Augmenter of liver regeneration: an important intracellular survival factor for hepatocytes.
OBJECTIVE
Augmenter of liver regeneration (ALR), a protein synthesized and stored in hepatocytes, is associated with mitochondria, and possesses sulfhydryl oxidase and cytochrome c reductase activities. We sought to determine the effects of ALR depletion in hepatocytes by antisense oligonucleotide transfection.


METHODS
Rat hepatocytes in primary culture were transfected with antisense oligonucleotide for ALR mRNA (ALR-AS) or scrambled oligonucleotide. Various analyses were performed at times up to 24h after transfection.


RESULTS
Treatment with ALR-AS caused a decrease in ALR mRNA, cellular depletion of ALR protein primarily from mitochondria, and decreased viability. Flow cytometric analysis of ALR-AS-transfected hepatocytes stained with annexin-Vcy3 and 7-aminoactinomycin D revealed apoptosis as the predominant cause of death up to 6h; incubation beyond this time resulted in necrosis in addition to apoptosis. ALR-AS-transfection caused release of mitochondrial cytochrome c, activation of caspase-3, profound reduction in the ATP content, and cellular release of LDH. Inhibition of caspase-3 inhibited the early phase of ALR-AS-induced death but not the late phase that included ALR and LDH release.


CONCLUSIONS
These results suggest that ALR is critically important for the survival of hepatocytes by its association with mitochondria and regulation of ATP synthesis.
Thirunavukkarasu
Chinnasamy
C
Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, E-1542 BST, 200 Lothrop Street, Pittsburgh, PA 15213, USA.
Wang
Lian Fu
LF
Harvey
Stephen A K
SA
Watkins
Simon C
SC
Chaillet
J Richard
JR
Prelich
John
J
Starzl
Thomas E
TE
Gandhi
Chandrashekhar R
CR
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D017209:Apoptosis; D002452:Cell Count; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D005434:Flow Cytometry; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000235:genetics / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D014162:Transfection; D050862:Oxidoreductases Acting on Sulfur Group Donors
D000255:Adenosine Triphosphate; D016376:Oligonucleotides, Antisense; D011506:Proteins; D012333:RNA, Messenger; C088825:GFER protein, rat; D050862:Oxidoreductases Acting on Sulfur Group Donors