Kuanoniamine C Suppresses Adipogenesis and White Adipose Tissue Expansion by Modulating Mitochondrial Function

Abstract

Obesity is characterized by the excessive accumulation of fat to adipose tissue, which is related to abnormal increasing white adipose tissue (WAT) in the body, and it upregulates the risk of multiple diseases. Here, kuanoniamine C, which is a pyridoacridine alkaloid, suppressed the differentiation of pre-adipose cells into white adipocytes via the modulation of mitochondrial function, and inhibited WAT expansion in the early phase of high-fat-diet-induced obesity model. Pharmacological analysis revealed that inhibition of mitochondrial respiratory complex II, which new target of kuanoniamine C, activated reactive oxygen species (ROS)–extracellular signal-regulated kinase (ERK)–β-catenin signaling, and this signaling was antagonized by insulin-, IBMX-, and dexamethasone-induced adipogenesis. Therefore, the kuanoniamine C might prevent abnormal WAT expansion even when eating a diet that is not calorie restricted.

INTRODUCTION

Obesity, defined as a body mass index of 30 kg/m² or greater, is a complex chronic disease characterized by the excessive accumulation of fat to adipose tissue in the body.^1,2) The WHO states that obesity is a common yet underestimated public health problem, and it can increase the risk of multiple diseases. The possible positive relationship between obesity and individual mortality has been recognized for more than 20 years, but the prevalence of obesity worldwide continues to increase, with the WHO estimating that one in five adults worldwide will be obese by 2025. The prevalence of obesity in children and adolescents is also increasing globally, exceeding 340 million as of 2016 (https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight). Thus, controlling obesity from childhood is necessary to prevent multiple diseases.

Abnormal hypertrophy and hyperplasia of adipocytes contribute to the increase in adipose tissue resulting from high caloric intake.³⁾ Differentiated adipocytes are post-mitotic cells; thus, hyperplasia is linked to an excessive increase in de novo adipogenesis.⁴⁾ Adipogenesis is induced in visceral fat from childhood through adolescence, and it occurs until about age 40, although the rate of adipogenesis induction declines.^5,6) Therefore, if adipogenesis can be controlled to suppress the abnormal increase in the number of white adipocytes, then the expansion of adipose tissue can be effectively suppressed, thereby suppressing obesity.

White adipocyte differentiation is regulated by complex gene expression through synergistic interactions between transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and the CCAAT/enhancer binding protein (C/EBPα, β, δ).⁷⁾ Various intracellular pathways, processes, and secreted factors modulate these transcription factors, affecting adipogenesis.⁸⁾ Accumulating data implicate that redox status, which is related to reactive oxygen species (ROS) production control, is closely associated with energy metabolism and direct or indirect affection of adipogenesis-regulated transcription factor activity.⁹⁾ Thus, the regulation of the redox system by controlling the mitochondrial function may be an important target for the control of adipogenesis.

A mitochondrion is an organelle responsible for energy and substance metabolisms in the cell, and it produces ROS during these metabolic processes. Although ROS are considered as important factors for the induction of normal cellular signaling as secondary messengers and maintenance of cellular homeostasis, they also damage various components of the cell, such as DNA, organelles, and proteins, and disrupt the normal function of these components.¹⁰⁾ The regulation of mitochondrial function is important for controlling ROS production-related redox status. However, some studies reported that increasing ROS production hinders adipose tissue development, whereas some reported that increasing ROS production is necessary to induce adipose tissue development.¹¹⁾ The relationship between mitochondrial function, ROS production, and adipogenesis-related signals remains to be clarified.

Kuanoniamine C is isolated from Oceanapia sp., which is known to have various bioactivities such as anti-microbe and anticancer activity.^12,13) Our study found that it enhances the anticancer activity of bortezomib, but its detailed bioactivities beyond anticancer and antibacterial effects remain unexplored and warrant further investigation.¹³⁾

Herein, kuanoniamine C was identified as an adipogenesis modulator, suppressing the differentiation of pre-adipose cells into white adipocytes under non-toxic conditions. Further analysis revealed that ROS induction, caused by partial mitochondrial inhibition, activated the β-catenin pathway through sustained extracellular signal-regulated kinase (ERK) phosphorylation. Moreover, in vivo experiments revealed that kuanoniamine C administration suppressed abnormal adipocyte hyperplasia and WAT expansion in the early phase of high-fat-diet (HFD) induced obesity model. Therefore, the present study suggests that the regulation of mitochondrial function, without inducing cellular toxicity, might suppress abnormal adipogenesis and adipose tissue expansion.

MATERIALS AND METHODS

Materials

Kuanoniamine C (sc-202682) purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Atopenin A5 (AG-CN2-0100), Rotenon (AG-CN2-0516) and Antimycin (19433) purchased from Funakoshi (Tokyo, Japan). FCCP (588-83231) and Oligomycin (O4532) purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Cell Lines and Differentiation into White Adipocytes

NIH3T3-L1 preadipocytes (JCRB9014, JCRB Cell Bank, Osaka, Japan) were maintained in Dulbecco’s modified Eagle’s medium (containing low glucose, L-glutamine, phenol red (FUJIFILM Wako Pure Chemical Corporation), 10% fetal bovine serum (FBS), 100 units/mL of penicillin G, and 100 units/mL of streptomycin). The cells were maintained at 37 °C in an atmosphere containing 5% CO₂ and cultured to confluence after 2 d, after which the medium was changed to Dulbecco’s modified Eagle’s medium (containing high glucose, L-glutamine, phenol red (FUJIFILM Wako Pure Chemical Corporation), 10% FBS, 100 units/mL of penicillin G, and 100 units/mL of streptomycin) with 10 µg/mL of Insulin, 100 µM of IBMX, and 10 µM of dexamethasone (DEX) to induce differentiation into white adipocytes (designated as day 0).

Oil Red Staining

Differentiated NIH3T3-L1 cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min. Fixed cells were washed with distilled water (DW) and stained with Oil Red O (FUJIFILM Wako Pure Chemical Corporation) for 20 min. Then, cells were rinsed three times for 5 min with DW and another 5 min with 60% isopropanol, washed and added with PBS, and photographed. After photographing, the stain in cells was extracted with 100% isopropanol, and absorbance was measured at 495 nm.

Real-Time Quantitative PCR (qRT–PCR)

qRT–PCR was performed as previously described.¹⁴⁾ Total RNA was normalized in each reaction using β-actin cDNA as an internal standard. The following forward and reverse primers for mouse were obtained: C/ebpα, 5′-TCTGCGAGCACGAGACGTC-3′ and 5′-GCCAGGAACTCGTCGTTGAA-3′; C/ebpβ, 5′-CACCACGACTTCCTCTCCGA-3′ and 5′-GTACTCGTCGCTCAGCTTGT-3′; C/ebpδ, 5′-CCATGTACGACGACGAGAGC-3′ and 5′-TGTGGTTGCTGTTGAAGAGG-3′; Pparγ, 5′-GCATGGTGCCTTCGCTGA-3′ and 5′-TGGCATCTCTGTGTCAACCATG-3′; Ucp1, 5′-CGACTCAGTCCAAGAGTACTTCTCTTC-3′ and 5′-GCCGGCTGAGATCTTGTTTC-3′; Glut4, 5′-GACGGACACTCCATCTGTTG-3′ and 5′-GCCACGATGGAGACATAGC-3′; Tfam, 5′-CAGGAGGCAAAGGATGATTC-3′ and 5′-CCAAGACTTCATTTCATTGTCG-3′; and β-actin, 5′-TTGCTGACAGGATGCAGAAG-3′ and 5′-ACATCTGCTGGAAGGTGGAC-3′.

Immunoblotting Analysis

Immunoblotting experiments were conducted as previously described.¹⁴⁾ The antibodies used for immunoblotting were specific for the following proteins: phosphorylated (P)-AKT, AKT (total), P-ERK1/2 (Thr202/Tyr204), total ERK1/2, P-glycogen synthase kinase 3β (GSK-3β) (Ser9) total GSK-3β, P-MEK (Ser217/221), total MEK and β-Catenin (Cell Signaling, Danvers, MA, U.S.A.), and β-actin (Sigma-Aldrich, St. Louis, MO, U.S.A.). The antibodies were diluted at a ratio of 1 : 1000, except for anti-β-actin (1 : 10000). Secondary antibodies were purchased from Promega (anti-rabbit and anti-mouse at 1 : 5000; Madison, WI, U.S.A.). Original images were shown in Supplementary Fig. S1.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT), Cell Counting Kit-8 (CCK-8) and Cell Count Assay

Cell viability was determined using cell count assay. In MTT, cells were administered with the indicated treatments and incubated with MTT solution (1 mg/mL) for 2 h. Then, isopropanol and HCl were added to final concentrations of 50% and 20 mM, respectively, and the absorbance was measured at 570 nm using a spectrophotometer. Cells were administered with the indicated treatments in 98-well plates; 10 µL/well of CCK-8 was added and incubated for 1 h. Afterward, absorbance was measured at 450 nm.

ATP Assay

Intracellular ATP levels were measured using the CellTiter-Glo 2.0 assay kit (Promega) following the manufacturers’ instructions.

ROS Measurements

Cells were cultured on black-bottom culture plates, washed with PBS to remove the medium and subsequently incubated for 10 min at 37 °C in 5 mM MitoSOX Red (Invitrogen, Waltham, MA, U.S.A.).¹⁵⁾ After incubation, the cells were washed with PBS. Fluorescence was measured using a microplate reader equipped with a 535 /590 nm filter pair.

HFD-Induced Obesity Model

The Animal Research Committee of Kochi university approved all experimental protocols and surgical procedures (Permit Number: P-00039). Animal experiments were conducted following the guidelines for animal experimentation by the Kochi university and the recommendations of the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All animals were housed and bred in an animal room and exposed to a 12 h light/dark cycle with free access to food and water. C57BL/6 mice (5 weeks) were fed a HFD (High Fat Diet 32, CLEA Japan Inc., Tokyo, Japan). Mice received 1 mg/kg of kuanoniamine C intraperitoneally every other day for 24 d. The body weight and amount of food intake were measured daily. After the administration period, mice were fasted for 16 h, and blood glucose and triglyceride levels were measured using GLUCOCARD G Black (GT1830; Arkray, Kyoto, Japan) and LabAssay Triglyceride (FUJIFILM Wako Pure Chemical Corporation). Each tissue was collected and stored in 4% paraformaldehyde or subjected to qPCR. Paraffin sections of each tissue were prepared and observed with hematoxylin–eosin (H&E) staining. The frequency of area and volume of adipocytes within the epididymal white adipose tissues (eWAT) was determined using Image J.¹⁶⁾ Cells smaller than 250 µm² and larger than 8000 µm² were excluded.

Statistical Analyses

Differences in mean values were evaluated using Student’s t-test for unpaired results to assess differences between two groups, and ANOVA (using more than two compounds) followed by the Tukey-HSD test. A p-value of <0.05 was used to indicate statistical significance (Mac statistical analysis software, Esumi Co., Ltd., Tokyo, Japan).

RESULTS

Kunanoniamine C Suppressed the Differentiation of Pre-adipose Cells into Full Function of White Adipocytes

We tried to reveal kuanoniamine C pharmacological effects and focused on adipogenesis signaling. Numerous reports suggested that an adipogenesis model, which uses insulin, IBMX, and DEX to chemically induce the differentiation of NIH3T3-L1 preadipocytes into white adipocytes, simulates the differentiation of white adipocytes in vivo.^7,17)

First, we examined whether kuanoniamine C affects lipid accumulation in differentiating NIH3T3-L1 cells using Oil Red O staining. As shown in Figs. 1a and b, kuanoniamine C treatment dose-dependently suppressed the IBMX–DEX–Insulin-dependent increase of lipid accumulation. The suppression of Oil Red staining might be due to the suppression of differentiation into white adipocytes (white adipocyte markers: PPARγ). As shown in Fig. 1c, kuanoniamine C treatment partially suppressed mRNA expression of PPARγ. These results indicated that kuanoniamine C partially suppressed IBMX–DEX–Insulin-induced differentiation of white adipocytes in NIH3T3-L1 cells.

Fig. 1. Kunanoniamine C Suppressed the Differentiation of NIH3T3-L1 Preadipocytes into Full Function of White Adipocytes

(a–c) NIH3T3-L1 preadipocytes were co-treated with IBMX–DEX–Insulin and DMSO or the indicated concentration of kuanoniamine C for 7 d (a, b) or 6 d (c). (a, b) Kuanoniamine C suppressed oil droplet formation. Oil red staining was followed by microscopic imaging (scale bar 50 µm) (a) and absorbance measurement (b). (c) Kuanoniamine C suppressed the mRNA expression of white adipocyte-related genes. The cells were subjected to qPCR. Data are presented as the mean ± standard deviation (S.D.) of three simultaneous experiments (b, c). Each p-value was calculated using ANOVA following Tukey-HSD test; * p < 0.05, ** p < 0.01 (b, c).

Kuanoniamine C Activates MEK-ERK-GSK3β Signaling in the Early Stage of Adipogenesis

Next, we tried to elucidate the molecular mechanism of kuanoniamine C–dependent inhibition of IBMX–DEX–Insulin-induced adipogenesis. As shown in Figs. 2a and b, kuanoniamine C treatment for 0 to 4 d suppressed lipid accumulation compared with treatment for 4 to 7 d, indicating that kuanoniamine C suppresses IBMX–DEX–Insulin-induced adipogenesis signaling from 0 to 4 d. Based on previous reports, IBMX–DEX–Insulin treatment induces adipogenesis by upregulation PPARγ and C/EBPα through activation of C/EBPδ and C/EBPβ, and IBMX–DEX–Insulin treatment also inhibits β-catenin signaling in the early phase of adipogenesis.^7,18) C/EBPδ and C/EBPβ are transcription factors controlling the mRNA expression of PPARγ and C/EBPα, and β-catenin suppresses the function of PPARγ and C/EBPα.^19,20) Kuanoniamine C suppressed mRNA expression level of C/EBPδ for 1 to 4 d, and also suppressed the mRNA expression of PPARγ and C/EBPα for 2 to 4 d (Fig. 2c). These results indicated that kuanoniamine C inhibited PPARγ and C/EBPα mRNA expression via the suppression of C/EBPδ expression. Kuanoniamine C did not completely suppress PPARγ and C/EBPα mRNA expression; thus, we focused whether kuanoniamine C affects β-catenin signaling. As shown in Fig. 2d, IBMX–DEX–Insulin suppressed β-catenin protein expression from day 3 and decreased the phosphorylation of GSK3β from day 1. In addition, kuanoniamine C treatment upregulated the phosphorylation of GSK3β and β-catenin protein expression. The phosphorylation of ERK and Akt can suppress the function of GSK3β (phosphorylation of GSK3β is inactive form), which promotes the upregulation of β-catenin expression.^21,22) As shown in Fig. 2d, the phosphorylation of MEK, ERK, and Akt decreased in a time-dependent manner by treating with IBMX–DEX–Insulin, but kuanoniamine C treatment increased the phosphorylation of MEK, ERK, and Akt compared with IBMX–DEX–Insulin treatment. Thus, kuanoniamine C increased the expression level of β-catenin protein by activation of the MEK-ERK and Akt pathway compared with IBMX–DEX–Insulin treatment only.

Fig. 2. Kunanoniamine C Attenuated the Activation of MEK-ERK-GSK3β-β-Catenin Signaling in the Early Stage of Adipogenesis

(a, b) Kuanoniamine C suppressed IBMX–DEX–Insulin-induced adipogenesis signaling in 0–4 d. NIH3T3-L1 preadipocytes were co-treated with IBMX–DEX–Insulin for 7 d and DMSO (0–7 d) or 5 µM of kuanoniamine C for the indicated days and subjected to Oil Red staining followed by microscopic imaging (left panel) and absorbance measurement (right panel). (c, d) Kuanoniamine C suppressed C/EBPδ-PPARγ signaling, and upregulated of p-ERK and β-catenin expression in IBMX–DEX–Insulin treated NIH3T3-L1 preadipocytes. NIH3T3-L1 preadipocytes were co-treated with IBMX–DEX–Insulin and DMSO (−) or 5 µM of kuanoniamine C (+) for the indicated days and subjected to qPCR (c) or immunoblotting (d: left panel) (d). In addition, the intensity of the P-ERK1/2, P-GSK3β, β-catenin, and P-Akt bands was determined (the levels of P-ERK1/2 are reported relative to those of ERK1/2; the levels of P-GSK3β are reported relative to those of GSK3β; the levels of P-Akt are reported relative to those of Akt, and the levels of β-catenin are reported relative to those of β-actin [d: right panel]). (e) Kuanoniamine C did not affect IBMX–DEX–Insulin-induced activation of ERK phosphorylation in very early phage of adipogenesis, but maintained ERK phosphorylation in the following phase. NIH3T3-L1 preadipocytes were co-treated with or without IBMX–DEX–Insulin and 5 µM of kuanoniamine C or DMSO (–) for the indicated time and subjected to immunoblotting (left panel) and the intensity of the P-ERK1/2 was determined (the levels of P-ERK1/2 was reported relative to those of ERK1/2 [e: right panel]). Data are presented as the mean ± S.D. of three simultaneous experiments (b–d). Each p-value was calculated using ANOVA following Tukey-HSD test (b–d) or student’s t-test (e); * p < 0.05, ** p < 0.01.

Previous report has suggested that a response in which a high level of ERK phosphorylation is induced in the first few minutes after treatment with IBMX–DEX–Insulin, and then the phosphorylation of ERK is reduced, is important for the accumulation of fat.²³⁾ Therefore, we confirmed whether kuanoniamine C affects the phosphorylation of ERK by IBMX–DEX–Insulin treatment in very early phase. As shown in Fig. 2e, IBMX–DEX–Insulin treatment increased the phosphorylation of ERK in 0.5 h and then decreased the phosphorylation of ERK for 1 to 4 h. Kuanoniamine C treatment also increased the phosphorylation of ERK in 0.5 h the same as IBMX–DEX–Insulin treatment, but suppressed reduction of the phosphorylation of ERK for 4 h compared with IBMX–DEX–Insulin treatment only, suggested that kuanoniamine C alters the transient induction of ERK phosphorylation by IBMX–DEX–Insulin, which is important for adipogenesis, and maintains a sustained state of ERK phosphorylation. These results indicated that kuanoniamine C inhibited adipogenesis via suppression of PPARγ mRNA expression by downregulation of C/EBPδ expression and upregulation of β-catenin by continuously activating the ERK signaling.

Kuanoniamine C Partially Suppresses Mitochondrial Functions

In verifying the cytotoxicity of kuanoniamine C, we performed a cell counting assay and found that kuanoniamine C showed no cytotoxicity up to 5 µM (Fig. 3a, upper panel). MTT and WST-8 (CCK-8) assays are also methods to examine cytotoxicity. However, MTT activity was reduced by kuanoniamine C treatment, but the CCK-8 assay was not affected (Fig. 3a, upper panel). Previous studies reported that MTT is localized in the mitochondria, and it is primarily converted to formazan by dehydrogenase, which is a mitochondrial complex II.²⁴⁾ In addition, CCK-8 contains WST-8, which is highly water soluble and localized outside the cell, and an electron transfer substance (1-Methoxy PMS) reduces WST-8 by passing electrons from whole cellular nicotinamide adenine dinucleotide (NAD)(P)H.²⁵⁾ Thus, MTT activity shows mitochondrial function, and WST-8 activity shows whole cellular production of NAD(P)H. We hypothesized that kuanoniamine C may inhibit mitochondrial complex II activity and performed cell count assay, MTT assay, and CCK-8 assay to determine whether atpenin A5, an inhibitor of mitochondrial complex II, has the same effect as kuanoniamine C. The results of Fig. 3a, lower panel, atpenin A5 only suppressed MTT activity as well as kuanoniamine C. As shown in Figs. 3b and c, ATP production was reduced by kuanoniamine C treatment, and the amount of succinate increased, which indicated that kuanoniamine C suppressed mitochondrial complex II function. Next, to determine whether kuanoniamine C induced total mitochondrial dysfunction, we examined the expression level of mitochondrial transcription factor A (Tfam). Previous reports suggested that Tfam binds to mitochondrial DNA and maintains its stability, implying that its expression is reduced when mitochondrial function is severely inhibited.^26,27) As shown in Fig. 3d, the treatment with kuanoniamine C did not affect the Tfam mRNA expression level. These results indicated that kuanoniamine C slightly suppressed mitochondrial function, despite the observed decrease in cell viability and evidence of mitochondrial dysfunction.

Fig. 3. Kunanoniamine C Inhibits Mitochondrial Function despite Cell Viability

(a) Kuanoniamine C and atpnin A5, which inhibitor of complex II only suppressed MTT activity. NIH3T3-L1 cells were treated with the indicated concentration of kuanoniamine C or atpenin A5 for 24 h (a–c). Cell viability was determined by using cell count assay. MTT assay represented mitochondrial complex II activity. WST-8 assay indicated total amount of NAD[P]H (a). (b) Kuanoniamine C slightly suppressed ATP production. The cells were subjected to ATP measurement. (c) Kuanoniamine C affected mitochondrial complex II activity. The cells were subjected to succinate measurement. (d) Kuanoniamine C did not affect Tfam mRNA expression level. NIH3T3-L1 preadipocytes were co-treated with IBMX–DEX–Insulin and DMSO or the indicated concentration of kuanoniamine C for 6 d. The cells were subjected to qPCR. Data are presented as the mean ± S.D. of three simultaneous experiments (a–d). Each p-value was calculated using ANOVA following Tukey-HSD test; n.s. not significant, * p < 0.05, ** p < 0.01.

Treatment of IBMX–DEX–Insulin-Induced Adipogenesis Signaling Is Modulated by Increasing ROS Production via Alteration of Mitochondrial Function

The reduction of the partial mitochondrial respiratory chain and the inhibition of membrane potential increase ROS production in the mitochondria.²⁸⁾ Atpenin A5 (complex II inhibitor) treatment significantly increased mitochondrial ROS production but not Rotenon (complex I inhibitor), Antimycin (complex III inhibitor), FCCP (membrane potential inhibitor), and Oligomycin (H⁺-ATPase inhibitor) by using a mitoSOX reagent in NIH3T3-L1 cells (Fig. 4a). These results indicated that mitochondrial complex II inhibition is important to accumulate ROS production in NIH3T3-L1 cells. We examined whether kuanoniamine C stimulates ROS production by using a mitoSOX and MnTBAP reagent, which is a superoxide scavenger.²⁹⁾ As shown in Fig. 4b, kuanoniamine C stimulated ROS production, and MnTBAP treatment eliminated kuanoniamine C-induced ROS, indicating that kuanoniamine C stimulates ROS production via the suppression of mitochondrial complex II function. Increasing ROS activates MEK-ERK signaling.³⁰⁾ Thus, we hypothesized that kuanoniamine C attenuated the phosphorylation of ERK because of the increase ROS production and then suppressed the differentiation of pre-adipose cells into white adipocytes. Inducing ERK phosphorylation and suppressing fatty acid accumulation by kuanoniamine C treatment were inhibited by MnTBAP treatment (Figs. 4c–e). Collectively, kuanoniamine C-activated ROS–ERK–β-catenin signaling is an important pathway, which suppressed IBMX–DEX–Insulin-induced differentiation of pre-adipose cells into white adipocytes.

Fig. 4. Kuanoniamine C Increased ROS Production-Modulated IBMX–DEX–Insulin-Induced Adipogenesis Signaling

(a–d) NIH3T3-L1 cells were co-treated with IBMX–DEX–Insulin and the indicated compound and concentration for 4 h (a, b), 24 h (b), or 7 d (c, d). The ROS level in cells was determined using the MitoSOX staining assay (a, b). (a) Atopenine A5 increased ROS production. (b) Kuanoniamine C stimulated ROS production and its ROS production is suppressed by MnTBAP (10 µM). (c, d) ROS scavenger recovered IBMX–DEX–Insulin-induced fatty acid droplet in kuanoniamine C-treated NIH3T3-L1 cells. Oil Red staining was followed by microscopic imaging (c) and absorbance measurement (d). (e) Kuanoniamine C induced the ROS-attenuated phosphorylation of ERK expression in IBMX–DEX–Insulin-treated NIH3T3-L1 cells. NIH3T3-L1 cells were co-treated with IBMX–DEX–Insulin or DMSO (−) and the indicated compound and concentration for 24 h. The cells were subjected to immunoblotting assay (left panel) and the intensity of the P-ERK1/2 was determined (the levels of P-ERK1/2 was reported relative to those of ERK1/2 [e: right panel]) (e). Data are presented as the mean ± S.D. of three simultaneous experiments (a, b, d, e). Each p value was calculated using ANOVA following Tukey-HSD test; n.s. not significant, * p < 0.05, ** p < 0.01.

Kuanoniamine C Prevents Abnormal Adipose Expansion in the Early Stage of HFD-Induced Obesity Mouse Model

Using an HFD-induced obesity mouse model, which induced adipogenesis in eWAT of young mice,⁴⁾ we investigated whether kuanoniamine C suppressed the increasing WAT. Five-week-old mice were fed with HFD for 24 d, which is the pre-obesity state, and 1 mg/kg of kuanoniamine C or dimethyl sulfoxide (DMSO) was intraperitoneally administered every 2 d and then subjected to blood glucose concentration, blood triglyceride concentration, and H&E staining in eWAT. The amount of food intake, body weight, fasting blood glucose concentration, and blood triglyceride concentration were not significantly changed by kuanoniamine C administration, but inguinal white adipose tissue (iWAT) and eWAT weight were significantly reduced by kuanoniamine C administration (Figs. 5a–d). We showed the H&E staining results of eWAT and calculated the size and volume of epididymal white adipose cells with or without kuanoniamine C administration (Figs. 5e–g). The administration of kuanoniamine C decreased WAT weight and, in adipocytes of specific sizes, treatment with kuanoniamine C increased the number of small adipocytes (250–400 µm²) and decreased the number of large adipocytes (4800–6000 µm²) (Fig. 5g). As shown in Fig. 5h, the administration of kuanoniamine C did not affect the expression of adipogenesis marker genes such as PPARγ and glucose transporter 4 (Glut4), nor the Tfam mRNA expression. This suggested that while kuanoniamine C decreased the total volume of WAT, there was no significant difference in the differentiation level of WAT in vivo, and it did not induce mitochondrial dysfunction. Consequently, the administration of kuanoniamine C effectively mitigated the enlargement of WAT by inhibiting the maturation of white adipocyte cells in HFD-induced obesity mouse model.

Fig. 5. Kuanoniamine C Suppressed WAT Expansion in the Early Stage of HFD-Induced Obesity Mouse Model

(a–h) Administration of kuanoniamine C suppressed HFD-induced weight increase of WAT. Mice (6 weeks) were fed HFD and received DMSO (n = 4) or 1 mg/kg of kuanoniamine C (n = 3) intraperitoneally every other day for 24 d. The body weight (a), amount of food intake (b), iWAT and eWAT weight (c), blood glucose and triglyceride concentration (d), and eWAT adipocyte were measured by using H&E staining (scale bar 50 µm) (e). The size and volume of white adipocytes in eWAT were analyzed using H&E staining sections (f, g). (f, g) Approximately 1000 adipocytes per mouse were counted using Image J, and then the distribution of adipose depot area was described. (g) Administration of kuanoniamine C increased small size of adipocyte and increased large size of adipocyte. The percentage of small (250–400 cm²) and large (4800–6000 cm²) white adipocytes in terms of size was compared between DMSO and kuanoniamine C. (h) Administration of kuanoniamine C did not affect adipogenesis marker and Tfam mRNA level. iWATs were subjected to qPCR. Each p-value was calculated using Student’s t test or ANOVA following Tukey-HSD test; n.s. not significant, * p < 0.05.

DISCUSSION

In this study, we found that the regulation of mitochondrial function is a key factor for the differentiation of pre-adipose cells into white adipocytes. In addition, the activation of the β-catenin pathway via ROS production suppressed adipogenesis signaling. We found that kuanoniamine C as a compound inhibits the differentiation of pre-adipose cells into white adipocytes by IBMX–DEX–Insulin treatment. Furthermore, kuanoniamine C suppressed adipose tissue expansion in the early stage of obesity in mouse models fed HFD. Therefore, this study suggests that the kuanoniamine C prevents adipogenesis of white adipocyte and adipose tissue expansion even under HFD intake conditions.

Many studies established that mitochondrial biogenesis during adipogenesis is necessary because adipocyte differentiation needs a large amount of ATP.⁸⁾ Inducing the impairment of mitochondrial function suppressed adipogenesis,^11,31,32) but the detail of the molecular mechanism, which related ROS production remained unknown. Here, we proposed that alteration of mitochondrial function disrupted crucial adipogenesis signaling via ROS production that does not affect cell viability. Moreover, activating the ROS–ERK–β-catenin pathway suppresses the adipogenesis.

Based on previous reports, partial mitochondrial inhibition increases ROS production,²⁸⁾ and the present study confirms that ROS is also increased by inhibiting mitochondria complex II function in NIH3T3-L1 cells. We identified kuanoniamime C is a unique compound that has little to no effect on cell viability or ATP production, and does not completely inhibit the function of the mitochondria. We also hypothesized that kuanoniamine C might inhibit mitochondrial complex II activity due to the increased amount of succinate and its function similar to that of atpenin A5. However, future studies are required to determine whether kuanoniamine C binds directly to mitochondrial complex II. Our results indicated that ROS scavenger suppressed kuanoniamine C-dependent upregulation of phosphorylation of ERK and β-catenin expression, then induction of ROS is necessary to kuanoniamine C-dependent suppression of adipogenesis signaling. Several separate studies showed that mitochondrial ROS production induced ERK phosphorylation,³⁰⁾ thereby increasing the β-catenin expression level,²¹⁾ which are consistent with our results. Controlled pre-adipocyte mitochondrial function might be a useful method to regulate the degree of differentiation into white adipocytes.

Normal WAT are important in maintaining nutritional homeostasis by taking in excess nutrients from the blood and storing them as lipids, but excessive lipid accumulation in WAT causes inflammatory and other harmful reactions that lead to obesity-related diseases.³⁾ The agents, which stimulates adipogenesis promote weight gain because of increased WAT, but such agents improve insulin resistance in a HFD obesity model. In addition, the agents, which inhibition of adipogenesis reduce nutrient stored in WAT because of decreased adipose tissue, but such agents are effective in ameliorating obesity symptoms and insulin resistance in HFD obesity model.³³⁾ These reports indicated that both suppression and stimulation of adipogenesis have therapeutic effect for diabetes symptoms, and suppression of adipogenesis especially inhibits obesity. We used the early phase of HFD obesity model, which induced adipogenesis, and kuanoniamine C suppressed adipose tissue gain and maturation of adipocytes without affecting blood glucose and triglyceride concentrations, although no change was observed in the total amount of food intake. Based on our in vitro results, kuanoniamine C might suppress abnormal expansion of WAT and disruption of WAT function in obesity model. Therefore, kuanoniamine C could be used as a prophylactic compound for obesity; however, the total amount of kuanoniamine C was too limited to create a long-term HFD obesity model and analyze mitochondrial function in WAT; thus, we must obtain sufficient kuanoniamine C and determine whether it has preventive and therapeutic effects for diabetes symptoms on a long-term HFD obesity model, as well as its therapeutic effect through the modulation of mitochondrial function.

This study suggests that the partial inhibition of mitochondrial function by kuanoniamine C treatment inhibited differentiation into white adipocytes via ROS–ERK–β-catenin signaling, and kuanoniamine C administration suppressed adipose tissue expansion in HFD-induced obesity model.

Acknowledgments

This work was partially supported by Kanae Foundation and Ono Medical Research Foundation. We thank the Division of Biological Research, Science Research Center, Kochi University for the use of research instruments.

Author Contributions

SO conducted most of the experimental work. SK and KM made experimental contributions. SO, KM, and TN designed the experimental plans and analyzed and interpreted the data. TN designed and directed the project. SO and TN wrote the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary Materials

This article contains supplementary materials.

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