UNIVERSIDAD AUTÓNOMA DE MADRID
Departamento de Biología Molecular Doctorado en Biociencias Moleculares
Changes in gut microbiota associated to inflammation during ageing and non-alcoholic steatohepatitis
Carmen Rubio Caballero
Madrid, 2018
A mis padres, Luciano y Carmen
“No pierdas el tiempo chocando contra una pared, con la esperanza de transformarla en una puerta”
Coco Chanel.
“…Pero desde que tiré las llaves ya no quiero entrar… desde que olvidé el teléfono en un bar, desde que no tengo nada parecido a un plan, te prometo hermano que mis suelas no tocan el suelo. Solté todo lo que tenía y fui feliz, solté las riendas y dejé pasar...”
Juan Gómez, “El Kanka”.
“Sé amable, pues cada persona con la que te cruzas está librando su ardua batalla”
Platón.
Qué difícil es escribir estas líneas, sobre todo cuando a lo largo de este duro camino te ha rodeado gente tan maravillosa.
En primer lugar agradecer a José el haberme dado la oportunidad de incorporarme a su grupo de investigación. Gracias por haber sido tan comprensivo en mis inicios, con mi falta de manejo en el laboratorio, con mis idas y venidas a veterinaria y con mi poca maña con las ratillas. Gracias por haberme acompañado en las primeras incursiones al animalario, por buscar quién podría enseñarme en todas partes, por adentrarme en el maravilloso mundo del metabolismo y de la microbiota (aunque me haya hecho sufrir tanto), por enseñarme que nuestras ratas no son gordas sino adiposas, por tu integridad y por ser una excelente persona.
A Ángela, por haberme acogido en su laboratorio con la mitad de mi beca ya vencida.
Por haber confiado en que nos daría tiempo a hacer tantas cosas en tan poco tiempo y en un campo desconocido para ambas. Gracias por enseñarme tanto de insulin y señalización, por ponerle tanta pasión a todo lo que haces, por enseñarme a cogerle el puntillo al western (bueno, en realidad lo sigo intentando), por estar siempre disponible para todos. Eres un ejemplo de esfuerzo y dedicación.
A la gente del CBMSO, donde comencé mis andanzas en esto de la ciencia.
A Jorgina Satrústegui, por permitirme empezar la tesis en su laboratorio, por interesarse siempre en mis avances en la tesis en mis visitas al lab.321 y por dejarme formar parte de un grupo tan excelente. Eres una gran líder, científica y personalmente, y se nota en el equipo que te acompaña.
Al lab 321, a Isabel, a Bárbara, a Elena, a Laura, las más veteranas, por hacer que el laboratorio realmente funcione. A Irene Pérez, con quien coincidí poco pero de la que solo oigo y veo virtudes. A Inés por enseñarme desde feminismo hasta por qué las cebras tienen rayas, por transmitirnos a todos su enorme pasión por la ciencia. A Irene Llorente, por esa comida rica que siempre estaba dispuesta a compartir, por ser encantadora incluso teniendo alojadas a 50 personas en su casa de Dublín, por su sonrisa que irradia bondad. Y sobre todo a mis dos P; por los viajes, por el apoyo estos últimos meses, por el intercambio de audios en el WhatsApp, por veniros a Benavente a mis 30. A Paloma, por no dejarme ser negativa en ningún momento, pero ni uno ni uno (ni aunque me haya caído 58447 veces de los esquíes), por su espíritu relajado
“surferillo style” y por enseñarme que la voluntad no se le quita a las personas. Me encanta tu forma de ver la vida. A Pauli, por ser la mejor compi de conciertos que se puede tener, bueno y de exposiciones, museos, de Starbucks, cines, óperas, noches de juegos,…Por ser tan buena gente y por hacer que tus quedadas contigo sean siempre un momento de desconexión. Sabes que formas parte de mi familia en Madrid. Os adoro a las 2.
nuestras queridas bestias y a sacarles sangre (1 ml en 30 min, ¡qué duro fue aquello!), por ser un excelente compañero tanto dentro como fuera del laboratorio.
Al personal del animalario y en especial a Cobeña, un paisano de los de toda la vida, por hacerme más amenas las horas en la “sala de rata”.
A toda la gente de la URJC, en especial a Daniel Horrillo, por enseñarme a perfundir y por todas las horas de auto-infligido ayuno que pasamos juntos.
A la gente del IIB Alberto Sols, el sitio de “mi primera estancia”:
A todo el laboratorio 1.10 (y el nuevo 2.4.2), a los que se fueron y a los que aún están.
Y en especial a mi Rada, por enseñarme todo en cultivos y en el lab en general, por trasmitir tanta sabiduría (tanto científica como de la vida en general), por tener siempre algún tema de conversación en mente, por enseñarme que siempre hay que llegar a un
“ten con ten”, por pegarme el “quitivoyadecir” así todo junto y porque me parto contigo,
¡qué gracia tienes maldita! ¡Eres una grande!
A la gente del Gregorio Marañón, la estancia de mi estancia:
A los hepatogastros y allegados. Qué puedo decir de vosotros…Formáis un equipo maravilloso. Siempre con un sonrisa en la cara y siempre dispuestos a ayudar en lo que sea. Gracias a Javier Vaquero, a Rafael Bañares y a Luis Menchén, por darme la oportunidad de colaborar con su equipo. A Christian, a Iris, a las Elenas, a Bea, a Maribel, a Laura, a Johanna, a Raquel, a Rafa Confocal (ya es tu nuevo apellido), por esos desayunos y comidas maravillosos, por los ratos buenos en el laboratorio y fuera de él, por preocuparse tanto los unos de los otros, por ofrecer siempre una mano cuando veis a alguien apurado, por ser personas tan geniales. Sois unos compis ejemplares.
Gracias a Juan UCM por su maña con los ratones y por hacer tan ameno el trabajo a tu lado.
Y sobre todo gracias a Marta, nuestra madre científica. Gracias por guardar la calma en todas las situaciones, por buscar soluciones a cualquier problema que surja en el camino (y encontrarlas), por los detalles maravillosos que tienes con todos nosotros, por las recetas que compartes (soy fan). Eres mi “influencer” favorita, te admiro cada día.
A la gente del MRC Institute of Metabolic Science, la estancia de la estancia de la estancia:
Thanks to Frank and Fiona for welcoming me in their lab and for allowing me to have such a great experience. Thanks to Van, for her patience with my English, for her great ideas and for always being there, ready to help.
Gracias a María, por acogerme bajo su ala protectora, por darme ánimos y por enseñarme un poquito de Cambridge.
distintas ciudades, por permitirme desahogarme cuando lo necesitaba y por hacerme sentir que puedo contar con vosotros cuando os necesite.
Y por último y lo más importante gracias a mi familia. A mis padres, aunque unas líneas no puedan expresar tanto agradecimiento.
A mi madre, por su paciencia, su dedicación y por cuidarnos tanto y tan bien a todos, haciendo que todo sea más fácil. Eres el pilar que sostiene nuestra familia.
A mi padre, que siempre se llena de orgullo al hablar de “sus dos Ferraris”.
Gracias por depositar tanta confianza en mí y en todo lo que hago. Haces que el esfuerzo merezca la pena, sin ti nada sería igual.
A mi hermano Pablo, a Baudi (nuestra segunda madre, que odia la palabra tía) y a Nines. A mis abuelos, que ya no están con nosotros. Aunque os perdimos demasiado pronto seguiréis aquí siempre.
Os quiero
1
INDEX ... 1
ABBREVIATIONS ... 9
RESUMEN ... 15
ABSTRACT ... 19
INTRODUCTION ... 23
1. The gut microbiota. ... 23
2. Emerging role of gut microbiota in metabolic diseases. ... 24
3. Intestinal architecture and gut barrier function. ... 24
4. Immune and energy homeostasis in the gut. ... 26
4.1 Gut immunity and its interactions with gut microbiota. ... 26
4.2 Gut hormones and the regulation of energy homeostasis. ... 28
5. Insulin signaling and role of the insulin receptor in the gut. ... 29
6. Ageing and gut-brain axis. ... 31
7. Non-alcoholic fatty liver disease and the gut-liver axis. ... 34
7.1 Non-alcoholic fatty liver disease (NAFLD). ... 34
7.2 Mouse models of non-alcoholic steatohepatitis. ... 35
7.3 Link between NASH and gut microbiota. ... 36
7.4 Gut-liver axis. ... 37
7.4.1 Gut microbiota, incretins and liver. ... 37
7.4.2 Crosstalk between bile acids and microbiota. ... 37
7.5 PTP1B knockout mouse model. ... 39
OBJECTIVES ... 45
MATERIALS AND METHODS ... 49
1. Animals. ... 49
1.1 Ageing rat model. ... 49
1.2 NASH murine model ... 49
2. Microbiome determination. ... 49
2.1 Accession numbers... 50
2.2 Microbiome analysis: Alpha and beta diversity. ... 50
2
2.3 PICRUSt analysis. ... 51
3. Magnetic resonance analysis. ... 51
4. Short term satiety test. ... 51
5. Triglyceride and cholesterol tissue content. ... 52
6. FITC-Dextran assay. ... 52
7. Endotoxemia. ... 52
8. Colon tissue collection. ... 53
9. Immunohistochemistry. ... 53
10. Colon mucosal integrity evaluation by light microscopy. ... 53
11. Colonic expression of neutral and acid mucins. ... 54
12. Measurement of colon edema. ... 54
13. RNA isolation and quantitative Real-Time PCR analysis. ... 55
14. GLP-1 measurement in plasma samples. ... 56
15. Bile acids profiling. ... 56
16. Macrophages and Enteroendocrine cells culture and procedures. ... 57
16.1 Raw 264.7 ... 57
16.2 STC-1. ... 57
16.2.1 Analysis of insulin signaling in pro-inflamatory conditions. ... 57
16.2.2 Analysis of pro-inflammatory-mediated signaling cascades... 57
16.2.3 Analysis of proglucagon expression after pro-inflammatory stimuli. .... 58
17. Preparation of total protein cellular extracts. ... 58
18. Determination of proteins by western blot. ... 58
19. Determination of TransEpithelial Electrical Resistance (TEER). ... 59
20. Lentiviral silencing of Ptpn1 in Caco-2 cells. ... 60
21. Organoid cultures. ... 60
21.1 2D organoid culture... 61
21.2 GLP-1 secretion. ... 61
22. Statistical analysis. ... 62
RESULTS ... 65
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AGEING RAT MODEL ... 65
1. Ageing gut microbiota characterization ... 65
1.1 Analysis of microbiome diversity and taxonomic changes during ageing in Wistar rats. ... 65
2. Changes in adiposity along ageing. ... 68
3. Effect of ageing on mucosal markers of intestinal integrity. ... 69
4. CCK satiating effect along ageing. ... 73
NASH MURINE MODEL ... 74
1. Characterization of gut microbiota in PTP1B WT and PTP1B KO mice in a preclinical model of NASH. ... 74
1.1 Analysis of fecal microbiota in PTP1B WT and PTP1B KO mice fed a chow or methionine and choline-deficient (MCD) diet. ... 75
1.2 Functional differences in gut microbiota between PTP1B WT versus PTP1B KO mice fed a chow or MCD diet. ... 79
2. .... Anatomical and histological analysis of the colon in PTP1B WT and PTP1B KO mice under NASH conditions. ... 80
3. Pro-inflammatory effects of NASH in colon tissue. ... 82
4. Effects of MCD diet on colon mucosal integrity in PTP1B WT and PTP1B KO mice. ... 84
5. Differential effects in the intestinal permeability, bacterial translocation and proteins involved in the intestine architecture in PTP1B WT and PTP1B KO mice during NASH. ... 86
6. Effect of PTP1B in the barrier properties in Caco-2 monolayer cell cultures by Transepithelial Electrical Resistance (TEER). ... 88
7. Effect of PTP1B deficiency in bile acids content in serum and faeces during NASH. ... 90
8. Effect of the MCD diet and genotype on GLP-1 serum levels. ... 92
9. Evaluation of the expression of proglucagon in enteroendocrine cells after pro- inflammatory stimuli. ... 93
10. .... Analysis of insulin signaling in the enteroendocrine cell line STC-1: effects of the pro-inflammatory environment and PTP1B inhibition. ... 95
11. .. Effect of the pro-inflammatory environment in the signaling pathways mediated by the stress-activated protein kinases in STC-1 cells. ... 97
DISCUSSION ... 103
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DISCUSSION: AGEING RAT MODEL ... 103
1. Ageing associates with changes in gut microbiota and gut barrier integrity. ... 103
2. Fat accretion related changes in microbiota. ... 105
3. Low CCK satiating effect reflects alterations in gut-brain axis in aged rats. ... 105
DISCUSSION: NASH MURINE MODEL ... 107
1. PTP1B deficiency protects against the shift in the gut microbiota composition during NASH. ... 107
2. Impact of PTP1B deficiency in the colonic immune responses and gut barrier function during NASH. ... 109
3. .. The pro-inflammatory environment during NASH progression modulates GLP-1 secretion. ... 111
4. The gut liver-axis during NASH: effect of PTP1B deficiency. ... 112
CONCLUSIONS ... 117
CONCLUSIONES ... 121
REFERENCES ... 125
FIGURES INDEX INTRODUCTION Figure 1. The intestinal epithelial barrier and immunity in the gut ... 27
Figure 2. GLP-1 secretion in a pro-inflammatory environment ... 29
Figure 3. Gut-Brain cross-talk in eating behaviour……… ... …33
Figure 4. NAFLD spectrum scheme……… 35
Figure 5. Bile acids effects on metabolic actions via TGR5 and FXR signaling ... 39
Figure 6. Scheme of PTP1B actions on the insulin signaling cascade ... 40
Figure 7. PTP1B regulates cytokine receptor–JAK–STAT signaling ... 41
MATERIALS & METHODS Figure 8. Thickness and area evaluation with Halo™ software ... 54
Figure 9. Scheme of TEER measurement ... 60
RESULTS Figure 10. Phylogenetic diversity of fecal microbiota from 4- and 24-month old rats .. 66
5 Figure 11. Differences in fecal microbiota at phylum level in 4- and 24-month old rats
….. ... 67
Figure 12: Fat deposition in 4- and 24-month old rats ... 68
Figure 13. Evaluation of colon mucosal integrity in young and aged rats ... 70
Figure 14. Differential mucin staining of colonic mucosal crypts from young (4 months) and old (24 months) rats ... 71
Figure 15. Acidic and neutral mucin individual analysis ... 72
Figure 16. Determination of inflammatory markers in serum samples ... 72
Figure 17. Short term satiety analysis ... 73
Figure 18. Alpha-diversity analysis for PTP1B WT and PTP1B KO fed a chow or MCD diet ... 76
Figure 19. Phylogenetic β-diversity of fecal microbiota in NASH murine model ... 77
Figure 20. Pyrosequencing analysis of phyla from PTP1B WT and PTP1B KO fed a chow or MCD diet for 4 weeks ... 78
Figure 21. Analysis of anatomical and histological parameters on colon samples in PTP1B WT and PTP1B KO mice under NASH ... 81
Figure 22. Histological analysis of H&E colonic stained sections in PTP1B WT and PTP1B KO mice ... 82
Figure 23. RNA expression levels of pro-inflammatory markers in colon samples of PTP1B WT and PTP1B KO mice ... 84
Figure 24. Mucin layer analysis (low magnification) in colonic samples from mice under NASH conditions ... 85
Figure 25. Mucin layer analysis (high magnification) in colonic samples from mice under NASH conditions ... 86
Figure 26. Alterations in the gut barrier permeability, translocation of microbial products and expression levels of intestine architecture-related genes during NASH in PTP1B WT and PTP1B KO mice ... 87
Figure 27. Effect of the pro-inflammatory environment on TEER values in Caco-2 Scrb and Caco-2 shPtpn1 monolayers ... 89
Figure 28. Bile acids levels in SERUM SAMPLES FROM PTP1B WT and PTP1B KO MICE ... 91
Figure 29. Fecal bile acids levels in PTP1B WT and PTP1B KO mice ... 92
Figure 30. GLP-1 levels in serum samples from PTP1B WT and PTP1B KO mice ... 93
Figure 31. Gcg expression in STC-1 cell line after stimulation with CM-LPS... 94
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Figure 32. GLP-1 release in colonic organoid cultures after stimulation with CM-LPS ... 95 Figure 33. Insulin signaling cascade under basal conditions or in the presence of a PTP1B inhibitor in the STC-1 cell line ... 96 Figure 34. Effect of the pro-inflammatory environment and PTP1B inhibition in insulin signaling in STC-1 cells ... 97 Figure 35. The pro-inflammatory environment induces the phosphorylation of JNK and p38 MAPK and degradation of IκBα in STC-1 cells ... 98
TABLES INDEX INTRODUCTION
Table 1. Classification of the different IECs subsets and their respective functions in the maintenance of the gut barrier integrity ... 25 MATERIALS & METHODS
Table 2. Primers list for RT-qPCR ... 55 Table 3. Antibody list for Western Blotting ... 59 RESULTS
Table 4. Changes in gut microbiota community at different taxonomic levels during ageing ... 66 Table 5. Changes in body weight, and in triglyceride and cholesterol deposition in liver and cardiac muscle, in 4- and 24-month old rats ... 69 Table 6. Changes in gut microbiota composition at different taxonomic levels after NASH insult in PTP1B WT and PTP1B KO mice ... 79 Table 7. TEER values of Caco-2 Scrb and Caco-2 siPtp1n cells under CM-C and CM- LPS stimuli ... 89
ABBREVIATIONS
9
ABBREVIATIONS
1% P/S: 100 U/ml penicillin, 100 µg/ml streptomycin
AB: Alcian blue
ADF: Advanced DMEM:F12 medium
A.I.: Arbitrary units
AT: Annealing temperature
bp: Base pair
CA: Cholic acid
Cdh1: E-Cadherin mouse gene
CCK: Cholecystokinin
Cd3g: T-cell surface glycoprotein CD3 γ
CDCA: Chenodeoxycholic acid
CM-C: Conditioned media control
CM-LPS: Conditioned media LPS
d: Days
DC: Dendritic cells
DPP-4 Dipeptidyl peptidase-4
EECs: Enteroendocrine cells.
F/B Firmicutes/Bacteroidetes
FBS: Fetal bovine serum.
FXR: Farnesoid X receptor
Gcg: Proglucagon gene
GIP: Glucose-dependent insulinotropic polypeptide
GLPs: Glucagon-like peptides
GLP-1: Glucagon-like peptide 1
Gzmb: granzyme B mouse gene
h: Hours
10
H&E: Hematoxylin and Eosin
IECs: Intestinal epithelial cells
IELs: Intestinal intraepithelial lymphocytes
IFNγ: Interferon gamma
Ifng: Interferon gamma mouse gene
IGF-1: Insulin-like growth factor 1
IIS: Insulin and Insulin-like growth factor 1 (IGF-1) signaling
IκBα: Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha
IL1β: Interleukin 1 beta
IR: Insulin receptor
IRS: Insulin receptor substrate
IS: Internal standard
iNOS: Nitric oxide synthase
LCA: Lithocholic acid
LPS: Lipopolysaccharide
M cells: Microfold cells
MCD: Methionine-Choline-Deficient (diet)
MCP-1: Macrophage chemotactic protein-1
min: Minutes
MW: Molecular weight
NAFLD: Non-alcoholic fatty liver disease
NASH: Non-alcoholic steatohepatitis
Nos2: Inducible nitric oxide synthase mouse gene
N.S: Non-significant differences
NTS: Adjacent nucleus tractus solitaries
Ocln: Occludin mouse gene
OTUs: Operational Taxonomic Units
PAS: Periodic acid Schiff
11
PBS: Phosphate-buffered saline
PI: Posphatidylinositol
PRRs: Pattern-recognition receptors.
PTP1B: Protein-tyrosine phosphatase 1B
PTP1B KO: Protein-tyrosine phosphatase 1B knockout mice PTP1B WT: Protein-tyrosine phosphatase 1B wild-type mice
PTP1B KO MCD: Protein-tyrosine phosphatase 1B knockout mice fed with MCD diet for 1 month.
PTP1B WT MCD: Protein-tyrosine phosphatase 1B wild-type mice fed with MCD diet for 1 month.
PVDF: Polyvinylidene fluoride
PYY: Peptide YY
RT-qPCR: Real-time quantitative PCR
SDS: Sodium dodecyl sulfate
SDS-PAGE: SDS polyacrylamide gel electrophoresis
sec: Seconds
SEM: Standard error of the mean
SGLT1: Sodium-glucose linked transporter 1 SIBO: Small intestinal bacterial overgrowth
TBS: Tris-buffered saline
TCA: Taurocholic acid
TCDCA: Taurochenodeoxycholic acid
TDCA: Taurodeoxycholic acid
TEER: Transepithelial electrical resistance
Tjp1: Tight junction protein 1 or Zonula occludens 1
TLRs: Toll-like receptors.
TNFα: Tumor necrosis factor
TTBS: TBS supplemented with 0.05% Tween 20
UDCA: Ursodeoxycholic acid
UHPLC: Ultra-High Performance Liquid Chromatography
12
Vil1: Villin mouse gene
ZO-1: Zonula occludens-1
RESUMEN
15 En esta Tesis se han estudiado los cambios en la microbiota y barrera intestinal en dos condiciones fisiopatológicas que concurren con inflamación sistémica, el envejecimiento y la esteatohepatitis no alcohólica (del inglés NASH).
El envejecimiento está caracterizado por alteraciones en la regulación del balance energético, lo que conduce a un incremento en el peso corporal. Puesto que la microbiota intestinal ha emergido como un factor clave en numerosos estados fisiopatológicos, el concepto del eje intestino-cerebro está alcanzando un gran interés.
Nuestros resultados muestran que el envejecimiento en ratas Wistar está asociado con alteraciones en la comunidad microbiana fecal, conduciendo a un aumento de bacterias productoras de LPS, una disminución de microorganismos anti-inflamatorios y un aumento de bacterias degradadoras de mucina. Además, el análisis histológico del colon muestra alteraciones en el ancho de la cripta y en el contenido en mucina, indicando la presencia de una barrera intestinal más débil en los animales envejecidos. En cuanto al eje intestino-cerebro, observamos una disminución del efecto saciante de la colecistoquinina durante el envejecimiento que podría aumentar la adiposidad. Algunos cambios observados en la microbiota intestinal de ratas envejecidas son similares a los de animales obesos, sugiriendo que envejecimiento y obesidad son dos formas distintas de aumentar la adiposidad.
La NASH cursa con un componente pro-inflamatorio tanto a nivel hepático como sistémico. La proteína tirosina fosfatasa 1B (PTP1B) tiene papeles distintos en células no inmunes e inmunes, en éstas últimas inhibiendo las repuestas pro-inflamatorias.
Hemos explorado el papel de PTP1B en la composición de la microbiota y en la función de barrera intestinal en un modelo murino de NASH inducido por un dieta deficiente en metionina y colina. Nuestros resultados muestran cambios en la microbiota, disrupción de la barrera intestinal, disminución en el contenido en mucinas y aumento de los ácidos biliares séricos durante la NASH. Sorprendentemente, a pesar del fenotipo inflamatorio de los ratones deficientes en PTP1B, puesto de manifiesto en el intestino por un incremento en la secreción de GLP-1, estos animales están protegidos frente a las alteraciones en la composición de la microbiota y en la función de barrera intestinal durante la NASH, un efecto que concurre con la preservación del contenido en mucina y con menores niveles de ácidos biliares séricos comparados con los ratones de genotipo salvaje. En conjunto, nuestros resultados sugieren un papel protector de PTP1B en el eje intestino-hígado durante la NASH.
ABSTRACT
19
ABSTRAC T
In this Thesis we have explored the changes in gut microbiota and several gut barrier features in two pathophysiological conditions that commonly concur with systemic inflammation, ageing and non-alcoholic steatohepatitis (NASH). In order to facilitate the description of our studies, the results of each model are presented separately.
Ageing is characterized by alterations in the homeostatic mechanisms that regulate energy balance leading to an increased body weight. Although the presence of a low-grade chronic inflammation has been proposed as the responsible of the homeostatic misbalance, the primary alteration has not been yet fully elucidated. Since gut microbiota has emerged as a key player in a wide range of physiological and pathological stages, the concept gut-brain axis is getting high interest. Using metagenomics we demonstrate that ageing in Wistar rats associates with alterations in the fecal microbial community leading to a higher presence of LPS-producing bacteria, a decrease of anti-inflammatory microorganisms and an increase in mucin-degrading bacteria. The histological analysis of the colon shows alterations in crypt width and mucin content, revealing a weaker gut barrier in aged animals. Gut-brain axis is also altered regarding impairment in cholecystokinin satiating effect that could contribute to the increased adiposity in old rats. Some of the observed changes in gut microbiota are also shared by obese animals, suggesting that ageing and obesity microbiota are likely two different ways to induce fat accretion.
NASH is characterized by a robust pro-inflammatory component at both hepatic and systemic levels. Protein tyrosine phosphatase 1B (PTP1B) plays distinct roles in non-immune and immune cells, in the latter inhibiting pro-inflammatory responses. In this Thesis we have explored the role of PTP1B in the composition of gut microbiota, as well as in gut barrier dynamics in methionine and choline-deficient (MCD) diet-induced NASH in mice. Our results revealed that a shift in the gut microbiota shape, disruption in gut barrier function, decrease in mucins abundance and higher levels of serum bile acids are gut features during NASH. Surprisingly, despite of the pro-inflammatory phenotype of global PTP1B deficiency in mice manifested by increased GLP-1 release by the gut, it protects against the alterations in gut microbiota composition and improves gut barrier function during NASH, an effect that concurred with preservation of mucin abundance and decreased serum bile acids compared to their wild-type mice counterparts. Altogether our results have unravelled a potential role of PTP1B in the gut-liver axis during NASH.
INTRODUCTION
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1. The gut microbiota.
The intestine harbours a complex dynamic ecosystem known as gut microbiota including bacteria, bacteriophage particles, viruses, fungi and archaea that have co- evolved with the host over thousands of years to form an intricate and mutually beneficial relationship [1]. This ecosystem is dominated by anaerobic bacteria mainly belonging to the phyla Firmicutes, Bacteroidetes, Actinobacteria, Verrucomicrobia and Proteobacteria [2].
To highlight the importance of gut microbiota thorough life, it must be considered that approximately 90% of the cells in the human body are microbes whilst only 10% are human cells. Robert Koch and Ilya Mechnikov were awarded with Physiology and Medicine Nobel prizes in 1905 and 1908, respectively, for their studies on the relationship between microbes and human health [3]. In fact, it was Mechnikov who postulated the “Intoxication theory of ageing” aiming that waste products from the intestines poison the human body, being those responsible of ageing and death later on [4].
After birth, gastrointestinal tract is rapidly colonised. Gut microbiota composition is conditioned by many factors such as the mode of delivery (vaginal or caesarean birth), genetics, diet, as well as environmental factors among others. Its composition stabilizes around the third year of life in humans [1]. Despite gut microbiota remains relatively stable during adulthood, it can be modified by many conditions such as ageing, diet or treatment with antibiotics [5, 6].
The host receives many benefits from microbiota through a range of physiological functions such as strengthening gut integrity or shaping the intestinal epithelium [7], protecting against pathogens colonization [8] and regulating host immunity [9].
Furthermore, gut microbiota is currently considered as a metabolic organ since not only facilitates harvesting of nutrients and energy from ingested food, but also it produces metabolites that interact with receptors regulating host metabolism. Gut microbiota plays an important role in digestion and vitamin synthesis [10]. However, an altered microbial composition, known as dysbiosis, can lead to a potential disruption in these mechanisms. Dysbiosis has been associated with the pathogenesis of many inflammatory diseases and infections [1].
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2. Emerging role of gut microbiota in metabolic diseases.
Large amounts of data concerning gut microbiota in obesity and metabolic disorders have been obtained by research consortia such as the Human Microbiome Project (HMP) [11] or Metagenomics of the human intestinal tract (MetaHIT) [12]. During the last decade these projects have demonstrated a relevant association of intestinal microbiota with obesity, type 2 diabetes and metabolic syndrome, leading to the search of targeted nutritional interventions. Besides, the role of microorganisms and their interactions with the host have been assessed through studies in preclinical mouse models which have become a key tool to analyze the molecular mechanisms underlying metabolic processes. In this regard, numerous studies have demonstrated that dysbiosis contributes to disease phenotypes [13]. Thus, conventionalizing of germ free mice results in an increase of fat mass despite lower food intake [14]. Moreover, fecal transplant from lean donors improves insulin sensitivity in patients with metabolic syndrome [15] and transplant of microbiota from discordant twins for obesity to germ- free mice induces a differential increase of their respective body weights in agreement with the obese phenotype of the donor [16].
Gut microbiota can affect host metabolism not only directly by fermentation processes and metabolites production, but also indirectly by modulating immune responses (for instance via toll-like receptor (TLR) 5 during hyperphagia [17]) and hormone signaling mediated by glucagon like-peptide 1 (GLP-1) that induces the stimulation of pancreatic insulin secretion (incretin effect), the inhibition of gastric emptying, an increased feeling of satiety, as well as other actions on peripheral tissues such as the liver [18].
Furthermore, it has been reported that changes in intestinal microbiota composition could be responsible for several harmful effects including the impairment of the gut- barrier integrity, the development of endotoxemia and the presence of inflammation in gut and other peripheral tissues that in turn might lead to the development of insulin resistance and lipid deposition in different organs [19].
3. Intestinal architecture and gut barrier function.
The gut is divided in two main regions, the small intestine (duodenum, ileum and jejunum) and the large intestine (cecum, colon and rectum). The intestinal epithelium is the largest mucosal surface in the body. It constitutes a barrier surface highly specialized and adapted to colonization by commensal bacteria which impact on the
25 development and role of the mucosal immune system. The intestinal epithelium is formed by a single layer of cells organized in crypts (tubular invaginations of the epithelium) and villi (projections of the epithelium into the lumen) [20]. This epithelium is constituted by the intestinal epithelial cells (IECs), which include not only enterocytes, but also subsets of specialized IEC lineages (Table 1).
IECs are organized forming a continuous single layer whose integrity depends on the tight junctions. More than 40 different proteins conform the tight junctions [22] that are associated to the actin and myosin in the cytoplasma, establishing a network which
IEC lineage Function
Enterocytes Nutrient absorption, resorption of unconjugated bile salts, secretion of IgA from plasma cells (an immune component of the epithelial barrier).
Enteroendocrine cells (EECs)
Secretion of hormones such as cholecystokinin (CCK), glucagon like peptides (GLPs) or PYY, among others, that are the link between central and enteric neuroendocrine system.
Goblet cells Secretion of mucins (highly glycosylated proteins) which constitute the first line of defence against microbial invasion.
Microfold cells (M cells)
Epithelial cells of the mucosa-associated lymphoid tissues that transport antigens from the lumen to cells of the immune system for the induction of efficient immune responses against mucosal antigens in Peyer’s patches.
Paneth cells Secretion of antimicrobial proteins (defensins, lysozyme, cathelicidins) to establish a biochemical barrier to microbiota.
This subset of cells is the unique, along with the stem cells, located at the base of the crypts.
Stem cells Located at the base in the crypts. They continuously renew the epithelium surface originating all the subsets of IECs which migrate up the crypt-villus axis (except Paneth cells).
Table 1. Classification of the different IECs subsets and their respective functions in the maintenance of the gut barrier integrity [20, 21].
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firmly links the intestinal epithelial cells in order to regulate paracelullar permeability [20].
The gut barrier function is defined as the intestine property that provides an essential impediment against the flux of harmful substances and pathogens from the external environment to the systemic circulation of the host while allowing the nutrient absorption [23]. The gut barrier function depends on physical, chemical and immunological factors. The gut physical barrier is constituted by the mucus layer secreted by the goblet cells, the glycocalyx covering the microvilli in the enterocytes and the tight junctions. Antimicrobial proteins, secreted mainly by Paneth cells, and bile acids, which inhibit small intestinal bacterial overgrowth (SIBO), constitute the gut chemical barrier. Finally, IgA produced by B cells acts by inhibiting pathogens from adhering to the mucus, thereby configuring the gut immune barrier [24, 25] (Figure 1A).
Studies conducted in mouse models with genetic defects in tight-junctions have shown that a disruption of the gut barrier function is not sufficient by itself to cause intestinal inflammatory bowel disease [26]. Thus, it has been suggested that in a situation of gut barrier dysfunction a compensatory immune mechanism is able to protect the host against the development of colitis [27], highlighting the importance of the immune system in the development of diseases related to gut barrier alterations and dysbiosis.
4. Immune and energy homeostasis in the gut.
4.1 Gut immunity and its interactions with gut microbiota.
The intestinal epithelium owns a complex system of luminal microbial signals recognition to establish a balance between homeostasis and inflammation in the context of host-microorganism symbiosis. IECs express pattern-recognition receptors (PRRs) and among them TLRs are differently segregated due to the polarized nature of the IECs. This polarization allows the TLRs to respond in a different way to apical and basolateral stimuli [28, 29] and, therefore, to distinguish between commensal (apical) and pathogens (basolateral) based on the localization of the stimuli. In addition, it has been proposed that IECs are able to discriminate between commensal and pathogen signals through viability-associated PAMPs (pathogen-associated molecular patterns) [30].
27 Peyer’s patches, located in the submucosa, are groups of lymphoid aggregates containing B cells, T cells and dendritic cells (DC). These lymphoid aggregates receive lumen antigen information through the microfold cells (M cells), a specialized group of epithelial cells (Table 1). Moreover, CX3CR1H1 are the intestine-resident macrophages, which in their steady stage remain close to IECs. Once CX3CR1H1 recognize the exogenous antigens, they form transepithelial dendrites to pass through the epithelial barrier [31], being this process initiated by TLR signaling [32]. This migratory cell population presents and activates secondary lymphoid tissues, as lymph nodes and Peyer’s patches (Figure 1B).
Figure 1. The intestinal epithelial barrier and immunity in the gut. A. The intestinal epithelial barrier. IECs form a biochemical, immune and physical barrier. Stem cells along with stromal and haemotopoietic cells control the renewal of the epithelial cell layer. Dashed arrows indicate migration direction, up in the crypt-villus axis (except Paneth cells). B. Immunity in the gut. Transport of luminal antigens and live bacteria across the epithelial barrier to dendritic cells (DC) is mediated by M cells. Resident macrophages take samples from exogenous antigens to activate lymphoid tissues. NOTE.
Adapted from “Intestinal epithelial cells: regulators of barrier function and immune homeostasis” by L.W. Peterson and D. Artis, 2014, Nat Rev Immunol 14(3):141-153 [20].
A B
28
Another notable immune cell population is represented by the intestinal intraepithelial lymphocytes (IELs). These are a differentiated group of cells that modulate innate immunity and participate in the control of the gut barrier function, epithelial turnover and the host response against enteric pathogens [33].
4.2 Gut hormones and the regulation of energy homeostasis.
The enteroendocrine system consists in a complex network of specialized endocrine cells known as enteroendocrine cells (EECs). EECs secrete hormones in response to nourishment that maintain energy homeostasis regulating food intake, digestion, absorption, satiation, storage and disposal of digested nutrients. Their effects are likely due to the fact that these hormones act as neurotransmitters stimulating enteric nerve fibers [34].
Cholecystoquinin (CCK) is a peptide synthesized by enteroendocrine I cells of the small intestine in response to dietary fat and proteins [35]. In addition to its effects on gall bladder contraction, pancreatic enzyme secretion and gastric emptying, CCK regulates meal size acting as a satiety factor [36]. GLP-1 is secreted by proglucagon-producing L cells predominantly located in the ileum and colon. This peptide controls gut motility and food intake. In addition, GLP-1 stimulates insulin and on the other hand inhibits glucagon secretion [37].
Recent interest has emerged on the interaction of EECs with the immune system. It has been reported that levels of some gut hormones, like GLP-1, increase after intestinal injury or in situations of mucosal inflammation [38]. GLP-1 could be released in response to elevations of endotoxin or cytokines and exert anti-inflammatory effects acting through the GLP-1 receptors (GLP-1R) in the IELs [39] (Figure 2).
29 Figure 2. GLP-1 secretion in a pro-inflammatory
environment. When endotoxin levels rise GLP-1 is secreted to exert an anti-inflammatory role controlling host mucosal immune responses through modulation of IELs.
NOTE. Adapted from “GLP-1R agonists modulate enteric immune responses through the intestinal intraepithelial lymphocyte GLP-1R” by B. Yusta, 2015, Diabetes 2015;64:2537–2549.[39]
5. Insulin signaling and role of the insulin receptor in the gut.
Insulin and Insulin-like growth factor 1 (IGF-1) signaling (IIS) play key roles in metabolic homeostasis, embryonic development and growth. Since both insulin receptor (IR) and IGF-1 receptor (IGF-1R) have ubiquitous expression patterns, their specific functions have been studied in multiple cell types and organs [40-42].
Insulin is a hormone produced by the beta cells of the pancreatic islets. Its main function is to control blood glucose levels in order to prevent hyperglycaemia. Given the importance of insulin signaling in maintaining glucose homeostasis, it must be tightly regulated. The cascade begins with the activation of the IR by autophosphorylation on several tyrosine residues leading to the recruitment and tyrosine phosphorylation of IRS (Insulin Receptor Substrate) proteins 1 and 2 [43, 44]. Then, phosphorylated IRSs bind proteins containing Src homology 2 (SH2) domains such as phosphatidylinositol (PI) 3-kinase [45], which has a central role in the metabolic actions elicited by insulin. The next critical node in the insuling signaling pathway is the protein kinase B (PKB/Akt). Akt is a serine/threonine kinase that phosphorylates the transcription factor forkhead box O1 (FoxO1), preventing its translocation to the
30
nucleus and, consequently, suppressing the transcription of gluconeogenic genes such as glucose 6-phosphatase and phosphoenolpyruvate carboxykinase [46]. In this manner, insulin negatively regulates hepatic glucose production through a main pathway involving IR, IRS1/2, PI 3-kinase and Akt. Likewise, insulin stimulates glucose uptake in peripheral skeletal muscle and adipose tissues via translocation of GLUT4 transporter from intracellular stores to the plasma membrane which is also mediated by IRS proteins and Akt [47].
Glucose is absorbed mainly in the small intestine through the sodium-glucose linked transporter 1 (SGLT1) located at the luminal membrane of the enterocytes.
Furthermore, this SGLT-mediated glucose uptake stimulates the expression and secretion of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) by EECs.
However, little is known about the role of IR function in the gut and how insulin affects the intestinal glucose absorption. A recent study of Kahn and colleagues [48] has revealed important findings about this topic. They found that in the gut IR is mainly expressed in the duodenum and colon and its deletion during development does not alter gut development or growth. These data contrast with previous studies suggesting that supplementation of infant formula with insulin (or IGF-1) could improve gut maturation [49]. However, they proved that the loss of the IR in IECs improves age-dependent glucose intolerance and causes decreased glucose absorption from the intestinal lumen by regulating glucose transporter activity, but not its expression. Furthermore, the loss of intestinal epithelial IR causes a reduction in GIP expression and release although GLP-1 expression and circulating levels were not affected [48]. Therefore, IR plays a specific role in the gut controlling glucose homeostasis by regulating glucose uptake and glucose-dependent incretin release.
GLP-1 acts on islet cells potentiating insulin secretion after meals; thereby it acts maintaining glucose homeostasis. GLP-1Rs are also widely expressed in other peripheral tissues such as the liver, where GLP-1 suppress hepatic glucose production [50], and the intestine where GLP-1 modulates gastrointestinal motility [51] and exerts an anti-inflammatory role as recently proposed and discussed above [39]. It is noteworthy to mention that in the hypothalamus GLP-1 agonism stimulates brown adipose tissue thermogenesis and browning through hypothalamic activation of AMPK [52].
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6. Ageing and gut-brain axis.
Ageing can be considered as a global functional decline that affects multicellular organisms in a time-dependent manner. This decline has been postulated to occur upon accumulation of cellular damage [53]. Recently, nine hallmarks of ageing have been reported to resume the functional time-dependent decline of most living organisms [54].
Deregulated nutrient-sensing is one of those hallmarks. It is classified as an antagonistic hallmark since it has opposite effects depending on its intensity; it mediates beneficial effects at low levels, but it becomes deleterious at high levels. Mice models with decreased insulin/IGF-I signaling (IIS) activity such as PTEN-overexpressing or hypomorphic PI3K mice show an increased longevity [55, 56]. Paradoxically a decreased IIS is characteristic during ageing both in non-pathological models and in mouse models of premature ageing [57] whilst it’s constitutively reduction extends longevity. According to this, aged organisms could decrease IIS in an attempt to extend their lifespan. In contrast, one of the main characteristics of ageing in rodents and humans is the development of insulin resistance, mainly due to increased adiposity and body weight [58]. This is considered as one of the main causes of age-associated pathologies such as type 2 diabetes, cardiovascular disease or a prothrombotic state that, in turn, results in reduced longevity. Although insulin resistance can be viewed as a defensive response against ageing-associated systemic damage to extend life span, it finally leads to a pathologic state that reduces longevity and, more importantly, healthy span [59]. Hence, a decrease in IIS as a defensive response against ageing could result in a deleterious and aggravating factor. Nevertheless, there is strong evidence that the anabolic signaling accelerates ageing and, conversely, a decrease in nutrient signaling extends longevity [60].
Studies in rats have demonstrated that an alteration of energy balance occurs during ageing, indicating that the homeostatic mechanisms are unable to counteract the increment of energy stores. Leptin is a well-known adipostat whose function is impaired in aged rats due to decreases in the hypothalamic expression and signaling of the leptin receptor [61-64] and a similar impairment of its anorectic function was observed for insulin [65]. A widely accepted hypothesis about the physiological control of body weight homeostasis in humans and rodents involves the gut-brain axis, a system
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constituted by peripheral hormones derived from gut that regulates the appetite modulatory centres located in the hindbrain and/or the hypothalamus.
The intertwined development of human brain and the gastrointestinal gut throughout evolution seems to be a consequence of the increasing availability of easily digestible energy and nutrient-rich food sources [66]. Gut derived humoral and neural signals are produced after nutrient intake. Central nervous system receives and integrates this information along with visual, taste and olfactory stimuli, as well as internal signals related to adiposity and stress among others in order to generate proper behavioural and endocrine responses completing the energy homeostatic loop [67]. The regulation of energy homeostasis is coordinated by a complex neurocircuit which comprises multiple brain regions. For instance, hypothalamus senses and integrates hormonal signals (insulin and leptin) related to the amount of fat stored in the adipose tissue whereas caudal brainstem receives information about composition and quantity of ingested nutrients. In particular, the area postrema integrates circulating metabolic signals such as leptin, amylin, CCK, GLP-1, PYY, ghrelin, and others, resulting in the regulation of satiety. The area postrema projects to the adjacent nucleus tractus solitaries (NTS), which in turn, relays visceral information to other brain areas [68]. NTS receives continuous signals from vagal afferent fibers of the intestinal wall and gastric mucosa that transmit information about the meal size and composition by secreting peptide hormones such as CCK and GLP-1 that act in a paracrine manner stimulating receptors located on the surface of the local vagal afferent fibers in the intestine. Those fibers transform these stimuli into electrochemical data before project them to the NTS [69]
(Figure 3).
33 Figure 3. Gut-Brain cross-talk in eating behaviour. Signals
generated from humoral factors (gut hormones) arrive to the brain through afferent neurons and together with cognitive processes and information arising from visual, gustatory and olfactory stimuli influence feeding behaviour.
PFC, prefrontal cortex; NAc, nucleus accumbens; VTA, ventral tegmental area; Hypo, hypothalamus; NTS, nucleus tractus solitaries.
NOTE. Adapted from “Gut-Brain Cross-Talk in Metabolic Control” by C. Clemmensen, 2017, Cell. 2017 Feb 23; 168(5):
758–774 [67].
Specifically, the research in this Thesis regarding ageing and gut brain axis is focussed in the satiety hormone CCK, whose functions have been described above (Epigraph
“Immune and energy homeostasis in gut”). Secreted CCK interacts with specific receptors in the vagal afferents innervating the intestinal area and, therefore, vagotomy of these fibers inhibits the satiety effect of CCK [36]. Leptin administered both centrally and peripherally sensitizes brain stem nuclei and vagal afferents to CCK action [70, 71].
Moreover, OLEFT rats lacking functional CCK receptors show obesity and hyperphagia, further supporting a role for the satiating effect of CCK in energy homeostasis [72]. Thus, this continuous flow of information between gut and brain allows a highly complex metabolic control system [67].
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Contradictory, current energy-dense and hyper-palatable food, as well as the increase in the meal size, has corrupted millions of years of biological optimization of metabolism in less than a century to transform rapidly Homo sapiens into an obese specie [67].
The emerging concept of the microbiota-gut-brain axis postulates that alterations in the microbial populations of the intestine also influence neurophysiological-governed behaviours including those related with appetite control [73]. Although many studies on different models of obesity and insulin resistance have been reported, little information is currently available on how ageing associates with changes in intestinal microbiota and alterations in gut-barrier integrity and gut structure. Also, there are no studies on the possible relationship of these changes with the gut-brain axis function.
7. Non-alcoholic fatty liver disease and the gut-liver axis.
7.1 Non-alcoholic fatty liver disease (NAFLD).
Non-alcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease in Western countries since it is associated with obesity, insulin resistance and cardiovascular complications and, consequently, it is considered as the hepatic manifestation of the metabolic syndrome [74]. NAFLD is a very slow progressive disease characterized by accumulation of different lipid species within the hepatocyte.
Therefore, free fatty acids together with pro-inflammatory cytokines are major triggers of NAFLD progression. In this regard, 10-40% of NAFLD patients progress to a more advanced stage of the disease termed non-alcoholic steatohepatitis (NASH), where hepatic tissue presents a robust pro-inflammatory component and hepatocytes injury/death. NASH, in turn, can progress to more severe and irreversible stages like fibrosis, cirrhosis (15-25%) or even hepatocellular carcinoma (7%) [75] (Figure 4).
It is known that insulin resistance plays an important role in the development of NAFLD [76]. Besides the genetic determinants, nutritional factors and lifestyle, soluble mediators produced by immune cells and adipocytes are involved in NAFLD manifestation and progression as well as in the regulation of insulin action [77].
35 Figure 4. NAFLD spectrum scheme. Progression of NAFLD from healthy liver to the irreversible stage cirrhosis. Blue circles contain histology representative schemes of healthy liver or NAFLD stage respectively.
In the past years it has been observed that intestinal microbiota and host metabolism can be tightly linked with NAFLD [78]. Consequently, a great interest has emerged around the study of gut microbiota related to liver diseases.
7.2 Mouse models of non-alcoholic steatohepatitis.
Animal models are helpful to study aspects of pathogenesis and progression of NASH. They are classified as toxin-induced, genetic approaches and nutritional (dietary), or even a combination of these factors.
Toxic-induced models: Intoxication of mice with the porphyrinogenic agents griseofulvin or 3,5-diethoxycarbonly-1,4-dihydrocollidine (DDC) that lead to steatohepatitis after prolonged treatment (>2 months) [79].
Genetic models: some examples of the most used transgenic mice that recapitulate NASH are db/db mice that are hyperphagic and present hyperglycemia, insulin resistance, hyperinsulinemia and macrovesicular steatosis, ob/ob mice that are inactive, hyperphagic and extremely obese and exhibit hyperglycemia, insulin resistance and hyperinsulinemia, develop intestinal bacterial overgrowth and overexpress tumor necrosis factor-α (TNF-α) or MTPa+/- mice (heterozygous mutation of mitochondrial trifunctional protein) that present elevation of serum alanine aminotransferase, insulin resistance, hyperinsulinemia, impaired glucose tolerance and progressive hepatic steatosis. [80].
Healthy liver NAFLD
Simple steatosis
NASH
Inflammation +/- Fibrosis
Cirrhosis
Advanced fibrosis Scar tissue
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Nutritional (dietary) models: dietary models that intend to reproduce a metabolic syndrome-like situation but most of them show differences to the human disease regarding clinical, metabolic or morphologic aspects.
o Fat-enriched diets: high fat and high fat/fructose/sucrose diet (+/−cholesterol).
o Atherogenic diet: high cholesterol and high cholate.
o Methionine-and choline-deficient (MCD) diet: one of the most widely- used mouse models to induce NASH [81]. This diet contains high amounts of sucrose (40%) and fat (10%) but lacks methionine and choline. The latter molecules are required for hepatic secretion of triglycerides (TGs) as very low density lipoproteins (VLDL) and are also involved in mitochondrial fatty acid β-oxidation. Although MCD diet has been criticized because it causes body weight loss (opposite to the situation in metabolic syndrome), it rapidly produces hepatic steatosis, focal inflammation, hepatocyte necrosis and fibrosis [82]
morphologically correlative to those observed in human NASH.
Morphologic features are macrovesicular steatosis, perisinusoidal fibrosis, mitochondrial abnormalities, hepatocyte ballooning, apoptosis and necroinflammation. [80].
7.3 Link between NASH and gut microbiota.
Since microbiota affects energy extraction from food, gut microbiota is considered nowadays as a major environmental factor contributing to obesity and its complications such as insulin resistance and NAFLD [83].
Several studies have shown that NASH is characterized by dysbiosis and systemic inflammation [84, 85]. When microbiome from healthy subjects and obese patients was analyzed, abundant differences were observed among them. However, these differences were fewer between microbiomes from obese and NASH individuals where only Protobacteria, Enterobacteriaceae and Escherichia exhibited significant differences [84]. Interestingly, NASH patients exhibit systemic ethanol levels; therefore, ethanol producing microbial strains might be involved in the pathogenesis of NASH [86].
Several studies analyzing gut microbiome among NASH, obese and healthy control individuals have been performed in the last decade in order to find a target for
37 intervention or a marker for disease [87-89]. Additionally, interventional studies with probiotics such as VSL#3 or Lactobacillus casei Shirota, and prebiotics like fermentable dietary fructo-oligosaccharides, have been reported, suggesting that their administration may improve NAFLD [90-92].
7.4 Gut-liver axis.
7.4.1 Gut microbiota, incretins and liver.
Lipotoxicity refers to the imbalance of the lipid environment and/or intracellular composition which can lead to cell injury or death. Lipotoxicity is a characteristic of metabolic syndrome, obesity and diabetes and, therefore, is a feature of NAFLD and NASH [93]. Recent studies have shown that gut microbiota impacts on NASH and lipotoxicity. Gut microbes produce short chain fatty acids (SCFAs) through dietary fiber (such as cellulose, xylans or inulin) fermentation. SCFAs can act as substrates in the liver increasing lipogenesis and gluconeogenesis. In addition, ligands of G-protein coupled receptors (such as SCFAs) can also induce incretin secretion i.e.GLP-1 in EECs [94]. SCFAs improve metabolism and their effects include the increase of energy expenditure and thermogenesis and the improvement of the gut barrier function to reduce metabolic endotoxemia [95]. Likewise, dysbiosis inhibits a lipoprotein lipase inhibitor known as fasting-induced adipocyte factor (FIAF) leading to a rise in fat deposition both in adipose tissue and liver [96]. Furthermore, translocation of bacterial products due to an altered gut barrier induced by dysbiosis specifically stimulates TLRs (for example LPS binding to TLR4) in the liver, activating pro-inflammatory pathways which lead to hepatocellular inflammation and eventually to fibrosis [97].
7.4.2 Crosstalk between bile acids and microbiota.
Currently, gut-liver axis is a concept that is acquiring a great impact in the study of liver diseases since it is indispensable for metabolic regulation, gut hormone release and immune responses [98].
Bile acids are responsible of the communication in the gut-liver axis. These are endogenous molecules synthesized in the liver from cholesterol through a complex process that includes at least 17 different enzymes, being later further metabolized by the gut microbiota. In rodents, four primary bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA) and muricholic acids (MCAs) can be found. These bile acids are conjugated with the aminoacid taurine
38
before being excreted. After a meal, bile acids are released into the duodenum, and conjugated bile acids will be then reabsorbed in the distal ileum by the apical sodium dependent bile acid transporter (ASBT). Bile acids will recirculate to the liver within portal blood through a process called enterohepatic circulation [99]. Gut microbiota not only regulates the expression of several of the enzymes involved in the primary bile acids synthesis (CYP7A1 or CYP27A1), the synthesis of taurine and the first enzyme required for bile acid conjugation (acyl-CoA-synthetase), but also it regulates the bile acid uptake through ASBT [100, 101].
A large number of gut bacteria from genera Lactobacilli, Bifidobacterium, Clostridium and Bacteroides express bile salt hydrolase activity. Through this mechanism, bile acids are deconjugated (i.e. taurine conjugate is removed), and, consequently, its active reuptake from small intestine via ASBT is avoided. Thus, deconjugated primary bile acids can enter the colon and be metabolized to lithocholic acid (LCA) from CDCA and deoxycholic acid (DCA) from CA. This microbial metabolism facilitates fecal elimination of bile acids (about 5%) since it leads to a more hydrophobic bile acid pool [102].
The link between gut microbiota, bile acids and host metabolism is established through the bile acid receptors farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (also known as TGR5), that also regulate the enterohepatic circulation [10].
FXR is a nuclear receptor expressed mainly in liver and ileum [103] which is activated by the primary bile acids CDCA, CA and LCA (from most potent ligand to less) and inhibited by UDCA. Bile acids synthesis is regulated by feed-back inhibition through the nuclear receptor FXR. In addition, FXR participates in immune responses, insulin signaling, and glucose and lipid homeostasis [102]. FXR seems to protect against hepatic steatosis and elevated triglyceride and bile acid levels [104].
Regarding TGR5, it is a transmembrane receptor widely expressed in gallbladder, intestine, liver, brown and white adipose tissue, among others. It is activated mainly by the secondary bile acids LCA and DCA and, consequently, it is an interesting target to study in the microbiota-bile acid interactions. TGR5 signaling is involved in thermogenesis, insulin signaling, inflammation, and energy homeostasis. Furthermore, it has been recently demonstrated that TGR5 activation can increase energy expenditure
39 by increasing GLP-1 release in intestinal L cells [105], and also that L cells express FXR and that FXR regulates GLP-1 synthesis either [106] (Figure 5).
Figure 5. Bile acids effects on metabolic actions via TGR5 and FXR signaling. Primary bile acids are represented in green circles, secondary bile acids in blue circles.
NOTE. Adapted from “Intestinal Crosstalk between Bile Acids and Microbiota and Its Impact on Host Metabolism” by A. Wahlström et al, 2016, Cell Metabolism Volume 24, Issue 1, 12 July 2016, Pages 41- 50 [102].
7.5 PTP1B knockout mouse model.
PTP1B is a protein tyrosine phosphatase ubiquitously expressed that acts as a negative regulator of insulin and leptin sensitivity since it dephosphorylates the IR and the leptin receptor-associated Janus kinase 2 (JAK2), respectively [107, 108]. In fact, PTP1B is the main negative regulator in the insulin signaling cascade as it acts directly by dephosphorylating tyrosine residues on different critical nodes (IR and IRSs) (Figure 6) [109].
Mice with a global deficiency in the Ptpn1 gene encoding PTP1B are resistant to diet-induced obesity [110] and do not show the typical alterations in metabolic parameters that appear during ageing-associated obesity as the fat mass accretion and increased adipocyte size, hepatic steatosis, islet hyperplasia and hyperinsulinemia [111].
At the molecular level, previous studies in the laboratory demonstrated that during ageing there is an activation of the stress kinases JNK and p38 MAPK in the liver of
40
wild-type mice, an effect absent in PTP1B KO mice. Hence, PTP1B plays a role in the development of chronic low grade inflammation and insulin resistance associated with obesity during ageing [60]. Consequently, PTP1B inhibition has been proposed as a promising drug target for obesity and type 2 diabetes.
Figure 6. Scheme of PTP1B actions on the insulin signaling cascade. Black bars represent points of dephosphorylation.
PTP1B is not only a metabolic modulator, but also an immune modulator. It controls cytokine-mediated signaling pathways by dephosphorylation of JAK2, the non- receptor tyrosine protein kinase 2 (TYK2), and signal transducer and activator of transcription 5 (STAT5) (Figure 7) [112-114]. Moreover, interleukin-4 (IL-4) induces PTP1B mRNA in PI3K-dependent manner and enhances PTP1B protein stability to suppress IL-4-induced STAT6 signaling [112]. It has been reported that PTP1B- deficient mice exhibit an increase in monocyte/macrophages in the spleen and the bone marrow [115]. This seems to be due to a decreased threshold of response to macrophage colony-stimulating factor (M-CSF) by enhancing tyrosine phosphorylation of the activation loop of its receptor (M-CSF1R). In addition, PTP1B modulates activation of monocytes/macrophages restricting the time of pro-inflammatory signaling in response to TLR and to both type I and type II interferon (IFN) signaling [115, 116].In fact, in the absence of PTP1B tissue macrophages exhibit an activated phenotype characterized
41 by the increased expression of CD80 that has a role in the maintenance of inflammation.
However, this effect might require developmental adaptations in macrophages since short-term treatment of mice with the PTP1B inhibitor suramin protects against apoptotic liver damage induced by CD95 and against endotoxic shock mediated by TNF-α [116].
Figure 7. PTP1B regulates cytokine receptor–JAK–STAT signaling. Black bar represent point of dephosphorylation.
Note: Adapted from Pike, K.A. and M.L. Tremblay, TC-PTP and PTP1B: Regulating JAK-STAT signaling, controlling lymphoid malignancies. Cytokine, 2016. 82: p. 52-7. [114]
Studies performed in peritoneal macrophages from PTP1B KO mice evidenced an exacerbated inflammatory response versus the wild-type in response to pro- inflammatory stimuli with increased expression of M1 polarization markers. Moreover, this enhanced activation in PTP1B KO macrophages was not restricted to TLR4 signaling, but also occurred in response to TLR2 (lipoteichoic acid, LTA) and TLR3 (polyI:C) challenge. Nevertheless, in the absence of PTP1B, macrophages showed an impaired response to IL-4 plus IL-13 resulting in attenuated M2 polarization that suggests a dual role for PTP1B in the regulation of M1/M2 commitment [117]. These data suggest that the broad use of PTP1B inhibitors, although with potential benefits over the insulin signaling pathway, might exert undesirable effects in response to stressors of the immune system including the fine tuning of the pro-inflammatory and pro-resolution balance. Taking these data together, it can be summarized that PTP1B
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has dual role; in non-immune cells such as hepatocytes promoting insulin resistance and cell death [118] and in immune cells (macrophages and B cells) inhibiting the pro- inflammatory responses [112, 113].
This duality of PTP1B effects is highly relevant in the inflammatory diseases of the liver due to the coexistence of both in immune and non-immune cells in this organ.
Global PTP1B deficiency protects against hepatic steatosis and improves hepatic regeneration in mice fed a HFD [87], whilst hepatocyte-specific PTP1B-deficient mice present a better glucose and lipid homeostasis [88] and attenuates diet-induced endoplasmic reticulum stress [89]. Importantly, during NASH progression, PTP1B restrains inflammation due to the above mentioned effects in immune cells (both resident and recruited), whilst in NASH reversion it targets the proliferative responses mediated by Met signaling in oval liver cells, the adult hepatic progenitor cells in rodents [90]. In particular, when challenged a MCD diet, PTP1B-deficient mice developed hepatic inflammation and NASH more rapid that their wild-type counterparts. By contrast, when mice with established NASH were switched to a chow diet those lacking PTP1B presented an accelerated recovery due at least in part to the effects on hepatic oval cells. Therefore PTP1B KO is a quite interesting mouse model to study NASH evolution due to the cell specific effects in non-immune and immune cells of this phosphatase.
In this Thesis we propose two hypotheses:
1. Since ageing is a stage characterized by a low-grade chronic inflammation and insulin and leptin resistance, it could also be associated to gut inflammation with an altered gut microbiota and CCK resistance and, hence, to an alteration in the gut-brain axis.
2. Due to the duality of the PTP1B effects, protecting against inflammation on immune cells and decreasing insulin sensitivity on non-immune cells, it could exert also dual effects on the gut since intestine owns both, immune and non- immune cells.
