Transcriptional Regulation of Transforming Growth Factor ß1 by Glucose: Investigation into the Role of the Hexosamine Biosynthesis Pathway.

BACKGROUND: The hexosamine biosynthesis pathway (HBP) is hypothesized to mediate many of the adverse effects of hyperglycemia. We have shown previously that increased flux through this pathway leads to induction of the growth factor transforming growth factor-α (TGF-α) and to insulin resistance in cultured cells and transgenic mice. TGF-ß is regulated by glucose and is involved in the development of diabetic nephropathy. We therefore hypothesized that the HBP was involved in the regulation of TGF-ß by glucose in rat vascular and kidney cells. METHODS: A plasmid containing the promoter region of TGF-ß1 cloned upstream of the firefly luciferase gene was electroporated into rat aortic smooth muscle, mesangial, and proximal tubule cells. Luciferase activity was measured in cellular extracts from cells cultured in varying concentrations of glucose and glucosamine. RESULTS: Glucose treatment of all cultured cells led to a time- and dose-dependent stimulation in TGF-ß1 transcriptional activity, with high (20 mM) glucose causing a 1.4- to 2.0-fold increase. Glucose stimulation did not occur until after 12 hours and disappeared after 72 hours of treatment. Glucosamine was more potent than glucose, with 3 mM stimulating up to a 4-fold increase in TGFß1-transcriptional activity. The stimulatory effect of glucosamine was also dose-dependent but was slower to develop and longer lasting than that of glucose. CONCLUSIONS: The metabolism of glucose through the HBP mediates extracellular matrix production, possibly via the stimulation of TGF-ß in kidney cells. Hexosamine metabolism therefore, may play a role in the development of diabetic nephropathy.

c-Myc is required for the glucose-mediated induction of metabolic enzyme genes.

Glucose exerts powerful effects on hepatocyte gene transcription by mechanisms that are incompletely understood. c-Myc regulates hepatic glucose metabolism by increasing glycolytic enzyme gene transcription while concomitantly decreasing gluconeogenic and ketogenic enzyme gene expression. However, the molecular mechanisms by which c-Myc exerts these effects is not known. In this study, the glucose-mediated induction of L-type pyruvate kinase and glucose-6-phosphatase mRNA levels was diminished by maneuvers involving recombinant adenoviral vectors that interfere with (i) c-Myc protein levels by antisense expression or (ii) c-Myc function through a dominant-negative Max protein. These results were obtained using both HL1C rat hepatoma cells and primary rat hepatocytes. Furthermore, a decrease in c-Myc abundance reduced glucose production in HL1C cells, presumably by decreasing glucose-6-phosphatase activity. The repression of hormone-activated phosphoenolpyruvate carboxykinase gene transcription by glucose was not affected by a reduction in c-Myc levels. The basal mRNA levels for L-pyruvate kinase and glucose-6-phosphatase were not altered to any significant degree by adenoviral treatment. Furthermore, adenoviral overexpression of the c-Myc protein induced glucose-6-phosphatase mRNA in the absence of glucose stimulation. We conclude that multiple mechanisms exist to communicate the glucose-derived signal and that c-Myc has a key role in the hepatic glucose signaling pathway.

Elements of the glucocorticoid and retinoic acid response units are involved in cAMP-mediated expression of the PEPCK gene.

Although many genes are regulated by the concerted action of several hormones, hormonal signaling to gene promoters has generally been studied one hormone at a time. The phosphoenolpyruvate carboxykinase (PEPCK) gene is a case in point. Transcription of this gene is induced by glucagon (acting by the second messenger, cAMP), glucocorticoids, and retinoic acid, and it is dominantly repressed by insulin. These hormonal responses require the presence of different hormone response units (HRUs), which consist of constellations of DNA elements and associated transcription factors. These include the glucocorticoid response unit (GRU), cAMP response unit (CRU), retinoic acid response unit (RARU), and the insulin response unit. HRUs are known to have functional overlap. In particular, the cAMP response element of the CRU is also a component of the GRU. The purpose of this study was to determine whether known GRU or RARU elements or transcription factors function as components of the CRU. We show here that the glucocorticoid accessory factor binding site 1 and glucocorticoid accessory factor binding site 3 elements, which are components of both the GRU and RARU, are an important part of the CRU. Furthermore, we find that the transcription factor, chicken ovalbumin upstream promoter-transcription factor, and two coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, participate in both the cAMP and glucocorticoid responses. This provides a further illustration of how the PEPCK gene promoter integrates different hormone responses through overlapping HRUs that utilize some of the same transcription factors and coactivators.