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ORIGINAL ARTICLE Tub Has a Key Role in and Signaling and Action In Vivo in Hypothalamic Nuclei Patrícia O. Prada, Paula G.F. Quaresma, Andrea M. Caricilli, Andressa C. Santos, Dioze Guadagnini, Joseane Morari, Laís Weissmann, Eduardo R. Ropelle, José Barreto C. Carvalheira, Lício A. Velloso, and Mario J.A. Saad

Mutation of tub in mice induces obesity, suggesting that tub Tub have been evaluated in only a few studies. could be an important regulator of energy balance. In the current Boggon et al. (22) suggested that the highly conserved study, we investigated whether insulin, leptin, and obesity can COOH-terminal portion of the protein is a DNA-binding modulate Tub in vivo in hypothalamic nuclei, and we investigated structure and that the NH2-terminal portion may be a reg- possible consequences on energy balance, expres- ulator of . They observed that on serotonin sion, and hepatic . Food intake, metabolic activation of 5-HT2c receptors, there was a translocation characteristics, signaling , and neuropeptide expression of Tub protein from the plasma membrane to the cellular were measured in response to fasting and refeeding, intracere- nucleus of primary cultured neurons, suggesting that Tub broventricular insulin and leptin, and Tub antisense oligonu- cleotide (ASO). Tub tyrosine phosphorylation (Tub-p-tyr) is may function as a transcription factor (22). modulated by nutritional status. Tub is a substrate of insulin re- In addition to being a transcription factor, in vitro ceptor (IRTK) and leptin (LEPR)–Janus studies suggested that Tub might act as an adaptor 2 (JAK2) in hypothalamic nuclei. After leptin or insulin downstream of the insulin receptor-signaling pathway stimulation, Tub translocates to the nucleus. Inhibition of Tub (23). Moreover, it was also demonstrated that insulin in- expression in hypothalamus by ASO increased food intake, duced Tub tyrosine phosphorylation in 3T3- adipocytes fasting blood glucose, and hepatic glucose output, decreased (24). Together, these studies suggest that Tub is a sub- O2 consumption, and blunted the effect of insulin or leptin strate of the insulin receptor, at least in vitro. However, the on , thyroid-releasing hormone, melanin- role of Tub in insulin action and signaling in vivo has not concentrating hormone, and expression. In hypothalamus fi of mice administered a high-fat diet, there is a reduction in leptin yet been clari ed. and insulin-induced Tub-p-tyr and nuclear translocation, which is In the hypothalamus, neuron populations of the arcuate reversed by reducing protein tyrosine phosphatase 1B expres- nucleus (Arc), ventromedial hypothalamus, paraventricular sion. These results indicate that Tub has a key role in the control nucleus (PVN), and lateral hypothalamus (LH) are re- of insulin and leptin effects on food intake, and the modulation of sponsive to insulin and leptin. Insulin acts through the Tub may contribute to insulin and leptin resistance in DIO mice. insulin receptor (IR), which is a protein with endogenous 62:137–148, 2013 tyrosine kinase activity, and in the hypothalamus insulin signals through IRS/PI3k/Akt/Foxo1 to control food intake (1–3,25–30). Leptin signals through the recruitment of the JAK2 to eptin and insulin signal adiposity to the hypo- the (LEPR), where it phosphorylates sev- thalamus and inhibit feeding, in part by reducing eral tyrosine residues on the LEPR. Signal transducer and the expression of orexigenic and activator of transcription (STAT) 3 proteins bind to the increasing the expression of anorexigenic neuro- L – phosphorylated LEPR and are phosphorylated by JAK2. RETRACTEDpeptides (1 5). However, the network of signaling proteins Phosphorylated ARTICLE STAT3 is translocated to the nucleus, induced by these hormones is not completely understood. where it is thought to bind to specific DNA sequences to The protein Tub is primarily expressed in the central regulate neuropeptide expression. Both leptin and insulin nervous system and has been implicated in energy balance change the membrane potential of target neurons to con- homeostasis (6–14). Mice with a spontaneous mutation of fi tub “ ” trol their ring rate, as well as neuropeptide and neuro- the gene were called tubby mice and had development transmitter release. In parallel, leptin and insulin signaling of late-onset obesity, , and sensory im- directly control transcription of neuropeptides in multiple pairments (15–20). tub fi hypothalamic nuclei. In the Arc, leptin and insulin increase Inactivation of the gene by gene targeting is suf - the transcription of proopiomelanocortin (POMC), which cient to induce the same phenotype as tubby mice; this is an anorexigenic neuropeptide (27,31–33) and inhibit suggests that the loss of Tub protein function is re- transcription of agouti-related (AgRP) and neuro- sponsible for the tubby mice phenotype (21). Functions of peptide Y (NPY), which are orexigenic neuropeptides (29). In the PVN, insulin and leptin induce an increase in thyroid- From the Department of Internal Medicine, State University of Campinas, releasing hormone (TRH) expression, which is a positive Campinas, Brazil. regulator of energy expenditure (31). In the ventromedial Corresponding author: Mario J.A. Saad, [email protected]. hypothalamus, steroidogenic factor 1–expressing neurons Received 4 October 2011 and accepted 7 May 2012. respond to insulin and leptin to regulate food intake DOI: 10.2337/db11-1388 This article contains Supplementary Data online at http://diabetes (34,35) and, in the LH, both hormones decrease the release .diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1388/-/DC1. of melanin-concentrating hormone (MCH) and orexin Ó 2013 by the American Diabetes Association. Readers may use this article as neuropeptides (36). 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 However, it has not yet been investigated whether, in the -nc-nd/3.0/ for details. hypothalamus, insulin and leptin can modulate Tub

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phosphorylation and nuclear translocation, or whether this A pool of 20 hypothalamic nuclei or a pool of four hypothalami were used for control might influence the expression of neuropeptides in plasma membrane and nucleus separation as described previously (43,44). The physiologic conditions and also in obesity. In the current procedure was performed at 4°C. Plasma membrane and nucleus fractions were analyzed by IP and immunoblotting with anti-Tub (M-19) antibody. study, we investigated whether insulin, leptin, and obesity RNA extraction and real-time PCR. Random fed or fasted (24 h) mice can modulate Tub in vivo in hypothalamic nuclei, and we treated for 5 days with Tub ASO or sense received ICV insulin or leptin in- investigated possible consequences on energy balance, jection, and the hypothalami or hypothalamic nuclei were harvested after 6 h, neuropeptide expression, and hepatic glucose metabolism. quickly frozen in liquid nitrogen, and stored at 280°C for processing. Total RNA was obtained using RNeasy Mini Kit (Cat. 74106; Qiagen). Real-time PCR was performed using TaqMan RT-PCR Master Mix (Applied Biosystems) in an RESEARCH DESIGN AND METHODS Mx3000P thermocycler (Stratagene). The Mx3000P software was used to All experiments were approved by the Ethics Committee of the State University calculate the cycle threshold for each reaction. Relative expression lev- of Campinas. Eight-week-old male C57BL/6 mice obtained from the University els were determined using the comparative cycle threshold method with of Campinas, São Paulo, Brazil, were assigned to receive a standard rodent normalization of target levels to 18s. Primers and probes chow or a high-fat diet (HFD) as previously described (37,38) and water ad were purchased from Applied Biosystems and were described as TRH, libitum. For fasting and refeeding experiments, fasted (24 h) mice were Mm01182425_g1; NPY, Mm00445771_m1; AgRP, Mm00475829_g1; POMC, allowed to refeed for 1 h or 2 h, and hypothalamic nuclei were dissected for Mm00435874_m1; MCH, Mm01242886_g1; and Ore, Mm01964031_s1 for protein studies, as described. All feeding tests were conducted between 8:00 mouse. The polymerase chain reaction conditions were 2 min at 50°C, 10 min A.M. and 10:00 A.M. at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. Real-time data Intracerebroventricular cannulation. Anesthetized mice were stereotaxi- were analyzed using the engine provided by Applied Biosystems. cally instrumented (Ultra Precise model 963; Kopf) to implant stainless steel Protein tyrosine phosphatase activity assay in immunoprecipitated Tub. cannulas (26-gauge; Plastics One) in the right lateral ventricle. The coordinates Protein tyrosine phosphatase (PTPase) activity was measured as previously used from the bregma were: anterior/posterior, 20.5 mm; lateral, 21.3 mm; described (45,46). and dorso/ventral, 22.2 mm. Mice were single-housed after surgery and were Statistical analysis. Results are expressed as means 6 SD. Significance was allowed to recover for 5–7 days. The correct implantation of cannulas was determined using two-tailed Student t test or one-way or two-way ANOVA checked by 10 ng angiotensin II intracerebroventricular (ICV) injection, which with Bonferroni posttest, as appropriate, and differences were considered elicits an intake of water (39). Animals that did not reach this criterion were significant if P , 0.05. We used GraphPad Prism (GraphPad Software, excluded from the experiments. San Diego, CA). Hypothalamic nuclei dissection. Arc, medial hypothalamus (MH; ventro- medial and dorsomedial), PVN, and LH were quickly dissected in a stainless steel matrix with razor blades as described previously (40) and frozen in liquid RESULTS nitrogen for further protein studies. ICV injections. To determine if insulin or leptin induces Tub tyrosine phos- Tub protein is expressed in hypothalamic nuclei and phorylation in vivo, overnight fasted mice fed chow or HFD received an ICV is regulated by fasting and refeeding. Previous studies injection of insulin (human recombinant insulin; Eli Lilly, Indianapolis, IN) or have shown that Tub is expressed in the central nervous recombinant leptin (Calbiochem, San Diego, CA), and hypothalamic nuclei system (13). Here, we confirmed that Tub was expressed were quickly dissected and frozen in liquid nitrogen for further protein studies. in multiple hypothalamic nuclei (Fig. 1A). The time and dose responses to ICV insulin or leptin on Tub tyrosine phos- The Tub protein abundance was not different in hypo- phorylation were also investigated. To inhibit Tub (59AGG AAC ACC TTC TTG CCA T 39) or protein tyrosine phosphatase 1B (PTP1B) (59CCC AGC AGC thalamic nuclei after fasting and refeeding states. How- GGC TTC TGC AT 39) expression in the hypothalamus, we developed anti- ever, Tub-p-tyr was strongly induced after 1 h of refeeding, sense oligonucleotide (ASO) and administered them by ICV injections in mice followed by a decrease after 2 h of refeeding in Arc, MH, fed chow or fed HFD, respectively. Both ICV ASO treatments were adminis- PVN, and LH in control mice (Fig. 1B). tered twice per day (8:00 A.M. and 5:00 P.M.) for 5 days. Body weight, epidid- Insulin and leptin induce Tub tyrosine phosphorylation ymal fat mass, cumulative food intake, 4-h to 8-h food intake in response to in hypothalamic nuclei in a time-dependent and dose- ICV insulin and leptin, fasting blood glucose and blood glucose levels in re- p sponse to pyruvate (pyruvate test) (41), and PEPCK expression in fasting dependent manner. Insulin-induced Tub- -tyr was dose- were measured in Tub ASO-treated mice. We also determined O2 con- dependent. The presence of phosphorylated Tub was sumption and respiratory exchange rate (38). Akt, FoxO1, JAK2, and STAT3 detectable after ICV injection of as little as 0.02 mg insulin, phosphorylation in response to ICV insulin or leptin, respectively, were eval- half-maximal stimulation occurred with 0.2 mg of the hor- uated as described. mone (Fig. 1C), and maximal stimulation was observed with fi RETRACTEDImmunoprecipitation and immunoblotting. A pool of ve to eight of each 2.0 mg insulin ARTICLE (n = 3). Insulin-induced Tub-p-tyr occurred nucleus (Arc, MH, PVN, LH) was homogenized in extraction buffer. Immu- noprecipitation (IP) and immunoblotting were performed as previously de- after 5 min of ICV insulin injection, and the maximal scribed (37–39) using the following antibodies: antiphosphotyrosine, anti-IRb phosphorylation was observed at 10 min (Fig. 1D). The (a-IR), anti-Tub (M-19), anti-b actin, anti-JAK2, anti-Akt, anti-STAT3, anti- presence of phosphorylated Tub was detectable after ICV FoxO1, anti-PTP1B, and anti-PEPCK (H-300) antibodies from Santa Cruz leptin injection of as little as 0.1 ng leptin, and the maximal Technology (Santa Cruz, CA); and antipAkt Ser473, antipSTAT3, antipJAK2, stimulation was observed with 10.0 ng leptin (Fig. 1E). and antipFoxO1 antibodies from . Leptin-induced Tub-p-tyr occurred after 2 min of ICV leptin Coimmunoprecipitation and tyrosine kinase assay using Tub as a substrate. The protocol described was adapted for ICV injections (42). injection, and the maximal phosphorylation was observed Briefly, a low dose of insulin (0.2 mg) or leptin (0.1 ng) was injected into the at 7 min (Fig. 1F). lateral ventricle to trigger IR and JAK2 . After 7–10 min, Next, we compared the maximal effect of insulin and Arc, MH, PVN, and LH were dissected and homogenized in extraction buffer leptin in Arc, MH, PVN, and LH hypothalamic nuclei, and (38) for IR or JAK2 IP. In another group of animals, Arc, MH, PVN, and LH the results showed that in all these nuclei insulin or leptin were dissected without insulin or leptin stimulation and samples underwent IP induced similar increases in Tub-p-tyr (Fig. 1G). with anti-Tub antibody. Samples were collected on protein A-Sepharose and combined as followed: IP IR plus IP Tub and IP JAK2 plus IP Tub. Then, the Tyrosine kinase assay of IR and JAK2 using Tub as kinase activity was performed as previously described (42). a substrate. The IR kinase activity was increased signifi- Translocation of Tub from plasma membrane to the nucleus in cantly in Arc lysates after ICV insulin in vivo and there was hypothalamic nuclei. Fasted animals received an ICV injection of insulin or a further increase after ATP addition in vitro in mice fed leptin or acetylcholine (Ach) (100 mmol/L; Sigma-Aldrich, St. Louis, MO). The chow. Most importantly, Tub-p-tyr was increased in Arc of fi b b m speci c phospholipase C (PLC ) inhibitor U73122 (1 mol/L; Sigma-Aldrich) mice injected with a low dose of insulin, with a further was previously injected to block Tub nuclear translocation induced by the hormones. The U73343 (1 mmol/L; Sigma-Aldrich), which weakly inhibits PLCb, increase after ATP addition in vitro, demonstrating en- was injected as a negative control. Hypothalamic nuclei or whole hypothalamus hanced IR kinase activity with Tub. These results were were quickly dissected and frozen in liquid nitrogen for further processing. also demonstrated in MH, PVN, and LH (Fig. 2A).

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FIG. 1. Expression of Tub, effect of nutritional states, and effect of insulin and leptin on Tub-p-tyr in hypothalamic nuclei. A: Representative blots of Tub protein expression in hypothalamic nuclei, including Arc, MH, PVN, LH, white adipose tissue (WAT), and . B: Effect of fasting (24 h) and refeeding on Tub-p-tyr in hypothalamic nuclei. Mice were killed at 10 min (C)orat7min(E) after hormone injections to determine the dose- response of ICV insulin or leptin on Tub-p-tyr. The different doses of insulin were: 0, 0.02, 0.2, and 2.0 mg; the different doses of leptin were: 0, 0.1, 1.0, 10, and 100 ng. To determine the time-course of Tub-p-tyr in response to insulin (D) or leptin (F), we used 2-mg(D) and 10-ng (F) doses, and mice were killed at different time points: 5, 10, and 15 min for insulin or 2, 5, 7, 10, 15, and 20 min for leptin. G: Tub-p-tyr is increased in response to ICV insulin (2 mg) or leptin (10 ng) in hypothalamic nuclei. Data are presented as means 6 SD from 8 mice per nucleus or whole hypothalamus from 8 to 10 mice. One-way ANOVA with Bonferroni posttest was used. *P < 0.05 vs. other groups, †P < 0.05 vs. fast. a-PY, antiphosphotyrosine; ICV, ICV injection; IB, immunoblotting.

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FIG. 2. Tyrosine kinase assay of IR and JAK2 using Tub as a substrate. A:0.2mg insulin or (B) 0.1 ng leptin was injected into lateral ventricle to increase IR or LEPR-associated JAK2 phosphorylation, respectively. Hypothalamic nuclei were dissected, and IP was performed with anti-IR antibody for insulin-stimulated mice (n = 7) or anti-JAK2 antibody for leptin-stimulated mice (n = 5). In another group of animals (n = 7), hy- pothalamic nuclei were dissected without insulin or leptin stimulation, and samples underwent IP with anti-Tub antibody. After combining IP samples (IR with Tub) for insulin-stimulated mice or (JAK2 with Tub) for leptin-stimulated mice, they were incubated with ATP and blotted with antiphosphotyrosine (a-PY). Representative blots show insulin-induced IR and Tub-p-tyr–induced and leptin-induced JAK2 and Tub-p-tyr in Arc, MH, PVN, and LH of mice fed chow. ICV, ICV injection.

Similarly to insulin, JAK2 kinase activity was increased blotted 30 mgofproteinfromtheplasmamembraneand significantly in Arc lysates in response to leptin injection, nucleus with anti-IR and anti-TATA-box antibodies, re- as demonstrated by an increase in JAK2 autophosphor- spectively, as controls for these preparations (data not RETRACTEDylation. The addition of Tub to the reaction also demon- shown). ARTICLE strated that after leptin injection, Tub was a substrate of Previous data showed that Ach stimulates translocation JAK2 tyrosine kinase in this nucleus. These results were of Tub in Neuro-2A cells, and this involves PLCb, which is also demonstrated in MH, PVN, and LH (Fig. 2B). We blocked by a specific PLCb inhibitor (U73122). We in- reprobed the membranes and confirmed that the bands vestigated whether Ach is able to induce Tub translocation blotted with antiphosphotyrosine antibody were IR, JAK2, in vivo in the hypothalamus of mice fed chow. We injected and Tub (Fig. 2A and B). Ach (100 mmol/L) alone or U73122 before Ach ICV injection, ICV insulin or leptin stimulation induces the nuclear or U73343, which weakly inhibits PLCb,asanegative translocation of Tub in hypothalamic nuclei. Previous control. We observed that Ach induced Tub nuclear trans- studies suggest that Tub acts as a transcription factor that location, an effect that was blocked by U73122, but not by can translocate from the plasma membrane to the nu- U73343 (Fig. 3E–G). To investigate whether insulin- or cleus after G-protein activation (47). Here, we observed leptin-induced Tub translocation also involved PLCb,we that ICV insulin induced the nuclear translocation of Tub injected insulin or leptin in mice previously treated with after 15 min in Arc, MH, and LH, and this translocation ICV U73122. The results showed that U73122 blocked was maximal after 30 min in these nuclei. In PVN, insulin- insulin-induced and leptin-induced Tub translocation in induced Tub translocation was delayed compared with vivo (Fig. 3H and I). It is interesting that ICV Ach altered other nuclei and was evident at 90 min after the hormone TRH expression in the hypothalamus. Further, insulin or injection. Leptin also was able to induce Tub nuclear leptin induced an increase in POMC and TRH and a re- translocationinArc,MH,LH,andinPVN(Fig.3A–D). In duction in orexin or MCH expression, respectively. The Arc, MH, and LH, leptin-induced Tub nuclear trans- ICV U73122 injection before Ach or insulin or leptin location was observed at 7 min, but in PVN the maxi- blunted these effects on neuropeptides (Supplementary mal translocation was observed 20 min after leptin. We Fig. 1A–C).

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FIG. 3. Tub nuclear translocation in response to hormones in hypothalamic nuclei. A pool of 20 mice was used for each time point (A–D). Arc, MH, PVN, and LH were dissected from the same mice, and the plasma membrane and nucleus fractions were obtained from the same samples. E–I: Whole hypothalami of 14 mice were used. Plasma membranes and nuclear lysates underwent IP and were blotted (IB) with anti-Tub antibody. Tub nuclear translocation in response to ICV (2 mg) insulin (0, 15, 30, 90 min) or (10 ng) leptin (0, 7, 20 min) was investigated in Arc (A), MH (B), PVN (C), and LH (D) of mice on chow. Tub nuclear translocation in response to ICV (E) 100 mmol/L acetylcholine (Ach) (0, 30, 120 min), (F)1mmol/L specific PLCb inhibitor (U73122) plus Ach (0, 10, 30, 120 min), (G)1mmol/L unspecific PLCb inhibitor (U73343) plus Ach (0, 10, 30, 120 min), U73122 plus insulin (0, 30, 120 min) (H), and U73122 plus leptin (0, 120 min) (I) were investigated in the hypothalamus of mice fed chow diet. U73122 and U73343 inhibitors were administered 30 min before hormone injections. ICV, ICV injection.

Metabolic characteristics of mice treated with Tub after pyruvate intraperitoneal injection at 15 and 30 min ASO. Control mice treated for 5 days with Tub ASO had and enhanced PEPCK expression in fasting livers of adecreaseof80–90% in Tub expression in the hypo- mice treated with ASO (Fig. 4H and I). These differ- thalamus compared with sense-treated mice (Fig. 4A). ences persisted in pair-fed mice (Supplementary Fig. Mice treated with ASO had higher body weight and ep- 2A–D). ididymal fat mass associated with an increase in food Food intake and hypothalamic neuropeptide expression ingestion compared with sense-treated mice (Fig. 4B– in response to insulin or leptin in mice treated with D). Additionally, there was a decrease in O2 consump- Tub ASO. The anorexigenic effect of ICV insulin or leptin tion without changes in the respiratory exchange rate was measured by recording 4-h and 8-h food intake. Food (Fig. 4E and F). There was also an increase in fasting intake was reduced in response to ICV insulin or leptin in blood glucose levels in ASO-treated mice (Fig. 4G). mice treated with sense, but not in mice treated with ASO Furthermore, we observed higher blood glucose levels (Fig. 5A and B).

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RETRACTED ARTICLE FIG. 4. Metabolic characteristics of mice treated with Tub ASO.A: ICV injection of Tub ASO for 5 days decreases 80–90% of Tub expression in the hypothalamus compared with sense controls. Body weight (B), epididymal fat mass (C), and cumulative food intake (D) are enhanced in Tub ASO- treated mice. O2 consumption (E) is decreased in Tub ASO-treated mice without change in respiratory exchange rate (F) (RER). Fasting blood glucose (G), blood glucose after a pyruvate load (H), and PEPCK protein expression (I) are enhanced in mice treated with Tub ASO. For pyruvate tolerance test, we injected 2 g/kg sodium pyruvate (intraperitoneal) in fasted mice, and blood glucose levels were measured using a glucometer at 0, 15, 30, 60, 90, and 120 min. Data are presented as means 6 SD from 8–10 mice. Two-tailed Student t test (A–C, G) or one-way ANOVA (D, F, I)or two-way ANOVA (E, H) with Bonferroni posttest were used. *P < 0.05 vs. sense.

Next, we investigated whether ASO treatment for 5 insulin-induced increases were blunted for POMC and days affects the influence of insulin or leptin on neu- TRH (Fig. 5E and H). ropeptide expression in the hypothalamus. As expected, In mice treated with ASO, insulin-induced Akt and Foxo1 fasting increased the expression of NPY, AgRP, MCH, phosphorylation and leptin-induced JAK2/STAT3 tyrosine and orexin, and insulin or leptin was able to markedly phosphorylation were not altered, indicating that the blunted reduce expression of these neuropeptides in sense- insulin and leptin effects on food intake in mice treated with treated mice (Fig. 5C, D, F,andG). However, in mice ASO are independent from these pathways (Fig. 5I–L). treated with ASO, leptin-induced reduction of MCH Insulin- and leptin-induced Tub-p-tyr in hypothalamic mRNA was blunted (Fig. 5F) and insulin-induced re- nuclei is blunted in DIO mice. We next investigated the duction of orexin was also blunted (Fig. 5G). In con- modulation of insulin-induced or leptin-induced Tub-p-tyr trast, fasting decreased the expression of POMC and in obese mice. Acute treatment with ICV insulin increased TRH, and insulin or leptin increased the expression of Tub-p-tyr in Arc, MH, PVN, and LH from control mice, and these neuropeptides in mice treated with sense; how- this effect was reduced in mice fed a HFD (Fig. 6A). ever, in mice treated with ASO, leptin-induced or Similarly, ICV injection of leptin increased Tub-p-tyr in

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RETRACTED ARTICLE FIG. 5. Food intake and hypothalamic neuropeptide expression in response to insulin or leptin in mice treated with Tub ASO. The 4-h (A)or8-h (B) food intake in response to ICV insulin or leptin is impaired in mice treated for 5 days with Tub ASO. C–I: Neuropeptide expression (mRNA levels) in response to ICV insulin and leptin are altered in the hypothalamus of ASO-treated mice. J and K: Akt and Foxo1 phosphorylation are increased in response to ICV insulin in the hypothalamus of Tub ASO-treated mice. L and M: JAK2 and STAT3 phosphorylation in response to leptin are increased in the hypothalamus of ASO-treated mice. Data are presented as means 6 SD from 8–10 mice. One-way ANOVA with Bon- ferroni posttest was used. Sense: ASO control. *P < 0.05 vs. sense; †P < 0.05 vs. saline sense; ‡P < 0.05 vs. saline and insulin sense; §P < 0.05 vs. saline, insulin, and leptin sense; ‖P < 0.05 vs. fed; ¶ vs. fast; # vs. fast with insulin (fast + I) and fast with leptin (fast + L) sense; ** vs. fast + L sense. AU, arbitrary units; ICV, ICV injection; Sal, saline; ins, insulin; lep, leptin; NPY, ; AgRP, agouti-related peptide.

Arc, MH, PVN, and LH in control mice, and this effect was association in mice fed a HFD, after leptin insulin blunted in mice fed a HFD (Fig. 6B). Insulin-induced or infusion, and the results showed that in obese mice there leptin-induced Tub nuclear translocation was blunted in was a marked increase in Tub/PTP1B association in the hypothalamus of DIO mice (Fig. 6C). response to insulin and leptin in Arc, MH, PVN, and LH PTP1B may play a role in the regulation of Tub-p-tyr nuclei (Fig. 7A–D). Then, we treated mice with ICV PTP1B in hypothalamic nuclei from DIO mice. Because PTP1B ASO for 5 days, which decreased PTP1B protein level in dephosphorylates proteins that bind the IR and JAK2, we the hypothalamus (Fig. 7E), and we measured PTPase next investigated if insulin and leptin can induce an in- activity associated with PTP1B (Fig. 7F) and with Tub crease in Tub/PTP1B association in hypothalamic nuclei (Fig. 7G). We observed an increase in PTP1B and Tub using coimmunoprecipitation. Our data showed that after associated with PTPase activity in the hypothalamus of these hormones were injected, there was an increase in mice fed a HFD, and this result was blunted by PTP1B- Tub/PTP1B interaction in all hypothalamic nuclei investi- ASO treatment, suggesting that PTP1B may have phos- gated (Fig. 7A–D). In addition, we investigated Tub/PTP1B phatase activity with Tub (Fig. 7F and G). Furthermore, we

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FIG. 6. Tub-p-tyr and translocation in response to insulin and leptin are impaired in mice fed HFD. Representative blots (n = 5 each nucleus) show insulin- (A) and leptin-induced (B) Tub-p-tyr in Arc, MH, PVN, and LH of mice fed chow or HFD. A pool of 20 mice was used for each time point. Plasma membranes and nuclear lysates underwent IP and were blotted (IB) with anti-Tub antibody. Tub nuclear translocation in response to ICV insulin (0, 15, 30, 90 min) or leptin (0, 7, 20 min) is investigated in the hypothalamus of mice fed a HFD. Data are presented as means 6 SD. One- RETRACTEDway ANOVA with Bonferroni posttest was used. a-PY, antiphosphotyrosine; *P < 0.05 vs. fastARTICLE on chow diet.

investigated the modulation of insulin-induced or leptin- in cumulative food intake, lower anorexigenic effect of induced Tub-p-tyr levels in mice fed a HFD. The results insulin and leptin, and reduced O2 consumption may showed that the reduction in PTP1B protein level in hy- contribute to explain enhanced adiposity in mice treated pothalamus was accompanied by an increase in Tub-p-tyr with Tub ASO. levels after leptin or insulin ICV treatment in DIO mice, Previous data showed that Tub overexpression, re- suggesting that PTP1B may have a role in the dephos- stricted to the Arc by a herpes simplex viral system, had no phorylation of Tub (Fig. 7H). effect on food intake in rats fed chow, suggesting that Tub expression beyond normal may not be functional (48). Here, we demonstrate that the inhibition of Tub expres- DISCUSSION sion by ASO in all hypothalamic nuclei enhanced food The results of the current study demonstrate that Tub is intake and adiposity. It is possible that the manipulation of a direct substrate of insulin and leptin in vivo. Because the the amount of Tub only in neurons from one nucleus in- insulin receptor has tyrosine kinase activity, it induced duced such a discrepant result. The regulation of food Tub tyrosine phosphorylation directly; however, for leptin, intake and adiposity is made by an interaction of different similar to other substrates, it uses JAK2 as a kinase to kinds of neurons in multiple brain regions (1–5). The induce Tub phosphorylation in Arc, MH, PVN, and LH. Our response is variable and depends on which neuron is data also show that Tub-p-tyr is decreased during fasting targeted (1–5). The effect of Tub inhibition on the con- and increased after refeeding, indicating that Tub is regu- trol of metabolism seen in our experiments with ASO lated by nutritional status. The combination of an increase may be a reflection of the circuit of leptin-responsive

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RETRACTED ARTICLE FIG. 7. Tub/PTP1B association in response to insulin or leptin is higher in hypothalamic nuclei of mice fed a HFD. A–D: Representative blots show Tub/PTP1B association in response to ICV insulin (2 mg) or leptin (10 ng) in mice fed chow. This effect is greater in mice fed a HFD in Arc, MH, PVN, and LH of mice fed chow and HFD. E: PTP1B expression is reduced in mice fed a HFD treated with PTP1B ASO for 5 days. F: PTPase activity is increased by HFD and is reduced by PTP1B ASO treatment in mice fed a HFD. G: Tub associated with PTPase activity is greater in mice fed a HFD and is decreased by PTP1B ASO treatment. In this experiment, hypothalami were homogenized and centrifuged, and the supernatants were immunoprecipitated with anti-Tub antibody. Then, immune complexes were incubated with pp60c-src COOH-terminal phosphoregulatory peptide (TSTEPQpYQPGENL; Biomol) for 1 h at 30°C. To determine PTPase activity in a spectrophotometer, 40-mL aliquots with 100 mL Biomol Green reagent (Biomol) were used. H: Tub tyrosine phosphorylation in response to insulin or leptin is higher in mice fed a HFD treated with PTP1B ASO than in sense-treated mice. Data are presented as means 6 SD from five mice per nucleus. One-way ANOVA with Bonferroni posttest was used. *P < 0.05 vs. sense, †P < 0.05 vs. mice fed chow treated with sense, ‡P < 0.05 PTP1B ASO groups. PTP1B, protein tyrosine phosphatase 1B; Sense, ASO control.

and insulin-responsive neurons in multiple hypothalamic translocation in the PVN cannot be related to a lack of nuclei. Further, although Tub acts as a transcription factor these hormones penetration into the area because PVN, moving from the plasma membrane to the nucleus (47), it as well as Arc, is periventricular. The IR expression in was not shown in this previous study (48) whether the PVN is higher than in LH, but PVN has the lowest LEPR elevated amount of Tub altered the translocation of Tub expression of all other nuclei, which may contribute to from the plasma membrane to the nucleus. explaining the delay in leptin-induced Tub translocation The results of the current study showed that after in- in this nucleus. In addition, PVN receives neuronal pro- sulin and leptin stimulation, there was a clear trans- jections from Arc; thus, we can speculate that modulation location of Tub from the plasma membrane to the of neural activity in Arc induced by insulin or by leptin nucleus in the Arc, MH, PVN, and LH being slower might delay the effect of these hormones on Tub trans- in PVN. The delay in insulin- and leptin-induced Tub location in PVN.

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Previously, it was demonstrated that activation of mRNA of POMC in Arc or the mRNA of TRH in PVN. G-protein aq by Ach releases Tub from the plasma mem- Furthermore, insulin and leptin were unable to decrease brane through the action of PLCb, triggering translocation the mRNA of orexin and MCH, respectively, in LH, sug- of Tub to the nucleus (47). In this regard, our data suggest gesting that Tub may have a role modulating neuro- that insulin and leptin also use PLCb to induce Tub expression. translocation, because an inhibitor of PLCb was able to The inhibition of Tub protein expression in mice treated block the effects of insulin and leptin on Tub translocation. with ASO induced an increase in fasting blood glucose, Here, we demonstrated that after reduction in Tub protein hepatic glucose production, and the PEPCK expression of expression, insulin or leptin was unable to increase the the liver in mice fed a chow diet. This phenomenon seems

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FIG. 8. Tub signaling in the context of other energy balance pathways. A: Insulin receptor has tyrosine kinase activity and is able to induce PI3K/ Akt/Foxo1 pathway activation, altering transcription of neuropeptides involved in the regulation of energy balance. Because LEPR has no tyrosine kinase activity, it recruits JAK2. The LEPR/JAK2 complex binds and activates STAT3, which migrates to the nucleus, regulating feeding through neuropeptide expression. Here, we suggest that phosphorylated IR and JAK2 are also able to induce Tub-p-tyr and translocation to the nucleus, accompanied by changes in neuropeptide expression and regulation of energy balance. In addition, we demonstrate that acetylcholine stimulates translocation of Tub via PLCb in vivo and insulin-induced and leptin-induced Tub translocation may occur via PLCb. B: Furthermore, in obesity insulin-induced or leptin-induced Tub-p-tyr in Arc, MH, PVN, and LH are blunted in parallel to enhanced PTP1B activation.

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