The growing body of evidence has shown that insulin resistance (IR) contributes to vascular endothelial dysfunction. Under the IR state, overproduction of reactive oxygen species (ROS), along with oxidative stress induced by IR, may be playing a major role in the pathogenesis of endothelial dysfunction, hypertension and atherosclerosis.1-4 Recently, increasing attention has been drawn to the beneficial effects of heme oxygenase-1 (HO-1) in the cardiovascular system. HO-1 catalyzes oxidative degradation of heme into biliverdin, carbon monoxide (CO) and ferrous ions. These breakdown products have proven highly anti-oxidative and vasomodulatory, which be important for the regulation and maintenance of angiostasis, anti-platelet effects and inhibition of smooth muscle cell proliferation.5-8 However, the effects of HO-1 on endothelial dysfunction in IR states and the mechanisms by which it works remain unclear.
In the present study, we treated the IR rats with hemin (an inducer of HO-1) and zinc protoporphyrin-IX (ZnPP-IX, an inhibitor of HO-1) and then examined the effects of HO-1 on vascular function of thoracic aorta in IR rats. Furthermore, the probable mechanisms of HO-1 against endothelial dysfunction in the IR state were also demonstrated.
Eight-week-old healthy male Sprague-Dawley (SD) rats weighing 220-260 g were obtained from the Experimental Animal Center of Hebei Province (Certificate No. 04064). The rats were kept at (22 ± 2)°C with a light cycle of 12-hour light/12-hour dark and were given free access to standard chow diet and tap water. After acclimation, these rats were randomly assigned to receive either standardized chow diet (18% fat, 33% protein, and 48% carbohydrate) as the normal control group or a high-fat diet (HFD, 59% fat, 21% protein, and 20% carbohydrate) as the IR group for 6 weeks. Body weight and blood glucose levels of all rats were determined weekly throughout the study. At week 6 the rats underwent a hyperinsulinemic-euglycemic clamp test for assessment of insulin sensitivity (see below).
Hyperinsulinemic-euglycemic clamp techniques
As previously described9 the rats were fasted for 12-14 hours overnight before the clamp study began the next morning. Rats were anesthetized by an intraperitoneal injection of amobarbital sodium (25 mg/kg) individualized to each subject’s body weight. Rats were cannulated in the right jugular vein for infusion of glucose and insulin (using separate lines) and in the left carotid artery for blood sampling. The glucose and insulin solutions were stored in two digital syringe pumps and jointed by a “Y” connector to the jugular catheter. Insulin (Novolin R, Novo Nordisk Pharmaceuticals, Denmark) was infused at a rate of 1.67 m·kg-1 min-1 through the jugular vein catheter from 0 to 120 minutes. Glucose concentrations were clamped at euglycemic levels ((5.0 ± 0.5) mmol/L) by a variable rate infusion of 10% glucose. Blood glucose (BG) levels were serially monitored with a Glucometer (Surestep, Johnson, USA), and glucose infusion rates (GIR) were adjusted every 5 to 10 minutes as needed to maintain the BG at euglycemic levels ((5.0 ± 0.5) mmol/L). Clamping was achieved by 90 minutes and maintained for 30 minutes. The mean GIR was calculated based on GIR readings corresponding to the last 7 samplings over 90-120 mg·kg-1 ·min-1.
The IR rats (n=44), after validation with the hyperinsulinemic-euglycemic clamp test, were further randomized into 3 subgroups; namely, the IR control group, hemin treated IR group and ZnPP-IX treated IR group. Subsequent treatments to the IR model rats and the normal control group were designed as follows: (1) The IR control group (n=26), 12 rats were killed immediately and evaluated for all study measures (0-week IR control group, n=12), whereas the remaining 14 were maintained on a high-fat diet and injected with normal saline intraperitoneally every other day for 4 weeks (4-week IR control group, n=14); (2) The hemin treated IR group (n=10), all IR rats continued on a high-fat diet and were given intraperitoneal injections of hemin (inducer of HO-1, 30 μmol/kg) every other day for 4 weeks; (3) The ZnPP-IX treated IR group (n=8), the IR rats resumed a high-fat diet and received intraperitoneal ZnPP-IX (inhibitor of HO-1, 10 μmol/kg) every other day for 4 weeks; (4) The normal control group (n=12), the rats remained on standardized chow diet and were treated with intraperitoneal injection of normal saline every other day for 4 weeks.
Measurement of systolic arterial blood pressure (SABP)
SABP was measured by tail-cuffed microphotoelectric plethysmography and reported as the mean value of 3 consecutive readings taken 2 minutes apart.
Measurement of biochemical indexes in blood
With the rat sedated with intraperitoneal injection of amobarbital sodium (25 mg/kg), 0.3 ml of blood was collected by cardiac puncture for blood gas analysis. The level of arterial CO was presented as % of HbCO to total hemoglobin (Hb), which was detected by a Chiron 855 analyzer (Chrion Diagnostics, Medfied, USA).
Nitrogen oxide (NO) production was assessed by spectrophotometric measurement of serum nitrite, a stable degradation product of NO, using a specific kit and following the manufacturer’s instructions (Jiancheng Bioengineering Ltd, Nanjing, China).
Total cholesterol and triglycerides
Total cholesterol (TC) and triglycerides (TG) contents were assayed in fasting serum samples by spectrophotometry using a specific kit following manufacturer’s instructions using a specific kit (Jiancheng Bioengineering Ltd).
Insulin was measured using a radioimmunoassay kit (China Institute of Atomic Energy, Beijing, China).
Measurement of biomarkers in thoracic aorta
Thoracic aorta tissues were homogenized in normal saline. The levels of NO, activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), total antioxidant capacity (TAOC), malondialdehyde (MDA) and superoxide dismutase (SOD) in the tissue supernatant were determined by specific detection kits according to the manufacturer’s instructions (Jiancheng Bioengineering Ltd).
Vasoreactive tensometry of thoracic aortic rings
Thoracic aortic rings (TARs) were tailored to 2-3 mm in width and positioned vertically in a tissue bath of 6 ml Krebs Ringer bicarbonate (KRB) solution (37°C, containing 10-5 mol/L indomethacin). The vascular tensometry was performed using a Model-3066 platform recorder (No. 4 Instrument Manufactory, Sichuan, China). The tension of TARS rised slowly to 2 g within 15 minutes and sustained for 60 minutes, during which, the tissue bath solution was renewed every 15 minutes. When baseline tension became stable the TARs was further studied for: (1) contractive responses to 10-6 mol/L phenylephrine (PE); (2) endothelium-dependent relaxation responses to 10-6 mol/L acetylcholine (ACh); (3) curve of cumulative dose responses to 10-8 -10-5 mol/L ACh upon a pre-contraction generated by 10-6 mol/L PE; (4) curve of cumulative dose responses to 10-8 -10-5 mol/L PE; (5) endothelium-independent relaxation responses to 10-6 mol/L sodium nitroprusside (SNP). After completion of all studies above, the TARs were dehydrated in a thermostat (60-70°C) until their weight was constant. We recorded the dry weight of TARs and used it to standardize the tension of the TARs. The results of the contractive response were presented as grams per milligram dry weight (g/mg) and those of the relaxation response as percent contractive responses to 10-6 mol/L PE.
Detection of HO-1 mRNA expression with semi-quantitative RT-PCR
Total RNA of thoracic aortas in each group of rats was extracted using TRIzol Reagent kits (Invitrogen Life Technologies, USA) according to the manufacturer’s instructions. Quantification and purity of RNA were assured by the ratio of OD260 to OD280 determined by a U-2000 ultraviolet spectrophotometer (Hitachi, Japan); where RNA samples with the OD260 to OD280 ratio between 1.8 and 2.0 were used for reverse transcription polymerase chain reaction amplification (RT-PCR) with the Geme Amp PCR System 9600 (Perkin-Elmer, USA). The first-strand cDNA was synthesized from the total RNA with Omniscript RT (Qiagen) following the manufacturer’s instructions. The cDNA products were amplified by PCR in a total volume of 50 μl containing 2.5 U Taq DNA polymerase (Promega) and 10 pmol each of the upstream and downstream primers. After predenaturation at 94°C for 2 minutes, 35 cycles were allowed to run for 45 seconds at 94°C, followed by 45 seconds at 56°C and 45 seconds at 72°C, and a final extension at 72°C for 5 minutes. The primers for HO-1 were sense 5′-CTGGAAGAGGAGATAGAGCGAA-3′, and the antisense 3′-TCTTAGCCTCTTCTGTCACCC-T-5′. Primers for β-actin were sense 5′-GAGACCTTC-AACACCCAGCC-3′, antisense 5′-GCGGGGCATCGG-AACCGCTCA-3′ (Shanghai Bioengineering Ltd., Shanghai, China). The predicted sizes of the amplified HO-1 and β-actin DNA products were 433 bps and 374 bps, respectively. Amplified products (5 μl) were loaded onto 1.5% agarose gels previously stained with 0.5 μg/ml ethidium bromide, electrophoresed at 100 V for 30 minutes and then examined under a UVP gel imaging system (UVP Co. USA). Images were analyzed with the Gel-Pro Analyzer Version 3.0, and the semi-quantitative measure of mRNA expression was expressed as the ratio of integrated optical density (IOD) with HO-1/β-actin.
Detection of HO-1 protein expression using Western blot
The thoracic aortas of each group of rats were lysed with buffer (Tris-Cl 50 mmol/L, pH 6.8; dithiothreitol (DTT) 100 mmol/L; 2% sodium dodecyl sulfate (SDS); 12% glycerol) for total protein extraction. Aliquots of each sample containing 180 μg of total protein were fractionated on 10% polyacrylamide-sodium dodecyl sulfate gels (SDS-PAGE) and transferred to a nitrocellulose membrane. Western blot was performed using the monoclonal antibody for HO-1 (Stressgen Co., Canada). GADPH protein was used as an internal control. Gels were analyzed with Gel-Pro Analyzer Version 3.0 and the semi-quantitative measure of protein expression was expressed as the ratio of IOD with HO-1 protein/GADPH.
Standard software (SPSS 11.5 for Windows; SPSS) was used for statistical analysis. Measurement data were expressed as mean±standard deviation (SD). The differences between the means were tested using the student’s t test or one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.
After 6 weeks the GIR of rats on HFD, as shown by hyperinsulinemic-euglycemic clamp, was lower than that of normal control rats on standardized chow diet ((7.63±2.85) mg·kg-1·min-1 vs (12.71±1.68) mg·kg-1·min-1, P<0.01), suggesting decreased insulin sensitivity and development of insulin resistance in HFD rats; hence a successful establishment of the IR models.
SABP and metabolic parameters in rats
Compared with normal controls, rats in the IR control groups progressively developed hypertension as evidenced by elevated SABP (P<0.01). In addition, the IR control group demonstrated metabolic abnormalities with significantly increased levels of BG, insulin, TC and TG as compared with the normal control group (P<0.01). The 4-week treatment with hemin (as given to the hemin group) showed no significant effect on BG, insulin, TC or TG compared with those in 4-week IR control group(P>0.05), however, it significantly decreased SABP (P<0.01) to a level comparable with that of the normal control group (P>0.05). The 4-week treatment with ZnPP-IX (as given to the ZnPP-IX group) significantly elevated SABP compared to the 4-week IR control group (P<0.05, Table 1).
Changes in blood CO and NO contents
Compared with normal control group, the blood CO contents were decreased in 0-week and 4-week IR control rats (P<0.01) and the serum NO levels of the IR control rats were markedly increased (P<0.01). After treatment with hemin in the IR rats, notably higher levels of blood CO (P<0.01) and decreased serum NO (P<0.05) were measured compared with the 4-week IR control rats. Alternatively, after treatment with ZnPP-IX in the IR rats the contents of blood CO further decreased (P<0.01) and the serum levels of NO significantly increased compared with the 4-week IR control rats (P<0.01, Table 2).
Changes in aortic NO contents, iNOS and eNOS activities
Compared with the normal control group, the NO contents in the aorta were increased in 0-week and 4-week IR control rats (P<0.05), and the iNOS activity was significantly enhanced (P<0.01), while eNOS activity was significantly inhibited (P<0.01). Treatment with hemin for 4 weeks in IR rats resulted in decreased NO contents (P<0.05), inhibition activity of iNOS (P<0.01) and increased activity of eNOS (P<0.01) in the aorta compared with 4-week IR control rats. ZnPP-IX treatment in IR rats caused a further increase in aortic contents of NO (P<0.05) and enhancement of iNOS activity (P<0.05), but had no effect on eNOS activity (P>0.05, Table 2).
Changes in intra-aortic biomarkers of oxidative damage
Compared with the normal control group levels of TAOC and SOD in the aorta at 0-week and 4-week in the IR control group were reduced (P<0.05 and P<0.01), whereas the contents of MDA progressively increased (P<0.01). After treatment with hemin for 4 weeks in the IR rats, the levels of TAOC and SOD were significantly increased (both P<0.01), while MDA decreased compared with the 4-week IR control group (P<0.05). Alternatively, after treatment with ZnPP-IX in the IR rats, lower contents of TAOC and SOD and higher levels of MDA in the aorta were measured compared with the normal control group (P<0.01), while there were no significant differences compared with the 4-week IR control group (P>0.05, Table 3).
Outcomes of vascular reactivity
Compared with the normal control group the endothelium-dependent relaxation responses to 10-6 mol/L ACh in 0-week and 4-week IR control rats were diminished (both P<0.01). Administration of hemin in IR rats appeared to improve the disordered vasorelaxation of TARs to ACh compared with what was found in the 4-week IR control group (P<0.01), while treatment with ZnPP-IX in IR rats aggravated the vasorelaxation disturbance (P<0.05). Significantly, the contractive responses of TARs to 10-6 mol/L PE in 0-week and 4-week IR control rats were higher compared with the normal control group (P<0.01). Compared with 4-week IR control rats, it was significantly reduced in the hemin treated IR group (P<0.01) to a level comparable with that of the normal control group, and ZnPP-IX treatment did not alter the contractions to PE in the aortas of IR rats(P>0.05). No changes were detected in endothelium-independent relaxation responses to 10-6 mol/L SNP among the four groups (P>0.05, Table 4).
HO-1 mRNA and HO-1 protein assays in aorta
Compared with the normal control rats, HO-1 mRNA and HO-1 protein expression did not show significant changes in the aorta in the 4-week IR control group nor in the ZnPP-IX treated IR group (P>0.05). However, increased expression of HO-1 mRNA and HO-1 protein were observed in the hemin treated IR group; where the IOD values were 2.01 fold (P<0.01) and 1.75 fold (P<0.05), respectively, higher than those in the normal control group (Figures 1 and 2).
In the present study, rats on HFD for 6 weeks exhibited typical signs of IR as featured by hyperglycemia, hypertriglyceridemia, hypercholesterolemia, hyperinsu-linaemia and hypertension. The GIR of HFD rats, as shown by hyperinsulinemic-euglycemic clamp, was lower than that of standardized chow fed rats, suggesting decreased insulin sensitivity and development of insulin resistance in HFD rats, hence successful establishment of the IR models.
Hyperlipemia and hyperglycemia under IR states have been shown to trigger oxidative stress,10-12 with down-regulation of several key antioxidases in vascular tissues, including SOD, catalase and glutathione peroxidase.11 MDA, a breakdown product from oxidation reactions of polyunsaturated fatty acids, can be a reliable marker of free radical-mediated lipid peroxidation, as well as one of the major indicators of free radical-mediated tissue injury.12 In our study, the higher level of MDA and lower levels of TAOC and SOD in the aorta of the IR control group animals compared with the normal control group outlined the presence of oxidative damage and impaired antioxidant capacity in IR rats. This may be one of the mechanisms underlying endothelium dysfunction.
In our experiment IR rats showed vascular dysfunction. The diminished endothelium-dependent relaxation responses and enhanced contractive responses to PE in the TARs led to an overall elevation of SABP, suggesting a much higher sensitivity of the aorta to adrenergic stimulants under the IR state. However, whether such enhanced vascular responses means a liability to hypertension warrants further study.
HO-1 is the initiator of, and rate-limiting enzyme involved in, heme metabolism, and catalyzes oxidative degradation of heme into biliverdin, CO and ferrous ions. These breakdown products have proved highly anti-oxidative and vasomodulatory, which may be important in protection of cells against oxidative stress damage.13,14 Upregulation of HO-1 gene expression in endothelial cells can be induced after administration of hemin. High expressions of HO-1 in aortas may, therefore, be important in maintaining high levels of SOD, thus diminishing extracellular O-2 and decreasing endothelial cell apoptosis.15,16 Our study demonstrated that MDA contents decreased while SOD activity and the TAOC level increased after hemin injection accompanying the increased expressions of both the HO-1 gene and protein, indicating the antioxidative effect of the HO-1 protein. Alternatively, after treatment with ZnPP-IX in IR rats, a lower contents of TAOC and SOD and a higher level of MDA in aorta were measured.
It is not clear from the present study whether the improvement of endothelial dysfunction in aorta is directly related to the antioxidation effects of HO-1 in IR states. However, the substantial regulation of HO-1 on vasoactive substances such as CO and NO may have a direct impact on the vascular endothelial function.
NO is one of the endothelium-generated vasoactive substances. Physiologically, eNOS accounts for producing most of the NO, which in turn participates in a wide range of normal biological events; including vasodilation, anti-platelet effects and inhibition of smooth muscle cell proliferation. NO production due to iNOS seems to be minimal under normal condition; however, when activated by some predisposing factors, this may present as persistent NO surges which give rise to cell damage and toxic effects.17 In IR states, the enhanced expression of iNOS and excessive NO production were associated with oxidative stress and activation of inflammatory pathways, which may lead to endothelial dysfunction.18-21 According to our study, iNOS activity and NO production were higher and eNOS activity was lower in IR rats than in the normal control group, suggesting that the iNOS/NO pathway may be related to endothelial dysfunction in the aorta of IR rats.
In our study, a lower level of blood CO was shown in the 4-week IR control rats. After treatment with hemin, a high level of CO, improved endothelium-dependent vasorelaxation disorder and the reversal of SABP were observed. As a gaseous messenger, CO mediates activation of soluble guanylate cyclase which leads to increased cGMP production in vascular tissues. The soluble guanylate cyclase-cGMP pathway has been implicated in the effects of CO on vascular relaxation and inhibition of platelet aggregation and smooth muscle cell proliferation.22,23 In addition, CO may dilate blood vessels by directly activating calcium-dependent potassium channels.24 A strong association between vasorelaxation disorder and development of hypertension has been documented. The results of our study suggest that, in IR states, a lower level of CO might be a factor that lead to arterial endothelium-dependent vasorelaxation disorder, which may be involved the development of hypertension. Therefore, we supposed that administration of inducer of HO-1 would be helpful for treatment of hypertension and improvement of vasculopathy due to facilitated CO production.
We observed that treatment with hemin, an inducer of HO-1, elevated the CO content, inhibited iNOS activity and decreased NO production, while it enhanced the expression of eNOS, thus improving aortic vasodilatation dysfunction. Alternatively, the reverse results were obtained after ZnPP-IX treatment. Taken together, these results suggested that the interaction of the HO-1/CO and NOS/NO systems could play a protective role against vascular injury in the IR state. Upregulation of HO-1 could promote CO production and eNOS expression, inhibit iNOS activity and decrease NO production, thus improving the vasodilatation dysfunction of the artery.
In summary, the findings of this study suggest that obvious oxidative damage and vasoactive substances are present in the IR state. The protective effects of HO-1 against endothelial dysfunction in the aorta may be due to its action as an antioxidant and regulation of vasoactive substances. It is proposed that hemin, inducer of HO-1, could be a potential therapeutic option for vascular dysfunction in the IR state.
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Keywords:© 2008 Chinese Medical Association
heme oxygenase-1; hemin; insulin resistance; oxidative stress