Newly developed primary culture of rat visceral adipocytes and their in vitro characteristics

Abstract

We have recently developed a primary culture system for visceral adipocytes (VAs) using stomal-vascular cells (SVCs) isolated from the mesenteric fat tissue of male Sprague-Dawley rats of 3-5 weeks of age. Modified Dulbecco's modified Eagle medium (DMEM)/F12 containing 17 microM pantothenic acid, 33 microM biotin, 100 microM ascorbic acid, 1 microM octanoic acid, 50 nM triiodothyronine, 10 microg/ml insulin, 10% newborn calf serum (NCS), 100 units/ml penicillin and 100 microg/ml streptomycin was used as a basal culture medium, which did not contain any synthetic compounds usually used to promote adipogenesis, such as indomethacin, dexamethasone, or peroxisome proliferator-activated receptor (PPAR)-gamma agonists. The SVCs differentiated and proliferated efficiently, and formed a confluent monolayer in 3 days. The VAs accumulated lipids droplets in their cytoplasm at approximately 7 days. The differentiation rate from applied SVCs to mature adipocytes was >80% per culture. Adiponectin concentration in the medium increased from Day 5 to Day 7. Application of lipid emulsion stimulated maturation of the SVCs into VAs, as well as subsequent lipid accumulation. Norepinephrine (2 x 10(-5) mM) reduced the size of lipid particles and decreased triglyceride (TG) content in the matured adipocytes at 30 min. These results indicate that the new culture system is sufficient to maintain the physiological activity of visceral adipose tissue similar to that in vivo, making it an appropriate and useful tool for basic and applied research on obesity.

Temporal and spatial variations of lipid droplets during adipocyte division and differentiation

“…Fresh culture medium was adjusted to 378C and pH 7.0, and a lipid emulsion (Intrafat; Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) containing 1.2 g/ 100 ml lecithin, 2.25 g/100 ml glycerol, and 20 g/100 ml soybean oil was added at 0.1% to the medium. The addition of lipid emulsion was reported to promote the enlargement of LDs during adipocyte differentiation (27), although the lipid emulsion was not essential for adipocyte differentiation. The dish was completely filled with the culture medium with the lipid emulsion, and was then sealed with a sterile coverslip and silicone grease (Dow Corning Toray Co., Ltd., Tokyo, Japan).…”

“…The culture system, comprising SVCs derived from the mesenteric adipose tissue of Sprague-Dawley rats and a culture medium based on DMEM/F12, was previously established by Shimizu et al (27). In their study, the culture medium was supplemented with 10% newborn calf serum, 17 mM pantothenic acid, 33 mM biotin, 100 mM ascorbic acid, 1 mM octanoic acid, 50 nM triiodothyronine, 2.5 mM nicotinamide, 10 mg/ml insulin, 100 U/ml penicillin, and 100 mg/ml streptomycin as optimal conditions for the induction of adipocyte differentiation from SVCs.…”

Sphingosine kinase is induced in mouse 3T3-L1 cells and promotes adipogenesis

“…II and data not shown). We also tested the effects of these SphK inhibitors in rat primary preadipocytes that undergo spontaneous adipogenesis at the day 7 of the culture (17,18). Both DMS (5 mM) and DHS (2.5 mM) significantly attenuated the degrees of lipid accumulation compared with vehicle (supplementary Fig.…”

“…These cells were commercially purchased from Primary Cell (Hokkaido, Japan). They reach confluence at day 3 of culture, and then spontaneously start differentiating into adipocytes when cultured in Adipocyte Differentiation Medium (supplied) (17,18).…”

Involvement of guanylin and GC-C in rat mesenteric macrophages in resistance to a high-fat diet

“…The culture system included the stromal vascular cell fraction from the mesenteric fat tissue of SD rats and a differentiation medium that was based on DMEM and F12; this medium did not include indomethacin, dexamethasone, or peroxisome proliferator-activated receptor ␥ (PPAR-␥ ) agonist ( 26 ). Coculture was performed by adding peritoneal macrophages (1 × 10 5 cells/well) of Tg or WT rats to wells containing undifferentiated primary adipocytes (1 × 10 5 cells/well; 24-well plate).…”