““New Approaches to Gene New Approaches to Gene Discovery with Animal Discovery with Animal Models of Obesity and Models of Obesity and

Diabetes”Diabetes”

Greg Collier, Ken Walder, Andrea De Greg Collier, Ken Walder, Andrea De Silva, Janette Tenne-Browm, Andrew Silva, Janette Tenne-Browm, Andrew

Sanigorski, David Segal, Lakshmi Sanigorski, David Segal, Lakshmi Kantham, and Guy AugertKantham, and Guy Augert

By Martha Raj

What is a disease model? What is a disease model?

To learn more about the diseaseTo learn more about the disease To study the factors important for To study the factors important for

the disease process. the disease process. To study disease treatment. To study disease treatment.

Methods of Modeling:Methods of Modeling:• Mathematical modelsMathematical models• Cell culture Cell culture • Volunteer patientsVolunteer patients• Animal ModelsAnimal Models

Examples of Animal ModelsExamples of Animal Models

Induction of cancer in a mouse to Induction of cancer in a mouse to simulate cancer in a human simulate cancer in a human

Studying a genetic disease in a pure-Studying a genetic disease in a pure-bred dog that is similar to a human bred dog that is similar to a human disease disease

Transplanting an organ between 2 Transplanting an organ between 2 pigs pigs

Why use animal models?Why use animal models?

Alternatives cannot answer all of the Alternatives cannot answer all of the questions scientists have.questions scientists have.

Computer Models and Cell Cultures Computer Models and Cell Cultures are limited. are limited.

DNA based approaches for the DNA based approaches for the discovery of genes have not been discovery of genes have not been very successful. very successful.

Studying patients is also not ideal.Studying patients is also not ideal.

Type 2 DiabetesType 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 For Glucose to be used by body cells, a hormone

produced by the pancreas, called insulin, is produced by the pancreas, called insulin, is needed.needed.

Diabetes Diabetes - - The pancreas produces little or no insulin.The pancreas produces little or no insulin.

- The cells throughout the body are unable to - The cells throughout the body are unable to respond to the insulin that is being produced. respond to the insulin that is being produced.

Type 2 Diabetes = Noninsulin dependent Type 2 Diabetes = Noninsulin dependent diabetes mellitus (NIDDM) or adult onset diabetes diabetes mellitus (NIDDM) or adult onset diabetes

- A combination of insulin resistance and a defect - A combination of insulin resistance and a defect in insulin production. in insulin production.

DNA Based ApproachesDNA Based Approaches

A complex disease such as Type 2 A complex disease such as Type 2 Diabetes cannot be easily studiedDiabetes cannot be easily studied

- gene-gene interactions.- gene-gene interactions.

- gene-environment interactions.- gene-environment interactions. Large numbers and variable Large numbers and variable

combinations of small gene defects combinations of small gene defects leading to the final diseaseleading to the final disease

AlternativesAlternatives

RNA (gene expression) based approachesRNA (gene expression) based approaches Proteomics based approachesProteomics based approaches

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

RNA based technologiesRNA based technologies ddPCR ddPCR -- Display polymerase chain Display polymerase chain

reaction.reaction.- - A widely used technique for identifying A widely used technique for identifying genes genes

differentially expressed between cell types.differentially expressed between cell types. SSH, Suppression subtractive SSH, Suppression subtractive

hybridization:hybridization:- A method for generating differentially regulated or - A method for generating differentially regulated or

tissue-specific cDNA probes and libraries. tissue-specific cDNA probes and libraries. cDNA microarrayscDNA microarrays

- Similar to the oligonucleotide arrays. Instead of short - Similar to the oligonucleotide arrays. Instead of short oligos on the DNA chip, each spot contains a cDNA oligos on the DNA chip, each spot contains a cDNA clone from a known gene.clone from a known gene.

RNA Based Technologies Cont’dRNA Based Technologies Cont’d

ddPCR & SSH – successfully used to ddPCR & SSH – successfully used to find novel genes in energy find novel genes in energy metabolism.metabolism.

- Beacon, a polypeptide involved - Beacon, a polypeptide involved in the regulation of energy balance, in the regulation of energy balance, was identified using ddPCR. This was identified using ddPCR. This gene is differetially expressed in the gene is differetially expressed in the hypothalamus of Israeli sand rats hypothalamus of Israeli sand rats ((Psammomys obesusPsammomys obesus) )

SSHSSH

Developed to overcome technical Developed to overcome technical limitations of traditional subtractive limitations of traditional subtractive methods.methods.

Key Advantage: Its ability to Key Advantage: Its ability to exponentially amplify and equalize exponentially amplify and equalize both rare and abundant differentially both rare and abundant differentially expressed transcripts while expressed transcripts while suppressing common sequences in suppressing common sequences in the population. the population.

SSH Cont’dSSH Cont’d

The generated cDNAs can be directly The generated cDNAs can be directly inserted into a cloning vector.inserted into a cloning vector.

The clones can be screened using cDNA The clones can be screened using cDNA dot blots to eliminate false positives. dot blots to eliminate false positives.

Then they can be sequenced or can be Then they can be sequenced or can be used to generate subtracted cDNA used to generate subtracted cDNA libraries for future microarray analysis. libraries for future microarray analysis.

cDNA libraries generated using SSH are cDNA libraries generated using SSH are quick and is species-and tissue-specific. quick and is species-and tissue-specific.

Disadvantages of SSH & ddPCRDisadvantages of SSH & ddPCR

Labor IntensiveLabor Intensive Identify only small numbers of Identify only small numbers of

differentially expressed genes.differentially expressed genes. Limitations caused by the binary Limitations caused by the binary

nature of the comparisons using nature of the comparisons using these techniques. these techniques.

-cannot be used in complex -cannot be used in complex experiments such as time-course or experiments such as time-course or dose response studies. dose response studies.

cDNA Microarray ExperimentscDNA Microarray Experiments

Can provide gene expression data for Can provide gene expression data for thousands of genes and large thousands of genes and large experimental samples.experimental samples.

Suitable for complex, multivariate Suitable for complex, multivariate analyses. analyses.

Current analysis techniques tend to Current analysis techniques tend to focus on clustering algorithms to focus on clustering algorithms to identify similarities between genes. identify similarities between genes.

The ideal combinationThe ideal combination

New technologies New technologies

++

Appropriate animal modelsAppropriate animal models

==

Successful understanding of a human Successful understanding of a human diseasedisease

Animal Models Animal Models for Obesity & Type 2 Diabetesfor Obesity & Type 2 Diabetes

Psammomys obesusPsammomys obesus (Israeli sand rat): (Israeli sand rat): Gerbil-like rodents found in the desert Gerbil-like rodents found in the desert regions of the Middle East and North regions of the Middle East and North America.America.

They are lean and free from diabetes in They are lean and free from diabetes in their native habitat. their native habitat.

Under study, they were fed standard Under study, they were fed standard rodent chow in the labs, which resulted in rodent chow in the labs, which resulted in obesity, insulin resistance, and Type 2 obesity, insulin resistance, and Type 2 diabetes developed.diabetes developed.

Why is Why is P.obesusP.obesus an Excellent an Excellent model?model?

Adult Adult P.obesusP.obesus have a wide range of body have a wide range of body weight and body fat content. weight and body fat content.

The body weight distribution closely The body weight distribution closely resembles that observed in human resembles that observed in human populations. populations.

Has a heterogeneous distribution of blood Has a heterogeneous distribution of blood glucose, plasma insulin and body weight, glucose, plasma insulin and body weight, and these are almost identical to the and these are almost identical to the patterns found in human populations. patterns found in human populations.

Why is Why is P.obesusP.obesus used? Cont’d used? Cont’d Genetic factors cause 51% of the variation Genetic factors cause 51% of the variation

in body weight, and 23-26% of the in body weight, and 23-26% of the variation in blood glucose and plasma variation in blood glucose and plasma insulin concentrations. insulin concentrations.

Various disturbances occur in the plasma Various disturbances occur in the plasma lipid profile, lipoprotein composition, and lipid profile, lipoprotein composition, and in liver cholesterol metabolism. in liver cholesterol metabolism.

The close resemblance of the phenotypic The close resemblance of the phenotypic pattern in P.obesus to the human pattern in P.obesus to the human population, makes it an excellent animal population, makes it an excellent animal model of obesity and type 2 diabetes.model of obesity and type 2 diabetes.

ResultResult

The Beacon GeneThe Beacon Gene

- Identified using ddPCR.- Identified using ddPCR.

- A gene product overexpressed - A gene product overexpressed in in the hypothalamus of obese, the hypothalamus of obese, diabetic animals. diabetic animals.

- Further studies showed that - Further studies showed that this this gene was expressed in direct gene was expressed in direct proportion to the body fat content. proportion to the body fat content.

Is this gene important in the Is this gene important in the development of diabetes?development of diabetes?

It takes 3 steps in determining the It takes 3 steps in determining the importance of a gene in any importance of a gene in any particular disease.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 Validation of the protein’s potential as a

therapeutic target.therapeutic target.

Validation of the Functional Role of Validation of the Functional Role of a Target Genea Target Gene

The target gene undergoes a lot of The target gene undergoes a lot of study:study:

- - Protein production in bacteria, Protein production in bacteria, yeast, insect, or mammalian cells.yeast, insect, or mammalian cells.

--In vitroIn vitro cellular studies. cellular studies.

- Protein/Protein interactions.- Protein/Protein interactions.

- - In vivoIn vivo animal studies. animal studies.

Validation of the Beacon gene Validation of the Beacon gene

Cell culture models – Reveal the Cell culture models – Reveal the nature of metabolic pathways.nature of metabolic pathways.

Protein-Protein interactions – Protein-Protein interactions – Provides clues for positioning the Provides clues for positioning the candidate protein in a defined signal candidate protein in a defined signal transduction pathway.transduction pathway.

• Yeast two-hybrid systemYeast two-hybrid system• Fluorescence and Bioluminescence Resonance Fluorescence and Bioluminescence Resonance

Energy Transfer.Energy Transfer.• BiacoreBiacore

Protein-Protein InterationProtein-Protein Interation

Yeast two-hybrid method helped Yeast two-hybrid method helped identify a novel kinase that interacts identify a novel kinase that interacts with beacon.with beacon.

This kinase could provide a novel candidate This kinase could provide a novel candidate pathway.pathway.

This kinase can be used as a target to This kinase can be used as a target to decrease beacon action, and with further decrease beacon action, and with further development could be used as a potential development could be used as a potential therapeutic agent in the treatment of therapeutic agent in the treatment of obesity. obesity.

In vivo Animal StudiesIn vivo Animal Studies

P.obesus was treated with beacon P.obesus was treated with beacon and observed for 7 days.and observed for 7 days.

Increase in food intakeIncrease in food intake Increase in body weight Increase in body weight

ConclusionConclusion

Beacon plays a major role in the Beacon plays a major role in the regulation of energy balance in regulation of energy balance in P.obesus.P.obesus.

Researches can use the defined Researches can use the defined pathway of beacon action to find new pathway of beacon action to find new therapeutic approaches to obesity therapeutic approaches to obesity and diabetes. and diabetes.