Regulation of glucose responsive protein (GRP) gene expression by insulin

Abstract

While screening for insulin-induced genes, we identified two members of a family of stress-induced genes referred to as glucose-regulated proteins (GRPs). GRPs are members of the stress-responsive superfamily of genes which also includes heat shock proteins (HSPs). The GRP proteins are not normally heat-inducible, but are overproduced when cells are starved of glucose. The two major GRP proteins, GRP78 and GRP94, are highly conserved among vertebrates. We have found that physiological concentrations of insulin stimulate the transcription of GRP78 and GRP94 in rat H4IIE hepatoma cells. The regulation of GRP78 transcription was rapid, with the first induction within minutes, and a further induction after several hours, and both occurred in the presence of glucose. GRP78 transcription was more greatly induced by insulin in the presence of SB202190, a specific p38-MAPK inhibitor. Transcription of GRP94 was also induced, but only after several hours. Calcimycin (A23187) and anisomycin were used to induce endoplasmic reticulum (ER)/cellular stress, and both induced GRP78 and GRP94 transcription.

Keywords: Anisomycin; Calcimycin; Chaperones; ER stress; Insulin.

Fig. 1

Time course of insulin stimulation of GRP78 and GRP94 transcription rates and the role of the p38 pathway. Serum-deprived rat H4 hepatoma cells were treated with insulin (10 nM) alone for the indicated times (A–C). Additional plates of cells were pretreated with SB202190 (10 μM) alone for 60 min, or insulin was added for the last 30 min of the 60 min (C). Transcription rates were then measured by nuclear run-on assays as described in the methods. The symbols or bars indicate mean ± SEM for at least 4 experiments at each time point of the GRP94 curve and at least 10 separate experiments for the GRP78 curve, and a minimum of 3 separate experiments with SB202190. The vehicle treated level was set to unity. Statistical significance: #P < 0.05 and $P < 0.01 vs. control (basal) levels, *P < 0.01 vs. insulin alone, and &P < 0.01 versus SB202190 alone, respectively

Fig. 2

Effects of calcimycin (A23187) on GRP78 and GRP94 transcription rates. Serum-deprived rat H4 hepatoma cells were treated with calcimycin (1 μM) alone for the indicated times (A–C) or calcimycin in the presence of insulin (10 nM) for 30 or 90 min (B, C). Transcription rates were then measured by nuclear run-on assays. The symbols or bars indicate mean ± SEM for at least 4 separate experiments for the curves and bar graph at each time point. The vehicle treated level was set to unity. Statistical significance: # and $, designate P < 0.05 or P < 0.01 vs. control (basal) levels, respectively

Fig. 3

Effects of anisomycin on GRP78 transcription rates. Serum-deprived rat H4 hepatoma cells were treated with anisomycin (30 μM) alone for the indicated times (A) or (B) were pretreated with anisomycin (30 μM) or insulin (10 nM) either for 30 min or anisomycin for 60 min and insulin for the last 30 min. GRP78 transcription rates were then measured by nuclear run-on assays. The symbols or bars indicate mean ± SEM for 3–9 separate experiments. The vehicle treated level was set to unity. Statistical significance: #P < 0.05 and $P < 0.01 vs. control (basal) levels, respectively