Animal Models of Endocrine Disease

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Animal Models of Endocrine Disease

Mark J. Hoenerhoff, DVM, PhD, DACVP Associate Professor, In Vivo Animal Core

Unit for Laboratory Animal Medicine

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Outline – Animal models

• History of Animal Models of Endocrine Disease

“The Discovery of Insulin” and Early Animal Studies

• Animal Models of Metabolic Endocrine Disease

• Diabetes mellitus models

• Models of diabetic complications

• Models of endocrine disruption

• Animal Models of Endocrine Neoplasia

• Primary endocrine tumors, Multiple Endocrine Neoplasia type 1

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Outline – Animal models

• History of Animal Models of Endocrine Disease

“The Discovery of Insulin” and Early Animal Studies

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“The Discovery of Insulin”

Aka, “the story of famous, almost famous and little-known people, of serendipities, discoveries and re-discoveries”

(Vecchio et al, 2018)

Discovery of insulin led to the end of the pre-insulin era, or also know

as the “era of frustration”

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“The Discovery of Insulin”

Aka, “the story of famous, almost famous and little-known people, of serendipities, discoveries and re-discoveries”

(Vecchio et al, 2018)

Discovery of insulin led to the end of the pre-insulin era, or also know as the “era of frustration”

Animal models played a critical role in the discovery and development

of insulin therapy in diabetic patients

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“The Discovery of Insulin”

• Diabetes is one of the most studied diseases in the history of medicine

• Ebers Papyrus Egyptian medical texts (~1500 BC)

“Too great emptying of the urine”

Prescribed a treatment made of a

“decoction of bones, wheat, grain, grit, green lead, and earth”

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“The Discovery of Insulin”

• Aretaeus of Cappadocia (~100AD), Greek physician

First to introduce the term “diabetes,” meaning “Passing through”

or “siphon”

Describes the disease of diabetes as “Melting down of the flesh and limbs into urine”

“…fluids do not remain in the body, but use the body only as a channel through which they may flow out. . . . The evil of the

affliction is that thirst and drink increase one another.”

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“The Discovery of Insulin”

• Indian physicians Sushruta and Charaka (400-500 AD)

“…a mysterious disease causing thirst, enormous urine output, and wasting away of the body with files and ants attracted to the

urine” – glucosuria

Identified type II diabetes, termed madhumeha, or “honey urine”

Differentiated type I (associated with youth) and type II diabetes (associated with being overweight)

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“The Discovery of Insulin”

• Thomas Willis (1674), British physician

Conducts urine taste test to connect diabetes and sweetness Describes disease as “p–ing evil” … “evil urination”

Renames disease diabetes mellitus (Greek for honey)

• Matthew Dobson (1776), Liverpool physician

Builds on Willis’ observations, evaporates a patient’s urine Leaves white powder that smells and tastes like brown sugar

Connects sugar in body fluids with diabetes mellitus

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“The Discovery of Insulin”

• Paul Langerhans (1869), PhD student

Describes pancreatic islets, but unable to explain their function

• Edward Albert Sharpey-Schafer (1910), London physician

Theorizes existence of single pancreatic substance causing diabetes Coins the term “insulin” (Latin “insula”, island), referencing

pancreatic islets

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“The Discovery of Insulin”

• Paul Langerhans (1869), PhD student

Describes pancreatic islets, but unable to explain their function

• Edward Albert Sharpey-Schafer (1910), London physician

Theorizes existence of single pancreatic substance causing diabetes Coins the term “insulin” (Latin “insula”, island), referencing

pancreatic islets

BUT…without the use of animal studies, the medical community could not

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Early Animal Studies

• Johann Brunner (1683), Swiss anatomist

Performs first partial pancreatectomy in dogs

Describes polyuria, polydipsia, polyphagia (diabetes)

• Claude Bernard (1856), French physiologist

Performs ductal occlusion in dogs and rabbits

Induces complete acinar atrophy, but fails to produce diabetes (islets unknowingly spared)

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Early Animal Studies

• German physicians Minkowski and von Mering (1889)

Perform first successful total pancreatectomy in dogs Find that dogs without pancreata develop diabetes

Irrevocably establishes the role of the pancreas in the disease

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Early Animal Studies

• German physicians Minkowski and von Mering (1889)

Perform first successful total pancreatectomy in dogs Find that dogs without pancreata develop diabetes

Irrevocably establishes the role of the pancreas in the disease

• BUT…the role of pancreatic islets is still a mystery…

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Early Animal Studies

UNTIL….

• Lydia Maria Adams DeWitt (1905), American pathologist

Performs ductal ligation in cats, isolates extract from Islets that possesses glycolytic properties

Most likely first to isolate and test this extract but was limited by facilities at the University of Michigan

Was unable to examine the effects of the extract on human patients

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Early Animal Studies

• Eugene Gley (1905), French endocrinologist

Isolates extracts from Islets of Langerhans of dogs Administers to diabetic dogs, and improves glycosuria Seals research findings in envelope given to the Societe Francaise de Biologie, to be opened only upon his request

Stops research for unknown reasons…

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Early Animal Studies

• 1921: Frederick Banting, Canadian surgeon and Charles Best, student (University of Toronto)

Repeat Eugene Gley’s experiments…

Isolated insulin from dogs, induced diabetes, then reversed hyperglycemia with injections of extract

Introduce insulin to the world

Banting receives the Nobel Prize in 1923!

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Early Animal Studies

• 1921: Frederick Banting, Canadian surgeon and Charles Best, student (University of Toronto)

Repeat Eugene Gley’s experiments…

Isolated insulin from dogs, induced diabetes, then reversed hyperglycemia with injections of extract

Introduce insulin to the world

Banting receives the Nobel Prize in 1923!

…..Eugene Gley never knew what he had.

“Marjorie”

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Early Animal Studies

• 1922: 14-year old Leonard Thompson

First person to be treated with insulin Saved from imminent death

• 1923: Eli Lilly and Company

Begins mass-producing insulin from pigs and cows Mass produces the drug, shipping around the world

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Outline – Animal models

• Animal Models of Metabolic Endocrine Disease

• Diabetes mellitus models

• Models of diabetic complications (cardiomyopathy, nephropathy, retinopathy)

• Models of endocrine disruption

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How do we model diabetes?

• Multifactorial Disease

• Diet, lifestyle, genetic predisposition, obesity  insulin resistance

• Animal models remain indispensable

• Discovery, validation, optimization of novel therapeutics

• Model selection and interpretation are critical (pathology)

• Multiple animal models and systems

• Non-mammalian to NHPs, each with advantages and limitations

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Rodent DM Models – Diet Induced

• Diet Induced Obesity (DIO) and Insulin Resistance (Rodents)

• Provided free access to high fat diets (HFD)

• Translationally relevant model of obesity and insulin resistance (IR)

• Assessment of certain drugs on progression/reversal of obesity and IR

• Cons: Costly, time consuming, interpretation influenced by multiple factors

• Strain, Sex, Age, Diet

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Intrinsic factors

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Extrinsic factors Intrinsic factors

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• Mechanical – Pancreatectomy (total/partial)

• Pros – lack of toxic effects of chemicals/drugs on other organs

• Cons – need for surgical expertise, confounding effects of loss of exocrine pancreatic enzymes and other islet hormones

• Chemical – Diabetogenic drugs (streptozocin, alloxan)

• Cytotoxic glucose analogs that target β-cells through GLUT2 transporter

• High dose STZ – destroy all β-cells  model for type I diabetes mellitus

• Low dose STZ – partial β-cell loss  more reminiscent of type II DM

Rodent DM Models – Mechanical/Chemical

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Rodent DM Models – Mechanical/Chemical

• HFD/STZ model: Modeling slow (adult) onset Type II diabetes mellitus

High Fat Diet (HFD)  DIO and insulin resistance

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Rodent DM Models – Mechanical/Chemical

• HFD/STZ model: Modeling slow (adult) onset Type II diabetes mellitus

IP low dose STZ

Partial loss of

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Rodent DM Models – Mechanical/Chemical

• HFD/STZ model: Modeling slow (adult) onset Type II diabetes mellitus

IP low dose STZ

Partial loss of β-cells

↓ INSULIN

T2DM

Hyperglycemia

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Rodent DM Models – Mechanical/Chemical

• HFD/STZ model: Modeling slow (adult) onset Type II diabetes mellitus

IP low dose STZ

Partial loss of

↓ INSULIN Hyperglycemia

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Rodent DM Models – Genetic

• Numerous spontaneous and targeted Tg/KO models

• (NOD, Lepr, Mc4r, Pomc Pcsk1, polygenic)

• Spontaneous mutants (NOD, Leptin)

• Non-obese diabetic mouse (NOD) – immune mediated type I diabetes

• Similar molecular alterations as in human disease (e.g. MHCII locus)

• Similar autoantibody response to the same islet antigens

• Limitations

• Phenotype more severe and progressive than humans

• Failures in translating therapies from NOD mice to human patients

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Rodent DM Models – Genetic

• 1960s – two stocks of mutant mice identified at JAX

• Phenotype of obesity (Ob) and diabetes (Db) – (Leptin, Lepr)

• ob/ob

^-/-

mouse – Severely obese, mildly diabetic

• Spontaneous mutation in Lep  Non-functional leptin

• db/db

^-/-

mouse – Moderately obese, severely diabetic

• Mutation in Lepr  Non-responsive to normal leptin

• Phenotype background strain-dependent

db/db^-/- ob/ob^-/-

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Rodent DM Models – Genetic

• 1960s – two stocks of mutant mice identified at JAX

• Phenotype of obesity (Ob) and diabetes (Db) – (Leptin, Lepr)

• ob/ob

^-/-

mouse – Severely obese, mildly diabetic

• Spontaneous mutation in Lep  Non-functional leptin

• db/db

^-/-

mouse – Moderately obese, severely diabetic

• Mutation in Lepr  Non-responsive to normal leptin

• Phenotype background strain-dependent

• similar phenotype when on same strain, phenotypes based on individual strain

db/db^-/- ob/ob^-/-

C57BL/6J C57BL/KsJ

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Rodent DM Models – Genetic

• Genetically engineered mice (GEM) models

• Used to understand specific pathways of β-cell dysfunction in humans

• Maturity onset of diabetes of the young (MODY)

• Heritable diseases caused by mutations in specific β-cell genes

• Encoding for β-cell identity, glucose sensing, and insulin secretion

• Kcnj11 mutation – hyperglycemia, β-cell de-differentiation - neonatal diabetes

• Gck and Hnf1a mutation – impaired glucose tolerance, deficient insulin secretion

• Pdx1 KO – loss of β-cell identity (lack insulin production) and hyperglycemia

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Diabetes Mellitus Models – Large Animals

• Canine model of pre-diabetes and type II diabetes mellitus

• Combination of high fat, high fructose (HFFD) with diabetogenic drugs

• Allows invasive measures/assessments not possible in humans or rodents

• Porcine models of obesity and diabetes mellitus

• Diets inducing obesity in pigs do not result in diabetes!

• Requires additional manipulations such as STZ or genetic manipulation

• Genetically engineered models of diabetes in pigs have provided

extremely valuable tailored models of pre-diabetes and Type II DM

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Diabetes Mellitus Models – Porcine Models

• Promising models to overcome gaps between proof-of-concept models and clinical studies in obesity and diabetes research

• Anatomic and physiologic similarities to humans

• High fertility rate and easy maintenance

• Possibility of chemical and surgical interventions

• Specific genetic modifications

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Diabetes Mellitus Models – Porcine Models

Also can serve as tissue donors for β-cell replacement therapies for insulin-dependent

diabetes mellitus

• Promising models to overcome gaps between proof-of-concept models and clinical studies in obesity and diabetes research

• Anatomic and physiologic similarities to humans

• High fertility rate and easy maintenance

• Possibility of chemical and surgical interventions

• Specific genetic modifications

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Diabetes Mellitus Models – Non-human Primates

• Type II diabetes, hyperlipidemia, atherosclerosis, fatty liver disease

• Rhesus, cynos, baboons, African green monkeys, marmosets

• More translationally relevant to humans than rodents

• Major site of de novo lipogenesis (adipose vs. liver)

• Major circulating lipoprotein subclasses

• Mechanisms of insulin-mediated glucose utilization and IR

• Diabetes mellitus occurs spontaneously in a number of NHPs

• obesity/aging  insulin resistance  β-cell failure and overt DM

• Islet cell amyloidosis seen in humans also occurs in diabetic NHPs

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Diabetes Mellitus Models – Non-human Primates

• Models involve dietary acceleration of metabolic disease

• High fat, high sugar diets

• Advantages

• Close genetic, physiologic relationship to humans (baboon, macaque sequenced)

• Larger size for instrumentation and imaging

• Sequential liver/adipose biopsy or serum collection

• Disadvantages

• Limited number of animals per study

• Expense of maintaining NHP colonies

• Limited number of organizations able to support NHP research

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Models of Diabetic Complications

• Generally use primary DM models to induce secondary disease

• Diabetic nephropathy

• Diabetic retinopathy

• Diabetic cardiomyopathy

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Models of Diabetic Complications

• Diabetic nephropathy

• DM most common cause of end-stage renal disease in humans

• Mesangial and GBM thickening, albuminuria

• Models of DM induced nephropathy

• PROS: Useful for modeling early stages of diabetic nephropathy

• CONS: Limited utility for understanding pathogenesis of advanced diabetic nephropathy

• Does not recapitulate all key features of human dz

• Glomerular nodular lesions

• Tubulointerstitial fibrosis

• Progressive renal insufficiency

Kitada et al., Int J Nephrol Renovasc Dis. 2016;9:279-290.

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Models of Diabetic Complications

• Diabetic retinopathy

• Leading cause of blindness in older adults

• Microangiopathy, hemorrhage, ischemia and neovascularization (VEGF)

• Rodents models most common

• Dogs develop lesions most similar morphologically to humans

• Pigs and zebrafish – normal retinal structures and vasculature similar to humans

• No model recapitulates the full pathophysiology of end stage diabetic retinopathy

• Neovascularization and proliferative angiopathy not induced by hyperglycemia

• Transgenic (VEGF) or direct implantation, vascular obstruction

• Should not be used for studies into etiology or development of pre-retinal neovascularization

• Cannot imitate later stages of diabetic retinopathy in humans

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Models of Diabetic Complications

• Diabetic cardiomyopathy

• Main cause of morbidity/mortality in diabetic patients is CV disease

• Diastolic dysfunction, interstitial fibrosis, myocardial hypertrophy

• Multifactorial disease, mechanisms poorly understood

• Multiple molecular alterations in the myofiber

• Mitochondrial, ER, and oxidative stress, FA and glucose oxidation

• Tg rodent models indispensable for mechanistic studies

• Cardiomyocyte-selective insulin receptor knockout (CIRKO) mice

• Effect of decreased insulin signaling in cardiomyocytes without metabolic disturbances

Aboumsallem et al., Int J Mol Sci.

2019;20(6):1273.

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Models of Endocrine Disruption

• Endocrine Disrupting Chemicals (EDCs)

• Alter the structure and function of the endocrine system

• Interferes with any aspect of hormone action, affects multiple systems

• Zebrafish – novel model with advantages over traditional rodents

• Easy and cheap to maintain, can be bred at high speed in large numbers

• Large scale automated analysis, non-invasive imaging, in vivo development

• Comprehensively annotated genome, easy to manipulate

• Effectively model some of the basic modes of endocrine disruption

• Many basic physiologic and metabolic systems homologous to mammals

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Endocrine Disruption – Zebrafish Models

• Zebrafish – Limitations

• Exposure via tank water (dermal/gills) – chemical update different than humans

• Tissue concentrations poorly understood

• Difficult to extrapolate concentration-response to dose-response in mammals

• Differences in metabolism or conservation of metabolic pathways

• Lack brown adipose – analysis of pathways in β-adrenergic system are limited

• Differences in response to EDCs may vary between laboratories, lab vs wild, etc.

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Endocrine Disruption – GEM Models

• Transgenic rodents

• Used to probe mode of action, conjugation, and metabolism of EDCs

• Estrogen receptor mutations and KOs – estrogenic endocrine disruption

• Humanized mouse models

• Overcome known species differences in toxicologic susceptibility

• Mutated or humanized AhR and PPARα _mice

• Limitations of transgenic animals for EDC research

• High cost and time required to generate and utilize

• Off-target gene expression, incomplete recapitulation of disease phenotype

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Endocrine Disruption – Population Models

• Collaborative Cross (CC)

• 83 inbred strains descended from 8 founder strains

• Diversity Outbred (DO)

• Outbred from same 8 founder strains as CC

• In contrast, DO mice are outbred

• Each individual is unique

• Hundreds of mice used in a single mapping study, enables precise genetic mapping

• Establish and maximize natural genetic diversity

• Able to capture individual differences in susceptibility

• Better model of diverse human populations

Saul et al., Trends Genet. 2019;35(7):501-514.

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Models of Endocrine Neoplasia

• Spontaneous

• Naturally occurring mutations, viral incorporations

• Some strains (C3H, Wistar Han, Sprague Dawley) more predisposed

• Less predictable

• Non-genetic

• Chronic hormone administration (estrogen to OVX rats)

• Carcinogen treatment (cadmium – estrogen mimic)

• Advantages: provide mechanistic insights into environmental factors

• Xenograft/Orthotopic

• Cell-derived (CDX), patient-derived (PDX)

• Do not mimic biology and progression well, but useful for drug development

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Models of Endocrine Neoplasia – Pituitary

• Pituitary Neoplasia

• Hereditary (Multiple Endocrine Neoplasia/MEN, familial) or Sporadic (95%)

• >40 transgenic and KO models have been generated

• Hormones: Growth hormone releasing hormone (GHRH)

• Pituitary adenomas with excessive GH expression

• Oncogenes: Polyoma large T antigen (PyLT)

• ACTH-secreting pituitary tumors – model for Cushing’s disease

• Overexpression of Pituitary Tumor Transforming Gene (PTTG1)

• Overexpressed in human pituitary tumors

• Transgenic mouse model develops pituitary hyperplasia enhanced by loss of Rb

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Models of Endocrine Neoplasia – Thyroid

• Sporadic papillary thyroid cancer (PTC)

• Multiple RET fusion genes discovered by genetic analysis

• Tissue-specific transgenic models

• Thyroglobulin or thyroid peroxidase promoters

• RET overexpression

• Locally invasive thyroid carcinomas, metastasis is rare

• BRAF overexpression

• Most common mutation in aggressive sporadic papillary carcinomas

• Activating mutations of TSH receptor

• Documented in subset of human tumors

• Thyroid hyperplasia and hyperthyroidism, no tumors in Tg mice

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Models of Endocrine Neoplasia – Parathyroid

• CCND1 – fusion gene with PTH

• Parathyroid specific overexpression of CCND1

• Parathyroid adenomas in humans, hyperplasias in mice

• RET, MEN1, CDKN1B, p27 - models of MEN1

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Models of Endocrine Neoplasia – Adrenal

• Prepubertal gonadectomy (GDX)-induced

• Uncouples pituitary-gonadal axis

• Loss of target cells (gonads)

Hypothalamus

Pituitary

Testis

Leydig cells Sertoli cells

Ovary Granulosa cells

LH FSH FSH

+ + +

+

^GnRH

-

✘ - ✘

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Models of Endocrine Neoplasia – Adrenal

• Prepubertal gonadectomy (GDX)-induced

• Loss of Inhibin negative feedback

• Ongoing gonadotroph stimulation

Hypothalamus

Pituitary

Testis

Testosterone Inhibin Estrogen

LH FSH FSH

+ + +

+

^GnRH

-

✘

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Models of Endocrine Neoplasia – Adrenal

• Prepubertal gonadectomy (GDX)-induced

• Loss of Inhibin negative feedback

• Ongoing gonadotroph stimulation

• Chronically increased LH

• Re-expression of LH receptors in AC

• Adrenal activated gonadal gene profile

• Adrenal cortical hyperplasia/neoplasia

Hypothalamus

Pituitary

Testis

LH

FSH FSH

+ + +

+

^GnRH

✘ ✘

AC hyperplasia

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Models of Endocrine Neoplasia – Adrenal

• Adrenocortical Neoplasia – Transgenic

• Sf1, Ctnnb1, Tp53, Igf1, Inha

• Inhibin KO models of GDX-induced neoplasia

• Inhibin-α (Inha) KO + gonadectomy

• Combination of elevated gonadotropins and loss of adrenal inhibin

• enhancement of tumorigenesis www.brown.edu/Courses

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Models of Endocrine Neoplasia – MEN1/2

• Multiple Endocrine Neoplasia type 1 (MEN1)

• Mutations in MEN1 (MENIN) tumor suppressor gene

• Parathyroid, pancreatic islets, duodenal endocrine cells, anterior pituitary tumors

• MEN1^-/+ (het) models: pituitary, parathyroid, islet cell, adrenocortical tumors

• Multiple Endocrine Neoplasia type 2 (MEN2)

• Three clinical subtypes: MEN2A, MEN2B, familial medullary thyroid carcinoma (FMTC)

• Tumors of thyroid gland, pheochromocytoma, hyperparathyroidism

• Transgenic mice with RET or cMOS mutation or overexpression

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Questions?

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ADRENAL FATIGUE

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References

1. Arjmand, B., et al., Zebrafish for Personalized Medicine; A More Predictive Humanized Model of Endocrine Disease. Front Endocrinol, 2020. 11: p. 396.

2. Vecchio, I., et al., The Discovery of Insulin: An Important Milestone in the History of Medicine. Front Endocrinol, 2018. 9: p. 613.

3. Riehle, C. and J. Bauersachs, Of mice and men: models and mechanisms of diabetic cardiomyopathy. Basic Res Cardiol, 2018. 114(1): p. 2.

4. Patisaul, H.B., S.E. Fenton, and D. Aylor, Animal models of endocrine disruption. Best Pract Res Clin Endocrinol Metab, 2018. 32(3): p. 283-297.

5. Kleinert, M., et al., Animal models of obesity and diabetes mellitus. Nat Rev Endocrinol, 2018. 14(3): p. 140-162.

6. Lines, K.E., M. Stevenson, and R.V. Thakker, Animal models of pituitary neoplasia. Mol Cell Endocrinol, 2016. 421: p. 68-81.

7. Kirschner, L.S., et al., Mouse models of thyroid cancer: A 2015 update. Mol Cell Endocrinol, 2016. 421: p. 18-27.

8. Basham, K.J., et al., Mouse models of adrenocortical tumors. Mol Cell Endocrinol, 2016. 421: p. 82-97.

9. Chatzigeorgiou, A., et al., The use of animal models in the study of diabetes mellitus. In Vivo, 2009. 23(2): p. 245-58.

10. Carlus, M., et al., Historical control data of neoplastic lesions in Wistar Han Rats in 2-year carcinogenicity studies. Exp Tox Pathol, 2013. 65(3): p. 243-53.

11. Jones, G.N., et al., Mouse models of endocrine tumours. Best Pract Res Clin Endocrinol Metab, 2010. 24(3): p. 451-60.

12. Kitada, M., Y. Ogura, and D. Koya, Rodent models of diabetic nephropathy: their utility and limitations. Int J Nephrol Renovasc Dis, 2016. 9: p. 279-290.

13. Lai, A.K. and A.C. Lo, Animal models of diabetic retinopathy: summary and comparison. J Diabetes Res, 2013. 2013: p. 106594.

14. Olivares, A.M., et al., Animal Models of Diabetic Retinopathy. Curr Diab Rep, 2017. 17(10): p. 93.

15. Reed, J.A., Aretaeus, the Cappadocian: history enlightens the present. Diabetes, 1954. 3(5): p. 419-21.

16. Saul, M.C., et al., High-Diversity Mouse Populations for Complex Traits. Trends Genet, 2019. 35(7): p. 501-514.

17. Gahete, M.D., et al., Mouse models in endocrine tumors. J Endocrinol, 2019. 240: p. 73-96.

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