KCTD10 regulates brown adipose tissue thermogenesis and metabolic function via Notch signaling

in Journal of Endocrinology
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Ming-sheng YeDepartment of Endocrinology, Endocrinology Research Center, Xiangya Hospital, Central South University, Changsha, Hunan, China

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Liping LuoDepartment of Endocrinology, Endocrinology Research Center, Xiangya Hospital, Central South University, Changsha, Hunan, China

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Qi GuoDepartment of Endocrinology, Endocrinology Research Center, Xiangya Hospital, Central South University, Changsha, Hunan, China

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Tian SuDepartment of Endocrinology, Endocrinology Research Center, Xiangya Hospital, Central South University, Changsha, Hunan, China

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Peng ChengDepartment of Gerontology, The First Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu, China

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Yan HuangDepartment of Endocrinology, Endocrinology Research Center, Xiangya Hospital, Central South University, Changsha, Hunan, China
National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha, Hunan, China

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Correspondence should be addressed to P Cheng or Y Huang: cphh@sohu.com or yanhuang1018@csu.edu.cn
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Brown adipose tissue (BAT) is emerging as a target to beat obesity through the dissipation of chemical energy to heat. However, the molecular mechanisms of brown adipocyte thermogenesis remain to be further elucidated. Here, we show that KCTD10, a member of the polymerase delta-interacting protein 1 family, was reduced in BAT by cold stress and a β3 adrenoceptor agonist. Moreover, KCTD10 level increased in the BAT of obese mice, and KCTD10 overexpression attenuates uncoupling protein 1 expression in primary brown adipocytes. BAT-specific KCTD10 knockdown mice had increased thermogenesis and cold tolerance protecting from high-fat diet (HFD)-induced obesity. Conversely, overexpression of KCTD10 in BAT caused reduced thermogenesis, cold intolerance, and obesity. Mechanistically, inhibiting Notch signaling restored the KCTD10 overexpression-suppressed thermogenesis. Our study presents that KCTD10 serves as an upstream regulator of Notch signaling pathway to regulate BAT thermogenesis and whole-body metabolic function.

Abstract

Brown adipose tissue (BAT) is emerging as a target to beat obesity through the dissipation of chemical energy to heat. However, the molecular mechanisms of brown adipocyte thermogenesis remain to be further elucidated. Here, we show that KCTD10, a member of the polymerase delta-interacting protein 1 family, was reduced in BAT by cold stress and a β3 adrenoceptor agonist. Moreover, KCTD10 level increased in the BAT of obese mice, and KCTD10 overexpression attenuates uncoupling protein 1 expression in primary brown adipocytes. BAT-specific KCTD10 knockdown mice had increased thermogenesis and cold tolerance protecting from high-fat diet (HFD)-induced obesity. Conversely, overexpression of KCTD10 in BAT caused reduced thermogenesis, cold intolerance, and obesity. Mechanistically, inhibiting Notch signaling restored the KCTD10 overexpression-suppressed thermogenesis. Our study presents that KCTD10 serves as an upstream regulator of Notch signaling pathway to regulate BAT thermogenesis and whole-body metabolic function.

Introduction

Obesity as a global disease has been shown to increase the risk of developing diabetes, cardiovascular disease, and tumor diseases (Ling & Rönn 2019, Su et al. 2019, Xiao et al. 2020, Petrus et al. 2020, Brestoff et al. 2021). Brown adipose tissue (BAT) dissipates energy in the form of heat by highly expressed uncoupling protein 1 (UCP1) protein to uncouple the mitochondrial proton gradient, which is important for thermogenesis and energy balance in mammals (Chouchani et al. 2019, Blondin et al. 2020, Chen et al. 2021, Fischer et al. 2021). In adult humans, very recent studies display that BAT deports are mainly located in the supraclavicular areas, axillar and paravertebral regions, though the variability across individuals and populations is still being worked out (Balaz et al. 2019, Fraum et al. 2019, Jung et al. 2019, Gnad et al. 2020). Thus, increasing BAT thermogenesis is a promising strategy to prevent diet-induced obesity (Cypess et al. 2015, Cereijo et al. 2018). However, the mechanisms underlying the regulation of thermogenesis and adipogenesis in BAT remain to be fully elucidated.

Notch signaling has been revealed as a novel regulator for metabolism (Bi & Kuang 2015), especially for energy expenditure. Bi et al. found that Notch signaling exerts an important role in regulating inguinal white adipose tissue (iWAT) browning (Bi et al. 2014). Consistently, the Notch target gene Hes1 (hes family bHLH transcription factor 1) suppresses thermogenic genes expression during the browning process. Recent studies showed that suppressing Notch signaling in liver or β cell-protected mice from obesity-induced glucose intolerance (Bartolome et al. 2019, Richter et al. 2020). In addition, inhibition of Notch signaling promoted mitochondrial biogenesis and reduced body weight in pigs (Huang et al. 2020a). However, how the Notch signaling is regulated during energy metabolism has been largely unknown.

Potassium channel tetramerization domain containing 10 (KCTD10), a novel polymerase delta-interacting protein 1-related protein, was found to be associated with HDL concentrations (Junyent et al. 2009) and induced in epididymal white adipose tissue (eWAT) of mice when fed with high-fat diet (HFD) (Jones et al. 2020), while, the function of KCTD10 in adipose tissue has not been investigated. Recently, KCTD10 was identified to play an important biological role in cardiovascular development via binding to the Notch (Ren et al. 2014, Pang et al. 2019). Considering the role of Notch signaling in energy metabolism, we hypothesize that KCTD10 might play an essential role in thermogenesis of fat tissue.

In the present study, we showed that KCTD10 in BAT decreased upon cold exposure and CL316243 treatment (a β3 adrenoceptor agonist that mimics cold stimulation), while increased in the BAT of HFD-induced obese mice and db/db mice (a transgenic mouse model of obesity). Moreover, BAT-special KCTD10 knockdown promotes thermogenesis and prevents diet-induced obesity. In contrast, KCTD10 overexpression mice showed decreased thermogenesis, obesity, and insulin resistance. Mechanistically, knockdown of KCTD10 increases UCP1 expression by suppressing Notch signaling. Taken together, these studies demonstrate that KCTD10 acts as a dominant regulator of thermogenesis and might become a promising target in treating obesity.

Materials and methods

Animals and treatment

Male and female C57BL/6J mice and diabetic mice (db/db) were purchased from Hunan SJA Laboratory Animal Company. Mice were housed and maintained in 12 h light:12 h darkness cycle at constant temperature (20–24°C) with free access to water and standard diet (SD). Mice were fed high-fat diet (D12492; contained 60% fat) for 2 months and then euthanized using CO2 euthanasia. In addition, all animal care protocols and experiments were reviewed and approved by the Animal Care and Use Committees of the Laboratory Animal Research Center at Xiangya Medical School of Central South University. This study was compliant with all relevant ethical regulations regarding animal research. For hematoxylin and eosin (H&E) staining, BAT, iWAT, eWAT, and liver were fixed with 4% paraformaldehyde. Detailed steps were conducted as per previous descriptions (Huang et al. 2020b).

Generation of BAT-specific KCTD10 knockdown and overexpression mice

Fabp4-dependent adeno-associated viral vector 9 (AAV9) for KCTD10 knockdown and overexpression were purchased from Hanbio Technology Corporation (Shanghai, China). Viruses were then diluted in sterile PBS and administrated at a dose of 1010–1011 vg per mice aged 2 months old via BAT in situ injection. One month after virus injection, the mice underwent metabolic phenotyping and were sacrificed for tissue collection and biochemical study.

Primary stromal vascular fractions isolation and differentiation of primary brown adipocytes

Stromal vascular fractions (SVFs) from BAT of 2 months old WT C57BL/6J mice were isolated and cultured as previously described (Yang et al. 2020). Briefly, the digested BAT was centrifuged at 100 g for 5 min to collect the SVFs. SVFs-containing preadipocytes were cultured in medium (DMEM with 10% FBS) and maintained at 37°C with 5% CO2 in a humidified atmosphere. Medium was changed every other day until the cells reached 100% confluence. Then, differentiation was induced in the cells by exposing them to a medium containing 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5 mM dexamethasone, 850 nM insulin, and 1 nM T3 for 2 days and maintaining the cells in a medium containing 850 nM insulin and 1 nM T3. After 7 days of differentiation, the adipocytes were collected for RNA or protein extraction.

Plasmid and siRNA transfection

The KCTD10 pcDNA3.1-HIS-C plasmid was purchased from YouBio Technology (Shanghai, China) Corporation and the KCTD10 siRNA was purchased from RiBoBio (Guangzhou, China) Biotechnology Corporation. The siRNA sequence is listed in Supplementary Table 1 (see section on supplementary materials given at the end of this article). Plasmid transfection in brown adipocytes was performed using Lipofectamine 2000 (Life Technologies, Thermo Fisher Scientific), according to the manufacturer’s instructions. Six hours later, the serum-free medium was changed with culture medium with 10% FBS for another 18 h. siRNA transfection in brown adipocytes was performed using Lipofectamine RNAiMAX according to the manufacturer’s instructions. Twelve hours later, the medium was changed with culture medium with 10% FBS for another 48 h. Functional validation was determined by Western blot (WB) and quantitative RT-PCR (qPCR).

Cold stress experiment and Cl316243 treatment

For cold stress, mice were individually housed in a cage at 4°C for 10 h every day and had free access to water and food for a week. Body temperature was examined by rectal probe. For Cl316243 treatment, mice were intraperitioneally injected with 1 mg/kg Cl316243 (C5976, Sigma-Aldrich) or equivalent volume of sterile saline every morning for a week.

Insulin tolerance test and glucose tolerance test

Insulin tolerance tests and glucose tolerance tests were conducted as described in our previous study (Liu et al. 2020).

Quantitative real-time PCR and Western blot analysis

qPCR and WB analysis were performed as previously described (Li et al. 2018, Yang et al. 2019). For qPCR, β-actin was used as an internal reference. For WB, α-tubulin was used as an internal reference. The sequences of primers are shown in Supplementary Table 1. Antibodies for WB were purchased from ABclonal (KCTD10; A11941, Wuhan, China), Cell Signaling Technology (UCP1, 14670s; FABP4, 2120s), Abcam (Notch; ab52627), and Proteintech (Alpha Tubulin; 11224-1-AP, Chicago, IL, USA).

Administration of Notch inhibitor

To inhibit Notch function, LY-411575 (S2714, Selleck Chemicals, Houston, TX, USA) dissolved in DMSO was used to treat primary brown adipocytes with different concentrations from 0.1 to 10 μM. A low concentration (1 μM) was found to significantly enhance UCP1 expression. The experiments involved in Notch signaling inhibition were performed by treating primary brown adipocytes with 1 μM LY411575 or DMSO for 24 h.

RNA sequencing

The SVFs from 2-month-old WT mice were transfected with Scramble or siRNA-KCTD10 for 24 h. Total cellular RNA was extracted and subjected to commercial RNA-seq in OE Biotech Co., Ltd. (Shanghai, China). P value <0.05 and fold change >1.2 or <0.83 was set as the threshold for significantly differential expression.

Oil red O staining

Oil red O staining was conducted as previously described (Wang et al. 2019). Briefly, the cells were fixed by 4% paraformaldehyde at room temperature for 30 min. Oil red O (Sigma-Aldrich) was dissolved at 0.5% in isopropanol, then diluted in water (3:2, v/v) to prepare working solution. The fixed cells were incubated in working solution at room temperature for 1 h. Images were acquired with a microscope for analysis. Then, the oil red O stained cells were solubilized in isopropanol to measure the optical density at 500 nm.

Statistical analysis

For cell study, data are representative of at least three independent experiments with a similar result. The WB images were quantified with the Image lab program. Data analysis was performed using GraphPad Prism 7.0. The statistical significance of the differences between various treatments or groups was measured by either Student’s t-test or ANOVA. Data are presented as mean ± s.e.m. *P < 0.05 and **P < 0.01 were considered as significant.

Results

KCTD10 expression is reduced by cold stress

KCTD10 was detected in BAT, iWAT, eWAT, and other tissues such as liver and colon in mice (Fig. 1A). The mRNA and protein level of KCTD10 were significantly higher in BAT than that in eWAT and iWAT (Fig. 1A and B), suggesting that KCTD10 may play a role in BAT function. To investigate whether KCTD10 expression is regulated during the activation of BAT, 12-week-old male mice were housed at 4 or 23°C for 7 days. We found that cold stress significantly decreased the mRNA and protein level of KCTD10 in BAT, iWAT, and eWAT, along with increased UCP1 expression in BAT and iWAT (Fig. 1C, D, E, and F). CL316243, a selective β3 adrenergic agonist, activates thermogenesis in BAT. Administration of CL316243 also suppressed Kctd10 mRNA level in fat tissues through intraperitoneally injecting 12-week-old male mice (C56BL/6 J) (Fig. 1G). In vitro, SVFs of BAT were isolated and brown adipocytes differentiation was induced (Supplementary Fig. 1A and B). The mRNA level of Kctd10 was gradually increased during differentiation as well as the expression of adipogenic genes such as Ucp1, Ppargc1α (Peroxisome proliferator-activated receptor gamma coactivator 1 alpha), Pparγ (Peroxisome proliferator-activated receptor), and Adipq (Adiponectin) (Fig. 1H). Thus, these results suggested that KCTD10 is involved in adipogenesis and adipose tissue function.

Figure 1
Figure 1

KCTD10 is highly expressed in BAT compared to WAT and decreases in cold stress and CL316243 treatment. (A) KCTD10 and UCP1 protein expression levels in the BAT, iWAT, eWAT, liver, muscle, and colon of 2-month-old male mice. (B) The mRNA expression of Kctd10 in BAT, iWAT, and eWAT of 2-month-old male mice. (C and D) The mRNA expression of Kctd10 and Ucp1 in BAT (C) and iWAT (D) of 2-month-old male mice after 1-week cold exposure. (E) The protein levels of UCP1 and KCTD10 in BAT, iWAT, and eWAT of mice after 1-week cold stress and quantification of specific protein bands relative to tubulin control using Image Lab software. (F) The mRNA level of Kctd10 in eWAT of mice after 1-week cold exposure by qPCR. (G) The mRNA expression level of KCTD10 in BAT, iWAT, and eWAT of 2-month-old male mice injected daily with saline or CL316243 for 1 week by qPCR. (H) The mRNA levels of Kctd10, Ucp1, Ppargc1α, Prdm16, Pparγ, and Adipq at days 0, 3, and 7 of brown adipocyte differentiation by qPCR.

Citation: Journal of Endocrinology 252, 3; 10.1530/JOE-21-0016

Knockdown of KCTD10 enhances UCP1 expression

To determine the role of KCTD10 in BAT function and adipogenesis, we performed RNA-Seq on BAT SVFs knocked down KCTD10 by siRNA interference. A total of 2570 differentially expressed genes were found, of which 1630 were downregulated and 940 were upregulated (Fig. 2A and B). As per our expectation, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis displayed that the pathways associated with thermogenesis such as PIK3-Akt signaling pathway and cAMP signaling pathway were enriched in KCTD10 knocked down cells (Fig. 2C and D). Consistent with the above results, RNA-seq analysis showed that thermogenic genes especially Ucp1 was increased (Fig. 2E). Moreover, KCTD10 silencing in primary brown adipocytes significantly upregulated the mRNA and protein level of UCP1 (Fig. 2F and H). By contrast, two adipogenic markers, Fabp4 (fatty acid-binding protein) and Adipq, were dramatically suppressed by KCTD10 deficiency as well as WAT-selective genes including Trim14 (tripartite motif-containing 14) and Agt (angiotensinogen), though there was no significant change in the expression of Prdm16, Dio2 (deiodinase, iodothyronine, type II), Cox7α (cytochrome c oxidase subunit 7α), Pparγ, and Pparα (Fig. 2E and F).

Figure 2
Figure 2

In primary brown adipocytes, knockdown of KCTD10 increases UCP1 and decreases adipogenic gene expression. Overexpression of KCTD10 reduces UCP1 and induces adipogenic gene expression. (A and B) Volcano plot of RNA-seq data in SVFs transfected with scrambled or siKCTD10 and its quantification. (C and D) Top 20 KEGG and GO-enrichment signaling pathways. (E) Heatmap of RNA-seq data showed thermogenic, WAT-selective, inflammatory, and adipogenic gene expression in cells described in A. (F) qPCR analysis of thermogenic, WAT-selective, and adipogenic gene expression in SVFs transfected with scrambled siRNA or siKCTD10 after 7 days of adipogenic differentiation. (G) qPCR analysis of thermogenic, WAT-selective, and adipogenesis gene expression in SVFs transfected with empty or KCTD10 plasmid after 7 days of adipogenic differentiation. (H) Western blot analysis of KCTD10, UCP1, and Prdm16 in SVFs transfected with scrambled siRNA or siKCTD10 after 7 days of adipogenic differentiation. (I) Western blot analysis of KCTD10, UCP1, and PRDM16 expression in SVFs transfected with empty or KCTD10 plasmid after 7 days of adipogenic differentiation.

Citation: Journal of Endocrinology 252, 3; 10.1530/JOE-21-0016

Overexpression of the KCTD10 gene in mature adipocytes-attenuated thermogenesis

Having established that knockdown of KCTD10 induces UCP1 expression, we wonder if overexpression of KCTD10 exerts a suppressive effect on thermogenic gene expression. In mature brown adipocytes, enhanced KCTD10 expression reduced UCP1 expression both in the protein and mRNA levels (Fig. 2G and I), suggesting that KCTD10 inhibits thermogenesis. In the meanwhile, the expression of Fabp4, Adipq, Trim14, and Agt were significantly induced in KCTD10 overexpressed adipocytes, without significant changes in Prdm16, Dio2, Cox7α, Pparγ, and Pparα (Fig. 2G and I).

BAT-specific knockdown of KCTD10 induces thermogenesis and protects mice from diet-induced obesity and insulin resistance

Lower BAT activity has been shown in obese mice (Liu et al. 2014). Here, we found that KCTD10 was highly expressed in the BAT of HFD-induced obese mice and db/db mice in comparison with their respective control mice (Fig. 3A, B, C, and D). Since KCTD10 knockdown in primary brown adipocytes affected the expression of genes in thermogenesis, we next investigated the potential link between KCTD10 and thermogenesis in vivo. To test this, BAT-specific KCTD10 knockdown mice (designated as BATK10KD) and control mice were generated by locally BAT injection with adeno-associated viral vectors, which carry FABP4 promoter-driven small hairpin RNAs targeting KCTD10 (AAV9-shKCTD10) or negative control (AAV9-shNC) with a Green fluorescent protein (GFP) reporter gene (Supplementary Fig. 1C). The green fluorescence could be found in BAT of BATK10KD mice (Supplementary Fig. 1D). Compared to the control mice, Kctd10 mRNA expression was markedly reduced (Fig. 3E), while the mRNA and protein levels of UCP1 were significantly induced in BAT of BATK10KD mice (Fig. 3E and F). However, no significant difference was found in the expression levels of Ppargc1α and Prdm16 between control and BATK10KD mice (Fig. 3E). In line with in vitro results, the protein level of FABP4 and mRNA level of Agt were decreased in BATK10KD mice (Fig. 3E and F). Furthermore, BATK10KD mice were able to resist acute cold stress in agreement with induced UCP1 level (Fig. 3G). In consistence with it, BATK10KD mice displayed higher body surface temperature compared with control mice (Fig. 3H), which is supported by increased UCP1 levels in their BAT (Fig. 3E and F). Next, we detected the role of KCTD10 in diet-induced obesity. Control and BATK10KD mice aged 8 weeks were fed with SD or HFD for 2 months. Although there was no significant difference in body weight between BATK10KD mice and controls fed with SD (Fig. 3I), BATK10KD mice were more tolerant of glucose and insulin challenge compared with control mice (Supplementary Fig. 1E and F). Under HFD-fed conditions, BATK10KD mice had clearly reduced body weight compared with controls, with no significant difference in food intake (Fig. 3I and Supplementary Fig. 1G). Consistently, HFD-fed BATK10KD mice showed lower body weight and fat mass, smaller adipocyte size, alleviated hepatosteatosis, and improved glucose tolerance and insulin sensitivity when compared with HFD-fed control mice (Fig. 3I, J, K, L, M, N and Supplementary Fig. 1H, I). Taken together, we determined that KCTD10 knockdown in BAT induces brown adipocytes thermogenesis and improves whole-body energy homeostasis.

Figure 3
Figure 3

BAT-special KCTD10 knockdown mice exhibits induced thermogenesis and resists HFD-induced obesity. (A and B) Western blot analysis of KCTD10 and UCP1 expression in BAT of HFD-induced mice (A) and db/db mice (B) compared to control mice, respectively. (C and D) qPCR analysis of Kctd10 and Ucp1 expression in BAT of HFD-induced mice (C) and db/db mice (D). (E) qPCR analysis of Kctd10, Ucp1, Prdm16, Ppargc1α, and Agt expression in BAT of mice injected with AAV-shKCTD10 (KD, n = 6) or AAV-Ctrl (Ctrl, n = 4). (F) Western blot analysis of FABP4 and UCP1 expression in BAT of mice described in E. (G) Rectal temperature of BATK10KD mice and their controls exposed to cold condition for 6 h; control, n = 4; KD, n = 6. (H) Body surface temperature measured with infrared thermometer of BATK10KD mice and their controls. (I) Body weight of control and BATK10KD mice during 2 months of SD or HFD feeding. (J) Gross morphology of BATK10KD mice and their controls after 2 months HFD feeding. (K) A representative image of BAT, iWAT, eWAT, and liver tissue of BATK10KD mice and their controls after 2 months of HFD. (L) A representative H&E staining of iWAT, eWAT of BATK10KD mice and their controls after 2 months HFD feeding, along with quantification of adipocyte diameter by the NIH ImageJ, scale bar: 100 μm. (M) A representative H&E staining of BAT, liver of BATK10KD mice, and their controls after 2 months HFD, scale bar, BAT: 100 μm; liver: 200 μm. (N) ITT and GTT were performed on BATK10KD mice and their controls after 2-month HFD feeding; control, n = 6; BATK10KD, n = 6.

Citation: Journal of Endocrinology 252, 3; 10.1530/JOE-21-0016

BAT-specific overexpression of KCTD10 reduces energy expenditure and predisposes mice to HFD-induced obesity

To further illustrate the regulatory role of KCTD10 in thermogenesis, we generated BAT-specific overexpression of KCTD10 (BATK10OE) by injecting BAT of mice aged 2 months with AAV9 vector carrying Fabp4-dependent KCTD10 gene. The efficiency of KCTD10 expression in BATK10OE mice was determined by qPCR analysis (Fig. 4A). Expectedly, the mRNA and protein expression levels of UCP1 were downregulated in BATK10OE mice compared with control mice (Fig. 4A and B). Besides, overexpression of KCTD10 induced the expression of adipogenic genes FABP4 and Agt, with little changes in the mRNA levels of Prdm16, Pparα, and Pparγ (Fig. 4A and B). Cold stress significantly induced UCP1 expression in both BATs of control and KCTD10OE mice (Fig. 4C and D). Whereas, the response to cold stress was markedly attenuated in BAT of BATK10OE mice, suggesting that KCTD10 suppressed cold-induced thermogenesis (Fig. 4C and D). In support of this, we found reduced body surface temperature in BATK10OE mice (Fig. 4E). The cold tolerance was mildly but significantly reduced in BATK10OE mice compared with controls fed with SD (Fig. 4F). However, this discrepancy was becoming more pronounced during 2-month HFD feeding (Fig. 4G). Then, we tested if overexpression of KCTD10 predisposes mice to diet-induced obesity. On a SD, the body weight did not show a clear difference between male and female BATK10OE mice and control mice (Supplementary Fig. 1J and K). No significant changes were found in glucose tolerance and insulin sensitivity between BATK10OE mice and control mice (Supplementary Fig. 1O and P). However, the body weight of BATK10OE mice was significantly greater than that of control mice when fed a HFD without affecting food intake (Fig. 4H and Supplementary Fig. 1L). As expected, the body weight, fat mass, fat cell size, and degree of hepatic steatosis of HFD-fed BATK10OE mice were increased compared with HFD-fed controls (Fig. 4I, J, K, L and Supplementary Fig. 1M, N). Consistently, impaired glucose tolerance and insulin sensitivity were detected in BATK10OE mice in comparison with controls under HFD feeding condition (Fig. 4M).

Figure 4
Figure 4

BAT-special KCTD10 overexpression mice exhibits reduced thermogenesis and predisposes mice to HFD-induced obesity. (A) qPCR analysis of Kctd10, Ucp1, Prdm16, Pparα, Pparγ, and Agt expression in BAT of mice injected with AAV-Control (Ctrl, n = 5) or AAV-Kctd10 (OE, n = 5). (B) Western blot analysis of FABP4 and UCP1 expression in BAT of mice described in A. (C and D) Cold stress-induced UCP1 expression in BAT of BATK10OE mice and their controls by qPCR (C) and Western blot (D). (E) Body surface temperature measured with infrared thermometer of BATK10OE mice and their controls. (F and G) Rectal temperature of BATK10OE mice and their controls exposed to cold condition for 6 h after 2 months of SD (F) and HFD (G) feeding. (H) Body weight of BATK10OE mice and their controls during 2 months of HFD feeding; control, n = 5; OE, n = 5. (I) Gross morphology of BATK10OE mice and their controls after 2 months of HFD feeding. (J) A representative image of BAT, iWAT, eWAT, and liver tissue of BATK10OE mice and their controls after 2 months of HFD. (K) A representative H&E staining of iWAT, eWAT of BATK10OE mice and their controls after 2 months HFD, along with quantification of adipocyte diameter by the NIH ImageJ, scale bar: 100 μm. (L) A representative H&E staining of BAT and liver of BATK10OE mice and their controls after 2 months of HFD, scale bar: 100 μm. (M) ITT and GTT were performed on BATK10OE mice and their controls after 2-month HFD feeding; control, n = 5; OE, n = 5.

Citation: Journal of Endocrinology 252, 3; 10.1530/JOE-21-0016

KCTD10 reduces thermogenic gene expression via the Notch signaling pathway in primary brown adipocytes

Previous studies showed that KCTD10 had involved in the regulation of Notch signaling pathway (Ren et al. 2014, Pang et al. 2019). In addition, our RNA sequencing analysis also displayed that Notch signaling was inhibited when knocking down KCTD10 in primary brown adipocytes (Fig. 5A). In line with this, knockdown of KCTD10 suppressed the mRNA levels of Notch1, Notch3, and Hey1 (Fig. 5B). Notch1 protein level was also decreased in KCTD10-silenced brown adipocytes (Fig. 5C). In contrast, forced expression of KCTD10 induced Notch1 protein expression (Fig. 5D). Interestingly, decreased Notch1 mRNA in BAT was observed upon cold exposure for 7 days (Fig. 5E). LY411575, a γ-secretase inhibitor to indirectly inhibit Notch pathway, was used to treat primary brown adipocyte with different concentrations. The results showed that LY411575 treatment suppressed the mRNA levels of Notch1, Notch3, Hes1, and Hey1, while significantly increased Ucp1 expression in a dose-dependent manner in primary brown adipocytes (Fig. 5F), suggesting that Notch signaling suppressed thermogenesis in BAT of mice. Then, we wondered whether impaired thermogenesis caused by KCTD10 overexpression was Notch signaling dependent. Treating brown adipocytes with LY411575 clearly restored KCTD10-suppressed thermogenic gene UCP1 expression (Fig. 5G). Taken together, these results indicated that KCTD10-suppressed thermogenesis is mediated by the activation of Notch signaling pathway.

Figure 5
Figure 5

KCTD10 reduces UCP1 expression via Notch-dependent signaling pathway in primary brown adipocytes. (A) Heatmap of RNA-seq data of Kctd10, Notch1, Notch3, and Hey1 mRNA expression in SVFs transfected with scrambled siRNA or siKCTD10. (B) qPCR analysis of Notch1, Notch3, Hes1, and Hey1 expression in primary brown adipocyte transfected with scrambled siRNA or siKCTD10 after 7 days of adipogenic differentiation. (C) Western blot analysis of Notch1 expression in primary brown adipocytes transfected with scrambled siRNA or siKCTD10 after 7 days of adipogenic differentiation. (D) Western blot analysis of Notch1 expression in primary brown adipocytes transfected with empty or KCTD10 plasmid after 7 days of adipogenic differentiation. (E) The Notch1 mRNA level of BAT in WT mice after 1-week cold exposure. (F) qPCR analysis of Notch1, Notch3, Hes1, Hey1, and Ucp1 expression in primary brown adipocytes treated with LY411575 as indicated concentration. (G) The protein levels of UCP1 in primary brown adipocytes transfected with empty or KCTD10 plasmid with or without 1μM LY411575 treatment. (H) A proposed model of how KCTD10 regulates thermogenesis.

Citation: Journal of Endocrinology 252, 3; 10.1530/JOE-21-0016

Discussion

Shivering is the first step to cope with cold stress and then non-shivering thermogenesis is enhanced when skeletal muscle becomes fatigue (Lee et al. 2014, Knuth et al. 2018). Non-shivering thermogenesis is classically executed by UCP1 activation (Chouchani et al. 2019). In the present study, we first demonstrated that KCTD10 was highly expressed in BAT in comparison with iWAT and eWAT of mice. Accordingly, we speculated that KCTD10 plays a certain role in BAT function. BAT thermogenesis is activated by cold stress to maintain body temperature (Leiria et al. 2019, Krisko et al. 2020). KCTD10 was decreased in fat tissue upon cold exposure and CL316243 treatment. These findings encourage us to investigate the relationship between KCTD10 and thermogenesis. Furthermore, knockdown of KCTD10 increased UCP1 expression in primary brown adipocytes. RNA-seq analysis also displayed that some thermogenic genes were upregulated in KCTD10 deficient SVFs. Most of the classical thermogenic genes were not differentially expressed, considering that we did not induce the SVFs used in RNA-Seq into mature brown adipocytes. KCTD10 was induced in BAT of HFD-induced obese mice and db/db mice. Therefore, we asked if KCTD10 predisposes mice to diet-induced obesity. We demonstrated that BAT-specific knockdown of KCTD10 led to body weight loss in mouse during HFD feeding. Although there was no significant difference in body weight during SD feeding, we found that insulin and glucose tolerances were improved in KCTD10 knockdown mice, indicating that inhibiting KCTD10 expression in BAT is beneficial to whole-body metabolism. In consistence with the important role of KCTD10 in the regulation of UCP1 expression. BATK10KD mice were protected from hypothermia following cold exposure. In contrast, BAT-specific overexpression of KCTD10 decreased UCP1 expression in BAT, reduced core body temperature upon acute cold stress and increased body weight of mice fed HFD. Furthermore, we also found that KCTD10 plays an essential role in regulating Fabp4 expression. The expressions of Fabp4 and Agt were induced in BATK10OE mice, suggesting that KCTD10 promotes WAT-selective genes expression. Though the energy metabolism of BATK10OE mice was impaired compared with controls on HFD, we did not completely exclude the effect of adipogenesis on the body weight gain of BATK10OE mice.

Notch signaling is crucial in the occurrence and development of tumor (Bensard et al. 2020, Ludikhuize et al. 2020). However, its role in adipose tissue remains to be further explored. Based on previous studies, suppression of Notch signaling was capable of promoting the browning of iWAT (Bi et al. 2014). However, the similar effects of Notch signaling on BAT were not found. Suppression of Notch signaling has been reported to reduce thermogenesis in the brown adipocytes (Meng et al. 2019). Instead, we found that inhibiting Notch signaling induced thermogenic genes expression in primary brown adipocyte, which is in agreement with a recent study that Notch inhibitor promoted beige adipogenesis and thermogenesis though it occurred in inguinal white adipocytes (Huang et al. 2020a). Notch signaling impacts cellular activity in variable manners depending on the signal and the cellular environment, suggesting a possible mechanism of Notch signaling regulated thermogenesis (Aster et al. 2017). Further investigation was required to reveal the role of Notch signaling in adipose tissue. Moreover, we found that the impaired thermogenesis caused by KCTD10 overexpression is Notch signaling dependent, revealing that KCTD10 serves as an upstream regulator of Notch signaling in the regulation of BAT thermogenesis.

What is the mechanism by which KCTD10 regulates Notch signaling? KCTD10 has been shown to play a negative role in regulating Notch signaling by recruiting proteins for ubiquitination and mediating subsequent degradation of the substrates in human umbilical vessel endothelial cells (Ren et al. 2014). However, we found that in BAT, KCTD10 is able to activate Notch signaling, suggesting that KCTD10 regulates Notch signaling via a ubiquitination-independent mechanism in brown adipocytes. The molecular mechanisms underlying the regulation of KCTD10 in Notch signaling remain under further investigation.

How Notch signaling regulates UCP1 expression in brown adipocytes remains unknown. It is well established that transcriptional co-repressor Hes1 directly binds to the promoter regions of Ppargc1α and Prdm16 (Bi & Kuang 2015). In our study, we found that KCTD10 knockdown significantly decreased hey1 expression but had little effect on hes1 expression. That may partially explain the results that KCTD10 has little effect on Ppargc1α and Prdm16 expression. Thus, it is possible that hey1 is a critical target for KCTD10 to activate Notch signaling and consequently regulate UCP1 expression, which needs to be further investigated.

In summary, we identified KCTD10 was an essential factor in thermogenesis and adipogenesis in BAT. KCTD10 deficiency in BAT induced UCP1 expression and prevented diet-induced obesity in a Notch signaling dependent manner. Further research will be required to explore the mechanisms of KCTD10 in brown adipogenesis.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/JOE-21-0016.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by National Natural Science Foundation of China (Grants 81700785, 81873643, 81900732, 81930022, 82000811).

Acknowledgements

The authors appreciate Prof Xiang-hang Luo for his good advice and guidance.

References

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    Figure 1

    KCTD10 is highly expressed in BAT compared to WAT and decreases in cold stress and CL316243 treatment. (A) KCTD10 and UCP1 protein expression levels in the BAT, iWAT, eWAT, liver, muscle, and colon of 2-month-old male mice. (B) The mRNA expression of Kctd10 in BAT, iWAT, and eWAT of 2-month-old male mice. (C and D) The mRNA expression of Kctd10 and Ucp1 in BAT (C) and iWAT (D) of 2-month-old male mice after 1-week cold exposure. (E) The protein levels of UCP1 and KCTD10 in BAT, iWAT, and eWAT of mice after 1-week cold stress and quantification of specific protein bands relative to tubulin control using Image Lab software. (F) The mRNA level of Kctd10 in eWAT of mice after 1-week cold exposure by qPCR. (G) The mRNA expression level of KCTD10 in BAT, iWAT, and eWAT of 2-month-old male mice injected daily with saline or CL316243 for 1 week by qPCR. (H) The mRNA levels of Kctd10, Ucp1, Ppargc1α, Prdm16, Pparγ, and Adipq at days 0, 3, and 7 of brown adipocyte differentiation by qPCR.

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    Figure 2

    In primary brown adipocytes, knockdown of KCTD10 increases UCP1 and decreases adipogenic gene expression. Overexpression of KCTD10 reduces UCP1 and induces adipogenic gene expression. (A and B) Volcano plot of RNA-seq data in SVFs transfected with scrambled or siKCTD10 and its quantification. (C and D) Top 20 KEGG and GO-enrichment signaling pathways. (E) Heatmap of RNA-seq data showed thermogenic, WAT-selective, inflammatory, and adipogenic gene expression in cells described in A. (F) qPCR analysis of thermogenic, WAT-selective, and adipogenic gene expression in SVFs transfected with scrambled siRNA or siKCTD10 after 7 days of adipogenic differentiation. (G) qPCR analysis of thermogenic, WAT-selective, and adipogenesis gene expression in SVFs transfected with empty or KCTD10 plasmid after 7 days of adipogenic differentiation. (H) Western blot analysis of KCTD10, UCP1, and Prdm16 in SVFs transfected with scrambled siRNA or siKCTD10 after 7 days of adipogenic differentiation. (I) Western blot analysis of KCTD10, UCP1, and PRDM16 expression in SVFs transfected with empty or KCTD10 plasmid after 7 days of adipogenic differentiation.

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    Figure 3

    BAT-special KCTD10 knockdown mice exhibits induced thermogenesis and resists HFD-induced obesity. (A and B) Western blot analysis of KCTD10 and UCP1 expression in BAT of HFD-induced mice (A) and db/db mice (B) compared to control mice, respectively. (C and D) qPCR analysis of Kctd10 and Ucp1 expression in BAT of HFD-induced mice (C) and db/db mice (D). (E) qPCR analysis of Kctd10, Ucp1, Prdm16, Ppargc1α, and Agt expression in BAT of mice injected with AAV-shKCTD10 (KD, n = 6) or AAV-Ctrl (Ctrl, n = 4). (F) Western blot analysis of FABP4 and UCP1 expression in BAT of mice described in E. (G) Rectal temperature of BATK10KD mice and their controls exposed to cold condition for 6 h; control, n = 4; KD, n = 6. (H) Body surface temperature measured with infrared thermometer of BATK10KD mice and their controls. (I) Body weight of control and BATK10KD mice during 2 months of SD or HFD feeding. (J) Gross morphology of BATK10KD mice and their controls after 2 months HFD feeding. (K) A representative image of BAT, iWAT, eWAT, and liver tissue of BATK10KD mice and their controls after 2 months of HFD. (L) A representative H&E staining of iWAT, eWAT of BATK10KD mice and their controls after 2 months HFD feeding, along with quantification of adipocyte diameter by the NIH ImageJ, scale bar: 100 μm. (M) A representative H&E staining of BAT, liver of BATK10KD mice, and their controls after 2 months HFD, scale bar, BAT: 100 μm; liver: 200 μm. (N) ITT and GTT were performed on BATK10KD mice and their controls after 2-month HFD feeding; control, n = 6; BATK10KD, n = 6.

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    Figure 4

    BAT-special KCTD10 overexpression mice exhibits reduced thermogenesis and predisposes mice to HFD-induced obesity. (A) qPCR analysis of Kctd10, Ucp1, Prdm16, Pparα, Pparγ, and Agt expression in BAT of mice injected with AAV-Control (Ctrl, n = 5) or AAV-Kctd10 (OE, n = 5). (B) Western blot analysis of FABP4 and UCP1 expression in BAT of mice described in A. (C and D) Cold stress-induced UCP1 expression in BAT of BATK10OE mice and their controls by qPCR (C) and Western blot (D). (E) Body surface temperature measured with infrared thermometer of BATK10OE mice and their controls. (F and G) Rectal temperature of BATK10OE mice and their controls exposed to cold condition for 6 h after 2 months of SD (F) and HFD (G) feeding. (H) Body weight of BATK10OE mice and their controls during 2 months of HFD feeding; control, n = 5; OE, n = 5. (I) Gross morphology of BATK10OE mice and their controls after 2 months of HFD feeding. (J) A representative image of BAT, iWAT, eWAT, and liver tissue of BATK10OE mice and their controls after 2 months of HFD. (K) A representative H&E staining of iWAT, eWAT of BATK10OE mice and their controls after 2 months HFD, along with quantification of adipocyte diameter by the NIH ImageJ, scale bar: 100 μm. (L) A representative H&E staining of BAT and liver of BATK10OE mice and their controls after 2 months of HFD, scale bar: 100 μm. (M) ITT and GTT were performed on BATK10OE mice and their controls after 2-month HFD feeding; control, n = 5; OE, n = 5.

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    Figure 5

    KCTD10 reduces UCP1 expression via Notch-dependent signaling pathway in primary brown adipocytes. (A) Heatmap of RNA-seq data of Kctd10, Notch1, Notch3, and Hey1 mRNA expression in SVFs transfected with scrambled siRNA or siKCTD10. (B) qPCR analysis of Notch1, Notch3, Hes1, and Hey1 expression in primary brown adipocyte transfected with scrambled siRNA or siKCTD10 after 7 days of adipogenic differentiation. (C) Western blot analysis of Notch1 expression in primary brown adipocytes transfected with scrambled siRNA or siKCTD10 after 7 days of adipogenic differentiation. (D) Western blot analysis of Notch1 expression in primary brown adipocytes transfected with empty or KCTD10 plasmid after 7 days of adipogenic differentiation. (E) The Notch1 mRNA level of BAT in WT mice after 1-week cold exposure. (F) qPCR analysis of Notch1, Notch3, Hes1, Hey1, and Ucp1 expression in primary brown adipocytes treated with LY411575 as indicated concentration. (G) The protein levels of UCP1 in primary brown adipocytes transfected with empty or KCTD10 plasmid with or without 1μM LY411575 treatment. (H) A proposed model of how KCTD10 regulates thermogenesis.

  • Aster JC, Pear WS & Blacklow SC 2017 The varied roles of Notch in cancer. Annual Review of Pathology 12 245275. (https://doi.org/10.1146/annurev-pathol-052016-100127)

    • Search Google Scholar
    • Export Citation
  • Balaz M, Becker AS, Balazova L, Straub L, Müller J, Gashi G, Maushart CI, Sun W, Dong H & Moser C et al.2019 Inhibition of mevalonate pathway prevents adipocyte browning in mice and men by affecting protein prenylation. Cell Metabolism 29 901.e8916.e8. (https://doi.org/10.1016/j.cmet.2018.11.017)

    • Search Google Scholar
    • Export Citation
  • Bartolome A, Zhu C, Sussel L & Pajvani UB 2019 Notch signaling dynamically regulates adult β cell proliferation and maturity. Journal of Clinical Investigation 129 268280. (https://doi.org/10.1172/JCI98098)

    • Search Google Scholar
    • Export Citation
  • Bensard CL, Wisidagama DR, Olson KA, Berg JA, Krah NM, Schell JC, Nowinski SM, Fogarty S, Bott AJ & Wei P et al.2020 Regulation of tumor initiation by the mitochondrial pyruvate carrier. Cell Metabolism 31 284 .e7300.e7. (https://doi.org/10.1016/j.cmet.2019.11.002)

    • Search Google Scholar
    • Export Citation
  • Bi P & Kuang S 2015 Notch signaling as a novel regulator of metabolism. Trends in Endocrinology and Metabolism 26 248255. (https://doi.org/10.1016/j.tem.2015.02.006)

    • Search Google Scholar
    • Export Citation
  • Bi P, Shan T, Liu W, Yue F, Yang X, Liang XR, Wang J, Li J, Carlesso N & Liu X et al.2014 Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity. Nature Medicine 20 911918. (https://doi.org/10.1038/nm.3615)

    • Search Google Scholar
    • Export Citation
  • Blondin DP, Nielsen S, Kuipers EN, Severinsen MC, Jensen VH, Miard S, Jespersen NZ, Kooijman S, Boon MR & Fortin M et al.2020 Human brown adipocyte thermogenesis is driven by β2-AR stimulation. Cell Metabolism 32 287 .e7300.e7. (https://doi.org/10.1016/j.cmet.2020.07.005)

    • Search Google Scholar
    • Export Citation
  • Brestoff JR, Wilen CB, Moley JR, Li Y, Zou W, Malvin NP, Rowen MN, Saunders BT, Ma H & Mack MR et al.2021 Intercellular mitochondria transfer to macrophages regulates white adipose tissue homeostasis and is impaired in obesity. Cell Metabolism 33 270.e8282.e8. (https://doi.org/10.1016/j.cmet.2020.11.008)

    • Search Google Scholar
    • Export Citation
  • Cereijo R, Gavaldà NA, Cairó M, Quesada-López T, Villarroya J, Morón-Ros S, Sánchez-Infantes D, Peyrou M, Iglesias R & Mampel T et al.2018 CXCL14, a brown adipokine that mediates brown-fat-to-macrophage communication in thermogenic adaptation. Cell Metabolism 28 750.e6763.e6. (https://doi.org/10.1016/j.cmet.2018.07.015)

    • Search Google Scholar
    • Export Citation
  • Chen S, Liu X, Peng C, Tan C, Sun H, Liu H, Zhang Y, Wu P, Cui C & Liu C et al.2021 The phytochemical hyperforin triggers thermogenesis in adipose tissue via a Dlat-AMPK signaling axis to curb obesity. Cell Metabolism 33 565 .e7580.e7. (https://doi.org/10.1016/j.cmet.2021.02.007)

    • Search Google Scholar
    • Export Citation
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