Priming with magnesium-deficient media inhibits preadipocyte differentiation via potential upregulation of tumor necrosis factor-alpha

The effect of priming stromal-vascular cells in primary cultures with magne sium-deficient (MgD) media on preadipocyte differentiation was studied. Cul tures were derived from dorsal subcutaneous fat tissue of young pigs and ma intained 3 d in serum-free control or MgD Dulbecco's modified Eagle's mediu m, 3 d in 10% fetal bovine serum and dexamethasone, and 6 d in insulin. At d 12 of culture, immunocytochemical and glycerophosphate dehydrogenase assa ys indicated depressed adipocyte differentiation in the MgD groups. Culture s were enriched for preadipocytes up to 50% of total cells. On the third da y of treatment with control and MgD medium, total cell lysates were isolate d and 50 mu g of them were run on two-dimensional gel electrophoresis. The separated proteins from both treatment groups showed similar patterns. Howe ver, spots of proteins with predicted molecular weight in the range of 25.8 -37.4 kDa and pi of 5.39-5.85 were sixfold denser from the MgD 10 groups th an from the controls. These proteins migrate similarly to tumor necrosis fa ctor-alpha (TNF-alpha). The amount of TNF-alpha in cell lysates from the Mg D group was about 2.35 times greater than controls determined by TNF-alpha- ELISA. It is likely that proteins upregulated by MgD medium are TNF-alpha i soforms.