Phosphorylation of Insig-2 mediates inhibition of fatty acid synthesis by polyunsaturated fatty acids

Significance

Fatty acid synthesis consumes energy. To prevent energy wastage, animals limit fatty acid synthesis by controlling the activity of sterol regulatory element-binding protein-1 (SREBP-1), a transcription factor that activates genes necessary to produce fatty acids. SREBP-1 is synthesized in the endoplasmic reticulum and processed proteolytically to generate the active nuclear fragment. Processing is inhibited by two Insig proteins, Insig-1 and Insig-2. This paper demonstrates that Insig-2 plays a specific role in limiting fatty acid synthesis. Addition of eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, to cultured cells leads to phosphorylation of Insig-2 but not Insig-1, causing it to block the processing of SREBP-1. This feedback prevents energy wastage by limiting fatty acid synthesis when a cell’s requirement for polyunsaturated fatty acids is satisfied.

Abstract

Insig-1 and Insig-2 are endoplasmic reticulum (ER) proteins that inhibit lipid synthesis by blocking transport of sterol regulatory element-binding proteins (SREBP-1 and SREBP-2) from ER to Golgi. In the Golgi, SREBPs are processed proteolytically to release their transcription-activating domains, which enhance the synthesis of fatty acids, triglycerides, and cholesterol. Heretofore, the two Insigs have redundant functions, and there is no rationale for two isoforms. The current data identify a specific function for Insig-2. We show that eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, inhibits fatty acid synthesis in human fibroblasts and rat hepatocytes by activating adenylate cyclase, which induces protein kinase A (PKA) to phosphorylate serine-106 in Insig-2. Phosphorylated Insig-2 inhibits the proteolytic processing of SREBP-1, thereby blocking fatty acid synthesis. Phosphorylated Insig-2 does not block the processing of SREBP-2, which activates cholesterol synthesis. Insig-1 lacks serine-106 and is not phosphorylated at this site. EPA inhibition of SREBP-1 processing was reduced by the replacement of serine-106 in Insig-2 with alanine or by treatment with KT5720, a PKA inhibitor. Inhibition did not occur in mutant human fibroblasts that possess Insig-1 but lack Insig-2. These data provide an Insig-2-specific mechanism for the long-known inhibition of fatty acid synthesis by polyunsaturated fatty acids.

Long-chain polyunsaturated fatty acids (PUFAs) have long been known to inhibit fatty acid synthesis in liver and in cultured cells (1). As a result, fish oils that contain PUFAs are used clinically to reduce plasma triglycerides. Many mechanisms of inhibition have been proposed, most of which implicate a reduction in the nuclear content of sterol regulatory element-binding protein-1 (SREBP-1), a transcription factor that activates all of the genes necessary to synthesize fatty acids (2, 3). More than 20 y ago, Hannah et al. (4) showed that incubation of cultured fibroblasts with PUFAs inhibits SREBP-1 activity by blocking its processing from its precursor state to its active nuclear state. In vivo, PUFA inhibition does not require exogenous PUFAs. Endogenously synthesized PUFAs exhibit a tonic negative inhibition of SREBP-1 activity in liver, as indicated by an increase in SREBP-1 activity in livers of mice that lack an elongase required for endogenous PUFA synthesis (5).

Animal cells produce three isoforms of SREBP (6). Two of these (SREBP-1c and SREBP-1a) are transcribed from a single gene using different promoters and first exons. Their sequences differ only in a short stretch of acidic amino acids at the amino terminal end. SREBP-1a is expressed primarily in growing cells where it activates genes for cholesterol and fatty acid synthesis (6). SREBP-1c is expressed primarily in liver, where it controls fatty acid synthesis. Available antibodies do not distinguish between SREBP-1a and SREBP-1c. These antibodies measure total levels of SREBP-1. The third isoform, SREBP-2, is found in liver and in growing cells where it primarily controls cholesterol synthesis.

All three SREBPs are synthesized as transmembrane precursors in the endoplasmic reticulum (ER). They bind to Scap, a membrane-bound protein that escorts them into coat complex proteins II (COPII)-coated vesicles that move to the Golgi (7). There, the SREBPs are processed by two proteases that release the transcription-activating beta-HLH Zip domains so that they can enter the nucleus. The ER location of SREBP precursors allows proteolytic processing to be inhibited by cholesterol. When the amount of cholesterol in ER membranes exceeds a threshold, the cholesterol binds to Scap, and this induces the Scap/SREBP complex to bind to ER retention proteins called Insigs (7). The SREBP/Scap/Insig complex can no longer bind to COPII proteins. As a result, the SREBP cannot reach the Golgi for processing, transcription of the SREBP target genes declines, and lipid synthesis falls.

Two isoforms of Insigs exist, Insig-1 and 2 (8). The human proteins are 59% identical and both block ER-to-Golgi movement of Scap/SREBP. Cultured fibroblasts express both forms of Insig. So far as is known, Insig-1 and Insig-2 have identical functions in inhibiting transport of all three SREBP isoforms (7) Thus, there is no rationale to explain the need for two different isoforms of Insig. In the present study, we provide an answer. Our data demonstrate that Insig-2, but not Insig-1, blocks the processing of SREBP-1, but not SREBP-2, when cultured cells are exposed to PUFAs. The PUFA effect is mediated through activation of adenylate cyclase, which activates protein kinase A to phosphorylate a specific serine residue that is present in Insig-2, but not in Insig-1. We conclude that Insig-2 has the unique role of blocking activation of SREBP-1 and inhibiting fatty acid synthesis when cellular levels of cyclic AMP (cAMP) rise.

Results

As discussed in the Introduction, fibroblasts express predominantly the SREBP-1a isoform, but they also express some SREBP-1c (6). In the experiments described below, we measured total SREBP-1 levels, which include both the 1a and 1c isoforms. We use EPA as a classic PUFA and deliver it to cells bound to fat-free bovine serum albumin (Materials and Methods). We initially determined the necessary dosage and time for EPA to diminish nuclear SREBP-1 (nSREBP-1) protein in SV-589 cells, an immortalized line of SV40-transformed human fibroblasts (Fig. 1). A noticeable decline in nSREBP-1 was observed at an EPA concentration of 3 μM (lane 3). The level dropped by 85% at 10 µM and became undetectable at 100 µM (Fig. 1A). In contrast, nSREBP-2 exhibited no decline in response to varying concentrations of EPA (Fig. 1B).

Fig. 1.

EPA-mediated reduction of nuclear SREBP-1 (nSREBP-1) in SV-589 cells. SV-589 cells were plated on day 0 as described in SI Appendix, Materials and Methods. On day 2, cells were washed once with PBS and then incubated for 16 h with 2 ml of Medium A and the indicated concentration of EPA (A and B), or with 10 µM EPA for the indicated time (C and D). The amounts of precursor and nuclear forms of SREBP-1 and SREBP-2 were assessed by immunoblotting with anti-SREBP-1 (A–C) and anti-SREBP-2 (B–D) as described in SI Appendix, Materials and Methods. Each lane contains the pooled extracts of cells from triplicate dishes. All experiments were performed twice on different days with similar results. P, precursor; N, nuclear.

In addition to EPA, other PUFAs at a concentration of 10 µM, including arachidonic acid and docosahexaenoic acid, inhibited the formation of nSREBP-1, but not SREBP-2 (SI Appendix, Fig. S1, lanes 5 and 6). Oleic acid, a monounsaturated fatty acid, inhibited SREBP-1 processing by ~40% (lane 3), whereas palmitic acid, a saturated fatty acid, showed no inhibition of SREBP-1 processing (lane 2).

The Fig. 1C shows the time course of the decline in nSREBP-1 in response to 10 μM EPA. The levels of nSREBP-1 protein diminished by 70% at 6 h as determined by LI-COR imaging of the immunoblots and became undetectable at 16 h, while precursor protein levels remained high. In contrast, there was no discernible reduction in nSREBP-2 protein levels even after 16 h (Fig. 1D).

In addition to blocking proteolytic processing, PUFAs have been reported to lower the amount of mRNA encoding SREBP-1 (4, 9). In our experiments, the SREBP-1 mRNA declined by only about 20% when fibroblasts were treated for 16 h with 10 μM EPA (SI Appendix, Fig. S2). At this concentration, there was no reduction in the SREBP-1 precursor but a profound reduction in the nuclear form (Fig. 1 A and C). Considered together with previous data (4), the current data indicate that the EPA-mediated reduction in nSREBP-1 is caused primarily by a block in proteolytic processing rather than a decrease in the synthesis of the precursor.

Previous studies demonstrated that EPA (10) and arachidonic acid (11) induce cAMP production and enhance PKA activity in rat ventricular myocytes (10) and 3T3-L1 cells (11). Fig. 2 A and B demonstrate that the level of cAMP and the activity of PKA increased in response to 10 μM EPA in SV-589 cells. cAMP content and PKA activity increased by 4-fold and 3-fold, respectively, at 1-hr post-EPA treatment, and increased by 58-fold and 7-fold at 6 h.

Fig. 2.

EPA elevates cAMP, activates PKA, and reduces nSREBP-1 in SV-589 cells. (A and B) Cells were plated on day 0 as described in Fig. 1. On day 2, the cells were washed once with PBS and incubated for the indicated time with Medium A in the absence or presence of 10 µM EPA, after which cells were harvested for measurement of cAMP levels (A) and PKA activity (B) as described in SI Appendix, Materials and Methods. Each bar represents the mean value of three dishes, with individual values shown as dots. (C and D) Effect of 8-Bromo-cAMP on precursor and nuclear forms of SREBP-1 and SREBP-2. On day 0, SV-589 cells were plated as described in SI Appendix, Materials and Methods. On day 2, the cells were washed once with PBS and incubated for 4 h with 2 ml of Medium A and the indicated concentration of 8-Bromo-cAMP (C) or with 300 μM 8-Bromo-cAMP for the indicated time (D). The amounts of precursor and nSREBP-1 or -2 were assessed by immunoblotting with anti-SREBP-1 and anti-SREBP-2 as indicated. (E) PKA inhibitor KT5720 abolishes EPA-mediated reduction in nSREBP-1. On day 0, SV-589 cells were plated as described in SI Appendix, Materials and Methods. On day 2, the cells were washed once with PBS and incubated for 30 min with Medium A with or without 1 µM KT5720, after which the cells received a direct addition of 2 μl of an albumin solution containing sufficient EPA to achieve a final concentration of 10 µM. The cells were then incubated for 16 h. Precursor and nSREBP-1 were measured by immunoblotting with anti-SREBP-1. (F) Proteins in panel E were quantified with the LI-COR Odyssey Infrared Imaging System; the bars represent the ratio of nSREBP-1 to its precursor. Experiments in panels A–E were each done twice on different days with similar results. P, precursor; N, nuclear.

Incubation of cells with 8-Bromo-cAMP, a cell-permeable cAMP analog, reduced the nuclear content of SREBP-1, but not SREBP-2 (Fig. 2 C and D). 8-Bromo-cAMP did not reduce the amount of mRNA for either SREBP-1 or SREBP-2 (SI Appendix, Figs. S3 A and B).

To confirm the requirement for PKA in the EPA-mediated decrease of nSREBP-1, we employed a PKA inhibitor, KT5720 (12) (Fig. 2 E and F). KT5720 prevented the reduction in nSREBP-1 in cells treated with 8-Bromo-cAMP (SI Appendix, Fig. S4). KT5720 also prevented the reduction in nSREBP-1 in cells treated with EPA (Fig. 2 E and F). In the absence of KT5720, EPA reduced the ratio of nuclear to precursor SREBP-1 by 90% as determined by LI-COR scans of the immunoblots (Fig. 2E, lanes 4 and 6 and Fig. 2F). This decrease was prevented by KT5720 (Fig. 2E, lanes 10 and 12, and Fig. 2F).

To further probe the requirements for EPA reduction of nSREBP-1, we measured the effect of EPA in mutant SV-589 cells lacking SCAP, Insig-1, and/ or Insig-2 (Fig. 3A). Triplicate dishes of SV-589 cells from each genotype were incubated with or without 10 μM EPA for 16 h. Extracts from the triplicate dishes were pooled and aliquots were subjected to SDS-PAGE and immunoblotted for precursor and nSREBP-1. The ratio of nuclear SREBP-1 to its precursor (N/P ratio) was determined by LI-COR scanning of the immunoblots. In wild-type (WT) cells, EPA reduced the N/P ratio from 0.8 to 0.3, a fall of 63% (lanes 1 and 2). As expected, there was no detectable nSREBP-1 in cells lacking SCAP (lanes 3 and 4). In cells lacking Insig-1, EPA retained the ability to reduce nSREBP-1; the N/P ratio decreased 67% from 0.6 to 0.2 (lanes 5 and 6). In contrast, EPA failed to lower nSREBP-1 significantly in cells lacking Insig-2 (lanes 7 and 8); the N/P ratio was 0.8 and 0.7 in the absence or presence of EPA, respectively. In cells lacking both Insigs, EPA produced a slight reduction in nSREBP-1 (lanes 9 and 10); the N/P ratio fell from 1.2 to 0.9. This 25% decrease was much less than the 63% decrease in the WT cells. These data indicate that the profound EPA-mediated reduction in SREBP processing requires Insig-2, but not Insig-1.

Fig. 3.

EPA-mediated decrease in nSREBP-1 by PKA-mediated phosphorylation of serine-106 of Insig-2. (A) Inhibition of SREBP-1 processing requires Insig-2. WT and the indicated mutant SV-589 cells were plated on day 0 in Medium A. On day 2, the cells were washed once with PBS and incubated for 16 h with Medium A containing 10 μM EPA, after which cell extracts were prepared and immunoblotted with anti-SREBP-1. Each lane contains pooled lysates from three dishes. The N/P ratio, determined by LI-COR imaging, indicates the ratio between nuclear and precursor SREBP-1. (B) Amino acid sequences of four potential PKA phosphorylation sites in human Insig-2 compared with human Insig-1. Potential phosphorylated residues are depicted in boxes and indicated by arrows. (C) Conservation across vertebrate species of the PKA phosphorylation site at serine-106 in Insig-2, and replacement with alanine at the corresponding site in Insig-1. (D and E) 8-Bromo-cAMP and EPA induce phosphorylation of Insig-2 at S106. On day 2 after plating, SV-589 cells lacking Insig-1 were washed once with PBS and refed with Medium A in the absence or presence of either 100 μM 8-Bromo-cAMP for 4 h (D) or the indicated concentration of EPA for 8 h (E). Cells were lysed and membranes were isolated as described in SI Appendix, Materials and Methods. Insig-2 was immunoprecipitated with mouse monoclonal anti-rat Insig-2 covalently attached to a resin (SI Appendix, Materials and Methods), after which the precipitated proteins were released by treatment with SDS and subjected to 4 to 12% SDS-PAGE. The gels were immunoblotted with affinity-purified rabbit anti-human phosphorylated Insig-2 peptide(100-111) or with rabbit monoclonal anti-rat Insig-2, and visualized with the LI-COR imaging system. (F) EPA-mediated inhibition of SREBP-1 processing requires phosphorylation of serine-106 of Insig-2. On day 0, Insig-1^−/−; Insig-2^−/− SV-589 cells were plated. On day 1, they were transfected with plasmids encoding the indicated WT or mutant Insig-2 as described in SI Appendix, Materials and Methods. Forty-eight hours after transfection, the cells were washed once with PBS and incubated for 16 h with Medium A with or without 10 μM EPA, after which the cells were harvested and whole cell lysates were prepared for immunoblotting with anti-SREBP-1, anti-SREBP2, anti-rat Insig-2 (IgG-4H2), or anti-GAPDH, as indicated. Each lane contains pooled lysates from triplicate dishes. (G) Levels of mRNAs produced by SREBP-1 target genes. Insig-1^−/−; Insig-2^−/− SV-589 cells were plated and subjected to transfection with WT Insig-2 or the S106A mutant as described for panel F. After 48 h, the cells were incubated for 16 h in Medium A with or without 10 μM EPA. The cells were harvested, and mRNA amounts were measured by quantitative RT-PCR as described in SI Appendix, Materials and Methods. Each value represents the relative amount of mRNA compared to cells receiving no EPA and expressing WT Insig-2. The P value was calculated by Student’s t test. The experiment depicted in panel A was performed three times, experiments in panel D–G were each performed twice with similar results. SCD-1, stearoyl CoA desaturase-1; ACC-1, acetyl CoA carboxylase-1; LDLR, LDL receptor; FAS, fatty acid synthase. P, precursor; N, nuclear.

The simplest explanation for the aforementioned data would be that EPA activates PKA and PKA phosphorylates a serine or threonine that is present in Insig-2, but not in Insig-1. Accordingly, we searched for a consensus PKA site in human Insig-2. PKA sites consist of a Ser/Thr residue with arginine at the −3 or −2 positions (either RXXS/T or RXS/T) (13). We identified four potential PKA phosphorylation sites in Insig-2: threonine-56 (T56), serine-106 (S106), serine-107 (S107), and serine-153 (S153) (Fig. 3B). As shown in Fig. 3B, S106 is exclusively present in Insig-2. At the corresponding position in human Insig-1 (amino acid 162), this serine is replaced with alanine. Conversely, T56, S107, and S153 are found in both Insig-1 and Insig-2. S106 in Insig-2 and A162 in the corresponding position in Insig-1 are totally conserved across vertebrate species, from humans to zebrafish (Fig. 3C). A previously published cryogenic electron microscopy study of Insig-2 (14) indicates that S106 is located at the cytosolic surface of the ER membrane, making it well-positioned for phosphorylation by a cytosolic kinase such as PKA.

To demonstrate directly that 8-Bromo-cAMP and EPA induce phosphorylation of Insig-2 at S106, we commissioned the preparation of an affinity-purified polyclonal rabbit antibody directed against a synthetic peptide corresponding to amino acids 100 to 111 of human Insig-2 in which S106 was phosphorylated. We treated SV-589 cells lacking Insig-1 with 8-Bromo-cAMP or EPA, immunoprecipitated with mouse monoclonal anti-rat Insig-2, and then blotted with rabbit monoclonal anti-rat Insig-2 (Fig. 3 D and E). The blot in Fig. 3D revealed that equal amounts of Insig-2 were precipitated in the absence and presence of 8-Bromo-cAMP (Lower panel). In cells treated with 8-Bromo-cAMP, the anti-phospho-Insig-2 stained the immunoprecipitated Insig-2 (Upper panel). In contrast, no staining was seen in the absence of 8-Bromo-cAMP treatment. Fig. 3E demonstrates that the level of phosphorylated Insig-2 in the SV-589 cells increased with increasing concentrations of EPA. EPA induced the phosphorylation of Insig-2 at serine-106 by 3.5-, 9.6-, and 12.5-fold at concentrations of 1, 3, and 10 μM, respectively, as determined by LI-COR imaging (Upper panel). In contrast, total Insig-2 levels remained unchanged (Lower panel).

In response to the reviewers’ suggestion, we performed a further test of the specificity of the polyclonal rabbit anti-phospho Insig-2 antibody (SI Appendix, Fig. S5). We used Insig-1/Insig-2-deficient SV589 cells that were transfected with cDNAs encoding WT Insig-2 or the S106A mutant. The cells were incubated in the absence or presence of 8-Bromo-cAMP after which they were lysed, and Insig-2 was immunoprecipitated. The immunoprecipitates were subjected to SDS-PAGE and blotted with the anti-phospho Insig-2 antibody as described in Materials and Methods. In cells expressing WT Insig-2, the antibody stained the immunoprecipitated Insig-2, but only after the cells were treated with 8-Bromo-cAMP (Upper panel, lanes 1 and 2). In contrast, the anti-phospho Insig-2 antibody failed to stain the S106A mutant Insig-2 even after 8-Bromo-cAMP treatment (lanes 3 and 4). Equal amounts of Insig-2 were present in all immunoprecipitates as revealed by staining of the gels with rabbit monoclonal anti-rat Insig-2 (Lower panel, lanes 1-4). This experiment confirms the specificity of the anti-phospho peptide antibody for detecting phosphorylated Insig-2 at S106.

To further test the role of S106 phosphorylation, we generated plasmids encoding Insig-2 in which serine-106 was replaced with alanine (S106A). We transfected WT and mutant plasmids into an SV-589 cell line that lacks all Insigs (Fig. 3F). After 48 h, the transfected cells were incubated for 16 h in the presence or absence of 10 μM EPA. Cell extracts from triplicate dishes were pooled, subjected to SDS-PAGE, and blotted with monoclonal rabbit anti-mouse SREBP-1 (Fig. 3F). In cells lacking all Insigs, EPA produced a minimal reduction in nSREBP-1. The N/P ratio was reduced by 9% from 1.1 to 1.0 as determined by LI-COR imaging (lanes 1 and 2, Upper panel). In contrast, EPA reduced the ratio by 72% (from 1.8 to 0.5) when the cells were transfected with a plasmid encoding WT Insig-2 (lanes 3 and 4). The inhibition was diminished when the transfected Insig-2 contained the S106A mutation (lanes 5 and 6). The N/P ratio was reduced by 20% from 2.0 to 1.6. These data suggest that phosphorylation of S106 is necessary for maximal inhibition of SREBP-1 processing.

To validate the functional significance of Insig-2(S106A) phosphorylation, we measured the levels of mRNA encoded by SREBP-1 target genes in EPA-treated Insig-1 and Insig-2 double knockout cells expressing WT or mutant Insig-2 proteins. As shown in Fig. 3G, EPA reduced SCD-1, ACC-1, LDLR, and FAS mRNA levels in cells expressing WT Insig-2, whereas there was no reduction in cells expressing the S106A mutant. As a result of these reductions, EPA led to a decrease in fatty acid synthesis in SV-589 cells, as determined by incubation with ¹⁴C-labeled acetate (SI Appendix, Fig. S6).

The liver serves as the primary site for the synthesis of fatty acids and plasma triglycerides (2, 3). Hence, we conducted experiments to determine whether our findings regarding EPA-mediated inhibition of SREBP-1 processing in human SV-589 cells could be extrapolated to rat liver cells. We examined SREBP-1c processing in primary cultures of hepatocytes obtained from transgenic rats expressing HA-tagged human SREBP-1c driven by a constitutive human apoE promoter/enhancer (15). This allowed us to investigate the effect of EPA on SREBP-1 processing in liver cells without the complication of transcriptional inhibition.

Exposure of freshly isolated transgenic hepatocytes to 10 μM EPA for 16 h led to an 80% reduction in nSREBP-1 protein levels, as determined by immunoblotting and LI-COR quantification (Fig. 4A). Treatment with the PKA activator, 8-Bromo-cAMP for 4 h, produced a similar decrease (Fig. 4B). The amount of full-length SREBP-1 precursor remained unchanged in response to either EPA or 8-Bromo-cAMP.

Fig. 4.

EPA and 8-Bromo-cAMP inhibit SREBP-1 processing in primary hepatocytes from transgenic rats expressing HA-tagged human SREBP-1. (A and B) Hepatocytes from transgenic HA-hSREBP-1 rats were prepared and plated on day 0 as described in SI Appendix, Materials and Methods. On day 1, the cells were incubated as follows: (A) with or without 10 μM EPA for 16 h; and (B) with or without 100 or 300 μM 8-Bromo-cAMP for 4 h. Cells were then harvested for immunoblotting with anti-SREBP-1 and anti-GAPDH as indicated. Experiments were performed twice with similar results. P, precursor; N, nuclear.

Discussion

The data in this paper uncover a unique mechanism by which polyunsaturated fatty acids (PUFAs) inhibit the processing of SREBP-1, thereby inhibiting the transcription of genes required for the synthesis of fatty acids and triglycerides. The experiments demonstrate that EPA, a PUFA, increases the cellular content of cAMP which activates PKA, which in turn phosphorylates serine-106 in Insig-2. Phosphorylated Insig-2 inhibits proteolytic processing of SREBP-1, thereby lowering the nuclear content of the active form of SREBP-1 and reducing transcription of lipogenic genes (7).

The SREBP pathway contains two pairs of closely related proteins with partially overlapping functions. The two SREBPs constitute one of these pairs. SREBP-1 preferentially activates fatty acid biosynthesis, whereas SREBP-2 activates cholesterol synthesis (2, 3). As shown in Fig. 1, EPA reduces the processing of SREBP-1, but not SREBP-2, thereby selectively reducing fatty acid biosynthesis. The other related pair is the Insigs. Both Insigs bind the Scap/SREBP complex and prevent the movement of SREBPs to the Golgi for proteolytic processing. EPA acts solely by activating Insig-2. As shown in Fig. 3A, EPA inhibits SREBP-1 processing profoundly in cells that express Insig-2 but only minimally in cells that express only Insig-1 (Fig. 3A Upper panel). Serine-106 in Insig-2 is essential for this inhibition (Fig. 3F). Serine-106 is located in a consensus site for PKA phosphorylation (Fig. 3B), and its replacement with alanine markedly reduced EPA-mediated inhibition of SREBP-1 processing (Fig. 3F). Immunoblotting with a polyclonal antibody directed against the Insig-2 peptides (amino acids 100-111) containing phosphor S106 demonstrates directly that EPA induces phosphorylation of this serine (Fig. 3E). Importantly, serine-106 is conserved in Insig-2 in all vertebrate species through zebrafish, but it is replaced by alanine in the equivalent position in the Insig-1 protein in all of these species (Fig. 3C). This universal replacement occurs even though the surrounding amino acid sequences are identical and highly conserved between the two Insig isoforms (Fig. 3C).

The role of PKA in Insig phosphorylation is supported by the experiments of Fig. 2. These experiments show that EPA increases the cellular content of cAMP (Fig. 2A) and the activity of PKA (Fig. 2B). Administration of 8-Bromo-cAMP, a cell permeable analog of cAMP, blocked SREBP-1 processing (Fig. 2 C and D). Moreover, EPA inhibition was blocked by KT5720, an inhibitor of PKA (Fig. 2E). In addition to their effects on cultured human fibroblasts, EPA and 8-Bromo-cAMP blocked the processing of SREBP-1 in primary cultures of rat hepatocytes (Fig. 4).

Insig-2-dependent inhibition of SREBP-1 processing is not the only mechanism by which PUFAs may lower the nuclear content of SREBP-1. The current paper shows that EPA activates PKA, and PKA has been shown to phosphorylate the liver X receptor (LXR) (16), which is required for the synthesis of SREBP-1c mRNA (17). Moreover, PUFAs also inhibit LXR directly (9). Other studies have implicated additional mechanisms by which PUFAs may reduce the nuclear content of SREBP-1 (3, 18). These mechanisms may account for the slight decline in nSREBP-1 that we observed when EPA was administered to mutant SV-589 cells that lack both Insig isoforms (Fig. 3F, lanes 1 and 2). These alternative mechanisms do not produce the profound reduction in nSREBP-1 that is produced by EPA in SV-589 cells that express WT Insig-2 (Fig. 3F, lanes 3 and 4).

The current results raise a fundamental question regarding the mechanism that allows phosphorylated Insig-2 to selectively block the processing of SREBP-1 but not SREBP-2. Previous biochemical and structural data indicate that Insig proteins block SREBP transport by binding to the Scap component of the Scap/SREBP complex (14, 19–21). There is no prior evidence that the conformation of Scap may be different when it binds to SREBP-1 as opposed to SREBP-2. How, then, does phosphorylated Insig-2 recognize the Scap/SREBP-1 complex but not the Scap/SREBP-2 complex? Further experiments are being conducted to answer this question.

Materials and Methods

SI Appendix includes descriptions of the following items: Key Resources Table, SV-589 Cells, Primary Rat Hepatocytes, Generation of Mutant SV-589 Cells, SREBP-1 and SREBP-2 Processing, Assays for cAMP and PKA, Insig-2 Phosphorylation, Generation of Insig-2(S106A) Mutant Plasmid and Transfection, Fatty Acid Synthesis, Immunoblotting, and Quantitative Real-Time PCR.

Preparation of Solutions.

Stock solutions containing 10 mM fatty acids bound to 1.5 mM fatty acid-free bovine serum albumin were prepared and stored under liquid nitrogen exactly as described previously (4). A 0.1 M stock solution of 8-Bromo-cyclic AMP was prepared in DMSO. A 1 mM stock solution of KT5720 was prepared in DMSO. All solutions were aliquoted into multiple tubes and stored at -20 °C.

For experiments, SV-589 cells and rat hepatocytes were cultured in 2 ml of medium (described below) to which was added 2 to 20 μl of the appropriate solution of albumin or DMSO with or without the indicated concentration of fatty acid, KT5720, or 8-Bromo-cAMP as described in the figure legends.

Antibodies.

Mouse monoclonal anti-rat Insig-2 (IgG-4H2) was produced by immunizing NZBWF1/J mice with purified recombinant His-tagged rat Insig-2 (full length) combined with the Sigma Adjuvant System. Hybridoma cells were prepared by fusion of splenic B lymphocytes isolated from immunized mice with SP2-mIL6 mouse myeloma cells. We screened hybridoma culture supernatants by ELISA and immunoblotting. A positive hybridoma (designated IgG-4H2) was purified through two rounds of serial dilution, and its antibody was purified by gravity-flow affinity chromatography on protein G Sepharose 4 Fast Flow columns.

Rabbit monoclonal anti-rat Insig-2 antibody (IgG-40D3) was generated by immunizing New Zealand White (NZW) rabbits with purified His-tagged rat Insig-2 (full length) combined with Incomplete Freund’s Adjuvant. Hybridoma cells were produced by fusion of splenic B lymphocytes isolated from immunized rabbits with 240E-1 rabbit myeloma cells (22). Screening, subcloning, and purification were carried out as described above.

A rabbit affinity-purified anti-phospho peptide antibody was prepared by Pacific Immunology Corp. by immunizing rabbits with a peptide corresponding to amino acids 100-111 of human Insig-2 in which serine-106 was phosphorylated. The sequence was KFKREW-pS-SVMRC.

All other antibodies were described previously or were purchased commercially as listed in the SI Appendix, Key Resources Table.

Reproducibility.

All experiments were repeated on multiple occasions on different days as indicated in the figure legends. Similar results were obtained with all replications.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.