Acute Stimulation of White Adipocyte Respiration by PKA-Induced Lipolysis

Diabetes Publish Ahead of Print, published online August 3, 2010

Running title: - Uncoupling in White Adipocytes Involves BAX

Einav Yehuda-Shnaidman1, Ben Buehrer2, Jingbo Pi1, Naresh Kumar1, Sheila Collins1,3*

1The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709; 2Zen-Bio, Inc. Research Triangle Park, NC 27709; 3Sanford-Burnham Medical Research Institute, Orlando, FL 32827

Address correspondence to: Dr. Sheila Collins Email: [email protected]

Submitted 19 February 2010 and accepted 19 July 2010.

Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org

This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org.

Objective - We examined the effect of β-adrenergic receptor (βAR) activation and cAMP elevating agents on respiration and mitochondrial uncoupling in human adipocytes, and probed the underlying molecular mechanisms. research design and methods - Oxygen consumption (OCR, aerobic respiration) and extracellular acidification (ECAR, anaerobic respiration) were examined in response to isoproterenol, forskolin and dibutyryl-cAMP, coupled with measurements of mitochondrial depolarization, lipolysis, kinase activities and gene targeting or knock-down approaches.

Results - Isoproterenol, forskolin or dibutyryl-cAMP rapidly increased oxidative and glycolytic respiration together with mitochondrial depolarization in human and mouse white adipocytes. The increase in OCR was oligomycin-insensitive and contingent on cAMP-dependent protein kinase (PKA)-induced lipolysis. This increased respiration and uncoupling were blocked by inhibiting the mitochondrial permeability transition pore (PTP) and its regulator BAX. Interestingly, compared to lean individuals, adipocytes from obese subjects exhibited reduced OCR and uncoupling capacity in response to isoproterenol.

Conclusions – Lipolysis stimulated by βAR activation or other maneuvers that increase cAMP levels in white adipocytes acutely induce mitochondrial uncoupling and cellular energetics, which are amplified in the absence of scavenging BSA. The increase in OCR is dependent on PKA-induced lipolysis and is mediated by the PTP and BAX. Since this effect is reduced with obesity, further exploration of this uncoupling mechanism will be needed to determine its cause and consequences.

dipose tissue is a key component electrochemical proton gradient that is the in the management of whole-body driving force for ATP synthesis, in a process Aenergy balance and metabolic termed ‘mitochondrial uncoupling’ [reviewed homeostasis. In mammals, adipose tissue is in (1; 2)]. Caatechecholamine stimulation composed of white and brown adipose tissue also increases Ucp1 gene expression and (WAT; BAT). Both tissues are similar in that mitochondrial mass, altogether resulting in they are highly responsive to insulin to store robust oxidation of FAs for heat production energy as triglyceride (TG), and both respond and energy expenditure. White adipocytes to catecholamines to catabolize these energy have fewer mitochondria and negligible reserves into their constituent fatty acids amounts of UCP1. Upon βAR stimulation of (FAs) and glycerol. However, the fate of WAT, FAs liberated by lipolysis are mostly released FAs from BAT and WAT is released into the circulation. While an different. Brown adipocytes possess a rich important source of energy for other tissues, complement of mitochondria and are the only chronically elevated circulating FAs in cell type to express uncoupling protein obesity are associated with insulin resistance (UCP1). Following catecholamine stimulation and progression to Type II diabetes (3). of the β-adrenergic receptors (βARs) UCP1 Because of recent evidence for the activation (by the released FAs) increases the existence of BAT in adult humans [reviewed proton conductance of the inner mitochondrial in (4) and see also (5-9)] there is renewed membrane (IMM) and dissipates the interest in the idea that mitochondrial

2 Uncoupling in White Adipocytes Involves BAX uncoupling could contribute to FA oxidation Interestingly, this βAR-stimulated respiration and weight reduction. However, it is not yet is reduced with obesity. Such compromised clear whether there are sufficient numbers of capacity could contribute to increased brown adipocytes to have a significant impact adipocyte size, elevated plasma FA levels and on body weight and energy expenditure, and oxidative stress; all of which exist in obesity most of the adipose tissue in adult humans and its metabolic complications. consists of white adipocytes. More recently, white adipocytes are appreciated to have a RESEARCH DESIGN AND METHODS greater complement of mitochondria than Materials/ Forskolin (FSK), isoproterenol previously thought (10), and there are recent (ISO), dibutyryl-cAMP (DB), N-[2-(p- reports showing that FAs in adipocytes can be bromocinnamylamino)-ethyl]-5-isoquinoline- oxidized in situ (11-13). Earlier suggestions sulfonamide (H89), GlutaMAX™, gelatin, in the literature also noted that rodent white carbonylcyanide-p-trifluoromethoxyphenyl- adipocytes can exhibit mitochondrial hydrazone (FCCP), cyclosporin-A (CSA), uncoupling following catecholamine rotenone, and FA-free BSA: Sigma-Aldrich, stimulation (14; 15). Also of note, previous St. Louis, MO. Oligomycin: Calbiochem, San experiments in mice with ectopic expression Diego, CA. Rp-cAMPS: Enzo Life Sciences of UCP1 in WAT from the aP2 promoter Inc, Farmingdale, NY. Tetramethylrhodamine documented the potential of mitochondrial methyl ester (TMRM) and MitoTracker Green uncoupling in vivo and resistance to dietary (MTG): Molecular Probes, Invitrogen, San obesity (16). The uncoupling role of FAs Diego, CA. released during white adipocyte lipolysis and Cells culture. Human pre-adipocytes (Zen- its molecular basis remain unclear, especially Bio, RTP, NC) were isolated from human in less commonly studied human adipocytes. subcutaneous adipose tissue using Therefore, a better understanding of the conventional techniques including enzymatic potential role for white adipocytes to engage dissociation, differential centrifugation and in metabolic fuel oxidation and uncoupling is plating. Cells were seeded into: 0.2% gelatin- warranted. covered 24-well XF24 plates (13,000 Using an approach combining cells/well, Seahorse Bioscience, North measures of oxygen consumption (aerobic Billerica, MA, #100777-004) for OCR and respiration), extracellular acidification ECAR experiments; 60 mm dishes (8 x 105 (anaerobic respiration or glycolysis), cells) for confocal microscopy; 6-well plates mitochondrial inner membrane potential and (4 x 105 cells/well) for Western blotting. biochemical measurements, we present Growth for 24-48 hours in preadipocyte evidence that human white adipocytes can media (PM-1, Zen-Bio) contained: acutely increase aerobic and anaerobic DMEM/F12 (1:1, v/v), HEPES (pH 7.4), 10% respiration in response to βAR and PKA- FBS and antibiotics Cells were differentiated dependent stimulation of lipolysis. Under for 7 days in media containing: DMEM/F12 conditions where the released FAs are not (1:1; v/v), HEPES (pH 7.4), 10% FBS, biotin, scavenged by BSA in the medium we show pantothenate, insulin, dexamethasone, IBMX that the increase in respiration results, in part, and a non-TZD PPARγ agonist (DM-2, Zen- from mitochondrial uncoupling. Moreover, Bio), followed by an additional week in we present evidence that the molecular adipocyte maintenance media (AM-1, Zen- mechanism mediating this uncoupling Bio: DM-2 without IBMX and PPARγ involves the mitochondrial permeability agonist). transition pore and its regulator protein BAX.

3 Uncoupling in White Adipocytes Involves BAX

Mouse adipocytes, 3T3-L1 and 3T3-F442A Mitochondrial membrane potential and cells were grown in high-glucose DMEM + mitochondrial mass by confocal 10% serum and differentiated as described microscopy. Differentiated human or mouse (17) for up to 10 days. Twenty-four hours 3T3-L1 adipocytes were trypsinized and before XF24 analysis, cells were trypsinized seeded onto 0.2 % gelatin coated glass-bottom and seeded at 25,000 cells/well in the plates. 10 mm culture dishes (MatTak Corporation, Animals. Age-matched male Ucp2+/+ and Ashland, MA). Following overnight Ucp2-/- mice (10-16 weeks old) on a incubation at 37°C, 5 % CO2, cells were C57BL/6J background were created in our lab either treated with ISO (1 µM, 30 min), or as described (19; 20). Animals were housed FSK (10 µM, 60 min), and subsequently and genotyped as previously described (20) stained with 20 nM TMRM or with 15 nM and fed standard rodent chow. All protocols MTG (45 min at 37 °C in the dark). Cells for animal use were approved by the IACUC were washed twice with 1 ml phenol red-free of The Hamner Institutes in accordance with DMEM (GIBCO, #31053) and visualized by NIH guidelines. laser scanning confocal microscopy (Zeiss, Cellular metabolic rate. Cells: Human or Thornwood, NY). FCCP (40 µM, 60 min) mouse adipocytes were washed with 1 ml XF- was used as a control for mitochondrial DMEM (#D5030, Sigma-Aldrich), containing uncoupling and depolarization. Fluorescence 1 mM sodium pyruvate, 2 mM GlutaMAX- intensities were calculated with ImagePro 1™, 17.5 mM glucose, 1.85 g/L NaCl, 15 Plus software (Media Cybernetics, Bethesda, mg/L phenol red, pH 7.4), and 500 µl were MD). added per well. Cellular oxygen consumption Lipolysis and protein kinase activity. rate (OCR) and extracellular acidification rate Glycerol release and PKA activity in (ECAR) were measured (XF24, Seahorse differentiated human adipocytes were Bio.) as described previously (21). Per measured as described (22) following ISO specific experiments, drugs were delivered to treatment (1 µM: 2 hours for lipolysis; 15 min final concentrations of: ISO (1 µM), FSK (10 for PKA activity). µM), oligomycin (Oligo, 1 µg/ml), FCCP (0.6 siRNA. Differentiated human adipocytes µM) and rotenone (3 µM). For some were transfected with siRNAs using experiments, cells were pretreated with H89 INTERFERinTM reagent (Polyplus- (10 µM), dibutyryl-cAMP (1 mM), Rp- transfection, Genesee Scientific, San Diego, cAMPS (0.5 mM), 5% FA-free BSA or CSA CA) per the manufacturer for 24 or 48 hours (5 µg/ml). Optimal drug concentrations were as detailed in Fig 8 and in Supp. Fig. 6 in the determined in preliminary experiments. online appendix (available at Tissues: Freshly isolated mouse gonadal http://diabetes.diabetesjournals.org). Total WAT was rinsed with XF-DMEM containing cellular RNA was extracted for RT-PCR, or 25 mM HEPES, cleaned of non-adipose respiration measurements were performed in material and cut into pieces (~10 mg). After the XF24. Optimal transfection conditions extensive washing, one piece of tissue was were determined in preliminary experiments placed in each well of a XF24-well plate for each gene target, which also included (Seahorse Bio. #101122-100) and covered siRNA positive control (Hs_MAPK1, with a customized screen that allows free Qiagen). perfusion while minimizing tissue movement. RNA extraction and real-time PCR. Total XF-DMEM (500 µl) was added per well and RNA was isolated using TRIzol® in samples were analyzed in the XF24. combination with PureLinkTM Micro-to-Midi Total RNA Purification System (Invitrogen,

4 Uncoupling in White Adipocytes Involves BAX

San Diego, CA). Reverse transcription was and kinetics are observed for freshly isolated performed using High-Capacity cDNA Kit adipose tissue or adipocytes or cell lines, with random primers on a Veriti® 96-well although the response in the cell lines was Thermal Cycler (Applied Biosystems). Real- weaker. time PCR (RT-PCR) was done using TaqMan The increase in ECAR was more rapid or SYBR Green (Applied Biosystems) in an than for OCR, reaching a peak after 10 min ABI PRISM® 7900HT Sequence Detection and then declining (Figure 1C). This rise in System. RT-PCR primers are described in ECAR was fully inhibited by the lactate Supp. Figure 6. All data were normalized to dehydrogenase inhibitor, oxamate (5 mM, not GAPDH content. shown) indicating an acute increase in Western blotting. Cells were lysed, protein glycolysis (21). The maximum percent concentrations measured by BCA (Pierce) and change in ECAR is summarized in Figure 1D. 2.5 µg of each sample was fractionated by ECAR was also increased in 3T3-L1 and 3T3- electrophoresis for Western blotting, all F44A adipocytes in response to ISO or FSK exactly as described (22). Primary and (Supp. Fig. 1C and Supp. Fig. 2B). secondary antibodies were obtained from Cell cAMP-induced mitochondrial uncoupling. Signaling Technology (Danvers, MA) or We next determined whether mitochondrial Santa Cruz Biotechnology (Santa Cruz, CA). uncoupling was a contributor to the increased Data analysis. Statistical analysis was OCR in response to cAMP-elevating agents performed by one-way repeated measure by using the ATP synthase inhibitor ANOVA with post hoc Newman-Keuls test. oligomycin (Oligo) and by examining cells For comparison of two groups, significance stained with the fluorescent dye TMRM, was analyzed by t-test. Results are which accumulates specifically in the represented as mean ± SE, unless otherwise mitochondrial matrix as a function of IMM noted in the figure legends. potential. As shown in Figures 2A-D, under basal conditions (CONT) Oligo inhibited RESULTS OCR by 50-60 %. However, in ISO, FSK or Elevated cAMP levels acutely induce DB-treated cells, OCR was less sensitive to respiration in white adipocytes. Continuous Oligo; being inhibited by less than 25 %. measurements of oxygen consumption rate This finding suggests that some of the cAMP- (OCR) and extracellular acidification rate induced OCR is uncoupled respiration. To (ECAR) were collected from human further assess percent of uncoupling, cells adipocytes over time. The addition of were treated with rotenone, which inhibits isoproterenol (ISO), forskolin (FSK) or respiratory chain activity at Complex I, with dibutyryl-cAMP (DB) led to a rapid increase the remaining OCR from non-mitochondrial in OCR, which gradually peaked after 20-30 sources. Figure 2D shows that about 20% of min (ISO) or 60 min (FSK, DB) and then total OCR remains in the presence of slowly declined (Figure 1A). Figure 1B rotenone, and is similar for all groups. After presents the average maximal OCR changes accounting for this non-mitochondrial OCR from several experiments, with maximal rates (shown by the dotted arrows in Fig. 2D, and of respiration measured by the mitochondrial illustrated as % uncoupling in Fig. 2E), uncoupler FCCP. Similar increases in OCR mitochondrial uncoupling accounts for 30 % in response to elevations in cAMP were also of OCR in untreated control cells, ~ 55 % of observed in mouse WAT (Supp. Fig. 1A) and ISO-induced respiration and 65-70 % of FSK cell lines (Supp. Figs. 1B and 2A). Note from or DB-stimulated respiration. We next a technical standpoint that the same responses analyzed TMRM fluorescence intensity by

5 Uncoupling in White Adipocytes Involves BAX confocal microscopy. Figure 3 shows that contribute to the cAMP-dependent TMRM intensity is significantly reduced in mitochondrial oxidative respiration of white either ISO- or FSK-treated human adipocytes adipocytes, consistent with other studies (15; relative to untreated control cells. Note that 25). Although these data do not clearly the changes in TMRM intensity occur distinguish between FAs as a substrate, specifically in the differentiated adipocytes, uncoupler, or both, two additional recognized by their lipid accumulation observations suggest that FAs contribute to (arrows in Fig. 3A) and occur in the presence both. First, in contrast to OCR, ECAR levels of serum-containing media. The changes in in the presence of BSA were even higher fluorescence intensity are summarized in following ISO or FSK (Fig. 5C, D and Supp. Figure 3B. Both ISO and FSK decreased Fig 2B) This suggests that the reduction of IMM potential by about 50 %. Similar results available FAs from lipolysis for oxidation were obtained in mouse 3T3-L1 adipocytes resulted in increased glycolysis. The cells (Supp. Fig. 3). All together these results respire well on other substrates (e.g. pyruvate) indicate that βAR stimulation or cAMP since they increase respiration in response to elevating agents can acutely increase FCCP and in the absence of cAMP elevation. mitochondrial uncoupling in white Second, a much earlier study in primary rat adipocytes. adipocytes reported that mitochondrial cAMP-induced OCR is FA-dependent. In depolarization observed following ISO adipocytes, βAR agonists stimulate lipolysis treatment was largely prevented by to generate FFAs. This response depends pretreatment with FA-free BSA (15). Also, largely on the activation of PKA (17). We the reduction in mitochondrial membrane examined the involvement of PKA as well as potential measured by confocal microscopy the liberated FAs on cAMP-induced changes was performed in the presence of complete in adipocyte respiration. We used two serum-containing media. mechanistically different inhibitors of PKA: To further evaluate the importance of the catalytic inhibitor H89 (23), and the lipolysis in this cAMP-induced respiration, competitive cAMP antagonist Rp-cAMPS we used a siRNA approach to suppress the (24). Figures 4A, B show that both H89 and expression of the important lipase, ATGL Rp-cAMPS inhibited ISO or FSK-induced (26). Knock-down of ATGL (Supp. Fig. 5B) OCR. Both inhibitors also effectively resulted in a dramatic inhibition of FSK- blocked the increase in ECAR (Figure 4C; induced OCR (Fig. 6A, B). The portion of average of several experiments presented in OCR that was not inhibited may be due to Figure 4D). To examine the role of the residual ATGL. FSK-induced ECAR was not liberated FAs on respiration, cells were changed with ATGL knock-down (Fig. 6C, provided with 5% BSA (FA-free) prior to ISO D). These data support the role of lipolysis or FSK injections (15). A sample data set of in the cAMP-induced oxidative respiration in OCR is shown in Figure 5A, and results from white adipocytes. several experiments are summarized in Figure cAMP-induced OCR is reduced with obesity. 5B. The increase in OCR induced by either Catecholamine responsiveness of adipose ISO or FSK was significantly reduced due to tissue to increase lipolysis has been reported scavenging by BSA of the FAs released from to become impaired with increasing BMI lipolysis. Similar results were obtained in [reviewed in (27; 28)]. Therefore, an mouse 3T3-F442A adipocytes (Supp. Fig. interesting question is whether adipocytes of 2A). Together, the data suggest that the FAs obese individuals have an equivalent capacity released by PKA-dependent lipolysis for this βAR-activated increase in cAMP-

6 Uncoupling in White Adipocytes Involves BAX dependent respiration. We compared ISO- We assessed the involvement, if any, induced OCR and ECAR in adipocytes from of major proteins in these groups in WAT that lean (BMI = 21.7-24.6) or obese (BMI = 30- could be candidates for mediating this cAMP- 35.5) donors. As seen in Figure 7, the triggered respiration in white adipocytes. increase in OCR by ISO was impaired in First, we measured their expression levels in adipocytes from obese subjects (Fig. 7A, B). human white adipocytes by real-time PCR. ECAR was not changed (Fig. 7C, D). This Expression of ANT3, PiC, UCP2, VDAC1, deficit in OCR response from obese BAX and to less extent ANT2/1 are relatively individuals was not due to impaired abundant in white adipocytes, while UCP1, differentiation of the adipocytes, as measured UCP5, ANT4, and AGC are very low (Supp. by Nile Red staining and the expression of Fig. 5A). Therefore, we focused on the former such differentiation markers as aP2, C/EBPα, group, measuring the OCR response to ISO PGC-1α. PPARγ2, HSL (not shown). There after targeted reduction of these candidates. were also no discernible differences in The involvement of UCP2 was assessed using mitochondrial mass, as measured by MTG WAT from Ucp2+/+ and Ucp2-/- mice (19; (Supp. Fig. 4A) or by gene expression of 20), and the increase in OCR was identical mitochondrial proteins (cytochrome C, ATP between the two genotypes (Supp. Fig. 5C). synthase and cytochrome C oxidase subunits; Expression of ANT3, ANT2, PiC, VDAC1 or not shown). Moreover, other parameters that BAX in human adipocytes was suppressed by were not different between lean and obese siRNAs. The efficiency of targeted knock- included ISO-stimulated: lipolysis (Supp. Fig. down was 75-85% vs. the scrambled siRNA 4B) or PKA activity (Supp. Fig. 4C); basal (Supp. Fig. 5B). The OCR response was OCR (Supp. Fig. 4D), and βAR subtype unchanged in cells receiving the siRNAs for expression (not shown). Interestingly, PiC, ANT3, ANT2, ANT2+ANT3 or VDAC1 maximal OCR evoked by FCCP (Supp. Fig. (Supp. Fig. 5C). However, knock-down of 4E) was also significantly reduced in BAX, as shown in Figures 8A, B, resulted in adipocytes from obese subjects, while basal a significant decrease in the OCR response to respiration was less sensitive to oligomycin ISO (Figs. 8C, D). Since BAX can increase (Supp. Fig 4F). mitochondrial uncoupling by opening the PTP cAMP-induced mitochondrial uncoupling is (31) we also treated cells with the specific mediated by BAX and the permeability PTP inhibitor CSA (30) prior to measuring transition pore. A number of mitochondrial cAMP-induced OCR. Figures 8E, F show proteins have been associated with that CSA similarly inhibited the OCR uncoupling induced by FAs (reviewed in 2; response to either ISO or FSK. Together 29). These include members of the these results suggest an important role for mitochondrial carrier family such as UCP1, BAX to regulate PTP opening for this cAMP the adenine nucleotide translocases (ANTs), and FA-induced mitochondrial uncoupling in phosphate carrier (PiC), aspartate/glutamate white adipocytes. carrier (AGC), and the permeability transition pore (PTP) complex. The subunit DISCUSSION composition of the PTP is not completely The ability of adipocytes to oxidize clear (29; 30). It is thought to consist of the FAs and uncouple mitochondrial respiration integral membrane proteins ANT and VDAC1 in response to βAR activation is best and regulatory proteins of the BCL-2 family, understood in brown adipocytes, where the which control the open-closed states of the high density of mitochondria and the presence pore (30; 31). of the unique protein UCP1 allow for robust

7 Uncoupling in White Adipocytes Involves BAX

OCR and energy expenditure. Obviously implying that glucose oxidation preceded FA white adipocytes have less oxidative capacity oxidation. Importantly, a major portion of than brown adipocytes and their ability to cAMP-induced OCR was oligomycin- oxidize FA is much lower (25; 32). insensitive, implying that some respiration Nevertheless, a case can be made for was independent of ATP synthase activity. assessing the relevance of WAT as a target These results, together with mitochondrial for increasing energy expenditure based on depolarization, represent mitochondrial the sheer amount of the tissue as a percentage uncoupling. of body mass. In fact, evidence exists in The physiological significance of rodent adipocytes for a moderate increase in mitochondrial uncoupling in white fat is not FA oxidation in response to βAR activation fully understood and is debated (13; 32; 37) (12; 25), and various futile cycles have been and reviewed in ref. (38). One consequence examined in adipocytes (18). of mitochondrial uncoupling in WAT is an Several years ago a collection of increase of the AMP/ATP ratio and activation unrelated reports suggested that epinephrine of AMP kinase to (i) promote ATP-generating could increase OCR in rat epididymal fat pads processes such as FA and glucose oxidation [(33) and refs therein] or primary cultures of (38; 39) and refs therein) and (ii) inhibit ATP- rat adipocytes (14). βAR-stimulated lipolysis consuming pathways such as lipogenesis, TG in these adipocytes was also associated with synthesis and, to some extent, lipolysis (36; both FA-dependent IMM depolarization (15; 38; 40; 41). Another outcome of uncoupling 34), and a decrease in ATP levels (14; 15; 34; in WAT could be protection against 35). A more direct link between lipolysis and mitochondrial-derived ROS and oxidative ATP levels was shown in 3T3-L1 cells in stress directly within the adipocyte, which can which the generic lipase inhibitor orlistat lead to cellular damage (12; 36; 42). suppressed FSK-stimulated lipolysis as well Interestingly, we observed that adipocytes as the drop in ATP content (36). Since a high derived from obese donors had a significantly OCR together with mitochondrial lower OCR response to βAR activation. In depolarization and ATP reduction are adipose tissue from obese individuals there is common characteristics of mitochondrial evidence for an impaired lipolytic response in uncoupling, all together these studies suggest vivo, and a reduced mitochondrial mass (10; there is mitochondrial uncoupling in response 43), with a variety of mechanisms proposed to βAR activation. However, no single study (reviewed in (27; 28)). Although we did not has presented cohesive evidence for βAR- observe a difference in lipolysis, our results induced uncoupling in white adipocytes, suggest that there is a difference in the ability especially in human adipocytes, and a of the mitochondria from obese vs. lean molecular mechanism was not identified. subjects to increase respiratory activity in Here we find an acute increase in the response to βAR stimulation, mirroring cellular energetics of human white adipocytes notions that an impaired response of WAT to in response to βAR activation or cAMP catecholamines at some level could be elevating agents that is dependent on the FAs significant in the development or maintenance liberated by lipolysis. In following cAMP- of obesity (28). induced changes in cellular respiration over The ability of FAs to uncouple time, the increases in both aerobic (measured mitochondrial respiration has been as OCR) and anaerobic (measured as ECAR) extensively investigated, particularly in respiration were immediate and transient. tissues such as BAT, muscle and liver The ECAR response was faster than OCR, because of their high aerobic capacity. The

8 Uncoupling in White Adipocytes Involves BAX molecular basis mediating FA-induced lean and obese, but will require further study uncoupling has been vigorously discussed and and must be extended to a larger sample set. a number of mitochondrial carriers have been Since white adipocytes comprise the suggested (2; 29; 44; 45). In BAT, the bulk of adipose tissue in the body, even a general consensus is that FAs allosterically moderate, sustained, ability of adipocytes to activate the ‘carrier’ or ‘channel’ properties of oxidize FAs could have several positive UCP1 (46) (mechanistically still debated in consequences. First, limiting the amount of some circles). By comparison, in WAT FAs released into the circulation (13; 25; 37) UCP1 levels are fairly negligible while the would be protective against lipotoxicity in UCP1 homologue, UCP2, is abundant (47). other tissues, and second, it might contribute UCP2 has periodically been proposed to to whole body energy expenditure. The mediate proton leak or mitochondrial concept of mitochondrial uncoupling as a uncoupling [(1) and refs therein) but this point strategy to reduce body weight has been is not fully agreed upon (48; 49)]. However, clearly demonstrated with the past clinical use regarding UCP2 there was no difference in of such strong global uncouplers as 2,4- the OCR response to ISO in WAT both wild- dinitrophenol, but that enterprise was not type and Ucp2-null mice. Moreover, the without major side effects, including fatalities contribution of ANT2, ANT3, PiC and [reviewed in (48)]. Therefore, greater VDAC1 were excluded. Instead, a significant understanding of the mechanism(s) portion of βAR and cAMP-induced OCR was responsible for this cAMP-dependent inhibited by the specific PTP inhibitor, CSA, uncoupling in WAT and the role of the PTP as well as by suppressing BAX expression; are warranted since the potential to identify altogether suggesting that the opening of the novel agents and/or processes that could be mitochondrial PTP by the regulatory protein harnessed for selectively stimulating BAX is important for the observed moderate uncoupling are needed to combat mitochondrial uncoupling in WAT. Given the metabolic disease epidemic. that the amplitude of the OCR and uncoupling response in the obese subjects was reduced, Author contributions. E.Y.S. performed we measured whether there was a change in experiments, wrote manuscript; B.B. expression of BAX and the PTP in obese performed experiments and contributed to WAT. In our sample set no differences were discussion; J.P. performed experiments and detected in the expression levels of BAX (or contributed to discussion; N.K. performed VDAC1, ANTs) between lean and obese experiments; S.C. wrote and edited WAT. However, proteins of the BCL-2 manuscript family are known to be post-translationally modified, which controls their activity and ACKNOWLEDGMENTS regulates PTP opening (31; 50). Such This study was supported by the North modifications in WAT mitochondria could Carolina Biotechnology Center (CFG- 8006). reasonably be a basis for the observed The authors greatly acknowledge Seahorse differences in mitochondrial function between Bioscience for their advice and technical support of this study.

REFERENCES 1. Brand MD: The efficiency and plasticity of mitochondrial energy transduction. Biochem Soc Trans 33:897-904, 2005

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2. Gonzalez-Barroso MM, Rial E: The role of fatty acids in the activity of the uncoupling proteins. Curr Chem Biol 3:180-188, 2009 3. Arner P: Insulin resistance in type 2 diabetes: role of fatty acids. Diabetes Metab Res Rev 18 Suppl 2:S5-9, 2002 4. Nedergaard J, Bengtsson T, Cannon B: Unexpected evidence for active brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab 293:E444-452, 2007 5. van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM, Kemerink GJ, Bouvy ND, Schrauwen P, Teule GJ: Cold-activated brown adipose tissue in healthy men. N Engl J Med 360:1500-1508, 2009 6. Cypess AM, Lehman S, Williams G, Tal I, Rodman D, Goldfine AB, Kuo FC, Palmer EL, Tseng YH, Doria A, Kolodny GM, Kahn CR: Identification and importance of brown adipose tissue in adult humans. N Engl J Med 360:1509-1517, 2009 7. Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi T, Taittonen M, Laine J, Savisto NJ, Enerback S, Nuutila P: Functional brown adipose tissue in healthy adults. N Engl J Med 360:1518-1525, 2009 8. Zingaretti MC, Crosta F, Vitali A, Guerrieri M, Frontini A, Cannon B, Nedergaard J, Cinti S: The presence of UCP1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue. Faseb J 23:3113-3120, 2009 9. Saito M, Okamatsu-Ogura Y, Matsushita M, Watanabe K, Yoneshiro T, Nio-Kobayashi J, Iwanaga T, Miyagawa M, Kameya T, Nakada K, Kawai Y, Tsujisaki M: High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity. Diabetes 58:1526-1531, 2009 10. Wilson-Fritch L, Nicoloro S, Chouinard M, Lazar MA, Chui PC, Leszyk J, Straubhaar J, Czech MP, Corvera S: Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone. J Clin Invest 114:1281-1289, 2004 11. Stenson BM, Ryden M, Steffensen KR, Wahlen K, Pettersson AT, Jocken JW, Arner P, Laurencikiene J: Activation of liver X receptor regulates substrate oxidation in white adipocytes. Endocrinology 150:4104-4113, 2009 12. Cho SY, Park PJ, Shin ES, Lee JH, Chang HK, Lee TR: Proteomic analysis of mitochondrial proteins of basal and lipolytically (isoproterenol and TNF-alpha)-stimulated adipocytes. J Cell Biochem 106:257-266, 2009 13. Maassen JA, Romijn JA, Heine RJ: Fatty acid-induced mitochondrial uncoupling in adipocytes as a key protective factor against insulin resistance and beta cell dysfunction: a new concept in the pathogenesis of obesity-associated type 2 diabetes mellitus. Diabetologia 50:2036-2041, 2007 14. Hepp D, Challoner DR, Williams RH: Respiration in isolated fat cells and the effects of epinephrine. J Biol Chem 243:2321-2327, 1968 15. Davis RJ, Martin BR: The effect of beta-adrenergic agonists on the membrane potential of fat-cell mitochondria in situ. Biochem J 206:611-618, 1982 16. Kopecky J, Clarke G, Enerback S, Spiegelman B, Kozak LP: Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity. J Clin Invest 96:2914-2923, 1995 17. Robidoux J, Kumar N, Daniel KW, Moukdar F, Cyr M, Medvedev AV, Collins S: Maximal beta3-adrenergic regulation of lipolysis involves Src and epidermal growth factor receptor- dependent ERK1/2 activation. J Biol Chem 281:37794-37802, 2006

10 Uncoupling in White Adipocytes Involves BAX

18. Ukropec J, Annunciado RP, Ravussin Y, Hulver MW, Kozak LP.:UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1-/- mice. J Biol Chem 281:31894- 31908, 2010 19. Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves-Guerra MC, Goubern M, Surwit R, Bouillaud F, Richard D, Collins S, Ricquier D: Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet 26:435-439, 2000 20. Pi J, Bai Y, Daniel KW, Liu D, Lyght O, Edelstein D, Brownlee M, Corkey BE, Collins S: Persistent oxidative stress due to absence of uncoupling protein 2 associated with impaired pancreatic beta-cell function. Endocrinology 150:3040-3048, 2009 21. Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, Lemire K, Orrell J, Teich J, Chomicz S, Ferrick DA: Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol Cell Physiol 292:C125-136, 2007 22. Kumar N, Robidoux J, Daniel KW, Guzman G, Floering LM, Collins S: Requirement of vimentin filament assembly for beta3-adrenergic receptor activation of ERK MAP kinase and lipolysis. J Biol Chem 282:9244-9250, 2007 23. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H: Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p- bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267-5272, 1990 24. Botelho LH, Rothermel JD, Coombs RV, Jastorff B: cAMP analog antagonists of cAMP action. Methods Enzymol 159:159-172, 1988 25. Wang T, Zang Y, Ling W, Corkey BE, Guo W: Metabolic partitioning of endogenous fatty acid in adipocytes. Obes Res 11:880-887, 2003 26. Zechner R, Kienesberger PC, Haemmerle G, Zimmermann R, Lass A: Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores. J Lipid Res 50:3-21, 2009 27. Dodt C, Lonnroth P, Wellhoner JP, Fehm HL, Elam M: Sympathetic control of white adipose tissue in lean and obese humans. Acta Physiol Scand 177:351-357, 2003 28. Jocken JW, Blaak EE: Catecholamine-induced lipolysis in adipose tissue and skeletal muscle in obesity. Physiol Behav 94:219-230, 2008 29. Di Paola M, Lorusso M: Interaction of free fatty acids with mitochondria: coupling, uncoupling and permeability transition. Biochim Biophys Acta 1757:1330-1337, 2006 30. Halestrap AP: What is the mitochondrial permeability transition pore? J Mol Cell Cardiol 46:821-831, 2009 31. Gross A, McDonnell JM, Korsmeyer SJ: BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899-1911, 1999 32. Frayn KN, Langin D, Karpe F: Fatty acid-induced mitochondrial uncoupling in adipocytes is not a promising target for treatment of insulin resistance unless adipocyte oxidative capacity is increased. Diabetologia 51:394-397, 2008 33. Ball EG, Jungas RL: On the action of hormones which accelerate the rate of oxygen consumption and fatty acid release in rat adipose tissue in vitro. Proc Natl Acad Sci U S A 47:932-941, 1961 34. Vallano ML, Lee MY, Sonenberg M: Hormones modulate adipocyte membrane potential ATP and lipolysis via free fatty acids. Am J Physiol 245:E266-272, 1983

11 Uncoupling in White Adipocytes Involves BAX

35. Angel A, Desai KS, Halperin ML: Reduction in adipocyte ATP by lipolytic agents: relation to intracellular free fatty acid accumulation. J Lipid Res 12:203-213, 1971 36. Gauthier MS, Miyoshi H, Souza SC, Cacicedo JM, Saha AK, Greenberg AS, Ruderman NB: AMP-activated protein kinase is activated as a consequence of lipolysis in the adipocyte: potential mechanism and physiological relevance. J Biol Chem 283:16514-16524, 2008 37. Maassen JA, Romijn JA, Heine RJ: Fatty acid-induced mitochondrial uncoupling in adipocytes as a key protective factor against insulin resistance and beta cell dysfunction: do adipocytes consume sufficient amounts of oxygen to oxidise fatty acids? Diabetologia 51:907- 908, 2008 38. Kopecky J, Rossmeisl M, Flachs P, Bardova K, Brauner P: Mitochondrial uncoupling and lipid metabolism in adipocytes. Biochem Soc Trans 29:791-797, 2001 39. Rognstad R, Katz J: The effect of 2,4-dinitrophenol on adipose-tissue metabolism. Biochem J 111:431-444, 1969 40. Hardie DG, Hawley SA, Scott JW: AMP-activated protein kinase--development of the energy sensor concept. J Physiol 574:7-15, 2006 41. Huber CT, Duckworth WC, Solomon SS: The reversible inhibition by carbonyl cyanide m- chlorophenyl hydrazone of epinephrine-stimulated lipolysis in perifused isolated fat cells. Biochim Biophys Acta 666:462-467, 1981 42. Subauste AR, Burant CF: Role of FoxO1 in FFA-induced oxidative stress in adipocytes. Am J Physiol Endocrinol Metab 293:E159-164, 2007 43. Kaaman M, Sparks LM, van Harmelen V, Smith SR, Sjolin E, Dahlman I, Arner P: Strong association between mitochondrial DNA copy number and lipogenesis in human white adipose tissue. Diabetologia 50:2526-2533, 2007 44. Andreyev A, Bondareva TO, Dedukhova VI, Mokhova EN, Skulachev VP, Tsofina LM, Volkov NI, Vygodina TV: The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria. Eur J Biochem 182:585-592, 1989 45. Wojtczak L, Schonfeld P: Effect of fatty acids on energy coupling processes in mitochondria. Biochim Biophys Acta 1183:41-57, 1993 46. Garlid KD, Jaburek M, Jezek P: The mechanism of proton transport mediated by mitochondrial uncoupling proteins. FEBS Lett 438:10-14, 1998 47. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, Warden CH: Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 15:269-272, 1997 48. Harper JA, Dickinson K, Brand MD: Mitochondrial uncoupling as a target for drug development for the treatment of obesity. Obes Rev 2:255-265, 2001 49. Nedergaard J, Ricquier D, Kozak LP: Uncoupling proteins: current status and therapeutic prospects. EMBO Rep 6:917-921, 2005 50. Yehuda-Shnaidman E, Kalderon B, Azazmeh N, Bar-Tana J: Gating of the mitochondrial permeability transition pore by thyroid hormone. Faseb J 24:93-104

12 Uncoupling in White Adipocytes Involves BAX

FIGURE LEGENDS Figure 1 - Elevated cAMP levels acutely induce respiration in white adipocytes Representative measurements of the percent increase in OCR (A) or ECAR (C), respectively, relative to baseline rates in response to isoproterenol, forskolin or dibutyryl-cAMP in human adipocytes. As indicated, at 0 min isoproterenol (ISO, 1 µM, squares), forskolin (FSK, 10 µM, triangles), dibutyryl-cAMP (DB, 1 mM, stars) or DMEM (CONT, circles) were injected. OCR (A) or ECAR (C) measurements before drug injection were set as 100%. Adipocytes were pooled from 5 different human subjects and each data point is a mean of 4-6 wells. Histograms summarizing the average maximal percent increase of OCR (B) or ECAR (D), respectively, over their baseline rates in response to ISO, FSK, DB and FCCP (0.6 µM). Data are collected from 8- 10 experiments, using a total of 20 subjects (BMI range of 21.7-35.5). All experiments included wells that received ISO, FSK or DB, and wells that received FCCP. *, p < 0.001; # p < 0.01 compared with CONT. Figure 2 - Elevated cAMP levels induce oligomycin-insensitive OCR Representative measurements of the percent change in OCR in human adipocytes following injections of DMEM (open symbols) or inhibitors (closed symbols): ATP synthase inhibitor oligomycin (Oligo, 1 µg/ml) or the complex 1 inhibitor, rotenone (Rot, 3 µM) as indicated by the dashed vertical lines. Oligo was injected 30-40 min after: (A) isoproterenol (ISO, 1 µM, squares), (B) forskolin (FSK, 10 µM, triangles), (C) dibutyryl-cAMP (DB, 1 mM, diamonds) or DMEM (CONT, circles). OCR before Oligo injection was set as 100%. The results are from adipocytes pooled from 5 subjects, and each data point is a mean of 4-5 wells. (D) Histogram summarizing the percent decrease in OCR in response to Oligo or Rot injections. Data is relative to OCR levels before Oligo injection (and after ISO, FSK or DB injections). The data are an average of 3-5 experiments using a total of 15 subjects (BMI range of 21.7-35.5). *, p < 0.001, compared with untreated CONT; ^, p < 0.01, compared with untreated ISO; #, p < 0.001, compared with CONT + Oligo. (E) The relative changes in OCR between Oligo and Rot are expressed as % uncoupling (calculated from the results presented in (D) – dotted arrows). Figure 3 - Elevated cAMP levels induce mitochondrial depolarization (A) Microscopy images of human adipocytes stained with TMRM and visualized under confocal microscopy or their phase-contrast (Ph-Cont) mode. Cells were treated with ISO (1 µM, 30 min) or FSK (10 µM, 60 min) in DMEM + 10% FBS, stained with TMRM and immediately analyzed, as described in Research Design and Methods. The scale bar in the images represents 20 microns. (B) Histogram summarizing TMRM fluorescence intensity of untreated (CONT) and ISO, FSK or FCCP (40 µM, 60 min) treated cells of 50 images collected from 3 independent experiments. TMRM intensity in the untreated cells (CONT) was set as 1. *, p < 0.001, compared with CONT. Figure 4 - cAMP-induced respiration is PKA-dependent Representative measurements of the percent increase in OCR (A) and ECAR (C), relative to baseline rates, in response to ISO or FSK in human adipocytes pretreated or not with H89. At 0 min, ISO (1 µM, squares), FSK (10 µM, triangles), or DMEM (CONT, circles) were injected to cells that were pretreated (closed symbols) or not (open symbols) with H89 (10 µM, 1 hour). Each data point is the mean of 4 wells, and error bars not visible are contained within the symbols. Histograms summarizing the maximum percent increase of OCR (B) or ECAR (D), over their baseline rates in response to DMEM, ISO or FSK. Cells were pretreated with or without H89 or Rp-cAMPS (0.5 mM, 1 hour). Results are the average of a total of 11 subjects (BMI range of 21.7-35.5), measured in 3 independent experiments. *, p < 0.05, compared with

13 Uncoupling in White Adipocytes Involves BAX untreated respective control; ^, p < 0.05, compared with respective ISO or FSK without H89; #, p < 0.05, compared with respective ISO or FSK without Rp-cAMPS. Figure 5 - cAMP-induced OCR is FA-dependent Representative measurements of the percent increase in OCR (A) and ECAR (C), relative to baseline rates, in response to ISO or FSK in adipocytes in the presence or absence of FA-free BSA. At 0 min, ISO (1 µM, squares), FSK (10 µM, triangles), or DMEM (CONT, circles) were injected to cells that were pretreated (closed symbols) or not (open symbols) with BSA (5 %, 1 hour). Each data point is the mean of 4 wells, and error bars not visible are contained within the symbols. Histograms summarizing the maximum percent increase of OCR (B) or ECAR (D) in response to DMEM (CONT), ISO, FSK or DB (1 mM) over their baseline rates. Data is an average of 11 subjects (BMI range of 21.7-35.5), measured in 3 independent experiments. *, p < 0.05, compared with CONT; #, p < 0.05, compared with respective samples without BSA. Figure 6 - cAMP-induced OCR is dependent upon ATGL Human adipocytes were transfected with siRNA targeted to ATGL or scrambled sequence (SCR), as described in Research Design and Methods, which resulted in 50% suppression (Supp Fig. 5B). Representative measurements of the percent increase in OCR (A) and ECAR (C) relative to baseline rates in response to FSK (10 µM), in human adipocytes treated with SCR (open symbols) or ATGL siRNA (close symbols). As indicated, at 0 min forskolin (FSK, triangles) or DMEM (CONT, circles) were injected. OCR measurements before FSK injection were set as 100 %. Adipocytes were pooled from 5 different human subjects and each data point is a mean of 6 wells. Histograms summarizing the maximum percent increase of OCR (B) or ECAR (D), respectively, relative to respective baseline rates, collected from 3 experiments using a total of 20 subjects (BMI range of 21.7-35.5). *, p < 0.01, compared with respective control samples; # p < 0.05, compared with FSK+SCR. Figure 7 - ISO-induced OCR is reduced with obesity Representative measurements of the percent increase in OCR (A) and ECAR (C), in response to ISO of adipocytes taken from lean or obese humans. Cells were pooled from 5 lean subjects (BMI 21.7-24.6, LEAN, circles) or 5 obese subjects (BMI 30-35.5, OBESE, squares). As indicated, at 0 min ISO (1 µM, open symbols), or DMEM (close symbols) were injected. Each data point is a mean of 6-8 wells, and error bars not visible are contained within the symbols. Histograms summarizing the maximum percent increase of OCR (B) or ECAR (D), in response to ISO and relative to baseline rates. Results are presented as fold vs. LEAN. Data is the mean of 12 LEAN and 9 OBESE samples; each was measured in 3 different experiments, containing 6- 8 replicates. *, p < 0.001, compared with LEAN. Figure 8 – cAMP-induced mitochondrial uncoupling is mediated by BAX and the Permeability Transition Pore (A, B)Human adipocytes were transfected with scrambled (SCR) or BAX siRNAs (30 nM, 48 hours). (A) BAX gene expression was measured by real-time PCR and normalized to GAPDH. (B) Representative Western blots (1 of 4 replicates) of BAX protein level compared to GAPDH. (C, E) Representative measurements of the percent increase in OCR relative to baseline rates, in response to ISO or FSK. Human adipocytes were transfected with scrambled (SCR; open symbols) or BAX (closed symbols) siRNAs (C) or pretreated (closed symbols) or not (open symbols) with CSA (5 µg/ml, 72 hours) (E). At 0 min, ISO (1 µM, squares), FSK (10 µM, triangles) or DMEM (CONT, circles) were injected to cells. Each data point is the mean of 4 wells; error bars not visible are contained within symbols. (D, F) Histograms summarizing the maximum percent increase of OCR over the baseline rates in response to DMEM (CONT), ISO

14 Uncoupling in White Adipocytes Involves BAX or FSK. In (D), results are the average of 6 subjects measured in 3 independent experiments. *, p < 0.001, compared with respective control; #, p < 0.001, compared with ISO+SCR. In (F), results are the average of a total of 12 subjects measured in 5 independent experiments. *, p < 0.001, compared with CONT; ^, p < 0.05, compared with respective ISO without CSA; #, p < 0.001, compared with respective FSK without CSA.

Figure 1

15 Uncoupling in White Adipocytes Involves BAX

Figure 2

16 Uncoupling in White Adipocytes Involves BAX

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Figure 8

A B 0.07

0.05 Ab: BAX Ab: GAPDH 0.03 SCR BAX

(vs. GAPDH) (vs. * siRNA

Gene expression Gene 0.01

SCR BAX siRNA

C D * 150% # 150% CONT + SCR * 125% CONT + BAX siRNA 100% ISO + SCR 100% ISO + BAX siRNA 75% 50% OCR 50% over baseline OCR baseline over % of baseline of % 25% increase % Maximum 0% 0% SCR SCR 0 14 28 42 56 + + O siRNA NT IS Time (min) BAX BAX siRNA CO + + O NT O IS C E F

200% CONT * CONT+CSA 150% * # 150% ISO ^ ISO+CSA 100% 100% FSK OCR FSK+CSA 50% 50% % of baseline

over baseline OCR over baseline 0% 0% % increase Maximum 0 7 14 21 28 35 42 CSA ISOCSAFSK CONT + Time (min) ISO FSK+CSA