Association of retinol binding protein4 (RBP4) and ghrelin plasma levels with insulin resistance and disease severity in non‐diabetic patients with hidradenitis suppurativa

Hidradenitis suppurativa (HS) is a chronic inflammatory disease associated with insulin resistance (IR). Retinol binding protein 4 (RBP4) and ghrelin are two bioactive proteins that have been involved in glucose metabolism and IR, but also in the regulation of immune and inflammatory processes. The aim of this study was to determine the serum levels of RBP4 and ghrelin in patients with HS, and to assess the possible relationship between these levels and IR, disease severity and HS risk. A total of 137 subjects (77 HS patients and 60 controls) without diabetes mellitus were enrolled in this cross‐sectional study. Patients with HS had significantly higher RBP4 but lower ghrelin plasma levels than controls, independently of body mass index (BMI). Serum RBP4 levels were positively correlated to disease severity and IR in HS patients. However, we found no association between ghrelin levels and any clinical or laboratory parameters. Moreover, high serum RBP4 and low ghrelin levels were associated with an increased risk for HS. Our results suggest that high RBP4 levels may be a surrogate biomarker for IR in patients with HS. Moreover, increased RBP4 and decreased ghrelin levels could also be independent risk factors for the development of HS.


| Participants and protocol
A cross-sectional study including 137 participants (77 HS patients and 60 controls) recruited from our Dermatology outpatient clinic at the University Hospital Marques de Valdecilla (Santander, Northern Spain). Patients were ≥18 years and fulfilled the diagnostic criteria for HS. [19] The control group was set up with hospital medical staff and subjects who had been admitted at the Dermatology Department because of non-inflammatory disorders. The research protocol was approved by the local ethics committee, and all the participants gave written informed consent.
Exclusion criteria, clinical evaluation and laboratory studies have been previously described. [3,10] Briefly, patients or controls with a history of CV events, DM and other endocrine diseases, chronic renal or liver failure and/or other inflammatory cutaneous or systemic diseases, or taking drugs (in the previous 6 months) affecting carbohydrate metabolism, were excluded from the study. The severity of HS was assessed by the HS Physician's Global Assessment (HS-PGA); HS was classified as moderate-severe-very severe (PGA ≥ 3) and as minimal-mild HS (PGA < 3).
All the participants provided information on demographic features and past medical history. Body height and weight, body mass index (BMI), waist circumference (WC), systolic blood pressure (BP) and diastolic BP were measured in all patients and controls. Body mass index (BMI) was calculated as weight (kg)/[height Blood samples were collected after overnight fasting. Glucose and insulin levels, glycated haemoglobin (HbA1c), triglycerides, serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c) and high-sensitivity C-reactive protein (hs-CRP) were assessed in all participants.
MS was diagnosed by the presence of three or more criteria according to the National Cholesterol Education Program's Adult Treatment Panel III (ATP III). [20] The degree of IR was calculated using the homeostatic model assessment for IR (HOMA-IR) expressed as fasting insulin level (µIU/mL) × fasting glucose level (mg/dL)/405. IR was diagnosed if HOMA-IR > 2.5. [10] Serum levels of RBP4 and ghrelin were analysed by enzyme-linked immunosorbent assay (Sigma-Aldrich Co. LLC). RBP4 and ghrelin levels were expressed as μg/mL and as ng/L, respectively. Intra-and inter-assay coefficients of variation were less than 10% for both analytes.

| Statistical analysis
Results were expressed as numbers (percentage), mean ± standard deviation (SD) or median and interquartile range (IQR), as appropriate. Mann-Whitney U-test and chi-squared or Fisher's exact tests were used to compare quantitative and qualitative variables, respectively. To evaluate the relationship between serum RBP-4 and ghrelin levels and HOMA-IR, Pearson's correlation was used. Moreover, forward stepwise multivariable logistic regression models were built to assess the potential association between both proteins and IR, HS risk and HS severity.

| Baseline features and serum RBP4 and ghrelin levels
Demographic, clinical and laboratory data of HS patients and controls are summarized in Table 1. There were no significant

| Association of RBP4 and ghrelin with IR and severity of HS
In patients with HS, we found a significantly positive correlation between RBP4 levels and IR (r = .390; P = .001) and disease severity (HS-PGA ≥ 3) (r = .639; P < .0001), once adjusting for age, sex and BMI. However, no correlation was found between plasma ghrelin concentrations and clinical or laboratory parameters.
Circulating RBP4 levels were significantly higher in patients with HS-PGA score ≥3 than in those with minimal-mild HS (HS-PGA score

| CON CLUS IONS
This is the first study, to our knowledge, that assesses RBP4 and ghrelin concentrations in patients with HS. We found that serum RBP4 levels were significantly increased in HS patients and positively correlated with disease severity. In this regard, increased plasma levels of this adipokine have also been found in other cutaneous diseases, such as contact dermatitis [21] and psoriasis. [22] Moreover, raised RBP4 concentrations in our study were associated with an increased risk for HS, suggesting that this adipokine might play a role in the pathogenesis of HS. In this respect, it should be noted that RBP4 acts as an immunomodulatory adipocytokine and may induce the release of pro-inflammatory mediators, including TNF-α, IL-1β and IL-6. [12] Furthermore, in our HS patients, serum RBP4 levels were positively associated with IR even after adjustment for BMI, suggesting that other factors independent of obesity might be implicated in such an association. It is known that RBP4 may inhibit insulin signalling in adipocytes by inducing the release of classic pro-inflammatory mediators from macrophages. [15] These facts suggest that RBP4 might represent a mechanistic link between the pathological inflammatory process of HS and the development of IR in patients with this disease.
On the other hand, ghrelin is a pleiotropic peptide-hormone/cytokine that exerts an anti-inflammatory function, [16,23] although its production may also be directly inhibited by pro-inflammatory mediators. [24] In our study, serum ghrelin levels were significantly lower in HS patients than in controls, irrespective of BMI. In this regard, the pro-inflammatory cytokines implicated in the chronic inflammatory process of HS might have played a role in the lower serum ghrelin levels. Regarding the association between ghrelin and IR, several studies have found an inverse correlation. [25] However, we did not find any relationship between serum ghrelin levels and IR in HS patients. In this sense, it is tempting to speculate that ghrelin secretion in HS patients might be influenced by an increase of pro-inflammatory cytokines, leading to a decrease in its serum levels. Besides, we have also found that serum ghrelin levels were negatively related to the risk for developing HS, although we cannot explain this protective finding through a beneficial effect on IR.
In conclusion, we found that serum RBP4 levels were significantly increased and serum ghrelin concentrations significantly decreased in HS patients. Furthermore, our results suggest that high RBP4 levels may be a surrogate biomarker for IR in patients with HS.
Finally, increased serum RBP4 and decreased ghrelin levels could be independent risk factors for the development of HS.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R S CO NTR I B UTI O N S
MAGL recruited patients for the study, contributed to the elaboration of the protocol of study, performed the study, helped in the in-