A comparative assessment of reference genes in mouse brown adipocyte differentiation and thermogenesis in vitro.

Adipogenic differentiation and thermogenesis in brown adipose tissue (BAT) undergo dynamic processes, altering phenotypes and gene expressions. Proper reference genes in gene expression analysis are crucial to mitigate experimental variances and ensure PCR efficacy. Unreliable reference genes can lead to erroneous gene expression quantification, resulting in data misinterpretation. This study focused on identifying suitable reference genes for mouse brown adipocyte research, utilizing brown adipocytes from the Ucp1-luciferase ThermoMouse model. Comparative analysis of gene expression data under adipogenesis and thermogenesis conditions was conducted, validating 13 housekeeping genes through various algorithms, including DeltaCq, BestKeeper, geNorm, Normfinder, and RefFinder. Tbp and Rer1 emerged as optimal references for Ucp1 and Pparg expression in brown adipogenesis, while Tbp and Ubc were ideal for the expression analysis of these target genes in thermogenesis. Conversely, certain conventional references, including Actb, Tubb5, and Gapdh, proved unstable as reference genes under both conditions. These findings stress the critical consideration of reference gene selection in gene expression analysis within specific biological systems to ensure accurate conclusions.

Caveolae-associated protein 3 promotes adipogenic differentiation of porcine preadipocytes by promoting extracellular signal-regulated kinase phosphorylation.

Fat deposition is one of the key factors affecting the economic development of pig husbandry. The aim of this study was to investigate the expression characteristics of caveolae-associated protein 3 (CAVIN3) and to elucidate its effect and mechanism on adipogenic differentiation of porcine preadipocytes. Cell transfection, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, and oil red O staining were used to detect the effect of CAVIN3 on the differentiation of porcine preadipocytes. The results showed that CAVIN3 was expressed in various tissues, with higher expression in adipose tissue, differentially expressed during cell adipogenic differentiation, and mainly distributed in the cytoplasm. Functional studies showed that, after CAVIN3 interference in preadipocytes, the expression of adipogenic factors and the content of lipid droplets were significantly decreased (p < 0.05). The results were reversed after CAVIN3 was overexpressed. The mechanism research showed that LY3214996 inhibited the extracellular signal-regulated kinase (ERK) phosphorylation and further inhibited lipogenic factors expression. Overexpression of CAVIN3 attenuates the inhibitory effect of LY3214996 on ERK phosphorylation and attenuates its inhibitory effect on adipogenic differentiation. Therefore, this study demonstrated that CAVIN3 promotes the differentiation of porcine preadipocytes by promoting ERK phosphorylation. The present study can lay a theoretical foundation for further studying the molecular mechanism of porcine fat deposition.

Adipogenesis of ear mesenchymal stem cells (EMSCs): adipose biomarker-based assessment of genetic variation, adipocyte function, and brown/brite differentiation.

Ear mesenchymal stem cells (EMSCs) have been investigated to differentiate into adipocytes, chondrocytes, and muscle cells in vitro. However, the factors controlling adipogenesis of this stem cell population in vitro, function, and type of adipocytes raised from them are still unclear. Here we found that genetics have a modest effect on adipogenic capacity of EMSCs. Adipocytes differentiated from EMSCs have a potential function in lipid metabolism as indicated by expression of lipogenic genes and this function of EMSC adipocytes is regulated by genetics. EMSCs failed to be differentiated into brite/brown adipocytes due to their lack of a thermogenic program, but adipocytes raised from EMSCs showed a fate of white adipocytes. Overall, our data suggest that EMSCs differentiate into functional white adipocytes in vitro and this is genetic-dependent.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Zinc transporter ZIP13 suppresses beige adipocyte biogenesis and energy expenditure by regulating C/EBP-ß expression.

Given the relevance of beige adipocytes in adult humans, a better understanding of the molecular circuits involved in beige adipocyte biogenesis has provided new insight into human brown adipocyte biology. Genetic mutations in SLC39A13/ZIP13, a member of zinc transporter family, are known to reduce adipose tissue mass in humans; however, the underlying mechanisms remains unknown. Here, we demonstrate that the Zip13-deficient mouse shows enhanced beige adipocyte biogenesis and energy expenditure, and shows ameliorated diet-induced obesity and insulin resistance. Both gain- and loss-of-function studies showed that an accumulation of the CCAAT/enhancer binding protein-ß (C/EBP-ß) protein, which cooperates with dominant transcriptional co-regulator PR domain containing 16 (PRDM16) to determine brown/beige adipocyte lineage, is essential for the enhanced adipocyte browning caused by the loss of ZIP13. Furthermore, ZIP13-mediated zinc transport is a prerequisite for degrading the C/EBP-ß protein to inhibit adipocyte browning. Thus, our data reveal an unexpected association between zinc homeostasis and beige adipocyte biogenesis, which may contribute significantly to the development of new therapies for obesity and metabolic syndrome.

Assay validation for the assessment of adipogenesis of multipotential stromal cells--a direct comparison of four different methods.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed. METHODS: Four previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4',6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls. RESULTS: Oil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ~5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ~13-fold, with significant correlations with oil red scoring observed for MSC from other sources. CONCLUSIONS: Flow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources.

Transcriptomic coordination in the human metabolic network reveals links between n-3 fat intake, adipose tissue gene expression and metabolic health.

Understanding the molecular link between diet and health is a key goal in nutritional systems biology. As an alternative to pathway analysis, we have developed a joint multivariate and network-based approach to analysis of a dataset of habitual dietary records, adipose tissue transcriptomics and comprehensive plasma marker profiles from human volunteers with the Metabolic Syndrome. With this approach we identified prominent co-expressed sub-networks in the global metabolic network, which showed correlated expression with habitual n-3 PUFA intake and urinary levels of the oxidative stress marker 8-iso-PGF(2α). These sub-networks illustrated inherent cross-talk between distinct metabolic pathways, such as between triglyceride metabolism and production of lipid signalling molecules. In a parallel promoter analysis, we identified several adipogenic transcription factors as potential transcriptional regulators associated with habitual n-3 PUFA intake. Our results illustrate advantages of network-based analysis, and generate novel hypotheses on the transcriptomic link between habitual n-3 PUFA intake, adipose tissue function and oxidative stress.

Uncoupling of inflammation and insulin resistance by NF-kappaB in transgenic mice through elevated energy expenditure.

To study the metabolic activity of NF-kappaB, we investigated phenotypes of two different mouse models with elevated NF-kappaB activities. The transcriptional activity of NF-kappaB is enhanced either by overexpression of NF-kappaB p65 (RelA) in aP2-p65 mice or inactivation of NF-kappaB p50 (NF-kappaB1) through gene knock-out. In these models, energy expenditure was elevated in day and night time without a change in locomotion. The mice were resistant to adulthood obesity and diet-induced obesity without reduction in food intake. The adipose tissue growth and adipogenesis were inhibited by the elevated NF-kappaB activity. Peroxisome proliferator-activator receptor gamma expression was reduced by NF-kappaB at the transcriptional level. The two models exhibited elevated inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) in adipose tissue and serum. However, insulin sensitivity was not reduced by the inflammation in the mice on a chow diet. On a high fat diet, the mice were protected from insulin resistance. The glucose infusion rate was increased more than 30% in the hyperinsulinemic-euglycemic clamp test. Our data suggest that the transcription factor NF-kappaB promotes energy expenditure and inhibits adipose tissue growth. The two effects lead to prevention of adulthood obesity and dietary obesity. The energy expenditure may lead to disassociation of inflammation with insulin resistance. The study indicates that inflammation may prevent insulin resistance by eliminating lipid accumulation.