Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c

Abstract

Sterol regulatory element-binding proteins (SREBPs) are transcription factors central to the regulation of lipid metabolism. The SREBPs are synthesized as precursor proteins that require proteolytic processing to become transcriptionally active. Whereas the regulation of SREBP-1a and -2 cleavage by cellular sterol content is well defined, much less is known about the regulation of SREBP-1c, the predominant SREBP isoform in the liver. Both insulin and liver X receptor alpha (LXRalpha) induce SREBP-1c transcription; however, the respective roles of these factors and the mechanism responsible for proteolytic cleavage of this SREBP isoform are not known. In this study, we compare the effects of insulin and LXR agonist TO-901317 on SREBP-1c expression and transcriptional activity in isolated rat hepatocytes. We report that full induction of the mature and transcriptionally active form of SREBP-1c protein requires insulin. Although activation of LXR leads to the induction of SREBP-1c gene expression and precursor protein, it has a very poor effect in inducing the mature nuclear form of the transcription factor. This may be due to the induction of insulin-induced gene-2a mRNA and protein by LXR activation. The LXR-induced SREBP-1c precursor, however, is rapidly cleaved on acute exposure to insulin via a phosphatidylinositol 3-kinase-dependent mechanism. Finally, we show through experiments in suckling mice that this acute action of insulin to stimulate the proteolytic processing of SREBP-1c is functional in vivo.

Fig. 1.

Relative expression of SREBP-1c, IRS-2, ABCA1, and GK mRNA in primary hepatocytes treated with insulin or the LXR agonist TO-901317. Hepatocytes were maintained overnight in basal medium before treatment with insulin (100 nM; open bars), TO-901317 (10 μM; black bars), or DMSO (vehicle control; hatched bars) for the time period indicated. Total RNA from triplicate plates of hepatocytes was extracted and analyzed for SREBP-1c (A), IRS-2 (B), ABCA1 (C), and GK (D). Data are means ± SEM. Representative of three independent experiments.

Fig. 2.

Differential induction of nuclear SREBP-1c by insulin and TO-901317. After 16 h in basal medium, hepatocytes were incubated for 6 h in either fresh basal medium or basal medium supplemented with insulin (100 nM; INS) or TO-901317 (10 μM; TO). Subsets of hepatocytes cultured in basal medium or with TO-901317 were treated with insulin (100 nM) for the final 30 min of this 6-h treatment period (INS pulse; lanes 4 and 5). (A) SREBP-1c mRNA expression. (B) Immunoblot of SREBP-1c precursor expressed in the microsomal fraction of hepatocytes. (C) Immunoblot of mature SREBP-1c in the nuclear extracts of hepatocytes. (D) GK mRNA expression. mRNA values represent the mean ± SEM of three independent experiments performed in triplicate. Immunoblots are representative of three independent experiments. ^*, P < 0.01; ^**, P < 0.001 compared with untreated control (lane 1).

Fig. 3.

Characterization of the acute accumulation of nuclear SREBP-1c with insulin. After 16 h in basal medium, hepatocytes were incubated for 6 h in basal medium supplemented with TO-901317 (10 μM) with additional treatments as detailed. (A) Time-course and effect of ALLN: cells were treated with insulin (100 nM; INS pulse) for the indicated time, or the calpain-1 inhibitor, ALLN (50 μg/ml; 30 min). (B) Dose-response to insulin: cells were treated with 0, 1, 10, or 100 nM insulin, as indicated (30 min; INS pulse). (C) Inhibition by wortmannin: wortmannin (100 nM) or vehicle was added to plates 15 min before the insulin pulse (100 nM, 30 min; INS pulse). At the end of the 6-h treatment period, cells were collected for preparation of microsomal membranes and nuclear extracts. Each blot is representative of two independent experiments.

Fig. 4.

Acute induction of SREBP-1c cleavage by insulin in liver of suckling mice. Suckling mice were administered 0.4 units of insulin or saline by i.p. injection. After 20 min, mice were killed and livers collected. (A) SREBP-1c mRNA expression. (B) Immunoblot of calnexin (90 kD) is provided as a loading control for microsomal membrane proteins. Immunoblot of SREBP-1c in hepatic microsomal membranes (SREBP-1c precursor) and nuclear extracts (mature SREBP-1). (C) EMSA performed with hepatic nuclear extracts from suckling mice. The position of the SREBP-1c specific complex is indicated. Each lane represents an individual animal.

Fig. 5.

Differential regulation of Insig expression by insulin and TO-901317. (A) Hepatocytes were treated as described in Fig. 2. Insig-1 and -2a mRNA were measured by RT-PCR; each value represents the mean ± SEM of three independent experiments performed in triplicate. ^*, P < 0.05; ^**, P < 0.01 compared with untreated control (lane 1). Protein expression of Insig-2 in hepatocyte microsomal membranes was measured by Western blot, as described in Methods. (B) Insig-2 protein in liver of adult mice force-fed with TO-901317 or vehicle, detected by Western blot. Each lane represents one individual animal.