The P2 promoter of rat PC gene contains glucose-responsive element(s) (GRE).

<p>A series of 5′-truncated P2-luciferase reporter constructs were transfected into INS-1 832/13 cells. The transfected cells were maintained in RPMI containing normal (5.5 mM) or high (25 mM) glucose for 24 h. The luciferase activity of each construct was normalized to the β-galactosidase activity and expressed as relative luciferase activity. Relative luciferase values obtained from transfected cells maintaining in high glucose medium were presented as fold change relative to those maintaining in normal concentration of glucose, each of which was arbitrarily set as 1. The statistical analysis was conducted using ANOVA test where **P<0.01.</p

Sp1 regulates glucose-induced PC expression through E-box-like element (E2) in P2 promoter of the PC gene.

<p><b>A</b>, The WT-P2(pGL-P2) or its mutation at E2-luciferase reporter constructs (MuE2) with plasmid overexpressing Sp1, Sp3 or empty vector (pcDNA) were transfected into INS-1 832/13 cells before they were maintained in medium containing low (5.5 mM) or high (25 mM) glucose before their luciferase activities were assayed. Relative luciferase activity of cells co-transfected with pGL-P2 (WT) or mutant promoter construct with empty vector (pcDNA), pSp1 or pSp3 maintained at 5.5 mM or 25 mM were normalized with that of cells co-transfected with pGL-P2 and empty vector maintained at 5.5 mM, which was arbitrarily set as 1. The statistical analysis was conducted using ANOVA test. Ψ (P<0.05) and ΨΨ (P<0.01), compared with cells transfected with pGL-P2 and pcDNA maintained at 5.5 mM glucose. # (P<0.05) and ## (P<0.01), compared with cells transfected with MuE2 maintained at 5.5 mM glucose. **P<0.01 (transactivation of WT promoter by Sp1 or Sp3 under 5.5 mM and 25 mM glucose). <b>B</b>, The Sp1-bound chromatin was prepared from INS-1 832/13 cells grown in low (5.5 mM) or high (25 mM) glucose, fragmented and immunoprecipitated with anti-Sp1 antibody and subjected to real time PCR. The fluorescence signals obtained from quantitation of immunoprecipitated fraction was normalized to the input levels. The input fraction was the sonicated Sp1-bound DNA before immunopreripitating with anti-Sp1 antibody. The statistical analysis was conducted by ANOVA test where *,ΨΨ P<0.01. <b>C</b>, Western blot analysis of nuclear (NC) and cysolic (CYT) extracts of INS-1 832/13 cells maintained under 5.5 or 25 mM glucose with anti-Sp1 antibody. Loading controls of the cytosolic and nuclear proteins were assessed by stripping the blot and re-probed with anti-tubulin and anti-lamin B antibody, respectively. <b>D</b>, A representative of Western blot analysis of nuclear extracts of INS-1 832/13 cells maintained in the presence of 5.5 or 25 m glucose with anti-Sp1, anti-phospho Sp1. Control loading was assessed by stripping the blot and re-probed with anti-actin antibody. E, The intensity of total Sp1, phospho-Sp1 bands was quantitated and normalized with that of actin band and shown as the ratios of total Sp1/actin, phospho-Sp1/actin and total Sp1/phospho-Sp1 from three independent experiments. The statistical analysis was conducted using ANOVA test where *P<0.05 **P<0.01.</p

USF1 and USF2 bind to the E1 in the P2 promoter of the PC gene.

<p><b>A.</b> Nucleotide sequences of M4, M5 and M5 pmut E2 probes. E-box and GC-box are underline. <b>B,</b> EMSA of M4 probe with INS-1 832/13 nuclear extract. Lane 1, M4 probe alone; lane 2, probe incubated with nuclear extract; lanes 3–4, nuclear extracts pre-incubated with anti-USF1 or anti-USF2 antibody before the probes were added into the reaction, respectively. Lanes 5–6, probe incubated with nuclear extract of INS-1 832/13 overexpressing USF1 or USF2, respectively. Lanes 7 and 8, nuclear extracts of INS-1 832/13 overexpressing USF1 pre-incubated with anti-USF1 antibody or overexpressing USF2 pre-incubated with anti-USF2 antibody before the probe was added into the reaction, respectively. Lane 9, INS-1 832/13 nuclear extract pre-incubated with anti-ChREBP antibody before the probe was added into the reaction. <b>C.</b> EMSA of M5 pmut E2 probe with an INS-1 832/13 nuclear extract. Lane 1, M5 pmut E2 probe alone; lane 2, probe incubated with nuclear extract; lanes 3–5, nuclear extracts pre-incubated with anti-USF1, anti-USF2 or anti-ChREBP antibody before the probes were added into the reaction, respectively. Arrows represent DNA-protein complexes.</p

High glucose enhances binding of USF2 and ChREBP to E4 in the P2 promoter of the PC gene and suppression of Sp1, USF2 and ChREBP expression blunts glucose-induced expression of endogenous PC expression in INS-1 8322/13.

<p><b>A</b>, <i>Top panel</i>, a schematic representation of the P2 promoter region with E-box4 and primer binding sites for quantitative real time PCR indicated. <i>Bottom panel</i>, the USF1-, USF2- or ChREBP-bound chromatin was prepared from INS-1 832/13 cells grown in low (5.5 mM) or high (25 mM) glucose was prepared, immunoprecipitated with their corresponding antibodies and subjected to real time PCR using the primers indicated above. The fluorescence signals obtained from the immunoprecipitated fractions were normalized to those obtained from the input fraction which was the sonicated transcription factor-bound DNA before immunoprecipitating with the antibodies. The statistical analysis was conducted by ANOVA test where **P<0.01 compared between low and high glucose concentrations. ^ΨΨP<0.001 compared with the fraction that was immunoprecipiated with no antibody at both low or high glucose concentration. <b>B</b>, Western blot analysis of nuclear (NC) and cysolic (CYT) extracts of INS-1 832/13 cells maintained under 5.5 or 25 mM glucose with anti- USF2 antibody. Loading controls of the cytosolic and nuclear proteins were assessed by stripping the blot and re-probed with anti-tubulin and anti-lamin B antibodies, respectively. <b>C</b>, INS-1 832/13 cells were mock- or transfected with siRNAs targeted to Sp1, USF1, USF2 and ChREBP. The transfected cells were cultured in the medium containing low (5.5 mM) or high (25 mM) glucose for the next 24 h before the expression of Sp1, USF1, USF2, ChREBP and PC mRNAs was measured by quantitative real time PCR and their expression levels were normalized with that of 18 s rRNA. The values obtained from scramble and each knocked down cells are expressed relative to that obtained from the mocked transfection, which was arbitrarily set as 100%. The values shown are means ± standard deviations of the three independent experiments (n = 3). The statistical analysis was conducted using ANOVA test where *P<0.05; **P<0.01.</p