Clostridium Butyricum CGMCC0313.1 Improve Diabetes And Associated Metabolic Dysfunction

Clostridium butyricum were considered as probiotics,which can change the gut environment to a less proinflammatory immunological milieu.Patients with type 2diabetes（T2D）have decreased butyrate-producing bacteria,and healthy children have ahigherproportionofbutyrate-producingbacteria in their microbiomes compared with children expressing at least one beta islet cell autoantibody.In addition,it has been earlier reported that butyrate improves insulin resistance,fasting hyperglycemia and reduces the incidence of diabetes.However,there is no evidence to date that any butyrate-producing bacteria protect against diabetes.Previous studies have shown an induction of colonic regulatory T cells（Tregs）by indigenous C lostridium species,and gut lymphocytes are able to migrate to pancreaticisletsin an inflammatory environment.Thus,wespeculate that supplementation with the butyrate-producing bacteria may exert benifical effects to diabetes.Diabetes is recognized as the world’s third largest fatal disease.Both the incidence of diabetes and the number of patients with diabetes around the world are increasing.In addition,diabetes can not only be improved,not cured.Our study suggests that CB0313.1 may act as a beneficial probiotic for the prevention and treatment of hyperglycemia and associated metabolic dysfunction.In our study,we explored the new probiotic role of butyrate-producing bacteria,and provide new strategies for improving diabetes.Clostridium butyricum CGMCC0313.1（CB0313.1）was administered daily by oral gavage to lep^db/db mice（T2D mice model）for 5 weeks starting from 3 weeks of age,and to HF diabetic mice induced by high fat diet（HFD）plus streptozotocin（STZ）in C57BL/6J mice for 13 weeks starting from 4 weeks of age.CB0313.1 improved diabetic markers（fasting glucose,glucose tolerance,insulin tolerance,GLP-1 and insulin secretion）,and decreased blood lipids and inflammatory tone.Furthermore,CB0313.1 reversed hypohepatias and reduced glucose output.We also found that CB0313.1 modulated gut microbiota composition,enhanced peroxisome proliferator-activated receptor-γ（PPARγ）,insulin signaling molecules and mitochondrial function markers,suggesting that CB0313.1 may act as a beneficial probiotic for the prevention and treatment of hyperglycemia and associated metabolic dysfunction.Next,C B0313.1 was administered daily to female NOD mice from 3 to 45 weeks of age.The control group received equal sterile water.Fasting glucose was measured twice a week.Pyrosequencing of the gut microbiota and flow cytometry of mesenteric lymph node（MLN）,pancreatic and splenic immune cells were performed to investigate the effect of C B0313.1 treatment.Early oral administration of CB0313.1mitigated insulitis,delayed the onset of diabetes and improved energy metabolic dysfunction.Protection was associated with increased Tregs and rebalanced Th1/Th2/Th17 cells in pancreas.Furthermore,metagenomics gene sequencing revealedthat CB0313.1 enhancedFirmicutes/Bacteroidetesratio,enriched Clostridium-subgroups and butyrate-producing bacteria subgroups.Next,the NOD mice were used to clarify the associated hypoglycemic mechanism.Flow cytometry of MLN,PLN were performed to investigate the effect of C B0313.1 ontheTregsmigration.Intranuclear staining from MLN cells showed an increasing trend of Foxp3⁺abundance at 6 weeks.Moreover,at 9 weeks,a similar proportion of CD4⁺Foxp3⁺cells were also observed in the two groups.However,at 13 weeks of age the percentage of Tregs of NOD-CB mice markedly decrease vs NOD controls.We next detected the percentage of Tregs in PLN and found a significant increase.In addition,an increase ofα4β7⁺（the gut homing receptor）Tregs in the pancreatic lymph node（PLN）suggests that part of the Tregs in PLN might come from gut and associated lymphoid tissues-primed Tregs via lymphocinesia and blood circulation,suggesting that lymphocytes may circulate between gut and pancreas in NOD mice.Increased Tregs in PLN indicate higher immune tolerance locally in the pancreas due to CB0313.1 treatment.To confirm if CB0313.1 promoted the migration of gut-primed Tregs to PLN,we administrated NOD mice with FTY720 by gavage,which inhibits T cells circulation and traps them in the lymph node.In the PLN,FTY720 suppressed the accumulation ofα4β7⁺Tregs,however,did not significantly change the total proportion of Tregs.This study demonstrated that C B0313.1 limits the development of T1D primarily by modulation of intestinal immune homeostasis and induction of pancreatic Tregs in the early life of NOD mice.Our results provide the basis for future clinical investigations in preventing T1D by oral CB0313.1 administration.