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. 2022 Mar 18;10(3):709.
doi: 10.3390/biomedicines10030709.

Thioredoxin Domain Containing 5 Suppression Elicits Serum Amyloid A-Containing High-Density Lipoproteins

Javier Sánchez-Marco  1 , Roberto Martínez-Beamonte  1   2   3 , Alicia De Diego  4 , Tania Herrero-Continente  1 , Cristina Barranquero  2   3 , Carmen Arnal  2   5 , Joaquín Surra  2   3   6 , María A Navarro  1   2   3 , Jesús Osada  1   2   3
Affiliations

Thioredoxin Domain Containing 5 Suppression Elicits Serum Amyloid A-Containing High-Density Lipoproteins

Javier Sánchez-Marco et al. Biomedicines. .

Abstract

Thioredoxin domain containing 5 (TXNDC5) is a protein disulfide isomerase involved in several diseases related to oxidative stress, energy metabolism and cellular inflammation. In a previous manuscript, a negative association between fatty liver development and hepatic Txndc5 expression was observed. To study the role of TXNDC5 in the liver, we generated Txndc5-deficient mice. The absence of the protein caused an increased metabolic need to gain weight along with a bigger and fatter liver. RNAseq was performed to elucidate the putative mechanisms, showing a substantial liver overexpression of serum amyloid genes (Saa1, Saa2) with no changes in hepatic protein, but discrete plasma augmentation by the gene inactivation. Higher levels of malonyldialdehyde, apolipoprotein A1 and platelet activating factor-aryl esterase activity were also found in serum from Txndc5-deficient mice. However, no difference in the distribution of high-density lipoproteins (HDL)-mayor components and SAA was found between groups, and even the reactive oxygen species decreased in HDL coming from Txndc5-deficient mice. These results confirm the relation of this gene with hepatic steatosis and with a fasting metabolic derive remedying an acute phase response. Likewise, they pose a new role in modulating the nature of HDL particles, and SAA-containing HDL particles are not particularly oxidized.

Keywords: HDL; RNAseq; SAA; Saa1; Saa2; TXNDC5; Txndc5-deficient mice; liver; serum amyloid; thioredoxin domain containing 5.

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Conflict of interest statement

The authors declare no conflict of interest and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Somatometric parameters and hepatic fat content. Animal weight gained (mg) in 4 weeks per food intake (kcal) (A). Liver mass percentage of body weight after 16 h fasting (B). Hepatic cholesterol (C) and triglyceride (D) contents. Representative liver micrographs from wild type (E) and Txndc5-deficient mice (F), bar denotes 20 μm. Data are means ± SD for each group (n = 13 and n = 10, respectively, for WT and KO). Statistical analyses were done according to Mann–Whitney’s U-test and *, p value < 0.05; **, p value < 0.01.
Figure 2
Figure 2
Differentially expressed genes. (A) Venn diagram of gene expression between groups. (B) Volcano plot of DEGs. X axis represents log2 transformed fold change. Y axis represents -log10 transformed significance. Red points represent upregulated DEGs. Blue points represent downregulated DEGs. Grey points represent non-regulated DEGs. Gray points represent genes with no changes. (C) Correlation analysis of 10 selected genes between RNAseq and RT-qPCR normalized to the invariant Pipb gene. The mean values obtained for signal log2 ratio (SL2R) from individual analyses were plotted against the RNAseq (Table 1). Good agreement between the procedures was seen (r = 0.96, p < 0.0001). (D) Changes in values of SL2R expression of both methods for the 10 selected genes.
Figure 3
Figure 3
Serum amyloid A protein level. (A) Western blot of three protein pools from hepatic tissue in wild type (WT) and Txndc5-deficient (KO) male mice where SAA (13 kDa) and β-ACTIN, used as loading control, were detected. (B) Western blot bands were quantified using ACTIN as reference. (C) Total plasma SAA quantified by ELISA. Data are means ± SD for each group (n = 13 and n = 10, respectively, for WT and KO). Statistical analyses were done according to Mann–Whitney’s U-test and **, p value < 0.01.
Figure 4
Figure 4
Influence of Txndc5 and SAA in functional plasma parameters. APOA1 (A) and APOA4 (B) were determined by ELISA while activity assays were done to determine paraoxonase 1 (C) and PAF-acetylhydrolase activities (D). All measurements were done in total serum collected from mice fed for 4 weeks on a chow diet and fasted 16 h prior to the sacrifice. Data are means ± SD for each group (n = 13 and n = 10, respectively, for WT and KO). Statistical analyses were done according to Mann–Whitney’s U-test and **, p value < 0.01.
Figure 5
Figure 5
Influence of Txndc5 deficiency on plasma FPLC chromatographs of male mice on a chow diet. Mice were 4 weeks on a chow diet and fasted 16 h prior sacrifice. Two pools per experimental group were prepared and data are represented as mean ± SD. Fluorometric assays were performed to determine total cholesterol (A), non-esterified cholesterol (B) and phosphatidylcholine (C). ELISA assays were used to measure APOA1 (D), APOA4 (E) and SAA (F).
Figure 6
Figure 6
ROS content of HDL subgroups in wild type and Txndc5-deficient male mice. Two pools of each group were separated by FPLC and fractions 16–19 and 20–22 were combined into large HDL (lHDL) and small HDL (sHDL), respectively. Data are means ± SD for each group. Statistical analyses were done according to Mann–Whitney’s U-test and **, p value < 0.01.
Figure 7
Figure 7
Comprehensive scheme displaying the experimental approaches. The preparation of knock-out mice, the analyses of livers and plasma and the main findings of hepatic RNAseq and plasma characterization are summarized.
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