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Discovery of a potent FKBP38 agonist that ameliorates HFD-induced hyperlipidemia via mTOR/P70S6K/SREBPs pathway

The mammalian target of rapamycin (mTOR)-sterol regulatory element-binding proteins (SREBPs) signaling promotes lipogenesis. However, mTOR inhibitors also displayed a significant side effect of hyperlipidemia. Thus, it is essential to develop mTOR-specific inhibitors to inhibit lipogenesis. Here, we screened the endogenous inhibitors of mTOR, and identified that FKBP38 as a vital regulator of lipid metabolism. FKBP38 decreased the lipid content in vitro and in vivo via suppression of the mTOR/P70S6K/SREBPs pathway. 3,5,6,7,8,3ʹ,4ʹ-Heptamethoxyflavone (HMF), a citrus flavonoid, was found to target FKBP38 to suppress the mTOR/P70S6K/SREBPs pathway, reduce lipid level, and potently ameliorate hyperlipidemia and insulin resistance in high fat diet (HFD)-fed mice. Our findings suggest that pharmacological intervention by targeting FKBP38 to suppress mTOR/P70S6K/SREBPs pathway is a potential therapeutic strategy for hyperlipidemia, and HMF could be a leading compound for development of anti-hyperlipidemia drugs.


Introduction
Hyperlipidemia is a common metabolic disorder characterized by an increase of plasma lipids and/or lipoproteins, and is a key risk factor for obesity, atherosclerosis, and type II diabetes 1 . Currently, anti-hyperlipidemic drugs mainly function as lanosterol synthase inhibitors, squalene epoxidase inhibitors, diacyl glycerol acyl transferase inhibitors, and ATP citrate lyase inhibitors. However, these anti-hyperlipidemic agents including statins and fibrates, are reported to have severe side effects on the muscles and liver 2 . It is not known whether other novel targets can be identified as antihyperlipidemic therapeutic agents.
In mammals, the biosynthesis of cholesterol, fatty acid (FA), and triglyceride (TG) are tightly regulated by sterol regulatory element-binding proteins (SREBPs) through activating the expression of rate-limiting lipogenic and cholesterogenic genes 3 . SREBPs activity is enhanced by the mammalian target of rapamycin (mTOR) to promote hepatic lipogenesis 4e6 . However, a significant side effect of hyperlipidemia has been observed in rodents and humans treated with mTOR inhibitor rapamycin 7,8 . Hence, there is an urgent need to develop mTOR-specific inhibitors which also can inhibit lipogenesis.
FKBP prolyl isomerase 38 (FKBP38, also known as FKBP8), an endogenous mTOR inhibitor 9 , is a member of FK506 binding proteins (FKBPs) which elicit their function through direct binding and altering conformation of their target proteins, hence acting as molecular switches 10 . Here, we found that FKBP38 plays a vital role in the regulation of mTOR/P70S6K/SREBPs pathway and lipid homeostasis. Knockdown of FKBP38 resulted in enhanced SREBP processing and lipid level both in vitro and in vivo. Importantly, we identified a natural flavonoid, 3,5,6,7,8,3ʹ,4ʹ-heptamethoxyflavone (HMF), binds to FKBP38 using docking approach, and demonstrated that HMF can activate FKBP38 to suppress mTOR/P70S6K/ SREBPs pathway, reduce lipid level, and potently ameliorate hyperlipidemia in high fat diet (HFD)-fed mice.
The procedures for experiments and animal care were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University (Nanjing, China). Animal testing and research conforms to all relevant ethical regulations. All institutional and national guidelines for the care and use of laboratory animals were followed.

Nile-red staining
Cells were fixed with 4% paraformaldehyde (PFA) for 30 min, stained with 0.5 mg/mL nile-red (Macklin, China) after wash for 30 min at room temperature. Images were captured with fluorescence microscopy (Nikon, Japan), and quantified by Image Pro Plus 6.0 (Media Cybernetics, USA).

Luciferase assays
Cells were plated in 96-well plates at a density of 2.5 Â 10 4 cells/well. After 18 h culture, cells were treated with or without 5, 10, or 20 mmol/L HMF with lipid depletion (LD) medium contained a 1:1 mixture of Ham's F-12 medium (GIBCO, USA) and DMEM supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, 5% lipoprotein-deficient serum (Kalen Biomedical, USA), 10 mmol/L compactin (Aladdin), and 50 mmol/L sodium mevalonate (Sigma, USA) for another 18 h. The luciferase activity was measured using luciferase assay kit (Promega, USA) and normalized by the concentration of total proteins using Enhanced BCA Protein Assay Reagent (Beyotime, China). 2.7. RNAs extraction, cDNA synthesis, and quantitative realtime reverse transcription PCR (qRT-PCR) Total RNAs 13 were extracted using TRIzol (Vazyme, China) and cDNA synthesis was carried out using high capacity cDNA reverse transcription kit (Vazyme) according to the manufacturer's instructions. Gene expressions were measured by the Roche LightCycler 96 System (Roche, Switzerland) using SYBR-green. The mRNA expressions of respective genes were normalized to the mRNA levels of Gadph (mice) and GAPDH (human), or b-actin, and quantified by the 2 ÀDDCt method. Primer sequences were described in Supporting Information Table S1.

Ultrafiltration-liquid chromatography/mass spectrometry (LC/MS) analysis
For ultrafiltration, HMF (50 mmol/L) was incubated with 50 mg/mL Recombinant human FKBP38 proteins (ImmunoClone, USA) in the phosphate buffered saline (PBS) buffer at room temperature for 1 h. The control was prepared by using the binding buffer substitute for PBS during incubation. After incubation, each sample was filtered through a 10 kDa molecular weight cut-off ultrafiltration membrane by centrifugation. Then, the filtrates were washed by 200 mL of PBS for three times to remove the free components. Afterwards, the solution of protein complexes retained on the ultrafiltration membrane was transferred to a new tube. The bound ligand with specific binding to FKBP38 was separated by culturing with 60% ice methanol for 10 min. After repeating three times, the above filtrates were dried by nitrogen and reconstituted in 50 mL of 60% methanol, and subsequently analyzed using high-performance liquid chromatographyequadrupole-quadrupole-quadrupoleeMS/MS (HPLC eQQQeMS/MS) 14,15 .

Thermal shift assay
The thermal shift assay was performed as previously described 16 . For the living cell temperature-dependent thermal shift assay, HL7702 hepatocytes were incubated with 20 mmol/L of HMF for 18 h, then the cells were heated at each temperature point from 37 to 60 C for 3 min. The samples were centrifuged at 20,000 Â g for 10 min at 4 C to separate the supernatants and pellets, and then separated on a 10% SDS-PAGE for immunoblotting analysis of FKBP38.

Metabolic analysis
Total cholesterol (TC), TG levels, low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) levels in mice serum, homogenized liver tissues, and homogenized mice feces were measured using TG, TC, LDL-c, and HDL-c kits (Applygen, China) according to the manufacturer's instructions. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice serum were measured using ALT and AST kits (Jiancheng, China).
An insulin tolerance test (ITT) was performed after a 6-h fast. Blood glucose level was measured before intraperitoneal injection of insulin (0.75 U/kg) and then 30, 60, 90, 120, or 150 min after injection. Glucose tolerance testing (GTT) was performed after overnight fasting. Blood glucose levels were measured before intraperitoneal injection of glucose (2 g/kg body weight) and then 15, 30, 60, 90, and 120 min after injection. Area under the curve (AUC) was calculated to quantify the oral glucose tolerance test (OGTT) and ITT results.

Histological analysis of liver and adipose
Livers and epididymal adipose tissues (WAT) were fixed in 4% PFA and embedded in paraffin wax. Paraffin sections (5 mm) were stained with haematoxylin and eosin (H&E). Sections were examined under digital pathological section scanner (Nano-Zoomer 2.0 RS, Hamamatsu, Japan).

Statistical analysis
All data are expressed as mean AE standard error of the mean (SEM). Comparisons between two groups were analyzed by using a two-tailed Student's t test, and those among three or more groups by using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc tests. Differences were considered significant at P < 0.05. Statistical significance analyses were performed using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA).

FKBP38 suppresses mTOR/P70S6K/SREBP signaling and lipid level
FKBPs regulate inflammation and adaptive immune responses through multiple mechanisms including inhibition of mTOR 10 . Given that mTOR plays an important role in lipid biosynthesis 17,18 , we reasoned that FKBPs are potential therapeutic targets for treatment of hyperlipidemia. To investigate whether FKBPs regulates lipid metabolism, we used specific siRNAs to knockdown five FKBPs (FKBP4, FKBP10, FKBP12, FKBP38, and FKBP51) transcripts, respectively (Supporting Information Fig. S1A) in HL7702 hepatocytes. Among these FKBPs, Comparisons between two groups were analyzed by using a two-tailed Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. knockdown of FKBP38 markedly elevated the level of lipid content as measured by Nile red fluorescence and BODIPY (493/ 503) staining ( Fig. 1A; Supporting Information Fig. S1B and S1C), indicating that FKBP38 is a negative regulator for lipid biosynthesis. We next examined whether FKBP38 regulates lipogenesis through mTOR activity. Consistent with the fact that FKBP38 bounds to mTOR and suppresses mTOR activity 19 , knockdown of FKBP38 significantly increased the phosphorylation of mTOR as well as its downstream targets P70S6K and 4EBP1 ( Fig. 1B and C). Since many lines of evidence suggest that mTOR-mediated control of lipogenesis through SREBPs in hepatocytes 20 , we further assessed whether SREBPs are involved in FKBP38-mediated lipogenesis. As shown in Fig. 1D, knockdown of FKBP38 conspicuously up-regulated the protein levels of n-SREBP-1 and n-SREBP-2, but did not activate pre-SREBP-1 and pre-SREBP-2. We also observed a significant increase of the mRNA levels of SREBP-1C, SREBP-2, and their target genes involved in lipid metabolism, such as FASN, ACC-1, SCD-1, HMGCR, and LDLR, by FKBP38 deletion (Fig. 1E and F). In line with the knockdown studies, overexpression of FKBP38 in HL7702 cells (Supporting Information Fig. S2A) significantly reduced the lipid level (Supporting Information Fig. S2B), dephosphorylated phosphorylated mTOR, phosphorylated P70S6K, and phosphorylated 4EBP1 (Supporting Information Fig. S2C), down-regulated the protein levels of n-SREBP-1 and n-SREBP-2 (Supporting Information Fig. S2D), as well as the mRNA levels of SREBP-1C, SREBP-2, and their target genes (Supporting Information Fig. S2E and S2F). These observations suggested that FKBP38 reduces hepatic lipid content through mTOReSREBPs signaling.
To further examine the function of FKBP38 in vivo, we deleted Fkbp38 in mice livers by administration of AAV8-shRNA followed by a challenge of HFD for 6 weeks to induce hyperlipidemia ( Fig. 1G and H). Liver-specific knockdown of Fkbp38 in HFD-fed mice led to augment in blood and liver TC and TG levels ( Fig. 1I and J; Supporting Information Fig. S1D and S1E). Similarly to the in vitro observations, Fkbp38 inactivation up-regulated the phosphorylation of mTOR, P70S6K, and 4EBP1 (Fig. 1K), increased the protein levels of n-SREBP-1 and n-SREBP-2 (Fig. 1L), as well as the mRNA levels of Srebp1-c, Srebp-2, and their target genes in liver tissues ( Fig. 1M and N). Taken together, we identified FKBP38 as a key regulator for FFA and cholesterol biosynthesis through the mTOR/P70S6K/SREBPs pathway.

HMF targets FKBP38 to decrease cellular lipid level
As FKBP38 is an attractive target for hyperlipidemia treatment, we sought to identify specific small molecules targeting FKBP38 by  Comparisons between two groups were analyzed by using a two-tailed Student's t test, and those among three or more groups by using one-way ANOVA followed by Dunnett's post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the NC þ DMSO group or Vector group; # P < 0.05, ## P < 0.01, #### P < 0.0001 vs. the Vector þ HMF group. ns, no significance. docking approach. From a small molecule pool containing 504 natural compounds (Supporting Information Table S2), HMF ( Fig. 2A), a citrus flavonoid, was found to fit into the protein pocket of FKBP38 with the lowest binding energy of À6.71 kcal/mol. HMF had a hydrogen bond at Gln97 with FKBP38, which maintained the binding stability (Fig. 2B), but it bound poorly with other FKBPs (Supporting Information Table S3). To further assess whether HMF bounds to FKBP38, we performed the ultrafiltration-LC/MS analysis and observed the binding of HMF and FKBP38, as reflected by the increase of MS response (Fig. 2C and D). The temperature-dependent cellular thermal shift assays demonstrated that HMF affected the thermal stability of FKBP38 (Fig. 2E), supporting that there is a direct interaction between FKBP38 and HMF. Collectively, these results indicate that FKBP38 is a direct target of HMF.
We next examined whether HMF can affect lipid metabolism. Similar with the effect of LOV, a well-known treatment of hyperlipidemia, HMF reduced lipid content in HL7702 hepatocytes as measured by Nile red fluorescence (Supporting Information Fig. S3A). In addition, HMF treatment reduced lipid droplets as measured by BODIPY (493/503) staining (Supporting Information Fig. S3B). Without discernible toxicity (Supporting Information Fig. S3C), HMF treatment also decreased the phosphorylation level of mTOR and P70S6K (Supporting Information Fig. S3D), protein expression of n-SREBP-1 and n-SREBP-2 (Supporting Information Fig. S3E), SREluciferase activity of SREBPs (Supporting Information Fig. S3F and Comparisons between two groups were analyzed by using a two-tailed Student's t test, and those among three or more groups by using one-way ANOVA followed by Dunnett's post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the HFD group. S3G), and mRNA levels of SREBP-1C, SREBP-2, as well as their target genes in a dose-dependent manner (Supporting Information Fig. S3H  and S3I). MHY1485, a potent mTOR activator, restored the activity of mTOR and P70S6K activity, expression of mature SREBP-1, SREBP-2, and their target genes in HMF-treated cells (Supporting Information Fig. S4AeS4C). Since the activity of mTOR is also regulated by AKT, AMPKa, and GSK-3b, we assessed whether these signal pathways are affected by HMF. However, HMF showed no discernible effect on the phosphorylation of AKT, AMPKa, and GSK-3b (Supporting Information Fig. S4D). Collectively, these data indicate that HMF may function as a FKBP38 activator to inhibit mTOR activity and regulate lipid synthesis.
To further investigate whether HMFeFKBP38 interaction is required for HMF-mediated reduce of lipogenesis, we knocked down FKBP38 in HMF-treated cells. As expected, FKBP38 inactivation diminished the reduction of lipid content by HMF ( Fig. 3A and B), reversed the HMF-induced dephosphorylation of mTOR and P70S6K (Fig. 3C), as well as the reduction of the expression of n-SREBP-1 and n-SREBP-2 (Fig. 3D). In contrast, FKBP38 overexpression augmented HMF-mediated reduction of lipid production ( Fig. 3E; Supporting Information Fig. S4E), dephosphorylation of mTOR and P70S6K (Fig. 3F), and reduction of n-SREBP-1 and n-SREBP-2 (Fig. 3G). Taken together, these data indicate that FKBP38 activation is required for HMFmediated reduction of lipogenesis.

HMF potently ameliorates diet-induced obesity and hyperlipidemia and improves insulin resistance in mice
We next investigate the effect of HMF on hyperlipidemia in vivo. We fed C57BL/6J mice with HFD for 6 weeks and supplemented HMF or saline in the diet daily. HFD feeding led to significant Comparisons between two groups were analyzed by using a two-tailed Student's t test, and those among three or more groups by using one-way ANOVA followed by Dunnett's post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the HFD þ NC shRNA group.
increases in body weight, serum TG, TC, and LDL-c compared with control, while HMF significantly reduced these hyperlipidemic traits in a dose-dependent manner (Fig. 4AeD). Also, the HDL-c was markedly increased in HMF-treated mice (Fig. 4E). No significant difference in food intake, fecal cholesterol, and TG was observed among HMF or saline treated HFD mice (Supporting Information Fig. S5AeS5C), indicating HMFameliorated hyperlipidemia is not due to the change of food consumption and lipid excretion. HMF treatment also lowered lipid accumulation in liver, reduced the cell size of white adipocyte tissue (Fig. 4FeH), and exhibited protective effects against the HFD-induced liver damage as supported by significantly lower plasma ALT and AST levels (Supporting Information Fig. S5D and E). Additionally, HMF-treated mice markedly restore glucose intolerance (Supporting Information Fig. S5F) and insulin resistance (Supporting Information Fig. S5GeS5J) induced by HFD. Consistent with in vitro results, immunohistochemistry analysis demonstrated that HMF inhibited the phosphorylation of mTOR and P70S6K (Fig. 4I), decreased the expression of SREBP-1 and SREBP-2 compared to HFD-fed group (Fig. 4J). For genes in free fatty acid (FFA) metabolism and cholesterol biosynthesis, HMF significantly decreased the mRNA levels of Fasn, Acc1, Scd-1, Hmgcr, Ldlr, Tnf-a, and Il-1b in the livers of HMF-treated mice (Supporting Information Fig. S5K). These results indicate that HMF exhibits robust efficacy against hyperlipidemia in HFD-fed mice.

Fkbp38 knockdown relieves the hypolipidemic effect of HMF in HFD mice
To assess whether HMF activates FKBP38 to relieve hyperlipidemia and improve insulin resistance, we knocked down Fkbp38 in mice liver by AAV8-Fkbp38 shRNA ( Fig. 5A and B) in HFD-fed mice. We observed negligible toxicity and food intake in HMF or AAV8-Fkbp38 shRNA treated HFD mice (Supporting Information Fig. S6A). Liver-specific knockdown of Fkbp38 abolished not only HMF-induced reduction of body-weight gain, lipid accumulation in blood and liver, liver damage ( Fig. 5CeJ; Supporting Information Fig. S6B and S6C), but also HMF-induced amelioration of blood glucose, glucose tolerance, and insulin resistance (Supporting Information Fig. S6DeS6H), suggesting that FKBP38 is essential for the hypolipidemic effects of HMF in HFD-fed mice. Liver-specific knockdown of Fkbp38 also blunted the decreased phosphorylation of mTOR and P70S6K as well as the inhibited expression of SREBPs and their target genes by HMF treatment (Fig. 5KeN). Together, these results indicate that the hypolipidemic effects of HMF are mostly ascribed to targeting FKBP38 to suppress the mTOR/P70S6K/SREBPs pathway.

Discussion
A large body of evidence indicates that mTOR signaling regulates specific transcription factors (e.g., SREBPs) and promotes lipogenesis 21e25 . mTORC1 can exert regulation on SREBP-1 activity via activation of S6 kinase 1 (S6K1) 26 . However, many researchers report that treatment of rodents or humans with rapamycin leads to hyperlipidemia 4 . From a clinical perspective, the development of mTOR-specific inhibitors should reduce the side effects associated with rapamycin. Here, we came up an alternative strategy to activate mTOR endogenous inhibitors and decrease hyperlipidemia. FKBP38 is a member of FKBPs, which binds and inhibits mTORC1 function 10 . However, its role in lipid metabolism remains unclear. In this study, we demonstrated that FKBP38 was actively involved in cholesterol, FA, and triglyceride biosynthesis by restraining mTOR/P70S6K pathway and SREBPs activity in vitro and in vivo. These results laid a solid foundation to identify FKBP38 as a potential target for hyperlipidemia treatment.
Although natural compounds are considered as an indispensable source of drugs and drug leads, it has proven difficult to identify their target proteins and elucidate the mechanism. Here, we reported a systematic strategy for screening of FKBP38targeted small molecules employing docking approach, ultrafiltration-LC/MS analysis and temperature-dependent cellular Figure 6 Proposed mechanism of HMF-mediated hypolipidemic effect. HMF targets FKBP38 to suppress mTOR/P70S6K/SREBPs pathway, which mediate the de novo synthesis of free fatty acid and cholesterol. thermal shift assays. We identified HMF, a polymethoxyflavone isolated from citrus peel, can potently bind to FKBP38. FKBP38 inactivation and overexpression in vitro and AAV8-Fkbp38 shRNA in vivo experiments confirmed that HMF targeted FKBP38 to produce hypolipidemic effects. Together, our study illustrates how integration of computational screening and functional data can identify mechanisms by which natural products regulate biological activities.
Recently, Feng et al. 27 reported that HMF prevented obesity in high-fat diet-induced rats by regulation of the expression of lipid metabolism-related and inflammatory response related genes. In this work, we found that HMF can bind and activate FKBP38 to inhibit phosphorylation of mTOR and P70S6K and SREBP activity, down-regulate the genes associated with cholesterol and FFA biosynthesis, and decrease the content of cellular lipids. We further confirmed that FKBP38 is required for the hypolipidemic effect of HMF as evidenced by elevated lipid level after Fkbp38 silencing. Statins, inhibitors of HMGCR, are the most widely prescribed drugs to treat hypercholesterolemia via blocking cholesterol biosynthesis. However, statin activates SREBPs and raises cholesterol and fatty acid biosynthetic enzymes in liver, which lead to adverse effects 28,29 . Notably, HMF ameliorated obesity, insulin resistance, fatty accumulation in liver and hyperlipemia without apparent side effects, indicating HMF is a prospective and safe drug target to lower lipid levels.
HMF has been reported to have wide biological activities including anti-tumor, anti-inflammation, and neuroprotective properties 30e32 . From this study, we demonstrated that HMF can function as mTOR inhibitor, which may expand its clinical potential in transplantation and autoimmune disease without hyperlipidemic side effects.

Conclusions
Our in vivo and in vitro results reveal that FKBP38 plays a crucial role in regulating mTOR/P70S6K/SREBPs pathway as well as cholesterol, FFA, and triglyceride biosynthesis. Interestingly, we found that the small molecule HMF specifically targeted FKBP38 to suppress the mTOR/P70S6K/SREBPs pathway, thereby decrease lipid content in vitro and ameliorate hyperlipidemia in HFD-fed mice (Fig. 6). Our study sheds some light on the key role of FKBP38 in hyperlipidemia, and indicates that HMF may serve as a leading compound for the pharmacological control of metabolic diseases.