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VOL. 76 (1), 23-44, 2010 BETA-CELL HYPERPLASIA INDUCED BY HEPATIC INSULIN...

2.4. Western blot analysis

Tissues were homogenized as described (11). Western blot
analyses of insulin signaling proteins were performed as described
(13). The antibodies used were anti-IRß (Ab-4) from Oncogene
(San Diego, CA), anti-PEPCK antibody was kindly provided by
Dr. DK. Granner (Vanderbilt University, TN), Anti-Glucokinase
antibody was a generous gift of Dr. S. Lenzen (Hannover Medical
School, Germany), anti-FAS antibody was purchased from BD
Transduction Laboratories (San Diego, CA) anti-ß-actin antibody
was from Sigma-Aldrich Corp. (St. Louis, MO). Anti-IGF-1 antibody
was purchased from Upstate Biotechnology (Lake Placid, NY).
Anti-phospho-p70-S6-Kinase (Thr421/Ser424), anti-phospho-p44/
p42-MAPK (Thr202/Tyr204), anti-phospho-Akt (Ser473) antibodies
were purchased from Cell Signaling (Beverly, MA). For in vivo insulin
signaling studies, mice were intraperitoneally injected with 1 U/kg
body weight of human insulin (Novo Nordisk, Denmark). After 10
minutes, the tissues were removed and frozen in liquid nitrogen.
Densitometric analysis of the autoradiograms was performed using
a GS-710 Imaging Densitometer and the Quantity One software (Bio-
Rad Laboratories, CA).

2.5. Analytical procedures

Insulin levels in serum were measured by radioimmunoassay (RIA)
using mouse insulin as a standard (Linco Research, MO). IGF-1 levels
in serum were measured by RIA using mouse IGF-1 as a standard
(Diagnostic Systems Laboratories, TX). Glucose tolerance and insulin
tolerance tests were performed as previously described (14).

2.6. Histological analysis

The immunohistochemical analysis of pancreata was preformed
as described (14). ß cell mass was evaluated by point counting
morphometry (15, 16). Staining of liver sections with hematoxylin/
eosin, periodic Acid-Schiff (PAS) and Masson reagents was
performed using standard techniques.

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