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Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice Anusha Jahagirdar a, c , Dandamudi Usharani b, Malathi Srinivasan a, c , Ram Rajasekharan a, c, * a b c
Lipidomics Center, Department of Lipid Science, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, 570020, India Food Safety & Analytical Quality Control Laboratory, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, 570020, India Academy of Scientific and Innovative Research (AcSIR), CSIR-Central Food Technological Research Institute, Mysore, Karnataka, 570020, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2018 Accepted 30 October 2018 Available online xxx
Brown adipose tissue (BAT) is the site of non-shivering thermogenesis in mammals, wherein energy is dissipated as heat. We observed that aqueous extract of black sesame seed triggers an increase in the expression of Uncoupling Protein 1 (UCP1) in brown adipocytes from mice. The active component from the extract was purified and identified to be sesaminol diglucoside (SDG). SDG treatment decreased mass of white fat pads and serum glucose levels and increased UCP1 levels in BAT thereby protecting mice against high fat induced weight gain. Further in silico and in vitro studies revealed that these effects are due to the agonist like behaviour of SDG towards beta 3 adrenergic receptors (b3-AR). Together, our results suggest that SDG induces BAT mediated thermogenesis through b3-AR and protects mice against diet-induced obesity. © 2018 Elsevier Inc. All rights reserved.
Keywords: Sesame Sesaminol diglucoside Brown adipose tissue Uncoupling protein 1 b3 adrenergic receptor
1. Introduction Obesity and metabolic disorders arise due to a mismatch between energy intake and energy expenditure. Once considered as a problem only in developed countries, the incidence of obesity has dramatically increased even in developing countries, especially in urban areas. Since obesity arises because of excess energy storage, increasing energy expenditure through thermogenesis can be harnessed to treat this state of energy imbalance [1]. Brown fat/adipose tissue (BAT) is a mitochondria-rich adipose tissue, specialized for non-shivering thermogenesis in mammals. It functions through the activity of uncoupling protein 1 (UCP1) that uncouples mitochondrial oxidative phosphorylation and produces heat to maintain body temperature. It is distinct from white adipose tissue (WAT), which is specialized to store triglycerides and functions as an energy store [2]. Studies have shown that BAT is activated not only to maintain body temperature, but also to dissipate the excess calories that accumulate due to prolonged consumption
Abbreviations: BAT, Brown adipose tissue; UCP1, Uncoupling Protein 1; SDG, Sesaminol diglucoside; HFD, High fat diet; b3-AR, beta 3 adrenergic receptor. * Corresponding author. Lipidomics Center, Department of Lipid Science, CSIRCentral Food Technological Research Institute, Mysuru, Karnataka, 570020, India. E-mail addresses: [email protected], [email protected] (R. Rajasekharan).
of nutrient-poor fat-rich diets [3]. Adult humans usually have about 50e80 g of BAT, and when activated it can burn up to 20 percent of daily energy intake which would be equivalent to reducing 4e20 kg weight in a year [4]. Additionally, BAT also reduces excessive glucose and triglyceride concentrations and hence can be used to alleviate obesity in humans [5]. Dietary molecules that increase UCP1 expression are therefore increasingly being considered as attractive anti-obesity therapies as they are safer for long-term consumption [6]. More recently, studies have reported an increase in energy expenditure due to various dietary components, which include butein, capsaicin, fucoxanthin, leucine, methionine and resveratrol [7e11]. Many food sources are traditionally known to induce thermogenesis and aid in maintaining body temperature. However, it is not evident whether this is through the activation of BAT mediated non-shivering thermogenesis. To this end, we screened various food sources for their effect on non-shivering thermogenesis and identified Sesamum indicum (Sesame) as a potent inducer of BAT activity. Sesame seeds contain a high amount of oil (up to 60%) [12], which is rich in polyphenolic lignans, sesamin, sesaminol and sesamolin. These lignans have been reported to exhibit various therapeutic properties such as reduction of hypertension, inflammation and carcinogenesis [13e15]. Sesame seed hulls are also an
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Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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abundant source of phenolic compounds, which possess considerable antioxidant activities [16]; however, biochemical properties of the water-soluble compounds in sesame seed and defatted sesame seed flour have attracted less attention. In the present study, we found that the aqueous extract of black sesame seeds induced UCP1 activity in brown adipocytes in vitro and in vivo in mice. The molecule responsible for this activity was identified as sesaminol diglucoside (SDG). SDG treatment in mice led to enhanced BAT metabolism, reduced serum glucose levels and protected the mice against high fat diet (HFD) induced obesity. Further studies revealed that these effects of SDG are mediated via beta 3 adrenergic receptors (b3-AR). Together, our results establish that SDG induces BAT activation through b3-AR and therefore, thermogenic food molecules represent a potential complementary and safe approach to combat diet-induced obesity and associated metabolic disease.
2. Materials and methods 2.1. Extract preparation 100 g of black sesame seeds sourced locally were ground in a blender and defatted with ten volumes of hexane (Merck-Millipore) for 24 h. The defatted seed powder was further extracted for 24 h with distilled water. The aqueous extract was lyophilized and used for further experiments.
2.2. SDG purification and characterization The aqueous extract was fractionated using preparative HPLC on a Waters Spherisorb C18 ODS (10m, 250 mm) semi-prep column with a mobile phase of methanol: water: 10:90 to 90:10 (v/v) for 60 min at a flow rate of 5ml/min; the wavelength used for detection was 280 nm. The fractions collected were screened for UCP1 inducing activity, and the positive fraction was further purified on a Biogel-P2 (Sigma-Aldrich) column and was eluted with water at a flow rate of 0.6ml/min for 17 h. 86 fractions were collected in total and were assayed for the presence of carbohydrates by the phenolsulphuric acid method [17]. The mass of the positive fractions was determined using an AB Sciex TripleTOF™ 5600 system. The MS and MS/MS scans were recorded.
2.3. Preparation of brown fat explant Interscapular BAT was excised from 2 months old male C57BL6/J mice and washed with sterile Phosphate Buffer Saline (PBS). The tissue was minced into small pieces approximately 1e2 mm and incubated in DMEM-F12 medium with 10% Foetal Bovine Serum (FBS) (ThermoFisher) and the extract, or compounds, for a period of 4 h at 37 C. For treatment with pan b - adrenergic antagonist propranolol (Sigma-Aldrich), explants were treated with 100 mM propranolol for 30 min [18] following which they were treated with SDG (Nacalai-Tesque).
2.5. Immunoblotting BAT explants and tissue sections were lysed in RIPA buffer containing 0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5. Cell lysates, normalized for protein concentration, were resolved on a 12% SDS-polyacrylamide gel and were transferred to a PVDF membrane (GE healthcare). The blots were probed with primary antibody overnight at 4 C and with horseradish peroxidase-conjugated secondary antibodies (SigmaAldrich) for 1 h at room temperature. 2.6. Animal studies Animal handling and procedures were conducted with the approval of the Institutional Animal Ethics Committee (IAEC, CSIRCFTRI). Eight weeks old C57BL/6J mice were fed a standard diet (AIN-93M) with 10% kcal fat or 60% kcal fat (Table S1) in the case of high fat diet (HFD) fed animals. The animals were housed at 22 C ± 2 under a standard 12 h light/dark cycle. Body weight, feed intake and surface temperature above interscapular BAT were monitored daily. Mice were fed standard diet (10% kcal fat) and treated with aqueous extract of sesame, or with vehicle by oral gavage (20 mg/kg BW) for two weeks. The treatment with purified SDG consisted of the following groups: normal diet (ND) control (10% kcal fat), HFD control (60% kcal fat) and SDG treated group (60% kcal fat along with SDG 5 mg/kg BW). The mice were injected intraperitoneally with SDG or vehicle for ten days. Post treatment mice were euthanized by carbon dioxide inhalation, and tissues and blood were harvested. Serum levels of glucose, triglycerides and cholesterol were estimated using kits from ERBA Mannheim Diagnostics Gmbh, Germany. 2.7. H & E staining BAT sections were fixed with 4% paraformaldehyde (SigmaAldrich) overnight and then sequentially dehydrated with increasing concentration of ethanol. The fixed and dehydrated tissues were sectioned after being embedded in paraffin. Multiple sections were used for hematoxylin-eosin (H& E) staining. 2.8. In silico modeling A theoretical model of b3-AR of mouse which is 60.07% homologous to b1 receptor (4AMJ) [19] was built using homology modeling methodology in MODELLER 9.14. The agonists such as noradrenaline, CL-316,243 and SDG molecules were quantum mechanically optimised at B3LYP/def-SVP basis set [20] using TURBOMOLE program package (TURBOMOLE V6.6, 2014). Molecular docking was carried out using Autodock 4.2.6. 2.9. Statistics Data are expressed as mean ± standard error of mean (SEM) and significance testing was performed by two-way ANOVA or Student's t-test. Statistical significance was set at p < 0.05 and data were analysed using GraphPad Prism software 5.0.
2.4. Real-time PCR
3. Results
Post treatment, the explants and cells were lysed in Trizol (Sigma-Aldrich), and RNA was isolated using Qiagen pure link RNA isolation kit. 1 mg RNA was reverse transcribed to cDNA using ABI high capacity cDNA synthesis kit. The cDNA was used for quantitative PCR in BioRad CFX96 Touch™ Real-Time PCR Detection System. The primers used for qPCR are listed in Table S2.
3.1. Sesame extract induces UCP1 in brown adipocytes To explore the relationship between thermogenic foods and BAT activity, several foods traditionally known to induce thermogenesis were screened for Ucp1 inducing activity (data not shown). Among the screened foods, we found that defatted aqueous extract of black
Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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sesame seeds, Sesamum indicum (Fig. 1A), triggered a significant increase in the expression of Ucp1 in BAT explants. Real-time PCR and immunoblot analysis of brown adipocytes treated for 4 h with aqueous extract of black sesame seeds revealed that Ucp1 mRNA expression nearly doubled (Fig. 1B) and UCP1 protein levels increased two-fold compared to untreated cells (Fig. 1C). These results indicate that black sesame seed extract induces UCP1 in vitro. To determine the UCP1 inducing effects in vivo, C57BL6/J mice were treated orally with 20 mg/kg BW aqueous extract of black sesame seeds for two weeks. Despite similar food intake, there was an 8% decrease in body mass in the sesame extract treated mice (Fig. 1D). The weight loss could be attributed to an increase in energy expenditure, which was reflected by the rise in surface body temperature over the interscapular BAT area (Fig. 1E). This result was further confirmed by H&E staining of paraformaldehyde-fixed BAT tissue sections (Fig. 1G) which displayed much smaller lipid droplets in BAT from treated mice. The analysis of lipid parameters in the serum of treated mice also showed a reduction in the levels of total cholesterol and triglycerides (Fig. 1F). Western blot analyses of protein from treated BAT displayed a marked increase in UCP1 protein levels as well (Fig. 1H), confirming the activation of BAT thermogenesis in sesame extract treated mice. 3.2. SDG purified from sesame extract triggers a dose-dependent increase in UCP1 in vitro Aqueous extract of black sesame seed was subjected to preparative HPLC analysis, to identify the active component responsible for the induction of UCP1 (Fig. 2B). The fractions were screened based on their ability to induce UCP1 protein in BAT explants in vitro. The selected fractions were further purified by Biogel-P2 size exclusion chromatography (Fig. 2C). The various fractions from Biogel-P2 were tested for UCP1 inducing ability, and the positive fraction was analysed by ESI-MS to determine its
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homogeneity and molecular mass. In positive mode, a major peak corresponding to m/z ratio of 717.1 (Fig. 2E) was observed. The peaks obtained (347.1, 555.1 and 484.9) on MS/MS analysis (Fig. 2F) matched with that of the reported molecule, Sesaminol diglucoside (MW: 694) which appears as a sodium adduct (MW: 717) in positive mode [21]. The structure of the purified molecule was studied by 1H NMR and 13C NMR and was validated as SDG by comparing with the NMR profile of commercially obtained standard (Fig. S1). On treating BAT explants with increasing concentrations of SDG for 4 h, we noticed a corresponding dose-dependent increase in the levels of UCP1 protein (Fig. 2D). Commercially, SDG is available in two isomeric forms SDG (1, 6) and SDG (1, 2). The increase in UCP1 expression was observed only with the SDG (1, 6) isomer (Fig. S2) and therefore future experiments were restricted to the (1, 6) isomer of SDG. 3.3. Treatment with SDG prevents weight gain in HFD fed mice Given the remarkable brown adipogenic activity of SDG in vitro, next, we investigated the effect of SDG in vivo. We administered male C57BL6/J mice with vehicle (saline) or SDG (5 mg/kg) i.p for ten days along with a HFD. SDG treated mice gained less body weight compared to control mice (Fig. 3A) despite no change in feed consumption (Fig. S3). Surface temperature measurements of the mice, right above the interscapular BAT region revealed a modest but significant increase in body temperature in treated mice (Fig. 3C). These data suggest that SDG probably decreases body weight through an increase in energy expenditure in vivo. To understand in detail the changes in body composition, the masses of the various fat pads were also recorded and examined at the end of the study. There was no significant change in the mass of the interscapular BAT, however inguinal and epididymal WAT masses significantly reduced in SDG treated mice (Fig. 3B). Furthermore, serum levels of glucose (Fig. 3D) reduced to a significant extent in treated mice, and H&E staining revealed much smaller lipid
Fig. 1. Sesame extract induces thermogenesis. (A) Schematic representation of the preparation of aqueous extract from black sesame seeds. (B) mRNA expression of Ucp1 gene on treating BAT explants with sesame extract for 4 h. (C) Western blot of UCP1 protein in sesame extract treated BAT explants. 2 month old C57BL6/J mice were treated with vehicle(saline) or sesame extract (20 mg/kg, BW) by oral gavage for two weeks (n ¼ 6). (D) Body weight and feed consumption. Bar graph represents body weight (g), and line graph represents feed intake (g) per animal per day (E) Surface temperature measured on the interscapular BAT area. (F) Total cholesterol and triglyceride levels (G) H & E staining of formalin fixed paraffin embedded (PPFE) BAT sections. (H) Immunoblot of UCP1 from BAT samples. Values are represented as mean ± SEM. p values were determined by Student's ttest. *p < 0.05, **p < 0.01.
Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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Fig. 2. Purification and characterization of Sesaminol diglucoside (SDG) from sesame extract. (A) Schematic representation of purification procedure of sesame extract (B) Preparative HPLC of aqueous extract from sesame seeds. (C) Major fractions observed with Biogel-P2 separation of selected peaks after preparative HPLC. (D) Dose-dependent effect of SDG on UCP1 expression in BAT explants. (E) MS spectrum of the purified compound in the positive mode with major peak at m/z ¼ 717. (F) Parent and fragment ion peaks in the MS/MS spectrum of SDG. (G) Structure of SDG.
Fig. 3. SDG treatment decreases body weight and lipid parameters in vivo. Two month old C57BL6/J mice received vehicle (saline) or SDG (5 mg/kg) for ten days i.p. (n ¼ 6 per group) along with an acute HFD challenge. (A) Body weight of vehicle-(saline) and treated mice. (B) Mass of BAT, iWAT and eWAT. (C) Surface temperature measured on the interscapular BAT area (D) Serum glucose (E) H & E staining of PPFE BAT sections. (F) Western blot analysis of UCP1 in BAT tissue. Values are mean ± SEM. p values were determined by two-way ANOVA. *p < 0.05. ND- Normal diet; HFD- High fat diet.
droplets in BAT of SDG treated mice (Fig. 3E), representing the overall favorable effect of SDG on glucose and lipid parameters of treated animals. Western blot analyses revealed that the levels of essential thermogenic gene Ucp1 increased significantly (Fig. 3F). Taken together, these data suggest that SDG exerts a protective effect against HFD induced phenotype by increasing energy expenditure through UCP1.
3.4. Induction of thermogenesis by SDG is beta-adrenergic dependent
b3-AR is a seven transmembrane helix containing protein, predominantly found in adipose tissue [22]. Multiple sequence alignment of human and mouse b3-ARs along with b2 and b1-ARs show that human and mouse b3 protein sequence has ~80% sequence
Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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similarity and active sites are conserved throughout (Fig. S4). Understanding the molecular interaction of SDG towards the mouse b3-AR in silico revealed that it embedded in the same active site pocket as the known agonists, noradrenaline and CL-316,243 (Fig. 4A). Ser208, Ser205 (TM5) and Asp114 (TM3) from the top and Asn329 (TM6 and TM7) from the bottom of the active site hold the agonists through various H-bond interactions as observed in b receptors [19,23]. SDG additionally gains hydrophobic interactions at the active site and was found to have similar binding energies as the synthetic agonists (Fig. 4B, Table S3). Whereas in human b3-AR receptor the glycoside moiety of SDG is facing towards the surface, resulting in lower binding energy (Fig. S5, Fig. 4B). This effect was validated in vitro by treating BAT explants with SDG in the presence of b-adrenergic receptor antagonist propranolol. Treatment of brown adipocytes with SDG in the presence of propranolol diminished the induction of Ucp1 mRNA (Fig. 4C) and protein expression (Fig. 4D). These results suggest that SDG has a promising binding affinity towards the b3-AR receptor in vitro. Together, our results suggest that SDG induces BAT activation and thermogenesis in mice, potentially in a b3-AR-dependent manner. 4. Discussion In sixty-five percent of the world's population, overweight and obesity kill more people than malnutrition [24]. Dieting and exercise are the most widely recommended treatments; however, the success rate for these range a meagre 2e20%, due to difficulty in adhering to these regimens in the long term. Other options currently available are bariatric surgery and oral prescription drugs. Oral prescription medications available in the market suffer from
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low success rates and serious side effects including gastric issues, nausea, dizziness and liver damage [25]. Therefore, dietary sources are a promising avenue to identify molecules that increase energy expenditure and are safer for long-term consumption. The current study indicates that aqueous extract of black sesame seed increases whole-body energy expenditure, which could be useful in preventing metabolic disorders. Our data demonstrate that black sesame seed extract increases the expression of the major thermogenic gene Ucp1 in brown adipocyte explants and induces weight loss in vivo in mice. The weight loss was accompanied by a concurrent increase in body temperature, which indicated that the weight loss was due to an increase in energy expenditure. Sesame seeds have been used over the years in traditional medicine to treat various health conditions [26]. However, no reports exist concerning its effect on BAT thermogenesis. The aqueous extract of sesame seeds is reported to contain lignan glucosides apart from other small molecules, which exert beneficial pharmacological effects such as anti-hypertensive effect in prehypertensive human patients and elevation of g-tocopherol levels [27,28]. Purification and activity-based assays revealed that SDG present in the aqueous fraction of sesame seeds mediated our observed effects on BAT metabolism. Our data demonstrate that SDG increases the expression of the major thermogenic gene Ucp1 in brown adipocytes and protects against HFD-induced weight gain in mice. SDG also displayed a positive effect on serum glucose levels. BAT activation clears excessive glucose in the plasma by increasing glucose uptake into BAT and using it as a substrate for thermogenesis thereby improving insulin sensitivity and weight loss [29]. These results
Fig. 4. SDG induces Ucp1 in a b3-AR dependent manner. (A) Binding poses of agonists such as noradrenaline, CL-316,243 and SDG in the active site of mouse b3-AR. Crucial active site residues and the respective transmembrane domains are highlighted. Yellow dotted lines represent the hydrogen bonding interactions between the agonists and the receptor. Note that all these molecules have crucial H-bonding interactions with Ser205/Ser209 of TM5 and residue numbering is according to 4AMJ crystal structure. (B) The estimated free energy of binding (DG, kcal/mol) of various agonists towards human and mouse b3-ARs were obtained from MM/GBSA calculations. BAT explants were treated with CL-316,243 or SDG in the presence or absence of propranolol (C) Relative Ucp1 mRNA as measured by RT-qPCR. (D) UCP1 protein levels assayed by Western blot. p values are represented with asterisks (*p < 0.05, **p < 0.01) and error bars represent SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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suggest that the increase in BAT thermogenic function through SDG treatment could contribute to the clearance of excessive glucose and lipids, which prevents weight gain on a HFD. b3-ARs present on the surface of brown adipocytes are one of the major contributors to cold-induced activation of BAT thermogenesis. Noradrenaline is the biological ligand for b3-AR, however many synthetic ligands including CL-316,243 have been identified [30]. Since b3-AR is the major activator of BAT thermogenesis, we evaluated the binding affinity of SDG towards b3-AR in silico. Our results revealed that SDG embeds in the same active site pocket as the known agonists noradrenaline and CL-316,243 with promising binding energies. In vitro assays in the presence of the b3-AR antagonist propranolol further confirmed the in silico results. However, since a complex adipogenic network exists in these cells, we cannot rule out the role of other transcription factors for observed energy expenditure in in vivo studies [31]. Together, our results suggest that SDG prevents weight gain on HFD by inducing b3-AR dependent BAT thermogenesis. Funding This study was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, under the 12th 5-year plan project LIPIC (BSC0401). AJ was supported by a fellowship from CSIR, New Delhi. RR is a recipient of the JC Bose national fellowship. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgements We are grateful to Mr. Shinde Vijay Sukhdeo for his help with the animal studies and Ms. Jayasundarnaidu Megha for initial in silico studies. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.195. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.195. References [1] J.O. Hill, H.R. Wyatt, J.C. Peters, Energy balance and obesity, Circulation 126 (2012) 126e132. [2] D.G. Nicholls, R.M. Locke, Thermogenic mechanisms in brown fat, Physiol. Rev. 64 (1984) 1e64. [3] N.J. Rothwell, M.J. Stock, B.P. Warwick, Energy balance and brown fat activity in rats fed cafeteria diets or high-fat, semisynthetic diets at several levels of intake, Metab., Clin. Exp. 34 (1982) 474e480. [4] K.A. Virtanen, M.E. Lidell, J. Orava, M. Heglind, R. Westergren, T. Niemi, M. Taittonen, J. Laine, N.J. Savisto, S. Enerback, P. Nuutila, Functional brown adipose tissue in healthy adults, N. Engl. J. Med. 360 (2009) 1518e1525. [5] A. Bartelt, O.T. Bruns, R. Reimer, H. Hohenberg, H. Ittrich, K. Peldschus, M.G. Kaul, U.I. Tromsdorf, H. Weller, C. Waurisch, A. Eychmuller, P.L. Gordts, F. Rinninger, K. Bruegelmann, B. Freund, P. Nielsen, M. Merkel, J. Heeren, Brown adipose tissue activity controls triglyceride clearance, Nat. Med. 17 (2011) 200e205. [6] I. Vermaak, A.M. Viljoen, J.H. Hamman, Natural products in anti-obesity therapy, Nat. Prod. Rep. 28 (2011) 1493e1533.
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Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice Anusha Jahagirdar a, c , Dandamudi Usharani b, Malathi Srinivasan a, c , Ram Rajasekharan a, c, * a b c
Lipidomics Center, Department of Lipid Science, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, 570020, India Food Safety & Analytical Quality Control Laboratory, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, 570020, India Academy of Scientific and Innovative Research (AcSIR), CSIR-Central Food Technological Research Institute, Mysore, Karnataka, 570020, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2018 Accepted 30 October 2018 Available online xxx
Brown adipose tissue (BAT) is the site of non-shivering thermogenesis in mammals, wherein energy is dissipated as heat. We observed that aqueous extract of black sesame seed triggers an increase in the expression of Uncoupling Protein 1 (UCP1) in brown adipocytes from mice. The active component from the extract was purified and identified to be sesaminol diglucoside (SDG). SDG treatment decreased mass of white fat pads and serum glucose levels and increased UCP1 levels in BAT thereby protecting mice against high fat induced weight gain. Further in silico and in vitro studies revealed that these effects are due to the agonist like behaviour of SDG towards beta 3 adrenergic receptors (b3-AR). Together, our results suggest that SDG induces BAT mediated thermogenesis through b3-AR and protects mice against diet-induced obesity. © 2018 Elsevier Inc. All rights reserved.
Keywords: Sesame Sesaminol diglucoside Brown adipose tissue Uncoupling protein 1 b3 adrenergic receptor
1. Introduction Obesity and metabolic disorders arise due to a mismatch between energy intake and energy expenditure. Once considered as a problem only in developed countries, the incidence of obesity has dramatically increased even in developing countries, especially in urban areas. Since obesity arises because of excess energy storage, increasing energy expenditure through thermogenesis can be harnessed to treat this state of energy imbalance [1]. Brown fat/adipose tissue (BAT) is a mitochondria-rich adipose tissue, specialized for non-shivering thermogenesis in mammals. It functions through the activity of uncoupling protein 1 (UCP1) that uncouples mitochondrial oxidative phosphorylation and produces heat to maintain body temperature. It is distinct from white adipose tissue (WAT), which is specialized to store triglycerides and functions as an energy store [2]. Studies have shown that BAT is activated not only to maintain body temperature, but also to dissipate the excess calories that accumulate due to prolonged consumption
Abbreviations: BAT, Brown adipose tissue; UCP1, Uncoupling Protein 1; SDG, Sesaminol diglucoside; HFD, High fat diet; b3-AR, beta 3 adrenergic receptor. * Corresponding author. Lipidomics Center, Department of Lipid Science, CSIRCentral Food Technological Research Institute, Mysuru, Karnataka, 570020, India. E-mail addresses: [email protected], [email protected] (R. Rajasekharan).
of nutrient-poor fat-rich diets [3]. Adult humans usually have about 50e80 g of BAT, and when activated it can burn up to 20 percent of daily energy intake which would be equivalent to reducing 4e20 kg weight in a year [4]. Additionally, BAT also reduces excessive glucose and triglyceride concentrations and hence can be used to alleviate obesity in humans [5]. Dietary molecules that increase UCP1 expression are therefore increasingly being considered as attractive anti-obesity therapies as they are safer for long-term consumption [6]. More recently, studies have reported an increase in energy expenditure due to various dietary components, which include butein, capsaicin, fucoxanthin, leucine, methionine and resveratrol [7e11]. Many food sources are traditionally known to induce thermogenesis and aid in maintaining body temperature. However, it is not evident whether this is through the activation of BAT mediated non-shivering thermogenesis. To this end, we screened various food sources for their effect on non-shivering thermogenesis and identified Sesamum indicum (Sesame) as a potent inducer of BAT activity. Sesame seeds contain a high amount of oil (up to 60%) [12], which is rich in polyphenolic lignans, sesamin, sesaminol and sesamolin. These lignans have been reported to exhibit various therapeutic properties such as reduction of hypertension, inflammation and carcinogenesis [13e15]. Sesame seed hulls are also an
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Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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abundant source of phenolic compounds, which possess considerable antioxidant activities [16]; however, biochemical properties of the water-soluble compounds in sesame seed and defatted sesame seed flour have attracted less attention. In the present study, we found that the aqueous extract of black sesame seeds induced UCP1 activity in brown adipocytes in vitro and in vivo in mice. The molecule responsible for this activity was identified as sesaminol diglucoside (SDG). SDG treatment in mice led to enhanced BAT metabolism, reduced serum glucose levels and protected the mice against high fat diet (HFD) induced obesity. Further studies revealed that these effects of SDG are mediated via beta 3 adrenergic receptors (b3-AR). Together, our results establish that SDG induces BAT activation through b3-AR and therefore, thermogenic food molecules represent a potential complementary and safe approach to combat diet-induced obesity and associated metabolic disease.
2. Materials and methods 2.1. Extract preparation 100 g of black sesame seeds sourced locally were ground in a blender and defatted with ten volumes of hexane (Merck-Millipore) for 24 h. The defatted seed powder was further extracted for 24 h with distilled water. The aqueous extract was lyophilized and used for further experiments.
2.2. SDG purification and characterization The aqueous extract was fractionated using preparative HPLC on a Waters Spherisorb C18 ODS (10m, 250 mm) semi-prep column with a mobile phase of methanol: water: 10:90 to 90:10 (v/v) for 60 min at a flow rate of 5ml/min; the wavelength used for detection was 280 nm. The fractions collected were screened for UCP1 inducing activity, and the positive fraction was further purified on a Biogel-P2 (Sigma-Aldrich) column and was eluted with water at a flow rate of 0.6ml/min for 17 h. 86 fractions were collected in total and were assayed for the presence of carbohydrates by the phenolsulphuric acid method [17]. The mass of the positive fractions was determined using an AB Sciex TripleTOF™ 5600 system. The MS and MS/MS scans were recorded.
2.3. Preparation of brown fat explant Interscapular BAT was excised from 2 months old male C57BL6/J mice and washed with sterile Phosphate Buffer Saline (PBS). The tissue was minced into small pieces approximately 1e2 mm and incubated in DMEM-F12 medium with 10% Foetal Bovine Serum (FBS) (ThermoFisher) and the extract, or compounds, for a period of 4 h at 37 C. For treatment with pan b - adrenergic antagonist propranolol (Sigma-Aldrich), explants were treated with 100 mM propranolol for 30 min [18] following which they were treated with SDG (Nacalai-Tesque).
2.5. Immunoblotting BAT explants and tissue sections were lysed in RIPA buffer containing 0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5. Cell lysates, normalized for protein concentration, were resolved on a 12% SDS-polyacrylamide gel and were transferred to a PVDF membrane (GE healthcare). The blots were probed with primary antibody overnight at 4 C and with horseradish peroxidase-conjugated secondary antibodies (SigmaAldrich) for 1 h at room temperature. 2.6. Animal studies Animal handling and procedures were conducted with the approval of the Institutional Animal Ethics Committee (IAEC, CSIRCFTRI). Eight weeks old C57BL/6J mice were fed a standard diet (AIN-93M) with 10% kcal fat or 60% kcal fat (Table S1) in the case of high fat diet (HFD) fed animals. The animals were housed at 22 C ± 2 under a standard 12 h light/dark cycle. Body weight, feed intake and surface temperature above interscapular BAT were monitored daily. Mice were fed standard diet (10% kcal fat) and treated with aqueous extract of sesame, or with vehicle by oral gavage (20 mg/kg BW) for two weeks. The treatment with purified SDG consisted of the following groups: normal diet (ND) control (10% kcal fat), HFD control (60% kcal fat) and SDG treated group (60% kcal fat along with SDG 5 mg/kg BW). The mice were injected intraperitoneally with SDG or vehicle for ten days. Post treatment mice were euthanized by carbon dioxide inhalation, and tissues and blood were harvested. Serum levels of glucose, triglycerides and cholesterol were estimated using kits from ERBA Mannheim Diagnostics Gmbh, Germany. 2.7. H & E staining BAT sections were fixed with 4% paraformaldehyde (SigmaAldrich) overnight and then sequentially dehydrated with increasing concentration of ethanol. The fixed and dehydrated tissues were sectioned after being embedded in paraffin. Multiple sections were used for hematoxylin-eosin (H& E) staining. 2.8. In silico modeling A theoretical model of b3-AR of mouse which is 60.07% homologous to b1 receptor (4AMJ) [19] was built using homology modeling methodology in MODELLER 9.14. The agonists such as noradrenaline, CL-316,243 and SDG molecules were quantum mechanically optimised at B3LYP/def-SVP basis set [20] using TURBOMOLE program package (TURBOMOLE V6.6, 2014). Molecular docking was carried out using Autodock 4.2.6. 2.9. Statistics Data are expressed as mean ± standard error of mean (SEM) and significance testing was performed by two-way ANOVA or Student's t-test. Statistical significance was set at p < 0.05 and data were analysed using GraphPad Prism software 5.0.
2.4. Real-time PCR
3. Results
Post treatment, the explants and cells were lysed in Trizol (Sigma-Aldrich), and RNA was isolated using Qiagen pure link RNA isolation kit. 1 mg RNA was reverse transcribed to cDNA using ABI high capacity cDNA synthesis kit. The cDNA was used for quantitative PCR in BioRad CFX96 Touch™ Real-Time PCR Detection System. The primers used for qPCR are listed in Table S2.
3.1. Sesame extract induces UCP1 in brown adipocytes To explore the relationship between thermogenic foods and BAT activity, several foods traditionally known to induce thermogenesis were screened for Ucp1 inducing activity (data not shown). Among the screened foods, we found that defatted aqueous extract of black
Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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sesame seeds, Sesamum indicum (Fig. 1A), triggered a significant increase in the expression of Ucp1 in BAT explants. Real-time PCR and immunoblot analysis of brown adipocytes treated for 4 h with aqueous extract of black sesame seeds revealed that Ucp1 mRNA expression nearly doubled (Fig. 1B) and UCP1 protein levels increased two-fold compared to untreated cells (Fig. 1C). These results indicate that black sesame seed extract induces UCP1 in vitro. To determine the UCP1 inducing effects in vivo, C57BL6/J mice were treated orally with 20 mg/kg BW aqueous extract of black sesame seeds for two weeks. Despite similar food intake, there was an 8% decrease in body mass in the sesame extract treated mice (Fig. 1D). The weight loss could be attributed to an increase in energy expenditure, which was reflected by the rise in surface body temperature over the interscapular BAT area (Fig. 1E). This result was further confirmed by H&E staining of paraformaldehyde-fixed BAT tissue sections (Fig. 1G) which displayed much smaller lipid droplets in BAT from treated mice. The analysis of lipid parameters in the serum of treated mice also showed a reduction in the levels of total cholesterol and triglycerides (Fig. 1F). Western blot analyses of protein from treated BAT displayed a marked increase in UCP1 protein levels as well (Fig. 1H), confirming the activation of BAT thermogenesis in sesame extract treated mice. 3.2. SDG purified from sesame extract triggers a dose-dependent increase in UCP1 in vitro Aqueous extract of black sesame seed was subjected to preparative HPLC analysis, to identify the active component responsible for the induction of UCP1 (Fig. 2B). The fractions were screened based on their ability to induce UCP1 protein in BAT explants in vitro. The selected fractions were further purified by Biogel-P2 size exclusion chromatography (Fig. 2C). The various fractions from Biogel-P2 were tested for UCP1 inducing ability, and the positive fraction was analysed by ESI-MS to determine its
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homogeneity and molecular mass. In positive mode, a major peak corresponding to m/z ratio of 717.1 (Fig. 2E) was observed. The peaks obtained (347.1, 555.1 and 484.9) on MS/MS analysis (Fig. 2F) matched with that of the reported molecule, Sesaminol diglucoside (MW: 694) which appears as a sodium adduct (MW: 717) in positive mode [21]. The structure of the purified molecule was studied by 1H NMR and 13C NMR and was validated as SDG by comparing with the NMR profile of commercially obtained standard (Fig. S1). On treating BAT explants with increasing concentrations of SDG for 4 h, we noticed a corresponding dose-dependent increase in the levels of UCP1 protein (Fig. 2D). Commercially, SDG is available in two isomeric forms SDG (1, 6) and SDG (1, 2). The increase in UCP1 expression was observed only with the SDG (1, 6) isomer (Fig. S2) and therefore future experiments were restricted to the (1, 6) isomer of SDG. 3.3. Treatment with SDG prevents weight gain in HFD fed mice Given the remarkable brown adipogenic activity of SDG in vitro, next, we investigated the effect of SDG in vivo. We administered male C57BL6/J mice with vehicle (saline) or SDG (5 mg/kg) i.p for ten days along with a HFD. SDG treated mice gained less body weight compared to control mice (Fig. 3A) despite no change in feed consumption (Fig. S3). Surface temperature measurements of the mice, right above the interscapular BAT region revealed a modest but significant increase in body temperature in treated mice (Fig. 3C). These data suggest that SDG probably decreases body weight through an increase in energy expenditure in vivo. To understand in detail the changes in body composition, the masses of the various fat pads were also recorded and examined at the end of the study. There was no significant change in the mass of the interscapular BAT, however inguinal and epididymal WAT masses significantly reduced in SDG treated mice (Fig. 3B). Furthermore, serum levels of glucose (Fig. 3D) reduced to a significant extent in treated mice, and H&E staining revealed much smaller lipid
Fig. 1. Sesame extract induces thermogenesis. (A) Schematic representation of the preparation of aqueous extract from black sesame seeds. (B) mRNA expression of Ucp1 gene on treating BAT explants with sesame extract for 4 h. (C) Western blot of UCP1 protein in sesame extract treated BAT explants. 2 month old C57BL6/J mice were treated with vehicle(saline) or sesame extract (20 mg/kg, BW) by oral gavage for two weeks (n ¼ 6). (D) Body weight and feed consumption. Bar graph represents body weight (g), and line graph represents feed intake (g) per animal per day (E) Surface temperature measured on the interscapular BAT area. (F) Total cholesterol and triglyceride levels (G) H & E staining of formalin fixed paraffin embedded (PPFE) BAT sections. (H) Immunoblot of UCP1 from BAT samples. Values are represented as mean ± SEM. p values were determined by Student's ttest. *p < 0.05, **p < 0.01.
Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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Fig. 2. Purification and characterization of Sesaminol diglucoside (SDG) from sesame extract. (A) Schematic representation of purification procedure of sesame extract (B) Preparative HPLC of aqueous extract from sesame seeds. (C) Major fractions observed with Biogel-P2 separation of selected peaks after preparative HPLC. (D) Dose-dependent effect of SDG on UCP1 expression in BAT explants. (E) MS spectrum of the purified compound in the positive mode with major peak at m/z ¼ 717. (F) Parent and fragment ion peaks in the MS/MS spectrum of SDG. (G) Structure of SDG.
Fig. 3. SDG treatment decreases body weight and lipid parameters in vivo. Two month old C57BL6/J mice received vehicle (saline) or SDG (5 mg/kg) for ten days i.p. (n ¼ 6 per group) along with an acute HFD challenge. (A) Body weight of vehicle-(saline) and treated mice. (B) Mass of BAT, iWAT and eWAT. (C) Surface temperature measured on the interscapular BAT area (D) Serum glucose (E) H & E staining of PPFE BAT sections. (F) Western blot analysis of UCP1 in BAT tissue. Values are mean ± SEM. p values were determined by two-way ANOVA. *p < 0.05. ND- Normal diet; HFD- High fat diet.
droplets in BAT of SDG treated mice (Fig. 3E), representing the overall favorable effect of SDG on glucose and lipid parameters of treated animals. Western blot analyses revealed that the levels of essential thermogenic gene Ucp1 increased significantly (Fig. 3F). Taken together, these data suggest that SDG exerts a protective effect against HFD induced phenotype by increasing energy expenditure through UCP1.
3.4. Induction of thermogenesis by SDG is beta-adrenergic dependent
b3-AR is a seven transmembrane helix containing protein, predominantly found in adipose tissue [22]. Multiple sequence alignment of human and mouse b3-ARs along with b2 and b1-ARs show that human and mouse b3 protein sequence has ~80% sequence
Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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similarity and active sites are conserved throughout (Fig. S4). Understanding the molecular interaction of SDG towards the mouse b3-AR in silico revealed that it embedded in the same active site pocket as the known agonists, noradrenaline and CL-316,243 (Fig. 4A). Ser208, Ser205 (TM5) and Asp114 (TM3) from the top and Asn329 (TM6 and TM7) from the bottom of the active site hold the agonists through various H-bond interactions as observed in b receptors [19,23]. SDG additionally gains hydrophobic interactions at the active site and was found to have similar binding energies as the synthetic agonists (Fig. 4B, Table S3). Whereas in human b3-AR receptor the glycoside moiety of SDG is facing towards the surface, resulting in lower binding energy (Fig. S5, Fig. 4B). This effect was validated in vitro by treating BAT explants with SDG in the presence of b-adrenergic receptor antagonist propranolol. Treatment of brown adipocytes with SDG in the presence of propranolol diminished the induction of Ucp1 mRNA (Fig. 4C) and protein expression (Fig. 4D). These results suggest that SDG has a promising binding affinity towards the b3-AR receptor in vitro. Together, our results suggest that SDG induces BAT activation and thermogenesis in mice, potentially in a b3-AR-dependent manner. 4. Discussion In sixty-five percent of the world's population, overweight and obesity kill more people than malnutrition [24]. Dieting and exercise are the most widely recommended treatments; however, the success rate for these range a meagre 2e20%, due to difficulty in adhering to these regimens in the long term. Other options currently available are bariatric surgery and oral prescription drugs. Oral prescription medications available in the market suffer from
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low success rates and serious side effects including gastric issues, nausea, dizziness and liver damage [25]. Therefore, dietary sources are a promising avenue to identify molecules that increase energy expenditure and are safer for long-term consumption. The current study indicates that aqueous extract of black sesame seed increases whole-body energy expenditure, which could be useful in preventing metabolic disorders. Our data demonstrate that black sesame seed extract increases the expression of the major thermogenic gene Ucp1 in brown adipocyte explants and induces weight loss in vivo in mice. The weight loss was accompanied by a concurrent increase in body temperature, which indicated that the weight loss was due to an increase in energy expenditure. Sesame seeds have been used over the years in traditional medicine to treat various health conditions [26]. However, no reports exist concerning its effect on BAT thermogenesis. The aqueous extract of sesame seeds is reported to contain lignan glucosides apart from other small molecules, which exert beneficial pharmacological effects such as anti-hypertensive effect in prehypertensive human patients and elevation of g-tocopherol levels [27,28]. Purification and activity-based assays revealed that SDG present in the aqueous fraction of sesame seeds mediated our observed effects on BAT metabolism. Our data demonstrate that SDG increases the expression of the major thermogenic gene Ucp1 in brown adipocytes and protects against HFD-induced weight gain in mice. SDG also displayed a positive effect on serum glucose levels. BAT activation clears excessive glucose in the plasma by increasing glucose uptake into BAT and using it as a substrate for thermogenesis thereby improving insulin sensitivity and weight loss [29]. These results
Fig. 4. SDG induces Ucp1 in a b3-AR dependent manner. (A) Binding poses of agonists such as noradrenaline, CL-316,243 and SDG in the active site of mouse b3-AR. Crucial active site residues and the respective transmembrane domains are highlighted. Yellow dotted lines represent the hydrogen bonding interactions between the agonists and the receptor. Note that all these molecules have crucial H-bonding interactions with Ser205/Ser209 of TM5 and residue numbering is according to 4AMJ crystal structure. (B) The estimated free energy of binding (DG, kcal/mol) of various agonists towards human and mouse b3-ARs were obtained from MM/GBSA calculations. BAT explants were treated with CL-316,243 or SDG in the presence or absence of propranolol (C) Relative Ucp1 mRNA as measured by RT-qPCR. (D) UCP1 protein levels assayed by Western blot. p values are represented with asterisks (*p < 0.05, **p < 0.01) and error bars represent SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195
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suggest that the increase in BAT thermogenic function through SDG treatment could contribute to the clearance of excessive glucose and lipids, which prevents weight gain on a HFD. b3-ARs present on the surface of brown adipocytes are one of the major contributors to cold-induced activation of BAT thermogenesis. Noradrenaline is the biological ligand for b3-AR, however many synthetic ligands including CL-316,243 have been identified [30]. Since b3-AR is the major activator of BAT thermogenesis, we evaluated the binding affinity of SDG towards b3-AR in silico. Our results revealed that SDG embeds in the same active site pocket as the known agonists noradrenaline and CL-316,243 with promising binding energies. In vitro assays in the presence of the b3-AR antagonist propranolol further confirmed the in silico results. However, since a complex adipogenic network exists in these cells, we cannot rule out the role of other transcription factors for observed energy expenditure in in vivo studies [31]. Together, our results suggest that SDG prevents weight gain on HFD by inducing b3-AR dependent BAT thermogenesis. Funding This study was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, under the 12th 5-year plan project LIPIC (BSC0401). AJ was supported by a fellowship from CSIR, New Delhi. RR is a recipient of the JC Bose national fellowship. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgements We are grateful to Mr. Shinde Vijay Sukhdeo for his help with the animal studies and Ms. Jayasundarnaidu Megha for initial in silico studies. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.195. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.195. References [1] J.O. Hill, H.R. Wyatt, J.C. Peters, Energy balance and obesity, Circulation 126 (2012) 126e132. [2] D.G. Nicholls, R.M. Locke, Thermogenic mechanisms in brown fat, Physiol. Rev. 64 (1984) 1e64. [3] N.J. Rothwell, M.J. Stock, B.P. Warwick, Energy balance and brown fat activity in rats fed cafeteria diets or high-fat, semisynthetic diets at several levels of intake, Metab., Clin. Exp. 34 (1982) 474e480. [4] K.A. Virtanen, M.E. Lidell, J. Orava, M. Heglind, R. Westergren, T. Niemi, M. Taittonen, J. Laine, N.J. Savisto, S. Enerback, P. Nuutila, Functional brown adipose tissue in healthy adults, N. Engl. J. Med. 360 (2009) 1518e1525. [5] A. Bartelt, O.T. Bruns, R. Reimer, H. Hohenberg, H. Ittrich, K. Peldschus, M.G. Kaul, U.I. Tromsdorf, H. Weller, C. Waurisch, A. Eychmuller, P.L. Gordts, F. Rinninger, K. Bruegelmann, B. Freund, P. Nielsen, M. Merkel, J. Heeren, Brown adipose tissue activity controls triglyceride clearance, Nat. Med. 17 (2011) 200e205. [6] I. Vermaak, A.M. Viljoen, J.H. Hamman, Natural products in anti-obesity therapy, Nat. Prod. Rep. 28 (2011) 1493e1533.
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Please cite this article as: A. Jahagirdar et al., Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.195