Characterization of insulin binding to slices of slow and fast twitch skeletal muscles in the rabbit.
Insulin binding was studied in rabbit semimembranosus proprius and psoas major muscles composed of slow-twitch oxidative (SO) and fast-twitch glycolytic (FG) fibers, respectively. For this purpose, we developed a technique using cryostat microtome muscle slices. Degradation of 125(I)-insulin during the incubation period was prevented by the addition of 1 mM bacitracin in the buffer. Specific binding to muscle slices plateaued by the 24 hrs. of incubation at 4 degrees C. It increased as a function of the amount of muscle, with a maximum binding occurring at about 5 mg of muscle slices. Triton X-100 has been shown to increase specific binding from a critical concentration of 10(-4) M with a maximum effect occurring at 3.3 10(-4) M. Under this condition, the binding was specific since displacement studies showed no inhibition of 125(I)-insulin binding by GH, HCG, ACTH and glucagon, whereas half maximal inhibition was achieved using 5 10(-10) M insulin, 3 10(-9) M IGF1 and 2 10(-8) M proinsulin. The analysis of the binding data yielded curvilinear Scatchard plots. The number of high affinity insulin receptors was higher in the SO muscle than in the FG muscle (4.3 +/- 0.7 vs 0.7 +/- 0.2 fmol/mg fresh muscle; P less than 0.001) with similar high affinity dissociation constants (Kd = 1.5 10(-10) M). Analogous results were obtained using muscle microsomal fractions. The differences in insulin binding might be related to the more intense metabolism of SO fibres which contract more often than FG fibres in vivo.
D000818:Animals; D001414:Bacitracin / Q000494:pharmacology; D001667:Binding, Competitive; D005260:Female; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D009119:Muscle Contraction; D009132:Muscles / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011817:Rabbits; D011972:Receptor, Insulin / Q000378:metabolism
D007328:Insulin; D007457:Iodine Radioisotopes; D001414:Bacitracin; D011092:Polyethylene Glycols; D017830:Octoxynol; D011972:Receptor, Insulin