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Tetradecylthioacetic acid prevents high fat diet induced adiposity and insulin resistance

Open AccessPublished:May 01, 2002DOI:https://doi.org/10.1016/S0022-2275(20)30116-4

      Abstract

      Tetradecylthioacetic acid (TTA) is a non-β-oxidizable fatty acid analog, which potently regulates lipid homeostasis. Here we evaluate the ability of TTA to prevent diet-induced and genetically determined adiposity and insulin resistance. In Wistar rats fed a high fat diet, TTA administration completely prevented diet-induced insulin resistance and adiposity. In genetically obese Zucker (fa/fa) rats TTA treatment reduced the epididymal adipose tissue mass and improved insulin sensitivity. All three rodent peroxisome proliferator-activated receptor (PPAR) subtypes were activated by TTA in the ranking order PPARα > PPARδ > PPARγ. Expression of PPARγ target genes in adipose tissue was unaffected by TTA treatment, whereas the hepatic expression of PPARα-responsive genes encoding enzymes involved in fatty acid uptake, transport, and oxidation was induced. This was accompanied by increased hepatic mitochondrial β-oxidation and a decreased fatty acid/ketone body ratio in plasma. These findings indicate that PPARα-dependent mechanisms play a pivotal role, but additionally, the involvement of PPARα-independent pathways is conceivable. Taken together, our results suggest that a TTA-induced increase in hepatic fatty acid oxidation and ketogenesis drains fatty acids from blood and extrahepatic tissues and that this contributes significantly to the beneficial effects of TTA on fat mass accumulation and peripheral insulin sensitivity.—Madsen, L., M. Guerre-Millo, E. N. Flindt, K. Berge, K. J. Tronstad, E. Bergene, E. Sebokova, A. C. Rustan, J. Jensen, S. Mandrup, K. Kristiansen, I. Klimes, B. Staels, and R. K. Berge. Tetradecylthioacetic acid prevents high fat diet induced adiposity and insulin resistance. J. Lipid Res. 2002. 43: 742–750.

      Supplementary key words

      Insulin resistance with ensuing hyperinsulinemia and dyslipidemia characterizes the metabolic syndrome, which eventually may develop into type II diabetes. Since most type II diabetic patients are obese, and obesity is virtually always associated with insulin resistance, a causal relationship has been suggested. Although it is generally assumed that high fat diets promote obesity, (
      • Golay A.
      • Bobbioni E.
      The role of dietary fat in obesity.
      ,
      • Berry E.M.
      Dietary fatty acids in the management of diabetes mellitus.
      ,
      • Vessby B.
      Dietary fat and insulin action in humans.
      ), it still remains uncertain how obesity may induce insulin resistance. In this respect, it is of interest that saturated and ω-6 unsaturated fatty acids in the diet may lead to insulin resistance in experimental animals, whereas ω-3 fatty acids prevents the development of insulin resistance (
      • Storlien L.H.
      • Kraegen E.W.
      • Chisholm D.J.
      • Ford G.L.
      • Bruce D.G.
      • Pascoe W.S.
      Fish oil prevents insulin resistance induced by high-fat feeding in rats.
      ,
      • Storlien L.H.
      • Jenkins A.B.
      • Chisholm D.J.
      • Pascoe W.S.
      • Khouri S.
      • Kraegen E.W.
      Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid.
      ,
      • Lichtenstein A.H.
      • Schwab U.S.
      Relationship of dietary fat to glucose metabolism.
      ). Particularly, polyunsaturated fatty n-3 acids have been shown to ameliorate metabolic dysfunctions, including improvement of insulin sensitivity (
      • Storlien L.H.
      • Kraegen E.W.
      • Chisholm D.J.
      • Ford G.L.
      • Bruce D.G.
      • Pascoe W.S.
      Fish oil prevents insulin resistance induced by high-fat feeding in rats.
      ,
      • Gasperikova D.
      • Klimes I.
      • Kolter T.
      • Bohov P.
      • Maassen A.
      • Eckel J.
      • Clandinin M.T.
      • Sebokova E.
      Glucose transport and insulin signaling in rat muscle and adipose tissue. Effect of lipid availability.
      ) and lowering of plasma triacylglycerol levels (
      • Harris W.
      n-3 fatty acids and serum lipoproteins: human studies.
      ).
      Results from both in vivo and in vitro experiments indicate that reduced triacylglycerol synthesis in and secretion from the liver due to increased fatty acid oxidation contribute to the hypolipidemic effect of n-3 fatty acids (
      • Wong S.H.
      • Nestel P.J.
      • Trimble R.P.
      • Stores G.B.
      • Illmann R.J.
      • Topping D.L.
      The adaptive effects of dietary fish and safflower oil on lipid and lipoprotein metabolism in perfused rat liver.
      ,
      • Rustan A.C.
      • Christiansen E.N.
      • Drevon C.A.
      Serum lipids, hepatic glycerolipid metabolism and peroxisomal fatty acid oxidation in rats fed ω−3 and ω−6 fatty acids.
      ,
      • Frøyland L.
      • Madsen L.
      • Vaagenes H.
      • Totland G.K.
      • Auwerx J.
      • Kryvi H.
      • Staels B.
      • Berge R.K.
      Mitochondrion is the principal target for nutritional and pharmacological control of triglyceride metabolism.
      ,
      • Berge R.K.
      • Madsen L.
      • Vaagenes H.
      • Tronstad K.J.
      • Göttlicher M.
      • Rustan A.C.
      In contrast to docosahexaenoic acid, eicosapentaenoic acid and hypolipidemic derivatives reduce hepatic synthesis and secretion of triacylglycerol by decreased 1,2-diacylglycerol acyltransferase activity and stimulation of fatty acid oxidation.
      ). Similarly, feeding the 3-thia substituted fatty acid {tetradecylthioacetic acid (TTA) [CH3-(CH2)13-S-CH2-COOH]} to rats causes a significant reduction of plasma triacylglycerol accompanied by increased mitochondrial and peroxisomal β-oxidation in the liver (
      • Berge R.K.
      • Hvattum E.
      Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-Thia fatty acids).
      ,
      • Vaagenes H.
      • Madsen L.
      • Asiedu D.K.
      • Lillehaug J.R.
      • Berge R.K.
      Early modulation of genes encoding peroxisomal and mitochondrial β-oxidation enzymes by 3-thia fatty acids.
      ). TTA is unable to undergo β-oxidation due to the sulfur substitution, but TTA is otherwise handled as a normal fatty acid and incorporated into triacylglycerols and phospholipids. Prolonged feeding of TTA to rats also changes the fatty acid composition in liver, heart, kidney, and adipose tissue (
      • Grav H.
      • Asiedu D.
      • Berge R.K.
      Gas chromatographic measurement of 3- and 4-thia fatty acids incorporated into various classes of rat liver lipids during feeding experiments.
      ,
      • Frøyland L.
      • Asiedu D.
      • Vaagenes H.
      • Garras A.
      • Lie Ø.
      • Totland G.K.
      • Berge R.K.
      Tetradecylthioacetic acid incorporated into very low density lipoprotein: changes in the fatty acid composition and reduced plasma lipids in cholesterol-fed hamsters.
      ,
      • Madsen L.
      • Frøyland L.
      • Grav H.
      • Berge R.K.
      Upregulated Δ9-desaturase gene expression by hypolipidemic peroxisome proliferating fatty acids resulted in increaced oleic acid content in liver and VLDL: accumulation of a Δ9-desaturated metabolite of tetradecylthioacetic acid.
      ).
      These observations prompted us to investigate whether TTA could prevent high fat diet induced insulin resistance in Wistar rats and reduce insulin resistance in obese Zucker (fa/fa) rats. In this report we demonstrate that TTA completely prevented high fat diet induced insulin resistance and adiposity. In obese Zucker (fa/fa) rats TTA also reduced adiposity and hyperglycemia, and markedly improved insulin sensitivity as determined by the intravenous glucose tolerance test. The exact mechanisms mediating these effects still remain to be deciphered. We show that TTA efficiently activates peroxisome proliferator-activated receptor (PPAR)α and PPARδ, whereas activation of PPARγ requires relatively high concentrations of TTA. In keeping with this we show that PPARα responsive genes, but not PPARγ responsive genes, are upregulated in TTA-treated rats, suggesting that TTA-dependent activation of PPARα is of importance.

      MATERIALS AND METHODS

      Animals

      All animal studies were conducted according to the Guidelines for the Care and Use of Experimental Animals, and the Local Animal Care Committees approved the protocols in the individual research centers.

      Obese Zucker (fa/fa) rats

      The young obese Zucker (fa/fa) rats (5 weeks old) used in this study were bred at the U465 INSERM animal facility from pairs originally provided by the Harriet G. Bird Laboratory (Stow, MA) and the old obese Zucker (fa/fa) rats (4 month old) were from IFFA-CREDO (France). The animals were maintained under a constant light-dark cycle (light from 7 AM to 7 PM) at 21 ± 1C° and were given free access to food and water. Three rats were housed per cage. Weight gain was recorded daily. In a first experiment, young male rats (5 weeks old) receiving a standard diet (UAR, Epinay/Orge, France) were given either 300 mg/kg/day TTA in 0.5% carboxymethyl cellulose (CMC) (n = 6) by oral gavage in the morning or an equal amount of CMC only (n = 6). After 11 days of treatment, rats were killed by cervical dislocation between 9 AM and 10 AM. Blood was collected. Liver and epididymal adipose tissue were dissected out and weighed. In a second series of experiments, the rats were given either a standard diet (n = 6) or a standard diet enriched with 0.15% TTA (n = 6) for 15 days (corresponding to approximately 150 mg/kg/day). Intravenous glucose tolerance tests were performed on these rats. In a third experiment, 4 months old obese Zucker rats were given a standard chow either with (n = 5) or without 0.15% wt/wt TTA in chow (n = 6) for 21 days.

      Wistar rats

      Male Wistar rats weighing 280–358 g were purchased from AnLab Ltd. (Prague, Czech Republic) and housed in wire-mesh cages at 22 ± 1°C with light from 7 AM to 7 PM. They were given free access to chow and water. Three rats were housed per cage. Weight gain and food intake were recorded daily. One group of animals was fed a standard pellet diet (ST1, Velaz Prague, Czech Republic) containing 10 cal% of fat, and is referred to as the control group. The second group received a high fat (HF) diet, containing 70 cal% fat prepared according to Storlien et al. (
      • Storlien L.H.
      • Baur L.A.
      • Kriketos A.D.
      • Pan D.A.
      • Cooney G.J.
      • Jenkins A.B.
      • Calvert G.D.
      • Campbell L.V.
      Dietary fats and insulin action.
      ). Fatty acid composition of the standard chow and the HF diets were as published earlier (
      • Klimes I.
      • Mitkova A.
      • Gasperikova D.
      • Ukropec J.
      • Liska B.
      • Bohov P.
      • Stanek J.
      • Sebokova E.
      The effect of the new oral hypoglycemic agnet A-4166 on glucose turnover in the high fat diet-induced and/or in the hereditary insulin resistance of rats.
      ). The third group received the HF diet with 0.4% wt/wt TTA in chow (corresponding to 400 mg/kg/day). Following 3 weeks of ad libitum feeding, rats were subjected to vascular surgery in preparation for in vivo measurements of insulin sensitivity (see below).
      In a second series of experiment, rats fed the same experimental diets for 3 weeks were used for the collection of blood and tissues.

      Physiological techniques

      Intravenous glucose tolerance tests

      Male Zucker (fa/fa) rats (5 weeks old) were fasted for 5 h and subsequently anesthetized at 2 PM by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The rats were injected with glucose (0.55 g/kg) in the saphenous vein and blood samples were collected from the tail vein in heparinized tubes at time 0, 5, 10, 15, 20, and 30 min after the glucose injection. Samples were kept on ice, centrifuged, and plasma was stored at −20 C° until analysis was performed.

      Hyperinsulinemic euglycemic clamp

      After 21 days on their respective diets (see above), the rats were anesthetized by injection of xylazine hydrochloride (Rometar SPOFA, Prague, Czech Republic; 10 mg/ml) and ketamine hydrochloride (Narkamon SPOFA, Prague, Czech republic; 75 mg/ml), and fitted with chronic carotid artery and jugular vein cannulas as described by Koopmans et al. (
      • Koopmans S.J.
      • Maassen A.J.
      • Radder J.K.
      • Frolich M.
      • Krans H.M.J.
      In vivo insulin resposiveness for glucose uptake at eu- and hyperglycemic levels in diabetic rats.
      ). The cannulated rats were allowed to recover for 2 days after surgery before the clamping studies, which were carried out according to Kraegen et al. (
      • Kraegen E.W.
      • James D.E.
      • Bennett S.P.
      • Chisholm D.J.
      Dose response curves for in vivo insulin sensitivity in individual tissues in rats.
      ). On the third day after surgery, unrestrained conscious rats were given a continuous infusion of porcine insulin (Actrapid, Novo Nordisk, Denmark) at a dose of 6.4 mU/kg/min to achieve plasma insulin levels in the upper physiological range. The arterial blood glucose concentration was clamped at the basal fasting level, by variable infusion of a 30% w/v glucose solution (Leciva, Prague, Czech Republic). Blood samples for determination of plasma glucose and insulin concentrations were collected every 15 min from the start of the glucose infusion. After 90 min, the rats were disconnected from the infusions and immediately decapitated. Blood was collected for plasma separation, and liver and epididymal adipose tissues were dissected out and weighed.

      Measurement of plasma parameters

      In Zucker (fa/fa) rats glucose (GLU, Boehringer Mannheim, Germany), free fatty acids (NEFA C ACS-ACOD kit; Wako Chemicals, Dalton, USA) and β-hydroxybutyrate (310-A kit; Sigma Diagnostics Inc., St. Louis) concentrations were measured using enzymatic methods, and insulin concentrations were determined with radioimmunoassay (CIS bio International, Gif sur Yvette, France) using rat insulin as standard. In the Wistar rats, plasma glucose concentrations were measured using a Beckman Glucose Analyzer (Fullerton, CA, USA). Plasma insulin levels were measured using a radioimmunoassay kit from Linco Research Inc. (St. Charles, MO, USA). Plasma triacylglycerol levels were measured using the Monotest triacylglycerol kit (Boehringer Mannheim, Germany)

      Preparation of post-nuclear and mitochondrial fractions and measurement of enzyme activities

      Freshly isolated livers from individual old Zucker rats were homogenized in ice-cold sucrose buffer [0.25 M sucrose, 10 mM HEPES (pH 7.4) and 2 mM EDTA]. Post-nuclear and mitochondrial fractions were prepared using preparative differential centrifugation according to DeDuve et al. (
      • De Duve C.
      • Pressman B.C.
      • Gianetto R.
      • Wattiaux R.
      • Appelmans F.
      Intracellular distribution patterns of enzymes in rat liver tissue.
      ). Acid soluble products were measured in post-nuclear and mitochondrial enriched fractions, using [1−14C]palmitoyl-CoA (Radiochemical Centre, Amersham, England) as substrates as described earlier (
      • Willumsen N.
      • Hexeberg S.
      • Skorve J.
      • Lundquist M.
      • Berge R.K.
      Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats.
      ). Carnitine palmitoyltransferase-I and -II activities were measured in the post-nuclear fractions essentially as described by Bremer (
      • Bremer J.
      The effect of fasting on the activity of liver carnitine palmitoyltransferase and its inhibition by malonyl-CoA.
      ) and 3-hydroxy-3-methylglutharyl-CoA synthase activity in the mitochondrial fractions was measured according to Clinkenbeard et al. (
      • Clinkenbeard K.D.
      • Reed W.D.
      • Mooney R.A.
      • Lane M.D.
      Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzyme A cycle enzymes in liver.
      ).

      RNA analysis

      RNA extraction (
      • Chomczynski P.
      • Sacchi N.
      Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
      ), and Northern blot and slot blot analysis were performed as earlier described (
      • Vaagenes H.
      • Madsen L.
      • Asiedu D.K.
      • Lillehaug J.R.
      • Berge R.K.
      Early modulation of genes encoding peroxisomal and mitochondrial β-oxidation enzymes by 3-thia fatty acids.
      ). The following cDNA fragments were used as probes: carnitine palmitoyltransferase (CPT)-I (
      • Esser V.
      • Britton C.H.
      • Weis B.C.
      • Foster D.W.
      • McGarry J.D.
      Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I.
      ) and CPT-II (
      • Woeltje K.F.
      • Esser V.
      • Weis B.C.
      • Sen A.
      • Cox W.F.
      • McPhaul M.J.
      • Slaughter C.A.
      • Foster D.W.
      • McGarry J.D.
      Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II.
      ). The relative levels of CPT-I and CPT-II RNA were determined by normalizing to the level of hybridization to 28S rRNA. The expression of liver fatty acid binding protein (L-FABP) and fatty acid transport protein (FATP) was analyzed by multiplex RT-PCR essentially as described (
      • Hansen J.B.
      • Petersen R.K.
      • Larsen B.M.
      • Bartkova J.
      • Alsner J.
      • Kristiansen K.
      Activation of peroxisome proliferator-activated receptor gamma bypasses the function of the retinoblastoma protein in adipocyte differentiation.
      ). Total RNA was reverse transcribed (M-MLV Reverse Transcriptase kit, Life Technologies) and selected mRNAs were amplified by 25 cycles of hot PCR with Tata binding protein serving as internal standard. The following primers were used. TBP: 5′-ACCCTTCACCAATGACTCCTATG-3′ and 5′-ATGATGACTGCAGCAAATCGC-3′; L-FABP: 5′-GCAAGTACCAACTGCAGAGC-3′ and 5′-CCAATGTCATGGTATTGGTGAT-3′; FATP: 5′-CATTGTGGTGCACAGCAGG-3′ and 5′-CATATTTCACCGATGTAGTGCAC-3′. Quantification was performed by phosphorimaging (Molecular Dynamics).

      Transfection analysis

      NIH-3T3 cells passaged in DMEM supplemented with 8% calf serum were transiently transfected at 50–60% confluence by the DC-Chol method (
      • Gao X.
      • Huang L.
      A novel cationic liposome reagent for efficient transfection of mammalian cells.
      ) with a total of 2.5 μg DNA per 9.6 cm2 well. The Gal4-mPPAR hinge region/ligand binding domain fusions used are described earlier (
      • Helledie T.
      • Antonius M.
      • Sørensen R.
      • Hertzel A.
      • Bernlohr D.
      • Kolvraa S.
      • Kristiansen K.
      • Mandrup S.
      Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm.
      ). One hundred twenty five nanograms of pcDNA1-Gal4-mPPAR, 500 ng 5xUAS-TK-luciferase reporter, and 50 ng pCMVβ (Clontech) were used per well. For pcDNA1-Gal4-mPPARδ, an equimolar amount of pCMX-mRXRα (kindly provided by R. M. Evans) (
      • Mangelsdorf D.J.
      • Borgmeyer U.
      • Heyman R.A.
      • Zhou J.Y.
      • Ong E.S.
      • Oro A.E.
      • Kakizuka A.
      • Evans R.M.
      Characterization of three RXR genes that mediate the action of 9-cis retinoic acid.
      ) was cotransfected. Empty expression vector was used to equalize plasmid load. Following exposure to liposomes for 6 h, cells were incubated for 20 h in DMEM supplemented with 10% resin-charcoal stripped CS and ligand/vehicle as indicated. β-galactosidase and luciferase activities from cell lysates were determined by use of the MicroLumat LB 96 P luminometer (EG&G Berthold). Luciferase values were normalized to the β-galactosidase values.

      RESULTS

      TTA prevents high fat diet induced increase in adipose tissue mass

      It is well established that high fat feeding induces obesity and may lead to the development of insulin resistance. Accordingly, the relative weight of both epididymal and retroperitoneal fat pads increased in Wistar rats fed a high fat diet (Table 1). Inclusion of TTA in the high fat diet prevented the relative increase in adipose tissue mass (Table 1) without a concomitant decrease in food consumption (high fat: 15.1 ± 1.1 vs. high fat + TTA: 14.8 ± 1.3 g/rat/day, n = 6). To further investigate the effect of TTA on fat accumulation, we treated obese Zucker (fa/fa) rats, a well-established genetic model for obesity and insulin resistance, with TTA. The body weight, as well as weight gain per day, was similar in control and TTA-treated rats during the 11 days of treatment (Table 1). However, the relative adipose tissue weight was lower in TTA-treated than in control rats (Table 2). The liver weight was, however, increased by TTA treatment in both animal models (Table 1 and 2), as observed in earlier studies (
      • Berge R.K.
      • Hvattum E.
      Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-Thia fatty acids).
      ).
      TABLE 1Influence of high fat diets with and without TTA supplement for 3 weeks on liver and adipose tissue weights in Wistar rats
      ParametersStandard
 Chow DietHigh Fat Diet
 −TTAHigh Fat Diet
 +TTA
      Epididymal Adipose Tissue/
 Body Weight (%)0.81 ± 0.03
      Different letters within a row indicates statistical significance (P < 0.05).
      1.29 ± 0.06
      Different letters within a row indicates statistical significance (P < 0.05).
      0.96 ± 0.07
      Different letters within a row indicates statistical significance (P < 0.05).
      Retroperitoneal Adipose
 Tissue/Body Weight (%)0.62 ± 0.08
      Different letters within a row indicates statistical significance (P < 0.05).
      1.36 ± 0.08
      Different letters within a row indicates statistical significance (P < 0.05).
      0.82 ± 0.05
      Different letters within a row indicates statistical significance (P < 0.05).
      Liver Weight (g)8.9 ± 0.6
      Different letters within a row indicates statistical significance (P < 0.05).
      9.8 ± 1.0
      Different letters within a row indicates statistical significance (P < 0.05).
      12.2 ± 1.4
      Different letters within a row indicates statistical significance (P < 0.05).
      Hepatic CPT-I Activity
 (nmol/min/mg protein)2.5 ± 0.2
      Different letters within a row indicates statistical significance (P < 0.05).
      3.1 ± 0.2
      Different letters within a row indicates statistical significance (P < 0.05).
      3.7 ± 0.3
      Different letters within a row indicates statistical significance (P < 0.05).
      Hepatic CPT-II Activity
 (nmol/min/mg protein)5.7 ± 0.6
      Different letters within a row indicates statistical significance (P < 0.05).
      7.0 ± 0.9
      Different letters within a row indicates statistical significance (P < 0.05).
      25.7 ± 2.5
      Different letters within a row indicates statistical significance (P < 0.05).
      Plasma Triacylglycerol (mM)0.66 ± 0.16
      Different letters within a row indicates statistical significance (P < 0.05).
      0.70 ± 0.16
      Different letters within a row indicates statistical significance (P < 0.05).
      0.40 ± 0.16
      Different letters within a row indicates statistical significance (P < 0.05).
      Data are means ± SEM of six animals in each group. Wistar rats (280–360 g) were fed three different diets (see Materials and Methods) for 3 weeks ad libitum. Afterwards, they were killed by decapitation; liver, retroperitoneal, and epididymal adipose tissue pads were dissected out and weighed.
      a,b Different letters within a row indicates statistical significance (P < 0.05).
      TABLE 2Influence of TTA on liver and adipose tissue weights, hepatic enzyme activities, and plasma parameters in young (A) and old (B) obese Zucker (fa/fa) rats
      ParametersControlTreated
      A.
      Epididymal Adipose Tissue/
 Body Weight (%)0.88 ± 0.020.78 ± 0.02
      P < 0.05 when compared to nontreated obese rats.
      Final Body Weight (g)186 ± 7177 ± 7
      Weight Gain (g/day)5.9 ± 0.46.2 ± 0.3
      Liver Weight (g)7.8 ± 0.310.6 ± 0.7
      P < 0.05 when compared to nontreated obese rats.
      B.
      FFA/Ketone Ratio0.40 ± 0.100.17 ± 0.09
      P < 0.05 when compared to nontreated obese rats.
      HMG-CoA Synthase Activity
 (nmol/min/mg protein)13 ± 427 ± 6
      P < 0.05 when compared to nontreated obese rats.
      Mitochondrial β-Oxidation
 (nmol/min/mg protein)1.3 ± 0.74.6 ± 1.2
      P < 0.05 when compared to nontreated obese rats.
      CPT-I
 Activity (nmol/min/mg protein)2.3 ± 0.54.5 ± 0.7
      P < 0.05 when compared to nontreated obese rats.
      mRNA (relative values)1.0 ± 0.22.4 ± 0.8
      P < 0.05 when compared to nontreated obese rats.
      CPT-II
 Activity (nmol/min/mg protein)14 ± 142 ± 13
      P < 0.05 when compared to nontreated obese rats.
      mRNA (relative values)1.0 ± 0.14.6 ± 0.9
      P < 0.05 when compared to nontreated obese rats.
      A: Five-week-old male obese Zucker (fa/fa) rats were fed 300 mg TTA/kg/day suspended in 0.5% CMC for 11 days. B: Four-month-old Zucker (fa/fa) rats were given a standard chow diet either with or without 0.15% TTA for 21 days. At the end of the experiments rats were killed by cervical dislocation; liver and epididymal adipose tissue pads were dissected out and weighed. Data are means ± SEM of six animals in each group.
      a P < 0.05 when compared to nontreated obese rats.

      TTA prevents high fat diet induced hyperinsulinemia

      Development of insulin resistance is known to be associated with hyperinsulinemia. Wistar rats fed a high fat diet to induce insulin resistance exhibited increased plasma insulin levels, compared with controls fed the standard chow diet (Fig. 1), yet the levels of plasma glucose were unchanged (not shown). The development of diet-induced hyperinsulinemia was completely prevented by inclusion of TTA in the diet (Fig. 1).
      Figure thumbnail gr1
      Fig. 1.Tetradecylthioacetic acid (TTA) treatment prevents high fat diet-induced hyperinsulinemia in Wistar rats. Wistar rats weighing 280–360 g were divided into three groups (n = 6) and fed with three different diets: standard chow, high fat diet (HF) and HF supplemented with TTA. After 21 days on the respective diets, blood was collected after an overnight fast from the tail vein. The data are presented as mean ± SEM. Results were analyzed by ANOVA and different letters denote statistical difference (P < 0.05).
      The obese Zucker (fa/fa) rats develop hyperinsulinemia spontaneously early in life and this defect worsens with age. TTA treatment reduced blood insulin concentrations in both 5 weeks and 4 months old obese Zucker (fa/fa) rats (Fig. 2). As expected, TTA had a marginal effect on plasma glucose levels in young normoglycemic animals (Fig. 2). The plasma glucose levels were, however, significantly reduced in 4 months old hyperglycemic obese rats treated with TTA (Fig. 2). Thus, the reduction in the insulin concentration observed after TTA treatment is not solely the result of decreased plasma glucose levels.
      Figure thumbnail gr2
      Fig. 2.TTA treatment reduces blood insulin and glucose concentrations in 5 weeks (A and B) and 4 months old (C and D) Zucker (fa/fa) rats. Five weeks old male obese Zucker (fa/fa) rats were fed 300 mg TTA kg/day suspended in 0.5% carboxymethyl cellulose (CMC) for 11 days. Control animals received CMC only. The 4 months old rats were given a standard chow either with (n = 5) or without (n = 6) 0.15% TTA for 21 days. At the end of both experiments blood was collected and the levels of insulin and glucose were measured. Data are means ± S.D. *Denotes statistical significant differences (Student's t-test) between control and TTA treated rats (P < 0.05).
      An intravenous glucose tolerance test performed in young normoglycemic obese Zucker rats demonstrated that TTA treatment resulted in a significantly lower plasma insulin response to glucose (Fig. 3A), whereas the kinetics of glucose clearance was similar in treated and untreated obese rats (Fig. 3B). This indicates that TTA improved insulin sensitivity.
      Figure thumbnail gr3
      Fig. 3.TTA treatment decreases the plasma insulin response to glucose in 5 weeks old Zucker (fa/fa) rats. The rats were given either a standard diet (open circle) (n = 6) or a standard diet enriched with 0.15% TTA (closed circle) (n = 6) for 15 days and subsequently submitted to an intravenous glucose tolerance test as described in Materials and Methods. Results are presented as the mean ± SEM. Area under the curve (AUC) for insulin: P < 0.01, AUC for glucose, not statistically significant.

      TTA prevents high fat diet induced insulin resistance in vivo

      To substantiate the insulin sensitizing effect of TTA, a 90 min euglycemic hyperinsulinemic clamp experiment was performed in Wistar rats fed a chow diet, a high fat diet, or a high fat diet supplemented with TTA. In keeping with the notion that high fat feeding leads to insulin resistance, the exogenous glucose infusion rate (GIR) required to maintain euglycemia in the high fat fed group was significantly reduced compared with that of the chow fed Wistar rats (Fig. 4). TTA completely prevented development of insulin resistance in these rats, as evidenced by a fully normal GIR in rats fed the high fat diet supplemented with TTA.
      Figure thumbnail gr4
      Fig. 4.TTA treatment prevents high fat diet-induced insulin resistance in Wistar rats. Rats weighing 330 ± 20 g were divided into three groups (n = 9) and fed with three different diets: standard rat chow, high fat diet (HF), and HF supplemented with TTA. After 21 days on the respective diets, a 90 min euglycemic hyperinsulinemic clamp was performed in unrestrained conscious animals as described in Materials and Methods. The glucose infusion rate (GIR) was determined after glucose levels had stabilized, i.e., between 45–90 min after initiation of the clamp. The data are presented as mean ± SEM. Results were analyzed by ANOVA and different letters denote statistical difference (P < 0.05).
      Elevated levels of plasma free fatty acids (FFA) characterize the fasted state of most obese subjects (
      • Gordon E.S.
      Non-esterified fatty acids in blood of obese and lean subjects.
      ,
      • Reaven G.M.
      • Hollenbeck C.
      • Jeng C.-Y.
      • Wu M.S.
      • Chen Y.-D.I.
      Measurment of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM.
      ) and have been associated with the development of insulin resistance (
      • Boden G.
      Role of fatty acids in the pathogenesis of insulin resistance and NIDDM.
      ,
      • Milburn J.L.
      • Hirose H.
      • Lee Y.H.
      • Nagasawa Y.
      • Ogawa A.
      • Ohneda M.
      • BeltrandelRio H.
      • Newgard C.B.
      • Johnson J.H.
      • Unger R.H.
      Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.
      ,
      • Boden G.
      Free fatty acids, insulin resistance, and type 2 diabetes mellitus.
      ). Interestingly, TTA prevented the increase in plasma FFA levels in fasted rats kept on a high fat diet (Fig. 5).
      Figure thumbnail gr5
      Fig. 5.TTA treatment reduces plasma levels of free fatty acids in Wistar rats fed a high fat diet. Rats weighing 330 ± 20 g were divided into three groups (n = 9) and fed with three different diets: standard rat chow, HF, and HF supplemented with TTA. The values are from decapitation after 16 h of fasting. The data are presented as % of control. Results were analyzed by ANOVA and different letters denote statistical difference (P < 0.05).

      TTA decreases plasma triacylglycerol levels and activates PPARα-dependent pathways in vivo

      It is suggested that elevated plasma triacylglycerol levels may inhibit peripheral glucose metabolism in humans (
      • Stouthard J.M.
      • Endert E.
      • Romijn J.A.
      Infusion of long-chain or medium-chain triglycerides inhibits peripheral glucose metabolism in men.
      ). TTA reduced the plasma triacylglycerol levels by 40% in high fat fed Wistar rats (Table 1) and by 35% in old genetically hypertriacylglycerolemic obese Zucker (fa/fa) rats (not shown). As expected, TTA-treatment led to increased mitochondrial β-oxidation as well as increased activities of mitochondrial CPT-I and II in both Wistar rats fed a high fat diet (Table 1) and Zucker (fa/fa) rats (Table 2). In Zucker (fa/fa) rats CPT-I and -II mRNA levels were concomitantly increased by TTA (Table 2). Similarly, TTA treatment increased the mRNA levels of PPARα target genes, such as L-FABP and FATP above those observed in the high fat fed rats (Fig. 6), suggesting in vivo activation of PPARα by TTA.
      Figure thumbnail gr6
      Fig. 6.TTA modulates gene expression of peroxisome proliferator-activated receptor (PPAR)α target genes in the liver of Wistar rats. Rats weighing 330 ± 20 g were divided into three groups (n = 9) and fed with three different diets: standard rat chow, high fat diet (HF) and HF supplemented with TTA. After 21 days on the respective diets, the livers were removed and RNA extracted. RNA-profiles for liver fatty acid binding protein (L-FABP) and fatty acid transport protein (FATP) were determined by phosphoimager analysis of multiple RT-PCR bands and normalized to TBP values. Determinations were repeated twice. Columns represent the average normalized values in the respective groups. Standard deviations are indicated. Results were analyzed by ANOVA and different letters denote statistical difference (P < 0.05).

      Activation of PPAR subtypes by TTA

      Simultaneous activation of PPARα and PPARγ has been shown to exert beneficial hypolipidemic and insulin sensitizing effects exceeding those observed with subtype-selective agonists (
      • Lefebvre A-M.
      • Peinado-Onsurbe J.
      • Leitersdorf I.
      • Briggs M.R.
      • Paterniti J.R.
      • Fruchart J.-C.
      • Fivert C.
      • Auwerx J.
      • Staels B.
      Regulation of lipoprotein metabolism by thiazolidinediones occurs through a distinct but complementary mechanism relative to fibrates.
      ,
      • Murakami K.
      • Tobe K.
      • Ide T.
      • Mochizuki T.
      • Ohashi M.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      A novel insulin sensitizer acts as a coligand for peroxisome proliferator-activated receptor-α (PPARα) and PPARγ.
      ,
      • Berger J.
      • Leibowitz M.D.
      • Doebber T.W.
      • Elbrecht A.
      • Zhang B.
      • Zhou G.
      • Biswas C.
      • Cullinan C.A.
      • Hayes N.S.
      • Li Y.
      • Tanen M.
      • Ventre J.
      • Wu M.S.
      • Berger G.D.
      • Mosley R.
      • Marquis R.
      • Santini C.
      • Sahoo S.P.
      • Tolman R.L.
      • Smith R.G.
      • Moller D.E.
      Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects.
      ). We have previously demonstrated that TTA is able to activate both human PPARα- and human PPARγ-mediated transactivation (
      • Raspé E.
      • Madsen L.
      • Lefebvre A.-M.
      • Leitersdorf I.
      • Peinado-Onsube J.
      • Dallongeville J.
      • Fruchart J.-C.
      • Berge R.
      • Staels B.
      Modulation of rat liver apoprotein gene expression and serum lipid levels by 3-thia fatty acids via PPARα activation.
      ), and hence, we evaluated the transactivation profile of TTA on rodent PPARs using Gal4-mouse PPAR hinge region/ligand binding domain chimeras for transient transfection analyses. Figure 7shows that TTA in a dose-dependent manner activated all three subtypes. However, the TTA concentrations needed to activate the PPARs were clearly subtype-dependent. PPARα-dependent transactivation was significantly enhanced by 10 μM TTA to a degree similar to or exceeding that observed by 100 μM WY14643. PPARδ required 100 μM TTA to achieve a 6–7-fold induction comparable to that obtained with 1 μM of the PPARδ-selective ligand L165041. Finally, activation of PPARγ required 100 μM TTA, and the level of transactivation was only approximately 20% of that observed with 1 μM BRL 49653. Thus, these results indicate that PPARα is a main target of TTA in the treated rats. It is possible that also PPARδ is activated, whereas activation of PPARγ at most is very modest. Thus, the potency and efficacy of TTA are reminiscent of those of naturally occurring polyunsaturated fatty acids, which have been shown to activate PPARα and PPARδ in the 10–100 μM range, whereas activation of PPARγ is either not detected or low (
      • Forman B.M.
      • Chen J.
      • Evans R.M.
      Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanois are ligands for peroxisome proliferator-activated receptors α and δ.
      ,
      • Kliewer S.A.
      • Sundseth S.S.
      • Jones S.A.
      • Brown P.J.
      • Wisely G.B.
      • Koble C.S.
      • Devchand P.
      • Wahli W.
      • Willson T.M.
      • Lenhard J.M.
      • Lehmann J.M.
      Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors α and γ.
      ).
      Figure thumbnail gr7
      Fig. 7.Mouse PPARs are activated by TTA NIH-3T3 cells were transiently transfected with a 5xUAS-TK-luciferase reporter, β-galactosidase-control, and either Gal4-mPPARα, Gal4-mPPARδ, or Gal4-mPPARγ expressing the Gal4 DNA binding domain fused to the hinge region/ligand binding domains of PPARα, PPARδ, or PPARγ, respectively. The retinoid X receptor (RXR) expression vector pCMX-mRXRα was included in transfection with Gal4-mPPARδ. Empty expression vector was included to achieve for equal plasmid load. Cells were treated with the vehicle alone (DMSO), 10, 30, or 100 μM TTA, or 100 μM Wy14643 (A), 1 μM L165041 (B), or 1.0 μM BRL49653 (C) as indicated. All transfections were performed a minimum of two times in triplicate and normalized to the β-galactosidase values. Results from one representative experiment are shown. The DMSO control values for each subtype were set equal to 1.
      In keeping with the results from the transfection experiments showing that a very high concentration of TTA was required to activate PPARγ, TTA treatment did not alter the expression of PPARγ target genes in adipose tissue. Northern blot analysis demonstrated that TTA did not affect the levels PEPCK mRNA (control; 100 ± 8 vs. TTA; treated 106 ± 12), leptin mRNA (control; 100 ± 9 vs. TTA treated; 97 ± 5) and lipoprotein lipase mRNA (control; 100 ± 9 vs. TTA treated; 108 ± 7) in epididymal adipose tissue of old obese Zucker rats.

      DISCUSSION

      Increased fat consumption is associated with a wide range of metabolic abnormalities, including hyperglycemia, dyslipidemia, and insulin resistance. In the present report, we demonstrate that inclusion of TTA in a high fat diet completely prevented the development of insulin resistance and adiposity. Moreover, we show that TTA markedly improved insulin sensitivity, as determined by intravenous glucose tolerance tests, and reduced adipose tissue mass in animal models of genetically determined and diet induced insulin resistance and obesity.
      While a clear link between adiposity and development of insulin resistance has been established, details of the underlying molecular mechanisms remain elusive. High levels of plasma FFA, a characteristic of most obese subjects, are suggested to act directly or indirectly as messengers (
      • Gordon E.S.
      Non-esterified fatty acids in blood of obese and lean subjects.
      ,
      • Reaven G.M.
      • Hollenbeck C.
      • Jeng C.-Y.
      • Wu M.S.
      • Chen Y.-D.I.
      Measurment of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM.
      ). In obese subjects, an increased rate of lipolysis from the expanded fat cell mass (
      • Bjorntorp P.
      • Bergman H.
      • Varnauskas E.
      Plasma free fatty acid turnover in obesity.
      ,
      • Jensen M.D.
      • Haymond M.W.
      • Rizza R.A.
      • Cryer P.E.
      • Miles J.M.
      Influence of body fat distribution on free fatty acid metabolism in obesity.
      ) would increase the plasma levels of FFA leading to inhibition of insulin action (
      • Boden G.
      Role of fatty acids in the pathogenesis of insulin resistance and NIDDM.
      ,
      • Milburn J.L.
      • Hirose H.
      • Lee Y.H.
      • Nagasawa Y.
      • Ogawa A.
      • Ohneda M.
      • BeltrandelRio H.
      • Newgard C.B.
      • Johnson J.H.
      • Unger R.H.
      Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.
      ,
      • Boden G.
      Free fatty acids, insulin resistance, and type 2 diabetes mellitus.
      ). Here we demonstrate that a high fat diet increased the plasma levels of FFA in the fasted state more than two fold, and notably, this increase was totally prevented by TTA. In a separate study, basal and β-adrenergic stimulated lipolysis in epididymal adipocytes from TTA-treated and untreated rats did not differ, and accordingly, no differences in the activity of hormone sensitive lipase were detected (A. Rustan and R. K. Berge, unpublished observations). Thus, we consider it unlikely that TTA reduced plasma FFA levels by decreasing lipolysis in fat. Increased fatty acid oxidation and ketone body formation accompanied by a decreased plasma FFA-ketone ratio rather suggest an increased flux of fatty acids to the liver. Increased fatty acid oxidation accompanied by reduced expression of apolipiprotein CIII (
      • Raspé E.
      • Madsen L.
      • Lefebvre A.-M.
      • Leitersdorf I.
      • Peinado-Onsube J.
      • Dallongeville J.
      • Fruchart J.-C.
      • Berge R.
      • Staels B.
      Modulation of rat liver apoprotein gene expression and serum lipid levels by 3-thia fatty acids via PPARα activation.
      ) as well as increased FABP and FATP mRNA levels will diminish the availability of substrates for triacylglycerol synthesis (
      • Berge R.K.
      • Hvattum E.
      Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-Thia fatty acids).
      ), thereby reducing the rate of fat accumulation.
      Increased triacylglycerol content in skeletal muscle is also related to insulin resistance and obesity (
      • Goodpaster B.H.
      • Kelley D.E.
      Role of muscle in triglyceride metabolism.
      ). By lowering plasma triacylglycerols, TTA may diminish the delivery of triacylglycerol to skeletal muscle. We have recently observed that TTA treatment reduced the volume fraction of fat droplets concomitantly with an induction of mitochondrial proliferation in skeletal muscle (
      • Totland G.K.
      • Madsen L.
      • Klementsen B.
      • Vaagenes H.
      • Kryvi H.
      • Frøyland L.
      • Hexeberg S.
      • Berge R.K.
      Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids.
      ). In agreement with this hypothesis, insulin stimulated glucose uptake was increased by 50% after TTA treatment in the epitrochlearis muscle in Zucker (fa/fa) rats (data not shown). Thus, the improved glucose homeostasis observed by TTA treatment might at least partly be explained by improved insulin action in skeletal muscle.
      How TTA exerts the observed effects on adiposity and insulin resistance is not yet known in detail. However, several of our findings clearly indicate that molecular mechanisms governing the action of TTA differ from those of thiazolidinediones, which are high affinity ligands of PPARγ and known to exert their effect as insulin sensitizers by virtue of their ability to activate this PPAR subtype. Thus, treatment of rats with thiazolidinediones markedly influences gene expression in adipose tissue (
      • Schoonjans K.
      • Peinado-Onsurbe J.
      • Lefebvre A.M.
      • Heyman R.A.
      • Briggs M.
      • Deeb S.
      • Staels B.
      • Auwerx J.
      PPARα and PPARγ activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene.
      ,
      • Hallakou S.
      • Doaré L.
      • Foufelle F.
      • Kergoat M.
      • Guerre-Millo M.
      • Berthault M.F.
      • Dugail I.
      • Morin J.
      • Auwerx J.
      • Ferré. P.
      Pioglitazone induces in vivo adipocyte differentiation in the obese Zucker fa/fa rat.
      ), whereas treatment with TTA, which we show is a poor PPARγ ligand, did not alter the expression of PPARγ target genes in adipose tissue in the obese Zucker rats. Moreover, thiazolidinedione treatment increases food intake and adipose tissue mass (
      • Hallakou S.
      • Doaré L.
      • Foufelle F.
      • Kergoat M.
      • Guerre-Millo M.
      • Berthault M.F.
      • Dugail I.
      • Morin J.
      • Auwerx J.
      • Ferré. P.
      Pioglitazone induces in vivo adipocyte differentiation in the obese Zucker fa/fa rat.
      ,
      • de Souza C.J.
      • Yu J.H.
      • Robinson D.D.
      • Ulrich R.G.
      • Meglasson M.D.
      Insulin secretory defect in Zucker fa/fa rats is improved by ameliorating insulin resistance.
      ,
      • 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 γ.
      ), whereas TTA did not change food intake and decreased adipose mass in obese Zucker rats.
      TTA treatment resulted in an increased expression of PPARα target genes. We show that TTA is a potent activator of rodent PPARα, and hence, PPARα-dependent processes most probably contribute significantly to the lowering of plasma FFA and plasma triacylglycerol, and the improved insulin sensitivity. Although it was recently reported that PPARα null mice were protected from insulin resistance (
      • Tordjman K.
      • Bernal-Mizrachi C.
      • Zemany L.
      • Weng S.
      • Feng C.
      • Zhang F.
      • Leone T.
      • Coleman T.
      • Kelly D.
      • Semenkovich C.
      PPARalpha deficiency reduces insulin resistance and atherosclerosis in apoE-null mice.
      ), our findings are in accordance with the reported beneficial effects on insulin sensitivity by administration of the PPARα activators of the fibrate class (
      • Matsui H.
      • Okumura K.
      • Kawakami K.
      • Hibino M.
      • Toki Y.
      • Ito T.
      Improved insulin sensitivity by bezafibrate in rats: relationship to fatty acid composition of skeletal-muscle triglycerides.
      ,
      • Ogawa S.
      • Takeuchi K.
      • Sugimura K.
      • Fukuda M.
      • Lee R.
      • Ito S.
      • Sato T.
      Bezafibrate reduces blood glucose in type 2 diabetes mellitus.
      ,
      • Guerre-Millo M.
      • Gervois P.
      • Raspé E.
      • Madsen L.
      • Poulain P.
      • Derudas B.
      • Herbert J.-M.
      • Winegar D.A.
      • Willson T.M.
      • Fruchart J.-C.
      • Berge R.K.
      • Staels B.
      Peroxisome proliferator-activated α activators improve insulin sensitivity and reduce adiposity.
      ,
      • Ye J.M.
      • Doyle P.J.
      • Iglesias M.A.
      • Watson D.G.
      • Cooney G.J.
      • Kraegen E.W.
      Peroxisome proliferator-activated receptor (PPAR)-alpha activation lowers muscle lipids and improves insulin sensitivity in high fat-fed rats: comparison with PPAR-gamma activation.
      ).
      Although PPARα appears to play an important role in the effects of TTA on lipid and glucose metabolism, it cannot be excluded that activation of other transcription factors, such as PPARδ, are involved in the regulation of glucose metabolism and improvement of insulin sensitivity. However, available evidence obtained in feeding experiments using PPARδ-selective ligands would argue that this is unlikely (
      • Berger J.
      • Leibowitz M.D.
      • Doebber T.W.
      • Elbrecht A.
      • Zhang B.
      • Zhou G.
      • Biswas C.
      • Cullinan C.A.
      • Hayes N.S.
      • Li Y.
      • Tanen M.
      • Ventre J.
      • Wu M.S.
      • Berger G.D.
      • Mosley R.
      • Marquis R.
      • Santini C.
      • Sahoo S.P.
      • Tolman R.L.
      • Smith R.G.
      • Moller D.E.
      Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects.
      ). It is likely, however, that TTA may exert PPAR independent effects. PPAR-independent effects of TTA on the growth of human keratinocytes (
      • Westergaard M.
      • Henningsen J.
      • Svendsen M.
      • Johansen C.
      • Jensen U.
      • Schroder H.
      • Kratchmarova I.
      • Berge R.
      • Iversen L.
      • Bolund L.
      • Kragballe K.
      • Kristiansen K.
      Modulation of keratinocyte gene expression and differentiation by PPAR-selective ligands and tetradecylthioacetic acid.
      ) and rat glioma cells (
      • Berge K.
      • Tronstad K.J.
      • Findt E.N.
      • Rasmussen T.H.
      • Madsen L.
      • Kristiansen K.
      • Berge R.K.
      Tetradecylthioacetic acid inhibits growth of rat glioma cells ex vivo and in vitro via PPAR-dependent and PPAR-independent pathways.
      ) have been recently documented. The effects of TTA on lipid homeostasis are in some aspects comparable to those of ω-3 polyunsaturated fatty acids (
      • Berge R.K.
      • Hvattum E.
      Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-Thia fatty acids).
      ). TTA is known to affect the lipid profile of cells and TTA in itself is a substrate for the Δ9 desaturase (
      • Madsen L.
      • Frøyland L.
      • Grav H.
      • Berge R.K.
      Upregulated Δ9-desaturase gene expression by hypolipidemic peroxisome proliferating fatty acids resulted in increaced oleic acid content in liver and VLDL: accumulation of a Δ9-desaturated metabolite of tetradecylthioacetic acid.
      ). Both TTA and its Δ9 desaturated product are incorporated into hepatic triacylglycerols and phospholipids (
      • Madsen L.
      • Frøyland L.
      • Grav H.
      • Berge R.K.
      Upregulated Δ9-desaturase gene expression by hypolipidemic peroxisome proliferating fatty acids resulted in increaced oleic acid content in liver and VLDL: accumulation of a Δ9-desaturated metabolite of tetradecylthioacetic acid.
      ). Therefore it is likely that other transcription factors and cellular signaling pathways that are affected by fatty acids are also influenced by TTA. Potential targets include the sterol regulatory element binding protein 1c (SREBP-1c) the activity of which is regulated by polyunsaturated fatty acids at the level of transcription, mRNA stability, and protein processing. Furthermore, it was recently demonstrated that mono and poly-unsaturated fatty acids could antagonize ligand-dependent liver X-activated receptors-mediated transactivation (
      • Ou J.
      • Tu H.
      • Shan B.
      • Luk A.
      • DeBose-Boyd R.
      • Bashmakov Y.
      • Goldstein J.
      • Brown M.
      Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR.
      ). Finally, as increased oxidative stress contributes to poor insulin action (
      • Paolisso G.
      • D'Amore A.
      • Volpe C.
      • Balbi V.
      • Saccomanno F.
      • Galzerano D.
      • Giugliano D.
      • Varricchio M.
      • D'Onofrio F.
      Evidence for a relationship between oxidative stress and insulin action in non-insulin dependent (type II) diabetes patients.
      ,
      • Gopaul N.K.
      • Änggård E.E.
      • Mallet A.I.
      • Betteridge D.J.
      • Wolff S.P.
      • Nourooz-Zadeh J.
      Plasma 8-epi-prostaglandin F levels are elevated in individuals with non-insulin dependent diabetes mellitus.
      ,
      • Rudich M.
      • Kozlovsky N.
      • Potashnic R.
      • Bashan N.
      Oxidant stress reduces insulin responsiveness in 3T3–L1 adipocytes.
      ), the antioxidant capacity of the sulfur atom (
      • Muna Z.A.
      • Doudin K.
      • Songstad J.
      • Ulvik R.J.
      • Berge R.K.
      Tetradecylthioacetic acid inhibits the oxidative modification of low density lipoprotein and 8-hydroxydeoxyguanosine formation in vitro.
      ) may contribute to the insulin sensitizing effect of TTA.
      Taken together, our findings indicate that PPARα-dependent mechanisms play a pivotal role, but additionally, the involvement of PPARα-independent pathways is conceivable. Our results suggest that TTA-induced increase in hepatic fatty acid oxidation and ketogenesis will drain fatty acids from blood and extrahepatic tissues and that this contribute significantly to the beneficial effects of TTA on fat mass accumulation and peripheral insulin sensitivity. The draining of fatty acids by the liver may relieve the fatty acid pressure on adipose tissue and muscle, where according to Randle (
      • Randle P.J.
      • Garland P.B.
      • Hales C.N.
      • Newsholme E.A.
      The glucose-fatty acid cycle: its role in insuline sensitivity and the metabolic disturbances of diabetes mellitus.
      ) glucose uptake and oxidation may be improved.

      Acknowledgments

      The authors acknowledge the technical contribution of Kari Williams, Kari Helland Mortensen, and Svein Kryger (Bergen, Norway), Bruno Derudas and Philippe Poulain (Lille, France), Alica Mitkova and Silvia Kuklova (Bratislava, Slovak), and Catherine Ilic, Virginie Guilter, and Jocelyne Andre (Paris, France). The work was supported by grants from Novo Nordisk Fonden, the Research Council of Norway, the Institut Pasteur de Lille, INSERM, the Région Nord-Pas de Calais, the Slovak Research Grant Agency (VEGA No. 2/4131/97), and grants from the Slovak Diabetes Association.

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