Howard S. Tager

PROCESSING MECHANISMS IN THE BIOSYNTHESIS OF PROTEINS

Limited proteolysis is a widely occurring mechanism in protein biosynthesis. Protein precursors can be classified according to their functions, localization within cell compartments, and enzymic cleavage mechanisms. The presecretory proteins represent an important class of very rapidly turning over precursors which play an early role in the sequestration of secretory products and whose cleavage appears to be intimately associated with structures formed at the ribosome-membrane junction during protein synthesis. A model is proposed which predicts that the prepeptide forms a beta-pleated sheet structure with other components of the membrane which results in the transfer of a loop of peptide across the microsomal membrane. Proinsulin is representative of the general class of proproteins that are processed post-translationally within their secretory cells either during the formation and maturation of secretory granules (peptides hormones and neurotransmitters, serum albumins) or during the assembly of macromolecular structures (virus capsules, membrane-associated enzyme complexes). The former group are cleaved by Golgi-associated proteases having tryptic and carboxypeptidase B-like specificity. Some precursors are secreted as such and processed extracellularly either in the circulation or at special sites (procollagens, zymogens, provenoms, vitellogenins).

Metabolism of C-peptide in the dog. In vivo demonstration of the absence of hepatic extraction.

The in vivo hepatic metabolism of connecting peptide (C-peptide) in relation to that of insulin has not been adequately characterized. A radioimmunoassay for dog C-peptide was therefore developed and its metabolism studied in conscious mongrel dogs, with sampling catheters chronically implanted in their portal and hepatic veins and femoral artery. The hepatic extraction of endogenous C-peptide under basal conditions was negligible (4.3 +/- 4.5%) and was similar to the hepatic extraction of C-peptide measured during the constant exogenous infusion of C-peptide isolated from dog pancreas. Simultaneously measured hepatic extraction of endogenous and exogenously infused insulin were 43.8 +/- 7.6 and 47.5 +/- 4.4%, respectively. The metabolic clearance rate of infused C-peptide was 11.5 +/- 0.8 ml/kg per min and was constant over the concentration range usually encountered under physiological conditions. In additional experiments, the effect of parenteral glucose administration on the hepatic extraction of C-peptide and insulin was investigated. The hepatic extraction of C-peptide (6.2 +/- 4.0%) was again negligible in comparison with that of insulin (46.7 +/- 3.4%). Parenteral glucose administration did not affect the hepatic extraction of either peptide irrespective of whether it was infused peripherally, intraportally, or together with an intraportal infusion of gastrointestinal inhibitory polypeptide. The fasting C-peptide insulin molar ratio in both the portal vein (1.2 +/- 0.1) and femoral artery (2.1 +/- 0.3) was also unaffected by the glucose stimulus. These results therefore indicate that, since the hepatic extraction of C-peptide is negligible and its clearance kinetics linear, the peripheral C-peptide concentration should accurately reflect the rate of insulin secretion. New approaches to the quantitation of hepatic extraction and secretion of insulin by noninvasive techniques are now feasible.

Lessons Learned From Molecular Biology of Insulin-Gene Mutations

Studies on naturally occurring and man-made mutations in the insulin gene have provided new insights into insulin biosynthesis, action, and metabolism. Ten families have been identified in which one or more members have single-point mutations in their insulin genes that result in amino acid substitutions within the proinsulin molecule. Six of these cause the secretion of biologically defective insulin molecules due to changes within the A or B chains. Replacing A3-Val with Leu, B24-Phe with Ser, or B25-Phe with Leu results in molecules that have essentially normal immunoreactivity but greatly reduced insulin-receptor-binding potency. Individuals with these mutations have a syndrome of mild diabetes or glucose intolerance, which is inherited in an autosomal-dominant mode and is associated with hyperinsulinemia and altered insulin-C-peptide ratios. Although affected individuals are heterozygous and coexpress both normal and abnormal molecules, the elevated circulating insulin consists mainly of the biologically defective form, which accumulates because it fails to be rapidly metabolized via receptor-mediated endocytosis. Four additional families have mutations that are associated with relatively asymptomatic hyperproinsulinemia. A point mutation affecting proinsulin occurs in 3 of the 4 families, leading to replacement of Arg-65 by His, which prevents recognition of the C-peptide-A-chain dibasic cleavage site by the appropriate (β-cell processing protease and results in the circulation of a type II proinsulin intermediate form (des 64, 65 HPI). Members of a fourth family with hyperproinsulinemia have a substitution of B10-His with Asp, resulting in a proinsulin that exhibitsmarkedly altered subcellular sorting behavior. A significant proportion of the newly synthesized Asp-10 proinsulin is secreted in an unprocessed form via an unregulated or constitutive secretory pathway. This syndrome has been modeled in transgenic mice by introduction of this abnormal gene into the germ line, resulting in its expression at high levels along with the normal mouse insulin genes in the β-cells. These animals have not only reproduced the hyperproinsulinemia syndrome, thus allowing us to examine its mechanism in considerable detail, but have also provided opportunities to examine other aspects of insulin-gene expression. Various molecular expression techniques are now available that allow normal or mutated insulin genes to be expressed via transfection of DNA in cultured cells, injections of in vitro-generated mRNA into Xenopus oocytes, or translation of mRNA in reticulocyte cell-free systems so that their altered properties can be assessed. Application of these and other molecular biological techniques to the expression of naturally occurring mutant proinsulins and others made in the laboratory has provided new forms of insulin for therapy of diabetes and a deeper understanding of the mechanisms of biosynthesis, intracellular sorting, processing, and secretion of insulin under normal and abnormal conditions.

Factitious hypoglycemia. Diagnosis by measurement of serum C-peptide immunoreactivity and insulin-binding antibodies.

In seven patients with factitious hypoglycemia due to the surreptitious injection of insulin, we made the diagnosis by measurements of plasma insulin and C-peptide immunoreactivity (in seven patients), facilitated by the finding of circulating insulin-binding antibodies (in two patients). The simultaneous demonstration of low plasma glucose, high immunoreactive insulin and suppressed C-peptide immunoreactivity represents a triad of results pathognomonic of exogenous insulin administration. Determination of plasma free C-peptide and free insulin permitted patients with high titers of insulin antibodies, including those with a history of insulin-treated diabetes, to be studied and diagnosed in a way similar to that in subjects who had no circulating insulin antibodies.

Three mutant insulins in man

We have previously identified a structurally abnormal insulin in the serum and pancreas of a middle-aged man with diabetes mellitus1,2 which arose from a leucine for phenylalanine substitution at position 24 or 25 of the insulin B chain; further analysis of the patients leukocyte DNA showed that one of the patients insulin alleles had undergone mutation resulting in loss of an MboII restriction site normally present in the human insulin gene3. Two additional and unrelated patients with the same clinical syndrome have now been identified (ref. 4 and unpublished results). All of these patients showed hyperglycaemia typical of diabetes and with marked hyperinsulinaemia typical of insulin resistance, but all three show normal tolerance to exogenously administered insulin. As the opportunity of examining pancreatic tissue from patients suspected of secreting insulin variants is rare, we have developed a method combining HPLC and radioimmunoassay to identify insulin variants isolated from human sera. By this method we have shown that all three patients noted above secrete structurally variant and chemically distinct insulins. In correction of our original assignment, one is identified as [LeuB25]insulin.

Identification and processing of proglucagon in pancreatic islets

Immunoprecipitation and tryptic peptide analysis of newly synthesised proteins from rat islets have identified an 18,000 molecular weight (MW) protein as proglucagon. Conversion of this precursor was kinetically similar to the conversion of proinsulin and resulted in the formation of both pancreatic glucagon and a 10,000-MW protein lacking this hormonal sequence.

The Limitations to and Valid Use of C-Peptide as a Marker of the Secretion of Insulin

The accuracy with which the secretion rate of insulin can be calculated from peripheral concentrations of C-peptide was investigated in conscious mongrel dogs. Biosynthetic human C-peptide and insulin were infused intraportally and their concentrations measured in the femoral artery. During steady-state infusions of C-peptide, the peripheral concentration changed in proportion to the infusion rate and the metabolic clearance rate (5.2 ± 0.3 ml/kg/min) remained constant over a wide range of plasma concentrations. Application of a two-compartment mathematical model, in which the model parameters were estimated from analysis of C-peptide decay curves after intravenous bolus injections, allowed the intraportal infusion rate of C-peptide to be derived from peripheral C-peptide concentrations, even under non-steady-state conditions. Estimates of the intraportal infusion rate based on this model were 102.4 ± 2.6% of the actual infusion rate as it was increasing and 102.3 ± 5.5% of this rate as it was falling. The peripheral C-peptide : insulin molar ratio was influenced by the rate at which equimolar intraportal infusions of C-peptide and insulin were changed. The baseline C-peptide : insulin molar ratio (4.1 ± 0.9) increased to peak values of 8.2 ± 0.6,10.3 ± 2.0, and 14.9 ±1.3 when the infusion rate was increased and then decreased rapidly. Peak values of only 5.7 ±1.2 were found if the intraportal infusion rate was changed slowly. In conclusion: (1) under steady-state conditions the secretion rate of insulin can be calculated as the product of the peripheral concentration of C-peptide and its MCR; (2) under non-steady-state conditions, however, application of more complex mathematical models, such as the two-compartment model used in the present study, allows insulin secretion rates to be accurately calculated at discrete time points; and (3) under non-steady-state conditions the C-peptide:insulin molar ratio is influenced not only by the extent of hepatic insulin extraction but by other factors, including the rate of change of insulin secretion and the clearance rate of C-peptide. Changes in this ratio should therefore not be assumed to reflect changes in hepatic insulin extraction.

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).

Tumor hypoglycemia: relationship to high molecular weight insulin-like growth factor-II.

The mechanism of tumor-associated hypoglycemia was examined in 11 patients with hepatocellular carcinoma, 6 of whom presented with severe hypoglycemia and 5 in whom plasma glucose was persistently normal. Serum insulin levels in the hypoglycemic patients were low. Although total serum insulin-like growth factor II (IGF-II) levels in both groups of tumor patients were lower than normal, tumor tissue from hypoglycemic patients contained levels of IGF-II mRNA that were 10-20-fold higher than those present in normal liver. IGF-II immunoreactivity consisted in all cases of a mixture of both higher molecular weight forms and material having the character of IGF-II itself. The former comprised a greater proportion of total IGF-II, in patients with hypoglycemia. Studies to characterize the interactions of IGF-II with serum proteins showed that (a) the radiolabeled peptide bound to an approximately 40,000-D protein in sera from both hypoglycemic patients and normal subjects, (b) sera from hypoglycemic patients and normal subjects had similar capacity to bind the radiolabeled peptide, and (c) the apparent affinities of serum binding proteins for IGF-II were the same for both hypoglycemic patients and normal subjects. Whereas, acid extracted, tumor-derived IGF-II immunoreactive peptides with low or intermediate molecular weights bound to serum proteins in a manner indistinguishable from that of IGF-II itself, the highest molecular weight IGF-II immunoreactive peptide exhibited negligible ability to compete for radiolabeled ligand binding to serum proteins. The low affinity of serum binding proteins for this component suggests that high molecular weight IGF-II immunoreactivity might circulate free and be available for interaction with cell-surface receptors.

Diabetes Due to Secretion of an Abnormal Insulin

A 51-year-old, nonobese man with diabetes mellitus had marked hyperinsulinemia (70 to 120 muU per milliliter; 502 to 860 pmol per liter) and fasting hyperglycemia (140 to 170 mg per 100 ml; 7.8 to 9.4 mmol per liter). Plasma proinsulin, glucagon, growth hormone, and cortisol levels were normal; insulin antibodies and insulin-receptor antibodies were not detected. The patient showed relatively normal insulin sensitivity, and insulin receptors on circulating monocytes were within the normal range. Insulin from the patients serum bound to IM-9 lymphocytes and rat adipocytes approximately 40 per cent as well as insulin standards. Its biologic activity on rat adipocytes averaged 15 per cent of that expected from its immunologic concentration. The impaired biologic activity of this patients circulating insulin was probably due to a structural abnormality. Subsequent studies of the patients insulin (fortuitously obtained from his pancreas during a laparotomy for a pancreatic cyst) have confirmed this conclusion. (N Engl J Med 302:129-135, 1980).