125I-insulin Binding Research Articles

Insulin-like receptors and carbohydrate metabolism in gills of the euryhaline crab Neohelice granulata: Effects of osmotic stress

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG.

Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality

Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant Phe(B24), one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [D-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the L-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [D-HisB24]-insulin is more flexible, and the reverse chirality of the B24C(α) atom swayed the D-His(B24) side chain into the solvent. Furthermore, the pocket vacated by Phe(B24) is filled by Phe(B25), which mimics the Phe(B24) side and main chains. The B25→B24 downshift results in a subsequent downshift of Tyr(B26) into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic L-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.

Repeated Hypoglycemia Caused by the Overproduction of Anti-insulin Antibodies and Isolated ACTH Deficiency in a Type 2 Diabetic Patient Receiving Insulin Therapy

A 46-year-old man with untreated type 2 diabetes visited our hospital. His plasma glucose and HbA1c levels were 429 mg/dL and 11.5%, respectively. He had no renal dysfunction. Insulin therapy (18 units aspart and 22 units biphasic insulin aspart 30/70) was initiated, and the HbA1c level improved (5.7–6.4%). One year later, he started experiencing frequent hypoglycemic attacks, which persisted despite significantly reducing his insulin dosage (total 9 units). Fasting plasma glucose, serum immunoreactive insulin (IRI), C-peptide, and HbA1c levels were 75 mg/dL, 420 μU/mL (reference, 1.7–10.4), 1.2 ng/mL (reference, 0.6–1.8), and 5.4%, respectively. His anti-insulin immunoglobulin G antibody (IA) titer (125I-insulin binding rate) was 95.5% (reference, 0–7.0); free and total IRI levels were 11.0 and >240 μU/mL, respectively. After insulin cessation, …

Ahsg-fetuin blocks the metabolic arm of insulin action through its interaction with the 95-kD β-subunit of the insulin receptor

We previously have shown that Ahsg, a liver glycoprotein, inhibits insulin receptor (InsR) tyrosine kinase (TK) activity and the ERK1/2 mitogenic signaling arm of insulin signaling. Here we show that Ahsg blocks insulin-stimulated GLUT4 translocation and Akt activation in intact cells (mouse myoblasts). Furthermore, Ahsg inhibits InsR autophosphorylation of highly-purified insulin holoreceptors in a cell-free, ATP-dependent system, with an IC50 within the range of single-chain Ahsg concentrations in human serum. Binding of 125I-insulin to living cells overexpressing the InsR shows a dissociation constant (KD) of 250pM, unaltered in the presence of 300 nM Ahsg. A mutant InsR cDNA encoding the signal peptide, the β-subunit and the furin processing site, but deleting the α-subunit, was stably expressed in HEK293 cells. Treatment with peroxovanadate, but not insulin, dramatically increased the 95 kD β-subunit tyrosine phosphoryation. The level of tyrosine phosphorylation of the 95-kD β-subunit can be driven down sharply by treatment of living HEK293 transfectant cells with physiological doses of Ahsg. Treatment of myogenic cells with Ahsg blunts insulin-stimulated InsR autophosphorylation and AKT phosphorylation. Taken together, we show that Ahsg antagonizes the metabolic functions initiated by InsR activation without interference in insulin binding. The experiments suggest a direct interaction of Ahsg with the InsR ectodomain β-subunit in a mode that does not significantly alter the high-affinity binding of insulin to the holoreceptor's two complementing α-subunits.

Diagnosis of Hypoglycemia due to Over-Dosage of Insulin Analog, Insulin Lispro

Serum insulin assay is used for the diagnosis of diabetes and hypoglycemia and is also used to quantify insulin for insulin pharmacokinetic evaluations. Serum C-peptide is usually measured in subjects who use insulin for evaluation of intrinsic insulin secretion. We experienced a hypoglycemic diabetic patient treated by premixed insulin lispro 75/25 (75% insulin lispro protamine suspension and 25% insulin lispro) who showed undetected serum levels of both insulin and Cpeptide. A 74-year-old type 2 diabetic man has been treated by injection of 26 units of premixed insulin lispro 75/25 before breakfast. In December 2010, his food intake decreased due to appetite loss, however, he injected 26 units of premixed insulin lispro 75/25, and subsequently developed disturbance of consciousness. He was admitted to our department, and his blood glucose level was 37 mg/dL. His consciousness promptly recovered after intravenous injection of glucose, and then he has been diagnosed as having hypoglycemia. We expected that his serum insulin level is high. However, serum insulin and also C-peptide were not detected. HbA1c is 5.5%, and anti-glutamic acid decarboxylase antibody was negative, and 125I-insulin binding rate was not too high (2.6%). His urinary C-peptide level was very low (4.0 μg/ day; normal range, 29.2 167.0 μg/day), suggesting the presence of severely decreased insulin secretion capacity. What is the cause of his hypoglycemia? Insulin analogs that are prepared with recombinant DNA technology are available for clinical use. One obvious question with insulin analogs is whether they are detectable by immunoassay. Serum insulin could not be detected by the chemiluminescent enzyme immunoassay (CLEIA) using Lumipulse Presto Insulin (Fuji Rebio, Tokyo, Japan) in our patient (Table 1). However, serum insulin could be measured by radioimmunoassay (RIA) using Insulin Eiken (Eiken Chemical Company, Tokyo, Japan). Lumipulse Presto Insulin could detect NPH human insulin, but, could not detect insulin lispro, and showed very low cross-reactivity with premixed insulin lispro 75/25 (Table 1). Insulin Eiken showed a high cross-reactivity with NPH human insulin, insulin lispro and premixed insulin lispro 75/25 (Table 1). Insulin lispro is a human insulin analog created by reversing the amino acids at positions 28 (Pro to Lys) and 29 (Lys to Pro) of the B chain of human insulin [1]. A sensitive RIA that is specific for insulin lispro has been developed [2]. Further, the combination of insulin assays that detect human insulin only or both human insulin and insulin analogs provides a new tool for studying pharmacokinetics of insulin lispro [3]. However, all of these assays are used mainly for research and are not usually used in clinical laboratories. Owen WE, et al studied cross-reactivity of insulin analogs with commercial insulin immunoassays [4]. Insulin lispro had a high cross-reactivity of 80% and 90% with the Access analyzer (Beckman Coulter) and the Advia Centaur analyzer (Bayer Diagnostics), respectively, whereas insulin lispro had a low cross-reactivity of 43% and 28% with the Coat-ACount (Diagnostic Products Corporation) and the IMMULITE 2000 analyzer (Diagnostics Products Corporation), respectively. The E170 method did not detect insulin lispro, indicating that the antibody used in this assay could not recognize an epitope that includes B28 and/or B29 of the B chain. The antibody in the assay of Lumipulse Presto InsuManuscript accepted for publication April 6, 2012

Insulin action in adipose tissue in type 1 diabetes

Background:Insulin action has been reported to be normal in type 1 diabetic patients. However, some studies have reported an insulin resistance state in these patients. The aim of this study was to investigate insulin resistance in a group of type 1 diabetic patients. We studied the insulin action in adipose tissue and analyzed the effects of duration of disease, body mass index (BMI), and glycosylated hemoglobin on insulin action at the receptor and postreceptor levels in adipocytes.Methods:Nine female type 1 diabetic patients with different durations of disease and eight nondiabetic female patients of comparable age and BMI were studied. 125I-insulin binding and U-[14C]-D-glucose transport was measured in a sample of subcutaneous gluteus adipose tissue obtained by open surgical biopsy from each subject.Results:The duration of disease was negatively correlated with both 125I-insulin binding capacity (r = −0.70, P < 0.05) and basal and maximum insulin-stimulated glucose transport (r = −0.87, P < 0.01, and r = −0.88, P < 0.01, respectively). Maximum specific 125I-insulin binding to the receptors in adipocytes was higher in the group of patients with a shorter duration of disease (P < 0.01). Basal and maximum insulin-stimulated glucose transport was significantly higher in the group with less than 5 years of disease (P < 0.01). No correlation was found between BMI and insulin action.Conclusion:Female type 1 diabetic patients have normal insulin action. There is a high glucose uptake in the early phase of the disease, although a longer duration of disease appears to be a contributing factor to a decrease in insulin action in these patients, and involving both receptor and postreceptor mechanisms.

Anti-BSA antibodies are a major cause of non-specific binding in insulin autoantibody radiobinding assays

Insulin autoantibodies (IAA) are usually the first risk-markers detected during the type 1 diabetes prodrome, but precise measurement is difficult as insulin binding is often low. Non-specific binding (NSB) of 125I-labelled insulin necessitates competitive displacement with unlabelled insulin to demonstrate specificity. NSB varies with different batches of label, suggesting that it is caused by impurities in the label. Addition of bovine serum albumin (BSA) can reduce NSB, so we investigated whether BSA antibodies cause lack of specificity in IAA assays.Samples from patients with newly-diagnosed type 1 diabetes, healthy schoolchildren previously found to have raised 125I-insulin binding (≥0.4units) and IAA-negative schoolchildren were re-assayed for IAA by radiobinding microassay using commercial 125I-insulin with and without 1g/dl BSA added to the buffer.Of 100 patients, 68 were IAA-positive on re-assay with BSA compared to 72 without BSA (p=0.125). Of 154 schoolchildren who previously had raised 125I-insulin binding, only 45 had 125I-insulin binding≥0.4units on re-assay with BSA compared to 90 without BSA (p<0.001). Following competitive displacement with unlabelled insulin, 40 were IAA-positive with BSA compared to 48 without BSA (p=0.02). No IAA-negative schoolchildren were IAA-positive on re-assay. Levels of NSB were associated with antibodies binding 125I-BSA and purification of labelled insulin reduced NSB.Addition of BSA to assay buffer improves the screening efficiency of the IAA assay without reducing disease sensitivity in patients. High titre BSA antibodies interfere with IAA measurement because of 125I-BSA present in some insulin labels. Improved purification of insulin labels should obviate the need for competitive displacement.

Two separate receptors for insulin and insulinlike growth factors on arterial smooth muscle cells.

Insulin and insulinlike growth factors (IGF) are related polypeptides that have similar biological activities. Both factors produce metabolic effects as well as growth effects. Most cells have separate receptors for IGF and insulin. In the present study we have demonstrated specific IGF receptors in cultured smooth muscle cells of rat aorta. The properties and specificity of these receptors were compared with those of the insulin receptor in the same cell system. The specific binding of both 125I-IGF and 125I-insulin binding in the order of potency: insulin proinsulin IGF. The potency of IGF in displacing 125I-insulin was rapid and reversible. Maximal 125I-IGF binding occurred at 20 degrees C with a specific binding of 10%. At this temperature specific 125I-insulin binding was 1.3% and occurred in biphase. The pH optimum for 125I-IGF binding was between pH 7 and 8. Both receptors show a high degree of specificity. IGF, insulin and proinsulin competed for 125I-IGF binding in the order of potency: IGF proinsulin insulin. The potency of insulin in displacing 125I-IGF was about 2000 times lower than that of IGF itself. In addition, IGF, insulin and proinsulin competed for 125I-insulin was about 450 times lower than that of insulin. These results indicate two separate binding sites for insulin and IGF on arterial smooth muscle cells.

Diurnal variation in specific insulin binding to erythrocytes.

We studied the insulin binding to erythrocytes during a 24-h period in 11 normal subjects. Compared with 0800 and 1600 h the specific 125I-insulin binding decreased significantly at midnight. These changes were mainly due to alterations in insulin binding affinity rather than a decrease in receptor number. An inverse finding was obtained concerning the plasma insulin levels. In fact, the highest insulin concentrations were observed at midnight. The results suggest that diurnal variations in insulin receptor function occur in response to fluctuations of plasma insulin levels.

Endogenous opiates do not influence glucose and lipid metabolism in rat adipocytes.

The effects of the opiates beta-endorphin, dynorphin, met-enkephalin, leu-enkephalin, morphine and of the opiate-antagonist naloxone on glucose metabolism and lipolysis were studied in isolated rat adipocytes. None of the tested substances was found to alter 125I-Insulin binding. The opiates did not influence 2-deoxy-glucose uptake or glucose incorporation into lipids. They also did not exhibit a lipolytic activity. We conclude that the disturbances of glucose homeostasis induced by endogenous opiates are not mediated by an effect on peripheral glucose metabolism.

Effects of glucagon-like peptide-1 (GLP-1) in RINm5F insulinoma cells. Stimulation of insulin secretion, insulin content, and insulin receptor binding.

Glucagon-like peptide-1 (GLP-1) is the major incretin hormone from the distal small intestine which stimulates basal and glucose-induced insulin secretion. Using the rat insulinoma cell line RINm5F (Gazdar et al. 1980) we investigated the effects of GLP-1 on insulin secretion, insulin content, and insulin receptor binding. During a 1 hour incubation, GLP-1 [1 nM] stimulated insulin secretion 2-fold (p < 0.01 vs controls). Incubating RINm5F for 24 h with GLP-1 [1 nM], a 1.6-fold higher cellular insulin content was observed (p < 0.01 vs controls). Moreover, GLP-1 induced a 2-fold higher capacity and a 15-fold higher affinity of 125I-insulin binding on the cell surface (p < 0.01 vs controls). Glucagon, known as a potent stimulator of insulin secretion, yielded a similar effect only in 1,000-fold higher concentrations, whereas the intracellular insulin content as well as insulin receptor binding was not increased. Taken together, in RINm5F insulinoma cells GLP-1 potently stimulates insulin secretion and insulin content, and improves insulin receptor binding.

Glucose tolerance in amyotrophic lateral sclerosis.

Blood glucose and plasma insulin during an oral glucose tolerance test were determined in 21 patients with amyotrophic lateral sclerosis and in 10 control patients matched for age, obesity and physical activity. In addition, 125I-insulin binding to circulating erythrocytes were studied in a subgroup of 4 ALS patients and 8 controls. Both impaired glucose tolerance and diabetes mellitus were evenly distributed between the study groups, and no difference in mean blood glucose levels during the OGTT was found between ALS and control patients. Fasting plasma immunoreactive insulin concentration was significantly higher in ALS patients as compared to controls, but plasma IRI increments to the glycemic stimulus were similar in the 2 groups. The number of insulin binding sites per cell appeared lower in patients with ALS, but the difference in receptor concentration was not statistically significant. In addition, the specific bound fraction of 125I-insulin showed no difference between ALS and control patients. In conclusion, we were unable to demonstrate any marked deterioration of glucose tolerance or increase in insulin resistance in patients with ALS.

Role of the insulin receptor in IGF-system signaling and biology.

Abstract Abstract #4055 The role of the type I insulin-like growth factor (IGF) receptor (IGF1R) in breast cancer has been extensively studied, and has led to the development of several IGF1R inhibitors currently in clinical trials. The closely related insulin receptor (IR) has been shown to contribute to IGF signaling through the formation of IGF1R/IR-hybrid receptors and through a homodimer of an alternatively spliced form of the IR (IR-A). Understanding the role of the IR in cancer biology will help determine which receptor(s) in the IGF system should be targeted to efficiently inhibit IGF signaling. Our previous work has shown that selective downregulation of IGF1R by siRNA resulted in enhanced sensitivity to insulin through increasing the numbers of insulin receptor homodimers. To determine how downregulation of IR affected IGF1R signaling, we used T47D cells, a breast cancer cell line expressing both the IR and IGF1R, as our model system. We transiently downregulated IR levels using siRNA and evaluated signaling. 5 nM of insulin activated downstream signaling molecules, such as insulin receptor substrate 1(IRS-1) and Akt in control cells, but this response was diminished in the IR-downregulated cells. In contrast, 5 nM of IGF-I did not result in differences in signaling between IR-downregulated cells and control cells. Using IR shRNA, we created stable clones with IR levels reduced by more than 72%. IR shRNA clones demonstrated decreased insulin binding, as measured by 125I-insulin binding assay. Additionally, clones showed decreased response to insulin signaling. 5 nM of insulin activated IRS-1 and Akt in parental T47D cells, but this response was attenuated in the IR-downregulated clones. Proliferation was also decreased in response to 5 nM insulin in the clones compared to parental cells. In contrast, IGF-I mediated signaling was not altered in clones when compared to vector control cells. Further, no change in proliferation was observed in response to IGF-I between wild-type and IR shRNA clones. We also generated stable IR shRNA clones using LCC6 cells. Insulin signaling was reduced in LCC6 clones when compared to wild-type LCC6 cells. However, in contrast to the T47D clones, IGF-I signaling was increased in LCC6 IR shRNA clones. Thus, the role of the insulin receptor in cancer biology may be dependent upon the ratio of IR, IGF1R, and the hybrid receptor. If cells contain mostly homodimers of IGF1R and IR, then downregulation of IR may affect only insulin response. However, if there are high levels of hybrid receptor, then downregulation of IR could affect, and even augment, IGF1R signaling. Our data suggest that the ratio and confirmation states of both IR and IGF1R should be understood to predict the effect of receptor downregulation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4055.

Up-Regulation of Cell Surface Insulin Receptor by Protein Kinase C-α in Adrenal Chromaffin Cells

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid ( 15 h) translocation of both conventional (c) protein kinase C-α (PKC-α) and novel PKC-e (but not atypical PKC-ζ) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-α. In the present study, chronic (≥12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4α-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the β-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor β-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor β-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Go6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-α up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Insulin Binding to Erythrocytes in Children with Type I Diabetes Mellitus before, and after Insulin Treatment

125I-insulin binding to erythrocytes was studied in 24 children with type I diabetes mellitus before institution of insulin therapy, and in 10 normals. Mean specific 125I-insulin binding in the diabetics was significantly lower than in the controls (10.2 +/- 0.4% (SEM), as against 11.6 +/- 0.5%, P less than 0.05). In 8 diabetics the study was repeated after 3-6 weeks of therapy, at which time the insulin binding had increased to normal (12.1%, range 10.7-13.6). No correlation was found between insulin binding and blood glucose, plasma insulin or plasma beta-hydroxybutyrate. We conclude that insulin binding to the receptors is altered in the early phase of type I diabetes.

Internalization of colloidal gold-labelled insulin complexes into cultivated human smooth muscle cells.

The binding and internalization of colloidal gold-labelled insulin complexes into cultivated human arterial smooth muscle cells was studied. We could demonstrate that the colloidal gold-labelled insulin complex was bound to specific receptors on the cell surface of smooth muscle cells and internalized after 30 min at 20 degrees C. The gold-labelled insulin complexes entered the cell via vesicles and escaped from them in the cytoplasm. The biological activity of the complexes was determined by measuring their effect on DNA synthesis in smooth muscle cells and incorporation of (14C)-glucose into isolated fat cells. The binding characteristics of the complexes were examined by competitive inhibition of 125I-insulin binding to isolated fat cells in the presence of increasing concentrations of gold-labelled insulin complexes or unlabelled insulin. The gold-labelled insulin complex showed a growth promoting and metabolic effect and was able to compete with 125I-insulin for its binding sites.

Type B insulin resistance syndrome induced by increased activity of systemic lupus erythematosus in a hemodialysis patient

A-23-year-old woman with systemic lupus erythematosus (SLE) and on hemodialysis for 5 years was admitted to the hospital because of severe general fatigue. On admission, laboratory findings showed that fasting plasma glucose levels ranged from 25 â 45 mg/dl, a test for antinuclear antibody (ANA) was positive (1 : 320, with a discrete-speckled pattern), serum C3 and C4 complement and CH50 level were remarkably depressed (22.5 mg/dl, < 5.1 mg/dl, < 13 U/ml, respectively), and insulin receptor antibodies were present (89.3% inhibition of 125I-insulin binding to cultured human lymphocytes by a binding inhibition assay). She also showed acanthosis nigricans. The patient was diagnosed to suffer from a Type B insulin resistance syndrome. The patient's serum insulin and C-peptide levels were markedly elevated during hypoglycemia without insulinoma (2,313.8 microU/ml, 55 ng/ml, respectively). Interestingly, not only postprandial hyperglycemia but also fasting hypoglycemia was observed. Treatment with steroid pulse and subsequent high dose of prednisolone resulted in restoration of euglycemia associated with disappearance of insulin receptor antibodies and improvement of both serum hypocomplementemia and the high titer of ANA. Later, the patient's course was complicated by hemorrhagic shock due to duodenal ulcer and she died of subsequent pneumocystis carinii pneumonia. The presented patient developed a Type B insulin resistance syndrome induced by increased activity of SLE after she had been treated with hemodialysis for 5 years. This is the first reported case with such a history and constellation. It is recommended that SLE patients on dialysis are carefully followed-up by clinical and serological monitoring. Type B insulin resistance syndrome must be considered in the differential diagnosis of hypoglycemia in SLE patients on dialysis.

AT1-IR-beta Association: A New Mechanism for the Inhibition of Insulin Receptor Function in Breast Cancer

Epidemiological evidence show that increased mortality in breast cancer is linked to hypertension and insulin resistance. Because Angiotensin II (Ang II), a hormone implicated in hypertension and insulin resistance, is a normal mitogen for breast tissue and elevated expression of the Ang II receptor AT1 is seen in breast cancer, we analyzed the effects of Ang II exposure on the functions of IR in human breast cancer cell line MCF-7. Exposure of MCF-7 to Ang II for 2 hours a) significantly reduced 125I-insulin binding to IR, and b) induced co-immuno-precipitation of the AT1 with IR-beta subunit. These Ang II-mediated effects on IR were inhibited by the AT1 antagonist losartan, and were not observed when exposure time was below 1-hour. These observations suggest extended exposure to Ang II have detrimental effects on insulin binding to IR that were not discovered in the previous studies where Ang II-exposure of insulin responsive cells was performed for periods less than one hour. In addition, they suggest a novel mechanism that involves AT1-IR-beta association for the inhibition of insulin binding to IR in response to extended exposure (2-hours) of breast cancer cells to elevated levels of Ang II (as seen in hypertensive patients), and provides a molecular link for the inhibition of normal IR signaling by Ang II in breast cancer.

Acute Liver Dysfunction Complicated with Uncontrollable Glycemia due to Insulin Antibody: Successful Treatment with Glucocorticoid and Lispro Insulin

Here, we report a case of acute liver dysfunction complicated with uncontrollable glycemia due to insulin antibody. The patient was admitted to our hospital due to diabetic ketoacidosis. He was administered insulin immediately, however, his fasting plasma glucose level remained unstable despite the insulin treatment. Blood biochemistry revealed severe liver dysfunction, although no markers including hepatitis virus or autoantibodies associated with autoimmune liver diseases were detected. The 125I-insulin binding rate was high (54%). The characteristics of insulin antibody in this patient were similar to the antibodies of IAS patients, therefore we administered oral glucocorticoid against insulin antibody. The reduction in the 125I-insulin binding rate and the binding capacity of the high affinity site of insulin antibodies were balanced after oral glucocorticoid therapy. In addition, preprandial subcutaneous regular insulin was switched to lispro insulin. Postprandial plasma glucose levels were relatively improved by lispro insulin. The etiology of acute liver dysfunction was unknown, however, we believe that the combination of oral glucocorticoid and lispro insulin was suitable and useful for preventing recurrent liver dysfunction in this patient.

Uremia and insulin resistance: N-carbamoyl-asparagine decreases insulin-sensitive glucose uptake in rat adipocytes

In uremia, urea-derived cyanate reacts with amino groups irreversibly forming carbamoyl amino acids (C-AA) and carbamoyl proteins. Carbamoylated molecules can affect binding and trafficking and alter metabolic pathways. The C-AA role in insulin-sensitive glucose transport has not been explored and may contribute to insulin resistance in uremia. Insulin-stimulated glucose uptake by cultured rat adipocytes was measured using both 3-minute and 3-second assays. Adipocytes were incubated for 24 hours in medium containing 0.5 micromol/mL of 15 different C-AA. 125I-insulin binding studies were done. C-asparagine in plasma from 10 uremic patients on continuous ambulatory peritoneal dialysis (CAPD) was measured using high-performance liquid chromatography (HPLC). Insulin-sensitive glucose uptake was reduced 34% by N-carbamoyl-l-asparagine, (N-C-Asn), in a dose-dependent manner with a half-maximally effective concentration of 0.15 micromol/mL. Fourteen other N-carbamoyl-amino acids as well as 0.5 micromol/mL of asparagine did not affect insulin sensitive glucose uptake. N-C-Asn, l-asparagine, and the other N-carbamoyl amino acids (0.5 micromol/mL) had no effect on basal glucose uptake. These data suggest that that N-C-Asn affects the insulin sensitive glucose transporter system. 125I-insulin binding studies demonstrated that N-C-Asn did not alter insulin binding. Glucose uptake measured using a 3-second assay showed that the glucose affinity of the transporter and glucose phosphorylation were not affected. In uremic patients managed by CAPD, the mean free N-C-Asn plasma level was 1.33 micromol/mL. These data suggest that N-C-Asn concentration may contribute to the insulin resistance seen in uremia.