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Reduction of leptin gene expression by dietary polyunsaturated fatty acids

Open AccessPublished:May 01, 2001DOI:https://doi.org/10.1016/S0022-2275(20)31636-9

      Abstract

      Supplementation with n-3 polyunsaturated fatty acids (PUFA) for 6 weeks did not alter plasma leptin concentrations in male smokers. Changes in dietary intake of saturated fatty acids (FA) correlated positively, whereas changes in the intake of PUFA correlated negatively to changes in plasma leptin levels. A 3-week n-3 PUFA-enriched diet, as compared with a 3-week lard-enriched diet, induced lower plasma leptin concentration and reduced leptin mRNA expression in rat epididymal adipose tissue. In the human throphoblast cell line (BeWo), n-3 PUFA had a dose- and time-dependent effect on leptin expression. One mM of eicosapentaenoic acid or docosahexaenoic acid (DHA) reduced leptin expression by 71% and 78%, respectively, as compared with control, after 72 h. There was no effect on expression of the signal transducing form of the leptin receptor. In BeWo cells transfected with the human leptin promoter, we found that n-3 PUFA reduced leptin promoter activity; in contrast saturated and monounsaturated FA had no effect on leptin promoter activity. The transcription factors peroxysomal proliferator activated receptor γ and sterol regulatory element binding protein-1 mRNAs were reduced after incubation with n-3 PUFA, whereas the expression of CCAAT/enhancer binding protein α was unchanged. DHA-reduced leptin expression was abolished in BeWo cells grown in cholesterol-free medium.
      In conclusion, n-3 FA decreased leptin gene expression both in vivo and in vitro. The direct effects of PUFA on leptin promoter activity indicate a specific regulatory action of FA on leptin expression.
      Leptin is a hormone of marked importance for regulation of the amount of body fat (
      • Zhang Y.
      • Proenca R.
      • Maffei M.
      • Barone M.
      • Leopold L.
      • Friedman J.M.
      Positional cloning of the mouse obese gene and its human homologue.
      ). It is expressed and secreted in proportion to adipose mass and circulates in plasma in a concentration highly correlated to body fat mass (
      • Maffei M.
      • Halaas J.
      • Ravussin E.
      • Pratley R.E.
      • Lee G.H.
      • Zhang Y.
      • Fei H.
      • Kim S.
      • Lallone R.
      • Ranganathan S.
      • Kern P.A.
      • Friedman J.M.
      Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects.
      ,
      • Considine R.V.
      • Sinha M.K.
      • Heiman M.L.
      • Kriaugiunas A.
      • Stephens T.W.
      • Nyge M.R.
      • Ohannesian J.P.
      • Margo C.C.
      • McKee L.J.
      • Bauer T.L.
      • Caro J.F.
      Serum immunoreactive-leptin concentrations in normal-weight and obese humans.
      ). Plasma leptin levels are acutely regulated by fasting and refeeding (
      • Kolaczynski J.W.
      • Considine R.V.
      • Ohannesian J.
      • Marco C.
      • Opentanova I.
      • Nyce M.R.
      • Myint M.
      • Caro J.E.
      Responses of leptin to short-term fasting and refeeding in humans: a link with ketogenesis but not ketones themselves.
      ,
      • Saladin R.
      • De Vos P.
      • Guerre-Millo M.
      • Leturque A.
      • Girard J.
      • Staels B.
      • Auwerx J.
      Transient increase in obese gene expression after food intake or insulin administration.
      ). A relationship between meals and diurnal variations of plasma leptin concentration has been reported, in which plasma leptin concentrations corresponded directly to the shift in meal timing (
      • Schoeller D.A.
      • Cella L.K.
      • Sinha M.K.
      • Caro J.F.
      Entrainment of the diurnal rhythm of plasma leptin to meal timing.
      ). Meal composition and intake of nutrients might also affect plasma leptin concentrations and thereby contribute to the large individual variations. Potential modifiers of plasma leptin levels are energy-yielding nutrients such as fatty acids (FA), carbohydrates, proteins, and alcohol. Cooling, Barth, and Blundell (
      • Cooling J.
      • Barth J.
      • Blundell J.
      The high-fat phenotype: is leptin involved in the adaptive response to a high fat (high energy) diet?.
      ) found a higher plasma leptin level among individuals on a high fat diet compared with those on a low fat diet, and observed a correlation between plasma leptin levels and the dietary intake of fat. They hypothesized that differences in plasma leptin concentration between individuals with similar body composition could be due to dietary intake of fat. Long-term changes in diet, including decreased intake of saturated and increased intake of polyunsaturated fat, over a period of one year, reduced plasma leptin concentration in humans beyond the reduction expected as a result of changes in fat mass (
      • Reseland J.E.
      • Anderssen S.A.
      • Solvoll K.
      • Hjermann I.
      • Urdal P.
      • Holme I.
      • Drevon C.A.
      Effect of long-term changes in diet and exercise on plasma leptin concentrations.
      ). The type of fat in the habitual diet has also been found to influence the plasma leptin concentration in humans (
      • Rojo-Martinez G.
      • Soriguer F.J.
      • Gonzalez-Romero S.
      • Tinahones F.
      • Moreno F.
      • de Adana S.R.
      • Garriga M.J.
      • Esteva I.
      • Garcia-Arnes J.
      • Gomez-Zumaquero J.M.
      • Garcia-Almeida J.M.
      Serum leptin and habitual fatty acid dietary intake in patients with type 1 diabetes mellitus.
      ).
      It is also well documented that dietary intake of marine n-3 FA as compared with saturated FA may affect energy metabolism in rats after long-term feeding (
      • Rustan A.C.
      • Hustvedt B.E.
      • Drevon C.A.
      Dietary supplementation of very long-chain n-3 fatty acids decreases whole body lipid utilization in the rat.
      ,
      • Rustan A.C.
      • Hustvedt B.
      • Drevon C.A.
      Postprandial decrease in plasma unesterified fatty acids during n-3 fatty acid feeding is not caused by accumulation of fatty acids in adipose tissue.
      ). High intake of n-3 FA promotes reduced plasma concentration of triacylglycerol, glycerol, and free FA, as well as reduced size in adipocytes in epididymis and perirenal tissue (
      • Parrish C.C.
      • Pathy D.A.
      • Parkes J.G.
      • Angel A.
      Dietary fish oils modify adipocyte structure and function.
      ).
      Eicosapentaenoic acid (EPA) and dicosahexaenoic acid (DHA) are poor substrates for enzymes responsible for esterification to triacylglycerol (
      • Rustan A.C.
      • Nossen J.O.
      • Christiansen E.N.
      • Drevon C.A.
      Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A:1,2-diacylglycerol acyltransferase.
      ). Marine FAs reduce synthesis and increase oxidation of fatty acid (
      • Willumsen N.
      • Skorve J.
      • Hexeberg S.
      • Rustan A.C.
      • Berge R.K.
      The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis.
      ). It has also been shown that n-3 FAs are ligands for peroxysomal proliferator activated receptor α (PPARα) and PPARγ and can heterodimerize with retinoid X receptor to increase expression of FA oxidizing enzymes, and reduce expression of sterol regulatory element binding protein (SREBP)-1, promoting reduced lipogenesis (
      • Clarke S.D.
      Polyunsaturated fatty acid regulation of gene transcription: a mechanism to improve energy balance and insulin resistance.
      ). Because n-3 FA seem to influence fatty acid metabolism markedly and leptin is important for adipose tissue size, we examined the effects of different FA on leptin expression in vivo and in vitro in men and rats.

      MATERIALS AND METHODS

      Subjects and study design

      Forty-two male smokers with combined hyperlipidemia were recruited through a continuous screening of risk factors at Ullevål hospital in Oslo, Norway. Inclusion criteria were males, 40–60 years of age, cholesterol levels of 6–9 mM, and triacylglycerol levels of 2–5 mM (
      • Brude I.R.
      • Drevon C.A.
      • Hjermann I.
      • Seljeflot I.
      • Lund-Katz S.
      • Saarem K.
      • Sandstad B.
      • Solvoll K.
      • Halvorsen B.
      • Arnesen H.
      • Nenseter M.S.
      Peroxidation of LDL from combined-hyperlipidemic male smokers supplied with omega-3 fatty acids and antioxidants.
      ). The study was carried out as a randomized, double-blinded, placebo-controlled trial aimed to assess the effects of marine n-3 FA and antioxidants, alone or in combination, over a period of 6 weeks. All the subjects were told to stop their intake of cod liver oil, fish oil, and vitamin supplementation at least 3 months prior to the start of the study. The participants were randomly allocated to one of four groups receiving supplementation with either 5 g of EPA (20:5 n-3) and DHA (22:6 n-3) (n = 11); a mixture of 75 mg vitamin E, 150 mg vitamin C, 15 mg β-carotene, and 30 mg Coenzyme Q10 (n = 11); a combination of n-3 FA and antioxidants (n = 11); or control oil (n = 8). The FA control capsule contained 5 g of oil with a FA pattern similar to an ordinary Norwegian diet, whereas the antioxidant control capsule contained peanut oil only. Counts of unused capsules indicated that overall compliance was satisfactory, with less than 2% of the provided capsules returned. Assessment of dietary intake of food and nutrients was performed by a self-administered, quantitative food-frequency questionnaire that has been extensively validated (
      • Nes M.
      • Frost A.L.
      • Solvoll K.
      • Sandstad B.
      • Hustvedt B.E.
      • Lovo A.
      • Drevon C.A.
      Accuracy of a quantitative food frequency questionnaire applied in elderly Norwegian women.
      ,
      • Andersen L.F.
      • Solvoll K.
      • Johansson L.R.
      • Salminen I.
      • Aro A.
      • Drevon C.A.
      Evaluation of a food frequency questionnaire with weighed records, fatty acids, and alpha-tocopherol in adipose tissue and serum.
      ,
      • Solvoll K.
      • Lundw-Larsen K.
      • Søyland E.
      • Sandstad B.
      • Drevon C.A.
      A quantitative food frequency questionnaire evaluated in a group of dermatologic outpatients.
      ). All the subjects smoked more than 10 cigarettes per day. To standardize the smoking prior to blood sampling, all blood samples were drawn after an overnight fast and 90 min after smoking 2–3 cigarettes, both at baseline and after 6 weeks of supplementation. One subject did not complete the study owing to non-fatal myocardial infarction; all others completed the study without major problems. One person refused to fill out the food-frequency questionnaire after 6 weeks of supplementation.
      Informed consent was obtained from all the subjects, and the study protocol was approved by the Regional Committee of Medical Ethics and by the Norwegian health authorities.

      Animals and diets

      Twelve male Wistar rats (SPF, Mol) (214.0 ± 1.7 g) were purchased from Møllegaard Breeding Centre (Ejby, Denmark). The temperature in the animal quarters was 24 ± 1°C, and the humidity was 55 ± 3%. The dark period was from 19:00–07:00. The rats had free access to tap water.
      After habituation to commercial rat chow for 10 days, the rats were randomly divided into two groups (n = 6 in each group), transferred to cages with 2 animals in each, and offered one of two semi-synthetic diets ad libitum. The diets were either lard (19.5%) (Agro Fellesslakteriet, Norges Kjøtt og Fleske sentral, Oslo, Norway) or very long-chain n-3 FAs (13% lard and 6.5% n-3 FA concentrate) (K85, Omacor, batch #1020 from Pronova A/S, Oslo, Norway) (
      • Rustan A.C.
      • Hustvedt B.E.
      • Drevon C.A.
      Dietary supplementation of very long-chain n-3 fatty acids decreases whole body lipid utilization in the rat.
      ) (Table 1). In addition, 1.5% soybeanoil was provided to both dietary groups to prevent essential FA deficiency. The composition of nutrients in weight as % of total was as follows: sucrose 20; cornstarch 31.5; casein 20; cellulose 1; vitamin mixture 1.5; salt mixture 5; fat 21.5. Both diets provided 40% of the energy from fat. Diets were stored at −20°C and given to the rats in one-day portions. The experimental protocol was essentially as described previously (
      • Rustan A.C.
      • Hustvedt B.E.
      • Drevon C.A.
      Dietary supplementation of very long-chain n-3 fatty acids decreases whole body lipid utilization in the rat.
      ,
      • Rustan A.C.
      • Hustvedt B.
      • Drevon C.A.
      Postprandial decrease in plasma unesterified fatty acids during n-3 fatty acid feeding is not caused by accumulation of fatty acids in adipose tissue.
      ,
      • Halvorsen B.
      • Rustan A.C.
      • Madsen L.
      • Reseland J.E.
      • Berge R.K.
      • Sletnes P.
      • Christiansen E.N.
      Effect of monounsaturated and long-chain n-3 fatty acids on fatty acid oxidation and lipid composition in rats.
      ).
      TABLE 1.FA composition of the semisynthetic rat diets (% of total FA)
      FALardn-3 FA
      14:01.61.2
      16:026.118.2
      16:1 (n-7)1.71.2
      18:016.310.9
      18:1 (n-9)34.223.8
      18:1 (n-7)2.21.6
      18:2 (n-6)15.011.6
      18:4 (n-3)0.8
      20:00.20.2
      20:2 (n-6)0.2
      20:4 (n-6)0.11.3
      20:4 (n-3)0.2
      20:5 (n-3)0.115.1
      22:4 (n-3)0.4
      22:5 (n-6)0.2
      22:5 (n-3)0.10.8
      22:6 (n-3)0.28.9
      Others2.13.9
      1.5% soybean oil was added to both diets. Fatty acid (FA) composition was determined by gas-liquid chromatography, and data are presented as average of two measurements (% of total FA). The lard diet contained 19.5% lard; the n-3 FA diet contained 13% lard, 6.5% K85. K85 is a very long-chain n-3 FA ethylester concentrate of EPA (54.2%) and DHA (32.8%) (Omacor, Pronova A/S).

      Plasma analysis

      Blood samples were collected with 0.2% EDTA and immediately chilled on ice. Plasma was prepared and stored at −70°C prior to analysis. Enzymatic kits from Sigma Diagnostics (St. Louis, MO) and Wako Chemicals GmbH (Neuss, Germany) were used to measure the plasma concentration of triacylglycerol and unesterified FA, respectively. Glycerol concentration in plasma was measured fluorimetrically (
      • Laurell S.
      • Tibbling G.
      An enzymatic fluorometric micromethod for the determination of glycerol.
      ). The results from these experiments are published elsewhere (
      • Halvorsen B.
      • Rustan A.C.
      • Madsen L.
      • Reseland J.E.
      • Berge R.K.
      • Sletnes P.
      • Christiansen E.N.
      Effect of monounsaturated and long-chain n-3 fatty acids on fatty acid oxidation and lipid composition in rats.
      ). Concentration of leptin in plasma was measured by a competitive radioimmunoassay (Linco Research, St. Charles, MO) with recombinant 125I-leptin as tracer (
      • Ma Z.
      • Gingerich R.L.
      • Santiago J.V.
      • Klein S.
      • Smith C.H.
      • Landt M.
      Radioimmunoassay of leptin in human plasma.
      ). Intra-assay variation was 5.5% for the human leptin kit and 1.4% for the rat leptin kit.

      Adipose rat tissue

      Adipose tissue samples (1.5–2 g) were taken from epididymal fat depots and immediately clamp frozen in liquid nitrogen. Adipose homogenate was prepared from 250 mg frozen epididymal tissue using an ultrasonic processor (Vibra cell) in 1 ml lysis/binding buffer. mRNA was isolated as described below.

      Cell culture

      3T3-L1 cells (American Type Culture Collection, ATCC) were cultured at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (Integrob, Zaandam, the Netherlands) and 50 U/ml penicillin and 50 μg/ml streptomycin (Biowhittaker, Walkerville, MD). Differentiation assays were performed as described by others (
      • Benito M.
      • Porras A.
      • Nebreda A.R.
      • Santos E.
      Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes.
      ), and the tests were performed 10 days after the start of differentiation. The human throphoblast cell line, BeWo (ATCC #CCL-98), was grown in Nutrient Mixture Ham's F-12 (Gibco BRL, Paisley, UK) supplemented with 10% FCS and 50 U/ml penicillin and 50 μg/ml streptomycin. For RT-PCR or transfection experiments, cells were split into 12-well culture plates and grown to 60% confluence prior to incubation with FA. Cells incubated for northern blot analysis were cultured in 75 cm2 culture flasks.
      The cells were incubated with medium containing either 0.2–0.4 mM bovine serum albumin (BSA) or 0.5–1 mM FA [palmitic acid (PA), 16:0; oleic acid (OA), 18:1 n-9; EPA, 20:5 n-3; and DHA, 22:6 n-3] with a FA:BSA ratio of 2.5:1. Fresh medium with FA was added every day, and the cells harvested after 24, 48, or 72 h. Viability tests showed that 95 ± 2% of the transfected cells were viable after 72-h incubation with either BSA or FA. The final results are presented as % of BSA control.

      Transient transfection

      BeWo cells were transfected using Lipofectamine Plus as described in the protocol from the manufacturer (Gibco BRL). Three-hundred eighty nanograms of a reporter vector with a human leptin promoter fragment inserted (pGL3-ob1 and pGL3-ob2, generous gifts from Prof. Johan Auwerx, Department d'Atherosclerose/U325 INSERM, Institute Pasteur, Lille, France) (
      • Miller S.G.
      • De Vos P.
      • Guerre-Millo M.
      • Wong K.
      • Hermann T.
      • Staels B.
      • Briggs M.R.
      • Auwerx J.
      The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression.
      ) and 130 ng transfection control plasmid (pSEAP2) (Clontech, Palo Alto, CA) were added to cells in each well and incubated for 3 h.

      Cell harvest and measurements of luciferase activity

      Aliquots of the medium were removed every day for determination of alkaline phosphatase activity from the transfection control plasmid. Medium samples were stored at −20°C prior to analysis using the Great EscAPe SEAP Chemiluminescence Detection kit (Clontech) in a luminometer (Turner Designs Luminometer Model TD-20). At 72 h the cells were washed twice with PBS and lysed in 150 μl Reporter Lysis buffer (Promega, Madison, WI). The lysate was centrifuged to remove cell debris and stored at −70°C until measurements were performed. Five μl of the cell extract was used in each assay with the Luciferase detection kit (Promega). The relative luciferase activity (LUC) was calculated as the LUC/SEAP activity. Each experiment was performed in triplicate and repeated more than three times.

      mRNA isolation and semi-quantitative RT-PCR

      Tissue homogenate and cells were lysed in lysis/binding buffer (100 mM Tris-HCl pH 8.0, 500 mM LiCl, 10 mM EDTA pH 8.0, 0.5 mM DTT, 1% SDS). mRNA was isolated using magnetic beads [oligo (dT)25] as described by the manufacturers (Dynal AS, GenoVision, Oslo, Norway). Beads containing mRNA were resuspended in 10 mM Tris-HCl pH 8.0 and stored at −70°C until use. One μl of the mRNA suspension was used in each RT-PCR. The GeneAmp EZ rTth RNA PCR kit (Perkin Elmer, Applied Biosystems, Foster City, CA) was used for the RT-PCR and 2 μCi α-32P-CTP was added in each reaction. Temperature cycles were as follows: 60°C for 30 min, 94°C for 1 min followed by 30–35 cycles of 94°C for 30 s, and 60°C for 1.5 min. At the end the samples were incubated at 60°C for 7 min.
      Oligonucleotide sequences of sense and antisense primers are as follows:
      Human leptin, estimated product size 197 bp: 5′-GGCTTTGGCCCTATCTTTTC-3′,5′-GGATAAGGTCAGGATGGGGT-3′
      Rat leptin was amplified using a murine leptin primer set, estimated product size 250 bp: 5′-AGCAGTGCCTATCCAGAAAGT-3′,5′-TTCTCCAGGTCATTGGCTAT-3′
      The signaling form of human leptin receptor, OB-Rb, estimated size 417 bp: 5′-GCCAGAGACAACCCTTTGTTAAA-3′,5′-TGGAGAACTCTGATGTC-CGTGAA-3′
      G3PDH, estimated product size 452 bp: 5′-ACCACAGTCCATGCCATCAC-3′,5′-TCCACCACCCTGTTGCTGTA-3′
      PPARγ, estimated size 400 bp: 5′-CAGTGGGGATGCTCATAA-3′,5′-CTTTTGGCATACTCTGTGAT-3′
      CCAAT/enhancer C/EBPα, estimated product size 638 bp: 5′-CCTTCAACGACGAGTTCCTG-3′,5′-CTCGTTGCTGTTCTTGTCCA-3′
      SREBP-1 (common region of SREBP 1a, b, and c), estimated size 601 bp: 5′-CGGAGAAGCTGCCTATCAAC-3′,5′-CAGGACAGGC AGAGGAAGAC-3′
      The G3PDH cDNA product was used to normalize the mRNA loading in the analysis. PCR products were analyzed on a 2% agarose gel, and the cDNA bands were excised from the gel and allowed to elute for 2 h in scintillation liquid before counting in a liquid scintillation counter. Relative abundance of the different mRNA was calculated as the ratio between the PCR products of the gene of interest and G3PDH for each sample, and presented as % of control.

      Northern blotting and hybridization

      mRNA from one 75 cm2 cell culture flask was separated on 1% agarose gels containing formaldehyde and blotted onto positively charged nylon membranes (Amersham-Pharmacia Biotech, Uppsala, Sweden). Probes that are used for hybridization were cloned from the PCR products described above using the Original TA cloning kit (Invitrogen, Carlsbad, CA). All probes were confirmed by DNA sequencing. Hybridization was performed in Church buffer (0.5 M Na-phosphate, 7% SDS, 1 mM EDTA) at 65°C. Hybridization signals were analyzed in a Phospho Imager (Molecular Dynamics, Sunnyvale, CA) and normalized to the signals for G3PDH.

      Activation of SREBP

      BeWo cells were starved of cholesterol using lipoprotein deficient serum (LPDS) (
      • Drevon C.A.
      • Engelhorn S.C.
      • Steinberg D.
      Secretion of very low density lipoproteins enriched in cholesteryl esters by cultured rat hepatocytes during simulation of intracellular cholesterol esterification.
      ) for 24 h. Sterols regulate the cleavage of SREBP (
      • Nohturfft A.
      • Brown M.S.
      • Goldstein J.L.
      Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N.
      ) into the active form of the protein. BeWo cells were incubated in Hams F12 medium containing 10% LPDS for 24 h prior to incubation with FA for a period of 48–72 h as described above. The expression of leptin mRNA was compared with cells incubated in media containing 10% FCS.

      Statistics

      Mean values (±SD) are presented. Non-parametric statistical methods were chosen, as most of the variables were skewed and the number of observations was limited. The Wilcoxon Signed Rank test was used when comparing data before and after supplementation, and correlations were tested using the Spearman Rank Sum test. When data were normally distributed, parametrical statistical tests were performed. Statistical significance was set to P < 0.05.

      RESULTS

      Effect of dietary FA on plasma leptin concentration and leptin expression in vivo

      We observed no significant effects of placebo-controlled dietary supplementation with n-3 FA and/or antioxidants on plasma leptin concentration or body weight in men.Plasma concentration of leptin at baseline ranged from 0.5 to 37 μg/l with a mean of 9.2 ± 7.9 μg/l. Baseline leptin concentration for all individuals (n = 41) correlated to body weight (r = 0.477, P < 0.001) and body mass index (BMI) (r = 0.534, P < 0.001).
      Plasma leptin concentration correlated negatively with dietary intake of PUFA, but failed to be statistically significant (P = 0.053, n = 41). No correlation was found with the intake of total energy or other nutrients (Table 2). Furthermore, we observed a positive correlation (P = 0.003) between the changes (difference between week 6 and baseline values) in dietary intake of saturated FA (as % of total fat) and the change in plasma leptin concentration in the n-3 FA group (n = 10) (Table 3). A significant negative correlation (P = 0.03) was found between changes in the intake of PUFA and changes in plasma leptin concentration. In the combined n-3 FA/antioxidant group, no correlation between change in plasma leptin concentration and change in dietary intake of fat was observed. When combining the two groups receiving n-3 FA supplementation (n = 21), the changes in dietary intake of saturated FA correlated positively (P = 0.03), and changes in dietary intakeof PUFA correlated negatively (P = 0.02), to changes in plasma leptin concentrations (Table 3).
      TABLE 2.Nutrient intake at baseline correlated (Spearman) to plasma leptin concentration
      VariablesBaseline (n = 40)rP
      Energy intake (MJ)10.0 ± 3.1−0.074ns
      Protein (g)97.1 ± 31.3−0.019ns
      Carbohydrate (g)262.5 ± 74.7−0.163ns
      Fat (g)92.0 ± 47.2−0.012ns
      Saturated fat (%)37.0 ± 5.70.201ns
      Monounsaturated fat (%)36.2 ± 5.70.163ns
      PUFA (%)19.1 ± 4.7−0.3040.053
      PUFA, n-6 (%)74.4 ± 6.20.100ns
      PUFA, n-3 (%)15.8 ± 4.9−0.196ns
      Alcohol (g)16.8 ± 15.80.022ns
      β-Carotene (mg)3.62 ± 3.47−0.103ns
      Retinol (mg)1.18 ± 0.83−0.142ns
      Vitamin C (mg)126 ± 780.003ns
      Vitamin D (mg)7.4 ± 5.8−0.168ns
      Calcium (mg)940 ± 437−0.110ns
      Iron (mg)11.9 ± 3.50.008ns
      n-6; (C18:2 + C20:4) in percentage of PUFA. n-3; (C18:3 + C20:5 + C22:5 + C22:6) in percentage of PUFA. ns, nonsignificant.
      TABLE 3.Change in dietary intake of fat and FA correlated to change in plasma leptin concentration in men after 6 weeks of supplementation with n-3 FA and antioxidants
      GroupsnFatSaturated FAPUFA
      g%%
      Control8nsnsns
      n-3 FA10ns0.83
      P < 0.001.
      −0.68
      P < 0.05.
      Antioxidants11nsnsns
      n-3 FA/antioxidants11nsnsns
      n-3 FA combined21ns0.47
      P < 0.05.
      −0.50
      P < 0.05.
      Antioxidant combined22nsnsns
      Data are presented as Spearman Rank Order correlation factors.
      a P < 0.05.
      b P < 0.001.
      Because changes in n-3 FA intake correlated negatively to changes in plasma leptin concentration among men, we examined the effect of n-3 FA-enriched diets on plasma leptin concentration in rats. We observed a lower plasma leptin level in rats fed an n-3 FA-enriched diet (8.6 ± 3.4 ng/ml, n = 6) as compared with a lard-enriched diet (10.6 ± 4.9 ng/ml, n = 6), but the difference did not reach significance (Fig. 1A). However, the relative expression of leptin mRNA in epididymal adipose tissue was lower in the n-3 FA group than in the lard group (P = 0.012) (Fig. 1B). No significant differences in weight gain (380.1 ± 33.9 g/2 animals in the lard group, 337.1 ± 34.9 g/2 animals in the n-3 FA group) was found between the two feeding groups.
      Figure thumbnail gr1
      Fig. 1.Plasma leptin concentration (A) and relative concentration of leptin mRNA (B) in epididymal adipose tissue in rats fed either lard-enriched or n-3 FA-enriched diets for 3 weeks.

      Effect of FA on leptin expression in vitro

      Because of the reducing effect of dietary PUFA on leptin mRNA concentration in vivo, we measured the effects of different FAs on leptin expression in vitro. For this purpose we used the murine 3T3-L1 and human placental throphoblast (BeWo) cell lines, both known to express leptin. In cultured BeWo cells we observed that both EPA (20:5 n-3) and DHA (22:6 n-3) reduced the relative expressionof leptin mRNA to 29 ± 9% (P < 0.001) and 22 ± 21% (P < 0.003) of control, respectively (Fig. 2). However, PA (16:0) (168 ± 72%) and OA (18:1 n-9) (81 ± 26%) had no significant effect on the expression of leptin mRNA. We observed similar results in the murine 3T3-L1 cells, where 0.5 mM EPA and DHA reduced the leptin mRNA to 64 ± 7% and 49 ± 3% of control after 48 h, respectively, whereas PA (77 ± 43%) and OA (94 ± 45%) had no significant effect (results not shown). Incubation with FA had no effect on the expression of the long signal transducing form of the human leptin receptor (OB-Rb) in BeWo cells (Fig. 2).
      Figure thumbnail gr2
      Fig. 2.RT-PCR amplified mRNAs of leptin, leptin receptor (OB-Rb), and G3PDH in BeWo cells after 24-, 48-, and 72-h incubation with: 1) control with medium, 2) 0.4 mM BSA, 3) 1 mM PA (16:0), 4) 1 mM OA (18:1 n-9), 5) 1 mM EPA (20:5 n-3), and 6) 1 mM DHA (22:6 n-3).
      Constructs of the leptin promoter linked to a luciferase reporter gene were used in transiently transfected BeWo cells to study human leptin promoter activity in response to different FAs. Leptin promoter activity of the pGL3-ob1 construct (containing 2.9 kb of the human leptin promoter) was reduced by incubation with EPA to 28 ± 19% (P < 0.001) and DHA to 17 ± 9% (P < 0.001) of control, whereas incubation with PA (106 ± 51%) and OA (121 ± 87%) had no significant effects (Fig. 3A). A similar expression pattern was found for the pGL3-ob2 construct (containing 0.2 kb of the proximal human leptin promoter), in which EPA reduced the luciferase activity to 28 ± 19% (P < 0.006) and DHA to 18 ± 19% (P < 0.002) of control, whereas PA (114 ± 50%) and OA (117 ± 24%) had no significant effects (Fig. 3B). The results indicate a specific reduction of leptin gene expression by n-3 PUFA as compared with saturated and monones.
      Figure thumbnail gr3
      Fig. 3.The effect of 1 mM PA (16:0), OA (18:1 n-9), EPA (20:5 n-3), and DHA (22:6 n-3) in BeWo cells transfected with the pGL3-ob1 (A) or pGL3-ob2 (B) constructs containing 2.9 and 0.2 kb of the human leptin promoter, respectively, linked to the luciferase gene (presented as % of control).
      The pGL3-ob2 construct of the leptin promoter contains an E-box (
      • Tontonoz P.
      • Kim J.B.
      • Graves R.A.
      • Spiegelman B.M.
      ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation.
      ,
      • Murre C.
      • McCaw P.S.
      • Baltimore D.
      A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins.
      ) with the potential to bind SREBP and a functional C/EBP element (
      • He Y.
      • Chen H.
      • Quon M.J.
      • Reitman M.
      The mouse obese gene. Genomic organization, promoter activity, and activation by CCAAT/enhancer-binding protein alpha.
      ), but no sequence similarity was observed to any known peroxisome proliferator-activated receptor element (PPRE) (
      • Hwang C.S.
      • Mandrup S.
      • MacDougald O.A.
      • Geiman D.E.
      • Lane M.D.
      Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.
      ). Extensive cross-talk between these transcriptional factors has been demonstrated (
      • Willumsen N.
      • Skorve J.
      • Hexeberg S.
      • Rustan A.C.
      • Berge R.K.
      The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis.
      ,
      • Kim J.B.
      • Wright H.M.
      • Wright M.
      • Spiegelman B.M.
      ADD1/SREBP1 activates PPARgamma through the production of endogenous ligand.
      ,
      • Hollenberg A.N.
      • Susulic V.S.
      • Madura J.P.
      • Zhang B.
      • Moller D.E.
      • Tontonoz P.
      • Sarraf P.
      • Spiegelman B.M.
      • Lowell B.B.
      Functional antagonism between CCAAT/Enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma on the leptin promoter.
      ,
      • Hwang C.S.
      • Loftus T.M.
      • Mandrup S.
      • Lane M.D.
      Adipocyte differentiation and leptin expression.
      ,
      • Raclot T.
      • Groscolas R.
      • Langin D.
      • Ferre P.
      Site-specific regulation of gene expression by n-3 polyunsaturated fatty acids in rat white adipose tissues.
      ). The PPARγ agonist BRL 49653 reduced the luciferase activity in BeWo cells transfected with the pGL3-ob2 construct in a time- and dose-dependent manner. After 72-h incubation with 1 and 10 μM BRL 49653, the activity of the proximal part of the leptin promoter was reduced to 53 ± 13% and 35 ± 3% of control, respectively. Northern blot analysis showed that 0.5 mM OA, EPA, and DHA for 48 h reduced the expression of PPARγ mRNA to 55 ± 7%, 28 ± 6%, and 31 ± 11%, respectively (Fig. 4). SREBP-1 expression was also reduced to 55 ± 15%, 24 ± 10%, and 27 ± 4% by OA, EPA, and DHA, respectively, whereas no significant effect on C/EBPα expression was observed. The mature, nuclear form of SREBP-1 is known to increase when cells are starved of cholesterol. After activation of SREBP by starving the cells for cholesterol in LPDS-supplemented medium, we observed that most of the effect of DHA on leptin mRNA expression was abolished (Fig. 5). DHA reduced the leptinmRNA level to 24 ± 15% in BeWo cells cultured in FCS-supplemented medium, whereas the leptin mRNA level was not significantly different from the basal expression (82 ± 16%) when DHA was added to the LPDS-supplemented medium. This indicates that leptin expression is not reduced by DHA when cells are grown under choleserol-deficient conditions.
      Figure thumbnail gr4
      Fig. 4.Northern blot analysis of mRNA from BeWo cells after incubation with BSA, or 0.5 mM PA (16:0), OA (18:1 n-9), EPA (20:5 n-3), and DHA (22:6 n-3) for 48 h, hybridized to PPARγ, C/EBPα, SREBP-1, β-actin, and G3PDH.
      Figure thumbnail gr5
      Fig. 5.RT-PCR of leptin mRNA in BeWo cells after incubation with 0.5 mM PA (16:0) and DHA (22:6 n-3) in control cells (FCS) or cells starved in LPDS (presented as % of control).

      DISCUSSION

      We present data indicating that dietary n-3 PUFAs reduce human leptin mRNA expression both in vivo and in vitro. We observed a negative correlation between changes in plasma leptin concentration and dietary intake of PUFA, and a reduction in leptin mRNA in epididymal adipose tissue in rats fed n-3 FA-enriched diet compared with lard-fed rats. Moreover, incubation with PUFA reduced leptin mRNA levels and leptin promoter activity in vitro, indicating a regulation of leptin transcription by individual dietary FA.
      Short-term supplementation with n-3 FA and antioxidants, alone or in combination, had no effect on plasma leptin concentration in men.
      The lack of effect might be due to large individual variationsin plasma leptin concentration and the limited number of subjects supplemented (
      • Considine R.V.
      Weight regulation, leptin and growth hormone.
      ). However, when analyzing the relationship between changes in dietary intake of FA and plasma leptin level during the period of supplementation, a significant positive correlation was found to changes in the intake of saturated FA and a negative correlation to changes in the intake of PUFA (Table 3). The same correlation to changes in leptin was found when combining the groups receiving n-3 FA supplementation, further strengthening the physiological validity of the observation. The concentration of serum leptin in patients with type 1 diabetes mellitus have also been found to be influenced by the type of dietary fat in the diet (
      • Rojo-Martinez G.
      • Soriguer F.J.
      • Gonzalez-Romero S.
      • Tinahones F.
      • Moreno F.
      • de Adana S.R.
      • Garriga M.J.
      • Esteva I.
      • Garcia-Arnes J.
      • Gomez-Zumaquero J.M.
      • Garcia-Almeida J.M.
      Serum leptin and habitual fatty acid dietary intake in patients with type 1 diabetes mellitus.
      ). The results are also in accordance with our previous findings on the long-term effect of changes in dietary intake of fat (
      • Reseland J.E.
      • Anderssen S.A.
      • Solvoll K.
      • Hjermann I.
      • Urdal P.
      • Holme I.
      • Drevon C.A.
      Effect of long-term changes in diet and exercise on plasma leptin concentrations.
      ). We concluded that increased intake of PUFA and reduced intake of saturated fat over a period of one year reduced the plasma leptin concentration beyond what would be expected due to changes in body fat mass in men.
      Rats fed n-3 FA-enriched diets had a weight gain similar to that of lard-fed rats during the 3-week feeding period, but the leptin mRNA level in epididymal tissue in n-3 FA-fed rats was lower than in lard-fed rats. The lack of significant differences in plasma leptin concentration between the two groups of rats in our study may be due to small sample size and large individual variations. In rodents, leptin mRNA levels may vary in different adipose depots, with the highest expression of leptin in epididymal adipose tissue (
      • Rayner D.V.
      • Trayhurn P.
      Ob (obese) gene expression in white adipose tissue of obese Zucker (fa/fa) rats.
      ). But it is unclear whether epidydymal tissue contributes markedly to plasma leptin levels. Cha and Jones (
      • Cha M.C.
      • Jones P.J.
      Dietary fat type and energy restriction interactively influence plasma leptin concentration in rats.
      ) found that PUFA-enriched diets gave higher plasma leptin levels than diets rich in saturated fat, and that the serum leptin level was normalized by mild energy restriction in rats. Their results, like ours, indicate that dietary FA may influence circulating leptin levels. However, the diet composition, feeding pattern, and type of experimental species might influence plasma leptin concentrations, giving inconclusive results. There may be both species differences between rodents and humans, and tissue differences in the regulation of leptin expression.
      Fasting may induce changes in expression and localization of the leptin receptor, and nutrient availability may cause rapid alteration in the autoregulation of leptin expression (
      • Wang J.
      • Liu R.
      • Liu L.
      • Chowdhury R.
      • Barzilai N.
      • Tan J.
      • Rossetti L.
      The effect of leptin on Lep expression is tissue-specific and nutritionally regulated.
      ). Changes in expression of the leptin receptor might influence the transcription of leptin, but we found no change in the expression of the leptin receptor (OB-Rb) in our study, suggesting that the effect of PUFA on leptin mRNA is regulated independently of OB-Rb.
      The pattern of FA might affect the nuclear receptor PPARγ, which regulates transcription of leptin and several adipocyte-specific genes (
      • Kallen C.B.
      • Lazar M.A.
      Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes.
      ). De Vos et al. (
      • De Vos P.
      • Lefebvre A.M.
      • Miller S.G.
      • Guerre-Millo M.
      • Wong K.
      • Saladin R.
      • Hamann L.G.
      • Staels B.
      • Briggs M.R.
      • Auwerx J.
      Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor gamma.
      ) found that activation of PPARγ in rats fed a fish oil-enriched diet reduced epidydymal leptin mRNA levels by more than 30%. We also observed a reduction in PPARγ expression in cultured BeWo cells incubated with EPA and DHA amounting to approximately 70% of that in the control. The proximal part of the human leptin promoter contains no known PPRE, but the PPARγ agonist BRL 49653 reduced the promoter activity in transfected BeWo cells. The effect of n-3 FA on leptin mRNA levels was accompanied by a reduction in PPARγ and SREBP-1 mRNA levels, but it had no effect on C/EBPα mRNA expression estimated by northern blot analysis.
      A decrease in C/EBPα mRNA and leptin levels has previously been found in retroperitoneal adipose tissue in rats fed DHA and mixed fish oils, but not in rats fed EPA (
      • Raclot T.
      • Groscolas R.
      • Langin D.
      • Ferre P.
      Site-specific regulation of gene expression by n-3 polyunsaturated fatty acids in rat white adipose tissues.
      ). Sloop et al. (
      • Sloop K.W.
      • Surface P.L.
      • Heiman M.L.
      • Slieker L.J.
      Changes in leptin expression are not associated with corresponding changes in CCAAT enhancer binding protein-a.
      ) found no change in C/EBPα expression in rats during fasting or conditions that altered leptin gene expression. However, C/EBPα may modulate leptin expression by mechanisms that do not require changes in its own expression, e.g., via post-transcriptional modification (
      • An M.R.
      • Hsieh C.C.
      • Reisner P.D.
      • Rabek J.P.
      • Scott S.G.
      • Kuninger D.T.
      • Papaconstantinou J.
      Evidence for posttranscriptional regulation of C/EBPalpha and C/EBPbeta isoform expression during the lipopolysaccharide-mediated acute-phase response.
      ).
      Xu et al. (
      • Xu J.
      • Nakamura M.T.
      • Cho H.P.
      • Clarke S.D.
      Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.
      ) showed that PUFA suppress the expression of SREBP-1 and regulate SREBP-1 at the post-transcriptional level. We observed PUFA-induced changes in SREBP-1 mRNA levels in BeWo cells. PUFA may also decrease the transcription of SRE-regulated genes as well as the level of mature SREBP-1 protein, presumably by increasing the intracellular regulatory pools of cholesterol (
      • Worgall T.S.
      • Sturley S.L.
      • Seo T.
      • Osborne T.F.
      • Deckelbaum R.J.
      Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein.
      ). Starving the cells for cholesterol in LPDS media will activate the enzymatic cleavage and increase the levels of mature SREBP-1 in the nucleus (
      • Nohturfft A.
      • Brown M.S.
      • Goldstein J.L.
      Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N.
      ). In combination with increased mature SREBP-1, n-3 FA did not reduce the leptin mRNA level, which leads us to speculate that the PUFA-induced effect on leptin expression may involve a reduction in mature SREBP-1.
      In conclusion, we have shown that dietary FA composition may modify plasma leptin concentration in humans. In addition we have shown that PUFA decrease leptin gene expression both in vivo and in vitro, by mechanisms associated with reduced PPARγ and SREBP-1 gene expression.

      Acknowledgments

      The authors thank the Research Council of Norway, The Norwegian Council of Cardiovascular Disease, Petter Möller Orkla ASA, Johan Trone-Holst Foundation, Freia Chocolade Fabrics Medical Foundation, and the Norwegian Cancer Research Society for financial support.

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