Effect of sterol regulatory element binding protein-1a on the mitochondrial protein pattern in human liver cells detected by 2D-DIGE.
Sterol regulatory element binding protein-1a (SREBP-1a) is a transcription factor that is a major player in lipid metabolism and insulin action. We have generated human liver cells (HepG2) overexpressing active SREBP-1a constitutively called SREBP-1a (+). These cells show massive intracellular lipid accumulation. To elucidate the effect of SREBP-1a on lipid metabolism at the level of the cellular protein network, we have analyzed the protein pattern of mitochondria using the novel technique two-dimensional difference gel electrophoresis (2D-DIGE). Mitochondria were enriched by subcellular fractionation using differential and isopyknic centrifugation. Proteins of isolated organelles were labeled with Cy dyes and separated on 2D gels. These gels revealed more than 100 protein spots, which were significantly different in their abundance between wild-type and SREBP-1a (+) cells. MALDI mass spectrometry showed that 68% of identified proteins belong to mitochondria. In SREBP-1a (+) cells, several enzymes involved in lipid metabolism were significantly altered, suggesting that cellular lipid metabolism is triggered by accumulation of fatty acids rather than by its degradation. To test the possible functional relevance of this finding, intracellular fatty acid (FA) patterns were analyzed by gas chromatography. The results showed a significant increase in total fatty acid content with a shift in composition to long-chain unsaturated FAs. Therefore, the detected protein differences might be an explanation for the observed intracellular lipid accumulation and might link SREBP-1a to features like steatosis hepatis.
Lehr
Stefan
S
Institute for Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Heinrich-Heine-University Düsseldorf, and Leibniz Center for Diabetes Research, Düsseldorf, Germany.
Kotzka
Jorg
J
Avci
Haluk
H
Knebel
Birgit
B
Muller
Stefan
S
Hanisch
Franz G
FG
Jacob
Sylvia
S
Haak
Claudia
C
Susanto
Fransiscus
F
Muller-Wieland
Dirk
D
D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D050356:Lipid Metabolism; D008854:Microscopy, Electron; D024101:Mitochondrial Proteins / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D012313:RNA; D004247:DNA