A Model of Type 2 Diabetes: BBZDR/Wor rat

Biomedical Research Models, Inc
Contract Discovery Research

BBZDR/Wor Rat
• Obese (dyslipidemic)
• Hyperleptinemic
• Insulin Resistant
(hyperinsulinemic)
• Hyperglycemic
• Hypertensive

BBZDR Rats Clinical Characteristics
Genetic Predisposition (fa/fa homozygous)
Obesity (Hyperleptinemia)
Insulin Resistance (Hyperinsulinemia)
Impaired Glucose Tolerance (IGT)
Type 2 Diabetes (Hyperglycemia)
Macrovascular Diabetic Complications:
Hypertension, Accelerated Stenosis
(Inducible), Atherosclerosis (Duration
Dependent
Microvascular Diabetic Complications:
Retinopathy, Neuropathy, and Nephropathy

Features of BBZDR/Wor Rats
OBESE LEAN
MALES FEMALES MALES FEMALES
Incidence of
Diabetes
98%
(N=225)
3%
(N=217)
0%
(N=729)
0%
(N=783)
Age at Onset 66.8± 2.7 72.1 ±4.6 N/A N/A
Hypertension + ++ – –
Insulin
Resistance + + – –
GLUT-2 – + + +

Normal Islets Lean Rat
InsulinGlucagon

Islets from an Obese Rat with IGT
InsulinGlucagon
Glut 2 Receptor

0
100
200
300
400
500
600
0 10 20 30 40 50
Time (min)
OBESE > 16
LEAN > 6
3.4 mmol/kg
glucose i.v.
1.7 mmol/kg
arginine i.v.
BloodGlucose(mg/dl)
Blood Glucose: Fasted BBZDR Females

Insulin Release: Fasted Female BBZDR Rats
0
20
40
60
80
100
0 10 20 30 40 50
Time (min)
OBESE > 16
LEAN > 6
3.4 mmol/kg
glucose i.v.
1.7 mmol/kg
arginine i.v.
Insulin(ng/ml)

BBZDR/Wor Obese Rat Islet:
Glut-2 Receptor(400x)

Islets from Type 2 Diabetic Rat
Glucagon Insulin
Glut-2 Receptor Glut-2 Control

Blood Glucose: Fasted Male BBZDR rats
0
100
200
300
400
500
600
700
0 10 20 30 40 50
Time (min)
OBESE
LEAN
3.4 mmol/kg
glucose i.v.
1.7 mmol/kg
arginine i.v.
BloodGlucose(mg/dl)

Insulin Release: Fasted BBZDR Males
0
20
40
60
80
100
0 10 20 30 40 50
Time (min)
Obese
Lean
3.4 mmol/kg
glucose i.v.
1.7 mmol/kg
arginine i.v.
Insulin(ng/ml)

Blood Chemistry Profile of BBZDR Rats
4 Months of age
0
100
200
300
400
500
600
700
800
Glucose Cholesterol Triglyceride HDL
mg/dl
Lean
Male
Obese
Female
*
*
*
*
*
* P<0.05
*
*
*
*

Atherosclerosis in Renal Artery
BBZDR Diabetic Rat (4mo duration)

Severe atherosclerosis:
10 months ~8 months duration of diabetes
Complete occlusion of renal artery, x100 (Left);
Arrows identify plaque, x400 (Both panels).

Measurement and Calculation of
Neointima Thickness
The % stenosis was calculated as the Internal Elastic Area (IEA)
minus the Residual Area (RA) divided by the IEA ( x 100)

Neointima Thickness in Lean and Obese Male
BBZDR Rats One Week Post Balloon Injury
BBZDR Lean Rats Type 2 Diabetic Rats

Neointima Thickness in Lean and Obese Male
BBZDR Rats 4 Weeks Post Balloon Injury
BBZDR Lean Rats Type 2 Diabetes Rats

Atherosclerosis Plaque in
BBZDR/Wor Obese Female Rats

Fatty Deposits and Inflammatory Cells in
Occluded Obese Female Carotid Artery

% of Stenosis In Balloon Injured Rat Carotid Artery
0
5
10
15
20
25
30
35
40
45
50
7 14 21 28
Days Post Balloon Injury
%Stenosis
Lean Rat
Obese Female
Obese Male
*
*
**

Clinical Phases
Microalbuminuria
Clinical proteinuria
Progression to ESRD

LEAN
BBZDR Kidney at 5 Months of age
Obese
Female
Obese
Diabetic

Lean rat: 5 months old
Normal Glomeruli: x100 (left); insert x400 (right).

Obese female: non-diabetic, 5 month old
Evidence of mild glomeruli enlargement and mesangial
expansion (arrows), x100 (Right); Insert: x400 (Right)

Aldose Reductase
Lean BBZDR Obese BBZDR
6 months 4months duration

Sorbitol Dehydrogenase in BBZDR Kidneys
Lean ControlObese T2 Diabetic

RAGE in BBZDR Rat Kidneys
Obese T2 Diabetic Lean Control

Summary
• The diabetic BBZDR male rat is a good model of
diabetic nephropathy.
• Supported by Clinical blood chemistry and
Histopathology
– Evidence of hyperfiltration and glomeruli
hypertrophy at 3months age
– Microalbumuria progressing to proteinuria from
3month through 12month
– Mesangial expansion, basement membrane thicking
and interstitial fibrosis
– Tubule expansion, aschemia and diffuse necrosis after
12months of age (10 months duration)

Neuropathy in BBZDR
• Experimental rat models of types 1 and 2 diabetes differ in
sympathetic neuroaxonal dystrophy. J Neuropathol Exp
Neurol. 2004 May;63(5):450-60
Schmidt RE, Dorsey DA, Beaudet LN, Parvin CA, Zhang W, Sima AA.
• C-peptide corrects endoneurial blood flow but not
oxidative stress in type 1 BB/Wor rats. Am J Physiol
Endocrinol Metab. 2004 Sep;287(3):E497-505.
Stevens MJ, Zhang W, Li F, Sima AA.
• A comparison of diabetic polyneuropathy in type II
diabetic BBZDR/Wor rats and in type I diabetic BB/Wor
rats. Diabetologia, 2000 Jun;43(6):786-93
Sima AA, Zhang W, Xu G, Sugimoto K, Guberski D, Yorek MA.

Type 1 and 2 Diabetes Type 1 Diabetes
↑ Hyperglycemia Insulin / C-peptide ↓
↑ Polyol Pathway
↓ NO
↓ Na+/K+- ATPase
↑ Oxidative Stress
↑ Nonenzymatic Glycation
↓ Neurotrophism
Apoptosis ?
Axonal
Degeneration /Loss
Impaired Regeneration Nodal / Paranodal
Degeneration
Acute
Reversible
NCV-Slowing
Chronic
Irreversible
NCV-Slowing
Glucose Sorbitol Fructose
NADPH NADP
Arginine Citrullin + NO ↓
GSSG GSH ↓
Pathogenetic Scheme of DPN
Sima, Cell Mol Life Sci 2003.

Diabetic Polyneuropathy (DPN)
Type 2 vs Type 1
Type 2 Type 1
• 35% /25 years
• Slow progression
• Mild axonal
degeneration
• No nodal changes
• Close to 100% /25years
• More rapid progression
• Severe axonal degeneration
and loss
• Progressive nodal/paranodal
• degeneration

Progression of Axo-glial Dysjunction
Axo-glialdysjunction(%)
A. BB/Wor – BBZDR/Wor- rats
Duration Of Diabetes
Baseline 4mo 6mo 8mo 12mo 14mo
0
10
20
30
40
50 Control
BB/Wor
BBZDR/ Wor
Axo-glialdysjunction(%)
20
40
60
20 30 40 50 60 70
Age (years)
Common slope: df =39, f=13.91, p< 0.001
Control: a= 0.24
b= 0.26
NIDM: a= 3.44
b= 0.26
IDDM: a= 25.43
b= 0.26
B. Human
Sima et al., J Clin Invest 1984 Sima et al., N Engl J Med, 1986

0
20
40
60
80
100
0
5
10
15
20
25
30
35
Teased Fiber Pathology
in Type 1 and Type 2 Diabetic Patients
Control Type 1 Type 2
n=19 n=11 n=17
Control Type 1 Type 2
n=19 n=11 n=17
p<0.001
p<0.005
p<0.001
p<0.001
Sima et al, JCI,1988

Structural Abnormalities In Sural Nerve
Axonal Degeneration Fiber Number
Nerve Regeneration
P<0.001
P<0.001
P<0.01
P<0.001
PercentageofAxonal
Degeneration(%)
0
5
10
15
20
25
C8 D1-8 D1CP D2-8
P<0.05
P=0.07
P<0.051000
800
600
400
200
0
NumberofMyelinated
Fibers
C8 D1-8 D1CP D2-8
P<0.001
P<0.001
P<0.05
C8 D1-8 D1CP D2-8
Fibers(%)
0
1
2
3
4
5
6
7
8
Sima et al., Diabetologia,2002

*: p<0.01, **: p<0.001 vs control.
Blood Glucose Serum Insulin Serum C-peptide
0
100
200
300
400
500
600
0
200
400
600
800
1000
0
100
200
300
400
500
600
700
C D1 D1+C D2 C D1 D1+C D2 C D1 D1+C D2
** ** **
*
** **
**
BloodGlucose(mmol/L)
Insulin(pmol/L)
C-peptide(pmol/L)
Glucose, insulin and C-peptide plasma
levels in BB/Wor- and BBZDR/Wor-rats
Sima et al: Diabetologia, 2001

Na+/K+-ATPaseActivity
(mmol/ADP/mg/wetwt./hr)
Control Sham 10µg 100µg 500µ 1000µg
hrC-peptide
0
200
400
600
800
1000
*
†
†
*: p<0.01, vs age-matched controls;
†: p<0.05 vs duration-matched sham-treated BB/W-rats.
The Effect of C-peptide on Neural Na+/K+- ATPase
Zhang et al: Exp. Diab.Res. 2001

35
40
45
50
55
60
65
70
75
Control
BB/Wor
BB/Wor+C
BBZDR/Wor
Onset 1 Week 2 Week 3 Week 4 Week 5 Week
Hyperglycemic
Component
Insulin/C-Peptide
Deficiency
Component
Acute Metabolic NCV Defects

Retinopathy in BBZDR
• N-3 polyunstaurated Fatty acids prevent diabetic
retinopathy by inhibition of retinal vascular damage and
enhanced endothelial progenitor cell reparative function.
Plos One. 2013;8(1):e55177. Epub 2013 Jan29.
Tikhonenko M, Lydic TA, Opreanu M, Li Calzi S, Bozack S, McSorley KM, Sochacki AL, Faber MS,
Hazra S, Duclos S, Guberski D, Reid GE, Grant MB, Busik JV
• Diabetic retinopathy is associated with bone marrow
neuropathy and a depressed peripheral clock. Journal of
Experimental Medicine. 2009 Dec21;206(13):2897-906
Busik JV, Tikhonenko M, Bhatwadekar A, Opreanu M, Yakubova N, Caballero S, Player D, Nakagawa T,
Afzal A, Kielczewski J, Sochacki A, Hasty S, Li Calzi S, Kim S, Duclos S, Segal MS, Guberski DL,
Esselman WJ, Boulton ME, Grant MB.
• Time course of NADH oxidase,inducible nitric oxide
synthase and peroxynitrite in diabetic retinopathy in the
BBZ/Wor rat. Nitric Oxide. 2002 May;6(3): 295-304.
Ellis,E.A., Guberski, D.L., Huston, B. and Grant, M.B.

Small Pressurized Artery Myography:
As close as it gets to true organ
perfusion or function
0 50 100 150 200 250
50
75
100
125
150
175
Diameter with tone
Passive diameter
WKY
Pressure (mmHg)
Diameter(µM)

Endothelial Dysfunction in BBZDR Rats
 Obese-Diabetic; O Lean control (age-matched)
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
Pre-diabetic age 3-4 Weeks of Diabetes
5 Months of Diabetes 10 Months of Diabetes

Levels of Blood Sugars and
Glycosylated Hemoglobin
Blood Sugars
(mg/dl)
Total
Glycosylated
Hemoglobin (%)
Diabetic (BBZ/Wor) 496 ± 15 13.0 ± 0.4
Non-Diabetic (BBDR
/Wor) 123 ± 3 5.3 ± 0.5

VEGF and H2O2 Correlation
H2O2% VEGF
CORRELATION
COEFFICIENT
DIABETIC 78.7 ± 4.84 24.67 ± 0.33 0.82*
NON-DIABETIC 39.0 ± 4.47 21.52 ± 0.43 0.83**
*p = 0.001
**p = 0.0001

Percentage of Blood Vessels
Positive for Peroxide
Central
Retina
Peripheral
Retina
Diabetic (BBZ/Wor) 80.00 ± 10.52 44.24 ± 12.79
Non-Diabetic (BB
DR
/Wor) 38.28 ± 9.68 26.47 ± 8.62

Basement Membrane Thickness
(nm) in Type 2 BBZDR Rats
Diabetic (BBZ/Wor) 96.0 ± 13.32nm
Non-Diabetic (BBDR
/Wor) 60.4 ± 5.62nm

Immunocytochemical
Localization of Fibronectin
Colloidal Gold Particles/um2
Diabetic (BBZ/Wor) 4.45 ± 1.09
Non-Diabetic (BBDR
/Wor) 1.56 ± 0.79

Animal Characteristics for iNOS
and Nitrotyrosine Study
ANIMAL
GROUP
AGE LENGTH OF
DIABETES
BLOOD
GLUCOSE
PRE-DIABETIC
(BBZ/Wor)
1.5 – 2 months ND 142 ± 29 md/dl
NEW ONSET
DIABETES
(BBZ/Wor)
2 months 2 – 6 days 383 ± 79 mg/dl
CHRONIC
DIABETES
(BBZ/Wor)
7 – 20 months 4.8 – 18 months 387 ± 96 mg/dl
NON-DIABETIC
CONTROLS
(BB
DR
/Wor)
7 – 20 months ND 130 ± 28 mg/dl

Colloidal gold localization of nitrotyrosine (arrows)
in retina of BBZ/Wor rat with diabetes for two days

eNOS and iNOS
Immunoreactivity in Retina
eNOS iNOS
BBZ/Wor 13.9 ± 2.3 part/50 µm
2
33.9 ± 10.0 part/50 µm
2
BBDR
/Wor 78.8 ± 6.0 part/50 µm
2
3.5 ± 2.8 part/50 µm
2

iNOS and Nitrotyrosine
Immunoreactivity
ANIMAL GROUP iNOS NITROTYROSINE
PRE-DIABETIC
(BBZ/Wor)
3.7 ± 0.81 %
C
8.2 ± 1.70 part/50 µm
2 C
NEW ONSET DIABETES
(BBZ/Wor)
69.6 ± 5.88 %
A
60.8 ± 16.91 part./50 µm
2 A
CHRONIC DIABETES
(BBZ/Wor)
49.9 ± 9.75 %
B
29.5 ± 4.31 part./50 µm
2 B
NON-DIABETIC
CONTROLS (BB DR
/Wor)
8.7 ± 4.66 %
C
9.0 ± 1.87 part./50 µm
2 C
A: p = 0.0001
B: p = 0.0001
Means with the same letter (A, B, C) are not significantly different.

Time Course of Vascular Changes in BBZDR/Wor
Rat With Duration of Hyperglycemia
Length of hyperglycemia 1 week 4-6 months 18-24 months
NADH oxidase
Free Radicals
iNOS
Nitrotyrosine
eNOS ¯ ¯ ¯
NOS cofactors - ¯ ¯
Vessel Diameter ¯ ¯

Isolated Intact Pressurized Resistance Arteries:
The heart of microvascular (patho) physiology
• Cerebral
• Coronary
• Renal
• Retinal, opthalmic

Inflow
T=37.4
outflow
The Arteriograph System:
Total control over the micro vascular environment
Pressure / flow
control
Open / close
micromanipulator

The Pressure Arteriograph System:
A controlled two cell system in native interaction

CONTROL
116 MICRONS
U73122
185 MICRONS
U73122+KCL
77 MICRONS
1.305 1.058 1.595
1.6
0.7
Quantitative Calcium Imaging (FURA-2)

10 mmHg
122 MICRONS
70 mmHg
108 MICRONS
130 mmHg
112 MICRONS
0.2
3.0
1.095 1.408 1.564
Quantitative Membrane Potential Imaging
(Di-8-ANNEPS)

0 0
-40 -47
-39
-40
-60
V
-60
-20 -20Arterial membrane
potential, mV:
Arterial diameter, µm:
video
edge
detection
Arterial wall [Ca ], nM:2+
60 mm Hg
105
189 245 68
205103
100 mm Hg
+
10 nM
Nisoldipine
100 mm Hg
8 1416 2012 18
Time (min) Time (min)V
Simultaneous Electrophysiology,
diameter and calcium Imaging

Long-term artery culture:
a physiological genomics platform
• Viral transduction
• Protein transduction
• Ribozyme, siRNA
• To VSM and/or ENDO

3-4 Weeks of DiabetesPre-diabetic age
0 25 50 75 100 125 150 175 200 225
0
20
40
60
Intraluminal pressure (mm Hg)
Myogenictone(%)
0 25 50 75 100 125 150 175 200 225
0
20
40
60
Intraluminal pressure (mm Hg)
Myogenictone(%)
0 25 50 75 100 125 150 175 200 225
0
20
40
60
Intraluminal pressure (mmHg)
Myogenictone(%)  Obese-Diabetic; O Lean control (age-matched)
0 25 50 75 100 125 150 175 200 225
0
20
40
60
Intraluminal Pressure (mmHg)
Myogenictone(%)
Characterization of Cerebral-arterial
Dysfunction in BBZ/Wor Rats

Endothelial Dysfunction
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone

Arterial smooth muscle hyper-reactivity
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
log[5-Hydroxy tryptamine] M
%KClConstriction
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
%KClConstriction
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
%KClConstriction
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
%KClConstriction

-9 -8 -7 -6 -5 -4
0
25
50
75
100
125
log[U-73122] M
%Myogenictone
-9 -8 -7 -6 -5 -4
0
25
50
75
100
125
log[U-73122] M
%Myogenictone
-9 -8 -7 -6 -5 -4
0
25
50
75
100
125
log[U-73122] M
%Myogenictone
-9 -8 -7 -6 -5 -4
0
25
50
75
100
125
log[U-73122] M
%Myogenictone
Arterial smooth muscle hyper-reactivity PLC activation

Autoregulation in rat (Sprague Dawley)
ophthalmic artery
0 30 60 90 120 150 180 210
100
150
200
250
300
Diameter with tone
Passive diameter
Myogenic tone
0
25
50
Intraluminal Pressure (mm Hg)
Lumendiameter(microns)
Myogenictone(%)

-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
Serotonin
Phenylephrine
Vasopressin
UK-14304
U-46619
log M [Vasoconstrictor]
%KCl(60mM)response
-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
0
50
100
150
log M [Endothelin-1]
%KCl(60mM)response
Effect of different vasoconstrictors
in ophthalmic artery
Order of efficacy:
Endothelin-1 > Vasopressin = Serotonin = Phenylephrine > U-46619 > UK 14304
Order of potency:
Endothelin-1 > Vasopressin = Serotonin > U-46619 > UK 14304 > Phenylephrine

-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
Histamine
Carbachol
Adenosine
CGRP
Isoprenaline
VIP
log M [Vasodilator]
Myogenictone(%)
Order of efficacy:
Carbachol > Isoprenaline > Histamine = CGRP = VIP = Adenosine
Order of potency:
CGRP > VIP > Carbachol = Isoprenaline > Adenosine = Histamine
Bradykinin, substance P and octreotide did not produce sustained dilatory responses
Effect of different vasodilators
in ophthalmic artery

-9 -8 -7 -6 -5 -4 -3
20
60
100
140
180
L-NAME
Indomethacin
log M [Inhibitor]
Myogenictone(%)
Effect of the inhibition of NOS
and prostaglandin synthesis on myogenic tone
in rat ophthalmic artery

CNS Controls Release of EPC’s
• Bone marrow derived epithelial
progenitor cells (EPCs) home to sites of
vascular injury and help to maintain
capillary integrity or re-perfuse acellular
capillaries
• The release of EPCs from bone marrow
follows a circadian rhythmicity in
response to local adrenergic signals
(Mendez-Ferrer et al. 2008). Circadian
rhythms are driven by a central clock
(suprachiasmaticnucleus, SCN) and
peripheral clocks (tissues)
• Clock genes induce circadian rhythm for EPC release by synchronous interplay of
positive (Clock, Bmal1) and negative (Per1, Per2, Cry1, Cry2) loops of the peripheral
circadian clock
J Busik et. al,. (2009) J. Exp. Med

Diabetes Decreases
Circadian Release of EPCs.

qPCR mRNA Expression
Analysis for Clock Genes

Decreased Adrenergic Function in T2D

Conclusions
• BBZ has most of the features of diabetic
retinopathy as observed in humans
• Evaluation of drug therapies can be
performed using
– Histological studies
– Immunological studies
– Functional studies using small vessels

A Model of Type 2 Diabetes: BBZDR/Wor rat

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