| Since Martin Rodbell published his collagenase method for isolating rat adipocytes from their stromal-vascular matrix in 1964, researchers have modified the procedure in an attempt to maximize results. These modifications are also purported to decrease the interlaboratory variability observed by researchers who utilize this procedure. Strict adherence to these modifications does not, however, necessarily result in robust, repeatable in vitro insulin-stimulated glucose oxidation. The purpose of this study was to optimize Rodbell's procedure for measuring in vitro glucose oxidation in rat adipocytes, and to measure the effect of meal feeding on this process.Adipocytes from rats in 26 optimization experiments showed that the observed lack of insulin-stimulated glucose oxidation was not caused by reagents, environmental factors, or strain/size of animal. They also showed that the cause was likely related to an uncontrolled factor associated with the animals (ie, the pattern of food intake).Unlike adipocytes from rats in the optimization experiments, adipocytes from rats in the meal-feeding experiments showed a robust, reliable, expected increase (two to three-fold) in insulin-stimulated glucose oxidation. The average fold response in insulin-stimulated glucose oxidation was 3.5-fold. There was no significant difference in nmol glucose oxidized to CO2 between the three time points, but a trend toward decreasing nmol glucose oxidized in the presence of insulin over time was observed (p = 0.15). The adipocytes also showed a decrease in variability of absolute insulin stimulation over time (coefficient of variation decreased from 53% at 0 H-fasted to 29% at 20 H-fasted).In conclusion, meal feeding rats for approximately three weeks resulted in reproducible, robust insulin stimulation of glucose oxidation of adipocytes. This is the first study to investigate the short-term effects of two hour meal-feeding on adipocyte glucose oxidation.For optimization experiments, adipocytes were isolated from the epididymal fat pads of 58 male rats (38 Sprague Dawley and 20 Wistar). For meal-feeding experiments, adipocytes were isolated from the epididymal fat pads of 32 male Wistar rats at one of three time points after feeding: 0 H fasting (n = 10 rats), 10 H fasting (n = 10 rats), 20 H fasting (n = 8 rats) and two pilot experiments at 5 H fasting (n = 4 rats). The adipose tissue from two rats was pooled for each experiment. Insulin sensitivity was measured after incubation with 6 mM D-[U-14C]-glucose and 10-7 M insulin, by measuring 14CO2 production. |
