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FoxO1 mediates insulin-dependent regulation of hepatic VLDL production in mice
Adama Kamagate, … , Marcia Meseck, H. Henry Dong
Adama Kamagate, … , Marcia Meseck, H. Henry Dong
Published May 22, 2008
Citation Information: J Clin Invest. 2008;118(6):2347-2364. https://doi.org/10.1172/JCI32914.
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Research Article

FoxO1 mediates insulin-dependent regulation of hepatic VLDL production in mice

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Abstract

Excessive production of triglyceride-rich VLDL is attributable to hypertriglyceridemia. VLDL production is facilitated by microsomal triglyceride transfer protein (MTP) in a rate-limiting step that is regulated by insulin. To characterize the underlying mechanism, we studied hepatic MTP regulation by forkhead box O1 (FoxO1), a transcription factor that plays a key role in hepatic insulin signaling. In HepG2 cells, MTP expression was induced by FoxO1 and inhibited by exposure to insulin. This effect correlated with the ability of FoxO1 to bind and stimulate MTP promoter activity. Deletion or mutation of the FoxO1 target site within the MTP promoter disabled FoxO1 binding and resulted in abolition of insulin-dependent regulation of MTP expression. We generated mice that expressed a constitutively active FoxO1 transgene and found that increased FoxO1 activity was associated with enhanced MTP expression, augmented VLDL production, and elevated plasma triglyceride levels. In contrast, RNAi-mediated silencing of hepatic FoxO1 was associated with reduced MTP and VLDL production in adult mice. Furthermore, we found that hepatic FoxO1 abundance and MTP production were increased in mice with abnormal triglyceride metabolism. These data suggest that FoxO1 mediates insulin regulation of MTP production and that augmented MTP levels may be a causative factor for VLDL overproduction and hypertriglyceridemia in diabetes.

Authors

Adama Kamagate, Shen Qu, German Perdomo, Dongming Su, Dae Hyun Kim, Sandra Slusher, Marcia Meseck, H. Henry Dong

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Figure 3

Molecular interaction between FoxO1 and MTP promoter.

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Molecular interaction between FoxO1 and MTP promoter.
(A) EMSA for assay...
(A) EMSA for assaying FoxO1 binding to DNA. HepG2 cells were transduced with FoxO1 vector at an MOI of 200 pfu/cell. Cells were lysed for the preparation of nuclear protein extracts 24 h after transduction. Aliquots of FoxO1 protein extracts (5 μg) were incubated with a biotin-labeled DNA probe, followed by chemiluminescent EMSA. DNA probe was derived from a 27-bp DNA covering the consensus IRE (–499/–473 nt) of the mouse MTP promoter, shown in lanes 1–4. A mutant DNA with an altered IRE motif was used as a control, shown in lanes 5–8. In addition, ChIP assay was used to assay for FoxO1 binding to DNA in cells. HepG2 cells were transfected with pGH11 in the presence of FoxO1 vector at an MOI of 100 pfu/cell in triplicate. After 24-h incubation, cells were cross-linked with 1% formaldehyde, followed by ChIP assay using rabbit anti-FoxO1 antibody (lanes 1 and 2) or preimmune IgG as a control (lanes 3 and 4). Immunoprecipitates were subjected to immunoblot assay using anti-FoxO1 antibody (B), and to PCR analysis using a pair of primers flanking the IRE sequence in the MTP promoter (C). As a positive control, aliquots of input DNA samples (1 μl) were used in PCR analysis. As a negative control, the immunoprecipitates were subjected to PCR analysis using a pair of off-target primers flanking a distal region (–3,528/–3,045 nt) devoid of the consensus IRE motif at 3 kb upstream of the MTP promoter.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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