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

Abstract

To investigate the effects of cytokines on adipocyte lipolysis, a macrophage cell line (RAW 264.7) was treated with Escherichia coli lipopolysaccharide (1 microgram/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-alpha (TNF alpha), and the direct addition of recombinant human TNF alpha (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) TNF alpha can account for most, or perhaps all, of this effect; 3) TNF alpha increases the rate of lipolysis by a mechanism that does not involve increased expression of HSL. Based on the time-dependent aspects of TNF alpha stimulation and the lack of change in immunoreactive HSL, the findings suggest a TNF-induced posttranslational modification of the enzyme.

Bacterial Endotoxin Stimulates Adipose Lipolysis via Toll-Like Receptor 4 and Extracellular Signal-regulated Kinase Pathway

“…An early study showed that the conditioned medium of LPS-stimulated macrophages increased lipolysis in adipocytes, an effect thought to result from elevated TNF-␣ secretion by LPS, not from LPS itself, although this conditioned medium contained at least 0.05 g/ml LPS (19). We observed that when original LPS residues in the conditioned medium of LPS-stimulated adipocytes were neutralized by the anti-endotoxin BNEP, the conditioned medium could not elevate lipolysis.…”

“…Adipocytokine or NFB Activation Is Not Involved in LPS-stimulated Lipolysis-An early study showed that the conditioned medium of LPS-stimulated macrophages increased lipolysis, but this effect may have resulted from increased TNF-␣ secretion from LPS-stimulated microphages (19). We therefore investigated whether the cytokine secretion from adipocytes accounts for LPS-stimulated lipolysis.…”

“…These observations imply that endotoxin could directly act on adipose tissues to modulate lipolysis. Nevertheless, other studies showed that short term (2-h) stimulation with LPS could not induce lipolysis in pig adipocytes (18), and incubation with a conditioned medium of LPS-stimulated macrophages increased lipolysis, but the effect was thought to result from LPS-elevated TNF-␣ secretion in the medium (19). Therefore, the direct effect of LPS in adipose lipolysis and its association with an elevated level of serum FFAs in endotoxemia remain unclear.…”

Relative Contribution of Adipose Triglyceride Lipase and Hormone-sensitive Lipase to Tumor Necrosis Factor-α (TNF-α)-induced Lipolysis in Adipocytes

“…Although a previous report indicates that TNF-␣ down-regulates ATGL and HSL mRNA (53), we find that TNF-␣ does not alter their protein levels in 3T3-L1 adipocytes. Parallel observations were obtained from primary rat adipocytes treated with TNF-␣ (39,54). Most importantly, we demonstrate that HSL and ATGL are both required for the lipolytic response to TNF-␣.…”

“…Statistical analysis was determined by Student's t test or one-way analysis of variance. (39,41,45,46). To examine the modulation of individual players in the lipolytic proteome, we treated fully differentiated 3T3-L1 adipocytes with TNF-␣ for various periods of time.…”