HDAC6 regulates thermogenesis of brown adipocytes ...

Biochemical and Biophysical Research Communications xxx (2018) 1e6

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HDAC6 regulates thermogenesis of brown adipocytes through activating PKA to induce UCP1 expression Suna Jung a, b, 1, Miae Han a, 1, Sovannarith Korm a, Se-in Lee a, Solhee Noh a, Sophors Phorl a, Rema Naskar a, Kye-Sung Lee c, Geon-Hee Kim a, c, Yun-Jaie Choi b, *, Joo-Yong Lee a, c, ** a

Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon, 305-764, Republic of Korea Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea c Division of Scientific Instrumentation, Korea Basic Science Institute, 169-148 Gwahak-ro, Yuseong-gu, Daejeon, 34133, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 May 2018 Accepted 7 June 2018 Available online xxx

Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). UCP1 increases the conductance of the inner mitochondrial membrane (IMM) for protons to make BAT mitochondria generate heat rather than ATP. HDAC6 is a cytosolic deacetylase for non-histone substrates to regulate various cellular processes, including mitochondrial quality control and dynamics. Here, we showed that the body temperature of HDAC6 knockout mice is slightly decreased in normal hosing condition. Interestingly, UCP1 was downregulated in BAT of HDAC6 knockout mice, which extensively linked mitochondrial thermogenesis. Mechanistically, we showed that cAMP-PKA signaling plays a key role in HDAC6-dependent UCP1 expression. Notably, the size of brown adipocytes and lipid droplets in HDAC6 knockout BAT is increased. Taken together, our findings suggested that HDAC6 contributes to mitochondrial thermogenesis in BAT by increasing UCP1 expression through cAMP-PKA signaling pathway. © 2018 Published by Elsevier Inc.

Keywords: Histone deacetylase 6 (HDAC6) Uncoupling protein 1 (UCP1) Mitochondrial thermogenesis Brown adipose tissue (BAT) cAMP PKA

1. Introduction The balance between energy intake and expenditure is important to keep energy homeostasis preserving a ‘set-point’ body weight. After that, metabolic rate, physical activity, and thermogenesis play important roles [1]. Among them, thermogenesis is the cellular dissipation of energy through heat production. This process has been extensively studied in brown adipose tissue (BAT). Unlike white adipocytes, brown adipocytes store lipids in multiple small droplets and contain many mitochondria with uncoupling protein 1 (UCP1) in the inner mitochondrial membrane. When activated, UCP1 creates a proton leak across the inner mitochondrial

* Corresponding author. Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea. ** Corresponding author. Chungnam National University, 99 Daehak-ro (St), Yusoeng-gu E2, Daejeon, 305-764, Republic of Korea. E-mail addresses: [email protected] (Y.-J. Choi), [email protected] (J.-Y. Lee). 1 These authors contributed equally to this paper.

membrane, diverting protons away from ATP synthase, thereby converting energy stored in the form of triglycerides into heat [2,3]. UCP1 gene transcription is largely controlled by noradrenaline released from the sympathetic terminals innervating the tissue in response to cold stress, acting through a b3-adrenergic receptor on the surface of brown adipocytes [3,4]. Stimulation of b3-adrenergic receptors by noradrenaline is transduced via Gs proteins and the cAMP-PKA signaling pathway, resulting in the transcriptional activation of UCP1 as well as lipolysis of intracellular triglyceride that provide free fatty acids as fuel for the electron-transport chain in mitochondria [3]. In addition, long-chain fatty acids bind UCP1 and increase its proton conductance, resulting in efficient thermogenesis of BAT [5]. HDAC6 belongs to class IIb HDACs [6], and is located mainly in cytoplasm [7] and deacetylates various cytoplasmic substrates, such as tubulin [8], Hsp90 [9,10], cortactin [11,12], and MFN1 [13]. Previously, we and others showed that HDAC6 regulates the cellular quality-control system for cytotoxic protein aggregates as well as damaged mitochondria through autophagy [11,14e16]. Under metabolic stress, such as glucose starvation and hypoxia, HDAC6

https://doi.org/10.1016/j.bbrc.2018.06.016 0006-291X/© 2018 Published by Elsevier Inc.

Please cite this article in press as: S. Jung, et al., HDAC6 regulates thermogenesis of brown adipocytes through activating PKA to induce UCP1 expression, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.016

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S. Jung et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e6

also regulates mitochondrial fusion, resulting in metabolic adaptation to stresses [13,17]. Accumulating evidences suggested that HDAC6 deficiency leads to mitochondrial dysfunction represented by reduced mitochondrial oxygen consumption [13,17]. However, it remains elusive how HDAC6 regulates mitochondiral energy metabolism. In this study, we showed that thermogenesis of BAT decreased in HDAC6 knockout (KO) mice because of the downregulated UCP1 expression in BAT. Mechanistically, we showed cAMP-PKA signaling is responsible for the decrease of UCP1 expression in brown adipocytes, which leads to lipid accumulation in BAT.

2.6. Preparation of tissue protein samples and Western blotting Tissues were homogenized in a cold PBS containing protease inhibitors and phosphatase inhibitors with zirconium oxide beads. The homogenates were exposed to a detergent solution (1% NP40 and 0.1% SDS) and centrifuged at 15,000 rpm for 15 min at 4 C. Then protein concentration was quantified using a BCA assay. Tissue extracts were subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk and incubated with primary antibodies at 4 C UCP1 (sc-6528, sc6529), HDAC6 (CST-7612), GAPDH (sc-32233), pHSL (CST-4126), pPLN (CST-4855), and PGC1 (sc-13067).

2. Material and methods 2.7. Primary brown adipocyte culture 2.1. Animals Wild-type and HDAC6 KO mice were housed in a pathogen-free mouse facility of Chungnam National University. Mice was maintained at ambient temperature with free access to food and water, and with a normal light/dark cycle. All procedures were approved by the Institutional Animal Care and Use Committee (CNU-00540). 2.2. Body temperature measurement Rectal temperature was measured using a digital thermometer with a mouse rectal-temperature probe (WPI Inc.). Skin temperature surrounding interscapular BAT depot was recorded with an infrared camera (ImageIR 8300, InfraTec, Inc.) after shaving hair in those areas. The skin temperature surrounding BAT for each animal was calculated as the average temperature recorded by analyzing those pictures. 2.3. Mitochondrial respiration assay Oxygen consumption rate (OCR) in MEF cells was measured using Seahorse Extracellular Flux (XF) 24 Analyzer and Seahorse XF Cell Mito Stress Test kit (Seahorse Bioscience). Mouse embryonic fibroblasts (MEFs) that were isolated from HDAC WT and KO embryos were cultured with DMEM supplemented with 10% fetal bovine serum. After establishing a baseline, glucose (10 mM), oligomycin (1 mM), FCCP (0.5 mM), and antimycin A (1 mM) were sequentially added, and OCR was analyzed by XF24 Flux Analyzer. The proton leak was calculated using the difference between the means of OCR in oligomycin treatment and those in antimycin A.

Brown adipocytes were isolated from newborn HDAC6 WT and KO interscapular BAT and cultured as described [19e22]. In brief, tissues were dissected and minced, followed by collagenase digestion for 30 min at 37 C in isolation buffer (123 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 100 Mm HEPES, and 4% BSA). Digested tissues were filtered with strainers and centrifuged at 700 g for 5 min. Cells were cultured with growth medium DMEM containing 10% calf serum, 4 nM insulin, 20 mM HEPES, and 25 mg/ ml sodium ascorbate. Cells were treated with forskolin for 24 h. 2.8. H&E staining and TEM analysis Interscapular BAT samples were isolated and fixed with formalin overnight, embedded in paraffin. The paraffin-embedded tissue was sectioned into 5-mm thick slices and stained with hematoxylin and eosin. For electron microscopy, BAT was isolated and fixed with 2.5 M glutaldehyde solution. Sample preparation and image acquisition were performed at the Korean Basic Science Institute TEM facility. 2.9. Statistical analysis All data are expressed as means ± S.D. Statistical significance between groups was assessed using Student's t-test or analysis of variance. A p < 0.05 was considered to be statistically significant (*: p < 0.05, **: p < 0.01 and ***: p < 0.001). 3. Results 3.1. Thermogenesis in BAT is reduced in HDAC6 KO

2.4. cAMP assay Intracellular cAMP level was measured using the cAMP-Glo™ Assay (Promega) as described previously [18]. In brief, cells are treated with a test compound for induction and lysed to release cAMP. Next, a cAMP detection solution that consists of PKA and Kinase-Glo Substrate is added. PKA reaction is completed by adding the Kinase-Glo Reagent. The remaining ATP is detected using a microplate reading luminometer. 2.5. Real-time PCR Total RNA was extracted from interscapular brown adipose tissue using a Reliaprep RNA tissue miniprep system (Promega). cDNA synthesis was performed using a ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Quantitative RT-PCR was performed using SYBR Green. Primers used were 50 -ATCAAACCYCGCTACACG30 and 50 - GGRGTMGTCCCTTTCCAAAG-30 ) for mouse UCP1 and 50 ATGACATCAAGAAGGTGGTG-30 and 50 - AAGGTGGARGAGTGGG-30 for mouse glyceraldehyde 3 phosphate dehydrogenase (GAPDH).

To address the role of HDAC6 in thermogenesis in BAT, we directly measured the surface temperature surrounding interscapular BAT depot using thermal imaging. As shown in Fig. 1AeB, wild-type mice showed 37.56 C ± 0.01 (S.E.); in contrast, HDAC6 KO mice showed 37.10 C ± 0.12 (S.E.). Although the difference is only 0.46 C, it is statistically significant (p < 0.01), showing that BAT thermogenesis is reduced in HDAC6 KO mice. To confirm the reduction of thermogenesis in HDAC6 KO mice, we also measured rectal temperature. As shown in Fig. 1C, wild-type mice showed 38.61 C ± 0.14 (S.E.); in contrast, HDAC6 KO mice showed 38.19 C ± 0.09 (S.E.), indicating that rectal temperature was reduced in HDAC6 KO mice (0.42 C). These results indicated that thermogenesis in BAT was decreased in HDAC6 KO mice. 3.2. HDAC6 positively regulates UCP1 expression HDAC6 has been reported to be an important regulator for the mitochondrial quality-control systems, such as mitophagy for damaged mitochondria, and mitochondrial fusion and fission. Thus,



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Fig. 1. Body temperature is reduced in HDAC6 knockout mice. (A) Representative thermal images of wild-type (n ¼ 13) and HDAC6 KO (n ¼ 13) mice with (B) Spot temperatures adjacent to interscapular BAT depot. (C) Rectal temperature was measured in 6-month-old HDAC6 WT (n ¼ 11) and HDAC6 KO (n ¼ 9) mice. Dots in (B) and (C) represent individual animals. Horizontal lines represent median group values with standard errors. **: p < 0.01, *: p < 0.05.

it is plausible that HDAC6 regulates mitochondrial thermogenesis in BAT. UCP1 is a key protein that mediates thermogenesis in brown fat cells by dissipating the mitochondrial proton gradient that drives ATP synthesis. As shown in Supplementary Figure S1, the relative amount of proton leak is reduced in HDAC6 KO MEFs, implying that mitochondrial heat generation is reduced. To directly address if HDAC6 regulates UCP1 mRNA transcription, we examined the amount of UCP1 mRNA in BAT from wild-type and HDAC6 KO mice using qPCR. As shown in Fig. 2A, the UCP1 mRNA level in HDAC6 KO BAT was significantly reduced by 40% compared to that in wild-type BAT. To confirm the reduction of UCP1 expression in HDAC6 KO BAT, we examined the protein level of UCP1. Consistent with qPCR result, the protein level of UCP1 is reduced in HDAC6 KO BAT. These results suggest that UCP1 is responsible for the reduction of thermogenesis in HDAC6 KO BAT.

3.3. HDAC6 regulates cAMP-dependent PKA activity to control UCP1 expression in BAT Activation of PKA regulates UCP1 expression in BAT [23,24]. Recently, it was reported that cAMP-PKA signaling promotes HDAC6 expression enhancing cell migration [25]. Vice versa, HDAC6 inhibition reduces intracellular cAMP and Ca2þ levels resulting in the inhibition of cystic cells [26,27]. Based on that, we

hypothesized that the cAMP-PKA signal is reduced in HDAC6 KO mice, thus reducing thermogenesis in BAT. To prove the hypothesis, we examined the level of cAMP in BAT from wild-type and HDAC6 KO mice. As shown in Fig. 3A, the level of cAMP in BAT from HDAC6 KO mice is reduced by about 20% compared to that in the wild type, suggesting that the cAMP-PKA signal is reduced in the BAT of HDAC6 KO mice. To further investigate the role of cAMP-PKA signaling in thermogenesis, we isolated primary brown adipocytes from the interscapular BAT of wild-type and HDAC6 KO mice, which were treated with forskolin to elevate the cAMP level [28] and monitored the UCP1 protein level by Western blotting. As shown in Fig. 3B, UCP1 protein level was reduced in primary brown adipocytes from HDAC6 KO mice (lane 2). Notably, UCP1 protein level is increased by forskolin treatment (lanes 4 and 6) and completely restored in HDAC6 KO primary brown adipocytes (lane 6). These results suggest that the reduction of the cAMP level in HDAC6 KO BAT is responsible for the decrease of UCP1 expression and thermogenesis.

3.4. HDAC6 deficiency leads to lipid accumulation in BAT UCP1 expression level and thermogenesis in BAT has been related to lipid accumulation [29,30]. To address the role of HDAC6

Fig. 2. Loss of HDAC6 reduces UCP1 expression. (A) qPCR analyses of UCP1 mRNA from the BAT of HDAC6 WT and HDAC6 KO mice. Fold change is calculated relative to gene expression in WT mice. Error bar: S.D. ***: p < 0.001 (B) Western blot analysis of UCP1, HDAC6, and GAPDH in BAT from WT and HDAC6 KO mice.


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Fig. 3. cAMP and PKA is responsible for the decrease of UCP expression in HDAC6 KO primary adipocytes. (A) Primary brown adipocytes were isolated from BAT of WT and HDAC6 KO mice and subjected to the analysis for cAMP. Means of three independent experiments were graphed with S.D. as error bar; *: p < 0.05. (B) Primary brown adipocytes were treated with forskolin (FSK, 5 mM and 10 mM for 24 h) to raise levels of cAMP and were subjected to Western blotting for UCP1, HDAC6, and GAPDH.

in lipid metabolism in BAT, we examined the histological morphology of brown adipose tissue of wild-type and HDAC6 KO mice. As shown in Fig. 4AeB, the size of brown adipocytes in BAT from HDAC6 KO mice is significantly increased, indicating lipid accumulation. We also examined the size of individual lipid droplets in BAT by transmission electron microscope imaging. As shown in Fig. 4C and supplemental Figure S2, we observed larger lipid droplet in HDAC6 KO BAT than in the wild type. These results suggest that the failure of mitochondrial thermogenesis in HDAC6

KO BAT leads to the accumulation of lipid. 4. Discussion Thermogenesis of BAT has been spotlighted as a therapeutic target for obesity and metabolic disease [3]. Activation of BAT thermogenesis markedly improved metabolic parameters, such as levels of free fatty acids and insulin sensitivity [31,32]. BAT have a unique thermogenic mechanism to convert the chemical energy of

Fig. 4. Lipid is accumulated in brown adipose tissue of HDAC6 knockout mice. (A) Representative H&E staining images of BAT from 6-month-old WT and HDAC6 KO male mice. Scale bar indicates 100 mm. (B) Brown adipocyte size were measured from H&E staining images of BAT from 6-month-old WT (n ¼ 3) and HDAC6 KO (n ¼ 3) mice. Means of 3 independent mice were graphed with S.D. as an error bar; *: p < 0.05. (C) Representative TEM images of BAT from 6-month-old WT and HDAC6 KO male mice. Scale bar is 2 mm.



fat into heat in mitochondria: UCP1, which can short-circuit the mitochondrial proton gradient, dissipates energy as heat. In this report, we have presented evidence that mitochondrial thermogenesis in BAT is reduced in HDAC6 KO mice because of downregulation of UCP1 expression. The loss of HDAC6 reduces the cellular cAMP level, a main regulator of UCP1 transcription, resulting in the accumulation of lipid in BAT. Our findings identify HDAC6 as a potential regulator of mitochondrial thermogenesis through cAMP-PKA signaling, related to lipid metabolism in BAT. BAT thermogenesis is controlled by the sympathetic nervous system [33]. Norepinephrine, released from the nerve terminals, activates b3-adrenergic receptors, transducing Gs proteins and stimulating adenylate cyclase to raise the cAMP level; cAMP binds to and activates PKA, resulting in the transcriptional activation of UCP1 as well as lipolysis of intracellular triglyceride that provides free fatty acids as fuel for the electron-transport chain in mitochondria [3]. The cellular level of cAMP is reduced in BAT of HDAC6 KO mice (Fig. 3A), which is responsible for the reduction of UCP1 expression (Figs. 2e3B). Other groups have also showed that pharmaceutical inhibition of HDAC6 leads to the decrease of the cAMP level in renal epithelial cells [26,27]. Interestingly, HDAC6 expression is also increased by PKA activation, suggesting a positive feedback mechanism [25]. Recently, it was reported that the levels of HDAC6 are markedly reduced in adipose tissues of obese animals and humans [34]. Mice with adipocyte-specific depletion of HDAC6 showed an increase of fat accumulation and a decrease of insulin sensitivity [34]. Mechanistically, HDAC6 deacetylates CIDEC (cell-death-inducing DFFAlike effector C), leading to destabilization, reduced lipid-droplet fusion, and the conversion of FAs to triacylglycerols [34]. We also showed that HDAC6-deficient BAT leads to enlarged lipid droplets, supporting their conclusion (Fig. 4C and Supplementary Figure S2). Although the HDAC6-CIDEC regulation axis directly controls lipiddroplet fusion to control its size, the HDAC6-cAMP-PKA regulation axis would be an additional mechanism to control lipolysis and energy expenditure. Notably, pharmaceutical inhibition of HDAC6 reduces cAMP levels and protein expression of adenylyl cyclase 6 in renal epithelial cells [26,27], supporting our conclusion. In summary, our study showed that HDAC6 plays a role in thermogenesis of BAT through cAMP-PKA signaling that activates the transcription of UCP1, which also controls lipid utilization in BAT. Our findings provide a basis for the HDAC6-dependent thermogenic regulation in BAT that has an impact on metabolic syndrome associated with obesity and diabetes. Acknowledgements This work is supported by a research fund of Chungnam National University, KBSI, NRF-2015R1D1A1A01058257, NRF2016M3A9E1918329, 2014R1A6A9064166 to J.Y. Lee. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.06.016. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.06.016. References [1] J.P. Fuller-Jackson, B.A. Henry, Adipose and skeletal muscle thermogenesis: studies from large animals, J. Endocrinol. 237 (2018) R99eR115.

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