Supporting Materials and Methods:
Partial Hepatectomy and Carbon Tetrachloride (CCl4) Treatment: Mice were subjected to twothirds partial hepatectomy using standard methodology as described previously (1-4). First, they
were sedated with inhaled Isoflurane (VEDCO, Inc., St. Joseph, MO) via anesthesia vaporizer,
then subjected to mid-ventral laparotomy with exposure, ligation and resection of the left and
median hepatic lobes, and closure of the peritoneal and skin wounds. Some animals were
treated with leptin supplementation using a regimen shown to rescue impaired regeneration in
ob/ob mice (see below and (5)); some were subjected to one-third partial hepatectomy, in which
only the median lobes of the liver were resected; and some were treated with one dose of CCl4
(2 l/g of 50% solution in mineral oil) or vehicle. At serial times after surgery or CCl4 treatment
animals were sacrificed by inhaled CO2 and plasma and liver tissue were harvested. Very little
morbidity or mortality occurred in experimental animals (summarized below). Blood glucose
was determined immediately prior to sacrifice as described (6). Three or more animals were
examined at each time point for each genotype, surgical, and treatment group. All experiments
were approved by the Animal Studies Committee of Washington University and conducted in
accordance with institutional guidelines and the criteria outlined in the “Guide for Care and Use
of Laboratory Animals” (NIH publication 86-23).
Leptin Supplementation: Some mice were treated twice daily with 50 g/kg recombinant mouse
leptin (R&D Systems, Minneapolis, MN) or vehicle (20 mM Tris-HCl, pH 8) by subcutaneous
injection beginning 12 hours prior to partial hepatectomy and continued through the time of
animal sacrifice.
Morbidity and Mortality after Partial Hepatectomy or CCl4 Treatment: A total of 5 out of 162
operated mice (4/60 C57Bl6/J, 0/60 +/+ or fld/+, and 1/42 fld/fld) exhibited morbidity (e.g.
decreased activity, tremor, pilorection prior to harvest and/or hepatic tissue necrosis at harvest)
and these animals were excluded from further analyses. Mortality after partial hepatectomy
occurred in 4 mice including 1/60 C57Bl6/J, 0/60 +/+ or fld/+ mice, and 3/42 fld/fld mice.
Mortality after CCl4 administration occurred in ¼ fld/+ and 4/4 fld/fld mice (see Results).
Histology and Immunohistochemistry: Liver histology and hepatocellular bromodeoxyuridine
(BrdU) incorporation were assessed as previously described (2;3). Briefly, animals were
injected with 100 mg/kg BrdU 1 hour prior to sacrifice. Formalin-fixed, paraffin-embedded liver
tissue was stained either with hematoxylin and eosin (H&E) or for nuclear BrdU incorporation.
Hepatocellular nuclear BrdU labeling and mitoses were quantified by examination of at least
three random 400x fields and at least 300 cells and nuclei in each tissue section.
Protein Expression Analysis: Tissue lysates were made and protein immunoblot studies
conducted as described previously (1;3).
Primary antibodies for these analyses included those
directed against STAT3, phospho-STAT3, p21 and p27 (Cell Signaling Technology, Beverly,
MA); adiponectin (Sigma, St. Louis, MO), and Cyclin D (Millipore, Burlington, MA).
Semiquantitative Real-Time RT-PCR Gene Expression Analysis: Total RNA was prepared from
mouse liver tissue, reverse-transcribed to cDNA, and analyzed for levels of expression of
specific genes of interest using real-time reverse-transcriptase polymerase-chain-reaction (RTPCR) as described previously (2). For each gene analyzed, an aliquot of cDNA was added to a
reaction mixture containing gene-specific forward and reverse primers, deoxynucleotides, Taq
DNA polymerase, and SYBR (Agilent, Santa Clara, CA). Quantification of cDNA was based on
monitoring increased SYBR fluorescence during exponential phase amplification in a Real-Time
PCR Machine (Agilent), and determination of the PCR cycle number at which the amplified
product exceeded a defined threshold (the ‘crossing threshold’). These data were also
standardized to the expression of 2-microglobulin, which is commonly used as a reference
gene in analyses of regulation of gene expression during liver regeneration (7-10), and exhibits
an expression pattern homologous to other genes used for this purpose (e.g. -actin (11) and
glyceraldehyde-3-phosphate dehydrogenase (12)). These standardized data were used to
calculate fold-differences in gene expression. Specificity of this assay was verified for each
gene under analysis by confirmation of predicted product size and uniformity using melt-curves
and agarose-gel electrophoresis of the PCR products. Specificity was further confirmed by
simultaneous analysis of a “reverse-transcribed” reaction mixture containing all components
except reverse transcriptase.
Gene specific primers: 2-microglobulin: forward 5’-TGGCTGCTTCTTTCGATTTCTG-3’,
reverse 5’-CCAGAAAACCCCTCAAATTCAAG-3’; cyclin D1: forward 5’GAAGGAGACCATTCCCTTGA-3’, reverse 5’- GTTCACCAGAAGCAGTTCCA-3’; C/EBP:
forward 5’-CCTGAGAGCTCCTTGGTCA-3’, reverse 5’-GAAACCATCCTCTGGGTCTC-3’;
C/EBP forward 5’-ACGACTTCCTCTCCGACCT-3’, reverse 5’GAGGCTCACGTAACCGTAGTC-3’.
Analysis of Body Composition: Total body fat and lean mass were determined by magnetic
resonance spectroscopy using a calibrated EchoMRI instrument (Echo Medical Systems,
Houston, TX) according to the instrument manufacturer’s instructions. The EchoMRI device
consists of a permanent magnet, transmitter/antenna, electronics instrumentation, and software,
and permits the direct evaluation of fat and lean tissue.
Hepatic Triglyceride Determination: Hepatic triglyceride content was determined by the
Washington University Medical School Nutrition Obesity Research Center Unit using Infinity
reagents (Thermo Scientific, Waltham, MA) according to manufacturer’s instructions.
Plasma Leptin and Insulin Determination: Plasma leptin and insulin levels were determined by
enzyme-linked-immunoassay (Millipore, Burlington, MA) according to manufacturer’s
instructions.
Reference List
1. Rudnick DA, Perlmutter DH, Muglia LJ. Prostaglandins are required for CREB activation
and cellular proliferation during liver regeneration. Proc Natl Acad Sci U S A 2001;
98(15):8885-8890.
2. Shteyer E, Liao Y, Muglia LJ, Hruz PW, Rudnick DA. Disruption of hepatic adipogenesis is
associated with impaired liver regeneration in mice. Hepatology 2004; 40(6):1322-1332.
3. Liao Y, Shikapwashya ON, Shteyer E, Dieckgraefe BK, Hruz PW, Rudnick DA. Delayed
hepatocellular mitotic progression and impaired liver regeneration in early growth
response-1-deficient mice. J Biol Chem 2004; 279(41):43107-43116.
4. Clark A, Weymann A, Hartman E, Turmelle Y, Carroll M, Thurman JM et al. Evidence for
non-traditional activation of complement factor C3 during murine liver regeneration. Mol
Immunol 2008; 45(11):3125-3132.
5. Leclercq IA, Field J, Farrell GC. Leptin-specific mechanisms for impaired liver regeneration
in ob/ob mice after toxic injury. Gastroenterology 2003; 124(5):1451-1464.
6. Weymann A, Hartman E, Gazit V, Wang C, Glauber M, Turmelle Y et al. p21 is required
for dextrose-mediated inhibition of mouse liver regeneration. Hepatology 2009; 50:207215.
7. Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V et al. Liver
failure and defective hepatocyte regeneration in interleukin-6- deficient mice. Science
1996; 274(5291):1379-1383.
8. Yamada Y, Kirillova I, Peschon JJ, Fausto N. Initiation of liver growth by tumor necrosis
factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor.
Proceedings of the National Academy of Sciences of the United States of America 1997;
94(4):1441-1446.
9. Yamada Y, Webber EM, Kirillova I, Peschon JJ, Fausto N. Analysis of liver regeneration in
mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not
type 2 receptor [comment]. Hepatology 1998; 28(4):959-970.
10. Greenbaum LE, Li W, Cressman DE, Peng Y, Ciliberto G, Poli V et al. CCAAT enhancerbinding protein beta is required for normal hepatocyte proliferation in mice after partial
hepatectomy. Journal of Clinical Investigation 1998; 102(5):996-1007.
11. Li W, Liang X, Kellendonk C, Poli V, Taub R. STAT3 contributes to the mitogenic response
of hepatocytes during liver regeneration. Journal of Biological Chemistry 2002;
277(32):28411-28417.
12. Sakamoto T, Liu Z, Murase N, Ezure T, Yokomuro S, Poli V et al. Mitosis and apoptosis in
the liver of interleukin-6-deficient mice after partial hepatectomy. Hepatology 1999;
29(2):403-411.