RESUMO
Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Microbioma Gastrointestinal/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Agmatina/metabolismo , Animais , Caenorhabditis elegans/microbiologia , Proteína Receptora de AMP Cíclico , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Humanos , Hipoglicemiantes/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Metformina/farmacologia , Nutrientes/metabolismoRESUMO
Individuals infected with human immunodeficiency virus type-1 (HIV-1) show metabolic alterations of CD4+ T cells through unclear mechanisms with undefined consequences. We analyzed the transcriptome of CD4+ T cells from patients with HIV-1 and revealed that the elevated oxidative phosphorylation (OXPHOS) pathway is associated with poor outcomes. Inhibition of OXPHOS by the US Food and Drug Administration-approved drug metformin, which targets mitochondrial respiratory chain complex-I, suppresses HIV-1 replication in human CD4+ T cells and humanized mice. In patients, HIV-1 peak viremia positively correlates with the expression of NLRX1, a mitochondrial innate immune receptor. Quantitative proteomics and metabolic analyses reveal that NLRX1 enhances OXPHOS and glycolysis during HIV-1-infection of CD4+ T cells to promote viral replication. At the mechanistic level, HIV infection induces the association of NLRX1 with the mitochondrial protein FASTKD5 to promote expression of mitochondrial respiratory complex components. This study uncovers the OXPHOS pathway in CD4+ T cells as a target for HIV-1 therapy.
Assuntos
Linfócitos T CD4-Positivos/virologia , Genômica , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Metaboloma , Metabolômica , Fosforilação Oxidativa , Proteoma , Transcriptoma , Replicação Viral , Animais , Antivirais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/imunologia , HIV-1/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Células Jurkat , Masculino , Metformina/farmacologia , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteômica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Carga Viral , Replicação Viral/efeitos dos fármacosRESUMO
We report here a simple and global strategy to map out gene functions and target pathways of drugs, toxins, or other small molecules based on "homomer dynamics" protein-fragment complementation assays (hdPCA). hdPCA measures changes in self-association (homomerization) of over 3,500 yeast proteins in yeast grown under different conditions. hdPCA complements genetic interaction measurements while eliminating the confounding effects of gene ablation. We demonstrate that hdPCA accurately predicts the effects of two longevity and health span-affecting drugs, the immunosuppressant rapamycin and the type 2 diabetes drug metformin, on cellular pathways. We also discovered an unsuspected global cellular response to metformin that resembles iron deficiency and includes a change in protein-bound iron levels. This discovery opens a new avenue to investigate molecular mechanisms for the prevention or treatment of diabetes, cancers, and other chronic diseases of aging.
Assuntos
Ferro/metabolismo , Metaloproteínas/metabolismo , Metformina/farmacologia , Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Teste de Complementação Genética , Humanos , Metaloproteínas/genética , Saccharomyces cerevisiae/genéticaRESUMO
Several metabolic alterations accumulate over time along with a reduction in biological fitness, suggesting the existence of a "metabolic clock" that controls aging. Multiple inborn defects in metabolic circuitries accelerate aging, whereas genetic loci linked to exceptional longevity influence metabolism. Each of the nine hallmarks of aging is connected to undesirable metabolic alterations. The main features of the "westernized" lifestyle, including hypercaloric nutrition and sedentariness, can accelerate aging as they have detrimental metabolic consequences. Conversely, lifespan-extending maneuvers including caloric restriction impose beneficial pleiotropic effects on metabolism. The introduction of strategies that promote metabolic fitness may extend healthspan in humans.
Assuntos
Envelhecimento/metabolismo , Longevidade , Envelhecimento/sangue , Animais , Restrição Calórica , Senescência Celular , Dieta , Dieta Ocidental , Exercício Físico , Humanos , Estilo de Vida , Metformina/administração & dosagem , Mitocôndrias/metabolismo , Estresse FisiológicoRESUMO
In this issue of Cell, Wu et al. employed C. elegans and human cell experiments to identify a pathway through which metformin increases lifespan and inhibits growth. A key transcriptional target, ACAD10, is activated when metformin induces nuclear exclusion of the GTPase RagC, thereby inhibiting mTORC1 through an unexpected mechanism.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Metformina/farmacologia , Transporte Ativo do Núcleo Celular , Envelhecimento/efeitos dos fármacos , Animais , Humanos , Neoplasias/tratamento farmacológicoRESUMO
Metformin has utility in cancer prevention and treatment, though the mechanisms for these effects remain elusive. Through genetic screening in C. elegans, we uncover two metformin response elements: the nuclear pore complex (NPC) and acyl-CoA dehydrogenase family member-10 (ACAD10). We demonstrate that biguanides inhibit growth by inhibiting mitochondrial respiratory capacity, which restrains transit of the RagA-RagC GTPase heterodimer through the NPC. Nuclear exclusion renders RagC incapable of gaining the GDP-bound state necessary to stimulate mTORC1. Biguanide-induced inactivation of mTORC1 subsequently inhibits growth through transcriptional induction of ACAD10. This ancient metformin response pathway is conserved from worms to humans. Both restricted nuclear pore transit and upregulation of ACAD10 are required for biguanides to reduce viability in melanoma and pancreatic cancer cells, and to extend C. elegans lifespan. This pathway provides a unified mechanism by which metformin kills cancer cells and extends lifespan, and illuminates potential cancer targets. PAPERCLIP.
Assuntos
Metformina/farmacologia , Acil-CoA Desidrogenase/genética , Envelhecimento , Animais , Tamanho Corporal , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Longevidade , Alvo Mecanístico do Complexo 1 de Rapamicina , Mitocôndrias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Neoplasias/tratamento farmacológico , Poro Nuclear/metabolismo , Fosforilação Oxidativa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1ß production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.
Assuntos
Trifosfato de Adenosina/metabolismo , DNA Mitocondrial/biossíntese , Inflamassomos/efeitos dos fármacos , Metformina/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia/prevenção & controle , Animais , COVID-19/metabolismo , COVID-19/prevenção & controle , Citocinas/genética , Citocinas/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Metformina/uso terapêutico , Camundongos , Núcleosídeo-Fosfato Quinase/metabolismo , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/prevenção & controle , SARS-CoV-2/patogenicidadeRESUMO
Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.
Assuntos
Plasticidade Celular , Cobre , Inflamação , Transdução de Sinais , Animais , Camundongos , Cobre/metabolismo , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , NAD/metabolismo , Transdução de Sinais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Peróxido de Hidrogênio/metabolismo , Epigênese Genética/efeitos dos fármacos , Metformina/análogos & derivados , Oxirredução , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/genética , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/genéticaRESUMO
The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.
Assuntos
Proteína 5 Relacionada à Autofagia/genética , Proteína de Domínio de Morte Associada a Fas/genética , Intestino Grosso/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Animais Recém-Nascidos , Proteína 5 Relacionada à Autofagia/deficiência , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular/genética , Proteína de Domínio de Morte Associada a Fas/deficiência , Regulação da Expressão Gênica , Glucose/antagonistas & inibidores , Glucose/farmacologia , Células HEK293 , Células HT29 , Humanos , Intestino Grosso/efeitos dos fármacos , Intestino Grosso/patologia , Células Jurkat , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/antagonistas & inibidores , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Transdução de Sinais , Sirolimo/farmacologia , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoAssuntos
Modelos Animais de Doenças , Disbiose/imunologia , Microbioma Gastrointestinal/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Congressos como Assunto , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos , Disbiose/microbiologia , Regulação da Expressão Gênica/imunologia , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/microbiologia , Metformina/uso terapêutico , Camundongos , RNA-Seq , Análise de Célula Única , Ubiquinona/análogos & derivados , Ubiquinona/uso terapêutico , Comunicação por VideoconferênciaRESUMO
The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy-and also to its side effects, which include folate deficiency and gastrointestinal upset.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Ácido Fólico/metabolismo , Hipoglicemiantes/farmacologia , Longevidade/efeitos dos fármacos , Metformina/farmacologia , Metionina/metabolismo , Adenilato Quinase/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Biguanidas/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Restrição Calórica , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Escherichia coli/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Metagenoma , Metformina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.
Assuntos
Hipoglicemiantes , Metformina , ATPases Vacuolares Próton-Translocadoras , Proteínas Quinases Ativadas por AMP/metabolismo , Adenosina Trifosfatases/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Caenorhabditis elegans/metabolismo , Diabetes Mellitus/tratamento farmacológico , Glucose/metabolismo , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Lisossomos/metabolismo , Proteínas de Membrana , Metformina/agonistas , Metformina/metabolismo , Metformina/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Metabolismo Energético , Galectinas/metabolismo , Lisossomos/enzimologia , Ubiquitina/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Adolescente , Adulto , Animais , Autofagia/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática , Feminino , Galectinas/genética , Células HEK293 , Células HeLa , Humanos , Hipoglicemiantes/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/microbiologia , Lisossomos/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Metformina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/patogenicidade , Transdução de Sinais , Células THP-1 , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Adulto JovemRESUMO
Despite being the frontline therapy for type 2 diabetes, the mechanisms of action of the biguanide drug metformin are still being discovered. In particular, the detailed molecular interplays between the AMPK and the mTORC1 pathway in the hepatic benefits of metformin are still ill defined. Metformin-dependent activation of AMPK classically inhibits mTORC1 via TSC/RHEB, but several lines of evidence suggest additional mechanisms at play in metformin inhibition of mTORC1. Here we investigated the role of direct AMPK-mediated serine phosphorylation of RAPTOR in a new RaptorAA mouse model, in which AMPK phospho-serine sites Ser722 and Ser792 of RAPTOR were mutated to alanine. Metformin treatment of primary hepatocytes and intact murine liver requires AMPK regulation of both RAPTOR and TSC2 to fully inhibit mTORC1, and this regulation is critical for both the translational and transcriptional response to metformin. Transcriptionally, AMPK and mTORC1 were both important for regulation of anabolic metabolism and inflammatory programs triggered by metformin treatment. The hepatic transcriptional response in mice on high-fat diet treated with metformin was largely ablated by AMPK deficiency under the conditions examined, indicating the essential role of this kinase and its targets in metformin action in vivo.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Metformina/farmacologia , Proteína Regulatória Associada a mTOR/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Genótipo , Hipoglicemiantes/farmacologia , Inflamação , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metabolismo/efeitos dos fármacos , Metformina/uso terapêutico , Camundongos , Fosforilação/efeitos dos fármacos , Proteína Regulatória Associada a mTOR/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmA and mfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103 M-1s-1 and KM for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.
Assuntos
Diabetes Mellitus Tipo 2 , Guanidina/análogos & derivados , Metformina , Microbiota , Ureia/análogos & derivados , Humanos , Metformina/metabolismo , Águas Residuárias , Níquel , Hidrolases/genética , Preparações FarmacêuticasRESUMO
Fragile X syndrome (FXS) is the most common genetic cause of autism spectrum disorder engendered by transcriptional silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Given the early onset of behavioral and molecular changes, it is imperative to know the optimal timing for therapeutic intervention. Case reports documented benefits of metformin treatment in FXS children between 2 and 14 y old. In this study, we administered metformin from birth to Fmr1-/y mice which corrected up-regulated mitogen-2 activated protein kinase/extracellular signal-regulated kinase and mammalian/mechanistic target of rapamycin complex 1 signaling pathways and specific synaptic mRNA-binding targets of FMRP. Metformin rescued increased number of calls in ultrasonic vocalization and repetitive behavior in Fmr1-/y mice. Our findings demonstrate that in mice, early-in-life metformin intervention is effective in treating FXS pathophysiology.
Assuntos
Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Metformina , Metformina/farmacologia , Metformina/uso terapêutico , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Síndrome do Cromossomo X Frágil/metabolismo , Animais , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Camundongos , Masculino , Camundongos Knockout , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Modelos Animais de Doenças , Transdução de Sinais/efeitos dos fármacosRESUMO
Tumor blood vessels are highly leaky in structure and have poor blood perfusion, which hampers infiltration and function of CD8T cells within tumor. Normalizing tumor vessels is thus thought to be important in promoting the flux of immune T cells and enhancing ant-tumor immunity. However, how tumor vasculature is normalized is poorly understood. Metformin (Met) combined with ant-PD-1 therapy is known to stimulate proliferation of and to produce large amounts of IFNγ from tumor-infiltrating CD8T lymphocytes (CD8TILs). We found that the combination therapy promotes the pericyte coverage of tumor vascular endothelial cells (ECs) to improve blood perfusion and that it suppresses the hyperpermeability through the increase of VE-cadherin. Peripheral node addressin(PNAd) and vascular cell adhesion molecule (VCAM)-1, both implicated to promote tumor infiltration of CD8T cells, were also increased. Importantly, tumor vessel normalization, characterized as the reduced 70-kDa dextran leakage and the enhancement of VE-cadherin and VCAM-1, were canceled by anti-CD8 Ab or anti-IFNγ Ab injection to mice. The increased CD8TILs were also abrogated by anti-IFNγ Ab injection. In vascular ECs, flow cytometry analysis revealed that pSTAT1 expression was found to be associated with VE-cadherin expression. Moreover, in vitro treatment with Met and IFNγ enhanced VE-cadherin and VCAM-1 on human umbilical vein endothelial cells (HUVECs). The Kaplan-Meier method revealed a correlation of VE-cadherin or VCAM-1 levels with overall survival in patients treated with immune checkpoint inhibitors. These data indicate that IFNγ-mediated cross talk of CD8TILs with tumor vessels is important for creating a better tumor microenvironment and maintaining sustained antitumor immunity.
Assuntos
Linfócitos T CD8-Positivos , Interferon gama , Metformina , Receptor de Morte Celular Programada 1 , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Animais , Interferon gama/metabolismo , Camundongos , Metformina/farmacologia , Metformina/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Linhagem Celular Tumoral , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Camundongos Endogâmicos C57BL , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Caderinas/metabolismo , Antígenos CD/metabolismo , Sinergismo FarmacológicoRESUMO
Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.
Assuntos
Peso Corporal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/metabolismo , Metformina/farmacologia , Administração Oral , Adulto , Idoso , Animais , Glicemia/análise , Glicemia/metabolismo , Dieta Hiperlipídica , Método Duplo-Cego , Ingestão de Energia/efeitos dos fármacos , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Feminino , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/antagonistas & inibidores , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/deficiência , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/sangue , Fator 15 de Diferenciação de Crescimento/deficiência , Fator 15 de Diferenciação de Crescimento/genética , Homeostase/efeitos dos fármacos , Humanos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Masculino , Metformina/administração & dosagem , Camundongos , Camundongos Obesos , Pessoa de Meia-Idade , Redução de Peso/efeitos dos fármacosRESUMO
Endoplasmic reticulum-associated degradation (ERAD) plays a crucial role in turnover of defective secretory proteins to maintain protein homeostasis. Cha et al. (2018) revealed that the anti-diabetic drug metformin induces ERAD of programmed death ligand (PD-L1), which attenuates tumor growth.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Metformina , Animais , Antígeno B7-H1 , Retículo Endoplasmático , Proteínas/genéticaRESUMO
Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.