RESUMO
Bariatric surgical procedures such as sleeve gastrectomy (SG) provide effective type 2 diabetes (T2D) remission in human patients. Previous work demonstrated that gastrointestinal levels of the bacterial metabolite lithocholic acid (LCA) are decreased after SG in mice and humans. Here, we show that LCA worsens glucose tolerance and impairs whole-body metabolism. We also show that taurodeoxycholic acid (TDCA), which is the only bile acid whose concentration increases in the murine small intestine post-SG, suppresses the bacterial bile acid-inducible (bai) operon and production of LCA both in vitro and in vivo. Treatment of diet-induced obese mice with TDCA reduces LCA levels and leads to microbiome-dependent improvements in glucose handling. Moreover, TDCA abundance is decreased in small intestinal tissue from T2D patients. This work reveals that TDCA is an endogenous inhibitor of LCA production and suggests that TDCA may contribute to the glucoregulatory effects of bariatric surgery.
Assuntos
Cirurgia Bariátrica , Ácidos e Sais Biliares , Diabetes Mellitus Tipo 2 , Microbioma Gastrointestinal , Intestino Delgado , Camundongos Endogâmicos C57BL , Obesidade , Microbioma Gastrointestinal/efeitos dos fármacos , Animais , Camundongos , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Ácidos e Sais Biliares/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Obesidade/cirurgia , Obesidade/metabolismo , Obesidade/microbiologia , Masculino , Ácido Litocólico/metabolismo , Glucose/metabolismoRESUMO
Recent studies have demonstrated that metabolites produced by commensal bacteria causally influence health and disease. The sulfated metabolome is one class of molecules that has recently come to the forefront due to efforts to understand the role of these metabolites in host-microbiome interactions. Sulfated compounds have canonically been classified as waste products; however, studies have revealed a variety of physiological roles for these metabolites, including effects on host metabolism, immune response and neurological function. Moreover, recent research has revealed that commensal bacteria either chemically modify or synthesize a variety of sulfated compounds. In this Review, we explore how host-microbiome collaborative metabolism transforms the sulfated metabolome. We describe bacterial and mammalian enzymes that sulfonate and desulfate biologically relevant carbohydrates, amino acid derivatives and cholesterol-derived metabolites. We then discuss outstanding questions and future directions in the field, including potential roles of sulfated metabolites in disease detection, prevention and treatment. We hope that this Review inspires future research into sulfated compounds and their effects on physiology.
Assuntos
Microbioma Gastrointestinal , Microbiota , Mamíferos , Metaboloma , SulfatosRESUMO
The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.
Assuntos
Deficiência de Vitamina D , Vitamina D , Humanos , VitaminasRESUMO
Altered host-microbe interactions and increased intestinal permeability have been implicated in disease pathogenesis. However, the mechanisms by which intestinal microbes affect epithelial barrier integrity remain unclear. Here, we investigate the impact of bacterial metabolism of host-produced bile acid (BA) metabolites on epithelial barrier integrity. We observe that rats fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) exhibit reduced intestinal abundance of host-produced conjugated BAs at early time points, coinciding with increased gut permeability. We show that in vitro, conjugated BAs protect gut epithelial monolayers from damage caused by bacterially produced unconjugated BAs through micelle formation. We then demonstrate that inhibition of bacterial BA deconjugation with a small-molecule inhibitor prevents the development of pathologic intestinal permeability and hepatic inflammation in CDAHFD-fed rats. Our study identifies a signaling-independent, physicochemical mechanism for conjugated BA-mediated protection of epithelial barrier function and suggests that rational manipulation of microbial BA metabolism could be leveraged to regulate gut barrier integrity.
Assuntos
Ácidos e Sais Biliares , Microbioma Gastrointestinal , Animais , Fígado , Micelas , Permeabilidade , RatosRESUMO
Human-associated microorganisms play a vital role in human health, and microbial imbalance has been linked to a wide range of disease states. In this Review, we explore recent efforts to progress from correlative studies that identify microorganisms associated with human disease to experiments that establish causal relationships between microbial products and host phenotypes. We propose that successful efforts to uncover phenotypes often follow a chain of evidence that proceeds from (1) association studies; to (2) observations in germ-free animals and antibiotic-treated animals and humans; to (3) fecal microbiota transplants (FMTs); to (4) identification of strains; and then (5) molecules that elicit a phenotype. Using this experimental 'funnel' as our guide, we explore how the microbiota contributes to metabolic disorders and hypertension, infections, and neurological conditions. We discuss the potential to use FMTs and microbiota-inspired therapies to treat human disease as well as the limitations of these approaches.
Assuntos
Doenças Transmissíveis/microbiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Microbiota/fisiologia , Animais , Anti-Infecciosos/farmacologia , Transplante de Microbiota Fecal , Vida Livre de Germes , HumanosRESUMO
Bile acids play crucial roles in host physiology by acting both as detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. Structure-activity relationship (SAR) studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in mouse and human fecal slurry, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo.
Assuntos
Amidoidrolases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Ácido Litocólico/análogos & derivados , Ácido Litocólico/farmacologia , Animais , Bactérias/efeitos dos fármacos , Ácidos e Sais Biliares/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/toxicidade , Fezes/química , Fezes/enzimologia , Humanos , Ácido Litocólico/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The activation of the Takeda G-protein receptor 5 (TGR5, also known as the G protein-coupled bile acid receptor 1, GPBAR1) in enteroendocrine L-cells results in secretion of the anti-diabetic hormone Glucagon-Like Peptide 1 (GLP-1) into systemic circulation. Consequently, recent research has focused on identification and development of TGR5 agonists as type 2 diabetes therapeutics. However, the clinical application of TGR5 agonists has been hampered by side effects of these compounds that primarily result from their absorption into circulation. Here we describe an in vitro screening protocol to evaluate the TGR5 agonism, GLP-1 secretion, and gut-restricted properties of small molecules. The protocol involves differentiating gut epithelial and endocrine cells together in transwells to assess both the pharmacodynamics of TGR5 agonists and the toxicity of compounds to the intestinal monolayer. As a proof of concept, we demonstrate the use of the protocol in evaluating properties of naturally occurring bile acid metabolites that are potent TGR5 agonists. This protocol is adapted from Chaudhari et al. (2021).
RESUMO
Bariatric surgery is the most effective treatment for type 2 diabetes and is associated with changes in gut metabolites. Previous work uncovered a gut-restricted TGR5 agonist with anti-diabetic properties-cholic acid-7-sulfate (CA7S)-that is elevated following sleeve gastrectomy (SG). Here, we elucidate a microbiome-dependent pathway by which SG increases CA7S production. We show that a microbial metabolite, lithocholic acid (LCA), is increased in murine portal veins post-SG and by activating the vitamin D receptor, induces hepatic mSult2A1/hSULT2A expression to drive CA7S production. An SG-induced shift in the microbiome increases gut expression of the bile acid transporters Asbt and Ostα, which in turn facilitate selective transport of LCA across the gut epithelium. Cecal microbiota transplant from SG animals is sufficient to recreate the pathway in germ-free (GF) animals. Activation of this gut-liver pathway leads to CA7S synthesis and GLP-1 secretion, causally connecting a microbial metabolite with the improvement of diabetic phenotypes.
Assuntos
Cirurgia Bariátrica , Microbioma Gastrointestinal/fisiologia , Fígado/metabolismo , Animais , Diabetes Mellitus Tipo 2 , Gastrectomia , Vida Livre de Germes , Peptídeo 1 Semelhante ao Glucagon , Células Hep G2 , Humanos , Íleo/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Calcitriol/genética , Sulfotransferases/metabolismoRESUMO
Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is associated with increased levels of the incretin hormone glucagon-like peptide-1 (GLP-1) and changes in levels of circulating bile acids. The levels of individual bile acids in the gastrointestinal (GI) tract after surgery have, however, remained largely unstudied. Using ultra-high performance liquid chromatography-mass spectrometry-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a Takeda G-protein receptor 5 (TGR5) agonist that increases Tgr5 expression and induces GLP-1 secretion. Furthermore, CA7S administration increases glucose tolerance in insulin-resistant mice in a TGR5-dependent manner. CA7S remains gut restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into the circulation. By studying changes in individual metabolites after surgery, the present study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.
Assuntos
Fármacos Antiobesidade/farmacologia , Cirurgia Bariátrica/métodos , Ácido Cólico/farmacologia , Obesidade/cirurgia , Receptores Acoplados a Proteínas G/genética , Animais , Fármacos Antiobesidade/metabolismo , Bile/química , Bile/metabolismo , Células CACO-2 , Ácido Cólico/biossíntese , Colo/metabolismo , Regulação da Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Teste de Tolerância a Glucose , Células HEK293 , Humanos , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , SulfatosRESUMO
Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.
Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Microbioma Gastrointestinal/fisiologia , Amidoidrolases/fisiologia , Animais , Bactérias/enzimologia , Ácidos e Sais Biliares/metabolismo , Desenho de Fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bibliotecas de Moléculas PequenasRESUMO
Fused, elongated mitochondria are more efficient in generating ATP than fragmented mitochondria. In diverse C. elegans longevity pathways, increased levels of fused mitochondria are associated with lifespan extension. Blocking mitochondrial fusion in these animals abolishes their extended longevity. The long-lived C. elegans vhl-1 mutant is an exception that does not have increased fused mitochondria, and is not dependent on fusion for longevity. Loss of mammalian VHL upregulates alternate energy generating pathways. This suggests that mitochondrial fusion facilitates longevity in C. elegans by increasing energy metabolism. In diverse animals, ATP levels broadly decreases with age. Substantial evidence supports the theory that increasing or maintaining energy metabolism promotes the survival of older animals. Increased ATP levels in older animals allow energy-intensive repair and homeostatic mechanisms such as proteostasis that act to prevent cellular aging. These observations support the emerging paradigm that maintaining energy metabolism promotes the survival of older animals.
Assuntos
Caenorhabditis elegans/fisiologia , Metabolismo Energético , Longevidade/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Proteínas Culina/genética , Mitocôndrias/metabolismo , Dinâmica MitocondrialRESUMO
Dafachronic acid (DA) is a bile acid-like steroid hormone that regulates dauer formation, heterochrony, and lifespan in C. elegans. Here, we describe that DA is an inhibitor of C. elegans germ stem cell proliferation in adult hermaphrodites. Using a C. elegans germ cell primary culture system, we show that DA inhibits the proliferation of germ cells in vitro. Exogenous DA reduces the frequency of large tumors in adult tumorous germline mutants and decreases the proliferation of wild-type germ stem cells in adult hermaphrodites. In contrast, DA has no appreciable effect on the proliferation of larval-stage germ cells in wild type. The inhibition of adult germ cell proliferation by DA requires its canonical receptor DAF-12. Blocking DA production by inactivating the cytochrome P450 DAF-9 increases germ cell proliferation in wild-type adult hermaphrodites and the frequency of large tumors in germline tumorous mutants, suggesting that DA inhibits the rate of germ cell proliferation under normal growth conditions.
Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Colestenos/metabolismo , Células-Tronco Germinativas Adultas/citologia , Animais , Ácidos e Sais Biliares , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proliferação de Células/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de SinaisRESUMO
The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016.
RESUMO
Mitochondria are dynamic organelles that undergo fusion and fission events. Mitochondrial dynamics are required for mitochondrial viability and for responses to changes in bioenergetic status. Here we describe an insulin-signaling and SCFLIN-23-regulated pathway that controls mitochondrial fusion in Caenorhabditis elegans by repressing the expression of the mitochondrial proteases SPG-7 and PPGN-1. This pathway is required for mitochondrial fusion in response to physical exertion, and for the associated extension in lifespan. We show that diverse longevity pathways exhibit increased levels of elongated mitochondria. The increased mitochondrial fusion is essential for longevity in the diverse longevity pathways, as inhibiting mitochondrial fusion reduces their lifespans to wild-type levels. Our results suggest that increased mitochondrial fusion is not a major driver of longevity, but rather is essential to allow the survival of older animals beyond their normal lifespan in diverse longevity pathways.Mitochondria can undergo shape changes as a result of fusion and fission events. Here the authors describe how insulin signalling regulates mitochondrial fusion in C. elegans, and show that mitochondrial fusion is necessary, but not sufficient, for longevity of worms with mutations that increase lifespan.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Longevidade , Dinâmica Mitocondrial , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Caenorhabditis elegans , Metabolismo Energético , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Here we describe an in vitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Proliferação de Células , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Células Germinativas/citologia , Células-Tronco/citologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Escherichia coli/citologia , Transportadores de Ácido Fólico/genética , Transportadores de Ácido Fólico/metabolismo , Células Germinativas/metabolismo , Células-Tronco/metabolismoRESUMO
Structured illumination microscopy provides twice the linear resolution of conventional fluorescence microscopy, but in thick samples, aberrations degrade the performance and limit the resolution. Here, we demonstrate structured illumination microscopy through 35 µm of tissue using adaptive optics (AO) to correct aberrations resulting in images with a resolution of 140 nm. We report a 60% minimum improvement in the signal-to-noise ratio of the structured illumination reconstruction through thick tissue by correction with AO.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Animais , Caenorhabditis elegans/química , Desenho de Equipamento , Microesferas , Razão Sinal-RuídoRESUMO
BACKGROUND: Humanized mice able to reconstitute a surrogate human immune system (HIS) can be used for studies on human immunology and may provide a predictive preclinical model for human vaccines prior to clinical trials. However, current humanized mouse models show sub-optimal human T cell reconstitution and limited ability to support immunoglobulin class switching by human B cells. This limitation has been attributed to the lack of expression of Human Leukocyte Antigens (HLA) molecules in mouse lymphoid organs. Recently, humanized mice expressing HLA class I molecules have been generated but showed little improvement in human T cell reconstitution and function of T and B cells. METHODS: We have generated NOD.Rag1KO.IL2RγcKO mice expressing HLA class II (HLA-DR4) molecules under the I-E(d) promoter that were infused as adults with HLA-DR-matched human hematopoietic stem cells (HSC). Littermates lacking expression of HLA-DR4 molecules were used as control. RESULTS: HSC-infused HLA-DR4.NOD.Rag1KO.IL-2RγcKO mice developed a very high reconstitution rate (>90%) with long-lived and functional human T and B cells. Unlike previous humanized mouse models reported in the literature and our control mice, the HLA-DR4 expressing mice reconstituted serum levels (natural antibodies) of human IgM, IgG (all four subclasses), IgA, and IgE comparable to humans, and elicited high titers of specific human IgG antibodies upon tetanus toxoid vaccination. CONCLUSIONS: Our study demonstrates the critical role of HLA class II molecules for development of functional human T cells able to support immunoglobulin class switching and efficiently respond to vaccination.