Diabetes Research and Clinical Practice - Volume 120 - Supplement 1 - October 2016

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inflammation. Here we investigate the role of TLR4 knockout
(TLR4KO) on insulin resistance, glucose intolerance, mito-
chondrial reactive oxygen species (ROS), oxidative stress,
mitochondrial biogenesis in VF using a high fat high sugar
(HFHS) diet-induced obesity mouse model. C57BL6 (B6) and
TLR4KO mice were fed with either control diet (CD) or HFHS
for six months, totally four experimental groups: B6 + CD,
B6 + HFHS, TLR4KO + CD and TLR4KO + HFHS. Compared to
B6 + CD, B6 + HFHS demonstrated significant increase in body-
weight (BW), VF accumulation, VF oxidative damage, VF
mitochondrial ROS level, VF inflammation markers and
development of insulin resistance as well as glucose intoler-
ance. TLR4KO + CD did not show differences in all physio-
logical and biomarker measurements from B6 + CD. In
contrast, TLR4KO + CD showed markedly increased BW and
subcutaneous fat (SF), but no difference in VF compared to
B6 + CD. On the other hand, TLR4KO + HFHS mice presented
significant improvement in VF oxidative damage and VF
mitochondrial ROS, insulin resistance and glucose intolerance,
as compared to B6 + HFHS. The TLR4KO + HFHS mice also
presented increased BWas compared to B6 + HFHS. Notably, SF
contributes higher proportion than VF in the increase of BWof
TLR4KO + HFHS mice. In addition, TLRKO hindered HFHS-
induced increasing mtDNA content in VF over time. Also,
TLR4KO mice exhibited decreased HFHS-induced inflamma-
tory markers in VF. Taken together, despite showing higher
BW, abrogation of TLR4 genemitigates obesity-induced insulin
resistance and glucose intolerance via reducing mitochondrial
ROS level, which is associated with induction of mitochondrial
biogenesis and inflammation in VF. Thus, our study provides a
critical insight in linking innate immunity to prevention of
insulin resistance. That paves the way to developing novel
therapeutic strategy for diabetes mellitus.
OL02-7
Interaction of TET-1 and OGT enzymes regulates epigenome
modification and high-glucose induced renal proximal tubular
cell injury
Tusty-Jiuan HSIEH1,2, Yen-Chu CHEN1, Pei-Hsuan HUNG 1,
Shyi-Jang SHIN 2,3*. 1Graduate Institute of Medicine, College of
Medicine, Kaohsiung Medical University, 2Lipid Science and Aging
Research Center, Kaohsiung Medical University, 3Division of
Endocrinology and Metabolism, Department of Internal Medicine,
Kaohsiung Medical University Hospital, Kaohsiung Medical
University, Taiwan
Diabetic kidney disease is a leading cause of chronic kidney
disease (CKD) and end-stage renal disease (ESRD) in Taiwan
and worldwide. Currently, available therapies have not been
fully effective in the treatment of CKD and ESRD, suggesting
that further understanding of the molecular mechanisms
underlying the pathogenesis of diabetic nephropathy (DN) is
necessary. Epigenetic mechanisms may underlie the renal cell
injury and the progression of diabetic kidney disease. Aberrant
DNA methylation has been observed in the renal proximal
tubules of diabetic mice. However, the mechanism is not fully
understood. Ten-eleven translocation (TET) enzymes can
oxidize the 5-methylcytosine (5-mC) of DNA to generate 5-
hydroxymethylcytosine (5-hmC) and dynamically regulate
global or locus specific 5-mC and 5-hmC levels by facilitating
active DNA demethylation. TETs are also found to interact
with O-GlcNAc transferase (OGT) for regulating histone
modification. Thus far, the function of TETs in kidney has
not been investigated. We hypothesize that high-glucose may
influence DNA demethylation and histone modification via
TETs in renal proximal tubular cells. In this study, we
demonstrated that 5-mC was decreased and 5-hmC was
increased in a time-dependent manner, indicating an active
DNA demethylation occurred in the human renal proximal
tubular HK-2 cells under 25 mM D-glucose stimulation. By
real-time PCR, we observed TET-1 mRNA was significantly
upregulated after high glucose stimulation. These results are
consistent with the immunohistochemistry data that showed
TET-1 proteinwas upregulated in the renal proximal tubules of
db/db mice. We also found methylation level of histone 3
lysine 4 (H3K4me) was increased in high-glucose stimulated
HK-2 cells and in renal proximal tubules of db/db mice. After
treated the HK-2 cells with TET-1 or/and OGT siRNA, we found
increase in high-glucose induced H3K4me and 5-hmC were
reversed. In addition, TET-1 and OGT could be co-immuno-
precipitated with either TET-1 or OGT antibody in HK-2 cells
treated with HG. The result suggests these two proteins may
have interaction in response to glucose stimulation. TET-1 and
OGT siRNA downregulated cleaved-caspase-3 protein level
that suggests an interaction of TET-1 and OGT may involve in
regulating tubular cell apoptosis induced by high glucose. Our
study reveals TET-1 may be a novel pathological molecule in
modifying epigenome in diabetic kidney disease.
OL02-8
Astaxanthin prevents and reverses insulin resistance and
steatohepatitis: A comparison with vitamin E
Liang XU1, Yinhua NI1, Mayumi NAGASHIMADA 1, Fen ZHUGE1,
Naoto NAGATA1, Shuichi KANEKO1, Tsuguhito OTA 1. 1Brain/
Liver Interface Medicine Research Center, Kanazawa University,
Kanazawa, Japan
Objective: Nonalcoholic steatohepatitis (NASH) and insulin
resistance frequently coexist in subjects with obesity and type
2 diabetes. Hepatic insulin resistance and NASH could be
caused by excessive hepatic lipid accumulation and peroxida-
tion. In our previous study, we developed a cholesterol- and
saturated fatty acid-induced model of lipotoxic NASH and
revealed that hepatic oxidative stress and insulin resistance
promoted hepatic inflammation and fibrosis. To date, vitamin
E has become a standard treatment for NASH. However,
additional more effective therapies are needed. Astaxanthin
is a carotenoid compound that is known to be approximately
500 times more potent in inhibiting lipid peroxidation than
vitamin E in vitro. In this study, we compared the preventative
and therapeutic effects of lipophilic antioxidants, astaxanthin
and vitamin E, in a lipotoxic model of NASH.
Methods: C57BL/6 mice were fed a high-cholesterol high-fat
(CL) diet or CLdiet eithercontaining 0.02%astaxanthin (CL + AX)
or 0.02% vitamin E (CL + VE). Liver histology, insulin sensitivity,
and intrahepatic immune cell numbers were examined.
Results: After 12 weeks on the CL diet, astaxanthin treatment
alleviated excessive hepatic lipid accumulation by reducing
hepatic TG, TC, and NEFA by 25%, 24%, and 31%, compared
that of 12%, 10%, and 23% by vitamin E. Although both of
astaxanthin and vitamin E suppressed lipid peroxidation
assessed by TBARS equivalently, by 37% and 33%, astaxanthin
reduced the accumulation of Kupffer cells and inhibited the
activation of hepatic stellate cells and fibrogenesis (hydro-
xyprolin content 10.4 ± 0.7 vs 7.1 ± 0.5 nmol/mg protein,
P < 0.05), in the liver of NASH to extents greater than did
vitamin E. Flow cytometry analysis revealed that CL + AX
and CL + VE mice exhibited a 56% and 33% reduced CD11c +
CD206−(M1) macrophages, respectively, whereas the number
of CD11c−CD206 + (M2)macrophageswas increased by 3.7- and
1.5-fold, respectively. These effects resulted in an M2-domin-
ant shift of macrophages/Kupffer cells in the livers of both
CL + AXandCL + VEmice, witha reductionof hepatic CD4 + and
CD8+T cell recruitment, which contributed to improved insulin
resistance and steatohepatitis. Importantly, astaxanthin
reversed insulin resistance as well as hepatic inflammation
and fibrosis, in pre-existing NASH more potently than did
vitamin E.
Conclusions: Overall, astaxanthin was more effective at
preventing and treating NASH compared with vitamin E in
mice, suggesting that astaxanthin might be a novel and
promising treatment for NASH.
Oral Presentations / Diabetes Research and Clinical Practice 120S1 (2016) S40–S64 S44