Restricted accessResearch articleFirst published online 2016-04
Malfunction in Mitochondrial β-Oxidation Contributes to Lipid Accumulation in Hepatocyte-Like Cells Derived from Citrin Deficiency-Induced Pluripotent Stem Cells
Citrin deficiency (CD) is a recessive genetic disorder caused by mutations in the citrin gene SLC25A13. CD causes various symptoms related to nutrient metabolism such as urea cycle failure, abnormal amino acid levels, and fatty liver. To understand the pathophysiology of CD, the molecular phenotypes were investigated using induced pluripotent stem cells derived from fibroblasts of CD patient (CD-iPSCs). In this study, we demonstrate that aberrant mitochondrial β-oxidation may lead to fatty liver in CD patients. CD-iPSCs normally differentiated into hepatocytes, similar to wild-type iPSCs (WT-iPSCs). However, hepatocytes derived from CD-iPSCs (CD-HLCs) did not exhibit ureogenesis. Cellular triglyceride and lipid granule levels were significantly increased in CD-HLCs compared with WT-HLCs. Peroxisome proliferator-activated receptor-α (PPAR-α) and its target genes which are involved in mitochondrial β-oxidation were downregulated in CD-HLCs, and treatment with a PPAR-α agonist partially reduced the lipid accumulation in CD-HLCs. In addition, the mitochondria in CD-HLCs exhibited abnormal morphologies. Based on these observations, we conclude that the lipid accumulation in CD-HLCs results from dysfunctional mitochondrial β-oxidation and abnormal mitochondrial structure.
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References
1.
KobayashiK, SinasacDS, IijimaM, BorightAP, BegumL, LeeJR, YasudaT, IkedaS, HiranoR, et al. (1999). The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial carrier protein. Nat Genet, 22:159–163.
2.
SinasacDS, CrackowerMA, LeeJR, KobayashiK, SahekiT, SchererSW and TsuiLC. (1999). Genomic structure of the adult-onset type II citrullinemia gene, SLC25A13, and cloning and expression of its mouse homologue. Genomics, 62:289–292.
3.
Del ArcoA, AgudoM and SatrusteguiJ. (2000). Characterization of a second member of the subfamily of calcium-binding mitochondrial carriers expressed in human non-excitable tissues. Biochem J, 345 (Pt 3):725–732.
4.
KobayashiK, ShaheenN, KumashiroR, TanikawaK, O'BrienWE, BeaudetAL and SahekiT. (1993). A search for the primary abnormality in adult-onset type II citrullinemia. Am J Hum Genet, 53:1024–1030.
5.
SahekiT, KobayashiK, IijimaM, NishiI, YasudaT, YamaguchiN, GaoHZ, JalilMA, BegumL and LiMX. (2002). Pathogenesis and pathophysiology of citrin (a mitochondrial aspartate glutamate carrier) deficiency. Metab Brain Dis, 17:335–346.
6.
TakagiH, HagiwaraS, HashizumeH, KandaD, SatoK, SoharaN, KakizakiS, TakahashiH, MoriM, et al. (2006). Adult onset type II citrullinemia as a cause of non-alcoholic steatohepatitis. J Hepatol, 44:236–239.
SinasacDS, MoriyamaM, JalilMA, BegumL, LiMX, IijimaM, HoriuchiM, RobinsonBH, KobayashiK, SahekiT and TsuiLC. (2004). Slc25a13-knockout mice harbor metabolic deficits but fail to display hallmarks of adult-onset type II citrullinemia. Mol Cell Biol, 24:527–536.
9.
SahekiT, IijimaM, LiMX, KobayashiK, HoriuchiM, UshikaiM, OkumuraF, MengXJ, InoueI, et al. (2007). Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency. J Biol Chem, 282:25041–25052.
10.
TiscorniaG, VivasEL and Izpisua BelmonteJC. (2011). Diseases in a dish: modeling human genetic disorders using induced pluripotent cells. Nat Med, 17:1570–1576.
11.
TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK and YamanakaS. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.
12.
ImI, JangMJ, ParkSJ, LeeSH, ChoiJH, YooHW, KimS and HanYM. (2015). Mitochondrial respiratory defect causes dysfunctional lactate turnover via AMP-activated protein kinase activation in human-induced pluripotent stem cell-derived hepatocytes. J Biol Chem, 290:29493–29505.
13.
ParkHJ, ChoiYJ, KimJW, ChunHS, ImI, YoonS, HanYM, SongCW and KimH. (2015). Differences in the epigenetic regulation of cytochrome P450 genes between human embryonic stem cell-derived hepatocytes and primary hepatocytes. PLoS One, 10:e0132992.
14.
OgawaS, SurapisitchatJ, VirtanenC, OgawaM, NiapourM, SugamoriKS, WangS, TamblynL, GuillemetteC, et al. (2013). Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development, 140:3285–3296.
15.
BatshawML, BrusilowS, WaberL, BlomW, BrubakkAM, BurtonBK, CannHM, KerrD, MamunesP, et al. (1982). Treatment of inborn errors of urea synthesis: activation of alternative pathways of waste nitrogen synthesis and excretion. N Engl J Med, 306:1387–1392.
16.
WalkerV. (2009). Ammonia toxicity and its prevention in inherited defects of the urea cycle. Diabetes Obes Metab, 11:823–835.
17.
OhuraT, KobayashiK, TazawaY, AbukawaD, SakamotoO, TsuchiyaS and SahekiT. (2007). Clinical pictures of 75 patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). J Inherit Metab Dis, 30:139–144.
18.
ZhangSR, WangJS, WangXH, ZhuQR and LiuLY. (2008). SLC25A13 gene mutations in Chinese infants with intrahepatic cholestasis and abnormal blood amino acids. Zhonghua Gan Zang Bing Za Zhi, 16:445–448.
19.
TanakaN, YazakiM and KobayashiK. (2007). A lean man with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol, 5:A32.
20.
AoyamaT, PetersJM, IritaniN, NakajimaT, FurihataK, HashimotoT and GonzalezFJ. (1998). Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem, 273:5678–5684.
21.
GulickT, CresciS, CairaT, MooreDD and KellyDP. (1994). The peroxisome proliferator-activated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression. Proc Natl Acad Sci U S A, 91:11012–11016.
22.
YoshikawaT, IdeT, ShimanoH, YahagiN, Amemiya-KudoM, MatsuzakaT, YatohS, KitamineT, OkazakiH, et al. (2003). Cross-talk between peroxisome proliferator-activated receptor (PPAR) alpha and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. I. PPARs suppress sterol regulatory element binding protein-1c promoter through inhibition of LXR signaling. Mol Endocrinol, 17:1240–1254.
23.
WatanabeK, FujiiH, TakahashiT, KodamaM, AizawaY, OhtaY, OnoT, HasegawaG, NaitoM, et al. (2000). Constitutive regulation of cardiac fatty acid metabolism through peroxisome proliferator-activated receptor alpha associated with age-dependent cardiac toxicity. J Biol Chem, 275:22293–22299.
24.
MutohK, KurokawaK, KobayashiK and SahekiT. (2008). Treatment of a citrin-deficient patient at the early stage of adult-onset type II citrullinaemia with arginine and sodium pyruvate. J Inherit Metab Dis, 31 (Suppl 2):S343–S347.
25.
KomatsuM, KimuraT, YazakiM, TanakaN, YangY, NakajimaT, HoriuchiA, FangZZ, JoshitaS, et al. (2015). Steatogenesis in adult-onset type II citrullinemia is associated with down-regulation of PPARalpha. Biochim Biophys Acta, 1852:473–481.
26.
KerstenS. (2014). Integrated physiology and systems biology of PPARalpha. Mol Metab, 3:354–371.
27.
KerstenS, SeydouxJ, PetersJM, GonzalezFJ, DesvergneB and WahliW. (1999). Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J Clin Invest, 103:1489–1498.
28.
HashimotoT, CookWS, QiC, YeldandiAV, ReddyJK and RaoMS. (2000). Defect in peroxisome proliferator-activated receptor alpha-inducible fatty acid oxidation determines the severity of hepatic steatosis in response to fasting. J Biol Chem, 275:28918–28928.
29.
MekhoubadS, BockC, de BoerAS, KiskinisE, MeissnerA and EgganK. (2012). Erosion of dosage compensation impacts human iPSC disease modeling. Cell Stem Cell, 10:595–609.
30.
ListerR, PelizzolaM, KidaYS, HawkinsRD, NeryJR, HonG, Antosiewicz-BourgetJ, O'MalleyR, CastanonR, et al. (2011). Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature, 471:68–73.
31.
DornGW2nd, VegaRB and KellyDP. (2015). Mitochondrial biogenesis and dynamics in the developing and diseased heart. Genes Dev, 29:1981–1991.
32.
HsuPD, LanderES and ZhangF. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157:1262–1278.
33.
DoudnaJA and CharpentierE. (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346:1258096.
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