Oleate-mediated stimulation of microsomal triglyceride transfer protein (MTP) gene promoter: implications for hepatic MTP overexpression in insulin resistance.
Hepatic lipoprotein overproduction in a fructose-fed hamster model of insulin resistance was previously shown to be associated with a significant elevation of intracellular mass of microsomal triglyceride transfer protein (MTP) and elevated plasma levels of free fatty acids (FFA). Here, we further establish that fructose feeding and development of an insulin resistant state result in higher levels of MTP mRNA, protein mass, and lipid transfer activity. MTP protein mass was increased in fructose-fed hamster hepatocytes to 161 +/- 35.8% of control (p < 0.05), while MTP mRNA levels and MTP lipid transfer activity were increased to 147.5 +/- 30.8% (p < 0.05) and 177.5 +/- 14.5% (p < 0.05) of control levels, respectively. To identify underlying mechanisms, we also investigated the potential link between enhanced FFA flux and hepatic MTP gene expression. Direct modulation of MTP gene transcription by fatty acids was investigated by transfecting HepG2 cells with a reporter (luciferase) construct containing various base pair regions of the human MTP promoter including pMTP124 (with the sterol response element (SRE)), pMTP116, pMTP109 and pMTP100 (no SRE), and pMTP124SREKO (SRE sequences mutated). Treatment of HepG2 cells with oleic acid (360 muM) significantly increased luciferase activities in cells transfected with pMTP124 (136.6 +/- 11.0%, p < 0.05) and pMTP124SREKO (153.9 +/- 11.1%, p < 0.01) compared with control. Luciferase activity was also increased in a time and dose-dependent manner in the presence of oleic acid when transfected with pMTP124SREKO but not pMTP109 and pMTP100. Furthermore, long-term oleic acid treatment of HepG2 cells (10 days) induced higher levels of MTP mRNA (p < 0.05) confirming transcriptional stimulation of the MTP gene by oleic acid. In contrast, palmitate, arachidonic acid or linoleic acid did not significantly stimulate pMTP124 or pMTP124SREKO luciferase activity (p > 0.05). These data demonstrate that (1) MTP gene transcription may be directly up-regulated by oleic acid; (2) up-regulation of MTP gene transcription by oleic acid is SRE sequence independent; and (3) the sequence -116 to -109 in the MTP promoter region is essential for oleic acid-mediated stimulation. Stimulation of MTP gene expression may be a novel mechanism by which certain FFAs can induce hepatic lipoprotein secretion in insulin resistant states.
Qiu
Wei
W
Division of Clinical Biochemistry, Department of Laboratory Medicine & Pathobiology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X8.
Taghibiglou
Changiz
C
Avramoglu
Rita Kohen
RK
Van Iderstine
Stephen C
SC
Naples
Mark
M
Ashrafpour
Homa
H
Mhapsekar
Shailen
S
Sato
Ryuichiro
R
Adeli
Khosrow
K
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007333:Insulin Resistance / Q000235:genetics*; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D023421:Models, Animal; D008969:Molecular Sequence Data; D019301:Oleic Acid / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D015183:Restriction Mapping
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; C106092:microsomal triglyceride transfer protein; D019301:Oleic Acid