The aim of the present study was to determine the respective roles of energy substrates and insulin on leptin secretion from white adipocytes. Cells secreted leptin in the absence of glucose or other substrates, and addition of glucose (5 mM) increased this secretion. Insulin doubled leptin secretion in the presence of glucose (5 mM), but not in its absence. High concentrations of glucose (up to 25 mM) did not significantly enhance leptin secretion over that elicited by 5 mM glucose. Similar results were obtained when glucose was replaced by pyruvate or fructose (both 5 mM). l-Glycine or l-alanine mimicked the effect of glucose on basal leptin secretion but completely prevented stimulation by insulin. On the other hand, insulin stimulated leptin secretion when glucose was replaced by l-aspartate, l-valine, l-methionine, or l-phenylalanine, but not by l-leucine (all 5 mM). Interestingly, these five amino acids potently increased basal and insulin-stimulated leptin secretion in the presence of glucose. Unexpectedly, l-glutamate acutely stimulated leptin secretion in the absence of glucose or insulin. Finally, nonmetabolizable analogs of glucose or amino acids were without effects on leptin secretion. These results suggest that 1) energy substrates are necessary to maintain basal leptin secretion constant, 2) high availability of glycolysis substrates is not sufficient to enhance leptin secretion but is necessary for its stimulation by insulin, 3) amino acid precursors of tricarboxylic acid cycle intermediates potently stimulate basal leptin secretion per se, with insulin having an additive effect, and 4) substrates need to be metabolized to increase leptin secretion.