High Affinity Insulin Receptors Research Articles

Insulin Resistance through High-sugar Diets and its Effects on the Endometrium in Swiss Albino Mice: An Experimental Study to Elicit the Beneficial Effects of Coconut Sugar

Introduction: Insulin resistance (IR) has an insidious onset and is mainly due to the consumption of high-energy diets. Refined sugars contain high fructose which predisposes to IR and affects the endometrium since endometrium exhibits high-affinity insulin receptors, and the natural unrefined sugars such as coconut sugar are proved to be beneficial, but scientific studies are scarce to validate the findings. Subjects and Methods: A cross-sectional study was carried out, in which 36 female Swiss albino mice were divided into control and high-fructose and high-coconut sugar diet groups. The respective diets were prepared daily and fed for a period of 12 weeks, and the animals were sacrificed by the administration of ether. Biochemical parameters were assessed, and the uterus was removed and processed for histopathological analysis. Results: Glucose, insulin, and homeostasis model assessment-IR were assessed, in which there was an increase in the levels in the high-fructose group than the control and coconut sugar groups. The values were statistically significant in the Kruskal–Wallis analysis (P < 0.001) and post hoc test showed a significant difference between the control and fructose diet groups. Hormonal assay showed elevated testosterone levels in the fructose group (Kruskal–Wallis test, P < 0.001), which showed significance in post hoc test when compared with the control group (P < 0.030). Histopathological examination revealed complex hyperplasia with nuclear atypia in the fructose group, whereas the findings were normal in the coconut sugar and control groups. Discussion: The present study proved coconut sugar to be beneficial, and there is a strong correlation of high-energy diets on the endometrium due to IR. Conclusion: The present study concludes that the intake of natural sugars does not cause damage to the endometrium which is sensitive to insulin resistance.

Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (KD: 2.3 ± 0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (KD: 2.1 ± 0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-β-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-β-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.

Probing the correlation between insulin activity and structural stability through introduction of the rigid A6–A11 bond

The development of fast-acting and highly stable insulin analogues is challenging. Insulin undergoes structural transitions essential for binding and activation of the insulin receptor (IR), but these conformational changes can also affect insulin stability. Previously, we substituted the insulin A6-A11 cystine with a rigid, non-reducible C=C linkage ("dicarba" linkage). A cis-alkene permitted the conformational flexibility of the A-chain N-terminal helix necessary for high-affinity IR binding, resulting in surprisingly rapid activity in vivo Here, we show that, unlike the rapidly acting LysB28ProB29 insulin analogue (KP insulin), cis-dicarba insulin is not inherently monomeric. We also show that cis-dicarba KP insulin lowers blood glucose levels even more rapidly than KP insulin, suggesting that an inability to oligomerize is not responsible for the observed rapid activity onset of cis-dicarba analogues. Although rapid-acting, neither dicarba species is stable, as assessed by fibrillation and thermodynamics assays. MALDI analyses and molecular dynamics simulations of cis-dicarba insulin revealed a previously unidentified role of the A6-A11 linkage in insulin conformational dynamics. By controlling the conformational flexibility of the insulin B-chain helix, this linkage affects overall insulin structural stability. This effect is independent of its regulation of the A-chain N-terminal helix flexibility necessary for IR engagement. We conclude that high-affinity IR binding, rapid in vivo activity, and insulin stability can be regulated by the specific conformational arrangement of the A6-A11 linkage. This detailed understanding of insulin's structural dynamics may aid in the future design of rapid-acting insulin analogues with improved stability.

Investigating New Therapeutic Strategies Targeting Hyperinsulinemia's Mitogenic Effects in a Female Mouse Breast Cancer Model

Epidemiological and experimental studies have identified hyperinsulinemia as an important risk factor for breast cancer induction and for the poor prognosis in breast cancer patients with obesity and type 2 diabetes. Recently it was demonstrated that both the insulin receptor (IR) and the IGF-IR mediate hyperinsulinemia's mitogenic effect in several breast cancer models. Although IGF-IR has been intensively investigated, and anti-IGF-IR therapies are now in advanced clinical trials, the role of the IR in mediating hyperinsulinemia's mitogenic effect remains to be clarified. Here we aimed to explore the potential of IR inhibition compared to dual IR/IGF-IR blockade on breast tumor growth. To initiate breast tumors, we inoculated the mammary carcinoma Mvt-1 cell line into the inguinal mammary fat pad of the hyperinsulinemic MKR female mice, and to study the role of IR, we treated the mice bearing tumors with the recently reported high-affinity IR antagonist-S961, in addition to the well-documented IGF-IR inhibitor picropodophyllin (PPP). Although reducing IR activation, with resultant severe hyperglycemia and hyperinsulinemia, S961-treated mice had significantly larger tumors compared to the vehicle-treated group. This effect maybe secondary to the severe hyperinsulinemia mediated via the IGF-1 receptor. In contrast, PPP by partially inhibiting both IR and IGF-IR activity reduced tumor growth rate with only mild metabolic consequences. We conclude that targeting (even partially) both IR and IGF-IRs impairs hyperinsulinemia's effects in breast tumor development while simultaneously sparing the metabolic abnormalities observed when targeting IR alone with virtual complete inhibition.

Expression and function of the insulin receptor in normal and osteoarthritic human chondrocytes: modulation of anabolic gene expression, glucose transport and GLUT-1 content by insulin

Chondrocytes respond to insulin, but the presence and role of the specific high affinity insulin receptor (InsR) has never been demonstrated. This study determined whether human chondrocytes express the InsR and compared its abundance and function in normal and osteoarthritis (OA) human chondrocytes. Cartilage sections were immunostained for detection of the InsR. Non-proliferating chondrocyte cultures from normal and OA human cartilage were treated with 1nM or 10nM insulin for various periods. InsR, insulin-like growth factor receptor (IGFR), aggrecan and collagen II mRNA levels were assessed by real time RT-PCR. InsR, glucose transporter (GLUT)-1, phospho-InsRbeta and phospho-Akt were evaluated by western blot and immunofluorescence. Glucose transport was measured as the uptake of [3H]-2-Deoxy-d-Glucose (2-DG). Chondrocytes staining positively for the InsR were scattered throughout the articular cartilage. The mRNA and protein levels of the InsR in OA chondrocytes were approximately 33% and 45%, respectively, of those found in normal chondrocytes. Insulin induced the phosphorylation of the InsRbeta subunit. Akt phosphorylation and 2-DG uptake increased more intensely in normal than OA chondrocytes. Collagen II mRNA expression increased similarly in normal and OA chondrocytes while aggrecan expression remained unchanged. The Phosphoinositol-3 Kinase (PI3K)/Akt pathway was required for both basal and insulin-induced collagen II expression. Human chondrocytes express functional InsR that respond to physiologic insulin concentrations. The InsR seems to be more abundant in normal than in OA chondrocytes, but these still respond to physiologic insulin concentrations, although some responses are impaired while others appear fully activated. Understanding the mechanisms that regulate the expression and function of the InsR in normal and OA chondrocytes can disclose new targets for the development of innovative therapies for OA.

Expression and localization of insulin receptors in dissociated primary cultures of rat Schwann cells

The objective of the present study was to examine for the presence of the IRs (insulin receptors) in the primary dissociated culture preparation of SCs (Schwann cells). This was achieved using immunological techniques using a rabbit polyclonal anti-IR antibody and at molecular level by RT (reverse transcription)-PCR. Light microscopic immune cytochemistry revealed that almost all SCs in cluster and associated neuritis exhibited positive immune reaction with the antibody, confirming the presence of IRs in them. Immunoblotting detected a prominent protein band of 90 kDa, which is consistent with those reported by the manufacturer. Like the peripheral nerve, primary SC cultures showed a predominantly high affinity IR mRNA lacking exon 11. Ultrastructural immune localization confined the presence of the IRs in the basal lamina, plasma membrane and the cytoplasmic processes of the SCs.

Insulin secretion, peripheral insulin sensitivity and insulin-receptor binding in subjects with different degrees of obesity

The aim of the present study was to investigate insulin secretion, insulin-receptor binding and peripheral insulin sensitivity in subjects with different degrees of obesity. 36 obese subjects with normal glucose tolerance and different degrees of obesity and 40 healthy normal-weight subjects participated in the study. Peripheral insulin sensitivity was measured by using the euglycaemic hyperinsulinaemic clamp technique, and insulin-receptor binding-on circulating mononuclear blood cells. Insulin secretion was studied during intravenous tolbutamide test. The subjects with I degree of obesity demonstrated a significant decrease in the number of total (p<0.0001) and high-affinity (p<0.01) insulin receptors per cell, as well as significantly higher insulin receptor affinity (p<0.01) as compared to the normal-weight subjects. The subjects with II degree of obesity also demonstrated a significant decrease in the number of total (p<0.0001) and high-affinity receptors (p<0.001) per cell as well as an increase (p<0.001) in insulin-receptor affinity as compared to the controls. The significantly decreased receptor number in the subjects with I and II degree of obesity was accompanied by an increase in insulin receptor affinity; thus their insulin-receptor binding being maintained similar to the controls. The subjects with III degree obesity presented a significant decrease (p<0.0001) in the number of both the total and high-affinity insulin receptors as well as a reduction in insulin receptor affinity as compared to the controls. Therefore the percentage of specifically bound insulin was significantly lower (p<0.01) as compared to that of the control group. Insulin resistance in the obese subjects is associated with secondary hyperinsulinaemia, which is present in subjects with I and II degree of obesity; while in severely obese subjects exhaustion of beta-cell secretory capacity is observed. We consider that III degree of obesity appears to be a risk factor for type 2 diabetes mellitus as the alterations in insulin sensitivity, insulin-receptor binding and beta-cell secretion are quite similar to the reported in diabetic patients.

Biochemical and molecular biological aspects of glucose intolerance in elderly persons

Changes in carbohydrate metabolism in elderly persons have drawn considerable attention but the findings from different studies are in contrast and are even controversial. The insulin receptors in erythrocytes were studied in elderly euglycaemic patients and in a middle-aged control group. The examined persons were also subjected to measurements of blood glucose, insulin and C-peptide concentrations, before and 3 hours after a dietetic meal. In the present study it was found that in spite of the maintained insulin level and C-peptide secretion, some structural and corresponding changes in membrane insulin receptors and the binding site caused differences in postreceptor responses in elderly persons. The examined groups, consisted of 29 males, average age of 66 years (65-70), with normal serum glucose level and 19 middle-aged males, average age of 42 years (32-48), with normal glycoregulation. In basal condition, elderly persons have both normal morning serum insulin (19.68 ? 4.00 mU/L) and C-peptide (2.04 ? 0.78 nmol/L) level. In elderly persons, the number of high affinity insulin receptors in erythrocytes membrane is 22.80 ? 6.18 but the formed insulin-high affinity receptors were not stable. Dissociation constant (Kd1) indicates its elevated dissociation (0.11 ? 0.04). At the same time the number of insulin low affinity binding sites is increased (13 273 ? 5 572) with a fast dissociation of the hormone (13.99 ? 3.37). Food intake raised the number of high affinity receptors compared to the basal value. Alteration in insulin binding affinity suggests the structural and corresponding changes in membrane receptors that may cause differences in postreceptors responses in elderly persons.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks.

Insulin is thought to elicit its effects by crosslinking the two extracellular alpha-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

Feeding different amounts of colostrum or only milk replacer modify receptors of intestinal insulin-like growth factors and insulin in neonatal calves

Colostrum intake influences growth and development of the gastrointestinal tract (GIT) in several species and colostral insulin-like growth factors I and II (IGF-I and IGF-II), and insulin are involved in neonatal intestinal tissue growth. We have studied IGF type 1, IGF type 2, and insulin receptors in the intestine of 8-day-old calves fed different amounts of colostrum or only milk replacer. Calves were fed colostrum of the first six milkings on the first 3 days and then milk replacer (GrC 6) or colostrum only once and then milk replacer (GrC 1) or they were fed only milk replacer from the beginning, i.e., no colostrum (GrM). Competitive binding studies and ligand blots confirmed the presence of IGF type 1, IGF type 2, and insulin receptors in mucosal cell membranes of duodenum, jejunum, ileum, and colon. The IGF type 1 receptor number in ileum and total intestine in GrC 6 was greater ( P<0.05) than in GrC 1 and in GrM, and IGF type 2 receptor number in total intestine was greater ( P<0.05) in GrC 6 than in GrM. Insulin binding was best fitted by a model with two binding sites. High affinity insulin receptor numbers in duodenum, ileum, and total intestine were greater ( P<0.05) in GrC 6 than in GrM. The data demonstrate that IGF type 1, IGF type 2, and insulin receptors in intestinal mucosa of neonatal calves are influenced by feeding.

Insulin receptor and insulin receptor substrate-I in rat retinae.

Insulin receptor substrate-I (IRS-I) is a major cytosolic substrate of the insulin receptor Expression of insulin receptor and IRS-I, and the distribution of these components of the insulin-signalling pathway, were investigated in rat retinae. Insulin receptor and IRS-I were located in retinal sections with anti-insulin receptor and anti-IRS-I antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) of retinal mRNA was performed with primers specific for insulin receptor and IRS-I gene sequences. Immunohistochemistry demonstrated distinct but closely associated staining patterns for insulin receptor and IRS-I throughout rat retinae. The RT-PCR product from rat retinal insulin receptor mRNA corresponded to the high affinity insulin receptor isoform. The RT-PCR product for retinal IRS-I mRNA agreed with that predicted from the gene sequence. The expression of IRS- I and insulin receptors indicates a signalling mechanism by which insulin can influence retinal metabolism or function.

Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities.

In the present study, we have examined the insulin-signaling pathways involved in myogenesis in mouse C2C12 skeletal muscle cell line, a cellular system that expresses high number of high affinity insulin receptors. Insulin (50 nM) rapidly (5 min) stimulated beta-chain insulin receptor, activated the phosphatidylinositol (PI) 3-kinase/Akt/p70S6-kinase signaling pathway, as well as phosphorylated both p44/p42- and p38-mitogen-activated protein kinases (MAPKs). Preconfluent cells were differentiated in a serum-free medium in response to 50 nM insulin for 72 h, as revealed by the formation of multinucleated myotubes and the induction of the creatine kinase activity. This differentiation process was also monitored by the inhibition of the PCNA content and induction of the cell cycle inhibitor p21. Furthermore, insulin induced nuclear factor-kappaB (NF-kappaB) DNA binding activity and down-regulated activating protein-1 (AP-1) DNA binding activity throughout the differentiation process. The use of specific inhibitors of the insulin-signaling pathways indicated that myogenesis was precluded by treatment for 72 h with LY294002 (an inhibitor of PI 3-kinase), rapamycin (a p70S6-kinase blocker), and SB203580 or PD169316 (p38-MAPK inhibitors). These inhibitors abolished insulin induction of NF-kappaB DNA binding activity and kappaB-chloramphenicol acetyltransferase (CAT) promoter activity, maintaining expressed cytosolic IkappaB-alpha protein, and increased AP-1 DNA binding activity and TRE-CAT promoter activity. These data suggest that insulin induces myogenesis in C2C12 through PI 3-kinase/ p70S6-kinase and p38-MAPK pathways, the signaling through p44/p42-MAPK being inhibited.

Insulin receptor in rat peripheral nerve: its localization and alternatively spliced isoforms.

Diabetic neuropathy accompanies both Type 1 and Type 2 diabetes, although it shows in both humans and animal models distinct differences between the two types of diabetes. Progressive paranodal degenerations occurring in Type 1, but not in Type 2, diabetes is believed to account for the more severe functional deficits in Type 1 diabetic rats. This suggests that factors other than hyperglycemia, such as insulin deficiency, may play a pathogenetic role. In this study, we investigated the immunolocalization of the insulin receptor (IR) and the expression of its two alternatively spliced isoforms in adult rat peripheral nerve. Adult male Wistar rats 6-8 months of age were examined. Both light and ultrastructural immunohistochemistry was employed for localization of IR. The antibody was a mouse monoclonal antibody raised against the beta-subunit of human IR. Reverse transcription polymerase chain reaction (RT-PCR) was used to identify the two IR isoforms in peripheral nerve and seven other organs. Localization of the mRNA message was assessed by in situ hybridization. IR was localized to paranodal terminal Schwann cell loops and microvilli and to the paranodal axolemma. Furthermore, IR immunoreactivity was also present in Schmidt-Lantermann incisures. Endoneurial vessels showed IR localization on plasma membranes and in endocytotic vesicles of endothelial cells and pericytes. A high intensity of immunostained IR was found in close proximity to interendothelial tight junctions. Peripheral nerve showed, like the brain, predominantly the high affinity IR lacking exon 11. The mRNA message was localized to Schwann cells, endothelial cells and pericytes. Peripheral nerve expresses predominantly the high affinity IR, which is localized to strategic structures associated with the blood-nerve barrier and the paranodal ion-channel barrier.

Effect of Isoflavone Genistein on Insulin Receptors in Perfused Liver of Ovariectomized Rats

The experiments were carried out on ovariectomized Wistar rats. Their livers were perfused with basic perfusion medium (BPM) or BPM supplemented with isoflavone genistein. insulin or combination of the two factors. The obtained results support the hypothesis that genistein influences the kinetics of insulin binding to cell membranes changing the number of insulin receptors and dissociation constant (Kd) BPM supplementation with genistein decreased number of high affinity insulin receptors (HAIR) both in livers treated and untreated with insulin. The amount of HAIR diminished significantly from 610±77 × 10-15 (no genistein) to 238±72 × 10-13 mol/mg of membrane protein (supplement of genistein). Similarly, genistein reduced slightly the amount of HAIR even when added together with insulin (372±59 × 10-13 mol/mg) in comparison to rats perfused with medium containing insulin but not the isoflavone (421±46 × 10-15 mol/mg). Simultaneously, genistein decreased significantly Kd for HAIR (perfusion with BPM - 1.44±0.18 × 10-9 mol/l; perfusion with BMP + genistein - 0.83±0.20 × 10-9 mol/l). Such effects of genistein during liver perfision did not take place when the liver membranes were in vitro incubated with this xenobiotic.

The Influence of Hypo- and Hyperthyreosis on Insulin Receptors and Metabolism

Changes in thyroid status affect metabolism not only directly, but influence it also by alterations in insulin secretion and action. Despite several investigations, these effects are, however, poorly characterised or even controversial. The aim of the studies was to investigate the effect of hyperthyreosis (HT) and hypothyreosis (HPT) on insulin binding by rat liver membranes. Some metabolic parameters reflecting insulin and thyroid hormones action were also determined. HT and HPT were developed by daily administration for 3 weeks of thyroxine (T 4) and thiouracil (TU), respectively. Experimental hyperthyreosis and hypothyreosis caused deep changes in metabolism. The greatest alterations were observed in body and thyroid glands weight, blood triiodothyronine (T 3), T 4, glucose, and insulin levels, liver glycogen amount and number of insulin receptors. HT reflected in rats in slower rate of growth and in smaller thyroid glands weight. In comparison to controls, T 4 concentration in HT was almost doubled and it was reduced by about 30% in HPT. Also, T 3, insulin and glucose levels in HT were heightened. Simultaneously, binding of insulin to liver membranes was elevated in HT and reduced in HPT. In HT the number of high affinity insulin receptors (HAIRs) and low affinity insulin receptors (LAIRs) was increased, whereas in HPT the amount of HAIRs was diminished. HT caused a drastic reduction of glycogen concentration in liver, but no changes were observed for muscle glycogen. Considering lipid metabolism, only free fatty acids (FFA) level in blood was changed (in HPT), but no differences were observed in serum concentration of triglycerides and cholesterol. Several metabolic changes observed in HT and HPT seem to be the dire ct consequence of alterations of thyroid hormone concentrations. These disturbances, together with the direct effect of HT or HPT on insulin secretion, binding and action lead, in turn, to changes in the other metabolic parameters. As a result of these disturbances the adaptive mechanisms appear. One of them is change in the number of insulin membrane receptors taking place even against the well known “down-regulation“ theory.

Acute leptin action on insulin blood level and liver insulin receptor in the rat

Aim of the study was to investigate acute leptin effect on insulin blood level and liver insulin binding in the rat. The administration of leptin induced time and dose dependent decrease in the insulin level, which was statistically significant in comparison to the control animals 120 min after administration of higher dose of peptide (0.30 ± 0.05 vs 0.14 ± 0.01 nmol l , respectively). Simultaneously, we have shown the attenuation of liver sensitivity to insulin 2 hours after higher leptin dose injection. This phenomenon was caused by the decrease of binding capacity of high affinity insulin receptor sites (HAIR), which was statistically significant after higher leptin dose administration at both time points (0.54 ± 0.13 vs 0.26 ± 0.03 and 0.71 ± 0.12 vs 0.40 ± 0.05 pmol mg protein for 1 and 2 h, respectively). The present study provides evidence that leptin, in addition to its inhibitory effect on insulin secretion, acts as a modulator of insulin receptor, through the decrease of binding capacity. It seems legitimate to suggest that leptin-induced decrease of insulin receptor binding capacity may be one of several causes of insulin resistance.

Insulin Promotes Pancreatic Cancer: Evidence for Endocrine Influence on Exocrine Pancreatic Tumors

Type-II diabetes is a risk factor for pancreatic cancer. In addition, diabetic patients present with more advanced tumors and have shortened survival compared to stage-matched counterparts. We hypothesize that the diabetic endocrine milieu, particularly elevated plasma insulin, favors pancreatic cancer growth. This study examines six human pancreatic cancer cell lines for the presence of insulin receptors and the influence of insulin on tumor proliferation. Classical competitive binding assays are performed using [125I]insulin. Cell proliferation assays are conducted over 3 days on cultured cell lines (n= 6 replicates) with increasing concentrations of insulin. Insulin receptors are demonstrated on all six cell lines and dose dependent increases in cell proliferation (15–120% of control) are demonstrated in response to insulin. Patients with type-II diabetes hypersecrete insulin. The presence of high-affinity insulin receptors and dose dependent increases in pancreatic cancer cell proliferation with insulin supports the hypothesis that insulin may be an important tumor growth promoter in diabetes, particularly if paracrine mechanisms are involved. Additional study is required to determine whether other islet peptides altered in diabetes influence tumor growth and whether elevated plasma insulin favors pancreatic cancer induction.

Tissue-related changes in insulin receptor number and autophosphorylation induced by starvation and diabetes in rats

Insulin action is subject to regulation at the level of the insulin receptor and at postreceptor levels. Starvation and diabetes are often associated with insulin resistance for glucose metabolism in various tissues. In muscle, fat, and liver, we examined whether changes in the functionality of the insulin receptor correlated with changes in insulin action in the starved and diabetic state. Insulin-stimulated receptor autophosphorylation reflects an early physiologic step in transmission of the insulin signal, and for that reason, changes in auto phosphorylation activity of the insulin receptor were used as a marker to determine the functionality of the insulin receptor. Glycoprotein fractions prepared from skeletal muscle, diaphragm, epididymal fat, and liver of control, 3-day starved, short-term 3-day (S) diabetic (streptozotocin, 70 mg/kg intravenously), and long-term 6-month (L) diabetic (neonatal streptozotocin 100 μwg/g intraperitoneally) rats were used in this study. Receptor activity was monitored by measuring insulin-stimulated [γ- 32P]adenosine triphosphate (ATP) receptor autophosphorylation. In addition, to obtain information about whether changes in receptor autophosphorylation are related to changes in receptor number, relative numbers of high-affinity insulin receptors were determined by affinity cross-linking of [ 125I]insulin to the receptor α-chain and quantitation of the yield of labeled receptor α-chain. Control, starved, S diabetic, and L diabetic rats had plasma insulin and glucose levels of 294 ± 42, 90 ± 24, 48 ± 12, and 216 ± 30 pmol/L and 6.7 ± 0.2, 4.1 ± 0.2, 23.3 ± 0.7, and 21.6 ± 2.9 mmol/L, respectively. In all tissues, insulin-stimulated receptor autophosphorylation was normal to increased (skeletal muscle > liver > diaphragm > fat), and these changes in receptor functionality did not correlate with changes in insulin action on glucose metabolism in muscle, fat, and liver of starved and diabetic rats. This indicates that insulin resistance for glucose metabolism, when present in starved and diabetic rats, is due to postreceptor defects rather than to a decreased functionality of the receptor.

Mechanisms of cellular insulin resistance in human pregnancy

OBJECTIVE: The cellular mechanism(s) of insulin resistance developed during pregnancy were studied by investigating the functionality of insulin receptors and glucose transport. STUDY DESIGN: Abdominal adipose tissue was obtained from eight lean pregnant and nine control subjects, matched for insulin resistance by intravenous glucose tolerance testing. Insulin receptor binding and glucose transport were measured in freshly isolated adipocytes. Receptor kinase activity was measured on partially purified receptors. Data were analyzed by Student t test. RESULTS: High-affinity insulin receptors were reduced in cells from pregnant compared with normal controls (2.0 ± 0.4 vs 5.8 ± 1.3 × 104 sites per cell, p < 0.05). Kinase activity of insulin receptors was unaltered in pregnancy. Adipocytes from pregnant subjects displayed a threefold decrease in insulin sensitivity for glucose transport (median effective concentration 324 ± 93 vs 93 ± 14 pmol/L, p < 0.025) and a reduction in maximal insulin-stimulated glucose transport (1.58 ± 0.15 vs 2.33 ± 0.24 pmol/105 cells/10 seconds, p < 0.025). CONCLUSION:These results show that adipocytes from pregnant subjects exhibit decreased insulin receptor number and an impaired insulin sensitivity in the absence of functional alterations of receptor kinase activity. (AM J OBSTET GYNECOL 1994;170:635-41.)

Insulin receptor binding to erythrocytes in the first half of pregnancy is increased in healthy pregnant women as compared with non-pregnant or gestational diabetic women

Insulin binding to erythrocytes was measured longitudinally by a competitive radioreceptor assay in 21 healthy pregnant (HP) and 20 well-controlled gestational diabetic women (GD) in 4-week intervals throughout pregnancy and at day 4 post-partum. Maximum insulin binding (maxbdg) at weeks 8–14 was increased ( P < 0.001) in HP (median: 6.0%) but not in GD (median: 2.7%) as compared with non-pregnant control subjects (C) (median: 3.6%; previously reported: Clin. Chim. Acta 1992;207:57–71) due to an increased number of high-affinity insulin receptors. Throughout gestation the binding decreased continuously, to reach at term the levels found in C. In GD maxbdg remained close to the level of C throughout pregnancy. Binding differences between HP and GD were independent of the body mass index. Maxbdg did not differ between diet- and insulin-treated patients. It was higher in women whose offspring had low umbilical cord insulin levels (< 10 μunits/ml). The findings suggest that (a) higher insulin binding in HP could contribute to the improved glucose tolerance in early pregnancy and (b) the lack of increase in insulin binding during early pregnancy in gestational diabetes might be one factor leading to the manifestation of the disease in late pregnancy. However, it must be kept in mind that insulin receptors on erythrocytes do not necessarily resemble those on the major target tissues of insulin.