Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases. In Western societies, NAFLD is clearly associated with the features of the metabolic syndrome, including obesity, type 2 diabetes, hypertension, and dyslipidemia. Hepatic steatosis is considered the first stage leading to more severe complications, such as steatohepatitis, cirrhosis, and hepatocellular carcinoma. Moreover, hepatic fat accumulation is associated with the development of hepatic insulin resistance, characterized by increased hepatic glucose production leading to fasting hyperglycemia.
Recent studies in humans pointed out the important role of de novo lipogenesis in the excessive accumulation of triglycerides in the livers of patients with NAFLD, since one-third of total triglycerides might originate from this pathway (1
). In addition, hepatic lipogenesis can also contribute to lipid accumulation by reducing fatty acid oxidation, as malonyl-CoA, generated in the lipogenic pathway, is a potent inhibitor of carnitine palmitoyl transferase I. Lipogenesis is highly dependent on nutritional conditions for its activation; it is induced by high-carbohydrate feeding and inhibited by fasting. SREBP-1c, a member of the SREBP family of transcription factors, and carbohydrate response element–binding protein (ChREBP) have emerged as transcription factors involved in the transcriptional effects of insulin and glucose, respectively, on lipogenic gene expression in the liver (2
). Activation of SREBP-1c by insulin during carbohydrate feeding involves 2 mechanisms: activation of SREBP-1c transcription and increase in proteolytic cleavage of the SREBP-1c precursor form embedded in the membranes of the ER (4
). Within the ER membranes, the inactive SREBP proteins are associated with 2 essential proteins that take part in the control of the cleavage process: SREBP cleavage–activating protein (SCAP) and insulin-induced gene (Insig). SCAP interacts on the one hand with newly synthesized SREBP precursor and on the other hand with Insig, which functions as a retention protein of the SCAP/SREBP complex into the ER (7
). In the presence of specific signals such as insulin, SCAP dissociates from Insig and escorts SREBPs in coatomer protein II (COPII) vesicles from the ER to the Golgi apparatus, where SREBPs are proteolytically processed to yield the transcriptionally active form. Once released, the SREBP mature transcription factors can enter into the nucleus and, for the SREBP-1c mature isoform, activate the expression of lipogenic genes. The critical role of SREBP-1c in lipogenesis is emphasized in loss- and gain-of-function studies in vitro and in vivo. Hepatocytes or mice overexpressing an active form of SREBP-1c develop steatosis as a result of activation of the entire lipogenic program (9
). In contrast, mice invalidated for hepatic SREBP-1c fail to induce lipogenic enzyme expression in response to fasting and refeeding (12
Although highly dependent on insulin for its activation, lipogenesis is paradoxically very active in the livers of obese rodents such as ob/ob
mice, which are characterized by severe hepatic insulin resistance resulting in glucose overproduction and hyperglycemia. Moreover, accumulation of nuclear SREBP-1c is detected in the livers of these animals (13
). The failure of insulin to suppress gluconeogenesis while lipogenesis is still activated could reflect a differential sensitivity of these pathways to insulin. It was proposed that decreased IRS-2 content caused by hyperinsulinemia could account for the resistance to insulin-mediated repression of gluconeogenic genes (14
) and that insulin could still activate lipogenic genes by IRS-1 or by another route (14
). However, IRS tyrosine phosphorylation in the livers of ob/ob
mice decreases for both IRS-1 and IRS-2 (15
). Thus the marked activation of SREBP-1c in the ob/ob
mouse liver cannot be explained by stimulated activity of insulin signaling.
Recent studies have demonstrated the presence of an unfolded protein response (UPR) in the liver and adipose tissue of insulin-resistant rodents, which, when counteracted, improves the insulin resistance of these animals (17
). Secreted and transmembrane proteins are folded in the ER, and the role of the UPR is to dynamically adjust the protein folding capacity to the cell requirements. Thus, any event that can disturb ER folding capacity, such as a load of unfolded proteins or altered redox state, calcium equilibrium, or glycosylation potential, will induce a UPR. The UPR is mediated by 3 transducer proteins that are integral membrane proteins of the ER: inositol-requiring kinase–1 (IRE1), an inositol-requiring kinase with endonuclease activity; activating transcription factor 6 (ATF6), a transcription factor that, like SREBP-1c and SREBP-2, is activated by proteolytic cleavage in the Golgi; and protein kinase RNA–like ER kinase (PERK). Each of these effectors activates specific pathways allowing for increased ER folding capacity, decreased protein folding load, induced degradation of misfolded proteins, and, ultimately, induced cell death (21
). It has been shown previously that a UPR induced by homocysteine is able to activate SREBP-1c and to induce lipogenic gene expression (23
). We therefore reasoned that in the livers of obese insulin-resistant animals, an ER stress, by inducing SREBP-1c cleavage, could be an alternative explanation for the ongoing lipid synthesis.
In the present study, we showed that SREBP-1c cleavage was induced by the ER stress pathway independently of insulin. We also demonstrated that attenuation of ER stress by molecular chaperones in livers of ob/ob mice slowed down lipogenesis by inhibiting SREBP-1c proteolytic cleavage and thus improved steatosis and insulin sensitivity of these animals. Finally, we showed that insulin-induced SREBP cleavage was inhibited by overexpression of the chaperone glucose-regulated protein 78 (GRP78) and that the SREBP-1c complex was able to bind GRP78. Our results indicate that ER stress is a major component of the hepatic steatosis and insulin resistance observed in obese insulin-resistant rodents.