In vivo regulation of acyl-CoA synthetase mRNA and activity by endotoxin and cytokines

Riaz A. Memon1, John Fuller1, Arthur H. Moser1, Pamela J. Smith2, Kenneth R. Feingold1, and Carl Grunfeld1

1 Department of Medicine, University of California, San Francisco 94143; Metabolism Section, Medical Service, Department of Veterans Affairs Medical Center, San Francisco, California 94121; and 2 Ross Products Division, Abbott Laboratories, Columbus, Ohio 43215

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Acyl-CoA synthetase (ACS) catalyzes the activation of fatty acids (FA) to acyl-CoA esters, which are further metabolized in either anabolic or catabolic pathways. Endotoxin [lipopolysaccharide (LPS)], tumor necrosis factor (TNF), and interleukin-1 (IL-1) enhance hepatic FA synthesis and reesterification and inhibit FA oxidation. LPS also decreases triglyceride storage in adipose tissue and inhibits the uptake of FA by heart and muscle. Therefore, in this study we examined the effects of LPS and cytokines on ACS (now also known as ACS1) mRNA expression and activity in multiple tissues in Syrian hamsters. LPS markedly decreased ACS1 mRNA levels in liver, adipose tissue, heart, and skeletal muscle. The inhibitory effects of LPS on ACS1 mRNA levels in liver and adipose tissue were observed as early as 2-4 h after administration, became maximal by 4-8 h, and were sustained for >= 24 h. Very low doses of LPS (0.1-1 µg/100 g body wt) were needed to reduce ACS1 mRNA levels in liver and adipose tissue. TNF and IL-1 mimicked the effect of LPS on ACS1 mRNA levels in liver and adipose tissue. LPS decreased ACS activity in adipose tissue, heart, and muscle. In liver, where ACS is localized in several subcellular organelles, both LPS and cytokines decreased mitochondrial ACS activity, whereas they increased microsomal ACS activity. Taken together, these results indicate that LPS and cytokines decrease ACS1 mRNA expression and ACS activity in tissues where FA uptake and/or oxidation is decreased during sepsis. In liver, where FA oxidation is decreased during sepsis but the reesterification of FA is increased, LPS and cytokines decrease ACS1 mRNA and mitochondrial ACS activity, which may inhibit FA oxidation, but increase microsomal ACS activity, which may support the reesterification of peripherally derived FA for triglyceride synthesis.

tumor necrosis factor; interleukin-1; sepsis; fatty acid oxidation; fatty acid transport

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACYL-COA SYNTHETASE (ACS) plays a central role in the regulation of fatty acid (FA) metabolism by catalyzing the activation of long-chain FA (LCFA) to acyl-CoA esters (37). These acyl-CoA esters are utilized either in the anabolic pathways that include synthesis of cellular lipids such as triglycerides, phospholipids, and cholesterol esters or in the catabolic pathway of beta -oxidation. Because FA transport across the biological membranes is a bidirectional process, the activation of FA to acyl-CoA esters by ACS prevents the efflux of FA and renders FA transport unidirectional (37). Acyl-CoA esters are also involved in several other functions, such as posttranslational modification of proteins, intracellular signaling, and regulation of gene expression (see review in Ref. 6).

ACS is localized in microsomes, outer mitochondrial membranes, and peroxisomes in rat liver (31, 43). Purified ACS from these fractions of rat liver is identical with respect to several characteristics, including molecular weight, amino acid composition, substrate specificities, and kinetic properties (31, 43), suggesting that the same enzyme is localized in three different subcellular organelles. In contrast to liver, ACS activity is localized primarily to microsomes in adipose tissue and to outer mitochondrial membrane in heart and skeletal muscle. Several genes coding for ACS have recently been cloned that are expressed to various degrees in different tissues and may vary in their substrate specificity (12, 13, 19, 41, 44). ACS1 is the well-characterized gene that has been cloned from rat liver and murine adipocytes (41, 44). The mRNA for ACS1 is abundant in liver, adipose tissue, heart, and skeletal muscle (41, 44).

Despite a critical role of ACS in fuel homeostasis, very few studies have demonstrated ACS regulation in vivo. ACS has been considered a constitutive enzyme because early studies on the regulation of ACS under different nutritional conditions found no significant difference in enzyme activity (1, 24). However, recent studies have shown that the mRNA levels for ACS1 are markedly increased in rat liver by the feeding of a high carbohydrate or high fat diet (41). ACS1 gene expression is induced by insulin and triiodothyronine and inhibited by tumor necrosis factor (TNF) in 3T3-L1 adipocytes (20, 44).

The host response to infection and inflammation is accompanied by many changes in FA metabolism, such as increased adipose tissue lipolysis, enhanced hepatic lipogenesis and reesterification of FA in the liver, and inhibition of FA oxidation in multiple tissues including liver, heart, and skeletal muscle (3, 11, 22, 23, 39, 42). These metabolic changes can be induced by the administration of endotoxin or lipopolysaccharide (LPS), which mimics gram-negative infections, and by proinflammatory cytokines such as TNF and interleukin-1 (IL-1), which mediate many of the metabolic responses that occur during the host response to infectious and inflammatory stimuli (see review in Ref. 15). For example, LPS and cytokines increase serum triglyceride levels and very low-density lipoprotein (VLDL) production by stimulating hepatic lipogenesis, suppressing FA oxidation, and increasing reesterification of peripherally derived FA in the liver (10, 26, 28, 33).

We have recently shown that LPS and cytokines decrease mRNA levels of FA transport protein (FATP) and FA translocase (FAT) in adipose tissue, heart, and muscle in Syrian hamsters (27). LPS and cytokine also downregulate FATP mRNA expression in liver but upregulate hepatic FAT mRNA expression (27). The expression of FATP and FAT in cultured fibroblasts enhances the uptake of LCFA (2, 36). FA are esterified by ACS once they are inside the cells to prevent their efflux. Therefore, ACS is a logical site for metabolic regulation. Hence, in this study we have examined the effects of LPS and cytokines on ACS1 mRNA levels in liver, adipose tissue, heart, and muscle. Because ACS is present in different subcellular organelles in liver that channel FA to different metabolic pathways, we also examined the effect of LPS and cytokines on hepatic microsomal as well as mitochondrial ACS activity. Finally, we have examined the effect of LPS on ACS activity in adipose tissue, heart, and muscle.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. [alpha -32P]dCTP (3,000 Ci/mmol) and [U-14C]palmitic acid (850 mCi/mmol) were purchased from New England Nuclear (Boston, MA). Endotoxin (Escherichia coli 55:B5) was purchased from Difco Laboratories (Detroit, MI) and was freshly diluted to desired concentrations in pyrogen-free 0.9% saline (Kendall McGraw Laboratories, Irvine, CA). Human TNF-alpha with a specific activity of 5 × 107 U/mg was kindly provided by Genentech (South San Francisco, CA). Recombinant human IL-1beta with a specific activity of 1 × 109 U/mg was generously provided by Immunex (Seattle, WA). IL-1beta was used in this study because it is the secreted form of IL-1, whereas IL-1alpha is primarily intracellular or membrane associated (5). The cytokines were freshly diluted to desired concentrations in pyrogen-free 0.9% saline containing 0.1% human serum albumin. The multiprime DNA labeling system was purchased from Amersham International (Amersham, UK); minispin G-50 columns were from Worthington Biochemical (Freehold, NJ); Oligo(dT) cellulose, type 77F, was from Pharmacia LKB Biotechnology (Uppsala, Sweden); nitrocellulose and Nytran were from Scleicher & Schuell (Keene, NH). Kodak XAR5 film was used for autoradiography. cDNA for ACS1 was prepared as described previously (44).

Animal procedures. Male Syrian hamsters (~160-180 g) were purchased from Simonsen Laboratories (Gilroy, CA). The animals were maintained on a normal light cycle (6 AM to 6 PM light, 6 PM to 6 AM dark) and were provided with rodent chow (Simonsen Laboratories) and water ad libitum. Animals (n = 5 hamsters per group) were injected intraperitoneally with either LPS, TNF, IL-1, or TNF plus IL-1 at the indicated doses or with 0.9% saline containing 0.1% human serum albumin. Our previous studies have shown that LPS and cytokines induce marked anorexia in Syrian hamsters (14). The induction of anorexia by LPS is acute in onset, with a 90% or more decrease in food intake during the first 18 h after a 10-100 µg/100 g body wt LPS dose, and the anorexigenic effect of LPS persists for >= 72 h (14). Hence, in this study food was withdrawn from both control and treated groups to avoid the effect of variations in food intake on ACS regulation. Animals were studied between 1.5 and 24 h after LPS or 8 h after cytokine administration. The doses of LPS used (0.1-100 µg/100 g body wt) are far below those required to cause death in rodents in our laboratory (lethal dose ~5 mg/100 g body wt) but have significant effects on triglyceride and cholesterol metabolism in Syrian hamsters (7, 9). Similarly, the doses of TNF and IL-1 used (17 µg/100 g body wt and 1 µg/100 g body wt, respectively) in these experiments were based on previous studies demonstrating that these doses have marked effects on lipid metabolism and reproduce many of the effects of LPS on lipid metabolism in Syrian hamsters (16, 17).

Isolation of RNA and Northern blotting. Total RNA was isolated by a variation of the guanidinium thiocyanate method (4), as described earlier (7). Total RNA from adipose tissue was used for Northern blotting, whereas poly(A)+ RNA from liver, heart, and muscle was isolated using oligo(dT) cellulose. Total or poly(A)+ RNA was quantified by measuring absorption at 260 nm. Equal amounts of total or poly(A)+ RNA were loaded on 1% agarose-formaldehyde gels and electrophoresed. The uniformity of sample applications was checked by ultraviolet visualization of the acridine orange-stained gels before transfer to Nytran membranes. We and others have found that LPS increases actin mRNA levels in liver by 2- to 5-fold in rodents (7, 32). TNF and IL-1 produce a 2-fold increase in actin mRNA levels. LPS also produced a 2-fold increase in hepatic mRNA levels for glyceraldehyde 3-phosphate dehydrogenase (G-3PD) and a 2.6-fold increase in cyclophilin mRNA (27). Therefore, the mRNA levels of actin, G-3PD, and cyclophilin, which are widely used for normalizing data, cannot be used to study LPS or cytokine-induced regulation of proteins in liver. However, the differing direction of the changes in mRNA levels for specific proteins after LPS or cytokine, the magnitude of the alterations, and the relatively small standard error of the mean make it unlikely that the changes observed are due to unequal loading of mRNA (7, 9, 16, 17, 27). cDNA probe hybridization was performed in 0.75 M sodium chloride, 0.075 M sodium citrate, 2% SDS, 10% dextran sulfate, 2 × Denhardt's solution, and 100 mg/ml sheared salmon sperm DNA at 65°C overnight. Blots were washed in 0.2× standard sodium citrate and 0.1% SDS at room temperature for 30 min and at 65°C for 1 h. The blots were exposed to X-ray films for various durations to ensure that measurements were done on the linear portion of the curve, and the bands were quantified by densitometry.

Acyl-CoA synthetase activity assay. Hepatic ACS activity was measured by a radioisotopic assay in liver homogenates, microsomes, or mitochondria by the method of Tanaka et al. (43). Postnuclear supernatants were used for measuring ACS activity in adipose tissue, heart, and skeletal muscle (38). Briefly, at indicated times after the administration of LPS or cytokines, the livers were removed and homogenized in a buffer [0.25 M sucrose, 1 mM dithiothreitol (DTT), and 1 mM EDTA], and mitochondria and microsomes were isolated by differential centrifugation. Similarly, adipose tissue, heart, and muscle were obtained and homogenized. The homogenates were centrifuged at 800 g for 5 min, and the resultant supernatants were used for measuring enzyme activity. As reported by others (43), our initial experiments also showed that the rate of reaction was linear up to 10 min and plateaued by 15 min. Hence, in all subsequent assays the reaction time was kept at 5 min. The assay mixture contained 0.1 M Tris · HCl buffer, pH 8.0, 5 mM DTT, 0.15 M KCl, 15 mM MgCl2, 1.6 mM Triton X-100, 10 mM ATP, 1 mM CoA, and 1 mM [U-14C]palmitic acid in a final reaction volume of 0.2 ml. The assay was initiated by addition of 5-10 µl of enzyme suspension (5 µg protein), and the reaction was carried out at 37°C for 5 min. The reaction was terminated by adding 2.5 ml of a mixture containing isopropanol-heptane-sulfuric acid 1 M (40:10:1, vol/vol/vol). The unreacted palmitic acid was extracted with heptane, and the radioactivity in the aqueous phase containing palmitoyl-CoA was measured (43). Control reactions containing no protein were run in parallel to correct for the background. ACS activity is expressed as nanomoles of palmitoyl-CoA formed per milligram protein per minute. Protein was assayed by the method of Bradford (Bio-Rad Laboratories).

Statistics. The results are presented as means ± SE. Statistical significance between two groups was determined by use of Student's t-test. Comparison among several groups was performed by analysis of variance, and statistical significance was calculated using Bonferroni's multiple comparison test.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACS1 mRNA levels in liver, adipose tissue, heart, and muscle in the fed and fasted states. It has been previously shown that short-term fasting has no significant effect on hepatic ACS1 mRNA levels in rats (41). In this study, we compared ACS1 mRNA levels in liver, adipose tissue, heart, and muscle from animals that were either fasted for 24 h or had free access to lab chow. Our results demonstrate that ACS1 mRNA levels are not significantly different between fed and 24-h-fasted animals in liver, heart, and muscle (Fig. 1). On the other hand, adipose tissue ACS1 mRNA levels were 70% lower in the fasted animals than in the fed group (Fig. 1).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of feeding and fasting on acyl-CoA synthetase (ACS)1 mRNA levels in liver, adipose tissue, heart, and muscle. Groups of Syrian hamsters had either free access to lab chow or were fasted for 24 h. Animals were euthanized and tissues were obtained for RNA isolation. ACS1 mRNA levels were determined by Northern blotting as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 for each group. * P < 0.001.

Effect of LPS on ACS1 mRNA levels in liver and adipose tissue. To investigate the effect of LPS on ACS1 mRNA levels in liver and adipose tissue, Syrian hamsters were injected with LPS (100 µg/100 g body wt) or saline (controls); both groups of animals were fasted to avoid the effect of variations in food intake on ACS1 mRNA level. Livers and adipose tissue were obtained at various time points, and total or poly(A)+ RNA was isolated from adipose tissue and liver, respectively, for Northern blot analysis. LPS markedly decreased ACS1 mRNA levels in both liver and adipose tissue (Fig. 2). ACS1 mRNA levels in liver were decreased by 50% at 90 min, 90% at 4 h, and 80% at 8 h after LPS administration (Fig. 2A). At 24 h after LPS, ACS1 mRNA levels were 40% of control values. In adipose tissue, ACS1 mRNA levels began to decrease by 4 h after LPS administration and were decreased by 87% at 8 h and 92% at 16 h after LPS treatment (Fig. 2B). At 24 h after LPS, ACS1 mRNA levels in adipose tissue were 23% of the control levels. These data demonstrate that LPS dramatically downregulates ACS1 mRNA levels in vivo in both liver and adipose tissue.


View larger version (16K):
[in this window]
[in a new window]
 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Time course of endotoxin [lipopolysaccharide (LPS)] effect on mRNA levels of ACS1 in liver (A) and adipose tissue (B). Syrian hamsters were injected ip either with saline (controls) or LPS (100 µg/100 g body wt), and food was removed from both groups. At the indicated times animals were euthanized and tissues were harvested for RNA isolation. ACS1 mRNA levels were determined by Northern blotting as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 for each time point. A: * P < 0.002; ** P < 0.001; B: * P < 0.001.

We next determined the dose response of LPS-induced decrease in ACS1 mRNA levels in liver and adipose tissue at 8 h after LPS treatment (Fig. 3). LPS doses as low as 1 and 10 µg (per 100 g body wt) were sufficient to induce a maximal decrease in ACS1 mRNA levels in liver and adipose tissue, respectively. Although the effect of LPS at a 100-µg dose was less in magnitude (65% decrease) than the effect of a 1- or 10-µg dose (83% decrease) on ACS1 mRNA in liver, the differences were not statistically significant. The half-maximal dose for LPS-induced decrease in ACS1 mRNA in liver was below 0.1 µg/100 g body wt, whereas the half-maximal dose for LPS-induced decrease in ACS1 mRNA levels in adipose tissue was 0.3 µg/100 g body wt (Fig. 3).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Dose response of LPS effect on ACS1 mRNA levels in liver and adipose tissue. Syrian hamsters were injected ip either with saline (controls) or different doses of LPS (0.1-100 µg/100 g body wt), and food was removed from all groups. Eight hours later animals were euthanized and tissues collected for RNA isolation. ACS1 mRNA levels were determined by Northern blotting as described in MATERIALS AND METHODS. Values are means ± SE; n = 4 for each dose. * P < 0.005, ** P < 0.001.

Effect of cytokines on ACS1 mRNA levels in liver and adipose tissue. Because TNF and IL-1 mediate many of the metabolic effects of LPS, we next examined the effects of TNF (17 µg/100 g body wt) and IL-1 (1 µg/100 g body wt) on ACS1 mRNA levels in liver and adipose tissue (Fig. 4). These doses have previously been shown to mimic the metabolic effects of LPS on lipid and lipoprotein metabolism in Syrian hamsters, although the magnitude of the effect may vary (16, 17, 27). Eight hours after administration, TNF produced a 60% decrease in ACS1 mRNA levels in liver, whereas IL-1 either alone or in combination with TNF decreased hepatic ACS1 mRNA levels by 90% (Fig. 4A). Similarly, TNF produced a 60% decrease in ACS1 mRNA levels in adipose tissue, whereas IL-1 either alone or in combination with TNF produced an 80% decrease in ACS1 mRNA levels in adipose tissue (Fig. 4B). It is possible that a higher dose of TNF may produce an effect that is comparable in magnitude to that of IL-1.


View larger version (34K):
[in this window]
[in a new window]
 


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of tumor necrosis factor (TNF), interleukin-1 (IL-1), and the combination of TNF and IL-1 (T + I) on ACS1 mRNA levels in liver (A) and adipose tissue (B). Syrian hamsters were injected with TNF (17 µg/100 g body wt), IL-1 (1 µg/100 g body wt), TNF plus IL-1, or saline (controls), and food was removed from all groups. Eight hours later animals were euthanized, and livers and adipose tissue were obtained for RNA isolation. ACS1 mRNA levels were determined by Northern blotting as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 in each group. A: * P < 0.002, ** P < 0.001; B: * P < 0.001.

Effect of LPS on ACS1 mRNA levels in heart and muscle. In addition to liver and adipose tissue, ACS1 is also abundantly expressed in heart and skeletal muscle. Moreover, both of these organs metabolize FA as their primary oxidative fuel; hence, we next examined the effect of high-dose LPS (100 µg/100 g body wt) on ACS1 mRNA levels in heart and skeletal muscle. Sixteen hours after LPS treatment, ACS1 mRNA levels in heart and skeletal muscle were decreased by 40 and 80%, respectively (Fig. 5).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of LPS on ACS1 mRNA levels in heart and muscle. Syrian hamsters were injected ip either with saline (controls) or LPS (100 µg/100 g body wt), and food was removed from both groups. Sixteen hours later animals were euthanized and tissues were harvested for RNA isolation. ACS1 mRNA levels were determined by Northern blotting as described in MATERIALS AND METHODS. Values are means ± SE; n = 4 for each group. * P < 0.001.

Effect of LPS and cytokines on ACS activity. Previous studies have shown that LPS and cytokines inhibit FA oxidation and stimulate triglyceride synthesis in the liver (10, 26, 28, 33). Because ACS is localized in several subcellular organelles in liver and directs FA into anabolic or catabolic pathways, we investigated the effect of LPS on ACS activity in liver homogenates as well as in microsomal and mitochondrial fractions. Eight hours after LPS treatment, ACS activity (nmol · mg protein-1 · min-1) in liver homogenates was decreased by only 20% (control 680 ± 29, LPS 544 ± 32; P < 0.02) despite a marked reduction in hepatic ACS1 mRNA after LPS treatment (Figs. 2A and 3). However, when the activity of ACS was examined in mitochondrial and microsomal fractions, we observed differential regulation of ACS activity. LPS decreased mitochondrial ACS activity by 61%, whereas there was a 55% increase in microsomal ACS activity (Fig. 6A). Differential regulation of ACS activity was also observed after cytokine treatment. TNF, IL-1, or the combination of both cytokines produced a 37, 45, and 52% decrease, respectively, in mitochondrial ACS activity (Fig. 6B). In contrast, IL-1 produced a 60% increase in microsomal ACS activity, whereas TNF had no effect on microsomal ACS activity and also did not synergize with IL-1. It is possible that a higher dose of TNF may be needed to produce an increase in microsomal ACS activity.


View larger version (34K):
[in this window]
[in a new window]
 


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of LPS (A) and cytokines (B) on ACS activity in liver. Syrian hamsters were injected ip with saline (controls), LPS (100 µg/100 g body wt), TNF (17 µg/100 g body wt), or TNF + IL-1 (1 µg/100 g body wt), and food was removed from all groups. Eight hours later animals were euthanized and livers were obtained. Mitochondrial and microsomal fractions were isolated by differential centrifugation, and ACS activity was measured as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 for each group. A: * P < 0.001; B: * P < 0.01, ** P < 0.001.

Because LPS or cytokines may influence the composition of cellular membranes or may affect protein levels in subcellular fractions, which could influence the ACS activity on a per milligram protein basis, we determined the effect of 8 h of LPS, TNF, and IL-1 treatment on total protein content in liver mitochondrial and microsomal fractions. LPS and cytokines produced no significant increase in protein content in microsomal fraction (control 58.4 ± 2.1, LPS 62.2 ± 1.2, TNF 64.8 ± 2.2, IL-1 65.7 ± 2.7 mg protein/g liver) and a small but statistically significant increase (P < 0.05 for all treatments) in mitochondrial protein content (control 44.6 ± 2.1, LPS 52.3 ± 2.2, TNF 53.1 ± 2.3, IL-1 52.7 ± 2.5 mg protein/g liver). Thus it is unlikely that LPS or cytokine-induced increases of such a small magnitude in both microsomal and mitochondrial protein content could account for the differential regulation of ACS activity in these fractions.

The data presented in Fig. 7 depict the effect of LPS treatment on ACS activity in postnuclear supernatants in adipose tissue, heart, and muscle in which ACS is only found in one subcellular fraction. LPS produced a significant decrease in ACS activity in heart (38%), muscle (51%), and adipose tissue (54%). LPS had no significant effect on total protein content in heart [control 56.1 ± 5.3, LPS 48.3 ± 1.8 mg protein/g heart; not significant (NS)], skeletal muscle (control 56.0 ± 4.4, LPS 56.2 ± 1.2 mg protein/g muscle tissue; NS), or adipose tissue (control 6.0 ± 0.5, LPS 5.1 ± 0.4 mg protein/g adipose tissue; NS).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of LPS on ACS activity in adipose tissue, heart, and muscle. Syrian hamsters were injected with either saline (controls) or LPS (100 µg/100 g body wt), and the food was removed. Sixteen hours later animals were euthanized and tissues were harvested. ACS activity was measured in postnuclear supernatants, as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 for each tissue in both groups. * P < 0.001.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

During infection and inflammation, marked changes occur in FA, triglyceride, and cholesterol metabolism in several tissues (15, 39). Studies in experimental models of sepsis have shown that on one hand there is an increase in hepatic lipogenesis, reesterification, and VLDL production, whereas on the other hand there is decreased FA oxidation and ketone body production in liver (11, 22, 23, 42). LPS decreases the storage of triglycerides in adipose tissue and increases the mobilization of FA (3, 11, 23). Finally, the uptake of LCFA by heart and skeletal muscle is markedly reduced after LPS administration (35, 40). Cytokines, particularly TNF and IL-1, have been shown to mimic many of these effects, suggesting that they may be the in vivo mediators of metabolic changes produced during infection or inflammation (15).

The results of our present study demonstrate that LPS downregulates ACS1 mRNA levels and activity in adipose tissue. This downregulation of ACS1 mRNA is rapid, sustained, and elicited by very low doses of LPS. IL-1 and TNF also decreased ACS1 mRNA levels in adipose tissue; however, the effect of IL-1 was greater in magnitude than that of TNF. ACS is primarily associated with microsomes in adipose tissue to support the synthesis of triglycerides for storage of energy (18). We have previously shown that LPS and cytokines decrease the expression of mRNAs for FATP and FAT in adipose tissue (27). A coordinate decrease in FA transport proteins, ACS1 mRNA expression, and ACS activity in adipose tissue will prevent the storage of FA in adipose tissue during infection and inflammation and will promote the mobilization of FA. This conclusion is reinforced by previous studies that have shown that LPS and cytokines stimulate adipose tissue lipolysis and increase the mobilization of FA in rodents (10, 11, 26). A decrease in ACS1 mRNA under conditions of nutrient deprivation such as fasting, as reported here, will also prevent the activation and subsequent storage of FA in adipose tissue and will enhance FA mobilization to support the energy demands of other tissues.

Our results also demonstrate that LPS decreased ACS1 mRNA expression and activity in heart and skeletal muscle. ACS is primarily associated with outer mitochondrial membranes in heart and muscle for beta -oxidation, as these tissues preferentially utilize FA as their oxidative fuel (34). We have previously shown that LPS decreased the mRNA expression for FATP and FAT in heart and muscle (27). Thus a coordinate decrease in both FA transport proteins, coupled with a decrease in ACS expression and activity, will decrease the uptake and utilization of FA in heart and muscle. Although FA are the preferred fuel substrate for heart and muscle under normal conditions, several studies have shown that, during sepsis, their fuel preference is altered. For example, Spitzer et al. (40) have shown that in vivo myocardial uptake and oxidation of LCFA are decreased after LPS treatment, whereas the uptake and utilization of lactate are elevated. The uptake and oxidation of LCFA by skeletal muscle are also reduced after LPS (35), whereas the uptake and utilization of glucose are increased in several organs, including muscle after LPS treatment (29).

Lipoprotein lipase (LPL) is the key enzyme responsible for hydrolyzing triglyceride-rich lipoproteins. The FA released by the hydrolysis of triglycerides are either oxidized or reesterified. The activity of LPL is highest in tissues that oxidize FA for energy production (heart and skeletal muscle) or esterify FA for storage (adipose tissue). Hence, like ACS, LPL also plays a key role in the regulation of adipocyte lipid storage and provision of fuel substrate for heart and muscle (21). We and others have previously shown that LPS and several cytokines decrease the activity of LPL in adipose tissue, heart, and muscle in rodents (3, 8, 22). A coordinate decrease in LPL and ACS during infection and inflammation will also decrease the storage of FA in adipose tissue and decrease their utilization by heart and muscle.

In the liver, both LPS and cytokines markedly downregulated ACS1 mRNA expression. As seen in adipose tissue, the effect of IL-1 was greater than that of TNF on ACS1 mRNA in the liver and was comparable to that of LPS. It is of interest to note that, in the liver, where ACS is present in multiple subcellular organelles to facilitate the activation of FA for different metabolic pathways, LPS produced only a small but statistically significant decrease in ACS activity in liver homogenates, a marked decrease in mitochondrial ACS activity, and an increase in microsomal ACS activity. Previous studies have shown that LPS and cytokines suppress FA oxidation and ketone body production but stimulate FA reesterification, triglyceride synthesis, and VLDL production in the liver (15, 39). LPS and cytokines inhibit FA oxidation in part by raising the levels of malonyl-CoA (26), which is the first committed intermediate in FA synthesis and is an allosteric inhibitor of carnitine palmitoyltransferase I, the rate-limiting enzyme in FA oxidation (25). It is important to note that both FA oxidation and reesterification are highly compartmentalized, with FA oxidation taking place in mitochondria and reesterification occurring in the cytosol. We have recently shown that, in contrast to a coordinate downregulation of FATP and FAT in adipose tissue, heart, and muscle by LPS, the mRNAs for these proteins are differentially regulated in the liver (27). LPS and cytokines downregulate hepatic FATP mRNA levels, whereas they upregulate FAT mRNA expression. These results have suggested that FATP may transport FA toward mitochondria for oxidation, which is suppressed in sepsis, whereas FAT may transport FA to cytosol for reesterification, which is enhanced in sepsis. Our present results on the differential regulation of ACS activity in the liver support such a possibility. Thus, whereas a decrease in hepatic mitochondrial ACS activity will be an additional mechanism for inhibiting FA oxidation in liver, an increase in microsomal ACS activity is likely to lead to an activation of FA for hepatic triglyceride synthesis, which is enhanced during sepsis.

Although the decrease in mitochondrial ACS activity in liver corresponds to the decrease in hepatic ACS1 mRNA expression, the increase in microsomal ACS activity in the presence of a profound decrease in hepatic ACS1 mRNA levels is surprising. The mechanism for the discordance between decreased ACS1 mRNA expression and increased microsomal ACS activity is not clear. It is possible that microsomal ACS may be regulated by LPS and cytokines at the posttranslational level by the modification of protein through one of the several known mechanisms, such as phosphorylation, dephosphorylation, acylation, or isoprenylation; however, no such mechanism has been reported for the regulation of ACS. The decrease in mitochondrial ACS, coupled with increased microsomal activity, might represent a compartmental shift of the enzyme; however, we are not aware of any such example of enzyme translocation between mitochondria and endoplasmic reticulum. It is also possible that there is increased activation of the enzyme because of a conformational change induced by substrate binding. Thus an increase in FA delivery to the microsomal compartment induced by LPS and cytokines (by upregulating FAT and downregulating FATP) could preferentially enhance microsomal ACS activity.

An increase in hepatic microsomal ACS activity, despite a profound decrease in ACS1 mRNA levels, also raises the possibility that microsomal ACS may be coded by a different ACS gene product. This hypothesis is supported by the fact that several ACS genes have been recently cloned that are expressed to various degrees in different tissues and may vary in their fatty acid specificity. ACS1, the mRNA studied here, is the well-characterized gene that is most abundant in liver, adipose tissue, heart, and muscle, with very low expression in brain, lung, and small intestine, and ACS1 has a broad FA specificity (41, 44). ACS2 is predominantly expressed in brain and to a much lesser degree in heart, but it is not detectable in other tissues (13). ACS3 is highly expressed in brain (12). ACS4 is abundantly present in steroidogenic tissues such as adrenal gland, ovary, and testis, but substantial ACS4 mRNA expression is also seen in liver, brain, and lung (19). Two distinct ACS genes have also been reported in yeast, with ACS1 being responsible for providing acyl-CoA exclusively for synthesis of cellular lipids and ACS2 for producing acyl-CoA for degradation via beta -oxidation (30). In light of this diversity of the ACS gene family in mammals as well as in yeast, it is possible that there may be another member of the ACS gene family present in liver that may code for the microsomal isozyme. Further studies will be needed to address these issues.

In summary, our results demonstrate that LPS and cytokines decrease ACS1 mRNA expression and activity in tissues where FA uptake and/or oxidation is decreased during sepsis. In the liver, where FA oxidation is suppressed but triglyceride synthesis is increased during sepsis, LPS and cytokines decrease ACS1 mRNA and mitochondrial ACS activity but increase microsomal ACS activity. This reciprocal regulation of ACS activity may inhibit FA oxidation and support the reesterification of peripherally derived FA for triglyceride synthesis.

    ACKNOWLEDGEMENTS

This work was supported by a grant from the Research Service of the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49448.

    FOOTNOTES

Address for reprint requests: R. A. Memon, Dept. of Veterans Affairs Medical Center, 4150 Clement St. (111F), San Francisco, CA 94121.

Received 8 December 1997; accepted in final form 9 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Aas, M., and L. N. W. Daae. Fatty acid activation and acyl transfer in organs from rats in different nutritional states. Biochim. Biophys. Acta 239: 208-216, 1971[Medline].

2.   Abumrad, N. A., M. R. El-Maghrabi, E. Z. Amri, E. Lopez, and P. A. Grimaldi. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long chain fatty acids that is induced during preadipocyte differentiation. Homology with human CD36. J. Biol. Chem. 268: 17665-17668, 1993[Abstract/Free Full Text].

3.   Bagby, G. J., C. B. Corll, and R. R. Martinez. Triacylglycerol kinetics in endotoxic rats with suppressed lipoprotein lipase activity. Am. J. Physiol. 253 (Endocrinol. Metab. 16): E59-E64, 1987[Abstract/Free Full Text].

4.   Chomczynski, P., and N. Sacchi. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

5.   Dinarello, C. A. Biological basis for interleukin-1 in disease. Blood 87: 2095-2147, 1996[Abstract/Free Full Text].

6.   Faergeman, N. M., and J. Knudsen. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling. Biochem. J. 323: 1-12, 1997[Medline].

7.   Feingold, K. R., I. Hardardottir, R. A. Memon, E. J. T. Krul, A. H. Moser, J. M. Taylor, and C. Grunfeld. Effect of endotoxin on cholesterol biosynthesis and distribution in serum lipoproteins in Syrian hamsters. J. Lipid Res. 34: 2147-2158, 1993[Abstract].

8.   Feingold, K. R., M. Marshall, R. Gulli, A. H. Moser, and C. Grunfeld. Effect of endotoxin and cytokines on lipoprotein lipase activity in mice. Arterioscler. Thromb. 14: 1866-1872, 1994[Abstract].

9.   Feingold, K. R., A. S. Pollock, A. H. Moser, J. K. Shigenaga, and C. Grunfeld. Discordant regulation of protein of cholesterol metabolism during the acute phase response. J. Lipid Res. 36: 1474-1482, 1995[Abstract].

10.   Feingold, K. R., M. Soued, S. Adi, I. Staprans, R. Neese, J. K. Shigenaga, W. Doerrler, A. H. Moser, C. A. Dinarello, and C. Grunfeld. Effects of interleukin-1 on lipid metabolism. Similarities to and differences from tumor necrosis factor. Arterioscler. Thromb. 11: 495-500, 1991[Abstract].

11.   Feingold, K. R., I. Staprans, R. A. Memon, A. H. Moser, J. K. Shigenaga, W. Doerrler, C. A. Dinarello, and C. Grunfeld. Endotoxin rapidly induces changes in lipid metabolism that produce hypertriglyceridemia: low doses stimulate hepatic triglyceride production while high doses inhibit clearance. J. Lipid Res. 33: 1765-1776, 1992[Abstract].

12.   Fujino, T., M. Kang, H. Suzuki, H. Iijima, and T. Yamamoto. Molecular characterization and expression of rat acyl-CoA synthetase 3. J. Biol. Chem. 271: 16748-16752, 1996[Abstract/Free Full Text].

13.   Fujino, T., and T. Yamamoto. Cloning and functional expression of a novel long-chain acyl-CoA synthetase expressed in brain. J. Biochem. (Tokyo) 111: 197-203, 1992[Abstract].

14.   Grunfeld, C., C. Zhao, J. Fuller, A. Pollock, A. Moser, J. Friedman, and K. R. Feingold. Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters. A role for leptin in the anorexia of infection. J. Clin. Invest. 97: 2152-2157, 1996[Abstract/Free Full Text].

15.   Hardardottir, I., C. Grunfeld, and K. R. Feingold. Effects of endotoxin and cytokines on lipid metabolism. Curr. Opin. Lipidol. 5: 207-215, 1994[Medline].

16.   Hardardottir, I., A. H. Moser, R. A. Memon, C. Grunfeld, and K. R. Feingold. Effects of TNF, IL-1, and the combination of both cytokines on cholesterol metabolism in Syrian hamsters. Lymphokine Cytokine Res. 13: 161-166, 1994[Medline].

17.   Hardardottir, I., J. Sipe, A. H. Moser, C. J. Fielding, K. R. Feingold, and C. Grunfeld. LPS and cytokines regulate extra-hepatic mRNA levels of apolipoproteins during the acute phase response in Syrian hamsters. Biochem. Biophys. Acta 1344: 210-220, 1997[Medline].

18.   Jason, C. J., M. A. Polokoff, and R. M. Bell. Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity. J. Biol. Chem. 251: 1488-1492, 1976[Abstract].

19.   Kang, M., T. Fujino, H. Sasano, H. Minekura, N. Yabuki, H. Nagura, H. Iijima, and T. Yamamoto. A novel arachidonate-perferring acyl-CoA synthetase is present in steroidogenic cells of rat adrenal, ovary and testis. Proc. Natl. Acad. Sci. USA 94: 2880-2884, 1997[Abstract/Free Full Text].

20.   Kansara, M. S., A. K. Mehra, J. Von Hagen, E. Kabotyansky, and P. J. Smith. Physiological concentrations of insulin and T3 stimulate 3T3-L1 adipocyte acyl-CoA synthetase gene transcription. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E873-E881, 1996[Abstract/Free Full Text].

21.   Ladu, M. J., H. Kapsas, and W. K. Palmer. Regulation of lipoprotein lipase in adipose and muscle tissues during fasting. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R953-R959, 1991[Abstract/Free Full Text].

22.   Lanza-Jacoby, S., E. Rosato, G. Braccia, and A. Tabares. Altered ketone body metabolism during gram-negative sepsis in rat. Metabolism 39: 151-157, 1990.

23.   Lanza-Jacoby, S., and A. Tabares. Triglyceride kinetics, tissue lipoprotein lipase, and liver lipogenesis in septic rats. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E678-E685, 1990[Abstract/Free Full Text].

24.   Lippel, K. Regulation of rat liver acyl-CoA synthetase activity. Biochim. Biophys. Acta 239: 384-392, 1971[Medline].

25.   McGarry, J. D., K. F. Woeltje, M. Kuwajima, and D. W. Foster. Regulation of ketogenesis and renaissance of carnitine palmitoyltransferase. Diabetes. Metab. Rev. 5: 271-284, 1989[Medline].

26.   Memon, R. A., K. R. Feingold, A. H. Moser, W. Doerrler, S. Adi, C. A. Dinarello, and C. Grunfeld. Differential effects of interleukin-1 and tumor necrosis factor on ketogenesis. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E301-E309, 1992[Abstract/Free Full Text].

27.   Memon, R. A., K. R. Feingold, A. H. Moser, J. Fuller, and C. Grunfeld. Differential regulation of fatty acid transport protein and fatty acid translocase mRNA levels by endotoxin and cytokines. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E210-E217, 1998[Abstract/Free Full Text].

28.   Memon, R. A., C. Grunfeld, A. H. Moser, and K. R. Feingold. Tumor necrosis factor mediates the effects of endotoxin on cholesterol and triglyceride metabolism in mice. Endocrinology 132: 2246-2253, 1993[Abstract].

29.   Meszaros, K., C. H. Lang, G. J. Bagby, and J. J. Spitzer. Contribution of different organs to increased glucose consumption after endotoxin administration. J. Biol. Chem. 262: 10965-10970, 1987[Abstract/Free Full Text].

30.   Mishina, M., T. Kamiryo, S. Tashiro, T. Hagihara, A. Tanaka, S. Fukui, M. Osumi, and S. Numa. Subcellular localization of two long-chain acyl-coenzyme-A synthetases in Candida lipolytica. Eur. J. Biochem. 89: 321-328, 1978[Abstract].

31.   Miyazawa, S., T. Hashimoto, and S. Yokota. Identity of long-chain acyl-CoA synthetase of microsomes, mitochondria and peroxisomes in rat liver. J. Biochem. 98: 723-733, 1985[Abstract].

32.   Morrow, J. F., R. S. Steraman, C. G. Peltzman, and D. A. Potter. Induction of hepatic synthesis of serum amyloid A and actin. Proc. Natl. Acad. Sci. USA 78: 4718-4722, 1981[Abstract].

33.   Nachiappan, V., D. Curtiss, B. E. Corkey, and L. Kilpatrick. Cytokines inhibit fatty acid oxidation in isolated rat hepatocytes. Synergy among TNF, IL-6 and IL-1. Shock 1: 123-129, 1994[Medline].

34.   Oram, J. F., J. I. Wenger, and J. R. Neely. Regulation of long-chain fatty acid activation in heart muscle. J. Biol. Chem. 250: 73-78, 1975[Abstract].

35.   Romanosky, A. J., G. J. Bagby, E. L. Bockman, and J. J. Spitzer. Free fatty acid utilization by skeletal muscle after endotoxin administration. Am. J. Physiol. 239 (Endocrinol. Metab. 2): E391-E395, 1980[Abstract/Free Full Text].

36.   Schaffer, J. E., and H. F. Lodish. Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein. Cell 79: 427-436, 1994[Medline].

37.   Schaffer, J. E., and H. F. Lodish. Molecular mechanisms of long chain fatty acid uptake. Trends Cardiovasc. Med. 5: 218-224, 1995.

38.   Shimomura, I., K. Tokunaga, K. Kotani, Y. Keno, M. Yanase-Fujiwara, K. Kanosue, S. Jiao, T. Funahashi, T. Kobatake, T. Yamamoto, and Y. Matsuzawa. Marked reduction of acyl-CoA synthetase activity and mRNA in intra-abdominal visceral fat by physical exercise. Am. J. Physiol. 265 (Endocrinol. Metab. 28): E44-E50, 1993[Abstract/Free Full Text].

39.  Spitzer, J. J., G. J. Bagby, C. Meszaros, and C. H. Lang. Alterations in lipid and carbohydrate metabolism in sepsis. J. Parenter. Enteral Nutr. 12, Suppl. 6: 53S-58S, 1988.

40.   Spitzer, J. J., A. A. Bechtel, L. T. Archer, M. R. Black, and B. L. Hinshaw. Myocardial substrate utilization in dogs following endotoxin administration. Am. J. Physiol. 227: 132-136, 1974[Medline].

41.   Suzuki, H., Y. Kawarabayasi, J. Kondo, T. Abe, K. Nishikawa, S. Kimura, T. Hashimoto, and T. Yamamoto. Structure and regulation of rat long-chain acyl-CoA synthetase. J. Biol. Chem. 265: 8681-8685, 1990[Abstract/Free Full Text].

42.   Takeyama, N., Y. Itoh, Y. Kitazawa, and T. Tanaka. Altered hepatic mitochondrial fatty acid oxidation and ketogenesis in endotoxic rats. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E498-E505, 1990[Abstract/Free Full Text].

43.   Tanaka, T., K. Hosaka, M. Hoshimaru, and S. Numa. Purification and properties of long-chain acyl-CoA synthetase from rat liver. Eur. J. Biochem. 98: 165-172, 1979[Medline].

44.   Weiner, F., P. J. Smith, S. Wertheimer, and C. S. Rubin. Regulation of gene expression by insulin and tumor necrosis factor in 3T3-L1 cells. Modulation of the transcription of genes encoding acyl-CoA synthetase and stearoyl-CoA desaturase-1. J. Biol. Chem. 266: 23525-23528, 1991[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 275(1):E64-E72