Sterol regulatory element-binding protein-1c (SREBP-1c) is a transcription factor that activates fatty acid synthesis and thereby induces fatty liver in obese rodents (Horton et al., 2002; Moon et al., 2012). The level of SREBP-1c in liver is controlled by insulin (Shimomura et al., 1999; Foufelle and Ferré, 2002). When animals fast and plasma insulin is low, the amount of SREBP-1c mRNA in liver is low. SREBP-1c mRNA levels rise 8–30 fold when insulin secretion is stimulated by refeeding a high carbohydrate diet to rodents that were previously fasted (Horton et al., 1998; Liang et al., 2002; Li et al., 2010). The resultant increase in nuclear SREBP-1c leads to the characteristic insulin-induced increase in fatty acid and triglyceride synthesis. Under conditions of obesity and insulin resistance, insulin loses its ability to suppress gluconeogenic mRNAs in liver, yet the hormone continues to stimulate SREBP-1c production and fatty acid synthesis in that organ (Brown and Goldstein, 2008; Titchenell et al., 2016). This phenomenon has been termed ‘selective insulin resistance’ and it leads to a pathologic state in which the liver produces glucose and fatty acids simultaneously, a phenomenon that results in hyperglycemia, hypertriglyceridemia, fatty liver and cirrhosis (Brown and Goldstein, 2008). Solving this problem will require an understanding of the mechanism by which insulin stimulates production of SREBP-1c mRNA. The current experiments were designed to increase this understanding.

Previous studies identified liver X receptors (LXRs) and C/EBPβ as transcription factors that are required for insulin stimulation of SREBP-1c mRNA production (Chen et al., 2004; Tian et al., 2016; Millward et al., 2007). Immunoprecipitation studies revealed that LXRα and C/EBPβ form a complex that binds to a region of the SREBP-1c promoter that contains two canonical LXR-binding sites (Tian et al., 2016). These sites were shown previously to be required for insulin induction of SREBP-1c transcription (Chen et al., 2004). Deficiency of LXR or C/EBPβ prevents insulin induction of SREBP-1c mRNA and fatty acid synthesis (Kalaany et al., 2005; Tian et al., 2016). Chromatin immunoprecipitation assays revealed that insulin does not activate the LXRα-C/EBPβ complex directly. Indeed, the complex is bound to the SREBP-1c promoter in the absence as well as the presence of insulin (Tian et al., 2016). This finding suggests that the liver must contain another protein that activates the pre-existing LXRα-C/EBPβ complex when insulin is present.

The current study was designed to identify an insulin-regulated factor that acts together with the LXRα-C/EBPβ complex to increase SREBP-1c transcription in the presence of insulin. Through quantitative mass spectrometry, we identified a single phosphorylated protein that was co-immunoprecipitated with LXRα and C/EBPβ in nuclear extracts of livers from refed but not fasted rats. The protein, which we designate as BHLHE40 (Basic Helix-Loop-Helix Family Member E40), has been observed by others in many organs and contexts and has received several other names, including SHARP-2, STRA13, and DEC1. We use the name BHLHE40 because that is the name given to its gene (http://www.genecards.org/cgi-bin/carddisp.pl?gene=BHLHE40). Previous studies showed that insulin induces production of BHLHE40/SHARP-2 in rat liver (Kanai et al., 2017; Teboul et al., 1995). However, this induction was not postulated to be involved in SREBP-1c gene activation. Here, we provide evidence that induction of BHLHE40 is a required step in insulin-mediated activation of the SREBP-1c gene, and therefore it is required for insulin-mediated stimulation of fatty acid synthesis.

To search for a factor that works together with LXRα and C/EBPβ to mediate insulin induction of SREBP-1c gene transcription, we sought to identify a nuclear protein that was enriched in livers of fed rats and that bound to both of these transcription factors. As shown in Figure 1A, we prepared nuclear extracts from livers of rats that were either fasted for 48 hr to reduce plasma insulin or fasted for 48 hr and then refed a high-carbohydrate diet for 6 hr in order to induce insulin secretion. Separate aliquots of the nuclear proteins were subjected to immunoprecipitation with either anti-LXRα or anti-C/EBPβ. The precipitated proteins were subjected to SDS-PAGE and identified by mass spectrometry. Using label-free quantification, we identified a total of 32 proteins that were enriched by at least threefold in the extracts from refed rats as compared with fasted rats and that were also precipitated by both anti-LXRα and anti-C/EBPβ (Figure 1B, shown in red). Inasmuch as insulin signaling is mediated by a cascade of phosphorylation reactions, we subjected the immunoprecipitates to LC-MS/MS analysis to determine whether any of the 32 refeeding-induced proteins was phosphorylated. Only one of the 32 proteins fulfilled this criterion, namely, BHLHE40, which was observed to be phosphorylated on serine 383 (Figure 1B–D). The mass spectroscopy data indicated that BHLHE40 protein was increased by 18-fold in the anti-LXRα immunoprecipitates from refed rats as compared with fasted rats. The increase was 9.8-fold in the anti-C/EBPβ immunoprecipitates (Figure 1C).

Figure 1

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Figure 2 shows an experiment in which we used real-time PCR to quantify mRNAs in liver extracts from fasted and refed rats. As shown previously (Liang et al., 2002), refeeding increased the SREBP-1c mRNA by 18-fold (Figure 2B). Refeeding increased the BHLHE40 mRNA by 8.4-fold (Figure 2A). The LXRα mRNA rose less than twofold (Figure 2C) and the C/EBPβ mRNA was not increased by refeeding (Figure 2D).

Figure 2

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To confirm the co-immunoprecipitation of BHLHE40 with LXRα and C/EBPβ, we prepared liver nuclear extracts from fasted and refed rats (Figure 3). The extracts were split into four equal portions. One portion was reserved as the input fraction, and the other three were incubated with agarose beads coated with anti-IgG, anti-LXRα or anti-C/EBPβ. Aliquots containing 30% of the input fraction and all of the proteins eluted from the beads were subjected to SDS-PAGE and immunoblotted with antibodies directed against LXRα, C/EBPβ, BHLHE40, SREBP-1c and CREB. SREBP-1c and CREB, like BHLHE40, are basic-helix-loop-helix transcription factors. They were included as controls for nonspecific interaction. In the extracts from refed rats, BHLHE40 was precipitated by anti-LXRα and anti-C/EBPβ (lanes 6 and 8), but not by the control anti-IgG (lanes 3 and 4). Neither SREBP-1c nor CREB was precipitated by anti-LXRα or anti-C/EBPβ (lanes 5–8). These data show that anti-LXRα and anti-C/EBPβ can precipitate BHLHE40 from refed rats, consistent with the formation of a LXRα-C/EBPβ-BHLHE40 complex.

Figure 3

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To study the binding of the LXRα-C/EBPβ-BHLHE40 complex to the SREBP-1c enhancer/promoter region, we performed ChIP assays on DNA from livers of fasted and refed rats (Figure 4). Slices of rat liver were incubated with formaldehyde to cross-link proteins to DNA. The tissue was disrupted, and the DNA was sheared by sonication. After preclearance with a protein A agarose/salmon sperm DNA resin, the supernatant was incubated with various antibodies, and the antibody-bound protein/DNA complexes were precipitated with protein A agarose. The precipitated DNA was subjected to PCR with primer pair 1 (Figure 4A), which amplifies the SREBP-1c promoter/enhancer region that includes two LXR-binding sites (Chen et al., 2004) or with primer pair 2, which amplifies a region 5 kb upstream. In liver samples amplified by primer pair 1, anti-BHLHE40 brought down more DNA from the fed than the fasted livers (Figure 4B, lanes 6 and 5). Anti-LXRα and anti-C/EBPβ brought down the same LXRE region in samples from both fasted and fed rats (lanes 7–10), as previously shown (Tian et al., 2016). This segment of DNA was not precipitated by a control anti-IgG (lanes 3 and 4). The BHLHE40, LXRα and C/EBPβ antibodies did not precipitate a segment of DNA that is 4700 bp upstream of the transcription start site, as visualized with primer pair 2 (lanes 5–10). Similar results were obtained in two other experiments. These data show that BHLHE40 binds to the SREBP-1c promoter/enhancer region that contains two LXR elements.

Figure 4

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We showed previously that the induction of SREBP-1c mRNA by insulin can be blocked by rapamycin, an inhibitor of the mTOR pathway (Li et al., 2010; Owen et al., 2012). Kanai et al. (2017) showed that insulin induction of BHLHE40 in cultured hepatoma cells is also inhibited by rapamycin. To determine whether rapamycin blocks insulin-mediated induction of BHLHE40 in vivo, we subjected rats to fasting or refeeding for 6 hr. At the time of refeeding, the rats were injected intraperitoneally with vehicle or vehicle containing rapamycin. Refeeding increased the SREBP-1c mRNA by 26-fold and this was reduced to 4.4-fold by rapamycin (Figure 5A). In vehicle-injected rats, the BHLHE40 mRNA rose by 7.2-fold after refeeding. This increase was reduced to 2.9-fold when the vehicle contained rapamycin (Figure 5B).

Figure 5

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The finding that rapamycin inhibits insulin induction of BHLHE40 mRNA raised the possibility that rapamycin blocks insulin induction of SREBP-1c mRNA by blocking induction of BHLHE40. To test this hypothesis, we treated fresh rat hepatocytes with or without rapamycin, stimulated them with insulin and then measured the levels of mRNAs encoding BHLHE40 and SREBP-1c (Figure 6). In the absence of rapamycin, insulin induced the BHLHE40 mRNA, and this induction was partially blocked by rapamycin. Infection with Lenti-B40, a recombinant lentivirus encoding BHLHE40, raised the level of BHLHE40 mRNA in the absence of insulin and prevented the rapamycin-mediated reduction of BHLHE40 mRNA in the presence of insulin (Figure 6A). As shown in Figure 6B, insulin induction of SREBP-1c mRNA was inhibited by rapamycin, and this inhibition persisted even when BHLHE40 mRNA was restored with Lenti-B40. These results indicate that mTORC1 plays two independent roles in the insulin-mediated induction of SREBP-1c mRNA, one pertaining to BHLHE40 and the other directly affecting SREBP-1c.

Figure 6

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The data of Figure 6 also indicate that BHLHE40 is not sufficient in itself to induce SREBP-1c mRNA. As shown in Figure 6A, in the absence of insulin, infection with Lenti-B40 increased the BHLHE40 mRNA to levels similar to those seen after insulin treatment. However, this increase did not lead to a substantial increase in SREBP-1c mRNA in the absence of insulin (Figure 6B).

If the insulin-mediated induction of BHLHE40 is a prerequisite for the induction of SREBP-1c, then the increase in BHLHE40 mRNA must occur before the increase in SREBP-1c mRNA. To test this hypothesis, we measured the amounts of these mRNAs in rat livers at various times after refeeding (Figure 7A–C). The BHLHE40 mRNA rose nearly threefold within 1 hr, which was the earliest time point tested (Figure 7A). The SREBP-1c mRNA was still low after 2 hr and rose dramatically thereafter (Figure 7B). Figure 7C compares the relative increases in the BHLHE40 and SREBP-1c mRNAs, showing clearly that the rise in BHLHE40 mRNA precedes the increase in SREBP-1c mRNA. We also conducted a time course study in primary rat hepatocytes (Figure 7D–F). The BHLHE40 mRNA rose within 1 hr after addition of insulin to the hepatocytes (Figure 7D), and this preceded the increase in SREBP-1c mRNA (Figure 7E). Figure 7F shows an experiment in which we measured mRNAs in hepatocytes at shorter times after adding insulin. The BHLHE40 mRNA rose within 30 min after adding insulin. The speed of the increase was similar to the fall in PEPCK mRNA, which is known to respond rapidly to insulin (Granner et al., 1983). In contrast, the SREBP-1c mRNA did not increase even at 1 hr. These findings identify BHLHE40 as an early response gene to insulin.

Figure 7

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To confirm that BHLHE40 is required for the insulin-mediated induction of SREBP-1c mRNA, we treated primary rat hepatocytes with two different siRNAs targeting the BHLHE40 mRNA and then added insulin (Figure 8). Both siRNAs (designated siB40A and siB40B) reduced the basal and insulin-stimulated level of BHLHE40 mRNA by about 60% (Figure 8A). The two siRNAs also reduced the basal level of SREBP-1c mRNA and diminished the response to insulin (Figure 8B). It should be noted that the fivefold stimulation of SREBP-1c mRNA in this transfection experiment is smaller than the 20-fold stimulation in the experiment of Figure 7E, which did not include transfection. We attribute this difference to a nonspecific effect of transfection in the primary hepatocytes. Neither of the two siRNAs decreased the expression of LXRα (Figure 8C) nor C/EBPβ (Figure 8D).

Figure 8

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To determine whether BHLHE40 is required in vivo for insulin induction of SREBP-1c, we produced knockout mice derived from embryonic stem cells that harbor a gene trap in the intron between exons 3 and 4 of the Bhlhe40 gene. This gene-trap mutation produces a truncated transcript that eliminates exons 4 and 5, which encode amino acids 86–411 of the 411-amino acid protein. Knockout mice homozygous for this mutation grew normally and had normal body weights. At 8 weeks of age, the body weights of WT and knockout mice were 20.0 and 19.8 g, respectively.

Figure 9A shows an immunoblot of nuclear extracts from the livers of wild type (WT) and Bhlhe40^-/- (KO) mice. As compared to WT liver, the KO liver lacks the 45.4 kDa BHLHE40 protein (lanes 1 and 2). Several bands in the 75–150 kDa range were observed in both the WT and KO samples, one of which was reduced in the KO liver. We believe that these are unrelated proteins recognized nonspecifically by the polyclonal anti-BHLHE40 antibody. Figure 9B and C show an experiment in which WT and Bhlhe40 KO mice were fasted overnight and then refed a high-carbohydrate diet for 4 hr. Hepatic mRNA levels were measured separately for male (Figure 9B) and female (Figure 9C) mice. In both genders, the BHLHE40 mRNA was undetectable in the livers from refed knockout mice, and the SREBP-1c mRNA was reduced dramatically. On the other hand, the knockout produced no significant change in the mRNAs encoding LXRα or C/EBPβ.

Figure 9

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To confirm that the reduction in SREBP-1c mRNA in the knockout mice was caused by the loss of BHLHE40, we injected the mice with a recombinant adeno-associated virus (AAV) encoding BHLHE40 or a nonspecific control protein. To determine whether phosphorylation at serine 383 is essential, we also injected an AAV encoding a mutant form of BHLHE40 in which serine 383 was replaced with alanine. Three weeks after virus injection, the mice were fasted and refed. In the refed knockout mice, BHLHE40 mRNA was undetectable (Figure 10B). Both BHLHE40 viruses restored BHLHE40 mRNA to levels that were approximately twofold above the levels seen in WT mice which are normalized to 1.0 in Figure 10A. In WT mice, the injection of virus encoding WT or BHLHE40 (S383A) raised the level of SREBP-1c mRNA by about 1.5-fold (Figure 10C). In the knockout mice, the level of SREBP-1c mRNA was low (Figure 10D). The level was increased by eightfold when the mice received virus encoding either WT or BHLHE40(S383A) (Figure 10D). The Bhlhe40 knockout had no effect on levels of mRNAs encoding LXRα (Figure 10F) or C/EBPβ (Figure 10H), and the viruses encoding BHLHE40 did not alter these levels significantly. A small decrease in LXRα expression (28%) was seen in WT mice receiving AAV-BHLHE40 (Figure 10E), the functional significance of which is not known.

Figure 10

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The current data fit one more piece into the puzzle of insulin regulation of SREBP-1c mRNA in liver. Solving this puzzle is important clinically because insulin stimulation of SREBP-1c is postulated to underly the fatty liver that occurs in obese people who have high plasma insulin levels (Brown and Goldstein, 2008; Moon et al., 2012). Previously we showed that two transcription factors are required for insulin induction of SREBP-1c, namely, LXRα and C/EBPβ (Chen et al., 2004; Tian et al., 2016). Now we add a third factor: BHLHE40, a basic-helix-loop-helix transcription factor that has been studied in many organs and has received multiple names as described in the Introduction.

Our current data show that the level of BHLHE40 mRNA in liver increases rapidly when fasted rats are refed (Figure 7A). In freshly isolated rat hepatocytes, insulin increased the amount of BHLHE40 within 30 min (Figure 7F). In both cases, the increase in BHLHE40 mRNA preceded the increase in SREBP-1c mRNA. BHLHE40 is essential for the subsequent insulin induction of SREBP-1c mRNA as revealed by experiments in which siRNAs were used to decrease the amount of BHLHE40 in fresh hepatocytes (Figure 8). Similarly, the refeeding-induced elevation of SREBP-1c mRNA was severely blunted in livers of Bhlhe40 knockout mice, and this was reversed when the mice were injected with an adeno-associated virus encoding BHLHE40 (Figure 10).

One reason for the relatively slow increase in SREBP-1c mRNA upon refeeding (Figure 7B and C) may lie in the necessity to destroy an inhibitor of SREBP-1c transcription. In this regard, Takeuchi et al. (2016) reported that KLF15, a ubiquitous transcription factor, forms a complex with LXR that is bound to the SREBP-1c promoter. KLF15 recruits RIP140, a transcriptional co-repressor. The KLF15 gene is activated by cyclic AMP, which is high in fasted liver, owing to the action of glucagon (Teshigawara et al., 2005). Upon refeeding, production of KLF15 mRNA is repressed, and KLF15 protein levels decline in concert with the increase in SREBP-1c mRNA (Takeuchi et al., 2016). Our laboratory showed previously that glucagon blocks the induction of SREBP-1c mRNA by insulin (Shimomura et al., 2000). The data in the current paper raise the possibility that glucagon increases KLF15 protein in livers of fasted rodents and that BHLHE40 cannot activate the SREBP-1c gene until KLF15 has been destroyed.

We identified BHLHE40 in a screen for phosphorylated proteins that were induced in rat liver by refeeding and were precipitated by antibodies against both C/EBPβ and LXRα. Mass spectrometry showed that BHLHE40 is phosphorylated on serine 383 (Figure 1). Experiments with fresh hepatocytes demonstrated that the increase in BHLHE40 is mediated by insulin, and it is required for insulin induction of SREBP-1c mRNA. However, the increase in BHLHE40 is not sufficient to induce SREBP-1c mRNA. Indeed, treatment of fresh hepatocytes with a lentivirus encoding BHLHE40 did not increase the amount of SREBP-1c mRNA in the absence of insulin (Figure 6B).

It was attractive to consider that insulin also increases phosphorylation of BHLHE40, and phosphorylation is required to increase transcription of the SREBP-1c gene. However, this is not the case as indicated by the finding that insulin responsiveness in BHLHE40-deficient mouse liver was restored by an adeno-associated virus encoding a mutant form of BHLHE40 in which serine 383 was changed to alanine (Figure 10). Thus, insulin-mediated induction of SREBP-1c transcription must require yet an additional factor that is activated by insulin. Further studies to identify this missing factor are under way.

All data generated or analysed during this study are included in the manuscript.

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Author details

1. Jing Tian

   Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Designed research, Performed Research, Wrote the paper, Analyzed the data, Approved the final version

   Competing interests

   No competing interests declared

2. Jiaxi Wu

   Department of Molecular Biology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Designed, Performed and analyzed data for LC-MS/MS analysis, Approved final version of paper

   Competing interests

   No competing interests declared

3. Xiang Chen

   Department of Molecular Biology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Designed, Performed and analyzed data for LC-MS/MS analysis, Approved final version of paper

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6608-3211

4. Tong Guo

   Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Performed research, Approved final draft of paper

   Competing interests

   No competing interests declared

5. Zhijian J Chen

   Department of Molecular Biology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Designed and analyzed LC-MS/MS analysis, Assisted in preparing manuscript, Approved final version of paper

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8475-8251

6. Joseph L Goldstein

   Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Designed experiments, Analyzed data, Wrote the paper, Supervised the research

   For correspondence

   joe.goldstein@utsouthwestern.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1894-9463

7. Michael S Brown

   Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Designed experiments, Analyzed data, Wrote the paper, Supervised the research

   For correspondence

   mike.brown@utsouthwestern.edu

   Competing interests

   No competing interests declared

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