Relationship Between Orexin-A and Insulin Resistance in Patients with Type 2 Diabetes Mellitus

Background: Orexin-A is an orexigenic hormone that plays an important role in the metabolism of blood glucose, insulin, and insulin resistance (IR). The pathogenesis of type 2 diabetes mellitus (T2DM) is related to the abnormality in insulin and IR. However, no sufficient studies to date have clearly shown the association of orexinA with biochemical parameters related to T2DM. Objectives: The aim of this study was to determine the relation of orexin-A with IR and how they associate with physiological changes in T2DM patients. Understanding this relation will offer some pharmacological tools to reduce some complications in diabetes. Materials and Methods: A total of 41 T2DM and 43 non-DM subjects, aged between 40-60 years with body mass index (BMI) ≤25 kg/m 2 , participated in the present study. Fasting serum orexin-A, IR, fasting blood glucose (FBG), glycated hemoglobin (HbA1C), lipid profile, liver enzymes (alanine aminotransferase (ALT) and aspartate transaminase (AST)), nitric oxide (NO), and malondialdehyde (MDA) parameters were evaluated. Orexin-A was evaluated by using enzyme-linked immunosorbent assay (ELISA). For statistical analysis, GraphPad Prism 7.0 and SPSS version 24.0 programs were used. Results: Orexin-A was positively correlated with blood pressure, FBG, HbA1c, insulin, and IR but inversely related to insulin sensitivity (IS), leptin, and gender. Stepwise multiple regression presented HOMA-IR, diastolic blood pressure, and very-low-density lipoprotein-cholesterol as predictors for orexin-A. The area under control value showed orexin-A, FBG, HbA1c, HOMA-IR, IS, ALT, AST, NO and MDA as biomarkers for T2DM disease. Conclusion: Orexin-A has a predictive ability to diagnose T2DM, as it is significantly associated with hyperglycemia, IR, and IS.


Introduction
Diabetes mellitus is a complex noncontagious chronic disease.T2DM is classified as the most common form of diabetes, which is characterized by hyperglycemia [1] due to insufficient insulin production, improper responses of the peripheral cells to insulin, or both [2]. The untreated hyperglycemia causes several serious complications [1]. The hormonal disturbance is one of the consequences of T2DM. Orexin-A (hypocretin) is an orexigenic hormone which has an important role in appetite, arousal, and energy homeostasis regulation [3]. Orexin-A hormone has an effectual role on the metabolic processes of the body, especially glucose metabolism [4], insulin, IR [5] and IS [6]. Orexin-A also affects other hormones, such as leptin and the anorexigenic hormone [5]. Furthermore, orexin-A is involved in increasing blood pressure, as Li and Nattie recorded the hypertensive effect of orexin on normal rats [7]. According to our knowledge, the mechanisms of the modulation of orexin-A and T2DM are not fully understood and more studies are needed to determine these mechanisms in T2DM. Thus, we have designed the current study to determine orexin-A association with insulin, IR, and some clinical and physiological changes in T2DM patients. Understanding of this relation will offer certain pharmacological tools to reduce certain complications in diabetes.

Subjects and Methods Subjects
The study was performed by enrolling eighty-four Kurdish subjects from Al-Sulaymaniyah city. Forty-one subjects (16 men and 25 women) with T2DM disease attending the Diabetic and Endocrine Clinical Center at Al-Sulaymaniyah city were included. Forty-three healthy subjects (14 men and 29 women) were randomly selected as a control group. The age of the participants ranged between 40-60 years, with BMI ≤ 25. They were free from any chronic diseases (hypertension, cancer, and cardiovascular, digestive, hepatic, renal, autoimmune, and thyroid diseases). Furthermore, none of them were pregnant, smokers, or alcoholic consumer subjects. The blood samples were collected from April to October 2018.

Clinical parameters
The clinical history of each participant was recorded, while their BMI and mean arterial pressure (MAP) values were calculated [8]. HOMA-IR was used as an insulin resistance index and calculated by applying the following homeostasis model assessment formula. HOMA-IR= (Fasting Serum Insulin (µIU/ml)×FBG (mg/dl)) / 405 In addition, the quantitative insulin sensitivity check index (QUICKI) was used for assessing IS [9].

Laboratory measurements
About 10ml of fasting blood was withdrawn from the antecubital vein of all participants. For estimation of all biochemical parameters, Diagnosticum Zrt (TOKYO BOEKI MEDISYS) Biolis kits were used. The amount of insulin was determined by utilizing MAGLUMI Insulin (CLIN) Snib kit. Ref. no. 130205002M (Shenzhen New Industries, Shenzhen, China). Friedewald's equation was applied for the calculation of very-low-density lipoprotein-cholesterol (VLDL-C) [10]. Serum NO was estimated by colorimetric method based on Griess reaction [11]. For the determination of serum MDA, a simple and sensitive spectrophotometric method and a 2-Thiobarbituric acid (TBA) reagent (Merck KGaA, Darmstadt, Germany) were used [12].
Hormonal tests (orexin-A, ghrelin, and leptin) were estimated by ELISA. Serum orexin-A and ghrelin levels were estimated by using Cloud-clone corp. USA kits, Cat. No.: CEA607Hu and CEA991, respectively. For leptin assessment, human leptin ELISA kit (Biovender research and diagnostic products, Cat no.: RD191001100, Czech Republic) was used.

Statistical analysis
GraphPad Prism 7 software (San Diego, CA, USA ) was used. To compare the sensitivity and specificity of variables in both control and patient groups, the receiver operating characteristic (ROC) curve was created. To analyze the correlation coefficient of orexin-A and all other biochemical parameters, Pearson correlation coefficient (r) was used. Stepwise multiple regression was performed to predict the relationship of orexin-A with all other variables. Statistical package for social science (SPSS) version 24.0 (IBM, Armonk, NY, USA) was used for all the analyses. A p-value <0.05 was considered statistically significant.

Ethical approval
The study was approved ethically by the ethical committee of the College of Medicine at University of Sulaimani-Kurdistan Region of Iraq.

Results Fasting serum orexin-A and several biochemical parameters are biomarkers of T2DM
A total of 84 participants (41 diabetic and 43 non-diabetic) were involved in the study. The results of the ROC curve analysis of all study subjects are demonstrated in Figure-

Fasting serum orexin-A is correlated with blood pressure, glucose, and insulin-related parameters
The results also demonstrated that orexin-A correlated significantly with systolic blood pressure (SBP), diastolic blood pressure (DBP), MAP, FBG, insulin, HOMA-IR, and HbA1c. Whereas it was inversely correlated with sex, QUICKI, and leptin level (Table-1).

IR, diastolic blood pressure (DBP), and VLDL-C are correlated with serum fasting orexin-A levels
Stepwise multiple regression was performed to test the relation of orexin-A with all the studied parameters, as shown in Table-2. The results demonstrated that the main independent predictor for orexin-A in all studied groups is HOMA-IR, followed by DBP, height, and VLDL-C.

Discussion
According to our knowledge, this is the first study that evaluates the prevalence of orexin-A hormone in T2DM patients and non-DM subjects in Kurdistan Region/Iraq.
In this study, Pearson correlation analysis determined a negative significant correlation of orexin-A with sex , as was also previously shown [13], but a positive significant positive relation with blood pressure, FBG, HbA1c, insulin, and HOMA-IR. The positive correlation of orexin-A with FBG and insulin may be related to the roles of orexin-A in the protection against pancreatic cell dysfunction and the stimulation of insulin secretion from the pancreatic beta cells in T2DM patients [14]. The high level of orexin and the disorder in its secretion were shown to be associated with insulin resistance [15]. The significant correlation of orexin-A with SBP, DBP, and MAP may be related to the effects of orexin-A on the sympathetic nervous activities, which causes an increased blood pressure [7]. It also may be due to an increase in adrenalin and noradrenalin hormones [16], through its role on the hypothalamic-pituitary-adrenal pathway [17].
A significant inverse correlation of serum orexin-A with leptin was recorded in our analysis, which support that both have an inverse regulatory effect on energy and metabolic homoeostasis maintenances [5]. This correlation might be due to the inhibitory effect of leptin on orexigenic neuron [15] or due to the effect of orexin on the neuroendocrine system, including leptin level's setting [18]. On the other hand, an inverse correlation of orexin-A with QUICKI was obtained. It can be suggested that the possible hypertension conditions and chronic psychological stress on our population may cause chronic over activity of the sympathetic nervous system, which may conduce to a decline in IS [19]. Lipid profile has no significant correlation with orexin-A, which may be due to the normal body weight of our study participants, as indicated by BMI values. This result is confirms that of Rani et al., who showed a negative correlation of orexin with triglyceride in obese diabetic patients [5].
ALT and AST are markers for liver injury that showed no significant relation with orexin-A in our group of T2DM patients, although Lin et al. found the involvement of orexin-A in hepatic reperfusioninduced liver injury in rats [20]. Ghrelin has stimulatory effects on orexin neuron, leading to the stimulation of orexin mRNA expression in the hypothalamus [21]. Nevertheless, we obtained no significant association of ghrelin with orexin-A in our patients with T2DM disease. NO is a free radical that has an inhibitory effect on orexin neurons, as recorded by Yamakawa et al., who found that the level of circulating NO regulates the activation of orexin neurons [22]. As related to serum MDA, Zwirska-Korczala et al. found that orexin-A caused an increase in MDA level [23]. However, we did not record any significant relation of orexin-A with serum NO and MDA. When the effects of all other parameters were excluded by stepwise multiple regression analysis, HOMA-IR was shown to be the first independent predictor of orexin-A (Table-2). This confirms the role of orexin-A in IR and T2DM disease [5,24] and that HOMA-IR is a strong predictor of T2DM development [25].
As presented in Figure-1, and in accordance with our results established by the AUC value, orexin-A [26], HOMA-IR, QUICKI, FBG and HbA1c were considered as hallmarks for T2DM disease [27,28]. Orexin-A is considered as a biomarker for T2DM disease due to its regulatory effect on glucose and insulin homeostasis. Orexin-A has a dual effect on blood glucose regulation through the autonomic nervous system, i.e. the sympathetic and parasympathetic nervous system. When the cytosolic energy is reduced, the orexin neuron is stimulated and causes an increase in blood glucose level through the sympathetic nervous system. This explanation applies to diabetes patients as they have a low level of cytosolic energy which leads to hepatic glucose production and, thus, hyperglycemia [4]. HOMA-IR is presented as a marker for T2DM disease in our study. This confirmed the critical role of insulin resistance in the development of T2DM disease [2]. Meanwhile, we found that MDA and NO are biomarkers for T2DM disease. MDA is a lipid peroxidation product which is evaluated as an oxidative stress marker and predicts T2DM [29]. NO has a potential role in the pathogenesis of type 2 DM [30].

Conclusions
Orexin-A possesses a linear correlation with hyperglycemia and insulin resistance, which indicates the stimulatory effects of orexin-A on glucose and insulin secretion from the liver. Accordingly, orexin-A and HOMA-IR may be suggested as predictors for the metabolic disorder and diabetic complications in our subjects. Furthermore, orexin-A can be considered as a good biomarker for T2DM disease. Financial support and sponsorship: Nil Conflict of interest: The authors declare that they have no conflict of interest