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Circulating plasma insulin and glucose levels are thought to be major regulators of leptin secretion. There is evidence from in vitro and animal experiments that glucose metabolism rather than insulin alone is a main determinant of leptin expression. Here, we tested the hypothesis that in humans also leptin secretion is primarily regulated by glucose uptake and only secondarily by plasma insulin and glucose. In 30 lean and healthy men we induced 4 experimental conditions by using the blood glucose clamp technique. A total of 60 hypoglycemic and euglycemic clamps, lasting 6 h each, were performed. During these clamps insulin was infused at either high (15.0 mU/min x kg) or low (1.5 mU/min x kg) rates, resulting in low-insulin-hypo, high-insulin-hypo, low-insulin-eu, and high-insulin-eu conditions. Serum leptin increased from 0-360 min by 20.5 +/- 4.1% in the low-insulin-hypo, 33.6 +/- 7.6% in the high-insulin-hypo, 39.6 +/- 6.0% in the low-insulin-eu, and 60.4 +/- 7.6% in the high-insulin-eu condition. Multiple regression analysis revealed a significant effect of circulating insulin (low vs. high insulin; P = 0.001) and blood glucose (hypoglycemia vs. euglycemia; P = 0.001) on the rise of serum leptin. However, when the total amount of dextrose infused during the clamp (grams of dextrose per kg BW) was included into the regression model, this variable was significantly related to the changes in serum leptin (P = 0.001), whereas circulating insulin and glucose had no additional effect. These findings in humans support previous in vitro data that leptin secretion is mainly related to glucose metabolism.