Laboratory of Apoptosis and Cancer Biology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Tianjin Key Laboratory of Protein Sciences, College of Life Sciences, Nankai University, Tianjin, China
College of Life Sciences, Nankai University, Tianjin 300071, or the State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
Potential conflict of interest: Nothing to report.
Normal hepatocytes do not express endogenous uncoupling protein 2 (UCP2) in adult liver, although Kupffer cells do, and it is strikingly induced in hepatocytes in steatotic liver and obese conditions. However, the direct link of UCP2 with the pathogenic development of liver diseases and liver injury remains elusive. Here we report that targeted expression of UCP2 to mouse liver increases susceptibility to acute liver injury induced by lipopolysaccharide (LPS) and galactosamine (GalN). UCP2 appears to enhance proton leak, leading to mild uncoupling in a guanosine diphosphate-repressible manner. Indeed, mitochondria from the genetically manipulated mouse liver have increased state 4 respiration, lower respiratory control ratio, and reduced adenosine triphosphate (ATP) levels, which altered mitochondrial physiology. To address the underlying mechanism of how UCP2 and the reduced energy coupling efficiency enhance cell death in mouse liver, we show that the reduced ATP levels lead to activation of 5′AMP-activated protein kinase (AMPK) and its downstream effector, c-Jun N-terminal kinase; thus, the increased sensitivity toward LPS/GalN-induces apoptosis. Importantly, we show that inhibition of UCP2 activity by its pharmacological inhibitor genipin prevents LPS/GalN-induced ATP reduction, AMPK activation, and apoptosis. Also, inhibition of ATP production by oligomycin promotes LPS/GalN-induced cell death both in vivo and in vitro. Conclusion: Our results clearly show that targeted expression of UCP2 in liver may result in compromised mitochondrial physiology that contributes to enhanced cell death and suggests a potential role of UCP2 in the development of liver diseases. (HEPATOLOGY 2009.)
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Mitochondrial dysfunction may play a critical role in the development of liver diseases stemming from metabolic disorders, fatty liver, nonalcoholic steatohepatitis, cryptogenic cirrhosis, and eventually hepatocellular carcinoma.1, 2 Under these pathological conditions, one key functional change in mitochondria is the inability to maintain sufficient levels of adenosine triphosphate (ATP). Uncoupling protein 2 (UCP2), a member of the anion carrier superfamily of mitochondrial inner membrane,3 negatively impacts cellular energy production when the availability of oxidizable substrates becomes limited, e.g., under conditions of acute stress like ischemia, partial hepatectomy, and lipopolysaccharide (LPS) exposure. This protein, like its homolog UCP1, which is mainly expressed in brown fat tissue, was observed to be able to increase the guanosine diphosphate (GDP)-sensitive proton conductance across the mitochondrial inner membrane.4, 5 The activation of UCP2 was observed to enhance the extent of mild uncoupling of the electrochemical gradient of mitochondria and reduce cellular ATP levels.6 UCP2 was also found to be significantly up-regulated in many liver disease conditions and linked to obesity and type 2 diabetes by affecting energy expenditure and beta cell functions.3, 6 Therefore, UCP2 may represent an important drug target of diseases for metabolic disorders and oxidative stresses. Additionally, inhibition of UCP2 activity by its pharmacological inhibitor genipin reverses the UCP2-mediated loss of glucose-sensing in beta cells.7
UCP2 was thought to be a negative regulator of reactive oxidative species (ROS) production and a physiological brake for ROS signaling owing to its possible role in reducing the protonic membrane potential of mitochondria.8 UCP2 attenuates oxidative stress and has a protective role in mouse cardiomyocytes,9 neuronal cells,10, 11 pancreatic alpha cells,12 and macrophages or monocytes13 in a transgenic mouse model. Overexpression of UCP2 produced a reduced inflammatory response following LPS treatment.14 Moreover, targeted expression of human UCP2 (hUCP2) in fly increases its life span, which is attributable to reduced ROS production.15 In contrast, UCP2 deficiency promotes oxidative stress and impairs liver regeneration in mice.16 Macrophages from UCP2 knockout (KO) mice have increased ROS production, persistent nuclear factor-kappa B (NF-κB) activation,17 and augmented LPS-induced mitogen-activated protein kinase (MAPK) activation.18 These mice have a higher incidence of colon tumor,19 likely due to the enhanced inflammatory response.20 However, there is also experimental evidence showing that there is no difference of proton leak in spleen or lung mitochondria from wildtype (Wt) mice and UCP2 KO mice.21 UCP2 KO mice do not show signs of obesity or cold sensitivity, as manifests in UCP1 KO mice.13 The physiological role of UCP2 in regulating mitochondrial ROS and related oxidative stress remains to be clarified. UCP2 may have paradoxical roles for regulating cell death. It reduces mitochondrial ROS production, which may protect against cell death.9–11 On the contrary, it may lead to ATP depletion or even energy crisis that may promote cell death.22
UCP2 protein is not expressed in hepatocytes in adult healthy liver but is restricted in Kupffer cell, although UCP2 messenger RNA (mRNA) is detected in hepatocytes.23 An interesting note is that UCP2 expression becomes significantly abundant in hepatocytes of fatty liver, steatotic liver, and chronic liver disease conditions.24–26 Under these conditions, hepatocytes are prone to ischemia/reperfusion-induced liver injury,27 implying that UCP2 has pathogenic roles in the development of liver diseases. It was found that UCP2 expression induced by obesity in hepatocytes promotes liver ATP depletion.25, 28 However, it remains to be addressed whether high expression of UCP2 and subsequent ATP depletion are causally linked with the higher sensitivity of liver injury. Here we took an approach to target expression of UCP2 in mouse liver and addressed the functional roles of UCP2 in acute liver injury. Our data show that enforced expression of UCP2 in vivo led to enhanced mitochondrial state 4 respiration and decreased respiratory control ratio (RCR, states 3/4), resulting in reduced ATP production by decreasing energy coupling efficiency of oxidative phosphorylation. Furthermore, our results show that altered mitochondrial physiology may contribute to the increased susceptibility toward LPS-induced cell death in the liver.
Transgenic mice with targeted expression of UCP2 in the liver were developed using the well-established albumin promoter-enhancer driven vector (see the detailed description in the supporting information). Transgenic mice harboring the Alb promoter/hUCP2 complementary DNA (cDNA) were identified by polymerase chain reaction (PCR) analysis using specific primers (see Supporting Table 1) with genomic DNA prepared from tail biopsies. The transgenic lines were maintained by backcrossing to the C57BL/6 Wt and 6 to 8-week-old mice were used in all the experiments with age- and sex-matched Wt mice as controls.
Tissue RNA Extraction and Reverse-Transcription PCR (RT-PCR).
Total RNA was purified from snap-frozen tissues with Trizol reagent (Invitrogen). First-strand cDNA was synthesized from 1 μg total RNA using Oligo dT (Promega) and reverse transcript II (Invitrogen). The resulting complementary DNAs (cDNAs) were amplified with specific primers (see Supporting Table 1).
Isolation of Liver Mitochondria and Measurement of Mitochondrial Function.
Mitochondria were isolated from mouse liver as described29 and protein content was determined by the microbiuret method. Isolated mitochondria were used for measuring mitochondrial oxygen consumption with a Clark oxygen electrode and estimating mitochondrial membrane potential (ΔΨm) using Rodamine 123 (R123, Molecular Probes) as before.30
Mice were maintained in specific pathogen-free (SPF) conditions and all studies were approved by the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences. For acute liver injury, mice were given an intraperitoneal injection of LPS (25 μg/kg, Echerichia coli 0111:B4, Sigma) alone or together with galactosamine (800 mg/kg, Calbiochem). Control animals were injected with saline. Other treatments of mice are described in the supporting information. After treatment, mice were sacrificed at the indicated times. Blood was obtained through the tail vein or cardiac puncture. Liver tissues were excised and snap-frozen in liquid nitrogen or processed for histological studies.
Histological, Immunohistochemistry (IHC) Analysis and In Situ Apoptosis Detection.
Liver specimens were fixed overnight in 10% formalin solution then dehydrated, embedded in paraffin, and cut into 4-μm sections. Sections were stained with hematoxylin and eosin (H&E). Expression of UCP2 in liver sections was detected as described.16 The in situ labeling of apoptotic cells was performed using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay with commercial kits (Roche).
Tissue Lac Z Staining.
Lac Z staining were performed as described.31 Stained tissues were mounted in paraffin and nuclear-counterstained with carmine.
Subcellular Fraction for Immunoblotting.
Mitochondrial and cytosolic protein fractions were obtained as described.32
SDS-PAGE and Western Blotting.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as described.33, 34 Immune complexes were detected with horseradish peroxidase (HRP)-conjugated second antibody and were visualized by enhanced chemiluminescence (ECL; Pierce). The sources of primary antibodies are provided in the supporting information.
Commercial kits were used to measure serum ALT and aspartate aminotransferase (AST) levels, to evaluate hepatic ATP contents, and to detect caspase-3 activation according to the manufacturer's instructions. The sources of the kits are provided in the supporting information.
Data are expressed as the means ± standard error of the mean (SEM). Student's t test was used for comparisons between two groups and differences at P < 0.05 were considered significant. *P < 0.05 and #P < 0.01 versus the corresponding are indicated.
Generation and Characterization of Targeted Expression hUCP in Mouse Liver.
In order to understand the physiological functions of UCP2 and its role in the development of liver disease, we generated transgenic mice carrying hUCP2 cDNA under the control of mice albumin promoter. For convenience of identification, we introduced a Lac Z reporter gene, which was together controlled by the albumin promoter. Founder mice were generated by injection of the construct into one-cell embryos and were identified by PCR analysis (Fig. 1A). Expression and transcript of the transgene in liver was first examined by RT-PCR. The sequences of primers for RT-PCR are located in exon 3 and exon 5 of hGH polyA and there are two introns between exon 3 and exon 5. If the transgene can be expressed and spliced in liver, a short fragment (548 base pairs [bp]) should be detected by using these primers for RT-PCR analysis. Otherwise, only a long fragment (898 bp) can be detectable (Fig. 1B). The protein expression of UCP2 in mouse liver was confirmed by immunoblot (Fig. 1C) and by in situ IHC staining using UCP2 antibodies or Lac Z staining (Fig. 1D). The specific expression of hUCP2 in liver but not in other tissues was further confirmed by RT-PCR analysis in the UCP2 transgenic (Tg) mouse. Spliced mRNA of transgene can only be detected in liver but not in the other tissues, although transgene can be detected in contaminated genomic DNA (no DNase digestion for extracted RNA) of all the tissues (Fig. 1E). Lac Z staining also showed no transgene was expressed in other tissues (Fig. 1F). Finally, three transgenic mice lines (G171, J492, and K459) were identified and two of them (G171 and J492) that produce an equivalent number of offspring like Wt mice were used for subsequent studies.
Altered Mitochondrial Parameters and Lower ATP Levels in UCP2 Transgenic Mice.
The Tg animals were phenotypically similar to their normal littermates and did not exhibit detectable histological changes in the liver. We first compared the respiration rate of the isolated mitochondria from Tg mice and its Wt littermates. UCP2 expression increased state 4 respiration (Fig. 2A, B) and decreased RCR (states 3/4) in Tg mice in comparison with that in Wt littermates (Fig. 2C). Importantly, this decrease of RCR was inhibited by GDP (1 mM) in Tg mice, but not in Wt mice (Fig. 2C). Furthermore, in the presence of oleic acid (200 μM), which is an activating substance of UCP2,35 mitochondrial state 4 respiration was significantly activated in Tg mice, but not in Wt mice (Fig. 2D,E). The presence of palmitic acid showed similar results (Supporting Fig. 1A,B). Also, there was a decrease of mitochondrial membrane potential (ΔΨm, 10≈20 mV reduction) (Fig. 2F,G). These data suggest that enforced expression of UCP2 may function in vivo to enhance the extent of proton leak leading to mild uncoupling and decreased energy coupling efficiency as indicated by decreased RCR. We next measured the ATP level of liver and found a significant decrease of ATP levels in Tg mice compared to Wt littermates (Fig. 2H). However, there was no essential difference between these two mice in mitochondrial ROS production (Supporting Fig. 2A,B). Taken together, enforced UCP2 expression in liver mitochondria have shown some physiological consequences in enhancing proton leak (expressed as an increase in state 4 respiration) and mild uncoupling, resulting in a decrease of energy coupling efficiency of oxidative phosphorylation (expressed as a decrease in RCR and ATP production); however, these changes in parameters of mitochondrial bioenergetics stemming from enforced expression of UCP2 in mitochondria may not be related to the production of ROS per se, or the interaction between ROS and UCP2 protein.8
UCP2 Promotes Hepatocyte Apoptosis in LPS/GalN-Induced Acute Liver Injury.
Having clearly demonstrated that targeted expression of UCP2 functions properly in vivo, we next addressed the question whether the forced expression of UCP2 could affect the cell death response in the liver. To our surprise, we found that liver damage was more severe in Tg mice than that of Wt mice from the same parenting after LPS/GalN treatment as revealed by serum transaminase changes (Fig. 3A,B) and liver H&E staining (Fig. 3C). TUNEL staining further revealed that the pronounced apoptosis in Tg mice liver was more than that in Wt mice liver after LPS/GalN treatment. LPS or GalN alone had no effect on apoptosis in hepatocytes (Fig. 3D). These results suggest that UCP2 has no protective effect on cell death, rather promoting cell death in liver.
Bcl-2 Family Proteins Changes, Cytochrome c Release, and Caspase-3 Activation in UCP2-Related Hepatocyte Apoptosis.
The above results raise an interesting question: How does the forced expression of UCP2 increase the susceptibility of hepatocytes? We first asked if Bcl-2 family proteins play a role in UCP2-related, LPS/GalN-induced apoptosis. We then checked the expression of both pro- and antiapoptotic Bcl-2 family proteins in mice liver following LPS/GalN treatment. We found that Bax protein expression was increased in Tg mice (Fig. 4A) and more Bax was translocated onto the mitochondria (Fig. 4B). There was also a greater reduction of Bcl-xL in Tg mice than in Wt mice. Also, Bid cleavage was more evident in Tg mice than in Wt mice. However, Mn-SOD, an important antioxidative protein in mitochondrial matrix, had no change (Fig. 4A). In addition, more cytochrome c was released from mitochondria in Tg mice liver after LPS/GalN treatment (Fig. 4C). As a result, casapse-3 activity was higher in Tg mice liver than in Wt mice one (Fig. 4D). These data clearly showed that UCP2 expression contributes to LPS/GalN-induced apoptosis of hepatocytes by shifting the balance toward a proapoptotic response.
Genipin Could Decrease Apoptosis of Hepatocytes in Acute Liver Injury Induced by LPS/GalN.
To further ascertain that UCP2 is indeed involved in regulating mitochondrial-dependent apoptosis, we tested whether genipin, a pharmacological inhibitor of UCP2,7 could block cell death of hepatocytes. Indeed, pretreatment of mice with genipin in vivo could potently protect mice from hepatocyte apoptosis induced by LPS/GalN, whereas genipin alone had no effect on liver injury. TUNEL staining showed fewer apoptotic hepatocytes in mice pretreated with genipin (Fig. 5A). We further checked the expression of Bcl-2 family proteins by Western blotting and found that the reduction of Bcl-xL and proapoptotic Bid were attenuated in mice pretreated with genipin (Fig. 5B). It was shown that genipin may protect LPS/GalN-induced liver injury by decreasing the tumor necrosis factor alpha (TNF-α) production of macrophage.36 To exclude the effect of genipin on TNF-α, we directly treated the mice with TNF-α/ActD to check whether genipin pretreatment can still protect hepatocytes from apoptosis in Tg mice. The results showed that even in TNF-α/ActD-induced liver injury, genipin pretreatment has greater protective effects on hepatic apoptosis in Tg mice but not in Wt mice (Fig. 5C). Taken together, these data further substantiate our conclusion that UCP2 expression in hepatocytes could have a proapoptotic role in acute liver injury and specific inhibition of UCP2 function could alleviate the proapoptotic responses.
Activation of 5′AMP-Activated Protein Kinase in Liver May Contribute to the Sensitivity to LPS/GalN-Induced Hepatocyte Apoptosis in Tg Mice.
To understand the mechanism of how UCP2 promotes liver injury, we asked whether the reduced levels of ATP contributed to the increased sensitivity to liver injury because a decrease of intracellular ATP levels was found to activate cellular signaling pathways and to promote cell death.22 We measured the ATP levels in the liver after LPS/GalN treatment and found a greater reduction of ATP levels in Tg mice livers (Fig. 6A). To determine if the reduced ATP indeed contributes to downstream events, we treated Wt mice with oligomycin and found that treated mice have decreased ATP levels in the liver (Fig. 6B). Concomitantly, these mice showed an enhanced apoptosis in hepatocytes as Tg mice after LPS/GalN administration (Fig. 6C).
The 5′AMP-activated protein kinase (AMPK) acts as an intracellular energy sensor and it is allosterically activated by AMP in response to cellular ATP depletion.37 We thus asked whether AMPK was activated in liver of Tg mice. Interestingly, the more phosphorylated form of AMPK was detectable in Tg mice than in Wt littermates under normal conditions (Fig. 6D,E). Following LPS/GalN treatment, AMPK was chronically activated in Tg mice liver than that in Wt mice. Genipin pretreatment in vivo could partially inhibit AMPK activation induced by LPS/GalN treatment (Fig. 6D). Also, further activation of AMPK was detected in Wt mice after treatment with oligomycin (Fig. 6E). Collectively, these data demonstrated that UCP2 expression in hepatocytes could alter mitochondrial parameters leading to activation of AMPK. To know whether direct activation of AMPK is indeed involved in hepatic apoptosis, we treated Wt and Tg mice with 5-amino-4-imidazolecarboxamine riboside (AICAR), an activator of AMPK. The results showed that AICAR could directly activate AMPK and activated AMPK was greater in Tg mice than in Wt mice (Supporting Fig. 3A). Moreover, AMPK activation can subsequently activate c-Jun N-terminal kinase (JNK) even in Wt mice (Supporting Fig. 3B). As a result, Wt mice pretreated with AICAR become sensitive to LPS/GalN-induced liver injury (Supporting Fig. 3C,D). These results indicate direct AMPK activation really contributes to the susceptibility to liver injury induced by LPS/GalN.
Activation of JNK in Tg Mice After LPS/GalN Treatment.
One of the downstream effectors of activated AMPK is JNK, which is implicated as a major mechanism of LPS-induced hepatic apoptosis. JNK activation could promote the apoptotic response, including Bax targeting to mitochondria.38 We thus checked the JNK activation in mice liver after LPS/GalN treatment. The results showed that the phosphorylated forms of JNK were more pronounced in Tg mice liver than in Wt mice (Fig. 7A), and genipin could potently inhibit this JNK activation (Fig. 7B). To know whether JNK activation induced by LPS/GalN is in hepatocytes, we checked the JNK phosphorylation by IHC using a specific antibody and found that activated JNK can be readily detected in apoptotic hepatocytes (Supporting Fig. 4). To know whether the ATP depletion contributed to JNK activation, we compared JNK activation in liver of oligomycin-treated and untreated Wt mice after LPS/GalN treatment. We found that JNK activation was more pronounced in liver of oligomycin-treated mice, which have lower ATP levels (Fig. 7C). To further confirm the role of JNK activation in LPS/GalN-induced hepatic apoptosis, SP600125, a selective JNK inhibitor, was used in vivo to inhibit the JNK activation. Western blots showed that JNK activation was partially inhibited after SP600125 treatment (Fig. 7D) and the number of apoptotic hepatocytes decreased in LPS/GalN-induced liver injury (Fig. 7E). However, SP600125 had a limited effect on AMPK activation (Fig. 7F).
UCP2 is highly induced in hepatocytes of fatty liver, steatotic liver, obesity, and other metabolic diseases conditions.24–26 Hepatocytes under these pathological conditions have higher susceptibility to liver injury. Indeed, in 1999 Chavin et al.25 showed that UCP2 mRNA have a marked induction in ob/ob mice liver and was further induced by LPS administration in these mice. As a result, there was a 10-fold higher serum ALT activity in LPS-treated obese mice than in LPS-treated lean mice. In this study, we first demonstrated that the expression of UCP2 has normal physiological functions to enhance mild mitochondrial uncoupling leading to the reduced ATP levels in hepatocytes in vivo. We showed that these changes of mitochondrial parameters by UCP2 contribute to the pronounced cell death of hepatocytes in mouse liver. Previous studies suggested that UCP2 promotes mitochondrial proton leak and increases susceptibility of liver to ischemia/reperfusion injury.27, 39 However, the underlying mechanisms of how UCP2 sensitizes hepatocytes to death are not fully understood. Our data for the first time suggest the possible mechanism of how the compromised energy metabolism signals the core apoptotic machinery (Fig. 8). We show there is increased Bax targeting to mitochondria and more JNK activation (Fig. 4A,B) in liver of UCP2 Tg mice after liver injury. This is consistent with previous reports suggesting that activated JNK could phosphorylate Bax, which promotes Bax targeting to mitochondria to induce apoptosis.38 We also showed that expression of UCP2 leads to AMPK activation (Fig. 6D,E) and sustained activation of AMPK could induce JNK activation (Fig. 7).40 Moreover, a decrease in ATP levels by the ATPase inhibitor oligomycin can promote LPS/GalN-induced cell death. In general, normally living cells maintain ATP levels around 3 mM, and a decrease of ATP levels could be the major stimulus for apoptosis in hypoxia/reperfusion. The apoptosis related to ATP depletion could be inhibited by JNK inhibition.22, 41 A better understanding of the mechanism of UCP2 in cell death holds promise for fighting liver diseases and metabolic disorders.
An early study by Negre-Salvayre et al.42 suggested that UCP2 is a negative regulator of ROS production due to the reduction of mitochondrial membrane potential. This suggestion was further extended by Brand and colleagues,8 who proposed that the activity of UCP was regulated by ROS. Despite great interest in the potential role of UCP2 as a protectant against oxidative damage, convincing in vivo data are still not available and there are also results indicating the detrimental effect of UCPs under oxidative stress.43 Experiments from KO mice are puzzling. Although there is increased ROS production in macrophages in UCP2 KO mice, there is no difference of proton leak and ROS production of splenic mitochondria.21 A previous study showed that UCP2 deficiency reduced Fas-mediated liver cell death in ob/ob mice.34 It is likely that the roles of UCP2 in ROS generation and cell death are tissue-specific. This is understandable because the impact of UCP2 on the redox status and signaling networking are likely to be cell type-specific, thus responses to oxidative stress. Our results do not support that upregulation of UCP2 protein leads to reduced ROS production in mitochondria. Alternatively, we suggest the possibility that reduced energy coupling efficiency for oxidative phosphorylation and compromised mitochondrial bioenergetics due to enforced UCP2 expression contribute to sensitivity to liver injury.
The physiological function of UCP2 is a subject of intense debate. Here we clearly show that targeted expression of UCP2 manifested all the characteristics of UCP functions for increased state 4 respiration and mild uncoupling in a GDP-repressible manner. It is argued that the uncoupling effect of UCP may be due to a gain of function of artifacts under overexpression.44 We have different lines of founder mice that express different levels of UCP2. Two lines of mice with significant differences in UCP2 show similar phenotypes. Both have similar mild uncoupling effects and similar levels of ATP and responses to LPS/GalN-induced liver injury. This suggests that UCP2 functions despite the UCP2 protein levels difference, at least in liver where hepatocytes lack endogenous UCP2. Further studies are needed to directly address the question of the roles of UCP2 in the development of liver diseases under other physiological and pathological conditions.
We thank Ali Azad, Eric Collard, and Professor Zhigang Tian for critical reading and comments on the article. We thank Xiaochen Pan and Lianjun Zhang for technical assistance.