RESUMO
The peptide hormone relaxin plays a critical role in tissue remodeling in a variety of tissues through activation of its cognate receptor, RXFP1. Relaxin's ability to modify extracellular matrices has provided a strong rationale for treating fibrosis in a variety of tissues. Treatment with recombinant relaxin peptides in clinical studies of heart failure has not yet proven useful, likely due to the short half-life of infused peptide. To circumvent this particular pharmacokinetic pitfall we have used a Protein-in-Protein (PiP) antibody technology described previously, to insert a single-chain human relaxin construct into the complementarity-determining region (CDR) of an immunoglobulin G (IgG) backbone, creating a relaxin molecule with a half-life of â¼4-5 days in mice. Relaxin-PiP biologics displaced Europium-labeled human relaxin in RXFP1-expressing cells and demonstrated full agonist activity on both human and mouse RXFP1 receptors. Relaxin-PiPs did not show signal transduction bias, as they activated cAMP in THP-1 cells, and cGMP and pERK signaling in primary human cardiac fibroblasts. In an induced carbon tetrachloride mouse model of liver fibrosis one relaxin-PiP, R2-PiP, caused reduction of liver lesions, ameliorated collagen accumulation in the liver with the corresponding reduction of Collagen1a1 gene expression, and increased cell proliferation in hepatic parenchyma. These relaxin biologics represent a novel approach to the design of a long-acting RXFP1 agonist to probe the clinical utility of relaxin/RXFP1 signaling to treat a variety of human fibrotic diseases.
Assuntos
Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Relaxina , Relaxina/farmacologia , Animais , Humanos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Camundongos , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Engenharia de Proteínas/métodos , Masculino , Camundongos Endogâmicos C57BL , Cricetulus , Células HEK293 , Células CHORESUMO
Signaling of BDNF via its TrkB receptor is crucial in regulating several critical aspects of the architecture and function of neurons both during development and in the adult central nervous system. Indeed, several neurological conditions, such as neurodevelopmental and neurodegenerative disorders are associated with alterations both in the expression levels of BDNF and TrkB, and in their intracellular signaling. Thus, the possibility of promoting BDNF/TrkB signaling has become relevant as a potential therapeutic intervention for neurological disorders. However, the clinical potential of BDNF itself has been limited due to its restricted diffusion rate in biological tissue, poor bioavailability and pharmacological properties, as well as the potential for unwanted side effects due to its ability to also signal via the p75NTR pathway. Several small molecule and biologic drug candidate TrkB agonists have been developed and are reported to have effects in rescuing both the pathological alterations and disease related symptoms in mouse models of several neurological diseases. However, recent side-by-side comparative studies failed to show their specificity for activating TrkB signaling cascades, suggesting the need for the generation and validation of improved candidates. In the present study, we examine the ability of the novel, fully human TrkB agonist antibody ZEB85 to modulate the architecture, activity and synaptic plasticity of hippocampal murine neurons under physiological conditions. Moreover, we show here that ZEB85 prevents ß-amyloid toxicity in cultured hippocampal neurons, in a manner which is comparable to BDNF.
RESUMO
In the central nervous system, most neurons co-express TrkB and TrkC, the tyrosine kinase receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down-regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Glicoproteínas de Membrana/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Regulação para Baixo , Humanos , Neurônios/metabolismo , Neurotrofina 3/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor trkB/genética , Receptor trkC/genética , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismoRESUMO
The diverse physiological roles of the neurotrophin family have long prompted exploration of their potential as therapeutic agents for nerve injury and neurodegenerative diseases. To date, clinical trials of one family member, brain-derived neurotrophic factor (BDNF), have disappointingly failed to meet desired endpoints. Contributing to these failures is the fact that BDNF is pharmaceutically a nonideal biologic drug candidate. It is a highly charged, yet is a net hydrophobic molecule with a low molecular weight that confers a short t1/2 in man. To circumvent these shortcomings of BDNF as a drug candidate, we have employed a function-based cellular screening assay to select activating antibodies of the BDNF receptor TrkB from a combinatorial human short-chain variable fragment antibody library. We report here the successful selection of several potent TrkB agonist antibodies and detailed biochemical and physiological characterization of one such antibody, ZEB85. By using a human TrkB reporter cell line and BDNF-responsive GABAergic neurons derived from human ES cells, we demonstrate that ZEB85 is a full agonist of TrkB, comparable in potency to BDNF toward human neurons in activation of TrkB phosphorylation, canonical signal transduction, and mRNA transcriptional regulation.
Assuntos
Comunicação Autócrina , Neurônios GABAérgicos/metabolismo , Biblioteca Gênica , Glicoproteínas de Membrana/agonistas , Receptor trkB/agonistas , Transdução de Sinais/efeitos dos fármacos , Anticorpos de Cadeia Única , Transcrição Gênica/efeitos dos fármacos , Linhagem Celular , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fosforilação/efeitos dos fármacos , Receptor trkB/genética , Receptor trkB/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacologiaRESUMO
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
BACKGROUND: SRT1720 and SRT2183 were described recently as activators of the NAD+-dependent deacetylase, SIRT1. These molecules enhanced metabolic function when administered to rodents at doses of 100-500 mg/kg/day, purportedly by activating SIRT1 enzymatic activity in various tissues; however, considerable controversy surrounds these claims. RESULTS: We find that these molecules do not activate SIRT1 deacetylase activity when tested in a variety of enzymatic assay formats and conditions. The compounds effectively decrease acetylated p53 in cells treated with DNA damaging agents but do so in cells that lack SIRT1, calling into question their designation as direct activators of SIRT1. In contrast, we find that the compounds inhibit p300 histone acetyltransferase activity in vitro, suggesting a possible mechanism for their effects in vivo. CONCLUSION: Structural features of these molecules may account for false-positive activation using fluorescence-based assays.
Assuntos
Ativadores de Enzimas/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Sirtuínas/metabolismo , Acetilação , Animais , Linhagem Celular , Ativadores de Enzimas/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Camundongos , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
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
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
Identification of genes and pathways that alter lifespan has allowed for new insights into factors that control the aging process as well as disease. While strong molecular links exist between aging and metabolism, we hypothesize that targeting the mechanisms involved in aging will also give rise to therapeutics that treat other devastating age-related diseases, such as neurodegeneration, cancer, inflammation and cardiovascular disease. Insulin sensitivity, glycemic control and adiposity are not only hallmarks of the major metabolic diseases, type 2 diabetes and obesity, but they also represent significant risk factors for the development of Alzheimer's Disease and cognitive impairment. Insulin/IGF-1 signaling is an important pathway regulating aging and disease in a variety of species, including mammals. Here we describe an important role for the gut-derived peptide ghrelin in upstream signaling through the insulin/IGF-1 pathway and exemplify modulation of ghrelin signaling as an approach to mechanistic treatment of multiple age-related diseases by virtue of its ability to regulate key metabolic functions.
Assuntos
Adiposidade , Envelhecimento/fisiologia , Glicemia , Resistência à Insulina , Longevidade/fisiologia , Animais , HumanosRESUMO
BACKGROUND: NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC 1 might be involved in the propagation of apoptotic signaling in neurons. METHODS: CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scanning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC 1 proapoptotic effect. RESULTS: Wild-type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF-sensitive component of cell death. NH(2)-terminal deletion constructs of NARC 1 had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAF. CONCLUSION: The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc 1 active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc 1 protein.
Assuntos
Apoptose/genética , Citometria de Varredura a Laser/métodos , Neurônios/patologia , Serina Endopeptidases/genética , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Caspase 3/metabolismo , Inibidores de Caspase , Células Cultivadas , Cerebelo/citologia , Cerebelo/enzimologia , Cerebelo/patologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Mutação Puntual , Pró-Proteína Convertase 9 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Serina , Serina Endopeptidases/metabolismo , TransfecçãoRESUMO
Molecular genetics in lower organisms has allowed the elucidation of pathways that modulate the aging process. In certain instances, evolutionarily conserved genes and pathways have been shown to regulate lifespan in mammals as well. Many gene products known to affect lifespan are intimately involved in the control of energy metabolism, including the fuel sensor AMP-activated protein kinase (AMPK). We have shown previously that over-expression of an AMPK alpha subunit in Caenorhabditis elegans, designated aak-2, increases lifespan. Here we show the interaction of aak-2 with other pathways known to control aging in worms. Lifespan extension caused by daf-2/insulin-like signaling mutations was highly dependent on aak-2, as was the lifespan extension caused by over-expression of the deacetylase, sir-2.1. Similarly, there was partial requirement for aak-2 in lifespan extension by mitochondrial mutations (isp-1 and clk-1). Conversely, aak-2 was not required for lifespan extension in mutants lacking germline stem cells (glp-1) or mutants of the eating response (eat-2). These results show that aging is controlled by overlapping but distinct pathways and that AMPK/aak-2 represents a node in a network of evolutionarily conserved biochemical pathways that control aging.
Assuntos
Envelhecimento/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/fisiologia , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Nucleotídeos de Adenina/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Comportamento Alimentar , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica no Desenvolvimento , Mutação em Linhagem Germinativa/genética , Larva/crescimento & desenvolvimento , Longevidade , Mitocôndrias/metabolismo , Modelos Biológicos , Ligação Proteica , Sirtuínas/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.
Assuntos
Dano ao DNA , Inibidores de Histona Desacetilases , Sirtuínas/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Catálise/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Sirtuína 1RESUMO
High-throughput screening against the human sirtuin SIRT1 led to the discovery of a series of indoles as potent inhibitors that are selective for SIRT1 over other deacetylases and NAD-processing enzymes. The most potent compounds described herein inhibit SIRT1 with IC50 values of 60-100 nM, representing a 500-fold improvement over previously reported SIRT inhibitors. Preparation of enantiomerically pure indole derivatives allowed for their characterization in vitro and in vivo. Kinetic analyses suggest that these inhibitors bind after the release of nicotinamide from the enzyme and prevent the release of deacetylated peptide and O-acetyl-ADP-ribose, the products of enzyme-catalyzed deacetylation. These SIRT1 inhibitors are low molecular weight, cell-permeable, orally bioavailable, and metabolically stable. These compounds provide chemical tools to study the biology of SIRT1 and to explore therapeutic uses for SIRT1 inhibitors.
Assuntos
Carbazóis/síntese química , Inibidores de Histona Desacetilases , Indóis/síntese química , Sirtuínas/antagonistas & inibidores , Animais , Disponibilidade Biológica , Células CHO , Carbazóis/química , Carbazóis/farmacologia , Permeabilidade da Membrana Celular , Cricetinae , Cricetulus , Estabilidade de Medicamentos , Fluorometria , Histona Desacetilases/química , Humanos , Técnicas In Vitro , Indóis/química , Indóis/farmacologia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , NAD/química , NAD+ Nucleosidase/química , Niacinamida/química , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Sirtuína 1 , Sirtuínas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We describe a microplate-based assay for NAD-dependent Class III histone deacetylases (also known as SIRTs) that measures the enzyme-catalyzed release of nicotinamide from radiolabeled NAD, using a boronic acid resin to selectively capture the NAD. This method avoids the need for fluorogenic or radiolabeled peptides or separation of the reaction products using solvent extraction. The protocol reported here is rapid and uses commercially available materials. The use of a simple microplate filtration device allows for the simultaneous processing of 96 samples, facilitating enzyme kinetic analyses and inhibition studies. Furthermore, monitoring nicotinamide release rather than peptide deacetylation obviates the need for chemical modification of protein and peptide substrates. This assay is applicable to SIRTs and other enzymes that cleave nicotinamide from NAD.
Assuntos
Resinas Acrílicas/química , Ácidos Borônicos/química , NAD/metabolismo , Niacinamida/metabolismo , Sirtuínas/metabolismo , Animais , Radioisótopos de Carbono/química , Catálise , Técnicas de Laboratório Clínico , Filtração/métodos , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Hidrólise , Cinética , Modelos Químicos , Estrutura Molecular , NAD/química , Niacinamida/química , Sirtuínas/químicaRESUMO
There has recently been significant progress in our understanding of the mechanisms that regulate ageing, and it has been shown that changes in single genes can dramatically extend lifespan and increase resistance to many diseases. Furthermore, many of these genes belong to evolutionarily conserved pathways that also control energy metabolism. In this review, we describe the shared molecular machinery that regulates ageing and energy metabolism. Although drugs to slow ageing face severe regulatory hurdles, it is likely that an understanding of ageing pathways will help to identify novel drug targets to treat metabolic disorders and other age-related diseases.
Assuntos
Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Desenho de Fármacos , Metabolismo/efeitos dos fármacos , Metabolismo/fisiologia , Proteínas Quinases Ativadas por AMP , Adipócitos/efeitos dos fármacos , Envelhecimento/genética , Animais , Ingestão de Alimentos/fisiologia , Humanos , Insulina/fisiologia , Expectativa de Vida , Metabolismo/genética , Complexos Multienzimáticos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologiaRESUMO
Resveratrol, a small molecule found in red wine, is reported to slow aging in simple eukaryotes and has been suggested as a potential calorie restriction mimetic. Resveratrol has also been reported to act as a sirtuin activator, and this property has been proposed to account for its anti-aging effects. We show here that resveratrol is a substrate-specific activator of yeast Sir2 and human SirT1. In particular, we observed that, in vitro, resveratrol enhances binding and deacetylation of peptide substrates that contain Fluor de Lys, a non-physiological fluorescent moiety, but has no effect on binding and deacetylation of acetylated peptides lacking the fluorophore. Consistent with these biochemical data we found that in three different yeast strain backgrounds, resveratrol has no detectable effect on Sir2 activity in vivo, as measured by rDNA recombination, transcriptional silencing near telomeres, and life span. In light of these findings, the mechanism accounting for putative longevity effects of resveratrol should be reexamined.