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Hexosamines Regulate Leptin Production in 3T3-L1 Adipocytes through Transcriptional Mechanisms

Division of Endocrinology and Metabolism, Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Robert V. Considine, Ph.D., Indiana University School of Medicine, 541 North Clinical Drive, Clinical Building 455, Indianapolis, Indiana 46202-5111. E-mail: rconsidi{at}iupui.edu

This study was undertaken to examine the regulation of leptin gene (LEP) transcription and leptin release by hexosamines in 3T3-L1 adipocytes. Glucosamine (1 mM), an intermediate in hexosamine biosynthesis, increased leptin release to 117.0 ± 7.3% (P = 0.0430; n = 9) and 134.6 ± 6.5% of the control value (P = 0.0367; n = 4) by 48 and 96 h, respectively. With 0.01 mM glucosamine, leptin release was increased to 120.0 ± 3.0% of the control value (P = 0.0069; n = 4) by 96 h of treatment. Glucose at 5 and 20 mM stimulated leptin release to 759 ± 227% and 1104 ± 316% of the control value over the 96-h culture period. Inhibition of hexosamine biosynthesis with 6-diazo-5-oxonorleucine (20 µM) reduced glucose-stimulated leptin release 13 ± 2.3% and 29.9 ± 6.6% at 24 and 96 h, respectively (n = 4; P < 0.05). A 24-h incubation in 5 mM glucose significantly increased (163.0 ± 19.3%; n = 7) the activity of a human LEP promoter electroporated into differentiated 3T3-L1 cells. Glucosamine (1 mM; 48 h) also increased LEP promoter activity 170.0 ± 13.0% (n = 5). Mutation of the three Sp1 binding sites in the LEP construct significantly reduced promoter activity. However, glucose (5 mM; 24 h) and glucosamine (1 mM; 48 h) increased the activity of the mutated promoter to 165 ± 40% (n = 8) and 143 ± 13% of the control value (n = 8). Glucosamine significantly increased O-glycosylation of Sp1 by 16.1 ± 4.5% (P = 0.0305; n = 3). These data demonstrate that glucose and hexosamines regulate leptin production through transcriptional mechanisms localized to the proximal portion of the LEP promoter. Hexosamine-mediated regulation of LEP gene expression does not depend on Sp1 binding to traditional sites on the promoter.