Activation of Bile Acid Signaling Shapes the Gut Microbiota to Improve Diabetes and Fatty Liver Disease

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1 Activation of Bile Acid Signaling Shapes the Gut Microbiota to Improve Diabetes and Fatty Liver Disease John Chiang, Ph.D. Northeast Ohio Medical University Rootstown, OH, USA International Conference on Diabetes and Metabolism Seoul, South Korea October 3, 28

2 Conflict of Interest Disclosure Nothing to disclose

3 Bile Acid Biology And Physiology End products of cholesterol catabolism in the liver: Classic and alternative pathways. Physiological detergents required for intestinal absorption of nutrients, drugs and xenobiotics. Activate bile acid receptors FXR and membrane G- protein-coupled receptor TGR5 (Gpbar). Intestinal FXR and TGR5 crosstalk and gut microbiota regulate lipid, glucose and energy homeostasis and protect against obesity and diabetes. Bile acids as therapeutic drugs for diabetes, obesity and NAFLD

4 Bile Acid Synthesis Entero-hepatic circulation CA CYP8B Classic Pathway: Cholesterol CYP27A Taurine/Glycine Conjugation CYP7A CDCA Alternative pathway: CYP27A Tα-MCA (3α, 6β, 7α) CYP7B Cyp2c7(mice) Epimerase Primary bile acid synthesis Tβ-MCA (3α, 6β, 7β) Ileum Colon TCA (3α,7α,2α) CA BSH 7α-HSDH DCA (3α,2α) LCA (3α) UDCA (3α, 7β) Microbiota TCDCA (3α, 7α) CDCA Epimerase Secondary bile acid synthesis

5 Mechanisms Of Bile Acid Feedback Regulation : The Gut To Liver Axis In Bile Acid Homeostasis Cholesterol Tight junction CYP7A ABCG5 Phospholipid CY8BABCG8 MDR3 CYP27A Bile Bile Hepatocytes ERK SHP CDCA CA FXR BSEP MRP2 Bilirubin Glutathione Bile acids NTCP FGFR4 β-klotho Na + OSTα/β MRPs OATPs Portal blood OSTα/β Sinusoidal side Enterocytes FGF9 CDCA FXR IBABP ASBT Na + Apical side

6 TGR5 Signaling In Metabolic Regulation TGR5 LCA,DCA NH 2 Gαs Protein-Coupled Receptor Intestine barrier: Anti-inflammation - TNFα, NFκB Protect against: Colitis, IBD, Atherosclerosis Intestine L cells: GLP- secretion GTP GDP Pancreatic Β cells: Increase insulin Sensitivity G s ATP COOH AC camp Adipose tissue browning: DIO 2, UCP-/2, PGC-, Prmd6. Energy Metabolism, Oxphor Brain: Growth hormone, Fatty acid oxidation Cholangiocytes: Proliferation, Inflammation Cholangiocarcinoma Reduce weight

7 Non-alcoholic Fatty Liver Diseases (NAFLD) Obesity The second leading cause of liver cancer (26% HCC in US) Metabolic syndromes T2D, CVD, Cancer NASH: Non Alcoholic Steatohepatitis First hits: High fat diet Obesity Second hits: Insulin resistance Visceral fat lipotoxicity Oxidative stress Cholesterol Inflammation Fibrosis J C Cohen et al. Science 2;332:59-523

8 Bile Acids are Therapeutic Agents FXR agonists anti-inflammation, cholestasis, NASH, diabetes, obesity - INT ethyl-CDCA; obeticholic acid (OCA): approved for PBC. - Fexaramine ()- an intestine-restricted FXR agonist FXR antagonist - Obesity, Diabetes - Gly-MCA, UDCA TGR5 agonists anti-inflammation; obesity, NASH - INT-777: 6α-ethyl-23(S)-methyl-CA Dual FXR and TGR5 agonists NASH? - INT-767 Dual FXR & TGR5 agonist: EC 5 =.3 μm, FXR.63 μm, TGR5 FXR agonist: OCA, OCALIVA EC 5 =.99 μm TGR5 agonist: EC 5 =.82 μm TGR5

9 FXR And TGR5 Dual Agonist INT-767 Improves Hepatic Bile Acid, Glucose And Lipid Metabolism INT767 activates FXR and TGR5 to stimulate intracellular Ca 2+ and camp, and GLP- secretion to improves hepatic glucose and lipid metabolism. INT 767 reduces TCA, while increases TMCAs, and reduce hydrophobicity of bile. FXR induces TGR5 expression and GLP- procession. INT-767 reduces weight and lipogenesis, and improve insulin sensitivity in DIO mice. Activation of both FXR and TGR5 may be an effective therapy for treating hepatic steatosis, obesity and diabetes. Pathak et al. J Biol Chem, 292:55, 27

10 Paradoxical Roles Of Intestinal FXR In Metabolic Regulation In conventionally-raised mice, intestinal FXR induces FGF5 to inhibit bile acid synthesis, while in germ-free mice, T-α/β-MCA are increased to antagonize intestinal FXR and increase bile acid synthesis (Sayin, et al., Cell Metab 23). Antioxidant, tempol remodels microbiome to increase T-α/β-MCA to antagonize intestinal FXR signaling and reduce DIO (Li, Nature Commun, 23). Deficiency of intestinal Fxr protects mice from DIO and diabetes (Jiang, et al., JCI, 25). Gly-MCA, an intestine-restricted FXR antagonist, inhibits Intestinal FXR and improves DIO and diabetes (Jiang, et al., Nature Comm. 25). Fexaramine activates intestinal FXR to promote adipose tissue browning and improve glucose and insulin tolerance (Fang, et al., Nature Med, 25).

11 Intestine FXR Agonist Fexaramine Shapes The Gut Microbiota To Regulate Liver Metabolism Rationale: Both intestinal FXR agonist and antagonist have been shown to improve metabolic disorders. What causes differential effects of intestinal FXR signaling in improving metabolism? Hypothesis: Intestinal FXR agonists and antagonists may shape the gut microbiota differently to improve hepatic metabolism and disorders. Approach: Study fexaramine () effects on gut microbiota and bile acid, lipid, glucose and adipose tissue energy metabolism in Wt, Fxr -/- and Tgr5 -/- and db/db mice. Pathak, et al. Hepatology, 28, 68:

12 Stimulated Intestinal FXR Target Gene, But Not Liver FXR-Target Gene Expression In WT Mice Ileum Liver Relative mrna Expression Relative mrna expression CYP7A CYP8B CYP7B Calnexin CYP27A GAPDH

13 Stimulated Glucose-Induced GLP- Section In WT, But Not In Fxr -/- And Tgr5 -/- Mice GLP- secretion assay Wt Tgr5 -/- Fxr -/- Serum GLP- (pm) min Serum GLP- (PM) min vehicle Serum GLP- (PM) min vehicle Both intestinal FXR and TGR5 are involved in -stimulated GLP- secretion from L cells.

14 Improved Glucose Tolerance And Hepatic Insulin Sensitivity In WT Mice Oral Glucose Tolerance Test Serum Glucose (mg/dl) Time (min) Insulin Tolerance Test Serum Glucose (mg/dl) Time (min) Hepatic insulin signaling pakt 473 AKT Histone pacc ACC pacc/acc) pakt/akt Serum FGF-2 (ng/ml) FGF-2

15 Fexaramine Promoted White Adipose Tissue Beiging In WT Mice WT IHC staining Relative mrna Expression H&E staining Relative mrna Expression Fxr -/ Tgr5 -/-

16 Dose Not Alter Total Bile Acid Pool Size But Increases TLCA In Bile In WT Mice Bile Acids (µmole) Gallbladder bile acid concentration Intestine Gallbladder Liver Total Gallbladder bile (mm) Hydrophobicity Index

17 FX Induced LCA-Producing Bacteria In The Gut Microbiota Of WT Mice A. PCA Analysis B. Heat map C. LCA producing Bacteria p=.6 Mouse # V Fexaramine Acetatifactor Bacteroides Genera

18 Synthesis Of LCA By Gut Bacteria 7 -dehydroxylase LCA (3α) TCDCA BSH CDCA Acetatifactor (3α,7α) Bacteroides 7 -dehydroxylase epimerase UDCA (3α, 7β) Genus Acetatifactor: anaerobic, Gram +, Phylum-Firmicutes, class-clostridia; high BSH and 7 - and 7 -dehydroxylase activities. Bacteroides: anaerobic, Gram -, Phylum-Bacteroidetes, class-bacteroidia; high BSH and 7 - and 7 -dehydroxylase activities.

19 Antibiotics Reversed -Stimulated GLP- Secretion and Reduced Bile Acid Pool in WT Mice Antibiotics (ABX): Ampicillin (g/l), Vancomycin (.5g/l), Neomycin (g/l), Metronidazole ( g/l) Serum GLP- (PM) min +ABX +ABX Serum glucose (mg/dl) min +ABX +ABX Bile acid (µmole) ABX +ABX Total bile acid pool (µmole) 2 5 5

20 Antibiotics Abolished DCA And LCA Production In WT Mice Gallbladder bile % Conjugated Gallbladder Bile Acid % Conjugated gallbladder bile acid % Unconjugated Gallbladder Bile Acid ABX +ABX Serum %Serum Bile Acid ABX +ABX Hydrophobicity Index ABX +ABX

21 Antibiotics Reversed Effects On White Adipose Tissue Beiging, But Not Intestinal FXR Activity In WT Mice iwat Relative mrna Expression.5 +ABX +ABX.5 Ileum Relative mrna Expression 7 +ABX 6 +ABX Liver Relative mrna Expression ABX +ABX CYP8B CYP7A CYP7B Calnexin

22 Stimulated GLP- Secretion, Improved Glucose And Insulin Sensitivity, and Ileum FXR Activity In db/db Mice GLP- secretion OGTT ITT Serum GLP- level (pm) db/db- db/db min Relative mrna expression Glucose (mg/dl) min db/db-vehicle db/db- vehicle Serum Glucose mg/dl db/db- db/db min Fxr Shp Fgf5 Ostα Ostβ Pc/3 Asbt Gcg

23 Reduced Serum and Hepatic Lipids in db/db Mice Fat-mass/bodyweight (g) db/db+vehicle db/db+ Lean-mass/bodyweight(g) Serum cholesterol (mg/dl) Hepatic Triglycerides ( mg/g) Serum Triglycerides Serum FGF-2 (ng/ml) FGF-2 Serum Free Fatty Acids (mmol/l) Liver Free Fatty Acids (mmol/mg)

24 Promoted Adipose Tissue Browning In db/db Mice BAT swat ewat iwat Relative mrna Expression 3 2 db/db+ db/db- Relative mrna Expression 2 db/db+.8 db/db Relative mrna Expression Relative mrna Expression Db/db +vehicle Db/db + ingwat iwat db/db+veh db/db+ UCP Tubulin epiwat

25 Serum cholesterol Antibiotics Reversed Beneficial Effects In db/db Mice Serum triglycerides (mg/dl) Liver triglycerides Liver cholesterol (mg/µg) Serum FGF-2 (ng/ml) Serum GLP-(pM) GLP- secretion db/db+abx- db/db+abx Min Serum glucose (mg/dl) OGTT db/db-abx+vehicle db/db-abx Min Serum glucose (mg/dl) ITT db/db+abx- db/db+abx Min

26 Antibiotics Reversed Effects on Adipose Tissue Browning in db/db Mice Ileum ingwat Liver db/db + ABX+vehicle Relative mrna expression Fxr db/db+abx+ Shp Fgf5 Ostα Ostβ Pc/3 Asbt Gcg Relative mrna expression Prdm6 Ucp- Ucp-2 Ucp-3 db/db+abx-vehicle db/db+abx- Cptβ Pgc-α Relative mrna expression Cyp8b Cyp7a Cyp27a Srebpc Dgat Fas Srebp2 Scd Shp Hnf4α

27 Fexaramine Shaped Gut Microbiota To Stimulate TGR5/ GLP- Signaling To Improve Hepatic Metabolism, Insulin Resistance And Reduce Weight Fex activated intestinal FXR and TGR5 signaling to stimulate GLP- secretion and improve insulin sensitivity. Fex promoted adipose tissue browning via activation of TGR5 to reduce weight. Fex induced BSH and LCA producing bacteria in gut microbiota. activation of TGR5/GLP- signaling may play a major role in improving lipid metabolism and metabolic disorders. Activation of intestinal TGR5 signaling may have therapeutic potential for treating metabolic diseases. Pathak, et al. Hepatology, 28, 68:

28 Conclusion FXR agonists and antagonists shape the gut microbiota differently: - Gly-MCA increases Firmicutes to Bacteroidetes ratio to inhibit BSH and increase T-α/βMCA to antagonize FXR/FGF5 signaling and decrease ceramides (Frank Gonzales Lab). - Fexaramine induces BSH and 7 /β-dehydroxylase activities to increase LCA, which activates TGR5/GLP- signaling and adipocyte beiging and energy metabolism (Pathak et al, Hepatology, 28). Both intestinal FXR agonists and antagonist may have beneficial effects on insulin resistance and obesity by shaping the gut microbiota differently.

29 Mechanisms of Intestinal FXR and TGR5 and Gut Microbiota In Improving Metabolism NTCP SHP Liver BA Chol CYP7A FXR BA Adipocyte Beiging: FGF2 FGFR4 BSEP GB FGF2 TGR5 LCA FGF5 BA OSTa/b TβMCA FXR DCA TCDCA TCA BA ASBT Ileum L cells LCA TGR5 camp colon GLP- Pancreas: Insulin sensitivity Acetatifactor Bacteroides

30 Acknowledgements Preeti Pathak, Ph.D. Shannon Boehme, M. S. Jessie Ferrell, Ph.D. Collaborators: Frank Gonzalez, Ph.D., (NCI) Andrew Patterson, Ph.D.,(PSU) RDK R37DK