Introduction

Adipose tissue is the major site in vivo for free fatty acid (FFA)¹ storage and release. The tissue responds to varying nutritional and hormonal conditions. Adipocytes also contain functional mitochondrial machinery that oxidizes FFA to CO₂ (1). The partitioning of FFA between oxidation and storage could play an important role in the regulation of body weight gain. The ultimate goal of our work was to determine the key steps that control this partitioning and their regulatory mechanisms. Among the enzymes that are involved in FFA metabolism, acyl coenzyme A synthetase (ACSL) catalyzes the first step: conversion of FFA to long-chain acyl-CoA (LC-CoA) esters.² These are further metabolized in either anabolic or catabolic pathways. Marked increases in ACSL-1 activity distribution and mRNA levels have been found in liver and adipose tissue of obese rats (2,3) and mice (4). In cultured 3T3L1 adipocytes, insulin elicits a 4-fold increase in expression of ACSL-1 mRNA (5), suggesting that transcription of ACSL-1 may be directly related to increased FFA storage in adipocytes. Furthermore, Sleeman et al. (6) have reported association of ACSL-1 activity with Glut4 vesicles in adipocytes. Reductions in ACSL-1 activity and mRNA levels in visceral fat tissue have been found in rats after physical exercise (7) and in hamsters during sepsis, when FFA uptake and conversion to triglyceride are suppressed (8). It has been postulated that this enzyme may have a potentially important control function in lipid metabolism (9) and may contribute to fat accumulation during the development of obesity (10).

There are several isoforms of ACSL in mammals, and these are expressed to various degrees in different tissues (11,12). Among these, ACSL-1 is the most abundant isoform in adipose tissue, liver, and heart, with broad fatty acid specificity (11). ACSL-1 is not expressed in adipocyte precursors but is markedly induced during differentiation, implying importance of this enzyme to fatty acid metabolism in adipose tissue (5). In contrast, ACSL-5 is present in preadipocytes at a moderately low level and remains unchanged during differentiation (13). In mature adipocytes, ACSL-1 is the dominant isoform, with minor contributions from ACSL-4 and ACSL-5 (14).

Early studies have shown that in liver, ACSL is associated with the outer membrane of mitochondria, endoplasmic reticulum (ER), and peroxisomes (15). Hence, ACSL at different sites may serve to activate FFA as required by local demands: phospholipid and triglyceride synthesis in ER, chain shortening in peroxisomes, and -oxidation in mitochondria. Recently, it has been found that ACSL activity distribution in liver was different in genetically obese (ob/ob) mice compared with control littermates, with reduced activity associated with mitochondria and increased activity associated with ER, where esterification occurs (4).

We show here that ACSL distribution changes in response to diabetes, fasting, acute insulin administration, and age. Changes in activity are positively correlated with changes of FFA oxidation and lipid synthesis.

Research Methods and Procedures

Animals

Sprague Dawley male rats were either young (60 5 grams, 26 days old) or adult (250 10 grams, 56 days old). They were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and maintained on standard chow and water ad libitum and a photoperiod of 12-/12-hour light/dark cycle at 26 °C. Fasted rats were deprived of food from 6 pm to 10 am. Diabetic animals were made diabetic by intraperitoneal injection of streptozotocin (STZ; 60 mg/kg) (16). Three days after injection, blood glucose and urinary glucose levels were tested to confirm that the rats were diabetic. After anesthesia with CO₂, the rats were exsanguinated, and epididymal and retroperitoneal fat pads were removed and pooled for preparation of adipocytes.

Preparation of Adipocytes

White adipocytes were prepared by a collagenase digestion procedure described previously (17,18). Briefly, cleaned pieces of tissue (3 to 6 grams) were minced and incubated for 30 minutes at 37 °C with a shaking frequency of 170 cycles/min in 3 to 6 mL of Krebs-Ringer-phosphate buffer (KRP; pH 7.4) containing: 2% bovine serum albumin (BSA), 2.5 mM d-glucose, 12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 1 mM CaCl₂, 1 mM Na₂HPO₄ plus NaH₂PO₄, and 3 to 5 mg collagenase/g tissue. The cells were filtered through a nylon filter (500 m) and washed twice with 10 mL of KRP buffer. The cells were suspended in 3 volumes of KRP, then preincubated for 10 minutes at 37 °C with shaking frequency of 25 cycles/min. Insulin treatment in whole cells consisted of incubation with 10 nM insulin for 15 minutes at 37 °C.

Cell Fractionation

Subcellular fractions were prepared as described previously (17,19). The cells were washed two to three times at 14 °C to 16 °C with HEPES, EDTA, sucrose (HES) buffer (20 mM HEPES, 1 mM EDTA, 250 mM sucrose, 5 mM benzamidine, 1 M phenylmethyl sulfonyl fluoride, 1 M pepstatin, 1 M leupeptin, and 1 M aprotinin, pH 7.4). The cells were homogenized with a Wheaton Potter-Elvejhem Teflon pestle tissue grinder (catalog no. 08-414-14c, Fisher Scientific Co., Pittsburgh, PA). HES buffer was used throughout the fractionation procedure. All centrifugation and handling of samples were carried out at 4 °C. The original homogenate was centrifuged at 18,000g to 20,000g for 20 minutes, the solidified lipid cake was carefully removed, and the supernatant was saved for preparation of the microsomal and cytosolic fractions. The initial pellet was resuspended in 1 mL of HES buffer and applied to the top of a 9-mL 1.12 M sucrose gradient in HES and ultracentrifuged at 126,000g to 132,000g for 60 minutes (SW 41 TI 34,600 rpm). The fraction containing mitochondria/nuclei (M/N) was collected as a pellet. The plasma membrane (PM) collected at the interface between 250 mM and 1.12 M sucrose was resuspended in 10 mL of phosphate-buffered saline and centrifuged at 48,000g for 45 minutes or 68,000g for 20 minutes, yielding a pellet of PM. The initial supernatant was centrifuged at 30,000g to 40,000g for 20 minutes, yielding a pellet of high-density microsomes (HDM) that were rich in ER. The supernatant was then ultracentrifuged at 210,000g, (Ti 70.1/Ti 70 48,000 rpm) for 75 minutes, yielding a second pellet of low-density microsomes (LDM) that were rich in vesicles and some Golgi. The last supernatant was cytosol. All pellets were resuspended in HES buffer and stored at -20 °C until use. Protein was assayed by the method of Bradford (Bio-Rad, Hercules, CA).

ACSL Activity Assay

The ACSL activity was measured in homogenate and different cell fractions by a radioisotopic assay of labeled palmitate incorporation into palmitoyl CoA (20).

The assay mixture contained 175 mM Tris-HCl, pH 7.4, 8 mM MgCl₂, 5 mM dithiothreitol, 1 mM ATP, 0.2 mM CoASH, and 50 M palmitate containing 0.1 Ci of [^3-H]-palmitic acid (NEN, Boston, MA) in a solution of 0.5 mM Triton X-100 with 10 M EDTA. The reaction was initiated by addition of 10 to 30 g of cell fraction protein. The total volume was 200 L in each assay. The reaction was terminated after 10 minutes at room temperature by addition of 1 mL Dole's reagent (isopropanol:heptane:1 M H₂SO₄ = 40:10:1 by volume). Two milliliters heptane and 0.5 mL H₂O were added, and the upper layer was removed. The lower layer was washed with 2 mL of heptane, and the top phase was removed. Scintillation fluid (4 mL) was added to the lower phase for counting. ACSL activity is expressed as nanomoles per milligram of protein per minute.

Western Blotting for ACSL and Glut4

Protein (5 to 15 g) from each subcellular fraction of rat adipocytes was separated by SDS-PAGE and transferred to a 0.2-m polyvinylidene difluoride membrane by a standard protocol (Bio-Rad catalog no. 162-0177). The membrane was blocked in buffer consisting of 10 mM phosphate, pH 7.4, 150 mM NaCl, and 0.05% Tween 20 (PBST) containing 10% fat-free milk for 1 to 2 hours at room temperature. The blot was then incubated with anti-ACSL-1 antibody at a 1:5000 dilution in 1% BSA-PBST overnight at 4 °C. The membrane was washed with PBST for 45 minutes, changing to fresh buffer every 15 minutes, and then incubated with the secondary antibody (antirabbit IgG-horseradish peroxidase conjugated, Sigma-Aldrich catalog no. A 6154; Sigma-Aldrich, St. Louis, MO) at a 1:1000 dilution in BSA-PBST for 1 hour at room temperature. After the membrane was washed with buffer as before, the membrane was developed in ECL detection reagent for 1 minute (catalog no. NEL 105; PerkinElmer Life and Analytical Sciences, Boston, MA) and exposed for 20 to 60 seconds to Bio Max Kodak film. Glut4 was detected by the same general procedure as has been described previously (21). ACSL-1 antibody was a gift from Dr. Paul Watkins (Johns Hopkins University, Baltimore, MD) and was raised against purified rat ACSL-1. It has recently been reported to cross-react with ACSL-4 and -5 (22). The monoclonal anti-Glut4 antibody, 1F8, has been previously described (23).

CO₂ Generation from Exogenous Oleic Acid

The reaction system was sealed with a rubber septum with attached plastic center well and contained 500 L of adipocytes (15 to 30 mg protein/mL). Cells were incubated with 2 mL of 1 mM [¹⁴C]-oleic acid (1Ci/mL, PerkinElmer Life and Analytical Sciences) complexed to 1% BSA in KRP buffer for 2 hours at 37 °C with gentle shaking (25 rpm). A rolled strip of Whatman No. 1 filter paper (1 cm²) was in the center well. After 2 hours of incubation, 0.5 mL of 6 N H₂SO₄ was injected into the media to release CO₂ by acidification, and 0.2 mL of -phenethylamine was injected into the center well to wet the filter paper and absorb CO₂. Trapping of CO₂ was complete in 1 hour at room temperature. The center well was then rapidly removed and adherent liquid wiped off. The well was placed in a scintillation vial containing 4 mL of scintillant, and counted in a -counter. The radioactivity collected from cell-free media incubated for 2 hours was used as a blank. The rate of CO₂ generation was calculated using the specific activity of [¹⁴C] oleic acid in the incubation media (160,000 dpm/nmole). Results are expressed as picomoles per milligram of protein per 2 hours.

Incorporation of Exogenous Oleic Acid into Cellular Lipids

Adipocytes (500 L containing 15 to 30 mg protein/mL) were incubated in 2 mL of 1 mM oleic acid and [¹⁴C]-oleic acid (1 Ci/mL) in 1% BSA-KRP buffer for 2 hours at 37 °C with gentle shaking (25 rpm). The reaction was stopped by washing the cells with 1% BSA-KRP buffer three times to remove the residual oleic acid. Four milliliters of scintillant were added to the cells, and the radioactivity was counted using a -counter. The rate of oleic acid incorporation into lipids was calculated using the specific activity of [¹⁴C]-oleic acid in the incubation media minus the blank. Results are expressed as picomoles per milligram of cell protein per 2 hours.

FFA Release from Adipocytes

Adipocytes were perifused in a column at 37 °C as described by Turpin et al. (24). After washing, 400 L of packed cells (2 to 4 million cells) were loaded onto a column preloaded with KRB containing 0.05% BSA and 2 mM glucose. The cells were perifused for 30 minutes to allow for equilibration. After the equilibration period, samples were taken to determine basal lipolysis. The cells were then perifused with insulin (5 U/mL) for 1 hour. Samples of the effluent were collected every minute and measured for FFA using a kit (NEFA C; Wako Pure Chemical Industries, Richmond, VA) that uses a colorimetric assay based on the acylation of CoASH. Due to the very low levels of FFA in the collected effluent, the samples were concentrated. Samples were dried down in a high-performance vacuum pump, reconstituted in distilled water, and then assayed.

LC-CoA Analysis

LC-CoA was measured in trichloroacetic acid precipitates of adipocyte suspensions after hydrolysis of LC-CoA to liberate free CoASH as described previously (25,26,27).

Chemicals

STZ, collagenase, and BSA (fraction V) were purchased from Roche Diagnostics (Mannheim, Germany). Dithiothreitol and CoASH were purchased from Sigma-Aldrich. Bovine insulin, lot 1102756, was purchased from Gibco BRL (Rockville, MD). Aprotinin, pepstatin A, and leupeptin hemisulfate were purchased from Calbiochem (San Diego, CA).

Statistical Analysis

InStat and DeltaGraph software were used to examine the data (F test).

Results

ACSL Activity in Adipocyte Homogenates: Effects of Fasting, Diabetes, and Donor Age

To determine whether total ACSL varied in response to nutritional change, activity was measured in homogenates of rat adipocytes isolated from young, adult, and diabetic rats that were either fasted or fed. ACSL activity decreased in response to fasting by 37% in adult (Figure 1A) and 35% in young (Figure 1B) rats. A similar reduction was also observed in diabetic rats (Figure 1A). ACSL activity was higher in young rats than in adult rats under all conditions.

Figure 1.

Comparison of total ACSL activity in homogenates from isolated rat adipocytes. Adipocytes were isolated from young (60 grams) or adult (250 grams) male Sprague Dawley rats that were fed ad libitum, fasted 16 hours, or made diabetic by treatment with STZ. ACSL activity was measured in the homogenate by a radioisotopic assay measuring labeled palmitate incorporation into palmitoyl CoA. Values represent the mean SE of four to six separate animals. (*) p < 0.05.

Full figure and legend (44K)

Activity of ACSL in the Intracellular Compartments of Adipocytes: The Effects of Fasting

To determine whether the changes observed in homogenates were general or specific to particular compartments, cells were fractionated, and the total and specific activity of individual fractions were determined. There was a wide distribution of ACSL activity detected among the M/N, HDM, LDM, PM, and cytosolic fractions (Figure 2). The highest total activity was found in the HDM in the fed state and in the M/N in the fasted state (Figure 2). The data further show that ACSL activity declined with fasting in all fractions except M/N, with the greatest percentage decrease in the HDM and PM fractions. Fasting overnight resulted in an increase in the M/N fraction from 23% to 43% of the total and a 100% increase in specific activity (Figure 3) with a simultaneous decrease in the HDM fraction from 34% to 25%. Decreases in total but not specific activities were observed in the other fractions. Diabetes caused similar activity changes in M/N and HDM as in fasting rats but no changes in the PM and cytosolic activity (data not shown). The specific activity at the subcellular level in the M/N, PM, and cytosolic fractions in both fed and fasted states was higher in the young rats (data not shown). In contrast, the specific activity associated with the LDM fraction was substantially higher in the adult (285 20 nmole/mg per minute) than in the young (197 15 nmole/mg per minute) rats.

Figure 2.

Effect of fasting on subcellular distribution of total ACSL activity in adipocyte fractions from adult fed and fasted rats. ACSL activity was determined in adipocytes separated into subcellular fractions using a sucrose gradient as described in "Research Methods and Procedures." Results are corrected for the total volume of each fraction. Values represent the mean SE of four to six separate animals. (*) p < 0.05.

Full figure and legend (92K)

Figure 3.

Effect of fasting on the specific activity of ACSL in subcellular fractions from adult fed and fasted rats. ACSL activity was determined in adipocytes separated into subcellular fractions using a sucrose gradient as described in "Research Methods and Procedures." Values represent the mean SE of four to six separate animals. (*) p < 0.05.

Full figure and legend (51K)

Insulin Treatment Increased PM-Associated ACSL Activity

It is known that ACSL activity is associated with the PM (20) and LDM (6) fractions. Large changes occur in vesicular trafficking in response to insulin (28), so these studies were undertaken to evaluate the effect of insulin on ACSL distribution in adult rats. Exposure of fat cells to insulin for 15 minutes caused the ACSL-specific activity in the PM fraction to significantly increase (Figure 4), whereas a modest increase occurred in specific activity in the LDM fraction. The total and specific activities associated with other subcellular fractions remained essentially unchanged.

Figure 4.

Effect of insulin on the specific activity of ACSL in subcellular fractions from adult fed rats. ACSL activity was determined in adipocytes separated into subcellular fractions using a sucrose gradient as described in "Research Methods and Procedures." Values represent the mean SE of four to six separate animals. (*) p < 0.05.

Full figure and legend (64K)

Changes in ACSL Activity Were Associated with Only Small Changes in ACSL Mass as Determined by Western Blot

To determine whether the insulin-induced change in activity was due to translocation or activation of ACSL, the effect of insulin on ACSL protein mass was compared with its effect on Glut4 (Figure 5). In response to insulin, Glut4 protein decreased in the LDM and increased 5-fold in the PM fraction, consistent with previous published reports (21,23,29). In contrast, the changes in ACSL localization to the PM increased to only 122 9% of control values with a commensurate decrease in the LDM (Figure 5), suggesting that translocation probably could not explain the large increase in PM activity, particularly because there was no compensatory decrease in other fractions (Figure 4). Thus, it seems likely that activation of ACSL occurred in the PM in response to insulin.

Figure 5.

Western blot illustrating the effect of insulin on ACSL and Glut4 distribution in LDM and PM fractions from adult fed rats. Adipocyte membrane fractions (5 to 15 g protein) were subjected to SDS-PAGE (9% gel) and Western blotting as described in "Research Methods and Materials." A similar distribution was found in four independent experiments.

Full figure and legend (71K)

Alterations of ACSL Activity with Fasting or Insulin in the M/N, HDM, and PM Fractions Were Associated with Changes in FFA Oxidation, Lipid Synthesis, and LC-CoA Esters

The relevance of observed activity changes depends on their functional consequences. Because HDM is associated with lipid synthesis, M/N with FFA oxidation, and PM with FFA movement into and out of the cell, these pathways were investigated in isolated intact adipocytes. The conversion of exogenous oleate to CO₂ increased with fasting and diabetes (Figure 6A) as did the activity of ACSL associated with the M/N fraction (Figure 3). Stimulation of CO₂ production during fasting was greater in young than in adult rats (data not shown), and LC-CoA levels were lower (81 10 in 150-gram rats vs. 200 12 pmol/mg protein in 350-gram rats). In the fed state, oleate incorporation into lipid was greater where high ACSL activity was associated with HDM (Figure 3). Lipid synthesis was significantly decreased by fasting and diabetes (Figure 6B), conditions also characterized by low ACSL activity associated with the HDM fraction (Figure 3). Insulin had little effect on FFA oxidation in either the fed or fasted states (data not shown) but nearly doubled FFA incorporation into lipid (Figure 6B) without a change in ACSL activity in the HDM fraction. The effectiveness of insulin is documented by the marked decrease in FFA efflux from adipocytes by 30 5% (basal, 1.00 0.03 vs. insulin, 0.70 0.04 ng/mL per minute, p < 0.05), presumably due to increased ACSL activity at the PM that promoted reesterification. Consistent with stimulation of esterification, in fasted young rats, insulin increased LC-CoA levels from 81 10 to 169 10 pmol/mg protein.

Figure 6.

Comparison of oxidation and lipid synthesis in isolated rat adipocytes from fed, fasted, insulin-treated, and diabetic rats. Intact isolated adipocytes were from adult rats that were fed ad libitum, treated with insulin for 15 minutes, fasted 16 hours, or made diabetic by treatment with STZ. FFA conversion to CO₂ and lipids was measured using radiolabeled palmitate. Values represent the mean SE of four to six separate animals. (*) p < 0.05.

Full figure and legend (92K)

Discussion

A wide distribution of ACSL was found among subcellular fractions, and high specific activity was expressed by M/N-containing mitochondria, HDM-containing ER, and LDM-containing vesicular fractions. Changes in ACSL activity occurred in response to fasting and diabetes. Interestingly, activity associated with M/N and HDM fractions varied inversely in fed and fasted rats. During fasting, there was greater ACSL activity associated with M/N fraction, and this was accompanied by increased CO₂ production from FFAs. In STZ diabetic rats, the changes were similar to fasted rats. These data suggest that during fasting, mitochondria increase in importance as a metabolic site involved in FFA partitioning and acquire enhanced ability to oxidize FFA. In the fed state, greater ACSL activity was associated with HDM fraction, along with increased lipid formation from FFA, consistent with active FFA metabolism to complex lipids at the ER. This ability is diminished after a fast. Thus, part of the adaptation to fasting or fed states may involve local changes in ACSL activity.

Addition of insulin to fat cells also altered the ACSL activity profile by increasing PM-associated ACSL activity. ACSL activity associated with the PM fractions was accompanied by decreased FFA efflux from cells. It was expected that, because ACSL is associated with Glut4 vesicles (6), it would be translocated to the PM through Glut4 vesicles. Western blots did not support major translocation of ACSL but rather showed only small changes compared with Glut4 movement. Thus, insulin-generated signals seem to activate ACSL possibly through phosphorylation or acylation to explain the greater increase in activity relative to mass. This was also evident in the LDM fraction where activity increased, whereas the protein shown on Western blots decreased slightly in response to insulin. Increased ACSL activity at the PM appears to facilitate conversion of FFA to LC-CoA at the normal site of FFA egress from the cell resulting in an increase in LC-CoA levels. This increase in substrate could explain the increase in lipid synthesis that occurs despite a lack of change of ACSL activity in the HDM fraction. Because FFA freely enter or leave the cell by simple diffusion (30,31,32,33), the presence of increased ACSL activity may serve to trap FFA in the cell as the nondiffusable LC-CoA product. It should also be noted that fasting decreased ACSL activity at the PM. Because efflux of FFA from the cell during fasting is essential to provide fuel for other cells in the body, this finding is consistent with a putative trapping role for ACSL associated with the PM.

In contrast with adult rats, the activity in the whole cell homogenates was higher in young rats. ACSL activity associated with M/N and HDM fractions also varied inversely in fed and fasted young rats as it did in adult rats. During fasting, greater ACSL activity was associated with the M/N fraction accompanied by increased CO₂ production from FFA. This is consistent with the higher metabolic rates seen in young animals (34,35). Previous studies of the effect of aging on lipid composition and metabolism in the adipose tissues of rats have demonstrated that the rate of oxidation is significantly greater in young adult rats (38 to 44 days old) than in old adult rats (420 to 647 days old) (34,35). An interesting observation was that, in the fed state, the specific activity associated with HDM from adult rats was higher than in young rats, whereas, in the fasted state, it was significantly lower than in young rats, suggesting a more active lipid synthetic process in adult rats. Consistent with a more active synthetic rate in the older animals, the level of LC-CoA was also higher than in the younger rats.

It will be of considerable interest to determine whether ACSL is associated with other enzymes or binding proteins at its different intracellular locations and whether changes in groups of enzymes also occur. The concept of a multi-enzyme complex including ACSL has been supported by data showing association of HSL (36) and adipocyte fatty acid binding protein and association of ACSL with different subcellular fractions in liver (12,37,38). We hypothesize that ACSL may play an important role in targeting FFA to specific metabolic pathways or acylation sites in adipocytes, thus acting as an integral part of the control mechanism regulating fuel partitioning.

Notes

¹ Nonstandard abbreviations: FFA, free fatty acid; CoASH, free coenzyme A; ACSL, acyl coenzyme A synthetase; LC-CoA, long-chain acyl coenzyme A; ER, endoplasmic reticulum; STZ, streptozotocin; KRP, Krebs-Ringer-phosphate buffer; BSA, bovine serum albumin; HES, HEPES, EDTA, sucrose; M/N, mitochondria/nuclei; PM, plasma membrane; HDM, high-density microsomes; LDM, low-density microsomes; PBST, 10 mM phosphate, pH 7.4, 150 mM NaCl, 0.05% Tween 20.

² Abbreviations used for ACSL are based on a recent revision to the nomenclature; see http://www.gene.ucl.ac.uk/nomenclature/genefamily/acs.html.

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Acknowledgments

We thank Rosalind Coleman for sparking our interest in this area and for subsequent informative discussions on ACSL activity and distribution. This work was supported by NIH Grants DK56690 and DK46200 (to B.E.C.) and DK30425 and DK56935 (to P.F.P.).