Circadian pathways as a mediator between alcohol and liver disease Shannon M. Bailey Department of Pathology University of Alabama at Birmingham Circadian Rhythm in GI Health & Disease Rush University Medical Center May 5 6, 2016 Chicago, IL
Talk Highlights Alcohol induced fatty liver disease Energy programs Glycogen, lipid, and mitochondrial metabolism in the liver Dynamic nature of liver metabolism & molecular circadian clock Alcohol disrupts the liver circadian clock Alcohol disrupts diurnal rhythms in energy metabolism processes Lipid and Glycogen Impact genetic disruption of the clock has on metabolism Hepatocyte specific BMAL1 knockout mouse model
Alcohol use & Alcoholic liver disease Alcohol use Top 10 cause of preventable death in US 4 th Place (2014, MMWR) Half of Americans drink alcohol 15 million heavy drinkers and 52 million binge drinkers Most prevalent cause of liver related illness and death 2 million alcohol related liver disease 15,000 20,000 deaths/year Increasing incidence of liver cancer No FDA approved therapies
Alcoholic liver disease complex spectrum of pathologies Early disease Late disease Steatosis: Fat accumulation Triglycerides Molecular Mechanisms of ALD: Steatohepatitis: Fat accumulation Inflammation Cell death Fibrosis/Cirrhosis: Excess ECM Collagen Lipid and glycogen alterations Oxidative stress Redox signaling Mitochondrial damage Bioenergetic stress Inflammation Fibrogenesis Circadian clocks
Alcohol disrupts energy metabolism pathways Lipid Triglyceride Carbohydrate Glycogen Mitochondria Bioenergetics Lipid and CHO Metabolism: Increased fat Decreased glycogen Bioenergetics: Decreased respiration and ATP mtdna damage Altered proteome Endpoints measured at one time point Snapshot of metabolism Liver metabolism is dynamic and changes during the day Liver is a different organ at different times of the day
Time of day dependent changes in metabolism Extrinsic and Intrinsic Factors BMAL1 : CLOCK PER & CRY Bass and Takahashi. Science (2010)
Many diseases are associated with a broken clock Desynchrony of Rhythms: Disrupted phase relationships Mismatch in metabolism = Disease Susceptible population Shift workers http://medicalxpress.com/news/2013 02 circadian clock linked obesitydiabetes.html Heart disease Dyslipidemia Hypertension Obesity Diabetes Insulin resistance Inflammation Cancer What about alcohol use disorders and pathologies?
Disruption in circadian rhythms and alcoholism Light dark phase shifts modulate voluntary ethanol intake in high alcohol drinking rats. Clark et al (2007), Alcohol Clin Exp Res Acute ethanol disrupts photic and serotonergic clock phase setting in mice. Brager et al (2011), Alcohol Clin Exp Res Clock gene polymorphisms in alcohol use disorders and alcohol consumption. Kovanen et al (2010), Alcohol Alcohol Increased alcohol consumption in shift work and long working hours. Schulter et al (2012), Int J Nurs Stud Virtanen et al (2015), BMJ Reduced expression of circadian clock genes in male alcoholic patients. Huang et al (2010), Alcohol Clin Exp Res Role of intestinal circadian genes in alcohol induced gut leakiness. Swanson et al (2011), Alcohol Clin Exp Res Summa et al (2013), PLoS One
Alcohol and Liver Clock Project Goal: Demonstrate the importance of disrupted clocks and metabolic rhythms in alcoholic liver disease Does alcohol consumption disrupt the liver molecular clock? What effect does chronic alcohol have on diurnal rhythms in energy metabolism? Lipid, glycogen, and mitochondrial metabolism Are these processes regulated by the liver clock? Hepatocyte specific BMAL1 Knockout (HBK) Mouse What impact does a disrupted clock have on alcohol related changes in metabolism?
Around the clock experimental design Male mice: C57BL/6J mice Hepatocyte specific BMAL1 KO Control littermates ZT 23 5 AM ZT 0 6 AM Lights on ZT 3 9 AM Control Diet Alcohol Diet Feeding 5 wk Lieber DeCarli Protocol 5 Dietwk Collect Tissues: 4 hr for 24 hr Diurnal oscillations (12 : 12 hr LD cycle) ZT 19 1 AM ZT 15 9 PM Active phase Lights off ZT 12 6 PM Inactive phase ZT 11 5 PM ZT 7 1 PM 3% or 4% ethanol (w/v) 22% or 29% daily calories Iso caloric pair fed controls Diets = 1 kcal/ml Six separate groups of mice ZT 3, 7, 11, 15, 19, and 23
Chronic alcohol alters clock gene rhythms in liver Relative Expression Bmal1 1.2 0.8 0.4 0.0-0.4 0 4 8 12 16 20 24 1.4 1.2 1.0 0.8 0.6 0.4 Clock 0 4 8 12 16 20 24 1.6 1.2 0.8 0.4 0.0-0.4 Rev-erba 0 4 8 12 16 20 24 Control Ethanol 8.0 Cry1 25 Per2 75 Dbp Relative Expression 6.0 4.0 2.0 0.0 0 4 8 12 16 20 24 ZT (hr) 20 15 10 5 0 0 4 8 12 16 20 24 ZT (hr) 60 45 30 15 0-15 0 4 8 12 16 20 24 ZT (hr) BMAL1 : CLOCK E box Gene Per1 3 Cry1 2 Rev erba Dbp Alcohol disrupts: Mesor & amplitude of all clock genes Acrophase of Cry1, Per2, Rev erba From: Filiano et al. PLoS One 2013
Alcohol and the Molecular Clock Chronic alcohol does not alter the central SCN clock no effect on clock gene rhythms Chronic alcohol influences the liver clock alters mean expression, amplitude, and phase (timing) of clock genes Chronic alcohol advances the liver clock SCN and Liver explants from Per2::Luc mice fed control and alcohol diets Per2 peaks earlier (~1.5 hr) in liver of alcohol fed mice No effect on SCN Per2 rhythms Chronic alcohol consumption desynchronizes the liver clock from the central clock in the SCN Circadian desynchrony may contribute to alcoholic liver injury
Chronic alcohol increases lipid and depletes glycogen in liver Control Our model: Lieber DeCarli mouse model of ALD Steatosis w/ mild inflammation Earliest stage of ALD Alcohol mg TAG/mg prot 5.0 4.0 3.0 2.0 1.0 Triglyceride * mg glycogen/g liver 60 50 40 30 20 10 Glycogen * 0.0 Control Ethanol 0 Control 1 Ethanol 2 Snapshot of metabolism Early morning, ZT 3 4 Inactive/sleep phase
Alcohol disrupts the diurnal rhythm in liver triglyceride 180 160 Control Ethanol 22 fold Liver triglycerides Control 30 Triglyceride ( g/mg liver) 140 120 100 80 60 40 2.4 fold TAGs ( g/mg liver) 25 20 15 10 5 0 0 4 8 12 16 20 24 ZT (hr) 20 0 0 3 6 9 12 15 18 21 24 ZT (hr) Diet, p<0.001 Time, p=0.011 D X T, p<0.001 n = 4 6 mice/time point/group
Summary Fattyacid trafficking genes in liver Many, many more targets Unpublished data From: DG Mashek, Adv Nutr 2013, 4
Glycogen storage and function Glucose Glycogen is the storage form of glucose in mammalian cells Stored predominantly in muscle and liver Glycogen Liver glycogen serves two major functions: Maintenance of blood glucose level Fuel reserve for ATP synthesis
Diurnal rhythms in glucose and glycogen metabolism genes 2.0 GLUT1 2.0 GLUT2 Relative Expression 1.5 1.0 0.5 Relative Expression 1.5 1.0 0.5 From: Udoh et al, AJP G&L, 2015 Relative Expression 0.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.0 0 4 8 12 16 20 24 ZT (hr) GCK Relative Expression 2.5 2.0 1.5 1.0 0.5 0 4 8 12 16 20 24 ZT (hr) GYS2 0.0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 ZT (hr) ZT (hr)
Alcohol disrupts the diurnal rhythm in liver glycogen 75 Control Ethanol Glycogen (mg/g liver) 60 45 30 15 0 0 4 8 12 16 20 24 ZT (hr) From: Udoh et al, AJP G&L, 2015 n = 4 6 mice/time point/group
Alcohol disrupts diurnal rhythm in liver glycogen Glycogen particle Dynamic organelle 40 50 proteins Glycogen targeted phosphatase Protein Phosphatase 1 PP1 GP and GS activities regulated by (de)phosphorylation pgp active & pgs inactive GP inactive & GS active Glycogen targeting proteins Seven GTPs Scaffold proteins target PP1 to glycogen particle From: Roach et al., Biochem J, 2012 Unpublished data
Summary Glycogen metabolism genes in liver Unpublished data Alcohol depletes liver glycogen Glycogen metabolism genes exhibit diurnal rhythms Rhythms are disrupted by chronic alcohol Glycogen
What effect does genetic disruption of the clock have on glycogen metabolism? Hepatocyte specific BMAL1 Knockout Mouse Model HBK mouse X BMAL1 CLOCK E box CG and CCG WT (Cre ): Alb Cre ( / ) Bmal1 (f/f) HBK (Cre +): Alb Cre (+/ ) Bmal1 (f/f) X Chow fed mice 12 wks of age, male BMAL1 Protein in Liver: BMAL1 Ponceau S n = 3 4 mice/time point/group Liver gene expression Clock controlled genes Fold Change 80 60 40 20 0 20 WT HBK DBP 0 4 8 12 16 20 24 ZT (hr)
Alcohol and clock mediated change in hepatic glycogen Unpublished data n = 4 7 mice/time point/group
Clock dependence of glycogen metabolism genes in liver 3 Factor ANOVA Results Alcohol Feeding Study H BMAL1 KO and control littermates 5 weeks Unpublished data
Summary Alcohol disrupts energy metabolism 1.2 0.8 0.4 0.0-0.4 Bmal1 0 4 8 12 16 20 24 Alcohol Consumption 12 Time of day 9 12 3 DeSynchrony = Disease Health 6 Triglyceride Accumulation Glycogen Depletion Fatty Liver Disease
Acknowledgements and Big Thanks! Lab members Telisha Swain Jennifer Valcin Marisol Gomez Umme Lena Rachel Finley Ashley Filiano Uduak Udoh Kelly Andringa Collaborators on Clock Project Karen Gamble Martin Young Collaborators at UAB Mito Group Scott Ballinger Gloria Benavides Corinne Griguer Claudia Oliva Victor Darley Usmar Maria De Luca Doug Moellering Jianhua Zhang Funding: www.uab.edu/medicine/pathology