H-STAT3KO mice are more susceptible to ethanol-induced fatty liver
documents the complete loss of STAT3 protein expression in hepatocytes from H-STAT3KO mice, confirming the deletion of STAT3 in these cells. Weak expression of the STAT3 protein in whole liver tissue of H-STAT3KO mice in is probably due to the expression of STAT3 in nonparenchymal cells. Expression of STAT1 protein was slightly higher in H-STAT3KO mice compared with WT mice, and was further increased after ethanol feeding. Expression of CYP2E1 was similar in H-STAT3KO and WT mice and, as expected, was elevated after ethanol feeding (). Next, the effects of alcohol feeding on liver injury were compared in H-STAT3KO mice and WT controls. As shown in , serum ALT and AST levels were similar in H-STAT3KO and WT mice, and higher in the ethanol-fed groups than in the respective pair-fed groups. H&E and oil-red-O staining revealed greater steatosis (fat deposition) in the livers of H-STAT3KO mice compared with WT mice after ethanol feeding (), which was also confirmed via biochemical analyses (). Furthermore, ethanol-fed H-STAT3KO mice showed significantly higher triglyceride levels in the serum than corresponding WT controls (). In contrast, no significant difference was found in cholesterol levels in the liver and serum between ethanol-fed H-STAT3KO and WT mice, although alcohol feeding increased the amount of cholesterol ().
To examine the time course of liver injury, we also investigated the effects of ethanol feeding for various weeks in H-STAT3KO and WT mice. There was no difference in serum ALT and AST levels between these 2 groups (), while the hepatic and serum triglyceride levels were significantly higher in H-STAT3KO mice at 2, 4, and 8 weeks post ethanol feeding (). Levels of hepatic cholesterol and serum cholesterol, glucose, and insulin were similar in these 2 groups at all time points post ethanol feeding ( and sFig. 8
Ethanol upregulates the expression of lipogenic genes in H-STAT3KO mice
To identify the mechanism by which ethanol induces more steatosis in H-STAT3KO mice than in WT mice, we profiled the expression of a group of fat metabolism-related genes. As shown in , mRNA expression of SREBP1c and the SREBP1c-regulated genes, FAS, ACC1, and SCD1, were higher in ethanol-fed H-STAT3KO mice than in WT mice. Expression of CPT1 and PPARα mRNA, which accelerate fatty acid lipid oxidation, was also upregulated in ethanol-fed H-STAT3KO mice compared to WT groups. Upregulation of SREBP1c protein was further confirmed by Western blotting. As shown in , consistent with previous reports,30
ethanol feeding increased mature form of nuclear SREBP1c protein expression about 2 fold in WT (white bars). Interestingly, the same feeding increased expression of nuclear SREBP1 protein about 10 fold in H-STAT3KO mice (grey bars). As expected, nuclear pSTAT3 protein was much lower in H-STAT3 KO mice than in WT mice ().
Figure 2 Upregulation of lipogenic gene expression in ethanol-fed H-STAT3KO mice. Liver samples from 4 week-fed WT and H-STAT3KO mice in were used to analyze the expression of lipid metabolism- and gluconeogenesis-related genes (mRNAs) by semi-quantitative (more ...)
In contrast, expression of gluconeogenesis-related genes such as PCK1, G6PC, and PGC-1α was similar in pair- and ethanol-fed WT and H-STAT3KO mice (). Finally, there was no significant difference in phospho-AMPKα expression between H-STAT3KO and WT mice; however, ethanol feeding increased phospho-AMPKα levels relative to the control diet ().
H-STAT3KO mice are resistant to ethanol-induced hepatic inflammation, expression of pro-inflammatory cytokines and chemokines
Ethanol feeding increased the number of inflammatory foci and neutrophils (myeloperoxidase [MPO]+ cells) in both H-STAT3KO and WT mice, but such increase was less pronounced in H-STAT3 KO mice (). This suggests that H-STAT3KO mice are not similarly prone to inflammation as WT mice after ethanol feeding.
Figure 3 H-STAT3KO mice are resistant to ethanol-induced hepatic inflammation. Feeding of mice for 4 weeks as in . Liver tissues were collected for H&E staining and MPO staining. (A) The number of inflammatory foci and the number of MPO positive (more ...)
Expression of CCR2 and F4/80, markers for monocytes and macrophages, respectively, were examined to detect the infiltration of monocytes/macrophages. As shown in , expression of CCR2 and F4/80 increased after ethanol feeding in WT mice, but such increase was less evident in H-STAT3KO mice. Taken together, this implies that ethanol-induced infiltration of inflammatory cells is attenuated in H-STAT3KO mice.
Expression of a variety of proinflammatiory cytokines in the liver was examined by real-time PCR. As shown in , ethanol feeding increased the hepatic expression of several proinflammatory cytokines and chemokines in WT mice (white bars); however, such increase was not observed, or was less pronounced, in H-STAT3KO mice (grey bars). In contrast, ethanol decreased hepatic IFN-γ mRNA expression in both WT and H-STAT3KO mice. Moreover, ethanol feeding decreased slightly the expression of hepatic complement 3 (C3) and C5 mRNA expression in WT mice, and the decrease in C5 was more pronounced in H-STAT3KO mice after ethanol feeding ().
Serum levels of several proinflammatory cytokines were also examined. As shown in , ethanol increased serum levels of TNF-α and IL-12p70, but decreased IFN-γ and MCP-1 in WT mice. No significant differences were found in TNF-α, IFN-γ, IL12p70 and MCP1 levels between WT and H-STAT3KO mice. Serum levels of IL-6 and IL-10 were under the assay detection limits in each group (data not shown).
Kupffer cells from ethanol-fed H-STAT3KO mice and WT mice respond similarly to LPS stimulation
shows that ethanol feeding induces ROS production by hepatocytes and Kupffer cells in WT mice. In hepatocytes from H-STAT3KO mice, ethanol-induced ROS was reduced, while Kupffer cells from WT and H-STAT3KO mice produced similar levels of ROS after ethanol feeding. Moreover, Kupffer cells from ethanol-fed mice produced higher basal levels of TNF-α compared with pair-fed mice (). Production of TNF-α by Kupffer cells from ethanol-fed mice further increased with LPS treatment. This increase was less evident in Kupffer cells from pair-fed mice, in agreement with previous findings that ethanol increases Kupffer cell sensitivity to LPS stimulation due to increased expression of the LPS receptor, CD14, after ethanol feeding.31
There was no difference in TNF-α production by Kupffer cells from either WT or H-STAT3KO mice ().
Figure 4 Kupffer cells from H-STAT3KO and WT mice produce similar levels of ROS and respond similarly to LPS stimulation. Feeding of mice for 4 weeks as in . (A) ROS production in hepatocytes and Kupffer from WT or H-STAT3KO mice. (B) Kupffer cells from (more ...)
M/N-STAT3 KO mice are more susceptible to ethanol-induced hepatic injury and inflammation
shows STAT3 protein expression was markedly reduced in peritoneal macrophages and hepatic macrophages/Kupffer cells from M/N-STAT3KO mice. The significant reduction of STAT3 activation (pSTAT3) in hepatic macrophages/Kupffer cells was also confirmed by flow cytometric analyses (sFig. 10
). shows that ethanol feeding elevated serum ALT/AST levels in both M/N-STAT3KO and WT groups, such increase was more evident in the M/N-STAT3KO group (grey bars). Although liver histology showed similar levels of fat deposition, greater inflammation was observed from livers of ethanol-fed M/N-STAT3KO versus WT mice (). The similar levels of hepatic fat deposition in these 2 groups were further confirmed by measurement of lipid contents (). Serum levels of triglyceride, cholesterol, glucose, and insulin were also similar in M/N-STAT3KO and WT mice (sFig. 9
). Finally, more hepatic inflammation in M/N-STAT3KO versus WT mice was further demonstrated by increased numbers of inflammatory foci and neutrophils, and increased expression of CCR2 and F4/80 mRNA ().
Figure 5 M/N-STAT3KO mice are more susceptible to ethanol-induced liver inflammation. (A) Western blot analyses of STAT3 protein expression in peritoneal macrophages and Kupffer cells. (B–F) WT and M/N-STAT3KO mice were given an ethanol-diet or pair-fed (more ...)
Upregulation of proinflammatory cytokines and chemokines in the liver and serum of ethanol-fed M/N-STAT3KO versus WT mice
shows that in pair-fed groups, basal levels of various hepatic pro-inflammatory cytokines and chemokines were higher in M/N-STAT3KO than in WT mice. Ethanol feeding slightly increased the expression of all of those cytokines and chemokines in WT mice, but dramatically increased their expression levels in M/N-STAT3KO mice. Interestingly, ethanol feeding decreased hepatic expression of IFN-γ in M/N-STAT3KO mice. Similarly, in pair-fed mice, serum levels of TNF-α, IFN-γ, and IL-6 were higher in M/N-STAT3KO than in WT mice (). Ethanol feeding further increased TNF-α and IL-6 but decreased IFN-γ serum levels in M/N-STAT3KO mice ().
Figure 6 Upregulation of proinflammatory cytokines and chemokines in M/N-STAT3 KO mice. Feeding of mice as in . (A) Real-time PCR analyses of gene expression in liver. (B) Serum cytokine and chemokine levels. Values represent means ± SD (N=6–10). (more ...)
Ethanol feeding induces greater production of ROS and TNF-α in Kupffer cells from M/N-STAT3KO compared to WT mice
Ethanol feeding enhanced ROS production by hepatocytes and Kupffer cells in WT mice (white bars in ). Basal levels of ROS production in pair-fed groups were higher in hepatocytes and Kupffer cells from M/N-STAT3KO compared with WT mice. The high basal levels of ROS found in M/N-STAT3KO mice were further increased after ethanol feeding. Similarly, as shown in , ethanol feeding increased basal and LPS-stimulated TNF-α production in Kupffer cells from WT mice (white bars), whereas in M/N-STAT3KO mice it only enhanced TNF-α production stimulated by 10 but not by 1 ng/mL of LPS, probably due to the very high basal levels of TNF-α (grey bars). Finally, basal and LPS-stimulated TNF-α production by Kupffer cells was greater in M/N-STAT3KO than in WT mice in pair-fed or ethanol-fed groups (grey bars vs. white bars).