“New Approaches to Gene Discovery with Animal Models of Obesity and Diabetes” Greg Collier, Ken Walder, Andrea De Silva, Janette Tenne-Browm, Andrew Sanigorski, David Segal, Lakshmi Kantham, and Guy Augert By Martha Raj

What is a disease model? To learn more about the disease To learn more about the disease To study the factors important for the disease process. To study the factors important for the disease process. To study disease treatment. To study disease treatment. Methods of Modeling: Methods of Modeling: Mathematical modelsMathematical models Cell cultureCell culture Volunteer patientsVolunteer patients Animal ModelsAnimal Models

Examples of Animal Models Induction of cancer in a mouse to simulate cancer in a human Induction of cancer in a mouse to simulate cancer in a human Studying a genetic disease in a pure- bred dog that is similar to a human disease Studying a genetic disease in a pure- bred dog that is similar to a human disease Transplanting an organ between 2 pigs Transplanting an organ between 2 pigs

Why use animal models? Alternatives cannot answer all of the questions scientists have. Alternatives cannot answer all of the questions scientists have. Computer Models and Cell Cultures are limited. Computer Models and Cell Cultures are limited. DNA based approaches for the discovery of genes have not been very successful. DNA based approaches for the discovery of genes have not been very successful. Studying patients is also not ideal. Studying patients is also not ideal.

Type 2 Diabetes Glucose is necessary for body growth and energy. Glucose is necessary for body growth and energy. For Glucose to be used by body cells, a hormone produced by the pancreas, called insulin, is needed. For Glucose to be used by body cells, a hormone produced by the pancreas, called insulin, is needed. Diabetes Diabetes - The pancreas produces little or no insulin. - The cells throughout the body are unable to respond to the insulin that is being produced. Type 2 Diabetes = Noninsulin dependent diabetes mellitus (NIDDM) or adult onset diabetes Type 2 Diabetes = Noninsulin dependent diabetes mellitus (NIDDM) or adult onset diabetes - A combination of insulin resistance and a defect in insulin production.

DNA Based Approaches A complex disease such as Type 2 Diabetes cannot be easily studied A complex disease such as Type 2 Diabetes cannot be easily studied - gene-gene interactions. - gene-environment interactions. Large numbers and variable combinations of small gene defects leading to the final disease Large numbers and variable combinations of small gene defects leading to the final disease

Alternatives RNA (gene expression) based approaches RNA (gene expression) based approaches Proteomics based approaches Proteomics based approaches Out of the two, RNA based approaches coupled with the appropriate animal bodies is a powerful tool in identifying diabetes candidate genes and understanding the underlying mechanisms of the disease.

RNA based technologies ddPCR - Display polymerase chain reaction. ddPCR - Display polymerase chain reaction. - A widely used technique for identifying genes differentially expressed between cell types. SSH, Suppression subtractive hybridization: SSH, Suppression subtractive hybridization: - A method for generating differentially regulated or tissue-specific cDNA probes and libraries. cDNA microarrays cDNA microarrays - Similar to the oligonucleotide arrays. Instead of short oligos on the DNA chip, each spot contains a cDNA clone from a known gene.

RNA Based Technologies Cont’d ddPCR & SSH – successfully used to find novel genes in energy metabolism. ddPCR & SSH – successfully used to find novel genes in energy metabolism. - Beacon, a polypeptide involved in the regulation of energy balance, was identified using ddPCR. This gene is differetially expressed in the hypothalamus of Israeli sand rats (Psammomys obesus)

SSH Developed to overcome technical limitations of traditional subtractive methods. Developed to overcome technical limitations of traditional subtractive methods. Key Advantage: Its ability to exponentially amplify and equalize both rare and abundant differentially expressed transcripts while suppressing common sequences in the population. Key Advantage: Its ability to exponentially amplify and equalize both rare and abundant differentially expressed transcripts while suppressing common sequences in the population.

SSH Cont’d The generated cDNAs can be directly inserted into a cloning vector. The generated cDNAs can be directly inserted into a cloning vector. The clones can be screened using cDNA dot blots to eliminate false positives. The clones can be screened using cDNA dot blots to eliminate false positives. Then they can be sequenced or can be used to generate subtracted cDNA libraries for future microarray analysis. Then they can be sequenced or can be used to generate subtracted cDNA libraries for future microarray analysis. cDNA libraries generated using SSH are quick and is species-and tissue-specific. cDNA libraries generated using SSH are quick and is species-and tissue-specific.

Disadvantages of SSH & ddPCR Labor Intensive Labor Intensive Identify only small numbers of differentially expressed genes. Identify only small numbers of differentially expressed genes. Limitations caused by the binary nature of the comparisons using these techniques. Limitations caused by the binary nature of the comparisons using these techniques. -cannot be used in complex experiments such as time-course or dose response studies.

cDNA Microarray Experiments Can provide gene expression data for thousands of genes and large experimental samples. Can provide gene expression data for thousands of genes and large experimental samples. Suitable for complex, multivariate analyses. Suitable for complex, multivariate analyses. Current analysis techniques tend to focus on clustering algorithms to identify similarities between genes. Current analysis techniques tend to focus on clustering algorithms to identify similarities between genes.

The ideal combination New technologies + Appropriate animal models = Successful understanding of a human disease

Animal Models for Obesity & Type 2 Diabetes Psammomys obesus (Israeli sand rat): Gerbil-like rodents found in the desert regions of the Middle East and North America. Psammomys obesus (Israeli sand rat): Gerbil-like rodents found in the desert regions of the Middle East and North America. They are lean and free from diabetes in their native habitat. They are lean and free from diabetes in their native habitat. Under study, they were fed standard rodent chow in the labs, which resulted in obesity, insulin resistance, and Type 2 diabetes developed. Under study, they were fed standard rodent chow in the labs, which resulted in obesity, insulin resistance, and Type 2 diabetes developed.

Why is P.obesus an Excellent model? Adult P.obesus have a wide range of body weight and body fat content. Adult P.obesus have a wide range of body weight and body fat content. The body weight distribution closely resembles that observed in human populations. The body weight distribution closely resembles that observed in human populations. Has a heterogeneous distribution of blood glucose, plasma insulin and body weight, and these are almost identical to the patterns found in human populations. Has a heterogeneous distribution of blood glucose, plasma insulin and body weight, and these are almost identical to the patterns found in human populations.

Why is P.obesus used? Cont’d Genetic factors cause 51% of the variation in body weight, and 23-26% of the variation in blood glucose and plasma insulin concentrations. Genetic factors cause 51% of the variation in body weight, and 23-26% of the variation in blood glucose and plasma insulin concentrations. Various disturbances occur in the plasma lipid profile, lipoprotein composition, and in liver cholesterol metabolism. Various disturbances occur in the plasma lipid profile, lipoprotein composition, and in liver cholesterol metabolism. The close resemblance of the phenotypic pattern in P.obesus to the human population, makes it an excellent animal model of obesity and type 2 diabetes. The close resemblance of the phenotypic pattern in P.obesus to the human population, makes it an excellent animal model of obesity and type 2 diabetes.

Result The Beacon Gene The Beacon Gene - Identified using ddPCR. - A gene product overexpressed in the hypothalamus of obese, diabetic animals. - Further studies showed that this gene was expressed in direct proportion to the body fat content.

Is this gene important in the development of diabetes? It takes 3 steps in determining the importance of a gene in any particular disease. Candidate gene identification.Candidate gene identification. Study of its physiological function.Study of its physiological function. Validation of the protein’s potential as a therapeutic target.Validation of the protein’s potential as a therapeutic target.

Validation of the Functional Role of a Target Gene The target gene undergoes a lot of study: - Protein production in bacteria, yeast, insect, or mammalian cells. -In vitro cellular studies. - Protein/Protein interactions. - In vivo animal studies.

Validation of the Beacon gene Cell culture models – Reveal the nature of metabolic pathways. Cell culture models – Reveal the nature of metabolic pathways. Protein-Protein interactions – Provides clues for positioning the candidate protein in a defined signal transduction pathway. Protein-Protein interactions – Provides clues for positioning the candidate protein in a defined signal transduction pathway. Yeast two-hybrid systemYeast two-hybrid system Fluorescence and Bioluminescence Resonance Energy Transfer.Fluorescence and Bioluminescence Resonance Energy Transfer. BiacoreBiacore

Protein-Protein Interation Yeast two-hybrid method helped identify a novel kinase that interacts with beacon. Yeast two-hybrid method helped identify a novel kinase that interacts with beacon. This kinase could provide a novel candidate pathway. This kinase could provide a novel candidate pathway. This kinase can be used as a target to decrease beacon action, and with further development could be used as a potential therapeutic agent in the treatment of obesity. This kinase can be used as a target to decrease beacon action, and with further development could be used as a potential therapeutic agent in the treatment of obesity.

In vivo Animal Studies P.obesus was treated with beacon and observed for 7 days. P.obesus was treated with beacon and observed for 7 days. Increase in food intake Increase in food intake Increase in body weight Increase in body weight

Conclusion Beacon plays a major role in the regulation of energy balance in P.obesus. Beacon plays a major role in the regulation of energy balance in P.obesus. Researches can use the defined pathway of beacon action to find new therapeutic approaches to obesity and diabetes. Researches can use the defined pathway of beacon action to find new therapeutic approaches to obesity and diabetes.