Wnt Signaling Regulates Blood Pressure by Downregulating a GSK-3B–Mediated Pathway to Enhance Insulin Signaling in the Central Nervous System

Diabetes Volume 64, October 2015 3413

Pei-Wen Cheng,1 Ying-Ying Chen,2 Wen-Han Cheng,1 Pei-Jung Lu,3 Hsin-Hung Chen,4 Bo-Rong Chen,1 Tung-Chen Yeh,5 Gwo-Ching Sun,3 Michael Hsiao,6 and Ching-Jiunn Tseng1,4,7

Wnt Signaling Regulates Blood Pressure by Downregulating a GSK-3b–Mediated Pathway to Enhance Insulin Signaling in the Central Nervous System

Diabetes 2015;64:3413–3424 | DOI: 10.2337/db14-1439

Aberrant Wnt signaling appears to play an important role The nucleus tractus solitarii (NTS), which is located in the in the onset of diabetes. Moreover, the insulin signaling dorsal medulla of the brainstem, is the primary site of blood pathway is defective in the nucleus tractus solitarii (NTS) pressure (BP) and sympathetic nerve activity modulation. of spontaneously hypertensive rats (SHRs) and fructose- The NTS participates in cardiovascular, gastric, and gusta- fed rats. Nevertheless, the relationships between Wnt tory regulation. Our previous studies revealed that several signaling and the insulin pathway and the related modu- neuromodulators are involved in BP control of the NTS, lation of blood pressure (BP) in the central nervous sys- including insulin (1,2) and nitric oxide (NO) (3). METABOLISM tem have yet to be established. The aim of this study Insulin is primarily produced by pancreatic b-cells. was to investigate the potential signaling pathways Margolis and Altszuler (4) reported that insulin can cross involved in Wnt-mediated BP regulation in the NTS. Pre- the blood-brain barrier, as demonstrated by sensitive and treatment with the LDL receptor–related protein (LRP) an- specific radioimmunoassay results. However, insulin mRNA tagonist Dickkopf-1 (DKK1) significantly attenuated the and immunoreactive insulin are present in the rat central Wnt3a-induced depressor effect and nitric oxide produc- nervous system (5,6). The insulin receptor (IR) is a tetra- tion. Additionally, the inhibition of LRP6 activity using DKK1 significantly abolished Wnt3a-induced glycogen synthase meric glycoprotein that belongs to the receptor tyrosine kinase 3b (GSK-3b)S9, extracellular signal–regulated kinase superfamily. Insulin can bind to the IR, leading to b kinases 1/2T202/Y204, ribosomal protein S6 kinaseT359/S363, the autophosphorylation of the -subunit of the IR at and AktS473 phosphorylation; and increased insulin recep- tyrosine residues, which induces the tyrosine phosphoryla- tor substrate 1 (IRS1)S332 phosphorylation. GSK-3b was tion of IR substrates (IRSs) (7). IRs/IRSs can activate the also found to bind directly to IRS1 and to induce the phos- following two major signaling pathways: the phosphatidy- phorylation of IRS1 at serine 332 in the NTS. By contrast, linositol 3-kinase (PI3K)-Akt pathway, and the Ras-mitogen- administration of the GSK-3b inhibitor TWS119 into the activated protein kinase pathway. Insulin has also been brain decreased the BP of hypertensive rats by enhancing implicated in the regulation of the baroreceptor reflex in IRS1 activity. Taken together, these results suggest that the NTS (8,9). We previously found (1,2) that insulin is in- the GSK-3b-IRS1 pathway may play a significant role in volved in the control of central BP via the PI3K-Akt-NO Wnt-mediated central BP regulation. synthase (NOS) signaling pathway in the NTS.

1Department of Medical Education and Research, Kaohsiung Veterans General Corresponding author: Ching-Jiunn Tseng, [email protected]. Hospital, Kaohsiung, Republic of China Received 21 September 2014 and accepted 8 April 2015. 2Department of Ophthalmology, Kaohsiung Veterans General Hospital, Kaohsiung, This article contains Supplementary Data online at http://diabetes Republic of China .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1439/-/DC1. 3Institute of Clinical Medicine, National Cheng Kung University, Tainan, Republic of China P.-W.C. and Y.-Y.C. contributed equally to this work. 4Institute of Clinical Medicine, National Yang-Ming University, Taipei, Republic of © 2015 by the American Diabetes Association. Readers may use this article as China long as the work is properly cited, the use is educational and not for proﬁt, and 5Department of Internal Medicine, Division of Cardiology, Kaohsiung Veterans the work is not altered. General Hospital, Kaohsiung, Republic of China See accompanying article, p. 3342. 6Genomics Research Center, Academia Sinica, Taipei, Republic of China 7Department of Medical Research, China Medical University Hospital, China Med- ical University, Taichung, Republic of China 3414 Cross Talk Between Wnt and Insulin Signaling in BP Regulation Diabetes Volume 64, October 2015

Wnt proteins are a family of secreted glycoproteins injection of aCSF as a vehicle control; 5) the WKY + Wnt that bind to the receptor Frizzled (Fzd) and to coreceptors group, in which the WKY rats received an intracerebro- referred to as LDL receptor–related protein (LRP) 5 and ventricular injection of Wnt; 6) the WKY + Wnt + DKK1 LRP6 (10). The canonical Wnt pathway involves the acti- group, in which the WKY rats received an intracere- vation of Fzd receptors and LRP5/6, which results in the broventricular injection of Wnt and DKK1; 7) the WKY stabilization of b-catenin and importation into the nucleus, group, in which the WKY rats received an intracerebro- where it acts as a cofactor of T-cell factor (TCF)/lymphoid ventricular injection of aCSF as a vehicle control; 8) the enhancer factor family transcription factors (11). Dickkopf Fructose group, in which the WKY rats were provided (DKK) molecules have been revealed to function as specific with 10% fructose water for 2 weeks; and 9) the Fructose antagonists of canonical Wnt signaling by directly binding + Wnt group, in which the fructose-fed WKY rats received to LRP5/6 (12). Wnt signaling regulates various biological an intracerebroventricular injection of Wnt. The rats were activities, including cancer (13,14), stem cell maintenance provided with normal rat chow (Purina, St. Louis, MO) (15), cardiac hypertrophy (16), and neural differentiation and tap water ad libitum. All animal research protocols (17,18). In addition, recent clinical and in vitro studies were approved by the Research Animal Facility Committee revealed that aberrant Wnt signaling appears to play an of Kaohsiung Veterans General Hospital. important role in the onset of hypertension and diabetes (19–23). Nevertheless, the relationships among Wnt signal- Intracerebroventricular Injection Procedure ing and the insulin pathway and the modulation of BP in Intracerebroventricular infusion experiments were per- the central nervous system have yet to be established. formed following a stabilization period of at least 30 min In the current study, we investigated whether Wnt stim- after the insertion of the microinjector into the ventricular- ulation promotes central insulin signaling in the NTS to guided cannula. The BP was monitored for 3 days after drug modulate BP. We clarified whether aberrant Wnt signal- infusion. As a vehicle control, the effect of intracerebro- ing causes a neuronal insulin signaling defect that in- ventricular injection of aCSF (142 mmol/L NaCl, 5 mmol/L duces hypertension. In addition, we investigated which KCl, 10 mmol/L glucose, and 10 mmol/L HEPES, pH 7.4) m molecular mechanisms are essential for the fructose-induced was analyzed. Wnt3a (0.9 pmol/day) and DKK1 (1 g/day) b hypertension-mediated dysfunction of the canonical Wnt were dissolved in aCSF; the GSK-3 inhibitor (TWS119, m signaling pathway in the NTS. Overall, our results suggest 0.173 g/day) was initially dissolved in DMSO and then fi that this neuronal insulin signaling defect is a core mech- diluted in aCSF at a nal concentration of 1% DMSO. The anism that induces hypertension and that activation of the basal BP was examined prior to injection. The daily intra- Wnt signaling pathway may alleviate this defect. cerebroventricular drug infusions were performed over a 2-min period and delivered as a single bolus with a final volume of 5 mL from day 0 to day 14. Wnt3a and the RESEARCH DESIGN AND METHODS inhibitors were injected simultaneously. Experimental Chemicals All experimental drugs were purchased from Sigma-Aldrich BP Measurement (St. Louis, MO), unless otherwise noted. Using a previously described tail-cuff method (Model MK- 2000 Storage Pressure Meter; Muromachi Kikai, Tokyo, Animals Japan), the systolic BP (SBP) and the heart rate were Twenty-week-old male WKY rats and spontaneously hyper- measured prior to Wnt3a or DKK1 treatment (day 0). The tensive rats (SHRs) were obtained from the National Science animals were placed in the chamber for 30 min. In this Council Animal Facility and were housed in the animal room method, the reappearance of pulsation on a digital display of Kaohsiung Veterans General Hospital (Kaohsiung, Re- of the BP cuff was detected using a pressure transducer public of China). Humane treatment was administered at all and was amplified and recorded as the SBP. During the times. The rats were maintained in individual cages in measurement, a series of 10 individual readings was rapidly a room in which the lighting was controlled (12 h on/12 h obtained. The highest and lowest readings were dropped off), and the temperature was maintained between 23°C and from consideration, and the remaining eight readings were 24°C.Theratswereacclimatized to the housing conditions averaged. for 1 week and were then trained for 1 week to acclimate the animals to the procedure of indirect BP measurement. Measurement of the NO and Insulin Levels in the NTS The rats were randomly assigned to nine groups of six Total protein was prepared by homogenizing the NTS rats per group, as follows: 1) the SHR group, in which the tissue in lysis buffer and was deproteinized using Microcon SHRs received an intracerebroventricular injection of arti- YM-30 centrifugal filter units (Millipore, Bedford, MA). ficial cerebrospinal fluid (aCSF) as a vehicle control; 2)the The total amount of NO in the samples was determined SHR + Wnt group, in which the SHRs received an intra- using a modified procedure based on the purge system of cerebroventricular injection of Wnt; 3) the SHR + Wnt + a Sievers Nitric Oxide Analyzer (NOA 280i; Sievers Instru- DKK1 group, in which the SHRs received an intracerebro- ments, Boulder, CO), which involves the use of chemilu- ventricular injection of Wnt and DKK1; 4) the WKY group, minescence (24). The samples (10 mL) were injected into in which the WKY rats received an intracerebroventricular areflux column containing 0.1 mol/L VCl3 in 1 mol/L HCl diabetes.diabetesjournals.org Cheng and Associates 3415 at 90°C to reduce any nitrates and nitrites to NO. Then, hematoxylin. Then, the sections were photographed us- the NO reacted with the O3 produced by the analyzer to ing a microscope equipped with a charge-coupled device form NO2. The resulting emission from the excited NO2 camera. was detected using a photomultiplier tube and was digi- Coimmunoprecipitation and In Vitro Kinase Assays tally recorded (in millivolts). Then, the emission values The Catch and Release Reversible Immunoprecipitation were interpolated to a concurrently determined standard System (Millipore) was used according to the manufac- concentration curve of NaNO . The measurements were 3 turer instructions. The proteins were eluted in 70 mL performed in triplicate for each sample. The measured NO elution buffer and subjected to immunoblotting analysis levels were corrected for the volume of the examined rats. using the anti–GSK-3b and anti–P-IRS1S332 antibodies. The NTS insulin levels were measured using an Ultrasen- For the in vitro kinase assay, coimmunoprecipitation sitive Rat Insulin ELISA kit (Mercodia, Uppsala, Sweden) using the anti–GSK-3b and anti-IRS1 antibodies was per- and detected with a Biochrom Anthos Zenyth 200rt formed, and the GSK-3b-IRS1 complex was eluted using Microplate Reader (Biochrom, Cambridge, U.K.). the Catch and Release Reversible Immunoprecipitation TOPflash/FOPflash Wnt Reporter Assay System. The kinase reaction was initiated by adding ki- 3 The cells were transfected with TOPflash or FOPflash nase buffer and the terminated by adding 2 sample Wnt reporter plasmids (Millipore) containing wild-type or buffer and boiling for 10 min. The phosphorylation of mutant TCF DNA binding sites by using Lipofectamine IRS1 was determined via immunoblotting analysis using – S332 2000. The cells were also cotransfected with b-galactosidase the anti P-IRS1 and anti-IRS1 antibodies. reporter plasmids. The reporter activity was analyzed as de- Immunofluorescence Staining Analysis scribed above. The brainstem sections were incubated in the anti–GSK-3b (1:200) and anti-IRS1S332 (1:50) antibodies. After washing Immunoblotting Analysis fl Total protein was prepared by homogenizing the NTS with PBS, the sections were incubated in green uorescent tissue in lysis buffer containing a protease inhibitor Alexa Fluor 488 anti-rabbit IgG (1:200; Invitrogen) at 37°C for 2 h. The sections were analyzed using a confocal micro- cocktail and a phosphatase inhibitor cocktail and sub- scope and LSM Image software (Carl Zeiss MicroImaging). sequently incubating the sample for 1 h at 4°C. The protein extracts (20 mg/sample; Pierce BCA Protein Assay; Statistical Analysis Life Technologies, Rockford, IL) were resolved on a 6% An unpaired Student t test was used to compare protein polyacrylamide gel and transferred to a polyvinylidene levels (SHR and WKY groups, WKY and fructose-treated fluoride membrane (GE Healthcare, Buckinghamshire, U.K.). groups, SHR and SHR + TWS119 groups, or fructose and The membranes were incubated in the appropriate anti– fructose + TWS119 groups) and the BP measurements (SHR phosphorylated (P)-LRP6S1490 (2568), anti–P-AktS473 (4060), andSHR+TWS119groupsorfructoseandfructose+ anti–P-ribosomal protein S6 kinase (RSK)T359/S363 (9344), TWS119 groups). One-way ANOVA followed by Scheffé anti–P-extracellular signal–related kinase (ERK)1/2T202/Y204 post hoc analysis were applied to compare the differences (4370), anti-LRP6 (2560), anti-Akt (9272), anti-RSK (9341), between groups. Differences in which P , 0.05 were con- anti-ERK1/2 (137F5), anti–P-IRS1S332 (2580) (Cell Signaling sidered to be significant. All data are expressed as the Technology, Beverly, MA); anti–P-GSK-3bS9 (05–643), anti– mean 6 SEM. GSK-3b (07–389), anti-IRS1 (05–784R), anti–unphosphorylated RESULTS (Un-P)-b-catenin (05–665), anti–b-catenin (AB19022) (EMD Millipore, Billerica, MA); and anti–P-b-cateninSer33 (SC-16743) Wnt Signaling Mediates the Effects of the NTS on the and anti-Dvl1 (Santa Cruz Biotechnology, Dallas, TX) anti- BP bodies. Then, the membranes were then incubated in To assess whether Wnt signaling is defective in hyper- an horseradish peroxidase (HRP)-labeled goat anti- tension, we determined the expression patterns of several rabbit secondary antibody at 1:10,000. The membranes molecules involved in Wnt signaling in genetic (SHR) and – were developed using the ECL Plus detection kit (GE dietary (fructose feeding induced) models of hyperten- b Healthcare). sion. Our results revealed decreased LRP6 and GSK-3 phosphorylation levels in the NTS of SHRs and fructose- Immunohistochemistry Analysis fed rats. The protein expression levels of Dvl1 and the The sections were deparaffinized, quenched in 3% unphosphorylated form of b-catenin were also reduced in H2O2/methanol, heated (using a microwave) in citrate buffer the SHRs and fructose-fed rats; however, the phosphory- (10 mmol/L, pH 6.0), blocked in 5% goat serum, and in- lated levels of b-cateninSer33 were induced in the SHRs and cubated in the anti–P-LRP6S1490 antibody overnight at 4°C. fructose-fed rats (Fig. 1A–C). Figure 1D–F shows that there Next, the sections were incubated in a biotinylated second- were fewer P-LRP6S1490–positive cells in the NTS of SHRs ary antibody (1:200; Vector Laboratories, Burlingame, CA) and fructose-fed rats. Then, we examined whether central for 1 h and in AB complex (1:100) for 30 min at room tem- administration of Wnt3a and/or DKK1, a negative regula- perature. The sections were visualized using a DAB Substrate tor of Wnt signaling, affects hypertension and found that Kit (Vector Laboratories) and were counterstained with SHRs that were intracerebroventricularly infused with Wnt3a 3416 Cross Talk Between Wnt and Insulin Signaling in BP Regulation Diabetes Volume 64, October 2015

Figure 1—Downregulation of Wnt signaling associated with NO decrease in NTS of hypertensive model. A: Immunoblot demonstrating reduced levels of Wnt signaling factors, including the proteins P-LRP6S1490, Dvl1, P-GSK-3bS9, b-cateninS33, and Un-P-b-catenin, in the NTS of SHRs and fructose-fed rats. T, total. B: Densitometric analysis of the Wnt signaling protein levels in the NTS of SHRs and WKY rats. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the WKY group. C: Densitometric analysis of the Wnt signaling protein levels in the NTS of fructose-fed rats and WKY rats. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the WKY group. D: In situ qualitative analysis of P-LRP6S1490–immunopositive cells in the NTS. The arrowheads indicate the central canal. The arrows indicate the P-LRP6S1490–expressing cells in the NTS. Scale bar, 200 mm. E: Graphs depicting the quantitative analysis of in situ P-LRP6S1490–expressing cells in the NTS. The percentage of P-LRP6S1490–positive cells was determined by counting the P-LRP6S1490– expressing cells in each hemisphere of the NTS at 3200 magnification. These values were divided by the total number of cells in the same paraffin section. *P < 0.05 (n = 6). F: Graph depicting the quantitative analysis of in situ P-LRP6S1490–expressing cells in the NTS. The percentage of P-LRP6S1490–positive cells was determined by counting the P-LRP6S1490–expressing cells in each hemisphere of the NTS at 3200 magnification. These counts were divided by the total number of cells in the same paraffin section. *P < 0.05 (n = 6). G: Time course of SBP after intracerebroventricular administration of Wnt and DKK1 for 2 weeks. The filled circles (●) represent the SHR group, the open circles (○) represent the SHR + Wnt group, and the inverted filled triangles (▼) represent the SHR + Wnt + DKK1 group. SBP was measured on days 0, 4, 7, 11, and 14. The data are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the SHRs and #P < 0.05 vs. the Wnt-treated SHRs. H: Time course of SBP after intracerebroventricular administration of Wnt and DKK1 for 2 weeks. The filled circles (●) represent the WKY group, the open circles (○) represent the WKY + Wnt group, and the inverted filled triangles (▼) represent the WKY + Wnt + DKK1 group. SBP was measured on days 0, 4, 7, 11, and 14. The data are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the Wnt-treated diabetes.diabetesjournals.org Cheng and Associates 3417 exhibited a depressor response. Coadministration of the simultaneous injection of DKK1 and Wnt3a reduced Wnt3a and DKK1 blocked the Wnt3a-induced depres- canonical Wnt signaling activity in the NTS of normo- sor effect and NO release in the NTS of SHRs (Fig. 1G tensive WKY rats. Interestingly, fructose administration and J). In contrast, Fig. 1H and K shows that intracere- resulted in a significant decrease in the serine phosphor- broventricular infusion of DKK1 increased BP and de- ylation levels of LRP6 and GSK-3b and in the expression creased NO production in the NTS of WKY rats. In the levels of Dvl1 and unphosphorylated levels of b-catenin; fructose-fed–induced hypertension model, the increase in however, there was a significant increase in the serine BP was attenuated by intracerebroventricular infusion of phosphorylation levels of b-catenin. Moreover, the inhibi- Wnt3a for 2 weeks (Fig. 1I). In addition, NO release in the tory effect of fructose on Wnt pathway activity was en- NTS was found to be restored following treatment with hanced following Wnt3a infusion in the brain (Fig. 2F). Wnt3a for 2 weeks (Fig. 1L). As shown in Fig. 1, elevating These results suggest that the activation of Wnt signaling the levels of Wnt3a in the brain resulted in an improve- may be required for NO release and BP regulation in the ment of hypertension. The reported results of the assay NTS. and immunofluorescence staining (Fig. 2A–C)demonstrate b The Induction of Wnt Signaling Is Associated With that the level of nuclear -catenin and its mediated tran- Insulin Activity in the NTS fi scriptional activity signi cantly increased (active) in the We found that rats exhibit a depressor response following A B NTS of SHRs. In Fig. 2 and , results from the confocal microinjection of insulin into the NTS (1). In addition, the b – microscopy experiments showed that active -catenin activation of Wnt signaling leads to an upregulation of the fl positive cells (labeled with green uorescence) were scarcely insulin pathway (23). Therefore, to determine whether Wnt A B detected in the NTSs of SHRs. (Fig. 2 and ,lane1). signaling interferes with the insulin pathway in the NTS, we Strikingly, the administration of Wnt3a resulted in an in- injected Wnt3a into the brain. Wnt3a administration and b fl A crease in active -catenin immuno uorescence (Fig. 2 coadministration of DKK1 and Wnt3a did not affect the B and ,lane2).Moreover,coadministrationofDKK1and phosphorylation of IRS2 at Serine (Ser) 731 in the NTS of b Wnt3a did not restore the active -catenin levels in nuclei SHRs, WKY rats, and fructose-fed rats (Supplementary Fig. A B fl (Fig. 2 and , lane 3). The data from the TOP ash/ 1A–C). These results indicate that IRS2 may not be involved fl FOP ash Wnt reporter assay showed that treatment in Wnt induced insulin signaling. Wnt3a administration fi b – with Wnt3a can signi cantly increase the -catenin significantly decreased the phosphorylation of IRS1 at mediated transcriptional activity in the NTSs of SHRs Ser332 and increased downstream insulin signaling in the C compared with the control group (SHRs) (Fig. 2 ,lane2). NTSs of SHRs and fructose-fed rats (Fig. 3A and C). Simi- Intracerebroventricular infusion of both DKK1 and Wnt3a larly, a reduction in the insulin levels in the NTSs of SHRs b – can reduce the Wnt3a-induced increase in the -catenin and fructose-fed rats was detected following Wnt3a injec- C mediated transcriptional activity (Fig. 2 , lane 3). Next, we tion (Fig. 3D and F). Furthermore, coadministration of investigated the expression pattern of Wnt signaling fac- DKK1 and Wnt3a enhanced the IRS1S332 phosphorylation tors in the NTSs of vehicle-, Wnt3a-, and DKK1-infused and insulin levels and significantly decreased insulin signal- fi rats. Treatment with Wnt3a signi cantly increased the ing in the NTSs of WKY rats (Fig. 3B and E and Supple- b phosphorylation levels of LRP6 and GSK-3 ,theprotein mentary Fig. 2B). These results indicate that Wnt promotes expression levels of Dvl1, and the unphosphorylated levels the activation of IR signaling, which, in turn, downregulates b of -catenin; however, the phosphorylation levels of insulin production in the NTSs of hypertensive rats. b-catenin did not change in the NTSs of SHRs. Intracere- broventricular infusion of both DKK1 and Wnt3a reduced The Interaction Between GSK-3b and IRS1 Plays the Wnt3a-induced increase in the phosphorylation a Crucial Role in the Wnt3a-Mediated Hypotensive and expression levels of LRP6, GSK-3b,andDvl1 Effects and the unphosphorylated levels of b-catenin, but the As shown above, our experiments indicated that treat- phosphorylation levels of b-cateninSer33 were reversed ment with Wnt3a leads to a decrease in IRS1S332 phos- (Fig. 2D and Supplementary Fig. 2A). As shown in Fig. 2E, phorylation in the NTSs of hypertensive rats. Next, we

WKY rats. I: Time course of SBP after intracerebroventricular administration of Wnt and DKK1 for 2 weeks. The filled circles (●) represent the WKY group, the open circles (○) represent the fructose group, and the inverted filled triangles (▼) represent the fructose + Wnt group. SBP was measured on days 0, 4, 7, 11, and 14. The data are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the WKY rats and #P < 0.05 vs. the fructose-fed WKY rats. J: Quantification of the NO concentrations in the NTS of rats. The bar graph displays the NO concentration (as micromoles nitrate per microgram of NTS protein). The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the SHRs and #P < 0.05 vs. the Wnt-treated SHRs. K: Quantification of the NO concentrations in the NTSs of rats. The bar graph displays the NO concentration (as micromoles nitrate per microgram protein) with the group treated with DKK1 exhibiting significantly decreased NO levels in the NTS compared with the WKY + Wnt group. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the Wnt-treated WKY rats. L: Quantification of the NO concentrations in the NTSs of rats. The bar graph displays the NO concentration (as micromoles nitrate per microgram of NTS protein). The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the WKY rats and #P < 0.05 vs. the fructose-fed rats. T, total. 3418 Cross Talk Between Wnt and Insulin Signaling in BP Regulation Diabetes Volume 64, October 2015

Figure 2—Wnt3a promotes Wnt signaling in the NTS of hypertensive rats. A: The immunofluorescence image displays the active level of b-catenin. The representative green fluorescence images indicate active b-catenin–positive cells in the NTS with or without the administration of Wnt3a and DKK1. The nuclei of the cells in the NTS were counterstained with DAPI, displaying blue fluorescence. The images were photographed at 3400 and 31,000 magnification. B: The bar graph is representative of the active b-catenin fluorescence intensity in the NTS of the indicated groups. The presented values are the mean 6 SEM (n = 6). *P < 0.05 vs. the control group and #P < 0.05 vs. the Wnt-treated SHRs. C: Luciferase reporter assay of b-catenin-Tcf/Lef activity in SHR primary culture cells transfected with the Wnt signaling reporter (TOPflash or the control FOPflash). TOP-Luc reporter vector was used for detecting b-catenin–dependent transcriptional activation. FOP-Luc reporter vector was used as negative control. The ratio of TOP/FOP-Luc is presented. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the SHRs and #P < 0.05 vs. the Wnt-treated SHRs. D: Quantitative immunoblotting analysis revealed that Wnt treatment increased the phosphorylation levels of LRP6, GSK-3b, and b-catenin and Un-P-b-catenin protein in the NTSs of SHRs. Densitometric analysis of the P-LRP6S1490, P-GSK-3bS9, and Un-P-b-catenin levels after administration of Wnt3a with or without DKK1. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the SHRs and #P < 0.05 vs. the Wnt-treated SHRs. E: Quantitative immunoblotting analysis revealed that DKK1 treatment decreased the phosphorylation levels of LRP6 and the GSK-3b and b-catenin and protein levels of Un-P-b-catenin in the NTSs of WKY rats. Densitometric analysis of the P-LRP6S1490,P-GSK-3bS9, b-cateninS33, and Un-P-b-catenin levels after administration of Wnt3a with or without DKK1. The values are presented as the mean 6 SEM; n =6. *P < 0.05 vs. the Wnt-treated WKY rats. F: Quantitative immunoblotting analysis revealed that Wnt treatment increased the phosphorylation levels of LRP6, GSK-3b,andb-catenin and Un-P-b-catenin in the NTSs of fructose-fed rats. Densitometric analysis of the levels of P-LRP6S1490, P-GSK-3bS9, b-cateninS33,andUn-P-b-catenin after the administration of Wnt3a. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the WKY rats and #P < 0.05 vs. the fructose-fed rats. T, total. diabetes.diabetesjournals.org Cheng and Associates 3419

Figure 3—P-IRS1S332 is a critical determinant of Wnt-mediated Akt, ERK1/2, RSK, and NOS phosphorylation and insulin production in the NTS. A: Quantitative immunoblotting analysis revealed that Wnt treatment increased the phosphorylation levels of Akt, ERK1/2, RSK, and endothelial NOS (eNOS) in the NTSs of SHRs. Densitometric analysis of the P-AktS473,P-ERK1/2T202/Y204,P-RSKT359/S363,andeNOSS1177 levels after administration of Wnt3a with or without DKK1. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the SHRs and #P < 0.05 vs. the Wnt-treated SHRs. B: Quantitative immunoblotting analysis revealed that DKK1 treatment decreased the phosphorylation levels of Akt, ERK1/2, RSK, and eNOS in the NTSs of WKY rats. DKK1 treatment elevated the IRS1S332 phosphorylation ratio. Densitometric analysis of the P-AktS473,P-ERK1/2T202/Y204,P-RSKT359/S363,andeNOSS1177 levels after administration of Wnt3a with or without DKK1. The values are presented as the mean 6 SEM; n =6.*P < 0.05, #P < 0.05 vs. the Wnt-treated WKY rats. C: Quantitative immunoblotting analysis revealed that Wnt treatment increased the phosphorylation levels of Akt, ERK1/2, RSK, and nNOS in the NTSs of fructose-fed rats. Densitometric analysis of the P-AktS473,P-ERK1/2T202/Y204,P-RSKT359/S363, and nNOSS1416 levels after administration of Wnt3a. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the WKY rats and #P < 0.05 vs. the fructose-fed rats. P-IRS1S,P-IRS1S332;P-IRS1Y, P-IRS1Y612;T,total.D: High insulin levels in the NTSs of SHRs. The reduced insulin level due to Wnt3a administration was increased by the addition of DKK1 in the NTS. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the SHRs and #P < 0.05 vs. the Wnt-treated SHRs. E: Low insulin levels in the NTSs of WKY rats. DKK1 administration elevated the insulin level in the NTS. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the Wnt-treated WKY rats. F: High insulin levels in the NTSs of fructose-fed rats. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the WKY rats and #P < 0.05 vs. the fructose-fed rats. 3420 Cross Talk Between Wnt and Insulin Signaling in BP Regulation Diabetes Volume 64, October 2015 examined the endogenous IRS1S332 phosphorylation lev- and fructose-fed rats as animal models in our current study els in the NTSs of hypertensive and normotensive rats. As to investigate the signaling mechanisms involved in Wnt- shown in Fig. 4A, there was a significant increase in the mediated central BP regulation in the NTS. In addition, our P-IRS1S332 levels in the NTSs of SHRs and fructose- present results reveal endogenous aberrant canonical Wnt fed rats compared with normotensive WKY rats. In addi- signaling in the NTSs of hypertensive rats. Phosphorylation tion, GSK-3b phosphorylates IRS1 at Ser332 to attenuate of LRP6 promotes canonical Wnt signaling activation. As downstream insulin signaling in cultured cells (25). To shown in Fig. 1A and D, on the basis of immunoblotting determine whether Wnt regulates the interaction between and immunohistochemistry analyses, the LRP serine phos- GSK-3b and P-IRS1S332 in the NTS, we double immunola- phorylation levels in both the SHRs and the fructose-fed beled for P-IRS1S332 and GSK-3b. Using confocal micros- rats were lower than in the normotensive rats. Interest- copy, P-IRS1S332 (red) immunoreactivity was abundantly ingly, our results show that central administration of detected in the NTSs of SHRs (Fig. 4B, lane 1). GSK-3b Wnt3a induced NO release from the NTS and a depressor (green) immunofluorescence colocalized to P-IRS1S332 im- effect in the SHRs (Fig. 1G). The fructose-mediated pressor munoreactivity (Fig. 4B, lane 1). Strikingly, the adminis- effect was also abolished by Wnt3a administration (Fig. 1I). tration of Wnt3a resulted in a decrease in P-IRS1S332 The inhibition of LRP6 activity using DKK1 in the presence immunofluorescence and in the colocalization of P-IRS1S332 of Wnt3a decreased LRP6 phosphorylation at Ser1490 in with GSK-3b (Fig. 4B, lane 2). Moreover, coadministration the NTS after the induction of IRS1 serine phosphorylation of DKK1 and Wnt3a restored the P-IRS1S332 levels and the and the development of hypertension (Fig. 1H). Our find- colocalization of P-IRS1S332 with GSK-3b (Fig. 4B, lane 3). ings in murine models support those obtained from Then, we performed coimmunoprecipitation assays to an- a clinical study, in which the subjects who carried a loss- alyze whether GSK-3b binds to P-IRS1S332. The NTS lysate of-function mutation in LRP6 displayed hypertension, in- was immunoprecipitated using either an anti–GSK-3b sulin resistance, and diabetes (19). Krüger et al. (31) or anti–P-IRS1S332 antibody and was subsequently probed revealed that stimulation of insulin increases LRP6 activa- using anti–GSK-3b and anti–P-IRS1S332 antibodies. The tion. Additionally, a mutation in LRP6 impairs insulin sig- results revealed that GSK-3b coimmunoprecipitated naling in skeletal muscle and leads to the development of with P-IRS1S332 (Fig. 4C, lanes 2 and 3). Next, an in vitro insulin resistance (32). In contrast to our findings, in kinase assay was performed to determine whether GSK-3b homozygotic LRP6 knockout mice, insulin signaling and directly phosphorylates IRS1S332 in vitro. We incubated IRS1 activity are enhanced in brown adipose tissue (33). purified IRS1 protein in GSK-3b immunoprecipitated Cruciat et al. (34) revealed that the (pro)renin receptor binds from the NTS lysate in the presence of ATP for the indi- to the Fzd receptor, phosphorylating LRP6 and activating cated reaction period. Figure 4D shows the time-dependent LRP6 phosphorylation. The mRNA levels of the (pro)renin increase in phosphorylation of IRS1 at Ser332 by GSK-3b. receptor were 45% higher in the NTSs of SHRs than in These results suggest that GSK-3b phosphorylates IRS1S332 those of WKY rats, suggesting that the (pro)renin recep- in the NTS. In addition, we determined whether GSK-3b is tor may contribute to the regulation of BP (35). The pos- involved in the Wnt-induced depressor response and NO sible mechanism connecting Wnt and the (pro)renin production. Intracerebroventricular infusion of the GSK- receptor in the NTS for BP regulation remains to be elu- 3b–specific inhibitor TWS119 decreased the BP in SHRs cidated. Our findings extend the findings of previous and partially blocked the fructose-induced depressor re- reports that Wnt signaling in the NTS might participate sponse in fructose-fed rats (Fig. 4E and F). As shown in BP regulation. in Fig. 4G, after intracerebroventricular treatment with IRS1 is a docking protein for the IR and insulin growth TWS119, the phosphorylation levels of IRS1 in the NTSs factor-1 receptor. IRS1 contains several serine/threonine of hypertensive rats were significantly decreased compared and tyrosine phosphorylation sites. The phosphorylation with the control rats; however, neuronal NOS (nNOS), Akt, of IRS1 at tyrosine residues occurs in response to insulin- ERK, and RSK in the NTSs of hypertensive rats were signif- stimulated activation. Moreover, most phosphorylation icantly increased compared with the control rats in SHRs events at serine/threonine residues are recognized as andfructose-fedrats. negative feedback regulators of insulin signaling. Many serine/threonine kinases, including GSK-3b, S6 kinase, DISCUSSION ERK1/2, c-Jun NH2-terminal kinase, mammalian target of Essential hypertension is the most common cardiovascu- rapamycin, AMPK, and protein kinase C (36,37), have been lar disease worldwide, being estimated to cause 13% of all shown to phosphorylate IRS1. GSK-3b has been identified deaths (26). The SHR is a widely used animal model of to phosphorylate IRS1 at Ser332, and high-glucose stimu- human essential hypertension and insulin resistance (27). lation activates GSK-3b to promote IRS1S332 phosphoryla- It has been reported (28–30) that various animal species tion (25,38). The current study also showed that GSK-3b that are fed a high-fructose diet or fructose-containing directly increases P-IRS1S332 in a time-dependent manner water for several weeks develop the metabolic syndrome, and identified GSK-3b as a key factor in the downregulation which includes insulin resistance, impaired glucose of IRS1 phosphorylation. Treatment with Wnt3a reduced homeostasis, and hypertension. Therefore, we used SHRs the interaction between GSK-3b and P-IRS1S332 in the diabetes.diabetesjournals.org Cheng and Associates 3421

Figure 4—Phosphorylation of IRS1 at Ser332 by GSK-3b in the NTS. A: Immunoblot demonstrating the low levels of P-IRSS332 in the NTSs of SHRs and fructose-fed rats. The values are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the WKY group. B: The immunofluorescence image displays the colocalization of GSK-3b and P-IRS1S332. The representative yellow fluorescence images indicate GSK-3b– colocalized and P-IRS1S332–colocalized cells in the NTS with or without the administration of Wnt3a and DKK1. Red fluorescence indicates the P-IRS1S332–positive cells in the NTS. Green fluorescence indicates the GSK-3b–positive cells in the NTS. The nuclei of the cells in the NTS were counterstained with DAPI, displaying blue fluorescence. The images were photographed at 3280 and 31,000 magnification. C and D: Lysates from the NTS were assayed and used for coimmunoprecipitation of IRS1 and GSK-3b. Total lysates were also analyzed via immunoblot using antibodies against GSK-3b and P-IRS1. GSK-3b–associated kinase activity was determined using IRS1 as a substrate. The P-IRS1 levels were analyzed on a 6% SDS-PAGE gel subjected to autoradiography. The values are presented as the 3422 Cross Talk Between Wnt and Insulin Signaling in BP Regulation Diabetes Volume 64, October 2015

NTSs of SHRs. Simultaneous intracerebroventricular infu- and the stimulation of Wnt-dependent transcription. These sion of DKK1 and Wnt3a restored this interaction between results establish the function of Ser21/Ser9 phosphoryla- GSK-3b and P-IRS1S332, suggesting that the administration tion in several processes in which GSK-3 inactivation has of Wnt3a downregulated the GSK-3b-P-IRS1S332 signaling previously been implicated (45). In Fig. 2D and E,our cascade in the NTSs of hypertensive rats. TWS119 is a po- data found that intracerebroventricular infusion of Wnt3a tent and cell-permeable GSK-3b inhibitor; it has been increases the phosphorylation and expression levels of reported (39) to promote Wnt signaling in CD8+ T cells. GSK-3bS9, which may be associated with Akt or Wnt3a Treatment with TWS119 decreased the SBP of SHRs stimulation. and ameliorated the fructose-induced pressor response Inhibition of DKK improves insulin sensitivity and (Fig. 4E and F). Our data revealed that inhibiting GSK- glucose tolerance in diabetic mice but not in nondiabetic 3b activity using TWS119 prevents IRS1S332 phosphoryla- mice (46). Epidemiological studies showed that mutations tion in the NTS. However, it has been reported (40) that of LRP5 may cause type 1 diabetes and obesity (21,47). membrane-associated GSK-3b activates LRP6 via phosphor- Furthermore, Wnt3a-stimulated insulin secretion is medi- ylation at Ser1490. GSK-3b is also negatively regulated by ated by LRP5 (48). LRP5 has also been shown to regulate insulin. Furthermore, insulin activates the PI3K-Akt cascade, LRP6 serine phosphorylation. Additionally, LRP5 may which leads to the phosphorylation of GSK-3b at Ser9, serve as a coreceptor in the Wnt and insulin signaling resulting in its inactivation (41). Together, these findings pathways to upregulate the insulin signaling cascade in indicate that GSK-3b is a critical factor in the cross talk preadipocytes (23). In the current study, we found de- between Wnt and insulin signaling. Our results indicate creased insulin levels in the NTS following intracerebro- that Wnt3a-mediated BP regulation may occur via the ventricular infusion of Wnt3a, which was restored by downregulation of the GSK-3b-P-IRS1S332 interaction, cotreatment with DKK1. Central administration of Wnt3a subsequently activating insulin signaling in the NTS. Fur- into the brain activated critical mediators of the insulin ther studies are required to determine the mechanisms by signaling network, including phosphorylated AktS473, which the stimulation of insulin signaling is associated ERK1/2T202/Y204, and RSKT359/S363 in the NTS of hyperten- with GSK-3b activity and the Wnt signaling cascade in sive rats, and these phosphorylation events were blocked the NTS to regulate BP. by the administration of DKK1 (Fig. 3A and B). Moreover, Epidemiologic studies (42) have established that diabe- antagonizing LRP6 phosphorylation prevented PI3K-Akt tes is estimated to affect nearly 24 million people in the and ERK1/2-RSK signaling activation and led to insulin U.S. This significant disease burden translates into a major production in the NTS of WKY rats. A mutation in LRP6 economic impact. Clinical intervention trials have estab- underlies insulin resistance and diminishes AktS473 phos- lished the key roles of the transcription factor 7-like 2 phorylation (32). Taken together, these data suggest that (TCF7L2) gene with type 2 diabetes in African (43) and Wnt administration reverses insulin signaling and im- African-derived populations (i.e., African Americans) (44). proves insulin release via the Wnt coreceptor LRP. How- The TCF7L2 gene product is a high-mobility transcription ever, Mani et al. (19) identified a missense mutation in factor that is associated with blood glucose homeostasis. LRP6, which substitutes cysteine for arginine at a highly It is via the Wnt signaling pathway that TCF7L2 regulates conserved residue of an epidermal growth factor–like do- the proglucagon gene expression in enteroendocrine cells main, that impairs Wnt signaling in vitro. These results (20). Recent reports (22) showed that Wnt stimulation link a single gene defect in Wnt signaling to coronary artery enhances IRS1 expression and triggers Akt activation. Be- disease and multiple cardiovascular risk factors (19). Sim- sides, Cross et al. (41) demonstrated that insulin is ilarly, rare mutations in other Wnt-related transcription thought to stimulate the dephosphorylation of glycogen factors cause maturity-onset diabetes of youth (49). In synthase at these residues by inducing the inactivation of a further perspective on patients with early coronary artery GSK-3a and GSK-3b via phosphorylation of an N-terminal disease, metabolic syndrome, and its components, we sug- Ser residue (Ser21 in GSK-3a and Ser9 in GSK-3b), which gest that Wnt signaling may provide new insight into dis- is catalyzed by Akt in muscle. McManus et al. (45) also ease pathophysiology or into approaches to prevent these found that Wnt3a induces normal inactivation of GSK-3 disorders and may be a potential therapeutic target for at Ser21/Ser9, as judged by the stabilization of b-catenin treating type 2 diabetes.

mean 6 SEM; n =6.*P < 0.05 vs. 0 h. E: Time course of SBP after intracerebroventricular administration of the GSK-3b inhibitor TWS119 for 2 weeks. The ﬁlled circles (●) represent the SHR group, and the open circles (○) represent the SHR + TWS119 group. SBP was measured on days 0, 4, 7, 11, and 14. The data are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the SHR group. F: Time course of SBP after intracerebroventricular administration of the GSK-3b inhibitor TWS119 for 2 weeks. The ﬁlled circles (●) represent the fructose group, and the open circles (○) represent the fructose + TWS119 group. The data are presented as the mean 6 SEM; n =6.*P < 0.05 vs. the fructose group. G: Immunoblot demonstrating the low P-IRSS332 levels and the increase in the P-nNOS, P-Akt, P-ERK1/2, and P-RSK levels in the NTSs of SHRs and fructose-fed rats after the administration of TWS119. The values are presented as the mean 6 SEM; n =6. *P < 0.05 vs. the SHR and WKY groups. T, total. diabetes.diabetesjournals.org Cheng and Associates 3423

Author Contributions. P.-W.C. conducted most of the studies and wrote the manuscript. Y.-Y.C., W.-H.C., and B.-R.C. assisted in the conduct of most of the studies. P.-J.L., M.H., and C.-J.T. conceived and designed the study. H.-H.C., T.-C.Y., and G.-C.S. contributed to the writing of the manuscript. C.-J.T. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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