The aim of the following experiments was to validate the downregulation of Agxt - observed in the genome-wide datasets - in liver samples of the ob/ob mouse model, in the in vitro steatosis systems of primary mouse hepatocytes and human hepatocellular carcinoma cell lines and in primary human hepatocytes. Confirmation of the downregulation of Agxt would justify the need for further experiments to investigate the consequences of the downregulation of Agxt.
3.5.1 Repressed Agxt expression in steatotic ob/ob mouse liver tissue
To confirm the downregulation of Agxt in steatosis as identified in the above delineated genome-wide studies, quantitative real-time PCR was performed using a specific Agxt Taqman assay. The quantitative real-time PCR revealed a 1.86 ± 0.34 fold downregulation of Agxt at the RNA level in livers of ob/ob mice (Figure 3.18 A) and thus, confirmed the gene array results used for the pipeline (showing a fold change of - 1.91). Furthermore, protein analysis using both the Western blot technique and immunohistochemistry showed reduced expression of the protein Agxt. In addition, for the first time a pericentral zonation of Agxt in mouse liver was discovered (Figure 3.18).
Figure 3.18: Livers of ob/ob mice display a reduced expression of Agxt. A) The RNA level of Agxt of ob/ob mouse
livers was analysed by quantitative real-time PCR relative to ob/+ mouse livers. Eif2a was used as the endogenous control (n = 5); B) The protein expression and zonation of Agxt were assessed by immunohistochemistry in paraffin embedded liver slides. The scale bars represent 200 µm, CV: central vein, PV: portal vein; C) Western blot and D) its densitometric quantification of Agxt expression in liver lysates of ob/+ and ob/ob mice (n = 6). β- Actin was used as a loading control. * indicates p < 0.05; ** indicates p < 0.01.
3.5.2 Reduced Agxt expression in in vitro models of steatosis
After having demonstrated the downregulation of Agxt in ob/ob mouse livers, the next step was the validation of Agxt expression in in vitro steatosis models of primary mouse hepatocytes and an additional liver cancer cell line. These results would provide evidence of a direct association between lipid accumulation in hepatocytes and the downregulation of Agxt. The Affymetrix gene array of lipid-loaded HepG2 cells and corresponding control cells revealed a significant repression of AGXT, starting after 72 h of oleic acid exposure that remained until 5 days of treatment.
First, the expression of Agxt was investigated in the in vitro steatosis system of primary mouse hepatocytes. Figure 3.19 shows the analysis of Agxt expression at the RNA level (A) and protein level (B). After 72 h incubation with OA/BSA, the expression of Agxt was reduced upon both applied concentrations of OA/BSA compared to BSA controls. 5 d stimulation with OA/BSA caused strong variations in the RNA levels of Agxt between the biological replicates, and the difference between BSA and OA/BSA incubation was no longer obvious. One potential explanation could be a cultivation sensitivity of Agxt after plating the primary mouse hepatocytes.
Protein levels of Agxt were not reduced after 72 h but after 5 d incubation with 1.5 mM OA/BSA. This result indicated a relative long cellular half-time of the Agxt protein, which appears stable for several days despite a reduction of its transcript.
Figure 3.19: Downregulation of Agxt in primary mouse hepatocytes upon OA/BSA incubation. A) RNA level of
Agxt of OA/BSA exposed primary mouse hepatocytes was analysed by quantitative real-time PCR relative to the BSA controls and time points; Gapdh was used as the endogenous control (n = 4); B) Representative Western blot of Agxt expression of FM and BSA or OA/BSA treated primary mouse hepatocytes. β-Actin was used as a loading control (n = 3).
All in all, the in vitro steatosis model of primary mouse hepatocytes indicated a lipid- dependent downregulation of Agxt. To confirm this observation, OA/BSA-induced lipid accumulation was applied in the additional human hepatocellular carcinoma cell line Huh7. In these cells, we could also demonstrate lipid droplet accumulation upon OA/BSA exposure, using the Oil Red O staining (Figure 3.20). In the subsequent experiments, AGXT expression in lean and lipid-loaded Huh7 cells was analysed at the RNA as well as at the protein level.
Figure 3.20: Induced lipid accumulation in Huh7 cells upon OA/BSA incubation. Oil Red O staining visualised
lipid droplets after 72 h and 5 d incubation with 0.5 mM or 1.5 mM OA/BSA. Scale bars represent 50 µm.
As demonstrated in Figure 3.21, there was a reduction of the AGXT expression at the RNA level after 72 h incubation with 0.5 mM or 1.5 mM OA/BSA. In contrast, there was no decrease in AGXT protein expression after 72 h incubation with OA/BSA. Instead, the protein level of AGXT was clearly reduced after 5 d exposure to 1.5 mM OA/BSA.
Figure 3.21: Downregulation of AGXT in Huh7 cells upon OA/BSA incubation. A) RNA level of AGXT of OA/BSA
incubated Huh7 cells was analysed by quantitative real-time PCR relative to the BSA control (0.25 mM) and time point, UBC was used as the endogenous control (n = 4); B) Representative Western blot of AGXT expression of FM and BSA (0.25 mM) or OA/BSA exposed Huh7 cells (n = 3). α-Tubulin was used as a loading control.
These experiments in primary mouse hepatocytes as well as in human Huh7 cells confirmed a time-dependent downregulation of Agxt expression upon OA/BSA stimulation. The protein level Agxt did not decrease concurrently with the RNA levels but exhibited a delay. In addition, only the highest concentration of OA/BSA was able to decrease Agxt at the protein level. Altogether, these experiments demonstrated successful a time- and concentration- dependent downregulation of Agxt upon OA/BSA incubation in both murine and human in vitro steatosis systems, suggesting a similar mechanism of downregulation.