SIRT4 regulates fatty acid oxidation and mitochondrial gene expression in liver and muscle cells

Abstract

SIRT4, a member of the sirtuin family, has been implicated in the regulation of insulin secretion by modulation of glutamate dehydrogenase. However, the role of this enzyme in the regulation of metabolism in other tissues is unknown. In this study we investigated whether depletion of SIRT4 would enhance liver and muscle metabolic functions. To do this SIRT4 was knocked down using an adenoviral shRNA in mouse primary hepatocytes and myotubes. We observed a significant increase in gene expression of mitochondrial and fatty acid metabolism enzymes in hepatocytes with reduced SIRT4 levels. SIRT4 knockdown also increased SIRT1 mRNA and protein levels both in vitro and in vivo. In agreement with the increased fatty acid oxidation (FAO) gene expression, we showed a significant increase in FAO in SIRT4 knockdown primary hepatocytes compared with control, and this effect was dependent on SIRT1. In primary myotubes, knockdown of SIRT4 resulted in increased FAO, cellular respiration, and pAMPK levels. When SIRT4 was knocked down in vivo by tail vein injection of a shRNA adenovirus, we observed a significant increase in hepatic mitochondrial and FAO gene expression consistent with the findings in primary hepatocytes. Taken together these findings demonstrate that SIRT4 inhibition increases fat oxidative capacity in liver and mitochondrial function in muscle, which might provide therapeutic benefits for diseases associated with ectopic lipid storage such as type 2 diabetes.

FIGURE 1.

SIRT4 knockdown increase FAO gene expression and FAO in mouse primary hepatocytes. Primary hepatocytes cells were silenced with a shRNA-SIRT4 adenovirus. A, quantitative real time PCR showing that SIRT4 was decreased by shRNA-SIRT4 compared with shRNA-control cells. B–F, mouse primary hepatocytes were transduced with control or shRNA-SIRT4 adenovirus. Cells were harvested after 48 h. RNA was extracted and used to measure the indicated gene expression using quantitative real time PCR analysis. G, FAO in control and SIRT4 knockdown cells was measured. Values represent the mean of quadruplicate samples. Error bars represent S.E. Statistical analyses were performed using analysis of variance. *, p < 0.05.

FIGURE 2.

SIRT4 knockdown increases mitochondrial gene expression in primary hepatocytes. A–E, mouse primary hepatocytes were transduced with control or shRNA-SIRT4 adenovirus. Cells were harvested after 48 h. FAO was measured, and RNA was extracted and used to measure the indicated gene expression using quantitative PCR analysis. Values represent the mean of quadruple samples. Error bar represent S.E. Statistical analyses were performed using analysis of variance. *, p ≤ 0.005.

FIGURE 3.

SIRT4 knockdown increases SIRT1 levels and activity. A, quantitative real time PCR shows SIRT1 was induced by shRNA-SIRT4 compared with shRNA-control cells. B, immunoblot of SIRT1 and actin (loading control) in cells that were silenced using a shRNA-SIRT4 adenovirus is shown. KD, kinase dead. C, mouse primary hepatocytes were transduced with control of shRNA-SIRT4 and/or shRNA SIRT1 adenovirus. Cells were harvested 48 h later. FAO was. Values represent the mean of quadruple samples. Error bars represent S.E. Statistical analyses were performed using analysis of variance. *, p ≤ 0.005. FAO was measured as described under “Experimental Procedures.”

FIGURE 4.

SIRT4 knockdown increases fatty acid oxidation genes in mouse liver. Mice were infected with shRNA-control or shRNA-SIRT4 adenovirus. Animals were sacrificed 4 days after adenovirus injection. Liver RNA was extracted and used to measure the indicated gene expression using quantitative real time PCR analysis (A), body weight (B), liver weight (C), and blood glucose (D). Data are presented as mean ± S.E. n = 5/group. Statistical analyses were performed using Student's t test. *, p ≤ 0.05.

FIGURE 5.

SIRT4 knockdown increases FAO, oxygen consumption, and pAMPK in mouse primary myotubes. Myotubes were transduced with an shRNA-SIRT4 adenovirus or a scramble shRNA-control adenovirus for 48 h. A, total RNA was extracted, and the mRNA levels of SIRT4 were determined by quantitative real time-PCR. B, fatty acid oxidation rates were measured. C, cellular oxygen consumption rates were determined by Seahorse XF-24. Values represent the means of quadruplicate samples in B and C. Error bars represent S.E. Statistical analyses were performed using analysis of variance. *, p < 0.05. D, SIRT4 was knocked down in primary myotubes, and the levels of pAMPK were measured by Western blot. pAMPK was measured under basal and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-stimulated conditions. E, quantification of experiment is shown in D.

FIGURE 6.

A, the average gene expression of SIRT4 in liver tissue of three diabetes mouse models versus their corresponding background controls is shown. The probe set used for SIRT4 is 1426847_at. The error bars indicate the S.E. for each group. B, shown is schematic representation of the possible mechanisms by which SIRT4 regulates FAO. LCAD, long chain acyl-CoA dehydrogenase.