Tumor necrosis factor increases the rate of lipolysis in primary cultures of adipocytes without altering levels of hormone-sensitive lipase

To investigate the effects of cytokines on adipocyte lipolysis, a macrophage cell line (RAW 264.7) was treated with Escherichia coli lipopolysaccharide (1 μg/ml) for 18 h to induce cytokine release. Conditioned medium (5%, vol/vol) from these cells was added to rat epididymal adipocytes isolated and incubated under sterile conditions. After incubation, the adipocytes were washed, and the rate of lipolysis (glycerol release) was determined after a further 1-h incubation. The conditioned medium caused an approximately 2.7-fold increase in lipolysis, detectable after 6-12 h, maximal by 24 h, and reversible by 48 h after washing the cells. The effect of conditioned medium was reversed by a neutralizing antibody to mouse tumor necrosis factor-α (TNFα), and the direct addition of recombinant human TNFα (0.1-50 ng/ml) reproduced the effect, with a half-maximally effective concentration of approximately 3 ng/ml. The effect of TNF on the expression of hormone-sensitive lipase (HSL; the rate-limiting enzyme for lipolysis) was investigated by Western immunoblots using an antibody raised to a bacterially expressed 96-amino acid portion of the HSL enzyme. TNF treatment did not alter the concentration of immunoreactive HSL. From these data we conclude that 1) macrophages release a cytokine(s) in response to lipopolysaccharide that stimulates lipolysis in freshly isolated adipocytes; 2) TNFα can account for most, or perhaps all, of this effect; 3) TNFα increases the rate of lipolysis by a mechanism that does not involve increased expression of HSL. Based on the time-dependent aspects of TNFα stimulation and the lack of change in immunoreactive HSL, the findings suggest a TNF-induced posttranslational modification of the enzyme.