Project: Research project

Project Details


The overall goal of this research proposal is to examine the insulin and
glucose-dependent regulation of the facilitative glucose transport
systems found in rat epididymal adipose tissue and primary isolated
adipocytes. We have recently observed that streptozotocin (STZ)-induced
diabetes results in a specific decrease in the expression of the insulin-
responsive (GLUT4) glucose transporter mRNA in rat adipose tissue without
significant effect on GLUT1 expression. In these studies, we will
ultimately address the molecular mechanisms responsible for the
regulation of adipose glucose transporter gene expression. This will be
accomplished by detailed analysis of GLUT4 promoter function and
identification of cis-regulatory elements. In addition, we propose to
characterize adipocyte-specific DNA binding factors responsible for the
hormonal/metabolic and tissue-specific regulation of this gene. To
address these issues, the following specific aims will be addressed.

1) In comparison to in vivo regulation of adipose tissue GLUT4, we will
examine the effect of in vitro insulin and glucose treatments in isolated
primary adipocytes from control, STZ-diabetic and insulin-treated STZ-
diabetic rats in terms of altered basal and insulin-stimulated glucose
transport activity and relative expression of glucose transporter protein
and mRNA levels.

2) We will determine whether the hormonal/metabolic regulation of GLUT4
mRNA levels in adipose tissue and isolated adipocytes results from
transcriptional events by nuclear run-on analysis.

3) To identify sequences important for the adipocyte-specific and
hormonal/metabolic-dependent transcriptional regulation of this gene,
fusions between the GLUT4 promoter region and a CAT reporter gene will
be used in transient transfection assays.

4) Based upon the information obtained in specific aims 2 and 3, several
promoter constructs will be tested for their ability to produce
appropriate tissue-specific and hormonal/metabolic-dependent regulation
in transgenic mice.

5) The specific binding of known transcription factors, which have
consensus DNA sequences in the GLUT4 control region, will be assessed
along with their effect on GLUT4 gene expression. In future studies, the
functional DNA sequences determined in specific aims 3 and 4 will be used
in mobility shift and DNase 1 footprint assays to identify trans-factors
in nuclear extracts which affect adipocyte-specific GLUT4 expression.
Effective start/end date9/30/918/31/95


  • Genetics
  • Molecular Biology


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