Role of protease inhibitors and acylation stimulating protein in the adipogenesis in 3T3-L1 cells

Abstract

Treatment of AIDS (HIV) and hepatitis C virus needs protease inhibitors (PI) to prevent viral replication. Uses of PI in therapy are usually associated with a decrease in body weight and dyslipidemia. Acylation stimulating protein (ASP) is a protein synthesized in adipocytes to increase triglycerides biosynthesis, for that the relation of PI and ASP to adipogenesis is tested in this work. ASP expression was increased during 3T3-L1 differentiation and reached a peak at day 8 with cell maturation. Addition of PI during adipocytes differentiation dose dependently and significantly (p < 0.5) inhibited the degree of triglycerides (TG) accumulation. Moreover, presence of ASP (450 ng/mL) in media significantly (p < 0.5) stimulated the degree of TG accumulation and there was additive stimulation for ASP when added with insulin (10 microg/mL). Finally, when ASP in different doses (Low, 16.7; Medium, 45 and High, 450 ng/mL) incubated with a dose of x150 PI, ASP partially inhibited the PI-inhibited adipogenesis and TG accumulation. The results in this study show that PI inhibit lipids accumulation and confirm role of ASP in TG biosynthesis and adipogenesis.

Fig. 1

Time dependent increase in acylation stimulating protein (ASP) expression during differentiation of 3T3-L1 cells. Every 2 days, RNA was extracted up to 12 days and reverse transcribed and RT-PCR analysis was carried out. (A) RT-PCR analysis for ASP expression, upper bands for ASP and the lower is for glyceraldehydes-3-phosphate dehydrogenase (G3PDH). (B) Densitometric analysis (fold increase) of ASP bands relative to G3PDH (internal control).

Fig. 2

Effect of PI on adipogenesis in 3T3-L1 cells. Cells were incubated with insulin for 4 days and then with PI in different dose to observe lipids accumulation. (A) Control; Showing fibroblast like cells without lipids accumulation. (B) Insulin alone (10 µg/mL); Cells became round and a marked increase in lipids was recorded. (C-F) C; Insulin plus PI (×300), D; Insulin plus PI (×200), E; Insulin plus PI (×150) and F; Insulin plus PI (×100). Oil red O stain, ×400.

Fig. 3

Inhibitory effect of PI on adipogenesis in 3T3-L1 cells. Lipids accumulation measured spectrophotometrically at OD 540 nm. The lipids were removed from cells after staining by oil red O and removed by isopropanol. Values are means ± SE obtained from 3 experiments. ^*p < 0.05 compared to control and ^†p < 0.05 compared to insulin.

Fig. 4

Effect of PI on ASP stimulated lipids accumulation in 3T3-L1 cells. Cells were incubated for 4 days with either insulin (10 µg/mL) or ASP in high dose (ASPH, 450 ng/mL) plus insulin (10 µg/mL). Also, cells were incubated with PI (×150), insulin and different doses of ASP, ASP in low dose (ASPL; 16.7 ng/mL), medium dose of ASP (ASPM; 45 ng/mL) and ASPH. (A) Control; Showing fibroblast like cells without lipids accumulation. (B) Insulin alone; Cells became round and marked increase in lipids accumulation (C) Insulin plus ASPH; Showing more lipids accumulation. (D) Insulin plus PI (×150); Lipids accumulation was moderately decreased in the presence. (E-G) E; Insulin plus PI (×150) and ASPL, F; Insulin plus PI (×150) and ASPM, G; Insulin plus PI (×150) and ASPH. Oil red O stain, ×400.

Fig. 5

Effect of PI on ASP stimulated lipids accumulation in 3T3-L1 cells. Mature cells were incubated for 4 days as described in Fig. 4. Lipid accumulation was measured spectrophotmetrically at OD 540 nm. The lipids were removed from cells after staining by oil red O and removal by isopropanol. Values are means ± SE obtained from 3 experiments. ^*p < 0.05 compared to control, ^†p < 0.05 compared to insulin, ^‡p < 0.05 compared to insulin plus PI and, ^§p < 0.05 compared to insulin and insulin plus ASP.