Fisiología Integral de la Obesidad: Obesidad y Diabetes Dario C. Ramirez Laboratorio de Medicina Experimental y Terapéuticas Cátedra de Genética Molecular Facebook: Dario C Ramirez E-mail: ramirezlabimibiosl@ymail.com

Obesidad: definición, etiología y prevalencia

MEDIDAS ANTROPOMÉTRICAS Peso del cuerpo (kg) Altura (m) (Las medidas fueron tomadas mientras los sujetos estaban descalzos y con ropas livianas) El índice de masa corporal (BMI) fue calculado como: IMC = peso del cuerpo [ = ] kg/m2 (altura)2 La circunferencia de cintura (cm) se mide a la altura del ombligo

Grados de Obesidad según IMC ( Kg/m2 ) (20 – 25 normal) GRADO DE SOBREPESO TIPO DE OBESIDAD 25 – 30 I Leve 30 – 35 II Moderada 35 – 40 III Grave 40 – 50 IV Mórbida > 50 V Super-obesidad

Modelo causal de la obesidad

Age-adjusted Percentage of U. S Age-adjusted Percentage of U.S. Adults Who Were Obese or Who Had Diagnosed Diabetes Obesity (BMI ≥30 kg/m2) 1994 2000 2008 No Data <14.0% 14.0-17.9% 18.0-21.9% 22.0-25.9% >26.0% Diabetes 1994 2000 2008 No Data <4.5% 4.5-5.9% 6.0-7.4% 7.5-8.9% >9.0% CDC’s Division of Diabetes Translation. National Diabetes Surveillance System available at http://www.cdc.gov/diabetes/statistics

La obesidad crece en Latinoamérica La obesidad crece en Latinoamérica. Entre los años 1995 y 2012 la población obesa mayor a 15 años creció un 99%, reflejando que un 26,54% de los latinoamericanos se pueden catalogar como  obesos. - See more at: http://blog.euromonitor.com/2013/06/obesidad-en-latinoam%C3%A9rica-factores-detr%C3%A1s-del-incremento.html#sthash.FLUY9bDH.dpuf "La prevalencia de diabetes en la población argentina es de 8.5%“ – ADA 2010

Complicaciones metabólicas de la obesidad

Con o sin tolerancia a la glucosa PERFILES LIPÍDICOS Perfiles lipídicos alterados OBESIDAD DMT2 Con o sin tolerancia a la glucosa Obesos y no obesos Anormalidades lipídicas: TG aumentados LDL aumentadas HDL disminuidas

Infecciones recurrentes (cutáneas, urinarias, etc.) Diabetes Mellitus Signos y síntomas Poliuria Polidipsia Polifagia Infecciones recurrentes (cutáneas, urinarias, etc.) Pérdida de peso o aumento de peso Prurito Sequedad de la boca Alteración visual Fatiga Gentileza Dras: Ojeda y Sweret (Laboratorio de Diabetes UNSL)

Examen de glucosa

Clasificación de la Diabetes

Síndrome metabólico

La inflamación del TA es la causante de las metabolopatías asociadas a la obesidad

Anatomical distribution of adipose tissue Subcutaneous adipose tissue : - abdominal - femoral Intraabdominal adipose tissue : - visceral (mesenteric and omental) - retroperitoneal (perirenal and perigonadic) Other depots : - intra and intermuscular - perivascular - epicardiac Different physiological and pathogenic roles of the fat depots

WAT BAT

Characteristics of brown and white adipocytes Brown adipocyte Multilocular adipocyte Lipid storage and mobilization (++) Mitochondria (+++) Fatty acid oxidation (+++) Respiratory chain (+++) UCP1 (+++) PGC-1a (+++) White adipocyte Unilocular adipocyte ( 200µm) Lipid storage and mobilization (+++) Mitochondria (+) Fatty acid oxidation (+) Respiratory chain (+) UCP1 (0) PGC-1a (+)

Differences in the fate of fatty acids between brown and white fat cells White adipocyte Brown adipocyte Glycerol Glycerol FA FA Glycerol Glycerol FA Lipolysis Lipolysis Triglycerides Triglycerides FA FA FA FA FA FA b oxidation FA esterification FA esterification Glycerol-3P Glycerol-3P Glucose, amino acids, lactate, pyruvate Glycerol, glucose

Adipose tissue development : beyond adipocyte differentiation Mature adipocytes Preadipocytes Endothelial cells Mature adipocytes ADIPOCYTE HYPERTROPHY & HYPERPLASIA ANGIOGENESIS INFLAMMATION Macrophages Preadipocytes

fat cell-specific gene expression Transcriptional control of adipocyte differentiation SREBP1c / ADD1 C/EBP b/d PPAR g C/EBP a RXRa PPAR b Wnt signaling GATA 2 & 3 proliferation differentiation fat cell-specific gene expression J. Lipid Res., 2002, 43, 835-860

Adrenergic control of metabolism in brown fat cells 1, 2, 3 A C Plasma membrane Gs T H E R M O G E N E S I S Mitochondrial biogenesis (PGC1) cAMP PKA UCP1 transcription UCP1 activation Lipolysis (FA) b oxidation

White adipose tissue as an endocrine organ

Hypertrophy of adipocytes & triglyceride overload Crosstalk between the cells ADIPOCYTE Hypertrophy of adipocytes & triglyceride overload Adiponectin NEFA others Leptin  HYPERTROPHIED ADIPOCYTE MCP-1 MIP-1a others TNF-a MCP-1 IL-1b others preadipocyte TNF-a others NEFAs  Macrophage TNF-a MIP-1a MCP-1 others Activation and infiltration of macrophages Cytokines 

Mecanismo central de regulación del peso corporal

FA, Other metabolites, Adipokines, … TNFa, Cytokines, Chemiokines, … Glycerol Lipolysis FA Triglycerides FA FA b oxidation FA esterification Glycerol-3P TNFa, Cytokines, Chemiokines, …

El rol de los macrófagos en la inflamación del TA en obesidad

Macrophages cause fat tissue inflammation Adp Lep Lep Adp Inflamed fat tissue Furukawa, S. et al. 2004. J. Clin. Invest. 114: 1752-1761 Minamino et al. 2009. Nat. Med. 15:1082-1087 Lumeng, C.N. et al. 2007. J. Clin. Invest. 117: 175-184

Modified from: Nature Clin. Pract. Endocrinol. Metab. (2008) 4, 619-626 Lumbeng, C.N.; et al. 2007. J. Clin. Invest. 117:175-184.

Caracteristicas de pacientes obesos insulin sensibles (IS) e insulino resistentes (IR) Kloting, N. et al. Am J Physiol Endocrinol Metab 299: E506-E515 2010 Fig. 3. Representative photographs for the insulin-sensitive (IS) and insulin-resistant (IR) morbidly obese phenotype and increased macrophage infiltration into omental adipose tissue in IR obesity. A: the photographs of 1 patient each for the IS and the IR obese subgroups should demonstrate the differences in abdominal fat distribution despite the same BMI of 45.2 kg/m2. B: hematoxylin and eosin staining of omental adipose tissue sections from representative study individuals. Initial magnification, x20. C: increased macrophage infiltration into omental compared with subcutaneous (sc) adipose tissue. D: CD68 mRNA expression differences between the fat depots and the IR and IS group. E: diameter distribution curves of isolated adipocytes from omental adipose tissue (pooled data from 20 individuals/group). F: insulin-stimulated glucose uptake was significantly lower in omental adipocytes from IR compared with IS obese individuals. *P < 0.05.

Diabetes. 60(12):3159-3168, December 2011. FIG. 2 Diabetes. 60(12):3159-3168, December 2011. FIG. 2 . Body weight change and adiposity in HFD-fed 4-1BB-deficient mice. WT and 4-1BB-deficient mice were fed an HFD for 9 weeks. A: Expression of 4-1BB mRNA in epididymal adipose tissue, liver, and skeletal muscle. Body weight changes and gross morphology of mice (B), energy intake (C), and adipose tissue weight (Ep, epididymal; Re, retroperitoneal; Me, mesenteric; and Sc, subcutaneous) of WT (n = 8) and 4-1BB-deficient mice (n = 8) fed an RD or HFD, and gross morphology of adipose tissues (D). Results are means +/- SEM. *P P P E: Histological analysis of epididymal adipose tissue and size distribution of adipocytes from WT and 4-1BB-deficient mice fed an HFD. Sections were stained with hematoxylin-eosin. Hypertrophied adipocytes are indicated by asterisks. Original magnification is x200 (scale bar = 50 [mu]m). The sizes of adipocytes in randomly chosen fields were measured with a microscope (magnification x200) and calculated using Axiovision AC software. #P < 0.005 compared with WT mice fed an HFD. WAT, white adipose tissue. (A high-quality color representation of this figure is available in the online issue.) FIG. 2 . Body weight change and adiposity in HFD-fed 4-1BB-deficient mice. WT and 4-1BB-deficient mice were fed an HFD for 9 weeks. A: Expression of 4-1BB mRNA in epididymal adipose tissue, liver, and skeletal muscle. Body weight changes and gross morphology of mice (B), energy intake (C), and adipose tissue weight (Ep, epididymal; Re, retroperitoneal; Me, mesenteric; and Sc, subcutaneous) of WT (n = 8) and 4-1BB-deficient mice (n = 8) fed an RD or HFD, and gross morphology of adipose tissues (D). Results are means +/- SEM. *P P P E: Histological analysis of epididymal adipose tissue and size distribution of adipocytes from WT and 4-1BB-deficient mice fed an HFD. Sections were stained with hematoxylin-eosin. Hypertrophied adipocytes are indicated by asterisks. Original magnification is x200 (scale bar = 50 [mu]m). The sizes of adipocytes in randomly chosen fields were measured with a microscope (magnification x200) and calculated using Axiovision AC software. #P < 0.005 compared with WT mice fed an HFD. WAT, white adipose tissue. (A high-quality color representation of this figure is available in the online issue.) 4

Aspectos bioquímicos de la obesidad y sus complicaciones

Role of adipokines and cytokines in obesity complications Subcutaneous adipose tissue Visceral adipose tissue macrophages IL-6 TNF-a Secretions : Leptin Adiponectin RBP4 TNF-a, IL-6… Skeletal muscle Liver utilisation glucose Other target tissues Vessels atherosclerosis, hypertension Non alcoholic CRP steatohepatitis PAI1 Glucose intolerance Insulin resistance Obesity complications

Coordination of the regulation of fat deposition and fat mobilization in white adipose tissue Fed Fasting Triglycerides LPL Fatty acids + Gut Esterification + Triglycerides - + Catecholamines Glycerol 3-phophate ANP, BNP Lipases Glucose Fatty Acids + Glycerol To liver, muscle Glut4 + To liver Pancreas Insulin

Dislipemia en el Síndrome Metabólico ASP ApoB VLDL TG ApoC II-III- E Ac. Grasos Adipocito Hígado Riñon ApoA1 VLDL CE HDL CE TG CETP CETP IR actividad de la lipoproteinlipasa IDL TG CETP CE TG LDL Lipasa hepatica LPL LDL pequeña Grundy MS et al. Am J Cardiol 1998; 81: 18

Role of fatty acids in obesity complications Subcutaneous adipose tissue Visceral adipose tissue Vessels Thrombosis Heart Myocardial performance Portal and visceral FA Abdominal FA FA hepatic flux Skeletal muscle Liver Hepatic glucose production Non alcoholic steatohepatitis Glucose utlization Pancreas Increased synthesis of TG-rich VLDL Glucose intolerance Insulin resistance Hyperinsulinemia Hyperlipidemia Obesity complications

Bases moleculares de la resistencia a la insulina en obesidad

Overview of fatty acid metabolism Alternate fuel source for brain and other organs Liver TG NEFA Ketone bodies and CO2 Adipose tissue VLDL TG LPL Lipases Chylomicrons (lymphatic circulation) NEFA LPL TG FA Muscle, myocardium, kidney cortex, etc. Intestinal absorption CO2 Essays Biochem. 2006;42:89-103.

Myocardial performance Deleterious effects of an excess of nonesterified fatty acids SNS activity Triglycerides Myocardial performance Increased lipolysis Thrombosis Antilipolytic effect HDL-C VLDL secretion NEFA Insulin resistance Glucose utilisation LDL particle size Glucose production Glucose intolerance Hyperinsulinemia Hypertension

Insulin’s command: “utilize glucose now, build proteins and store energy for the future” De Luca & Olefsky, FEBS Lett. 2008,582(1): 97–105

Journal of Clinical Investigation. 116(7):1793-1801, July 2006.

Nature. 444(7121):860-867, December 14, 2006. 7 Figure 6 Therapeutic targets at the interface between metabolic and inflammatory pathways. The pathways are divided into peptide- and lipid-mediated targets for practical purposes and do not represent an exhaustive list. Treating several loci involved in the disease process by targeting organelles such the ER and mitochondria represents a new approach to treating metabolic diseases.

Nature. 444(7121):860-867, December 14, 2006. Figure 5 Molecular pathways integrating stress and inflammatory responses with insulin action. IRS-1 and 2 are crucial signalling molecules in insulin action. Activation of JNK by cytokine signalling, lipid products, ROS or through IRE1 during ER stress leads to serine phosphorylation of IRS-1 and 2, and consequently inhibits insulin signalling. Similar signals, including PERK, also activate IKK and inhibition of insulin action through a series of transcriptional events mediated by NF-[kappa]B. JNK also regulates transcription through AP-1. Lipid-activated transcriptional events are mediated by nuclear hormone receptors PPAR and LXR. The biological activities of lipids are regulated by FABPs that function as chaperones. Mitochondria and the ER can both contribute to ROS production. ATF6 and XBP1 are critical regulators of ER function and its adaptive responses. 6

Genetics of obesity and its complications

Niveles de la regulación de la expresión génica Chromosome GENE RNA transcript mRNA in nucleus mRNA in cytoplasm Polypeptide ACTIVE PROTEIN Exon Intron Tail Cap NUCLEUS Flow through nuclear envelope CYTOPLASM Breakdown of mRNA Translation Broken-down mRNA Broken-down protein Cleavage/modification/ activation Breakdown of protein DNA unpacking Other changes to DNA TRANSCRIPTION Addition of cap and tail Splicing

Polygenic obesity Rare monogenic mutation KCNJ11 Increased energy APOE Challenge Discover pertinent genes, gene combinations and interactions GNB3 APOE AGT KCNJ11 Increased energy HSL LPL MC4R ADRB3 MCR4 represents an intermediate situation between rare monogenic and polygenic obesity. Multiple genes may operate in multiple pathways and interact in multiple ways with the environment. PPARG HNF1A UCP1 mtDNA GYS INS-VNTR Decreased energy expenditure LEPR, POMC, PCSK1 SIM1, LEP, others MC4R 4

Identifying Disease Genes Family Based Linkage Studies Population Based Association Studies Members of a family affected by the Same disease share Identical disease genes Distribution of disease alleles is Different between Cases and Controls

Genetics of human obesity Dissection and strategy Environment Genes Monogenic High penetrance Monogenic Low penetrance Variable expression Polygenic The knowledge brought by the genetic approach is summarized on this slide. First obesity development can be strongly influenced by a limited number of genes in monogenic situations of obesity. The genetic approach in rare obesity cases or syndromes led to the identification of new genes. Then if you go from the left side to the right side of the slide, multiple genes start to interact with environmental factors. So here we have to deal with genetic and phenotypic heterogeneity. Population study and genetic epidemiology have been the approaches to find the causal genes. However, in polygenic forms of obesity, expression gene screening can help to identify the key drivers of obesity physiopathology. The next slides show examples of knowledge brought by the genetic approach. Rare cases Syndromes Population study Genetic epidemiology Tissue investigation in clinical trials “omic studies” Example: Adipose tissue analysis

Candidate genes in obese populations Food intake-peripheral Pancreatic peptides; Isl1, CCK receptors A&B, GLP1-R Food intake-central Monoamines, Peptides&receptors : CART, DRD2, NPY, NPYR, MC3R, POMC, HT2A, AGRP, MC3R, MC4R Morbid obesity Life span Weight gain Obesity onset Fat mass Glucid values Lipid values Food intake Physical activity Thermogenesis bAR1, 2, 3, a AR, CAPN10 UCP1, 2, 3 And others .. FAT and glucose metabolism leptin, leptinR,insulin, InsR, SUR, PTP1b, IRS1, Isl1, GCK LPL, HSL, GRL, DGAT, CPT-1 apoA4, B, E, CD36, FABP2, LDLR, LIPE, GRL, TNFa , TNF-R, adiponectin Master genes ? PPARg, CDX3, SREBP1 30 Positive associations 49 Negative associations

Fisiología integral de la obesidad y sus complicaciones

SNP Databases dbSNP http://www.ncbi.nlm.nih.gov/SNP/index.html Human Genome Variation Database (HGVbase) http://hgvbase.cgb.ki.se/ TSC: The SNP Consortium http://snp.cshl.org/

Nuevos enfoques

Nutritional, genetic and environmental factors contribute to the incidence of type 2 diabetes in obese patients NUTRITION (Imbalance between energy intake and expenditure ) GENOME (SNPs in the MPO promoter) ENVIRONMENT (endotoxin inhalation) OBESITY Adipocyte hypertrophy and hyperplasia Hypoxia Recruitment of Type M1 macrophages (M) M synthesize and release MPO and IR-inducing cytokines Adipocytes take up MPO FAT TISSUE DYSFUNCTION  homing of neutrophils in the lung LUNG INFLAMMATION (Synthesis of IR-inducing mediators)  Circulating pro-inflammatory and IR-inducing cytokines INSULIN RESISTANCE Type 2 diabetes