Branched Chain Amino Acids Cause Liver Injury in Obese/Diabetic Mice by Promoting Adipocyte Lipolysis and Inhibiting Hepatic Autophagy

Abstract

The Western meat-rich diet is both high in protein and fat. Although the hazardous effect of a high fat diet (HFD) upon liver structure and function is well recognized, whether the co-presence of high protein intake contributes to, or protects against, HF-induced hepatic injury remains unclear. Increased intake of branched chain amino acids (BCAA, essential amino acids compromising 20% of total protein intake) reduces body weight. However, elevated circulating BCAA is associated with non-alcoholic fatty liver disease and injury. The mechanisms responsible for this quandary remain unknown; the role of BCAA in HF-induced liver injury is unclear. Utilizing HFD or HFD+BCAA models, we demonstrated BCAA supplementation attenuated HFD-induced weight gain, decreased fat mass, activated mammalian target of rapamycin (mTOR), inhibited hepatic lipogenic enzymes, and reduced hepatic triglyceride content. However, BCAA caused significant hepatic damage in HFD mice, evidenced by exacerbated hepatic oxidative stress, increased hepatic apoptosis, and elevated circulation hepatic enzymes. Compared to solely HFD-fed animals, plasma levels of free fatty acids (FFA) in the HFD+BCAA group are significantly further increased, due largely to AMPKα2-mediated adipocyte lipolysis. Lipolysis inhibition normalized plasma FFA levels, and improved insulin sensitivity. Surprisingly, blocking lipolysis failed to abolish BCAA-induced liver injury. Mechanistically, hepatic mTOR activation by BCAA inhibited lipid-induced hepatic autophagy, increased hepatic apoptosis, blocked hepatic FFA/triglyceride conversion, and increased hepatocyte susceptibility to FFA-mediated lipotoxicity. These data demonstrated that BCAA reduces HFD-induced body weight, at the expense of abnormal lipolysis and hyperlipidemia, causing hepatic lipotoxicity. Furthermore, BCAA directly exacerbate hepatic lipotoxicity by reducing lipogenesis and inhibiting autophagy in the hepatocyte.

Keywords: AMP-activated protein kinase; Branched chain amino acids; Lipolysis; Lipotoxicity; Mammalian target of rapamycin; Non-alcoholic fatty liver disease.

Fig. 1

BCAA supplementation reduced fat mass and body weight gain in HFD-induced obese/diabetic mice. (A–B) Adult male C57BL6J mice were fed 12 weeks of normal diet (ND) or high-fat diet (HFD). (A) Plasma BCAA was determined via commercial kit and plasma BCKA was determined via high performance liquid chromatography (HPLC). (B) Leucine tolerance test results in ND and HFD mice. (C to F) ND and HFD-fed mice were then orally administered BCAA (ND + BCAA, HFD + BCAA) or vehicle (ND, HFD) for 12 weeks. (C) Quantification of plasma BCAA. (D) Body weight was determined every two weeks. (E) Lean and fat mass, and (F) adipocyte size were determined. n = 8–10 per group. *P < 0.05 versus ND; ^#P < 0.05 versus HFD. Data presented as mean + SEM.

Fig. 2

BCAA supplementation suppressed hepatic de novo lipogenesis in HFD-induced obese/diabetic mice via mTOR signaling. (A–C) Adult C57BL6J mice were fed on high-fat diet (HFD), high-fat plus BCAA diet (HFD + BCAA), or rapamycin (RAPA, 1 mg/kg body weight intraperitoneal injection, every other day) atop HFD + BCAA (HFD + BCAA + RAPA) for 12 weeks. (A) Upper: HE staining in liver sections (n = 10 per group). Lower: hepatic TG content (n = 10 per group). (B) Expression of hepatic SREBP-1c (full length, fl), SREBP-1c (partial, p), p-AKT, AKT, p-mTOR, and mTOR by Western blot (n = 6 per group). (C) Expression of hepatic ACC1, FASN, SCD1, ELOVL6, and DGAT1 mRNA by real-time PCR (n = 6 per group). For (A, B, and C): *P < 0.05 versus HFD and ^#P < 0.05 versus HFD + BCAA. Data presented as mean + SEM. (D–E) Cultured primary hepatocytes were treated with bovine serum albumin (BSA) or oleic acid (OA, 1 mM) for 12 h. Effects of BCAA (5 mM), rapamycin (RAPA, 1 μM), and AKT inhibitor (AKTi, 10 μM) co-treatment upon hepatocyte lipogenesis were determined. (D) Expression of SREBP-1c (fl), SREBP-1c (p), and p-AKT/AKT ratio by Western blot. Left panel: representative blots; right three panels: quantification data. (E) Intracellular TG content, as measured via commercial kit. For (D and E): n = 4 per group. *P < 0.05. Data presented as mean + SEM.

Fig. 3

BCAA caused significant liver damage in HFD mice. (A) Plasma ALT and AST were measured in HFD and HFD + BCAA group (n = 10 per group). (B) Hepatic proinflammatory cytokines mRNA levels were determined, which was compared to normal diet (dotted line), n = 10 per group. (C) Hepatic FFA content in HFD and HFD + BCAA group (n = 6 per group). (D) Hepatic MDA and 4-HNE (two common indices of lipid peroxidation, n = 8 per group). (E) Hepatic apoptosis determined by TUNEL staining (n = 8 per group). (F) Hepatic cleaved and non-cleaved caspase-3 expression levels determined by Western blot (n = 6 per group). *P < 0.05 versus HFD. Data presented as mean + SEM.

Fig. 4

BCAA exacerbated HFD-induced hyperlipidemia and insulin resistance, due to aberrant lipolysis. HFD-fed mice were given BCAA daily for 12 weeks. (A) After 6 hour-fasting, plasma TG and FFA levels were determined. (B) In vivo lipolysis was determined in HFD and HFD + BCAA group. n = 6 per group. (C) In vitro lipolysis was performed in cultured adipocytes with or without BCAA (5 mM) co-treatment for 6 h per Methods. n = 6 per group. (D to F) After 6 hour-fasting, glucose homeostasis was determined in HFD and HFD + BCAA mice. (D) Glucose tolerance test (GTT) and insulin release during GTT. (E) Insulin tolerance test (ITT). (F) HOMA-IR indexes, calculated after GTT and ITT. n = 6 per group. *P < 0.05. Data presented as mean + SEM.

Fig. 5

AMPKα2, but not mTOR, signaling mediates BCAA-enhanced lipolysis in adipocytes. (A) Cultured adipocytes were treated with vehicle (Veh), BCAA (5 mM), or BCAA + rapamycin (RAPA, 1 μM) for 6 h. Insulin (10 ng/ml) was added to culture medium for 10 min, followed by 90 minute-stimulation by isoprenaline (ISO, 10 μM). Expression of p-mTOR and mTOR in adipocytes by Western blot (upper panel); lipolytic rates were measured by glycerol release into supernatant (lower panel). n = 6 per group. (B) Expression of p-AMPK, AMPK, p-ACC, and ACC in white adipose tissue isolated from HFD and HFD + BCAA group mice (n = 6 per group). (C to E) Cultured adipocytes were treated with vehicle (Veh), BCAA, or BCAA + Compound-C (CC, 10 μM) for 6 h. Insulin and ISO were added to culture medium as described in Fig. 5A. (C) Effects of different doses of BCAA upon expression of p-AMPK and AMPK in adipocytes (left panel) and glycerol release in supernatant (right panel). (D) PDE activity in adipocytes (upper panel) and glycerol release in supernatant (lower panel). (E) p-AMPK, AMPK, p-HSL, HSL, and ATGL expression in adipocytes by Western blot (n = 6 per group). (F) On Day 5 after differentiation, adipocytes treated by control siRNA (siCon), AMPKα1 siRNA (siAMPKα1), or AMPKα2 siRNA (siAMPKα2). 48 h after siRNA transfection, expression of AMPKα1 and AMPKα2 was determined by Western blot (left two panels). Effects of BCAA upon lipolysis were determined in siRNA-transfected adipocytes (right panel). n = 5 per group. *P < 0.05. All data presented as mean + SEM.

Fig. 6

Inhibition of lipolysis abolished BCAA-enhanced hyperglycemia and weight loss, improved insulin sensitivity, but failed to normalize BCAA-induced liver injury. Adult C57BL6J mice were treated with HFD, HFD + BCAA, or HFD + BCAA + acipimox (10 mg/kg body weight, daily IP injection) for 12 weeks. (A) Left panel, body weight was monitored every two weeks (n = 10 per group). Lean and fat mass (middle panel), and adipocyte size (right panel) were determined (n = 10 per group). (B) Quantification of plasma TG and FFA (n = 8 per group). (C) Glucose homeostasis (GTT, insulin release during GTT, and ITT) was evaluated (n = 6 per group). (D) Quantification of plasma ALT and AST (n = 8 per group). (E) Determination of hepatic MDA and 4-HNE (n = 8 per group). (F) Hepatic apoptosis determined by TUNEL staining (n = 8 per group). *P < 0.05 versus HFD and ^#P < 0.5 versus HFD + BCAA. All data presented as mean + SEM.

Fig. 7

BCAA inhibited hepatic autophagy in response to lipid exposure by activating mTOR signaling. GFP-LC3 transgenic mice were treated with ND, HFD, HFD + BCAA, or HFD + BCAA + RAPA (rapamycin, 1 mg/kg body weight, IP injection every other day) for 12 weeks. (A) GFP-LC3 droplets were analyzed in the liver (n = 10 per group). (B) Hepatic p-mTOR, mTOR, LC3-I/II, and p62 protein expression were determined by Western blot from ND, HFD, and HFD + BCAA groups (n = 4 per group). (C) Hepatic LC3-I/II and p62 expression were determined by Western blot from HFD + BCAA and HFD + BCAA + RAPA groups (n = 4 per group). (D) Cultured hepatocytes were treated with different doses of BCAA (0, 1, 5, 10 mM) in the absence or presence of palmitate (500 μM) for 24 h. LC3-I/II protein expression was evaluated by Western blot and normalized against β-actin expression (n = 3 per group). (E) Cultured hepatocytes were treated with or without BCAA (5 mM) or rapamycin (1 μM) after 24 h of palmitate (500 μM) exposure. Expression of p-mTOR, mTOR, and LC3-I/II were determined by Western blot (n = 3 per group). *P < 0.05. All data presented as mean + SEM.

Fig. 8

In vivo mTOR inhibition had no significant effect upon BCAA-induced plasma FFA elevation, but significantly attenuated liver injury. Adult C57BL6J mice were treated with HFD, HFD + BCAA, or HFD + BCAA + RAPA (rapamycin, 1 mg/kg body weight, IP injection every other day) for 12 weeks. (A) Plasma and hepatic FFA were determined. (B) Plasma ALT and AST were determined. (C) Quantification of hepatic MDA and 4-HNE. (D) TUNEL staining on liver sections. (E) Hepatic mRNA expression levels of inflammatory cytokines were determined by real time-PCR. n = 10 per group. *P < 0.05 versus HFD group and ^#P < 0.05 versus HFD + BCAA group. Data presented as mean + SEM. (F) Schematic reviewing proposed mechanism underlying BCAA-aggravated hepatic lipotoxicity. HFD induces BCAA catabolic dysfunction in mice. Under HFD conditions, increased BCAA consumption increases circulating BCAA. BCAA-induced ROS promotes AMPK phosphorylation and AMPKα2 triggers lipolysis. BCAA-enhanced lipolysis induces hyperlipidemia. Elevated circulating FFA results in insulin resistance and lipotoxic liver injury. Meanwhile, BCAA activate hepatic mTOR signaling, inhibit lipogenesis and autophagy, therefore increasing hepatic susceptibility to FFA-mediated lipotoxicity. Overall, increased BCAA consumption aggravates HFD-induced hepatic lipotoxicity, providing mechanistic insight regarding diet-induced steatohepatitis pathogenesis.