Cardiovascular Diabetology BioMed Central Original investigation

Accelerated atherosclerosis is one of the major vascular complications of diabetes. Factors including hyperglycemia and hyperinsulinemia may contribute to accelerated vascular disease. Among the several mechanisms proposed to explain the link between hyperglycemia and vascular dysfunction is the hexosamine pathway, where glucose is converted to glucosamine. Although some animal experiments suggest that glucosamine may mediate insulin resistance, it is not clear whether glucosamine is the mediator of vascular complications associated with hyperglycemia. Several processes may contribute to diabetic atherosclerosis including decreased vascular heparin sulfate proteoglycans (HSPG), increased endothelial permeability and increased smooth muscle cell (SMC) proliferation. In this study, we determined the effects of glucose and glucosamine on endothelial cells and SMCs in vitro and on atherosclerosis in apoE null mice. Incubation of endothelial cells with glucosamine, but not glucose, significantly increased matrix HSPG (perlecan) containing heparin-like sequences. Increased HSPG in endothelial cells was associated with decreased protein transport across endothelial cell monolayers and decreased monocyte binding to subendothelial matrix. Glucose increased SMC proliferation, whereas glucosamine significantly inhibited SMC growth. The antiproliferative effect of glucosamine was mediated via induction of perlecan HSPG. We tested if glucosamine affects atherosclerosis development in apoE-null mice. Glucosamine significantly reduced the atherosclerotic lesion in aortic root. (P < 0.05) These data suggest that macrovascular disease associated with hyperglycemia is unlikely due to glucosamine. In fact, glucosamine by increasing HSPG showed atheroprotective effects.


Background
Sympathetic and parasympathetic components of neurovegetative system regulate cardiac activity. Spectral analysis of heart rate variability (HRV) is a non invasive metodic used to assess cardiac autonomic activity. The autonomic activities can be assessed in HRV by the relative distribution (evaluated in normalized units) of low frequency (LF), as an index of sympathetic modulation and of high frequency (HF), as an index of parasympathetic modulation. The analysis of HRV has been used for evaluate the autonomic cardiac activity in numerous pathophisyologic conditions [1][2][3][4][5][6][7][8][9]: an impaired heart rate variation is a marker of autonomic neuropathy [10,11] as in diabetic subjects [12][13][14][15][16].
Several studies have showed a sympathetic overactivity also in non diabetic insulin resistant group [17][18][19]. However, little is known about the association between the familiarity for type 2 diabetes mellitus and the autonomic activity. An increased LF/ HF ratio (low frequency/high frequency ratio) has identified in only insulin resistant offsprings of type 2 diabetic subjects [20]. De Angelis et al [21] has showed this condition at rest but not under stimulated conditions. Laitinen et al [22] has identified a sympathetic overactivity during acute hyperinsulinemia both in insulin resistant and non insulin resistant offsprings of type 2 diabetic subjects.
The aim of the present study was to evaluate, by heart rate variability with 24-hours ECG Holter registration, the autonomic activity and circadian autonomic rhythm in offspring of type 2 diabetic subjects and to evaluate the possible impact of sympathetic activity on insulin resistance. We tested the hypothesis that in non insulin resistant offsprings of type 2 diabetic subjects an alteration of circadian rhythm of autonomic activity are present and that the impairment of autonomic system activity could precede the insulin resistance.

Subjects and methods
70 consecutive caucasian offsprings of type 2 diabetic subjects, admitted to our department, were screened. In all subjects, after an overnight fast, oral glucose tolerance tests (OGTTs) was performed: samples blood for glucose and plasma insulin were collected before and 2 h after a glucose load consisting of 75 g glucose anhydrate in 300 ml of water ingested over the course of 5 min. Also, fasting plasma insulin was measured to evaluate the insulinresistance by the homeostasis model assessment-index (HOMA-I). Among them, 50 caucasian subjects (age: 47.71 ± 9.96 years, 32 men and 18 women), with normal OGTTs, were admitted in this study and were divided in two groups: offsprings with insulin resistance and offsprings without insulin resistance: Subjects with hypertension [23], diabetes mellitus, impaired fasting glycemia, impaired glucose tolerance [24], obesity, dyslipidemia, cardiac arrhythmias, microalbuminuria and with drug treatment or diseases that could potentially disturbs carbohydrate metabolism (glucocorticoids, furosemide, beta-blockers, etc.) and cardiac autonomic activity (beta-blockers, anti-arrhythmics, ACEinhibitors) were excluded.
The control group consisted of 25 sex and age matched healthy non insulin resistant subjects with normal OGTTs and without familiarity for type 2 diabetes mellitus.
Height, weight and body circumferences were measured on all subjects. Body mass index (BMI, kg/m 2 ) was calculated as weight divided by height squared. Waist-to-hip ratio (WHR) was defined as waist circumference divided by hip circumference.
Informed consent was obtained from all participants; all the investigations were performed in accordance with the participants of the Declaration of Helsinki.

Insulin resistance
The insulin-resistance was evaluated by the homeostasis model assessment index (HOMA-I) [25][26][27][28]. The HOMA-I was calculated by the formula: fasting plasma glucose (mmol/L) x fasting plasma insulin (µU/ml)/ 22,5, as described by Matthews and coworkers [29]. Insulin-resistance was defined as the third and fourth quartiles of HOMA-I. The index subject were subdivide into two groups based on HOMA-I: 1) group of insulin-resistant offsprings (IR); 2) group of non insulin-resistant offsprings (NIR).

HRV assessment
Autonomic nervous activity was evaluated by heart rate variability (HRV) analysis during 24-hour ECG recording. All Holter recordings were performed using a three-channel recorder. Autonomic nervous activity was analysed following the recommendations of the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology [30]. Spectral estimates of R-R interval were obtained from stationary regions free of ectopic beats and technical artefacts. The standard deviation of normal-to-normal RR intervals (SDNN) (ms) and the square root of the mean of the sum of the squares of differences between adjacent NN intervals (RMS-SD), correlated with parasympathetic system, were calculated and were divided in two periods: night (0 am -6 am) and day (7 am -9 pm). Fast Fourier Transform was used to obtain power spectral estimates of HRV. Total power in the frequency range (0 -0.40 Hz) was divided into low frequency (LF: 0.04 -0.15 Hz, modulated by sympathetic system) and high frequency (HF: 0.15 -0.40 Hz, modulated by parasympathetic system). The power of LF and HF components was considered in normalized units (n.u.). Subjects were analysed for 24 hours, at 10 minutes interval. Artificial data and arrhythmic were excluded. The day was divided in four periods: night (0 am -6 am), morning (7 am -12 am), afternoon (1 pm -6 pm), evening (7 pm -11 pm). Data analyses were performed with software Del Mar Avionics Accuplus 363, Irvine California, USA.

Statistical analysis
All analysis were done with SPSS 12.0 (SPSS Inc., Chicago, IL, USA) for Windows XP. Data are presented as means + SD. For data with multiple time points, variables were analysed by the general linear model ANOVA and simple regression analyses were carried out by standard techniques 95% confidence intervals (CT) were calculated for regression coefficient. Means values were considered significant at p < 0.05.

Clinical characteristics
The groups had not statistically significant difference in age, sex and anthropometrics parameters (i.e. BMI, waist and hip circumference, waist to hip ratio, table 1). Tables 2 and 3 show the means of autonomic function measures for each group.  1). Total SDNN were reduced in IR group when compared with (NIR) NIR group (p 0.003). These results showed a total activity reduction of autonomic system in insulin resistant and non insulin resistant offsprings of type 2 diabetic patients. The autonomic activity reduction is major in IR group than NIR group.

Time domain
RMS-SD in night time not increased in IR group. Therefore insulin-resistance was associated with alteration of circadian rhythm of parasympathetic component (at the night).  n.u.) and NIR (24.28 ± 9.08 n.u.) groups than control group (35.75 ± 9.14 n.u.), respectively p: 0.001 and p: SDNN value in offsprings of type 2 diabetic patients and controls

Discussion
The data of our study suggested that an autonomic impairment is associated with the familiarity for type 2 diabetes independently of insulin resistance. In frequency domain, the analysis of sympathetic (LF) and parasympathetic (HF) component and the symphatovagal balance (LF/HF) evidenced an association between the familiarity and a sympathetic overactivity, especially in nocturnal period, demonstrated by increase of LF ( figure 2 and figure 3) and LF/HF ratio ( figure 4). This autonomic impairment is major in insulin resistant offsprings than non insulin resistant offsprings of type 2 diabetic patients. Moreover, our study had demonstrated, in time domain analysis of HRV, a significant reduction of the total autonomic system activity in both groups, expressed by progressive decrease of SDNN value from NIR to IR groups. These results indicated that the familiarity of type 2 diabetes mellitus is related to a global reduction of autonomic nervous system and that the dysautonomia increases if offsprings are insulin resistant.
In summary, a global reduction and alteration of circadian rhythm of autonomic activity are present in offspring of type 2 diabetic patients, without and with insulin resistance ( figure 2, 3).
A long term observation can answer the question whether autonomic abnormality precede the occurence of insuline resistence and play a role in the complex pathogenesis of the insulin resistance and type 2 diabetes mellitus. Others studies occurs to explain a possible pathogenetic role of autonomic dysfunction in the development of insulin resistance and type 2 diabetes mellitus.
LF value in offsprings of type 2 diabetic patients and control

Limitations
A limitation in this study is the use of the HOMA index as a conventional indicator of insulin resistance. The best method for assessment of insulin resistance is the glucose clamp technique, however, the HOMA model has proved be a robust clinical and epidemiological tool in descriptions of the pathophysiology of diabetes, already quoted in > 500 publications, it has become one of standard tools in the armamentarium of the clinical physiologist [23].
Other study are necessary to determine the mechanism whereby insulin-resistance may be related to autonomic dysfunction.

List of abbreviations
BMI: body mass index; DM: type 2 diabetes mellitus; HF: high frequency; HOMA-I: the homeostasis model assessment-index; HRV: heart rate variability; LF: low frequency; OGTTs: oral glucose tolerance tests; RMS-SD: the square root of the mean of the sum of the squares of differences between adjacent NN intervals; SDNN: The standard deviation of normal-to-normal RR intervals; WHR: waist-to-hip ratio.