Prevalence and incidence of type 2 diabetes mellitus in patients with adrenal incidentalomas: a study of 709 cases

To evaluate the prevalence and incidence of type 2 diabetes in patients with nonfunctioning adrenal incidentalomas (NFAI) or adrenal incidentalomas (AI) with autonomous cortisol secretion (ACS). In this single-center retrospective study, all patients with adrenal incidentalomas ≥1 cm and ACS or NFAI studied between 2013 and 2020 were included. ACS was defined by a post-dexamethasone suppression test (DST) serum cortisol concentration ≥1.8 μg/dl, in the absence of signs of hypercortisolism, and NFAI was defined as a DST < 1.8 μg/dl without biochemical evidence of hypersecretion of other hormones. Inclusion criteria were met by 231 patients with ACS and 478 with NFAI. At diagnosis, type 2 diabetes was present in 24.3% of patients. No differences were found in the prevalence of type 2 diabetes (27.7 vs. 22.6%, P = 0.137) between patients with ACS and NFAI. However, fasting plasma glucose values and glycated hemoglobin levels were significantly higher in patients with ACS than with NFAI (112 ± 35.6 vs. 105 ± 29 mg/dl, P = 0.004; and 6.5 ± 1.4 vs. 6.1 ± 0.9%, P = 0.005, respectively). Furthermore, patients with type 2 diabetes had higher urinary free cortisol (P = 0.039) and late-night salivary cortisol levels (P = 0.010) than those without type 2 diabetes. After a median follow-up of 28 months, no differences were found in the incidence of type 2 diabetes between the groups (HR 1.17, 95% 0.52–2.64). Type 2 diabetes was present in one fourth of our cohort. We found no differences in its prevalence or incidence between the groups. However, glycemic control might be worse among diabetic patients with ACS. Higher concentrations of urinary and salivary cortisol were found in patients with than without type 2 diabetes.


Introduction
Adrenal incidentalomas (AIs) are asymptomatic adrenal masses detected on an imaging procedure performed for reasons different than a suspicion of adrenal disease [1]. Based on computed tomography (CT) scan findings the prevalence of AIs is estimated at 5-10% of the population [1][2][3].
The discovery of an adrenal mass requires ruling out malignancy and evaluating its functionality to guide management [1]. Adrenocortical adenomas show biochemical evidence of adrenal hormone excess in 50-60% of cases, most commonly mild autonomous cortisol secretion (ACS) [4]. Guidelines from the European Society of Endocrinology and the European Network for the Study of Adrenal Tumors (ESE/ENSAT) recommend a 1 mg overnight dexamethasone suppression test (DST) to exclude excessive cortisol secretion [1]. Thus, cortisol levels after DST < 50 nmol/L (1.8 µg/dL) exclude ACS [1]. Post-dexamethasone serum cortisol levels between 51 and 138 nmol/L (1.9-5.0 µg/dL) are regarded as 'possible ACS'; and cortisol levels post dexamethasone >138 nmol/L (>5.0 µg/dL) confirm ACS, provided that signs and symptoms of hypercortisolism are absent [1].
It is well known that ACS is associated with metabolic disorders such as diabetes mellitus, obesity, dyslipidemia, and hypertension; and with cardiovascular diseases that could increase mortality rates in these patients [1,3]. Similarly, a high prevalence of cardiometabolic disease at diagnosis and an increased risk of prediabetes and type 2 diabetes incidence during follow-up has been observed in patients with nonfunctioning AI (NFAI) [3]. Such observation might be related to a subtle cortisol -and/or other glucocorticoids-hypersecretion that goes unrecognized by the 1 mg DST as has been previously suggested [5].
The aim of this study was to evaluate the differences in the prevalence and incidence of type 2 diabetes among patients with NFAI and AI with ACS recruited from a cohort of AIs consecutively evaluated in a tertiary hospital during a predefined period (2013-2020). Moreover, we analyzed differences in the hypothalamic-pituitary-adrenal axis in patients with and without type 2 diabetes and adrenal incidentalomas.

Methods
The study was approved by the Local Ethics Committee of the Hospital Universitario Ramón y Cajal.

Study population
The electronic records of the Hormone Laboratory of Hospital Universitario Ramón y Cajal were searched for patients undergoing a DST between the 1th of January 2013 and 31th of December of 2020 [5]. Then, the medical records of all retrieved patients were evaluated. We included all patients aged 18-90 years old who presented with unilateral or bilateral AI of at least 10 mm in greatest diameter without any of the following exclusion criteria: (i) patients diagnosed with hereditary syndromes associated with adrenal tumors [1]; (ii) patients on chronic glucocorticoid (GC) treatment or who had received GC treatment with a dose ≥10 mg/day of prednisone or equivalent for 3 months during the 3 months prior to the hormonal evaluation, (iii) patients on oral contraceptives (treatment had to be suspended at least 6 weeks before performing the DST test), (iv) patients in whom the imaging test was performed in the context of the extension study of a primary extra-adrenal cancer; (v) patients diagnosed with overt adrenal hormone hypersecretion: Cushing's syndrome (n = 2), primary hyperaldosteronism (n = 12), and pheochromocytoma (n = 4); (vi) diagnosis of adrenocortical carcinoma (n = 2) and (vii) patients with missing values in the initial DST (n = 1). A total of 709 out of 730 patients were finally included in the study.

Clinical evaluation
Medical records were retrospectively reviewed to extract demographic information such as age and sex; medical history of ACS-related comorbidities such as hypertension, type 2 diabetes mellitus, obesity, dyslipidaemia, cerebrovascular and cardiovascular diseases; and physical examination variables, including body mass index (BMI) and blood pressure. These parameters were collected at baseline and at the last follow-up visit. Hypertension was defined as blood pressure 140/90 mmHg or higher, or active treatment with antihypertensive drugs. Diagnoses of type 2 diabetes and dyslipidaemia were based on established criteria [6] [7]. Obesity was defined as a BMI of 30 kg/m 2 or higher. Cardiovascular disease was defined as ischemic heart disease or heart failure. Information on the treatment of type 2 diabetes (oral antidiabetic drugs or insulin, including doses) was recorded.

Hormonal and biochemical evaluation
All patients underwent a DST and a 24 h urine test with measurement of normetanephrine and metanephrine. Other hormones such as serum cortisol, adrenocorticotropic hormone (ACTH), dehydroepiandrosterone sulfate (DHEA-S), and 24 h urinary free cortisol (UFC) were also measured in some patients at the discretion of the treating physician. In hypertensive or hypokalemic patients, the aldosterone/renin ratio was calculated. Baseline serum 17-hydroxyprogesterone and cortisol concentrations were used to rule out non-classic congenital adrenal hyperplasia and adrenal insufficiency, respectively, in patients with bilateral AI. The DST was requested again at the follow-up visit. Other hormones were also determined at the discretion of the treating physician.
In the absence of specific signs of hypercortisolism (myopathy, ecchymosis, and/or skin atrophy), ACS was defined as a serum cortisol concentration ≥1.8 µg/dL at 8 am after a single oral dose of 1 mg dexamethasone taken at 11 pm the night before sampling. When the hormonal evaluation was normal (post-DST serum cortisol < 1.8 µg/dl, and aldosterone/renin ratio and urinary metanephrine concentrations were within the reference ranges), AIs were considered NFAI.
All patients underwent a complete laboratory analysis with a biochemistry profile after an overnight fast, both at the initial evaluation and at the last follow-up visit including fasting glucose levels, total cholesterol LDL and HDL cholesterol, and triglyceride concentrations. HbA1c was requested at the discretion of the treating physician depending on glucose concentrations and whether or not the patient had been diagnosed with diabetes.

Statistical analysis
For statistical analysis we used version 15 of STATA. Continuous variables were expressed as mean ± standard deviation (SD) or median and interquartile range (IQR) depending on whether or not a normal distribution was met. Categorical variables were expressed as counts and percentages. Normality was assessed by the Shapiro-Wilk test and the homogeneity of variances was assessed using Levene's test. Odds ratios (with 95% confidence intervals were calculated using a logistic regression model. Unpaired t-tests or Mann-Whitney test were used to compare differences in continuous parameters between NFAI and ACS as appropriate. To compare baseline values with those obtained at the end of the follow-up we used paired t-tests or McNemar's tests, as needed. Cox regression analysis was performed to estimate risk ratios during follow-up. Pearson's or Spearman's correlation analyses served to evaluate the associations between continuous variables. Chi-square tests were performed to compare categorical variables between groups. In all cases, a two-sided p value < 0.05 was considered as statistically significant.

Baseline characteristics
Of the 709 included patients, the mean age was 63.4 ± 10.8 years, 56.1% were women and 27.9% had bilateral adrenal tumors. At the AI diagnosis, type 2 diabetes was present in 172 (24.3%) patients: 13 were being treated with life-style changes (diet and exercise); 98 with oral antidiabetic drugs; 16 with insulin in monotherapy; and 45 with insulin and oral antidiabetic drugs. Mean HbA1c and fasting plasma glucose levels were of 6.2 ± 1.1% and 107 ± 31 mg/dL, respectively, in the entire cohort.
In patients with type 2 diabetes, the mean HbA1c was of 6.8 ± 1.3%. Most of them (63%) had HbA1c levels below 7%. However, only 57% had LDL-c levels <100 mg/dL and 21% had LDL-levels <70 mg/dl. Moreover, in those patients with associated cardiovascular disease, LDL was below 70 mg/dl in only 38% of the cases.

Differences in the prevalence and incidence of type 2 diabetes between ACS and NFAI
Of all patients with AIs, 478 (67.4%) were NFAI and 231 (32.6%) had ACS. We did not find significant differences in the prevalence of type 2 diabetes between the two groups (OR 1.31, 95% 0.92-1.88) ( Table 1).
The proportion of diabetic patients with HbA1c above 7% and above 8% was similar in both groups. However, HbA1c and fasting plasma glucose levels were significantly higher in patients with ACS than with NFAI ( Table 1). The proportion of patients with type 2 diabetes under insulin therapy was similar in both groups (33.9 vs. 23.1% in patients with ACS and NFAI, respectively, P = 0.13).
Differences in the degree of hypercortisolism between patients with or without type 2 diabetes Patients with type 2 diabetes (n = 172) had higher levels of urinary free cortisol (44.2 ± 29.83 vs. 37.3 ± 24.61 µg/dL; P = 0.039) and late-night salivary cortisol (5.0 ± 5.7 vs. 3.6 ± 3.5 nmol/L, P = 0.010) than patients without type 2 diabetes (n = 537) ( In addition, post-DST cortisol levels tended to be higher in diabetic patients (2.2 ± 2.3 vs. 1.9 ± 2.0 µg/dL, P = 0.091) (Fig. 1). However, no differences were found in ACTH and DHEAS levels between diabetic and non-diabetic patients, although this information was missing in many cases. A positive yet weak correlation was found between fasting plasma glucose and UFC, late-night salivary cortisol, and DST (Table 3).

Discussion
This retrospective study conducted in 709 patients consecutively evaluated for AIs at a single institution found no differences in the prevalence or incidence of type 2 diabetes between patients with NFAI and patients with ACS. However, our study suggests that glycemic control might be worse in patients with ACS; and that this might be directly associated with the degree of hypercortisolism.
The prevalence of type 2 diabetes in our cohort was 24%, which is within the range reported in previous studies [3,8]. The average prevalence of type 2 diabetes reported in a recent meta-analysis on 32 studies and 4121 patients with AIs was 18.1% [8]. Furthermore, that meta-analysis found that type 2 diabetes was twice as prevalent in patients with ACS (28.1%) than in patients with NFAI (14.4%); and that worsening of pre-existing diabetes mellitus was much more frequent in patients with ACS (9.2%) than in NFAI (0.0%) [8]. Our study, on the other hand, found no differences in the prevalence of type 2 diabetes between these groups. These differences may be related to the fact the threshold of 1.8 µd/dL was employed for the definition of ACS and only 34 out of the 231 patients with ACS had confirmed ACS (DST values > 5.0 µg/dL). On the other hand, patients with NFAI had a mean DST of 1.2 µg/dL, quite close to 1.8 µg/dL threshold that differentiate ACS and NFAI. Therefore, the possibility of finding differences between both groups was reduced. We identified 24 new cases of type 2 diabetes during a median follow-up time of 28 months. However, no differences in the incidence rates  between patients with NFAI and ACS were found. This observation is in line with the results of a previous metaanalysis [8]. Nonetheless, the risk of incident type 2 diabetes in patients with AIs might double that of patients with healthy adrenals, as observed in another study with a mean follow-up time of 7.2 years [9]. In agreement with our findings, other series reported no differences in the burden of cardiovascular comorbidity such as prevalence of diabetes, hypertension, obesity or dyslipidemia, between these two groups of AIs: NFAI and ACS [3]. Nevertheless, we are aware that a follow-up period of 28 months is shorter that the reported in the Elhassan (50.2 months) [8] and Lopez studies (7.7 years) [9], and these differences may also explain the differences detected regarding the incidence of type 2 diabetes in AIs, including ACS and NFAI. This study also found that patients with type 2 diabetes had higher UFC (P = 0.039) and late-night salivary cortisol levels (P = 0.010) than patients without type 2 diabetes. Type 2 diabetes is a systemic metabolic disease in which hyperinsulinism and insulin resistance constitute the fundamental pathophysiological mechanisms. Several studies have shown that there is a correlation between insulin resistance and NFAI [3,[9][10][11][12][13][14]. It has been postulated that the relationship between AI and type 2 diabetes could be reciprocal and progressive [15]. In fact, patients with adrenocortical adenomas seem to have insulin resistance [11]; and its severity might be dependent on the size of the adrenal mass [12]. This could suggest that NFAI patients might have mild cortisol secretion that would go unrecognized by diagnostic tests presently used [3,[8][9][10][11][12][13][14][15][16]. This possibility is supported by the findings of a recent study on the urinary steroid profile measured by gas chromatography coupled to mass spectrometry which showed a subtle glucocorticoid excess in patients with NFAI [5]. Mild glucocorticoid excess can lead to changes in body composition, including increased visceral fat [17]. Higher BMI, and larger waistlines; thus, increasing insulin resistance and the likelihood of developing diabetes [3]. In addition, the higher degree of cortisol hypersecretion in patients with type 2 diabetes compared to patients without diabetes may be related with the stress induced by diabetes mellitus, especially when it is not well-controlled, since type 2 diabetes is considered a chronic inflammatory state.
The pathophysiological pathways of insulin resistance leading to the development of an adrenal mass is not as well-understood, although mechanisms for this pathway have been proposed. There are several hypotheses that relate insulin resistance, typical of type 2 diabetes, and the development of AIs, as follows. Insulin is an anabolic hormone and, along with insulin-like growth factor 1 (IGF- 1), is involved in growth [14]. IGF-1 receptors (IGF-1R), M6P/IGF-2 receptors, and insulin receptors are found in the adult adrenal cortex and play an important role in the activation of different receptors in the adrenal glands that lead to their growth and development [14,15,[18][19][20][21]. Since IGF-1 and insulin receptors are similar in structure, insulin may bind to both receptors, albeit with lower affinity for the former [14,22]. Furthermore, high insulin levels may also promote hepatic growth hormone receptor activity, leading to increased IGF-1 secretion [14,23]. Both mechanisms, would lead to the initiation of downstream pathways such as PI3K and MAPK [14,24,25], inducing cell growth and proliferation and, perhaps, tumor initiation as has been described in several organs [26]. Moreover, elevated IGF-2 levels and IGF-1R overexpression have been implicated as a common occurrence related to adrenocortical tumors [15]. Thus, it can be hypothesized that the compensatory hyperinsulinemia resulting from insulin resistance could contribute to adrenal tumor growth [14]. Unfortunately, previous studies have not been able to establish the direction of this AI-insulin resistance relationship. Strengths of our study are that our cohort is similar to that expected according to previous studies (prevalence of diabetes, age distribution, and sex), that the patients were consecutively evaluated in a given period through the laboratory, and that the results obtained are consistent with previous studies. In addition, this is the largest Spanish series of patients with AIs focused on diabetes evaluation reported in the literature. Furthermore, both prevalence and incidence of type 2 diabetes has been evaluated, not only in patients with ACS, also in those with NFAI.
Within the limitations we found that it is a single-center retrospective study, and an inherent bias of these retrospective studies is that diseased individuals are more likely ACTH adrenocorticotropic hormone, DST dexamethasone suppression test, DHEAS dehydroepiandrosterone sulfate. LNSC late-night salivary cortisol, UFC urinary free cortisol a Fasting plasma glucose levels to undergo imaging tests than healthy individuals and thus the association between AIs and conditions such as diabetes, hypertension, and metabolic syndrome can be explained to some extent by a selection bias [3]. Furthermore, HbA1c was not available in all patients without diabetes and, therefore, the data in this group of patients are not reliable which could lead to a type 2 error in the incidence. Thus, the average A1c in the cohort, and especially in non-diabetic patients, is probably overestimated, since this data is absent in the majority of patients without diabetes in whom it is expected that it should be lower.

Conclusion
We found no significant differences in the prevalence or incidence of type 2 diabetes between patients with ACS and patients with NFAI. In diabetic patients, however, glycemic control was worse among patients with ACS than with NFAI. Thus, suggesting a possible deleterious impact of glucocorticoid excess. Furthermore, higher levels of salivary and urinary free cortisol were found in patients with than without type 2 diabetes.

Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Ethical approval All procedures performed in the participants of the study were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the Ethics Committee of Hospital Universitario Ramón y Cajal on February 14 of 2019.
Informed consent The Ethical committee of Hospital Universitario Ramón y Cajal approved the waiver for informed consent given the retrospective nature of the study.