RESUMO
Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.
Assuntos
Doença de Huntington/metabolismo , Doença de Huntington/patologia , Fator de Crescimento Insulin-Like II/metabolismo , Agregação Patológica de Proteínas/metabolismo , Animais , Humanos , Fator de Crescimento Insulin-Like II/farmacologia , Camundongos , Camundongos Transgênicos , Agregados Proteicos/efeitos dos fármacosRESUMO
Ghrelin influences a variety of metabolic functions through a direct action at its receptor, the GhrR (GhrR-1a). Ghrelin knockout (KO) and GhrR KO mice are resistant to the negative effects of high-fat diet (HFD) feeding. We have generated several classes of small-molecule GhrR antagonists and evaluated whether pharmacologic blockade of ghrelin signaling can recapitulate the phenotype of ghrelin/GhrR KO mice. Antagonist treatment blocked ghrelin-induced and spontaneous food intake; however, the effects on spontaneous feeding were absent in GhrR KO mice, suggesting target-specific effects of the antagonists. Oral administration of antagonists to HFD-fed mice improved insulin sensitivity in both glucose tolerance and glycemic clamp tests. The insulin sensitivity observed was characterized by improved glucose disposal with dramatically decreased insulin secretion. It is noteworthy that these results mimic those obtained in similar tests of HFD-fed GhrR KO mice. HFD-fed mice treated for 56 days with antagonist experienced a transient decrease in food intake but a sustained body weight decrease resulting from decreased white adipose, but not lean tissue. They also had improved glucose disposal and a striking reduction in the amount of insulin needed to achieve this. These mice had reduced hepatic steatosis, improved liver function, and no evidence of systemic toxicity relative to controls. Furthermore, GhrR KO mice placed on low- or high-fat diets had lifespans similar to the wild type, emphasizing the long-term safety of ghrelin receptor blockade. We have therefore demonstrated that chronic pharmacologic blockade of the GhrR is an effective and safe strategy for treating metabolic syndrome.
Assuntos
Resistência à Insulina/fisiologia , Insulina/metabolismo , Receptores de Grelina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Fármacos Antiobesidade/farmacologia , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , Gorduras na Dieta/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Grelina/antagonistas & inibidores , Grelina/farmacologia , Técnica Clamp de Glucose , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/tratamento farmacológico , Receptores de Grelina/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
BACKGROUND: We and others have demonstrated previously that ghrelin receptor (GhrR) knock out (KO) mice fed a high fat diet (HFD) have increased insulin sensitivity and metabolic flexibility relative to WT littermates. A striking feature of the HFD-fed GhrR KO mouse is the dramatic decrease in hepatic steatosis. To characterize further the underlying mechanisms of glucose homeostasis in GhrR KO mice, we conducted both hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI-E) clamps. Additionally, we investigated tissue glucose uptake and specifically examined liver insulin sensitivity. RESULTS: Consistent with glucose tolerance-test data, in HG clamp experiments, GhrR KO mice showed a reduction in glucose-stimulated insulin release relative to WT littermates. Nevertheless, a robust 1st phase insulin secretion was still achieved, indicating that a healthy ß-cell response is maintained. Additionally, GhrR KO mice demonstrated both a significantly increased glucose infusion rate and significantly reduced insulin requirement for maintenance of the HG clamp, consistent with their relative insulin sensitivity. In HI-E clamps, both LFD-fed and HFD-fed GhrR KO mice showed higher peripheral insulin sensitivity relative to WT littermates as indicated by a significant increase in insulin-stimulated glucose disposal (Rd), and decreased hepatic glucose production (HGP). HFD-fed GhrR KO mice showed a marked increase in peripheral tissue glucose uptake in a variety of tissues, including skeletal muscle, brown adipose tissue and white adipose tissue. GhrR KO mice fed a HFD also showed a modest, but significant decrease in conversion of pyruvate to glucose, as would be anticipated if these mice displayed increased liver insulin sensitivity. Additionally, the levels of UCP2 and UCP1 were reduced in the liver and BAT, respectively, in GhrR KO mice relative to WT mice. CONCLUSIONS: These results indicate that improved glucose homeostasis of GhrR KO mice is characterized by robust improvements of glucose disposal in both normal and metabolically challenged states, relative to WT controls. GhrR KO mice have an intact 1st phase insulin response but require significantly less insulin for glucose disposal. Our experiments reveal that the insulin sensitivity of GhrR KO mice is due to both BW independent and dependent factors. We also provide several lines of evidence that a key feature of the GhrR KO mouse is maintenance of hepatic insulin sensitivity during metabolic challenge.
Assuntos
Técnica Clamp de Glucose/métodos , Resistência à Insulina/genética , Insulina/sangue , Receptores de Grelina/deficiência , Animais , Gorduras na Dieta/administração & dosagem , Teste de Tolerância a Glucose/métodos , Índice Glicêmico/genética , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
To define the relationship between the respiratory quotient (RQ) and energy intake (EI) and to determine the impact of spontaneous locomotor activity (LMA) in the development of diet-induced obesity (DIO), we fed C57BL/6 mice a high-fat diet (HFD) for either 4 days or 17 wk and analyzed them using indirect calorimetry. Importantly, changes in body mass during calorimetry (DeltaM(b)) significantly covaried with RQ and EI; adjusting the data for DeltaM(b) permitted an analysis of the energy-balanced state. The 24-h RQ strongly predicted 24-h EI, and the slope of this relationship was diet dependent (HFD or chow) but independent of the HFD feeding period. Early-stage DIO was characterized by dark-period hyperphagia and fat storage, offset by greater light-period lipid oxidation; later stage DIO mice had a milder hyperphagia and lower substrate flexibility. Consequently, whereas 24-h RQ equaled the food quotient of the HFD in both early- and late-stage DIO, the range of RQ values was negatively correlated with, and mostly explained by, 24-h EI only in late-stage DIO. Lean and early-stage DIO mice had similar LMA values that were reduced in late-stage DIO. However, LMA significantly explained variance in total energy expenditure (EE) in only early-stage DIO mice. This indicated that the link between LMA and EE was a transient adaptive response to early DIO, whereas the later loss of LMA did not explain body weight gain in C57BL/6 DIO mice.
Assuntos
Peso Corporal/fisiologia , Metabolismo Energético/fisiologia , Hiperfagia/metabolismo , Obesidade/metabolismo , Consumo de Oxigênio/fisiologia , Animais , Calorimetria Indireta , Gorduras na Dieta/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Valor Preditivo dos TestesRESUMO
The orexigenic peptide ghrelin has been shown to have prokinetic activity in the gastrointestinal (GI) system of several species, including humans. In this series of experiments, we have evaluated the prokinetic activity of novel, small-molecule ghrelin receptor (GhrR) agonists after parenteral and peroral dosing in mice and rats. Gastric emptying, small intestinal transport, and fecal output were determined after intraperitoneal and intracerebroventricular dosing of GhrR agonists, using ghrelin as a positive control. These same parameters were evaluated after oral gavage dosing of the synthetic agonists. Regardless of dose route, GhrR agonist treatment increased gastric emptying, small intestinal transit, and fecal output. However, fecal output was only increased by GhrR agonist treatment if mice were able to feed during the stimulatory period. Thus, GhrR agonists can stimulate upper GI motility, and the orexigenic action of the compounds can indirectly contribute to prokinetic activity along the entire GI tract. The orexigenic and prokinetic effects of either ghrelin or small-molecule GhrR agonists were selective for the GhrR because they were absent when evaluated in GhrR knockout mice. We next evaluated the efficacy of the synthetic GhrR agonists dosed in a model of opiate-induced bowel dysfunction induced by a single injection of morphine. Oral dosing of a GhrR agonist normalized GI motility in opiate-induced dysmotility. These data demonstrate the potential utility of GhrR agonists for treating gastrointestinal hypomotility disorders.
Assuntos
Motilidade Gastrointestinal/efeitos dos fármacos , Grelina/administração & dosagem , Grelina/farmacologia , Hormônios Peptídicos/administração & dosagem , Hormônios Peptídicos/farmacologia , Receptores de Grelina/agonistas , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Doença de Bowen/induzido quimicamente , Doença de Bowen/tratamento farmacológico , Doença de Bowen/fisiopatologia , Sistema Nervoso Central/efeitos dos fármacos , Defecação/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Esvaziamento Gástrico/efeitos dos fármacos , Trânsito Gastrointestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Hormônios Peptídicos/sangue , Ratos , Ratos Sprague-Dawley , Receptores de Grelina/genética , Receptores de Grelina/metabolismoRESUMO
Stimulation of the ghrelin receptor (GhrR) by ghrelin results in a variety of metabolic changes including increased food intake, fat storage and insulin resistance. Loss of ghrelin signaling is protective against diet-induced obesity, suggesting that ghrelin plays a significant homeostatic role in conditions of metabolic stress. We examined glycemic control in GhrR -/- mice fed a high-fat diet, and used indirect calorimetry to assess fuel substrate usage and energy expenditure. GhrR -/- mice fed a high-fat diet had several measures of greater insulin sensitivity, including: lower fasted blood glucose and plasma insulin, lower %Hb(A1c), lower insulin levels during glucose tolerance tests, and improved performance in hyperinsulinemic-euglycemic and hyperglycemic clamp studies. GhrR -/- mice fed a high-fat diet did not develop hepatic steatosis and had lower total cholesterol, relative to controls. Furthermore, GhrR -/- mice demonstrated a lower intestinal triglyceride secretion rate of dietary lipid. GhrR -/- mice have higher respiratory quotients (RQ), indicating a preference for carbohydrate as fuel. The range of RQ values was wider in GhrR -/- mice, indicating greater metabolic flexibility and insulin sensitivity in these animals. We therefore propose that loss of ghrelin signaling promotes insulin sensitivity and metabolic flexibility, and protects against several fatty diet-induced features of metabolic syndrome due to convergent changes in the intake, absorption and utilization of energy.
Assuntos
Gorduras na Dieta/metabolismo , Metabolismo Energético/fisiologia , Resistência à Insulina/fisiologia , Receptores de Grelina/genética , Animais , Glicemia/análise , Calorimetria Indireta/métodos , Colesterol/metabolismo , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Jejum , Teste de Tolerância a Glucose , Hemoglobinas Glicadas/análise , Hemoglobinas Glicadas/metabolismo , Insulina/sangue , Camundongos , Camundongos Knockout , Triglicerídeos/metabolismoRESUMO
This study reports the characterization of a recombinant adenoviral vector containing a tetracycline-regulatable promoter, driving the bicistronic expression of the human H2 preprorelaxin (hH2) cDNA and enhanced green fluorescent protein, via an internal ribosomal entry site. An hH2 ELISA was used to measure the secreted levels of recombinant hH2 in transfected canine (CF33.Mt) and human (MDA-MB-435) mammary cancer cell lines over a 6-d period; secreted peptide peaked on d 2 and 4 for the canine and human cell types, respectively. An unprocessed hH2 immunoreactive form of approximately 18 kDa was identified by Western blotting analysis and confirmed by mass spectrometry, suggesting that prorelaxin remains unprocessed in these cell types. The biological activity of the adenovirally expressed human prorelaxin was measured in the established human monocytic cell line THP-1 cAMP ELISA and in an in vitro Transwell cell migration system. Exogenous recombinant hH2 and adenovirally-mediated delivery of prorelaxin to CF33.Mt cells conferred a significant migratory action in the cells, compared with controls. Cell proliferation assays were performed to discount the possibility that the effect of relaxin was mitogenic. Thus, we have demonstrated that prorelaxin has the ability to facilitate cell migration processes exclusive of its ability to stimulate cell proliferation. In validating this adenovirus-based system, we have created a potential tool for further exploration of the physiology of relaxin in mammalian systems.
Assuntos
Adenoviridae/genética , Neoplasias Mamárias Animais/patologia , Invasividade Neoplásica , Precursores de Proteínas/genética , Relaxina/genética , Animais , Western Blotting , Divisão Celular , Linhagem Celular , Movimento Celular , Meios de Cultivo Condicionados/química , Cães , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Espectrometria de Massas , Monócitos/metabolismo , Precursores de Proteínas/análise , Precursores de Proteínas/fisiologia , Proteínas Recombinantes/análise , Relaxina/análise , Relaxina/fisiologia , TransfecçãoRESUMO
To identify new drug targets for the treatment of obesity, we employed a degenerate reverse transcriptasepolymerase chain reaction technique to isolate novel members of the G-protein coupled receptor superfamily from mouse hypothalamus. One of our clones was found to encode a protein with 90% amino acid identity to human GPR10, which was previously identified as the receptor for prolactin-releasing peptide (PrRP) and has been implicated in lactation, the regulation of food intake and other physiological functions. To investigate the role of GPR10 in food intake and body weight homeostasis, we generated mice carrying a targeted deletion of the GPR10 gene. First, using these knockout animals, we confirmed that GPR10 is the principle receptor for PrRP in the mouse hypothalamus because deletion of GPR10 completely abolished PrRP binding to isolated hypothalamic cell membranes. Second, we investigated the effect of normal and high-fat diets on energy intake, body weight, and glucose homeostasis in wild-type and GPR10 knockout mice. After fasting and refeeding, food intake in knockout animals was unchanged relative to control littermates. However, beginning at 16 wk of age on a normal diet, knockout mice became hyperphagic, obese, and showed significant increases in body fat and the levels of leptin and insulin, as well as decreased glucose tolerance. This metabolic profile was similar to the effect of a high-fat diet on wild-type animals. Our findings provide direct evidence that GPR10 is the receptor for PrRP and that it is involved in the regulation of energy balance in mice. GPR10 knockout mice will also prove useful for investigating other proposed activities for PrRP.
Assuntos
Regulação do Apetite/genética , Homeostase/genética , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/metabolismo , Neuropeptídeos/metabolismo , Receptores Acoplados a Proteínas G/genética , Animais , Peso Corporal/genética , Restrição Calórica , Membrana Celular/genética , Membrana Celular/metabolismo , DNA Complementar/genética , Alimentos Formulados , Glucose/metabolismo , Humanos , Hiperfagia/genética , Hiperfagia/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Obesidade/genética , Obesidade/metabolismo , Hormônio Liberador de Prolactina , Ligação Proteica/genética , Receptores Acoplados a Proteínas G/isolamento & purificação , Receptores Acoplados a Proteínas G/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The goal of this study was to examine factors that contribute to energy balance in female GHR -/- mice. We measured energy intake, energy expenditure (EE), fuel utilization, body mass (M(b)) changes and physical activity in 17month-old female GHR -/- mice and their age-matched wild type littermates. The GHR -/- mice were smaller, consumed more food per unit M(b), had greater EE per unit M(b) and had an increase in 24-h EE/M(b) that was similar to the increase in their surface-area-to-volume ratio. Locomotor activity (LMA) was reduced in the GHR -/- mice, but the energetic cost associated with their LMA was greater than in wild type controls. Furthermore, M(b) and LMA were independent explanatory covariates of most of the variance in EE, and when adjusted for M(b) and LMA, the GHR -/- mice had higher EE during both the light and dark phases of the daily cycle. Respiratory quotient was lower in GHR -/- mice during the light phase, which indicated a greater utilization of lipid relative to carbohydrate in these mice. Additionally, GHR -/- mice had higher ratios of caloric intake to EE at several intervals during the dark phase, and this effect was greater and more sustained in the final 3h of the dark phase. Therefore, we conclude that GHR -/- mice are able to overcome the substantial energetic challenges of dwarfism through several mechanisms that promote stable M(b). Relative to wild type mice, the GHR -/- mice consumed more calories per unit M(b), which offset the disproportionate increase in their daily energy expenditure. While GHR -/- mice oxidized a greater proportion of lipid during the light phase in order to meet their energy requirements, they achieved greater energy efficiency and storage during the dark phase through a combination of higher energy consumption and lower LMA.
Assuntos
Metabolismo Energético/fisiologia , Receptores da Somatotropina/metabolismo , Ciclos de Atividade/fisiologia , Animais , Nanismo/metabolismo , Ingestão de Energia/fisiologia , Feminino , Metabolismo dos Lipídeos/fisiologia , Camundongos , Camundongos Mutantes , Atividade Motora/fisiologia , Receptores da Somatotropina/genética , Receptores da Somatotropina/fisiologiaRESUMO
Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis, NFkappaB activation, and cytokine regulation. In this study we identified a novel human protein, CARD-8, which contains a C-terminal CARD domain with high similarity to the CARD domain of caspase-1/ICE. We demonstrate that CARD-8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1beta generation in the THP-1 monocytic cell line. CARD-8 binds also to ICEBERG and pseudo-ICE, two other recently identified proteins, which bind to the CARD domain of caspase-1 and negatively regulate its activity. Reverse transcriptase-PCR analysis revealed that CARD-8 is expressed mainly in monocytes, placenta, lymph nodes, and spleen. This pattern of expression is consistent with caspase-1 expression in the same cells and tissues. CARD-8 was also found to negatively regulate NF-kappaB activation by TNF-alpha stimulation and by ectopically expressed RICK, suggesting that this protein may control cell survival. Consistent with these results, stable expression of CARD-8 in U937 or THP-1 cells sensitizes the cells to differentiation-induced apoptosis. Overexpression of CARD-8 can also induce apoptosis in transfected cells. The results suggest that CARD-8 represents a new signaling molecule involved in the regulation of caspase-1 and NF-kappaB activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Proteínas de Neoplasias , Núcleosídeo-Fosfato Quinase/metabolismo , Sequência de Aminoácidos , Proteínas Adaptadoras de Sinalização CARD , Linhagem Celular , DNA Complementar/metabolismo , Ativação Enzimática , Humanos , Interleucina-1/metabolismo , Luciferases/metabolismo , Dados de Sequência Molecular , NF-kappa B/metabolismo , Testes de Precipitina , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The PYRIN domain is a recently identified protein-protein interaction domain that is found at the N terminus of several proteins thought to function in apoptotic and inflammatory signaling pathways. We report here that PYPAF1 (PYRIN-containing Apaf1-like protein 1) is a novel PYRIN-containing signaling protein that belongs to the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signaling proteins. The expression of PYPAF1 is highly restricted to immune cells, and its gene maps to chromosome 1q44, a locus that is associated with the rare inflammatory diseases Muckle-Wells syndrome and familial cold urticaria. To identify downstream signaling partners of PYPAF1, we performed a mammalian two-hybrid screen and identified ASC as a PYRIN-containing protein that interacts selectively with the PYRIN domain of PYPAF1. When expressed in cells, ASC recruits PYPAF1 to distinct cytoplasmic loci and induces the activation of NF-kappaB. Furthermore, coexpression of PYPAF1 with ASC results in a potent synergistic activation of NF-kappaB. These findings suggest that PYPAF1 and ASC function as upstream activators of NF-kappaB signaling.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , NF-kappa B/metabolismo , Sequência de Aminoácidos , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Dados de Sequência Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-kappa B. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1 beta. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-kappa B and cytokine processing.