The Research On Lipid-regulating And Glucose-regulating Mechanisms Of Lactobacillus Acidophilus NX2-6

Unhealthy dietary habits easily disrupt lipid and carbohydrate metabolism,subsequently leading to increased levels of lipid and glucose in the blood and the pathogenesis of obesity and insulin resistance.Nowadays,obesity and insulin resistance have been major health concerns all over the world.Meanwhile,obesity and insulin resistance also pose a great threat to human health and trigger other metabolic diseases.Traditional approaches to lose weight and reduce blood sugar include medication,exercise,dieting and insulin injection.Although these methods efficaciously reduce blood sugar and lipid profile,these approaches still have several limitations.With the development of molecular biology,many investigators have revealed the close relationship between the alteration in gut microbiota composition and the pathogenesis of obesity or insulin resistance.Therefore,improvement in intestinal micro-environment is an effective strategy to prevent or ameliorate diet-induced obesity and insulin resistance.Probiotics are health-promoting microorganisms that can improve intestinal micro-environment.Accumulating evidence has revealed that several probiotics possess ability to prevent and/or ameliorate diet-induced metabolic diseases.Screening for lipid-and glucose-lowering probiotics and exploring their ameliorative mechanisms are necessary to develop and utilize functional foods.1.In vitro lipid-and glucose-regulating mechanisms of Lactobacillus acidophilus NX2-6In this study,oleic acid(OA)was used to treat HepG2 cells to construct hepatic steatosis model and these cells were also treated with cell-free extract(CFE)from lactic acid bacteria.We discovered that CFE from Lactobacillus acidophilus NX2-6 significantly reduced triglyceride content,but did not obviously change glucose uptake and cholesterol content in the cells.Western blot analysis exhibited that CFE notably decreased the expression levels of FAS,Srebp-lc,ACC,SCD-1 and DGAT1,but increased the protein levels of HSL and CPT-1α in the hepatic cells,suggesting that CFE inhibited de novo lipogenesis and triglyceride synthesis,and promoted fatty acid oxidation and triglyceride hydrolysis.CFE also enhanced mitochondrial biogenesis and respiratory chain function,subsequently improving energy metabolism.CFE augmented CAT activity and mitochondrial membrane potential,and reduced intracellular ROS production by upregulating Nrf-2 expression,further suppressing oxidative stress in the cells.Moreover,CFE reduced the secretion of TNF-α,IL-1βand IL-6 as well as suppressed inflammatory responses by downregulating the expression of NF-κB p50 and MAPK p-ERK.On the other hand,CFE promoted autophagy and mitophagy by upregulating the expression levels of Beclin,Bnip3 and ATG7,which indirectly alleviated OA-induced oxidative stress and improved energy metabolism.2.In vivo lipid-regulating mechanism of Lactobacillus acidophilus NX2-6In this study,high-fat diet(HFD)-fed mice were administered with 109 cfu of live(LHLA)or dead(DHLA)L.acidophilus NX2-6 once a day,respectively.In vivo experiment exhibited that LHLA and DHLA significantly reduced body weight,fat weight and liver index,and improved serum lipid profile in the HFD-fed mice.The oil red and H&E staining images as well as biochemical assay also showed that LHLA and DHLA decreased lipid content in the liver.Western blot analysis confirmed that LHLA and DHLA promoted triglyceride hydrolysis and fatty acid oxidation,but suppressed de novo lipogenesis and adipogenesis in the adipose tissue.Protein analysis also determined that LHLA and DHLA reduced hepatic lipid content by promoting fatty acid oxidation and inhibiting de novo lipogenesis in the liver.Meanwhile,LHLA and DHLA activated Adiponectin/AMPKα/PGC-1α/UCP1 and Parkin/PINK pathway,resulting in white adipose tissue browning,enhancing mitophagy and improving energy metabolism in the adipose tissue.LHLA and DHLA also promoted energy expenditure,autophagy and mitophagy in the liver by acting on FGF21/AMPKα/PGC-1α/NRF1,Parkin/PINK,Bnip3 and ATG5/Beclin-1 signaling pathway,eventually suppressing hepatic lipid accumulation.Moreover,LHLA and DHLA enhanced the synthesis and excretion of cholesterol and bile acids,both of which were involved in lipid metabolism.LHLA and DHLA did not obviously regulate key proteins involved in endoplasmic reticulum stress and antioxidant system in the adipose tissue.However,LHLA and DHLA upregulated key proteins related to antioxidant system in the liver,indicating that LHLA and DHLA effectively ameliorated HFD-induced non-alcoholic fatty acid liver(NAFLD).3.In vivo glucose-regulating mechanism of Lactobacillus acidophilus NX2-6In this study,high-fat diet(HFD)-fed mice were administered with 109 cfu of live(LHLA)or dead(DHLA)L.acidophilus NX2-6 once a day,respectively.In vivo experiment exhibited LHLA and DHLA significantly reduced fasting glucose level,HOMA-IR index and insulin concentration,but augmented glucose tolerance and serum FGF21 level in the HFD-fed mice.However,LHLA and DHLA did not obviously change the concentration of glycosylated hemoglobin and PYY in the serum.Western blot analysis revealed that LHLA and DHLA activated insulin signaling pathway in the liver and skeletal muscle,subsequently increasing insulin sensitivity.However,LHLA and DHLA did not regulate insulin signaling pathway in the adipose tissue.LHLA and DHLA also increased protein levels of GLUT2 and Glucokinase in the small intestine,which promoted glucose uptake and glycolysis.Meanwhile,LHLA notably decreased hepatic PEPCK expression and increased PEPCK level in the small intestine,suggesting that LHLA promoted gluconeogenesis in the small intestine and suppressed hepatic gluconeogenesis.Surprisingly,LHLA and DHLA did not obviously change glycogen content in the liver and muscle.Moreover,LHLA and DHLA did not regulate key proteins involved in function,apoptosis,inflammatory responses,endoplasmic reticulum stress and autophagy in the pancreas.These results fully elucidated that LHLA and DHLA reduced blood glucose by enhancing insulin sensitivity and promoting glycolysis,independent of insulin secretion.Immunofluorescent technique and western blot analysis showed that LHLA and DHLA increased protein levels of colonic tight junction proteins including ZO-1 and Occludin,which strengthened colonic barrier integrity and suppressed the translocation of opportunistic pathogens and free fatty acids into the blood.LHLA and DHLA also protected colonic tissues by suppressing inflammatory responses,and enhancing antioxidant capacity and promoting autophagy.High-throughput sequencing analysis showed that LHLA and DHLA increased microbial richness and diversity in the colon.In detail,LHLA and DHLA increased relative abundance of S24-7,Rikenella and Parabacteroides,and decreased the ratio of Firmicutes to Bacteroidetes,and the proportion of Paraprevotellaceae,Odoribacteraceae,Desulfovibrionaceae,Oscillospira,Alistipes,Odoribacter,Akkermansia,Desulfovibrio,Allobaculum and Helicobacter.Moreover,LHLA also promoted the proliferation of Bacteroidetes,Verrucomicrobia,Lactobacillaceae and Lactobacillus,but suppressed the growth of Firmicutes,Proteobacteria and Mucispirillum,suggesting that LHLA possessed a more desirable ability to regulate colonic microbiota.LHLA and DHLA also increased butyric acid production in the colon,a microbial molecule that can maintain intestinal health.LHLA and DHLA downregulated the expression levels of key proteins involved in NF-κB and MAPK signaling pathway in the liver and muscle,which further reduced the production of inflammatory cytokines and alleviated inflammatory responses.However,LHLA and DHLA did not change the expression of key proteins involved in NF-κB and MAPK pathway,cytokines and chemokines in the adipose tissue.In conclusion,L.acidophilus NX2-6 enhanced intestinal barrier integrity,suppressed colonic inflammation,promoted the proliferation of commensals,reduced the relative abundance of endotoxin-producing microorganisms and increased butyric acid production,which prevented translocation of antigens into the blood and subsequently suppressed systemic inflammation.On the other hand,L.acidophilus NX2-6 also suppressed inflammatory responses in the liver and muscle,leading to increased insulin sensitivity and glycolysis.4.In vivo lipid-and glucose-regulating mechanisms of Lactobacillus acidophilus NX2-6-fermented yogurtFirstly,the pasteurized milk was fermented by Lactobacillus acidophilus NX2-6 to obtain yogurt.During the 20-day storage period,the viable microorganisms,water-holding capacity,viscosity,acidity,intensity of puncture and main odour of Lactobacillus acidophilus NX2-6-fermented yogurt were not obviously changed at 4 ℃.In vivo animal experiment determined that L.acidophilus NX2-6-fermented yogurt not only reduced body weight,fat index and liver index,but also improved serum lipid profile.L.acidophilus NX2-6-fermented yogurt significantly reduced fasting blood sugar level and HOMA-IR index,but enhanced glucose tolerance.However,L.acidophilus NX2-6-fermented yogurt did not obviously change the concentration of cholesterol and total bile acids in the serum,and the content of glycogen in the liver and muscle.ELISA analysis showed that L.acidophilus NX2-6-fermented yogurt decreased the levels of endotoxin,TNF-α and IL-1β,but increased adiponectin level in the serum.Western blot analysis also revealed that L.acidophilus NX2-6-fermented yogurt upregulated the expression levels of fatty acid oxidation-related proteins(e.g.CPT-la and CPT-2),and downregulated the expression of key protein(FAS)involved in de novo lipogenesis,subsequently decreasing lipid content in the liver and adipose tissue.L.acidophilus NX2-6-fermented yogurt also activated Adiponectin/AMPKα/PGC-1α/UCP1 and Adiponectin/AdipoR1/APPL1/AMPKα/PGC-1α signaling pathway in the adipose tissue or liver and skeletal muscle,respectively,which improved energy metabolism.Moreover,L.acidophilus NX2-6-fermented yogurt reduced blood sugar level by initiating insulin signaling pathway in the liver and muscle,and suppressing insulin resistance.These observations highlighted the potential application of L.acidophilus NX2-6-fermented yogurt in suppressing lipid accumulation and reducing blood sugar concentration.