Long-chain acyl-CoAs (LCACoA) are an activated lipid species that are key metabolites in lipid metabolism; they also have a role in the regulation of other cellular processes. However, few studies have linked LCACoA content in rat and human muscle to changes in nutritional status and insulin action. Fasting rats for 18 h significantly elevated the three major LCACoA species in muscle (P < 0.001), whereas high-fat feeding of rats with a safflower oil (18:2) diet produced insulin resistance and increased total LCACoA content (P < 0.0001) by specifically increasing 18:2-CoA. The LCACoA content of red muscle from rats (4–8 nmol/g) was 4- to 10-fold higher than adipose tissue (0.4–0.9 nmol/g,P < 0.001), suggesting that any contamination of muscle samples with adipocytes would contribute little to the LCACoA content of muscle. In humans, the LCACoA content of muscle correlated significantly with a measure of whole body insulin action in 17 male subjects (r 2 = 0.34, P = 0.01), supporting a link between muscle lipid metabolism and insulin action. These results demonstrate that the LCACoA pool reflects lipid metabolism and nutritional state in muscle. We conclude that the LCACoA content of muscle provides a direct index of intracellular lipid metabolism and its links to insulin action, which, unlike triglyceride content, is not subject to contamination by closely associated adipose tissue.
- adipose tissue
- insulin action
high-fat feeding of ratsfor 3 wk results in accumulation of triglyceride (TG) and significant insulin resistance in skeletal muscle (12). A significant negative relationship between insulin action and skeletal muscle-associated TG has also been described in human subjects (23, 22). However, there is continuing controversy as to whether changes in the TG content of muscle samples reflect alterations in intramyocellular TG or result from an increased amount of closely associated adipose tissue (33,22). It is also unclear how increased TG content affects insulin action in muscle, although it has been suggested that increased TG content indicates an increased availability of fatty acid or fatty acid derivatives that interfere with insulin signaling and glucose metabolism. Recent studies have shown a strong correlation between insulin action and the tissue content of long-chain acyl-CoA (LCACoA) in rat skeletal muscle (20, 21). LCACoAs are intermediates in lipid biosynthesis and fatty acid oxidation and may play a role as regulatory ligands both for key enzymes associated with glucose metabolism (3, 6) and for nuclear transcription factors regulating gene expression (9). Acute changes in tissue lipid metabolism are also likely to be better reflected by changes in the content of this metabolically available form of fatty acid than by changes in TG content, which is the storage form of lipids. However, the extent to which the measurement of LCACoA in muscle might be contaminated by the LCACoA content of infiltrating white adipose tissue has not been investigated.
Furthermore, little is known about the LCACoA content of human muscle and whether these lipid species are a useful indicator of lipid metabolism and insulin action in human muscle. Total LCACoA content has been determined indirectly by measurements both of enzyme activity (1) and of coenzyme A formation after extraction and alkaline hydrolysis of the LCACoA esters (5,29). Alternately, HPLC allows direct quantitation of individual fatty acyl-CoA species (18, 25,36). The aims of this study were 1) to assess the LCACoA content of individual muscle and adipose tissue samples from rats and humans using HPLC and 2) to determine whether the LCACoA content of muscle reflected changes in lipid metabolism induced in rats by high-fat feeding, fasting, and insulin administration.
All surgical and experimental procedures performed were approved by the Garvan Institute/St. Vincent's Hospital Animal Experimentation Ethics Committee and were in accordance with the National Health and Medical Research Council of Australia guidelines on animal experimentation. Male Wistar rats (300–350 g) were maintained in a temperature-controlled room (22°C) with a 12:12-h light-dark cycle (lights on at 0600) and, unless otherwise stated, had free access to rodent chow diet and water.
Manipulation of animals.
Rats were randomly assigned to either a high-fat diet for 3 wk as previously described (21), overnight fasting, or insulin stimulation via a euglycemic-hyperinsulinemic clamp with radioactive tracer administration to assess glucose uptake in vivo (12). The high-fat diet was provided as a fixed daily ration of 59% fat, 20% carbohydrate, and 21% protein (on a calorie basis). The fat content of the diet was supplied as safflower oil, of which the major constituents were linoleic (70%) and oleic acid (20%) (27). This ration contained approximately the same caloric content as the average daily energy intake of the chow diet-fed animals (350 kJ/day). The macronutrient composition of the chow diet, expressed as a percentage of total calories, was 3.5% fat, 76.5% carbohydrate, and 20% protein. Animals were euthanized with a lethal dose of pentobarbitone (60 mg iv), and the red and white quadriceps muscle, red gastrocnemius muscle, liver, and heart tissues were rapidly collected and freeze clamped. Epididymal and inguinal fat depots were also collected and immersed in liquid N2. All tissues were stored at −70°C for subsequent lipid and enzymatic assays.
Blood and plasma glucose levels were measured with a glucose analyzer (YSI, Yellow Springs, OH). Plasma nonesterified fatty acids (NEFA) were measured with a colorimetric assay method (Wako Chemical, Osaka, Japan). Total neutral lipid was extracted from ∼30 mg tissue in CHCl3-MeOH (2:1), and 0.6% NaCl (wt/vol) was added to separate the phases. The organic extracts were dried down, reconstituted in ethanol, and assayed for triglycerides by measuring the glycerol liberated after enzymatic hydrolysis of triglycerides (GPO-PAP kit, Boehringer Mannheim, Mannheim, Germany).
Determination of individual long-chain fatty acids in the triglyceride fraction of skeletal muscle.
An aliquot (100 μl) of the reconstituted muscle lipid extract used for measurement of triglycerides was hydrolyzed, and the fatty acids were converted to LCACoA derivatives with the use of reagent A from the NEFA assay kit (Wako Chemical). The LCACoAs were extracted, purified, and quantitated using the method for measurement of LCACoA described below. The proportion of each of palmitoyl (16:0), palmitoleoyl (16:1), linolenoyl (18:3), linoleoyl (18:2), and oleoyl (18:1) long-chain fatty acids in muscle TG was calculated as a percentage of the total.
Tissue LCACoA measurement.
LCACoAs were quantitated by a combination of methods previously described by Corkey (3) and Mancha et al. (17). These methods involved extraction and purification of LCACoA from tissues and quantitation by HPLC. Muscle, liver or heart (250 mg), or adipose tissue (1,000 mg) was dismembered and homogeneously suspended in isopropanol with 50 mM KH2PO4 (pH 7.2). A known amount of lauroyl-CoA was added to act as an internal standard for the extraction and detection procedures, and the homogenate was acidified with glacial acetic acid (12.5 μl/ml of extract). The suspension was extracted with petroleum ether saturated with 50% aqueous isopropanol to remove free fatty acids and less polar lipids. Other lipids, including LCACoA, were extracted from the aqueous layer with saturated (NH4)2SO4 (25 μl/ml of extract) and CHCl3-MeOH (1:2 vol/vol) followed by further CHCl3-MeOH (1:2) extraction. The resulting two supernatants were combined and lyophilized overnight and then reconstituted in 1 ml 50 mM KH2PO4 (pH 5.3). Extracts were loaded onto C18 Novapak columns (Waters, Milford, MA) that had been activated with 5 ml of MeOH followed by 5 ml of Milli-Q H2O and then 5 ml 50 mM KH2PO4 (pH 5.3). Each column was washed with 5 ml of 50 mM KH2PO4 (pH 5.3)-acetonitrile (ACN) (80:20), and LCACoAs were eluted with 5 ml of 50 mM KH2PO4 (pH 5.3)-ACN (30:70) followed by 2 ml MeOH-50 mM KH2PO4 (pH 5.3; 3:1). A Nova-Pak C18 4-μm reverse-phase column (3.9 × 100 mm) and a 24-min gradient of 40–62% ACN with 25 mM KH2PO4 (pH 5.3) buffer was used to separate LCACoAs. Individual LCACoA peaks were detected at 254 nm by means of photodiode array detection (Waters) and quantitated by comparing sample peak area with that of a mixture of LCACoA standards. The concentrations of each separate LCACoA standard were determined spectrophotometrically at 254 nm with a millimolar extinction coefficient of 15.4. The LCACoA content was calculated as the sum of the major CoA species, namely palmitoyl (16:0), linoleoyl (18:2), and oleoyl (18:1). The amount of individual CoA species from each sample was corrected for loss during extraction and analysis by adjusting for the recovery of the internal standard (lauroyl-CoA) in each individual sample. Final recoveries of the lauroyl-CoA standard with this method in muscle, liver, heart, and white adipose tissues were high and reproducible (68 ± 1%, n = 59).
Acyl-CoA synthase activity.
Acyl-CoA synthase (ACS) activity was measured according to the method of Shimomura et al. (26). Liver, heart, muscle, and inguinal adipose tissues (∼50 mg) were hand-homogenized in 10–40 vol of buffer containing 300 mM Tris · HCl and 150 mM MgCl2, pH 7.4. Inguinal tissue homogenates were centrifuged at 1,000 rpm, and the supernatant was removed for the assay. Homogenates were preincubated for 5 min at 37°C. Reaction buffer, consisting of 300 mM Tris·HCl and 150 mM MgCl2 (pH 7.4), Triton X-100, 200 μM [1-14C]palmitate (0.01 μCi/assay), 20 mM ATP, 2.25 mM glutathione, and 0.2 mM CoASH, was added to each sample, and the reaction was terminated after 2, 5, or 10 min by adding 1 ml of Dole's reagent (40 isopropanol-10 hexane-1 0.5 M H2SO4). Water (0.35 ml) and hexane (0.6 ml) were added to achieve phase separation, and after vortexing, the upper layer was removed, and the lower layer was washed twice with 0.6 ml of hexane. The radioactivity in 0.7 ml of the lower layer was counted. ACS activity was expressed as palmitoyl-[14C]CoA formed per minute per gram of wet weight tissue.
Seventeen men undergoing elective knee surgery volunteered for the study after all procedures were explained, and written informed consent was obtained from each subject. The protocol was approved by the St. Vincent's Hospital Human Research Ethics Committee. The age of the subjects ranged from 56 to 75 yr, (median 68 yr), and the body mass index ranged from 24 to 38 kg/m2 (median 29 kg/m2). All subjects were ambulatory but sedentary, as assessed by the Framingham activity questionnaire (8). Of the 17 subjects, eight were of normal glucose tolerance, six had impaired fasting plasma glucose (>6.1 mM) and three were classified as type 2 diabetics. Two of the diabetic subjects were treated with oral hypoglycemic agents and one was treated with insulin (52 U/day). These medications were omitted 24 h before the euglycemic-hyperinsulinemic clamp and before surgery. Four weeks before surgery, insulin sensitivity, defined as glucose infusion rate (GIR) per kilogram of lean mass, was determined with a 150-min hyperinsulinemic- (0.05 U · kg−1 · h−1) euglycemic (5 mM) clamp. Whole body insulin action was calculated as the clamp plateau GIR normalized for fat-free mass (determined by dual-energy X-ray absorptiometry) and the increase above basal in plasma insulin during the clamp.
A 1-cm3 area of the vastus lateralis muscle was biopsied from each subject at the beginning of surgery, before any compression or ischemia, for the determination of muscle TG and LCACoA content. Muscle samples were immediately frozen in liquid N2 and stored at −70°C for subsequent lipid analysis. Fat tissue (∼1 g) lying proximal to the biopsy site was removed from each of four subjects, immersed in liquid N2, and stored at −70°C. Muscle and adipose tissue triglyceride and LCACoA contents were then measured as described above for rat tissues. Possible relationships between insulin sensitivity and muscle lipid content were investigated by correlating whole body insulin action against measures of muscle LCACoA and triglyceride.
Data were analyzed by one- or two-way ANOVA, or linear regression where appropriate, by use of the commercial software package StatView (Abacus Concepts/Brainpower, Berkeley, CA). All results are expressed as means ± SE. P < 0.05 was considered statistically significant.
Fatty acid content of triglyceride in rat muscle.
Palmitic acid (16:0) was the most abundant fatty acid in the TG fraction of rat skeletal muscle from chow-fed rats (49.4 ± 1.4% of total; Table 1). Oleate (18:1) and linoleate (18:2) acids were the next major fatty acids in muscle TG, whereas linolenate (18:3) and palmitoleate (16:1) each contributed <5% to the total fatty acid content of TG. The proportion of 18:2 in TG from muscle of rats fed a high-fat diet, of which the predominant component was linoleate (18:2), was almost three times higher (33.0 ± 3.1%) than that of muscle from chow-fed rats (12.5 ± 0.6%; Table 1).
Intertissue differences in total LCACoA vs. TG content.
Basal levels of LCACoA in heart (7.4 ± 0.2 nmol/g) and liver (7.7 ± 0.4 nmol/g) of chow-fed rats were both significantly higher than in red muscle, white muscle, and fat tissues (P < 0.001; Table 2). The total LCACoA content of red muscle (4.1–4.4 nmol/g) was two- to threefold that of white quadriceps muscle (P < 0.05) and sixfold higher than that of epididymal and inguinal adipose tissues (P < 0.001), where LCACoA levels were <1 nmol/g. The TG contents of liver, heart, red quadriceps, red gastrocnemius, and white quadriceps muscle from chow-fed rats ranged between 2.0 and 6.9 μmol/g and were all significantly lower (P < 0.001; Table 2) than in white adipose tissue, which was >70% (wt/wt) TG.
Effect of high-fat feeding, fasting, and insulin on muscle LCACoA content.
Rats fed a high-fat diet containing 59% of calories as fat, of which 70% was linoleate (18:2), had significantly increased levels of LCACoA esters in liver and red muscle compared with chow-fed controls (P < 0.02; Table 2). The 93% increase in total LCACoA levels of red gastrocnemius muscle was caused mainly by a fourfold increase in the content of linoleoyl-CoA (Fig.1). High-fat feeding also increased the TG content (Table 2) and the maximal activity of acyl-CoA synthase in liver and muscle tissues (Fig. 2). Rats fasted for 18 h had significantly higher total LCACoA levels in the red gastrocnemius muscle compared with fed rats (P= 0.001), which resulted from an increase in all major LCACoA species (16:0-, 18:1-, and 18:2-CoAs) (P < 0.01; Fig.3). Chow-fed rats infused with insulin during the euglycemic clamp had significantly lower muscle LCACoA levels than basal animals (3.4 ± 0.2 and 4.4 ± 0.4 nmol/g, respectively, P < 0.05). Insulin stimulation also suppressed the total LCACoA content in muscle of fat-fed rats from 8.3 ± 1.3 to 5.9 ± 0.1 nmol/g (P < 0.001). However, the LCACoA level after insulin stimulation in fat-fed rats was still higher than that in basal or insulin-stimulated rat muscle from control animals. The higher level of LCACoA in muscle of fat-fed rats in the basal and insulin-stimulated states was associated with a decreased ability of insulin to stimulate glucose uptake into red gastrocnemius muscle (control glucose metabolic index 25.7 ± 1.7 μmol · 100 g−1 · min−1; fat-fed glucose metabolic index 17.5 ± 1.8 μmol · 100 g−1 · min−1,P = 0.006).
Fatty acid content of triglyceride in muscle.
In the human vastus lateralis muscle biopsies, the proportions of fatty acids in the TG fraction of human skeletal muscle were similar to those of chow-fed rats, with palmitate (16:0), oleate (18:1), and linoleate (18:2) acids contributing almost 90% of the total fatty acid content (Table 1).
Total LCACoA vs. triglyceride content in muscle and fat.
In agreement with the results in rat tissues (Table 2), the samples of human vastus lateralis muscle contained a significantly higher level of LCACoA than human adipose tissue (Table3). The variation in LCACoA content in the human muscle samples ranged between 2.2 and 6.7 nmol/g muscle, whereas the measured TG content of the same muscle samples ranged from 6.6 to 91.4 μmol/g muscle. This indicates that some samples were probably contaminated with adipose tissue. The GIR during a hyperinsulinemic euglycemic clamp in these subjects varied between 1.8 and 10.2 mg · min−1 · kg lean mass−1. A significant relationship (r 2 = 0.34, P = 0.01) was observed between whole body insulin action (adjusted for incremental increase in clamp plasma insulin) and muscle LCACoA (Fig.4). In contrast, there was no significant correlation (r 2 = 0.03, P = 0.5) observed between GIR and muscle TG in this group of subjects (data not shown).
The HPLC technique used in this study was sufficiently sensitive to detect differences in LCACoA content of tissues with different capacities for fatty acid utilization (e.g., muscle, heart, liver). More importantly, differences could be detected in muscle with only 150–250 mg of tissue, thus overcoming the need to pool tissue samples, as has been described in other studies (19,25). The low levels of LCACoA detected in white adipose tissue from both rats and humans, as well as the LCACoA content of human muscle, had not previously been documented. A major concern of studies relating intracellular fat availability with insulin action in muscle tissue has been determining the extent to which adipocytes between muscle fibers contribute to the lipid content measured in a muscle biopsy sample. Because adipose tissue is at least 70% TG (wt/wt), <1% contamination of a muscle sample with adipose tissue could account for all the TG content normally attributed to muscle. However, by measuring LCACoA content, a more accurate index of intramuscular (myocyte) lipid metabolism is obtained, because a 1% contamination of muscle sample with adipocytes, if present, would result in a negligible (<0.5%) increase in sample LCACoA content.
The present study shows that, by measuring LCACoA content of muscle samples, intracellular lipid metabolism can be determined with little risk of significant contamination from closely associated adipose tissue, making it a more robust measure of cellular lipid metabolism than TG content. Because LCACoA species have been shown to interact with insulin signaling pathways and glucose metabolism by modulating enzyme activity (35,31) and gene transcription (9), measurement of LCACoA has potential importance, because these metabolites may provide a mechanistic link between increased lipid availability and impaired glucose utilization in tissues.
Although the values of LCACoAs in rat tissue have been reported to vary by up to 50-fold, depending on tissue type and metabolic state (see Ref. 6 for review), the data in this study are consistent with the increases in LCACoA content of red muscle and liver previously reported for rats fed a high-fat diet for 10 wk (2). The increased activity of acyl-CoA synthase in the liver, heart, and muscle of fat-fed rats suggests that the induction of this enzyme is an important factor in the increase in the LCACoA pool, which in turn could lead to increased TG storage and β-oxidation. The fourfold increase in the content of linoleoyl-CoA in muscle with high-fat feeding reflected dietary fat composition, because ∼70% of the safflower oil used for the diet is in the form of linoleic acid. The fatty acid compositions of muscle phospholipid (28) and adipose tissue (34) have also been shown to reflect the composition of the diet. It is therefore possible that the specific change in an individual LCACoA level is relevant to the mechanism by which the high-fat diet used in this study produces muscle insulin resistance (27). The observation that total LCACoA levels correlate with insulin-stimulated glucose uptake in muscle (20) and are elevated in muscle of insulin-resistant 4-day glucose-infused rats (16) provides additional evidence that an increase in this intracellular pool of lipid may have a role in the manifestation of muscle insulin resistance. Significantly increased levels of LCACoA in red muscle from rats after 18-h fasting have also been documented in liver and heart tissue (32, 36). This increase likely reflects the upregulation of fatty acid utilization due to increased fatty acid supply from systemic and intracellular sources. Fasting led to an increase in several 16–18 carbon LCACoA species, which reflected the composition of TG in the tissue. There was, therefore, no difference in the ratio of the different LCACoA species, as was the case with high-fat feeding.
The current study shows that correlations between LCACoA levels and insulin resistance previously found in rodents (20,21) are likely to be relevant to humans. We demonstrate that measurement of the LCACoA content in human muscle samples is possible and that LCACoA content in muscle correlates with the ability of insulin to stimulate whole body glucose turnover in the same subjects when insulin levels are raised by means of a euglycemic-hyperinsulinemic clamp. Although a significant association between human muscle TG content and measures of insulin sensitivity has been obtained with larger numbers of subjects (22,23), the problems have been raised of erroneous results caused by possible contamination of the biopsy samples with adipose tissue (33). The measurement of LCACoA in biopsy samples may overcome this problem, although a new methodology for measuring intramuscular TG may also help. Noninvasive methods for directly determining intramyocellular TG, such as computed tomography and nuclear magnetic resonance spectroscopy (NMRS), are now becoming available (11, 14, 15). Although computed tomography has shown a correlation in obese subjects between reduced muscle attenuation and insulin resistance (11), the measure is an indirect estimation of muscle lipid content. NMRS is a relatively sensitive and precise method that can distinguish between intra- and extramyocellular TG by resolving different resonances from methylene and methyl protons of triglyceride acyl chains, depending on the immediate intracellular environment of the lipid. Although these methods can give an estimation of intramyocellular TG content, one can only presume that increased TG content reflects increased levels of lipid metabolism and metabolites. The measurement of LCACoA in muscle may therefore provide a more useful measure of lipid metabolism than TG content, because the LCACoAs themselves may influence insulin action through mechanisms other than substrate competition via the classic glucose-fatty acid cycle (10, 24). Possible mechanisms include a role for LCACoA in modulating protein kinase C isozyme and other kinase activity (6) and in regulating glycogen synthase (35), glucokinase (30, 31), and gene transcription (9).
In summary, LCACoA content can be used as an index of intracellular lipid metabolism in muscle that is unlikely to be influenced by contamination from closely associated adipose tissue. Changes in LCACoA content parallel changes in lipid metabolism, such as those that occur during fasting, insulin stimulation, or feeding a high-fat diet in rats. In human muscle, the measurement of LCACoA provides a similar indicator of lipid metabolism that is inversely correlated with insulin action and may act as a better indicator of lipid metabolism and glucose utilization than measurements of TG content.
The authors gratefully acknowledge the expertise and assistance of the Staff of the Biological Testing Facility at the Garvan Institute.
This study was supported by a Block Grant to the Garvan Institute by the National Health and Medical Research Council of Australia.
Address for reprint requests and other correspondence: B. Ellis, Garvan Inst. of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010 Australia (E-mail:).
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