Triheptanoin: long-term effects in the very long-chain acyl-CoA dehydrogenase-deficient mouse
2016; Elsevier BV; Volume: 58; Issue: 1 Linguagem: Inglês
10.1194/jlr.m072033
ISSN1539-7262
AutoresSara Tucci, U Floegel, Frauke Beermann, Sidney Behringer, Ute Spiekerkoetter,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoA rather new approach in the treatment of long-chain fatty acid oxidation disorders is represented by triheptanoin, a triglyceride with three medium-odd-chain heptanoic acids (C7), due to its anaplerotic potential. We here investigate the effects of a 1-year triheptanoin-based diet on the clinical phenotype of very long-chain-acyl-CoA-dehydrogenase-deficient (VLCAD−/−) mice. The cardiac function was assessed in VLCAD−/− mice by in vivo MRI. Metabolic adaptations were identified by the expression of genes regulating energy metabolism and anaplerotic processes using real-time PCR, and the results were correlated with the measurement of the glycolytic enzymes pyruvate dehydrogenase and pyruvate kinase. Finally, the intrahepatic lipid accumulation and oxidative stress in response to the long-term triheptanoin diet were assessed. Triheptanoin was not able to prevent the development of systolic dysfunction in VLCAD−/− mice despite an upregulation of cardiac glucose oxidation. Strikingly, the anaplerotic effects of triheptanoin were restricted to the liver. Despite this, the hepatic lipic content was increased upon triheptanoin supplementation. Our data demonstrate that the concept of anaplerosis does not apply to all tissues equally. A rather new approach in the treatment of long-chain fatty acid oxidation disorders is represented by triheptanoin, a triglyceride with three medium-odd-chain heptanoic acids (C7), due to its anaplerotic potential. We here investigate the effects of a 1-year triheptanoin-based diet on the clinical phenotype of very long-chain-acyl-CoA-dehydrogenase-deficient (VLCAD−/−) mice. The cardiac function was assessed in VLCAD−/− mice by in vivo MRI. Metabolic adaptations were identified by the expression of genes regulating energy metabolism and anaplerotic processes using real-time PCR, and the results were correlated with the measurement of the glycolytic enzymes pyruvate dehydrogenase and pyruvate kinase. Finally, the intrahepatic lipid accumulation and oxidative stress in response to the long-term triheptanoin diet were assessed. Triheptanoin was not able to prevent the development of systolic dysfunction in VLCAD−/− mice despite an upregulation of cardiac glucose oxidation. Strikingly, the anaplerotic effects of triheptanoin were restricted to the liver. Despite this, the hepatic lipic content was increased upon triheptanoin supplementation. Our data demonstrate that the concept of anaplerosis does not apply to all tissues equally. Mitochondrial β-oxidation is essential for energy production from fat. Deficiency of one of the enzymes involved is associated with life-threatening events and death. Very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency (OMIM 609575) is the second most common disorder of fatty acid oxidation in Europe and the USA, with an incidence of 1:25,000–1:100,000 newborns (1Arnold G.L. Van Hove J. Freedenberg D. Strauss A. Longo N. Burton B. Garganta C. Ficicioglu C. Cederbaum S. Harding C. et al.A Delphi clinical practice protocol for the management of very long chain acyl-CoA dehydrogenase deficiency.Mol. Genet. Metab. 2009; 96: 85-90Crossref PubMed Scopus (124) Google Scholar, 2Lindner M. Hoffmann G.F. Matern D. Newborn screening for disorders of fatty-acid oxidation: experience and recommendations from an expert meeting.J. Inherit. Metab. Dis. 2010; 33: 521-526Crossref PubMed Scopus (127) Google Scholar, 3Spiekerkoetter U. Sun B. Zytkovicz T. Wanders R. Strauss A.W. Wendel U. MS/MS-based newborn and family screening detects asymptomatic patients with very-long-chain acyl-CoA dehydrogenase deficiency.J. Pediatr. 2003; 143: 335-342Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 4Therrell Jr., B.L. Lloyd-Puryear M.A. Camp K.M. Mann M.Y. Inborn errors of metabolism identified via newborn screening: ten-year incidence data and costs of nutritional interventions for research agenda planning.Mol. Genet. Metab. 2014; 113: 14-26Crossref PubMed Scopus (76) Google Scholar). Pathophysiological mechanisms responsible for the development of symptoms include i) severe energy deficiency due to a deficient fatty acid oxidation and subsequent impairment of ketone body biosynthesis and ii) accumulation of toxic long-chain acylcarnitines. Therefore, catabolic situations in which the organism mainly relies on fatty acid oxidation induce symptoms and severe metabolic derangement. The clinical phenotype is very heterogeneous and presents with different severity and age of onset (3Spiekerkoetter U. Sun B. Zytkovicz T. Wanders R. Strauss A.W. Wendel U. MS/MS-based newborn and family screening detects asymptomatic patients with very-long-chain acyl-CoA dehydrogenase deficiency.J. Pediatr. 2003; 143: 335-342Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), involving organs and tissues that mostly rely on fatty acid β-oxidation for energy production. To date, treatment recommendations (5Spiekerkoetter U. Bastin J. Gillingham M. Morris A. Wijburg F. Wilcken B. Current issues regarding treatment of mitochondrial fatty acid oxidation disorders.J. Inherit. Metab. Dis. 2010; 33: 555-561Crossref PubMed Scopus (97) Google Scholar) include a long-chain fat-restricted and fat-modified diet in which long-chain fatty acids are fully or in part replaced by medium-chain triglycerides (MCTs) (1Arnold G.L. Van Hove J. Freedenberg D. Strauss A. Longo N. Burton B. Garganta C. Ficicioglu C. Cederbaum S. Harding C. et al.A Delphi clinical practice protocol for the management of very long chain acyl-CoA dehydrogenase deficiency.Mol. Genet. Metab. 2009; 96: 85-90Crossref PubMed Scopus (124) Google Scholar, 5Spiekerkoetter U. Bastin J. Gillingham M. Morris A. Wijburg F. Wilcken B. Current issues regarding treatment of mitochondrial fatty acid oxidation disorders.J. Inherit. Metab. Dis. 2010; 33: 555-561Crossref PubMed Scopus (97) Google Scholar) and the avoidance of prolonged fasting. In contrast to long-chain fatty acids, medium-chain fatty acids (MCFAs) are oxidized by medium-chain acyl-CoA dehydrogenase, bypassing VLCAD; therefore, MCTs may be fully metabolized, supplying the organism with the required energy. Many reports confirm the effectiveness of MCT application in the treatment of cardiomyopathy in long-chain fatty acid oxidation disorders (FAODs) (6Brown-Harrison M.C. Nada M.A. Sprecher H. Vianey-Saban C. Farquhar Jr., J. Gilladoga A.C. Roe C.R. Very long chain acyl-CoA dehydrogenase deficiency: successful treatment of acute cardiomyopathy.Biochem. Mol. Med. 1996; 58: 59-65Crossref PubMed Scopus (75) Google Scholar, 7Lund A.M. Dixon M.A. Vreken P. Leonard J.V. Morris A.A. What is the role of medium-chain triglycerides in the management of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency?.J. Inherit. Metab. Dis. 2003; 26: 353-360Crossref PubMed Scopus (8) Google Scholar, 8Pervaiz M.A. Kendal F. Hegde M. Singh R.H. MCT oil-based diet reverses hypertrophic cardiomyopathy in a patient with very long chain acyl-coA dehydrogenase deficiency.Indian J. Hum. Genet. 2011; 17: 29-32Crossref PubMed Scopus (19) Google Scholar, 9Sharef S.W. Al-Senaidi K. Joshi S.N. Successful treatment of cardiomyopathy due to very long-chain acyl-CoA dehydrogenase deficiency: first case report from Oman with literature review.Oman Med. J. 2013; 28: 354-356Crossref PubMed Scopus (15) Google Scholar). In patients with exercise-induced muscle pain, the application of an MCT bolus immediately prior to physical exercise has been proven effective (1Arnold G.L. Van Hove J. Freedenberg D. Strauss A. Longo N. Burton B. Garganta C. Ficicioglu C. Cederbaum S. Harding C. et al.A Delphi clinical practice protocol for the management of very long chain acyl-CoA dehydrogenase deficiency.Mol. Genet. Metab. 2009; 96: 85-90Crossref PubMed Scopus (124) Google Scholar, 5Spiekerkoetter U. Bastin J. Gillingham M. Morris A. Wijburg F. Wilcken B. Current issues regarding treatment of mitochondrial fatty acid oxidation disorders.J. Inherit. Metab. Dis. 2010; 33: 555-561Crossref PubMed Scopus (97) Google Scholar). Our own studies on the VLCAD-deficient (VLCAD−/−) mouse showed the beneficial effects of MCTs when applied during increased demand (10Primassin S. Tucci S. Herebian D. Seibt A. Hoffmann L. ter Veld F. Spiekerkoetter U. Pre-exercise medium-chain triglyceride application prevents acylcarnitine accumulation in skeletal muscle from very-long-chain acyl-CoA-dehydrogenase-deficient mice.J. Inherit. Metab. Dis. 2010; 33: 237-246Crossref PubMed Scopus (24) Google Scholar). Although an MCT diet is considered to be a safe dietary intervention and is applied in different FAODs for longer periods of time, recent reports highlight the adverse effects of an MCT diet in the murine model of VLCAD deficiency (11Tucci S. Flogel U. Sturm M. Borsch E. Spiekerkoetter U. Disrupted fat distribution and composition due to medium-chain triglycerides in mice with a beta-oxidation defect.Am. J. Clin. Nutr. 2011; 94: 439-449Crossref PubMed Scopus (32) Google Scholar, 12Tucci S. Herebian D. Sturm M. Seibt A. Spiekerkoetter U. Tissue-specific strategies of the very-long chain acyl-CoA dehydrogenase-deficient (VLCAD−/−) mouse to compensate a defective fatty acid beta-oxidation.PLoS One. 2012; 7: e45429Crossref PubMed Scopus (29) Google Scholar, 13Tucci S. Primassin S. Spiekerkoetter U. Fasting-induced oxidative stress in very long chain acyl-CoA dehydrogenase-deficient mice.FEBS J. 2010; 277: 4699-4708Crossref PubMed Scopus (22) Google Scholar, 14Tucci S. Primassin S. Ter Veld F. Spiekerkoetter U. Medium-chain triglycerides impair lipid metabolism and induce hepatic steatosis in very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient mice.Mol. Genet. Metab. 2010; 101: 40-47Crossref PubMed Scopus (36) Google Scholar). A rather new therapeutic approach for the treatment of FAODs is represented by the application of MCTs in the form of triheptanoin, a triglyceride with three medium odd-chain heptanoic acids (C7). The rationale behind the application of triheptanoin is that it has a potential anaplerotic effect, supplying the citric acid cycle (CAC) with the required substrates (15Roe C.R. Mochel F. Anaplerotic diet therapy in inherited metabolic disease: therapeutic potential.J. Inherit. Metab. Dis. 2006; 29: 332-340Crossref PubMed Scopus (99) Google Scholar). Our study on the long-term effects of triheptanoin supplementation in VLCAD−/− mice showed that, similarly to MCTs, this diet strongly stimulates lipogenesis, resulting in a disturbed fatty acid composition of plasma membranes in liver, heart, and skeletal muscle (16Tucci S. Behringer S. Spiekerkoetter U. De novo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase- (VLCAD) deficient mice supplemented with odd or even medium-chain fatty acids.FEBS J. 2015; 282: 4242-4253Crossref PubMed Scopus (29) Google Scholar). Whether triheptanoin is able to better supply all tissues with the required energy by additionally replenishing the CAC with substrates is unknown. An FDA phase 2 clinical trial with double blind comparison of physiologic effects of MCT oil versus triheptanoin in patients with long-chain FAOD is ongoing. However, in patients it is not possible to evaluate metabolic adaptations in tissues in response to dietary modifications over a long period of time. Here we explored how a triheptanoin-based diet, applied as a regular diet long term and not to correct acute metabolic decompensation, affects tissue metabolism in mice. We measured biochemical parameters such as blood lipids, glucose, insulin, and FFAs in serum and assessed cardiac morphology and function by in vivo MRI. The data were complemented by the evaluation of the cardiac metabolic adaptation in response to triheptanoin. Furthermore, lipid accumulation and markers of oxidative stress were examined in the liver in addition to anaplerotic effects in heart and liver. Experiments were performed on intercrosses of C57BL6+129sv VLCAD genotypes. Littermates served as controls, and genotyping of mice was performed as described previously by Exil et al. (17Exil V.J. Roberts R.L. Sims H. McLaughlin J.E. Malkin R.A. Gardner C.D. Ni G. Rottman J.N. Strauss A.W. Very-long-chain acyl-coenzyme a dehydrogenase deficiency in mice.Circ. Res. 2003; 93: 448-455Crossref PubMed Scopus (96) Google Scholar). Serum parameters were determined under standard conditions. Blood from mice at the age of 3 and 12 months was drawn after 5 h of food withdrawal. Mice were euthanized by CO2 asphyxiation. Blood samples were collected by heart puncture. Serum was obtained by centrifugation at 16,000 g for 10 min and stored at −80°C for further analysis. Tissues were rapidly removed and immediately frozen in liquid nitrogen. All animal studies were performed with the approval of the University's Institutional Animal Care and Use Committee and in accordance with the Committees' guidelines (approval number: 35-9185.81/G-14/20). At 5 weeks of age, mice of each genotype were divided in two groups and fed with different diets for 1 year. The first group received a normal purified mouse diet containing 5% crude fat in the form of long-chain TGs, corresponding to 12% of metabolizable energy as calculated with Atwater factors (ssniff® EF R/M Control; ssniff Spezialdiäten GmbH, Soest, Germany). The treatment recommendation for long-chain fatty acid oxidation diseases includes a strict fat-modified diet in which the normal long-chain fatty acids are completely replaced by even- and medium-chain MCTs (C8 and C10 chains) (1Arnold G.L. Van Hove J. Freedenberg D. Strauss A. Longo N. Burton B. Garganta C. Ficicioglu C. Cederbaum S. Harding C. et al.A Delphi clinical practice protocol for the management of very long chain acyl-CoA dehydrogenase deficiency.Mol. Genet. Metab. 2009; 96: 85-90Crossref PubMed Scopus (124) Google Scholar, 18Spiekerkoetter U. Lindner M. Santer R. Grotzke M. Baumgartner M.R. Boehles H. Das A. Haase C. Hennermann J.B. Karall D. et al.Treatment recommendations in long-chain fatty acid oxidation defects: consensus from a workshop.J. Inherit. Metab. Dis. 2009; 32: 498-505Crossref PubMed Scopus (153) Google Scholar). To evaluate the effects of triheptanoin, which is an odd medium-chain triglyceride (C7 chain), we prepared a strict diet in which triheptanoin fully replaced the normal long-chain fatty acids, with the exception of the essential fatty acids. Therefore, the second group was fed a diet corresponding as well to 12% of total metabolizable energy. Here, 4.4% from a total of 5% fat was Triheptanoin (CREMER OLEO GmbH and Co. KG, basis GmbH, Hamburg, Germany), and the remaining 0.6% was derived from soybean oil, providing the required essential long-chain fatty acids. The necessary amount of essential long-chain fatty acids was calculated in accordance to the Nutrient Requirements of Laboratory Animals (Subcommittee on Laboratory Animal Nutrition, Committee on Animal Nutrition, Board on Agriculture, National Research Council). Both diets were based on purified feed ingredients and contained the same nutrient concentration as follows: 94.8% dry matter, 17.8% crude protein (N × 6.25), 5% crude fat, 5% crude fiber, 5.3% crude ash, 61.9% nitrogen-free extract, 36.8% starch, 14.8% dextrin, and 11% sugar. The detailed fatty acid composition of the diets is reported in supplementary Table S1. In both diets, the carbohydrate and protein contents corresponded to 69% and 19% of metabolizable energy, respectively. The mice were fed control diet or triheptanoin-based diet either over 5 weeks or over 12 months. All mouse groups received water ad libitum. Data were recorded on an AvanceIII 9.4 Tesla Wide Bore (89 mm) nuclear magnetic resonance spectrometer (Bruker, Billerica, MA) operating at frequencies of 400.13 MHz for 1H as previously described (11Tucci S. Flogel U. Sturm M. Borsch E. Spiekerkoetter U. Disrupted fat distribution and composition due to medium-chain triglycerides in mice with a beta-oxidation defect.Am. J. Clin. Nutr. 2011; 94: 439-449Crossref PubMed Scopus (32) Google Scholar, 19Flogel U. Jacoby C. Godecke A. Schrader J. In vivo 2D mapping of impaired murine cardiac energetics in NO-induced heart failure..Magn. Reson. Med. 2007; 57: 50-58Crossref PubMed Scopus (35) Google Scholar). Tissues were homogenized in CelLytic MT Buffer (Sigma-Aldrich, Steinheim, Germany) in the presence of 1 mg/ml protease inhibitors and centrifuged at 4°C and 16,000 g for 10 min to pelletize any cell debris. The clear supernatant was immediately used for the enzyme assays or stored at –80°C. Pyruvate kinase (PK) activity was performed in duplicate using the Pyruvate Kinase Assay Kit (BioVision, Mountain View, CA) as recommended by the manufacturer. Pyruvate dehydrogenase (PDH) assay was measured using the PDH Assay Kit (Abcam, Cambridge, UK) following the manufacturer's protocol. α-Ketoglutarate-CoA dehydrogenase (αKGDH) activity was assayed by monitoring the rate of NAD+ reduction at 340 nm upon addition of 5.0 mM MgCl2, 40 μM rotenone, 2.5 mM α-ketoglutarate, 0.1 mM CoA, 0.2 mM thymine pyrophosphate, and 1.0 mM NAD to protein homogenate as previously described (20Nulton-Persson A.C. Szweda L.I. Modulation of mitochondrial function by hydrogen peroxide.J. Biol. Chem. 2001; 276: 23357-23361Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The measurement of citrate synthase is performed by assessing the reduction of 5,5′-dithiobis-nitrobenzoic acid in the presence of acetyl-CoA and oxaloacetate (21Trounce I.A. Kim Y.L. Jun A.S. Wallace D.C. 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Antioxidant enzymes in hypercholesterolemia and effects of vitamin E in rabbits.Atherosclerosis. 1993; 101: 135-144Abstract Full Text PDF PubMed Scopus (144) Google Scholar). FFA, triacylglycerides (TAGs), and lipoprotein concentrations were measured in duplicate in serum samples as described previously (13Tucci S. Primassin S. Spiekerkoetter U. Fasting-induced oxidative stress in very long chain acyl-CoA dehydrogenase-deficient mice.FEBS J. 2010; 277: 4699-4708Crossref PubMed Scopus (22) Google Scholar). Glucose and ketone bodies were determined with a Precision Xceed blood sugar meter (Abbott, Wiesbaden, Germany). The enzymatic method used for detection of C4 ketone bodies did not allow the detection of C5 species. Insulin was measured in duplicate by using the Ultrasensitive Mouse Insulin ELISA Kit (Mercodia AB, Uppsala Sweden). Insulin resistance was calculated by the homeostasis model assessment (HOMA) formula (24Matthews D.R. Hosker J.P. Rudenski A.S. Naylor B.A. Treacher D.F. Turner R.C. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man.Diabetologia. 1985; 28: 412-419Crossref PubMed Scopus (25502) Google Scholar). The HOMA of insulin resistance index, as described by Matthews et al. (24Matthews D.R. Hosker J.P. Rudenski A.S. Naylor B.A. Treacher D.F. Turner R.C. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man.Diabetologia. 1985; 28: 412-419Crossref PubMed Scopus (25502) Google Scholar), is the most easily obtained measurement of insulin resistance and can be used as a reliable surrogate measure of in vivo insulin sensitivity because this method correctly differentiates between insulin sensitivity and insulin resistance (25Lee S. Muniyappa R. Yan X. Chen H. Yue L.Q. Hong E.G. Kim J.K. Quon M.J. Comparison between surrogate indexes of insulin sensitivity and resistance and hyperinsulinemic euglycemic clamp estimates in mice.Am. J. Physiol. Endocrinol. Metab. 2008; 294: E261-E270Crossref PubMed Scopus (124) Google Scholar). HOMA Index was calculated with glucose and insulin concentrations obtained after 5 h of fasting using the following formula: fasting blood glucose (md/dl) × fasting insulin (µU/ml)/22.5. Lipoprotein concentrations were measured in duplicate in serum samples by using enzymatic kits (EnzyChrom HDL and VLDL/LDL Assay kit; BioTrend, Cologne, Germany) on an Infinite M200 Tecan (Crailsheim, Germany) plate reader. The intrahepatic lipid content was measured gravimetrically according to a method by Folch et al. (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) and was modified as previously reported (14Tucci S. Primassin S. Ter Veld F. Spiekerkoetter U. Medium-chain triglycerides impair lipid metabolism and induce hepatic steatosis in very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient mice.Mol. Genet. Metab. 2010; 101: 40-47Crossref PubMed Scopus (36) Google Scholar). Fatty acid profiles have been analyzed as previously described (16Tucci S. Behringer S. Spiekerkoetter U. De novo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase- (VLCAD) deficient mice supplemented with odd or even medium-chain fatty acids.FEBS J. 2015; 282: 4242-4253Crossref PubMed Scopus (29) Google Scholar). Thiobarbituric acid reactive substances have been analyzed as previously reported (11Tucci S. Flogel U. Sturm M. Borsch E. Spiekerkoetter U. Disrupted fat distribution and composition due to medium-chain triglycerides in mice with a beta-oxidation defect.Am. J. Clin. Nutr. 2011; 94: 439-449Crossref PubMed Scopus (32) Google Scholar). Analysis of acylcarnitines was performed as described previously (10Primassin S. Tucci S. Herebian D. Seibt A. Hoffmann L. ter Veld F. Spiekerkoetter U. Pre-exercise medium-chain triglyceride application prevents acylcarnitine accumulation in skeletal muscle from very-long-chain acyl-CoA-dehydrogenase-deficient mice.J. Inherit. Metab. Dis. 2010; 33: 237-246Crossref PubMed Scopus (24) Google Scholar, 27Vreken P. van Lint A.E. Bootsma A.H. Overmars H. Wanders R.J. van Gennip A.H. Rapid diagnosis of organic acidemias and fatty-acid oxidation defects by quantitative electrospray tandem-MS acyl-carnitine analysis in plasma.Adv. Exp. Med. Biol. 1999; 466: 327-337Crossref PubMed Google Scholar). Briefly, acylcarnitines were extracted from dried blood spots and tissues with acetonitrile/water (80/20% v/v) in the presence of [2H3]-free carnitine, [2H3] octanoyl-carnitine, and [2H3] palmitoyl-carnitine as internal standards. The extracted supernatant was dried, and the butylated acylcarnitines were analyzed by ESI-MS/MS. All even- and odd-chain C0–C19 acylcarnitines (saturated and unsaturated) were measured. Protein expression in the different tissues was performed by Western blot analysis. Protein homogenate (20–40 µg) from tissue lysate was separated on a gradient (4–12%) SDS polyacrylamide gel and transferred to nitrocellulose. Detection was carried out with anti-propionyl-CoA carboxylase antibody (monoclonal mouse; Santa Cruz Biotechnology, Dallas, TX), anti-αKGDH antibody (polyclonal rabbit; Abcam), and anti-succinyl-CoA synthetase (SCoA) antibody (polyclonal rabbit; Cell Signaling, Danvers, MA) used at a dilution 1:1,000–1:2,000. Anti-GAPDH and anti-actin were used as a loading control at 1:4,000. HRP-conjugated secondary antibodies were used at 1:5,000. Samples have been analyzed as pool (n = 10–12). Signals were detected and quantified Fusion FX Analyzer (Peqlab, Erlangen, Germany). Total RNA from heart was isolated with the RNeasy mini kit (Qiagen, Hilden, Germany). Forward and reverse primers were designed with the Primer Design Tool from NCBI (http://www.ncbi.nlm.nih.gov/tools). Gene function and primer sequences are reported in supplementary Table S2. RT-PCR was performed in a single-step procedure with the iTaq™ Universal SYBR® Green Supermix (Biorad, München, Germany) on a CFX96 Touch (Biorad). Gene coding for the 18S ribosomal subunit was used as reference. MRI data are reported as means ± SD. All other reported data are presented as means ± SEM; n denotes the number of animals tested. Analysis for the significance of differences was performed using Student's t-tests for paired and unpaired data. To test the effects of the variables, diet and genotype two-way ANOVA with Bonferroni posttest was performed (GraphPad Prism 5.0; GraphPad Software, San Diego, CA). Differences were considered significant if p < 0.05. We did not observe genotype- or diet-dependent effects on the body weight of mice after either short- or long-term supplementation (Table 1).TABLE 1Clinical parameters in WT and VLCAD−/− mice either under control or triheptanoin diet at the age of 3 and 12 monthsAgeWTVLCAD−/−ControlC7ControlC7Clinical phenotype3 monthsBody weight (g)21.6 ± 0.423.1 ± 1.222.7 ± 0.823 ± 0.9Serum lipidsFFA (µM)317 ± 43369 ± 47382 ± 26377 ± 35TAG (mg/dl)26.3 ± 5.633.4 ± 4.552.2 ± 6.6aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.66.1 ± 16.1aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.Cholesterol total (mg/dl)58.2 ± 5.663.6 ± 678.7 ± 5.2aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.87 ± 4.5aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.HDL (mg/dl)35.7 ± 339.8 ± 3.755.2 ± 3.3aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.65.3 ± 4.6aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.VLDL/LDL (mg/dl)14.6 ± 117.1 ± 1.718.8 ± 0.921.7 ± 1.8Serum variablesGlucose (mg/dl)217 ± 19218 ± 25245 ± 11bSignificant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen.301 ± 13aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.Insulin (pmol/L)107 ± 11106 ± 1672 ± 9105 ± 18bSignificant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen.HOMA Index8.2 ± 1.18.3 ± 1.96.2 ± 0.811.2 ± 2bSignificant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen.Ketone bodies C4 (mmol/L)0.77 ± 0.11.07 ± 0.21.26 ± 0.1aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.0.95 ± 0.1Clinical phenotype12 monthsBody weight (g)27.6 ± 2.528.6 ± 3.727.4 ± 2.930.1 ± 4.4Serum lipidsFFA (µM)277 ± 14319 ± 36308 ± 29260 ± 22TAG (mg/dl)19.6 ± 1.829.6 ± 4.2bSignificant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen.37.2 ± 3.2aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.40.2 ± 3.2aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.Cholesterol total (mg/dl)69.3 ± 5.378.4 ± 4.384.5 ± 6.5aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.76.3 ± 8.9HDL, mg/dl59.5 ± 5.662.6 ± 5.367.5 ± 4.563.4 ± 8.6VLDL/LDL (mg/dl)27.4 ± 2.423.4 ± 1.525.7 ± 2.324.2 ± 2.3Serum variablesGlucose (mg/dl)196 ± 22213 ± 22256 ± 29308 ± 16abInsulin (pmol/L)104 ± 15154 ± 19bSignificant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen.184 ± 25aSignificant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.162 ± 16HOMA Index7.5 ± 1.712.2 ± 2.6bSignificant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen.9.8 ± 1.518 ± 1.8abKetone bodies (mmol/L)1.19 ± 0.10.63 ± 0.09bSignificant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen.1.25 ± 0.100.78 ± 0.05bSignificant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen.Values are mean ± SEM (n = 10–12).a Significant difference (P < 0.05, two-way ANOVA and Student's t-test) between WT and VLCAD−/− mice under the same dietary regimen.b Significant differences (P < 0.05, two-way ANOVA and Student's t-test) between mice of the same genotype under different dietary regimen. Open table in a new tab Values are mean ± SEM (n = 10–12). Blood lipid analysis revealed no alteration due to a triheptanoin diet, with the exception of the serum TAGs and total cholesterol. In line with previous reports (11Tucci S. Flogel U. Sturm M. Borsch E. Spiekerkoetter U. Disrupted fat distribution and composition
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