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1.
Curr Genomics ; 19(6): 464-482, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30258277

RESUMO

Aging is a complex phenomenon, where damage accumulation, increasing deregulation of biological pathways, and loss of cellular homeostasis lead to the decline of organismal functions over time. Interestingly, aging is not entirely a stochastic process and progressing at a constant rate, but it is subject to extensive regulation, in the hands of an elaborate and highly interconnected signaling network. This network can integrate a variety of aging-regulatory stimuli, i.e. fertility, nutrient availability, or diverse stresses, and relay them via signaling cascades into gene regulatory events - mostly of genes that confer stress resistance and thus help protect from damage accumulation and homeostasis loss. Transcription factors have long been perceived as the pivotal nodes in this network. Yet, it is well known that the epigenome substantially influences eukaryotic gene regulation, too. A growing body of work has recently underscored the importance of the epigenome also during aging, where it not only undergoes drastic age-dependent changes but also actively influences the aging process. In this review, we introduce the major signaling pathways that regulate age-related decline and discuss the synergy between transcriptional regulation and the epigenetic landscape.

2.
In Vivo ; 37(4): 1672-1679, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37369466

RESUMO

BACKGROUND/AIM: Obesity is associated with the structural and functional disorders related to the molecules of the tissues, cells, and membranes. This study aimed to examine the alterations in the secretion of inflammatory cytokines and metabolic factors and structural changes in inguinal (IF) and gonadal (GF) adipose tissues at the molecular level. MATERIALS AND METHODS: The IF and GF tissues of Berlin Fat Mouse Inbred (BFMI) lines namely BFMI852, BFMI856, BFMI860, BFMI861 obese and DBAJ control mouse lines were used for mRNA expression and Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy (ATR-FTIR) studies. The mRNA levels of inflammatory cytokines including leptin, interleukin 6 (IL-6), tumor necrosis factor-alpha (Tnf-α), and insulin-like growth factor-1 (Igf-1), and peroxisome proliferator-activated receptor gamma 2 (Pparγ-2), were investigated using quantitative reverse transcriptase real-time PCR (qRT-PCR). Infrared spectroscopy does not provide information about specific proteins, instead, it gives information about overall (total) proteins, which is called global information. Therefore, in the current study, adequate information about secondary structures of adipose tissues proteins was obtained using artificial neural network (ANN) and secondary derivative-vector normalization methods based on the spectral profiles. RESULTS: According to the mRNA expression studies, high leptin resistance was found in all BFMI lines. Differences were observed in the levels of measured factors except for Igf-1 among BFMI lines. Protein secondary structure studies showed an increase in random coil contents, especially for BFMI860, which indicates denaturation of the proteins. CONCLUSION: Among the spontaneous obese BFMI mouse lines, the BFMI860 line is the most suitable for obesity studies. Obesity-induced effect on the adipose tissues varies considerably with location, type of adipose tissue, and animal line.


Assuntos
Fator de Crescimento Insulin-Like I , Leptina , Camundongos , Animais , Leptina/genética , Fator de Crescimento Insulin-Like I/genética , Camundongos Obesos , Tecido Adiposo/metabolismo , Obesidade/complicações , Citocinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Commun Biol ; 6(1): 208, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36813870

RESUMO

αKlotho (Klotho) has well established renoprotective effects; however, the molecular pathways mediating its glomerular protection remain incompletely understood. Recent studies have reported that Klotho is expressed in podocytes and protects glomeruli through auto- and paracrine effects. Here, we examined renal expression of Klotho in detail and explored its protective effects in podocyte-specific Klotho knockout mice, and by overexpressing human Klotho in podocytes and hepatocytes. We demonstrate that Klotho is not significantly expressed in podocytes, and transgenic mice with either a targeted deletion or overexpression of Klotho in podocytes lack a glomerular phenotype and have no altered susceptibility to glomerular injury. In contrast, mice with hepatocyte-specific overexpression of Klotho have high circulating levels of soluble Klotho, and when challenged with nephrotoxic serum have less albuminuria and less severe kidney injury compared to wildtype mice. RNA-seq analysis suggests an adaptive response to increased endoplasmic reticulum stress as a putative mechanism of action. To evaluate the clinical relevance of our findings, the results were validated in patients with diabetic nephropathy, and in precision cut kidney slices from human nephrectomies. Together, our data reveal that the glomeruloprotective effects of Klotho is mediated via endocrine actions, which increases its therapeutic potential for patients with glomerular diseases.


Assuntos
Nefropatias Diabéticas , Podócitos , Humanos , Camundongos , Animais , Glomérulos Renais , Nefropatias Diabéticas/metabolismo , Rim/metabolismo , Albuminúria/metabolismo , Camundongos Transgênicos , Camundongos Knockout
4.
Curr Protoc ; 1(7): e187, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34242493

RESUMO

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) has become one of the most popular methods to study protein-DNA interactions and can be used, for instance, to identify the binding sites of transcription factors or to determine the distributions of histones with specific post-translational modifications throughout the genome. Although standard ChIP-seq protocols work well in most experimental systems, there are exceptions, and one of these is the popular model organism Caenorhabditis elegans. Even though this system is very amenable to genetic and cytological methods, biochemical approaches are challenging. This is due to both the animals' cuticle, which impairs lysis as well as penetration by cross-linkers, and the rather low protein and chromatin content per body weight. These issues have rendered standard ChIP-seq protocols inefficient in C. elegans and raised a need for their improvement. Here, we describe improved protocols, with the most important advances being the efficient breakage of the C. elegans cuticle by freeze-grinding and the use of a very sensitive sequencing library construction procedure, optimized for the relatively low DNA content per body weight of C. elegans. The protocols should therefore improve the reproducibility, sensitivity, and uniformity across tissues of ChIP-seq in this organism. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth and harvesting of synchronized Caenorhabditis elegans Basic Protocol 2: Chromatin immunoprecipitation (ChIP) Basic Protocol 3: Library construction for Illumina sequencing.


Assuntos
Caenorhabditis elegans , Sequenciamento de Cromatina por Imunoprecipitação , Animais , Caenorhabditis elegans/genética , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Reprodutibilidade dos Testes
5.
Mol Metab ; 54: 101329, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34454092

RESUMO

OBJECTIVE: The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. METHODS: The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. RESULTS: We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. CONCLUSIONS: Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.


Assuntos
Proteína Forkhead Box O1/metabolismo , Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Adulto , Animais , Desdiferenciação Celular , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade
6.
Nat Commun ; 11(1): 138, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919361

RESUMO

In C. elegans, the conserved transcription factor DAF-16/FOXO is a powerful aging regulator, relaying dire conditions into expression of stress resistance and longevity promoting genes. For some of these functions, including low insulin/IGF signaling (IIS), DAF-16 depends on the protein SMK-1/SMEK, but how SMK-1 exerts this role has remained unknown. We show that SMK-1 functions as part of a specific Protein Phosphatase 4 complex (PP4SMK-1). Loss of PP4SMK-1 hinders transcriptional initiation at several DAF-16-activated genes, predominantly by impairing RNA polymerase II recruitment to their promoters. Search for the relevant substrate of PP4SMK-1 by phosphoproteomics identified the conserved transcriptional regulator SPT-5/SUPT5H, whose knockdown phenocopies the loss of PP4SMK-1. Phosphoregulation of SPT-5 is known to control transcriptional events such as elongation and termination. Here we also show that transcription initiating events are influenced by the phosphorylation status of SPT-5, particularly at DAF-16 target genes where transcriptional initiation appears rate limiting, rendering PP4SMK-1 crucial for many of DAF-16's physiological roles.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Envelhecimento/genética , Animais , Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/genética , Longevidade/genética , Complexos Multiproteicos/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Estresse Fisiológico/genética , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/genética
7.
Cell Rep ; 27(2): 467-480.e6, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970250

RESUMO

Aging strongly influences human morbidity and mortality. Thus, aging-preventive compounds could greatly improve our health and lifespan. Here we screened for such compounds, known as geroprotectors, employing the power of transcriptomics to predict biological age. Using age-stratified human tissue transcriptomes and machine learning, we generated age classifiers and applied these to transcriptomic changes induced by 1,309 different compounds in human cells, ranking these compounds by their ability to induce a "youthful" transcriptional state. Testing the top candidates in C. elegans, we identified two Hsp90 inhibitors, monorden and tanespimycin, which extended the animals' lifespan and improved their health. Hsp90 inhibition induces expression of heat shock proteins known to improve protein homeostasis. Consistently, monorden treatment improved the survival of C. elegans under proteotoxic stress, and its benefits depended on the cytosolic unfolded protein response-inducing transcription factor HSF-1. Taken together, our method represents an innovative geroprotector screening approach and was able to identify a class that acts by improving protein homeostasis.


Assuntos
Envelhecimento/efeitos dos fármacos , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Macrolídeos/farmacologia , Envelhecimento/genética , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcriptoma
8.
Nat Commun ; 9(1): 4400, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30353013

RESUMO

The ability to perceive and respond to harmful conditions is crucial for the survival of any organism. The transcription factor DAF-16/FOXO is central to these responses, relaying distress signals into the expression of stress resistance and longevity promoting genes. However, its sufficiency in fulfilling this complex task has remained unclear. Using C. elegans, we show that DAF-16 does not function alone but as part of a transcriptional regulatory module, together with the transcription factor HLH-30/TFEB. Under harmful conditions, both transcription factors translocate into the nucleus, where they often form a complex, co-occupy target promoters, and co-regulate many target genes. Interestingly though, their synergy is stimulus-dependent: They rely on each other, functioning in the same pathway, to promote longevity or resistance to oxidative stress, but they elicit heat stress responses independently, and they even oppose each other during dauer formation. We propose that this module of DAF-16 and HLH-30 acts by combinatorial gene regulation to relay distress signals into the expression of specific target gene sets, ensuring optimal survival under each given threat.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Longevidade/fisiologia , Estresse Fisiológico , Animais , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Modelos Genéticos , Regiões Promotoras Genéticas , Ligação Proteica
9.
Appl Spectrosc ; 69(6): 679-88, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26054332

RESUMO

The current study aims to determine lipid profiles in terms of the content and structure of skeletal muscle and adipose tissues to better understand the characteristics of juvenile-onset spontaneous obesity without high fat diet induction. For the purposes of this study, muscle (longissimus, quadriceps) and adipose (inguinal, gonadal) tissues of 10-week-old male DBA/2J and Berlin fat mouse inbred (BFMI) lines (BFMI856, BFMI860, BFMI861) fed with a standard breeding diet were used. Biomolecular structure and composition was determined using attenuated total reflection Fourier transform (ATR FT-IR) spectroscopy, and muscle triglyceride content was further quantified using high-performance liquid chromatography (HPLC) coupled with an evaporative light scattering detector (ELSD). The results revealed a loss of unsaturation in BFMI860 and BFMI861 lines in both muscles and inguinal adipose tissue, together with a decrease in the hydrocarbon chain length of lipids, especially in the BFMI860 line in muscles, suggesting an increased lipid peroxidation. There was an increase in saturated lipid and triglyceride content in all tissues of BFMI lines, more profoundly in longissimus muscle, where the increased triglyceride content was quantitatively confirmed by HPLC-ELSD. Moreover, an increase in the metabolic turnover of carbohydrates in muscles of the BFMI860 line was observed. The results demonstrated that subcutaneous (inguinal) fat also displayed considerable obesity-induced alterations. Taken together, the results revealed differences in lipid structure and content of BFMI lines, which may originate from different insulin sensitivity levels of the lines, making them promising animal models for spontaneous obesity. The results will contribute to the understanding of the generation of insulin resistance in obesity without high fat diet induction.


Assuntos
Tecido Adiposo/química , Lipídeos/análise , Músculo Esquelético/química , Obesidade Infantil/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Lipídeos/química , Camundongos , Camundongos Endogâmicos DBA
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