RESUMEN
Blood plasma is one of the most commonly analyzed and easily accessible biological samples. Here, we describe an automated liquid-liquid extraction (LLE) platform that generates accurate, precise, and reproducible samples for metabolomic, lipidomic, and proteomic analyses from a single aliquot of plasma while minimizing hands-on time and avoiding contamination from plasticware. We applied mass spectrometry to examine the metabolome, lipidome, and proteome of 90 plasma samples to determine the effects of age, time of day, and a high-fat diet in mice. From 25 µL of mouse plasma, we identified 907 lipid species from 16 different lipid classes and subclasses, 233 polar metabolites, and 344 proteins. We found that the high-fat diet induced only mild changes in the polar metabolome, upregulated Apolipoproteins, and induced substantial shifts in the lipidome, including a significant increase in arachidonic acid (AA) and a decrease in eicosapentaenoic acid (EPA) content across all lipid classes.
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Systemic lupus erythematosus is a common autoimmune inflammatory disease which is associated with increases in autoantibodies and immune complexes that deposit in the kidney. The MRL-lpr mouse is a common mouse model used for the study of lupus and immune complex glomerulonephritis but very little is known about the plasma proteome changes in this model. We performed in-depth quantitative proteome profiling on MRL-lpr and control (strain MpJ) mice to investigate the changes in the proteome, immunoglobulins and their glycoproteome as well as protein and immune complexes. Methodologies used included immunohistochemistry, immunoglobulin isotyping, multiplexed proteome profiling, immunoglobulin immunoprecipitation with glycoproteome profiling, and size exclusion chromatography (SEC) profiling to enable a comprehensive proteome profiling of proteins and protein complexes. We also used a novel native multiplexed plasma proteome profiling (NativeMP3) method that relies on native enrichment of plasma proteins enabling ultra-deep single shot profiling where we identified 922 plasma proteins at 1% false discovery rate (FDR) in a single shot mass spectrometry run. We observed many large plasma protein differences between the MRL-lpr and control strain including differences in the immunoglobulins, immunoglobulins against specific antigens, chemokines, and proteases as well as changes in protein complexes such as the immunoproteasome.
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Enfermedades Autoinmunes , Enfermedades del Complejo Inmune , Ratones , Animales , Ratones Endogámicos MRL lpr , Complejo Antígeno-Anticuerpo , Proteómica , Proteoma , Autoanticuerpos , Modelos Animales de Enfermedad , Péptido HidrolasasRESUMEN
The connection between an altered gut microbiota and metabolic disorders such as obesity, diabetes, and cardiovascular disease is well established. Defects in preserving the integrity of the mucosal barriers can result in systemic endotoxaemia that contributes to chronic low-grade inflammation, which further promotes the development of metabolic syndrome. Interleukin (IL)-22 exerts essential roles in eliciting antimicrobial immunity and maintaining mucosal barrier integrity within the intestine. Here we investigate the connection between IL-22 and metabolic disorders. We find that the induction of IL-22 from innate lymphoid cells and CD4(+) T cells is impaired in obese mice under various immune challenges, especially in the colon during infection with Citrobacter rodentium. While innate lymphoid cell populations are largely intact in obese mice, the upregulation of IL-23, a cytokine upstream of IL-22, is compromised during the infection. Consequently, these mice are susceptible to C. rodentium infection, and both exogenous IL-22 and IL-23 are able to restore the mucosal host defence. Importantly, we further unveil unexpected functions of IL-22 in regulating metabolism. Mice deficient in IL-22 receptor and fed with high-fat diet are prone to developing metabolic disorders. Strikingly, administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. IL-22 shows diverse metabolic benefits, as it improves insulin sensitivity, preserves gut mucosal barrier and endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues. In summary, we identify the IL-22 pathway as a novel target for therapeutic intervention in metabolic diseases.
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Diabetes Mellitus/inmunología , Diabetes Mellitus/metabolismo , Inmunidad Mucosa , Interleucinas/inmunología , Interleucinas/metabolismo , Enfermedades Metabólicas/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Crónica , Citrobacter rodentium/efectos de los fármacos , Citrobacter rodentium/inmunología , Citrobacter rodentium/fisiología , Colon/efectos de los fármacos , Colon/inmunología , Colon/microbiología , Diabetes Mellitus/patología , Dieta Alta en Grasa , Femenino , Hiperglucemia/dietoterapia , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Inmunidad Mucosa/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Insulina/metabolismo , Resistencia a la Insulina , Interleucina-23/inmunología , Interleucina-23/metabolismo , Interleucina-23/farmacología , Interleucinas/farmacología , Interleucinas/uso terapéutico , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Enfermedades Metabólicas/dietoterapia , Enfermedades Metabólicas/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/metabolismo , Receptores de Interleucina/deficiencia , Receptores de Interleucina/metabolismo , Receptores de Leptina/deficiencia , Receptores de Leptina/metabolismo , Interleucina-22RESUMEN
FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.
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Factores de Crecimiento de Fibroblastos/metabolismo , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Endopeptidasas , Factores de Crecimiento de Fibroblastos/química , Gelatinasas/genética , Eliminación de Gen , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genéticaRESUMEN
BACKGROUND: Platelet-derived growth factor C (PDGF-C) has been reported to promote angiogenesis independently of vascular endothelial growth factor (VEGF), although its significance in postnatal angiogenesis in vivo remains poorly understood. VEGF has been employed as a major molecular tool to induce therapeutic angiogenesis. However, VEGF therapy is not very effective in models of cardiovascular diseases associated with diabetes, and the mechanisms of this phenomenon still remain to be elucidated. METHODS: We used a murine model of hind limb ischemia and of streptozotocin-induced diabetes. RESULTS: Expression of PDGF-C and its receptor PDGFR-α were markedly upregulated in ischemic limbs. Treatment with a neutralizing antibody against PDGF-C significantly impaired blood flow recovery and neovascularization after ischemia almost to the same extent as a VEGF-neutralizing antibody. Mice deficient in PDGF-C exhibited reduced blood flow recovery after ischemia compared with wild-type mice, confirming a strong proangiogenic activity of PDGF-C. Next, we injected an expression vector encoding PDGF-C into ischemic limbs. Blood flow recovery and neovascularization after ischemia were significantly improved in the groups treated with PDGF-C compared with controls. Attenuation of angiogenic responses to ischemia has been reported in patients with diabetes even after VEGF treatment, although a precise mechanism remains unknown. We hypothesized that PDGF-C might relate to the impaired angiogenesis of diabetes. We tested this hypothesis by inducing diabetes by intraperitoneal injection of streptozotocin. Expression levels of PDGF-C at baseline and after ischemia were significantly lower in limb tissues of diabetic mice than in those of control mice, whereas expression levels of other members of the PDGF family and VEGF were not changed or were even higher in diabetic mice. Introduction of VEGF complementary DNA expression plasmid vector into ischemic limbs did not improve blood flow recovery. However, these changes were effectively reversed by additional introduction of the PDGF-C complementary DNA plasmid vector. CONCLUSIONS: These results indicate that downregulation of PDGF-C expression in limb tissues of diabetic mice contributes to impaired angiogenesis and suggest that introduction of PDGF-C might be a novel strategy for therapeutic angiogenesis, especially in the diabetic state. CLINICAL RELEVANCE: Angiogenesis and arteriogenesis after ischemia are attenuated in most diabetic patients, although the precise mechanisms remain unclear. Platelet-derived growth factors (PDGFs) have a variety of functions on many cell types, and PDGF-C stimulates angiogenesis and revascularizes ischemic tissues. This study indicates the role for PDGF-C as a critical regulator of impaired angiogenesis of diabetes and suggests that PDGF-C might be a novel target for the treatment of ischemic cardiovascular diseases in diabetes.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Angiopatías Diabéticas/metabolismo , Isquemia/metabolismo , Linfocinas/metabolismo , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Anticuerpos Neutralizantes/administración & dosificación , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatología , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/fisiopatología , Angiopatías Diabéticas/terapia , Técnicas de Transferencia de Gen , Miembro Posterior , Isquemia/genética , Isquemia/fisiopatología , Isquemia/terapia , Linfocinas/antagonistas & inhibidores , Linfocinas/deficiencia , Linfocinas/genética , Linfocinas/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropilina-1/metabolismo , Neuropilina-2/metabolismo , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/deficiencia , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/inmunología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Recuperación de la Función , Flujo Sanguíneo Regional , Transducción de Señal , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.
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Colesterol/sangre , Furina/metabolismo , Proproteína Convertasas/metabolismo , Proteolisis , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Anticuerpos Monoclonales de Origen Murino/química , Colesterol/genética , Furina/genética , Células Hep G2 , Humanos , Hígado/metabolismo , Ratones , Ratones Noqueados , Proproteína Convertasa 9 , Proproteína Convertasas/genética , Estructura Secundaria de Proteína , Receptores de LDL/genética , Serina Endopeptidasas/genéticaRESUMEN
Immunological targeting of pathological cells has been successful in oncology and is expanding to other pathobiological contexts. Here, we present a flexible platform that allows labeling cells of interest with the surface-expressed model antigen ovalbumin (OVA), which can be eliminated via either antigen-specific T cells or newly developed OVA antibodies. We demonstrate that hepatocytes can be effectively targeted by either modality. In contrast, pro-fibrotic fibroblasts associated with pulmonary fibrosis are only eliminated by T cells in initial experiments, which reduced collagen deposition in a fibrosis model. This new experimental platform will facilitate development of immune-based approaches to clear potential pathological cell types in vivo.
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Anticuerpos , Fibrosis Pulmonar , Humanos , Fibroblastos , Hepatocitos , CinéticaRESUMEN
We present a method to infer the 3D pose of mice, including the limbs and feet, from monocular videos. Many human clinical conditions and their corresponding animal models result in abnormal motion, and accurately measuring 3D motion at scale offers insights into health. The 3D poses improve classification of health-related attributes over 2D representations. The inferred poses are accurate enough to estimate stride length even when the feet are mostly occluded. This method could be applied as part of a continuous monitoring system to non-invasively measure animal health, as demonstrated by its use in successfully classifying animals based on age and genotype. We introduce the Mouse Pose Analysis Dataset, the first large scale video dataset of lab mice in their home cage with ground truth keypoint and behavior labels. The dataset also contains high resolution mouse CT scans, which we use to build the shape models for 3D pose reconstruction.
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Modelos Animales , Grabación en Video , Animales , Ratones , Extremidades , Pie , GenotipoRESUMEN
Partial pluripotent reprogramming can reverse features of aging in mammalian cells, but the impact on somatic identity and the necessity of individual reprogramming factors remain unknown. Here, we used single-cell genomics to map the identity trajectory induced by partial reprogramming in multiple murine cell types and dissected the influence of each factor by screening all Yamanaka Factor subsets with pooled single-cell screens. We found that partial reprogramming restored youthful expression in adipogenic and mesenchymal stem cells but also temporarily suppressed somatic identity programs. Our pooled screens revealed that many subsets of the Yamanaka Factors both restore youthful expression and suppress somatic identity, but these effects were not tightly entangled. We also found that a multipotent reprogramming strategy inspired by amphibian regeneration restored youthful expression in myogenic cells. Our results suggest that various sets of reprogramming factors can restore youthful expression with varying degrees of somatic identity suppression. A record of this paper's Transparent Peer Review process is included in the supplemental information.
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Envejecimiento , Reprogramación Celular , Animales , Reprogramación Celular/genética , Expresión Génica , Mamíferos/genética , RatonesRESUMEN
Nicotinamide adenine dinucleotide (NAD) levels decline during aging, contributing to physical and metabolic dysfunction. The NADase CD38 plays a key role in age-related NAD decline. Whether the inhibition of CD38 increases lifespan is not known. Here, we show that the CD38 inhibitor 78c increases lifespan and healthspan of naturally aged mice. In addition to a 10% increase in median survival, 78c improved exercise performance, endurance, and metabolic function in mice. The effects of 78c were different between sexes. Our study is the first to investigate the effect of CD38 inhibition in naturally aged animals.
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Longevidad , NAD , ADP-Ribosil Ciclasa 1/metabolismo , Envejecimiento/metabolismo , Animales , Ratones , NAD/metabolismo , NAD+ Nucleosidasa/metabolismoRESUMEN
Pregnancy-associated plasma protein-A (PAPP-A) is a secreted metalloprotease that increases insulin-like growth factor (IGF) availability by cleaving IGF-binding proteins. Reduced IGF signaling extends longevity in multiple species, and consistent with this, PAPP-A deletion extends lifespan and healthspan; however, the mechanism remains unclear. To clarify PAPP-A's role, we developed a PAPP-A neutralizing antibody and treated adult mice with it. Transcriptomic profiling across tissues showed that anti-PAPP-A reduced IGF signaling and extracellular matrix (ECM) gene expression system wide. The greatest reduction in IGF signaling occurred in the bone marrow, where we found reduced bone, marrow adiposity, and myelopoiesis. These diverse effects led us to search for unifying mechanisms. We identified mesenchymal stromal cells (MSCs) as the source of PAPP-A in bone marrow and primary responders to PAPP-A inhibition. Mice treated with anti-PAPP-A had reduced IGF signaling in MSCs and dramatically decreased MSC number. As MSCs are (1) a major source of ECM and the progenitors of ECM-producing fibroblasts, (2) the originating source of adult bone, (3) regulators of marrow adiposity, and (4) an essential component of the hematopoietic niche, our data suggest that PAPP-A modulates bone marrow homeostasis by potentiating the number and activity of MSCs. We found that MSC-like cells are the major source of PAPP-A in other tissues also, suggesting that reduced MSC-like cell activity drives the system-wide reduction in ECM gene expression due to PAPP-A inhibition. Dysregulated ECM production is associated with aging and drives age-related diseases, and thus, this may be a mechanism by which PAPP-A deficiency enhances longevity.
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Homeostasis , Longevidad , Células Madre Mesenquimatosas/metabolismo , Proteína Plasmática A Asociada al Embarazo/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/metabolismo , Médula Ósea/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Mielopoyesis , Osteoblastos/metabolismo , Osteogénesis , Proteína Plasmática A Asociada al Embarazo/metabolismo , Transducción de Señal , Somatomedinas/metabolismoRESUMEN
Diabetic foot ulcers (DFU) are one of the major complications in type II diabetes patients and can result in amputation and morbidity. Although multiple approaches are used clinically to help wound closure, many patients still lack adequate treatment. Here we show that IL-20 subfamily cytokines are upregulated during normal wound healing. While there is a redundant role for each individual cytokine in this subfamily in wound healing, mice deficient in IL-22R, the common receptor chain for IL-20, IL-22, and IL-24, display a significant delay in wound healing. Furthermore, IL-20, IL-22 and IL-24 are all able to promote wound healing in type II diabetic db/db mice. Mechanistically, when compared to other growth factors such as VEGF and PDGF that accelerate wound healing in this model, IL-22 uniquely induced genes involved in reepithelialization, tissue remodeling and innate host defense mechanisms from wounded skin. Interestingly, IL-22 treatment showed superior efficacy compared to PDGF or VEGF in an infectious diabetic wound model. Taken together, our data suggest that IL-20 subfamily cytokines, particularly IL-20, IL-22, and IL-24, might provide therapeutic benefit for patients with DFU.
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Diabetes Mellitus Tipo 2/genética , Pie Diabético/genética , Interleucinas/genética , Receptores de Interleucina/genética , Cicatrización de Heridas/genética , Animales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/terapia , Pie Diabético/patología , Pie Diabético/terapia , Regulación de la Expresión Génica , Humanos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Interleucinas/administración & dosificación , Ligandos , Ratones , Ratones Endogámicos NOD , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/genética , Infección de Heridas/terapia , Interleucina-22RESUMEN
OBJECTIVE: Fibroblast Growth Factor 21 (FGF21) is a potent stimulator of brown fat thermogenesis that improves insulin sensitivity, ameliorates hepatosteatosis, and induces weight loss by engaging the receptor complex comprised of Fibroblast Growth Factor Receptor 1 (FGFR1) and the requisite coreceptor ßKlotho. Previously, recombinant antibody proteins that activate the FGFR1/ßKlotho complex were proposed to act as an FGF21-mimetic; however, in vivo action of these engineered proteins has not been well studied. METHODS: We investigated the mechanism by which anti-FGFR1/ßKlotho bispecific antibody (bFKB1) stimulates thermogenesis in UCP1-expressing brown adipocytes using genetically engineered mice. Anti-FGFR1 agonist antibody was also used to achieve brown adipose tissue restricted activation in transgenic mice. RESULTS: Studies with global Ucp1-deficient mice and adipose-specific Fgfr1 deficient mice demonstrated that bFKB1 acts on targets distal to adipocytes and indirectly stimulates brown adipose thermogenesis in a UCP1-independent manner. Using a newly developed transgenic system, we also show that brown adipose tissue restricted activation of a transgenic FGFR1 expressed under the control of Ucp1 promoter does not stimulate energy expenditure. Finally, consistent with its action as a FGF21 mimetic, bFBK1 suppresses intake of saccharin-containing food and alcohol containing water in mice. CONCLUSIONS: Collectively, we propose that FGFR1/ßKlotho targeted therapy indeed mimics the action of FGF21 in vivo and stimulates UCP1-independent brown fat thermogenesis through receptors outside of adipocytes and likely in the nervous system.
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Proteínas de la Membrana/inmunología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/inmunología , Termogénesis/fisiología , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Anticuerpos/metabolismo , Metabolismo Energético/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Klotho , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Mitocondriales/metabolismo , Obesidad/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/agonistas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Termogénesis/genética , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Pérdida de PesoRESUMEN
We introduce neutron-encoded (NeuCode) amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode, we characterize an inducible knockout mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1 null mice present with fully penetrant perinatal lethality, severe hypoglycemia, and hepatic lipid deficiency. This work reveals Bap1 as a metabolic regulator in liver and pancreas, and it establishes NeuCode as a reliable proteomic method for deciphering in vivo biology.
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Proteómica/métodos , Proteínas Supresoras de Tumor/fisiología , Ubiquitina Tiolesterasa/fisiología , Animales , Hematopoyesis , Histonas/metabolismo , Marcaje Isotópico , Metabolismo de los Lípidos , Lisina/metabolismo , Masculino , Redes y Vías Metabólicas , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias Hepáticas/metabolismo , Páncreas/metabolismo , Proteoma/metabolismo , UbiquitinaciónRESUMEN
Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and ß-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into ß-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.
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Aminoácidos/metabolismo , Glucagón/metabolismo , Hígado/citología , Hígado/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proliferación Celular , Metabolismo , Ratones , Transducción de SeñalRESUMEN
Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/ßKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/ßKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/ßKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/ßKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/ßKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.
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Tejido Adiposo Pardo/metabolismo , Anticuerpos Biespecíficos/farmacología , Insulina/farmacología , Proteínas de la Membrana/agonistas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/agonistas , Adiponectina/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Animales , Línea Celular , Metabolismo Energético/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Células HEK293 , Humanos , Proteínas Klotho , Macaca fascicularis , Masculino , Proteínas de la Membrana/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Obesos , Unión Proteica/efectos de los fármacos , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Termogénesis/efectos de los fármacos , Pérdida de Peso/efectos de los fármacosRESUMEN
The phosphaturic hormone Fibroblast Growth Factor 23 (FGF23) controls phosphate homeostasis by regulating renal expression of sodium-dependent phosphate co-transporters and cytochrome P450 enzymes involved in vitamin D catabolism. Multiple FGF Receptors (FGFRs) can act as receptors for FGF23 when bound by the co-receptor Klotho expressed in the renal tubular epithelium. FGFRs also regulate skeletal FGF23 secretion; ectopic FGFR activation is implicated in genetic conditions associated with FGF23 overproduction and hypophosphatemia. The identity of FGFRs that mediate the activity of FGF23 or that regulate skeletal FGF23 secretion remains ill defined. Here we report that pharmacological activation of FGFR1 with monoclonal anti-FGFR1 antibodies (R1MAb) in adult mice is sufficient to cause an elevation in serum FGF23 and mild hypophosphatemia. In cultured rat calvariae osteoblasts, R1MAb induces FGF23 mRNA expression and FGF23 protein secretion into the culture medium. In a cultured kidney epithelial cell line, R1MAb acts as a functional FGF23 mimetic and activates the FGF23 program. siRNA-mediated Fgfr1 knockdown induced the opposite effects. Taken together, our work reveals the central role of FGFR1 in the regulation of FGF23 production and signal transduction, and has implications in the pathogenesis of FGF23-related hypophosphatemic disorders.
Asunto(s)
Factores de Crecimiento de Fibroblastos/biosíntesis , Hipofosfatemia/inmunología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/inmunología , Animales , Secuencia de Bases , Densidad Ósea , Células Cultivadas , Cartilla de ADN , Factor-23 de Crecimiento de Fibroblastos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Local delivery of therapeutic angiogenic agents that stimulate blood vessel formation represents a promising strategy for the treatment of peripheral vascular disease (PVD). At present, requirements for temporal and spatial parameters for localized delivery are unclear, with a variety of sustained delivery approaches being examined. Two polymer-based sustained formulations containing the 165 amino acid isoform of human recombinant vascular endothelial growth factor-A (rhVEGF(165)) were evaluated for their potential application in the treatment of PVD following intramuscular injection. Microspheres prepared from a 50:50 ratio of polylactic-co-glycolic acid (PLGA) and a gel of PLGA polymer solubilized in N-methyl pyrrolidone (PLGA:NMP) were each loaded with rhVEGF(165) and tested in vitro and in vivo. PLGA microspheres averaged â¼30 µm in diameter and contained 8.9% (w/w) rhVEGF(165), while the PLGA:NMP gel was formulated with varying amounts of spray freeze-dried rhVEGF(165) to result in final gel formulations having concentrations of 0.36, 0.72, or 3.6 mg/mL rhVEGF(165). In vitro release of rhVEGF(165) from PLGA microspheres showed â¼10% cumulative release by day 6, whereas the cumulative release of rhVEGF(165) from the PLGA:NMP gel matrices (0.65% w/w loading) was less than 0.25% at this same time point. While the in vitro release characteristics of these two sustained release formulations were broadly different, the plasma rhVEGF(165) concentration-time profiles following hind-limb intramuscular (IM) injection of these formulations in non-compromised rats revealed similar in vivo pharmacokinetics. Three-dimensional resin casts of vascular architecture were prepared at days 3, 7, 14, 21, 28, 60, and 75 following a single IM dosing of these sustained release microsphere and gel matrix formulations in the gastrocnemius muscle of immune-compromised mice. Scanning electron microscopic visualization of these vascular casts demonstrated spatial arrangement of capillary sprouts and vessel enlargement consistent with profound vascular changes occurring within 3 days of dosing that persisted for 2 months, approximately 1 month beyond the anticipated completion of rhVEGF(165) release from these sustained delivery formulations. Vascular re-modeling events were correlated with histological and immunohistochemical parameters attributed to known biological actions of rhVEGF(165) signaling. Together, these pharmacokinetic and pharmacodynamic results support the use of sustained release PLGA-based formulations for the local delivery of rhVEGF(165) to achieve a durable vascular re-modeling response.
Asunto(s)
Modelos Animales de Enfermedad , Neovascularización Patológica/prevención & control , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Femenino , Humanos , Ratones , Ratones Desnudos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Factor A de Crecimiento Endotelial Vascular/químicaRESUMEN
Clinical use of recombinant fibroblast growth factor 21 (FGF21) for the treatment of type 2 diabetes and other disorders linked to obesity has been proposed; however, its clinical development has been challenging owing to its poor pharmacokinetics. Here, we describe an alternative antidiabetic strategy using agonistic anti-FGFR1 (FGF receptor 1) antibodies (R1MAbs) that mimic the metabolic effects of FGF21. A single injection of R1MAb into obese diabetic mice induced acute and sustained amelioration of hyperglycemia, along with marked improvement in hyperinsulinemia, hyperlipidemia, and hepatosteatosis. R1MAb activated the mitogen-activated protein kinase pathway in adipose tissues, but not in liver, and neither FGF21 nor R1MAb improved glucose clearance in lipoatrophic mice, which suggests that adipose tissues played a central role in the observed metabolic effects. In brown adipose tissues, both FGF21 and R1MAb induced phosphorylation of CREB (cyclic adenosine 5'-monophosphate response element-binding protein), and mRNA expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1α) and the downstream genes associated with oxidative metabolism. Collectively, we propose FGFR1 in adipose tissues as a major functional receptor for FGF21, as an upstream regulator of PGC-1α, and as a compelling target for antibody-based therapy for type 2 diabetes and other obesity-associated disorders.