The Acyl-CoA-binding domain-containing protein (ACBD3) plays multiple roles across the cell. Although generally associated with the Golgi apparatus, it operates also in mitochondria. In steroidogenic cells, ACBD3 is an important part of a multiprotein complex transporting cholesterol into mitochondria. Balance in mitochondrial cholesterol is essential for proper mitochondrial protein biosynthesis, among others. We generated ACBD3 knock-out (ACBD3-KO) HEK293 and HeLa cells and characterized the impact of protein absence on mitochondria, Golgi, and lipid profile. In ACBD3-KO cells, cholesterol level and mitochondrial structure and functions are not altered, demonstrating that an alternative pathway of cholesterol transport into mitochondria exists. However, ACBD3-KO cells exhibit enlarged Golgi area with absence of stacks and ribbon-like formation, confirming the importance of ACBD3 in Golgi stacking. The glycosylation of the LAMP2 glycoprotein was not affected by the altered Golgi structure. Moreover, decreased sphingomyelins together with normal ceramides and sphingomyelin synthase activity reveal the importance of ACBD3 in ceramide transport from ER to Golgi.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• biologický transport fyziologie   MeSH
• ceramidy metabolismus   MeSH
• cholesterol metabolismus   MeSH
• glykosylace MeSH
• Golgiho aparát metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• membránový protein 2 asociovaný s lyzozomy metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• transferasy pro jiné substituované fosfátové skupiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

• MeSH
• asparagin farmakologie   MeSH
• autofagie účinky léků   MeSH
• chlorochin farmakologie   MeSH
• diglyceridy metabolismus   MeSH
• hepatocyty metabolismus   patologie   účinky léků   MeSH
• játra metabolismus   patologie   účinky léků   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• kyseliny mastné neesterifikované metabolismus   MeSH
• lipolýza MeSH
• lipoproteiny VLDL metabolismus   MeSH
• lyzozomy metabolismus   patologie   účinky léků   MeSH
• membránový protein 2 asociovaný s lyzozomy metabolismus   MeSH
• oxidace-redukce MeSH
• potkani Wistar MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• sirolimus farmakologie   MeSH
• sterolesterasa metabolismus   MeSH
• triglyceridy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH