University of Groningen Lipotoxicity in Non-alcoholic Fatty Liver Disease: Mechanisms and Prevention in Experimental Models Geng, Yana

University of Groningen

Lipotoxicity in Non-alcoholic Fatty Liver Disease: Mechanisms and Prevention in Experimental

Models

Geng, Yana

DOI:

10.33612/diss.130260314

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2020

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Geng, Y. (2020). Lipotoxicity in Non-alcoholic Fatty Liver Disease: Mechanisms and Prevention in

Experimental Models. University of Groningen. https://doi.org/10.33612/diss.130260314

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Chapter 8

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SUMMARY

The disturbance of lipid metabolism accompanying NAFLD, leads to increased lipid accumulation in the liver and an imbalance between non-toxic and toxic lipids. This imbalance results in lipotoxicity and a deleterious long-term prognosis. Lipotoxicity, as one important hallmark of NASH, helps us to understand the transition from benign steatosis to NASH, the (chronic) inflammatory stage of NAFLD. In Chapter 2 of this thesis, a detailed

explanation of the disturbed lipid metabolism and the molecular mechanisms of lipotoxicity is presented and discussed. It is now recognized that some lipid species or metabolites are more toxic than others, e.g. some free fatty acids (FFAs), lysophosphatidylcholine (LPC), ceramide and eicosanoid metabolites, which cause subcellular damage including mitochondrial dysfunction, ER stress, oxidative stress and the aberrant activation of signaling pathways. Eventually, this cellular injury leads to cell death and eventually inflammation and fibrogenesis thus contributing to the transition from steatosis to NASH [1-3].

In Chapter 3 and 4, we focused on mitochondria and ER, two major organelles

that are affected by lipotoxicity, with metformin and hesperetin respectively, to protect against toxic fatty acid-induced hepatic cell death. In Chapter 3, we showed that partial

inhibition of mitochondrial complex Ι, by metformin or low dose rotenone, protected against toxic fatty acid-induced cell death in hepatocytes. This protective effect is associated with the correction of mitochondrial dysfunction demonstrated by the re-polarization of the mitochondrial membrane potential and restoration of mitochondrial respiration. Furthermore, we demonstrated that the restored mitochondrial function was associated with reduced oxidative stress and increased SOD2 expression [4]. In Chapter 4, we showed that targeting

ER and alleviating ER stress could also protect against palmitate-induced cell death in hepatocytes. Hesperetin, a flavonoid derivative, activated the sXBP1/GRP78 signaling pathway, one of the three branches of the UPR signaling pathway and subsequently increased expression of the ER chaperone protein GRP78 that inhibited the activation of the apoptotic UPR branch eIF2α/CHOP. Meanwhile, we also observed that high concentrations of hesperetin could induce hepatic cell death by activating the apoptotic UPR signaling pathways.

Palmitate and stearate are the two most frequently used fatty acids to induce lipotoxicity in in vitro experiments. However, using the combination of saturated and unsaturated fatty acids (for example, palmitate and oleate) might better represent the steatotic stage of NAFLD in vitro. The combination of palmitate and oleate causes a more pronounced intracellular lipid accumulation, but much less cellular damage than palmitate or stearate alone [5, 6]. In Chapter 6, we used the palmitate and oleate-induced fat-laden

hepatocyte model and found that hypoxia is an important risk factor that aggravates the toxic effects of fatty acids. In fat-laden hepatocytes, the hypoxic condition significantly induced pyroptosis and increased the production of EVs from hepatocytes. Importantly, these

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increased expression of cytokines and chemokines [7].

As mentioned before, unlike palmitate, oleate does not exhibit lipotoxic effects and protects against palmitate-induced lipotoxicity. This phenomenon is, however, cell-type specific, since in Chapter 5, we found that oleate caused necrotic cell death in liver

sinusoidal endothelial cells (LSECs). Specifically, comparing the actions of oleate with or without palmitate in human umbilical cord endothelial cells (HUVECs) and LSECs, we demonstrated that oleate exhibited no toxicity to HUVECs but it did induce necrotic cell death in LSECs. Its toxic effects are related to decreased mitochondrial respiration and subsequent ATP depletion. On the other hand, the effects of palmitate and the combination of oleate and palmitate are consistent in both HUVECs and LSECs: palmitate induced apoptotic cell death whereas the combination of these two fatty acids was non-toxic. One important observation was that the non-toxic conditions are always accompanied by increased TG synthesis and lipid droplet formation. Whether the increased TG synthesis and lipid droplet formation underlie the protective mechanism against oleate or palmitate is currently under investigation.

Finally, in Chapter 7, we studied the communication between hepatic stellate cells

(HSCs) and KCs. In the progression of NAFLD, the inflammatory phenotype of hepatic macrophages is an important indicator of NASH. Several studies have shown that both fatty acid and lipopolysaccharides (LPS or endotoxin) play major roles in inducing an inflammatory response in hepatic macrophages [8-10]. In Chapter 7, we showed that,

besides fatty acids and LPS, HSC-derived EVs induce an inflammatory response in KCs. Moreover, the inflammatory effects of HSC-derived EVs are dependent on the Toll-like receptor-4 (TLR4) on KCs.

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PERSPECTIVES

The heterogeneity of fatty acids

As already discussed in this thesis, the biological properties of fatty acids vary greatly due to differences in their degree of saturation, chain length and cis-trans isomerism. Based on the number of double bonds in their chemical structure, fatty acids can be classified into saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). Thanks to the substantial amount of studies on lipids from the last two decades, it is now recognized that, roughly speaking, saturated fatty acids (especially long-chain and very long-chain) are toxic; whereas unsaturated fatty acids are mostly non-toxic or even beneficial. It has been shown that saturated fatty acids and unsaturated fatty acids are subjected to different metabolic pathways in cells. For example, palmitate is more prone to undergo β-oxidation or to be incorporated into phospholipids, whereas oleate is more readily incorporated into TG [11-16]. The differences in intracellular partitioning of fatty acids (either to mitochondria or to lipid droplets) may account for the opposite cell fates. The different metabolic outcomes of fatty acids will also cause the activation or inhibition of distinct signaling pathways, including the insulin signaling pathway and inflammatory signaling cascades [17-20]. Furthermore, a study from Chausse et al. showed that the differential modulation of inflammation by palmitate and oleate was related to their effect on intracellular PUFA trafficking and metabolism. Specifically, palmitate promotes the incorporation of PUFAs into membrane phospholipids, which made PUFAs more susceptible to lipid peroxidation. Meanwhile, oleate induces the storage of PUFAs into lipid droplets, which prevents their lipid peroxidation and subsequent oxidative stress [21].

The heterogeneity of fatty acids is not only defined by their degree of saturation. The chain length also causes difference in their properties and metabolism. For example, the members of the acyl-CoA thioesterases (Acots) superfamily, which reside in mitochondria and hydrolyze fatty acyl-CoA esters, exhibit substrate specificity based on the fatty acid chain length [22]. Because Acots are expressed at different levels in different cells, the fatty acyl-CoA esters are hydrolyzed to different extents in different cell types. Moreover, exogenous or endogenous fatty acids also demonstrate differential metabolic preferences. As indicated by Yao et al., proliferating fibroblasts preferentially use exogenous fatty acids as the source of membrane lipids [23]. Taken together, these differences in cellular metabolism of fatty acids may explain the cell-type specific effects of fatty acids. As described In Chapter 6, oleate was either protective or detrimental in HUVECs and LSECs,

respectively, which might be associated to differences in metabolism between these two cell types.

In summary, the heterogeneity of fatty acids and their metabolism may lead to opposite metabolic outcomes. Although lipotoxicity is mostly associated to SFAs and their derivatives, such as LPC and ceramide, under certain conditions beneficial, unsaturated fatty acids can also induce cellular toxicity. Therefore, understanding the heterogeneity of

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aspects of them.

Targeting lipid metabolism in the treatment of NAFLD

With the advances in our understanding of the pathogenesis of NAFLD, regaining the balance between lipid accumulation (including lipid synthesis) and utilization in the liver has become an important pharmacological target in the development of treatments for NAFLD. Several promising therapeutic targets are currently explored and many drugs/compounds are now investigated in clinical trials. So far, the molecular targets that directly modulate lipid metabolism range from key enzymes regulating de novo lipogenesis in the liver (acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD1) and ATP-citrate lyase (ACLY)), to crucial nuclear hormone receptors (peroxisome proliferator-activated receptors (PPARs), farnesoid X receptors (FXRs) and thyroid hormone receptor-β (THR-β)) and to fibroblast growth factors (FGF21 and FGF19) [24-31]. In addition, there are also two glycemic modulators that show improvement of hepatic steatosis, liver inflammation and injury: the gut-derived hormone glucagon-like peptide-1 (GLP-1) and renal sodium glucose co-transporter 2 (SGLT2).

Based on the outcomes of the clinical trials, these modulators of lipid metabolism demonstrated a significant reduction in liver fat content and improvement of liver function. However, discrepant beneficial effects with regard to NASH resolution and fibrosis regression and non-negligible side effects still exist [3, 26, 28]. Current efforts are aimed at tackling these problems and discrepancies, for example using combination therapy aimed at multiple targets rather than monotherapy. The combination therapy might be a solution to the existing problems. However, there is still a grey area with regard to the usefulness of the previously mentioned lipid modulators. It is not known whether these lipid modulators also restore the non-diseased phenotypes of non-parenchymal cells (KCs, HSCs and LSECs). Non-parenchymal cells are the principal cells regulating inflammation and fibrogenesis. Whether the drugs also improve non-parenchymal cell function has so far been poorly addressed, although this will be important to assess the therapeutic potential of these drugs. On the other hand, lipotoxicity is not necessarily correlated with hepatic TG level. Although most of these lipid-modulating agents reduced hepatic steatosis, it remains important to assess whether the reduction of steatosis leads to a balance between healthy lipids and toxic lipids.

Although all of the previously mentioned lipid-balancing strategies show promising therapeutic results against NAFLD, more detailed cell-specific and lipid-specific studies, are necessary to establish their therapeutic potential and clinical use.

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CONCLUSION

Although the molecular basis of lipotoxicity has been substantially explored, there are still many grey areas with regard to lipotoxic effects and lipid metabolism. Specific areas of interest are the mode of fatty acid-induced cell death and the activation of inflammatory pathways. Moreover, as the most abundant cells in the liver, hepatocytes are mainly studied in the context of NAFLD. However, the role of non-parenchymal cells, which govern inflammation and fibrogenesis in NASH, should receive more attention. Overall, besides counteracting lipotoxicity, a better understanding of lipid metabolism, including its cell-specific character, is important in developing treatments for NAFLD and to prevent the progression of NAFLD to more advanced stages.

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