Carnitine Deficiency in OCTN2？/？ Newborn Mice Leads to a Severe Gut and Immune Phenotype with Widespread Atrophy, Apoptosis and a Pro-Inflammatory Response
We have investigated the gross, microscopic and molecular effects of carnitine deficiency in the neonatal gut using a mouse model with a loss-of-function mutation in the OCTN2 (SLC22A5) carnitine transporter. The tissue carnitine content of neonatal homozygous (OCTN2？/？) mouse small intestine was markedly reduced; the intestine displayed signs of stunted villous growth, early signs of inflammation, lymphocytic and macrophage infiltration and villous structure breakdown. Mitochondrial β-oxidation was active throughout the GI tract in wild type newborn mice as seen by expression of 6 key enzymes involved in β-oxidation of fatty acids and genes for these 6 enzymes were up-regulated in OCTN2？/？ mice. There was increased apoptosis in gut samples from OCTN2？/？ mice. OCTN2？/？ mice developed a severe immune phenotype, where the thymus, spleen and lymph nodes became atrophied secondary to increased apoptosis. Carnitine deficiency led to increased expression of CD45-B220+ lymphocytes with increased production of basal and anti-CD3-stimulated pro-inflammatory cytokines in immune cells. Real-time PCR array analysis in OCTN2？/？ mouse gut epithelium demonstrated down-regulation of TGF-β/BMP pathway genes. We conclude that carnitine plays a major role in neonatal OCTN2？/？ mouse gut development and differentiation, and that severe carnitine deficiency leads to increased apoptosis of enterocytes, villous atrophy, inflammation and gut injury.
References
[1]
Neu J, Walker WA (2011) Necrotizing enterocolitis. N Engl J Med 364: 255–264.
[2]
Patole S (2007) Prevention and treatment of necrotising enterocolitis in preterm neonates. Early Hum Dev 83: 635–642.
[3]
Patole S (2005) Prevention of necrotising enterocolitis: year 2004 and beyond. J Matern Fetal Neonatal Med 17: 69–80.
[4]
Schulzke SM, Deshpande GC, Patole SK (2007) Neurodevelopmental outcomes of very low-birth-weight infants with necrotizing enterocolitis: a systematic review of observational studies. Arch Pediatr Adolesc Med 161: 583–590.
[5]
Bisquera JA, Cooper TR, Berseth CL (2002) Impact of necrotizing enterocolitis on length of stay and hospital charges in very low birth weight infants. Pediatrics 109: 423–428.
[6]
Rees CM, Pierro A, Eaton S (2007) Neurodevelopmental outcomes of neonates with medically and surgically treated necrotizing enterocolitis. Arch Dis Child Fetal Neonatal Ed 92: F193–198.
[7]
Soraisham AS, Amin HJ, Al-Hindi MY, Singhal N, Sauve RS (2006) Does necrotising enterocolitis impact the neurodevelopmental and growth outcomes in preterm infants with birthweight < or = 1250 g? J Paediatr Child Health 42: 499–504.
[8]
Neu J (2005) Neonatal necrotizing enterocolitis: an update. Acta Paediatr Suppl 94: 100–105.
[9]
Sisk PM, Lovelady CA, Dillard RG, Gruber KJ, O'Shea TM (2007) Early human milk feeding is associated with a lower risk of necrotizing enterocolitis in very low birth weight infants. J Perinatol 27: 428–433.
Shekhawat P, Bennett MJ, Sadovsky Y, Nelson DM, Rakheja D, et al. (2003) Human placenta metabolizes fatty acids: implications for fetal fatty acid oxidation disorders and maternal liver diseases. Am J Physiol Endocrinol Metab 284: E1098–1105.
[12]
Shekhawat PS, Srinivas SR, Matern D, Bennett MJ, Boriack R, et al. (2007) Spontaneous development of intestinal and colonic atrophy and inflammation in the carnitine-deficient jvs (OCTN2(？/？)) mice. Mol Genet Metab 92: 315–324.
[13]
Koizumi A, Nozaki J, Ohura T, Kayo T, Wada Y, et al. (1999) Genetic epidemiology of the carnitine transporter OCTN2 gene in a Japanese population and phenotypic characterization in Japanese pedigrees with primary systemic carnitine deficiency. Hum Mol Genet 8: 2247–2254.
[14]
Wilcken B, Wiley V, Sim KG, Carpenter K (2001) Carnitine transporter defect diagnosed by newborn screening with electrospray tandem mass spectrometry. J Pediatr 138: 581–584.
[15]
Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B (2001) The Metabolic and Molecular basis of Inherited Diseases; Roe C, Ding J, editor. New York: McGraw Hill. 2297–2326 p.
[16]
Wang Y, Ye J, Ganapathy V, Longo N (1999) Mutations in the organic cation/carnitine transporter OCTN2 in primary carnitine deficiency. Proc Natl Acad Sci U S A 96: 2356–2360.
[17]
Wang Y, Kelly MA, Cowan TM, Longo N (2000) A missense mutation in the OCTN2 gene associated with residual carnitine transport activity. Hum Mutat 15: 238–245.
[18]
Spiekerkoetter U, Huener G, Baykal T, Demirkol M, Duran M, et al. (2003) Silent and symptomatic primary carnitine deficiency within the same family due to identical mutations in the organic cation/carnitine transporter OCTN2. J Inherit Metab Dis 26: 613–615.
[19]
Cederbaum SD, Koo-McCoy S, Tein I, Hsu BY, Ganguly A, et al. (2002) Carnitine membrane transporter deficiency: a long-term follow up and OCTN2 mutation in the first documented case of primary carnitine deficiency. Mol Genet Metab 77: 195–201.
[20]
Rinaldo P, Raymond K, al-Odaib A, Bennett MJ (1998) Clinical and biochemical features of fatty acid oxidation disorders. Curr Opin Pediatr 10: 615–621.
[21]
Boles RG, Buck EA, Blitzer MG, Platt MS, Cowan TM, et al. (1998) Retrospective biochemical screening of fatty acid oxidation disorders in postmortem livers of 418 cases of sudden death in the first year of life. J Pediatr 132: 924–933.
[22]
Chace DH, DiPerna JC, Mitchell BL, Sgroi B, Hofman LF, et al. (2001) Electrospray tandem mass spectrometry for analysis of acylcarnitines in dried postmortem blood specimens collected at autopsy from infants with unexplained cause of death. Clin Chem 47: 1166–1182.
[23]
Peltekova VD, Wintle RF, Rubin LA, Amos CI, Huang Q, et al. (2004) Functional variants of OCTN cation transporter genes are associated with Crohn disease. Nat Genet 36: 471–475.
[24]
Newman B, Gu X, Wintle R, Cescon D, Yazdanpanah M, et al. (2005) A risk haplotype in the Solute Carrier Family 22A4/22A5 gene cluster influences phenotypic expression of Crohn's disease. Gastroenterology 128: 260–269.
[25]
Babusukumar U, Wang T, McGuire E, Broeckel U, Kugathasan S (2006) Contribution of OCTN variants within the IBD5 locus to pediatric onset Crohn's disease. Am J Gastroenterol 101: 1354–1361.
[26]
Bene J, Magyari L, Talian G, Komlosi K, Gasztonyi B, et al. (2006) Prevalence of SLC22A4, SLC22A5 and CARD15 gene mutations in Hungarian pediatric patients with Crohn's disease. World J Gastroenterol 12: 5550–5553.
[27]
Ferraris A, Torres B, Knafelz D, Barabino A, Lionetti P, et al. (2006) Relationship between CARD15, SLC22A4/5, and DLG5 polymorphisms and early-onset inflammatory bowel diseases: an Italian multicentric study. Inflamm Bowel Dis 12: 355–361.
[28]
Gazouli M, Mantzaris G, Archimandritis AJ, Nasioulas G, Anagnou NP (2005) Single nucleotide polymorphisms of OCTN1, OCTN2, and DLG5 genes in Greek patients with Crohn's disease. World J Gastroenterol 11: 7525–7530.
[29]
Fisher SA, Hampe J, Onnie CM, Daly MJ, Curley C, et al. (2006) Direct or indirect association in a complex disease: the role of SLC22A4 and SLC22A5 functional variants in Crohn disease. Hum Mutat 27: 778–785.
[30]
Akisu M, Ozmen D, Baka M, Habif S, Yalaz M, et al. (2002) Protective effect of dietary supplementation with L-arginine and L-carnitine on hypoxia/reoxygenation-induced necrotizing enterocolitis in young mice. Biol Neonate 81: 260–265.
[31]
Kabaroglu C, Akisu M, Habif S, Mutaf I, Turgan N, et al. (2005) Effects of L-arginine and L-carnitine in hypoxia/reoxygenation-induced intestinal injury. Pediatr Int 47: 10–14.
[32]
Schmidt-Sommerfeld E, Werner D, Penn D (1988) Carnitine plasma concentrations in 353 metabolically healthy children. Eur J Pediatr 147: 356–360.
[33]
Sachan DS, Smith RB, Plattsmier J, Lorch V (1989) Maternal, cord, and neonatal carnitine correlations and lipid profiles of various birthweight infants. Am J Perinatol 6: 14–17.
[34]
Shenai JP, Borum PR (1984) Tissue carnitine reserves of newborn infants. Pediatr Res 18: 679–682.
[35]
Shenai JP, Borum PR, Mohan P, Donlevy SC (1983) Carnitine status at birth of newborn infants of varying gestation. Pediatr Res 17: 579–582.
[36]
Meyburg J, Schulze A, Kohlmueller D, Linderkamp O, Mayatepek E (2001) Postnatal changes in neonatal acylcarnitine profile. Pediatr Res 49: 125–129.
[37]
Meyburg J, Schulze A, Kohlmueller D, Poschl J, Linderkamp O, et al. (2002) Acylcarnitine profiles of preterm infants over the first four weeks of life. Pediatr Res 52: 720–723.
[38]
Chace DH, Pons R, Chiriboga CA, McMahon DJ, Tein I, et al. (2003) Neonatal blood carnitine concentrations: normative data by electrospray tandem mass spectometry. Pediatr Res 53: 823–829.
[39]
Penn D, Bobrowski PJ, Zhang L, Schmidt-Sommerfeld E (1997) Neonatal nutritional carnitine deficiency: a piglet model. Pediatr Res 42: 114–121.
[40]
Penn D, Zhang L, Bobrowski PJ, Quinn M, Liu X, et al. (1998) Carnitine deprivation adversely affects cardiovascular response to bacterial endotoxin (LPS) in the anesthetized neonatal pig. Shock 10: 377–382.
[41]
Larsson LE, Olegard R, Ljung BM, Niklasson A, Rubensson A, et al. (1990) Parenteral nutrition in preterm neonates with and without carnitine supplementation. Acta Anaesthesiol Scand 34: 501–505.
[42]
Crill CM, Christensen ML, Storm MC, Helms RA (2006) Relative bioavailability of carnitine supplementation in premature neonates. JPEN J Parenter Enteral Nutr 30: 421–425.
[43]
Nezu J, Tamai I, Oku A, Ohashi R, Yabuuchi H, et al. (1999) Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter. Nat Genet 21: 91–94.
[44]
Stevens RD, Hillman SL, Worthy S, Sanders D, Millington DS (2000) Assay for free and total carnitine in human plasma using tandem mass spectrometry. Clin Chem 46: 727–729.
[45]
Wang J, Wang X, Yang H, Wu D, Wang L, et al. (2011) Contribution of the IBD5 locus to inflammatory bowel disease: a meta-analysis. Hum Genet 129: 597–609.
[46]
Fortin G, Yurchenko K, Collette C, Rubio M, Villani AC, et al. (2009) L-carnitine, a diet component and organic cation transporter OCTN ligand, displays immunosuppressive properties and abrogates intestinal inflammation. Clin Exp Immunol 156: 161–171.
[47]
Gasbarrini G, Mingrone G, Giancaterini A, De Gaetano A, Scarfone A, et al. (2003) Effects of propionyl-L-carnitine topical irrigation in distal ulcerative colitis: a preliminary report. Hepatogastroenterology 50: 1385–1389.
[48]
Laviano A, Molfino A, Seelaender M, Frascaria T, Bertini G, et al. (2011) Carnitine administration reduces cytokine levels, improves food intake, and ameliorates body composition in tumor-bearing rats. Cancer Invest 29: 696–700.
[49]
Yuan Y, Guo H, Zhang Y, Zhou D, Gan P, et al. (2011) Protective effects of L-carnitine on intestinal ischemia/reperfusion injury in a rat model. J Clin Med Res 3: 78–84.
[50]
Mikhailova TL, Sishkova E, Poniewierka E, Zhidkov KP, Bakulin IG, et al. (2011) Randomised clinical trial: the efficacy and safety of propionyl-L-carnitine therapy in patients with ulcerative colitis receiving stable oral treatment. Aliment Pharmacol Ther 34: 1088–1097.
[51]
Thangasamy T, Jeyakumar P, Sittadjody S, Joyee AG, Chinnakannu P (2009) L-carnitine mediates protection against DNA damage in lymphocytes of aged rats. Biogerontology 10: 163–172.
[52]
Ferrara F, Bertelli A, Falchi M (2005) Evaluation of carnitine, acetylcarnitine and isovalerylcarnitine on immune function and apoptosis. Drugs Exp Clin Res 31: 109–114.
[53]
Giancaterini A, Mingrone G, De Gaetano A, Capristo E, Calvani M, et al. (2001) Effects of propyonil-l-carnitine topical irrigation in distal ulcerative colitis: a preliminary report. Am J Gastroenterol 96: 2275–2276.
[54]
Johnson JA (1992) L-carnitine for treatment of distal ulcerative colitis. Gastroenterology 103: 1709–1710.
[55]
Johnson JA (1993) L-carnitine for treatment of nonspecific proctosigmoiditis. Dis Colon Rectum 36: 518.
[56]
Ohtsuka Y, Sanderson IR (2000) Transforming growth factor-beta: an important cytokine in the mucosal immune response. Curr Opin Gastroenterol 16: 541–545.
[57]
Radtke F, Clevers H (2005) Self-renewal and cancer of the gut: two sides of a coin. Science 307: 1904–1909.
[58]
De Santa Barbara P, Williams J, Goldstein AM, Doyle AM, Nielsen C, et al. (2005) Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development. Dev Dyn 234: 312–322.
[59]
Sakuraba H, Ishiguro Y, Yamagata K, Munakata A, Nakane A (2007) Blockade of TGF-beta accelerates mucosal destruction through epithelial cell apoptosis. Biochem Biophys Res Commun 359: 406–412.
[60]
Batts LE, Polk DB, Dubois RN, Kulessa H (2006) Bmp signaling is required for intestinal growth and morphogenesis. Dev Dyn 235: 1563–1570.
[61]
Maheshwari A, Kelly DR, Nicola T, Ambalavanan N, Jain SK, et al. (2011) TGF-beta2 suppresses macrophage cytokine production and mucosal inflammatory responses in the developing intestine. Gastroenterology 140: 242–253.
[62]
Lahjouji K, Elimrani I, Wu J, Mitchell GA, Qureshi IA (2002) A heterozygote phenotype is present in the jvs +/？ mutant mouse livers. Mol Genet Metab 76: 76–80.
