Date of Award

4-2005

Degree Name

Doctor of Philosophy

Department

Biological Sciences

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), an important enzyme involved in glucose homeostasis, 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 showed that G6PDH is regulated by glucose in primary 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, Sterol Regulatory Element Binding Protein or SP1. These factors may induce transcription through an E-box consisting of the sequence 5 ' -CACGTG-3' , of which thefirst 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 its glucose-regulated expression. Using primary hepatocytes transfected with G6PDH promoter constructs, we clearly established not only theregulation of G6PDH expression by insulin, but also by glucose. The two 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. Insertion ofE-box sequences into 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 showed a significant increase in specific hepatic nuclear protein binding to an E-box oligonucleotide in response to ratsfed 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 hypothesis that glucose, or a metabolite of glucose, such as glucose-6-phosphate may actually be thecarbohydrate 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.

Access Setting

Dissertation-Open Access

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