Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes

Abstract

Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli-type chickpeas, which are used worldwide. The effects were similar, but of much lesser magnitude compared to those of ECP described above. Thus, Desi-type chickpeas may prove to be effective for the treatment of diabetes, as they can alter the lipid content, thus reducing fat storage.

Figure 1

Effect of extracts of chickpeas (ECP) on lipid droplet formation and lipid contents. (A) Large droplets were observed after an 8-day culture for differentiation in the absence of ECP, whereas only small droplets were observed in the presence of ECP at 0.1% (scale bar, 20 µm). (B) After staining with Oil Red O, the absorbance of the dye at 510 nm was then measured and the absorbance/µg protein was calculated (^**p<0.0001; Jonckheere-Terpstra test). (C) The protein expression of perilipin was inhibited in the presence of ECP at 0.1% (^**p=0.0002 -ECP vs. + ECP; unpaired Student's t-test, values are the means ± SD of triplicate experiments).

Figure 2

Changes in lipid composition by extracts of chickpeas (ECP) and the sum total of fatty acids. Total cellular lipids were extracted from cells cultured with or without ECP for 8 days, hydrolyzed to fatty acid, and then esterified to methyl ester form. The composition of the fatty acid methyl ester was analyzed by gas chromatography. Lipid contents for palmitate (C16:0), palmitoleate (C16:1), stearate (C18:0), oleate (C18:1n-9) and vaccinate (C18:1n-7) and the sum total are shown. (Values are the means ± SD of triplicate experiments. ^**p<0.01 for C16:1 and C18:1n-7 and sum total. ^*p<0.05 for C18:1n-9. No significant difference for C16:0 or C18:0. All p-values were obtained by the Jonckheere-Terpstra test).

Figure 3

Effects of extracts of chickpeas (ECP, shown as CP) on stearoyl-CoA desaturase 1 (SCD1) and elongation of very long chain fatty acids family member 6 (ELOVL6).(A) Western blot analysis was performed for SCD1 and ELOVL6. (B and C) Densitometric analysis was performed by reading individual protein bands thereafter. Each value was corrected with an internal standard (RPS18), and the value relative to day 0 was then calculated [means ± SD of triplicate experiments; (B) SCD1, ^**p=0.0002; (C) ELOVL6, p=0.1333; Jonckheere-Terpstra test].

Figure 4

Effects of extracts of chickpeas (ECP) on the phosphorylation of liver kinase B1 (LKB1) and AMPK. Western blot analysis was performed for (A) LKB1 and (C) AMPK. (B and C) Densitometric analysis was performed by reading individual protein bands thereafter. Densitometric readings were performed on the bands of (B) LKB1 and p-LKB1 and those of (D) AMPK and p-AMPK. Each value was corrected with the internal standard RPS18, and each ratio (p-LKB1/LKB1 or p-AMPK/AMPK) was then calculated. Values are the means ± SD of triplicate experiments (Jonckheere-Terpstra test: p-LKB1/LKB1, ^**p=0.0002; p-AMPK/AMPK, ^**p= 0.0011).

Figure 5

Effects of extracts of chickpeas (ECP) on the phosphorylation of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). (A) Western blot analysis was performed for ACC, p-ACC and FAS. (B and C) Densitometric analysis was performed by reading individual protein bands thereafter. Densitometric readings were performed on the bands of (B) ACC and p-ACC and those of (C) FAS. Each value was corrected with the internal standard RPS18, and the ratios (p-ACC/ACC) or values relative to day 0 (FAS) were then calculated. Values are the means ± SD of triplicate experiments (Jonckheere-Terpstra test: p-ACC/ACC, ^**p<0.0001; FAS, ^**p= 0.005).

Figure 6

Effects of extracts of chickpeas (ECP) on carnitine palmitoyltransferase 1 (CPT1) and UCP2. Western blot analysis was performed for (A) CPT1 and (C) UCP2. (B and D) Densitometric analysis was performed by reading individual protein bands thereafter. Each value was corrected with the internal standard RPS18, and the value relative to day 0 was then calculated (means ± SD of triplicate experiments; Jonckheere-Terpstra test: CPT1, ^*p=0.0124; UCP2, ^*p= 0.0369).

Figure 7

Effects of ethanol extracts of Kabuli-type chickpeas on stearoyl-CoA desaturase 1 (SCD1) and lipid contents. (A) After 8 days of culture with and without ethanol extracts of the Kabuli type or Desi type, the amount of SCD1 was examined by a western blot analysis (means ± SD of triplicate experiments; ^**p<0.05, unpaired Student's t-test). (B) Densitometric analysis was performed by reading individual protein bands thereafter. (C) The lipid contents contained in 3T3-L1 cells after 8 days of culture with or without ethanol extracts of the Kabuli or Desi type were detected by measuring the absorbance at 510 nm with a fluorometer [means ± SD of triplicate experiments; one-way ANOVA (Tukey), 0 vs. 0.1% Desi type ^**p=0.0037; 0 vs. 0.1% Kabuli type ^*p= 0.0429].

Figure 8

Hypothetical regulation of lipogenesis and lipolysis by stearoyl-CoA desaturase 1 (SCD1) and other enzymes. The hypothetical regulation of lipogenesis and lipolysis by extracts of chickpeas (ECP), based on our findings in this study. Three enzymes, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and SCD1, participating in lipogenesis in each step are described. SCD1 promotes the desaturation of saturated fatty acid (SFA) of liver kinase B1 (LKB1), causing an increase in the phosphorylation of AMP activated protein kinase (AMPK). The phosphorylated form of AMPK then promotes the phosphorylation of ACC to inactivate ACC, thereby suppressing the reaction from acetyl-CoA to malonyl-CoA. The depletion of malonyl-CoA simulates the transportation of fatty acids into the mitochondria by carnitine palmitoyltransferase 1 (CPT1). SCD1 itself catalyzes the reaction from SFA (16:0, 18:0) to MUFA (16:1, 18:1), as shown in the balloon. p-AMPK, phospho-AMPK; p-ACC, phospho-ACC; ACL, ATP citrate lyase; ELOVL6, elongation of very long chain fatty acids family member 6; MUFA, monounsaturated fatty acid.