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Keywords:

  • gastrointestinal motility;
  • HO/CO;
  • NOS/NO;
  • rosiglitazone

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information

Background  Thiazolidinediones (TZDs) including rosiglitazone (ROSI) are insulin sensitizing agents with beneficial gastrointestinal effects. However, no studies are available on TZDs effect in gastrointestinal motility. We evaluated the effects of ROSI on gastrointestinal inhibitory neurotransmission focusing on the modulatory roles of nitric oxide synthase/nitric oxide (NOS/NO) and heme oxygenase/carbon monoxide (HO/CO) pathways.

Methods  Spontaneously hypertensive rats (SHR) were used as model of insulin resistance. Duodenal strips were obtained from vehicle-treated SHR, ROSI-treated SHR (5 mg kg−1 by gavage daily per 6 weeks), and Wistar Kyoto (WKY). Inhibitory responses to electrical field stimulation (EFS) were evaluated in the presence of HO inhibitor zinc protoporphyrin IX (ZnPPIX, 10 μmol L−1) or NOS inhibitor NG-nitro-l-arginine (L-NNA, 100 μmol L−1), alone and in combination. Protein levels of HO and NOS isoforms were evaluated by immunohistochemistry and western blot analysis.

Key Results  Basal responses to EFS were significantly increased in duodenum strips from vehicle-treated SHR vs WKY. This effect was reversed in ROSI-treated SHR. The EFS-mediated relaxation was comparably reduced by ZnPPIX in WKY and SHR, but not in ROSI-treated SHR animals. The L-NNA reduced EFS response to a similar extent in WKY and ROSI -treated SHR, but its effect was significantly higher in vehicle-treated SHR. Expression of HO-1 protein was significantly lower, whereas HO-2 protein levels were unchanged in ROSI-treated SHR with respect to vehicle-treated SHR. Finally, increased levels of nNOS in vehicle-treated SHR were reduced in ROSI-treated SHR.

Conclusions & Inferences  Chronic ROSI treatment reverses increased SHR duodenal inhibitory response acting on CO and NO components.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information

Thiazolidinediones (TZDs) are insulin sensitizing agents acting as exogenous ligands of peroxisome proliferator-activated receptor gamma (PPARγ) receptors,1 which are highly expressed in the gastrointestinal tract as well as in kidney, liver, and pancreas.2 Despite side effects, including edema, weight gain, macular edema, heart failure, and increased bone fracture risk3 that may limit therapeutic advantages of TZDs administration, clinical and experimental evidence supports the beneficial effects of TZDs on gastrointestinal system. For example, co-treatment with rosiglitazone (ROSI) significantly improves clinical outcome in patients with ulcerative colitis with respect to 5-aminosalycilate treatment alone.4 In experimental studies, administration of TZDs limits the extent of gastric ulceration induced by intraluminal application of acetic acid,5 protects from ischemia/reperfusion injury in a rat model of gastric damage,6 and has beneficial effect in acute trinitrobenzenesulphonic acid (TNBS) – induced colitis.7

Pretreatment with ROSI markedly improves intestinal contractility in postoperative gut dysmotility.8 On this regard, it has also been shown that pretreatment with carbon monoxide (CO), an end-product of the heme oxygenase (HO) signaling, significantly diminishes the incidence and severity of postoperative ileus in mice.9–11 Up-regulation of HO-1, the inducible isoform of HO, or inhalation of CO significantly reduce cellular damage and prevent diabetic gastroparesis in non-obese diabetic mice.12 In line with these findings, we13 and others12 have reported that the HO/CO signaling pathway is involved in the altered gastrointestinal motility during experimental type 1 diabetes. Taken together, these observations suggest that some effects of TZDs in the gastric system may be related, at least partially, to the ability of modulating the HO/CO pathway.

In rodent gastrointestinal system, it has been suggested that CO and NO act as co-neurotransmitters.14,15 The NO represents one of the main inhibitory mediators within the enteric nervous system,16–18 as underlined by studies carried out in NOS knockout mice14 or in patients with pathological conditions leading to impairment of nitrergic transmission such as diabetes.19 On this regard, the ability of TZDs to significantly modulate vascular smooth muscle contractility in genetic hypertensive rats has been associated with TZDs effects on release or availability of NO.20

However, to the best of our knowledge, no attempt has been made to evaluate the effect of chronic administration of TZDs on gastrointestinal motility during the metabolic syndrome.

Therefore, the aim of the present investigation was to study the possible effects of chronic ROSI treatment on gastrointestinal motility of spontaneously hypertensive rat (SHR), a rat model of genetic hypertension with associated insulin resistance.21 More specifically, we explored the ability of ROSI treatment to modulate the HO/CO pathway alongside the nitric oxide synthase/nitric oxide (NOS/NO) signaling on the gastrointestinal motility of insulin-resistant animals. The involvement of HO/CO and NOS/NO pathways in non-adrenergic/non-cholinergic (NANC) duodenal inhibitory neurotransmission was evaluated in the absence and in the presence of HO inhibitor zinc protoporphyrin IX (ZnPPIX) alone or in combination with the NOS inhibitor NG-nitro-l-arginine (L-NNA) in control (WKY), vehicle-treated SHR, and ROSI-treated SHR. Based on results from functional studies, the expression of HO-1/HO-2 and neuronal or inducible NOS (nNOS/iNOS) isoforms was analyzed in duodenum samples from Wistar Kyoto (WKY), vehicle-treated SHR and ROSI-treated SHR.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information

Animal model and treatment protocol

All procedures in animals were performed in accordance with Guidelines and Authorization for the Use of Laboratory Animals (Italian Government, Ministry of Health).

Male 8-week-old SHR and age-matched normotensive WKY control rats weighing 220–250 g at arrival (Charles River, Calco, Milan, Italy) were used. Prior to treatment, SHR were randomly assigned into vehicle-treated (ethanol) or ROSI-treated (5 mg kg−1) group and administered drugs by gavage daily for 6 weeks. Wistar Kyoto rats were administered with vehicle only. All rats were treated between 08:00 a.m. and 08:30 a.m. and were housed in an animal facility with monitored temperature and light (12 h cycle and 21 ± 2 °C). All animals were handled and trained for 1 week to reduce the possible stress induced by gavage administration and blood pressure measurements. In all groups, systolic blood pressure (SBP) was measured non-invasively using a tail-cuff BP-2000 Blood Pressure Analysis SystemTM, (Visitech System Inc, Apex, NC, USA). Systolic blood pressure values reported are the average of three sequential blood pressure measurements that were within 10 mmHg of each other. During the treatment, SBP was monitored twice weekly, the last time 24 h before sacrifice. Body weight was measured daily, last time just before sacrifice. After 6 weeks, blood samples obtained from overnight fasted rats were collected in test tubes with or without EDTA and centrifuged at 845 g for 10 min at 4 °C. Serum or plasma was aliquoted and frozen at −70 °C until the time of biochemical assay. Then, the animals were heparinized (200 IU ip, Pfizer) and killed by decapitation under ether anesthesia. Serum insulin was measured by ELISA kit (Linco Research, St. Charles, MO, USA). Plasma glucose concentrations were determined with a diagnostic glucometer (Accu-Chek Active, Roche Diagnostics, Germany). Insulin sensitivity was assessed using the quantitative insulin-sensitivity check index (QUICKI = 1/[log (insulin) + log (glucose)]).22

Tensiometric studies

A 3-cm section of duodenum was obtained through a midline incision of the abdomen. Specimens (1 cm in length) were immediately placed in a cooled modified Krebs’ solution (pH = 7.4) of the following composition (mmol L−1): NaCl 113, KCl 4.8, MgSO4 1.2, CaCl2 (H2O) 2.2, NaH2PO4 1.2, NaHCO3 25, glucose 5.5, and ascorbic acid 5.5. Specimens were then cleaned, rinsed, and mounted longitudinally in an organ bath (20 mL) filled with modified Krebs’ solution, maintained at 37 °C and aerated with a mixture of 95% O2 and 5% CO2. One end of the longitudinal duodenum was connected to a metal rod whereas the other end was attached to a strain gauge transducer (cat. 7003 Basile, Milan, Italy). Isometric tension was measured by the PowerLab data acquisition system and recorded using Chart 5.5.5 (ADInstruments, Castle Hill, Australia). The tissue was allowed to equilibrate for at least 20 min prior to the start of the experiment. An initial load of 1.0 g tension was applied to the preparation. It was then subjected to transmural stimulation (Electrical Field Stimulation, EFS) at frequencies of 1, 3, 5, 10 Hz (14 V, 1 ms, pulse trains lasting 30 s) through two parallel platinum electrodes connected to a stimulator (Electrical Stimulator, Type 215/T, Hugo Sachs Elektronic, Germany). To obtain a non-adrenergic, non-cholinergic (NANC) inhibitory response, all experiments were carried out in the presence of atropine (3 μmol L−1) and guanethidine (3 μmol L−1). To evaluate the CO- and NO-component of NANC inhibitory response, EFS-induced relaxation was measured before and after incubation with ZnPPIX (60 min, 10 μmol L−1) or L-NNA (20 min, 100 μmol L−1), alone and in combination. At the end of each evaluation curve, specimens were stimulated with sodium nitroprusside (SNP, 50 μmol L−1). The inhibitory responses obtained with each stimulation were calculated as area under the curve (AUC) and expressed as percentage of the maximal effect elicited by SNP.

Immunoblotting detection of HO-1, HO-2 and nNOS isoforms

Duodenal preparations were homogenized and approximately 70 μg of total proteins from supernatant fractions were loaded into 12% SDS-polyacrylamide gels to separate HO-1 and HO-2 proteins. Ten percent SDS-polyacrylamide gels were used to separate nNOS protein. After electrophoresis, proteins were transferred to a nitrocellulose membrane. Immunostaining of HO-1, HO-2, and nNOS was achieved using polyclonal rabbit antibodies directed against rat HO-1 and HO-2 (Stressgen Biotechnologies Corp., Victoria, BC, Canada) and mouse monoclonal antibody against rat nNOS (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) followed by incubation with HRP-conjugated secondary antirabbit or antimouse antibodies, as appropriate (Amersham, Amersham Biosciences, Little Chalfont, Buckinghamshire, England). The blot was detected using an enhanced chemiluminescence assay (ECL, Amersham Biosciences, Little Chalfont, Buckinghamshire, England) and evaluated by densitometry (Image J, National Institutes of Health, Bethesda, MD).

Immunohistochemistry

Duodenum sections (1 cm) were isolated from rats 6 weeks after the beginning of treatment, and rinsed with cooled modified Krebs’ solution. Samples were then fixed in 4% paraformaldehyde and transverse whole duodenum sections (10 μm) obtained on a cryostat. Randomly selected sections were blocked (1 h at room temperature) in phosphate-buffered saline (PBS) solution containing 3% Bovine Serum Albumin (BSA) and 0.5% Triton X-100. Samples were immunolabeled overnight with primary antibodies against HO-1 (SC-1796 – Goat anti-HO-1 polyclonal antibody, Santa Cruz Biotechnology, Inc, Santa Cruz), HO-2 (OSA- 200 – Rabbit anti-HO-2 polyclonal antibody, Stressgen Biotechnologies Corp), nNOS (SC-5302 – mouse monoclonal antibody, Santa Cruz Biotechnology, Inc, Santa Cruz), iNOS(SC-7271 – mouse monoclonal antibody, Santa Cruz Biotechnology, Inc, Santa Cruz), and PGP9.5 (ab10419 – guinea pig polyclonal antibody, Abcam, Cambridge, UK) in PBS containing 0.3% Triton X-100. Alexa Fluor 568-conjugated donkey antigoat IgG, Alexa Fluor 488-conjugated donkey antirabbit, Alexa Fluor 568-conjugated goat antimouse IgG, Alexa Fluor 488-conjugated goat antimouse, Alexa Fluor 568-conjugated goat antiguinea pig, Alexa Fluor 488-conjugated goat antiguinea pig (Invitrogen, Eugene, OR, USA) were used as secondary antibodies, respectively.

In double labeling experiments, specimens were incubated with a mix of primary antibodies followed by corresponding mix of secondary antibodies. Negative controls were obtained by omitting the primary antibodies (Figure. S1). Images were visualized by an inverted epifluorescent microscope (Zeiss Axiovert 200) with appropriate filters and camera. Images of the myenteric plexus ganglia were captured with Axiovision Software (Carl Zeiss). The exposures time chose for immunofluorescence images were as follows (ms): HO-1: rhodamine 398; FITC 694; DAPI 62; HO-2: rhodamine 962; FITC 654; DAPI 84; nNOS: rhodamine 1397; FITC 350; DAPI 63; iNOS: rhodamine 1880; FITC: 409; DAPI 109.

Quantitative analysis of fluorescence staining for HO-1, HO-2, nNOS, and iNOS was performed with Image J software in three randomly selected areas of interest from three different sections for each group.

Drugs and chemicals

The following drugs were used: atropine sulphate, guanethidine monosulphate, SNP, L-NNA, ZnPPIX dissolved in saline solution containing 5 mmol L−1 Na2CO3 (Sigma Chemical Co., St. Louis, MO, USA). Stock solutions of ROSI (Alexis Biochemicals, Enzo Life Sciences, Inc., Farmingdale, NY, USA) were in ethanol (1%). Final dilutions were in drinking water (about 4 × dilution). Vehicle-treated WKY and vehicle-treated SHR received the same amount of ethanol as drug-treated animals.

Statistical analysis

Statistical analysis was performed by means of two-way analysis of variance, as appropriate, followed by paired t-test or Newman-Keuls multiple comparison test. The level of significance was set at < 0.05. Unless specified, results are expressed as the mean ± SEM from 8 to 12 preparations for each experiment.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information

Effect of rosiglitazone on physiologic and biochemical parameters

When compared with control age-matched WKY, SHR treated with vehicle alone for 6 weeks were heavier (< 0.001) and hypertensive (< 0.001) with substantial fasting hyperinsulinemia (but normoglycemia) (< 0.0001, Table 1). Insulin sensitivity as assessed by the surrogate index QUICKI22 was significantly reduced in vehicle-treated SHR (< 0.0001, Table 1). When compared with vehicle-treated SHR, SHR treated with ROSI (5 mg kg−1 day−1) for 6 weeks had comparable fasting glucose levels, but significantly lower fasting plasma insulin levels that were even below those in WKY rats (< 0.0001, Table 1). This reflected enhanced insulin sensitivity that was greater than that of WKY rats. Treatment of SHR with ROSI was also associated with a significant reduction in SBP (< 0.001 vs SHR, Table 1) and did not further increase body weight (Table 1). Thus, consistent with literature data, SHR exhibit metabolic and cardiovascular abnormalities resembling features of the human metabolic syndrome.23 The ROSI treatment significantly improved insulin sensitivity and systemic blood pressure without inducing weight gain in SHR.

Table 1.   Physiological and biochemical parameters obtained from 16-week-old WKY and SHR treated with vehicle or rosiglitazone (ROSI, 5 mg kg−1 day−1) for 6 weeks
ParametersWKYSHR-vehicleSHR + ROSI
  1. ADMA, asymmetric dimethylarginine; ROSI, Rosiglitazone; QUICKI, quantitative insulin-sensitivity check index; SHR, Spontaneously hypertensive rats; WKY, Wistar Kyoto.

  2. *< 0.001 vs WKY; < 0.0001 vs WKY; < 0.001 vs SHR; §< 0.0001 vs SHR (one-way anova followed by Newman-Keuls multiple comparison test).

Number of rats242424
Body weight (g)325 ± 7.5380 ± 3.5*387 ± 3.4*
Systolic blood pressure (mmHg)139 ± 1222 ± 1*187 ± 2
Fasting glucose (mg dL−1)109 ± 4.31107 ± 1.64105 ± 3.1
Fasting insulin (ng mL−1)1.55 ± 0.2713.3 ± 2.20.77 ± 0.2§
QUICKI0.279 ± 0.0060.218 ± 0.0040.334 ± 0.005§
ADMA (μmol L−1)1.59 ± 0.102.15 ± 0.200.84 ± 0.053

Although a statistically significant difference was not reached, circulating levels of asymmetric dimethylarginine (ADMA, endogenous inhibitor of NO synthase and circulating marker of oxidative stress) tended to be higher in vehicle-treated SHR than in WKY rats. A significant reduction of ADMA levels was observed in SHR treated with ROSI with respect to vehicle-treated SHR (< 0.001, Table 1).

Effect of rosiglitazone on basal EFS-induced relaxation

Colonic longitudinal muscle segments were stimulated by electrical field stimulation (EFS, 1-10 Hz; 14 V, 1 ms, pulse trains lasting 30 s) in the presence of atropine (3 μmol L−1) and guanethidine (3 μmol L−1) to obtain a NANC inhibitory response.13 For each condition, EFS-induced relaxation was measured as the AUC and calculated as percentage of maximal relaxation elicited by SNP (50 μmol L−1).

To exclude that ROSI treatment might affect the sensitivity to SNP, the inhibitory response to SNP (50 μmol L−1) was preliminarily analyzed in the absence and in the presence of L-NNA (100 μmol L−1). Results obtained show no difference in SNP-mediated inhibitory responses between groups [AUC (g × s) WKY = −5.78 ± 0.40; SHR = −6.37 ± 0.57; SHR + ROSI = −4.43 ± 0.73].

Under these conditions, basal inhibitory responses to EFS were significantly greater in vehicle-treated SHR with respect to either WKY or ROSI-treated SHR (*< 0.05; §< 0.05, Fig. 1).

image

Figure 1.  Basal duodenal longitudinal smooth muscle inhibitory responses to Electrical Field Stimulation (EFS) (1–10 Hz; 14 V, 1 ms, pulse trains lasting 30 s) in WKY, vehicle-treated SHR (SHR), and ROSI-treated SHR (SHR ROSI). Basal inhibitory responses to EFS were significantly increased in duodenum strips from vehicle-treated SHR vs WKY (< 0.05), but not in ROSI-treated SHR. The results are expressed as percentage of maximal relaxation induced by sodium nitroprusside (SNP, 50 μmol L−1). Values are expressed as mean ± SEM of 8–12 experiments. *< 0.05 vs controls; §< 0.05 vs SHR. (Two-way anova).

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Interestingly, EFS-mediated relaxation was not significantly different between WKY and ROSI-treated SHR (> 0.22) (Fig. 1). These observations suggest that EFS-dependent relaxation is differentially modulated in duodenal longitudinal smooth muscle strips from SHR and WKY rats, and that ROSI treatment partially restores the altered inhibitory responses measured in SHR.

Effect of rosiglitazone on inhibition of EFS-induced relaxation by ZnPPIX and L-NNA

To evaluate the role of HO/CO and NOS/NO signaling, modulation of EFS-induced relaxation was measured before and after 60-min incubation with the inhibitor of HO activity ZnPPIX (10 μmol L−1), before and after 20-min incubation with the inhibitor of NOS activity L-NNA(100 μmol L−1), and before and after the sequential incubation of both ZnPPIX and L-NNA. At the concentration used, ZnPPIX is able to selectively inhibit HO enzymes without affecting soluble guanilate cyclase (sGC) activity.13 When compared with basal EFS-induced inhibitory responses, pretreatment with ZnPPIX significantly reduced the inhibitory response in duodenal preparations from WKY rats (*< 0.05, Fig. 2A). This is consistent with previous studies showing that HO/CO signaling plays a role in the relaxation induced by EFS.13 A similar significant decrease in the EFS-induced relaxation was found in vehicle-treated SHR (*< 0.05, Fig. 2B). Thus, despite divergent basal levels of relaxation inhibition of the HO/CO signaling results in comparable inhibition of EFS-induced relaxation in both WKY and vehicle-treated SHR. Conversely, the inhibitory response to EFS was not affected by pretreatment with ZnPPIX in SHR treated with ROSI (Fig. 2C). This result suggests that chronic administration of ROSI may affect the HO/CO pathways in duodenal specimen from SHR. As far as the contribution of NOS/NO signaling pathway is concerned, incubation with L-NNA alone or in combination with ZnPPIX resulted in a very relevant and significant reduction of EFS-induced relaxation across the three study groups (*,§< 0.05, Fig. 2A, B, C and in-sets); this confirms the renowned role of NO as one of the main inhibitory neurotransmitter in the gut.24 Interestingly, the reduction of inhibitory response was higher in vehicle-treated SHR with respect to WKY (Fig. 2A,B and in-sets). Furthermore, the effect of L-NNA was comparable between WKY and ROSI-treated SHR suggesting that chronic treatment with ROSI was able to reduce the increased nitrergic signaling in SHR (Fig. 2A,C and in-sets).

image

Figure 2.  Duodenal longitudinal smooth muscle inhibitory responses to Electrical Field Stimulation (EFS) (1–10 Hz; 14 V, 1 ms, pulse trains lasting 30 s) after in vitro NG-nitro-l-arginine (L-NNA) (in-sets) or zinc protoporphyrin IX (ZnPPIX) and subsequent L-NNA administration in WKY, vehicle-treated SHR (SHR), and ROSI-treated SHR (SHR ROSI). (A) ZnPPIX, a HO-1 inhibitor, significantly reduces duodenal longitudinal smooth muscle inhibitory responses to EFS in WKY. Addition of L-NNA, a NOS inhibitor, further reduces this response. In-set figure shows that in vitro treatment with L-NNA alone significantly reduces inhibitory response to EFS. Values expressed as mean ± SEM of 8–10 experiments; *< 0.05 vs WKY, §< 0.05 vs WKY ZnPPIX (Two-way anova). (B) ZnPPIX significantly reduces duodenal longitudinal smooth muscle inhibitory response to EFS in vehicle-treated SHR. Addition of L-NNA further reduces this response. In-set figure shows that in vitro treatment with L-NNA alone significantly reduces inhibitory response to EFS. Values are expressed as mean ± SEM of 8–10 experiments; *< 0.05 vs SHR, §< 0.05 vs SHR ZnPPIX (Two-way anova). (C) ZnPPIX does not reduce duodenal longitudinal smooth muscle inhibitory responses to EFS in ROSI-treated SHR rats. Addition of L-NNA significantly reduces this response. In-set figure shows that in vitro treatment with L-NNA alone significantly reduces inhibitory response to EFS. Values are expressed as mean ± SEM of 8–10 experiments; *< 0.05 vs SHR ROSI; §< 0.05 vs SHR ROSI + ZnPPIX (Two-way anova).

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Effect of rosiglitazone on HO-1, HO-2, and nNOS protein expression

To gain further insights into mechanisms involved in ROSI-mediated effects on HO/CO pathways, protein levels of HO-1 and HO-2 isoforms were analyzed in tissue homogenates of duodenum from WKY, vehicle-treated SHR, and ROSI-treated SHR. No significant differences in HO-1 protein levels were observed by western blot analysis between samples from WKY and vehicle-treated SHR (Fig. 3A), whereas a significant decrease in HO-1 expression was detected in SHR treated chronically with ROSI (*< 0.05). Conversely, no significant change in protein levels of constitutive HO-2 isoform was detected in samples from WKY, vehicle-treated or ROSI-treated SHR (Fig. 3B).

image

Figure 3.  Expression of HO-1, HO-2, and nNOS in duodenum from WKY, vehicle-treated SHR (SHR), and ROSI-treated SHR rats (SHR ROSI). Representative anti-HO-1 (A), anti-HO-2 (B), and anti-nNOS (C) immunoblots of duodenal homogenates in WKY, vehicle-treated SHR, and ROSI-treated SHR (upper panels) and quantitative densitometry (lower panels). Similar expression of HO-1 was found in WKY and vehicle-treated SHR and reduced expression of HO-1 was observed in ROSI-treated SHR. No difference in HO-2 expression was found among groups. Increased levels of nNOS in vehicle-treated SHR were reduced in ROSI-treated SHR. Values are expressed as mean ± SEM of 3–5 experiments. *< 0.05 vs WKY (Newman-Keuls multiple comparison test). (M = markers; C = positive controls).

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Moreover, increased levels of nNOS (*< 0.05) were observed in vehicle-treated SHR, whereas nNOS levels were reduced below control in ROSI-treated SHR (§< 0.05, Fig. 3C).

HO-2 co-localizes with neural marker PGP9.5

To ascertain neuronal localization of HO isoforms, duodenum sections from all experimental groups were double-stained for HO-1 or HO-2, and neuronal marker PGP9.5. Fig. 4A (left and right panels) shows representative images of immunofluorescent detection in specimen obtained from WKY, vehicle-treated SHR, and ROSI-treated SHR.

image

Figure 4.  Expression, quantitative analysis of HO-1 and HO-2 and possible co-localization with PGP9.5 and DAPI in duodenum from WKY, vehicle-treated SHR, and ROSI- treated SHR rats. (A) Left panel Representatives photomicrographs of duodenum sections immunostained with antibodies against HO-1 (C, G, K), PGP9.5 (D, H, L), DAPI (E, I, M), and superimposed images (F, J, N). Visualization by inverted microscopy shows: (first row) immunoreactivity red fluorescence-associated staining for HO-1 in nerve cell bodies of myenteric plexus between the circular (cm) and the longitudinal muscle (lm) layers in duodenum sections from WKY (C), vehicle-treated SHR (G), and ROSI-treated SHR rats (K); second row shows green fluorescence-associated staining for PGP9.5, neuronal marker, in duodenum section from WKY (D), vehicle-treated SHR (H), and ROSI-treated SHR rats (L); third row shows blue fluorescence-associate staining for DAPI, nuclear marker, in duodenum section from WKY (E), vehicle-treated SHR (I), and ROSI-treated SHR rats (M); fourth row shows merge of green, red, and blue fluorescence indicative of possible co-localization for HO-1, PGP9.5 (yellow fluorescence), and DAPI in WKY (F), vehicle-treated SHR (J), and ROSI-treated SHR rats (N). ×400 magnification. Right panel Representatives photomicrographs of duodenum sections immunostained with antibodies against HO-2 (O, S, W), PGP9.5 (P, T, X), DAPI (Q, U, Y), and superimposed images (R, V, Z). Visualization by inverted microscopy shows: (first row) immunoreactivity red fluorescence-associated staining for HO-2 in nerve cell bodies of myenteric plexus between the circular (cm) and the longitudinal muscle (lm) layers in duodenum sections from WKY (O), vehicle-treated SHR (S), and ROSI-treated SHR rats (W); second row shows green fluorescence-associated staining for PGP9.5, neuronal marker, in duodenum section from WKY (P), vehicle-treated SHR (T), and ROSI-treated SHR rats (X); third row shows blue fluorescence-associate staining for DAPI, nuclear marker, in duodenum section from WKY (Q), vehicle-treated SHR (U), and ROSI-treated SHR rats (Y); fourth row shows merge of green, red, and blue fluorescence indicative of co-localization for HO-2, PGP9.5 (yellow fluorescence), and DAPI in WKY (R), vehicle-treated SHR (V), and ROSI-treated SHR rats (Z). ×400 magnification. (B) Semi-quantitative analysis showing a similar relative HO-1 immunofluorescence in WKY and vehicle-treated SHR that was reduced in ROSI-treated SHR (left panel). No difference of relative HO-2 immunofluorescence was measured (right panel) among groups. Values are expressed as mean ± SEM of three experiments. *< 0.05 vs WKY (Newman-Keuls multiple comparison test).

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The HO-1 expression was found significantly lower in ROSI-treated SHR compared with WKY and vehicle-treated SHR, as indicated by panels C, G, K of Fig 4A and by semi-quantitative analysis of red fluorescence (Fig 4B; bar graphs). When overlapped with green fluorescent staining of neuronal marker PGP9.5 (Fig 4A; D,H,L), merging yellow fluorescence images (Fig. 4A; F,J,N) may suggest, but not clearly indicate that HO-1 co-localizes in the nerve cell body of myenteric plexus between the circular and the longitudinal muscle layers (See also Figure. S2). However, co-localization was found for HO-2 in duodenal sections from WKY, vehicle-treated SHR, and ROSI-treated SHR (Fig 4A; R,V,Z). No difference in HO-2 expression among groups was revealed by semi-quantitative analysis of red fluorescence (Fig. 4B, bar graphs).

Effect of rosiglitazone on nNOS and iNOS expression

Similar to HO isoforms, nNOS protein was found predominantly expressed in the nerve cell body of myenteric plexus between the circular and the longitudinal muscle layers of duodenal sections from WKY, vehicle-treated SHR, and ROSI-treated SHR (Fig. 5A; F,J,N). When compared with levels in WKY, nNOS expression was found significantly increased in vehicle-treated SHR (Fig. 5B, bar graphs), and was similar to WKY levels in SHR treated with ROSI (Fig. 5B, bar graphs).

image

Figure 5.  Expression, quantitative analysis of nNOS and iNOS, and co-localization with PGP9.5 and DAPI in duodenum from WKY, vehicle-treated SHR, and ROSI-treated SHR rats. (A) Left panel Representatives photomicrographs of duodenum sections immunostained with antibodies against nNOS (C, G, K), PGP9.5 (D, H, L), DAPI (E, I, M), and superimposed images (F, J, N). Visualization by inverted microscopy shows: (first row) immunoreactivity red fluorescence-associated staining for nNOS in nerve cell bodies of myenteric plexus between the circular (cm) and the longitudinal muscle (lm) layers in duodenum sections from WKY (C), vehicle-treated SHR (G), and ROSI-treated SHR rats (K); second row shows green fluorescence-associated staining for PGP9.5, neuronal marker, in duodenum section from WKY (D), vehicle-treated SHR (H), and ROSI-treated SHR rats (L); third row shows blue fluorescence-associate staining for DAPI, nuclear marker, in duodenum section from WKY (E), vehicle-treated SHR (I), and ROSI-treated SHR rats (M); fourth row shows merge of green, red, and blue fluorescence indicative of co-localization for nNOS, PGP9.5 (yellow fluorescence), and DAPI in WKY (F), vehicle-treated SHR (J), and ROSI-treated SHR rats (N). Right panel Representatives photomicrographs of duodenum sections immunostained with antibodies against iNOS (O, S, W), PGP9.5 (P, T, X), DAPI (Q, U, Y), and superimposed images (R, V, Z). Visualization by inverted microscopy shows: (first row) immunoreactivity red fluorescence-associated staining for iNOS in nerve cell bodies of myenteric plexus between the circular (cm) and the longitudinal muscle (lm) layers in duodenum sections from WKY (O), vehicle-treated SHR (S), and ROSI-treated SHR rats (W); second row shows green fluorescence-associated staining for PGP9.5, neuronal marker, in duodenum section from WKY (P), vehicle-treated SHR (T), and ROSI-treated SHR rats (X); third row shows blue fluorescence-associate staining for DAPI, nuclear marker, in duodenum section from WKY (Q), vehicle-treated SHR (U), and ROSI-treated SHR rats (Y); fourth row shows merge of green, red, and blue fluorescence indicative of co-localization for iNOS, PGP9.5 (yellow fluorescence), and DAPI in WKY (R), vehicle-treated SHR (V), and ROSI-treated SHR rats (Z). (B) Semi-quantitative analysis shows increased levels of relative nNOS immunofluorescence in WKY and vehicle-treated SHR (left panel). No difference of relative iNOS immunofluorescence (right panel) was detected among groups. Values expressed as mean ± SEM of three experiments. *< 0.05 vs WKY (Newman-Keuls multiple comparison test).

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Despite low fluorescent intensity, iNOS expression co-localized with neuronal marker PGP9.5 (Fig. 5A; R,V,Z). No significant differences in iNOS levels between groups were found by semi-quantitative analysis of red fluorescence (Fig. 5B, bar graphs).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information

The purpose of the present study was to investigate whether the gastrointestinal motility of SHR, commonly considered an animal model of genetic hypertension with features of the metabolic syndrome,23 could be affected by chronic administration of ROSI, a widely used drug in patients affected by type 2 diabetes.

In line with literature data, our study shows that vehicle-treated SHRs were heavier with respect to age-matched control rats; however, administration of ROSI did not result in further increase in weight gain, an adverse effect mainly due to fluid retention reported in patients treated with this drug.3 As expected, vehicle-treated SHRs showed high levels of systolic blood pressure, hyperinsulinemia but normoglycemia with a reduced insulin sensitivity as indicated by the surrogate index QUICKI.25,26 In agreement with several scientific reports, these metabolic parameters were efficiently corrected by chronic administration of ROSI. In fact, it has been shown that TZDs are able to restore insulin sensitivity as well as to lower SBP in both animals and humans.23,27–29 Moreover, the recently shown ability of ROSI to decrease oxidative stress in SHR30 was confirmed by the markedly reduced levels of ADMA (circulating marker of oxidative stress) in ROSI-treated SHRs.

To evaluate the role of HO/CO and NOS/NO pathways in the NANC inhibitory neurotransmission of duodenal motility after chronic treatment with ROSI, we first studied the basal inhibitory response to transmural stimulation in the presence of atropine and guanethidine. Results obtained indicate that, when compared with WKY, the inhibitory response to EFS is significantly different in vehicle-treated SHRs, but not in ROSI-treated SHRs. This suggests that (i) the type of group affects NANC inhibitory response to EFS and (ii) the last effect is reversed by administration of ROSI.

In line with these observations, Pattern et al., (2004) have shown that in vitro contractile responses may depend on the type of the group, and that isolated ileum and colon of SHR had a lower contractile response to PGF and PGE2. In part, the altered basal gastrointestinal motility found in vehicle-treated SHRs has been ascribed to the deleterious effects of dietary fat on gut contractility in this animal model.31

It was of interest to find that the difference of the basal inhibitory response to EFS in vehicle-treated SHRs was ameliorated by chronic treatment with ROSI. To the best of our knowledge, this study is the first to acknowledge the positive effects of ROSI on altered inhibitory response of SHRs.

To evaluate whether the two main inhibitory gastrointestinal neurotransmitters i.e. CO and NO may be involved in the effect of ROSI, we explored the role of HO/CO pathway by in vitro administration of ZnPPIX, an HO inhibitor. As expected,13 the exclusion of one of the inhibitory neurotransmitters (i.e. CO) of NANC neurotransmission significantly reduced the inhibitory response to EFS in both WKY and vehicle-treated SHRs. Conversely, this effect was not found in duodenal preparations from ROSI-treated SHRs. This finding could be explained by a reduced CO production and therefore a limited effect of CO on its downstream target.

Blocking the endogenous production of NO confirmed the main role of NO as inhibitory neurotransmitter16–18: in all the experimental groups, the inhibitory response to EFS was more markedly reduced under incubation with the NOS inhibitor L-NNA than with the HO inhibitor ZnPPIX. The reduction was higher in vehicle-treated SHR, but similar in WKY and ROSI-treated SHR. This finding substantiates the hypothesis that increased basal inhibitory response to EFS in specimens from vehicle-treated SHRs can be ascribed to NO, whose relative production is higher in SHR than in WKY. On the other hand, these same findings underline the comparable importance of CO on regulation of gut contractility in vehicle-treated SHRs and WKY. Treatment with ROSI significantly ameliorated the increased inhibitory response to EFS in SHRs by both reducing the NO-mediated component and by abolishing the CO-mediated dependent effects.

Although the consequences of these in vitro observations were not evaluated in terms of changes in duodenal or intestinal transit in vivo, other studies suggest a direct dependence between response to EFS in vitro and duodenal/intestinal transit in vivo: Zakhary et al. (1997) have shown that, in NANC conditions, ileal segments from mice with targeted deletion of HO or nNOS exposed to EFS (2, 4, 8, 16 Hz) relaxed less than segments from wild-type mice. In parallel, the intestinal transit in HO-2 knockout mice was significantly slower than in wild-type controls, whereas gastric emptying was substantially delayed in nNOS knockout mice.32 Taken together, these data further support the hypothesis that impaired relaxation in duodenal or ileal musculature may result in delayed gastrointestinal transit.

To verify whether the minimal effect of ZnPPIX incubation in ROSI-treated SHR could be ascribed to a reduced expression of HO, protein levels of both HO isoforms were measured in our samples. Interestingly, a significant reduction in HO-1 levels was observed only in duodenum homogenates from ROSI-treated SHR, whereas the levels of HO-2 were not affected by both types of group or treatment. Our data are in agreement with De Backer et al.(2009), showing that preadministration of ROSI restores levels of HO-1 increased after surgical manipulation.8 In line with results from western blot analysis, a decreased expression of HO-1 was found by immunochemistry in the nerve cell bodies of myenteric plexus from duodenal preparations of ROSI-treated SHR. Thus, in an animal model of metabolic syndrome, prolonged administration of ROSI may qualitatively and quantitatively modulate the NANC HO/CO component of duodenal inhibitory neurotransmission.

Similarly, evaluation of NOS expression in duodenal tissues and homogenates of WKY and SHR animal supports the findings of functional experiments. Indeed, the higher ability of L-NNA to counteract the EFS inhibitory response in vehicle-treated SHRs is corroborated by the increased levels of nNOS protein found by western blot and immunohistochemical analysis. Moreover, and consistent with the reduced response to L-NNA on the inhibitory response to transmural stimulation, a lower expression of nNOS was found in duodenal preparations of ROSI-treated SHR.

Increased levels of iNOS have been reported in several tissues of SHR compared with WKY.33 Overproduction of NO by increased iNOS expression may enhance the inhibitory effect of this mediator and lead to reduced colon contractility.34 In our experiments, iNOS levels were not found significantly increased in SHR. In part, this may be due to technical limits of western blot or immunofluorescent methods. Nevertheless, a significant increase in nNOS levels was observed in duodenal samples from vehicle-treated SHR. As far as we are concerned, this is the first time that increased levels of constitutive nNOS isoform is documented in gastrointestinal tissues from SHR. Therefore, in our conditions, the increased levels of NO might depend on higher activity of enhanced nNOS expression. Interestingly, along with improved inhibitory response, a significantly reduced expression of nNOS was found in SHR treated with ROSI. This finding is in agreement with other studies documenting the ability of ROSI to reduce the increased expression of NOS isoforms35 and to ameliorate the intestinal motility by reducing NO overproduction.8

In conclusion, our study further expands the functional effects of TZDs in an animal model of metabolic syndrome, and suggests a possible role for HO/CO and nNOS/NO signaling in ROSI-mediated modulation of gastrointestinal motility.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information

This work was supported, in part, by grant ORBA10DLZD from Ministero Italiano Università e Ricerca to M.A.P, M.M. and M.A.D.

Author Contribution

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information

LD, SG, AZ, and MAP performed the research; MM contributed to analyses of data and writing the manuscript; MAD designed the research study, analyzed the data and wrote the manuscript.

References

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Competing Interests
  9. Author Contribution
  10. References
  11. Supporting Information

Figure S1. Green and red immunofluorescence negative controls.

Figure S2. Expression of HO-1 and possible colocalization with PGP9.5 and DAPI in duodenum from WKY, vehicle-treated SHR, and ROSI- treated SHR rats. Representatives photomicrographs of duodenum sections immunostained with antibodies against HO-1 (C, G, K), PGP9.5 (D, H, L), DAPI (E, I, M), and superimposed images (F, J, N). Visualization by inverted microscopy shows: (first row) immunoreactivity red fluorescence-associated staining for HO-1 in nerve cell bodies of myenteric plexus between the circular (cm) and the longitudinal muscle (lm) layers in duodenum sections from WKY (C), vehicle-treated SHR (G), and ROSI-treated SHR rats (K); second row shows green fluorescence-associated staining for PGP9.5, neuronal marker, in duodenum section from WKY (D), vehicle-treated SHR (H), and ROSI-treated SHR rats (L); third row shows blue fluorescence-associated staining for DAPI, nuclear marker, in duodenum section from WKY (E), vehicle-treated SHR (I), and ROSI-treated SHR rats (M); fourth row shows merge of green, red, and blue fluorescence indicative of co-localization for HO-1, PGP9.5 (yellow fluorescence), and DAPI in WKY (F), vehicle-treated SHR (J), and ROSI-treated SHR rats (N). ×630 magnification.

FilenameFormatSizeDescription
NMO_1798_sm_FigS1.TIF134KSupporting info item
NMO_1798_sm_FigS2.tif285KSupporting info item

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