Insulin inhibits transcription of the human gene for insulin-like growth factor-binding protein-1.

D.R Powell,A Suwanichkul,M.L Cubbage,L.A Depaolis,M.B Snuggs,P.D Lee

doi:10.1016/s0021-9258(18)55144-9

Abstract

Insulin rapidly lowers serum insulin-like growth factor-binding protein-1 (IGFBP-1) levels in vivo. In studies reported here, HEP G2 cells were used as a model system to investigate how insulin achieves this effect. When HEP G2 cells were incubated with 100 nM insulin for 6, 14, or 24 h, IGFBP-1 protein levels in conditioned medium fell to approximately 50% of control values. This apparently was due to a fall in the rate of IGFBP-1 protein synthesis, since HEP G2 cells incorporated 46% less [35S]methionine into IGFBP-1 during a 4-h incubation with 100 nM insulin. IGFBP-1 mRNA levels were similarly affected by 100 nM insulin, falling to 45% of control values after 2 h, and to 9% of control values after 4 h of incubation with this hormone. The fall in IGFBP-1 mRNA level is consistent with data from nuclear transcription assays. HEP G2 nuclei isolated from cells that were incubated with 100 nM insulin for 2 h synthesized only approximately 1/3 the number of IGFBP-1 transcripts as did control nuclei. Further evidence that insulin decreases IGFBP-1 gene transcription comes from transient transfections using chimeric IGFBP-1 promoter-chloramphenicol acetyltransferase reporter gene constructs. IGFBP-1 promoter activity fell to approximately 50% of control values when HEP G2 cells transfected with a construct containing the first 1205 base pairs of the IGFBP-1 promoter were incubated with 100 nM insulin for 6, 14, or 24 h. Insulin lowered both IGFBP-1 protein levels and promoter activity in a dose-dependent manner. A half-maximal effect was found at approximately 1 nM insulin and a maximal effect was found at approximately 10 nM insulin in each instance. Transfections with constructs containing smaller IGFBP-1 promoter fragments showed that the region spanning from 103 to 529 base pairs 5' to the IGFBP-1 mRNA cap site was necessary to demonstrate the inhibitory effect of insulin. These studies indicate that insulin lowers IGFBP-1 protein levels, at least in part, by rapidly decreasing the rate of IGFBP-1 gene transcription, and suggest that this insulin-mediated fall in transcription is conferred through a specific region of the IGFBP-1 promoter.

Highlights

When HEP G2 cells were incubated with 100 nM insulin for 6, 14, or 24 h, Insulin-like growth factor-bindingprotein-1 (IGFBP-1) protein levels in conditioned medium fell to -50% of control values
HEP G2 cells transfected with a construct containing and reversible changes in unsaturated serum IGFBP-1 conthe first 1205 base pairs of the IGFBP-1 promoter centrations,insulin may coordinatethe anaboliceffects of were incubated with 100 nM insulin for6, 14, or 24 h. unbound, circulating IGF-I with the nutritional statusof the Insulin lowered both IGFBP-1 protein levelsand pro- individual
Insulin induceda dose-dependent decreasein IGFBP-1 primer complementary to sequence 8-33 bp upstream of the CAT protein levels in medium conditioned by H E P G2 cells

Summary

RESULTS

Measured by CATassay of total cellular protein(fordetails, see "ExperimentalProcedures"). Insulin decreased the activityof p529CAT to 45 f 18% of control values, but increased the activity of pl03CAT to188 f 77% of control values, suggesting that the insulin effect is confered in the region between 103 and 529 bp 5' to the IGFBP-1 mRNA cap site. When HEP G2 cells were transfected with p1205LUC and incubated with 100 nM insulin for tion of 5"IGFBP-1 promoter sequence results in major, po- 14 h, luciferase activity fell to 59 & 12% of control values. Panel R, HEP G2 cells were transfected wit.h either p529CAT or pl03CAT and incubated wiotrhwithout 100 0.2-fold higher than control (84% inhibition), and IGFBP-1 n~ insulin for 14 h. Data for p529CAT of insulin on IGFBP-1 productionamisplified, and the results and pl03CAT represent the mean k S.D. of three and seven experi- are again consistent with an inhibitory effect of insulin on ments, respectively. From HEP G2 cells transfected with pl03CAT and incubated for 24 h without (lane I ) or with (lane2 ) 100 nM insulin, and from HEP G2 cells transfected with p1205CAT and incubated for 24 h with 100nM insulin (lane 3).A ‘”P-labeled, 25-base oligonucleotide

DISCUSSION

Findings

GFBP- 1 mRNA Levels