RESUMEN
The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas Serina-Treonina Quinasas , Ratones , Humanos , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citocinas , Inflamación/tratamiento farmacológico , Isoformas de Proteínas , Antiinflamatorios/farmacología , Inmunidad Innata , Factores de TranscripciónRESUMEN
Inflammatory bowel disease (IBD) is an umbrella term for two conditions (Crohn's Disease and Ulcerative Colitis) that is characterized by chronic inflammation of the gastrointestinal tract. The use of pre-clinical animal models has been invaluable for the understanding of potential disease mechanisms. However, despite promising results of numerous therapeutics in mouse colitis models, many of these therapies did not show clinical benefits in patients with IBD. Single cell RNA-sequencing (scRNA-seq) has recently revolutionized our understanding of complex interactions between the immune system, stromal cells, and epithelial cells by mapping novel cell subpopulations and their remodeling during disease. This technology has not been widely applied to pre-clinical models of IBD. ScRNA-seq profiling of murine models may provide an opportunity to increase the translatability into the clinic, and to choose the most appropriate model to test hypotheses and novel therapeutics. In this review, we have summarized some of the key findings at the single cell transcriptomic level in IBD, how specific signatures have been functionally validated in vivo, and highlighted the similarities and differences between scRNA-seq findings in human IBD and experimental mouse models. In each section of this review, we highlight the importance of utilizing this technology to find the most suitable or translational models of IBD based on the cellular therapeutic target.
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Colitis Ulcerosa , Colitis , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Animales , Ratones , Enfermedades Inflamatorias del Intestino/terapia , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , ARNRESUMEN
CD4+CD25highFoxp3+ regulatory T-cells (Tregs) are functionally characterized for their ability to suppress the activation of multiple immune cell types and are indispensable for maintaining immune homeostasis and tolerance. Disruption of this intrinsic brake system assessed by loss of suppressive capacity, cell numbers, and Foxp3 expression, leads to uncontrolled immune responses and tissue damage. The conversion of Tregs to a pathogenic pro-inflammatory phenotype is widely observed in immune mediated diseases. However, the molecular mechanisms that underpin the control of Treg stability and suppressive capacity are incompletely understood. This review summarizes the concepts of Treg cell stability and Treg cell plasticity highlighting underlying mechanisms including translational and epigenetic regulators that may enable translation to new therapeutic strategies. Our enhanced understanding of molecular mechanism controlling Tregs will have important implications into immune homeostasis and therapeutic potential for the treatment of immune-mediated diseases.
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Autoinmunidad , Factores de Transcripción Forkhead , Factores de Transcripción Forkhead/metabolismo , Homeostasis , Tolerancia Inmunológica , Linfocitos T ReguladoresRESUMEN
Tumor necrosis factor (TNF) and interleukin (IL)-17A are pleiotropic cytokines implicated in the pathogenesis of several autoimmune diseases including rheumatoid arthritis (RA) and psoriatic arthritis (PsA). JNJ-61178104 is a novel human anti-TNF and anti-IL-17A monovalent, bispecific antibody that binds to both human TNF and human IL-17A with high affinities and blocks the binding of TNF and IL-17A to their receptors in vitro. JNJ-61178104 also potently neutralizes TNF and IL-17A-mediated downstream effects in multiple cell-based assays. In vivo, treatment with JNJ-61178104 resulted in dose-dependent inhibition of cellular influx in a human IL-17A/TNF-induced murine lung neutrophilia model and the inhibitory effects of JNJ-61178104 were more potent than the treatment with bivalent parental anti-TNF or anti-IL-17A antibodies. JNJ-61178104 was shown to engage its targets, TNF and IL-17A, in systemic circulation measured as drug/target complex formation in normal cynomolgus monkeys (cyno). Surprisingly, quantitative target engagement assessment suggested lower apparent in vivo target-binding affinities for JNJ-61178104 compared to its bivalent parental antibodies, despite their similar in vitro target-binding affinities. The target engagement profiles of JNJ-61178104 in humans were in general agreement with the predicted profiles based on cyno data, suggesting similar differences in the apparent in vivo target-binding affinities. These findings show that in vivo target engagement of monovalent bispecific antibody does not necessarily recapitulate that of the molar-equivalent dose of its bivalent parental antibody. Our results also offer valuable insights into the understanding of the pharmacokinetics/pharmacodynamics and target engagement of other bispecific biologics against dimeric and/or trimeric soluble targets in vivo.
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Anticuerpos Biespecíficos/inmunología , Interleucina-17/inmunología , Trastornos Leucocíticos/inmunología , Pulmón/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Interleucina-17/antagonistas & inhibidores , Interleucina-17/metabolismo , Trastornos Leucocíticos/metabolismo , Trastornos Leucocíticos/prevención & control , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Macaca fascicularis , Ratones , Inhibidores del Factor de Necrosis Tumoral/inmunología , Inhibidores del Factor de Necrosis Tumoral/farmacocinética , Inhibidores del Factor de Necrosis Tumoral/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.
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Artritis Experimental/metabolismo , Criopreservación/métodos , Pie/patología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/genética , Colágeno/toxicidad , Perfilación de la Expresión Génica , Inflamación/genética , Inflamación/metabolismo , Masculino , Ratones , ProteómicaRESUMEN
The safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of JNJ-61178104, a novel anti-tumor necrosis factor-alpha (TNFα) and anti-interleukin-17A (IL-17A) bispecific antibody, were investigated in a placebo-controlled, first-in-human study. Healthy subjects (n = 54) received a single dose of JNJ-61178104 by either intravenous infusion (0.1, 0.3, 1, 3, and 10 mg/kg) or subcutaneous injection (1 mg/kg). Blood samples for measurement of serum JNJ-61178104 concentrations, total IL-17A, total TNFα, and detection of antidrug antibodies were collected for up to 16 weeks after dosing and assessed using electrochemiluminescence immunoassays. PK parameters were calculated by noncompartmental analysis and estimated by nonlinear mixed-effects modeling. JNJ-61178104 was generally well tolerated in healthy subjects. For the intravenous cohorts, mean maximum concentration, and area under the concentration-time curve values increased in a dose-proportional manner. Mean clearance ranged from 6.73 to 9.99 mL/day/kg, mean volume of distribution at terminal phase after intravenous administration ranged from 51.0 to 91.9 mL/kg, and mean half-life ranged from 4.3 to 9.7 days following intravenous administration. After a single subcutaneous dose of 1 mg/kg, median time to maximum concentration was 4.0 days, mean bioavailability was 52.0% and mean half-life was 5.3 days. A linear 2-compartment population model with first-order elimination adequately characterized the pharmacokinetics with parameters consistent with noncompartmental analysis estimates. Body weight and antidrug antibodies were significant covariates on JNJ-61178104 clearance. The time to reach mean maximum serum total TNFα and total IL-17A concentrations appeared to be dose dependent across the 0.1 mg/kg to 10 mg/kg IV dose groups. All subjects who received active treatment were antidrug antibody positive after dosing with JNJ-61178104.
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Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/farmacocinética , Interleucina-17/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adulto , Área Bajo la Curva , Femenino , Semivida , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The cytokines TNF-α and IL-17A are elevated in a variety of autoimmune diseases, including rheumatoid arthritis. Both cytokines are targets of several biologic drugs used in the clinic, but unfortunately many patients are refractory to these therapies. IL-17A and TNF-α are known to mediate signaling synergistically to drive expression of inflammatory genes. Hence, combined blockade of TNF-α and IL-17A represents an attractive treatment strategy in autoimmune settings where monotherapy is not fully effective. However, a major concern with this approach is the potential predisposition to opportunistic infections that might outweigh any clinical benefits. Accordingly, we examined the impact of individual versus combined neutralization of TNF-α and IL-17A in a mouse model of rheumatoid arthritis (collagen-induced arthritis) and the concomitant susceptibility to infections that are likely to manifest as side effects of blocking these cytokines (oral candidiasis or tuberculosis). Our findings indicate that combined neutralization of TNF-α and IL-17A was considerably more effective than monotherapy in improving collagen-induced arthritis disease even when administered at a minimally efficacious dose. Encouragingly, however, dual cytokine blockade did not cooperatively impair antimicrobial host defenses, as mice given combined IL-17A and TNF-α neutralization displayed infectious profiles and humoral responses comparable to mice given high doses of individual anti-TNF-α or anti-IL-17A mAbs. These data support the idea that combined neutralization of TNF-α and IL-17A for refractory autoimmunity is likely to be associated with acceptable and manageable risks of opportunistic infections associated with these cytokines.
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Artritis Reumatoide/inmunología , Factores Inmunológicos/efectos adversos , Interleucina-17/antagonistas & inhibidores , Infecciones Oportunistas/epidemiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Artritis Experimental/inmunología , Progresión de la Enfermedad , Huésped Inmunocomprometido/inmunología , Ratones , Infecciones Oportunistas/etiologíaRESUMEN
CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding affinity and ability to block NF-κB signaling, however their clearance rates in monkeys are very different. The pharmacokinetics profiles could be epitope dependent. To identify the epitopes, we determined the crystal structure of the ternary complex between CD27 and the Fab fragments of these non-competing antibodies. The structure reveals the binding modes of the antibodies suggesting that their mechanisms of action are distinctly different and provides a possible explanation of the in vivo data.
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Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Ligando CD27/química , Ligando CD27/inmunología , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Semivida , Humanos , Macaca fascicularis , RatonesRESUMEN
Recent advances in genetics have spurred rapid progress towards the systematic identification of genes involved in complex diseases. Still, the detailed understanding of the molecular and physiological mechanisms through which these genes affect disease phenotypes remains a major challenge. Here, we identify the asthma disease module, i.e. the local neighborhood of the interactome whose perturbation is associated with asthma, and validate it for functional and pathophysiological relevance, using both computational and experimental approaches. We find that the asthma disease module is enriched with modest GWAS P-values against the background of random variation, and with differentially expressed genes from normal and asthmatic fibroblast cells treated with an asthma-specific drug. The asthma module also contains immune response mechanisms that are shared with other immune-related disease modules. Further, using diverse omics (genomics, gene-expression, drug response) data, we identify the GAB1 signaling pathway as an important novel modulator in asthma. The wiring diagram of the uncovered asthma module suggests a relatively close link between GAB1 and glucocorticoids (GCs), which we experimentally validate, observing an increase in the level of GAB1 after GC treatment in BEAS-2B bronchial epithelial cells. The siRNA knockdown of GAB1 in the BEAS-2B cell line resulted in a decrease in the NFkB level, suggesting a novel regulatory path of the pro-inflammatory factor NFkB by GAB1 in asthma.
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Antiasmáticos/farmacología , Asma/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antiasmáticos/uso terapéutico , Asma/tratamiento farmacológico , Asma/metabolismo , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Inflamación/genética , Inflamación/metabolismo , Modelos Genéticos , FN-kappa B/genética , FN-kappa B/metabolismo , Mapeo de Interacción de Proteínas , Transducción de SeñalAsunto(s)
Asma/inmunología , Fibroblastos/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Interleucina-13/inmunología , Interleucina-17/inmunología , Pulmón/patología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Asma/terapia , Células Cultivadas , Humanos , Inmunoterapia , Subunidad alfa2 del Receptor de Interleucina-13/genética , Interleucina-4/inmunología , Ratones , Terapia Molecular Dirigida , Regulación hacia ArribaRESUMEN
The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules.
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Cartílago Articular/lesiones , Cartílago Articular/metabolismo , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Interleucina-6/inmunología , Adulto , Animales , Cartílago Articular/inmunología , Bovinos , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Transporte de Proteínas , Estrés Mecánico , Factor de Necrosis Tumoral alfa/uso terapéuticoRESUMEN
OBJECTIVE: In addition to improve glucose intolerance, recent studies suggest that glucagon-like peptide-1 (GLP-1) receptor agonism also decreases triglyceride (TG) levels. The aim of this study was to evaluate the effect of GLP-1 receptor agonism on very-low-density lipoprotein (VLDL)-TG production and liver TG metabolism. EXPERIMENTAL APPROACH: The GLP-1 peptide analogues CNTO3649 and exendin-4 were continuously administered subcutaneously to high fat diet-fed APOE*3-Leiden transgenic mice. After 4 weeks, hepatic VLDL production, lipid content, and expression profiles of selected genes involved in lipid metabolism were determined. RESULTS: CNTO3649 and exendin-4 reduced fasting plasma glucose (up to -30% and -28% respectively) and insulin (-43% and -65% respectively). In addition, these agents reduced VLDL-TG production (-36% and -54% respectively) and VLDL-apoB production (-36% and -43% respectively), indicating reduced production of VLDL particles rather than reduced lipidation of apoB. Moreover, they markedly decreased hepatic content of TG (-39% and -55% respectively), cholesterol (-30% and -55% respectively), and phospholipids (-23% and -36% respectively), accompanied by down-regulation of expression of genes involved in hepatic lipogenesis (Srebp-1c, Fasn, Dgat1) and apoB synthesis (Apob). CONCLUSION: GLP-1 receptor agonism reduces VLDL production and hepatic steatosis in addition to an improvement of glycemic control. These data suggest that GLP-receptor agonists could reduce hepatic steatosis and ameliorate dyslipidemia in patients with type 2 diabetes mellitus.
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Apolipoproteína E3/metabolismo , Hígado Graso/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptores de Glucagón/metabolismo , Animales , Apolipoproteína E3/genética , Apolipoproteínas B/metabolismo , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Dislipidemias/sangre , Exenatida , Hígado Graso/terapia , Receptor del Péptido 1 Similar al Glucagón , Insulina/metabolismo , Lipogénesis , Hígado/patología , Masculino , Ratones , Ratones Transgénicos , Péptidos/química , Péptidos/metabolismo , Ponzoñas/metabolismoRESUMEN
Patients treated with recombinant human Epo demonstrate an improvement in insulin sensitivity. We aimed to investigate whether CNTO 530, a novel Epo receptor agonist, could affect glucose tolerance and insulin sensitivity. A single administration of CNTO 530 significantly and dose-dependently reduced the area under the curve in a glucose tolerance test in diet-induced obese and diabetic mice after 14, 21, and 28 days. HOMA analysis suggested an improvement in insulin sensitivity, and this effect was confirmed by a hyperinsulinemic-euglycemic clamp. Uptake of (14)C-2-deoxy-D-glucose indicated that animals dosed with CNTO 530 transported more glucose into skeletal muscle and heart relative to control animals. In conclusion, CNTO530 has a profound effect on glucose tolerance in insulin-resistant rodents likely because of improving peripheral insulin sensitivity. This effect was observed with epoetin-α and darbepoetin-α, suggesting this is a class effect, but the effect with these compounds relative to CNTO530 was decreased in duration and magnitude.
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Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Receptores de Eritropoyetina/agonistas , Proteínas Recombinantes de Fusión/farmacología , Animales , Darbepoetina alfa , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/fisiopatología , Grasas de la Dieta/efectos adversos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Epoetina alfa , Eritropoyetina/análogos & derivados , Eritropoyetina/farmacología , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Resistencia a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/complicaciones , Obesidad/etiología , Obesidad/metabolismo , Proteínas Recombinantes , Factores de TiempoRESUMEN
The release of small intestinal hormones by constituents of ingested food, such as fatty acids, is integral to post-prandial responses that reduce food intake. Recent evidence suggests that small intestinal electrical stimulation reduces food intake, although the mechanism of action is debated. To test the hypothesis that intestinal stimulation directly alters hormone release locally we used isolated rat distal ileum and measured glucagon-like peptide-1 (GLP-1) released in the presence or absence of linoleic acid (LA) and electrical field stimulation (EFS). Intact segments were oriented longitudinally between bipolar stimulating electrodes in organ bath chambers containing modified Krebs-Ringers bicarbonate (KRB) buffer including protease inhibitors. Incubation in LA (3 mg ml(-1)) for 45 min increased GLP-1 concentration (21.9 +/- 2.6 pM versus KRB buffer alone 3.6 +/- 0.1 pM). Eleven electrical stimulation conditions were tested. In the presence of LA none of the stimulation conditions inhibited LA-evoked GLP-1 release, whereas two high frequency short pulse widths (14 V, 20 Hz, 5 ms and 14 V, 40 Hz, 5 ms) and one low frequency long pulse width (14 V, 0.4 Hz, 300 ms) EFS conditions enhanced LA-evoked GLP-1 release by >250%. These results are consistent with a local effect of intestinal electrical stimulation to enhance GLP-1 release in response to luminal nutrients in the intestines. Enhancing hormone release could improve the efficacy of intestinal electrical stimulation and provide a potential treatment for obesity and metabolic conditions.
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Alimentos , Péptido 1 Similar al Glucagón/metabolismo , Íleon/metabolismo , Animales , Estimulación Eléctrica , Motilidad Gastrointestinal/fisiología , Íleon/fisiología , Técnicas In Vitro , Vías Nerviosas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
CNTO736 is a glucagon-like peptide (GLP) 1 receptor agonist that incorporates a GLP-1 peptide analog linked to the Mimetibody platform. We evaluate the potential of acute and chronic CNTO736 treatment on insulin sensitivity and very low-density lipoprotein (VLDL) metabolism. For acute studies, diet-induced insulin-resistant C57BL/6 mice received a single intraperitoneal injection of CNTO736 or vehicle. Chronic effects were studied after 4 weeks of daily intraperitoneal administration. A hyperinsulinemic-euglycemic clamp monitored insulin sensitivity. A single dose of CNTO736 reduced fasting plasma glucose levels (CNTO736, 4.4 +/- 1.0; control, 6.3 +/- 2.4 mM) and endogenous glucose production (EGP) (CNTO736, 39 +/- 11; control, 53 +/- 13 micromol/min/kg) and increased insulin-mediated glucose uptake (CNTO736, 76 +/- 25; control, 54 +/- 13 micromol/min/kg). Chronic administration of CNTO736 reduced fasting glucose levels (CNTO736, 4.1 +/- 0.8; control 6.0 +/- 1.0 mM), improved insulin-dependent glucose uptake (CNTO736, 84 +/- 19; control, 61 +/- 15 micromol/min/kg), and enhanced inhibition of EGP (CNTO736, 91 +/- 18; control, 80 +/- 10% inhibition). In addition, chronic dosing with CNTO736 reduced fasting EGP (CNTO736, 39 +/- 9; control, 50 +/- 8 micromol/min/kg) and VLDL production (CNTO736, 157 +/- 23; control, 216 +/- 36 micromol/h/kg). These results indicate that CNTO736 reinforces insulin's action on glucose disposal and production in diet-induced insulin-resistant mice. In addition, CNTO736 reduces basal hepatic glucose and VLDL output in these animals. The data suggest that CNTO736 may be a useful tool in the treatment of type 2 diabetes.
Asunto(s)
Grasas de la Dieta/farmacología , Resistencia a la Insulina/fisiología , Lipoproteínas VLDL/sangre , Receptores de Glucagón/agonistas , Proteínas Recombinantes de Fusión/farmacología , Alimentación Animal , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Clonación Molecular , Citomegalovirus/genética , Receptor del Péptido 1 Similar al Glucagón , Glucosa/farmacología , Técnica de Clampeo de la Glucosa , Hiperinsulinismo , Infusiones Intravenosas , Insulina/administración & dosificación , Insulina/sangre , Insulina/farmacología , Lipoproteínas VLDL/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Receptores de Glucagón/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Triglicéridos/metabolismoRESUMEN
OBJECTIVE: We have developed a novel platform for display and delivery of bioactive peptides that links the biological properties of the peptide to the pharmacokinetic properties of an antibody. Peptides engineered in the MIMETIBODY platform have improved biochemical and biophysical properties that are quite distinct from those of Fc-fusion proteins. CNTO736 is a glucagon-like peptide 1 (GLP-1) receptor agonist engineered in our MIMETIBODY platform. It retains many activities of native GLP-1 yet has a significantly enhanced pharmacokinetic profile. Our goal was to develop a long-acting GLP-1 receptor agonist with sustained efficacy. RESEARCH DESIGN AND METHODS: In vitro and in vivo activity of CNTO736 was evaluated using a variety of rodent cell lines and diabetic animal models. RESULTS: Acute pharmacodynamic studies in diabetic rodents demonstrate that CNTO736 reduces fasting and postprandial glucose, decreases gastric emptying, and inhibits food intake in a GLP-1 receptor-specific manner. Reduction of food intake following CNTO736 dosing is coincident with detection of the molecule in the circumventricular organs of the brain and activation of c-fos in regions protected by the blood-brain barrier. Diabetic rodents dosed chronically with CNTO736 have lower fasting and postprandial glucose and reduced body weight. CONCLUSIONS: Taken together, our data demonstrate that CNTO736 produces a spectrum of GLP-1 receptor-dependent actions while exhibiting significantly improved pharmacokinetics relative to the native GLP-1 peptide.
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Tejido Adiposo/metabolismo , Glucosa/metabolismo , Lactoferrina/farmacología , Ingeniería de Proteínas/métodos , Receptores de Glucagón/fisiología , Transferrina/farmacología , Tejido Adiposo/efectos de los fármacos , Secuencia de Aminoácidos , Alimentación Animal , Animales , Línea Celular , Receptor del Péptido 1 Similar al Glucagón , Homeostasis , Humanos , Riñón , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Obesidad/etiología , Obesidad/fisiopatología , Receptores de Glucagón/agonistas , Receptores de Glucagón/efectos de los fármacosRESUMEN
Exercise training decreases insulin resistance and increases glucose tolerance in conditions of prediabetes and overt Type 2 diabetes. However, the adaptive responses in skeletal muscle at the molecular and genetic level for these effects of exercise training have not been clearly established in an animal model of prediabetes. The present study identifies alterations in muscle gene expression that occur with exercise training in prediabetic, insulin-resistant obese Zucker rats and insulin-sensitive lean Zucker rats and are associated with a well-defined metabolic outcome. Treadmill running for up to 4 wk caused significant enhancements of glucose tolerance as assessed by the integrated area under the curve for glucose (AUCg) during an oral glucose tolerance test. Using microarray analysis, we identified a set of only 12 genes as both significantly altered by exercise training (>1.5-fold change; P < 0.05) and significantly correlated (P < 0.05) with the AUCg. Two genes, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) and protein kinase C-zeta (PKC-zeta), are involved in the regulation of muscle glucose transport, and we provide the first evidence that PKC-zeta gene expression is enhanced by exercise training in insulin-resistant muscle. Protein expression of PGC-1alpha and PKC-zeta were positively correlated with the mRNA expression for these two genes. Overall, we have identified a limited number of genes in soleus muscle of lean and obese Zucker rats that are associated with both decreased insulin resistance and increased glucose tolerance following endurance exercise training. These findings could guide the development of pharmaceutical "exercise mimetics" in the treatment of insulin-resistant, prediabetic, or Type 2 diabetic individuals.
Asunto(s)
Regulación de la Expresión Génica , Músculo Esquelético/metabolismo , Obesidad/genética , Condicionamiento Físico Animal/fisiología , Delgadez/genética , Animales , Femenino , Perfilación de la Expresión Génica , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Resistencia a la Insulina/genética , Obesidad/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Zucker , Delgadez/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Human FIZZ3 (hFIZZ3) was identified as an ortholog of mouse resistin (mResistin), an adipocyte-specific secreted factor linked to insulin resistance in rodents. Unlike mResistin, hFIZZ3 is expressed in macrophages and monocytes, but is undetectable in adipose tissue. The profound macrophage infiltration of adipose that occurs during obesity suggests that hFIZZ3 may play an important role in adipocyte biology. Using a recombinant protein produced in Escherichia coli, we report here that chronic treatment of cultured human adipocytes with hFIZZ3 results in hypotropic cells with smaller lipid droplets. Recombinant hFIZZ3 facilitates preadipocyte proliferation and stimulates adipocyte triglyceride lipolysis, whereas recombinant mResistin inhibits adipocyte differentiation, with no detectable effect on proliferation or lipolysis. In addition, insulin-stimulated glucose uptake and Akt phosphorylation are not altered in hFIZZ3-treated adipocytes, indicating an intact insulin response. In mouse adipose explants, hFIZZ3 accelerates simultaneously triglyceride lipolysis and fatty acid reesterification, as assessed by measurement of glycerol and fatty acid release. Consistent with the in vitro findings, acute administration of recombinant hFIZZ3 into normal mice caused a significant increase in serum glycerol concentration with no elevation in free fatty acid at 45 min post injection. Taken together, the data suggest that recombinant hFIZZ3 can influence adipose metabolism by regulating preadipocyte cell number, adipocyte lipid content, and energy expenditure via accelerating the fatty acid/triglyceride futile cycle.