The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.

Leprechaunism is a rare genetic disorder characterized by severe growth retardation and insulin resistance. Maximal epidermal growth factor (EGF) binding was reduced in fibroblasts from three unrelated patients with leprechaunism (Ark-1, Can-1, and Minn-1) compared with control (0.8-2.2%/mg protein vs. 5.5%/mg protein). This was due to a decrease in receptor affinity in Ark-1 and Can-1 and a decrease in receptor number in Minn-1. In all cell lines, EGF-stimulated receptor autophosphorylation was also decreased to 18-60% of control, whereas EGF internalization and degradation was normal. Sphingosine (40 microM), a protein kinase C inhibitor, increased EGF receptor affinity twofold in control cells and six- to nine-fold in cells of leprechaunism. However, sphingosine did not enhance EGF-stimulated receptor autophosphorylation in either the controls or the patients' cells. By contrast, only one of the three cell lines of patients with the type A syndrome demonstrated a decrease in EGF binding and all demonstrated normal or near normal EGF-stimulated receptor autophosphorylation. These data indicate that in patients with leprechaunism, there are functional abnormalities of the EGF receptor, as well as of the insulin receptor, that may contribute to the severity of the syndrome. These data also suggest a role for the insulin receptor in maintaining normal EGF receptor function in these cells.

Obesity is associated with insulin resistance and type II diabetes mellitus. In the present study, we have characterized hepatic insulin receptor function in two animal models of obesity: the Zucker fatty rat (ZFR), a model of genetic obesity with severe hyperinsulinemia, and the Sprague-Dawley rat with dietary obesity, a model of acquired obesity. Zucker fatty rats were also treated with streptozotocin (STZ) in an effort to examine the effects of relative insulin deficiency and hyperglycemia in the setting of obesity. Using wheat germ agglutinin-purified insulin receptor extracted from liver, no significant difference in insulin binding was identified in either model of obesity. beta-Subunit autophosphorylation was significantly decreased in both obese models relative to that in controls (72% in the obese ZFR and 49% in the overfed Sprague-Dawley model). Kinase activity, as measured by phosphorylation of the 1142-1153 synthetic peptide, was also decreased in both models of obesity by 22% and 64%, respectively. In the Zucker rat, STZ treatment led to an 80% increase in receptor concentration and a further 70% increase in beta-subunit autophosphorylation per receptor, whereas tyrosine kinase activity toward substrate was not altered. Since kinase activity is closely linked to autophosphorylation, we determined the fraction of autophosphorylated (activated) receptors vs. non-phosphorylated (inactive) receptors by using antiphosphotyrosine antibody to precipitate receptors bound with [125I]insulin. There was no significant difference in the percentage of activated insulin receptors in the dietary obese, ZFR, or STZ-treated Zucker rat vs. that in the controls. In all models, the percentage of activated receptors ranged from 32-46% of the total receptor pool. These data suggest that in genetic and acquired obesity, autophosphorylation of the beta-subunit is reduced and is a limiting factor in insulin receptor activation. A similar fraction of all receptors appears to undergo some level of autophosphorylation; however, full autophosphorylation and, thus, activation of the receptor do not occur, and this results in a decrease in kinase activity. This block in autophosphorylation may account for significant reductions in insulin receptor kinase function in obesity.

Insulin and insulin-like growth factor-I (IGF-I) effects on protein phosphorylation were investigated in intact skeletal muscle cells at different stages of differentiation. In undifferentiated L6 myoblasts, stimulation by either insulin or IGF-I, but not IGF-II, led to a 3- to 5-fold increase in phosphorylation of insulin and IGF receptor beta-subunits and the appearance of a 175,000 mol wt (Mr) phosphoprotein (pp175). These effects reached a maximum within 3 min, were maintained for 12 min, and then declined. Dose-response curves for pp175 phosphorylation in response to insulin (ED50 = 2 nM) and IGF-I (ED50 = 0.2 nM) were consistent with occupancy and stimulation of each receptor kinase by its specific hormone. The 175,000 Mr phosphoprotein was not precipitated by antireceptor antibodies, and the phosphoamino acid composition differed markedly from that of insulin and IGF-I receptors, with a 10-fold lower phosphotyrosine/phosphoserine ratio after insulin stimulation. In contrast to insulin and IGF-I receptors, pp175 was not extracted by the nonionic detergent Triton X-100, but required sodium dodecyl sulfate for solubilization. When experiments were carried out with L6 cells after differentiation into skeletal muscle myotubes, hormone-induced phosphorylation of pp175 was almost undetectable. We conclude that pp175 is a phosphoprotein distinct from insulin and IGF-I receptors that is involved in the early phosphorylation events that follow the activation of the insulin and IGF-I receptor kinases. Its disappearance after terminal differentiation of the L6 cells is consistent with a role in hormonal stimulation of cell proliferation.

Phosphotyrosine phosphatase (PTPase) activity in rat liver was measured using a phosphopeptide substrate containing sequence identity to the major site of insulin receptor autophosphorylation. PTPase activity was detected in both cytosolic and particulate fractions of rat liver and produced linear dephosphorylation over a 15-min time course. In rats made insulin-deficient diabetic by streptozotocin treatment (STZ), cytosolic PTPase activity increased to 180% of the control values after 2 d of diabetes and remained elevated at 30 d (P less than 0.02). Gel filtration on Sephadex-75 revealed a single peak of activity in the cytosol in both control and diabetic animals and confirmed the increased levels. In BB diabetic rats, another model of insulin deficiency, the PTPase activity in the cytosolic fraction was increased to approximately 230% of control values. PTPase activity in the particulate fraction of liver was also increased by 30 and 80% after 2 and 8 d of STZ diabetes, respectively. However, this increase was not sustained and after 30 d of STZ diabetes, PTPase activity associated with the particulate fraction in the BB diabetic rat was reduced to approximately 70% of the control levels. Treatment of STZ diabetic rats with subcutaneous insulin or vanadate in their drinking water for 3 d reduced tyrosine PTPase activity in the particulate, but not in the cytosolic fraction. This was associated with a change in blood glucose toward normal. These data indicate insulin deficient diabetes is accompanied by significant changes in hepatic PTPase activity. Since tyrosine phosphorylation plays a central role in the cellular action of insulin receptor, an increase in PTPase activity may be an important factor in the altered insulin action associated with these diabetic states.

Leprechaunism is a genetic form of insulin resistance characterized by severe growth retardation and early death. To clarify the molecular basis of the insulin resistance, we investigated the insulin binding and kinase properties of the insulin receptor and the receptor gene in cultured skin fibroblasts of two patients (Ark-1 and Ark-2) with leprechaunism and in those of three of their parents. Specific insulin binding to fibroblasts was markedly reduced (less than 25% of control) in both patients with leprechaunism but was essentially normal in the parents. In contrast, insulin receptor autophosphorylation in 1% Triton X-100 cell lysates was reduced in both patients and parents. In Ark-1, the 70% reduction in autophosphorylation correlated with the decrease in binding, whereas in Ark-2 and in the three parents included in the study, autophosphorylation of the insulin receptor was reduced below the level accounted for by a change in receptor content. Analysis of the insulin receptor gene by hybridization with the receptor cDNA probes revealed no gross defect in either Ark-1 or Ark-2. Both parents of Ark-2 were heterozygous for a restriction fragment length polymorphism in the beta-subunit detected with Bam HI digestion (observed in 15% of controls). Ark-2 was homozygous for the more common allele of this polymorphism (observed in 84% of controls). Thus, we have biochemically characterized a new family of leprechaunism (Ark-2) and have found insulin receptor phosphorylation defects in their phenotypically normal parents.

We used a ribonuclease cleavage assay to screen for insulin receptor mRNA sequence alterations in 12 patients with syndromes of severe insulin resistance. Uniformly labeled [32P]antisense RNA probes complementary to insulin receptor mRNA were prepared by an SP6 or T7 RNA polymerase transcription reaction. Four probes ranging in size from 670-1470 bases were used to examine the entire 4.2-kilobase receptor protein-coding region. Patient RNA samples were hybridized to individual probes in solution, and mismatched sequences were detected by susceptibility to cleavage by a mixture of RNAses A and T1. The method was validated with insulin receptor mRNAs from cells transfected with cDNA constructs bearing known point and deletion mutations. Alterations in the insulin receptor mRNA sequence of two patients were detected. A patient with the type A syndrome of severe insulin resistance (A2-Boston) had a mutation in the insulin receptor beta-subunit mRNA sequence that localized to the region coding for amino acid residues 1174-1211 near the tyrosine kinase domain. The second alteration was a sequence polymorphism in the insulin receptor alpha-subunit mRNA in a patient with lipoatropic diabetes (LA-2) that localized to a region within amino acids 268-272. Direct sequence analysis revealed that the ribonuclease cleavage sites in patients A2-Boston and LA-2 were due to distinct single base changes in the insulin receptor gene and mRNA. Additional insulin receptor mRNA sequence polymorphisms were also identified as mismatches between the labeled RNA probes used and mRNA from several cultured human cell types. This study demonstrates that ribonuclease cleavage can rapidly detect and localize insulin receptor mRNA sequence mutations and polymorphic variations as small as single base changes. Further analysis of insulin receptor mRNA sequence alterations identified in this way may elucidate a possible genetic basis for functional insulin receptor defects in patients with severe insulin resistance and can also reveal some insulin receptor sequence polymorphisms that occur in the population at large.

A patient with type II diabetes associated with hyperproinsulinemia has been shown to have a point mutation in one insulin gene allele, resulting in replacement of histidine with aspartic acid at position 10 of the B-chain. To investigate the basis of the proinsulin processing defect, we introduced an identical mutation in the rat insulin II gene and expressed both the normal and the mutant genes in the AtT-20 pituitary corticotroph cell line. Cells expressing the mutant gene showed increased secretion of proinsulin relative to insulin and rapid release of newly synthesized proinsulin. Moreover, the mutant cell lines did not store the prohormone nor did they release it upon stimulation with secretagogues. These data indicate that a significant fraction of the mutant prohormone is released via the constitutive secretory pathway rather than the regulated pathway, thereby bypassing granule-related processing and regulated release.