The impairment of endothelial-dependent vasodilatations has been reported for diabetic patients (1,2) and animal diabetic models (3-6). In streptozotocin-induced diabetic rats, we previously reported a close relation between endothelial dysfunction and the control of the disease, estimated by measuring the levels of glycosylated hemoglobin (HbA1c) in blood (7,8).
Among the mechanisms proposed as mediators of diabetic endothelial dysfunction, the enhanced generation of oxygen-derived free radicals is considered a crucial factor (9). Some studies suggested that the oxidative stress observed in diabetic animals and patients also can be related to the control of hyperglycemia (10,11).
Acarbose improved glycemic control in type 2 diabetic patients (12) or diabetic animal models (13). Acarbose acts as a potent inhibitor of intestinal α-glucosidase, leading to a delay in carbohydrate digestion and reducing the postprandial increase of glycemia (13). In addition, acarbose does not cross enterocytes, and its effects are limited to intestinal glucosidases, lacking any direct metabolic effect on diabetes evolution (12,13). In this study, we aimed to analyze whether a single reduction of hyperglycemia and the derivatives from nonenzymatic protein glycosylation may be effective in reducing the development of diabetic endothelial dysfunction. For this, we used a model of insulin-dependent diabetes (the streptozotocin-induced diabetic rats) well established in our laboratory (7,8) receiving a treatment with acarbose. Although this is an inappropriate diabetic model for this kind of drug, the reason for using it was precisely to discard any other action but the reduction of blood glucose by acarbose. Furthermore, a comparative analysis with a low-insulin treatment was performed.
Insulin-dependent diabetes was induced in 16-week-old male Sprague-Dawley rats (300-350 g) by a unique intraperitoneal injection of streptozotocin (60 mg/kg, in citric acid-trisodic citrate, 0.1 M, pH 4.5). Diabetes may be diagnosed 72 h after injection by blood glucose assay and considered successful for a glycemia >11 mM. The animals were acclimatized for a 2-week period before starting the protocol, housed at the facilities of the Facultad de Medicina de la Universidad Autónoma de Madrid, and fed with regular chow, having free access to water. Two different protocols were arranged. In the first protocol, immediately after diabetes induction, four different groups of rats were established. The first one was maintained without any treatment. The second one was treated orally with acarbose mixed in the rat chow (10 mg/kg/day). The third one received a low amount of insulin (1 unit/day), whereas the fourth one was treated with acarbose plus low insulin. This protocol was maintained for 6 weeks before killing of the animals and isolation of the vessels. In the second protocol, 6 weeks without any treatment was allowed after diabetes induction, and then the described treatments were established for 3 additional weeks before killing and isolation of the vessels. In each protocol, nondiabetic rats of matched age were used as controls but, as all parameters measured in nondiabetic animals were similar, they were pooled and expressed in just one group of control nondiabetic animals. Insulin was administered by subcutaneous implants of bovine insulin (Linplant, Scarborough, Ontario, Canada), which contain palmitic as excipient, introduced without sutures under the dorsal skin of the rats slightly anesthetized with ketolar (30 mg/kg).
Blood pressure measurements
Before killing, the rats were anesthetized with 40 mg/kg ketolar, 3.2 mg/kg diazepam, and 0.16 mg/kg atropine (i.p.). The animals were mechanically ventilated with a respirator (New England Medical Instruments, Medway, MA, U.S.A.), and their temperature maintained at 37°C with a thermic table (RTC1; Cibertec, Madrid, Spain). The right carotid artery was cannulated, and systemic blood pressure and heart rate were recorded through a pressure transducer (P23XL; Spectramed, Oxnard, CA, U.S.A.) connected to a polygraph (7D; Grass, Quincy, MA, U.S.A.). Afterward the animals were killed by bleeding. Then aorta and mesenteric microvessels were carefully excised, cleaned of excess fat and connective tissue, placed in a petri dish containing Krebs-Henseleit solution (KHS), and divided into cylindrical segments 4-5 mm in length for aortic segments and 1-1.5 mm in length for mesenteric microvessels.
Isometric tension recording for aortic segments
Each vascular cylinder was set up in an organ bath according to the method previously described (7). The organ chamber contained 5 ml of KHS at 37°C, continuously bubbled with a 95% O2/5% CO2 mixture, which gives a pH of 7.4. Two horizontally arranged stainless steel pins were passed through the lumen of the vascular cylinder, one connected to the organ-bath wall and the other to a strain gauge for isometric tension recording. The isometric contractions were recorded through a force-displacement transducer (FTO3C; Grass) connected to a Grass model 7D polygraph. The segments were subjected to a tension of 1.5 g (optimal resting tension), which was readjusted every 15 min during a 90-min equilibration period before drug administration. The vessels were exposed to 75 mM K+ to check their functional integrity. After a washout period, each segment was contracted with 10-30 nM norepinephrine (NE). Once a stable plateau was reached, a concentration-response curve to acetylcholine (ACh; 10 nM-10 μM) was performed. Endothelium removal was checked by testing the loss of ACh-induced relaxations. In some aortic segments from every rat, the curve to ACh was performed in the presence of 100 U/ml superoxide dismutase (SOD), added to the organ bath 15 min in advance.
Isometric tension recording for mesenteric microvessels
To obtain arteries of approximately equal diameter, thirdorder mesenteric branches were dissected free of connective tissue under light microscope and mounted as ring preparations on a small-vessel myograph (7) capable of measuring isometric tension (600M; J.P. Trading, Aarhus, Denmark), connected to a digital recorder (MacLab/8e; ADInstruments, Castle Hill, Australia). The segments were subjected to an optimal tension (90% of that tension equivalent to a intramural pressure of 100 mm Hg). After 30-min equilibration, the vessels were exposed to 125 mM K+ to check their functional integrity. After a washout period, each segment was contracted with 10-30 nM NE. Once a stable plateau was reached, concentration-response curves to ACh (1 nM-10 μM) were performed. Endothelium removal was checked by testing the loss of ACh-induced relaxations. In some mesenteric microvessels from every rat, the curve to ACh was performed in the presence of 100 U/ml SOD, added to the medium 15 min in advance.
Blood was sampled at death to determine blood glucose, measured with a glucometer (Accutrend; Boehringer Mannheim, Mannheim, Germany), and glycosylated hemoglobin (HbA1c) was measured by immunoturbidimetric assay (14). In addition, advanced glycosylation end product (AGE) formation was analyzed according to a previously described protocol (15) using polyclonal antiserum to AGE epitopes, which were formed in vitro after the incubation of bovine pancreatic ribonuclease in the presence of 0.5 mM glucose for 60 days. The antiserum was kindly provided by Dr. R. Bucala (Rockefeller University, New York, NY, U.S.A.). It was obtained from female New Zealand White rabbits receiving four primary and one booster immunization of ribonuclease or AGE ribonuclease emulsified in Freund's complete adjuvant. Antibody response was monitored by enzyme-linked immunosorbent assay (ELISA; EL-340; Bio-Tek Instruments Inc., Winooski, VT, U.S.A.). A standard curve of the antibody binding to antigen was already established using bovine seroalbumin AGEs as a standard antigen. Then results of serum AGEs were expressed as equivalents of bovine seroalbumin AGEs per mg of proteins (U/mg prot). In some cases, the plasma levels of insulin were determined by radioimmunoassay (kit Insik-5; Diasorin, Italy).
Data management and statistical analysis, drugs, ethical considerations
The different curves to ACh were performed in a nonpaired way. Only one curve was performed in every segment. Deviations from the mean regarding the curves to ACh were analyzed using factorial two-way analysis of variance (ANOVA). Student's t test were used for other comparisons. pD2 values were calculated as the negative log of the effective dose required to produce half of the maximal effect. Significance was considered as a value of p < 0.05.
The composition of KHS (mM) was NaCl, 115; CaCl2, 25; KCl, 4.6; KH2PO4, 1.2; MgSO4 · 7H2O, 1.2; NaHCO3, 25; glucose, 11.1; and Na2EDTA, 0.03. Drugs used were norepinephrine hydrochloride, acetylcholine chloride, CuZn superoxide dismutase (EC 184.108.40.206) from bovine erythrocytes (all of them from Sigma), and acarbose (Bayer). Drug solutions were made in distilled water except norepinephrine, which was prepared in saline (0.9% NaCl)-ascorbic acid (0.01% wt/vol).
This work was performed according to the European regulations. The study was approved by the Local Committee of Investigation.
Biologic parameters of streptozotocin-induced diabetic rats
After 6 or 9 weeks of diabetes evolution, untreated diabetic rats showed a significant reduction in plasma insulin (from 66.8 ± 7.9 μU/ml in control nondiabetic to 9.3 ± 0.7 μ/Uml in untreated diabetic; p < 0.001) and body weight. Conversely, glycemia, HbA1c, and AGEs were enhanced, whereas mean arterial pressure did not change (Table 1). Treatment of the animals only with 10 mg/kg acarbose after diabetes induction slightly improved hyperglycemia and HbA1c. When a low dose of insulin (1 U/day) was administered, alone or in combination with acarbose, both hyperglycemia and HbA1c were improved, as well as body weight (Table 1). After applying these treatments during 3 weeks after 6 previous weeks of untreated diabetes, acarbose was not effective at reducing glycemia and HbA1c, low-insulin improved body weight and HbA1c, whereas combined administration of the drugs improved body weight, blood glucose, and HbA1c(Table 1). The blood levels of AGEs were enhanced after diabetes induction in all the studied groups, but there were no differences among groups depending on the received treatments (Table 1). Conversely, in all groups, there were no changes in mean arterial pressure (Table 1).
ACh-induced relaxations after 6 weeks of diabetes with treatment after diabetes induction
The administration of ACh on vessels previously contracted with NE caused concentration-dependent relaxations, which were abolished when the vascular segments were previously mechanically deendothelialized with saponin (data not shown). The relaxant responses obtained in aortic segments and mesenteric microvessels from 6-week evolution in untreated streptozotocin-induced diabetic rats were significantly lower than those observed in vessels from nondiabetic animals (Figs. 1 and 2). In these vessels, significant improvements of ACh-evoked relaxations were obtained when 100 U/ml SOD was administered to the organ bath (Tables 2 and 3). In 6-week evolution diabetes rats receiving acarbose after diabetes induction, the vasodilatations to ACh in aortic or mesenteric vessels were significantly higher than those from untreated diabetic rats, although they did not reach the values observed in vessels from control nondiabetic animals (Figs. 1 and 2). Thus, further enhancements of vasodilatory responses was obtained in the presence of SOD (Tables 2 and 3). Similar effects were observed when a low dose of insulin was administered, either alone or in combination with acarbose (Figs. 1 and 2; Tables 2 and 3).
ACh-induced relaxations after 6 weeks of untreated diabetes plus 3 additional weeks with treatments
After 9 weeks of untreated streptozotocin-induced diabetes, significant impairment of endothelium-dependent relaxations was found in aortic segments and mesenteric microvessels, similar to those obtained in animals with 6 weeks of untreated diabetes (Figs. 3 and 4; Tables 2 and 3). An analogous improvement of the relaxation was observed when 100 U/ml SOD was previously administered to the organ bath (Tables 2 and 3). When diabetic rats received acarbose during the 3 final weeks, some improvement of the ACh-evoked vasodilations could be still observed in the aortic segments but not in the mesenteric microvessels (Figs. 3 and 4; Tables 2 and 3). In aortic segments, the administration of low doses of insulin did not produce a further improvement, whereas in mesenteric microvessels, only the combined treatment with acarbose and low insulin induced a significant enhancement of the relaxant responses (Figs. 3 and 4; Tables 2 and 3).
Several studies have demonstrated the impairment of endothelium-dependent relaxations associated with diabetes (1-3). In streptozotocin-induced diabetic rats, the reduction of endothelium-dependent vasodilatations has been observed either in experiments with isolated aortic rings (5,7,16) and mesenteric microvessels (7,17,18), or in studies analyzing hindquarter vasoactive responses (6,8), indicating that diabetic endothelial dysfunction affects both the conductance and the resistance vasculature. In our study, the results obtained both in aortic segments and mesenteric microvessels showed a similar impairment of the endothelium-mediated responses in a 6- or 9-week evolution of streptozotocin-induced diabetes. However, there was no alteration of mean arterial blood pressure, which also agrees with previously reported data (8).
As exposed earlier, it is widely accepted that increased oxidation occurs in diabetes (19,20), which can be detected by the presence of peroxidation products (21) or by an enhanced generation of free radicals, mainly superoxide anion (22). Studies from several laboratories, including ours, indicated that SOD improves ACh-induced endothelium-dependent relaxations in vessels from diabetic animals (7,19,23). We further confirm this fact in this study in both conductance and resistance vessels. Conversely, at the concentrations used in this study, SOD had no effect on the relaxations evoked by ACh in vessels from nondiabetic rats, suggesting that the response to SOD may be an appropriate indicator for the existence of oxidative stress in diabetes.
The main objective of the study, however, was to determine whether a discrete reduction in carbohydrate absorption may be effective in preventing diabetic endothelial dysfunction. Acarbose is an inhibitor of intestinal α-glucosidase that improved glycemic control by decreasing carbohydrate enteric absorption, being effective in type 2 diabetic patients (12). This drug has not direct metabolic effects; therefore it seems reasonable to propose that its effects in these experimental conditions, applied to a model of type 1 diabetes, are limited to a reduction of total glucose absorption. Acarbose treatment after diabetes induction caused a small reduction in glycemia and HbA1c levels, whereas no changes from untreated diabetes were observed when the drug was introduced after 6 weeks of diabetes induction. For comparative purposes, some animals received a rather low dose of insulin, a more appropriate treatment for a model of type 1 diabetes, which produced similar reductions in blood glucose and HbA1c levels but also exerted metabolic actions, as reflected in a better preserved body weight.
We first analyzed the ACh-evoked endothelium-dependent relaxations in both aortic segments and mesenteric microvessels from diabetic rats treated either with acarbose, low insulin, or both, after diabetes induction with streptozotocin. In those animals receiving only acarbose, we observed a partial recovery of the relaxant responses in comparison with untreated animals; this improvement was in the same range as that observed in the rats treated with low insulin or with acarbose plus insulin. As previously reported by us in this experimental model (7), SOD was effective in recovering defective endothelium-dependent relaxations, indicating that this kind of endothelial dysfunction is related to superoxide anion production. It is worth remarking that the responses in the conductance vessels, the aortic segments, were more easily recovered than were those from the resistance vasculature.
The effects of drugs were clearly lower when the agents were applied after 6 weeks of diabetes without any treatment, when the diabetic endothelial dysfunction was already established. Thus, in aortic vessels, acarbose treatment slightly improved endothelium-dependent relaxations, whereas no significant action could be detected in mesenteric microvessels. In these resistance vessels, only the treatment with both acarbose and low insulin was able to cause some partial recovery of the relaxant responses. Again, diabetic endothelial dysfunction could be fully recovered in the presence of SOD, suggesting a predominant role for reactive oxygen species (9,19-23). As previously described, acarbose is an inappropriate treatment for this experimental model of diabetes, lacking any metabolic action on disease evolution. Therefore, its effects can only be related to its ability to reduce carbohydrate absorption, lowering the levels of blood glucose. It is worth noting that the improvement of ACh-induced relaxations by this drug was observed solely when reductions in blood glucose or HbA1c levels occurred, which only happened if acarbose were administered after diabetes induction.
Although related to hyperglycemia, the specific mechanism that mediates the impairment of endothelium-dependent relaxations is not clearly defined. Some authors suggested that these alterations may be directly caused by elevated glucose (24-26), but an important role for the nonenzymatic protein glycosylation has been also proposed. The process of glycosylation is related to two main factors: the glucose concentration in the medium and the duration of hyperglycemia. Different products are formed, which may be the mediators of the endothelial dysfunction. Therefore, the terminal adducts of nonenzymatic glycosylation of proteins, called AGEs, have been proposed to quench nitric oxide and to induce alterations of endothelial function (27-29). This is not apparently the case in our experimental conditions, as the plasma levels of AGEs, increased during streptozotocin-induced diabetes, were not affected by any of the tested treatments.
Earlier products of protein glycosylation also can be involved in diabetic endothelial dysfunction. Previous work from our laboratory indicated that early Amadori products of protein glycosylation, highly glycosylated oxyhemoglobin, may interfere with endothelium-dependent relaxations, at concentrations that can be found free in plasma, by a mechanism involving enhanced production of superoxide anions (30,31). Furthermore, we have found a strong correlation in streptozotocin-induced diabetic rats between the levels of blood HbA1c and the development of endothelial dysfunction (7,8). Therefore, we suggest that the effects of acarbose or low insulin in this study are linked to the reduction of HbA1c produced by both agents, although from these results, we cannot discard a role for high glucose itself, as proposed by others (24,26).
Taken together all these results, we conclude that the endothelial dysfunction observed in streptozotocin-induced diabetic rats is associated with hyperglycemia and elevation of HbA1c levels, which generate an enhancement of oxidative stress. The treatment of the animals with acarbose may produce a partial improvement of endothelium-dependent relaxations. This effect is likely related to the ability of the drug to reduce glucose absorption, leading to lower hyperglycemia and HbA1c levels, whereas the plasma AGEs are unchanged.
Acknowledgment: We thank to Carmen Fernández-Criado and José M. Badajoz-Martínez for animal care. This work was supported by grants from Bayer España S.A., CICYT (SAF 98-0010), FISS (99/0246), CAM (08.4/0027/1998), and FEDER (2FD97-0445-CO2). Dr. Peiró is the recipient of a postdoctoral fellowship from Comunidad Autónoma de Madrid.
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