Novel Function of TREK-1 in Regulating Adipocyte Differentiation and Lipid Accumulation

doi:10.21203/rs.3.rs-4909738/v1

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Novel Function of TREK-1 in Regulating Adipocyte Differentiation and Lipid Accumulation

https://doi.org/10.21203/rs.3.rs-4909738/v1

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K2P (two-pore domain potassium) channels, a diversified class of K⁺-selective ion channels, have been found to affect a wide range of physiological processes in the body. Despite their established significance in regulating proliferation and differentiation in multiple cell types, K2P channels' specific role in adipogenic differentiation (adipogenesis) remains poorly understood. In this study, we investigated the engagement of K2P channels, specifically KCNK2 (also known as TREK-1), in adipogenesis using primary cultured adipocytes and TREK-1 knockout (KO) mice. Our findings showed that TREK-1 expression in adipocytes decreases substantially during adipogenesis. This typically causes an increased Ca²⁺ influx and alters the electrical potential of the cell membrane. Furthermore, we observed a reduction in differentiation and lipid accumulation in both 3T3-L1 cell lines and primary cultured adipocytes when the TREK-1 activity was blocked with Spadin, the specific inhibitors, and TREK-1 shRNA. Finally, our findings revealed that mice lacking TREK-1 gained more weight and had worse glucose tolerance when fed a high-fat diet (HFD) compared to the wild-type controls. These findings imply that TREK-1 plays an important role in the control of adipogenesis and could be a potential target for the development of therapeutic treatments for obesity.

Biological sciences/Biochemistry/Ion channels/Potassium channels

Health sciences/Diseases/Metabolic disorders

K2P channels

TREK-1

adipocyte

Calcium influx

membrane potential

obesity

Adipocytes, the primary cells in adipose tissue, are essential in maintaining energy balance by storing excess energy or regulating thermogenesis. The number and size of these cells might fluctuate depending on a person's diet and physical condition [1]. The differentiation of pre-adipocytes, into mature adipocytes is central to adipose tissue development and metabolism regulation [2]. Notably, stem cell proliferation is linked to obesity, which is defined by an increase in white adipose tissue (WAT) [3]. Ion channels have emerged as key components affecting the development of adipocytes. The determination of adipocyte fate is primarily regulated by voltage-dependent potassium channels (e.g., TRPV, TRPM) and calcium channels (e.g., Cav3.1) [4]. These channels respond to changes in membrane potential by influencing calcium, an intracellular messenger crucial for adipocyte differentiation and proliferation. Compared to resting cells, proliferating or differentiating cells exhibit distinct ion channel properties and expression patterns, providing a new perspective on adipocyte differentiation and the onset of obesity. Voltage-gated K⁺ currents (Kv) have been identified in human preadipocytes and in brown and white adipose tissue, suggesting their potential role in adipogenesis [5–8]. Additionally, calcium signaling via voltage-gated calcium channels (VGCCs) in adipocytes is essential for adipogenesis, governing key functions such as proliferation, differentiation, and energy metabolism. Dysregulation of cytosolic calcium has been linked to metabolic abnormalities associated with human obesity [9].

Adipogenesis is a complex process in which preadipocytes differentiate into mature adipocytes, which store lipid droplets as triglycerides for future energy release. Hormones, growth factors, and transcriptional regulators all play roles in this process, controlling gene expression and cell fate decisions [10, 11]. Membrane potentials are crucial for regulating cell proliferation, differentiation, and signaling, with potassium ion channels being an important component of the cellular machinery that governs membrane potential [12]. Specifically, K2P channels contribute to leaking K⁺ currents and help stabilize negative resting membrane potentials in many cell types [13]. Although some members of the K2P potassium channel family have been reported to be expressed in adipose tissue or adipocytes [14, 15], the expression changes, and mechanisms of K2P channels during adipogenesis remain poorly understood.

In this study, we present results from experiments using cell lines, cultured primary adipocytes, and animal models induced by a high-fat diet. We also demonstrated experimentally that changes in membrane potential mediated by TREK-1 can induce alterations in intracellular calcium signaling via voltage-gated calcium channels. Furthermore, transcriptome data confirmed that these changes were replicated during the differentiation of adipose stem cells derived from human adipose tissue [16]. Thus, we propose that TREK-1 could be a novel therapeutic target for obesity.

2.1. Chemicals

Spadin was purchased from TOCRIS (Tocris Bioscience, Bristol, UK) and stored as a stock solution at − 20°C. It was diluted to the required concentration in a standard bath solution immediately before experimentation. Nifedipine, also purchased from TOCRIS, was stored as stock solutions at − 4°C and similarly diluted to the required concentrations in a standard bath solution immediately before use. ML402 was obtained from TOCRIS and stored at − 4°C. Before the start of the experiment, it was diluted to the desired concentration and used. GO6983 was purchased from TOCRIS, stored as stock solutions at − 20°C, and diluted to the required concentrations in a standard bath solution immediately before experimentation.

2.2. Cell culture and adipocyte differentiation

3T3-L1 cells were purchased from ATCC (American Type Culture Collection) and cultured in Dulbecco’s Modified Eagle Medium (Gibco, NY, USA) with 10% (v/v) bovine serum (Gibco, NY, USA) and 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific, NJ, USA). Cultures were maintained in a humidified 5% CO₂ incubator at 37°C. After reaching 80% confluence, 3T3L1 cells were induced differentiation by replacing the medium with DMEM containing 10% FBS plus 1 µg/ml insulin, 0.5mM isobutyl-1-methylxanthine (IBMX), 1 µM and 2 µM Rosiglitazone for 3 days. Afterward, the medium was changed with DMEM containing 10% FBS plus 1ug/ml insulin, and the cells were further cultured for 2 or 4 days.

2.3. Primary adipocyte culture

Primary white adipocytes were isolated from the inguinal WAT 6–8 weeks-old mouse(C57BL/6). Wash the extracted WAT in a 15 ml tube. The enzyme solution was mixed with Type II collagenase (Worthington Industries, Columbus, OH, USA) and PBS (Gibco, NY, USA). Incubate the tissue in 20ml enzyme solution in an incubator at 37°C for 1hour After filtering, put it in a new 50 ml tube and centrifuge at 10000 rpm for 10 minutes. Adipocytes floating in the solution were transferred to a new tube using a pipette. The adipocytes were centrifuged again in DMEM/F12 (Gibco, NY, USA) containing 10% (v/v) FBS, and the fat layer was transferred to a cell culture flask and filled with the DMEM/F12 + mixture. Invert the flask, incubate it in a 37°C, CO₂ incubator, and change the medium after about 5 days.

2.4. Real-time PCR

Total RNA was isolated from ND and D5 cells using an RNA purification kit (GeneAll) according to the manufacturer’s instructions. The cDNAs were synthesized from 1µg total RNA through reverse transcription using a SensiFAST™ cDNA Synthesis Kit (BIOLINE) according to the manufacturer’s instructions. Real-time PCR was performed using a SensiFAST™ Probe Hi-ROX kit (BIOLINE, London, UK). Primer sets for KCNK1, KCNK2, KCNK3, KCNK4, KCNK5, KCNK6, KCNK9, KCNK10, KCNK12, KCNK13, KCNK16, KCNK18, and GAPDH were purchased from IDT (PrimeTime qPCR assays). GAPDH was used as the reference gene. The 2-ΔΔCt method was used to calculate fold changes in gene expression. All experiments were repeated at least three times.

2.5. Western blot

Cells were treated with the samples, collected, washed twice with cold PBS, and lysed in cell lysis buffer (Cell signaling Biotechnology, Beverly, MA, USA) on ice for 10-15min. The lysate was clarified by centrifugation, and protein concentration was measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) following the manufacturer’s instructions. For western blotting, cells were lysed with lysis buffer (1% NP-40, 0.5% Sodium deoxycholate, 0.2% SDS, 150 mM NaCl, and 50 mM Tris, containing a protease inhibitor cocktail, pH 7.5). Total proteins were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk and then blotted with the appropriate antibodies: anti-TREK-1 (Santa Cruz, sc-11556), anti-PPARɣ (Cell Signaling Technology, #2435), anti-C/EBPɑ (Cell Signaling Technology, #2295), anti-p-ERK (Cell Signaling Technology, #4370), anti-ERK (Cell Signaling Technology, #4695), anti-p-PKCɑ (Santa Cruz, sc-377565), anti-PKCɑ (Abcam, ab4124), anti-p-AMPK, anti-AMPK, anti-FABP4 (Cell Signaling Technology, #2120), anti-Actin (Sigma-Aldrich, A5441). The membranes were then washed and incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch). The blots were detected using an ECL Western Blotting Substrate (Thermo Fisher Scientific).

2.6. Lipid staining (Oil red O staining)

Lipid staining was performed using Oil Red O (Sigma-Aldrich). Saint Louis, USA). Oil Red O dye was dissolved in isopropanol and free water to stain the loaded lipid droplets on the adipocytes, which were then fixed with 4% formaldehyde for 15 min. Next, the cells were washed twice with PBS, and adipocytes were incubated with Oil Red O solution at room temperature for 15–30 min. The cells were then washed twice with PBS for 5 min. The cell fragments were observed under a microscope.

2.7 Electrophysiology

Whole-cell voltage-clamp recordings were performed using a MultiClamp 700 B amplifier. using the pClamp10 software. Whole-cell patch-clamp recordings were performed using glass pipettes prepared with a 2-stage vertical pipette puller (P-1000, Shutter Instruments). The pipette resistance value was between 5–10 MΩ. For recording potassium current, the pipettes were filled with a solution having the following composition (in mM): 140.0 K-gluconate, 10.0 HEPES, 0.5 EGTA, 10.0 glucose, 2.0 Na-ATP, and 0.5 Na-GTP; pH was adjusted to 7.2. standard solution of the pipetted sample (mM). The current-voltage (I–V) relations were measured by applying 1s ramp pulses (from − 60 mV to + 140 mV) from a holding potential at − 40 mV. The Digidata 1440A interface (Axon Instruments, Union City, CA, USA) was used to convert digital to analog signals between the amplifier and the computer. Whole-cell currents were recorded 15–20 min after changing the spadin-containing solution.

2.8 Calcium imaging

3T3-L1 96well plate 2x10^4 seeding. Intracellular Ca²⁺ concentration was monitored by loading cultured 3T3L1 with fluo-4AM dye (Thermo Fisher, Carlsbad, USA). 3T3-L1 was incubated with 5 µM Fluo-4AM in medium and incubated for 30 min. was used in the experiments within 1 h. The bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose at pH 7.4. Imaging was performed using a microscope to detect changes in fluorescence signals.

2.9. Generation and genotyping of TREK-1 knock-out mice

TREK-1 knock-out (KO) mice were generated using the CRISPR/Cas9 system. Briefly, Cas9 mRNA and sgRNA were microinjected into the fertilized embryos of C57BL/6 mice (Korea Bio Co., Inc., Korea). Mutations in Kcnk2 were confirmed using sequencing (Korea Bio Co., Inc.). TREK-1 KO mice were genotyped by genomic DNA PCR analysis using genomic DNA prepared from their tails. Wild-type littermates were used as controls. TREK-1 KO forward: 5’-aatccttagccatccatgc-3,’ TREK-1 KO reverse: 5’-ctatctcagatggctttgacc-3.’

2.10. Animals and High fat diets

The study used 7-week old male C57BL/6J WT and TREK-1 KO mice were used in this study. Animal care and handling were performed according to the institutional guidelines of Institutional Animal Care and Use Committee (IACUC, KIST-2020-068) at the Korea Institute of Science and Technology (Seoul, Korea). The mice were housed in a temperature (22–26℃) and humidity-controlled animal room and maintained on a 12-h light/ 12-h dark cycle (light from 07:00 a.m. to 07:00 p.m.) with food and water provided during the experiments. One week before the high-fat diet, the mice were randomly distributed into the following four groups: (i) control mice fed a normal chow diet (10 kcal% fat content; Research Diets, D12450B); (ii) mice fed an HFD (60 kcal% fat content; Research Diets, D12492); (iii) TREK-1 KO mice fed a normal chow diet; (iv) TREK-1 KO mice fed an HFD. This study was conducted over 13 weeks. Food intake and body weight were measured thrice per week. NMR (Minispec LF90Ⅱ, Bruker Bioscience Corp.) was used to investigate body composition.

2.11. Oral glucose tolerance test (OGGT)

Two days before euthanasia, the oral glucose tolerance test (OGGT) was performed. Fasted animals (18 h) were orally administered glucose (2 g/kg), which was measured before and after 15, 30, 60, and 120 min using a glucometer (Accu-Chek, Roche). The area under the curve (AUC) was then calculated.

2.10. Plasma leptin (ELISA)

Plasma leptin levels were quantified using a mouse leptin quantitative ELISA kit (R&D Systems, MOB00B), which was used according to manufacturer’s instructions.

2.12. H&E staining and analysis

iWAT samples were collected for histological analyses. Immediately after harvesting, the tissue was washed with saline, placed in histological cassettes, and fixed in 10% neutral buffered formalin solution. After 24 h, tissues were processed and embedded in paraffin. Multiple sections (4 µm) were prepared and stained with hematoxylin and eosin (H&E) for morphological analysis. Images were captured using a Leica microscope (Leica DMi8). AdipoCount (csbio, version 1.0) was used to measure the cell count and size of adipocytes [17].

2.13. ShRNA transfection

An shRNA against TREK-1 was used as previously reported [18]. Briefly, TREK-1 shRNA was transfected into iWAT SVC 24 hours before induction of adipogenic differentiation. 9 days after induction of adipocyte differentiation, electrophysiology and ORO staining were performed to analyze RMP and lipid accumulation

2.14. Statistical analysis

The data are represented by the mean ± SEM. The importance of comparing two groups was determined using Student’s t-test, and the importance of multiple comparisons (more than two groups) was evaluated by one-way ANOVA with a post-hoc test. Two-way analysis of variance (ANOVA) and Tukey’s post-hoc tests were used for experiments with two independent variables. The significance levels are specified as * P < 0.05, ** P < 0.01, and *** P < 0.001.

3 − 1. Reduction in K ⁺ currents and increase in membrane potentials observed in adipogenesis in 3T3-L1 cell line

We investigated the modulation of K⁺ channels during adipogenic differentiation (Fig 1A). Using voltage-clamp recording techniques, we confirmed the expression of functional K⁺ channels in adipocytes and found that K⁺ current conductance gradually decreased after adipocyte differentiation at D3, D5, and D7 in 3T3-L1 adipocytes (Fig. 1B-D). The resting membrane potential (RMP) of 3T3L-1 adipocytes was significantly reduced after adipocyte differentiation (Fig. 1E). These results showed that the membrane potential significantly decreased as preadipocyte differentiated into mature adipocytes.

3 − 2. Adipogenic differentiation modulates the functional expression of TREK-1 in adipocytes.

Next, we aimed to identify the ion channels that reduce the cell membrane potential during adipocyte maturation. Previous studies have shown that K2P channels are important for maintaining cell membrane potential, and changes in the expression of K2P channels have been confirmed using a transcriptome database (GSE227819) that compared the differentiation process of pre-adipocytes. The change in TREK-1 was the most significant among the K2P channels (Supplementary Fig. 1). Real-time PCR analysis revealed that the mRNA levels of KCNK2 (also known as TREK-1), a member of the K2P family, were predominantly expressed in pre-adipocytes and were significantly reduced on day 5 after the induction of adipocyte differentiation (Fig. 2A). These results suggest that K⁺-conductive ion channels, particularly TREK-1, may play a role in adipogenic differentiation by altering their properties or being downregulated during the process. Following a significant reduction in TREK-1 expression during adipocyte differentiation, we aimed to determine whether this reduction was due to downregulation of TREK-1 in adipocytes. To verify the functional expression of TREK-1, we measured the K⁺ currents in pre-differentiated (ND) and mature (D7) adipocytes. In the presence of 1 µM spadin, a TREK-1 antagonist, voltage-step currents were significantly decreased in ND but not in D7 (Fig. 2B-E). Spadin-sensitive currents measured in both pre- and mature adipocytes suggested that the majority of the reduction in whole-cell currents were due to TREK-1 function. Moreover, we found that the inhibition of TREK-1 by spadin induced the depolarization of RMP in pre-adipocytes (Fig. 1E), indicating that adipogenic differentiation modulates the functional expression of TREK-1 and RMP in adipocytes. To investigate the phenotypic effects of TREK-1 on adipogenesis, we evaluated cytosolic lipid accumulation using Oil Red O (ORO) staining. We observed that the inhibition of TREK-1 by spadin treatment significantly increased lipid droplet formation during adipogenic differentiation (Fig. 2F-G). These findings suggested that the absence of TREK-1 makes adipocytes more susceptible to differentiation into mature cells.

3–3. Adipogenic biomarkers were upregulated by TREK-1 inhibition in adipocytes.

To investigate the impact of TREK-1 inhibition on adipogenic differentiation, we performed western blot analysis to detect changes in adipogenic markers after differentiation induction with spadin treatment (Fig. 3A). Initially, we confirmed the downregulation of TREK-1 following differentiation, and spadin treatment further enhanced this downregulation (Fig. 3B). Our findings revealed a significant upregulation of PPARγ and C/EBPα expression upon inhibition of TREK-1 by spadin treatment, indicating a potential regulatory role of TREK-1 in these adipogenic markers (Fig. 3C-D). Additionally, we observed a significant increase in protein kinase C alpha (PKCα) expression with spadin treatment, while the level of phosphorylated ERK remained mostly unchanged (Fig. 3E-F). Notably, a significant decrease in AMPK levels was observed during adipogenic differentiation, which was not further reduced by spadin treatment (Fig. 3G). Furthermore, we detected upregulation of FABP4, a late marker of adipogenic differentiation, during adipocyte differentiation (Fig. 3H). Overall, our results suggest that the inhibiting of the TREK-1 channel by spadin treatment in 3T3-L1 adipocytes modulates the expression of key biomarkers involved in adipogenic differentiation.

3–4. Inhibition of TREK-1 promotes adipogenesis via induction of intracellular Ca increase through VDCC.

We showed adipogenic markers, including C/EBPα and PPARγ, which were increased upon spadin treatment in differentiated adipocytes. Moreover, we found that the phosphorylated form of PKCα was elevated following spadin treatment. Interestingly, we observed that the membrane potential of adipocytes was significantly depolarized during cell differentiation. Based on these findings, we hypothesized that the inhibiting of TREK-1 during adipogenic differentiation could lead to an increase in cytosolic Ca²⁺, an essential molecule in adipogenesis. Calcium imaging experiments were performed to test this hypothesis using Fluo-4-loaded pre-adipocytes and an epifluorescence microscope. We observed that Ca²⁺ signals were evoked by the application of spadin and significantly blocked by the co-application of nifedipine, an L-type calcium channel blocker (Fig. 4A-C). While this was not a complete inhibition of the Ca²⁺ increase, these results partially support our hypothesis that downregulation of TREK-1 during adipogenic differentiation can lead to an increase in cytosolic Ca²⁺ in adipocytes, which is critical for adipogenesis. To further understand the effect of increased Ca²⁺ on PKC and TREK-1, we examined the expression levels of PKC and TREK-1 in the presence of high Ca²⁺ levels in the media. It has been reported that the activation of PKCα can downregulate TREK-1 expression in pre-adipocytes[19]. Consistent with this, we found that the phosphorylated form of PKCα was upregulated, whereas TREK-1 was downregulated in the presence of high Ca²⁺ stimulation (Fig. 4D). We verified this protein change using whole-cell patch-clamp experiments, which showed that the K⁺ current in preadipocytes was reduced. The RMP was depolarized in the presence of high Ca²⁺ stimulation. The PKCα inhibitor, GO6983, blocked the reduction of the K⁺ current and RMP depolarization in high Ca²⁺-stimulated pre-adipocytes (Fig. 4E-G). To investigate the impact of increased Ca²⁺ on adipogenic differentiation, we examined whether the inhibition of Ca²⁺ increase, mediated by TREK-1 downregulation via VDCCs, could inhibit phenotypic changes, such as lipid accumulation during adipogenic differentiation, using ORO staining in 3T3-L1 cells. To isolate the effect of TREK-1 inhibition on adipogenesis, we cultured 3T3-L1 cells in an MDI medium without rosiglitazone to slow differentiation, as spadin treatment accelerated adipogenic differentiation. We found that spadin-induced lipid accumulation increased at D5 and D7 compared to that in the control, and this increase was blocked by co-treatment with nifedipine (Fig. 4H-J). Taken together, our findings demonstrate that the inhibition of TREK-1 promotes adipogenesis through an increase in intracellular calcium levels via VDCCs.

3–5. TREK-1 activation prevented adipogenesis

Based on our finding that the inhibition of TREK-1 promotes adipogenesis, we investigated the effect of TREK-1 activation on adipocyte differentiation using the TREK-1 agonist ML402. First, we determined the concentration of ML402 required to activate TREK-1 by whole-cell patch clamping of HEK293 cells fully transfected with TREK-1 (Fig. 5A). Our results revealed that over 10 µM ML402 was required to saturate the increase in TREK-1-mediated currents (Fig. 5B). We then divided the cells into two groups to evaluate the preventive and therapeutic effects of TREK-1 activation on adipocyte differentiation (Fig. 5C). In the first group, ML402 was administered at an early stage of differentiation induction. We observed a significant reduction in lipid accumulation at all doses on day 7 (D7), and ORO staining showed no difference compared to the untreated group on day 3 (D3) (Fig. 5D-E). In the second group, ML402 was administered 3 days after the induction of differentiation. We observed a significant reduction in lipid accumulation on days 5, 7, and 9 (D5, D7, and D9) compared to that in the untreated group (Fig. 5F-G). These results suggested that activating TREK-1 in adipocytes has preventive and therapeutic effects on adipogenesis.

3–6. TREK-1 knockdown promotes adipogenesis in iWAT Adipocytes

We further investigated the role of TREK-1 in adipogenesis using primary adipocytes derived from inguinal white adipose tissue stromal vascular cells (iWAT SVCs). To assess the functionality of TREK-1 in primary iWAT SVCs, we recorded voltage-induced ramp currents and applied the TREK-1 antagonist, spadin, which significantly reduced the amplitude of the currents (Fig. 6A-B). Additionally, a significant decrease in the K + current was confirmed by the application of TREK1 shRNA (Fig. 6C). Our results indicated the presence of functional TREK-1 channels in primary adipocytes. We transfected primary iWAT SVCs with TREK-1 shRNA and induced adipocyte differentiation to investigate the effect of TREK-1 on adipogenic differentiation. After nine days, we performed ORO staining to analyze lipid accumulation and observed a significant increase in lipid droplets in the shTREK-1 transfected group compared to the SC group (Fig. 6D-E). Our results suggest that TREK-1 plays a crucial role in primary iWAT SVCs and 3T3-L1 cells. The consistency between the ex vivo and in vitro results further strengthens the evidence supporting the role of TREK-1 in adipogenesis.

3.7 HFD-fed TREK-1 KO mice increased adipogenic phenotypes

Next, to evaluate the systemic effects of TREK-1 on the development of obesity, we subjected wild-type (WT) and TREK-1 KO mice to either a normal chow diet (NCD) or a high-fat diet (HFD) from 8 to 20 weeks of age (Fig. 7A). Body weight gradually increased in the HFD groups of both WT and KO mice; however, there was no significant difference in the NCD group (Fig. 7B). Food intake decreased in TREK-1 KO mice fed an HFD compared to WT mice fed an HFD (Fig. 7C), but calorie intake did not show significant differences (Fig. 7D). Fat mass was significantly increased in the HFD groups of both mice. Interestingly, TREK-1 KO mice showed a significant increase in fat mass and a decrease in lean body mass, even in the NCD group (Fig. 7E-F). We also observed decreased glucose disposal in the oral glucose tolerance test (OGTT) in TREK-1 KO mice compared to WT mice on NCD and HFD (Fig. 7G). The plasma leptin level was much higher in KO mice fed HFD than in WT mice fed HFD (Fig. 7H). In the HFD group of TREK-1 KO mice, we observed a significant increase in adipocyte size compared to that in WT mice. Adipocyte size and area were increased in TREK-1 KO mice fed an HFD (Fig. 7I-K). These results suggest that TREK-1 deficiency may play a role in the development of obesity and related metabolic disorders.

In this study, we found that potassium currents were significantly reduced during the differentiation of preadipocytes into mature adipocytes. Among the K2P ion channel family, the expression of TREK-1 was dramatically reduced during adipocyte differentiation. Spadin, a TREK-1 antagonist, increased lipid droplet accumulation and decreased adipogenic markers during adipogenesis through PKCα modulation. It also increased the RMP and calcium influx through voltage-dependent calcium channels. We also demonstrated that increased calcium can activate PKCα, further suppressing TREK-1 expression. This suggests that TREK-1 may serve as an upstream regulator of adipogenesis via increased intracellular calcium. Conversely, TREK-1 activators were shown to suppress lipid accumulation during both the early and late stages of adipocyte maturation. As expected, TREK-1-deficient mice fed HFD showed increased body weight, WAT, and insulin resistance compared to WT mice on the same diet.

Adipogenesis is a complex process involving the differentiation of preadipocytes into mature adipocytes and is regulated by various signaling pathways, including the critical insulin signaling pathway[10]. In this study, we investigated the role of K2P ion channels, which are known to be important for the resting membrane potential in adipocyte differentiation, using 3T3-L1 preadipocytes. While previous studies have reported that TREK-2 may be involved in the early stages (3–6 hrs) of adipocyte differentiation [20] and that TASK-1 is important for the β-adrenergic response in brown adipocytes[21], no study has verified the expression of all K2P family members. We observed a decrease in potassium currents during adipocyte differentiation, and while the expression of all K2P ion channels was confirmed, only TREK-1 was selectively decreased. These findings align with recent analyses of K2P expression changes in transcriptome databases from human adipose tissue (human adipose tissue stem cells induced to differentiate into adipocytes for 9 days) (Supplementary Fig. 1) [16]. Therefore, these findings may have important implications for the pathogenesis of obesity in adults.

In this study, the function of TREK-1 in adipocyte differentiation was investigated for the first time. Real-time PCR analysis showed that TREK-1 was the most abundantly expressed K2P channel in 3T3-L1 cells before differentiation, and its expression was downregulated during the process. This study also revealed that TREK-1 plays a critical role in regulating potassium currents in preadipocytes, as evidenced by treatment with spadin, a TREK-1-selective inhibitor. The study clearly demonstrates that TREK-1 is crucial for regulating intracellular calcium influx and suppressing excessive adipogenesis.

These findings underscore the vital role of TREK-1 in regulating adipogenesis and suggest that TREK-1 modulators could be potential targets for therapeutic interventions. This study demonstrated the influence of spadin and ML402, both known TREK-1 modulators, on adipocytes. Spadin, a TREK-1 inhibitor, promoted adipogenesis, whereas ML402, a TREK-1 activator, suppressed it. Two strategies, prevention and treatment, were used to regulate adipogenesis based on the presence of TREK-1. These results provide evidence for the efficacy of TREK-1 activation in preventing and treating accelerated adipocyte differentiation. TREK-1 activation through ML402 treatment emerges as a potential target for preventing and treating accelerated adipogenesis.

TREK-1 regulates the excitability of the cell's resting membrane potential. Interestingly, we found that TREK-1 inhibition depolarized preadipocytes, increasing intracellular calcium influx. Previous studies have established that intracellular calcium influx plays a crucial role in cell differentiation and proliferation [9, 20, 22, 23]. In this study, we investigated whether TREK-1 inhibition led to changes in intracellular calcium influx using calcium imaging in preadipocytes. Spadin increased intracellular calcium influx, which was inhibited when combined with nifedipine, an L-type calcium channel blocker. In cellular Ca²⁺ signaling, conventional PKCs (cPKCs) are important modulators of finely-tuned Ca²⁺ responses [24]. PKC-dependent phosphorylation regulates TREK-1 function [19, 25]. In summary, depolarization caused by TREK-1 inhibition increases intracellular calcium, which in turn phosphorylates the S300 site of TREK-1 via PKCα activation, further inhibiting TREK-1. This creates a feedback loop, where depolarization by TREK-1 inhibition enhances calcium influx and PKC phosphorylation, thereby accelerating the inhibition of TREK-1. Additionally, these results show that this process regulates adipogenesis by activating the expression of C/EBPα and PPARγ, which are critical transcription factors in adipogenesis [26, 27] .

TREK-1 is also a well-known mechano- and temperature-activated background potassium channel [28, 29]. It opens steadily and reversibly under heat or cold conditions [28]. A 10°C increase enhances the TREK-1 current by approximately sevenfold [28, 30]. It has been reported that mechano-activated TRAKK and TREK-1 channels control both warm and cold sensations in polymodal pain perception [31, 32]. Unlike white fat, brown adipose tissue (BAT) can dissipate significant amounts of chemical energy through non-shivering thermogenesis [33, 34]. This process is mediated by major heat-generating factors, and BAT can be activated by specific stimuli such as cold exposure, adrenergic compounds, or genetic modifications [35]. While it remains undetermined whether these TREK-1 characteristics affect BAT, our results suggest that TREK-1 may be associated with BAT thermogenesis.

The dysfunction of K2P channels has been associated with obesity and metabolic disorders. The modulation of K2P channels has also been implicated in insulin secretion, beta-cell dysfunction, and type 2 diabetes [36, 37]. In this study, we demonstrated that TREK-1 KO mice exhibit increased adiposity, hyperleptinemia, and impaired glucose tolerance. In vivo, the higher body weight gain, fat mass, insulin resistance, and leptin resistance observed in TREK-1 KO mice fed an HFD indicate that TREK-1 KO accelerates the development of metabolic disorder-like phenotypes. Without an HFD, TREK-1 KO mice still showed higher fat mass, even though they did not exhibit differences in food intake or weight gain. Taken together, these results suggest that TREK-1 is a powerful modulator of adipogenesis and metabolic syndromes, including obesity.

Competing Interest:

The authors have declared no competing interests.

Funding:

This work was supported by the following funds and foundations: The Main Research Program (E00210201-04; JL) of the Korea Food Research Institute funded by the Ministry of Science and ICT.

Author Contributions:

EMH and JL supervised the project and wrote the manuscript. JY performed most experiments and analysis in vitro experiments. AK performed in vivo studies and analyzed the data. HL and JJ performed in vitro experiments. YK and JYP contributed to the discussion and critically reviewed and edited the manuscript.

Rosen, E.D. and B.M. Spiegelman, Adipocytes as regulators of energy balance and glucose homeostasis. Nature, 2006. 444(7121): p. 847-53.
Zhang, K., et al., Molecular Mechanism of Stem Cell Differentiation into Adipocytes and Adipocyte Differentiation of Malignant Tumor. Stem Cells Int, 2020. 2020: p. 8892300.
Gray, S.L. and A.J. Vidal-Puig, Adipose tissue expandability in the maintenance of metabolic homeostasis. Nutr Rev, 2007. 65(6 Pt 2): p. S7-12.
Vasconcelos, L.H., et al., Ion Channels in Obesity: Pathophysiology and Potential Therapeutic Targets. Front Pharmacol, 2016. 7: p. 58.
Lucero, M.T. and P.A. Pappone, Voltage-gated potassium channels in brown fat cells. J Gen Physiol, 1989. 93(3): p. 451-72.
Russ, U., T. Ringer, and D. Siemen, A voltage-dependent and a voltage-independent potassium channel in brown adipocytes of the rat. Biochim Biophys Acta, 1993. 1153(2): p. 249-56.
Ramírez-Ponce, M.P., et al., Voltage-dependent potassium channels in white adipocytes. Biochem Biophys Res Commun, 1996. 223(2): p. 250-6.
Bai, X., et al., Electrophysiological properties of human adipose tissue-derived stem cells. Am J Physiol Cell Physiol, 2007. 293(5): p. C1539-50.
You, M.H., et al., Voltage-gated K+ channels in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells. Acta Pharmacol Sin, 2013. 34(1): p. 129-36.
Sarjeant, K. and J.M. Stephens, Adipogenesis. Cold Spring Harb Perspect Biol, 2012. 4(9): p. a008417.
Ghaben, A.L. and P.E. Scherer, Adipogenesis and metabolic health. Nature Reviews Molecular Cell Biology, 2019. 20(4): p. 242-258.
Sundelacruz, S., M. Levin, and D.L. Kaplan, Role of membrane potential in the regulation of cell proliferation and differentiation. Stem Cell Rev Rep, 2009. 5(3): p. 231-46.
Enyedi, P. and G. Czirják, Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev, 2010. 90(2): p. 559-605.
Chen, Y., et al., Crosstalk between KCNK3-Mediated Ion Current and Adrenergic Signaling Regulates Adipose Thermogenesis and Obesity. Cell, 2017. 171(4): p. 836-848.e13.
Nishizuka, M., et al., KCNK10, a tandem pore domain potassium channel, is a regulator of mitotic clonal expansion during the early stage of adipocyte differentiation. Int J Mol Sci, 2014. 15(12): p. 22743-56.
Potolitsyna, E., et al., Cytoskeletal rearrangement precedes nucleolar remodeling during adipogenesis. Commun Biol, 2024. 7(1): p. 458.
Zhi, X., et al., AdipoCount: A New Software for Automatic Adipocyte Counting. Front Physiol, 2018. 9: p. 85.
Hwang, E.M., et al., A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes. Nat Commun, 2014. 5: p. 3227.
García, G., et al., PKC- and PKA-dependent phosphorylation modulates TREK-1 function in naïve and neuropathic rats. J Neurochem, 2021. 157(6): p. 2039-2054.
Shi, H., et al., Role of intracellular calcium in human adipocyte differentiation. Physiol Genomics, 2000. 3(2): p. 75-82.
Pisani, D.F., et al., The K+ channel TASK1 modulates β-adrenergic response in brown adipose tissue through the mineralocorticoid receptor pathway. Faseb j, 2016. 30(2): p. 909-22.
Zhai, M., et al., Involvement of calcium channels in the regulation of adipogenesis. Adipocyte, 2020. 9(1): p. 132-141.
Henao, J.C., et al., TRPM8 Channel Promotes the Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells. Front Cell Dev Biol, 2021. 9: p. 592946.
Lipp, P. and G. Reither, Protein kinase C: the "masters" of calcium and lipid. Cold Spring Harb Perspect Biol, 2011. 3(7).
Hayoz, S., et al., Protein kinase A and C regulate leak potassium currents in freshly isolated vascular myocytes from the aorta. PLoS One, 2013. 8(9): p. e75077.
Rosen, E.D., et al., PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell, 1999. 4(4): p. 611-7.
Darlington, G.J., S.E. Ross, and O.A. MacDougald, The role of C/EBP genes in adipocyte differentiation. J Biol Chem, 1998. 273(46): p. 30057-60.
Maingret, F., et al., TREK-1 is a heat-activated background K(+) channel. Embo j, 2000. 19(11): p. 2483-91.
Lamas, J.A., L. Rueda-Ruzafa, and S. Herrera-Pérez, Ion Channels and Thermosensitivity: TRP, TREK, or Both? Int J Mol Sci, 2019. 20(10).
Rueda-Ruzafa, L., et al., Are TREK Channels Temperature Sensors? Front Cell Neurosci, 2021. 15: p. 744702.
Noël, J., et al., The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception. Embo j, 2009. 28(9): p. 1308-18.
Alloui, A., et al., TREK-1, a K+ channel involved in polymodal pain perception. Embo j, 2006. 25(11): p. 2368-76.
Mulya, A. and J.P. Kirwan, Brown and Beige Adipose Tissue: Therapy for Obesity and Its Comorbidities? Endocrinol Metab Clin North Am, 2016. 45(3): p. 605-21.
Nakamura, Y. and K. Nakamura, Central regulation of brown adipose tissue thermogenesis and energy homeostasis dependent on food availability. Pflugers Arch, 2018. 470(5): p. 823-837.
Fenzl, A. and F.W. Kiefer, Brown adipose tissue and thermogenesis. Horm Mol Biol Clin Investig, 2014. 19(1): p. 25-37.
Vierra, N.C., et al., Type 2 Diabetes-Associated K+ Channel TALK-1 Modulates β-Cell Electrical Excitability, Second-Phase Insulin Secretion, and Glucose Homeostasis. Diabetes, 2015. 64(11): p. 3818-28.
Cho, Y.S., et al., Meta-analysis of genome-wide association studies identifies eight new loci for type 2 diabetes in east Asians. Nat Genet, 2011. 44(1): p. 67-72.

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Novel Function of TREK-1 in Regulating Adipocyte Differentiation and Lipid Accumulation

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Chemicals

2.2. Cell culture and adipocyte differentiation

2.3. Primary adipocyte culture

2.4. Real-time PCR

2.5. Western blot

2.6. Lipid staining (Oil red O staining)

2.7 Electrophysiology

2.8 Calcium imaging

2.9. Generation and genotyping of TREK-1 knock-out mice

2.10. Animals and High fat diets

2.11. Oral glucose tolerance test (OGGT)

2.10. Plasma leptin (ELISA)

2.12. H&E staining and analysis

2.13. ShRNA transfection

2.14. Statistical analysis

3. Results

3 − 2. Adipogenic differentiation modulates the functional expression of TREK-1 in adipocytes.

3–3. Adipogenic biomarkers were upregulated by TREK-1 inhibition in adipocytes.

3–4. Inhibition of TREK-1 promotes adipogenesis via induction of intracellular Ca increase through VDCC.

3–5. TREK-1 activation prevented adipogenesis

3–6. TREK-1 knockdown promotes adipogenesis in iWAT Adipocytes

3.7 HFD-fed TREK-1 KO mice increased adipogenic phenotypes

4. Discussion

Declarations

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References

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Supplementary Files

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