Obesogenic and Diabetogenic Effects of High-Calorie Nutrition Require Adipocyte BK Channels

Diabetes Volume 65, December 2016 3621

Julia Illison,1 Lijun Tian,2 Heather McClafferty,2 Martin Werno,3 Luke H. Chamberlain,3 Veronika Leiss,4 Antonia Sassmann,5 Stefan Offermanns,5 Peter Ruth,1 Michael J. Shipston,2 and Robert Lukowski1

Diabetes 2016;65:3621–3635 | DOI: 10.2337/db16-0245

Elevated adipose tissue expression of the Ca2+- and suggesting that BK channels are promising drug targets voltage-activated K+ (BK) channel was identified in mor- for pharmacotherapy of metabolic disorders and obesity. bidly obese men carrying a BK gene variant, supporting the hypothesis that K+ channels affect the metabolic responses of fat cells to nutrients. To establish the role Obesity is caused by high calorie intake, typically calories of endogenous BKs in fat cell maturation, storage of derived from dietary fats or sugars. Over time, an imbalance excess dietary fat, and body weight (BW) gain, we studied between consuming nutrients and burning calories leads to a gene-targeted mouse model with global ablation of the a massive increase in fat mass, which is—among other STUDIES OBESITY L1/L1 BK channel (BK ) and adipocyte-specific BK-deficient factors—a major cause of insulin resistance and diabetes L1/L2 (adipoqBK ) mice. Global BK deficiency afforded (1,2). In addition, genetic mutations may cause excessive protection from BW gain and excessive fat accumula- lipid accumulation and thereby a morbid increase in body tion induced by a high-fat diet (HFD). Expansion of white weight (BW), emphasizing the multifactorial etiology of – BKL1/L1 adipose tissue derived epididymal preadipocytes chronic excessive weight gain (3,4). It has become increas- fi and their differentiation to lipid- lled mature adipocytes ingly clear that a variety of K+ ion channels—that is, K+ in vitro, however, were improved. Moreover, BW gain and channels expressed within the brain and in the periphery, total fat masses of usually superobese ob/ob mice were possibly by complex effects on appetite and satiety, significantly attenuated in the absence of BK, together energy expenditure, glucose balance, and/or fat cell supporting a central or peripheral role for BKs in the function—are involved in the pathophysiology of obesity regulatory system that controls adipose tissue and weight. adipoqBKL1/L2 and related disorders (5). Accordingly, a genome-wide as- Accordingly, HFD-fed mutant mice pre- fi sented with a reduced total BW and overall body fat mass, sociation study recently identi ed the human KCNMA1 smaller adipocytes, and reduced leptin levels. Protection gene, encoding for the pore-forming subunit of the large- 2+ + from pathological weight gain in the absence of adipo- conductance Ca -activated K channel (BK), as a novel sus- cyte BKs was beneficial for glucose handling and related ceptibility locus for obesity (6). BK channel mRNA levels in to an increase in body core temperature as a result of higher variant carriers were significantly increased in white adipose levels of uncoupling protein 1 and a low abundance of the tissue (WAT) and adipose tissue–derived cells, suggesting proinflammatory interleukin-6, a common risk factor for a pathogenic role for fat cell BK in metabolism. Others diabetes and metabolic abnormalities. This suggests that reported electrophysiological evidence for BK channels in adipocyte BK activity is at least partially responsible human preadipocytes, and BK knockdown, or its pharmaco- for excessive BW gain under high-calorie conditions, logical inhibition, further revealed a possible link between

1Pharmakologie, Toxikologie und Klinische Pharmazie, Institut für Pharmazie, Corresponding author: Robert Lukowski, [email protected]. Tübingen, Germany Received 26 February 2016 and accepted 16 August 2016. 2Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, This article contains Supplementary Data online at http://diabetes University of Edinburgh, Edinburgh, U.K. .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0245/-/DC1. 3Strathclyde Institute of Pharmacy and Biomedical Sciences, Strathclyde Univer- sity, Glasgow, U.K. © 2016 by the American Diabetes Association. Readers may use this article as 4Department of Pharmacology and Experimental Therapy, Institute of Experimen- long as the work is properly cited, the use is educational and not for profit, and the tal and Clinical Pharmacology and Toxicology, University Hospital Tübingen, work is not altered. More information is available at http://www.diabetesjournals Tübingen, Germany .org/content/license. 5Department of Pharmacology, Max-Planck-Institute for Heart and Lung Re- search, Bad Nauheim, Germany 3622 Adipocyte BK Protects From Overwhelming BW Gain Diabetes Volume 65, December 2016 channel activity and the proliferative capacity of these Department of Pharmacology, Toxicology and Clinical cells (7), whereas Ca2+-activated K+ channels of the IK type Pharmacy, University of Tübingen, Tübingen, Germany, seem to be dominant in the widely studied murine preadi- as described previously (22). To produce BKL1/L1 mice pocyte model 3T3-L1 (8). However, because both peripheral that are also unable to produce functional leptin (genotype and central organs involved in the control of metabolism BKL1/L1;B6.V-Lepob/ob [BKL1/L1;ob/ob]), BKL1/+ animals were express BK, it is difficult to estimate the impact of pre- crossed with mice carrying a heterozygous mutation of the adipocyte BKs on fat cell formation and adiposity in vivo. leptin gene B6.V-Lep+/ob (ob/+)obtainedfromCharlesRiver For example, a direct modulation of BK by fatty acids and Laboratories (Sulzfeld, Germany). Intercrossing of double- their metabolites seems to provide a possible link between heterozygous animals (genotype BKL1/+;B6.V-Lepob/+ [BKL1/+; lipid-mediated effects on the channel and altered vascular ob/+]) produced homozygous obese BKL1/L1;ob/ob,obese functions in hypertriglyceridemia secondary to obesity BK+/+ (genotype BK+/+;B6.V-Lepob/ob [BK+/+;ob/ob]), lean (9–13). Previous analyses of global BK knockout mice imply BKL1/L1 (BKL1/L1;B6.V-Lep+/+ [BKL1/ L1; +/+]), and lean BK+/+ that both glucose-induced insulin secretion and endocrine (BK+/+;B6.V-Lep+/+ [BK+/+;+/+]) offspring from the same lit- output from the hypothalamic-pituitary-adrenal (HPA) axis ters.Toobtaintissue-specific mutants lacking BK in various require endogenous BK channels (14,15). However, it has adipocyte populations, heterozygous BK mice (BKL1/+)ona not been determined whether these dysregulations in C57BL6/N background, carrying a tamoxifen-inducible adi- b-cell or HPA function caused metabolic abnormalities pose tissue–specific Cre recombinase (genotype adiponectin- in vivo (16). Moreover, several studies found that BK pro- CreERT2tg/+) under control of the adiponectin promoter moted the inhibitory effects of leptin signaling, via phos- (23), were first mated to homozygous floxed BK ani- phoinositide 3-kinase in hippocampal neurons (17) and in mals (genotype BKL2/L2). Adipocyte-specificcontrols(geno- mouse chromaffincells(18),suggestingthatneuronalcir- type adiponectin-CreERT2tg/+;BK+/L2 [adipoqBK+/L2]) and cuits controlling appetite and energy expenditure may also premutant BK animals (genotype adiponectin-CreERT2tg/+; depend on functional BK. A malfunction of neuronal circuits BKL1/L2 [adipoqBKL1/L2]) derived from this breeding were in- has been widely appreciated as causing excessive fat storage jected with tamoxifen (1 mg/day) for 5 consecutive days to (19), and indeed a number of studies found evidence for BK induce Cre-mediated recombination at an age of 8 weeks in the brainstem, different hypothalamic nuclei, parts of the (Figs. 3 and 4C–H)or19weeks(Fig.4I). cortex and limbic system—hence in brain regions that are The specificity and efficiency of the adiponectin-CreERT2- implicated in the central control of food intake, appetite, and derived recombination was supported by studying double- energy expenditure (20,21). fluorescent ROSA26-Tomato reporter mice (genotype The recent discovery of functional channels with prop- ROSA26mTomato,mEGFP/+ [tom/+]) obtained from Charles River erties similar to BK in fat cell progenitors (7), together with (24); these mice expressed the adipoqCreERT2 transgene the link between high-fat-cell BK mRNA expression levels (genotype adiponectin-CreERT2tg/+;ROSA26mTomato,mEGFP/+; and morbid adiposity (6), suggest that BK activity in the [adipoqtg/+;tom/+]). Double-transgenic animals were either adipose tissue plays physiologically and pathophysiologi- injected with tamoxifen for 5 days (as described) to induce cally important roles for weight control, although these the recombination of the reporter allele—that is, a switch specific roles remain to be discovered. To establish endog- from cell membrane-localized red fluorescence to green enous BK channels as potential modulators of fat deposi- fluorescence proteins—or received solvent. Genotyping tion and excessive weight gain, herein we assess the was performed using specific primers to identify the Cre susceptibility of global and adipocyte-specific BK mouse transgene (23), the single nucleotide mutation in the leptin mutants to genetic and dietary causes of adiposity. Our gene (according to a protocol for stock no. 000632 from approach to validate BK as a novel player in the response The Jackson Laboratory), and the L1, L2, and wild-type to obesogenic factors in vivo should also direct future studies alleles of the BK on DNA samples obtained either from on the pharmacotherapy of adiposity and related disorders. different fat depots, control tissue, or tail-tip biopsies from mice that were left untreated or received tamoxifen, RESEARCH DESIGN AND METHODS as described previously (22). Animals and Diets Before administering the different research diets, all All animal experiments were performed with the permission experimental mice received ad libitum tap water and a of the local authorities and conducted in accordance with commercial chow obtained from Altromin (Lage, Germany). German and/or accordingly U.K. legislation on the pro- Dietary feeding trials were performed in male mice that were tection of animals. Animals were housed in cages under allowed to adapt to a defined control diet (CD) formulation controlled environmental conditions, with temperatures containing 10% of calories from fat for 2 weeks before the maintained between 21°C and 24°C, humidity at 45–55%, long-term feeding experiments. Ten-week-old experimental and 12-h light/12-h dark cycle. mice received either a high-fat diet (HFD) containing 45% or Global BK channel–deficient mice (genotype BKL1/L1) 60% of calories from fat or continued feeding on the CD for and their heterozygous (genotype BKL1/+) and wild-type another 18 weeks. Progressive BW gain in the monogenetic littermates (genotype BK+/+) on a C57BL/6N background model of obesity was monitored between 4 and 24 weeks of were generated and maintained at the Institute of Pharmacy, age while eating normal chow diet. diabetes.diabetesjournals.org Illison and Associates 3623

Food Intake and Core Body Temperature 4.8 (Carl Zeiss, Jena, Germany). Cell growth and proliferation Measurements assays were analyzed in real time using the xCELLigence The core body temperature and the locomotor activity impedance setup (Omni Life Science, Bremen, Germany) dur- were measured using a telemetry setup (ETA-F10 with ing the first 60 h upon plating in the medium (DMEM/Ham two-lead electrocardiogram transmitters of the implant F12with5%FCS).Datawereacquiredandanalyzedusing removed because they were not required to monitor the RTCA software version 1.2.1 (ACEA Biosciences Inc.). activity and temperature; Data Sciences, Inc.). The surgical procedure was described previously (25). In brief, Electrophysiological Recordings anesthesia was induced by inhalation of 4% isoflurane The conventional whole-cell mode of patch clamp electro- in oxygen and was then maintained with 1–2% isoflurane. physiology was used to analyze BK currents in undifferen- The implants were placed in the peritoneal cavity using an tiated and differentiated 3T3-L1 cells, as well as from aseptic technique and the skin was closed with 5-0 surgical preadipocytes and induced matured adipocytes prepared silk. During recovery, mice were placed on a warming pad from primary cultures of mouse eWAT. The pipette solution before they were returned to their home cages for at least contained 140 mmol/L KCl, 10 mmol/L HEPES, 30 mmol/L 3 days before recordings began. Activity and core body glucose, 1 mmol/L 1,2-Bis(2-Aminophenoxy)ethane- 9 9 temperature were assessed continuously for 96 h in experi- N,N,N ,N -tetra acetic acid (BAPTA), 2 mmol/L MgCl, and m mental mice that had received the HFD for 18 weeks. free calcium buffered to 0.5 mol/L at pH 7.2. The stan- Food intake was measured using a nonautomated and dard bath (extracellular) solution contained: 140 mmol/L modified metabolic cage setup. To reduce environmental NaCl, 5 mmol/L KCl, 10 mmol/L HEPES, 20 mmol/L glu- stress in the feeding chamber of the metabolic cage, mice cose,2mmol/LMgCl,and1mmol/LCaCl;pHwasadjusted housed individually were placed on paper bedding that to 7.4. Electrophysiological recordings were performed at – was renewed daily. After 2 days of acclimatization, food room temperature (18 22°C) using pCLAMP 9 (Molecular fi consumption was assessed for 4 days using an analytic Devices), with a sampling rate of 10 kHz and ltering at balance. Food pellets were replaced every 24 h. The amount 2 kHz. Patch pipettes were fabricated from borosilicate V of food consumed by each individual mouse was deter- glass (Garner) with resistances between 2 and 3 mol/L . mined by calculating the weight differences between the Drugs were applied to cells using a gravity perfusion system fl – fl initial weight of the pellets and the final weight 24 h later. with a ow rate of 1 2 mL/min to minimize ow-induced – Food pieces dropped by the animals on the paper bedding artifacts. For analysis of BK currents, the BK channel fi m were thoroughly collected and included in the final weight speci c blocker paxilline (1 mol/L) was applied and the determination. Food intake was assessed in a subset of paxilline-sensitive outward current was analyzed at the fi 10-week-old experimental mice before the dietary feeding membrane voltages indicated in the respective gure legends. or after the CD or HFD feeding protocols. Adipose Tissue Histology For histological examination, tissues were fixed in 4% Growth and Maturation of Preadipocytes In Vitro paraformaldehyde for 1.5 h directly after harvesting and Preadipocytes derived from inguinal (iWAT) or epididy- were then embedded in NEG 50 (Richard-Allan Scientific, mal WAT (eWAT) of 10-week-old BK+/+ and BKL1/L1 mice Thermo Scientific) for sectioning following sucrose-gradient were isolated using an established collagenase liberation cryoprotection. Serial 14-mm cryosections of different protocol (26). Upon 50 min of digestion in collagenase WAT depots were prepared using an HM 560 cryostat type I/HEPES buffer at 37°C, 2 3 104 or 5 3 104 cells/cm2 (Thermo Scientific, Waltham, MA) and stored at 280°C from iWAT or eWAT, respectively, were plated in until further processing. To determine the size of the fat DMEM/Ham F12 with 5% FCS. At 90–100% cell conflu- cells before and after the administration of different di- ence, maturation was induced by adding induction me- ets, their perimeter was determined from digital images of dium containing insulin (170 nmol/L), dexamethasone the iWAT depot using the AxioVision software package (1 mmol/L), isobutylmethylxanthin (500 mmol/L), and indo- version 4.8 (Carl Zeiss, Oberkochen, Germany). The methacin (30 mmol/L) to eWAT cultures. Differentiation of uncoupling protein 1 (UCP1) expression pattern was com- iWAT preadipocytes was stimulated by adding rosiglitazone pared between iWAT cryosections obtained from HFD-fed (2.5 mmol/L) and triiodothyronine (1 nmol/L) to the adipoqBKL1/L2 and adipoqBK+/L2 mice using a commercial eWAT induction medium. After 48 h of induction, the antibody (dilution 1:400; Santa Cruz Biotechnology, Dallas, medium was exchanged with insulin (170 nmol/L)–containing TX). BK immunodetection was performed in iWAT and medium in DMEM/Ham F12 with 10% FCS for another 48h, eWAT sections derived from 10-week-old global and adi- followed by maintenance medium (DMEM/Ham F12 with pocyte-specific BK mutants and control litters using a 10% FCS). Maintenance medium was changed every second primary antibody specific for the BK channel a-subunit day until cells were mature, usually 14 to 16 days after plating. (dilution 1:1,000) and hormone-sensitive lipase (dilution Triglyceride incorporation was detected by Oil Red O (ORO) 1:400), according to a previously published protocol (27). staining. ORO content was quantified by photometry using In brief, antigen-antibody complexes were detected by the Implen system at 518 nm. Digital images of the matura- a fluorescence-labeled secondary antibody (antirabbit tion process were acquired using AxioVision software version AlexaFluor568; dilution 1:500) using the AxioCam MR system 3624 Adipocyte BK Protects From Overwhelming BW Gain Diabetes Volume 65, December 2016

2DD (Carl Zeiss) or by appropriate secondary antibodies con- RT-PCR was performed using the comparative 2 C(t) method, jugated to horseradish peroxidase (dilution 1:500). as above, with mRNA levels normalized to importin-8 (Ipo8). Respective primers were used: BK forward: 59-GTCTCCAAT Adipose Tissue Mass and Total Body Composition GAAATGTACACAGAATATC-39; BK reverse: 59-CTATCATCAG To determine total fat mass and mass of different individual GAGCTTAAGCTTCACA-39; GLUT4 using the Quantitect As- fi depots, fat pads were dissected un xed upon dietary feeding say (Mm_Slc2a4_2_SG); and Ipo8 using Quantitect as- then weighed, immediately immersed in liquid nitrogen, and say (Mm_Ipo8_2_SG) from Qiagen. finally stored at 280°C for further analysis. For analysis of total body composition, total BW was determined before and 3T3-L1 Culture and Differentiation fi after the corpse was dried for 24 h at 85°C. Water content Undifferentiated (UD) 3T3-L1 broblasts were maintained in was calculated as the difference between dried corpse weight DMEM containing 10% FBS and 1% penicillin/streptomycin fi and total weight of the corpse at death. Fat content was at 37°C at 10% CO2; the broblasts were passaged every – fl determined in dried corpses using the Soxhlet procedure 4 days after reaching 60 80% con uence. Differentiation fl according to a previously published protocol (28). was initiated in con uent monolayers in DMEM/FBS containing 1 mg/mL insulin, 0.25 mmol/L dexamethasone, Blood Parameters and Glucose Tolerance Test 1mmol/L troglitazone, and 500 mmol/L isobutylmethyl- Blood was collected from mice before or after dietary feeding. xanthine for 3 days, followed by DMEM/FBS containing Leptin, interleukin (IL)-6, and adiponectin concentrations 1 mg/mL insulin and 1 mmol/L troglitazone. After 3 days were measured in serum samples using mouse immunoassay the cell medium was replaced with DMEM/FBS, and the kits (Merck Millipore, Darmstadt, Germany), according to medium was refreshed every 3 days until differentiated (DF) manufacturer’s instructions. Blood glucose was determined cells were analyzed (typically 10–14 days after differentia- using the GlucoCheck ADVANCE system (TREND Pharma tion was initiated). GmbH, Saalfeld/Saale, Germany) immediately before an in- Immunoblot Analysis of iWAT- and 3T3-L1–Derived traperitoneal glucose challenge (2 g/kg of body-weight) and Protein Lysates 15, 30, 60, and 120 min after the injection in mice fasted iWAT was dissected and processed as described above to overnight. At each time point, additional blood (25 mL) was generate total protein lysates for subsequent immunoblot collected via the tail vein for subsequent insulin determi- analyses (27). Total protein was obtained from UD and nation. Plasma insulin was measured using the Ultrasen- DF 3T3-L1 fibroblasts. The proteins were separated by sitive Mouse Insulin ELISA (Mercodia, Uppsala, Sweden), molecular weight via gel electrophoresis using 12% SDS according to the manufacturer’s instructions. gels. For immunodetection, various primary antibodies were mRNA Expression Analysis used: UCP1 (dilution 1:1,000; Santa Cruz Biotechnology), Total mRNA was extracted from WAT samples by a GAPDH (dilution 1:1,000; Cell Signaling Technologies), guanidineisothiocyanat-phenol-chloroform extraction protocol GLUT4 (1:1,000; a gift from Gwyn Gould, Glasgow, using peqGold RNAPure (Peqlab Biotechnologie, Erlangen, U.K.), BK (1:1,000; NeuroMab, University of California, b Germany), according to the manufacturer’s instructions. Davis, Davis, CA), and -actin (1:500; Sigma-Aldrich, Following DNase treatment for 30 min to remove traces St. Louis, MO). of genomic DNA, RNA samples were quantified; 0.5 mg Statistical Analysis RNA was used to generate cDNA using an iScript cDNA Statistical analysis was performed on experimental data synthesis kit (Bio-Rad, Hercules, CA). Quantitative real-time using a two-tailed Student t test for paired or unpaired 2DDC(t) PCR was performed in triplicate using the comparative 2 comparison or ANOVA with the post hoc Dunnett test for method, with C(t) indicating the cycle number at which multiple comparisons, where appropriate. All data are pre- the signal of the PCR product crosses the threshold, set sented as mean 6 SEM. For all tests, P values less than within the exponential phase of the detected fluorescence 0.05 were considered as significant. signal. BK and IL-6 levels were normalized to b-actin to determine their relative quantities. Respective primer sequences RESULTS were designated: BK forward: 59-ACGCCTCTTCATGGTCTTC- BW gain was investigated in global BK-deficient (BKL1/L1) 39, BK reverse: 59-TAGGAGCCCCCGTATTTCTT-39; IL-6 and age- and litter-matched male wild-type mice (BK+/+) forward: 59-CTTCAACCAAGAGGTAAAAG-39,IL-6reverse: (Fig. 1A) receiving either a purified, low-fat CD or an HFD. 59-CCAGCTTATCTGTTAGGAGAG-39. To extract total mRNA Before the different diets were administered, we observed from 3T3-L1 cells or preadipocytes and differentiated adi- a small but highly significant (P , 0.001) difference in the pocytes in culture, the Roche High Pure mRNA Isolation BWs between the two genotypes, as previously reported Kit was used according to the manufacturer’s instructions (29,30). Starting with 5 weeks of HFD feeding, however, a following shearing of cells in lysis buffer using a 25-gauge substantial weight gain in male BK+/+ mice became appar- needle. Following DNase treatment as described above, ent, resulting in a total BW gain of 16.44 6 0.83 g for 0.25 mg RNA was used to generate cDNA using the Roche BK+/+ mice at the end of an 18-week HFD feeding protocol Transcriptor High Fidelity cDNASynthesisKitwithrandom (Supplementary Fig. 1A), whereas BKL1/L1 mutants gained hexamersandoligo-dTusedata2:1ratio.Quantitative 8.37 6 0.45 g during the same observation period. diabetes.diabetesjournals.org Illison and Associates 3625

Figure 1—Global lack of BK channels protects from overwhelming BW gain and excessive fat accumulation. A:BWincreaseofBK+/+ (blue) and BKL1/L1 (red) mice that received a CD (n =12–14 mice per time point; open data points) or 45% HFD (n =23–25 mice per time point; solid data points) for 18 weeks. B: Body composition of BK+/+ and BKL1/L1 mice after 18 weeks of dietary feeding was analyzed using the Soxhlet extraction method (n =4–7micepergenotype).C: Representative images of BK+/+ and BKL1/L1 mice after 18 weeks of CD or HFD feeding. D:Fatmassof different WAT and BAT depots of BK+/+ and BKL1/L1 mice fed either a CD (n =6–9 mice per fat depot) or HFD (n =9–16 mice per fat depot). Fat masses were normalized to TL (Supplementary Fig. 1F) to overcome growth differences between BK+/+ and BKL1/L1 mice that are not related to an increase in body adiposity. Relative mRNA expression of the BK-channel a-subunit in eWAT (E) and subcutaneous iWAT (F) of 10-week-old BK+/+ and BKL1/L1 mice. GAPDH mRNA levels were used as a reference to normalize the data, which are presented as means 6 SEMs. The statistical difference between the two genotypes is indicated: *P < 0.05, **P < 0.01. Representative cryosections of eWAT (G)andiWAT(H)stainedfortheBK a-subunit (top panels) using a speciﬁc BK channel antibody. Frozen tissue sections without the primary BK antibody were processed as a negative control (bottom panels). Scale bars = 200 mm. Data were analyzed using two-way ANOVA (A)ortheStudentt test (B and D–F) and are presented as means 6 SEMs. Statistical difference between the two genotypes is indicated: *P < 0.05, **P < 0.01, ***P < 0.001. intWAT, interscapular WAT; intBAT, interscapular BAT; mesWAT, mesenteric WAT.

Importantly, the differences in BW gain among BKL1/L1 and HFD-fed BKL1/L1 mice did not differ (Fig. 1B), sup- mutants were about ;10% for the groups fed the CD porting the notion that the genotype-specific differences (33.34 6 2.38%) and the HFD (44.26 6 2.68%), whereas in BW upon CD and HFD feeding (Fig. 1A) are largely the the respective difference in the diet-induced BW gain for result of reduced accumulation of fat in the absence of BK. BK+/+ mice was 27.76% (Supplementary Fig. 1B). Neither Less total body fat mass of CD- and HFD-fed BKL1/L1 mice daily food intake of BK+/+ and BKL1/L1 mice under CD or was accompanied by a small but significant shift toward HFD conditions (Supplementary Fig. 1C) nor the activity lower dry mass values and reduced tibial length (TL) (Sup- or body temperature of the animals under HFD conditions plementary Fig. 1F), both of which confirm the lower over- differed significantly (Supplementary Fig. 1D and E), sug- all body size in the global absence of BK channels (Fig. 1C). gesting that the lean phenotype of the BKL1/L1 mice was, Analyses of fat mass normalized to TL revealed significant although not primarily, the result of abnormal hyperactiv- weight differences for various WAT depots as well as ity or dysfunction in the central control of peripheral cir- interscapular brown adipose tissue (BAT), indicating cuits regulating food intake and reward behaviors. Body that the lean phenotype of the BKL1/L1 mice was related composition revealed that the HFD did not affect wet to a decrease in multiple cell populations in adipocyte body mass and carcass dry mass, but rather stimulated tissue (Fig. 1D). So far, the data suggest that high-caloric a substantial increase in the total body fat mass in BK+/+ nutrition-induced BW gain requires functional BK channels. mice, whereas the respective body composition of CD- In addition, the reduced TL in the global BK knockouts is in 3626 Adipocyte BK Protects From Overwhelming BW Gain Diabetes Volume 65, December 2016 accordance with previous reports, which collectively imply was induced at 90–100% confluence of BK+/+ and BKL1/L1 a mild growth defect that may contribute, at least in part, precursor cells derived from either eWAT or iWAT depots. to the differences observed between BK+/+ and BKL1/L1 ORO incorporated into lipid droplets revealed strong accu- BW during dietary feeding (30). We next tested whether mulation of lipid-filled adipocyte-like cells under adipogenic BK plays a role in the excessive fat accumulation in the maintenance conditions in cell cultures derived from both monogenic ob/ob model of morbid obesity. BW gain (Sup- BK-negative and BK+/+ eWAT (Fig. 2A–C) and iWAT (Fig. plementary Fig. 2A), body composition (Supplementary 2D–F). In iWAT-derived cultures we did not observe differ- Fig. 2B and C), and individual weights of different fat ences in adipogenic differentiation among genotypes (Fig. depots (Supplementary Fig. 2D) were studied in BK-negative 2D–F), whereas significantly more ORO was incorporated ob/ob double-mutants and age- and litter-matched BK+/+ ob/ob in BKL1/L1 than BK+/+ cultures established from eWAT controls.InlinewiththeHFD-fedBK+/+ and BKL1/L1 mice, we (Fig. 2A–C), suggesting a depot-specificroleforBK in found that key parameters of the progressively developing in vitro lipid storage. Moreover, genetic and pharmacolog- superobese phenotype, including BW, body composition, ical blockades of BK channels enhanced the growth of and fat depot masses, are attenuated in the absence of eWAT-derived preadipocytes (Fig. 2G and H). BK (Supplementary Fig. 2A–D). By contrast, differences To further validate differentiated adipocytes from eWAT in BW gain between lean control mice in the absence express functional BK channels, we assayed BK mRNA, pro- (D16.83 6 0.55 g) and presence (D15.73 6 0.55 g) of tein, and ionic currents in the differentiated cultures. BK BK were not significant (P = 0.17). Protection against over- channel, GLUT4 transporter, mRNA, and protein were whelming BW gain in the BK-deficient ob/ob model was strongly upregulated in differentiated cells compared with related to a consistent effect on total fat masses and nonfat precursor cells (Fig. 2I and J). Importantly, outward potas- components of the body (Supplementary Fig. 2B). Indeed, sium currents that are sensitive to the specificBKchannel lower initial and final BWs of BK-negative lean and ob/ob blocker paxilline (1 mmol/L) were significantly upregulated double-mutant mice (Supplementary Fig. 2A), as well as in differentiated adipocytes (Fig. 2K). Increases in BK chan- differences in the mean TL (Supplementary Fig. 2E), sug- nel mRNA, protein, and currents were also observed in the gest that BK plays a role in normal growth (Supplementary established 3T3-L1 preadipocyte cell model following the Fig. 2) and morbid obesity resulting from leptin deficiency. induction of differentiation (Supplementary Fig. 4A–C). In Given the previous reports of the role of K+ channels in addition, the inhibition of BK channels with paxilline in- adipocytes (6,7) and our consistent observation of lower creased the growth of 3T3-L1 preadipocytes and the fat masses of multiple fat depots (Fig. 1D and Supplemen- amount of ORO incorporated into lipid droplets (Supple- tary Fig. 2D), we considered that protection against over- mentary Fig. 4D and E). Taken together, these data suggest whelming weight gain stemmed from the adipocyte itself. that fat cell BK channels control preadipocyte expansion To address this possibility, we first assessed the BK mRNA and adipogenic conversion of preadipocytes in an adipocyte and protein expression in iWAT and eWAT. Fat pads were depot–specific manner. studied using primer pairs, allowing for specificdetection Because homeostasis of mature adipocytes and adipo- and quantification of BK mRNA (Fig. 1E and F). Compared cyte differentiation in vivo are distinct from the above- with the internal reference, BK levels in iWAT and eWAT studied in vitro models, we next studied the lean depots were low in BK+/+ mice, but reliable amplification phenotype by generating an adipocyte-specific BK knock- was accomplished in all samples tested. Importantly, signal out mouse model (adipoqBKL1/L2). In an initial series of traces detected in the respective fat depots derived from experiments we confirmed the tissue-specificrecombina- BKL1/L1 mice were well within the range of nonspecificPCR tion efficacy of the recently established adiponectin product formation in the absence of reverse transcriptase promotor–driven, tamoxifen-inducible CreERT2 mouse (Supplementary Fig. 3A and B). Because adipose tissue is model (23) using a two-color fluorescent Cre reporter heterogeneous, comprising fat and nonfat cells, we next system (24). Before Cre-mediated excision, we observed examined the cellular distribution of BK channel protein ubiquitous expression of the cell membrane–targeted in frozen adipose tissue sections obtained from 10-week-old red fluorescent protein Tomato (mT) in eWAT, iWAT, BK+/+ and BKL1/L1 mice. These analyses revealed evidence and all other tissues studied (Fig. 3A and B and data not for BK in unilocular BK+/+ cells, characteristic of white shown). Tamoxifen-induced Cre-mediated excision of adipocytes, whereas BKL1/L1 iWAT and eWAT remained the floxed mT led to an almost complete switch to green BK-negative (Fig. 1G and H). Importantly, our specific fluorescent protein (mG) expression in eWAT and iWAT BK antibodies and an antibody for the white fat cell fatcells,whereaschangesinmTlabelingwerenotob- marker hormone-sensitive lipase marked the same cells served in various other organs such as the brain, skel- in BK+/+ eWAT (Supplementary Fig. 3C), supporting the etal muscle, and liver (data not shown). High efficiency notion that mature adipocytes express BK channels. and specificity of the adiponectin-CreERT2 approach was To explore the functional attributes of endogenous BK further confirmed by a BK-specific primer set designed channels in adipocytes, we adopted a previously established to identify the three different BK alleles—wild-type protocol of primary culture and differentiation of murine (+), floxed (L2), and knockout (L1)—within one sam- adipocyte precursor cells (26). Adipocyte differentiation ple. In line with the Cre reporter assay (Fig. 3A and B), diabetes.diabetesjournals.org Illison and Associates 3627

Figure 2—Accelerated growth of BK-negative eWAT-derived preadipocytes. Representative ORO staining of eWAT-derived preadipocytes at a high magnification (A) and as an overview screen (B) after 14 days of adipogenic differentiation, revealing higher numbers of mature adipocytes in BKL1/L1 in comparison with BK+/+ cells. C: The increased adipogenic potential in BKL1/L1 mice (red) was confirmed by quantitatively analyzing ORO incorporation at 518 nm (n = 3 independent in vitro assays per genotype). *P < 0.05. D and E: Representative ORO staining of iWAT-derived preadipocytes after 14 days of adipogenic differentiation. Scale bars = 100 mm(A and D). E and F: On the basis of the ORO incorporation assay, maturation of iWAT-derived BK+/+ and BKL1/L1 preadipocytes was not different between the genotypes (n = 7–8 independent in vitro assays per genotype). G: Representative growth of eWAT-derived preadipocytes examined in real time using an impedance-based setup. Lack of BK channels (red) in eWAT-derived preadipocytes resulted in accelerated growth properties at different cell concentrations tested compared with wild-type cells (blue). H: A pharmacological approach to blocking the BK channel using 1 mmol/L paxilline (PAX) confirmed the BK-dependent growth capacity of eWAT-derived preadipocytes. Upregulation of GLUT4 and BK channel mRNA (I)andprotein(J) levels following the differentiation of eWAT murine preadipocytes (preadipo) to adipocytes (adipo) (n = 3 independent experiments per group). K: Preadipocyte cells display a small paxilline-sensitive (1 mmol/L) outward whole-cell BK potassium current determined under physiological potassium gradients (n = 8 cells) that was significantly increased upon adipogenic differentiation for 14 days (n = 12 cells). neg. Ctr, negative control. 3628 Adipocyte BK Protects From Overwhelming BW Gain Diabetes Volume 65, December 2016

Figure 3—Spatiotemporal ablation of fat cell BK channels using the adiponectin-CreERT2tg/+ recombination system. Recombination efficiency was determined by a change from red (cyanine dye [Cy3] channel) to green (fluorescein isothiocyanate [FITC] channel) fluorescence using the ROSA26mTomato,mEGFP/+ (tom/+) Cre-reporter mouse line. Analysis of eWAT (A) and subcutaneous iWAT (B) derived from double-transgenic adiponectin-CreERT2tg/+;ROSA26mTomato,mEGFP/+ (tg/+;tom/+) mice with or without tamoxifen (TAM) injections compared with wild-type age- matched littermate male control mice (+/+;+/+). C: Genomic PCR analysis of the adiponectin-CreERT2tg/+-mediated recombination of the premutant BK gene locus in different tissues. PCR products amplified from the loxP-flanked (L2 allele with two loxP sites; two black triangles flanking a black box), wild-type (+ allele; solid black box), and knockout (L1 allele with one loxP site; one black triangle) alleles of representative adiponectin-CreERT2tg/+;BK+/L2 (adipoqBK+/L2) animals. Conversion of the loxP-flanked L2 allele to the L1 allele was only observed in fat cell depots after injecting TAM (+), whereas with saline (2)treatmentwedidnotfind evidence for unspecific Cre activity. Recombination was analyzed 14 days after beginning the TAM or saline injections in 10-week-old mice. The size of the PCR amplicons were 577, 466, and 132 bp for the floxed, wild-type, and knockout alleles, respectively. P, pancreas; L, liver; H, hypothalamus; B, bulb; C, cerebellum; Br, rest of brain; He, heart; S, skeletal muscle; D, duodenum; M, marker. D: Quantitative mRNA analysis of the BK mRNA in eWAT and iWAT of TAM-injected adiponectin-CreERT2tg/+;BK+/L2 control mice (n = 5 for eWAT and iWAT) and age- and litter-matched adiponectin-CreERT2tg/+;BKL1/L2 tissue- specific mutants (n =7foreWAT;n =4foriWAT).*P < 0.05; **P < 0.01. Analysis of the frequency and distribution of the Cre-mediated recombination after TAM injections in eWAT (E) and iWAT (F) of adiponectin-CreERT2tg/+;BK+/L2 (adipoqBK+/L2) and adiponectin-CreERT2tg/+; BKL1/L2 (adipoqBKL1/L2)miceusingaspecific BK channel antibody (+anti-BK; top panels). A parallel analysis of serial eWAT and iWAT sections from both genotypes was performed to establish the background fluorescence in the absence of BK-specific antibodies (anti-BK; bottom panels). Scale bars = 50 mm.

PCR products indicative of a recombination event were conditions in the absence of changes in satiety or adipos- only observed in WATs and BATs of adiponectin-CreERT2 ity signals arising from endocrine or neuronal circuits transgenic BK+/L2 mice (adipoqBK+/L2), whereas analysis of potentially involving hypothalamic BK channels, among multiple other cell types and organ systems did not reveal others. To induce site-specific recombination of the L2 significant Cre activity, regardless of whether tamoxifen BK gene locus, adipoqBKL1/L2 mice and adipoqBK+/L2 con- was applied (Fig. 3C). Compared with adipoqBK+/L2 control age-matched mice and littermates were subjected to trols, BK mRNA expression levels in eWAT and iWAT the tamoxifen injection 2 weeks before the experimental were reduced in tissue-specific adipoqBKL1/L2 mutants upon diets were administered, that is, a CD or an HFD with 60% tamoxifen application (Fig. 3D). Accordingly, adiponectin- of its calories derived from fat (Fig. 4A and B). Consistent CreERT2 activation by tamoxifen resulted in a nearly com- with our previous findings in the global BKL1/L1 model plete ablation of the BK protein in the different WAT (Supplementary Fig. 1C), the tissue-specificablationof tissues (Fig. 3E and F). Hence the adipocyte-specificknock- BK did not affect the amount of food (CD or HFD) con- out model of BK should allow us to test the role of fat cell sumed by the animals at the start or end of the feeding BK channels for BW gain under CD and HFD feeding experiment (Fig. 4C and D). Starting BWs and BW gain in diabetes.diabetesjournals.org Illison and Associates 3629

Figure 4—Reduced BW gain in adipoqBKL1/L2 mice receiving a 60% HFD. A: CD feeding protocol. Male adipoqCreERT2tg/+;BKL1/L2 (adipoqBKL1/L2) and adipoqCreERT2tg/+;BK+/L2 (adipoqBK+/L2) 8-week-old mice were injected with tamoxifen (TAM) before the dietary feeding for 5 consecutive days, which induced a specific and efficient ablation of the BK channel in various adipose tissues (see also Fig. 3). Upon 2 weeks of adaption to the CD, CD feeding was continued or mice revived an HFD for 18 weeks (prevention study) (B). In an alternative approach, additional groups of adipoqBKL1/L2 and adipoqBK+/L2 mice were subjected to the same feeding protocol, but TAM L1/L2 +/L2 was injected at an age of 19 weeks (t19) when overweight was already established. Food intake of adipoqBK (red) and adipoqBK (blue) mice were analyzed using modified metabolic cages, as described in the RESEARCH DESIGN AND METHODS, either at the beginning (n =4–6 mice per genotype) (C) or at the end (n =3–5 mice per genotype) of the respective dietary feeding (D). BW development under CD feeding (n =12–20 per genotype per time point) (E) and HFD feeding (n =24–29 mice per genotype per time point) (F). Difference (DBW) between the initial BW (at week 10) and the BW at week 30 in adipoqBKL1/L2 (red) and adipoqBK+/L2 (blue) mice under CD (G) and HFD (H) feeding conditions (n =12–15 per genotype for CD; n =24–26 per genotype for HFD). I: TAM was injected for 5 consecutive days when the mice were 19 weeks old, when an HFD-induced BW gain in adipoqBKL1/L2 (red) and adipoqBK+/L2 (blue) mice was already established, to test the therapeutic potential of WAT BK inactivation (n =17–22 mice per genotype per time point). The final BW between the two genotypes was significantly different (*P < 0.05). the CD-fed group were not different between adipoqBK+/L2 at an age of 19 weeks, when the adipoqBK+/L2 control and and adipoqBKL1/L2 mice (Fig. 4E and G), whereas the lack of adipoqBKL1/L2 pre mutants had reached a BW of 32.35 6 1.05 adipocyte BK channels afforded partial protection against and 31.91 6 0.89 g, respectively. Total BW gain was not HFD-induced BW gain (Fig. 4F and H), an effect that different between age- and litter-matched adipoqBK+/L2 and fi L1/L2 reached a signi cant level after 4 weeks of HFD feeding adipoqBK mice before the tamoxifen treatment (weeks10–19 +/L2 and was maintained until the mice reached their final BWs 8.18 6 1.00 g for adipoqBK ;weeks10–19 7.71 6 0.76 g (Fig. 4F), indicative of a lower susceptibility of adipoqBKL1/L2 for adipoqBKL1/L2 [P = 0.7]), whereas upon five repetitive mutant mice to high-calorie challenges. tamoxifen injections, the extent of the weight gain showed We next tested whether excess BW gain is also affected a clear tendency toward lower values in adipoqBKL1/L2 mice L1/L2 by fat cell BK in mice that have already gained significant (Fig. 4I)(weeks20–30 5.33 6 0.56 g for adipoqBK ; +/L2 weight. To test this, tamoxifen treatment was commenced weeks20–30 7.40 6 1.03 g for adipoqBK [P , 0.06]). 3630 Adipocyte BK Protects From Overwhelming BW Gain Diabetes Volume 65, December 2016

In-depth analyses of the individual fat depots (Fig. 5A adipocytes were smaller upon CD and HFD feeding (Fig. and G) and the total fat mass (Fig. 5B) did not reveal 6A and C). statistical weight differences between adipoqBK+/L2 and Because enlarged, hypertrophic fat cells in the adipose adipoqBKL1/L2 mice fed a CD diet. HFD feeding, however, depot relate to obesity, inflammation,andinsulinresistance, confirmed protection against excessive fat storage in we next tested whether the observed changes in adipocyte adipoqBKL1/L2 mice. Interestingly, the lack of adipocyte morphology (Fig. 6A–C) were associated with a more BK was associated with smaller total fat masses and proinflammatory state. Indeed, we found reduced IL-6 smaller masses of various fat depots (Fig. 5C–G). mRNA expression in iWAT of HFD-fed adipoqBKL1/L2 Because fat mass is determined by both adipocyte mice and tendentially lower serum IL-6 concentrations (P = number and size, we next studied fat storage at the cellular 0.11) (Fig. 6E). Moreover, HFD-fed adipoqBKL1/L2 mice level. iWAT cell size was calculated by assessing the cell exhibited lower serum leptin concentrations as a marker perimeters in different cryosectional areas obtained from of body mass (Fig. 6F), whereas adiponectin concentrations adipoqBK+/L2 and adipoqBKL1/L2 mice. Before the different were not different between diets or genotypes (Fig. 6G). In diets were administered (i.e., at an age of 10 weeks) accordance with the markers of inflammation, small adipose genotype-specific differences in fat cell size were not de- tissue mass, and low fat cell hypertrophy, adipoqBKL1/L2 tectable in different areas of the iWAT sections (Fig. 6A mice showed improved glucose clearance (Fig. 6H)with and C). Compared with adipoqBK+/L2 fat cells, BK-deficient lower insulin concentrations (Fig. 6J)uponintraperitoneal

Figure 5—Reduced fat accumulation and smaller fat depots in adipoqBKL1/L2 mice fed a 60% HFD. Mass of different WAT and BAT depots of male adipoqBKL1/L2 (red) and adipoqBK+/L2 (blue) mice examined after 18 weeks of CD (A) or HFD (B) feeding (n =12–15 per genotype for CD; n =24–26 per genotype for HFD). Body composition of adipoqBK+/L2 and adipoqBKL1/L2 at an age of 30 weeks after CD (C) and HFD (D) feeding. Total body fat was extracted using the Soxhlet extraction method (n = 3 per genotype for CD; n =4–7 per genotype for HFD). E– G: Representative images of adipoqBK+/L2 and adipoqBKL1/L2 mice and selected fat depots after 18 weeks on CD: views from the back (E), the front with an open abdominal cavity (F), and magniﬁcation of BAT, eWAT, and iWAT (G). All data were analyzed using the Student t test and are presented as means 6 SEMs (*P < 0.05; **P < 0.01). Scale bars = 2 cm. intAT, total interscapular adipose tissue; intBAT, interscapular BAT; intWAT, interscapular WAT; mesWAT, mesenteric WAT; total, total investigated adipose depots. diabetes.diabetesjournals.org Illison and Associates 3631

Figure 6—Fat cell hyperplasia and improved glucose handling in adipoqBKL1/L2 mice. A: Average cell size of iWAT of male adipoqBK+/L2 and adipoqBKL1/L2 mice after 18 weeks of HFD was examined in serial cryosections by measuring the circumference of cells in the central and peripheral regions of the respective depots. B: Classiﬁcation of average cell sizes (as an indicator of adipose cell number) in adipoqBK+/L2 and adipoqBKL1/L2 iWAT, before a diet (PD) and after 18 weeks of the CD or 60% HFD feeding. C: Representative cryosections of iWAT derived from adipoqBK+/L2 (blue) and adipoqBKL1/L2 (red) PD or after 18 weeks of either CD or HFD. Scale bar = 100 mm. Relative mRNA-expression (D) and serum concentrations (E) of IL-6, a marker for adipose tissue inﬂammation, and serum concentrations of leptin (F) and adiponectin (APN) (G) determined after 18 weeks of CD (open bars) and/or HFD (solid bars) feeding in adipoqBK+/L2 (blue) and adipoqBKL1/L2 (red) mice. Blood glucose (BG) concentrations were monitored before as well as 15, 30, 60, and 120 min after intraperitoneal injection of 2 g/kg glucose after 18 weeks of HFD feeding (H) and before administering the respective diet (I). J: Plasma insulin concentrations at baseline (0 min) and during the intraperitoneal glucose tolerance test (15 to 120 min after injection) in adipoqBKL2/+ (n = 6; blue) and adipoqBKL1/L2 (n =8;red)miceafter 18 weeks of HFD feeding. All data (A, D–J) were examined using the Student t test and are presented as means 6 SEMs. *P < 0.05, **P < 0.01.

glucose challenge after HFD feeding, whereas a lack of adi- brown adipocyte–like fat cells. Higher levels of UCP1 protein pocyte BK did not affect glucose handling before the feeding under HFD conditions were also reﬂected by a signiﬁcant protocol (Fig. 6I). Change in the cellularity of the iWAT may increase in core body temperature during the night (Fig. also be considered an indicator for accelerated browning to 7D), whereas during the day, while the mice were sleeping, promote energy expenditure, which counters obesity and its body temperatures of adipoqBKL1/L2 and adipoqBK+/L2 metabolic consequences. Lack of fat cell BK resulted in mice were lower, suggesting a reduced burning of stored strong induction of UCP1 (Fig. 7A and C) as a hallmark of fats, with no apparent differences between the two ge- uncoupled respiration and heat production by brown or notypes (Fig. 7D). 3632 Adipocyte BK Protects From Overwhelming BW Gain Diabetes Volume 65, December 2016

DISCUSSION Using combined analysis of different BK channel mutant mouse lines we uncovered a novel function for adipocyte BKs in fat cell biology and metabolism under different nutritional conditions. We found resistance to HFD-induced BW gain, a smaller total fat mass, and thereby improved glucose handling upon ablation of endogenous BK channels in various adipose depots in different parts of the body. These data imply that the development of obesity caused by nutrient excess is promoted by the BK channel (Fig. 4). Accordingly, a previously identified BK gene variant was associated with elevated levels of fat cell BK mRNA in morbidly obese human subjects, suggesting a causal relationship between the amount of adipocyte BKs and weight gain (6). To the best of our knowledge, BK levels in other organ systems of the affected patients have not been assessed; therefore, it remains unclear whether the obesogenic effects attributed to amplified BK expression resulted exclusively from an effect on adipocyte cell function or whether BK channels present in nonadipocyte cells contributed to morbid weight gain. Interestingly, our HFD-fed BKL1/L1 mutants did not exhibit any changes in dietary food consumption or body temperature (Supplemen- tary Fig. 1C and D), whereas the respective fat cell–specific mutants exhibited a lean phenotype that was related to an increase in UCP1 level and energy expenditure (Fig. 7). It is therefore tempting to speculate that BK channels in the brain or other nonfat cells do not play a role in the chemical and neural signals regulating calorie intake and/or energy expenditure and thereby body composition. For several reasons, however, we find it is too early to draw such conclusions.

1) Our previous analyses of the global BKL1/L1 mouse Figure 7—Increased UCP1 levels and body temperature in HFD-fed model revealed multiple defects in various systems of adipoqBKL1/L2 mice. A: UCP1 expression analysis (brown staining these animals, including in b-cells (15), the HPA axis in the left panels) in frozen tissue sections derived from HFD-fed (14), and cerebellar neurons (29), which separately or +/L2 L1/L2 adipoqBK and adipoqBK iWAT. Sections derived from the together could affect energy balance and BW develop- same fat pads were processed in the absence of the primary UCP1 antibody as the negative control (right panels). Scale bars = 100 mm. ment. For example, a lack of cerebellar BK channels B: Representative UCP1 immunoblot of iWAT protein lysates derived in Purkinje cells, among other deficits, was shown to +/L2 L1/L2 from HFD-fed adipoqBK and adipoqBK mice. Equal loading cause motor learning impairment and signs of ataxia, of the gel was verified by codetection of GAPDH. C: Quantification of the immunoblot data shown in B. UCP1 protein levels were normal- with the latter being related to muscle shivering and ized to the expression of GAPDH in adipoqBK+/L2 (n =3;solidblue trembling (29), a common cause of increased energy ex- bars) and adipoqBKL1/L2 (n = 4; solid red bars) iWAT samples. D: penditure. Irrespective of the dietary fat content, we did Telemetric sensors were used to measure the core body temper- +/L2 L1/L2 not observe an increase in the core body temperatures of ature of a subset of adipoqBK and adipoqBK mice that L1/L1 werefedtheHFD.Alldata(C and D) were assessed using the BK mice (Supplementary Fig. 1D), suggesting that Student t test and are presented as means 6 SEMs. *P < 0.05, their very low body fat (Fig. 1) allows them to maintain **P < 0.01. their body temperature at a physiological level. Potential changes in body temperature regulation (i.e., adjustments due to changes in [non]shivering thermogenesis) may be superimposed by the loss of heat in BKL1/L1 mutants. Yet In summary, adipocyte BKs promote fat cell size and fat BKL1/L1 mice present with a lean phenotype with CD and pad mass in vivo. Adipocyte tissue growth in the presence HFDfeedingregimes,andweobservedalowerpropen- of endogenous BK channels was related to a noninfectious sity of the BK/ob double-mutants to accumulate excessive activation of adipose tissue inflammation and metabolically BW and fat mass. This suggests that the global lack of BK unfavorable effects on fat cell functions, which together channels for the entire life span prevents excessive BW may result in insulin resistance and amplified HFD-induced gain,aneffectthatwasmoreobviousinthepresenceof adiposity. genetic or nutritional risk factors for developing obesity. diabetes.diabetesjournals.org Illison and Associates 3633

In this complex mouse model, the dysregulation of mul- both eWAT fat cell precursors and the preadipocyte 3T3- tiple pathways (e.g., in fat cells, hypothalamus, liver, L1 cell line (Fig. 2K and Supplementary Fig. 4C). Pharma- sympathetic neurons, b-cells) involving BK may be re- cological blockade of BK channels stimulated both growth lated to this phenotype. and lipid incorporation, supporting a role for BK channels 2) The protection in terms of BW gain and fat accumulation as regulators of cell cycle progression in human preadipo- waslesspronouncedinadipocyte-specificBKknockout cytes (7). By assessing BK-deficient eWAT- and iWAT-derived groups compared with the BKL1/L1 or BK/ob double-mutant preadipocytes in vitro, however, we recognized fat depot– models (compare Fig. 1A and Supplementary Fig. 2A and specific functions for endogenous BK channels potentially Fig. 4F), implying that nonfat cell BKs are also impor- involved in the control of cell growth and lipid accumu- tant for weight control in vivo. Indeed, dietary-related lation (Fig. 2). Because adiposity is usually induced changes in energy expenditure involving, for example, through hypertrophic expansion of existing adipocytes, hypothalamic control mechanisms may be positively or leading to dysfunctional cells, an increase in the number negatively regulated by BK channels of neuronal nuclei of fat cells (i.e., hyperplasia) is proposed as a mechanism that respond to satiety or hunger signals. Lack of GIRK4, that preserves metabolic fitness (39). Accordingly, lower aGprotein–gated, inwardly rectifying K+-channel, in mice, levels of the proinflammatory cytokine IL-6 (Fig. 6D and for instance, resulted in late-onset obesity through hypo- E), significantly lower BW (Fig. 4F) and smaller iWAT cell thalamic mechanisms (31), whereas ATP-sensitive K+ chan- size together support the notion that a lack of BK leads to “ ” nel (KATP) knockout mice showed hyperphagia but were the production of more healthy adipocytes that may resistant to HFD-induced BW and visceral fat mass gains afford protection against excessive BW gain, thereby (32).Alongtheselines,KATP conductance in diet-induced maintaining a regular response to the action of insulin. obesity has been reported in pro-opiomelanocortin-positive Adipocytes reportedly produce and release IL-6 (40). In nuclei of the hypothalamus, and neuronal excitability obesity the fraction of adipose tissue–derived IL-6 may and thereby the release of peptides that control food promote a chronic state of low-grade inflammation, af- intake and BW were sensitive to central KATP inhibition fecting metabolism and the development of insulin resis- L1/L2 (33). In addition to KATP,BKchannelshavebeenshown tance (41). In comparison with adipoqBK mutant to modulate the excitability of hypothalamic neurons in mice, we found higher IL-6 adipose tissue mRNA (P , response to insulin and leptin under physiological con- 0.05) and nonsignificantly elevated serum concentrations ditions (34), suggesting they may be involved in central (P = 0.11) in HFD-fed adipoqBK+/L2 control mice; hence, it circuits regulating BW via energy intake and expendi- seems that fat cell BK channel activity is positively related ture. Given the complexity of the different metabolic to the adipose tissue IL-6 pathway, providing a link be- pathways, future studies are needed to clarify the func- tween BK and obesity-related comorbidities such insulin tional roles of hypothalamic and other nonfat cell BK resistance (Fig. 6D, E, and H). Interestingly, high plasma channels in adiposity and thereby the mechanism(s) and adipose tissue concentrations of IL-6 were previously underlying the lean phenotype of BKL1/L1 mice. Resis- reported for the ob/ob mouse model (42), suggesting that tance to genetic and diet-induced models of obesity BK channels may interfere with both the genetic and dietary (i.e., hypothalamic-driven obesity) has also been re- causes of adiposity through an IL-6-dependent mode of ac- ported in mice with a Shaker family voltage-dependent tion. Along the same lines, HFD-induced overnutrition K+ channel gene disruption in Kv1.3 (35,36), further among adipoqBKL1/L2 mice revealed smaller total fat mass highlighting the importance of K+ channels for the and smaller fat cells in iWAT depots. Of note, iWAT cells hypothalamic-regulation of BW (37). More recently, and depots differed in size and mass only in vivo (Fig. 6), however, the therapeutic benefits on obesity and insulin whereas in (pre)adipocyte cultures originating from iWAT resistance upon Kv1.3 inhibition using a blocker that pads, we did not detect differences in terms of various adi- was unable to cross the blood-brain barrier have been pogenic parameters (Fig. 2). The reason for this apparent attributed to peripheral mechanisms stemming from discrepancy is unclear, although several mechanisms could changes in liver and WAT metabolism and BAT (38). be involved. First, the adipocyte-specificlackofBKchannels Obviously, peripheral and central K+ channels affect in vivo results in a decrease in the iWAT depot, but not the the development of adiposity. eWAT depot, fat mass. A “normal” mass of the adipoqBKL1/L2 eWAT depot in vivo (Fig. 5A and C) may result from direct Studying adipocyte-specific BK knockout mice, we did effects of BK on the lipid storage and cell growth capacities; not find any evidence for tamoxifen-induced recombina- both parameters were augmented in the respective BKL1/L1- tion in different brain regions, including the hypothala- derived fat cells in vitro (Fig. 2A–C, G,andH). By contrast, a mus and other tissues involved in total body metabolism, smaller eWAT depot mass in the global BK knockouts such as muscle and liver (Fig. 3), suggesting that partial (Fig. 1D) may result from dysregulation of BK-dependent protection against diet-induced obesity (Fig. 4F) results mechanisms in nonadipocytes. Interestingly, our observa- from the lack of BK channels in adipocytes (Fig. 3). Fur- tions in HFD-fed adipoqBKL1/L2 and adipoqBK+/L2 mice im- ther, we observed amplified BK expression and currents ply a small but significant increase (approximately 0.3°C) with adipogenic differentiation of mouse adipocytes from in the body’s core temperature in the absence of fat cell 3634 Adipocyte BK Protects From Overwhelming BW Gain Diabetes Volume 65, December 2016

BKs (Fig. 7D), which indicates that these channels are reduce the pathological features of excessive weight gain involved in the thermogenic program of adipose cells. The and related disorders. presence of UCP1 in iWAT is characteristic of a process known as “browning,” whereby this depot acquires brown adipocyte–like catabolic functions (43); we found the Acknowledgments. The authors thank Clement Kabagema-Bilan, Isolde adipocyte-specific ablation of BK channels to result in Breuning, Michael Glaser, and Katrin Junger (all from the Department of Pharmacology, elevated mRNA (data not shown) and protein levels of Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, iWAT UCP1 (Fig. 7A–C). The burning of stored fats by Tübingen, Germany) for excellent technical help. expansion or activation of BAT or by browning of WAT Funding. This work was funded in part by the Deutsche Forschungsgemein- schaft (to P.R. and R.L.), the Wellcome Trust (to P.R. and M.J.S.), and Diabetes may result in a metabolically favorable phenotype (44). UK (to L.H.C. and M.J.S.). Accordingly, we observed a large fraction of smaller fat cells Duality of Interest. No potential conflicts of interest relevant to this article in iWAT, smaller iWAT and total fat depot weights, as well were reported. as an improved glucose homeostasis and insulin sensitivity Author Contributions. J.I., L.T., H.M., M.W., and M.J.S. conducted L1/L2 in HFD-fed adipoqBK mice (Fig. 7A–C and H–J). experiments. J.I., M.J.S., and R.L. analyzed data and wrote the manuscript. L.H.C., Clearly, the mechanistic details underlying the lean V.L., S.O., and P.R. contributed to discussions and edited the manuscript. V.L., A.S., phenotype of HFD-fed adipoqBKL1/L2 mutants require future and S.O. contributed new tools. P.R., M.J.S., and R.L. designed the research. All investigation. In general, it is widely accepted that BK chan- authors approved the final manuscript. M.J.S. and R.L. are the guarantors of this nels are activated either by an increase in intracellular Ca2+ work and, as such, had full access to all the data in the study and take 2+ 2+ responsibility for the integrity of the data and the accuracy of the data analysis. ([Ca ]i) or by membrane depolarization. [Ca ]i dynamics have a multifaceted role in brown adipocyte–like functions References and in the formation of mature fat-laden adipocytes 1. Gesta S, Tseng YH, Kahn CR. Developmental origin of fat: tracking obesity 2+ (45–52); however, relatively little is known about Ca sig- to its source. Cell 2007;131:242–256 naling in white and brown adipocytes despite its suggested 2. Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin importance (53). Yet the efflux of K+ via BK in nonexcitable resistance and type 2 diabetes. Nature 2006;444:840–846 cells such as adipocytes may serve as a positive regulator for 3. Choquet H, Meyre D. Genetics of obesity: what have we learned? Curr Ca2+ entry as well as a number of Ca2+-dependent processes, Genomics 2011;12:169–179 whereas in excitable cells BK is usually part of a negative 4. Ramachandrappa S, Farooqi IS. Genetic approaches to understanding – feedback loop limiting the influx of Ca2+ via voltage-dependent human obesity. J Clin Invest 2011;121:2080 2086 5. Sohn JW. Ion channels in the central regulation of energy and glucose Ca2+ channels (54). Transient receptor potential vanilloid fi homeostasis. Front Neurosci 2013;7:85 4 (TRPV4) channels have recently been identi ed as a 6. Jiao H, Arner P, Hoffstedt J, et al. Genome wide association study identifies fl 2+ major feature of the in ux of extracellular Ca into fat KCNMA1 contributing to human obesity. BMC Med Genomics 2011;4:51 cells (55). TRPV4 seems to negatively regulate the ther- 7. Hu H, He ML, Tao R, et al. Characterization of ion channels in human mogenic and proinflammatory programs in adipocytes, preadipocytes. J Cell Physiol 2009;218:427–435 and mice with a TRPV4 ablation were protected from 8. Zhang XH, Zhang YY, Sun HY, Jin MW, Li GR. Functional ion channels and diet-induced obesity; hence TRPV4 deficiency results in cell proliferation in 3T3-L1 preadipocytes. J Cell Physiol 2012;227:1972–1979 a phenotype very similar to that caused by adipocyte- 9. Dopico AM, Bukiya AN. Lipid regulation of BK channel function. Front specific BK ablation. A functional coupling of BK and Physiol 2014;5:312 TRPV4 was reported in different systems (56,57). Al- 10. Martín P, Moncada M, Enrique N, et al. Arachidonic acid activation of BKCa b though this study did not examine this link, we hypoth- (Slo1) channels associated to the 1-subunit in human vascular smooth muscle cells. Pflugers Arch 2014;466:1779–1792 esize that BK may suppress the acquisition of brown 11. Hoshi T, Tian Y, Xu R, Heinemann SH, Hou S. Mechanism of the modulation adipocyte–like features in white fat cells by an effect 2+ of BK potassium channel complexes with different auxiliary subunit compositions on Ca entry and subsequent signaling pathways involv- by the omega-3 fatty acid DHA. Proc Natl Acad Sci U S A 2013;110:4822–4827 2+ ing TRPV4 or other Ca -permeable members of the 12. Lynch FM, Withers SB, Yao Z, et al. Perivascular adipose tissue-derived large TRP channel protein family (50,51,58). adiponectin activates BK(Ca) channels to induce anticontractile responses. Am J Finally, BK channels are common targets of various intra- Physiol Heart Circ Physiol 2013;304:H786–H795 cellular signaling molecules including cGMP and cAMP 13. Climent B, Simonsen U, Rivera L. Effects of obesity on vascular potassium (59,60). It is well established that both cAMP and cGMP path- channels. Curr Vasc Pharmacol 2014;12:438–452 ways play differential roles in lipolysis, browning of WAT, and 14. Brunton PJ, Sausbier M, Wietzorrek G, et al. Hypothalamic-pituitary-adrenal brown fat cell thermogenesis (44,61), among other processes, axis hyporesponsiveness to restraint stress in mice deficient for large-conductance which should allow for complex adjustments of BK function calcium- and voltage-activated potassium (BK) channels. Endocrinology 2007;148: – in the different fat depots in vivo under (patho)physiological 5496 5506 15. Düfer M, Neye Y, Hörth K, et al. BK channels affect glucose homeostasis conditions. and cell viability of murine pancreatic beta cells. Diabetologia 2011;54:423–432 Together, the data presented here show that loss of fat 16. Peters A, Schweiger U, Fruhwald-Schultes B, Born J, Fehm HL. The neuroen- cell BK channels supports the browning of fat cells to docrine control of glucose allocation. Exp Clin Endocrinol Diabetes 2002;110:199–211 enhance thermogenesis in response to nutritional excess, 17. Shanley LJ, O’Malley D, Irving AJ, Ashford ML, Harvey J. Leptin inhibits thereby limiting excessive weight gain. Targeting fat cells epileptiform-like activity in rat hippocampal neurones via PI 3-kinase-driven with BK channel inhibitors may thus have the potential to activation of BK channels. J Physiol 2002;545:933–944 diabetes.diabetesjournals.org Illison and Associates 3635

18. Gavello D, Vandael D, Gosso S, Carbone E, Carabelli V. Dual action of leptin 39. Rutkowski JM, Stern JH, Scherer PE. The cell biology of fat expansion. on rest-firing and stimulated catecholamine release via phosphoinositide J Cell Biol 2015;208:501–512 3-kinase-driven BK channel up-regulation in mouse chromaffin cells. J Physiol 40. Sopasakis VR, Sandqvist M, Gustafson B, et al. High local concentrations 2015;593:4835–4853 and effects on differentiation implicate interleukin-6 as a paracrine regulator. 19. Schneeberger M, Gomis R, Claret M. Hypothalamic and brainstem neuronal Obes Res 2004;12:454–460 circuits controlling homeostatic energy balance. J Endocrinol 2014;220:T25–T46 41. Eder K, Baffy N, Falus A, Fulop AK. The major inflammatory mediator 20. Sausbier U, Sausbier M, Sailer CA, et al. Ca2+ -activated K+ channels of interleukin-6 and obesity. Inflamm Res 2009;58:727–736 the BK-type in the mouse brain. Histochem Cell Biol 2006;125:725–741 42. Harkins JM, Moustaid-Moussa N, Chung YJ, et al. Expression of interleukin-6 21. Chang CP, Dworetzky SI, Wang J, Goldstein ME. Differential expression of the is greater in preadipocytes than in adipocytes of 3T3-L1 cells and C57BL/6J and alpha and beta subunits of the large-conductance calcium-activated potassium ob/ob mice. J Nutr 2004;134:2673–2677 channel: implication for channel diversity. Brain Res Mol Brain Res 1997;45:33–40 43. Bartelt A, Heeren J. Adipose tissue browning and metabolic health. Nat Rev 22. Sausbier M, Arntz C, Bucurenciu I, et al. Elevated blood pressure linked to Endocrinol 2014;10:24–36 primary hyperaldosteronism and impaired vasodilation in BK channel-deficient 44. Pfeifer A, Hoffmann LS. Brown, beige, and white: the new color code of fat and mice. Circulation 2005;112:60–68 its pharmacological implications. Annu Rev Pharmacol Toxicol 2015;55:207–227 23. Sassmann A, Offermanns S, Wettschureck N. Tamoxifen-inducible Cre- 45. Neal JW, Clipstone NA. Calcineurin mediates the calcium-dependent in- mediated recombination in adipocytes. Genesis 2010;48:618–625 hibition of adipocyte differentiation in 3T3-L1 cells. J Biol Chem 2002;277:49776– 24. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent 49781 Cre reporter mouse. Genesis 2007;45:593–605 46. Hu R, He ML, Hu H, et al. Characterization of calcium signaling pathways in 25. Lukowski R, Rybalkin SD, Loga F, Leiss V, Beavo JA, Hofmann F. Cardiac human preadipocytes. J Cell Physiol 2009;220:765–770 hypertrophy is not amplified by deletion of cGMP-dependent protein kinase I in 47. Shi H, Halvorsen YD, Ellis PN, Wilkison WO, Zemel MB. Role of intracellular cardiomyocytes. Proc Natl Acad Sci U S A 2010;107:5646–5651 calcium in human adipocyte differentiation. Physiol Genomics 2000;3:75–82 26. Jennissen K, Haas B, Mitschke MM, Siegel F, Pfeifer A. Analysis of cGMP 48. Oguri A, Tanaka T, Iida H, et al. Involvement of CaV3.1 T-type calcium signaling in adipocytes. Methods Mol Biol 2013;1020:175–192 channels in cell proliferation in mouse preadipocytes. Am J Physiol Cell Physiol 27. Leiss V, Illison J, Domes K, Hofmann F, Lukowski R. Expression of cGMP- 2010;298:C1414–C1423 dependent protein kinase type I in mature white adipocytes. Biochem Biophys 49. Jensen B, Farach-Carson MC, Kenaley E, Akanbi KA. High extracellular Res Commun 2014;452:151–156 calcium attenuates adipogenesis in 3T3-L1 preadipocytes. Exp Cell Res 2004; 28. Leiss V, Friebe A, Welling A, Hofmann F, Lukowski R. Cyclic GMP kinase I 301:280–292 modulates glucagon release from pancreatic a-cells. Diabetes 2011;60:148–156 50. Sun W, Uchida K, Suzuki Y, et al. Lack of TRPV2 impairs thermogenesis in 29. Sausbier M, Hu H, Arntz C, et al. Cerebellar ataxia and Purkinje cell dys- mouse brown adipose tissue. EMBO Rep 2016;17:383–399 function caused by Ca2+-activated K+ channel deficiency. Proc Natl Acad Sci U S A 51. Ma S, Yu H, Zhao Z, et al. Activation of the cold-sensing TRPM8 channel 2004;101:9474–9478 triggers UCP1-dependent thermogenesis and prevents obesity. J Mol Cell Biol 30. Soltysinska E, Bentzen BH, Barthmes M, et al. KCNMA1 encoded cardiac BK 2012;4:88–96 channels afford protection against ischemia-reperfusion injury. PLoS One 2014;9: 52. Zhang LL, Yan Liu D, Ma LQ, et al. Activation of transient receptor potential e103402 vanilloid type-1 channel prevents adipogenesis and obesity. Circ Res 2007;100: 31. Perry CA, Pravetoni M, Teske JA, et al. Predisposition to late-onset obesity 1063–1070 in GIRK4 knockout mice. Proc Natl Acad Sci U S A 2008;105:8148–8153 53. Worrall DS, Olefsky JM. The effects of intracellular calcium depletion on 32. Park YB, Choi YJ, Park SY, et al. ATP-sensitive potassium channel-deficient mice insulin signaling in 3T3-L1 adipocytes. Mol Endocrinol 2002;16:378–389 show hyperphagia but are resistant to obesity. Diabetes Metab J 2011;35:219–225 54. Yang H, Cui J. BK channels. In Handbook of Ion Channels. Zheng J, Trudeau 33. Plum L, Ma X, Hampel B, et al. Enhanced PIP3 signaling in POMC neurons MC, Eds. Boca Raton, FL, CRC Press, 2015, p. 227–240 causes KATP channel activation and leads to diet-sensitive obesity. J Clin Invest 55. Ye L, Kleiner S, Wu J, et al. TRPV4 is a regulator of adipose oxidative 2006;116:1886–1901 metabolism, inflammation, and energy homeostasis. Cell 2012;151:96–110 34. Yang MJ, Wang F, Wang JH, et al. PI3K integrates the effects of insulin and 56. Earley S, Heppner TJ, Nelson MT, Brayden JE. TRPV4 forms a novel Ca2+ leptin on large-conductance Ca2+-activated K+ channels in neuropeptide Y signaling complex with ryanodine receptors and BKCa channels. Circ Res 2005; neurons of the hypothalamic arcuate nucleus. Am J Physiol Endocrinol Metab 97:1270–1279 2010;298:E193–E201 57. Fernández-Fernández JM, Andrade YN, Arniges M, et al. Functional cou- 35. Tucker K, Overton JM, Fadool DA. Kv1.3 gene-targeted deletion alters pling of TRPV4 cationic channel and large conductance, calcium-dependent longevity and reduces adiposity by increasing locomotion and metabolism in potassium channel in human bronchial epithelial cell lines. Pflugers Arch 2008; melanocortin-4 receptor-null mice. Int J Obes 2008;32:1222–1232 457:149–159 36. Xu J, Koni PA, Wang P, et al. The voltage-gated potassium channel Kv1.3 58. Che H, Yue J, Tse HF, Li GR. Functional TRPV and TRPM channels in human regulates energy homeostasis and body weight. Hum Mol Genet 2003;12:551–559 preadipocytes. Pflugers Arch 2014;466:947–959 37. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS. Interacting appetite- 59. Zhou XB, Arntz C, Kamm S, et al. A molecular switch for specific stimulation of regulating pathways in the hypothalamic regulation of body weight. Endocr Rev the BKCa channel by cGMP and cAMP kinase. J Biol Chem 2001;276:43239–43245 1999;20:68–100 60. Zhou XB, Wulfsen I, Utku E, et al. Dual role of protein kinase C on BK 38. Upadhyay SK, Eckel-Mahan KL, Mirbolooki MR, et al. Selective Kv1.3 channel regulation. Proc Natl Acad Sci U S A 2010;107:8005–8010 channel blocker as therapeutic for obesity and insulin resistance. Proc Natl Acad 61. Carmen GY, Víctor SM. Signalling mechanisms regulating lipolysis. Cell Sci U S A 2013;110:E2239–E2248 Signal 2006;18:401–408