Leptin- leptos means thin.Leptin produced from adipocytes, released in pulsatile manner, inversely related to cortisol level. Leptin acts on hypothalamic nuclei, at specific leptin receptors2gps of nuclei in arcuate nucleus. AGRP agouti related peptide. POMC preproopiomelanocortin
Epidemic of obesity-globesityOverwt grading I BMI 25-30 , II 30-40, III >40
Body composition is estimated: carcasses oven dried at 95*C for 6-9 days till constant wt is reached. Lipid content is measured in gonadal and retroperitoneal fat pads. For this, adipose tissue is homogenised with 2:1 chloroform-methanol mixture and washed with water. The resulting mixture separates into two phases, lower one has pure lipid extract.
Screening of anti obesity drugs
SCREENING OF ANTI- OBESITY DRUGS Feb 2011
Objectives To understand the need for new anti obesity drugs To understand the pathophysiology To understand how to manipulate in vivo models To know basic in vitro tests
Overview Introduction Pathophysiology Problems in animal models Parameters assessed In vitro methods In vivo methods
Obesity Energy intake> Energy expenditure BMI>30 kg/m2 Multifactorial Orexigenic peptide- NPY, AgRP, Orexin A &B, galanin, endorphin, NE, GH-RH Anorectic peptide- Corticotrophin-RH, MSH, CCK, GLP1,CGRP, bombesin
Need for anti obesitydrugs? One billion adults are overweight >300 million are obese Prevalent in low income countries. Obesity are linked to more deaths worldwide than underweight.
Need for anti obesity drugs? Cont’d… In late 2009, $1.1 billion market anti-obesity drugs could nearly triple to reach $3.1 billion by 2016 No new anti-obesity drug FDA approved since 1999
Why mouse models ? Represenative for human disease Genome sequenced Acceptable reproduction time Large numbers can be handled Identification of disease genes easier than in humans
Lack of Ideal model Obesity – a complex disorder Exact pathology - unknown Humans tend to enjoy eating and are not forced to eat high fat diet Humans do not have induced gene mutations No single animal model can display interplay of behaviour, environment and genetic factors.
Animal models-Parameters Food intake- intake and spillage Body weight Adipose tissue cell size and number- osmium fixation method Body composition Locomotor /physical activity Plasma lipids, insulin and glucose levels
Animal models In vivo In vitro Diet Virus Hypothalainduce induce Genetic mic d d Monoge Polyge nic nic
1. Diet Induced Obesity Rationale: calorie foods Animal: Adult female rat 230g Procedure: Animals given cafeteria diet. Body wt, food intake, locomotor activity and serum insulin measured. After 3months, rats sacrificed and adipose tissue cell size, body composition and lipid content is determined
2. Hypothalamic Obesity Rationale: Hypothalamus regulates food intake. Venteromedial hypothalamic lesions food intake- obesity in 3-4 months. Surgical Chemical
a).Surgically inducedhypothalamic obesity Animal: female Sprague Dawley rats 190g Procedure: high fat diet for 5-9 days. The cuts are made 1mm lateral to the midline, extended from 8.5-5.5mm anterior to ear bars and from 3mm dorsally from the base of the brain.
b). Chemically inducedhypothalamic obesity Animals: Mice/Rat (2-40 d old) parameters Observation of x 3months Procedure:Daily inj Monosodium-L-glutamate 2g/Kg , S/C x 5d ORSingle Inj of Gold thioglucose 30-40mg/Kg , I/P ORSingle Inj Bipiperidyl mustard 5-50mg/Kg, I/P OR
3. Virus induced obesity Rationale: Some specific viruses target hypothalamus leading to virus induced disruption of critical brain CA pathways, leading to obesity Animals: Mice Procedure: Mice infected with canine distemper virus, develops obesity in 8-10 weeks. Other viruses: Rous-associated virus-7 Avian adenovirus SMAM-I Ad-36
4. Genetic models of obesity Monogenic Polygenic
Yellow obese mouse (Aya) Rationale: Obesity inherited through dominant gene, on Chromosome 2 at linkage group 5, agouti locus.
Obese Diabetesmouse mouse Autosomal recessive Autosomal recessive mutation on mutation on chromosome 6 chromosome 4 Inbred stock of Inbred stock of C57BL/6J strain C57BL/KsJ strain Obesity, Obesity, hyperglycaemia, insulin hyperglycaemia, resistance
Fat mouse Tubby mouse Late onset obesity Late onset Autosomal recessive Autosomal recessive ‘Fat mutation’ Tub mutation Chromosome 8 C57BL/6J inbred strain coding for CPE Additional: CPE involved in sensorineural insulin metabolism deafness,retinal Additional: infertility degradation
Fatty rat Obese SHR rat Zucker fatty rat Mating SHR female Most widely used rat (kyoto wistar)with Autosomal recessive normotensive Fa/fa homozygous Sprague Dawley rat Inbred strains after Obese by 3-5 weeks age several generation Substrain JCR: LA Corpulent rat Vascular complications
WDF/TA-FA rat Wistar fatty rat Tranfer of fatty gene (fa) from Zucker rat to Wistar Kyoto rat
Polygenic ModelsJapanese KK mouse NZO mouse Most suitable New Zealand obese Large body size mouse mice inbred 6month age- renal Yellow obesity(AY) disease, autoimmune transferred to KK disorder mice KK-Ay mice Delayed onset obesity
Other polygenic models OLETF rat Otsuka-Long evans-Tokushima-Fatty rat nephropathy model BSB model AKR/J x SWR/J model
Transgenic modelsRationale: genes regulating energy homeostasis are manipulatedProcedure – gene for diphtheria toxin A chain is used to link to the gene chosen to be knocked out KO 3 gene – in white and brown adipose tissue KO Uncoupling protein in brown fat (thermogenesis) KO mice lacking Steriodogenic factor I (SF-I) Overexpression of corticotropin releasing factor gene, GLUT-4 gene, human agouti-related protein complementary DNA Genes for leptin, leptin receptor, growth hormone,
In vitro assays1. To study metabolic activity in brown adipose tissue Assay for uncoupling protein and GLUT4 Procedure: OLETF rat, 10 weeks age are given test drug OD S/C . Rats sacrificed at 14 weeks age. Brown and white fat removed. UCP and GLUT4 determined with western blot analysis.2. To study 3 agonist activity they induce wt. loss by increased thermogenesis, suppression of leptin gene expression c-AMP response element-luciferase receptor gene assay for agonist.
In vitro assaysContd’…..3. Assay for Neuropeptide Y It stimulates appetite. Six receptors Y 1-6 Y5,Y1 antagonist- new drug targets4. Role of leptin Ob gene product. Receptor: lepr or OB-R a)Assay for leptin mRNA level in adipose tissue - Northern blot analysis b) RIA for measurement of plasma leptin
Isolated adipocyte cell linesFor leptin and leptin mRNA:1. Rat Preadipocytes- epididymal fat pad2. Rat primary cultured mature adipocytes3. 3T3-L1 adipocytes- mouse fibroblasts
Practical Implications Dietary models- represent behaviour and environmental factors Genetic models- for understanding genetics of human obesity Polygenic models- human obesity is also polygenic
References Drug screening methods - S K Gupta Drug Discovery and Evaluation - Vogel Pharmacology- Rang and Dale Biology of Obesity: Lessons from Animal Models of Obesity. Journal of Biomedicine and Biotechnology doi:10.1155/2011/197636