| BackgroundEndoplasmic reticulum(ER)is a central organelle for protein synthesis and folding.Under physiologic conditions,there is an equilibrium between the protein synthesis and its folding capacity,and these proteins are correctly folded and assembled by chaperones in the ER.When the protein synthesis rate exceeds the folding capacity,unfolded proteins accumulate in ER.The perturbation in ER homeostasis causes ER stress and leads to activation of the complex signaling cascade called the unfolded protein response(UPR).Activation of UPR signaling causes a transcriptional activation of ER chaperones and a reduction in protein synthesis,which aid in restoring the ER homeostasis.When the UPR fails to restore the ER homeostasis,the cells may undergo a pro-apoptotic ER stress response pathway.ER stress is a crucial link among obesity,insulin resistance and diabetes.It is closely associated with hepatic insulin resistance and plays important roles in altered glucose homoeostasis in type 2 diabetes.Obesity causes ER stress,hepatic steatosis and insulin resistance.Obesity-induced ER stress inhibits insulin signaling via hyperactivation of c-Jun N-terminal kinase(JNK)and subsequent se rine phosphorylation of insulin receptor substrate-1(IRS-1).ER stress has been proposed as a novel mechanism for the development of insulin resistance in obesity.Insulin signaling in liver is attenuated in diabetes-evoked ER stress,indicated by reduced Akt phosphorylation and IRS-1 tyrosine phosphorylation Inhibition of cytochrome P4504A reduces hepatic ER stress and insulin resistance in mice.Similarly,chemical chaperones reduce ER stress and restore glucose homeostasis in type 2 diabetes.ER stress plays a crucial role in the insulin resistance and could be apotential therapeutic target for diabetesβ-aminoisobutyric acid(BAIBA)is a nonprotein β-amino acid,a catabolite of thymine,which is further degraded into propionyl-CoA,methylmalonyl-CoA and succinyl-CoA within mitochondria,especially in liver.BAIBA can also be generated by catabolism of the branched-chain amino acid valine.BAIBA reduces body fat percentage through increased fatty acid oxidation(FAO)and decreased de novo lipogenesis in liver in mice.Recently,BAIBA is identified as a small molecule myokine secreted from myocytes with forced expression of peroxisome proliferator-activated receptor gamma coactivator-1 a(PGC-1 a).BAIBA enhances the browning of white adipose tissue and FAO in the liver via peroxisome proliferator-activated receptor α(PPARa),and may contribute to exercise-induced protection from metabolic diseases.More recently,it has been foun d that BAIBA attenuates insulin resistance,inhibits inflammation and induces FAO in skeletal muscle via AMPK-PPARδ pathway.However,whether BAIBA is involved in ER stress remains unknown.In the present study,the therapeutic effects of BAIBA on hepatic ER stress,apoptosis and glucose/lipid metabolic disturbance in type 2 diabetes were investigated.Objective1.To determin wheather BAIBA improve glucose/lipid metabolic disturbance,ER stress and apoptosis in type 2 diabetic mice;2.To determin wheather BAIBA have the improving effects on ER stress in HepG2 cells;3.To discuss the molecular mechanism of BAIBA on improving ER stress.MethodsMouse model of type 2 diabetes was induced in mice by combination of low-dose of STZ and HFD:Mice were randomly divided into three groups(n=7 for each group).The mice in one group were us ed as control;the mice in other two groups were used to induce type 2 diabetes,which received 4-hour’s fasting and subsequent injection of low-dose of STZ(120 mg/kg in 10 mM citrate buffer,pH 4.0,i.p.),and 3 weeks later,normal diet.Eight weeks after injection of STZ,the mice in two groups were mixed into one group,and then randomly were re-divided into two groups,which respectively received normal drinking water(STZ/HFD group)and 150 mg/kg/day of BAIBA dissolved in drinking water(STZ/HFD-BAIBA group)for 4 weeks.In tunicamycin-induced ER stress mouse model:Mice were injected with saline or BAIBA(300 mg/Kg,i.p.)0.5 h before DMSO or tunicamycin(2.5 mg/Kg,i.p.).The livers were collected for measurement 2 h after administration of DMSO or tunicamycin.In Ga1N/LPS-induced apoptosis mouse model:Mice were injected with saline or BAIBA(300 mg/Kg,i.p.)0.5 h before PBS or Ga1N(700 mg/kg,i.p.)plus LPS(100(μg/kg,i.p.).The livers were collected for measurement 6 h after administration of PBS or Ga1N/LPS.To induce insulin resistance,HepG2 cells were incubated with glucosamine(18 mM)for 18 h in serum-free medium,and followed by treatment with BAIBA(10 μM)for 24 or 48 h in the presence of glucosamine.To mimic the hyperglycemia in type 2 diabetic mice,high glucose was used to induce ER stress in HepG2 cells.The HepG2 cells were treated with high concentrations of glucose(30 mM)for 4 h,and then treated with BAIBA(10 μM)for 48 h in the presence of high glucose.To induce ER stress,HepG2 cells were incubated with thapsigargin(1 μM)or tunicamycin(3 μM)for 4 h in DMEM containing 10%FBS,and subsequently treated with different concentrations of BAIBA(1,10 or 100 μM)for 48 h in the presence of thapsigargin or tunicamycin.The main experimental methods including siRNA transfection,cell apoptosis analysis,insulin tolerance test(ITT),glucose tolerance test(GTT),serum sample preparation and biochemical test and analysis,liver TG test,protein extraction and western blot analysis,RNA extraction and Real-time analysis,oil red O staining,H&E staining,immunohistochemistry and TUNEL staining.Results1.Liver weight and the ratio of liver weight to body weight in STZ/HFD-BAIBAmice were reduced compared with STZ/HFD mice;2.The fast blood glucose and expressions of two key enzymes of gluconeogenesis in STZ/HFD-BAIBA mice were reduced compared with STZ/HFD mice,the phosphorylation of Akt(Ser473)、IRS-1(Tyr632)、IRS-1(Ser307)in STZ/HFD-BAIBA mice were restored compared with STZ/HFD mice;3.The concentration of serum TG、TCH、FFA and LDL-C,hepatic lipid accumulation and expressions of key makers of lipogenesis in STZ/HFD-BAIBA mice were reduced compared with STZ/HFD mice;4.The protein levels of CHOP、GRP78 and ATF4,the phosphorylation of eIF2a and JNK,the ratio of Bax to Bcl-2 and the mRNA levels of caspase-3 and caspase-9 in the liver of STZ/HFD-BAIBA mice were reduced compared with STZ/HFD mice;5.BAIBA reduced the protein levels of ER stress markers in glucosamine-,thapsigargin-,tunicamycin-or high glucose-induced ER stress in HepG2 cells;6.The eIF2a phosphorylation and ATF4 expression or caspase-3 and caspase-9 protein levels were increased in the liver of tunicamycin-treated mice or Ga1N/LPS-induced mice,which were restored by the pretreatment with BAIBA;7.The inhibitory effects of BAIBA on eIF2a phosphorylation and GRP78 expression were reversed by knockdown of AMPK with siRNA in the glucosamine-induced ER stress in HepG2 cells.Conclusions1.BAIBA attenuates hepatic ER stress,apoptosis and glucose/lipid metabolic disturbance in the mice with STZ/HFD-induced type 2 diabetic mice;2.BAIBA alleviates glucosamine-,high glucose-,thapsigargin-or tunicamycin-induced ER stress in HepG2 cells.AMPK signaling is involved to the role of BAIBA in attenuating ER stress;... |
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