Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance and sustained hyperglycemia. Over time, chronic hyperglycemia has been shown to contribute significantly to the development of both macrovascular and microvascular complications, particularly through its deleterious effects on the endothelium and autonomic nervous system regulation [1]. These complications play a pivotal role in increasing the morbidity and mortality associated with T2DM. Endothelial dysfunction is an early marker of vascular injury and is closely linked to the pathogenesis of diabetic cardiomyopathy and atherosclerosis. Persistent hyperglycemia promotes oxidative stress, inflammation, and impaired nitric oxide bioavailability, which collectively compromise endothelial integrity [2]. In the context of diabetes, this endothelial impairment leads to reduced vasodilation, increased vascular stiffness, and heightened cardiovascular risk [4]. Equally concerning is the impact of chronic hyperglycemia on the autonomic nervous system. Diabetic autonomic neuropathy, particularly cardiac autonomic neuropathy (CAN), is a well-established complication in long-standing T2DM, associated with increased risk of arrhythmias, silent myocardial ischemia, and sudden cardiac death [3]. The pathophysiology of CAN involves

hyperglycemia-induced damage to autonomic nerve fibres that regulate cardiovascular function, including heart rate variability and baroreflex sensitivity [6]. Moreover, there is growing evidence that poor glycemic control is linked with microvascular dysfunction, further compounding autonomic impairment and vascular dysregulation [4]. Importantly, therapeutic interventions such as metformin have demonstrated potential in improving endothelial function, highlighting the reversible nature of some of these vascular insults when glycemic control is optimized [5].

This study aims to investigate the relationship between chronic hyperglycemia, endothelial dysfunction, and autonomic regulation in patients with long-standing T2DM. By characterizing these interrelated pathophysiological mechanisms, the research seeks to inform clinical strategies that mitigate cardiovascular risk in this vulnerable population.

Aims and Objectives

The aim of this study was to evaluate the effect of chronic hyperglycemia on vascular endothelial function and cardiovascular autonomic regulation in patients with long-standing type 2 diabetes mellitus (T2DM).

The specific objectives were:

1. To assess the extent of endothelial dysfunction in patients with long-standing T2DM using flow-mediated dilation (FMD).
2. To evaluate cardiovascular autonomic regulation through standardized autonomic reflex function tests.
3. To determine the association between chronic hyperglycemia (as measured by HbA1c) and the degree of endothelial and autonomic dysfunction.

Study Design and Setting

This was a cross-sectional, observational study conducted at the Department of Medicine, SMS Medical College, Jaipur, India, over a 12-month period from January 2023 to December 2023. The study protocol was approved by the institutional ethics committee, and all participants provided written informed consent.

Study Population

A total of 100 patients with a diagnosis of type 2 diabetes mellitus for ≥10 years were recruited from outpatient and inpatient services. Participants were between 40 and 65 years of age and had no known macrovascular complications at enrollment.

Inclusion Criteria:

• Confirmed diagnosis of T2DM for ≥10 years
• HbA1c ≥7.0% at the time of enrollment
• Age between 40 and 65 years
• No history of overt cardiovascular disease

Exclusion Criteria:

• Type 1 diabetes mellitus
• Use of medications known to affect endothelial or autonomic function (e.g., β-blockers, autonomic drugs)
• Diagnosed neuropathies unrelated to diabetes
• Chronic kidney disease (eGFR <60 mL/min/1.73 m²)
• Active infection or systemic illness

Clinical and Laboratory Evaluation

All participants underwent a thorough clinical examination, including anthropometry, blood pressure, and heart rate measurements. A venous blood sample was collected after overnight fasting to measure:

• Fasting blood glucose
• HbA1c (using HPLC method)
• Lipid profile
• Serum creatinine
• Inflammatory markers (hs-CRP)

Assessment of Endothelial Function

Endothelial function was assessed noninvasively using flow-mediated dilation (FMD) of the brachial artery via high-resolution ultrasonography, following standardized protocols. The percentage change in arterial diameter after reactive hyperaemia was calculated as a marker of endothelial function.

Assessment of Autonomic Function

Autonomic regulation was evaluated using a battery of cardiovascular autonomic reflex tests, including:

• Heart rate variability during deep breathing
• Valsalva manoeuvre
• Orthostatic heart rate and blood pressure response
• 30:15 ratio test

Results were interpreted using age-adjusted normative values, and a composite autonomic score was calculated to quantify autonomic dysfunction severity.

Statistical Analysis

Descriptive statistics were calculated for all variables. Continuous variables were reported as mean ± standard deviation, and categorical data as

frequencies or percentages. Pearson or Spearman correlation coefficients were used to assess associations between HbA1c and endothelial/autonomic parameters. Comparisons between groups were performed using Student’s t-test or Mann–Whitney U test. A two-tailed p-value of <0.05 was considered statistically significant. All analyses were conducted using SPSS version 26.0.

Section 1: Baseline Clinical and Biochemical Characteristics

The study included 100 participants with long-standing type 2 diabetes mellitus. The mean age of the cohort was 53.4 ± 7.5 years. Sex distribution was balanced, with 52% males and 48% females. The average body mass index (BMI) was 27.0 ± 3.5 kg/m², reflecting a trend toward overweight status. Mean systolic and diastolic blood pressures were 138.4 ± 11.6 mmHg and 83.4 ± 8.4 mmHg, respectively. The resting heart rate averaged 78.7 ± 8.7 beats per minute.

Biochemically, the mean fasting plasma glucose was 165.3 ± 30.5 mg/dL, and the average HbA1c was 8.4 ± 1.2%, confirming poor glycemic control among participants. Lipid profile showed a mean LDL of 110.2 ± 25.7 mg/dL, HDL of 42.3 ± 9.0 mg/dL, and triglycerides of 179.2 ± 61.5 mg/dL. Mean serum creatinine was 0.9 ± 0.15 mg/dL, with high-sensitivity C-reactive protein (hs-CRP) averaging 2.8 ± 1.1 mg/L, indicating a low-grade inflammatory state.

Table 1. Baseline Clinical and Biochemical Characteristics (N = 100)

Sex Distribution:

Male: 52 (52.0%)
Female: 48 (48.0%)

Section 2: Endothelial Function (FMD%)

Endothelial function was assessed using flow-mediated dilation (FMD) of the brachial artery. The overall mean FMD in the study population was 5.2 ± 1.5%. When stratified by HbA1c quartiles, a progressive decline in FMD was observed with increasing levels of chronic hyperglycemia. Participants in the highest HbA1c quartile (Q4, HbA1c ≥ 9.2%) exhibited a mean FMD of 4.4 ± 1.2%, whereas those in the lowest quartile (Q1, HbA1c ≤ 7.5%) had a mean FMD of 6.0 ± 1.4%. This inverse relationship suggests a potential link between poor long-term glycemic control and impaired endothelial-dependent vasodilation.

Table 2. Flow-Mediated Dilation (FMD%) by HbA1c Quartiles

Section 3: Autonomic Function Parameters

Cardiovascular autonomic regulation was evaluated through standardized reflex tests. The mean heart rate variability during deep breathing was 12.1 ± 5.1 beats per minute, while the mean Valsalva ratio was 1.20 ± 0.21. The orthostatic heart rate response averaged 15.7 ± 6.1 bpm, and the orthostatic systolic blood pressure drop was 12.3 ± 4.8 mmHg. The 30:15 ratio, a marker of parasympathetic reactivity, showed a mean value of 1.03 ± 0.09. The composite autonomic score, ranging from 0 (normal) to 4 (severe dysfunction), had a mean of 2.1 ± 1.3 in this cohort, suggesting prevalent subclinical or mild autonomic dysfunction.

Table 3. Summary of Autonomic Function Parameters

Section 4: Correlation Analyses

To assess the relationship between chronic hyperglycemia and physiological dysfunction, Pearson correlation analyses were performed. HbA1c showed a negative correlation with endothelial function as measured by flow-mediated dilation (r = –0.49), indicating that higher HbA1c levels were associated with impaired vasodilation. Additionally, a positive correlation was observed between HbA1c and autonomic dysfunction score (r = 0.44), suggesting greater autonomic impairment in those with poorer glycemic control. Individual autonomic test parameters also showed modest correlations with HbA1c, including HRV during deep breathing (r = –0.31), 30:15 ratio (r = –0.28), and Valsalva ratio (r = –0.25). These findings reinforce the pathophysiological link between chronic hyperglycemia and multisystem dysregulation.

Table 4. Correlation of HbA1c with Endothelial and Autonomic Parameters

Section 5: Statistical Significance and Group Comparison

To further evaluate the relationship between glycemic control and physiological outcomes, participants were stratified into tertiles based on their HbA1c levels. Group comparisons were made for key endothelial and autonomic parameters using one-way ANOVA. The mean FMD did not significantly differ across HbA1c tertiles (F = 0.24, p = 0.7835), and the effect size was small (Cohen’s d = 0.10 for lowest vs highest tertile). Similarly, autonomic dysfunction scores showed no statistically significant difference between groups (F = 0.67, p = 0.5141), with a small effect size (Cohen’s d = –0.27). These findings suggest that while correlation analyses indicated modest associations, the between-group differences based on tertile stratification were not robust enough to reach statistical significance.

Table 5. Comparison of Physiological Parameters Across HbA1c Tertiles

This cross-sectional study assessed endothelial and autonomic function in individuals with long-standing type 2 diabetes mellitus (T2DM), focusing on the impact of chronic hyperglycemia. Our findings reveal a moderate inverse correlation between glycemic control, as measured by HbA1c, and both endothelial function (r = –0.49) and autonomic regulation (r = 0.44), suggesting that persistent hyperglycemia may drive multisystem dysregulation in T2DM patients.

Endothelial dysfunction was quantified using flow-mediated dilation (FMD), which showed a mean of 5.2 ± 1.5% across the cohort. Stratification by HbA1c quartiles revealed a clear decremental pattern: the highest HbA1c group exhibited an FMD of 4.4 ± 1.2%, compared to 6.0 ± 1.4% in the lowest quartile. Although between-group differences did not reach statistical significance (F = 0.24, p = 0.7835; Cohen’s d = 0.10), this trend is clinically relevant. Prior studies have shown similar impairment. Rask-Madsen et al. (2001) reported improvement in endothelial function with insulin therapy in diabetic patients [12], while Beckman et al. (2003) highlighted a lack of endothelial response to antioxidants in type 2 diabetes, underscoring an irreversible component of vascular injury [15].

Cardiovascular autonomic dysfunction was also evident in our cohort. Mean autonomic dysfunction score was 2.1 ± 1.3, indicating mild-to-moderate dysregulation. Significant inverse correlations were observed for HRV during deep breathing (r = –0.31) and the 30:15 ratio (r = –0.28). These values reflect a progressive autonomic decline with increasing HbA1c. Pop-Busui (2012) emphasized that cardiovascular autonomic neuropathy remains underdiagnosed despite its prognostic importance in diabetes [7]. Supporting our results, Dhumad et al. (2021) demonstrated strong associations between long-standing T2DM and reduced Valsalva and orthostatic response scores [8].

Interestingly, although individual correlation coefficients between HbA1c and physiological indices were moderate (r = –0.49 for FMD, r = 0.44 for autonomic score), ANOVA comparisons across HbA1c tertiles failed to reach significance for both FMD (F = 0.24, p = 0.7835) and autonomic dysfunction (F = 0.67, p = 0.5141). This discrepancy likely reflects interindividual variability and highlights the limitations of tertile stratification. Fuchsjäger-Mayrl et al. (2002) similarly noted high variability in endothelial function among patients with similar glycemic profiles [17]. Furthermore, Lefrandt et al. (2010) proposed that autonomic decline in diabetes may occur independently of overt cardiovascular pathology [9].

The clinical implications of our findings are notable. Kamimura and Tamura (2023) suggested that resting heart rate may serve as a surrogate marker for subclinical autonomic dysfunction [10], while Serhiyenko et al. (2022) identified abnormal circadian arterial stiffness patterns in diabetics with advanced autonomic neuropathy [11]. Together, these reinforce the value of early physiologic testing. Our findings support integrating noninvasive vascular and autonomic assessments into routine evaluation for high-risk T2DM patients.

This study highlights that long-standing type 2 diabetes mellitus is associated with measurable impairments in both vascular endothelial and autonomic function. Higher HbA1c levels correlate with worsening physiological indices, underscoring the importance of sustained glycemic control. Noninvasive assessments such as flow-mediated dilation and autonomic reflex testing may serve as valuable adjuncts in identifying high-risk individuals for early intervention. Future studies should explore longitudinal patterns and potential reversibility of these changes with targeted therapies.

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