The increased availability of saturated lipids has been correlated with development of insulin resistance, although the basis for this impairment is not defined. This work examined the interaction of saturated and unsaturated fatty acids (FA) with insulin stimulation of glucose uptake and its relation to the FA incorporation into different lipid pools in cultured human muscle. It is shown that basal or insulin-stimulated 2-deoxyglucose uptake was unaltered in cells preincubated with oleate, whereas basal glucose uptake was increased and insulin response was impaired in palmitate- and stearate-loaded cells. Analysis of the incorporation of FA into different lipid pools showed that palmitate, stearate, and oleate were similarly incorporated into phospholipids (PL) and did not modify the FA profile. In contrast, differences were observed in the total incorporation of FA into triacylglycerides (TAG): unsaturated FA were readily diverted toward TAG, whereas saturated FA could accumulate as diacylglycerol (DAG). Treatment with palmitate increased the activity of membrane-associated protein kinase C, whereas oleate had no effect. Mixture of palmitate with oleate diverted the saturated FA toward TAG and abolished its effect on glucose uptake. In conclusion, our data indicate that saturated FA-promoted changes in basal glucose uptake and insulin response were not correlated to a modification of the FA profile in PL or TAG accumulation. In contrast, these changes were related to saturated FA being accumulated as DAG and activating protein kinase C. Therefore, our results suggest that accumulation of DAG may be a molecular link between an increased availability of saturated FA and the induction of insulin resistance.
- insulin sensitivity
- skeletal muscle
- glucose uptake
- saturated fatty acids
skeletal muscle insulin resistance, which is associated with obesity and type 2 diabetes, has been correlated to the increase in the availability of lipids and, more recently, with an increased intramuscular lipid accumulation. The largest body of evidence comes from dietary studies. High-fat diets are known to affect glucose metabolism; however, the impact on insulin sensitivity in particular depends on the dietary fatty acid (FA) composition. Diets rich in saturated FA were found to decrease insulin sensitivity (13, 27, 28), whereas replacement of part of the fat by polyunsaturated FA (PUFA) prevented such effect (27). This observation was correlated to an increase in percentage of PUFA in skeletal muscle phospholipids (PL), leading to the hypothesis that the content of PUFA in PL determines insulin action (4, 28). In contrast, another study reported no correlation between changes in the FA composition of PL and insulin resistance, but rather with the content and FA composition of triacylglycerides (TAG) (17).
Support for the relationship between intramuscular TAG accumulation and insulin sensitivity comes mainly from type 2 diabetic patients, in which a strong correlation has been shown between intramuscular levels of TAG and insulin resistance, independent of the degree of obesity (15, 18, 20, 28). Similarly, rodent models of obesity and insulin resistance, such as the Zucker and Goko-Kakizaki (GK) rats, display elevated intramuscular TAG content (2). Nevertheless, it has been noted that, in rodent diabetic models, not only TAG but also diacylglycerol (DAG) levels are increased in muscle. This phenomenon has been correlated to persistent translocation and activation of protein kinase C (PKC) (2, 21), suggesting involvement of a DAG-PKC signaling pathway in the generation of muscle insulin resistance. Likewise, Schmitz-Peiffer et al. (25) showed that reduction in insulin sensitivity of rats fed with a high-fat diet was associated with the elevation in TAG and DAG levels, as well as an increased proportion of membrane-associated PKC in muscle. Finally, additional support for this hypothesis has been found by elevation of plasma free FA through lipid and/or heparin infusion (10), which caused skeletal muscle insulin resistance associated with chronic activation of PKC.
With use of different cell lines, in vitro studies have been undertaken to elucidate the mechanism by which FA impair insulin response, leading to the notion that saturated and unsaturated FA act through different pathways (12, 24). In C2C12skeletal muscle cells, palmitate has been shown to reduce insulin-stimulated glycogen synthesis through the inhibition of protein kinase B phosphorylation without modulation of the insulin receptor substrate-1 (IRS-1) function (24). Ceramide, a derivative of palmitate, has been proposed to be responsible for these inhibitory effects and the induction of insulin resistance (24).
In summary, there is limited information about the specific metabolism of FA in muscle cells. Intramuscular TAG accumulation in vivo is promoted after the feeding of high-fat diets (17, 28); likewise, in vitro studies, with perfused (6) or isolated muscle (8), have demonstrated a positive correlation between extracellular FA and muscle TAG content. Nevertheless, differences in individual FA incorporation into TAG have been noted. After infusion of 13C-labeled FA, distinct profiles in rat muscle FA content were observed between intramuscular TAG and nonesterified fatty acids (NEFA) (11). For instance, the PUFA linoleate was found to be more highly incorporated into TAG than into NEFA, whereas the opposite occurred for the saturated FA stearate. Moreover, optimal TAG accumulation was reported with unsaturated long-chain FA in the myogenic L6 cell line (23). Thus FA composition, and not only FA availability, has been suspected to influence intramuscular lipid metabolism.
Therefore, there has been no clear understanding of the relationship between muscle insulin resistance and FA accumulation in different pools, PL, DAG, or TAG. The aim of the present study was to examine, in a cultured human muscle model, the influence of saturated and unsaturated FA on the insulin stimulation of glucose uptake and its relation to their incorporation into lipid pools. It is shown that supplementation with saturated FA impaired insulin stimulation of glucose uptake, whereas loading cells with the unsaturated FA oleate had no effect. Insulin insensitivity was not correlated to a modification of the FA composition of PL or TAG or to the accumulation of TAG. In contrast, our data suggest a role of DAG accumulation in the saturated FA-induced insulin resistance.
MATERIALS AND METHODS
Human muscle primary cultures.
Human muscle primary cultures were initiated from a bank of satellite cells of muscle biopsies obtained from patients considered free of muscle disease (biopsies were obtained with informed consent and approval of the Human Use Committee of the Hospital Clinic, Barcelona). Aneural muscle cultures were established in a monolayer through an explant-reexplantation technique as described in Ref. 1. Cultures were grown in a DMEM-M-199 medium, 3:1, supplemented with 10% fetal bovine serum (FBS), 10 μg/ml insulin, 2 mM glutamine, 25 ng/ml fibroblast growth factor, and 10 ng/ml epidermal growth factor (Becton-Dickinson, Franklin Lakes, NY). Immediately after myoblast fusion, cells were rinsed in Hanks' balanced salt solution (HBSS), and a medium devoid of FGF, EGF, and glutamine was added. Muscle cultures were maintained in this medium for up to 4 wk.
FA handling and treatment.
Sodium salts of FA (octanoic, palmitic, stearic, oleic, and linoleic acids) were prepared immediately before utilization by dissolving the FA in deionized water containing 1.2 equivalents of NaOH at 70°C, until an optically clear dispersion was obtained. The FA salt solution was immediately added to a DMEM medium containing 25 mM glucose and FA-free bovine serum albumin (BSA; Sigma, St. Louis, MO) with continuous agitation to avoid precipitation. Afterward, this medium was added to the cells. The FA-to-BSA molar ratio was of 2.5:1 or 5:1 as stated.
Measurement of 2-deoxy-d-[3H]glucose uptake.
Cells were grown in 24-well cluster plates (16 mm each) under the conditions mentioned above. Before assay, monolayers were washed three times in PBS, 0.1 mmol/l CaCl2, and 0.1% BSA, and 450 μl of the same buffer were added to each well. Insulin was included when stated, and the mixture was incubated for 30 min at 37°C. 2-Deoxy-d-[3 H]glucose (2-DG) uptake was then measured by the addition of 0.5 μCi 2-DG/well and unlabeled 2-deoxyglucose at the concentration of 0.5 mmol/l, followed by three washes in ice-cold PBS after 6 min of exposure. Cells were subsequently lysed with 1% SDS, and aliquots were measured for radioactivity in 5 ml of the cocktail (Optiphase, LKB).
To determine the TAG content, extracts were prepared by scraping cell monolayers in a buffer consisting of 50 mM Tris, 100 mM KCl, 20 mM KF, 0.5 mM EDTA, and 0.05% Lubrol PX, pH 7.9, and they were sonicated three times for 5 s (9). Homogenates were centrifuged at 11,000 g for 15 min, and the resulting supernatants were collected. Protein concentration was measured with the aid of Bio-Rad protein assay reagent. Total TAG were measured enzymatically with a Cobas-Bio autoanalyzer with a GPO-Trinder (Sigma) kit, with triolein resuspended in the extraction buffer as a standard.
Lipids in muscle cells were evidenced by histochemical staining with Sudan III in 70% ethanol.
Incorporation of [14C]acetate and14C-FA into lipid fractions: thin-layer chromatography and mass spectrometry analysis.
Cells were incubated with 1 mM concentrations of [1-14C]acetate, [1-14C]palmitic acid, [1-14C]stearic acid, [1-14C]oleic acid, or [1-14C]linoleic acid (1 mCi/mmol; Amersham Life Science, Dubendorf, Switzerland) (FA-to-BSA molar ratio of 2.5:1) for 20 h. The cell monolayers were then washed three times in HBSS, and the lipids were extracted twice with hexane-isopropanol (3:2). After drying under nitrogen, the residual lipid extract was redissolved in chloroform-methanol (2:1) and separated on thin-layer chromatography (TLC; Polygra SIL G; Macherey-Nagel, Duren, Germany) by use of hexane-diethyl ether-acetic acid (70:30:1). The lipid spots were identified by iodine vapor and counted in an Instant Imager 2024 (Camberra Packard, Zurich, Switzerland). Lipid spots having a retention factor (Rf) of 0.25 and comigrating with 1.2 diolein (Sigma, Buchs, Switzerland) were mechanically scraped from the TLC plate for further identification purposes. Silica particles from these zones were extracted four times with a hexane-isopropanol (3:2) solvent mixture and then dried under nitrogen in glass vials. Silylation of the samples was then performed with a mixture of BSTFA-TMCS-pyrine (80:20:10, vol/vol/vol) and heating to 60°C for 30 min. Subsequent gas chromatography-mass spectrometry (GC-MS) analysis was carried out, according to standard procedures, on a Finnigan MAT-8430 mass spectrometer connected to an HP-5890 gas chromatograph (Finnigan MAT, Bremen, Germany) equipped with a DB-5 capillary column. The resulting mass spectra clearly identified the presence of glycerol-dipalmitate at this Rf.
TAG and PL FA analyses.
Lipids from cultured human muscle cells were extracted according to the method of Bligh and Dyer (3) and were separated by TLC as described above. The FA from the PL and TAG were converted to their methyl esters in a 3% H2SO4 methanolic reagent for 3 h at 80°C and then extracted with hexane, dried under N2, and resuspended into hexane. FA methyl ester separation was performed by automated gas-liquid chromatography (HP-6890 series) by use of a DB-Wax column (30 m × 0.32 mm ID; J&W Scientific, Folsom, CA) with a 2-m retention gap. Helium (2.9 ml/min, constant flow) was used as the carrier gas with a splitless injection mode. The initial oven temperature of 40°C, maintained for 2 min, was increased by 15°C/min up to 145°C and held for 1 min; then it was increased by 4°C/min up to 227°C and held for 5 min before a further increase of 1.5°C/min up to 250°C. This final temperature was held for 24 min. FA methyl esters were detected by flame ionization detector (280°C); authentic standard mixtures of FA methyl esters were injected to identify FA methyl ester peaks.
Subcellular fractionation and PKC activity assay.
Cytosolic and membrane PKC fractions were prepared and PKC activities assayed as follows. Human muscle cells (1.5 × 106) were incubated in duplicate in 60-mm dishes. After the incubation, cells were washed twice in ice-cold PBS and picked by scraping, and the content of every two plates was homogenized (15 passes) in 0.35 ml ofbuffer A (2 mM EDTA, 0.5 mM EGTA, 20 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 20 mM Tris · HCl, pH 7.5) in a glass Potter-Elvehjem homogenizer. The homogenate was centrifuged at 200,000g for 90 min, and the supernatant was designated as the cytosolic fraction. The pellet was washed once in 0.5 ml ofbuffer A and then resuspended in 100 μl of buffer A containing 0.1% Nonidet P-40. The suspension was rocked for 30 min and then centrifuged at 200,000 g for 30 min, all at 4°C. The resulting supernatant was designated as the membrane fraction. PKC activities were determined in both cytosolic and membrane fractions by measuring the transfer of 32P from [γ-32P]ATP to histone III-S. The reaction mixture (60 μl) contained the enzymatic source (20 μl), 24 μg histone III-S, 10 mM magnesium acetate, 50 μM [γ-32P]ATP (200–400 counts · min−1 · pmol−1) and 20 mM Tris · HCl, pH 7.5, in the presence of either 1 mM EGTA or 0.5 mM CaCl2, 3.5 μg phosphatidylserine, and 0.7 μg 1-oleoyl-2-acetyl-rac-glycerol. After incubation at 30°C for 4 min, the reaction was stopped by spotting 50 μl of the reaction mixture onto phosphocellulose P81 Whatman chromatography papers (2 × 2 cm) and immediately placing them in 75 mM ice-cold phosphoric acid. The papers were washed thrice in the same solution, dried, and counted in an LKB scintillation counter after addition of 5 ml Biogreen. PKC activity was calculated by subtracting the incorporation of phosphate in the absence of Ca2+ and PL from that in the presence of these effectors. One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the transfer of 1 pmol of phosphate from ATP to histone per min at 30°C. Protein was determined by the Bradford method (4a), with BSA as standard, to calculate PKC specific activity.
Effect of FA on basal and insulin-stimulated glucose uptake.
2-DG uptake was determined in myotubes preincubated with 0.25 mM FA, either saturated (palmitate and stearate) or monounsaturated (oleate) (Fig. 1). Compared with that in FA-deprived cells, basal glucose uptake was higher (∼20%) in cells supplemented with both saturated FA, whereas no modification was noted with oleate supplementation. Insulin increased 2-DG uptake by 20% in myotubes incubated in the absence of FA, as well as in cells treated with oleate. On the contrary, no effect of insulin was observed in cells preincubated with saturated FA, either palmitate or stearate. Incubation of cells with a mixture of 1:1 of palmitate and oleate led to basal or insulin-stimulated glucose uptake similar to that of control or oleate-treated cells.
FA and acetate incorporation into lipid pools.
Because saturated and unsaturated FA appeared to affect basal and insulin-stimulated glucose uptake differently, the metabolic fate of these FA was studied. Incorporation of 14C-FA into the muscle cell lipids was analyzed by TLC after supplementation with a maximal concentration of 1 mM [1-14C]palmitic acid, -stearic acid, -oleic acid, or -linoleic acid in the absence or presence of glucose. In the absence of glucose, the major part of total FA incorporated into cell lipids [palmitic acid (220 ± 25 pmol/mg protein), stearic acid (141 ± 9 pmol/mg protein), oleic acid (175 ± 19 pmol/mg protein) and linoleic acid (103 ± 8 pmol/mg protein)] was found in the PL fraction (>70% of radioactivity), whereas 1,2-DAG and TAG labeling represented a low percentage (Table 1). Supplementation with glucose, as a source of glycerol 3-phosphate for FA esterification, caused a marked rise in the total incorporation of each FA in muscle cell lipids: palmitic acid (2.3-fold), stearic acid (1.9-fold), oleic acid (2.0-fold), or linoleic acid (2.2-fold). However, in cells incubated with saturated FA, the maximal net increase was found in 1,2-DAG labeling (30-fold and 10-fold for palmitic and stearic acids, respectively), with minor changes in TAG (5- and 2.5-fold for palmitic and stearic acids, respectively) or PL (∼2-fold). In contrast, with unsaturated FA, [1-14C]oleic or [1-14C]linoleic acid, the highest increase (∼8-fold) was in TAG labeling, which reached a percentage of ∼40% (Table 1), but with minor change in the incorporation of these FA into 1,2-DAG (∼2-fold) or PL (1.3-fold). These results indicate that, when glucose is available, unsaturated FA are readily used for TAG synthesis, whereas saturated FA accumulated mainly as 1,2-DAG. The capacity of muscle cells to synthesize lipids de novo was assessed by measuring [1-14C]acetate incorporation into lipid fractions. The majority of the radiolabeled acetate was found to be incorporated into PL, whereas <5% was associated with either the DAG or TAG fractions, irrespective of the presence or absence of glucose (Table 1), confirming that muscle cells have a limited lipogenic activity.
Because the saturated FA seemed to be poorly incorporated into TAG when provided alone, the fate of 0.25 mM [1-14C]palmitic acid mixed with oleate at varying concentrations up to 0.25 mM was evaluated (Table2). It was found that the total incorporation of [1-14C]palmitic was relatively constant in all conditions tested (data not shown). The highest proportion of radioactivity incorporation into DAG, and the lowest into TAG, occurred when palmitic acid was provided alone. Nevertheless, the accumulation of DAG reached with 0.25 mM palmitate was substantially lower than that detected with 1 mM palmitate, suggesting a dose-response effect. When a mixture of [1-14C]palmitate and unlabeled oleate was applied to the cells, FA incorporation in DAG decreased, whereas TAG labeling progressively increased in parallel with oleate concentration. These data indicated that unsaturated FA facilitate the diversion of saturated FA from DAG to TAG.
Dependence of TAG accumulation on FA degree of unsaturation and concentration.
The importance of FA degree of unsaturation for net TAG accumulation was further studied by measuring intracellular TAG content by use of the lipase method (9). No increase in the content of TAG was observed after supplementation with the medium-chain-length FA octanoate (C8:0) (46.3 ± 7.1 μg/mg protein) compared with that of FA-deprived cells (45.7 ± 8.5 μg/mg protein). In contrast, the concentration of TAG was raised after addition of 0.5 mM long-chain FA, according to the series: oleate C18:1(n-9), (506 ± 24 μg/mg protein) > linoleate, C18:2(n-6) (408 ± 42 μg/mg protein) > stearate, C18:0 (274 ± 36 μg/mg protein) > palmitate C16:0 (152 ± 7 μg/mg protein). To study the dependence of TAG synthesis on FA concentration (Fig.2), cells were incubated with varying palmitate or oleate concentrations at a fixed molar FA-to-BSA ratio (5:1). Accumulation of TAG, from both oleate and palmitate, was dose dependent, and maximal increases were observed with FA concentrations ranging from 0 to 0.5 mM. A saturation tendency was shown at higher concentrations up to 2 mM. Again, marked differences between oleate and palmitate were found along the range of concentrations tested.
Further evidence for the FA type dependence of TAG accumulation was provided by microscopic analysis of lipid droplets after histochemical staining with Sudan III (Fig. 3). No lipid droplets could be detected in cells incubated in the absence of FA (Fig. 3 A) or in the presence of octanoate, C8:0 (Fig.3 B), whereas barely visible droplets were observed after incubation with 0.5 mM saturated long-chain FA palmitate C16:0 and stearate C18:0 (Fig. 3, C and D). In contrast, cells incubated with oleate C18:1(n-9) or linoleate C18:2(n-6) showed clearly visible lipid droplets (Fig. 3, E and F).
Analysis of FA composition of TAG and PL fractions.
The FA composition of the PL and TAG in myotubes was assessed by gas chromatography (Table 3). In cells incubated in FA-deprived medium, the predominant FA in PL was C18:1(n-9) (33%), followed by C16:0, C18:0, and C20:4(n-6), with relative contributions of 19, 16, and 8%, respectively. Addition of 0.05 mM palmitic acid to the medium slightly raised the proportion of C16:0 (up to 24%). Similarly, incubation with 0.05 mM oleic acid modestly increased its relative content up to 38%. In contrast, cells supplemented with 0.05 mM linoleic acid showed a 500% increase in the proportion of C18:2(n-6). The total FA content in PL was similarly increased after incubation with all exogenous FA tested, i.e., palmitic (1.3-fold), oleic (1.8-fold), and linoleic acid (1.6-fold), compared with that in FA-deprived cells (402 ± 29 nmol/mg protein). These results show that supplementation with NEFA did not qualitatively influence the FA composition of PL, whereas the essential FA linoleic acid was strongly incorporated as such.
The FA content in the TAG fraction (7.35 ± 0.5 nmol/mg protein) was very low compared with that in PL as assessed in cells deprived of FA (Table 3). In these cells, the most abundant FA were C16:0 and C18:1(n-9), which each represented ∼35% of the total. The major differences between the FA profile in the lipid pools were the lower contents of PUFA, particularly C18:2(n-6), in PL than in TAG. Addition of 0.05 mM palmitic, oleic, or linoleic acid, caused a 2.5-, 7-, or 5-fold increase, respectively, in the total FA content of TAG compared with FA-deprived cells. These data corroborate an enhanced incorporation of unsaturated compared with saturated FA into TAG. The individual FA profile in TAG, in contrast to PL, was clearly affected by the provision of FA. In cells incubated with C16:0, C18:1(n-9), or C18:2(n-6), the proportion of every FA increased up to ∼50%. In cells supplemented with linoleic acid, the content of C18:3(n-6) and C20:3(n-6) increased, suggesting the presence of delta-6 desaturase activity.
Effect of FA on translocation and activity of PKC.
Because it was found that saturated FA specifically promoted the chronic accumulation of DAG, which could translocate and activate PKC, we next analyzed the effect of palmitate and oleate on the subcellular distribution of PKC activity (Fig. 4). In control cells incubated in the absence of FA, the major part of the calcium and lipid-dependent phosphorylation activity was found in the cytosolic fraction. PKC activity distribution was not modified by incubation with oleate. In contrast, in cells incubated with palmitate, there was a twofold increase in the PKC activity present in the membrane fraction at the expense of the activity in the cytosolic fraction, which was decreased. These data indicated that palmitate specifically promoted persistent translocation of PKC activity to the muscle cell membrane.
In this work, the impact of FA accumulation on glucose uptake in human skeletal muscle cells was investigated. It is shown that, whereas incubation of cells with oleate had no impact on basal or insulin-stimulated glucose uptake, cells preincubated with saturated FA, palmitate, or stearate exhibited increased basal glucose uptake and impaired response to insulin. Likewise, exposure of rat adipocytes to saturated FA was shown to specifically stimulate glucose transport and induce insulin resistance (12).
To understand the differential effects of FA in human muscle cells, their utilization and incorporation into different lipid pools were analyzed. In the presence of glucose, differences in lipid accumulation were noted between saturated and unsaturated FA. For saturated FA, C16:0 and C18:0, the highest increase in incorporation, which was dose dependent, was associated with the 1,2-DAG fraction, whereas unsaturated FA, C18:1(n-9) and C18:2(n-6), were found to be diverted mainly toward TAG, with minor increases in the DAG pool. Further evidence for this metabolic difference came from enzymatic determination of muscle cell TAG content. Varying TAG levels were observed after incubation with several long-chain FA types, so that they were higher in cells provided with unsaturated compared with saturated FA. Moreover, supplementation with unsaturated FA was correlated with the appearance of lipid droplets throughout the cytoplasm, whereas in cells incubated with saturated FA, lipid droplets were only faintly visible. One possible explanation for this preferential usage of unsaturated FA for TAG synthesis and accumulation of saturated FA as DAG might be the affinity of acyl-CoA:diacylglycerol acyltransferase (DGAT), the enzyme that catalyzes the final conversion of DAG and acyl-CoA to TAG, for different DAG. It has been previously shown that DGAT isolated from fat cells had maximal activity with diolein and minimal saturable activity when dipalmitin was used as substrate (7). Likewise, DGAT from L6 rat skeletal myotubes (23) was found to have optimal activity in vitro when long-chain unsaturated acyl-CoAs were used as substrates. As an attempt to evaluate this hypothesis, the fate of palmitate was examined when it was added to the muscle cells as a mixture with the unsaturated FA oleate (from 1:0 to 1:1). It was observed that the incorporation of palmitate into DAG progressively declined, whereas that in TAG rose, in parallel with oleate concentration. Thus the presence of oleate was able to divert palmitate incorporation from DAG into TAG. When extrapolating these data to an in vivo setting, it is suggested that the amount of saturated FA accumulated as DAG in skeletal muscle may vary depending on the relative FA composition of serum lipids as well as total FA concentration.
It was also investigated whether differential effects of palmitate and oleate on glucose uptake could be related to changes in the FA profile of TAG or PL. The analysis of individual FA content in TAG by gas chromatography confirmed that unsaturated FA were more readily incorporated into this lipid fraction at a concentration as low as 0.05 mM. Nevertheless, the relative FA composition of TAG was evenly affected by supplementation with palmitic, oleic, or linoleic acids, so that the proportion of the added FA represented up to 50% of the total FA. Palmitic, oleic, and linoleic acids were incorporated with similar efficiency into PL, whereas the relative FA composition was poorly affected by the FA tested except by the essential PUFA linoleic acid, which markedly changed the profile. In relation to that, the PUFA content in PL was much higher than in TAG (20% in PL compared with 3% in TAG), even in cells deprived of FA. Overall, these data suggested that the FA composition of PL is highly preserved and depends on endogenous FA metabolism rather than exogenous FA uptake, except for the essential FA. Our results are consistent with those analyzing the impact of dietary fat on the muscle lipid profile, because dietary fat composition has been shown to affect the FA composition of PL in a tightly controlled manner while overtly modifying the FA profile in TAG (17, 19). Thus no specific changes in the FA profile of either TAG or PL were found by provision of palmitate or oleate.
Therefore, these data support the conclusion that the effects of saturated FA, palmitate and stearate, on glucose uptake and insulin sensitivity could be associated with their preferential accumulation as DAG. Consistent with that, when palmitate was provided together with oleate (in proportion 1:1), a condition during which palmitate was diverted from DAG to TAG, basal glucose uptake was not increased, and insulin response was not impaired. In this same line of evidence, it was shown that the insulin resistance that was specifically induced by a saturated fat diet (13, 28) could be prevented by the inclusion of the PUFA linolenate (28). In this study, the FA profile in PL was analyzed, and the PUFA content was found to be increased; therefore, it was inferred that the improvement in insulin response was due to this modification. The alteration of PL profile, namely the rise in PUFA, was also observed in the present study after incubation with the essential FA linoleate. It is shown here that the reversion of palmitate-induced insulin insensitivity is also accomplished by inclusion of the nonessential unsaturated FA oleate. Oleate does not modify the FA profile in PL but rather redirects palmitate metabolism from DAG to TAG. Thus these experiments clearly demonstrate that the recovery of insulin sensitivity may occur by reducing DAG accumulation without modifying the FA profile in PL. The mechanism by which increased DAG content may interfere with insulin sensitivity could be mediated by PKC activity. We show that incubation with palmitate, together with the accumulation of DAG, causes chronic translocation of PKC activity from the cytosolic to the membrane fractions of human muscle cells, whereas oleate, which does not raise DAG, does not modify PKC cellular distribution. PKC appears to be a major signal in insulin-stimulated glucose transport (5a, 14). Moreover, elevated expression of DAG-sensitive PKC delta (5) or chronic increase in DAG content (26) is known to cause activation of basal glucose transport and to impair further increase by insulin stimulation (26). In addition, the increase in DAG content associated with persistent translocative membrane activation of several PKC isoforms has been proposed to be the cause of the insulin resistance in obese Zucker and GK diabetic rat models (2). In this context, our data show that activation of the DAG-PKC mechanism may underlie the impairment in insulin sensitivity caused by an increased lipid availability and that this mechanism is promoted specifically by saturated FA. Nevertheless, we cannot rule out the possibility that saturated FA metabolism may also lead to the accumulation of other second messenger molecules, such as ceramide, as previously shown (24). Ceramide could be acting on DAG-insensitive PKC zeta (16), as well as on other kinases such as protein kinase B (24), involved in the insulin signal on glucose transport (5, 29). In this sense, Schmitz-Peiffer et al. (24) demonstrated that palmitate reduced insulin stimulation of glycogen synthase kinase 3 and protein kinase B, whereas it did not modify tyrosine phosphorylation, p85 association, or phosphatidylinositol 3-kinase activity in IRS-1 immunoprecipitates. Further work will be necessary to elucidate the precise contribution of the different second messengers, and the corresponding lipid-sensitive kinases, on the effect of saturated FA metabolism in insulin-stimulated glucose uptake.
Another conclusion implicit in our data is that the accumulation of TAG in this muscle cell model does not modify insulin sensitivity, because incubation with unsaturated FA, which elevated TAG, did not alter basal or insulin-stimulated glucose uptake. Nevertheless, this does not rule out the possibility that an increased TAG content, as a source of oxidizable FA, may interfere with glucose oxidation and consumption at other control steps. In this sense, it is well known that situations favoring FA oxidation decrease glucose usage because of increased acetyl-CoA levels, as described by Randle et al. (22). In summary, our results suggest that saturated FA may specifically induce a desensitization to the insulin stimulation of glucose uptake. Whereas our data do not support the contention that this effect is due to a modification in the FA profile of PL or TAG accumulation, it suggests that it is related to a rise in DAG. Thus the data provide a molecular link between an increased availability of saturated FA and the establishment of insulin resistance in skeletal muscle.
We gratefully acknowledge Irene Zbinden and Alexandra Arias for technical assistance, and Prof. J. B. German for critical reading of the manuscript. We thank Dr. Sancho Navarro (Hospital de Sant Pau i de la Santa Creu, Barcelona, Spain) for assistance with the muscle culture.
E. Montell was the recipient of a fellowship Formación Personal Investigador from the Ministerio de Educación y Cultura (Spain).
Address for reprint requests and other correspondence: A. M. Gómez-Foix, Departament de Bioquı́mica i Biologia Molecular, Universitat de Barcelona, Martı́ i Franqués 1, 08028-Barcelona, Spain (E-mail:).
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- Copyright © 2001 the American Physiological Society