Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor.

Cells of the rat pheochromocytoma clone PC12 possess receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), thus enabling the study of the interaction of these receptors in the regulation of proliferation and differentiation. Treatment of the cells with NGF induces a progressive and nearly total decrease in the specific binding of EGF beginning after 12 h and completed within 4 d. Three different measures of receptor show that the decreased binding capacity represents, in fact, a decreased amount of receptor: (a) affinity labeling of PC12 cell membranes by cross-linking of receptor-bound 125I-EGF showed a 60-90% decrease in the labeling of 170- and 150-kD receptor bands in cells treated with NGF for 1-4 d; (b) EGF-dependent phosphorylation of a src-related synthetic peptide or EGF receptor autophosphorylation with membranes from NGF-differentiated cells showed a decrease of 80 and 90% in the tyrosine kinase activity for the exogenous substrate and for receptor autophosphorylation, respectively; (c) analysis of 35S-labeled glycoproteins isolated by wheat germ agglutinin-Sepharose chromatography from detergent extracts of PC12 membranes showed a 70-90% decrease in the 170-kD band in NGF-differentiated cells. These findings permit the hypothesis that long-term heterologous down-regulation of EGF receptors by NGF in PC12 cells is mediated by an alteration in EGF receptor synthesis. It is suggested that this heterologous down-regulation is part of the mechanism by which differentiating cells become insensitive to mitogens.

Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing.

A point mutation in the human insulin receptor gene in a patient with type A insulin resistance alters the amino acid sequence within the tetrabasic processing site of the proreceptor molecule from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser. Epstein-Barr virus-transformed lymphocytes from this patient synthesize an insulin receptor precursor that is normally glycosylated and inserted into the plasma membrane but is not cleaved to mature alpha and beta subunits. Insulin binding to these cells is severely reduced but can be increased about fivefold by gentle treatment with trypsin, accompanied by the appearance of normal alpha subunits. These results indicate that proteolysis of the proreceptor is necessary for its normal full insulin-binding sensitivity and signal-transducing activity and that a cellular protease that is more stringent in its specificity than trypsin is required to process the receptor precursor.

The NOD mouse: recessive diabetogenic gene in the major histocompatibility complex.

Examination of the histocompatibility region of the nonobese diabetic (NOD) mouse with antibodies against class II glycoproteins (products of immune response genes of the major histocompatibility complex I-A and I-E), hybrid T-cell clones, and mixed-lymphocyte cultures and analysis of restriction fragment length polymorphisms indicate that the NOD mouse has a unique class II major histocompatibility complex with no expression of surface I-E, no messenger RNA for I-E alpha, and an I-A not recognized by any monoclonal antibodies or hybrid T-cell clones studied. In crosses of NOD mice with control C3H mice, the development of diabetes was dependent on homozygosity for the NOD mouse's unique major histocompatibility region.

Defective processing of insulin-receptor precursor in cultured lymphocytes from a patient with extreme insulin resistance.

We have studied a patient with extreme insulin resistance, acanthosis nigricans, and decreased erythrocyte insulin binding. EBV-transformed lymphocytes from this patient exhibited markedly reduced binding of 125I-insulin. Radioiodination of cell surface receptors followed by immunoprecipitation with anti-receptor antibodies revealed the presence of increased amounts of a 210-kD protein but no detectable alpha or beta subunits. Continuous labeling with 2-[3H]mannose revealed the synthesis of a 190-kD precursor and a 210-kD protein. The 210-kD protein was phosphorylated in an insulin-dependent manner at high insulin concentrations. These results suggest that in this patient the biosynthesis of 190-kD receptor precursor, its terminal glycosylation, and intracellular transport to the cell surface proceed normally, while proteolytic maturation to alpha and beta subunits does not occur. We postulate that this defect either results from mutation(s) within the insulin-receptor gene, which render the precursor resistant to cleavage, or from a defect in the receptor processing enzyme.

Kinetics of human connecting peptide in normal and diabetic subjects.

The metabolic clearance rate (MCR) of synthetic human connecting peptide (C-peptide) was measured with a single-dose injection technique in six normal and seven diabetic subjects and with a constant infusion technique in one normal subject. The MCR of C-peptide did not differ in normal subjects (4.4 ml/min per kg; range, 3.7-4.9) and in diabetic subjects (4.7 ml/min per kg; range, 3.7-5.8). Employment of both techniques in one subject gave similar MCR. The average half-life of C-peptide in plasma calculated from the last 1-h period of the single-dose injection studies was longer in the insulin-dependent diabetics (42.5 min; range, 39.4-48.5) than in the normal subjects (33.5 min; range, 24.9-45.3). These results indicate that the beta-cell secretory capacity of normal and insulin-dependent diabetic subjects can be compared by measuring the C-peptide concentration in peripheral venous plasma. The difference in the half-life of C-peptide in plasma between diabetics and normals suggests an altered kinetics of the disappearance of the peptide, while the overall metabolism, as expressed by the MCR, is similar.

Reduced expression of the leptin gene (ob) by catecholamine through a G(S) protein-coupled pathway in 3T3-L1 adipocytes.

Leptin, a recently identified hormone, is believed to reduce appetite and maintain body weight. The mRNA of leptin is expressed only in mature adipose cells. To clarify the regulation of leptin gene expression in adipocytes, 3T3-L1 adipocytes were treated for 16 h with various agents known to modulate lipid metabolism, and then the leptin mRNA was measured by the reverse transcription-polymerase chain reaction method. Interestingly, both norepinephrine and isoproterenol reduced the level of leptin mRNA to about 20% of that found in untreated control cells in a dose-dependent fashion. The maximum reduction occurred at 100 nmol/l of either norepinephrine or isoproterenol, and the half-maximal effect was observed at approximately 3 nmol/l norepinephrine and approximately 1 nmol/l isoproterenol. Propranolol reversed about 50% of the reduction by either norepinephrine or isoproterenol. In contrast, phentolamine did not inhibit the reduction by either norepinephrine or isoproterenol. Moreover, both cholera toxin and dibutyryl cAMP decreased the level of leptin mRNA to about 10% of that in control cells in a dose-dependent fashion. The maximum effect was elicited at 100 ng/ml cholera toxin and 100 micromol/l dibutyryl cAMP. The concentration producing the half-maximal effect was approximately 1 ng/ml cholera toxin and approximately 50 micromol/l dibutyryl cAMP. Dibutyryl cGMP, however, did not affect leptin gene expression. These results suggest that a signaling pathway that results in the activation of protein kinase A regulates leptin gene expression in 3T3-L1 adipocytes.

On the preparation and characterization of standard materials for natural human proinsulin and C-peptide.

The preparation and characterization of intact native human proinsulin and C-peptide to serve as reference standards in immunologic and biologic assays is described. The major difficulty encountered in the preparation of human proinsulin from crystalline human insulin is the rather low yield (0.08 per cent), which may be due to postmortem autolysis in the pancreatic specimens. Under such circumstances, minor degrees of contamination of batches by fresh pancreases from other species, such as cows or pigs, may be magnified considerably. Such contamination can be readily detected with various currently available proinsulin- or C-peptide-specific antisera. Autolysis before or during preparation may also affect the yields and quality of human C-peptide preparations. Some of these C-peptide by-products are described, along with methods for their purification and characterization.

Starvation-induced changes of somatostatin, glucagon, and insulin secretion from the isolated perfused rat pancreas.

The effect of 16- and 48-h fasting on pancreatic somatostatin, insulin, and glucagon secretion was studied, using the isolated perfused rat pancreas. In the presence of 4.4 mM glucose, basal somatostatin and insulin concentrations in the perfusate were significantly lower in 48-h fasted rats than in fed animals, whereas basal glucagon secretion was significantly elevated in fasted rats. The infusion of 19 mM arginine significantly augmented secretion of somatostatin and glucagon and attenuated insulin secretion in 48-h fasted rats. It is concluded that fasting causes a decrease in basal pancreatic somatostatin secretion in vitro, although the response to arginine is rather exaggerated. Insulin and glucagon secretion also changed during the fasting. These results suggest that not only insulin and glucagon, but also somatostatin contribute to nutrient homeostasis.

Somatostatin-like immunoreactivity in human peripheral plasma measured by radioimmunoassay following affinity chromatography.

Somatostatin-like immunoreactivity (SLI) concentrations were determined in human peripheral plasma using affinity chromatography followed by radioimmunoassay. In normal subjects, fasting SLI ranged from 2.9 to 22.0 pg/ml with a mean +/- SE value of 10.2 +/- 2.1 pg/ml. In totally pancreatectomized or gastrectomized patients, fasting SLI levels were not different from the values in normal subjects. In patients with medullary thyroid carcinoma, fasting SLI ranged from 11.8 to 71.0 pg/ml with a mean of 29.3 +/- 12.3 pg/ml, which was significantly higher than normal values (P less than 0.01). Following meal ingestion, plasma SLI increased significantly in normal subjects from a basal level of 9.1 +/- 2.1 pg/ml to a peak value of 15.4 +/- 2.9 pg/ml (P less than 0.02). These results indicate that radioimmunoassay combined with affinity chromatography provides an accurate method of measuring SLI in human plasma.

Determination of free and total insulin and C-peptide in insulin-treated diabetics.

Serum levels of free and total insulin as well as total C-peptide immunoreactivity (C-peptide and proinsulin) and C- peptide were measured in insulin-treated diabetics with circulating insulin antibodies by the addition of polyethylene glycol (PEG) before and after acidification. PEG resulted in complete precipitation of insulin antibodies from serum and made it possible to measure free insulin in the supernatant. Incubation of serum at 37 degrees C. for two hours before addition of PEG resulted in values for free insulin that probably resembled the in-vivo levels most closely. The same method could also be used to remove proinsulin bound to circulating insulin antibodies and permitted the measurement of C-peptide in the supernatant. Clinical studies using this approach indicate that combined measurements of serum free and total insulin and C-peptide provide information that is helpful in understanding the contribution of endogenous and exogenous insulin to the course and metabolic control of insulin-requiring diabetic patients.

Trial of insulinlike growth factor I therapy for patients with extreme insulin resistance syndromes.

Extreme insulin resistance occurs in patients with primary defects in insulin action at the receptor or postreceptor levels. The condition commonly is associated with acanthosis nigricans and ovarian masculinization. Despite a marked increase in insulin secretion, some patients develop frank diabetes mellitus that does not respond adequately to insulin therapy. Insulinlike growth factor I exerts metabolic effects similar to those of insulin. This study assessed the potential effectiveness of IGF-I as a blood glucose lowering agent in patients with extreme insulin resistance syndromes, including type A insulin resistance, congenital generalized lipodystrophy, and leprechaunism. Among the 11 patients studied, some exhibited mutated insulin receptors, whereas others were suspected to have defects in postreceptor sites. In each patient, plasma glucose levels decreased in response to subcutaneous injections of recombinant human IGF-I (0.1-0.3 mg/kg body wt). The degree of the decrease was roughly comparable with that observed in normal individuals. IGF-I also reduced plasma insulin concentrations. A long-term trial of IGF-I (up to 16 mo) showed that IGF-I (0.1-0.4 mg/kg body wt twice daily) is effective in lowering both fasting and postprandial plasma glucose concentrations with decreases in both fructosamine and HbA1c values. Improvement of acanthosis nigricans was observed in some of the patients. These results suggest that recombinant human IGF-I could be used clinically as a hypoglycemic agent in diabetic patients with extreme insulin resistance in whom insulin treatment is ineffective.

Regulation of glucose-transporter gene expression by insulin in cultured human fibroblasts.

To clarify the effect of insulin on glucose-transporter (GT) biosynthesis, we determined GT mRNA levels in human cultured skin fibroblasts, using HepG2 GT cDNA as a probe. Insulin specifically increased the GT mRNA level in a time- and dose-dependent manner. Time-course study demonstrated that the mRNA level peaked within 3 h of insulin (1 x 10(-7) M) addition. After remaining elevated for several hours, mRNA decreased and returned to the basal level after 24 h. In the cell strains from seven normal subjects, the mean (+/- SE) GT mRNA level determined after 3 h of treatment with 1 x 10(-7) M insulin was 164.3 +/- 8.5% of the level found in untreated control cells. The insulin dose-response curve of GT mRNA levels showed that the maximum stimulation was elicited at 1 x 10(-7) M, and the half-maximum stimulation occurred at approximately 5 x 10(-10) M. Degradation rates of GT mRNA determined in the presence of actinomycin D were not different between insulin-treated and untreated cells. These results suggest that insulin increases GT gene expression in cultured human fibroblasts.

Heterogeneity of circulating human C-peptide.

Values reported for serum C-peptide immunoreactivity in healthy individuals vary considerably. To assess factors that contribute to this finding, three human C-peptide assay systems were developed utilizing three distinct antisera that react differently with various C-peptide fragments. Preparations of natural pancreatic and synthetic human C-peptide standards were compared immunologically in these systems. The curves of the natural C-peptide and the synthetic preparations were not identical. The relative immunoreactivity of each standard varied, depending on the particular antiserum used. Serum C-peptide concentrations varied when measured in the different assay systems. Furthermore, the results of dilution and recovery tests and stability of the C-peptide during storage showed differences among the three assays. Gel filtration of serum indicated heterogeneity within the major C-peptide peak, and in addition, a smaller peak of lower molecular weight material was present in some samples. Although degradation of serum C-peptide may occur during storage, fragments of C-peptide may also be secreted or arise during in-vivo metabolism. Thus, the present studies indicate the need for careful standardization and checking of each particular assay system before its use in clinical studies.

Characterization of seven C-peptide antisera.

The plasma C-peptide immunoreactivity (CPR) in 10 normal subjects varied considerably when measured with different antisera in parallel assays. The CPR level correlated with the blank "CPR" value measured in plasma devoid of C-peptide and to a lesser degree with the sensitivity of the standard curves obtained with the individual antisera. Storage of plasma samples at different temperatures and for different lengths of time before the analyses were carried out resulted in further variation in the CPR results. This was caused by a time- and temperature-dependent fall in CPR, which was more pronounced with some antisera than with others. This sensitivity to storage of plasma did not correlate with the antigenic characteristics of the antisera as determined by their reactivity with 11 specific fragments of the C-peptide molecule. The contribution of human proinsulin to the CPR concentration relative in normal subjects was considered to be negligible even though the relative immunoreactivity of human proinsulin and C-peptide ranged from 11 to 143 per cent among these antisera. These results suggest that differences in C-peptide antisera are a major reason for the variation in the concentration of circulating CPR as measured in different C-peptide immunoassays.

A new approach to the detection of autoantibodies against insulin receptors that inhibit the internalization of insulin into human cells.

It has been shown that the conjugate of the fragment A of diphtheria toxin to insulin is cytotoxic to cultured cells bearing insulin receptors, apparently through the endocytosis of fragment A. We examined the effect of autoantibodies against insulin receptors on the cytotoxicity of the conjugate. The conjugate was cytotoxic to a rat fibroblast cell line that was resistant to the intact toxin, and the cytotoxicity was inhibited by exogenous insulin, indicating that the fragment A underwent endocytosis through insulin receptors. Immunoglobulins from three patients with type B syndrome of insulin-resistant diabetes blocked the cytotoxicity of the conjugate to Chang's liver cells in a dose-dependent manner. When the cells were pretreated with the immunoglobulins, cytotoxicity of the conjugate was also blocked. These results suggest that autoantibodies against insulin receptors interfere with the binding of the conjugate to insulin receptors or with the endocytosis of fragment A after binding. This assay system seems useful for detecting autoantibodies against the determinants that are involved in the internalization of the ligand-receptor complex.

Differential activation of mitogen-activated protein kinase by insulin and epidermal growth factor in 3T3-L1 adipocytes: a possible involvement of PI3-kinase in the activation of the MAP kinase by insulin.

Mitogen-activated protein (MAP) kinase plays crucial roles in cell growth and differentiation. It has recently been shown that the MAP kinase cascade in growth factor signaling diverges and cross-talks with other signaling pathways. In the present study, we examined the effects of wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), on the activation of Ras, Raf-1 kinase, and MAP kinase by insulin and epidermal growth factor (EGF). The effect of LY294002, a structurally distinct PI3-kinase inhibitor, on the activation of Raf-1 kinase by both ligands was also examined. In 3T3-L1 adipocytes, 25 nmol/l wortmannin inhibited the insulin-induced activation of Raf-1 kinase to the basal level, whereas the same dose of wortmannin had little effect on the EGF-induced activation of Raf-1 kinase. One hundred micromol/l LY294002 blocked insulin-induced activation of Raf-1 kinase without affecting EGF-induced activation of this kinase. Twenty-five nmol/l wortmannin inhibited the insulin-induced activation of MAP kinase to the basal level with no effect on the EGF-induced activation of this kinase. But the same dose of wortmannin did not affect the formation of guanosine 5'-triphosphate (GTP)-bound Ras stimulated by either ligand. In KB cells, results similar to those in 3T3-L1 adipocytes were obtained. In contrast, in Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-HIR cells), neither wortmannin nor LY294002 inhibited the insulin-induced activation of Raf-1 kinase, and wortmannin had little effect on the activation of MAP kinase by insulin. These results indicate that 1) PI3-kinase or wortmannin-sensitive molecules are involved in the interaction between activated Ras and Raf-1 kinase in the insulin signaling in 3T3-L1 adipocytes, 2) the involvement of PI3-kinase or wortmannin-sensitive molecules in the insulin-induced activation of MAP kinase appears to be cell-type specific, and 3) differential mechanisms to activate Raf-1 kinase and MAP kinase by insulin and EGF exist.

Clinical significance of circulating C-peptide in diabetes mellitus and hypoglycemic disorders.

Proinsulin is converted to insulin and C-peptide in the pancreatic in the pancreatic beta cells: the latter two peptides are secreted in equimolar concentrations. Thus, measurements of serum C-peptide provide a means of assessing pancreatic beta cell function in addition to that of insulin. This technique has proved particularly useful in insulin treated diabetic patients in whom the development of circulating insulin antibodies interferes with the radioimmunoassay of the hormone. The C-peptide assay has also been used to facilitate the diagnosis of various hypoglycemic conditions, including islet cell tumors and factitious injection of insulin. The extraction of C-peptide in the urine reflects average serum values over a period of time and urine C-peptide measurements are especially useful in children or individuals in whom repeated blood sampling is difficult.

A novel substrate for insulin-sensitive serine/threonine kinase in intact cells.

We have studied insulin-stimulated threonine phosphorylation of cellular proteins by immunoblotting and immunoprecipitation using antiphosphothreonine antibody (anti-P-Thr). A 50-kilodalton protein (p50) was found to be greatly phosphorylated on threonine residues upon insulin stimulation in intact rat hepatoma cells (Fao) and Chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR). Insulin induced threonine phosphorylation of this protein in a dose-dependent manner, with an ED50 of 3-6 x 10(-9) M. The 50-kilodalton phosphoprotein (pp50) was detectable 20 min after exposure of the cells to insulin, and phosphorylation reached a maximum after 90 min. Immunoprecipitation of pp50 with anti-P-Thr required extraction of the cellular proteins with sodium dodecyl sulfate and dithiothreitol, and subcellular fractionation of the cells revealed that pp50 is present in the membrane fraction, implying that pp50 is a protein integrated into the membrane component in the cells. Tryptic phosphopeptide mapping of the pp50 was distinct from that of the insulin receptor beta-subunit. Phosphoamino acid analysis of the pp50 demonstrated that insulin increased phosphorylation, mainly of threonine and moderately of serine, whereas pp50 did not contain phosphotyrosine. Cycloheximide, a protein synthesis inhibitor, did not affect the insulin-induced appearance of pp50 in the cells. pp50 was not detectable in A431 cells and KB cells stimulated by epidermal growth factor. These data suggest that p50 is a novel endogenous substrate for insulin-sensitive serine/threonine kinase in intact cells.

Heterogeneity of circulating C-peptide.

Serum C-peptide levels vary when measured with different immunoassay systems. In order to assess the factors contributing to this finding, serum C-peptide was measured in two assays utilizing different antisera, but the same standards and labeled peptide. The antisera were characterized with synthetic C-peptide fragments and their reactivities towards some of these fragments differed. The results of dilution and recovery tests and stability of the C-peptide during storage showed differences between the two assays. Gel filtration experiments indicated heterogeneity within the major C-peptide peak, and, in addition, a smaller peak of lower molecular weight material was present in some sera. Although degradation of serum C-peptide may occur during storage or with freezing and thawing, fragments of C-peptide may also be secreted or arise during in vivo metabolism.

Hypoglycemia and endogenous hyperinsulinism complicating diabetes mellitus. Application of the C-peptide assay to diagnosis and therapy.

Described here is a patient with insulin-requiring diabetes mellitus in whom spontaneous fasting hypoglycemia developed. Endogenous hyperinsulinism was considered after the systematic exclusion of other causes of hypoglycemia, and this possibility was confirmed by measurement of serum C-peptide reactivity (CPR), an indicator of beta cell secretory function. Subtotal pancreatectomy relieved the fasting hypoglycemia, and was associated with a marked decline in CPR levels. The pancreatic islets showed hyperplasia and the beta cells were degranulated.