The three insulin response sequences in the glucose-6-phosphatase catalytic subunit gene promoter are functionally distinct.
Glucose-6-phosphatase catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. In HepG2 cells, the maximum repression of basal glucose-6-phosphatase catalytic subunit (G6Pase) gene transcription by insulin requires two distinct promoter regions, designated A (located between -231 and -199) and B (located between -198 and -159), that together form an insulin response unit. Region A binds hepatocyte nuclear factor-1, which acts as an accessory factor to enhance the effect of insulin, mediated through region B, on G6Pase gene transcription. We have previously shown that region B binds the transcriptional activator FKHR (FOXO1a) in vitro. Chromatin immunoprecipitation assays demonstrate that FKHR also binds the G6Pase promoter in situ and that insulin inhibits this binding. Region B contains three insulin response sequences (IRSs), designated IRS 1, 2, and 3, that share the core sequence T(G/A)TTTT. However, detailed analyses reveal that these three G6Pase IRSs are functionally distinct. Thus, FKHR binds IRS 1 with high affinity and IRS 2 with low affinity but it does not bind IRS 3. Moreover, in the context of the G6Pase promoter, IRS 1 and 2, but not IRS 3, are required for the insulin response. Surprisingly, IRS 3, as well as IRS 1 and IRS 2, can each confer an inhibitory effect of insulin on the expression of a heterologous fusion gene, indicating that, in this context, a transcription factor other than FKHR, or its orthologs, can also mediate an insulin response through the T(G/A)TTTT motif.
Vander Kooi
Beth T
BT
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
Streeper
Ryan S
RS
Svitek
Christina A
CA
Oeser
James K
JK
Powell
David R
DR
O'Brien
Richard M
RM
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D020134:Catalytic Domain / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D005952:Glucose-6-Phosphatase / Q000235:genetics*; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D007004:Hypoglycemic Agents; D007328:Insulin; D018970:Insulin-Like Growth Factor Binding Protein 1; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C074639:Foxo1 protein, rat; D015500:Chloramphenicol O-Acetyltransferase; D005952:Glucose-6-Phosphatase