Palmitate-Induced S1P Signaling Pathway Attenuates by Cchicoric Acid in Human Peripheral Blood Mononuclear Cells

Objective: Sphingosine 1-phosphate (S1P) signaling pathway is involved in the pathogenesis of type 2 diabetes (T2D). So, targeting S1P signaling pathway could be considered as potential therapeutic target for T2D. The aim of this study was to investigate the effects of palmitate and chicoric acid (CA) on S1P signaling pathway in peripheral blood mononuclear cells (PBMCs) from newly diagnosed patients with T2D and healthy subjects. Materials and Methods: 20 newly diagnosed patients with T2D and 20 healthy subjects, aged 40-60 years, were enrolled in the study. PBMCs were isolated and treated with palmitate and CA. Then, Sphingosine kinase 1 (SPHK1) and Sphingosine 1-phosphate receptor 1 (S1PR1) genes expression were evaluated by real-time PCR and S1PR1 protein levels were quantied using ELISA. Results: Palmitate signicantly increased SPHK1 and S1PR1 genes expression and S1PR1 protein levels in PBMCs of both patients and healthy subjects. However, CA ameliorates palmitate-increased SPHK1 and S1PR1 genes expression and S1PR1 levels in these cells. Furthermore, a signicant positive correlation between SPHK1 and S1PR1 genes expression with the S1PR1 protein levels was observed. Conclusions: These data indicate that CA could be considered as a novel S1P signaling pathway inhibitor through down regulation of SPHK1 and S1PR1.


Introduction
Type 2 diabetes (T2D) is an endocrine disorder worldwide. The prevalence of T2D has increased markedly around the world and constitutes one of the major public healthcare problem [1]. Peripheral blood mononuclear cells (PBMCs) consist of lymphocytes and monocytes. These cells are helpful in revealing the complex nature of T2D [2]. Increasing evidence has implicated that in ammation is essential contributors to T2D. The signaling pathways involved in the production of in ammatory mediators play crucial role in the induction of in ammation in tissues, followed by progression of diseases [3]. Now, understanding the molecular mechanism involved in the in ammation has attracted interest of researchers. Undoubtedly, knowing the molecular details of the in ammatory pathways lead to identify and develop appropriate treatment.
Sphingosine signaling pathway is one of the newly pathways involved in the tissue in ammation and plays an important role in the activation of tissue macrophages and induction of in ammation. Sphingosine 1 phosphate (S1P) is a bioactive lipid with structural role in plasma membranes. In addition to its structural function, it has been demonstrated that S1P has a critical role in many cellular processes such as in ammation and immunity, cell survival and proliferation, cell migration, metastases and etc [4].
The role of S1P in modulating the relationship between monocytes and endothelium in diabetic and healthy mice has been evaluated and it has been shown that S1P in diabetic mice increases the adhesion of monocytes to the endothelial wall [5]. Today, S1P recognized as a vital regulator of many pathological processes including diabetes, cancer, atherosclerosis and osteoporosis [6]. S1P is generated from sphingosine by two kind of sphingosine kinase (SPHK) including SPHK1 and SPHK2. SPHK1 present mainly in the cytosol. However, SPHK2 localized in the several intracellular compartments [6]. It has been found that SPHK1 gene expression increases in high-fat diet [7]. Various studies clearly showed that inhibition of SPHK1 is a therapeutic target for in ammatory diseases [6]. S1P is secreted out of cells and regulates cellular processes through binding to its receptors. Five speci c cell surface G protein-coupled receptors, named S1PR1-5, have been recognized for S1P. Binding of S1P to S1PR1 activates SIPR1. Activated S1PR1 plays an important role in the PBMCs tra cking and induces in ammation. Therefore, the regulation of S1P signaling pathway could be a good therapeutic strategy for T2D [6].
Palmitate has been proposed to induce activation of in ammatory cascade in PBMCs [2]. Palmitate increases CCl4 levels in monocytes and macrophages by activating NF-κB and PI3K signaling pathway [8]. CCl4 increases the production of in ammatory cytokines such as IL-6. Increased CCl4 and in ammatory cytokines production leads to metabolic in ammation. Metabolic in ammation plays a vital role in many diseases such as diabetes, atherosclerosis, cancer and etc. There are several reports that the circulating level of free fatty acids (FFAs), especially palmitate increases during T2D [8,9].
Indeed, palmitate is a major risk factor for the development of T2D. Recently, it has been reported that palmitate increases S1P through up regulation of SPHK1 [10].
Chicoric acid (CA) is a phenolic compound with anti-in ammatory, anti-oxidants and anti-hyperglycemic effects [11]. CA found in several widely used herbal medicinal plants including Cichorium intybusL., Echinacea purpurea and Orthosiphon stamineus B. Recently, the bene cial effects of CA on T2D has been demonstrated [12,13]. However, the exact molecular mechanism underlying the effect of CA on T2D remains unexplored.
In this study, we aimed to evaluate the effects of palmitate and CA on SPHK1 and S1PR1 genes expression, as well as protein levels of S1PR1 in PBMCs from newly diagnosed patients with T2D and healthy subjects.

Participants and study design
A total of 40 subjects (20 newly diagnosed patients with T2D and 20 healthy subjects) with an age between 40 and 60 years old were recruited in this study. Diagnosis of T2D was performed based on American Diabetes Association (ADA) criteria [14]. Subjects with fasting blood sugar (FBS) ≥ 126 mg/dl were considered to be diabetic and subjects with FBS < 100 mg/dl were grouped as healthy subjects. It should be noted that all patients with T2D had a rst-time diagnosis of T2D at time of sampling and do not took any anti-diabetic medication. Patients with acute and chronic in ammatory diseases, cardiovascular disease, uncontrolled hypertension, T1DM and gestational diabetes were excluded from the study. Also, none of the studied subjects have smoking and alcohol consumption. All participants gave written informed consent before the study. The protocol of present study approved by Ethics Committee of Shahrekord University of Medical Sciences (code: IR.SKUMS.REC.1399.115). After an overnight fast, anthropometric measurements including age, height, weight, BMI (weight/height 2 ), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded. Then, venous blood samples were collected for biochemical parameters analysis and PBMCs isolation. FBS, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine were measured by colorimetric methods (Pars Azmoon, Tehran, Iran) on a BIOLIS24i Premium autoanalyzer (Tokyo Boeki Machinery Ltd., Japan). Insulin was detected by enzyme-linked immunosorbent assay (ELISA) kit (Monobind Inc., CA, USA). Homeostasis model assessment of insulin resistance (HOMA-IR) was measured using the formula: fasting insulin (µIU/ml) × FBS (mg/dl) / 405. Hemoglobin A1C (HbA1C) was measured with HPLC method using Tosoh G8 instrument (South San Francisco, CA).
Fold change in SPHK1 and S1PR1 genes expression were calculated using the 2 −ΔΔCt method.

Determination of S1PR1 levels
Concentrations of S1PR1 in the PBMCs were measured by an ELISA kit with a sensitivity of 0.057 ng/ml, according to the protocol provided by the manufacturer (catalog number: MBS2021994).

Statistical analysis
Data are reported as mean ± standard deviations (SD). All data were analyzed using SPSS software (version 16.0, Chicago, IL, USA) and GraphPad Prism software (version 7, San Diego, CA, USA).
Comparisons between data were made by Two-way analysis of variance (Two-way ANOVA). Pearson's correlation test was used to examine the relationship between the variables. A value of p < 0.05 was considered signi cant.

Anthropometric and Biochemical ndings
Anthropometric characteristics of the studied subjects are illustrated in Table 1. Age, height, weight, BMI, SBP and DBP of the subjects were not signi cantly differences between two groups. As shown in Table 2 the patients with T2D had signi cantly higher FBS, insulin, HOMA-IR, HbA1C and TG as compared to healthy subjects (p < 0.001). However, other biochemical parameters including TC, LDL-C, HDL-C, urea, creatinine, AST and ALT were not signi cantly differences between the two studied groups.   3.2. Effects of palmitate and CA on SPHK1 and S1PR1 genes expression To elucidate the effects of palmitate and CA on S1P signaling pathway, we investigated SPHK1 and S1PR1 genes expression in response to palmitate and CA. As shown in Fig. 1 Our ndings revealed that treatment of PBMCs with palmitate increases S1PR1 gene expression in nondiabetic subjects and patients with T2D approximately 1.93 and 1.80 fold, respectively (p < 0.001). Unlike palmitate, treatment of PBMCs with CA caused a decrease in S1PR1 gene expression in healthy volunteers (0.72 fold) and patients with T2D (0.68 fold) (p < 0.001). Moreover, we found that in healthy subjects increased S1PR1 gene expression induced by palmitate signi cantly has been restored via CA (from 1.93 fold to 1.46) (p < 0.05). In the current study, we did not detect signi cant difference in S1PR1 gene expression in untreated PBMCs from healthy and diabetic groups (Fig. 2).

Effects of palmitate and CA on S1PR1 protein levels
We also evaluated the effects of palmitate and CA on S1PR1 protein levels. Fig. 3 showed that S1PR1 protein levels is dramatically increased in PBMCs exposed to palmitate as compared to untreated cells in healthy volunteers and patients with T2D (p < 0.001). However, CA signi cantly decreased S1PR1 protein levels in PBMCs of healthy subjects (p < 0.001). Furthermore, our study revealed that CA signi cantly ameliorates palmitate-increased S1PR1 protein levels in PBMCs from healthy subjects (p < 0.05).
3.4. Association between SPHK1 and S1PR1 genes expression with S1PR1 protein levels Analysis of the relationships of SPHK1 and S1PR1 genes expression with S1PR1 protein levels showed that S1PR1 protein levels had a signi cant positive association with SPHK1 gene expression in palmitatetreated PBMCs from patients with T2D (r = 0.498, p = 0.004) as well as S1PR1 gene expression in chicoric acid-treated PBMCs from healthy subjects (r = 0.753, p = 0.028).

Discussion
It is well known that in ammation impairs cellular function and leads to several disease such as T2D, IR, atherosclerosis, cancer and etc [16]. Increased FFAs stimulate in ammation-related pathways such as S1P signaling pathway [10]. Based on the data from previous studies, CA exerts the anti-in ammatory effects. However, the exact molecular mechanism in which CA regulates anti-in ammatory pathways has not been recognized [2,11]. Here, we investigated the effects of CA on S1P signaling pathway.
We rst showed that SPHK1 gene expression in untreated cells of T2D was signi cantly lower as compared to healthy subjects. However, the gene expression and protein levels of S1PR1 in untreated PBMCs of patients with T2D is not signi cantly difference from healthy subjects. Consistent with our results, it was showed that HFD-induced muscle IR is attenuated in SPHK1 transgenic mice. SPHK1 prevents ceramide accumulation through promoting its metabolism into S1P and, therefore, protects against the development of IR. Accumulation of ceramide impairs the insulin signaling pathway through activating JNK [17]. In another study, it was demonstrated that injection of an adenoviral vector containing the human SphK1 cDNA in diabetic mice improves glucose tolerance and decreases total cholesterol, triglycerides, and LDL [18]. Also, it was showed that administration of the S1P analogue FTY720 in HFD animals attenuates IR [19]. However, another studies reported the opposite results. It was demonstrated that SPHK1 gene expression in subcutaneous adipose tissue from ob/ob mice was elevated [20]. Also, Blachnio-Zabielska and et al, found that S1P levels was increased in subcutaneous adipose tissue of obese subjects as compared to lean subjects [21].
We examined the effects of palmitate and CA on S1P signaling pathway in PBMCs. Our study indicates for the rst time that SPHK1 and S1PR1 genes expression and the protein levels of S1PR1 are signi cantly increased in response to palmitate. In fact, palmitate, which has a crucial role in the development of IR stimulates S1P signaling via increasing SPHK1 and S1PR1. This is in accordance with the fact that palmitate is necessary for the de novo synthesis of sphingosine. Also, palmitate increases the S1P formation by inducing SPHK1 gene expression [22]. In this regard, it was shown that palmitate increases SPHK1 expression and activity in C2C12 myotubes [4]. This may suggest that SPHK1 and S1PR1 mediate pathological effects of palmitate. Elevated S1P in response to upregulation of SPHK1 not only plays a role in the impairment of insulin pathway but also increases in ammation [7]. In a study by Wang et al. on adipocytes of HFD-mice it was demonstrated that SPHK1 de ciency, which leads to reduced S1P, increases anti-in ammatory cytokines and decreases proin ammatory cytokines [23]. On the other hand, based on our previous results that palmitate induces in ammatory cytokine production, we can suggest that palmitate increases in ammation through up regulation of S1P signaling. In accordance with our result, Jin et al. reported that palmitate and LPS synergistically increases S1P through stimulation of SPHK1 expression. Also, their result reveal increased proin ammatory cytokine expression in response to palmitate and LPS [10].
To evaluate the molecular mechanism underlying the effect of CA on diabetes, we investigated S1P pathway proteins including SPHK1 and S1PR1. Our study provides the rst evidence that CA decreases the SPHK1 and S1PR1 genes expression and S1PR1 protein levels. Binding of S1P to its receptors induces in ammatory cytokine production. Increased in ammation is contribute to development of IR [23]. These results were accompanied by previous studies that implicated resveratrol, as a poly phenol, inhibits SPHK1 activity in HEK 293 cells [24]. Here, we provide novel evidence that CA could be as a new SPHK1 inhibitor. Down regulation of SPHK1 gene expression maybe due to changes in SPHK1 protein turnover. Like other SK1 inhibitors, CA probably stimulates the SPHK1 ubiquitin-proteasomal degradation or lysosomal-cathepsin B catalysed proteolysis or changes in activity of gene promoter [25,26]. Together, anti-in ammatory effects of CA might be a consequence of the inhibition of S1P signaling pathway. Therefore, inhibition of S1P signaling pathway by CA could be considered as a novel therapeutic target for the improvement of diabetes [6].
Another nding in our study was positive correlation between SPHK1 and S1PR1 genes expression with S1PR1 protein levels. These data suggest that SPHK1 and S1PR1 are essential in S1P signaling pathway [6].

Conclusion
In conclusion, our data identify CA as an important regulator of S1P signaling pathway by decreasing SPHK1 and S1PR1 expression. Since it is known that activated S1P signaling pathway exacerbates T2D, thereby targeting S1P signaling pathway by CA may improve T2D. However, more works are needed to evaluate the e cacy of CA in the management of T2D.

Declarations
Compliance with Ethical Standards Figure 1 Effects of palmitate and CA on SPHK1 gene expression in PBMCs of healthy subjects and patients with T2D. Left side of the graph shows mRNA expression (2−ΔCt) and right side of the graph shows mRNA fold change (2−ΔΔCt). The non-dotted and dotted lines show fold change for healthy subjects and patients with T2D, respectively. Data are shown as mean ± SD. *p < 0.05 (between group), +p < 0.05 compare to untreated cells (within group), #p < 0.05 compare to palmitate treated cells (within group).

Figure 2
Effects of palmitate and CA on S1PR1 gene expression in PBMCs of healthy subjects and patients with T2D. Left side of the graph shows mRNA expression (2−ΔCt) and right side of the graph shows mRNA fold change (2−ΔΔCt). The non-dotted and dotted lines show fold change for healthy subjects and patients with T2D, respectively. Data are shown as mean ± SD. +p < 0.05 compare to untreated cells (within group), #p < 0.05 compare to palmitate treated cells (within group).