Obesity is defined as a state of excessive adipose tissue expansion arising from an imbalance between energy intake and expenditure [1
]. Due to economic development and changes in diet and lifestyle, obesity has become a global epidemic. Obesity is regarded as a major health problem that predisposes toward type 2 diabetes, cardiovascular disease, hypertension, stroke, atherosclerosis, non-alcoholic fatty liver disease, and various cancers [3
In mammals, energy balance involves two types of adipose tissue: white adipose tissue (WAT), which stores energy in the form of triglycerides and can expand through changes in cell size (hypertrophy) and cell number (hyperplasia) [5
], and brown adipose tissue (BAT), which dissipates stored energy as heat through the expression of uncoupling protein 1 (UCP1) [7
]. UCP1 promotes proton leakage in the mitochondrial membrane, thereby dissipating the energy released by oxidation instead of using it for ATP synthesis. Recently, it has been shown that there is also a third type of adipocyte, in which UCP1 expression can be induced by cold exposure or β3-adrenoceptor agonists, and which is referred to as a beige or BAT-like adipocyte [10
An increase in WAT-to-BAT transdifferentiation is an important mechanism that is used to combat fat accumulation. This “browning” involves mitochondrial expansion and fatty acid oxidation (FAO), which increase energy expenditure. Fatty acids are required for the UCP1-mediated uncoupling mechanism, and numerous studies have shown that cold stimulates FAO in mice, and this is required for thermogenesis [11
]. Carnitine palmitoyltransferase 1 (CPT1), which is an enzyme required for the transport of long-chain fatty acids from the cytoplasm into the mitochondria, is a rate-limiting factor in FAO [14
]. In addition, the browning process requires high levels of expression of PR domain-containing 16 (PRDM16) and peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC1α) as regulators. Both these transcription factors promote the expression of mitochondrial genes, including UCP1, and mitochondrial biogenesis [15
]. Therefore, the identification of a nutritional activator of UCP1 expression in WAT may lead to the development of a new class of therapeutic agents for obesity [18
Acid-hydrolyzed silk peptide (SP), derived from Bombyx mori
cocoons, is a dietary biomaterial that has various applications in biotechnology, bio-pharmacology, cosmetic, and food industries in Asian countries [19
]. It is known to be biocompatible and its administration has not been associated with any documented side effects. Recently, in vitro and in vivo studies have shown that SP has beneficial effects on metabolism and health [20
]. For instance, it has been reported that treatment with silk fibroin enhances insulin sensitivity and glucose uptake in 3T3-L1 adipocytes and type 2 diabetic mice [22
]. In addition, silk fibroin proteins have been shown to attenuate adipogenesis in adipocytes and C57BL/6N mice [24
], and to increase fat oxidation in exercising mice [26
]. However, the molecular mechanisms whereby SP may induce browning and fatty acid oxidation in WAT have yet to be established. Therefore, we aimed to determine the effects of dietary SP on the metabolism of high-fat diet (HFD)-induced obese mice and in subcutaneous white adipose tissue (sWAT)-derived primary cells.
2. Materials and Methods
2.1. Preparation of SP from Bombyx Mori
Dietary SP (lot number, 1803002) was prepared from the cocoons of Bombyx mori
and was obtained from Worldway Co., Ltd. (Sejong, Korea). As shown in Figure 1
, raw cocoons were acid-hydrolyzed, and the resulting solution was neutralized, decolorized, filtered, desalted, and freeze-dried to obtain a pale yellow powder. The nutrient composition of the SP was analyzed by International official methods of analysis (AOAC) methods, and the results are presented in Table 1
. In detail, carbohydrates and sugars in an activated charcoal column and a later elution with different proportions of ethanol (AOAC 954.11) to fractionate them selectively according to their degree of polymerization. Crude fat of SP were extracted using the soxhlet apparatus with hexane for 4 h (AOAC 920.153) and determined gravimetrically. Crude protein was estimated by AOAC 968.06 method, through an acid digestion and nitrogen distillation using Kjeldahl method. Lastly, sodium content in SP was conducted following the AOAC 984.27 method. The mean molecular weights of SP were measured by MicroQ-TOF III mass spectrometry (Bruker Daltonics, Hamburg, Germany). The SP sample which is dissolved in 10 mM Sodium phosphate buffer with methanol (4:1) was then injected into an UltiMate 3000 high-performance liquid chromatography (HPLC) system (Dionex, Sunnyvale, CA, USA), and a Poroshell 120 EC-C18 column (2.1 mm × 100 mm, 2.7 μm) was used to analyze. Acetonitrile containing 0.2% formic acid and 0.2% formic acid in water were used as the mobile phases (at ratios of 95:5 to 5:95 (v/v
), with a flow rate of 0.2 mL/min), and the results are presented in Figure 2
. Lastly, the free amino acid composition of SP was determined using a 2695 HPLC system (Waters, Milford, MA, USA). An AccQ-Tag Amino acid analysis column (silica C18
, 3.9 mm × 150 mm) and a Waters 2475 Multiλ Fluorescence detector were used for the SP analysis, which is described in Figure 3
2.2. Cell Culture and Treatment
The stromal vascular fraction was obtained from the sWAT of 6–8-week-old male institute of cancer research (ICR) mice. As described previously [28
], isolated sWAT pads were minced and digested in phosphate-buffered saline (PBS) containing 1.5 U/mL of collagenase D (Roche Diagnostics GmbH, Mannheim, Germany), 2.4 U/mL of Dispase II (Roche, Basel, Switzerland), and 10 mM CaCl2
for 1 h. The digest was then filtered through 40 μm cell strainers (SPL Life Science, Gyeonggi-do, Korea) three times, washed in PBS, and centrifuged at 1000× g
for 10 min.
Glutamax DMEM/F12 medium containing 10% FBS and 1% penicillin/streptomycin solution (P/S) were used for the maintenance of primary sWAT cells. Two days after the cells reached confluence, this medium was replaced by differentiation medium (DMEM supplemented with 10% FBS, 1% P/S, 100 μM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 5 μg/mL insulin) for two days, and subsequently, the differentiated adipocytes were maintained in DMEM containing 10% FBS, 1% P/S, and 5 μg/mL insulin.
SP was prepared as a 400 mM stock solution in distilled water and then diluted with medium to 25, 50, 100, 200, or 400 μM. To induce browning in primary sWAT-derived adipocytes, the cells were incubated in differentiation medium containing 10 nM triiodothytonine (T3) and 1 μM rosiglitazone (Rsg). The compounds 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-]pyrimidine (dorsomorphin) (5 μM) or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) was added to the differentiation medium to determine the role of AMPK.
2.3. Animals and Diet
Four-week-old male ICR mice were purchased from Joong-Ah Bio (Suwon, Korea). The animal experiments and care were approved by the Institutional Animal Care and Use Committee (IACUC) of CHA University (IACUC approval number, 180161). Mice were housed for 1 week under a 12 h light/dark cycle for adaptation. Mice were randomly allocated to four groups (n
= 10 per group). The first group was fed a chow diet (CD, 10% of calories derived from fat, D12450B, Research Diets, New Brunswick, NJ, USA), the second group was fed an HFD (60% of calories derived from fat, D12492, Research Diets), the third group was fed an HFD and administered 50 mg/kg/day SP (HFD + SP50), and the final group was fed an HFD and administered 200 mg/kg/day SP (HFD + SP200). Each SP concentration was derived from the human doses (0.25 g/60 kg/day and 1 g/60 kg/day) in mathematical table, as described [29
]. SP was orally administered to the mice by gavage daily for 6 weeks. The body mass, food intake, and water consumption of the mice were measured weekly. At the end of the treatment period, the mice were euthanized, and their tissues were collected for analysis and weighed.
2.4. Rectal Temperature Measurement
At the end of the 6 week treatment period, the rectal temperatures of the mice were measured three times using a Testo 925 Type Thermometer (Testo, Lenzkirch, Germany).
2.5. Serum Biochemistry
After the treatment, the mice were fasted overnight and blood samples were collected by cardiac puncture. Serum samples were separated by centrifugation at 4 °C and 4000× g for 10 min after the blood had clotted. Commercial enzyme-linked immunosorbent assay (ELISA)/calorimetric assay kits (Abcam and Biocompare, Burlingame, CA, USA) were used to measure serum triglyceride (ab65336), total cholesterol (ab65390), high-density lipoprotein (HDL)-cholesterol (EKC37055), low-density lipoprotein (LDL)-cholesterol (EKC41016), leptin (ab100718), alanine aminotransferase (ALT, ab105134), aspartate aminotransferase (AST, ab133878), and creatinine (ab65340) concentrations. Absorbances were measured at appropriate wavelengths using a plate reader (BioTek Instruments Inc. Winooski, VT, USA).
2.6. Histological Analysis and Immunofluorescence Staining
After euthanasia, sWAT and visceral WAT (vWAT) were rapidly collected and fixed in 4% paraformaldehyde solution for 48 h. The tissues were then paraffin-embedded and the resulting blocks were cut into 5–10 µm sections and stained with hematoxylin and eosin (H&E) to assess adipose histology. Photomicrographs were obtained using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan).
For immunofluorescence analysis, fixed cells and WAT sections were stained with rabbit anti-UCP1 (dilution, 1:200) or anti-CPT1 (dilution, 1:1000) antibodies overnight at 4 °C in a moist chamber. Fluorescein isothiocyanate (FITC)-conjugated (dilution, 1:1000) and Alexa Fluor™ 594-conjugated (dilution, 1:1000) secondary antibodies were used. Mitochondria were identified by staining with 1 mM MitoTracker Red (Cell Signaling Technology, Danvers, MA, USA), according to the manufacturer’s protocol, and nuclei were stained using DAPI (Thermo Fisher Scientific, Waltham, MA, USA) fluorescence. After mounting using ProLong Gold Antifade reagent (Thermo Fisher Scientific), fluorescence images were captured using a Zeiss confocal laser scanning microscope (LSM880; Carl Zeiss, Oberkochen, Germany) and Zen 2012 software (Carl Zeiss).
2.7. Oil Red O Staining
After 8–10 days of differentiation of the primary cells, they were fixed in 10% formaldehyde for 1 h at room temperature and then washed twice with PBS. The fixed cells were stained with Oil red O (ORO) solution in 6:4 (v/v) isopropanol:distilled water for 30 min. After rinsing and drying, the stained adipocytes were imaged and the stain was eluted using 100% isopropanol. The absorbances of these eluates were then measured at 490 nm. Cryostat sections (5 µm) of liver were also stained with 0.1% (w/v) ORO solution to visualize hepatic lipid accumulation.
2.8. Western Blot Analysis
Cells or tissue was lysed in lysis buffer (iNtRON Biotechnology, Seoul, Korea) supplemented with phosphatase and protease inhibitors. The total protein contents of the lysates were quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA) and then equalized. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. These membranes were blocked using 5% non-fat dried milk for 1 h and then incubated overnight at 4 °C with primary antibodies targeting AMPKα, phospho-AMPKα (Thr172), phosphor-hormone-sensitive lipase (p-HSL, Ser563), adipose triglyceride lipase (ATGL, Cell Signaling Technology), fatty acid-binding protein 4 (FABP4), CCAAT enhancer-binding protein α (C/EBPα), diacylglycerol acyltransferase 1 (DGAT1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PGC1α, peroxisome proliferator-activated receptor alpha (PPARα), PRDM16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), sterol regulatory element-binding transcription factor 1 (SREBP1), CPT1, or UCP1 (Abcam, Cambridge, UK). After washing, the membranes were incubated for 4 h with secondary antibodies conjugated with horseradish peroxidase (1:1000, Santa Cruz Biotechnology) in 5% non-fat dried milk. Reactive band was obtained by chemiluminescence and LAS image software (Fuji, Valhalla, NY, USA).
2.9. Quantitative Reverse Transcription Polymerase Chain Reaction Analysis
RNA was isolated from adipocytes or homogenized tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was then obtained from 1 μg RNA on a thermal cycler (Bio-Rad) using a Maxime RT PreMix (iNtRON Biotechnology, Seongnam, Korea) for 60 min. The cDNA was amplified using SYBR Green (Roche Diagnostics GmbH, Mannheim, Germany) and a Bio-Rad CFX96 Real-Time Detection System (Bio-Rad). Expression data were normalized to 18S. The mRNA levels were calculated as a ratio, using the 2−ΔΔCT
method by using Bio-Rad software (Quantity One 4.62; Bio-Rad), for comparing the relative mRNA expression levels between different groups in the qPCR. The sequences of the primers used in this study are listed in Table 2
2.10. Statistical Analysis
Protein expression data and relative cell diameter data are summarized as mean ± SD and were analyzed using one-way ANOVA and Duncan’s test (SPSS, Chicago, IL, USA). Values with different letters are significantly different; p < 0.05 (a > b > c > d > e). Tissue masses and qPCR data are expressed as mean ± SD, and Student’s t-test was used to analyze the data. Organ mass and mRNA data were analyzed using Student’s t-test, and values were considered significant when * p < 0.05 and ** p < 0.01.
There has been a great deal of recent interest in methods of enhancing energy expenditure in BAT or inducing the transdifferentiation of white to BAT-like adipocytes because these strategies have the potential to prevent the metabolic complications of obesity. The latter mechanism, referred to as “browning”, crucially involves the induction of UCP1 expression in WAT. A number of small molecules, such as berberine, curcumin, and capsaicin, have been shown to activate thermogenic transcriptional factors or regulate key signaling cascades in adipocytes and thus induce browning [43
]. In the present study, we aimed to determine whether acid-hydrolyzed SP could reduce lipid accumulation by stimulating energy expenditure, browning, and FAO in WAT via AMPK activation.
Dietary SP is a natural material isolated from Bombyx mori
that is known to have effects on metabolism [27
]. In recent studies, it is reported that hydrolyzed proteins are efficiently absorbed in the small intestine [48
]. Since SP contains dipeptides and tripeptides including amino acids, dietary SP also good source of nutrients for Intestinal Absorption. It has been shown that silk fibroin inhibits adipocyte differentiation by reducing C/EBPα expression in the 3T3-L1 cell line [49
], and a variety of silk proteins are also known to have a protective effect against metabolic diseases [50
]. However, the molecular mechanisms of the beneficial effects of SP have yet to be established, and whether it might induce transdifferentiation of WAT is of particular interest. Therefore, we administered SP to HFD-induced obese mice and determined whether this would induce a switch in WAT toward a BAT-like phenotype.
In the present study, we have shown a novel effect of SP to reduce adipogenesis and obesity in HFD-fed mice. As expected, mice fed the HFD became obese and deposited more lipid, including in both the sWAT and vWAT. However, SP treatment markedly reduced the body mass and the adipocyte size of both the sWAT and vWAT of HFD-fed mice. Mature adipocytes in WAT store triglyceride, which is released by lipolysis. We also confirmed that the serum concentrations of triglyceride, total cholesterol, and LDL-cholesterol are higher, and that of HDL-cholesterol is lower in HFD-fed mice, but these defects are ameliorated by SP treatment. Therefore, we also determined the effects of SP on adipogenic genes/proteins and found that the expression of C/EBPα and FABP4 was much lower in SP-treated HFD-fed obese mice and differentiated primary sWAT-derived adipocytes. Thus, the present data suggest that SP can prevent HFD-induced obesity by inhibiting adipogenesis in WAT.
WAT serves as a regulator of a variety of physiologic processes and plays an important role in the regulation of energy balance and lipid homeostasis [51
]. In the present study, we have also shown that SP induces the browning of WAT. In vivo and ex vivo experiments showed that SP increased the white adipocyte expression of UCP1, a key marker of browning, as well as that of other vital regulators of this process: PRDM16, PGC1α, and UCP3. The thermogenic capacity of BAT-like adipocytes is conferred by UCP1, which is located in the inner mitochondrial membrane [11
], while PGC1α upregulates fatty acid and lipid catabolism, and PRDM16 activates the BAT-selective expression program in white adipocytes [18
]. In addition, SP induced mitochondrial biogenesis, as indicated by greater expression of NRF1 and TFAM in both WAT depots. The mitochondria are the site of a number of metabolic pathways in the adipocyte, including β-oxidation, and an enhancement of mitochondrial biogenesis is a crucial feature of WAT browning, because enhancement of mitochondrial biogenesis induces WAT to a BAT-like phenotype [52
]. In summary, our data demonstrate that SP induces browning in the WAT of HFD-induced obese mice.
These findings were corroborated by the fact that SP administration also increased the rectal temperature of HFD-fed mice, which probably indicates greater loss of energy as heat. Many researchers have previously demonstrated thermogenesis in animals using indirect thermometers [53
]. The dissipation of energy as heat is the result of uncoupling in BAT-like adipocytes and involves the transfer of fatty acids from the TGs stored in fat droplets to the mitochondria for FAO [31
In beige adipocytes, AMPK activation induces the expression of key thermogenic transcription factors, which upregulate the transcription of browning and FAO genes [55
]. In SP-treated mice, p-AMPK/AMPK protein levels and CPT1 expression were normalized. FAO is required for the upregulation of UCP1 expression, which is indicative of the phenotypic change from WAT to beige adipocytes [32
]. Moreover, mitochondrial biogenesis is accompanied by an upregulation of FAO, and SP treatment increased the expression of the β-oxidation genes Acox1
, and Cyp4a14
. ACOX1 is the first enzyme in the peroxisomal fatty acid β-oxidation pathway, and it has recently been reported that it mediates the increase in energy expenditure and the lean phenotype that results from PPARα activation [56
]. Furthermore, CYP4A10 and CYP4A14 are involved in the β- and ω-oxidation of fatty acids in adipose tissue. These genes are induced by PPARα and mediate the conversion of fatty acids to acyl-CoA, which are transported toward the mitochondrial FAO through the action of CPT1 [31
]. Thus, our data are consistent with the notion that SP induces the formation of BAT-like adipocytes in WAT by increasing the expression of FAO genes.
Interestingly, SP induced higher expression levels of PRDM16, PGC1α, UCP1, and CPT1 in sWAT than in vWAT. Immunofluorescence analysis also showed that CPT1 and UCP1 expression was much higher in sWAT than in vWAT. Consistent with this, numerous previous mouse studies have shown that sWAT has a larger mitochondrial mass than vWAT and a higher respiratory capacity, attributable to higher expression of mitochondrial respiratory chain components [57
]. Thus, SP treatment of HFD-fed mice induces browning more efficiently in sWAT than in vWAT.
Lipolysis is accompanied by a potent stimulation of mitochondrial β-oxidation in WAT and is necessary for the browning of WAT. Free fatty acids released by lipolysis can be oxidized in mitochondria [52
], and ATGL and HSL are the rate-limiting enzymes in intracellular lipid catabolism. According to a recent report, ATGL-overexpressing mice demonstrate an upregulation of both lipolysis and FAO in WAT, which is accompanied by an upregulation of thermogenesis, resulting in greater energy expenditure [31
]. In the present study, SP treatment increased ATGL and HSL expression, implying an upregulation of lipolysis, which would provide substrates for FAO in WAT.
Liver is a key tissue for whole body energy homeostasis but develops non-alcoholic fatty liver disease and specifically steatosis in obese mice and humans [59
]. In the present study, hepatic lipid accumulation was ameliorated by SP treatment. Furthermore, SP-treated mice had lower hepatic SREBP1c and DGAT1 expression than HFD-fed control mice, whereas p-AMPK and CPT1 levels were higher in the SP-treated groups. To summarize, SP appears to upregulate FAO and ameliorate fat accumulation in the liver, in addition to improving lipid metabolism in adipose tissue.
We also compared the direct browning effect of the conventional inducers T3 and Rsg and that of SP by measuring the expression levels of p-AMPK, CPT1, and BAT-specific genes, such as PRDM16 and UCP1, in primary sWAT-derived adipocytes. T3 and Rsg are known to regulate thermogenesis, as well as energy balance, by increasing UCP1 expression. In the present study, SP had a synergistic effect with Rsg and T3 to induce the expression of browning-related genes. In addition, differentiated primary cells were assessed to determine whether SP increases mitochondrial biogenesis and UCP1 expression. Immunofluorescence staining for UCP1, and MitoTracker staining to identify mitochondria, were strongly enhanced in cells treated with browning inducers T3 and Rsg. Taking these data together, SP directly stimulates mitochondrial function and upregulates UCP1 expression in white adipocytes.
Finally, we determined whether AMPK phosphorylation is required for the induction of browning and FAO in WAT by SP. AMPK is an essential energy sensor in adipocytes and regulates body mass, liver lipid content, and the thermogenic program in BAT and sWAT [60
]. SP treatment of primary adipocytes cultured with AICAR or dorsomorphin caused an increase in AMPK phosphorylation. In addition, the expression level of UCP1 was regulated by both the AMPK inducer and inhibitor in SP-treated cells. Furthermore, the size of lipid droplet in primary cells treated with both AICAR and SP was greatly reduced. These data suggest that AMPK activation induces adipocyte browning by regulating UCP1 expression and fat storage. In conclusion, SP could not only inhibit fat accumulation, but also enhances energy metabolism by regulating UCP1 expression.