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1.
bioRxiv ; 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38293175

RESUMO

Metabolic dysfunction-associated steatohepatitis (MASH) can progress to cirrhosis and liver cancer. There are no approved medical therapies to prevent or reverse disease progression. Fructose and its metabolism in the liver play integral roles in MASH pathogenesis and progression. Here we focus on mannose, a simple sugar, which dampens hepatic stellate cell activation and mitigates alcoholic liver disease in vitro and in vivo . In the well-validated FAT-MASH murine model, oral mannose supplementation improved both liver steatosis and fibrosis at low and high doses, whether administered either at the onset of the model ("Prevention") or at week 6 of the 12-week MASH regimen ("Reversal"). The in vivo anti-fibrotic effects of mannose supplementation were validated in a second model of carbon tetrachloride-induced liver fibrosis. In vitro human and mouse primary hepatocytes revealed that the anti-steatotic effects of mannose are dependent on the presence of fructose, which attenuates expression of ketohexokinase (KHK), the main enzyme in fructolysis. KHK is decreased with mannose supplementation in vivo and in vitro, and overexpression of KHK abrogated the anti-steatotic effects of mannose. Our study identifies mannose as a simple, novel therapeutic candidate for MASH that mitigates metabolic dysregulation and exerts anti-fibrotic effects.

2.
Hepatol Commun ; 6(7): 1711-1724, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35315595

RESUMO

The mechanisms underlying liver fibrosis are multifaceted and remain elusive with no approved antifibrotic treatments available. The adult zebrafish has been an underutilized tool to study liver fibrosis. We aimed to characterize the single-cell transcriptome of the adult zebrafish liver to determine its utility as a model for studying liver fibrosis. We used single-cell RNA sequencing (scRNA-seq) of adult zebrafish liver to study the molecular and cellular dynamics at a single-cell level. We performed a comparative analysis to scRNA-seq of human liver with a focus on hepatic stellate cells (HSCs), the driver cells in liver fibrosis. scRNA-seq reveals transcriptionally unique populations of hepatic cell types that comprise the zebrafish liver. Joint clustering with human liver scRNA-seq data demonstrates high conservation of transcriptional profiles and human marker genes in zebrafish. Human and zebrafish HSCs show conservation of transcriptional profiles, and we uncover collectin subfamily member 11 (colec11) as a novel, conserved marker for zebrafish HSCs. To demonstrate the power of scRNA-seq to study liver fibrosis using zebrafish, we performed scRNA-seq on our zebrafish model of a pediatric liver disease with mutation in mannose phosphate isomerase (MPI) and characteristic early liver fibrosis. We found fibrosis signaling pathways and upstream regulators conserved across MPI-depleted zebrafish and human HSCs. CellPhoneDB analysis of zebrafish transcriptome identified neuropilin 1 as a potential driver of liver fibrosis. Conclusion: This study establishes the first scRNA-seq atlas of the adult zebrafish liver, highlights the high degree of similarity to human liver, and strengthens its value as a model to study liver fibrosis.


Assuntos
Transcriptoma , Peixe-Zebra , Animais , Criança , Humanos , Cirrose Hepática/genética , Manose-6-Fosfato Isomerase , Fenótipo , Transcriptoma/genética , Peixe-Zebra/genética
3.
Hepatology ; 70(6): 2107-2122, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31016744

RESUMO

The growing burden of liver fibrosis and lack of effective antifibrotic therapies highlight the need for identification of pathways and complementary model systems of hepatic fibrosis. A rare, monogenic disorder in which children with mutations in mannose phosphate isomerase (MPI) develop liver fibrosis led us to explore the function of MPI and mannose metabolism in liver development and adult liver diseases. Herein, analyses of transcriptomic data from three human liver cohorts demonstrate that MPI gene expression is down-regulated proportionate to fibrosis in chronic liver diseases, including nonalcoholic fatty liver disease and hepatitis B virus. Depletion of MPI in zebrafish liver in vivo and in human hepatic stellate cell (HSC) lines in culture activates fibrotic responses, indicating that loss of MPI promotes HSC activation. We further demonstrate that mannose supplementation can attenuate HSC activation, leading to reduced fibrogenic activation in zebrafish, culture-activated HSCs, and in ethanol-activated HSCs. Conclusion: These data indicate the prospect that modulation of mannose metabolism pathways could reduce HSC activation and improve hepatic fibrosis.


Assuntos
Células Estreladas do Fígado/fisiologia , Cirrose Hepática/etiologia , Manose-6-Fosfato Isomerase/fisiologia , Manose/farmacologia , Animais , Células Cultivadas , Glicosilação , Humanos , Masculino , Fator de Crescimento Derivado de Plaquetas/fisiologia , Transdução de Sinais/fisiologia , Peixe-Zebra
4.
Elife ; 62017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28644127

RESUMO

Rapid cellular proliferation in early development and cancer depends on glucose metabolism to fuel macromolecule biosynthesis. Metabolic enzymes are presumed regulators of this glycolysis-driven metabolic program, known as the Warburg effect; however, few have been identified. We uncover a previously unappreciated role for Mannose phosphate isomerase (MPI) as a metabolic enzyme required to maintain Warburg metabolism in zebrafish embryos and in both primary and malignant mammalian cells. The functional consequences of MPI loss are striking: glycolysis is blocked and cells die. These phenotypes are caused by induction of p53 and accumulation of the glycolytic intermediate fructose 6-phosphate, leading to engagement of the hexosamine biosynthetic pathway (HBP), increased O-GlcNAcylation, and p53 stabilization. Inhibiting the HBP through genetic and chemical methods reverses p53 stabilization and rescues the Mpi-deficient phenotype. This work provides mechanistic evidence by which MPI loss induces p53, and identifies MPI as a novel regulator of p53 and Warburg metabolism.


Assuntos
Acetilglucosamina/metabolismo , Manose-6-Fosfato Isomerase/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular Tumoral , Frutosefosfatos/metabolismo , Glicólise , Humanos , Peixe-Zebra/embriologia
5.
Mol Biol Cell ; 27(8): 1220-34, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26912795

RESUMO

Activation of the unfolded protein response (UPR) can be either adaptive or pathological. We term the pathological UPR that causes fatty liver disease a "stressed UPR." Here we investigate the mechanism of stressed UPR activation in zebrafish bearing a mutation in thetrappc11gene, which encodes a component of the transport protein particle (TRAPP) complex.trappc11mutants are characterized by secretory pathway defects, reflecting disruption of the TRAPP complex. In addition, we uncover a defect in protein glycosylation intrappc11mutants that is associated with reduced levels of lipid-linked oligosaccharides (LLOs) and compensatory up-regulation of genes in the terpenoid biosynthetic pathway that produces the LLO anchor dolichol. Treating wild-type larvae with terpenoid or LLO synthesis inhibitors phenocopies the stressed UPR seen intrappc11mutants and is synthetically lethal withtrappc11mutation. We propose that reduced LLO level causing hypoglycosylation is a mechanism of stressed UPR induction intrappc11mutants. Of importance, in human cells, depletion of TRAPPC11, but not other TRAPP components, causes protein hypoglycosylation, and lipid droplets accumulate in fibroblasts from patients with theTRAPPC11mutation. These data point to a previously unanticipated and conserved role for TRAPPC11 in LLO biosynthesis and protein glycosylation in addition to its established function in vesicle trafficking.


Assuntos
Oligossacarídeos/metabolismo , Resposta a Proteínas não Dobradas , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Atorvastatina/farmacologia , Dolicóis/biossíntese , Dolicóis/genética , Glicosilação , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Larva/efeitos dos fármacos , Larva/metabolismo , Lipídeos/química , Fígado/metabolismo , Fígado/patologia , Mutação , Oligossacarídeos/química , Terpenos/metabolismo , Terpenos/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Proteínas de Transporte Vesicular/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
6.
FASEB J ; 28(4): 1854-69, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24421398

RESUMO

Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (~15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1-2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ~50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.


Assuntos
Cegueira/etiologia , Suplementos Nutricionais/toxicidade , Manose-6-Fosfato Isomerase/metabolismo , Manose/toxicidade , Animais , Cegueira/genética , Cegueira/metabolismo , Western Blotting , Células Cultivadas , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Olho/embriologia , Olho/crescimento & desenvolvimento , Olho/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Manose/sangue , Manose/metabolismo , Manose-6-Fosfato Isomerase/genética , Manosefosfatos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Placenta/efeitos dos fármacos , Placenta/embriologia , Placenta/metabolismo , Gravidez
7.
Nat Med ; 18(1): 71-3, 2011 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-22157680

RESUMO

Congenital disorder of glycosylation-Ia (CDG-Ia, also known as PMM2-CDG) is caused by mutations in the gene that encodes phosphomannomutase 2 (PMM2, EC 5.4.2.8) leading to a multisystemic disease with severe psychomotor and mental retardation. In a hypomorphic Pmm2 mouse model, we were able to overcome embryonic lethality by feeding mannose to pregnant dams. The results underline the essential role of glycosylation in embryonic development and may open new treatment options for this disease.


Assuntos
Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/prevenção & controle , Desenvolvimento Embrionário/efeitos dos fármacos , Manose/uso terapêutico , Fosfotransferases (Fosfomutases)/genética , Cuidado Pré-Natal/métodos , Animais , Defeitos Congênitos da Glicosilação/genética , Modelos Animais de Doenças , Feminino , Genes Letais , Glicosilação/efeitos dos fármacos , Humanos , Camundongos , Gravidez
8.
Proc Natl Acad Sci U S A ; 105(5): 1674-9, 2008 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-18227516

RESUMO

Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth factor stimuli. Here, we use a mouse model in which ATF2 was selectively deleted in keratinocytes. Crossing the conditionally expressed ATF2 mutant with K14-Cre mice (K14.ATF2(f/f)) resulted in selective expression of mutant ATF2 within the basal layer of the epidermis. When subjected to a two-stage skin carcinogenesis protocol [7,12-dimethylbenz[a]anthracene/phorbol 12-tetradecanoate 13-acetate (DMBA/TPA)], K14.ATF2(f/f) mice showed significant increases in both the incidence and prevalence of papilloma development compared with the WT ATF2 mice. Consistent with these findings, keratinocytes of K14.ATF2(f/f) mice exhibit greater anchorage-independent growth compared with ATF2 WT keratinocytes. Papillomas of K14.ATF2(f/f) mice exhibit reduced expression of presenilin1, which is associated with enhanced beta-catenin and cyclin D1, and reduced Notch1 expression. Significantly, a reduction of nuclear ATF2 and increased beta-catenin expression were seen in samples of squamous and basal cell carcinoma, as opposed to normal skin. Our data reveal that loss of ATF2 transcriptional activity serves to promote skin tumor formation, thereby indicating a suppressor activity of ATF2 in skin tumor formation.


Assuntos
Fator 2 Ativador da Transcrição/fisiologia , Papiloma/genética , Neoplasias Cutâneas/genética , Proteínas Supressoras de Tumor/fisiologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Fator 2 Ativador da Transcrição/análise , Fator 2 Ativador da Transcrição/genética , Animais , Apoptose , Carcinógenos/toxicidade , Proliferação de Células , Ciclina D1/metabolismo , DNA/biossíntese , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Epiderme/patologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Knockout , Papiloma/induzido quimicamente , Papiloma/patologia , Presenilina-1/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Receptor Notch1/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/toxicidade , Análise Serial de Tecidos , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/genética , beta Catenina/metabolismo
9.
J Biol Chem ; 281(9): 5916-27, 2006 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-16339137

RESUMO

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.


Assuntos
Perda do Embrião , Embrião de Mamíferos/fisiologia , Manose-6-Fosfato Isomerase/deficiência , Manose/metabolismo , Manosefosfatos , Trifosfato de Adenosina/metabolismo , Animais , Erros Inatos do Metabolismo dos Carboidratos , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/patologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Marcação de Genes , Genótipo , Idade Gestacional , Hexoquinase/metabolismo , Humanos , Masculino , Manose/administração & dosagem , Manose/toxicidade , Manose-6-Fosfato Isomerase/genética , Manose-6-Fosfato Isomerase/metabolismo , Manosefosfatos/metabolismo , Manosefosfatos/toxicidade , Camundongos , Camundongos Knockout , Polissacarídeos/biossíntese , Gravidez
10.
Cell ; 112(3): 355-67, 2003 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-12581525

RESUMO

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. Here, we demonstrate that mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3(-)(/)(-) embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling.


Assuntos
Padronização Corporal/genética , Polaridade Celular/genética , Embrião de Mamíferos/embriologia , Chaperonas Moleculares/metabolismo , Receptores de LDL/metabolismo , Animais , Células COS , Diferenciação Celular/genética , Anormalidades Congênitas/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Feminino , Genes Letais/genética , Proteínas Relacionadas a Receptor de LDL , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Mutação/genética , Fenótipo , Proteínas/genética , Proteínas/metabolismo , Receptores de LDL/genética , Transdução de Sinais/genética , Proteínas Wnt , Proteína Wnt3
11.
Toxicol Sci ; 69(1): 117-24, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12215665

RESUMO

It is well established that dioxins cause a variety of toxic effects and syndromes including alterations of lymphocyte development. Exposure to the prototypical dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to severe thymic atrophy in all species studied. It has been shown that most of this toxicity is due to TCDD binding to and activating the aryl hydrocarbon receptor (AHR). Upon activation, the AHR enters the nucleus, dimerizes with the AHR nuclear translocator (ARNT), and this heterodimer modulates a number of genes that mediate toxicity. The AHR and ARNT are members of the basic-helix-loop-helix-Per, ARNT, and Sim homology (bHLH-PAS) family of transcription factors. In this study, we wanted to determine if another bHLH-PAS transcription factor, ARNT2, which has high amino acid sequence identity to ARNT and has been shown to dimerize with the TCDD-activated AHR, is involved in mediating TCDD's effect on lymphocyte development. We determined by RT-PCR that ARNT2 is expressed at a low level in whole thymus, thymocytes, and bone marrow lymphocytes. We created hemopoietic chimeras by lethally irradiating C57BL/6 mice and reconstituting them with fetal liver stem cells that either have or are deficient in a portion of chromosome 7 that contains ARNT2. Regardless of whether chimeras possessed or lacked this chromosome fragment, equal sensitivity to TCDD-induced thymic atrophy was observed despite expression of ARNT2 in the thymus. Furthermore, the absence of ARNT2 (or any other genes found on this portion of chromosome 7) did not confer any protection against TCDD-induced alterations in bone marrow B-cell subsets. These data indicate that in this model system the effects of TCDD-induced thymic atrophy and alterations in B-cell maturation are not dependent on an AHR-ARNT2 heterodimer.


Assuntos
Quimera/fisiologia , Poluentes Ambientais/toxicidade , Sistema Hematopoético/citologia , Linfócitos/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Timo/citologia , Fatores de Transcrição/genética , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto , Atrofia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Contagem de Células , Separação Celular , Citometria de Fluxo , Sistema Hematopoético/efeitos dos fármacos , Sistema Hematopoético/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/efeitos dos fármacos , Timo/patologia , Fatores de Transcrição/deficiência
12.
Glycobiology ; 12(7): 435-42, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12122025

RESUMO

Phosphomannose isomerase (PMI) interconverts fructose-6-P (Fru-6-P) and mannose-6-P (Man-6-P), linking energy metabolism to protein glycosylation. We have cloned the mouse Mpi cDNA, analyzed its genomic organization, and studied the expression in different tissues. The Mpi gene has eight exons covering 7.2 kb. The structure and intron-exon boundaries are essentially the same as its human ortholog with 85% amino acid identity. Mpi is alternatively spliced at the 3' end, resulting in three messages with different 3'-untranslated regions. Mpi expression is regulated at both the transcription and translation levels, with the highest expression level in testis. Rabbit antibodies prepared against mouse PMI expressed in E. coli recognize a single 47-kDa band. Immunohistochemistry of mouse tissues shows general cytosolic staining in all cells. In testis, staining is intense in round spermatids and residual bodies, moderate in pachytene spermatocytes, and weak in spermatogonia and spermatozoa. In contrast, northern blot analysis shows comparable transcripts of 1.8 and 1.6 kb in pachytene spermatocytes and round spermatids, suggesting delayed translation of PMI. The stage-specific expression of PMI in testis may be important for KDN synthesis, which requires Man-6-P, or it may be needed to ensure sufficient glycosylation precursors in cells that do not utilize glucose and instead rely on lactate and pyruvate.


Assuntos
Manose-6-Fosfato Isomerase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Éxons , Imuno-Histoquímica , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Espermatozoides/enzimologia
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