Liver mitochondrial β-oxidation of LCFAs (long-chain fatty acids) is tightly regulated through inhibition of CPT1A (carnitine palmitoyltransferase 1A) by malonyl-CoA, an intermediate of lipogenesis stimulated by glucose and insulin. Moreover, CPT1A sensitivity to malonyl-CoA inhibition varies markedly depending on the physiopathological state of the animal. In the present study, we asked whether an increase in CPT1A activity solely or in association with a decreased malonyl-CoA sensitivity could, even in the presence of high glucose and insulin concentrations, maintain a sustained LCFA β-oxidation and/or protect from triacylglycerol (triglyceride) accumulation in hepatocytes. We have shown that adenovirus-mediated expression of rat CPT1wt (wild-type CPT1A) and malonyl-CoA-insensitive CPT1mt (CPT1AM593S mutant) in cultured fed rat hepatocytes counteracted the inhibition of oleate β-oxidation induced by 20 mM glucose/10 nM insulin. Interestingly, the glucose/insulin-induced cellular triacylglycerol accumulation was prevented, both in the presence and absence of exogenous oleate. This resulted from the generation of a metabolic switch allowing β-oxidation of de novo synthesized LCFAs, which occurred without alteration in glucose oxidation and glycogen synthesis. Moreover, CPT1mt expression was more effective than CPT1wt overexpression to counteract glucose/insulin effects, demonstrating that control of CPT1A activity by malonyl-CoA is an essential driving force for hepatic LCFA metabolic fate. In conclusion, the present study highlights that CPT1A is a prime target to increase hepatic LCFA β-oxidation and that acting directly on the degree of its malonyl-CoA sensitivity may be a relevant strategy to prevent and/or correct hepatic steatosis.

INTRODUCTION

The hepatic isoform of CPT1 (carnitine palmitoyltransferase 1; EC 2.3.1.21) (termed CPT1A) is known to be the key regulatory enzyme in liver mitochondrial β-oxidation of LCFAs (long-chain fatty acids) [1,2]. By converting LC-CoA (long-chain acyl-CoA) into acylcarnitine, CPT1A catalyses the rate-limiting step in the entry of cytosolic LC-CoA into mitochondria where β-oxidation takes place. Under physiological conditions, lipogenesis and LCFA β-oxidation are tightly regulated in the liver [1]. Malonyl-CoA, the first intermediate in lipogenesis, is synthesized by ACC (acetyl-CoA carboxylase) and is the substrate of FAS (fatty acid synthase) for de novo LCFA synthesis. Malonyl-CoA is also the physiological allosteric inhibitor of CPT1A [3]. Therefore, after feeding a carbohydrate-rich meal, the presence of both high plasma glucose and insulin concentrations stimulates liver glucose oxidation, glycogen storage and lipogenesis, allowing convertion of excess glucose into LCFAs. The resulting increase in malonyl-CoA level inhibits CPT1A activity. Both exogenous LCFAs taken up by the liver and endogenous LCFAs generated by lipogenesis are then esterified into TAGs [triacylglycerols (triglycerides)] and partly secreted as VLDLs (very-low-density lipoproteins). Conversely, in the fasted state when lipogenesis is low, CPT1A is retrieved from malonyl-CoA inhibition. Hepatic β-oxidation of LCFAs released from adipose tissue can then occur to produce energy, cofactors required for optimal gluconeogenesis and ketone bodies used as fuels by extrahepatic tissues [1,2].

Disturbance of this key regulatory malonyl-CoA–CPT1A partnership might contribute to hepatic steatosis. Inherited CPT1A deficiency in humans is associated with hypoglycaemia and hypoketonaemia, as well as hepatic steatosis during fasting [4,5]. Similarly to drug-induced impairment of mitochondrial β-oxidation [6], pharmacological inhibition of CPT1A activity by irreversible inhibitors, such as tetradecylglycidic acid [7] or etomoxir [8], induces mitochondrial injury leading to steatosis and inflammation. Whereas the consequences of a CPT1A activity defect are relatively well known, the metabolic impact of an increased CPT1A activity itself had, until recently, never been reported in liver cells. During the course of the present study, it was published that CPT1A overexpression in rat hepatocytes cultured in the presence of a low glucose concentration increased LCFA β-oxidation capacity, leading to metabolic reorientation of exogenous LCFAs taken up by the cells toward oxidation at the expense of esterification [9]. However, whether such an increased capacity to oxidize exogenous LCFAs could be maintained in conditions under which this pathway is usually abolished, e.g. in the presence of high glucose and insulin concentrations known to stimulate de novo lipogenesis [10], had never been investigated in any model. Moreover, a unique feature of CPT1A is that its sensitivity to malonyl-CoA inhibition varies markedly depending on the physiological state in adult rats. For example, it is increased by refeeding carbohydrate to fasted rats, by obesity state or after insulin administration to diabetic rats, whereas it is decreased by starvation and diabetes [1115].

Thus the aim of the present study was to clearly decipher whether a decrease in CPT1A malonyl-CoA sensitivity represents an efficient strategy to enhance mitochondrial LCFA β-oxidation in liver cells. By overexpressing CPT1As with distinct malonyl-CoA sensitivity in cultured rat hepatocytes, we have demonstrated that expression of a malonyl-CoA-insensitive CPT1A is more effective than overexpressing CPT1wt (wild-type CPT1A) to counteract the glucose/insulin inhibitory effect on exogenous LCFA oxidation flux and to prevent glucose/insulin-induced TAG accumulation. Moreover, modulation of the malonyl-CoA–CPT1A partnership generates a metabolic switch allowing β-oxidation of de novo synthesized LCFAs, hence preventing their esterification into TAGs. Taken together, these results highlight that control of CPT1A activity by malonyl-CoA is an essential driving force for hepatic LCFA metabolic fate and that, acting directly on the degree of CPT1A malonyl-CoA sensitivity may be a relevant strategy to prevent and/or reduce liver steatosis.

EXPERIMENTAL

Materials

Collagenase used to isolate rat hepatocytes was purchased from Roche Diagnostics. Cell culture reagents (medium M199 with Earl salts, glutamine and Ultroser G) were obtained from Invitrogen. [14C]sodium bicarbonate was purchased from PerkinElmer. [1-14C]oleate, D-[U-14C]glucose, L-[methyl-3H]carnitine and [1-14C]acetate were purchased from GE Healthcare. TOFA [5-(tetradecyloxy)-2-furoic acid] was a gift from Dr A. Richardson (Merrel National Laboratories, Cincinnati, OH, U.S.A.). TLC silica plates and dexamethasone were purchased from Merck Chemicals. Insulin was obtained from Novo Nordisk. Other biochemicals were purchased from Sigma–Aldrich.

Construction of recombinant adenovirus

CPT1mt (the CPT1AM593S mutant) was constructed using the QuikChange® site-directed mutagenesis kit (Stratagene) using pYeDP1/8-10 containing the full-length rat CPT1A cDNA [16] as a template, and the forward (5′-CCTCACATATGAGGCCTCCAGTACCCGGCTCTTCCGAGAAGG-3′) and reverse (5′-CCTTCTCGGAAGAGCCGGGTACTGGAGGCCTCATATGTGAGG-3′) primers. Human adenovirus serotype 5 vectors (denoted as Ad) encoding either β-galactosidase (Ad-βgal), CPT1wt (Ad-CPT1wt) or CPT1mt (Ad-CPT1mt) under the control of the CMV (cytomegalovirus) promoter were produced by the Laboratoire de Thérapie Génique (INSERM U649, Nantes, France).

Isolation of rat hepatocytes

Male Wistar rats (200–300 g) were purchased from Elevage Janvier and adapted to the environment for at least 1 week prior to the experiment. Rats were housed in plastic cages in a temperature-controlled (21 °C) and ventilated room with a 12 h light/dark cycle (light from 15:00 h to 03:00 h). Animals had free access to water and were fed ad libitum a standard rodent chow (A03, SAFE). All procedures were carried out according to the French guidelines for the care and use of experimental animals, and were approved by the Direction Départementale des Services Vétérinaires de Paris. Hepatocytes were isolated at 09:00 h by collagenase perfusion into the portal vein as described previously [10]. Isolated hepatocytes were resuspended in M199 medium containing 5 mM glucose, 100 i.u/ml penicillin, 100 μg/ml streptomycin, 0.1% (w/v) BSA and 2 mM glutamine (basal medium). From cell attachment, irrespective of the experiment performed, 1 mM carnitine was systematically added to avoid a limitation in CPT1A activity which could alter LCFA oxidation. Cell viability estimated by Trypan Blue exclusion was always greater than 80%.

Hepatocyte culture and adenovirus infection

Hepatocytes were plated at a density of 8×106 cells/100 mm Petri dish (for isolation of mitochondria), at 0.5×106 cells/well of 12-well plates {for the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay} or at 2.5×106 cells/25 cm2 flask (for all other experiments) and cultured for 4 h at 37 °C in an incubator equilibrated with air/CO2 (95:5) in basal medium supplemented with 100 nM dexamethasone, 10 nM insulin and 2% (v/v) Ultroser G before infection with adenovirus. After cell attachment, the medium was removed and hepatocytes were incubated for 2 h with 1 ml (flask), 250 μl (12-well plate) or 3 ml (dish) of basal medium containing either 0, 2.5 or 5 infectious particles/cell of Ad-βgal, Ad-CPT1wt or Ad-CPT1mt. Thereafter, the infection medium was removed and cells were further cultured for another 40 h, the experiments being performed during the last 24 h.

Immunoblot analysis

Aliquots of proteins were subjected to SDS/PAGE [17] (7% gel) and transferred on to nitrocellulose membrane. Detection of proteins was performed as described previously [16] using the ECL (enhanced chemiluminescence) Pierce detection system (Perbio Sciences SAS). The antibodies used were against rat CPT1A [16], Escherichia coli βgal (Rockland), rat FAS (a gift from Dr I. Dugail, INSERM U465, Paris, France), and human ACC2 (Upstate). For the generation of an anti-CPT2 polyclonal antibody, a peptide corresponding to the last 20 C-terminal residues of human CPT2 was synthesized, conjugated to keyhole limpet haemocyanin, and used to immunize New Zealand White rabbits (Neosystem). The immunoblots were quantified using a chemigenius apparatus (Syngene).

Immunofluorescence assay

Hepatocytes (7×105 cells/well of six-well plates) were cultured on coverslips coated with 4% (v/v) collagen in 0.001% acetic acid/PBS. At 40 h after infection (5 infectious particles/cell), cells were fixed with 2% (v/v) formaldehyde, permeabilized with 0.1% SDS and non-specific binding of antibodies was blocked by incubation with 10% (v/v) FBS (foetal bovine serum)/PBS for 20 min. Cells were stained with polyclonal anti-rat CPT1A and mouse monoclonal anti-cytochrome c (BD Biosciences) antibodies which were detected by a goat anti-rabbit IgG conjugated with Alexa Fluor® 488 and a goat anti-mouse IgG conjugated with Alexa Fluor® 594 (Molecular Probes) respectively. After incubation with 2 μM Hoechst 33342 for DNA staining, coverslips were mounted on to glass slides with fluoromount G (Clinisciences). Fluorescent staining was viewed in a confocal laser-scanning microscope (Leica TCS SP2 AOBS). The captured images were processed using ImageJ software (Wayne Rasband).

Isolation of mitochondria and CPT activity assay

At 40 h after infection, cells were washed and scraped with ice-cold PBS. Mitochondria were isolated in an isolation buffer [0.3 M sucrose, 5 mM Tris/HCl and 1 mM EGTA (pH 7.4)] using differential centrifugation [16]. The protein concentration was determined using the Lowry method with BSA as a standard [18]. CPT1 activity was assayed in intact isolated mitochondria as palmitoyl-L-[methyl-3H]carnitine formed from L-[methyl-3H]carnitine (400 μM; 10 Ci/mol) and palmitoyl-CoA (600 μM) in the presence of 1% (w/v) BSA. Estimation of the malonyl-CoA IC50 value (the concentration of malonyl-CoA required for 50% inhibition) and CPT2 activity were measured as described previously [16].

Measurement of fatty acid metabolism

During the last 24 h of culture, the medium was replaced by a medium containing either 5 mM glucose or 20 mM glucose plus 10 nM insulin. Fatty acid oxidation and esterification were determined during the last 2 h of culture in the presence of 0.3 mM [1-14C]oleate (0.5 Ci/mol) bound to 1% (w/v) defatted BSA. After 2 h of incubation, medium was collected to determine 14CO2, [14C]ASP (acid-soluble products), unlabelled ketone body production (acetoacetate and β-hydroxybutyrate) and lactate and pyruvate concentrations. Cells were washed and scraped in PBS to determine [14C]TAG and [14C]PL (phospholipid) production as previously described [19,20]. For the determination of VLDL secretion, medium (3 ml) was collected, immediately adjusted to 0.005% gentamicin, 1 mM EDTA, 0.04% sodium azide and 0.02% sodium (ethylomercurithio)-2 benzoate, and was layered under 5 ml of 0.15 M NaCl. After centrifugation at 43000 rev./min for 18 h at 10 °C (in a Beckman 70 Ti rotor), the 1 ml top fraction containing VLDLs was counted in 10 ml of scintillation liquid in a scintillation counter (Packard).

Measurement of intracellular TAG and Oil Red O staining

For measurement of unlabelled TAGs, cellular lipids were extracted in chloroform/methanol (2:1, v/v) with vigorous shaking for 10 min. After centrifugation for 25 min at 1200 g, the lower organic phase was collected, dried, and solubilized in chloroform/methanol. Lipid classes were separated by TLC on silica-gel plates by using petroleum ether/diethyl ether/acetic acid (85:15:0.5, v/v/v) as the mobile phase. Lipids were visualized with iodine vapour. Bands were scraped and TAGs were extracted from silica in chloroform/methanol. After centrifugation for 45 min at 1500 g to precipitate silica, and evaporation, TAGs were measured with a PAP 150 TAG kit (Biomerieux). Lipids droplets were detected in hepatocytes fixed with 3% (w/v) paraformaldehyde in PBS and coloured with Oil Red O [21].

Measurement of ACC, FAS and MCD (malonyl-CoA decarboxylase) enzyme activity

Cells were scraped in ice-cold 0.25 M saccharose, 1 mM DTT (dithiothreitol), 1 mM EDTA and protease inhibitors, and cytosolic fractions obtained by differential centrifugation (10000 g for 10 min at 4 °C, followed by 100000 g for 1 h at 4 °C) were used for ACC [22] and FAS activity [23] assays. MCD activity was assayed as described previously [24].

Measurement of glucose metabolism

During the last 24 h of culture, the medium was replaced by a medium containing either 5 mM glucose or 20 mM glucose plus 10 nM insulin in the absence or presence of 200 μM TOFA, a specific ACC inhibitor [25]. Glucose oxidation and glycogen synthesis were determined during the last 2 h of culture in the presence of either 5 mM [U-14C]D-glucose (133 μCi/mmol) or 20 mM [U-14C]D-glucose (87.5 μCi/mmol) plus 10 nM insulin, without or with 0.3 mM oleate bound to 1% (w/v) defatted BSA. After 2 h of incubation, the medium was collected to determine 14CO2 and unlabelled β-hydroxybutyrate production, whereas cells were washed three times with ice-cold PBS and scraped in 1 ml of 5 M KOH. Non-radioactive glycogen carrier (2.5 mg) was added to the lysates (600 μl) and samples were boiled for 30 min. Glycogen was precipitated overnight at −20 °C with two volumes of ice-cold 100% ethanol. Precipitated glycogen was centrifuged at 10000 g for 10 min. Pellets were washed once with 70% ethanol, resuspended in 0.5 ml of water, and counted by scintillation counting. The protein concentration was determined using the Bradford method (Bio-Rad) using BSA as a standard. The lipogenesis rate from 5 mM [1-14C]acetate (10 μCi per flask) was determined during the last 2 h of culture as described previously [21]. Briefly, cells were rinsed twice with ice-cold PBS, immediately frozen in liquid nitrogen and scraped off in 30% (v/v) KOH. Labelled fatty acids were then extracted and counted by scintillation counting.

Measurement of mitochondrial activity

The MTS assay was performed in 12-well plates using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer's instructions.

Statistical analysis

Results are expressed as means±S.E.M. ANOVA was carried out using the StatView program (Abacus) to determine differences between groups. When significant differences were detected, a posteriori comparisons between means were conducted using the Fisher LSD (least significant difference) test (α=0.05).

RESULTS

Adenovirus-mediated expression of CPT1wt and CPT1mt in rat hepatocytes

To investigate whether an increase in CPT1A activity solely or in association with a decreased malonyl-CoA sensitivity could modulate hepatic LCFA oxidation, primary rat hepatocytes were infected with 2.5 or 5 infectious particles/cell of adenovirus encoding either CPT1wt or CPT1mt that is active but insensitive to malonyl-CoA inhibition [2628]. This led to an increase in CPT1A protein expression in an adenovirus concentration-dependent manner in comparison with uninfected hepatocytes (Figure 1a). The protein level of another enzyme of the mitochondrial carnitine-shuttle system, CPT2, whose activity is insensitive to malonyl-CoA inhibition [1], was not modified (Figure 1a). As a control, hepatocytes were also infected with an adenovirus-encoding βgal. The resulting βgal expression had no effect on CPT1A and CPT2 protein levels when compared with uninfected cells (Figure 1a). Considering these results, the amount of 5 infectious particles/cell was chosen for subsequent infection experiments. Immunofluorescence analysis of infected hepatocytes showed that both overexpressed CPT1wt and CPT1mt proteins were co-localized with cytochrome c, a mitochondrial marker protein (Figure 1b), indicating, as expected, a mitochondrial localization. Moreover, infection with Ad-CPT1wt and Ad-CPT1mt respectively led to a 15- and 11-fold increase in mitochondrial CPT1A protein level and a 7- and 3-fold increase in CPT1A activity when compared with mitochondria isolated from either Ad-βgal-infected or uninfected hepatocytes (Figure 2a). In agreement with Figure 1(a), CPT1wt and CPT1mt overexpression had no repercussion on both mitochondrial CPT2 protein level and activity (Figure 2a).

Mitochondrial localization of the overexpressed CPT1wt and CPT1mt proteins in cultured rat hepatocytes

Figure 1
Mitochondrial localization of the overexpressed CPT1wt and CPT1mt proteins in cultured rat hepatocytes

Primary rat hepatocytes were infected with either 0, 2.5 or 5 infectious particles/cell of the indicated adenovirus and cultured for 40 h in 5 mM glucose. (a) Immunoblot analysis of cellular extracts (30 μg of protein) using specific antibodies for βgal, CPT1A and CPT2. Western blots are representative of four independent experiments performed in duplicate. (b) Immunofluorescence analysis detection of the overexpressed CPT1 proteins. Hepatocytes were double-immunostained with antibodies raised against cytochrome c (Cyt c) and CPT1A. The right-hand panels (merge) represent the overlay of the two left-hand panels. Co-localization of cytochrome c and CPT1A appears as yellow spots reflecting the merge of red (Cyt c) and green (CPT1A) fluorescence. Hoechst 33342 was used to stain the hepatocyte nuclei. Scale bar=50 μm.

Figure 1
Mitochondrial localization of the overexpressed CPT1wt and CPT1mt proteins in cultured rat hepatocytes

Primary rat hepatocytes were infected with either 0, 2.5 or 5 infectious particles/cell of the indicated adenovirus and cultured for 40 h in 5 mM glucose. (a) Immunoblot analysis of cellular extracts (30 μg of protein) using specific antibodies for βgal, CPT1A and CPT2. Western blots are representative of four independent experiments performed in duplicate. (b) Immunofluorescence analysis detection of the overexpressed CPT1 proteins. Hepatocytes were double-immunostained with antibodies raised against cytochrome c (Cyt c) and CPT1A. The right-hand panels (merge) represent the overlay of the two left-hand panels. Co-localization of cytochrome c and CPT1A appears as yellow spots reflecting the merge of red (Cyt c) and green (CPT1A) fluorescence. Hoechst 33342 was used to stain the hepatocyte nuclei. Scale bar=50 μm.

Effects of CPT1wt and CPT1mt overexpression on CPT1A protein level and activity (a) and malonyl-CoA sensitivity (b) in mitochondria isolated from infected hepatocytes

Figure 2
Effects of CPT1wt and CPT1mt overexpression on CPT1A protein level and activity (a) and malonyl-CoA sensitivity (b) in mitochondria isolated from infected hepatocytes

Mitochondria were isolated 40 h after infection of hepatocytes with 5 infectious particles/cell of the indicated adenovirus. (a) Immunoblot analysis of mitochondrial proteins using specific antibodies for CPT1A and CPT2. Western blots are representative of three independent experiments with mitochondria isolated from separate hepatocyte cultures. CPT1A and CPT2 activities were measured in either intact (CPT1A) or solubilized (CPT2) mitochondria. Results are means±S.E.M. of three independent experiments with separate isolated mitochondria. (b) Malonyl-CoA sensitivity of CPT1A was measured in intact isolated mitochondria. Results are means±S.E.M. of two independent experiments with separate isolated mitochondria. *P<0.01, compared with Ad-βgal; #P<0.01, CPT1wt compared with CPT1mt.

Figure 2
Effects of CPT1wt and CPT1mt overexpression on CPT1A protein level and activity (a) and malonyl-CoA sensitivity (b) in mitochondria isolated from infected hepatocytes

Mitochondria were isolated 40 h after infection of hepatocytes with 5 infectious particles/cell of the indicated adenovirus. (a) Immunoblot analysis of mitochondrial proteins using specific antibodies for CPT1A and CPT2. Western blots are representative of three independent experiments with mitochondria isolated from separate hepatocyte cultures. CPT1A and CPT2 activities were measured in either intact (CPT1A) or solubilized (CPT2) mitochondria. Results are means±S.E.M. of three independent experiments with separate isolated mitochondria. (b) Malonyl-CoA sensitivity of CPT1A was measured in intact isolated mitochondria. Results are means±S.E.M. of two independent experiments with separate isolated mitochondria. *P<0.01, compared with Ad-βgal; #P<0.01, CPT1wt compared with CPT1mt.

To evaluate the capacity of CPT1mt to exhibit enzyme activity despite the presence of malonyl-CoA, malonyl-CoA sensitivity was then measured in isolated mitochondria. In uninfected hepatocytes and Ad-βgal- or Ad-CPT1wt-infected hepatocytes, CPT activity was almost completely suppressed (80%) by the highest malonyl-CoA concentration used (150 μM) (Figure 2b). This was indicative of a good membrane integrity of the isolated mitochondria and suggested that only CPT1A activity was measured without any significant contribution of CPT2. Their corresponding IC50 values for malonyl-CoA were not significantly different (uninfected, 2.5±0.7 μM; Ad-βgal, 2.4±0.6 μM; Ad-CPT1wt, 12.2±8.7 μM). By contrast, in Ad-CPT1mt-infected hepatocytes, CPT1A activity was not inhibited by 1–10 μM malonyl-CoA (Figure 2b). Moreover, even in the presence of 150 μM of malonyl-CoA, only 43% of the activity was inhibited, which probably corresponds to the inhibition of endogenous CPT1A. Taken together, these results confirm that CPT1wt and CPT1mt overexpression in rat hepatocytes increases mitochondrial CPT1A protein levels and activity, CPT1mt being insensitive to malonyl-CoA inhibition.

CPT1wt and CPT1mt overexpression counteracts glucose and insulin effects on lipid metabolism

CPT1wt and CPT1mt overexpression in cultured rat hepatocytes increased LCFA β-oxidation capacity under basal conditions of culture (5 mM glucose), leading to metabolic reorientation of exogenous LCFAs toward oxidation at the expense of esterification (Supplementary Figure S1 at http://www.BiochemJ.org/bj/420/bj4200429add.htm). We then evaluated their metabolic effects in the presence of high concentrations of glucose and insulin, conditions known to promote hepatic lipogenesis [10], hence preventing mitochondrial LCFA oxidation [1]. In uninfected hepatocytes, a 24 h-exposure to 20 mM glucose plus 10 nM insulin markedly stimulated the expression and the activity of lipogenic ACC and FAS enzymes (Supplementary Figures S2a–S2c at http://www.BiochemJ.org/bj/420/bj4200429add.htm), de novo lipogenesis (see below Figure 6b) and suppressed [1-14C]oleate oxidation by more than 90% (Figures 3a and 3b). Similar results were obtained in Ad-βgal-infected hepatocytes. Overexpression of CPT1wt and CPT1mt did not alter the glucose/insulin-stimulated expression and activity of ACC and FAS, nor the enzymatic activity of MCD which catalyses the degradation of malonyl-CoA into acetyl-CoA (Supplementary Figure S2). However, their overexpression led to a marked increase in [1-14C]oleate oxidation. Indeed, 14CO2 and [14C]ASP production were increased by 4- and 6.8-fold respectively, for Ad-CPT1wt-infected hepatocytes, and by 6.6- and 13.6-fold respectively, for Ad-CPT1mt-infected hepatocytes, when compared with Ad-βgal-infected hepatocytes (Figures 3a and 3b). Conversely, only CPT1mt expression significantly decreased by 44% [1-14C]oleate esterification into TAGs when compared with Ad-βgal-infected cells (Figure 3c), without any significant modification of its esterification into PLs (Figure 3d). In the presence of glucose and insulin, the total amount of [1-14C]oleate metabolized was similar for all infections (uninfected, 103±21 nmol/2 h per mg of protein; Ad-βgal, 104±18 nmol/2 h per mg of protein; Ad-CPT1wt, 115±12 nmol/2 h per mg of protein; Ad-CPT1mt, 138±17 nmol/2 h per mg of protein). Whereas [1-14C]oleate oxidation rate represented only 5% of [14C]oleate metabolized in Ad-βgal-infected hepatocytes, CPT1wt and CPT1mt overexpression increased this rate up to 30% and 47% respectively (Figure 3e). To determine whether the decrease in [14C]TAG production, and hence in esterification flux, was not due to an increased export of TAG as VLDL, [1-14C]oleate incorporation into VLDLs secreted into the culture medium was also measured. [1-14C]VLDL secretion was increased by 50% and 80% in hepatocytes overexpressing CPT1wt and CPT1mt respectively (Figure 3f). However, the corresponding increased amount of incorporated oleate into VLDL (1.5 and 1.8 nmol/2 h per mg of protein) was 10- and 20-fold lower than the observed reduction in oleate incorporation into TAGs (18 and 35 nmol/2 h per mg of protein). Consequently, oleate incorporated into VLDLs can probably explain no more than 10% of the decrease in TAG esterification. Thus, in the presence of glucose and insulin, CPT1A overexpression decreased exogenous oleate esterification into TAGs by enhancing hepatic LCFA oxidation capacity, the effects of CPT1mt expression always being significantly higher than those observed for CPT1wt.

CPT1wt and CPT1mt overexpression counteracts the glucose/insulin effects on [1-14C]oleate metabolism

Figure 3
CPT1wt and CPT1mt overexpression counteracts the glucose/insulin effects on [1-14C]oleate metabolism

At 16 h after infection of hepatocytes with 5 infectious particles/cell of the indicated adenovirus, cells were cultured for 24 h in the presence of either 5 mM glucose (G5) or 20 mM glucose plus 10 nM insulin (G20+ins). During the last 2 h of culture, 0.3 mM [1-14C]oleate bound to 1% defatted BSA was added. Oleate oxidation into CO2 (a) and ASP (b), and oleate esterification into cellular TAGs (TG; c) and PLs (d) were measured at the end of the 2 h-incubation period. (e) Metabolic orientation of oleate as a percentage of the total nmol of metabolized [1-14C]oleate. (f) [1-14C]Oleate incorporation into VLDLs secreted in the culture medium during the 2 h incubation period. Results are means±S.E.M. of duplicate flasks from four independent experiments. *P<0.05 and **P<0.01, compared with Ad-βgal; #P<0.05, CPT1wt compared with CPT1mt.

Figure 3
CPT1wt and CPT1mt overexpression counteracts the glucose/insulin effects on [1-14C]oleate metabolism

At 16 h after infection of hepatocytes with 5 infectious particles/cell of the indicated adenovirus, cells were cultured for 24 h in the presence of either 5 mM glucose (G5) or 20 mM glucose plus 10 nM insulin (G20+ins). During the last 2 h of culture, 0.3 mM [1-14C]oleate bound to 1% defatted BSA was added. Oleate oxidation into CO2 (a) and ASP (b), and oleate esterification into cellular TAGs (TG; c) and PLs (d) were measured at the end of the 2 h-incubation period. (e) Metabolic orientation of oleate as a percentage of the total nmol of metabolized [1-14C]oleate. (f) [1-14C]Oleate incorporation into VLDLs secreted in the culture medium during the 2 h incubation period. Results are means±S.E.M. of duplicate flasks from four independent experiments. *P<0.05 and **P<0.01, compared with Ad-βgal; #P<0.05, CPT1wt compared with CPT1mt.

To determine whether this decrease in exogenous oleate esterification was reflected in intracellular lipid content, Oil Red O staining of hepatocytes, which allows visualization of neutral lipids within the cells, and measurement of intracellular TAG content were performed. A 24 h-exposure to glucose and insulin induced a huge increase in lipid droplets in Ad-βgal-infected cells, which was much lower in hepatocytes expressing CPT1mt (Figure 4a). Moreover, whereas the presence of glucose and insulin induced a 5-fold increase in TAG content both in uninfected and Ad-βgal-infected hepatocytes, CPT1mt expression decreased the TAG content by 43% when compared with Ad-βgal-infected hepatocytes (Figure 4b, left-hand panel). CPT1wt overexpression led to an intermediate decrease in TAG content (Figure 4b, left-hand panel). In these culture conditions, cellular TAG could result from esterification of both LCFAs produced by de novo lipogenesis and exogenous oleate. To determine the contribution of TAGs coming from lipogenesis, intracellular TAGs were quantified after a 24 h-treatment with glucose and insulin, but in the absence of oleate. In these conditions, the TAG content was also markedly increased in both uninfected and Ad-βgal-infected hepatocytes (Figure 4b, right-hand panel). CPT1wt and CPT1mt overexpression led to a 25% and 50% decrease respectively in the glucose/insulin-stimulated TAG accumulation when compared with Ad-βgal-infected hepatocytes, with a significant difference between CPT1wt and CPT1mt (Figure 4b, right-hand panel). Whether exogenous oleate was present or not, the TAG accumulation induced by glucose and insulin was not statistically different in hepatocytes infected with Ad-βgal (Figure 4b). This indicated that accumulated cellular TAGs mostly resulted from esterification of LCFAs newly synthesized from glucose. Thus the increased capacity for LCFA oxidation due to CPT1wt and CPT1mt overexpression counteracts the glucose/insulin-induced accumulation of TAGs coming from lipogenesis.

CPT1mt overexpression decreases the glucose/insulin-induced TAG accumulation in cultured hepatocytes

Figure 4
CPT1mt overexpression decreases the glucose/insulin-induced TAG accumulation in cultured hepatocytes

At 16 h after infection with 5 infectious particles/cell of the indicated adenovirus, hepatocytes were cultured for 24 h in the presence of either G5 or G20+ins. (a) Oil Red O staining of hepatocytes to detect neutral lipid droplets. Oleate (0.3 mM) was added during the last 2 h of culture. Original magnification×200. (b) Measurement of cellular TAG content. When indicated, 0.3 mM oleate was added during the last 2 h of culture. Results are means±S.E.M. of duplicate flasks from three independent experiments. *P<0.05 and **P<0.01 compared with Ad-βgal in G20+ins; #P<0.05, CPT1wt compared with CPT1mt.

Figure 4
CPT1mt overexpression decreases the glucose/insulin-induced TAG accumulation in cultured hepatocytes

At 16 h after infection with 5 infectious particles/cell of the indicated adenovirus, hepatocytes were cultured for 24 h in the presence of either G5 or G20+ins. (a) Oil Red O staining of hepatocytes to detect neutral lipid droplets. Oleate (0.3 mM) was added during the last 2 h of culture. Original magnification×200. (b) Measurement of cellular TAG content. When indicated, 0.3 mM oleate was added during the last 2 h of culture. Results are means±S.E.M. of duplicate flasks from three independent experiments. *P<0.05 and **P<0.01 compared with Ad-βgal in G20+ins; #P<0.05, CPT1wt compared with CPT1mt.

Effect of CPT1wt and CPT1mt overexpression on glucose metabolism

To understand how the level of cellular TAGs resulting from lipogenesis could be decreased, two hypotheses were explored. The first one was that CPT1wt and CPT1mt overexpression could induce a decrease in glucose utilization, leading to a lower cellular TAG content. To answer this question, [14C]glucose oxidation and [14C]glycogen synthesis were measured during the last 2 h of culture in the absence of exogenous oleate. As expected, glucose and insulin markedly stimulated glucose oxidation into CO2 and glycogen synthesis (Figures 5a and 5b, left-hand panels). Infection of hepatocytes with either Ad-βgal, Ad-CPT1wt or Ad-CPT1mt had no significant effect on 14CO2 production and [14C]glycogen synthesis (Figures 5a and 5b, left-hand panels). A trend of decreased 14CO2 production was observed in hepatocytes infected with Ad-CPT1wt or Ad-CPT1mt. However, pyruvate, lactate, malate and oxaloacetate levels were not altered by CPT1wt and CPT1mt overexpression (results not shown). These results suggested that, in our experimental conditions, CPT1wt and CPT1mt overexpression did not alter glucose utilization. Similar results were observed in the presence of oleate (Figures 5a and 5b, right-hand panels), indicating that, even when LCFA oxidation flux was further enhanced by addition of exogenous LCFAs, glucose metabolism was also not altered.

CPT1wt and CPT1mt overexpression does not alter the glucose/insulin effects on [U-14C]glucose metabolism

Figure 5
CPT1wt and CPT1mt overexpression does not alter the glucose/insulin effects on [U-14C]glucose metabolism

At 16 h after infection with 5 infectious particles/cell of the indicated adenovirus, hepatocytes were cultured for 24 h in the presence of either G5 or G20+ins. During the last 2 h, a tracer amount of [U-14C]glucose was added in the absence or in the presence of 0.3 mM oleate. The medium was then collected to measure [U-14C]glucose oxidation into CO2 (a). Cells were washed and scraped to measure incorporation of [U-14C]glucose into glycogen (b). Results are means±S.E.M. of duplicate flasks from five independent experiments. §P<0.01, compared with G5.

Figure 5
CPT1wt and CPT1mt overexpression does not alter the glucose/insulin effects on [U-14C]glucose metabolism

At 16 h after infection with 5 infectious particles/cell of the indicated adenovirus, hepatocytes were cultured for 24 h in the presence of either G5 or G20+ins. During the last 2 h, a tracer amount of [U-14C]glucose was added in the absence or in the presence of 0.3 mM oleate. The medium was then collected to measure [U-14C]glucose oxidation into CO2 (a). Cells were washed and scraped to measure incorporation of [U-14C]glucose into glycogen (b). Results are means±S.E.M. of duplicate flasks from five independent experiments. §P<0.01, compared with G5.

The second hypothesis was that CPT1wt and CPT1mt overexpression, which leads to an increase in LCFA oxidation capacity, might allow the oxidation of LCFAs newly synthesized from glucose. To test this hypothesis, β-hydroxybutyrate production was measured in hepatocytes cultured in the absence or presence of 0.3 mM oleate during the last 2 h of culture. As expected, in basal conditions of culture (5 mM glucose) and in the absence of exogenous oleate, the β-hydroxybutyrate level remained low in uninfected hepatocytes, whereas it increased by 2.8-fold upon oleate addition (Figure 6a). In uninfected and Ad-βgal-infected hepatocytes, a 24 h-exposure to glucose and insulin decreased β-hydroxybutyrate production to the same basal level whether oleate was present or not (Figure 6a). By contrast, in the presence of oleate, the β-hydroxybutyrate level increased by 2.5- and 3.2-fold respectively in CPT1wt- and CPT1mt-overexpressing hepatocytes, hence abrogating the glucose/insulin inhibitory effect on ketogenesis (Figure 6a, right-hand panel). The observation that a 1.7-fold (CPT1wt) and 2.3-fold (CPT1mt) increase in β-hydroxybutyrate production occurred in the absence of exogenous oleate (Figure 6a, left-hand panel) strongly suggested that LCFAs arising from lipogenesis are oxidized to produce ketone bodies. To confirm this, hepatocytes were cultured for 24 h in the presence of TOFA, a specific ACC inhibitor [25]. As expected, TOFA treatment of uninfected hepatocytes totally inhibited both basal and glucose/insulin-stimulated de novo lipogenesis (Figure 6b), as well as glucose/insulin-induced TAG accumulation (Figure 6c). In these conditions, the increase in β-hydroxybutyrate production previously observed in CPT1wt- and CPT1mt-overexpressing hepatocytes was fully abrogated (Figure 6a), confirming that the produced β-hydroxybutyrate arose from the oxidation of de novo synthesized LCFAs. Thus the higher induced increase in β-hydroxybutyrate production observed in the presence of exogenous oleate (Figure 6a) resulted from the oxidation of both exogenous and de novo synthesized LCFAs. As observed previously, CPT1mt expression had, both in the absence and presence of oleate, a significantly higher effect than CPT1wt overexpression. Taken together, these results indicate that the observed decrease in cellular TAG content in the absence of exogenous LCFA (Figure 4b) is not due to an alteration in glucose utilization following CPT1wt or CPT1mt overexpression, but results from the oxidation of LCFAs newly synthesized from glucose.

CPT1wt and CPT1mt overexpression allows oxidation of de novo synthesized LCFAs

Figure 6
CPT1wt and CPT1mt overexpression allows oxidation of de novo synthesized LCFAs

At 16 h after infection with 5 infectious particles/cell of the indicated adenovirus, hepatocytes were cultured for 24 h in the presence of either G5 or G20+ins, in the absence or presence of 200 μM TOFA. When indicated, 0.3 mM oleate was added during the last 2 h of culture. (a) The medium was collected to measure β-hydroxybutyrate production. (b) The lipogenesis rate from 5 mM [1-14C]acetate was measured, in the absence of exogenous oleate, in uninfected cells during the last 2 h of culture. (c) Measurement of cellular TAG content. Results are means±S.E.M. of duplicate flasks from three to seven independent experiments. †P<0.01, compared without oleate; *P<0.05 and **P<0.01, compared with Ad-βgal in G20+ins; #P<0.01, as indicated on the Figure.

Figure 6
CPT1wt and CPT1mt overexpression allows oxidation of de novo synthesized LCFAs

At 16 h after infection with 5 infectious particles/cell of the indicated adenovirus, hepatocytes were cultured for 24 h in the presence of either G5 or G20+ins, in the absence or presence of 200 μM TOFA. When indicated, 0.3 mM oleate was added during the last 2 h of culture. (a) The medium was collected to measure β-hydroxybutyrate production. (b) The lipogenesis rate from 5 mM [1-14C]acetate was measured, in the absence of exogenous oleate, in uninfected cells during the last 2 h of culture. (c) Measurement of cellular TAG content. Results are means±S.E.M. of duplicate flasks from three to seven independent experiments. †P<0.01, compared without oleate; *P<0.05 and **P<0.01, compared with Ad-βgal in G20+ins; #P<0.01, as indicated on the Figure.

DISCUSSION

The present study provides the first proof of concept that, even in conditions under which lipogenesis is stimulated, a direct modulation of the malonyl-CoA–CPT1A partnership in liver cells is a relevant strategy to maintain a high mitochondrial LCFA β-oxidation flux which protects against TAG accumulation.

In agreement with previous in vitro studies performed in pancreatic β-cells [27], muscle cells [29] and hepatocytes [9], the present study showed that overexpression of rat CPT1wt or expression of CPT1mt in cultured rat hepatocytes increased LCFA β-oxidation capacity under basal conditions of culture. To challenge whether this increased oxidative capacity could be maintained in conditions under which LCFA β-oxidation is usually abolished, we chose to expose primary cultured rat hepatocytes to 20 mM glucose plus 10 nM insulin, conditions known to elicit hepatic lipogenesis [10]. As expected, this markedly increased glucose oxidation and glycogen synthesis, stimulated ACC and FAS expression and enzymatic activity, as well as de novo lipogenesis, and led to TAG accumulation, whereas it abolished exogenous oleate oxidation and ketogenesis. We have shown that CPT1A overexpression partially (CPT1wt) or fully (CPT1mt) abolished the inhibitory effect of glucose/insulin on oleate oxidation. This allowed, in the case of CPT1mt expression, the maintenance of an oxidation flux and a ketone body production from exogenous LCFAs similar to those observed in basal conditions of culture. It is noteworthy that this was accomplished without alteration in the stimulatory effects of glucose/insulin on lipogenic enzyme expression and activity and on glucose utilization. Moreover, the higher effectiveness of CPT1mt than CPT1wt overexpression to counteract the glucose/insulin effects on hepatic lipid metabolism occurred despite a lower increase in both mitochondrial CPT1A protein level and activity. The biological relevance of this finding is that it represents the converse experimental demonstration of the physiological role of malonyl-CoA in controlling hepatic LCFA metabolic fate [13]. Although liver mitochondrial glycerol-3-phosphate acyltransferase 1 isoform, which catalyses the first step in glycerolipid synthesis, may be important for LCFA metabolic channeling by competing with CPT1A for cytosolic LC-CoAs [30,31], our results clearly demonstrated that control of CPT1A activity by malonyl-CoA is an essential driving force for exogenous LCFA β-oxidation flux. Additionally, they strengthened our previous findings that changes in the degree of CPT1A malonyl-CoA sensitivity, which occur In vivo in different physiopathological situations [1115], play a critical role in the regulation of hepatic LCFA oxidation flux [19,32]. Therefore this observation allowed us to predict that strategies aiming to increase only CPT1A activity might be limited in situations where malonyl-CoA levels remain high. This may explain why a compensatory increase in CPT1A gene expression might be insufficient to prevent hepatic TAG accumulation when lipogenesis is exacerbated, such as in mice overexpressing acyl-CoA:diacylglycerol acyltransferase 2 in the liver [33].

The key finding in the present study is that, in the absence of exogenous LCFAs, CPT1mt expression still markedly protects liver cells from glucose/insulin-induced TAG accumulation. In these culture conditions, TAG can only be produced by esterification of LCFAs de novo synthesized from glucose through lipogenesis. By deciphering the metabolic impact of CPT1mt expression, we clearly showed that the decreased TAG content directly resulted from β-oxidation of the newly synthesized LCFAs, as reflected by the increased β-hydroxybutyrate production. Thus an increased CPT1A activity in association with a decreased malonyl-CoA sensitivity allowed hepatocytes to oxidize LCFAs whatever their origin, i.e. exogenous or endogenous (Figure 7). According to Randle's glucose/fatty acid cycle [34], one would predict that increased LCFA β-oxidation may have an effect on the metabolic fate of glucose. However, previous studies have reported that CPT1wt and CPT1mt overexpression did not affect glucose metabolism in pancreatic β-cells [27] and in muscular cells [28]. Similarly, in the present study, an increased LCFA β-oxidation flux in hepatocytes did not alter the insulin-induced glucose oxidation and glycogen synthesis. Additionally, oxidation of both glucose and LCFAs is associated with conversion of oxidized cofactors into reduced cofactors which are then re-oxidized by the mitochondrial respiratory chain in order to permit other cycles of fuel oxidation [35]. The lactate/pyruvate and β-hydroxybutyrate/acetoacetate ratio respectively reflect the cytosolic and mitochondrial NADH/NAD+ ratio. In the presence of glucose plus insulin, CPT1wt and CPT1mt overexpression did not modify the lactate/pyruvate ratio, suggesting no alteration in the cytosolic redox state. The β-hydroxybutyrate/acetoacetate ratio increased upon CPT1wt and CPT1mt overexpression, but did not exceed the one measured in basal conditions of culture (results not shown). Moreover, the mitochondrial activity reflected by an MTS assay was not modified by CPT1wt and CPT1mt overexpression (Supplementary Figure S3 at http://www.BiochemJ.org/bj/420/bj4200429add.htm). This suggests that mitochondria, in our experimental conditions, are able to handle efficiently the re-oxidation of NADH when both glucose and LCFAs are oxidized. As illustrated in the present study, the fundamental role of the hepatic malonyl-CoA–CPT1A partnership is to provide a powerful regulatory mechanism for the metabolic switch between carbohydrate and lipid utilization as an energy fuel. In the present study, we report for the first time that the malonyl-CoA–CPT1A interaction is also essential to preserve the cells from wasting energy by diverting the newly de novo synthesized LCFAs from mitochondrial oxidation (Figure 7). This may greatly contribute to the physiological function of the liver to convert excess dietary carbohydrates into TAGs.

Importance of the malonyl-CoA–CPT1A partnership in the liver

Figure 7
Importance of the malonyl-CoA–CPT1A partnership in the liver

In physiological situations, inhibition of CPT1A by malonyl-CoA preserves liver cells from wasting energy by preventing β-oxidation of LCFAs de novo synthesized from glucose. Indeed, retrieving CPT1A from malonyl-CoA inhibition (expression of a malonyl-CoA-insensitive CPT1A, i.e. CPT1mt) induces a metabolic switch towards β-oxidation of LCFAs independently of their origin (exogenous or de novo synthesized), and prevents TAG (TG) accumulation in liver cells. Therefore the malonyl-CoA–CPT1A partnership greatly contributes to the physiological function of the liver to convert excess dietary carbohydrates into TAGs.

Figure 7
Importance of the malonyl-CoA–CPT1A partnership in the liver

In physiological situations, inhibition of CPT1A by malonyl-CoA preserves liver cells from wasting energy by preventing β-oxidation of LCFAs de novo synthesized from glucose. Indeed, retrieving CPT1A from malonyl-CoA inhibition (expression of a malonyl-CoA-insensitive CPT1A, i.e. CPT1mt) induces a metabolic switch towards β-oxidation of LCFAs independently of their origin (exogenous or de novo synthesized), and prevents TAG (TG) accumulation in liver cells. Therefore the malonyl-CoA–CPT1A partnership greatly contributes to the physiological function of the liver to convert excess dietary carbohydrates into TAGs.

Because hepatic steatosis is a risk factor for non-alcoholic steatohepatitis and Type 2 diabetes, research efforts have focused on a decreased capacity for de novo lipogenesis in an attempt to prevent and/or reduce hepatic steatosis. Among them, liver-specific suppression of rat ACC isoform expression [36], inhibition of the nuclear factor carbohydrate-responsive element-binding protein gene expression [37], which decreased ACC and FAS gene expression, and hepatic MCD overexpression [38] were effective in decreasing TAG content in fed animals and/or hepatic steatosis and insulin resistance in obese animals. Owing to the existence of the malonyl-CoA–CPT1A partnership, these strategies also stimulated LCFA β-oxidation flux through the release of CPT1A from malonyl-CoA inhibition [3638]. Direct evidence that an enhanced capacity to oxidize LCFAs, independently of a reduced lipogenesis flux, may contribute to decrease liver TAG content was recently provided by In vivo overexpression of CPT1wt in rat liver [9]. However, in high-fat-diet-induced obese rats, hepatic CPT1A overexpression led only to a slight increase in LCFA β-oxidation capacity and a substantial reduction in hepatic TAG accumulation, whereas no improvement of insulin sensitivity could be observed [9]. Such moderate effects might be due to the presence of an elevated malonyl-CoA concentration which, despite an increased CPT1A expression, can limit In vivo CPT1A activity since the latter remains malonyl-CoA sensitive. Therefore we propose that, in addition to an increase in CPT1A expression, acting directly on the degree of CPT1A malonyl-CoA sensitivity might be a more relevant strategy in situations of insulin resistance which are associated with an increase in LCFA delivery to the liver (increased consumption of a high-fat diet and/or lipolysis) and in endogenous LCFAs (excessive lipogenesis).

In summary, a direct modulation of the malonyl-CoA–CPT1A interaction in liver cells, independently of the lipogenic pathway, allows mitochondrial oxidation of both exogenous LCFAs taken up by the liver and endogenous LCFAs generated by lipogenesis, without alteration in glucose utilization. Thus, whereas CPT1A inhibition by malonyl-CoA is essential to avoid, in physiological situations, a waste of energy, modulation of its malonyl-CoA sensitivity may be a useful therapeutic strategy to prevent and/or reduce liver steatosis.

AUTHOR CONTRIBUTION

Marie Akkaqui, Jean Gira and Carina Prip-Buus designed the research; Marie Akkaqui, Isabelle Cohen, Catherine Esnous, Véronique Lenoir, Martin Sournac and Carina Prip-Buus performed the research; Isabelle Cohen contributed new analytic tools; Marie Akkaqui, Isabelle Cohen and Carina Prip-Buus analysed results and wrote the paper.

We thank France Demaugre (INSERM U785, Villejuif, France) and Abdelhak Mansouri for critical review of the manuscript prior to submission, and Sylvie Demignot (CNRS UMR 505, Paris, France) for helpful suggestions for TAG and VLDL measurements. We thank the Vector Core of the University Hospital of Nantes supported by the AFM (Association Française contre les Myopathies) for providing the adenovirus vectors.

Abbreviations

     
  • ACC

    acetyl-CoA carboxylase

  •  
  • ASP

    acid-soluble products

  •  
  • βgal

    β-galactosidase

  •  
  • CPT

    carnitine palmitoyltransferase

  •  
  • CPT1mt

    CPT1AM593S mutant

  •  
  • CPT1wt

    wild-type CPT1A

  •  
  • FAS

    fatty acid synthase

  •  
  • LC-CoA

    long-chain acyl-CoA

  •  
  • LCFA

    long-chain fatty acid

  •  
  • MCD

    malonyl-CoA decarboxylase

  •  
  • MTS

    3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

  •  
  • PL

    phospholipid

  •  
  • TAG

    triacylglycerol

  •  
  • TOFA

    5-(tetradecyloxy)-2-furoic acid

  •  
  • VLDL

    very-low-density lipoprotein

FUNDING

This work was supported, in part, by the Ministère de la Recherche “ACI Biologie du Développement et Physiologie Intégrative” [grant number 0220527], ALFEDIAM-AstraZeneca, INSERM “Programme National de Recherches sur le Diabète” [grant number ASE04179KSA] and the Agence Nationale de la Recherche “Cardiovasculaire, Obésité, Diabète” [grant number APV05051KSA]. M.A. is a recipient of a MENRT (Ministère de l'Education Nationale, de la Recherche et de la Technologie) fellowship.

References

References
1
McGarry
 
J. D.
Brown
 
N. F.
 
The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis
Eur. J. Biochem.
1997
, vol. 
244
 (pg. 
1
-
14
)
2
Zammit
 
V. A.
 
Carnitine palmitoyltransferase 1: central to cell function
IUBMB Life
2008
, vol. 
60
 (pg. 
347
-
354
)
3
McGarry
 
J. D.
Mannaerts
 
G. P.
Foster
 
D. W.
 
A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis
J. Clin. Invest.
1977
, vol. 
60
 (pg. 
265
-
270
)
4
Bougneres
 
P. F.
Saudubray
 
J. M.
Marsac
 
C.
Bernard
 
O.
Odievre
 
M.
Girard
 
J. R.
 
Decreased ketogenesis due to deficiency of hepatic carnitine acyl transferase
N. Engl. J. Med.
1980
, vol. 
302
 (pg. 
123
-
124
)
5
Bonnefont
 
J. P.
Djouadi
 
F.
Prip-Buus
 
C.
Gobin
 
S.
Munnich
 
A.
Bastin
 
J.
 
Carnitine palmitoyltransferases 1 and 2: biochemical, molecular and medical aspects
Mol. Aspects Med.
2004
, vol. 
25
 (pg. 
495
-
520
)
6
Fromenty
 
B.
Pessayre
 
D.
 
Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity
Pharmacol. Ther.
1995
, vol. 
67
 (pg. 
101
-
154
)
7
Grefhorst
 
A.
Hoekstra
 
J.
Derks
 
T. G.
Ouwens
 
D. M.
Baller
 
J. F.
Havinga
 
R.
Havekes
 
L. M.
Romijn
 
J. A.
Kuipers
 
F.
 
Acute hepatic steatosis in mice by blocking β-oxidation does not reduce insulin sensitivity of very-low-density lipoprotein production
Am. J. Physiol. Gastrointest. Liver Physiol.
2005
, vol. 
289
 (pg. 
G592
-
G598
)
8
Vickers
 
A. E.
Bentley
 
P.
Fisher
 
R. L.
 
Consequences of mitochondrial injury induced by pharmaceutical fatty acid oxidation inhibitors is characterized in human and rat liver slices
Toxicol. in vitro
2006
, vol. 
20
 (pg. 
1173
-
1182
)
9
Stefanovic-Racic
 
M.
Perdomo
 
G.
Mantell
 
B. S.
Sipula
 
I. J.
Brown
 
N. F.
O'Doherty
 
R. M.
 
A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels
Am. J. Physiol. Endocrinol. Metab.
2008
, vol. 
294
 (pg. 
E969
-
E977
)
10
Prip-Buus
 
C.
Perdereau
 
D.
Foufelle
 
F.
Maury
 
J.
Ferre
 
P.
Girard
 
J.
 
Induction of fatty-acid-synthase gene expression by glucose in primary culture of rat hepatocytes. Dependency upon glucokinase activity
Eur. J. Biochem.
1995
, vol. 
230
 (pg. 
309
-
315
)
11
Ontko
 
J. A.
Johns
 
M. L.
 
Evaluation of malonyl-CoA in the regulation of long-chain fatty acid oxidation in the liver. Evidence for an unidentified regulatory component of the system
Biochem. J.
1980
, vol. 
192
 (pg. 
959
-
962
)
12
Bremer
 
J.
 
The effect of fasting on the activity of liver carnitine palmitoyltransferase and its inhibition by malonyl-CoA
Biochim. Biophys. Acta
1981
, vol. 
665
 (pg. 
628
-
631
)
13
Grantham
 
B. D.
Zammit
 
V. A.
 
Restoration of the properties of carnitine palmitoyltransferase I in liver mitochondria during re-feeding of starved rats
Biochem. J.
1986
, vol. 
239
 (pg. 
485
-
488
)
14
Grantham
 
B. D.
Zammit
 
V. A.
 
Role of carnitine palmitoyltransferase I in the regulation of hepatic ketogenesis during the onset and reversal of chronic diabetes
Biochem. J.
1988
, vol. 
249
 (pg. 
409
-
414
)
15
Faye
 
A.
Borthwick
 
K.
Esnous
 
C.
Price
 
N. T.
Gobin
 
S.
Jackson
 
V. N.
Zammit
 
V. A.
Girard
 
J.
Prip-Buus
 
C.
 
Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity
Biochem. J.
2005
, vol. 
387
 (pg. 
67
-
76
)
16
Prip-Buus
 
C.
Cohen
 
I.
Kohl
 
C.
Esser
 
V.
McGarry
 
J. D.
Girard
 
J.
 
Topological and functional analysis of the rat liver carnitine palmitoyltransferase 1 expressed in Saccharomyces cerevisiae
FEBS Lett.
1998
, vol. 
429
 (pg. 
173
-
178
)
17
Laemmli
 
U. K.
 
Cleavage of structural proteins during the assembly of the head of bacteriophage T4
Nature
1970
, vol. 
227
 (pg. 
680
-
685
)
18
Lowry
 
O. H.
Rosebrough
 
N. J.
Lewis Farr
 
A.
Randall
 
R. J.
 
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
1951
, vol. 
193
 (pg. 
265
-
275
)
19
Prip-Buus
 
C.
Pegorier
 
J. P.
Duee
 
P. H.
Kohl
 
C.
Girard
 
J.
 
Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Perinatal development and effects of pancreatic hormones in cultured rabbit hepatocytes
Biochem. J.
1990
, vol. 
269
 (pg. 
409
-
415
)
20
Ferre
 
P.
Pegorier
 
J. P.
Williamson
 
D. H.
Girard
 
J.
 
Interactions In vivo between oxidation of non-esterified fatty acids and gluconeogenesis in the newborn rat
Biochem. J.
1979
, vol. 
182
 (pg. 
593
-
598
)
21
Guillet-Deniau
 
I.
Pichard
 
A. L.
Kone
 
A.
Esnous
 
C.
Nieruchalski
 
M.
Girard
 
J.
Prip-Buus
 
C.
 
Glucose induces de novo lipogenesis in rat muscle satellite cells through a sterol-regulatory-element-binding-protein-1c-dependent pathway
J. Cell Sci.
2004
, vol. 
117
 (pg. 
1937
-
1944
)
22
Fujioka
 
T.
Tsujita
 
Y.
Shimotsu
 
H.
 
Induction of fatty acid synthesis by pravastatin sodium in rat liver and primary hepatocytes
Eur. J. Pharmacol.
1997
, vol. 
328
 (pg. 
235
-
239
)
23
Halestrap
 
A. P.
Denton
 
R. M.
 
Insulin and the regulation of adipose tissue acetyl-coenzyme A carboxylase
Biochem. J.
1973
, vol. 
132
 (pg. 
509
-
517
)
24
Antinozzi
 
P. A.
Segall
 
L.
Prentki
 
M.
McGarry
 
J. D.
Newgard
 
C. B.
 
Molecular or pharmacologic perturbation of the link between glucose and lipid metabolism is without effect on glucose-stimulated insulin secretion. A re-evaluation of the long-chain acyl-CoA hypothesis
J. Biol. Chem.
1998
, vol. 
273
 (pg. 
16146
-
16154
)
25
Panek
 
E.
Cook
 
G. A.
Cornell
 
N. W.
 
Inhibition by 5-(tetradecyloxy)-2-furoic acid of fatty acid and cholesterol synthesis in isolated rat hepatocytes
Lipids
1977
, vol. 
12
 (pg. 
814
-
818
)
26
Morillas
 
M.
Gomez-Puertas
 
P.
Bentebibel
 
A.
Selles
 
E.
Casals
 
N.
Valencia
 
A.
Hegardt
 
F. G.
Asins
 
G.
Serra
 
D.
 
Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition. Mutation of methionine 593 abolishes malonyl-CoA inhibition
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
9058
-
9063
)
27
Herrero
 
L.
Rubi
 
B.
Sebastian
 
D.
Serra
 
D.
Asins
 
G.
Maechler
 
P.
Prentki
 
M.
Hegardt
 
F. G.
 
Alteration of the malonyl-CoA/carnitine palmitoyltransferase I interaction in the beta-cell impairs glucose-induced insulin secretion
Diabetes
2005
, vol. 
54
 (pg. 
462
-
471
)
28
Sebastian
 
D.
Herrero
 
L.
Serra
 
D.
Asins
 
G.
Hegardt
 
F. G.
 
CPT I overexpression protects L6E9 muscle cells from fatty acid-induced insulin resistance
Am. J. Physiol. Endocrinol. Metab.
2007
, vol. 
292
 (pg. 
E677
-
E686
)
29
Perdomo
 
G.
Commerford
 
S. R.
Richard
 
A. M.
Adams
 
S. H.
Corkey
 
B. E.
O'Doherty
 
R. M.
Brown
 
N. F.
 
Increased β-oxidation in muscle cells enhances insulin-stimulated glucose metabolism and protects against fatty acid-induced insulin resistance despite intramyocellular lipid accumulation
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
27177
-
27186
)
30
Linden
 
D.
William-Olsson
 
L.
Ahnmark
 
A.
Ekroos
 
K.
Hallberg
 
C.
Sjogren
 
H. P.
Becker
 
B.
Svensson
 
L.
Clapham
 
J. C.
Oscarsson
 
J.
Schreyer
 
S.
 
Liver-directed overexpression of mitochondrial glycerol-3-phosphate acyltransferase results in hepatic steatosis, increased triacylglycerol secretion and reduced fatty acid oxidation
FASEB J.
2006
, vol. 
20
 (pg. 
434
-
443
)
31
Linden
 
D.
William-Olsson
 
L.
Rhedin
 
M.
Asztely
 
A. K.
Clapham
 
J. C.
Schreyer
 
S.
 
Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis
J. Lipid Res.
2004
, vol. 
45
 (pg. 
1279
-
1288
)
32
Prip-Buus
 
C.
Bouthillier-Voisin
 
A. C.
Kohl
 
C.
Demaugre
 
F.
Girard
 
J.
Pegorier
 
J. P.
 
Evidence for an impaired long-chain fatty acid oxidation and ketogenesis in Fao hepatoma cells
Eur. J. Biochem.
1992
, vol. 
209
 (pg. 
291
-
298
)
33
Monetti
 
M.
Levin
 
M. C.
Watt
 
M. J.
Sajan
 
M. P.
Marmor
 
S.
Hubbard
 
B. K.
Stevens
 
R. D.
Bain
 
J. R.
Newgard
 
C. B.
Farese
 
R. V.
, et al 
Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver
Cell Metab.
2007
, vol. 
6
 (pg. 
69
-
78
)
34
Randle
 
P. J.
 
Regulatory interactions between lipids and carbohydrates: the glucose fatty acid cycle after 35 years
Diabetes Metab. Rev.
1998
, vol. 
14
 (pg. 
263
-
283
)
35
Fromenty
 
B.
Robin
 
M. A.
Igoudjil
 
A.
Mansouri
 
A.
Pessayre
 
D.
 
The ins and outs of mitochondrial dysfunction in NASH
Diabetes Metab.
2004
, vol. 
30
 (pg. 
121
-
138
)
36
Savage
 
D. B.
Choi
 
C. S.
Samuel
 
V. T.
Liu
 
Z. X.
Zhang
 
D.
Wang
 
A.
Zhang
 
X. M.
Cline
 
G. W.
Yu
 
X. X.
Geisler
 
J. G.
, et al 
Reversal of diet-induced hepatic steatosis and hepatic insulin resistance by antisense oligonucleotide inhibitors of acetyl-CoA carboxylases 1 and 2
J. Clin. Invest.
2006
, vol. 
116
 (pg. 
817
-
824
)
37
Dentin
 
R.
Benhamed
 
F.
Hainault
 
I.
Fauveau
 
V.
Foufelle
 
F.
Dyck
 
J. R.
Girard
 
J.
Postic
 
C.
 
Liver-specific inhibition of ChREBP improves hepatic steatosis and insulin resistance in ob/ob mice
Diabetes
2006
, vol. 
55
 (pg. 
2159
-
2170
)
38
An
 
J.
Muoio
 
D. M.
Shiota
 
M.
Fujimoto
 
Y.
Cline
 
G. W.
Shulman
 
G. I.
Koves
 
T. R.
Stevens
 
R.
Millington
 
D.
Newgard
 
C. B.
 
Hepatic expression of malonyl-CoA decarboxylase reverses muscle, liver and whole-animal insulin resistance
Nat. Med.
2004
, vol. 
10
 (pg. 
268
-
274
)

Author notes

1

This manuscript is dedicated to Professor Denis J. McGarry.

Supplementary data