Therefore, this study aimed to investigate the effects of pro-inflammatory cytokines, TNF-α and IL-6, on ghrelin expression. To investigate the effects of pro-inflammatory cytokines TNF-α and IL-6 on ghrelin mRNA expression in AR42J cells. To investigate the effects of pro-inflammatory cytokines TNF-α and IL-6 on ghrelin protein expression in AR42J cells.
Growth Hormone Secretagogue and Its Receptor 4
Ghrelin 5
- Ghrelin and Des-acyl Ghrelin 5
- Ghrelin Structure and Gene Expression 8
- Distribution of Ghrelin 11
- Regulation of Ghrelin Production 11
The acylation process is important as it is essential for the binding of ghrelin to its receptor, GHS-R1a. The difference in the amino acid sequence of ghrelin gene between human and other species is shown in Table 2.1.
Physiological Functions of Ghrelin 13
- Growth Hormone 13
- Appetite Regulation 14
- Gastrointestinal Function 14
- Immunoregulation 15
- Cardiovascular Function 16
Adenosine 5' monophosphate activated protein kinase (AMPK) plays an important role in the orexigenic effect of ghrelin. This property of ghrelin and also its involvement in regulating energy expenditure is used to treat cachectic patients such as those with anorexia nervosa, bulimia nervosa and cancer cachexia (DeBoer et al., 2007). Furthermore, intravenous administration of ghrelin increases gastric acid secretion through activation of the vagus nerve (Masuda et al., 2000).
Thus, much research has been done on the effects of ghrelin on the immune system, and to date, the functional role of ghrelin in the immune system has not been well defined. Ghrelin inhibits the release of TNF-α, IL-6 and IL-1α through the GHS-R-specific pathway, alleviating the inflammatory responses (Sung et al., 2008). Ghrelin also improves experimentally induced arthritis by decreasing IL-6 synthesis (Sung et al., 2008).
This anti-inflammatory effect of ghrelin can be used as a treatment for harmful diseases, arthritis and aging. 2002) revealed the presence of ghrelin and GHS-R1a mRNA in the heart and aorta. In addition to ameliorating levels of inflammation, ghrelin action in a heart failure model improves left ventricular function and attenuates the development of cardiac cachexia (Smith et al., 2005).
Pro-inflammatory cytokines 17
Cytokine: Tumour Necrosis Factor Alpha (TNF-α) 18
It is a homotrimeric protein of 17 kDa that is produced by various types of cells such as mast cell, macrophage, endothelial cell, adipocyte, astrocyte, smooth muscle cell and fibroblast (Wacknik, Eikmeier, Simone, Wilcox, & Beitz, 2005). Of all the cells involved in the production of TNF-α, macrophages are the main producer of TNF-α. It is initially synthesized as a 26 kDa transmembrane monomer and then undergoes proteolytic cleavage by TNF-α converting enzyme (TACE) to form a 17 kDa protein (Cawthorn & Sethi, 2007).
There are two receptors identified as the TNF-α receptor (TNF-R), the 55 kDa TNF-R1 and the 75 kDa TNF-R2. TNF-R1 is mainly expressed in all somatic cells while TNF-R2 is commonly found in hematopoietic cells (Apostolaki, Armaka, Victoratos, & Kollias, 2010). Activation of TNF-R2 is associated with thymocyte proliferation, while the cytotoxic effect of TNF-α is mediated through both receptors (Soliven & Szuchet, 1995).
19 It is produced only after bacterial lipopolysaccharide stimulation and other bacterial products (Mukai et al., 2009). Overproduction of this cytokine would cause arthritis, inflammatory diseases and multiple sclerosis (Apostolaki et al., 2010).
Cytokine: Interleukin-6 (IL-6) 19
In the immune system, it functions to stimulate the acute phase response, acts as a chemoattractant for neutrophils, increases the adhesion molecule on endothelial cells for neutrophil migration, and stimulates phagocytosis (Morjaria, 2008). In the central nervous system, TNF-α stimulates corticotropin-releasing hormone production by the hypothalamus, suppresses appetite, and causes fever ( Endo, Masaki, Seike, & Yoshimatsu, 2007 ). It is released during infection, burns, trauma and neoplasia in response to bacterial endotoxin, IL-1 and TNF-β.
In the immune system, IL-6 stimulates the acute phase response, proliferation and differentiation of B cells and T cells and also stimulates the release of interferon from B cells and T cells. Similar to hematopoiesis, IL-6 stimulates the proliferation of murine pluripotential hematopoietic progenitors and together with IL-3 stimulate megakaryocyte maturation (Kishimoto, 2003). This role of IL-6 links it to Fanconi anemia, as reported by Rosselli, Sanceau, Wietzerbin, and Moustacchi (1992), where this disease is caused by a deficiency in IL-6 production.
Other roles of IL-6 include stimulating the release of anterior pituitary hormones such as prolactin and growth hormone, enhancing the development of osteoclasts, inducing insulin resistance, and repairing hepatocytes (Kishimoto, 2003).
Materials 21
Cell Culture Media 23
Stock Solutions 23
Coomassie Blue Staining Solution 50% Methanol 10% Acetic Acid 0.05 g Coomassie Blue Coomassie Blue Destaining Solution 50% Methanol.
Preparation of Glassware and Plasticware 25
Some solutions used in the project, especially solutions in RNA and protein work were autoclaved before use as well.
Cell Culture Techniques 26
- Maintenance of Cells in Culture 26
- Subculturing of Cells 26
- Preserving and Storing of Cells 27
- Thawing of Frozen Cells 28
- Treatment of AR42J Cells with Cytokines 28
After centrifugation, the supernatant was removed and the pellet was gently resuspended in 2 ml of complete growth medium. The supernatant was discarded and the cells were resuspended in 10% DMSO, 30% FBS and 60% growth medium. A cryo vial was removed from the liquid nitrogen container and placed in a 37°C water bath until the cells were thawed.
The supernatant was discarded and the cells were resuspended in 2 ml of growth medium and plated in the tissue culture flask. The next day, the cells were washed twice with PBS and cultured in the appropriate medium. Before treatment of cells with cytokine, the growth medium was first discarded and the cells were washed twice with PBS.
The cells were then pre-incubated for one hour with 4 ml of complete growth medium but with a reduced amount of heat-inactivated fetal bovine serum (HI-FBS) which was from 20% to 1% (v/v) HI-FBS. After the incubation, the growth medium was discarded and the cells were washed again twice with PBS.
RNA-Associated Techniques 29
- Isolation of Total Cellular RNA Using TRI-Reagent ® LS 29
- Electrophoresis of RNA on Denaturing
- DNase Treatment of RNA 30
- Real Time Reverse Transcriptase- Polymerase Chain
- Primers Used 32
1 µL sample of each RNA sample was added to the measuring tip of the spectrophotometer and the optical density was measured at 230 nm, 260 nm and 280 nm. Samples and RNA loading dye were prepared in a PCR tube and then incubated at 65°C for 15 minutes, cooled on ice before loading onto the gel. DNase treatment of RNA was performed by adding 2 µg RNA to 1 µl RQ1 buffer and 2 µl RNase-free DNase in a final volume of 10 µl.
After incubation, 1 µL of RQ1 DNase stop solution was added to the sample to stop the reaction of DNase. Real-Time RT-PCR was performed using the QIAGEN Quantifast SYBR Green RT-PCR Kit (Bio-Rad) according to the manufacturer's instructions. The samples were placed in the iCycler IQ5 Real-Time PCR Detection System (Bio-Rad) for quantitative PCR (Q-PCR).
32 The amount of target mRNA was normalized against a housekeeping gene, β-actin, which served as an internal control for determining the relative mRNA expression of the target gene. The nucleotide sequences of the primers used for real-time RT-PCR are shown in Table 3.9.
Western Blot Analysis 32
- Preparation of Protein Samples 32
- Bio-Rad Protein Assay 33
- Sodium Dodecyl Sulfate – Polyacrylamide Gel
- Coomassie Blue Staining of the SDS-PAGE Gel 35
- Western Blotting 36
- Probing of Blotted PVDF Transfer Membranes 36
- Chemiluminescent Detection of Membrane Bound
- Stripping of the Antibody Blotted Membrane 38
- Densitometry analysis of Western Blotting Results 38
First, the soluble gel was poured up to 2.5 cm from the upper edge of the inner glass plate. After the polymerization of the stacking gel, the comb was removed and the wells were washed with distilled water. During this period, four filter papers and PVDF membrane were cut to the size of the gel.
The membrane was assembled correctly on the block holder, which was previously wet with the distillate. ImmobilonTM Western detection reagent (Millipore, USA), which was prepared as shown in Table 3.6, was added to the membrane surface. After the detection of the antigen-antibody complex, the membrane-bound antibody was stripped off using stripping reagent (Table 3.6).
Then, the membrane was washed twice with physiological Tris-buffer by shaking also for 10 min each. The washing solution was then removed and the membrane was briefly immersed in methanol, air-dried and stored at -80˚C.
AR42J Cell Culture 39
Total Cellular RNA Extraction 41
Concentration and Purity of Total Cellular RNA
Integrity of Total Cellular RNA Extracted 42
Numbers labeled above the figure indicate the concentration of cytokine used for stimulation.
Real-Time RT-PCR 44
PCR Amplification of TNF-α and IL-6 Treated AR42J
The sigmoid curve of the PCR amplification diagram shows that there was an increase in the fluorescence of the sample.
Melting Curve Analysis for β-actin and Ghrelin 46
Ghrelin mRNA Expression in TNF-α and IL-6 Treated
Protein Analysis 48
Concentration of Total Cellular Protein Extracted 48
Coomassie Blue Staining of SDS-PAGE Gel 49
Some of the protein bands were very faint and could not be observed due to the low concentrations of sample added to each well.
Ghrelin Protein Expression in TNF-α and IL-6 Treated
Lai, Cheng, Ko, and Leung (2005) successfully detected the presence of ghrelin mRNA and its receptor in this cell line. This suggests that this cell line could be used as a model system to study ghrelin gene expression. Therefore, the levels of these three substances should be investigated together with the expression of the ghrelin gene in cells stimulated with TNF-α and IL-6.
Since the relationship between TNF-α and IL-6 with ghrelin gene expression had been established, more detailed studies could be performed. The exact form of ghrelin affected by the cytokines can be studied as each molecular form of ghrelin has different functions. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans.
Expression of ghrelin gene in peripheral blood mononuclear cells and plasma concentrations of ghrelin in patients with metabolic syndrome. For the quantification of relative mRNA expression of ghrelin using Real-Time RT-PCR, the delta delta Ct formula was used (Dharmaraj, 2011).
Comparison of Ghrelin mRNA and Protein Expression
AR42J Cells as the Model of Study 52
Studies of these cytokines have been performed on this particular cell line and these cytokines were shown to be involved in the progression of acute pancreatitis (Boole, 2006; Chan & Leung, 2009; Langley, 2008). Since TNF-α and IL-6 are involved in the progression of acute pancreatitis and ghrelin was found to exert anti-inflammatory effects on these cytokines, the cell line was used as a study model to investigate ghrelin gene expression in a condition similar to acute pancreatitis; whereby the levels of TNF-α and IL-6 are high in this disease. In this study, many factors are suggested to have caused the decrease in ghrelin expression by TNF-α and IL-6.
It is also suggested that both cytokines act indirectly in downregulating ghrelin gene expression. Since there are some ambiguities about insulin itself or because the degree of insulin resistance plays a role in influencing ghrelin gene expression, further studies need to be conducted. Serum levels of interleukin-6, C-reactive protein, vascular cell adhesion molecule 1 and monocyte chemotactic protein 1 in relation to insulin resistance and glucose intolerance - the Chennai Urban Rural Epidemiology Study (CURES).
Expression, regulation and biological effects of growth hormone (GH) and ghrelin in the immune system. Roles of plasma interleukin-6 and tumor necrosis factor and FFA and TG in the development of insulin resistance induced by high-fat diet.
