2.3. Cell preparationFL83B cells we

2.3. Cell preparation
FL83B cells were incubated in F12K medium without (basal) or
with 30 ng/mL TNF-a for 5 h to induce insulin resistance.
Then, the cells were transferred to F12K medium containing
5 mM glucose without (basal) or with insulin (5 lg/mL) and
with caffeic acid (12.5 lM) or cinnamic acid (12.5 lM) for 3 h
at 37 C.
2.4. Determination of glycogen
The accumulation of glycogen in FL83B cells was determined
using a glycogen assay kit (Biovision Corp., Mountain View,
CA, USA). Briefly, the cells were collected, washed twice with
ice-cold PBS, and homogenized in 200 lL deionized water. The
homogenates were boiled for 5 min to inactivate enzymes
and centrifuged at 13,000g for 5 min to remove pellet. Fifty
microlitres of supernatant of each sample were mixed with
2 lL of Hydrolysis Enzyme Mix in a 96-well plate, and the
plate was incubated at room temperature for 10 min. A 50-
lL aliquot of the reaction mix (46 lL Development Buffer,
2 lL Development Enzyme Mix, 2 lL OxiRed Probe) was added
to each well, and the plate was incubated at room temperature
for 30 min in the dark. Absorbance at 570 nm was measured
using a microplate reader (Sunrise, TECAN, Salzburg,
Austria). A standard glycogen curve (0, 0.4, 0.8, 1.2, 1.6, and
2.0 lg/well) was calculated by the above method.
2.5. Phosphoenolpyruvate carboxylase (PEPCK) activity
assay
PEPCK activity was determined using the spectrophotometric
assay developed by Greenway and Storey (2000) with a slight
modification. Cells that were pretreated with TNF-a were
homogenized in the extraction buffer [20 mM imidazole–
HCl, pH 7.2; 100 mM NaF; 5 mM ethylenediaminetetraacetic
acid (EDTA); 5 mM 2-mercaptoethanol; and 20 lM phenylmethanesulphonyl
fluoride (PMSF)] and centrifuged (13,000g,
20 min) to remove cell debris. Each 25-lL aliquot of the supernatant
was mixed with 125 lL 100 mM imidazole–HCl (pH 6.6),
125 lL 0.5 mM nicotinamide adenine dinucleotide (NADH),
125 lL 1 mM MnCl2, 50 lL 30 mM 2-mercaptoethanol, 150 lL
1.25 mM inositol-diphosphate, 125 lL 50 mM NaHCO3, 125 lL
2.5 U/mL malate dehydrogenase, and 150 lL 5 mM phosphoenol
pyruvate in series. The final reaction volume was 1 mL.
Enzyme activity was determined at 25 C for 5 min by measuring
the decrease in absorbance at 340 nm.

Hasil (Bahasa Indonesia) 1:

2.3. sel persiapanSel-sel FL83B yang diinkubasi di F12K media tanpa (basal) ataudengan 30 ng/mL TNF-a untuk 5 h untuk menginduksi resistensi insulin.Kemudian, sel-sel dipindahkan ke F12K medium yang mengandung5 mM glukosa tanpa (basal) atau dengan insulin (5 lg/mL) dandengan asam caffeic (12,5 lM) atau Asam sinamat (12,5 lM) selama 3 jamdi 37 C.2.4. penentuan glikogenAkumulasi glikogen dalam sel-sel FL83B ditentukanmenggunakan glikogen assay kit (Biovision Corp, Mountain View,CA, USA). Secara singkat, sel-sel dikumpulkan, dicuci dua kali denganPBS dingin, dan homogen di 200 lL dideionisasi air. Thehomogenates yang direbus selama 5 menit untuk menonaktifkan enzimdan disentrifugasi di 13.000 g selama 5 menit untuk menghapus pelet. Lima puluhmicrolitres dari supernatant dari setiap sampel dicampur dengan2 lL hidrolisis enzim Mix di piring 96-baik, danpiring diinkubasi pada suhu kamar selama 10 menit. 50-lL aliquot dari campuran reaksi (46 lL pengembangan Buffer,2 lL pengembangan enzim campuran, 2 lL OxiRed Probe) ditambahkanbaik untuk setiap, dan piring diinkubasi pada suhu kamaruntuk 30 menit dalam gelap. Absorbansi di 570 nm diukurmenggunakan microplate reader (Sunrise, TECAN, Salzburg,Austria). Kurva standar glikogen (0, 0.4, 0.8, 1.2, 1.6, danlg 2.0/sumur) dihitung dengan metode di atas.2.5. Phosphoenolpyruvate carboxylase (PEPCK) kegiatanujiPEPCK kegiatan ditentukan menggunakan Spektrofotometriassay yang dikembangkan oleh Greenway dan lantai (2000) dengan sedikitmodifikasi. Sel-sel yang telah pretreated dengan TNF-a yanghomogen dalam buffer ekstraksi [20 mM imidazol-HCl, pH 7.2; 100 mM NaF; etilenadiaminatetraasetat 5 mMAcid (EDTA); 5 mM 2-mercaptoethanol; dan 20 lM phenylmethanesulphonylfluorida (PMSF)] dan disentrifugasi (13, 000 g,20 min) untuk menghapus puing-puing sel. Masing-masing 25-lL aliquot dari supernatantdicampur dengan 125 lL 100 mM imidazol-HCl (pH 6.6),lL 125 0.5 mM nikotinamida adenina dinukleotida (NADH),125 lL 1 mM MnCl2, 50 lL 30 mM 2-mercaptoethanol, 150 lL1.25 mM inositol-diphosphate, 125 lL 50 mM NaHCO3, 125 lL2.5 U/mL malate dehidrogenase, dan 150 lL 5 mM phosphoenolpiruvat seri. Volume akhir reaksi adalah 1 mL.Aktivitas enzim ditentukan 25 C selama 5 menit dengan mengukurpenurunan absorbansi di 340 nm.