Insulin activates human sterol-regulatory-element-binding protein-1c (SREBP-1c) promoter through SRE motifs

Abstract

In the present study, we aimed to decipher the mechanisms involved in the transcriptional effect of insulin on the SREBP-1c specific promoter of the human srebf-1 gene. Using luciferase reporter gene constructs in HEK-293 cells (human embryonic kidney cells), we demonstrated that the full effect of insulin requires the presence of SREs (sterol response elements) in the proximal region of the promoter. Furthermore, insulin increases the binding of SREBP-1 (sterol-regulatory-element-binding protein-1) to this promoter region in chromatin immunoprecipitation assay. We also found that the nuclear receptors LXRs (liver X receptors) strongly activate SREBP-1c gene expression and identified the LXRE (LXR-response element) involved in this effect. However, our results suggested that these LXREs do not play a major role in the response to insulin. Finally, using expression vectors and adenoviruses allowing ectopic overexpressions of the human mature forms of SREBP-1a or SREBP-1c, we demonstrated the direct role of SREBP-1 in the control of SREBP-1c gene expression in human skeletal-muscle cells. Altogether, these results strongly suggest that the SREBP-1 transcription factors are the main mediators of insulin action on SREBP-1c expression in human tissues.

Figure 1. Effects of insulin infusion on the mRNA levels of SREBP-1 isoforms in skeletal muscle and adipose tissue

The mRNA levels of SREBP-1a and SREBP-1c were determined by quantitative real-time PCR in skeletal-muscle (upper panel) and adipose tissue (lower panel) biopsies of healthy subjects before (open bars) and at the end (black bars) of a 3 h hyperinsulinaemic euglycaemic clamp. Results are presented as means±S.E.M. *P<0.05 after 3 h of insulin infusion versus before 3 h of insulin infusion; **P<0.01 after 3 h of insulin infusion versus before 3 h of insulin infusion.

Figure 2. Activation of human SREBP-1c promoter by insulin

HEK-293 cells were transfected with a luciferase reporter gene driven by different constructs of the human SREBP-1c gene promoter. Cells were incubated for 24 h with (black bars) or without insulin (10⁻⁷ mol/l) (open bars). Relative luciferase activity was calculated using a dual luciferase assay as indicated in the Materials and methods section. Results are expressed by reference to the basal luciferase activity of p1c-E and presented as the means±S.E.M. for at least three independent transfection experiments. *P<0.05 in the presence of insulin versus basal conditions. Inset: effect of insulin (10⁻⁷ mol/l) on the mRNA levels of SREBP-1a, SREBP-1c and SREBP-2 in HEK-293 cells. The mRNA levels were determined by quantitative real-time PCR. Results are presented as means±S.E.M. (n=4). *P<0.05 compared with untreated cells.

Figure 3. Mutations of the LXREs in the SREBP-1c promoter reduce its response to T0901317 but not to insulin

The luciferase reporter gene p1c-E1 and LXRE mutated constructs were obtained as described in the Materials and methods section. After transfection, HEK-293 cells were incubated for 24 h with or without T0901317 (1 μM) (A) and with or without insulin (10⁻⁷ mol/l) (B). Results are expressed by reference to the basal activity of p1c-E1 and are presented as the means±S.E.M. (n=3). *P<0.05 in the presence of T0901317 or insulin versus basal conditions.

Figure 4. Mutations of the SREs in the proximal SREBP-1c promoter suppress transcriptional stimulation by insulin

The luciferase reporter gene p1c-E2 and SRE mutated constructs were obtained as described in the Materials and methods section. After transfection, HEK-293 cells were incubated for 24 h with or without insulin (10⁻⁷ mol/l). Results are expressed by reference to the basal activity of p1c-E2 and are presented as the means±S.E.M. (n=3). *P<0.05 in the presence of insulin versus basal conditions.

Figure 5. ChIP assay of SREBP-1 protein association with the SREBP-1c promoter

HEK-293 cells were incubated for 6 h in control medium or in medium supplemented with insulin (10⁻⁷ mol/l). After cross-linking chromatin DNA to the interacting proteins, specific immunoprecipitations with anti-SREBP-1 or anti-SREBP-2 or non-specific (IgG) antibodies were performed as indicated in the Materials and methods section. The PCR products were generated by the amplifications of the SREBP-1c promoter, the SREBP-1a promoter, the FAS promoter, the LDL receptor (LDLr) promoter and the SREBP-1 exons 4/5 (negative control). Amplification products were resolved in 3%-agarose gels stained with ethidium bromide. The Figure is representative of three independent experiments. Abbreviations: Mock, no antibody condition; C, untreated cells; I, cells treated with insulin. The lower part of the Figure shows the quantitative aspect of the PCR amplification using serial dilutions of the input.

Figure 6. Effects of human SREBP-1a or SREBP-1c overexpression on the SREBP-1c promoter

Each reporter gene construct was co-transfected with an empty expression vector pcDNA3.1 (control condition) or an expression vector for mature form of the human SREBP-1a (pCMV-hSREBP1a) or the human SREBP-1c (pCMV-hSREBP1c) in HEK-293 cells. Luciferase activity was measured 24 h after transfection. Results are expressed by reference to the basal activity of p1c-E2 and are presented as the means±S.E.M. (n=3). *P<0.05 in the presence of SREBP-1a or SREBP-1c versus control conditions.

Figure 7. Effects of adenoviral expression of human SREBP-1a or SREBP-1c in differentiated skeletal-muscle cells

Human skeletal-muscle cells were infected with increasing concentrations of adenovirus expressing SREBP1a or SREBP1c (Ad-SREBP1a or Ad-SREBP1c) as indicated in the Materials and methods section. Total cell lysates were prepared, 24 h after infection, and analysed for the presence of precursor and mature forms of SREBP-1 and for INSIG-1 using specific antibodies (A). α-Tubulin is shown as a loading control. In parallel, total RNA was prepared from muscle cells overexpressing the SREBP-1 isoforms (24 h after adenofection) and analysed for the expression of SREBP-1a and SREBP-1c transcripts using RT–qPCR (B). Due to sequence identities between the endogenous gene and the adenoviruses, SREBP-1c transcripts were measured when SREBP-1a adenovirus was used and, conversely, SREBP-1a mRNA was determined when SREBP-1c mature protein was overexpressed. Results are presented as means±S.E.M. (n=3). *P<0.05.