The long term objectives of our program are to determine the physiologic, biochemical, and molecular mechanisms responsible for the nutritional and hormonal regulation of hepatic gene expression. The studies in this application will delineate the mechanisms that regulate the transcription and processing of a hepatic mRNA, mRNA-S14. We have taken advantage of the rapid response of this mRNA to dietary carbohydrate to show that the transcriptional response reflects the intracellular metabolism of sugars and not a response to insulin. Therefore, this MRNA is an excellent model to study the effects of nutritional signals on the regulation of gene expression. Further, the induction by feeding a sucrose containing diet or the administration of thyroid hormone is similar to that of the hepatic lipogenic enzymes. Therefore, this mRNA is also an excellent model to study the response of the lipogenic enzyme mRNAs. The dietary response is due both to enhanced transcription of the gene and increased efficiency of processing of pre-mRNA to mature mRNA. We can reproduce the transcriptional regulation of mRNA-S14 in primary hepatocyte cultures by altering the concentration of glucose presented to the hepatocyte. To define the mechanisms responsible for the dietary regulation of this hepatic gene, we propose to introduce a series of deletion mutations in the 5'flanking DNA of the S14 gene. The mutant constructs will be transfected into primary hepatocyte cultures and the reporter luciferase activity measured as an indicator of S14 gene transcription. These studies will define the specific DNA sequences responsible for the regulation of gene transcription by dietary carbohydrate. To explore the mechanism of the synergistic interaction between thyroid hormone and dietary carbohydrates we will create constructs containing a thyroid response element and a carbohydrate response element of the S14 gene and test the ability of these constructs to show a synergistic response to thyroid hormone and carbohydrates. These studies will define the minimum DNA sequences that lead to interaction between thyroid hormone and nutritional signals. We will also define new hepatic transcription factors that are responsible for the carbohydrate regulation of mRNA-S14. The time course and magnitude of response of these proteins to dietary and hormonal stimuli will be measured. Lastly, we will use in vitro splicing reactions to demonstrate that dietary carbohydrate enhances the efficiency of pre-mRNA-S14 processing. The studies outlined in this proposal should further our understanding of the molecular and physiologic basis of dietary regulation of hepatic gene expression. Since altered glucose metabolism leads to changes in gene expression in many tissues, these studies have major implications for the pathophysiologic mechanisms that lead to complications associated with diabetes mellitus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK032885-10
Application #
2138905
Study Section
Nutrition Study Section (NTN)
Project Start
1984-04-01
Project End
1997-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
10
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Ota, Yasuhiro; Mariash, Cary N (2003) Paradoxical triiodothyronine suppression of S14 transcription in permanent hepatic cell lines. Thyroid 13:437-45
Zhu, Q; Mariash, A; Margosian, M R et al. (2001) Spot 14 gene deletion increases hepatic de novo lipogenesis. Endocrinology 142:4363-70
Ercan-Fang, S; Schwartz, H L; Mariash, C N et al. (2000) Quantitative assessment of pituitary resistance to thyroid hormone from plots of the logarithm of thyrotropin versus serum free thyroxine index. J Clin Endocrinol Metab 85:2299-303
Liu, B; Li, W; Mariash, C N (1999) Two different gene elements are required for glucose regulation of S14 transcription. Mol Cell Endocrinol 148:11-9
Kirschner, L S; Mariash, C N (1999) Adipose S14 mRNA is abnormally regulated in obese subjects. Thyroid 9:143-8
Ota, Y; Mariash, A; Wagner, J L et al. (1997) Cloning, expression and regulation of the human S14 gene. Mol Cell Endocrinol 126:75-81
Harmon, J S; Mariash, C N (1996) Identification of a carbohydrate response element in rat S14 gene. Mol Cell Endocrinol 123:37-44
Walker, J D; Burmeister, L A; Mariash, A et al. (1996) Insulin increases the processing efficiency of messenger ribonucleic acid-S14 nuclear precursor. Endocrinology 137:2293-9
Sudo, Y; Mariash, C N (1996) Lowering glucose depletes a thapsigargin-sensitive calcium pool and inhibits transcription of the S14 gene. Endocrinology 137:4677-84
Sudo, Y; Mariash, C N (1994) Two glucose-signaling pathways in S14 gene transcription in primary hepatocytes: a common role of protein phosphorylation. Endocrinology 134:2532-40

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