K.Y. Wong

Degradation of Insulin by Isolated Mouse Pancreatic Acini: Evidence for Cell Surface Protease Activity

In the present study, we have used isolated mouse pancreatic acini to investigate the relationship between 125I-insulin binding and its degradation in order to probe the nature and cellular localization of the degradative process. In these cells, the proteolysis of 125I-insulin was dependent on time and cell concentration, and was saturated by unlabeled insulin with a Km of 290 nM. Since this value was much higher than the Kd for insulin binding to its receptor (1.1 nM), the data indicated that 125I-insulin degradation by acini occurred primarily via nonreceptor mechanisms. Several lines of evidence suggested that insulin was being degraded by the neutral thiol protease, insulin degrading enzyme (IDE). First, insulin degradation was inhibited by thiolreacting agents such as N-ethylmaleimide and p-chloromercuribenzoate. Second, the Km for degradation in acini was similar to the reported Km for IDE in other tissues. Third, the enzyme activity had a relative mol wt of approximately 130,000 by gel filtration, a value similar to that reported for purified IDE. Fourth, the degrading activity was removed with a specific antibody to IDE. Other lines of evidence suggested that enzymes located on the cell surface played a role in insulin degradation by acini. First, the nonpenetrating sulfhydryl reacting agent 5,5′dithiobis-2-nitrobenzoic acid blocked 125I-insulin degradation. Second, a specific antibody to IDE identified the presence of the enzyme on the cell surface. Third, chloroquine, leupeptin and antipain, agents that inhibit lysosomal function, did not influence 125I-insulin degradation. Fourth, highly purified pancreatic plasma membranes degraded 125I-insulin.

Insulin receptors in isolated mouse pancreatic acini

Abstract Specific insulin receptors were measured in isolated mouse pancreatic acini. Scatchard analyses revealed a high affinity binding site with a K d of 1.67 nM and a lower affinity site with a K d of 83 nM. Binding of insulin to these receptors was rapid, one-half maximal binding occurring at 2 min and maximal binding at 30 min. Insulin stimulated the uptake of the glucose analogue 2-deoxy-D-glucose; maximum effects were detected at 1.67 μM. Insulin, in contrast, had no direct effects on alpha-aminoisobutyric acid uptake. The finding of high affinity insulin receptors in pancreatic acinar cells supports the hypothesis that insulin may directly regulate specific functions in the exocrine pancreas.

The CCK receptor on pancreatic plasma membranes: binding characteristics and covalent cross-linking

The cholecystokinin (CCK) receptor in purified plasma membranes prepared from mouse pancreatic acini had a binding affinity of 1.8 nM, an acid pH optimum between 6.0 and 6.5, and an analog specificity of CCK8 > CCK33 > desulphated CCK8 > CCK4. Binding of CCK to its receptor was abolished by pretreatment of plasma membranes with trypsin. When [125I]CCK was cross‐linked to its receptors with disuccinimidyl suberate, and the preparation solubilized and subjected to gel electrophoresis and autoradiography, the hormone was associated with M r 80000 protein in both the presence and absence of the reducing agent dithiothretol.

Sequence and Analysis of Promoter Region of Human Insulin-Receptor Gene

The promoter region of the human insulin-receptor (HINSR) gene was isolated from a human chromosome 19 bacteriophage library. With S1 nuclease mapping and primer-extension analysis, we identified multiple transcription-initiation sites. Dexamethasone, a known inducer of HINSR transcription, enhanced transcription of all major transcription-initiation sites. DNA sequence analysis indicated that the HINSR promoter has neither a TATA box nor a CAAT box. The HINSR promoter region contains six GGGCGG sequences that may be binding sites for the transcription factor Sp1. In addition, there were three TCCC sequences that were putative promoter regulatory regions. The HINSR gene promoter has structural similarity to the epidermal growth factor receptor gene promoter and has some features of the promoter of the meglutol (hydroxymethylglutaryl, HMG) CoA reductase gene and the early promoter of simian virus 40.

Regulation of Insulin-Receptor mRNA Levels by Glucocorticoids

We found with IM-9 human cultured lymphocytes, that the glucocorticoid dexamethasone increased insulinreceptor mRNA levels. This increase correlated in a time- and dose-dependent manner with the increase in the biosynthesis of the insulin-receptor precursor. In addition, in AR42J cultured rat pancreatic acinar cells, dexamethasone increased insulin-receptor mRNA levels. These studies suggest, therefore, that an increase in mRNA levels is an early step in the regulation of the insulin receptor by glucocorticoids in several cell types.

Production of Inhibitor of Insulin-Receptor Tyrosine Kinase in Fibroblasts From Patient With Insulin Resistance and NIDDM

Although non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in insulin action, the molecular basis of this resistance is unknown. We studied fibroblasts from a markedly insulin-resistant patient with NIDDM but without acanthosis nigricans. Her fibroblasts were resistant to insulin when α-aminoisobutyric acid uptake was measured. Fibroblasts from this patient demonstrated normal insulin-receptor content as measured by both insulin-receptor radioimmunoassay and by Scatchard analysis. However, when compared with nondiabetic control subjects, insulin-receptor kinase assays of wheat-germ–purified receptors prepared from her fibroblasts showed very low basal and no insulin-stimulated tyrosine kinase activity. The insulin receptor was then removed from the wheat-germ fraction by monoclonal antibody affinity chromatography. This insulin-receptor–deficient fraction inhibited both basal and insulin-stimulated tyrosine kinase activity of highly purified insulin receptors. When the specificity of this inhibition was tested, less inhibition was seen with insulinlike growth factor I–receptor tyrosine kinase, and even less inhibition was seen with the proto-oncogene p60c-src tyrosine kinase. Thus, these studies indicate that fibroblasts from an insulin-resistant patient with NIDDM produce a relatively specific glycoprotein inhibitor of insulin-receptor tyrosine kinase. Therefore, these studies raise the possibility that this inhibitor may play an important role in the insulin resistance seen in this patient.

Monoclonal antibodies to the human insulin receptor mimic a spectrum of biological effects in transfected 3T3/HIR fibroblasts without activating receptor kinase.

The effects of four monoclonal antibodies to the alpha subunit of the human insulin receptor were studied in transfected mouse 3T3 fibroblasts expressing human insulin receptors (3T3/HIR). Three antibodies, MA-5, MA-20, and MA-51, mimicked insulin stimulation of the uptake of both 2-deoxy-D-glucose and alpha-aminoisobutyrio acid, and S6 kinase activity. Antibody MA-5 also mimicked insulin stimulation of [3H]thymidine incorporation and cell growth. Although these antibodies mimicked insulin stimulation of biological effects, they failed to significantly activate insulin receptor tyrosine kinase activity. These studies suggest, therefore, that the insulin receptor can signal a variety of cellular functions without stimulation of receptor kinase activity.

Production of antibodies that inhibit the binding of insulin to its receptor

In this study, we report a procedure for producing antisera that block the binding of 125I-insulin to its receptor. After 2 injections with intact IM-9 cultured human lymphocytes, the antisera from 8 of 17 BalbC mice inhibited the binding of 125I-insulin to its receptor on IM-9 cells by 50% or greater. One antiserum at dilutions of 1:200 and 1:50 inhibited the binding of 125I-insulin by 50% and 80%, respectively. Four lines of evidence indicated that the inhibition of 125I-insulin binding by this antiserum was due to a specific immunoglobulin directed against the insulin receptor. First, removal of the immunoglobulin fraction of the antiserum resulted in a complete loss of its inhibitory activity. Second, the antiserum inhibited the binding of 125I-insulin to its receptor on both human cultured lymphocytes and human placenta particles. Third, the antisera bound solubilized insulin-receptor complexes. Finally, the antiserum did not inhibit the binding of 125I-human growth hormone to its receptor on IM-9 lymphocytes. These studies demonstrate therefore, a simple method for producing antibodies that block the binding of 125I-insulin to the human insulin receptor.

Growth factor receptor regulation in the Minn-1 leprechaun: Defects in both insulin receptor and epidermal growth factor receptor gene expression

Leprechaunism is a disorder characterized by intrauterine growth retardation, distinctive dysmorphology, and extreme insulin resistance due to structural abnormalities of the insulin receptor (IR). In addition to the IR, it has been suggested that abnormalities of the other growth factor receptors may occur in this syndrome. Using fibroblasts from the Minn-1 leprechaun, we have now investigated the expression of three different growth factor receptor genes: the IR, the insulin-like growth factor-I receptor (IGF-IR), and the epidermal growth factor receptor (EGFR). In agreement with previous studies, we found decreased insulin binding to fibroblasts from the Minn-1 leprechaun. In these cells, the IR transcription rate was not decreased, and sequence analysis of the IR promoter region of the patient showed no abnormalities. Both single-stranded conformational polymorphism analysis (SSCP) and DNA sequencing confirmed a previously reported nonsense mutation in one of the patients two IR alleles at exon 14. mRNA levels for the IR were markedly decreased, suggesting that IR mRNA turnover was enhanced. We then studied the expression of the closely related IGF-IR Ligand binding, mRNA content, and transcription rate were all normal. In contrast to the IGF-IR, when the EGFR was studied, ligand binding and mRNA content were markedly decreased. These studies therefore raise the possibility that the phenotypic expression of leprechaunism results from defects in the expression of both the IR and the EGFR.

ENTRY OF INSULIN INTO TARGET CELLS IN VITRO AND IN VIVO

At one time it was believed that insulin and other polypeptide hormones did not enter the interior of target cells. Electron microscope autoradiographic and other types of studies have recently indicated, however, that insulin enters the interior of lymphocytes in vitro and hepatocytes in vivo. This entry is time dependent and presumably is mediated by insulin receptors located on the plasma membrane of these cells. Electron microscope autoradiographs reveal that insulin, after entry into lymphocytes and hepatocytes, interacts with specific intracellular organelles, including the endoplasmic reticulum and nuclear envelope in both cell types, and the Golgi in hepatocytes. This interaction of insulin with intracellular organelles may represent one mechanism whereby insulin regulates intracellular functions. In addition, there is a vesicular uptake system for insulin in hepatocytes that degrades insulin and then deposits the degraded hormone into the bile.