Zinc mediates the SREBP-SCD axis to regulate lipid metabolism in Caenorhabditis elegans[S]

Maintenance of lipid homeostasis is crucial for cells in response to lipid requirements or surplus. The SREBP transcription factors play essential roles in regulating lipid metabolism and are associated with many metabolic diseases. However, SREBP regulation of lipid metabolism is still not completely understood. Here, we showed that reduction of SBP-1, the only homolog of SREBPs in Caenorhabditis elegans, surprisingly led to a high level of zinc. On the contrary, zinc reduction by mutation of sur-7, encoding a member of the cation diffusion facilitator (CDF) family, restored the fat accumulation and fatty acid profile of the sbp-1(ep79) mutant. Zinc reduction resulted in iron overload, which thereby directly activated the conversion activity of stearoyl-CoA desaturase (SCD), a main target of SREBP, to promote lipid biosynthesis and accumulation. However, zinc reduction reversely repressed SBP-1 nuclear translocation and further downregulated the transcription expression of SCD for compensation. Collectively, we revealed zinc-mediated regulation of the SREBP-SCD axis in lipid metabolism, distinct from the negative regulation of SREBP-1 or SREBP-2 by phosphatidylcholine or cholesterol, respectively, thereby providing novel insights into the regulation of lipid homeostasis.

In spite of the above established regulatory circuits of SREBP-2 by cholesterol (6), SREBP-1 by PC (7), and Drosophila (d)SREBP by phosphatidylethanolamine (27), it is still arguable that other unknown factors may participate in the SREBPs' regulation of lipid metabolism. The activities and functions of SREBPs in lipid metabolism are highly evolutionarily conserved across metazoans. The model organism, Caenorhabditis elegans, contains only one SREBP family member encoded by sbp-1. Similar to its mammalian homologs, SBP-1 also regulates fatty acid (28)(29)(30)(31), PC (7), and cholesterol metabolism (32,33). Depletion of sbp-1 by RNA interference (RNAi) knockdown (29,32,34) or deletion (28) leads to altered fatty acid profiles and lowered fat stores. Through a forward genetic screen of suppressors of the sbp-1(ep79) mutant, we identified that mutations in sur-7, encoding a member of the cation diffusion facilitator (CDF) family, could restore the altered lipid profiles of the sbp-1(ep79) mutant. Furthermore, we revealed a distinct zinc-mediated SREBP-stearoyl-CoA desaturase (SCD) regulatory circuit to maintain lipid homeostasis.

Strains and culture conditions
C. elegans strains were maintained on NGM plates with Escherichia coli OP50 under standard culture conditions, unless otherwise specified. The WT strain was N2. CB4856 is a WT isolated from Hawaii used for SNP mapping. The organisms and strains used in this study are shown in supplemental Table S1.

RNAi
RNAi was performed by feeding bacterial strains from the Ahringer C. elegans RNAi library, as we described previously (35). Young adult animals were harvested for further analysis.

Genome screening and isolation of sbp-1 suppressors
Genome screening of sbp-1(ep79) suppressors was carried out following a previously described method (36,37). Briefly, L4 sbp-1(ep79) worms were treated with ethyl methanesulfonate. Individual F2 worms that displayed morphologies of growth, body size, and color obviously close to the WT N2 were singled out and continuously cultured to the F3 generation. Then, F3 worms were harvested for analysis of fat accumulation by Nile red staining of fixed cells and analysis of fatty acid composition by GC, as well as quantification of the growth rate, to confirm the real suppressors of the sbp-1(ep79) mutant.

Nile red staining of fixed worms and quantification of lipid droplet size
Nile red staining of fixed worms was performed as previously described (28,41). The images were captured using identical settings. At least 20 worms were visualized. The quantification of lipid droplet size was as previously described (42). Approximately six to eight worms were measured for each worm strain or treatment.

Analysis of fatty acid composition and triacylglycerol
C. elegans lipid extraction, the separation of triacylglycerol (TAG) and phospholipids by TLC, and the determination of fatty acids by GC were performed as previously described (42). Fatty acid compositions were analyzed with an Agilent 7890A gas chromatograph equipped with a 15 m × 0.25 mm × 0.25 m DB-WAX column.

Analysis of quantitative real-time PCR and semiquantitative PCR
Total RNA and cDNA preparation, quantitative real-time PCR (QPCR), and semi-QPCR were performed as previously described (43). For semi-quantitative PCR, there were 25 PCR cycles for sbp-1. For QPCR, the relative abundance was determined using the Ct method and the reference gene, act-5, was used as a control for template levels.

Supplementation or sequestration of zinc
ZnSO 4 supplementation and zinc reduction by N,N,N′,N′tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) were analyzed as described previously (44,45). In brief, ZnSO 4 and TPEN were added to NGM plates to a final concentration of 50 and 5 M, respectively. Later, synchronized L1 worms were placed and cultivated on NGM plates supplied with either ZnSO 4 or TPEN. L4 worms or young adults were harvested for analysis.

Zinpyr-1 staining and visualization
Zinpyr-1 staining was performed as described previously (46). The fluorescence of Zinpyr-1 was visualized under an OLYMPUS BX53 fluorescence microscope (Olympus, Japan). Images were captured using identical settings and exposure times, unless specifically noted. The fluorescence intensity was quantified using Photoshop software.

Visualization of GFP fluorescence
At least 20 GFP worms were picked and mounted on an agarose pad and anesthetized using 10 mM sodium azide. GFP fluorescence was visualized under an OLYMPUS BX53 fluorescence microscope (Olympus, Japan). GFP reporter expression was quantified using Photoshop software. At least six worms were used for each biological sample.

Statistical analysis
The data were presented as the mean ± SEM, except when specifically indicated. Statistical analyses included the t-test or ANOVA. All figures were generated using GraphPad Prism 6 (GraphPad Software, La Jolla, CA).
The top two mutations, kun82 and kun84, significantly increased lipid droplet size and TAG content, restored the fatty acid profiles, and partially rescued the growth rate in the sbp-1(ep79) mutant background (Fig. 1). Therefore, they were chosen to identify the mutation in a specific gene by classical mapping of snip-SNP (38,39) and WGS (40). Eventually, kun82 was mapped to chromosome I and identified as a missense mutation in smg-2 (GGT to GAT, G805D) (supplemental Fig. S4A), while kun84 was mapped to chromosome X and identified as a missense mutation in sur-7 (CCT to CTT, P191L) ( Fig. 2A).
SMG-2 is an ortholog of human UPF1 (UPF1 regulator of nonsense transcripts in yeast) and is required for nonsense-mediated mRNA decay or mRNA surveillance (48,49). Because ep79 is a reduction-of-function allele of sbp-1 (28), we thought that the smg-2(kun82) mutation might affect the mRNA expression of sbp-1. Indeed, QPCR and semi-QPCR showed that the mRNA expression level of sbp-1 was obviously increased to the WT level in sbp-1(ep79);smg-2(kun82) mutants, but not in sbp-1(ep79); sur-7(kun84) mutants, compared with the sbp-1(ep79) single mutant alone (supplemental Fig. S4B, C). Therefore, increasing or stabilizing the expression of truncated sbp-1 by mutation of smg-2 may be able to restore its biological functions.

Inactivation of SUR-7 restores lipid profiles of sbp-1(ep79) mutant
The sur-7 encodes a member of the CDF family (50,51). At least two additional alleles, tm6523 and ku119, of sur-7 are available from Wormbase (http://www.wormbase.org). The tm6523 mutation contains a 566 bp deletion, and ku119 has a substitution mutation (GT-AT) near the 5′splicing site in the third intron that leads to an altered transcript ( Fig. 2A, supplemental Fig. S5). To confirm the suppression of sbp-1(ep79) by sur-7(kun84), we then crossed the tm6523 and ku119 alleles into the sbp-1(ep79) mutant background, respectively. In accordance with the kun84 mutation, both mutant alleles significantly increased lipid droplet size, fat content (TAG), the level of C18:1(n-9), and conversion activity of SCD, but decreased the level of C18:0, as well as improved the growth rates of the sbp-1(ep79) mutant ( Fig. 2B-H). In addition, both sur-7(tm6523) and sur-7(ku119) mutants accumulated slightly, but significantly, higher fat contents than the WT (Fig. 2D), suggesting a slight difference among the three alleles of the sur-7 gene. Because the phenotypes of sur-7(tm6523) and sur-7(ku119) mutants were similar ( Fig. 2B-H), we then used tm6523 and kun84 mutations for the rest of the experiments for convenience. Meanwhile, unlike the kun82 mutation of smg-2 (supplemental Fig. S4B, C), mutations of tm6523 and kun84 did not affect the mRNA expression of sbp-1 (supplemental Fig. S6), implying a distinct mechanism from SMG-2 to suppress the defects of the sbp-1(ep79) mutant. Taken together, these results indicate that disruption of SUR-7 apparently alleviates the defects of the sbp-1(ep79) mutant.

SBP-1 functions to reduce zinc level
SUR-7 was reported to likely localize to the endoplasmic reticulum membrane and play a role in zinc metabolism (46,50), raising the question of whether SBP-1 has a role in zinc homeostasis. MTL-1, one of two C. elegans metallothioneins, was reported to have high zinc binding ability (52). In fact, the mRNA expression of mtl-1 was very responsive to the zinc level, in which it was significantly induced by supplementation with ZnSO 4 or reduced by the zinc chelator, TPEN (Fig. 3A). Interestingly, the expression of mtl-1 was significantly increased in the sbp-1(ep79) mutant, but was reversely inhibited in the sur-7(tm6523) and sur-7(kun84) mutants (Fig. 3B).

Zinc bypasses SBP-1 to negatively regulate fat accumulation
Because the Zinpyr-1 fluorescence was negatively correlated with fat accumulation in both sbp-1 and sur-7 mutants, we hypothesized that zinc might affect fat accumulation. Dietary supplementation with ZnSO 4 had no obvious effect on fat accumulation, indicated by Nile red staining of fixed worms and lipid droplet size in both N2 and sbp-1(ep79) worms (Fig. 3F, G). However, compared with the control, Representative animals, the anterior is on the right and the posterior is on the left. Scale bar represents 10 m. B: Lipid droplet size in the posterior region of intestines from six to seven worms of each worm strain. The data are presented as scatter dot plot and mean. C: Percentage of TAG in total lipids (TAG/TL). The data are presented as the mean ± SEM of three to four biological repeats. D-G: Percentage of saturated fatty acids (SFAs) (C16:0 + C18:0) (D), MUFAs [C16:1(n-7) + C18:1(n-9) + C18:1(n-7)] (E), C18:0 (F), and C18:1(n-9) (G) in total fatty acids. The data are presented as the mean ± SEM of at least four biological repeats. H: The conversion activity of SCD presented as the ratio of C18:1(n-9)/ C18:0. The data are presented as the mean ± SEM of four biological repeats. I: The growth rate of worms. The data are presented as the mean ± SEM of three biological repeats of which at least 300 worms were counted for each worm strain. Statistical analysis was performed using ANOVA. ***P < 0.001, significant difference between sbp-1(ep79) mutant and a specific worm strain. # P < 0.05, ### P < 0.001, significant difference between WT N2 and a specific worm strain. ZnSO 4 treatment apparently reduced the fat accumulation and lipid droplet size in strains with a sur-7 mutant background, including sur-7(tm6523), sur-7(tm6523);sbp-1 (ep79), sur-7(kun84), and sur-7(kun84);sbp-1(ep79) mutants (Fig. 3F, G). On the contrary, reduction of zinc by TPEN treatment remarkably exacerbated the fat accumulation and increased lipid droplet size in all tested strains (Fig. 3F, G). In brief, these data explicitly supported that zinc negatively affected fat accumulation, and the restored fat accumulation of the sbp-1(ep79) mutant was due to the reduction in zinc levels.

Zinc negatively regulates the activity of SCD, a main target of SREBP
SCD introduces the first double bond and converts saturated fatty acids (C16:0 and C18:0) to MUFAs [C16:1(n-7) and C18:1(n-9)], which are the primary substrates for the biosynthesis of TAGs, phospholipids, and cholesterol esters (53). SCD is a main target of SREBP, which transcriptionally regulates SCD expression from C. elegans to mammals (28,53,54). Although the expression of sbp-1 was not affected in sur-7(tm6523);sbp-1(ep79) and sur-7(kun84);sbp-1(ep79) mutants, as in the sbp-1(ep79) mutant alone (supplemental Fig. S6), the level of C18:1(n-9) (Fig. 2F), which is the de novo synthesized fatty acid by SCD in C. elegans (54,55), and the conversion activity of SCD were significantly elevated in sur-7;sbp-1 double mutants compared with the sbp-1(ep79) mutant (Fig. 2G), implying a mechanism independent of SBP-1 transcription to regulate SCD conversion activity. In fact, dietary ZnSO 4 slightly, but significantly, decreased both the level of C18:1(n-9) and the conversion activity of SCD (Fig. 4A, B) in the sur-7 mutant background. In contrast, zinc reduction by TPEN treatment dramatically increased the C18:1(n-9) level and the conversion activity of SCD in all tested strains except the fat-6(tm331);fat-7(wa37) mutants (Fig. 4A, B), which lack SCD enzymes for the conversion of C18:0 to C18:1(n-9) in C. elegans (54). Altogether, these results suggest that the suppression of the sbp-1(ep79) mutant is due to increased conversion activity of SCD and its converted product, C18:1(n-9). Furthermore, dietary supplementation with oleic acid [C18:1(n-9)] improved fat accumulation with increased lipid droplet size and the growth rate in the sbp-1(ep79) mutant (supplemental Fig. S7), which was consistent with a previous report (32). To examine whether the increased level of C18:1(n-9) and SCD conversion activity by TPEN treatment was due to upregulated expression of scds, we investigated the mRNA and GFP expression of fat-5, fat-6, and fat-7, encoding three separate SCDs, in C. elegans (54). Interestingly, the GFP expression of all three SCDs and the mRNA expression of fat-5 and fat-7 were significantly reduced in TPEN-treated worms compared with the control worms, while ZnSO 4 treatment slightly, but significantly, increased the fluorescence intensity of FAT-6::GFP and FAT-7::GFP (Fig. 4C-E). Moreover, TPEN treatment obviously reduced the GFP expression of GFP::SBP-1 (KQ377) in the nucleus compared with the control (Fig. 4F), whereas it did not affect the mRNA expression of sbp-1 (Fig. 4G). Therefore, zinc reduction The kun84 is a missense mutation (P191L) in the fourth exon, the tm6523 mutation has a 566 bp deletion, and ku119 is a substitution mutation (GT→AT) neighboring the splicing site. B: Nile red staining of fixed worms. Representative animals, the anterior is on the right and the posterior is on the left. Scale bar represents 10 m. C: Lipid droplet size in the posterior region of intestines from eight worms of each worm strain. The data are presented as mean ± SEM. D: Percentage of TAG in total lipids (TAG/TL). The data are presented as the mean ± SEM of four biological repeats. E, F: Percentage of C18:0 (E) and C18:1(n-9) (F) in total fatty acids. The data are presented as the mean ± SEM of four biological repeats. G: The conversion activity of SCD presented as the ratio of C18:1(n-9)/ C18:0. The data are presented as the mean ± SEM of four biological repeats. H: The growth rate of worms. The data are presented as the mean ± SEM of three biological repeats of which at least 300 worms were counted for each worm strain. Statistical analysis was performed using ANOVA. **P < 0.01, ***P < 0.001, significant difference between sbp-1(ep79) mutant and a specific worm strain. # P < 0.05, ## P < 0.01, significant difference between WT N2 and a specific worm strain. The data are presented as the mean ± SEM of three to four biological repeats. C: Fluorescence microscopy of live animals stained with Zinpyr-1. Scale bar represents 100 m for the whole animal and 20 m for the enlarged body part, respectively. Images were captured using identical settings and exposure times. D: Quantitation of the fluorescence intensity from (C). The data are presented as the mean ± SEM from three worms of each worm strain. E: Relative mRNA expression of sur-7 by QPCR. The data are presented as the mean ± SEM of four biological repeats. F: Nile red staining of fixed worms. Representative animals, the anterior is on the right and posterior is on the left. Scale bar represents 10 m. G: Lipid droplet size in the posterior region of intestines from eight worms of each worm strain. The data are presented as mean ± SEM. The concentration of TPEN is 5 M and of ZnSO 4 is 50 M. Statistical analysis was performed using the t-test. # P < 0.05, ## P < 0.01, ### P < 0.001, significant difference between WT N2 and a specific condition. $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, significant difference between the control and either TPEN treatment or ZnSO 4 treatment. *P < 0.05, significant difference between a specific worm strain and sbp-1(ep79) mutant. might repress the nuclear translocation of SBP-1 and then downregulate the transcriptional and translational expression of scds. However, these results were contradicted by the increased level of C18:1(n-9) and the conversion activity of SCD (Fig. 4A, B).
In spite of that, RNAi knockdown of fat-6, which had a stronger RNAi effect than fat-7 RNAi to some degree, apparently decreased fat accumulation with decreased lipid droplet size in all test strains (Fig. 4H, J), even under TPEN treatment (Fig. 4I, K). In addition, although the fat-6(tm331);fat-7(wa37) double mutants were similar to the sbp-1(ep79) mutant in terms of fat accumulation, they were partially resistant to TPEN-induced fat accumulation (Fig. 4L, M). Altogether, these results suggest that zinc reduction-induced fat accumulation probably depends on the SCD function, in which its conversion activity was elevated, but its transcriptional and translational expression was repressed inversely.

Zinc antagonizes iron to determine SCD conversion activity and fat accumulation
To explore genes responding to zinc reduction, we performed RNA-seq analysis in worms treated with 2.5 and 5 M TPEN. Transcriptome profiles revealed that the expression of 78 genes was responsive to TPEN in a concentration-dependent manner (Fig. 5A, B; supplemental Table  S2). Consistent with our previous QPCR results (Fig. 4E), the expression of fat-5 and fat-7 was also downregulated in the RNA-seq data (Fig. 5B). Surprisingly, we found that the expression of ftn-1 and smf-3, encoding ferritin and divalent metal-ion transporter 1 (DMT1), respectively, was reversely responsive to zinc concentration. Previous reports, including ours, show that the expression of the ftn-1 gene and protein are induced; in contrast, the expression of smf-3 is suppressed to reduce iron uptake under iron overload (56)(57)(58). QPCR confirmed that the mRNA expression of ftn-1 and smf-3 indeed displayed an opposite pattern in response to zinc concentrations (Fig. 5C, D). Consistently, TPEN treatment dramatically increased the fluorescent expression of FTN-1::GFP, while dietary ZnSO 4 had a negative effect (Fig. 5E). Thus, zinc had an antagonizing role to affect iron homeostasis.
SCDs are iron-containing enzymes that require iron for their activity (53,59). Because zinc reduction increased FTN-1 expression and SCD conversion activity, we hypothesized that the elevated conversion activity of SCD by zinc reduction was probably due to iron overload. The iron chelator, 2,2ʹ-dipyridyl (BP), was used to reduce the iron level (60). Indeed, the level of C18:0 was increased and the conversion activity of SCD was decreased in a BP concentration-dependent manner (Fig. 5F, G). However, TPEN treatment significantly increased the level of C18:1(n-9) and the conversion activity of SCD, even under BP treatment (Fig. 5F, G). Although either TPEN or BP treatment apparently reduced the growth rate of worms, TPEN treatment obviously improved the slow growth rate of worms treated with BP (Fig. 5H). In addition, compared with the control, TPEN treatment led to high fat accumulation with increased lipid droplet size, while BP treatment had the opposite effect in worms (Fig. 5I, J). Moreover, TPEN treatment restored the low level of fat accumulation in Fig. 4. Zinc reduction increases the conversion activity of SCD. A: Percentage of C18:1(n-9) in total fatty acids. The data are presented as the mean ± SEM of three to four biological repeats. B: The conversion activity of SCD presented as the ratio of C18:1(n-9)/C18:0. The data are presented as the mean ± SEM of three to four biological repeats. C: Fluorescence microscopy of FAT-5::GFP, FAT-6::GFP, and FAT-7::GFP. Scale bar represents 100 m. The concentration of TPEN is 5 M and of ZnSO 4 is 200 M. Images were captured using identical settings and exposure times. D: Quantitation of the fluorescence intensity from (C). The data are presented as the mean ± SEM. At least five worms were analyzed for each treatment. E: Relative mRNA expression of fat-5, fat-6, and fat-7 by QPCR. The data are presented as the mean ± SEM of four biological repeats. Statistical analysis was performed using the t-test. F: Fluorescence intensity of GFP::SBP-1 (KQ377) treated by TPEN. The red arrows indicate nuclear. Images were captured by confocal microscopy using identical settings and exposure times. G: Relative mRNA expression of sbp-1 by QPCR. The data are presented as the mean ± SEM of four biological repeats. H, I: Nile red staining of fixed worms. Representative animals, the anterior is on the right and the posterior is on the left. Scale bar represents 10 m. J, K: Lipid droplet size in the posterior region of intestines from four worms of each condition. The data are presented as mean ± SEM. L: Percentage of TAG in total lipids (TAG/TL). The data are presented as the mean ± SEM of four biological repeats. M: The increased percentage of TAG between TPEN treatment and nontreatment in a specific worm strain. The data are presented as the mean ± SEM of four biological repeats. The concentration of TPEN is 5 M and of ZnSO 4 is 50 M. The statistical analysis was performed using ANOVA unless specifically indicated. # P < 0.05, ## P < 0.01, ### P < 0.001, significant difference between WT (N2) and a specific worm strain. **P < 0.01, significant difference between a specific worm strain and sbp-1(ep79) mutant. $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, significant difference between treatment (TPEN or ZnSO 4 ) and nontreatment (Control) in the same worm strain. ns., no significant difference between the two groups. BP-treated worms (Fig. 5I, J). Altogether, these lines of evidence consistently suggest that iron competes with zinc to determine the SCD conversion activity and fat accumulation, and zinc reduction-induced fat accumulation depends on the iron-activated SCD conversion activity.

DISCUSSION
The evolutionarily conserved transcription factors, SREBPs, play central roles to transcriptionally regulate genes involved in fatty acid, triglyceride, and cholesterol metabolism, from C. elegans to mammals. To our great surprise, we found that the sbp-1(ep79) mutant displayed an elevated zinc level, as indicated by Zinpyr-1 fluorescence and mtl-1 expression (Fig. 3A-D). In contrast, zinc reduction repressed the nuclear translocation of GFP::SBP-1 (Fig. 4F), consequently downregulating the transcriptional expression of the scd genes, fat-5, fat-6, and fat-7 ( Fig. 4C-E). Furthermore, zinc was sufficient to directly regulate SCD function and lipid homeostasis, independent of SREBP function (Fig. 6). Thus, we uncovered a distinct zinc-mediated regulation of SREBP-SCD in lipid metabolism, in which SREBP repressed the zinc level to upregulate SCD conversion activity, while, reciprocally, zinc affected the trans activation of SREBP on scd genes (Fig. 6), although the details need to be further characterized.
SCD1 catalysis is dependent on its di-iron center (53,61). Interestingly, the recent crystal structure of mouse SCD1 showed that zinc, instead of iron, was the predominant ion in the structure (62). We found that zinc reduction led to iron overload, as well as increased SCD conversion activity and fat accumulation, consistent with a recent report that zinc deficiency promotes insulin resistance by exacerbating iron overload in the liver and induces hepatic steatosis in patients (63). On the other hand, . Scale bar represents 100 m. F: Percentage of C18:0 and C18:1(n-9) in total fatty acids. The data are presented as the mean ± SEM of three to four biological repeats. G: The conversion activity of SCD presented as the ratio of C18:1(n-9)/C18:0. The data are presented as the mean ± SEM of at least three to four biological repeats. H: The light field of the growth of WT N2 worms under different treatments. Synchronized L1 worms were cultured on NGM plates containing TPEN (5 M) or BP (0.1 or 0.05 M) at 0 h, and the photographs were taken at 72 or 132 h. Scale bar represents 500 m. I: Nile red staining of fixed worms. Representative animals, the anterior is on the right and the posterior is on the left. Synchronized L1 worms were cultured on NGM plates containing TPEN (5 M) or BP (0.1 or 0.05 M), harvested, and stained when they reached adulthood. Scale bar represents 10 m. I: Lipid droplet size in the posterior region of intestines from six worms of each condition. The data are presented as mean ± SEM. Statistical analyses were performed using the t-test (F, G, J) or ANOVA (C, D). *P < 0.05, **P < 0.01, ***P < 0.001, significant difference between treatment (TPEN or BP) and nontreatment (Control). $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, significant difference between two indicated treatments (either TPEN or BP). the reduction of iron by BP indeed decreased the SCD conversion activity and fat accumulation, whereas it was absolutely reversed by zinc reduction (Fig. 5F-J). Thus, our results, together with those of others, convincingly revealed an evolutionarily antagonistic role between zinc and iron to determine SCD conversion activity and fat accumulation from C. elegans to humans.
Zinc has been reported to play an important role in lipid metabolism. The zinc content was significantly lower in patients with alcoholic liver disease (64). In contrast, zinc supplementation significantly reduced total cholesterol, LDL cholesterol, and triglycerides in a meta-analysis of 24 studies on humans (65) and reversed alcoholic steatosis in mice (66).
Here, we showed that zinc reduction promoted SCD conversion activity, but increased levels of zinc conversely impaired SCD conversion activity to positively or negatively affected fat accumulation in C. elegans (Fig. 6). Altogether, these results revealed an evolutionarily conserved role of zinc that negatively regulated fat accumulation, from C. elegans to humans. Reduction in iron or targeting SCD may hold potential promise for the treatment of zinc-or iron-related metabolic diseases. Some strains were provided by the CGC, which is funded by National Institutes of Health Office of Research Infrastructure Program. Fig. 6. A working model of how zinc mediates the SBP-1-SCD axis to regulate lipid metabolism. Under normal zinc level, zinc promotes SBP-1 (SREBP homolog) nuclear translocation to transcriptionally regulate SCD expression, whereas zinc competitively replaces iron to inactivate the enzymatic activity of SCD to reduce biosynthesis of MUFAs and maintain normal fat accumulation. In contrast, zinc reduction leads to the removal of zinc from the SCD enzyme, allowing iron to incorporate into SCD to directly activate its catalyzing function, promoting the biosynthesis of MUFAs and fat accumulation. Zinc reduction may feed back to repress the nuclear translocation of SBP-1 and reduces its transcriptional activation of SCD.