H2O2 was estimated fluorometrically by oxidation of Amplex Red (non-fluorescent) to resofurin (fluorescent), as described by Schönfeldt et al. 161 with modifications. Briefly, isolated mitochondria were added (~0.3 mg/mL) to a respiration buffer containing malate + glutamate or palmitoyl-L-carnitine (PC) and oligomycin (as in respiration
measurements). For estimation of H2O2 release, 50 µmol/L Amplex Red and 2 IU/mL of horseradish peroxidase were added to the suspension, allowing Amplex Red oxidation by H2O2. Superoxide (O2
·-) release was estimated fluorometrically as described by Johnson-Caldwell et al. 162. Briefly, the same mitochondria suspension as for H2O2 release was prepared and 5 µmol/L MitoSOXred was added. All samples were then added to a 96 well microplate and O2·- and H2O2 were measured simultaneously in separate wells. Release of O2
was monitored at ex 510 nm/em 580 nm and release of H2O2 was monitored at ex 571 nm/em 585 nm at 30 °C, using a spectrofluorometer (Spectramax Gemini EM). The production of H2O2 was calculated as the increase in relative fluorescence (RF)/min over a time period of 30 min and O2·- release was estimated as RF after 15 min incubation.
43 8. SUMMARY OF RESULTS
8.1. Paper I
In paper I, we established insulin dose-response curves for hepatic and peripheral insulin sensitivity (Substudy B) (Fig. 8) and verified the applicability of this technique for assessment of acute insulin resistance in anesthetized pigs (Substudy C) by performing a series of studies with 3-step HEC in combination with glucose tracer infusion and labelled glucose infusates (Fig. 7).
Figure 7 Step-clamp timelines during step hot-GINF HEC procedures (n=8). GIR, glucose infusion rate;
APE, glucose tracer atom percent enrichment; S-glucose, serum glucose. Data are means ± SE.
We further found that instrumentation and dissection needed for combination of the technique with other investigative methods such as regional blood flow measurements and sampling did not elicit alterations in the hormonal milieu (Table 1) or glucose homeostasis that would change the premises for correct labelling of the glucose infusates (Table 1). Higher serum insulin concentrations, probably caused by decreased metabolic insulin clearance rate were found after surgery (Fig. 8), which should be kept in mind when utilizing the described method for measurement of acute insulin resistance in porcine research models.
44
Figure 8 Calculated endogenous glucose release (EGR) and whole-body glucose disposal (WGD) from both step hot-GINF HEC procedures in substudy B (anaesthesia only; n=8) and the step hot-GINF HEC performed after major surgery (surgery; n=8) plotted vs. serum insulin (S-insulin) levels. Data are means ± SE
Figure 9 Relative changes in EGR and WGD from basal at low (clamp 2 in substudy B vs.
clamp 1 in substudy C) and high (clamp 6 in substudy B vs. clamp 2 in substudy C) serum insulin (n=8). Data are means ± SE. *P<0.01;
**P=0.001; ***P<0.001 vs. anaesthesia only.
Table 1 Substudy A: circulating hormone concentrations
Table 2 Substudy A: tracer enrichment and glucose kinetics
45 8.2. Paper II
In paper II , the pilot study revealed that a dose of ~0.85 g CHO pr kg bodyweight, which is equivalent to the standard morning dose given to humans, induces hyperglycaemia and a concomitant uniform rise in serum insulin similar to the levels seen in human studies
140,163
(Fig. 10).
Figure 10 Serum glucose and insulin in four separate pigs plotted against time after administration of an oral carbohydrate (CHO) load of 25 g (arrow).
In the main study, the same single CHO dose was then shown to increase the GIR needed to maintain euglycaemia during the last 40 min of each step-clamp in the CHO/surgery compared with the fasting/surgery group (Fig. 11A), indicating increased postoperative whole-body insulin sensitivity after preoperative CHO supplementation. The non-operated control groups exhibited equal whole-body insulin sensitivity (Fig. 11B), indicating that the effect of CHO is limited to surgically stressed animals.
46
Figure 11 Glucose infusion rate (GIR) and serum insulin during three consecutive hyperinsulinaemic-euglycaemic clamps at stepwise increasing insulin infusion rates (0.4, 1.2 and 2.0 mU/kg/min) plotted against time in fasted and CHO treated operated (A) and non-operated control animals (B). Each group n=8.
Data are means ± SE. *Significantly different from fasting surgery, one symbol P<0.05; two symbols P<0.02 (Student’s independent t-test).
#Significantly different from corresponding control group, P<0.001 (Student’s independent t-test). The fasting/surgery group is the same group shown in figure 7.
Due to different insulin serum concentrations during clamping (Fig. 11), insulin
sensitivity in the fasting and CHO treated groups was compared at ~40 µU/ml (i.e. clamp 2 in the surgery groups vs. clamp 3 in control groups). A 29% lower SS GIR was seen after surgery in fasted pigs whereas the decline in CHO treated pigs did not reach statistical significance (-15%, P=0.088) (Fig. 12A). Compared to their non-operated controls, surgery induced a 41% decline in insulin-stimulated WGD at ~40 µU/ml serum
47
insulin in fasted pigs. The postoperative decline was reduced to a mere 16% in CHO treated pigs (P=0.180) (Fig. 12B). No effect of CHO treatment was seen on hepatic insulin sensitivity (Fig. 12C) or glycogen content in liver and muscle after surgery.
Figure 12 Postoperative glucose infusion rate (GIR) (A) and insulin stimulated whole-body glucose disposal (WGD) (B) during clamping at
~40 µU/ml s-insulin in fastedand carbohydrate treated animals (CHO) compared to their respective control groups. Each group n=8. Data are means ± SE. ***Significant difference by Student´s independent t-test, P<0.001.
48
Preoperatively fasted and CHO supplemented pigs displayed an equal increase in serum cortisol concentrations during the experiments compared to their respective non-operated controls. Glucagon was increased in CHO/surgery compared to CHO/control during the low insulin clamp (Fig. 13).
Figure 13 Counter-regulatory hormone profiles during the experiments. Each group n=8. Data are means ± SE. *Significant difference from preoperative, P<0.02; #Significant difference by group, one symbol P<0.02, two symbols P=0.01 (two-way (time and group) repeated measures ANOVA).
49
A strong negative correlation between mean cortisol and insulin stimulated WGD was seen in fasted (Fig. 14A), but not in CHO treated animals (Fig. 14B).
Figure 14 Correlation between mean cortisol after surgery/control and the insulin stimulated increase in whole-body glucose disposal (i.e. peripheral insulin sensitivity) in fasted (A) and carbohydrate treated animals (B). Pearson’s correlation was used (n = 16).
FFA concentrations were reduced in the CHO/surgery group compared to the fasting/surgery group both perioperatively and during the step-clamp procedures.
Compared with the fasting/control group there was a similar, but statistically non-significant (P=0.09) trend towards lower perioperative FFA in the CHO/control group, whereas no difference was seen during the step-clamps (Fig. 15).
50
Figure 15 Free fatty acid time course perioperatively (left) and during three step clamping (right) in the surgery (A) and control (B) groups. Each group n=8. Data are means ± SE. *Significantly different by group, one symbol P<0.05, Two symbols P<0.02 (two-way (time and group) repeated measures ANOVA)