Cell surface alteration in Epstein-Barr virus-transformed cells from patients with extreme insulin resistance.

An abnormality was detected in the morphology of the cell surface of Epstein-Barr virus-transformed lymphocytes of patients with genetic forms of insulin resistance. In cells from two patients with leprechaunism and two patients with type A extreme insulin resistance, scanning electron microscopy demonstrated a decrease in the percentage of the cell surface occupied by microvilli in cells from the patients with leprechaunism and type A insulin resistance compared with control cells. When cells from a healthy control subject and one of the patients with leprechaunism (Lep/Ark-1) were incubated with 125I-labeled insulin, there was a decrease in the percentage of 125I-insulin associated with microvilli on the cell surface. Thus, the decreased localization of insulin receptors with the microvillous region of the cell surface was in proportion to the decrease in microvilli.

Atypical antiinsulin receptor antibodies in a patient with type B insulin resistance and scleroderma.

We studied a 23-yr-old woman with scleroderma and type B insulin resistance. The association with autoimmune disease suggested that the insulin resistance resulted from autoantibodies to the insulin receptor. However, in preliminary studies, serum antireceptor antibodies were not detected in an assay that measures the ability of the antibodies to inhibit insulin binding to the insulin receptor. Antireceptor antibodies were subsequently detected by their ability to immunoprecipitate affinity-labeled receptors. After the patient had received immunosuppressive therapy with prednisone and cyclophosphamide for 3 months, her insulin resistance remitted, and she developed hypoglycemia. Simultaneously with the remission of insulin resistance, the titer of serum antireceptor antibody (measured by the immunoprecipitation assay) fell to less than 1% of the previous level. In a series of 21 patients, this is the first patient with antireceptor antibodies that bound to the insulin receptor without inhibiting insulin binding.

Defects in human insulin receptor gene expression.

The insulin receptor plays a central role in mediating the biological actions of insulin. We have used Epstein-Barr virus-transformed lymphocytes (EBV-lymphocytes) to investigate the receptor defects in patients with genetic forms of insulin resistance. Within the normal population, we found a close correlation between the number of insulin receptors on the surface of EBV-lymphocytes and the cellular content of insulin receptor mRNA. In addition, we have used the cloned human insulin receptor cDNA to investigate the nature of the mutations causing the reduction in the number of insulin receptors in EBV-lymphocytes from three insulin resistant patients. One patient with leprechaunism has a marked reduction in the level of receptor mRNA, which probably accounts for the extremely slow rate of receptor biosynthesis measured in this patient's cells. The remaining two patients with type A extreme insulin resistance are sisters, the products of a consanguineous marriage, who have normal levels of insulin receptor mRNA. We have previously shown that the insulin receptor precursor is synthesized at a normal rate in these patients' cells, thus suggesting a defect in the posttranslational processing of the receptor or in its translocation to the plasma membrane.

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]glucosamine, 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 Mr of 190,000. It undergoes endopeptidase cleavage and further posttranslational 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 rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin receptor biosynthesis in cultured lymphocytes from insulin-resistant patients.

In some patients with genetic forms of extreme insulin resistance, the cause of insulin resistance is a marked (greater than or equal to 90%) reduction in the number of insulin receptors on the cell surface. In the present work, we describe studies of insulin receptor biosynthesis in Epstein-Barr virus (EBV)-transformed lymphocytes from three patients (A-1, A-5, and A-8) with type A extreme insulin resistance. Insulin receptors are composed of two major glycoprotein subunits (apparent molecular weight [Mr] of 135 and 95 kD), which are both derived from a common precursor molecule with Mr of 190 kD. In one patient (A-1), there was a marked reduction in the biosynthesis of both the 190-kD precursor and the mature receptor. Thus, in this patient, the defect appears to occur early in the biosynthetic pathway (i.e., before the synthesis of the 190-kD precursor). In contrast, in two sisters (A-5 and A-8) with type A extreme insulin resistance, biosynthesis of the 190-kD precursor proceeds at a normal rate. However, there appears to be a defect subsequent to the biosynthesis of the 190-kD precursor, but before the insertion of the mature receptor in the plasma membrane. Our observations suggest the existence of at least two distinct types of biosynthetic defects which may give rise to a marked reduction in the number of insulin receptors on the cell surface. In addition, for comparison, we have studied receptor biosynthesis in cultured EBV lymphocytes from a fourth patient (A-7) with type A extreme insulin resistance. Although the cells of patient A-7 have a normal number of insulin receptors, we have detected subtle abnormalities in the posttranslational processing of the insulin receptor precursor, which may be a biochemical marker for a postbinding defect that causes insulin resistance in this patient.

An in vitro binding assay which differentiates benzodiazepine 'agonists' and 'antagonists'.

An in vitro test which discriminates benzodiazepine "agonists' and 'antagonists' has been developed by exploiting the apparent differences in modulation of the benzodiazepine receptor by these classes of compounds. In the presence of 10 microM GABA, the potency of benzodiazepine 'agonists' (i.e., compounds which bind to the benzodiazepine receptor with a relatively high affinity and share pharmacologic properties with benzodiazepines) to displace [3H]3-carboethoxy-beta-carboline is significantly increased. In contrast, the potency of benzodiazepine 'antagonists' (compounds which have been demonstrated to antagonize some of the pharmacologic actions of benzodiazepines) is not altered by GABA. Several chemically diverse classes of compound have been examined in this test, and within the limited number of compounds examined, this test accurately predicts 'agonist' and 'antagonist' actions in vivo.