The mechanism of leptin and IGF-1 in the diabetic rheumatoid arthritis Iraqi patients

We aimed here to study the impact of leptin on insulin like growth factor-1 (IGF-1) level to imply its antidiabetic effect on Iraqi rheumatoid arthritis’s, who was with and without diabetes mellitus. Rheumatoid arthritis is a chronic inflammatory disease, primarily targets the synovium and articular cartilage, which causes joint damage. Although the role of adipocytokines in mediating damages of joint has recently suggested, it is still a matter of considerable debate. METHODS: Patients diagnosed as diabetic rheumatoid arthritis aged from 33-60 years and others diagnosed as non-diabetic rheumatoid arthritis were compared with healthy control (aged 33-46 years). Some biochemical parameters have determined, such as fasting serum glucose, glycated hemoglobin, lipid profile, serum fasting insulin, IGF-1, and leptin, using ELISA and immune radiometric assay. Results indicated an elevation in some biochemical parameters in diabetic rheumatoid arthritis’s when compared with patients without diabetes. There was a significant increase in fasting serum glucose and some of the lipid components in diabetic rheumatoid arthritis patients compared to that non-diabetic. A significant elevation of leptin in diabetic rheumatoid arthritis patients compared to control (p<0.003). However, a non-significant difference of leptin was detected between diabetic rheumatoid arthritis and non-diabetic patients. In addition, some other parameters were significantly reduced in diabetic rheumatoid arthritis patients when compared to non-diabetic patients, such as high-density lipoprotein and IGF-1. This study highlights that leptin could act as pro-inflammatory mediator in rheumatoid arthritis. The negative correlation between leptin level and IGF-1 plays an important role in understanding the metabolic pathogenesis of rheumatoid arthritis.


Introduction
Rheumatoid Arthritis (RA) is a chronic systemic autoimmune inflammatory disease affects mainly the peripheral joints, frequently foremost to damage of these joints [1]. RA has many symptoms such as symmetrical synovitis, advanced joint destruction, pain, fatigue, and walk disability. The occurrence of RA ranges from gentle remitting appearances to rapidly advanced forms with increased mortality [2]. Chronic inflammation in RA activates some disorders, such as fat mobilization, enhanced gluconeogenesis, alter protein catabolism, and negative nitrogen balance [3]. RA causes a damage of cartilage and bone, as well as, a damage of cardiovascular, pulmonary and endocrine system [4]. The insulin like growth factor-1 (IGF-1) is a protein of 70mer stimulated by the liver as a response to the stimulus of growth hormone. Therefore, IGF-1 is frequently used to measure the body's natural biochemical foundation for proper bone, muscle and tissue [5]. It has been reported that insulin resistance (IR) is associated with some metabolic disorders, such as diabetes mellitus-type 2 (DM-T2), obesity and hypertension [6]. IR usually refers to the disability of insulin to adequately control the metabolism of glucose in the peripheral tissues. IR has been associated with 1.7-fold increase in the risk of cardiovascular diseases (CVD) and contributed to the progress of  [7]. Leptin is the major recognized adipocytokine. It composed of 167mer, and it is mainly expressed in adipose tissue. Leptin controls energy homeostasis and restricts by numerous neuroendocrine and immune functions. Leptin and its receptors (Ob-R) segment acts as structural and functional resemblances with cytokines family and their receptors [8]. Although arthritis does not lead to DM, diabetes does sometimes correlate with joint symptoms, where lifestyle and obesity contribute to or deteriorate both. As well as, corticosteroids have shown to restrict with glucose metabolism [9]. The pro inflammatory influences, DM and hypertension have been hypothesized as mean causes of arthritis [10]. Arthritis was previously described as a deteriorating disease, but recently is considered as a metabolically dynamic process amenable to treatment [11]. This study designed to deeply investigate the contribution of leptin hormone on the pathogenesis and mechanism of RA patients suffered DM. The interference of leptin with some biochemical parameters was studied using ELISA and immune radiometric assay.

Patients
This study included RA patients suffered DM-type2 (diabetic RA), who were diagnosed by specialist physicians and weren't under any treatment. In order to understand the role of leptin as antidiabetic, other RA patients without DM were included in the study (non-diabetic RA). Fifty RA patients with and without DM (18 males and 32 females) aged between (33-60) and 40 healthy volunteers (15 males and 25 females) aged between 33-46 were involved. All participates (ninety) were attended at the National Diabetic Center (NDC) of Mustansiriya University and Al-Yarmok Teaching Hospital in Baghdad. An informed consent was obtained from all patients and volunteers participants. The board of the National center for Diabetic and treatment research of Mustansiriyah University has approved the project.

Blood Collection
Ten mL blood of fasting patients and volunteers were collected from the median cubital. The blood was allowed to stand for 30 min for clotting. Serum samples were collected after centrifuge for 5 min at 10000 rpm and stored at ultra-freezer at -40C, for further analysis.

Methods
Determination of FBG, Serum TC, serum TAG and serum HDL-C: Each biochemical parameter was determined by its kit method using automated analyzer (BIOLABO Kenza 240TX). Determination of serum LDL-C: LDL was calculated indirectly using the Friedewald ̓ s equation [17]. LDL-C = TC − [HDL-C + TAG/5]. This equation is only accurate when: TG levels are below 400 mg/dl. The Bio-Rad variant hemoglobin A1c (HbA1c): The ion-exchange high-performance liquid chromatography was used for an automatic and accurate separation of glycated hemoglobin (HbA1c) using Vearent kit.
Determination of serum Insulin and leptin: They were measured using the DRG insulin ELISA kit. Determination of serum HOMA2-IR: This was calculated using HOMA2 calculator software. Determination of serum IGF-1: Serum IGF-1 concentration was measured using the DIASORIN kit.

Statistical analysis
Microsoft office excel 2010 work sheet was assisted for the statistical analyses. Data were expressed as means ± standard deviation SD and differences between variables considered of statistical significance in the t-test at P level <0.05.

RESULTS
The diabetic and non-diabetic RA patients were introduced to some biochemical tests to investigate the chemical interference of leptin level as antidiabetic hormone with some biochemical parameters of arthritis`s. Results in figure 1 showed a significant increase in FSG, TC, TAG, LDL-C, VLDL, and leptin in RA patients when compared with controls, (p<0.03). A significant decrease in fasting insulin level, HDL-C, and IGF-1 in RA patients were detected when compared with controls, (p<0.03). Results in figure 2 showed the characteristics of diabetic and non-diabetic RA patients, where a significant increase in FSG, TC, TAG, and LDL-C was indicated in diabetic RA patients rather than those without DM. However, a non-insignificant elevation in HbA1C, HOMA2-IR, VLDL, and leptin in diabetic RA patients when compared with those without DM. In addition, HDL-C and IGF-1 have significantly decreased in diabetic RA patients when compared with non-diabetic RA patients, (p<0.05). see also Table 1.  Table 2 showed a correlation between leptin with other biochemical parameters in RA patients with and without DM. Results indicated that there was a significant high positive correlation between leptin level with FSG (p<0.05), TC, TAG, and LDL-C in diabetic RA patients (p<0.03). Whilst, a significant negative correlation was detected between serum leptin with HDL-C and IGF-1 in diabetic and nondiabetic RA.

DISCUSSION
RA is a prolonged inflammatory disease attacked the articular, as well as, the extra-articular structures. It is usually characterized by synovial hyperplasia, inflammatory cell recruitment and the advanced stage of RA in cartilage and bone destruction. Chronic inflammation in RA` patients is associated with metabolic syndrome and atherosclerosis [12]. It also and contributes to insulin resistance [13], which leads to some other disorders such as hyperglycemia and dyslipidemia that indirectly associated to atherosclerosis and CVD. Thus, sequences mechanisms starting from inflammation, IR, or dyslipidemia could increase the burden of CVD risk in RA patients (14). Herein, we indicated higher levels of FSG, TC, and TAG, but lower HDL-C levels in RA patients when compared to controls. Interestingly, these levels were high in diabetic RA patients more than that of non-diabetic patients. High levels of lipids in RA`s was previously reported [15, 16, 17 and 18]. Studies in the field showed discrepancies in the lipid values, which could be attributed to the difference in the studied population, as well as, the level of the disease activity [19]. Non-diabetic RA patients showed an increased in HOMA-IR index when compared with controls, which was in agreement with others [20]. Results here suggest that the early secretion of insulin could damage the cell after glucose has stimulated. On the contrary, the HOMA-levels were higher in RA patients, which could be due to overproduction of insulin and HOMA-to maintained normal FSG levels. Therefore, the increase in the HOMA-index is not to improve the function of -cell, but merely to compensate the decrease of insulin sensitivity [21]. Results in Figure 1 showed non-significant low levels of serum insulin in RA patients, significant low levels of IGF-1 in diabetic RA patients compared with that of control or of non-diabetic RA. The mitogenic effects of insulin regulate the utilization of carbohydrates. IGF-1 as an important growth factor involve in cell growth, differentiation, and survival of cells [22]. Insulin along with IGF-1 contribute to the pathology of some inflammatory diseases including RA [23]. Therefore, the insulin resistance/IGF-1R may play a significant role in the development of the disease. IGF-1 contributes to the synthesis of cartilage and bone extracellular matrix proteins within RA synovium [24,25]. Previous studies showed that high IGF-1 is related with low-grade inflammation, and that production of IGF-1 is repressed in patients with RA [26,27]. Serum leptin level of RA patients was significantly more than that of controls (p<0.02), in agreement with others [28]. However, no significant differences between leptin of diabetic RA patients and that of non-diabetic RA patients. Leptin increases the production of the pro-inflammatory cytokines through activation of monocyte/macrophage cells [29]. Accordingly, we were able to suggest a mechanism of leptin and IGF-1 in diabetic RA based on the statistical results of this study, see figure 3. Diabetic RA patients had a significant high TC, TG and LDL, as well as hyperglycemia. However, insulin and IR were within normal (see figure 1), which could indicate high gluconeogenesis and lipogenesis in the liver and therefore, higher level in the blood and adipose tissue. This will stimulate more secretion of leptin, which will send a positive feedback to the hypothalamus to store the excess of glucose and lipids in the adipose tissue and muscle and leads to obesity RA patients. Diabetic RA patients had a significant low IGF-1 in the serum (figure 2), which may result from unsteady state of the cartilage due to the chronic inflammatory caused by RA and immune dysfunction. Low IGF-1 in the serum was negatively correlated with leptin (Table 2), thus high leptin level in the serum (figure 2). Another study on RA patients revealed on decrease in serum leptin concentration and a shift towards Th2 cytokine production. These alterations observed in RA patients and the experimental models suggest that leptin may play a role in inflammatory mechanisms of arthritis [29,30]. On the other hand, female appeared to suffer RA more than male of around 3:1 (figure 1b). Genetic factors and hormonal features are probably involved [31], however, sex variance in functional capacity for RA patients has been previously distinguished [32,33].

CONCLUSION
The present study found that leptin act as pro-inflammatory mediator in RA. Results also showed that females had higher chance to have RA than male. The negative correlation between leptin and IGF-1 level in diabetic and non-diabetic RA patients could play an important role in the metabolic disease or the disease activity of RA.