Cell Metabolism Article

Hepatic Oxidative Stress Promotes Insulin-STAT-5 Signaling and Obesity by Inactivating Tyrosine Phosphatase N2

Esteban N. Gurzov,1 Melanie Tran,1 Manuel A. Fernandez-Rojo,1 Troy L. Merry,1 Xinmei Zhang,1 Yang Xu,2,3 Atsushi Fukushima,1 Michael J. Waters,4 Matthew J. Watt,5 Sofianos Andrikopoulos,6 Benjamin G. Neel,2,3 and Tony Tiganis1,* 1Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia 2Princess Margaret Cancer Center, University Health Network, 101 College Street, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada 3Department of Medical Biophysics, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada 4Institute for Molecular Bioscience, Services Road, University of Queensland, St. Lucia, QLD 4072, Australia 5Department of Physiology, Monash University, Wellington Road, Clayton, VIC 3800, Australia 6Department of Medicine Heidelberg Repatriation Hospital, 300 Waterdale Road, Heidelberg West, The University of Melbourne, VIC 3081, Australia *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cmet.2014.05.011

SUMMARY and Kahn, 2001). AKT2, in turn, phosphorylates Forkhead box protein O1 (Foxo1) and prevents its nuclear translocation, Hepatic insulin resistance is a key contributor to the thereby repressing Foxo1-mediated transcription. The resultant pathogenesis of obesity and type 2 diabetes (T2D). inhibition of Foxo1-mediated expression of the gluconeogenic Paradoxically, the development of insulin resistance phosphoenolpyruvate carboxykinase (PEPCK) and in the liver is not universal, but pathway selective, glucose 6-phosphatase (G6P) helps to suppress hepatic glucose such that insulin fails to suppress gluconeogenesis production and prevent postprandial hyperglycemia and but promotes lipogenesis, contributing to the hyper- glucose toxicity. Insulin also acts in the liver via AKT2 to promote sterol-regulatory element-binding protein 1c (SREBP-1c) glycemia, steatosis, and hypertriglyceridemia that expression and processing (Foretz et al., 1999; Leavens et al., underpin the deteriorating glucose control and 2009; Li et al., 2010; Lu et al., 2012; Yecies et al., 2011). Pro- microvascular complications in T2D. The molecular cessed SREBP-1c in turn promotes the transcription of basis for the pathway-specific insulin resistance re- involved in triglyceride (TAG) synthesis, including acetyl-coen- mains unknown. Here we report that oxidative stress zyme A carboxylase (ACC), stearoyl-coenzyme A desaturase accompanying obesity inactivates protein-tyrosine (SCD-1), and fatty acid synthase (FAS) (Horton et al., 2002). phosphatases (PTPs) in the liver to activate select Obesity is a key contributor to the development of insulin resis- signaling pathways that exacerbate disease progres- tance and T2D (Johnson and Olefsky, 2013). Insulin resistance is sion. In obese mice, hepatic PTPN2 (TCPTP) inacti- associated with defects or aberrations in signaling downstream vation promoted lipogenesis and steatosis and of the IR PTK. In particular, under conditions of insulin resistance, insulin-STAT-5 signaling. The enhanced STAT-5 PI3K/AKT signaling is attenuated, diminishing insulin’s metabolic effects (Defronzo, 2009; Saltiel and Kahn, 2001). Defective insu- signaling increased hepatic IGF-1 production, which lin-induced PI3K/AKT signaling has been attributed to various suppressed central growth hormone release and interrelated factors including intracellular lipid accumulation, exacerbated the development of obesity and T2D. inflammation, ER stress, and oxidative stress (Johnson and Olef- Our studies define a mechanism for the development sky, 2013; Samuel and Shulman, 2012). Hepatic insulin resis- of selective insulin resistance with wide-ranging tance is thought to be an early event in the development of implications for diseases characterized by oxidative T2D (Michael et al., 2000; Turner et al., 2013). The diminished in- stress. sulin-induced suppression of hepatic gluconeogenesis contrib- utes to the fasting hyperglycemia evident in patients with T2D. Paradoxically, insulin-induced lipid synthesis in the liver is not INTRODUCTION diminished, but rather elevated in the insulin-resistant state, exacerbating hepatic steatosis (Biddinger et al., 2008; Brown The liver is an essential organ in glucose homeostasis serving to and Goldstein, 2008; Haas et al., 2012; Leavens et al., 2009). produce glucose during periods of fasting to prevent hypoglyce- Although the basis for the apparent selective insulin resistance mia and maintain brain function and survival. After a meal, insulin and the contrary effects on hepatic gluconeogenesis versus acts in the liver via the (IR) protein-tyrosine lipogenesis in obesity and T2D remain to be resolved, the bifur- kinase (PTK) to phosphorylate IR substrate-1/2 and activate cation of insulin signaling appears to be downstream of AKT2 the phosphatidylinositol 3-kinase (PI3K)/AKT pathway (Saltiel (Leavens et al., 2009). De novo lipogenesis also occurs via

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insulin-independent pathways, particularly by carbohydrates diet-induced obesity promotes the oxidation of hepatic PTPs, after feeding. Dietary fatty acids and the flux of peripheral fats age-matched C57BL/6 mice were fed either a high-fat diet from white adipose tissue to the liver can also drive steatosis. (23% fat) or standard chow diet for 24 weeks, and oxidative Increased lipogenesis and TAG accumulation can, in turn, result stress and PTP oxidation were assessed. Consistent with the in increased very-low-density lipoprotein secretion and uptake in development of oxidative stress, blood oxidized glutathione muscle, which exacerbates the development of insulin resis- (GSSG) levels were increased and the reduced glutathione tance and contributes to the cardiovascular complications of (GSH):GSSG ratio was decreased in mice rendered obese by the disease (Postic and Girard, 2008; Samuel and Shulman, high-fat feeding when compared with chow-fed lean controls 2012). (Figure 1A). The systemic oxidative stress was, in turn, associ-

Chronic reactive oxygen species (ROS) generation and oxida- ated with increased H2O2 production by hepatocytes (measured tive stress occur in a wide variety of human diseases, including in live cells using the H2O2-selective probe Amplex red) isolated À d obesity and T2D (Newsholme et al., 2007). Superoxide (O2 )is from high-fat-fed (HFF) versus chow-fed mice (Figure 1B); H2O2 a natural byproduct of mitochondrial oxidative phosphorylation, production by hepatocytes from HFF mice was significantly but under normal conditions this is rapidly converted to higher than that occurring in insulin-stimulated hepatocytes (Fig- hydrogen peroxide (H2O2) by superoxide dismutase. H2O2 is ure 1B). To assess whether PTPs may be oxidized in the livers of thereon eliminated by antioxidant enzymes such catalase, per- HFF mice, we took advantage of a monoclonal antibody (PTPox) oxiredoxins, and glutathione peroxidase 1 (Gpx1) (Fisher-Well- raised against the signature motif peptide of PTP1B with its cat- man and Neufer, 2012). In obesity, the chronic uptake and oxida- alytic cysteinyl residue oxidized to the irreversible sulfonic acid tion of energy substrates, particularly lipids, is thought to result in (Persson et al., 2005). Previous studies have established that À d enhanced mitochondrial O2 and H2O2 generation (Fisher-Well- this antibody can detect virtually all classical tyrosine-specific man and Neufer, 2012). ER stress and the unfolded protein PTPs when they are oxidized to the sulfonic (ÀSO3H) state (Kar- response in obesity and the increased expression and activation isch et al., 2011). H2O2 promotes the reversible oxidation of PTPs of NADPH oxidases (NOXs) contribute further to ROS generation to the sulfenic (ÀSOH) state, which is accompanied in many (Furukawa et al., 2004; Jiang et al., 2011). Compelling evidence classical PTPs by the formation of a sulfenylamide intermediate links oxidative stress to the promotion of insulin resistance in hu- that limits further oxidation to the irreversible sulfinic (ÀSO2H) mans and rodents (Anderson et al., 2009; Furukawa et al., 2004; and ÀSO3H states. At first, we monitored the oxidation status Houstis et al., 2006), and oxidative stress in the liver exacerbates of PTPs by immunoblot analysis (Figure 1C). Livers from HFF inflammation and hepatic fibrosis (Paik et al., 2011; Rolo et al., obese and chow-fed lean mice were homogenized in an anaer- 2012). obic chamber in the presence of N-ethylmaleimide (NEM) to pre- PTPs are important targets for ROS (Tiganis, 2011). Classical vent postlysis oxidation and to alkylate all reduced and active tyrosine-specific PTPs contain a conserved signature motif PTPs. Reversibly oxidized PTPs were reduced and hyperoxi-

([I/V]HCSXGXGR[S/T]G) in their catalytic domains wherein the dized to the ÀSO3H state for detection with PTPox by immuno- invariant Cys is essential for catalytic activity. The low pKa of blot analysis. We noted an increase in the oxidation status of the active site Cys facilitates nucleophilic attack of the substrate several PTPs in the livers from HFF mice (Figure 1C); PTP oxida- phosphotyrosine, but also renders PTPs highly susceptible to tion was largely reversible, as several PTPox species were either oxidation by ROS such as H2O2 (Tiganis, 2011). Oxidation of not detected or decreased when NEM-treated tissue extracts the active site Cys abrogates its nucleophilic properties and in- were immunoblotted without further processing (Figure S1A). duces conformational changes that inhibit PTP activity and pre- Oxidized hepatic PTPs in HFF mice included those comigrating vent substrate binding. PTP oxidation is thought to occur under with the prototypic PTP1B (PTPN1) and the closely related both physiological and pathological conditions to promote tyro- TCPTP (PTPN2), both of which have been implicated in the regu- sine phosphorylation-dependent signaling (Tiganis, 2011). lation of hepatic insulin sensitivity and gluconeogenesis (Tiganis, Here we report that oxidative stress in high-fat diet-induced 2013). PTP oxidation was not increased further when HFF mice obesity results in pronounced hepatic PTP oxidation. In partic- were administered insulin, and hepatic PTP oxidation was not ular, we demonstrate that under conditions of obesity and insulin altered in chow-fed mice subjected to hyperinsulinemic-eugly- resistance, when the PI3K/AKT pathway is otherwise attenuated, cemic clamps (insulin infused for 4 hr; Figures S1B and S1C). PTPN2 (also known as PTP: TCPTP) is oxidized and inac- Thus, we propose that the heightened hepatic PTP oxidation in tivated to promote lipogenesis and insulin-induced STAT-5 obese mice occurs as a consequence of the systemic oxidative signaling. We report that the selective activation of the insulin- stress and the elevated ROS production in the liver parenchyma. STAT-5 pathway may be instrumental in the promotion of obesity Hepatocytes constitute roughly 80% of the liver volume, with through the STAT-5 transcriptional target insulin-like growth fac- the remainder comprised of nonparenchymal cells including tor-1 (IGF-1) and the repression of growth hormone (GH) release Kupffer cells, endothelial cells, and stellate cells. We determined from the pituitary. Our studies have defined a liver-centric mech- whether the increased PTP oxidation in the livers of HFF mice anism contributing to the development of obesity and T2D. could be ascribed to changes in the oxidation status of PTPs in hepatocytes. PTP oxidation was assessed in hepatocytes RESULTS that were freshly isolated from chow-fed versus HFF mice (Fig- ure 1D); several PTPox species were increased in hepatocytes Hepatic PTP Oxidation in High-Fat-Fed Mice from HFF mice (Figure 1D). Thus, the increased PTP oxidation Obesity is associated with systemic oxidative stress in both hu- in livers of HFF mice may, at least in part, be due to changes in mans and rodents (Furukawa et al., 2004). To determine whether PTP oxidation in hepatocytes.

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Figure 1. Oxidative Stress and Hepatic PTP Oxidation (A) Eight-week-old male C57BL/6 mice were chow-fed or HFF for 24 weeks. Blood GSH and GSSG levels and GSH:GSSG ratios were determined.

(B) Hepatocytes from 12 week chow-fed or HFF mice were either left untreated or serum-starved and stimulated with 100 nM insulin for 30 min and H2O2 production measured. (C–F) Livers from 24 week chow-fed or HFF mice were processed for an assessment of total PTP oxidation by (C) immunoblot analysis with the PTPox antibodyor

(E) mass spectrometry determining relative PTP oxidation (PTP-SO3H), or (F) processed for immunoblot analysis for the indicated PTPs. (D) Hepatocytes were processed immediately after isolation for an assessment of total PTP oxidation. In (C) and (D), arrows correspond to TCPTP. Results are means ± SEM for the indicated number of mice (A, E, F) or experimental repeats (B).

To determine the extent to which individual hepatic PTPs are 2011). In this approach, hyperoxidized PTPs are digested, en- oxidized in HFF obese versus chow-fed, lean mice, we utilized riched by PTPox immunoprecipitation, and quantified by mass a recently developed, quantitative proteomic method for the spectrometry (Karisch et al., 2011). Using this approach, 6 of global assessment of classical PTP oxidation (Karisch et al., 17 known nonreceptor and 9 of 21 known receptor PTPs were

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(legend on next page)

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identified (Figure 1E; data not shown). Of the hepatic PTPs de- (p-IRb) phosphorylation (Figure 2A; Figure S2A). On the other tected in chow-fed and HFF livers, only 8 exhibited an increase hand, basal p-AKT was reduced (Figure 2A), in line with the in their oxidation status in HFF mice (Figure 1E). These included development of pathway-specific insulin resistance. These re- the nonreceptor phosphatases TCPTP (PTPN2) and PTP1B sults are consistent with high-fat diet-induced obesity resulting (PTPN1) and several transmembrane receptor-type PTPs, in in the promotion of STAT-1/STAT-3/STAT-5 signaling. particular CD45 (PTPRC), which marks hematopoietic cells, and DEP-1 (PTPRJ), which is expressed in immune and nonim- Selective Activation of Insulin-Induced STAT-5 mune cells alike (Hermiston et al., 2009). CD45 protein levels Signaling were increased in the livers of HFF mice (Figure 1F), consistent We next assessed the potential impact of hepatic PTP oxidation with the infiltration of immune cells that accompanies hepatos- on insulin signaling in the liver by comparing responses to bolus teatosis. These results demonstrate that PTPs can be exten- insulin administration (0.65 mU/g, intraperitoneal, 10 min) in sively oxidized in vivo in the context of systemic oxidative stress 24 week chow-fed versus HFF C57BL/6 mice (Figure 2A). We and raise the possibility that PTP oxidation may contribute to he- monitored for p-IRb, activation of PI3K/AKT signaling with anti- patic pathophysiology in obesity/T2D. bodies to Ser-473-phosphorylated AKT, and the activation sta- tus of STAT-1, STAT-3, and STAT-5 using appropriate phospho- Hepatic STAT-1/STAT-3/STAT-5 Are Hyperactivated specific antibodies. IR phosphorylation was not overtly altered, in HFF Mice whereas AKT phosphorylation was reduced consistent with the Since PTP1B and TCPTP are implicated in liver physiology (De- development of insulin resistance (Figure 2A). Surprisingly, we libegovic et al., 2009; Fukushima et al., 2010), we focused our found that insulin-induced p-STAT-5 was robust in the livers attention on the signaling pathways regulated by these two from HFF mice and not detectable in chow-fed control mice (Fig- phosphatases. TCPTP and PTP1B attenuate both IR signaling ure 2A). By contrast, although basal p-STAT-1 and p-STAT-3 and Janus-activated kinase (JAK)/STAT signaling (Tiganis, were elevated in livers from HFF mice, they were not induced 2013). Both PTPs dephosphorylate and inactivate the IR and further by insulin (Figure 2A). To determine whether the insulin- JAK family PTKs: PTP1B dephosphorylates JAK-2 and Tyk2 induced activation of the p-STAT-5 pathway was unique to whereas TCPTP dephosphorylates JAK-1 and JAK-3. TCPTP HFF mice, or whether high-fat feeding and obesity exacerbated also dephosphorylates STAT family members (including the normal response to insulin, we also challenged 8- to 10- STAT-1, STAT-3 and STAT-5) (Tiganis, 2013). To assess the po- week-old chow-fed mice with increasing concentrations of insu- tential impact of hepatic PTP oxidation on cellular signaling and lin and monitored for the status of p-STAT-5. We found that with biological responses in HFF mice, we assessed hepatic IR and higher concentrations of insulin (1.3 mU/g), p-STAT-5 was de- JAK/STAT signaling in liver homogenates from chow-fed versus tected in liver homogenates from chow-fed mice (Figure S2C). HFF mice. JAK/STAT signaling was assessed by monitoring GH acts in the liver via JAK-2 and STAT-5 to increase IGF-1 STAT-1 Y701 (p-STAT-1), STAT-3 Y705 (p-STAT-3), and production (Lichanska and Waters, 2008). As insulin lowers STAT-5 Y694 (p-STAT-5) phosphorylation. We found that blood glucose and hypoglycemia can in turn promote the release p-STAT-1 and p-STAT-3 were elevated in liver homogenates of GH from the pituitary (Jaffe et al., 1999), the enhanced insulin- from HFF (24 week) fasted mice (Figure 2A; Figure S2A); basal induced hepatic p-STAT-5 in chow-fed mice (administered p-STAT-5 was also elevated with more prolonged (40 week) 1.3 mU/g insulin) could have been a consequence of enhanced high-fat feeding (Figure S2A). By contrast, we noted no overt dif- GH release and signaling. To address this possibility, we sub- ferences in general tyrosine phosphorylation in liver homoge- jected chow-fed C57BL/6 mice to hyperinsulinemic-euglycemic nates from fasted (4 hr) chow-fed versus HFF mice as assessed clamps, wherein a high dose of insulin was infused constantly with phospho-tyrosine (p-Tyr)-specific antibodies (Figure S2B). (60 mU/ml insulin, 10–40 ml/min for 4 hr), while euglycemia was These results demonstrate that enhanced PTP oxidation per se maintained by the coinfusion of glucose, and assessed GH does not result in constitutive tyrosine phosphorylation-depen- levels, p-STAT-5 signaling, and hepatic Igf-1 mRNA levels (Fig- dent signaling in fasted livers. In keeping with this, there were ure 2B; Figure S1C). It is well established that insulin can mimic no overt differences in mitogen-activated protein kinase IGF-1 in the suppression of GH release from the anterior pitui- signaling, as assessed with antibodies to phosphorylated tary, a negative feedback loop that normally prevents the exces- ERK1/2, nor any increase in basal IR b-subunit Y1162/Y1163 sive release of GH (Yamashita and Melmed, 1986). Accordingly,

Figure 2. Oxidative Stress Promotes Insulin-Induced STAT-5 (A) Eight-week-old male C57BL/6 mice were chow-fed or HFF for 24 weeks, fasted, and injected with PBS or insulin, and livers were processed for immunoblotting. (B) Twenty-week-old male C57BL/6 mice were subjected to hyperinsulinemic-euglycemic clamps (4 hr). Plasma GH levels were measured and livers were processed for immunoblotting or quantitative real-time PCR to measure Igf-1 mRNA. (C–G) Hepatocytes from chow-fed mice were treated with vehicle or 0.5 mM palmitate-BSA. Hepatocytes were serum-starved, stimulated with 100 nM insulin, and processed for immunoblotting (C, E–G). (D) H2O2 production was measured in live cells. Where indicated, hepatocytes were pretreated with vehicle, the JAK inhibitor CMP6 (10 mM), mitoTempol (10 mM), or SS31 (50 mM) for 2 hr. In (F), hepatocytes were transfected with GFP control or Jak-2-specific siRNAs prior to palmitate treatment.

(H and I) H2O2 production and (I) insulin (100 nM)-induced p-STAT-5 signaling in Gpx1+/+ and Gpx1–/– hepatocytes. (J) Eight-week-old male C57BL/6 mice were HFF for 12 weeks and treated with vehicle or SS31 for 10 days. Mice were fasted and injected with PBS or insulin (0.5 mU/g, 10 min), and livers were extracted for immunoblotting. (K) Gpx1+/+ and Gpx1–/– male mice were HFF for 24 weeks, then fasted, and livers were processed for immunoblotting. Representative and quantified (means ± SEM) results are shown.

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we noted significantly reduced serum GH levels in clamped mice hepatocytes (Figure 2H), and this was accompanied by a striking (Figure 2B). Despite these lower GH levels, p-STAT-5 was increase in basal and insulin-induced p-STAT-5 signaling (Fig- increased significantly in livers from clamped mice when ure 2I), which was increased further when cells were treated compared to saline-infused controls (Figure 2B). Igf-1 levels with palmitate (data not shown). also were increased in clamped mice, compared with controls, Having established that H2O2 can promote insulin-induced and the increase in Igf-1 expression correlated largely with the p-STAT-5 signaling in hepatocytes ex vivo, we next assessed degree of p-STAT-5 (Figure 2B; numbers in lanes correspond whether this might occur in vivo. First we determined if the mito- to points in panel below). Complementing these in vivo studies, chondrial-targeted SS31 could attenuate insulin-induced we found that insulin stimulation also resulted in a modest, but p-STAT-5 signaling in HFF mice. Mice that had been HFF for reproducible increase in STAT-5 activation in isolated primary 12 weeks were administered vehicle control or SS31 hepatocytes from C57BL/6 mice (Figure 2C). Taken together, (2.0 mg/kg/day intraperitoneal) for 12 days, and insulin-induced these results demonstrate that the insulin-induced p-STAT-5 (0.5 mU/g) STAT-5 signaling was assessed (Figure 2J). Insulin- pathway occurs in hepatocytes to promote the expression of induced p-STAT-5 signaling trended lower in SS31-treated STAT-5 target genes such as Igf-1 and that the STAT-5 pathway mice (Figure 2J), consistent with mitochondrial-derived oxidative is exacerbated in HFF obese mice, when insulin-induced PI3K/ stress contributing to the promotion of STAT-5 signaling. We AKT signaling is otherwise attenuated. next determined if exacerbating oxidative stress could have the opposite effect and enhance insulin-induced STAT-5 ROS Promote Insulin-Induced STAT-5 Signaling signaling. For this we took advantage of Gpx1À/À mice and as-

Having established that insulin signals via STAT-5 in the liver and sessed if Gpx1 deficiency and elevated H2O2 could exacerbate that this is exacerbated in obesity, we next sought to define the high-fat diet-induced oxidative stress, PTP oxidation, and basis for the exacerbation of the IR-STAT-5 pathway in HFF p-STAT-5 signaling (Figures S1F and S1G). Blood GSH:GSSG mice. In particular, we asked whether the hepatic oxidative ratios in Gpx1–/– versus Gpx1+/+ HFF (24 week) mice were stress associated with obesity might inactivate PTPs for the se- reduced (Figure S1F), consistent with the promotion of systemic lective promotion of insulin-induced STAT-5 signaling. To this oxidative stress; alterations in GSH:GSSG ratios were not end we isolated hepatocytes from chow-fed lean mice and as- evident in chow-fed mice. The increased oxidative stress was sessed the impact of incubating them with the fatty acid palmi- in turn associated with elevated hepatic PTP oxidation (Fig- tate, which promotes insulin resistance and generates high rates ure S1G), as assessed by immunoblot analysis with the PTPox of mitochondrial H2O2 emission (Fisher-Wellman and Neufer, antibody; the increase in PTP oxidation occurred for several spe- 2012). We found that treatment with palmitate-BSA promoted cies including one comigrating with TCPTP (Figure S1G).

H2O2 production and resulted in increased insulin-induced Furthermore, the increased hepatic PTP oxidation coincided p-STAT-5 (Figures 2C and 2D). The increase in p-STAT-5 was with the detection of hepatic p-STAT-5 in the livers of fasted accompanied by a decrease in PI3K/AKT signaling (Figure 2C), Gpx1–/– mice (Figure 2K). Taken together these results provide consistent with palmitate promoting insulin resistance. The insu- evidence for hepatic oxidative stress promoting insulin-induced lin-induced p-STAT-5 in untreated and palmitate-treated hepa- hepatic p-STAT-5 signaling in vivo. Moreover, these results are tocytes was reliant on JAK-2, since a highly selective JAK PTK consistent with the possibility that the ROS-mediated promotion inhibitor (Figure 2E), or JAK-2 knockdown using specific of insulin-p-STAT-5 signaling might be reliant on the oxidation of siRNAs (Figure 2F), attenuated insulin-induced p-STAT-5 PTPs such as TCPTP. signaling. To determine if the palmitate-mediated increase in in- sulin-induced p-STAT-5 was mediated by the increase in Hepatic TCPTP Deficiency Exacerbates Obesity, mitochondrial ROS emission, we took advantage of the mito- Steatosis, and Insulin Resistance chondrial-targeted antioxidant mitoTEMPOL and the mitochon- To ascertain if the inactivation of hepatic PTPs might contribute drial-targeted peptide SS31 (Bendavia, Stealth Peptides) that to the promotion of STAT-1, STAT-3, and STAT-5 signaling and accumulates in mitochondria and reduces mitochondrial the development of obesity and T2D, we sought to delete those À O2, /H2O2 generation/release (Anderson et al., 2009). We found hepatic PTPs that were oxidized in HFF mice. We focused our that treating hepatocytes with mitoTEMPOL or SS31 signifi- attention on TCPTP for several reasons. First, the relative in- cantly attenuated the palmitate-induced increase in p-STAT-5 crease in TCPTP oxidation in HFF obese mice was greater signaling in response to insulin (Figure 2G). Finally, we assessed than that of other nontransmembrane PTPs (Figures 1E and whether the oxidation status of PTPs may be increased in 1F). Second, TCPTP has the capacity to attenuate STAT-5 response to palmitate in hepatocytes. For these experiments signaling (Loh et al., 2011), and third, TCPTP has been implicated we took advantage of nontransformed AML12 hepatocytes. in the attenuation of hepatic STAT-3 signaling (Fukushima et al., lox/lox Treatment of AML12 cells with palmitate increased both H2O2 2010). Ptpn2 (lox/lox) mice were bred with Alb-Cre mice to production and PTP oxidation (Figure S1E). generate liver-specific TCPTP knockout (Alb-Cre;Ptpn2lox/lox:

To further explore the role of H2O2 in the promotion of insulin- LTKO) mice (Figure S3A). Seven-week-old LTKO mice appeared induced p-STAT-5 signaling, we took advantage of mice lacking normal and did not exhibit differences in body weight/adiposity the antioxidant Gpx1, which converts H2O2 to water. or overt alterations in glucose tolerance (data not shown). To Hepatocytes were isolated from 12-week-old male Gpx1À/À assess the impact of hepatic TCPTP deficiency on the develop- and Gpx1+/+ mice, and H2O2 levels and insulin-induced ment of obesity and T2D, 6-week-old male and female lox/lox p-STAT-5 signaling were assessed (Figures 2H and 2I). Gpx1 and LTKO mice were fed standard chow versus high-fat diets deficiency resulted in a 2-fold increase in H2O2 production in for 12 weeks, and their body weights and metabolic status

90 Cell Metabolism 20, 85–102, July 1, 2014 ª2014 Elsevier Inc. Cell Metabolism Oxidative Stress and Selective Insulin Resistance

were assessed (Figure 3; Figure S4). Strikingly, both male and fe- not in HFF LTKO HET mice (Figure 4C; data not shown). SREBP- male LTKO mice fed a high-fat diet gained more weight than their 1c promotes the expression of lipogenic enzymes such as FAS lox/lox counterparts, and this was associated with increased and SCD-1 (Horton et al., 2002) and PPAR-g promotes the tran- adiposity, as assessed by measuring fat pad weights and by scription of fatty acid transporters such as CD36/FAT to promote monitoring body composition by dual-energy X-ray absorptiom- the uptake of FFAs (Barclay et al., 2011). SCD-1 and FAS protein etry (DEXA) (Figures 3A–3H). Although liver weights were higher levels were elevated in the livers of HFF male and female LTKO in HFF LTKO mice, gross differences in body length were not mice (Figure 4D), as was the expression of Cd36 mRNA in female evident, and no significant differences were noted in lean mass mice (Figure 4E). Moreover, hepatic lipogenesis, as assessed in or bone density (Figures 3A–3H; data not shown). The increased liver slices ex vivo also was increased in HFF LTKO mice (Fig- adiposity was accompanied by decreased energy expenditure in ure 4F). Taken together, these results demonstrate that the dele- both sexes and decreased respiratory exchange ratio (RER) in tion of TCPTP in the liver promotes diet-induced obesity and male mice (Figure 3I; Figure S4A). No differences were seen insulin resistance as well as hepatic lipogenesis and steatosis. in ambulatory activity (Figure 3I; Figure S4A) or food intake (data not shown), arguing against overt differences in the central Hepatic TCPTP Deficiency Promotes STAT-1/STAT-3/ control of body weight. Increased weight gain and adiposity and STAT-5 Signaling decreased energy expenditure were also observed in female To understand how hepatic TCPTP deletion/inactivation might LTKO mice fed a standard chow diet, but not in chow-fed male be promoting obesity/T2D and steatosis, we sought to delin- mice (Figures S4B and S4C; data not shown). These results indi- eate the tyrosine phosphorylation-dependent pathways that cate that TCPTP deficiency in the liver promotes diet-induced might be altered in 12 week HFF LTKO mice and 20 week weight gain. Moreover, male mice with heterozygous hepatic LTKO HET mice. We did not observe any significant change TCPTP deficiency (Alb-Cre;Ptpn2lox/+; LTKO HET) also gained in global tyrosine phosphorylation, basal ERK1/2, or p38 activa- more weight when fed a high-fat diet, and this was associated tion, or in the activation of the ER stress response, as moni- with increased adiposity (Figure 3J). Thus, even partial hepatic tored with antibodies to Thr-980 phosphorylated PERK or TCPTP deficiency, or TCPTP inactivation, as might occur as a Ser-51 phosphorylated eIFa in the livers of fasted mice (Fig- consequence of oxidation, is sufficient to promote obesity. In ure S6A; data not shown). Similarly, TCPTP deficiency did not keeping with their increased adiposity, HFF LTKO and LTKO result in a global increase in the phosphorylation of TCPTP sub- HET mice exhibited decreased insulin sensitivity, as monitored strates, including that of Src family kinases (SFKs), as moni- in insulin tolerance tests, and glucose intolerance, as assessed tored with antibodies to the Y416 site on c-Src (Figure S6A), in glucose tolerance tests (Figure 3K; Figures S4D, S4E, and and JAK-1, as monitored with antibodies to the Y1022/Y1023 S4G). HFF LTKO mice also exhibited elevated fasting blood PTK activation loop site (Figure S5F). Thus, hepatic signaling glucose levels, accompanied by elevated plasma insulin levels in fasted mice was not elevated in general as a consequence in fed (female) and fasted (male and female) mice (Figure 3L; Fig- of TCPTP deficiency. Previous studies have established that ure S4F) and decreased insulin-induced PI3K/AKT signaling in STAT-1, STAT-3, and STAT-5 may serve as substrates of liver and muscle (Figure 3M; Figures S4H and S4I). TCPTP (Tiganis, 2013; Tiganis and Bennett, 2007). We found Next we assessed the impact of hepatic TCPTP deletion on that p-STAT-1, p-STAT-3, and p-STAT-5 were elevated in the liver morphology (Figure 4A; Figure S5A). As noted earlier, livers livers of fasted male and female LTKO mice that were HFF from male and female 12 week HFF LTKO mice were heavier for 12 weeks, when compared to lox/lox controls (Figures 5A than their lox/lox counterparts. This was associated with and 5E; Figures S5D and S5E). The elevated STAT-1/STAT-3/ increased steatosis, as assessed by gross morphology, histol- STAT-5 signaling in HFF LTKO liver homogenates was not ogy (hematoxylin and eosin staining), and oil Red O staining associated with overt alterations in the expression of PTP1B, (stains lipid droplets) and by measuring hepatic lipids (Figures SHP-1, and SHP-2 (Figure S6B) that have also been implicated 4A and 4B). The steatotic phenotype was not accompanied by in the control of JAK/STAT signaling. Importantly, interferon alterations in circulating free fatty acids (FFAs) and TAGs (Fig- (IFN) g-induced p-STAT-1 was increased in hepatocytes iso- ure S5B). The increased steatosis was not evident in 20 week lated from LTKO mice, p-STAT-3 and STAT-5 were elevated HFF LTKO HET livers (Figure 4C), even though these mice ex- constitutively in LTKO hepatocytes (Figure 5B), and p-STAT-1 hibited increased diet-induced weight gain and insulin resistance and STAT-5 were elevated in C57BL/6 hepatocytes after (Figure 3J; Figure S4G). These findings suggest that the promo- TCPTP knockdown (Figure S6C), consistent with the enhanced tion of obesity and the development of steatosis in HFF LTKO signaling in vivo being hepatocyte intrinsic. Interestingly, only mice might be regulated by independent processes. p-STAT-5 was elevated in the livers of 20 week HFF LTKO Steatosis in obesity is accompanied by increased hepatic HET mice, highlighting the sensitivity of the STAT-5 pathway levels of the transcription factors SREBP-1c and peroxisome to Ptpn2 dosage in the context of obesity (Figure 5C). proliferator-activated receptor g (PPAR-g)(Horton et al., 2002; Moreover, as p-STAT-1 and p-STAT-5 were not detected in Memon et al., 2000). Hepatic overexpression of SREBP-1c or the livers of 7-week-old chow-fed LTKO male mice (Figure 5D), PPAR-g is sufficient to promote steatosis (Shimano et al., these results point toward TCPTP deficiency promoting 1997; Yu et al., 2003), whereas PPAR-g deletion in hepatocytes p-STAT-1 and p-STAT-5 only in the context of high-fat can attenuate both genetic and high-fat diet-induced fatty liver feeding/obesity. disease (Matsusue et al., 2003; Mora´ n-Salvador et al., 2011). Our studies indicate that high-fat feeding of C57BL/6 mice SREBP-1 and PPAR-g were elevated in the livers of HFF male promotes hepatic insulin-induced p-STAT-5 signaling. Accord- and female LTKO mice (Figure 4D; Figure S5C), but interestingly ingly, we next examined whether the inactivation of hepatic

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TCPTP by ROS in diet-induced obesity might be responsible for was not altered. Thus, changes in PDK4 expression may not the promotion of insulin-induced p-STAT-5 signaling. To this end be sufficient to influence the development of obesity and insulin we assessed the impact of TCPTP deficiency on hepatic insulin resistance in LTKO mice. signaling in mice that had been HFF for 12 weeks (Figure 5E; Fig- STAT-5 is both necessary and sufficient for the GH-induced ure S5E). Despite insulin-induced PI3K/AKT signaling being transcription of Igf-1 in the liver (Cui et al., 2007). IGF-1 can in attenuated in HFF LTKO livers (Figure 3M; Figure S4H), we found turn feed back onto hypothalamic neurons and somatotrophs that the induction of p-STAT-5 was significantly enhanced in to inhibit GH release from the pituitary (Romero et al., 2010). male and female LTKO mice (Figure 5E; Figure S5E). Although We found that serum IGF-1 levels were increased in male and fe- IR b-subunit Y1162/Y1163 phosphorylation was elevated in the male mice fasted overnight (Figure 4J). Moreover, in keeping livers of fasted HFF LTKO male mice (not female mice), insulin- with TCPTP’s potential to regulate STAT-5 signaling and Igf-1 induced IR phosphorylation (Figure 5E; Figure S5E) was not expression, we found that Ptpn2 knockdown using siRNAs altered by TCPTP deficiency. These results are consistent with increased Igf-1 mRNA expression in C57BL/6 hepatocytes (Fig- previous studies demonstrating that TCPTP regulates basal ure 4I). Finally, consistent with circulating IGF-1 levels being but not insulin-induced IR phosphorylation in the liver (Fukush- increased, we found that serum GH levels were decreased in fe- ima et al., 2010). Moreover, while insulin-induced STAT-5 male mice and trended lower in male mice (Figure 4J); the differ- signaling was enhanced in LTKO livers, JAK-1 Y1022/Y1023 ence in GH levels is in keeping with the continuous versus pulsa- (p-JAK-1) and JAK-2 Y1007/Y1008 (p-JAK-2) phosphorylation tile nature of GH secretion in female versus male mice (Lichanska was not altered (Figures S5F and S5G). Therefore, these results and Waters, 2008). GH levels are reduced in obese rodents and are consistent with the induction of STAT-5 signaling in response humans and this can promote fat accumulation (Lichanska and to insulin occurring downstream of the IR and JAK PTKs. Taken Waters, 2008). Thus, elevated hepatic STAT-5 signaling associ- together, our results indicate that the inactivation of TCPTP ated with hepatic TCPTP deficiency/inactivation could promote might contribute to the promotion of basal STAT-1 and STAT-3 IGF-1 expression for the suppression of GH release and the pro- signaling and insulin-induced STAT-5 signaling in the context motion of adiposity in nonhepatic tissues. of obesity and insulin resistance. Hepatic Stat-5 Heterozygosity Corrects Obesity but Not Hepatic TCPTP Deficiency Alters the IGF-1/GH Axis Steatosis We next explored the potential molecular mechanisms by To assess the importance of hepatic STAT-5 signaling in the which hepatic TCPTP deficiency might promote obesity and development of obesity and T2D, we determined the effect of T2D. We noted that p-STAT-5, but not p-STAT-1 or p-STAT- superimposing STAT-5 deficiency on LTKO mice. To this 3, was elevated in the livers of HFF LTKO HET mice (Figure 5D). end we crossed male and female LTKO mice onto a hepatic Because HFF LTKO HET male mice gained more weight and Stat-5lox/+ (Cui et al., 2007) background (generating LTKO; exhibited greater insulin resistance than controls, we reasoned Stat-5lox/+ mice) to specifically reduce STAT-5 in the liver by that the enhanced p-STAT-5 might be sufficient to drive the 50%, and then fed mice a high-fat diet for 12 weeks (Figure 6; expression of genes that promote obesity and T2D. In partic- Figure S7A). Remarkably, hepatic Stat-5 heterozygosity largely ular, we focused on two STAT-5 transcriptional targets, Igf-1 corrected the obesity phenotype in LTKO male and female (Cui et al., 2007) and the gene encoding pyruvate dehydroge- mice (Figure 6A; Figure S7B). The decreased weight gain in nase kinase 4 (Pdk4)(White et al., 2007). PDK4 phosphorylates LTKO;Stat-5lox/+ mice was attributable to decreased adiposity, pyruvate dehydrogenase complex to conserve glucose and as assessed by DEXA and by weighing fad pads, and was divert three carbon compounds for gluconeogenesis (Kim accompanied by increased energy expenditure (Figure 6A; Fig- et al., 2012). In keeping with the elevated hepatic p-STAT-5 ure S7B), but unaltered food intake and activity (data not in HFF LTKO mice, we found that PDK4 protein and Pdk4 shown). Moreover, in female mice the decreased adiposity in mRNA were increased in both male and female mice (Figures LTKO;Stat-5lox/+ mice was accompanied by improved glucose 4G and 4H; Figures S5F and S5G). Furthermore, Ptpn2 knock- homeostasis, as reflected by the reduced fasted blood glucose down in hepatocytes using siRNAs increased Pdk4 message levels in females (Figure 6B); fed and fasted plasma insulin levels (Figure 4I), consistent with the increased PDK4 in vivo being also trended lower in both male and female mice (Figure 6C). directly attributable to TCPTP deficiency. Despite the increase Notably, when we assessed p-STAT-5 levels in 4 hr fasted in PDK4, we did not detect any differences in the expression of LTKO;Stat-5lox/+ mice we found that p-STAT-5 approximated the gluconeogenic genes for PEPCK (Pck1) or G6P (G6pc)in that seen in lox/lox and lox/lox;Stat-5lox/+ controls (Figure 6D). HFF LTKO livers (Figure 4E), nor differences in glucose excur- In keeping with the decreased STAT-5 signaling, we found that sions in response to the gluconeogenic substrate pyruvate the otherwise increased hepatic PDK4 levels in LTKO mice (data not shown), suggesting that hepatic glucose production were corrected by Stat-5 heterozygosity (Figure 6D), as were

Figure 3. Hepatic TCPTP Deletion Promotes Obesity and Insulin Resistance (A–I) Seven-week-old lox/lox and LTKO mice were HFF for 12 weeks and body weights (A and E), gross morphology (B and F), tissue weights (C and G), body composition (D and H), and oxygen consumption, RER, energy expenditure and ambulatory activity (I) were assessed. (J) Seven-week-old lox/+ and LTKO HET mice were HFF for 20 weeks, and body weights and composition were determined. (K and L) HFF mice were subjected to insulin tolerance tests (K), and fed and fasted blood glucose levels and plasma insulin levels were determined (L). (M) Fasted HFF mice were injected with insulin, and livers were extracted for immunoblot analysis. Representative and quantified (means ± SEM) results are shown.

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Figure 4. LTKO Mice Exhibit Steatosis and Alterations in IGF-1 and GH (A–H) Seven-week-old male lox/lox and LTKO or lox/+ and LTKO HET mice were HFF for 12 and 20 weeks, respectively. Livers were extracted from fasted mice and processed for histology (A and C), lipid analyses (B), immunoblotting (C, D, and G), or real-time PCR (E and H). Lipogenesis was assessed ex vivo in liver slices (F). (I) Hepatocytes from chow-fed C57BL/6 were transfected with GFP-orPtpn2-specific (TCPTP#1) siRNAs and, after 48 hr, processed for real-time PCR. (J) Lox/lox and LTKO mice were HFF for 12 weeks, then fasted overnight, and serum IGF-1 and GH levels were determined. Representative and quantified results (means ± SEM) results are shown.

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circulating IGF-1 (in male and female mice) and GH levels (in fe- capacity of ROS in obesity to inactivate hepatic PTPs for the acti- male mice; levels in male mice not determined) (Figures 7A and vation of select signaling pathways in an otherwise insulin-resis- 7B). Therefore, the heightened STAT-5 signaling in HFF LTKO tant state. In particular, we demonstrate that ROS inactivate mice alters IGF-1 and GH levels and promotes obesity. TCPTP to promote the insulin-STAT-5 pathway and exacerbate In contrast to the effects on obesity, Stat-5 heterozygosity did the development of obesity and insulin resistance and thus the not correct the steatotic phenotype evident in HFF LTKO male hasten progression toward morbid obesity and T2D. In addition, mice (Figure 6E); in female HFF LTKO;Stat-5lox/+ mice, steatosis our studies suggest that the oxidation of PTPs such as TCPTP in appeared to be decreased (Figure 6E) and hepatic TAG levels obesity might also function through independent pathways, trended lower, but this was not significant (Figure 6F). Moreover, possibly in concert with insulin-induced and AKT2-mediated p-STAT-1 and p-STAT-3 signaling as well as PPAR-g and signaling (Biddinger et al., 2008; Haas et al., 2012; Leavens SREBP-1 protein levels remained elevated and were indistin- et al., 2009), to promote lipogenesis and the development of fatty guishable from those in LTKO mice (Figure 6D), in line with these liver disease. being STAT-5-independent events. Interestingly, we found that The inhibition of PTPs by ROS is thought to represent a key heterozygous Stat-5 deletion alone in the liver (Alb-Cre; mechanism for the regulation of PTP function and the coordina- Stat-5lox/+) promoted steatosis and modestly increased tion of tyrosine phosphorylation-dependent cellular signaling (Ti- adiposity in HFF mice (Figures S7C–S7E); thus, the decreased ganis, 2011). Cell-based studies have established that the inhibi- lox/+ adiposity in HFF LTKO;Stat-5 mice was not due to an unre- tion of PTPs by ROS such as H2O2 is essential for tyrosine lated effect of STAT-5 deficiency. The increased adiposity and phosphorylation-dependent signaling in response to growth fac- steatosis in HFF Alb-Cre;Stat-5lox/+ mice is consistent with the tors, , hormones, and cell adhesion (Tiganis, 2011). results of previous studies (Cui et al., 2007). Irrespective, these Moreover, increases in H2O2 in muscle, occurring as a conse- results indicate that the promotion of hepatic STAT-5 signaling quence of Gpx1 deficiency, can promote insulin signaling in vivo in LTKO mice promotes obesity, whereas alternate pathways to attenuate the development of insulin resistance (Loh et al., promote steatosis. 2009). However, to date there is no direct evidence for PTPs be- ing oxidized in vivo in response to a physiological stimulus. Simi- The Altered IGF-1/GH Axis in LTKO Mice Promotes larly, although others have reported that PTPs can be oxidized Obesity under pathological conditions in vivo, including ischemia/reper- Having established the importance of STAT-5 signaling in the fusion injury and hepatocarcinogenesis, evidence for this has development of obesity, we next sought to determine the extent been indirect, relying on the measurement of phosphatase to which perturbations in the IGF-1/GH axis might contribute to activity (He et al., 2010; Sandin et al., 2011), or undertaken in the obesity phenotype. In particular, we determined if the mice with a deficiency in Gpx1 (Merry et al., 2013). Our studies elevated IGF-1 and lower GH levels in LTKO mice were respon- directly assess the oxidation status of PTPs in the context of sible for the high-fat diet-induced obesity by asking whether the an important disease, demonstrating that classical PTPs in the promotion of endogenous GH secretion could correct the liver undergo substantial oxidation in obese mice characterized increased weight gain (Figures 7C–7E). These studies were un- by systemic and hepatic oxidative stress. Prominent nontrans- dertaken in female mice where GH secretion is continuous, membrane PTPs undergoing oxidation included PTP1B and rather than in male mice where interventions could be TCPTP. The differential oxidation of PTPs may result from confounded by the pulsatile nature of GH secretion (Lichanska intrinsic differences in PTP sensitivity to oxidation (Lou et al., and Waters, 2008). HFF LTKO mice were administered either 2008; Ross et al., 2007) and/or reflect differences in PTP versus vehicle control or the synthetic GH releasing hormone agonist antioxidant enzyme subcellular localizations. JI-38 (Kanashiro-Takeuchi et al., 2010). JI-38 was administered Notably, the oxidation of such PTPs did not result in global in- at the beginning of the and dark cycles for 20 days and creases in tyrosine phosphorylation in fasted mice. Although we body weights were monitored every 4–5 days. We found that cannot exclude additional tyrosine phosphorylation-dependent JI-38 administration promoted GH release and corrected the pathways being elevated in response to specific stimuli, our find- obesity phenotype in LTKO mice, preventing weight gain and ings are consistent with hepatic PTP oxidation in HFF mice pro- the increase in adiposity without affecting lean mass or bone moting the activation of select signaling pathways. In particular, density (Figures 7C–7E). Importantly, the effects of JI-38 admin- we found that basal p-STAT-1, p-STAT-3, and insulin-induced istration were reversible, and HFF LTKO mice regained weight p-STAT-5 were increased in the livers of HFF mice. This is in upon cessation of JI-38 administration (data not shown). Taken keeping with TCPTP being one of the most abundantly oxidized together, these results are consistent with the STAT-5/IGF-1/ PTPs in the livers of HFF mice and TCPTP’s capacity to dephos- GH axis playing a pivotal role in the development of obesity in phorylate STAT family members (Tiganis, 2013; Tiganis and Ben- LTKO mice. nett, 2007). Although in this study we focused our attention on TCPTP, it is probable that the oxidation of multiple PTPs contrib- DISCUSSION utes to the promotion of hepatic STAT-1/STAT-3/STAT-5 signaling in obesity. Previous studies have reported that It has long been known that the development of insulin resis- STAT-5 can interact directly with the IR and that the IR directly tance in the liver can be pathway selective, such that insulin fails or indirectly via JAK-2 can phosphorylate and activate STAT-5 to suppress gluconeogenesis but promotes lipogenesis, contrib- in cell lines (Chen et al., 1997; Le et al., 2002). Our results indicate uting to the development of fatty liver disease, dyslipidemia, and that JAK-2 is essential for the ROS-mediated and insulin- atherosclerosis (Brown and Goldstein, 2008). We establish the induced activation of STAT-5 in hepatocytes. We speculate

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that the concomitant oxidation of PTP1B and TCPTP in the livers IGF-1 (Moran et al., 2002) are exaggerated and associated of HFF mice might promote the coordinated activation of hepatic with decreased GH levels (Vanderschueren-Lodeweyckx, JAK-2 and STAT-5 in response to insulin. This is in keeping with 1993). Our studies indicate that the oxidative stress and the inac- previous studies that have shown that PTP1B and TCPTP can tivation of hepatic PTPs in obesity might promote the insulin- function in concert to attenuate hypothalamic JAK-2/STAT-3 STAT-5-IGF-1 pathway to reduce GH levels and exacerbate signaling in vivo (Loh et al., 2011). Although liver-specific the development of obesity and T2D. Our studies also provide PTP1B deficiency does not promote obesity (Delibegovic et al., a potential explanation for the enhanced growth rates seen in 2009), PTP1B knockout mice fed a high-fat diet for 19.5 weeks obese children, where the hyperinsulinemia of obesity coupled are more steatotic than controls (Miraldi et al., 2013). Thus the with inactivation of PTPs may provide sufficient active STAT-5 oxidation of PTP1B and TCPTP may at least cooperate in the to promote IGF-1 for increased linear growth in the presence promotion of steatosis. of relative GH deficiency. Our studies indicate that the selective activation of the insulin- Although the enhanced hepatic insulin-induced STAT-5 STAT-5 pathway in the livers of HFF/obese mice was attributable pathway exacerbated the development of obesity, our studies to the accompanying oxidative stress, since (1) palmitate- indicate that other pathways might contribute to the enhanced induced mitochondrial H2O2 generation in isolated hepatocytes lipogenesis and steatosis in LTKO mice. We observed elevated enhanced insulin-induced p-STAT-5, while repressing PI3K/ STAT-1 signaling, which can drive PPAR-g to AKT signaling; (2) basal and insulin-induced p-STAT-5 were promote lipogenesis via SREBP-1c, SCD-1, and ACC, and FFA exacerbated by the increased H2O2 accompanying Gpx1 defi- uptake via CD36 (Barclay et al., 2011; Matsusue et al., 2003; ciency; and (3) insulin-induced hepatic p-STAT-5 in HFF mice Mora´ n-Salvador et al., 2011). Thus, the increased steatosis in was at least partially attenuated by SS31. It remains unclear if LTKO mice might result from the high p-STAT-1, occurring as mitochondria are solely responsible for the ROS generation, a direct consequence of TCPTP deficiency, promoting lipogen- PTP oxidation, and promotion of p-STAT-5 in HFF mice. esis and, at least in female mice, increasing FFA uptake via Increased NOX activity and expression can contribute to hepatic CD36. The heightened STAT-3 signaling in LTKO mice might ROS generation in the fibrotic liver (Aoyama et al., 2012; Paik also contribute to steatosis, since STAT-3 overexpression in- et al., 2011), but it remains to be seen if NOXs contribute to he- creases ACC and FAS and inhibits acyl-CoA oxidase (involved patic oxidative stress in obesity. in b-oxidation) expression and promotes steatosis (Kinoshita Aberrations in the IGF-1/GH axis have been implicated in the et al., 2008). Obesity is associated with systemic inflammation, development of obesity. GH levels are markedly reduced in and there is evidence for alteration of the inflammatory obese humans, with lower GH levels correlating with increased milieu in the livers of obese rodents (Li et al., 2005). Determining visceral adiposity (Rasmussen, 2010). Similarly, GH deficiency the extent to which TCPTP oxidation/inactivation might exacer- (Donahue and Beamer, 1993) and defects in GH receptor- bate inflammatory STAT-1/STAT-3 signaling in the liver to pro- STAT-5 signaling (Rowland et al., 2005) in mice are associated mote steatosis will require further study. with the development of obesity. The precise mechanisms by The results of this study identify a mechanism for the develop- which decreased GH levels contribute to obesity are not fully ment of selective hepatic insulin resistance, define the contribu- understood, but may be attributable to defective lipolytic re- tions of the insulin-STAT-5 pathway in the liver to the develop- sponses and decreased fatty acid oxidation (Lichanska and Wa- ment obesity and T2D, and highlight the potential utility of ters, 2008). In humans the increased adiposity may also be due targeted antioxidants in combating the associated hepatic com- to decreases in energy expenditure (Møller and Jørgensen, plications. Many human diseases, including inflammatory and 2009; Snel et al., 1995; Wolthers et al., 1996). This is consistent neurological disorders, cancer, obesity, and T2D, are character- with the decreased energy expenditure seen in HFF LTKO mice; ized by oxidative stress. Thus the results of this study may have although corrected by Stat-5 heterozygosity, it remains to be wide-ranging implications highlighting the potential for oxidative determined if the effects on energy expenditure were mediated stress and PTP oxidation to alter cellular signaling in diverse by the IGF-1/GH axis. The underlying cause(s) of the lower GH pathological contexts. levels in human obesity is/are contentious and include the sup- pression of GH secretion by elevated plasma insulin (Steyn EXPERIMENTAL PROCEDURES et al., 2013) and IGF-1 (Romero et al., 2010). Total plasma IGF- 1 is not reduced to the degree predicted by the low GH levels, Mice We maintained mice on a 12 hr light-dark cycle in a temperature-controlled and circulating IGF-1 is in the normal-high range in obese hu- high-barrier facility with free access to food and water. Age- and sex-matched mans (Frystyk et al., 1999; Nam et al., 1997). The association be- mice were used for experiments. Ptpn2lox/lox (C57BL/6) described previously tween IGF-1 and GH is especially evident in adolescent obesity, (Loh et al., 2012; Wiede et al., 2011) were mated with Alb-Cre (C57BL/6) where physiological insulin resistance and elevated circulating mice (JAX) for the postnatal deletion of Ptpn2 in hepatocytes. STAT-5lox/lox

Figure 5. Hepatic TCPTP Deletion Exacerbates Insulin-Induced STAT-5 Signaling (A) Seven-week-old male lox/lox and LTKO mice were HFF for 12 weeks. Livers from 4 hr fasted mice were processed for immunoblotting. (B) Hepatocytes were serum-starved and stimulated with either 50 U/ml INF-g, 100 U/ml IL-6, or 100 nM insulin and processed for immunoblotting. (C) Seven-week-old male lox/+ and LTKO HET mice were HFF for 20 weeks. Livers from fasted mice were processed for immunoblotting. (D) Livers from 7-week-old fasted male lox/lox and LTKO mice were processed for immunoblotting. (E) Seven-week-old male lox/lox and LTKO mice were HFF for 12 weeks, fasted, and injected with PBS or insulin, and livers were processed for immunoblotting. Representative and quantified (means ± SEM) results are shown.

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Figure 7. JI-38 Corrects the Obesity in LTKO Mice (A and B) Seven-week-old lox/lox, lox/lox;STAT-5 lox/+, LTKO, and LTKO;Stat-5lox/+ mice were HFF for 12 weeks, then fasted overnight, and serum IGF-1 and GH levels (at 8 a.m.) were measured. (C–E) Seven-week-old female lox/lox and LTKO mice were HFF for 24 days, administered vehicle or the synthetic GH releasing hormone agonist JI-38 (50 mg/kg in 100 ml) subcutaneously twice daily (8 a.m. and 6 p.m.) for 20 days, and body weights were recorded. Serum GH levels (8 a.m.; 30 min after JI-38) and body composition were deter- mined. Results are means ± SEM; significance in (A) and (B) (* LTKO and lox/lox, $ LTKO and lox/ lox;Stat-5lox/+, # LTKO and LTKO;Stat-5lox/+) and (C) and (D) (* LTKO and lox/lox, # LTKO + JI-38 and lox/lox) were determined using one-way ANOVA.

free BSA overnight and then serum starved and stimulated as indicated. Ptpn2 or Jak-2 was knocked down transiently in primary murine hepa- tocytes using Ptpn2- or Jak2-specific siRNAs; enhanced GFP siRNA was used as a control.

ROS Determinations

H2O2 production in live hepatocytes was deter- mined using the Amplex Red hydrogen peroxide assay (Invitrogen). Total (GSH) and oxidized (GSSG) glutathione levels were measured using a BIOXYTECH GSH/GSSG-412 assay kit (Oxis International).

PTP Oxidation Total (reversible and irreversible) PTP oxidation was assessed essentially as described previously (Karisch et al., 2011) with some modifications. Briefly, frozen liver tissue or freshly isolated hepa- tocytes were homogenized under anaerobic condi- tions in degassed, ice-cold PTPox lysis buffer (C57BL/6) mice described previously (Cui et al., 2007) were mated with containing 10 mM N-ethylmaleimide to prevent postlysis oxidation and to Alb-Cre;Ptpn2lox/lox mice. Gpx1–/– (C57BL6) mice have been described previ- alkylate all reduced and active PTPs and incubated for 1 hr at 4C. Cell ously (Loh et al., 2009). Mice were fed a standard chow or a high-fat diet (23% lysates and liver homogenates were clarified by centrifugation at 16,000 g fat) as indicated. Experiments were approved by the Monash University and 50,000 g, respectively, for 20 min, and the buffer was exchanged School of Biomedical Sciences Animal Ethics Committee. (NAP-5 columns, GE Healthcare) with 20 mM HEPES containing 10 mM DTT to reduce oxidized PTPs. Reduced PTPs were then hyperoxidized to their sul-

Metabolic Measures fonic (ÀSO3H) state by exchanging the buffer to 20 mM HEPES containing Insulin and glucose tolerance tests and hyperinsulinemic-euglycemic clamps 100 mM pervanadate, resolved by SDS-PAGE, and immunoblotted with PTPox were performed as described previously (Loh et al., 2009). Metabolic mea- antibody. Alternatively, pervanadate-treated samples were made to 9 M urea sures were undertaken in an environmentally controlled Comprehensive Lab and 4.5 mM DTT, incubated at 60C for 30 min, and then treated with 10 mM Animal Monitoring System (Columbus Instruments) and body composition iodoacetamide before being diluted to a final concentration of 2 M urea for was measured by DEXA (Lunar PIXImus2; GE Healthcare). digestion with TPCK-trypsin (Thermo Scientific) and processing for PTPox immunoprecipitation and analysis by mass spectrometry as described previ- Cell Culture and RNA Interference ously (Karisch et al., 2011). Hepatocytes were isolated by a two-step collagenase A (0.05% w/v; Roche Di- agnostics) perfusion method as described previously (Fukushima et al., 2010). Statistical Analysis Where indicated, cells were serum starved in M199 medium for 2–4 hr and then Statistical significance was determined by a two-tailed paired Student’s t test stimulated with bovine insulin, GH, IFN-g, or IL-6. Alternatively, cells were or, where indicated, ANOVA with Bonferroni correction. p values < 0.05 were treated with 0.5 mM sodium palmitate in the presence of 1% w/v fatty acid- considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6. Liver-Specific Stat-5 Heterozygosity Corrects Obesity but Not Steatosis (A–F) Seven-week-old male lox/lox, Ptpn2lox/lox;Stat-5lox/+ (lox/lox;STAT-5 lox/+), LTKO, and Alb-Cre;Ptpn2lox/lox;Stat-5lox/+ (LTKO;Stat-5lox/+) mice were HFF for 12 weeks and body and tissue weights, body composition, and energy expenditure were determined (A); fed and fasted blood glucose and plasma insulin were measured (B and C); and livers were extracted for immunoblotting (D), histology (E), or lipid analyses (F). Results shown are means ± SEM; significance (* LTKO and lox/lox, $ LTKO and lox/lox;STAT-5 lox/+, # LTKO and LTKO;Stat-5lox/+) was determined using a one-way ANOVA (A–C) or a two-tailed Student’s t test (F).

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SUPPLEMENTAL INFORMATION expression of glucokinase and lipogenesis-related genes. Proc. Natl. Acad. Sci. USA 96, 12737–12742. Supplemental Information includes Supplemental Experimental Procedures Frystyk, J., Skjaerbaek, C., Vestbo, E., Fisker, S., and Orskov, H. (1999). and seven figures and can be found with this article online at http://dx.doi. Circulating levels of free insulin-like growth factors in obese subjects: the org/10.1016/j.cmet.2014.05.011. impact of type 2 diabetes. Diabetes Metab. Res. Rev. 15, 314–322. Fukushima, A., Loh, K., Galic, S., Fam, B., Shields, B., Wiede, F., Tremblay, ACKNOWLEDGMENTS M.L., Watt, M.J., Andrikopoulos, S., and Tiganis, T. (2010). T-cell protein tyro- sine phosphatase attenuates STAT3 and insulin signaling in the liver to regulate We thank A. Ziegler, T. Butt, Teresa Tiganis, and C. Nelson for technical assis- gluconeogenesis. Diabetes 59, 1906–1914. tance. The work was supported by NHMRC Australia (T.T., M.J. Waters, M.J. Furukawa, S., Fujita, T., Shimabukuro, M., Iwaki, M., Yamada, Y., Nakajima, Y., Watt, and S.A.), AICR (T.T.; 12-1172), NIH (B.G.N.; R37 CA49152), the Ontario Nakayama, O., Makishima, M., Matsuda, M., and Shimomura, I. (2004). Ministry of Health and Long Term Care, and the Princess Margaret Cancer Increased oxidative stress in obesity and its impact on metabolic syndrome. Foundation (B.G.N.). B.G.N. is a Canada Research Chair, Tier 1. T.T., J. Clin. Invest. 114, 1752–1761. M.J. Waters, M.J. Watt, and S.A. are NHMRC Fellows. Haas, J.T., Miao, J., Chanda, D., Wang, Y., Zhao, E., Haas, M.E., Hirschey, M., Received: December 12, 2013 Vaitheesvaran, B., Farese, R.V., Jr., Kurland, I.J., et al. (2012). Hepatic insulin Revised: March 23, 2014 signaling is required for obesity-dependent expression of SREBP-1c mRNA Accepted: April 28, 2014 but not for feeding-dependent expression. Cell Metab. 15, 873–884. 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