Diacylglycerol acyltransferase-1 inhibition enhances intestinal fatty acid oxidation and reduces energy intake in rats
2013; Elsevier BV; Volume: 54; Issue: 5 Linguagem: Inglês
10.1194/jlr.m035154
ISSN1539-7262
AutoresGudrun Schober, Myrtha Arnold, Susan Birtles, Linda K. Buckett, Gustavo Pacheco‐López, Andrew V. Turnbull, Wolfgang Langhans, Abdelhak Mansouri,
Tópico(s)Adipose Tissue and Metabolism
ResumoAcyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final step in triacylglycerol (TAG) synthesis and is highly expressed in the small intestine. Because DGAT-1 knockout mice are resistant to diet-induced obesity, we investigated the acute effects of intragastric (IG) infusion of a small molecule diacylglycerol acyltransferase-1 inhibitor (DGAT-1i) on eating, circulating fat metabolites, indirect calorimetry, and hepatic and intestinal expression of key fat catabolism enzymes in male rats adapted to an 8 h feeding-16 h deprivation schedule. Also, the DGAT-1i effect on fatty acid oxidation (FAO) was investigated in enterocyte cell culture models. IG DGAT-1i infusions reduced energy intake compared with vehicle in high-fat diet (HFD)-fed rats, but scarcely in chow-fed rats. IG DGAT-1i also blunted the postprandial increase in serum TAG and increased β-hydroxybutyrate levels only in HFD-fed rats, in which it lowered the respiratory quotient and increased intestinal, but not hepatic, protein levels of Complex III of the mitochondrial respiratory chain and of mitochondrial hydroxymethylglutaryl-CoA synthase. Finally, the DGAT-1i enhanced FAO in CaCo2 (EC50= 0.3494) and HuTu80 (EC50= 0.00762) cells. Thus, pharmacological DGAT-1 inhibition leads to an increase in intestinal FAO and ketogenesis when dietary fat is available. This may contribute to the observed eating-inhibitory effect. Acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final step in triacylglycerol (TAG) synthesis and is highly expressed in the small intestine. Because DGAT-1 knockout mice are resistant to diet-induced obesity, we investigated the acute effects of intragastric (IG) infusion of a small molecule diacylglycerol acyltransferase-1 inhibitor (DGAT-1i) on eating, circulating fat metabolites, indirect calorimetry, and hepatic and intestinal expression of key fat catabolism enzymes in male rats adapted to an 8 h feeding-16 h deprivation schedule. Also, the DGAT-1i effect on fatty acid oxidation (FAO) was investigated in enterocyte cell culture models. IG DGAT-1i infusions reduced energy intake compared with vehicle in high-fat diet (HFD)-fed rats, but scarcely in chow-fed rats. IG DGAT-1i also blunted the postprandial increase in serum TAG and increased β-hydroxybutyrate levels only in HFD-fed rats, in which it lowered the respiratory quotient and increased intestinal, but not hepatic, protein levels of Complex III of the mitochondrial respiratory chain and of mitochondrial hydroxymethylglutaryl-CoA synthase. Finally, the DGAT-1i enhanced FAO in CaCo2 (EC50= 0.3494) and HuTu80 (EC50= 0.00762) cells. Thus, pharmacological DGAT-1 inhibition leads to an increase in intestinal FAO and ketogenesis when dietary fat is available. This may contribute to the observed eating-inhibitory effect. Obesity is a global epidemic and a major risk factor for type 2 diabetes, hypertension, cardiovascular disease, and other ailments. The development of obesity is multifactorial (1Ogden C.L. Yanovski S.Z. Carroll M.D. Flegal K.M. The epidemiology of obesity.Gastroenterology. 2007; 132: 2087-2102Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar, 2Wilborn C. Beckham J. Campbell B. Harvey T. Galbreath M. La Bounty P. Nassar E. Wismann J. Kreider R. Obesity: prevalence, theories, medical consequences, management, and research directions.J. Int. Soc. Sports Nutr. 2005; 2: 4-31Crossref PubMed Google Scholar). Often an overconsumption of energy-dense, fat-rich food leads to excessive triacylglycerol (TAG) accumulation in adipose and nonadipose tissue. This can result in insulin resistance and nonalcoholic fatty liver disease, emphasizing the need for pharmacotherapeutic approaches that are effective when a high-fat diet (HFD) is consumed (3Speakman J.R. Obesity: the integrated roles of environment and genetics.J. Nutr. 2004; 134: 2090S-2105SCrossref PubMed Google Scholar). In the small intestine dietary TAGs are hydrolyzed to 2-monoacylglycerols (MAGs) and fatty acids (FAs) that are absorbed. Specific binding proteins in the enterocytes shuttle MAG and FFA to the endoplasmic reticulum for reesterification. This involves the acylation from MAG to sn-1,2-diacylglycerol (DAG) by MAG acyltransferase and the acylation from DAG to TAG catalyzed by acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) (EC 2.3.1.20), the rate-limiting step in TAG synthesis (4Shi Y. Burn P. Lipid metabolic enzymes: emerging drug targets for the treatment of obesity.Nat. Rev. Drug Discov. 2004; 3: 695-710Crossref PubMed Scopus (265) Google Scholar). This MAG pathway is important after eating, when intestinal MAG levels are high (5Shi Y. Cheng D. Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E10-E18Crossref PubMed Scopus (155) Google Scholar). DGAT-1 is one of two known DGAT enzymes (6Farese Jr, R.V. Cases S. Smith S.J. Triglyceride synthesis: insights from the cloning of diacylglycerol acyltransferase.Curr. Opin. Lipidol. 2000; 11: 229-234Crossref PubMed Scopus (121) Google Scholar) in tissues associated with TAG synthesis, including the small intestine (6Farese Jr, R.V. Cases S. Smith S.J. Triglyceride synthesis: insights from the cloning of diacylglycerol acyltransferase.Curr. Opin. Lipidol. 2000; 11: 229-234Crossref PubMed Scopus (121) Google Scholar, 7Cases S. 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Abumrad N.A. Farese Jr, R.V. DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis.J. Biol. Chem. 2002; 277: 25474-25479Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Expression of DGAT-1 only in the intestine of dgat-1−/− mice reverses the resistance to DIO and hepatic steatosis, suggesting that the beneficial effects of DGAT-1 inhibition are primarily due to an intestinal action (12Lee B. Fast A.M. Zhu J. Cheng J.X. Buhman K.K. Intestine-specific expression of acyl CoA:diacylglycerol acyltransferase 1 reverses resistance to diet-induced hepatic steatosis and obesity in Dgat1−/− mice.J. Lipid Res. 2010; 51: 1770-1780Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Selectively inhibiting intestinal DGAT-1 might therefore be a promising approach to treat hypertriglyceridemia and obesity (13King A.J. Judd A.S. Souers A.J. Inhibitors of diacylglycerol acyltransferase: a review of 2008 patents.Expert Opin. Ther. Pat. 2010; 20: 19-29Crossref PubMed Scopus (28) Google Scholar, 14Birch A.M. Buckett L.K. Turnbull A.V. DGAT1 inhibitors as anti-obesity and anti-diabetic agents.Curr. Opin. Drug Discov. Devel. 2010; 13: 489-496PubMed Google Scholar). Some effects of selective small molecule DGAT-1 inhibitors mimic the features of genetic DGAT-1 deletion, including the prevention of weight gain and the reduction of plasma TAG excursions after oral lipids, but the effects of transgenic deletion and pharmacological inhibition of DGAT-1 on eating appear to be different. Whereas dgat-1−/− mice display an increase in food intake, DGAT-1 inhibitors do not stimulate eating (14Birch A.M. Buckett L.K. Turnbull A.V. DGAT1 inhibitors as anti-obesity and anti-diabetic agents.Curr. Opin. Drug Discov. Devel. 2010; 13: 489-496PubMed Google Scholar, 15Zhao G. Souers A.J. Voorbach M. Falls H.D. Droz B. Brodjian S. Lau Y.Y. Iyengar R.R. Gao J. Judd A.S. et al.Validation of diacyl glycerolacyltransferase I as a novel target for the treatment of obesity and dyslipidemia using a potent and selective small molecule inhibitor.J. Med. Chem. 2008; 51: 380-383Crossref PubMed Scopus (120) Google Scholar, 16Qian Y. Wertheimer S.J. Ahmad M. Cheung A.W. Firooznia F. Hamilton M.M. Hayden S. Li S. Marcopulos N. McDermott L. et al.Discovery of orally active carboxylic acid derivatives of 2-phenyl-5-trifluoromethyloxazole-4-carboxamide as potent diacylglycerol acyltransferase-1 inhibitors for the potential treatment of obesity and diabetes.J. Med. Chem. 2011; 54: 2433-2446Crossref PubMed Scopus (36) Google Scholar). We examined the effects of a small molecule DGAT-1 inhibitor (DGAT-1i; Compound 2) (17Birch A.M. Birtles S. Buckett L.K. Kemmitt P.D. Smith G.J. Smith T.J. Turnbull A.V. Wang S.J. Discovery of a potent, selective, and orally efficacious pyrimidinooxazinyl bicyclooctaneacetic acid diacylglycerol acyltransferase-1 inhibitor.J. Med. Chem. 2009; 52: 1558-1568Crossref PubMed Scopus (65) Google Scholar) on food intake in rats fed HFD or standard chow and started to explore its mechanism of action. To test whether the eating inhibition observed after DGAT-1i administration in HFD-fed rats was related to metabolic effects, we measured circulating metabolites, respiratory quotient (RQ), and energy expenditure (EE) as well as protein expression of hepatic and intestinal enzymes related to lipid catabolism after DGAT-1i administration. In addition, we measured the effect of DGAT-1i on fatty acid oxidation (FAO) in intestinal epithelial cell culture models. A stimulatory effect of DGAT-1 inhibition on enterocyte FAO together with a reduction in food intake would be interesting because peripheral, in particular enterocyte, FAO has been implicated in the control of eating (18Langhans W. Leitner C. Arnold M. Dietary fat sensing via fatty acid oxidation in enterocytes: possible role in the control of eating.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2011; 300: R554-R565Crossref PubMed Scopus (57) Google Scholar). Male Sprague-Dawley rats (6–8 weeks old; Charles River, Sulzfeld, Germany) were housed individually in an air conditioned testing room (22 ± 2°C) with a 12 h light/12 h dark cycle (lights off at 1000 h). During at least 1 week adaptation to housing conditions before catheter implantation (see below) animals were fed ad libitum standard chow or a lard-enriched HFD (60% energy from fat) (Table 1). All experiments were performed in rats adapted to an 8 h feeding/16 h deprivation schedule (food access from 1000 to 1800 h) with continuous access to water unless otherwise indicated. Daily 8 h food intake and body weight (BW) were recorded. All procedures were approved by the Zurich Cantonal Veterinary Office.TABLE 1Diet content of the regular chow and the lard enriched high fat dietMajor NutrientsRegular Chow Diet,No. 3433aDiets purchased from Provimi Kliba Nafag, Kaiseraugst, Switzerland.High Fat Diet (60% kcal % fat), No. 2127aDry matter (%)88.092.0Crude protein (%)18.523.9Crude fat (%)4.535.0Crude fiber (%)4.54.9NFE (%)54.223.2Starch (%)351Crude ash (%)6.35.0Gross energy (MJ/kg)16.123.8Metabolizable energy (MJ/kg)13.222.0NFE, nitrogen-free extract.a Diets purchased from Provimi Kliba Nafag, Kaiseraugst, Switzerland. Open table in a new tab NFE, nitrogen-free extract. After adaption to the housing conditions, rats (BW about 300 g) were equipped with chronic intragastric (IG) and jugular vein (JV) catheters as described previously (19Steffens A.B. A method for frequent sampling of blood and continuous infusion of fluids in the rat without disturbing the animal.Physiol. Behav. 1969; 4: 833-836Crossref Scopus (456) Google Scholar) with some modifications (20Ferrari B. Arnold M. Carr R.D. Langhans W. Pacini G. Bodvarsdottir T.B. Gram D.X. Subdiaphragmatic vagal deafferentation affects body weight gain and glucose metabolism in obese male Zucker (fa/fa) rats.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 289: R1027-R1034Crossref PubMed Scopus (24) Google Scholar). The rats had ad libitum access to tap water until 1 h before surgery and they were food deprived for a few hours. Catheters were implanted using sterile techniques. For infection prophylaxis and analgesia, rats were pretreated with 5 mg/kg trimethoprim/20 mg/kg sulfadoxin (i.e., 200 μl Borgal® 24%; Intervet, Veterinaria AG, Zurich, Switzerland) diluted 1:6 with saline (NaCl 0.9%; B. Braun Medical AG, Sempach, Switzerland) subcutaneously (SC) 3 h prior to anesthesia. Fifteen to 30 min prior to anesthesia, animals were pretreated with 100 μl/100 g atropine SC (0.05 mg/kg/day) (Sintetica S.A, Mendrisio, Switzerland) diluted 1:10 with saline. Inhalation anesthesia was induced with 4–5% isoflurane (Minrad Inc., Provet AG) in oxygen 1,000 ml/min (Pan Gas, Dübendorf, Switzerland). When surgical tolerance was reached, anesthesia was maintained with 1–3% isoflurane, oxygen, and dinitrogenoxide, 300 ml/min each. Eye ointment (Vitamin A 15,000 IE/g; Bausch and Lomb, Steinhausen, Switzerland) was applied, and the animals were kept on heating pads (39°C) throughout the surgery to prevent hypothermia. The depth of anesthesia was checked frequently by testing the interdigital reflexes. One to two hours after surgery and on the following 2 days rats received once daily 100 μl/100 g carprofen (Rimadyl® Pfizer AG, Zurich, Switzerland) diluted 1:10 with saline (5 mg/kg/day) SC for analgesia and, in addition, trimethoprim/sulfadoxine (5/20 mg/kg) on the day after surgery. During the first week after surgery IG and JV catheters were flushed with sterile saline daily, during the second week every second day, and thereafter every third day. After flushing, all JV catheters were locked with 100 μl sterile heparinized [Heparin-Na (25,000 IU/5 ml); B. Braun] polyvinylpyrrolidone (PVP) solution (PVP-10; Sigma, St. Louis, MO), 21 g PVP/12 ml 0.9% NaCl. All animals were allowed to recover for at least 10 days before experiments began. Both JV and IG catheters consisted of silastic tubing [ID, 0.508 mm; OD, 0.939 mm; length 11.5 cm (JV) and ID, 0.762 mm; OD, 1.651 mm; length 18 cm (IG) respectively; Gore W.L., Newark, DE] with one end slipped on a 20 gauge (JV) or 18 gauge (IG) Vacutainer cannula (Becton-Dickinson, Basel, Switzerland) that was bent into an L shape (>100°) for JV catheters or a U shape for IG catheters. The connection between tubing and cannula was shielded with silicon tubing [ID, 1.016 mm; OD, 2.159 mm; length 1.3 cm (JV) and ID, 1.47 mm; OD, 1.159 mm; length 1.5 cm (IG)] and the proximal ends of both catheters were fixed with 3/0 silk thread (B. Braun, Melsungen, Germany) on a small piece of nonabsorbable polypropylene surgical mesh (2.2 × 3 cm, rounded edges; Bard, NJ). The cannula of the IG catheter protruded cranially from the mesh with a 7–10 mm cranio-caudal distance to the cannula of the JV catheter. In addition, one small JV and three bigger IG bands of silicon (Silicone Adhesive; Cook Veterinary Products, Queensland, Australia) were applied around the tubing at 4 cm (JV) and 0.7, 0.8, and 2.5 cm (IG) respectively, away from the distal end. Two holes were made 0.5 mm away from the distal end of the JV tubing with a 20 gauge needle to reduce adhesion of the catheter to the wall of the atrium during blood aspiration. The cannula ends of the JV and IG catheters were led from a 1.5 cm interscapular incision cranially and exteriorized through a stab wound. The mesh with the cannulas was placed subcutaneously between the scapulae. The distal end of the JV catheter was inserted into the linguofacial vein 2 mm rostral to the bifurcation with the external jugular and maxillary vein and advanced until the silicon band reached the point of perforation. The distal end of the catheter thus lay 2 mm inside the right atrium. The catheter was secured in place by two ligatures, and the cervical skin incision was closed in two layers. Finally, the JV catheter was filled with 100 μl of heparinized PVP solution (see above). The distal end of the IG catheter was then led subcutaneously to a 3 cm midline laparotomy. A small stab wound was made in the wall of the stomach along the greater curvature, and the catheter was inserted 5 mm into the stomach (until the first silicon band was inside the stomach) and fixed in place with a purse-string suture (4/0 nonabsorbable silk; Johnson and Johnson, Spreitenbach, Switzerland). In addition, the catheter was fixed to the abdominal wall using the third silicon band and nonabsorbable 4/0 silk. The abdominal muscle layer and the skin incision were closed with 3/0 and 5/0 absorbable Vicryl (Ethicon® Johnson and Johnson) respectively. Small pieces (3–4 cm) of polyethylene tubing [0.76 × 1.22 mm (JV); 1.14 × 1.57 (IG); Smiths Medical, London, UK] were slipped on the cannulas of both catheters and subsequently sealed with a stainless steel pin ("stopper"), which was made out of an obdurate blunt 20 gauge needle. IG catheter position and patency was verified post mortem or by X-ray scans [Small Animal Computed Tomography scanner, Zinsser Analytic, La Theta 2.10, Aloka, Tokyo, Japan (settings: slice, multi; number, 10; pitch, 1 mm; speed, fast; position, face down-head front)] (supplementary Fig. I). For the latter procedure rats were fasted overnight and the X-ray scans were taken under light isoflurane inhalation anesthesia (see above). Diluted contrast agent (1:2; Accupaque TM, GE Healthcare, Chalfont St. Giles, UK; 0.6 ml, 2 parts Accupaque + 1 part saline) was IG infused and scans were taken immediately. Thereafter, the IG catheter was flushed with 0.5 ml saline. All rats recovered from anesthesia within minutes. The DGAT-1i [Compound 2 (17Birch A.M. Birtles S. Buckett L.K. Kemmitt P.D. Smith G.J. Smith T.J. Turnbull A.V. Wang S.J. Discovery of a potent, selective, and orally efficacious pyrimidinooxazinyl bicyclooctaneacetic acid diacylglycerol acyltransferase-1 inhibitor.J. Med. Chem. 2009; 52: 1558-1568Crossref PubMed Scopus (65) Google Scholar)] was suspended in the vehicle, consisting of 0.5% w/v hydroxypropyl methylcellulose powder (Methocel E4M; Colorcon Limited, Kent, UK) in 0.1% w/v Tween 80 solution (Sigma-Aldrich, Steinheim, Germany) to yield concentrations of 3, 9, or 10 mg DGAT-1i/5 ml vehicle. The suspension was always prepared the day before the experiment and continuously stirred at room temperature (RT) overnight to ensure adequate particle size reduction. The suspension was physically stable for at least 7 days when stored at RT, protected from light, and stirred continuously. IG DGAT-1i or vehicle infusions (5 ml/kg BW; <1 min) were always performed 1 h prior to the 8 h food access beginning at dark onset (1000 h). All rats were adapted to the IG infusion procedure for at least 2 weeks before the experiments. Cumulative energy intake (EI) was measured manually (± 0.1 g) during the 8 h feeding period (1, 3, 5, 7, and 8 h after food access) after IG vehicle (control) or DGAT-1i (3 and 9 mg/kg BW) administration (1 h before food access) in rats adapted to chow (n = 4) or HFD (n = 5) (see supplementary Fig. IIA). The effects of the different DGAT-1i doses versus vehicle in both diet groups were tested in two separate within-subjects cross-over trials (1 day between crossovers and 5 days between trials). An additional experiment was performed in another batch of rats adapted to HFD only (n = 15) under the same conditions with some modification. In this experiment, EI in vehicle- and DGAT-1i-treated (10 mg/kg) rats was measured electronically every 30 s, as previously described (21Baumgartner I. Pacheco-Lopez G. Ruttimann E.B. Arnold M. Asarian L. Langhans W. Geary N. Hillebrand J.J. Hepatic-portal vein infusions of glucagon-like peptide-1 reduce meal size and increase c-Fos expression in the nucleus tractus solitarii, area postrema and central nucleus of the amygdala in rats.J. Neuroendocrinol. 2010; 22: 557-563Crossref PubMed Scopus (48) Google Scholar), and EI data from defined time points (1, 2, 4, 6, and 8 h after food access) on the experimental day and on the subsequent day without treatment (post-vehicle vs. post-DGAT-1i) were analyzed. The DGAT-1i dose versus vehicle was tested in a within-subjects crossover trial with 1 day between trials. To assess whether rats avoid flavors paired with IG DGAT-1i infusions (10 mg/kg), a two-bottle choice test (22K., Ackroff, A., Sclafani, 2001. Conditioned flavor preferences: evaluating postingestive reinforcement by nutrients. Accessed March 2013, at http://onlinelibrary.wiley.com/doi/10.1002/0471142301.ns0806fs05/abstract;jsessionid=7BB886FC0EC2A8220BB752B28420B223.d02t04Google Scholar) was performed in 16 naïve HFD-fed rats. According to a previously described paradigm (23Labouesse M.A. Stadlbauer U. Weber E. Arnold M. Langhans W. Pacheco-Lopez G. Vagal afferents mediate early satiation and prevent flavour avoidance learning in response to intraperitoneally infused exendin-4..J. Neuroendocrinol. 2012; 24: 1505-1516Crossref PubMed Scopus (46) Google Scholar) (with some modifications), rats were adapted for at least 4 days to a drinking schedule with 22.5 h water deprivation and 60 min access to tap water in the morning during the light phase (experimental drinking session; 2–1 h prior to dark onset, i.e., at 0800–0900 h) and 30 min in the afternoon (7.5–8 h after dark onset, i.e., 1730–1800 h); during this afternoon drinking session rats had access to food (see Fig. 2A). During the drinking sessions water was always provided in two bottles simultaneously (e.g., A+B) and the positions of the bottles were switched at mid-fluid access to avoid potential place associations. This drinking regimen was maintained throughout all adaption and experimental stages, and fluid intake was always monitored by weighing the bottles before and after each drinking session. To encourage drinking during short sessions, a 0.2% (w/v) saccharin solution was added to the flavors that were offered during experimental stages (see below). To avoid possible neophobia to saccharin, rats were adapted to the saccharin solution for 2 days. On association days (trials 1 and 2), rats had access to two different flavored solutions [i.e., grape or cherry flavor (0.05% w/v) in 0.2% saccharin solution, unsweetened powdered Kool-Aid drink mix; Kraft Foods, White Plains, NY] during the experimental drinking session in the morning. Shortly after this drinking session, rats were IG infused with either vehicle or DGAT-1i (10 mg/kg BW) 1 h before dark onset (i.e., 0900 h). Both flavors were counterbalanced with respect to drug order and with respect to flavor/drug combinations across the two association days separated by one intervening day (e.g., grape/cherry paired with DGAT-1i (CS+)/vehicle (CS−) on association trail 1, cherry/grape paired with DGAT-1i/vehicle on association trial 2. Two days after the second association trial, a two-bottle choice test was performed in which rats had simultaneous access to both flavors during the experimental drinking session in the morning. The locations (right vs. left) of the two flavored bottles were similarly counterbalanced and bottle positions were switched at mid-fluid access. No IG vehicle or DGAT-1i infusions were performed on this test day and rats were then returned to ad libitum water access. For each rat the percentage of fluid intake from the DGAT-1i-paired flavor (CS+) was calculated using the following equation: CS+ preference (%) = [VCS+/(VCS++ VCS−)] × 100, where VCS+ is the volume of DGAT-1i-paired flavor intake, and VCS− is the volume of vehicle-paired flavor intake. Vehicle and DGAT-1i flavor preference ratios (CS− preference%:CS+ preference%) from each rat were averaged and a paired t-test was used to determine whether differences in preference for vehicle-paired versus DGAT-1i-paired flavors during the choice test were statistically significant. A preference ratio of 50%:50% was considered equal for both flavors (no effect of flavor pairing on intake). A shift in the ratio toward the vehicle-paired flavor (e.g., 75%:25%) was interpreted as evidence for conditioned flavor avoidance (CFA) (24Rinaman L. Dzmura V. Experimental dissociation of neural circuits underlying conditioned avoidance and hypophagic responses to lithium chloride.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 293: R1495-R1503Crossref PubMed Scopus (46) Google Scholar). The effect of acute IG DGAT-1i infusions (9 mg/kg) on serum and plasma fat metabolites was tested in rats that were refed with a 3 g HFD or 5 g isocaloric chow test meal at dark onset after 16 h of food deprivation. All rats were well adapted to this procedure, and the size of the test meal was chosen based on preliminary trials showing that under these conditions all rats finished 5 g chow within 10 min. The experiment was designed as a within-subjects crossover test in each diet group with the treatments given in random order and with 2 days between trials. Blood samples (400 μl each) were taken via the JV catheter in the fasted state shortly before IG infusions at about 0900 h (baseline) and at 2 (1100 h), 3 (1200 h), and 5 h (1400 h) after IG infusions (see supplementary Fig. IIB). Blood (250 μl) was transferred into 0.6 ml Eppendorf tubes containing 9 μl EDTA solution (Titriplex 3, Merck, Germany; 180 mg dissolved in 3.0 ml distilled water), mixed gently, put on wet ice, and centrifuged (10 min, 10,000 rpm, 4°C) within 20 min. The plasma was transferred into Cobas-Mira analyzer cups (Hoffmann La Roche, Basel, Switzerland) and stored at −20°C until analysis (see below). The remaining 150 μl of blood were transferred into a glass tube (Schmidlin Labor and Service AG, Neuheim, Switzerland) and allowed to clot for 4–5 h at RT. The serum was transferred into Cobas-Mira analyzer cups and stored at 4°C until analysis (see below) later on the same day. Fasting and postprandial serum TAG, plasma free glycerol, nonesterified fatty acid (NEFA), and β-hydroxybutyrate (BHB) concentrations were measured using standard colorimetric and enzymatic methods adapted for the Cobas MIRA® autoanalyzer (Hoffman LaRoche) (25Langhans W. Hepatic and intestinal handling of metabolites during feeding in rats.Physiol. Behav. 1991; 49: 1203-1209Crossref PubMed Scopus (24) Google Scholar). After an IG DGAT-1i (10 mg/kg) or vehicle infusion (1 h before food access) the feces of 18 rats (n = 9/group) fed a HFD were collected every 2 h during the 8 h food access period in the dark phase and 12 and 24 h after food access during the light phase (see supplementary Fig. IIC). The feces were dried overnight, ground, and fecal fat was extracted via the Folch extraction method (26Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The extracted lipids were then dissolved in chloroform:methanol (2:1) and separated by thin-layer chromatography (TLC Silica Gel 60 F254 Aluminum sheets, 20 × 20 cm; Merck, Germany) using petrol ether (boiling point 40–60°C):diethyl ether:acetic acid (84.5:15:0.5) as migration solvent. After the separation, the silica gel plate was taken out of the hermetically closed glass chamber to dry it and to let the solvent mixture evaporate. The different lipid fractions were visualized using iodine vapor, which colors them brownish/yellowish. Sunflower oil (100 mg in 10 μl chloroform/methanol (2:1)/mg) was used as reference (see Fig. 4B). The silica gel plate was cut to collect the NEFA fractions for each animal by scratching off the silica gel of the single pieces. Subsequently, the NEFAs contained in the scratched silica were reextracted with acetone and quantified with an enzymatic colorimetric diagnostic kit (NEFA-HR(2) R1 + R2 Set; Wako Chemicals GmbH). Indirect calorimetry was performed in 16 Plexiglas airtight metabolic cages (42 × 42 × 30 cm) in an open-circuit indirect calorimetry system (AccuScan Instruments Inc., Columbus, OH) as previously described (with modifications) (27Riediger T. Cordani C. Potes C.S. Lutz T.A. Involvement of nitric oxide in lipopolysaccharide induced anorexia.Pharmacol. Biochem. Behav. 2010; 97: 112-120Crossref PubMed Scopus (26) Google Scholar). In a within-subjects crossover design (DGAT-1i vs. vehicle) with two intervening days between trials, rats were single housed on a layer of wood shavings under the same light, temperature, and food access conditions as described above, except that powdered HFD (No. 2127; Provimi Kliba AG) was used to allow for the detecti
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