Biologic and clinical importance of proinsulin.
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Adolescent↗
Biomedical subjects
H S Tager
Publications and source records attributed to H S Tager.
Heterogeneity of glucagon receptors of rat hepatocytes: a synthetic peptide probe for the high affinity site.
A glucagon analog with the following sequence has been synthesized: His- Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg -Leu-Gln-Glu-Phe-Leu-Gln-Trp-Ala-Leu-Gln-Thr. When interacting with rat hepatocytes, the analog mimics, in part, the activities of glucagon in receptor binding and inhibition of carbohydrate incorporation into glycogen. Comparison of the binding of the analog with that of glucagon demonstrates the existence of two distinct homogeneous populations of glucagon receptors. The synthetic analog acts as a specific probe for those receptors that have a high affinity for glucagon.
Amino Acid Sequence↗
Human pancreatic icosapeptide: isolation, sequence, and immunocytochemical localization of the COOH-terminal fragment of the pancreatic polypeptide precursor.
In dogs, the COOH-terminal part of the pancreatic polypeptide precursor gives rise to a stable icosapeptide product against which an antiserum has been raised. By immunohistochemistry, icosapeptide immunoreactivity was localized in human pancrease exclusively to cells that also stored pancreatic polypeptide. Analytical peptide chemistry demonstrated that a peptide corresponding to the canine icosapeptide could be extracted from the pancreatic polypeptide-rich duodenal part of the human pancreas. The human pancreatic icosapeptide was isolated by acid ethanol extraction, gel filtration, anion-exchange chromatography, and reverse-phase high-performance liquid chromatography. The COOH-terminal sequence of the human icosapeptide is very similar to that of the canine icosapeptide, whereas none of the first nine amino acid residues are identical. When the primary structure of peptides from three different species are compared, it is apparent that the pancreatic polypeptide part of the common precursor is a well-conserved sequence as compared to the icosapeptide part, although 8 out of 11 residues in the COOH-terminal sequence of the icosapeptide are identical in all three species.
Amino Acid Sequence↗
Familial hyperproinsulinemia. Two cohorts secreting indistinguishable type II intermediates of proinsulin conversion.
Familial hyperproinsulinemia, a hereditary syndrome in which individuals secrete high amounts of 9,000-mol wt proinsulin-like material, has been identified in two unrelated cohorts. Separate analysis of the material from each of the two cohorts had suggested that the proinsulin-like peptide was a conversion intermediate in which the C-peptide remained attached to the insulin B-chain in one case, whereas it was a conversion intermediate in which the C-peptide remained attached to the insulin A-chain in the other. To reinvestigate this apparent discrepancy, we have now used chemical, biochemical, immunochemical, and physical techniques to compare in parallel the structures of the immunoaffinity chromatography-purified, proinsulin-like peptides isolated from the serum of members of both families. Our results show that affected individuals in both cohorts secrete two-chained intermediates of proinsulin conversion in which the COOH-terminus of the C-peptide is extended by the insulin A-chain and from which the insulin B-chain is released by oxidative sulfitolysis. Analysis of the conversion intermediates by reverse-phase high-performance liquid chromatography using two different buffer systems showed that the proinsulin-related peptides from both families elute at a single position very near that of the normal intermediate des-Arg31, Arg32-proinsulin. Further, treatment of these peptides with acetic anhydride prevented trypsin-catalyzed cleavage of the C-peptide from the insulin A-chain, a result demonstrating the presence of Lys64 and the absence of Arg65 in both abnormal forms. We conclude that individuals from both cohorts with familial hyperproinsulinemia secret very similar or identical intermediates of proinsulin conversion in which the C-peptide remains attached to the insulin A chain and in which Arg65 has been replaced by another amino acid residue.
Acetic Anhydrides↗
Human insulin B24 (Phe----Ser). Secretion and metabolic clearance of the abnormal insulin in man and in a dog model.
We have already demonstrated that a hyperinsulinemic, diabetic subject secreted an abnormal insulin in which serine replaced phenylalanine B24 (Shoelson S., M. Fickova, M. Haneda, A. Nahum, G. Musso, E. T. Kaiser, A. H. Rubenstein, and H. Tager. 1983. Proc. Natl. Acad. Sci. USA. 80:7390-7394). High performance liquid chromatography analysis now shows that the circulating insulin in several other family members also consists of a mixture of the abnormal human insulin B24 (Phe----Ser) and normal human insulin in a ratio of approximately 9.5:1 during fasting. Although all affected subjects show fasting hyperinsulinemia, only the propositus and her father are overtly diabetic. Analysis of the serum insulin from two nondiabetic siblings revealed that normal insulin increased from approximately 2 to 15% of total serum insulin after the ingestion of glucose and that the proportion of the normal hormone plateaued or fell while the level of total insulin continued to rise. Animal studies involving the graded intraportal infusion of equimolar amounts of semisynthetic human [SerB24]-insulin and normal human insulin in pancreatectomized dogs (to simulate the secretion of insulin due to oral glucose in man) also showed both a rise in the fraction of normal insulin that reached the periphery and the attainment of a brief steady state in this fraction while total insulin levels continued to rise. Separate experiments documented a decreased hepatic extraction, a decreased metabolic clearance rate, and an increased plasma half-life of human [SerB24]-insulin within the same parameters as those determined for normal human insulin. These results form a basis for considering (a) the differential clearance of low activity abnormal insulins and normal insulin from the circulation in vivo, and (b) the causes of hyperinsulinemia in both diabetic and nondiabetic individuals who secrete abnormal human insulins.
Adult↗
C-peptide and insulin secretion. Relationship between peripheral concentrations of C-peptide and insulin and their secretion rates in the dog.
Estimation of the insulin secretory rate from peripheral C-peptide concentrations depends upon the following characteristics of C-peptide kinetics: (a) equimolar secretion of insulin and C-peptide by pancreatic beta cells; (b) negligible hepatic extraction of C-peptide; (c) constant metabolic clearance rate (MCR) of C-peptide over a physiological and pathophysiological range of plasma levels; and (d) proportional changes in the secretion rate of C-peptide and its peripheral concentrations under varying physiological conditions. In the present experiments, the relationship between a variable intraportal infusion of C-peptide and its concentration in the femoral artery was explored in 12 pancreatectomized dogs. As the infusion of C-peptide was rapidly increased, the magnitude of its peripheral concentration initially increased less than the infusion rate by 20-30%. After an equilibration period of approximately 30 min, however, further increases and decreases in the intraportal infusion were accompanied by nearly proportional changes in its peripheral concentration. Estimates of the amount of C-peptide infused during the experiment based on the steady state C-peptide MCR and its peripheral concentration were within 20% of the amount of C-peptide actually infused. These experiments demonstrate that the portal delivery rate of C-peptide can be calculated from its MCR and peripheral concentration in the dog. They also provide a basis for testing the validity of more complicated models of insulin secretion based on peripheral C-peptide concentrations in the dog as well as other species, including man. Finally, we have shown that the hepatic extraction of endogenously secreted C-peptide is negligible in the basal state (3.1 +/- 6.1%), and does not change after oral glucose ingestion. The MCR of exogenous dog C-peptide was similar whether measured by constant peripheral intravenous infusion (12.3 +/- 0.7 ml/kg per min), constant intraportal infusion (13.4 +/- 0.6 ml/kg per min), or analysis of the decay curve after a bolus injection (13.5 +/- 0.7 ml/kg per min).
Animals↗
Isolation and amino-acid sequence determination of monkey insulin and proinsulin.
Insulin has been isolated and purified from rhesus monkey pancreas by means of acid-ethanol extraction, gel filtration and ion exchange chromatography. The complete amino-acid sequence of the hormone has been determined by amino-acid analysis of the oxidized A- and B-chains, by end group determination, by the identification of the C-terminal residues (AsnA21 and ThrB30) by carboxypeptidase A digestion and by Edman degradation of the S-carboxymethylated A- and B-chains. The 51-residue monkey insulin was shown to be identical to human insulin. From the known insulin and C-peptide sequence the primary sequence of monkey proinsulin has been proposed.
Amino Acid Sequence↗
Lilly lecture 1983. Abnormal products of the human insulin gene.
Heritable abnormalities in the insulin gene have often been considered in terms of their potential for contributing to diabetes. It is only within the last 5 yr, however, that evidence has demonstrated the existence of insulin gene mutations in man and the secretion of abnormal human insulins in affected individuals. HPLC analysis of insulin purified from serum by immuno-affinity chromatography and detection of insulin by radioimmunoassay have documented abnormal insulins in subjects from three separate families. HPLC analysis of these natural insulins and of semisynthetic insulin analogues have identified the accompanying amino acid substitutions in two of these cases: in one, leucine replaces phenylalanine B25; in the other, serine replaces phenylalanine B24. Both substitutions occur in an invariant tetrapeptide sequence within the active site of the hormone. Studies of the biologic activities of these analogues further suggest that replacements at position B25 result in the loss of an important side chain contact between the hormone and its receptor, whereas those at position B24 result in conformational changes in the insulin molecule as a whole. Two additional individuals have been identified to secrete abnormal intermediates of proinsulin conversion in which the C-peptide remains attached to the insulin A-chain and in which Arg65 has been replaced by a nonbasic amino acid. This result emphasizes the importance of dibasic amino acid pairs at prohormone conversion sites and provides clues about the evolution of hormone precursors. Thus, studies of the products of abnormal human insulin genes have provided insights into subjects as varied as insulin biosynthesis, structure-activity relationships in insulin recognition by receptors, and the physiology of insulin action.
Amino Acid Sequence↗
Glucagon receptors on isolated hepatocytes and hepatocyte membrane vesicles. Discrete populations with ligand- and environment-dependent affinities.
Analysis of glucagon and deshistidine glucagon binding to isolated canine hepatocytes and to hepatocyte membrane vesicles (formed by budding of hepatocytes in hypotonic medium) reveals two separate populations of hormone binding sites. Mathematical modeling further shows that the high affinity population represents 1% of the total in all four cases. Although calculated dissociation constants for hormone binding range from 0.2 to 400 nM, whether considering glucagon or deshistidine glucagon binding, or binding to the high affinity or low affinity receptor populations, receptor affinity increases 2- to 100-fold in the environment of the membrane vesicle; concomitant with this alteration in receptor affinity, receptor selectivity for the structure of the native hormone decreases 1.5- to 40-fold in hepatocyte-derived vesicles. Consideration of receptor affinity in relation to receptor number suggests that hepatocyte glucagon binding is distributed about equally between high and low affinity receptor populations at typical portal hormone levels. Nevertheless, consideration of receptor binding in relation to biological activity suggests that the activity of glucagon in inhibiting carbohydrate flux into glycogen is attributable to occupancy of the high affinity receptor population.
Animals↗
[LeuB24]insulin is an insulin agonist at the liver in vivo.
A mutant insulin isolated from the plasma of a diabetic patient has been reported to antagonize insulin action in vitro and was thought to be [LeuB24]insulin. This study examines the ability of [LeuB24]insulin to antagonize insulin action at the liver in vivo in anesthetized dogs. Antagonism of insulin action was first simulated by decreasing the intraportal insulin infusion 50%. This resulted in a significant increase in both glucose production (Ra) (delta = + 0.30 +/- 0.08 mg X kg-1 X min-1) and the glucose level in arterial plasma (delta = +6.5 +/- 2.8 mg/dl), validating the responsiveness of the preparation to partial insulin antagonism. [LeuB24]insulin was infused intraportally, at molar ratios of 1:1, 1:2, 1:4, and 1:10 (50, 25, 12.5, and 5 ng/min, respectively) with insulin (54 ng/min). Infusion at all but the lowest dose resulted in a significant drop in glucose production (delta = -0.44 +/- 0.07, -0.35 +/- 0.06, and -0.28 +/- 0.08 mg X kg-1 X min-1 for4 analogue infusions of 50, 25, and 12.5 ng/min, respectively) and plasma glucose levels (delta = -7 +/- 3 and -3 +/- 1 mg/dl for analogue infusions of 50 and 25 ng/min, respectively). No change in Rd (glucose disposal) was observed for either insulin withdrawal of [LeuB24]insulin infusion. We conclude that, at the liver in vivo, [LeuB24]insulin does not antagonize insulin action but rather acts as an insulin agonist. Its hepatic effects would not contribute to a diabetic hyperglycemia.
Animals↗
Identification of a point mutation in the human insulin gene giving rise to a structurally abnormal insulin (insulin Chicago).
Both insulin gene alleles of a diabetic patient with a mutant insulin were cloned in a lambda vector and their nucleotide sequences were determined. Nucleotide sequence analysis revealed, in one allele, a C (cytidylate) to G (guanylate) transversion in the codon for phenylalanine at position 25 of the insulin B-chain. This point mutation leads to the substitution of a leucine for phenylalanine accompanied by the loss of a restriction endonuclease Mboll recognition site and the creation of a new Rsal cleavage site at this position.
Amino Acid Sequence↗
Peptide intermediates in the cellular metabolism of insulin.
We have studied the metabolism of insulin to peptide intermediates which arise during the early stages of cellular hormone processing. [125I]Iodoinsulin (prepared by direct radioiodination of the hormone) and [(125I)iodotyrosylB1]insulin (prepared by semisynthetic procedures and indirect radioiodination) were used as specific probes for the metabolic fates of the A and B chain domains of the hormone, respectively. Incubation of the A chain-labeled probe with isolated rat hepatocytes and subsequent analysis of the cell-associated products revealed low amounts of several insulin-derived peptides. Mapping of these insulin fragments by chemical and enzymatic modification, electrophoresis, and autoradiography showed that they result from specific cleavages in both chains of the hormone. Two A chain-labeled fragments containing residues A14 to A20-21 in disulfide linkage to COOH-terminal portions of the insulin B chain were identified among the cell-associated metabolites. Other products contained intact A chains yet their elution positions during gel filtration were distinct from the position of [125I]iodoinsulin. Incubation of [(125I)iodotyrosylB1]insulin with hepatocytes resulted in cell-associated products containing a set of B chain-labeled fragments complementary to those described above. Structural analysis showed that these metabolites result from sequential cleavage of the insulin molecule at three sites between the interchain disulfide bonds of the B chain domain. Taken together, these A and B chain-labeled peptides form a series of cell-associated and structurally related metabolites. They likely represent the intermediate products which arise during the cellular processing of insulin.
Animals↗
[LeuB24]insulin and [AlaB24]insulin: altered structures and cellular processing of B24-substituted insulin analogs.
We have used insulin analogs having leucine or alanine substitutions at positions B24 and B25 to examine the structural basis for insulin binding and insulin metabolism by isolated rat hepatocytes. Apparent receptor binding affinities for the analogs were in the order insulin greater than [LeuB24]insulin greater than [LeuB25]insulin = [AlaB24]insulin. Incubation of the corresponding 125I-labeled peptides with hepatocytes followed by analysis of the cell-associated products showed that [125I]iodoinsulin and [125I]iodo-[LeuB25]insulin were processed to a peptide intermediate which appeared as an ascending shoulder on the peak of cell-associated hormone during gel filtration; similar incubations using [125I]iodo-[LeuB24]insulin or [125I]iodo-[AlaB24]insulin failed to yield detectable amounts of the intermediate. In addition, assessment of the structures of insulin and the three insulin analogs by tyrosine radioiodination showed that [LeuB24]insulin and [AlaB24]insulin maintain similar solution conformations which differ from the conformations taken by insulin and [LeuB25]insulin. We conclude that (a) alterations in side-chain bulk at position B24 result in long-range structural perturbations in the insulin molecule, (b) these structural alterations lead to an altered cellular processing of the two B24 insulin analogs, and (c) the selectivity of this processing arises from events subsequent to ligand-receptor recognition.
Animals↗
Surface receptors for pancreatic hormones in dog and rat hepatocytes: qualitative and quantitative differences in hormone-target cell interactions.
In order to evaluate potential differences in the kinetics of peptide hormone-receptor interactions in hepatocytes of different species, we developed a simple procedure for the isolation of canine hepatocytes. Cells (obtained by collagenase perfusion of an extirpated dog liver lobe) were isolated with uniform high viability and yield. In addition, isolated dog hepatocytes tolerated incubation for at least 4 hr in defined medium with only a slight decrease in viability and with no change in the kinetics of [125I]iodoinsulin or [125I]iodoglucagon binding to cell-surface receptors. Comparisons of peptide hormone interactions with isolated dog and rat hepatocytes showed that (i) [125I]iodoglucagon associated with specific membrane receptors more rapidly than did [125I]iodoinsulin, for both rat and dog hepatocytes and at both 30 degrees C and 37 degrees C; (ii) the steady-state binding of [125I]iodoglucagon at 30 degrees C was greater than that of [125I]iodoinsulin in dog hepatocytes, but the reverse relationship held in rat hepatocytes; (iii) the rate of dissociation of [125I]iodoinsulin from hepatocytes of both species was enhanced by the presence of the unlabeled hormone, whereas the rate of dissociation of receptor-bound [125I]iodoglucagon was enhanced by the presence of unlabeled glucagon only in hepatocytes derived from the dog; and (iv) [125I]iodopancreatic polypeptide bound to neither rat nor dog hepatocytes, although the [125I]iodotyrosylated peptide bound to rat hepatocytes with an unusually high apparent dissociation constant. While confirming essential findings of pancreatic hormone binding to isolated hepatocytes, this comparison suggests that both qualitative and quantitative aspects of hormone-target cell interactions can show interspecies variability.
Animals↗
Isolation and biogenesis of a new peptide from pancreatic islets.
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Amino Acid Sequence↗
Relationship between negative cooperativity and insulin action.
We have compared the respective abilities of porcine insulin and [LeuB25]insulin to enhance the rate of dissociation of receptor-bound [125I]insulin from IM-9 lymphocytes and isolated rat adipocytes and to increase the rate of adipocyte glucose transport and oxidation. Although porcine insulin (100 ng/mL) greatly enhanced the dissociation rate of previously bound [125I]insulin, [LeuB25]insulin (at a concentration yielding equivalent receptor occupancy) had no effect. Nevertheless, the analogue fully stimulated adipocyte glucose transport and oxidation at concentrations consistent with its reduced (1.7% of normal) intrinsic binding affinity. Activation of glucose transport by the analogue was rapid, and the corresponding rate of activation was indistinguishable from that produced by native insulin. The increased dissociation rate observed with increasing receptor occupancy by native porcine insulin has been interpreted as evidence for negative cooperative site-site interactions between occupied receptors. According to this formulation, [LeuB25]insulin is a "noncooperative" insulin analogue. Since this [LeuB25]insulin retains full biologic activity, the enhancement of the insulin dissociation rate at high levels of receptor occupancy does not reflect a phenomenon inherent in insulin's action to augment glucose metabolism.
3-O-Methylglucose↗
A human proinsulin variant at arginine 65.
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Adult↗
[(125I]IodotyrosylB1]insulin. Semisynthesis, receptor binding, and cell-mediated degradation of a B chain-labeled insulin.
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Animals↗