Differentiation Of Stromal-vascular Cells Research Articles

Hygiene of housing conditions and proinflammatory signals alter gene expressions in porcine adipose tissues and blood cells.

Adipose tissue is an organ with metabolic, endocrine and immune functions. In this tissue, the expressions of genes associated with several metabolic pathways, including lipid metabolism, have been shown to be affected by genetic selection for feed efficiency, an important trait to consider in livestock. We hypothesized that the stimulation of immune system caused by poor hygiene conditions of housing impacts the molecular and cellular features of adipose tissue and that the impact may differ between pigs that diverge in feed efficiency. At the age of 12 weeks, Large White pigs from two genetic lines divergent for residual feed intake (RFI) were housed in two contrasting hygiene conditions (good vs poor). After six weeks of exposure, pigs were slaughtered (n=36). Samples of blood, subcutaneous (SCAT) and perirenal (PRAT) adipose tissues were collected for cell response and gene expression investigations. The decrease in the relative weight of PRAT was associated with a decline in mRNA levels of FASN, ME, LCN2 and TLR4 (P<0.05) in pigs housed in poor conditions compared with pigs housed in good conditions for both RFI lines. In SCAT, the expressions of only two key genes (PPARG and TLR4) were significantly affected by the hygiene of housing conditions. Besides, the mRNA levels of both LCN2 and GPX3 were influenced by the RFI line (P<0.05). Because we suspected an effect of poor hygiene at the cellular levels, we investigated the differentiation of stromal vascular cells isolated from SCAT in vitro in the absence or presence of a pro-inflammatory cytokine, Tumor Necrosis Factor-α (TNF-α). The ability of these cells to differentiate in the absence or presence of TNF-α did not differ among the four groups of animals (P>0.05). We also investigated the expressions of genes involved in the immune response and lipid metabolism in whole blood cells cultured in the absence and presence of LPS. The hygiene conditions had no effect but, the relative expression of the GPX3 gene was higher (P<0.001) in high RFI than in low RFI pigs while the expressions of IL-10 (P=0.027), TGFβ1 (P=0.023) and ADIPOR2 (P=0.05) genes were lower in high RFI than in low RFI pigs. Overall, the current study indicates that the hygiene of housing had similar effects on both RFI lines on the expression of genes in adipose tissues and on the features of SCAT adipose cells and whole blood cells in response to TNF-α and LPS. It further demonstrates that the number of genes with expression impacted by housing conditions was higher in PRAT than in SCAT. It suggests a depot-specific response of adipose tissue to the current challenge.

A genome-wide transcriptomic analysis of embryos fathered by obese males in a murine model of diet-induced obesity

Paternal obesity is known to have a negative impact on the male’s reproductive health as well as the health of his offspring. Although epigenetic mechanisms have been implicated in the non-genetic transmission of acquired traits, the effect of paternal obesity on gene expression in the preimplantation embryo has not been fully studied. To this end, we investigated whether paternal obesity is associated with gene expression changes in eight-cell stage embryos fathered by males on a high-fat diet. We used single embryo RNA-seq to compare the gene expression profile of embryos generated by males on a high fat (HFD) versus control (CD) diet. This analysis revealed significant upregulation of the Samd4b and Gata6 gene in embryos in response to a paternal HFD. Furthermore, we could show a significant increase in expression of both Gata6 and Samd4b during differentiation of stromal vascular cells into mature adipocytes. These findings suggest that paternal obesity may induce changes in the male germ cells which are associated with the gene expression changes in the resulting preimplantation embryos.

Pre-Adipocyte Determination and Adipocyte Differentiation of Stromal Vascular Cells Isolated From Intramuscular Tissue of Hanwoo Beef Cattle Treated by Acetate and Propionate

The effects of acetate and propionate in enhancing intramuscular fat (marbling) deposition in beef cattle are poorly understood. This study aimed to evaluate the effects of acetate and propionate on pre-adipocyte determination and adipocyte differentiation in stromal vascular cells (SVC) isolated from the intramuscular tissue of Hanwoo beef cattle. SVC were isolated and treated with different concentrations of acetate and propionate (0.125 to 1 mM). Pre-adipocyte determination was observed through the Zfp423 (Zinc finger protein 423) and Pref-1 (Pre-adipocyte factor-1) genes expressions, which were important transcription factors used to identify committed pre-adipocytes. Adipocyte differentiation was determined based on the lipid accumulation, triglyceride contents, and their molecular activities related to adipogenic markers. The results suggest that acetate supports pre-adipocyte determination in SVC which is observed to increase the expression of Zfp423 and Pref-1 along with the increasing acetate concentration. Propionate treatment was seen to significantly affect lipid accumulation as well as triglyceride content in SVC, compared to acetate treatment. Furthermore, propionate treatment very significantly increased the expressions of PPARγ, C/EBPα, SREBP-1c, and FABP4. In conclusion, acetate likely enhances pre-adipocyte determination, while propionate effectively promotes adipogenesis in SVC isolated from the intramuscular tissue of Hanwoo beef cattle.

The effects of acetate on enhancing the proliferation and preadipocyte differentiation of stromal vascular cells isolated from Hanwoo beef cattle

We investigated the effect of acetate on the proliferation and differentiation of bovine stromal‐vascular cells (SVC). SVC was isolated from the longissimus muscle collected directly from approximately 30‐month‐old Hanwoo beef cattle steers. After confluence, the differentiation was induced by 1‐methyl‐3‐isobutyl‐xanthine, dexamethasone, insulin, and rosiglitaszone for 2 days, and then subsequently cultured for 14 days. The cells were treated with different concentrations of acetate (0.125 to 2 mMol) throughout the differentiation period. Cell viability assay revealed a dose‐dependent increase in the proliferation of SVC. Monitoring of the differentiation through Oil Red‐O staining showed that treatment with acetate up to 1mMol significantly increased cell differentiation and fat accumulation. Supplementation of acetate in the media throughout the differentiation period also enhanced the induction of all differentiation markers. Acetate treatment unregulated the mRNA and protein expression of peroxisome proliferator‐activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα), Sterol regulatory element‐binding protein 1c (SREBP‐1c), fatty acid binding protein 4 (FABP 4) and fatty acid synthase (FAS). These data suggests that acetate can enhance adipocyte differentiation from SVC isolated from Hanwoo beef cattle and possibly improve meat palatability through increased intramuscular fat deposition and reduce meat toughness through decreased fibrogenic differentiation.Support or Funding InformationThis work was carried out by the support of the Rural Development Administration Grant (Project No. PJ01268703).

AMP‐activated protein kinase (AMPK) α1 is required for brown adipogenesis (LB843)

Brown adipose tissue (BAT) burns fatty acids to generate heat, holding the promise to treat obesity and type 2 diabetes. However, mechanisms regulating brown adipocyte formation remain poorly defined. AMP‐activated protein kinase (AMPK) is a key regulator of energy metabolism. We hypothesized that AMPK regulates the development of brown fat, an important organ involved in energy catabolism. To test, we employed AMPKα1 floxed mice which were crossed with ER‐Cre mice to conditionally knockout AMPKα1. AMPKα1 knockout was induced by 4‐hydrotamoxifen. Abrogation of AMPKα1 blunted the brown adipogenic differentiation of stromal vascular cells isolated from brown fat. Correspondingly, the expression of UCP‐1 and Prdm16, two markers of brown adipogenesis, was pronouncedly suppressed during differentiation (around 4 fold and 3 fold reduction respectively, P = 0.03). Moreover, adipogenic progenitor cells were separated from brown stromal vascular cells by FACS using surface markers (sca1+/CD34+/CD45‐). Consistently, the absence of AMPKα1 blocked brown adipogenesis in purified progenitors. Taken together, these data suggest that the AMPKα1 is required for brown adipogenesis (The work was funded by NIH R01HD067449).Grant Funding Source: Supported by NIH R01HD067449

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Proliferation and Differentiation of Stromal-Vascular Cells in Primary Culture Differ between Neonatal Pigs Consuming Maternal or Formula Milk

Proliferation and differentiation of preadipocytes from 7-d-old pigs consuming maternal or formula milk were examined in primary culture of stromal-vascular (s-v) cells derived from subcutaneous adipose tissue. Unsuckled pigs were bottle-fed isoenergetically with colostrum and then sow's milk (SM) or with formula milk alone (F) from birth to 7 d. Isolated cells were exposed to serum-supplemented medium and serum-free medium to determine proliferation and differentiation, respectively. Proliferation estimated between d 3 and 4 of culture was higher (P<0.05) in cells from F than SM pigs. In addition, the number of s-v cells isolated from 1 g of adipose tissue was higher (P<0.01) in F than SM pigs. Variables assessing differentiation were also affected. The percentage of differentiating cells and lipoprotein lipase (LPL) activity were lower (P<0.05) in F than SM pigs, whereas malic enzyme (ME) activity did not differ significantly between the two groups. In conclusion, formula milk increased the number of s-v cells and their capacity for proliferation, whereas the potential for cell differentiation was lower compared with cells from the maternal milk group. Further studies are required to identify the growth and/or nutritional factors that are implicated in the observed differences and to determine whether subsequent development of adipose tissue is affected.