CRTC2 modulates hepatic SREBP1c cleavage by controlling Insig2a expression during fasting

Dysregulation of hepatic lipid and glucose production results in obesity, diabetes and nonalcoholic fatty liver disease. De novo lipogenesis and gluconeogenesis in the liver contribute, at least in part, to the dynamic homeostasis of lipid and glucose levels (Rui, 2014; Han and Wang, 2017). These processes are regulated at the transcriptional level by different transcription factors in response to various environmental cues, including hormones, nutrition and stress. Regulators of lipogenesis include the sterol regulatory element-binding proteins (SREBPs), which are members of the basic helix-loop-helix leucine zipper transcription factor family (Goldstein et al., 2006). There are three closely related SREBPs, SREBP1a, SREBP1c and SREBP2, among which SREBP1c and SREBP2 are the major isoforms expressed in the liver. SREBP1c mainly regulates fatty acid synthesis, while SREBP2 controls cholesterol production (Goldstein et al., 2006). SREBPs are synthesized as inactive precursors bound to the endoplasmic reticulum (ER), where they associate with the sterol cleavage activating protein (SCAP) (Goldstein et al., 2006). Upon sensing sterol demand, the SREBP/SCAP complex buds from the ER in a COPII-dependent manner and is transported to the Golgi, where SREBP processing occurs. The mature SREBP then translocates to the nucleus and activates the transcription of lipid biosynthesis genes. In the presence of excess sterol levels, the SREBP/SCAP complex binds to insulin-induced gene (INSIG) and is restricted to the ER (Goldstein et al., 2006). Thus, SREBP processing, shuttling of SREBP to the nucleus and transcriptional control of genes involved in cholesterol and fatty acid synthesis are tightly regulated in response to nutrition conditions. SREBP1c processing is regulated not only by nutrient status but also by hormonal signaling during feeding and fasting (Horton et al., 1998; Li et al., 2010). These features distinguish it from SREBP2, processing of which is regulated by sterol levels. During feeding, insulin signaling promotes SREBP1c activity through two parallel pathways, one dependent on mTOR and the other dependent on Insig2a (Yecies et al., 2011; Han and Wang, 2017). The Insig2 gene produces two alternatively spliced transcripts, Insig2a and Insig2b, which have different 5′-UTRs but share the same coding region (Yabe et al., 2003). Previous studies have shown that expression of Insig2a, the predominant Insig2 isoform in liver, is enhanced during fasting and attenuated during feeding (Yabe et al., 2003). Furthermore, insulin signaling decreases Insig2a expression with concomitant enhancement of SREBP1c activity (Yabe et al., 2003; Yellaturu et al., 2009). Together, these results suggest that Insig2a is a potent regulator of SREBP1c activity during both feeding and fasting in the liver. Although both fasting and/or glucagon signaling can attenuate SREBP1c activity (Horton et al., 1998; Yabe et al., 2003), the molecular mechanism linking fasting to Insig2a expression is unclear. In contrast to SREBP1c-mediated hepatic lipogenesis, gluconeogenesis is activated during fasting and shut down during feeding. Gluconeogenesis is partially regulated by cAMP response element-binding protein (CREB) and CREB regulated transcription coactivators (CRTCs) (Altarejos and Montminy, 2011). CRTC2, which is highly expressed in the liver, is dephosphorylated and shuttled to the nucleus during fasting, where it binds to CREB and induces gluconeogenic gene expression (Altarejos and Montminy, 2011). During feeding, CRTC2 is phosphorylated and sequestered in the cytoplasm through phosphorylation-dependent interactions with 14-3-3 proteins (Altarejos and Montminy, 2011). Our previous results showed that CRTC2 mediates mTOR-controlled SREBP1c processing and lipogenesis during feeding (Han et al., 2015). However, it is unclear whether CRTC2 affects SREBP1c processing during fasting. To investigate the effect of CRTC2 on SREBP1c processing during fasting, we fasted mice for 12 h and evaluated SREBP1c processing. We found that higher levels of mature SREBP1c accumulate in Crtc2 mice than in Crtc2 mice following fasting (Fig. 1A). Expression of two SREBP1c-targeted lipogenic genes (Fasn, Scd1) was also elevated in Crtc2 mice as judged by real-time quantitative PCR (qPCR) (Fig. 1B). In contrast to SREBP1c, levels of SCAP are comparable in Crtc2 and Crtc2 mice under fasted conditions (Fig. 1A). In addition to SCAP, INSIG family proteins, including INSIG1 and INSIG2, also play an


Dear Editor,
Dysregulation of hepatic lipid and glucose production results in obesity, diabetes and nonalcoholic fatty liver disease. De novo lipogenesis and gluconeogenesis in the liver contribute, at least in part, to the dynamic homeostasis of lipid and glucose levels (Rui, 2014;Han and Wang, 2017). These processes are regulated at the transcriptional level by different transcription factors in response to various environmental cues, including hormones, nutrition and stress. Regulators of lipogenesis include the sterol regulatory element-binding proteins (SREBPs), which are members of the basic helix-loop-helix leucine zipper transcription factor family (Goldstein et al., 2006). There are three closely related SREBPs, SREBP1a, SREBP1c and SREBP2, among which SREBP1c and SREBP2 are the major isoforms expressed in the liver. SREBP1c mainly regulates fatty acid synthesis, while SREBP2 controls cholesterol production (Goldstein et al., 2006). SREBPs are synthesized as inactive precursors bound to the endoplasmic reticulum (ER), where they associate with the sterol cleavage activating protein (SCAP) (Goldstein et al., 2006). Upon sensing sterol demand, the SREBP/SCAP complex buds from the ER in a COPII-dependent manner and is transported to the Golgi, where SREBP processing occurs. The mature SREBP then translocates to the nucleus and activates the transcription of lipid biosynthesis genes. In the presence of excess sterol levels, the SREBP/SCAP complex binds to insulin-induced gene (INSIG) and is restricted to the ER (Goldstein et al., 2006). Thus, SREBP processing, shuttling of SREBP to the nucleus and transcriptional control of genes involved in cholesterol and fatty acid synthesis are tightly regulated in response to nutrition conditions. SREBP1c processing is regulated not only by nutrient status but also by hormonal signaling during feeding and fasting Li et al., 2010). These features distinguish it from SREBP2, processing of which is regulated by sterol levels. During feeding, insulin signaling promotes SREBP1c activity through two parallel pathways, one dependent on mTOR and the other dependent on Insig2a (Yecies et al., 2011;Han and Wang, 2017). The Insig2 gene produces two alternatively spliced transcripts, Insig2a and Insig2b, which have different 5′-UTRs but share the same coding region . Previous studies have shown that expression of Insig2a, the predominant Insig2 isoform in liver, is enhanced during fasting and attenuated during feeding . Furthermore, insulin signaling decreases Insig2a expression with concomitant enhancement of SREBP1c activity Yellaturu et al., 2009). Together, these results suggest that In-sig2a is a potent regulator of SREBP1c activity during both feeding and fasting in the liver. Although both fasting and/or glucagon signaling can attenuate SREBP1c activity Yabe et al., 2003), the molecular mechanism linking fasting to Insig2a expression is unclear.
In contrast to SREBP1c-mediated hepatic lipogenesis, gluconeogenesis is activated during fasting and shut down during feeding. Gluconeogenesis is partially regulated by cAMP response element-binding protein (CREB) and CREB regulated transcription coactivators (CRTCs) (Altarejos and Montminy, 2011). CRTC2, which is highly expressed in the liver, is dephosphorylated and shuttled to the nucleus during fasting, where it binds to CREB and induces gluconeogenic gene expression (Altarejos and Montminy, 2011). During feeding, CRTC2 is phosphorylated and sequestered in the cytoplasm through phosphorylation-dependent interactions with 14-3-3 proteins (Altarejos and Montminy, 2011). Our previous results showed that CRTC2 mediates mTOR-controlled SREBP1c processing and lipogenesis during feeding (Han et al., 2015). However, it is unclear whether CRTC2 affects SREBP1c processing during fasting.
Considering that Insig2a is a predominant isoform of In-sig2 in the liver  and CRTC2-dependent transcriptional effect on Insig2a expression, we tested whether the CRTC2/CREB complex directly regulates Insig2a expression. Overexpression of wild-type (WT) and constitutively active (CRTC2/AA) (Han et al., 2015) CRTC2 in the liver increased Insig2a expression; knockdown of Crtc2 by short hairpin RNA decreased its expression ( Fig. 2A and   2B), which is consistent with the results from Crtc2 knockout mice (Fig. 1B). In addition, overexpression of ACREB, a dominant-negative form of CREB (Herzig et al., 2003), decreased Insig2a expression (Fig. 2C). Together, these results suggest that the CRTC2/CREB complex, as a whole, controls Insig2a expression.
To further demonstrate whether the CRTC2/CREB complex autonomously regulates Insig2a, we used reporter assays in mouse primary hepatocytes. Overexpression of CRTC2 and CRTC2/AA enhanced Insig2a-luc activity following glucagon (Gcg) treatment, while Crtc2 deficiency by knockdown or knockout attenuated Insig2a-luc activity (Fig. 2D-F). As shown in Figure 2G, overexpression of ACREB decreased Insig2a-luc activity in hepatocytes, consistent with the results in liver (Fig. 2C). These results show that the CRTC2/CREB complex autonomously controls In-sig2a expression.
Having obtained evidence for a regulatory effect of the CRTC2/CREB complex on Insig2a, we performed chromatin immunoprecipitation (ChIP) studies to determine whether Insig2a is a direct target of CRTC2/CREB. Supporting this notion, glucagon stimulation dramatically increased both CRTC2 and CREB occupancy on the Insig2a promoter (Fig. 2H). CREB regulates gene expression by binding to palindromic (TGACGTCA) or to half-site (TGACG/CGTCA) cAMP-responsive elements (CREs) positioned within 1 kb of the transcriptional start site (Altarejos and Montminy, 2011). When we mutated a CRE half-site located at position −859