N A L A R T I C L E

Circulating Endothelin-1Levels Increase DuringEuglycemicHyperinsulinemic Clamp inLean NIDDM MenCLAUDIO FERRI, MDANTONIO CARLOMAGNO, MDSIMONETTA COASSIN, MDROBERTA BALDONCINI, MDMARIA R. CASSONE FALDETTA, MD

ORIANA LAURENTI, MDGLULIANA PROPERZI, MDANNA SANTUCCI, MDGLANCARLO DE MATT1A, MD

OBJECTIVE— To evaluate whether or not insulin stimulates endothelin (ET)-lsecretion in vivo.

RESEARCH DESIGN AND METHODS— Plasma El 1 levels were evalu-ated in 16 lean normotensive men with non-insulin-dependent diabetes mellitus(NIDDM) (mean age 50.3 ±4 .1 years) during either a 2-h euglycemic hyperinsuline-mic clamp (40 mU insulin • m~2 • min"1) or placebo infusion (50 ml isotonic saline)according to a single-blind randomized crossover protocol.

RESULTS— Circulating ET-1 levels increased during the euglycemic hyperinsu-linemic clamp (from 0.88 ± 0.38 pg/ml at time 0 to 1.66 ± 0.22 pg/ml and 1.89 ±0.99 pg/ml at 60 and 120 min, respectively [P < 0.05 vs. time 01) and returned tobaseline levels after the discontinuation of insulin infusion (0.71 ± 0.22 pg/ml after a30-min period of recovery [NS]). Compared with placebo, the euglycemic hyperinsu-linemic clamp induced a significant increase in plasma ET-1 levels at 60 min (P <0.0001) and 120 min (P < 0.0001). Changes in basal insulin levels and correspondingchanges in circulating ET-1 levels after a 2-h euglycemic hyperinsulinemic clamp weresignificantly correlated (r = 0.771, P < 0.0001). A possible unfavorable effect of ET-1on the tissue sensitivity to insulin-stimulated glucose uptake was suggested by thepresence of a negative correlation between total glucose uptake and baseline ET-1levels (r = -0.498, P < 0.05).

CONCLUSIONS — Our findings indicate that circulating ET-1 levels significantlyincrease during euglycemic hyperinsulinemic clamp in men with NIDDM. The nega-tive correlation between total glucose uptake and circulating ET-1 levels suggests thatthe peptide might exert negative effects on the insulin sensitivity of target tissues. Theconsequent increase in insulin secretion as well as the insulin-related ET-1 release fromendothelial cells could favor the development of diabetes-related vascular lesions.

From the Istituto di I Clinica Medica, Fondazione Andrea Cesalpino, Universita "La Sapienza,"Rome; and the Department of Internal Medicine (G.P., A.S.), University of L'Aquila, L'Aquila,Italy.

Address correspondence and reprint requests to Claudio Ferri, MD, Universita "La Sapienza,"Istituto di I Clinica Medica, Fondazione Andrea Cesalpino, 00161 Rome, Italy.

Received for publication 23 May 1994 and accepted in revised form 22 September 1994.NIDDM, non-insulin-dependent diabetes mellitus; ET, endothelin.

I nsulin resistance and the presence ofelevated insulin levels have been re-cently indicated as potential contribu-

tors to the development of atherosclerosisin non-insulin-dependent diabetes melli-tus (NIDDM) (1), impaired glucose toler-ance (1-4), obesity (2,5), and essentialhypertension (2,6,7). Hyperinsulinemiacould promote vascular damage (6)through increased platelet (8), sympa-thetic nervous system (9,10), Na+/Li+

countertransport (11), and Na+/H+ anti-port activities (11-13). Furthermore, in-sulin decreases fibrinolysis (14) and in-duces vascular smooth muscle cellproliferation (15).

Recently, studies have indicatedthat insulin may stimulate synthesis andsecretion of the potent vasoconstrictiveand mitogenic peptide endothelin (ET)-l(16,17) from cultured bovine (18) andporcine endothelial cells (19). Accord-ingly, elevated plasma ET-1 levels havebeen found in insulin-treated diabetic pa-tients (20) and in experimental models ofdiabetes during insulin treatment(18,21). In acute conditions, Wolpert etal. (22) have demonstrated a significantincrease in circulating ET-1 levels in eightobese women during a euglycemic hyper-insulinemic clamp. However, the studywas not placebo-controlled and was con-ducted in unselected patients, three ofwhom had chronic hypertension andwere being treated with hypotensiveagents. Moreover, blood samples forplasma ET-1 evaluations were not re-peated after recovery from insulin infu-sion, and no data on the range of circulat-ing ET-1 in nonobese normotensivesubjects were given, thus limiting the po-tentially exciting implications of the re-sults.

This was a single-blind, random-ized, crossover study, aiming to evaluatethe effect of either a euglycemic hyperin-sulinemic clamp or placebo (50 ml iso-tonic saline) on circulating ET-1 levels ina selected group of male NIDDM patientswithout signs of diabetes-related organdamage and with normal body weight

226 DIABETES CARE, VOLUME 18, NUMBER 2, FEBRUARY 1995

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and blood pressure levels. Moreover, toassess the impact of NIDDM on circulat-ing ET-1, the basal levels of the peptidewere also evaluated in age-matched non-diabetic men.

RESEARCH DESIGN ANDMETHODS — The study protocol wasapproved by our ethics committee. Afterinformed consent, 16 NIDDM men (Ta-ble 1) underwent either euglycemic hy-perinsulinemic clamp or placebo infusion(50 ml isotonic saline) at 1-week inter-vals, according to a single-blind, random-ized, crossover design. Patients were ad-mitted to the study after a 1-week periodon a normocaloric diet containing 120mmol NaCl and 60 mmol K+ per day.During the study, no subjects took anydrug except oral hypoglycemic agents (allpatients were treated with sulfonylureas).All patients had good metabolic control(i.e., HbAlc <7%, plasma glucose levels<7.8 mmol/1 at fast and <10.0 mmol/1 2and 4 h after breakfast and lunch, and noglycosuria).

Other inclusion criteria werebody mass index <25 kg/m2, supine sys-tolic blood pressure <90 mmHg, serumcreatinine <100 mmol/1, normality ofboth 99mTc-diethylenetriaminepentaace-tic acid and [131I]o-iodohippurate scin-tirenograms, absence of proteinuria andmicroalbuminuria <20 ^Lg/min on threeconsecutive 24-h urine collections, andabsence of cardiovascular defects as eval-uated on the basis of clinical and ultra-sound studies. All patients were whitenonsmokers, and none of them con-sumed >10 g of alcohol per day.

Euglycemic hyperinsulinemicclamp studies were performed in our out-patient unit after an overnight fast, with-out administering the morning dose oforal hypoglycemic agents, using themethod described by DeFronzo et al.(23). Briefly, at 0800, an intravenous he-parin-lock catheter system for blood sam-pling was located in a hand vein in retro-grade fashion while the patient wasrecumbent and comfortable in an air-conditioned room. An antecubital vein of

the contralateral forearm was temporarilycannulated for infusion. The hand bear-ing the blood-sampling system was main-tained in a thermoregulated box at 70°Cto allow measurement of arterialized ve-nous glucose levels (24) and to avoid theET-1 release that usually follows venouscannulation and blood stasis (25). For thesame reason, the basal blood sampling forET-1 measurement was done after 1 hfrom the locations of both intravenouslines. After the collection of blood sam-ples at time 0, a 10-min priming insulininfusion was started, followed by a con-stant insulin infusion (40 mU • m~2 •min"1 over a period of 110 min).Throughout the clamp, blood samples forplasma insulin determinations were takenat 20-min intervals. Plasma glucose con-centrations were measured at 5-min in-tervals and maintained at euglycemic lev-els by a variable infusion of 20%D-glucose. After time 0, blood samples forplasma ET-1 determination were takenagain at 60 and 120 min during insulininfusion and then at 150 min (i.e., after30 min of recovery). Throughout the test,blood pressure and heart rate were mon-itored every 10 min by a Riva-Roccisphygmomanometer and a stethoscope.On the morning of the study, patientsbrought their 24-h urine collections. A1-h urine collection was then obtainedfrom each patient before the clamp wasstarted. Furthermore, urine samples werecollected at the end of the clamp, i.e., at120 min.

The procedure followed for pla-cebo infusion was identical, but each pa-tient received, in single-blind fashion, 50ml isotonic saline (25 ml/h for 2 h). Inparticular, randomization assigned eightpatients to receive first the placebo andthen the euglycemic hyperinsulinemicclamp, while the remaining eight patientsstarted with the clamp and then were sub-jected to placebo infusion. In all cases, a1-week interval occurred between thetwo infusions. For ethical reasons, theclamp studies were performed in single-blind fashion. Therefore, each researcherknew when each patient was infused with

placebo or insulin, but was unaware ofthe study purpose, design, and results.

Assessment of normal range forplasma ET-1 levelsFor the range of normality, plasma ET-1levels were assessed in 12 age-matchedhealthy men with normal NaCl intakes at0900, 1 h after the subjects assumed thesupine position and underwent venouscannulation. All normal subjects were re-cruited from normotensive (i.e., bloodpressure constantly < 140/90 mmHg)members of our medical staff who hadnormal glucose tolerance, serum lipidlevels, body mass index (i.e., >19 and<25 kg/m2), renal and cardiac functions,and no concomitant pathological condi-tions. All volunteers were nonsmokers,and none of them consumed more than10 g of alcohol per day. One week beforeblood sampling for plasma ET-1 determi-nation, each subject was asked to followthe same diet prescribed for the NIDDMsubjects; the diet consisted of 120 mmolNa+ and 60 mmol K+ per day.

ET-1, insulin, and laboratorymeasurementsFor circulating ET-1 determination, eachplasma sample was injected onto C18 oe-tyloecylsilane columns (Pharmacia) pre-viously activated with 0.1% trifluoroaee-tic acid. To separate ET-1 from the otherET isoforms and big ET-1, each eluatewas then freeze-dried, reconstituted instarting high-pressure liquid chromatog-raphy buffer, and eluted by reverse-phasehigh-pressure liquid chromatographyover 70 min with the use of a linear gra-dient of 15-75% acetonitrile/0.1% triflu-oroacetic acid in distilled water. Thechromatographic separation of plasmaeluates identified a single peak of ET-1,perfectly corresponding to the elution po-sition of the human ET-1 standard. Frac-tions corresponding to ET-1 were col-lected each minute and evaporated beforereconstitution in assay buffer (50 mmol/lphosphate buffer, pH 7.4, containing0.9% NaCl, 0.05% NaNv and 0.4% bo-vine serum albumin). ET-1 immunoreac-

DIABETES CARE, VOLUME 18, NUMBER 2, FEBRUARY 1995 227

Endothelin-1 and insulin

tivity was assayed on reconstituted sam-ples by a sensitive radioimmunoassay(Peninsula). Human ET-1 (Peptide Insti-tute) was used as a standard. Interassayand intra-assay variations were <10%.Cross-reactivity of the ET-1 antibodywith ET-2 and ET-3 was <7%.

Plasma insulin levels were assayedby a commercial radioimmunoassay kit(Ares Serono). Plasma glucose during theeuglycemic clamp studies were assayedby the glucose oxidase method with theuse of the Glucose Analyzer II (Beckman).Other routine chemical tests, includingurinary sodium and potassium, were per-formed by standard laboratory methods.All of the above laboratory procedures aswell as all blood sampling for ET-1 mea-surements during either clamp or placebowere performed by two separate staffs ofresearchers, who where unaware of thestudy design and purpose.

Statistical analysisData were stored in an M380 Olivetticomputer and analyzed by the softwareSPSS (SPSS). The statistical tests usedwere Student's t test, analysis of variance,and Pearson's coefficient. Repeated mea-sures were tested by analysis of variancefollowed by Bonferroni's test and Stu-dent-Newman-Keuls test. Statistical sig-nificance was considered as P < 0.5. Alldata are presented as means ± SD.

RESULTS

Baseline comparison betweennormal and NIDDM menTo compare baseline ET-1 levels betweenNIDDM patients and nondiabetic controlsubjects, plasma ET-1 levels were evalu-ated in 12 nondiabetic men and in 16NIDDM men 1 h after venous cannula-tion, in the supine position. Our resultsshowed that plasma ET-1 levels were sig-nificantly higher (P < 0.05) in NIDDMpatients (0.88 ± 0.38 pg/ml) than in con-trol subjects (0.64 ± 0.10 pg/ml) (Fig. 1).Fasting insulin levels (P < 0.05) were alsohigher in NIDDM patients than in controlsubjects (Table 1), while serum choles-

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NON-INSULIN-DEPENDENT NON-DIABETICDIABETIC PATIENTS (N.I6) SUBJECTS (N.12)

Figure 1—Plasma ET-1 levels (means ± SD) inlean NIDDM men (D, n = 16) and age-matchednondiabetic control men (EH, n = 12).

terol and triglyceride levels were not sig-nificantly different between the twogroups. Baseline ET-1 levels were directlycorrelated with fasting insulin levels inNIDDM men (P < 0.0001). In contrast,plasma ET-1 levels did not correlate withbody mass index, serum lipid levels, orsystolic and diastolic blood pressure.

Circulating ET-1 during aeuglycemic hyperinsulinemic clampIn each NIDDM man, a euglycemic hy-perinsulinemic clamp was started imme-

diately after blood sampling for baselineplasma ET-1 assessment. During theclamp, plasma insulin was maintained ata plateau ranging from 574 ± 16.4 pmol/1at 40 min to 558.3 ± 14.2 pmol/1 at 120min (Fig. 2A). Plasma glucose levels re-mained stable throughout the 2 h of theclamp (Fig. IB). Plasma ET-1 levels sig-nificantly increased during the 2-h insu-lin infusion (P < 0.05 vs. time 0 at 60 and120 min) (Fig. 3). The discontinuation ofinsulin infusion was followed by the re-turn of plasma ET-1 to pretest levels (Fig.3).

Compared with placebo, euglyce-mic hyperinsulinemic clamp induced asignificant increase in plasma ET-1 levelsat 60 min (P < 0.0001) and 120 min (P <0.0001). It was of particular interest thatchanges in circulating ET-1 levels duringthe clamp (i.e., from baseline to 120 min)directly correlated with the correspond-ing changes in circulating insulin (r =0.771, P < 0.0001).

A quantitative estimate of the in-sulin sensitivity was provided by themean glucose infusion rate during the eu-glycemic clamp (22). Total glucose up-take was 20.4 ± 8.6 jamol • kg"1 • min"1

(the normal range in our diabetes unit forage-matched healthy men being 42.9 ±4.0 jamol • kg"1 • min"1; P < 0.0001 vs.the level for NIDDM men). A possible re-

Table 1—Clinical and laboratory features of NIDDM men and nondiabetic control men

Mean age (years)Duration of diabetes (years)Weight (kg)Body mass index (kg/m2)Blood pressure (mmHg)

SystolicDiastolic

Fasting insulin (pmol/1)Fasting glucose (mmol/1)Total cholesterol (mmol/1)Triglycerides (mmol/1)Serum creatinine (mmol/1)

Diabetic patients(n = 16)

50.3 ± 4 . 17.3 ± 6.2

73.9 ± 9.923.2 ± 0.5

128.9 ± 8 . 181.9 ±6 .2

116.7 ±63.16.1 ± 0.34.9 ± 1.21.2 ± 0.5

88.4 ± 4.4

Control subjects(n = 12)

49.5 ± 4.6—

72.1 ± 2.423.5 ± 0.6

125.0 ± 4.278.5 ± 2.5

71.16 ±29.15.4 ± 0.64.4 ± 0.21.1 ±0.1

79.6 ± 8.8

P<

———

——

0.05————

Data are means ± SD.

228 DIABETES CARE, VOLUME 18, NUMBER 2, FEBRUARY 1995

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Figure 2—Graphs show plasma insulin (A), glucose (B), and blood pressure levels (C) in lean NIDDMmen (n = 16) during either a 2-h euglycemic hyperinsulinemic clamp (•) or placebo (50 ml isotonicsaline) infusion (•).

lationship between circulating ET-1 lev-els and glucose metabolism was sug-gested by the presence of a negativecorrelation between total glucose uptakeand baseline ET-1 levels (r = -0.498, P< 0.05) (Fig. 4).

No patients complained of anyproblems during the euglycemic hyperin-sulinemic clamp. Neither insulin-stimu-lated nor recovery ET-1 levels were corre-lated with corresponding blood pressurelevels. In particular, insulin infusion wasnot accompanied by significant changesin blood pressure (Fig. 2C), heart rate,and serum sodium and potassium levels.On the contrary, both urinary sodium(from 85.1 ± 7.3 jutmol/min to 39.7 ±16.8 jLtmol/min, P < 0.0001) and potas-sium excretion (from 47.7 ± 15.0 to 28.1± 18.1 /Ltmol/min, P < 0.05) significantlydecreased during the clamp.

CONCLUSIONS— Our resultsshowed that circulating ET-1 levels sig-nificantly increased during insulin infu-sion in NIDDM men. Insulin and ET-1increments observed during the euglyce-mic hyperinsulinemic clamp were di-rectly correlated, thus further supportingthe role of insulin as a promoter of ET-1secretion in vivo. Accordingly, baselineET-1 levels, as evaluated by a method thatallows distinguishing ET-1 from the othertwo isoforms and big ET-1 (26,27), weresignificantly higher in NIDDM men thanin nondiabetic control subjects.

As previously reported, ET-1 se-cretion is stimulated in vitro by severalsubstances, such as adrenaline (16), an-giotensin II (28), vasopressin (29), trans-forming growth factor-j3 (30), interleu-kin-1 (31), and thrombin (32,33). Withregard to insulin, both ET-1 gene ex-pression (34) and release (18,19) havebeen demonstrated after insulin stimu-lation in bovine- (18,34) and porcine-(19) cultured endothelial cells. In accor-dance with these in vitro data, elevatedplasma ET-1 levels have been describedin insulin-treated rats with strcptozoein-induced diabetes (18,21) and in nondia-betic Sprague-Dawley rats after subcuta-

DIABETES CARE, VOLUME 18, NUMBER 2, FEBRUARY 1995 229

Endoihelin-1 and insulin

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Figure 3—Plasma ET-1 levels (means ± SD) in lean NIDDM men (n = 16) receiving either a 2-heuglycemic hyperinsulinemic clamp (J3) or placebo (50 ml isotonic saline) infusion (•). Horizontal barindicates the time of insulin or placebo infusion. Statistical significance values are given versus baseline(*P < 0.05 vs. baseline and 150 min) and (into the dotted lines) versus placebo.

neous abdominal implantation of aninsulin pellet (18). In humans, the result-ing plasma ET-1 levels were significantlyhigher in insulin-dependent and NIDDMpatients than in nondiabetic control sub-jects (20).

The presence of a direct correla-tion between insulin and ET-1 levels andthe significant ET-1 increment during eu-glycemic hyperinsulinemia are in com-plete agreement with these data. Never-theless, the physiological explanation ofour findings seems to be problematic,since ET-1 has potent contractile and pro-liferative effects on vascular smooth mus-cle cells (16,17,35-37). Indeed, our pa-tients were all normotensive, and nosignificant blood pressure changes oc-curred during changes in circulating ET-1levels.

From a theoretical point of view,our findings seem to suggest that circulat-ing ET-1 plays no role in the regulation ofarterial pressure in NIDDM patients. Ac-cording to this hypothesis, the secretionof ET-1 is polarized, and more than 80%of newly generated ET-1 is released intothe basolateral compartment (38). There-fore, circulating ET-1 could not corre-

spond to the local concentrations into thevascular wall and may not reflect the ef-fective pathophysiological role of ET-1. Incomplete agreement with this interpreta-

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tion of our results, Wolpert et al. (22) in-dicated that ET-1 levels increase, whileblood pressure remains unchanged, dur-ing a euglycemic hyperinsulinemic clampperformed in obese women. Further-more, Takahashi et al. (20) reported in-creased circulating ET-1 levels in NIDDMpatients compared with age-matchednondiabetic subjects, but no differenceswere found between normotensive andhypertensive subjects suffering fromNIDDM (20). Therefore our and Taka-hashi's data seem to suggest that diabetesrather than hypertension may determinean increase in plasma ET-1 level.

On the other hand, experimentalfindings have demonstrated that mild va-sodilation occurs during insulin infusion(39). Therefore, insulin itself might coun-teract the vasoconstrictive action of ET-1,at least when infused during a short-termperiod. Moreover, it should be empha-sized that a rapid increase in circulatingET-1 levels is followed by a rapid decre-ment in peripheral vascular resistance(16,35,37,40,41), mediated by the acti-vation of ETB receptors on the endothelial

DD

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8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42TOTAL GLUCOSE UPTAKE (gmol/Kg/min)

Figure 4—Relationship between total glucose uptake and basal circulating ET-1 levels in leanNIDDM men (n = 16). r = -0A98; P < 0.05.

230 DIABETES CARE, VOLUME 18, NUMBER 2, FEBRUARY 1995

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surface and the consequent release ofprostacyclin (42) and nitric oxide (42-45). On the contrary, the sustained vaso-constriction due to ET-1 binding to ETA

receptors on the smooth muscle cell sur-face is delayed in both in vitro and in vivoconditions (16,35,46) and can be furthercounteracted by the simultaneous activa-tion of both paracrine and endocrine va-sodilating agents (42-45,47).

According to these data, a causalrelationship between a persistent eleva-tion of circulating ET-1 and increase inblood pressure levels has been reportedonly in patients affected by ET-1-secret-ing tumors (48), while either elevated(49-51) or normal ET-1 levels (52,53)have been described in essential hyper-tensive patients. Therefore, it does notseem arbitrary to state that a rise in bloodpressure is not per se combined with apersistent rise in circulating ET-1 and viceversa.

On the other hand, insulin itselfmay exert some hemodynamic actions,inducing a mild increase in blood flow ininsulin-sensitive tissues (39,54). In thiscontext, Laakso et al. (54) have recentlysuggested that an impaired ability of in-sulin to stimulate skeletal muscle bloodflow could contribute to insulin resis-tance in obese men. Moreover, a strongcorrelation between muscle blood flowand insulin sensitivity (55) already hasbeen demonstrated in animals (56) and inhuman normotensive (57,58) and hyper-tensive (58) subjects. In keeping with thishypothesis, we demonstrated a negativecorrelation between total glucose uptakeand circulating ET-1 levels. This findingsuggests the possibility that ET-1 couldimpair the ability of insulin to increasetissue blood supply, thus reducing the in-sulin sensitivity of target tissues. The con-sequent increase in insulin secretioncould itself favor the development of car-diovascular disease due to higher insulinlevels in the bloodstream.

Another finding of this study wasrepresented by the significant decrease inrenal sodium and potassium excretion oc-curring during the euglycemic hyperinsu-

linemic clamp. Similar data have been pre-viously described in both animal andhumans (6,59-62). However, it is interest-ing to note that these acute tubular effects ofinsulin were not followed by significantchanges in blood pressure and serum elec-trolytes. Therefore, insulin may regulate so-dium and potassium excretion, at leastwhen given at high doses and under acuteconditions, but its influence on blood pres-sure levels seems to be inconsistent. Inkeeping with this hypothesis, our NIDDMmale patients were significantly resistant toinsulin-mediated glucose uptake and hadincreased fasting insulin levels, but their ar-terial pressure was absolutely normal, evenduring euglycemic hyperinsulinemic clampstudies.

In conclusion, in this study weprovide evidence that short-term insulininfusion has no effect on blood pressureregulation while it is able to stimulateET-1 release in NIDDM male patients. Al-though the physiological meaning of theinsulin-related ET-1 release remains to beelucidated, the negative correlation evi-dent between baseline ET-1 levels and to-tal glucose uptake suggested that ET-1may have unfavorable effects on the insu-lin sensitivity of target tissues. In futurestudies, the possible benefit deriving fromET-1 antagonism could be tested by spe-cific monoclonal antibodies against theET-1 receptor.

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