Oversupply of lipids to skeletal muscle causes insulin resistance by promoting the accumulation of lipid-derived metabolites that inhibit insulin signaling. In this study, we tested the hypothesis that overexpression of carnitine palmitoyltransferase I (CPT I) could protect myotubes from fatty acid-induced insulin resistance by reducing lipid accumulation in the muscle cell. Incubation of L6E9 myotubes with palmitate caused accumulation of triglycerides, diacylgycerol, and ceramide, produced an activation of PKCθ and PKCζ, and blocked insulin-stimulated glucose metabolism, reducing insulin-stimulated PKB activity by 60%. Transduction of L6E9 myotubes with adenoviruses encoding for liver CPT I (LCPT I) wild-type (WT), or a mutant form of LCPT I (LCPT I M593S), which is insensitive to malonyl-CoA, produced a twofold increase in palmitate oxidation when LCPT I activity was increased threefold. LCPT I WT and LCPT I M593S-overexpressing L6E9 myotubes showed normal insulin-stimulated glucose metabolism and an improvement in PKB activity when pretreated with palmitate. Moreover, LCPT I WT- and LCPT I M593S-transduced L6E9 myotubes were protected against the palmitate-induced accumulation of diacylglycerol and ceramide and PKCθ and -ζ activation. These results suggest that LCPT I overexpression protects L6E9 myotubes from fatty acid-induced insulin resistance by inhibiting both the accumulation of lipid metabolites and the activation of PKCθ and PKCζ.
- carnitine palmitoyltransferase I
- protein kinase C
skeletal muscle is responsible for 70–80% of whole body insulin-stimulated glucose uptake (13) and is therefore generally considered the most important site of insulin resistance. Insulin resistance in skeletal muscle plays a major role in the pathogenesis of type 2 diabetes (51), although the mechanism responsible remains unclear. Intramuscular lipid accumulation is evident in a wide set of experimental models, including insulin resistance induced acutely by lipid infusion in both humans (5) and rodents (11), genetic forms of obesity such as Zucker rats (29), high-fat-fed rats (12, 41), and muscle cells in culture incubated with high concentrations of fatty acids (35, 44, 55). These observations have promoted the so-called lipotoxic model of skeletal muscle insulin resistance, which proposes that high plasma fatty acid levels observed in the obese or insulin-resistant state lead to muscle lipid accumulation, which in turn causes a predisposition toward decreased insulin sensitivity and worsening of the disease (57).
Although the association between increased intramyocellular lipid and the development of insulin resistance is compelling, the molecular mechanisms linking free fatty acids (FFAs) to the inhibition of insulin action remain unclear. Recent studies in either humans (6), rodent models of obesity and/or insulin resistance (60), or cultured cells (44, 50, 55) indicate that fatty acids disrupt one or more of the early steps in insulin signal transduction. The mechanism for this disruption involves the accumulation of fatty acids in lipid-derived metabolites such as long-chain acyl-CoA (LCACoA) species (16), diacylglycerol (DAG) (23, 49), and ceramides (2, 56). Each of these compounds has been implicated in the activation of isoforms of PKC, leading to impairment of phosphatidylinositol 3-kinase (PI3K) activity and inhibition of the activation of PKB (Akt/PKB), decreasing insulin-induced glucose transport and glycogen synthesis (20, 23, 44, 49, 50, 56).
In addition to a fatty acid overload, the accumulation of lipid-derived metabolites within the muscle fiber could be a result of decreased mitochondrial fatty acid oxidative capacity. Insulin resistance is associated with a decreased mitochondrial number, unusual morphology, lower levels of mitochondrial oxidative enzymes, and lower ATP synthesis in both in vivo studies in humans (25, 43) and ex vivo human muscle biopsies (18, 26, 52). Moreover, activity of carnitine palmitoyltransferase (CPT I), the rate-limiting enzyme in fatty acid import into mitochondria for oxidation, is decreased in muscle of obese/insulin-resistant humans (26), and pharmacological inhibition of muscle CPT I activity leads to intramuscular lipid accumulation and insulin resistance in rats (14). It is possible, therefore, that an impaired ability to transport and oxidize fatty acids in skeletal muscle mitochondria could be a primary defect either leading to lipid accumulation in muscle or exacerbating lipid accumulation in the context of elevated plasma levels of fatty acids.
To examine whether a greater increase in β-oxidation could reduce lipid accumulation and enhance the protective effect of CPT I overexpression in fatty acid-induced insulin resistance, we decided to overexpress a mutant form of LCPT I (LCPT I M593S) that is insensitive to malonyl-CoA (36), its physiological inhibitor. Overexpression of both LCPT I WT and LCPT I M593S in L6E9 myotubes increased CPT I activity and palmitate oxidation. Palmitate oxidation was higher in LCPT I M593S-overexpressing cells than in cells overexpressing LCPT I WT only at high-glucose concentrations. LCPT I overexpression prevented palmitate incorporation into cellular lipids and DAG and ceramide accumulation after palmitate treatment. As a result, insulin sensitivity was completely restored. Overall, these data provide compelling evidence that overexpression of LCPT I protects muscle cells from fatty acid-induced insulin resistance by decreasing the accumulation of lipid metabolites.
RESEARCH DESIGN AND METHODS
Materials and reagents.
Protran nitrocellulose membranes for protein analysis were from Scheicher & Schuell (Keene, NH). The enhanced chemifluorescence detection kit from Amersham Biosciences was used for Western blot analysis. The Bradford solution for protein assay was from Bio-Rad Laboratories (Hercules, CA). FBS, DMEM, and antibiotics were from GIBCO-Invitrogen. Defatted BSA, palmitate, 2-deoxyglucose, insulin, and other chemicals were purchased from Sigma-Aldrich. [1-14C]Palmitic acid, [1-14C]acetyl-CoA, l-[methyl-3H]carnitine hydrochloride, 2-deoxy-d-[2,6-3H]glucose, d-[U-14C]glucose, [γ-32P]ATP, and sn-1,2-DAG Biotrak Assay System were from Amersham Biosciences. [1-14C]Sodium octanoate was purchased from American Radiolabeled Chemicals. Antibodies used included rabbit polycolonal anti-Akt/PKB and rabbit polyclonal anti-PKCθ and -PKCζ antibodies from Santa Cruz Biotechnology and rabbit polyclonal anti-phospho-Akt/PKB (Ser473) antibody from Cell Signaling. Silica gel 60 TLC plates were from Merck (Rahway, NJ). Etomoxir was provided by Dr. H. P. O. Wolf (GMBH, Allensbach, Germany).
The L6E9 rat skeletal muscle cell line was cultured in a humidified atmosphere containing 5% CO2 in DMEM containing 10% FBS, 100 units/ml penicillin G, 100 μg/ml streptomycin, and 25 mmol/l HEPES (pH 7.4; growth medium). Preconfluent myoblasts (80–90%) were induced to differentiate by lowering FBS to a final concentration of 2% (vol/vol; differentiation medium). Cells were completely differentiated after 4 days in this medium.
Adenoviral preparation and transduction of L6E9 cells.
Ad-LCPT I WT encoding rat LCPT I WT, Ad-LCPT I M593S encoding a malonyl-CoA-insensitive LCPT I M593S, and Ad-LacZ, which expresses bacterial β-galactosidase, were constructed as previously described (21, 47). Adenoviruses were amplified using the human embryonic kidney cell line (HEK-293) as host. Lysates obtained were titrated using the Adeno-X Rapid Titer kit (BD Biosciences) and used directly for cell transduction. The titers of the lysates used in this study were 2.3 × 109 pfu/ml for Ad-LacZ, 1 × 1010 pfu/ml for Ad-LCPT I WT, and 1.4 × 109 pfu/ml for Ad-LCPT I M593S. Adenoviral transduction was performed in differentiated L6E9 myotubes in serum-free medium for 30 h at the multiplicity of infection indicated in the figure legends. After this time, the infection medium was removed and cells were incubated with serum-free medium for an additional 16 h.
Sodium salt of palmitic acid was conjugated with fatty acid-free BSA in a 5:1 palmitate-BSA ratio, and a stock solution of 2.5 mmol/l was made up. Palmitate was dissolved in 0.1 mol/l NaOH at 80°C until an optically clear solution was obtained. The palmitate salt solution was immediately added to a fatty acid-free BSA solution in 0.9% NaCl maintained at 45°C, with continuous stirring to avoid precipitation. Cells were incubated with this solution at 0.25 mmol/l palmitate-BSA, or with 0.325% BSA alone as a control, for 16 h in serum-free medium (DMEM).
Cells were cultured on six-well plates, differentiated, and transduced with the adenovirus as described above. After transduction, cells were treated with palmitate as indicated and 2-deoxyglucose uptake was measured as described previously (24).
[14C]Glucose incorporation into glycogen.
Transduced L6E9 myotubes were incubated on six-well plates with palmitate or BSA as a control for 16 h. On the day of the experiment cells were washed in PBS and incubated in serum-free medium plus 4 μCi/ml d-[U-14C]glucose in the absence or presence of 1 μmol/l insulin for 1 h, and glucose incorporation into glycogen was determined as described elsewhere (50).
CPT I activity assay.
L6E9 cells were grown in 100-mm dishes and differentiated as described above. After transduction with Ad-LacZ, Ad-LCPTI WT, or Ad-LCPTI M593S, cells were washed in Krebs-Ringer bicarbonate HEPES (KRBH) buffer (135 mmol/l NaCl, 3.6 mmol/l KCl, 0.5 mmol/l NaH2PO4, 0.5 mmol/l MgSO4, 1.5 mmol/l CaCl2, 2 mmol/l NaHCO3, and 10 mmol/l HEPES, pH 7.4) that contained 0.1% (wt/vol) defatted BSA, preincubated at 37°C for 30 min in KRBH plus 1% BSA without glucose in the absence or presence of 200 μmol/l etomoxir, and washed again in KRBH 0.1% BSA. Mitochondria-enriched cell fractions were obtained as previously described (47). LCPT I activity was assayed in these preparations in which mitochondria remain largely intact (53). CPT activity of 10 μg of protein was determined by the radiometric method using l-[methyl-3H]carnitine and palmitoyl-CoA as substrates, as previously described (37). For malonyl-CoA inhibition assays, 10 μg of mitochondria-enriched cell fractions were preincubated for 1 min at 30°C with different amounts of malonyl-CoA before the CPT I activity assay.
Fatty acid oxidation.
Palmitate oxidation to CO2 and acid-soluble products (ASPs), essentially acyl-carnitine, Krebs cycle intermediates, and acetyl-CoA (59), were measured in L6E9 cells grown in 25-cm2 flasks, differentiated, and transduced as described above. On the day of the assay, cells were washed in KRBH 0.1% BSA, preincubated at 37°C for 30 min in KRBH 1% BSA, and washed again in KRBH 0.1% BSA. Cells were then incubated for 3 h at 37°C with fresh KRBH containing 2.5, 15, or 25 mmol/l glucose and 0.8 mmol/l carnitine plus 0.25 mmol/l palmitate and 1 μCi/ml [1-14C]palmitate bound to 1% BSA. Oxidation measurements were performed as previously described (21). Octanoate oxidation to CO2 was measured using sodium [1-14C]octanoate with the same procedure as for palmitate oxidation.
Palmitate incorporation into cellular lipids.
Palmitate incorporation into complex lipids was measured in L6E9 cells that were cultured on six-well plates and pretreated as described above. Cells were incubated for 16 h at 37°C in serum-free medium containing 0.25 mmol/l palmitate and 1 μCi/ml [1-14C]palmitate bound to 1% BSA. On the day of the assay cells were washed in PBS, and lipids were extracted as described previously (47). Total lipids were dissolved in 30 μl of chloroform and separated by TLC to measure the incorporation of labeled fatty acid into phospholipids (PL), DAG, triglycerides (TG), and nonesterified labeled palmitate (NE palm), as described elsewhere (21).
Triacylglycerol content measurement.
Cells were seeded on six-well plates, differentiated, and transduced for 30 h. After this time, cells were treated with palmitate for 16 h. On the day of the assay lipids were extracted (47) and dissolved in 50 μl of tert-butanol/Triton X-100/methanol (3:1:1). Triacylglycerols were measured using the Sigma 337-B GPO-Trinder Triglyceride kit.
Ceramide and DAG assay.
Cells were treated and lipids extracted as described above. Lipid extracts were stored at −20°C under N2 until the moment of the assay. Both ceramide and DAG content in the extracts were determined using a radiometric DAG assay kit (Amersham Biosciences) according to the manufacturer's instructions.
L6E9 myotubes were cultured on 150-mm dishes, differentiated, and transduced as described above. Cells were then incubated for 30 min at 37°C with fresh KRBH that contained 2.5, 15, or 25 mmol/l glucose. Malonyl-CoA was extracted as described previously (38) and assayed by a radioenzymatic method (33) using [1-14C]acetyl-CoA. The fatty acid synthetase required for the assay was isolated from rat liver as described previously (30).
Western blot analysis.
Cells were grown on six-well plates, differentiated, and transduced with the adenoviruses as described above. Cells were collected in 40 μl of 1× SDS sample buffer and sonicated for 5 s. Proteins were subjected to SDS-PAGE (8% gels) and transferred onto nitrocellulose membranes. For LCPT I detection, membranes were incubated with the LCPT I-specific polyclonal antibody against amino acids 317–430 of the rat LCPT I (45) (1/6,000 dilution) for 4 h at room temperature. For muscle (M)CPT I detection, the MCPT I-specific antibody against amino acids 259–760 of the rat MCPT I (58) was used (1/1,000 dilution), and membranes were incubated overnight at 4°C. For phospho-PKB (Ser473) detection a rabbit polyclonal anti-phospho-PKB (Ser473) antibody was used at 1/2,500 dilution, and membranes were incubated overnight at 4°C. For total PKB detection, membranes were stripped and incubated with the rabbit polyclonal anti-PKB antibody at 1/1,000 dilution overnight at 4°C. Detection was carried out with the enhanced chemifluorescence immunoblotting detection system (Amersham Biosciences) using the anti-rabbit IgG alkaline phosphatase goat antibody (1/10,000 dilution) and with the enhanced chemiluminiscence immunoblotting detection system using the anti-sheep IgG peroxidase-linked whole antibody (1/5,000 dilution) for MCPT I protein.
PKC kinase assay.
L6E9 cells were grown on 150-mm plates, differentiated, and transduced with the adenoviruses. After transduction, cells were incubated in the presence or absence of 0.25 mmol/l palmitate for 16 h. Cells were collected in PBS and homogenized with a douncer in 500 μl of lysis buffer (50 mmol/l Tris, pH 7.5, 150 mmol/l NaCl, 1% Triton X-100, 2 mmol/l EDTA, 1 mmol/l EGTA, 1 μmol/l PMSF, 25 μg/ml leupeptin, and 25 μg/ml aprotinin) and centrifuged at 700 g for 10 min to remove nuclei, cellular debris, and floating cells. After that, 500 μg of protein were immunoprecipitated with 5 μg/ml of anti-PKCθ or anti-PKCζ antibodies (54). Immunoprecipitates were collected on protein A-Sepharose G beads, washed 5 times in lysis buffer and twice in kinase buffer (35 mmol/l Tris, pH 7.5, 10 mmol/l MgCl2, 0.5 mmol/l EGTA, 1 μmol/l Na3VO4), and incubated for 30 min at 30°C in 20 μl of kinase buffer containing 1 μCi of [γ32P]ATP, 60 μmol/l ATP, and 1 μg of myelin basic protein (MBP) as substrate. The kinase reaction was terminated by the addition of 4× SDS-PAGE sample buffer, and the mixture was then boiled for 5 min at 95°C (9). Samples were resolved in 12% SDS-PAGE, gels were dried out, and bands corresponding to phosphorylated MBP were quantified with a Storm 840 Laser scanning system (Molecular Dynamics, Amersham Pharmacia Biotech).
Data are expressed as means ± SE. The significance of differences was assessed by the unpaired Student's t-test. Different experimental groups were compared with a one-way ANOVA followed by Tukey's test for comparisons post hoc. A probability level of P < 0.05 was considered to be statistically significant.
CPT I activity is not inhibited by malonyl-CoA in L6E9 cells expressing LCPT I M593S.
In a previous study, we described an LCPT I M593S mutant that was insensitive to malonyl-CoA when expressed in yeast (36) or in pancreatic β-cells (47). In the present study, malonyl-CoA sensitivity of LCPT I in L6E9 myotubes was evaluated in cells overexpressing LCPT I WT and LCPT I M593S. Mitochondria-enriched fractions of cells infected with Ad-LCPT I WT or Ad-LCPT I M593S were incubated with different amounts of malonyl-CoA, and CPT I activity was determined. In the presence of 100 μmol/l of malonyl-CoA, mitochondria-enriched fractions of cells that were infected with Ad-LCPT I M593S retained most of their activity, whereas that of the LCPT I WT was inhibited by 80% (Fig. 1).
CPT I protein levels and activity in L6E9 cells transduced with Ad-LCPT I WT and Ad-LCPT I M593S.
Western blot analysis using antibodies against MCPT I or LCPT I showed that the endogenous CPT I protein expressed in L6E9 myotubes was the liver isoform (LCPT I; Fig. 2A), consistent with that described in other cultured muscle cell lines (42). To determine the capacity of the adenovirus to increase CPT I activity, L6E9 myotubes were infected with different amounts of Ad-LCPT I WT and Ad-LCPT I M593S, and CPT I activity assay was performed with mitochondria-enriched cell fractions (Fig. 2B). In both cases, CPT I activity increased proportionally to the quantity of virus used, to a maximum of 25-fold compared with the endogenous LCPT I, calculated from Ad-LacZ-infected cells (2.5 ± 0.6 nmol·mg protein−1·min−1, means ± SE of 4 experiments). No saturation effect was seen with the amounts of virus used. To evaluate whether the overexpressed LCPT I could be inhibited by etomoxir, cells were infected with the amount of adenovirus LCPT I WT (40 pfu/cell) and LCPT I M593S (20 pfu/cell) that increased CPT I activity sixfold (15 nmol·mg protein−1·min−1), with respect to control Ad-LacZ, and incubated for 30 min in the absence or presence of 200 μmol/l etomoxir. This irreversible CPT I inhibitor reduced CPT I activity in both Ad-LacZ and Ad-LCPT I WT and, to a lesser extent, in Ad-LCPT I M593S-overexpressing cells (Fig. 2C). Consistent with the activity assays, Western blot showed similar amounts of protein in Ad-LCPT I WT- and Ad-LCPT I M593S-infected cells and a similar increase over the Ad-LacZ-infected cells (control; Fig. 2D).
Effect of LCPT I WT and LCPT M593S overexpression on fatty acid oxidation in L6E9 myotubes.
To evaluate the metabolic effects of LCPT I overexpression, we measured fatty acid oxidation in L6E9 myotubes. First, we measured palmitate oxidation at increasing levels of CPT I activity in both Ad-LCPT I WT- and Ad-LCPT I M593S-infected cells (Fig. 3A). Palmitate oxidation to CO2 increased up to twofold for a threefold increase in CPT I activity, followed by saturation at higher levels of CPT I activity. On the basis of these results, we decided to use the amount of adenoviruses that increased CPT I activity threefold (20 pfu/cell for Ad-LCPT I WT and 10 pfu/cell for Ad-LCPT I M593S) for subsequent experiments. Palmitate oxidation was measured at 2.5 mmol/l glucose in L6E9 cells infected with this amount of adenovirus (Fig. 3B). Fatty acid oxidation to CO2, ASPs, and their sum (total oxidation) was increased 1.6-, 2.2-, and 2.2-fold, respectively, in Ad-LCPT I WT-infected cells and 2.1-, 2.1-, and 2.2-fold, respectively, in Ad-LCPT I M593S-infected cells compared with Ad-LacZ control cells, with no significant differences between LCPT I WT- and LCPT I M593S-overexpressing cells. The ratio of incomplete (ASPs) vs. complete (CO2) oxidation was similar in Ad-LacZ-, Ad-LCPT I WT-, and Ad-LCPT I M593S-infected cells (Fig. 3C), indicating that the increases in fatty acid transport into the mitochondria obtained after LCPT I overexpression were followed by increased β-oxidation of these fatty acids. At 25 mmol/l glucose, palmitate oxidation decreased by 40% in all cases (Fig. 3D), consistent with a 30% increase in malonyl-CoA levels (0.22 ± 0.02 nmol/mg protein at 2.5 mmol/l glucose and 0.30 ± 0.04 nmol/mg protein at 25 mmol/l glucose; Fig. 3E), but LCPT I M593S-overexpressing cells showed 30% higher palmitate oxidation than LCPT I WT-overexpressing cells. The oxidation of the medium-chain fatty acid octanoate, which enters the mitochondrion independently of the CPT pathway, was unaffected by the overexpression of both CPT I enzymes (Fig. 3F).
LCPT I WT and LCPT I M593S overexpression protects L6E9 myotubes from fatty acid-induced insulin resistance.
In the absence of palmitate incubation, insulin stimulated glucose uptake and incorporation into glycogen 1.5- and twofold, respectively, in Ad-LacZ-infected cells (Fig. 4). Basal and insulin-stimulated glucose metabolism were not affected by overexpression of LCPT I WT or LCPT I M593S in these conditions (Fig. 4). Incubation of control cells with 0.25 mmol/l palmitate totally blocked insulin-stimulated glucose uptake and incorporation into glycogen, indicating fatty acid-induced insulin resistance. However, those cells overexpressing LCPT I WT or LCPT I M593S presented a normal response to insulin in both glucose uptake (Fig. 4A) and incorporation into glycogen (Fig. 4B). These data show that LCPT I overexpression protected these cells from palmitate-induced insulin resistance. Insulin activates a signaling pathway that leads to Akt/PKB phosphorylation and induction of glucose transport and glycogen synthesis. In L6E9 myotubes, insulin caused a ninefold increase in Ser473 phosphorylation of Akt/PKB (Fig. 5). After incubation of cells with 0.25 mmol/l palmitate, PKB phosphorylation was reduced by 60% in control cells, whereas in Ad-LCPT I WT- and Ad-LCPT I M593S-infected cells, Akt/PKB phosphorylation was reduced by only 30%.
Effect of LCPT I WT and LCPT M593S overexpression on lipid partitioning in L6E9 myotubes.
To examine the effect of the increase in fatty acid oxidation on the availability of fatty acids for esterification, we measured the incorporation of [1-14C]palmitate into different lipid species. Incorporation to TG, DAG, and PL was decreased by 30, 34, and 33%, respectively, for Ad-LCPT I WT-infected cells and by 53, 30, and 45%, respectively, for Ad-LCPT I M593S-infected cells (Fig. 6, A–C), both compared with Ad-LacZ-infected cells (control). The levels of NE palm, an indirect measurement of cytosolic LCACoA (46), were reduced by 40% in both LCPT I WT- and LCPT I M593S-overexpressing cells compared with control (Fig. 6D). Palmitate treatment induced a twofold increase in TG content in Ad-LacZ-infected cells, and no changes were observed after the overexpression of LCPT I WT or LCPT I M593S (Fig. 7A). This is probably because differences in the incorporation of LCACoA into TG are not reflected in the large and stable total TG pool. Palmitate incubation also produced a 2.5-fold increase in DAG content in Ad-LacZ-infected cells and only a 1.7-fold increase in LCPT I WT- and LCPT I M593S-overexpressing cells (Fig. 7B). In the case of ceramide, palmitate incubation produced a 1.9-fold increase in control cells but not in LCPT I WT- or LCPT I M593S-overexpressing cells (Fig. 7C).
PKCθ and -ζ activation is inhibited in L6E9 myotubes expressing LCPT I WT or LCPT I M593S.
DAG, ceramide, and LCACoA have been implicated in the activation of PKCθ and -ζ, which are involved in impairment of insulin signaling (20, 23, 44, 49). To assess whether palmitate induces activation of these kinases in L6E9 cells, and whether the decrease of DAG, ceramide, and LCACoA observed in Ad-LCPT I WT- and Ad-LCPT I M593S-infected cells is associated with the alteration in PKCθ and -ζ activity, we determined the activity of both PKC isoforms by in vitro kinase assay. Palmitate produced a 2.5- and 1.5-fold increase in PKCθ and PKCζ activity, respectively, in control cells. However, LCPT I WT or LCPT I M593S overexpression significantly reduced this activation (Fig. 8).
Lipid accumulation in skeletal muscle has been implicated in insulin resistance and type 2 diabetes (34, 57). Either increased lipid supply or defects in fatty acid metabolism in skeletal muscle can lead to accumulation of lipid metabolites such as LCACoA, DAG, or ceramide, which have been implicated in the disruption of insulin signaling (2, 16, 20, 23, 49, 56, 60). In addition, the insulin-resistant phenotype is associated with decreased oxidative capacity in muscle (25, 43), including lower CPT I activity (26, 52). CPT I is the rate-limiting enzyme in fatty acid import into mitochondria for oxidation (32). We therefore examined whether overexpression of a malonyl-CoA-insensitive mutant form of LCPT I (LCPT I M593S) (36) would increase fatty acid oxidation. This in turn would lower the cellular lipid content and thus provide protection against fatty acid-induced insulin resistance in muscle cells.
We used L6E9 rat skeletal muscle cells, which display a robust response to insulin in terms of glycogen synthesis and glucose uptake (40). A recent study (42) showed that several muscle cell lines, such as L6 and C2C12 cells, express the liver isoform of CPT I (LCPT I) instead of the muscle isoform (MCPT I). This pattern was also confirmed in L6E9 cells by Western blot (Fig. 2A). This finding, together with the report (42) that palmitate oxidation did not increase after MCPT I overexpression in L6 myotubes, suggests that CPT I isoform expression and malonyl-CoA concentrations are deregulated coordinately during the establishment of these cultured cell lines. Malonyl-CoA levels are thus appropriate for the regulation of LCPT I (IC50∼3–10 μmol/l) and high enough to inhibit the MCPT I overexpressed (IC50∼0.03 μmol/l). This is consistent with the malonyl-CoA concentrations measured in our system, which ranged from 2.5 μmol/l at low glucose to 3.5 μmol/l at high glucose.
In L6E9 myotubes, the increase in fatty acid import into the mitochondria by overexpression of both LCPT I WT and LCPT I M593S increased fatty acid oxidation rates at all glucose concentrations tested. When glucose concentration was increased, palmitate oxidation rates were reduced in accordance with the increases in malonyl-CoA levels, which could inhibit CPT I activity. Only at high glucose concentrations, when the levels of malonyl-CoA were elevated, did overexpression of LCPT I M593S produce a higher increase in palmitate oxidation compared with Ad-LCPT I WT-infected cells. This illustrates the effectiveness of the malonyl-CoA-insensitive LCPT I M593S. It has been suggested (39) that an increase in fatty acid import into mitochondria could lead to the accumulation of some fatty acylcarnitines that can be mediators of insulin resistance. This accumulation would result from the lack of a coordinate induction in β-oxidation and other downstream metabolic pathways such as the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) (39). In our study, the ratio of incomplete vs. complete fatty acid oxidation was maintained after LCPT I overexpression, with respect to control cells. This indicates that the fatty acids that enter the mitochondria as a consequence of LCPT I overexpression can be oxidized and do not overaccumulate in incompletely oxidized molecules (i.e., palmitoyl carnitine). Moreover, the fact that the rate of complete octanoate oxidation is 10-fold higher than that for palmitate oxidation suggests that the β-oxidation pathways behind CPT I (i.e., TCA cycle and ETC) are not limiting steps.
In the absence of palmitate incubation, LCPT I overexpression affected neither basal nor insulin-stimulated glucose metabolism. After 16-h incubation with palmitate, insulin no longer induced glucose uptake or incorporation into glycogen in L6E9 control cells. This loss of sensitivity was correlated with the accumulation of TG, DAG, and ceramide, which is consistent with that reported in L6 myotubes (44), C2C12 cells (50), cultured human muscle cells (35), high-fat-fed rats (49, 56), and humans (2, 23). However, in the context of LCPT I WT or LCPT I M593S overexpression, insulin induced glucose metabolism after palmitate treatment to the same extent as in control cells without palmitate incubation. This indicates that LCPT I overexpression protected cells from palmitate-induced insulin resistance. This protection was associated with a reduction in the incorporation of palmitate into cellular lipids (TG, DAG, and PL) and a blockage in the increase in DAG and ceramide content and probably of LCACoA species (measured indirectly by palmitate incorporation into NE palm). In an earlier study, Perdomo et al. (42) showed that LCPT I overexpression stimulated insulin-induced glucose metabolism after palmitate treatment without completely restoring insulin sensitivity and with no reduction in lipid accumulation. They suggested that distinct fatty acid/acyl-CoA pools might be available for β-oxidation or lipid synthesis and that CPT I overexpression could affect the oxidative pool without affecting the biosynthetic pool. However, in our study, LCPT I overexpression reduced lipid accumulation after palmitate treatment, which could account for the stronger protection from palmitate-induced insulin resistance observed in our system. Furthermore, the protective effect against palmitate-induced insulin resistance in these cells is due only to the overexpressed LCPT I WT or LCPT I M593S protein and not to the increase in CPT I mRNA levels observed in β-cells (4) and L6 myotubes (28) after palmitate treatment for long times, since the control Ad-LacZ-infected cells were also incubated with palmitate and were insulin resistant.
Activation of Akt/PKB is necessary for insulin to stimulate glucose uptake and glycogen synthesis. In this study, the fatty-acid induced impairment of insulin-stimulated glucose metabolism in L6E9 cells is associated with decreased insulin-induced Akt/PKB activity. We have shown that accumulation of lipid metabolites activated two serine kinases, PKCθ and PKCζ. Activation of PKCθ by DAG induces insulin resistance in muscle (20, 49), and activation of PKCζ by ceramide induces insulin resistance in L6 myotubes (44). PKCθ phosphorylates IRS-1 in serine residues, impairing its phosphorylation in tyrosines, the association of PI3K, and the activation of Akt/PKB (60). Although some studies have shown that PKCζ can phosphorylate and inhibit Akt/PKB directly (7, 15, 27), other studies have shown that the activation of PKCζ mediates insulin-stimulated glucose transport in muscle and adipocytes (reviewed in Ref. 17). We cannot explain these discrepant observations, but in our study, palmitate treatment induced the activation of PKCζ, consistent with that previously reported by others in L6 myotubes (44). The overexpression of LCPT I WT or LCPT I M593S blocked the activation of these two kinases, consistent with the decrease in DAG, ceramide, and LCACoA levels, and protected L6E9 cells from fatty acid-induced impairment in Akt/PKB activity.
Interestingly, we found that CPT I overexpression did not affect total TG levels after palmitate treatment in L6E9 myotubes. However, palmitate incorporation into TG was significantly reduced in LCPT I-overexpressing cells, indicating that the differences in the incorporation of LCACoA into TG may not be reflected in the stable and probably metabolically more regulated total TG pool. Therefore, insulin sensitivity was restored independently of TG levels after LCPT I overexpression in L6E9 myotubes. It has previously been shown (19) that well-trained individuals have increased muscle TG content despite being insulin sensitive. Thus it has been suggested (19) that intramyocellular TG per se does not directly influence insulin action in skeletal muscle, with other lipid metabolites such as LCACoA, DAG, and ceramides being the mediators of insulin resistance. Our results in L6E9 myotubes are consistent with this hypothesis.
Overexpression of the malonyl-CoA-insensitive form of LCPT I (LCPT I M593S) did not produce any improvement in palmitate-induced insulin resistance compared with LCPT I WT-overexpressing cells. This could be explained by the finding that the increase in palmitate oxidation to CO2 observed in LCPT I M593S-overexpressing cells was only 1.3-fold higher than that observed for LCPT I WT, and so there was no further reduction in DAG, ceramide, or LCACoA levels. In an earlier study, we tested the effect of LCPT I M593S overexpression on insulin secretion in the clonal β-cell line INS(832/13) (21). In that study, LCPT I M593S overexpression produced a twofold increase in palmitate oxidation compared with LCPT I WT overexpression. This discrepancy between the two cell types suggests that, in L6E9 muscle cells, other factors might control fat oxidation independently of malonyl-CoA/CPT I interaction. Moreover, the fact that palmitate oxidation did not increase more than 2.5-fold and LCPT I activity increased up to 25-fold without saturation suggests that another factor besides CPT I might control the entrance of fatty acids into the mitochondrial matrix, since β-oxidation is not the limiting step, as demonstrated by the higher oxidation rates obtained using octanoate. Such a mechanism has been suggested by several authors. Recently, it has been postulated (10) that the fatty acid translocase FAT/CD36 could be implicated in fatty acid transport into the mitochondria in tandem with CPT I in skeletal muscle. Another study (31) suggests a role for UCP3 in fatty acid oxidation in muscle, hypothesizing that UCP3 exports the excess of fatty acid anions from the mitochondrial matrix to the intermembrane space and cytosol when the entry of fatty acids to the mitochondria exceeds their oxidation rate. It is possible, therefore, that these other proteins could limit the increases in palmitate oxidation produced by overexpression of both LCPT I WT and LCPT I M593S. Further research is needed to elucidate the role of these other factors in fatty acid oxidation in muscle cells.
A recent study (8) has shown that endurance training in obese humans improved glucose tolerance by increasing CPT I activity and fatty acid oxidation and reducing DAG and ceramide levels, which is consistent with the results obtained in our study. Studies using other strategies to increase fatty acid oxidation showed similar results. It has been postulated (48) that any treatment that decreases malonyl-CoA concentration and thus activates CPT I could improve insulin sensitivity in muscle. For example, mutant mice with reduced expression of acetyl-CoA carboxylase (ACC)2 (1) or rats overexpressing malonyl-CoA decarboxylase (MCD) (3) presented low levels of malonyl-CoA, leading to increased CPT I activity and protection from fat-induced insulin resistance. Moreover, pharmacological activation of AMP-activated protein kinase, which inhibits ACC and activates MCD, leading to a reduction in malonyl-CoA levels, enhanced fatty acid oxidation and protected from fatty acid-induced insulin resistance in high-fat-fed rats (22).
In conclusion, this study shows for the first time that LCPT I overexpression reduces lipid accumulation in muscle cells, thus protecting them from fatty acid-induced insulin resistance. These results, together with those cited herein, strongly favor the hypothesis that any strategy designed to enhance fatty acid oxidation in muscle should protect cells from lipid-induced insulin resistance, which makes CPT I a therapeutic target for the treatment of type 2 diabetes.
This study was supported by grant SAF-2004-06843-C03 from the Ministerio de Educación y Ciencia, by Grant C3/08 from the Fondo de Investigación Sanitaria of the Instituto de Salud Carlos III, by Grant CIBER CB06/03/0026 from the Ministry of Health, Madrid, Spain, and by the Ajut de Suport als Grups de Recerca de Catalunya (2005SGR-00733), Spain. D. Sebastián and L. Herrerro are recipients of fellowships from the University of Barcelona and the Ministry of Education and Science, Spain, respectively.
We thank Drs. Carina Prip-Buus and Victor Zammit for supplying anti-rat liver CPT I and anti-rat muscle CPT I antibodies, respectively. The L6E9 rat skeletal muscle cell line was kindly provided by Dr. A. Zorzano (University of Barcelona). We are also grateful to Robin Rycroft of the Language Service for valuable assistance in the preparation of the manuscript.
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