The adipocyte is the principal cell type for fat storage. CPT1 (carnitine palmitoyltransferase-1) is the rate-limiting enzyme for fatty acid β-oxidation, but the physiological role of CPT1 in adipocytes remains unclear. In the present study, we focused on the specific role of CPT1A in the normal functioning of adipocytes. Three 3T3-L1 adipocyte cell lines stably expressing hCPT1A (human CPT1A) cDNA, mouse CPT1A shRNA (short-hairpin RNA) or GFP (green fluorescent protein) were generated and the biological functions of these cell lines were characterized. Alteration in CPT1 activity, either by ectopic overexpression or pharmacological inhibition using etomoxir, did not affect adipocyte differentiation. However, overexpression of hCPT1A significantly reduced the content of intracellular NEFAs (non-esterified fatty acids) compared with the control cells when adipocytes were challenged with fatty acids. The changes were accompanied by an increase in fatty acid uptake and a decrease in fatty acid release. Interestingly, CPT1A protected against fatty acid-induced insulin resistance and expression of pro-inflammatory adipokines such as TNF-α (tumour necrosis factor-α) and IL-6 (interleukin-6) in adipocytes. Further studies demonstrated that JNK (c-Jun N terminal kinase) activity was substantially suppressed upon CPT1A overexpression, whereas knockdown or pharmacological inhibition of CPT1 caused a significant enhancement of JNK activity. The specific inhibitor of JNK SP600125 largely abolished the changes caused by the shRNA- and etomoxir-mediated decrease in CPT1 activity. Moreover, C2C12 myocytes co-cultured with adipocytes pre-treated with fatty acids displayed altered insulin sensitivity. Taken together, our findings have identified a favourable role for CPT1A in adipocytes to attenuate fatty acid-evoked insulin resistance and inflammation via suppression of JNK.
- carnitine palmitoyltransferase 1A (CPT1A)
- c-Jun N-terminal kinase (JNK)
- fatty acid
- insulin resistance
- pro-inflammatory adipokine
Adipocytes, traditionally thought to be an inert cell type for lipid storage, are now recognized as an essential player orchestrating the overall energy metabolism of the body . Insulin sensitivity, adipokine production and inflammatory status in adipocytes are central to global energy homoeostasis. Adipocyte dysfunction underlies the initiation and development of a panel of metabolic disorders, including insulin resistance, hypertension, atherosclerosis and stroke. It is now evident that pro-inflammatory adipokines secreted from adipocytes systemically control glucose and lipid metabolism in the body. In obese subjects, the expansion of adipose tissue leads to the secretion of excess amounts of fatty acids and pro-inflammatory adipokines, such as TNF-α (tumour necrosis factor-α), A-FABP (adipocyte fatty acid-binding protein), MCP-1 (monocyte chemoattractant protein-1), PAI-1 (plasminogen-activator inhibitor-1), lipocalin-2 and RBP4 (retinol-binding protein 4), to name a few, but also causes a decrease in the production of adiponectin, an insulin-sensitizing adipokine with anti-diabetic, anti-inflammatory, and cardioprotective and vasculoprotective properties . Additionally, insulin resistance in adipocytes manifests as a blunted GLUT4 (glucose transporter 4) translocation in response to insulin, which is followed by the whole-body glucose intolerance .
How abnormal adipokine production is evoked in the context of obesity and subsequently leads to insulin insensitivity still needs to be fully elucidated. Obesity begins with the overexpansion of adipose tissues both in cell number (hyperplasia) and size (hypertrophy), which is essentially a result of an imbalance between lipid storage and lipid utilization. Although it is generally believed that enlarged adipose tissue is predisposed to adipocyte dysfunction, the exact role of fatty acid oxidation in adipocyte functioning remains unclear.
CPT1 (carnitine palmitoyltransferase 1) is the key regulatory enzyme in fatty acid oxidation. It is anchored in the outer membrane of mitochondria and catalyses the formation of long-chain acyl-carnitine, which is enabled to traverse the inner mitochondrial membrane and thus committed to β-oxidation in the mitochondria. There are three genes that code for CPT1: CPT1A, which is the most abundant form in liver, CPT1B, which is the major form in muscle, and CPT1C, which is mainly present in the brain. Deletion of CPT1A or CPT1B in mouse is lethal in the early development [4,5], therefore the exact role of CPT1A or CPT1B in energy homoeostasis remains unresolved. However, the essential role of CPT1A has been inferred by several studies. In patients with unusual spontaneous mutations in the CPT1A gene, hepatic deficiency of CPT1A leads to recurrent episodes of hypoketotic hypoglycaemia, hepatomegaly, seizures and coma. In the pancreas, inhibition of fatty acid oxidation by a CPT1-specific inhibitor, etomoxir , or by non-metabolizable fatty acid analogues  changes GSIS (glucose-stimulated insulin secretion). Additionally, blockade of CPT1 activity by its selective inhibitors protected hearts from fatty acid-induced ischaemic injury [8–10]. Alteration of hypothalamic CPT1 activity by molecular and pharmacological approaches significantly affects endogenous glucose production [11,12]. In addition, studies by Gao et al.  and Wolfgang et al.  have shown that mice with Cpt1c deleted are more sensitive to high-fat diet-induced insulin resistance.
CPT1 is expressed in adipocytes [15–17], yet little is known about the functional role of CPT1 in this cell type, and whether CPT1 serves as an active regulator for adipocyte function is yet to be elucidated. Thus it is relevant and important to examine the role of CPT1 in adipocytes, where fat storage and accumulation are believed to be the main fate for fatty acids. In the present study, we examined the alterations in the normal functioning of adipocytes after using strategies to provide gain or loss of CPT1 function. In addition, the underlying mechanisms for the observed effects were also investigated. Our findings demonstrate that CPT1 is a key regulator in adipocyte inflammation and insulin signalling.
Cell culture and treatment
Mouse 3T3-L1 pre-adipocytes were cultured and differentiated into mature adipocytes using a standard protocol. Briefly, at 2 days post-confluence (day 0), cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum) (Gibco), 0.5 mM 1-methyl-3-isobutylxanthine (Sigma), 1 μM dexamethasone (Sigma) and 10 μg/l insulin (Sigma) for 48 h. For days 2–4, the medium was supplemented only with 10 μg/ml insulin. The cells were then switched to DMEM containing 10% FBS until fully differentiated at day 10. For fatty acid treatment, equal molar quantities of palmitate and oleate (Sigma) were dissolved in 95% ethanol at 60°C and then mixed with pre-warmed fatty acid-free BSA (3:1 molar ratio) to yield a fatty acid stock concentration of 5 mM. Fatty acid was added to the medium and incubated with cells for 6 h. Etomoxir (Sigma) and SP600125 (Merck) were dissolved in DMSO and added to the culture medium as indicated.
Generation of stable cell lines for hCPT1A (human CPT1A) overexpression and endogenous CPT1A knockdown
cDNA encoding hCPT1A and GFP (green fluorescent protein) were cloned into a PB (PiggyBac) expression vector and named PB-CAG-hCPT1A-IRES-puro and PB-CAG-GFP-IRES-puro respectively.
For RNAi (RNA interference) experiments, annealed oligonucleotides were cloned into the BamHI/XhoI sites of the pRNAT-U6.2/Lenti vector (GenScript) and were designed to target the coding sequence of mouse CPT1A from nucleotides 290 to 318. The sequence of the oligonucleotides was: 5′-GGATCCCGACTCACGATGTTCTTCGTCTGGCTTGACATTGATATCCGTGTCAAGCCAGACGAAGAACATCGTGAGTTTTTTTCCAACTCGAG-3′. Thereafter the 700-bp PCR fragment containing the U6.2 promoter and CPT1A shRNA (short-hairpin RNA) construct were produced and subcloned into PB-CAG-GFP-IRES-puro vector before the CAGG promoter and behind 3′-LTR (long terminal repeat) to form PB-shRNA-CAG-GFP-IRES-puro.
To obtain stable cells, 3×106 3T3-L1 pre-adipocytes were harvested and electroporated using an Amaxa Protocol T-030 with 2 μg of plasmid (PB-CAG-hCPT1A-IRES-puro, PB-shRNA-CAG-GFP-IRES-puro and PB-CAG-GFP-IRES-puro) mixed with 1 μg of PB-transposase vector plasmid respectively. After 24 h of culture, the selection medium containing 14 μg/ml puromycin (Merck) was added for 2 weeks with the medium exchanged every 2–3 days. Isolated cell clones were picked and expanded for use in subsequent experiments.
Real-time PCR analysis
Total RNA was isolated from cells using the TRIzol® reagent (Invitrogen) and reverse-transcribed using the Superscript III reverse transcriptase kit (Invitrogen). For real-time PCR analysis, cDNA samples were used in a quantitative PCR in the presence of fluorescent dye SYBR®Green (Bio-Rad). The following PCR conditions were applied: 5 min at 95°C, and 40 rounds of 10 s at 95°C, 20 s at 60°C and 1 s at 70°C each. After each elongation step, the reaction was quantified in a reading step and the product quality was tested by melting curve analysis. Relative abundance of mRNA was calculated after normalization to 18S RNA. The sequences for the primers used in the present study are shown in Supplementary Table S1 (at http://www.BiochemJ.org/bj/435/bj4350723add.htm).
Analysis of cellular glucose and fatty acid uptake
Following treatment with 0.5 mM fatty acid (palmitate/oleate mixture, two of the most abundant nutritional fatty acids) for 6 h, adipocytes were glucose-starved for 30 min in KRH (Krebs–Ringer–Hepes) buffer [120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 10 mM Hepes, 1.2 mM KH2PO4, 1.2 mM MgSO4 and 0.1% BSA (pH 7.5)]. After incubation for 15 min without or with insulin (100 nM), the tracer [1,2-3H]deoxy-D-glucose (0.93 GBq/mmol; Amersham Pharmacia) was then added for 15 min and glucose uptake was assayed in triplicate for each condition. Fatty acid uptake assays were initiated by incubating the cells for 20 min in KRH buffer containing 5.4 mM glucose and [1–14C]oleic acid (2.04 GBq/mmol; Amersham Pharmacia) bound to fatty acid-free BSA. The cells were washed and cellular incorporated 3H and 14C radioactivity was determined by liquid-scintillation counting. The abundance of radioactivity was normalized to protein content.
Western blot and ELISA analyses
Isolated cell lysates were resolved by SDS/PAGE, electroblotted on to PVDF membranes (Millipore) and immunoblotted with antibodies, including rabbit anti-Myc, rabbit anti-Akt, rabbit anti-(phospho-Akt), rabbit anti-JNK (c-Jun N-terminal kinase) and mouse anti-(phospho-JNK) (all from Cell Signaling Technology). Bound antibodies were detected using ECL (enhanced chemiluminescence) reagents (GE Healthcare). TNF-α levels in the medium were determined using mouse-specific ELISA kits (Pierce Endogen), according to the manufacturer's protocols.
Mitochondrial CPT1 activity assay
Mitochondria of 3T3-L1 cells were prepared using the procedure described previously . Mitochondrial protein concentrations were determined using Bradford assay and CPT1 activity was assayed as described in .
Analysis of lipolysis and cellular fatty acid content
After treatment with 0.5 mM fatty acid for 6 h, adipocytes were starved in low-serum medium (0.1%) overnight and incubated with DMEM containing 450 μmol/l BSA and 25 mM Hepes. After incubation for 3 h, the culture medium was completely removed for NEFA (non-esterified fatty acid) and glycerol assays. For the cellular NEFA content assay, cellular lipids were extracted with chloroform/methanol (2:1, v/v). The organic phase was dried and re-solubilized in a minimal volume of chloroform. NEFA and glycerol concentrations were determined using colorimetric kits (NEFA and Glycerol kits; Kexin). Protein concentrations were used to normalize the sample values. For Oil Red O staining, differentiated adipocytes in 24-well plates were fixed in fresh formalin solution and stained with Oil Red O dye (Amresco). The lipid was quantified by extracting the dye with propan-2-ol and determining the absorbance at 520 nm.
Co-culture of differentiated 3T3-L1 adipocytes with C2C12 myocytes
Mouse C2C12 cells were maintained at a subconfluent density in DMEM supplemented with 10% FBS. To induce differentiation, confluent cells grown on a transwell plate were shifted to a medium containing DMEM supplemented with 2% horse serum for 6 days. After in vitro differentiation of C2C12, the myocytes were washed twice with PBS and then incubated in DMEM containing 10% FBS. Differentiated adipocytes grown on individual membrane inserts were pre-treated with fatty acid or BSA for 6 h and subsequently transferred to the culture plates containing the differentiated myocytes. This resulted in an assembly of the two cell types sharing the culture medium, but separated by the membrane of the insert. The distance from the bottom of the culture dish to the membrane was 0.9 mm. Co-culture was conducted for 18 h and the glucose uptake assay and Western blot analysis of C2C12 myocytes were performed as described above.
Comparisons were analysed by unpaired two-tailed Student's t test. All calculations were performed using SPSS version 13.0 for Windows. A P value of < 0.05 was considered significant.
CPT1A expression does not influence differentiation of 3T3-L1 adipocytes
In order to investigate the biological function of CPT1A in adipocytes, we adopted a PB delivery system  to deliver hCPT1A cDNA (hCPT1A-adipocytes) and shRNA against endogenous mouse CPT1A (shRNA-adipocytes) into proliferating 3T3-L1 pre-adipocytes and selected for stably transduced cell lines with puromycin. Control cells were 3T3-L1 pre-adipocytes transfected with the PB vector inserted with GFP [WT (wild-type)], ruling out the possibility of the PB insertion and/or antibiotic selection-mediated toxicity as the source of the observed phenotypic variation. The results showed that transfection of CPT1A and mouse shRNA effectively resulted in the overexpression or knocking-down of CPT1 in 3T3-L1 pre-adipocytes respectively compared with control (Figure 1A and Supplementary Figure S1A at http://www.BiochemJ.org/bj/435/bj4350723add.htm), as determined by both Western blotting and quantitative real-time PCR. Consistently, CPT1 activities mirrored the changes in CPT1A expression (Figure 1B).
Next, we investigated whether CPT1A regulated adipogenesis in 3T3-L1 cells. hCPT1A, shRNA and WT 3T3-L1 pre-adipocytes were subjected to adipogenic induction medium and their adipogenic differentiation was monitored. Neither overexpression nor knockdown of CPT1A led to any obvious changes in lipid droplet accumulation (Figure 1C). Consistently, quantitative real-time PCR analysis revealed that expression of some key adipogenic transcription factors, including PPARγ (peroxisome-proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-binding protein α) and their target gene A-FABP/aP2, were not influenced (Figure 1D).
Pharmacological inhibition of endogenous CPT1 activity does not influence differentiation of 3T3-L1 pre-adipocytes
To exclude the possibility that lipid accumulation in 3T3-L1 cells was altered by the cloning procedures rather than the stably expressing constructs, we tested the effects of an irreversible CPT1-specific inhibitor etomoxir  on cell differentiation and lipid accumulation in naïve 3T3-L1 pre-adipocytes. Similar to previous reports [19a], etomoxir significantly inhibited CPT1 activity in a dose-dependent manner, as determined by the amount of CO2 released from adipocytes (Supplementary Figure S1B). However, results for lipid accumulation (Figure 2A) and adipogenic marker gene expression (Figure 2B) indicated that the differentiation of 3T3-L1 pre-adipocyte was not affected by etomoxir treatment. These results demonstrated that CPT1 expression is not critically involved in adipocyte differentiation in 3T3-L1 adipocytes.
CPT1A alters lipid metabolism in adipocytes
Considering CPT1A is the key enzyme in fatty acid catabolism, we sought to investigate whether CPT1A interferes with lipid storage and metabolism in adipocytes. To address this question, the release of glycerol and NEFAs, which are two major products of lipolysis, was determined under both normal and fatty acid-stimulated conditions. There were no significant differences in glycerol release among WT adipocytes, hCPT1A-adipocytes and shRNA-adipocytes (Figure 3A), suggesting that lipid hydrolysis is not significantly affected by CPT1 activity under both basal and fatty acid-treatment conditions. NEFA release was increased in WT adipocytes pre-treated with fatty acid. In contrast, the release of NEFAs was largely unaltered in 3T3-L1 adipocytes overexpressing hCPT1A, whereas knockdown of CPT1A caused an even higher level of NEFA release into cell culture medium (Figures 3A and 3B) in response to pre-treatment with fatty acid. Fatty acid uptake was also examined and we found that hCPT1A-adipocytes exhibited a much higher level of fatty acid uptake than did control adipocytes, whereas in shRNA-adipocytes this was reduced significantly compared with the WT adipocytes (Figure 3C). In agreement with this observation, triacylglycerol content was enhanced in hCPT1A-adipocytes and attenuated in shRNA-adipocytes respectively, as compared with WT adipocytes. This difference was not observed under BSA treatment conditions (results not shown). Taken together, the uncoupling between glycerol and NEFA release and varied fatty acid uptake in tight association with CPT1A expression levels in adipocytes suggested that CPT1A interferes with the intracellular lipid availability. To test this hypothesis, the intracellular lipids were extracted and the amount of NEFAs was measured. We found that treatment of fatty acid increased the intracellular level of NEFAs (Figure 3D). However, this effect was completely abolished in hCPT1A-adipocytes. Conversely, knockdown of CPT1A caused an even higher level of NEFAs inside the cell (Figure 3D), which is similarly observed in adipocytes treated with the CPT1-specific inhibitor (Supplementary Figures S1C–S1F), clearly indicating the relevance of CPT1 in the regulation of lipid flux in adipocytes.
CPT1A rescued fatty acid-induced insulin resistance in adipocytes
Fatty acids have a well established role as a principal mediator of adipocyte dysfunction, including insulin resistance and activation of inflammatory responses. In line with our finding that CPT1A promoted the depletion of intracellular fatty acid, it is highly possible that overexpression of CPT1A in adipocytes may rectify fatty acid-induced adipocyte dysfunction. To this end, we investigated the effect of altered CPT1 activity on adipocyte insulin sensitivity. We evaluated basal and insulin-stimulated glucose uptake without (BSA control) or with fatty acid treatment.
Under normal conditions, there were no significant differences in both basal and insulin-stimulated glucose uptake and activation of Akt signalling among the three cell types used in the present study (results not shown). Incubation with fatty acid attenuated insulin-induced glucose uptake in WT mature 3T3-L1 adipocytes (Figure 4A). Activation of Akt was also significantly decreased by ~30% (Figure 4B). However, overexpression of hCPT1A rendered cells resistant to the impairment of glucose uptake and Akt phosphorylation by fatty acids (Figures 4A and 4B). In contrast, knockdown of endogenous CPT1A resulted in an even more blunted response to insulin compared with WT cells. These results demonstrated that maintaining adequate CPT1A activity in adipocytes can ameliorate fatty acid-induced insulin resistance.
Pro-inflammatory cytokine expression and secretion are suppressed upon CPT1A overexpression
We next examined whether manipulation of CPT1A activity in adipocytes led to alterations in inflammation in adipocytes. To address this question, we quantified the expression levels of two pro-inflammatory adipokines, TNF-α and IL-6, by quantitative real-time PCR. As shown in Figure 5(A), fatty acid treatment caused a significant elevation in mRNA levels of both TNF-α and IL-6. The changes were largely abolished in hCPT1A-adipocytes. In contrast, shRNA-adipocytes were more sensitive to fatty acid-evoked up-regulation of these two pro-inflammatory adipokines. The expression of TNF-α was also confirmed by ELISA (Figure 5B).
CPT1A exert its beneficial effects in adipocytes via suppression of JNK
It is well established that fatty acids play a negative role in eliciting pro-inflammatory responses primarily via the activation of JNK and NF-κB (nuclear factor κB), which in turn induce the expression of various pro-inflammatory cytokines. In addition, activation of JNK has been shown to block insulin signalling via the phosphorylation of IRS1 (insulin receptor substrate 1) and then Akt . We therefore investigated whether these mechanisms participated in CPT1A-related insulin resistance and inflammation in adipocytes. Consistent with previous reports , we found that incubation with fatty acids led to a significant activation of JNK, as assessed by the ratio of phosphorylation of JNK1/2 to total JNK1/2 (Figure 6A). More interestingly, this effect was attenuated considerably in hCPT1A-adipocytes and enhanced in shRNA-adipocytes respectively (Figure 6A). Furthermore, inhibition of JNK by its selective inhibitor SP600125 potently protected against CPT1A-deficiency-related insulin resistance in shRNA-adipocytes. To be more specific, the impairment of insulin-induced glucose uptake (Figure 6B) and Akt signalling (Figure 6C) by CPT1A knockdown was completely reversed upon pre-incubation with 5 μM SP600125. In addition, inhibition of JNK activity in shRNA-adipocytes abolished the fatty acid-induced increase in TNF-α and IL-6 gene expression (Figure 6D). On the other hand, NF-κB was also activated following fatty acid treatment, but there was no significant difference among the three adipocyte cell lines used (results not shown). Hence these results suggested that JNK plays a central role that exacerbated adipocyte dysfunction in response to decreased CPT1A expression.
Pharmacological inhibition of endogenous CPT1 activity exacerbates fatty acid-induced insulin insensitivity and inflammation
To rule out the possibility that the cellular cloning procedure is responsible for the observed phenotypes in our cloned adipocyte lines, we treated the naïve 3T3-L1 adipocytes with the CPT1-specific inhibitor etomoxir. As shown in Figure 7, etomoxir significantly enhanced the phosphorylation of JNK1/2 (Figure 7A) and exacerbated the negative effects of fatty acids on insulin sensitivity (Figures 7B and 7C) and expression of pro-inflammatory cytokines (Figure 7D). Similarly, the JNK inhibitor SP600125 completely reversed the impairment in insulin-induced glucose uptake (Figure 7B) and Akt signalling (Figure 7C). Again, the fatty acid-induced increase in TNF-α and IL-6 gene expression in etomoxir-treated adipocytes was abolished by inhibiting JNK activity with SP600125 (Figure 7D).
Adipocyte CPT1A modulates insulin sensitivity in co-cultured C2C12 myocytes
Adipose tissue has been well recognized as one of the largest endocrine organs in the body. We next investigated whether alteration in CPT1A expression within adipocytes systemically affected other tissues or cell types. Differentiated C2C12 myocytes were co-cultured with the three adipocyte cell lines and the insulin signalling in the cultured myocytes was examined. Consistent with a previous report , myocytes co-cultured with adipocytes showed a decreased insulin sensitivity (results not shown). There was no significant difference in basal and insulin-stimulated glucose uptake (Figure 8A) and phosphorylation status of Akt (results not shown) among C2C12 myocytes co-cultured with the three adipocyte cell lines. However, when these adipocytes were pre-treated with fatty acid, the C2C12 myocytes co-cultured with WT adipocytes displayed a modest reduction in insulin-stimulated glucose uptake (Figure 8A), whereas the effect was significantly alleviated in myocytes co-cultured with hCPT1A-adipocytes and aggravated in myocytes co-cultured with shRNA-adipocytes (Figure 8A). The insulin-stimulated phosphorylation of Akt in differentiated myocytes co-cultured with fatty acid-pre-treated adipocytes mirrored the changes in glucose uptake (Figure 8B).
Adipocytes are no longer regarded as an inert site for fat storage and we designed the present study on the plausible hypothesis that fatty acid metabolism in adipocytes intricately regulates the function of the cell. Fatty acids, which are elevated in obese individuals, have been firmly established as one of the major factors triggering insulin resistance in peripheral metabolic tissues. Although the detailed mechanisms are still unclear, mounting evidence suggests that fatty acid overload causes intracellular accumulation of fatty acid-derived metabolic products [23,24]. These derivatives activate the key molecules in pro-inflammatory pathways, such as JNK, which blunt insulin signalling via direct phosphorylation on the threonine residue in IRS1 and then Akt [20,25]. In the present study, we show that the key enzyme in fatty acid oxidation, CPT1A, protected adipocytes from fatty acid-induced insulin resistance and inflammation via suppression of JNK activity.
The accumulation of fatty acid derivatives, such as in the acyl-CoA form, are capable of activating JNK, and this mechanism is of great significance in the pathogenesis of fatty acid-induced insulin resistance and inflammation in adipocytes. This is supported by our present findings that the differences among three cell lines with various degrees of CPT1A activity were more evident when the cells were challenged with fatty acid. We hypothesize (Figure 9) that, under conditions of lipid overload, CPT1 catalyses the transesterification reaction between LCA-CoA (long-chain acyl-CoA) and acyl-carnitine esters, and thus changes the availability of LCA-CoA inside the cell. In addition, it is also key substrate for the synthesis of several complex lipid derivatives which act as key signalling molecules in the activation of inflammation and insulin resistance. Notably, it has been shown previously that knockdown of LCA-CoA synthase, a key enzyme for LCA-CoA synthesis, remarkably attenuated insulin signalling in 3T3-L1 adipocytes . Furthermore, knockdown of LCA-CoA synthase in adipocytes leads to reduced fatty acid uptake, which is largely reminiscent of our present findings that CPT1 overexpression caused an increase in fatty acid uptake. There are also reports showing that mitochondrial oxidation dysfunction in adipocytes has a close correlation with the states of insulin resistance and was considered as a target for treatment of diabetes [27,28]. CPT1 is a mitochondrial oxidative enzyme, thus lowered CPT1 expression might contribute to the pathogenesis of obesity-related inflammation and insulin resistance.
Our present co-culture study demonstrated that elevated expression of CPT1A in adipocytes also increased insulin sensitivity in the co-cultured muscle cells, as compared with myocytes co-cultured with WT adipocytes. This paracrine effect was possibly mediated by the pro-inflammatory cytokines and fatty acids secreted from adipocytes. Indeed, the master role of adipocytes in systemic glucose and lipid metabolism has been implicated by several adipocyte-specific knockout mice models [29,30]. Adipocyte-specific knockout or transgenic animal models for CPT1 would be especially informative to elucidate its biological actions in vivo.
In summary, our present study has uncovered a protective role for CPT1 in fatty acid-evoked adipocyte dysfunction. Pharmacological activation of CPT1 might represent a promising strategy for the prevention and treatment of obesity-related metabolic diseases.
Xuefei Gao, Kuai Li and Xiaoyan Hui designed and performed the experiments. Gary Sweeney, Yu Wang and Aimin Xu contributed to writing the manuscript. Xiangping Kong, Maikun Teng and Pentao Liu contributed to discussion about the manuscript. Donghai Wu designed the experiments, and wrote and edited the manuscript prior to submission.
This work was supported, in part, by the National Basic Research Program of China (973 Program) [grant numbers 2011CB504004, 2010CB945500], the National Natural Science Foundation of China [grant numbers 31000353, 30970637], and the Knowledge Innovation Program of the Chinese Academy of Sciences and Guangzhou Administration of Science and Technology [grant number 2007Z2-E4021].
Abbreviations: A-FABP, adipocyte fatty acid-binding protein; C/EBPα, CCAAT/enhancer-binding protein α; CPT1, carnitine palmitoyltransferase 1; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GFP, green fluorescent protein; hCPT1A, human CPT1A; hCPT1A-adipocyte, adipocyte overexpressing hCPT1A; IL-6, interleukin 6; IRS1, insulin receptor substrate 1; JNK, c-Jun N-terminal kinase; KRH, Krebs–Ringer–Hepes; LCA-CoA, long-chain acyl-CoA; NEFA, non-esterified fatty acid; NF-κB, nuclear factor κB; PB, PiggyBac; PPARγ, peroxisome-proliferator-activated receptor γ; shRNA, short-hairpin RNA; shRNA-adipocyte, adipocyte with CPT1A knocked down by shRNA; TNF-α, tumour necrosis factor-α; WT, wild-type
- © The Authors Journal compilation © 2011 Biochemical Society