To investigate the effects of iptakalim on endothelial dysfunction induced by insulin resistance (IR) and to determine whether iptakalim improved IR associated with hypertension in fructose-fed rats (FFRs) and spontaneously hypertensive rats (SHRs).
Human umbilical vein endothelial cells (HUVECs) were used for in vitro study. The levels of endothelial vasoactive mediators and eNOS protein expression were determined using radioimmunoassays, ELISAs, colorimetric assays or Western blotting. Sprague-Dawley rats were fed with a high-fructose diet. In both FFRs and SHRs, tail-cuff method was used to measure systolic blood pressure (SBP), and hyperinsulinemic- euglycemic clamp was used to evaluate IR states.
(1) Cultured HUVECs incubated with the PI3-kinase inhibitor wortmannin (50 nmol/L) and insulin (100 nmol/L) induced endothelial dysfunction characterized by significantly reduced release of NO and expression of eNOS protein, and significantly increased production of ET-1. Pretreatment with iptakalim (0.1–10 μmol/L) could prevent the endothelial dysfunction. (2) In FFRs, the levels of SBP, fasting plasma glucose and insulin were significantly elevated, whereas the glucose infusion rate (GIR) and insulin sensitive index (ISI) were significantly decreased, and the endothelium-dependent vascular relaxation response to ACh was impaired. These changes could be prevented by oral administration of iptakalim (1, 3, or 9 mg·kg−1·d−1, for 4 weeks). The imbalance between serum NO and ET-1 was also ameliorated by iptakalim. (3) In 2–4 month-old SHRs (IR was established at the age of 4 months), oral administration of iptakalim (1, 3, or 9 mg·kg−1·d−1, for 8 weeks) significantly ameliorated hypertension and increased the GIR to the normal level.
These results demonstrate that iptakalim could protect against IR-induced endothelial dysfunction, and ameliorate IR associated with hypertension, possibly via restoring the balance between NO and ET-1 signaling.
Epidemiological evidence has demonstrated that insulin resistance (IR) and compensatory hyperinsulinemia play important roles in the onset and persistence of essential hypertension1, 2, 3, 4. IR has been reported in several animal models of hypertension, including spontaneously hypertensive rats (SHRs) and fructose-fed hypertensive rats (FFRs)5. Humans with a genetic predisposition to hypertension tend to develop IR with hyperinsulinemia6. Because IR and hypertension often co-existed, the effects of antihypertensive drugs on IR have been highlighted. Of the first-line antihypertensive drugs, only angiotensin-converting enzyme (ACE) inhibitors and angiotensin II (Ang II) type 1 (AT1) receptor antagonists have been reported to have benefits in patients with IR7. However, despite intense research, studies identifying drugs able to lower blood pressure and reverse IR are still lacking.
Recent research indicates that endothelial dysfunction plays a key role in linking the pathogenesis of hypertension and IR, and the vicious circle involving hypertension, IR, and endothelial dysfunction has been documented8, 9, 10, 11. Endothelial dysfunction associated with hypertension and IR is often characterized by an imbalance between NO and ET-1 signaling12. Therefore, restoration of endothelial function may be a useful way to inhibit the development of IR in hypertensive patients.
The pharmacological approach to restoring the balance between nitric oxide (NO) and endothelin-1 (ET-1) signaling in the endothelium has been well documented13. A promising candidate in this field is iptakalim, a novel adenosine triphosphate (ATP)-sensitive potassium (KATP) channel opener that has been shown to have antihypertensive effects in different animals models of hypertension and in humans14, 15. Our laboratory has recently demonstrated that iptakalim can protect against endothelial dysfunction induced by cardiovascular risk factors, such as homocysteine, hyperglycemia, and hyperuricemia, by activating the SUR2B/Kir6.1 subtype of KATP channels in endothelial cells16, 17, 18, 19. Because iptakalim has highly efficacious antihypertensive and potent endothelial protective effects, we hypothesized that iptakalim treatment has beneficial effects on the IR associated with hypertension, through protecting endothelial function.
Here, we first investigated the protective effects of KATP activation by iptakalim on endothelial dysfunction induced by insulin and wortmannin in human umbilical vein endothelial cells (HUVECs) and then explored its effects on IR associated with hypertension in fructose-fed rats (FFRs) and spontaneously hypertensive rats (SHRs). We found that iptakalim improved the IR associated with hypertension by restoring the balance between nitric oxide and endothelin-1 signaling, which was a different mechanism than that of drugs affecting the renin-angiotensin system (RAS). These investigations provide experimental evidence for a novel strategy to treat the IR associated with hypertension.
Materials and methods
Iptakalim was synthesized by the Thadweik Academy of Medicine (Beijing, China). Rosiglitazone, insulin, wortmannin, and glibenclamide were purchased from Sigma-Aldrich (St Louis, MO, USA). Benazepril was a gift from Novartis (Beijing, China).
Endothelial dysfunction induced by insulin resistance with hyperinsulinemia in vitro
Human umbilical vein cells (HUVECs) were obtained from Cascade Biologics (Portland, OR, USA). Cells from the third to fourth passage were used for experiments. To mimic IR, wortmannin, a partial inhibitor of phosphoinositide 3-kinase (PI3-kinase), and an high concentration of insulin were added to the cells20. Briefly, HUVECs were preincubated with or without iptakalim (10 nmol/L–100 μmol/L) for 6 h and then incubated with wortmannin (50 nmol/L) and insulin (100 nmol/L) for 20 h. Glibenclamide (10−5 mol/L) was added 1 h before iptakalim pretreatment. The medium was collected for the NO, ET-1, 6-Keto-PGF1α (namely 6-keto, a PGI2 metabolite) and PAI-1 assays. The level of NO, including nitrite and nitrate, was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); PAI-1 was measured by ELISA using a commercial kit (Beijing BIO-LAB Materials Institute, Beijing, China); and the other substances were measured using a commercial radioimmunoassay kit (Eastern Asia Radioimmunity Research Institute, Beijing, China) according to the manufacturer's instructions. The expression of eNOS protein was detected using Western blot analysis. The methods for cell lysis and Western blot analysis have been described previously21. The monoclonal anti-eNOS antibody was used at the manufacturers' suggested concentration. A secondary antibody (rat anti-rabbit IgG-horseradish peroxidase) and an enhanced chemiluminescence kit were used to visualize the immunoreactive bands. Multiple exposures of films were obtained to determine the optimal exposure time. The protein bands were scanned by a densitometer, and the relative intensities were quantified using ImageQuant software.
Animals and drug treatments
Six-week-old male Sprague-Dawley (SD) rats were provided by the Experimental Animal Center of the Beijing Institute of Pharmacology and Toxicology. Male SHRs and normotensive Wistar-Kyoto (WKY) rats of various ages were obtained from the Institute of Cardiovascular Diseases, Chinese Academy of Medical Sciences (Beijing, China). The rats were housed in groups of five, under a 12-h light/dark cycle at a temperature of 24±1 °C and relatively humidity of 56%±10%, with free access to water and a normal diet (22% protein). All animal procedures were performed in accordance with the Declaration of the National Institutes of Health Guide and Use of Laboratory Animals (Publication 85–23, revised 1985) and approved by the local animal care and use committee.
Six-week-old male Sprague-Dawley rats were divided into 7 groups (n=14 per group) and fed ad libitum one of the following diets for 8 weeks: standard chow (control rats), standard chow with iptakalim (9 mg·kg−1·d−1), a high-fructose diet (fructose-fed rats), or a high-fructose diet with iptakalim (1, 3, or 9 mg·kg−1·d−1) or rosiglitazone (5 mg·kg−1·d−1). During the last 4 weeks, all the medications were given daily by gavage. In a separate study, the effect of iptakalim (3 mg/kg) on endothelial function in the fructose-fed rats during the last 4 weeks was compared to enalapril (3 mg/kg). The high-fructose diet contained 60% fructose, 13% fat, and 20% protein by calories. Body weight, systolic blood pressure (SBP), and heart rate (HR) were determined every week throughout the study. The SBP and HR were measured using a tail-cuff method in conscious rats.
Both SBP and HR were elevated, while IR was not established in the 2- and 3-month-old SHRs. IR was established in the 4-month-old SHRs. The therapeutic arrangement was carried out before (2–3 months of age) and after (4 months of age) IR was established. SHRs aged 2, 3, or 4 months were randomized into five groups (n=5 rats/group) and treated daily for 8 weeks by gavage with iptakalim (1, 3, or 9 mg·kg−1·d−1), benazepril (6 mg·kg−1·d−1), or vehicle. Six age-matched WKY rats were given vehicle alone for 8 weeks.
Euglycemic-hyperinsulinemia clamp analysis was performed as described previously22. Briefly, at the end of 8 weeks, rats were fasted for 12 h before the start of the experimental protocol. Then, they were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg). The left carotid artery (for blood sampling) and both femoral veins (left femoral vein for exogenous glucose infusion and right femoral vein for insulin infusion) were cannulated with silastic catheters. The catheters were flushed periodically with heparinized saline to maintain patency. At the beginning, blood samples were collected to measure basal plasma glucose concentrations, and then a constant infusion of insulin at a rate of 10 mU·kg−1·min−1 was performed. Meanwhile, a variable infusion of 10% dextrose was adjusted based on 5-min blood samples to maintain the plasma glucose at the target value. A stable plasma glucose concentration and an exogenous glucose infusion rate (GIR) were generally achieved within 70 to 90 min, at which time 6 blood samples (200 μL) were collected at 5-min intervals to determine the GIR.
Metabolic and endothelial function measurements
Blood samples were obtained from the retro-orbital sinus from rats that were fasted overnight and euthanized with ether. Plasma glucose levels were determined with an automated biochemical analyzer (7600-DDP-ISE; Hitachi Software Engineering, Yokohama, Japan). The concentration of insulin, NO, ET-1, and Ang II levels in the plasma were measured using a commercial radioimmunoassay kit (Eastern Asia Radioimmunity Research Institute, Beijing, China) according to the manufacturer's instructions. The NO level was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Insulin sensitivity was assessed using the quantitative insulin sensitivity index [ISI=-ln(fasting glucose level×fasting insulin level)].
Determination of the endothelium-dependent vascular relaxation
The thoracic aorta was isolated and removed from high-fructose diet rats after 4 weeks of vehicle or drug therapy. Acetylcholine-induced endothelium-dependent vascular relaxation was tested as described previously23.
The data are expressed as the mean±SD, and n indicates the number of experiments. For multiple comparisons, the results were analyzed by factorial ANOVA using the Statistical Analysis System software (SAS8.00). A value of P<0.05 was considered to be statistically significant.
Activation of KATP reverses endothelial dysfunction in vitro
The experiments were carried out in cultured HUVECs to investigate the improvement in KATP channel activation by iptakalim in vitro. Endothelial dysfunction was induced with wortmannin to block PI3-kinase-dependent signaling, and meanwhile, the cells were exposed to high concentrations of insulin20, 24. The release of NO (Figure 1A) and 6-keto-F1α (Figure 1B) decreased, and the levels of ET-1 (Figure 1C) and PAI-1 (Figure 1D) increased in the HUVECs incubated with wortmannin and insulin at concentrations of 50 and 100 nmol/L, respectively, for 20 h. Pretreatment with iptakalim at concentrations ranging from 10 nmol/L–10 μmol/L for 6 h increased NO release (Figure 1A) and prevented ET-1 formation (Figure 1C). Iptakalim had mild effects on 6-keto-F1α release only at the high concentration of 10 μmol/L, which was 100 times greater than the effective concentration for increasing NO release (Figure 1B) but had no effect on the API-1 level (Figure 1D). The effect of iptakalim on NO release was abolished by glibenclamide (10 μmol/L), a specific blocker of KATP channels (Figure 1E). In addition, eNOS protein expression in HUVECs was decreased when incubated with wortmannin and insulin, while pretreatment with iptakalim could significantly increase eNOS expression (Figure 2). These results suggested that iptakalim protects against endothelial dysfunction mainly by restoring the balance between NO and ET-1 via opening the SUR2B/Kir6.1 subtype of KATP channel in the endothelium, which is consistent with a previous report from our lab16.
Iptakalim improves IR associated with hypertension in vivo
First, the effect of KATP activation by iptakalim on the progression of IR associated with hypertension was investigated in SHRs. SBP, HR, and the GIR were compared between the SHRs and age-matched Wistar-Kyoto rats (WKY). SBP and GIR in the 1-month-old SHRs and age-matched WKY rats were comparable. SBP was elevated, and GIR was normal in the 2-3-month-old SHRs. SBP was elevated, the GIR was decreased, and hypertension and IR developed in the 4-6-month-old SHRs (Figure 3A). The effect of treatment with iptakalim at doses of 1, 3, or 9 mg·kg−1·d−1 for 8 weeks in SHRs before or after IR had developed was evaluated. SBP decreased, and GIR increased in the SHRs treated with iptakalim at 2, 3, or 4 months of age. Treatment with iptakalim improved IR associated with hypertension, whereas treatment with benazepril at a dose of 6 mg·kg−1·d−1 for 8 weeks decreased the SBP but did not improve the decrease in the GIR (Figure 3B).
Second, we investigated the effects of iptakalim on IR associated with hypertension in the FFRs. Food intake was significantly lower in the FFRs compared to the control rats. Iptakalim treatment did not affect food consumption in either the normal chow-fed or fructose-fed rats, but rosiglitazone treatment significantly increased food intake in the fructose-fed rats. Iptakalim treatment did not affect body weight or food consumption in either the normal chow-fed or fructose-fed rats (data not shown). IR associated with hypertension in the FFRs was characterized by an elevated SBP, decreased GIR, and insulin sensitive index (ISI) with diabetic manifestations of increased fasting blood levels of insulin (FBI) and glucose (FBG). Treatment with iptakalim at a dose of 1, 3, or 9 mg·kg−1·d−1 or the PPARγ agonist rosiglitazone at 5 mg·kg−1·d−1 for 4 weeks prevented IR associated with hypertension and hyperinsulinemia, and the SBP (Figure 4A), FBI (Figure 4B), FBG (Figure 4C), GIR (Figure 4D), and ISI (Figure 4E) were normalized. In addition, iptakalim had no effect on the above parameters in the control rats fed a normal diet.
Activation of KATP ameliorates fructose-induced endothelial dysfunction in vivo
Endothelial dysfunction was characterized by impaired vascular relaxation induced by acetylcholine in aorta from the FFRs. The endothelium-dependent vascular relaxation induced by acetylcholine at concentrations of 0.1, 1, 10, or 100 μmol/L was significantly decreased in the in vitro aortic preparations isolated from the FFRs. This change was significantly improved by treatment with iptakalim at the doses of 1, 3, or 9 mg·kg−1·d−1 for 4 weeks or by treatment with rosiglitazone (Figure 5). Serum levels of NO (Figure 6A) decreased, whereas ET-1 (Figure 6B) increased in the FFRs, which was improved by treatment with iptakalim at a dose of 3 mg·kg−1·d−1 for 4 weeks. These findings suggest that endothelial dysfunction can be restored by treatment with iptakalim in vivo.
In addition, serum levels of angiotensin II increased from 597.63±55.97 (n=10) to 796.48±113.62 (n=10) ng/mL (P<0.01 vs normal control group) in the FFRs. However, these increased levels of angiotensin II were not influenced by treatment with iptakalim at a dose of 3 mg/kg po qd for 4 weeks, and the serum content of angiotensin II remained high at 808.30±87.03 ng/mL (n=10, P>0.05 vs model group). However, the levels of angiotensin II decreased to 589.78±67.96 (10) ng/mL (P<0.01 vs model group; P>0.05 vs normal control group) with treatment with enalapril at a dose of 3 mg·kg−1·d−1 (Figure 6C). These results are consistent with those observed in hypertensive renal damage and renal injury induced by hyperuricemia25, 26. Because iptakalim has no effect on the renin-angiotensin system, its benefit on IR associated with hypertension is entirely different from those of drugs acting on the RAS.
Essential hypertension and insulin resistance are inextricably linked1. Approximately 50 percent of patients with hypertension can be considered to have insulin resistance and hyperinsulinemia27. Studies in both hypertensive humans and rodent models of hypertension suggest that IR and/or compensatory hyperinsulinemia have an important role in blood pressure regulation and may predispose individuals to develop hypertension. However, an antihypertensive drug that reduces BP does not necessarily improve IR. Of the current first-line antihypertensive agents, β-adrenergic receptor blockers and diuretics can impair insulin sensitivity, and the metabolic effects of calcium channel blockers are still controversial28. Only angiotensin-converting enzyme (ACE) inhibitors and angiotensin II (Ang) II type 1 (AT1) receptor antagonists have been reported to be beneficial in patients with IR7. Drugs that can lower blood pressure and reverse the associated IR are still lacking, which reveals the need for developing new drugs that can reduce blood pressure and improve IR.
Recent studies have demonstrated that endothelial dysfunction is likely to be one of the most important underlying pathophysiological mechanisms that might serve as the link between IR and hypertension. The endothelium releases many vasoactive mediators that regulate cardiovascular function and, importantly, contribute to maintaining homeostasis. Endothelial dysfunction has been attributed to various cardiovascular risk factors and is mainly characterized by an imbalance between NO and ET-1 signaling, which contributes to further progression of hypertension. Insulin resistance is characterized by pathway-specific impairment in PI3-kinase-dependent signaling, which in the endothelium, may also cause an imbalance between NO and endothelin-1, leading to decreased blood flow and worsening insulin resistance. Therapeutic interventions in animal models and human studies have indicated that improving endothelial dysfunction ameliorates insulin resistance, whereas improving insulin sensitivity ameliorates endothelial dysfunction11. Thus, if an antihypertensive agent protects against endothelial dysfunction, it is very likely to improve IR in hypertensive individuals.
Iptakalim is a novel KATP channel opener with potent antihypertensive properties, which have been confirmed in phase 3 clinical trials in China, and has selective activation of the SUR2B/Kir6.1 channel, resulting in the protection of endothelial function14, 15, 16, 17, 18. Iptakalim has protective effects against endothelial dysfunction induced by low-density lipoprotein, homocysteine, hyperglycemia, and hypertension16. In this study, we demonstrated for the first time that iptakalim improves IR associated with hypertension by restoring the balance between NO and ET-1 signaling in the endothelium.
First, we performed experiments to mimic IR-induced endothelial dysfunction. Simultaneous treatment of endothelial cells with wortmannin and high insulin levels blunts the PI3-kinase-dependent effects of insulin, such as the induction of eNOS expression and the release of NO, and increases the production of ET-1, which is consistent with previous reports20, 24. Iptakalim rebalanced the release of NO and the production of ET-1 but had little effects on PGI2 and PAI-1. The effect of iptakalim on NO release was abolished by glibenclamide, a specific blocker of KATP channels. Furthermore, iptakalim was able to increase eNOS protein expression in endothelial cells. These observations confirm previous results indicating that iptakalim potently protects against endothelial dysfunction16, 17, 18, 19. Iptakalim can improve IR-induced endothelial dysfunction and ameliorate the impaired insulin-PI3-Kinase-NO signaling pathway via opening the SUR2B/Kir6.1 subtype of KATP channel. Thus, we hypothesized that iptakalim could ameliorate IR in vivo through endothelial protection induced by activating the SUR2B/Kir6.1 channel.
Several animal models have been used to study the relationship between IR and the development of hypertension, including SHRs and FFRs29, 30, 31, 32. The SHR is a widely used genetic model of essential hypertension and the associated metabolic disturbances, including IR and endothelial dysfunction. Recent advances in the genetics of the SHR have revealed that there are some specific genes that influence blood pressure and IR33. The FFR is another useful model of acquired systolic hypertension that displays numerous features of the metabolic syndrome including IR. Rats fed a high fructose diet exhibit IR/hyperinsulinemia and hypertension, which is independent of genetic contributions34. In our study, these two different animal models were used to identify the effect of iptakalim on IR. Because of the effects of ACEIs and γ peroxisome proliferator-activated receptors (PPARγ) agonists on insulin-resistance states have been well documented35, we chose different ACEIs (benazepril for the SHRs and enalapril for the FFRs), and a PPARγ agonist (rosiglitazone for the FFRs) as positive controls to demonstrate the reliability of the rat models of IR associated with hypertension.
Although growing evidence demonstrates that several related mechanisms for IR contribute to the pathogenesis of hypertension in the SHRs, the exact cause of this disorder remains unclear. In addition, whether IR is a “reason” or a “result” for the progression of hypertension is yet to be elucidated. Our results indicate that hypertension develops before IR. Furthermore, we have shown for the first time that the GIR was improved after 8 weeks of iptakalim treatment in SHRs. There are at least two reasons that may explain this phenomenon. First, the lower blood pressure may result from iptakalim directly. Another possibility is that the lower blood pressure results from the restoration of endothelial function by iptakalim. Therefore, it is very important to further explore the molecular mechanism by which iptakalim affects IR associated with hypertension in SHRs.
A substantial number of studies suggest a relationship among the occurrence of IR, endothelial dysfunction and hypertension in FFRs, but the cause and effect among them has yet to be elucidated. Katakam and colleagues reported that hyperinsulinemia occurs following 3 d of high fructose feeding, IR develops after 7–10 d, endothelial dysfunction was observed following 18 d and hypertension was found after 4 weeks33, 36, 37. Based on the order in which they develop, we designed this study such that the iptakalim treatments were performed after hypertension had developed after 4 weeks of the diet. We found that treating the FFRs with iptakalim for 4 weeks resulted in lower blood pressures and restored the balance between the production of NO and secretion of ET-1. Furthermore, the impaired ACh-induced endothelium-dependent relaxation of aortic tissue derived from the FFRs was improved significantly after 4 weeks of iptakalim administration. In agreement with other reports31, 38, we have demonstrated that iptakalim has a direct endothelial effect. Thus, the similar in vivo and in vitro effects of iptakalim indicate that the positive effects of iptakalim on vascular function are mainly due to its direct effects on endothelial function.
Interestingly, in this study, we have demonstrated that iptakalim treatment improves insulin sensitivity, normalizes the GIR, and decreases plasma insulin levels in FFRs. Because therapeutic interventions in animal models and human studies have confirmed that improving endothelial dysfunction ameliorates insulin resistance11, it is reasonable to postulate that improvements in IR by iptakalim are due to its amelioration of endothelial dysfunction.
It is well known that ACE inhibitors may directly improve endothelial dysfunction by blocking local endothelial angiotensin II production. Our results showed that enalapril can inhibit the increased serum concentration of Ang II, while iptakalim has no direct effect on this change in the FFRs, which suggests that the mechanism for iptakalim-induced improvement in IR differs from that of enalapril and occurs through a novel molecular pathway.
Based upon the above investigation, it appears reasonable to conclude that iptakalim, a KATP channel opener, protects against IR-induced endothelial dysfunction by restoring the balance between NO and ET-1 signaling, and ameliorates the IR associated with hypertension.
Iptakalim can protect endothelial cells by activating KATP through the preferential activation of the SUR2B/Kir6.1 subtypes of KATP expressed in the endothelium. In this study, we first investigated the effects of iptakalim on IR in SHRs and FFRs and then explored the molecular mechanism by which it protects against endothelial dysfunction in vivo and in vitro. These findings suggest that iptakalim improves the IR associated with hypertension through restoring the balance between NO and ET-1 signaling and that targeting the SUR2B/Kir6.1 subtype of KATP is a new strategy for ameliorating the IR associated with hypertension.
This study was supported by grants from the National New Drug Research and Development Key Project (No 2008ZX09101-006, 2008ZXJ09004-018 and 2009ZX09301-002), the State Key Basic Research and Development from the Ministry of Science and Technology of China (No GT1998051112), the 863-High Technology Research and Development Program Plan (No 2002AA2Z3137), the National 1035 Project (No 969010101) of China and the New Drug Development of Beijing Key Project (No D0204003040721).
ATP-sensitive potassium channel
spontaneously hypertensive rat
glucose infusion rate
systolic blood pressure
human umbilical vein endothelial cells
- Ang II
angiotensin-converting enzyme inhibitor
plasminogen activator inhibitor-1
γ peroxisome proliferator-activated receptors
About this article
Acta Pharmacologica Sinica (2012)