RESUMEN
Adipose tissue is highly plastic, as illustrated mainly by the transdifferentiation of white adipocytes into beige adipocytes, depending on environmental conditions. However, during gestation and lactation in rodent, there is an amazing phenomenon of transformation of subcutaneous adipose tissue into mammary glandular tissue, known as pink adipose tissue, capable of synthesizing and secreting milk. Recent work using transgenic lineage-tracing experiments, mainly carried out in Saverio Cinti's team, has demonstrated very convincingly that this process does indeed correspond to a transdifferentiation of white adipocytes into mammary alveolar cells (pink adipocytes) during gestation and lactation. This phenomenon is reversible, since during the post-lactation phase, pink adipocytes revert to the white adipocyte phenotype. The molecular mechanisms underlying this reversible transdifferentiation remain poorly understood.
Asunto(s)
Tejido Adiposo , Lactancia , Animales , Humanos , Femenino , Tejido Adiposo/fisiología , Tejido Adiposo/metabolismo , Tejido Adiposo/citología , Lactancia/fisiología , Embarazo , Transdiferenciación Celular/fisiología , Glándulas Mamarias Animales/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Adipocitos Blancos/fisiología , Adipocitos Blancos/metabolismo , Adipocitos Blancos/citología , Plasticidad de la Célula/fisiología , Glándulas Mamarias Humanas/fisiología , Glándulas Mamarias Humanas/crecimiento & desarrollo , Glándulas Mamarias Humanas/citología , Adipocitos/fisiología , Adipocitos/citologíaAsunto(s)
Adipocitos Beige , Adipocitos Marrones , Adipocitos Blancos , Tejido Adiposo Blanco , Humanos , Adipocitos Marrones/fisiología , Adipocitos Blancos/fisiología , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Animales , Adipocitos Beige/fisiología , Adipocitos Beige/metabolismo , Adipocitos Beige/citología , Tejido Adiposo Blanco/fisiología , Tejido Adiposo Blanco/citología , Tejido Adiposo Pardo/fisiología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo/fisiología , Tejido Adiposo/metabolismo , Tejido Adiposo/citología , Obesidad/patología , Adipocitos/fisiologíaRESUMEN
Protein crotonylation plays a role in regulating cellular metabolism, gene expression, and other biological processes. NDUFA9 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9) is closely associated with the activity and function of mitochondrial respiratory chain complex I. Mitochondrial function and respiratory chain are closely related to browning of white adipocytes, it's speculated that NDUFA9 and its crotonylation are associated with browning of white adipocytes. Firstly, the effect of NDUFA9 on white adipose tissue was verified in white fat browning model mice, and it was found that NDUFA9 promoted mitochondrial respiration, thermogenesis, and browning of white adipose tissue. Secondly, in cellular studies, it was discovered that NDUFA9 facilitated browning of white adipocytes by enhancing mitochondrial function, mitochondrial complex I activity, ATP synthesis, and mitochondrial respiration. Again, the level of NDUFA9 crotonylation was increased by treating cells with vorinostat (SAHA)+sodium crotonate (NaCr) and overexpressing NDUFA9, it was found that NDUFA9 crotonylation promoted browning of white adipocytes. Meanwhile, the acetylation level of NDUFA9 was increased by treating cells with SAHA+sodium acetate (NaAc) and overexpressing NDUFA9, the assay revealed that NDUFA9 acetylation inhibited white adipocytes browning. Finally, combined with the competitive relationship between acetylation and crotonylation, it was also demonstrated that NDUFA9 crotonylation promoted browning of white adipocytes. Above results indicate that NDUFA9 and its crotonylation modification promote mitochondrial function, which in turn promotes browning of white adipocytes. This study establishes a theoretical foundation for the management and intervention of obesity, which is crucial in addressing obesity and related medical conditions in the future.
Asunto(s)
Adipocitos Blancos , Mitocondrias , Animales , Ratones , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Adipocitos Blancos/metabolismo , Adipocitos Blancos/efectos de los fármacos , Adipocitos Blancos/citología , Masculino , Ratones Endogámicos C57BL , Termogénesis/efectos de los fármacos , Adipocitos Marrones/metabolismo , Adipocitos Marrones/efectos de los fármacos , Células 3T3-L1 , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/citología , Acetilación/efectos de los fármacosRESUMEN
The role of mitochondria in white adipocytes (WAs) has not been fully explored. A recent study revealed that brown adipocytes contain functionally distinct mitochondrial fractions, cytoplasmic mitochondria, and peridroplet mitochondria. However, it is not known whether such a functional division of mitochondria exists in WA. Herein, we observed that mitochondria could be imaged and mitochondrial DNA and protein detected in pellets obtained from the cytoplasmic layer and oil layer of WAs after centrifugation. The mitochondria in each fraction were designated as cytoplasmic mitochondria (CMw) and peridroplet mitochondria (PDMw) in WAs, respectively. CMw had higher ß-oxidation activity than PDMw, and PDMw was associated with diacylglycerol acyltransferase 2. Therefore, CMw may be involved in ß-oxidation and PDMw in droplet expansion in WAs.
Asunto(s)
Adipocitos Blancos , Citoplasma , Mitocondrias , Mitocondrias/metabolismo , Mitocondrias/genética , Animales , Citoplasma/metabolismo , Adipocitos Blancos/metabolismo , Adipocitos Blancos/citología , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , Ratones , Oxidación-Reducción , Diacilglicerol O-Acetiltransferasa/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genéticaRESUMEN
Family with sequence similarity 83 A (FAM83A) is a newly discovered proto-oncogene that has been shown to play key roles in various cancers. However, the function of FAM83A in other physiological processes is not well known. Here, we report a novel function of FAM83A in adipocyte differentiation. We used an adipocyte-targeting fusion oligopeptide (FITC-ATS-9R) to deliver a FAM83A-sgRNA/Cas9 plasmid to knockdown Fam83a (ATS/sg-FAM83A) in white adipose tissue in mice, which resulted in reduced white adipose tissue mass, smaller adipocytes, and mitochondrial damage that was aggravated by a high-fat diet. In cultured 3T3-L1 adipocytes, we found loss or knockdown of Fam83a significantly repressed lipid droplet formation and downregulated the expression of lipogenic genes and proteins. Furthermore, inhibition of Fam83a decreased mitochondrial ATP production through blockage of the electron transport chain, associated with enhanced apoptosis. Mechanistically, we demonstrate FAM83A interacts with casein kinase 1 (CK1) and promotes the permeability of the mitochondrial outer membrane. Furthermore, loss of Fam83a in adipocytes hampered the formation of the TOM40 complex and impeded CK1-driven lipogenesis. Taken together, these results establish FAM83A as a critical regulator of mitochondria maintenance during adipogenesis.
Asunto(s)
Adipocitos Blancos , Adipogénesis , Quinasa de la Caseína I , Mitocondrias , Proteínas de Neoplasias , Proto-Oncogenes , Animales , Ratones , Células 3T3-L1 , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Adipogénesis/genética , Quinasa de la Caseína I/metabolismo , Diferenciación Celular , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
The adipose organ comprises two main fat depots termed white and brown adipose tissues. Adipogenesis is a process leading to newly differentiated adipocytes starting from precursor cells, which requires the contribution of many cellular activities at the genome, transcriptome, proteome, and metabolome levels. The adipogenic program is accomplished through two sequential phases; the first includes events favoring the commitment of adipose tissue stem cells/precursors to preadipocytes, while the second involves mechanisms that allow the achievement of full adipocyte differentiation. While there is a very large literature about the mechanisms involved in terminal adipogenesis, little is known about the first stage of this process. Growing interest in this field is due to the recent identification of adipose tissue precursors, which include a heterogenous cell population within different types of adipose tissue as well as within the same fat depot. In addition, the alteration of the heterogeneity of adipose tissue stem cells and of the mechanisms involved in their commitment have been linked to adipose tissue development defects and hence to the onset/progression of metabolic diseases, such as obesity. For this reason, the characterization of early adipogenic events is crucial to understand the etiology and the evolution of adipogenesis-related pathologies, and to explore the adipose tissue precursors' potential as future tools for precision medicine.
Asunto(s)
Adipocitos Blancos/citología , Adipogénesis , Diferenciación Celular , Obesidad/fisiopatología , Termogénesis , Animales , HumanosRESUMEN
The development of hepatic insulin resistance (IR) is a critical factor in developing type 2 diabetes (T2D), where insulin fails to inhibit hepatic glucose production but retains its capacity to promote hepatic de novo lipogenesis leading to hyperglycemia and hypertriglyceridemia. Improving insulin sensitivity can be effective in preventing and treating T2D. However, selective control of glucose and lipid synthesis has been difficult. It is known that excess white adipose tissue is detrimental to insulin sensitivity, whereas brown adipose tissue transplantation can restore it in diabetic mice. However, challenges remain in our understanding of liver-adipose communication because the confounding effects of hypothalamic regulation of metabolic function cannot be ruled out in previous studies. There is a lack ofin vitromodels that use primary cells to study cellular-crosstalk under insulin resistant conditions. Building upon our previous work on the microfluidic primary liver and adipose organ-on-chips, we report for the first time, the development of an integrated insulin resistant liver-adipose (white and brown) organ-on-chip. The design of the microfluidic device was carried out using computational fluid dynamics; the experimental studies were conducted by carrying out detailed biochemical analysis RNA-seq analysis on both cell types. Further, we tested the hypothesis that brown adipocytes (BAC) regulated both hepatic insulin sensitivity and de novo lipogenesis. Our results show that BAC effectively restored insulin sensitivity and supressed hepatic glucose production and de novo lipogenesis suggesting that the experimental platform could be useful for identifying potential therapeutics to treat IR and diabetes.
Asunto(s)
Adipocitos Marrones , Adipocitos Blancos , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Análisis de Matrices Tisulares , Adipocitos Marrones/citología , Adipocitos Marrones/metabolismo , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Dispositivos Laboratorio en un Chip , Lipogénesis/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Análisis de Matrices Tisulares/instrumentación , Análisis de Matrices Tisulares/métodosRESUMEN
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.
Asunto(s)
Adipocitos Marrones/citología , Adipocitos Blancos/citología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Prednisona/farmacología , Receptores Adrenérgicos beta 3/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adipocitos Marrones/efectos de los fármacos , Adipocitos Marrones/metabolismo , Adipocitos Blancos/efectos de los fármacos , Adipocitos Blancos/metabolismo , Animales , Antiinflamatorios/farmacología , Regulación de la Expresión Génica , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Receptores Adrenérgicos beta 3/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
The present study evaluated the anti-obesity effect of sulforaphane (SFN) and glucoraphanin (GRN) in broccoli leaf extract (BLE) on 3T3-L1 adipocytes and ob/ob mice. Based on Oil Red O staining and triglyceride (TG) assay, SFN and BLE significantly reduced (P<.05) both lipid accumulation and TG content in the differentiated 3T3-L1 adipocytes. SFN and BLE increased 2-NBDG uptake by 3T3-L1 adipocytes in a dose-dependent manner. Western blot analysis confirmed that SFN and BLE increased the phosphorylation levels of both AMPK (Thr172) and ACC (Ser79), and reduced the expression of HMGCR in liver and white adipose tissues of ob/ob mice. Histological analysis revealed that SFN and BLE ameliorated hepatic steatosis, and reduced the size of adipocyte in ob/ob mice. Treatment with SFN and BLE significantly reduced (P<.05) TG content, low-density lipoprotein (LDL) cholesterol, total cholesterol (TC), and glucose in the serum of ob/ob mice. RNA sequencing analysis showed that up- or down-regulation of 32 genes related to lipid metabolism was restored to control level in both SFN and BLE-treated ob/ob mice groups. A protein-protein interaction (PPI) network was constructed via STRING analysis, and Srebf2, Pla2g2c, Elovl5, Plb1, Ctp1a, Lipin1, Fgfr1, and Plcg1 were located in the functional hubs of the PPI network of lipid metabolism. Overall results suggest that the SFN content in BLE exerts a potential anti-obesity effect by normalizing the expression of genes related to lipid metabolism, which are up- or down-regulated in ob/ob mice.
Asunto(s)
Adipocitos/metabolismo , Fármacos Antiobesidad/farmacología , Brassica/química , Isotiocianatos/farmacología , Obesidad/metabolismo , Extractos Vegetales/farmacología , Sulfóxidos/farmacología , Células 3T3-L1 , Proteínas Quinasas Activadas por AMP/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos Blancos/citología , Animales , Glucemia/metabolismo , Glucosa/metabolismo , Glucosinolatos/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Obesos , Obesidad/tratamiento farmacológico , Obesidad/patología , Oximas/farmacología , Fosforilación , Hojas de la Planta/química , Transcriptoma , Triglicéridos/metabolismoRESUMEN
Adipose tissue has been classified based on its morphology and function as white, brown, or beige/brite. It plays an essential role as a regulator of systemic metabolism through paracrine and endocrine signals. Recently, multiple adipocyte subtypes have been revealed using RNA sequencing technology, going beyond simply defined morphology but also by their cellular origin, adaptation to metabolic stress, and plasticity. Here, we performed an in-depth analysis of publicly available single-nuclei RNAseq from adipose tissue and utilized a workflow template to characterize adipocyte plasticity, heterogeneity, and secretome profiles. The reanalyzed dataset led to the identification of different subtypes of adipocytes including three subpopulations of thermogenic adipocytes, and provided a characterization of distinct transcriptional profiles along the adipocyte trajectory under thermogenic challenges. This study provides a useful resource for further investigations regarding mechanisms related to adipocyte plasticity and trans-differentiation.
Asunto(s)
Adipocitos Blancos/citología , Tejido Adiposo Blanco/citología , Núcleo Celular/metabolismo , Plasticidad de la Célula , RNA-Seq , Termogénesis/fisiología , Animales , Ratones , Temperatura , Proteína Desacopladora 1/metabolismoRESUMEN
We studied the effect of bacterial pathogen-associated molecular patterns and myokines on the secretion of adipokines by mesenchymal stem cells (MSC) and products of their adipogenic differentiation. The secretion of adiponectin, adipsin, leptin, and insulin by adipogenically differentiated cell cultures was quantitatively determined using multiplex ELISA. MSC obtained from the stromal vascular fraction of human subcutaneous adipose tissue were shown to secrete a known adipokine adipsin. The ability of white adipocytes to secrete significant amounts of insulin (in vitro) has been shown for the first time. Control cultures of white adipocytes secreted much higher levels of adiponectin, leptin, and insulin when compared to other adipocytes cultures. On the other hand, beige and brown adipocyte cultures secreted more adipsin than white adipocyte cultures. The influence of myokine ß-aminoisobutyric acid on the secretion of adipsin in MSC, white, beige, and brown adipocytes was also studied.
Asunto(s)
Adipocitos Beige/efectos de los fármacos , Adipocitos Marrones/efectos de los fármacos , Adipocitos Blancos/efectos de los fármacos , Adipoquinas/farmacología , Ácidos Aminoisobutíricos/farmacología , Flagelina/farmacología , Lipopolisacáridos/farmacología , Adipocitos Beige/citología , Adipocitos Beige/metabolismo , Adipocitos Marrones/citología , Adipocitos Marrones/metabolismo , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Adiponectina/genética , Adiponectina/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/cirugía , Diferenciación Celular/efectos de los fármacos , Factor D del Complemento/genética , Factor D del Complemento/metabolismo , Regulación de la Expresión Génica , Humanos , Insulina/genética , Insulina/metabolismo , Leptina/genética , Leptina/metabolismo , Lipectomía/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Especificidad de Órganos , Cultivo Primario de CélulasRESUMEN
Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration when muscle tissue is injured. However, these cells can be induced to differentiate into adipocytes once exposed to PPARγ activator like EPA and DHA that are highly suggested during pregnancy. The objective of this study aims at determining the identity of trans-differentiated cells by exploring the effect of EPA and DHA on C2C12 undergoing differentiation into brown and white adipocytes. DHA but not EPA committed C2C12 cells reprograming into white like adipocyte phenotype. Also, DHA promoted the expression of lipolysis regulating genes but had no effect on genes regulating ß-oxidation referring to its implication in lipid re-esterification. Furthermore, DHA impaired C2C12 cells differentiation into brown adipocytes through reducing the thermogenic capacity and mitochondrial biogenesis of derived cells independent of UCP1. Accordingly, DHA treated groups showed an increased accumulation of lipid droplets and suppressed mitochondrial maximal respiration and spare respiratory capacity. EPA, on the other hand, reduced myogenesis regulating genes, but no significant differences were observed in the expression of adipogenesis key genes. Likewise, EPA suppressed the expression of WAT signature genes indicating that EPA and DHA have an independent role on white adipogensis. Unlike DHA treatment, EPA supplementation had no effect on the differential of C2C12 cells into brown adipocytes. In conclusion, DHA is a potent adipogenic and lipogenic factor that can change the metabolic profile of muscle cells by increasing myocellular fat.
Asunto(s)
Adipocitos Blancos/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Adipocitos Marrones/efectos de los fármacos , Adipocitos Blancos/citología , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/efectos de los fármacos , Animales , Línea Celular , Transdiferenciación Celular/efectos de los fármacos , Transdiferenciación Celular/genética , ADN Mitocondrial , Regulación de la Expresión Génica/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Lipólisis/efectos de los fármacos , Ratones , Mioblastos/citología , Mioblastos/efectos de los fármacosRESUMEN
Increased browning of white adipocytes (beiging) is considered a promising therapeutic strategy to fight obesity and its associated metabolic complications. However, the molecular mechanism modulating brown and beige fat-mediated thermogenesis is not fully elucidated. Here, we identified the lymphocyte cytosolic protein 1 (LCP1) as a factor that obstructs fat browning in white adipocytes. LCP1 plays a vital role in non-hematopoietic malignancies, and is also a well-known tumor biomarker; however, evidence regarding its function in adipocytes remains to be elucidated. The current study explores the physiological role of LCP1 in cultured 3T3-L1 white adipocytes, by applying the loss-of-function study using siRNA. Induction of fat browning by LCP1 depletion was evidenced by evaluating the gene and protein expression levels of brown fat-associated markers through real-time qRT-PCR and immunoblot analysis, respectively. We observed that deficiency of LCP1 promotes mitochondrial biogenesis, and significantly enhances expressions of the core brown fat-specific genes (Cd137, Cidea, Cited1, Tbx1, and Tmem26) and proteins (PGC-1α, PRDM16, and UCP1). In addition, deficiency of LCP1 promotes lipid catabolism as well as suppresses adipogenesis and lipogenesis. Loss of LCP1 also ameliorates cellular stress by downregulating JNK and c-JUN in adipocytes, and stimulates apoptosis. A mechanistic study revealed that deficiency of LCP1 induces browning in white adipocytes, independently via ß3-AR and the ERK signaling pathway. The current data reveals a previously unknown mechanism of LCP1 in browning of white adipocytes, and highlights the potential of LCP1 as a pharmacotherapeutic target for treating obesity and other metabolic disorders.
Asunto(s)
Adipocitos Blancos/citología , Tejido Adiposo Pardo/citología , Proteínas de Microfilamentos/deficiencia , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Estrés Fisiológico , Células 3T3-L1 , Adipocitos Blancos/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Lipogénesis , Lipólisis , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Receptores Adrenérgicos beta 3/genética , Transducción de Señal , TermogénesisRESUMEN
The aim of the study was to investigate the mechanisms of Ca2+ oscillation generation upon activation of connexin-43 and regulation of the lipolysis/lipogenesis balance in white adipocytes through vesicular ATP release. With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels-carbenoxolone, octanol, proadifen and Gap26-as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.
Asunto(s)
Adipocitos Blancos/metabolismo , Calcio/metabolismo , Conexina 43/fisiología , Lipogénesis , Lipólisis , Adipocitos Blancos/citología , Animales , Señalización del Calcio , Células Cultivadas , RatonesRESUMEN
Heparan sulfate (HS), a highly sulfated linear polysaccharide, is involved in diverse biological functions in various tissues. Although previous studies have suggested a possible contribution of HS to the differentiation of white adipocytes, there has been no direct evidence supporting this. Here, we inhibited the synthesis of HS chains in 3T3-L1 cells using CRISPR-Cas9 technology, resulting in impaired differentiation of adipocytes with attenuated bone morphogenetic protein 4 (BMP4)-fibroblast growth factor 1 (FGF1) signaling pathways. HS reduction resulted in reduced glucose uptake and decreased insulin-dependent intracellular signaling. We then made heterozygous mutant mice for the Ext1 gene, which encodes an enzyme essential for the HS biosynthesis, specifically in the visceral white adipose tissue (Fabp4-Cre+::Ext1flox/WT mice, hereafter called Ext1Δ/WT) to confirm the importance of HS in vivo. The expression levels of transcription factors that control adipocyte differentiation, such as peroxisome proliferator-activated receptor gamma, were reduced in Ext1Δ/WT adipocytes, which contained smaller, unilocular lipid droplets, reduced levels of enzymes involved in lipid synthesis, and altered expression of BMP4-FGF1 signaling molecules. Furthermore, we examined the impact of HS reduction in visceral white adipose tissue on systemic glucose homeostasis. We observed that Ext1Δ/WT mice showed glucose intolerance because of insulin resistance. Our results demonstrate that HS plays a crucial role in the differentiation of white adipocytes through BMP4-FGF1 signaling pathways, thereby contributing to insulin sensitivity and glucose homeostasis.
Asunto(s)
Adipocitos Blancos/citología , Diferenciación Celular/fisiología , Glucosa/metabolismo , Heparitina Sulfato/fisiología , Homeostasis , Resistencia a la Insulina , Células 3T3-L1 , Adipocitos Blancos/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Sistemas CRISPR-Cas , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Ratones , Transducción de SeñalRESUMEN
To investigate whether the beiging process changes the interactive effects of salt and MEK6 gene on inflammatory adipogenesis, the salt treatment (NaCl 50 mM) and MEK6 transfection of Tg(+/+) cells were performed with white adipocytes (WAT) and beige-like-adipocytes (BLA). BLA induced by T3 were confirmed by UCP-1 expression and the MEK6 protein was 3.5 times higher in MEK6 transfected WAT than the control. The adipogenic genes, PPAR-γ and C/EBP-α, were 1.5 times more highly expressed in the salt-treated groups than the non-salt-treated groups, and adipogenesis was greatly increased in Tg(+/+) WAT compared to non-transfected Tg(-/-). The adipogenesis induced by salt treatment and MEK6 transfection was significantly reduced in BLA. The inflammatory adipocytokines, TNF-α, IL-1ß, and IL-6, were increased in the salt-treated Tg(+/+) WAT, but an anti-inflammation biomarker, the adiponectin/leptin ratio, was reduced in Tg(+/+), to tenth of that in Tg(-/-). However, the production of adipocytokines in WAT was strongly weakened in BLA, although a combination of salt and MEK6 transfection had the most significant effects on inflammation in both WAT and BLA. Oxygen consumption in mitochondria was maximized in salt-treated and MEK6 transfected WAT, but it was decreased by 50% in BLA. In conclusion, beiging controls the synergistic effects of salt and MEK6 on adipogenesis, inflammation, and energy expenditure.
Asunto(s)
Adipocitos Beige/metabolismo , Adipocitos Blancos/metabolismo , Adipogénesis , MAP Quinasa Quinasa 6/metabolismo , Cloruro de Sodio/farmacología , Células 3T3-L1 , Adipocitos Beige/citología , Adipocitos Blancos/citología , Adipocitos Blancos/efectos de los fármacos , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Metabolismo Energético , MAP Quinasa Quinasa 6/genética , Ratones , PPAR gamma/genética , PPAR gamma/metabolismoRESUMEN
Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)-dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors' interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5-15% vs. 30-80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity.
Asunto(s)
Adipocitos Blancos/metabolismo , Señalización del Calcio , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Obesidad/metabolismo , Adipocitos Blancos/citología , Adipocitos Blancos/efectos de los fármacos , Animales , Señalización del Calcio/efectos de los fármacos , Tamaño de la Célula , Células Cultivadas , Diabetes Mellitus Tipo 2/etiología , Dieta Alta en Grasa/efectos adversos , Epidídimo , Proteínas de Unión al GTP/metabolismo , Masculino , Ratones , Óxido Nítrico Sintasa de Tipo III/metabolismo , Obesidad/inducido químicamente , Palmitoilcarnitina/farmacología , Periodicidad , Cultivo Primario de Células , Fosfolipasas de Tipo C/metabolismoRESUMEN
AIMS: Browning induction (beiging) of white adipocytes is an emerging prospective strategy to defeat obesity and its related metabolic disorders. Cytochrome P450 2E1 (CYP2E1), a membrane protein which belongs to the cytochrome P450 superfamily, reportedly functions in the xenobiotic metabolism in the body, especially ethanol metabolism. Although previous studies have reported the effect of CYP2E1 on obesity in animal models, the data remains controversial. In the current study, we investigate for the first time, the role of CYP2E1 in lipid metabolism in 3T3-L1 white adipocytes, with a focus on fat browning. METHODS: 3T3-L1 white adipocytes and Cyp2e1 siRNA were applied to investigate the role of CYP2E1 in white adipocytes. After that, cells were seperately exposed to ß3-AR agonist, ß3-AR antagonist and p38 inhibitor to identify the pathway which CYP2E1 was involved in to regulate browning event in white adipocytes. KEY FINDINGS: We found that CYP2E1 deficiency results in reduced adipogenesis and lipogenesis as well as brown adipocyte-like phenotype induction. A mechanistic study to identify the molecular signals for CYP2E1 regulation in the browning of white adipocytes revealed that CYP2E1 inhibition deters the ß3-adrenergic receptor activation and its downstream targets. SIGNIFICANCE: Our data unveilved a previously unknown mechanism in the regulation of browning by CYP2E1 in 3T3-L1 white adipocytes, suggesting that CYP2E1 is a promising molecular target for the treatment of obesity and its related diseases.
Asunto(s)
Adipocitos Marrones/metabolismo , Adipocitos Blancos/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Lipólisis , Células 3T3-L1 , Adipocitos Marrones/citología , Adipocitos Blancos/citología , Adipogénesis , Animales , Citocromo P-450 CYP2E1/genética , Silenciador del Gen , Metabolismo de los Lípidos , RatonesRESUMEN
Genistein, a naturally occurring phytoestrogen and a member of the large class of compounds known as isoflavones, exerts protective effects in several diseases. Recent studies indicate that genistein plays a critical role in controlling body weight, obesity-associated insulin resistance, and metabolic disorders, but its target organs in reversing obesity and related pathological conditions remain unclear. In this study, we showed that mice supplemented with 0.2% genistein in a high-fat diet for 12 weeks showed enhanced metabolic homeostasis, including reduced obesity, improved glucose uptake and insulin sensitivity, and alleviated hepatic steatosis. We also observed a beiging phenomenon in the white adipose tissue and reversal of brown adipose tissue whitening in these mice. These changes led to enhanced resistance to cold stress. Altogether, our data suggest that the improved metabolic profile in mice treated with genistein is likely a result of enhanced adipose tissue function.
Asunto(s)
Tejido Adiposo Beige/efectos de los fármacos , Tejido Adiposo Beige/metabolismo , Respuesta al Choque por Frío/efectos de los fármacos , Respuesta al Choque por Frío/fisiología , Genisteína/farmacología , Adipocitos Blancos/citología , Adipocitos Blancos/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Aumento de la Célula/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Ingestión de Alimentos/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Resistencia a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Obesidad/patología , Fitoestrógenos/farmacología , Sustancias Protectoras/farmacologíaRESUMEN
High uncoupling protein 1 (Ucp1) expression is a characteristic of differentiated brown adipocytes and is linked to adipogenic differentiation. Paracrine fibroblast growth factor 8b (FGF8b) strongly induces Ucp1 transcription in white adipocytes independent of adipogenesis. Here, we report that FGF8b and other paracrine FGFs act on brown and white preadipocytes to upregulate Ucp1 expression via a FGFR1-MEK1/2-ERK1/2 axis, independent of adipogenesis. Transcriptomic analysis revealed an upregulation of prostaglandin biosynthesis and glycolysis upon Fgf8b treatment of preadipocytes. Oxylipin measurement by LC-MS/MS in FGF8b conditioned media identified prostaglandin E2 as a putative mediator of FGF8b induced Ucp1 transcription. RNA interference and pharmacological inhibition of the prostaglandin E2 biosynthetic pathway confirmed that PGE2 is causally involved in the control over Ucp1 transcription. Importantly, impairment of or failure to induce glycolytic flux blunted the induction of Ucp1, even in the presence of PGE2 . Lastly, a screening of transcription factors identified Nrf1 and Hes1 as required regulators of FGF8b induced Ucp1 expression. Thus, we conclude that paracrine FGFs co-regulate prostaglandin and glucose metabolism to induce Ucp1 expression in a Nrf1/Hes1-dependent manner in preadipocytes, revealing a novel regulatory network in control of Ucp1 expression in a formerly unrecognized cell type.