Association of IGF-1 Level with Low Bone Mass in Young Patients with Cushing's Disease

Purpose Few related factors of low bone mass in Cushing's disease (CD) have been identified so far, and relevant sufficient powered studies in CD patients are rare. On account of the scarcity of data, we performed a well-powered study to identify related factors associated with low bone mass in young CD patients. Methods This retrospective study included 153 CD patients (33 males and 120 females, under the age of 50 for men and premenopausal women). Bone mineral density (BMD) of the left hip and lumbar spine was measured by dual energy X-ray absorptiometry (DEXA). In this study, low bone mass was defined when the Z score was −2.0 or lower. Results Among those CD patients, low bone mass occurred in 74 patients (48.37%). Compared to patients with normal BMD, those patients with low bone mass had a higher level of serum cortisol at midnight (22.31 (17.95-29.62) vs. 17.80 (13.75-22.77), p=0.0006), testosterone in women (2.10 (1.33–2.89) vs. 1.54 (0.97–2.05), p=0.0012), higher portion of male (32.43% vs. 11.54%, p=0.0016) as well as hypertension (76.12% vs. 51.67%, p=0.0075), and lower IGF-1 index (0.59 (0.43–0.76) vs. 0.79 (0.60–1.02), p=0.0001). The Z score was positively associated with the IGF-1 index in both the lumbar spine (r = 0.35153, p < 0.0001) and the femoral neck (r = 0.24418, p=0.0057). The Z score in the femoral neck was negatively associated with osteocalcin (r = −0.22744, p=0.0229). Compared to the lowest tertile of the IGF-1 index (<0.5563), the patients with the highest tertile of the IGF-1 index (≥0.7993) had a lower prevalence of low bone mass (95% CI 0.02 (0.001–0.50), p=0.0002), even after adjusting for confounders such as age, gender, duration, BMI, hypertension, serum cortisol at midnight, PTH, and osteocalcin. Conclusions The higher IGF-1 index was independently associated with lower prevalence of low bone mass in young CD patients, and IGF-1 might play an important role in the pathogenesis of CD-caused low bone mass.


Introduction
Cushing's disease (CD), caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, is a rare disease with approximately 1.2 to 2.4 new cases per million people each year [1].
Osteoporosis has been recognized as a serious consequence of endogenous hypercortisolism since the frst description in 1932 [2]. Te prevalence of osteoporosis is around 38-50%, and the rate of atraumatic compression fractures is 15.8% in CD patients [3]. After cortisol normalization and appropriate treatment, recovery of the bone impairment occurs slowly (6-9 years) and partially [4,5]. Hypercortisolemia impairs bone quality through multiple mechanisms [6]. Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) play a crucial role in bone growth and development [7]. IGF-1 is considered essential for the longitudinal growth of bone, skeletal maturity, and bone mass acquisition not only during growth but also in the maintenance of bone in adults [8]. Previous research studies revealed that low serum IGF-1 levels were associated with a 40% increased risk of fractures [9,10], and serum IGF-1 levels could be clinically useful for evaluating the risk of spinal fractures [11]. In Marl Hotta's research, extremely low or no response of plasma GH to recombinant human growth hormone (hGRH) injection was noted in CD patients. Tis result suggested that the diminished hGRH-induced GH secretion in patients with Cushing's syndrome might be caused by the prolonged period of hypercortisolemia [12]. Other surveys indicated that glucocorticoids, suppressing GH-IGF-1 and the hypothalamic-pituitary-gonadal axes, lead to decreased number and dysfunction of osteoblast [13].
However, the exact mechanism is still unclear, and few risk factors for osteoporosis in CD have been identifed so far. Until now, relevant and sufciently powered studies in CD patients have been rare [14,15]. Early recognition of the changes in bone mass in CD patients contributes to early diagnosis of bone mass loss and prompt treatment, which could help minimize the incidence of adverse events such as fractures.
On account of the scarcity of data and pressing open questions concerning risk evaluation and management of osteoporosis, we performed a well-powered study to identify the related factors associated with low bone mass in young CD patients at the time of diagnosis.

Subjects.
Tis retrospective study enrolled 153 CD patients (33 males and 120 females) from the Department of Endocrinology and Metabolism of Huashan Hospital between January 2010 and February 2021. All subjects were evaluated by the same group of endocrinologists for detailed clinical evaluation. Tis study, which was in complete adherence to the Declaration of Helsinki, was approved by the Human Investigation Ethics Committee at Huashan Hospital, Fudan University (No. 2017M011). We collected data on demographic characteristics, laboratory tests, and bone mineral density.
Inclusion criteria included the following: (1) willingness to participate in the study; (2) premenopausal women ≥18 years old, men ≥18 years old but younger than 50 years old, and young women (<50 years old) with menstrual abnormalities who were associated with CD after excluding menstrual abnormalities caused by other causes; (3) diagnosis of CD according to the updated diagnostic criteria [16]; and (4) pathological confrmation after transsphenoidal surgery (positive immunochemistry staining with ACTH). Exclusion criteria included Cushing's syndrome other than pituitary origin.
Te IGF-1 index was defned as the ratio of the measured value to the respective upper limit of the reference range for age and sex. Body mass index (BMI) was calculated using the following formula: weight (kg)/height 2 (m 2 ). Te bone mineral density (BMD) measuring instrument was Discovery type W dual energy X-ray absorptiometry from the American HOLOGIC company. Quality control tests were conducted every working day. Before examination, the date of birth, height, weight, and menopause date of the examiner were accurately recorded, and then BMD (g/cm 2 ) of the left hip and lumbar spine were measured by DEXA. Z value was used for premenopausal women and men younger than 50 years old, and Z-value � (measured value − mean bone mineral density of peers)/standard deviation of BMD of peers [17,18]. In this study, low bone mass was defned as a Z-value of −2.0 or lower.

Statistical Analysis.
Te baseline characteristics were compared between CD patients with and without low bone mass by using the Student's t-test for continuous variables and the χ 2 test for category variables. Bone turnover markers, alanine aminotransferase (ALT), triglyceride (TG), IGF-1 index, thyroid stimulating hormone (TSH), free triiodothyronine (FT3), free thyroxine (FT4), testosterone (T), 24 hours of urine cortisol (24 h UFC), and serum cortisol at 8 a.m. (F8 am) and at midnight (F24 pm) were not in normal distribution, so variables mentioned above were Log10transformed, which could be used as continuous variables during statistical analysis. Participants were categorized into three groups according to tertiles of the IGF-1 index: <0.5986, 0.5986-0.8380, and >0.8380. Te linear trend across IGF-1 index tertiles was tested using linear regression analysis for continuous variables and the Cochran-Armitage test for categorical variables. We used a multivariate logistic regression model to identify related factors that are independently associated with the risk of low bone mass. Variables included in the multivariate logistic regression model were selected based on the Spearman rank correlation analysis and established traditional low bone mass risk factors as priors. Te results were presented as odds ratios (OR) and the corresponding 95% confdence intervals (CI). Signifcance tests were two-tailed, with P value <0.05 considered statistically signifcant for all analyses. Statistical analysis was performed using SAS version 9.3 (SAS Institute Inc, Cary, NC, USA).

Te Prevalence of Low Bone Mass in Young
Cushing's Disease Patients. From the inpatient system of Huashan hospital, a total of 153 CD patients under the age of 50 for men and premenopausal women (some with menstrual abnormalities were associated with CD) were included, aged from 13 to 49 years, with an average age of 34.25 ± 8.39 years. Tere were 33 males (21.57%) and 120 females (78.43%).
Tese CD patients included newly diagnosed CD, recurrences of CD, and CD without remission after treatment. Tere were no diferences in the prevalence of diferent statuses of CD between the two groups (Table 1).
Among these CD patients, low bone mass occurred in 74 patients (48.37%), including 24 men and 50 women. Te prevalence of low bone mass was 41.67% and 72.73% in female and male CD patients, respectively, and 42 (56.76%) patients sufered from low bone mass in the lumbar spine only, while 10 (13.51%) patients had low bone mass in the femoral neck only, and 22 (29.73%) patients had low bone mass in both parts.
In female patients with low bone mass, 27 (54%) had low bone mass in the lumbar region only, 9 (18%) in the femoral neck only, and 14 (28%) had low bone mass in both parts. For male patients with low bone mass, 16 (66.67%) patients had low bone mass only in the lumbar region, and the rest (8, 33.33%) had low bone mass in both parts.
Ten patients had a history of fragility fractures (6 ribs, 3 vertebrae, 1 femoral neck, and ribs), and all of them achieved low bone mass in BMD.

Baseline Characteristics of Cushing's Disease Patients with and without Low Bone Mass.
Tese CD patients were divided into two groups with and without low bone mass ( , p � 0.0169) than those without low bone mass. CD patients without low bone mass were more likely to have serum IGF-1 above the upper limit of the normal reference range (ULN) with age-adjusted (18, 26.87% vs. 3, 4.84%, p � 0.0007). No diferences of bone turnover makers were found between the two groups.
In the logistic regression analysis of the related factors of low bone mass, most of the potentially relevant factors were put into this model; only the IGF-1 index was still signifcantly negatively associated with the prevalence of low bone mass after adjusting for covariables. Te results indicated that compared to the patients in the lowest tertile of the IGF-1 index (<0.5563), those with the highest tertile of the IGF-1 index (≥0.7993) had a lower prevalence of low bone mass (95% CI 0.16 (0.06-0.41), p � 0.0002). After adjusting for age, gender, and BMI, the patients in the highest tertile of the IGF-1 index still conferred a lower prevalence of low bone mass (95% CI 0.15 (0.06-0.42), p � 0.0003). Te association between the IGF-1 index and low bone mass still existed (95% CI 0.02 (0.001-0.5), p � 0.0209) even after adjusting for age, gender, CD duration, BMI, hypertension, dyslipidemia, diabetes, ALT, Scr, FT3, F24 pm, PTH, and osteocalcin (Table 3). In comparison to the reference population, the participants in the middle tertile of the IGF-1 index (0.5563-0.7993) had no diferent risk of low bone mass.

Discussion
Our results revealed that low bone mass occurred in around half of young CD patients, afecting more males than females, and mostly in the lumbar spine. Te CD patients in International Journal of Endocrinology our study had a high prevalence (48.37%) of low bone mass at the baseline. Tis was in accordance with the fndings of previous research, and the reported prevalence of osteoporosis due to excess endogenous cortisol ranges from 22% to 59% [19][20][21][22][23][24][25]. In this study, CD patients' lumbar vertebrae were more severely afected than the neck of the femur. It is reported that lumbar vertebrae, containing more trabecular bone than femur neck, were more vulnerable to endogenous cortisol [26].
Our results also indicated that men were more prone to low bone mass than women in CD, which was in accordance with several other studies [23,27,28]; possibly, the deleterious efect of cortisol excess on BMD might overrule the protective efects of sex hormones, and men were more often hypogonadal compared with women in CD patients. In our study, patients with low bone mass had a signifcantly higher level of F24 pm. Both cortisol levels in the morning and at midnight, were negatively associated with the Z-score of BMD in the lumbar spine at diagnosis. But these results were not seen in the femoral neck at diagnosis. Tis further indicated that lumbar vertebrae were more vulnerable to endogenous cortisol. BMI was considered to be associated with bone mass [29]. In our study, higher BMI was associated with higher BMD at diagnosis in the femur neck but not in the lumbar vertebrae, consistent with other studies [30]. Interestingly, besides the above known related factors, we also found that a higher level of the IGF-1 index was strongly associated with a lower prevalence of low bone mass, both in the vertebra lumbalis and the neck of the femur, independently of age, gender, duration, BMI, hypertension, dyslipidemia, diabetes, level of ALT, creatinine, FT3, and F24 pm. Te IGF-1 index was also positively associated with the BMD Z-score, both in the lumbar spine and the femoral neck. So far, there have been few studies concerning the association between IGF-1 and low bone mass in Cushing's disease patients. As we know, GH [31,32] and IGF-1 [33] have been demonstrated to increase both bone formation (e.g., collagen synthesis) and bone resorption. However, in CD patients, glucocorticoids resulted in decreased number and dysfunction of osteoblasts by inhibiting GH-IGF-1 axes [34,35]. In vitro studies suggested that at high concentrations of glucocorticoids, a decreased release of GHRH had been reported [36][37][38]; therefore, GH-IGF-1 axes were inhibited. IGF-1 possessed anabolic mitogenic actions in osteoblasts while reducing the anabolic actions of TGF-β [39]. Te decrease in IGF-1 might be a risk factor for low bone mass in CD patients. In vitro studies had also indicated that the suppressive efects of glucocorticoids on osteoblast function can be partially reversed by GH or IGF treatment [8]. In recent years, some studies have also shown that patients with untreated Cushing's disease may have elevated IGF-1, and mildly elevated IGF-1 in Cushing's disease does not imply pathological growth hormone excess. Higher IGF-1 levels could predict better outcomes in CD [40,41]. Possible mechanisms were not clear, which might involve changes in IGF binding proteins (IGFBPs), interference in IGFBP fragments, IGF-1 synthesis or clearance, and/or the efects of hyperinsulinism induced by excess glucocorticoids. In our study, the results also showed that IGF-1 was an independent protective factor for low bone mass in CD patients.
Our study was one of the few well-powered research studies on the association of IGF-1 levels with low bone mass in young CD patients. Tese represented important strengths of our study, especially given the rarity of CD. Te main limitation of this study was its retrospective nature. Tis could not prove causality. A prospective study should be conducted to explore the causality between IGF-1 and osteoporosis in CD patients. In addition, this study lacked morphometric data for spinal fractures in all patients, which may underestimate the incidence of fractures and osteoporosis. However, our study indicated that a lower IGF-1 index level was signifcantly associated with low bone mass in young CD patients, which might provide a new aspect to understand the possible risk factors and mechanism of osteoporosis in CD patients.      In conclusion, our study found that a higher IGF-1 index was independently and signifcantly associated with decreased prevalence of low bone mass in young CD patients, drawing attention to the role of IGF-1 in the pathogenesis of CD-caused low bone mass and may support the exploration of this pathway in therapeutic agent development in antiosteoporosis in CD.

Data Availability
Te data used to support the fndings of the study are available on request from the authors.

Additional Points
Trough a retrospective study of a large sample of Cushing's disease (CD) patients from a single center, we found that a higher IGF-1 index was independently associated with a lower prevalence of low bone mass in young CD patients and IGF-1 might play an important role in the pathogenesis of CD-caused low bone mass.

Disclosure
Wanwan Sun and Quanya Sun were the co-frst authors.