The Effect of Long-Term Taurine Supplementation and Fructose Feeding on Glucose and Lipid Homeostasis in Wistar Rats - Advances in Experimental Medicine and Biology - page 39

administration (time −30 min) in blood from the tail vein followed by a blood
sample of 100 ml for plasma fasting insulin. Blood glucose levels were furthermore
determined in blood from tail vein at 0, 30, 60, 90, and 120 min after glucose
administration using two different automated Accu-Check Glucometers (Roche,
Basal, Switzerland), thus measuring blood glucose at all time points in duplicate.
The area under the curve (AUC) of the glucose tolerance test measurements was
calculated from between 0 and 120 min (following glucose administration) from
baseline (before glucose bolus).

5.2.3 Insulin Signaling

Overnight fasted rats were sedated with a mixture of Hypnorm (active ingredients
fentanyl and fluanisone at a concentration of 0.079 and 2.5 mg/ml, respectively) and
Dormicum (active ingredient midazolam at a concentration of 1.25 mg/ml) in water
given as 0.3 ml per 100 g of body weight. Quadriceps muscle was dissected from
one leg, and the rat was cut open. A liver lobe was removed by disconnecting the
blood supply using suture. An 18 G catheter was inserted into vena portae for injec-
tion of 4.2 nmol/kg of insulin in a 2 ml volume of Krebs-Henseleit bicarbonate
buffer (KHB) (an aqueous solution at pH 7.4 of 4.74 mM KCl, 1.18 mM KH 2 PO 4 ,
1.18 mM MgSO 4 , 118.5 mM NaCl, 24.7 mM NaHCO 3 , 2.5 mM CaCl 2 ) with 0.1%
BSA (free from free fatty acids, Sigma) to directly measure insulin signaling in vivo.
After 5 min of insulin stimulation, another liver lobe and the other quadriceps mus-
cle were dissected. All tissues were quick frozen in liquid nitrogen and stored at
−80°C for further analysis.

5.2.4 Quantitative Real-Time PCR

RNA was extracted from liver tissue using Qiazol (Qiagen, Valencia, CA, USA) as
described by the manufacturer. In short, samples were homogenized in Qiazol with
5 mm steal beads, using a Qiagen Tissuelyzer (Qiagen) three times for 1 min at
30 Hz. Upon lysis, RNA was extracted with chloroform (Sigma) and precipitated
using isopropanol (Sigma). Finally, precipitated RNA was washed with 75% ethanol
and dissolved in 30 ml diethyl pyrocarbonate-treated water (DEPC water) (Sigma).
Quality of RNA was assured using a Bioanalyzer (Agilent, Santa Clara, CA, USA)
as described by the manufacturer. RNA concentrations were measured at a Saveen
Werner Nanodrop Spectrophometer ND-1000 (Saveen Werner, Limhamn, Sweden).
Total RNA was mixed (at a concentration >0.15 m g/ ml for a total of 2 mg RNA in
20 ml volume) with reverse transcriptase, random hexamer primers and nucleotides,
and cDNA synthesis performed using the High Capacity cDNA Reverse Transcription
Kit with RNase inhibitor (Applied Biosystems, Carlsbad, CA, USA) according to
the manufacturer's instruction employing an Eppendorf Thermo cycler (Applied