Abstract
Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). To determine whether the hexosamine biosynthesis pathway (HBP) mediates glucose regulation of mRNA expression, we treated primary cultured adipocytes for 18 h with insulin (25 ng/ml) and either glucose (20 mm) or glucosamine (2 mm). A ribonuclease protection assay was used to quantitate mRNA levels for FAS, ACC, and glycerol-3-P dehydrogenase (GPDH). Treatment with insulin and various concentrations of d-glucose increased mRNA levels for FAS (280%), ACC (93%), and GPDH (633%) in a dose-dependent manner (ED50 8-16 mm). Mannose similarly elevated mRNA levels, but galactose and fructose were only partially effective. l-glucose had no effect. Omission of glutamine from the culture medium markedly diminished the stimulatory effect of glucose on mRNA expression. Since glutamine is a crucial amide donor in hexosamine biosynthesis, we interpret these data to mean that glucose flux through the HBP is linked to regulation of lipogenesis through control of gene expression. Further evidence for hexosamine regulation was obtained using glucosamine, which is readily transported into adipocytes where it directly enters the HBP. Glucosamine was 15-30 times more potent than glucose in elevating FAS, ACC, and GPDH mRNA levels (ED50 approximately 0.5 mm). In summary: 1) GPDH, FAS, and ACC mRNA levels are upregulated by glucose; 2) glucose-induced up-regulation requires glutamine; and 3) mRNA levels for lipogenic enzymes are up-regulated by glucosamine. Hyperglycemia is the hallmark of diabetes mellitus and leads to insulin resistance, impaired glucose metabolism, and dyslipidemia. We postulate that disease pathophysiology may have a common underlying factor, excessive glucose flux through the HBP.
Highlights
Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC)
The current studies were initiated to examine the hypothesis that the glucose-mediated up-regulation of lipogenic enzymes in isolated adipocytes is mediated by glucose flux through the hexosamine biosynthesis pathway (HBP) and the subsequent regulation of lipogenic enzyme mRNA levels
Regulation of Acetyl-CoA Carboxylase mRNA Levels by Glucose and Glucosamine—To explore whether the HBP may control other mRNAs involved in lipid metabolism, we evaluated the regulation of mRNA for the lipogenic enzyme ACC and the lipolytic enzyme hormone-sensitive lipase (HSL)
Summary
Materials—Sources of materials were as follows: Porcine insulin, Sigma; collagenase, Worthington Biochemicals, Freehold, NJ; bovine serum albumin (CRG-7), Armour Company, Kankakee, IL.; penicillinstreptomycin and Dulbecco’s modified Eagle’s medium (DMEM), Invitrogen; RNAzol B, Cinna/Biotecx, Friendswood, TX. Preparation of Sterile Isolated Adipocytes—Isolated adipocytes were obtained from the epididymal fat pads of male Sprague-Dawley rats (180 –225 g) by collagenase digestion (15) as described previously (16). At the end of the digestion period, cells were filtered through nylon mesh (1000 m) and washed three times in Hepes-buffered balanced saline solution (HBSS). HBSS contains 25 mM Hepes, 120 mM NaCl, 0.8 mM MgSO4, 2 mM CaCl2, 5.4 hexosamine biosynthesis pathway; TGF, transforming growth factor; DMEM, Dulbecco’s modified Eagle’s medium; HBSS, Hepes-buffered balanced saline solution; Glc-6-P, glucose-6-phosphate; Fru-6-P, fructose-6-phosphate
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