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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Publication date:
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 1
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NON-ALCOHOLIC FATTY LIVER DISEASE
Non-alcoholic fatty liver disease (NAFLD) poses an increasing threat globally and is believed to be the most frequent liver disease leading to mortality and the most frequent cause for liver transplantation in the near future. NAFLD comprises a spectrum of clinicopathological phenomena, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and eventually may develop into cirrhosis and hepatocellular carcinoma. The current prevalence of NAFLD is estimated to be around 25% globally with the lowest rate of NAFLD in Africa (13.5%) and the reported prevalence of NASH ranges from 1.5-6.5% [1-4]. Of note, the prevalence of NAFLD in obese or type 2 diabetes mellitus (T2DM) patients is even higher: the global prevalence of NAFLD among patients with T2DM has been reported to be 55.5% and among morbidly obese subjects who underwent weight reduction surgery more than 95% [1, 5, 6]. Since the progression of NAFLD is not linear, an increased rate of severity in the long-term outcomes of patients with NAFLD in the years to come can be expected.
Despite the significant progress in our understanding of this disease, there are still major gaps in our knowledge on the diagnosis and clinical management of NAFLD and the pathophysiology of NALFD. Nowadays, the diagnosis of NAFLD is usually based on abdominal ultrasound, which demonstrates fat infiltration in the liver, or magnetic resonance spectroscopy (MRS), which allows quantification of hepatic steatosis. However, the gold standard for diagnosis and assessment of liver fibrosis is still liver biopsy, which is an invasive procedure and may cause serious complications [3, 4]. To address this issue, several potential biomarkers have been suggested that may distinguish NASH from simple steatosis and predict disease activity for the whole spectrum of NAFLD. These biomarkers include cytokeratin-18 (CK-18), fibroblast growth factor 21 (FGF21) and insulin-like growth factor 2/epidermal growth factor receptor (IGF-2/EGFR) [7-10]. However, the accuracy and reproducibility of these non-invasive tests still need to be further investigated. In addition, several non-invasive scoring assessments combing non-invasive clinical and laboratory data have been suggested to grade hepatic fibrosis, such as NAFLD activity score (NAS), Fib-4 score, BARD score etc. [1, 3, 4]. Therefore, a simple, reliable and accurate test is still needed. Apart from the lack in diagnostic tools, to date, there are no effective FDA approved drugs for the treatment of NAFLD.
The commonly used definition of NAFLD is increased fat accumulation in the liver (>5% of liver volume) without secondary causes of fatty liver disease, such as excessive alcohol intake. It is now generally recognized that the development of NAFLD is a multifactorial process, which is currently described as “the multiple parallel-hit hypothesis” [11, 12]. For example, obesity and T2DM are two important and independent factors associated with NAFLD. Although the progression from steatosis to NASH was initially conceived as a sequential process, steatosis and NASH may correspond to two different entities [11]. Pathogenically, the development of steatosis is closely related to the imbalance of lipid metabolism, with lipid synthesis exceeding its utilization in the liver and with insulin resistance (IR) playing an important role. Importantly, some lipid species or metabolites
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demonstrate toxic features that lead to cellular damage [13-15]. This toxic effect of lipids istermed lipotoxicity. It is believed that lipotoxicity plays a critical role in the initiation of NASH. In Chapter 2, a comprehensive overview of the mechanism(s) of hepatic lipid accumulation
and a detailed explanation of lipotoxic effects in the liver is presented.
Although it is now recognized that several factors contribute to the progression from steatosis to NASH, the determining triggers are still not clear. Factors related to the progression of NASH include toxic lipid-driven inflammation, fibrogenesis and cell death [13-15]. Indeed, attenuation of lipotoxicity has been shown to prevent the development of NAFLD. In this thesis, we aim to study the role of fatty acids in the development of NAFLD using a cell-based approach in order to obtain a better understanding of the pathogenesis of NAFLD. We have also examined the protective effects of two compounds (metformin, a first-line anti-diabetic medicine and hesperetin, a flavonoid derivative) against lipotoxicity.
Figure 1. Illustration of hepatic lobule and sinusoid
A. Hepatic lobule. (Image courtesy of https://mda06el.webnode.com/liver-lobule/. This image is in the public domain and thus free of any copyright restrictions.) B. Liver sinusoid. (Image courtesy of https://en.wikipedia.org/wiki/Liver_sinusoid)
CELL-BASED STUDIES OF NAFLD
The liver has a very sophisticated architecture, containing different types of cells. The basic unit of the liver is the hepatic lobule, which has a hexagonal shape with a central vein in the center and portal triads at the vertices. Collectively, these channels and ducts compose the circulatory system of the liver (Fig 1). The blood supply from the portal vein (roughly 75%) and hepatic artery (roughly 25%) terminate into the sinusoids where the
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exchange of nutrients and waste products takes place [16]. Sinusoids form the vascular channels within the lobule. After exchanging nutrients and waste products with hepatocytes, the blood flow continues to the central vein, subsequently to the hepatic veins and eventually leaves the liver via the vena cava. The bile duct is the third channel of the portal triad and together with the gall bladder forms the biliary system of the liver. The most important cell types in the hepatic lobule are: hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells and Kupffer cells. Together these cells maintain the homeostasis of the liver. In this thesis, we studied the roles hepatic cells in the development and progression of NAFLD. We focused our studies on lipotoxicity, with special emphasis on the role of fatty acids in the development of NAFLD. In addition, we identified potential therapeutic interventions for NAFLD.
Hepatocytes
Hepatocytes, the parenchymal cells in the liver, constitute around 90% of the total volume of liver cells as revealed by stereological analysis [17]. The major functions of hepatocytes include metabolism of proteins, carbohydrates and lipids and detoxification, modification and excretion of endogenous and exogenous substances. In the progression of NAFLD, the increased lipid flux causes the accumulation of toxic lipids in hepatocytes, which induces cell stress as demonstrated by mitochondrial dysfunction, endoplasmic reticulum (ER) stress, oxidative stress and dysfunctional signaling pathways. This cellular stress promotes inflammation and fibrogenesis and contributes to the onset of NASH [13, 14, 18-20]. In our studies, we found that restoring both mitochondrial function and alleviating ER stress prevents fatty acid-induced hepatic cell death.
In Chapter 3, we show that moderate inhibition of mitochondrial complex Ι by
metformin or rotenone protects against toxic fatty acid-induced cell death. The protective mechanism is independent of the activation of AMPK, a target of metformin, but it is related to the restoration of mitochondrial function and reduction of oxidative stress [21]. Of note, several studies from other groups also imply that mitochondria are an interesting therapeutic target and that restoring mitochondrial function might attenuate the progression of NAFLD [22-24].
In Chapter 4, we show that the endoplasmic reticulum (ER) could be another
therapeutic target in the treatment of NAFLD. Homeostasis of the ER is largely supervised by the unfolded protein response (UPR) [25, 26]. Once there is a disturbance in ER homeostasis, the UPR will be activated to restore and/or maintain ER function. In this study, we used the natural product and flavonoid derivative hesperetin to treat palmitate-induced lipotoxicity and demonstrate that hesperetin inhibits palmitate-induced ER stress by activating adaptive UPR signaling, thus rescuing hepatocytes from lipotoxic cell death.
In Chapter 6, we study the effects of obstructive sleep apnea syndrome
(OSAS)-induced hypoxia on lipotoxicity in the context of NAFLD. We observe that subjecting hepatocytes to hypoxia clearly aggravates fatty acid-induced toxicity and promotes the release of (pro-inflammatory) extracellular vesicles (EVs) from hepatocytes. Importantly,
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these hepatocyte-derived EVs serve as extracellular signals that promote an inflammatoryphenotype in Kupffer cells [27].
Kupffer cells
Kupffer cells (KCs) are the liver resident macrophages with important roles in immune surveillance and scavenging of toxic compounds in the liver. They line the walls of sinusoids and are the first immune cells to respond in the progression of NAFLD [28, 29]. Because of their high plasticity, KCs can polarize (differentiate) into different phenotypes: the M1 and M2 phenotype and respond to the changes in the microenvironment. Once activated, KCs excrete immune-regulatory mediators and recruit blood-derived monocytes to the liver, a critical event in the initiation of NASH [30-32]. As already mentioned, in
Chapter 6, we investigate the communication between hepatocytes and KCs in the context
of NAFLD and show that hypoxia increases the release of EVs from fat-laden hepatocytes and that these EVs act as pro-inflammatory agents and induce the inflammatory phenotype in KCs. In Chapter 7, we also describe the communication between hepatic stellate cells
(HSCs) and KCs, focusing on the early activation stage of HSCs. We demonstrate that communication between HSCs and KCs is not unidirectional [33]. HSCs, in the early stage of activation, also promote the inflammatory phenotype of KCs via EVs.
Liver sinusoidal endothelial cells
The liver sinusoidal endothelial cell (LSEC) is a unique member of the endothelial cell family. As highly specialized endothelial cells, LSECs not only mediate the exchange of nutrients between the blood compartment and hepatocytes but also exhibit high endocytic ability and immune regulatory capacity. Under physiological conditions, LSECs are perforated with fenestrae organized in sieve plates and lack a basement membrane. However, in pathological conditions, LSECs capillarize, dedifferentiate and participate in the initiation and progression of liver diseases, including NAFLD [34-36]. It has been demonstrated that LSEC dysfunction correlates with the pathogenesis of NAFLD. Moreover, it has been reported that LSECs are the first cells to respond in the onset of NAFLD [37, 38]. However, the exact roles of LESCs in the development of NAFLD are largely elusive. In
Chapter 5, we study the effects of fatty acids on LSECs in parallel to vascular endothelial
cells.
Hepatic stellate cells
Hepatic stellate cells (HSCs) reside in the space of Disse between hepatocytes and sinusoids. In the normal liver, they are maintained in a quiescent state and store lipids, mainly retinyl esters. In response to liver injury, HSCs lose their retinol stores and became activated. Long-term chronic injury can lead to uncontrolled activation of HSCs resulting in liver fibrosis [39-41]. The activation of HSCs is regulated by many factors. The cytokines and chemokines produced by Kupffer cells are the main contributors to HSCs activation [42-44]. In Chapter 7, we observe that during the early activation stage of HSCs, HSCs and
Chapter 1
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inflammatory response of Kupffer cells. This positive feedback loop is mediated via the release of EVs from activating HSCs.
In summary, this thesis includes a collection of studies aimed at elucidating the role of fatty acids in the progression of NAFLD and describes some potential therapeutic drugs/targets for the treatment of NAFLD. The ultimate goal is to obtain a better understanding of the natural history of NAFLD and to provide novel insights into drug design and development. A summary and perspectives of this work are presented in Chapter 8.
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