Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation

Maria Ida Malandrino, Raquel Fucho, Minéia Weber, María Calderon-Dominguez, Joan Francesc Mir, Lorea Valcarcel, Xavier Escoté, María Gómez-Serrano, Belén Peral, Laia Salvadó, Sonia Fernández-Veledo, Núria Casals, Manuel Vázquez-Carrera, Francesc Villarroya, Joan J Vendrell, Dolors Serra, and Laura Herrero Department of Biochemistry and Molecular Biology, Institut de Biomedicina de la Universitat de Barcelona, Universitat de Barcelona, Barcelona, Spain; Institut de Biomedicina de la Universitat de Barcelona Fisiopatología de la Obesidad y la Nutrición, Instituto de Salud Carlos III, Madrid, Spain; Endocrinology and Diabetes Unit, Joan XXIII University Hospital, Institut d’Investigació Sanitària Pere i Virgili, Universitat Rovira i Virgili, Tarragona, Spain; Institut de Biomedicina de la Universitat de Barcelona de Diabetes y Enfermedades Metabólicas Asociadas, Instituto de Salud Carlos III, Madrid, Spain; Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain; Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry and Institut de Biomedicina de la Universitat de Barcelona, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain; and Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Barcelona, Spain

Over the last two decades, adipose tissue has gained crucial importance in the mechanisms involved in obesity-related disorders.The energy-storing white adipose tissue (WAT) is well vascularized and contains adipocytes, connective tissue, and numerous immune cells such as macrophages, T and B cells, mast cells, and neutrophils that infiltrate and increase their presence during obesity (22).Macrophages were the first immune cells reported to participate in obesity-induced insulin resistance (56).This highlights their pathological role in adipose tissue in addition to their traditional involvement in tissue repair and in response to dead and dying adipocytes (5,14).Fat is an active endocrine tissue that secretes hormones such as leptin, adiponectin, or resistin and inflammatory cytokines such as TNF-␣, IL-6, IL-1␤, etc. in response to several stimuli.It is therefore a complex organ controlling energy expenditure, appetite, insulin sensitivity, endocrine and reproductive functions, inflammation, and immunity (53).
Strenuous efforts are being made by the research community to elucidate the mechanisms involved in the pathophysiology of obesity-related disorders.However, an alternative strategy could be to act upstream by preventing the accumulation of lipids and the progression of obesity.In addition to reducing caloric intake, a potential effective approach to combating obesity would be to increase energy expenditure in key metabolic organs such as adipose tissue.Obese individuals and those with T2D are known to have lower fatty acid oxidation (FAO) rates and lower electron transport chain activity in muscle (17,19,37), together with higher glycolytic capacities and enhanced cellular FA uptake compared with nonobese and nondiabetic individuals (44).Thus, any strategy able to eliminate the excess of lipids found in obesity could be beneficial for health.Lipid levels can be reduced by inhibiting synthesis and transport or by increasing oxidation; here, we focus on the latter.
Malonyl-CoA, derived from glucose metabolism and the first intermediate in lipogenesis, regulates FAO by inhibiting carnitine palmitoyltransferase 1 (CPT1).This makes CPT1 the rate-limiting step in mitochondrial FA ␤-oxidation.Thus, in high-energy conditions, malonyl-CoA inhibits oxidation, diverting FAs' fate into TG accumulation.There are three CPT1 isoforms, with different tissue expressions: CPT1A (liver, kidney, intestine, pancreas, ovary, and mouse and human WAT), CPT1B (brown adipose tissue, skeletal muscle, heart, and rat and human WAT), and CPT1C (brain and testis) (2,36).The fact that CPT1 controls FAO makes it a very attractive target to reduce lipid levels and fight against obesity and T2D.Despite their excess fat, obese individuals have reduced visceral WAT CPT1 mRNA and protein levels (20).This prompted our group and others to overexpress CPT1 in liver (26,29,45), muscle (3,33,40), and white adipocytes (9), which led to a decrease in triglyceride (TG) content and an improvement in insulin sensitivity.
Here, we show that CPT1A expression was higher in human adipose tissue macrophages than in mature adipocytes and that it was differentially expressed in visceral vs. subcutaneous adipose tissue.To further investigate the role of CPT1A in both adipocytes and macrophages, we used a permanently active mutant form of CPT1A, CPT1AM, which is insensitive to its inhibitor malonyl-CoA (27), to achieve continuous oxidation of lipids.When cells were incubated with palmitate to mimic obesity, CPT1AM restored most of the palmitate-induced imbalances.An increase in FAO in adipocytes and macrophages reduced TG content and inflammatory levels, improved insulin sensitivity in adipocytes, and reduced ER stress and ROS damage in macrophages.

MATERIALS AND METHODS
Human cohorts: selection of patients.Adipose tissue was selected from an adipose tissue biobank collection of the University Hospital Joan XXII (Tarragona, Spain).All subjects were of Caucasian origin and reported that their body weight had been stable for at least 3 mo before the study.They had no systemic disease other than obesity or T2D, and all had been free of any infections in the previous month before the study.Liver and renal diseases were specifically excluded by biochemical work-up.Appropriate Institutional Review Board approval and adequate biobank informed consent were obtained from all participants.Biobanking samples included plasma and total and fractionated adipose tissue from subcutaneous and visceral origin.All patients had fasted overnight before collection of blood and adipose tissue samples.Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) samples were obtained during surgical procedures that included laparoscopic surgery for hiatus hernia repair or cholecystectomy.Samples were selected according stratification by age, sex, and BMI and grouped into two cohorts: Obesity cohort.Subjects were classified by BMI according to the World Health Organization criteria (WHO, 2000).The study included 19 lean, 28 overweight, and 15 obese nondiabetic subjects matched for age and sex (Table 1).
T2D cohort.Patients were classified as having T2D according to American Diabetes Association criteria (1997).Variability in metabolic control was assessed by stable glycated hemoglobin A 1c (HbA1c) values during the previous 6 mo.These criteria having been gathered, there were 11 T2D subjects.As a control group, we selected 36 subjects without diabetes from the obesity cohort, matched for age, BMI, and sex (Table 2).No patient was being treated with thiazolidinedione.
Anthropometric measurements.Height was measured to the nearest 0.5 cm and body weight to the nearest 0.1 kg.BMI was calculated as weight (kilograms) divided by height (meters) squared.Waist circumference was measured midway between the lowest rib margin and the iliac crest.
Collection and processing of human samples.Samples from VAT (omental) and SAT (anterior abdominal wall) from the same individual were obtained during abdominal elective surgical procedures (cholecystectomy or surgery for abdominal hernia).All patients had fasted overnight at least 12 h before the surgical procedure.Blood samples were collected before the surgical procedure from the antecubital vein, 20 ml of blood with EDTA (1 mg/ml), and 10 ml of blood in silicone tubes.Collected blood (15 ml) was used for the Adipose tissue fractionation.Adipose tissue biopsies were processed immediately.The adipose tissue was finely diced into small pieces (10 -30 mg), washed in PBS, and incubated in Medium 199 (Life Technologies) supplemented with 4% BSA plus 2 mg/ml collagenase type I (Sigma) for 1 h in a shaking water bath at 37°C.After digestion, mature adipocytes (ADI) were separated from tissue matrix by filtration through a 200-m mesh fabric (Spectrum Laboratories).The filtrated solution was centrifuged for 5 min at 1,500 g.The mature adipocytes were removed from the top layer and the stromal vascular fraction (SVF) cells remained in the pellet.Cells were washed four times in PBS and processed for RNA and protein extraction.
Analytic methods.Glucose, cholesterol, and TG plasma levels were determined in an autoanalyzer (Hitachi 737, Boehringer Mannheim) using the standard enzyme methods.High-density lipoprotein (HDL) cholesterol was quantified after precipitation with polyethylene glycol at room temperature (PEG-6000).Plasma insulin was determined by radioimmunoassay (Coat-A-Count insulin; Diagnostic Products).Nonesterified free fatty acid (NEFA) serum levels were determined in an autoanalyzer (Advia 1200, Siemens) using an enzymatic method developed by Wako Chemicals.Plasma glycerol levels were analyzed by using a free glycerol determination kit, a quantitative enzymatic determination assay (Sigma-Aldrich).Intra-and interassay CVs were less than 6% and less than 9.1%, respectively.The degree of insulin resistance was determined by homeostasis model assessment (HOMA), as [glucose (mmol/l) ϫ insulin (mIU/l)]/22.5.(24).
Immunohistochemistry. Five-micrometer sections of formalin-fixed paraffin-embedded adipose tissue were deparaffinized and rehydrated prior to antigen unmasking by boiling in 1 mM EDTA, pH 8.Sections were blocked in normal serum and incubated overnight with rabbit anti-CPT1A (Sigma-Aldrich) at 1:50 dilution.Secondary antibody staining was performed using a VECTASTAIN ABC kit (Vector Laboratories) and detected with diaminobenzidine (Vector Laboratories).Sections were counterstained with hematoxylin prior to dehydration and coverslip placement and examined under a Nikon Eclipse 90i microscope.As a negative control, the procedure was performed in the absence of primary antibody.
Immunofluorescence.Five-micrometer sections of formalin-fixed paraffin-embedded adipose tissue were blocked in normal serum and incubated overnight with rabbit anti-CPT1A antibody (Sigma-Aldrich) at 1:50 dilution and with mouse anti-CD68 (Santa Cruz Biotechnology) at 1:50 dilution, washed, and visualized using Alexa fluor 546 goat anti-rabbit, and Alexa fluor 488 goat anti-mouse antibodies, respectively (1:500, Molecular Probes).The slides were counterstained with DAPI (4,6-diamidino-2-phenylindole) to reveal nuclei and were examined under a Nikon Eclipse 90i fluorescent microscope.As a negative control, the assay was performed in the absence of primary antibody.
Materials.Sodium palmitate, sodium oleate, BSA, and L-carnitine hydrochloride were purchased from Sigma Aldrich.DMEM, FBS, and penicillin-streptomycin mixture were purchased from Life Technologies.
Cell culture.Murine 3T3-L1 CAR⌬1 preadipocytes, kindly given by Dr. Orlicky (Department of Pathology, UCHSC at Fitzsimons, Aurora, CO), were cultured and differentiated into mature adipocytes following the published protocol (31).Mature adipocytes were used for experiments at day 8 postdifferentiation.Murine RAW 264.7 macrophages were obtained from ATCC and were maintained in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin mixture.Simpson-Golabi-Behmel Syndrome (SGBS) human cells were cultured and differentiated to adipocytes as previously described (55).
Adenovirus infection.At day 8 of differentiation, 3T3-L1 CAR⌬1 cells were infected with adenoviruses AdGFP [100 moi (multiplicity of infection)] and AdCPT1AM (13) (100 moi) for 24 h in serum-free DMEM, and then the medium was replaced with complete DMEM for additional 24 h.RAW 264.7 macrophages were infected with AdGFP (100 moi) and AdCPT1AM (100 moi) for 2 h in serum-free DMEM and then replaced with complete medium for an additional 72 h.Adenovirus infection efficiency was assessed in AdGFP-infected cells (see Fig. 3, A and B).The same batch of adenoviruses stored in 50-l aliquots was used throughout the experiments.
Adipocyte and macrophage viability.3T3-L1 CAR⌬1 adipocytes and RAW 264.7 macrophages were infected as previously described and incubated for 24 h with 1 or 0.3 mM palmitate, respectively.Cells were washed twice with PBS and lifted from the surface with trypsin followed by 2 min of incubation at 37°C.Trypsinization was stopped with 10% FBS-containing medium, and equal volumes of cell suspension were mixed with 0.4% Trypan blue staining.Trypan bluepositive and -negative cells were counted using a Neubauer chamber for adipocytes and a Countess Automated Cell Counter (Invitrogen) for macrophages.Percentage of viability was determined normalizing viable cells of each group to viable cells of BSA GFP group.Statistical significance was assessed using two-way ANOVA of three individual experiments (*P Ͻ 0.05).
CPT1 activity.Mitochondria-enriched fractions were obtained from cell culture grown in 10-cm 2 dishes, and CPT1 activity was measured by a radiometric method as described (13).
TG content.Cells were grown in 12-well plates, differentiated, and infected as described above.After 24 h (3T3-L1 CAR⌬1 adipocytes) or 18 h (RAW 264.7 macrophages) of FA treatment, cells were collected for lipid extraction following a protocol of Gesta et al. (10) with minor modifications: after cell lysis with 0.1% SDS, 1/2/0.12(vol/vol/vol) methanol-chloroform-0.5 M 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, and TGs were quantified using a TG Ttriglyceride kit (Sigma) according to the manufacturer's instructions.Protein concentrations were used to normalize sample values.
Oil red O staining.RAW 264.7 macrophages grown on coverslips were infected as described above and incubated with 0.75 mM palmitate for 18 h.After this time, cells were rinsed twice with PBS, fixed in 10% paraformaldehyde for 30 min at room temperature, and washed again with PBS.Then, cells were rinsed with 60% isopropanol for 5 min to facilitate the staining of neutral lipids and were stained with filtered Oil red O working solution (0.3% Oil red O in isopropanol) for 15 min.After several washes with distilled water, the coverslips were removed and mounted on a drop of mount medium.The intracellular lipid vesicles stained with Oil red O were identified by their bright red color under the microscope.
Analysis of intracellular protein oxidation.RAW 264.7 macrophages were cultured in 12-well plates and infected as described before.After FA treatment, cell extracts were prepared and analyzed for protein oxidative modifications (i.e., carbonyl group content) with a OxyBlot Protein Oxidation Detection kit (Millipore), following the manufacturer's instructions.
Analysis of mRNA expression by quantitative real-time PCR.Total RNA was extracted from cultured cells grown in 12-well plates using Illustra Mini RNA Spin kit (GE Healthcare), and cDNA was obtained using a Transcriptor First Strand cDNA Synthesis kit (Roche).Quantitative real-time PCR was performed using a SYBR Green PCR Master Mix Reagent Kit (Life Technologies).Levels of mRNA were normalized to those of ␤-actin and expressed as fold change.Forward/ reverse primers for several genes were used (Table 3; other sequences are available upon request): Frozen human adipose tissue (400 -500 mg) was homogenized with an Ultra-Turrax 8 (Ika).Total RNA from adipose biopsies, SVF, and isolated adipocytes were extracted by using an RNeasy Lipid Tissue Midi Kit (QIAGEN) following the manufacturer's instructions, and total RNA was treated with 55 U of RNase-free DNase (QIAGEN) to avoid contamination with genomic DNA.Between 0.2 and 1 g of total RNA was reverse-transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems) and subsequently diluted with nuclease-free water (Sigma) to 20 ng/l cDNA.For adipose tissue gene expression analysis, real-time quantitative PCR was performed, with duplicates, on a 7900HT Fast Real-Time PCR System using commercial Taqman Assays (Applied Biosystems).SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems) were used to analyze the results with the comparative threshold cycle (C T) method (2 ⌬⌬CT ).CT values for each sample were normalized with an optimal reference gene (cyclophilin) after testing of three additional housekeeping genes: ␤-actin and RNA 18S.A panel of genes involved in the adipocyte differentiation and metabolism was selected in the study of CPT1A gene expression (Table 4).
Cytokine measurement in culture media.Cytokine protein levels in culture medium of 3T3-L1 CAR⌬1 adipocytes and RAW 264.Analysis of cellular redox status.To detect ROS (superoxide) formation, MitoSOX Red (M36008, Life Techonologies) fluorescence was measured by flow cytometry.RAW 264 cells were infected with 100 moi AdCPT1AM (or AdGFP as control) for 48 h; then, 16 h prior to ROS measurement, macrophages were treated with 0.75 mM palmitate BSA-conjugated (or with BSA as control).Medium was removed, and cells were incubated for 30 min with PBS containing 5 M MitoSOX Red.The labeled macrophages were washed three times with HBSS-Ca-Mg, pelleted, resuspended in 300 l of HBSS-Ca-Mg, and fixed by adding 1.2 ml of absolute ethanol and keeping them at Ϫ20°C for 5 min.Cells were pelleted again and resuspended in HBSS-Ca-Mg containing 3 M DAPI to mark their nuclei.Then macrophages were analyzed by flow cytometry (Gallios Cytometer, Beckman-Coulter).The fluorescence intensity of MitoSOX Red was measured using excitation at 510 nm and emission at 580 nm.
Statistical analysis.Data are expressed as means Ϯ SE and analyzed statistically using Student's t-test (column analysis) or two-way ANOVA (grouped analysis).All figures and statistical analyses were generated using GraphPad Prism 6. P Ͻ 0.05 was considered statistically significant.For human data, statistical analyses were performed with SPSS 12.0.Results are expressed as means Ϯ SD.The nonnor-Table 3. Forward and reverse primers

RESULTS
CPT1A expression pattern in human adipose tissue from obese and diabetic patients.Visceral and subcutaneous adipose tissue (VAT and SAT, respectively) were analyzed from both an obesity cohort (lean, overweight and obese patients) and a T2D cohort (control and T2D patients).Tables 1 and 2 show the phenotypic and metabolic characteristics and CPT1A expression levels of the subjects.No differences in CPT1A gene expression levels either in SAT or in VAT depots were observed when comparing with the nonobese or the nondiabetic counterparts (Fig. 1, A and B; Tables 1 and 2).However, in the obesity cohort, CPT1A mRNA expression was significantly higher in lean and overweight VAT than in SAT (Fig. 1A); this difference was lost in the obese patients.These results were corroborated by Western blot with human adipose tissue of several lean and obese individuals (Fig. 1, C and D, P ϭ 0.015).Similar results were obtained in the T2D cohort, where control subjects showed significantly higher CPT1A mRNA levels in VAT vs. SAT (Fig. 1B); however, this difference disappeared in T2D patients.Despite T2D patients showing a trend to express higher CPT1A levels in SAT and VAT compared with controls (the opposite of that in the obese subjects), this difference was nonsignificant.Since the CPT1B isoform is also expressed in human adipose tissue, we analyzed CPT1B mRNA (Fig. 1, E and F) and protein (data not shown) levels in human VAT and SAT of the obesity and the T2D cohort.No differences were seen among the groups.
To establish the main relationship between CPT1A gene expression and key adipocyte genes involved in differentiation and metabolic pathways, we explored a panel of genes (see MATERIALS AND METHODS) both in SAT and in VAT depots of the obesity cohort.Results are shown from those genes that changed the most (up or down; Tables 5 and 6).Simple association analysis showed an inverse correlation between CPT1A and peroxisome proliferator-activated receptor-␥ (PPAR␥) in SAT (r ϭ Ϫ0.38, P ϭ 0.002; Table 5).Positive CPT1A correlation in both VAT and SAT was found with 1-acylglycerol-3-phosphate O-acyltransferase 5 (AGPAT5; phospholipid biosynthesis), sterol regulatory element binding transcription factor 1 (SREBF1; glucose and lipid metabolism), B cell CLL/lymphoma 2 (BCL2; antiapoptosis), and CD163 (macrophage marker) (Table 5).
To study the main determinants of CPT1A gene expression levels, a stepwise multiple regression analysis was performed, including the aforementioned bivariate associations and confounding factors such as BMI, age and sex.This model showed that SAT CPT1A was positively associated with AGPAT5, SREBF1, and CD163 and that VAT CPT1A was positively correlated with SREBF1 and CD163 and negatively with age and PPAR␥ (Table 6).The inverse relationship between CPT1A and PPAR␥ was corroborated with the human adipocyte cell line SGBS.CPT1A mRNA expression dropped to a new steady state in adipocytes that was 11% of its expression in fibroblasts (data not shown).
CPT1A is highly expressed in human adipose tissue macrophages.To determine the cellular distribution of CPT1A gene and protein in human adipose tissue biopsies, we performed qRT-PCR and immunostaining analysis on both adipose and SVF.CPT1A mRNA levels were 42.6-and 43.4-fold increased in the SVF compared with mature adipocytes in both human SAT (P Ͻ 0.05) and VAT (P Ͻ 0.05), respectively (Fig. 2A).Immunohistological examination of SAT from obese subjects revealed CPT1Aϩ cells mostly in the SVF (Fig. 2B).Immunofluorescence detection showed a bright staining pattern in cells resembling adipose tissue macrophages.Costaining analysis using CPT1A and CD68 (a macrophage marker) antibodies confirmed the expression of CPT1A in macrophages (Fig. 2C).Macrophages seemed to localize forming the so-called "crown-like structures" surrounding the adipocytes.CPT1AM-expressing adipocytes show enhanced FA oxidation and reduced TG content.To further study the role of CPT1A in adipocytes and macrophages, we decided to con-tinue with in vitro studies.Since 3T3-L1 adipocytes are inefficiently infected with adenovirus, we decided to use the high-infection efficiency white adipocyte cell culture line, 3T3-L1 CAR⌬1 adipocytes (31) (Fig. 3A).Cells were transduced for the first time with adenoviruses carrying the CPT1AM gene or GFP as a control.Interestingly, CPT1AM-

A B
SAT VAT   expressing adipocytes were partially protected from palmitateinduced cell death (Fig. 3C).
CPT1A mRNA, protein, and activity levels were increased in CPT1AM-expressing adipocytes compared with GFP control cells (Fig. 4, A-C).CPT1AM-expressing adipocytes retained most of the CPT1 activity after incubation with the CPT1A inhibitor malonyl-CoA (Fig. 4C).The FA oxidation (FAO) rate was concordantly enhanced (1.37-fold increase, P Ͻ 0.05) in CPT1AM-expressing adipocytes (Fig. 4D).FA undergoing ␤-oxidation yield acetyl-CoA moieties that have two main possible fates: 1) complete oxidation to CO 2 and ATP production or 2) conversion to ketone bodies (mainly in the liver).Here, the total FAO rate was calculated as the sum of acid-soluble products plus CO 2 oxidation.CPT1AM expression blocked the palmitate-induced increase in TG content (Fig. 4E).
Enhanced adipocyte FAO improves insulin sensitivity and reduces inflammation.We examined the effect of increased FAO on insulin sensitivity and inflammatory responses in 3T3-L1 CAR⌬1 adipocytes infected with AdCPT1AM.Palmitate-induced decrease in insulin-stimulated Akt phosphorylation and insulin receptor-␤ (IR␤) protein levels was partially restored in CPT1AM-expressing adipocytes (Fig. 4, F-H).Palmitate-induced increase of proinflammatory markers [IL-1␤, monocyte chemoattractant protein-1 (MCP-1), and IL-1␣] mRNA and protein levels was blunted in CPT1AM-expressing adipocytes (Fig. 4, I-K).Several palmitate concentrations and times of incubation were used to better fit the different dose and time responses of the cytokines and parameters measured.Consistent with previous studies (9,11), palmitate incubation raised cytokines expression two-to threefold.
Increased FAO in CPT1AM-expressing macrophages protects from palmitate-induced TG accumulation.Since CPT1A was highly expressed in the SVF, and particularly in macrophages, of human adipose tissue, we decided to further analyze the effect of increased FAO on cultured macrophages.RAW 264.7 macrophages were efficiently infected with AdCPT1AM (Fig. 3B).CPT1AM-expressing macrophages were protected from palmitate induced cell death (Fig. 3D).CPT1AM-expressing macrophages showed a 2.4-fold (P Ͻ 0.01) increase in CPT1A mRNA levels, a 6.6-fold (P Ͻ 0.01) increase in protein levels, and a 2.2-fold (P Ͻ 0.05) increase in activity levels (Fig. 5, A-C).In addition, we showed that malonyl-CoA did not inhibit CPT1 activity in CPT1AM-expressing macrophages (Fig. 5C).CPT1AM-expressing macrophages showed a 1.5fold increase in FAO rate compared with GFP control cells (Fig. 5D, P Ͻ 0.05) and a total restoration in palmitate-induced enhancement of TG content (Fig. 5, E and F).

DISCUSSION
The obesity epidemic has put a spotlight on adipose tissue as a key player in obesity-induced insulin resistance (38).Obese individuals and those with T2D have lower FAO rates (17,19,37).Although these data were reported in skeletal muscle, we expected to see reduced CPT1A expression levels in the adipose tissue of both obese and T2D patients.However, no differences were seen in CPT1A mRNA expression between the obese or T2D patients and their respective controls either in VAT or in SAT.Other authors have reported a decrease in VAT CPT1 mRNA and protein levels in obese individuals (20).However, those authors did not specify which of the CPT1 isoforms was measured in VAT, CPT1A or CPT1B.We showed that CPT1A expression is higher in adipose tissue macrophages than in mature adipocytes.Since the obese adipose tissue has higher infiltration of immune cells such as macrophages, we postulate that the putative decrement of CPT1A expression in obese individuals could be compensated for by increased expression from the infiltrated macrophages and thus that no differences are seen between the groups.The CPT1B isoform is also expressed in human adipose tissue, and it has been shown to raise FAO in metabolic tissues such as skeletal muscle (3).Thus, we measured mRNA and protein levels in the obese and T2D cohorts.However, no differences were seen among the groups, indicating CPT1B expression is not changed by obesity and T2D.We found that, in insulin-sensitive individuals (control and overweight patients from the obese cohort and control patients from the T2D cohort), CPT1A mRNA expression was higher in VAT than in SAT.However, no differences between VAT and SAT were seen in the more insulin-resistant individuals with a more proinflammatory environment: obese and T2D patients.A similar phenomenon was described for T regulatory cells, described to have anti-inflammatory properties and to improve obesity-induced insulin resistance (7).Those authors reported that the VAT and SAT of healthy individuals had similar low numbers of T regulatory cells at birth, with a progressive accumulation over time in the VAT, though not in the SAT.Our results suggest a CPT1A expression balance between SAT and VAT depots that may be disturbed in obese and T2D patients.The difference in CPT1A expression between these two fat depots is potentially crucial, given the association of VAT, but not SAT, with insulin resistance (1,52).It might indicate, in healthy individuals, a potential protective role of CPT1A in the more insulin-resistant associated VAT.
Gene expression analysis revealed a negative association between CPT1A and the adipocyte marker of differentiation PPAR␥.This is consistent with the fact that while white adipocytes mature they shift their lipid preferences to storage rather than oxidation.Aging was associated with reduced CPT1A expression in VAT.This might reflect a potential protective role of CPT1A expression in VAT that is lost with age.Considering that VAT accretion is a hallmark of aging and especially that it is a stronger risk factor for comorbidities and mortality (23), we speculate a favorable role of enhanced CPT1A expression in age metabolic decline and related pathological conditions.Positive correlation in both VAT and SAT CPT1A was found with AGPAT5, SREBF1, Bcl2, and CD163.
These results may indicate a potential role of CPT1A in lipid biosynthesis processes (AGPAT5), glucose and lipid metabolism (SREBF1), and protecting adipose tissue from apoptosis (Bcl2).The positive association between CPT1A and CD163 (macrophage marker) was not surprising given the higher CPT1A expression in macrophages than in adipocytes (Fig. 2).We are aware that many of the aforementioned associations may be secondary to obesity or T2D and that no causal relationship may be inferred with this study design.To prove the causality of some of these observations, we performed in vitro studies directly targeting adipocytes and macrophages to burn off the excess lipids through an increase in FAO.We used the high-infection efficiency adipocyte cell line 3T3-L1 CAR⌬1 (31) to express for the first time CPT1AM through adenoviral infection.Noteworthy, white adipocytes are designed to store lipids rather than to oxidize them.Thus, CPT1 activity in WAT is lower than in other tissues (6).However, CPT1AM-expressing adipocytes showed a 4.3-fold increase in CPT1 activity that was not inhibited despite incubation with high concentrations of malonyl-CoA.Since increased lipid accumulation, inflammation, ER stress, and ROS-induced protein damage trigger metabolic diseases, we decided to measure TG content, inflammation, ER stress, and ROS damage as important mechanisms that could explain the potential protec- tive effect of CPT1AM expression.Enhanced FAO led to complete restoration of TG content, improved insulin signaling (measured as pAkt), increased IR␤ expression and cell viability, and reduced inflammation in palmitate-incubated CPT1AM-expressing adipocytes.CPT1AM-expressing adipocytes showed a general improvement in lipid-induced derangements as a consequence of increased FA flux through mitochondria.However, enhanced FA flux in the absence of a concomitant dissipation of FAO metabolites has been associated with increased ROS damage (35) and inflammation (8,21,43).Interestingly, although no differences were seen in ER or oxidative stress (data not shown), CPT1AM-expressing adipocytes showed a significant decrease in proinflammatory mediators such as IL-1␤ and MCP-1.The favorable role of CPT1A in adipocytes to attenuate FA-evoked insulin resistance and inflammation has been also described to act via suppression of JNK (9).These results suggest that factors other than a FAO increase per se are responsible for ROS production and inflammation.Accumulation of toxic substances (diacylglycerol or ceramides) (49), hypoxia (15), as well as cytokines (42) might participate in the induction of ROS damage and the inflammatory state.Several researchers have demonstrated that enhanced FAO through CPT1A or CPT1AM expression results in a decrease in relevant lipid mediators involved in inflammation and insulin resistance such as diacylglycerol, intracellular NEFAs, free FA, ceramides, and TG (3,9,13,26,29,40,45).Although some authors (3) did not see changes in skeletal muscle acylcarnitines' profile, our group has shown an increase in several acylcarnitines in CPT1AM-expressing neurons (25).FA undergoing ␤-oxidation yield acetyl-CoA moieties that have two main possible fates: 1) entry to the TCA cycle for complete oxidation and ATP production or 2) conversion to ketone bodies (mainly in the liver).We observed increased FAO to CO 2 and acid-soluble products in CPT1AM-expressing adipocytes and macrophages.CPT1AM expression in liver has been shown to enhance ATP and ketone body production with no changes in glucose oxidation (13,29).All together, this indicates a metabolic rate switch toward FA.
Monocytes were the first immune cells reported to infiltrate obese adipose tissue, differentiate to macrophages, produce inflammatory cytokines, and trigger insulin resistance (56,57).Thus, we examined whether CPT1AM expression could play a protective role in obesity-induced macrophage derangements.We found that, in human WAT, CPT1A is highly expressed in SVF compared with adipocytes.This happened in both human VAT and SAT.A closer histological and immunofluorescence examination showed that macrophages present in the adipose tissue expressed CPT1A.This does not rule out CPT1A expression in other immune cells also present in the adipose tissue such as T and B cells, T regulatory cells, and mast cells.
Given the high CPT1A expression in human adipose tissue macrophages, we decided to study the effect of CPT1AM in RAW 264.7 macrophages.A permanently enhanced FAO rate in CPT1AM-expressing macrophages led to a complete restoration of palmitate-induced increase in TG content and a decrease in inflammation and ER and oxidative stress without affecting cell viability.Recent data show that FAO is capable of regulating the degree of acyl chain saturation in ER phospholipids (28).Since increasing the degree of saturation in ER phospholipids has been described to directly activate ER stress and inflammation (28), this might provide a mechanistic link to how FAO alleviates ER stress under palmitate loading.Thus, enhancing CPT1A expression in macrophages may be a potential approach to fight against obesity-induced disorders.
In conclusion, we have shown that CPT1A expression was higher in human adipose tissue macrophages than in mature adipocytes and that it was differentially expressed in VAT vs. SAT.Further in vitro studies demonstrated that an increase in FAO in lipid-treated adipocytes and macrophages reduced TG content and inflammatory levels, improved insulin sensitivity in adipocytes, and reduced ER stress and ROS damage in macrophages.Adipocyte-specific knockout or transgenic animal models for CPT1A would be especially relevant to elucidate its potential protection against obesity-induced insulin resistance in vivo.Our data support the hypothesis that pharmacological or genetic strategies to enhance FAO may be beneficial for the treatment of chronic inflammatory pathologies such as obesity and T2D.

CFig. 1 .
Fig. 1.Carnitine palmitoyltransferase 1A (CPT1A) gene and protein expression in human adipose tissue.A and B: CPT1A relative mRNA levels in human visceral (VAT) and subcutaneous adipose tissue (SAT) of the obesity (A) or the type 2 diabetes (T2D; B) cohort.Numbers of individuals: 19 lean, 28 overweight, 15 obese, 36 control, and 11 T2D (see Tables1 and 2for more details).C and D: CPT1A protein levels in human VAT and SAT of 7 lean individuals (P1-P7; C) and 3 obese individuals (D).E and F: CPT1B relative mRNA levels in human VAT and SAT of the obesity (E) or the T2D (F) cohort.*P Ͻ 0.05.

Fig. 2 .
Fig. 2. CPT1A is highly expressed in human adipose tissue macrophages.A: CPT1A mRNA levels in both adipose (Ad) and stromal-vascular fraction (SVF) of human VAT and SAT; n ϭ 4, *P Ͻ 0.05.B: immunohistochemical detection of CPT1A (brown) in SAT of obese subjects.C: immunofluorescence staining of CPT1A (red) and CD68 (green) proteins in SAT of obese individuals.Counterstaining of nuclei (DAPI) is shown in blue.Images are representative of adipose tissue preparations collected from 3 subjects.

Fig. 5 .Fig. 6 .
Fig. 5. Enhanced FAO and reduced TG content in CPT1AM-expressing RAW 264.7 macrophages.Relative CPT1A mRNA expression (A) and protein levels (B) in AdGFP-or AdCPT1AM-infected macrophages.C: CPT1 activity from mitochondria-enriched cell fractions incubated (or not) with 100 M malonyl-CoA.D: total FAO rate measured as the sum of acid-soluble products plus CO2 oxidation.TG content (E) and Oil red O staining (F) of macrophages treated for 18 h with 0.75 mM PA. Shown are representative experiments out of 3; n ϭ 3-6, *P Ͻ 0.05.

Table 2 .
Clinical, analytic, and CPT1A gene expression analysis of the T2D cohortValues are expressed as means Ϯ SD or median (interquartile range) for non-Gaussian distributed variables.Differences vs. controls: *P Ͻ 0.001; ¶P Ͻ 0.05.Differences between SAT and VAT in the same group: †P ϭ 0.03.

Table 4 .
Gene symbols, denominations, and assay ID numbers

Table 6 .
Multiple regression analysis for CPT1A in VAT and SAT as dependent variable in the obesity cohort Independent variables included in the model: age, sex, BMI, PPAR␣, PPAR␥, AGPAT5, SREBF1, BCL2, and macrophage and monocyte marker (CD163) gene expression levels.␤ st, standardized ␤-coefficient.CI, confidence interval.