MANASH publishable summary
In Europe, 80 billion euros are spent annually on the health care costs associated with metabolic
syndromes. This collection of diseases includes obesity and type 2 diabetes. According to the
International Diabetes Foundation, the projections for the number of adults with diabetes/metabolic
syndrome will increase by 20% from 2010 to 2030 in Europe and by 54% worldwide. Non-alcoholic
steatohepatitis (NASH) is a major health problem and is rapidly becoming one the most common
liver diseases worldwide. The prevalence of NASH in the general population of Western countries
is significantly high and ever increasing, not only in over-weight children and adults but also in
insulin-resistant individuals of normal weight. Due to the rapidly progressing epidemics of obesity
and diabetes a large segment of the population is at risk for NASH. Particularly worrisome is the
emergence of NASH with significant fibrotic disease in developing countries even in patients of
normal weight or who are underweight. Therefore it is of utmost importance to find treatment
options against NASH.
Impaired energy metabolism and the chronic
inflammation of metabolic tissues
following from lipid overload are hallmarks of the metabolic syndrome. NASH is the hepatic event
of the metabolic syndrome and is characterized by steatosis and inflammation. Although our
understanding of the disease mechanisms has progressed, the therapy options for treatment of
NASH remain poor. The mechanism(s) underlying the development of hepatic steatosis include
excess dietary fat, increased delivery of free fatty acids to the liver, inadequate fatty acid oxidation,
and increased de novo lipogenesis. The benign condition that precedes NASH is termed nonalcoholic fatty liver disease (NAFLD) and refers to reversible steatosis, whereas in NASH (by
unknown mechanisms) the liver progresses to inflammation leading to fibrosis, cirrhosis and
eventually liver failure.
AMP-activated protein kinase (AMPK) is an important integrator of signals managing
energy balance and acts as a protective response to energy stress during metabolic deregulations.
A significant amount of literature points at AMPK to be a vital cog in regulating the metabolism of
carbohydrates and lipids as well as protein synthesis and cell growth in metabolically active
tissues. In the liver, AMPK activates fatty acid oxidation and inhibits lipid biogenesis via its effects
on transcription factors and directly by enzyme activation/inhibition. AMPK may also be activated
by pathological stresses (i.e. oxidative stress), exercise and tissue-derived hormones. Recent
evidence suggests that AMPK limits inflammation by
acting in immune cells. Accordingly, liver-targeted
activation of AMPK could provide with a new
therapeutic option for the treatment of NASH by
reducing the inflammation and ameliorating the
associated insulin resistance. In metabolic
syndromes AMPK activity is down-regulated and,
thus, attempts have focused on the development of
direct pharmacological activators. Upstream kinases
of AMPK phosphorylate AMPKα at Thr172 and this
phosphorylation is the key activation mechanism of
AMPK.
MSP inhibits hepatic gluconeogenesis
and toll-like receptor signaling via activation of
AMPK signaling. MSP is constitutively secreted by
the liver into the circulating blood as a single chain
biologically inactive pro-MSP. Mature MSP is
generated through proteolytic cleavage by trypsinlike serine proteases at extravascular sites to target
macrophages and other cell types. MSP is a ligand
for the Recepteur d’Origine Nantais (RON) receptor
tyrosine kinase, expressed in several tissues
including liver and brain. MSP/RON has inhibitory
roles in inflammatory responses, such as production
of nitric oxide in stimulated macrophages. Downstream of RON, phosphoinositide-3-kinase
(PI3K)/Akt, mitogen activated protein kinase (MAPK), β-catenin and others have been suggested
to mediate the effect. However, an obvious link to explain AMPK activation is missing at present.
Remarkably, on normal diet MSP-deficient mice develop steatosis. Furthermore, MSP is both
necessary and sufficient to induce macrophage polarization into the anti-inflammatory M2
phenotype (opposed to the pro-inflammatory M1 activation), which assists in attenuation of
inflammation. The possible implication of the MSP/RON-AMPK axis in NASH has not been
investigated before. Therefore, we elucidated and evaluated MSP/RON-mediated activation of
AMPK in the context of NASH (Figure 1).
The main objectives of this study were (1) to investigate the molecular mechanism of
MSP-mediated AMPK activation and (2) to assess the potential of MSP for ameliorating NASH.
The results obtained clearly show the beneficial effects of MSP in the context of
NASH-inducing conditions in vitro and ex vivo. In primary hepatocytes, MSP treatment leads to
significant activation of AMPK signaling pathway. This activation is sustained even in the presence
of lipogenic as well as inflammatory stimuli. Concomitantly, we observe that lipogenic/inflammatory
stimulus-induced expression of several pro-inflammatory cytokine genes was significantly reduced
upon MSP treatment. To confirm this observation, we analysed pro- and anti-inflammatory cytokine
production level under these conditions. Our results demonstrate that MSP reduces production of
pro-inflammatory cytokine and elevates anti-inflammatory production level in this set-up.
Additionally, MSP reduces lipogenic stimulus-induced lipid accumulation in these cells.
Furthermore, MSP treatment of bone marrow derived macrophages (BMDMs), which were
exposed to NASH-mimicking stimuli, led to significant reduction in pro-inflammatory genes
expression. These results strongly support the anti-inflammatory role of MSP and indicate a
potential beneficial effect of MSP in treating NASH. Knockdown of RON receptor expression in
human hepatic HepG2 cell line dramatically reduced the effect of MSP as was evidenced by a
significant decrease in AMPK activation. After knockdown of RON, MSP failed to inhibit the free
fatty acid-induced increase in pro-inflammatory genes expression under this condition. These
results clearly demonstrate that an intact MSP/RON-AMPK axis is required to counter NASH.
In conclusion, MSP has been verified as a potential treatment strategy against NASH.
However, further analysis of in vivo data and long-term studies will be required to substantiate the
efficacy and safety of MSP in mice and other experimental models. In long-term we foresee clinical
testing of recombinant MSP for amelioration of human NASH. Given the severity and rising
incidence of NASH, identifying potential ways of ameliorating the disease directly correlates to the
well being of the society from the economical (keeping in mind the rising costs of healthcare in
Europe) till the humanitarian point of view.