The Effect of Glucose Loading on Adiponectin Levels in Diabetes Mellitus Type 2: Implications for Endothelial Dysfunction

Research Article

J Dis Markers. 2014;1(2): 1006.

The Effect of Glucose Loading on Adiponectin Levels in Diabetes Mellitus Type 2: Implications for Endothelial Dysfunction

Katerina Koniari1, Dimitris Tousoulis1, Charalambos Antoniades1, Aggeliki Nikolopoulou1, Evangelos Voltyrakis1, Nikolaos Papageorgiou1, Marina Noutsou2, Christodoulos Stefanadis1 and Konstantinos Makris3*

1Cardiology Department, Athens University Medical School, Greece

2Department of Medicine, University of Athens Medical School, Greece

3Department of Clinical Biochemistry, General Hospital, Greece

*Corresponding author: Konstantinos Makris, EurClinChem, Clinical Biochemist, Clinical Biochemistry, Laboratory of metabolic bone diseases, KAT General Hospital, 2 Nikis street, 14561 Kifissia Athens, Greece

Received: August 01, 2014; Accepted: Aug 19, 2014; Published: Aug 21, 2014


Aim: Adiponectin levels are decreased in patients with type II Diabetes Mellitus (DM) but it is unclear whether Impaired Glucose Tolerance (IGT) affects adiponectin's release, or whether glucose intake modifies its release from adipocytes. We examined the effect of glucose loading on serum adiponectin and endothelial function in patients with newly diagnosed DM, IGT subjects and healthy individuals.

Methods: Seventy nine patients with newly diagnosed DM, eighteen with IGT, and sixteen healthy individuals were recruited. All participants received 75g oral glucose. Endothelium-Dependent Dilation (EDD) was evaluated by gaugestrain plethysmography before glucose loading (baseline) and every 1h until 3h post-loading. Blood samples were obtained at baseline and at 3 hours postloading and serum adiponectin and insulin levels were measured.

Results: Glucose loading significantly increased adiponectin levels in healthy and IGT but not in diabetic individuals. Although insulin was correlated with adiponectin both at baseline (r=-0.375, p=0.0001) and at 3h (r=-0.286, p=0.006), insulin variations did not follow the same pattern in our three groups. There was no association between the changes of insulin and those of adiponectin (p=NS). EDD was similarly decreased in healthy, in IGT and DM patients after glucose loading (p=NS for all).

Conclusion: Glucose intake increases adiponectin levels in healthy individuals and subjects with IGT, but not in those with DM. This effect is independent of insulin variations. On the contrary, endothelial function was similarly impaired after glucose intake in all study groups, suggesting that deregulation of adiponectin's expression after glucose loading precedes the development of endothelial dysfunction in diabetes mellitus type II.

Keywords: Adiponectin; Acute hyperglycemia; Diabetes mellitus; Impaired glucose tolerance; Endothelium


DM: Diabetes Mellitus; TNF-a: Tumor Necrosis Factor-alpha; IGT: Impaired Glucose Tolerance; EDD: Endothelium Dependent Dilation; FBF: Forearm Blood Flow; ApoAI: Apolipoprotein AI; ApoB: Apolipoprotein B; ApoE: Apolipoprotein E; Lp (a): Lipoprotein (a); HbA1c: Glycosylated Haemoglobin A1c; HPLC: High-performance Liquid Chromatography; ELISA: Enzyme-Linked Immuno Sorbent Assay; HOMA: Homeostasis Model Assessment; AGEs: Advanced Glycation End Products; NO: Nitric Oxide; AMPK: Adenosine Monophosphate-activated Protein Kinase


Diabetes Mellitus (DM) is nowadays recognized as a metabolic disorder which is characterized by endothelial dysfunction and accelerated atherosclerosis [1-5]. Evidence suggests that chronic lowgrade inflammation plays a key role in the process of atherogenesis in diabetes [6,7]. Inflammation begins with lipid per oxidation and secretion of chemo attractants, proinflammatory cytokines and growth factors, which then induce the expression of cell surface adhesion molecules on endothelial cells, mediate monocytes adhesion to the endothelium and lead to atherogenesis [8-10].

Adipose tissue plays an active role in the normal metabolic and vascular homeostasis as well as in the development of type 2 diabetes, dyslipidemia and atherosclerosis [11]. These actions are mediated by secreting many biologically active molecules referred to as adipokines, such as Tumour Necrosis Factor-alpha (TNF-a), lepton, resist in and Adiponectin. Adiponectin is an adipose tissue-specific protein with beneficial effects on vascular function. It inhibits the inflammatory processes of atherosclerosis by suppressing the expression of adhesion and cytokine molecules in vascular endothelial cells and macrophages, respectively. Adiponectin concentrations correlate negatively with glucose, insulin, triglyceride serum levels, liver fat content and body mass index and positively with high-density lipoprotein-cholesterol levels, hepatic insulin sensitivity and insulinstimulated glucose disposal. Adiponectin has been shown to increase insulin sensitivity and decrease plasma glucose by increasing tissue fat oxidation [12-14]. It is well-known that Adiponectin levels are decreased in patients with DM [11], however it is unclear whether Impaired Glucose Tolerance (IGT) affects adiponectin's release, or whether glucose intake modifies its release from adipocytes.

In the present study we examined whether glucose loading affects serum Adiponectin and insulin concentrations in subjects with impaired glucose tolerance, diabetes mellitus type 2 and normal individuals. We also evaluated the impact of acute hyperglycemia on endothelial function in the above three groups of our study population.

Materials and Methods


The protocol was approved by the Institutional Ethics Committee and an informed consent was given by each subject. All parts of the study were performed in accordance with the guidelines in the Declaration of Helsinki. One hundred and thirteen subjects (46 men and 67 women) were enrolled in this study. Eighteen patients had impaired glucose tolerance (IGT, age 55.4±3.3 years), 79 patients had newly diagnosed DM (receiving no treatment, aged 56.6±1.3 years, p=NS vs. IGT) and 16 healthy young subjects had normal glucose tolerance (aged 38.0±3.8 years). All subjects were selected from the registry of Cardiology and Diabetic Department in Hippokration Hospital of Athens. Diabetes mellitus and IGT were defined in accordance with the National Data Group Criteria [15]. The inclusion criteria were that they had never been treated with any anti-diabetic or hypolipidemic agent, and that they were receiving no cardiovascular medication. All subjects underwent glucose loading with 75g oral glucose. Endothelium-Dependent Dilation (EDD, measured by venous occlusion gauge-strain plethysmography) was evaluated before glucose loading and every 1 hour until 3 hours postloading. Blood samples were obtained before glucose loading (after a 12-hour overnight fast) and at 3 hours post-loading (just before the last EDD measurement). Blood glucose was measured 1 min before each new plethysmography using a finger stick and a One Touch Profile (Johnson & Johnson, Life scan Benelux). The demographic characteristics of the participants are presented in Table1.

Forearm blood flow measurements

All studies were performed between 08:00 and 12:00. All participants were asked to proceed after a 12 hour overnight fast. Vasoactive agents (such as caffeine, tobacco and alcohol) were also avoided for the last 12 hours before the examination. They were also asked to refrain from eating and drinking during the whole study. Subjects were rested in a supine position, in a dark quiet room under constant temperature 22-25oC, for 15 minutes prior to the study. Forearm Blood Flow (FBF) was measured using gauge-strain plethysmography (EC-400, D.E. Hokanson Inc) [16,17]. The FBF output signal was transmitted to a personal computer (Hokanson NIVP3 software). FBF was finally calculated as the % change of arm volume/100 ml tissue/minute. Forearm vasodilator response to reactive hyperaemia (endothelium-dependent dilation-EDD) induced by 5-minutes ischemic occlusion of the distal forearm, was defined as the percent change of FBF from baseline to the maximum flow during reactive hyperaemia [16,17]. EDD was determined before glucose loading and every 1 hour until 3 hours post-loading.

Biochemical measurements

Venous blood samples were obtained at baseline (before plethysmography and glucose loading were performed), as well as at 3 hours post-loading (just before the last EDD measurement). After centrifugation at 3500 rpm at 4o C for 15 minutes, plasma or serum was collected and stored at -80 °C until assayed. Routine biochemical methods were used to determine serum concentrations of total cholesterol, lipoproteins [ApoAI, ApoB, ApoE, Lp(a)], triglycerides and glucose on an automated clinical chemistry analyser (Architect-8200, Abbott, Il, USA). HbA1c was measured by an automated analyzer (Menarini-Akray HA8160; Menarini, Florence, Italy) using the HPLC technique, calibrated using standards traceable to National Glucosylation Standardization Program (NGSP). Adiponectin was measured by an enzyme linked immunosorbent assay (Mercodia Laboratory Medicine Inc, Sweden). Serum insulin levels were measured by a micro particle enzyme immunoassay technique on an automated immunoassay analyzer (Axsym, Abbott Diagnostics, Abbott Park, Il, USA). Free fatty acids were measured with an enzymatic method (Wako Diagnostics, Osaka, Japan) on an automated clinical chemistry analyser (Architect-8200, Abbott, Il, USA). All the laboratory measurements were performed blindly.

Statistical analysis

Variables were tested for normal distribution by Kolmokorov- Smirnov test. Comparisons of demographic characteristics between the 3 groups were performed by using Pearson's chi square test or one-way ANOVA for multiple comparisons as appropriate. Continuous variables are expressed as means±SEM. Comparisons between variables were performed by using bivariate analysis and Pearson's correlation coefficient was estimated.

The effect of glucose loading on each variable between the 3 groups was examined by two-way ANOVA for repeated measures, and as there were significant differences in age between the 3 groups, the statistical interactions of these effects with age were also tested. The statistical analysis was performed by using SPSS 20.0 statistical program.


The baseline characteristics of our three study groups are shown in Table 1. There were significant differences in risk factor profile (BMI, W/H, hypercholesterolemia, hypertension). At baseline HOMAInsulin Resistance index was significantly lower in the normal group compared to IGT (p=0.045) and diabetes (p=0.0001) groups but there were not significant differences between IGT individuals and diabetic patients (p=0.641). At baseline (Table 1) serum Adiponectin levels were not significantly different between IGT and diabetes group (p=0.720). Although healthy controls exhibit higher baseline values compared to IGT and diabetes groups, these differences are not statistically significant (p=0.072 and 0.080 respectively). In addition, free fatty acid levels were significantly higher in the diabetic and IGT subjects compared to healthy controls (p=0.001 and 0.045 respectively), although there was no significant difference between the diabetic patients and the IGT subjects (p=0.238).