Abstract
The regulation of lipid homeostasis by insulin is mediated in part by the enhanced transcription of the gene encoding sterol regulatory element-binding protein-1c (SREBP-1c). The nascent SREBP-1c is embedded in the endoplasmic reticulum (ER) and must be transported to the Golgi where two sequential cleavages generate its NH(2)-terminal fragment, nSREBP-1c. We have shown recently that in primary cultures of rat hepatocytes, insulin rapidly and selectively stimulates proteolytic processing of the nascent SREBP-1c by enhancing the affinity of the SREBP cleavage-activating protein (SCAP).SREBP-1c complex for coatomer protein complex II (COPII) vesicles. The SCAP.SREBP complex is retained in the ER by Insig proteins. We report here that insulin persistently stimulates controlled proteolysis of the nascent SREBP-1c by selectively reducing the level of Insig-2a protein via accelerated degradation of its cognate mRNA. Insulin enhanced the rate of turnover of Insig-2a mRNA via its 3'-untranslated region. Insulin-induced depletion of Insig-2a promotes association of the SCAP.SREBP-1c complex with COPII vesicles and subsequent migration to the Golgi where site-1 and site-2 proteases process the nascent SREBP-1c. Consistent with this mechanism, experimental knockdown of Insig-2a expression with small interfering RNA mimicked insulin-induced proteolysis of the nascent SREBP-1c, whereas exogenous expression of Insig-2a in hepatocytes led to reduced intramembrane proteolysis of the newly synthesized SREBP-1c. The action of insulin on the processing of the nascent SREBP-1c via Insig-2a was highly selective, as proteolysis of the newly synthesized SREBP-2 remained unchanged under identical conditions. On the basis of these data, we propose that the stimulation of SREBP-1c processing by insulin is mediated by a selective depletion of Insig-2a protein by promoting decay of its cognate mRNA. Thus, insulin-induced reduction in Insig-2a protein leads to an enhanced export of the SCAP.SREBP-1c complex from ER to the Golgi.
Highlights
Sterol-regulatory element-binding proteins (SREBPs),3 represented by SREBP-1a, SREBP-1c, and SREBP-2, are key transcription factors that control a suite of genes involved in the homeostatic regulation of cholesterol and lipid metabolism [1,2,3,4,5,6,7]
We carried out experiments to test the hypothesis that insulin-induced signals enhance the intramembrane proteolysis of SREBP-1c by altering its association with endoplasmic reticulum (ER)-bound Insig and/or complex II (COPII) proteins
Consistent with previously published data, nuclear extracts prepared from insulin-treated hepatocytes were more highly enriched in nSREBP-1c compared with extracts from untreated cells; in insulin-treated hepatocytes, the nuclear SREBP-1c was increased by more than 3-fold (Fig. 1)
Summary
Sterol-regulatory element-binding proteins (SREBPs), represented by SREBP-1a, SREBP-1c, and SREBP-2, are key transcription factors that control a suite of genes involved in the homeostatic regulation of cholesterol and lipid metabolism [1,2,3,4,5,6,7]. Within the ER, SREBPs are associated with two integral membrane proteins, SCAP (SREBP cleavage-activating protein) and Insig-1 (insulin-induced gene product-1) or Insig-2 [1, 2] Following their transport from the ER to the Golgi, the nascent SREBPs undergo intramembrane proteolysis, which liberates their transcriptionally active NH2-terminal fragments. The molecular details of the putative mechanisms by which insulin enhances the biogenesis of nSREBP-1c via more efficient posttranslational proteolysis are unclear Both SREBP-1c and -2 isoforms are associated with the SCAP/Insig complex and undergo ER-to-Golgi transport and proteolytic processing, they are selectively regulated by insulin and sterols, respectively [16]. The mechanism of this specificity is not clearly defined. We report that insulin mediates depletion of Insig-2a protein by degradation of its cognate mRNA, promoting the association between the SCAP1⁄7SREBP-1c complex and Sec231⁄7Sec24-containing COPII vesicles and their more rapid transport to the Golgi
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