Summary
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° C. It increased as a function of the amount of muscle, with a maximum binding
occuring 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 < 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.
Key-Words
Insulin Receptor
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Insulin Binding
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Skeletal Muscle
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Rabbit
