Ginger prevents obesity through regulation of energy metabolism and activation ofbrowning in high-fat diet-induced obese mice☆
Jing Wanga,b, c,d,e, Daotong Lia,b, c,d, Pan Wanga,b, c,d, Xiaosong Hua,b, c,d, e, Fang Chena,b, c,d,⁎aCollege of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
bNational Engineering Research Center for Fruit and Vegetable Processing, China Agricultural University, Beijing, ChinacKey Laboratory of Fruit and Vegetable Processing, Ministry of Agriculture, China Agricultural University, Beijing, China
dEngineering Research Centre for Fruit and Vegetable Processing, Ministry of Education, China Agricultural University, Beijing, ChinaeBeijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
Received 28 January 2019; received in revised form 8 April 2019; accepted 8 May 2019
Keywords: Ginger; Obesity; Energy metabolism; Browning; AMP-activated protein kinase (AMPK)
1. Introduction
Obesity is increasing at an alarming rate and has become one of thegreatest public health concerns all over the world. Estimates indicatethat 38% of the world's adult population will be overweight in 2030without effective intervention [1]. Obesity is caused by an imbalanceof energy intake and expenditure, and thus, the best way to treatobesity is to cause a decrease in the former and an increase in the latter[2]. Adipose tissue is a major metabolic tissue and plays a key role inenergy homeostasis. Currently, three types of adipose tissue areknown-white adipose tissue (WAT), brown adipose tissue (BAT), andbeige adipose tissue. WAT is optimized to store energy in the form oflarge droplets of triglycerides (TGs) for later use and regulate thewhole-body homeostasis [3]. BAT contains a high density ofmitochondria with uncoupling protein-1 (UCP1) that uncouplesmitochondrial oxidative respiration from ATP production to produce
☆ Conflict of interest statement: All authors have declared no conflicts of intere⁎ Corresponding author at: College of Food Science andNutritional Engineering, Ch
heat [4,5]. Original white adipocytes can be transformed into beigeadipocytes (browning process), which have similar characteristics tobrown adipocytes, such as higher respiratory rate, thermogenesis rateand energy consumption rate [6]. Recent studies indicated thatbrowning of WAT is regulated by several genes, such as UCP1, PRdomain containing 16 (PRDM16), cell death-inducing DFFA-likeeffector a (CIDEA) and PGC-1α. The expression of PGC-1α is regulatedby AMPK and SIRT1, which are two important nutrient and energysensors [7]. Thus, SIRT1, AMPK and PGC-1α form an energy sensingnetwork that controls energy expenditure in mitochondria [8]. Thus,promoting adipose tissue browning may offer a viable approach tocounteracting obesity.
Currently, several phytochemicals and plant-derived foods, such ascapsaicin [9], curcumin [10], cranberry [11], and rose [12], are knownto affect energy homeostasis by browning of WAT and reduce the riskof overweight and obesity. Ginger, derived from the rhizome of
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Zingiber officinaleRosco (Zingiberaceae), is awell-known spice that hasbeen widely used for culinary purposes [13]. It contains variousphytochemicals and biologically active components, such as gingerolsand shogaols [14]. Apart from other traditional medical uses, gingerand itsmain bioactive constituents were reported to attenuate obesityboth in rodent animal models and cell lines [15]. For example, gingerextract administration can prevent HFD-induced obesity [16] andfructose overconsumption-induced insulin resistance in rats [17] byregulating peroxisome proliferator-activated receptors (PPARs) andthe inflammatory signaling pathway in adipocytes. Moreover, 6-gingerol and 6-shogaol, two major bioactive constituents of ginger,also exhibited antiobesity effects by altering the activities of some lipidmetabolismmarker enzymes and decreasing the expression of variouslipogenicmarker proteins in both rats and cell lines [18–21]. However,whether ginger supplementation can influence WAT browning inobese mice has never been explored.
In the present study, we analyzed the effects of ginger on energymetabolites involved in glycolysis and the TCA cycle and investigatedthe changes in some key gene and protein expression levels followingginger treatment. Finally, we proposed the molecular mechanism ofthe ginger-induced browning effect. The present study could providenew insight into the antiobesity function of ginger.
2. Materials and methods
2.1. Ethics statement
All animal experimental procedures followed in this study werereviewed and approved by the Biomedical Ethical Committee ofPeking University (Beijing, China) with an approval number(LA2018288).
2.2. Preparation and analysis of chemical components of ginger
Fresh ginger rhizomes (Zingiber officinale Rosco) were purchasedfrom a local market in Laiwu, Shandong Province, China, in February2018. Ginger samples were authenticated by Prof. Fang Chen based onthe monograph of ginger documented in the Chinese Pharmacopeia(2015 edition). A total of 2 kg of fresh ginger rhizomes were peeled,washed, further cut into pieces and then dried with a freeze-dryer(LGJ-10C, Beijing, China) for 48 h. The dried samples were subse-quently milled using a commercial hand-carry milling machine toproduce ginger powder, which was then stored in a moisture-controlled cabinet.
The protein, lipid, water and total sugar content of ginger weredetermined by the Kjeldahlmethod, the Soxhlet extractormethod, theambient pressure drying method and the dinitrosalicylic acid (DNS)colorimetricmethod, respectively. The ash contentwasmeasured by acombination of ashing and gravimetric procedures. The dietary fibercontent of ginger was determined by using a combination ofenzymatic and gravimetric procedures according to AOAC. The totalphenolic and flavonoid contents were determined by using the Folin–Ciocalteu method and the aluminum chloride colorimetric method,respectively (Table 1).
2.3. Identification and quantification of the main phenolic compounds inginger by reversed-phase high-performance liquid chromatography(HPLC)
HPLC was performed to identify and quantitate the majorcompounds in ginger using an RF-10AXL HPLC system (ShimadzuCo., Japan). Chromatographic separations were performed on areversed-phase column (Sunfire™ C18, 250×4.6 mm ID, 5 μm). The
mobile phases consisted of 2 phases: water (A) and acetonitrile (B).The gradient program forHPLCwas as follows: 0–5min, 0–20%B; 5–45min, 20–90% B; and 45–70 min, 100% B. The injection volume was 20μL with a 1 mL/min flow. The column temperature was maintained at30°C, and the detection wavelength was 280 nm. The chromatogramsof the major compounds from ginger are presented in SupplementaryFig. S1, and the main phenolic compounds concentrations are shownin Table 1. The 6-, 8-, and 10-gingerols and 6-shogaol standards(purityN98%) were purchased from Chromadex (Irvine, CA, USA).Unless noted, all chemicals were purchased from Sigma-Aldrich (St.Louis, MO, USA).
2.4. Animals and experimental design
Five-week-old C57BL/6 Jmalemice (provided by PekingUniversityAnimal Breeding Unit) were housed under standard laboratoryconditions (25±2°C, 12-h light/dark cycle) with free access to foodand drinking water. After acclimatization for one week, animals wererandomly divided into four dietary groups (n=8 per group). Twogroups were fed with a normal control diet (NCD; 10% kcal from fat,D12450B; Research Diets) or an identical diet supplemented withginger (NCD-G). The other two groups were fed a HFD (60% kcal fromfat, D12492; Research Diets) or an identical diet supplemented withginger (HFD-G). The ingredients and energy densities of theexperimental diets are shown in Supplementary Table S2. In thisstudy, we investigated the effects of ginger onWAT browning in obesemice with the dose of 500mg/kg according to the previous study [22].In each feeding condition, treatment groups were orally administered500 mg/kg body weight ginger once daily, whereas the other twogroups were used as controls and gavaged with an equal volume ofnormal saline. Body weight and food intake were assessed once perweek. After 16 weeks, mice were euthanized by carbon dioxideanesthesia followed by cervical dislocation. Blood was drawn andimmediately centrifuged (3000 rpm, 10 min). Epididymal whiteadipose tissue (Epi-WAT), perirenal white adipose tissue (Per-WAT),inguinal subcutaneous adipose tissue (Ing-SAT) and BAT were rapidlyisolated, weighed and stored at−80°C until further use.
2.5. Biochemical analysis
For biochemical analyses, the blood samples were centrifuged at3000 rpm for 10 min at 4°C. The supernatant was stored at −80°C. A3100 automatic biochemistry analyzer (Hitachi Ltd., Tokyo, Japan)wasused to determine serum concentrations of glucose (GLU), totalcholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL),high-density lipoprotein (HDL), alanine aminotransferase (ALT), andaspartate aminotransferase (AST).
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2.6. Histology and immunofluorescence staining of tissue sectionsanalysis
After mice were sacrificed, BAT and Epi-WAT were collected andfixed in 4% paraformaldehyde; then the tissues were paraffin-embedded and sectioned at 5–7 μm. Hematoxylin and eosin (H&E)staining was performed according to a standard method. Forimmunofluorescence, briefly, BAT and Epi-WAT sections wereincubated with UCP1 (Sigma), diluted at 1:100, at 4°C for 12 h, andthen incubated with a secondary antibody for 1 h. Stained slides wereviewedunder a Zeiss Observer.A1Microscope (Carl Zeiss, Oberkochen,Germany) at ×200magnification, and an Olympus C4000 zoomdigitalcamera (Nikon DS-L1, Nikon, Tokyo, Japan) was used to capture theimages.
2.7. Metabolic phenotyping
Two days before tissue sample collection, mice were placed inmetabolic chambers (Columbus Instruments, Columbus, OH) andacclimated over a 24 h period. Then, the metabolic rate, includingoxygen consumption (VO2) and carbon dioxide production (VCO2),was monitored every 13 min for the duration of the next 24 h. Therespiratory exchange ratio (RER) was calculated as the ratio of carbondioxide volume versus oxygen volume (VCO2/VO2). In addition, heatand spontaneous locomotor activity were closely monitored for theduration of the measurement period.
2.8. GC–MS analysis of target TCA metabolites in serum
A total of 20 μL of thawed serum and 80 μL of cold methanolincluding an internal standard (10 μg/mL dulcitol)were combined andvortexed for 60 s. After centrifugation at 14,000 × g and 4°C for 15min, 70 μL of supernatant was evaporated to dryness under a nitrogenstream. The residue was reconstituted in 30 μL of 20 mg/mLmethoxyamine hydrochloride in pyridine, and the resulting mixturewas incubated at 37°C for 90 min. Next, 30 μL of N,O-bis(trimethylsi-lyl)trifluoroacetamide (BSTFA) (with 1% trimethylchlorosilane(TMCS)) was added to the mixture and derivatized at 70°C for 60min prior to GC–MS analysis.
A mixed standard solution, containing 20 μg/mL succinic acid,fumaric acid, malic acid, citric acid, cis-aconitic acid, 2-ketoglutaricacid, pyruvic acid, lactic acid, glutamic acid, glutamine, pantothenicacid, ribose-5-phosphate, glucose, glucose-6-phosphate and fructose-6-phosphate (Sigma-Aldrich), was prepared in water and seriallydiluted to 0.02–10 μg/mL. Then, 70 μL of the standardmixture, 35 μL ofthe quality control (QC) sample and 28 μL of 10 μg/mL dulcitol weremixed and evaporated to dryness under a nitrogen stream. The othersteps were the same as those for serum.
An instrumental analysis was performed on an Agilent 7890A gaschromatography system coupled to an Agilent 5975C inert MSDsystem (Agilent Technologies, CA, USA). An OPTIMA® 5 MS Accentfused-silica capillary column (30 m×0.25 mm×0.25 μm; MACHEREY-NAGEL, Düren, GER) was utilized to separate the derivatives. Helium(N 99.999%) was used as a carrier gas at a constant flow rate of 1 mL/min through the column. Injection volume was 1 μL in a split ratio of10:1 for glucose and lactic acid and 1 μL in splitless mode for the othermetabolites. The solvent delay time was 5.5 min. The initial oventemperature was held at 60°C for 1 min, ramped to 240°C at a rate of12°C/min, to 320°C at 40°C/min and finally held for 4 min. Thetemperatures of the injector, transfer line, and electron impact ionsource were set to 250°C, 260°C, and 230°C, respectively. The impactenergy was 70 eV, and data were collected in both full-scan (m/z50–600) and SIM mode. Energy metabolites were analyzed by MSDChemStation Version E.02.02.1431 (Agilent Technologies). The quan-titation ions for all target compounds, including the internal standard,
are shown in Supplementary Table S3, which also includes thechromatographic retention times and relative standard deviation (%RSD).
Total RNAwas extracted fromdifferent adipose tissues using TRIzolreagent (Invitrogen, Carlsbad, USA) according to the manufacturer'sinstructions. The quantification and qualitative ratiometric analysis ofRNA was performed using a Nanodrop 2000 (Thermo Scientific,Wilmington, DE, USA). For each sample, 1 μg of total RNAwas reverse-transcribed with a High-Capacity cDNA Reverse Transcription kit(Tiangen Biotech Co., Ltd). Gene expression was quantitated using aFast Start Essential DNA Green Light Master kit (Roche, Indianapolis,IN, USA) in a Light Cycler 480 real-time PCR system (Roche). The PCRconditionswere as follows: 10min at 95°C, followed by 40 cycles of 10s at 95°C, annealing for 10 s at 60°C and extension for 10 s at 72°C. Dataanalysis was conducted by the 2-ΔΔCT method. β-Actin was used asthe reference gene for normalization. The sequences of the sense andantisense primers used for amplification are shown in SupplementaryTable S4.
2.10. Western blot analysis
Tissues were incubated in RIPA lysis buffer (Thermo Fisher,Waltham, MA, USA) and phosphatase inhibitor (Sigma-Aldrich, SaintLouis, MN, USA) at pH 7.4 for 30 min. The samples were subsequentlysonicated at 4°C. After centrifuging the samples (12,000×g for 15 minat 4°C), the protein concentrations of the supernatant were measuredusing a BCA protein assay kit (Pierce, Rockford, IL, USA) according tothe manufacturer's protocol. Equal amounts of protein (40 μg) fromeach sample were loaded and separated by SDS-PAGE and transferredonto a PVDF membrane using Bio-RAD electrophoresis equipment.Membranes were blocked with 5% nonfat milk in Tris-buffered salineand Tween 20 (TBST) for 1 h at room temperature. Themembranewasthen rinsed three times consecutively with TBST buffer, followed byincubation for 1 h with 1:1000 dilutions of primary monoclonalantibodies: anti-UCP1 (Abcam), anti-β-actin, anti-p-AMPK, anti-AMPK, anti-SIRT1, anti-PRDM16 and anti-PGC-1α (Cell SignalingTechnology)-in TBST buffer containing 1% skim milk. After threewashes, the membrane was incubated for 1 h with horseradishperoxidase-conjugated anti-rabbit IgG secondary antibody (1:2000,Cell Signaling Technology) in TBST buffer containing 1% skimmilk. Allsignals were visualized and analyzed by densitometric scanning(Image Quant TL7.0, GE Healthcare Bio-Sciences AB). The density ofthe bandswas quantified using ImageJ Software (National Institutes ofHealth, USA). The ratio of the intensity of the target protein to that ofthe β-actin loading control was calculated to represent the expressionlevel of the protein.
2.11. Statistical analysis
All data are expressed as themean±standard error of themean (S.E.M.). Statistical analyses were conducted using SPSS software (IBMCorporation, NY, USA) version 20. Differences between groups wereanalyzedwith one-way analysis of variance (ANOVA), followed by theleast significant difference (LSD) post hoc test. P values less than .05were considered indicative of significance.
3. Results
3.1. Effect of ginger on obesity-induced metabolic disorders in mice
After the 16-week feeding period, ginger supplementation showeda protective role against the development of obesity in mice (Table 2).
Table 2Effect of ginger administration on obesity-related parameters.
Variables/Groups NCD NCD-G HFD HFD-G
Final body weight (g) 27.98±0.33a 26.00±0.41a 36.39±0.66c 32.43±0.60b
Weight gain (g) 9.25±0.28a 7.44±0.39a 17.04±0.83c 13.90±0.43b
Total energy intake(Kcal)
9 4 4 . 5 0 ±14.50a
9 4 8 . 1 6 ±7.00a
1 1 8 8 . 7 7±5.35b
1 1 7 9 . 5 0±9.48b
Energy efficiency (g/Kcal)
0 . 0 2 9 6 ±0.0003b
0 . 0 2 7 4 ±0.0004a
0 . 0 3 0 6 ±0.0005b
0 . 0 2 7 4 ±0.0005a
Epi-WAT/body weight(mg/g)
17.57±1.11a 16.18±0.74a 54.48±2.41c 31.87±2.00b
Per-WAT/body weight(mg/g)
4.91±0.69a 4.47±0.52a 24.44±2.37c 11.85±0.88b
Ing-SAT/body weight(mg/g)
9.10±1.31a 7.22±1.01a 26.14±1.80b 10.83±1.53a
All data are expressed as the mean±S.E.M. (n=8). Statistical analysis was performedusing ANOVA. The means with different superscripts are considered significantlydifferent (Pb.05)
Table 3Serum biochemical analyses fromNCD and HFDmice with or without ginger treatment.
Values are expressed as the mean±S.E.M. (n=8). Statistical analysis was performedusing ANOVA. The means with different superscripts are considered significantlydifferent (Pb.05).
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First, no significant difference in body weight was found between theNCD-G group and NCD group. However, the final body weight, bodyweight gain and energy efficiency tended to be lower in the HFD-Ggroup than in the HFD group.We then isolated the threemain types ofwhite adipose tissue frommice: Epi-WAT, Per-WAT, and Ing-SAT (Fig.1). Consistent with the body weight gain, weights of all three types ofadipose tissues and the ratio of various adipose tissues normalized tobody weight were clearly increased by HFD and decreased by gingersupplementation in mice, indicating a protective role of ginger in thedevelopment of obesity. Moreover, ginger was highly effective inrecovering theHFD-induced increase in levels of serumglucose, TC, TGand HDL-C as well as liver AST and ALT levels (Table 3).
3.2. Effect of dietary ginger on energy expenditure in mice
The metabolic rates of mice were further analyzed by open-circuitindirect calorimetry. Compared with the NCD group, the HFD grouppresented lower O2 consumption and CO2 production. However,ginger supplementation significantly elevated themetabolic rate bothin NCD and HFD mice (Fig. 2A, B). Next, RER was used to estimate therelative contribution of fat and carbohydrates to whole-body energymetabolism in mice. As expected, mice treated with ginger revealed asmall increase during the nocturnal activity phase in both diet models(Fig. 2C), and heat production was also increased (Fig. 2D). Consistent
Fig. 1. Representative image
with the elevation in metabolic rates, ginger supplementationsignificantly increased the ambulatory activity of the animals duringthe night phase (Fig. 2E). These data indicate that ginger attenuatedHFD-induced obesity due to enhanced energy expenditure.
3.3. Effect of dietary ginger on serum metabolites involved in glycolysisand the TCA cycle in mice
In this study, a novel, specific and sensitive GC–MS assay formetabolites involved in glycolysis and the TCA cycle was developed.The GC–MS chromatograms of the 13 target compounds in thestandard solutions and a representative serum sample are shown inSupplementary Fig. S5. The concentrations of the major intermediatesinvolved in glycolysis, such as fructose-6-phosphate (F6P), glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P), were increased inHFD rats compared with NCD rats, and ginger administrationcorrected the changes in glycolysis induced by a HFD. In addition,HFD-fed mice also displayed reduced concentrations of TCA cycleintermediates in serum, such as pyruvic acid, lactic acid, succinic acid,glutamate, glutamic acid, fumaric acid and malic acid. Clearly,treatment with ginger reversed the levels of intermediates (Fig. 3),suggesting that ginger positively modulated the TCA cycle. Consider-ing the above results, we speculated that ginger improved metabolicimpairments via the glycolysis/gluconeogenesis-TCA cycle.
s of WATs and BAT.
Fig. 2. Changes inwhole-body energy balance in response to ginger supplementation. Indirect calorimetrymeasures of 24-h (A) oxygen consumption, (B) carbon dioxide production, (C)RER, (D) heat production and (E) voluntary activity. The results are expressed as themeans±S.E.M. (n=3). Differenceswere assessed byANOVA anddenoted as follows:&Pb.05,&&Pb.01,&&&Pb.001, HFD-G vs HFD; #Pb.05, ##Pb.01, ###Pb.001, HFD vs. NCD; ⁎Pb.05,⁎⁎Pb.01, ⁎⁎⁎Pb.001, NS not significant, NCD-G vs NCD.
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3.4. Ginger can promote browning of WAT and activate BAT in mice
Further, we determined whether dietary ginger stimulated thegeneration of brown-like adipocytes. The results showed that gingersupplementation upregulated the expression of a series of brownadipocyte-specific genes (UCP1, PRDM16 and CIDEA) and beigeadipocyte-selective marker genes (TMEM26 and CITED1) in bothWAT and BAT of obese mice. Moreover, ginger increased the mRNAexpression of PGC-1α, the master regulator of mitochondrialbiogenesis and oxidative phosphorylation. Consistently, ginger pro-moted the expression of genes involved in mitochondrial biogenesis,such as TFAM and NRF1 (Fig. 4A, B).
The immunofluorescence staining results showed that the expres-sion of UCP1 in the ginger-treated groups was higher than that in thecorresponding control groups (Fig. 4G). This finding was in accordwith the Western blotting results demonstrating that the UCP1protein level was increased in WAT and BAT after ginger supplemen-tation (Fig. 4C-F). Moreover, the protein expression of PRDM16 andPGC-1αwas also markedly increased in both WAT and BAT of ginger-treated mice (Fig. 4C-F). These data indicated that ginger could
increase the BAT thermogenic progress and induce adipocytebrowning in WAT. Additionally, HFD decreased AMPK phosphoryla-tion without an effect on total AMPK levels, resulting in a decrease inthe p-AMPK/AMPK ratio, which was recovered by ginger. A similarmodulation of SIRT1 was observed after ginger treatment (Fig. 4E, F).Together, these results provide evidence that administration of gingerinduced browning in WAT of mice by activating AMPK.
4. Discussion
Browning of WAT can increase energy expenditure through UCP1-mediated thermogenesis. Thus, pharmacologic or nutritional strate-gies for promoting adipocyte browning and thermogenesis mayprovide a defense against obesity and associated metabolic diseases[23]. Growing evidence indicates that ginger exerts beneficial effectsagainst obesity and relatedmetabolic syndromes [15,16,24], but only asmall number of studies focus on energy metabolism. Dietary gingerextract was previously shown to increase energy expenditure andattenuate diet-induced obesity in C57BL/6 Jmice through activation ofthe PPAR-δ pathway [16]. Moreover, an in vitro mechanistic study
Fig. 3. Potential metabolic pathways disturbed in HFD-induced obesemice and alterations by ginger treatment. The results are expressed as themeans±S.E.M. (n=8). Differences wereassessed by ANOVA and denoted as follows: &Pb.05, &&Pb.01, &&&Pb.001, HFD-G vs HFD; #Pb.05, ##Pb.01, ###Pb.001, HFD vs. NCD; ⁎Pb.05,⁎⁎Pb.01, ⁎⁎⁎Pb.001, NS not significant, NCD-G vsNCD. No mark = no significant difference.
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showed that 6-gingerol dose-dependently increased AMPKα-subunitphosphorylation accompanied by a time-dependent marked increasein PGC-1αmRNA expression and mitochondrial content in L6 skeletalmuscle cells [25]. Nevertheless, whether ginger supplementation caninfluenceWAT browning in obese mice and if so, what the underlyingmechanisms are remain unclear.
The present study demonstrated that ginger prevented obesity byenhancing energy expenditure and activating browning and thermo-genic functions. First, ginger noticeably lowered HFD-induced in-creases in body weight gain, fat accumulation, and serum glucose, TC,TG and LDL-C levels. Consistently, ginger supplementation signifi-cantly alleviated the HFD-induced increase in the size of adipocytes. Inaddition, ginger treatment dramatically increased RER and heatproduction.
Next, we evaluated the impact of ginger supplementation onenergy metabolism. Recently, many nontargeted metabolomicsanalyses have suggested that obesity is associated with the levels ofmetabolic intermediates in energy metabolism pathways, includingglycolysis and the TCA cycle [26,27]. Metabolites such as pyruvate,lactate, and citratewere themain discriminatingmetabolites betweenthe obese and the healthy models [28]. Therefore, the present studyshowed that HFD clearly distorted glucose metabolism by increasingthe concentrations of the major intermediates involved in thephosphogluconate pathway. Moreover, certain other key metabolitesrelated to the TCA cycle were reduced in obese rats. These findings areconsistent with previous results obtained in nontargeted metabolo-mics studies of obesity [26,29]. Interestingly, ginger supplementationsignificantly lowered the concentrations of the metabolic intermedi-ates involved in glucose metabolism, such as R5P, G6P and F6P. Theseresults demonstrated that ginger could improve distorted glucosemetabolism, which was further supported by the reduction in serumglucose levels. Moreover, dietary ginger could greatly attenuatemetabolic impairments by ameliorating the blood lipid profile and
reducing lipid accumulation, as well as elevating concentrations ofsome metabolites involved in the TCA cycle, such as pyruvate andlactate. Among these metabolites, pyruvate plays a key role in glucoseaerobic oxidation and energy production [30], as it not only is a keymetabolite in glucose metabolism but also can serve as an importantsource of acetyl-CoA to fuel the TCA cycle. As the product of pyruvate,lactate in particular was increased after ginger treatment of obesemice. Notably, lactate can induce UCP1 gene expression in whiteadipocytes and the expression of genes associated with fatty acidoxidation andmitochondrial function, indicative of the recruitment ofa thermogenic profile [31]. Consistently, Morinda citrifolia L. leafextract, commonly called noni or Indian mulberry, improved HFD-induced perturbations in various metabolic pathways, predominantlyin the glucose and TCA cycle, as reflected by positive modulation oflactate, pyruvate, and glucose levels [32]. Another important inter-mediate in the TCA cycle is citric acid, which is regulated by insulin,glucose level, fatty acid utilization, and cholesterol synthesis [33]. Theincreased level of citric acid in obese mice supplemented with gingerfurther indicated a restoration of the glycolytic and energy metabolicdysfunction induced by HFD [34]. Alterations in some other metab-olites (such as 2-ketoglutaric acid, succinic acid, fumaric acid,glutamine and malic acid levels) caused by HFD-induced obesity incertain metabolic pathways were also notably improved upontreatment with ginger. Therefore, ginger could prevent energymetabolism impairment caused by HFD.
The TCA cycle is coupled with ATP production and proton leak,which can release energy in the form of heat [35]. UCP1, one of themain mitochondrial proteins, can dissipate the proton gradientgenerated by the NADH-powered pumping of protons from themitochondrial matrix to the mitochondrial intermembrane space andlet H+directly pass through the inner-membrane ofmitochondria andrelease heat [36]. Furthermore, BAT thermogenesis is dependent onUCP1 since it confers to brown adipocytes the specific capacity to
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dissipate oxidation energy as heat [37]. Improvement of thermogeniccapacity through activation of BAT may be a highly beneficialtherapeutic strategy for counteracting obesity and its complications[38,39]. Many natural compounds and food ingredients, such asresveratrol, cinnamaldehyde, sesamol and spirulina maxima extract,have shown antiobesity effects through activation of browning andincreases in energy expenditure [39–42]. Evidences from the presentstudy indicated that the beneficial effects of ginger on suppression ofobesity-related pathological factors were associated with BAT activa-tion. The expression of UCP1 was increased in BAT and emerged inWAT after ginger treatment, which was further confirmed by UCP1immunofluorescence staining. Moreover, ginger administration up-regulated several other dominant transcriptional regulators of
Fig. 4. Dietary ginger supplementation induced brown fat-like changes inWAT and BAT of mice(B) (n=6); Protein level of brown adipocyte-specificmarker expression in BAT (C andD) andWnuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) (scale: 50 μm). The renormalized to β-actin (used as reference gene/protein). Data from the NCD group were set toANOVA and denoted as follows: &Pb.05, &&Pb.01, &&&Pb.001, HFD-G vs HFD; #Pb.05, ##Pb.01, ###Pdifference.
browning in adipocytes, such as PGC-1α, PRDM16 and CIDEA.Among them, PRDM16 is a determinant factor of brown-fat develop-ment and brings about the induction of BAT-specific genes byassociating with the transcriptional coactivators PGC-1α [43,44].PGC-1α controls respiration through induction of UCPs and regulationof nuclear respiratory factors [45]. In muscle-specific PGC-1αtransgenic mice, UCP1 and CIDEA mRNA expressions were up-regulated in WAT [46]. Meanwhile, adipose PGC-1α deficiency inmice results in a blunted expression of these thermogenic genes inWAT [47]. Furthermore, some beige adipocyte markers, includingTMEM26 and CITED1, also showed significant upregulation afterginger administration. These findings strongly suggested that gingercould activate the BAT thermogenic progress and induce adipocyte
. mRNA expression of brown and beige adipocyte-selective markers in BAT (A) andWATAT (E and F) (n=6); Immunofluorescence staining inWAT and BAT sections (G) (n=3);sults are expressed as the means±S.E.M. The ratios of specific mRNA or protein levels100%, and the rest of the values were normalized to this. Differences were assessed byb.001, HFD vs. NCD; ⁎Pb.05, ⁎⁎Pb.01, ⁎⁎⁎Pb.001, NCD-G vs NCD. Nomark= no significant
Fig. 4. (continued).
112 J. Wang et al. / Journal of Nutritional Biochemistry 70 (2019) 105–115
Fig. 4. (continued).
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browning in WAT. Additionally, the thermogenesis of BAT is wellknown to be closely related to the number and function ofmitochondria. TFAM is a key regulator of mitochondrial replicationand biogenesis, and PGC-1α is the master regulator of mitochondrialbiogenesis by controlling the expression of TFAM by interacting withNRF1 [45]. Moreover, activation of PGC-1α plays an important role inmitochondrial biogenesis, and SIRT1-mediated PGC-1α deacetylationand activation are critical for mitochondrial function in BAT [48]. Inthis study, we found that ginger treatment couldmarkedly elevate theexpression of genes involved in mitochondrial biogenesis, such asPGC-1α, TFAM and NRF1, both in BAT and WAT. Moreover, theexpression of core proteins (SIRT1 and PGC-1α) were significantlyupregulated after ginger administration. Certainly, these resultsindicated that ginger supplementation could activate the thermogen-esis of BAT through upregulating mitochondrial biogenesis andfunction.
AMPK, a key regulator of energy metabolism, can affect theexpression of the key factors involved inmitochondrial biogenesis andenergy expenditure [49]. Furthermore, AMPK and SIRT1 act togetherwith PGC-1α to regulate energy homeostasis [8]. Activation of PGC-1αfurther upregulates the expression of PRDM16 and UCP1, thedominant regulator of energy expenditure [43]. Interestingly, anumber of phytochemicals (such as raspberry and resveratrol)promoteWAT browning and nonshivering thermogenesis by increas-ing expression and activation of AMPK and SIRT1, along with PGC-1α[40,50]. Consistent results in this study showed that ginger couldeffectively activate AMPK-promoted PGC-1α and SIRT1 proteinexpression and improve mitochondrial biogenesis, resulting inbrowning of WAT. These results indicated that ginger amelioratedHFD-induced obesity by promoting the browning of WAT andactivating the thermogenic program through the SIRT1/AMPK/PGC-1α signaling pathway in HFD-induced obese mice. Future studies onthe molecular mechanisms involved in energy metabolism regulationare required to elucidate the contribution of ginger to the preventionand treatment of obesity and related metabolic disorders. However,
due to the complex composition of ginger, its precise phytochemicalscontributing to its effects on energy metabolism remain unknown.Thus, more evidence is required to fully assess the bioactivesubstances in ginger that prevent obesity by regulating energymetabolism.
In conclusion, oral ginger supplementation significantly preventedand improved HFD-induced obesity, triggered the energy metabolismremodeling and enhanced browning-related gene and proteinexpression both in WAT and BAT. Briefly, ginger can regulate theglycolysis/gluconeogenesis-TCA cycle pathway and stimulate thebrowning program by the SIRT1/AMPK/PGC-1α pathway. Based onthese findings, we concluded that ginger is an edible plant that plays arole in the therapeutic treatment of obesity and related disorders.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jnutbio.2019.05.001.
Acknowledgments
This work was supported by the grant from National KeyTechnologies R&D Program [grant number 2017YFD0400700] andthe Beijing Municipal Science and Technology Project [grant numberD16110500540001].
Competing interests
The authors declare no competing interests.
Author contributions
J. W. designed and executed the experiments, analyzed the data,and wrote the manuscript; X-SH, P. W. and F. C. contributed to theexperimental design; D-TL contributed to performing experiments;All authors contributed to and have approved the final manuscript.
114 J. Wang et al. / Journal of Nutritional Biochemistry 70 (2019) 105–115
Abbreviations
BAT brown adipose tissueWAT white adipose tissueEpi-WAT epididymal white adipose tissuePer-WAT perirenal white adipose tissueIng-SAT inguinal subcutaneous adipose tissueNCD normal chow dietHFD high-fat dietNCD-G normal chow diet supplemented with gingerHFD-G high-fat diet supplemented with gingerTCA tricarboxylic acid cycleGC–MS gas chromatography–mass spectrometryACTB beta-actinSIRT1 sirtuin-1AMPK AMP-activated protein kinasePGC-1α peroxisome proliferator-activated receptorγ coactivator 1αUCP1 uncoupling protein-1CIDEA Cell death-inducing DNA fragmentation factor alpha-like
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