ZDSD Overview a model of obesity, metabolic syndrome and diabetes, 28 June 2013

INDEX 1. Introduction Page 2- 2. Spontaneous or Synchronous Diabetes Page 7- 3. Metabolic Syndrome Elements Page 10-

A. Visceral Obesity Page 13- B. Insulin Resistance Page 19- C. Clamp Study Page 28- D. Dyslipidemia Page 34- E. Hypertension Page 37-

4. Eating Behavior Page 39- 5. Beta cell Failure Page 40- 6. Renal Injury Page 43-

A. Urinary biomarkers, Exp. 1, 2 Page 46- B. RBM Biomarkers – Renal, Exp 3 Page 57- C. Glomerular Pathology, EM, Exp 4 Page 66- D. Synchronized Nephropathy, Exp 5 Page 78-

7. Osteoporosis Page 88- 8. Wound Healing Page 94- 9. RBM Biomarkers – Pro-Thrombotic Page 98- 10. RBM Biomarkers - Inflammation Page 104- 11. Therapeutic Efficacy

A. Common Anti-diabetic Compounds Page 112- B. Rimonabant Page 124- C. Niacin Page 132-

12. Summary Page 134- 1

→

The ZDSD Rat as a Translational Model for the Development of Drugs for Obesity, Metabolic

Syndrome and Diabetes that Demonstrates Many of the Serious Complications of Diabetes.

PreClinOmics, Inc.

2

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PDF Return to Index, For Web Use Refresh Above

• Most rodent models of type 2 diabetes have a

monogenetic mutation that is responsible for the

initiation of obesity and subsequent insulin

resistance.

• The two most common obesity causing mutations

are

– the leptin receptor

• Zucker Fatty; ZF rat

• Zucker Diabetic Fatty; ZDF rat

• db/db mouse

– the leptin molecule

• ob/ob mouse

• Both leptin and leptin receptor mutations are rare in

humans.

• The ZDSD rat does not have these mutations but still

has obesity metabolic syndrome and diabetes.

Background

3

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Development Scheme:

Zucker Diabetic-Sprague Dawley Rat (ZDSD)

• Produced by crossing diet induced obese (DIO) rats

derived from the Crl:CD(SD) strain (exhibiting

polygenetic obesity and insulin resistance) with

homozygous lean ZDF/Crl rats (which expresses

beta cell failure with the Leprfa/Leprfa genotype).

• Selectively bred for obesity and diabetes.

• Selected for genetically matched breeders to

develop phenotypic homogeneity.

• Studied male rats at different ages.

4

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Characteristics of the ZDSD Rat

• Unique translational model for obesity, metabolic

syndrome/type II diabetes - 35 generations inbred

• Polygenic obesity and phenotype can be modulated by diet.

• Phenotype is expressed in the presence of a functional leptin

pathway.

• Insulin resistance development starts at an early age.

• Early onset of hyperglycemia and slower progression to frank

diabetes when compared to the ZDF rat.

– Slower deterioration of beta cell function.

• Manifests diabetic complications:

Diabetic nephropathy Hypertension

Cardiovascular markers Inflammation

Osteoporosis Delayed Wound Healing

• In Production

5

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ZDSD

Obesity Metabolic Syndrome

Diabetes

Obesity Model

before diabetes develops, 5-16 weeks of age

Metabolic Syndrome

Insulin Resistance

Hyperlipidemia

Obesity

Hypertension

Delayed Wound Healing

Diabetes Models

Spontaneous

Development

(LabDiet 5008; Slower & more random)

Diabetic Nephropathy

Osteoporosis

Cardiovascular/ Inflammatory Biomarkers


Diet

Synchronized

(RD D12468 or TestDiet 5SCA)


Osteoporosis

Cardiovascular/ Inflammatory Biomarkers


The ZDSD Rat: One Rodent – Many Models

6

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Spontaneous

Development of

Diabetes

Age (weeks)

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Body w

eig

ht

(g)

100

200

300

400

500

600

700

The ZDSD Rat when

maintained on Lab Diet 5008

chow will spontaneously

develop diabetes as it ages

beyond 16 wks. As fed serum

glucose levels begin to

increase above ~350 mg/dl,

body weight begins to

decrease.

Age (weeks)

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Seru

m g

lucose (

mg/d

L)

100

150

200

250

300

350

400

450

7

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Synchronization of

Diabetic Onset

The ZDSD rat can be placed on

either D12468 (Research Diets)

or 5SCA (LabDiet) to synchronize

the onset of diabetes.

When the ZDSD rat was placed

on either diet at 17 wks of age,

the plasma glucose levels of the

animals averaged over 450 mg/dl

within 1 week. Following a return

to LabDiet 5008 at 19 wks of age,

the animals maintained the

diabetic state.

PCO recommends a 3 week

synchronization protocol

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

0

200

400

600

SD Male Rats

ZDSD Males

ZDSD Females

Age (wks)

Glu

co

se (

mg

/dl)

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

0

200

400

600

SD Male Rats

ZDSD Males

ZDSD Females

Age (wks)

Weig

ht

(g)

Area shaded in grey indicates time frame of diabetogenic diet

8

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Body composition

changes in response

to diabetogenic diet

(5SCA or D12468).

% Body Fat by QNMR

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

0

5

10

15

20

25

30

SD Male Rats

ZDSD Males

ZDSD Females

Age (wks)

% B

od

y F

at

Area shaded in grey indicates

time frame of diabetogenic diet

Synchronization

of Diabetic Onset

9

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Metabolic Syndrome affects a large proportion of the population and is

becoming increasingly important in adolescents. The syndrome has many

components including central obesity, insulin resistance, dyslipidemia and

hypertension. In addition, the syndrome features a chronic low grade

inflammatory state, vascular endothelial dysfunction, and a prothrombotic

environment. Long standing metabolic syndrome can thus pre-dispose to

atherosclerosis, microvasculature disease (retina), stroke, renal injury and

diabetes. Due to the complicated mechanisms involved in the syndrome and

its sequelae, current standard of care reflects poly-pharmacy and is aimed at

controlling atherogenic dyslipidemia, hyperglycemia and hypertension as well

as intervening in secondary diseases such as renal dysfunction, stroke, and

micro-vascular disease related to retinopathy. Development of new chemical

entities with the potential to control more than one risk factor is hampered by

currently available animal models. To that end, the ZDSD rat was designed

to spontaneously develop a phenotype that mimics many aspects of the

human metabolic syndrome, including hypertension and the progression to

frank diabetes with long-standing disease.

ZDSD as a Preclinical Model of Metabolic Syndrome

10

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Metabolic Syndrome

• Metabolic syndrome is most frequently

defined by a presence of certain traits,

including:

– abdominal obesity

– insulin resistance

– dyslipidemia

– elevated blood pressure and

– pro-thrombotic and pro-inflammatory

states

11

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Characteristics of Metabolic Syndrome

seen in the ZDSD Rat

• Increased body weight with increased abdominal fat

• Increased fed and fasting glucose and HbA1c levels

• Insulin resistance / Glucose intolerance

• Hyperlipidemia

• Increased blood pressure --> Hypertension

• Increased Serum Biomarkers of Coagulation,

Inflammation and Vascular Disease

12

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A prominent component of metabolic syndrome is insulin resistance which is

thought to be mediated by an increase in metabolically active visceral fat.

Visceral fat accumulation occurs in human patients in the presence of a

functional leptin pathway as leptin deficiencies and receptor defects are

rarely reported. According to published growth charts for male leptin

resistant ZDF rats, the new ZDSD rats are heavier when fed a normal diet

(PMI 5008) and exhibit a body composition (increased % fat) comparable to

age matched DIO-LE model which is a mainstay for anti-obesity research.

In addition, the ZDSD rat responds to a common reference anti-obesity

agent (rimonabant) with significant loss of body fat. Interestingly, ZDSD rats

are not typically nocturnal in that they exhibit significant feed intake during

the daylight hours. Exogenously administered leptin results in an acute

anorexic effect quite similar to normal SD rats and indicates the presence of

a functioning leptin pathway.

Visceral Obesity

13

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Comparative Growth Curves in SD and ZDSD Rat Fed 5008 chow

ZDSD rats were significantly (15%) heavier than their SD counterparts at 8 weeks of age. In addition, the rate of body weight gain was increased in ZDSD rats as evidenced by an 82% vs 62% weight gain when compared to SD rats over 24 weeks of observation.

Study # 09-550-170

All time points statistically different

14

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Spontaneous Development of Obesity in

ZDSD Rats Fed 5008 Chow

Study # 09-550-170


Body composition was

assessed using QNMR .

The percentage of body

weight identified as fat was

50 % higher in ZDSD rats

compared to SD controls as

early as 8 weeks of age.

Body fat percentage

continued to increase

throughout the study and

remained significantly

higher than control rats at

each time-point.

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Visceral Obesity in the ZDSD Rat

Subcutaneous fat

Retroperitoneal fat

Visceral fat

16

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CT Scan

Terminal Body Weight Comparison

Terminal animal weights in diabetic and control animals.

100

200

300

400

500

600

700 CRL-SD, CD

+/fa

ZDF

ZDSD, Diabetic 12-21 weeks


Weig

ht

(g)

17

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Terminal Comparison

Liver Weight Food & Water Consumption

Terminal liver weights, water intake and food consumption are highest in the diabetic

groups.

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0

5

10

15

20

25

30

Weig

ht (g

ram

)

CRL-SD, CD +/fa

ZDF



Water Food

0

50

100

150

200

250

300

350

CRL-SD, CD +/fa

ZDF

ZDSD, Diabetic 12-21 weeks ZDSD, Diabetic 7-11 weeks

Am

ou

nt/

rat (g

ram

)

Development of Insulin Resistance in

the ZDSD Rat on 5008 Purina chow

• Rats tested started at 8 weeks of age (SD & ZDSD)

• Weight, glucose and insulin measured weekly

• Animals fasted every two weeks for OGTT

• Data analyzed:

– Weight

– Body composition

– Glucose levels

– OGTT glucose and insulin

– Glucose disposal

– HOMA-IR

19

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Comparative Growth Curves

in SD and ZDSD Rat Fed 5008 chow

ZDSD rats were significantly

(15%) heavier than their SD

counterparts at 8 weeks of

age. In addition, the rate of

body weight gain was

increased in ZDSD animals

as evidenced by an 82% vs

62% weight gain in SD

animals during the 24

weeks.

Study # 09-550-170


20

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Spontaneous Development of Obesity in

ZDSD Rats Fed 5008 Chow

Study # 09-550-170


Body composition was

assessed using QNMR. The

percentage of body weight

identified as fat was 50 %

higher in ZDSD compared

to SD controls as early as 8

weeks of age. Body fat

percentage continued to

increase throughout the

study and remained

significantly higher than

control rats at each time

point.

21

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Spontaneous Development of Hyperglycemia

in ZDSD Rats Fed 5008 Chow

Study # 09-550-170

All time points statistically different 22

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Spontaneous Development of Glucose Intolerance

Shown by OGTT in ZDSD Rats Fed 5008 Chow

23

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Spontaneous Development of Insulin Resistance

Shown by OGTT in ZDSD Rats Fed 5008 Chow

24

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Spontaneous Development of Impairment in

Glucose Disposal in ZDSD Rats as

demonstrated by OGTT (AUC)

Study # 09-550-170

Impairment in glucose

disposal as

represented by the

area under the

glucose curve during

an OGTT developed

spontaneously in

ZDSD rats and was

evident as early as 8

weeks of age (fed

Purina 5008 chow).


25

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ZDSD rats become

increasingly more insulin

resistant with age as

evidenced by the calculated

HOMA-IR. The insulin

resistance is evident

compared to SD rats as

early as 8 weeks of age

(fed Purina 5008 chow).

Progressive Development of Insulin

Resistance (HOMA-IR) in ZDSD Rats


26

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Glucose and Glycated Hemoglobin Levels in

CD and Prediabetic ZDSD Rats

27

→


←

Glucose in CD vs ZDSD

16 weeks of age

CD ZDSD

100

110

120

130

140

150 CD

ZDSD

Blo

od

Glu

co

se

(m

g/d

L)

Glycated Hb in CD vs ZDSD

16 weeks of age

CD ZDSD

3.0

3.2

3.4

3.6

3.8

4.0 CD

ZDSD

Gly

ca

ted

Hb

(%

)

28

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AM and PM Glucose Levels in the ZDSD Rat:

18-19 weeks old

Days

0

100

200

300

1 2 3 4 5

6:00 am 6:00 pm

Wh

ole

Blo

od

Glu

co

se

(m

g/d

L)

29

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Glucose Levels at Different Times

of the Day in the ZDSD Rat

Time of Day

0

100

200

300

Food Intake at Different Times

of the Day in the ZDSD Rat

F o

o d

C o

n s u

m p t i o

n (g

)

Time of Day

0

2

4

6

8

10

Wh

ole

Blo

od

Glu

co

se (

mg

/dL

)

Pre-diabetic Insulin Sensitivity,

Hyperinsulinemic- Euglycemic Glucose Clamp

Design: – Rosiglitazone treatment: 3 mg/kg

PO, QD for 2 weeks

– Comparison of insulin sensitivity at 9

wks of age in

• The ZDSD Rat,

• Zucker Fatty (ZF), and

• Sprague Dawley (SD) rats

– Assessed by exogenous glucose

infusion rate (GIR) during

hyperinsulinemic (25 mU/kg/min)-

euglycemic glucose clamp

30

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Body Weight and Basal Glucose of Rats Before Undergoing Glucose Clamp

* P<0.05 compared to SD rat (age matched) group # P<0.05 compared to vehicle treated group

31

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SD (age) SD (wt) ZDSD ZF 0

100

200

300

400

500

*

#

Rat Strain (SD rats are age or weight matched)

Body W

eig

ht (g

)

* *


40

80

120

160

200 Vehicle (n=6-8) Rosiglitazone (3 mg/kg p.o., n=6-8)


Basal B

lood G

lucose (

mg/d

l)

ZDSD and ZF Rats are Insulin Resistant which

improved with Rosiglitazone Treatment


32

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10

20

30

40

50

60 Vehicle (n=6-8)

Rosiglitazone (3 mg/kg PO, n=6-8)

*

#

#


Glu

cose Infu

sio

n R

ate

(m

g/k

g/m

in)

*

ZDSD Rats Have Modest Visceral and

Whole Body Obesity Unlike ZF Rats


33

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2

4

6

8

10

12 Vehicle (n=6-8)

Rosiglitazone (3 mg/kg PO, n=6-8)

#


Epid

idym

al F

at

Pad (

g)

* *

*


10

20

30

40

Vehicle (n=6-8)

#

Rat Strain (SD rats are age or weight matched) B

od

y F

at

(%)

*

*

Patients with Type II diabetes and metabolic syndrome often present with

dyslipidemia including elevated cholesterol and triglycerides and decreased

HDL-C. These lipids have been shown to impact cardiovascular and renal

co-morbidities. Hypertriglyceridemia expresses as early as 12 weeks of

age in ZDSD rats when maintained on a normal diet and levels progress up

to 500 mg/dL by 15 weeks. Similar to the fructose fed rat, a model

commonly used for the study of dyslipidemia, the spontaneous nature of the

ZDSD lipid abnormality may provide a relevant model for the examination

of compounds affecting the up-regulated lipogenic pathway seen in

metabolic syndrome. The dyslipidemia in this model responds to classic

reference agents including rosiglitazone. Increases in cholesterol are not as

dramatic and may be induced by feeding a high fat diet

Dyslipidemia

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Terminal Comparison of Models

Glucose, triglyceride and cholesterol levels are elevated in all of the diabetic

groups (ZDF and ZDSD). The model and duration of diabetes did not have a

significant effect on these measurements.

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Glucose Triglycerides Cholesterol

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300 A

na

lyte

s (

mg

/dL

) CRL-SD, CD +/fa

ZDF



Treatment of Dyslipidemia in ZDSD with Niacin

7 Days of Treatment 36

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Fed Fasted Fed Fasted 0

200

400

600

Before Treatment After Treatment

We

igh

t (g

ram

s)




200

400

600

Vehicle Niacin


Tri

gly

ce

rid

e (

mg

/dL

)

Fed Fasted Fed Fasted 0.0

0.5

1.0

1.5


FF

A (

mE

q/L

)

Effect of 7 Days of Niacin Treatment

50

100

150

200

250

Glu

co

se

(m

g/d

L)

*

*

* * *

High blood pressure is a key symptom of metabolic syndrome and is a

major contributor to the increased risk of cardiovascular disease, kidney

disease and ischemic stroke seen in these patients. Examination of the

interactions of all the components of the syndrome in rats is complicated

by the absence of high blood pressure in current models (i.e., Zucker fatty

rat). Indirect evidence of probable elevated pressure in the form of

elevated biomarkers for an activated RAAS ,endothelial dysfunction and

aberrant vasoconstriction is noted in ZDSD rats. Direct evidence of

Hypertension has been confirmed in the pre-diabetic state via the tail-cuff

method.

Hypertension

37

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Blood pressure data produced in collaboration with

Dr. Subah Packer’s Laboratory, IU School of Medicine

Blood Pressure in ZDSD vs CD Rats

8-16 weeks of age

38

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60 70 80 90 100 80

100

120

140

160 ZDSD

CD

Age in Days

Systo

lic B

P (

mm

Hg

)

Leptin Physiology

Food intake of ZDSD rats is

more evenly divided between day and night

Food Intake of ZDSD rats is

Reduced in Response to Leptin Indicating a Functioning Leptin Pathway. Leptin was given just before the start of the dark cycle and food intake was measured for the first 4 dark hours.

SD ZDSD

Fo

od

in

take

(g/p

erio

d)

0

5

10

15

20

25

30

dark cycle

light cycle

daily total

ANOVA/pooled t (p<0.05) compared

to SD animals

Assessment of leptin pathway function as determined

by feeding response to leptin injection (1 mg/kg, IP)

SD-saline SD-leptin ZDSD-saline ZDSD-leptin

Fo

od

in

take

4 h

rs a

fte

r tr

ea

tme

nt

0

1

2

3

4

5

6

7

8

9

10ANOVA/pooled t (p<0.05) compared to corresponding saline control

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Insulin Levels Decline as Diabetes

Progresses

Glucose Insulin

Insulin levels of the group that become diabetic between 11-17 weeks of age.

The animals that become diabetic earlier have higher insulin levels than those

who become diabetic later.

40

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5 7 9 11 13 15 17 19 21 23 25 27 29 0

100

200

300

400

500

600

11-17 WEEKS

Age (wks)

Glu

co

se

(m

g/d

l)

5 7 9 11 13 15 17 19 21 23 25 27 29 0

1

2

3

4

5

6

7

8

11-13 Weeks 15 Weeks 17 Weeks

Age (wks)

Ins

uli

n (

ng

/ml)

The glucose levels for ZDSD

rats were followed from 16 to

28 weeks of age (upper figure).

At 28 weeks of age

approximately 75% of the

animals were overtly diabetic.

The average glucose levels for

each animal (16 to 28 weeks)

were correlated with insulin

content of the pancreas when

the animals were terminated at

about 28 weeks of age (lower

figure). Higher average

glucose levels were associated

with lower insulin content in the

pancreas.

Pancreatic Insulin Content

41

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Average Glucose % Diabetic

15 20 25 30 0

100

200

300

400

500

3.6% 13.1% 18.0% 32.8% 52.5% 62.3% 70.5% 75.8%

Age (weeks)

Glu

co

se

(m

g/d

L)

Pancreatic Insulin versus

Blood Glucose Level

0 200 400 600 800

0

200

400

600

Insulin (ng/g)

Glu

co

se

(m

g/d

L)

Representative Islets from ZDSD Rats

Pre-diabetic Diabetic

42

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Obesity and metabolic syndrome are clear

predictors of chronic kidney disease largely due to

the potentiation of chronic inflammation by insulin

resistance. In addition, the lipoprotein

abnormalities, increased hemodynamics,

hypercoagulability and vascular dysfunction

associated with metabolic syndrome have all been

implicated as causative for renal disease.

Biomarkers for renal dysfunction (i.e., IL6, TNF-

α,NGAL,KIM-1, VEGF etc.) as well as significant

albuminuria , elevated free fatty acids with

oxidative stress, and histological analysis have

shown the ZDSD rat to exhibit nephropathy that

closely mimics that observed in obese insulin

resistant patients.

Renal Injury

43

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Diabetic Nephropathy in

the ZDSD Rat

• Increased kidney weight

• Increased urinary markers for kidney

disease

• Increased serum markers for kidney disease

• Glomerular sclerosis

• Nodular sclerosis, KW nodules

• Thickening basement membrane of

glomerular capillaries

• Podocyte effacement on capillaries

44


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Terminal kidney weights are highest in the ZDSD rat groups. These increased kidney weights

and high urinary volume along with increased micro-albumin concentration and the total amount of micro-albumin indicate that there may be significant diabetic nephropathy in the ZDSD rat

model.

Terminal Comparison

Kidney Weight Urine Analysis

0

1

2

3

4

5

6

7 CRL-SD, CD +/fa

ZDF



We

igh

t (g

)

45

← →


0

50

100

150

200

250

300

CRL-SD, CD

+/fa

ZDF



Experiment 1

ZDSD Diabetic Nephropathy

Spontaneous Diabetes

ELISA Analysis of Markers

46

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47

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Weight

10 12 14 16 18 20 22 24 26 28 30 300

400

500

600

SD

ZDSD

Age (weeks)

We

igh

t (g

) Glucose

10 12 14 16 18 20 22 24 26 28 30 0

200

400

600 SD

ZDSD

Age (weeks)

Glu

co

se

(m

g/d

L)

Urine Volume

10 20 22 24 26 30 0

50

100

150

200 SD

ZDSD

Age (weeks)

Uri

na

ry V

olu

me

(m

l/2

4h

r)

48

← →


Urinary albumin

10 20 22 24 26 30 0

25

50

75

100

125

150 SD

ZDSD

Age (weeks)

Uri

na

ry a

lbu

min

(m

g/d

ay)

beta-2 microglobulin

10 20 22 24 26 30 0

500

1000

1500

2000 SD

ZDSD

Age (weeks)

Uri

na

ry b

-2 m

icro

glo

bu

lin

( m g

/da

y)

Cystatin C

10 20 22 24 26 30 0

10

20

30 SD

ZDSD

Age (weeks)

Uri

na

ry c

ysta

tin

C (

m g

/da

y)

KIM-1

10 20 22 24 26 30 0.0

2.5

5.0

7.5

10.0

12.5

15.0 SD

ZDSD

Age (weeks)

Uri

na

ry K

IM-1

(n

g/d

ay)

Experiment 2 Urine BioMarkers of Renal Disease

Study Details

• Male ZDSD rats were allowed to become diabetic

spontaineously on Purina 5008 and aged to 33 weeks. Two groups of animals were selected for further study: animals

that were diabetic for longer than 16 weeks and animals that

were diabetic for less than 8 weeks.

• Mesoscale (MSD) urine panels were run on urine

(Argutus AKI test, Kidney Injury Panel 1 and Rat Clusterin)

• Pathological evaluation of the kidneys was done.

49

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Data From Urinary Excretion Study

50

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Urinary Excretion of Kidney Markers

51

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Urinary Excretion of Kidney Markers

52

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Pathological Evaluation of Kidney • Glomerulopathy: Changes in the renal glomeruli consisted of one or more of the following:

increased cellularity in the mesangium; increased in mesangial connective tissue; thickening of

Bowman’s capsule; hypertrophy of capsular epithelium; dilation of the capsular space. Individual

glomeruli appeared moderately enlarged. The lesions were highly variable within individual

glomeruli and between glomeruli within a kidney. The changes were most usually segmental,

although a rare glomeruli was fibrotic (condensed). Expanded mesangial material stained

positively with the PAS stain and to a lesser extent with the Trichrome stain.

• Tubular dilation/degeneration: This change was mainly in the cortex and consisted of irregularly

dilated, empty tubules, that sometimes were lined by cuboidal epithelium that stained basophilic

compared to the expected normal eosinophilic tubular epithelium. In some individual tubules the

epithelium were flattened. These dilated/degenerate tubules were randomly scattered throughout

the cortex, and sometimes were associated with protein casts and/or non-suppurative inflammation

(see below). Focal mild increases in fibrous connective tissue within the interstitial space was

present, frequently in association with the interstitial inflammatory response, but not restrictively so.

• Protein casts: Individual tubules contained acellular, uniformly staining eosinophilic material

consistent with protein. These protein casts were present in the cortex and in the medulla, as well

as at the cortico-medullary junction in various sections. Often, several such dilated tubules

containing protein casts were clustered together, usually in the cortex.

• Inflammation: The inflammatory process consisted of focal collections of lymphocytes and

macrophages, which were seen in the cortical interstitial space, adjacent to individual glomeruli

and individual blood vessels, and in association with the renal pelvic epithelium.

53

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54

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55

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Kidney Histopathology of the ZDSD Rat

A Novel Animal Model of Diabetes

56

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Glomerulopathy Tubular dilation Protein casts Inflammation

His

top

ath

olo

gy S

co

re (

0-5

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 Non-diabetic

Diabetic

* * * *

* compared to Non-diabetic animals (t-test)

/degeneration

Experiment 3

Serum BioMarkers of Renal Disease

RBM Collaboration

Rules Based Medicine Analysis

57

← →


Obesity and metabolic syndrome are clear predictors of chronic

kidney disease largely due to the potentiation of chronic

inflammation by insulin resistance. In addition, the lipoprotein

abnormalities, increased hemodynamics, hypercoagulability and

vascular dysfunction associated with metabolic syndrome have all

been implicated as causative for renal disease. Biomarkers for

renal dysfunction (i.e., IL6, TNF-α, NGAL, KIM-1, VEGF) as well

as significant albuminuria, elevated free fatty acids with oxidative

stress, and histological analysis have shown the ZDSD rat to

exhibit nephropathy that closely mimics that observed in obese

insulin resistant patients.

Renal Injury

58

← →


Experimental details

• Male ZDSD rats were placed on a high-fat diet

(RD12468) between 17 and 19 weeks of age.

• Fifteen out of 21 animals in this experiment developed

diabetes during this period (this is usually 90%+).

• Rules Based Medicine panels (Rat Metabolic MAP, Rat

Kidney MAP and RodentMAP™) were run on serum

samples that were collected:

– before diabetes developed (14 weeks)

– while diabetic on the high fat diet (18 weeks) and

– one week after they were taken off the high fat diet

(20 weeks).

59

← →


Neutrophil Gelatinous Associated

Protein

• also called lipocalin2

• levels up-regulated during

inflammation

• protective protein can trigger

nephrogenesis

• associated with obesity, insulin

resistance and hyperglycemia

60

Serum Biomarkers of Renal Disease

Age (weeks)

14 18 20

Seru

m N

GA

L (

ng/m

l)

200

400

600

800

1000

1200

1400 Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

14 weeks = non-diabetic

18 weeks = on diabetogenic diet one week

20 weeks = off diabetogenic diet one week

← →


Beta-2-microglobulin

• a protein present in all nucleated

cells which is normally reabsorbed

in renal tubules

• increased circulating levels

indicate glomerular membrane

disease and inflammation

61

Age (weeks)

14 18 20

Se

rum

be

ta-2

-mic

roglo

bu

lin (

ug/m

l)

50

55

60

65

70

75

80

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

#

compared to SD

# Diabetic vs. non-diabetic





← →


Age (weeks)

14 18 20

Se

rum

Kim

-1 (

ng/m

l)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

#

compared to SD

# diabetic vs. non-diabetic

Kidney-Injury Molecule-1

• a membrane protein, not normally

present but appears in urine in

response to acute kidney tubular

injury

• highly sensitive predictor of renal

injury when elevated in urine

62





← →


Age (weeks)

14 18 20

Seru

m G

ST

-alp

ha (

ng/m

l)

0

10

20

30

40

50

60

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

#


Glutathione-S-transferase-alpha

• enzyme that reduces toxin levels

by conjugation with glutathione

• localized in proximal convoluted

tubules, medullary tubules and

loop of Henle

• in diabetes, hyperglycemia

triggers oxidative stress which

increases the renal excretion of

this enzyme and therefore

removes this protective function

and increases blood levels.

• Biomarker for tubular kidney

disease

63


14 weeks = non-diabetic 18 weeks = on diabetogenic diet one week 20 weeks = off diabetogenic diet one week

← →


Clusterin

• (apolipoprotein J) is a protein

highly correlated with apoptosis

and the clearance of cellular debri

• It is elevated in glomeruli and

tubules of diabetic kidneys

64


← →





Age (weeks)

14 18 20

Se

rum

Clu

ste

rin

(m

g/m

l)

100

200

300

400

500

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

#


compared to SD

von Willebrand factor

• required for platelet adhesion,

makes platelets "sticky“

• vWF binds inactive Factor VIII,

protecting it from degradation

• defiency leads to bleeding disorders

• increased levels predispose to

stroke

• increases precede microalbuminuria

in diabetic nephropathy

65

Age (weeks)

14 18 20

Se

rum

vo

n W

ille

bra

nd

Fa

cto

r (n

g/m

l)

0

50

100

150

200

250

300

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

#

#






← →


Experiment 4

Diabetic Nephropathy,

EM of Glomerular Pathology

66

← →



• Male ZDSD rats allowed to become spontaneously

diabetic.

• Animals were terminated and perfused fixed at about

35 weeks of age.

– Control CD rats

– ZDSD rats that had been diabetic from 12-13

weeks

– ZDSD rats that had been diabetic from 16-17

weeks

• Took pictures of glomerular capillaries and BM

– Measured GBM thickness

– Evaluated podocyte morphology

67

← →


68

Glomerular Capillary Control, Age Matched Diabetic, 12 Weeks

68

← →


Glomerular Capillary, Basement Membrane

Control, Age Matched Diabetic, 12 Weeks

69

← →


70

← →



Control, Age Matched Diabetic, 12 Weeks

71

Glomerular Capillary Control, Age Matched Diabetic, 16.5 Weeks

71

← →



Control, Age Matched Diabetic, 16.5 Weeks

72

← →


73

← →


Glomerular Basement Membrane Thickness

Time of Diabetes in the ZDSD Rat

Th

ickn

ess (

mm

)

CD Control 12 Weeks 16.5 Weeks

0

100

200

300

400

500

74

← →


Scanning Microscopy Glomerular Capillaries

Control Diabetic

75

← →


Scanning Microscopy

Control Glomerular Capillary with Normal Podocyte Foot Processes

76

← →


Scanning Microscopy

Diabetic Glomerular Capillaries Demonstrating Effacement

77

← →


Transmission Microscopy Glomerular Mesangium, Advanced Diabetic Changes

Experiment 5

Diabetic Nephropathy,

Synchronized Diabetes:

Clinical Data and

LM of Glomerular Pathology

78

← →



• Male ZDSD rats synchronized to become diabetic

by feeding them Purina 5SCA.

• Animals were put on 5SCA at 19 weeks of age

and were diabetic by 20 weeks of age. They were

monitored until they were 47 weeks old. We

evaluated the following groups:

– ZDSD rats that had been diabetic for 27 weeks

(14)

– ZDSD rats that failed to become diabetic (4)

• Graphed terminal data and evaluated pictures of

glomeruli and other kidney pathology

79

← →


80

← →


Body weight

0

7

14

21

28

35

42

70

105

126

155

172

185

196 350

400

450

500

550

600 Diabetic

Non-diabetic

Day of study

Bo

dy w

eig

ht (g

)

Glucose

0

7

14

21

28

35

42

70

105

126

155

172 0

200

400

600

800 Diabetic

Non-diabetic

Day of study

Glu

co

se (m

g/d

L)

Diabetic

Non-diabetic

43 47 0

5

10

15 * t-test

* *

Age (weeks)

Hb

A1c (

%)

HbA1c

43

0.0

0.2

0.4

0.6

0.8

1.0 Diabetic

Non-diabetic

Age (weeks)

NE

FA

(m

Eq

/L)

NEFA

81

← →


Diabetic Non-diabetic 0

5

10

15

20

25 Diabetic

Non-diabetic

*

* t-test L

iver

weig

ht

(g)

47 w

eeks o

f ag

e

Liver Weight

Diabetic Non-diabetic 0

2

4

6 Diabetic

Non-diabetic

Kid

ney w

eig

ht (g

)

47 w

eeks o

f ag

e

* t-test

*

Kidney Weight

43 47 0

100

200

300 Diabetic

Non-diabetic

Age (weeks)

Uri

ne v

olu

me (m

ls/2

4 h

r)

Urine Volume

43 0

20

40

60

80 Diabetic

Non-diabetic

*

* t-test

Age (weeks)

Uri

ne a

lbu

min

(m

g/d

ay)

Urinary Albumin

Blood Chemistry

82

← →


43 45 47 0

10

20

30 Diabetic

Non-diabetic

* t-test

* * *

Age (weeks)

Seru

m B

UN

(m

g/d

L)

BUN

45 47 0.0

0.1

0.2

0.3

0.4

0.5 Diabetic

Non-diabetic

Age (weeks)

Seru

m c

reati

nin

e (m

g/d

L)

Creatinine

43 47 0

50

100

150

200 Diabetic

Non-diabetic * t-test

* *

Age (weeks)

Seru

m c

ho

leste

rol (m

g/d

L)

Cholesterol

43 47 0

500

1000

1500 Diabetic

Non-diabetic * t-test

* *

Age (weeks)

Seru

m t

rig

lyceri

des (m

g/d

L)

Triglyceride

47 Week-old, 27 Weeks Diabetes

83

83

← →


Non-Diabetic Diabetic



Diabetic Diabetic 84

← →


85


85

← →



86


86

← →




87

← →


Osteoporosis in the ZDSD Rat

Reference: Skeletal changes associated with

the onset of type 2 diabetes in the ZDF and

ZDSD rodent models. Susan Reinwald,

Richard G. Peterson, Matt R. Allen, and David

B. Burr. Am J Physiol Endocrinol Metab 296:

E765–E774, 2009.

88

← →


0

100

200

300

400

500

600

700

800

7 9 11 13 15 17 19 21 23 25 27 29 31 33

Glu

cose

(m

g/d

l)

Age (weeks old)

ZDSD (diabetic)

Control (non-diabetic)

*

*

*

***

Reference range for controls

A

0

100

200

300

400

500

600

700

800

23 25 27 29 31 33

Age (weeks old)

ZDF fa/fa (diabetic)

ZDF fa/+ (non-diabetic)

B

Comparative Glucose Concentrations

Despite a later increase in blood glucose levels in the

ZDSD rats, by 21-wks-old the average glucose

concentrations are >500 mg/dl.

Mean ±SEM

Mean ±SEM n=12-17/group

89

← →


L4 Vertebra – dimensions not affected by

differences in growth in ZDSD rats


90

← →


0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

ZDF

(fa/fa)

ZDF

(+/fa)

L4 c

ross-s

ectional are

a (

mm

)

p<0.05

0

1

2

3

4

5

6

7

8

ZDF

(fa/fa) ZDF

(+/fa)

L4

ve

rte

bra

l bo

dy h

eig

ht (m

m)

p<0.05

0.27

0.28

0.29

0.30

0.31

0.32

0.33

0.34

0.35

ZDSD Controls

L4

cro

ss-s

ectio

na

l are

a (

mm

)

0

1

2

3

4

5

6

7

8

ZDSD Controls

L4

ve

rte

bra

l bo

dy h

eig

ht (m

m)

Structural Properties L4 vertebrae P-values for differences in diabetic rats vs. respective controls


91

← →

PDF Return to Index, For Web Use Refresh Above 0

10

20

30

40

50

60

70

80

90

ZDF (fa/fa) ZDF (+/fa)

Energ

y to

Ultim

ate

Load (

mJ)

*

Biomechanical Test – axial compression ZDF ZDSD

Yield Force (N) p<0.050 p<0.001

Stiffness (N/mm) p<0.001 p<0.005

Ultimate Load (N) p<0.001 p<0.001

Energy to Ultimate Load ( mJ) p<0.050 p<0.001

0

10

20

30

40

50

60

70

80

90

ZDSD Controls

Energ

y t

o U

ltim

ate

Loa

d (

mJ)

*

Material Properties L4 vertebrae P-values for differences in diabetic rats vs. respective controls


92

← →


Parameters Normalized to BV/TV ZDF ZDSD

Ultimate Stress (N/mm2) p<0.050 p<0.050

Modulus (N/mm2) p<0.051 p<0.050

Toughness (mJ/mm3) p=0.657 p<0.051

Post yield Toughness (mJ/mm3) p=0.224 p=0.101

0.0

0.5

1.0

1.5

2.0

2.5

ZDSD Controls

*

0.0

0.5

1.0

1.5

2.0

2.5


Toughness/[B

V/T

V]

(mJ/m

m3)

Toughness/[B

V/T

V]

(mJ/m

m3)

Glycated Hemoglobin Evidence of a high level of non-enzymatic glycation (NEG, or

cross-linking) occurring in the ZDSD rats.

The large decline in vertebral mechanical toughness in the ZDSD model

may be attributable to an accumulation of NEGs in the bone collagen

matrix. This possibility is currently under investigation.

93

← →



0

1

2

3

4

5

6

7

8

9

10


HbA

1c (

%)

*

0

1

2

3

4

5

6

7

8

9

10

ZDSD Controls

*

Hb

A1c (

%)

Delayed Wound Healing in ZDSD

• Rats were all put on 5SCA for 2 weeks (age 17-19)

• Diabetic, non-diabetic and SD rats were wounded with a

6mm punch

• Animals were followed and pictures were taken on days 1, 4,

7, 9, 11 and 14

• Wounds were analyzed by evaluating and measuring the

healing process. The diameter of open wound or thin skin

(reddish in color) was measured

• Data were graphed

• There are statistical differences in wound healing between

ZDSD (diabetic and non-diabetic) and SD rats.

94

← →


Data at 11 days after Wound

95

← →


WEIGHT

Weig

ht

(g)

ZDSD

Diabetic

ZDSD

Non-Diabetic

0

200

400

600

SD

GLUCOSE

Glu

co

se (

mg

/dl)

ZDSD

Diabetic

ZDSD

Non-Diabetic

0

100

200

300

400

500

SD

96

Control SD animals 233019

Day 11

Day 14

233023

Day 11

Day 14

Diabetic ZDSD animals 233003

233006

Day 11

Day 14

Day 11

Day 14

96 Scale in mm is to the left of each picture. There is a visible difference in healing at 14 days.

← →


Wound Healing in the ZDSD Rat

This figure demonstrates the wound healing in the three groups of

animals. There were no differences between diabetic and non-

diabetic ZDSD animals. There were several statistically significant

differences between the SD group and the ZDSD groups (*). Since

the data were not different in the ZDSD groups there were also

analyzed as a combined group.

97

← →


Separated Diabetic and Non-Diabetic ZDSD

4 7 9 11 14

-100

-80

-60

-40

-20

0 SD

Diabetic ZDSD

Non-Diabetic ZDSD

* *

* *

*

*

*

*compared to SD (Dunnett's)

Time(days) post-wounding

% C

ha

ng

e F

rom

In

iita

l W

ou

nd

Combined ZDSD Data

4 7 9 11 14

-100

-80

-60

-40

-20

0 SD

ZDSD

*

*

* *

*

*compared to SD (t-test)

Time(days) post-wounding

% C

ha

ng

e F

rom

In

iita

l W

ou

nd

The dysregulation of hemostasis is a common feature of metabolic

syndrome and T2DM. Endothelial dysfunction, platelet hyperactivity, high

platelet count, hypercoagulability and decreased fibrinolysis have all been

positively correlated with insulin resistance. Elevated levels of biomarkers

of endothelial dysfunction (vWF, PAI-1, sVCAM), platelet hyperactivity (p-

selectin, β-thromboglobulin), hypercoagulability (fibrinogen) and

decreased fibrinolysis (PAI-1) have been reported in patients with this

syndrome. Similarly, many biomarkers known to indicate a prothrombotic

state, including PAI-1, sVCAM, VWF, have been observed in ZDSD rats.

Interestingly, an elevation in thrombopoietin was also noted in ZDSD and

may indicate an over-production of platelets.

Pro-Thrombotic Environment.

98

← →


Vascular Endothelial Growth Factor

• promotes angiogenesis

• increased in atherosclerosis

• increased in diabetic

retinopathy

• contributes heavily to renal

endothelial dysfunction

• elevated with insulin

resistance

Serum Biomarkers of Coagulation and Vascular Disease

Confidential


99

← →


Age (weeks)

14 18 20

Se

rum

VE

GF

(p

g/m

l)

150

200

250

300

350

400

450

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

von Willebrand factor

• required for platelet adhesion

• binds inactive Factor VIII,

protecting it from degradation

• deficiency leads to bleeding

disorders

• increased levels predispose to

stroke

• increases precede micro-

albuminuria in diabetic

nephropathy



100

← →


Age (weeks)

14 18 20

Se

rum

vo

n W

ille

bra

nd

Fa

cto

r (n

g/m

l)

0

50

100

150

200

250

300

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

†

Diabetic vs. Non-diabetic † †

Thrombopoietin

• increases platelet count and size

• high levels pre-dispose to

thrombosis and contribute to

platelet activation



101

← →


Age (weeks)

14 18 20

Se

rum

Th

rom

bo

po

ieti

n (

ng

/ml)

0

50

100

150

200

250

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

Factor VII

• central protein in the coagulation

cascade called the “stable factor”

• upon injury or trauma, complexes

with tissue factor to activate

factor X which initiates cascade

• vitamin K dependent clotting

factor



102

← →


Age (weeks)

14 18 20

Se

rum

Fa

cto

r V

II (

ng

/ml)

0

1

2

3

4

5

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

Plasminogen Activator Inhibitor-1

• inhibits clot breakdown by

urokinase and tPA

• high levels are present in

obesity, metabolic

syndrome and indicate

hypercoagulability

• excessive restriction of clot

dissolution results in

thrombosis and increases

fibrosis

• stimulated by angiotensin II



103

← →


Age (weeks)

14 18 20

Se

rum

PA

I-1

(n

g/m

l)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

†

Diabetic vs. non-diabetic †

Chronic inflammation contributes to multiple organ

dysfunctions in the presence of insulin resistance and

obesity. Inflammatory mediators such as IL-6, TNF-α,

CRP and resistin are elevated in patients with metabolic

syndrome in conjunction with decreased circulating

levels of anti-inflammatory adipokines such as

adiponectin. Evaluation of circulating biomarkers in the

ZDSD rat revealed a similar pattern of low-mid range

chronic inflammation which was present in animals

before frank diabetes develops.

Low-grade inflammatory state

104

← →


Monocyte Chemotactic Protein-3

• produced by tumor cells

and macrophages

• closely related to MCP-1

Serum Biomarkers of Inflammation


105

← →


Age (weeks)

14 18 20

Seru

m M

CP

-3 (

pg

/ml)

300

350

400

450

500

550

600

650

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

†

Diabetic vs. non-diabetic †

Lymphotactin

• produced by activated

thymic and peripheral

blood CD8+ T cells

• involved in angiogenesis,

inflammation

• produced by T helper

cells 1 which can infiltrate

into pancreas and destroy

beta cells





106

← →


Age (weeks)

14 18 20

Seru

m L

ym

ph

ota

cti

n (

pg

/ml)

0

20

40

60

80

100

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

Interleukin -11

• IL-11 treatment decreases

glomerular NF-kappa B

activity

• reduces renal injury in

experimental

glomerulonephritis



107

← →


Age (weeks)

14 18 20

Seru

m IL

-11 (

pg

/ml)

0

50

100

150

200

250

300 Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

Macrophage Inflammatory Protein-1

• produced by

macrophages

• activated as response

to bacterial endotoxins

• activates granulocytes

(neutrophils,basophils

& eosinophils) to

produce acute

inflammation





108

← →


Age (weeks)

14 18 20

Seru

m M

IP-1

alp

ha (

ng

/ml)

0.0

0.5

1.0

1.5

2.0

2.5

3.0 Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

Myeloperoxidase

• enzyme most abundant in

neutrophils

• elevated in ischemic heart

disease

• elevated by

hypercholesterolemia

• lowered by rosiglitazone

• chemically produces

hypochlorous acid which

is cytotoxic and is used

by neutrophils to kill

bacteria

• presence of antibodies

against MPO associated

with glomerulonephritis



109

← →


Age (weeks)

14 18 20

Seru

m M

PO

(n

g/m

l)

0

10

20

30

40

50

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

Eotaxin

• cytokine that selectively

recruits eosinophils as a

mediator of allergic

response

• increased expression in

pancreatic beta cells in

pre-diabetic rats



110

← →


Age (weeks)

14 18 20

Seru

m E

ota

xin

(p

g/m

l)

400

500

600

700

800

900

1000

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

†

Diabetic vs. non-diabetic compared to SD †

CD40-Ligand

• pro-inflammatory cytokine

elevated in diabetes and

atherosclerosis



111

← →


Age (weeks)

14 18 20

Seru

m C

D40-l

igan

d (

pg

/ml)

0

50

100

150

200

250

300

350

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

Drug Efficacy

• Objective

o Examine if anti-diabetic reference compounds

could prevent the onset of diabetes

• Design o Age 17 weeks

o Treatments

• Metformin 150mg/Kg BID

• Rosiglitazone 3mg/Kg BID

• Exenatide 1µg/rat BID o Measurements

• Weight

• Glucose

• Other results (not reported here)

o Triglycerides ↑ with diabetes

o Cholesterol ↑ with diabetes

o FFA ↑ with diabetes

o Insulin ↓ with diabetes

112

← →


Anti-Diabetic Drug Treatment Prevents Diabetes and Weight Loss in ZDSD Rats (5008 Chow)

113

← →


17 19 21 23 25 27 0

100

200

300

400

500

600 Metformin (150mg/Kg BID; n=7)

Vehicle (BID; n=4-6)

Rosiglitazone (3mg/Kg BID; n=7)

Exenatide (1 µg/rat BID; n=6)

Age (weeks)

Glu

co

se (

mg

/dL

)

17 19 21 23 25 27 0

100

200

300

400

500

600

700

Metformin (150 mg/Kg BID; n=7)

Vehicle (BID; n=6)

Rosiglitazone (3 mg/Kg BID; n=7)

Exenatide (1 µg/rat BID; n=6)

Age (weeks)

Weig

ht

(gra

m)

The Effect of Acute DPP-IV Inhibition

and Sulfonylurea Treatment on

Glucose Disposal in 21 Week-Old ZDSD Rats

114

← →


Effect of DPP-IV on OGTT in ZDSD Rats:

Sitagliptin (3mg/kg)

115

← →


-30 0 30 60 90 120 0

100

200

300

400

Vehicle

Sitagliptin (3 mg/kg, p.o)

Glucose (2 g/kg)

Time (min) post-glucose

Glu

co

se (

mg

/dL

)

116

← →


Effect of the Sulfanylurea, Glyburide, on IPGTT in ZDSD rats

Glyburide 5mg/kg, IPGTT

-30 0 30 60 90 120 0

100

200

300

400

Vehicle

Glyburide

(5 mg/kg, p.o.)


Glu

co

se (

mg

/dL

)

Effect of Sulfanylurea in ZDSD Glyburide, IPGTT

-30 0 30 60 90 120 0

100

200

300

400

Vehicle

Glyburide 10mg/kg

Glyburide 30mg/kg

Glyburide 100mg/kg


Glu

co

se (

mg

/dL

)

Glucose (1 g/kg)

Glucose (1 g/kg)

Experimental Design

– Male rats, age 15 weeks

– Pre-treat with compounds for two weeks before feeding 5SCA

– Treatments • Vehicle/Vehicle on 5008

• Vehicle/Vehicle on 5SCA

• Compound X/Vehicle on 5SCA

• Vehicle/Exenatide on 5SCA

• Compound X/Exenatide on 5SCA

– Measurements • Weight

• Glucose

• Triglycerides

• Cholesterol

• FFA

• OGTT

Evaluating the Efficacy of a New Compound in Diabetes Prevention in the ZDSD Rat

117

← →


Glucose Levels before and after 5SCA

118

← →


Vehicle/Vehicle, 5008

Vehicle/Vehicle, 5SCA

Comp. X/Vehicle, 5SCA

Vehicle/Exenatide, 5SCA

Comp. X/Exenatide, 5SCA

Time (days)

Glu

co

se (

mg

/dL

)

0 7 14 21 28 35 42 0

200

400

600

All rats on 5008 Rats on 5008 and 5SCA

Body Weights before and after 5SCA

119

← →


Time (days)

Bo

dy W

eig

ht

(g)

0 7 14 21 28 35 42 450

500

550

600







Cholesterol before and after 5SCA

120

← →


Time (days)

Ch

ole

ste

rol

(mg

/dL

)

0 10 20 30 40 0

50

100

150

200







Triglyceride before and after 5SCA

121

← →


Time (days)

Tri

gly

ceri

de

(m

g/d

L)

0 7 14 21 28 35 42 0

500

1000







NEFA before and after 5SCA

122

← →


Time (days)

NE

FA

(m

Eq

/L)

0 7 14 21 28 35 42 0.0

0.5

1.0

1.5







OGTT, 16hr fast, glucose 2g/kg

123

← →


Minutes After Glucose Challenge

Glu

co

se (

mg

/dL

)

0 30 60 90 120 0

200

400

600






Rimonabant Treatment

Treatment started at 11 weeks of age

124

← →


Slower weight gain with high dose Rimonabant (5008 chow).

Rimonabant in ZDSD (11-14 weeks old)

125

← →


0 5 10 15 20 350

400

450

500

Vehicle

Rimonabant (3mg/kg/day)

Rimonabant (10mg/kg/day)

Days of Treatment

Bo

dy W

eig

ht

(g)

Slight but significant and dose-dependent decreases in body fat at 22 days (5008 chow).

Rimonabant

Vehicle 3mg/kg 10 mg/kg

De

lta

bo

dy f

at

(%)

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

compared to vehicle (Dunnett's)

Baseline body fat was 11.48

Rimonabant in ZDSD (14 weeks old) Body Composition

126

← →


Rimonabant Treatment

Treatment started at 20 weeks of age

127

← →


Rimonabant in ZDSD (20-27 weeks old) Body Weight

Vehicle 13.1% 3 mg/kg 12.5% 10 mg/kg 10.7%

QNMR Data 12.8%

128

← →


Time (weeks)

Baseline 1 2 3 4 5 6 7

Bo

dy w

eig

ht

(g)

520

540

560

580

600

620

Acacia

Rimonabant (3 mg/kg)


diet 12468 diet 5008 16

hr fa

st

diet 5008


Glucose

129

← →


600

Time (weeks)

Baseline 1 2 3 4 5 6 7 8

Pla

sm

a g

luc

ose (

mg

/dL

)

100

200

300

400

500

Acacia



diet 12468 diet 5008 16hr

fast

diet 5008


Cholesterol

130

← →


Time (weeks)

Baseline 1 2 3 4 5 6 7 8

Pla

sm

a c

ho

leste

rol (m

g/d

L)

40

60

80

100

120

140

160

Acacia

Rimonabant 3mg/kg

Rimonabant 10 mg/kg

diet 12468 diet 5008

16

hr

fast

diet 5008


Triglyceride

131

← →


Time (weeks)

Baseline 1 2 3 4 5 6 7 8

Pla

sm

a T

rig

lycerid

es (

mg/d

L)

0

200

400

600

800

1000

1200

1400

1600

Acacia

Rimonabant 3 mg/kg

Rimonabant 10 mg/kg

diet 12468 diet 5008

16hr fa

st

diet 5008

Niacin Treatment

The effect of 7 days of niacin

treatment on glucose, TG and FFA

132

← →


Treatment of Dyslipidemia in ZDSD with Niacin

7 days of treatment

133

← →



200

400

600 Vehicle

Niacin


We

igh

t (g

)


50

100

150

200

250 Vehicle

Niacin


Glu

co

se

(m

g/d

L)


200

400

600 Vehicle

Niacin


Tri

gly

ce

rid

e (

mg

/dL

)

Fed Fasted Fed Fasted 0.0

0.5

1.0

1.5 Vehicle

Niacin


FF

A (

mE

q/L

)

*

*

* * *

ZDSD Obesity

Metabolic Syndrome Diabetes

Obesity Model before

diabetes develops, 5-16 weeks of age

Metabolic Syndrome

Insulin Resistance Hyperlipidemia

Obesity Hypertension


Diabetes Model

Natural/Spontaneous

Development (LabDiet 5008)

Slower & more random


Osteoporosis

Cardiovascular/ Inflammatory

Biomarkers


Diet Synchronized

(RD D12468 or Purina Test Diet 5SCA)


Osteoporosis

Cardiovascular/ Inflammatory

Biomarkers


The ZDSD Rat:

One rodent – Many Models

134

← →


Summary of ZDSD Characteristics • Conforms to the FDA’s guidelines for development of therapeutics for obesity,

metabolic syndrome, and type II diabetes

• Intact Leptin Pathway

• Insulin resistance, elevated glucose levels and glucose intolerance develop early

• Mirrors the progression of Type II Diabetes in humans

• Progresses through Insulin Resistance, Hypertension, Dyslipidemia, Obesity &

Diabetes

• Diet sensitive

• Responsive to: TZDs, Metformin, Exenatide, Sitaglipin, Niacin, Rimonabant &

Glyburide

• Exhibits diabetic complications: nephropathy, osteoporosis, delayed wound

healing, and increased cardiovascular/inflammatory markers

• Complications of diabetes develop over reasonable timeframes

135

← →


Interested in the ZDSD Rat?

Contact us at

[email protected]

www.PreClinOmics.com ←


mailto:[email protected]

http://www.preclinomics.com/

Health & Medicine

ZDSD Overview a model of obesity, metabolic syndrome and diabetes, 28 June 2013