Candidate: Daryl Arkwright-Keeler

Degree of: Doctor of Philosophy

Department: Biological Sciences

Title: The Glucose and Insulin Responsiveness of the Rat Glucose-6-phosphate Dehydrogenase Promoter

Committee: Dr. Susan Stapleton, Chair
Dr. John Geiser
Dr. William Jackson
Dr. Alfredo Tomasselli

Date: Friday, December 10, 2004 3:00 p.m. – 5:00 p.m.
1710 Wood Hall

Abstract: The regulation of expression of some enzymes involved in glucose homeostasis and fat metabolism requires both insulin and glucose to elicit an effect, while the regulation of others requires only one. Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme involved in glucose homeostasis. It has been shown to be transcriptionally regulated by carbohydrate feeding in rat liver, a diet regime that increases the levels of both glucose and insulin. Our lab had previously shown that insulin induces expression of G6PDH, thus we wanted to determine if glucose also induced G6PDH expression and if so, are the glucose and insulin responses mediated through identical or separate mechanisms. Preliminary studies demonstrated that G6PDH is regulated by glucose in primary rat hepatocyte cultures, however the mechanism responsible for this regulation is not clear.
Several key metabolic enzymes are regulated transcriptionally by glucose and possibly involve the transcription factors Upstream Stimulatory Factor (USF), Sterol Regulatory Element Binding Protein (SREBP) or SP1. These transcription factors may induce transcription through an E-box consisting of the sequence 5'-CACGTG-3', of which the first four bases appear to be the most critical for the glucose response. The G6PDH promoter contains a single E-box, thus we investigated whether this E-box is involved in the regulation of its expression by glucose. Using primary rat hepatocytes transfected with G6PDH promoter constructs, we clearly established not only the regulation of G6PDH expression by insulin, but also by glucose. The glucose and insulin responses are additive, suggesting that they act though separate mechanisms. Mutation of the first four bases of the E-box in the G6PDH promoter resulted in not only significantly reduced glucose but also insulin responses. Interestingly, insertion of E-box sequences into glucose and insulin non-responsive constructs instilled modest, but significant insulin responsiveness, but not glucose responsiveness.
Mutation of a putative SP1 site near the E-box had no effect on the promoter response to insulin or glucose, indicating that this site is likely not involved in these responses.
Electrophorectic mobility shift assays (EMSA) showed a significant increase in specific hepatic nuclear protein binding to an E-box oligonucleotide in response to rats fed a high carbohydrate diet. Supershift studies revealed that USF is involved in this protein binding. We also examined whether the promoter response to carbohydrate was specific for glucose. Hepatocytes were treated with pyruvate, which did not elicit a response. This result is in agreement with the hypoTheses that glucose, or a metabolite of glucose, such as glucose-6-phosphate or xyulose-5-phosphate may actually be the carbohydrate signaling molecule.
In summary, our results suggest that the E-box is necessary, but probably not sufficient for the G6PDH glucose and insulin responses and that USF is involved in the hepatic nuclear protein binding to the E-box in response to a high carbohydrate diet.

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