Rats were raised as described in section 2.1.
3.2.2 In vivo experiments
Hyperinsulinemic euglycemic clamps were performed in fasted anesthetized rats as described in section 2.2. Once the surgery was completed, a 60-min equilibration period was allowed so that leg blood flow and blood pressure could become stable and constant. Femoral blood flow in one leg was continuously measured from a Transonic® flow probe positioned around the femoral artery. Rats were then allocated into either control (saline), T-1032 or euglycemic insulin clamp (insulin alone or T-1032 + insulin) group (n = 5-7 in each group). Glucose (30% w/v solution) was infused to maintain blood glucose levels at or above basal whilst infusing insulin for a period of 120 min. T-1032 was dissolved in 1 mM HCl in saline. At the end of the experiment samples were taken from the femoral artery and vein. Hindleg glucose uptake and 1-MX metabolism were calculated from the arterio-venous difference multiplied by the flow. 1-MX metabolism was an indicator of perfused capillary surface area. At 45 min prior to the completion of the experiment, a 100 µCi bolus of [14C]2-DG was administrated. Plasma samples (25µl) were collected at
freeze clamped in liquid nitrogen and stored at –20°C until assayed for 2-DG radioactivity as described in section 2.6.
3.2.3 Experimental protocols
Completion of the surgical procedures was followed by a 60-min equilibration period to allow leg blood flow and blood pressure to become constant. Rats were then subjected to Protocol A or B (Fig. 1), where they were infused with saline or T-1032 for 3 h (some received T-1032 for only 2 h starting at 0) and underwent euglycemic insulin clamp (3 mU/kg/min, Humulin R, Eli Lilly and Co., Indianapolis), or saline alone for the final 2 h. T-1032 was infused at 1 μg/min/kg in protocol A1 and at 10 μg/min/kg in protocol A2 and B. T-1032 was also infused 1h prior to insulin clamp (protocol B).
- 6 0 - 4 0 - 2 0 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 6 6 6 6 6 6 6 6 6 6 6
+
6 6 6 B lo o d g lu c o s e d e te r m in a tio n B o lu s A llo p u r in o l ( 1 0 µ m o le /k g ) 1 - M X ( 0 .5 m g / m in /k g ) 3 m U /m in /k g In s u lin c la m p*
2 - D G B o lu s 1 0 0 µ C i 6 A - V T - 1 0 3 2 1 µ g /m in /k g in A 1 a n d 1 0 µ g /m in /k g in A 2 P R O T O C O L A 1 & A 2 : C o - in f u s io n - 6 0 - 4 0 - 2 0 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 6 6 6 6 6 6 6 6 6 6 6+
6 6 6 B lo o d g lu c o s e d e te r m in a tio n B o lu s A llo p u r in o l ( 1 0 µ m o le /k g ) 1 - M X ( 0 .5 m g / m in /k g ) 3 m U /m in /k g In s u lin c la m p*
2 - D G B o lu s 1 0 0 µ C i 6 A - V T - 1 0 3 2 1 0 µ g /m in /k g P R O T O C O L B : P r e - in f u s io n Study design. Fig. 1. Arterial and venous samples were collected at times indicated asA-V for HPLC analysis and plasma glucose determination. Arterial blood glucose were
determined at time 6. Venous infusion periods are indicated by bars. Bolus infusion
periods are indicated by
+.
T-1032 was infused at a dose of 1 μg/min/kgin protocol A1 and at a dose of 10 μg/min/kg in protocol A2 and B. n = 5-7.3.2.4 Plasma T-1032 assay
A novel assay was developed for the measurement of plasma T-1032 by reverse- phase HPLC using 5 µm C-18 reverse-phase column (Luna) with 64% methanol in 50 mM NH4H2PO4 buffer as the mobile phase at a flow rate of 1.2 ml/min. 1 ml of plasma
was mixed with 3 ml of ethanol to precipitate the proteins and centrifuged at 3500 rpm for 15 minutes. To the 3.4 ml of supernatant 10.2 ml chloroform was added, mixed and briefly centrifuged (30 sec). The lower chloroform layer was then air dried at 60°C, the residue re-dissolved in HPLC buffer (100 μl) and an aliquot (50 μl) injected into the HPLC system to measure the plasma concentration of T-1032. A standard curve was constructed using different concentrations of spiked plasma samples that were treated in the same manner as above.
3.2.5 Plasma insulin assay
Rat insulin levels at the end of the euglycemic insulin clamp (and other groups) were determined from arterial plasma samples by ELISA assay (Mercodia rat insulin ELISA) using rat insulin standards.
3.2.6 Muscle cGMP assay
Muscle cGMP levels (soleus) were determined using a Biotrak cGMP enzyme immunoassay kit (Amersham Pharmacia Biochem., UK) on trichloroacetic acid extracts of muscle according to the instructions provided.
3.2.7 Plasma free fatty acid assay
Plasma free fatty acid were determined using an enzymatic colorimetric assay kit (Wako Pure Chemical Industries Ltd).
3.2.8 Muscle glucose-6-phosphate
Muscle glucose-6-phosphate was determined enzymatically in the neutralized perchlorate extracts of muscle. Extracted muscle glucose-6-phosphate was treated with glucose-6-phosphate dehydrogenase in the presence of NADP+. Change in absorbance at
340 nM after formation of NADPH+ was proportional to the levels of glucose-6-
phosphate.
3.2.9 Data analysis
All data are expressed as means ± SEM. Data-analysis was done as described in section 2.9.
3.2.10 Statistical analysis
Repeated measures two-way analysis of variance was used to test the hypothesis that there was no difference among treatment groups for femoral blood flow, blood pressure, heart rate, vascular resistance, 1-MX, and oxypurinol concentrations throughout the time course. When a significant difference (P < 0.05) was found, pair wise comparisons by the Student-Newman-Keuls test were used to determine at which individual times the differences were significant. Statistical differences between the treatments for arterial glucose and 1-MX, hindleg glucose extraction and uptake, and hindleg 1-MX extraction and disappearance were determined by one way ANOVA. These tests were performed using the SigmaStatTM statistical program (Jandel Software, version 2.03).