Impact of hepatocyte-specific deletion of staphylococcal nuclease and tudor domain containing 1 (SND1) on liver insulin resistance and acute liver failure of mice

Figures & data

Figure 1. Construction of SND1 LKO mice

(a) The mating strategy for the construction of SND1 LKO mice (b-c). We extracted the DNA from the primary hepatocytes of the wild-type (WT) control, SND1 Flox/Flox, SND1 Flox/Flox-albumin-Cre⁺ (#1, #2) mice, and performed a genotyping PCR assay. (d) We extracted the liver, spleen, pancreas, and kidney tissues from the WT (#1, #2, #3) and SND1 LKO (#4, #5, #6) mice, respectively. SMMC-7721 cells of SND1 WT and knockout (KO) were used as the controls. (e) We also extracted mouse primary hepatocytes. Western blotting was then performed using anti-SND1 or anti-β-actin antibodies. (f) The gross morphology of WT and LKO mice.

Figure 2. Effect of SND1 hepatocyte-specific deletion on weight of mice

(a) The body weight of WT or LKO mice with chow diet (CD) was measured every week, respectively. Then, the statistical analysis of two-way ANOVA with Sidak’s multiple comparisons was performed. The P values of Sidak’s multiple comparisons at the different time points were indicated. (b-c) At the 24 w of CD in mice, the weights of extracted liver tissue and white adipose tissue (WAT) were measured, respectively. And the ratio value of liver/body weight, or WAT/body weight, was calculated. Student’s t-test was then performed. (d-f) A similar measure was performed for the mice with a high-fat diet (HFD).

Figure 3. Effect of SND1 hepatocyte-specific deletion on Akt pathway

At the 24 w of chow diet in mice, SND1 WT (a) and LKO (b) mice were fasted overnight and anesthetized. After the injection of the insulin solution, the phosphorylation level of Akt protein in the liver tissue was analyzed by a western blotting assay at the time points of 0 min and 5 min. The band density was digitized by the Image J 2X software. The value of p-Akt/Total Akt was indicated.

Figure 4. Effect of SND1 hepatocyte-specific deletion on glucose homeostasis of mice

(a) After the fasting treatment of mice with 4 w CD for 16 h, the chow diet was restored. The blood glucose levels at the time points of 0 h, 0.5 h, 1 h, 2 h, 4 h, and 6 h were measured, respectively. (b) After the fasting treatment for 16 h, 1.5 g/kg glucose solution was injected intraperitoneally into WT or SND1 LKO mice with 4 w chow diet. The blood glucose levels at the time points of 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min were measured, respectively. (c) 0.75 U/kg insulin was injected intraperitoneally into WT or SND1 LKO mice with 16 w chow diet. The blood glucose levels at the time points of 0 min, 15 min, 30 min, 45 min, 60 min, and 90 min were measured. (d-f) The similar measure was performed for the mice with HFD. Two-way ANOVA with Sidak’s multiple comparison test was performed. In addition, the AUC value of the above was calculated, respectively. Statistical difference was analyzed by a Student’s t-test.

Figure 5. Cholesterol level and hepatic steatosis in WT and LKO mice with a high-fat diet

(a-c) At 24 w of a chow or high-fat diet, the levels of liver total cholesterol and liver-free cholesterol were measured, respectively. The ANOVA followed by Tukey’s multiple comparison test was performed for the statistical difference among different groups. (d) Liver sections were subjected to H&E staining, and images were captured by an optical microscope. Bar scale, 250 μm.

Figure 6. Effect of SND1 hepatocyte-specific deletion on acute liver failure induced by LPS/D-GalN

WT and LKO mice were administered 5 mg/kg LPS and 100 mg/kg D-GalN intraperitoneally. Normal saline (NS) was used as the control. Serum activities of ALT (a) and AST (b) were measured after LPS/D-GalN treatment for 6 h. The mRNA levels of IL-6 (c), IL-1β (d), and TNF-α (e) in the liver tissues were also measured by a qPCR assay. The ANOVA followed by Tukey’s multiple comparison test was performed. (f) H&E staining images of representative liver samples were provided. Scale bar, 250 μm.

Figure 7. SND1 expression in liver tissues of insulin resistance-related GEO datasets

(a) For GSE23343, the expression difference of human SND1 mRNA in the liver tissues between the T2D patients with insulin resistance (n = 7) and controls (n = 10) was analyzed using a wilcox.test. (b-d) We also analyzed the statistical difference of SND1 expression in liver tissue between the CD and HFD of mice (Mus musculus) or rats (Rattus norvegicus). (e) For GSE13271, we performed the two-way ANOVA with Sidak’s multiple comparison test to analyze the expression difference of SND1 in liver tissues of GotoKakizake or WistarKyoto rats between CD and HFD at the time points of 4 w, 8 w, 12 w, 16 w, 20 w. The positive P value was indicated.

Figure 8. SND1 expression in liver tissues of ALF-related GEO datasets

We performed a wilcox.test to analyze the statistical difference of SND1 expression in the liver tissues between ALF and control, based on the datasets of GSE62026 (a), GSE120652 (b), and GSE38941 (c).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The authors gratefully acknowledge the contributions of datasets within GEO database, including GSE23343, GSE160646, GSE120243, GSE48794, GSE13271, GSE151268, GSE62026, GSE120652, GSE38941.