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After the addition of insulin to monolayers of chick fibroblasts previously incubated in serum-free medium, the rates of protein and RNA synthesis increase continuously during the first 8-10 h. Little stimulation of DNA synthesis or mitosis results with the addition of insulin alone in contrast to the addition of fresh serum which stimulates both markedly. The stimulation in RNA synthesis does not result from expansion of the nucleotide pool but is correlated with increases in RNA polymerase activity. All major classes of RNA are stimulated; processing of preribosomal RNA to 28S and 18S and the association of this mature RNA with ribosomes appear to occur normally. The kinetics of stimulation of 5S RNA differ from those of the synthesis of 4S and of ribosomal RNA. Insulin and serum appear to affect the synthesis or stability of certain transcripts differentially.

Two methods are described for the preparation of NalphaB1,Nepsilon29-Boc2-insulin from Nalpha A1-trifluoroacetyl-insulin and Nalpha A1-citraconyl-insulin in 80 - 90% and 65% yields, respectively. Removal of the Boc protections afforded the fully active insulin. Application of this derivative was demonstrated by the preparations of des-GlyA1-insulin and [A1-guanidinoacetyl]insulin. The former compount exhibited 2% activity in the in vitro free fat cell assay and the latter 88 +/- 5% while NalphaB1-NepsilonB29-Boc2-insulin showed 45 +/- 3% activity only.

ATP was synthesized in presence of insulin or insulin and prostaglandin E2 by rat skeletal muscle particulate preparation enriched in plasmatic membranes. Isolation of the ATP was carried out using column chromatography on Dowex 1 x 8/cl-form, 100--200 mesh). ATP was formed within 1 min in a medium containing Tris-HCl buffer, pH 7.5, ADP, Mg2+, inorganic phosphate, NaF during NADH-related oxidation involving cytochrome c and O2 in amounts of 100--300 pmoles per mg of protein. Quantitative estimation of ATP in the lyophilized product was carried out by means of spectrophotometry at 340 nm of NADPH formed during a coupled enzymatic reactions involving hexokinase and glycose-6-phosphate dehydrogenase. This products identified by descending paper chromatography on Whatman NI in the system containing ethanol-ammonium citrate pH 4.4 and pH 7.5. Identification of ATP was also performed by thin-layer chromatography. The product was tested for content of ribose (orcinol method) and of inorganic phosphate after acid hydrolysis within 7.5 min at 100 degrees. In the product obtained adenine was identified by UV-spectrophotometry at 260 nm. A salt of ATP was synthesized from the product obtained.

We have studied a major product in the degradation of insulin by insulin proteinase (EC 3.4.22.11). Semisynthetic [[3H]PheB1]insulin and [[3H]GlyA1]insulin were used in the experiments. The structure of the fragment was deduced by observing the chromatographic and electrophoretic migration of the label both before and after further digestion of the fragment with proteinases of known specificity, with and without additional treatment by performic acid. Ambiguities were resolved by studying the behaviour of authentic fragments of known structure, isolated and characterized after digestion of intact insulin by proteinases of known specificity. We conclude that a major product in the degradation of insulin by insulin proteinase consists of a truncated section of the A chain, joined by the disulphide bridge B7-A7 to a truncated section of the B chain. The A-chain fragment consists most probably of residues A1-A13, and the B-chain fragment consists most probably of residues B1-B9. The similarity between this fragment and that found by other workers when insulin is degraded by intact hepatocytes is significant in the light of proposals that insulin proteinase is a possible participant in the physiological degradation of insulin by target cells.

A study was made of the early actions of insulin on uridine metabolism in mammary glang explants. A stimulation of both labeled uridine uptake and its incorporation into RNA was demonstrated as early as 15 min after addition of insulin to medium bathing the tissue; these effects persisted for several hours. The metabolic fate of [3-H] uridine to UMP, UDP, and UTP was observed, whereas insulin had no effect on the quantity of 3-H present as uridine or uracil in these tissues. Further studies were performed in which insulin was also shown to have a rapid stimulatory effect on the incorporation of [32-P] phosphate into RNA; however, the uptake of the labeled phosphate was not affected by insulin. Experiments were also carried out to determine whether the effects of insulin on labeled uridine uptake require ongoing RNA and protein synthesis and whether uridine incorporation depends on concomitant protein synthesis. Incubation of explants with antibiotics which inhibit protein synthesis resulted in the complete suppression of the effects of insulin on labeled uridine uptake and its incorporation into RNA. In contrast, the effect of insulin on labeled uridine uptake does not appear to require ongoing RNA synthesis, since this effect persisted when RNA synthesis was significantly reduced by the presence of actinomycin D.

Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.

(1) We [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637 and Davies, Muir & Offord (1986) Biochem. J. 240, 609-612] have previously identified a major product in the degradation of insulin by insulin proteinase (the N-terminal fragment produced by cleavage between residues LeuA13 and TyrA14, SerB9 and HisB10) together with evidence for a minor cleavage site between HisB10 and LeuB11 or between LeuB11 and ValB12. (2) We now present evidence for minor sites of cleavage between TyrA14 and GlnA15, GluB13 and AlaB14 as well as HisB10 and LeuB11.

1. Insulin deficiency induced by anti-insulin serum or streptozotocin increased glucose absorption, as measured in everted sacs of rat upper ileum incubated for 30 min with oxygenated Krebs-Henseleit bicarbonate medium.2. Everted sacs prepared from the terminal ileum of insulin-deficient rats were able to accumulate glucose against a concentration gradient (i.e. development of active glucose transport).3. In experimental diabetes induced by streptozotocin, everted sacs of upper ileum showed increased 3-methyl glucose active transport, and sacs of terminal ileum showed development of 3-methyl glucose active transport.4. Lactic acid formation during the absorption of both glucose and 3-methyl glucose was increased approximately twofold in everted sacs of insulin-deficient animals.5. Insulin added at 100 mu./ml. to the incubating media of everted sacs prepared from insulin-deficient rats did not result in a reduction of glucose absorption or reverse the other effects.6. Fluoride (5 x 10(-3)M) added to the serosal and mucosal media of sacs of terminal ileum prepared from insulin-deficient rats decreased [(14)C]CO(2) formation from [U-(14)C]glucose and lactate formation during glucose absorption, but was unable to reverse the effect of insulin deficiency on glucose active transport.7. The effects of insulin deficiency induced by streptozotocin were more striking than those induced by anti-insulin serum.8. Everted sacs prepared from rats starved for 3 days showed increased glucose active transport accompanied by diminished conversion of [U-(14)C]glucose to [(14)C]CO(2).9. The possible role of hexokinase is discussed in relation to these findings.

The destruction of (125)I-labelled insulin by an enzyme system from rat adipose tissue was studied. The system was located in the particulate fraction. Activity was assayed by the amount of (125)I-labelled degradation products rendered soluble in trichloroacetic acid. The system was heat-labile, with an alkaline pH optimum. The velocity of the reaction varied directly with the enzyme concentration. Paper chromatography of the degradation products showed six ninhydrin-sensitive areas with radioactivity coinciding with three of them. Albumin inhibited the system; ribonuclease did not. Although only 25% of the total (125)I-label was detected by this assay, results with insulin-specific assays suggested that most (80-90%) of the hormone was inactivated. Possible interpretations of these results are discussed. The particulate fractions of other tissues were also studied.