科技部補助專題研究計畫報告
代謝型與情感型疾病交互作用的機制研究-探討對三磷酸腺苷敏
感的鉀離子通道在邊緣情緒多巴胺系統的影響(3/3)
報 告 類 別 ： 成果報告 計 畫 類 別 ： 整合型計畫 計 畫 編 號 ： MOST 108-2320-B-006-002-執 行 期 間 ： 108年08月01日至109年07月31日 執 行 單 位 ： 國立成功大學生理學科暨研究所 計 畫 主 持 人 ： 陳珮君 計畫參與人員： 此計畫無其他參與人員本研究具有政策應用參考價值：■否 □是，建議提供機關
（勾選「是」者，請列舉建議可提供施政參考之業務主管機關）
本研究具影響公共利益之重大發現：□否 □是
中 華 民 國 109 年 10 月 13 日
中 文 摘 要 ： 背景 肥胖是代謝紊亂的關鍵特徵，與醫學抑鬱症有關。最近的證據表明 ，棕色脂肪組織（BAT）的活動可能會導致情緒障礙，三磷酸腺苷 （ATP）敏感的K +（KATP）通道調節BAT交感神經的活動。但是 ，BAT活動影響情緒控制的機制仍然未知。我們假設BAT通過販運 KATP通道參與抑鬱症的調節。 方法 用高脂飲食（HFD）餵養12週的八周大B6雄性小鼠表現出代謝紊亂的 特徵，包括高血糖，高胰島素血症和高脂血症，以及抑鬱症狀。在 這項研究中，我們通過手術移除了小鼠的肩inter間BAT，與其他小 鼠相比，這些小鼠在強迫游泳試驗中表現出固定性，對糖水的偏好 性更低。為了描述KATP通道在BAT活性調節中的作用，我們將包含 KATP通道阻滯劑glibenclamide（GB）的微型滲透泵植入了HFD餵養 小鼠的肩inter間BAT中。 結果 GB輸注可改善葡萄糖穩態，胰島素敏感性和抑鬱症狀。餵高脂飲食 的小鼠中的KATP通道表達低於餵食高脂飲食的小鼠。值得注意的是 ，HFD餵養的小鼠中的GB注入恢復了KATP通道的表達。 結論 KATP通道在BAT中功能性表達，抑制BAT-KATP通道可改善代謝綜合徵 ，並通過β-3腎上腺素受體介導的蛋白激酶A信號傳導降低抑鬱症狀 。 中 文 關 鍵 詞 ： 蛋白質移動,肥胖,憂鬱症,三磷酸腺甘敏感性的鉀離子通道,棕色脂 肪組織 英 文 摘 要 ： Background
Obesity, a critical feature in metabolic disorders, is associated with medical depression. Recent evidence reveals that brown adipose tissue (BAT) activity may contribute to mood disorders, Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels regulate BAT sympathetic nerve activity. However, the mechanism through which BAT activity affects mood control remains unknown. We hypothesized the BAT is involved in depression regulation by trafficking KATP channels.
Methods
Eight-week-old male B6 mice fed with a high-fat diet (HFD) for 12 weeks exhibited characteristics of metabolic
disorders, including hyperglycemia, hyperinsulinemia, and hyperlipidemia, as well as depressive symptoms. In this study, we surgically removed interscapular BAT in mice, and these mice exhibited immobility in the forced swim test and less preference for sugar water compared with other mice. To delineate the role of KATP channels in BAT activity regulation, we implanted a miniosmotic pump containing glibenclamide (GB), a KATP channel blocker, into the interscapular BAT of HFD-fed mice.
Results
GB infusion improved glucose homeostasis, insulin sensitivity, and depressive symptoms. KATP channel
expression was lower in HFD-fed mice than in chow-fed mice. Notably, GB infusion in HFD-fed mice restored KATP channel expression.
Conclusion
KATP channels are functionally expressed in BAT, and inhibiting BAT-KATP channels improves metabolic syndromes and reduces depressive symptoms through beta-3-adrenergic receptor-mediated protein kinase A signaling.
英 文 關 鍵 詞 ： protein trafficking, obesity, depression, KATP channels, brown adipose tissue
科技部補助專題研究計畫成果報告
（□期中進度報告/□期末報告）
（計畫名稱）
計畫類別：□個別型計畫 □整合型計畫
計畫編號：MOST 108 －2320 － B －006 － 002 －
執行期間： 2019 年 8 月 1 日至 2020 年 7 月 31 日
執行機構及系所：國立成功大學醫學院生理學研究所
計畫主持人：陳珮君
共同主持人：
計畫參與人員：
本計畫除繳交成果報告外，另含下列出國報告，共 ___ 份：
□執行國際合作與移地研究心得報告
□出席國際學術會議心得報告
□出國參訪及考察心得報告
本研究
具有政策應用參考價值：
□否 □是，建議提供機關_______
(勾選「是」者，請列舉建議可提供施政參考之業務主管機關)
本研究具影響公共利益之重大發現：□否 □是
附件一Glibenclamide restores dopaminergic reward circuitry in obese mice through interscauplar
brown adipose tissue
Yi-Ying Kuo2, 5, Jie-Kuan Lin2, Ya-Tin Lin1, Jin-Chung Chen1, Yu-Ming Kuo3,5, Po-See Chen4,5, Sheng-Nan Wu1,5, Pei-Chun Chen1,5#
1
Department of Physiology and Pharmacology, Graduate Institute of Biomedical Sciences, Chang-Gung University, 2Department of Physiology, 3Department of Cell Biology and Anatomy,4Department of Psychiatry, National Cheng Kung University Hospital, 5Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan
Running title: brown fat and KATP channel trafficking
#
To whom correspondence may be addressed: Pei-Chun Chen, Department of Physiology, Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, No.1 University Road, Tainan 701, Taiwan, Tel. 011886-6-2353535 ext 5423.; Fax: 011886-6-2362780 ; Email: [email protected]
Abstract
Background
Obesity, a critical feature in metabolic disorders, is associated with medical depression. Recent evidence reveals that brown adipose tissue (BAT) activity may contribute to mood disorders, Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels regulate BAT sympathetic nerve activity. However, the mechanism through which BAT activity affects mood control remains unknown. We hypothesized the BAT is involved in depression regulation by trafficking KATP channels.
Methods
Eight-week-old male B6 mice fed with a high-fat diet (HFD) for 12 weeks exhibited characteristics of metabolic disorders, including hyperglycemia, hyperinsulinemia, and hyperlipidemia, as well as depressive symptoms. In this study, we surgically removed interscapular BAT in mice, and these mice exhibited immobility in the forced swim test and less preference for sugar water compared with other mice. To delineate the role of KATP channels in BAT activity regulation, we implanted a miniosmotic pump containing glibenclamide (GB), a KATP channel blocker, into the interscapular BAT of HFD-fed mice.
Results
GB infusion improved glucose homeostasis, insulin sensitivity, and depressive symptoms. KATP channel expression was lower in HFD-fed mice than in chow-fed mice. Notably, GB infusion in HFD-fed mice restored KATP channel expression.
Conclusion
KATP channels are functionally expressed in BAT, and inhibiting BAT-KATP channels improves metabolic syndromes and reduces depressive symptoms through beta-3-adrenergic receptor-mediated protein kinase A signaling.
Keywords: protein trafficking, obesity, depression, KATP channels, brown adipose tissue
1. INTRODUCTION
Depression affects more than 120 million people worldwide and has highlighted the association between mortality and morbidity (Peng et al., 2015). Obesity changes metabolic health states and is a risk factor for mood disorders. Notably, a meta-analysis conducted in 2010 indicated that both depression and obesity have a bidirectional interaction, with people with obesity having a 55% higher risk of depression and those with depression having a 58% increased risk of obesity (Luppino et al., 2010). The nucleus accumbens (NAc) and its dopaminergic input from the ventral tegmental area (VTA) constitute the mesolimbic dopamine (DA) system that is mostly associated with the rewarding effects of food and drug addiction (Koob and Le Moal, 2001; Weiss and Koob, 2001). However, in depression, the VTA–NAc represents a ‘reward deficit’; this has been supported by research that has reported that manipulating a host of genes engendered considerable effects in relatively sophisticated animal models of depression as well as by human brain imaging investigations that have demonstrated abnormal VTA–NAc functioning in depression (Ackermann et al., 2014; Nestler, 2001; Wang et al., 2017).
Brown adipose tissue (BAT) contributes to thermogenesis and guides the circadian rhythm of core temperature. Recent evidence reveals that cold-stimulated BAT activity is as high as 100% in young adults and indicates that BAT activity may contribute to mood disorders, resulting in an increase in suicide mortality around the time of puberty (Partonen, 2015). Furthermore, the efferent
sympathetic and afferent sensory brain–BAT neural circuits control BAT functions (Ryu et al., 2015). Notably, studies have indicated that the dysregulation of proteins encoded by circadian clock genes may affect depression (Bunney and Potkin, 2008). Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple the metabolic state (i.e., the intracellular ATP/adenosine diphosphate [ADP] ratio) of a cell to membrane excitability (Ashcroft and Ashcroft, 1990; Nichols, 2006; Noma, 1983). The pancreatic beta (β)-cell KATP channel is a complex of four regulatory sulfonylurea receptor (SUR1) subunits and four potassium pore-forming (Kir6.2) subunits, forming an octamer (Clement et al., 1997; Inagaki et al., 1997; Mikhailov et al., 2005; Shyng and Nichols, 1997) of the same subtype as that found in the brain (Karschin et al., 1997). KATP channel activity inhibition by glibenclamide (GB), an antidiabetic drug in pancreatic β cells, improves glucose homeostasis and reduces lipotoxicity upon palmitic acid treatment (Ruan et al., 2018). A notable study reported that the loss of Kir6.2 adipocytes increases glucose uptake, implicating the role of KATP channels are involved in glucose homeostasis in adipose tissues (Miki et al., 2002). However, whether BAT is involved in depression regulation by trafficking KATP channels requires further investigation.
This study determined whether obesity and BAT are associated with the development of depressive symptoms in mice fed with a high-fat diet (HFD) or subjected to surgical removal of interscapular BAT (iBATX). We implanted miniosmotic pumps containing GB into the interscapular BAT of obese mice; we found that GB improved insulin sensitivity and glucose homeostasis and reduced depressive symptoms by increasing KATP channel expression. The increased KATP channels correlated with an upregulation of sympathetic nerve activity, which in turn activated
beta-3-adrenergic receptors (β3 ARs), causing protein kinase A (PKA) phosphorylation and stimulating forward trafficking of KATP channels. In conclusion, our study demonstrated that HFD causes the loss of KATP channels in BAT, resulting in depression and increased surface expression of KATP channels in mice with BAT-reversed HFD-induced depression, highlighting appropriate KATP channel trafficking in BAT.
2. METHODS
2.1 Animals
C57BL/6N mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA; RRID: IMSR_JAX:005304) and were maintained in the Laboratory Animal Center of National Cheng Kung University (NCKU). Male mice weighing 18–20 g were housed in cages (five mice per cage) under a 12-hour light/12-hour dark cycle (lights on at 8:00 am and lights off at 8:00 pm) at a constant temperature (25°C) and controlled humidity under the supervision of qualified caretakers in the Laboratory Animal Center for at least 1 week before the experiments. Mice were allowed free access to food and water. All experimental procedures were performed during the light period of the light-dark cycle. The ethical guidelines provided by the NCKU Animal Care and Use Committee (ethical approval reference number: 106058) were followed throughout the study. To reduce and refine our animal experiments, we employed the variable-criteria sequential stopping rule along with Student’s t-test and one-way analysis of variance (ANOVA) to determine the sample size. Additional details can be found in the paper by (Ruan et al., 2018). Mice were fed either a standard diet (chow: laboratory autoclavable rodent diet 5010, LabDiet, St. Louis, MO, USA) or an HFD
containing 60% kcal fat (58Y1, TestDiet, St. Louis, MO, USA) for 12 weeks.
2.2 Chronic drug infusion
After being fed the chow diet or HFD for 12 weeks, mice were anesthetized using a gas vaporizer (oxygen and isoflurane). Subsequently, miniosmotic pumps (Cat# 1002, Alzet Osmotic Pumps, Cupertino, CA, USA) containing vehicle or GB were implanted into the interscapular BAT for 2 weeks. The GB was dissolved in 88% polyethylene glycol (PEG) 400, 10% dimethyl sulfoxide
(DMSO), and 2% Tween 80.
2.3 Removal surgery for interscapular BAT
We followed a previously described surgical protocol for BAT removal (Grunewald et al., 2018). We subjected 8-week-old mice to a sham treatment or bilateral iBATX. Mice were anesthetized with isoflurane during surgery and were injected with ketoprofen (subcutaneous, 5 mg/kg) to reduce pain after surgery. Briefly, in the sham group, the interscapular BAT was exposed to air and seamed back. In the iBATX group, the interscapular BAT was removed, reducing total brown fat by approximately 70%. An absorbable surgical suture was used to close the incision. Mice were maintained at constant temperature (25°C) after surgery to reduce thermal stress.
2.4 Drugs and antibodies
GB (Cat#: G2539) and fluoxetine hydrochloride (F132, RRID: SCR_000037) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Uncoupling protein 1 (UCP1; Cat#: 14670, RRID: AB_2687530) for immunoblotting was obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). UCP1 (Cat#: AB10983, RRID: AB_2241462) for immunohistochemistry was purchased from
MA, USA). Kir6.2 (Cat#: AGP-067, RRID: AB_2756615) was obtained from Alomone Labs (Jerusalem, Israel). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cat#: 60004-1-Ig, RRID: AB_2107436) antibodies were purchased from Proteintech Group (Rosemont, IL, USA). Alexa 488 (goat anti-rabbit immunoglobulin G [IgG] [H+L], Cat#: A-11008, RRID: AB_143165), Alexa 488 (goat anti-mouse IgG [H+L], Cat#: A-11001, RRID: AB_2534069), Alexa 555 (goat anti-mouse IgG [H+L], Cat#: A-21422, RRID: AB_141822), Alexa 555 (goat anti–guinea pig IgG [H+L], Cat# A-21435, RRID: AB_1500610), and goat anti–guinea pig IgG (Cat#: A18769, RRID: AB_2535546) were obtained from Invitrogen (Carlsbad, CA, USA). Sheep ant-imouse IgG (Cat# NA931V) and goat antirabbit IgG (Cat#: NA934V) were purchased from GE Healthcare Life Science (Marlborough, MA, USA).
2.5 Immunohistochemical examination
A tissue section was dewaxed using xylene and rehydrated in ddH2O. The antigen retrieval conditions depended on the primary antibody. The details can be found in the paper by (Ruan et al., 2018). For the immunofluorescence experiment, Alexa 554 or Alexa 488 was used as a secondary antibody at 1:500 dilution and incubated for 1 hour. For the colorimetric assay, an alkaline phosphatase substrate (Cat# SK-5100; Vector Laboratories, Burlingame, CA, USA) was used. Images were acquired using a Zeiss light microscope (20× objective; Axioskop 2 plus).
2.6 Western blotting
Cells or tissue sections were lysed in triple lysis buffer containing 20 nM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 4 mM
ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Igepal, 0.1% sodium dodecyl sulfate, and 0.04% deoxycholic acid (pH = 7.2) at 4°C. Subsequently, they were cleared through centrifugation at 14,000 rpm for 15 minutes at 4°C. Lysates were cleared through centrifugation at 14,000 rpm for 10 minutes at 4°C. Small aliquots of the lysates were analyzed for protein determination using the Lowry method (Pierce, Rockford, IL, USA), with bovine serum albumin [BSA] serving as the standard. Details can be found in the paper by (Ruan et al., 2018).
2.7 Primary adipocyte culture
The primary brown adipocytes were cultured using the protocol described by (Lehr et al., 2009). The interscapular BAT was dissected from 4-week-old male C57BL/N mice, and cells were isolated in enzymatic digestion buffer (0.25% collagenase I, 5 mM glucose, and 1.5% BSA in phosphate-buffered saline). Cells were then cultured in Dulbecco's Modified Eagle’s Medium/Ham’s F-12 (DMEM/F12) medium containing 10% fetal bovine serum (Cat#:12003C, Sigma-Aldrich), 200 μM ascorbic acid (Cat#:A-1417, Sigma-Aldrich), 20 nM insulin (Cat#:I0516, Sigma-Aldrich), 0.2 nM T3 (Cat#:T6397, Sigma-Aldrich), and 1% penicillin and streptomycin (Cat#: CC502-0100, Simply, Miaoli, Taiwan) for 8 days, allowing complete differentiation.
2.8 High-performance liquid chromatography coupled with electrochemical detection
The striatum (STR) tissues were collected and frozen immediately to −80oC, followed by sonication in 0.1 N HClO4 on ice and centrifugation at 11,600 × g for 30 minutes at 4 oC. We collected and
filtered supernatants for high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD). The HPLC-ECD system consisted of a pump (model 125S, Beckman Coulter, Brea, CA, USA), a 5-µm C-18 column (4.6 × 150 mm) (Alltech ApolloTM
columns, Grace, Deerfield, IL, USA), and an electron capture detector (BASi, West Lafayette, IN, USA). The elutes were analyzed using System Gold software (Beckman Coulter). The mobile phase, 9% methanol, 0.105% glacial acetic acid, 29.9 mM citric acid, 50 mM sodium acetate, 52.5 mM NaOH, and 1.85 mM 1-octanesulfonic acid, was used after filtration and degassing. We detected the concentrations and metabolites of catecholamines and indoleamine by setting the oxidation potential at +650 mV.
2.9 Glucose and insulin tolerance test
The mice received an insulin tolerance test (ITT) after 4 hours of fasting and a glucose tolerance test (GTT) after 12 hours of fasting. Blood glucose was detected by a blood glucose meter (Lifescan, Milpitas, CA, USA). Additional details can be found in the paper by (Ruan et al., 2018).
2.91 Plasma triglyceride, leptin, and insulin levels
Blood was collected and centrifuged at 3600 rpm for 15 minutes at 4°C to obtain plasma. The obtained plasma was used to determine triglyceride and leptin levels through a triglyceride colorimetric assay (K622, Biovision, Milpitas, CA, USA) and enzyme-linked immunosorbent assay (ELISA) Kit (RD291001200R, Biovendor, Czech republic), respectively. To detect fasting insulin levels, blood was collected from the mice through orbital sinus sampling after they fasted for 12 hours. Insulin levels were detected using a mouse insulin ELISA Kit (10-1247-01, Mercodia,
Uppsala, Sweden).
2.92 Open-field test
We performed an open-field test using the protocol described by (Chen et al., 2007). Mice were placed in a white plastic chamber (40 × 40 × 45 cm3) and recorded for 30 minutes. The distance traveled was measured and analyzed using ANY-maze software (Stoelting, Wood Dale, IL, USA).
2.93 Forced swim test
We conducted a forced swim test according to the protocol described by (Tsai et al., 2018). The percentage of immobility time was calculated during final 4 minutes of the test, and it was analyzed using ANY-maze software (Stoelting, Wood Dale, IL, USA).
2.94 Sucrose preference
Mice were free to access one bottle of tap water and one bottle of 2% sucrose water for 24 hours. After 12 hours, the bottles were switched to avoid place preference. The percentage of sucrose intake was calculated by weighing the bottles after 24 hours. We followed the protocol described by (Tsai et al., 2018).
2.95 Electrophysiological measurements
We conducted electrophysiological measurements using bath solution (i.e., normal Tyrode’s solution) containing 136 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES-NaOH buffer (pH 7.4). Single KATP channel activity was measured by filling a pipette with a solution containing 145 mM KCl, 2 mM MgCl2, and 5 mM HEPES-KOH buffer (pH 7.2). To record K+ currents and avoid contamination of Cl− currents, a patch pipette was filled with a
solution containing 130 mM K-aspartate, 20 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer (pH 7.2). The examined cells were immersed in normal Tyrode’s solution containing 1.8 mM CaCl2 at room temperature (20ºC–25ºC). Patch-clamp recordings were obtained in a cell-attached configuration using an RK-400 (Biol-Logic, Claix, France) amplifier. The digitized data were analyzed using either pCLAMP or OriginPro 2016 (OriginLab, Northampton, MA). Experimentally measured single KATP channel currents in BAT cells were analyzed using pCLAMP 10.2. Single-channel amplitudes were virtually determined by fitting Gaussian distributions to the amplitude histograms of the closed and open states. Channel open probability was defined as NˑPO (Chen et al., 2018).
2.96 Image analysis
All fluorescence images were analyzed using NIS-Elements software version 4.50 (Nikon). Before measurement, each image was calibrated using a distance assigned to different images. We used regions of interest, followed by graticule measurement.
2.97 Statistical analysis
All data were analyzed using GraphPad PrismVI. Results are expressed as mean ± standard error of the mean. Differences were tested using either one-way or two-way ANOVA, followed by a post hoc Dunnett’s test for multiple comparisons. To compare only two groups, the unpaired Student’s t-test was used. Statistical significance was set at p < 0.05.
3 RESULTS
We followed a 12-week HFD-feeding protocol to induce obesity in mice (Tsai et al., 2018). The body weight of HFD-fed mice increased significantly compared with that of chow-fed mice since the third week (Fig. 1a). We compared the calories of food intake for these mouse groups and did not observe a difference between the groups in terms of the quantity of pellets consumed (Fig. 1b). Furthermore, mice were subjected to a GTT and ITT. Intraperitoneal (i.p.) glucose challenge (2 g/kg body weight) after overnight fasting significantly increased the blood glucose concentration in HFD-fed mice compared with chow-fed mice (Fig. 1c). Mice received an i.p. injection of insulin (0.75 unit/kg body weight) for the ITT test; HFD-fed mice exhibited marked insulin intolerance than did chow-fed mice (Fig. 1d). Next, we evaluated whether obesity induces depression in mice by performing standard behavioral tests, including sucrose preference and forced swim tests. First, weight gain did not affect mouse locomotion in both groups (Fig. 1e). Sucrose consumption decreased significantly in HFD-fed mice compared with chow-fed mice (Fig. 1f). In the forced swim test, the immobility duration was longer in HFD-fed mice compared with chow-fed mice. To confirm the depressive behavior, mice were injected with fluoxetine (FLX; 20 mg/kg body weight), a typical antidepressant, before the forced swim test. Immobility decreased significantly in HFD-fed mice with FLX treatment compared with HFD mice without FLX treatment (Fig. 1g). These data indicate that 12 weeks of HFD feeding is sufficient to induce weight gain and metabolic syndrome as well as depression-like behaviors without affecting locomotion.
3.2 Neurochemical changes of DA and serotonin occur in the NAc of mice with HFD-induced
The dorsal STR contains caudate/putamen and is involved in motor control. The ventral STR, also called the NAc, is involved in mood control, including motivation and reward perception (Gunaydin and Kreitzer, 2016; Zhang et al., 2018). Reduced DA turnover has been reported in patients with depression (Bowden et al., 1997), along with a decreased serotonin (5-HT) level in the NAc (Kitaichi et al., 2010). We used HPLC-ECD to analyze the total levels of DA,
3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) in the caudate/putamen and NAc. The DOPAC-to-DA ratio (DOPAC/DA) is an indicator of DA activity (Izzo et al., 2005). We found no difference in the DA, DOPAC, DOPAC/DA, 5-HT, or 5-HIAA levels between the STR of chow-fed mice and that of HFD-fed mice (Fig. 2a). Conversely, the DOPAC level and DOPAC-to-DA ratio were reduced in the NAc of HFD-fed mice. Furthermore, the 5-HT and 5-HIAA levels were reduced in HFD-fed mice compared with chow-fed mice (Fig. 2b). In summary, HFD-induced depression correlated with a decrease in DA activity and the serotonin level in the NAc.
3.3 Depression arises from compromised control of brown fat activity.
To directly test whether BAT controls mood, we conducted iBATX on mice, followed by the sucrose preference and forced swim tests. Fig. 3a depicts the timeline and photos of sham and iBATX mice after surgery. No difference in body weight or food intake was observed between iBATX and sham mice (Fig. 3b, 3c). The sucrose preference test showed that iBATX mice exhibited a reduced preference for sweet water compared with sham mice (Fig. 3d). Furthermore, the forced swim test revealed that the immobility percentage was significantly higher in iBATX mice than in sham mice
(Fig. 3e). A neurochemical analysis for DA, DOPAC, and 5-HT in the NAc revealed lower DOPAC and 5-HT levels and a reduced DOPAC-to-DA ratio in iBATX mice (Fig. 3f–3i). Collectively, BAT is crucial in emotion control.
3.4 Chronic infusion of KATP channel blocker into BAT improves insulin tolerance and
reduces depression-like behaviors in HFD-fed mice.
To determine whether GB exerts a similar effect in pancreatic β cells, we implanted a single miniosmotic pump containing GB (0.4 mg/kg body weight) into the interscapular BAT—where most BAT depots are found—of the mice (Frontini and Cinti, 2010; Mo et al., 2017). We divided the mice into the following groups according to GB infusion: chow, HFD, chow+GB (CG), and HFD+GB (HG) groups. The results showed that neither body weight (Fig. 4a) nor food intake (Fig. 4b) was affected by GB in chow- or HFD-fed mice. The HG group exhibited higher glucose (Fig. 4c) and insulin tolerance (Fig. 4d) levels compared with the HFD group. In particular, the triglyceride, leptin, insulin, and fasting insulin concentrations were significantly reduced in the HG group relative to the HFD group (Fig. 4e–4h). Notably, compared with the HFD group, the HG group exhibited reduced immobility in the forced swim test and preferred sugar water (Fig. 5a, 5b). GB infusion did not affect locomotor activity among four groups (Fig. 5c). We also analyzed catecholamine in the NAc. The results indicated a decrease in DOPAC, DA neuronal activity, and 5-HT in HFD-fed mice. GB infusion restored DA neuronal activity and 5-HT content in HFD-fed mice, supporting the antidepressive effects of GB (Fig. 5d–5g). By contrast, miniosmotic pumps containing GB that were implanted into the gastrointestinal tract resulted in the correction of metabolic syndromes only but
did not reduce depressive behaviors (Supplementary Fig. 1). Hence, to alleviate depressive symptoms, GB infusion into the interscapular BAT is vital.
3.5 GB infusion restores surface expression of KATP channels and BAT activity in HFD-fed
mice.
We performed immunoblotting to determine whether chronic infusion of GB affects KATP channel expression in BAT obtained from the chow, HFD, CG, and HG groups. The results indicated that the KATP channel levels in BAT were significantly reduced in the HFD group relative to the chow group and that GB infusion considerably restored KATP channel expression in the HG group compared with the HFD group (Fig. 6a). The KATP channel subunits in BAT obtained from the chow, HFD, CG, and HG groups were stained with two antibodies: SUR1 (in green) and Kir6.2 (in red). Our previous study indicated that GB protects pancreatic β cells from palmitic acid–induced lipotoxicity by increasing KATP channel expression (Ruan et al., 2018). A decrease in KATP channel expression was observed in BAT from the HFD group compared with that from the chow group. In particular, KATP channel expression was relatively high in BAT from the HG group (Fig. 6b). Additionally, the innervation of sympathetic nerves was investigated using tyrosine hydroxylase (TH) as an indicator (Zeng et al., 2015). As expected, TH expression was lower in BAT from the HFD group but was restored in BAT from the HG group compared with the expression observed in the CG group (Fig. 6c). This indicates that chronic GB infusion might induce more norepinephrine (NE) release by activating sympathetic nerve innervation in BAT.
We examined whether KATP channels are expressed in BAT. We isolated white adipose tissue (WAT) and BAT separately and performed immunoblotting to assess protein expression. In contrast to WAT, SUR1, Kir6.2, and UCP1 expression was noted in BAT (Fig. 7a). Furthermore, we cultured and differentiated primary brown and white adipocytes and then treated them with β3 AR agonist (Cl316, 243) for 3, 5, and 7 days. Inmmunostaining revealed that unlike adipocytes, differentiated primary brown adipocytes were multilocular cells with a spindle-like shape, exhibiting stronger UCP1 signals after 7 days of treatment with β3 AR (Fig. 7b). We also observed that the KATP channel current was sensitive to diazoxide stimulation as well as GB inhibition (Fig. 7c). Norepinephrine binds β3 ARs, activates adenylyl cyclase, and induces PKA phosphorylation upon sympathetic nerve stimulation (Bargut et al., 2016; Chernogubova et al., 2004). We conducted immunostaining to investigate whether β3 AR activation increases KATP channel expression. An increase in SUR1 protein (red fluorescence) was observed in β3 AR agonist–treated cells compared with control-treated cells (Fig. 7d). To examine whether β3 AR–mediated PKA signaling increases KATP channel expression, WT-1 brown preadipocytes were differentiated for 10 days, followed by various drug treatments and immunoblotting. Quantitative polymerase chain reaction analysis suggested that the mRNA levels of UCP1, SUR1, and Kir6.2 were significantly higher in differentiated than undifferentiated Wilms tumor 1 (WT-1) cells (Fig. 7e). β3 AR agonist–treated WT-1 cells exhibited a time-dependent increase in SUR1 and Kir6.2 proteins; this increase was inhibited by 10 µM PKA. An increase in SUR1 protein expression was observed after WT-1 cells were treated with either forskolin (FSK) or GB for 30 minutes (Fig. 7f). Collectively, KATP channels are present in
4. DISC
USSION
Obesity and metabolic disorders can be risk factors for neurodegenerative diseases. The content of several lipid species, such as total triglycerides, cholesterol, and ceramides, is elevated in the brains of HFD-fed mice (Charradi et al., 2017). Because dopamine deficiency was observed in obese subjects, the DA system regulates body mass index (Chen et al., 2008). Moreover, DA can reduce glucose uptake in WAT through ARs (Lee et al., 1998). Accordingly, restoring the dopamine level and coactivating dopamine receptors increase protein mass and reduce triglyceride, free fatty acid, and glucose concentrations in the blood (Cincotta et al., 1997). Hypodopaminergic activity was observed in HFD-fed mice; such activity may be involved in the development metabolic syndromes such as hyperlipidemia, hyperglycemia, and insulin intolerance (Fig. 1d, 1e and Fig. 2). However, GB infusion in BAT restored the DA level, engendered improved glucose homeostasis and insulin sensitivity, and reduced blood triglyceride levels in HFD-fed mice (Fig. 4c–4h and Fig. 5d–5g). Additionally, the midbrain dopaminergic neurons play a pivotal role in reward information processing. Striatal dopaminergic activity was noted to be reduced in depressed patients as compared with controls (Belujon and Grace, 2017). GB infusion in HFD-fed mice caused in an increase in DA levels in the NAc, resulting in relief from depression (Fig. 5a, b).
KATP channels are expressed in catecholaminergic neurons, including the locus coeruleus (LC), which control the sympathetic outflow to BAT (Dunn-Meynell et al., 1998). A previous study
demonstrated that the expression of mutant KATP channels, which are resistant to inhibition by ATP, in the catecholaminergic neurons of the LC reduces the activity of sympathetic nerves in BAT and enhances diet-induced obesity (Tovar et al., 2013). We also found less TH staining in BAT from HFD-fed mice compared with that from chow-fed mice. GB infusion into BAT from HFD-fed mice restored TH fluorescent intensity, resulting in an improved glucose homeostasis, insulin sensitivity, and blood triglyceride concentration (Fig. 6c). Another study demonstrated that the stimulation of KATP channels in the sympathetic nerve endings of the human and guinea pig atrium modulated NE release (Oe et al., 1999). KATP channel closure by GB leads to membrane potential depolarization, in turn increasing sympathetic nerve activity and promoting NE release. Leptin increases the surface expression of KATP channels through adenosine monophosphate–activated protein kinase
(AMPK) and PKA in pancreatic β cells to regulate insulin secretion (Chen et al., 2013). As presented in Fig. 7e and 7f, we speculate that GB increases KATP channels in BAT by promoting NE release for activating β3 AR followed by PKA phosphorylation.
A previous study demonstrated that a bilateral excision of the interscapular BAT in mice at 25°C did not disrupt systemic glucose metabolism, which is a limited thermal stress condition (Grunewald et al., 2018); no apparent failure in sustaining the core temperature when exposed to an environment at 4°C for 24 hours was noted in mice receiving iBATX (Connolly et al., 1982). The water temperature in the forced swim test in the present study was approximately 23°C– 25°C, as suggested in the literature (Can et al., 2012). We observed that mice receiving iBATX exhibited increased immobility in the water tank, which was unlikely due to hypothermia or hypoglycemia (Fig.
3e). Anhedonia is a depressive symptom, and rodents are born with an interest in sweet foods or solutions. Mice receiving iBATX exhibited a decreasing trend of preference for drinking sugar water compared with those that underwent sham surgery (Fig. 3d). We speculate that some mice could be neophobic, having fear of unknown substances; hence, inability of distinguishing anhedonia from fear is a limitation of the sucrose preference test (Serchov et al., 2016). The DA, DOPAC, and 5-HT levels in the NAc were lower in mice that underwent iBATX, supporting the importance of BAT in mood control. The role of BAT in obesity and glucose homeostasis is well established (Poher et al., 2015; Stanford et al., 2013). Twik-related acid-sensitive potassium channel-1, a pH-sensitive K+ channel, controls thermogenic activity in BAT through β AR signaling (Pisani et al., 2016). KATP channels not only couple membrane electrical activity to energy metabolism in a variety of cells but also are involved in the pathogenesis of depression. (Esmaeili et al., 2018; Fan et al., 2016).
KATP channels are functionally expressed in BAT, and the inhibition of BAT-KATP channels improves metabolic syndromes and reduces depressive symptoms in HFD-fed mice. Furthermore, we postulate that the closure of BAT-KATP channels induces the release of some endocrine factors from BAT to communicate to the brain. This novel function of BAT-KATP channels emphasizes a previously underappreciated role of BAT in mood control, and manipulating KATP channel levels in BAT can be a possible therapeutic avenue for treating obesity-induced depression (Fig. 8).
Abbrevations
Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels, sulfonylurea receptor 1 (SUR1), brown adipose tissue (BAT), inwardly rectifying potassium channel 6.2 (Kir6.2), glibenclamide (GB), Uncoupling protein 1 (UCP1), white adipose tissue (WAT)
Declarations
Ethical Approval and Consent to participate
The ethical guidelines was approved by the NCKU Animal Care and Use Committee (ethical approval reference number: 106058).
Consent for publication
None.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author, PCC, upon reasonable request.
Funding
This research received financial support from the Ministry of Science and Technology (MOST105-2628-B-006-006-MY3, MOST106-2320-B-006-050, and MOST107-2320-B-006-014), offered to Pei-Chun Chen.
Author contributions
YYK contributed to the drafting and design of the study, acquisition of data, and analysis and interpretation of data. JKL, YYK, and YTL contributed to the acquisition and analysis of data. SNW contributed to patch-clamp recording and single-channel analysis. JCC, YMK, and PSC revised the manuscript critically for intellectual content. PCC revised the manuscript, approved the final version to be published, and agreed to be accountable for all the aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Acknowledgments
We thank Dr. Yu-Hua Tseng to provide WT-1 cells at the Joslin Diabetes Center at Harvard Medical School, the staff of the Advanced Light Microscopy Core at National Cheng Kung University Hospital, and Wallace Academic Editing for their help with this manuscript.
Conflicts of interest
Figure legends
Fig. 1: High-fat diet (HFD) feeding for 12 weeks successfully induced metabolic disorders and depressive symptoms. (a) Timeline for animal study, indicating 8-week-old mice were fed either standard diet (chow) or HFD for 12 weeks. During 12th to 14th weeks, mice were subjected to behavioral tests or metabolism-related assays. Tissues were harvested after the fourteenth week. (b) The body weight of each mouse in the chow or HFD group was measured and averaged. In the third week, HFD-fed mice started exhibiting significant weight gain in comparison with chow-fed mice. (c) Food intake represents average kilocalories consumed per day per mouse in chow- or HFD-fed groups. (d) Glucose tolerance of the mouse groups was compared after fasted mice were challenged with glucose (1.5 g/kg). (e) Insulin tolerance of mouse groups was compared after fasted mice were challenged with insulin (0.75 unit/kg). (f) Representative bar graph indicating effect of chow and HFD on the locomotion distance in the open field test. (g) Representative bar graph indicating effect of chow and HFD on the immobility in the forced swim test before and after acute fluoxetine (FLX, 20 mg/kg) injection. (g) Representative bar graph indicating the intake ratio of sucrose to total intake in sucrose preference test. (Data are presented as means ± SEM; n = 10 per group; *p < 0.05 compared with the chow group; #p < 0.05 compared with the HFD group;
differences are evaluated using either unpaired t-test or one-way analysis of variance and Dunnett’s post hoc test).
Fig. 2: High-fat diet (HFD)-induced obesity resulted in the catecholamine level dysregulation in the nucleus accumbens. (a) Representative bar graphs indicating the catecholamine level in the caudate-putamen. (b) Representative bar graphs indicating the catecholamine level in the nucleus accumbens. (Data are presented as means ± SEM; n = 8 per group; *p < 0.05 compared with the control group; differences are evaluated using unpaired t-test).
Fig. 3: Mice subjected to surgical removal of interscapular brown adipose tissue (iBATX) developed depressive symptoms. (a) Top: Experiment timeline presenting the periods of surgery and indicated tests. Bottom: photographs of the interscapular BAT after sham and iBATX procedures conducted on mice. (b) Average body weight of each mouse. (c) Food intake represented as average kilocalories consumed per day per mouse in the sham or iBATX groups. (d) Representative bar graph indicating the intake ratio of sucrose to total intake in the sucrose preference test. (e) Representative bar graph indicating the effect of sham or iBATX removal surgery on the immobility through the forced swim test. (f-i) Representative bar graphs indicating the catecholamine level in the nucleus accumbens. (Data are presented as means ± SEM; n = 8 per group; *p < 0.05 compared with the control group; differences are evaluated using unpaired t-test).
Fig. 4: Chronic administration of glibenclamide reduced metabolic disorder characteristics without changing body weight and food intake in mice with high-fat diet (HFD)-induced depression. Top: Timeline indicating the drug regimen. Bodyweight (a) and food intake (b) measured during the 2
weeks of implantation. The detection of the blood glucose level after fasting in glucose- and insulin-challenged mice with glucose tolerance test (GTT) (c) and insulin tolerance test (ITT) (d) tests, respectively. The triglyceride (e), leptin (f), insulin (g), and fasting insulin (h) levels in the four (i.e., chow, HFD, CG, and HG) groups of mice. (Data are presented as means ± SEM; n = 10 per group; *p < 0.05 compared with the control group; #p < 0.05 compared with the HFD group; differences are evaluated either using one-way or two-way analysis of variance and Dunnett’s post hoc test).
Fig. 5: Chronic administration of glibenclamide attenuated depressive symptoms in high-fat diet (HFD)-induced obese mice. (a) Representative bar graph indicating the intake ratio of sucrose to total intake in sucrose preference test. (b) Representative bar graph indicating the effect of chow or HFD on the immobility in the forced swim test. (c) Representative bar graph indicating the effect of chow or HFD on the locomotion distance in the open field test. Representative bar graphs indicating the catecholamine level, including dopamine (DA) (d), 3,4-dihydroxyphenylacetic acid
(DOPAC) (e), DOPAC to DA ratio (f), and serotonin (5-HT) (g), in the nucleus accumbens. (Data are presented as means ± SEM; n = 10 per group; *p < 0.05 compared with the control group; #p < 0.05 compared with the HFD group; differences are evaluated using one-way analysis of variance and Dunnett’s post hoc test).
Fig. 6: Chronic administration of glibenclamide (GB) affected the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel expression in the interscapular brown adipose tissue. (a) Top: Representative blotting images indicating the levels of sulfonylurea receptor 1 (SUR1), potassium pore-forming (Kir6.2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the four (i.e., chow, high-fat diet [HFD], chow + GB [CG], and HFD + GB [HG]) groups of mice. Bottom: Representative bar graph indicating the SUR1 to Kir6.2 ratio. (b) Immunofluorescence staining of the paraffin embedded BAT sections from the four groups of mice with SUR1 (green) and Kir6.2 (red) antibodies. The scale bar is 100 µm. (c) Immunohistochemistry analysis of the paraffin embedded BAT sections from the four groups of mice using tyrosine hydroxylase antibody. The signal for TH is dark brown. The scale bar is 100 µm. (Data are presented as means ± SEM; n = 10 per group; *p < 0.05 compared with the control group; #p < 0.05 compared with the HFD group; differences are evaluated using one-way analysis of variance and Dunnett’s post hoc test).
Fig. 7: Examination of KATP channels in differentiated primary brown adipocytes and WT-1 cell line. (a) Representative blotting images indicating the presence of sulfonylurea receptor 1 (SUR1), potassium pore-forming (Kir6.2), and Uncoupling protein 1 (UCP1) proteins in the differentiated primary brown (BAT), but not in the white adipocytes (WAT). (b) The morphological examination by immunofluorescence staining using UCP1 antibody (green) and DAPI for staining of nucleus. of primary brown and white adipocytes culture treated with beta-3-adrenergic receptors (β3 ARs) agonist for 3, 5, and 7 days. The scale bar is 100 µm. (c) The electrophysiological recording
presenting the KATP channel current in the primary brown adipocytes. This current was increased by diazoxide and inhibited by glibenclamide. (d) Top: Differentiated primary brown adipocytes treated with β3 AR agonist for 7 days and subjected to immunostaining for SUR1 protein (red). Bottom: Representative bar graph indicating the quantification of the immunofluorescence intensity of SUR1 protein. The scale bar is 10 µm. (e) Representative bar graph indicating the relative expression of SUR1, Kir6.2, and UCP1 proteins in undifferentiated and differentiated WT-1 cells. (Data are presented as means ± SEM; n = 8 per group; *p < 0.05 compared with the corresponding undifferentiated group). (f) Top: Representative blotting images indicating the SUR1 level after WT-1 cells were treated with various indicated drugs. Bottom: Representative bar graph indicating the changes in SUR1 expression after drug treatments. (Data are presented as means ± SEM; n = 3 per group; *p < 0.05 compared with the vehicle-treated group; #p < 0.05 compared with the β3 AR agonist-treated (for 30 minutes) group; $p < 0.05 compared with the β3 AR agonist-treated (60 minutes) group; differences are evaluated using one-way ANOVA and Dunnett’s post hoc test).
Fig. 8: Schematic of glibenclamide-increased KATP channel expression in BAT. GB infusion recovered activities of sympathetic nerves by closing KATP channels to stimulate depolarization, in turn increasing norepinephrine (NE) release. NE bound to beta-3-adrenergic receptors activated protein kinase A phosphorylation and promoted trafficking of KATP channels. GB served as the chemical chaperone for assisting the KATP channel folding. Overall, more KATP channels were
expressed in brown adipocytes. The other function of GB is to close KATP channels, possibly inducing some endocrine factors released from brown adipose tissue to exert anti-depression.
Supplementary Fig. 1: High-fat diet (HFD) feeding for 12 weeks successfully induced metabolic disorders and depressive symptoms. (a) Timeline for animal study, indicating 8-week-old mice were fed either standard diet (chow) or HFD for 12 weeks. During 12th to 14th weeks, mice were subjected to behavioral tests or metabolism-related assays. Tissues were harvested after the fourteenth week. (b) The body weight of each mouse in the chow or HFD group was measured and averaged. In the third week, HFD-fed mice started exhibiting significant weight gain in comparison with chow-fed mice. (c) Food intake represents average kilocalories consumed per day per mouse in chow- or HFD-fed groups. (d) Glucose tolerance of the mouse groups was compared after fasted mice were challenged with glucose (1.5 g/kg). (e) Insulin tolerance of mouse groups was compared after fasted mice were challenged with insulin (0.75 unit/kg). (f) Representative bar graph indicating effect of chow and HFD on the locomotion distance in the open field test. (g) Representative bar graph indicating effect of chow and HFD on the immobility in the forced swim test before and after acute fluoxetine (FLX, 20 mg/kg) injection. (g) Representative bar graph indicating the intake ratio of sucrose to total intake in sucrose preference test. (Data are presented as means ± SEM; n = 10 per group; *p < 0.05 compared with the chow group; #p < 0.05 compared with the HFD group; differences are evaluated using either unpaired t-test or one-way analysis of variance and Dunnett’s post hoc test).
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