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T-Cell Protein Tyrosine Phosphatase Attenuates STAT3 and Insulin

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T-Cell Protein Tyrosine Phosphatase Attenuates STAT3 and Insulin ORIGINAL ARTICLE T-Cell Protein Tyrosine Phosphatase Attenuates STAT3 and Insulin Signaling in the Liver to Regulate Gluconeogenesis Atsushi Fukushima,1 Kim Loh,1 Sandra Galic,1 Barbara Fam,2 Ben Shields,1 Florian Wiede,1 Michel L. Tremblay,3 Matthew J. Watt,4 Sofianos Andrikopoulos,2 and Tony Tiganis1 OBJECTIVE—Insulin-induced phosphatidylinositol 3-kinase (PI3K)/Akt signaling and interleukin-6 (IL-6)-instigated JAK/ STAT3-signaling pathways in the liver inhibit the expression of ype 2 diabetes has reached epidemic propor- gluconeogenic genes to decrease hepatic glucose output. The tions, afflicting roughly 170 million people world- insulin receptor (IR) and JAK1 tyrosine kinases and STAT3 can wide. Although the underlying genetic causes serve as direct substrates for the T-cell protein tyrosine phos- Tand the associated pathologic symptoms are phatase (TCPTP). Homozygous TCPTP-deficiency results in peri- heterogenous, a common feature is high blood glucose due natal lethality prohibiting any informative assessment of to peripheral insulin resistance. Circulating insulin re- TCPTP’s role in glucose homeostasis. Here we have used leased from ␤-cells in the pancreas serves to lower blood Ptpn2ϩ/Ϫ mice to investigate TCPTP’s function in glucose glucose by triggering the translocation of the facilitative homeostasis. GLUT4 to the plasma membrane in muscle and adipose RESEARCH DESIGN AND METHODS—We analyzed insulin tissue (1). Insulin also acts in the liver to promote glycogen sensitivity and gluconeogenesis in chow versus high-fat–fed synthesis and lipogenesis and to suppress hepatic glucose (HFF) Ptpn2ϩ/Ϫ and Ptpn2ϩ/ϩ mice and insulin and IL-6 production (HGP) by inhibiting gluconeogenesis and gly- signaling and gluconeogenic gene expression in Ptpn2ϩ/Ϫ and cogenolysis (1). Elevated HGP caused by defective sup- Ptpn2ϩ/ϩ hepatocytes. pression of gluconeogenesis is one of the primary defects ϩ Ϫ contributing to fasting hyperglycemia in patients with type RESULTS—HFF Ptpn2 / mice exhibited lower fasted blood 2 diabetes (2–4). glucose and decreased hepatic glucose output as determined in Glucose-6-phosphatase (G6Pase; encoded by G6pc) and hyperinsulinemic euglycemic clamps and by the decreased blood glucose levels in pyruvate tolerance tests. The reduced hepatic phosphoenolpyruvate carboxykinase (PEPCK; encoded by glucose output coincided with decreased expression of the Pck1) are key enzymes involved in the rate-limiting steps gluconeogenic genes G6pc and Pck1 and enhanced hepatic of gluconeogenesis (1). The overexpression of PEPCK or STAT3 phosphorylation and PI3K/Akt signaling in the fasted G6Pase in rodent models results in hyperinsulinaemia, state. Insulin-induced IR-␤–subunit Y1162/Y1163 phosphoryla- insulin resistance, and glucose intolerance (5–7), and in at tion and PI3K/Akt signaling and IL-6–induced STAT3 phosphor- least one instance, PEPCK overexpression has been ylation were also enhanced in isolated Ptpn2ϩ/Ϫ hepatocytes. shown to promote weight gain (8). PEPCK catalyzes the The increased insulin and IL-6 signaling resulted in enhanced conversion of oxaloacetate to phosphoenolpyruvate, suppression of G6pc and Pck1 mRNA. whereas G6Pase catalyzes the dephosphorylation of glu- cose 6-phosphate to free glucose, the final step of both CONCLUSIONS—Liver TCPTP antagonises both insulin and gluconeogenesis and glycogenolysis. The expression of STAT3 signaling pathways to regulate gluconeogenic gene ex- these key gluconeogenic enzymes is controlled by signal- pression and hepatic glucose output. Diabetes 59:1906–1914, 2010 ing pathways that are activated by insulin, glucagon, and IL-6. Although insulin and IL-6 suppress G6pc and Pck1 expression, glucagon stimulates their expression (1,9–11). Insulin exerts its effects via the PI3K/Akt pathway. Insulin binds to its cell surface receptor to stimulate intrinsic protein tyrosine kinase (PTK) activity, resulting in the phosphorylation of the insulin receptor (IR) and several IR From the 1Department of Biochemistry and Molecular Biology, Monash substrates (IRS), such as IRS-1. IRS-1 tyrosine phosphor- University, Victoria, Australia; the 2Department of Medicine, Heidelberg ylation allows for the recruitment of PI3K, which catalyzes Repatriation Hospital, The University of Melbourne, Victoria, Australia; the formation of lipid phosphatidylinositol-3,4,5-triphos- 3Goodman Cancer Centre and the Department of Biochemistry, McGill University, Montreal, Quebec, Canada; and the 4Department of Physiology, phate (PIP3) at the plasma membrane (1). Increases in Monash University, Victoria, Australia. PIP3 activate several Ser/Thr protein kinases, including Corresponding author: Tony Tiganis, [email protected]. Akt, which phosphorylates and prevents the translocation Received 14 September 2009 and accepted 4 May 2010. Published ahead of print at http://diabetes.diabetesjournals.org on 18 May 2010. DOI: 10.2337/ of the transcription factor Foxo1a to the nucleus, where it db09-1365. otherwise functions in concert with peroxisome prolifera- Current address for S.G. is St Vincent’s Institute, Victoria 3065, Australia. tor-activated receptor g coactivator 1a (PGC1a) to in- © 2010 by the American Diabetes Association. Readers may use this article as crease the transcription of the gluconeogenic genes G6pc long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by and Pck1 (1,12,13). Several studies have also implicated -nc-nd/3.0/ for details. signal transducer and activator of transcription-3 (STAT3) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance in the PGC1a-independent suppression of hepatic glu- with 18 U.S.C. Section 1734 solely to indicate this fact. coneogenic gene expression (11,14). In particular, hypo- 1906 DIABETES, VOL. 59, AUGUST 2010 diabetes.diabetesjournals.org A. FUKUSHIMA AND ASSOCIATES thalamic control of hepatic IL-6 generation and JAK Mice. Mice were maintained on a 12-h light-dark cycle with free access to food (Janus activated kinase)/STAT3 signaling has emerged and water. Age- and sex-matched mice were used for all experiments. Ptpn2ϩ/Ϫ mice on a 129sv x BALB/c mixed background (32) were back- as an important mechanism for the regulation of HGP crossed onto BALB/c background for six generations and genotyped as (11,15–17). described previously (32). Mice were fed a standard chow diet (19% protein, Several protein tyrosine phosphatases (PTPs) have been 4.6% fat, and 4.8% crude fiber; Specialty Feeds, Australia) or a high-fat diet implicated in the modulation of glucose homeostasis in (19% protein, 60% fat, and 4.7% crude fiber; Specialty Feeds, Australia) as vivo, including the prototypic protein tyrosine phospha- indicated. tase 1B (PTP1B) (18–22). PTP1B dephosphorylates the IR Metabolic measurements. Insulin tolerance tests and pyruvate or glucose PTK in liver and muscle to regulate glucose homeostasis tolerance tests were performed on 4- and 6-h fasted mice, respectively, by injecting human insulin (0.75–1.5 mU/g body weight), D-glucose (1–2 mg/g (18,19,21,22). PTP1B also dephosphorylates and inacti- body weight), or pyruvate (1–2 mg/g body weight) intraperitoneally and vates the JAK2 PTK in the hypothalamus to antagonize measuring glucose in tail blood as described previously (34). Euglycemic leptin-induced JAK2/STAT3 signaling and thus leptin’s hyperinsulinemic clamps were performed on overnight-fasted and anesthe- effects on body mass and peripheral insulin sensitivity tized mice as described previously (34). Fed and fasted blood glucose and (20,23). PTP1B dephosphorylates the IR-␤ subunit Y1162/ corresponding plasma insulin levels were determined as described previ- ously (34). Y1163 autophosphorylation site, which is necessary for IR Cell culture. The generation and culture conditions of control HeLa cells and activation, as well as the Y972 site that contributes to IRS-1 those expressing TCPTP-specific shRNA have been described previously (31). recruitment (24). Muscle or liver-specific PTP1B knockout Hepatocytes from 8- to 12-week-old Ptpn2Ϫ/Ϫ and Ptpn2ϩ/ϩ mice were mice exhibit increased insulin-induced IR Y1162/Y1163 isolated by a two-step collagenase A (0.05% wt/vol; Roche Diagnostics, phosphorylation and PI3K/Akt signaling and concomitant Germany) perfusion as described previously (34). Hepatocytes were cultured improved glucose tolerance associated with enhanced in M199 medium (Invitrogen, Carlsbad, CA) containing 10% (vol/vol) heat- inactivated FBS, 100 units/ml penicillin, 100 ␮g/ml streptomycin, 10 nmol/l glucose uptake and decreased HGP respectively (21,22). dexamethasone, 50 nmol/l insulin and 20 ng/ml EGF (R&D Systems, Minne- The T-cell protein tyrosine phosphatase (TCPTP) (en- apolis, MN) for no more than 3 days. Cells were starved in M199 medium alone coded by Ptpn2) is a ubiquitous tyrosine-specific phospha- for 4 h, and then stimulated with 10 nmol/l insulin or 1 ng/ml IL-6, as indicated. tase (25). The catalytic domains of PTP1B and TCPTP Biochemical analyses. Tissues were mechanically homogenized in ice cold share a high degree of primary (72% identity) and tertiary RIPA lysis buffer (50 mmol/l Hepes [pH 7.4], 1% (vol/vol) Triton X-100, 1% structure similarity and have similar active sites. In partic- (vol/vol) sodium deoxycholate, 0.1% (vol/vol) SDS, 150 mmol/l NaCl, 10% (vol/vol) glycerol, 1.5 mmol/l MgCl2, 1 mmol/l EGTA, 50 mmol/l sodium ular, both PTPs share a second “phosphotyrosine-binding fluoride, leupeptin (5 ␮g/ml), pepstatin A (1 ␮g/ml), 1 mmol/l benzamadine, 2 pocket” that allows for the selective recognition of mmol/l phenylmethysulfonyl fluoride, 1 mmol/l sodium vanadate) and clarified tandem tyrosyl phosphorylated substrates (26,27), such by centrifugation (100,000g for 20 min at 4°C). Tissue and cell lysates were as the IR (24,26,28) and JAK PTKs (JAK1–3 and TYK2) resolved by SDS-PAGE and immunoblotted. Lipid analyses were performed as (29). Despite their similarity, TCPTP and PTP1B exhibit a described previously (34).
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