The role of leptin mediating the relationship between ‘Somatic Anxiety’ symptoms and major depressive disorder

Anxiety’ Abstract BackgroundLeptin is a multifunctional hormone with influences on neural circuitry in emotional processing, and it may play a role in the pathophysiology of major depressive disorder (MDD). In this study, we aimed to investigate whether leptin levels were differentiated in patients with MDD and those at genetic high risk of MDD (GHR-MDD) and the relationship between leptin and clinical symptoms. Methods: Participants (18 drug-naïve MDD, 15 GHR-MDD and 40 healthy controls) completed clinical assessments and provided blood samples for measurement of leptin levels. Leptin levels were compared across all groups and associations between leptin and clinical symptoms were explored and mediation models tested. Results: We found that leptin was increased in MDD. We also found a correlation between leptin and ‘Somatic Anxiety’ symptoms in MDD and that leptin was a significant and independent mediator of clinical state and ‘Somatic Anxiety’ symptoms. Conclusions: MDD patients occured with dysregulation of leptin. Additionally, there was a correlation between leptin and ‘Somatic Anxiety’ symptoms in MDD. The finding of leptin as a significant and independent mediator of clinical state and ‘Somatic Anxiety’ symptoms suggested leptin plays an indirect effect in somatic depressive symptoms in MDD.

more likely to suffer from depressive disorder [3]. However, despite the increased genetic risk, most people at genetic high risk of MDD (GHR-MDD) do not develop MDD. Novel methods of comparing diseased populations and healthy controls could potentially indicate candidate markers of vulnerability and progression to MDD, as well as potential markers for resilience to the disorder.
There is a bidirectional relationship between depressive disorder and obesity: the presence of one disorder increases the risk of developing the other [4]. Furthermore, patients with obesity and their first-degree relatives frequently experience depression, anxiety, and other psychiatric disturbances [5,6]. Leptin was discovered by Zhang et al [7], as a 16-kD hormone secreted by adipose tissue, that plays a core role in regulating energy intake and expenditure. Leptin can permeate the blood brain barrier (BBB), and paly a roles in synaptic activity, neuronal morphology, and neuronal development in the central nervous system [8,9]. The leptin distributed in the brain is related to emotional and cognitional processes, such as the hypothalamus and hippocampus, which has sparked increasing interest in mood disorder [9][10][11]. Animal models suggest that impaired leptin production may contribute to depression [10,12]. However, preliminary research in humans has provided conflicting clinical results with both high and low levels of leptin found in depressed patients [13][14][15]. Additionally, no consensus on the relationship between leptin levels and the severity of depression symptoms has been reached. These mixed results are likely due to the influence of antidepressants. Therefore, we determined to include only untreated patient with MDD.
Taken together, preliminary research findings indicate that dysregulation of peripheral metabolic markers play an important role in the pathophysiology of MDD, conferring factors influencing vulnerability to MDD to the children who have parents that suffer from MDD.
In this study, we examined the plasma leptin levels of people with untreated MDD, GHR-MDD and in healthy controls (HCs) and evaluated the correlation between leptin and MDD symptoms. In addition, we examined whether blood biomarkers mediated the association between clinical state (MDD, GHR-MDD) and symptoms, based on correlation analyses. We Demographic and clinical details are presented in Table 1.

Plasma leptin determination
Blood collection was carried out according to standardized protocols, with samples taken

Statistical analyses
We separated the participants into three groups (HC, GHR-MDD and MDD). Either one-way analysis of variances (ANOVAs) or chi-square tests were used to examine the demographic characteristics (age, gender and body mass index [BMI]) and clinical characteristics (duration of illness, first episode, and HAMD and HAMA scores) of the participants. Leptin concentrations were analyzed using one-way analysis of covariance (ANCOVA), with age, gender and BMI as covariates. Post-hoc analyses were performed among the HC, GHR-MDD, and MDD groups using a general linear model.
We used Spearman correlation to analyze the correlation between leptin levels and clinical symptoms in the MDD group. Based on previous correlation analyses, we then used multiple regression analyses to examine the effects of leptin on clinical symptom scores after accounting for age, gender and BMI. Based on these results, a mediation analysis was used to explore whether leptin (as mediator variable) potentially influenced the association between clinical state-MDD and GHR-MDD (causal variable) and clinical symptoms (outcome variable). For the mediation analysis, the PROCESS procedure for SPSS Version 3.2 (Written by Andrew F. Hayes, Ph.D., www.afhayes.com) was used, with a 5000 bias-corrected bootstrap sample for significance testing. We summarized mediators using mean, standard deviation (SD),, and 95% confidence interval (CI)..
Significance was set at p<0.05 (two-tailed) for all tests. All analyses were performed using SPSS 22.0.

Demographic and clinical characteristics
There were significant differences in age (p = 0.014) among the HC, GHR-MDD, and MDD groups but no significant differences in gender and BMI (p>0.05). The effect of diagnosis on HAMD (Somatic Anxiety, Psychic Anxiety, Core Depressive, Anorexia, Total scores) [16] and HAMA scores were significant among the HC, GHR-MDD, and MDD groups (all p-values<0.001; Table 1).

Comparison of plasma concentrations
After controlling for age, gender and BMI, significant group effects were observed in leptin level in the three-group analysis (p = 0.004). Post-hoc analysis revealed a significantly higher leptin level in MDD, compared with GHR-MDD (p = 0.003) and HC (p = 0.005) but no significant difference in leptin between GHR-MDD and HC (Fig. 1).

Correlation between leptin level and clinical symptoms in MDD
In the MDD group, correlation analysis identified a significant positive correlation between leptin level and 'Somatic Anxiety' score on the HAMD (r = 0.550, p = 0.024; Fig. 2) and no significant correlation between leptin and scores for 'Psychic Anxiety', 'Core Depressive', 'Anorexia', as well as 'Total' scores on the HAMD and HAMA ( Table 2). Multiple regression analyses showed that leptin predicted 'Somatic Anxiety' (β = 0.520, t = 2.355, p = 0.033).

Mediation analysis
Based on the results of the correlation analyses, we found a correlation between leptin and 'Somatic Anxiety' in the MDD group. We found that leptin (Path AB, β = -0.4752; 95% CI: -1.0395 to -0.0062) significantly mediated group differences in 'Somatic Anxiety' as measured by the HAMD. As shown in Fig. 3

Discussion
To the best of our knowledge, this was the first study to investigate alterations of leptin in MDD and GHR-MDD. We found that leptin was increased in participants with MDD.
Subsequently, we found a correlation between leptin and 'Somatic Anxiety' in MDD, with leptin as a significant and independent mediator of clinical state and 'Somatic Anxiety' symptom.

Dysregulation of leptin in MDD
We found that leptin was increased in the MDD group but failed to find the same change in the GHR-MDD group, suggesting that raised leptin may be a potential vulnerable factor related to MDD. Important symptoms of MDD include loss of appetite and weight loss, and leptin is known to regulate food intake and weight. Although leptin is an anti-obesity hormone associated with decreases in individual weight, patients with obesity appear to display high levels of leptin [17]. The results of studies of leptin levels in MDD have been inconsistent, with both reduced [18,19] and elevated [20][21][22] leptin levels found. The variability of these results may be due to subtype heterogeneity in MDD. Clinical symptoms in MDD patients may vary, such as appetite and/or weight increasing and/or decreasing, with subsequent alteration in leptin levels. Furthermore, sexual dimorphism may affect leptin levels, with leptin higher in females than males [23,24]. One metaanalysis indicated that males who expressed lower adiponectin and high leptin levels had a higher likelihood of developing MDD, but the same was not found in females [25]. One viewpoint put forward is that dysfunction of leptin signaling to the central nervous system, rather than the absolute concentration of leptin, affects mood [26].

The relationship between leptin and symptoms in MDD
In this study, we found a positive correlation between leptin and 'Somatic Anxiety' but not for other symptoms. Additionally, leptin significantly and independently mediated the association between diagnosis and 'Somatic Anxiety'. The 'Somatic Anxiety' psychopathological dimension of MDD in the HAMD-17 includes somatic anxiety, hypochondria, sleep disturbance, general somatic symptoms, and gastrointestinal symptoms [16]. Chirinos et al. demonstrated that leptin was positively associated with somatic depressive symptoms [27]. In another study, a sex difference in the correlation between leptin and severity of depression in type 2 diabetes was found, with a positive association in men but not in women [28]. In a mouse study, leptin was found to reduce depressive behaviors [29]. Evidence from preclinical studies has clearly indicated that leptin exerts antidepressant and anxiolytic effects [30]. These conflicting findings may be explained by the complexity of leptin response as a function of obesity, which is often associated with high levels of leptin. Taken together, the finding suggests that leptin plays a potential role in mediating somatic anxiety symptom in MDD.

Limitations
There are limitations to our study. Firstly, we selected medication-naïve MDD patients to minimize the influence of treatment, resulting in a small sample size that may limit the generalizability of our results as well as our ability to detect relationships between blood biomarkers and symptoms. Future studies with larger sample sizes will be important to further understand the pathophysiology of MDD. Second, this was a cross-sectional study; therefore, subsequent developments in participants with GHR-MDD were unknown. A longitudinal study of GHR-MDD with long-term follow-up is required, with comparisons between individuals who do and do not develop MDD, allowing for the possibility of developing a better mechanism to determine genetic susceptibility or protective factors for the disorder.

Conclusion
In summary, we found that leptin was increased in MDD and leptin as a vulnerable factor can influence MDD status. Additionally, we found a correlation between leptin and

Consent for publication
Not applicable.

Availability of data and materials
The datasets we applied are available from the corresponding author, provided reasonable requests.

Competing interests
The authors declare that they have no conflict of interest.     Leptin significantly mediated the association between diagnosis (MDD/GHR-MDD) and somatic anxiety, providing further evidence that there was an indirect way to influence patient somatic anxiety symptom by leptin. Path C represents the variance in diagnosis associated with somatic anxiety symptom, and Path C'

Funding
represents the association between diagnosis and somatic anxiety symptom after taking into account leptin as a mediator. Path AB in the mediation effect and is significant at P < 0.05 based on confidence intervals from bias-corrected bootstrapping of 5000 samples.