β-Elemene inhibits adipogenesis in 3T3-L1 cells by regulating AMPK pathway

Abstract

The prevalence of childhood obesity in global is quickly augmented, resulting into grievous public health problems and influencing adolescent development. β-Elemene is a sesquiterpene, and can extracted from traditional Chinese medicine-Curcuma longa L. β-Elemene has been discovered to display regulatory functions in multiple diseases, but it’s roles in obesity need further investigations. The purpose of this work is to investigate the regulatory impacts of β-elemene on obesity progression and associated pathways. In this study, it was revealed that the heightened lipid accumulation in 3T3-L1 cells triggered by 3-isobutyl-1-methylxanthine + dexamethazone + insulin (MDI) can be restrained by β-elemene. Furthermore, β-elemene can modulate lipid metabolism in 3T3-L1 cells mediated by MDI. The glucose consumption was descended after insulin resistance treatment, but this impact was reversed after β-elemene treatment. At last, it was illustrated that the AMPK pathway was retarded after β-elemene induction, but this change was offset after β-elemene treatment. To sum up, our results manifested that β-elemene inhibited adipogenesis in 3T3-L1 cells, and evoked the AMPK pathway. This project may supply serviceable insights of β-elemene in the progression of obesity.

Introduction

Obesity is occurred due to the imbalance of energy ingestion and utilization in white adipose tissue.⁽¹⁾ The prevalence of childhood obesity has been augmented year by year.⁽²⁾ The childhood obesity is tanglesome, and environmental and genetic factors both participate into the pathogenesis of this disease.⁽³⁾ The obesity in children is one primary global health problem, and it can result into the markedly increased risk to happen morbid obesity or diabetes mellitus in later life.⁽⁴⁾ Besides, obesity acts as the most familiar cause for insulin resistance (IR) in children, that is also correlative with dyslipidemia and vascular complications.^(5,6) Therefore, ameliorating obesity has become the key point, and searching useful drugs for obesity treatment is deemed as the top priority.

Natural products from herbs have become hopeful drugs, and own improvement roles in obesity.⁽⁷⁾ β-Elemene is one sesquiterpene, and can extracted from traditional Chinese medicine-Curcuma longa L.⁽⁸⁾ β-Elemene has been ascertained to display regulatory functions in diversified diseases. For instance, β-Elemene can strengthen lncRNA H19 in non-small cell lung cancer to intensify erlotinib sensitivity.⁽⁹⁾ Moreover, β-elemene modulates the JAK/STAT3/NF-κB pathway to improve cardiac inflammation and remodeling evoked by hyperglycemia.⁽¹⁰⁾ β-Elemene restrains HSP70 to accelerated hyperthermia-triggered cell apoptosis in colorectal carcinoma.⁽¹¹⁾ In addition, β-elemene regulates autophagy to facilitate M2 polarization, thereby ameliorating ischemic stroke.⁽¹²⁾ Importantly, it has been testified that β-Elemene can refrain imbalance of microbiota/gut/brain in obesity.⁽¹³⁾ Besides, β-elemene can regulate pro-inflammatory factors to modulate M1/M2 macrophage balance in obesity.⁽¹⁴⁾ However, the regulatory impacts and interrelated pathways of β-elemene on lipogenesis and IR in obesity progression remain indistinct.

In this work, it was disclosed that β-elemene inhibited adipogenesis in 3T3-L1 cells, and evoked the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway. This work suggested that β-elemene may be helpful on improving obesity progression.

Materials and Methods

Cell lines and cell culture

The mouse 3T3-L1 cells bought from iCell Bioscience Inc. (Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco; Thermo Fisher Scientific, Waltham, MA) with 10% Newborn Calf Serum (NBS) in a humidified incubator of 5% CO₂ at 37°C. 3T3-L1 cells were treated with 3-isobutyl-1-methylxanthine + dexamethazone + insulin (MDI) (0.5 ‍mM 3-isobutyl-1-methylxanthine, 1 ‍μM dexamethasone, 10 ‍μg/ml insulin). The 3-isobutyl-1-methylxanthine and dexamethasone were withdrawn at day 2, and then insulin was withdrawn at day 4. Next, 3T3-L1 cells were incubated in DMEM (changed every 2 days) until day 8.

β-Elemene (0, 5, 10, 20, 40, and 80 ‍μM; Apexbio, Boston, MA) was employed to treat 3T3-L1 cells.

For IR cell model, on day 8, 3T3-L1 cells were treated with 1 ‍μM dexamethasone for 72 ‍h. The IR-3T3-L1 cells were treated with β-elemene (5, 10, and 20 ‍μM) for 48 ‍h.

CCK-8 assay

3T3-L1 cells (1,000 cells/well) placed in the 96-well plate were cultivated. CCK-8 solution (10 ‍μl; Dojindo Laboratories, Kumamoto, Japan) was mixed into each well for 2 ‍h. Eventually, cell viability was determined through the spectrophotometer (Thermo Fisher Scientific).

Oil red O staining

After washing, 3T3-L1 cells were fixed by 4% formalin. Next, further washing, 3T3-L1 cells were subjected to air drying. Then, oil red O solution was mixed into each well for 30 ‍min. Post being washed with distilled water, images of stained cells were gained through the inverted fluorescence microscopy (Nikon, Tokyo, Japan).

Detection of TG

3T3-L1 cells were lysed, next the TG level was measured through the intracellular TG kit (Nanjing Jiancheng, Nanjing, China).

RT-qPCR

The RNAs from 3T3-L1 cells was obtained through using TRIzol reagent (Invitrogen, Carlsbad, CA). The cDNA was generated from RNAs under the SuperScript^TM II Reverse Transcriptase Kit (Invitrogen). Then, qRT-PCR was executed through the SYBR Premix Ex Taq^TM Kit (Takara, Shanghai, China). At last, the mRNA expressions were determined through the 2^−∆∆Ct method.

The primer sequences were as followed:

FABP4: F: 5'-GAGGCGGATGAGAACAAGCA-3', R: 5'-CTCCCAGCAGCTACCATGGA-3';

C/EBPα: F: 5'-CAAGAACAGCAACGAGTACCG-3', R: 5'-GTCACTGGTCAACTCCAGCAC-3';

PPARγ: F: 5'-TCTCCTGTTGACCCAGAGCAT-3', R: 5'-TGGGCCAGAATGGCATCT-3';

ACC: F: 5'-GCCTCAGGAGGATTTGCTGT-3', R: 5'-AGGATCTACCCAGGCCACAT-3';

FAS: F: 5'-GGAAGTTGCCCGAGTCAGAG-3', R: 5'-CTTTCCAGACCGCTTGGGTA-3';

ATGL: F: 5'-ACTCACATCTACGGAGCCTCG-3', R: 5'-TCCTTGGACACCTCAATAATGTTG-3';

HSL: F: 5'-TCAATGGAGACACTTGGCCC-3', R: 5'-TTGGCTTCAGCCTCTTCCTG-3';

GLUT-4: F: 5'-GTTCTTTCATCTTCGCCGCC-3', R: 5'-TTCCCCATCTTCGGAGCCTA-3';

IRS-1: F: 5'-AGAGGACCGTCAGTAGCTCA-3', R: 5'-ACTGAAATGGATGCATCGTACC-3';

Adiponectin: F: 5'-ACTGCAGTCTGTGGTTCTGA-3', R: 5'-CATGACCGGGCAGAGCTAAT-3';

β-actin: forward, 5'-TCTGGCACCACACCTTCTACAA-3', reverse, 5'-TTTTCACGGTTGGCCTTAGG-3'.

Detection of glucose uptake

The glucose consumption was confirmed through the Glucose oxidase-peroxidase kit (Shanghai Rongsheng Biotech, Shanghai, China). The non-esterified fatty acid (NEFA) level was determined through NEFA kit (Nanjing Jiancheng).

Western blot

Proteins from 3T3-L1 cells were gained through the RIPA buffer (Beyotime, Shanghai, China). SDS-PAGE (10%) was adopted for proteins’ separation. Then, proteins were put onto PVDF membranes (Beyotime). Post sealing (non-fat milk), primary antibodies were placed into membranes. Post 12 ‍h incubation, the appropriate secondary antibody (1:1,000, ab7090) was also appended. Finally, the chemiluminescence detection kit (Thermo Fisher Scientific) was utilized for measuring the protein bands.

The primary antibodies: GLUT-4 (1:1,000, ab313775; Abcam, Shanghai, China), Adiponectin (2 ‍μg/ml, ab22554), IRS-1 (1:500, ab131487), p-AMPK (1:500, ab131357), AMPK (1:1,000, ab32047) and β-actin (1:1,000, ab8227).

Statistical analysis

Data were expressed as mean ± SD. GraphPad Prism Software 9 (GraphPad Software, San Diego, CA) was utilized for performing statistical analysis. The comparisons were executed through one-way analysis of variance (ANOVA). The p<0.05 was thought as statistically significant.

Results

β-Elemene restrained lipid accumulation in 3T3-L1 cells

The cell viability was receded after β-elemene treatment (40 and 80 ‍μM), but it was not changed after β-elemene treatment (5, 10, and 20 ‍μM) (Fig. 1A). Furthermore, β-elemene treatment (5, 10, and 20 ‍μM) was utilized for next experiments. Through oil red O staining, it was uncovered that adipogenesis was strengthened after MDI treatment, but this phenomenon was offset after β-elemene treatment (5, 10, and 20 ‍μM) (Fig. 1B). In addition, the TG level was risen after MDI induction, but this change was alleviated after β-elemene treatment (Fig. 1C). In short, β-elemene restrained lipid accumulation in 3T3-L1 cells.

Fig. 1.

β-Elemene restrained lipid accumulation in 3T3-L1 cells. (A) The cell viability was confirmed with different concentrations of β-elemene (0, 5, 10, 20, 40, and 80 ‍μM) through CCK-8 assay. (B) The adipogenesis was examined through oil red O staining in the Control, MDI, MDI + β-ele (5 ‍μM), MDI + β-ele (10 ‍μM), and MDI + β-ele (20 ‍μM) groups. (C) The TG level was tested through the TG kit in the Control, MDI, MDI + β-ele (5 ‍μM), MDI + β-ele (10 ‍μM), and MDI + β-ele (20 ‍μM) groups. *p<0.05, ***p<0.001.

Effects of β-elemene on lipid metabolism in 3T3-L1 cells

The mRNA expressions of FABP4, C/EBPα, PPARγ, ACC, and FAS were all elevated as well as ATGL and HSL were declined after MDI stimulation, but these impacts were counteracted after β-elemene treatment (10 and 20 ‍μM) (Fig. 2). In general, β-elemene can modulate lipid metabolism in 3T3-L1 cells mediated by MDI.

Fig. 2.

Effects of β-elemene on lipid metabolism in 3T3-L1 cells. Groups were separated into the Control, MDI, MDI + β-ele (5 ‍μM), MDI + β-ele (10 ‍μM), and MDI + β-ele (20 ‍μM) group. The mRNA expressions of FABP4, C/EBPα, PPARγ, ACC, FAS, ATGL, and HSL were tested through RT-qPCR. *p<0.05, **p<0.01, ***p<0.001.

β-Elemene aggrandized glucose consumption in IR-3T3-L1 cells

The glucose consumption was descended and NEFA was ascended after IR treatment, but these changes were reversed after β-elemene treatment (10 and 20 ‍μM) (Fig. 3A). Besides, the mRNA expressions of GLUT-4, IRS-1, and adiponectin were all decreased after IR induction, but these influences were rescued after β-elemene treatment (Fig. 3B). The same changes of protein expressions for GLUT-4, Adiponectin and IRS-1 were found in Fig. 3C and D. Taken together, β-elemene aggrandized glucose consumption in IR-3T3-L1 cells.

Fig. 3.

β-Elemene aggrandized glucose consumption in IR-3T3-L1 cells. Groups were separated into the Control, IR, IR + β-ele (5 ‍μM), IR + β-ele (10 ‍μM), and IR + β-ele (20 ‍μM) group. (A) The glucose consumption and NEFA were measured through the commercial kits. (B) The mRNA expressions of GLUT-4, IRS-1, and adiponectin were assessed through RT-qPCR. (C) The protein expressions of GLUT-4 and Adiponectin were evaluated through Western blot. (D) The protein expression of IRS-1 was verified through Western blot. *p<0.05, **p<0.01, ***p<0.001.

β-Elemene evoked the AMPK pathway

The protein level of p-AMPK/AMPK was lessened after MDI stimulation, but this impact was neutralized after β-elemene treatment (10 and 20 ‍μM) (Fig. 4), hinting that β-elemene can evoke the AMPK pathway.

Fig. 4.

β-Elemene evoked the AMPK pathway. Groups were separated into the Control, MDI, MDI + β-ele (5 ‍μM), MDI + β-ele (10 ‍μM), and MDI + β-ele (20 ‍μM) group. The protein expressions of p-AMPK and AMPK were determined through Western blot. ***p<0.001.

Discussion

Obesity is generally correlated with lipid accumulation in adipose tissues.⁽¹⁵⁾ Many reports have concentrated on the modulation of lipid accumulation in obesity. For example, in obesity, berberine can positively regulate PPARδ expression to attenuate lipid accumulation.⁽¹⁶⁾ Additionally, liquiritigenin modulates autophagy mechanism in 3T3-L1 cells to against lipid accumulation.⁽¹⁷⁾ Geraniin inhibits lipogenesis to cut down lipid accumulation in 3T3-L1 adipocytes through affecting CaMKK2 expression.⁽¹⁸⁾ Furthermore, papain can evoke AMPK to weaken lipid accumulation in obesity.⁽¹⁹⁾ β-Elemene exhibits ameliorative properties in multiple diseases,^(9–12) can take part into obesity,^(13,14) and its’ roles in lipid accumulation and metabolism need further investigations. In this work, it also was revealed that the heightened lipid accumulation in 3T3-L1 cells triggered by MDI can be restrained by β-elemene. Furthermore, β-elemene can modulate lipid metabolism in 3T3-L1 cells mediated by MDI.

IR and glucose consumption are closely involved into obesity, and they have gained more and more attentions. For instance, cistanche tubulosa phenylethanoid glycosides alleviates adipogenesis and improved IR in obesity.⁽²⁰⁾ Additionally, Bax inhibitor-1 can ameliorate IR and glucose intolerance in obesity.⁽²¹⁾ The JNK-1 signaling can influence glucose homeostasis and IR in obesity mice.⁽²²⁾ In addition, miR-592/FOXO1 relieves hyperglycemia and IR in obesity.⁽²³⁾ Similarly, in this work, it was uncovered that the glucose consumption was descended after IR treatment, but this impact was reversed after β-elemene treatment.

AMPK can join into multiple lipid metabolic pathways, making it to be one potential target for intervention in childhood obesity.^(24,25) AMPK phosphorylates multiple substrates to intensify fatty acid oxidation, thereby receding lipid storage.⁽²⁶⁾ AMPK directly phosphorylates and restrains sterol regulatory element binding protein 1C (SREBP1C), and SREBP1C largely contributes to adipogenesis.⁽²⁷⁾ Therefore, AMPK can serve as one pivotal upstream regulator to suppress lipogenesis and adipogenesis, thereby joining into obesity.⁽²⁸⁾ Interestingly, it has been proved that β-elemene can stimulate the AMPK pathway.^(29,30) However, the regulatory impacts on the AMPK pathway in obesity keep unclear. In this work, it also was illustrated that the AMPK pathway was retarded after β-elemene induction, but this change was offset after β-elemene treatment, manifesting that β-elemene can evoke the AMPK pathway.

In conclusion, it was testified manifested that β-elemene inhibited adipogenesis in 3T3-L1 cells, and evoked the AMPK pathway. Nevertheless, in this project, some limitations are still exhibited. Deeply investigations for β-elemene in obesity progression will be executed in the future.

Author Contributions

All authors contributed to the study conception and design. Material preparation and the experiments were performed by XD. Data collection and analysis were performed by ZL. The first draft of the manuscript was written by SY and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the Scientific Research Fund of Chengdu Fifth People’s Hospital (Grant No. KYJJ2021-036).

Availability of Data and Materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Conflict of Interest

No potential conflicts of interest were disclosed.