β-aminoisobutyric acid attenuates hepatic endoplasmic reticulum stress and glucose/lipid metabolic disturbance in mice with type 2 diabetes

Abstract

β-aminoisobutyric acid (BAIBA) is a nature thymine catabolite, and contributes to exercise-induced protection from metabolic diseases. Here we show the therapeutical effects of BAIBA on hepatic endoplasmic reticulum (ER) stress and glucose/lipid metabolic disturbance in diabetes. Type 2 diabetes was induced by combined streptozotocin (STZ) and high-fat diet (HFD) in mice. Oral administration of BAIBA for 4 weeks reduced blood glucose and lipids levels, hepatic key enzymes of gluconeogenesis and lipogenesis expressions, attenuated hepatic insulin resistance and lipid accumulation, and improved insulin signaling in type 2 diabetic mice. BAIBA reduced hepatic ER stress and apoptosis in type 2 diabetic mice. Furthermore, BAIBA alleviated ER stress in human hepatocellular carcinoma (HepG2) cells with glucosamine-induced insulin resistance. Hepatic AMPK phosphorylation was reduced in STZ/HFD mice and glucosamine-treated HepG2 cells, which were restored by BAIBA treatment. The suppressive effects of BAIBA on glucosamine-induced ER stress were reversed by knockdown of AMPK with siRNA. In addition, BAIBA prevented thapsigargin- or tunicamycin-induced ER stress, and tunicamycin-induced apoptosis in HepG2 cells. These results indicate that BAIBA attenuates hepatic ER stress, apoptosis and glucose/lipid metabolic disturbance in mice with type 2 diabetes. AMPK signaling is involved to the role of BAIBA in attenuating ER stress.

Figure 1. BAIBA attenuates glucose metabolic disturbance and improves insulin resistance in STZ/HFD-induced type 2 diabetes in mice.

(A), body weight, liver weight and the ratio of liver weight to body weight. (B), Accumulated food intake in the last week (g/week and KJ/week). (C), fasting blood glucose levels. (D), expressions of key enzymes of gluconeogenesis (PEPCK, G6pase) in livers. (E), glucose tolerance test (GTT) and insulin tolerance test (ITT). (F), serum insulin levels. (G), phosphorylation of Akt. (H), phosphorylation of insulin receptor substrate-1 (IRS-1) at Tyr632 and at Ser307. *P < 0.05 and **P < 0.01 vs. Control, ^†P < 0.05 and ^††P < 0.01 vs. STZ/HFD. n = 7 for each group in (A–C,E,F); n = 4 for each group in (D, G, H).

Figure 2. BAIBA attenuates lipid metabolic derangements in mice with type 2 diabetes.

(A), Serum triglyceride (TG), total cholesterol (TCH) and free fatty acid (FFA) levels. (B), Serum low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) levels. (C), HE staining of liver sections. (D), Oil red O staining of liver sections. (E), Liver triglyceride (TG) levels. (F), Expressions of lipogenesis genes (Srebp-1c, Fas, Acc1, Scd1) in livers. *P < 0.05 and **P < 0.01 vs. Control; ^†P < 0.05 and ^††P < 0.01 vs. STZ/HFD. n = 7 for each group in (A,B,E); n = 4 for each group in (C,D,F).

Figure 3. BAIBA improves hepatic ER stress in type 2 diabetes of mice.

(A), Immunohistochemistry of liver sections for CHOP (an ER stress marker). (B), Western blot analysis for phosphorylation of eIF2α and JNK, and expression of GRP78 and ATF4 protein (ER stress markers) in liver. (C) Expression of Bax and Bcl-2 proteins and ratio of Bax to Bcl-2 (apoptosis markers) in liver. (D), TUNEL staining showing apoptosis in liver. (E), Expression of caspase-3 and caspase-9 (apoptosis markers) in liver. (F), Serum ALT and AST levels (markers of hepatic injury). *P < 0.05 and **P < 0.01 vs. Control, ^†P < 0.05 and ^††P < 0.01 vs. STZ/HFD. n = 4 for each group in (A–D); n = 7 for each group in (E,F).

Figure 4. BAIBA improves glucosamine-induced ER stress in HepG2 cells.

The HepG2 cells were treated with glucosamine (18 mM) for 18 h, and then treated with BAIBA (10 μM) for 24 h or 48 h. The phosphorylation of eIF2α and JNK, and the expressions of GRP78 and CHOP were used as the markers of ER stress. *P < 0.05 and **P < 0.01 vs. Control; ^†P < 0.05 vs. Glucosamine. ^‡P < 0.05 vs. 24 h. n = 4 for each group.

Figure 5. BAIBA improves thapsigargin- or tunicamycin-induced ER stress in HepG2 cells.

HepG2 cells were treated with thapsigargin (1 μM) or tunicamycin (3 μM) and different doses of BAIBA (1, 10 or 100 μM) for 48 h, followed with thapsigargin (1 μM) or tunicamycin (3 μM) for 4 h. The phosphorylation of eIF2α and JNK, and the expressions of GRP78 and CHOP were used as the markers of ER stress. (A), Effect of BAIBA on thapsigargin-induced ER stress. (B), Effect of BAIBA on tunicamycin-induced ER stress. (C), Effect of BAIBA on apoptosis in tunicamycin-induced apoptosis determined with flow cytometric analysis in HepG2 cells. HepG2 cells were incubated with tunicamycin (3 μM) for 4 h, then treated with BAIBA (100 μM) for 48 h. *P < 0.05 and **P < 0.01 vs. Control; ^†P < 0.05 and ^††P < 0.01 vs. thapsigargin; ^‡P < 0.05 and ^‡‡P < 0.01 vs. tunicamycin. n = 4 for each group.

Figure 6. AMPK mediated the role of BAIBA in attenuating ER stress.

(A), Effect of BAIBA on AMPK phosphorylation in livers of STZ/HFD mice. **P < 0.01 vs. Control; ^††P < 0.01 vs. STZ/HFD; (B), Effect of BAIBA on AMPK phosphorylation in glucosamine-treated HepG2 cells. **P < 0.01 vs. Control; ^†P < 0.05 vs. glucosamine; (C), Effects of knockdown of AMPK on AMPK and GRP78 expressions and eIF2α phosphorylation in glucosamine-treated HepG2 cells. HepG2 cells were transfected with scrambled-siRNA (Scr-siRNA) or AMPK-siRNA for 24 h, and then cultured in the presence of glucosamine for 18 h and treated with BAIBA for 48 h. *P < 0.05 and **P < 0.01 vs. Scr-siRNA; ^†P < 0.05 vs. Scr-siRNA/BAIBA. n = 4 for each group.