José A. Hedo

Insulin-Receptor Biosynthesis in Cultured Lymphocytes From an Insulin-Resistant Patient (Rabson-Mendenhall Syndrome): Evidence for Defect Before Insertion of Receptor Into Plasma Membrane

In some patients with genetic forms of extreme insulin resistance, there is a marked decrease in the number of insulin receptors on the cell surface. We studied an insulin-resistant patient (RM-1) with the Rabson-Mendenhall syndrome. As judged by insulin-binding studies, Epstein-Barr virus–transformed lymphocytes from patient RM-1 exhibit a 90% decrease in the number of insulin receptors. Similarly, with either lactoperoxidase-catalyzed radioiodination of cell surface receptors or biosynthetic labeling of receptors with [3H]glucos-amine, we demonstrated an 80–90% decrease in the number of insulin receptors in cells from patient RM-1. Previous studies have shown that the marked decrease in insulin receptors of the Rabson-Mendenhall patient is not due to accelerated receptor degradation. Therefore, we investigated the possibility that a slow rate of receptor biosynthesis might account for the 90% reduction of insulin receptors in cells from this patient. Insulin-receptor biosynthesis proceeds through a glycoprotein precursor with an apparent M, of 190,000. It undergoes endopeptidase cleavage and further post-translational processing to yield the mature 135,000- and 95,000-Mr glycoprotein subunits. We studied the biosynthesis of the 190,000-Mr precursor and mature receptor subunits by a pulse-chase labeling technique with [2-3H]mannose. The time course of insulin-receptor biosynthesis appeared normal in cells from patient RM-1, despite a 10-fold reduction in the number of receptors on the cell surface. Parallel pulse-chase experiments with either [2-3H]mannose or [35S]methionine yielded the same results regardless of which label was employed. Thus, the receptor precursor in the Rabson-Mendenhall patient seems to be synthesized at a normal rate. Moreover, once the receptor is inserted in the membrane, it appears to be degraded at a normal rate. Therefore, we propose that there may be a defect subsequent to the synthesis of the receptor but before insertion of the receptor into the plasma membrane.

Fluctuations of Human Pancreatic Polypeptide in Plasma: Effect of Normal Food Ingestion and Fasting

Summary In this work we have examined the daily fluctuations of circulating hPP in normal individuals subjected to a conventional meal schedule (breakfast, lunch, and dinner) as well as during food deprivation for 84 hr. In addition, we have tested the effect of ingestion of a low-calorie, fiber-rich salad as well as 500 ml of tap water on hPP secretion. Ingestion of each meal was followed by a sustained hPP elevation. Between meals, circulating hPP did not return to basal values. Both the vegetable meal and the water load evoked hPP release, suggesting that the hPP response to food intake is partially a nonspecific effect. In the fasted group, plasma hPP rose significantly 24 hr after the last meal and persisted elevated for the remainder of the experimental period. Moreover, in this condition hPP showed circadian variations, with higher values in the late evening than in the preceding and subsequent morning. Since pancreatic polypeptide is suspected to possess gastrointestinal functions, its elevation in plasma throughout the daytime in conditions of normal feeding may be thought to exert a tonic influence on some digestive process. On this basis, the increase of hPP during prolonged fasting appears paradoxical and, indeed, the explanation of this phenomenon awaits a better knowledge of the biological activity of this peptide.

GLUCAGON-RELEASING ACTIVITY OF GUANIDINE COMPOUNDS IN MOUSE PANCREATIC ISLETS

1. Introduction Structure-function studies performed to investigate the betacytotropic effect of arginine, have suggested that the guanidino group may be involved in triggering insulin release, since several compounds that contain this radical within their molecule are capable of stimulating the secretion of insulin, both in vivo [l] and in vitro [2] . Given that arginine also behaves as a potent glucagon secretagogue, whose mechanism of action remains unknown, we have examined the effect of guanidine and two guanidine derivatives (guanidino- acetic acid and creatine) on the secretory activity of the alpha-cell in a preparation of isolated mouse pancreatic islets. 2. Methods and materials Four-week old Swiss male mice, fasted for 14 h, were used as pancreas donors. In each experiment a pool of 8 pancreases was employed. The islets were isolated by the collagenase technique [3] as modified by Coll-Garcia and Gill [4]. Batches of 10 islets were incubated for 60 min in 2 ml of a bicarbonate-buffered salt solution [S] (gas phase, 95:5, O2 t COa) contain- ing 0.2% bovine serum albumin (Cohn fraction IV), 2000 U of kallikrein-trypsin inhibitor (Trasylol, Bayer) and 3.3 mM glucose (‘basal medium’). In order to verify the releasing activity of the islets, on each day arginine hydrochloride was used as a control secretagogue. All incubations were performed in a water bath at 37°C and at 60 strokes per min. At the end of the incubation, an aliquot of each sample was 52 diluted 1:4 in 0.2 M glycine containing 0.25% human albumin and 1% normal sheep serum (pH 8.8) and immediately frozen at -20°C until the time of analysis. Full details of this method will be published elsewhere [6]. Radioimmunoassay was used to estimate glucagon [7] and insulin [8]. The values were read against beef-pork glucagon (Eli Lilly) and mouse insulin (Novo) standards. Statistical differences were calcu- lated by unpaired t-test analysis. Collagenase was purchased from Boehringer Mannheim GmbH; arginine hydrochloride, guanidine HCl, guanidinoacetic acid and bovine serum albumin were obtained from Sigma Chem. Co. Glucagon antiserum (30K) was a generous gift from Dr R. H. Unger, Dallas. 3. Results As expressed in table 1, glucagon release by mouse pancreatic islets incubated in the basal medium, was fairly constant in the three groups of experiments. The addition of 10 mM arginine induced, in all cases, a marked increase of glucagon output, the increments ranging from 140% to 410% of the basal values. When 10 mM guanidine was incorporated into the medium, glucagon release amounted to 110% above the basal value. Guanidinoacetic acid and creatin, both at 10 mM, were also effective stimuli of glucagon secretion, the increments being 50% and 140%, respectively. At the glucose concentration employed (3.3 mM), 10 mM arginine elicited significant increments of insulin release, varying from 180% to 590%. While

Heterogeneity of pancreatic polypeptide immunoreactivity in human plasma.

Chance et al. [l] have recently isolated a nearly identical polypeptide from bovine, ovine, porcine and human pancreata. These pancreatic polypeptides represent the mammalian counterpart of avian pancreatic polypeptide (APP) [2,3]. Human pancreatic polypeptide (HPP) contains 36 amino acids and has an approximate molecular weight of 4200 [ 11. Immunoassayable HPP is present in blood and its levels increase in response to food ingestion and insulin-induced hypoglycemia [4,51. Since a considerable number of peptide hormones appear to circulate in blood in multiple immunoreactive forms [6], this work was undertaken to examine the behaviour of plasma immunoreactive HPP on molecular sieve chromatography. We have fractionated the HPP immunoreactivity present in plasma of normal subjects, both in the basal state and after stimulation of HPP release by insulininduced hypoglycemia.