Insulin regulates cellular proteins important for normal metabolism and growth. This regulation can occur by modifying enzymatic activity, changing the synthesis or stability of specific proteins, or altering the synthesis or stability of their specific mRNAs. Our goal is to understand insulin's control of gene expression. The objectives of this application are to study in detail the transcriptional regulation by insulin of 2 specific genes in rat H4 hepatoma cells, the tyrosine aminotransferase (TAT) gene, which is rapidly inhibited by insulin, and the beta-actin gene, which is induced. The effects of insulin on the activity and synthesis of the hepatic enzyme TAT are controversial. In rat H4 hepatoma cells, we found that insulin reduces TAT transcription within minutes. This effect of insulin was biphasic, with an induction of transcription by 10-12 hours. Preliminary evidence is presented which indicates that insulin also rapidly decreased the expression of a chimeric gene constructs containing an upstream, non-transcribed fragment of the TAT gene placed 5' to a reporter gene (chloramphenicol acetyl transferase; CAT). Thus, we have isolated a 0.3 kb region of the TAT gene that imparts the rapid inhibition of the TAT gene by insulin (e.g., contains a putative insulin responsive element; IRE) demonstrated in the transcription studies. This element will be identified and the role of this and other elements in the biphasic action of insulin will be investigated. We have also found that insulin rapidly induces transcription of the beta-actin gene. In other recent experiments insulin stimulated expression of several chimeric genes comprised of 3, and possibly more, distinct 5' untranscribed regions of the beta-actin gene placed in a CAT expression vector. Several of the beta-actin gene regions contain a putative serum-responsive elements (SREs). However, other regions of the beta-actin gene are insulin responsive and do not contain SREs or other known regulatory elements. One of these regions is within intron 1 and another is in the 3' untranslated region (3'UTR) of the beta-actin gene.
The aims of this work are to isolate and identify these IREs and to determine the exact base sequences that are necessary for insulin's actions. We also wish to study the interaction of insulin with other regulators of these genes, such as phorbol esters, and determine whether they exert their effects through common regulatory elements. Using a number of techniques we plan to study DNA binding proteins, the transcriptional regulatory factors, that associate with the IREs of these genes. Objectives include examining how insulin regulates the amount, binding and/or activity of these proteins. These studies are necessary to understand possible interactions between multiple DNA elements and multiple transcriptional regulatory factors in the regulation of expression of the TAT and beta-actin genes. By using this model system we hope to gain insight into the mechanisms by which insulin controls the expression of different genes. An understanding of how insulin acts to regulate gene expression is necessary if we are to understand the short- and long-term effects of insulin on cellular metabolism and growth and the origins of many of the problems that occur in diabetes when insulin secretion or activity is abnormal.
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