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Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125 Ileptin-serum complex demonstrated 125 I-leptin eluting in significant proportion associated with macromolecules. The 125 I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED 50 : 0.73 Ϯ 0.09 nM, mean Ϯ SEM, n ϭ 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80-or 100-kD proteins could be the soluble leptin receptor as ‫ف‬ 10% of the bound 125 I-leptin was immunoprecipitable with leptin receptor antibodies.Significantly higher ( P Ͻ 0.001) proportions of total leptin circulate in the bound form in lean (46.5 Ϯ 6.6%) compared with obese (21.4 Ϯ 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean ( P Ͻ 0.0005) and three obese ( P Ͻ 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 Ϯ 1.9 vs. 1.3 Ϯ 0.4 ng/ml) compared with those in obese subjects (28.3 Ϯ 9.8 vs. 14.7 Ϯ 5.3). No significant ( P Ͼ 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.

We investigated the response of leptin to short-term fasting and refeeding in humans. A mild decline in subcutaneous adipocyte ob gene mRNA and a marked fall in serum leptin were observed after 36 and 60 h of fasting. The dynamics of the leptin decline and rise were further substantiated in a 6-day study consisting of a 36-h baseline period, followed by 36-h fast, and a subsequent refeeding with normal diet. Leptin began a steady decline from the baseline values after 12 h of fasting, reaching a nadir at 36 h. The subsequent restoration of normal food intake was associated with a prompt leptin rise and a return to baseline values 24 h later. When responses of leptin to fasting and refeeding were compared with that of glucose, insulin, fatty acids, and ketones, a reverse relationship between leptin and beta-OH-butyrate was found. Consequently, we tested whether the reciprocal responses represented a causal relationship between leptin and beta-OH-butyrate. Small amounts of infused glucose equal to the estimated contribution of gluconeogenesis, which was sufficient to prevent rise in ketogenesis, also prevented a fall in leptin. The infusion of beta-OH-butyrate to produce hyperketonemia of the same magnitude as after a 36-h fast had no effect on leptin. The study indicates that one of the adaptive physiological responses to fasting is a fall in serum leptin. Although the mediator that brings about this effect remains unknown, it appears to be neither insulin nor ketones.

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