MOLECULAR BASIS OF INFLAMMATION AND INSULIN RESISTANCE IN OBESITY: CONTRIBUTION OF THE GUT MICROBIOTA. Mario J A Saad FCM-UNICAMP

(1)

MOLECULAR BASIS OF INFLAMMATION

AND INSULIN RESISTANCE IN OBESITY:

CONTRIBUTION OF THE GUT MICROBIOTA.

Mario J A Saad

FCM-UNICAMP

(2)

INSULIN

RESISTANCE

Hypothalamus

GIT

Muscle

Spleen

Adipose

Bone Marrow

INSULIN RESISTANCE IN DIO

(3)

IRS1/2 PO₄ Ras/Raf MEK ERK PI 3 kinase insulin PO₄ P P PIP₂ PIP₃ PP _{P PP} PDK AKT PO₄ PO₄ PO₄ FoxO1 GSK3 aPKC Glucose Uptake Gliconeogenesis Gene Transcription Protein Synthesis Cell Growth, Differentiation Glycogen Synthesis PO₄ PO₄ PO₄ SOS Grb2 Shc

INSULIN SIGNALING

(4)

0 100 150 200 250 300 350 C * (n mo l/g/m in ) 50 HFD

MOLECULAR MECHANISMS OF INSULIN RESISTANCE IN HIGH FAT DIET – 3 DAYS

Glucose uptake 0 20 40 60 80 100 120 C * (% ) pIRS-1 0 20 40 60 80 100 120 C * (% ) pAKT MUSCLE HFD HFD

(5)

MOLECULAR MECHANISMS OF INSULIN RESISTANCE – MODULATORS OF INSULIN SIGNALING

PI 3 Kinase S - S a S-S b ATP insulin S-S PP PO₄

IKK

b

JNK

IL6 – TNFa – FFA - AII HIGH FAT DIET

PO₄ PO₄ PO₄ PO₄ PO₄ P P

INSULIN

RESISTANCE

IRS1/2 PO₄

(6)

LPS

0 0,5 1,0 2,5 1,5 2,0 (EU /m L) HFD 3days *

LPS CIRCULATING LEVELS IN MICE ON HFD - 3 DAYS

(7)

IKK

b

JNK

HIGH FAT DIET

LPS

PO₄ PO₄ PO₄ PO₄ PO₄ P P

INSULIN

RESISTANCE

IRS1/2 PO₄

(8)

Duodenum101_-103 _cfu/ml Stomach 101_-103 _cfu/ml Jejunum 104_-107 _cfu/ml Cólon 1011_-1012 _cfu/ml

(9)

Another bacteria Firmicutes Firmicutes Bacteroidetes Bacteroidetes

LEAN

Another bacteria

OBESE

PROPORTION OF FIRMICUTES AND BACTEROIDETES IN LEAN AND OBESE MICE

(10)

PHENOTYPE

(11)

PHENOTYPE

(12)

MECHANISMS BY WHICH MICROBIOTA CAN

INFLUENCE OBESITY

(13)

LIVER

MECHANISMS BY WHICH MICROBIOTA CAN

INFLUENCE OBESITY

(14)

pAKT 0 20 40 100 60 80 - + - + GF (%) CONTROL *

LIVER

EFFECT OF HIGH FAT DIET -3DAYS ON INSULIN SIGNALING IN LIVER GERM-FREE AND CONTROL MICE

pIRS-1 0 20 40 100 60 80 - + - + GF (%) CONTROL *

(15)

pJNK 0 20 40 100 60 80 GF (%) CONTROL pIKK 0 20 40 100 60 80 GF (% ) CONTROL pIRS-1 Ser307 0 20 40 100 60 80 GF (%) CONTROL * * * Liver

EFFECT OF HIGH FAT DIET -3 DAYS ON INSULIN SIGNALING IN LIVER GERM-FREE AND CONTROL MICE

(16)

LPS

0 0,5 1,0 2,5 1,5 2,0 GF (EU /mL ) CONTROL *

SCFA- Acetate

0 1.0 2,0 5.0 3.0 4,0 GF (mmo l/L ) CONTROL *

EFFECT OF HIGH FAT DIET -3 DAYS ON

LPS

AND

SCFA

ON GERM-FREE AND CONTROL MICE

(17)

CLAUDIN OCCLUDIN ACTIN ZO-1 MIOSIN (% )

(18)

CLAUDIN OCCLUDIN ACTIN ZO-1 MIOSIN

ZO-1

0 20 40 60 80 100 120 GF CONTROL * (% )

ZO-1 EXPRESSION IN INTESTINE OF GERM-FREE MICE ON HIGH FAT DIET

(19)

6 sem

WEIGHT

0 5 10 15 20 25 30 TOM g TOM TLM TLM TOM 14 sem 600 400 200 0 0 40 80 120 Time (min) Bl oo d Glucose mg dl -1 ) TLM

GTT

METABOLIC CHARACTERISTICS OF LEAN MICE SUBMITTED TO MICROBIOTA TRANSPLANTATION FROM OBESE MICE

(20)

LPS

0 0,2 0,4 0,6 0,8 1 1,2 TFO C

pAkt

0 20 40 60 80 100 120

TFO _AntibTFO C TFO Antib TFO C _AntibTFO 0 10 15 20 25 30 35 (m g/kg /min ) 5 40 Glucose Clamp

INSULIN SENSITIVITY IN LEAN IN MICE SUBMITTED TO MICROBIOTA TRANSPLANTATION FROM OBESE MICE

EFEITO DE ANTIBIMTNZ+AMP+NEOM)

(EU/ml)

(21)

IKK

b

JNK

HIGH FAT DIET

LPS

PO₄ PO₄ PO₄ PO₄ PO₄ P P

INSULIN

RESISTANCE

IRS1/2 PO₄

(22)

INSULIN RESISTANCE IN HIV PATIENTS

(23)

Indivíduo obeso

Flora

Comensal

Epitélio Intestinal

Fígado _{Tec. Adiposo} _{Hipotal Muscle}

LPS LPS Indivíduo obeso Flora Comensal Fígado Adipose tissue

OBESE

Intestinal Micorbiota Liver

Short chain fatty acids:

ACETATE, BUTIRATE, PROP.

INSULIN

RESISTANCE

MECHANISMS BY WHICH MICROBIOTA CAN

INFLUENCE OBESITY

(24)

0 2 4 6 _* A CET A T O ( ng /ul )

ACETATE

CONT OB

CORRELATION BETWEEN SCFA LEVELS –

ACETATE- AND AMPK ACTIVITY IN OBESE MICE

0 20 40 60 _* A MP K A CTIVITY( a .u .) CONT OB 80

AMPK

(25)

EFFECT OF ACETATE ON AMPK ACTIVITY

0 30 60 90 * UA

CONT 30min 60min

ACETATE GAVAGE IV 30min

*

(26)

PI 3 quinase

AKT

S - S a S-S b ATP insulina

EFFECT OF AMPK ON GLUCOSE AND LIPID METABOLISM

S-S P P P P PO₄ PO₄ PO₄ PO₄ IRS1/2

AMPK

AGCC ACETATO GLUCOSE TRANSPORT

HEPATIC GLUCOSE OUTPUT LIPID

(27)

INSULIN RESISTANCAE AND LPS LEVELS IN OVERWEIGHT AND OBESE HUMANS

0 1 2 3 * 4 ** INSULIN LPS

(28)

(29)

MORPHOLOGY IMMUNOHISTOCHEMISTRY

EFECTO OF HIGH FAT DIET ON ADIPOSE

(30)

Adipose Tissue - LEAN Adipokines (adiponectin, leptin) M2 - Alternatively Activated Inflammatory Molecules (TNFα, IL-6, iNOS) Weight Gain

POLARIZATION OF MACROPHAGE IN ADIPOSE TISSUE

Adipose Tissue - OBESE

M1 - Classicaly Activated

(31)

M2

ACETATE

M1

EFFECT OF ACETATE ON POLARIZATION OF MACROPHAGE IN ADIPOSE TISSUE

Inflammatory Molecules (TNFα, IL-6, iNOS)

(32)

M2

EFFECT OF ACETATE ON M2 MACROPHAGE INFILTRATION IN

ADIPOSE TISSUE OF MICE ON HFD

Lean Obese

Obese + ACETATE

M2 macrophage

(33)

Adipose Tissue - LEAN Adipokines (adiponectin, leptin) M2 - Alternatively Activated Inflammatory Molecules (TNFα, IL-6, iNOS)

TREATMENT OF INSULIN RESISTANCE

-MODULATE THE POLARIZATION OF MACROPHAGE

Adipose Tissue - OBESE

M1 - Classicaly Activated

(34)

0 2 4 6 * Perc ent age -%

Akkermansia muciniphila

8 CONT DH DH+MET

EFFECT OF METFORMINE ON THE EXPRESSION OF DE AKKERMANSIA MUCINIPHILA IN COLON

(35)

0 2 4 6 * Perc ent age -%

Akkermansia muciniphila

8

CONT CLOZAPINE OLANZAPINE

EFFECT OF ANTIPSICOTHIC DRUGS ON THE

EXPRESSION OF DE AKKERMANSIA MUCINIPHILA

(36)

(37)

Bruno Geloneze Candida Parisi Kleber Franchini Lauro Kubota Lício Velloso

Maria Helena M Lima Patrícia Prada

Rui Curi

Sr. Luiz Janeri Sr. Jósimo Pinheiro

AGRADECIMENTOS

Alexandre Gabarra Oliveira Andrea Cariccili

Angélica Souza

Bruno de Melo Carvalho Dioze Guadagnine

Fernanda Pace Francine Mitzetneir Kelly Lima Calixto Natalia Tobar

Renata Bagarolli Tiago Araújo

(38)

RESIST À INSULINA (HOMA) E NÍVEIS DE LPS EM ADOLESCENTES COM SOBREPESO E OBESOS

0 1 2 3 * 4 ** HOMA

(39)

25 20 40 35 30 15 10 5 0 1974 1980 1986 1992 1998 2004 Prevalence of obesity Prevalence of ascaridiasis % Year

(40)

Cont. do apetite AKT HIPOTÁLAMO Prod. de glicose AKT FÍGADO Capt de glicose AKT MÚSCULO ADIPOSO Infiltração de macrófagos VASOS eNOS RESISTÊNCIA À INSULINA

RESISTÊNCIA À INSULINA – PAPEL DO TGI E ADIPOSO

TNF

a

IL6

TGI

LPS

(41)

INFILTRAÇÃO DE CÉLULAS NO ESTROMA

VASCULAR DO TECIDO ADIPOSO

Macrófago M2

Macrófago M1

Neutrófilo

Eosinófilo

(42)

Tec. Adiposo - MAGRO Adipocinas (adiponectina, leptina) M2 - Ativado Alternativamente M1 - Ativado Classicamente

Tec. Adiposo - OBESO

Moléculas pro-inflamatórias (TNFα, IL-6, iNOS)

Ganho de Peso

TRANSFORMAÇÃO DE MACRÓFAGOS

(43)

Flora

Comensal

Ácidos graxos de cadeia curta: ACETATO, BUTIRATO, PROP.

Fígado _{Tec. Adiposo} _Hipotál _Músculo

MECANISMOS QUE LIGAM FLORA INTESTINAL E RESISTÊNCIA À INSULINA – OBESIDADE

Flora

Comensal

Fígado Tec. Adiposo

Indivíduo obeso Flora Comensal Fígado

RESISTÊNCIA

À INSULINA

(44)

Flora

Comensal

Treg

LPS

Flora

Comensal

Flora

Comensal

Fígado

(45)

INFILTRAÇÃO DE CÉLULAS NO ESTROMA

VASCULAR DO TECIDO ADIPOSO

Macrófago M2

Macrófago M1

Neutrófilo

Cel T (Treg)

(46)

M2 - Ativado Alternativamente

M1 - Ativado Classicamente

TRANSFORMAÇÃO DE MACRÓFAGOS

INFLAMATÓRIOS-M1 EM REGENERATIVOS-M2.

AGCC

(47)

TRANSFORMAÇÃO DE MACRÓFAGOS INFLAMATÓRIOS –M1 EM REGENERATIVOS-M2

M2

Regenerativo

M1

Inflamatório

Treg

Acetato

(48)

M2

Treg

INFECÇÃO PELO STRONGYLOIDES VENEZUELENSIS A INFILTRAÇÃO DE M2 NO TECIDO ADIPOSO DE OBESOS

CONTROLE _OBESO OBESO + SV

(49)

EFEITO DA INFILTRAÇÃO DE CÉLS

Treg

NA DIFERENCIAÇÃO DO MACRÓFAGO EM M2 OU M1

M2

Treg

M1

Treg

(50)

ESTUDOS DE METAGENOMA DE MICROBIOTA EM DM2 145 F 345 M e F N, IG e DM2 N e DM2 Bactérias produtoras de butirato: Roseburia e F Praunitzi Bactérias produtoras de butirato: Roseburia e F Praunitzi Proteobactéria – LPS Bactérias - stress oxidativo Karlsson et al. Qi et al.

(51)

DROGAS QUE MODULAM FLORA

NOVAS ASSOCIAÇÕES E PERPECTIVAS NO ESTUDO DA MICROBIOTA

METFORMINA

ANTIPSICOTICOS ATÍPICOS – Clozapina - Olanzapina

DROGAS MODULADAS PELA FLORA

DIGOXINA

DOENÇAS CRÔNICAS –

Possível associaçao

OSTEOPOROSE – Massa Óssea HIPERTENSÃO ARTERIAL

(52)

25 20 40 35 30 15 10 5 0 1974 1980 1986 1992 1998 2004 OBESITY ASCARIDIASIS % YEARS

PREVALENCE OF ASCARIDIASIS AND

OBESITY IN CHILDREN IN BRAZIL

(53)

EFFECT OF ACUTE EXERCISE ON TLR4 ACTIVATION AND INSULIN SIGNALING IN MUSCLE OF MICE ON HFD

C _HFD 2h 16h HFD+EXE 0 20 120 AU 40 * 80 60 100 TLR4-MyD88

Muscle

C HFD 2h 16h HFD+EXE 0 20 120 U n id ad es ar b itr ár ia s 40 _* 80 60 100 # # pAKT

(54)

0 4 12 L/ min -1 8 Basal Exercise

EFFECT OF ACUTE EXERCISE ON BLOOD FLOW IN DIFFERENT ORGANS

(55)

LPS

0 0,25 0,5 0,75 1,0 1,25 C * HFD (EU /m L) HFD + EXE 2h 16h

EFFECT OF ACUTE EXERCISE ON CIRCULATING

(56)

Flora

Comensal

Flora

Comensal

Indivíduo obeso Flora Comensal Fígado

RESISTÊNCIA

À INSULINA

(57)

0 10 15 20 25 30 35 CONT * (m g/kg /min ) 5 STRG

EFFECT OS STRONGYLOIDES VENEZUELENSIS INFECTION ON INSULIN SENSITIVITY IN MICE ON HFD

40 CONT * STRG CHOW HFD

Glucose Clamp

(58)

0 30 40 50 60 70 80 CONT * (% ) 20 STRG 90 CONT * STRG

LPS

0 10 15 20 25 30 35 CONT * (EU/ L) 5 STRG 40 CONT * STRG

% Firmicutes

(lactobacilus)

CHOW HFD _CHOW HFD

EFFECT OF STRONGYLOIDES VENEZUELENSIS INFECTION ON INTESTINAL MICROBIOTA AND ON LPS CIRCULATING LEVELS

(59)

HL HL+ ANT C pAMPK 0 20 40 60 80 100 120 HL C 0 20 40 60 80 100 120 HL+ ANT pAMPK

FÍGADO

MÚSCULO

ATIVAÇÃO DA AMPK EM FÍGADO E MÚSCULO DE ANIIMAIS OBESOS TRATADOS COM ANTIBIÓTICOS

(60)

Alexandre G Oliveira Andréa Moro Caricilli Angélica Camacho

Bruno de Melo Carvalho Dioze Guadagnini

Francine Mittestainer Fernanda Pace

Kelly Lima Calixto Natalia Tobar

Patty Karoll Picardi Renata Bagarolli Tiago Araujo José B C Carvallheira Eduardo Ropelle Lício A Velloso A C Boschero Rui Curi Patrícia O Prada CNPq – FAPESP Instituto Nacional de Obesidade e Diabetes ACKNOWLEDGEMENTS

(61)

(62)

MODULAÇÃO DA FLORA INTESTINAL

DIETA _PROBIÓTICOS MICROBIOTA TRANSPLANTE DE FLORA ANTIBIÓTICOS

(63)

MODULATION OF GUT MICROBIOTA BY PROBIOTICS

COMPOSITION OF PROBIOTIC

Lactobacillus rhamnosus,

Lactobacillus acidophillus

Bifidobacterium bifidum.

(64)

0 5 10 15 * G luco se Utilization (mg/kg/min)

Hyperinsulinemic

Euglycemic Clamp

20 CONT HFD HFD+PB

EFFECT OF PROBIOTICS ON INSULIN SENSITIVITY IN MICE ON HFDC

(65)

HF HF+PB C

pJNK

0 20 40 60 80 100 120 0 200 600 1200 AU 800 * 1000 400 - + - ₊ - + Cont Insulin HF _{HF + PB}

pAKT

AU

EFFECT OF PROBIOTICS ON INSULIN SIGNALINGIN MUSCLE OF MICE ON HFD

(66)

BACTEROIDETES

EFFECT OF PROBIOTICS ON THE MICROBIOTA OF MICE ON HFD

(67)

ESTUDOS DE METAGENOMA DE MICROBIOTA EM DM2 145 F 345 M e F N, IG e DM2 N e DM2 Bactérias produtoras de butirato: Roseburia e F Praunitzi Bactérias produtoras de butirato: Roseburia e F Praunitzi Proteobactéria – LPS Bactérias - stress oxidativo Karlsson et al. Qi et al.

(68)

ER STRESS AND INSULIN RESISTANCE

PO₄ PO₄

INSULIN

RESISTANCE

IRE1

ATF6 PERK

IKK

b

, JNK

iNOS

PO₄ IRS1/2 P P IRS1/2 PI 3 kinase NO

(69)

0 5 10 15 * G luco se flu x (m g/kg /min )

Glucose Clamp

20 CONT OB OB+PB

EFFECT OF PROBIOTICS ON INSULIN SENSITIVITY IN OBESE MICE

(70)

0 10 15 20 25 30 35 CONT * (m g/kg /min ) 5 STRG 40 CONT * STRG LEAN OBESE

Glucose Clamp

EFFECT OF STRONDILOGIDIASIS ON MICROBIOTA AND ON INSULIN SENSITIVITY IN OBESE MICE

(71)

0 30 40 50 60 70 80 CONT * (% ) 20 STRG

EFFECT OF STRONDILOGIDIASIS ON MICROBIOTA AND ON SERUM LPS IN LEAN AND OBESE MICE

90 CONT * STRG LEAN OBESE

SERUM LPS

0 10 15 20 25 30 35 CONT * (EU/ L) 5 STRG 40 CONT * STRG LEAN OBESE

% Firmicutes

(lactobacilus)

(72)

0 1 2 4 * 3 INSULIN (ng/dL) HFD HFD+PB 0 50 100 200 * 150 GLUCOSE (mg/dL) HFD HFD+PB 0 10 15 25 * 20 GLP1 (pmol/L) HFD HFD+PB

EFFECT OF PROBIOTICS ON PLASMA GLUCOSE HORMONAL PARAMETERS IN MICE ON HFD

(73)

CLAUDIN OCCLUDIN ACTIN ZO-1 MIOSIN

ZO-1

EFFECT OF PROBIOTICS ON ZO-1 EXPRESSION IN INTESTINE OF OBESE MICE

OB OB+PB C 0 20 40 60 80 100 120

(74)

0 10 15 20 25 30 35 (m g/kg /min ) 5

EFFECT OF HIGH FAT DIET ON INSULIN SENSITIVITY AND SIGNALING IN MUSCLE OF TLR4 -/- MICE

40 * CONT TLR4-/-CONT TLR4-/-Insulin

pJNK

GLUCOSE UPTAKE

CONT

TLR4-/-IRS1

ser307

pAKT

CHOW HFD

(75)

LPS - FFA MYD88 MAL/TIRAP IRAK TRAF6 TAK1

JNK

IKK / I

k

B / NF

k

B

TNF

a

IL-6 iNOS

TLR4

LPS SIGNALING – TLR4

(76)

INTERACTION BETWEEN INNATE IMMUNE

SYSTEM AND INSULIN RESISTANCE

DIET AND

PATHOGENS

INNATE IMMUNE SYSTEM

INSULIN

(77)

0 50 100 150 200 250 300 C HFD

EFFECT OF HFD ON THE INTESTINAL MICROBIOTA AND CIRCULATING LEVELS OF LPS

0 5 10 15 20 25 C HFD Gl ic e mia mg/dl A U LPS levels Plasma Glucose Bac teró ides 65% Bif id ob_ac tér_ias 20_% F u s o b a c te ria s 1 2 % Clo st ríd_io s 3% 0 20 40 60 80 100 C HFD Body w e ight (g) 5m HFD