Hepatocyte glutathione peroxidase-1 deficiency improves hepatic glucose metabolism and decreases steatohepatitis in mice
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In obesity oxidative stress is thought to contribute to the development of insulin resistance, non-alcoholic fatty liver disease and the progression to non-alcoholic steatohepatitis. Our aim was to examine the precise contributions of hepatocyte-derived H2O2 to liver pathophysiology.
Glutathione peroxidase (GPX) 1 is an antioxidant enzyme that is abundant in the liver and converts H2O2 to water. We generated Gpx1 lox/lox mice to conditionally delete Gpx1 in hepatocytes (Alb-Cre;Gpx1 lox/lox) and characterised mice fed chow, high-fat or choline-deficient amino-acid-defined (CDAA) diets.
Chow-fed Alb-Cre;Gpx1 lox/lox mice did not exhibit any alterations in body composition or energy expenditure, but had improved insulin sensitivity and reduced fasting blood glucose. This was accompanied by decreased gluconeogenic and increased glycolytic gene expression as well as increased hepatic glycogen. Hepatic insulin receptor Y1163/Y1163 phosphorylation and Akt Ser-473 phosphorylation were increased in fasted chow-fed Alb-Cre;Gpx1 lox/lox mice, associated with increased H2O2 production and insulin signalling in isolated hepatocytes. The enhanced insulin signalling was accompanied by the increased oxidation of hepatic protein tyrosine phosphatases previously implicated in the attenuation of insulin signalling. High-fat-fed Alb-Cre;Gpx1 lox/lox mice did not exhibit alterations in weight gain or hepatosteatosis, but exhibited decreased hepatic inflammation, decreased gluconeogenic gene expression and increased insulin signalling in the liver. Alb-Cre;Gpx1 lox/lox mice fed a CDAA diet that promotes non-alcoholic steatohepatitis exhibited decreased hepatic lymphocytic infiltrates, inflammation and liver fibrosis.
Increased hepatocyte-derived H2O2 enhances hepatic insulin signalling, improves glucose control and protects mice from the development of non-alcoholic steatohepatitis.
KeywordsFibrosis Glutathione peroxidase-1 Hepatocyte Inflammation Insulin resistance Non-alcoholic fatty liver disease Non-alcoholic steatohepatitis Protein tyrosine phosphatase Reactive oxygen species Type 2 diabetes
Forkhead box protein O1
Hepatic glucose production
Non-alcoholic fatty liver disease
Protein tyrosine phosphatase
Reactive oxygen species
Small heterodimer partner
Tyrosine-protein phosphatase non-receptor type 2
Obesity is a major risk factor for the development of insulin resistance, a key factor in the aetiology of the metabolic syndrome and type 2 diabetes [1, 2, 3]. Obesity and insulin resistance promote the development of non-alcoholic fatty liver disease (NAFLD), which in a subset of individuals can progress to non-alcoholic steatohepatitis (NASH) . NASH is characterised by fatty liver and overt inflammation that can lead to the death of steatotic hepatocytes, instigating reparative responses that result in fibrosis . Oxidative stress accompanying the obese state is considered a key factor in the development of insulin resistance [5, 6, 7] and an important contributor to the development of NAFLD and NASH [4, 8, 9, 10]. Systemic and hepatic oxidative stress is evident in obesity and there is direct evidence for the involvement of reactive oxygen species (ROS) in the promotion of insulin resistance, NAFLD and NASH in obesity/type 2 diabetes in rodents [5, 6, 7, 8, 9, 10, 11, 12, 13].
Mitochondria are thought to be the primary contributors to oxidative stress in obesity. Superoxide (O2•–) is a natural byproduct of the single-electron transport chain [14, 15]. In obesity, the chronic uptake and oxidation of energy substrates is thought to generate reducing equivalents that exceed the rate of ATP utilisation, thus enhancing the generation of O2•– [14, 15]. In addition, anaplerotic/cataplerotic pathways in hepatocytes in NAFLD induce mitochondrial O2•– generation and inflammation , whereas in NASH, increased NADPH oxidase (NOX)-4 expression may also contribute to hepatic oxidative stress . O2•– is converted to H2O2 by superoxide dismutase (SOD) and thereafter eliminated by antioxidant enzymes such as catalase, peroxiredoxins (PRDXs) and glutathione peroxidase (GPX) [14, 15]. Oxidative stress ensues when the production of O2•–/H2O2 exceeds the antioxidant capacity of a cell.
Transient, localised H2O2 generation can occur in response to physiological stimuli such as growth factors and hormones . Both NOX and mitochondria have been implicated in the generation of O2•– and H2O2 in response to physiological stimuli [14, 16, 17]. An increase in H2O2 in response to stimuli such as insulin can facilitate signalling by oxidising and inactivating protein tyrosine phosphatases (PTPs) . Several PTPs, including the tyrosine-specific PTP1B and the dual-specificity phosphatase and tensin homologue, can be oxidised in response to insulin to promote insulin receptor (IR) activation and downstream phosphatidylinositol 3-kinase (PI3K) signalling, respectively [16, 19, 20, 21, 22].
GPX1 is a ubiquitous selenoenzyme that uses glutathione to catalyse the conversion of H2O2 into H2O . Gpx1 –/– mice are healthy and fertile and do not show any obvious abnormalities [23, 24]. Indeed, when Gpx1 –/– mice are fed a high-fat diet that promotes moderate adiposity and insulin resistance, but not hyperglycaemia , GPX1 deficiency promotes insulin signalling in muscle and protects mice from the development of insulin resistance , indicating that increases in H2O2 in muscle may be beneficial. Moreover, even in the context of morbid obesity and hyperglycaemia, global GPX1 deficiency protects mice from the development of steatohepatitis and liver damage and improves glucose metabolism . Given the extensive tissue crosstalk in the control of glucose and lipid homeostasis and the capacity of H2O2 to diffuse across membranes, it is difficult to definitively ascribe the effects of global GPX1 deficiency and heightened H2O2 on hepatic pathophysiology to any cell type or tissue and to exclude potential detrimental effects being masked by the global deletion of GPX1. Therefore we have ‘floxed’ the Gpx1 allele to allow us to explore the hepatocyte-specific contributions of GPX1 to the regulation of hepatic insulin sensitivity and the development of NAFLD and NASH.
Gpx1 lox/+ mice on a C57BL/6 J background were generated by the Monash Gene Targeting Facility as described in electronic supplementary material (ESM) Methods. Alb-Cre (C57BL/6 J) mice have been described previously . Mice were maintained on a 12 h light–dark cycle in a temperature-controlled high-barrier facility with free access to food (6% wt/wt fat) and water. Where indicated, mice were fed an obesogenic diet (23.5% wt/wt fat, SF04-027; Specialty Feeds, Glen Forest, WA, Australia) or a choline-deficient amino-acid-defined (CDAA) diet (SF13-103; Specialty Feeds). Experiments were conducted on age-matched male mice and experimentors were blind to outcome assessment. Experiments were approved by the Monash University Animal Ethics Committee.
Blood was collected for analysis of fed and fasted blood glucose and plasma insulin as described previously . Insulin and pyruvate tolerance tests were performed in mice fasted for 4–6 h and hyperinsulinaemic–euglycaemic clamps were performed as described previously . Other metabolic analyses and NASH scoring are described in ESM Methods.
Tissue homogenates were analysed as described previously . Immunoblotting antibodies are described in ESM Methods. Hepatic glycogen was extracted and debranched with amylo-α-l,4-α-1,6-glucosidase and glucose units were analysed using an enzymatic fluorometric method described previously .
For the analysis of PTP oxidation, frozen liver samples were homogenised under anaerobic conditions in the presence of N-ethylmaleimide to alkylate all reduced PTPs and then subsequently reduced and hyperoxidised to the sulfonic state as described previously . Immunoblots were probed with a mouse monoclonal antibody (PTPox) raised against the signature motif of the prototypic PTP1B oxidised to the irreversible sulfonic state, which detects tyrosine-specific PTPs oxidised to the sulfonic state .
Total and oxidised glutathione levels in tissue supernatant fractions or blood were measured as described previously .
Hepatocytes were isolated using a two-step collagenase A perfusion method [13, 28]. Hepatocytes were serum-starved in low-glucose DMEM (ThermoFisher, Waltham, MA, USA) containing 0.1% (vol./vol.) FBS for 2 h and incubated in PBS containing 100 μmol/l Amplex Red reagent and 1 U/ml HRP (Amplex Red hydrogen peroxide assay kit; ThermoFisher) in the presence or absence of 100 nmol/l insulin and fluorescence measured and normalised to protein.
Hepatic levels of Gpx1, Gpx3, Gpx4, Ho1 (also known as Hmox1), Txn1, Trxrd1 (Txnrd1), G6pc, Pck1, Gck, Pdk4, Lpk (Col2a1), Ppargc1α (Ppargc1a), Cpt1, Acadl, Srebp1c (Srebf1), Fasn, Saa1, Crp, Il1α (Il1a), Ifnγ (Ifng), Il6, Tnf, Mcp-1 (Ccl2), αSma (Acta2) and Tgfβ (Tgfb1) were assessed by quantitative real-time PCR (ΔΔCt) performed using the TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (ThermoFisher Scientific) as described previously .
Analyses were performed using the two-tailed Student’s t test or ANOVA. A p value of < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).
Hepatocyte-specific GPX1-deficient mice
H2O2 generation in HGKO mice
At 4–10 weeks of age no differences were evident in body weight or body/tissue composition between lox/lox and HGKO mice (ESM Fig. 1a–c). Food intake, oxygen consumption, ambulatory activity and energy expenditure were unaltered, as were respiratory exchange ratios, consistent with unaltered energy homeostasis and fuel utilisation (ESM Fig. 1d). To determine whether hepatocyte GPX1 deficiency results in oxidative stress we monitored alterations in GSH and GSSG in blood or liver homogenates from chow-fed lox/lox and HGKO mice. No alterations were evident in blood or liver as a consequence of GPX1 deficiency (Fig. 2a,b). Similarly, no changes in GSH or GSSG were evident in epididymal fat (Fig. 2c). Interestingly total GSH and GSSG were elevated in muscle of HGKO mice, but the GSH:GSSG ratio was unaltered (Fig. 2d), consistent with the increase in GSH being compensatory to counter any muscle oxidative stress that may otherwise occur as a result of decreased muscle catalase expression (Fig. 1e). In keeping with this, muscle H2O2 levels were not altered (Fig. 2e). To directly assess whether hepatic GPX1 deficiency results in increased H2O2, we isolated hepatocytes from chow-fed lox/lox and HGKO mice; we found that GPX1 deficiency resulted in increased hepatocyte H2O2 production (Fig. 2f). Therefore, GPX1 deficiency increases H2O2 in hepatocytes but the levels do not exceed the antioxidant capacity of the liver and do not promote overt hepatic or systemic oxidative stress in chow-fed mice.
Glucose homeostasis in HGKO mice
We next examined glucose homeostasis in chow-fed C57BL/6 (+/+), lox/lox and HGKO mice. Although blood glucose levels of fed mice and mice fasted for 6 h remained the same, 12 h fasted blood glucose levels were significantly reduced in HGKO mice compared with +/+ or lox/lox mice (Fig. 3e). The reduced blood glucose levels in mice fasted for 12 h is consistent with HGP being reduced in HGKO mice. No differences were observed in plasma insulin levels of fed mice (Fig. 3e), consistent with unaltered pancreatic insulin secretion, but 12 h fasted plasma insulin levels were reduced, consistent with improved insulin sensitivity; no differences were observed between +/+ and lox/lox mice. To assess the effect on HGP we performed pyruvate tolerance tests; pyruvate increases blood glucose by promoting gluconeogenesis. Administration of pyruvate increased blood glucose in +/+ and lox/lox mice, but the increase was attenuated in HGKO mice (Fig. 3f; ESM Fig. 2a). Next we assessed insulin sensitivity in insulin tolerance tests. We found that insulin responses were moderately improved in HGKO mice (Fig. 3f); no differences were observed between +/+ and lox/lox mice (ESM Fig. 2b). These results point towards hepatocyte-specific GPX1 deficiency repressing HGP and enhancing insulin sensitivity in chow-fed mice.
To further characterise glucose homeostasis we subjected chow-fed lox/lox and HGKO mice to hyperinuslinaemic–euglycaemic clamps (Fig. 3g). We found that the glucose infusion rate necessary to maintain euglycaemia was significantly increased in HGKO mice (Fig. 3g), consistent with enhanced whole-body insulin sensitivity. Moreover glucose disappearance, which reflects hepatic and peripheral glucose metabolism, tended to be higher in HGKO mice than in lox/lox mice (p = 0.06) (Fig. 3g). Finally, the extent to which endogenous glucose production was suppressed by insulin tended to be higher in the HGKO mice (Fig. 3g). These results indicate that hepatic GPX1 deficiency enhances insulin sensitivity by potentiating the suppression of gluconeogenic enzymes and by promoting hepatic glucose storage.
Insulin signalling in HGKO mice
PTP oxidation in HGKO mice
Oxidative stress in obese HGKO mice
Glucose homeostasis in obese HGKO mice
Reduced inflammation and NASH in HGKO mice
Global GPX1-deficient mice fed an obesogenic diet are protected from the development of steatohepatitis and ensuing liver damage . Although this was previously attributed to decreased pancreatic insulin secretion and attenuated insulin-mediated hepatic lipogenesis , a hepatocyte-intrinsic contribution could not be excluded. Here, we confirm that hepatocyte GPX1 deficiency does not alter the development of steatohepatitis in mice fed either an obesogenic diet that promotes NAFLD or a CDAA diet that promotes NASH. Strikingly, despite the unaltered steatosis, hepatic GPX1 deficiency was associated with decreased hepatic and systemic inflammation and reduced liver lymphocytic infiltration and fibrosis in mice fed a CDAA diet. Although the molecular mechanism by which hepatocyte GPX1 deficiency may decrease inflammation and ensuing NASH remains to be resolved, one possibility is that this may be linked to increased PTP1B oxidation, since hepatic PTP1B deficiency represses hepatocyte endoplasmic reticulum stress and inflammation [33, 34].
Altered oxidative phosphorylation and increased ROS levels are reported in patients with NASH [4, 11] and there is compelling evidence from rodent models linking oxidative stress with NAFLD or NASH [10, 35, 36]. Hepatocyte-specific SOD1-deficiency or combined SOD1/2 deficiencies increase steatohepatitis in HFF mice [37, 38]. This is consistent with superoxide being important in the promotion of steatohepatitis and liver disease. In keeping with this, NOXs are elevated in models of fibrogenic disease , whereas NOX1/4 inhibition or hepatocyte NOX4 deficiency protect mice against NASH [8, 40]. In contrast, a recent study has shown that the deletion of the haem oxygenase-1 gene in hepatocytes promotes the generation of H2O2 and attenuates high-fat diet-induced insulin resistance and hepatic inflammation and damage . Our findings also challenge the concept that hepatocyte-derived H2O2 per se is a driver of liver disease.
We previously reported that global GPX1 deficiency promotes hepatic insulin signalling and repression of HGP in the obese state . However, it was unclear whether these effects were intrinsic to hepatocytes or reflected reduced pancreatic insulin secretion , since hyperinsulinaemia can drive insulin resistance [42, 43, 44] and reduction in circulating insulin can protect mice from diet-induced obesity, insulin resistance and steatosis . In this study we found that insulin-induced H2O2 generation and insulin signalling were increased in hepatocytes from HGKO mice, accompanied by increased hepatic IR activation and signalling even in the context of obesity. Why does GPX1 deficiency in HFF mice not promote insulin resistance? One argument could be that a sufficient level of H2O2 was not achieved in hepatocytes. However, H2O2 generation by hepatocytes isolated from HFF HGKO mice far exceeded levels normally achieved in response to insulin. We propose that either H2O2 per se is not detrimental or that hepatic oxidative stress and the promotion of insulin resistance may also be reliant on contributions from non-parenchymal cells. In keeping with the latter premise, mice overexpressing PRDX4, a secreted enzyme that scavenges ROS, are protected from steatohepatitis .
Reactive and potentially modulatory cysteines exist in many proteins [14, 15]. However, the low thiol pKa of the catalytic cysteine in PTPs renders them highly susceptible to oxidation and inactivation . In this study we report that the oxidation of select hepatic PTPs, including PTP1B, SHP-1 and TCPTP, is increased in HGKO mice. Previous studies have established that PTP1B and SHP-1 can dephosphorylate the IR to regulate hepatic insulin sensitivity [31, 33, 47]. Similarly, heterozygous TCPTP deficiency enhances hepatocyte IR phosphorylation and represses HGP [28, 48]. We speculate that the oxidation of such PTPs would promote insulin signalling to regulate hepatic glucose metabolism, although we cannot exclude the contribution of other pathways given the growing number of metabolic proteins that can be oxidised, including pyruvate kinase M2  and pyruvate kinase 2 .
Our findings warrant a redress of the contributions of ROS such as H2O2 to hepatic pathophysiology in obesity and suggest that increases in hepatocyte H2O2 may in fact represent a compensatory and beneficial response to attenuate disease progression.
We thank E. N. Gurzov (St Vincent’s Institute, Australia) for reading the manuscript.
This work was supported by the National Health and Medical Research Council of Australia (TT and SA) and the Diabetes Australia Research Trust (TT).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript
All authors contributed to the study conception and design, acquisition of data or analysis and interpretation of data. TT supervised all aspects of the study and drafted the manuscript. All authors participated in the critical revision of the manuscript and approved the final version. TT is the guarantor of this work.
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