Potensi Whey Kefir Susu Kambing (Whey-Ksk) Sebagai Anti Obesitas Melalui Penghambatan Sintesis Trigliserida, Kolesterol Dan Phosphoenolpyruvate Carboxykinase (Pepck) Pada Model Sel Adiposit 3T3-L1

Hati, Dian Laksamana and Prof. Dr. dr. Sri Andarini and Prof. Dian Handayani S.K.M (2022) Potensi Whey Kefir Susu Kambing (Whey-Ksk) Sebagai Anti Obesitas Melalui Penghambatan Sintesis Trigliserida, Kolesterol Dan Phosphoenolpyruvate Carboxykinase (Pepck) Pada Model Sel Adiposit 3T3-L1. Magister thesis, Universitas Brawijaya.

Abstract

Obesitas merupakan masalah kesehatan kronik dikenal sebagai new world syndrom epidemi global. Obesitas didefinisikan kelebihan energi dalam bentuk lemak tubuh, berhubungan dengan sindrom metabolik dislipidemia yaitu perubahan konsentrasi triasilgliserida, kolesterol dan aktivitas enzim PEPCK. Pemecahan masalah obesitas dapat dilakukan melalui pendekatan adipogenesis sel model preadiposit 3T3-L1 yang berdiferensiasi menjadi sel adiposit 3T3-L1. Proses diferensiasi sel preadiposit diinduksi dengan DMI mengandung dexamethasone, IBMX dan insulin, sehingga sel menjadi sel adiposit, yang mampu mengkonversi glukosa dan protein dari medium menjadi TG, kolesterol yang disimpan didalam droplet lipid. Perubahan jumlah lipid intraselular diikuti dengan aktivitas PEPCK pada adiposit yang dapat digunakan sebagai indikator obesitas. Penggunaan obat untuk menurunkan obesitas mempunyai akibat buruk, oleh karena itu dikembang komponen pangan yang dapat menurunkan obesitas. Whey-KSK merupakan produk fermentasi susu mengandung komponen bioaktif peptida, asam organik, eksopolisakarida, enzim β-galatosidase dan α-glukan. Komponen tersebut berfungsi dalam meningkatkan kesehatan. Tujuan penelitian adalah menganalisis pemberian whey-KSK terhadap adipogenesis sel adiposit 3T3-L1. Tujuan khusus adalah (1) Menentukan dosis optimum pemberian whey-KSK terhadap penghambatan sintesis TG, TC dan PEPCK pada sel adiposit 3T3-L1. (2) Menganalisis pemberian dosis whey-KSK yang berbeda terhadap perbedaan kadar TG, TC dan PEPCK pada sel adiposit 3T3-L1, (3) Menganalisis hubungan pemberian dosisi whey-KSK yang berbeda terhadap penurunan kadar TG, TC dan PEPCK pada sel adiposit 3T3-L1. Metode penelitian adalah true eksperimental rancangan acak lengkap. Materi ekperimen adalah sel model adiposit 3T3-L1 (Mouse Mus musculus embryonic fibroblast, ditumbuhkan pada DMEM yang mengandung gk\lulosa, penicillin−streptomycin, FBS, diinduksi dengan DMI (Biovision K579-100), setiap sampel mengandung sel adiposit 105 sel/sumur, 24 piring kultur. Kelompok KN (Kontrol - ), KP (Kontrol +), kelompok yang diberi whey-KSK adalah P1, P2, P3, P4 berturut-turut 25, 50, 75 dan P4 100g/mL). Variabel diukur adalah TG, TC dan aktivitas PEPCK yang masing-masing berdasarkan uji kuantitatif colorimetric assay dengan kit Seri Biovision TG (K622-100), TC (K578- 100), aktivitas PEPCK (K603-100), pewarnaan lipid (Oil Red O K580-100), sel lisis (NP- 40 detergent surfac-Amp). Hasil uji menunjukkan TG sel adiposit 3T3-L1 pada berturut-turut pada kelompok KN, KP, P1, P2, P3 dan P4 adalah 1.19 0,03 nmol/L, 2,91 0,03 nmol/L, 1,88 0,08 nmol/L, 1,79 0,05 nmol/L, 1,56 0,06 nmol/L, 1,30±0,05 nmol/μL. Berdasarkan nilai TG menunjukkan bahwa pemberian whey-KSK semakin tinggi mengakibatkan nilai TG semakin rendah. Hasil regresi pemberian dosis whey-KSK berbeda pada sel adiposit berpengaruh secara negatif dan signifikan terhadap TG. Berdasarkan uji statistika (t- hitung 8,89 > t-tabel 2,10), pada (α <5%), dengan koefisien regresi -0,01, setiap peningkatan satu angka variabel dosis whey-KSK dapat menurunkan TG sebesar 0,01 secara signifikan. Koefisien determinasi R square 0,82, menunjukkan besarnya kontribusi pengaruh variabel dosis Whey-KSK terhadap variabel TG sebesar 82% dan pengaruh variabel bebas lainnya sebesar 18%. Rata-rata kadar TG tertinggi pada perlakuan KP sebesar 2,91±0,03 mnol/L, dan rata-rata angka TG terrendah pada perlakuan KN sebesar 1,30±0,05 mmol/L. Pemberian whey-KSK 25–100 μg/mL dapat menurunkan 35,39–55,32% TG sel adiposit 3T3-L1. Hasil uji kandungan TC sel adiposit 3T3-L1 pada kelompok perlakuan KN (1,11  0,01μg/ L), KP (4,99 0,09 μg/L), P1(3,47 0,09 μg /L), P2 (, 2,99 0,04 μg /L), P3 (2,11 0,09 μg/L), dan P4 (1,94±0,09 μg/μL). Berdasarkan nilai TC menunjukkan bahwa pemberian whey-KSK semakin tinggi mengakibatkan nilai TC rendah. Hasil regresi pemberian dosis Whey-KSK yang berbeda berpengaruh secara negatif dan signifikan terhadap TC. Hasil uji statistika ( t-hitung 13,956 < t-tabel 2,101), pada (α<0,05). Koefisien regresi -0,03 berarti setiap peningkatan satu angka variabel dosis whey-KSK dapat menurunkan variabel TC sebesar 0,03 angka secara signifikan. Koefisien determinasi dengan R square sebesar 0,92, menunjukkan besarnya kontribusi pengaruh variabel dosis Whey-KSK terhadap variabel TC sebesar 92%, pengaruh variabel bebas lainnya sebesar 8,5%. Rata-rata ninai TC tertinggi pada KP sebesar 4,99 0,09 μg/L, dan terrendah pada KN sebesar 1,11 0,01μg/ L Pemberian whey-KSK 25-100 μg/mL dapat menurunkan TC (10,42-61,12%), Aktivitas PEPCK sel adiposit 3T3-L1 menunjukkan KN (0,03  0,01mU/μL), KP (0,44 0,02mU/μL), P1 (0,32 0,00mU/μL), P2 (0,29 0,00 mU/μL), P3 (0,19 0,00 mU/μL), dan P4 (0,07±0,00 mU/μL). Aktivitas spesifik PEPCK adalah KN (0,08  0,00 mU/mg, KP(0,89 0,13 mU/mg), P1 (0,59 0,00 mU/mg), P2 (0,50 0,00 mU/mg), P3 (0,39 0,00 mU/mg), P4 (0,32±0,14 μg/mg). Berdasarkan aktivitas PEPCK menunjukkan bahwa pemberian whey-KSK semakin tinggi mengakibatkan nilai aktivitas PEPCK semakin rendah, sehingga pemberian whey-KSK dapat menurukan aktivitas PEPCK sel adiposit dibanding dengan KP. Pemberian whey-KSK menunjukkan penghambatan aktivitas PEPCK (56,81–84,09%) dan penghambatan aktivitas spesifik PEPCK (56,17- 64,04%) dibanding kontrol positif. Kontribusi variabel dosis Whey-KSK terhadap variabel aktivitas PEPCK sebesar 72,2%, dan pengaruh variabel lain 27,8%. Kontribusi variabel dosis whey-KSK terhadap variabel aktivitas spesifik PEPCK sebesar 43,0% dan pengaruh variabel lainnya sebesar 57,0%. Kesimpulan pemberian whey-KSK pada sel model obesitas dapat menghambat sisntesis TG, TC dan aktivitas enzim PEPCK yang merupakan indikator obesitas. Whey- KSK potinsial sebagai alternatif komponen diet anti obesitas.

English Abstract

Obesity becomes a chronic health problem alleged as the new world syndrome of a global epidemic. Obesity is defined as an accumulation excess of energy in the form of body fat, associated with metabolic syndrome such as dyslipidemia, indicating an alteration in triacylglyceride concentrations, cholesterol and PEPCK enzyme activity. Problem related to obesity is overcome through the differentiation approach of 3T3-L1 preadipocyte model cells into 3T3-L1 adipocytes. The process of cell differentiation was induced by DMI consisting of dexamethasone, 3-isobutyl-1-methylxanthine (IBMX) and insulin, thereby maturing the adipocyte cells containing lipid droplets. Lipid droplet was visualized with Oil red O (ORO) staining. Additionally, preadipocytes converted glucose and protein generated from the medium into TG, cholesterol and stored into lipid droplets. Changes in the amount of intracellular lipids and PEPCK activity in adipocytes were utilized as indicators of obesity. The administration of drugs to reduce obesity, however, presents inverse consequences, thereby encouraging the development of food components reducing obesity. Whey-KSK is a fermented milk product containing bioactive components which play a singular or synergistic role in improving health, including: peptides, organic acids, exopolysaccharides, -galatosidase enzymes and α-glucans. This research aims to analyze the administration of whey-KSK on adipogenesis of 3T3-L1 adipocyte cells. Particularly, this research aims (1) to determine the dose of whey-KSK exposure to inhibition of TG, TC and PEPCK synthesis in 3T3-L1 adipocyte model cells; (2) to analyze the differences in the levels of TG, TC and PEPCK between treatment groups on the administration of different doses of whey-KSK; and (3) to analyze the relationship between decreased levels of TG, TC and PEPCK between treatment groups on the administration of different doses of whey-KSK. This study applied a true experimental study method, designed with a completely randomized design. Experimental material included 3T3-L1 adipocyte model cells (Mouse Mus musculus embryonic fibroblast), cultured on DMEN with glucose, penicillin−streptomycin, FBS, induced with DMI differentiation kit (Biovision K579- 100), and each sample contained adipocyte model cells of 105 cells/well on 24 culture plates. The treatment groups included KN (Negative Control), KP (Positive Control), treated with different whey-KSK doses (P1: 25 g/ml, P2: 50 g/ml, P3: 75 g/ml and P4: 100 g/ml. The measured variables included TG, TC and PEPCK activity, each based on a quantitative colorimetric assay test with the Biovision TG Series kit (K622-100 ), TC (K578-100), PEPCK activity (K603-100), Lipid staining (Oil Red O K580-100), cell lysis (NP-40 detergent surfac-Amp). The test results of TG content in 3T3-L1 adipocyte cells in the treatment groups (KN, KP, P1, P2, P3 and P4) were 1.190.03 nmol/L, 2.910.03 nmol/L, 1.880.08 nmol/L, 1.790.05 nmol/L, 1.560.06 nmol/L, and 1.30±0.05 g/μL respectively. TG value indicated that higher value of whey-KSK led to lower TG value.The regression results of whey-KSK at different doses to TG value exhibited that the t-count value was greater than the t-table (8.89 > 2.10), and the p-value was smaller (α < 5%), with a coefficient of regression of -0.01, denoting that every one increase in the variable number of whey-KSK dose significantly reduced TG value by 0.01. The coefficient of determination R square was 0.82, indicating the contribution of whey-KSK dose variable to TG variable of 82%, while the remaining 18% was influenced by other independent variables. The highest average TG levels in KP treatment was 2.91±0.03 mnol/L, and the lowest mean of TG value in KN treatment was 1.30 ± 0.05 mmol/L. Administration of whey-KSK of 25 – 100 g/mL reduced 35.39 – 55.32% of TG in 3T3-L1 adipocyte cells. The test results of TC content in 3T3-L1 adipocyte cells in the treatment groups (KN, KP, P1, P2, P3, and P4) were 1.11, 0.01μg/ L, 4.990.09 g/L, 3.470.09 g/L, iv 2.990.04 g/L, 2.110.09 g/L, and 1.94±0.09 g/μL respectively. TC value indicated that higher whey-KSK resulted in lower TC value. The regression results of whey-KSK at different doses had a negative and significant effect on TC. The results of statistical tests indicated that t-count value was greater than t-table (13.956 > 2.101), and p-value was smaller (α < 0.05 ). The regression coefficient was -0.03 denoting that every one increase in whey-KSK dose variable significantly reduced TC variable by 0.03. The coefficient of determination with R square 0.92, indicated the contribution of whey-KSK dose variable to TC variable of 92%, while the remaining 8.5% was influenced by other independent variables. Thus, whey-KSK dose of 25-100 g/mL reduced TC (10.42-61.12%). PEPCK activity of 3T3-L1 adipocytes in the treatment groups (KN, KP, P1, P2, P3, and P4) which were 0.030.01mU/L, 0.440.02 mU/L, 0.320.00 mU/L, 0.290.00 mU/L, 0.19  0.00 mU/L, and 0.07±0.00 mU/μL respectively. The specific activity of PEPCK in a row was 0.08  0.00, 0.890.13 mU/mg, 0.590.00 mU/mg, 0.500.00 mU/mg, 0.390.00 mU/mg, 0.32±0.14 mU/mg. Based on PEPCK activity, it was apparent that higher administration dose of whey-KSK resulted in lower value of PEPCK activity, signifying that the administration of whey-KSK decreased PEPCK activity of adipocytes compared to KP. The administration of whey-KSK indicated inhibition of PEPCK activity (56.81– 84.09%) and inhibition of PEPCK specific activity (56.17-64.04%) compared to positive control. The contribution of Whey-KSK dose variable to PEPCK activity variable was 72.2%, while the remaining 27.8% was influenced by other independent variables. The contribution of whey-KSK dose variable to PEPCK-specific activity variable was 43.0%. while the remaining 57.0% was influenced by other independent variables. In conclusion, the administration of whey-KSK to obese model cells inhibited the synthesis of TG, TC and PEPCK enzyme activity as an indicator of obesity. Thus, whey-KSK was evident to be a potential alternative as an anti-obesity diet agent.

Item Type:	Thesis (Magister)
Identification Number:	0422060094
Uncontrolled Keywords:	Fermentasi Susu, Trigliserida, Kolesterol, Aktivitas Enzim,-Fermented Milk, Triglycerides, Cholesterol, Enzyme Activity
Subjects:	600 Technology (Applied sciences) > 616 Diseases > 616.02 Special topics of disease > 616.025 Medical emergencies / Emergency medicine / Emergency nursing / Triage (Medicine)
Divisions:	S2/S3 > Magister Ilmu Biomedis, Fakultas Kedokteran
Depositing User:	Endang Susworini
Date Deposited:	24 Jan 2023 06:54
Last Modified:	24 Jan 2023 06:54
URI:	http://repository.ub.ac.id/id/eprint/196887

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