3.3. Hepatic proteomic and phosphoproteomic analyses revealed
insulin- and glucose- independent effects of linagliptin in
OSI-906-induced hepatic steatosis
We also performed hepatic quantitative proteomic and phosphoproteomic
analyses of the livers from OSI-906- or linagliptin-treated mice (Figure
3a). We identified a total of 1884 proteins, and differentially
expressed proteins were analyzed using Progenesis QI software to compare
expressions in the OSI-906 vs. vehicle groups and the OSI-906 + Lina vs.
OSI-906 groups (Figure 3b).
We focused on proteins with
expressions that were significantly altered by the administration of
OSI-906 and for which the alterations were reversed by the treatment
with linagliptin (Figure 3c and Supplementary Table 2) to address the
amelioration of hepatic steatosis by linagliptin in the OSI-906-treated
model. Among the proteins that were significantly up-regulated or
down-regulated in response to the administration of OSI-906
(Supplementary Tables 2-4), the abundances of perilipin-2 (PLIN2) and
cytochrome P450 2b10 (CYP2B10) were reduced by OSI-906 and were restored
by treatment with linagliptin. In contrast, the abundance of major
urinary protein 20 (MUP20) was increased by OSI-906 and was also
restored by treatment with linagliptin. Those results indicate that
these molecules might contribute to the process of the
linagliptin-induced amelioration of hepatic steatosis. A previous study
reported that the expressions of cytochrome P450 enzymes were
down-regulated and those of major urinary proteins (MUPs) were
up-regulated by treatment with resveratrol, a sirtuin activator, in the
liver (Baur et al., 2006). We also found that the protein expression of
nicotinamide N-methyltransferase (NNMT) was significantly up-regulated
in OSI-906-treated liver. Recently, the contribution of NNMT activation
to the development of hepatic steatosis has been reported (Komatsu et
al., 2018). Therefore, these findings suggest that sirtuin activity is
regulated by OSI-906 administration and might contribute to the
improvement in hepatic steatosis (Pissios, 2017). We validated these
findings using immunoblotting or gene expression analyses. The protein
expressions of PLIN2 and NNMT were increased in OSI-906-treated liver,
and these increases were reversed by linagliptin (Figure 4a). In
addition, the increased pan acetyl-lysine levels in OSI-906-treated
liver were also reversed by linagliptin, implying an enhancement of
sirtuin activity by linagliptin in this OSI-906-induced hepatic
steatosis model (Figure 4a). The hepatic gene expression ofCyp2b10 was also induced by OSI-906 and was deceased by treatment
with linagliptin (Figure 4b). A canonical pathway analysis of
differentially expressed proteins in the OSI-906 vs. vehicle groups and
the OSI-906 + Lina vs. OSI-906 groups revealed that the administration
of OSI-906 activated the cholesterol biosynthesis pathway and inhibited
the LXR/RXR pathway, whereas linagliptin inhibited the cholesterol
biosynthesis pathway (Figure 5).
In a phosphoproteomic analysis of liver samples from the four groups,
more than 3700 sites of phosphorylation were identified. The top
increased or decreased phosphopeptides when the OSI-906 vs. vehicle
groups or the OSI-906 + Lina vs. OSI-906 groups were compared are shown
in Supplementary Tables 5-8.
A canonical pathway analysis of the phosphoproteomics data identified
the inhibition of insulin receptor signaling and mTOR signaling in
OSI-906-treated liver, consistent with the dual inhibition of
insulin/IGF-1 receptors by OSI-906 (Figure 6). Treatment with
linagliptin did not seem to affect insulin signaling. An upstream
regulator analysis indicated that PPARα and XBP1 were the top 2
predicted transcription activators involved in the process of hepatic
steatosis induced by OSI-906, whereas pathways related to SREBF2 and
lysophosphatidylcholine were identified in the linagliptin-treated group
(Supplementary Table 9).