Effect of PKM2 on Osteogenic and Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Myeloma Bone Disease / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs.@*METHODS@#BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs.@*RESULTS@#Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2.@*CONCLUSION@#The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.

Bone Marrow Adipocytes Promote the Survival of Multiple Myeloma Cells and Up-Regulate Their Chemoresistance / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM.@*METHODS@#Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor.@*RESULTS@#The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells.@*CONCLUSION@#The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.

Gene cloning and sequence analysis of the RPL29 gene and its effect on lipogenesis in goat intramuscular adipocytes / 生物工程学报

The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P < 0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P < 0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P < 0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P < 0.01) and a significant increase in FASN gene expression (P < 0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.

Cloning, identification and functional analysis of the goat transcription factor c-fos / 生物工程学报

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.

Influencia de ácidos grasos en parámetros sanguíneos y adipogénesis. Estudio experimental en pollos eclosionados / Influence of fatty acids on blood parameters and formation of adipocytes at early age

Introducción: La alimentación en la primera infancia influencia la instauración del tejido adiposo y el desarrollo de diversas patologías en la edad adulta. Objetivo: Evaluar la influencia del consumo de tres fuentes de ácidos grasos sobre parámetros sanguíneos y tejido adiposo en pollos recién eclosionados. Materiales y métodos: Se utilizaron 76 pollitos Cobb 500 distribuidos aleatoriamente en cuatro tratamientos, que fueron alimentados durante siete días con una de las cuatro dietas (T1: 97% Dieta basal (DB); T2: DB +3% de manteca vegetal parcialmente hidrogenada; T3: DB +3% de aceite de quinua y T4: DB +3% de aceite de pescado). Al finalizar, se evaluó en sangre glucosa, colesterol, triglicéridos y tamaño de adipocitos del tejido adiposo subcutáneo y visceral. Se aplicó ANOVA considerando 0,05 de significancia y en el caso de variables no distribuidas normalmente, se aplicó el test no paramétrico de Kruskal-Wallis mediante el programa R-Studio. Resultados: Se obtuvieron diferencias significativas con disminución de los niveles de glucosa y colesterol en animales suplementados con elevada proporción de aceites insaturados (T3 y T4) en comparación a T2. Los tratamientos T3 y T4 promovieron una formación hiperplásica de adipocitos, diferenciándose significativamente de T2, que promovió la hipertrofia en dichas células, esta respuesta fue similar en ambos depósitos subcutáneos. Conclusiones: El consumo de aceite de quinua y aceite de pescado promueve la formación de tejido adiposo saludable, y reducen los niveles de glucosa y colesterol. Contrariamente el consumo de manteca vegetal propicia la hipertrofia de adipocitos de gran tamaño e incrementa los parámetros bioquímicos evaluados(AU)

Introduction: Feeding in early childhood influences the establishment of adipose tissue, and therefore also in the development of various pathologies in adulthood. Objective: To evaluate the influence of the consumption of three sources of fatty acids on blood parameters and adipose tissue at an early age. Materials and methods: 76 Cobb 500 chicks randomly distributed in four treatments were used, who were fed for seven days with one of the four diets (T1: Basal diet; T2: DB +1.0% vegetable shortening partially hydrogenated; T3: DB +1.0% quinoa oil and DB +1.0% fish oil) until the seventh day of life. At the end, glucose, cholesterol, triglycerides and adipocyte size of the subcutaneous and visceral adipose tissue were evaluated. A completely randomized design with ANOVA considering 0.05 significance was applied and in the case of non-normally distributed variables, the non-parametric Kruskal-Wallis test was applied using the R-Studio program. Results: Significant differences were obtained with a decrease in glucose and cholesterol levels in animals supplemented with unsaturated oils (T3 and T4) compared to T2. Regarding the size of adipocytes, treatments T3 and T4 promoted a hyperplastic formation of adipocytes, differing significantly from T2, which promoted hypertrophy in these cells, this response was similar in both subcutaneous deposits. Conclusions: The consumption of quinoa oil and fish oil promote the formation of healthy adipose tissue, in addition to reducing glucose and cholesterol levels. In contrast, the consumption of vegetable shortening favors the hypertrophy of large adipocytes and increases the biochemical parameters evaluated(AU)

Microbiota intestinal y modulación del tejido adiposo en la patogénesis de la obesidad / Gut microbiota and modulation of adipose tissue in the pathogenesis of obesity

Las investigaciones realizadas durante el último siglo relacionadas con la descripción de la Microbiota Intestinal (MI) sugieren una relación concreta entre su composición y la salud del huésped. Su desregulación denominada disbiosis intestinal ha sido asociada a distintos tipos de enfermedades gastrointestinales, metabólicas, oncológicas e incluso psiquiátricas. Destacan numerosos reportes que han informado la condición de disbiosis en la obesidad, tanto en modelos animales como humanos de distintos grupos etarios y regiones del mundo. A su vez, la composición del microbioma también ha logrado asociarse a las diferentes comorbilidades de la obesidad, postulando que la MI posee influencia en la disfunción del tejido adiposo (TA), entendiendo que corresponde al principal modulador de la patogénesis de la obesidad. Sin embargo, aún no es posible establecer una explicación mecanicista plausible. Actualmente, la utilización de tecnologías multiómicas, junto con la evaluación de variables fisiológicas, nos podrían proporcionar una mejor comprensión a la incógnita planteada. Frente a esto, el presente trabajo tiene como objetivo revisar los últimos avances en la comprensión de la influencia de la microbiota intestinal en el TA y su contribución a los mecanismos relacionados con la patogénesis de la obesidad. Entre los principales mecanismos identificados, la evidencia reporta nexos fisiológicos entre la composición de la MI y la modulación de inflamación, permeabilidad intestinal y adipogénesis. Las vías implicadas derivan de la influencia de la disbiosis intestinal en el accionar de ácidos grasos de cadena corta, claudinas, macrófagos, oligosacáridos, entre otros. Los mecanismos implicados, principalmente estudiados en modelos animales, deberían ser considerados para su evaluación en próximos estudios longitudinales y experimentales en humanos con el fin de obtener una mayor comprensión sobre la implicancia de cada mecanismo en la patogenia global de la obesidad(AU)

The investigations carried out during the last century related to the description of the Gut Microbiota (GM) suggest a concrete relationship between its composition and the health of the host. Its deregulation called intestinal dysbiosis has been associated with different types of gastrointestinal, metabolic, oncological and even psychiatric diseases. Numerous reports that have described the condition of dysbiosis in obesity stand out, both in animal and human models of different age groups and regions of the world. In turn, the composition of the microbiome has also been associated with the different comorbidities of obesity, postulating that MI has an influence on adipose tissue (AT) dysfunction, understanding that it corresponds to the main modulator of the pathogenesis of obesity. However, it is not yet possible to establish a plausible mechanistic explanation. Currently, the use of multi-omics technologies, together with the evaluation of physiological variables, could provide us with a better understanding of the question raised. In view of this, this review aims to review the latest advances in understanding the influence of the intestinal microbiota on AT and its contribution to the mechanisms related to the pathogenesis of obesity. Among the main mechanisms identified, the evidence reports physiological links between the composition of GM and the modulation of inflammation, intestinal permeability and adipogenesis. The pathways involved derive from the influence of intestinal dysbiosis on the action of short-chain fatty acids, claudins, macrophages, oligosaccharides, among others. The mechanisms involved, mainly studied in animal models, should be considered for evaluation in future longitudinal and experimental studies in humans in order to obtain a better understanding of the implication of each mechanism in the global pathogenesis of obesity(AU)

Research progress in Th17 cells and the relevant cytokines in Graves ' ophthalmopathy / 中南大学学报(医学版)

Graves' ophthalmopathy is the most common clinical orbital disease, and T helper (Th) cells play an important role in the development of Graves' ophthalmopathy. Th17 cells are a major subpopulation of Th cells and abnormally highly expressed in patients with Graves' ophthalmopathy. Th17 cells and the related cytokines interleukin (IL)-17A, IL-21 and IL-23 are involved in regulating the inflammatory response, fibrosis and adipogenesis. Th17 cells are unstable and exhibit a degree of plasticity, and they can differentiate into IL-17A and interferon (IFN)-γ dual-producing Th17.1 cells, which exacerbate the pathogenicity of Th17 cells. In addition, Th17 cells and the relevant factors are strongly associated with disease activity and severity in Graves' ophthalmopathy.

Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells / 北京大学学报(医学版)

OBJECTIVE@#To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.@*RESULTS@#The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.@*CONCLUSION@#lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.

The effect of fat mass and obesity associated proteins mediated mRNA m6A modification on animal fat deposition and its application prospects / 生物工程学报

In the process of animal fat deposition, the proliferation and differentiation of pre-adipocytes and the change of lipid droplet content in adipocytes are regulated by a series of transcription factors and signal pathways. Although researchers have conducted in-depth studies on the transcriptional regulation mechanisms of adipogenesis, there are relatively few reports on post-transcriptional modification on mRNA levels. The modification of mRNA m6A regulated by methyltransferase, demethylase and methylation reading protein is a dynamic and reversible process, which is closely related to fat deposition in animals. Fat mass and obesity associated proteins (FTO) act as RNA demethylases that affect the expression of modified genes and play a key role in fat deposition. This article summarized the mechanism of FTO-mediated demethylation of mRNA m6A in the process of animal fat deposition, suggesting that FTO may become a target for effective treatment of obesity. Moreover, this review summarized the development of FTO inhibitors in recent years.

C/EBPb converts bovine fibroblasts to adipocytes without hormone cocktail induction

BACKGROUND: Adipogenesis and fibrogenesis can be considered as a competitive process in muscle, which may affect the intramuscular fat deposition. The CCAAT/enhancer-binding protein beta (C/EBPb) plays an important role in adipogenesis, which is well-characterized in mice, but little known in bovine so far. RESULTS: In this study, real-time qPCR revealed that the level of C/EBPb was increased during the developmental stages of bovine and adipogenesis process of preadipocytes. Overexpression of C/EBPb promoted bovine fibroblast proliferation through mitotic clonal expansion (MCE), a necessary process for initiating adipogenesis, by significantly downregulating levels of p21 and p27 (p < 0.01). Also, the PPARc expression was inhibited during the MCE stage (p < 0.01). 31.28% of transfected fibroblasts adopted lipid-laden adipocyte morphology after 8 d. Real-time qPCR showed that C/EBPb activated the transcription of early stage adipogenesis markers C/EBPa and PPARc. Expression of ACCa, FASN, FABP4 and LPL was also significantly upregulated, while the expression of LEPR was weakened. CONCLUSIONS: It was concluded C/EBPb can convert bovine fibroblasts into adipocytes without hormone induction by initiating the MCE process and promoting adipogenic genes expression, which may provide new insights into the potential functions of C/EBPb in regulating intramuscular fat deposition in beef cattle.

Inhibition of preadipocyte differentiation by Lycium barbarum polysaccharide treatment in 3T3-L1 cultures

BACKGROUND: Lycium barbarum (also called wolfberry), a famous Chinese traditional medicine and food ingredient, is well recognized for its significant role in preventing obesity; however, the molecular mechanisms underlying its preventive effects on fat accumulation are not well understood yet. The aim of this study was to determine the effects and mechanism of Lycium barbarum polysaccharides (LBP) on the proliferation and differentiation of 3T3-L1 preadipocytes. MTT was used to detect the proliferation of 3T3-Ll preadipocytes. Oil red O staining and colorimetric analysis were used to detect cytosolic lipid accumulation during 3T3-L1 preadipocyte differentiation. Real-time fluorescent quantitative PCR (qPCR) technology was used to detect peroxisome proliferator-activated receptor c (PPARc), CCAAT/enhancer-binding protein a (C/EBPa), adipocyte fatty-acid-binding protein (aP2), fatty acid synthase (FAS), and lipoprotein lipase (LPL) expression. RESULTS: The concentration of LBP from 25 to 200 lg/mL showed a tendency to inhibit the growth of preadipocytes at 24 h, and it inhibited the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. In the preadipocytes treated with 200 lg/mL LBP, there were reduced lipid droplets in the cytoplasm, and its effect was opposite to that of rosiglitazone (ROS), which significantly reduced the PPARc, C/EBPa, aP2, FAS, and LPL mRNA expression of adipocytes. CONCLUSIONS: LBP exerts inhibitive effects on the proliferation and differentiation of 3T3-L1 preadipocytes and decreases the cytoplasm accumulation of lipid droplets during induced differentiation of preadipocytes toward mature cells. Above phenomenon might link to lowered expression of PPARc, C/EBPa, aP2, FAS, and LPL after LBP treatment. Thus, LBP could serve as a potential plant extract to treat human obesity or improve farm animal carcass quality via adjusting lipid metabolism.

Effect of dietary nutrition on tail fat deposition and evaluation of tail-related genes in fat-tailed sheep

BACKGROUND: The effects of dietary nutrition on tail fat deposition and the correlation between production performance and the Hh signaling pathway and OXCT1 were investigated in fat-tailed sheep. Tan sheep were fed different nutritional diets and the variances in tail length, width, thickness and tail weight as well as the mRNA expression of fat-related genes (C/EBPα, FAS, LPL, and HSL) were determined in the tail fat of sheep at three different growth stages based on their body weight. Furthermore, the correlations between tail phenotypes and the Hedgehog (Hh) signaling pathway components (IHH, PTCH1, SMO, and GLI1) and OXCT1 were investigated. RESULTS: C/EBPα, FAS, LPL, and HSL were expressed with differences in tail fat of sheep fed different nutritional diets at three different growth stages. The results of the two-way ANOVA showed the significant effect of nutrition, stage, and interaction on gene expression, except the between C/EBPα and growth stage. C/EBPα, FAS, and LPL were considerably correlated with the tail phenotypes. Furthermore, the results of the correlation analysis demonstrated a close relationship between the tail phenotypes and Hh signaling pathway and OXCT1. CONCLUSIONS: The present study demonstrated the gene-level role of dietary nutrition in promoting tail fat deposition and related tail fat-related genes. It provides a molecular basis by which nutritional balance and tail fat formation can be investigated and additional genes can be identified. The findings of the present study may help improve the production efficiency of fat-tailed sheep and identify crucial genes associated with tail fat deposition.

Predicting the Benefit of Adjuvant Aromatase Inhibitor Therapy in Postmenopausal Breast Cancer Patients with Phosphorylated S6K1 Expression Status / 한국유방암학회지

PURPOSE: Phosphorylated ribosomal S6 kinase 1 (pS6K1) is a major downstream regulator of the mammalian target of rapamycin (mTOR) pathway. Recent studies have addressed the role of S6K1 in adipogenesis. pS6K1 may affect the outcome of estrogen depletion therapy in patients with hormone-sensitive breast cancer due to its association with adipogenesis and increased local estrogen levels. This study aimed to investigate the potential of pS6K1 as a predictive marker of adjuvant aromatase inhibitor (AI) therapy outcome in postmenopausal or ovarian function-suppressed patients with hormone-sensitive breast cancer.METHODS: Medical records were retrospectively reviewed in postmenopausal or ovarian function-suppressed patients with estrogen receptor-positive and node-positive primary breast cancer. pS6K1 expression status was scored on a scale from 0 (negative) to 3+ (positive) based on immunohistochemical analysis.RESULTS: A total of 428 patients were eligible. The median follow-up duration was 44 months (range, 1–90). In patients with positive pS6K1 expression, AIs significantly improved disease-free survival (DFS) compared to selective estrogen receptor modulators (SERMs) (5 year-DFS: 83.5% vs. 50.7%, p = 0.016). However, there was no benefit of AIs on DFS in the pS6K1 negative group (5 year-DFS 87.6% vs. 91.4%, p = 0.630). On multivariate analysis, AI therapy remained a significant predictor for DFS in the pS6K1 positive group (hazard ratio, 0.39; 95% confidence interval, 0.16–0.96; p = 0.041). pS6K1 was more effective in predicting the benefit of AI therapy in patients with ages < 50 (p = 0.021) compared to those with ages ≥ 50 (p = 0.188).CONCLUSION: pS6K1 expression may predict AI therapy outcomes and serve as a potential predictive marker for adjuvant endocrine therapy in postmenopausal and ovarian function-suppressed patients with hormone-sensitive breast cancer. AIs may be more effective in patients with pS6K1 positive tumors, while SERM could be considered an alternative option for patients with pS6K1 negative tumors.

Tribbles pseudokinase 3 inhibits the adipogenic differentiation of human adipose-derived mesenchymal stem cells / 北京大学学报(医学版)

OBJECTIVE@#To identify the role of Tribbles pseudokinase 3 (TRIB3) during the process of adipogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs), and to provide a new target and a novel idea for the application of hASCs in adipose tissue engineering and soft tissue regeneration.@*METHODS@#TRIB3-knockdown hASCs (shTRIB3) and TRIB3-overexpression hASCs (TRIB3-over) were established using lentivirus transfection technique. The transfection effect was estimated by the visible presence of green fluorescence as the expression of green fluorescent protein (GFP) in the transfected hASCs. The lentiviral transfection efficiency was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. After adipogenic induction, Oil Red staining and quantification, as well as qRT-PCR about several specific adipogenic markers were used to evaluate the adipogenic differentiation ability of hASCs.@*RESULTS@#In TRIB3-knockdown hASCs, the TRIB3 mRNA expression level decreased by about 84.3% compared with the control group (P<0.01), and the TRIB3 protein level also showed obvious reduction. Oppositely, in TRIB3-overexpression hASCs, the TRIB3 mRNA expression level increased by approximately 160% compared with the control group (P<0.01), and the TRIB3 protein level also showed a significant increase. These results indicated a successful construction of TRIB3-knockdown hASCs and TRIB3-overexpression hASCs. The Oil Red staining results showed that the down-regulation of TRIB3 significantly promoted lipid droplets formation in hASCs, consistent with Oil Red quantification. On the other hand, the up-regulation of TRIB3 suppressed lipid droplets formation in hASCs, consistent with Oil Red quantification. After adipogenic induction, adipogenesis-related genes, including peroxisome proliferator-activated receptor γ (PPARγ), cluster of differentiation 36 (CD36) and CCAAT/enhancer binding protein α (C/EBPα), were increased significantly in TRIB3-knockdown hASCs compared with the control group (P<0.01). Oppositely, PPARγ, CD36 and lipoprotein lipase (LPL) were significantly decreased in TRIB3-overexpression hASCs compared with the control group (P<0.01).@*CONCLUSION@#TRIB3 inhibited the adipogenic differentiation of hASCs. Knockdown of TRIB3 promoted the ability of adipogenesis of hASCs, while overexpression of TRIB3 inhibited the adipogenic differentiation of hASCs. Considering the important role of PPARγ in the adipogenis process, the molecular mechanism of the regulatory function of TRIB3 may be related with PPARγ signal pathway.

Effects of Lonicera caerulea extract on adipocyte differentiation and adipogenesis in 3T3-L1 cells and mouse adipose-derived stem cells (MADSCs) / 한국영양학회지

PURPOSE: Obesity is a major health problem of global significance because it is clearly associated with an increased risk of health problems, such as nonalcoholic fatty liver disease (NAFLD), diabetes, cardiovascular diseases, and cancer. Lonicera caerulea (LC) originates from high mountains or wet areas and has been used as a traditional medicine in northern Russia, China, and Japan. LC contains a range of bioactive constituents, such as vitamins, minerals, and polyphenols. This study examined the anti-obesity effects of LC during differentiation in preadipocytes. METHODS: The cell viability assay was performed after the differentiation of 3T3-L1 cells for 7 days. Oil Red O staining was used to visualize the changes in lipid droplets in 3T3-L1 cells and mouse adipose-derived stem cells (MADSCs). The mRNA expression of obesity-related genes was determined by quantitative real-time PCR. RESULTS: According to the results of Oil Red O staining, the lipid levels and size of lipid droplets in the adipocytes were reduced and the LC extract (LCE, 0.25–1 mg/mL) markedly inhibited adipogenesis in a dose-dependent manner. The treatment of LCE also decreased the mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα), and sterol regulatory element binding protein 1 (SREBP1) in 3T3-L1 cells. Western blot analysis showed that the PPARγ, C/EBPα, and SREBP1 protein levels in both 3T3-L1 and MADSC were reduced in a dose-dependent manner. CONCLUSION: These results suggest that LCE can inhibit adipogenic differentiation through the regulation of adipogenesis-related markers.

Recent Progress on Branched-Chain Amino Acids in Obesity, Diabetes, and Beyond

Branched-chain amino acids (BCAAs) are essential amino acids that are not synthesized in our body; thus, they need to be obtained from food. They have shown to provide many physiological and metabolic benefits such as stimulation of pancreatic insulin secretion, milk production, adipogenesis, and enhanced immune function, among others, mainly mediated by mammalian target of rapamycin (mTOR) signaling pathway. After identified as a reliable marker of obesity and type 2 diabetes in recent years, an increasing number of studies have surfaced implicating BCAAs in the pathophysiology of other diseases such as cancers, cardiovascular diseases, and even neurodegenerative disorders like Alzheimer's disease. Here we discuss the most recent progress and review studies highlighting both correlational and potentially causative role of BCAAs in the development of these disorders. Although we are just beginning to understand the intricate relationships between BCAAs and some of the most prevalent chronic diseases, current findings raise a possibility that they are linked by a similar putative mechanism.

Low-Frequency Intermittent Hypoxia Suppresses Subcutaneous Adipogenesis and Induces Macrophage Polarization in Lean Mice

BACKGROUND: The relationship between obstructive sleep apnoea (OSA) and metabolic disorders is complex and highly associated. The impairment of adipogenic capacity in pre-adipocytes may promote adipocyte hypertrophy and increase the risk of further metabolic dysfunction. We hypothesize that intermittent hypoxia (IH), as a pathophysiologic feature of OSA, may regulate adipogenesis by promoting macrophage polarization. METHODS: Male C57BL/6N mice were exposed to either IH (240 seconds of 10% O₂ followed by 120 seconds of 21% O₂, i.e., 10 cycles/hour) or intermittent normoxia (IN) for 6 weeks. Stromal-vascular fractions derived from subcutaneous (SUB-SVF) and visceral (VIS-SVF) adipose tissues were cultured and differentiated. Conditioned media from cultured RAW 264.7 macrophages after air (Raw) or IH exposure (Raw-IH) were incubated with SUB-SVF during adipogenic differentiation. RESULTS: Adipogenic differentiation of SUB-SVF but not VIS-SVF from IH-exposed mice was significantly downregulated in comparison with that derived from IN-exposed mice. IH-exposed mice compared to IN-exposed mice showed induction of hypertrophic adipocytes and increased preferential infiltration of M1 macrophages in subcutaneous adipose tissue (SAT) compared to visceral adipose tissue. Complementary in vitro analysis demonstrated that Raw-IH media significantly enhanced inhibition of adipogenesis of SUB-SVF compared to Raw media, in agreement with corresponding gene expression levels of differentiation-associated markers and adipogenic transcription factors. CONCLUSION: Low frequency IH exposure impaired adipogenesis of SAT in lean mice, and macrophage polarization may be a potential mechanism for the impaired adipogenesis.

Biological Properties of Mesenchymal Stem Cells Derived from Induced Pluripotent Stem Cells / 中国实验血液学杂志

OBJECTIVE@#To obtain induced pluripatent stem cells (iPSC) from peripheral blood mononucleated cells and further induce differentiation into mesenchymal stem cells (MSC), and to compare the biological characteristics of iPSC-derived MSC and other-derived MSC.@*METHODS@#Peripheral blood mononucleated cells were obtained and transduced with reprogramming factors by sendai virus vector. Induced differentiation of MSC was performed in 1 strain of iPSC that completed all identification, and their cell morphology and immunophenotype were identified by immu-nohistochemistry and flow cytometry. Adipogenic and osteogenic media were used to induce the adipogenic and osteogenic differentiation. The expression of immune-related transcription factors was identified by PCR to systematically elucidate the biological characteristics of iPSC-induced MSC.@*RESULTS@#After transfection with sendai virus with reprogramming factor, the fate of peripheral blood mononucleated cells was reversed, initiating the expression of stem cell characteristics, the iPSC was successfully cloned, amplified, and purified, and finally the stable proliferation of iPSC was obtained. Mesenchymal stem cells derived from iPSC, had morphology consistent with other-derived MSC, and the immunophenotypes met the standard. iPSC-MSC possessed the ability of lipogenic and osteogenic differentiation. RT-PCR showed that iPSC-MSC was high expression to PDL1, and low expression to A20; besides, the expression level of STAT3 was equal to BM-MSC; and also as to the expression level of HIF1α and UC-MSC, which was lower than BM-MSC.@*CONCLUSION@#Peripheral blood mononucleated cells successfully initiated the expression of stem cell characteristics after the transduction of sendai virus vector with reprogramming factors, and obtained multi-competent iPSC. iPSC can successfully be induced to the differentiation of MSC, and the iPSC-MSC have standard cell morphology, immunophenotype and differentiation ability. High expression of PDL1 and low expression of A20 in iPSC-MSC suggest that iPSC-derived cells have different biological characteristics in cell proliferation and immune regulation.

MicroRNA-138 Suppresses Adipogenic Differentiation in Human Adipose Tissue-Derived Mesenchymal Stem Cells by Targeting Lipoprotein Lipase

PURPOSE: Adipogenic differentiation of adipose tissue-derived mesenchymal stem cells (AMSCs) is critical to many disease-related disorders, such as obesity and diabetes. Studies have demonstrated that miRNA-138 (miR-138) is closely involved in adipogenesis. However, the mechanisms affected by miR-138 remain unclear. This work aimed to investigate interactions between miR-138 and lipoprotein lipase (LPL), a key lipogenic enzyme, in AMSCs. MATERIALS AND METHODS: Human AMSCs (hAMSCs) isolated from human abdomen tissue were subjected to adipogenic differentiation medium. Quantitative real-time polymerase chain reaction and Western blot assay were applied to measure the expressions of miR-138, LPL, and the two adipogenic transcription factors cytidine-cytidine-adenosine-adenosine-thymidine enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). The relationship between miR-138 and LPL was predicted utilizing the miRTarBase database and validated by dual luciferase reporter assay. RESULTS: Showing increases in C/EBPα and PPARγ expression levels, hAMSCs were induced into adipogenic differentiation. During adipogenesis of hAMSCs, miR-138 expression was significantly downregulated. Overexpression of miR-138 by transfection inhibited hAMSCs adipogenic differentiation in vitro. Mechanically, LPL was a target of miR-138. LPL expression was upregulated during adipogenesis of hAMSCs, and this upregulation was reversed by miR-138 overexpression. Functionally, silencing of LPL by transfection exerted similar inhibition of the expressions of C/EBPα and PPARγ. Meanwhile, LPL ectopic expression was able to partly abolish the suppressive effect of miR-138 overexpression on adipogenic differentiation of hAMSCs. CONCLUSION: Upregulation of miR-138 inhibits adipogenic differentiation of hAMSCs by directly downregulating LPL.

Three-Dimensional Spheroid Formation of Cryopreserved Human Dental Follicle-Derived Stem Cells Enhances Pluripotency and Osteogenic Induction Properties

BACKGROUND: Enhancement and maintenance of the stemness of mesenchymal stem cells (MSCs) is one of the most important factors contributing to the successful in vivo therapeutic application of these cells. In this regard, three-dimensional (3D) spheroid formation has been developed as reliable method for increasing the pluripotency of MSCs. Moreover, using a new protocol, we have previously shown that dental tissues of extracted wisdom teeth can be effectively cryopreserved for subsequent use as a source of autologous stem cells. The main purpose of this study is to analyze the stemness and in vitro osteogenic differentiation potential of 3D spheroid dental MSCs compared with conventional monolayer cultured MSCs. METHODS: In this study, MSC-characterized stem cells were isolated and cultured from long-term cryopreserved dental follicles (hDFSCs), and then 2D hDFSCs were cultured under 3D spheroid-forming conditions using a newly designed microchip dish. The spheroids (3D hDFSCs) thus produced were investigated and characterized with respect to stemness, MSC marker expression, apoptosis, cell cycle analysis, extracellular matrix (ECM) production, and osteogenic and adipogenic differentiation properties. RESULTS: In terms of MSC and senescence markers, spheroid cells showed no difference when compared with 2D hDFSCs; however, 3D hDFSCs were observed to have a higher proportion of cell cycle arrest and a larger number of apoptotic cells. Moreover, spheroids showed substantially increased levels of pluripotency marker (early transcription factors) and ECM protein expression. Compared with 2D hDFSCs, there was also a notable enhancement in the osteogenic induction potential of spheroids, although no differences were observed with respect to in vitro adipogenesis. CONCLUSION: To the best of our knowledge, this is the first study to demonstrate the application of a spheroid culture system for dental follicle-derived stem cells using a microchip dish. Although further studies are needed, including in vivo transplantation, the results obtained in this study indicate that spheroid hDFSCs derived from cryopreserved dental follicle tissues could be used as a valuable source of autologous stem cells for bone tissue regeneration.