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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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This study was undertaken to investigate the changes in obesity (OB) gene expression and production of leptin in response to insulin in vitro and in vivo under euglycemic and hyperglycemic conditions in humans. Three protocols were used: 1) euglycemic clamp with insulin infusion rates at 40, 120, 300, and 1,200 mU / m / min carried out for up to 5 h performed in 16 normal lean individuals, 30 obese individuals, and 31 patients with NIDDM; 2) 64-to 72-h hyperglycemic (glucose 12.6 mmol/l) clamp performed on 5 lean individuals; 3) long-term (96-h) primary culture of isolated abdominal adipocytes in the presence and absence of 100 nmol/l insulin. Short-term hyperinsulinemia in the range of 80 to > 10,000 microU/ml had no effect on circulating levels of leptin. During the prolonged hyperglycemic clamp, a rise in leptin was observed during the last 24 h of the study (P < 0.001). In the presence of insulin in vitro, OB gene expression increased at 72 h (P < 0.01), followed by an increase in leptin released to the medium (P < 0.001). In summary, insulin does not stimulate leptin production acutely; however, a long-term effect of insulin on leptin production could be demonstrated both in vivo and in vitro. These data suggest that insulin regulates OB gene expression and leptin production indirectly, probably through its trophic effect on adipocytes.

As one of the postulated roles of the ob gene product, leptin, is regulation of energy balance and preservation of normal body composition, we investigated the effect of acute and chronic calorie excess (weight gain) on serum leptin in humans. Two protocols were employed: 1) acute (12-h) massive (120 Cal/kg) voluntary overfeeding of eight healthy individuals; and 2) chronic overfeeding to attain 10% weight gain, with its subsequent maintenance for additional 2 weeks, involving six normal males. In the acute experiments (protocol 1), circulating leptin rose by 40% over baseline (P < 0.01) during the final hours of overfeeding; this increase persisted until the next morning. At the point of achievement and the 2-week maintenance of 10% weight gain (protocol 2), a more than 3-fold rise in the basal leptin concentration was observed (P < 0.01). A direct linear relationship was found between the magnitude of the leptin response to weight gain and the percent gain of body fat (r = 0.88; P < 0.01). In summary, 1) in contrast to normal food intake (8), short term massive overfeeding is associated with a moderate elevation of circulating leptin levels that persists until next feeding cycle is initiated; and 2) a 10% weight gain causes different changes in the body composition, and the resulting rise in circulating leptin parallels the increase in the percentage of body fat. In conclusion, these studies document acute elevation of leptin in response to positive energy balance and suggest that developing resistance to leptin is associated with bigger fat deposition during weight gain in humans.

We have studied the effect of prolonged hyperinsulinemia and hyperglycemia on serum leptin levels in young nonobese males during 72-h euglycemic-hyperinsulinemic and hyperglycemic ( ‫ف‬ 8.5 and 12.6 mM) clamps. Hyperinsulinemia increased serum leptin concentrations (by RIA) dosedependently. An increase in serum insulin concentration of Ͼ 200 pM for Ͼ 24 h was needed to significantly increase serum leptin. An increase of ‫ف‬ 800 pM increased serum leptin by ‫ف‬ 70% over 72 h. Changes in plasma glucose concentrations (from ‫ف‬ 5.0 to ‫ف‬ 12.6 mM) or changes in plasma FFA concentrations (from Ͻ 100 to Ͼ 1,000 M) had no effect on serum leptin. Serum leptin concentrations changed with circadian rhythmicity. The cycle length was ‫ف‬ 24 h, and the cycle amplitude (peak to trough) was ‫ف‬ 50%. The circadian leptin cycles and the circadian cycles of total body insulin sensitivity (i.e., GIR, the glucose infusion rates needed to maintain euglycemia during hyperinsulinemic clamping) changed in a mirror image fashion. Moreover, GIR decreased between Days 2 and 3 (from 11.4 Ϯ 0.2 to 9.8 Ϯ 0.2 mg/kg min, P Ͻ 0.05) when mean 24-h leptin levels reached a peak. In summary, we found ( a ) that 72 h of hyperinsulinemia increased serum leptin levels dose-dependently; ( b ) that hyperglycemia or high plasma FFA levels did not affect leptin release; ( c ) that leptin was released with circadian rhythmicity, and ( d ) that 24-h leptin cycles correlated inversely with 24-h cycles of insulin sensitivity. We speculate that the close positive correlation between body fat and leptin is mediated, at least in part, by insulin. ( J. Clin. Invest. 1997. 100:1107-1113.) Key words: hyperglycemia • free fatty acids • circadian leptin release • insulin resistance