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Cultured L6 myoblasts afford considerable advantages for identifying and studying the insulin-like actions of test substances in a muscle-derived line. We have used this system to examine the interaction of the oral hypoglycemic sulfonylurea glyburide with bovine insulin on protein degradation and synthesis as well as on thymidine incorporation (as a measure of DNA synthesis) in these cells. Bovine insulin, at doses of 0.1 μg/mL to 10 μg/mL, produced a dose-dependent inhibition of protein degradation (measured by release of trichloracetic acid (TCA)-soluble 14C-tyrosine from myoblasts into the culture medium) and increase in total protein content in the cultured myoblasts. At concentrations of 10 μg/mL, insulin achieved its maximal suppression of protein degradation (by nearly 50%) and increased cellular protein content (by 15%) over levels observed in the absence of added insulin. Glyburide, at concentrations at or above 1 μg/mL, significantly suppressed protein degradation (up to 14%) and slightly augmented protein content of the cells. The effects of glyburide on protein degradation were additive with those of submaximally but not maximally effective concentrations of insulin, suggesting a common mechanism of action of the compounds. Both insulin and glyburide, at maximally effective doses, significantly depressed protein degradation as early as 2 to 6 hours after exposure. In addition, in a 24-hour labeling experiment, insulin stimulated tyrosine incorporation into TCA-insoluble protein and thymidine incorporation into DNA in the cells, whereas glyburide did not enhance these processes and, under certain conditions, inhibited them. These results demonstrate that glyburide, either alone or in concert with insulin, is capable of significantly inhibiting protein turnover in skeletal muscle-derived cells. The physiological implications of these observations are discussed.

Studies were performed to examine the effects of various fractions of pineal gland origin upon rates of immunoreactive insulin release during short-term incubations of pancreatic islets from pinealectomized rats. Fractions of cerebral cortex were employed in control incubations. An ultrafiltrate of pineal extracts containing materials of molecular weight less than or equal to 1000 daltons stimulated insulin release while fractions of greater molecular weight were without effect. The stimulation of insulin release observed with the lower molecular weight pineal fraction was seen with both nonstimulatory (2 mM) and stimulatory (10 and 30 mM) medium glucose concentrations, but was abolished in the presence of 2,4-dinitrophenol (200 microM). Upon further fractionation of the low molecular weight pineal extract, fractions I (estimated molecular weight range 700-1000 daltons) and 11 (estimated molecular weight range 180-700 daltons) exhibited comparable stimulatory effects upon islet insulin release: similar effects were observed with cerebral cortical fractions in these molecular weight ranges. However, pineal fraction III (estimated molecular weight range less than 180 daltons) exhibited a striking inhibitory effect upon rates of insulin release compared to cerebral cortical fraction III. Potassium, dopamine, norepinephrine and epinephrine concentrations in both pineal and cortical tissue fractions were similarly low. We conclude that insulinotropic constituent(s) of similar molecular weight occur in pineal gland and cerebral cortex, and that the pineal insulinotropic activity stimulates active insulin secretion by the islets independently of concomitant stimulation by glucose; however, lower molecular weight insulinostatic constituent(s) are notable only in the pineal. It is suggested that mild hyperinsulinemia seen in pinealectomized rats under some circumstances may occur as a result of loss of the pineal insulinostatic factor(s).

A method is presented for the treatment of serum or other biological fluids to remove interfering substances prior to radioimmunoassay. Studies with radioimmunoassay systems for measuring serum levels of insulin revealed the presence of such interfering substances, which could be removed by utilizing small columns of Sephadex G-25 prepared in disposable pipet tips. The columns were centrifuged at low, and finally, higher speeds, to recover the sample volume with essentially no alteration in insulin activity. Validity of the technique was evaluated by both parallelism and recovery studies. This system offers considerable advantages over previous extraction and chromatography procedures in terms of simplicity, reproducibility, and recovery.