Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes

The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders.


Introduction
Obesity is a major public health problem and a worldwide epidemic contributing to the development of associated pathological conditions, such as insulin resistance, type 2 diabetes, cardiovascular disease, nonalcoholic fatty liver disease, and some forms of cancer among others [1,2]. Adipose tissue has gained a crucial role in the study of the mechanisms involved in (CIBEROBN) (Grant CB06/03/0001 to DS), the Centro de Investigación Biomédica en red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), the Generalitat de Catalunya (2014SGR465 to DS, and 2014SGR48 to AZ), the Comunidad de Madrid (S2010/BMD-2423 to MJO and AMV), INTERREG IV-B-SUDOE-FEDER (DIOMED, SOE1/P1/E178) to AZ, and the European Foundation for the Study of Diabetes (EFSD)/ Janssen-Rising Star and L'Oréal-UNESCO "For Women in Science" research fellowships to LH. AZ is a recipient of ICREA-Academia (Generalitat de Catalunya) and MW is a recipient of the Ciência sem Fronteiras-CNPq fellowship (237976/2012-9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.

Materials and Methods Materials
Norepinephrine, sodium palmitate, sodium oleate and FA-free BSA were purchased from Sigma Aldrich (St. Louis, MO). DMEM, FBS and penicillin/streptomycin mixture were purchased from Life Technologies (Carlsbad, CA). Etomoxir was kindly provided by Dr. H. P. O. Wolf (GMBH, Allensbach, Germany).

Cell culture and differentiation of brown adipocytes
Immortalized rBA from neonatal rats were generated as previously described [35]. rBA preadipocytes were grown in DMEM supplemented with 10% fetal serum, 1% penicillin/streptomycin and 2 mM HEPES. rBA were differentiated in DMEM supplemented with 10% fetal serum, 1 nM T3 and 20 nM insulin (differentiation media) until confluence (day 0). Then, cells were incubated with induction medium containing 1 μM rosiglitazone, 0.5 μM dexamethasone, 0.125 μM indomethacin and 0.5 mM isobutylmethylxanthine (IBMX) for two days. Finally, cells were incubated for a further three days in differentiation media, after which they exhibited a fully differentiated phenotype with numerous multilocular lipid droplets in their cytoplasm.
Oil Red O staining rBA were grown and differentiated on coverslips. They were rinsed twice in PBS, fixed in 10% paraformaldehyde for 30 minutes at room temperature, and washed again in PBS. Then, cells were rinsed in 60% isopropanol for 5 min to facilitate the staining of neutral lipids, and stained with filtered Oil Red O working solution (0.3% Oil Red O in isopropanol) for 15 minutes. After several washes in distilled water, the coverslips were removed and fixed with mount medium. The intracellular lipid vesicles stained with Oil Red O were identified by their bright red color under the microscope.
Western blot analysis rBA were cultured in 12-well plates, differentiated and infected as described above. Cells were harvested in lysis buffer (RIPA), and protein concentration was determined using a BCA protein assay kit (Thermoscientific). Samples were separated on 8% and 12% SDS-PAGE gels, for CPT1A and UCP1 respectively, and then transferred onto PVDF membranes (Millipore). The following primary antibodies were used: CPT1A (1/6,000) [24], Tim 44 (1/5,000; BD Bioscience), UCP1 (1/1,000; Abcam) and b-actin (I-19; 1/4,000; Santa Cruz). Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare) and developed on an Image-Quant LAS4000 mini Fuji luminescence imagining system. For the analysis of protein expression, bands from at least three independent experiments were quantified by densitometry using Image J analysis software.

Analysis of mRNA expression by quantitative real-time PCR
Total RNA was isolated from rBA using an Illustra MiniRNA Spin kit (GE Healthcare), and cDNA was obtained using a Transcriptor First Strand cDNA Synthesis kit (Roche), following the manufacturer's instructions. Relative quantification of mRNA was performed using a LightCycler 1 480 instrument (Roche) in 10 μL of reaction medium by using 6.5 ng of cDNA, forward and reverse primers at 100 nM each, and a SYBR Green PCR Master Mix Reagent kit (Life Technologies). Primer pairs are shown in Table 1. The mRNA levels were normalized to those of b-actin and expressed as fold change.

Fatty acid oxidation assay
The FAO rate was measured in rBA grown in 25-cm 2 flasks, differentiated and infected as described above. On the day of the assay, cells were washed in KRBH 0.1% BSA (FA free), preincubated at 37°C for 30 min in KRBH 1% BSA, and washed again in KRBH 0.1% BSA. Cells were then incubated for 3 hours at 37°C with fresh KRBH containing 11 mM glucose, 0.8 mM carnitine plus 0.2 mM [1-14 C] oleate (Perkin Elmer). Oxidation was measured as previously described [21].
Glycerol release rBA were seeded, differentiated in 12-wells plates and infected as described above or incubated in the presence or absence of 5 μM norepinephrine and 180 μM etomoxir for 6 hours. Then, the cell medium was removed and glycerol levels were measured using the TG Triglyceride kit (Sigma-Aldrich), according to the manufacturer's instructions. Samples were normalized by the protein concentration present in each well.  Lipid extraction from rBA rBA were seeded, differentiated in 12-wells plates and infected as described above. Then, cells were collected for lipid extraction following Gesta et al protocol [36] with minor modifications: after cell lysis with 0.1% SDS, 1/2/0.12 (v/v/v) methanol/chloroform/0.5M KCl solution was added, the two phases were separated by centrifugation and the upper phase was dried with N 2 . Finally, lipids were resuspended in 100% isopropanol.

Measurement of nonesterified fatty acids
Nonesterified fatty acids (NEFAs) were measured in rBA lipid extracts by NEFA-HR detection kit (Wako Diagnostics) according to the manufacturer's instructions. NEFA levels were normalized against protein content.

Seahorse bioanalyzer
The Seahorse XF24 (Seahorse Bioscience, www.seahorsebio.com) was used to measure oxygen consumption rate (OCR) in mature brown adipocytes. For a typical bioenergetic profile we used oligomycin to block ATP synthase; the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) to measure maximal respiratory capacity; followed by rotenone, the complex 1 inhibitor, and antimycin-A to leave only non-mitochondrial activity to be measured (all from Sigma-Aldrich). Cells were differentiated in customized Seahorse 24-well plates and infected as described above. Before the measurement, cells were incubated for 1 hour with XF Assay Medium (Seahorse Bioscience) plus 5 mM glucose. During the assay, we injected the fol-

Fatty acid (FA) treatment
Sodium palmitate was conjugated with free BSA in a 5:1 ratio to yield a stock solution of 2.5 mM [27]. Cells were incubated with 0.3 mM or 1 mM of this solution or only BSA (basal control) for 24 hours.

Triglycerides measurement
Triglycerides (TG) were quantified in rBA lipid extracts using TG Triglyceride kit (Sigma), according to the manufacturer's instructions. Protein concentrations were used to normalize sample values.

Statistical analysis
Data are expressed as the mean ± SEM. Control and experimental groups were compared using Student's t-test (column analysis) or two-way ANOVA (grouped analysis). P<0.05 was considered statistically significant. All figures and statistical analyses were generated using GraphPad Prism 6 software.

Characterization of rBA differentiation stage
A time-course study was performed to assess the differentiation stage of rBA. Differentiation was evaluated by Oil Red O staining, mRNA expression of BAT differentiation markers, and protein levels of UCP1. Oil Red O staining showed an increase in neutral lipid accumulation, starting at day 6 of differentiation (Fig 1A and 1B). The mRNA levels of the brown adipocyte differentiation markers CIDEA, PGC1a, PRDM16 and Zic1 increased at day 3, and peaked at day 6 of differentiation ( Fig 1C). The mRNA levels of the beige adipocyte marker CD137 were unchanged (Fig 1C), which indicates that rBA did not show a beige phenotype. The proteins levels of the classical BAT marker, UCP1, were maximal at day 6 of differentiation ( Fig 1D). Therefore, day 6, when cells were completely differentiated and had reached maturity, was chosen to perform the expression of CPT1AM.
In addition, we analyzed the mRNA expression pattern of CPT1 isoforms during differentiation ( Fig 1E). As expected, CPT1B (the isoform most expressed in BAT), showed higher mRNA changes than the other two isoforms starting at day 3 of differentiation. CPT1A was also increased at day 3 and 6 of differentiation compared to day 0, but to a lesser extent than CPT1B. At day six of differentiation, the three isoforms were significantly increased compared to undifferentiated rBA (day 0).

Enhanced FAO in CPT1AM-expressing rBA
Fully differentiated and mature rBA (day 6) were infected with adenoviruses carrying the CPT1AM gene or GFP as a control. The efficiency of the infection was evaluated in rBA transduced with AdGFP (Fig 2A upper panel; 52.8% infected cells). CPT1A mRNA and protein levels were 9.82-and 3.52-fold higher, respectively, in CPT1AM-expressing rBA than in GFP control cells (Fig 2A lower panel and Fig 2B). The mRNA expression levels of the other CPT1 isoforms (CPT1B and CPT1C) were unaltered in CPT1AM-expressing rBA (S1A Fig and S1 Table). In turn, the FAO rate was 3.42-fold higher in CPT1AM-expressing rBA (Fig 2C). To evaluate whether the increase in FAO was related to an increase in mitochondrial content, we measured the expression of Tim 44 (translocase of mitochondrial inner membrane 44, a mitochondrial content marker), PGC1aα (a mitochondrial biogenesis marker) and Mfn2 (target gene of PGC1a). No changes were seen in any of these markers, which indicate that CPT1AM expression did not affect mitochondrial content or mitochondrial biogenesis (Fig 2D-2F). Interestingly, the mRNA expression of the mitochondrial electron transport respiratory chain (ETC) complexes such as mitochondrial NADH dehydrogenase 1 (Mt-Nd1, complex I), cytochrome b (Mt-Cyb, complex III), cytochrome C oxidase I (Mt-Co1, complex IV component), and ATP synthase 6 (Mt-ATP6, complex V), were increased in CPT1AM-expressing rBA (Fig 2G).

CPT1AM expression increases lipolysis and UCP1 protein levels
The effects of increased FAO on lipolysis and UCP1 protein levels were measured as markers of activation of thermogenesis. We measured intracellular nonesterified fatty acids (NEFAs) and glycerol release, which are two major products of lipolysis, a conventional method to quantify thermogenesis. NEFAs and glycerol levels were increased in CPT1AM-expressing rBA, indicating that CPT1AM expression stimulates lipolysis (Fig 3A and 3B). CPT1AMexpressing cells showed a 1.6-fold increase in UCP1 protein levels compared to GFP-infected cells (Fig 3C).  To further examine the role of FAO in rBA thermogenesis, rBA were stimulated with norepinephrine (NE) and incubated with or without etomoxir, an irreversible CPT1-specific inhibitor. Glycerol release was measured after 6 hours of treatment. The NE-induced increase in lipolysis was blunted in etomoxir-treated rBA (Fig 3E).

Increased mitochondrial respiration in CPT1AM-expressing rBA
The CPT1AM-induced increase in FAO, UCP1 protein levels and lipolysis prompted us to evaluate potential changes in rBA energetics. Bioenergetic studies were performed using a XF24 Extracellular Flux Analyzer (Seahorse Biosciences) to measure the mitochondrial activity. First, basal respiration was measured, followed by exposure to oligomycin, an inhibitor of ATP synthase, which allowed the measurement of ATP synthesis-coupled respiration and H + leak. Then, the uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone [FCCP] was added to measure the maximal respiratory capacity, followed by the Complex I inhibitor rotenone and complex III inhibitor antimycin A, which left only non-mitochondrial respiration to be measured (Fig 4A). Interestingly, the bioenergetic profile of CPT1AM-expressing rBA revealed significant increases in overall mitochondrial respiration (Fig 4A-4F). CPT1AMexpressing rBA showed higher basal respiration (Fig 4B), H + leak (Fig 4C), maximal respiratory capacity (Fig 4D), ATP synthesis-coupled respiration (Fig 4E), and reserve capacity (Fig 4F) than GFP control cells.

CPT1AM expression reduces palmitate-induced derangements
The palmitate-induced increase in triglyceride (TG) accumulation was blunted in CPT1AMexpressing rBA (Fig 5A). The mRNA levels of the obesity (FABP4) and inflammatory (TNFa) markers were increased after palmitate treatment. However, CPT1AM-expressing rBA were protected from a palmitate-induced increase in FABP4 and TNFa (Fig 5B and 5C). The mRNA levels of the ER stress markers CHOP and EDEM were decreased in CPT1AM-expressing rBA compared to GFP-control cells (S1B Fig and S1 Table).

Discussion
Obesity and associated diseases such as diabetes have become a worldwide health threat. Dyslipidemia is a common characteristic shared by these metabolic disorders, and the lipid-central point of view has become popular in recent years. The finding that humans have active brown fat has raised high expectations for the treatment of obesity-induced diseases, since brown fat can dissipate caloric energy and reduce both obesity and diabetes in experimental animals [8][9][10][11][12][37][38][39][40]. In fact, after short-term cold exposure, brown fat is the main lipid clearance organ [16]. Thus, recent years have seen increasing interest in the regulation of BAT thermogenesis. Most of the strategies have been focused on the role of UCP1 and attempts to enhance heat production, although other authors argue that additional genes may cooperate in the thermogenic function [41].
The finding that BAT is the tissue with the highest FAO rate [42] and that the activity of the FAO rate-limiting enzyme, CPT1, is decreased in BAT of diabetic rats [32] led us to hypothesize that BAT thermogenic power could be enhanced by increasing its FAO. Thus, we overexpressed the constitutively active mutant form, CPT1AM, in rBA.
The assessment of rBA differentiation by Oil Red O staining, UCP1 protein levels and mRNA expression of several BAT differentiation markers showed that the rBA were fully differentiated and mature at day 6. Thus, day 6 was chosen for further experiments. The expression pattern of the three CPT1 isoforms (CPT1A, CPT1B and CPT1C) indicated that all were significantly increased during differentiation, especially CPT1B. Although further studies will

CPT1 in Brown Adipocytes
be necessary to identify the specific function of each isoform in BAT, these results implicate all three isoforms in the differentiation of brown adipocytes.
Despite BAT being the tissue with the highest FAO rate [42], here we were able to further increase FAO in rBA by the expression of CPT1AM. This was achieved without changes in mitochondrial content or biogenesis. CPT1AM-expressing cells had higher lipolysis and UCP1 protein expression, usually considered as increased thermogenesis. This indicates that CPT1AM-mediated increase in FAO could be an alternative pathway for the regulation of thermogenesis. Initially, BAT thermogenesis requires a plentiful supply of FAs as substrates [43,44]. Therefore, lipolysis is activated during this process, releasing FAs from TG. FAs could be transported into the mitochondria, be involved in the regulation of UCP1 or directly activate UCP1, which uncouples oxidative phosphorylation from ATP production, generating heat [45,46]. Although further experiments will be needed to determine UCP1 regulation during CPT1AM expression, here we have shown that FAO enhancement not only increases lipolysis in rBA, but it is also associated with increased UCP1 protein expression, whose levels are ratelimiting for thermogenesis [47]. Since FA constitute the primary substrate for BAT [48] we hypothesize that CPT1AM expression increases the provision of BAT FA by: 1) directly introducing them into the mitochondria and 2) enhancing lipolysis. The liberated FA then activate and bind to UCP1 in the inner mitochondrial membrane resulting in proton transport, heat generation, and oxygen consumption [48,49]. Thus, CPT1AM may directly or indirectly trigger lipid mobilization to provide BAT mitochondria with fuel (FA).
Several studies have demonstrated the importance of FAO in BAT. Ji et al. showed that CPT1B +/mice developed fatal hypothermia as a result of their inability to perform thermogenesis [50]. More recently, a study in adipocyte-specific CPT2 KO mice indicated that FAO is required for the induction of thermogenic genes in BAT and cold adaptation [29]. In addition, Chondronikola et al. have shown that humans with higher amounts of BAT have also higher FAO during cold exposure and that BAT volume is associated with increased lipid metabolism (whole-body FA turnover and oxidation) and adipose tissue insulin sensitivity [51,52]. Here we show that etomoxir (a CPT1-specific inhibitor) blocks TG breakdown in NE-stimulated rBA. These observations indicate that lipolysis supplies substrates for thermogenesis, but when CPT1 is inhibited, free FAs are not transported into the mitochondria and lipolysis is blunted. This would support previous observations that CPT1 inhibitors suppress mitochondrial respiration [53], and that FAO plays an important role during thermogenesis. CPT1AM-expressing rBA displayed increased basal respiration, H + leak, maximal respiratory capacity, and reserve capacity. The increase in H + leak is consistent with the increase seen in UCP1 protein expression. CPT1AM-expressing rBA showed increased lipolysis (FA substrate availability) and mitochondrial ETC complexes mRNA expression. This could have contributed to the enhanced maximal respiratory capacity [54]. Reserve capacity is the difference between maximal respiratory capacity and basal respiration, and it is a useful qualitative indicator of mitochondrial energetic status [54,55]. Thus, our data indicate that enhanced FAO in brown adipocytes potentiates mitochondrial activity.
Finally, we evaluated whether enhanced FAO by CPT1AM expression in the context of obesity would improve lipid-induced derangements. Thus, we decided to mimic an obese phenotype in our cellular rBA model. Traditionally, FA incubation has been used to activate thermogenesis [56]. We tested several palmitate concentrations and different times of incubation to achieve a lipid-induced increase in TG accumulation and FABP4 and TNFa mRNA expression, similar to that previously observed in obese and lipodystrophic mice [16,57,58]. Enhanced FAO in rBA reduced TG content and FABP4 and TNFa mRNA levels, thereby protecting brown adipocytes from obesity and inflammation. These observations highlight CPT1AM-mediated activation of FAO in brown adipocytes as a potential strategy to treat obesity-induced diseases.
In summary, we have shown that CPT1AM expression in rBA increases FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Incubation of rBA with the CPT1-specific inhibitor, etomoxir, blocks NE-stimulated lipolysis. Furthermore, enhanced FAO restored the palmitate-induced increase in TG accumulation and the expression levels of obese and inflammatory markers. We conclude that enhancing the fat-burning power of brown adipocytes through CPT1AM expression may protect them from lipid-induced derangements. Thus, CPT1AM-mediated increase in lipolysis, UCP1 protein expression and mitochondrial activity in brown adipocytes may lead to a new treatment of obesity and related disorders.