Ahlke Heydemann University of Illinois at Chicago Physiology and Biophysics ahlkeh@uic

Ahlke HeydemannUniversity of Illinois at Chicago

Physiology and [email protected]

The MRL mouse strain is naturally resistant to high fat diet-induced

hyperglycemia

MRLC57

day 3

3d

ay 2

0d

ay 9

day 1

Clark 1998

The super healing wild type MRL mouse

1) Heal ear punches used for identification

Heal all tissues; cartilage, blood vessels, glands, hair follicles, nerves

2) No fibrosis when breed to have muscular dystrophy

Mouse tissues stained with Masson’s trichrome at 8 weeks old. Blue represents fibrotic scar tissue. Very rare in the MRL mice on the right.

Heydemann 2012

Mull 2014

B6 CD B6 HFD MRL CD MRL HFD0

5

10

15

20

25

30

35

40

45

Perc

ent b

ody

fat m

ass

p<0.01

MRL HFDMRL CD

B6 CD B6 HFD

p<0.05

spinespine

spine spine

fem

ur

femur

femur

femur

3) The MRL and B6 mice gain equal amounts of fat and weight.

When put on a 12 week high fat diet. Fat appears white in dual energy X-ray (top). Quantified in the bottom panel.

But they are…

Mull 2014

4) Resistant to HFD mediated type 2 diabetes pathology

When challenged with a glucose bolus the HFD MRL mice cleared the glucose quickly, like the control diet mice.


100

200

300

400

500

600

700

AU

C 1

.5 h

our

blo

od g

lucose

Mull, 2014

p<0.0001

p<0.0001

The MRL mice

5) Are also susceptible to autoimmune diseases.

6) Contain 2 mitochondrialheteroplasmies (Sachadyne2008)

Serum analysis of 6hour fasted animals at end of 12 week

diets


0.2

0.4

0.6

0.8

1

1.2

1.4

1.6In

sulin

ng/

ml

P=0.001

A


1

2

3

4

5

6

7

adip

onec

tin n

g/m

l

Bp<0.0001

p<0.01

p<0.0005


0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

free

fatt

y ac

id p

Mol

es/u

l

C p<0.05

Mull, 2014

Mull, 2014


0.5

1

1.5

2

2.5

3

3.5

tric

ep p

AMPK

arbi

trar

y un

its


0.5

1

1.5

2

2.5

3

3.5

quad

ricep

s pAM

PKar

bitr

ary

units

p<0.001

p<0.0001

C

D

pAMPK quantitation of 6 hour fasted

animals

Mull, 2014

Quadriceps Tissue

AMPK

pAMPK

B6 MRL

CD CDHFD HFD

pAMPK

Rel

ativ

e p

AM

PK


5

10

15

20

p=0.0194 AMPK

Rel

ativ

e A

MP

K


5

10

15

20

25p=0.0194

Liver tissue of 6 hour fasted animals at end of 12 week diets

Glycogen Assay

um

ole

s g

luc

os

yl

un

its

pe

r g

ram

we

t w

eig

ht

B6 CD B6 HFD MRL CD MRL HFD D2 CD D2 HFD0

20

40

60

80

p=0.0029p=0.0191

Liver Glycogen Assayafter 12 week diet, 6 hour fast

B6 CD (n=8)

B6 HFD (n=18)

MRL CD (n=7)

MRL HFD (n=15)

0

1

2

3

4

5

6

7

8LV

Mas

s /

Tibi

a Le

ngth

p=0.015

Heart size

B6 CD B6 HFD MRL CD MRL HFD

pAM

PK (a

rbita

ry u

nits

)


AMPK

(arb

itary

uni

ts)

*

*

Cardiac; pAMPK comparison (n=9)

* p<0.05

**


HK

II (

arb

itary

un

its)

***

***

*

Cardiac; HKII comparison (n=6)

* p<0.05, *** p<0.0005


GLU

T4 (a

rbitr

ary

units

)Cardiac; Glut 4 comparison (n=3)

*

*

* p<0.05

CD B6

HFD MRLCD MRL

HFD B6

** +

+

Heart

MRL mitochondrial heteroplasmies

• One mitochondria and cell containing more than one mitochondrial genome.

• MRL mice have 2; T3900C and an adenine tract length insert (Sachydyn 2008).–MRL cytosine at 3900–MRL more than 8 adenines in the tract

• Inherited from the mother• Inherited independently• Polymorphisms in tRNAMet and tRNAArg

Quadriceps Heart Diaphragm Abdominals0

5

10

15

20

25

D2

MRL/D2 0%MRL mito

MRL/D2 100%MRL mito

hyd

roxyp

rolin

e n

M/m

g t

issu

eMuscular dystrophy

fibrosis

6 7 8 9 10 11 12 130

0.5

1

1.5

2

Remaining ear wound sizeand adenine tract length

CD MRL, p<0.05

Adenine tract length

mm

2

10 20 30 40 50 60 70 80 90 100200

220

240

260

280

300 AUC weekly weightAnd % cytosines, HFD MRL, p<0.01

% Cytosine at 3900

wei

ghts

(g x

wee

k)

0 10 20 30 40 50 60 70 80100

120

140

160

Average weekly blood glucose and % cytosines, HFD MRL, p<0.02

% Cytosine at 3900

we

ek

ly f

asti

ng

BG

, m

g/d

L

0 10 20 30 40 50 60 70 80 90 10050

100

150

200Average blood glucose post ITT

and % cytosines, CD MRL, p<0.01

% Cytosine at 3900

2 ho

ur a

verg

ae b

lood

glu

cose

Mitochondrial DNA Correlations

Four pure-breeding resultant mouse lines;nucMmitoM Sgcg +/-nucMmitoD Sgcg +/-nucDmitoM Sgcg +/-nucDmitoD Sgcg +/-

Early generation sib-crosseswild type mice; ear wound healing (2mm ear punch)HFD, metabolic profile *Sgcg-/- mice; Muscular Dystrophy panel ** genome wide association studies with mitochondrial content as a covariate

Late generation sib-crosseswild type mice; ear wound healing HFD, metabolic profileSgcg-/- mice; Muscular Dystrophy panel

Male FemaleP1 D Sgcg-/- x MRL

F1 nucM50D50mitoM, Sgcg+/- x MRL

N1 (nucM75D25mitoM, Sgcg+/-)2 xMRLN2 nucM88D12mitoM, Sgcg+/- x MRL

N3 nucM94D6mitoM, Sgcg+/- x MRL

N4 (nucM97D3mitoM, Sgcg+/-)2 x MRL

Current andfuture plans

Tirsit K. BerhanuNathan W. Roberts

Aaron J. MullJenan Holley-Cuthrell

Magdalis Vega-Gonsalez

ConclusionsWild type MRL mice can heal ear wounds, heal cardiac

cryo-injuries, resist muscular dystrophy-mediated fibrosis, resist high fat diet-mediated type 2 diabetes.

Multiple pieces of data indicate MRL-introducedmitochondrial heteroplasmies beneficially alter metabolism.

Mull, Figure 2


1015202530354045

Fat m

ass/

Body

mas

s %p<0.01

p<0.05

femur

MRL HFDMRL CD

B6 CD B6 HFD spinespine

spine spine

femur

femur

femur

Rel

ativ

e p

AM

PK

/AM

PK

Rat

io


1

2

3

4

pAMPK/AMPK Ratio

p=0.0192

p=0.0252

Upstream stimulatory factors and downstream targets of AMPK in the heart.

Beauloye C et al. Cardiovasc Res 2011;90:224-233

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Ahlke Heydemann University of Illinois at Chicago Physiology and Biophysics ahlkeh@uic