Stimulation of leptin release by arachidonic acid and prostaglandin E(2) in adipose tissue from obese humans.

The purpose of this study was to examine the effect of arachidonic acid and its metabolites on leptin formation by explants of human adipose tissue over a 48-hour incubation in primary culture. We found that arachidonic acid or prostaglandin E(2) (PGE(2)) stimulated leptin release by explants of subcutaneous adipose tissue from obese humans. The stimulatory effect of arachidonic acid on leptin formation was blocked by NS-398, a cyclooxygenase-2 (COX-2) inhibitor. There was appreciable release of PGE(2) to the medium over 48 hours, and this was inhibited by 99% in the presence of 200 nmol/L dexamethasone or 5 micromol/L NS-398. The increase in PGE(2) release correlated with induction of COX-2 activity during the 48-hour incubation. The increase in COX-2 activity was blocked by 200nmol/L dexamethasone. The level of leptin mRNA at 48 hours was reduced by 28% if PGE(2) was added in the absence of dexamethasone, while in the presence of dexamethasone, the amount of leptin mRNA was enhanced by 156%. These data suggest that when upregulation of COX-2 is blocked by dexamethasone, exogenous PGE(2) enhances both leptin release and leptin mRNA accumulation by explants of human adipose tissue in primary culture.

Regulation of leptin release by troglitazone in human adipose tissue.

In pieces of human subcutaneous adipose tissue incubated in primary culture for 48 hours, the release of leptin was stimulated by 50% in the presence of 3.3 micromol/L troglitazone. Insulin (0.1 nmol/L) and dexamethasone (200 nmol/L) stimulated leptin release by 30% and 300%, respectively. Troglitazone in combination with either insulin or dexamethasone had no effect on leptin release. Instead, troglitazone inhibited leptin release in the presence of both dexamethasone and insulin. The stimulatory effect of troglitazone on leptin release was also mimicked by 1 micromol/L 15-deoxy-delta(12-14)prostaglandin J2 (dPGJ2). However, if the concentration of dPGJ2 was increased to 10 micromol/L in the presence of dexamethasone, there was a decrease in leptin release, as well as of lactate formation and lipolysis. These data indicate that both stimulatory and inhibitory effects of troglitazone and dPGJ2 can be seen on leptin release by human adipose tissue.

Synergism between insulin and low concentrations of isoproterenol in the stimulation of leptin release by cultured human adipose tissue.

The release of leptin by pieces of human adipose tissue incubated in primary culture for 24 or 48 hours in the presence of dexamethasone was reduced by isoproterenol. An inhibition of leptin release was observed at 24 hours in the presence of isoproterenol and was mediated by beta1-adrenergic receptors, since it was blocked by the specific beta1-adrenoceptor antagonist CGP-20712A. The inhibitory effect of 33 nmol/L isoproterenol on leptin release was reversed in the presence of 0.1 nmol/L insulin to a 2-fold stimulation of leptin release. These data suggest that the primary mechanism by which insulin stimulates leptin release is to blunt the inhibitory effects of beta1-adrenergic receptor agonists, and low concentrations of catecholamines actually enhance the stimulation of leptin release by insulin.

Reduced galactosyltransferase mRNA levels are associated with the agalactosyl IgG found in arthritis-prone MRL-lpr/lpr strain mice.

MRL-lpr/lpr strain mice have defectively glycosylated IgG. This may be related to the rheumatoid arthritis (RA)-like disease that occurs in these mice, because a similar glycosylation defect is seen in human subjects with RA. Whilst it is known that this defect is associated with reduced activity of the beta-1,4-galactosyltransferase (beta-1,4-GalTase) enzyme, the cause of this reduced activity is at present unknown. We have therefore examined the molecular genetics of beta-1,4-GalTase in MRL-lpr/lpr mice. Using 10 different restriction endonucleases we found no evidence for a polymorphic variant of the gene in glycosylation-defective mice. However, the level of mRNA for beta-1,4-GalTase was lowest in the MRL-lpr/lpr mice, the strain with the most poorly galactosylated IgG of the four strains examined. Thus, the reduced level of IgG oligosaccharide galactosylation found in MRL-lpr/lpr strain mice appears to be related to either an altered transcriptional level of, or altered mRNA stability for, beta-1,4-GalTase in lymphocytes from these mice.

Regulation of the p58GTA cell division control-related protein kinase during phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells.

We describe the regulation of expression, activity and subcellular localization of a cell cycle control-related kinase, p58GTA, during the withdrawal from the cell cycle of U937 human leukemic cells induced by phorbol esters. Our studies indicate that steady-state mRNA, protein levels, transcription and subcellular localization of this kinase are affected in distinctly different manners by phorbol esters (phorbol 12-myristate 13-acetate, PMA). Steady-state mRNA levels increase dramatically within 1 h of PMA treatment, while steady-state protein levels increase only slightly. However, within 24 h of PMA treatment both steady-state p58GTA mRNA and protein levels decrease markedly. Assays of p58GTA protein kinase activity show that, even though steady-state protein levels are relatively constant, protein kinase activity increases within 30 min of PMA treatment, and then peaks at 2 h and 12 h after PMA treatment. Once again, p58GTA protein kinase activity decreases by 48 h to levels similar to unstimulated cells. These results suggest that the expression of the p58GTA protein kinase gene and, quite possibly, its post-translational modification are affected by phorbol esters in a complex manner.

Beta 1-4-galactosyltransferase gene expression is regulated during entry into the cell cycle and during the cell cycle.

Mammalian glycosyltransferases have been implicated in a wide variety of functions besides N-linked glycosylation, including developmental processes. For this reason, we studied the effects of cell cycle and entry into the cell cycle on beta 1-4-galactosyltransferase gene expression. In this study we report that beta 1-4-galactosyltransferase (GalTase) gene expression is, indeed, regulated during the normal cell cycle, peaking during late G1-, S, and early G2 phase of the cell cycle. In addition, GalTase gene expression is regulated in a manner that resembles other "early response" genes such as jun and fos upon reentry into the cell cycle from quiescence. Finally, we show that the GalTase gene is differentially expressed during murine embryogenesis and in terminally differentiated adult tissues. It is most abundant in testis, followed by skeletal muscle and spleen. The reasons for this pattern of differential expression in adult tissues are unknown. These studies should provide important new information regarding GalTase gene expression, its regulation, and its potential link to other developmental functions.