2. Materials and Methods
2.2. In vitro study
2.2.2. Human liver cells (C3A) cell culture
42 Chemilumescence was detected using Chemidoc-XRS imager. Blots were analyzed using quantity one 1D software from Bio-Rad. Beta actin was used as a housekeeping protein.
Table 2.6: List of primary and secondary anti-bodies, percentage of gel used and dilution used for Western blot analysis.
Differentiated C2C12 muscle cells % gel Dilution
Phosphorylated 5’adenosine monophosphate activated protein kinase (AMPK)
10 1:800
Glucose transporter 4 (GLUT4) 10 1:500
Phosphorylated Threonine kinase B (AKT ser 473) 10 1:1000
Total Threonine kinase B (AKT) 10 1:1000
Total 5’adenosine monophosphate activated protein kinase (AMPK ser 172)
10 1:800
Donkey anti-Rabbit 10 1:4000:
Goat NTI-mouse 10 1:4000
Beta actin 10 1:800
43 2.2.2.1. Cytotoxicity assay
Cells were treated with different concentrations of A. phylicoides and subjected to an MTT assay as described in section 2.2.1.4. For assay purposes, the different concentrations (0.01, 0.1, 1 and 2 mg/ml) of A. phylicoides were dissolved in DMEM without phenol red and pyruvate containing 8 mM glucose and cells were incubated for 3 hrs under standard culture conditions.
Figure 2.3: Experimental outline for C3A cell line.
PA - palmitic acid, Ctrl - control, Met - metformin, AP - A. phylicoides.
2.2.2.2. Induction of insulin resistance and treatment with extracts
Treatments for inducing insulin resistance were prepared and dissolved in DMEM as described in section 2.2.1.3. The cells were incubated under standard cell culture conditions for 16 hrs in order to induce insulin resistance. After 16 hrs, treatments were prepared as described above (see section 2.2.1.3) in DMEM containing 8 mM glucose.
The cells were treated with 300 µl of the treatments and incubated under standard culture conditions for 3 hours and insulin was added to yield a 2 µM final concentration 30 minutes before the end of the incubation time (see Figure 2.3)
44 Glucose uptake assay using 2-deoxy-[3H]-D-glucose uptake method. Treatments were removed from cells and washed with 250 µl of ice cold DPBS, thereafter, they were treated as described in section 2.2.1.5. Treatments were prepared in glucose free DMEM containing 0.5 µCi/ml 2-deoxy-[3H]-D-glucose with or without 2 µM insulin.
2.2.2.3. Protein determination-Bradford assay
Total protein concentration was determined using the Bradford assay. Standards and actual protein concentrations in a lysate were determined as described in section 2.2.1.6.
2.2.3. 3T3-L1 adipocytes as a model of insulin resistance
Pre-adipocytes 3T3-L1 cell line (ATTC Cat No. CL-173) were grown and maintained in DMEM supplemented with 10 %new born calf serum (NCS). After reaching a 70 % confluency, the cells were sub-cultured and seeded into 96 well plates (TC treated flat bottom, Eppendorf, Hamburg, Germany tissue and Corning® CellBIND®, sigma Aldrich, St Louis, USA) for MTT assays and Oil Red O stain (4000 cells/well, 20 000 cells/ml), respectively, and 75 cm2 flask maintenance (splitting ratio of 1:6).
2.2.3.1. Adipocyte differentiation
After 3T3-L1 fibroblasts had reached a confluency of 100 %, the growth medium was replaced with differentiating medium (DMEM containing 10 % FBS, 1 µM Dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 1 µg/ml insulin). Cells were refreshed with freshly prepared differentiating medium every 24 hrs for 3 days.
Thereafter, the differentiating medium was replaced by adipocyte maintenance medium (refer to table 2.1). The cells were refreshed with freshly prepared adipocytes maintenance medium every 24 hrs for two days. After 2 days, adipocyte maintenance medium was replaced by normal growth medium (DMEM supplemented with 10 % FBS), which was changed daily for 3 days. Full differentiation was confirmed by observing fully formed lipid droplets in each well under an inverted light microscope (Olympus CKX41, Olympus, Tokyo, Japan).
45 Figure 2.4: experimental outline for 3T3-L1 cell line.
PA - palmitic acid, Ctrl - control, Met - metformin, AP - A. phylicoides
2.2.3.2. Induction of insulin resistance and treatment
Insulin resistance was induced as described in section 2.2.1.3. Cells were serum starved and treated as described above (2.2.1.3). Treatments were dissolved in DMEM without phenol red containing 25 mM glucose, 2 % BSA, 1 % ethanol, with and without 1µM insulin and with and without 750 µM palmitic acid. Treated cells were incubated for 24hrs under standard culture conditions. Treatment blanks, which are leftovers of all the different treatments that were prepared, were added into a clear plate (polysterine TPP® 96 U-base, Zellkultur und labortechnologies, Switzerland) and they were incubated under the same conditions as the cells.
2.2.3.3. Glucose uptake assay using mutarotase-GOD method
LabAssayTM glucose kit was used to conduct uptake assays. The enzyme mutarotase converts α-D-glucose to β-D-glucose. The enzyme glucose oxidase oxidizes β-D- glucose yielding hydrogen peroxide. Hydrogen peroxidase reacts with phenol and 4- aminoantipyrine in the presence of hydrogen peroxide, yielding a red pigment.
Glucose can then be assayed by measuring the absorbance of the red pigment.
46 After 24 hrs, treatments were removed from the wells and transferred into a clear plate (polystyrene TPP® 96 U-base, Zellkultur und labortechnologies, Switzerland). A standard curve was prepared according to manufacturer’s recommendations yielding 6 different concentrations (50, 100, 200, 300, 400 and 500 mg/dl). Treatments, treatment blanks and standards (2 µl) were added to the assay plate, followed by 300 µl of the chromogen reagent. The plate was incubated at 37 ºC for 5 min and the absorbance was measured at a wavelength of 490 nm using BioTek® ELX800 plate reader (BioTek, Vermont, USA).
2.2.3.4. Lipid quantification using Oil Red O staining
The Oil Red O (ORO) stain is a fat-soluble dye with a maximum absorbance at 518 nm. The stain can be used to stain triglycerides, frozen lipid sections and freshly prepared samples of mammalian cells. After media was removed from the cells, the cells were washed with 100 µl of DPBS. In order to fix the cells, DPBS was removed and replaced by 50 µl of neutral buffered formalin (10 % v/v), followed by a 15 min incubation at room temperature. Thereafter, neutral buffered formalin was removed and cells were washed twice with 100 µl of DPBS. After washing, 50 µl of 0.7 % (v/v) ORO was added and incubated at room temperature for 30 min. After incubation, the ORO staining solution was removed and cells were washed 3 times with ddH2O.
Double distilled water was aspirated, followed by the addition of 50 µl of isopropanol into each well. The plate was gently agitated by gentle tapping the plate on the side in order to aid the extraction of the ORO stain. The extracted ORO stain (50 µl) was transferred to a clear assay plate and the absorbance was measured at 490 nm in a BioTek® ELX800 plate reader. After extracting the ORO stain, all wells were washed with 50 µl of 70 % ethanol. The ethanol was aspirated and it was followed by the addition of 80 µl of 0.5 % crystal violet (CV) stain into each well. Stained cells were incubated at room temperature for 5min. After 5 min, the staining solution was aspirated and cells were washed 3 times with 80 µl of DPBS. The DPBS was removed by aspiration and this was followed by the addition of 70 % ethanol (50 µl). The stain was extracted by gently tapping the plate on the side. Thereafter, 80 µl of the extracted stain was transferred into an assay plate and the absorbance of the plate was measured at 570 nm in a BioTek® ELX800 plate reader. Results for both stains were expressed as percentage of control. The CV stain is a measure of viability, therefore values obtained from the absorbance were used to normalize the ORO stain.
47 2.2.3.5. Cytotoxity assay-MTT assay
Mature 3T3-L1 adipocytes were subjected to an MTT assay to assess viability after they were treated with different concentration of A. phylicoides for 3 hrs without the addition of insulin. Treatments were prepared in DMEM containing 25 mM glucose and cells were treated as described in section 2.2.1.4. The absorbance was read at 570 nm in a BioTek® ELX800 plate reader. Results were calculated and expressed as percentage of control.