4. DISCUSSION
In this study, we reported that the DPP-4 inhibitor linagliptin
ameliorated hepatic steatosis elicited by the dual IR/IGF1R inhibitor
OSI-906. We previously reported that a DPP-4 inhibitor,
des-fluoro-sitagliptin, prevented diet-induced adipose tissue
inflammation and hepatic steatosis (Shirakawa et al., 2011b). In another
study, linagliptin reportedly improved insulin sensitivity and hepatic
steatosis in diet-induced obese (DIO) mice (Kern et al., 2012). DPP-4
inhibition with linagliptin has been shown to decrease the expressions
of SREBP1c, SCD-1 and FAS, all of which are known to be related tode novo lipogenesis, and increased the expression of PPARα in the
livers of DIO mice (Kern et al., 2012). On the other hand, the
expressions of genes involved in hepatic fatty acid oxidation were not
affected by treatment with linagliptin in DIO or NASH mouse models (Kern
et al., 2012; Klein et al., 2014). However, the mechanism by which DPP-4
inhibitors improve fatty liver in the presence of diet-induced obesity
or diabetes remains unclear.
IR and IGF-1R-mediated signaling play a crucial role in hepatic insulin
action, which regulates glucose and fatty acid metabolism. OSI-906
administration induces the acute inhibition of IR/IGF1R signaling and
evokes hepatic steatosis (Tajima et al., 2017). In our mouse model, the
dual inhibition of IR and IGF-1R with OSI-906 resulted in impaired
insulin signaling in the liver. The liver weight and liver TG contents
were significantly increased by the administration of OSI-906 for 7
days. Linagliptin significantly reversed the OSI-906-induced increase in
liver weight and TG content. The OSI-906 group exhibited increased
gluconeogenic gene expression, compared with the vehicle group, and
treatment with linagliptin did not affect the expressions of these
genes, consistent with an impairment in hepatic insulin action. The
expressions of Gck , Srebp1c , Fas , and PPARα ,
which were also regulated by insulin signaling, showed no significant
alteration by treatment with linagliptin in OSI-906 treated mice. Hence,
the effect of linagliptin on hepatic steatosis seemed to be independent
of the IR/IGF1R signaling pathway. Our data indicated that although
insulin signaling is involved in the development of hepatic steatosis,
an alternative pathway that improves liver steatosis without altering
insulin signals likely exists in the OSI-906-treated model. The OSI-906
+ Lina group had lower glucose levels at 4 hours after OSI-906
administration on days 1 and 2, compared with the levels in the OSI-906
group, but these effects were abolished after day 3. Hyperinsulinemia
evoked by OSI-906 was also observed in the OSI-906 + Lina group.
Therefore, the glucose-lowering effects of linagliptin did not
contribute to the improvement in OSI-906-induced hepatic steatosis. In
patients with metabolic syndrome, fatty liver occurs because of an
increased uptake of fatty acids accompanied by an impairment in insulin
action in adipose tissue. In this study, linagliptin did not reduce the
plasma free fatty acid levels and hepatic expression of Cd36 ,
which is a free fatty acid transporter, in OSI-906-treated mice.
Therefore, linagliptin seemed to ameliorate OSI-906-induced hepatic
steatosis independently of the fatty acid flux to the liver, consistent
with the regulation of fatty acid metabolism via insulin. Thus,
linagliptin improved OSI-906-induced hepatic steatosis via a pathway
that was independent of insulin signaling, glucose levels, or free fatty
acid metabolism.
Liver-specific IR knockout (LIRKO) mice showed severe insulin resistance
and glucose intolerance with increased hepatic glucose production,
impaired glucose utilization in the liver, and abnormal mitochondrial
function because of the failure of insulin activity (Cheng et al., 2009;
Michael et al., 2000; Titchenell, Chu, Monks & Birnbaum, 2015). The
loss of FoxO1 increases lipogenesis and decreases fatty acid oxidation
in hepatocytes. Thus, liver FoxO1-null mice developed hepatic steatosis,
accompanied by the upregulation of lipogenic genes. This regulation of
lipogenesis by FoxO1 requires intact hepatic insulin signaling. The
systemic inhibition of IR with S961 blunted the effects of insulin on
hepatic glucose production in liver-specific IR/FoxO1 double knockout
LIRFKO mice (I et al., 2015). Thus, systemic insulin signaling might be
required to regulate hepatic glucose production and de novolipogenesis. On the other hand, lipodystrophy in adipose tissue-specific
IR knockout (F-IRKO) mice causes progressive NAFLD (Softic et al.,
2016). Treatment with OSI-906 also induced lipodystrophy and hepatic
steatosis, similar to the F-IRKO mouse model. These findings indicate
that not only hepatic insulin signaling, but also insulin signaling in
adipose tissue plays a crucial role in the development in hepatic
steatosis. However, in this study, linagliptin did not reverse the
lipodystrophy induced by OSI-906 administration, implying that
linagliptin exerts its effect on OSI-906-induced hepatic steatosis
without altering either hepatic insulin signaling or insulin signaling
in adipose tissue.
Chronic low-grade inflammation is known to contribute to the development
of steatosis (Tilg & Moschen, 2010). Interestingly, the inhibition of
IR and IGF1R by OSI-906 showed a tendency to decrease inflammatory gene
expressions. These results indicate that OSI-906-induced hepatic
steatosis is not provoked by the inflammatory response, unlike high-fat
diet-induced hepatic steatosis. We previously reported that DPP-4
inhibition prevented diet-induced hepatic steatosis and adipose tissue
inflammation in a diabetic mouse model (Shirakawa et al., 2011a). We
also showed that the PAI-1 expression levels in adipose tissue were
elevated in diet-induced hepatic steatosis (Shirakawa et al., 2011a). In
the present study, PAI-1 expression in the liver tended to decrease with
linagliptin treatment in the absence or presence of OSI-906, though a
significant difference was not observed. Linagliptin reportedly reduced
advanced glycation end products (AGE)-related oxidative stress in the
kidneys of a type 1 diabetes mouse model (Nakashima, Matsui, Takeuchi &
Yamagishi, 2014), whereas no significant differences in the expressions
of genes related to oxidative stress were observed in the current study.
Thus, in this OSI-906-induced hepatic steatosis model, the
identification of a new molecular mechanism for hepatic steatosis via
insulin resistance, other than inflammation or oxidative stress, might
be useful for understanding the development of hepatic steatosis.
Proteomic and phosphoproteomic analyses revealed some possible
mechanisms underlying OSI-906-induced progression in hepatic steatosis
and its amelioration by treatment with linagliptin. We focused on
molecules such as PLIN2 and sirtuins, which were suggested by a
proteomic analysis to be responsible for the effect of linagliptin on
hepatic steatosis. PLIN2, a lipid droplet-coating protein, is related to
lipid accumulation in the liver and promotes hepatic steatosis (Hall et
al., 2010; Libby, Bales, Orlicky & McManaman, 2016; Najt et al., 2016).
PPAR γ is also known to be required for the induction of PLIN2. In a
quantitative proteomic analysis, OSI-906 administration significantly
up-regulated PLIN2, whereas treatment with linagliptin almost canceled
the OSI-906-induced elevation in PLIN2. These results imply that the
regulation of PLIN2 in the liver is associated with the development of
hepatic steatosis induced by OSI-906 and its recovery as a result of
DPP-4 inhibition using linagliptin. Similarly, CYP2B10 was up-regulated
in OSI-906-treated liver, and treatment with linagliptin reversed this
induction. Regarding MUP20, its expression was reduced by OSI-906 and
restored by linagliptin. The regulation of Cyp2b10 and Mup20 were
consistent with the context reported for high-fat-diet-induced steatosis
and its recovery by treatment with resveratrol (Baur et al., 2006).
Cyp2b10 is related to lipid metabolism in the liver, and one of the
regulators of Cyp2b10 is a constitutive androgen receptor (CAR) (Ohno,
Kanayama, Moore, Ray & Negishi, 2014). Major urinary proteins (MUPs),
which are secreted proteins, belong to the lipocalin family and are
predominantly produced by the liver. A previous report showed that MUP1
increases energy expenditure and improves glucose tolerance (Zhou, Jiang
& Rui, 2009). Since resveratrol acts as a sirtuin activator, these data
suggest that treatment with linagliptin improves OSI-906-induced hepatic
steatosis by regulating sirtuin activity and its upstream and downstream
metabolic pathways.
Sirtuin, a nutrient sensor, activates AMPK and downstream fatty acid
oxidation in the liver. In fact, NNMT was significantly up-regulated in
OSI-906-treated liver in a quantitative proteomic analysis, supporting
the impact of sirtuin-mediated metabolic regulation in these mice.
Treatment of a human hepatocyte cell line with exendin-4 reportedly
increased the transcript levels of sirtuin1 (SIRT1) (Lee et al., 2012).
There are several signal transduction pathways that have been proposed
to improve hepatic steatosis as a result of incretin-based therapy, such
as cAMP-PKA signaling, PI3K-PDK1-Akt/PKB signaling, and AMPK signaling
(Samson & Bajaj, 2013). Whether incretin exerts direct effects or
facilitates indirect pathways affecting hepatocyte metabolism remains
controversial. Moreover, a recent report showed the importance of
hepatocyte-derived DPP-4 and obesity on adipose inflammation and insulin
resistance (24).
To address the conflicts in the actions of DPP-4 inhibitors and the
GLP-1 receptor agonist on the liver, we examined the effect of
linagliptin and the GLP-1 analog liraglutide on hepatocyte AML-12 cells.
Linagliptin did not inhibit gluconeogenic gene expressions in
OSI-906-treated AML-12 cells, consistent with an insulin
signaling-independent action of linagliptin on hepatic steatosis. On the
other hand, liraglutide showed a tendency to reduce the expressions of
gluconeogenic genes in OSI-906-treated cells. These results suggest that
the DPP-4 inhibitor and the GLP-1 analog had different effects on
insulin signaling in hepatocytes. In addition, the influence of
linagliptin on Tnf expression differed between in vivo andin vitro studies, implying an indirect action of linagliptin on
hepatocytes that does not involve the regulation of DPP-4 enzymatic
activity.
Taken together, these results show an effect of DPP-4 inhibition on
hepatic steatosis that is induced by the acute inhibition of IR/IGF1R
signaling through an insulin signaling-independent pathway (Figure 8).
Our findings support the non-canonical pleiotropic effects of DPP-4
inhibitors without a glucose-lowering effect and suggest the potential
of DPP-4 inhibitors as a new treatment for NAFLD. Further investigation
of the underlying mechanism is required.