studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 375A
subjected to cold stress, Them1 was strongly localized to lipid
droplets. By contrast, Them1 in hepatocytes was visualized in
cytosol, and this was not influenced by high fat feeding. PKCβ
was the predicted kinase for phosphorylated sites (S15, S18,
S25) near the N-terminus of Them1. Activation of PKCβ using
phorbol 12-myristate 13-acetate led to the rapid redistribution
of Them1-EGFP in a pattern consistent with lipid droplet association.
The subcellular localization of Them1-EGFP was profoundly
disrupted by the deletion of a short peptide fragment
containing the phosphorylation sites. Conclusions: Our data
demonstrate that cellular localization of Them1 is tissue specific
and is regulated at least in part by phosphorylation. The tissue
specific control of Them1 localization may play a key role in
the pathogenesis of NAFLD.
Disclosures:
David E. Cohen - Advisory Committees or Review Panels: Merck, Aegerion,
Genzyme; Consulting: Intercept
The following authors have nothing to disclose: Yue Li, Susan J. Hagen
323
Longitudinal monitoring of hepatic endoplasmic reticulum
calcium changes in rats
Emily Wires, Mark Henderson, Kathleen Trychta, Brandon K. Harvey;
National Institute on Drug Abuse, Baltimore, MD
Obesity is a major health concern in developed countries; an
estimated 1.5 billion individuals are considered overweight
or obese worldwide. In stressed conditions, such as chronic
obesity, hepatocytes accumulate excess long chain fatty acids,
leading to the development of nonalcoholic fatty liver disease
(NAFLD), representing a spectrum of liver disorders. Moreover,
obese patients typically exhibit metabolic disorders, cardiovascular
disease and obesity-related mortality. While the exact
mechanisms of obesity-associated liver diseases are poorly
understood, disruptions to endoplasmic reticulum (ER) homeostasis
have been implicated in disease pathogenesis. The ER
contains the highest level of intracellular calcium, an estimated
1,000-10,000 fold greater than cytosolic levels. Perturbations
to ER calcium levels are associated with a variety of pathologies,
and have been predicted as a contributing factor to
the development and progression of obesity-associated disorders.
Recently, our lab developed a novel secreted ER calcium
monitoring protein (SERCaMP), to longitudinally monitor ER
calcium levels in vivo by measuring small volumes of blood;
a notable enhancement from calcium-specific fluorescent dyes
that become easily saturated in high levels of calcium and
are unable to be utilized for in vivo studies. In the present
study, rats expressing SERCaMP appended to Gaussia luciferase
(GLuc-SERCaMP) in the liver were fed highly palatable
foods, colloquially referred to as a cafeteria diet (e.g. cookies,
pepperoni, potato chips), to mimic dietary intake observed
in obese individuals. Additional groups included ad libitum
access to chow and restricted access. Rats exposed to cafeteria
diet items and ad libitum chow showed significant weight gain
when compared to restricted rats. Blood samples indicated
increased levels of circulating GLuc-SERCaMP in both cafeteria
diet and ad libitum chow, suggesting that a dietary intake, particularly
over eating, alters ER calcium homeostasis in the liver.
The half-life of GLuc-SERCaMP is estimated to be less than 10
minutes; eliminating the possible confound of sensor accumulation
in the blood. Our study is the first to use a secreted ER calcium
probe to demonstrate that dietary intake alters ER calcium
homeostasis in the liver. Furthermore, parallel work has been
done to monitor hepatic ER stress in order to establish a temporal
profile between ER calcium dysregualtion and ER stress.
Disclosures:
The following authors have nothing to disclose: Emily Wires, Mark Henderson,
Kathleen Trychta, Brandon K. Harvey
324
Mouse CD11b + Kupffer cells recruited from bone marrow
accelerate liver regeneration after partial hepatectomy
Hiroyuki Nakashima 1 , Kiyoshi Nishiyama 2 , Masahiro Nakashima 1 ,
Manabu Kinoshita 1 , Shuhji Seki 1 ; 1 Immunology and Microbiology,
National Defense Medical College, Saitama, Japan; 2 Surgery,
National Defense Medical College, Saitama, Japan
Background and Aims: Innate immune cells are known to
contribute to the liver regeneration after partial hepatectomy
(PHx). Although TNF and Fas/FasL are the vital components
for hepatocyte regeneration, the role of Kupffer cells is still in
controversy. Liver F4/80 + Kupffer cells are classified into two
subsets; resident radio-resistant CD68 + cells with phagocytic
and bactericidal activity, and recruited radio-sensitive CD11b +-
cells with cytokine-producing capacity. The aim of this study is
to investigate the role of these Kupffer cells in the liver regeneration
after PHx in mice. Material and Methods: C57BL/6 mice
were subjected to PHx (70%) or Sham operation. The proportion
of Kupffer cell subsets in the remnant liver was examined
by flow cytometry. To examine the role of CD11b + Kupffer cells,
mice were depleted of these cells before PHx by non-lethal 5 Gy
irradiation with or without bone marrow transplantation (BMT).
To investigate the mechanism of macrophage (Mφ) accumulation
into liver, mice were injected with CCR2 (MCP-1 receptor)
antagonist and liver regeneration was evaluated. Results: The
proportion of CD11b + Kupffer cells was greatly increased at
three days after PHx when the hepatocytes vigorously proliferate,
whereas the proportion of CD68 + Kupffer cells did not
significantly change after PHx. Serum TNF levels peaked one
day after PHx, and intracellular TNF staining revealed that
CD11b + Kupffer cells are the main source of TNF. Additionally,
the CD11b + Kupffer cells expressed surface FasL at three days
after PHx. Serum MCP-1 levels (produced by CD68 + resident
Kupffer cells) peaked at twelve hours after PHx. Irradiation eliminated
the CD11b + Kupffer cells for approximately two weeks
in the liver, while CD68 + Kupffer cells, NK cells and NKT cells
remained, and hepatocyte regeneration was retarded. However,
BMT partially restored CD11b + Kupffer cells and recovered
the liver regeneration. Furthermore, CCR2 antagonist
treatment decreased the CD11b + Kupffer cells and significantly
retarded liver regeneration. Conclusion: The CD11b + Kupffer
cells recruited from bone marrow via the MCP-1/CCR2 and
TNF and FasL produced by these cells play a pivotal role in the
liver regeneration after PHx. The possibility is raised that BMT
can be applied to patients with hepatocellular carcinoma after
partial hepatectomy for the acceleration of liver regeneration
and prevention of hepatic failure.
Disclosures:
The following authors have nothing to disclose: Hiroyuki Nakashima, Kiyoshi
Nishiyama, Masahiro Nakashima, Manabu Kinoshita, Shuhji Seki