Role Of Gut Microbiota In The Glucocorticoid-induced Glycolipid Metabolism Disorder

Background: Glucocorticoids(GCs)are widely used in the treatment of a variety of clinical diseases.However,both endogenous hypercortisolism(Cushing’s syndrome)and exogenous long-term/highdose glucocorticoid use result in abdominal obesity,hypertension,hyperglycemia,hyperlipidemia and other metabolic disorders,which severely limit the clinical application of glucocorticoids.Gut microbiota and its metabolites have been reported involved in the development of various metabolic diseases,but its role in glucocorticoid-induced glycolipid metabolism disorder remains unclear.Objective: To investigate the role and possible mechanism of gut microbiota and its metabolites in glucocorticoid-induced glycolipid metabolism disorder.Methods:(1)Twenty-three patients with Cushing’s syndrome(18female and 5 men,age 47.24±12.99 years)and 30 age-,sex-and BMImatched healthy controls(18 female and 12 men,age 45.03±6.69 years)were consecutively recruited.The differences in gut microbiota and plasma SCFAs concentrations between Cushing’s syndrome and controls were analyzed by 16 S r RNA sequencing and gas chromate-chromato-mass spectrometry(GC-MS).(2)A glucocorticoid-induced metabolic disorder mouse model was established.16 S r RNA sequencing and GC-MS were used to analyze the differences of gut microbiota,fecal metabolomics and serum short-chain fatty acid levels between model mice(Dex group,n=6)and control mice(Conrtol group,n=6),respectively.Glucose tolerance was evaluated by abdominal glucose tolerance test and insulin resistance was evaluated by insulin tolerance test.The expressions of the colon G proteincoupled receptors(GPRs),Proglucagon(Gcg)and proprotein convertase subtilisin/kexin type 1(Pcsk1)were detected by q PCR and the expressions of UCP1 and PRDM16 of subcutaneous adipose tissue were detected by immunohistochemistry,and the serum GLP-1 levels were detected by ELISA.(3)Antibiotic cleaning mouse model(ABX mouse)was constructed by broad-spectrum antibiotics.The glucose tolerance,insulin tolerance,fat distribution,intestinal flora distribution and other indicators of glucocorticoid-treated ABX mice(Abx+dex group,n=6),glucocorticoid-treated wild-type mice(Dex group,n=6),PBS treated ABX mice(Abx group,n=6)and PBS treated wild-type mice(Conrtol group,n=6)were compared.(4)ABX receptor mice were transplanted fecal samples from glucocorticoid-induced metabolic disorder model mice(dex+dex group,n=6)or PBS treated mice(dex+control group,n=6)to observe the effects of fecal bacteria transplantation on the recipient mice.Results:(1)Compared with Control group,Simpson and pielou＿e indexes in CS group were significantly decreased(P < 0.05);and the relative abundance of Firmicutes in CS group was significantly decreased,while the relative abundance of Proteobacteria was significantly increased(P<0.05).At the generic level,the relative abundance of EscherichiaShigella and Parabacteroides were significantly higher in CS patients.While Agathobacter,Anaerostipes,Eubacterium＿eligens＿group,Eubacterium＿hallii＿group,Lachnospiraceae＿ND3007＿group,Faecalibacterium and Lachnospira decreased significantly.Spearman analysis showed that the levels of Hb A1 c,SBP,DBP and cortisol were significantly positively correlated with Proteobacteria and EscherichiaShigella and significantly negatively correlated with Agathobacter,Anaerostipes,Eubacterium＿hallii＿group and Lachnospira.Cushing’s syndrome patients had lower serum concentration of propionic acid(0.151±0.054 vs.0.205±0.032μg/m L,P=0.039).And propionic acid level was negatively correlated with systolic blood pressure and cortisol levels(P<0.05).(2)(1)The contents of inguinal subcutaneous adipose tissue and epididymis white adipose tissue and the ratio of white adipose tissue to net body weight in Dex group were significantly higher than those in Control group(P<0.05);The blood glucose level and area under the curve in Dex group were significantly higher than those in Control group at 0min,15 min,30min,60 min,90min and 120 min of IPGTT test(P <0.01).Compared with Control group,the decrease range of blood glucose in Dex group was significantly decreased at 15 min,30min and 60min(P <0.01),and the area under the curve of ITT was significantly increased(P=0.019).(2)Compared with Control group,the Observed＿species index,Shannon index,ACE index and Beta diversity of Dex group were significantly decreased(P<0.05).At the phylum level,the relative abundance level of Proteobacteria in Dex group was significantly increased(8.9% vs 1.6%,P=0.009).LEFse analysis suggested that the dominant bacteria of mice in Dex group were Parasuttrerella and Parabacteroides.The dominant bacteria genera in the control group were Lachnospiraceae＿NK4A136＿group,Lachnospiraceae＿UCG＿006,Faecalibacterium and Odoribacter.There was significant positive correlation between Parasutterella and fasting blood glucose,inguinal and epididymal adipose tissue.Lachnospiraceae＿NK4A136＿group,Odoribacter was significantly negatively correlated with fasting blood glucose,the contents of inguinal and epididymal adipose tissue,while Faecalibacterium was significantly negatively correlated with fasting blood glucose.(3)Fecal metabolomics analysis showed that 196 kinds of negative ion metabolites and 250 kinds of positive ion metabolites were up-regulated in the feces of mice with glucocorticoid-induced glycolipid metabolism disorder,while the downregulated negative ion metabolites were 27,positive ion metabolites 99 in Dex group.The levels of tryptophan and related metabolites,succinic acid and branched amino acids,Lysophosphatidylcholine(LPC),phosphatidylcholine(PC)and butyric acid derivative-4-phenylbutyric acid were significantly up-regulated.(4)Serum acetic acid(2.72±0.43 vs 4.51±0.82 ug/ml,P=0.005),propionic acid(0.07±0.01 vs 0.10±0.02 ug/ml,P=0.011)and amount of total short chain fatty acids(2.91± 0.44 vs 4.73± 0.84 ug/ml,P=0.005)in Dex group were significantly decreased and significantly negatively correlated with fasting glucose.(5)The m RNA expression level of GPR41 receptor and Pcsk1 in colon,serum GLP-1 and UCP1 and PRDM16 expression levels in subcutaneous adipose tissue were significantly decreased in mice with metabolic disorder induced by glucocorticoids.(3)The contents of body weight,inguinal subcutaneous adipose tissue and epididymis adipose tissue in Abx+dex group were significantly lower than those in Dex group(P<0.05).The glucose tolerance test showed that the blood glucose values of Abx+dex group at 0,15,30,60 and 90 minutes and the area under the curve was significantly lower than that of Dex group(P<0.05).LEFse analysis showed that compared with Abx+dex group,Proteobacteria,Parasutterella and Parabacteroides were the dominant bacteria of mice in Dex group.(4)Compared with the dex+dex group,the contents of inguinal adipose tissue and epididymis adipose tissue of mice in dex+control group were significantly reduced and the m RNA expression of GPR41 and Pcsk1 in colon were significantly increased.Glucose tolerance test showed that the blood glucose at 30 min,60min,90 min,120min and the area under the curve in dex+control group were significantly lower than those in dex+dex group(P<0.05).Insulin tolerance test showed that the decrease range of blood glucose in dex+control group was significantly increased at 60 min,90min and 120 min,and the area under the curve was significantly decreased(all P <0.05).The relative abundance of Proteobacteria and Parasutterella of mice in dex+dex group was significantly increased,while the abundance of Lachnospiraceae and Lachnospireaceae-NK4A136-group was significantly lower than that in dex+control group.Conclusion:(1)Our study firstly found that both endogenous glucocorticoid increase in patients and exogenous glucocorticoid administration in mice could lead to gut microbiota dysbiosis,which was characterized by the increased abundance of Proteobacteria and decreased abundance of beneficial bacteria,especially short-chain fatty acid producing bacteria resulting in a decrease in serum propionic acid levels.(2)Our study firstly confirmed that glucocorticoids can cause changes in a variety of metabolites in mouse feces,resulting in significant increasement of succinic acid,tryptophan and related metabolites,amino acids,and lipid metabolites.(3)Gut microbiota might be involved in the occurrence of glucocorticoid-induced glycolipid metabolism disorder.Glucocorticoid induced glycolipid metabolism disorder may be associated with increased relative abundance of Parasutterella and decreased acetic acid/propionic acid levels caused by decreased abundance of short-chain fatty acid producing bacteria,especially Lachnospiraceae-NK4A136-group.Our study highly suggested the role of gut microbiota and metabolites in glucocorticoid-induced metabolic disorders,and provided new ideas for the prevention and treatment of glucocorticoid-induced metabolic disorders.