RESUMEN
Therapies based on glucagon-like peptide-1 (GLP-1) long-acting analogs and insulin are often used in the treatment of metabolic diseases. Both insulin and GLP-1 receptors are expressed in metabolically relevant brain regions, suggesting a cooperative action. However, the mechanisms underlying the synergistic actions of insulin and GLP-1R agonists remain elusive. In this study, we show that insulin-induced hypoglycemia enhances GLP-1R agonists entry in hypothalamic and area, leading to enhanced whole-body fat oxidation. Mechanistically, this phenomenon relies on the release of tanycyctic vascular endothelial growth factor A, which is selectively impaired after calorie-rich diet exposure. In humans, low blood glucose also correlates with enhanced blood-to-brain passage of insulin, suggesting that blood glucose gates the passage other energy-related signals in the brain. This study implies that the preventing hyperglycemia is important to harnessing the full benefit of GLP-1R agonist entry in the brain and action onto lipid mobilization and body weight loss.
Asunto(s)
Glucemia , Factor A de Crecimiento Endotelial Vascular , Humanos , Glucemia/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Insulina/metabolismo , Homeostasis , Encéfalo/metabolismoRESUMEN
Insulin acts on neurons and glial cells to regulate systemic glucose metabolism and feeding. However, the mechanisms of insulin access in discrete brain regions are incompletely defined. Here we show that insulin receptors in tanycytes, but not in brain endothelial cells, are required to regulate insulin access to the hypothalamic arcuate nucleus. Mice lacking insulin receptors in tanycytes (IR∆Tan mice) exhibit systemic insulin resistance, while displaying normal food intake and energy expenditure. Tanycytic insulin receptors are also necessary for the orexigenic effects of ghrelin, but not for the anorexic effects of leptin. IR∆Tan mice exhibit increased agouti-related peptide (AgRP) neuronal activity, while displaying blunted AgRP neuronal adaptations to feeding-related stimuli. Lastly, a highly palatable food decreases tanycytic and arcuate nucleus insulin signalling to levels comparable to those seen in IR∆Tan mice. These changes are rooted in modifications of cellular stress responses and of mitochondrial protein quality control in tanycytes. Conclusively, we reveal a critical role of tanycyte insulin receptors in gating feeding-state-dependent regulation of AgRP neurons and systemic insulin sensitivity, and show that insulin resistance in tanycytes contributes to the pleiotropic manifestations of obesity-associated insulin resistance.
Asunto(s)
Proteína Relacionada con Agouti/metabolismo , Células Ependimogliales/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Neuronas/metabolismo , Transducción de Señal , Proteína Relacionada con Agouti/química , Animales , Biomarcadores , Barrera Hematoencefálica/metabolismo , Calcio , Metabolismo Energético , Técnica del Anticuerpo Fluorescente , Ghrelina/metabolismo , Glucosa/metabolismo , Resistencia a la Insulina , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Modelos Biológicos , Fragmentos de Péptidos/metabolismo , Receptor de Insulina/metabolismoRESUMEN
INTRODUCTION: Insulin icodec is a novel, long-acting insulin analog designed to cover basal insulin requirements with once-weekly subcutaneous administration. Here we describe the molecular engineering and the biological and pharmacological properties of insulin icodec. RESEARCH DESIGN AND METHODS: A number of in vitro assays measuring receptor binding, intracellular signaling as well as cellular metabolic and mitogenic responses were used to characterize the biological properties of insulin icodec. To evaluate the pharmacological properties of insulin icodec in individuals with type 2 diabetes, a randomized, double-blind, double-dummy, active-controlled, multiple-dose, dose escalation trial was conducted. RESULTS: The long half-life of insulin icodec was achieved by introducing modifications to the insulin molecule aiming to obtain a safe, albumin-bound circulating depot of insulin icodec, providing protracted insulin action and clearance. Addition of a C20 fatty diacid-containing side chain imparts strong, reversible albumin binding, while three amino acid substitutions (A14E, B16H and B25H) provide molecular stability and contribute to attenuating insulin receptor (IR) binding and clearance, further prolonging the half-life. In vitro cell-based studies showed that insulin icodec activates the same dose-dependent IR-mediated signaling and metabolic responses as native human insulin (HI). The affinity of insulin icodec for the insulin-like growth factor-1 receptor was proportionately lower than its binding to the IR, and the in vitro mitogenic effect of insulin icodec in various human cells was low relative to HI. The clinical pharmacology trial in people with type 2 diabetes showed that insulin icodec was well tolerated and has pharmacokinetic/pharmacodynamic properties that are suited for once-weekly dosing, with a mean half-life of 196 hours and close to even distribution of glucose-lowering effect over the entire dosing interval of 1 week. CONCLUSIONS: The molecular modifications introduced into insulin icodec provide a novel basal insulin with biological and pharmacokinetic/pharmacodynamic properties suitable for once-weekly dosing. TRIAL REGISTRATION NUMBER: NCT02964104.
Asunto(s)
Diabetes Mellitus Tipo 2 , Insulina , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Hipoglucemiantes/farmacología , Insulina de Acción Prolongada , Insulina Regular HumanaRESUMEN
Translation across species of immunoassay results is often challenging due to the lack of cross-species reactivity of antibodies. In order to investigate the biology of insulin and IGF1 receptors, we generated new versatile monoclonal assay antibodies using the extracellular domain of the insulin/IGF1 hybrid receptor as the bait protein in the Adimab yeast antibody discovery platform and as the antigen in a rabbit monoclonal antibody platform. The resulting antibody clones were screened for receptor specificity as well as cross-species reactivity to both tissue and cell line derived samples. Using these strategies, we were able to identify highly specific insulin receptor monoclonal antibodies that lack cross-reactivity to the IGF1 receptor using the Adimab platform and a highly specific IGF1 receptor monoclonal antibody that lacks cross-reactivity to the insulin receptor using the rabbit antibody platform. Unlike earlier monoclonal antibodies reported in the literature, these antibodies show cross-species reactivity to the extracellular domains of mouse, rat, pig, and human receptors, indicating that they bind conserved epitopes. Furthermore, the antibodies work well in several different assay formats, including ELISA, flow cytometry, and immunoprecipitation, and therefore provide new tools to study insulin and IGF1 receptor biology with translation across several species and experimental model systems.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptor IGF Tipo 1/inmunología , Receptor de Insulina/inmunología , Animales , Anticuerpos Monoclonales/química , Reacciones Cruzadas , Células HCT116 , Humanos , Ratones , Conejos , Ratas , Especificidad de la Especie , PorcinosRESUMEN
Insulin treatment is associated with increased adipose mass in both humans and mice. However, the underlying dynamic basis of insulin induced lipid accumulation in adipose tissue remains elusive. To assess this, young female C57BL6/J mice were fed a low fat diet for 3 weeks, treated subsequently with 7 days of constant subcutaneous insulin infusion by osmotic minipumps and compared to mice with only buffer infused. To track changes in lipid deposition during insulin treatment, metabolic labeling was conducted with heavy water for the final 4 days. Blood glucose was significantly lowered within one hour after implantation of insulin loaded mini pumps and remained lower throughout the study. Insulin treated animals gained significantly more weight during treatment and the mean weight of the subcutaneous adipose depots was significantly higher with the highest dose of insulin. Surprisingly, de novo palmitate synthesis within the subcutaneous and the gonadal depots was not affected significantly by insulin treatment. In contrast insulin treatment caused accumulation of triglycerides in both depots due to either deposition of newly synthesised triglycerides (subcutaneous depot) or inhibition of lipolysis (gonadal depot).
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/crecimiento & desarrollo , Insulina/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Palmitatos/metabolismo , Animales , Glucemia , Peso Corporal , Conducta Alimentaria/efectos de los fármacos , Femenino , Insulina/administración & dosificación , Lipólisis , Ratones , Tamaño de los Órganos/efectos de los fármacos , Triglicéridos/metabolismoRESUMEN
Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B'29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1-10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints ((3)H-thymidine incorporation), and not on metabolic endpoints ((14)C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity ((3)H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B'29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.
Asunto(s)
Insulina/metabolismo , Péptidos/farmacología , Receptor de Insulina/agonistas , Receptor de Insulina/antagonistas & inhibidores , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células CHO , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Femenino , Glucosa/metabolismo , Humanos , Insulina/química , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.
Asunto(s)
Hígado/metabolismo , Receptores Nucleares Huérfanos/genética , PPAR alfa/genética , Receptores X Retinoide/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , Femenino , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Nucleares Huérfanos/deficiencia , Receptores Nucleares Huérfanos/metabolismo , PPAR alfa/agonistas , PPAR alfa/metabolismo , Receptor Cross-Talk , Receptores X Retinoide/metabolismoRESUMEN
Chemical modifications of proteins are increasingly important in the development of protein drugs with fine-tuned properties. Regioselective modification, such as the chemoselective alkylation of an unpaired cysteine residue, is a prerequisite for obtaining homogenous protein products. The introduction of an unpaired Cys into the Cys-rich protein, insulin, was investigated by using a Cys scan. This was challenging as the introduced Cys could interfere with insulin's three existing disulfide bonds. However, eight insulin precursors were expressed in Saccharomyces cerevisiae with good yields. Although extensive post-translational modifications of the unpaired Cys were observed, the majority could be removed by selective reduction. An example Cys(7) insulin analogue was modified with a PEGylated maleimide moiety. The new variant was active in in vitro and in vivo models. Our results show that even small Cys-rich proteins can be expressed with additional unpaired Cys in meaningful yields and further chemically modified, while maintaining their biological activity.
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Cisteína/química , Insulina/análogos & derivados , Alquilación , Animales , Cromatografía Líquida de Alta Presión , Disulfuros/química , Insulina/genética , Insulina/metabolismo , Masculino , Maleimidas/química , Polietilenglicoles/química , Ratas , Ratas Wistar , Estereoisomerismo , Espectrometría de Masas en TándemRESUMEN
The melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R-MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and α-melanocyte stimulating hormone (α-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSH analogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (PKB), adenosine 5' monophosphate activated protein kinase (AMPK) and Jun-amino-terminal kinase (JNK) in MCR mediated lipolysis were studied. Interestingly, results obtained in 3T3-L1 cells suggest that lipolysis stimulated by α-MSH, NDP-α-MSH, MT-II, SHU9119 and PG-901 is mediated through MC5R in a cAMP independent manner. Finally, we identify essential differences in MCR mediated lipolysis when using 3T3-L1 cells compared to primary adipocytes.
Asunto(s)
Adipocitos/metabolismo , Lipólisis , Sistema de Señalización de MAP Quinasas , Receptor de Melanocortina Tipo 2/metabolismo , Receptores de Melanocortina/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipogénesis , Hormona Adrenocorticotrópica/farmacología , Hormona Adrenocorticotrópica/fisiología , Animales , Unión Competitiva , AMP Cíclico/metabolismo , Epidídimo/citología , Epidídimo/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Expresión Génica , Hormonas/farmacología , Hormonas/fisiología , Masculino , Melanocortinas/farmacología , Melanocortinas/fisiología , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Melanocortina Tipo 2/agonistas , Receptor de Melanocortina Tipo 2/genética , Receptores de Melanocortina/genéticaRESUMEN
OBJECTIVE: Bexarotene (Targretin) is a clinically used antitumoral agent which exerts its action through binding to and activation of the retinoid-X-receptor (RXR). The most frequent side-effect of bexarotene administration is an increase in plasma triglycerides, an independent risk factor of cardiovascular disease. The molecular mechanism behind this hypertriglyceridemia remains poorly understood. METHODS AND RESULTS: Using wild-type and LXR alpha/beta-deficient mice, we show here that bexarotene induces hypertriglyceridemia and activates hepatic LXR-target genes of lipogenesis in an LXR-dependent manner, hence exerting a permissive effect on RXR/LXR heterodimers. Interestingly, RNA analysis and Chromatin Immunoprecipitation assays performed in the liver reveal that the in vivo permissive effect of bexarotene on the RXR/LXR heterodimer is restricted to lipogenic genes without modulation of genes controlling cholesterol homeostasis. CONCLUSIONS: These findings demonstrate that the hypertriglyceridemic action of bexarotene occurs via the RXR/LXR heterodimer and show that RXR heterodimers can act with a selective permissivity on target genes of specific metabolic pathways in the liver.
Asunto(s)
Colesterol/metabolismo , Proteínas de Unión al ADN/fisiología , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores X Retinoide/fisiología , Tetrahidronaftalenos/farmacología , Triglicéridos/metabolismo , Animales , Bexaroteno , Proteínas de Unión al ADN/química , Dimerización , Femenino , Homeostasis , Lipogénesis , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/química , Receptores X Retinoide/químicaRESUMEN
The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of adipocyte differentiation in vivo and ex vivo and has been shown to control the expression of several adipocyte-specific genes. In this study, we used chromatin immunoprecipitation combined with deep sequencing to generate genome-wide maps of PPARgamma and retinoid X receptor (RXR)-binding sites, and RNA polymerase II (RNAPII) occupancy at very high resolution throughout adipocyte differentiation of 3T3-L1 cells. We identify >5000 high-confidence shared PPARgamma:RXR-binding sites in adipocytes and show that during early stages of differentiation, many of these are preoccupied by non-PPARgamma RXR-heterodimers. Different temporal and compositional patterns of occupancy are observed. In addition, we detect co-occupancy with members of the C/EBP family. Analysis of RNAPII occupancy uncovers distinct clusters of similarly regulated genes of different biological processes. PPARgamma:RXR binding is associated with the majority of induced genes, and sites are particularly abundant in the vicinity of genes involved in lipid and glucose metabolism. Our analyses represent the first genome-wide map of PPARgamma:RXR target sites and changes in RNAPII occupancy throughout adipocyte differentiation and indicate that a hitherto unrecognized high number of adipocyte genes of distinctly regulated pathways are directly activated by PPARgamma:RXR.
Asunto(s)
Adipogénesis/fisiología , PPAR gamma/metabolismo , ARN Polimerasa II/metabolismo , Receptores X Retinoide/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Animales , Dimerización , Genoma , Ratones , Datos de Secuencia Molecular , Familia de MultigenesRESUMEN
The C/EBPalpha transcription factor regulates growth and differentiation of several tissues during embryonic development. Several hypotheses as to how C/EBPalpha inhibits cellular growth in vivo have been derived, mainly from studies of tissue culture cells. In fetal liver it has been proposed that a short, centrally located, 15-amino-acid proline-histidine-rich region (PHR) of C/EBPalpha is responsible for the growth-inhibitory function of the protein through its ability to interact with CDK2 and CDK4, thereby inhibiting their activities. Homozygous Cebpa(DeltaPHR/DeltaPHR) (DeltaPHR) mice, carrying a modified cebpa allele lacking amino acids 180 to 194, were born at the Mendelian ratio, reached adulthood, and displayed no apparent adverse phenotypes. When fetal livers from the DeltaPHR mice were analyzed for their expression of cell cycle markers, bromodeoxyuridine incorporation, cyclin-dependent kinase 2 kinase activity, and global gene expression, we failed to detect any cell cycle or developmental differences between the DeltaPHR mice and their control littermates. These in vivo data demonstrate that any C/EBPalpha-mediated growth repression via the PHR as well as the basic region is dispensable for proper embryonic development of, and cell cycle control in, the liver. Surprisingly, control experiments performed in C/EBPalpha null fetal livers yielded similar results.