Palm-based tototrienol-rich fraction (TRF) supplementation modulates cardiac sod1 expression, fxr target gene expression and tauro-conjugated bile acid levels in aleptinemic mice fed a high-fat diet

Palm-based tototrienol-rich fraction (TRF) supplementation modulates cardiac sod1 expression, fxr target gene expression and tauro-conjugated bile acid levels in aleptinemic mice fed a high-fat diet | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Palm-based tototrienol-rich fraction (TRF) supplementation modulates cardiac sod1 expression, fxr target gene expression and tauro-conjugated bile acid levels in aleptinemic mice fed a high-fat diet Nur Aliah Natasha Md Shahrulnizam, Mohd Danial Mohd Efendy Goon, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3757079/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2024 Read the published version in Genes & Nutrition → Version 1 posted 7 You are reading this latest preprint version Abstract Tocotrienol-rich fraction (TRF) has been reported to protect the heart from oxidative stress-induced inflammation. It is, however, unclear whether the protective effects TRF against oxidative stress involve the activation of farnesoid-x receptor ( fxr ), a bile acid receptor, and the regulation of bile acid metabolites. In the current study, we investigated the effects of TRF supplementation on antioxidant activities, expression of fxr and its target genes in cardiac tissue, and serum untargeted metabolomics of high-fat diet-fed mice. Mice were divided into high-fat diet (HFD) with or without TRF supplementation (control) for six weeks. At the end of the intervention, weight (BW), waist circumference (WC), and random blood glucose were measured. Heart tissues were collected, and the gene expression of sod1, sod2, gpx and fxr and its target genes shp and stat3 was determined. Serum was subjected to untargeted metabolomic analysis using UHPLC-Orbitrap. In comparison to the control, the WC of the TRF-treated group was significantly higher (p > 0.05) than that of the HFD-only group, but there was no significant difference in weight or random blood glucose level. Downregulation of sod1, sod2 and gpx expression was observed in TRF-treated mice; however, only sod1 was significant when compared to the HFD only group. The expression of cardiac fxr and shp was significantly upregulated, but stat3 was significantly downregulated in the TRF-treated group compared to the HFD-only group. Biochemical pathways found to be influenced by TRF supplementation include bile acid secretion, primary bile acid biosynthesis, and biotin and cholesterol metabolism. In conclusion, TRF supplementation in HFD-fed mice affects antioxidant activities, and more interestingly, TRF also acts as a signaling molecule that is possibly involved in several bile acid-related biochemical pathways accompanied by an increase in cardiac fxr shp expression. This study provides new insight into TRF in deregulating bile acid receptors and metabolites in high-fat diet-fed mice. tocotrienols farnesoid-x receptor animal model high-fat diet antioxidant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cardiovascular disease (CVD) is still considered as the major cause of death worldwide accounting for about 54% of all noncommunicable disease (NCD) mortality which is about 18 million of death in 2019 [ 1 ]. Effective preventive measures are crucial to reduce the number of deaths caused by CVD, this includes management and control of CVD risk factors. There are two classes of risk factors for CVD which is nonmodifiable and modifiable risk factors. Modifiable risk factors include behavioural, socioeconomic, psychosocial, and metabolic risk factors. Metabolic risk factors include raised blood pressure, overweight and obesity, hyperglycaemia (high blood glucose levels), and hyperlipidaemia (high levels of fat in the blood). Hyperlipidaemia with increased low-density lipoprotein (LDL), which has been reported as an atherogenic lipoprotein, possesses a strong CVD risk factor [ 2 ]. In laboratory setting, rats fed with a high-fat diet developed significantly greater adipose tissue, insulin resistance and hyperleptinaemia, which are associated with obesity [ 3 , 4 , 5 ] suggesting relationship between body fat and dietary fat although the exact mechanisms for the relationship are unclear. Several mechanisms have been proposed, and one possible mechanism is altered fat-induced satiation in response to prolonged fat ingestion and reduced sensitivity to cholecystokinin [ 6 ]. Farnesoid X receptor (FXR) is a nuclear receptor subfamily 1, group H, member 4 (NR1H4). It was first discovered in mice and rats as an orphan nuclear receptor [ 7 , 8 ] and later is identified as a nuclear receptor for bile acid that is highly expressed in the liver, gastrointestinal tract, adipose tissue, pancreas and kidney [ 9 , 10 ]. It is known to act as a key metabolic regulator for the regulation of systemic energy. Activation of FXR induces its target gene, small heterodimer partner (SHP), which accounts for the inhibition of cholesterol 7α-hydroxylase (CYP7A1), phosphoenolpyruvate carboxykinase (PEPCK) and sterol regulatory element binding-protein 1c (SREBP-1c). Additionally, studies have shown that the induction of FXR plays significant roles in inflammatory pathways. Activation of FXR in immune cells inhibits tumor necrosis factor-α (TNF-α) production and suppresses nuclear factor kappa-light-chain-enhancer-of activated B cells (Nf-kB) and interferon gamma (IFN-γ)-related genes in macrophages. The inflammatory mediators of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interferon-γ-inducible protein 10 (IP-10) induced by administration of lipopolysaccharides are repressed following FXR receptor activation [ 11 ]. FXR and its target genes, SHP and phospholipid transfer protein (PLTP) were first discovered in the cardiovascular system in 2004 [ 12 ]. It was present in the normal vascular smooth muscle of the coronary artery, aorta, cardiac muscle and diseased hypertrophic heart, heart failure and myocardial infarction. It was also expressed in neonatal rat ventricular myocytes, H9c2 cardiac cells and neonatal rat cardiac fibroblasts [ 13 ]. FXR expression was reported in the whole heart and cardiac vessels of obese fa/fa Zucker rats and neonatal cardiomyocytes isolated from Wistar rats [ 14 ]. The palm-based tocotrienol-rich fraction (TRF) is a vitamin E mixture that consists of 25% α-tocopherol and 75% tocotrienols. Both tocopherol (T) and tocotrienol (T3) consist of four isomers: alpha (α), beta (β), gamma (γ) and delta (δ) [ 15 ]. Tocotrienol supplementation has been proven to prevent the development of atherosclerosis in studies using animal models and is evident in human studies. In an earlier study, heterozygous apoE knockout mice fed an atherogenic diet supplemented with a vitamin E mixture derived from palm oil observed a substantial decrease in atherosclerotic lesion formation [ 16 ]. Recently, a study has reported that low-dose supplementation with TRF causes decreased endothelial activation and inflammation and reduced atherosclerotic lesions in the aorta of rabbits with induced atherosclerosis [ 17 ]. Similar findings were observed in apolipoprotein E (apoE −/− ) knockout mice fed an atherogenic diet following TRF supplementation [ 18 ]. In addition, a study showed that palm oil tocotrienol-rich extract was able to restore the endothelial function of aortic rats in the presence of oxidative stress by scavenging superoxide radicals produced by hypoxanthine/xanthine oxidase [ 19 ] while another study demonstrated that pre- and post-treatment with palm TRF significantly increased neonatal rat cardiomyocyte cell viability after exposure to H 2 O 2 [ 20 ]. Numerous studies have demonstrated the cardioprotective roles of TRF and FXR. However, the roles of FXR in mediating the effects of TRF remain unclear, especially in the cardiac tissue of mice subjected to an increased risk of CVD. Therefore, in the present study, the effects of TRF supplementation on the cardiac function of aleptinemic mice subjected to HFD were investigated in terms of body weight, waist circumference, random blood glucose and antioxidant activities comprising superoxide dismutase 1 ( sod1 ), superoxide dismutase 2 ( sod2 ) and glutathione peroxidase 1 ( gpx1 ) gene expression. In addition, the expression of cardiac fxr and its target genes shp and signal transducer and activator of transcription 3 ( stat3 ) were also measured. Finally, serum untargeted metabolomics was assessed and compared between the TRF-supplemented group and the control group. Determining the gene expression and metabolites altered by TRF supplementation would provide insights into the role and mechanism of action of TRF supplementation on the cardiac tissue of mice with an increased risk of CVD. Methods Animals and reagents. Fourteen six-week-old male mice B6.Cg-LepOb/J strain (leptin-deficient mice) was purchased from Jackson Laboratory (Maine, United States of America, USA). Mice were housed individually under controlled temperature (23 ± 1℃) and humidity of 50 ± 5% under a strict 12:12 light/dark cycle. Mice were provided with food and reverse osmosis water ad libitum . After two weeks of acclimatization, mice were randomly divided into two groups (n = 7 per group). Throughout this study, both groups were fed a high-fat diet (HFD; Altromin, Germany) consisting of 60% fat, 20% carbohydrates and 20% protein. One group was fed a high-fat diet without any intervention (HFD group). Another group was supplemented with tootrienol-rich fraction (TRF) at 200 mg/kg/day on top of their HFD for six weeks. During the study, the body weights of the mice were measured weekly at the beginning of every week. While the waist circumference was measured once at the end of the study. Random blood glucose levels of the mice were also measured at the end of the study, using a digital glucometer (Accu-cek, Roche Diagnostic, Switzerland) before being euthanized with rapid cervical dislocation. Blood was collected by cardiac aspiration using a 26 G needle (Terumo, Japan) and placed in a plain blood tube (red-top tube; Becton Dickinson, USA). The blood was settled at room temperature for 20 minutes before being centrifuged at 1500 g and 4°C for 10 minutes. The supernatant formed was transferred into microtubes and stored at -80°C for metabolomics analysis. Cardiac tissue from every mouse was dissected out by incising the ascending aorta, cleaned using phosphate-buffered saline (PBS) and snap-frozen using liquid nitrogen. Frozen cardiac tissue was stored at -80°C until further analysis. All procedures performed in mice were approved by the Universiti Teknologi MARA (UiTM) Committee on Animal Research and Ethics. The study was conducted at the Laboratory of Animal Care Unit, Faculty of Medicine, UiTM Sungai Buloh Campus, Selangor, Malaysia. Unless otherwise specified, all reagents are purchased from Sigma-Aldrich. Cardiac RNA Extraction, cDNA synthesis and RT-qPCR Total RNA was extracted from cardiac tissues from both groups using the GeneJet RNA Purification Kit (Thermo Scientific, USA). Each cardiac tissue sample weighing 30 mg was homogenized in a 1.5 ml centrifuge tube containing 300 µL of lysis buffer supplemented with 14.3 M β-mercaptoethanol using a rotor-stator homogenizer for 40 seconds. Briefly, all extractions were performed according to the manufacturer’s protocol to collect purified RNA in the final step. The concentration of RNA was measured and recorded using a Nanodrop. RNA was stored at -80°C for further use. The extracted total RNA (20 ng) was reverse transcribed into complementary DNA (cDNA) using a Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, USA). A reaction mixture was prepared by combining all components for the RT reaction into a sterile, RNase-free tube as indicated in Table S1 . The reaction mixture was mixed gently and centrifuged before being incubated for 10 minutes at 25°C followed by 15 minutes at 50°C. The reaction was terminated by heating at 85°C for 5 minutes. Subsequently, RT‒qPCR was carried out using Maxima SYBR Green qPCR Master Mix (Thermo Scientific, USA). Sense and antisense primers were designed using Primer Premier Software (Table S2). Housekeeping genes comprised rpl4, β-actin and gapdh . Target genes comprised of sod1, sod2, gpx1, fxr, shp and stat3 . First, Maxima SYBR Green qPCR Master Mix components were thawed, vortexed gently and briefly centrifuged. Then, the reaction master mix (Table S3) was prepared by adding all components (except template cDNA) for each 25 µL reaction to a tube at room temperature. Template cDNA was added (≤ 500 ng) to the individual PCR tubes containing the master mix. The reactions were gently mixed without creating bubbles. The CFX96 Bio-Rad Thermal Cycler was programmed according to the manufacturer’s recommendations (Table S4), and PCR tubes were placed in the real-time cycler to initiate the cycling program. Untargeted Metabolomics Analysis by UHPLC‒MS All sera collected by cardiac aspiration were thawed prior to preparation. Once thawed, 100 µL of each serum sample was individually transferred into sterile 1.5 mL microtubes on ice. A total of seven sera from the TRF group (n = 7) and five sera from the HFD group (n = 5) were prepared for untargeted metabolomics analysis. The sera were mixed with 300 µL of methanol (Optima® LC/MS, Fisher Chemical, USA) for deproteination. The mixture was vortexed for 15 seconds and centrifuged for 15 minutes at 15,800 G and 4°C. The formed supernatant was carefully pipetted and transferred into a sterile 2 mL microtube. Finally, the supernatant was dried using a concentrator (Concentrator Plus, Eppendorf, Germany) in VAQ mode for four hours. The dried samples were reconstituted by ultrahigh-performance liquid chromatography‒mass spectrometry (UHPLC‒MS) untargeted metabolomics analysis with 100 µL of LCMS-grade water (W6-4 Water, Optima® LC/MS, Fisher Chemical, USA), vortexed for 15 seconds and transferred into glass vials by filtering with a 0.22 µm cellulose membrane filter. Triplicates of blank samples were prepared by pipetting and filtering 200 µL of LCMS-grade water. All prepared samples and blanks were analysed using UHPLC (UltiMate™ 3000, Thermo Scientific™, USA) and MS (Q Exactive HF Orbitrap-MS, Thermo Fisher Scientific, USA). LCMS-grade water with 0.1% formic acid was used as mobile phase A, and acetonitrile (ACN) with 0.1% formic acid was used as mobile phase B. The UHPLC was equipped with a C18 column (100 mm x 2.1 mm, 1.7 µm; Synchronis™, Thermo Scientific™, USA). Chromatographic separation was carried out at a flow rate of 450 µL/min. The column temperature was set to 55°C and 2 µL per injection. The elution gradient was performed as outlined in Table S5. Mass spectrometry (Q Exactive HF Orbitrap-MS, Thermo Fisher Scientific, USA) scanning was conducted at 50 arbitrary unit (AU) sheath gas flow rate (GFR), 18 AU auxiliary GFR, 0 AU sweep GFR, 55 AU S-lens, capillary temperature at 320°C, and auxiliary gas heater temperature at 300°C. Electron-spray ionization was performed in positive and negative mode. In positive mode, the spray voltage was set at 3.5 kV, while 3.0 kV of spray voltage was set for negative mode. A resolution of 60,000 with a scan range of 100-1,000 (m/z) was set for MS scanning followed by MS/MS scans at a resolution of 15,000 with stepped normalized collision energies of 20, 40, and 60 AU. The generated spectra were preprocessed by Xcalibur™ version 3.1 (Thermo Fisher Scientific, USA). Metabolite Features Annotation Prior to annotation of metabolites of the generated spectra from each sample, statistical analysis was carried out on both the HFD and TRF groups to identify metabolites that were significantly important. Data normalization, statistical analysis, and chemometric and univariate analyses were performed using MetaboAnalyst [ 21 ]. Chemometric analysis using principal component analysis (PCA) was carried out to project the PCA score of variation between TRF and HFD and plot. Univariate analysis using a volcano plot where fold-change above 1.5 and p value less than 0.05 (p < 0.05) were set as the parameters when generating the plot. Using these parameters, a list of metabolite features in the TRF group against the HFD group that were significant with a fold change above 1.5 was generated. The metabolite features were annotated using the m/z cloud, chemspider, CEU Mass Mediator (CEUMM), Human Metabolome Database and METLIN. Statistical analysis Body weight, waist circumference, random blood glucose and the levels of gene expression were determined and compared by unpaired Student’s t test using SPSS. A P < 0.05 was considered statistically significant. Unless otherwise specified, all the data are presented as mean ± SEM. Results Effect of tocotrienol-rich fraction (TRF) supplementation on body weight, waist circumference and random blood glucose of high-fat diet mice. Mice were fed a high-fat diet (HFD) for eight weeks. The body weight of all mice gradually increased over time. Feeding with HFD increased body weight; however, supplementation with TRF while on HFD limited the body weight gain after week five (Fig. 1 ). However, the difference in body weight recorded at the end of the study between the groups was not significant (p > 0.05). At the end of the study, the waist circumference (WC) for the HFD group was 11.79 cm ± 0.34, while that of the TRF-supplemented group was 12. 86 cm ± 0.26 (Fig. 2 A). The WC measured was significantly larger in the TRF group than in the HFD group (p < 0.05). The mean random blood glucose of the high-fat diet-fed mice supplemented with TRF in comparison with the HFD group is shown in Fig. 2 B. The random blood glucose levels measured in the HFD and TRF groups were 13.24 mMol/L ± 0.74 and 16.40 mMol/L ± 3.89, respectively, with no significant difference (p > 0.05). Effects of tocotrienol-rich fraction (TRF) supplementation in the heart of high-fat diet-fed mice on superoxide dismutase ( sod1 and sod2 ) and glutathione peroxidase ( gpx1 ) gene expression. There was a significant downregulation of sod1 expression with TRF supplementation (0.27-fold lower) compared to the HFD group (p < 0.05) (Fig. 3 A). Meanwhile, Sod2 expression was 1.48-fold higher in TRF mice (Fig. 3 B), but no significant difference was found in the expression of sod2 in the TRF group compared to the HFD group. Relative to the HFD group, upregulation (1.04-fold) in gpx1 expression was observed in the TRF group but was not significant (p > 0.05; Fig. 3 C). Effects of tocotrienol-rich fraction supplementation on the fxr and its target genes ( shp and stat3 ) expression in the heart of high fat diet-fed mice. The expression of fxr was upregulated in the heart of high-fat diet-fed mice supplemented with TRF (5.17-fold) compared to the HFD group, but the difference was not significant (Fig. 3 D). The significant upregulation of heart expression of shp, a fxr target gene, was observed in the TRF group (3.66-fold) compared to the HFD group (p > 0.05; Fig. 3 E). A significant downregulation of stat3 was observed in the TRF group (0.41-fold) compared to the HFD group (p < 0.05; Fig. 3 F). Principal Component Analysis (PCA) of TRF Group Against HFD Group. A total of 31248 peaks were generated in UHPLC‒MS positive mode when comparing the TRF group against the HFD group. From the total of seven samples per group, the average total peaks detected in each sample was 2604, which gave rise to 2060 peak groups. The PCA score generated a total variation of 40.9%, whereby its principal component one (PC1) score was 24.6%, while PC2 showed 16.3% variation in the TRF group when compared against the HFD group (Fig. 4 A). In negative mode analysis, a total peak of 15204 peaks was detected. The average number of peaks per sample was 1267. This generated a total peak group of 1001. PCA showed a total variation of 43%, where the score on PC1 was 26.4% and that on PC2 was 16.6% (Fig. 4 B). Further analysis using univariate analysis and volcano plots identified a total of 141 metabolic features when comparing the TRF group against the HFD group in positive mode analysis and 55 metabolic features in negative mode analysis Annotation of Metabolic Features in TRF Group Against HFD Group A total of 42 metabolites were successfully annotated from 196 metabolic features (Table S6). The metabolites comprised bile acids, lipids, sphingosine and alkaloids. Metabolites such as isovaltrate (7.64-fold higher), (+)-abscisic acid beta-D-glucopyranosyl ester (5.98-fold higher) and paucin (5.19-fold higher) were found to be the top three metabolites upregulated in the TRG group compared to the HFD group. N-Undecanoylglycine (6.19-fold lower), phytosphingosine (4.92-fold lower) and 2-amino-hexadecanoic acid (2.78-fold lower) were found to be the top three metabolites that were most downregulated in the TRF group compared with the HFD group. Joint-pathway analysis of annotated metabolites and gene expression ( sod1, sod2, gpx1, fxr, shp and stat3 ) in the TRF group Joint-pathway analysis from the list of annotated metabolites and genes assessed generated a total of 12 possible biochemical pathways that were influenced by TRF supplementation in mice fed a HFD (Table S7). The most likely and significant biochemical pathway modulated by TRF was found to be bile secretion (p < 0.0001), where fxr (mmu:20186) and shp (mmu:23957) and metabolites composed of taurocholic acid (cpd:C05122), taurochenodeoxycholic acid (cpd:C05465), and ouabain (cpd:C01443) were involved in the biochemical pathway (Fig. 5 ). Similarly, primary bile acid biosynthesis was also found to be a significant biochemical pathway involved (p < 0.05). The hit features were taurochenodeoxycholic acid (cpd:C05465) and taurocholic acid (cpd:C05122). Another significant biochemical pathway involved was biotin metabolism, where L-lysine (cpd:C00047) and biotin sulfone (cpd:C20387) were found to be involved. Apart from that, cholesterol metabolism was also found to be a significant biochemical pathway involved (p < 0.05), in which taurocholic acid (cpd:C05122) and taurochenodeoxycholic acid (cpd:C05465) were found to be involved. Discussions This study investigates the effects of tocotrienol-rich fraction (TRF) supplementation in aleptinaemic mice that were fed a high-fat diet (HFD) for eight weeks. Feeding a HFD increases the risk of cardiovascular diseases by promoting cardiac injury, dyslipidemia and oxidative stress [ 22 , 23 ]. Mice that were subjected to HFD had an excessive increase in weight [ 24 ] and increased random blood glucose levels [ 25 ], both of which are risk factors for CVD. In this study, parameters of mice comprising body weight, random blood glucose and waist circumference were measured, while the effects of TRF supplementation on mice fed a HFD on genes related to antioxidants, including sod1, sod2 and gpx1 , were investigated in cardiac tissues. Furthermore, the role of the cardiac fxr, shp , and stat3 genes was assessed by measuring their level of expression in TRF-supplemented mice. Finally, systemic metabolomics alterations were analysed to provide insights into the metabolites that were most likely modulated or altered with TRF supplementation. Mice subjected to HFD have been well reported to develop large body weight and increased blood glucose levels due to the consistent supply of surplus daily calories [ 26 ]. Although caloric restriction has been shown to control increases in body weight and improve blood glucose levels [ 27 ], TRF supplementation has also demonstrated similar effects on body weight [ 28 ] and blood glucose levels [ 29 ]. In the present study, the body weight of mice was shown to decrease after 5 weeks of TRF supplementation. However, the body weight in this group exhibited an upwards trend after week 5, which may be contributed by the excessive calories delivered by HFD, overcoming the effect of TRF. The δ-tocotrienol, as the major component of TRF has been previously reported not able to significantly reduce body weight however promote smaller adipose tissue formation [ 30 ] and this possibly explain our finding on TRF supplementation and body weight. Therefore, TRF supplementation under caloric restriction would promote promising properties by delivering optimal weight reduction in mice subjected to prior HFD feeding, which would further decrease the risk of CVD. However, the waist circumference measured in the TRF group did not reflect the loss of body weight after week five, as the mean was significantly higher than that of the control group. The significant increase in waist circumference would most likely due to the confounding factor which higher starting body weight in the TRF group than in the HFD group. However, TRF supplementation seems to be able to control further expansion of the mouse waist, resulting in a mean difference of 1.07 cm. Apart from the positive outcome on body weight, TRF has been reported to improve fasting blood glucose (FBG) [ 31 ]. In this study, random blood glucose (RBG) was measured instead of FBG due to limitations in this study, where blood sera were dedicated for untargeted metabolomics analysis. The RBG level was higher in the TRF group than in the HFD group. A previous study demonstrated that TRF primarily exhibited antioxidant properties rather than exerting hypoglycemic effects in diabetic animals [ 32 ]. The present study recorded higher cardiac sod2 expression in the TRF group than in the HFD group, which suggests increased antioxidant activity in the cardiac tissue of mice supplemented with TRF. Similarly, a study using an in vitro model demonstrated a higher sod2 expression when supplemented with TRF [ 33 ]. Increased sod2 activities with TRF supplementation suggest improved oxidative stress in mitochondria, as the gene is highly expressed in the organelle [ 34 ]. The antioxidant activities exerted by TRF were most likely limited to sod2 , as sod1 was found to be downregulated and gpx1 was found to be only slightly increased. Downregulation of sod1 following TRF supplementation was also reported in other studies [ 33 , 35 ], suggesting that sod1 is most likely not modulated by TRF in promoting minimal gpx1 activity. However, the positive effect of TRF supplementation was observed to be not limited to sod2 , but it may act as a signaling molecule that affects several biochemical pathways based on findings in this study. Joint pathway analysis combining the fold changes of investigated genes ( sod1, sod2, gpx1, fxr, shp and stat3 ) and metabolites as listed in Table S7 generated a total of 12 important biochemical pathways. The most significant biochemical pathway (p < 0.001) with a 4.39-fold higher fold change compared to the HFD group was bile secretion (Fig. 5 ). The genes involved in bile secretion with TRF supplementation were fxr (5.17-fold higher in TRF group) and shp (3.66-fold higher), and the metabolites involved were taurocholic acid (fold change: 3.28) and taurochenodeoxycholic acid (fold change: 3.84). The increase in fxr gene expression, most likely from TRF supplementation, promotes the expression of shp as a result of nuclear translocation [ 36 ]. The increase in shp promotes the inhibition of bile acid reuptake by suppressing the Na + taurocholate cotransporting polypeptide (NTCP) transporter. Compared to other target organs, the action of bile acids (BAs) on cardiomyocytes is indirect [ 37 ] in regulating myocardial function, and the expression of fxr has been reported to be lower than that in vascular smooth muscles [ 12 ]. However, other conjugated bile acids, such as taurocholic acid (TCA) and taurochenodeoxycholic acid (TCDCA), were found to be increased above 3-fold in the TRF group compared to the HFD group. The presence and increase in these bile acids (BAs), apart from being involved in BA secretion and primary BA synthesis (2.32-fold increase), were most likely suggestive of anti-inflammatory properties exerted by these BAs [ 38 , 39 ] on the mice. Therefore, TRF supplementation in mice subjected to HFD was most likely elicited as a signaling molecule on fxr , thus modulating shp and stat3 gene expression. Conclusion In conclusion, TRF supplementation at 200 mg/kg/day for 8 weeks does not affect the body weight, waist circumference, blood pressure or random blood glucose of HFD-fed mice. However, the expression of sod1 was reduced significantly in TRF-supplemented mice, suggesting low superoxide dismutase 1 activity, possibly due to low radical oxygen species levels, reflecting the role of TRF as an exogenous antioxidative substance. According to the metabolomic analysis, supplementation of TRF in HFD-fed mice may also act as a signaling molecule affecting several biochemical pathways, such as bile acid biosynthesis and secretion. Although the plasma level of bile acids was not measured in this study, the significant increase in fxr and its target gene shp following TRF supplementation suggests an interesting involvement of TRF in bile acid signaling. Declarations Acknowledgement We thank the Research Management Centre team of Universiti Teknologi MARA for their support and aid in completing this project. Funding The project described was funded by the Malaysia Ministry of Higher Education with grant code RACER/1/2019/SKK08/UITM//9. Author Contribution Conceptualization: S.H.S.A.K., S.A.R., E.I. ; Methodology: S.H.S.A.K., S.A.R., E.I. ; Formal Analysis: M.D.M.E.G. ; Investigation: M.D.M.E.G., N.A.N.M.S. ; Resources: S.H.S.A.K. ; Data Curation: M.D.M.E.G. ; Writing – Original Draft: M.D.M.E.G., N.A.N.M.S. ; Writing – Reviewing & Editing: S.H.S.A.K., S.A.R., E.I. ; Visualization: M.D.M.E.G. ; Supervision: S.H.S.A. K; Funding Acquisition: S.H.S.A. K Declaration of Interest All authors declare no conflicts of interest. References World health statistics 2023: monitoring health for the SDGs, Sustainable Development Goals. Geneva: World Health Organization; 2023. Licence: CC BY‑NC‑SA 3.0 IGO Cromwell WC, Otvos JD, Keyes MJ, Pencina MJ, Sullivan L, Vasan RS, Wilson PW, D'Agostino RB. LDL Particle Number and Risk of Future Cardiovascular Disease in the Framingham Offspring Study - Implications for LDL Management. 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Mencarelli A, Cipriani S, Renga B, D'Amore C, Palladino G, Distrutti E, Baldelli F, Fiorucci S. FXR activation improves myocardial fatty acid metabolism in a rodent model of obesity-driven cardiotoxicity. Nutr Metab Cardiovasc Dis. 2013;23(2):94-101. doi: 10.1016/j.numecd.2011.06.008. Ghosh N, Das A, Khanna S. Vitamin E: Tocopherols and Tocotrienol and Their Role in Health and Disease. Essential and Toxic Trace Elements and Vitamins in Human Health. 2019; 283-293. Accessed November 9, 2023. https://doi.org/10.1016/B978-0-12-805378-2.00020-6. Black TM, Wang P, Maeda N, Coleman RA. Palm tocotrienols protect ApoE +/- mice from diet-induced atheroma formation. J Nutr. 2000 Oct;130(10):2420-6. doi: 10.1093/jn/130.10.2420. Razak AA, Omar E, Muid S, & Nawawi H. Low dose palm tocotrienol-rich fraction reduces aortic tissue endothelial activation in severely atherosclerotic rabbits. Pertanika Journal of Science and Technology .2017; 25 (S8), 63–72. Shibata A, Kobayashi T, Asai A, Eitsuka T, Oikawa S, Miyazawa T, Nakagawa K. High purity tocotrienols attenuate atherosclerotic lesion formation in apoE-KO mice. J Nutr Biochem. 2017;48:44-50. doi: 10.1016/j.jnutbio.2017.06.009 Ali SF, Woodman OL. Tocotrienol Rich Palm Oil Extract Is More Effective Than Pure Tocotrienols at Improving Endothelium-Dependent Relaxation in the Presence of Oxidative Stress. Oxid Med Cell Longev. 2015;2015:150829. doi: 10.1155/2015/150829 Abdul Khalid NSA & Jubri Z. The protective effect of palm tocotrienol-rich fraction against H 2 O 2 - induced oxidative stress in neonatal rat cardiomyocytes. 2017. PeerJ Preprints 5:e3333v1 https://doi.org/10.7287/peerj.preprints.3333v1 Pang Z, Chong J, Zhou G, de Lima Morais DA, Chang L, Barrette M, Gauthier C, Jacques PÉ, Li S, Xia J. MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights. Nucleic Acids Res. 2021;49(W1):W388-W396. doi: 10.1093/nar/gkab382. 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Nutr Metab (Lond). 2020;17:42. doi: 10.1186/s12986-020-00458-8. Irandoost P, Mesri Alamdari N, Saidpour A, Shidfar F, Roshanravan N, Asghari Jafarabadi M, Farsi F, Asghari Hanjani N, Vafa M. The effects of royal jelly and tocotrienol-rich fraction on impaired glycemic control and inflammation through irisin in obese rats. J Food Biochem. 2020;44(12):e13493. doi: 10.1111/jfbc.13493. Allen L, Ramalingam L, Menikdiwela K, Scoggin S, Shen CL, Tomison MD, Kaur G, Dufour JM, Chung E, Kalupahana NS, Moustaid-Moussa N. Effects of delta-tocotrienol on obesity-related adipocyte hypertrophy, inflammation and hepatic steatosis in high-fat-fed mice. J Nutr Biochem. 2017;48:128-137. doi: 10.1016/j.jnutbio.2017.07.003. Budin SB, Othman F, Louis SR, Bakar MA, Das S, Mohamed J. The effects of palm oil tocotrienol-rich fraction supplementation on biochemical parameters, oxidative stress and the vascular wall of streptozotocin-induced diabetic rats. Clinics (Sao Paulo). 2009;64(3):235-44. doi: 10.1590/s1807-59322009000300015. Matough FA, Budin SB, Hamid ZA, Abdul-Rahman M, Al-Wahaibi N, Mohammed J. Tocotrienol-rich fraction from palm oil prevents oxidative damage in diabetic rats. Sultan Qaboos Univ Med J. 2014;14(1):e95-e103. doi: 10.12816/0003342. Khor SC, Wan Ngah WZ, Mohd Yusof YA, Abdul Karim N, Makpol S. Tocotrienol-Rich Fraction Ameliorates Antioxidant Defense Mechanisms and Improves Replicative Senescence-Associated Oxidative Stress in Human Myoblasts. Oxid Med Cell Longev. 2017;2017:3868305. doi: 10.1155/2017/3868305. Karnati S, Lüers G, Pfreimer S, Baumgart-Vogt E. Mammalian SOD2 is exclusively located in mitochondria and not present in peroxisomes. Histochem Cell Biol. 2013;140(2):105-17. doi: 10.1007/s00418-013-1099-4. Durani LW, Jaafar F, Tan JK, Tajul Arifin K, Mohd Yusof YA, Wan Ngah WZ, Makpol S. Targeting genes in insulin-associated signalling pathway, DNA damage, cell proliferation and cell differentiation pathways by tocotrienol-rich fraction in preventing cellular senescence of human diploid fibroblasts. Clin Ter. 2015;166(6):e365-73. doi: 10.7417/T.2015.1902. Claudel T, Staels B, Kuipers F. The Farnesoid X receptor: a molecular link between bile acid and lipid and glucose metabolism. Arterioscler Thromb Vasc Biol. 2005;25(10):2020-30. doi: 10.1161/01.ATV.0000178994.21828.a7. Khurana S, Raufman JP, Pallone TL. Bile acids regulate cardiovascular function. Clin Transl Sci. 2011;4(3):210-8. doi: 10.1111/j.1752-8062.2011.00272.x. Qi YC, Duan GZ, Mao W, Liu Q, Zhang YL, Li PF. Taurochenodeoxycholic acid mediates cAMP-PKA-CREB signaling pathway. Chin J Nat Med. 2020 Dec;18(12):898-906. doi: 10.1016/S1875-5364(20)60033-4. Talebian R, Hashem O, Gruber R. Taurocholic acid lowers the inflammatory response of gingival fibroblasts, epithelial cells, and macrophages. J Oral Sci. 2020;62(3):335-339. doi: 10.2334/josnusd.19-0342. Additional Declarations No competing interests reported. Supplementary Files SupplimentaryTABLE.docx Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2024 Read the published version in Genes & Nutrition → Version 1 posted Editorial decision: Revision requested 04 Jan, 2024 Reviews received at journal 22 Dec, 2023 Reviewers agreed at journal 19 Dec, 2023 Reviewers invited by journal 19 Dec, 2023 Editor assigned by journal 18 Dec, 2023 Submission checks completed at journal 18 Dec, 2023 First submitted to journal 15 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3757079","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":260712500,"identity":"7d6f4855-9269-4bd7-9d1d-3e54022b67b7","order_by":0,"name":"Nur Aliah Natasha Md Shahrulnizam","email":"","orcid":"","institution":"Universiti Teknologi MARA (UiTM)","correspondingAuthor":false,"prefix":"","firstName":"Nur","middleName":"Aliah Natasha Md","lastName":"Shahrulni","suffix":"Md"},{"id":260712501,"identity":"267eaafb-797a-4e87-a6fd-26403d9eee62","order_by":1,"name":"Mohd Danial Mohd Efendy Goon","email":"","orcid":"","institution":"Universiti Teknologi MARA (UiTM)","correspondingAuthor":false,"prefix":"","firstName":"Mohd","middleName":"Danial Mohd Efendy","lastName":"Goon","suffix":""},{"id":260712502,"identity":"e4b8343e-b63e-4bbb-a9ec-53fb12c3c4a2","order_by":2,"name":"Sharaniza Ab Rahim","email":"","orcid":"","institution":"Universiti Teknologi MARA (UiTM)","correspondingAuthor":false,"prefix":"","firstName":"Sharaniza","middleName":"Ab","lastName":"Rahim","suffix":""},{"id":260712503,"identity":"07b9408c-4c6f-4a8b-bf37-c3756a31c641","order_by":3,"name":"Sook Weih Lew","email":"","orcid":"","institution":"Universiti Teknologi MARA (UiTM)","correspondingAuthor":false,"prefix":"","firstName":"Sook","middleName":"Weih","lastName":"Lew","suffix":""},{"id":260712505,"identity":"c36ce379-cfce-4077-9957-1ec350dfbeb2","order_by":4,"name":"Effendi Ibrahim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYDAC5oMPGBjYJOTs+5sPMCSAhRIIaGFLNgBpMTaQOJbAkJBAvBaGxA0MOQZQ1QS08LMxMz4uKLNI3M5w5pvEwx/bGPjZcwyYbrbh1iLZxsxsPOOchPHO5t5tEgkJtxkke94YMOfi0WJwv/+YNG+bhGzDgbMQLQY3cvBrsT/GzP4bqIWx4UDOM7AWe0JaDNiY2ZiBWhQ3HMhhg9giQUCLxDFmZmkeoF8kZxwztkhIu80jceZZweGcc7i18LcxM37mKauT4+dvfnjzh81tOf725I2Pc8pwa8EAPCDiACMbCVqg4A/pWkbBKBgFo2DYAgBDsU8oMDxH9AAAAABJRU5ErkJggg==","orcid":"","institution":"Universiti Teknologi MARA (UiTM)","correspondingAuthor":true,"prefix":"","firstName":"Effendi","middleName":"","lastName":"Ibrahim","suffix":""},{"id":260712506,"identity":"afdc8e31-e35d-4b32-9bb4-cb11d9e02c99","order_by":5,"name":"Siti Hamimah Sheikh Abdul Kadir","email":"","orcid":"","institution":"Universiti Teknologi MARA (UiTM)","correspondingAuthor":false,"prefix":"","firstName":"Siti","middleName":"Hamimah Sheikh Abdul","lastName":"Kadir","suffix":""}],"badges":[],"createdAt":"2023-12-15 06:44:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3757079/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3757079/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12263-024-00742-9","type":"published","date":"2024-02-27T15:02:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":48558411,"identity":"b8ca7bae-9ac9-4a6b-b9d8-75e28ae6fb92","added_by":"auto","created_at":"2023-12-20 19:38:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20758,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mousebody weight for eight weeks of HFD. Mice fed a HFD supplemented with TRF showed a decrease in body weight after five weeks of supplementation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3757079/v1/6fbbf396d3b893839e1985b0.png"},{"id":48558410,"identity":"628e0455-d0fd-453e-be24-cd73e017b4f7","added_by":"auto","created_at":"2023-12-20 19:38:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23622,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of waist circumference (A) and random blood glucose (B) at the end of the study between theTRF and HFD groups. The waist circumference of mice supplemented with TRF was found to be higher than that of the HFD group at the end of the study (A). The waist circumference measured was found to be significantly higher in theTRF group than in theHFD group (*p \u0026lt; 0.05). The random blood glucose level measured in the TRF group (16.40 ± 3.89) was higher than that in the HFD group (13.24 ± 0.74) (B). The mean difference was not significant (p \u0026gt; 0.05). Values are expressed as the mean ± SEM.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3757079/v1/469f925dfe4ec554d0e0bb95.png"},{"id":48559161,"identity":"5271f996-2736-45c5-b6cb-963cdd272905","added_by":"auto","created_at":"2023-12-20 19:46:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51974,"visible":true,"origin":"","legend":"\u003cp\u003eFold-change of cardiac gene expression of sod1 (A), sod2 (B), gpx1 (C), fxr (D), shp (E) and stat3 (F) in TRF group compared to HFD group. The gene expression of sod1 and stat3 was found to be significantly downregulated in the TRF group (*p \u0026lt; 0.05). Other genes, such as sod2, gpx1 and fxr, were upregulated but not significantly (p \u0026gt; 0.05) except for shp (*p \u0026lt; 0.05). Values are expressed as the mean fold-change (FC) with standard error of the mean (SEM).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3757079/v1/c5a408053b732ae80937c52a.png"},{"id":48558414,"identity":"a0d6884e-20cd-4f23-aed5-02c7032485de","added_by":"auto","created_at":"2023-12-20 19:38:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65512,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3757079/v1/c65a5fe5e8ce6f1859b7ea4d.png"},{"id":48558412,"identity":"0944a265-d282-4f4c-b5b1-87bf4064a38f","added_by":"auto","created_at":"2023-12-20 19:38:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":266713,"visible":true,"origin":"","legend":"\u003cp\u003eBile secretion pathway possibly modulated by TRF supplementation in HFD-fed mice (p \u0026lt; 0.0001). Hit features are shown in red. Nr1h4: farnesoid-x receptor (FXR), Nr0b2: small heterodimer protein (SHP); C05122: taurocholic acid; C00695: cholic acid\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3757079/v1/5d81a88113fb33881ee1f478.png"},{"id":51958468,"identity":"e149e99b-c3ea-4274-bfab-876486c75da9","added_by":"auto","created_at":"2024-03-04 15:16:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":605085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3757079/v1/7beaaa0c-63a7-4a59-be9f-d7d2566913f7.pdf"},{"id":48558415,"identity":"3f91020c-4f1d-41ce-aabc-e4ba5b2b3f85","added_by":"auto","created_at":"2023-12-20 19:38:22","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25032,"visible":true,"origin":"","legend":"","description":"","filename":"SupplimentaryTABLE.docx","url":"https://assets-eu.researchsquare.com/files/rs-3757079/v1/782c103c76bb4e30f5bc0408.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Palm-based tototrienol-rich fraction (TRF) supplementation modulates cardiac sod1 expression, fxr target gene expression and tauro-conjugated bile acid levels in aleptinemic mice fed a high-fat diet","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular disease (CVD) is still considered as the major cause of death worldwide accounting for about 54% of all noncommunicable disease (NCD) mortality which is about 18\u0026nbsp;million of death in 2019 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Effective preventive measures are crucial to reduce the number of deaths caused by CVD, this includes management and control of CVD risk factors. There are two classes of risk factors for CVD which is nonmodifiable and modifiable risk factors. Modifiable risk factors include behavioural, socioeconomic, psychosocial, and metabolic risk factors. Metabolic risk factors include raised blood pressure, overweight and obesity, hyperglycaemia (high blood glucose levels), and hyperlipidaemia (high levels of fat in the blood). Hyperlipidaemia with increased low-density lipoprotein (LDL), which has been reported as an atherogenic lipoprotein, possesses a strong CVD risk factor [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In laboratory setting, rats fed with a high-fat diet developed significantly greater adipose tissue, insulin resistance and hyperleptinaemia, which are associated with obesity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] suggesting relationship between body fat and dietary fat although the exact mechanisms for the relationship are unclear. Several mechanisms have been proposed, and one possible mechanism is altered fat-induced satiation in response to prolonged fat ingestion and reduced sensitivity to cholecystokinin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFarnesoid X receptor (FXR) is a nuclear receptor subfamily 1, group H, member 4 (NR1H4). It was first discovered in mice and rats as an orphan nuclear receptor [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and later is identified as a nuclear receptor for bile acid that is highly expressed in the liver, gastrointestinal tract, adipose tissue, pancreas and kidney [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It is known to act as a key metabolic regulator for the regulation of systemic energy. Activation of FXR induces its target gene, small heterodimer partner (SHP), which accounts for the inhibition of cholesterol 7α-hydroxylase (CYP7A1), phosphoenolpyruvate carboxykinase (PEPCK) and sterol regulatory element binding-protein 1c (SREBP-1c). Additionally, studies have shown that the induction of FXR plays significant roles in inflammatory pathways. Activation of FXR in immune cells inhibits tumor necrosis factor-α (TNF-α) production and suppresses nuclear factor kappa-light-chain-enhancer-of activated B cells (Nf-kB) and interferon gamma (IFN-γ)-related genes in macrophages. The inflammatory mediators of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interferon-γ-inducible protein 10 (IP-10) induced by administration of lipopolysaccharides are repressed following FXR receptor activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. FXR and its target genes, SHP and phospholipid transfer protein (PLTP) were first discovered in the cardiovascular system in 2004 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It was present in the normal vascular smooth muscle of the coronary artery, aorta, cardiac muscle and diseased hypertrophic heart, heart failure and myocardial infarction. It was also expressed in neonatal rat ventricular myocytes, H9c2 cardiac cells and neonatal rat cardiac fibroblasts [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. FXR expression was reported in the whole heart and cardiac vessels of obese fa/fa Zucker rats and neonatal cardiomyocytes isolated from Wistar rats [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe palm-based tocotrienol-rich fraction (TRF) is a vitamin E mixture that consists of 25% α-tocopherol and 75% tocotrienols. Both tocopherol (T) and tocotrienol (T3) consist of four isomers: alpha (α), beta (β), gamma (γ) and delta (δ) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Tocotrienol supplementation has been proven to prevent the development of atherosclerosis in studies using animal models and is evident in human studies. In an earlier study, heterozygous apoE knockout mice fed an atherogenic diet supplemented with a vitamin E mixture derived from palm oil observed a substantial decrease in atherosclerotic lesion formation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Recently, a study has reported that low-dose supplementation with TRF causes decreased endothelial activation and inflammation and reduced atherosclerotic lesions in the aorta of rabbits with induced atherosclerosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Similar findings were observed in apolipoprotein E (apoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) knockout mice fed an atherogenic diet following TRF supplementation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, a study showed that palm oil tocotrienol-rich extract was able to restore the endothelial function of aortic rats in the presence of oxidative stress by scavenging superoxide radicals produced by hypoxanthine/xanthine oxidase [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] while another study demonstrated that pre- and post-treatment with palm TRF significantly increased neonatal rat cardiomyocyte cell viability after exposure to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated the cardioprotective roles of TRF and FXR. However, the roles of FXR in mediating the effects of TRF remain unclear, especially in the cardiac tissue of mice subjected to an increased risk of CVD. Therefore, in the present study, the effects of TRF supplementation on the cardiac function of aleptinemic mice subjected to HFD were investigated in terms of body weight, waist circumference, random blood glucose and antioxidant activities comprising \u003cem\u003esuperoxide dismutase 1\u003c/em\u003e (\u003cem\u003esod1\u003c/em\u003e), \u003cem\u003esuperoxide dismutase 2\u003c/em\u003e (\u003cem\u003esod2\u003c/em\u003e) and \u003cem\u003eglutathione peroxidase 1\u003c/em\u003e (\u003cem\u003egpx1\u003c/em\u003e) gene expression. In addition, the expression of cardiac \u003cem\u003efxr\u003c/em\u003e and its target genes \u003cem\u003eshp\u003c/em\u003e and \u003cem\u003esignal transducer and activator of transcription 3\u003c/em\u003e (\u003cem\u003estat3\u003c/em\u003e) were also measured. Finally, serum untargeted metabolomics was assessed and compared between the TRF-supplemented group and the control group. Determining the gene expression and metabolites altered by TRF supplementation would provide insights into the role and mechanism of action of TRF supplementation on the cardiac tissue of mice with an increased risk of CVD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eAnimals and reagents.\u003c/p\u003e \u003cp\u003eFourteen six-week-old male mice B6.Cg-LepOb/J strain (leptin-deficient mice) was purchased from Jackson Laboratory (Maine, United States of America, USA). Mice were housed individually under controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃) and humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% under a strict 12:12 light/dark cycle. Mice were provided with food and reverse osmosis water \u003cem\u003ead libitum\u003c/em\u003e. After two weeks of acclimatization, mice were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;7 per group). Throughout this study, both groups were fed a high-fat diet (HFD; Altromin, Germany) consisting of 60% fat, 20% carbohydrates and 20% protein. One group was fed a high-fat diet without any intervention (HFD group). Another group was supplemented with tootrienol-rich fraction (TRF) at 200 mg/kg/day on top of their HFD for six weeks. During the study, the body weights of the mice were measured weekly at the beginning of every week. While the waist circumference was measured once at the end of the study. Random blood glucose levels of the mice were also measured at the end of the study, using a digital glucometer (Accu-cek, Roche Diagnostic, Switzerland) before being euthanized with rapid cervical dislocation. Blood was collected by cardiac aspiration using a 26 G needle (Terumo, Japan) and placed in a plain blood tube (red-top tube; Becton Dickinson, USA). The blood was settled at room temperature for 20 minutes before being centrifuged at 1500 g and 4\u0026deg;C for 10 minutes. The supernatant formed was transferred into microtubes and stored at -80\u0026deg;C for metabolomics analysis. Cardiac tissue from every mouse was dissected out by incising the ascending aorta, cleaned using phosphate-buffered saline (PBS) and snap-frozen using liquid nitrogen. Frozen cardiac tissue was stored at -80\u0026deg;C until further analysis. All procedures performed in mice were approved by the Universiti Teknologi MARA (UiTM) Committee on Animal Research and Ethics. The study was conducted at the Laboratory of Animal Care Unit, Faculty of Medicine, UiTM Sungai Buloh Campus, Selangor, Malaysia. Unless otherwise specified, all reagents are purchased from Sigma-Aldrich.\u003c/p\u003e \u003cp\u003eCardiac RNA Extraction, cDNA synthesis and RT-qPCR\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from cardiac tissues from both groups using the GeneJet RNA Purification Kit (Thermo Scientific, USA). Each cardiac tissue sample weighing 30 mg was homogenized in a 1.5 ml centrifuge tube containing 300 \u0026micro;L of lysis buffer supplemented with 14.3 M β-mercaptoethanol using a rotor-stator homogenizer for 40 seconds. Briefly, all extractions were performed according to the manufacturer\u0026rsquo;s protocol to collect purified RNA in the final step. The concentration of RNA was measured and recorded using a Nanodrop. RNA was stored at -80\u0026deg;C for further use. The extracted total RNA (20 ng) was reverse transcribed into complementary DNA (cDNA) using a Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, USA). A reaction mixture was prepared by combining all components for the RT reaction into a sterile, RNase-free tube as indicated in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The reaction mixture was mixed gently and centrifuged before being incubated for 10 minutes at 25\u0026deg;C followed by 15 minutes at 50\u0026deg;C. The reaction was terminated by heating at 85\u0026deg;C for 5 minutes. Subsequently, RT‒qPCR was carried out using Maxima SYBR Green qPCR Master Mix (Thermo Scientific, USA). Sense and antisense primers were designed using Primer Premier Software (Table S2). Housekeeping genes comprised \u003cem\u003erpl4, β-actin\u003c/em\u003e and \u003cem\u003egapdh\u003c/em\u003e. Target genes comprised of \u003cem\u003esod1, sod2, gpx1, fxr, shp\u003c/em\u003e and \u003cem\u003estat3\u003c/em\u003e. First, Maxima SYBR Green qPCR Master Mix components were thawed, vortexed gently and briefly centrifuged. Then, the reaction master mix (Table S3) was prepared by adding all components (except template cDNA) for each 25 \u0026micro;L reaction to a tube at room temperature. Template cDNA was added (\u0026le;\u0026thinsp;500 ng) to the individual PCR tubes containing the master mix. The reactions were gently mixed without creating bubbles. The CFX96 Bio-Rad Thermal Cycler was programmed according to the manufacturer\u0026rsquo;s recommendations (Table S4), and PCR tubes were placed in the real-time cycler to initiate the cycling program.\u003c/p\u003e \u003cp\u003eUntargeted Metabolomics Analysis by UHPLC‒MS\u003c/p\u003e \u003cp\u003eAll sera collected by cardiac aspiration were thawed prior to preparation. Once thawed, 100 \u0026micro;L of each serum sample was individually transferred into sterile 1.5 mL microtubes on ice. A total of seven sera from the TRF group (n\u0026thinsp;=\u0026thinsp;7) and five sera from the HFD group (n\u0026thinsp;=\u0026thinsp;5) were prepared for untargeted metabolomics analysis. The sera were mixed with 300 \u0026micro;L of methanol (Optima\u0026reg; LC/MS, Fisher Chemical, USA) for deproteination. The mixture was vortexed for 15 seconds and centrifuged for 15 minutes at 15,800 G and 4\u0026deg;C. The formed supernatant was carefully pipetted and transferred into a sterile 2 mL microtube. Finally, the supernatant was dried using a concentrator (Concentrator Plus, Eppendorf, Germany) in VAQ mode for four hours. The dried samples were reconstituted by ultrahigh-performance liquid chromatography‒mass spectrometry (UHPLC‒MS) untargeted metabolomics analysis with 100 \u0026micro;L of LCMS-grade water (W6-4 Water, Optima\u0026reg; LC/MS, Fisher Chemical, USA), vortexed for 15 seconds and transferred into glass vials by filtering with a 0.22 \u0026micro;m cellulose membrane filter. Triplicates of blank samples were prepared by pipetting and filtering 200 \u0026micro;L of LCMS-grade water. All prepared samples and blanks were analysed using UHPLC (UltiMate\u0026trade; 3000, Thermo Scientific\u0026trade;, USA) and MS (Q Exactive HF Orbitrap-MS, Thermo Fisher Scientific, USA). LCMS-grade water with 0.1% formic acid was used as mobile phase A, and acetonitrile (ACN) with 0.1% formic acid was used as mobile phase B. The UHPLC was equipped with a C18 column (100 mm x 2.1 mm, 1.7 \u0026micro;m; Synchronis\u0026trade;, Thermo Scientific\u0026trade;, USA). Chromatographic separation was carried out at a flow rate of 450 \u0026micro;L/min. The column temperature was set to 55\u0026deg;C and 2 \u0026micro;L per injection. The elution gradient was performed as outlined in Table S5. Mass spectrometry (Q Exactive HF Orbitrap-MS, Thermo Fisher Scientific, USA) scanning was conducted at 50 arbitrary unit (AU) sheath gas flow rate (GFR), 18 AU auxiliary GFR, 0 AU sweep GFR, 55 AU S-lens, capillary temperature at 320\u0026deg;C, and auxiliary gas heater temperature at 300\u0026deg;C. Electron-spray ionization was performed in positive and negative mode. In positive mode, the spray voltage was set at 3.5 kV, while 3.0 kV of spray voltage was set for negative mode. A resolution of 60,000 with a scan range of 100-1,000 (m/z) was set for MS scanning followed by MS/MS scans at a resolution of 15,000 with stepped normalized collision energies of 20, 40, and 60 AU. The generated spectra were preprocessed by Xcalibur\u0026trade; version 3.1 (Thermo Fisher Scientific, USA).\u003c/p\u003e \u003cp\u003eMetabolite Features Annotation\u003c/p\u003e \u003cp\u003ePrior to annotation of metabolites of the generated spectra from each sample, statistical analysis was carried out on both the HFD and TRF groups to identify metabolites that were significantly important. Data normalization, statistical analysis, and chemometric and univariate analyses were performed using MetaboAnalyst [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Chemometric analysis using principal component analysis (PCA) was carried out to project the PCA score of variation between TRF and HFD and plot. Univariate analysis using a volcano plot where fold-change above 1.5 and p value less than 0.05 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were set as the parameters when generating the plot. Using these parameters, a list of metabolite features in the TRF group against the HFD group that were significant with a fold change above 1.5 was generated. The metabolite features were annotated using the m/z cloud, chemspider, CEU Mass Mediator (CEUMM), Human Metabolome Database and METLIN.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eBody weight, waist circumference, random blood glucose and the levels of gene expression were determined and compared by unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test using SPSS. A P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Unless otherwise specified, all the data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eEffect of tocotrienol-rich fraction (TRF) supplementation on body weight, waist circumference and random blood glucose of high-fat diet mice.\u003c/p\u003e\n\u003cp\u003eMice were fed a high-fat diet (HFD) for eight weeks. The body weight of all mice gradually increased over time. Feeding with HFD increased body weight; however, supplementation with TRF while on HFD limited the body weight gain after week five (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e). However, the difference in body weight recorded at the end of the study between the groups was not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). At the end of the study, the waist circumference (WC) for the HFD group was 11.79 cm\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34, while that of the TRF-supplemented group was 12. 86 cm\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eA). The WC measured was significantly larger in the TRF group than in the HFD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The mean random blood glucose of the high-fat diet-fed mice supplemented with TRF in comparison with the HFD group is shown in Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eB. The random blood glucose levels measured in the HFD and TRF groups were 13.24 mMol/L\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74 and 16.40 mMol/L\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89, respectively, with no significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eEffects of tocotrienol-rich fraction (TRF) supplementation in the heart of high-fat diet-fed mice on \u003cem\u003esuperoxide dismutase\u003c/em\u003e (\u003cem\u003esod1\u003c/em\u003e and \u003cem\u003esod2\u003c/em\u003e) and \u003cem\u003eglutathione peroxidase\u003c/em\u003e (\u003cem\u003egpx1\u003c/em\u003e) gene expression.\u003c/p\u003e\n\u003cp\u003eThere was a significant downregulation of \u003cem\u003esod1\u003c/em\u003e expression with TRF supplementation (0.27-fold lower) compared to the HFD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eA). Meanwhile, \u003cem\u003eSod2\u003c/em\u003e expression was 1.48-fold higher in TRF mice (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eB), but no significant difference was found in the expression of \u003cem\u003esod2\u003c/em\u003e in the TRF group compared to the HFD group. Relative to the HFD group, upregulation (1.04-fold) in gpx1 expression was observed in the TRF group but was not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eEffects of tocotrienol-rich fraction supplementation on the \u003cem\u003efxr\u003c/em\u003e and its target genes (\u003cem\u003eshp\u003c/em\u003e and \u003cem\u003estat3\u003c/em\u003e) expression in the heart of high fat diet-fed mice.\u003c/p\u003e\n\u003cp\u003eThe expression of \u003cem\u003efxr\u003c/em\u003e was upregulated in the heart of high-fat diet-fed mice supplemented with TRF (5.17-fold) compared to the HFD group, but the difference was not significant (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eD). The significant upregulation of heart expression of shp, a fxr target gene, was observed in the TRF group (3.66-fold) compared to the HFD group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eE). A significant downregulation of stat3 was observed in the TRF group (0.41-fold) compared to the HFD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003ePrincipal Component Analysis (PCA) of TRF Group Against HFD Group.\u003c/p\u003e\n\u003cp\u003eA total of 31248 peaks were generated in UHPLC‒MS positive mode when comparing the TRF group against the HFD group. From the total of seven samples per group, the average total peaks detected in each sample was 2604, which gave rise to 2060 peak groups. The PCA score generated a total variation of 40.9%, whereby its principal component one (PC1) score was 24.6%, while PC2 showed 16.3% variation in the TRF group when compared against the HFD group (Fig. \u003cspan\u003e4\u003c/span\u003eA). In negative mode analysis, a total peak of 15204 peaks was detected. The average number of peaks per sample was 1267. This generated a total peak group of 1001. PCA showed a total variation of 43%, where the score on PC1 was 26.4% and that on PC2 was 16.6% (Fig. \u003cspan\u003e4\u003c/span\u003eB). Further analysis using univariate analysis and volcano plots identified a total of 141 metabolic features when comparing the TRF group against the HFD group in positive mode analysis and 55 metabolic features in negative mode analysis\u003c/p\u003e\n\u003cp\u003eAnnotation of Metabolic Features in TRF Group Against HFD Group\u003c/p\u003e\n\u003cp\u003eA total of 42 metabolites were successfully annotated from 196 metabolic features (Table S6). The metabolites comprised bile acids, lipids, sphingosine and alkaloids. Metabolites such as isovaltrate (7.64-fold higher), (+)-abscisic acid beta-D-glucopyranosyl ester (5.98-fold higher) and paucin (5.19-fold higher) were found to be the top three metabolites upregulated in the TRG group compared to the HFD group. N-Undecanoylglycine (6.19-fold lower), phytosphingosine (4.92-fold lower) and 2-amino-hexadecanoic acid (2.78-fold lower) were found to be the top three metabolites that were most downregulated in the TRF group compared with the HFD group.\u003c/p\u003e\n\u003cp\u003eJoint-pathway analysis of annotated metabolites and gene expression (\u003cem\u003esod1, sod2, gpx1, fxr, shp and stat3\u003c/em\u003e) in the TRF group\u003c/p\u003e\n\u003cp\u003eJoint-pathway analysis from the list of annotated metabolites and genes assessed generated a total of 12 possible biochemical pathways that were influenced by TRF supplementation in mice fed a HFD (Table S7). The most likely and significant biochemical pathway modulated by TRF was found to be bile secretion (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), where fxr (mmu:20186) and shp (mmu:23957) and metabolites composed of taurocholic acid (cpd:C05122), taurochenodeoxycholic acid (cpd:C05465), and ouabain (cpd:C01443) were involved in the biochemical pathway (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e). Similarly, primary bile acid biosynthesis was also found to be a significant biochemical pathway involved (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The hit features were taurochenodeoxycholic acid (cpd:C05465) and taurocholic acid (cpd:C05122). Another significant biochemical pathway involved was biotin metabolism, where L-lysine (cpd:C00047) and biotin sulfone (cpd:C20387) were found to be involved. Apart from that, cholesterol metabolism was also found to be a significant biochemical pathway involved (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), in which taurocholic acid (cpd:C05122) and taurochenodeoxycholic acid (cpd:C05465) were found to be involved.\u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003eThis study investigates the effects of tocotrienol-rich fraction (TRF) supplementation in aleptinaemic mice that were fed a high-fat diet (HFD) for eight weeks. Feeding a HFD increases the risk of cardiovascular diseases by promoting cardiac injury, dyslipidemia and oxidative stress [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Mice that were subjected to HFD had an excessive increase in weight [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and increased random blood glucose levels [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], both of which are risk factors for CVD. In this study, parameters of mice comprising body weight, random blood glucose and waist circumference were measured, while the effects of TRF supplementation on mice fed a HFD on genes related to antioxidants, including \u003cem\u003esod1, sod2\u003c/em\u003e and \u003cem\u003egpx1\u003c/em\u003e, were investigated in cardiac tissues. Furthermore, the role of the cardiac \u003cem\u003efxr, shp\u003c/em\u003e, and \u003cem\u003estat3\u003c/em\u003e genes was assessed by measuring their level of expression in TRF-supplemented mice. Finally, systemic metabolomics alterations were analysed to provide insights into the metabolites that were most likely modulated or altered with TRF supplementation.\u003c/p\u003e \u003cp\u003eMice subjected to HFD have been well reported to develop large body weight and increased blood glucose levels due to the consistent supply of surplus daily calories [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although caloric restriction has been shown to control increases in body weight and improve blood glucose levels [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], TRF supplementation has also demonstrated similar effects on body weight [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and blood glucose levels [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the present study, the body weight of mice was shown to decrease after 5 weeks of TRF supplementation. However, the body weight in this group exhibited an upwards trend after week 5, which may be contributed by the excessive calories delivered by HFD, overcoming the effect of TRF. The δ-tocotrienol, as the major component of TRF has been previously reported not able to significantly reduce body weight however promote smaller adipose tissue formation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and this possibly explain our finding on TRF supplementation and body weight. Therefore, TRF supplementation under caloric restriction would promote promising properties by delivering optimal weight reduction in mice subjected to prior HFD feeding, which would further decrease the risk of CVD. However, the waist circumference measured in the TRF group did not reflect the loss of body weight after week five, as the mean was significantly higher than that of the control group. The significant increase in waist circumference would most likely due to the confounding factor which higher starting body weight in the TRF group than in the HFD group. However, TRF supplementation seems to be able to control further expansion of the mouse waist, resulting in a mean difference of 1.07 cm.\u003c/p\u003e \u003cp\u003eApart from the positive outcome on body weight, TRF has been reported to improve fasting blood glucose (FBG) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, random blood glucose (RBG) was measured instead of FBG due to limitations in this study, where blood sera were dedicated for untargeted metabolomics analysis. The RBG level was higher in the TRF group than in the HFD group. A previous study demonstrated that TRF primarily exhibited antioxidant properties rather than exerting hypoglycemic effects in diabetic animals [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The present study recorded higher cardiac \u003cem\u003esod2\u003c/em\u003e expression in the TRF group than in the HFD group, which suggests increased antioxidant activity in the cardiac tissue of mice supplemented with TRF. Similarly, a study using an in vitro model demonstrated a higher \u003cem\u003esod2\u003c/em\u003e expression when supplemented with TRF [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Increased \u003cem\u003esod2\u003c/em\u003e activities with TRF supplementation suggest improved oxidative stress in mitochondria, as the gene is highly expressed in the organelle [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The antioxidant activities exerted by TRF were most likely limited to \u003cem\u003esod2\u003c/em\u003e, as \u003cem\u003esod1\u003c/em\u003e was found to be downregulated and \u003cem\u003egpx1\u003c/em\u003e was found to be only slightly increased. Downregulation of \u003cem\u003esod1\u003c/em\u003e following TRF supplementation was also reported in other studies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], suggesting that \u003cem\u003esod1\u003c/em\u003e is most likely not modulated by TRF in promoting minimal \u003cem\u003egpx1\u003c/em\u003e activity. However, the positive effect of TRF supplementation was observed to be not limited to \u003cem\u003esod2\u003c/em\u003e, but it may act as a signaling molecule that affects several biochemical pathways based on findings in this study.\u003c/p\u003e \u003cp\u003eJoint pathway analysis combining the fold changes of investigated genes (\u003cem\u003esod1, sod2, gpx1, fxr, shp and stat3\u003c/em\u003e) and metabolites as listed in Table S7 generated a total of 12 important biochemical pathways. The most significant biochemical pathway (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) with a 4.39-fold higher fold change compared to the HFD group was bile secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The genes involved in bile secretion with TRF supplementation were \u003cem\u003efxr\u003c/em\u003e (5.17-fold higher in TRF group) and \u003cem\u003eshp\u003c/em\u003e (3.66-fold higher), and the metabolites involved were taurocholic acid (fold change: 3.28) and taurochenodeoxycholic acid (fold change: 3.84). The increase in \u003cem\u003efxr\u003c/em\u003e gene expression, most likely from TRF supplementation, promotes the expression of \u003cem\u003eshp\u003c/em\u003e as a result of nuclear translocation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The increase in \u003cem\u003eshp\u003c/em\u003e promotes the inhibition of bile acid reuptake by suppressing the Na\u003csup\u003e+\u003c/sup\u003e taurocholate cotransporting polypeptide (NTCP) transporter. Compared to other target organs, the action of bile acids (BAs) on cardiomyocytes is indirect [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] in regulating myocardial function, and the expression of \u003cem\u003efxr\u003c/em\u003e has been reported to be lower than that in vascular smooth muscles [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, other conjugated bile acids, such as taurocholic acid (TCA) and taurochenodeoxycholic acid (TCDCA), were found to be increased above 3-fold in the TRF group compared to the HFD group. The presence and increase in these bile acids (BAs), apart from being involved in BA secretion and primary BA synthesis (2.32-fold increase), were most likely suggestive of anti-inflammatory properties exerted by these BAs [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] on the mice. Therefore, TRF supplementation in mice subjected to HFD was most likely elicited as a signaling molecule on \u003cem\u003efxr\u003c/em\u003e, thus modulating \u003cem\u003eshp\u003c/em\u003e and \u003cem\u003estat3\u003c/em\u003e gene expression.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, TRF supplementation at 200 mg/kg/day for 8 weeks does not affect the body weight, waist circumference, blood pressure or random blood glucose of HFD-fed mice. However, the expression of \u003cem\u003esod1\u003c/em\u003e was reduced significantly in TRF-supplemented mice, suggesting low superoxide dismutase 1 activity, possibly due to low radical oxygen species levels, reflecting the role of TRF as an exogenous antioxidative substance. According to the metabolomic analysis, supplementation of TRF in HFD-fed mice may also act as a signaling molecule affecting several biochemical pathways, such as bile acid biosynthesis and secretion. Although the plasma level of bile acids was not measured in this study, the significant increase in \u003cem\u003efxr\u003c/em\u003e and its target gene \u003cem\u003eshp\u003c/em\u003e following TRF supplementation suggests an interesting involvement of TRF in bile acid signaling.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank\u0026nbsp;the\u0026nbsp;Research Management Centre team of Universiti Teknologi MARA for their support and aid in completing this project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project described was funded by\u0026nbsp;the\u0026nbsp;Malaysia Ministry of Higher Education with grant code RACER/1/2019/SKK08/UITM//9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: S.H.S.A.K., S.A.R., E.I. ; Methodology: S.H.S.A.K., S.A.R., E.I. ; Formal Analysis: M.D.M.E.G. ; Investigation: M.D.M.E.G., N.A.N.M.S. ; Resources: S.H.S.A.K. ; Data Curation: M.D.M.E.G. ; Writing \u0026ndash; Original Draft: M.D.M.E.G., N.A.N.M.S. ; Writing \u0026ndash; Reviewing \u0026amp; Editing: S.H.S.A.K., S.A.R., E.I. ; Visualization: M.D.M.E.G. ; Supervision: S.H.S.A. K; Funding Acquisition: S.H.S.A. K\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no\u0026nbsp;conflicts\u0026nbsp;of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld health statistics 2023: monitoring health for the SDGs, Sustainable Development Goals. Geneva: World Health Organization; 2023. Licence: CC BY‑NC‑SA 3.0 IGO\u003c/li\u003e\n\u003cli\u003eCromwell WC, Otvos JD, Keyes MJ, Pencina MJ, Sullivan L, Vasan RS, Wilson PW, D\u0026apos;Agostino RB. LDL Particle Number and Risk of Future Cardiovascular Disease in the Framingham Offspring Study - Implications for LDL Management. J Clin Lipidol. 2007;1(6):583-92. doi: 10.1016/j.jacl.2007.10.001\u003c/li\u003e\n\u003cli\u003eWoods SC, Seeley RJ, Rushing PA, D\u0026apos;Alessio D, Tso P. A controlled high-fat diet induces an obese syndrome in rats. 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Ecotoxicol Environ Saf. 2021;222:112461. doi: 10.1016/j.ecoenv.2021.112461. \u003c/li\u003e\n\u003cli\u003eNagarajan V, Gopalan V, Kaneko M, Angeli V, Gluckman P, Richards AM, Kuchel PW, Velan SS. Cardiac function and lipid distribution in rats fed a high-fat diet: in vivo magnetic resonance imaging and spectroscopy. Am J Physiol Heart Circ Physiol. 2013;304(11):H1495-504. doi: 10.1152/ajpheart.00478.2012.\u003c/li\u003e\n\u003cli\u003eAvtanski D, Pavlov VA, Tracey KJ, Poretsky L. Characterization of inflammation and insulin resistance in high-fat diet-induced male C57BL/6J mouse model of obesity. Animal Model Exp Med. 2019;2(4):252-258. doi: 10.1002/ame2.12084\u003c/li\u003e\n\u003cli\u003eFraulob JC, Ogg-Diamantino R, Fernandes-Santos C, Aguila MB, Mandarim-de-Lacerda CA. A Mouse Model of Metabolic Syndrome: Insulin Resistance, Fatty Liver and Non-Alcoholic Fatty Pancreas Disease (NAFPD) in C57BL/6 Mice Fed a High Fat Diet. 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Nutr Metab (Lond). 2020;17:42. doi: 10.1186/s12986-020-00458-8.\u003c/li\u003e\n\u003cli\u003eIrandoost P, Mesri Alamdari N, Saidpour A, Shidfar F, Roshanravan N, Asghari Jafarabadi M, Farsi F, Asghari Hanjani N, Vafa M. The effects of royal jelly and tocotrienol-rich fraction on impaired glycemic control and inflammation through irisin in obese rats. J Food Biochem. 2020;44(12):e13493. doi: 10.1111/jfbc.13493.\u003c/li\u003e\n\u003cli\u003eAllen L, Ramalingam L, Menikdiwela K, Scoggin S, Shen CL, Tomison MD, Kaur G, Dufour JM, Chung E, Kalupahana NS, Moustaid-Moussa N. Effects of delta-tocotrienol on obesity-related adipocyte hypertrophy, inflammation and hepatic steatosis in high-fat-fed mice. J Nutr Biochem. 2017;48:128-137. doi: 10.1016/j.jnutbio.2017.07.003.\u003c/li\u003e\n\u003cli\u003eBudin SB, Othman F, Louis SR, Bakar MA, Das S, Mohamed J. 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Histochem Cell Biol. 2013;140(2):105-17. doi: 10.1007/s00418-013-1099-4.\u003c/li\u003e\n\u003cli\u003eDurani LW, Jaafar F, Tan JK, Tajul Arifin K, Mohd Yusof YA, Wan Ngah WZ, Makpol S. Targeting genes in insulin-associated signalling pathway, DNA damage, cell proliferation and cell differentiation pathways by tocotrienol-rich fraction in preventing cellular senescence of human diploid fibroblasts. Clin Ter. 2015;166(6):e365-73. doi: 10.7417/T.2015.1902.\u003c/li\u003e\n\u003cli\u003eClaudel T, Staels B, Kuipers F. The Farnesoid X receptor: a molecular link between bile acid and lipid and glucose metabolism. Arterioscler Thromb Vasc Biol. 2005;25(10):2020-30. doi: 10.1161/01.ATV.0000178994.21828.a7.\u003c/li\u003e\n\u003cli\u003eKhurana S, Raufman JP, Pallone TL. Bile acids regulate cardiovascular function. Clin Transl Sci. 2011;4(3):210-8. doi: 10.1111/j.1752-8062.2011.00272.x.\u003c/li\u003e\n\u003cli\u003eQi YC, Duan GZ, Mao W, Liu Q, Zhang YL, Li PF. Taurochenodeoxycholic acid mediates cAMP-PKA-CREB signaling pathway. Chin J Nat Med. 2020 Dec;18(12):898-906. doi: 10.1016/S1875-5364(20)60033-4.\u003c/li\u003e\n\u003cli\u003eTalebian R, Hashem O, Gruber R. Taurocholic acid lowers the inflammatory response of gingival fibroblasts, epithelial cells, and macrophages. J Oral Sci. 2020;62(3):335-339. doi: 10.2334/josnusd.19-0342.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genes-and-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gnnu","sideBox":"Learn more about [Genes \u0026 Nutrition](http://genesandnutrition.biomedcentral.com)","snPcode":"12263","submissionUrl":"https://submission.nature.com/new-submission/12263/3","title":"Genes \u0026 Nutrition","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"tocotrienols, farnesoid-x receptor, animal model, high-fat diet, antioxidant","lastPublishedDoi":"10.21203/rs.3.rs-3757079/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3757079/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTocotrienol-rich fraction (TRF) has been reported to protect the heart from oxidative stress-induced inflammation. It is, however, unclear whether the protective effects TRF against oxidative stress involve the activation of farnesoid-x receptor (\u003cem\u003efxr\u003c/em\u003e), a bile acid receptor, and the regulation of bile acid metabolites. In the current study, we investigated the effects of TRF supplementation on antioxidant activities, expression of \u003cem\u003efxr\u003c/em\u003e and its target genes in cardiac tissue, and serum untargeted metabolomics of high-fat diet-fed mice. Mice were divided into high-fat diet (HFD) with or without TRF supplementation (control) for six weeks. At the end of the intervention, weight (BW), waist circumference (WC), and random blood glucose were measured. Heart tissues were collected, and the gene expression of \u003cem\u003esod1, sod2, gpx\u003c/em\u003e and \u003cem\u003efxr\u003c/em\u003e and its target genes \u003cem\u003eshp\u003c/em\u003e and \u003cem\u003estat3\u003c/em\u003e was determined. Serum was subjected to untargeted metabolomic analysis using UHPLC-Orbitrap. In comparison to the control, the WC of the TRF-treated group was significantly higher (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) than that of the HFD-only group, but there was no significant difference in weight or random blood glucose level. Downregulation of \u003cem\u003esod1, sod2\u003c/em\u003e and \u003cem\u003egpx\u003c/em\u003e expression was observed in TRF-treated mice; however, only \u003cem\u003esod1\u003c/em\u003e was significant when compared to the HFD only group. The expression of cardiac \u003cem\u003efxr\u003c/em\u003e and \u003cem\u003eshp\u003c/em\u003e was significantly upregulated, but stat3 was significantly downregulated in the TRF-treated group compared to the HFD-only group. Biochemical pathways found to be influenced by TRF supplementation include bile acid secretion, primary bile acid biosynthesis, and biotin and cholesterol metabolism. In conclusion, TRF supplementation in HFD-fed mice affects antioxidant activities, and more interestingly, TRF also acts as a signaling molecule that is possibly involved in several bile acid-related biochemical pathways accompanied by an increase in cardiac \u003cem\u003efxr shp\u003c/em\u003e expression. This study provides new insight into TRF in deregulating bile acid receptors and metabolites in high-fat diet-fed mice.\u003c/p\u003e","manuscriptTitle":"Palm-based tototrienol-rich fraction (TRF) supplementation modulates cardiac sod1 expression, fxr target gene expression and tauro-conjugated bile acid levels in aleptinemic mice fed a high-fat diet","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-20 19:38:17","doi":"10.21203/rs.3.rs-3757079/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-04T09:03:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-12-22T16:29:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d6e1173a-9ad8-4ab4-a848-9572372dd9ef","date":"2023-12-19T12:38:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-19T10:47:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-18T05:23:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-18T05:23:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genes \u0026 Nutrition","date":"2023-12-15T06:43:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genes-and-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gnnu","sideBox":"Learn more about [Genes \u0026 Nutrition](http://genesandnutrition.biomedcentral.com)","snPcode":"12263","submissionUrl":"https://submission.nature.com/new-submission/12263/3","title":"Genes \u0026 Nutrition","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"39422d24-49b1-4816-9f4e-ad2d21ddf341","owner":[],"postedDate":"December 20th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-04T15:09:39+00:00","versionOfRecord":{"articleIdentity":"rs-3757079","link":"https://doi.org/10.1186/s12263-024-00742-9","journal":{"identity":"genes-and-nutrition","isVorOnly":false,"title":"Genes \u0026 Nutrition"},"publishedOn":"2024-02-27 15:02:06","publishedOnDateReadable":"February 27th, 2024"},"versionCreatedAt":"2023-12-20 19:38:17","video":"","vorDoi":"10.1186/s12263-024-00742-9","vorDoiUrl":"https://doi.org/10.1186/s12263-024-00742-9","workflowStages":[]},"version":"v1","identity":"rs-3757079","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3757079","identity":"rs-3757079","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}