Regulation of liver regeneration by suppressors of cytokine signaling-3

S12

Ratio of rates (RR) ⌬Fluorescenceb Number of cells

Alimentary Tract I

a

J Am Coll Surg

Rotenone control

Rotenone (10uM)

FCCP control

FCCP (10uM)

Thapsigargin control

Thapsigargin (1uM)

Oligomycin control

0.83⫾0.11

1.29⫾0.29ⴱ

0.80⫾0.06

3.18⫾1.17ⴱ

1.00⫾0

1.08⫾0.10

1.00⫾0

0.23⫾0.084 67

0.48⫾0.16ⴱ 41

0.29⫾0.05 92

0.74⫾0.13ⴱ 84

0.16⫾0.05 28

0.09⫾0.04 40

0.18⫾0.08 24

Oligomycin (5ug/mL)

RU360 control

RU360 (10uM)

2.1⫾0.47ⴱ

0.80⫾0.14

0.67⫾0.14

0.41⫾0.12ⴱ 55

0.23⫾0.07 31

0.26⫾0.08 49

a

RR represents the ratio of the rate of fluorescence change 10 minutes after inhibitor addition to rate of fluorescence change 10 minutes prior to inhibitor addition. b ⌬Fluorescence represents the difference in absolute fluorescence intensity at the end of inhibitor exposure and at the beginning of inhibitor addition. ⴱindicates p⬍0.05 compared to control

RESULTS: Linear increases in Fluozin3 fluorescence were observed during [Zn2⫹]o (60uM) exposure. When inhibitors were introduced at 10min (during 20min exposure), changes in rate (RR) and absolute change (delta-f ) of Zn2⫹ accumulation were significantly accelerated during exposure to rotenone, FCCP, and oligomycin but not thapsigargin or RU360.

CONCLUSIONS: Socs3 acts as a critical negative regulator of liver regeneration by preventing prolonged STAT3 activation. Loss of Socs3 enhances hepatocyte proliferation, indicating that Socs3 may act as a tumor suppressor gene in the liver.

CONCLUSIONS: Buffering of intracellular Zn2⫹ accumulation depends on mitochondrial electron transport proton gradient and mitochondrial ATP production but is influenced by disturbances in intracellular Ca2⫹ movements.

Insulin administration to insulin-resistant mice worsens in vivo gallbladder emptying

Regulation of liver regeneration by suppressors of cytokine signaling-3 Kimberly J Riehle MD,* ,** Ben Croker PhD, Jean Campbell PhD, Nelson Fausto MD University of Washington, Seattle, WA INTRODUCTION: We have previously shown that suppressor of cytokine signaling 3 (SOCS3) is induced by an IL-6-dependent pathway after partial hepatectomy (PH) in mice. Here we tested the hypothesis that SOCS3 is an important regulator of liver regeneration using hepatocyte-specific Socs3 knockout (Socs3 h-KO) mice. METHODS: We performed 2/3 PH on Socs3 h-KO mice and control littermates, and evaluated liver regeneration by progression through the cell cycle and mass restitution. Serum IL6 levels (ELISA) and STAT3 activation (phosphoimmunoblotting) were also determined, and differential gene expression investigated by cDNA microarrays. Primary hepatocytes were isolated from Socs3 h-KO and control mice, and adenoviruses expressing wt-Socs3, shRNA-Socs3, and shRNA-Stat3 were used to manipulate cytokine signaling pathways and hepatocyte proliferation in vitro. RESULTS: Liver regeneration is markedly enhanced in Socs3 h-KO mice, as shown by increased BrdU incorporation, mitotic indices, and expression of cell cycle proteins, and an earlier restoration of liver mass after PH. IL6 secretion is unaltered in Socs3 h-KO mice, but activation of STAT3 and the expression of STAT3 target genes are significantly prolonged. Socs3-deficient hepatocytes hyperproliferate in culture, with and in the absence of growth factor stimulation, again in association with enhanced STAT3 activation. Manipulation of Socs3 or STAT3 expression by shRNA accordingly affects hepatocyte proliferation.

Hayder H Al-Azzawi MD, Abhishek Mathur MD, Debao Lu MD, Deborah Swartz-Basile PhD, Attila Nakeeb MD, Henry A Pitt MD Indiana University, Indianapolis, IN INTRODUCTION: Obesity and diabetes are associated with an increased incidence of gallstones. Recent data suggest that insulin resistance is associated with increased gallbladder volume and/or impaired gallbladder emptying. However, nothing is known about the effect of insulin on gallbladder emptying in insulinresistant animals. Therefore, we tested the hypothesis that injecting insulin to insulin-resistant obese mice would lower serum glucose, decrease resting gallbladder volume and improve in vivo gallbladder emptying. METHODS: 20 eight-week old insulin-resistant obese mice were fed a lithogenic diet for 4 weeks. 10 mice received daily intraperitoneal injections of NPH insulin (200 Unit/Kg). The remaining mice received an equal volume of saline intraperitoneally. At 7 and 11 weeks mice were fasted overnight and underwent an ultrasonography of the gallbladder to determine in vivo resting volume and residual volume after an IP injection of 1mmol/kg CCK. At 12 weeks cholecystectomies were performed, and in vitro gallbladder response to neurotransmitters was determined. RESULTS: Saline injections did not alter serum glucose or in vivo gallbladder ejection fraction. Insulin did not alter in vitro gallbladder responses. Results for serum glucose, resting volume, residual volume, and gallbladder ejection fraction in the insulin group were: Glucose (mg/dl)

Resting Residual Ejection Volume (ul) Volume (ul) Fraction (%)

At 7 weeks

200⫾17

30⫾3

At 11 weeks

109⫾14ⴱ

78⫾5ⴱ

1⫾0.3 14⫾5†

97⫾1 81⫾6†

ⴱ p⬍0.01 vs. 7 wks, †p⬍0.05 vs. 7 wks

CONCLUSIONS: These data suggest that in insulin-resistant diabetic mice daily insulin 1) decreases serum glucose, 2) increases resting and residual gallbladder volume, 3) decreases in vivo gallbladder ejection fraction, but 4) does not alter in vitro gallbladder contractility. Therefore, we conclude that insulin paradoxically increases