Associations of stress hormones, autonomic function, and endothelial function in patients with diabetes

Abstract

Endothelial dysfunction is an early feature of diabetic vascular injury. Stress-induced activation of the hypothalamic–pituitary–adrenal (HPA) axis, together with autonomic imbalance, may contribute to endothelial dysfunction. However, its clinical relevance in diabetes remains to be fully elucidated. We retrospectively analyzed 60 patients with diabetes who underwent both flow-mediated dilation (FMD) of the brachial artery and the coefficient of variation of the R–R interval (CVRR) measurements. The primary objective was to examine the association between FMD and stress-related hormones, including adrenocorticotropic hormone (ACTH) and cortisol. The secondary objective was to evaluate the association with autonomic function (CVRR) and other clinical variables. Mean FMD was 5.10 ± 2.32%. FMD correlated inversely with ACTH (r = –0.41, p = 0.0015) and cortisol (r = –0.39, p = 0.0024), and positively with CVRR (r = 0.49, p < 0.001). These relationships were stronger in patients with BMI ≥27 kg/m², among whom the correlation between FMD and ACTH was particularly pronounced (r = –0.68, p = 0.0007). No significant associations were found between FMD and hemoglobin A1c or other lipid parameters. Given the cross-sectional design, these findings indicated associations rather than causality. Our results suggest that the HPA axis activity and autonomic function may be associated with endothelial dysfunction in diabetes, particularly in individuals with a higher BMI. This study was registered in the UMIN Clinical Trials Registry (UMIN-CTR; UMIN000060540).

Graphical Abstract

1. Introduction

Diabetes promotes endothelial dysfunction, which represents a pivotal early step in the pathogenesis of atherosclerosis. It is closely linked to the development of cardiovascular complications. Chronic hyperglycemia, dyslipidemia, and oxidative stress contribute to vascular injury through inflammatory and metabolic pathways, ultimately impairing nitric oxide (NO)-mediated vasodilation and promoting atherogenesis [1, 2].

Flow-mediated dilation (FMD) of the brachial artery is a well-established, noninvasive technique used to assess endothelium-dependent vasodilation, which is considered a sensitive marker of early vascular dysfunction [3]. Chronic psychological or physical stress, as well as obesity, can activate the hypothalamic–pituitary–adrenal (HPA) axis, resulting in increased secretion of adrenocorticotropic hormone (ACTH) and cortisol. Excess cortisol inhibits endothelial NO synthesis and augments oxidative stress, thereby impairing vasodilatory responses [4]. A recent study reported that stress-induced cortisol reactivity correlates with decreased FMD in patients with depression [5], and elevated serum cortisol levels are also associated with microvascular complications in diabetes [6]. Despite these insights, few studies have systematically investigated the association between HPA axis activity (ACTH and cortisol levels) and vascular endothelial function in patients with diabetes. Therefore, the primary hypothesis of this study is that activation of the HPA axis, reflected by circulating ACTH and cortisol levels, is associated with endothelial dysfunction in patients with diabetes. The primary objective of this study was to evaluate the association between flow-mediated dilation (FMD) and stress-related hormones (ACTH and cortisol) levels. Secondary analyses included associations between FMD and autonomic function (CVRR) and arterial stiffness (CAVI), and subgroup analyses stratified by body mass index (BMI). Given that obesity is associated with chronic activation of the HPA axis, autonomic imbalance, and endothelial dysfunction, we hypothesized that the association between neuroendocrine markers and endothelial function would be more pronounced in individuals with a higher BMI.

2. Methods

2.1. Study Participants

We enrolled 60 patients with diabetes who underwent both FMD and CVRR measurements at our hospital between April 2023 and July 2025. Diabetes mellitus was diagnosed based on the criteria established by the Japan Diabetes Society [7]. Diabetic neuropathy was diagnosed based on clinical symptoms, decreased vibration sensation, reduced or absent Achilles tendon reflexes, and autonomic function tests, including CVRR. Nerve conduction testing using the DPNCheck (sural nerve conduction test) was performed in all patients [8]. Diabetic nephropathy was defined according to the Japan Diabetes Society classification, based on urinary albumin excretion and estimated glomerular filtration rate (eGFR) [9]. Diabetic retinopathy was diagnosed based on ophthalmologic examination and classified as simple, preproliferative, or proliferative according to the Davis classification [10].

2.2. Inclusion and Exclusion Criteria

Eligible participants met all of the following conditions:

(1) a confirmed diagnosis of diabetes,

(2) available measurements for both FMD and CVRR, and

(3) sufficient clinical information recorded in the electronic medical records.

Patients were excluded if they met any of the following criteria:

(1) missing data for either FMD or CVRR,

(2) incomplete clinical data required for analysis, or

(3) current treatment with oral or injectable glucocorticoids.

2.3. Clinical and Laboratory Measurements

Clinical information was retrieved from the electronic medical records.

The following variables were collected:

Patient characteristics: age, sex, height, weight, and body mass index (BMI);

Vascular and autonomic indices: FMD, CVRR, and cardio–ankle vascular index (CAVI);

Diabetic complications: stage of retinopathy, presence of neuropathy or nephropathy, and urinary albumin levels;

Laboratory parameters: hemoglobin A1c (HbA1c), uric acid, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), insulin-like growth factor-1 (IGF-1), growth hormone (GH), ACTH, and serum cortisol levels.

Information on glucose-lowering medications, including oral agents and insulin, was obtained from electronic medical records and summarized descriptively. Blood samples for measuring adrenocorticotropic hormone (ACTH) and cortisol levels were collected early in the morning (approximately 6:00–7:00) while patients were fasting and resting in the hospital. All patients rested for at least 30 min after waking before blood sampling. These measurements were obtained as part of routine clinical care, and the timing of blood sampling was standardized, although it was not necessarily performed on the same day as the FMD measurements.

2.4. Measurement of Flow-Mediated Dilation

FMD was measured according to the guidelines of the Japan Society for Vascular Failure (2010 Revised Edition) and the Japan Atherosclerosis Society Guidelines (2022). After ≥10 min of rest, the subjects remained supine while the right brachial artery was imaged at the antecubital fossa using a high-resolution linear ultrasound probe (7.5–10 MHz). The baseline arterial diameter was continuously recorded, after which a pneumatic cuff was inflated on the upper arm to 30 mmHg above the systolic blood pressure for 5 min.

Reactive hyperemia was induced by rapid cuff deflation, and the maximal arterial diameter measured 40–90 s after release was expressed as a percentage increase from baseline (FMD%).

Measurements were performed under standardized conditions in a temperature-controlled room (22–25°C). Participants were instructed to avoid conversation and movement during the procedure. FMD measurements were performed in hospitalized patients in the late afternoon (approximately 16:00–17:00) before dinner, and the timing was standardized across all participants.

2.5. Measurement of CVRR

The CVRR was determined from a 5-min electrocardiographic recording in the supine resting position. The standard deviation of the R–R intervals was divided by the mean R–R interval, and the result is expressed as a percentage [8, 11].

2.6. Statistical Analysis

All analyses were conducted using EZR (version 1.68; Saitama Medical Center, Jichi Medical University), a graphical interface for R. The normality of the data was assessed prior to analysis. Pearson’s correlation coefficient was used for normally distributed variables, and Spearman’s rank correlation coefficient was used for nonparametric data. Associations between FMD and clinical parameters, including ACTH, cortisol, HbA1c, lipid profile, renal indices, CAVI, and diabetic complications, were assessed using correlation analyses. Continuous variables were compared using the Student’s t-test or Mann–Whitney U test, while categorical variables were compared using the χ² test. The analyses were further stratified according to BMI (<25 and ≥25 kg/m²) and again by BMI (<27 and ≥27 kg/m²). Differences in correlation coefficients between groups were examined using Fisher’s z test. BMI-stratified analyses were performed to explore the potential modifying effects of adiposity on the relationship between neuroendocrine markers and endothelial function. A BMI cut-off of 25 kg/m² was selected in accordance with the World Health Organization definition of overweight. In addition, a threshold of 27 kg/m² was applied to identify individuals with more advanced obesity-related metabolic and vascular risks, based on evidence that cardiovascular risk increases progressively across the overweight range [12, 13].

All tests were two-tailed, and p < 0.05 was considered statistically significant. Patients with missing data were excluded from each pairwise analysis. The primary outcome was the association between FMD, ACTH, and cortisol levels. Secondary outcomes included associations between FMD and autonomic function (CVRR) and arterial stiffness (CAVI), as well as subgroup analyses stratified by BMI. In addition, multivariable linear regression analyses were performed to identify independent determinants of FMD. ACTH or cortisol was included as the primary explanatory variable in separate models, and age, sex, BMI, HbA1c level, systolic blood pressure, LDL-C level, and diabetes duration were included as covariates. To account for potential confounding factors by pharmacological treatment, additional analyses were performed, including SGLT2 inhibitor and GLP-1 receptor agonist use as covariates.

2.7. Ethical Considerations

The study protocol was approved by the Ethics Committee of the Kagawa University Faculty of Medicine (approval No. 2025-141). As this retrospective observational study used existing clinical data and test results, the requirement for written informed consent was waived. Information regarding the study was disclosed on the hospital website and outpatient notice boards, allowing participants the opportunity to opt out. This study was registered in the UMIN Clinical Trials Registry (UMIN-CTR; UMIN000060540). The study was conducted solely for academic purposes and was judged to pose no undue risk to the participants’ rights or welfare.

3. Results

A total of 60 patients with diabetes who underwent both FMD and CVRR measurements between April 2023 and July 2025 were included in the analysis. The cohort comprised 50 patients with type 2 diabetes, six with type 1 diabetes, one with mitochondrial diabetes, one with pancreatogenic diabetes, and two with post-pancreatectomy diabetes. The mean age was 60.6 ± 18.1 years (range: 21–88 years); 40 patients (66.7%) were male and 20 (33.3%) were female. The mean height, body weight, and BMI were 161.2 ± 9.1 cm, 66.4 ± 14.7 kg, and 25.5 ± 5.8 kg/m², respectively. BMI categories were <25 in 31 (51.7%), 25–<27 in 8 (13.3%), 27–<30 in 9 (15.0%), 30–<35 in 5 (8.3%), and ≥35 in 7 (11.7%).

3.1. Baseline Characteristics (Table 1)

Table 1. Baseline characteristics of the study participants (n = 60).

Variable	Value
Age (years)	60.57 ± 18.08
BMI (kg/m2)	25.51 ± 5.83
HbA1c (%)	10.85 ± 2.38
HDL-C (mg/dL)	49.58 ± 19.62
LDL-C (mg/dL)	109.48 ± 34.31
ACTH (pg/mL)	44.14 ± 20.82
Cortisol (μg/dL)	13.08 ± 3.28
FMD (%)	5.10 ± 2.32
CAVI	8.02 ± 2.42
CVRR (%)	2.45 ± 1.22
Uric acid (mg/dL)	5.19 ± 1.49
IGF-1 (ng/mL)	92.41 ± 44.91
GH (ng/mL)	1.38 ± 2.13
Urinary albumin (mg/gCr)	89.47 ± 253.71
Sex (male), n (%)	40 (66.7)
Type 2 diabetes, n (%)	50 (83.3)
Other diabetes types, n (%)	10 (16.7)
Any retinopathy, n (%)	20 (33.3)
Neuropathy, n (%)	31 (51.7)
Nephropathy stage ≥2, n (%)	20 (33.3)

Data are presented as mean ± standard deviation (SD) or number (%). BMI, body mass index; HbA1c, glycated hemoglobin; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ACTH, adrenocorticotropic hormone; FMD, flow-mediated dilation; CAVI, cardio-ankle vascular index; CVRR, coefficient of variation of R–R intervals; IGF-1, insulin-like growth factor-1; GH, growth hormone.

The mean HbA1c was 10.9 ± 2.4%. HDL-C and LDL-C levels were 49.6 ± 19.6 mg/dL and 109.5 ± 34.3 mg/dL, respectively. ACTH and cortisol levels averaged 44.1 ± 20.8 pg/mL and 13.1 ± 3.3 μg/dL, respectively. Vascular and autonomic indices were as follows: FMD 5.10 ± 2.32%, CAVI 8.02 ± 2.42, and CVRR 2.45 ± 1.22%. Serum uric acid was 5.19 ± 1.49 mg/dL, IGF-1 92.4 ± 44.9 ng/mL, GH 1.38 ± 2.13 ng/mL, and urinary albumin 89.5 ± 253.7 mg/gCr. Neuropathy was identified in 31 patients (51.7%) and diabetic nephropathy (stage ≥2) in 20 (33.3%). Based on the diabetic nephropathy classification, stages 1, 2, 3, and 4 were observed in 40 (66.7%), 14 (23.3%), 3 (5.0%), and 3 (5.0%) patients, respectively. Participants were treated with a variety of glucose-lowering agents, including DPP-4 inhibitors, SGLT2 inhibitors, metformin, GLP-1 receptor agonists, and insulin (Supplementary Table 1).

3.2. Primary analysis: Associations between FMD and stress-related hormones (Fig. 1)

Fig. 1. Correlations between flow-mediated dilation (FMD) and clinical variables (Spearman’s correlation).

Points denote Spearman’s rank correlation coefficients (ρ); horizontal lines indicate 95% confidence intervals. The vertical line marks ρ = 0. Analyses for adrenocorticotropic hormone (ACTH), cortisol, growth hormone (GH), and insulin-like growth factor-1 (IGF-1) were performed in 58 patients, and those for other variables in 60 patients. Significant correlations (p < 0.05) were observed for the coefficient of variation of R–R intervals (CVRR) and cardio–ankle vascular index (CAVI; positive and negative, respectively), as well as ACTH, and cortisol (negative).

In the primary analysis, FMD was significantly negatively correlated with ACTH (r = –0.407; p = 0.0015) and cortisol (r = –0.391; p = 0.0024).

3.3. Secondary analysis: Associations between FMD and autonomic and vascular parameters (Fig. 1)

FMD was positively correlated with CVRR (r = 0.494; p < 0.001) and negatively correlated with CAVI (r = –0.506; p < 0.001). These relationships were consistent when analyzed using Spearman’s rank correlation, indicating distribution-independent robustness.

GH showed a weak positive correlation with FMD (r = 0.308; p = 0.019), and IGF-1 exhibited a mild positive trend (r = 0.261; p = 0.048). No significant correlations were observed between FMD and HbA1c (r = 0.161; p = 0.219), uric acid (r = –0.213; p = 0.102), HDL-C (r = –0.191; p = 0.144), or LDL-C (r = –0.134; p = 0.308). No significant correlations were observed between CVRR and ACTH (r = –0.14, p = 0.28) or cortisol (r = –0.17, p = 0.21). Collectively, these findings indicate that endothelial function is associated with the HPA axis activity, autonomic function, and arterial stiffness. In a supplementary analysis, CVRR was significantly lower in patients with diabetic neuropathy than in those without neuropathy (median 1.94 [IQR 1.57–2.46] vs. 2.58 [1.90–3.10], p = 0.029).

3.4. Stratified Analyses by BMI (Fig. 2, Supplementary Fig. 1)

Fig. 2. Correlations between flow-mediated dilation (FMD) and hormonal/autonomic parameters stratified by BMI.

Pearson’s correlation coefficients (r) are shown for patients with body mass index (BMI) <27 kg/m² (blue) and ≥27 kg/m² (red). Horizontal lines indicate 95% confidence intervals and the vertical line indicates r = 0. The correlation between adrenocorticotropic hormone (ACTH) and cardio–ankle vascular index (CAVI) tended to be stronger in the higher BMI group, although the between-group difference was not significant (Fisher’s z test, p > 0.05).

When stratified by BMI (<27 vs. ≥27 kg/m²), FMD correlated positively with CVRR in both groups, indicating better endothelial function in individuals with preserved autonomic activity. FMD was negatively correlated with cortisol, ACTH, and CAVI, with these associations being stronger in the BMI ≥27 group. The correlation between FMD and ACTH was particularly pronounced in the obese subgroup (r = –0.68; p = 0.0007), although the between-group difference was not statistically significant (p = 0.061). A similar trend was observed for CAVI (p = 0.074). Analyses using a BMI of 25 kg/m² as the cutoff yielded comparable results (Supplementary Table 1).

3.5. Subgroup Analyses According to HbA1c, LDL-C, and Diabetes Duration (Table 2)

Table 2. Subgroup analyses according to HbA1c, LDL-C, and diabetes duration.

Stratification	Variable	Low group	High group	p-value
HbA1c	FMD (%)	4.45 [3.42–5.88]	5.00 [3.58–7.12]	0.408
	CVRR (%)	1.94 [1.64–2.65]	2.27 [1.72–2.80]	0.756
LDL-C	FMD (%)	4.70 [3.42–7.50]	4.55 [3.60–6.02]	0.416
	CVRR (%)	2.04 [1.71–2.71]	2.29 [1.63–2.77]	0.877
Diabetes duration	FMD (%)	5.70 [4.10–6.88]	4.35 [2.70–5.57]	0.053
	CVRR (%)	2.53 [1.91–2.81]	1.92 [1.56–2.50]	0.041

Patients were stratified according to the median values of HbA1c (10.55%), LDL-C (110.5 mg/dL), and diabetes duration (9 years). Data are expressed as median [interquartile range]. Comparisons between groups were performed using the Mann–Whitney U test. HbA1c, glycated hemoglobin; LDL-C, low-density lipoprotein cholesterol; CVRR, coefficient of variation of R–R intervals.

Additional subgroup analyses were performed according to the median values of HbA1c (10.55%), LDL-C (110.5 mg/dL), and diabetes duration (9 years) (Table 3). When patients were stratified by HbA1c level, no significant differences in FMD or CVRR were observed between the low- and high-HbA1c groups (FMD, p = 0.408; CVRR, p = 0.756). Similarly, the LDL-C stratification did not reveal significant differences between the FMD and CVRR groups (FMD, p = 0.416; CVRR, p = 0.877). In contrast, diabetes duration was associated with autonomic function. Patients with longer diabetes duration showed significantly lower CVRR values than those with shorter duration (median 1.92 vs. 2.53, p = 0.041). FMD also tended to be lower in the longer-duration group, although this difference did not reach statistical significance (median 4.35% vs. 5.70%, p = 0.053).

Table 3. Comparison of FMD and CVRR according to the presence of diabetic complications.

Complication	Variable	No	Yes	p-value
Retinopathy	FMD (%)	5.44 ± 2.46	4.41 ± 1.88	0.078
Neuropathy	FMD (%)	5.57 ± 2.52	4.65 ± 2.05	0.128
	CVRR (%)	2.58 [1.90–3.10]	1.94 [1.57–2.46]	0.029
Nephropathy	FMD (%)	4.62 ± 1.70	5.34 ± 2.56	0.198

Data are presented as mean ± standard deviation or median [interquartile range]. Comparisons between groups were performed using Student’s t-test or the Mann–Whitney U test, as appropriate. FMD, flow-mediated dilation; CVRR, coefficient of variation of R–R intervals.

3.6. Comparison of FMD according to Diabetic Microangiopathy (Table 3)

FMD tended to be lower in patients with retinopathy or neuropathy than in those without these complications, although the differences were not statistically significant (retinopathy: p = 0.078; neuropathy: p = 0.128). No significant difference in FMD was observed between patients with and without nephropathy (p = 0.198). Consistent with the findings described above, CVRR was significantly lower in patients with neuropathy than in those without (median 1.94 vs. 2.58, p = 0.029).

3.7. Multivariable analysis

Multivariate linear regression analyses were performed, adjusting for age, sex, BMI, HbA1c level, systolic blood pressure, LDL-C level, and diabetes duration. In the model including ACTH (model 1), ACTH was not significantly associated with FMD (β = –0.019, 95% CI: –0.048 to 0.010, p = 0.186). Age (β = –0.042, 95% CI: –0.074 to –0.009, p = 0.013) and LDL-C (β = –0.021, 95% CI: –0.038 to –0.004, p = 0.019) were significantly and inversely associated with FMD, whereas male sex showed a trend toward lower FMD (β = –1.20, 95% CI: –2.41 to 0.008, p = 0.051). In the model including cortisol (model 2), cortisol was independently and inversely associated with FMD (β = –0.187, 95% CI: –0.352 to –0.023, p = 0.026). In addition, age (β = –0.042, 95% CI: –0.073 to –0.012, p = 0.008), male sex (β = –1.35, 95% CI: –2.47 to –0.24, p = 0.019), and LDL-C (β = –0.020, 95% CI: –0.037 to –0.003, p = 0.021) were also independently associated with FMD. These results remained unchanged after additional adjustments for SGLT2 inhibitor and GLP-1 receptor agonist use. The results of the multivariate regression analyses are summarized in Table 4.

Table 4. Multivariate linear regression analysis for determinants of FMD.

Variable	Model 1 (ACTH) β (95% CI)	p-value	Model 2 (Cortisol) β (95% CI)	p-value
ACTH	–0.019 (–0.048 to 0.010)	0.186	—	—
Cortisol	—	—	–0.187 (–0.352 to –0.023)	0.026
Age	–0.042 (–0.074 to –0.009)	0.013	–0.042 (–0.073 to –0.012)	0.008
Sex (male)	–1.20 (–2.41 to 0.008)	0.051	–1.35 (–2.47 to –0.24)	0.019
BMI	0.039 (–0.060 to 0.138)	0.430	0.041 (–0.055 to 0.136)	0.394
HbA1c	0.077 (–0.164 to 0.318)	0.524	0.115 (–0.119 to 0.348)	0.328
SBP	–0.007 (–0.035 to 0.021)	0.626	–0.010 (–0.037 to 0.018)	0.476
LDL-C	–0.021 (–0.038 to –0.004)	0.019	–0.020 (–0.037 to –0.003)	0.021
Diabetes duration	–0.539 (–1.233 to 0.155)	0.125	–0.401 (–1.088 to 0.285)	0.246

Regression coefficients (β) with 95% CI are presented. Model 1 included ACTH, and Model 2 included cortisol. Both models were adjusted for age, sex, BMI, HbA1c level, SBP, LDL-C level, and diabetes duration. Additional analyses, including the use of SGLT2 inhibitors and GLP-1 receptor agonists, yielded unchanged results. FMD, flow-mediated dilation; ACTH, adrenocorticotropic hormone; BMI, body mass index; SBP, systolic blood pressure; CI, confidence interval; HbA1c, glycated hemoglobin.

4. Discussion

In this study, endothelial function, as assessed by FMD, was inversely correlated with ACTH and cortisol levels and positively correlated with CVRR in patients with diabetes. These associations were stronger in patients with higher BMI (≥27 kg/m²). Taken together, our data indicate that markers of HPA axis activity and autonomic imbalance are associated with endothelial dysfunction in diabetes beyond the effects of metabolic abnormalities alone. As this study was based on cross-sectional analyses, the findings should be interpreted as associations rather than causal relationships. Endothelial dysfunction is an early and critical step in the development of atherosclerosis and is widely recognized as a hallmark of vascular complications in diabetes [1, 2, 14].

Hyperglycemia, dyslipidemia, oxidative stress, and chronic inflammation collectively impair NO-dependent vasodilation and promote arterial stiffening [2, 14]. In line with earlier reports, the mean FMD in our cohort (5.10 ± 2.32%) was low, supporting the notion that endothelial impairment is prevalent among patients with diabetes [2, 3, 15]. In the present study, the association between neuroendocrine markers and endothelial function was more pronounced in patients with higher BMI. This is consistent with previous evidence showing that cardiometabolic risk increases from a BMI of approximately 25 kg/m² and continues to increase even within the overweight range [12]. Furthermore, recent longitudinal data in patients with type 2 diabetes have demonstrated that BMI ≥27 kg/m² is associated with increased mortality risk, supporting the clinical relevance of this threshold [16].

Clinical studies have shown that patients with type 2 diabetes exhibit increased HPA axis activity. Elevated plasma ACTH and cortisol levels, as well as a flattened diurnal cortisol rhythm, have also been observed in healthy subjects. Hyperactivation may contribute to insulin resistance, impaired glucose tolerance, and metabolic complications [17].

In patients with diabetes, particularly type 2, chronic hyperactivation of the HPA axis is thought to result from a combination of metabolic, neuroendocrine, and inflammatory mechanisms.

Insulin resistance and hyperglycemia may attenuate central insulin and leptin signaling in the hypothalamus, while obesity-related inflammation (via interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) further stimulates HPA activity and impairs glucocorticoid feedback [18, 19]. Elevated cortisol levels are associated with insulin resistance, visceral fat accumulation, and endothelial dysfunction, potentially leading to a vicious cycle that worsens metabolic and vascular complications [4, 18].

Excess cortisol has been reported to aggravate glucose intolerance by promoting hepatic gluconeogenesis, inhibiting peripheral glucose uptake, and impairing β-cell function, creating a self-perpetuating cycle that reinforces both hyperglycemia and HPA axis overactivity [18].

Factors that influence endothelial NO synthesis, such as oxidative stress, inflammation, insulin resistance, and glucocorticoid excess, are expected to correlate with FMD values. Thus, a reduced FMD indicates impaired NO bioavailability and reflects endothelial dysfunction [1, 2].

The activation of the HPA axis increases circulating cortisol levels, which suppress endothelial NO production [17]. Cortisol may influence the expression of endothelial NO synthase and NO bioavailability [4]. Consequently, chronic hyperactivity of the HPA axis may be associated with endothelial dysfunction and vascular complications frequently observed in diabetes [15]. However, these mechanisms were not directly evaluated in this study.

We observed negative correlations between FMD and both ACTH and cortisol, and a positive correlation with CVRR, supporting the concept that endocrine and autonomic stress responses may be associated with endothelial dysfunction in diabetes. Notably, in multivariate analyses, cortisol remained independently associated with FMD, whereas ACTH was not (Table 4), suggesting that cortisol may more directly reflect vascular impairment. These associations remained consistent after additional adjustments for SGLT2 inhibitor and GLP-1 receptor agonist use. Chronic neuroendocrine activation and autonomic imbalances are likely to act in concert with the pathogenesis of endothelial dysfunction in diabetes. In contrast, no significant correlations were observed between the CVRR and ACTH or cortisol levels. Although sympathetic activation may theoretically reduce CVRR while activating the HPA axis, CVRR mainly reflects parasympathetic (vagal) activity, whereas ACTH and cortisol levels represent HPA axis activation. Therefore, these physiological systems capture different aspects of stress regulation and may not necessarily be directly correlated. Endothelial function assessed by FMD may represent a more integrated marker influenced by both neuroendocrine and autonomic pathways. The CVRR is a widely used index of autonomic function; however, it may also be affected by diabetic neuropathy. In the present study, the CVRR was significantly lower in patients with neuropathy, suggesting that part of the observed association between the CVRR and endothelial function may be mediated by neuropathy. Nevertheless, the significant association between FMD and CVRR observed in this study indicates that autonomic dysfunction may also contribute to endothelial dysfunction. Therefore, both diabetic neuropathy and autonomic imbalance should be considered when interpreting our findings. Recent experimental findings have suggested that activation of N-methyl-D-aspartate receptors in pancreatic β-cells impairs insulin secretion, potentially linking neuroendocrine stress to β-cell dysfunction [20].

Obesity is associated with chronic low-grade inflammation, oxidative stress, and insulin resistance, promoting vascular injury [19]. In our analysis, the relationships between FMD and ACTH, cortisol, and CAVI were stronger in patients with BMI ≥27 kg/m², indicating that obesity amplified the impact of neuroendocrine stress and arterial stiffening on endothelial function. CAVI reflects structural and functional arterial stiffness and has been reported to progress in parallel with FMD impairment [21]. Cytokines released from adipose tissue, including IL-6 and TNF-α, may further activate the HPA axis and sympathetic nervous system, creating a self-perpetuating cycle that links metabolic stress, neuroendocrine activation, and endothelial dysfunction.

Additional subgroup analyses according to HbA1c, LDL-C, and diabetes duration (Table 2) provided further insights into the determinants of endothelial and autonomic function in diabetes. HbA1c and LDL-C stratifications showed no significant differences in FMD or CVRR. In contrast, patients with a longer diabetes duration exhibited significantly lower CVRR values, whereas FMD tended to be lower in this group. These findings suggest that longer exposure to metabolic stress may contribute to progressive autonomic dysfunction and vascular impairment in diabetes, independent of current glycemic control.

Hyperactivation of the HPA axis in diabetes may exacerbate diabetic neuropathy [22]. Elevated cortisol levels promote oxidative stress, endothelial dysfunction, and microvascular impairment, reducing nerve perfusion and contributing to axonal damage. Chronic activation of the HPA axis may also enhance systemic inflammation and impair neuronal repair mechanisms. Collectively, these effects increase the risk and severity of peripheral and autonomic neuropathy in patients with diabetes, linking neuroendocrine dysregulation with microvascular and neural complications [19, 21].

Diabetic neuropathy may alter HPA axis activity via impaired autonomic and sensory nerve signaling. Damage to the afferent vagal and spinal pathways can disrupt the hypothalamic feedback loop, leading to altered secretion of corticotropin-releasing hormone and ACTH. Autonomic neuropathy may reduce modulation of stress responses, resulting in dysregulated cortisol rhythms [22]. Consequently, neuropathic damage may perpetuate HPA axis dysfunction, creating a bidirectional relationship between neuroendocrine imbalance and diabetic complications.

In patients with type 2 diabetes and obesity, hyperactivation of the HPA axis may be associated with endothelial dysfunction [23]. Elevated cortisol levels decrease NO bioavailability and increase oxidative stress, thereby impairing vasodilation. These findings may have implications for future therapeutic strategies, including lifestyle interventions such as weight loss, aerobic exercise, and dietary optimization to improve insulin sensitivity, reduce visceral fat, and normalize HPA activity. Pharmacological agents such as metformin or GLP-1 receptor agonists can enhance glycemic control and endothelial function, while angiotensin-converting enzyme inhibitors and statins support vascular health [24]. Stress-reduction techniques may further modulate cortisol levels and improve FMD. Further studies are needed to determine whether monitoring FMD and cortisol profiles may be useful in clinical practice [25].

This study has several limitations. First, it was a single-center retrospective analysis with a modest sample size, and selection bias could not be fully excluded. Second, subgroup analyses—particularly among patients with a BMI ≥35 kg/m²—had limited statistical power. Although additional analyses, including SGLT2 inhibitor and GLP-1 receptor agonist use, were performed, the potential influence of other medications, such as statins, could not be fully evaluated, which remains a limitation. In addition, the timing of the hormone measurements was not always identical to that of the FMD assessments. Finally, potential confounders such as medication use, inflammation, and lifestyle factors were not fully controlled; therefore, the results should be interpreted as associative rather than causal. Future multicenter and interventional studies are warranted to clarify the causal relationships among HPA axis activity, autonomic function, and endothelial impairment in diabetes.

In conclusion, FMD was inversely associated with ACTH and cortisol levels and positively correlated with CVRR, suggesting that HPA axis activation and autonomic imbalance may reflect endothelial dysfunction in diabetes. These effects were more evident in individuals with obesity, indicating that neuroendocrine stress and metabolic factors jointly influence vascular health. Evaluating and targeting stress-related endocrine and autonomic functions may offer practical avenues for protecting vascular health in diabetes. These findings are summarized in the Graphical Abstract.

Graphical Abstract.

Conflict of Interest Statement

The authors declare no conflict of interest.

Funding Sources

This study received no specific grant from any funding agency.

Data accessibility statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval

This study was approved by the Ethics Committee of the Kagawa University Faculty of Medicine (approval No. 2025-141).

Author Contributions

Takanobu Saheki: Conceptualization; Methodology; Investigation; Writing – Original Draft; Resources.

Keisuke Takahashi: Resources.

Eri Harada: Methodology; Resources.

Takafumi Yoshimura: Methodology; Resources.

Toshihiro Kobayashi: Methodology; Resources.

Kensaku Fukunaga: Methodology; Investigation; Resources.

Hitomi Imachi: Conceptualization; Methodology; Supervision.

Koji Murao: Conceptualization; Methodology; Writing – Review & Editing; Supervision.

Supplementary Material

Supplementary Table 1

Glucose-lowering medications used by the study participants (n = 60)

Supplementary Fig. 1

Correlations between flow-mediated dilation (FMD) and hormonal/autonomic parameters stratified by BMI (<25 vs. ≥25).