iron loading in diabetic obese mice

1. A HIGH FAT DIET AND PARENTERAL IRON CAUSES
INCREASED INFLAMMATION AND OXIDATIVE STRESS
BUT LESS STEATOSIS IN AN OBESE, DIABETIC MOUSE
MODEL OF NAFLD
James E. Nelson1, Bryan Maliken1, Barjinder Dhillon1, Heather Klintworth1, Matthew M. Yeh2, Kris V. Kowdley 1,2
1Center for Liver Disease, Digestive Disease Institute, and Benaroya Research Institute, Virginia Mason Medical Center Seattle WA. 2Dept of
Pathology, University of Washington Medical Center, Seattle WA
METHODS
Leprdb/db mice were fed either a 71% high-fat diet (61% unsaturated fat) or normal chow
for 8 weeks.
A subset of mice on both diets were administered a single dose of 1.25 mg/g wt Fe-
dextran by IP injection at the start of the study.
Histological features of NASH and iron deposition were scored using NASH CRN
scoring criteria.
Malondialdehyde (MDA), iron and hydroxyproline were assessed in liver tissue.
Liver and adipose tissue were collected for expression of proinflammatory, lipid and
oxidative stress genes, determined by RT-PCR, normalized to GAPDH mRNA
0
0.5
1
1.5
2
2.5
3
3.5
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
MeanScore
Steatosis (0-3)
#
0
0.5
1
1.5
2
2.5
3
3.5
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
MeanScore
Lobular Inflammation (0-3)
†#†#
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
MeanScore
Hepatocellular Ballooning (0-2)
0
1
2
3
4
5
6
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
NAFLD Activity Score (0-8)
†#
† p<0.05 vs Normal Chow; # p<0.05 vs HFD
High Fat Diet (20x) High Fat Diet + Iron (20x)
0
0.5
1
1.5
2
2.5
3
3.5
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
RelativeIL6/GAPDH
mRNA
Hepatic IL6 expression
†#
0
0.5
1
1.5
2
2.5
3
3.5
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
RelativeTNFα/GAPDH
mRNA
Hepatic TNFα expression †#
† p<0.05 vs Normal Chow; # p<0.05 vs HFD
0
0.5
1
1.5
2
2.5
3
3.5
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
RelativeSrebp1c/GAPDH
mRNA
Hepatic Srebp1c
†#
#
0
2000
4000
6000
8000
10000
12000
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
HICµgiron/glivertissue
Hepatic Iron Concentration
†#
†#
0
1
2
3
4
5
6
7
8
9
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
RelativeHAMP/GAPDHmRNA
Hepatic HAMP expression
†#
†
BACKGROUND
Iron is thought to contribute to NAFLD progression by increasing oxidative stress as a
catalyst for the production of reactive oxygen species. Iron may also potentiate
progression of NAFLD by altering insulin signaling and lipid metabolism. The aim of this
study was to examine the effects of parenteral iron loading in an obese, diabetic murine
model of NAFLD.
FIGURE 1: Histology Scores
FIGURE 2: Liver Histology and Iron Staining
H&E stain
Iron stain
FIGURE 3: Relationship of Iron and Hepcidin Expression
0
0.5
1
1.5
2
2.5
3
3.5
4
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
RelativeHO-1/GAPDHmRNA
Hepatic Heme Oxygenase-1
Expression
†#
†#
0
10
20
30
40
50
60
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
IronMDA(umoles/gtissue)
Hepatic Malondialdehyde †#
†#
0
2
4
6
8
10
12
Normal
Chow
HFD Parenteral
Iron
HFD
+Parenteral
Iron
ugHP/gramtissue
Hepatic Hydroxyproline Assay
†#
†
† p<0.05 vs Normal Chow; # p<0.05 vs HFD
FIGURE 5: Relationship of Iron, Oxidative Stress
and Collagen Production
FIGURE 4: Relationship of Iron and Inflammatory and Lipid Gene
Expression
B
CONCLUSIONS
PI administration causes a strong inflammatory response
PI administration leads to KC activation and phagocytosis of iron
PI caused decreased steatosis
PI induces oxidative stress and initiates fibrogenesis
Combination of HFD and PI led to more inflammation, oxidative stress and
collagen production than either iron or HFD alone