Acta Physiol 2014, 212, 28–38
Involvement of two distinct signalling pathways in IGF-1-mediated central control of hypotensive effects in normotensive and hypertensive rats P.-W. Cheng,1 B.-H. Kang,2 P.-J. Lu,3 S.-S. Lin,4 W.-Y. Ho,5 H.-H. Chen,6 L.-Z. Hong,7 Y.-S. Wu,1 M. Hsiao8 and C.-J. Tseng1,3,5,9 1 2 3 4 5 6 7 8 9
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan Department of Diving Medicine, Zouying Branch of Kaohsiung Armed Forces General Hospital Kaohsiung, Kaohsiung, Taiwan Institute of Clinical Medicine, National Cheng-Kung University, Tainan, Taiwan Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan Division of General Internal Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan Department of Medical Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan Genomics Research Center, Academia Sinica, Taipei, Taiwan Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan
Received 15 January 2014, revision requested 14 February 2014, revision received 26 June 2014, accepted 1 July 2014 Correspondence: C.-J. Tseng, MD, PhD, Department of Medical Education and Research, Kaohsiung Veterans General hospital, 386, Ta-Chung 1st Rd., 813 Kaohsiung, Taiwan. E-mail: [email protected]
Pei-Wen Cheng and Bor-Hwang Kang contributed equally to this work.
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Abstract Aims: Insulin-like growth factor-1 (IGF-1) is abundantly expressed in the nucleus tractus solitarii (NTS). In a previous study, we revealed that the induction of nitric oxide (NO) production in the NTS reduces blood pressure (BP). It is well known that both acute administration and chronic administration of IGF-I reduce BP. The aim of this study was to evaluate the short-term hypotensive effect of IGF-1 in the NTS and to delineate the underlying molecular mechanisms of IGF-1 in the NTS of normotensive WKY rats and spontaneously hypertensive rats (SHRs). Method: Microinjections of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the MAP kinase-ERK kinase (MEK) inhibitor PD98059 into the NTS in WKY rats and SHRs were used to study the involvement of IGF-1-induced depressor effects. Result: An IGF-1 (7.7 pmol) injection into the NTS resulted in a significant decrease in BP and HR in WKY rats and SHRs. Immunoblotting and immunohistochemical analysis showed that the microinjection of LY294002 (0.6 pmol) or PD98059 (3.0 pmol) into the NTS attenuated the IGF-1induced depressor effects and Akt or ERK phosphorylation in WKY rats. An attenuation effect of LY294002, but not PD98059, was found in the SHRs. However, the mRNA and protein expression levels of the IGF-1R showed no significant differences in the NTS of the WKY rats and the SHRs. Conclusion: These results suggest that distinct Akt and ERK signalling pathways mediated the IGF-1 control of the central depressor effects in WKY rats and SHRs. ERK signalling defects may be associated with the development of hypertension. Keywords Akt, central cardiovascular regulation, ERK, IGF-1, nucleus tractus solitarii.
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12340
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Insulin-like growth factor-1 (IGF-1), a small single peptide that consists of 70 amino acids, shares homology with human proinsulin (Rinderknecht & Humbel 1978), which controls metabolism, growth and survival in many mammalian tissues. However, IGF-1 and human proinsulin have distinct physiological roles. The effects of IGF-1 are mediated by the IGF-1 receptor (IGF-1R), a member of the growth factor tyrosine kinase receptor family that signals through the PI3K/Akt and the MEK1/ERK pathways (Taniguchi et al. 2006, Youngren 2007). The nucleus tractus solitarii (NTS) is located in the dorsal medulla of the brain stem and is the primary integrative centre for cardiovascular control in the central nervous system. Our previous studies showed that the induction of nitric oxide (NO) production in the NTS reduces blood pressure (BP), and this effect was induced with several neuromodulators, including adenosine (Ho et al. 2008), nicotine (Cheng et al. 2011), angiotensin II (Cheng et al. 2010) and insulin (Huang et al. 2004). However, IGF-1 plays an important role in stimulating NO production. Recently, it has been shown that the peripheral and central influences of IGF-1 on cardiovascular regulation are due to its influence on the sympathetic nervous system (Ford et al. 2000). These results suggest that IGF-1 may participate in central cardiovascular regulation. Previously, IGF-1 was found to contribute to the regulation of vascular tone (Sowers 1997, Izhar et al. 2000). NO is thought to be an important mediator of IGF-1-induced vascular relaxation (Sowers 1997, 2004). In addition to the liver, the brain and spinal cord also synthesize IGF-1 (Anderson et al. 2002). IGF-1R has been identified in mammalian brain regions (Holzenberger et al. 2000). Vascular relaxation in response to the activation of PI3K/ Akt-dependent signalling by IGF-1 is mediated, in part, by endothelial cell production of NO (Isenovic et al. 2003). Both insulin and IGF-1-induced stimulation of NO production are mediated via PI3K-dependent Akt activation on Ser473, which involves the phosphorylation of eNOS at Ser1177 (Isenovic et al. 2003). The spontaneously hypertensive rat (SHR) is a genetic model of the development of hypertension and has become one of the most widely used animal models of cardiovascular disease. The arterial pressure in SHRs begins to rise 2–3 weeks after birth and reaches hypertensive levels 12–14 weeks later. The high blood pressure in SHRs appears to be related to increased sympathetic activity and insulin resistance (Judy et al. 1979). To date, a limited number of studies have indicated that the regulation of vascular function by IGF-1 is dysfunctional in some cardiovascular disorders, such
· IGF-1 signalling pathway in the NTS of WKY and SHR
as hypertension and obesity (McCallum et al. 2005). The roles of the IGF-1 depressor effect and the underlying mechanisms have not been fully investigated. In this study, we attempted to confirm whether the both PI3K and MEK signalling pathways are involved in IGF-1-mediated blood pressure regulation in normotensive WKY rats and SHRs. In this study, we evaluated the short-term depressor effect of IGF-1 in the NTS and delineated the underlying molecular signalling mechanisms of IGF-1 in the NTS of WKY rats and SHRs. Our results showed that both PI3K/Akt and MEK/ERK signalling pathways may play important roles in the IGF-1-mediated central hypotensive effects of WKY rats, whereas the hypotensive effects of IGF-1 in SHRs were due to the differences in ERK protein expression. Based on these findings, we suggest that ERK may play an important role in the central regulation of blood pressure and the development of hypertension.
Materials and methods Animals This study was reviewed and approved by the Research Animal Facility Committee and was conducted according to the Guidelines for Animal Experiments of Kaohsiung Veterans General Hospital. Animal studies were performed on normotensive male WKY rats or hypertensive SHR at 8 and 16 weeks of age that weighed 250–300 g.
Intra-NTS Microinjection and Hemodynamic Measurements Rats were anesthetized with urethane (1.0 g kg 1 IP and 0.3 g kg 1 IV if necessary). The preparation of animals for intra-NTS microinjection and the methods used to locate the NTS have been described previously (Tseng et al. 1996). In this study, each microinjection volume was restricted to 60 nL. To investigate whether PI3K or MEK signalling participates in the depressor effect of IGF-1 in the NTS, the electrode was filled with one of the following chemicals: Mouse recombinant IGF-1 (7.7 pmol, Sigma Chemical, MO, USA) was dissolved in Opti-MEM (Invitrogen, Carlsbad, CA, USA). PI3K inhibitor LY294002 (0.6 pmol) (Vlahos et al. 1994) and MEK inhibitor PD98059 (3 pmol) (Mohr et al. 1998) was dissolved in reduced serum medium (Opti-MEM; Invitrogen, Carlsbad, Calif, Carlsbad, CA, USA). The injection volume in the NTS was restricted to 60 nL and made within 10 s so as to provide an effective compromise between excessive spread of the drug and coverage of the area
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being examined and injections were limited to 6–8 times in a rat.
considered significant. All collected data were expressed as means SEM.
Immunoblotting analysis
Results
The NTS tissues were separated carefully under the microscopy examination for protein extraction and Western blot procedures as described (Huang et al. 2004). The blot was incubated with anti-p-AKT S473 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), anti-AKT (1 : 1000), anti-p-ERK T202/Y204 antibody (1 : 500), anti-ERK (1 : 1000), anti-IGF-1Rb (1 : 1000, Santa Cruz Biotechnology, Paso Robles, CA, USA), or anti-a-tubulin antibody (1 : 10 000, Sigma) in PBST with 5% BSA and incubated for 1 h at room temperature. Peroxidase-conjugated anti-rabbit or anti-mouse antibody (1 : 5000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as secondary antibody. The membrane was detected with an ECL-Plus protein detection kit (Amersham, Piscataway, NJ, USA) on film.
Real-time PCR analysis Total cellular RNA was isolated from NTS, using RNAqueous kit (Invitrogen). Aliquots of 1 lg of RNA were reverse-transcribed with the SuperScript III firststrand synthesis kit from Invitrogen according to the manufacturer’s instructions. All primers were synthesized by Operon Technologies (Alameda, CA, USA).
Immunohistochemistry analysis The rat NTS was fixed in 4% formaldehyde overnight and embedded in paraffin. The NTS were sectioned coronally at 5 lm thickness. The sections were dewaxed, quenched in H2O2/methanol, microwaved in citric buffer (10 mM, pH 6.0), blocked in 5% goat serum, and incubated in antiphospho-AKT (1 : 100), antiphospho-ERK, (1 : 50), or anti-IGF-1R, (1 : 100) overnight at 4 °C. Afterwards, sections were incubated with biotinylated secondary antibody (1 : 200) for 1 h and in AB complex (1 : 100) for 30 min at room temperature. The sections were visualized with the DAB substrate kit (Vector Laboratories, Burlingame, CA, USA) and counterstained with haematoxylin.
Statistical analysis A paired t-test (before and after pre-treatments), unpaired t-test (comparing control and study group), or repeated-measures ANOVA followed by the post hoc analysis of Dunnett’s test were applied to compare the group differences. Probability value of P < 0.05 was 30
The depressor effects of IGF-1 microinjection into the NTS The cardiovascular effects of IGF-1 in the NTS of urethane-anaesthetized WKY rats and SHRs were determined. A prior injection of L-glutamate was used to locate the NTS area, which is suitable for measuring the cardiovascular response, as previously described (Tseng et al. 1989). Figure 1a shows that the unilateral microinjection (60 nL) of IGF-1 (7.7 pmol) into the NTS induced hypotension and bradycardia in 8-week-old WKY rats and SHRs (Fig. 1b; 34 1 mmHg and 77 4 bpm and 27 1 mmHg and 54 3 bpm, respectively; *P < 0.05, unpaired t-test).
PI3K and MEK signalling pathways are involved in IGF1-induced hypotension and bradycardia in WKY rats, but not in SHRs We explored the role of IGF-1 and the involvement of the Akt/PKB pathway centrally by injecting LY294002, a PI3K-specific inhibitor, into the NTS of WKY rats and SHRs. We examined whether the Akt/ PKB pathway was influenced by IGF-1 in the NTS of 8-week-old WKY rats and SHRs using LY294002, a PI3K-specific inhibitor. Representative tracing results of the cardiovascular effects of unilateral microinjection of IGF-1 into the NTS of 8-week-old WKY rats and SHRs are shown in Fig. 2a. A prior injection of LY294002 (0.6 pmol) into the NTS for 10 min significantly attenuated the depressor and bradycardic responses to IGF-1 in the WKY rats and the SHRs (*P < 0.05, paired t-test). However, a prior injection of PD98059 (3.0 pmol) into the NTS for 10 min significantly attenuated the depressor and bradycardic responses to IGF-1 only in the WKY rats, not in the SHRs (*P < 0.05, paired t-test).
Akt phosphorylation is induced by IGF-1 injection in the NTS of WKY rats and SHRs To elucidate the effects of IGF-1 on Akt activity in the NTS, Akt phosphorylation was determined by Western blot analysis using a phospho-Ser473 Akt antibody. The results shown in Fig. 3 reveal a significant increase in Akt phosphorylation after IGF-1 injection into the NTS of 8-week-old WKY rats compared with the control (Fig. 3a, left panel; lane 2 vs. lane 1, *P < 0.05). As expected, LY294002 can significantly block the increase in Akt phosphorylation
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(a)
(b)
Figure 1 (a) Depressor effects after unilateral microinjection of IGF-1 (7.7 pmol) in the NTS of WKY rats and SHRs. (b) Comparing the effects on MBP and HR responses induced by injection of IGF-1 into unilateral NTS in WKY rats and SHRs. Summary data means SEM, *P < 0.05 compared with WKY, n = 12 are shown in the graph. *indicate SHR group compare with WKY group and has significant differences.
induced by IGF-1 (Fig. 3a, left panel; lane 3 vs. lane 2, #P < 0.05). Similarly, IGF-1 microinjection into the NTS produced a significant increase in Akt phosphorylation in the SHRs compared with the control (Fig. 3b, right panel; lane 2 vs. lane 1, *P < 0.05). LY294002 significantly inhibited Akt phosphorylation in the 8-week-old SHRs after IGF-1 injection (#P < 0.05, lane 3 vs. lane 2). In contrast, the MEK inhibitor PD98059 did not inhibit Akt phosphorylation after IGF-1 injection into the 8-week-old WKY rats or the SHRs (Fig. 3b, lane 4). We further determined whether Akt phosphorylation occurred in situ after IGF-1 injection; paraffin sections of the NTS were subjected to an immunohistochemical staining analysis using a phospho-473 Akt antibody. Figure 3b shows a significant increase in in situ Akt phosphorylation in the NTS of 8-week-old WKY rats and SHRs (Fig. 3b, left panel) after IGF-1 injection (*P < 0.05), whereas pre-treatment with LY294002 significantly reduced Akt phosphorylation (#P < 0.05). These results suggest that the PI3K/Akt pathway may be participated in the cardiovascular regulation of
IGF-1 in the NTS of normotensive WKY rats and hypertensive SHRs.
ERK1/2 phosphorylation is induced by IGF-1 injection in the NTS of WKY rats, but not SHRs To determine whether ERK1/2 was activated after IGF-1 injection into the NTS, Western blot analysis was used to determine the phosphorylation of ERK1/2 using a phospho-Thr202/Tyr204 ERK antibody that has been shown to correlate with enzyme activity. Figure 4 shows a significant increase in the ERK1/2 phosphorylation levels after IGF-1 microinjection into the NTS of 8-week-old WKY rats compared with the control (Fig. 4a, left panel; lane 2 vs. lane 1, *P < 0.05). The increase in ERK1/2 phosphorylation was not due to the increase in total ERK1/2 proteins because there were no significant differences in total ERK1/2 protein levels between the groups. Furthermore, the MEK inhibitor PD98059 significantly inhibited the IGF1-induced increase in ERK1/2 phosphorylation (Fig. 4a, left panel; lane 4 vs. lane 2, #P < 0.05).
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(a)
(b)
Figure 2 Depressor effects of the unilateral microinjection of IGF-1 (7.7 pmol) into the NTS before and after LY294002 (0.6 pmol) or PD98059 (3.0 pmol) injection into WKY rats and SHRs. (a) Representative tracing results show the effects of MBP by injected IGF-1 into NTS pre-treated with PI3K inhibitor LY294002 in WKY rats and SHRs. (b) Representative tracing results of MBP and HR after injecting IGF-1 and pre-treatment with PD98059 in WKY rats and SHRs. Bars represent the mean SE of four independent experiments (n = 8). *P < 0.05 compared with the control value. *indicate WKY LY294002 group compare with WKY control group and has significant differences and #indicate SHR LY294002 group compare with SHR control group and has significant differences.
IGF-1 microinjection into the NTS in 8-week-old SHRs, however, did not induce ERK1/2 phosphorylation compared with the control (Fig. 4a, right panel; lane 2 vs. lane 1, P > 0.05). PD98059 also did not have a significant effect on the inhibition of ERK1/2 32
phosphorylation in the SHRs after IGF-1 injection (lane 4 vs. lane 2, P > 0.05). Moreover, the PI3K inhibitor LY294002 did not block IGF-1-induced ERK1/2 phosphorylation in the WKY rats (Fig. 4, lane 3).
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(a)
(b)
Figure 3 Western blot and immunohistochemical analyses demonstrating high levels of Akt phosphorylation in the NTS after IGF-1 and LY294002 microinjections in WKY rats and SHRs. (a) Western blotting shows p-Akt protein expression in the NTS of WKY rats and SHRs after IGF-1 microinjection (lane 2) or pre-treatment with LY294002 (lane 3) or PD98059 (lane 4) followed by IGF-1 microinjection. Note the IGF-1 injection significantly increased Akt phosphorylation in WKY rats and SHRs. Bars represent the mean SE of four independent experiments. *P < 0.05 vs. lane 1; #P < 0.05 vs. lane 2. (b) Immunohistochemical staining of p-Akt expressed cells in the NTS after IGF-1 and LY294002 microinjections. High-power field views of WKY rats and SHRs in the NTS after immunohistochemical staining with an anti-p-Akt antibody (400X). Note increased numbers of p-Akt-positive cells in the NTS after IGF-1 injection compared with the control. Also note the decreased numbers of p-Akt-positive cells after pre-treatment with the PI3K inhibitor LY294002. Graphs depict the quantitative analysis of in situ p-Akt expressed cells in 8-week-old WKY rats and SHRs after the injection of IGF-1 and LY294002 + IGF-1. The percentage of p-Akt-positive cells was determined by counting the p-Akt-positive stained cells in the entire NTS at 1009 power field divided by all cells in the same section. The significantly increase in the in situ p-Akt protein expression levels induced by IGF-1 in the NTS of WKY rats and SHRs. Also note that pre-treatment with LY294002 significantly decreased the p-Akt-positive cells in the NTS of WKY rats and SHRs. Statistical analysis was performed using a paired t-test. *P < 0.01, #P < 0.01 (n = 32).
Figure 4b shows a significant increase in in situ ERK1/2 phosphorylation in the NTS of 8-week-old WKY rats after IGF-1 injection (left panel, *P < 0.05), whereas pre-treatment with PD98059 significantly reduced ERK1/2 phosphorylation (#P < 0.05). No induction of ERK1/2 phosphorylation was detected in the 8-week-old SHRs after IGF-1 injection into the NTS (Fig. 4b, right panel, P > 0.05). These results suggest that the MEK1-ERK signalling pathway may be particulate in the cardiovascular regulation of IGF-1 in the NTS of normotensive WKY rats, but not hypertensive SHRs.
No differences in IGF-1 receptor expression in the NTS of WKY rats and SHRs The different hypotension and bradycardia effects of IGF-1 in the NTS between WKY rats and SHRs suggested that a defective IGF-1R expression might participate in the molecular pathogenesis of hypertension in the SHRs. To confirm this hypothesis, the localiza-
tion of IGF-1R was determined by real-time PCR, immunoblotting and immunohistochemical analysis in both strains of rats. Strikingly, the distribution of IGF-1R in the rat brain stem revealed that most of the cells with neuronal characteristics were strongly positive for IGF-1R immunostaining in the NTS (Fig. 5b). Interestingly, the results showed that the IGF-1R was equally expressed in the membranes of neuronal cells in the NTS of all age groups of WKY rats and SHRs (Fig. 5a and c). The results showed that IGF-1R mRNA and protein levels were equivalent in the NTS of the WKY rats and the SHRs, which suggests that the IGF-1 signalling discrepancy between the WKY rats and the SHRs might not be due to the differences in IGF-1 receptor expression.
Discussion In this study, we attempted to confirm whether the PI3K and MEK signalling pathways are involved in IGF-1-mediated blood pressure regulation in normo-
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(a)
(b)
Figure 4 Western blot and immunohistochemical analyses of ERK phosphorylation in the NTS after IGF-1 and PD98059 microinjections in WKY rats and SHRs. (a) Western blotting shows the p-ERK1 (44 kDa) and p-ERK2 (42 kDa) protein expression levels in the NTS of WKY rats and SHRs after IGF-1 microinjection (lane 2) or pre-treatment with LY294002 (lane 3) or PD98059 (lane 4) followed by IGF-1 microinjection. Bar graph demonstrates the densitometric analysis results of p-ERK1 and p-ERK2 levels after IGF-1 and LY294002 or PD98059 injections in WKY rats and SHRs. Note that the IGF-1 injection significantly increased ERK1 and ERK2 phosphorylation in WKY rats, but not in SHRs. Bars represent the mean SE of four independent experiments. *P < 0.05 vs. lane 1; #P < 0.05 vs. lane 2. (b) Immunohistochemical staining of p-ERK expressed cells in the NTS after IGF-1 and PD98059 microinjections. High-power field views of WKY rats and SHRs NTS after immunohistochemical staining with an anti-p-ERK antibody (400X). Note the increased numbers of p-ERK-positive cells in the WKY rats’ NTS after IGF-1 injection compared with the control. Also note the decreased numbers of p-ERK-positive cells after pre-treatment with the MEK1 inhibitor PD98059. Graphs depict the quantitative analysis of in situ p-ERK expressed cells after the injection of IGF-1 and LY294002 + IGF-1 in WKY rats and SHRs. The percentage of p-ERK-positive cells was determined by counting the p-ERK-positive stained cells in the entire NTS at 1009 power field divided by all cells in the same section. The significantly increase in the in situ p-ERK protein expression levels induced by IGF-1 in the NTS of WKY rats, but not in SHRs. Also note that pre-treatment with PD98059 significantly decreased the p-ERK-positive cells in the NTS of WKY rats. Statistical analysis was performed using a paired t-test. *P < 0.01, #P < 0.01 (n = 32).
tensive WKY rats and hypertensive SHRs. The major findings of this study are as follows: (i) IGF-1-modulated depressor responses in the NTS occur through the PI3K/Akt/NO and MEK/ERK/NO signalling pathways in WKY rats; (ii) IGF-1-modulated depressor responses in the NTS occur only through the PI3K/ Akt/NO axis in SHRs. It was reported previously that both acute and chronic treatments of IGF-1 reduce BP (Pete et al. 1996, Tivesten et al. 2001). Nevertheless, endogenous IGF-1-related modulation of BP has not been established. Lembo and colleagues demonstrated previously that the BP was significantly increased, which showed that 30% of IGF-1 levels have decreased in tissue and serum in IGF-1 mutant group comparing with wildtype group (Lembo et al. 1996). IGF-1 mutant mice 34
exhibit increased systolic BP and GH (growth hormone) secretion, which is due to the loss of feedback inhibition controls of the production of GH from circulating liver-derived IGF-1 (LI-IGF-1)(Wallenius et al. 2001). However, previous studies showed that no significant changes in BP after administration of GH in human (Thuesen et al. 1988). Approximately onethird of acromegalic patients have hypertension; they are high in serum GH secretion and low levels of IGF1(Sacca et al. 1994). Therefore, IGF-1 levels are low in patients with hypertension and cardiovascular diseases. Tivesten et al. (2002) also found that at the age of 4 weeks, LI-IGF-1 was inactivated and resulted in a 79% reduction in serum levels of IGF-1. At 16 weeks of age, the SBP was increased and NOS was significantly decreased in LI-IGF-1 knockout mice compared
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(b)
(c) a
c
e
g
b
d
f
h
Figure 5 IGF-IR RNA and protein expression in both WKY rats and SHRs in the NTS. (a) Real-time PCR analysis showed abundant IGF-1R RNA expressed in the NTS of WKY rats (lane 1, 8 weeks; lane 2, 16 weeks) and SHRs (lane 3, 8 weeks; lane 4, 16 weeks). (b) Western blot analysis showed abundant IGF-1R protein expressed in the NTS of WKY rats (lane 1, 8 weeks; lane 2, 16 weeks) and SHRs (lane 3, 8 weeks; lane 4, 16 weeks). (c) Immunohistochemical analysis of the IGF-1 receptor (IGF1R) revealed positive staining in both 16-week-old WKY rats (a, b for 8 weeks; c, d for 16 weeks) and SHRs (e, f for 8 weeks; g, h for 16 weeks) in the NTS. The percentage of positive cells in four groups is shown. Arrows indicate the strong expression of IGF-1R at the membrane level (original magnification 9100, 400). Densitometric analysis of the relative IGF-1R protein expression levels in both WKY rats and SHRs in the NTS. In the IGF-1 receptor RNA and protein levels, there were no significant differences among the four groups. Bars represent the mean SE of four experiments.
with control mice. The authors reported that the BP has significantly increased in LI-IGF-1 knockout mice, which suggests that the liver production of IGF-1 participates in BP regulation. Our results clearly showed that the microinjection of low-dose IGF-1 (7.7 pmol) into the NTS produced depressor and bradycardic effects (Fig. 1) in both WKY rats and SHRs, predominantly through the significant induction of Akt phosphorylation (Figs 2 and 3). The peripheral intravenous injection of high-dose IGF-1 induced bradycardic effects but only a moderate increase in Akt phosphorylation in a dose-dependent manner. Our results indicate that IGF plays a role in the central control of depressor effects. The IGF-1R is a transmembrane receptor that is activated by a hormone called IGF-1/2. IGF-1 binds to at least two cell surface receptors: IGF-1R and the insulin receptor (IR) (Jones & Clemmons 1995). The signalling receptor for cognate ligands: IGF-1 is bound with higher affinity; however, IGF-2 and insulin are bound with much lower affinity than IGF-1 (Baserga 2000). Binding of IGF-1 to IGF-1R activates receptor tyrosine kinase activity, in which stimulation of signalling cascades that involved the IRS-1/PI3K/Akt and Ras/MAPK pathways (Wang & Sun 2002). However,
due to the similarity of the structures of IGF-1R and IR, especially in the regions of the ATP-binding site and the tyrosine kinase regions, the synthesis of selective inhibitors of IGF-1R remains difficult. IGF-1R tyrosine kinase inhibitor, NVP-AEW541 (Novartis Pharma, Basel, Switzerland), inhibits autophosphorylation activity of IGF-1R signalling pathway (Garcia-Echeverria et al. 2004). Attias-Geva et al. (2011) demonstrated that the IGF-I-stimulated IGF-IR phosphorylation was reversed by NVPAEW541, whereas it abolished IGF-I-IGF-IR-AKT and ERK phosphorylation specifically in ECC-1 and USPC-1 cells. In the present study, we demonstrated that the PI3K inhibitor LY294002 attenuated IGF-1induced depressor effects, which suggested a possible involvement of Akt signalling in the mediation of the depressor effects elicited by IGF-1. However, no experiments allowing the identification of the PI3K/ Akt signalling pathway involved in the IGF-1-mediated depressor effects in the NTS have been conducted under physiological condition. Given the evidence supporting the role of downstream kinase activation in the participation of IGF-1-induced depressor and bradycardic effects, our present data demonstrated that IGF-1 induced Akt phosphorylation
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in the NTS. Similar results were found in SHRs and WKY rats. Furthermore, the observation in the present study demonstrated that microinjections of the MEK inhibitor PD98059 into the NTS attenuated the IGF-1-mediated depressor and bradycardic effects in WKY rats, but not in SHRs. It is unresolved whether the MEK/ ERK pathway is obligatory for the IGF-1-induced cardiovascular response of the SHRs in the NTS. Importantly, in the NTS of WKY rats, the activation of ERK by IGF-1 was altered by the presence of PD98059, which was demonstrated by immunohistochemical and Western blot analyses. Our data suggest that the activation of the ERK cascade by IGF-1 might be responsible, at least in part, for the IGF-1-induced depressor effects in the NTS of WKY rats, but not in SHRs (Fig. 4). Therefore, the possible mechanism connecting ERK1/2 in the NTS of SHRs remains to be elucidated. The insulin receptor family is composed of three members: the insulin receptor, the IGF-1R and the insulin receptor-related receptors, such as epidermal growth factor receptor (EGFR); all receptors exert activities similar to classic receptor tyrosine kinase activity (Medema & Bos 1993, Roudabush et al. 2000). Kagiyama et al. (2003) demonstrated that EGFR activities mediate the MEK/ERK pathway. Interestingly, phosphorylated ERK significantly declined from 5 to 20 weeks of age in SHRs. However, Kagiyama et al. (2003) also found significantly increased left ventricular weight/body weight and blood pressure in young SHRs compared with adult SHRs. In the adult SHRs, EGFR-AS (antisense oligonucleotide) did not affect the left ventricular weight/ body weight or blood pressure. Furthermore, an agedependent decrease in the phosphorylation of ERK in the heart has been observed in stroke-prone SHRs (Izumi et al. 1998). An IGF-IR signalling deficiency is, therefore, thought to precede hypertension and cardiac disorder in SHRs and SPSHRs (Kuo et al. 2005). Moreover, ageing is associated with increased vascular oxidative stress and vascular disease, which suggests that IGF-1 may exert beneficial effects on vascular ageing processes (Sparks et al. 2005, Dong et al. 2007, Higashi et al. 2012). Zhang et al. (2012) revealed that IGF-1 alleviates high-fat diet-induced cardiac dysfunction despite persistent cardiac remodelling, which may be due to increasing cell survival, mitochondrial function and insulin signalling. This finding and conclusion are consistent with our interpretation that impaired cardiac IGF-I signalling initiates hypertension. In conclusion, we propose a model to explain the signalling pathways involved in the IGF-1-mediated central control of depressor effects in normotensive 36
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Figure 6 Proposed signal transduction pathways involved in the IGF-1-mediated central control of BP and HR in WKY rats and SHRs.
and hypertensive rats (Fig. 6). IGF-1 induces both PI3K and ERK signalling pathways, which function in controlling central depressor effects in WKY rats, whereas the PI3K, but not the ERK, signalling pathway is functional in SHRs. Our study also provides evidence that the malfunction of the IGF-1-MEKERK system is not secondary to the regulation of depressor effects. The results further indicate that restoring functional MEK/ERK signalling in SHRs may hold an advantage over PI3K/Akt in maintaining the normotensive status of the SHRs. Therefore, we propose that ERK protein defects might be one of the causes underlying the development of hypertension or the central regulation of blood pressure in SHRs. Our findings provide new insight into the CNS regulation of essential hypertension and may facilitate the further development of therapy for this disease.
Conflict of interest None. The authors gratefully acknowledge the technical assistance and invaluable input and support of Mr. Bo-Zone Chen. This work was supported by grants from the National Science Council (NSC102-2320-B-075B-002-MY3, NSC101-2320-B075B-002-MY3) and Kaohsiung Veterans General Hospital (VGHKS 103-104) provided to Dr. Ching-Jiunn Tseng and the Zouying Branch of Kaohsiung Armed Forces General
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Hospital (ZAFGH 102-15) provided to Dr. Bor-Hwang Kang.
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© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12340
Involvement of two distinct signalling pathways in IGF-1-mediated central control of hypotensive effects in normotensive and hypertensive rats P.-W. Cheng,1 B.-H. Kang,2 P.-J. Lu,3 S.-S. Lin,4 W.-Y. Ho,5 H.-H. Chen,6 L.-Z. Hong,7 Y.-S. Wu,1 M. Hsiao8 and C.-J. Tseng1,3,5,9 1 2 3 4 5 6 7 8 9
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan Department of Diving Medicine, Zouying Branch of Kaohsiung Armed Forces General Hospital Kaohsiung, Kaohsiung, Taiwan Institute of Clinical Medicine, National Cheng-Kung University, Tainan, Taiwan Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan Division of General Internal Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan Department of Medical Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan Genomics Research Center, Academia Sinica, Taipei, Taiwan Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan
Received 15 January 2014, revision requested 14 February 2014, revision received 26 June 2014, accepted 1 July 2014 Correspondence: C.-J. Tseng, MD, PhD, Department of Medical Education and Research, Kaohsiung Veterans General hospital, 386, Ta-Chung 1st Rd., 813 Kaohsiung, Taiwan. E-mail: [email protected]
Pei-Wen Cheng and Bor-Hwang Kang contributed equally to this work.
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Abstract Aims: Insulin-like growth factor-1 (IGF-1) is abundantly expressed in the nucleus tractus solitarii (NTS). In a previous study, we revealed that the induction of nitric oxide (NO) production in the NTS reduces blood pressure (BP). It is well known that both acute administration and chronic administration of IGF-I reduce BP. The aim of this study was to evaluate the short-term hypotensive effect of IGF-1 in the NTS and to delineate the underlying molecular mechanisms of IGF-1 in the NTS of normotensive WKY rats and spontaneously hypertensive rats (SHRs). Method: Microinjections of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the MAP kinase-ERK kinase (MEK) inhibitor PD98059 into the NTS in WKY rats and SHRs were used to study the involvement of IGF-1-induced depressor effects. Result: An IGF-1 (7.7 pmol) injection into the NTS resulted in a significant decrease in BP and HR in WKY rats and SHRs. Immunoblotting and immunohistochemical analysis showed that the microinjection of LY294002 (0.6 pmol) or PD98059 (3.0 pmol) into the NTS attenuated the IGF-1induced depressor effects and Akt or ERK phosphorylation in WKY rats. An attenuation effect of LY294002, but not PD98059, was found in the SHRs. However, the mRNA and protein expression levels of the IGF-1R showed no significant differences in the NTS of the WKY rats and the SHRs. Conclusion: These results suggest that distinct Akt and ERK signalling pathways mediated the IGF-1 control of the central depressor effects in WKY rats and SHRs. ERK signalling defects may be associated with the development of hypertension. Keywords Akt, central cardiovascular regulation, ERK, IGF-1, nucleus tractus solitarii.
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Insulin-like growth factor-1 (IGF-1), a small single peptide that consists of 70 amino acids, shares homology with human proinsulin (Rinderknecht & Humbel 1978), which controls metabolism, growth and survival in many mammalian tissues. However, IGF-1 and human proinsulin have distinct physiological roles. The effects of IGF-1 are mediated by the IGF-1 receptor (IGF-1R), a member of the growth factor tyrosine kinase receptor family that signals through the PI3K/Akt and the MEK1/ERK pathways (Taniguchi et al. 2006, Youngren 2007). The nucleus tractus solitarii (NTS) is located in the dorsal medulla of the brain stem and is the primary integrative centre for cardiovascular control in the central nervous system. Our previous studies showed that the induction of nitric oxide (NO) production in the NTS reduces blood pressure (BP), and this effect was induced with several neuromodulators, including adenosine (Ho et al. 2008), nicotine (Cheng et al. 2011), angiotensin II (Cheng et al. 2010) and insulin (Huang et al. 2004). However, IGF-1 plays an important role in stimulating NO production. Recently, it has been shown that the peripheral and central influences of IGF-1 on cardiovascular regulation are due to its influence on the sympathetic nervous system (Ford et al. 2000). These results suggest that IGF-1 may participate in central cardiovascular regulation. Previously, IGF-1 was found to contribute to the regulation of vascular tone (Sowers 1997, Izhar et al. 2000). NO is thought to be an important mediator of IGF-1-induced vascular relaxation (Sowers 1997, 2004). In addition to the liver, the brain and spinal cord also synthesize IGF-1 (Anderson et al. 2002). IGF-1R has been identified in mammalian brain regions (Holzenberger et al. 2000). Vascular relaxation in response to the activation of PI3K/ Akt-dependent signalling by IGF-1 is mediated, in part, by endothelial cell production of NO (Isenovic et al. 2003). Both insulin and IGF-1-induced stimulation of NO production are mediated via PI3K-dependent Akt activation on Ser473, which involves the phosphorylation of eNOS at Ser1177 (Isenovic et al. 2003). The spontaneously hypertensive rat (SHR) is a genetic model of the development of hypertension and has become one of the most widely used animal models of cardiovascular disease. The arterial pressure in SHRs begins to rise 2–3 weeks after birth and reaches hypertensive levels 12–14 weeks later. The high blood pressure in SHRs appears to be related to increased sympathetic activity and insulin resistance (Judy et al. 1979). To date, a limited number of studies have indicated that the regulation of vascular function by IGF-1 is dysfunctional in some cardiovascular disorders, such
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as hypertension and obesity (McCallum et al. 2005). The roles of the IGF-1 depressor effect and the underlying mechanisms have not been fully investigated. In this study, we attempted to confirm whether the both PI3K and MEK signalling pathways are involved in IGF-1-mediated blood pressure regulation in normotensive WKY rats and SHRs. In this study, we evaluated the short-term depressor effect of IGF-1 in the NTS and delineated the underlying molecular signalling mechanisms of IGF-1 in the NTS of WKY rats and SHRs. Our results showed that both PI3K/Akt and MEK/ERK signalling pathways may play important roles in the IGF-1-mediated central hypotensive effects of WKY rats, whereas the hypotensive effects of IGF-1 in SHRs were due to the differences in ERK protein expression. Based on these findings, we suggest that ERK may play an important role in the central regulation of blood pressure and the development of hypertension.
Materials and methods Animals This study was reviewed and approved by the Research Animal Facility Committee and was conducted according to the Guidelines for Animal Experiments of Kaohsiung Veterans General Hospital. Animal studies were performed on normotensive male WKY rats or hypertensive SHR at 8 and 16 weeks of age that weighed 250–300 g.
Intra-NTS Microinjection and Hemodynamic Measurements Rats were anesthetized with urethane (1.0 g kg 1 IP and 0.3 g kg 1 IV if necessary). The preparation of animals for intra-NTS microinjection and the methods used to locate the NTS have been described previously (Tseng et al. 1996). In this study, each microinjection volume was restricted to 60 nL. To investigate whether PI3K or MEK signalling participates in the depressor effect of IGF-1 in the NTS, the electrode was filled with one of the following chemicals: Mouse recombinant IGF-1 (7.7 pmol, Sigma Chemical, MO, USA) was dissolved in Opti-MEM (Invitrogen, Carlsbad, CA, USA). PI3K inhibitor LY294002 (0.6 pmol) (Vlahos et al. 1994) and MEK inhibitor PD98059 (3 pmol) (Mohr et al. 1998) was dissolved in reduced serum medium (Opti-MEM; Invitrogen, Carlsbad, Calif, Carlsbad, CA, USA). The injection volume in the NTS was restricted to 60 nL and made within 10 s so as to provide an effective compromise between excessive spread of the drug and coverage of the area
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being examined and injections were limited to 6–8 times in a rat.
considered significant. All collected data were expressed as means SEM.
Immunoblotting analysis
Results
The NTS tissues were separated carefully under the microscopy examination for protein extraction and Western blot procedures as described (Huang et al. 2004). The blot was incubated with anti-p-AKT S473 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), anti-AKT (1 : 1000), anti-p-ERK T202/Y204 antibody (1 : 500), anti-ERK (1 : 1000), anti-IGF-1Rb (1 : 1000, Santa Cruz Biotechnology, Paso Robles, CA, USA), or anti-a-tubulin antibody (1 : 10 000, Sigma) in PBST with 5% BSA and incubated for 1 h at room temperature. Peroxidase-conjugated anti-rabbit or anti-mouse antibody (1 : 5000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as secondary antibody. The membrane was detected with an ECL-Plus protein detection kit (Amersham, Piscataway, NJ, USA) on film.
Real-time PCR analysis Total cellular RNA was isolated from NTS, using RNAqueous kit (Invitrogen). Aliquots of 1 lg of RNA were reverse-transcribed with the SuperScript III firststrand synthesis kit from Invitrogen according to the manufacturer’s instructions. All primers were synthesized by Operon Technologies (Alameda, CA, USA).
Immunohistochemistry analysis The rat NTS was fixed in 4% formaldehyde overnight and embedded in paraffin. The NTS were sectioned coronally at 5 lm thickness. The sections were dewaxed, quenched in H2O2/methanol, microwaved in citric buffer (10 mM, pH 6.0), blocked in 5% goat serum, and incubated in antiphospho-AKT (1 : 100), antiphospho-ERK, (1 : 50), or anti-IGF-1R, (1 : 100) overnight at 4 °C. Afterwards, sections were incubated with biotinylated secondary antibody (1 : 200) for 1 h and in AB complex (1 : 100) for 30 min at room temperature. The sections were visualized with the DAB substrate kit (Vector Laboratories, Burlingame, CA, USA) and counterstained with haematoxylin.
Statistical analysis A paired t-test (before and after pre-treatments), unpaired t-test (comparing control and study group), or repeated-measures ANOVA followed by the post hoc analysis of Dunnett’s test were applied to compare the group differences. Probability value of P < 0.05 was 30
The depressor effects of IGF-1 microinjection into the NTS The cardiovascular effects of IGF-1 in the NTS of urethane-anaesthetized WKY rats and SHRs were determined. A prior injection of L-glutamate was used to locate the NTS area, which is suitable for measuring the cardiovascular response, as previously described (Tseng et al. 1989). Figure 1a shows that the unilateral microinjection (60 nL) of IGF-1 (7.7 pmol) into the NTS induced hypotension and bradycardia in 8-week-old WKY rats and SHRs (Fig. 1b; 34 1 mmHg and 77 4 bpm and 27 1 mmHg and 54 3 bpm, respectively; *P < 0.05, unpaired t-test).
PI3K and MEK signalling pathways are involved in IGF1-induced hypotension and bradycardia in WKY rats, but not in SHRs We explored the role of IGF-1 and the involvement of the Akt/PKB pathway centrally by injecting LY294002, a PI3K-specific inhibitor, into the NTS of WKY rats and SHRs. We examined whether the Akt/ PKB pathway was influenced by IGF-1 in the NTS of 8-week-old WKY rats and SHRs using LY294002, a PI3K-specific inhibitor. Representative tracing results of the cardiovascular effects of unilateral microinjection of IGF-1 into the NTS of 8-week-old WKY rats and SHRs are shown in Fig. 2a. A prior injection of LY294002 (0.6 pmol) into the NTS for 10 min significantly attenuated the depressor and bradycardic responses to IGF-1 in the WKY rats and the SHRs (*P < 0.05, paired t-test). However, a prior injection of PD98059 (3.0 pmol) into the NTS for 10 min significantly attenuated the depressor and bradycardic responses to IGF-1 only in the WKY rats, not in the SHRs (*P < 0.05, paired t-test).
Akt phosphorylation is induced by IGF-1 injection in the NTS of WKY rats and SHRs To elucidate the effects of IGF-1 on Akt activity in the NTS, Akt phosphorylation was determined by Western blot analysis using a phospho-Ser473 Akt antibody. The results shown in Fig. 3 reveal a significant increase in Akt phosphorylation after IGF-1 injection into the NTS of 8-week-old WKY rats compared with the control (Fig. 3a, left panel; lane 2 vs. lane 1, *P < 0.05). As expected, LY294002 can significantly block the increase in Akt phosphorylation
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(a)
(b)
Figure 1 (a) Depressor effects after unilateral microinjection of IGF-1 (7.7 pmol) in the NTS of WKY rats and SHRs. (b) Comparing the effects on MBP and HR responses induced by injection of IGF-1 into unilateral NTS in WKY rats and SHRs. Summary data means SEM, *P < 0.05 compared with WKY, n = 12 are shown in the graph. *indicate SHR group compare with WKY group and has significant differences.
induced by IGF-1 (Fig. 3a, left panel; lane 3 vs. lane 2, #P < 0.05). Similarly, IGF-1 microinjection into the NTS produced a significant increase in Akt phosphorylation in the SHRs compared with the control (Fig. 3b, right panel; lane 2 vs. lane 1, *P < 0.05). LY294002 significantly inhibited Akt phosphorylation in the 8-week-old SHRs after IGF-1 injection (#P < 0.05, lane 3 vs. lane 2). In contrast, the MEK inhibitor PD98059 did not inhibit Akt phosphorylation after IGF-1 injection into the 8-week-old WKY rats or the SHRs (Fig. 3b, lane 4). We further determined whether Akt phosphorylation occurred in situ after IGF-1 injection; paraffin sections of the NTS were subjected to an immunohistochemical staining analysis using a phospho-473 Akt antibody. Figure 3b shows a significant increase in in situ Akt phosphorylation in the NTS of 8-week-old WKY rats and SHRs (Fig. 3b, left panel) after IGF-1 injection (*P < 0.05), whereas pre-treatment with LY294002 significantly reduced Akt phosphorylation (#P < 0.05). These results suggest that the PI3K/Akt pathway may be participated in the cardiovascular regulation of
IGF-1 in the NTS of normotensive WKY rats and hypertensive SHRs.
ERK1/2 phosphorylation is induced by IGF-1 injection in the NTS of WKY rats, but not SHRs To determine whether ERK1/2 was activated after IGF-1 injection into the NTS, Western blot analysis was used to determine the phosphorylation of ERK1/2 using a phospho-Thr202/Tyr204 ERK antibody that has been shown to correlate with enzyme activity. Figure 4 shows a significant increase in the ERK1/2 phosphorylation levels after IGF-1 microinjection into the NTS of 8-week-old WKY rats compared with the control (Fig. 4a, left panel; lane 2 vs. lane 1, *P < 0.05). The increase in ERK1/2 phosphorylation was not due to the increase in total ERK1/2 proteins because there were no significant differences in total ERK1/2 protein levels between the groups. Furthermore, the MEK inhibitor PD98059 significantly inhibited the IGF1-induced increase in ERK1/2 phosphorylation (Fig. 4a, left panel; lane 4 vs. lane 2, #P < 0.05).
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(a)
(b)
Figure 2 Depressor effects of the unilateral microinjection of IGF-1 (7.7 pmol) into the NTS before and after LY294002 (0.6 pmol) or PD98059 (3.0 pmol) injection into WKY rats and SHRs. (a) Representative tracing results show the effects of MBP by injected IGF-1 into NTS pre-treated with PI3K inhibitor LY294002 in WKY rats and SHRs. (b) Representative tracing results of MBP and HR after injecting IGF-1 and pre-treatment with PD98059 in WKY rats and SHRs. Bars represent the mean SE of four independent experiments (n = 8). *P < 0.05 compared with the control value. *indicate WKY LY294002 group compare with WKY control group and has significant differences and #indicate SHR LY294002 group compare with SHR control group and has significant differences.
IGF-1 microinjection into the NTS in 8-week-old SHRs, however, did not induce ERK1/2 phosphorylation compared with the control (Fig. 4a, right panel; lane 2 vs. lane 1, P > 0.05). PD98059 also did not have a significant effect on the inhibition of ERK1/2 32
phosphorylation in the SHRs after IGF-1 injection (lane 4 vs. lane 2, P > 0.05). Moreover, the PI3K inhibitor LY294002 did not block IGF-1-induced ERK1/2 phosphorylation in the WKY rats (Fig. 4, lane 3).
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(a)
(b)
Figure 3 Western blot and immunohistochemical analyses demonstrating high levels of Akt phosphorylation in the NTS after IGF-1 and LY294002 microinjections in WKY rats and SHRs. (a) Western blotting shows p-Akt protein expression in the NTS of WKY rats and SHRs after IGF-1 microinjection (lane 2) or pre-treatment with LY294002 (lane 3) or PD98059 (lane 4) followed by IGF-1 microinjection. Note the IGF-1 injection significantly increased Akt phosphorylation in WKY rats and SHRs. Bars represent the mean SE of four independent experiments. *P < 0.05 vs. lane 1; #P < 0.05 vs. lane 2. (b) Immunohistochemical staining of p-Akt expressed cells in the NTS after IGF-1 and LY294002 microinjections. High-power field views of WKY rats and SHRs in the NTS after immunohistochemical staining with an anti-p-Akt antibody (400X). Note increased numbers of p-Akt-positive cells in the NTS after IGF-1 injection compared with the control. Also note the decreased numbers of p-Akt-positive cells after pre-treatment with the PI3K inhibitor LY294002. Graphs depict the quantitative analysis of in situ p-Akt expressed cells in 8-week-old WKY rats and SHRs after the injection of IGF-1 and LY294002 + IGF-1. The percentage of p-Akt-positive cells was determined by counting the p-Akt-positive stained cells in the entire NTS at 1009 power field divided by all cells in the same section. The significantly increase in the in situ p-Akt protein expression levels induced by IGF-1 in the NTS of WKY rats and SHRs. Also note that pre-treatment with LY294002 significantly decreased the p-Akt-positive cells in the NTS of WKY rats and SHRs. Statistical analysis was performed using a paired t-test. *P < 0.01, #P < 0.01 (n = 32).
Figure 4b shows a significant increase in in situ ERK1/2 phosphorylation in the NTS of 8-week-old WKY rats after IGF-1 injection (left panel, *P < 0.05), whereas pre-treatment with PD98059 significantly reduced ERK1/2 phosphorylation (#P < 0.05). No induction of ERK1/2 phosphorylation was detected in the 8-week-old SHRs after IGF-1 injection into the NTS (Fig. 4b, right panel, P > 0.05). These results suggest that the MEK1-ERK signalling pathway may be particulate in the cardiovascular regulation of IGF-1 in the NTS of normotensive WKY rats, but not hypertensive SHRs.
No differences in IGF-1 receptor expression in the NTS of WKY rats and SHRs The different hypotension and bradycardia effects of IGF-1 in the NTS between WKY rats and SHRs suggested that a defective IGF-1R expression might participate in the molecular pathogenesis of hypertension in the SHRs. To confirm this hypothesis, the localiza-
tion of IGF-1R was determined by real-time PCR, immunoblotting and immunohistochemical analysis in both strains of rats. Strikingly, the distribution of IGF-1R in the rat brain stem revealed that most of the cells with neuronal characteristics were strongly positive for IGF-1R immunostaining in the NTS (Fig. 5b). Interestingly, the results showed that the IGF-1R was equally expressed in the membranes of neuronal cells in the NTS of all age groups of WKY rats and SHRs (Fig. 5a and c). The results showed that IGF-1R mRNA and protein levels were equivalent in the NTS of the WKY rats and the SHRs, which suggests that the IGF-1 signalling discrepancy between the WKY rats and the SHRs might not be due to the differences in IGF-1 receptor expression.
Discussion In this study, we attempted to confirm whether the PI3K and MEK signalling pathways are involved in IGF-1-mediated blood pressure regulation in normo-
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(a)
(b)
Figure 4 Western blot and immunohistochemical analyses of ERK phosphorylation in the NTS after IGF-1 and PD98059 microinjections in WKY rats and SHRs. (a) Western blotting shows the p-ERK1 (44 kDa) and p-ERK2 (42 kDa) protein expression levels in the NTS of WKY rats and SHRs after IGF-1 microinjection (lane 2) or pre-treatment with LY294002 (lane 3) or PD98059 (lane 4) followed by IGF-1 microinjection. Bar graph demonstrates the densitometric analysis results of p-ERK1 and p-ERK2 levels after IGF-1 and LY294002 or PD98059 injections in WKY rats and SHRs. Note that the IGF-1 injection significantly increased ERK1 and ERK2 phosphorylation in WKY rats, but not in SHRs. Bars represent the mean SE of four independent experiments. *P < 0.05 vs. lane 1; #P < 0.05 vs. lane 2. (b) Immunohistochemical staining of p-ERK expressed cells in the NTS after IGF-1 and PD98059 microinjections. High-power field views of WKY rats and SHRs NTS after immunohistochemical staining with an anti-p-ERK antibody (400X). Note the increased numbers of p-ERK-positive cells in the WKY rats’ NTS after IGF-1 injection compared with the control. Also note the decreased numbers of p-ERK-positive cells after pre-treatment with the MEK1 inhibitor PD98059. Graphs depict the quantitative analysis of in situ p-ERK expressed cells after the injection of IGF-1 and LY294002 + IGF-1 in WKY rats and SHRs. The percentage of p-ERK-positive cells was determined by counting the p-ERK-positive stained cells in the entire NTS at 1009 power field divided by all cells in the same section. The significantly increase in the in situ p-ERK protein expression levels induced by IGF-1 in the NTS of WKY rats, but not in SHRs. Also note that pre-treatment with PD98059 significantly decreased the p-ERK-positive cells in the NTS of WKY rats. Statistical analysis was performed using a paired t-test. *P < 0.01, #P < 0.01 (n = 32).
tensive WKY rats and hypertensive SHRs. The major findings of this study are as follows: (i) IGF-1-modulated depressor responses in the NTS occur through the PI3K/Akt/NO and MEK/ERK/NO signalling pathways in WKY rats; (ii) IGF-1-modulated depressor responses in the NTS occur only through the PI3K/ Akt/NO axis in SHRs. It was reported previously that both acute and chronic treatments of IGF-1 reduce BP (Pete et al. 1996, Tivesten et al. 2001). Nevertheless, endogenous IGF-1-related modulation of BP has not been established. Lembo and colleagues demonstrated previously that the BP was significantly increased, which showed that 30% of IGF-1 levels have decreased in tissue and serum in IGF-1 mutant group comparing with wildtype group (Lembo et al. 1996). IGF-1 mutant mice 34
exhibit increased systolic BP and GH (growth hormone) secretion, which is due to the loss of feedback inhibition controls of the production of GH from circulating liver-derived IGF-1 (LI-IGF-1)(Wallenius et al. 2001). However, previous studies showed that no significant changes in BP after administration of GH in human (Thuesen et al. 1988). Approximately onethird of acromegalic patients have hypertension; they are high in serum GH secretion and low levels of IGF1(Sacca et al. 1994). Therefore, IGF-1 levels are low in patients with hypertension and cardiovascular diseases. Tivesten et al. (2002) also found that at the age of 4 weeks, LI-IGF-1 was inactivated and resulted in a 79% reduction in serum levels of IGF-1. At 16 weeks of age, the SBP was increased and NOS was significantly decreased in LI-IGF-1 knockout mice compared
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(b)
(c) a
c
e
g
b
d
f
h
Figure 5 IGF-IR RNA and protein expression in both WKY rats and SHRs in the NTS. (a) Real-time PCR analysis showed abundant IGF-1R RNA expressed in the NTS of WKY rats (lane 1, 8 weeks; lane 2, 16 weeks) and SHRs (lane 3, 8 weeks; lane 4, 16 weeks). (b) Western blot analysis showed abundant IGF-1R protein expressed in the NTS of WKY rats (lane 1, 8 weeks; lane 2, 16 weeks) and SHRs (lane 3, 8 weeks; lane 4, 16 weeks). (c) Immunohistochemical analysis of the IGF-1 receptor (IGF1R) revealed positive staining in both 16-week-old WKY rats (a, b for 8 weeks; c, d for 16 weeks) and SHRs (e, f for 8 weeks; g, h for 16 weeks) in the NTS. The percentage of positive cells in four groups is shown. Arrows indicate the strong expression of IGF-1R at the membrane level (original magnification 9100, 400). Densitometric analysis of the relative IGF-1R protein expression levels in both WKY rats and SHRs in the NTS. In the IGF-1 receptor RNA and protein levels, there were no significant differences among the four groups. Bars represent the mean SE of four experiments.
with control mice. The authors reported that the BP has significantly increased in LI-IGF-1 knockout mice, which suggests that the liver production of IGF-1 participates in BP regulation. Our results clearly showed that the microinjection of low-dose IGF-1 (7.7 pmol) into the NTS produced depressor and bradycardic effects (Fig. 1) in both WKY rats and SHRs, predominantly through the significant induction of Akt phosphorylation (Figs 2 and 3). The peripheral intravenous injection of high-dose IGF-1 induced bradycardic effects but only a moderate increase in Akt phosphorylation in a dose-dependent manner. Our results indicate that IGF plays a role in the central control of depressor effects. The IGF-1R is a transmembrane receptor that is activated by a hormone called IGF-1/2. IGF-1 binds to at least two cell surface receptors: IGF-1R and the insulin receptor (IR) (Jones & Clemmons 1995). The signalling receptor for cognate ligands: IGF-1 is bound with higher affinity; however, IGF-2 and insulin are bound with much lower affinity than IGF-1 (Baserga 2000). Binding of IGF-1 to IGF-1R activates receptor tyrosine kinase activity, in which stimulation of signalling cascades that involved the IRS-1/PI3K/Akt and Ras/MAPK pathways (Wang & Sun 2002). However,
due to the similarity of the structures of IGF-1R and IR, especially in the regions of the ATP-binding site and the tyrosine kinase regions, the synthesis of selective inhibitors of IGF-1R remains difficult. IGF-1R tyrosine kinase inhibitor, NVP-AEW541 (Novartis Pharma, Basel, Switzerland), inhibits autophosphorylation activity of IGF-1R signalling pathway (Garcia-Echeverria et al. 2004). Attias-Geva et al. (2011) demonstrated that the IGF-I-stimulated IGF-IR phosphorylation was reversed by NVPAEW541, whereas it abolished IGF-I-IGF-IR-AKT and ERK phosphorylation specifically in ECC-1 and USPC-1 cells. In the present study, we demonstrated that the PI3K inhibitor LY294002 attenuated IGF-1induced depressor effects, which suggested a possible involvement of Akt signalling in the mediation of the depressor effects elicited by IGF-1. However, no experiments allowing the identification of the PI3K/ Akt signalling pathway involved in the IGF-1-mediated depressor effects in the NTS have been conducted under physiological condition. Given the evidence supporting the role of downstream kinase activation in the participation of IGF-1-induced depressor and bradycardic effects, our present data demonstrated that IGF-1 induced Akt phosphorylation
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in the NTS. Similar results were found in SHRs and WKY rats. Furthermore, the observation in the present study demonstrated that microinjections of the MEK inhibitor PD98059 into the NTS attenuated the IGF-1-mediated depressor and bradycardic effects in WKY rats, but not in SHRs. It is unresolved whether the MEK/ ERK pathway is obligatory for the IGF-1-induced cardiovascular response of the SHRs in the NTS. Importantly, in the NTS of WKY rats, the activation of ERK by IGF-1 was altered by the presence of PD98059, which was demonstrated by immunohistochemical and Western blot analyses. Our data suggest that the activation of the ERK cascade by IGF-1 might be responsible, at least in part, for the IGF-1-induced depressor effects in the NTS of WKY rats, but not in SHRs (Fig. 4). Therefore, the possible mechanism connecting ERK1/2 in the NTS of SHRs remains to be elucidated. The insulin receptor family is composed of three members: the insulin receptor, the IGF-1R and the insulin receptor-related receptors, such as epidermal growth factor receptor (EGFR); all receptors exert activities similar to classic receptor tyrosine kinase activity (Medema & Bos 1993, Roudabush et al. 2000). Kagiyama et al. (2003) demonstrated that EGFR activities mediate the MEK/ERK pathway. Interestingly, phosphorylated ERK significantly declined from 5 to 20 weeks of age in SHRs. However, Kagiyama et al. (2003) also found significantly increased left ventricular weight/body weight and blood pressure in young SHRs compared with adult SHRs. In the adult SHRs, EGFR-AS (antisense oligonucleotide) did not affect the left ventricular weight/ body weight or blood pressure. Furthermore, an agedependent decrease in the phosphorylation of ERK in the heart has been observed in stroke-prone SHRs (Izumi et al. 1998). An IGF-IR signalling deficiency is, therefore, thought to precede hypertension and cardiac disorder in SHRs and SPSHRs (Kuo et al. 2005). Moreover, ageing is associated with increased vascular oxidative stress and vascular disease, which suggests that IGF-1 may exert beneficial effects on vascular ageing processes (Sparks et al. 2005, Dong et al. 2007, Higashi et al. 2012). Zhang et al. (2012) revealed that IGF-1 alleviates high-fat diet-induced cardiac dysfunction despite persistent cardiac remodelling, which may be due to increasing cell survival, mitochondrial function and insulin signalling. This finding and conclusion are consistent with our interpretation that impaired cardiac IGF-I signalling initiates hypertension. In conclusion, we propose a model to explain the signalling pathways involved in the IGF-1-mediated central control of depressor effects in normotensive 36
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Figure 6 Proposed signal transduction pathways involved in the IGF-1-mediated central control of BP and HR in WKY rats and SHRs.
and hypertensive rats (Fig. 6). IGF-1 induces both PI3K and ERK signalling pathways, which function in controlling central depressor effects in WKY rats, whereas the PI3K, but not the ERK, signalling pathway is functional in SHRs. Our study also provides evidence that the malfunction of the IGF-1-MEKERK system is not secondary to the regulation of depressor effects. The results further indicate that restoring functional MEK/ERK signalling in SHRs may hold an advantage over PI3K/Akt in maintaining the normotensive status of the SHRs. Therefore, we propose that ERK protein defects might be one of the causes underlying the development of hypertension or the central regulation of blood pressure in SHRs. Our findings provide new insight into the CNS regulation of essential hypertension and may facilitate the further development of therapy for this disease.
Conflict of interest None. The authors gratefully acknowledge the technical assistance and invaluable input and support of Mr. Bo-Zone Chen. This work was supported by grants from the National Science Council (NSC102-2320-B-075B-002-MY3, NSC101-2320-B075B-002-MY3) and Kaohsiung Veterans General Hospital (VGHKS 103-104) provided to Dr. Ching-Jiunn Tseng and the Zouying Branch of Kaohsiung Armed Forces General
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Hospital (ZAFGH 102-15) provided to Dr. Bor-Hwang Kang.
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