Cell culture studies of a patient with congenital lipoatrophic diabetes—Normal insulin binding with alterations in intracellular glucose metabolism and insulin action

Insulin binding and the action of insulin on several aspects of glucose metabolism have been investigated in cultured fibroblasts derived from a patient with congenital lipoatrophic diabetes and compared to cultures from 9 nondiabetic controls. Incorporation of glucose was elevated in the patient's cells at glucose levels above 0.1 mM. When distribution of labelled glucose was examined, cell associated glycogen and acid soluble material were increased, but the greatest increment was in lactate production. Insulin binding, as indicated by maximum specific ¹²⁵I-insulin binding and concentration of unlabelled insulin for 50% displacement, was normal, although insulin regulation of the insulin receptor was diminished. Insulin stimulation of total glucose incorporation was reduced in cells from the patient. When insulin stimulation of glycogen synthase was measured directly, the response to insulin was also attenuated. On the other hand, insulin stimulation of hexose transport appeared to be unimpaired. The data indicate alterations in both cell glucose metabolism and insulin response which may be related to the observed insulin resistance of this disorder.     

Fluctuation of insulin resistance in a leprechaun with a primary defect in insulin binding

A 3-month-old female leprechaun demonstrated extreme insulin resistance with hyperinsulinemia (330 mumol/L) and resistance to exogenous insulin. Insulin binding to erythrocytes, cultured lymphocytes, and fibroblasts from the patient were decreased to less than 20% of normal, whereas insulin-like growth factor I binding to fibroblasts was normal. Antiinsulin receptor antibody binding to cultured lymphocytes was also decreased to 20% of normal, indicating a decreased concentration of insulin receptors on the cell surface. The ability of insulin to stimulate D-[14C]glucose uptake was decreased to 35% of normal in the patient's fibroblasts, and the dose-response curve was shifted to the right. With time, the insulin resistance fluctuated from near normal (fasting insulin, 244.0 pmol/L) to severe resistance (fasting insulin, 5740-9328 pmol/L), and an insulin tolerance test revealed amelioration of insulin resistance during remission. However, insulin binding to erythrocytes and adipocytes was decreased persistently to 20% of normal. These results indicate that the patient had a primary defect in her insulin receptors, i.e. decreased insulin receptor concentration. The variable degree of insulin resistance was possibly due to variable receptor function in the signal transmission process.     

Mediation of insulin-like growth factor actions by the insulin receptor in H-35 rat hepatoma cells

We have used H-35 rat hepatoma cells to test whether the type II insulin-like growth factor (IGF) receptor mediates metabolic responses to IGF-II. On the basis of both affinity cross-linking experiments and competition binding experiments, H-35 cells display insulin and type II IGF receptors, but not type I IGF receptors. IGF-II and multiplication-stimulating activity (MSA; the rat homolog of IGF-II) stimulate tyrosine aminotransferase, amino acid transport, and glycogen synthase activities to the same magnitude as insulin. However, MSA and IGF-II stimulate these metabolic responses only at high concentrations, indicating that these peptides are acting through the insulin receptor. Incubation of H-35 cells with MSA also induces a state of unresponsiveness to the further actions of both MSA and insulin. There is no associated loss of either insulin or IGF-II binding, indicating that desensitization occurs at a postbinding step in hormone action. The high concentration of MSA necessary to induce desensitization is also consistent with MSA acting through the insulin receptor. We conclude that in H-35 cells, the insulin receptor, rather than the type II IGF receptor, mediates the metabolic responses stimulated by MSA and IGF-II as well as the MSA-induced desensitization to insulin and MSA action.     

Expression of atypical and classical insulin receptors in Chinese hamster ovary cells transfected with cloned cDNA for the human insulin receptor

Human placenta and IM-9 lymphocytes contain subpopulations of atypical insulin receptors which differ from classical insulin receptors in their higher binding affinity for insulin-like growth factors I and II (IGF-I and IGF-II). Both types of insulin receptors may be derived from different but related genes, or may represent alternative post-translational modifications of the same gene product. To test these possibilities, we have examined the IGF binding characteristics of the human insulin receptors expressed in Chinese hamster ovary (CHO) cells which had been stably transfected with cloned human insulin receptor cDNA (CHO-T cells). The parent CHO cells contained 3 x 10(3) rodent insulin receptors/cell, and the CHO-T cells, 2.0 x 10(6) human insulin receptors/cell. Competition binding studies showed that the binding of [125I]IGF-I and [125I]multiplication stimulating activity (MSA/rat IGF-II) to parent CHO cells was primarily to type I and II IGF receptors, which cross-react poorly or not at all with insulin. However, competition binding studies with CHO-T cells showed that [125I]IGF-I binding was displaced 60-70%, and [125I]MSA binding, 50-55%, by low concentrations of insulin (20 ng/ml) and no further by higher concentrations of insulin (500 ng/ml). The insulin-insensitive IGF binding sites corresponded to the rodent type I and II IGF receptors; the insulin-sensitive IGF binding sensitive sites resembled the human atypical insulin receptors in that they bound IGF-I and MSA with moderately high affinity and reacted with insulin, MSA, and IGF-I in that order of potency. Atypical insulin receptors were also demonstrated by insulin-sensitive [125I]IGF-I and [125I]MSA binding to solubilized CHO-T proteins adsorbed to microtiter wells coated with monoclonal antibodies specific for the human insulin receptor. These results suggest that atypical human insulin receptors are generated by differential post-translational processing of the same gene product as classical human insulin receptors.     

Activation of glucose transport by a natural mutation in the human insulin receptor.

Naturally occurring mutations in the insulin receptor gene cause heritable severe insulin resistance. These mutations usually impair insulin receptor signaling in cells cultured from affected individuals. However, fibroblasts cultured from a patient with intrauterine growth restriction and severe insulin resistance (leprechaun Atl-1) had normal amounts of insulin receptor protein and defective insulin binding but constitutive activation of insulin-receptor autophosphorylation and kinase activity and of glucose transport. In the same fibroblasts, growth was impaired. Homozygosity for a mutation in the insulin receptor gene was suspected, since he inherited identical DNA haplotypes for this gene from both related parents. Here we report that the proband was homozygous and both parents were heterozygous for a point mutation in the insulin receptor gene converting the Arg86 codon (CGA) to Pro (CCA) (R86P). The R86P substitution is contiguous to the hydrophobic beta-sheet of the receptor alpha subunit implicated by DeMeyts et al. [DeMeyts, P., Gu, J.-L., Shymko, R. M., Kaplan, B. E., Bell, G. I. & Whittaker, J. (1990) Mol. Endocrinol. 4, 409-416] in the binding of aromatic residues of the insulin molecule. The R86P mutant insulin receptor cDNA was inserted into a plasmid under control of a simian virus 40 promoter and transfected into Chinese hamster ovary (CHO) cells. In contrast with fibroblasts from patient Atl-1, which had normal insulin receptor processing, CHO cells stably transfected with the R86P mutant cDNA (CHO-R86P) had altered posttranslational processing. The R86P mutant receptor failed to bind insulin but caused a significant increase in basal glucose transport in CHO cells. As in fibroblasts cultured from the patient, the R86P mutant insulin receptor did not stimulate growth in transfected CHO cells. These results suggest that the R86P mutation in the insulin receptor activates glucose transport without promoting cell growth and that distinct cell types process this mutant insulin receptor differently.     

Decreased insulin binding to cultured cells from a patient with the Rabson-Mendenhall syndrome: dichotomy between studies with cultured lymphocytes and cultured fibroblasts

[125I]Insulin binding has been studied with cultured cells from an insulin-resistant patient with the Rabson-Mendenhall syndrome. Previously, this patient has been demonstrated to have decreased insulin binding to circulating blood cells. Similarly, [125I]insulin binding to cultured lymphocytes and cultured fibroblasts was markedly decreased. Whereas the decrease in [125I]insulin binding to cultured lymphocytes appeared to result from a decrease in receptor number in cultured lymphocytes, there appeared to be a reduction in the affinity of [125I]insulin binding to cultured fibroblasts. This discrepancy between the observations with the two cell types is a general phenomenon which has been described in patients with a variety of syndromes of extreme insulin resistance. It is possible that the interpretation of the studies with the fibroblasts is complicated by the confounding influence of receptors for insulin-like growth factors on the surface of those cells. If the number of insulin receptors is sufficiently reduced on fibroblasts from the patient with extreme insulin resistance, then the low level of residual [125I]insulin binding may involve primarily receptors for insulin-like growth factors, which bind insulin with relatively low affinity.     

Regulation of insulin receptors in the human ovary: in vitro studies

The association between insulin resistance and ovarian hyperstimulation has led to a hypothesis that insulin stimulates ovarian steroidogenesis. This possible effect of insulin on the ovary could be mediated by either the insulin receptor or the type I insulin-like growth factor (IGF) receptor, both of which have been described in the human ovary. We examined the in vitro regulation of insulin receptors by LH, FSH, multiplication-stimulating activity (MSA), and insulin in ovarian stromal fragments from 24 women. [125I]Insulin binding was measured in the presence and absence of increasing concentrations of insulin, IGF-I, LH, and FSH. Neither LH nor FSH competed with [125I]insulin for binding sites, but preincubation with LH or FSH reduced [125I]insulin binding by 19-38%. Preincubation with MSA reduced [125I]insulin binding by 34-48%. The affinity of the insulin receptors, determined by Scatchard analysis, did not change (Ka = 3.3 X 10(8) mol-1). A concentration of 10 ng/mL insulin in the preincubation medium reduced [125I]insulin binding by 40%, whereas an insulin concentration of 50 or 500 ng/mL completely obliterated specific [125I]insulin binding. [125I]Insulin binding fully recovered, however, 4 h after termination of tissue exposure to insulin. The specificity of [125I] insulin binding was confirmed by studies with IGF-I. We conclude that of the hormones examined, insulin is the most potent regulator of human ovarian insulin receptors in vitro. Down-regulation of insulin receptors by insulin was reversed within 4 h after withdrawal of insulin. MSA, FSH, and LH also down-regulated the number of human ovarian insulin receptors in vitro, but were less potent. These phenomena, if also present in vivo, could be important factors in the regulation of ovarian function by insulin in normal and pathological states.     

Defects in insulin binding and autophosphorylation of erythrocyte insulin receptors in patients with syndromes of severe insulin resistance and their parents

Genetic forms of severe insulin resistance are often characterized by alterations in binding and/or kinase properties of the insulin receptor. To evaluate whether alterations in insulin receptor kinase of erythrocytes can be used as genetic markers, we studied patients with two apparently inherited conditions of severe insulin resistance (leprechaunism and the type A syndrome of insulin resistance) and their families. In the two propositi, [125I]insulin binding to intact erythrocytes was decreased by 64% and 45%, respectively. This was primarily due to a decrease in receptor number and was found in intact cells and solubilization of the receptors. Similar insulin binding defects were found on monocytes. Insulin-stimulated tyrosine kinase activity of the solubilized receptor from erythrocytes was also decreased and to a similar extent as binding. Parents of neither patient had clinical manifestations of leprechaunism or the type A syndrome. Furthermore, no alterations in insulin receptor binding or kinase activity were found in erythrocytes from the mothers. Insulin binding in the father of the type A patient was also normal, whereas the father of the leprechaun had decreased receptor affinity. Receptors extracted from the both fathers' cells had a 40-60% decrease in maximal insulin-stimulated phosphorylation and significant rightward shifts of the insulin dose-response curves (ED50, 141 and 42 ng/mL, respectively; control ED50, 16 ng/mL). The finding of biochemical defects in insulin receptor kinase activity in clinically unaffected parents of patients suggests that these alterations may be useful genetic markers and more sensitive than insulin binding studies for studying pattern of inheritance of these diseases.     

Partial insulin resistance in the mouse BC3H-1 cell line: absent hexose-independent actions of insulin

We have studied the regulation of glycogen metabolism by insulin in the insulin-sensitive nonfusing muscle cell line BC3H-1. The basal percentage of glycogen synthase I activity was not altered by insulin alone at any concentration, time of exposure, or age of cells tested. The addition of glucose or 2-deoxyglucose to the glucose- and serum-free incubation medium caused a 2-fold increase in glycogen synthase I activity over basal levels, and the effect was enhanced to 3-fold if insulin was added to the medium. Glycogen phosphorylase a activity was not altered by incubation in the presence of insulin, but was lowered by the addition of 2-deoxyglucose. This effect was also enhanced in the presence of insulin. The effect of exogenously added sugar occurred only if a 6-phosphorylatable hexose was used. The effect seen with 2-deoxyglucose was stable to Sephadex G-25 desalting, suggesting that activation of glycogen synthase was the result of a stable (covalent) modification of the enzyme. We were also able to demonstrate the presence of glucose-6-phosphate-activatable glycogen synthase phosphatase activity in the myocytes. The effect of 2-deoxyglucose in the presence or absence of insulin could be completely reversed by including cytochalasin B in the medium, suggesting that both the effect of hexose and the insulin enhancement of its effect were entirely dependent on carrier-mediated hexose uptake. Four insulin-mimetic agents, H2O2 Concanavalin A, Na orthovanadate, and antiinsulin receptor B2 serum, were also tested. Despite different mechanisms of action, each agent qualitatively mimicked insulin in the myocytes. All stimulated hexose transport, glucose incorporation into glycogen, and hexose-dependent activation of glycogen synthase in a manner not additive with insulin, but none increased basal glycogen synthase I activity in the absence of hexose. These results suggest that although insulin is capable of regulating glycogen metabolism both by increasing the uptake of sugar and by altering the activation state of glycogen synthase and phosphorylase, these effects are entirely due to the stimulation of hexose uptake, and hexose-independent actions of insulin are absent in BC3H-1 cells.     

Severe resistance to insulin and insulin-like growth factor-I in cells from a patient with leprechaunism as a result of two mutations in the tyrosine kinase domain of the insulin receptor

We studied the biological properties of insulin receptors (IRs) and insulin-like growth factor-I (IGF-I) receptors in cultured fibroblasts from a patient with leprechaunism (leprechaun Par-1). Patient cells displayed normal insulin binding capacity and affinity. Basal in vivo auto phosphorylation and in vitro exogenous kinase activity of patient IRs were elevated twofold to threefold compared with control receptors, and insulin had no further effect on these processes. Moreover, patient IRs were unable to promote the stimulation of metabolic and mitogenic pathways. IR substrate-1 (IRS-1) and mitogen-activated protein (MAP) kinase tyrosine phosphorylation and glycogen and DNA synthesis were not increased in the basal state in patient fibroblasts and were also insensitive to the stimulatory effect of insulin. As for IGF-I, although binding and receptor kinase activity were normal, the ability to stimulate glycogen and DNA synthesis was altered in patient cells. Two mutant alleles of the IR gene were detected by denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The maternal allele contained a point mutation in exon 18 encoding the tryptophan-for-arginine substitution at position 1092, and the paternal allele had a point mutation in exon 20 substituting lysine for glutamic acid at codon 1179. Thereby, leprechaun Par-1 was a compound heterozygote for two missense mutations located in the IR β-subunit. The present investigation provides the first evidence that leprechaunism can be causally related to structural alterations in the tyrosine kinase domain of the IR. These alterations result in severe impairment of insulin and IGF-I action.     

Epidermal growth factor stimulates glycogen synthesis in fetal rat hepatocytes: comparison with the glycogenic effects of insulin-like growth factor I and insulin

The effects of epidermal growth factor (EGF) on glycogen metabolism and the binding of [125I]iodo-EGF to receptors in fetal rat hepatocytes have been examined. The actions of EGF have been compared with those of insulin-like growth factor I (IGF-I) and insulin. EGF (0.1-45 nM) stimulated dose-dependent increases in [14C]glucose incorporation into glycogen (8.8-31.1%, P less than 0.01) and total cellular glycogen content (5.6-21.4%, P less than 0.05). The concentration of EGF causing half-maximal stimulation of glycogen synthesis was 2 ng/ml, and maximal stimulation occurred at 1 h of incubation. EGF had no effect on the uptake of the nonmetabolizable monosaccharide [14C]O-methyl-D-glucose, suggesting that the glycogenic effect of EGF was not mediated through stimulation of glucose transport. Although IGF-I (1-100 nM) and insulin (14 nM to 10 microM) also stimulated glycogen synthesis in fetal liver, the maximal effects of these hormones occurred at 2 h incubation, and the dose-response curves of IGF-I and insulin were not parallel to that of EGF. In addition, the maximal glycogenic effect of EGF was only 40% that of insulin or IGF-I, and the effects of EGF and insulin on [14C]glucose incorporation were additive. These findings suggest that EGF stimulates glycogen synthesis through a mechanism distinct from that of IGF-I or insulin. The binding of [125I]iodo-EGF to fetal hepatocytes was specific, saturable, and time- and temperature-dependent. Maximal specific binding occurred at 1 h of incubation at 37 C or at 24 h of incubation at 4 C. Unlabeled EGF (0.05-250 ng/ml) caused a dose-dependent inhibition of the binding of [125I]iodo-EGF to fetal hepatocytes, with half-maximal displacement of [125I]iodo-EGF by 1.7 ng unlabeled EGF/ml. The specific binding of [125I] iodo-EGF was not inhibited by high concentrations of insulin or IGF-I, suggesting that the differences in the mechanisms by which EGF, insulin, and IGF-I stimulate glycogenesis may be explained in part by differences in the binding of these hormones to fetal liver receptors. In addition to having mitogenic effects in fetal tissue, EGF or other EGF-like growth factors may have acute effects on fetal hepatic intermediary metabolism and may contribute to the accumulation of liver glycogen in the mammalian fetus during late gestation.     

The effects of diabetes and insulin on glycoprotein metabolism by rat liver

The hepatic metabolism of [125I]agalactoorosomucoid [( 125I]AGOR) was studied in normal and streptozotocin-induced diabetic rats. The blood clearance, hepatic transport time and rate of catabolism of [125I]AGOR were calculated from data of the blood [125I]AGOR disappearance rates and the appearance in blood of acid-soluble catabolites. In control rats the blood clearance of [125I]AGOR was rapid (8.7 +/- 0.6 ml/min) and the hepatic transport time of the ligand was 12.8 +/- 0.7 min. Insulin prolonged the hepatic transport time (18.1 +/- 1.9 min) and depressed ligand catabolism. Chloroquine had similar effects. Diabetes impaired hepatic [125I]AGOR uptake as judged by the prolonged blood clearance rate and depressed ligand catabolism but did not alter ligand transport time. The measured parameters returned to normal when diabetic animals were rendered acutely normoglycaemic. Diabetic rats, in which implanted osmotic insulin pumps had maintained normoglycaemia for 3 days, cleared [125I]-AGOR from the blood more rapidly than controls. This effect appeared to be due to the lower blood glucose levels in this group. The experiments have shown the complexity of the effects of insulin and diabetes mellitus on the uptake and processing of a glycoprotein by the hepatic mannose receptor.     

Impaired skeletal muscle glycogen synthase activation by insulin in the Goto-Kakizaki (G/K) rat

Summary The Goto-Kakizaki (G/K) rat is an animal model of non-insulin-dependent diabetes mellitus, with early hyperglycaemia, hyperinsulinaemia, and insulin resistance. We have studied the effect of insulin on the activation of glycogen synthase in the G/K rat and in the original parent strain, the Wistar rat. After insulin injection, glycogen synthase I activity, glycogen synthase phosphatase activity and glucose 6-phosphate content in skeletal muscle were significantly increased in the Wistar rats. In the G/K rats, insulin injection resulted in a reduced activation of skeletal muscle glycogen synthase, which was not significant when compared with the control rats without insulin, and no increases in glycogen synthase phosphatase and glucose 6-phosphate were seen. In adipose tissue the activation of glycogen synthase by insulin was normal in the G/K rats. Previous investigations have shown that glucose disappearance rates are low in the G/K rat. However, stimulation of glucose transport was reported to be normal in the G/K rat. A defective activation of glucose accumulation into glycogen by skeletal muscle may contribute to explain the hyperglycaemia in the G/K rat.Abbreviations DTT Dithiothreitol - TLCK tosyl lysyl chloromethyl ketone - GSPase glycogen synthase phosphatase - G3PAT glycerol-3-phosphate acyl transferase - G/K Goto-Kakizaki rat - IPG Inositolphosphoglycan     

Fibroblasts from a leprechaun patient have defects in insulin binding and insulin receptor autophosphorvlation

Summary Leprechaunism is an inherited human disorder associated with an extreme resistance of the target cells towards the action of insulin. We have examined the properties of the insulin receptor in fibroblasts from a leprechaun patient (Geldermalsen, the Netherlands). In vitro, severe insulin resistance is reflected by a low level of insulin stimulated uptake of 2-deoxyglucose by these fibroblasts. This defect seems to be caused by a combination of two factors: a low level of insulin binding to intact cells and a strong decrease of insulin stimulated autophosphorylation of the receptor. The stimulation of autophosphorylation by insulin was approximately six-fold in control subjects and less than two-fold in the patient. No abnormalities were observed in the total number of insulin receptors in these cells and the molecular weights of the receptor subunits. In addition, the insulin concentration required for half maximal autophosphorylation is similar for the solubilised receptor from control and patient fibroblasts.     

Oxidative stress impairs insulin internalization in endothelial cells in vitro

Aims/hypothesis. Because oxidative stress has been suggested to be a significant contributing factor in the development of endothelial dysfunction and insulin resistance, we investigated whether reactive oxygen species contribute to insulin resistance by impairing insulin uptake through an effect on endothelial insulin receptor function. Methods. Following a 2-h pro-oxidant challenge with xanthine oxidase, we examined the temporal pattern of insulin processing in the human umbilical endothelial cell line Ea.Hy926 and bovine aortic endothelial cells equilibrated with [¹²⁵I]-insulin. Insulin receptor mRNA concentrations were analysed by RT-PCR and insulin receptor tyrosine phosphorylation and protein concentrations were estimated by western blotting. Results. Xanthine oxidase exposure resulted in a major reduction in total insulin receptor-mediated [¹²⁵I]-insulin internalization over a 1-h period in both Ea.Hy926 and bovine aortic endothelial cells. After 15 min, untreated bovine aortic endothelial cells internalized fivefold more cell-bound [¹²⁵I]-insulin than pro-oxidant treated cells. The [¹²⁵I]-insulin disappeared from the cell surface at a similar rate in both pro-oxidant and untreated cells, with relatively more [¹²⁵I]-insulin being released into the medium in pro-oxidant treated cells. Although xanthine oxidase reduced insulin receptor mRNA and protein concentrations, cell surface insulin binding capacity was not affected. Following 5 min insulin exposure, insulin receptor auto-phosphorylation was considerably reduced in cells challenged with xanthine oxidase for 2 h, which could be important for insulin receptor activation and internalization. Conclusion/interpretation. Oxidative stress impairs insulin endocytosis in both arterial and venous endothelial cell lines. This was not a consequence of modified insulin binding capacity but could involve insufficient insulin receptor activation. [Diabetologia (2001) 44: 605–613] Received: 2 October 2000 and in revised from: 18 December 2000     

Acromegaloid patients with type A insulin resistance: parallel defects in insulin and insulin-like growth factor-I receptors and biological responses in cultured fibroblasts

A subset of patients with the syndrome of acanthosis nigricans and insulin resistance type A is characterized by acromegaloid features in addition to hyperinsulinemia, hyperandrogenemia, and an inherent defect in insulin receptor function. It has been proposed that the acromegaloid features result from the interaction of insulin at concentrations encountered in vivo, with a functionally intact insulin-like growth factor-I (IGF-I) receptor closely related to the insulin receptor. We investigated this possibility by examining binding and hormone-stimulated [14C]glucose uptake, [3H]thymidine uptake, and receptor autophosphorylation by both insulin and IGF-I in cultured fibroblasts from two affected patients. In comparison to normal fibroblasts, [125I]insulin binding, insulin-stimulated [14C]glucose, and [3H]thymidine uptake, and insulin-stimulated autophosphorylation were each reduced by approximately 50-60% of the absolute values in controls. In contrast to expectation, each of these apparent defects in insulin binding and action were mirrored by a parallel decrease in IGF-I binding and action. Thus, [125I]IGF binding was approximately 50%, IGF-I stimulated [3H]thymidine uptake was approximately 40% and 60% of the control value, and IGF-I-stimulated receptor autophosphorylation was reduced by 40%. Incubation of fibroblasts with insulin at 25 ng/mL reduced subsequent binding of [125I]IGF-I by approximately 20% and did not enhance maximal stimulation of [3H]thymidine incorporation. We conclude that in some patients with acanthosis nigricans and acromegaloid features, IGF-I receptors of cultured fibroblasts may share the inherent defects of insulin receptor function. These in vitro data do not explain the acromegaloid features observed in vivo, suggesting that acromegaloid features are mediated by other mechanisms.     

Selective effects of inhibitors of hormone processing on insulin action in isolated hepatocytes

Several compounds known to alter receptor-mediated endocytosis were tested for their effect on insulin action in isolated rat hepatocytes. Dansylcadaverine blocked the inhibitory effect of insulin on hepatic protein degradation, but was without effect on the inhibition produced by high concentrations of amino acids. Dansylcadaverine also was without effect on the stimulatory action of insulin on glucose incorporation into glycogen, but totally blocked the insulin stimulation of alanine uptake. No inhibition of basal or (Bu)2cAMP-stimulated alanine uptake was observed with dansylcadaverine. These results indicate that dansylcadaverine has selective effects on some but not all of insulin's actions. Methylamine also blocked insulin stimulation of alanine transport, although at higher doses, methylamine decreased basal and (Bu)2cAMP-stimulated uptake due to cellular toxicity. Chloroquine, an inhibitor of intracellular insulin processing, had no effect on insulin-stimulated alanine transport. To examine the site of action of dansylcadaverine on insulin processing, the binding (cell-associated radioactivity) and degradation of [125I]iodoinsulin by hepatocytes were examined in the presence and absence of dansylcadaverine. Dansylcadaverine delayed the time required to achieve maximal insulin binding and produced a dose-dependent decrease in cell-mediated insulin degradation. Acid dissociation and proteolytic digestion methods were used to differentiate between [125I]iodoinsulin bound to the cell surface vs. that which had been internalized. Dansylcadaverine did not block internalization as measured by either of these criteria, but did block the increase in cell-associated radioactivity produced by chloroquine, suggesting that dansylcadaverine blocks insulin processing after internalization but before the chloroquine-sensitive step. These data show that some of the hepatic actions of insulin, namely inhibition of protein degradation and stimulation of alanine transport, are blocked by certain inhibitors of cellular hormone processing, suggesting that these actions of insulin may require postreceptor events involving insulin metabolism.     

Transfer of functional insulin receptors to receptor-deficient target cells

Purified human insulin receptors incorporated into phospholipid vesicles have previously been shown to retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activity. These vesicles were used as a vehicle to transfer receptors to the insulin receptor-deficient Madin-Darby canine kidney (MDCK) cell line. Fusion of control [14C] dioleoylphosphatidylcholine-labeled phospholipid vesicles with MDCK cells was found to be dependent on both the amount of time and the concentration of polyethylene glycol used for fusion. Optimal insulin receptor transfer, as determined by recovery of [125I]insulin binding, occurred when MDCK cells were incubated for 45 min at 37 C in the presence of 15% polyethylene glycol plus receptor-containing vesicles. Scatchard analyses for insulin receptor binding before and after vesicle fusion demonstrated no postfusion alteration in insulin receptor affinity and a 10-fold increase in the number of insulin receptors present in the MDCK cells. Fusion transfer of insulin receptors to MDCK cells rendered the cells sensitive to insulin (10-100 nM) for stimulation of glycogen synthesis. Chloroquine (0.1 mM) was found to block endosomal processing of receptor-bound [125I] insulin within 1 h. These findings indicate that insulin receptors function as dissociable units which can be inserted into target plasma membranes with resultant recoupling to cellular systems.     

A case of leprechaunism with extreme insulin resistance due to a primary defect in insulin receptors

This report describes a 3-month-old female infant with the typical physical features of leprechaunism. The patient demonstrated glucose intolerance and marked hyperinsulinemia (4600 microU/ml). Since an intravenous insulin injection (actrapid insulin: 0.15 U/kg) caused no significant decrease in the blood glucose level, the presence of insulin resistance was suggested. Neither insulin antibodies nor insulin receptor antibodies were were found in the patient's plasma, and other circulating insulin antagonists such as glucagon, growth hormone, and cortisol were within normal limits. [125I]Insulin binding to the erythrocytes from the patient was as low as 1.02% (control infants: 4.89 +/- 1.08% [mean +/- SD]). [125I]Insulin binding to the cultured transformed lymphocytes from the patient was similarly reduced to 3.58% (control: 20.9 +/- 2.71% [mean +/- SD]). From these findings we concluded that the insulin resistance was due to a primary defect in insulin receptors. Interestingly, transient remissions of the patient's glucose intolerance and hyperinsulinemia were observed during a year of follow-up study. The insulin tolerance test which was performed at the remission period showed an improvement in insulin resistance. However, the insulin binding defect to erythrocytes remained unchanged even at the remission period. The exact cause of these remissions was not clear and remained to be elucidated.     

Insulin resistance due to a defect distal to the insulin receptor: Demonstration in a patient with leprechaunism

We have studied a 2-year-old girl with acanthosis nigricans, glucose intolerance, marked hyperinsulinemia, and somatic features characteristic of the leprechaunism syndrome. Circulating plasma insulin levels were increased up to 50-fold and the patient showed a blunted hypoglycemic response to an injection of exogenous insulin (0.2 units/kg), indicating the presence of severe insulin resistance. Insulin purified from the patient's plasma was normal on the basis of chromatographic, electrophoretic, and immunologic criteria. Furthermore, the purified insulin competed effectively with (125)I-labeled insulin for binding to insulin receptors on cultured IM-9 lymphocytes and rat fat cells and also exhibited normal biological potency when tested on rat fat cells. Anti-insulin receptor and anti-insulin antibodies were not detected in the patient's plasma, and plasma levels of glucagon, growth hormone, and cortisol were normal. Insulin binding to the patient's circulating monuclear leukocytes was only slightly depressed into the low normal range and could not account for the severe insulin resistance. Studies on the patient's fibroblasts revealed normal levels of insulin receptors but a total absence of insulin's ability to accelerate glucose transport. Because rates of glucose transport and metabolism were normal in the basal state in the absence of insulin, we conclude that this patient's insulin resistance is due to an inherited cellular defect in the coupling mechanism between occupied insulin receptors and the plasma membrane glucose transport system.