ASH patients were assessed. In the in vitro cellular experiment, we transfected siRNAs, oligonucleotides or plasmids into ethanol-induced AML-12 mouse hepatocytes to alter NEAT1 and miR-129-5p expression, and inflammatory factors and lipid content were determined. In the in vivo animal experiment, we injected lentiviruses carrying siRNAs, oligonucleotides or plasmids onto ASH mice (ASH induced by feeding mice a Lieber-DeCarli ethanol diet) to alter NEAT1 and miR-129-5p expression through the tail vein. Serum liver function, blood lipids and inflammatory factors were detected; liver histopathology, liver cell apoptosis, and fibrosis were observed. The relationship between NEAT1 and miR-129-5p, or between miR-129-5p and SOCS2 was verified. Results MiR-129-5p was reduced while NEAT1 and SOCS2 were elevated in ASH. Inhibited NEAT1 or elevated miR-129-5p suppressed the elevated lipid metabolism and restrained inflammation reaction in ethanol-stimulated AML-12 cells. The promoted miR-129-5p and inhibited NEAT1 could improve the liver function and repress blood lipid, inflammation reaction, hepatocyte apoptosis and liver fibrosis in ethanol-induced ASH mice. Furthermore, NEAT1 could negatively regulate miR-129-5p to target SOCS2. Conclusion We have found that the inhibited NEAT1 could suppress liver fibrosis in ASH mice by promoting miR-129-5p and restraining SOCS2, thereby decelerating the development of ASH.

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Determinating the role of the E3 Ubiquitin Ligase Ariadne 2 in mammalian systemss

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2863 ◽

2007 ◽

Vol 30 (4) ◽

pp. 87

Author(s):

A. E. Lin ◽

A. Wakeham ◽

A. You-Ten ◽

G. Wood ◽

T. W. Mak

Keyword(s):

Function Analysis ◽

Critical Role ◽

Ring Finger ◽

E3 Ligase ◽

Signalling Pathways ◽

Loss Of Function ◽

In Vivo Models

Ubiquitination is a eukaryotic process of selective proteolysis, where a highly conserved ubiquitin protein is selectively added as a chain to the targeted to a protein for degradation. In recent years, the process of ubiquitination has been shown to be a critical mechanism that can affect essential signalling pathways, including apoptosis, cell cycle arrest and induction of the inflammatory response. Thus, alterations in the ubiquitination process can alter signalling pathways pivotal to numerous disease pathologies. This is clearly demonstrated in perturbations of ubiquitination in the NFκB giving rise to cancer and other immunological disease processes. To gain insight into pathways that require regulation by ubiquitination, our lab has directed focus on the highly conserved E3 ligase, Ariadne 2. Ariadne 2 is characterized as a putative RING finger E3 ligase and is part of the family of highly conserved RBR (RING-B-Box-RING) superfamily. The role of Ariadne 2 has been well studied in Drosophila melanogaster, however, little is known of the function of Ariadne 2 in mammalian systems. Therefore, the main objectives of the project are as follows: To determine the biological role of Ariadne 2, the role of Ariadne 2 in development and differentiation, and the consequences of in vivo loss of Ariadne 2 expression. We are currently investigating the role of Ariadne 2 as an E3 ligase and its involvement in the immune response. To date, we have shown that Ariadne 2 is ubiquitously expressed, especially in the brain, heart, spleen and thymus. For in vivo loss of function analysis, mice were generated by homologous recombination to be deficient for Ariadne 2. These deficient mice die prematurely soon after birth, suggesting a critical role for Ariadne 2 in development and survival. We are currently focusing on the role of Ariadne 2 in development and it’s role in immune pathologies, in particular, spontaneous autoimmunity, using both in vitro studies and in vivo models.

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C9orf72 loss-of-function: a trivial, stand-alone or additive mechanism in C9 ALS/FTD?

Acta Neuropathologica ◽

10.1007/s00401-020-02214-x ◽

2020 ◽

Vol 140 (5) ◽

pp. 625-643 ◽

Cited By ~ 1

Author(s):

Elke Braems ◽

Bart Swinnen ◽

Ludo Van Den Bosch

Keyword(s):

Repeat Expansion ◽

Loss Of Function ◽

In Vivo Models ◽

Disease Phenotypes ◽

Potential Link ◽

Multiple Hit ◽

Protein Toxicity ◽

Lateral Sclerosis

Abstract A repeat expansion in C9orf72 is responsible for the characteristic neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in a still unresolved manner. Proposed mechanisms involve gain-of-functions, comprising RNA and protein toxicity, and loss-of-function of the C9orf72 gene. Their exact contribution is still inconclusive and reports regarding loss-of-function are rather inconsistent. Here, we review the function of the C9orf72 protein and its relevance in disease. We explore the potential link between reduced C9orf72 levels and disease phenotypes in postmortem, in vitro, and in vivo models. Moreover, the significance of loss-of-function in other non-coding repeat expansion diseases is used to clarify its contribution in C9orf72 ALS/FTD. In conclusion, with evidence pointing to a multiple-hit model, loss-of-function on itself seems to be insufficient to cause neurodegeneration in C9orf72 ALS/FTD.

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In Vitro and In Vivo Models of Non-Alcoholic Fatty Liver Disease: A Critical Appraisal

Journal of Clinical Medicine ◽

10.3390/jcm10010036 ◽

2020 ◽

Vol 10 (1) ◽

pp. 36

Author(s):

Pierre-Antoine Soret ◽

Julie Magusto ◽

Chantal Housset ◽

Jérémie Gautheron

Keyword(s):

Liver Disease ◽

Fatty Liver ◽

Fatty Liver Disease ◽

Preclinical Research ◽

In Vivo Models ◽

Alcoholic Fatty Liver ◽

Cellular Models ◽

Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), represents the hepatic manifestation of obesity and metabolic syndrome. Due to the spread of the obesity epidemic, NAFLD is becoming the most common chronic liver disease and one of the principal indications for liver transplantation. However, no pharmacological treatment is currently approved to prevent the outbreak of NASH, which leads to fibrosis and cirrhosis. Preclinical research is required to improve our knowledge of NAFLD physiopathology and to identify new therapeutic targets. In the present review, we summarize advances in NAFLD preclinical models from cellular models, including new bioengineered platforms, to in vivo models, with a particular focus on genetic and dietary mouse models. We aim to discuss the advantages and limits of these different models.

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LOXL2 attenuates osteoarthritis through inactivating Integrin/FAK signaling

Scientific Reports ◽

10.1038/s41598-021-96348-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Caixia Zhang ◽

Mengjiao Zhu ◽

Huijuan Wang ◽

Juan Wen ◽

Ziwei Huang ◽

...

Keyword(s):

Mechanical Stress ◽

Structural Damage ◽

Critical Role ◽

Joint Disease ◽

In Vivo Model ◽

Matrix Degradation ◽

Loss Of Function ◽

In Vivo Models

AbstractTemporomandibular joint OA (TMJOA) is a common degenerative joint disease, leads to structural damage and ultimately loss of function. Matrix degradation is one of the first pathogenesis during the progression of OA, it was effective to inhibit matrix degradation to block the development of OA. In this study, an in vivo model (compressive mechanical force) and an in vitro model (IL-1β) were used to induce OA-like changes in TMJ cartilage and chondrocytes. We revealed lysyl oxidase like-2 (LOXL2) play a critical role in TMJOA. LOXL2 expression decreased in mechanical stress/IL-β induced TMJOA-like lesions in both in vivo models and in vitro models. Furthermore, recombinant LOXL2 (rhLOXL2) treatment ameliorated the degenerative changes induced by mechanical stress in vivo, including the thinning cartilage, down-expression of collagen II and proteoglycan, and over-expression of TNF-a, while LOXL2 antibody (anti-LOXL2) treatment exacerbated these changes. Mechanistically, the protection of LOXL2 in chondrocytes was induced partly through activation of the Integrin/FAK pathway. The inhibition of the Integrin/FAK pathway could neutralized the effects caused by rhLOXL2. Collectively, our study suggests that the LOXL2 plays a protective role in mechanical stress induced TMJOA-like changes, and the Integrin/FAK pathway may be a key downstream pathway in this process.

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Fatty Acids and Effects on In Vitro and In Vivo Models of Liver Steatosis

Current Medicinal Chemistry ◽

10.2174/0929867324666170518101334 ◽

2019 ◽

Vol 26 (19) ◽

pp. 3439-3456 ◽

Cited By ~ 3

Author(s):

Laura Vergani

Keyword(s):

Fatty Acids ◽

Animal Models ◽

Fatty Liver ◽

Liver Steatosis ◽

In Vivo Models ◽

Alcoholic Fatty Liver ◽

Cellular Models ◽

Advantages And Disadvantages

Background: Fatty liver, or steatosis, is a condition of excess accumulation of lipids, mainly under form of triglycerides (TG), in the liver, and it is the hallmark of non-alcoholic fatty liver disease (NAFLD). NAFLD is the most common liver disorder world-wide and it has frequently been associated with obesity, hyperlipidemia and insulin resistance. Free fatty acids (FA) are the major mediators of hepatic steatosis; patients with NAFLD have elevated levels of circulating FA that correlate with disease severity. Methods: Steatosis is a reversible condition that can be resolved with changed behaviors, or that can progress towards more severe liver damages such as steatohepatitis (NASH), fibrosis and cirrhosis. In NAFLD, FA of exogenous or endogenous origin accumulate in the hepatocytes and trigger liver damages. Excess TG are stored in cytosolic lipid droplets (LDs) that are dynamic organelles acting as hubs for lipid metabolism. Results: In the first part of this review, we briefly reassumed the main classes of FA and their chemical classification as a function of the presence and number of double bonds, their metabolic pathways and effects on human health. Then, we summarized the main genetic and diet-induced animal models of NAFLD, as well as the cellular models of NAFLD. Conclusions: In recent years, both the diet-induced animal models of NAFLD as well as the cellular models of NAFLD have found ever more application to investigate the mechanisms involved in NAFLD, and we referred to their advantages and disadvantages.

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Modeling Non-Alcoholic Fatty Liver Disease (NAFLD) Using “Good-Fit” Genome-Editing Tools

Cells ◽

10.3390/cells9122572 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2572

Author(s):

Uijin Kim ◽

Nahyun Kim ◽

Ha Youn Shin

Keyword(s):

Liver Disease ◽

Fatty Liver ◽

Genome Editing ◽

Fatty Liver Disease ◽

Genome Engineering ◽

In Vivo Models ◽

Alcoholic Fatty Liver ◽

Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD), which affects both adults and children, is the most common liver disorder worldwide. NAFLD is characterized by excess fat accumulation in the liver in the absence of significant alcohol use. NAFLD is strongly associated with obesity, insulin resistance, metabolic syndrome, as well as specific genetic polymorphisms. Severe NAFLD cases can further progress to cirrhosis, hepatocellular carcinoma (HCC), or cardiovascular complications. Here, we describe the pathophysiological features and critical genetic variants associated with NAFLD. Recent advances in genome-engineering technology have provided a new opportunity to generate in vitro and in vivo models that reflect the genetic abnormalities of NAFLD. We review the currently developed NAFLD models generated using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. We further discuss unique features of CRISPR/Cas9 and Cas9 variants, including base editors and prime editor, that are useful for replicating genetic features specific to NAFLD. We also compare advantages and limitations of currently available methods for delivering genome-editing tools necessary for optimal genome editing. This review should provide helpful guidance for selecting “good fit” genome-editing tools and appropriate gene-delivery methods for the successful development of NAFLD models and clinical therapeutics.

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DIPG-53. CHARACTERIZING THE ROLE OF PPM1D MUTATIONS IN THE PATHOGENESIS OF DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGS)

Neuro-Oncology ◽

10.1093/neuonc/noaa222.098 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii297-iii297

Author(s):

Prasidda Khadka ◽

Zachary Reitman ◽

Sophie Lu ◽

Graham Buchan ◽

Rachel Hartley ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Radiation Treatment ◽

Ectopic Expression ◽

Loss Of Function ◽

In Vivo Models ◽

Cycle Arrest ◽

Diffuse Intrinsic Pontine Gliomas

Abstract INTRODUCTION We have previously found that up to 15% of all DIPGs harbor mutations in PPM1D, resulting in the expression of an activated and truncated PPM1D (PPM1Dtr). Here we evaluate the mechanisms through which PPM1Dtr enhances glioma formation and identify its associated therapeutic vulnerabilities. METHODS We have developed multiple in vitro and in vivo models of PPM1D-mutant DIPGs and applied quantitative proteomic and functional genomic approaches to identify pathways altered by PPM1Dtr and associated dependencies. RESULTS PPM1D mutations are clonal events that are anti-correlated to TP53 mutations. We find ectopic expression of PPM1Dtr to be sufficient to enhance glioma formation and to be necessary in PPM1D-mutant DIPG cells. In addition, endogenous truncation of PPM1D is sufficient to enhance glioma formation in the presence of mutant H3F3A and PDGFRA. PPM1Dtr overexpression attenuates g-H2AX formation and suppresses apoptosis and cell-cycle arrest in response to radiation treatment. Deep scale phosphoproteomics analyses reveal DNA-damage and cell cycle pathways to be most significantly associated with PPM1Dtr. Furthermore, preliminary analysis of genome-wide loss-of-function CRISPR/Cas9 screens in isogenic GFP and PPM1Dtr overexpressing mouse neural stem cells reveal differential dependency on DNA-damage response genes in the PPM1Dtr overexpressing cells. Consistent with PPM1D’s role in stabilizing MDM2, PPM1D-mutant DIPG models are sensitive to a panel of MDM2 inhibitors (Nutlin-3a, RG7388, and AMG232). CONCLUSION Our study shows that PPM1Dtr is both an oncogene and a dependency in PPM1D- mutant DIPG, and there are novel therapeutic vulnerabilities associated with PPM1D that may be exploited.

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