Hexosamines regulate leptin production in 3T3-L1 adipocytes through transcriptional mechanisms.
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 microM) 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.
Zhang
Peili
P
Division of Endocrinology and Metabolism, Indiana University School of Medicine, 541 North Clinical Drive, Indianapolis, IN 46202, USA.
Klenk
Ellen S
ES
Lazzaro
Marc A
MA
Williams
Lloyd B
LB
Considine
Robert V
RV
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D003980:Diazooxonorleucine / Q000494:pharmacology; D005944:Glucosamine / Q000494:pharmacology; D006031:Glycosylation; D006595:Hexosamines / Q000502:physiology*; D006801:Humans; D020738:Leptin / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
D006595:Hexosamines; D020738:Leptin; D016329:Sp1 Transcription Factor; D003980:Diazooxonorleucine; D005944:Glucosamine