Journal of Hepatology 50 (2009) 857–860 www.elsevier.com/locate/jhep
Editorial
Uncoupling proteins and non-alcoholic fatty liver disease q Helena Cortez-Pinto*, Mariana V. Machado Servicßo de Gastrenterologia, Hospital Santa Maria, Unidade de Nutricßa˜o e Metabolismo, Instituto de Medicina Molecular (IMM), Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, Lisboa 1649-035, Portugal
See Article, pages 1019–1028
1. Introduction Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by lipid deposition in hepatocytes, in the absence of excessive alcohol consumption. It is now considered part of metabolic syndrome, with insulin resistance as a primary underlying derangement. It is widely accepted that a first hit leads to hepatic steatosis, and further hits to necro-inflammation and fibrosis (steatohepatitis), with oxidative stress, reactive oxygen species (ROS) and endoplasmatic reticulum stress playing a major role [1]. Mitochondrial dysfunction seems to be crucial in the pathogenesis of NAFLD, with less fatty acid oxidation favoring fat accumulation [2], and also being the major source of ROS contributing to necro-inflammation [3–5]. In fact, NAFLD has been considered a mitochondrial disease [6,7]. 2. Uncoupling proteins Uncoupling proteins (UCPs) are mitochondrial inner-membrane proteins, whose main function is to mediate proton leak across the inner membrane and to uncouple substrate oxidation from ATP synthesis [8]. The liver is the largest metabolic organ in the human body, and mitochondrial proton leak accounts for 20– 30% of the oxygen consumption of isolated resting hepaAssociate Editor: C.P. Day q The authors declare that they do not have anything to disclose regarding funding from industries or conflict of interest with respect to this manuscript. * Corresponding author. Tel.: +351 217985187; fax: +351 217985142. E-mail address:
[email protected] (H. Cortez-Pinto).
tocytes [9]. Due to the uncoupling character of UCPs, it is plausible that they might participate in hepatic mitochondrial proton leak and certain deregulated metabolic pathways. There are several UCP homologues, UCP1 expressed in brown adipose tissue, UCP3 expressed in brown adipose tissue and skeletal muscle and UCP4 and 5 expressed in the brain [10]. UCP2 is rather ubiquitous, however in the liver it has been mostly localized to Kupffer cells, with very low or undetectable levels in hepatocytes [11]. Nonetheless, UCP2 expression was detected in healthy and diseased hepatocytes in human percutaneous liver biopsy specimens [12]. The fact that UCP2 expression in normal hepatocytes is so unusually low remains perplexing, raising the possibility of the existence of other proteins involved in hepatocyte proton conductance. In fact, a novel liver-specific uncoupling protein was isolated in 2004, termed HDMCP (hepatocellular carcinoma down-regulated mitochondrial carrier protein), that exhibits reduced expression in hepatocellular carcinoma and shows evolutionary conservation between humans, mice, and rats [13]. In this issue of the Journal, Jin et al. report new and exciting data about the potential role of this new uncoupling protein in NAFLD [14]. 3. Uncoupling proteins and NAFLD UCPs may provide a beneficial mechanism that permits an adaptation to increased fatty acid supply, enhancing fatty acids (FA) oxidation and thus conferring a theoretical benefit in preventing hepatic steatosis. In fact, there are several mechanisms (Fig. 1), through which the up-regulation of uncoupling proteins, can alleviate hepatic steatosis and the ensuing lipotoxicity
0168-8278/$36.00 Ó 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2009.02.019
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H. Cortez-Pinto, M.V. Machado / Journal of Hepatology 50 (2009) 857–860 LCFA
LCFA-CoA CoASH CPT
UCP
to β-oxidation
CoASH
to Krebs cycle
LCFA
LCFA-CoA
Acetyl-CoA Beta Oxidation O2.-
Krebs Cycle
H+ H+
ADP ATP
NADH/FADH 2 mitochondrial inner membrane
UCP
H+
I
II
III
IV
ATPase
H+
Fig. 1. Proposed UCP related mechanisms. NADH/FADH2 reduced during fatty acids oxidation, function as electron donors to the phosphorylative respiration which leads to the oxygen dependent production of ATP. The electron transport chain has four enzyme complexes in the inner mitochondrial membrane, the last being cytochrome c oxidase which gives two electrons to one O2 molecule and in combination with 2H+, converts it to H2O. That electron transport is coupled to a movement of protons (H+) from the mitochondrial matrix to the intermembrane space producing an electrochemical gradient. When energy is needed, H+ reenter the mitochondrial matrix, and the energy produced by that H+ movement is used by ATP synthase to convert ADP in ATP. However, mitochondrial hyperpolarization increases half life of mobile electron carriers, allowing incomplete electron transport and partial reduction of O2 molecules, with the production of oxygen reactive species such as O2 . Uncoupling proteins (UCPs) are mitochondrial innermembrane proteins that mediate H+ leak across the inner membrane and uncouple substrate oxidation from ATP synthesis. UCPs enhance fatty acids oxidation through enhancing recycling of oxidised NAD+/FAD+; allowing entrance of CoASH (rate-limiting coenzyme to beta oxidation and Krebs cycle) through a cycle of fatty acids inflow of acyl-CoA, cleavage of CoASH and outflow of fatty acids. Also, removal of fatty acids from mitochondria and lipid peroxide anions, protects further mitochondrial injury (adapted from McLellan et al. [15]). [This figure appears in colour on the web.]
in the event of an excess of fuel: (1) the proton leak may allow increased beta-oxidation of FA in the mitochondria, supporting ongoing fatty acid oxidation as opposed to accumulation, through reoxidation of NADH to NAD+; (2) it may provide a translocation mechanism that prevents accumulation of non-esterified fatty acids (NEFA) and their harmful effects in the mitochondrial matrix; (3) mediate free fatty acid (FFA) outflow from the mitochondrial matrix, allowing re-entry as acyl-CoA, functioning as CoASH donor necessary for beta-oxidation [15]; (4) activate AMPK (AMP-activated protein kinase) expression [16,17], that powerfully steers metabolic pathways from energy accumulation to energy expenditure and fuel preference from glucose to fatty acids [18]. AMPK promotes fatty acid oxidation and ketogenesis, and inhibits peripheral lipolysis and lipogenesis [18]. Interestingly, it was found that in UCP2 knockout mice, fasting initially promotes peripheral lipolysis and hepatic fat accumulation at less than expected rates, but culminates in protracted steatosis, indicating diminished hepatic utilization and clearance of FAs [19]. Concerning NAFLD, an increased hepatic expression of UCP2 in animal models was found [20], as well as a
trend for increased expression in the liver biopsies from non-alcoholic steatohepatitis (NASH) patients [21]. A large number of studies on various tissues confirmed that UCP2 expression is generally increased in response to elevated plasma FA levels [20,22,23]. In vitro exposure of primary cultured rat hepatocytes to lipids resulted in up-regulation of UCP2, indicating that excess UCP2 may indeed originate in hepatocytes as opposed to non-parenchymal liver cells [24]. However, until now, a protective effect of UCPs in the prevention of hepatic steatosis has not been demonstrated. In fact, Baffy et al. found no difference in the degree of steatosis in ob/ob mice knockout for UCP2 and ob/ob wild-type mice [23]. A possible role of UCPs in the second hit of NAFLD is also appealing. UCPs may unleash the flow of electrons in the respiratory chain, thereby preventing the over-reduction of respiratory complexes and excessive mitochondrial ROS formation [25], protecting the cell from lipoperoxidation and diminishing necro-inflammation. Furthermore, UCPs can protect the mitochondria from lipid peroxides formed by the reaction of FA and ROS, either through a decrease in ROS production or through a direct flip flop of those lipid peroxides across
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the mitochondrial membrane [17]. However, in animal models of NAFLD, oxidative stress and ROS production persists, despite an increase in UCP2 [4]. It may be speculated that even increased amounts of UCP2 are not sufficient to control the exacerbated intracellular ROS generation in fatty liver. Also, the increased expression of UCPs, may compromise ATP synthesis, increasing the vulnerability to energy depletion [26]. That might explain the increased susceptibly to ischemia–reperfusion [20], that is markedly reduced in UCP2 knockouts [27], and to Fas-mediated liver injury [28] in fatty liver. When translating these data to a potential effect in NASH, once again, Baffy et al. did not find any difference in steatohepatitis and fibrosis score in ob/ob mice, knockout for UCP2, and wild-type ob/ob mice [23]. In a report from Jin et al. in this issue it is suggested that the novel uncoupling protein, HDMCP may have a protective effect concerning steatosis development. In a series of elegant studies, the authors first confirmed that HDMCP had an uncoupling effect on yeast mitochondrial respiration, being more concentrated in the mitochondria. Next, using sequence analysis, they demonstrated the similarity between UCPs and HDMCP sequences. Furthermore, and more relevant for the issue in discussion, a significantly increased expression of HDMCP was seen in a high-fat rat NAFLD model. Sequentially, by in vitro exposition of hepatocytes to a mixture of oleic and palmitic acid (HFFA), a time dependent increase in the levels of HDMCP was observed in the steatotic hepatocytes; when the expression of HDMCP was partially silenced by HDMCP-ShRNA, steatosis was aggravated. Simultaneously, ATP levels of NAFLD livers and steatotic hepatocytes were significantly decreased, the latter negatively correlating with HDMCP protein levels; this ATP reduction was partially antagonized by silencing HDMCP. Interestingly, the incubation of hepatocytes with HFFA resulted in diminished H2O2 production, that was to some extent reversed when HDMCP was partially knocked out [14]. Accordingly, the authors claim the potential benefits of the adaptive effect of HDMCP in protecting hepatocytes from steatosis, considering it as an attractive drug target for therapeutic purposes [14]. However, one should question why did this uncoupling protein have an effect on the degree of steatosis, when UCP2 modulation did not influence the severity of fatty liver disease [23]. One potential explanation was recently proposed by Baffy [17]. In contrast with normal liver, where the expression of UCP2 dominates in Kupffer cells while it is rather limited in hepatocytes [11], this pattern appears to be the opposite in fatty liver, where there is an increased hepatocellular UCP2 expression. Conversely, a down-regulation of UCP2 in macrophages and increased ROS production has been shown in obese mice, a change that presumably
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extrapolates to Kupffer cells [29]. This cell-specific upregulation of UCP2 can also partially explain the absence of perceivable differences in obesity related fatty liver of mice with and without UCP2 [23]. If the regulation of HDMCP is not cell-specific, its up-regulation might have a much more profound effect. On the other hand, when considering the potential benefits of uncoupling agents, such as HDMCP, on NAFLD that should be tempered by the recognition of their potential to increase the risk of necrosis. The fact that Jin et al. reported a significant negative correlation between HDMCP and ATP implies an increased risk for necrosis when HDMCP is increased. In fact, establishing a parallel with the UCP2, in two studies in obese mice, the down-regulation of UCP2, either related with metformin treatment [30] or with, probiotics [31] was associated with an increased recovery of hepatocyte ATP stores and an improvement in necroinflammation. These observations suggest that the effect of uncoupling proteins may be deleterious, potentially leading to an increased risk of necro-inflammation/steatohepatitis. In fact, as acknowledged by the authors, there is the possibility that HDMCP may have an effect in preventing steatosis, although simultaneously increasing the risk for NASH [14]. Conversely, studies have shown that uncoupling proteins tend to protect from apoptosis [32,33], probably through a reduction of ROS production [34]. This could be beneficial, since apoptosis is a significant pathological feature of steatohepatitis [21,35]. However, it has been suggested that the persistent activation of UCP2, by reducing apoptosis, might contribute to the increased risk of hepatocellular carcinoma observed in NAFLD [17]. Even so, the effect of HDMCP on apoptosis and its potential consequences on carcinogenesis is still unknown. In conclusion, the possibility of modulating mitochondrial respiration and the fate of excessive fatty acids with this novel uncoupling protein is very interesting and promising. It should however be regarded with caution, since these are interacting mechanisms with unpredictable effects. References [1] Cortez-Pinto H, de Moura MC, Day CP. Non-alcoholic steatohepatitis: from cell biology to clinical practice. J Hepatol 2006;44:197–208. [2] Ibdah JA, Perlegas P, Zhao Y, Angdisen J, Borgerink H, Shadoan MK, et al. Mice heterozygous for a defect in mitochondrial trifunctional protein develop hepatic steatosis and insulin resistance. Gastroenterology 2005;128:1381–1390. [3] Perez-Carreras M, Del Hoyo P, Martin MA, Rubio JC, Martin A, Castellano G, et al. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology 2003;38:999–1007. [4] Yang S, Zhu H, Li Y, Lin H, Gabrielson K, Trush MA, et al. Mitochondrial adaptations to obesity-related oxidant stress. Arch Biochem Biophys 2000;15:259–268.
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