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 macr ophage cell line (RAW 264.7) was treated with Escherichia coli lipopol ysaccharide (1 mu g/ml) for 18 h to induce cytokine release. Condition ed medium (5%, vol/vol) from these cells was added to rat epididymal a dipocytes isolated and incubated under sterile conditions. After incub ation, the adipocytes were washed, and the rate of lipolysis (glycerol release) was determined after a further 1-h incubation. The condition ed medium caused an approximately 2.7-fold increase in lipolysis, dete ctable after 6-12 h, maximal by 24 h, and reversible by 48 h after was hing the cells. The effect of conditioned medium was reversed by a neu tralizing antibody to mouse tumor necrosis factor-alpha (TNF alpha), a nd the direct addition of recombinant human TNF alpha(0.1-50 ng/ml) re produced 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 in vestigated by Western immunoblots using an antibody raised to a bacter ially expressed 96-amino acid portion of the HSL enzyme. TNF treatment did not alter the concentration of immunoreactive HSL. From these dat a we conclude that 1) macrophages release a cytokine(s) in response to lipopolysaccharide that stimulates lipolysis in freshly isolated adip ocytes; 2) TNF alpha can account for most, or perhaps all, of this eff ect; 3) TNF alpha increases the rate of lipolysis by a mechanism that does not involve increased expression of HSL. Based on the time-depend ent aspects of TNF alpha stimulation and the lack of change in immunor eactive HSL, the findings suggest a TNF-induced posttranslational modi fication of the enzyme.