2. LA EMISORA
2.6 Legislación aplicable y situación tributaria
Analysis of ileal fibroblast growth factor 15 (fgf15), the mouse equivalent of human FGF-19 showed significantly higher levels in our lean NAFLD, compared to the non-lean NAFLD mice (Figure 35A). The increased fgf15 levels activates the FGF receptor in the liver to stimulate hepatic FXR activity (Figure 35B).
Figure 35. Serum fgf15 levels (A) and FXR mRNA levels (B) in
mice fed a cholesterol rich (ChR) or a high sucrose (HS) diet.
The x-axis shows the cholesterol rich (ChR, blue, n=5) and high sucrose (HS, red, n=6) diet fed mice. The y-axis showed the mean serum fgf15 levels in pg/ml in A, and mRNA levels in folds in B, using 36B4 as housekeeping gene. Results are expressed as mean ± SEM and P value was calculated using the Mann-Whitney non-parametric t- test. P < 0.05, ** P<0.01, *** P<0.001.A.
5.4 DISCUSSION
We used mice fed a cholesterol rich diet (ChR) to replicate human lean NAFLD and mice fed a high sucrose (HS) diet to replicate non-lean NAFLD patients. Mice fed the ChR diet like in human lean NAFLD maintained their weight throughout the period, as opposed to mice fed a HS diet. The latter gained significant amounts of weight during the experiment. Furthermore, mice in the ChR diet group had significantly better fasting glucose levels as well as a better response to the glucose tolerance test suggesting better insulin sensitivity. Histologically, both mice groups developed steatosis, however, the lean NAFLD mice demonstrated increased inflammatory infiltrates.
Like in human NAFLD, the gut microbiota profile in the mice models showed similar patterns between the groups. There was increased Bacteroidetes and decreased Firmicutes in the lean NAFLD compared to the non-lean NAFLD mice. Previous studies have demonstrated possible roles of certain families of microbiota including in Bacteroidaceae and Erysipelotrichaceae with BA metabolism. Increased BA levels are associated with increased levels of these families (Zietak 2016). This may explain our finding of increased BA production through up-regulation of BA synthetic enzyme mRNA. Furthermore, certain taxa belonging to the family Erysipelotrichaceae and Coriobacteriaceae have been shown to be decreased in non-obese humans or those who have lost weight after gastric bypass compared to obese humans (Zhang et al. 2009). This is similar to our mice where the levels of the taxa belonging to Erysipelotrichaceae was lower in the lean compared to the non-lean NAFLD mice.
We were able to demonstrate increased levels of bile acids in lean NAFLD mice which is similar to what was seen in human NAFLD patients. Further testing also suggested that the increased level of bile acid is due to increased bile acid production, as evidenced by the up regulation of BA synthetic enzymes involved in both the classical and alternative pathways.
The increased levels of ileal bile acid transporter mRNAs for ASBT and OSTβ (which accounts for more than 95% of intestinal BA reabsorption from the distal ileum enterocytes (Chavez-Talavera et al. 2017)), despite not being significant, may reflect the better metabolic adaptation seen in lean NAFLD mice, as in our human lean patients. Bile acids stimulate secretion of several hormones including fgf15 (mouse equivalent of human FGF-19) from the enteroendocrine cells which has positive metabolic effects, including improved glucose tolerance. In addition, fgf15 is involved in metabolic adaptation through mediating bile acid synthesis in the liver, acting via the FXR pathway.
Despite the many similarities and benefits of using mice models to study human disease in terms of their homogeneity and more standardized nutritional manipulation, there are also differences between mice and humans. Thus , results from mice models must be interpreted with caution. Mice for example have distinct bile acid homeostasis compared to humans. Whereas in humans the primary bile acid pool is made up of cholic acid (CA) and chenodeoxycholic acid (CDCA) and their conjugates, in mice the majority of the primary BA pool is comprised of T-βmuricholic acid (T-βMCA) and
T-αmuricholic acid (T-αMCA) which are formed through hydroxylation of CDCA and UDCA respectively, via the enzyme CYP2c70 (McGlone et al. 2019). This difference in BA pool composition also affects the physiology seen in the two species, as MCA are FXR antagonists, whereas CDCA is a potent FXR agonist (McGlone et al. 2019). In addition, there are also differences in the homeostasis of BA in humans and mice. In mice, bile acids are almost exclusively conjugated with taurine which has greater solubility, whereas in humans, the majority of bile acids are conjugated with glycine, with only a small proportion being conjugated to taurine (McGlone et al. 2019).
5.5 CONCLUSION
Lean NAFLD is a unique sub-group of NAFLD with unclear pathophysiology. In this study we demonstrated using mice models fed a cholesterol rich or a high sucrose diet, certain similarities to human NAFLD patients, namely in their histological and metabolic profile, as well as in bile acid homeostasis. We also demonstrated a distinct and improved metabolic adaptation in the lean mice similar to human patients with respect to increased FXR activity. Although the results suggests the potential to undertake interventional experiments on NAFLD pathophysiology, including lean NAFLD, mice models have key differences with respect to their bile acid physiology and results must therefore be interpreted with caution.