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. 2011 Jan;60(1):127-37.
doi: 10.2337/db09-1806. Epub 2010 Oct 7.

Direct recruitment of insulin receptor and ERK signaling cascade to insulin-inducible gene loci

Joel D Nelson  1 , Renée C LeBoeuf, Karol Bomsztyk
Affiliations

Direct recruitment of insulin receptor and ERK signaling cascade to insulin-inducible gene loci

Joel D Nelson et al. Diabetes. 2011 Jan.

Abstract

Objective: Insulin receptor (IR) translocates to the nucleus, but its recruitment to gene loci has not been demonstrated. Here, we tested the hypothesis that IR and its downstream mitogenic transducers are corecruited to two prototypic insulin-inducible genes: early growth response 1 (egr-1), involved in mitogenic response, and glucokinase (Gck), encoding a key metabolic enzyme.

Research design and methods: We used RNA and chromatin from insulin-treated rat hepatic tumor cell line expressing human insulin receptor (HTC-IR) and livers from lean and insulin-resistant ob/ob glucose-fed mice in quantitative RT-PCR and chromatin immunoprecipitation studies to determine gene expression levels and associated recruitment of RNA polymerase II (Pol II), insulin receptor, and cognate signaling proteins to gene loci, respectively.

Results: Insulin-induced egr-1 mRNA in HTC-IR cells was associated with corecruitment of IR signaling cascade (IR, SOS, Grb2, B-Raf, MEK, and ERK) to this gene. Recruitment profiles of phosphorylated IR, B-Raf, MEK, and Erk along egr-1 transcribed region were similar to those of elongating Pol II. Glucose-feeding increased Gck mRNA expression in livers of lean but not ob/ob mice. In lean mice, there was glucose feeding-induced recruitment of IR and its transducers to Gck gene synchronized with elongating Pol II. In sharp contrast, in glucose-fed ob/ob mice, the Gck recruitment patterns of active MEK/Erk, IR, and Pol II were asynchronous.

Conclusions: IR and its signal transducers recruited to genes coupled to elongating Pol II may play a role in maintaining productive mRNA synthesis of target genes. These studies suggest a possibility that impaired Pol II processivity along genes bearing aberrant levels of IR/signal transducers is a previously unrecognized facet of insulin resistance.

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Figures

FIG. 1.
FIG. 1.
Insulin-induced early growth response 1, egr-1, expression, and Pol II recruitment in HTC-IR cells overexpressing IR. A: Serum-deprived hepatocyte HTC-IR cells were treated with insulin (10−8 M) for 5, 10, 20, 30, and 60 min or left untreated. Total cellular RNA from these cells was used in RT reactions with random hexamers. cDNAs were used in real-time PCR with primers to the last exon of egr-1 or the first exon of gapdh. Data represent the mean ± SEM of three independent experiments. B: Schematic of the egr-1 locus. Boxes represent exons (black boxes are translated regions and white boxes are untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by egr-1 primers are represented by black bars. C: Cells were treated as in (A), cross-linked, and then used in ChIP with antibodies to Pol II. ChIPed DNA was used in PCR with primers to −5.3 kb, −252 bp, +232 bp, and +3.1 kb with respect to the TSS of egr-1. Data represent the mean ± SEM for three independent experiments. (A high-quality color representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Insulin receptor signaling complex at the egr-1 locus in HTC-IR cells. A: Schematic of one arm of the IR signaling pathway including some components of the IR signaling complex (highlighted) and MAPK pathway components. B: Schematic of the egr-1 locus. C: Cells were treated as in Fig. 1A, cross-linked, and used in ChIP with antibodies to the β subunit of IR (IR-β), IR-β phosphorylated at Y1146 (pIR), Grb2, and SOS. ChIPed DNA was used in PCR as in Fig. 1C. Data represent mean ± SEM for three independent experiments.
FIG. 3.
FIG. 3.
MAPK Erk pathway at the egr-1 locus in HTC-IR cells. A: Schematic of one arm of the IR signaling pathway with MAPK pathway highlighted. B: Cells were treated as in Fig. 1A, cross-linked, and used in ChIP with antibodies to Raf-B, Raf-B phosphorylated at T598 and S601 (pRaf-B), MEK1/2, MEK1/2 phosphorylated at S217/S221 (pMEK), ERK 1, and ERK1/2 phosphorylated at T202/Y204 (pERK). ChIPed DNA was used in PCR as in Fig. 1C. Data represent mean ± SEM for three independent experiments.
FIG. 4.
FIG. 4.
Feeding induces an increase in glucose, insulin, Gck expression, and synchronized Pol II and IR pathway recruitment in mice. A: Schematic of the Gck locus. B: Blood and serum from glucose-fed, fasted mice were used to determine glucose and insulin concentrations, respectively (top and middle). Total RNA from livers of glucose-fed, fasted mice was used in RT reactions with random hexamers. cDNA was used in PCR with primers to glucokinase (Gck). Data represent mean ± SEM of five mice for each time point. C: Chromatin from livers of glucose-fed, fasted mice was used in ChIP assays with antibodies to the indicated proteins. ChIPed DNA was used in PCR with primers to the upstream (first TSS) and downstream (second TSS) transcription start sites and the extreme 3′ end of Gck. Data represent mean fraction of input ± SEM of five mice for each time point. (A high-quality color representation of this figure is available in the online issue.)
FIG. 5.
FIG. 5.
Fasted obese mice have higher glucose, insulin, Gck expression, and Pol II and IR pathway recruitment than lean mice. A: Blood and serum from fasted lean and ob/ob mice were used to determine glucose and insulin concentrations, respectively (top and middle). Total RNA from livers of fasted lean and ob/ob mice was used in RT reactions with random hexamers. cDNA was used in PCR with primers to Gck and ribosomal protein L32 gene as a control for total RNA. B: Chromatin from the livers of fasted lean and ob/ob mice was used in ChIP assays with antibodies to the indicated proteins and histone H3 covalent modification. ChIPed DNA was used in PCR with primers to the indicated regions. C: Comparison of RT-PCR and ChIP results for Gck of fasted and 60-min glucose-fed lean and obese mice. Data (A–C) represent mean ± SEM of four mice for each time point.
FIG. 6.
FIG. 6.
Glucose feeding fails to induce Gck expression and synchronized Pol II and IR pathway recruitment in obese mice. A: Blood glucose, serum insulin, and liver Gck expression were measured in fasted, glucose-fed lean and obese mice as in Fig. 5A. B: Chromatin from the livers of fasted, glucose-fed lean and ob/ob mice was used in ChIP assays as in Fig. 5B.
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