Effects of sulfur amino acids, L: -methionine, L: -cystine and L: -cysteine on lipoprotein lipase and hormone-sensitive lipase in differentiated mouse 3T3-L1 adipocytes

Abstract

Rats subcutaneously implanted with AH109A hepatoma cells show hyperlipidemia with high concentrations of serum triglyceride and nonesterified fatty acid, suppression of lipoprotein lipase (LPL), and elevation of hormone-sensitive lipase (HSL) activities during the growth of the hepatoma. Supplementation of the diet with sulfur amino acids such as L: -methionine (Met) and L: -cystine (Cys) improved hyperlipidemia by restoring LPL and HSL activities. In the present study, we have attempted to examine the effects of sulfur amino acids on the activity and mRNA level of LPL and the activity of HSL using 3T3-L1 cells, which are known to differentiate to adipocytes. The adipocytes were incubated with various concentrations of Met, Cys or L: -cysteine (CysH) in the absence or presence of tumor necrosis factor-alpha (TNF-alpha). LPL activity was suppressed by TNF-alpha. In the absence of TNF-alpha, Met, Cys and CysH did not change the LPL activity. In the presence of TNF-alpha, Met and Cys significantly increased the LPL activity, and Met also enhanced the LPL mRNA level. HSL activity was also suppressed by TNF-alpha. In the absence of TNF-alpha, Met enhanced the HSL activity. In the presence of TNF-alpha, Met, Cys and CysH suppressed the HSL activity. Sulfur amino acids such as Met, Cys and CysH affected the LPL activity, mRNA level, and HSL activity in 3T3-L1 adipocytes. Some of these effects of sulfur amino acids were different between LPL and HSL, between the absence and the presence of TNF-alpha, and between 3T3-L1 adipocytes and the adipose tissue from rats.

Fig. 1

a Effect of the addition of tumor necrosis factor-α (TNF-α) on LPL activity in 3T3-L1 adipocytes. Each value represents % of control (=No addition of TNF-α group). Each point and vertical bar represents the means and SEM for 4 wells. A total of 50% inhibitory concentration (IC₅₀) of TNF-α against LPL activity is 10.0 pM. b Effect of the culture time on LPL activity in 3T3-L1 adipocytes without (None) or with (concentration in serum from AH109A-bearing rats (Serum) or IC₅₀ against LPL activity (IC₅₀)) TNF-α. Each value represents % of control (=TNF-α-free and 0 h group). Each point and vertical bar represents the means and SEM for 4 wells. In a same culture time, * represents significant difference from the TNF-α-free group at p < 0.05 by Dunnett’s pairwise multiple comparison t test

Fig. 2

Effect of the deprivation of sulfur amino acids (l-methionine (Met) and l-cystine (Cys) or Met and l-cysteine (CysH)) on LPL activity in 3T3-L1 adipocytes without (None) or with (concentration in serum from AH109A-bearing rats (Serum) or IC₅₀ against LPL activity (IC₅₀)) TNF-α. Each value represents % of control (=TNF-α-free and complete group). Each value and vertical bar represents the means and SEM for 4 wells. * Represents significant difference from the complete group at p < 0.05 by Dunnett’s pairwise multiple comparison t test

Fig. 3

Effect of the addition of sulfur amino acids (l-methionine (Met), l-cystine (Cys) or l-cysteine (CysH)) on LPL activity in 3T3-L1 adipocytes without (None) or with (concentration in serum from AH109A-bearing rats (Serum) or IC₅₀ against LPL activity (IC₅₀)) TNF-α. Each value represents % of control (=TNF-α-free and 200 μM (Met and Cys) or 400 μM (CysH) of sulfur amino acid-containing group). Each point and vertical bar represents the means and SEM for 4 wells. In a same line, * represents significant difference from the basal group (200 μM (Met and Cys) or 400 μM (CysH) of sulfur amino acid-containing group) at p < 0.05 by Dunnett’s pairwise multiple comparison t test

Fig. 4

Effect of sulfur amino acids (l-methionine (Met), l-cystine (Cys) or l-cysteine (CysH)) on LPL mRNA level in 3T3-L1 adipocytes without (None) or with (concentration in serum from AH109A-bearing rats (Serum) or IC₅₀ against LPL activity (IC₅₀)) TNF-α. Each value represents % of control (=TNF-α-free and 200 μM (Met and Cys) or 400 μM (CysH) of sulfur amino acid-containing group). Each point and vertical bar represents the means and SEM for 4 samples. In a same line, * represents significant difference from the basal group (200 μM (Met and Cys) or 400 μM (CysH) of sulfur amino acid-containing group) at p < 0.05 by Student’s t test

Fig. 5

a Effect of the addition of tumor necrosis factor-α (TNF-α) on HSL activity in 3T3-L1 adipocytes. Each value represents % of control (=No addition of TNF-α group). Each point and vertical bar represents the means and SEM for 4 wells. A total of 50% inhibitory concentration (IC₅₀) of TNF-α against HSL activity is 0.7 nM. b Effect of the culture time on HSL activity in 3T3-L1 adipocytes without (None) or with (concentration in serum from AH109A-bearing rats (Serum) or IC₅₀ against HSL activity (IC₅₀)) TNF-α. Each value represents % of control (=TNF-α-free and 0 h group). Each point and vertical bar represents the means and SEM for 4 wells. In a same culture time, * represents significant difference from the TNF-α-free group at p < 0.05 by Dunnett’s pairwise multiple comparison t test

Fig. 6

Effect of the deprivation of sulfur amino acids (l-methionine (Met) and l-cystine (Cys) or Met and l-cysteine (CysH)) on HSL activity in 3T3-L1 adipocytes without (None) or with (concentration in serum from AH109A-bearing rats (Serum) or IC₅₀ against HSL activity (IC₅₀)) TNF-α. Each value represents % of control (=TNF-α-free and complete group). Each value and vertical bar represents the means and SEM for 4 wells. * Represents significant difference from the complete group at p < 0.05 by Dunnett’s pairwise multiple comparison t test

Fig. 7

Effect of the addition of sulfur amino acids (l-methionine (Met), l-cystine (Cys) or l-cysteine (CysH)) on HSL activity in 3T3-L1 adipocytes without (None) or with (concentration in serum from AH109A-bearing rats (Serum) or IC₅₀ against HSL activity (IC₅₀)) TNF-α. Each value represents % of control (=TNF-α-free and 200 μM (Met and Cys) of or 400 μM (CysH) of sulfur amino acid-containing group). Each point and vertical bar represents the means and SEM for 4 wells. In a same line, * represents significant difference from the basal group (200 μM (Met and Cys) of or 400 μM (CysH) of sulfur amino acid-containing group) at p < 0.05 by Dunnett’s pairwise multiple comparison t test