zdsd overview a model of obesity, metabolic syndrome and diabetes, 24 may-2011

INDEX1. Introduction Page 2-2. Spontaneous or Synchronous Diabetes Page 7-3. Metabolic Syndrome Elements 0-

A. Visceral Obesity Page 13-B. Insulin Resistance Page 19-C. Clamp Study Page 28-D. Dyslipidemia Page 32-E. Hypertension Page 35-

4. Eating Behavior Page 37-5. Beta cell Failure Page 38-6. Renal Injury Page 41-

A. Urinary biomarkers, Exp. 1, 2 Page 44-B. RBM Biomarkers – Renal, Exp 3 Page 55-C. Glomerular Pathology, EM, Exp 4 Page 64-D. Synchronized Nephropathy, Exp 5 Page 76-

7. Osteoporosis Page 86-8. Wound Healing Page 92-9. RBM Biomarkers – Pro-Thrombotic Page 96-10. RBM Biomarkers - Inflammation Page 102-11. Therapeutic Efficacy

A. Common Anti-diabetic Compounds Page 110-B. Rimonabant Page 115-C. Niacin Page 123-

12. Summary Page 125- 1

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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.

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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

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Development SchemeZucker Diabetic-Sprague Dawley Rat (ZDSD)

• Produced by crossing diet induced obese (DIO) rat derived from the Crl:CD(SD) strain (exhibiting polygenetic obesity and insulin resistance) with homozygous lean ZDF/Crl rat (which will express 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.

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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 leptinpathway.

• 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 HypertensionCardiovascular markers InflammationOsteoporosis Delayed Wound Healing

• In production

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ZDSDObesity

Metabolic SyndromeDiabetes

Obesity Modelbefore

diabetes develops,5-16 weeks of age

Metabolic Syndrome

Insulin Resistance

Hyperlipidemia

Obesity

Hypertension

Delayed Wound Healing

Diabetes Model

Natural/Spontaneous

Development (LabDiet 5008)

Slower & more random

Diabetic Nephropathy

Osteoporosis

Cardiovascular/ Inflammatory

Biomarkers


Diet Synchronized

(RD D12468 or Purina Test Diet 5SCA)


Osteoporosis


Biomarkers


The ZDSD Rat:

One rodent – Many Models

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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 onsetThe 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 now 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

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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

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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

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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

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Characteristics of Metabolic Syndrome Seen in the ZDSD Rat

• Increased body weight with increased abdominal fat

• Insulin resistance / Glucose intolerance

• Hyper-lipidemia

• Increased blood pressure / Hypertension

• Increased Serum BioMarkers of Coagulation inflamation and Vascular Disease

• Increased fed and fasting glucose and HbA1c levels

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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 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

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Comparative Growth Curvesin 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

All time points statistically different14

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Spontaneous Development of Obesity in ZDSD Rats Fed 5008 Chow

Study # 09-550-170

All time points statistically different

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.

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Visceral Obesity in the ZDSD RatCT Scan

Sub-cutaneous fat

Retroperitoneal fat

Visceral fat

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Terminal Body Weight Comparison

Terminal animal weights in diabetic and control animals.

100

200

300

400

500

600

700CRL-SD, CD

+/fa

ZDF

ZDSD, Diabetic 12-21 weeks


Weig

ht (g

)

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Terminal ComparisonLiver Weight Food & Water Consumption

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

0

5

10

15

20

25

30

CRL-SD, CD

+/fa

ZDF



We

igh

t (g

ram

)

0

50

100

150

200

250

300

350

CRL-SD, CD+/fa

ZDF

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

Am

ou

nt/

rat

(gra

m)

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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

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Comparative Growth Curvesin 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


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Spontaneous Development of Obesity in ZDSD Rats Fed 5008 Chow

Study # 09-550-170

All time points statistically different

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.

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Spontaneous Development of Hyperglycemia in ZDSD Rats Fed 5008 Chow

Study # 09-550-170


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Spontaneous Development of Glucose Intolerance Shown by OGTT in ZDSD Rats Fed 5008 Chow

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Spontaneous Development of Insulin Resistance Shown by OGTT in ZDSD Rats Fed 5008 Chow

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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 oral glucose tolerance test developed spontaneously in ZDSD rats and was evident as early as 8 weeks of age (fed Purina 5008 chow).


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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


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Glucose in CD vs ZDSD16 weeks of age

CD

ZDSD

100

110

120

130

140

150CD

ZDSD

Blo

od

Glu

co

se m

g/d

L

Glycated Hb in CD vs ZDSD16 weeks of age

CD

ZDSD

3.0

3.2

3.4

3.6

3.8

4.0CD

ZDSD

Gly

cate

d H

b

Glucose and Glycated Hemoglobin Levels in CD and Prediabetic ZDSD Rats

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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

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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

SD (age) SD (wt) ZDSD ZF0

100

200

300

400

500

#

Rat Strain (SD rats are age or weight matched)

Bo

dy W

eig

ht

(g)


40

80

120

160

200Vehicle (n=6-8)

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


Ba

sa

l B

loo

d G

luco

se

(m

g/d

l)

29

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ZDSD and ZF Rats are Insulin Resistant which

Improved with Rosiglitazone Treatment



10

20

30

40

50

60 Vehicle (n=6-8)


#

#


Glu

co

se

In

fusio

n R

ate

(mg

/kg

/min

)

30

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ZDSD Rats Have Modest Visceral and

Whole Body Obesity Unlike ZF Rats



2

4

6

8

10

12Vehicle (n=6-8)


#


Ep

idid

ym

al F

at

Pa

d (

g)


10

20

30

40

Vehicle (n=6-8)

#


Bod

y F

at

(%)

31

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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

Glu

cose TG

CHOL

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

An

aly

tes (

mg

/dL

)

Glucose, triglyceride and cholesterol levels. 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.

100

200

300

400

500

600

700 CRL-SD, CD+/fa

ZDF


Weig

ht (g

)

33

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Weight

Fed Fasted Fed Fasted0

200

400

600Vehicle

Niacin

Before Treatment After Treatment

Weig

ht

(gra

ms)

Glucose


50

100

150

200

250 Vehicle

Niacin


Glu

co

se (

mg

/dL

)

Triglyceride


200

400

600 Vehicle

Niacin


Tri

gly

ceri

de (

mg

/dL

)

Free Fatty Acids

Fed Fasted Fed Fasted0.0

0.5

1.0

1.5 Vehicle

Niacin


FF

A (

mE

q/L

)

Effect of 7 Days of Niacin Treatment

*

*

* * *

Treatment of Dyslipidemia in ZDSD with Niacin

7 days of treatment 34

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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

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Blood pressure data produced in collaboration with Dr. Subah Packer’s Laboratory, IU School of Medicine

60 70 80 90 10080

100

120

140

160ZDSD

CD

Age in Days

Systo

lic B

P

Blood pressure in ZDSD vs CD Rats

8-16 weeks of age

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Leptin PhysiologyFood 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

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)

Insu

lin

(n

g/m

l)

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.

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 (

mg

/dl)

38

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Average Glucose% Diabetic

15 20 25 300

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 (

mg

/dL

)

Correlation BetweenPancreatic Insulin andBlood Glucose Level

0 200 400 600 800

0

200

400

600

Insulin ng/g

Glu

co

se (

mg

/dL

)

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

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Representative Islets from ZDSD Rats

Pre-diabetic Diabetic

40

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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

41

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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

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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 ComparisonKidney Weight Urine Analysis

0

1

2

3

4

5

6

7

CRL-SD, CD+/fa

ZDF


We

igh

t (g

ram

)

0

50

100

150

200

250

300

CRL-SD, CD

+/fa

ZDF



43

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Experiment 1ZDSD Diabetic Nephropathy

Spontaneous DiabetesELISA Analysis of Markers

44

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WEIGHT

10 12 14 16 18 20 22 24 26 28 30300

400

500

600SD

ZDSD

Age (weeks)

We

igh

t (g

ram

s)

GLUCOSE

10 12 14 16 18 20 22 24 26 28 300

200

400

600SD

ZDSD

Age (weeks)

Glu

co

se (

mg

/dL

)

Urine volume

10 20 22 24 26 300

50

100

150

200SD

ZDSD

Age (weeks)

Uri

nary

vo

lum

e (

mls

/24h

r)

45

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Urinary albumin

10 20 22 24 26 300

25

50

75

100

125

150SD

ZDSD

Age (weeks)

Uri

nary

alb

um

in (

mg

/day)beta-2 microglobulin

10 20 22 24 26 300

500

1000

1500

2000SD

ZDSD

Age (weeks)

Uri

nary

-2

mic

rog

lob

uli

n (

g/d

ay)

Cystatin C

10 20 22 24 26 300

10

20

30SD

ZDSD

Age (weeks)

Uri

nary

cysta

tin

C (

g/d

ay)

KIM-1

10 20 22 24 26 300.0

2.5

5.0

7.5

10.0

12.5

15.0SD

ZDSD

Age (weeks)

Uri

nary

KIM

-1 (

ng

/day)

46

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Experiment 2Urine 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 (ArgutusAKI test, Kidney Injury Panel 1 and Rat Clusterin)

• Pathological evaluation of the kidneys was done.

47

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Data From Urinary Excretion Study

48

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Urinary Excretion of Kidney Markers

49

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Urinary Excretion of Kidney Markers

50

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

51

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52

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53

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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

4.0non-diabetic

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

/degeneration

Kidney Histopathology of the ZDSD Rat

A Novel Animal Model of Diabetes

54

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Experiment 3Serum BioMarkers of Renal Disease

RBM Collaboration

Rules Based Medicine Analysis

55

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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

56

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Experimental details

• Male ZDSD rats were placed on a high-fat diet (RD12468) between 17 and 19 weeks of age. 15 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).

57

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Neutrophil Gelatinous Associated Protein

•also called lipocalin2

•levels up-regulated during inflammation

•protective protein can trigger nephrogenesis

•associated with obesity, insulin resistance and hyperglycemia

58

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-diabetic18 weeks = on diabetogenic diet one week20 weeks = off diabetogenic diet one week

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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

59

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

60



← →


Age (weeks)

14 18 20S

eru

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

61



← →


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

62


Age (weeks)

14 18 20

Se

rum

Clu

ste

rin

(

g/m

l)

100

200

300

400

500

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

#


comapred 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 microalbuminuriain diabetic nephropathy

63

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 4Diabetic Nephropathy,

EM of Glomerular Pathology

64

← →



• Male ZDSD rats allowed to become spontaneously diabetic.

• Animals were terminated by perfusion at about 35 weeks of age. We evaluated the following groups:

– 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

65

← →


66

Glomerular CapillaryControl, Age Matched Diabetic, 12 Weeks

66

← →


Glomerular Capillary, Basement MembraneControl, Age Matched Diabetic, 12 Weeks

67

← →


Glomerular Capillary, Basement MembraneControl, Age Matched Diabetic, 12 Weeks

68

← →


69

Glomerular CapillaryControl, Age Matched Diabetic, 16.5 Weeks

69

← →


Glomerular Capillary, Basement MembraneControl, Age Matched Diabetic, 16.5 Weeks

70

← →


71

Glomerular BasementMembrane Thickness

Time of diabetes in the ZDSD Rat 09-550-187

Th

ickn

ess in

nm

CD C

ontrol

12 W

eeks

16.5

Wee

ks

0

100

200

300

400

500

← →


72

← →


Scanning MicroscopyGlomerular Capillaries

Control Diabetic

73

← →


Scanning MicroscopyControl Glomerular Capillary with Normal Podocyte Foot Processes

74

← →


Scanning MicroscopyDiabetic Glomerular Capillaries Demonstrating Effacement

75

← →


Transmission MicroscopyGlomerular Mesangium, Advanced Diabetic Changes

Experiment 5Diabetic Nephropathy,Synchronized Diabetes:

Clinical Data andLM of Glomerular Pathology

76

← →



• 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

PCO 10-550-212

77

← →


78

Body weight (g)0 7

14

21

28

35

42

70

105

126

155

172

185

196

350

400

450

500

550

600Diabetic

Non-diabetic

Day of study

Bo

dy w

eig

ht

(g)

Glucose (mg/dL)

0 7

14

21

28

35

42

70

105

126

155

172

0

200

400

600

800Diabetic

Non-diabetic

Day of study

Glu

co

se (

mg

/dL

)

43 470

5

10

15Diabetic

Non-diabetic

* t-test

**

Age (weeks)

Hb

A1c (

%)

430.0

0.2

0.4

0.6

0.8

1.0Diabetic

Non-diabetic

Age (weeks)

NE

FA

(m

Eq

/L)

← →


Diabetic Non-diabetic0

5

10

15

20

25Diabetic

Non-diabetic

*

* t-testL

iver

weig

ht

(g)

47 w

eeks o

f ag

e

Diabetic Non-diabetic0

2

4

6Diabetic

Non-diabetic

Kid

ney w

eig

ht

(g)

47 w

eeks o

f ag

e

* t-test

*

43 470

100

200

300Diabetic

Non-diabetic

Age (weeks)

Uri

ne v

olu

me (

mls

/24 h

r)

430

20

40

60

80Diabetic

Non-diabetic

*

* t-test

Age (weeks)

Uri

ne a

lbu

min

(m

g/d

ay)

79

← →


Blood Chemistry

80

43 45 470

10

20

30Diabetic

Non-diabetic

* t-test

***

Age (weeks)

Seru

m B

UN

(m

g/d

L)

45 470.0

0.1

0.2

0.3

0.4

0.5Diabetic

Non-diabetic

Age (weeks)

Seru

m c

reati

nin

e (

mg

/dL

)

43 470

50

100

150

200Diabetic

Non-diabetic

* t-test

**

Age (weeks)

Seru

m c

ho

leste

rol (m

g/d

L)

43 470

500

1000

1500Diabetic

Non-diabetic* t-test

**

Age (weeks)

Seru

m t

rig

lyceri

des (

mg

/dL

)

← →


47 Week-old, 27 Weeks DiabetesNon-Diabetic Diabetic

81

81

← →


47 Week-old, 27 Weeks Diabetes

Non-Diabetic Diabetic

Diabetic Diabetic 82

← →


83


83

← →


84


84

← →


47 Week-old, 27 Weeks Diabetes

85

← →


Osteoporosis in the ZDSD Rat

Reference: Skeletal changes associated with theonset of type 2 diabetes in the ZDF and ZDSDrodent models. Susan Reinwald, Richard G.Peterson, Matt R. Allen, and David B. Burr. Am JPhysiol Endocrinol Metab 296: E765–E774, 2009.

86

← →


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 ±SEMn=12-17/group

87

← →


L4 Vertebra – dimensions not affected by

differences in growth in ZDSD rats

0

1

2

3

4

5

6

7

8

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

L4

ve

rte

bra

l b

od

y h

eig

ht (m

m)

p<0.05

0

1

2

3

4

5

6

7

8

ZDSD Controls

L4

ve

rte

bra

l b

od

y h

eig

ht (m

m)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35


L4

cro

ss-s

ect

ion

al a

rea

(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

ect

ion

al a

rea

(m

m)


88

← →


Structural Properties L4 vertebrae

0

10

20

30

40

50

60

70

80

90


En

erg

y to

Ultim

ate

Lo

ad

(m

J)

*

ZDF ZDSD

p < 0.001 p < 0.005

p < 0.050 p < 0.001

p < 0.001 p < 0.001

p <0.050 p < 0.010

0

10

20

30

40

50

60

70

80

90

ZDSD Controls

En

erg

y to

Ultim

ate

Lo

ad

(m

J)

*

P-values for differences in diabetic rats vs. respective controls

[Biomechanical Test – axial compression]

Yield Force, N

Stiffness, N/mm

Ultimate Load, N

Energy to Ultimate Load, mJ


89

← →


Material Properties L4 vertebrae

Ultimate Stress, N/mm2

Modulus, N/mm2

Toughness, mJ/mm3

Postyield Toughness, mJ/mm3

ZDF ZDSD[Parameters Normalized to BV/TV]

p < 0.051 p < 0.050

p < 0.050 p < 0.050

p = 0.657 p < 0.051

p = 0.224 p < 0.101

P-values for differences in diabetic rats vs. respective controls

0.0

0.5

1.0

1.5

2.0

2.5


To

ug

hn

ess/[B

V/T

V] (m

J/m

m3

)

0.0

0.5

1.0

1.5

2.0

2.5

ZDSD Controls

Tou

gh

ne

ss/[B

V/T

V] (

mJ/m

m3

)

*Mean ±SEMn=12-17/group

90

← →


Glycated hemoglobin

0

1

2

3

4

5

6

7

8

9

10


Hb

A1

c (

%)

*

0

1

2

3

4

5

6

7

8

9

10

ZDSD Controls

Hb

A1

c (%

)

*

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.


91

← →


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. 92

← →


Data at 11 days after woundWEIGHT

Weig

ht

in G

ram

s

Dia

betic

ZDSD

Non-D

iabet

ic Z

DSD

SD

0

200

400

600

GLUCOSE

Glu

co

se in

mg

/dl

Dia

betic

ZDSD

Non-D

iabet

ic Z

DSD

SD

0

100

200

300

400

500

93

← →


94

Control SD animals233019

Day 11

Day 14

233023

Day 11

Day 14

Diabetic ZDSD animals233003

233006Day 11

Day 14

Day 11

Day 14

94Scale 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.

95

Separated Diabetic and Non-Diabetic ZDSD

4 7 9 11 14-100

-80

-60

-40

-20

0SD

Diabetic ZDSD

non-diabetic ZDSD**

**

*

*

*

*compared to SD (Dunnett's)

Time(days) post-wounding

% C

han

ge

Fro

m In

ital W

ou

nd

Combined ZDSD Data

4 7 9 11 14-100

-80

-60

-40

-20

0SD

ZDSD

*

*

* *

*

*compared to SD (t-test)

Time(days) post-wounding

% C

han

ge

Fro

m In

ital 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 markers 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 markers known to indicate a prothrombotic state, including PAI-1, sVCAM, VWF, have been observed in ZDSD rats. Interestingly, an elevation in thrombopoeitin

was also noted in ZDSD and may indicate an over-production of platelets.

Pro-thrombotic environment.

96

← →


Vascular endothelial growth factor

•promotes angiogenesis

•increased in atherosclerosis

•increased in diabetic retinopathy

•contributes heavily to renal endothelial dysfunction

•elevated with insulin resistance

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

Serum BioMarkers of Coagulation and Vascular Disease

Confidential


97

← →


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-albuminuriain diabetic nephropathy

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

#

#




98

← →


Age (weeks)

14 18 20

Seru

m T

hro

mbopoie

tin (

ng/m

l)0

50

100

150

200

250

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

Thrombopoietin

•increases platelet count and size

•high levels pre-dispose to thrombosis and contribute to platelet activation



99

← →


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.

Age (weeks)

14 18 20

Se

rum

Fa

cto

r V

II (

ng/m

l)

0

1

2

3

4

5

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15) compared to SD



100

← →


Plasminogen activator inhibitor-1

•inhibits clot breakdown by urokinaseand tPA

•high levels are present in obesity , metabolic syndrome and indicate hyper-coagulability

•excessive restriction of clot dissolution results in thrombosis and increases fibrosis

•stimulated by angiotensin II

Age (weeks)

14 18 20

Seru

m P

AI-

1 (

ng/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

#




101

← →


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

102

← →


Monocyte chemotactic protein-3

•produced by tumor cells and macrophages

•closely related to MCP-1

Age (weeks)

14 18 20

Se

rum

MC

P-3

(p

g/m

l)

300

350

400

450

500

550

600

650

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

#


Serum BioMarkers of Inflammation


103

← →


Age (weeks)

14 18 20

Se

rum

Lym

ph

ota

ctin

(p

g/m

l)

0

20

40

60

80

100

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD

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



104

← →


Interleukin -11

•IL-11 treatment decreases glomerularNF-kappa B activity

•reduces renal injury in experimental glomerulonephritis

Age (weeks)

14 18 20

Se

rum

IL

-11

(p

g/m

l)

0

50

100

150

200

250

300

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD



105

← →


Macrophage inflammatory protein-1

•produced by macrophages

•activated as response to bacterial endotoxins

•activates granulocytes (neutrophils,basophils & eosinophils) to produce acute inflammation

Age (weeks)

14 18 20

Se

rum

MIP

-1a

lph

a (

ng/m

l)

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



106

← →


Myeloperoxidase

•enzyme most abundant in neutrophils

•elevated in ischemic heart disease

•elevated by hypercholesterolemia

•lowered by rosiglitazone

•chemically produces hypochlorousacid which is cytotoxic and is used by neutrophils to kill bacteria

•presence of antibodies against MPO associated with glomerulonephritis

Age (weeks)

14 18 20

Seru

m M

PO

(ng/m

l)

0

10

20

30

40

50

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD



107

← →


Eotaxin

•cytokine that selectively recruits eosinophils as a mediator of allergic response

•increased expression in pancreatic beta cells of pre-diabetic rats

Age (weeks)

14 18 20

Se

rum

Eo

taxin

(p

g/m

l)

400

500

600

700

800

900

1000

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

#

# Diabetic vs. non-diabeticcompared to SD



108

← →


CD40-Ligand

pro-inflammatory cytokine elevated in diabetes and atherosclerosis

Age (weeks)

14 18 20

Se

rum

CD

40

-lig

an

d (

pg/m

l)

0

50

100

150

200

250

300

350

Sprague-Dawley (5)

ZDSD (6)

Diabetic ZDSD (15)

compared to SD



109

← →


Drug Efficacy

• Objective

– Examine if anti-diabetic reference compounds could prevent the onset of diabetes

• Design– Age 17 weeks– Treatments

• Metformin 150mg/Kg BID• Rosiglitazone 3mg/Kg BID• Exenatide 1µg/rat BID

– Measurements • Weight• Glucose

– Other results (not reported here)

• Triglycerides ↑ with diabetes• Cholesterol ↑ with diabetes• FFA ↑ with diabetes• Insulin ↓ with diabetes

110

← →


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

PCO 083A: Glucose

17 19 21 23 25 27

0

100

200

300

400

500

600Metformin 150mg/Kg BID (N=7)

Vehicle BID (N=4/6)

Rosiglitazone 3mg/Kg BID (N=7)

Exenatide 1µg/rat BID (N=6)

Age (week)

Glu

co

se (

mg

/dL

)

PCO 083A: Body weight

17 19 21 23 25 27

0

100

200

300

400

500

600

700

Metformin 150mg/Kg BID (N=7)

Vehicle BID (N=6)

Rosiglitazone 3mg/Kg BID (N=7)

Exenatide 1µg/rat BID (N=6)

Age (week)

Weig

ht

(gra

m)

111

← →


The Effect of Acute DPP-IV Inhibition and Sulfonylurea

Treatment on Glucose Disposal in 21 Week-Old ZDSD Rats

112

← →


Effect of DPP-4 in ZDSDSitagliptin 3mg/kg, OGTT

-30 0 30 60 90 1200

100

200

300

400

Vehicle

SitagliptinGlucose dose2g/kg 0-time

Time (min) post-glucose

Glu

co

se (

mg

/dL

)

Effect of DPP-IV in ZDSD Rats

Sitagliptin 3mg/kg, OGTT

113

← →


Effect of Sulfanylurea in ZDSDGlyburide 5mg/kg, IPGTT

-30 0 30 60 90 1200

100

200

300

400

Vehicle

Glyburide

Glucose dose1g/kg 0-time


Glu

co

se (

mg

/dL

)

Effect of Sulfanylurea in ZDSDGlyburide, IPGTT

-30 0 30 60 90 1200

100

200

300

400

Vehicle

Glyburide 10mg/kgGlucose dose1g/kg 0-time Glyburide 30mg/kg

Glyburide 100mg/kg


Glu

co

se (

mg

/dL

)

114

← →


Rimonabant Treatment

Treatment started at 11 weeks of age

115

← →


Weight Data

0 5 10 15 20350

400

450

500

Vehicle

Rimonabant 3mg/kg/day

Rimonabant 10mg/kg/day

Days of Treatement

Bo

dy W

eig

ht

in G

ram

s

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

Rimonabant in ZDSD (11-14 weeks old)

116

← →


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

117

← →


Rimonabant Treatment

Treatment started at 20 weeks of age

118

← →


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

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

Rimonabant 10 mg/kg

diet 12468diet 500816hr

fast

diet 5008

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

QNMR Data 12.8%

119

← →


Time (weeks)

0 1 2 3 4 5 6 7 8

Fe

d P

lasm

a g

luco

se

(m

g/d

L)

100

200

300

400

500

600

Acacia

Rimonabant 3 mg/kg

Rimonabant 10 mg/kg

diet 12468diet 5008

16hr

fast

diet 5008

Rimonabant in ZDSD (20-27 weeks old)Glucose

120

← →


Time (weeks)

0 1 2 3 4 5 6 7 8

Fed P

lasm

a c

hole

ste

rol (m

g/d

L)

40

60

80

100

120

140

160

Acacia

Rimonabant 3mg/kg

Rimonabant 10 mg/kg

diet 12468diet 5008

16hr

fast

diet 5008

Rimonabant in ZDSD (20-27 weeks old)Cholesterol

121

← →


Time (weeks)

0 1 2 3 4 5 6 7 8

Fe

d P

lasm

a T

rigly

ce

rid

es (

mg/d

L)

0

200

400

600

800

1000

1200

1400

1600

Acacia

Rimonabant 3 mg/kg

Rimonabant 10 mg/kg

diet 12468diet 5008

16

hr

fast

diet 5008

Rimonabant in ZDSD (20-27 weeks old)Triglyceride

122

← →


Niacin Treatment

The effect of 7 days of niacin treatment on glucose, TG and FFA

123

← →


Weight


200

400

600Vehicle

Niacin


Weig

ht

(gra

ms)

Glucose


50

100

150

200

250 Vehicle

Niacin


Glu

co

se (

mg

/dL

)

Triglyceride


200

400

600 Vehicle

Niacin


Tri

gly

ceri

de (

mg

/dL

)

Free Fatty Acids

Fed Fasted Fed Fasted0.0

0.5

1.0

1.5 Vehicle

Niacin


FF

A (

mE

q/L

)

Effect of 7 Days of Niacin Treatment

*

*

* * *

Treatment of Dyslipidemia in ZDSD with Niacin

7 days of treatment 124

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ZDSDObesity

Metabolic SyndromeDiabetes

Obesity Modelbefore

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


Biomarkers


Diet Synchronized

(RD D12468 or Purina Test Diet 5SCA)


Osteoporosis


Biomarkers


The ZDSD Rat:

One rodent – Many Models

125

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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/inflamatory markers.

• Complications of diabetes develop over reasonable timeframes.126

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Interested in the ZDSD Rat?

Contact us at

[email protected]

www.PreClinOmics.com←


mailto:[email protected]

http://www.preclinomics.com/

zdsd overview a model of obesity, metabolic syndrome and diabetes, 24 may-2011

Technology