Short-term variations in serum leptin in response to changes in nutritional status are likely to be critical to leptin's ability to modulate appetite and metabolism in advance of alterations in body fat. Although the abundance of leptin mRNA is important in the long-term regulation of leptin, post-transcriptional mechanisms appear to account for short term changes in leptin secretion. Little is known, however, about the cellular regulation of leptin. We will use newly-developed methods for pulse-labelling leptin with 35S-methionine to analyze relative rates of leptin synthesis, turnover and secretion as a function of hormonal and nutritional state. Our preliminary studies and that insulin can stimulate the release of leptin without affecting leptin mRNA levels. Two mechanisms appear to be involved: a potentiation of the rate of leptin secretion, and an increase in relative rates of teptin biosynthesis, i.e. a translational effect. To enable detailed studies of the mechanisms regulating the post-transcriptional regulation of leptin, we will turn to a rat adipocyte model. Specific Aim 1 will assess the effects of insulin on leptin biosynthesis, turnover and secretion using pulse-chase analysis. The role of the long 3'-UTR of leptin mRNA in the insulin regulation of translation will be determined using transient transfections of reporter constructs. Glucocorticoids also increase leptin synthesis over the short term and its effects are additive with insulin; thus, we will investigate the mechanisms involved. Specific Aim 2 will address the hypothesis that short-term decrease in leptin with fasting are mediated by the ability of the catecholamines to decrease leptin release without affecting leptin synthesis. Experiments will also address the hypothesis that the balance between insulin and adrenergic agonists regulate leptin synthesis, turnover, and secretion via opposing effects of cyclic AMP. Specific Aim 3 will use pulse-chase studies to investigate the mechanisms underlying the increased basal and blunted nutritional responses of leptin in spontaneously obese rats. Focused studies of human adipocytes will follow-up on findings in the rat model. This work may lead to new insights into the nutritional regulation of leptin.