Generalised vasodilation is observed early in the course of diabetes mellitus. This hyperperfusion has been demonstrated in skeletal muscle (1), in the renal vasculature (2-4), in the retinal circulation (5), and in the cutaneous microvasculature (6). This increased microvascular blood flow is believed to be involved in the pathogenesis of diabetic microangiopathy and has led to the development of the haemodynamic hypothesis of diabetic microvascular disease (7-9). This hypothesis contends that an initial increase in microvascular flow and pressure leads to exudation of plasma proteins and endothelial damage with subsequent microvascular sclerosis. These structural adaptations of the microvasculature result in disturbed autoregulation with impaired vasodilatory reserve and microangiopathy. The mechanisms by which the metabolic abnormalities of diabetes lead to hyperperfusion remain uncertain.
Although improved glycaemic control causes a reduction in blood flow (5,10,11), blood flow remains elevated compared with normal subjects (11), suggesting hyperglycaemia alone does not cause this hyperperfusion. This is supported by the finding that microvascular complications develop in subjects with impaired glucose tolerance who have minimally elevated glucose and that such complications do not develop in some diabetic patients despite poor long-term control (12,13).
The vascular endothelium plays a central role in the regulation of vascular tone through the production of several vasoactive substances, including the vasodilators nitric oxide and prostacyclin and the vasoconstrictors thromboxane A2 and endothelin-1 (ET-1). An imbalance in the production and action of these endothelium-derived vasoactive substances could cause hyperperfusion. As it has been demonstrated that endothelium-dependent vasodilation is impaired in type 2 diabetes (14,15), it is unlikely that hyperperfusion occurs as a result of enhanced action of endothelium-derived vasodilators. However, recent work has focused on the role of ET-1 in the control of vascular tone (16). As ET-1 contributes to basal tone (16), impaired responses to ET-1 could result in hyperperfusion. In general, reduced responsiveness to ET-1 has been reported both in vitro (17) and in animal (18-20) models of diabetes. Impaired responsiveness to ET-1 has also been demonstrated in vivo in patients with type 2 diabetes in a preliminary study from our group (21). However, this work had several limitations in that it was unblinded, only a single low dose of ET-1 was examined, and there was no placebo treatment arm. It remains uncertain if responses to ET-1 are blunted rather than absent in type 2 diabetes. The purpose of this study was to extend our understanding of the role of ET-1 in forearm vascular responses in patients with type 2 diabetes.
MATERIALS AND METHODS
Ten patients (six men, four women) with type 2 diabetes of mean age 55 years (range, 43-64 years) were recruited. Diabetic control was achieved by diet alone or by diet and oral hypoglycaemic agents (sulphonylurea and/or biguanide preparation). Subjects with a history of atherosclerosis, hypertension [blood pressure (BP) >160/90 mm Hg], hypercholesterolaemia (>6 mM), cigarette smoking, renal impairment (creatinine >140 μM), or if taking any cardiovascular drugs were excluded. Nine healthy volunteers (five men, four women) of mean age 57 years (range, 41-68 years) were recruited from the local community to act as a control group (Table 1). All patients underwent a full medical history and examination including a resting ECG and Doppler examination of the lower limbs.
Each diabetic patient was assessed for the presence of microvascular disease. Fundoscopy was performed to assess for retinopathy. Timed overnight urinary albumin excretion was measured to detect microalbuminuria. Vibration perception was measured at the ankle and great toe using a biothesiometer. A value greater than the 95th percentile compared with an age-related value obtained from a healthy population was defined as abnormal (22). Measurement of heart rate variability by electrocardiography during deep breathing was used to assess cardiac autonomic function. Heart rate variability was compared with previously published age-related normal values obtained from a normal population (23), and heart rate responses below the 5th percentile were taken as abnormal.
All subjects gave written informed consent for all procedures, and this study was approved by the local ethical committee of the Queen's University of Belfast.
Each subject fasted for 10 h before the study. Alcohol- and caffeine-containing products were prohibited during this time. The studies took place in the morning in a temperature-controlled room (24-26°C) with the subject supine and the arms resting on a support slightly above the level of the heart. Under local anaesthesia (1% lignocaine; Antigen Pharmaceuticals, Roscrea, Ireland), a 20-gauge polyethylene cannula (Vygon leader cath, Ecouen, France) was inserted into the non-dominant brachial artery. Forearm blood flow (FBF) was measured by strain-gauge venous occlusion plethysmography. The hand was excluded from the circulation by inflating a wrist cuff to 200 mm Hg for 1 min before and during the FBF measurements. An upper arm cuff was inflated to 40 mm Hg for 5-10 s to achieve venous occlusion. A mercury-in-Silastic strain gauge was coupled to an electronically calibrated plethysmograph (model SPG16; Medasonics, CA, U.S.A.). The voltage output was transferred to a Macintosh personal computer (Performa 630; Apple Computer Inc., CA, U.S.A.) with a MacLab analog-to-digital converter and CHART software (v. 3.4.3; AD Instruments, Hastings, U.K.). FBF was measured in both arms using the noncannulated arm as a control to demonstrate that local vasoactive drug administration did not have a systemic action. The mean of five consecutive FBF measurements was taken for statistical evaluation. FBF was expressed as ml/100 ml forearm volume/min.
After a period of ≥30-min rest, during which 0.9% saline was infused at a rate of 1 ml/min, basal FBF was measured.
All drugs used were freshly prepared before each study. ET-1 (Clinalfa, Weidenmattweg, Switzerland) and sodium nitroprusside (Faulding DBL, Leamington SPA, U.K.) were diluted in 0.9% saline. Sodium nitroprusside was protected from exposure to light.
Local intraarterial drug infusions were performed in the experimental forearm to determine the direct effect of the vasoactive drug. Systemic administration was avoided, as this is associated with autoregulatory systemic reflexes, which can modify direct vascular responses. The doses used have previously been shown to have no systemic effect on heart rate, BP, or FBF (14,24,25). All infusions were administered at a rate of 1 ml/min using a constant-rate infusor (Braun Perfusor pump, Melsungen, Germany).
The study was carried out on two separate days ≥1 week apart. Each subject received a local intraarterial infusion of ET-1 at three doses of 5, 10, and 20 pmol/min and 0.9% saline placebo. Two infusions were administered on each day in a balanced double-blind randomised manner. From previous work, it has been found that the maximal response to ET-1 occurs at 35-45 min, with no evidence of a delayed onset in patients with type 2 diabetes (21). In view of this, each infusion was administered for a 45-min period. As ET-1 has a prolonged duration of action with an offset time of between 60 and 90 min (24,26), a 90-min washout period was allowed between each infusion. FBF was measured during the first 5 min of the infusion and at 5-min intervals thereafter until completion of the drug infusion. On the second day before the first ET-1 infusion, sodium nitroprusside, to assess nonspecific smooth muscle function, was infused in four increasing doses (3, 6, 9, and 12 nmol/min) each for 3 min, with FBF measured in the last minute of each infusion. A washout period of ≥20 min was allowed before ET-1 was administered.
Unless otherwise stated, data are expressed as mean with 95% confidence intervals (CI). The clinical and biochemical characteristics of the patient and control groups were compared using unpaired Student's t tests.
Basal FBF was analysed using a two-way analysis of variance. For responses to ET-1, as serial measurements were made with each infusion, summary measures were used to avoid making multiple comparisons (27).
The paired Student's t test was used to compare basal and mean FBF over the drug infusion period for within-group dose effects. Change in FBF from baseline in response to drug infusion as area under the curve (AUC) was analysed using a two-way analysis of variance for between-group differences with post hoc analysis using the unpaired Student's t test to compare each dose between groups. Responses to sodium nitroprusside were analysed using a two-way analysis of variance. Differences were considered significant at a value of p < 0.05.
Fasting blood sugar was significantly higher in the diabetic group (p < 0.001). No other significant differences were found in the clinical and biochemical characteristics given in Table 1 (p < 0.05). One of the diabetic subjects had microalbuminuria, and another had evidence of peripheral neuropathy. Fasting blood sugar and glycosylated haemoglobin were not significantly different in the diabetic patients between the study days (p > 0.05). Basal FBF did not differ significantly between the diabetic and control groups (p > 0.05). There was no significant difference in basal FBF before each drug infusion (p > 0.05).
FBF in the control arm did not change significantly in response to infusion of any study drug (p > 0.05), confirming that drug effects were confined to the experimental infused forearm. There was no significant response to 0.9% saline in either group (p > 0.05). Control subjects showed vasoconstriction to ET-1 at 5 (p < 0.05), 10 (p < 0.05), and 20 pmol/min (p < 0.01). In the diabetic group, there was no significant reduction in FBF from baseline in response to ET-1 at 5 pmol/min (3.0, 95% CI 2.1-3.8, to 2.9, 95% CI 2.0-3.8 ml/100 ml/min; p > 0.05); however, significant vasoconstriction developed in response to ET-1, 10 (p < 0.01) and 20 pmol/min (p < 0.01; Table 2).
Change in FBF from baseline in response to ET-1 at 5 pmol/min was significantly different between the diabetic and control groups (−0.1, 95% CI −0.3-1.2 vs. −0.8, 95% CI −1.5-−0.1 ml/100 ml/min, respectively; p < 0.05; Fig. 1). There was no significant difference in response to ET-1 at either 10 (p > 0.05) or 20 pmol/min (p > 0.05; Fig. 2). There was no significant order effect (p > 0.05).
Responses to sodium nitroprusside were similar in the diabetic and control groups (p > 0.05; Fig. 3).
This study has demonstrated a reduction in FBF in response to local intraarterial infusion of ET-1 in healthy subjects, as has previously been shown (24). In contrast, patients with type 2 diabetes have demonstrated a blunted vasoconstrictor response to ET-1. Vasoconstriction was absent in response to locally infused ET-1 at 5 pmol/min, with a normal vasoconstrictor response to higher doses. This is in keeping with in vitro studies using animal models of diabetes, which have demonstrated impaired responsiveness to ET-1 (17-20). We have previously reported a preliminary study in patients with type 2 diabetes, which demonstrated impaired vasoconstriction to ET-1 at 5 pmol/min (21). This current placebo-controlled dose-response study confirms these findings but further extends this work, showing that the response to ET-1 in diabetic subjects is blunted rather than absent.
Several possible mechanisms may underlie the impaired responsiveness to ET-1 in patients with type 2 diabetes. Decreased availability of endothelin receptors, due to either receptor downregulation, blockade, or modification, diminished responsiveness of signal-transduction mechanisms coupled to endothelin receptors, or a nonspecific impairment of vascular smooth muscle cell function could result in blunting of the vasoconstrictor responses to ET-1. As ET-B receptors situated on the vascular endothelium mediate the release of endothelium-derived vasodilators, it is possible that increased production of a vasodilator could oppose the vasoconstrictor action of ET-1.
Endothelin receptor downregulation could explain the impaired response to ET-1 seen at low but not higher concentrations of ET-1. As ET-1 has been reported to cause receptor downregulation in both vascular smooth muscle (28,29) and glomerular mesangial cells (30), elevated ET-1 (31,32) reported in patients with type 2 diabetes could lead to receptor downregulation. Alternatively, increased concentrations of big ET-1, which is a weak agonist for endothelin receptors, also could cause receptor downregulation.
Insulin and hyperglycaemia may modify endothelin-receptor expression. It is unlikely, however, that insulin and hyperglycaemia play a significant role in mediating impaired responses to ET-1 through changes in receptor expression, as the ET-A receptor, which predominantly mediates the vasoconstrictor response to ET-1, does not appear to be affected by either hyperglycaemia or insulin (33,34).
Competitive endothelin-receptor blockade, which could be due to a macromolecule such as big ET-1 (35), also could explain the impaired response to ET-1 seen at low but not higher concentrations of ET-1. Alternatively, hyperglycaemia could result in modification of receptor proteins by glycation and cross-linking, impairing receptor-ligand interaction (36). Impaired responsiveness to ET-1 also may be due to a defect in postreceptor signal-transduction mechanisms coupled to endothelin receptors (17).
Vasodilation in response to ET-A receptor antagonism is impaired in the forearm vasculature of patients with type 2 diabetes, indicating that vasoconstriction mediated by endogenous endothelin acting through the ET-A receptor is impaired (37). This provides support that reduced vasoconstrictor responses are due either to a receptor- or postreceptor-mediated defect.
A nonspecific impairment of smooth muscle contraction could explain these findings. This is unlikely, as previous studies have shown no difference in pressor responses to noradrenaline, angiotensin II, and high-dose serotonin in diabetes (38,39). As responses to sodium nitroprusside were preserved in the patients with type 2 diabetes, this argues against structural abnormalities leading to nonspecific impairment of smooth muscle function. In addition, as the vasoconstrictor response to ET-1 is blunted rather than absent, with normal responses at higher doses, this implies a functional rather than a structural abnormality.
Upregulation or impaired downregulation (40) of endothelial ET-B receptors, resulting in increased production of endothelium-derived vasodilators, could oppose the vasoconstrictor response to ET-1 in type 2 diabetes. However, in vitro studies in streptozotocin-induced diabetic rats indicated that the impaired vasoconstrictor responses to ET-1 are endothelium independent (18,19), indicating that blunting of ET-1 responses is unrelated to the release of endothelium-derived vasodilators. This is in keeping with previous studies that have demonstrated impaired endothelium-dependent vasodilation in patients with type 2 diabetes (14,41).
Additional studies would help to elucidate further the mechanisms involved in the impaired responsiveness to ET-1 in diabetes. Further in vitro studies examining the effect of diabetes on ET-1 receptor binding sites and second messenger generation will help to confirm whether the defect is at a receptor or postreceptor level.
It has been argued in the haemodynamic hypothesis of diabetic microangiopathy that early hyperperfusion is the initiating step in the development of microvascular disease. It is possible that abnormal regulation of vascular tone by ET-1 could result in hyperperfusion. It should, however, be noted that, although not significant, forearm blood flow was lower in the patients with diabetes, whereas blood pressure was higher. Accordingly, forearm vascular resistance may in fact be higher in the patients with type 2 diabetes compared with control subjects, indicating that these patients studied were unlikely to be in the early hyperperfusion stage. Therefore, these results cannot necessarily be extrapolated to patients in the early hyperperfusion phase of diabetes, and obviously, further study specifically in this group of patients is required. In conclusion this study confirms impaired responsiveness to ET-1 in type 2 diabetes in vivo. Impaired regulation of vascular tone by the endothelin system in type 2 diabetes suggests a previously unrecognised mechanism for the hyperperfusion that precedes the development of diabetic microangiopathy and merits further investigation.
Acknowledgment: This study was supported by a Lilly Research Grant.
1. Halkin A, Benjamin N, Doktor HS, Todd SD, Viberti G, Ritter JM. Vascular responsiveness and cation exchange in insulin-dependent diabetes. Clin Sci
2. Ditzel J, Junker K. Abnormal glomerular filtration rate, renal plasma flow, and renal protein excretion in recent and short-term diabetics. BMJ
3. Vora JP, Dolben J, Dean JD, et al. Renal hemodynamics in newly presenting non-insulin dependent diabetes mellitus
. Kidney Int
4. Ishida K, Ishibashi F, Takashina S. Comparison of renal haemodynamics in early non-insulin-dependent and insulin-dependent diabetes mellitus
. J Diabetes ... Complications
5. Grunwald JE, Riva CE, Martin DB, Quint AR, Epstein PA. Effect of an insulin-induced decrease in blood glucose on the human diabetic retinal circulation. Ophthalmology
6. Tooke JE. Capillary pressure in non-insulin-dependent diabetes. Int Angiol
7. Parving H-H, Viberti GC, Keen H, Christiansen JS, Lassen NA. Hemodynamic factors in the genesis of diabetic microangiopathy
8. Zatz R, Brenner BM. Pathogenesis of diabetic microangiopathy
: the hemodynamic view. Am J Med
9. Tooke JE. Microvascular haemodynamics in diabetes mellitus
. Clin Sci
10. Mogensen CE, Andersen MJK. Increased kidney size and glomerular filtration rate in untreated juvenile diabetics: normalization by insulin treatment. Diabetologia
11. Vora JP, Dolben J, Williams JD, Peters JR, Owens DR. Impact of initial treatment on renal function in newly-diagnosed type 2 (non-insulin-dependent) diabetes mellitus
12. Nelson RG, Kunzelman CL, Pettitt DJ, Saad MF, Bennett PH, Knowler WC. Albuminuria in type 2 (non-insulin-dependent) diabetes mellitus
and impaired glucose tolerance in Pima Indians. Diabetologia
13. Kuroda N, Taniguchi H, Baba S, Yamamoto M. The pupillary light reflex in borderline diabetics. J Int Med Res
14. McVeigh GE, Brennan GM, Johnston GD, et al. Impaired endothelium-dependent and independent vasodilation in patients with type 2 (non-insulin-dependent) diabetes mellitus
15. Williams SB, Cusco JA, Roddy MA, Johnstone MT, Creager MA. Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus
. J Am Coll Cardiol
16. Haynes WG, Webb DJ. Contribution of endogenous generation of endothelin-1
to basal vascular tone. Lancet
17. Chakravarthy U, McGinty A, McKillop J, Anderson P, Archer DB, Trimble ER. Altered endothelin-1
induced contraction and second messenger generation in bovine retinal microvascular pericytes cultured in high glucose medium. Diabetologia
18. Hodgson WC, King RG. Effects of glucose, insulin or aldose reductase inhibition on responses to endothelin-1
of aortic rings from streptozotocin-induced diabetic rats. Br J Pharmacol
19. Fulton DJ, Hodgson WC, Sikorski BW, King RG. Attenuated responses to endothelin 1, KCl and CaCl, but not noradrenaline of aortae from rats with streptozotocin induced diabetes mellitus
. Br J Pharmacol
20. Bursell S, Clermont A, Oren B, King G. The in vivo effect of endothelins on retinal circulation in nondiabetic and diabetic rats. Invest Ophthalmol Vis Sci
21. Nugent AG, McGurk C, Hayes JR, Johnston GD. Impaired vasoconstriction to endothelin 1 in patients with NIDDM. Diabetes
22. Bloom S, Till S, Sonkensen P, Smith S. Use of a biothesiometer to measure vibration thresholds and their variation in 519 non-diabetic subjects. BMJ
23. O'Brien IAD, O'Hare JP, Lewin IG, Corrall RJM. The prevalence of autonomic neuropathy in insulin-dependent diabetes mellitus
: a controlled study based on heart rate variability. Q J Med
24. Kiowski W, Luscher TF, Linder L, Buhler FR. Endothelin-1
-induced vasoconstriction in human: reversal by calcium channel blockade but not by nitrovasodilators or endothelium-derived relaxing factor. Circulation
25. Haynes WG, Strachan FE, Webb DJ. Endothelin ETA
and ETB receptors
cause vasoconstriction of human resistance and capacitance vessels in vivo. Circulation
26. Clarke JG, Benjamin N, Larkin SW, Webb DJ, Davies GJ, Maseri A. Endothelin is a potent long-lasting vasoconstrictor in men. Am J Physiol
27. Matthews JNS, Altman DG, Campbell MJ, Royston P. Analysis of serial measurements in medical research. BMJ
28. Roubert P, Gillard V, Plas P, Chabrier PE, Braquee P. Downregulation of endothelin-1
binding sites in rat vascular smooth muscle cells. Am J Hypertens
29. Hirata Y, Yoshimi H, Takaichi S, Yanagisawa M, Masaki T. Binding and receptor down regulation of a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. FEBS Lett
30. Baldi E, Dunn MJ. Endothelin binding and receptor down regulation of glomerular mesangial cells. J Pharmacol Exp Ther
31. Takahashi K, Ghatei MA, Lam HC, O'Halloran DJ, Bloom SR. Elevated plasma endothelin in patients with diabetes mellitus
32. Kawamura M, Ohgawara H, Naruse M, et al. Increased plasma endothelin in NIDDM patients with retinopathy. Diabetes Care
33. McDonald D, Bailie J, Archer D, Chakravarthy U. Molecular characterization of endothelin receptors
and the effect of insulin on their expression in retinal microvascular pericytes. J Cardiovasc Pharmacol
34. Awazu M, Parker RE, Harvie BR, Ichikawa I, Kon V. Downregulation of endothelin-1 receptors
by protein kinase C in streptozotocin diabetic rats. J Cardiovasc Pharmacol
35. Naylor WG, Liu WG, Panagiotopoulos S, Casley DJ. Streptozotocin-induced diabetes reduces the density of [125
I]-endothelin-binding sites in rat cardiac membranes. Br J Pharmacol
36. Tarsio JF, Reger LA, Furcht LT. Decreased interaction of fibronectin, type IV collagen and heparin due to non-enzymatic glycation: implications for diabetes mellitus
37. McAuley DF, Nugent AG, McGurk C, Maguire S, Hayes JR, Johnston GD. Vasoconstriction mediated by endothelin ETA receptors
is impaired in patients with non insulin dependent diabetes [Abstract]. Diabet. Med
38. Christlieb AR, Janka HU, Kraus B, et al. Vascular reactivity to angiotensin II and to norepinephrine in diabetic subjects. Diabetes
39. Nugent AG, McGurk C, Rutherford R, Johnston GD. Peripheral haemodynamic effects of serotonin in type 2 diabetes mellitus
[Abstract]. Br J Clin Pharmacol
40. Sakurai T, Yanagisawa M, Masaki T. Molecular characterization of endothelin receptors
. Trends Pharmacol Sci
41. Caballero AE, Arora S, Saouaf R, et al. Microvascular and macrovascular reactivity is reduced in subjects at risk for type 2 diabetes. Diabetes