Effects of Amino Acids Supplementation on Lipid and Glucose Metabolism in HepG2 Cells

Abstract

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2DM) are representing symptoms of metabolic syndrome (MetS), which is often accompanied with hepatic fat (triglycerides) accumulation and insulin resistance (IR). S-adenosylmethionione (SAMe), taurine, and betaine are sulfur-containing amino acids (SCAAs) and cofactor involved in an essential SCAA, methionine metabolism. SCAA seems to be effective for MetS related diseases such as NAFLD and T2DM, but its effect on metabolic disorders has not been fully elucidated. Branched-chain amino acids (BCAAs), namely valine, leucine and isoleucine, are essential amino acids. Several studies have shown that BCAAs mediate the activation of many important hepatic metabolic signaling pathways such as, insulin signaling and glucose regulation, but other studies have shown that BCAA can aggravate insulin resistance. Since liver is the major site of glucose and lipid metabolism, this study was aimed to understand the effects of SCAAs and BCAAs supplementations on glucose and lipid metabolism in HepG2 cells. These cells were pre-treated with SAMe (0.5 mM), betaine (1 mM), taurine (10 mM), BCAA (4mM) or butylated hydroxytoluene (BHT) (25 μM, antioxidant drugs as a positive control) for 24 hours followed by treatments of high concentration of glucose (50 mM) or palmitic acid (PA, 0.5 mM) for 48 hours to simulate high-glucose and high-fat environments. Then the cell viability, lipid accumulation, mRNA expression levels of key enzymes involved in glucose and lipid metabolism were determined. Glucose and PA treatment led to significant decrease in cell survival. Taurine and betaine significantly increased cell viability, and SAMe only increased cell viability in glucose treated group. BCAA reduced cell viability in a high-glucose and high-fat environment. In the experiment of Oil Red O staining, the groups treated with PA had obvious lipid droplets and the pretreatment of BCAA and SCAAs inhibited the fat accumulation. At the transcriptional level, glucose and PA treatment led to significant increase of mRNA expression of glucokinase (GK), mediating phosphorylation of glucose to glucose-6-phosphate (G6P), glucose-6-phosphatase (G6Pc) and phosphoenolpyruvate carboxykinase 1 (PCK1). Those are the key enzymes for maintaining blood glucose balance. BCAA increased the expression of GK and PCK1 in the high-glucose and high-fat environment. SAMe decreased GK and inhibited glucose synthesis by reducing the level of gluconeogenic enzyme G6Pc and PCK1. After SAMe pretreatment, the mRNA expression of lipogenic enzymes, such as acetyl-CoA carboxylase 2 (ACC2), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD1) were decreased. Taurine and BCAA also reduced FASN expression. The peroxisome proliferator activated receptor gamma (PPAR-γ) expression was increased after glucose and PA treatment, BCAA pretreatment toward a further increase in the expression, SAMe not only down-regulated the expression of PPAR-γ, but also inhibited the expression of carbohydrate response element binding protein (ChREBP) and sterol regulatory element-binding protein-1c (SREBP-1c). SAMe down-regulated the expression of GLUT2 and PCK1 by inhibiting PPAR-γ, decreased the expression of GK, ACC2, FASN and SCD1 by down-regulating SREBP-1c, and decreased G6Pc by down-regulating ChREBP. Through the above gene regulation affect glucose and fat metabolism, inhibit fat accumulation. Taken together in this study, the effect of SAMe on glucose and lipid metabolism is significant. SAMe has the effect of inhibiting hepatic lipogenesis and gluconeogenesis.;비알코올성지방간 (Nonalcoholic fatty liver disease, NAFLD) 및 제2형 당뇨병 (type 2 diabetes, T2DM)은 간지방 축적 (triglycerides accumulation) 및 인슐린 저항성 (insulin resistance, IR)을 동반하는 대사 증후군 (MetS)의 전형적인 증상을 나타낸다. S-adenosylmethionione (SAMe) 및 taurine 은 황함유 아미노산 (sulfur-containing amino acids, SCAAs) 이며 betaine는 필수 SCAA인 methionine 대사에 관여하는 보조 인자이다. SCAA는 NAFLD및 T2DM과 같은 MetS 관련 질병에 효과적인 것으로 보이지만 대사 장애에 어떤 메커니즘으로 영양을 미치는 지는 뚜렷하게 밝혀지지 않고 있다. 분지쇄아미노산 (branched-chain amino acids, BCAAs), 즉 valine, leucine 및 isoleucine은 필수 아미노산이다. 여러 연구에서 BCAA가 인슐린 신호 및 포도당 조절과 같은 많은 중요한 간 대사 신호 경로의 활성화를 매개한다는 것이 밝혀졌지만 또다른 연구에서는 BCAA가 IR을 악화시킬 수 있음을 보여주었다. 간은 포도당 및 지질 대사의 주요 기관이기 때문에, 본 연구는 HepG2 세포에서 포도당 및 지질 대사에 대한 SCAA 및 BCAA의 효과를 이해하는 것을 목표로 하였다. HepG2 세포를 사용하여 SAMe (0.5 mM), Betaine (1 mM), taurine (10 mM), BCAA (4 mM) 또는 BHT (25 μM)로 24 시간 동안 전처리한 후, 고농도 포도당 (glucose, 50mM) 또는 palmitic acid (PA, 0.5mM)를 48 시간 동안 처리하였다. 그 후, cell viability, TG accumulation, 포도당 및 지질 대사에 관여하는 주요 효소의 mRNA expression 을 측정하였다. Glucose 및 PA 처리 후 HepG2 세포의 생존율은 현저하게 감소하였다. 그러나 전처리한 taurine과 betaine은 세포 생존율을 유의하게 증가시켰으며, SAMe은 glucose 처리군에서 세포 생존율을 증가시켰다. BCAA는 glucose + PA 처리군에서 세포 생존율을 감소시켰다. Oil Red O staining 실험에서, PA 처리군은 lipid droplets을 나타냈으며 BCAA 및 SCAA의 전처리는 이러한 지방의 축적을 억제 하였다. 전사 수준에서 glucose 및 PA 처리에는 glucokinase (GK), glucose-6-phosphatase (G6Pc) 및 phosphoenolpyruvate carboxykinase 1 (PCK1) mRNA expression를 증가시켰다. 이들은 혈당 균형을 유지하기위한 주요 효소이다. BCAA는 glucose + PA 처리군에서 GK 및 PCK1 mRNA expression을 증가시켰다. SAMe은 GK mRNA expression을 감소시키고 글루코겐 생성 효소인 G6Pc 및 PCK1 mRNA의 수준을 감소시켜 포도당 합성을 억제했다. SAMe 전처리 후, ACC2, FASN 및 SCD1과 같은 lipogenesis 효소의 mRNA expression이 감소되었다. Taurine과 BCAA도 FASN expression을 감소시켰다. Glucose 및 PA 처리 후 peroxisome proliferator activated receptor gamma (PPAR-γ) expression은 증가되고, BCAA 전처리 후에 mRNA expression 이 더욱 증가하였다. SAMe은PPAR-γ expression 뿐만 아니라 carbohydrate response element binding protein (ChREBP) 및 sterol regulatory element-binding protein-1c (SREBP-1c) expression도 억제했다. SAMe은 PPAR-γ를 억제함으로써 GLUT2 및 PCK1 mRNA expression을 감소시키고, SREBP-1c 또한 억제시킴으로써 GK, ACC2, FASN 및 SCD1 mRNA expression을 downregulation을 유발했으며, ChREBP를 억제함으로써 G6Pc mRNA 발현을 감소시켰다. 이와 같은 경로로 포도당과 지방 대사에 영향을 미쳐 지방 축적을 억제한 것으로 판단이 된다. 이러한 결과는 포도당 및 지질 대사에 대한 SAMe의 효과가 유의하다는 것을 간접적으로 증명하며, SAMe은 간의 lipogenesis 및 gluconeogenesis를 억제하는 효과와 함께 대사 증후군을 예방하는 데 유리할 수 있다고 예상할수 있다. 그러나 이에 관한 분자생물학적, 영양학적 메타니쯤은 추가적인 연구가 필요하다.