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Research article
Echinacoside Induces UCP1- and ATP-Dependent Thermogenesis in Beige Adipocytes via the Activation of Dopaminergic Receptors
Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk 38453, Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2023; 33(10): 1268-1280
Published October 28, 2023 https://doi.org/10.4014/jmb.2306.06041
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract
Introduction
Obesity is characterized by an imbalance in energy intake and expenditure. Recent advances in molecular biotechnology have brought fresh insights into the involvement of brown and beige fat in obesity and the elucidation of ways to prevent obesity and consequent metabolic disorders [1]. In this regard, the identification of beige subtypes led to the understanding of the additional potential of the brown adipose tissue (BAT) in dissipating energy as heat by the combustion of nutrients during ATP generation via uncoupling protein 1 (UCP1) [2].
UCP1, a unique thermogenic indicator, is one of the 53 members of the mitochondrial carrier family [3] that short-circuits the mitochondrial proton gradient to produce heat via the oxidative phosphorylation of metabolic fuels such as fatty acids [4]. Several lines of evidence suggest that brown and beige adipocytes, despite possessing UCP1, also induce energy expenditure via ATP-consuming Ca2+ futile cycles [5, 6]. Catecholamines, under appropriate stimuli, can be engaged to increase intracellular Ca2+ cycling by stimulating the ryanodine receptors (RyR) and promoting Ca2+ export from the endoplasmic reticulum (ER). Calcium export then raises the activity of the ER Ca2+ (sarco/endoplasmic reticulum Ca²+-ATPase2, SERCA2), allowing Ca2+ to be imported back into the ER [7]. This cyclic import and export of Ca2+ into and from the ER results in the consumption of ATP, which eventually causes energy dissipation that helps to reduce the occurrence of weight gain and protects against obesity.
Many of the synthetic drugs that are still in use today are based on naturally occurring chemicals derived from plants. In the past three decades, around 35% of drugs approved in the global market have been made either directly or indirectly from natural substances [8]. A growing body of evidence suggests that phytochemicals mostly in the group of terpenoids, polyphenols, and alkaloids are excellent alternatives to synthetic drugs that promote adipose tissue browning and may be promising candidates for obesity management [9]. These studies exemplify the role of bioactive compounds in inducing white adipose browning in cultured adipocytes in vitro and in animal models, and this browning effect is linked to various metabolic mechanisms that may help prevent obesity [10, 11].
ECH is a naturally occurring phenylethanoid glycoside with several therapeutic properties, isolated from the perennial herb
Despite its diverse metabolic and therapeutic functions, the functional properties of ECH in fat browning and ATP-dependent futile cycle processes as well as its interaction with the dopaminergic receptors, have not yet been explored. The aim of this study was, therefore, to examine the functional significance of ECH in UCP1- and ATP-dependent thermogenesis in adipocytes in vitro and in mice in vivo, as well as its interaction with D1-like dopaminergic receptors.
Materials and Methods
Chemicals
ECH and prazosin (an antagonist of α1-adrenergic receptors [AR]) were acquired from Sigma-Aldrich (USA). The D1-like dopaminergic receptor agonist SKF38393 was obtained from Abcam (UK). SCH 23390 (DRD1/5 antagonist) and cirazoline (an α1-AR agonist) were purchased from Tocris Bioscience (UK). All other chemicals employed in this research were of analytical grade.
MTT Assay
The MTT colorimetric assay, in accordance with the manufacturer's instructions, was employed to determine the percentage of apoptotic cells. Briefly, 3T3-L1 cells were cultured in 24-well plates containing 500 μl Dulbecco’s Modified Eagle’s Medium (DMEM). Following the treatment with different concentrations of ECH, the DMEM medium in each well was substituted with a 200 μl solution containing 0.5 mg/ml MTT. The cells were then incubated at 37°C for 4 h. Afterward, the media were discarded, and the formazan blue generated within the cells was dissolved in 1 ml of DMSO. Optical density was then measured at 540 nm.
Cell Culture and Differentiation
3T3-L1 preadipocytes obtained from ATCC (USA) were cultured in DMEM (Thermo Fisher Scientific Inc., USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc.) and 100 μg/ml penicillin-streptomycin (Invitrogen, USA). The cells were cultured for 7-10 passages at 37°C in a 5% CO2 incubator. A suitable quantity of confluent 3T3-L1 cells was cultured in the differentiation induction medium composed of 10 μg/ml insulin (Sigma-Aldrich), 0.25 mM dexamethasone (Sigma-Aldrich), and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) in DMEM for 48 h. Subsequently, the cells were cultured for an additional 72 h in a DMEM maturation medium supplemented with 10% FBS and 10 μg/ml insulin.
Animal Experiments
For all the experiments, five-week-old C57BL/6 mice were purchased from Hyochang Science (Korea) and housed individually. The mice were maintained under a 12-h light/dark cycle at a temperature of 27°C with 34%humidity. They were acclimatized to a regular chow diet for 1 month, followed by a high-fat diet (HFD, 60% fat) for 5 weeks. Afterward, the mice were divided into two groups: HFD-fed control mice (HFD,
Hematoxylin and Eosin Staining
A histological examination was conducted on the iWAT, eWAT, and BAT. The tissues were fixed in 10% neutral-buffered formalin, followed by washing with phosphate-buffered saline (PBS), and then embedded in paraffin wax. Sections of the paraffin-embedded tissues, approximately 3-5 μm thick each, were deparaffinized, rehydrated, and stained with hematoxylin (Vector Laboratories Inc., Canada) and eosin (H&E, Daejung, Korea). Optical microscopy using an Olympus IX51 microscope (Japan) was employed to conduct the histopathological analysis of the tissues.
Quantitative Real-Time RT-PCR
Total RNA was isolated from the iWAT using a total RNA isolation kit (RNA-spin, iNtRON Biotechnology, Korea). Subsequently, cDNA synthesis was performed using 1 μg of RNA and a Maxime RT premix (iNtRON Biotechnology). The mRNA levels were quantitatively assessed using qPCR (Stratagene 246 mx 3000p QPCR System, Agilent Technologies, USA) with SYBR Green (Roche, Switzerland) staining. All experiments were conducted with technical triplicates, and the mRNA expression levels were normalized to the β-actin gene levels in the corresponding RNA samples. The primer sequences used in this study are provided in Table 1.
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Table 1 . Primer sequences used for real-time quantitative RT-PCR.
Gene Forward Reverse Drd1 AGGGGTTTTGGGAGAAGTGAC AGTCACTTTTCGGGGATGCTG Drd5 GGGGTTTTGGGAGAAGTGAC AGTCACTTTTCGGGGATGCTG Cited1 GGGGTAAAAGATCGCAAGGC TGGTAGAAGGGGTGGCAGTA Ppargc1α ATGAATGCAGCGGTCTTAGC AACAATGGCAGGGTTTGTTC Prdm16 GATGGGAGATGCTGACGGAT TGATCTGACACATGGCGAGG Tbx 1AGCGAGGCGGAAGGGA CCTGGTGACTGTGCTGAAGT Ucp1 CCTGCCTCTCTCGGAAACAA GTAGCGGGGTTTGATCCCAT Cd137 GGTCTGTGCTTAAGACCGGG TCTTAATAGCTGGTCCTCCCTC Cidea CGGGAATAGCCAGAGTCACC TGTGCATCGGATGTCGTAGG Cebpa CCCTCTCCCCTAGTTGTCCA CTTTCCAGACCCAGAGCCAG Pparγ ACCATGGTAATTTCAGTAAAGGGT CCTGACGGGGTCTCGGT Mest AGCAGCTTTCCTCTGCGG GATACAGGATCGGAGGTGGC Srbf1 CCTGACAGGTGAAATCGGCG GTTGTTGATGAGCTGGAGCA Fasn CACTGCCTTCGGTTCAGTCTC ACACCCTCCAAGGAGTCTCAC Scd1 TGGAGACGGGAGTCACAAGA ACACCCCGATAGCAATATCCAG Slc2a4 CTCTGACGTAAGGATGGGGA ACCTTCTGTGGGGCATTGAT Lipe CTTGGGGAGCTCCAGTCGGAAG TGTCTTCTGCGAGTGTCACCAG Pnpla2 CTGCTGTAAACCCCTGGTCT CCACGGATGGTCTTCACCAG Mfn1 TCCCCTCTTTCGGGAGGATG GTCCGGAGCTCGAAGGTCA Mfn2 GACACGGGACGGTTACCAG CTCTGAACGCTGTCACCTCA PDK4 TGAACACTCCTTCGGTGCAG TCGAACTTTGACCAGCGTGT Tfam TAGGCACCGTATTGCGTGAG GTGCTTTTAGCACGCTCCAC Cpt1 GACTCCGCTCGCTCATTCC TTGAGGGCTTCATGGCTCAG Nrf1 GCTAATGGCCTGGTCCAGAT CTGCGCTGTCCGATATCCTG Cycs CACCGACACCGGTACATAGG TAATTCGTTCCGGGCTGGTC Ryr2 CAGAAGGGACTGCTCAAGGT TCGAAGGCCAGTTTGTCAGT Tfam ATGTGGAGCGTGCTAAAAGC GGATAGCTACCCATGCTGGAA Cox4 ACATTCAGGGTGCCTCTTTG CATGGCAGAAGTGGGAGATT Atp2a1b TCAGGGCAGGAGCATCATTC ATGTAGCGCTGTCAGTCAAGA
Western Blot Analysis
Cell lysates were prepared by adding a radioimmunoprecipitation assay buffer (RIPA buffer, Sigma–Aldrich), followed by homogenization and centrifugation at 13,000 ×
Immunocytochemical Analysis
To directly detect the expression of UCP1 in the control and ECH-treated adipocytes, immunocytochemical analysis was performed on formalin-fixed 3T3-L1 cells. The obtained sections were incubated overnight at 4°C with the primary UCP1 antibody (dilution 1:1000, Santa Cruz Biotechnology), followed by incubation with the appropriate fluorescein isothiocyanate (FITC) goat anti-mouse secondary antibody at room temperature for 4 h. The mitochondria were stained by adding MitoTracker red (1 mM, Cell Signaling Technology, USA) to PBB-T (PBS+1% BSA, and 0.1% Tween 20) at a concentration of 200 nM, and incubating the cells for 2 h at 37°C. Following incubation, the cells were washed with PBS and subjected to immunostaining. The morphological findings were observed using an optical microscope at ×40 magnification.
Detection of Intracellular Ca2+
Calcium Quantification Kit-Red Fluorescence (Abcam, cat#: ab112115) was employed to measure intracellular calcium. Briefly, the calcium standard was first prepared following the manufacturer’s protocol by diluting the appropriate amount of the 300 mM calcium standard in deionized water to produce a calcium concentration ranging from 0 to 3 mM (12 mg/dl). Next, 50 μl of serially diluted calcium standard was added into each well. Then, 50 μl of the assay reaction mixture was added to each well of the calcium standard, blank control, and test samples to make up the total calcium assay volume of 100 μl/well. Finally, the reaction was incubated for 20–30 min at room temperature, protected from light. Fluorescence intensity was monitored with a fluorescence plate reader at excitation/emission (Ex/Em) wavelengths of 540/590 nm.
Statistical Analysis
All the data are presented as the mean ± standard deviation (SD). At least three independent experiments were performed for each data set. The Statistical Package of Social Science (SPSS, version 17.0; SPSS Inc., USA) tool was used to examine the statistical significance among several groups. The Student’s
Results
Echinacoside Reduces Diet-Induced Obesity in Mice
We first applied an MTT assay to assess the cytotoxicity of ECH (Fig. 1A) on 3T3-L1 cells and found no cytotoxicity up to 20 μM. A final concentration of 10 μM was selected as the working amount for subsequent in vitro experiments (Fig. 1B). Next, we investigated the effect of ECH on body weight and adipose tissue in vivo. 25 mg/kg of ECH was intraperitoneally injected into the HFD-fed mice, twice a week, for 6 weeks and a substantial decline in relative body weight was observed starting from the second week (Fig. 1C). We also measured the weights of various tissues. There was a significant reduction in eWAT and iWAT mass in HFD-fed mice after the ECH treatment, whereas a significant increase in BAT and skeletal muscle mass was observed as indicated in Fig. 1D.
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Fig. 1. Echinacoside reduces diet-induced obesity in mice.
Structure of ECH (A). Cytotoxicity of ECH on 3T3-L1 cells as determined by an MTT assay (B). Body weight of mice observed over 6 weeks (C). Relative weight of various tissues in HFDfed mice and ECH-treated HFD-fed mice (D). Histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using the Statistical Package for the Social Sciences (SPSS; version 17.0; SPSS Inc., USA) to perform ANOVA followed by Tukey's post-hoc tests. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *
p < 0.05 or **p < 0.01.
Echinacoside Induces Beiging in 3T3-L1 Adipocytes
Subsequently, we investigated the impact of ECH on key marker proteins associated with browning (UCP1, PGC-1α, and PRDM16). Our findings demonstrated that ECH treatment resulted in a significant and dose-dependent increase in the expression of these core browning marker proteins (Fig. 2A and 2B). In addition, the administration of ECH to HFD-fed mice displayed rapid upregulation of BAT-specific genes (
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Fig. 2. Echinacoside induces beiging in 3T3-L1 adipocytes.
Effect of ECH on key browning proteins (A and B). mRNA expressions of the core set of beige-fat marker genes in ECH-treated HFD-fed mice (C). Immunohistochemical staining in 3T3-L1 cells (D) (×40 magnification; scale bar = 100 μm). Histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using the Statistical Package for the Social Sciences (SPSS; version 17.0; SPSS Inc., USA) to perform ANOVA followed by Tukey's post-hoc tests. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *
p < 0.05 or **p < 0.01.
Echinacoside Promotes Lipid Metabolisms and Fat Oxidation
Building upon its application, we examined whether ECH affects lipid metabolism in 3T3-L1 white adipocytes. To investigate this, we first determined the expression level of acetyl-CoA carboxylase, which was downregulated after ECH treatment, with a concurrent increase in phosphorylated (p-ACC) and p-AMPK. Fatty acid synthase (FAS) was also downregulated after the ECH treatment of 3T3-L1 white adipocytes (Fig. 3A). In addition, adipogenic transcriptional factors (C/EBP and PPARγ) were downregulated following the ECH treatment of the 3T3-L1 cells (Fig. 3B). The mRNA expression levels of key genes associated with adipogenesis and lipogenesis were also downregulated following the ECH treatment in HFD-fed mice (Fig. 3C). This was further elucidated by the histopathological analysis determined by H & E staining in iWAT, eWAT, and BAT, which showed a reduction in lipid cell size in the ECH + HFD treated group compared with the HFD counterparts as indicated in Fig. 3D. The expression levels of lipolysis and fat oxidation-related proteins were also examined. ECH increased lipolysis by raising the levels of phosphorylated hormone-sensitive lipase (p-HSL) and adipose triglyceride lipase (ATGL). It also resulted in a notable increase in the expression of acyl-coenzyme A oxidase 1 (ACOX1), carnitine palmitoyltransferase 1 (CPT1), and PPARα, suggesting an enhanced ability of ECH to facilitate fat oxidation (Fig. 3E). Moreover, core genes for the lipolysis and fat oxidation processes were also substantially upregulated with the ECH treatment of the HFD-fed mice (Fig. 3F).
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Fig. 3. Echinacoside promotes lipid metabolism in 3T3-L1 white adipocytes.
Effect of ECH in the expression levels of lipogenesis (A) and adipogenesis markers (B). Effect of ECH on the mRNA expression of key adipogenesis and lipogenesis genes in ECH-treated HFD-fed mice (C). H & E staining for inguinal white adipose tissue (iWAT), epididymal adipose tissue (eWAT), and brown adipose tissue (BAT) (D). Effect of ECH in lipolysis and fat oxidation markers (E) in 3T3-L1 cells. mRNA expression of key lipolysis and fatty acid oxidation genes in ECH-treated HFD-fed mice (F). Histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using Student’s
t -test. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *p < 0.05 or **p < 0.01.
Echinacoside Upregulates Calcium Cycling Proteins, Increases Intracellular Calcium, and Facilitates Mitochondrial Biogenesis
We next sought to understand the role of ECH in the regulation of calcium-cycling proteins and intracellular calcium levels as well as mitochondrial biogenesis and found that ECH upregulated voltage-dependent anion (VDAC), mitochondrial calcium uniporter (MCU), ATP synthase F1 subunit beta (ATP5B), and cytochrome C (CYT-C) in 3T3-L1 cells (Fig. 4A). Intracellular calcium was also found to be significantly increased following the ECH treatment of 3T3-L1 cells (Fig. 4B). As shown in Fig. 4C, OXPHOS complexes (I, II, III, IV, and V) were upregulated in the ECH-treated 3T3-L1 cells. This was collectively elucidated in vivo by measuring the mRNA expression levels of mitochondrial biogenesis genes including
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Fig. 4. Echinacoside facilitates mitochondrial biogenesis.
Effect of ECH on the mitochondrial and ATP synthesis effectors (A). Effect of ECH on intracellular calcium (B). Effect of ECH on mitochondrial oxidative phosphorylation (OXPHOS) complexes (I, II, III, IV, and V) (C). In vivo effect of ECH on mitochondrial biogenesis genes (D). Histograms display triple independent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using Student's
t -test. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *p < 0.05 or **p < 0.01.
Echinacoside Induces Browning of White Adipocytes via the DRD1/5/PKA/p38 MAPK Pathway
To unravel the underlying mechanisms by which ECH contributes to the beiging of white adipocytes, we conducted further investigations. Following this, ECH treatment substantially increased the expression levels of signaling molecules, such as DRD1, DRD5, protein kinase A (PKA), p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 (p-p38), extracellular signal-regulated kinase (ERK)1/2, phosphorylated (p)-ERK1/ 2, cAMP-response element binding protein (CREB), p-CREB, activating transcription factor 2 (ATF2) and p-ATF2 in 3T3-L1 adipocytes (Fig. 5). A mechanistic study was undertaken to elucidate the participation of these pathway molecules by treating cells with SKF38393 (DRD1/5 agonist) and SCH 23390 (DRD1/5 antagonist) alone or together with ECH. The results showed that SKF38393 stimulated the expression levels of these molecules along with the browning effectors (UCP1, PGC-1α, and PRDM16), whereas SCH 23390 reversed the agonistic action of SKF38393 (Fig. 6).
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Fig. 5. Echinacoside upregulates browning pathway markers in 3T3-L1 adipocytes.
Effect of ECH on beiging pathway molecules. The histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. The differences between the groups were determined using the Student’s t-test. Statistical significance between control and ECH-treated 3T3-L1 cells is indicated by *
p < 0.05 or **p < 0.01. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *p < 0.05.
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Fig. 6. Mechanistic studies for the effect of echinacoside on browning effectors in 3T3-L1 cells.
Effect of ECH or SKF38393 (DRD5 agonist) and SCH 23390 (DRD1 antagonist) in pathway molecules. The histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. The differences between the groups were determined using the Statistical Package for the Social Sciences (SPSS; version 17.0; SPSS Inc., USA) to perform ANOVA, followed by Tukey’s post-hoc tests. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *
p < 0.05 or **p < 0.01.
Echinacoside Stimulates α1-AR-Mediated ATP-Dependent Thermogenesis by the Activation of DRD1/5
Finally, we investigated the impact of ECH on the ATP-dependent thermogenic mechanisms in 3T3-L1 cells. ECH treatment substantially increased the expression levels of the ryanodine receptor (RyR)2, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)2b, mitochondrial creatine kinase (CKmt), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) as well as their upstream receptor, α1-AR (Fig. 7A). The mRNA expressions of the genes involved in the ATP-dependent thermogenesis were also dramatically increased in the ECH-treated HFD-fed mice (Fig. 7B). This was further demonstrated by a mechanistic study showing that SKF38393 (DRD1/5 agonist) alone or together with ECH stimulated the expression levels of these proteins, whereas SCH 23390 (DRD1/5 antagonist) reversed the agonistic action of SKF38393 (Fig. 7C). We also sought to investigate the interaction of DRD1 and DRD5 with α1-AR and found that prazosin (α1-AR antagonist) downregulated DRD1, DRD5, and their common downstream effectors while cirazoline (α1-AR agonist) reversed the antagonistic effect of prazosin (Fig. 7D).
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Fig. 7. Echinacoside stimulates ATP-dependent thermogenesis.
Effect of ECH on ATP-dependent thermogenic mechanism effectors in 3T3-L1 cells (A). Effect of ECH on the mRNA expressions of genes involved in UCP1-independent thermogenesis (B). Effect of ECH and or SKF38393 (DRD1/5 agonist) as well as SCH 23390 (DRD1/5 antagonist) on ATPdependent thermogenic mechanism effectors (C). Effect of prazosin (α1-AR antagonist) cirazoline (α1-AR agonist) on DRD1 and DRD5 as well as ATP-dependent thermogenic mechanisms effectors (D). Histograms represent the results of tripleindependent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using the Statistical Package for the Social Sciences (SPSS; version 17.0; SPSS Inc., USA) to perform ANOVA followed by Tukey's post-hoc tests or Student's
t -test. Statistical significance between control and ECH-treated 3T3-L1 cells/ tissue is shown by *p < 0.05 or **p < 0.01.
Discussion
A growing body of evidence indicates that phytochemicals mediate thermogenesis via the stimulation of the β3-AR [9, 26, 27]. These molecules target various pathways that are intricately linked to the process of adipogenesis, lipogenesis, lipolysis, and the fat oxidation process, all of which provide a fertile environment for high energy expenditure in the metabolic machinery [11, 28]. As a part of this energy dissipation process, we aimed to investigate alternative receptors that promote thermogenesis and the ATP-dependent futile cycle process when activated with similar natural compounds. In this study, we found that ECH, a naturally occurring phenylethanoid glycoside isolated from
AMP-activated protein kinase (AMPK), the primary energy sensor that monitors the catabolic and anabolic energy balance [29], phosphorylates ACC, a cascade that inhibits fatty acid synthesis and enhances fatty acid oxidation [30]. In this study, the ECH treatment decreased ACC and FAS while increasing p-AMPK and p-ACC, indicating its negative regulation of lipogenesis-related proteins in 3T3-L1 preadipocytes. The current study also revealed that the ECH treatment downregulated the core adipogenic transcription factors (C/EBPα and PPARγ), suggesting its inhibiting role in adipogenesis. The effect of ECH in our current study is consistent with the role of various phytochemicals in the literature that inhibits adipogenesis and lipogenesis [9, 26, 27].
The cyclic AMP/protein kinase A/hormone sensitive lipase (cAMP/PKA/HSL) signaling pathway plays a major role in lipolysis which leads to the formation of free fatty acids (FFA) [31]. This pathway generates FFAs through lipolysis which regulates the energy expenditure of the body and serves as fuel for energy generation in adipocytes [32]. In our study, ECH upregulated the major lipolysis and fat oxidation mediating molecules as well as effectors suggesting its positive role in lipid catabolism.
The cAMP/PKA/p38 MAPK/ERK signaling pathway is the most familiar pathway for the β-oxidation of fatty acids and browning of white adipocytes [33,34]. In this process, catecholamines stimulate adenylyl cyclase (AC), which raises cAMP and subsequently binds to PKA. The PKA-mediated phosphorylation cascade enhances lipolysis, activates p38 MAPK and CREB, and this signaling pathway consequently promotes a thermogenic program. In parallel, the p38 MAPK phosphorylates and activates the transcription factor ATF2 and core browning effectors, causing the transcription of downstream thermogenic genes to be activated [33]. In our study, ECH stimulated the browning of 3T3-L1 adipocytes via the activation of DRD1/5, suggesting that dopaminergic receptors could be an alternative target for anti-obesity drugs in the adipose tissue. This is consistent with our recent findings that L-dihydroxyphenylalanine stimulated the browning of 3T3-L1 adipocytes
One of the major findings of our study was the elucidation of the role of ECH and DRD1/5 in mitochondrial biogenesis and related activities. Mitochondria are the primary source of energy for cellular activity through ATP generation via oxidative phosphorylation, and to operate the various intracellular signaling cascades, mitochondrial biogenesis must occur continuously through self-renewal [40]. The decrease in mitochondrial biogenesis, oxidative metabolic pathways, and OXPHOS complexes in adipose tissue indicate that mitochondrial dysfunction or low mitochondrial number and activity are risk factors in obesity and related metabolic disorders [41]. In this study, ECH upregulated mitochondrial biogenesis genes and proteins as well as OXPHOS complexes
Another remarkable finding from our study is that ECH positively regulated the creatine-dependent ADP/ATP substrate cycling and α1-AR-mediated ATP-dependent thermogenesis in beige adipocytes. The creatine kinase/phosphocreatine (CK/PCr) shuttle enhances energy transfer between mitochondrial and/or glycolytic ATP supply and consumption at the ER by SERCA2b [47]. In addition, UCP1-independent thermogenesis depends on the ATP-dependent Ca2+ cycling fashion in which SERCA2b and RyR2 monitor Ca2+ import/export, which eventually causes energy dissipation [5, 48, 49]. Our present study revealed that ECH stimulated DRD1/5 and activated ATP-dependent thermogenesis
Our study also revealed the positive association between ECH, DRD1, and DRD5 as well as calcium regulatory proteins in beige adipocytes. Ca2+ signaling is essential in improving the metabolic apparatus by boosting calorie intake, thereby minimizing the manifestation of obesity [50, 51]. The association between calcium signaling proteins (VDAC and MCU) is already established, with VDAC being upstream of MCU, as Ca2+ must travel through the outer mitochondrial membrane, where VDAC is located before reaching the MCU [52]. In our study, ECH substantially increased VDAC and MCU as well as the intracellular Ca2+ level in 3T3-L1 cells. In line with our study, impaired dopamine homeostasis remarkably lowered the VDAC1 and VDAC2 levels in human neuroblastoma SH-SY5Y cells [41], suggesting the positive modulation of dopaminergic receptors to VDAC even if the specific dopaminergic subtypes responsible to mediate this process were not clarified. Other earlier studies also reported that the pharmacological activation of DRD1 and DRD5 modified the synaptic function of the hippocampal and neocortical neurons
Finally, our study revealed that ECH positively regulated ATP5B in beige adipocytes. This could be because of the constant entry of cytosolic Ca2+ through the VDAC/MCU gate, and once within the mitochondria, these calcium ions modulate cell survival and ATP production [55]. Taken together, our study indicated that ECH not only directly enhances ATP5B expression, but also led to the expectation that increased levels of VDAC and MCU could positively regulate ATP5B. In accordance with the objective of our study, we made efforts to address the limitations; however, the unavailability of pharmacological drugs that can entirely distinguish between DRD1 and DRD5 prevented us from determining the specific affinity of ECH for either receptor. Nevertheless, this study explores the activity of ECH and the functional roles of dopaminergic receptors in browning activities. This research provides the concept that dopaminergic receptors can serve as alternative receptors for the thermogenesis process, presenting a potential alternative strategy for combating obesity. However, to establish a more comprehensive understanding, further investigations, including detailed
Conclusion
Our study is the first to reveal the phytochemical activation of dopaminergic receptors and their regulatory roles in the browning of 3T3-L1 adipocytes and the ATP-dependent thermogenic process. This study may provide important evidence for comprehensive in vivo functional studies in the future. Overall, our findings show that the thermogenic action of ECH
Acknowledgment
This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT, No. 2019R1A2C2002163).
Ethics Approval Statement
All the procedures were performed according to the guidelines approved by the National Institutes of Health. All animal experiments were approved by the Committee for Laboratory Animal Care and Use of Daegu University (DUIACC-2022-15-0901-015).
Author Contributions
K. Haddish performed experimental design, conducted experiments, analyzed the data, performed the statistical analysis, and wrote the manuscript, and J.W. Yun carried out scientific support, critically reviewed the manuscript and experimental design, and approved the manuscript version to be published.
Abbreviations
Name Memo
ACC Acyl-CoA carboxylase
ACOX Acyl-coenzyme A oxidase 1
AMPK AMP-activated protein kinase
ATF2 Activating transcription factor 2
ATGL Adipose triglyceride lipase
AR Adrenergic receptor
C/EBP/
ATP5B ATP synthase F1 subunit beta
BAT Brown adipose tissue
CKmt/
CPT1/
CYT-C/
CREB cAMP-response element binding protein
CaMKII Ca2+/calmodulin-dependent protein kinase II
DRD1 Dopamine receptor D1
DRD5 Dopamine receptor D5
eWAT Epididymal white adipose tissue
SM Skeletal muscle
MCU Mitochondrial calcium uniporter
ERK Extracellular signal-regulated kinase
FAS Fatty acid synthase
HSL Hormone-sensitive lipase
iWAT Inguinal white adipose tissue
OXPHOS Oxidative phosphorylation I~V
PGC-1α/
p38 MAPK p38 mitogen-activated protein kinase
PKA Protein kinase A
PPAR Peroxisome proliferator-activated receptor
PRDM16/
PDE4 Phosphodiesterase-4
RyR/Ryr Ryanodine receptor/encoding gene
SERCA/
UCP1/
VDAC Voltage-dependent anion channel.
GM Gastrocnemius muscle
Conflict of Interest
Supplemental Materials
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Related articles in JMB
Article
Research article
J. Microbiol. Biotechnol. 2023; 33(10): 1268-1280
Published online October 28, 2023 https://doi.org/10.4014/jmb.2306.06041
Copyright © The Korean Society for Microbiology and Biotechnology.
Echinacoside Induces UCP1- and ATP-Dependent Thermogenesis in Beige Adipocytes via the Activation of Dopaminergic Receptors
Kiros Haddish and Jong Won Yun*
Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk 38453, Republic of Korea
Correspondence to:Jong Won Yun, jwyun@daegu.ac.kr
Abstract
Echinacoside (ECH) is a naturally occurring phenylethanoid glycoside, isolated from Echinacea angustifolia, and this study aimed to analyze its effect on thermogenesis and its interaction with dopaminergic receptors 1 and 5 (DRD1 and DRD5) in 3T3-L1 white adipocytes and mice models. We employed RT-PCR, immunoblot, immunofluorescence, a staining method, and an assay kit to determine its impact. ECH showed a substantial increase in browning signals in vitro and a decrease in adipogenic signals in vivo. Additionally, analysis of the iWAT showed that the key genes involved in beiging, mitochondrial biogenesis, and ATP-dependent thermogenesis were upregulated while adipogenesis and lipogenesis genes were downregulated. OXPHOS complexes, Ca2+ signaling proteins as well as intracellular Ca2+ levels were also upregulated in 3T3-L1 adipocytes following ECH treatment. This was collectively explained by mechanistic studies which showed that ECH mediated the beiging process via the DRD1/5-cAMP-PKA and subsequent downstream molecules, whereas it co-mediated the α1-AR-signaling thermogenesis via the DRD1/5/SERCA2b/RyR2/CKmt pathway in 3T3-L1 adipocytes. Animal experiments revealed that there was a 12.28% reduction in body weight gain after the ECH treatment for six weeks. The effects of ECH treatment on adipose tissue can offer more insights into the treatment of obesity and metabolic syndrome.
Keywords: 3T3-L1, dopaminergic receptors, echinacoside, obesity, thermogenesis
Introduction
Obesity is characterized by an imbalance in energy intake and expenditure. Recent advances in molecular biotechnology have brought fresh insights into the involvement of brown and beige fat in obesity and the elucidation of ways to prevent obesity and consequent metabolic disorders [1]. In this regard, the identification of beige subtypes led to the understanding of the additional potential of the brown adipose tissue (BAT) in dissipating energy as heat by the combustion of nutrients during ATP generation via uncoupling protein 1 (UCP1) [2].
UCP1, a unique thermogenic indicator, is one of the 53 members of the mitochondrial carrier family [3] that short-circuits the mitochondrial proton gradient to produce heat via the oxidative phosphorylation of metabolic fuels such as fatty acids [4]. Several lines of evidence suggest that brown and beige adipocytes, despite possessing UCP1, also induce energy expenditure via ATP-consuming Ca2+ futile cycles [5, 6]. Catecholamines, under appropriate stimuli, can be engaged to increase intracellular Ca2+ cycling by stimulating the ryanodine receptors (RyR) and promoting Ca2+ export from the endoplasmic reticulum (ER). Calcium export then raises the activity of the ER Ca2+ (sarco/endoplasmic reticulum Ca²+-ATPase2, SERCA2), allowing Ca2+ to be imported back into the ER [7]. This cyclic import and export of Ca2+ into and from the ER results in the consumption of ATP, which eventually causes energy dissipation that helps to reduce the occurrence of weight gain and protects against obesity.
Many of the synthetic drugs that are still in use today are based on naturally occurring chemicals derived from plants. In the past three decades, around 35% of drugs approved in the global market have been made either directly or indirectly from natural substances [8]. A growing body of evidence suggests that phytochemicals mostly in the group of terpenoids, polyphenols, and alkaloids are excellent alternatives to synthetic drugs that promote adipose tissue browning and may be promising candidates for obesity management [9]. These studies exemplify the role of bioactive compounds in inducing white adipose browning in cultured adipocytes in vitro and in animal models, and this browning effect is linked to various metabolic mechanisms that may help prevent obesity [10, 11].
ECH is a naturally occurring phenylethanoid glycoside with several therapeutic properties, isolated from the perennial herb
Despite its diverse metabolic and therapeutic functions, the functional properties of ECH in fat browning and ATP-dependent futile cycle processes as well as its interaction with the dopaminergic receptors, have not yet been explored. The aim of this study was, therefore, to examine the functional significance of ECH in UCP1- and ATP-dependent thermogenesis in adipocytes in vitro and in mice in vivo, as well as its interaction with D1-like dopaminergic receptors.
Materials and Methods
Chemicals
ECH and prazosin (an antagonist of α1-adrenergic receptors [AR]) were acquired from Sigma-Aldrich (USA). The D1-like dopaminergic receptor agonist SKF38393 was obtained from Abcam (UK). SCH 23390 (DRD1/5 antagonist) and cirazoline (an α1-AR agonist) were purchased from Tocris Bioscience (UK). All other chemicals employed in this research were of analytical grade.
MTT Assay
The MTT colorimetric assay, in accordance with the manufacturer's instructions, was employed to determine the percentage of apoptotic cells. Briefly, 3T3-L1 cells were cultured in 24-well plates containing 500 μl Dulbecco’s Modified Eagle’s Medium (DMEM). Following the treatment with different concentrations of ECH, the DMEM medium in each well was substituted with a 200 μl solution containing 0.5 mg/ml MTT. The cells were then incubated at 37°C for 4 h. Afterward, the media were discarded, and the formazan blue generated within the cells was dissolved in 1 ml of DMSO. Optical density was then measured at 540 nm.
Cell Culture and Differentiation
3T3-L1 preadipocytes obtained from ATCC (USA) were cultured in DMEM (Thermo Fisher Scientific Inc., USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc.) and 100 μg/ml penicillin-streptomycin (Invitrogen, USA). The cells were cultured for 7-10 passages at 37°C in a 5% CO2 incubator. A suitable quantity of confluent 3T3-L1 cells was cultured in the differentiation induction medium composed of 10 μg/ml insulin (Sigma-Aldrich), 0.25 mM dexamethasone (Sigma-Aldrich), and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) in DMEM for 48 h. Subsequently, the cells were cultured for an additional 72 h in a DMEM maturation medium supplemented with 10% FBS and 10 μg/ml insulin.
Animal Experiments
For all the experiments, five-week-old C57BL/6 mice were purchased from Hyochang Science (Korea) and housed individually. The mice were maintained under a 12-h light/dark cycle at a temperature of 27°C with 34%humidity. They were acclimatized to a regular chow diet for 1 month, followed by a high-fat diet (HFD, 60% fat) for 5 weeks. Afterward, the mice were divided into two groups: HFD-fed control mice (HFD,
Hematoxylin and Eosin Staining
A histological examination was conducted on the iWAT, eWAT, and BAT. The tissues were fixed in 10% neutral-buffered formalin, followed by washing with phosphate-buffered saline (PBS), and then embedded in paraffin wax. Sections of the paraffin-embedded tissues, approximately 3-5 μm thick each, were deparaffinized, rehydrated, and stained with hematoxylin (Vector Laboratories Inc., Canada) and eosin (H&E, Daejung, Korea). Optical microscopy using an Olympus IX51 microscope (Japan) was employed to conduct the histopathological analysis of the tissues.
Quantitative Real-Time RT-PCR
Total RNA was isolated from the iWAT using a total RNA isolation kit (RNA-spin, iNtRON Biotechnology, Korea). Subsequently, cDNA synthesis was performed using 1 μg of RNA and a Maxime RT premix (iNtRON Biotechnology). The mRNA levels were quantitatively assessed using qPCR (Stratagene 246 mx 3000p QPCR System, Agilent Technologies, USA) with SYBR Green (Roche, Switzerland) staining. All experiments were conducted with technical triplicates, and the mRNA expression levels were normalized to the β-actin gene levels in the corresponding RNA samples. The primer sequences used in this study are provided in Table 1.
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Table 1 . Primer sequences used for real-time quantitative RT-PCR..
Gene Forward Reverse Drd1 AGGGGTTTTGGGAGAAGTGAC AGTCACTTTTCGGGGATGCTG Drd5 GGGGTTTTGGGAGAAGTGAC AGTCACTTTTCGGGGATGCTG Cited1 GGGGTAAAAGATCGCAAGGC TGGTAGAAGGGGTGGCAGTA Ppargc1α ATGAATGCAGCGGTCTTAGC AACAATGGCAGGGTTTGTTC Prdm16 GATGGGAGATGCTGACGGAT TGATCTGACACATGGCGAGG Tbx 1AGCGAGGCGGAAGGGA CCTGGTGACTGTGCTGAAGT Ucp1 CCTGCCTCTCTCGGAAACAA GTAGCGGGGTTTGATCCCAT Cd137 GGTCTGTGCTTAAGACCGGG TCTTAATAGCTGGTCCTCCCTC Cidea CGGGAATAGCCAGAGTCACC TGTGCATCGGATGTCGTAGG Cebpa CCCTCTCCCCTAGTTGTCCA CTTTCCAGACCCAGAGCCAG Pparγ ACCATGGTAATTTCAGTAAAGGGT CCTGACGGGGTCTCGGT Mest AGCAGCTTTCCTCTGCGG GATACAGGATCGGAGGTGGC Srbf1 CCTGACAGGTGAAATCGGCG GTTGTTGATGAGCTGGAGCA Fasn CACTGCCTTCGGTTCAGTCTC ACACCCTCCAAGGAGTCTCAC Scd1 TGGAGACGGGAGTCACAAGA ACACCCCGATAGCAATATCCAG Slc2a4 CTCTGACGTAAGGATGGGGA ACCTTCTGTGGGGCATTGAT Lipe CTTGGGGAGCTCCAGTCGGAAG TGTCTTCTGCGAGTGTCACCAG Pnpla2 CTGCTGTAAACCCCTGGTCT CCACGGATGGTCTTCACCAG Mfn1 TCCCCTCTTTCGGGAGGATG GTCCGGAGCTCGAAGGTCA Mfn2 GACACGGGACGGTTACCAG CTCTGAACGCTGTCACCTCA PDK4 TGAACACTCCTTCGGTGCAG TCGAACTTTGACCAGCGTGT Tfam TAGGCACCGTATTGCGTGAG GTGCTTTTAGCACGCTCCAC Cpt1 GACTCCGCTCGCTCATTCC TTGAGGGCTTCATGGCTCAG Nrf1 GCTAATGGCCTGGTCCAGAT CTGCGCTGTCCGATATCCTG Cycs CACCGACACCGGTACATAGG TAATTCGTTCCGGGCTGGTC Ryr2 CAGAAGGGACTGCTCAAGGT TCGAAGGCCAGTTTGTCAGT Tfam ATGTGGAGCGTGCTAAAAGC GGATAGCTACCCATGCTGGAA Cox4 ACATTCAGGGTGCCTCTTTG CATGGCAGAAGTGGGAGATT Atp2a1b TCAGGGCAGGAGCATCATTC ATGTAGCGCTGTCAGTCAAGA
Western Blot Analysis
Cell lysates were prepared by adding a radioimmunoprecipitation assay buffer (RIPA buffer, Sigma–Aldrich), followed by homogenization and centrifugation at 13,000 ×
Immunocytochemical Analysis
To directly detect the expression of UCP1 in the control and ECH-treated adipocytes, immunocytochemical analysis was performed on formalin-fixed 3T3-L1 cells. The obtained sections were incubated overnight at 4°C with the primary UCP1 antibody (dilution 1:1000, Santa Cruz Biotechnology), followed by incubation with the appropriate fluorescein isothiocyanate (FITC) goat anti-mouse secondary antibody at room temperature for 4 h. The mitochondria were stained by adding MitoTracker red (1 mM, Cell Signaling Technology, USA) to PBB-T (PBS+1% BSA, and 0.1% Tween 20) at a concentration of 200 nM, and incubating the cells for 2 h at 37°C. Following incubation, the cells were washed with PBS and subjected to immunostaining. The morphological findings were observed using an optical microscope at ×40 magnification.
Detection of Intracellular Ca2+
Calcium Quantification Kit-Red Fluorescence (Abcam, cat#: ab112115) was employed to measure intracellular calcium. Briefly, the calcium standard was first prepared following the manufacturer’s protocol by diluting the appropriate amount of the 300 mM calcium standard in deionized water to produce a calcium concentration ranging from 0 to 3 mM (12 mg/dl). Next, 50 μl of serially diluted calcium standard was added into each well. Then, 50 μl of the assay reaction mixture was added to each well of the calcium standard, blank control, and test samples to make up the total calcium assay volume of 100 μl/well. Finally, the reaction was incubated for 20–30 min at room temperature, protected from light. Fluorescence intensity was monitored with a fluorescence plate reader at excitation/emission (Ex/Em) wavelengths of 540/590 nm.
Statistical Analysis
All the data are presented as the mean ± standard deviation (SD). At least three independent experiments were performed for each data set. The Statistical Package of Social Science (SPSS, version 17.0; SPSS Inc., USA) tool was used to examine the statistical significance among several groups. The Student’s
Results
Echinacoside Reduces Diet-Induced Obesity in Mice
We first applied an MTT assay to assess the cytotoxicity of ECH (Fig. 1A) on 3T3-L1 cells and found no cytotoxicity up to 20 μM. A final concentration of 10 μM was selected as the working amount for subsequent in vitro experiments (Fig. 1B). Next, we investigated the effect of ECH on body weight and adipose tissue in vivo. 25 mg/kg of ECH was intraperitoneally injected into the HFD-fed mice, twice a week, for 6 weeks and a substantial decline in relative body weight was observed starting from the second week (Fig. 1C). We also measured the weights of various tissues. There was a significant reduction in eWAT and iWAT mass in HFD-fed mice after the ECH treatment, whereas a significant increase in BAT and skeletal muscle mass was observed as indicated in Fig. 1D.
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Figure 1. Echinacoside reduces diet-induced obesity in mice.
Structure of ECH (A). Cytotoxicity of ECH on 3T3-L1 cells as determined by an MTT assay (B). Body weight of mice observed over 6 weeks (C). Relative weight of various tissues in HFDfed mice and ECH-treated HFD-fed mice (D). Histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using the Statistical Package for the Social Sciences (SPSS; version 17.0; SPSS Inc., USA) to perform ANOVA followed by Tukey's post-hoc tests. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *
p < 0.05 or **p < 0.01.
Echinacoside Induces Beiging in 3T3-L1 Adipocytes
Subsequently, we investigated the impact of ECH on key marker proteins associated with browning (UCP1, PGC-1α, and PRDM16). Our findings demonstrated that ECH treatment resulted in a significant and dose-dependent increase in the expression of these core browning marker proteins (Fig. 2A and 2B). In addition, the administration of ECH to HFD-fed mice displayed rapid upregulation of BAT-specific genes (
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Figure 2. Echinacoside induces beiging in 3T3-L1 adipocytes.
Effect of ECH on key browning proteins (A and B). mRNA expressions of the core set of beige-fat marker genes in ECH-treated HFD-fed mice (C). Immunohistochemical staining in 3T3-L1 cells (D) (×40 magnification; scale bar = 100 μm). Histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using the Statistical Package for the Social Sciences (SPSS; version 17.0; SPSS Inc., USA) to perform ANOVA followed by Tukey's post-hoc tests. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *
p < 0.05 or **p < 0.01.
Echinacoside Promotes Lipid Metabolisms and Fat Oxidation
Building upon its application, we examined whether ECH affects lipid metabolism in 3T3-L1 white adipocytes. To investigate this, we first determined the expression level of acetyl-CoA carboxylase, which was downregulated after ECH treatment, with a concurrent increase in phosphorylated (p-ACC) and p-AMPK. Fatty acid synthase (FAS) was also downregulated after the ECH treatment of 3T3-L1 white adipocytes (Fig. 3A). In addition, adipogenic transcriptional factors (C/EBP and PPARγ) were downregulated following the ECH treatment of the 3T3-L1 cells (Fig. 3B). The mRNA expression levels of key genes associated with adipogenesis and lipogenesis were also downregulated following the ECH treatment in HFD-fed mice (Fig. 3C). This was further elucidated by the histopathological analysis determined by H & E staining in iWAT, eWAT, and BAT, which showed a reduction in lipid cell size in the ECH + HFD treated group compared with the HFD counterparts as indicated in Fig. 3D. The expression levels of lipolysis and fat oxidation-related proteins were also examined. ECH increased lipolysis by raising the levels of phosphorylated hormone-sensitive lipase (p-HSL) and adipose triglyceride lipase (ATGL). It also resulted in a notable increase in the expression of acyl-coenzyme A oxidase 1 (ACOX1), carnitine palmitoyltransferase 1 (CPT1), and PPARα, suggesting an enhanced ability of ECH to facilitate fat oxidation (Fig. 3E). Moreover, core genes for the lipolysis and fat oxidation processes were also substantially upregulated with the ECH treatment of the HFD-fed mice (Fig. 3F).
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Figure 3. Echinacoside promotes lipid metabolism in 3T3-L1 white adipocytes.
Effect of ECH in the expression levels of lipogenesis (A) and adipogenesis markers (B). Effect of ECH on the mRNA expression of key adipogenesis and lipogenesis genes in ECH-treated HFD-fed mice (C). H & E staining for inguinal white adipose tissue (iWAT), epididymal adipose tissue (eWAT), and brown adipose tissue (BAT) (D). Effect of ECH in lipolysis and fat oxidation markers (E) in 3T3-L1 cells. mRNA expression of key lipolysis and fatty acid oxidation genes in ECH-treated HFD-fed mice (F). Histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using Student’s
t -test. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *p < 0.05 or **p < 0.01.
Echinacoside Upregulates Calcium Cycling Proteins, Increases Intracellular Calcium, and Facilitates Mitochondrial Biogenesis
We next sought to understand the role of ECH in the regulation of calcium-cycling proteins and intracellular calcium levels as well as mitochondrial biogenesis and found that ECH upregulated voltage-dependent anion (VDAC), mitochondrial calcium uniporter (MCU), ATP synthase F1 subunit beta (ATP5B), and cytochrome C (CYT-C) in 3T3-L1 cells (Fig. 4A). Intracellular calcium was also found to be significantly increased following the ECH treatment of 3T3-L1 cells (Fig. 4B). As shown in Fig. 4C, OXPHOS complexes (I, II, III, IV, and V) were upregulated in the ECH-treated 3T3-L1 cells. This was collectively elucidated in vivo by measuring the mRNA expression levels of mitochondrial biogenesis genes including
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Figure 4. Echinacoside facilitates mitochondrial biogenesis.
Effect of ECH on the mitochondrial and ATP synthesis effectors (A). Effect of ECH on intracellular calcium (B). Effect of ECH on mitochondrial oxidative phosphorylation (OXPHOS) complexes (I, II, III, IV, and V) (C). In vivo effect of ECH on mitochondrial biogenesis genes (D). Histograms display triple independent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using Student's
t -test. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *p < 0.05 or **p < 0.01.
Echinacoside Induces Browning of White Adipocytes via the DRD1/5/PKA/p38 MAPK Pathway
To unravel the underlying mechanisms by which ECH contributes to the beiging of white adipocytes, we conducted further investigations. Following this, ECH treatment substantially increased the expression levels of signaling molecules, such as DRD1, DRD5, protein kinase A (PKA), p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 (p-p38), extracellular signal-regulated kinase (ERK)1/2, phosphorylated (p)-ERK1/ 2, cAMP-response element binding protein (CREB), p-CREB, activating transcription factor 2 (ATF2) and p-ATF2 in 3T3-L1 adipocytes (Fig. 5). A mechanistic study was undertaken to elucidate the participation of these pathway molecules by treating cells with SKF38393 (DRD1/5 agonist) and SCH 23390 (DRD1/5 antagonist) alone or together with ECH. The results showed that SKF38393 stimulated the expression levels of these molecules along with the browning effectors (UCP1, PGC-1α, and PRDM16), whereas SCH 23390 reversed the agonistic action of SKF38393 (Fig. 6).
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Figure 5. Echinacoside upregulates browning pathway markers in 3T3-L1 adipocytes.
Effect of ECH on beiging pathway molecules. The histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. The differences between the groups were determined using the Student’s t-test. Statistical significance between control and ECH-treated 3T3-L1 cells is indicated by *
p < 0.05 or **p < 0.01. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *p < 0.05.
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Figure 6. Mechanistic studies for the effect of echinacoside on browning effectors in 3T3-L1 cells.
Effect of ECH or SKF38393 (DRD5 agonist) and SCH 23390 (DRD1 antagonist) in pathway molecules. The histograms represent the results of triple-independent experiments for immunoblot analysis and are presented as the mean ± SD. The differences between the groups were determined using the Statistical Package for the Social Sciences (SPSS; version 17.0; SPSS Inc., USA) to perform ANOVA, followed by Tukey’s post-hoc tests. Statistical significance between control and ECH-treated 3T3-L1 cells/tissue is shown by *
p < 0.05 or **p < 0.01.
Echinacoside Stimulates α1-AR-Mediated ATP-Dependent Thermogenesis by the Activation of DRD1/5
Finally, we investigated the impact of ECH on the ATP-dependent thermogenic mechanisms in 3T3-L1 cells. ECH treatment substantially increased the expression levels of the ryanodine receptor (RyR)2, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)2b, mitochondrial creatine kinase (CKmt), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) as well as their upstream receptor, α1-AR (Fig. 7A). The mRNA expressions of the genes involved in the ATP-dependent thermogenesis were also dramatically increased in the ECH-treated HFD-fed mice (Fig. 7B). This was further demonstrated by a mechanistic study showing that SKF38393 (DRD1/5 agonist) alone or together with ECH stimulated the expression levels of these proteins, whereas SCH 23390 (DRD1/5 antagonist) reversed the agonistic action of SKF38393 (Fig. 7C). We also sought to investigate the interaction of DRD1 and DRD5 with α1-AR and found that prazosin (α1-AR antagonist) downregulated DRD1, DRD5, and their common downstream effectors while cirazoline (α1-AR agonist) reversed the antagonistic effect of prazosin (Fig. 7D).
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Figure 7. Echinacoside stimulates ATP-dependent thermogenesis.
Effect of ECH on ATP-dependent thermogenic mechanism effectors in 3T3-L1 cells (A). Effect of ECH on the mRNA expressions of genes involved in UCP1-independent thermogenesis (B). Effect of ECH and or SKF38393 (DRD1/5 agonist) as well as SCH 23390 (DRD1/5 antagonist) on ATPdependent thermogenic mechanism effectors (C). Effect of prazosin (α1-AR antagonist) cirazoline (α1-AR agonist) on DRD1 and DRD5 as well as ATP-dependent thermogenic mechanisms effectors (D). Histograms represent the results of tripleindependent experiments for immunoblot analysis and are presented as the mean ± SD. Differences between groups were determined using the Statistical Package for the Social Sciences (SPSS; version 17.0; SPSS Inc., USA) to perform ANOVA followed by Tukey's post-hoc tests or Student's
t -test. Statistical significance between control and ECH-treated 3T3-L1 cells/ tissue is shown by *p < 0.05 or **p < 0.01.
Discussion
A growing body of evidence indicates that phytochemicals mediate thermogenesis via the stimulation of the β3-AR [9, 26, 27]. These molecules target various pathways that are intricately linked to the process of adipogenesis, lipogenesis, lipolysis, and the fat oxidation process, all of which provide a fertile environment for high energy expenditure in the metabolic machinery [11, 28]. As a part of this energy dissipation process, we aimed to investigate alternative receptors that promote thermogenesis and the ATP-dependent futile cycle process when activated with similar natural compounds. In this study, we found that ECH, a naturally occurring phenylethanoid glycoside isolated from
AMP-activated protein kinase (AMPK), the primary energy sensor that monitors the catabolic and anabolic energy balance [29], phosphorylates ACC, a cascade that inhibits fatty acid synthesis and enhances fatty acid oxidation [30]. In this study, the ECH treatment decreased ACC and FAS while increasing p-AMPK and p-ACC, indicating its negative regulation of lipogenesis-related proteins in 3T3-L1 preadipocytes. The current study also revealed that the ECH treatment downregulated the core adipogenic transcription factors (C/EBPα and PPARγ), suggesting its inhibiting role in adipogenesis. The effect of ECH in our current study is consistent with the role of various phytochemicals in the literature that inhibits adipogenesis and lipogenesis [9, 26, 27].
The cyclic AMP/protein kinase A/hormone sensitive lipase (cAMP/PKA/HSL) signaling pathway plays a major role in lipolysis which leads to the formation of free fatty acids (FFA) [31]. This pathway generates FFAs through lipolysis which regulates the energy expenditure of the body and serves as fuel for energy generation in adipocytes [32]. In our study, ECH upregulated the major lipolysis and fat oxidation mediating molecules as well as effectors suggesting its positive role in lipid catabolism.
The cAMP/PKA/p38 MAPK/ERK signaling pathway is the most familiar pathway for the β-oxidation of fatty acids and browning of white adipocytes [33,34]. In this process, catecholamines stimulate adenylyl cyclase (AC), which raises cAMP and subsequently binds to PKA. The PKA-mediated phosphorylation cascade enhances lipolysis, activates p38 MAPK and CREB, and this signaling pathway consequently promotes a thermogenic program. In parallel, the p38 MAPK phosphorylates and activates the transcription factor ATF2 and core browning effectors, causing the transcription of downstream thermogenic genes to be activated [33]. In our study, ECH stimulated the browning of 3T3-L1 adipocytes via the activation of DRD1/5, suggesting that dopaminergic receptors could be an alternative target for anti-obesity drugs in the adipose tissue. This is consistent with our recent findings that L-dihydroxyphenylalanine stimulated the browning of 3T3-L1 adipocytes
One of the major findings of our study was the elucidation of the role of ECH and DRD1/5 in mitochondrial biogenesis and related activities. Mitochondria are the primary source of energy for cellular activity through ATP generation via oxidative phosphorylation, and to operate the various intracellular signaling cascades, mitochondrial biogenesis must occur continuously through self-renewal [40]. The decrease in mitochondrial biogenesis, oxidative metabolic pathways, and OXPHOS complexes in adipose tissue indicate that mitochondrial dysfunction or low mitochondrial number and activity are risk factors in obesity and related metabolic disorders [41]. In this study, ECH upregulated mitochondrial biogenesis genes and proteins as well as OXPHOS complexes
Another remarkable finding from our study is that ECH positively regulated the creatine-dependent ADP/ATP substrate cycling and α1-AR-mediated ATP-dependent thermogenesis in beige adipocytes. The creatine kinase/phosphocreatine (CK/PCr) shuttle enhances energy transfer between mitochondrial and/or glycolytic ATP supply and consumption at the ER by SERCA2b [47]. In addition, UCP1-independent thermogenesis depends on the ATP-dependent Ca2+ cycling fashion in which SERCA2b and RyR2 monitor Ca2+ import/export, which eventually causes energy dissipation [5, 48, 49]. Our present study revealed that ECH stimulated DRD1/5 and activated ATP-dependent thermogenesis
Our study also revealed the positive association between ECH, DRD1, and DRD5 as well as calcium regulatory proteins in beige adipocytes. Ca2+ signaling is essential in improving the metabolic apparatus by boosting calorie intake, thereby minimizing the manifestation of obesity [50, 51]. The association between calcium signaling proteins (VDAC and MCU) is already established, with VDAC being upstream of MCU, as Ca2+ must travel through the outer mitochondrial membrane, where VDAC is located before reaching the MCU [52]. In our study, ECH substantially increased VDAC and MCU as well as the intracellular Ca2+ level in 3T3-L1 cells. In line with our study, impaired dopamine homeostasis remarkably lowered the VDAC1 and VDAC2 levels in human neuroblastoma SH-SY5Y cells [41], suggesting the positive modulation of dopaminergic receptors to VDAC even if the specific dopaminergic subtypes responsible to mediate this process were not clarified. Other earlier studies also reported that the pharmacological activation of DRD1 and DRD5 modified the synaptic function of the hippocampal and neocortical neurons
Finally, our study revealed that ECH positively regulated ATP5B in beige adipocytes. This could be because of the constant entry of cytosolic Ca2+ through the VDAC/MCU gate, and once within the mitochondria, these calcium ions modulate cell survival and ATP production [55]. Taken together, our study indicated that ECH not only directly enhances ATP5B expression, but also led to the expectation that increased levels of VDAC and MCU could positively regulate ATP5B. In accordance with the objective of our study, we made efforts to address the limitations; however, the unavailability of pharmacological drugs that can entirely distinguish between DRD1 and DRD5 prevented us from determining the specific affinity of ECH for either receptor. Nevertheless, this study explores the activity of ECH and the functional roles of dopaminergic receptors in browning activities. This research provides the concept that dopaminergic receptors can serve as alternative receptors for the thermogenesis process, presenting a potential alternative strategy for combating obesity. However, to establish a more comprehensive understanding, further investigations, including detailed
Conclusion
Our study is the first to reveal the phytochemical activation of dopaminergic receptors and their regulatory roles in the browning of 3T3-L1 adipocytes and the ATP-dependent thermogenic process. This study may provide important evidence for comprehensive in vivo functional studies in the future. Overall, our findings show that the thermogenic action of ECH
Acknowledgment
This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT, No. 2019R1A2C2002163).
Ethics Approval Statement
All the procedures were performed according to the guidelines approved by the National Institutes of Health. All animal experiments were approved by the Committee for Laboratory Animal Care and Use of Daegu University (DUIACC-2022-15-0901-015).
Author Contributions
K. Haddish performed experimental design, conducted experiments, analyzed the data, performed the statistical analysis, and wrote the manuscript, and J.W. Yun carried out scientific support, critically reviewed the manuscript and experimental design, and approved the manuscript version to be published.
Abbreviations
Name Memo
ACC Acyl-CoA carboxylase
ACOX Acyl-coenzyme A oxidase 1
AMPK AMP-activated protein kinase
ATF2 Activating transcription factor 2
ATGL Adipose triglyceride lipase
AR Adrenergic receptor
C/EBP/
ATP5B ATP synthase F1 subunit beta
BAT Brown adipose tissue
CKmt/
CPT1/
CYT-C/
CREB cAMP-response element binding protein
CaMKII Ca2+/calmodulin-dependent protein kinase II
DRD1 Dopamine receptor D1
DRD5 Dopamine receptor D5
eWAT Epididymal white adipose tissue
SM Skeletal muscle
MCU Mitochondrial calcium uniporter
ERK Extracellular signal-regulated kinase
FAS Fatty acid synthase
HSL Hormone-sensitive lipase
iWAT Inguinal white adipose tissue
OXPHOS Oxidative phosphorylation I~V
PGC-1α/
p38 MAPK p38 mitogen-activated protein kinase
PKA Protein kinase A
PPAR Peroxisome proliferator-activated receptor
PRDM16/
PDE4 Phosphodiesterase-4
RyR/Ryr Ryanodine receptor/encoding gene
SERCA/
UCP1/
VDAC Voltage-dependent anion channel.
GM Gastrocnemius muscle
Conflict of Interest
Supplemental Materials
Fig 1.
Fig 2.
Fig 3.
Fig 4.
Fig 5.
Fig 6.
Fig 7.
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Table 1 . Primer sequences used for real-time quantitative RT-PCR..
Gene Forward Reverse Drd1 AGGGGTTTTGGGAGAAGTGAC AGTCACTTTTCGGGGATGCTG Drd5 GGGGTTTTGGGAGAAGTGAC AGTCACTTTTCGGGGATGCTG Cited1 GGGGTAAAAGATCGCAAGGC TGGTAGAAGGGGTGGCAGTA Ppargc1α ATGAATGCAGCGGTCTTAGC AACAATGGCAGGGTTTGTTC Prdm16 GATGGGAGATGCTGACGGAT TGATCTGACACATGGCGAGG Tbx 1AGCGAGGCGGAAGGGA CCTGGTGACTGTGCTGAAGT Ucp1 CCTGCCTCTCTCGGAAACAA GTAGCGGGGTTTGATCCCAT Cd137 GGTCTGTGCTTAAGACCGGG TCTTAATAGCTGGTCCTCCCTC Cidea CGGGAATAGCCAGAGTCACC TGTGCATCGGATGTCGTAGG Cebpa CCCTCTCCCCTAGTTGTCCA CTTTCCAGACCCAGAGCCAG Pparγ ACCATGGTAATTTCAGTAAAGGGT CCTGACGGGGTCTCGGT Mest AGCAGCTTTCCTCTGCGG GATACAGGATCGGAGGTGGC Srbf1 CCTGACAGGTGAAATCGGCG GTTGTTGATGAGCTGGAGCA Fasn CACTGCCTTCGGTTCAGTCTC ACACCCTCCAAGGAGTCTCAC Scd1 TGGAGACGGGAGTCACAAGA ACACCCCGATAGCAATATCCAG Slc2a4 CTCTGACGTAAGGATGGGGA ACCTTCTGTGGGGCATTGAT Lipe CTTGGGGAGCTCCAGTCGGAAG TGTCTTCTGCGAGTGTCACCAG Pnpla2 CTGCTGTAAACCCCTGGTCT CCACGGATGGTCTTCACCAG Mfn1 TCCCCTCTTTCGGGAGGATG GTCCGGAGCTCGAAGGTCA Mfn2 GACACGGGACGGTTACCAG CTCTGAACGCTGTCACCTCA PDK4 TGAACACTCCTTCGGTGCAG TCGAACTTTGACCAGCGTGT Tfam TAGGCACCGTATTGCGTGAG GTGCTTTTAGCACGCTCCAC Cpt1 GACTCCGCTCGCTCATTCC TTGAGGGCTTCATGGCTCAG Nrf1 GCTAATGGCCTGGTCCAGAT CTGCGCTGTCCGATATCCTG Cycs CACCGACACCGGTACATAGG TAATTCGTTCCGGGCTGGTC Ryr2 CAGAAGGGACTGCTCAAGGT TCGAAGGCCAGTTTGTCAGT Tfam ATGTGGAGCGTGCTAAAAGC GGATAGCTACCCATGCTGGAA Cox4 ACATTCAGGGTGCCTCTTTG CATGGCAGAAGTGGGAGATT Atp2a1b TCAGGGCAGGAGCATCATTC ATGTAGCGCTGTCAGTCAAGA
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