Cold Insulin Research Articles

Why 100-Years After the Discovery of Insulin and the Appearance of Insulin-Induced Lipodystrophy: Are We Still Struggling with this Nasty Complication?

Since a century ago, insulin extracted from the animal pancreas has been used for therapeutic purposes. Lipoatrophy (LA) lesions came up first due to the impurities of preparations. However, as technology has progressed, we have had more purified insulin preparations available, now reduced to less than 5%. The 95% of local complications was represented by lipohypertrophy (LH), due to the recourse to narrow areas of skin, reuse of the same needle, injection of cold insulin. The average frequency of LH in the world is 47%, with peaks of 75%, reflecting the inappropriate educational activity of the diabetic teams. In this brief review, the causes of this failure are described, and feasible rehabilitative solutions are proposed.

142-OR: Unique Role of the a4 Component for S6-Kinase Activity in Metabolic Regulation and Anti-apoptotic Effect in Brown Adipose Tissue

Insulin and IGF1 signaling regulate function of brown and white adipose tissues. Recently we created adipocyte-specific inducible IR/IGF1R knockout mouse (Ai-DKO) and demonstrated the critical role of insulin signaling in maintenance of BAT and WAT. To further investigate downstream targets of these hormones regulating adipose tissue mass in adult animals, we compared gene expression patterns in WAT and BAT by using Ai-DKO mice, and found α4 expression was significantly reduced in Ai-DKO BAT but not in WAT compared with those of control. Knockdown of α4 in brown preadipocytecell lines using shRNA did not affect insulin-stimulated phosphorylation of IR, IRS1 or Akt; however, phosphorylation of ribosomal S6 protein was significantly increased in α4 KD cells after insulin stimulation. To investigate the impact of α4in mature adipocytesin vivo, we created inducible adipocyte-specific knockout of α4 gene (Ai-α4 KO) using floxed mice and mice carrying a tamoxifen-inducible Cre-ERT2transgene on the adiponectin promoter. Within 7 days, Ai-α4KO displayed an apparent decrease in SC-WAT and BAT mass, but not a significant effect on PG-WAT mass. Ai-α4 KO revealed extensive losses of adipocytes in SC-WAT and BAT depots with a significant increase in stromovascular cells in histological analysis with elevation of F4/80, TNF-α and IL-6, while no apparent changes were observed in PG-WAT. This appears to be due to a rapid development of an apoptotic process with increased TUNEL and cleaved caspase-3.Interestingly, Ai-α4KO showed marked reduction of BAT-associated functional gene UCP1 as well as PRDM16, Tfam and PGC1α mRNA compared to control. Consistent with these changes in mRNA and the decrease in BAT mass, Ai-α4 KO mice showed marked cold intolerance, glucose intolerance and insulin resistance. Thus, α4 is one of the components in control of insulin signaling and preferentially BAT mass, and has a unique role in regulating anti-apoptotic effect in brown adipocytes. Disclosure M. Sakaguchi: None. S. Okagawa: None. S. Kitano: None. T. Kondo: None. E. Araki: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Merck & Co., Inc., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi. Funding Japan Society for the Promotion of Science; Takeda Foundation; MSD Foundation; Kenzou Suzuki Foundation; Ono Foundation; Motida Foundation

Doubly Reactive INS-IGF2 Autoantibodies in Children with Newly Diagnosed Autoimmune (type 1) Diabetes.

The splice variant INS-IGF2 entails the preproinsulin signal peptide, the insulin B-chain, eight amino acids of the C-peptide and 138 unique amino acids from an ORF in the IGF2 gene. The aim of this study was to determine whether levels of specific INS-IGF2 autoantibodies (INS-IGF2A) were related to age at diagnosis, islet autoantibodies, HLA-DQ or both, in patients and controls with newly diagnosed type 1 diabetes. Patients (n = 676), 0-18 years of age, diagnosed with type 1 diabetes in 1996-2005 and controls (n = 363) were analysed for specific INS-IGF2A after displacement with both cold insulin and INS-IGF2 to correct for non-specific binding and identify double reactive sera. GADA, IA-2A, IAA, ICA, ZnT8RA, ZnT8WA, ZnT8QA and HLA-DQ genotypes were also determined. The median level of specific INS-IGF2A was higher in patients than in controls (P < 0.001). Irrespective of age at diagnosis, 19% (126/676) of the patients had INS-IGF2A when the cut-off was the 95th percentile of the controls (P < 0.001). The risk of INS-IGF2A was increased among HLA-DQ2/8 (OR = 1.509; 95th CI 1.011, 2.252; P = 0.045) but not in 2/2, 2/X, 8/8, 8/X or X/X (X is neither 2 nor 8) patients. The association with HLA-DQ2/8 suggests that this autoantigen may be presented on HLA-DQ trans-heterodimers, rather than cis-heterodimers. Autoantibodies reactive with both insulin and INS-IGF2A at diagnosis support the notion that INS-IGF2 autoimmunity contributes to type 1 diabetes.

Autoimmunity against INS-IGF2 Protein Expressed in Human Pancreatic Islets*♦

Insulin is a major autoantigen in islet autoimmunity and progression to type 1 diabetes. It has been suggested that the insulin B-chain may be critical to insulin autoimmunity in type 1 diabetes. INS-IGF2 consists of the preproinsulin signal peptide, the insulin B-chain, and eight amino acids of the C-peptide in addition to 138 amino acids from the IGF2 gene. We aimed to determine the expression of INS-IGF2 in human pancreatic islets and autoantibodies in newly diagnosed children with type 1 diabetes and controls. INS-IGF2, expressed primarily in beta cells, showed higher levels of expression in islets from normal compared with donors with either type 2 diabetes (p = 0.006) or high HbA1c levels (p < 0.001). INS-IGF2 autoantibody levels were increased in newly diagnosed patients with type 1 diabetes (n = 304) compared with healthy controls (n = 355; p < 0.001). Displacement with cold insulin and INS-IGF2 revealed that more patients than controls had doubly reactive insulin-INS-IGF2 autoantibodies. These data suggest that INS-IGF2, which contains the preproinsulin signal peptide, the B-chain, and eight amino acids of the C-peptide may be an autoantigen in type 1 diabetes. INS-IGF2 and insulin may share autoantibody-binding sites, thus complicating the notion that insulin is the primary autoantigen in type 1 diabetes.

Advances in insect physiology and endocrinology through genomics, peptidomics, and related technologies1Introduction to the virtual symposium on recent advances in understanding a variety of complex regulatory processes in insect physiology and endocrinology, including development, metabolism, cold hardiness, food intake and digestion, and diuresis, through the use of omics technologies in the postgenomic era.

This series of the Canadian Journal of Zoology brings together scientists actively working on insect physiology and endocrinology in this postgenomic era. This issue is timely and appropriate. Timely, because of the pace of change brought about by genome projects, functional genomics and genetics (omics technologies), including gene microarrays, mutations, RNAi, and sophisticated mass spectrometry techniques, which are helping to unravel complex regulatory processes. Appropriate, because Canada, and the Canadian Journal of Zoology, has a rich history and strong tradition of cutting-edge research in insect biology—with particular strengths in insect physiology and endocrinology. The first review illustrates how these very modern omics technologies can be embraced and applied to insect physiology and endocrinology, and the subsequent reviews illustrate this in practice, with regard to insect cold hardiness, insulin signaling and stress, peptidergic control of food intake and digestion, endocrine control of diuresis, and finally allatoregulatory peptides. These reviews set the scene and context for the exciting era that we find ourselves in, and the depth of understanding that has come from this postgenomic revolution.

Autoimmune Hypoglycemia in a Patient with Characterization of Insulin Receptor Autoantibodies

BackgroundType B insulin resistance syndrome is a manifestation of autoantibodies to the insulin receptor that results in severe hyperglycemia and acanthosis nigricans. However, the mechanisms by which these autoantibodies induce hypoglycemia are largely unknown. In this paper, we report the case of patient with type B insulin resistance syndrome who presented with frequent severe fasting hypoglycemia and acanthosis nigricans.MethodsTo evaluate the mechanism of hypoglycemia, we measured the inhibition of insulin binding to erythrocytes and IM9 lymphocytes in a sample of the patient's dialyzed serum before and after immunosuppressive therapy.ResultsIn the patient's pre-treatment serum IgG, the binding of 125I-insulin to erythrocytes was markedly inhibited in a dose-dependent manner until the cold insulin level reached 10-9 mol/L. We also observed dose-dependent inhibition of insulin binding to IM9 lymphocytes, which reached approximately 82% inhibition and persisted even when diluted 1:20. After treatment with glucocorticoids, insulin-erythrocyte binding activity returned to between 70% and 80% of normal, while the inhibition of insulin-lymphocyte binding was reduced by 17%.ConclusionWe treated a patient with type B insulin resistance syndrome showing recurrent fasting hypoglycemia with steroids and azathioprine. We characterized the patient's insulin receptor antibodies by measuring the inhibition of insulin binding.

A Report of the Vancouver Forum on the Care of the Live Organ Donor: Lung, Liver, Pancreas, and Intestine Data and Medical Guidelines

An international conference of transplant physicians, surgeons, and allied health professionals was held in Vancouver, Canada, on September 15 and 16, 2005 to address the care of the live lung, liver, pancreas, and intestine organ donor. The Vancouver Forum was convened under the auspices of the Ethics Committee of The Transplantation Society. Forum participants included over 100 leaders in organ transplantation, representing many countries from around the world, including participants from the following continents: Africa, Asia, Australia, Europe, North and South America. The objective of the Vancouver Forum was to develop an international standard of care for the live lung, liver, pancreas and intestinal organ donor. This Vancouver Forum followed a conference convened in Amsterdam on the care of the live kidney donor (1, 2). There were four organ specific work groups at the Vancouver Forum: lung, liver, pancreas and intestine. Each organ work group addressed the following topics in concert and reported their findings in a plenary presentation to all participants:

A sensitive radioimmunoassay of insulin autoantibody: Reduction of non-specific binding of [ 125I]insulin

There has been a lack of consensus among results of assays for insulin autoantibody (IAA) carried out by different laboratories, despite a reduction in the non-specific effect using a cold insulin competitor in radioimmunoassays (RIAs) for IAA in type I diabetes. We speculated that the discrepancies are partly a result of the non-specific binding (NSB) of [ 125I]insulin to unidentified molecules in serum on polyethylene glycol separation, and tried to improve IAA RIAs. The molecular weight of a candidate for the factor causing NSB was estimated to be about 700 kDa by gel filtration analysis, resembling that of alpha 2-macroglobulin (a2M). Further, the addition of purified a2M to the assay resulted in an increase in NSB. Screening revealed that heterocyclic compounds, such as isothiazolinone derivatives (ProClin300), were greatly effective at reducing NSB in control subjects from 2.904 ± 0.909% to 1.347 ± 0.254% ( n = 283, mean ± SD, p < 0.0001). Using our newly developed IAA RIA with ProClin300, the sensitivity for newly diagnosed type I diabetes patients ( n = 55) was 32.7% and 30.9% with or without insulin competition, respectively, whereas that of the former assay without ProClin300 was only 20.0%.

In insulin-autoantibody-positive children from the general population, antibody affinity identifies those at high and low risk

Insulin autoantibodies (IAA) precede and predict the onset of type 1 diabetes, but not all children with IAA develop the disease. In affected families, IAA affinity can identify IAA-positive children who are more likely to progress to diabetes. The purpose of this study was to determine whether affinity is a useful marker to stratify type 1 diabetes risk in IAA-positive children from the general population. IAA affinity was determined by competitive binding to 125I-insulin with increasing concentrations of cold insulin and with cold proinsulin in sera from 46 IAA-positive children identified in the Karlsburg Type 1 Diabetes Risk Study of a Normal Schoolchild Population in north-eastern Germany. IAA affinity ranged between 5 x 10(6) and 1.2 x 10(11) l/mol. IAA affinity was higher in 24 children who developed multiple islet autoantibodies or diabetes (median 3.5 x 10(9) l/mol; interquartile range [IQR] 2.1x10(9) to 2.1 x 10(10) l/mol) than in 22 children who did not develop multiple islet autoantibodies or diabetes (median 1.3 x 10(8) l/mol; IQR 3.8 x 10(7) to 7.2 x 10(8) l/mol; p<0.0001). Using a threshold of > or = 10(9) l/mol, 22 of the 24 children who developed multiple islet autoantibodies or diabetes were correctly identified by high-affinity IAA and 18 of 22 who did not develop multiple islet autoantibodies or diabetes were correctly identified by low-affinity IAA. IAA affinity was significantly higher in samples with proinsulin reactive IAA (p<0.0001). IAA affinity measurement provides robust identification of IAA associated with high diabetes risk.

A novel high-throughput method for accurate, rapid, and economical measurement of multiple Type 1 diabetes autoantibodies

Prediction of Type 1 diabetes for study of preventive therapies requires screening the general population, where 85% of new cases occur. Even with HLA-based prescreening, nearly 20% of all children will need multiple serum autoantibody testings. High-throughput, economical, and accurate methods are therefore essential. We have developed such a radiobinding method, using 96-well microtiter plates and a novel immune complex capture method via membrane-bound Protein A. Each microtiter plate contained a standard negative control serum, and low-, medium-, and high-level positive control sera. All sera were evaluated in triplicate. This readily allowed quality control criteria both for triplicates of individual sera and for each 96-well plate. Inter-assay coefficients of variation (CVs) were all ≤16%, while intra-assay CVs were all ≤10%. The assay was found to be sensitive (to detect autoantibodies in patients) and specific (low reactivity in thousands of healthy volunteers). The format worked well using diverse antigens such as 35S-met-GAD65, 35S-met-ICA512/IA2, 35S-met-Phogrin, and 125I-insulin, and could be used for simultaneous screening of reactivity to both GAD65 and ICA512/IA2 in the same well. Diagnostic accuracy compared favorably with microcentrifuge tube-based Protein A-agarose GAD65 and IA2 autoantibody radiobinding assays and with acid–charcoal–polyethylene glycol (PEG) based competitive insulin autoantibody assays. In the case of 125I-insulin, comparing signal in the absence versus presence of cold insulin competitor was not necessary. Total serum volumes required were only 6 μl for GAD and ICA512, and only 15 μl for IAA. The method costs less than all other commonly used formats, and should be useful for population screening.

Insulin and IGF-I Binding in Isolated Trout Cardiomyocytes

Insulin and IGF-I receptor binding were characterized in cardiac muscle cells isolated from the brown trout,Salmo trutta fario.Cardiomyocyte suspensions obtained by perfusion of ventricles with collagenase showed a high degree of viability as judged by trypan blue exclusion, LDH leakage, and morphology. Specific insulin binding was 2.88 ± 0.28%/10 mg cells after overnight incubation at 4°. Scatchard analysis indicated the presence of high affinity insulin binding sites with an apparent dissociation constant (Kd) of 0.285 ± 0.043 nMand a binding site density of 1.61 ± 0.19 × 108/mg cells. Specificity of insulin binding was determined by displacing labeled insulin with increasing concentrations of IGF-I, and theKdvalue obtained was 4.77 ± 2.82 nM, 17-fold higher thanKdvalues for displacement of insulin tracer by nonlabeled insulin. The percentage of IGF-I specific binding (6.70±1.42%/10 mg cells), affinity (Kd= 0.163 ± 0.023 nM), and binding site density (4.00 ± 1.13 × 108/mg cells) were higher than those of insulin. Displacement curves of labeled IGF-I with nonlabeled insulin (Kd= 33.6 ± 9.9 nM), indicated a high specificity of the IGF-I binding site. High concentrations of cold insulin and IGF-I were able to decrease markedly the specific binding to their own receptor. Incubation with cold IGF-I also induced a diminution in insulin binding in agreement with the lower specificity of the insulin receptor. These data suggest that insulin and IGF-I are able to down-regulate their own receptor number in cardiac muscle cells. The present results demonstrate that the isolated cardiac myocyte preparation from brown trout is a useful model for studying insulin and IGF-I binding in fish heart tissue.

High affinity binding sites for proinsulin in human IM-9 lymphoblasts.

Proinsulin and insulin binding in IM-9 lymphoblasts show curvilinear Scatchard plots, which may be explained by two binding sites, negative cooperativity of receptors, or both. Using flow-cytometric analysis of insulin binding, we were able to distinguish and separate two different IM-9 cell fractions. In both fractions, Scatchard plots for specific binding of insulin and proinsulin were linear, suggesting the presence of two distinct populations of receptors. Type 1 cells showed low capacity but high affinity of insulin binding (16,300 +/- 3,000 sites/cell; Kd 0.4 +/- 0.1 nmol/l). Proinsulin and insulin-like growth factor 1 (IGF-1) were significantly less potent in competition. MA-20, a specific antibody against human insulin receptors, inhibited insulin binding by 80%, while the specific antibody against human IGF-1 receptors, alpha IR-3, had no effect. Pretreatment with insulin decreased insulin binding by 90%. 125I-insulin displayed stepwise dissociation with the rate markedly enhanced by cold insulin. Type 2 cells exhibited significantly different binding characteristics with higher capacity but lower affinity of 125I-insulin binding (430,000 +/- 25,000 sites/cell, p < 0.001 vs type 1; Kd 2 +/- 0.4 nmol/l, p < 0.02 vs type 1). Proinsulin competed with similar potency for insulin binding, while IGF-1 was still less potent. 125I-proinsulin showed a significantly higher binding affinity than 125I-insulin (Kd 0.5 +/- 0.3 nmol/l, p < 0.05) with 50,000 +/- 10,000 binding sites/cell. C-peptide was able to compete for 125I-proinsulin, but not for 125I-insulin binding. MA-20 did not influence 125I-proinsulin binding, but inhibited 125I-insulin binding by 50%, whereas alpha IR-3 increased proinsulin binding 1.5-fold with no effect on insulin binding. Preincubation with insulin decreased insulin binding by 50% and proinsulin binding by 10%. The dissociation of 125I-proinsulin showed linear first-order kinetics and was not significantly accelerated by cold proinsulin. Furthermore, the tyrosine phosphorylation of a 65 kDa protein was stimulated to a significantly greater extent by proinsulin than by insulin, indicating activation of different signalling cascades. DNA analysis revealed that type 1 cells were predominantly in the G1 phase, whereas type 2 cells were in the S and G2 + M phases of the cell cycle. We conclude, that IM-9 lymphoblasts were separated by flow-cytometry into one fraction with typical insulin receptors and a second fraction with high affinity binding sites for proinsulin. High affinity proinsulin binding sites were distinguished from typical insulin receptors by: 1) higher affinity for proinsulin than insulin, 2) inhibition of proinsulin binding by C-peptide but not by the insulin receptor antibody MA-20, 3) non-co-operative first order dissociation kinetics of proinsulin binding, 4) resistance to down-regulation by insulin, and 5) differences in signal transduction.

Abundant Insulin-like Growth Factor-1 (IGF-1) Receptor Binding in Fish Skeletal Muscle

Insulin and insulin-like growth factor-1 (IGF-1) receptors were quantified in glycoprotein fractions prepared by wheat germ agglutinin-agarose affinity chromatography from skeletal muscle of several species of salmonid fish and carp. Insulin binding in fish skeletal muscle was lower than insulin binding found in rat. IGF-1-specific binding was two- to sixfold higher than insulin binding (16.0 ± 3.0 versus 5.8 ± 0.75%/50 μg glycoprotein in brown trout, 15.5 ± 0.9 versus 2.2 ± 0.5%/50 μg in coho salmon, and 39.7 ± 7.6 versus 16.0 ± 3.0%/50 μg in carp muscle). Specific IGF-1 binding in fish skeletal muscle presented values similar to those in rat muscle. IGF-1 receptor binding was, in addition, highly specific. Cold IGF-1 displaced radiolabeled IGF-1 binding in doses 100-to 1000-fold lower than cold insulin (ED 50 of IGF-1 binding to carp receptor preparations was 0.24 ± 0.04 nM when displaced with cold IGF-1 and 368 ± 83 nM when displaced with insulin). On the other hand, cold insulin displaced radiolabeled insulin binding at concentrations 10- to 100-fold lower than cold IGF-1. Receptor tyrosine kinase activity was stimulated over basal values in a dose-dependent manner by both insulin and IGF-1, although IGF-1 was more potent than insulin. Basal rates of phosphorus transferred to the artificial exogenous substrate poly(Glu 4;Tyr 1) were similar in all salmonid species (200-320 fmol P/μg protein) and higher in carp (1840 ± 300 fmol P/μg protein). Maximum percentage of stimulation of kinase activity by insulin and IGF-1 was in the same range in all salmonid species and carp (130-200% for insulin, 160-232% for IGF-1). Abundance of IGF-1 receptors in fish skeletal muscle contrasts with the pattern observed in higher vertebrates, in which insulin receptors prevail over IGF-1 receptors.

Partition of insulin-like growth factor (IGF)-binding sites between the IGF-I and IGF-II receptors and IGF-binding proteins in the human kidney.

Quantitative ligand binding autoradiography and in situ hybridization were employed to analyze [125I]insulin-like growth factor-I ([125I] IGF-I) and [125I]IGF-II-binding sites in human kidney sections. Binding sites for both ligands were concentrated in the inner medulla and glomeruli, with low levels present in the tubulo-interstitial cortex. Competition with cold IGF-I, IGF-II, and insulin was used to determine nonspecific binding and differentiate binding of ligands to the IGF-I and IGF-II receptors and IGF-binding proteins (IGFBPs). Nonspecific binding was less than 20% of the total for both ligands. Insulin (10(-5) mol/L), which binds to the IGF-I receptor, but not to the IGF-II receptor or IGFBPs, displaced 39 +/- 8% of [125I]IGF-I binding in glomeruli, 60 +/- 7% in the tubulo-interstitial cortex, and 32 +/- 7% in the medulla. Insulin produced no detectable decrease in [125I]IGF-II binding in any region. IGF-I (10(-8) mol/L), which binds strongly to IGFBPs, but not appreciably to the IGF-II receptor, produced reductions of 46 +/- 9%, 35 +/- 8%, and 39 +/- 12% in [125I]IGF-II binding in glomeruli, tubulo-interstitial cortex, and medulla, respectively. In situ hybridization showed that IGFBP-1-5 mRNAs were all expressed in glomeruli. IGFBP-2 mRNA was abundant in medullary collecting duct epithelium, whereas IGFBP-3, -4, and -5 mRNAs were localized in interstitial and vascular cells throughout the kidney. IGF-I and -II receptor mRNAs were widely distributed in renal epithelium. The abundance of local IGFBP gene expression was positively correlated with insulin-nondisplaceable IGF binding in specific kidney regions. In summary, [125I]IGF-I binding appears to be partitioned largely to IGFBPs in glomeruli and largely to the IGF-I receptor in the tubulo-interstitial cortex, with binding in the medulla more evenly divided. The proportion and regional distribution of [125I]IGF-II binding to IGFBPs are similar, but the balance appears to be primarily associated with the IGF-II, rather than the IGF-I, receptor. Finally, this study shows that [125I]IGF binding autoradiography combined with in situ hybridization can be used to localize and potentially quantitative expression of IGFBPs in tissue sections.

Hypoglycaemia caused by atypical insulin antibodies in a patient with benign monoclonal gammopathy

We describe a 48-year-old woman with recurrent severe hypoglycaemia apparently caused by a paraprotein with insulin-binding capacity. Very high fasting values were found for serum insulin (170 and > 250 mU l-1) as well as for proinsulin 125 pmol l-1 and an insulinoma was suspected. Hypoglycaemia developed after an oral glucose tolerance (OGTT) test but not during fasting for 48 h. Free insulin and C-peptide were normal during OGTT whereas serum insulin was very high. 125I-insulin binding to serum, determined with a polyethylene glycol (PEG) precipitation method was high (40%), and equally high after addition of 1.7 x 10(-5) mol l-1 cold insulin to estimate non-specific binding. By adding very high concentrations of cold insulin, displacement of 125I-insulin bound to serum was found (50% displacement at 4 x 10(-5) mol l-1). No immunoglobulin G (IgG) insulin antibodies were detected by radioimmunoelectrophoresis. On agarose electrophoresis a small paraprotein (4 g l-1) in the gamma-globulin fraction was detected. 125I-insulin binding to this paraprotein was demonstrated. We conclude that if insulin autoantibodies are suspected as a cause of hypoglycaemia screening for insulin antibodies should always be done with a PEG-precipitation method.

Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [(125)I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [(125)I]-insulin binding reached a steady state within 20-30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (Kd1 = 5.5 pM) and low capacity (Bmax1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (Kd2 = 2.4 nM) and high capacity (Bmax2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [(125)I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC50 values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis.

Increased specificity and sensitivity of insulin antibody measurements in autoimmune thyroid disease and type I diabetes

Insulin autoantibodies (IAA), a marker for insulin-dependent diabetes mellitus (IDDM), have been reported in other diseases such as thyroid disease and after treatment with sulfhydryl containing medications. Reported prevalences of IAA in non-diabetics vary widely, probably due in part to methodological differences between laboratories. In addition, certain sera may have a high non-specific binding to insulin. We compared a radioimmunossay (RIA) for IAA which included non-specific binding with an RIA that incorporated a competitive displacement with cold insulin to remove non-specific binding. Using the RIA which measured specific plus non-specific binding, IAA positivity was found 22/92 (23.9%) of sera from thyroid disease patients, 16/124 (12.9%) of random masked sera from a hospital laboratory, 27/335 (8.1%) of first degree relatives of IDDM patients, 63/178 (35.4%) of subjects with newly diagnosed IDDM, and 0/92 (0%) of normal controls. Insulin antibodies (IA) were found in 80/99 (80.8%) of insulin-treated diabetic subjects. In contrast, using the displacement assay which allowed measurement of specific binding, the frequency of IAA positivity was lower for subjects with thyroid disease (7/92 (7.6%)), random hospital sera (12/124 (9.8%)), and for first degree relatives of IDDM patients (8/335 (2.4%)), while higher for subjects with newly diagnosed IDDM (71/178 (39.9%)). Subjects with insulin-treated diabetes (78/99 (78.8%)) and normal subjects (1/92 (1.1%)) showed little change. Strikingly, three of the eight (37.5%) relatives of IDDM patients that were positive in RIA measuring specific binding were detected only because cold displacement was utilized. We conclude: (1) subjects with thyroid disease and first degree relatives of IDDM patients frequently have high non-specific binding for IAA in an RIA not employing a cold displacement step, (2) in some newly diagnosed IDDM patients and first degree relatives of IDDM patients, IAA may be missed by an assay not optimized to measure specific binding, and (3) displacement with cold insulin increases both the specificity and sensitivity of RIAs measuring insulin autoantibodies.

Insulin Receptor Number and Binding Affinity in Newborn Dogs

The insulin resistance in newborn mammals may be caused by a receptor or postreceptor defect. Although liver and umbilical cord blood monocytes have increased numbers of insulin receptors, there is a paucity of information about other neonatal tissues. Glucose disposal takes place primarily in the skeletal muscle; therefore, it is important to evaluate this tissue for an insulin receptor defect. To determine the role of insulin receptors in neonatal insulin resistance, neonatal and adult canine skeletal muscle, heart, and liver were compared for numbers of insulin receptors and their affinity for insulin. Partially purified receptors from four animals in each group were obtained by wheat germ lectin affinity chromatography and used in competition binding studies. Specific binding (mean +/- SE) in the absence of cold insulin was increased in newborn skeletal muscle (9.7 +/- 0.8 versus 4.8 +/- 0.5%, p less than 0.001) and heart (8.1 +/- 1.2 versus 5.5 +/- 0.6%, p less than 0.05). High-affinity insulin receptor number (mean +/- SEM) was increased in newborn skeletal muscle (183 +/- 40 versus 120 +/- 29 pM, p less than 0.002) and heart (264 +/- 94 versus 157 +/- 51 pM, p less than 0.05) as estimated from the X intercept of the Scatchard plot. Using half-maximal binding to estimate affinity, there were no differences between adults and newborns among all tissues studied. High-affinity receptor number and percentage of specific binding were similar for newborn and adult liver tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in insulin receptors on methylcholanthrene-induced sarcoma during growth

Methylcholanthrene-induced sarcomas (MCA-S) have different growth patterns in diabetic (D) and nondiabetic (ND) rats. Diabetes delays the early phase of tumor growth and prolongs survival. This study evaluated MCA-S growth and its relation to insulin receptors (IR) and glucose uptake. Fisher 344 rats 150–200 g were assigned to two groups: Diabetic tumor bearers (DTB, n = 26) and nondiabetic tumor bearers (NDTB, n = 18). Diabetes was induced with iv streptozocin (40 mg/kg); MCA-S was inoculated (1 × 10 6 cells) subcutaneously 10 days later. Animals were sacrificed during early growth (tumor volume ⩽ 20 cc) or logarithmic growth (tumor volume > 20 cc). IR assay was performed (0–10 5 ng/ml cold insulin, 25 × 10 3 cpm/tube A14 125I-insulin, 90 min, 15°C, pH 7.8) on a single cell preparation. Serum glucose milligrams per deciliter and insulin nanograms per milliliter were assayed. Glucose uptake (dpm/g tissue/hr) was assayed 2 hr after an ip injection of 0.5 μCi 3- O[ 14C]methylglucose. Diabetic, tumor-bearing animals had a significantly increased number of insulin receptors at the small [⩽20 cc, 28.7 (D) vs 8.3 (ND)] and large [>20 cc, 82.8 (D) vs 27.8 (ND)] tumor volumes. Glucose uptake was increased in the tumor at both volumes in the nondiabetic animals compared to the diabetic animals. The increase in insulin receptor number on the surface of the tumors of diabetic animals did not result in an increase in glucose uptake. Substrate (glucose) deprivation due to hypoinsulinemia remains the most likely explanation for the early phase growth delay in the tumors of diabetic animals.

Basolateral and apical binding, internalization, and degradation of insulin by cultured kidney epithelial cells

In vivo, filtered insulin is absorbed and degraded in proximal tubules after binding to the apical membrane. Peritubular removal also occurs and involves basolateral receptor binding and degradation. Whether basolateral degradation proceeds within the cell or on the cell surface is unknown. Because of the difficulties in addressing this question in vivo, this study was carried out with a cultured opossum kidney epithelium cell line with proximal-like features and insulin receptors. Cells were grown in partitioned wells on polycarbonate filters and, when confluent, the monolayer effectively separated the culture well into apical and basolateral compartments. Apical and basolateral binding, internalization, and degradation were studied separately by incubating monolayers with 125I-insulin added to either the apical or basal compartment. At 37 degrees C insulin associated with either pole in a time-dependent manner. This interaction was specific, for it was competitively inhibited by cold insulin but not by unrelated peptides. Separation of surface-bound from internalized insulin was achieved by lowering extracellular pH. At 4 degrees C, 92% of the radioactivity added to either side of the monolayer was surface-bound, whereas at 37 degrees C and after 1 h, 57% was surface-bound and 43% internalized. Affinity of apical and basolateral receptors were similar (1-2 nM), but basolateral receptor number was greater, for at high insulin concentrations (5 x 10(-8) M) basolateral membrane binding exceeded apical by fivefold (250 +/- 81 vs. 56 +/- 11 fm/10(6) cells). Degradation followed exposure to either pole of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)