Comparison of the expression and activity of the lipogenic pathway in human and rat adipose tissue
2003; Elsevier BV; Volume: 44; Issue: 11 Linguagem: Inglês
10.1194/jlr.m300235-jlr200
ISSN1539-7262
AutoresDominique Letexier, Claudie Pinteur, V. Large, Vincent Fréring, M. Beylot,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoLipogenesis is considered less active in human than in rat adipose tissue. This could be explained by different nutritional conditions, namely high-carbohydrate (HCHO) diet in rats and high-fat (HF) diet in humans. Adipose tissue was sampled (postabsorptive state) in rats and humans receiving HCHO or HF diets, ad libitum fed humans, and obese subjects. We measured 1) mRNA concentrations of fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), sterol regulatory element binding protein 1c (SREBP-1c), and carbohydrate response element binding protein (ChREBP), 2) SREBP-1c protein, and 3) FAS activity. FAS, ACC1, ChREBP, and SREBP1-c mRNA concentrations were unaffected by diet in humans or in rats. FAS and ACC1 mRNA levels were lower in humans than in rats (P < 0.05). FAS activity was unaffected by diet and was lower in humans (P < 0.05). SREBP-1c mRNA concentrations were similar in rats and humans, but the precursor and mature forms of SREBP-1c protein were less abundant in humans (P < 0.05). ChREBP mRNA concentrations were lower in humans than in rats.In conclusion, the lipogenic capacity of adipose tissue is lower in humans than in rats. This is not related to differences in diet and is probably explained by lower abundance of SREBP-1c protein. A decreased expression of ChREBP could also play a role. Lipogenesis is considered less active in human than in rat adipose tissue. This could be explained by different nutritional conditions, namely high-carbohydrate (HCHO) diet in rats and high-fat (HF) diet in humans. Adipose tissue was sampled (postabsorptive state) in rats and humans receiving HCHO or HF diets, ad libitum fed humans, and obese subjects. We measured 1) mRNA concentrations of fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), sterol regulatory element binding protein 1c (SREBP-1c), and carbohydrate response element binding protein (ChREBP), 2) SREBP-1c protein, and 3) FAS activity. FAS, ACC1, ChREBP, and SREBP1-c mRNA concentrations were unaffected by diet in humans or in rats. FAS and ACC1 mRNA levels were lower in humans than in rats (P < 0.05). FAS activity was unaffected by diet and was lower in humans (P < 0.05). SREBP-1c mRNA concentrations were similar in rats and humans, but the precursor and mature forms of SREBP-1c protein were less abundant in humans (P < 0.05). ChREBP mRNA concentrations were lower in humans than in rats. In conclusion, the lipogenic capacity of adipose tissue is lower in humans than in rats. This is not related to differences in diet and is probably explained by lower abundance of SREBP-1c protein. A decreased expression of ChREBP could also play a role. Adipose tissue and liver are the two main sites of de novo lipogenesis (DNL), which is the synthesis of fatty acid molecules from nonlipid substrates, mainly carbohydrates. DNL is considered a minor metabolic pathway in humans. Indeed, in healthy humans, hepatic DNL is a minor contributor to the fatty acids used for liver triglyceride (TG) synthesis and secretion (usually less than 1 g/day) (1Diraison F. Beylot M. Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification.Am. J. Physiol. 1998; 274: E321-E327PubMed Google Scholar, 2Diraison F. Yankah V. Letexier D. Dusserre E. Jones P. Beylot M. Differences in the regulation of adipose tissue and liver lipogenesis by carbohydrates in humans.J. Lipid Res. 2003; 44: 846-853Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 3Faix D. Neese R. Kletke C. Wolden S. Cesar D. Countlangus M. Shackleton C. Hellerstein M. 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To determine whether the differences between the two species previously reported were or were not related to differences in diet, rats were studied while receiving an HCHO or high-fat (HF) diet, and humans while receiving either a moderate HCHO or a moderate HF diet. Obese human subjects were also included. Because sterol regulatory element binding protein 1c (SREBP-1c) is a key transcription factor controlling the expression of the lipogenic pathway (18Foufelle F. Ferré P. New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c.Biochem. J. 2002; 366: 377-391Crossref PubMed Scopus (407) Google Scholar, 22Gondret F. Ferré P. Dugail I. ADD1/SREBP-1c is a major determinant of tissue differential lipogenic capacity in mammalian and avian species.J. Lipid Res. 2001; 42: 106-113Abstract Full Text Full Text PDF PubMed Google Scholar, 23Stoeckman A. Towle H. 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Finally, a new transcription factor mediating the stimulatory effect of glucose on the expression of lipogenic genes was recently purified from rat liver and called carbohydrate response element binding protein (ChREBP) (29Yamashita H. Takenoshita M. Sakurai M. Bruick R. Henzel W. Shillinglaw W. Arnot D. Yeda K. A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver.Proc. Natl. Acad. Sci. USA. 2001; 98: 9116-9121Crossref PubMed Scopus (548) Google Scholar, 30Uyeda K. Yamashita H. Kawaguchi T. Carbohydrate responsive element-binding protein (ChREBP): a key regulator of glucose metabolism and fat storage.Biochem. Pharm. 2002; 63: 2075-2080Crossref PubMed Scopus (170) Google Scholar). To date, the role of ChREBP has been firmly established only in rat and mouse liver and for the l-pyruvate kinase gene. To our knowledge, no data on the presence and possible role of ChREBP in adipose tissue are available. Therefore, we checked for and measured the mRNA concentrations of ChREBP in human and rat adipose tissue. Male Sprague Dawley rats (4 weeks old) were obtained from Iffacredo (l'Arbresle, France). After acclimating to the animal facility, they were separated into two groups. One group (n = 5) received a standard diet (rHCHO) (20% proteins, 10% lipids, 70% starch) and the other group (n = 5) an HF diet (rHF) {20% proteins, 60% lipids [74% PUFA; 20% monounsaturated fatty acid (MUFA); 6% saturated fatty acid (SFA)], 20% starch} for 2 weeks. All rats had free access to food. Rats on the HF diet did not gain more weight than those in the HCHO group during the 2 weeks. For each diet, rats were studied in the postabsorptive state (6 h after food withdrawal). All rats were anaesthetized with pentobarbital (6 μg/100 g body weight). Perirenal adipose tissue was quickly removed and snap frozen in liquid nitrogen. Samples were stored at −80°C until analysis. Written informed consent was obtained from 12 healthy subjects and seven obese patients after explanation of the nature, purpose, and possible risks of the study. The control group consisted of seven women and five men [aged 24 ± 2 years, body mass index (BMI) 21 ± 1]. No control subject had a personal or familial history of diabetes or obesity or was taking any medication; all had normal physical examinations and normal plasma glucose and lipid concentrations (Table 1). Subjects with unusual dietary habits were excluded. The obese group consisted of seven women (aged 29 ± 4 years, BMI 42 ± 2) with normal physical examinations except for excessive body weight. All had plasma glucose, lipid, and cholesterol levels within normal values but slightly above values of the control group (P < 0.05) (Table 1).TABLE 1BMI, hormonal, and metabolic parameters of human subjects measured in the postabsorptive stateControl SubjectsObese SubjectshHCHO SubjectshHF SubjectsSex (M/F)3/40/72/32/3Age (years) 24 ± 1.9929.33 ± 4.26 26.8 ± 2.8623.8 ± 1.47BMI (Kg/m2)21.36 ± 0.7642.56 ± 2.49*P < 0.05 versus all groups.20.12 ± 0.9221.3 ± 1.01Glucose (mM) 4.71 ± 0.14 5.60 ± 0.33*P < 0.05 versus all groups. 4.45 ± 0.114.47 ± 0.08Insulin (pM) 54 ± 9ND 43 ± 7 43 ± 8Cholesterol (mM) 4.37 ± 0.23 5.51 ± 0.42*P < 0.05 versus all groups. 3.96 ± 0.464.06 ± 0.45TG (mM) 0.83 ± 0.08 1.76 ± 0.18*P < 0.05 versus all groups. 0.75 ± 0.100.66 ± 0.16TG, triglyceride; BMI, body mass index; ND, not determined; HCHO, high-carbohydrate; HF, high-fat.* P < 0.05 versus all groups. Open table in a new tab TG, triglyceride; BMI, body mass index; ND, not determined; HCHO, high-carbohydrate; HF, high-fat. The protocols of the study were approved by the Ethical Committee of Lyon and the Institut National de la Santé et de la Recherche Médicale, and the study was conducted according to the Hurriet law. A first group of seven control subjects was studied once while consuming its usual diet (i.e., fed ad libitum). The other five control subjects were studied twice after 3 weeks of a controlled diet, either an HCHO diet (hHCHO) or an HF diet (hHF), with 4 months between the two studies. The order of the diets was randomized. The hHCHO and hHF diets were isoenergetic. The hHCHO diet provided 55% of total energy as carbohydrate (20–25% simple and 30–35% complex carbohydrate) and 30% as fat (10% SFAs, 10% MUFAs, and 10% PUFAs). The hHF diet provided 45% of total energy as lipids (15% SFAs, 15% MUFAs, and 15% PUFAs) and 40% as carbohydrate (same proportions of simple and complex carbohydrates as in the hHCHO diet). These diets were provided by a dietitian who met with each subject before each diet period to obtain a report of the subject's usual diet and to establish the subject's diet during the HF and HCHO controlled diet period. The dietitian met again with each subject at the end of the controlled diet periods. A detailed report of each subject's dietary intake during the last week of the controlled diet period was obtained, and the actual intakes were calculated using the Cuqual tables. For women, the test was performed during the first 10 days of the menstrual cycle in order to take into account the known variations of lipogenesis during the menstrual cycle (there are no menstrual variations for cholesterol synthesis) (3Faix D. Neese R. Kletke C. Wolden S. Cesar D. Countlangus M. Shackleton C. Hellerstein M. Quantification of menstrual and diurnal periodocities in rates of cholesterol and fat synthesis in humans.J. Lipid Res. 1993; 34: 2063-2075Abstract Full Text PDF PubMed Google Scholar). All subjects abstained from alcohol or heavy physical activity the week before the study. The evening before the test, in order to measure hepatic lipogenesis, the subjects drank a loading dose of deuterated water (3 g/kg body water; one-half after the evening meal and one-half at 10 PM). Until the end of the study, they drank only water enriched with 2H2O (4.5 g 2H2O/l of drinking water). All tests were initiated in the postabsorptive state after an overnight fast. At 7:30 AM, an indwelling catheter was placed in a forearm vein and blood samples were drawn for the various concentration and enrichment measurements. Thereafter, a sample of abdominal subcutaneous (SC) adipose tissue (150–250 mg) was obtained by needle biopsy under local anesthesia and immediately stored in liquid nitrogen. For obese patients, samples of omental (Om) and abdominal SC adipose tissues were obtained during the surgical placement of an adjustable gastric ring by laparoscopy under general anesthesia and immediately stored in liquid nitrogen until analysis. Metabolites were assayed using enzymatic methods on neutralized perchloric extracts of plasma (glucose) or on plasma (TG, cholesterol) (9Diraison F. Dusserre E. Vidal H. Sothier M. Beylot M. Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity.Am. J. Physiol. 2002; 282: E46-E51Crossref PubMed Google Scholar). Plasma insulin and glucagon concentrations were determined by radioimmunoassay. Measurements of deuterium enrichment in the palmitate of plasma triglycerides were performed as described in detail previously (1Diraison F. Beylot M. Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification.Am. J. Physiol. 1998; 274: E321-E327PubMed Google Scholar, 9Diraison F. Dusserre E. Vidal H. Sothier M. Beylot M. Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity.Am. J. Physiol. 2002; 282: E46-E51Crossref PubMed Google Scholar, 31Diraison F. Pachiaudi C. Beylot M. Measuring lipogenesis and cholesterol synthesis in humans with deuterated water: use of simple gas chromatography mass spectrometry techniques.J. Mass Spectrom. 1997; 32: 81-86Crossref PubMed Scopus (109) Google Scholar). Deuterium enrichment in plasma water was measured by the method of Yang et al. (32Yang D. Diraison F. Beylot M. Brunengraber Z. Samols M. Brunengraber H. Assay of low deuterium enrichment of water by isotopic exchange with [U-13C]acetone and gas chromatography mass spectrometry.Anal. Biochem. 1998; 258: 315-321Crossref PubMed Scopus (98) Google Scholar). The contribution of hepatic lipogenesis to the plasma pool of TG was calculated as previously described (1Diraison F. Beylot M. Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification.Am. J. Physiol. 1998; 274: E321-E327PubMed Google Scholar, 9Diraison F. Dusserre E. Vidal H. Sothier M. Beylot M. Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity.Am. J. Physiol. 2002; 282: E46-E51Crossref PubMed Google Scholar). Total RNA was extracted from adipose tissue samples using the RNeasy Mini Kit (Qiagen, Coutaboeuf, France). Concentration and purity were verified by measuring optimal density at 260 and 280 nm. Their integrity was checked by 1% agarose gel electrophoresis (Tebu, Le Peray en Yvelines, France). Id1, Id2, Id3, Nfy γ, Sp1, TF1, and YY1 mRNA concentrations were measured by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) using β-actin as a reference. Primer sequences are shown in Table 2. For each target mRNA, RT was performed from 0.04 μg of total RNA with 2.5 U of the thermostable reverse transcriptase (Tth DNA polymerase, Promega Corp., Charbonnières France) in 10 mM Tris-HCl (pH 8.3), 90 mM KCl, and 10 mM MnCl2 buffer with 4 pmol deoxynucleoside triphosphate and 15 pmol of the specific antisense primer, in a final volume of 20 μl. The reaction consisted of 10 min at 32°C, then 3 min at 60°C, followed by 15 min at 72°C and 5 min at 99°C. After chilling, 5 μl were used for the PCR reaction. The 5 μl of RT medium was added to 45 μl of PCR mix [10 mM Tris-HCl (pH 8.3), 10 mM KCl, 0.75 mM EGTA, 0.05% Tween®, 5% glycerol, and 25 mM MgCl2] containing 8 pmol of deoxynucleoside triphosphate, 2.5 U of Taq polymerase (Invitrogen, Cergy Pontoise, France), 15 pmol of corresponding antisense primers, and 22.5 pmol of sense primers. The PCR conditions were 2 min at 94°C followed by 35 cycles (1 min at 94°C, 1 min at 60°C, 1 min at 72°C) and 10 min at 72°C. The β-actin sense primer was added after five complete cycles of PCR. For the determination of ChREBP mRNA concentration, the number of cycles were 33 and 23 for ChREBP and β actin, respectively. Products were analyzed on agarose gel, post-stained with Gelstar® (Tebu). For quantitation of relative band intensities, scanning was performed with a fluorimager (Molecular Dynamics Instruments, CA), and the ratio of each target to β actin was determined for each sample with Gel Grab software (Clara Vision, Paris, France).TABLE 2Sequence of primers used for RT-PCRSenseAntisenseSize(5′ 3′)bpβ-actinGACGAGGCCCAGAGCAAGAGAGGGTGTTGAAGGTCTCAAACA225h Id1TGTCTGTCTGAGCAGAGCGTCTGATCTCGCCGTTCAGGGT310r Id1ATGAAGGTCGCCAGTAGCAGTCTGATCTCGCCGTTCAGGGT391Id2AACAGCCTGTCGGACCACAGTGCAAGGACAGGATGCTGAT314Id3TGCCTGTCGGAACGTAGCCTCTCCTCTTGTCCTTGGAGAT304Nfy γAGCAGCAGTGATGCCCAGCAAAGCTGCTGACCTTCTCCAACCTGCAT572Sp1TAGGAACAGCAACAACTCCCATGCACCTGGTATGATCTGTA408TF1GCAAGCACTACGGCCAATTCAGCTCGCAGATGTTCTCGAT384YY1CAGAAGCAGGTGCAGATCAAGATGTGCACAGACGTGGACTCTG370h SREBP-1cGCGGAGCCATGGATTGCACCTCTTCCTTGATACCAGGCCC311r SREBP-1cACGACGGAGCCATGGATTGTTTGATTGGAGGCCCAGGGG308ChREBPCTGGTGTCTCCCAAGTGGAACACCGCTGAAGAGGGAGTCAACCA702FASGGCCTGGACTCGCTCATGGGTGGGCCTGCAGCTGGGAGCA514ACC1GTTGCACAAAAGGATTTCAGCGCATTACCATGCTCCGCAC504r, rat; h, human; SREBP-1c, sterol regulatory element binding protein 1c; ChREBP, carbohydrate response element binding protein; FAS, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1. Open table in a new tab r, rat; h, human; SREBP-1c, sterol regulatory element binding protein 1c; ChREBP, carbohydrate response element binding protein; FAS, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1. Fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and SREBP-1c mRNA concentrations in human adipose tissue were measured by RT followed by competitive PCR as published previously (9Diraison F. Dusserre E. Vidal H. Sothier M. Beylot M. Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity.Am. J. Physiol. 2002; 282: E46-E51Crossref PubMed Google Scholar, 33Letexier D. Diraison F. Beylot M. Addition of inulin to a moderately high-carbohydrate diet reduces hepatic lipogenesis and plasma triacylglycerol concentration in humans.Am. J. Clin. Nutr. 2003; 77: 559-564Crossref PubMed Scopus (199) Google Scholar). For measurements of FAS and ACC1 mRNA concentrations in rat adipose tissue, we utilized the same method used with the human adipose tissue measurement, as the primer was designed for use in both species. We verified that the expected decrease in rat liver mRNA concentrations during fasting was observed for FAS (postabsorptive state: 103 ± 25 attomoles/μg total RNA, 24 h fasted: 1.1 ± 0.2) and ACC1 (postabsorptive state: 46 ± 11 attomoles/μg total RNA, 24 h fasted: 5 ± 2). For SREBP-1c mRNA, the competitor and the antisense primer used are the same for rats and humans, but the sense primer is different (see Table 2). We verified also that the expected decrease of SREBP-1c mRNA in the liver of fasted rats was found using this method of measurement (postabsorptive state: 6.9 ± 2.4 attomoles/μg total RNA, 24 h fasted: 0.9 ± 0.2) Amounts of Sp1 and SREBP-1c proteins were quantified by immunoblotting. Frozen tissue samples (∼200 mg) were crushed in liquid nitrogen and then homogenized in 0.6 ml of 0.25 M sucrose with 1 mM dithioerythritol, 1 mM EDTA, 20 μg/ml leupeptin, 20 μg/ml antipain, and 1 μg/ml pepstatin A, at pH 7 and 4°C. Fat-depleted infranatants were obtained after centrifugation at 12,000 g and 4°C for 3 h. Total protein infranatant was measured using BCA protein assay (Pierce, Rockford, IL). Aliquots of the infranatants (200 μg of total proteins) were adjusted to final concentrations of 0.008% bromophenol blue (w/v), 1% sodium dodecyl sulfate (w/v), and 7% glycerol (v/v) and applied to 8% polyacrylamide gels according to the Laemmli method under reducing conditions (3% β-mercaptoethanol). Prestained precision protein standards (Bio-Rad, Marne-la-coquette, France) were used as references. Afterward, electrophoresis proteins were transferred to nitrocellulose membrane. The blot was incubated in blocking buffer (2 h) and then with a primary antibody (1 h). Finally, the blot was incubated with a phosphatase alkaline-conjugated secondary antibody for 1 h and then briefly incubated with ECF (Amersham Pharmacia Biotechnology, UK). The relative amounts of immunodetectable proteins contained in each lane were determined by scanning with a fluorimager and Gel Grab software. The following primary antibodies were used: a rabbit polyclonal anti-Sp1 antibody (1:500) (Santa Cruz Biotechnology, CA) and a mouse monoclonal anti-SREBP-1c antibody (1:200) (IgG-2A4) produced by cell line (American Type Culture Collection, Manassas, VA) and purified from conditioned medium by protein G-Sepharose affinity chromatography as described by the manufacturer (Amersham Pharmacia Biotech). The primary antibodies were visualized with anti-rabbit (1:10,000) for Sp1 or anti-mouse IgG (1:20,000) for SREBP-1c. The activity of the FAS was determined with the method of Linn (34Linn T. Purification and crystallization of rat liver fatty acid synthase.Arch. Biochem. Biophys. 1981; 209: 613-619Crossref PubMed Scopus (92) Google Scholar). Briefly, frozen tissue samples (∼100 mg) were crushed in liquid nitrogen and then homogenized in 0.3 ml of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 250 mM sucrose, and protease inhibitor cocktail. Fat-depleted infranatants were obtained after centrifugation at 800 g and 4°C for 10 min. A second centrifugation was perf
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