Prolonged treatment (12-24 h) of adipocytes with tumor necrosis factor ␣ (TNF␣) stimulates lipolysis. We have investigated the hypothesis that TNF␣ stimulates lipolysis by blocking the action of endogenous adenosine. Adipocytes were incubated for 48 h with TNF␣, and lipolysis was measured in the absence or presence of adenosine deaminase. Without adenosine deaminase, the rate of glycerol release was 2-3-fold higher in the TNF␣-treated cells, but with adenosine deaminase lipolysis increased in the controls to approximately that in the TNF␣-treated cells. This suggests that TNF␣ blocks adenosine release or prevents its antilipolytic effect. Both N 6 -phenylisopropyl adenosine and nicotinic acid were less potent and efficacious inhibitors of lipolysis in treated cells. A decrease in the concentration of ␣-subunits of all three G i subtypes was detected by Western blotting without a change in G s proteins or -subunits. G i2 ␣ was about 50% of control, whereas G i1 ␣ and G i3 ␣ were about 20 and 40% of control values, respectively. The time course of G i down-regulation correlated with the stimulation of lipolysis. Furthermore, down-regulation of G i by an alternative approach (prolonged incubation with N 6 -phenylisopropyl adenosine) stimulated lipolysis. These findings indicate that TNF␣ stimulates lipolysis by blunting endogenous inhibition of lipolysis. The mechanism appears to be a G i protein down-regulation.TNF␣ 1 is a multifunctional cytokine important in many pathological and physiological states (1). A series of recent reports has demonstrated that adipose tissue expresses TNF␣ mRNA and protein. Furthermore, adipose tissue from obese animals and humans expresses considerably more TNF␣ than does tissue from their lean counterparts (2, 3). This excess expression of TNF␣ in adipose tissue may form a link between obesity and development of insulin resistance, which often leads to type 2 diabetes in obese subjects (4). However, TNF␣ is not measurable in the circulation of obese subjects and is therefore considered an autocrine or paracrine regulator of adipose tissue metabolism.TNF␣ has several effects on adipocytes that may be related to the development of type 2 diabetes in obese subjects. It has been reported that TNF␣ induces insulin resistance possibly by inducing serine phosphorylation of insulin receptor substrate-1 and converting it to an inhibitor of the insulin receptor tyrosine kinase (5). In addition, TNF␣ causes a net depletion of the adipose tissue triglyceride. Initially, this was thought to be largely because of a decrease in the activity of lipoprotein lipase (6). However, we and others have reported that TNF␣ also stimulates lipolysis both in rat adipocytes maintained in primary culture (7) and in 3T3-L1 adipocytes (8 -10). We found that TNF␣-induced stimulation of lipolysis in primary adipocytes is chronic in nature, taking approximately 6 -12 h before a measurable effect is observed (7). Furthermore, neither the rate of isoproterenol-stimulated lipolysis nor the concentration of hormone-sens...
The porcine P-450 cholesterol side-chain cleavage enzyme gene (P450scc) contains a 30-bp region [insulin-like growth factor response element (IGFRE)] that mediates insulin-like growth factor I (IGF-I)-stimulated gene expression and binds Sp1. In this study, we showed that polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF), an RNA-binding component of spliceosomes, binds to the IGFRE. Southwestern analysis with an IGFRE oligonucleotide showed that a protein (from Sp1-immunodepleted HeLa extract) fractionated on SDS-PAGE at 100 kDa. Microsequence analysis of 100-kDa band HeLa proteins detected PSF. DNA affinity chromatography, using an IGFRE mutant oligonucleotide that does not bind Sp1, isolated a protein that immunoreacted with PSF antibody. Deoxyribonuclease I (DNase I) footprint analysis showed recombinant PSF binds 5' of the Sp1-binding GC box of the IGFRE, and mutant oligonucleotides further delineated this region to a palindrome, CTGAGTC. Functional analysis of these mutants by transfection experiments in a cell line overexpressing the IGF-I receptor (NWTb3) found that an inability to bind PSF significantly increased the IGFRE transcriptional activity, while retaining responsiveness to IGF-I. Moreover, transfection of expression vectors for Sp1 and PSF in porcine granulosa cells found that Sp1 expression stimulated IGFRE transcriptional activity while PSF inhibited activity even with coexpression of Sp1. In conclusion, we identified PSF as an independent, inhibitory regulator of the transcriptional activity of the porcine P450scc IGFRE.
Troglitazone (a thiazolidinedione that improves insulin resistance) lowers elevated androgen concentrations in women with polycystic ovarian syndrome. In this study, we assessed the direct effects of troglitazone on steroidogenesis in porcine granulosa cells. Troglitazone inhibited progesterone production in a dose- and time-dependent manner (earliest effects at 4 h, maximum at 24 h) without affecting cell viability. Progesterone production was also inhibited by troglitazone in the presence of 25-hydroxycholesterol, indicating that the drug does not affect intracellular cholesterol transport. Troglitazone also inhibited FSH- and forskolin-stimulated progesterone secretion. The reduced progesterone production was accompanied by marked elevations of pregnenolone concentrations, suggesting inhibition of 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The activity of 3beta-HSD in troglitazone-treated granulosa cells was decreased by more than 60%, compared with controls after 24 h. Troglitazone did not affect aromatase activity in porcine granulosa cells. In summary, troglitazone has direct effects on porcine granulosa cell steroidogenesis. The drug specifically inhibits 3beta-HSD activity, resulting in impaired progesterone production. The clinical relevance of this direct in vitro effect on steroidogenesis needs further investigation.
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