RESUMO
In vivo genetic modification of neural stem cells is necessary to model the origins and pathogenesis of neurological disorders. Electroporation is a technique that applies a transient electrical field to direct charged molecules into living cells to genetically modify the mouse brain. Here, we provide a protocol to electroporate the neural stem cells surrounding the neonatal ventricles. We describe subsequent steps to isolate and prepare nuclei from the cells and their cellular progeny for single-nuclei omics. For complete details on the use and execution of this protocol, please refer to Riley et al.1.
Assuntos
Eletroporação , Células-Tronco Neurais , Animais , Camundongos , Eletroporação/métodos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Núcleo Celular/metabolismo , Separação Celular/métodos , Análise de Célula Única/métodos , Ventrículos Cerebrais/citologiaRESUMO
Betaine-homocysteine S-methyltransferase (BHMT) is one of the most abundant proteins in the liver and regulates homocysteine metabolism. However, the molecular mechanisms underlying Bhmt transcription have not yet been elucidated. This study aimed to assess the molecular mechanisms underlying Bhmt transcription and the effect of BHMT deficiency on metabolic functions in the liver mediated by liver receptor homolog-1 (LRH-1). During fasting, both Bhmt and Lrh-1 expression increased in the liver of Lrh-1f/f mice; however, Bhmt expression was decreased in LRH-1 liver specific knockout mice. Promoter activity analysis confirmed that LRH-1 binds to a specific site in the Bhmt promoter region. LRH-1 deficiency was associated with elevated production of reactive oxygen species (ROS), lipid peroxidation, and mitochondrial stress in hepatocytes, contributing to hepatic triglyceride (TG) accumulation. In conclusion, this study suggests that the absence of an LRH-1-mediated decrease in Bhmt expression promotes TG accumulation by increasing ROS levels and inducing mitochondrial stress. Therefore, LRH-1 deficiency not only leads to excess ROS production and mitochondrial stress in hepatocytes, but also disrupts the methionine cycle. Understanding these regulatory pathways may pave the way for novel therapeutic interventions against metabolic disorders associated with hepatic lipid accumulation.
Assuntos
Betaína-Homocisteína S-Metiltransferase , Hepatócitos , Fígado , Metionina , Camundongos Knockout , Espécies Reativas de Oxigênio , Receptores Citoplasmáticos e Nucleares , Triglicerídeos , Animais , Fígado/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Betaína-Homocisteína S-Metiltransferase/metabolismo , Betaína-Homocisteína S-Metiltransferase/genética , Hepatócitos/metabolismo , Metionina/metabolismo , Triglicerídeos/metabolismo , Regiões Promotoras Genéticas/genética , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Peroxidação de LipídeosRESUMO
In this editorial, we comment on an article by Liao et al published in the current issue of the World Journal of Diabetes. We focus on the clinical significance of tibial transverse transport (TTT) as an effective treatment for patients with diabetic foot ulcers (DFU). TTT has been associated with tissue regeneration, improved blood circulation, reduced amputation rates, and increased expression of early angiogenic factors. Mechanistically, TTT can influence macrophage polarization and growth factor upregulation. Despite this potential, the limitations and conflicting results of existing studies justify the need for further research into its optimal application and development. These clinical implications highlight the efficacy of TTT in recalcitrant DFU and provide lasting stimuli for tissue re-generation, and blood vessel and bone marrow improvement. Immunomodulation via systemic responses contributes to its therapeutic potential. Future studies should investigate the underlying molecular mechanisms to enhance our understanding and the efficacy of TTT. This manuscript emphasizes the potential of TTT in limb preservation and diabetic wound healing and suggests avenues for preventive measures against limb amputation in diabetes and peripheral artery disease. Here, we highlight the clinical significance of the TTT and its importance in healing DFU to promote the use of this technique in tissue regeneration.
RESUMO
Tetrabutylammonium bromide (TBAB) is a widely used industrial reagent and is commonly found in our aquatic ecosystem as an industrial byproduct. In humans, the ingestion of TBAB causes severe neurological impairments and disorders such as vertigo, hallucinations, and delirium. Yet, the extent of environmental risk and TBAB toxicity to human health is poorly understood. In this study, we aim to determine the developmental toxicity of TBAB using zebrafish embryos as a model and provide novel insights into the mechanism of action of such chemicals on neurodevelopment and the overall embryonic program. Our results show that exposure to TBAB results in impaired development of the brain, inner ear, and pharyngeal skeletal elements in the zebrafish embryo. TBAB treatment resulted in aberrations in the specification of the neural crest precursors, hindbrain segmentation, and otic neurogenesis. TBAB treatment also induced a surge in apoptosis in the head, tail, and trunk regions of the developing embryo. Long-term TBAB exposure resulted in cardiac edema and craniofacial defects. Further, in silico molecular docking analysis indicated that TBAB binds to AMPA receptors and modulates neural developmental genes such as olfactomedin and acetylcholinesterase in the embryonic brain. To summarize, our study highlights the novel effects of TBAB on embryonic brain formation and segmentation, ear morphogenesis, and craniofacial skeletal development.
Assuntos
Crista Neural , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/metabolismo , Crista Neural/metabolismo , Acetilcolinesterase/metabolismo , Ecossistema , Simulação de Acoplamento Molecular , Encéfalo/metabolismo , Proteínas de Peixe-Zebra/genética , Neurogênese , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Colchicine has been used for therapeutic purposes and has attracted considerable attention because of its association with tubulin and the inhibition of small tubular polymerization. Although several studies have examined the possible preventive role of colchicine in metabolic diseases, its role in adipocytes is largely unknown. This study examined the novel functional role of colchicine in adipocytes demonstrating that colchicine stimulates browning in cultured white adipocytes. Colchicine stimulates browning by increasing the brown- and beige fat-specific markers in 3T3-L1 white adipocytes. Interestingly, colchicine decreased the expression of the main lipolytic proteins (ATGL, p-HSL) while it activated Ces3, suggesting a possibility for supplying essential fatty acids for inducing thermogenesis. Molecular docking analysis showed that colchicine has a strong affinity against GABA-BR and ß3-AR, and its binding activity with GABA-BR (-26.52 kJ/mol) was stronger than ß3-AR (-20.71 kJ/mol). Mechanistic studies were conducted by treating the cells separately with agonists and antagonists of GABA-BR and ß3-AR to understand the molecular mechanism underlying the browning effect of colchicine. The results showed that colchicine stimulates browning via the antagonism of GABA-BR and the agonism of ß3-AR in 3T3-L1 white adipocytes. The colchicine-mediated activation of ß3-AR stimulated the PKA/p38 MAPK signaling pathway, where consequently ATF2 acted as a positive regulator, but AFT4 was a negative regulator for the induction of browning.
Assuntos
Adipócitos Brancos , Receptores de GABA , Células 3T3-L1 , Adipócitos Marrons/metabolismo , Animais , Colchicina/metabolismo , Colchicina/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Receptores Adrenérgicos/metabolismo , Receptores de GABA/metabolismo , Transdução de Sinais , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
BACKGROUND: Natural compounds with medicinal properties are part of a strategic trend in the treatment of obesity. The vitamin A agent, ß-carotene, is a well-known carotenoid, and its numerous functions in metabolism have been widely studied. The activation of thermogenesis by stimulating white fat browning (beiging) has been identified as a treatment for obese individuals. PURPOSE: The current study was undertaken to unveil the browning activity of ß-carotene in 3T3-L1 white adipocytes. METHODS: The effects of ß-carotene were evaluated in 3T3-L1 white adipocytes, and gene/protein expressions were determined by performing quantitative real-time PCR, immunoblot analysis, immunofluorescence assessment, and molecular docking techniques. RESULTS: ß-carotene strikingly increased the expression levels of brown-fat-specific marker proteins (UCP1, PRDM16, and PGC-1α) and beige-fat-specific genes (Cd137, Cidea, Cited1, andTbx1) in 3T3-L1 cells. Exposure to ß-carotene also elevated the expressions of key adipogenic transcription factors C/EBPα and PPARγ in white adipocytes but decreased the expressions of lipogenic marker proteins ACC and FAS. Moreover, lipolysis and fat oxidation were regulated by ß-carotene via upregulation of ATGL, pHSL, ACOX, and CPT1. In addition, molecular docking studies revealed ß-carotene activation of the adenosine A2A receptor and ß3-AR. ß-Carotene increased the expressions of mitochondrial biogenic markers, stimulated the ß3-AR and p38 MAPK signaling pathways and its downstream signaling molecules (SIRTs and ATF2), thereby inducing browning. CONCLUSIONS: Taken together, our results indicate the potential of ß-carotene as a natural-source therapeutic anti-obesity agent.
Assuntos
Sirtuínas , beta Caroteno , Células 3T3-L1 , Adipócitos Marrons , Adipócitos Brancos , Animais , Humanos , Camundongos , Simulação de Acoplamento Molecular , Transdução de Sinais , Termogênese , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
AIMS: Prednisone is a corticosteroid-derived drug which is widely used for its role in immunosuppression and treatment of lung disorders. The current study reports, for the first time, the critical role of prednisone in the induction of white fat browning, thereby promoting thermogenic effect in cultured white adipocytes. MAIN METHODS: The fat-browning activity of prednisone was evaluated in 3T3-L1 cells by quantitative real-time PCR, immunoblot analysis, immunofluorescence, and molecular docking techniques. KEY FINDINGS: Exposure to prednisone stimulated browning in 3T3-L1 white adipocytes by increasing the expressions of core fat browning marker proteins (UCP1, PGC-1α and PRDM16) as well as beige-specific genes (Cd137, Cidea, Cited1, and Tbx1) via ATF2 and CREB activation mediated by p38 MAPK and ERK signaling, respectively. Prednisone exposure also resulted in the robust activation of lipolytic and fatty acid oxidation marker proteins, thereby increasing mitochondrial biogenesis. In addition, prednisone treatment resulted in reduced expression levels of adipogenic transcription factors while elevating SIRT1, as well as attenuation of lipogenesis and lipid droplets formation. Furthermore, molecular docking and mechanistic studies demonstrated the recruitment of beige fat by prednisone via the ß3-AR/p38 MAPK/ERK signaling pathway. SIGNIFICANCE: Taken together, these results indicate the unique role of prednisone as a fat-browning stimulant, and demonstrate its therapeutic potential in the treatment of obesity by enhancing thermogenesis.
Assuntos
Adipócitos Marrons/citologia , Adipócitos Brancos/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Prednisona/farmacologia , Receptores Adrenérgicos beta 3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Regulação da Expressão Gênica , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Receptores Adrenérgicos beta 3/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Microbial fermentation of grape-skin extracts is found to synthesize anthocyanin oligomers (AO), which are more active than the monomeric anthocyanins that are effective for some metabolic diseases such as diabetes and obesity. This study investigated the functional role of AO in 3T3-L1 white adipocyte metabolism, with a focus on inducing browning. To achieve this, we determined the expressions of core genes and protein markers responsible for browning and lipid metabolism in response to AO treatment of 3T3-L1 white adipocytes. AO exposure significantly increases the expressions of beige-specific genes (Cidea, Cited1, Ppargc1α, Prdm16, Tbx1, Tmem26, and Ucp1) and brown-fat signature proteins (UCP1, PRDM16, and PGC-1α), and suppresses the expressions of lipogenic marker proteins while enhancing the protein levels of lipolysis in white adipocytes. The mechanistic study revealed stimulation of white fat browning via activation of the ß3-AR/PKA/p38 axis and ERK/CREB signaling pathway subsequent to AO treatment. In conclusion, our current findings indicate the beneficial effects of AO for the treatment of obesity with interesting properties such as regulating the browning of adipocytes and increasing thermogenic activity. Although further research based on animal models or clinical trials remains, AO treatment can bring more insights into the treatment of obesity and metabolic syndrome.
Assuntos
Adipócitos Brancos , Antocianinas , Células 3T3-L1 , Adipócitos Marrons , Animais , Antocianinas/farmacologia , Sistema de Sinalização das MAP Quinases , Camundongos , Receptores Adrenérgicos , Transdução de Sinais , TermogêneseRESUMO
Embryos and microscopes share a long, remarkable history and biologists have always been intrigued to watch how embryos develop under the microscope. Here we discuss the advances in microscopy which have greatly influenced our current understanding of embryogenesis. We highlight the evolution of microscopes and the optical technologies that have been instrumental in studying various developmental processes. These imaging modalities provide mechanistic insights into the dynamic cellular and molecular events which drive lineage commitment and morphogenetic changes in the developing embryo. We begin the journey with a brief history of microscopy to study embryos. First, we review the principles and optics of light, fluorescence, confocal, and electron microscopy which have been key techniques for imaging cellular and molecular events during embryonic development. Next, we discuss recent key imaging modalities such as light-sheet microscopy, which are suitable for whole embryo imaging. Further, we highlight imaging techniques like multiphoton and super resolution microscopy for beyond light diffraction limit, high resolution imaging. Lastly, we review some of the scattering-based imaging methods and techniques used for imaging human embryos.
Assuntos
Desenvolvimento Embrionário , Microscopia , Embrião de Mamíferos , Feminino , Humanos , GravidezRESUMO
Induction of white fat browning (beiging) and activation of brown fat has been considered a promising strategy to treat obesity and associated metabolic complications. However, the molecular mechanisms regulating brown and beige fat-mediated thermogenesis remains unclear. Our study aimed to identify genes with a hitherto unknown mechanism in the metabolic functions of adipocytes and identified family with sequence similarity 107, member A (FAM107A) as a factor that interferes with fat browning in white adipocytes. We explored physiological roles of FAM107A in cultured 3T3-L1 white adipocytes and HIB1B brown adipocytes by using FAM107A-deficient adipocytes. Significant loss in FAM107A gene functionality induced fat browning was evidenced by evaluating the gene and protein expression level of brown fat-associated markers through real-time qRT-PCR and immunoblot analysis, respectively. Deficiency of FAM107A promoted mitochondrial biogenesis and significantly upregulated core fat-browning marker proteins (PGC-1α, PRDM16, and UCP1) and beige-specific genes (Cd137, Cited1, Tbx1, and Tmem26). Furthermore, FAM107A increased adipogenesis and negatively regulated lipid metabolism in 3T3-L1 adipocytes. In addition, in-silico analysis revealed a strong interaction between FAM107A and ß3-AR based on their energy binding score. Next, mechanistic study revealed that specific knockdown of FAM107A induces browning in white adipocytes via activation of ß3-AR, AMPK and p38 MAPK-dependent signaling pathways. Our data unveiled a previously unknown mechanism of FAM107A in the regulation of lipid metabolism and identified its significant role in metabolic homeostasis. This highlighted the potential of FAM107A as a pharmacotherapeutic target in treating obesity and related metabolic disorders.
Assuntos
Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Antígenos de Diferenciação/biossíntese , Regulação da Expressão Gênica , Termogênese , Proteínas Supressoras de Tumor/deficiência , Células 3T3-L1 , Animais , Metabolismo dos Lipídeos/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Proteínas Supressoras de Tumor/metabolismoRESUMO
AIMS: The bone-adipose axis requires complex homeostasis in energy and global metabolism. The bioenergetics of bone establishes the necessary energy balance to coordinate endocrine functions that are affected by various factors and is not limited to matrix proteins only. UCP1 is an uncoupling protein of adipocytes, commonly known for its unique feature of promoting thermogenesis, mainly in brown fat; however, the effects of UCP1 in other cell types remain unreported. MAIN METHODS: In the current study, we determined the roles of UCP1 in osteoblasts by silencing the Ucp1 gene in MC-3T3-E1 cells, as well as C3H10T1/2 mesenchymal stem cells, and explored its functional activities. KEY FINDINGS: Our results demonstrate for the first time the presence of UCP1 in osteoblast cells. We identified that UCP1 regulates ATP and oxidative phosphorylation in MC-3T3-E1 cells. In addition, our data reveal that the lack of Ucp1 results in reduced expressions of regulatory proteins involved in scavenging of ROS by enhancing an autophagic event to balance osteogenic differentiation. SIGNIFICANCE: In conclusion, this study highlights a novel perspective on the importance of UCP1 in bone cells.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Osteoblastos/fisiologia , Osteogênese , Fosforilação Oxidativa , Proteína Desacopladora 1/metabolismo , Animais , Autofagia , Células Cultivadas , Camundongos , Osteoblastos/citologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Bone morphogenetic protein-11 (BMP11), also known as growth differentiation factor-11 (GDF11), is implicated in skeletal development and joint morphogenesis in mammals. However, its functions in adipogenesis and energy homeostasis are mostly unknown. The present study investigates crucial roles of BMP11 in cultured 3T3-L1 white and HIB1B brown adipocytes, using Bmp11 gene depletion and pharmacological inhibition of BMP11. The silencing of Bmp11 markedly decreases the expression levels of brown-fat signature proteins and beige-specific genes in white adipocytes and significantly down-regulates the expression levels of brown fat-specific genes in brown adipocytes. The deficiency of Bmp11 reduces the expressions of lipolytic protein markers in white and brown adipocytes. Moreover, BMP11 induces browning of 3T3-L1 adipocytes via coordination of multiple signalling pathways, including mTORC1-COX2 and p38MAPK-PGC-1α as non-canonical pathways, as well as Smad1/5/8 as a canonical pathway. We believe this study is the first to provide evidence of the potential roles of BMP11 for improvement of lipid catabolism in both cultured white and brown adipocytes, as well as the effect on browning of white adipocytes. Taken together, these results demonstrate the therapeutic potential for the treatment of obesity.
Assuntos
Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Termogênese , Animais , Proteínas Morfogenéticas Ósseas/deficiência , Proteínas Morfogenéticas Ósseas/genética , Células Cultivadas , Fatores de Diferenciação de Crescimento/deficiência , Fatores de Diferenciação de Crescimento/genética , Camundongos , Mitocôndrias/metabolismoRESUMO
Trigonelline, a major alkaloid component of fenugreek, has been demonstrated to have several biological activities, including antidiabetic and anticancer effects. This study aimed to examine the possible application of trigonelline as an anti-obesity compound based on an investigation of its enhancement of lipid catabolism and induction of browning in white adipocytes. Trigonelline induces browning of 3T3-L1 white adipocytes by enhancing the expressions of brown-fat signature proteins and genes as well as beige-specific genes, including Cd137, Cited1, Tbx1, and Tmem26. Trigonelline also improves lipid metabolism in white adipocytes by decreasing adipogenesis and lipogenesis as well as promotes lipolysis and fatty acid oxidation. Moreover, trigonelline increases the expression of Cox4, Nrf1, and Tfam genes that are responsible for mitochondrial biogenesis. Mechanistic studies revealed that the browning effect of trigonelline in 3T3-L1 white adipocytes is mediated by activating ß3-AR and inhibiting PDE4, thereby stimulating the p38 MAPK/ATF-2 signaling pathway. Considering its high bioavailability in humans and the results of this study, trigonelline may have potential as an anti-obesity compound.
Assuntos
Células 3T3-L1/metabolismo , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/efeitos dos fármacos , Alcaloides/uso terapêutico , Obesidade/tratamento farmacológico , Alcaloides/farmacologia , Animais , Humanos , CamundongosRESUMO
The nonsteroidal anti-inflammatory drug (NSAID) ketoprofen is commonly used as a pain reliever, but its role in mediating the energy metabolism in lipids is unclear. This paper reports for the first time the critical role of ketoprofen in ameliorating white fat browning and alleviating diet-induced obesity. The effects of ketoprofen were evaluated using C57BL/6 mice fed a high fat diet and the expression levels of the target genes and proteins in the lipid metabolisms were determined by quantitative real-time PCR, immunoblot analysis, histopathology study, immunofluorescence, and molecular docking techniques. Ketoprofen induced browning in cultured 3T3-L1 white adipocytes and inguinal white adipose tissue by increasing the expression of core fat browning marker proteins as well as beige-specific genes through COX-2 activation. Ketoprofen also led to the robust activation of brown adipocytes and enhanced brown fat adipogenesis. In addition, ketoprofen elevated lipolysis, thereby increasing mitochondrial biogenesis resulting in higher fat oxidation. Furthermore, the molecular docking and mechanistic study demonstrated the recruitment of beige fat by ketoprofen via mTORC1-p38-mediated activation of the COX-2 pathway. Overall, these results indicate the unique role of ketoprofen in body weight reduction by enhancing thermogenesis, suggesting its therapeutic potential in the treatment of obesity.
Assuntos
Tecido Adiposo Branco/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Ciclo-Oxigenase 2/metabolismo , Cetoprofeno/uso terapêutico , Obesidade/tratamento farmacológico , Biogênese de Organelas , Células 3T3-L1 , Tecido Adiposo Branco/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Dieta Hiperlipídica/efeitos adversos , Cetoprofeno/farmacologia , Lipólise , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
SPARC, also known as osteonectin, is well known for its physiological roles in bone formation and tissue remodeling, as well as in cancer pathology; however, evidence regarding its function in adipocytes is lacking. The present study explored the physiological role of SPARC in cultured 3T3-L1 white and HIB1B brown adipocytes of murine cell lines. Treatment of recombinant SPARC upregulated the fat browning marker proteins and genes in white adipocytes and activated brown adipocytes. Conversely, knockdown of Sparc markedly reduced these genes and proteins in both cell lines. In addition, recombinant SPARC inhibited expression of adipogenic and lipogenic proteins but elevated lipolytic and fatty acid oxidation proteins. Furthermore, in silico analysis revealed that SPARC directly interacted and regulated VEGF in adipocytes. In conclusion, SPARC acts as a regulatory protein in both white and brown adipocytes by controlling thermogenesis and is thus regarded as a possible therapeutic target for treatment of obesity.
Assuntos
Adipócitos Marrons/fisiologia , Adipócitos Brancos/fisiologia , Osteonectina/fisiologia , Termogênese/genética , Células 3T3-L1 , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Camundongos , Osteonectina/farmacologia , Proteínas Recombinantes/farmacologia , Termogênese/efeitos dos fármacosRESUMO
PURPOSE: Modern science has given much attention to the treatment of obesity by activating brown adipose tissue (BAT) and browning of white adipose tissue (WAT). Recent studies have identified theobromine, a derivative of cocoa, as a potent natural component actively browning white fat cells. Here, we aimed to deduce the anti-obesity effect of theobromine involving phosphodiesterase (PDE) dependent-regulatory pathway in obese animal models. METHODS: For examining activity of theobromine, C57BL/6 mice were fed with high fat diet and treated with theobromine to determine the expression levels of protein markers by immunoblot analysis and gene targets by quantitative real-time PCR. Other methods used include histopathological studies, immunofluorescence and molecular docking approaches. RESULTS: Theobromine alleviated diet-induced obesity in mice by browning of iWAT and activating BAT. Further, theobromine actively interacted with PDE4D and inhibited its activity in adipose tissues and cells potentiating energy expenditure. Moreover, the regulatory action of theobromine via inhibition of PDE4D was mediated by ß3-AR signaling pathway. CONCLUSION: Altogether, the current results signifies critical role of theobromine in reducing obesity by regulation of lipid metabolism through inhibition of PDE4, indicating its potential as a major therapeutic medicinal compound.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Teobromina , Tecido Adiposo Marrom , Tecido Adiposo Branco , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Dieta Hiperlipídica/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Obesidade/tratamento farmacológicoRESUMO
Recently, pharmacological activation of brown fat and induction of white fat browning (beiging) have been considered promising strategies to treat obesity. To search for natural products that could stimulate the process of browning in adipocytes, we evaluated the activity of trans-cinnamic acid (tCA), a class of cinnamon from the bark of Cinnamomum cassia, by determining genetic expression using real time reverse transcription polymerase chain reaction (RT-PCR) and protein expression by immunoblot analysis for thermogenic and fat metabolizing markers. In our study tCA induced brown like-phenotype in 3T3-L1 white adipocytes and activated HIB1B brown adipocytes. tCA increased protein content of brown-fat-specific markers (UCP1, PRDM16, and PGC-1α) and expression levels of beige-fat-specific genes (Cd137, Cidea, Cited1, Tbx1, and Tmen26) in 3T3-L1 white adipocytes, as well as brown-fat-specific genes (Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. Furthermore, tCA reduced expression of key adipogenic transcription factors C/EBPα and PPARγ in white adipocytes, but enhanced their expressions in brown adipocytes. In addition, tCA upregulates lipid catabolism. Moreover, mechanistic study revealed that tCA induced browning in white adipocytes by activating the ß3-AR and AMPK signaling pathways. tCA can induce browning, increase fat oxidation, reduce adipogenesis and lipogenesis in 3T3-L1 adipocytes, and activate HIB1B adipocytes, suggesting its potential to treat obesity.
Assuntos
Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/efeitos dos fármacos , Cinamatos/farmacologia , Células 3T3-L1 , Animais , Cinamatos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Estrutura MolecularRESUMO
The role of carboxylesterase 3 (Ces3) in the lipolysis of adipocytes has been overlooked, as 2 major lipolytic enzymes, hormone-sensitive lipase and adipose triglyceride lipase, play more powerful roles in lipolysis. In this study, we explored the effects of Ces3 in lipid metabolism by activating and inhibiting, as well as silencing, Ces3-encoding gene in 3T3-L1 cell model. Our results demonstrated that activation of Ces3 increased adipogenesis, and attenuated lipogenesis, whereas it promoted lipolysis and fatty acid oxidation. In addition, activated Ces3 led to enhanced expression of core fat browning marker genes and proteins, suggesting that Ces3 may play a pivotal role in fat browning and thermogenesis. In contrast, deficiency of Ces3 nullified the browning effect in white adipocytes, along with decreased adipogenesis in 3T3-L1 adipocytes. Interestingly, the expression pattern of adipose triglyceride lipase was in line with Ces3, whereas hormone-sensitive lipase was independently regulated irrespective of Ces3 expression levels, suggesting that Ces3 may play an important and compensatory role in the breakdown of triglycerides in white adipocytes. In conclusion, we provide the first evidence that activation of Ces3 contributes in the browning of white adipocytes, and maintains a balance in lipid metabolism, which could be a potential strategy in fighting against obesity.
Assuntos
Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Carboxilesterase/metabolismo , Metabolismo dos Lipídeos/fisiologia , Células 3T3-L1 , Adipogenia , Animais , Carboxilesterase/antagonistas & inibidores , Carboxilesterase/genética , Ativação Enzimática , Técnicas de Silenciamento de Genes , Lipogênese , Camundongos , TermogêneseRESUMO
To treat obesity, suppression of white adipose tissue (WAT) expansion and activation of brown adipose tissue (BAT) are considered as potential therapeutic targets. Recent advances have been made in the induction of brown fat-like adipocytes (beige) in WAT, which represents an attractive potential strategy for the management and treatment of obesity. Use of natural compounds for browning of white adipocytes can be considered as a safe and novel strategy against obesity. Here, we report that trans-anethole (TA), a flavoring substance present in the essential oils of various plants, alleviated high fat diet (HFD)-induced obesity in mice models via elevation of the expression of beige-specific genes such as Ppargc1α, Prdm16, Ucp1, Cd137, Cited1, Tbx1, and Tmem26. TA also regulated lipid metabolism in white adipocytes via reduction of adipogenesis and lipogenesis as well as elevation of lipolysis and fat oxidation. Moreover, TA exhibited thermogenic activity by increasing mitochondrial biogenesis in white adipocytes and activating brown adipocytes. In addition, molecular docking analysis enabled us to successfully predict core proteins for fat browning such as ß3-adrenergic receptor (ß3-AR) and sirtuin1 (SIRT1) based on their low binding energy interactions with TA for promotion of regulatory mechanisms. Indeed, agonistic and antagonistic studies demonstrated that TA induced browning of 3T3-L1 adipocytes through activation of ß3-AR as well as the AMPK-mediated SIRT1 pathway regulating PPARα and PGC-1α. In conclusion, TA possesses potential therapeutic implications for treatment of obesity by playing multiple modulatory roles in the induction of white fat browning, activation of brown adipocytes, and promotion of lipid catabolism.
Assuntos
Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/efeitos dos fármacos , Anisóis/farmacologia , Aromatizantes/farmacologia , Obesidade/prevenção & controle , Células 3T3-L1 , Adipócitos Bege/efeitos dos fármacos , Adipócitos Bege/metabolismo , Adipócitos Marrons/metabolismo , Adipócitos Marrons/fisiologia , Adipócitos Brancos/metabolismo , Adipócitos Brancos/fisiologia , Derivados de Alilbenzenos , Animais , Dieta Hiperlipídica , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Obesidade/patologia , Obesidade/fisiopatologia , Termogênese/efeitos dos fármacosRESUMO
Induction of the brown adipocyte-like phenotype in white adipocytes (browning) is considered as a novel strategy to fight obesity due to the ability of brown adipocytes to increase energy expenditure. Here, we report that L-rhamnose induced browning by elevating expression levels of beige-specific marker genes, including Cd137, Cited1, Tbx1, Prdm16, Tmem26, and Ucp1, in 3T3-L1 adipocytes. Moreover, L-rhamnose markedly elevated expression levels of proteins involved in thermogenesis both in 3T3-L1 white and HIB1B brown adipocytes. L-rhamnose treatment in 3T3-L1 adipocytes also significantly elevated protein levels of p-HSL, p-AMPK, ACOX, and CPT1 as well as reduced levels of ACC, FAS, C/EBPα, and PPARγ, suggesting its possible role in enhancement of lipolysis and lipid catabolism as well as reduced adipogenesis and lipogenesis, respectively. The quick technique of efficient molecular docking provided insight into the strong binding of L-rhamnose to the fat-digesting glycine residue of ß3 -adrenergic receptor (AR), indicating strong involvement of L-rhamnose in fat metabolism. Further examination of the molecular mechanism of L-rhamnose revealed that it induced browning of 3T3-L1 adipocytes via coordination of multiple signaling pathways through ß3 -AR, SIRT1, PKA, and p-38. To the best of our knowledge, this is the first study to demonstrate that L-rhamnose plays multiple modulatory roles in the induction of white fat browning, activation of brown adipocytes, as well as promotion of lipid metabolism, thereby demonstrating its therapeutic potential for treatment of obesity. © 2018 IUBMB Life, 70(6):563-573, 2018.