Assessing metabolic plasticity in diet-induced obese mice upon lifestyle intervention. An integrative approach

[eng] Type 2 Diabetes Mellitus (T2DM) is the metabolic disorder that accounts for the presence of hyperglycemia within insulin resistance (IR). The International Diabetes Federation estimated in 2013 that 382 million people (8.3% of world society ) had diabetes and that this number is set to rise beyond 592 million people in the next 22 years. T2DM accounts for 90% of people with diabetes (WHO 1999). Obesity is considered a major risk factor for developing T2DM over time. The World Health Organization (WHO) stated in 2014 that more than 1.9 billion adults were overweight and of these, over 600 million were obese (body mass index (BMI) > 30 kg/m2). Besides healthcare costs, WHO projects that diabetes will be the 7th leading cause of death in 2030 (Mathers & Loncar 2006). Once T2DM is diagnosed, the first therapeutic approach is by lifestyle counselling consisting of an increase in physical activity and changes in the patient dietary habits. The aim of this project is to study and integrate the metabolic responses that regulate systemic glucose homeostasis. We are not aware of other works describing in a holistic way the different metabolic processes regulating glucose homeostasis in different tissues that play an important role during the development and onset of diet-induced T2DM. With this approach, we will gain more insight and a better knowledge of the metabolic alterations taking place during an obese state induced by high-fat diet, as well as assess the degree of reversibility that can be reached by undergoing a lifestyle intervention, known as “metabolic plasticity”. For this purpose a diet-induced obese animal model of T2DM is used. To achieve the presented aims, a phenotypical and funcional study is performed at systemic level in order to complete a more experimental and detailed approach afterwards in each of the studied tissues: pancreas, white adipose tissue, liver, oxidative and glycolytic skeletal muscle, and hypothalamus. This experimental approach encompasses tissue-specific-functional analysis, gene expression studies, protein content determination and signalling, metabolomics and RNAseq. Likewise, systems biology tools have been developed and have allowed to measure several correlations as well as perform different types of multivariant analysis with the studied parameters. Three experimental groups are defined representing the metabolic stages of interest: control group (Ctrl); pathologic group (HFD, that mimic diet-induced T2DM after 16 wks on HFD; and in which animals showed overweight, and fasting hyperglycemia and hyperinsulinemia); and a third group (Int) that follows a lifestyle intervention consisting of caloric restriction, modification of the fatty acid source and carbohydrates in the diet, and the performance of an exercise training programme. The diet-induced obese experimental group (HFD group) reported the typical physiological features of the pathological state: overweight, fasting hyperglycemia, hyperinsulinemia and hyperleptinemia, increase in fat mass and volume, increase of white adipose tissue, liver and pancreas weight, increase of liver and oxidative skeletal muscle triglycerice levels, glucose intolerance, insulin resistance, increase in beta-cell mass along with hypertrophic enlarged islets and dysfunction in glucose-stimulated insulin secretion in vivo and in vitro, and a diminishment in oxygen consumption, heat production and scapular temperature. Lifestyle intervention was enough to revert most of the disruptions reported in the pathological group. However, certain irreversibility degree was still observed in particular studied parameters: (1) alteration in fasting glucose and glucose-stimulated insulin secretion in vivo, (2) increment in pancreatic beta-cell area, (3) affectation in the epididymal white adipose tissue with inflammation and immune cell infiltration, as well as (4) mitochondria dysfunction, already observed in the pathological state. Taken all this together, we can conclude that the pathological state left a certain degree of metabolic irreversibility does not allow a total recovery of the phenotype across the different tissues studied, at least with this type of intervention and timings. The development and application of systems biology tools have allowed the study the irreversibility degree in an integrative mode, the correlations among certain parameters at a multiorganic level, the gene expression patterns of complexes described from a protein-protein interaction (PPI) network. These strategies and computational approaches have led to the identification of most of the altered tissues and metabolic pathways in the different states under study. [cat] La Diabetes Mellitus del tipus 2 (DM2) es una malaltia que es caracteritza per uns nivells elevats de glucosa i insulina circulants ocasionats per un estat de resistencia a la insulina. Segons la International Diabetes Federation, el 2013 382 milions de persones van ser diagnosticades de diabetes (8.3% de la poblacio mundial), i d’acord amb les prediccions aquesta xifra augmentara fins els 592 milions en els proxims 22 anys. La DM2 explica el 90% dels casos de diabetes (WHO 1999). L’obesitat es un factor de risc per la DM2 i avui en dia suposa una epidemia: el 2014 la OMS va xifrar en 1.9 bilions la poblacio adulta amb sobrepes i 600 milions amb obesitat. A part del cost economic que suposa per a la societat, la OMS va projectar que el 2013 la DM2 sera la setena causa de mort al mon (Mathers & Loncar 2006). Un cop diagnosticada, la primera aproximacio en l’assessorament al pacient amb DM2 o en un estat de risc consisteix en una intervencio en l’estil de vida: incrementant l’activitat fisica i portant a terme una dieta equilibrada i saludable. Aquest projecte te com a objectiu l’estudi i la integracio de les respostes metaboliques responsables de regular l’homeostasi de la glucosa a nivell sistemic. Avui en dia, no existeix cap treball que descrigui de forma holistica els diferents processos metabolics que regulen l’homeostasi de la glucosa en tots els teixits que juguen un paper determinant durant el desenvolupament de la DM2 associada a l’obesitat. D’aquesta manera, es preten guanyar coneixement sobre les alteracions metaboliques que tenen lloc en un estat d’obesitat induit per una dieta rica en greixos, i aixi mateix valorar el grau de reversibilitat que es pot assolir mitjancant una intervencio en l’estil de vida, al que ens referim com a “plasticitat metabolica”. Utilitzem un model animal d’obesitat i DM2 induida per una dieta alta en greixos. Per aconseguir els objectius plantejats inicialment es realitza un estudi fenotipic i funcional a nivell sistemic per mes tard realizar una aproximacio experimental mes exhaustiva en cadascun dels teixits d’interes: pancrees, teixit adipos blanc, fetge, muscul esqueletic oxidatiu i glicolitic, i hipotalem. Aquesta aproximacio experimental engloba analisis funcionals-teixit especific, estudis d’expressio genica, determinacio del contingut proteic i les vies de senyalitzacio, metabolomica i RNAseq. Aixi mateix, s’han desenvolupat eines de biologia de sistemes que han permes calcular diferents correlacions i fer diferents tipus d’analisis multivariant amb tots els parametres estudiats. Es defineixen tres grups experimentals d’animals que concreten els estats metabolics d’interes: grup control (Ctrl); grup patologic (HFD) (que simula la DM2 induida per la dieta grassa durant 16 setmanes, i en que els animals tenen sobrepres i la glucosa i insulina circulants elevades en deju); i un tercer grup que segueix una intervencio en l’estil de vida (Int) que consisteix en restriccio calorica, una modificacio de la font d’acids grassos i hidrats de carboni de la dieta, juntament amb la realitzacio d’un programa d’exercici. El grup experimental d’obesitat induida per una dieta grassa (grup HFD) presenta les caracteristiques fisiologiques propies de l’estat patologic: sobrepes, hiperglucemia, hiperinsulinemia i hiperleptinemia en deju, augment de la massa i el volum de greix, augment del pes del teixit adipos blanc, el fetge i el pancrees, augment dels nivells de triglicerids en fetge i muscul oxidatiu, intolerancia a la glucosa, resistencia a la insulina, augment de la massa de cel·lula beta en el pancrees juntament amb illots hipertrofics engrandits i disfuncio de la secrecio d’insulina estimulada per glucosa in vivo i in vitro, i disfuncio del consum d’oxigen, generacio de calor i temperatura escapular. La intervencio va ser suficient per revertir gran part de les alteracions observades en el grup patologic. No obstant, encara s’observa cert grau d’irreversibilitat en determinats parametres estudiats: (1) alteracio de la glucosa en deju i la resposta insulinica davant d’un estimul de glucosa in vivo, (2) increment en l’area de cel·lula beta pancreatica, (3) afectacio en el teixit adipos blanc epididimal amb la presencia d’inflamacio i infiltracio de cel·lules immunes, aixi com (4) disfuncio mitocondrial, ja observats en l’estat patologic. Amb tot, podem concloure que l’estat patologic deixa un cert grau d’irreversibilitat metabolica no permetent aixi una recuperacio total del fenotip en tots els teixits estudiats, almenys, amb aquest tipus d’intervencio i aquests periodes de temps. El desenvolupament i l’aplicacio d’eines de biologia de sistemes han permes estudiar el grau de reversibilitat d’una manera integrada, les correlacions entre parametres concrets a nivel multiorganic, el patro d’expressio genica de complexes descrits en una xarxa de protein-protein-interaction (PPI). Aquestes estrategies computacionals han permes identificar aquells teixits i vies metaboliques mes alterades en els diferents estats estudiats.