RESUMO
Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage in pre-malignant cells. Accumulated glycogen undergoes liquid-liquid phase separation, which results in the assembly of the Laforin-Mst1/2 complex and consequently sequesters Hippo kinases Mst1/2 in glycogen liquid droplets to relieve their inhibition on Yap. Moreover, G6PC or another glycogenolysis enzyme-liver glycogen phosphorylase (PYGL) deficiency in both human and mice results in glycogen storage disease along with liver enlargement and tumorigenesis in a Yap-dependent manner. Consistently, elimination of glycogen accumulation abrogates liver growth and cancer incidence, whereas increasing glycogen storage accelerates tumorigenesis. Thus, we concluded that cancer-initiating cells adapt a glycogen storing mode, which blocks Hippo signaling through glycogen phase separation to augment tumor incidence.
Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Glicogênio/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glucose-6-Fosfatase/metabolismo , Glicogênio Fosforilase/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Via de Sinalização Hippo , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Transição de Fase , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina-Treonina Quinase 3/metabolismo , Proteínas de Sinalização YAP/metabolismoRESUMO
The gut microbiota contributes to the development of normal immunity but, when dysregulated, can promote autoimmunity through various non-antigen-specific effects on pathogenic and regulatory lymphocytes. Here, we show that an integrase expressed by several species of the gut microbial genus Bacteroides encodes a low-avidity mimotope of the pancreatic ß cell autoantigen islet-specific glucose-6-phosphatase-catalytic-subunit-related protein (IGRP206-214). Studies in germ-free mice monocolonized with integrase-competent, integrase-deficient, and integrase-transgenic Bacteroides demonstrate that the microbial epitope promotes the recruitment of diabetogenic CD8+ T cells to the gut. There, these effectors suppress colitis by targeting microbial antigen-loaded, antigen-presenting cells in an integrin ß7-, perforin-, and major histocompatibility complex class I-dependent manner. Like their murine counterparts, human peripheral blood T cells also recognize Bacteroides integrase. These data suggest that gut microbial antigen-specific cytotoxic T cells may have therapeutic value in inflammatory bowel disease and unearth molecular mimicry as a novel mechanism by which the gut microbiota can regulate normal immune homeostasis. PAPERCLIP.
Assuntos
Autoantígenos/imunologia , Bacteroides/imunologia , Colite/imunologia , Microbioma Gastrointestinal , Glucose-6-Fosfatase/imunologia , Adulto , Animais , Bacteroides/classificação , Bacteroides/enzimologia , Colite/microbiologia , Feminino , Glucose-6-Fosfatase/genética , Humanos , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Mimetismo Molecular , Linfócitos T/imunologiaRESUMO
Insulin resistance is a hallmark of diabetes and an unmet clinical need. Insulin inhibits hepatic glucose production and promotes lipogenesis by suppressing FOXO1-dependent activation of G6pase and inhibition of glucokinase, respectively. The tight coupling of these events poses a dual conundrum: mechanistically, as the FOXO1 corepressor of glucokinase is unknown, and clinically, as inhibition of glucose production is predicted to increase lipogenesis. Here, we report that SIN3A is the insulin-sensitive FOXO1 corepressor of glucokinase. Genetic ablation of SIN3A abolishes nutrient regulation of glucokinase without affecting other FOXO1 target genes and lowers glycemia without concurrent steatosis. To extend this work, we executed a small-molecule screen and discovered selective inhibitors of FOXO-dependent glucose production devoid of lipogenic activity in hepatocytes. In addition to identifying a novel mode of insulin action, these data raise the possibility of developing selective modulators of unliganded transcription factors to dial out adverse effects of insulin sensitizers.
Assuntos
Proteína Forkhead Box O1/antagonistas & inibidores , Glucose/metabolismo , Hepatócitos/metabolismo , Resistência à Insulina , Acetilação , Animais , Células Cultivadas , Proteína Forkhead Box O1/química , Glucoquinase/genética , Glucoquinase/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Células HEK293 , Hepatócitos/enzimologia , Histona Desacetilases/metabolismo , Humanos , Lipogênese/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fosforilação , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
Sepsis is an often lethal syndrome resulting from maladaptive immune and metabolic responses to infection, compromising host homeostasis. Disease tolerance is a defense strategy against infection that preserves host homeostasis without exerting a direct negative impact on pathogens. Here, we demonstrate that induction of the iron-sequestering ferritin H chain (FTH) in response to polymicrobial infections is critical to establish disease tolerance to sepsis. The protective effect of FTH is exerted via a mechanism that counters iron-driven oxidative inhibition of the liver glucose-6-phosphatase (G6Pase), and in doing so, sustains endogenous glucose production via liver gluconeogenesis. This is required to prevent the development of hypoglycemia that otherwise compromises disease tolerance to sepsis. FTH overexpression or ferritin administration establish disease tolerance therapeutically. In conclusion, disease tolerance to sepsis relies on a crosstalk between adaptive responses controlling iron and glucose metabolism, required to maintain blood glucose within a physiologic range compatible with host survival.
Assuntos
Glucose/metabolismo , Ferro/metabolismo , Sepse/metabolismo , Animais , Apoferritinas/genética , Apoferritinas/metabolismo , Ceruloplasmina/metabolismo , Gluconeogênese , Glucose-6-Fosfatase/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Soluble dietary fibers promote metabolic benefits on body weight and glucose control, but underlying mechanisms are poorly understood. Recent evidence indicates that intestinal gluconeogenesis (IGN) has beneficial effects on glucose and energy homeostasis. Here, we show that the short-chain fatty acids (SCFAs) propionate and butyrate, which are generated by fermentation of soluble fiber by the gut microbiota, activate IGN via complementary mechanisms. Butyrate activates IGN gene expression through a cAMP-dependent mechanism, while propionate, itself a substrate of IGN, activates IGN gene expression via a gut-brain neural circuit involving the fatty acid receptor FFAR3. The metabolic benefits on body weight and glucose control induced by SCFAs or dietary fiber in normal mice are absent in mice deficient for IGN, despite similar modifications in gut microbiota composition. Thus, the regulation of IGN is necessary for the metabolic benefits associated with SCFAs and soluble fiber.
Assuntos
Gluconeogênese , Mucosa Intestinal/metabolismo , Intestinos/inervação , Animais , Encéfalo/metabolismo , Gorduras na Dieta/metabolismo , Fibras na Dieta/metabolismo , Ácidos Graxos Voláteis/metabolismo , Glucose/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Homeostase , Resistência à Insulina , Camundongos , Microbiota , Obesidade/metabolismo , Oligossacarídeos/metabolismo , RatosRESUMO
CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained, and the molecular programmes defining the autoimmune T cell state, are unknown. In type 1 diabetes, ß-cell-specific CD8 T cells destroy insulin-producing ß-cells. Here we followed the fate of ß-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas, where they differentiate further and destroy ß-cells. Whereas transplantation of as few as 20 autoimmune progenitors induced type 1 diabetes, as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived, and stem-like autoimmune progenitors must continuously seed the pancreas to sustain ß-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Células Secretoras de Insulina/imunologia , Células-Tronco/patologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Autorrenovação Celular , Células Clonais/imunologia , Células Clonais/metabolismo , Células Clonais/patologia , Modelos Animais de Doenças , Feminino , Glucose-6-Fosfatase/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Células Secretoras de Insulina/patologia , Linfonodos/imunologia , Masculino , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Célula Única , Transplante de Células-Tronco , Células-Tronco/imunologia , Células-Tronco/metabolismo , TranscriptomaRESUMO
Protein structures can provide invaluable information, both for reasoning about biological processes and for enabling interventions such as structure-based drug development or targeted mutagenesis. After decades of effort, 17% of the total residues in human protein sequences are covered by an experimentally determined structure1. Here we markedly expand the structural coverage of the proteome by applying the state-of-the-art machine learning method, AlphaFold2, at a scale that covers almost the entire human proteome (98.5% of human proteins). The resulting dataset covers 58% of residues with a confident prediction, of which a subset (36% of all residues) have very high confidence. We introduce several metrics developed by building on the AlphaFold model and use them to interpret the dataset, identifying strong multi-domain predictions as well as regions that are likely to be disordered. Finally, we provide some case studies to illustrate how high-quality predictions could be used to generate biological hypotheses. We are making our predictions freely available to the community and anticipate that routine large-scale and high-accuracy structure prediction will become an important tool that will allow new questions to be addressed from a structural perspective.
Assuntos
Biologia Computacional/normas , Aprendizado Profundo/normas , Modelos Moleculares , Conformação Proteica , Proteoma/química , Conjuntos de Dados como Assunto/normas , Diacilglicerol O-Aciltransferase/química , Glucose-6-Fosfatase/química , Humanos , Proteínas de Membrana/química , Dobramento de Proteína , Reprodutibilidade dos TestesRESUMO
Neuropeptides (NPs) and their cognate receptors are critical effectors of diverse physiological processes and behaviors. We recently reported of a noncanonical function of the Drosophila Glucose-6-Phosphatase (G6P) gene in a subset of neurosecretory cells in the central nervous system that governs systemic glucose homeostasis in food-deprived flies. Here, we show that G6P-expressing neurons define six groups of NP-secreting cells, four in the brain and two in the thoracic ganglion. Using the glucose homeostasis phenotype as a screening tool, we find that neurons located in the thoracic ganglion expressing FMRFamide NPs (FMRFaG6P neurons) are necessary and sufficient to maintain systemic glucose homeostasis in starved flies. We further show that G6P is essential in FMRFaG6P neurons for attaining a prominent Golgi apparatus and secreting NPs efficiently. Finally, we establish that G6P-dependent FMRFa signaling is essential for the build-up of glycogen stores in the jump muscle which expresses the receptor for FMRFamides. We propose a general model in which the main role of G6P is to counteract glycolysis in peptidergic neurons for the purpose of optimizing the intracellular environment best suited for the expansion of the Golgi apparatus, boosting release of NPs and enhancing signaling to respective target tissues expressing cognate receptors.
Assuntos
Drosophila melanogaster , FMRFamida , Glucose-6-Fosfatase , Glicogênio , Neurônios , Neuropeptídeos , Transdução de Sinais , Animais , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , FMRFamida/metabolismo , Glucose/metabolismo , Glucose-6-Fosfatase/metabolismo , Glucose-6-Fosfatase/genética , Glicogênio/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Músculos/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Neuropeptídeos/genéticaRESUMO
Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (TPEX) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear whether and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using nonobese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs (PLO). More IGRP-specific T cells resided in the PLO than in islets. To examine the impact of extraislet antigen exposure on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen-presenting cells. Antigen exposure in the extraislet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce interferon (IFN)γ, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes.
Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Camundongos , Animais , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/prevenção & controle , Proteínas/metabolismo , Exaustão das Células T , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Camundongos Transgênicos , Camundongos Endogâmicos NOD , Ilhotas Pancreáticas/metabolismo , Linfócitos T CD8-PositivosRESUMO
Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that is responsible for transporting G6P into the endoplasmic reticulum. GSD-Ib is characterized by disturbances in glucose homeostasis, neutropenia, and neutrophil dysfunction. Although some studies have explored neutrophils abnormalities in GSD-Ib, investigations regarding monocytes/macrophages remain limited so far. In this study, we examined the impact of G6PT deficiency on monocyte-to-macrophage differentiation using bone marrow-derived monocytes from G6pt-/- mice as well as G6PT-deficient human THP-1 monocytes. Our findings revealed that G6PT-deficient monocytes exhibited immature differentiation into macrophages. Notably, the impaired differentiation observed in G6PT-deficient monocytes seemed to be associated with abnormal glucose metabolism, characterized by enhanced glucose consumption through glycolysis, even under quiescent conditions with oxidative phosphorylation. Furthermore, we observed a reduced secretion of inflammatory cytokines in G6PT-deficient THP-1 monocytes during the inflammatory response, despite their elevated glucose consumption. In conclusion, this study sheds light on the significance of G6PT in monocyte-to-macrophage differentiation and underscores its importance in maintaining glucose homeostasis and supporting immune response in GSD-Ib. These findings may contribute to a better understanding of the pathogenesis of GSD-Ib and potentially pave the way for the development of targeted therapeutic interventions.
Assuntos
Doença de Depósito de Glicogênio Tipo I , Animais , Camundongos , Humanos , Doença de Depósito de Glicogênio Tipo I/complicações , Doença de Depósito de Glicogênio Tipo I/metabolismo , Doença de Depósito de Glicogênio Tipo I/terapia , Glucose-6-Fosfatase/metabolismo , Glucose/metabolismo , Macrófagos/metabolismoRESUMO
BACKGROUND: Congenital neutropenias are characterized by severe infections and a high risk of myeloid transformation; the causative genes vary across ethnicities. The Israeli population is characterized by an ethnically diverse population with a high rate of consanguinity. OBJECTIVE: To evaluate the clinical and genetic spectrum of congenital neutropenias in Israel. METHODS: We included individuals with congenital neutropenias listed in the Israeli Inherited Bone Marrow Failure Registry. Sanger sequencing was performed for ELANE or G6PC3, and patients with wild-type ELANE/G6PC3 were referred for next-generation sequencing. RESULTS: Sixty-five patients with neutropenia were included. Of 51 patients with severe congenital neutropenia, 34 were genetically diagnosed, most commonly with variants in ELANE (15 patients). Nine patients had biallelic variants in G6PC3, all of consanguineous Muslim Arab origin. Other genes involved were SRP54, JAGN1, TAZ, and SLC37A4. Seven patients had cyclic neutropenia, all with pathogenic variants in ELANE, and seven had Shwachman-Diamond syndrome caused by biallelic SBDS variants. Eight patients (12%) developed myeloid transformation, including six patients with an unknown underlying genetic cause. Nineteen (29%) patients underwent hematopoietic stem cell transplantation, mostly due to insufficient response to treatment with granulocyte-colony stimulating factor or due to myeloid transformation. CONCLUSIONS: The genetic spectrum of congenital neutropenias in Israel is characterized by a high prevalence of G6PC3 variants and an absence of HAX1 mutations. Similar to other registries, for 26% of the patients, a molecular diagnosis was not achieved. However, myeloid transformation was common in this group, emphasizing the need for close follow-up.
Assuntos
Síndrome Congênita de Insuficiência da Medula Óssea , Mutação , Neutropenia , Humanos , Neutropenia/genética , Neutropenia/congênito , Neutropenia/epidemiologia , Neutropenia/diagnóstico , Masculino , Israel/epidemiologia , Feminino , Criança , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea/diagnóstico , Pré-Escolar , Adolescente , Predisposição Genética para Doença , Adulto , Transplante de Células-Tronco Hematopoéticas , Lactente , Consanguinidade , Glucose-6-Fosfatase/genética , Alelos , Sistema de Registros , Sequenciamento de Nucleotídeos em Larga Escala , Adulto Jovem , Fenótipo , Estudos de Associação GenéticaRESUMO
G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit that modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). A common single-nucleotide polymorphism (SNP) in G6PC2, rs560887 is an important determinant of human FBG variability. This SNP has a subtle effect on G6PC2 RNA splicing, which raises the question as to whether nonsynonymous SNPs with a major impact on G6PC2 stability or enzyme activity might have a broader disease/metabolic impact. Previous attempts to characterize such SNPs were limited by the very low inherent G6Pase activity and expression of G6PC2 protein in islet-derived cell lines. In this study, we describe the use of a plasmid vector that confers high G6PC2 protein expression in islet cells, allowing for a functional analysis of 22 nonsynonymous G6PC2 SNPs, 19 of which alter amino acids that are conserved in mouse G6PC2 and the human and mouse variants of the related G6PC1 isoform. We show that 16 of these SNPs markedly impair G6PC2 protein expression (>50% decrease). These SNPs have variable effects on the stability of human and mouse G6PC1, despite the high sequence homology between these isoforms. Four of the remaining six SNPs impaired G6PC2 enzyme activity. Electronic health record-derived phenotype analyses showed an association between high-impact SNPs and FBG, but not other diseases/metabolites. While homozygous G6pc2 deletion in mice increases the risk of hypoglycemia, these human data reveal no evidence that the beneficial use of partial G6PC2 inhibitors to lower FBG would be associated with unintended negative consequences.
Assuntos
Glicemia , Jejum , Glucose-6-Fosfatase , Animais , Camundongos , Glicemia/metabolismo , Jejum/sangue , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Glucose-6-phosphatase catalytic subunit 1 (G6PC1) plays a critical role in hepatic glucose production during fasting by mediating the terminal step of the gluconeogenesis and glycogenolysis pathways. In concert with accessory transport proteins, this membrane-integrated enzyme catalyzes glucose production from glucose-6-phosphate (G6P) to support blood glucose homeostasis. Consistent with its metabolic function, dysregulation of G6PC1 gene expression contributes to diabetes, and mutations that impair phosphohydrolase activity form the clinical basis of glycogen storage disease type 1a. Despite its relevance to health and disease, a comprehensive view of G6PC1 structure and mechanism has been limited by the absence of expression and purification strategies that isolate the enzyme in a functional form. In this report, we apply a suite of biophysical and biochemical tools to fingerprint the in vitro attributes of catalytically active G6PC1 solubilized in lauryl maltose neopentyl glycol (LMNG) detergent micelles. When purified from Sf9 insect cell membranes, the glycosylated mouse ortholog (mG6PC1) recapitulated functional properties observed previously in intact hepatic microsomes and displayed the highest specific activity reported to date. Additionally, our results establish a direct correlation between the catalytic and structural stability of mG6PC1, which is underscored by the enhanced thermostability conferred by phosphatidylcholine and the cholesterol analog cholesteryl hemisuccinate. In contrast, the N96A variant, which blocks N-linked glycosylation, reduced thermostability. The methodologies described here overcome long-standing obstacles in the field and lay the necessary groundwork for a detailed analysis of the mechanistic structural biology of G6PC1 and its role in complex metabolic disorders.
Assuntos
Glucose-6-Fosfatase , Doença de Depósito de Glicogênio Tipo I , Animais , Domínio Catalítico , Glucose/metabolismo , Glucose-6-Fosfatase/química , Glucose-6-Fosfatase/metabolismo , Doença de Depósito de Glicogênio Tipo I/enzimologia , Doença de Depósito de Glicogênio Tipo I/metabolismo , Camundongos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismoRESUMO
Elevated fasting blood glucose (FBG) is associated with increased risks of developing type 2 diabetes (T2D) and cardiovascular-associated mortality. G6PC2 is predominantly expressed in islets, encodes a glucose-6-phosphatase catalytic subunit that converts glucose-6-phosphate (G6P) to glucose, and has been linked with variations in FBG in genome-wide association studies. Deletion of G6pc2 in mice has been shown to lower FBG without affecting fasting plasma insulin levels in vivo. At 5 mM glucose, pancreatic islets from G6pc2 knockout (KO) mice exhibit no glucose cycling, increased glycolytic flux, and enhanced glucose-stimulated insulin secretion (GSIS). However, the broader effects of G6pc2 KO on ß-cell metabolism and redox regulation are unknown. Here we used CRISPR/Cas9 gene editing and metabolic flux analysis in ßTC3 cells, a murine pancreatic ß-cell line, to examine the role of G6pc2 in regulating glycolytic and mitochondrial fluxes. We found that deletion of G6pc2 led to â¼60% increases in glycolytic and citric acid cycle (CAC) fluxes at both 5 and 11 mM glucose concentrations. Furthermore, intracellular insulin content and GSIS were enhanced by approximately two-fold, along with increased cytosolic redox potential and reductive carboxylation flux. Normalization of fluxes relative to net glucose uptake revealed upregulation in two NADPH-producing pathways in the CAC. These results demonstrate that G6pc2 regulates GSIS by modulating not only glycolysis but also, independently, citric acid cycle activity in ß-cells. Overall, our findings implicate G6PC2 as a potential therapeutic target for enhancing insulin secretion and lowering FBG, which could benefit individuals with prediabetes, T2D, and obesity.
Assuntos
Diabetes Mellitus Tipo 2 , Glucose-6-Fosfatase , Glucose , Células Secretoras de Insulina , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Camundongos , Camundongos Knockout , OxirreduçãoRESUMO
BACKGROUND & AIMS: Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis. METHODS: To build a taxonomy model, we assessed 165 disorders. Genes were prioritized based on penetrance of IBD and disease phenotypes were integrated with multi-omics datasets. Monogenic IBD genes were classified by (1) overlapping syndromic features, (2) response to hematopoietic stem cell transplantation, (3) bulk RNA-sequencing of 32 tissues, (4) single-cell RNA-sequencing of >50 cell subsets from the intestine of healthy individuals and patients with IBD (pediatric and adult), and (5) proteomes of 43 immune subsets. The model was validated by addition of newly identified monogenic IBD defects. As a proof-of-concept, we explore the intersection between immunometabolism and antimicrobial activity for a group of disorders (G6PC3/SLC37A4). RESULTS: Our quantitative integrated taxonomy defines the cellular landscape of monogenic IBD gene expression across 102 genes with high and moderate penetrance (81 in the model set and 21 genes in the validation set). We illustrate distinct cellular networks, highlight expression profiles across understudied cell types (e.g., CD8+ T cells, neutrophils, epithelial subsets, and endothelial cells) and define genotype-phenotype associations (perianal disease and defective antimicrobial activity). We illustrate processes and pathways shared across cellular compartments and phenotypic groups and highlight cellular immunometabolism with mammalian target of rapamycin activation as one of the converging pathways. There is an overlap of genes and enriched cell-specific expression between monogenic and polygenic IBD. CONCLUSION: Our taxonomy integrates genetic, clinical and multi-omic data; providing a basis for genomic diagnostics and testable hypotheses for disease functions and treatment responses.
Assuntos
Doenças Inflamatórias Intestinais/classificação , Doenças Inflamatórias Intestinais/genética , Idade de Início , Antiporters/genética , Células Cultivadas , Classificação , Perfilação da Expressão Gênica , Estudos de Associação Genética , Genótipo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfato/metabolismo , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Macrófagos , Metabolômica , Proteínas de Transporte de Monossacarídeos/genética , Penetrância , Fenótipo , Transdução de Sinais/genéticaRESUMO
Glycogen storage disease type Ib (GSD1b) and G6PC3-deficiency are rare autosomal recessive diseases caused by inactivating mutations in SLC37A4 (coding for G6PT) and G6PC3, respectively. Both diseases are characterized by neutropenia and neutrophil dysfunction due to the intracellular accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases. We recently showed that the use of SGLT2 inhibitor therapy to reduce tubular reabsorption of its precursor, 1,5-anhydroglucitol (1,5-AG), a glucose analog present in blood, successfully restored the neutropenia and neutrophil function in G6PC3-deficient and GSD1b patients. The intra-individual variability of response to the treatment and the need to adjust the dose during treatment, especially in pediatric populations, can only be efficiently optimized if the concentration of 1,5-AG in blood is monitored during treatment, together with the patients' clinical signs and symptoms. Monitoring the 1,5-AG levels would be greatly simplified if it could be performed on dry blood spots (DBS) which are easy to collect, store and transport. The challenge is to know if a suitable method can be developed to perform accurate and reproducible assays for 1,5-AG using DBS. Here, we describe and validate an assay that quantifies 1,5-AG in DBS using isotopic dilution quantitation by LC-MS/MS that should greatly facilitate patients' follow-up. 1,5-AG levels measured in plasma and DBS give comparable values. This assay was used to monitor the levels of 1,5-AG in DBS from 3 G6PC3-deficient and 6 GSD1b patients during treatment with SGLT2 inhibitors. We recommend this approach to verify the adequate therapeutical response and compliance to the treatment in G6PC3-deficient and GSD1b patients treated with SGLT2 inhibitors.
Assuntos
Desoxiglucose , Doença de Depósito de Glicogênio Tipo I , Neutropenia , Inibidores do Transportador 2 de Sódio-Glicose , Criança , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem , Doença de Depósito de Glicogênio Tipo I/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/complicações , Neutropenia/genética , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Monoéster Fosfórico Hidrolases , Proteínas de Transporte de Monossacarídeos , AntiportersRESUMO
Animals suffer hypoxia when their oxygen consumption is larger than the oxygen available. Hypoxia affects the white shrimp Penaeus (Litopenaeus) vannamei, both in their natural habitat and in cultivation farms. Shrimp regulates some enzymes that participate in energy production pathways as a strategy to survive during hypoxia. Glucose-6-phosphatase (G6Pase) is key to maintain blood glucose homeostasis through gluconeogenesis and glycogenolysis. We previously reported a shrimp G6Pase gene (G6Pase1) and in this work, we report a second isoform that we named G6Pase2. The expression of the two isoforms was evaluated in oxygen limited conditions and during silencing of the transcription factor HIF-1. High G6Pase activity was detected in hepatopancreas followed by muscle and gills under good oxygen and feeding conditions. Gene expression of both isoforms was analyzed in normoxia, hypoxia and reoxygenation in hepatopancreas and gills, and in HIF-1-silenced shrimp. In fed shrimp with normal dissolved oxygen (DO) (5.0 mg L- 1 DO) the expression of G6Pase1 was detected in gills, but not in hepatopancreas or muscle, while G6Pase2 expression was undetectable in all three tissues. In hepatopancreas, G6Pase1 is induced at 3 and 48 h of hypoxia, while G6Pase2 is down-regulated in the same time points but in reoxygenation, both due to the knock-down of HIF-1. In gills, only G6Pase1 was detected, and was induced by the silencing of HIF-1 only after 3 h of reoxygenation. Therefore, the expression of the two isoforms appears to be regulated by HIF-1 at transcriptional level in response to oxygen deprivation and subsequent recovery of oxygen levels.
Assuntos
Glucose-6-Fosfatase , Penaeidae , Animais , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Penaeidae/genética , Penaeidae/metabolismo , Hipóxia/metabolismo , Oxigênio/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
Impairment of gluconeogenesis is a key factor responsible for hyperglycemia in patients with type 2 diabetes. As an important member of the suppressors of cytokine signaling (SOCS) protein family, many physiological functions of cytokine-inducible SH2-containing protein (CISH) have been described; however, the role of hepatic CISH in gluconeogenesis is poorly understood. In the present study, we observed that hepatic CISH expression was reduced in fasted wild-type (WT) mice. Overexpression of CISH decreased glucose production in mouse primary hepatocytes, while silencing of CISH had the opposite effects. In addition, adenovirus-mediated hepatic CISH overexpression resulted in improved glucose tolerance and decreased gluconeogenesis in WT and leptin receptor-deficient diabetic (db/db) mice. In contrast, adenovirus-mediated hepatic CISH knockdown impaired glucose tolerance and increased gluconeogenesis in WT mice. We also generated liver-specific CISH knockout (LV-CISH KO) mice and discovered that these mice had a similar phenotype in glucose tolerance and gluconeogenesis as mice injected with adenoviruses that knockdown CISH expression. Mechanistically, we found that CISH overexpression decreased and CISH knockdown increased the mRNA and protein levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase 1 (PEPCK), two key enzymes involved in gluconeogenesis, in vitro, and in vivo. Moreover, we discovered that the phosphorylation of cAMP-responsive element binding protein 1 (CREB), a transcription factor of G6pase and Pepck, was required for regulating gluconeogenesis by CISH. Taken together, this study identifies hepatic CISH as an important regulator of gluconeogenesis. Our results also provide important insights into the metabolic functions of the SOCS protein family and the potential targets for the treatment of diabetes.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Gluconeogênese , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Glucose-6-Fosfatase/genética , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.
Assuntos
Glucose-6-Fosfatase , Doença de Depósito de Glicogênio Tipo I , Animais , Humanos , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/química , Glucose-6-Fosfatase/metabolismo , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/metabolismo , Cinética , Glucose/metabolismo , Aminoácidos , Mamíferos/metabolismoRESUMO
Glycogen storage disease type-Ia (GSD-Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). Using the G6pc-R83C mouse model of GSD-Ia, we explored a CRISPR/Cas9-based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end-joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild-type mRNA encoding the native G6Pase-α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with two doses of LNP-dsODN at age 2 and 4 weeks. The G6pc-R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype.