The impact of high fat dietinduced gut microbiota on circadian rhythm and obesity Vanessa A. Leone, Ph.D. Postdoctoral scholar Impact of circadian dysrhythmia in gastrointestinal diseases Circadian rhythms in GI health and diseases Chicago, IL
Prevalence of New Age disorders has increased over the past half century Obesity (BMI 3kg/m 2 ) Obesity (BMI 3kg/m 2 ) 1985 1994 2 21 213 No Data <1% 1-13.9% <14% 14 17.9% 18 21.9% 22.% 25.9% 26.% Type 2 Diabetes 1994 2 21 15% <2% 2% <25% 25% <3% 3% <35% 35% No Data <4.5% 4.5% 5.9% 6.% 7.4% 7.5% 8.9% >9.% Source: Behavioral Risk Factor Surveillance System, CDC 2
Development of obesity is multi-factorial and complex Sleep 3
Diet shapes gut bacteria profiles in humans Gut bacteria profiles A Different dietary intake results in differences in gut bacteria B A.) Burkina Faso, Africa Dietary intake, ages 1-6 672.2 996.1 kcal/day» Protein: 3.9-4.2g» Fat: 18.9-31.2g» Carbohydrate: 12.6 148.6g B.) European Union, Italy Dietary intake, ages 1-6 168.7 1512.7 kcal/day» Protein: 41.9 66.7g» Fat: 56.1 73.9g» Carbohydrate: 19. 29.g De Filippo et al. Proc Natl Acad Sci USA. 17. 14691 (21) 4
Germ-free mice are resistant to diet-induced obesity Standard environment + Microbes Germ-free environment - Microbes Low Fat diet High Fat diet Low fat or High Fat diet Lean Obese Lean 5
Western dietary intake shifts gut microbes altering host metabolism Pediatr Gastroenterol Hepatol Nutr. 213 March; 16(1): 22 27. 6
Hepatic genes up-regulated in germ-free (GF) vs. conventionalized mice Androgen and Estrogen Metabolism PXR/RXR Activation Methionine Metabolism LXR/RXR Activation Fatty Acid Metabolism Linoleic Acid Metabolism Circadian Rhythm Signaling Xenobiotic Metabolism Signaling Metabolism of Xenobiotics by Cytochrome P45 Biosynthesis of Steroids LPS/IL-1 Mediated Inhibition of RXR Function p<.5 3 6 9 Leone, et al., Cell Host & Microbe. 215 7
Relationship between circadian clock and nuclear receptors Circadian genes Nuclear receptors Leone, et al., Cell Host & Microbe. 215 8
Circadian clock networks regulate daily metabolic functions Bass, J. Nature 212 9
Hypothesis Diet-induced gut microbiota provide critical inputs that regulate circadian gene networks, affecting metabolic outcome. Do GF mice exhibit altered circadian gene expression patterns on low fat (regular chow) and high fat diets? Do gut microbes exhibit diurnal patterns, and are these patterns altered under high-fat feeding conditions? Bass, J. Nature 212 If so, how are these patterns regulated? 1
1 28: ; 9+: <86+=>? Germ-free mice are resistant to high fat (HF) diet-induced obesity D<E5F8G+G564HI J9856+=3G<EK7<L? "#& "#! ""& ""! "! & "!! RC Body weight RC ( ) * +,+- * ( ) * +,+. * / * +,+- * / * +,+. * ' &! " # $ % & 1 2234+56+7829 "C "B "A "& "% "$ "# Caloric consumption "" ( ) * +,+- RC * ( ) * +,+. * / * +,+- RC * / * +,+. * "!! @& "@! "@& #@! #@& $@! $@& %@! %@& &@! 1 2234+56+7829 Leone, et al., Cell Host & Microbe. 215 n = 17 or 18 age-matched, individually housed male mice/trt group Bass, J. Nature 212 ***p<.1; **p<.1; *p<.5 11
Bmal1 expression (relative GAPDH) 3 4 5 &6$) 78.) 99*: ; $1.) <=+*>) $? 5 " @' 2 -./ 1("23' "44#/ 5(+' ".6$#7"(8 9 : ; <, Bmal1 3 4 5 &6$) expression 78.) 99*: ; $1.) (relative <=+*>) $? GAPDH) 5 " @' 2 -./ 1("23' "44#/ 5(+' ".6$#7"(8 9 : ; <, " ).A$) 78.) 99*: ; $1.) <=+*>) $? 5 " @' 2 - Cry1 ' =>("23' expression "44#/ 5(+' ".6$#7"(8 (relative 9 GAPDH) : ; <, " ).A$) 78.) 99*: ; $1.) <=+*>) $? 5 " @' 2 Cry1 - ' =>("23' expression "44#/ 5(+' (relative ".6$#7"(8 GAPDH) 9 : ; <, Bmal1 3 4 5 &6$) expression 78.) 99*: ; $1.) (relative <=+*>) $$? GAPDH) 5 " @' 2 -./ 1("23' "44#/ 5(+' ".6$#7"(8 9 : ; <, Bmal1 3 4 5 &6$) expression 78.) 99*: ; $1.) (relative <=+*>) $$? GAPDH) 5 " @' 2 -./ 1("23' "44#/ 5(+' ".6$#7"(8 9 : ; <, " ).A$) 78.) 99*: ; $1.) <=+*>) $? 5 " @' 2 Cry1 - ' =>("23' expression "44#/ 5(+' (relative ".6$#7"(8 GAPDH) 9 : ; <, " ).A$) 78.) 99*: ; $1.) <=+*>) $? 5 " @' 2 Cry1 - ' =>("23' expression "44#/ 5(+' (relative ".6$#7"(8 GAPDH) 9 : ; <, Germ-free mice exhibit altered central and peripheral circadian gene expression 3 =9:?64/ ) B*=<$' * "@#6.(< C8: +D=<= =3/ $A6.6* E9 B4! '! "! '! (! '! %! '! $! '! # SPF! " #$%$&# - RC( ) * +,+-. / 12 ( ) * +,+3 45612! SPF " #$%$' - HF #! '! &! '!!! " # $% $& %! %"! " # $% $& %! %"! "#$%"&"' () #* "(+! ), ( ) *+, ) - ).$/ * ) $1( / 2! '! %#! SPF!'! " 8#$%$&# - RC! '! %" SPF! " #$%$' - HF #( ) * +,+-. / 12 ( ) * +,+3 45612! '! %!! '! "! '! $&! '! $%! '! "! '! % 3 =<6$%$! " # 3 =<6$%$? # " ).A$%$! " # -./ 1(D(E: F Bmal1-./- 1(D(8 GF F Cry1 - ' =>(D(E: - SPF F " ).A$%$? - ' # =>(D(8 F! '!! #! '! $! '!! "! '!!!! '!!! " # $% $&! " # $% $& %! ( ) *+, ) -! ) "#$%"&"'.$/ * ) $1( () / #* 2 "(+! ), %! %" %"! '! "! '! (! '! %! '! $! '!? # GF #$%$&# - RC 9 * +,+-. / 12 GF? #$%$' - HF # 9 * +,+3 45612! '! &! '!!! "! " # # $% $% $& $& %! %! %" %" ( ) *+,! ) "#$%"&"' - ).$/ * () ) $1( #* / "(+! 2 ), &*>). C#7"' Bmal1 - SPF Bmal1 - GF -./ 1(D(E: F -./ 1(D(8 F! '! 7! '! % Bmal1 Mediobasal Hypothalamus Liver Bmal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ass, J. Nature 212 n = 2 or 3 mice/time point/trt group; Zeitgeber (ZT) = lights on, 6am; ZT12 = lights off, 6pm! '! #! '! &! '! "! '! % 3 =<6$%$! " # 3 =<6$%$? # " ).A$%$! - ' =>(D(E: " # F " ).A$%$? - ' # =>(D(8 F! '! 7! '! %! '! $! '( %! " #$%$&#! '! %!! " #$%$' #( SPF ) * +,+- - RC. / 12! '%"! '! $& ( SPF ) * +,+3 - HF 45612! '$&! '! #! '! $%! '!! #! '!! "! '!!!! " # $% $& %! %"! " #! "#$%"&"' $& () #* "(+! %! ), %" ( ) *+, ) - ).$/ * ) $1( / 2! '! $%! '!! #! '!! " Cry1 - SPF Cry1! '!!! '!!!! '!!! '!!!! "! #" $% # $% $& $& %! %! %" %"! "! # " $% # $% $& $& %! %! %" %" ( ) *+, ) -! )"#$%"&"'.$/ * ) $1( () #* / 2"(+! ), ( ) *+, ) -!) "#$%"&"'.$/ * ) $1( ()/#* 2 "(+! ),! '! "! '! %! '( %! '%"! '$&! '! #! '! $%! '!! #! '!! "? #$%$&#! '! %!? #$%$' #! '! $& GF 9 * +,+- - RC. / 12! '! $% GF 9 * +,+3 - HF 45612! '!! #! '!! " Cry1 - GF Cry1 - GF Leone, et al., Cell Host & Microbe. 215 12
PC2 13.9% High fat diet elicits shifts in gut microbial membership PC2 13.9% PCoA: PC1 versus PC2 H F RC PCoA: PC1 versus PC2 HF RC Zeitgeber Time (ZT) = ZT 2 = ZT 6 = ZT 1 = ZT 14 = ZT 18 = ZT 22 PC1 56.2% PC1 56.2% Leone, et al., Cell Host & Microbe. 215 Bass, J. Nature 212 n = 2 or 3 mice/time point/trt group; Zeitgeber (ZT) = lights on, 6am; ZT12 = lights off, 6pm 13
Relative abundance Relative abundance High fat diet alters diurnal oscillations of specific 16s rrna operational taxanomic units (OTUs) Relative abundance Relative abundance Relative abundance Relative abundance Relative abundance Relative abundance RC oscillating OTUs 14 12 1 8 6 4 2 OTU #3666 - Lachnospiraceae SPF - RC SPF - HF 4 8 12 16 2 24 25 2 15 1 2 1 Zeitgeber Time (ZT) HF oscillating OTUs OTU #265793 - Oscillospira SPF - RC SPF - HF 4 8 12 16 2 24 Zeitgeber Time (ZT) * 125 1 75 5 25 OTU #2374993 - Lachnospiraceae SPF - RC SPF - HF 4 8 12 16 2 24 125 1 75 5 25 Zeitgeber Time (ZT) OTU #263272 - Ruminococcus SPF - RC SPF - HF 4 8 12 16 2 24 Zeitgeber Time (ZT) OTU #267411 - Lachnospiraceae 4 8 12 16 2 24 Leone, et al., Cell Host & Microbe. 215 Bass, J. Nature 212 n = 2 or 3 mice/time point/trt group; Zeitgeber (ZT) = lights on, 6am; ZT12 = lights off, 6pm 25 2 15 1 3 2 1 15 125 1 75 5 25 Zeitgeber Time (ZT) OTU #16734 - Oscillospira SPF - RC SPF - HF SPF - RC SPF - HF 4 8 12 16 2 24 Zeitgeber Time (ZT) 15 125 1 75 5 25 4 3 2 1 OTU #261365 - Ruminococcus SPF - RC SPF - HF 4 8 12 16 2 24 175 15 125 1 75 5 25 Zeitgeber Time (ZT) OTU #18535 - Oscillospira SPF - RC SPF - HF 4 8 12 16 2 24 Zeitgeber Time (ZT) 14
PC2 PC2 High fat diet alters diurnal oscillations of gut microbiota function PCoA: PC1 versus PC2 RC LF HF PCoA: PC1 versus PC2 RC LF HF Zeitgeber Time (ZT) = ZT 2 = ZT 6 = ZT 1 = ZT 14 = ZT 18 = ZT 22 Substrate RC Osillating HF Non-oscillating 1% 41% 59% 9% Sensitivity chemical RC Oscillating HF Non-oscillating 8% 36%$ 64%$ 92% Oscillating Non-oscillating Oscillating Non-oscillating PC1 PC1 Leone, et al., Cell Host & Microbe. 215 Bass, J. Nature 212 n = 2 or 3 mice/time point/trt group; Zeitgeber (ZT) = lights on, 6am; ZT12 = lights off, 6pm 15
High fat diet impacts diurnal patterns of known microbially-produced metabolites : ; 2<5=2)8! 7 >?@3)>A)B>C2C2D9 "$# "#! ""# "!! %$# %#! %"# %!! $# #! "#!.... &' ( )*)+, &' ( )*)- (! " # $% $& %! %" / 12345)617 )8/ 69... - # &)8! 7 : ;<3): =)>:?2?2@9 %"! $"! #"!.. &' ( )*)+,. &' ( )*)- (! "!! " # $% $& %! %" / 12345)617 )8/ 69 Leone, et al., Cell Host & Microbe. 215 Bass, J. Nature 212 n = 2 or 3 mice/time point/trt group; Zeitgeber (ZT) = lights on, 6am; ZT12 = lights off, 6pm 16
Per2 expression (relative GAPDH) Bmal1 expression (relative GAPDH) Microbial metabolites elicit a direct impact on hepatic circadian gene expression in vitro Butyrate H 2 S.7.6.5.4.3.2.1 * *** No trt 5mM Butyrate 8 16 24 32 4 48 Time after serum-shock (hours).7.6.5.4.3.2.1 ** No trt 1mM NaHS 8 16 24 32 4 48 Time after serum-shock (hours) Hepatic organoid.1.8.6.4 No trt 5mM Butyrate * *.1.8.6.4 No trt 1mM NaHS.2.2 8 16 24 32 4 48 Time after serum-shock (hours) Bass, J. Nature 212 8 16 24 32 4 48 Time after serum-shock (hours) Leone, et al., Cell Host & Microbe. 215 17
! "#$%&' ( )* %1 6! 7%-/ 84! "#$%&' ( )* %1 6! 7%-/ 84 Butyrate elicits a direct impact on hepatic circadian gene expression in vivo <. =84>/?/ @%AB$4A/ @/ 1 5? 214! " #$%&%' (' ) * 12! " #$%&%' (+* ) 14 312 Control Saline (PBS) Saline (PBS) 314 BUT ZT2 BUT ZT14 BUT Saline (PBS) ZT2 Saline (PBS) BUT ZT14 ZT 6 12 18 24 Bass, J. Nature 212. 12 C8D. -! " #$%& ' ( )*+*, -. ' ( )*+*, - /, -. / 1. 2%3-45$.. 9$%&%' (' +:. 4 / 6! " # $%&%' (; : + / 5 / /. / 4 6 5. 4! " #$%& ' ( )*+*, -. ' ( )*+*, - /, -. / 1. 2%3-45$ Leone, et al., Cell Host & Microbe. 215 18
Gut microbes sense dietary cues that translate into outputs that maintain circadian networks Dietary intake When How much What Gut microbiota Shift work Sleep apnea Jet lag Circadian Networks Microbial Oscillations Microbial signals Metabolome High fat diet Bass, J. Nature 212 19
High-fat diet induced gut microbiota lack outputs that are required for maintenance of circadian networks Dietary intake When How much What Gut microbiota Circadian Networks Microbial Oscillations signals Metabolome 2
University of Chicago Dr. Eugene Chang Dr. Mark Musch Dr. Brian Prendergast Dr. Betty Theriault Dr. Joseph Pierre Dr. Candace Cham Dr. Aaron Dinner Dr. Kristina Martinez Dr. Edmond Huang Dr. Yong Huang Dr. Sean Gibbons Dr. Anuradha Nadimpalli Acknowledgements Alan Hutchison Nathaniel Hubert, M.S. Elizabeth Zale Chang lab members Gnotobiotic animal care staff UW-Madison Dr. Kenneth Kudsk Dr. Aaron Heneghan Argonne National Lab Dr. Jack Gilbert Funding NIDDK P3 NIDDK DDRCC Gastro-intestinal Research Foundation (GIRF) Sunrise from the Knapp Center for Biomedical Discovery after a long circadian study 21
What might be contributing to the increasing prevalence of Western disorders? 22
FMT from lean donors improves insulin sensitivity in obese subjects 6 weeks *Improved symptoms of Type II Diabetes and Metabolic syndrome Lean male donors Obese male recipients BMI 3 Vrieze et al., Gastro. (212) 143:913-916 23
Germ-free mice exhibit altered metabolic response to Western diets compared to controls Backhed et al, 24; Backhed et al, 27 24