© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Altered paracrine effect of endothelin in blood vessels of the hyperinsulinemic, insulin resistant obese Zucker rat
Department of Pharmacology and the Cardiovascular Risk Factor Reduction Unit, College of Medicine, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, SK, Canada S7N 5E5
* Corresponding author. Tel.: +1-306-966-6294; fax: +1-306-966-6220 gopal{at}sask.usask.ca
Received 12 October 1999; accepted 1 December 1999
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Earlier, we reported that high insulin incubation in vitro leads to increased ETA receptor expression in cultured rat aortic smooth muscle cells (Diabetes 1998, 47: 934–944). Our later observation of enhanced endothelin-1 evoked vasoconstriction in aorta from the hyperinsulinemic obese Zucker rat indicated that this interaction might also be relevant in vivo. To further examine the relationship between insulinemia and endothelin, we characterized endothelin receptor expression and endothelin-1 peptide levels in vascular tissues and plasma from young and old obese Zucker rats. Methods: 12 and 40-week-old Zucker obese and lean rats were used. Plasma endothelin-1 levels and endothelin-1 peptide content in the mesenteric artery and in the thoracic aorta were examined by radioimmunoassay. Messenger RNA levels of endothelin-1 peptide and ETA and ETB receptors were examined in the aortic and mesenteric vessels using RT-PCR. Results: Obese rats from both age groups had significantly higher plasma levels of insulin (4–10 fold), total cholesterol (2–3 fold), triglycerides (10-fold), and glucose (
1.5 fold) than their lean counterparts. There was a trend toward worsening lipoproteinemia and glycemia, but improved insulinemia with age in the obese rats. In association with these changes, obese rats exhibited attenuated endothelin-1 peptide and preproET-1 mRNA levels, but conversely elevated ETA and ETB receptor mRNA levels in both aortic and mesenteric vessels. Conclusion: These data suggest that vascular tissue from the metabolically dysregulated obese Zucker rat exhibits attenuated endothelin-1 peptide production and elevated endothelin receptor levels. Since elevated insulin levels have been linked to increased endothelin receptor expression, it is plausible that hyperinsulinemia upregulates endothelin receptors contributing to elevated vasoconstrictor responses to endothelin-1 in this model of obesity and hypertension.
KEYWORDS Endothelins; Vasoactive agents
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The association of hypertension, dyslipidemia, obesity, and impaired glucose tolerance, referred to as Syndrome X [1], is well established as a cumulative risk factor for cardiovascular morbidity in man. The obese Zucker rat, a genetic model of obesity and hypertension, exhibits metabolic characteristics representative of this syndrome, including hyperinsulinemia, insulin resistance, hyperglycemia, obesity, and dyslipidemia [2]. There are many reports demonstrating functional changes in endothelium and smooth muscles from metabolically dysregulated animals, including the obese Zucker rat [3–6]. Specifically, exaggerated vasconstrictor responses to vasoactive agonists, including endothelin-1 (ET-1), have been observed in vascular smooth muscle (VSM) isolated from this strain [4–6]. Previous studies have linked this phenomenon to alterations in VSM intracellular calcium handling [4,5]. However, other studies suggest that such changes may be related to specific changes in receptor characteristics for certain vasoactive agonists [3,6].
The powerful endothelial derived vasoconstrictor and mitogenic peptide, ET-1, has been implicated in a variety of cardiovascular disease states, including diabetes mellitus [7] and hypertension [8]. Changes in plasma levels of ET-1 are widely studied in attempts to link this peptide to vascular pathologies. However, widely variable results have been obtained from many of these studies [7] and the reliability of current assay kits for ET-1 has been questioned [9]. Moreover, circulating ET-1 is unlikely to reach pharmacologically active levels [10]. A more viable measure of ET-1 activity is the local production of ET-1 and its receptors at the vascular tissue level, since the primary actions of ET-1 are modulated through autocrine and paracrine regulation of VSM tone subsequent to abluminal secretion of ET-1 from endothelial cells [11]. Indeed, this is cited as a major limitation to our understanding of the role of ET-1 in diabetic vascular complications [3,7]. Elevated local ET-1 content and up-regulated ET-1 receptors have recently been observed in vascular tissue from insulin deficient streptozotocin-induced diabetic rats [12]. However, to date, no studies have examined local and systemic ET-1 release and action in the obese Zucker rat.
High insulin has been shown to increase ET receptor expression in vitro [6,13] and considerable evidence demonstrates that this relationship might also hold true in vivo in hyperinsulinemic states [6,13,14]. Earlier, using functional studies, we demonstrated a two-fold increase in ETA receptor expression and exaggerated vasoconstrictor responses to ET-1 in aorta from obese Zucker rats [6]. However, a detailed examination of ET-1 peptide and receptor levels in conjunction with an examination of links between ET receptor expression and insulinemia in this animal model has not yet been investigated. The present study examines ET-1 peptide and mRNA levels as well as ETA and ETB receptor subtype levels in conduit (thoracic aorta) and resistance (mesenteric arteries) vessels present in the obese Zucker rat.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Animals
This investigation conforms to the regulations stipulated by the University of Saskatchewan Animal Care Committee which follows the protocol outlined in The Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1996). Groups of 16 male obese and lean Zucker rats were kept under standard conditions with a 12-h light–dark cycle and given free access to food and tap water until they reached 12 and 40 weeks of age. Two different age groups were utilized since there are distinct differences in metabolic function between old and young obese rats [15]. Rats were subsequently weighed and then killed by decapitation. The trunk blood was collected immediately into pre-chilled tubes containing EDTA. Blood samples were immediately centrifuged at 2000 g for 10 min and the supernatant plasma was stored at –70°C until insulin, lipoprotein, and ET-1 measurements were undertaken. Blood glucose was determined at the time of sacrifice by a glucose oxidase method (One Touch Basic, Lifescan, Vancouver, Canada). Plasma insulin levels were determined using a specific RIA kit (Amersham, Oakville, ON, Canada). The thoracic aorta and mesenteric vessels were quickly excised, frozen in liquid nitrogen and stored at –70°C until mRNA extraction. Plasma cholesterol and triglycerides were measured using standard enzymatic methods [16,17].
2.2 Extraction and measurement of ET-1 peptide from tissues and plasma
The frozen, excised blood vessels were homogenized in 2 M acetic acid and boiled for 10 min. The homogenate was centrifuged for 20 min (17 000 g at 4°C). The supernatant containing ET-1 peptide was lyophilized and stored at –20°C until RIA. Plasma ET-1 was extracted by according to the method described previously [18]. ET-1 levels in the extracts of plasma, mesenteric artery bed and thoracic aorta were measured using a specific RIA kit [18].
2.3 Extraction of tissue total RNA
Total RNA was extracted as previously described [12]. Briefly, thoracic aortae and mesenteric vessels were homogenized in 1 ml Trizol reagent (Gibco-Brl, Burlington, ON, Canada) using a polytron homogenizer. A 0.2-ml volume of chloroform was then added to the homogenate, vortexed and incubated at room temperature for 3 min. The samples were subsequently centrifuged for 15 min (10 000 g at 4°C) and the aqueous phase was collected and mixed with an equal volume of isopropanol. Total RNA was precipitated by centrifugation for 10 min (10 000 g at 4°C). The RNA pellet was then washed with 1 ml 70% ethanol and centrifuged. The pellet was air-dried and dissolved in TE buffer (pH 7.5) and stored at –70°C until mRNA assay.
2.4 Reverse transcription-polymerase chain reaction (PCR)
Total RNA samples were treated with DNase I. A random primed cDNA library was prepared from 1 µg total RNA with SuperScriptTMII RNase H-reverse transcriptase according to the manufacturer's recommendations (Gibco-Brl). The cDNAs were amplified using amplification primers based on previously reported sequences (Table 1). Taq DNA polymerase (5.0 U) was used in each PCR reaction. ETA and ETB cDNAs were co-amplified in the same tube while ET-1 and β-actin cDNAs were co-amplified in another tube. The PCR condition was optimized to ensure that the reaction was in the linear range. For the ETA and ETB cDNAs, PCR was programmed for twenty-five cycles. Alternatively, since preproET-1 mRNA is less abundant than β-actin mRNA, ET-1 cDNA was initially amplified for eight cycles and then co-amplified with β-actin cDNA for twenty-two cycles. The PCR condition was: (i) 95°C for 3 min (initial melt) followed by; (ii) 95°C for 1 min (melt); (iii) 60°C for 1 min (anneal); (iv) 72°C for 1 min (extend) and (v) 72°C for 10 min (final extension).
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2.5 Southern blot analysis of PCR products
The PCR products were size-fractionated on 1% agarose gel together with DNA size markers ranging from 72 to 1353 base pairs. The gels were blotted onto nylon membranes in 10xSSC solution. The blotted DNA was fixed using a UV-cross linker. The membranes were then baked at 80°C for 2 h. Oligoprobes (antisense DNA) positioned between 5' and 3' primers were synthesized to hybridize PCR products. The sequences of ETA, ETB, preproET-1 and β-actin oligoprobes are shown in Table 1. The synthetic oligonucleotide for hybridization was end-labeled with [
-32P]ATP (3000 Ci/mmol) using a 5' oligonucleotide labeling kit. Prehybridization (3 h) and hybridization (overnight) were carried out at 50°C. After hybridization, membranes were washed in 2xSSC (with 0.5% SDS) and then processed for autoradiography. The membranes were then exposed to X-ray film. The intensities of the signals from each band were analyzed using a Phospho-Imager (Molecular Dynamics).
2.6 Statistical analysis
Data are presented as mean±S.E.M. Two-tailed unpaired Student t-tests were used to compare the groups and differences were considered significant at P<0.05.
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3 Results |
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3.1 Body weight and metabolic parameters
Body weight and metabolic variables are shown in Table 2. The 40-week-old rats in each group weighed significantly more than 12-week-old rats, and the obese rats weighed more than their lean counterparts at both ages. Obese rats from both age groups had considerably higher plasma levels of insulin (4–10 fold), total cholesterol (2–3 fold), triglycerides (10-fold), and glucose (
1.5 fold) levels than their lean counterparts in both age groups. Furthermore, plasma glucose, triglycerides, and total cholesterol were higher, while plasma insulin levels were lower, in 40- compared with 12-week-old obese rats indicating age dependency in metabolic function in these rats.
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3.2 ET-1 levels in the plasma
There was no significant difference in plasma ET-1 level amongst any of the groups (Fig. 1). A correlational analysis of either plasma insulin and plasma ET-1 levels or plasma lipoproteins and plasma ET-1 levels was not completed in the present study. However, it is highly unlikely that a correlation between these parameters would exist since plasma ET-1 level remained unaltered in all the groups despite the highly significant changes observed in plasma insulin and lipoprotein levels.
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3.3 ET-1 peptide and preproET mRNA levels in the thoracic aorta
ET-1 peptide levels were lower in both aorta (Fig. 2a) and mesenteric vessels (Fig. 3a) from obese compared to lean rats from both age groups. In association with this, preproET mRNA levels were also attenuated in both aorta (Fig. 2b) and mesenteric vessels (Fig. 3b) indicating that attenuated ET-1 peptide levels result from suppressed production of ET at the gene level.
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3.4 ETA and ETB mRNA levels in aorta and mesenteric arteries
ETA and ETB mRNA levels in both thoracic aorta (Fig. 4) and mesenteric arteries (Fig. 5) of obese rats were consistently higher than in age-matched lean control rats. Moreover, ETA receptors were upregulated to a greater extent than ETB receptors in both aorta (1.6–2.0 ETA vs. 1.5–1.6 ETB) and mesenteric vessels (2.4–2.6 fold ETA vs. 1.5–1.7 fold ETB) of obese rats. There also was a trend towards higher ETA levels in both aorta (2.0 fold, 12-week vs. 1.6 fold, 40-week) and mesenteric vessels (2.6 fold, 12-week vs. 2.4 fold, 40-week) from 12- compared to the 40-week-old obese rats.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Obese rats at both age groups exhibited signs of significant metabolic dysfunction. Plasma insulin, glucose, triglycerides, cholesterol, and body weight were all significantly higher in obese rats at both age groups. There is some controversy as to the plasma glucose status of obese Zucker rats. In general, plasma glucose levels were only slightly elevated in obese rats from both age groups — indicating a moderately diabetic state. In support of previous studies [15], the present study also indicates that there might be age dependency in levels of hyperglycemia in these rats. Older obese rats also exhibit higher plasma triglycerides and cholesterol, but lower plasma insulin levels than their younger counterparts. Lower plasma insulin levels in late type II diabetes in humans has been attributed to a loss of insulin secretory capacity resulting from the toxicity of chronic hyperglycemia on β-cells [19,20]. Thus, lower plasma insulin levels in older obese rats might result from a decreased insulin secretory capacity resulting from long term exposure to worsening hyperglycemia.
Both ET-1 peptide and mRNA levels are attenuated in aorta and mesenteric arteries from obese rats. Tissue content of ET-1 is thought to represent a balance between synthesis and release of the peptide [12]. Thus, decreased vessel content of ET-1 could be due to either decreased synthesis or increased release of the peptide from the blood vessel. Accordingly, the observation that both peptide and mRNA levels of ET-1 are attenuated in blood vessels from obese rats indicates that production of the peptide is suppressed.
Locally produced ET-1 in blood vessels exerts its effects in an autocrine and paracrine fashion to constrict blood vessels by acting on underlying VSM cells. Many studies use plasma ET-1 as a measure of vascular ET-1 activity in vivo [7]. However, circulating ET-1 is thought to play only a minor role in the regulation of vascular tone [10]. While it is possible that circulating ET-1 represents spillover of the peptide from the vascular endothelium [7], there is no conclusive evidence of a link between local production and plasma levels of ET-1. Indeed, the present results suggest quite the opposite — despite attenuated preproET mRNA production in both mesenteric and aortic vascular beds of obese Zucker rats, ET-1 plasma levels remain unchanged. The presence of unchanged circulating ET-1 levels despite attenuated local production of the peptide might be related to the predominant basolateral secretion of the peptide by endothelial cells, to its short plasma half-life of 1–4 min, or to altered clearance of plasma ET-1 [10].
Interactions of glucose, lipoproteins, and insulin with the ET system are well documented, both in vitro and in vivo. Accordingly, ET-1 production appears to be stimulated by insulin, glucose, and oxidized lipoproteins [7]. However, in the present study, plasma levels of ET-1 are unaltered and local production and expression of the peptide in aorta and mesenteric arteries is attenuated in both young and old obese Zucker rats — despite the presence of insulinemia, glycemia, and lipoproteinemia. This somewhat surprising result might be explained by the fact that these metabolic abnormalities might affect other variables that could negatively modulate ET-1 production. Specifically, since ET-1 is primarily released by endothelial cells, damage to the endothelium resulting from exposure to such metabolic dysfunction might impair ET-1 production counter to the direct effects of each of these variables on stimulating production of the peptide [7]. In addition, the obese Zucker rat is known to be severely insulin resistant [2]. Thus, it is also plausible that the obese rat is not only resistant to the metabolic actions of insulin, but also to the ET-1 stimulating actions of insulin. Indeed, insulin resistance in VSM manifests not only as deficiency of glucose utilization but also as a partial loss of insulin-mediated vasodilation [21]. This might also explain observations that plasma ET-1 levels are normal in hyperinsulinemic type II diabetic patients [22,23].
Hyperinsulinemia has also been shown to increase ETA receptor expression in VSM cells in vitro [6]. Furthermore, there is some evidence that this interaction might be relevant in vivo as well [6,13,14]. In the present study, ETA mRNA levels are dramatically increased, and ETB mRNA slightly increased, in both aorta and mesenteric arteries from both 12- and 40-week-old obese Zucker rats with hyperinsulinemia. Two lines of evidence indicate that this insulinemia might have contributed to upregulated ETA receptor expression in blood vessels from the obese rat. First of all, ETA receptor expression was increased to a greater extent than ETB in vessels from both young and old obese rats — indicating some degree of selectivity in the upregulation of ETA receptors. Since insulin has previously been shown to selectively upregulate ETA receptors [6], it is plausible that insulinemia contributed to this selective increase in vivo. Secondly, ETA levels were slightly higher in both aorta and mesenteric arteries from the 12-week obese rats compared to 40-week obese rats. Similarly, plasma insulin levels were 10-fold higher in the younger group but only 4-fold higher in the 40-week group. Thus, levels of vascular ETA expression appear to correspond with levels of plasma insulin. The observation that ETB mRNA is also increased in the obese rat from both age groups might be explained by the non-specific upregulation of ET receptors on VSM and endothelial cells resulting from attenuated ET-1 production. Indeed, ET-1 has been shown to modulate its own receptors [24,25].
Previous studies from our laboratory demonstrated increased vasoconstrictor responses to ET-1 in aortic rings from obese Zucker rats [6,26,27]. Results from the present study indicate that this might be due to upregulated ET receptor expression in vascular tissue from this strain. Other studies indicate that Ca2+ handling is altered in VSM from obese rats contributing to a generalized elevation in vascular reactivity [4,5]. Since many studies indicate that obese Zucker rats are hypertensive [2,27], it is possible that increased vasoreactivity to vasoconstrictor agonists, including ET-1, contributes to elevated blood pressure in this strain.
In conclusion, the present study demonstrates that alterations in ET-1 release and action are present in the metabolically dysregulated obese Zucker rat. ET-1 plasma levels are unchanged, local ET-1 production in aortic and mesenteric vessels is decreased, and ETA and ETB receptor expression is increased. It is likely that alterations in metabolic parameters including plasma insulin, glucose, and lipoproteins contribute to these abnormalities, since all of them have been shown to variably affect ET-1 release and action. Notably, insulinemia appears to contribute to elevated ETA receptor expression in blood vessels from the obese Zucker rat. Further studies correlating individual metabolic variables, including insulinemia, with ET-1 peptide and ET receptor mRNA levels are required to determine the contribution of each to such changes. Importantly, our results provide a plausible explanation for previous observations of enhanced vasoconstrictor responses to ET-1 in vascular tissues isolated from Zucker obese rats ex vivo [6,26,27]. Moreover, it is possible that exaggerated vasoconstrictor responses to ET-1 contribute to elevated blood pressure in the obese Zucker rat.
Time for primary review 20 days.
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Acknowledgements |
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This work was supported by a grant from the Medical Research Council (MRC) of Canada (Grant No. MT-14377 to V.G.). Dr. Sheng-Qian Wu and Mr. Rob L. Hopfner gratefully acknowledge Post-Doctoral Fellowship and Doctoral Research Scholarship support provided by the Health Services Utilization and Research Commission of Saskatchewan and the MRC, respectively.
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