Patients with NAFLD present mitochondrial ultrastructural alterations that might reflect mitochondrial impairment and subsequent hepatocyte apoptosis [17
]. Although there is mounting evidence for a mitochondrial role during liver disease, few studies have focused on the role of this organelle during HSC activation [18
]. To our knowledge, this is the first demonstration that mild mitochondrial uncoupling can reduce several aspects of HSC activation.
Mitochondria can elicit cell signalling and influence cell function by adapting metabolism, altering cell response to cytokines and nuclear gene expression [20
]. Although initially counter intuitive, several studies indicate that uncoupling of the mitochondrial electron transport chain can elicit beneficial cell adaptations and improve several pathologies. Heart ischemia/reperfusion detrimental effects can be ameliorated through pre-conditioning with mild uncoupling using chemical uncouplers while studies on mice show that uncoupler treatment can increase life span, reduce oxidative stress and improve insulin levels [21
]. Remarkably, a study by Rohas et al
. has shown that once mild uncoupling is set, cells trigger a compensatory mechanism where PGC-1α is activated and orchestrate a signalling cascade to compensate the reduction in ATP levels [22
]. We also observe an increase in PGC-1α expression during HSC mild uncoupling and a protective effect against cell necrosis and apoptosis in both human and mouse derived HSCs.
ROS and consequent oxidative stress participate in HSC activation through several mechanisms such as glutathione depletion and activation of transcription factors [23
]. Indeed, the collagen type I promoter can be regulated by ROS generation in HSCs, demonstrating a fundamental role for oxidative stress in liver fibrosis perpetuation [24
]. We observed a decrease in ROS levels in cells treated with uncouplers, suggesting ROS reduction as one possible mechanism by which these molecules inhibit HSC activation. Additionally, this result also indicates that treatment with FCCP and Valinomycin induces only mild uncoupling, since harmful uncoupling is associated with high amounts of ROS and oxidative stress [25
]. Curiously, FCCP and Valinomycin show different antioxidant efficiencies. Valinomycin had a stable and less efficient effect on reducing ROS levels, while FCCP reduced ROS drastically after 30 minutes, reaching control levels after 24 hours. FCCP and Valinomycin uncouple mitochondria by different mechanisms. While FCCP is a proton mobile carrier that acidifies the mitochondrial matrix, Valinomycin acts as a specific potassium ionophore leading to alkalinisation of the mitochondrial matrix. This different effect on matrix pH can influence the dissimilar modulation of ROS levels [27
]. Since both chemical uncouplers affect ATP levels by the same extend, it also indicates that the mode by which they affect matrix pH does not influence the net result on mitochondrial uncoupling.
Transcriptional regulation by the so-called adipogenic transcription factors is essential to keep HSC quiescence and has been shown to, once stimulated, inhibit several aspects of HSC activation [28
]. Hence, it is hypothesized that signalling pathways leading to adipocyte differentiation similarly act on HSC activation [29
]. Brown adipocytes also share analogous signalling pathways during differentiation, but opposite to white adipocytes, they present small disperse lipid droplets in the cytoplasm and activate mitochondrial uncoupling when thermogenesis is needed. We show that chemical uncoupling inhibits HSC activation and consequently stimulates the maintenance of the quiescent phenotype. Curiously, treatment of white adipocytes with chemical uncouplers induces the opposite of what is seen in HSCs, stimulating de-differentiation, (i.e.
loss of lipid droplet and inducing a fibroblastic phenotype) [8
]. Moreover, AMP-activating kinase (AMPK), a kinase with a main role in maintaining cellular energy levels, induces differentiation of brown adipocytes when chronically activated, while it inhibits the differentiation of white adipocytes [30
]. In HSCs, AMPK inhibits several aspects of HSC activation and stimulates quiescent characteristics [31
]. Together, these data and the results observed in this study indicate that the HSC activation process shares analogous signalling pathways with the differentiation of brown adipocytes, cells that present mitochondrial uncoupling activity and, more importantly, a lipogenic phenotype similar to the one of quiescent HSCs.
We show that different aspects of human and mouse HSC activation such as α-SMA expression and proliferation are inhibited by chemical mitochondrial uncoupling. One important aspect of activated HSC is the capacity to proliferate and consequently aggravate chronic liver injury. HO-1, an enzyme involved in the metabolism of heme, can reduce HSC proliferation mainly due to the production of bilirubin, an antioxidant end product of heme group degradation [16
]. Curiously, both uncouplers inhibit the down regulation of HO-1 seen during activation, but HO-1 is only responsible for the anti-proliferative effect of FCCP and not Valinomycin, as shown using the HO-1 inhibitor SnPP. It has been reported that Valinomycin is able to inhibit the proliferation of different cell lines, although the mechanism is still not fully understood [32
]. Our results show that, in general, uncoupling can inhibit HSC proliferation, but the mechanism diverges depending on the mode of mitochondrial uncoupling.
TGF-β plays an essential role during liver fibrosis, and has been intensely investigated as a target for therapy. This cytokine activates HSC in vivo
, and TGF-β KO models have shown a dramatic reduction in α-SMA positive cells in liver fibrosis with a consecutive decrease in collagen deposition [33
]. In this study we observed an increase in the expression of several fibrogenic genes when HSCs were treated with TGF-β which was completely abrogated by co-treatment with chemical uncouplers. The inhibition of TGF-β induced early-immediate genes Smad6/7 by the uncouplers suggests a direct influence on TGF-β signalling. Although it is not clear how mild mitochondrial uncoupling can influence TGF-β signalling, it is known that this cytokine can induce the release of mitochondrial calcium stores and that this process is necessary for the activation of protein kinases and downstream signalling of TGF-β [34
]. Chemical uncoupling can influence calcium release from mitochondria [35
] and therefore might influence TGF-β signalling through modulation of mitochondrial calcium stores.
Intriguingly, the observations in this study add a new perspective to the action of some molecules that are already known to inhibit HSC activation. For example, curcumin has been shown to be a powerful mitochondrial uncoupler in the same concentration range known to inhibit HSC fibrogenic features [36