Sirtuin 3 Restores Synthesis and Secretion of Very Low-Density Lipoproteins in Cow Hepatocytes Challenged with Nonesterified Fatty Acids In Vitro

Abstract

Fatty liver is closely associated with elevated concentrations of nonesterified fatty acids (NEFA) and a low level of very low-density lipoproteins (VLDL) in blood of dairy cows. High NEFA inhibit the VLDL synthesis and assembly, and cause hepatic triacylglycerol (TAG) deposition. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, antagonizes NEFA-induced TAG accumulation through modulating expressions of fatty acid synthesis and oxidation genes in cow hepatocytes. However, the role of SIRT3 in the VLDL synthesis and assembly was largely unknown. Here we aimed to test whether SIRT3 would recover the synthesis and assembly of VLDL in cow hepatocytes induced by high NEFA. Primary cow hepatocytes were isolated from 3 Holstein cows. Hepatocytes were infected with SIRT3 overexpression adenovirus (Ad-SIRT3), SIRT3-short interfering (si) RNA, or first infected with Ad-SIRT3 and then incubated with 1.0 mM NEFA (Ad-SIRT3 + NEFA). Expressions of key genes in VLDL synthesis and the VLDL contents in cell culture supernatants were measured. SIRT3 overexpression significantly increased the mRNA abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100 (ApoB100) and ApoE (p < 0.01), and raised VLDL contents in the supernatants (p < 0.01). However, SIRT3 silencing displayed a reverse effect in comparison to SIRT3 overexpression. Compared with NEFA treatment alone, the Ad-SIRT3 + NEFA significantly upregulated the mRNA abundance of MTP, ApoB100 and ApoE (p < 0.01), and increased VLDL contents in the supernatants (p < 0.01). Our data demonstrated that SIRT3 restored the synthesis and assembly of VLDL in cow hepatocytes challenged with NEFA, providing an in vitro basis for further investigations testing its feasibility against hepatic TAG accumulation in dairy cows during the perinatal period.

Keywords: ApoB100; ApoE; MTP; animal metabolic diseases; fat metabolism; perinatal period.

Figure 1

Effects of SIRT3 overexpression on the synthesis and secretion of Very Low-Density Lipoproteins (VLDL) in cow hepatocytes. Primary cow hepatocytes were cultured and treated with Ad-SIRT3 at 0, 25, 50, and 100 multiplicity of infection (MOI) for 6 h. Hepatocytes treated with Ad-GFP were used as the negative controls. After another 42 h of incubation with RPMI1640 medium, cells were harvested. The mRNA abundance of key proteins involved in the synthesis and assembly of VLDL, MTP (A), ApoB (B), and ApoE (C), were measured by qRT-PCR. The VLDL content in supernatants was measured by a commercial kit (D). For all bar plots shown, data are expressed as the mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001 by the one-way ANOVA with Bonferroni correction. Results are representative of at least three independent measurements.

Figure 2

Effects of SIRT3 silencing on the synthesis and secretion of Very Low-Density Lipoprotein (VLDL) in cow hepatocytes. Primary cow hepatocytes were treated with si-SIRT3 by the lipofectamine 2000 regent for 6 h. Hepatocytes treated with si-RNA were used as the negative controls. After another 42 h of incubation with RPMI1640 medium, cells were harvested. The mRNA abundance of MTP (A), ApoB (B), and ApoE (C) were measured by qRT-PCR. The VLDL content in supernatants was measured by a commercial kit (D). NC, negative controls; C, blank controls; si-SIRT3, si-SIRT3 treatment group. For all bar plots shown, data are expressed as the mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01 by the one-way ANOVA with Bonferroni correction. Results are representative of at least three independent measurements.

Figure 3

Effects of SIRT3 overexpression on the synthesis and secretion of Very Low-Density Lipoprotein (VLDL) in cow hepatocytes treated with nonesterified fatty acids (NEFA). Primary cow hepatocytes were treated with Ad-SIRT3 at 100 MOI for 30 h, serum starved for 6 h, and then treated with 1.0 mM NEFA for 12 h before cell harvest. Hepatocytes treated with Ad-GFP were used as the negative controls. The blank controls were hepatocytes incubated with RPMI1640 growth medium in parallel with the Ad-SIRT3 + NEFA group. The mRNA abundance of microsomal triglyceride transfer protein (MTP) (A), ApoB (B), and ApoE (C) were measured by qRT-PCR. The VLDL content in supernatants was measured by a commercial kit (D). For all bar plots shown, data are expressed as the mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01 by the one-way ANOVA with Bonferroni correction. Results are representative of at least three independent measurements.