Structural analysis of normal and mutant insulin receptors in fibroblasts cultured from families with leprechaunism.

Leprechaunism is an inherited disorder characterized by insulin resistance and intrauterine growth restriction. In this study we analyze insulin binding and subunit structure of the insulin receptor in dermal fibroblasts cultured from three unrelated families whose probands (Ark-1, Atl, and Minn) were affected by leprechaunism. Cells cultured from all three probands had markedly reduced insulin binding at equilibrium. Fibroblasts cultured from the parents of Ark-1 and Atl had partial and differing degrees of impairment in insulin binding. The structure of the alpha subunit of insulin receptors was analyzed by cross-linking 125I-insulin to plasma membranes. A major band of 350 kilodaltons (kD) (corresponding to the heterotetrameric insulin receptor alpha 2 beta 2) was observed in control and leprechaun fibroblasts. The relative amount of radioactivity cross-linked to plasma membranes reflected the genetic variations seen in insulin binding to intact cells. In reducing gels, 125I-insulin was cross-linked equally to a 250-kD (alpha-alpha dimer) and a 125-kD (alpha monomer) protein in cells from controls, the parents of Ark-1 and Atl, and probands Atl and Minn. By contrast, cells from the Ark-1 proband had diminished cross-linking of alpha-alpha dimers. The ratio of dimer to monomer in cells from controls was 0.93 +/- 0.06, and that in cells from Ark-1 was 0.31 +/- 0.19 (P less than .01). Beta-subunit structure and function was analyzed by studying insulin-enhanced autophosphorylation. Although maximal stimulation of beta-subunit phosphorylation was reduced to 30% in proband Ark-1 fibroblasts, this reduction was quantitatively related to reduced insulin binding. These results indicate that mutations causing severe insulin resistance and defective insulin binding are transmitted with autosomal recessive patterns of inheritance and that heterogeneity exists for these mutations. The mutation in pedigree Ark-1 most likely produces conformational changes in alpha-subunit interaction.     

Molecular genetics of severe insulin resistance

Leprechaunism and type A diabetes represent inborn errors of insulin resistance whose phenotypes suggested causation by mutations in the insulin receptor gene. Cells cultured from patients with leprechaunism specifically lacked high-affinity insulin binding. Partial but different degrees of impairment were observed in cells cultured from first-degree relatives. Different mutations in the insulin receptor's alpha subunit were proposed in different families (Ark-1, Atl, Minn, Mount Sinai) based on phenotype, cellular insulin binding, and insulin receptor structure. Molecular cloning and sequencing of mutant insulin receptor cDNA from family Ark-1 confirmed that the proband inherited a maternal missense and a paternal nonsense mutation in the alpha subunit and was a compound heterozygote. The insulin receptor was immunologically present on the plasma membrane of fibroblasts cultured from patients Ark-1 and Atl but was markedly reduced in cells from patients Minn and Mount Sinai. In cells from patient Minn, but not from patient Mount Sinai, the decreased number of insulin receptors was associated with reduced insulin receptor mRNA. In two families with the less severe form of insulin resistance, type A diabetes, mutations altered post-translational processing of the insulin receptor molecule. At a cellular level, these mutations of the alpha subunit of the insulin receptor shared defective binding and impaired stimulation of sugar transport by insulin. In family Atl, however, glucose uptake was constitutively increased. Thus, genetic variation in the insulin receptor gene causes a spectrum of inherited insulin-resistant syndromes and altered cellular signaling.     

Analysis of expression of growth factor receptors in replicatively and oxidatively senescent human fibroblasts

Replicatively and oxidatively senescent human fibroblasts demonstrate an impaired response to mitogens. To investigate whether this is due to downregulation of growth factor receptors we examined their expression in these two types of senescence. mRNA and protein levels of the insulin receptor and platelet-derived growth factor (PDGF) alpha-receptor decreased in replicatively senescent cells. The PDGF beta-receptor and insulin-like growth factor 1 receptor at the protein level also decreased but remained readily detectable. However, these major growth factor receptors remained unchanged in oxidatively premature senescent cells. This suggests that mechanisms underlying diminished responsiveness to mitogens might be different in replicative senescence and oxidatively premature senescence.     

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.     

A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance.

We have determined the primary structure of a mutant insulin receptor of a leprechaun patient born from a consanguineous marriage. A characteristic feature of leprechaunism is an extreme resistance to insulin. In this patient the insulin resistance seems to result from an observed lack of insulin binding to intact cells. Solubilization of cells in non-ionic detergents leads to the appearance of insulin receptors which can bind insulin. However, the insulin-stimulated autophosphorylation of the receptor's beta subunit is markedly reduced. Cloning and sequencing of cDNA derived from insulin receptor mRNA of this patient revealed a leucine-to-proline mutation at position 233 in the alpha subunit. By means of DNA amplification we found that the patient is homozygous for this mutation and that the parents and two grandparents from the consanguineous line are heterozygous. The heterozygous individuals all show decreased insulin binding to cultured fibroblasts. In addition, they are mildly insulin resistant in vivo. These observations show a linkage between the leucine-to-proline mutation and the observed insulin resistance in this family. We therefore conclude that the mutation in the homozygous form is responsible for the extreme insulin resistance in the leprechaun patient. The mutation for the first time characterizes a region in the insulin receptor which seems to be involved in transmitting the insulin binding signal to the tyrosine kinase domain.     

Modulation of expression of insulin and IGF-I receptor by Epstein-Barr virus and its gene products LMP and EBNA-2 in lymphocyte cell lines

The receptors for insulin and insulin-like growth factor I (IGF-I) are two closely related integral membrane glycoproteins involved in signalling of cell growth and metabolism. We have used the unique paradigm of pairs of Burkitt lymphoma cell lines (BLO2, BL30, BL41) with or without Epstein-Barr Virus (EBV) infection and cells transfected with EBV-related genes to examine effects of EBV on expression of these receptors at the gene and protein functional level. In BL30 and BL41 cells, EBV infection increased surface insulin binding and total receptor number by 2-and 18-fold, respectively. By contrast, EBV infection decreased total IGF-I receptors by 29 to 87% in all three cell lines. In general, there was a correlation between total receptor concentration and the level of insulin or IGF-I receptor mRNAs, although in one cell line insulin binding increased while receptor mRNA levels decreased slightly, suggesting posttranslational effects. BL41 cells transfected with a vector expressing the EBV latent membrane protein (LMP) exhibited a 2.6- to 3.2-fold increase in insulin receptor expression, whereas cells transfected with EBNA-2 (one of the EBV nuclear antigens) alone exhibited no effect. However, EBNA-2 appears to be required for the EBV effect on insulin receptor expression since cells infected with a mutant virus, P3JHRI, which lacks the EBNA-2 gene failed to show an increase in insulin receptor number. These data indicate that EBV infection of lymphocytes increases expression of insulin receptors while simultaneously decreasing expression of IGF-I receptors. The magnitude and sometimes even the direction of change, depends on host cell factors. A maximal increase in insulin receptors appears to require the coordinate action of several of the EBV proteins including LMP and EBNA-2. © 1993 Wiley-Liss, Inc.     

Insulin-receptor autophosphorylation and kinase activity are constitutively increased in fibroblasts cultured from a patient with heritable insulin-resistance

Mutations in the insulin receptor gene have been described in families with the inherited insulin-resistant syndrome leprechaunism. At a cellular level, these mutations result in decreased insulin binding and impaired insulin stimulation of receptor autophosphorylation and sugar transport. By contrast, we previously found that fibroblasts cultured from leprechaun patient Atl had constitutively increased sugar transport, even though insulin binding was markedly reduced. Here we report that these fibroblasts have basal insulin-receptor autophosphorylation and kinase activity constitutively increased above insulin-stimulated control cells.     

Reduced mRNA and a nonsense mutation in the insulin-receptor gene produce heritable severe insulin resistance.

Leprechaunism is an autosomal recessive syndrome of severe insulin resistance and is characterized by intrauterine growth restriction, acanthosis nigricans, hirsutism, and loss of glucose homeostasis. Here we report a new female patient of Hispanic and Afro-American descent whose fibroblasts and lymphoblasts had markedly impaired insulin binding (less than 10% of that in controls). Insulin binding to lymphoblasts established from both unrelated parents was partially impaired. Insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) binding to the patient's fibroblasts were within the normal range. Insulin stimulation of receptor autophosphorylation and kinase activity was markedly reduced in the patient's fibroblasts. The patient's fibroblasts had both a reduced number of immunoreactive insulin receptor (6% of those in controls) and concomitantly reduced amounts of insulin-receptor mRNA, suggesting that both mutations inherited by the patient reduced insulin-receptor mRNA. Sequencing of the insulin-receptor gene and cDNA indicated that the patient was heterozygous for a paternally derived mutation at bp 1333, converting Arg372 to a STOP codon. This nonsense mutation was observed in the insulin-receptor gene, but not in cDNA, indicating reduced amounts of mRNA for the allele containing this mutation. The coding sequence of the maternally inherited insulin-receptor allele was normal. Both the marked reduction in insulin-receptor mRNA in the compound heterozygous fibroblasts of the proband and the partially reduced insulin binding in maternal cells suggest that the maternally derived mutation is located in an insulin-receptor gene sequence that controls cellular mRNA content.     

Coupling of insulin-responsive glucose transport to receptors for insulin-like growth factor 1 in primary human fibroblasts

We have recently described an insulin-resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine----proline mutation at amino acid position 233 in the alpha-chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2-deoxyglucose uptake by insulin and insulin-like growth factor 1 (IGF-1). Insulin hardly stimulates basal 2-deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2-deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF-1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF-1. With both types of fibroblasts, maximal IGF-1 response is reached at about 10 nM IGF-1, the ED50 being approximately 4 nM. The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF-1. Insulin binds with an approximately 200-fold lower affinity to IGF-1 receptors, compared to homologous IGF-1 binding. As an insulin concentration of 10 microM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF-1, it seems that occupation of IGF-1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.     

Downregulation of Wnt-Mediated ROS generation is causally implicated in leprechaunism

Although mutations in the insulin receptor have been causally implicated with leprechaunsim, the full pathophysiology of the syndrome cannot be accounted for by malfunction of this gene alone. We sought to characterize a connection between Wnt-mediated cell signaling and the production of reactive oxygen species (ROS) which revealed a novel mechanistic basis for understanding the pathogenesis of leprechaunism. To identify candidate genes involved in this process, a PCR-based subtractive hybridization was performed. Candidate genes were examined for interaction with the Wnt signaling pathway and ROS generation. We found that Dickkopf 1 (Dkk1), a Wnt inhibitor, is overexpressed in skin fibroblast cells derived from three leprechaunism patients and that the cells showed an impaired response to Wnt2 in terms of β-catenin-Tcf activation. Knockdown of Dkk1 in the patient cell lines rescued Wnt2-mediated Tcf activation. Concerted action of Wnt2 and knockdown of Dkk1 resulted in enhanced Nox4 expression and PDGF-induced ROS generation compared to parental patient cells. Furthermore, we found that NFATc2 was activated in response to Wnt2 stimulation and directly activates Nox4 expression. These data show a crosstalk between Wnt and ROS pathways which in turn provides new mechanistic insights at the molecular level into the pathogenesis of leprechaunism.     

Developmental regulation of insulin receptor gene in sciatic nerves and role of insulin on glycoprotein P0 in the Schwann cells

In view of the observations that Schwann cells contain insulin receptors, in the present study, we have investigated the developmental regulation of insulin receptor gene in the sciatic nerves of different postnatal age group rats. We have also investigated the role of insulin in the expression of the major PNS myelin glycoprotein P zero (P0) in normal as well as high glucose conditions in primary rat Schwann cells. The expression of insulin receptor gene in sciatic nerves appeared to be differentially regulated. The steady-state levels of insulin receptor mRNA increased remarkably during development and after postnatal day 10, when the peak of myelin structural gene (P0) expression occur and slowly increased further until at least postnatal day 90 in parallel with the growth of the myelin sheath. By employing immunofluorescence and RT-PCR, we observed significant increase in the P0 protein and mRNA levels in Schwann cells in response to the insulin than in insulin deprived counterparts. The presence of insulin in the high glucose medium ameliorated the altered protein and mRNA of P0 in Schwann cells compared to the insulin deprived counterparts. These studies demonstrate the importance of insulin and its receptor as possible regulatory factors in the PNS and also emphasizes their novel therapeutic applications in demyelinating diseases, especially in diabetic poly-neuropathy.     

Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: Relation to human somatomedins and insulin

The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component ot adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.     

A leucine to proline mutation at position 233 in the insulin receptor inhibits cleavage of the proreceptor and transport to the cell surface

We have previously shown that a homozygous mutation encoding a substitution of proline for leucine at position 233 in the insulin receptor is linked with the syndrome of leprechaunism, being a lethal form of insulin resistance in newborn children. Specific binding of insulin and insulin-stimulated autophosphorylation of the insulin receptor are nearly absent in fibroblasts from the leprechaun patient. To examine the molecular basis of the observed insulin receptor abnormalities, CHO cell lines overexpressing mutant insulin receptors were made by transfection. The results show that the mutation inhibits cleavage and transport of the proreceptor from intracellular sites to the cell surface. As the mutant receptor is poorly precipitated by two different monoclonal antibodies recognizing epitopes on undenatured wild-type alpha-subunits, the mutation probably affects overall folding of the alpha-subunit. The mutant proreceptor is unable to bind insulin and exhibits no insulin-stimulated autophosphorylation. These data explain the abnormalities seen in the patient's fibroblasts. Pulse-chase labeling experiments on transfected cells show that the mutant precursor has an extended half-life (approximately 5 h) compared to the precursor of wild-type insulin receptors (approximately 2 h). This mutation is the first example of a naturally occurring mutation in the insulin receptor which completely blocks cleavage of the proreceptor and transport to the cell surface.     

Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man

Two previously reported insulin receptor cDNA sequences differ by 36 base pairs (bp) in the distal alpha-subunit, suggesting that alternative mRNA splicing within the coding region may occur (two insulin receptor isoforms). We developed a quantitative modification of the polymerase chain reaction technique in order to detect and characterize differential mRNA splicing at this site within the distal alpha-subunit. Using RNA derived from a variety of human cell types, we detected two polymerase chain reaction-amplified cDNA species reflecting the presence or absence of the above 36 nucleotides. Identity of the two cDNA species was confirmed by Southern blots, the use of a BANI restriction site present only in the 36 base pair segment and dideoxy sequencing. The relative expression of the two mRNA forms varied markedly in a tissue-specific manner. Buffy coat leukocytes and Epstein-Barr virus-transformed lymphocytes express only the shorter mRNA. Placenta expresses both species equally; muscle, isolated adipocytes and cultured fibroblasts express somewhat more of the longer mRNA (relative ratios of mRNA abundance of 1.51, 3.18, and 2.77, respectively); liver expresses mostly the longer mRNA (relative ratio of 9.8). In RNA derived from cultured and fresh cells from patients with several states of insulin resistance, the relative expression of the two mRNA species was similar to results obtained with comparable normal tissues. Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.     

Loss of insulin binding and insulin receptor mRNA in a transformed human fetal fibroblast cell line

Insulin binding and insulin receptor gene expression have been assessed in cultured fetal (WI38) and SV40 transformed fetal (WI38/VA13) human fibroblasts to determine whether transformation influences the expression of insulin receptors. The transformed cell line had virtually no insulin binding and extremely low levels of insulin receptor mRNA. No apparent gene deletion or rearrangement was detected and therefore the marked decrease in insulin receptor gene expression seen in WI38/VA13 cells is an important example of negative regulation of insulin receptor gene expression. This cell line could serve as a model for studies of the mechanism for negative regulation of insulin receptor gene expression. Overexpression of the insulin receptor gene in these cells may reveal insights into the role of the insulin receptor in tumor biology.