Effects of Taurine Supplementation Upon Food Intake and Central Insulin Signaling in Malnourished Mice Fed on a High-Fat Diet - Advances in Experimental Medicine and Biology - page 92

10.2.4 Intraperitoneal Glucose Tolerance Tests

For intraperitoneal glucose tolerance tests (ipGTT), blood glucose levels (time 0)
were measured in overnight fasted mice using a glucose analyzer (Accu-Chek
Advantage, Roche Diagnostic, Switzerland). A glucose load of 2 g/Kg body weight
was then administered by ip injection and additional blood samples were collected
at 15, 30, 60, and 120 min.

10.2.5 Western Blot

For protein expression experiments, after 12h of fasting the hypothalamus from all
mice groups were removed and immediately homogenized in buffer containing 100
mmol/L Tris pH 7.5, 10 mmol/L sodium pyrophosphate, 100 mmol/L sodium
fluoride, 10 mmol/L EDTA, 10 mmol/L sodium vanadate, 2 mmol/L PMSF, and 1%
Triton X-100. The extracts were then centrifuged at 12,000 rpm at 4°C for 40 min
to remove insoluble material. The protein concentration was assayed using the
Bradford dye method (Bradford, 1976), using BSA as a standard curve and Bradford
reagent (Bio-Agency Lab., São Paulo, SP, BRA). For SDS gel electrophoresis and
Western blot analysis, the samples were treated with a Laemmli sample buffer con-
taining dithiothreitol. After heating to 95°C for 5 min, the proteins were separated
by electrophoresis (70 mg protein/lane, 10% gels). Following electrophoresis, pro-
teins were transferred to nitrocellulose membranes. The nitrocellulose filters were
treated overnight with a blocking buffer (5% nonfat dried milk, 10 mmol/L Tris, 150
mmol/L NaCl, and 0.02% Tween 20) and were subsequently incubated with a poly-
clonal antibody against pAkt (1:1,000, cat. sc-7985R, Santa Cruz Biotechnology),
Akt (1:1000, cat. sc-8313, Santa Cruz Biotechnology), pIRS1 (1:1,000, cat abcam-
4888), and IRS-1 (1:100, cat abcam-653-200). Detection was performed after 2-h
incubation with a horseradish peroxidase-conjugated secondary antibody (1:10,000,
Invitrogen, São 5 Paulo, SP, BRA). The band intensities were quantified by optical
densitometry using the free software, Image Tool ( http://ddsdx.uthscsa.edu/dig/
itdesc.html ). Densitometry values obtained from phosphorylated proteins (pAkt and
pIRS-1) were normalized by total protein expression (Akt and IRS-1), as previously
described (Batista et al. 2012 ; Ribeiro et al. 2012 ) .

10.2.6 Statistical Analysis

Results are presented as means + SEM for the number of determinations (n) indi-
cated. The statistical analyses were carried out using a one-way analysis of vari-
ance (ANOVA) followed by the Newman-Keuls post hoc test ( P < 0.05) and
performed using GraphPad Prism version 5.00 for Windows (GraphPad Software,
San Diego, CA, USA).