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Journal of Lipid Research, Vol. 47, 2028-2041, September 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology
,1
* Department of Physiology, Michigan State University, East Lansing, MI 48824
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824
Department of Horticulture, Michigan State University, East Lansing, MI 48824
** Department of Veterinary Science and Biomedical Sciences, Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University, University Park, PA 16802
Published, JLR Papers in Press, June 21, 2006.
1 Yun Wang, Daniela Botolin, and Jinghua Xu contributed equally to this work. 
2 To whom correspondence should be addressed. e-mail: jump{at}msu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PPAR)-deficient mice establish that PPAR was required for WY14643-mediated induction of fatty acid elongase-5 (Elovl-5), Elovl-6, and all three desaturases [
5 desaturase (5D), 6D, and 9D]. Increased nuclear sterol-regulatory element binding protein-1 (SREBP-1) correlated with enhanced expression of Elovl-6, 5D, 6D, and 9D. Only 9D was also regulated independently by liver X receptor (LXR) agonist. Glucose induction of L-type pyruvate kinase, 9D, and Elovl-6 expression required the carbohydrate-regulatory element binding protein/MAX-like factor X (ChREBP/MLX) heterodimer. Suppression of Elovl-6 and 9D expression in livers of streptozotocin-induced diabetic rats and high fat-fed glucose-intolerant mice correlated with low levels of nuclear SREBP-1. In leptin-deficient obese mice (Lepob/ob), increased SREBP-1 and MLX nuclear content correlated with the induction of Elovl-5, Elovl-6, and 9D expression and the massive accumulation of monounsaturated fatty acids (18:1,n-7 and 18:1,n-9) in neutral lipids. Diabetes- and obesity-induced changes in hepatic lipid composition correlated with changes in elongase and desaturase expression. In conclusion, these studies establish a role for PPAR, LXR, SREBP-1, ChREBP, and MLX in the control of hepatic fatty acid elongase and desaturase expression and lipid composition.
Supplementary key words peroxisome proliferator-activated receptor • sterol-regulatory element binding protein-1 • carbohydrate-regulatory element binding protein • MAX-like factor X • liver X receptor
Abbreviations: ACC, acetyl-coenzyme A carboxylase; ChoRE, carbohydrate-regulatory element; ChREBP, carbohydrate-regulatory element binding protein; 5D, 5 desaturase; Elovl-1, fatty acid elongase-1; HNF-4, hepatic nuclear factor-4; L-PK, L-type pyruvate kinase; Luc, luciferase; LXR, liver X receptor; MLX, MAX-like factor X; PPAR, peroxisome proliferator-activated receptor ; qRT, quantitative real-time; SREBP-1, sterol-regulatory element binding protein-1; T1317, T0901317; T3, triiodothyronine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of the various pathways known to affect fatty acid structure, physiological control of fatty acid elongation remains poorly defined. The predominant pathway for fatty acid elongation occurs in the endoplasmic reticulum and uses malonyl-CoA and fatty acyl-CoA as substrates for the addition of two carbons to fatty acids. Elongases are condensing enzymes that interact with 3-keto acyl-CoA reductase, a dehydratase, and trans-2,3-enoyl-CoA reductase to elongate fatty acids (7–9). The rate of fatty acid elongation is determined by the activity of the elongase (condensing enzyme) and not the reductases or the dehydratase. Seven distinct fatty acid elongase subtypes [fatty acid elongase-1 (Elovl-1) through Elovl-7] are present in the mouse, rat, and human genomes (www.ensembl.org). Elovl-1 (Ssc1) and Elovl-6 (LCE, FACE, rElo2) elongate saturated and monounsaturated fatty acids. Elovl-6 is induced in transgenic mice overexpressing sterol-regulatory element binding protein-1 (SREBP-1) (10–12). Elovl-2 (Ssc2) substrates include C20–22 PUFAs, whereas Elovl-5 (FAE1, Relo1, Helo1) uses a broad substrate array, C16–22 (10, 13). Elovl-2 and Elovl-5 likely play a role in endogenous PUFA synthesis [i.e., the conversion of the essential fatty acid precursors linoleic acid (18:2,n-6) and -linolenic acid (18:3,n-3) to arachidonic acid (20:4,n-6) and docosahexaenoic acid (22:6,n-3)] (11). Elovl-3 (Cig30, Elo3) and Elovl-4 (Elo4) are expressed in the skin (14) and retina (15), respectively. Both Elovl-3 and Elovl-4 elongate a broad array of fatty acids (
C26). Elovl-3 is induced in brown adipose tissue after exposure of animals to cold (16). Elovl-7 has not been characterized.
We recently reported on the tissue-specific, nutritional, and developmental regulation of fatty acid elongases in the rat (17). The outcome of those studies suggested that hepatic elongase expression might be controlled by at least two transcription factors, peroxisome proliferator-activated receptor (PPAR) and SREBP-1. This report extends those findings by examining SREBP-1, PPAR, liver X receptor (LXR), carbohydrate-regulatory element binding protein (ChREBP), and MAX-like factor X (MLX) control of elongase and desaturase expression. Our analysis also includes in vivo studies to determine whether changes in hepatic lipid composition induced by diabetes or obesity correlated with changes in elongase and desaturase expression. Overall, these studies establish a role for specific transcriptional regulatory networks in the control of hepatic desaturase and elongase gene expression and hepatic lipid composition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
All procedures for the use and care of animals for laboratory research were approved by the All University Committee for Animal Use and Care at Michigan State University.
Streptozotocin-induced diabetes
Male Sprague-Dawley rats (200–250 g; Charles River Laboratories, Kalamazoo, MI) were maintained on Harlan-Teklad laboratory chow (No. 8640) and water ad libitum. Rats were injected intraperitoneally with streptozotocin (7.5 mg/100 g body weight) and 3 ml of 25% glucose (18). Three weeks later, blood glucose was measured in animals receiving no streptozotocin (control) or streptozotocin (diabetic; blood glucose 
120 mg/dl). Blood glucose was measured in isoflurane-anesthetized rats using a glucose meter (Freestyle Flash; Thera Sense, Inc., Alameda, CA). Control and diabetic rats were euthanized (isoflurane anesthesia and exsanguination) for recovery of blood (plasma) and liver.
High-fat feeding of C57BL/6 mice Male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME), 2 months of age, were fed diets containing 10% lard (D12450B) or 60% lard (D12492; Research Diets, Inc.) ad libitum for 10 weeks. Four days before euthanasia, mice were subjected to a glucose tolerance test. Briefly, mice were injected with glucose (2 g/kg ip). Blood was withdrawn from the tail vein before and after glucose treatment. Blood glucose was measured using a hand-held glucose meter (Freestyle Flash; Thera Sense, Inc.). Mice were euthanized using isoflurane and exsanguinated; blood and liver were recovered. Livers were used for RNA and lipid extraction.
Lean and obese mice Livers from lean and obese C57BL/6 mice were obtained from Drs. D. Romsos and K. Claycomb (Department of Food Science and Human Nutrition, Michigan State University). Male lean (C57BL/6J-Lepob/+) and obese (C57BL/6J-Lepob/ob) mice (B6.V-Lep ob/J, No. 000632; Jackson Laboratories) were maintained on a Harlan-Teklad laboratory chow (No. 8640) diet and water ad libitum. Livers were used for RNA, lipid, and protein extraction.
Wild-type and PPAR-deficient mice
Homozygous wild-type and PPAR-deficient (PPAR–/–) mice on a Sv/129 genetic background (19, 20) were fed either a control diet or one containing WY14643 (at 50 or 500 mg/kg; Bio-Serv, Piscataway, NJ) for 1 week. Mice were euthanized, livers were removed, and RNA was isolated for analysis of gene expression.
Primary rat hepatocytes Male Sprague-Dawley rats (Charles River Laboratories) were maintained on Harlan-Teklad laboratory chow (No. 8640) and water ad libitum. Rat primary hepatocytes were prepared from Teklad chow-fed (ad libitum) male Sprague-Dawley rats and cultured on BioCoat (type 1 collagen) plates (Beckon Dickinson, Bedford, MA) (21). For RNA and protein extraction, cells were plated onto 100 mm type 1 collagen-coated plates (BD Bioscience, Bedford, MA) at 107 cells/plate in Williams E (Gibco/Invitrogen, Carlsbad CA), 10 mM lactate, 10 nM dexamethasone, 1 µM insulin (Sigma, St. Louis, MO), and 10% fetal bovine serum (Gibco/Invitrogen). For adenoviral infection studies, cells were plated in the same medium onto six-well type 1 collagen-coated plates at 1.5 x 106 cells/well. The ratio of culture medium to cell number was maintained constant for the different plating conditions. For treatments, hepatocytes were incubated in medium [Williams E + 10 nM dexamethasone with or without 1 µM (0.17 U/ml) insulin and/or 25 mM glucose].
RNA extraction, Northern analysis, and quantitative real-time PCR
Total RNA was extracted from primary hepatocytes or liver samples and used as a template for quantitative real-time (qRT) PCR or Northern analysis as described previously (17, 22). Specific primers for each gene (Table 1
) were designed using Primer Express software (Applied Biosystems, Foster City, CA). First-strand cDNA was synthesized using SuperScript II RNase H reverse transcriptase (Invitrogen). Synthesized cDNA was mixed with 2x SYBR Green PCR Master Mix (Applied Biosystems) and various sets of gene-specific forward and reverse primers and subjected to real-time PCR quantification using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). All reactions were performed in triplicate. The relative amounts of mRNAs were calculated using the comparative threshold cycle method (User Bulletin No. 2; Applied Biosystems). Cyclophilin was used as a control, and all results were normalized to the abundance of cyclophilin mRNA. Primers used for qRT-PCR are listed in Table 1.
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Immunoblotting
Liver microsomal and nuclear extracts were prepared as described previously (17, 23). Proteins (50–100 µg) extracted from microsomal or nuclear fractions were separated electrophoretically by SDS-PAGE (NuPAGE 4–12% polyacrylamide Bis-Tris; Invitrogen) and transferred to nitrocellulose membranes. Membranes were incubated with antibodies for SREBP-1 (IgG-2A4, sc-13551; Santa Cruz Biotechnology, San Cruz, CA) and SREBP-2 (IgG-7D4, monoclonal antibody). Hepatic nuclear factor-4 (HNF-4; C-19), MLX (N-17), and anti-goat and anti-rabbit antibodies were obtained from Santa Cruz Biotechnology. ChREBP antibody was obtained from Novus Biologicals (Littleton, CO). Anti-mouse and anti-rabbit secondary antibodies were obtained from Bio-Rad; anti-goat antibodies were obtained from Santa Cruz Biotechnology. The SuperSignal West Pico chemiluminescence kit (Pierce) detection system was used.
Recombinant adenovirus
Cloning of cDNA for Elovl-2, Elovl-5, and Elovl-6 was described previously (17). The coding region for each transcript was ligated into the Ad-Easy XL adenoviral vector system (Stratagene), recombined in BJ 5183 cells, and propagated in XL10 Gold ultracompetent cells. Ad-DNA was packaged into adenoviral particles in Ad-293 cells. The resulting adenovirus was amplified in HEK293 cells.
Recombinant adenovirus-expressing dominant negative MLX (Ad-dnMLX) and doxycycline-inducible nuclear SREBP-1c (Ad-nSREBP-1) were obtained from H. Towle (University of Minnesota, Minneapolis, MN) (24, 25). Adenovirus was propagated and amplified in HEK293 cells. Viral lysates were titered using the Adeno-X Rapid Titer Kit (Clontech). Confluent primary hepatocytes were infected (5–10 plaque-forming units/cell). Using Ad-green fluorescent protein as a control for infection, >80% of primary hepatocytes expressed functional protein at the 5–10 plaque-forming units/cell level.
In vitro fatty acid elongation assay
Rat liver microsomes were isolated by differential centrifugation (23). Elongation reactions were carried out with modifications to the procedure described by Moon et al. (10). Briefly, reaction mixtures contained 50 µg of microsomal proteins in a total reaction volume of 100 µl. The reaction constituents were as follows: 50 mM potassium phosphate buffer, pH 6.5, 5 µM rotenone (Sigma), 40 µM fatty acyl-CoA [Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL) and Sigma], 60 µM malonyl-CoA (Sigma), 6.5 dpm/pmol [2-14C]malonyl-CoA (Perkin-Elmer), 1 mM NADPH (Sigma), and 20 µM BSA (fatty acid-free). Reactions (at 37°C) were initiated with the addition of NADPH. When fatty acids were used as substrate, NaOH-neutralized fatty acid (40 µM) replaced fatty acyl-CoA. Coenzyme A (CoASH) (100 µM), MgCl2 (1 mM), and ATP (1 mM) were added to the reaction to generate fatty acyl-CoA. Elongase reactions were terminated after 20 min with the addition of 100 µl of 5 N KOH plus 10% methanol; lipids were saponified for 1 h at 65°C. The saponification reaction was acidified with 100 µl of 5 N HCl; 100 µl of ethanol was added to aid hexane extraction of fatty acids. Elongated fatty acids were collected by two independent extractions with hexane (800 µl). Hexane extracts were pooled, and 14C radioactivity was quantified by ß-scintillation counting. Results are expressed as elongase activity units (nmol [14C]malonyl-CoA incorporated/mg protein/20 min). Formation of reaction products was dependent on the presence of NADPH and the fatty acid-CoA. Fatty acid elongation products were verified by reverse-phase HPLC using a flow-through ß-scintillation counter (6).
Statistical analysis
Statistical analysis used Student's t-test and ANOVA plus post hoc Tukey's honestly significant difference test (http://faculty.vassar.edu/lowry/VassarStats.html).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5 desaturase (5D) and 6D are expressed at comparable levels in livers of all three species. 9D is highly expressed in mouse liver compared with rat or human livers. These results reveal important species differences in both elongase and desaturase expression in rat, mouse, and human liver.
Role of PPAR in the control of hepatic elongase and desaturase expression
Feeding rats the PPAR agonist, WY14643, induces certain hepatic fatty acid elongases and desaturases and promotes changes in hepatic and plasma lipid composition (17). Here, we further evaluated the role that PPAR plays in the control of hepatic elongase and desaturase expression. The effect of WY14643 on elongase and desaturase expression in wild-type and PPAR–/– mice was examined. Mice were fed a control diet or one containing WY14643 (at 50 or 500 mg WY14643/kg diet) for 1 week (Fig. 2
). Of the four elongases expressed in mouse liver, WY14643 (500 mg/kg) induced only Elovl-5 and Elovl-6 (14- and 3.6-fold, respectively). Previous studies established that the PPAR agonist increased desaturase expression (26). Our results indicate that 6D and 9D transcript levels were induced 
6-fold by WY14643 (500 mg/kg), whereas 5D was weakly induced. Elovl-1 and Elovl-2 mRNA abundance was unresponsive to WY14643 treatment. The absence of a WY14643 effect on elongase and desaturase expression in PPAR–/– mice indicates that PPAR plays a role in controlling both elongase (Elovl-5 and Elovl-6) and desaturase (6D and 9D) expression.
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Regulation of elongases and desaturases by insulin and LXR agonist Insulin regulates hepatic lipid synthesis, at least in part, by controlling SREBP-1 nuclear abundance (21, 29). LXR agonist stimulates lipogenesis through direct and indirect mechanisms (30). LXR/retinoid X receptor heterodimers bind LXR regulatory element in promoters of responsive lipogenic genes. LXR agonist also induces lipogenic gene expression through the induction of SREBP-1c gene transcription (29, 30). The effect of insulin and T1317 on hepatocyte elongase and desaturase expression was examined.
In the absence of insulin or T1317, SREBP-1 nuclear abundance in hepatocytes was low (Fig. 3
, inset). Treatment of rat primary hepatocytes with insulin or T1317 induced nuclear SREBP-1 (4-fold) but had no effect on SREBP-2 nuclear abundance. T1317 (1 µM) had no significant effect on Elovl-1, Elovl-2, or Elovl-5 expression in rat primary hepatocytes and only modestly induced Elovl-6 1.5-fold (Fig. 3). In contrast, all three desaturases were induced between 2- and 15-fold; 9D was most responsive. Insulin induced Elovl-6 and 5D 1.5-fold, whereas 6D and 9D were induced >3-fold. Cotreatment with insulin and T1317 had no additive effect on SREBP-1 nuclear abundance or the expression of any elongase or desaturase.
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5D, 6D, and 9D by insulin and T1317 likely involves the control of SREBP-1 nuclear abundance. Although others have reported that insulin induces LXR in primary hepatocytes (31), we found no evidence for an insulin effect on either LXR or LXRß mRNA abundance (data not shown). Glucocorticoids, T3, and leptin had no effect on elongase expression in primary rat hepatocytes. None of the hormones tested induced hepatic Elovl-3, Elovl-4, or Elovl-7 (data not shown).
Effect of overexpressed nuclear SREBP-1c on elongase and desaturase expression
To further evaluate the SREBP-1c control of elongase and desaturase expression, primary hepatocytes were infected with a recombinant adenovirus containing a doxycycline-inducible nuclear form of SREBP-1c (Ad-nSREBP-1c) (25). These cells received no insulin or LXR agonist. Treatment of primary hepatocytes with doxycycline significantly induced the nuclear form of SREBP-1c (data not shown) as well as the endogenous SREBP-1 transcript (Fig. 4
). The endogenous SREBP-1c promoter contains an SRE and is activated by increased levels of nuclear SREBP (29). Overexpressed nSREBP-1c induced transcripts encoding Elovl-2, Elovl-6, 5D, 6D, and 9D 3-fold at a doxycycline dose of 250 ng/ml. Modest induction (>1.5-fold) was detected with doxycycline at 25 ng/ml.
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5D, 6D, and 9D transcripts were induced 3.6-, 4.8-, and 3.1-fold, respectively. Addition of insulin had no other effect on the expression of any elongase or desaturase. Addition of T1317 induced only 9D (4-fold) (data not shown). The expression of no other elongase or desaturase was affected by T1317. Based on these results, the induction of Elovl-6, 5D, and 6D by insulin and T1317 (Fig. 4) is attributable to increased SREBP-1 nuclear abundance. Only 9D expression was induced by increased SREBP-1 nuclear abundance and independently by LXR agonist. Glucose, ChREBP, and MLX regulation of elongase and desaturase expression Insulin and glucose are potent inducers of glycolysis and lipogenesis (32, 33). Insulin-stimulated glucose metabolism induces the translocation of ChREBP to the nucleus, where the ChREBP/MLX heterodimer binds carbohydrate-regulatory elements (ChoREs) in promoters of glucose-responsive genes involved in glycolysis and lipogenesis (32, 34). The effect of glucose on elongase and desaturase expression in primary hepatocytes was examined.
Primary hepatocytes were maintained in medium containing lactate (20 mM) and insulin (1 µM) or switched to medium containing 25 mM glucose plus insulin. This approach is consistent with the one used in our previous studies and by other investigators to examine glucose-regulated gene expression in primary hepatocytes (22, 24, 35–43). Glucose and insulin induced the accumulation of ChREBP in hepatocyte nuclei with no effect on MLX nuclear abundance (Fig. 5A
). L-type pyruvate kinase (L-PK), a glucose-responsive transcript, is controlled at the transcriptional level by binding the ChREBP/MLX heterodimer to its promoter (24). Switching hepatocytes from lactate to glucose induced mRNAL-PK 30-fold (Fig. 5). Elovl-6 and 9D mRNAs were induced 7.6- and 10-fold, respectively. No other elongase or desaturase was affected by glucose.
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Infecting cells with Ad-Luc had no effect on the glucose induction of L-PK, Elovl-6, or 9D (Fig. 5). Infection of cells with Ad-dnMLX completely attenuated the glucose induction of L-PK, Elovl-6, and 9D. Ad-Luc or Ad-dnMLX expression in primary hepatocytes had no effect on Elovl-1, Elovl-2, Elovl-5, 5D, or 6D (data not shown). These studies indicate that glucose regulates both Elovl-6 and 9D expression by mechanisms that control the nuclear abundance of ChREBP and MLX. ChREBP and MLX play no role in the control of Elovl-1, Elovl-2, Elovl-5, 5D, or 6D expression.
Metabolism of fatty acids by fatty acid elongases
Fatty acid elongases have overlapping substrate specificities (7, 44). Here, the substrate specificity of three fatty acid elongases was examined. Ad-Luc, Ad-Elovl-2, Ad-Elovl-5, and Ad-Elovl-6 were used to overexpress these enzymes in rat primary hepatocytes. Substrate specificity was examined using saturated (16:0), monounsaturated (16:1,n-7), and polyunsaturated (18:3,n-6, 20:5,n-3, and 22:5,n-3) fatty acids. Ad-Luc-infected cells served as a control for basal elongase activity using the various substrates. Ad-Elovl-2-infected hepatocytes elongated only 20:5,n-3 and 22:5,n-3 (Fig. 6
). Ad-Elovl-5-infected cells elongated 16:1,n-7, 18:3,n-6, and 20:5,n-3. Ad-Elovl-6-infected cells elongated only 16:0 and 16:1,n-7 (Fig. 6).
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, SREBP-1, and ChREBP/MLX (Figs. 2–5). To determine whether changes in elongase and desaturase activity affect hepatocyte fatty acid composition, primary hepatocytes incubated with insulin (induces SREBP-1), PPAR agonists, and glucose (induces ChREBP) were examined for the capacity to elongate and desaturate [14C]16:0. Primary hepatocytes were maintained in Williams E medium with lactate and no insulin overnight. This treatment effectively decreases SREBP-1 and ChREBP nuclear abundance (21, 22). The next day, cells were treated with lactate- or glucose-containing medium in the absence or presence of insulin and WY14643. All cells received 100 µM [14C]16:0. After 24 h of treatment, cells were harvested for lipid extraction and analysis of elongation and desaturation products by reverse-phase HPLC. Cells maintained in lactate medium with no insulin or WY14643 did not desaturate 16:0 (Fig. 7 ), but these cells had the capacity to elongate 16:0 to 18:0. Addition of insulin or WY14643 to lactate-treated cells induced the formation of 16:1,n-7, whereas only WY14643 induced the formation of 18:1 (n-7 and n-9). The combination of these treatments had no additive effect.
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9D, measuring changes in 18:0 may not accurately reflect changes in elongation activity. In an effort to reveal an effect on elongation, we examined the effect of glucose, insulin, and WY14643 on the 18:1-to-16:1 ratio (Fig. 7D). This ratio (elongation index) would remain constant if these treatments did not regulate elongation. The most impressive effect on the 18:1-to-16:1 ratio was seen in cells maintained in lactate-containing medium supplemented with WY14643. Such studies indicate that hepatocyte levels of 18:1 (n-7 and n-9) are controlled by both elongation and desaturation pathways. The elongation pathway is not a constitutive pathway. Cellular levels of 18:1 are not determined solely by changes in 9D expression.
Regulation of elongase and desaturase expression in animal models of metabolic disease
PPAR (45), SREBP-1 (46) and glucose metabolism, ChREBP and MLX (32), and LXR (47) play important roles in metabolic diseases such as diabetes and obesity. Here, we sought to determine whether changes in hepatic lipid metabolism and composition induced by diabetes and obesity can be attributed to changes in elongase and desaturase expression. Three metabolic disorders were examined: streptozotocin-induced diabetes, glucose intolerance induced by high-fat diets, and obesity induced by leptin deficiency. Nuclear levels of SREBP-1, ChREBP, MLX, and HNF-4 were monitored. We wanted to determine whether changes in the nuclear content of these transcription factors correlated with changes in elongase or desaturase expression.
Streptozotocin-induced diabetes
Rats made diabetic using streptozotocin had high blood glucose (378 ± 21 mg/dl) compared with control animals (77.9 ± 5.2 mg/dl). Liver nuclei from diabetic rats contained little detectable nuclear SREBP-1 and suppressed levels of MLX, but there was no significant change in ChREBP or HNF-4 (Fig. 8A
). Diabetes suppressed the expression of lipogenic and glycolytic genes (e.g., fatty acid synthase and L-PK) by 70% (18), whereas expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase was induced 3-fold (Fig. 8). The decline in the nuclear abundance of SREBP-1 and MLX correlated with an 45% decline in Elovl-6 and a >95% decline in 9D mRNA abundance. Other elongases and desaturases remained unaffected. Examination of hepatic lipid composition revealed a significant 30% decline in 16:0 in diabetic animals but no change in other saturated, monounsaturated, or polyunsaturated fatty acids (data not shown). The decline in 16:0 is consistent with the decline in de novo lipogenesis in livers of diabetic rats (18).
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29 g and had blood glucose and insulin levels within the normal range, 121 mg/dl and 0.5 ng/ml (Table 2
). Animals fed the high-fat diet were 44 g with increased blood glucose (152 mg/dl) and insulin (3.9 ng/ml). Compared with the low-fat-fed group, high-fat-fed animals were glucose-intolerant (Fig. 9A
) and had fatty livers (48). Nuclear levels of SREBP-1 and MLX were suppressed 60% and 50% (Fig. 9B), whereas ChREBP and HNF-4 nuclear levels remained unchanged.
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5D or 6D but significantly suppressed 9D expression (80%). High-fat diets significantly suppressed fatty acid elongation of 16:0-CoA and 20:4-CoA but not 24:0-CoA (Fig. 9D).
Palmitic (16:0), stearic (18:0), oleic (18:1,n-9), and linoleic (18:2,n-6) acids represent the major fatty acids in both diets. Analysis of hepatic lipid composition indicated that 18:2,n-6 accumulated relative to 20:4,n-6 in livers of high-fat-fed animals (data not shown). 18:2,n-6, an essential fatty acid, is converted to 20:4,n-6. Because neither diet contained 20:4,n-6, the appearance of 20:4,n-6 in the livers required conversion from 18:2,n-6 to 20:4,n-6 by elongation (Elovl-2 and Elovl-5) and desaturation (5D and 6D). In the low-fat diet, the ratio of 20:4,n-6 to 18:2,n-6 was 0.75. In the high-fat diet, the ratio was 0.3. Failure to convert 18:2,n-6 to 20:4,n-6 is consistent with a decline in Elovl-5 expression, a key enzyme involved in PUFA synthesis.
Elongase and desaturase expression in livers of lean (Lepob/+) and obese (Lepob/ob) mice
Defective leptin expression in C57BL/6J-Lepob/ob mice leads to hyperphagia, hyperinsulinemia, insulin resistance, and obesity (50). Compared with their lean (Lepob/+) littermates, obese animals were heavier and had increased blood levels of glucose and insulin (Table 2). The livers of obese mice were massively engorged with lipid, predominantly as neutral lipid. Hepatic nuclei derived from obese mice had a 2.6- and 2-fold increase in SREBP-1 and MLX nuclear abundance but no change in ChREBP or HNF-4 compared with those of lean littermates. Expression levels of Elovl-5, Elovl-6, 5D, 6D, and 9D were increased in livers of obese mice compared with their lean littermates. Obesity also resulted in the induction of lipogenic gene expression (ACC, FAS) and PPAR target genes, such as cytochrome P450 4A(Cyp4A) and acyl CoA oxidase (data not shown). Fatty acid elongation of 16:0-CoA was increased 2-fold, whereas elongation of 20:4-CoA and 24:0-CoA was not affected (Fig. 10C
).
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9-fold increase in total esterified fatty acid. Much of this increase is in the form of neutral lipid esterified with 18:1 (n-7 and n-9) (Fig. 10D). Although monounsaturated fatty acid abundance increased, hepatic PUFA content (i.e., 18:2,n-6, 20:4,n-6, and 22:6,n-3) declined. This might appear inconsistent with the observed increase in fatty acid elongase activity and elongase and desaturase expression. Lepob/ob mice, however, are hyperphagic; ingestion of excessive calories as carbohydrate increased plasma insulin (Table 2) and enhanced de novo lipogenesis and monounsaturated fatty acid synthesis. Induction of Elovl-5, Elovl-6, and 9D expression by activated PPAR and increased SREBP-1 and MLX nuclear content facilitated monounsaturated fatty acid (18:1,n-7 and 18:1,n-9) synthesis, which was assimilated into neutral lipid. Elongation and desaturation are key steps for converting end products of de novo lipogenesis, such as 16:0, to monounsaturated fatty acids. These studies indicate that fatty acid elongation is induced along with de novo lipogenesis and 9-desaturation. Induction of these pathways contributes to the fatty liver phenotype characteristic of Lepob/ob mice. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9D (stearoyl-CoA desaturase-1), has emerged as a key enzyme in the control of whole body lipid composition (52).
In contrast to the desaturases, fatty acid elongases have only recently been recognized as proteins regulated at the pretranslational level (11, 44, 53). Fatty acid elongases are regulated by tissue-specific and nutritional factors and during postnatal development (17). Such studies implicated certain transcription factors, such as SREBP-1 and PPAR, as regulators of both elongase and desaturase expression. This report extends those previous observations by evaluating the role of several hormones (insulin, T3, glucocorticoids, and leptin), transcription factors (SREBP-1c, PPAR, LXR, ChREBP, and MLX), and nutrients (glucose and fat) in the control of hepatic elongase and desaturase gene expression, fatty acid elongase activity, and lipid composition. Finding the involvement of these transcription factors in the control of elongase expression prompted studies to evaluate how these enzymes were regulated in metabolic disease. The outcome of these studies has provided new information on how changes in both elongase and desaturase expression in metabolic disease contribute to hepatic lipid composition.
Seven fatty acid elongase subtypes (Elovl-1 to Elovl-7) have been identified in the genomes of the rat, mouse, and human (www.ensembl.org). Of these, four elongase subtypes are expressed in rat, mouse, and human liver (Fig. 1). The hierarchy for hepatic expression of these enzymes is similar in all three species: Elovl-5 > Elovl-1 = Elovl-2 = Elovl-6. Analysis of elongase activity indicates that mouse liver has the highest elongase activity.
Elovl-1
Elovl-1 is a low-abundance elongase in liver of all three species. Based on studies in yeast, Elovl-1 elongates a broad array of saturated and monounsaturated fatty acids. Elovl-1 expression, however, is not regulated by any physiological manipulation used to date in this or our previous study (17). Thus, changes in hepatic lipid composition induced during postnatal development or in association with fasting and refeeding, diabetes, obesity, dietary fat, LXR, or PPAR agonist cannot be attributed to changes in Elovl-1 activity. Hepatic Elovl-1 appears to be expressed constitutively.
Elovl-2
Elovl-2 is also a low-abundance elongase in liver of all three species. In contrast to other elongases, Elovl-2 has a very narrow substrate preference: it elongates C20 and C22 PUFAs (Fig. 6). As such, Elovl-2 participates in the conversion of essential fatty acid precursors to end products of PUFA synthesis (i.e., 20:4,n-6 and 22:6,n-3). Like Elovl-1, Elovl-2 is not regulated by any factors examined in this or our previous report (17). The exception to this is the induction of Elovl-2 mRNA after overexpression of SREBP-1c (Fig. 3). Because insulin, LXR agonist, and glucose fail to induce this transcript, we feel that the induction of Elovl-2 by overexpressed SREBP-1c may have limited physiological significance in vivo.
Elovl-5
Elovl-5 is the most abundant elongase transcript in all three species. It also is expressed in many tissues, induced during postnatal development, and suppressed by feeding rats n-3 PUFA-enriched diets (17). Several hormones (insulin, T3, glucocorticoids, and leptin) and transcription factors (SREBP-1c, LXR, ChREBP, and MLX) have no impact on hepatic Elovl-5 expression. Only PPAR, n-3 PUFA-enriched diets (17), high-fat diets (Fig. 9), and obesity (Fig. 10) affect Elovl-5 expression. The regulation of Elovl-5 is physiologically significant. Feeding rats a high-carbohydrate diet supplemented with olive oil plus WY14643 significantly increased mead acid (20:3,n-9) production (17). Mead acid is an elongation and desaturation product of 18:1,n-9, the predominant fatty acid in olive oil. WY14643 induction of Elovl-5 likely contributes to the formation of 20:3,n-9. Elovl-5 also converts 16:1,n-7, but not 16:0, to an 18 carbon monounsaturated fatty acid (18:1,n-7) (Fig. 6) as well as the elongation of an intermediate (18:3,n-6) in the pathway for n-6 PUFA synthesis (20:4,n-6) (Fig. 6). Suppression of Elovl-5 in high-fat-fed mice correlates with a decreased hepatic 20:4,n-6-to-18:2,n-6 ratio (Fig. 9). Enhanced Elovl-5 expression correlates with the increased content of 18 carbon monounsaturated fatty acids in livers of obese mice (Lepob/ob) (Fig. 10). Many PPAR-regulated transcripts, such as acyl-CoA oxidase and Cyp4A, are induced in livers of Lepob/ob mice (data not shown). Induction of Elovl-5 in livers of obese mice is likely attributable to PPAR activation.
Despite the role that Elovl-5 plays in PUFA synthesis and its increase in livers of obese mice, hepatic lipids in obese animals are not enriched in PUFAs. In fact, obese livers are depleted of PUFA relative to other fatty acids, such as 18:1 (Fig. 10D). 5D, 6D, and 9D are induced in livers of obese mice, but to differing extents (Fig. 10D). 5D and 6D are induced in obese liver as a result of the increased nuclear abundance of SREBP-1 and activation of PPAR. 9D is induced by these same transcription factors, plus increased nuclear ChREBP/MLX (Fig. 10D). Thus, hyperphagia resulting from defective leptin production, coupled with the ingestion of the high-carbohydrate diet, stimulates de novo lipogenesis and monounsaturated fatty acid synthesis. In this instance, Elovl-5 substrates, in particular 16:1,n-7 (Fig. 6), are end products of de novo lipogenesis and 9D. Increased expression of Elovl-5, Elovl-6, and 9D, coupled with enhanced production of end products of de novo lipogenesis, increases 18:1,n7 and 18:1,n9 production.
Elovl-6
Elovl-6 is expressed at low levels in livers of all three species. Like Elovl-2, Elovl-6 has a narrow substrate preference (i.e., C12–16 saturated and unsaturated fatty acids) (Fig. 6) (10). In contrast to other elongases, Elovl-6 is regulated by multiple factors. Insulin and LXR agonist increase SREBP-1 nuclear abundance, which leads to induced Elovl-6 expression (Figs. 3, 4). Insulin-induced glucose metabolism increases ChREBP nuclear content, and the ChREBP/MLX heterodimer regulates glucose-regulated genes, including L-PK, ACC, FAS, and 9D. Elovl-6 is among these glucose-regulated genes (Fig. 5). PPAR activation also induces Elovl-6 (Fig. 2). Elovl-6 is regulated during postnatal development, but unlike Elovl-5, Elovl-6 expression declines at birth and is induced at weaning. Elovl-6 expression during early postnatal development parallels SREBP-1 nuclear abundance (17). The finding that both Elovl-6 and 9D are induced along with L-PK and FAS (Fig. 10) indicates that these enzymes play a role in the hepatic response to excess carbohydrate consumption.
Excess carbohydrate is channeled to de novo lipogenesis via enhanced L-PK activity. Insulin-stimulated glucose metabolism induces ChREBP translocation into hepatic nuclei (32, 54, 55). ChREBP and MLX heterodimers bind ChoREs in promoters of responsive genes, such as L-PK, ACC, and FAS. Insulin also increases SREBP-1 nuclear abundance, leading to increased promoter occupancy of SREBP-1 on SRE in target genes (e.g., ACC, FAS, and 9D). Consistent with this scenario is the increased nuclear abundance of SREBP-1 and MLX in livers derived from obese animals (Fig. 10). The end product of de novo lipogenesis, 16:0, is elongated (Elovl-6) and desaturated (9D) to yield 18:1, the fatty acid that accumulates in livers of obese mice. In this metabolic scheme, there appears to be a tight coordination between glycolysis, de novo lipogenesis, fatty acid elongation (Elovl-6), and desaturation (9D) that involves three transcription factors: ChREBP, MLX, and SREBP-1c.
Although these studies provide a link between ChREBP, MLX, SREBP-1, and PPAR in the control of elongase expression, the mechanism(s) for this control remains undefined. Whether this control involves direct interaction of these transcription factors with regulatory elements in the promoters of the elongases or indirect control through other mechanisms will require detailed analysis of the promoters for Elovl-5 and Elovl-6. Such studies are beyond the scope of this report.
In conclusion, we have established that specific hepatic fatty acid elongases, Elovl-5 and Elovl-6, are regulated in liver by nutrients (glucose and fat), hormones (insulin), and nuclear receptor agonists (i.e., LXR and PPAR agonist). ChREBP, MLX, SREBP-1, PPAR, and LXR control both elongase and desaturase expression. Only 9D is independently regulated by LXR. Metabolic diseases, such as diabetes and obesity, induce changes in hepatic lipid composition by controlling the function of key transcription factors that affect elongase and desaturase expression. These studies support the notion that the regulation of both fatty acid elongase and desaturase expression may play an important role in managing hepatic lipid composition in response to changes in dietary and hormonal status.
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ACKNOWLEDGMENTS |
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Manuscript received April 19, 2006 and in revised form June 9, 2006 and in re-revised form June 20, 2006.
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REFERENCES |
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(PPAR) in HEK293 cells. J. Biol. Chem. 277: 39243–39250. in rat primary hepatocytes. J. Biol. Chem. 278: 35931–35939.
