The Possible Protective Role ofGlucagon-LikePeptide1onEndotheliumDuring theMeal and Evidencefor an “Endothelial Resistance” toGlucagon-Like Peptide 1 in DiabetesANTONIO CERIELLO, MD1

KATHERINE ESPOSITO, MD2

ROBERTO TESTA, MD3

ANNA RITA BONFIGLI, PHD3

MAURIZIO MARRA, PHD3

DARIO GIUGLIANO, MD2

OBJECTIVE—Glucagon-like peptide 1 (GLP-1) stimulates insulin secretion. However, GLP-1also improves endothelial function in diabetes.

RESEARCHDESIGNANDMETHODS—Sixteen type 2 diabetic patients and 12 controlsubjects received a meal, an oral glucose tolerance test (OGTT), and two hyperglycemic clamps,with or without GLP-1. The clamps were repeated in diabetic patients after 2 months of strictglycemic control.

RESULTS—During the meal, glycemia, nitrotyrosine, and plasma 8-iso prostaglandin F2a(8-iso-PGF2a) remained unchanged in the control subjects, whereas they increased in diabeticpatients. Flow-mediated vasodilation (FMD) decreased in diabetes, whereas GLP-1 increased inboth groups. During the OGTT, an increase in glycemia, nitrotyrosine, and 8-iso-PGF2a and adecrease in FMD were observed at 1 h in the control subjects and at 1 and 2 h in the diabeticpatients. In the same way, GLP-1 increased in both groups at the same levels of the meal. Duringthe clamps, in both the control subjects and the diabetic patients, a significant increase in nitro-tyrosine and 8-iso-PGF2a and a decrease in FMD were observed, effects that were significantlyreduced by GLP-1. After improved glycemic control, hyperglycemia during the clamps was lesseffective in producing oxidative stress and endothelial dysfunction and the GLP-1 administrationwas most effective in reducing these effects.

CONCLUSIONS—Our data suggest that during the meal GLP-1 can simultaneously exert anincretin effect on insulin secretion and a protective effect on endothelial function, reasonablycontrolling oxidative stress generation. The ability of GLP-1 in protecting endothelial functionseems to depend on the level of glycemia, a phenomenon already described for insulin secretion.

Diabetes Care 34:697–702, 2011

O ral administration of glucose is amore potent secretory stimulus forinsulin than its intravenous infu-sion (1). This observation gave rise to the“incretin effect” concept, i.e., stimulationof insulin secretion as a response to foodbefore an increase in blood glucose levels.An incretin hormone is the glucagon-likepeptide 1 (GLP-1).

Type 2 diabetes mellitus is increasingall over the world. Patients with diabetes

have an increased risk of cardiovasculardisease. Recently, much attention hasbeen paid to evidence that abnormalitiesof the postprandial state are importantcontributing factors to the developmentof atherosclerosis, even in diabetes (2). Indiabetic subjects, the combination ofpostprandial hyperglycemia and post-prandial hypertriglyceridemia has beenrecently proposed as an independentrisk factor for cardiovascular disease (2).

The response-to-injury hypothesisof atherosclerosis states that the initialdamage affects the arterial endothelium,leading to endothelial dysfunction (3). In-deed, endothelial dysfunction has beendemonstrated in patients with diabetes,and hyperglycemia has been implicatedas a cause of endothelial dysfunction innormal and diabetic subjects (2). It hasbeen suggested that hyperglycemia indu-ces an endothelial dysfunction throughthe production of an oxidative stress (2).

GLP-1 is now being used in clinics toenhance insulin secretion and reducebody weight in patients with type 2 di-abetes mellitus (4), in whom a defect ofGLP-1 secretion/action in response to themeal has often been reported (5). GLP-1has been shown to lower postprandialand fasting glucose and HbA1c, to sup-press the elevated glucagon level, and tostimulate glucose-dependent insulin syn-thesis and secretion (4).

Apart from the well-documented in-cretin effect of GLP-1, its role in thecardiovascular system also arouses inter-est. GLP-1 effects on the cardiovascularsystem may include a direct action on theendothelium, where the presence of spe-cific receptors for GLP-1 has been demon-strated (6). GLP-1 has been demonstratedto improve endothelial function in diabe-tes (7). However, the explanation of whyGLP-1 may have such a relevant physio-logic role on cardiovascular system stillremains unknown. A possible explanationwould be to consider GLP-1 as an endog-enous protective factor for the vascularsystem when this protection is especiallyneeded: during a meal. As pointed out byZilversmit (8) many years ago, atheroscle-rosis could be considered to be a prandialphenomenon. Therefore, it is clearly plau-sible that GLP-1, on the one hand, canhelp during a meal (glucose homeostasis,appetite control, fat metabolism), and onthe other, can protect the endotheliumagainst the possible damaging effect ofthe meal. This protective effect should be

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From the 1Insititut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain; the 2Division ofMetabolic Diseases, Center of Excellence for Cardiovascular Diseases, Second University of Naples, Naples,Italy; and the 3Metabolic and Nutrition Research Centre on Diabetes, INRCA, Ancona, Italy.

Corresponding author: Antonio Ceriello, aceriell@clinic.ub.es.Received 14 October 2010 and accepted 21 December 2010.DOI: 10.2337/dc10-1949© 2011 by the American Diabetes Association. Readers may use this article as long as the work is properly

cited, the use is educational and not for profit, and thework is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

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exerted improving the antioxidant defen-ses of the endothelium (9), thereby pro-tecting the vascular system against theoxidative stress that increases afteringesting a meal (2).

The aim of this study is to prove thatGLP-1 physiologically protects the endothe-lial function during a meal and, morespecifically, protects the endothelial func-tion from the hyperglycemia-induced alter-ations, and that this effect is mediated bylowering oxidative stress. Moreover, a fur-ther aim is to explore this aspect in diabetes.

RESEARCH DESIGN ANDMETHODS—Sixteen type 2 diabeticpatients and 12 matched healthy controlsubjects participated in the study. Base-line characteristics of the study groups areshown in Table 1.

The study was approved by the ethicscommittee of Institut d’Investigacions Bio-mèdiques August Pi i Sunyer (IDIBAPS),and written consent from the study sub-jects was obtained.

Ten patients were on diet alone, andthe other six patients were on metformin,which was discontinued at least 4 weeksbefore the study. None of the type 2 di-abetic patients had retinopathy, nephropa-thy, or neuropathy. Five patients hadhypertension treated with an angiotensin-converting enzyme inhibitor, which waswithheld on the study days. None of thesubjects were receiving statin or antioxi-dant supplementation.

Synthetic GLP-1 (7–36) amide waspurchased from PolyPeptide Laboratories

(Wolfenbuttel, Germany), and the samelot number was used in all studies.

Study designBoth healthy control subjects and type 2diabetic patients underwent the followingstudies: a standard meal according toVollmer et al. (10) and an oral glucosetolerance test (OGTT; 75 g glucose in300 mL water) in randomized order, ondifferent days. These tests were followedin a randomized order and on differentdays by two hyperglycemic clamps (11)with or without GLP-1. GLP-1 was in-fused at a rate aiming to have the sameplasma concentration reached duringthe OGTT (0.4 pmol z kg21 z min21) ac-cording to Nauck et al. (12). During thehyperglycemic clamp, the level of glyce-mia was settled at the same level as thatof mean glycemia reached at 1 h (controlsubjects 8.5 mmol/L; diabetic patients15 mmol/L) and 2 h (control subjects5 mmol/L; diabetic patients 12.8 mmol/L)during the OGTT.

At the end of these studies, diabeticpatients were treated intensively with in-sulin for 2 months to improve glycemiccontrol. The clamp studies were thenrepeated randomly with the same levelsof glycemia and GLP-1 infusion rate.

At baseline and at 1 and 2 h, duringthe meal test and the OGTT, and duringeach clamp, glycemia, insulin, endothelialfunction (flow-mediated vasodilation[FMD]), plasma nitrotyrosine and plasma8-iso prostaglandin F2a (8-iso-PGF2a)(both markers of oxidative stress), and

GLP-1 (active 7–36) plasma levels weremeasured.

Biochemical measurementsCholesterol and triglycerides were mea-sured enzymatically (Roche Diagnostics,Basel, Switzerland). HDL cholesterol wasestimated after the precipitation of apoli-poprotein B with phosphotungstate/magnesium (13). LDL cholesterol was cal-culated after lipoprotein separation (13).Plasma glucose was measured by theglucose-oxidase method, HbA1c wasmeasured by high-performance liquidchromatography, and insulin was mea-sured by microparticle enzyme immuno-assay (Abbott Laboratories, Wiesbaden,Germany). Nitrotyrosine plasma concen-tration was assayed by enzyme-linkedimmunosorbent assay, recently validatedby our laboratory (13).

A commercially available kit was usedto measure 8-iso-PGF2a (Cayman Chem-ical, Ann Arbor, MI). GLP-1 (active 7–36)was measured by a radioimmunoassay kit(Peninsula Laboratories, Belmont, CA).The detection limit is 10 pg/mL, and theintra- and interassay coefficient of varia-tion are 8 and 18% at 50 pg/mL and 5 and13% at 300 pg/mL, respectively.

Endothelial functionFMD was evaluated (14). At the end ofeach test, sublingual nitroglycerin (0.3 mg)was administered, and 3 min later the lastmeasurements were performed to mea-sure endothelium-independent vasodila-tion.

Statistical analysisData are expressed as mean 6 SE. Thesample size was selected according to pre-vious studies (7,10). The Kolmogorov–Smirnov algorithmwas used to determinewhether each variable had a normal dis-tribution. Comparisons of baseline dataamong the groups were performed usingunpaired Student t test or Mann-WhitneyU test, where indicated. The changes invariables during the tests were assessedby two-way ANOVA with repeated meas-ures or Kolmogorov–Smirnov test, whereindicated. If differences reached statisticalsignificance, post hoc analyses withpaired, two-tailed t test or Wilcoxonsigned rank test for paired comparisonswere used to assess differences at individ-ual time periods in the study. Statisticalsignificance was defined as P , 0.05.

RESULTS—As expected, basal glyce-mia, insulin, HbA1c, nitrotyrosine, and

Table 1—Baseline characteristics of the control and type 2 diabetic subjects

Control subjects (n 5 12)Type 2 diabeticsubjects (n 5 16)

Sex 6M/6F 9M/7FAge, years 50.5 6 2.5 51.3 6 2.6BMI, kg/m2 28.5 6 3.1 29.5 6 3.3Duration of diabetes, years 5.5 6 1.3Fasting plasma glucose, mmol/L 4.5 6 0.3 7.8 6 2.2*HbA1c, % 4.8 6 0.2 8.4 6 0.3*Resting systolic blood pressure, mmHg 117.3 6 5.5 123.4 6 6.4Resting diastolic blood pressure, mmHg 77.5 6 2.2 80.2 6 3.6Total cholesterol, mmol/L 4.5 6 0.6 5.1 6 0.8Triglycerides, mmol/L 0.9 6 0.2 1.2 6 0.4HDL cholesterol, mmol/L 1.4 6 0.2 1.2 6 0.3LDL cholesterol, mmol/L 2.5 6 0.3 2.6 6 0.4FMD, % 11.7 6 0.7 5.9 6 0.6*Nitrotyrosine, mmol/L 0.24 6 0.05 0.52 6 0.03*8-iso-PGF2a, pg/mL 32.6 6 4.6 65.0 6 4.5*Fasting insulin, pmol/L 73.3 6 4.4 107.3 6 15.2*Data are expressed as means 6 SE. *P , 0.001 vs. control subjects.

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8-iso-PGF2a were increased in diabetes,and FMD was decreased (Table 1). Basal,fasting level of GLP-1 was not differentbetween control subjects and diabeticpatients (Table 1).

During the meal, all test parameters,except those for GLP-1 and insulin, re-mained unchanged in the control sub-jects, whereas a significant increase at 1and 2 h of glycemia, insulin, nitrotyro-sine, and 8-iso-PGF2a and a decrease inFMD were observed in type 2 diabeticpatients compared with their basal values.In both control subjects and diabeticpatients, GLP-1 increased at 1 and 2 hin a similar manner (Fig. 1).

In the control subjects, during theOGTT, an increase of glycemia, insulin,nitrotyrosine, and 8-iso-PGF2a and a de-crease of FMD were observed at 1 h,whereas at 2 h all the parameters returnedto the basal values (Fig. 1). In the diabeticpatients, at both 1 and 2 h, a significant

increase of glycemia, insulin, nitrotyro-sine, and 8-iso-PGF2a and a decrease ofFMD were observed compared with thebasal values (Fig. 1). In both control sub-jects and diabetic patients, during theOGTT, GLP-1 increased at 1 and 2 hin a similar manner (Fig. 1), and the val-ues were not different from those reachedduring the meal test (Fig. 1).

In diabetic patients, at both 1 and2 h, a significant increase in glycemia (P,0.01), insulin (P , 0.01), nitrotyrosine(P , 0.05), and 8-iso-PGF2a (P , 0.05),and a decrease in FMD (P , 0.05) wereobserved compared with the valuesreached during theOGTT, whereas no dif-ference was found for GLP-1 (Fig. 1).

According to the values observedduring the OGTT, during the clamps gly-cemia was maintained for the first hourat 8.5 mmol/L in control subjects and at15 mmol/L 1 h in diabetic patients,whereas for the second hour glycemia

was maintained at 5 mmol/L in controlsubjects and at 13 mmol/L in diabeticpatients. During the clamps, performedwith placebo, GLP-1 concentration re-mained unchanged during the study pe-riod in both control subjects and diabeticpatients, whereas its concentration wasalmost equivalent to that observed duringthe meal test and the OGTT when it wasconstantly infused (Fig. 2). Insulin con-centration increased in both control sub-jects and diabetic patients during thehyperglycemic clamp, and its increasewas significantly higher during GLP-1 in-fusion (Fig. 2). During both the clamps,with or without GLP-1, in the control sub-jects, an increase in nitrotyrosine and8-iso-PGF2a and a decrease in FMDwere observed at 1 h, whereas at 2 h, allthe parameters returned to their basal val-ues (Fig. 2). Similarly, in diabetic patients,at both 1 and 2 h during both the clamps,a significant increase in nitrotyrosine and8-iso-PGF2a and a decrease in FMD wereobserved compared with the basal values(Fig. 2). However, in the control subjects,at 1 h, the values of nitrotyrosine and8-iso-PGF2a significantly increased, andthe values of FMD significantly decreasedin the clamp with placebo compared withthe values observed during the clamp withGLP-1 (Fig. 2). Similarly, in diabetic pa-tients at both 1 and 2 h, the values of nitro-tyrosine and 8-iso-PGF2a significantlyincreased, and the values of FMD signifi-cantly decreased in the clamp with pla-cebo compared with the values observedduring the clamp with GLP-1 (Fig. 2).

Two months of insulin treatment re-sulted in a significant decrease in HbA1c(8.46 0.3 vs. 7.26 0.4%, P, 0.01) andan improvement of fasting glycemia(8.2 6 2.0 vs. 6.4 6 1.8 mmol/L, P ,0.01), insulin (110.3 6 17.2 vs. 86.2 613.2 pmol/L, P , 0.01), nitrotyrosine(0.52 6 0.03 vs. 0.39 6 0.06 mmol/L,P , 0.05), 8-iso-PGF2a (65.0 6 4.5 vs.44.2 6 2.5 pg/mL, P , 0.05), and FMD(5.9 6 0.6 vs. 7.8 6 0.7%, P , 0.05) indiabetic patients.

During the two clamps, in diabeticpatients, at 1 and 2 h, a significant in-crease in nitrotyrosine and 8-iso-PGF2aand a decrease in FMD (P , 0.01) wereobserved compared with the basal values(Fig. 3). As before the improvement ofglycemic control, at both 1 and 2 h, thevalues of nitrotyrosine and 8-iso-PGF2asignificantly increased and the values ofFMD significantly decreased in the clampwith placebo compared with the valuesobserved during the clamp with GLP-1

Figure 1—Changes of glycemia, GLP-1, FMD, plasma nitrotyrosine, 8-iso-PGF2a, and insulinduring the meal and the OGTT in normal healthy control subjects and type 2 diabetic patients.Data are expressed as mean6 SE;△, meal test controls;▲, OGTT controls;○, meal test type 2diabetes;●, OGTT type 2 diabetes; *P, 0.001 vs. basal; †P, 0.01 vs. basal; ‡P, 0.05 vs. basal;§P , 0.01 vs. OGTT; ||P , 0.05 vs. OGTT.

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(Fig. 3). However, the same values of gly-cemia were less effective in producing ox-idative stress and endothelial dysfunctionafter 2 months of improved glycemic con-trol. Because the basal values before andafter tight glycemic control were signifi-cantly different, we have compared the Dbetween the basal value and the value at 1and 2 h, during each clamp, respectively:Dnitrotyrosine 1 h 0.426 0.04 vs. 0.2060.05, P, 0.01;Dnitrotyrosine 2 h 0.3260.03 vs. 0.15 6 0.05, P , 0.01; D 8-iso-PGF2a 1 h 68.96 4.1 vs. 30.36 3.2, P,0.05; D 8-iso-PGF2a 2 h 50.5 6 3.1 vs.20.3 6 1.2, P , 0.01; D FMD 1 h 4.1 60.5 vs. 2.36 0.2, FMD, P, 0.05; D FMD2 h 3.7 6 0.5 vs. 2.2 6 0.2, FMD, P ,0.05 in the study performed with pla-cebo, compared with that in the previousclamp. Of particular interest is that GLP-1administration was most effective in

this condition of improved metaboliccontrol than in previous experiments:Dnitrotyrosine 1 h 0.21 6 0.02 vs.0.08 6 0.02, P , 0.01; Dnitrotyrosine2 h 0.15 6 0.04 vs. 0.03 6 0.02, P ,0.01; D 8-iso-PGF2a 1 h 31.5 6 4.1 vs.15.36 3.7, P, 0.05; D 8-iso-PGF2a 2 h20.3 6 2.1 vs. 10.2 6 1.5, P , 0.05; DFMD 1 h 1.5 6 0.4 vs. 0.6 6 0.2, FMD,P, 0.05; D FMD 2 h 2.16 0.3 vs. 0.360.2, FMD, P, 0.05 in the study performedwith placebo, compared with the previousclamp. GLP-1 infusion after optimized gly-cemic control was accompanied by a sig-nificant increase in insulin secretion at both1 and 2 h (Fig. 3). No difference was foundin endothelium-independent vasodilata-tion in all the studies.

CONCLUSIONS—This study dem-onstrated that the presence of GLP-1

during hyperglycemia significantly pro-tects endothelial function and decreaseshyperglycemia-induced oxidative stressgeneration. This evidence clearly emergeswhen we compare the effect of hypergly-cemia during the clamps in both normaland diabetic patients. In the absence ofGLP-1, hyperglycemia induces endothe-lial dysfunction and oxidative stress,whereas the concomitant infusion ofGLP-1 significantly prevents this effect.It has already been largely demonstratedthat hyperglycemia induces endothelialdysfunction through the generation of anoxidative stress (2) and that GLP-1 canreduce oxidative stress (9). GLP-1 alsoimproves endothelial dysfunction in dia-betes (7). Therefore, our data suggest thatGLP-1 may protect endothelia functionduring hyperglycemia, reducing oxida-tive stress generation.

Our data also support a possiblephysiologic protective role of GLP-1 onendothelial function. However, the effectof GLP-1 on endothelial function seemsto be dependent on the level of hypergly-cemia. The same plasma levels of GLP-1have a different effect on the endothelialfunction and oxidative stress in normalsubjects during the test meal and theOGTT. Consistent with a previous study,in normal control subjects the levels ofGLP-1 are almost superimposable duringthe meal and the OGTT (9). However,during the meal test, when glycemia re-mains constantly in the normal range, en-dothelial function and oxidative stressremain unaltered. However, at 1 h, duringthe OGTT, as already reported (15), whenhyperglycemia appears, endothelial func-tion and oxidative stress also appear.These data suggest that in the presenceof hyperglycemia, GLP-1 partly loses itsprotective effect on endothelial functionand oxidative stress.

This finding is also supported by thedata in diabetic patients. As previouslyreported (10), the plasma levels of GLP-1were not different between the meal testand the OGTT. As expected, the levels ofglycemia were significantly different be-tween the meal test and the OGTT, andthis was accompanied by a parallel wors-ening of both oxidative stress and endo-thelial function.

The possibility that hyperglycemiamay condition the protective effects ofGLP-1 on endothelial function and oxi-dative stress is also supported by the datawith clamps in type 2 diabetic patientsafter a period of improved glycemic con-trol.

Figure 2—Changes of glycemia, GLP-1, FMD, plasma nitrotyrosine, 8-iso-PGF2a, and insulinduring the hyperglycemic clamp with or without GLP-1 infusion in normal healthy control sub-jects and type 2 diabetic patients. Data are expressed as mean6 SE;△, hyperglycemic clamp +placebo control subjects; ▲, hyperglycemic clamp + GLP-1 control subjects; ○, hyperglycemicclamp + placebo type 2 diabetes;●, hyperglycemic clamp + GLP-1 type 2 diabetes; *P, 0.001 vs.basal; †P , 0.01 vs. basal; ‡P , 0.05 vs. basal; §P , 0.01 vs. placebo; ||P , 0.05 vs. placebo.

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As previously reported, improvingglycemic control partly restored basalendothelial function and oxidative stress(16), as well as the response to the samelevel of glycemia during the clamp interms of oxidative stress and endothelialfunction. However, the protective effectof infused GLP-1 was more pronouncedin this situation of improved glycemiccontrol compared with the previousstudy.

These data together support the hy-pothesis that the endothelium becameless sensitive to GLP-1 in hyperglycemia,more than GLP-1 itself loses its activity.

This concept has already been pro-posed for insulin secretion, where evi-dence suggests that the impaired GLP-1incretin effect is mostly related to animpairment of b-cell function than to an

impairment of incretin secretion (5).Højberg et al. (17) reported that a near-normalization of blood glucose has no ef-fect on postprandial GLP-1 secretion, butit augments b-cell responsiveness, similarto our data for endothelial function.

A possible direct influence of insulinconcentration on our results cannot beexcluded. Insulin by itself has antioxidantand vasodilatory effects (18), and GLP-1infusion during any clamp was accompa-nied by a significant increase of insulinconcentration compared with the pla-cebo. However, it has been reported thatacute hyperglycemia during a clamp, as inour case, blunts the vasodilatory effect ofinsulin (19). Moreover, at 2 h during theclamps the effect of GLP-1 on FMD wasalmost similar to that at 1 h; even at 2 h,insulin concentration was significantly

reduced compared with 1 h (Fig. 3). Inaddition, insulin can induce an endothe-lial dysfunction (20,21), evidence thatsupports the possibility that GLP-1 has adirect beneficial effect on endothelialfunction.

In conclusion, our data suggest thatduring the meal, GLP-1 can simulta-neously exert an incretin effect on insulinsecretion and have a protective effect onthe endothelial function, reasonably con-trolling oxidative stress generation. As forinsulin secretion, the ability of GLP-1 inprotecting endothelial function seems tobe dependent on the level of glycemia (5).Hyperglycemia may induce at the endo-thelial level, as well as at the level of theb-cells, a resistance to the GLP-1 action.

Acknowledgments—No potential conflicts ofinterest relevant to this article were reported.A.C. and K.E. researched data, wrote the

article, and researched and reviewed the arti-cle. R.T. researched and reviewed the article.A.R.B. and M.M. reviewed the article. D.G.researched data, wrote the article, and re-searched and reviewed the article.

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