Improving Diabetes Research: Moving Beyond Animal Models Charu Chandrasekera, Ph.D. Anne Bunner, Ph.D. July 19, 2014
From Bench-to-Bedside Sulfonylurea Biguanide Dipeptidyl peptidase-4 inhibitor Glucagon-like peptide-1 receptor agonist
From Bench-to-Bedside Januvia DPP- 4 GLP-1 Victoza GLP-1 PI3K Akt Rap Erk1/2 Glyburide Insulin Secretion Pancreatic β-cell
Type 2 Diabetes Research 1. Animal models of human type 2 diabetes 2. Limitations of animal models 3. Humanizing type 2 diabetes research
1. Animal Models of Type 2 Diabetes in Basic Research
Animal Models of Diabetes
Deciphering Diabetes The Problem Research Questions: - How? - What? - Why? - Why not? - When? The Solution! - Where? - What else?
Rodent Models this is how we do it
Rodent Models: Surgical Induction Non-obese diabetic model Partial (10 90%) or Complete pancreatectomy Extent of pancreatectomy governs severity of disease
Rodent Models: Chemical Induction Streptozotocin Glucose GLUT2 Irreversible β-cell cytotoxicity Non-obese model Dosage governs severity of disease Alloxan
Rodent Models: Dietary Modification Cafeteria Diet
Rodent Models: Dietary Modification Pre-Defined High-Fat Diets 30% 20% Fat content 50% 40% 70% 60% Fat types Olive oil Butter Coconut oil Fish oil Lard Beef tallow Vegetable shortening Obese diabetic models Diet type influences disease phenotype Other Components High Sucrose High Fructose Protein Micronutrients
Rodent Models: Genetic Models Obese diabetic models Naturally occurring Spontaneous Genetic Models Leptin and leptin receptor mutations e.g., ob/ob mouse, db/db mouse, Zucker fatty rat, Zucker diabetic fatty rat
Rodent Models: Genetic Models Knock-out Genetically Engineered Models Obese & non-obese Disease severity depends on genetic alteration Knock-in Transgenic
Rodent Models of Diabetes > 50 publications / month Chandrasekera et al. ALTEX 2014; 31:157-76. ob/ob db/db ZDF Other Rats Spontaneously Chemical Transgenic Diabetic Dietary Surgical
2. Limitations of Animal Models
Deciphering Diabetes In order for a rodent model to have relevance to human diabetes, it should display causes natural history pathophysiology manifestations complications drug responses in a manner similar to what is observed in humans.
Deciphering Diabetes Human T2DM = multifactorial & complex Type 2 Diabetes
Of Rodents and Men 1. Experimental Limitations 2. Biological Limitations 3. Species Differences
1. Experimental Limitations: Surgical Induction Sudden pancreatic loss Extent governs disease severity e.g., 80% resection gives mild hyperglycemia β-cell regeneration & full recovery Loss of counter-regulation e.g., glucagon Chandrasekera et al. ALTEX 2014; 31:157-76
1. Experimental Limitations: Chemical Induction Dosage dependence Streptozotocin Extrapancreatic cytotoxic effects e.g., disruption of the HPG axis Human β-cells resistant to streptozotocin in rodents, enter cells via GLUT2 GLUT1 GLUT3 Chandrasekera et al. ALTEX 2014; 31:157-76
1. Experimental Limitations: Dietary Induction Diet type Diet components Control diet Age of onset of diet Duration of diet Lai et al. Nutr Diabetes, 2014
1. Experimental Limitations: Genetic Induction Leptin mutations rare in humans Wang et al. Curr Diab Rev. 2014; 10:131-45 Single-gene effect Exaggerated & compensatory effects Developmental defects Bunner et al. World J Diab. 2014; 5:146-159
2. Biological Limitations Age young adult old Sex No disease phenotype Prominent disease phenotype Strain C57BL/6J, AKR/J, FVB/N, 129SeVe
2. Biological Limitations Heterogeneity among strains 60% weight gain Heterogeneity within one strain 30% weight gain 10% weight gain Control High Fat Diet Weight gain Plasma glucose Plasma insulin Serum lipid Glucose tolerance
2. Biological Limitations Health of laboratory animals is poor by human standards Control laboratory rodents are metabolically morbid: why it matters Obese Insulin resistant Hypertesive On a trajectory to premature death profound differences in experimental outcome confound data interpretation and outcomes of human studies Martin et al. PNAS 2010; 107:6127-33
Chandrasekera et al. ALTEX 2014; 31:157-76 3. Species Differences
3. Species Differences
3. Species Differences α δ β
3. Species Differences
Critiques of Rodent Models Of rodents and men: species specificity of glucose regulation and type 2 diabetes research. Chandrasekera et al. ALTEX 2014; 31(2):157-76. Leptin- and leptin receptor-deficient rodent models: relevance for human type 2 diabetes. Wang et al. Curr Diab Rev. 2014; 10(2):131-45. Knockout mouse models of insuling signaling: relevance past and future. Bunner et al. World J Diabetes. 2014; 5(2):146-159. You are what you eat, or are you? The challenges of translating high-fatfed rodents to human obesity and type 2 diabetes. Lai et al. Accepted for publication in Nutrition & Diabetes, 2014 From animal models to clinical practicality: lessons learned from current translational progress of diabetic peripheral neuropathy. Li et al. In: Souayah N, ed. Peripheral Neuropathy - A New Insight into the Mechanism, Evaluation and Management of a Complex Disorder. New York, NY 2013.
Limitations of Rodent Models: The Case of ZnT8 Zn 2+ insulin secretory granules ZnT8 zinc transporter Genetic study in a small French population identified novel risk loci for T2D: Sladek et al. Nature 2007; 445:881-5. insulin secretory granules
Limitations of Rodent Models: The Case of ZnT8 ZnT8 zinc transporter ZnT8 Gene Deletion ZnT8 Knockout
Limitations of Rodent Models: The Case of ZnT8
Limitations of Rodent Models: The Case of ZnT8 ZnT8 Knockout Variability Age Sex Strain Weight gain Blood glucose Plasma insulin Glucose tolerance Insulin secretion Diet Disease Severity No Change Significant Increase Marked Reduction
Limitations of Rodent Models: The Case of ZnT8 Pound et al. PLoS One 2012; 7:e40972
Limitations of Rodent Models: The Case of ZnT8 intriguing differences between the phenotypes of the animal models 150,000 humans with protein-truncating mutations loss of ZnT8 increased disease risk loss of ZnT8 reduced disease risk Rutter GA. Islets 2010; 2:49-50. Flannnick et al. Nat Genet 2014; 46:357-63.
Limitations of Rodent Models causes natural history pathophysiology manifestations complications drug responses
Deciphering Diabetes NOT The Solution! The Problem
3. Human-based Methods
Human Data Modeling complex interactions in silico Algorithms for pancreas function were used to develop a closedloop insulin pump. Kovatchev B, et al. (2009) J Diabetes Sci Technol 3, 44 55. Kovatchev B, et al. (2010) J Diabetes Sci Technol 4, 1374 1381.
Genome-Wide Association Studies Patients Nonpatients Disease-specific markers Patient DNA Compare differences to discover genetic markers associated with diseases Non-patient DNA Non-disease markers
Human Populations Population-based studies reveal genetic and environmental factors.
Odds Ratio Dietary factors AHS-2 study 41,000 people asked about diets and health Followed for 2 years 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Diabetes Incidence * * * * 95% CI shown Tonstad S, et al. (2013) Nutr Metab Cardiovasc Dis 23, 292-9.
Human Beings Safe and ethical PET and functional MRI can be used to monitor glucose and insulin in volunteers. Bertoldo A, et al. (2006) Diabetes 55, 3028-3037. Tesfaye and Selvarajah (2012) Diab/Metab Res Rev 28, S8-S14. Kullmann, et al. (2012) Human Brain Mapping 33, 1052 1061
GLP-1 and insulin secretion Glucagon-like peptide 1 (GLP-1) is a peptide important for normal glucose tolerance Victoza is a GLP-1 agonist Does GLP-1 work the same way in diabetes patients as in healthy volunteers? Drug that temporarily blocks GLP-1 activity drug Healthy volunteers drug Type 2 Diabetes patients GLP-1 GLP-1 Insulin secretion Insulin secretion Salehi M, et al. (2010) Diabetes 59, 1330-1337.
Human Organs Microfluidic systems mimic architecture of tissues and organs Commercially available liver cells http://www.medicyte.com/products/human-upcyte-hepatocytes.html http://kirkstall.org/index.php/quasi-vivo-system/
Human Tissues Tissues from: cadavers surgical patients
Human islets for drug screening Cultured human islets from cadavers Islets produced insulin in response to glucose Islets exposed to 1,280 compounds Used automated technology to detect changes in insulin secretion Functional cultured islets indicated by white arrowheads Walpita D, et al. (2012) J Biomol Screen 17, 509-518.
Human islets for drug screening Functional cultured islets indicated by white arrowheads Walpita D, et al. (2012) J Biomol Screen 17, 509-518.
Human Cells A human pancreatic β- cell line is now available for insulin secretion studies. Ravassard P, et al. (2011) J Clin Invest 121, 3589-3597.
Inflammation and insulin resistance in human skeletal muscle How does inflammation contribute to insulin resistance? What genes are involved? Researchers used muscle cells from volunteers Turned off a certain gene in some samples to observe differences in response Stimulus Signaling protein Austin RL, et al. (2008) Diabetes 57, 2066-73. Insulin resistance Bouzakri K and Zierath JR (2007) J Biol Chem 282, 7783-7789.
Many options for human studies Human studies are: Reliable Relevant Practical
Thank you for your attention! Questions?