Biological Effects Of Insulin Research Articles

Insulin-mimetic peptides in sports drug testing.

Because of its influence on carbohydrate metabolism and, at the same time, anti-catabolic effects, the misuse of the peptide hormone insulin and its synthetic analogs is prohibited in sports at all times according to the regulations of the World Anti-Doping Agency (WADA). The biological effects of insulin and its analogs are mediated through binding to the insulin receptor, which was also found to be activated by different peptides structurally largely unrelated to insulin. Such insulin-mimetic peptides or selective-insulin receptor modulators (SIRMs) represent a novel class of potential performance-enhancing agents, which is currently not explicitly mentioned on the WADA Prohibited List. Within this research project, advanced solid-phase extraction (SPE) and liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) were employed to develop a fast, reliable, and specific assay for the detection of the insulin-mimetic peptides S597 and S519 from plasma. Method validation demonstrated a detection limit of 0.5ng/mL and successfully illustrated the applicability of the approach to routine sports drug testing programs. Moreover, sophisticated and comprehensive in vitro metabolism experiments were conducted, and several metabolic degradation products were identified, which will enhance the information generated from future analyses of doping control samples.

Abstract P1-05-03: Isoform specific targeting of insulin receptor

Abstract The insulin receptor (InsR) exists in both an A and B isoform. InsR-B differs from InsR-A by the inclusion of exon 11, which encodes 12 amino acid residues at the C-terminus of the InsR alpha-subunit. Increased InsR-A expression is associated with mitogenic signaling pathways while InsR-B is linked to insulin-mediated metabolic functions. Predominant InsR-A expression may therefore be important in growth and fetal development of embryos, whereas predominant InsR-B expression has a role in metabolic insulin action in adult life. Increased InsR-A expression is seen in breast cancer. In endocrine resistant breast cancer, InsR-A is expressed at high levels (Gradishar, et al. Clin Cancer Res 22:301 2016 PMID: 26324738). Thus, developing InsR-A specific inhibitors could be a useful therapy for breast cancer. We have previously published InsR specific binders using a T7 phage gene 2 protein (Gp2), a small protein scaffold (Chan, et al. Mol Cancer Ther 16:1324 2017 PMID: 28468775), with the long-term goal of creating effective InsR inhibitors and diagnostics. Using yeast display and directed evolution, we identified three Gp2 variants (Gp2 #1, #5, and #10) with low nanomolar affinity and specific binding to cell surface InsR. We have shown that these Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and resistant breast cancer, but did not downregulate InsR expression. To further characterize the specificity of Gp2 variants, we used two techniques. HEK293T cells were infected with lentiviral vectors expressing either InsR-A tagged with mCherry or InsR-B tagged with eGFP. Using these cells, we performed “mock panning” and showed the Gp2 #5 variant bound both InsR-A and InsR-B, but had higher affinity for InsR-B. We also incorporated Gp2 #5 into the capsid of a tropism-null adeno-associated virus (AAV). Using this Gp2-AAV, we infected HEK293T-InsR-A or InsR-B cells at a number of different multiplicities of infection. These data were consistent with panning data and showed specific Gp2-AAV infection of cells expressing high levels of InsR-B, but not InsR-A. Thus, our data show that Gp2 variants we created have a higher affinity for InsR-B than InsR-A. Despite this preferred affinity, these Gp2 binders have sufficient binding to InsR-A to disrupt the biological effects of insulin in breast cancer cells. Thus, even relatively low affinity binding to InsR-A can disrupt its function. Further development of InsR-A Gp2 binders may be developed and provide more specific targeting of the breast cancer specific isoform of InsR. Citation Format: LaPara K, Chan JY, Zdechlik A, Ljunggren K, Schmidt D, Hackel B, Yee D. Isoform specific targeting of insulin receptor [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-03.

Comparative research of effects of permeability enhancers on the effectiveness of model protein (insulin) administered via the inhalation route

Relevance.Inhalation administration of compounds with peptide and protein structure may be promising in provision of emergency medical assistance in emergency situations, including radiation injuries. The development of inhalation dosage forms for compounds with peptide and protein structure requires the selection of excipients to increase their bioavailability.Intention.To assess effects of permeability enhancers on the effectiveness of a model biologically active substance of protein nature (insulin) by inhalation.Methods.The experiments were carried out in outbred white male rats with initial weight of 180–220 g. To assess the effectiveness of insulin and permeability enhancers, they were administered via intranasal and intratracheal routes using a MicroSprayer® IA-2B instillation probe (Penn Century, USA). The list of biological membrane permeability enhancers under investigation (Sigma-Aldrich, USA) includes the following substances: methyl-β-cyclodextrin, dimethyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, Tween-80, sodium methyl oleate, oleic acid, sodium taurocholate, sodium glycocholate, Pluronic F-68, tesit, Brij-35, saponin, cetylpyridine.Results and Discussion. Insulin bioavailability without permeability enhancers does not exceed 0.5% when administered intranasally. When assessing effects of multiple doses of permeability enhancers on food and water consumption in animals over time, decreased parameters under study were observed in the control and experimental groups compared to intact animals. Multiple intratracheal administration of solutions to rats can lead to some infectious complications.Conclusion. Such excipients as sodium taurocholate, dimethyl-β-cyclodextrin and Тween-80 in a concentration of 5 % increase bioavailability and intensity of biological effects of insulin when administered intranasally. The surfactants Brij-35, Тween-80 and sodium taurocholate in a concentration of 0.25 % increase the duration of biological effects of insulin when administered intratracheally. Sodium taurocholate and Tween-80 in a concentration of 0.25 % have the lowest toxicity during multiple intratracheal administration.

Insulinska rezistencija kod krava u peripartalnom periodu (pregled)

Insulin resistance is a state in which the biological effect of insulin is reduced when its concentration decreases or when a compensatory mechanism increases its concentration. Insulin resistance is characterized by reduced insulin response to glucose, i.e. decreased pancreatic beta cell function (insulin hyporesponsiveness) and/or reduced sensitivity of glucose to insulin (reduced intake of glucose by peripheral tissues under the influence of insulin - eng. insulin sensitivity). Methods for estimating insulin resistance include direct methods (hyperinsulinaemic-euglycaemic clamp, golden standard), indirect methods (intravenous glucose tolerance test) and surrogate methods (indices calculated from basal concentrations of insulin, glucose, NEFA and BHB-HOMA, QUICKI, RQUICKI and RQUICKI- BHB). Surrogate indices show correlations with direct and indirect test results but they are inconsistent. Inconsistency occurs because the dependence of glucose concentrations on the degree of hepatic gluconeogenesis should be kept in mind when evaluating insulin resistance in ruminants. Therefore, the hyperinsulinaemic-euglicaemic clamp method is particularly suitable as it excludes gluconeogenesis in hepatocytes from analysis. Our results have shown a correlation between HOMA, QUICKI and RQUICKI indices and metabolic profile parameters. The correlation between dynamic and basal responses of NEFA, BHB, insulin, glucose and inorganic phosphorus is significantly dependent on RQUICKI-BHB index values in ketotic cows. RQUICKI index of insulin resistance is substantially dependent on NEFA values in early lactation as well as on insulin and glucose values in the dry period. Further investigations should focus on the practical evaluation of the use of insulin resistance indices.

Adipogenic function and other biologic effects of insulin

Studies on experimental animals with knockout of the insulin receptor gene Insr (in the whole body or in certain tissues) and/or related genes encoding proteins involved in realization of insulin signal transduction in target cells, have made an important contribution to the elucidation of insulin regulation of metabolism, particularly fat metabolism. Since the whole insulin secreted by b-cells, together with the products of gastrointestinal tract digestion of proteins, fats, and carbohydrates reach the liver, the latter is the first organ on which this hormone acts. The liver employs released amino acids for synthesis of proteins, including apoproteins for various lipoproteins. Glucose is used for synthesis of glycogen, fatty acids, and triglycerides, which enter all the organs in very low density lipoproteins (VLDL). The LIRKO mice with knockout of the Insr gene in the liver demonstrated inhibition of synthesis of macromolecular compounds from amino acids, glucose, and fatty acids. Low molecular weight substances demonstrated increased entry to circulation, and together with other disorders induced hyperglycemia. In LIRKO mice blood glucose levels and glucose tolerance demonstrated time-dependent normalization and at later stages the increase in glucose levels was replaced by hypoglycemia. These changes can be well explained if we take into consideration that one of the main functions of insulin consists in stimulation of energy accumulation by means of activation of triglyceride deposition in adipose tissue. FIRKO mice with selective knockout of adipose tissue Insr were characterized by decreased uptake of glucose in adipocytes, and its transformation into lipids. However, the level of body fat in animals remained normal, possibly due to preserved insulin receptor in the liver and insulin-induced activation of triglyceride production which maintained normal levels of body fat stores, the effective functioning of adipose tissue and secretion of leptin by adipocytes during inhibition of glucose transformation into triglyceride in adipose tissue. Knockout of the Insr gene in muscles blocked glucose uptake by myocytes, but it did not induce hyperglycemia, probably due to the increase in glucose uptake by other organs, which retained the insulin receptor, and induced some increase in fat resources in adipose tissue. Similar results were obtained in mice with knockout the glucose transporter 4 GLUT4 in muscle and/or adipose tissue. Insulin microinjections in the brain, in the cerebral ventricle 4 (ICV) and mediobasal hypothalamus (MBH) did not affect the insulin levels in the general circulation, but effectively activated lipogenesis and inhibited lipolysis in adipose tissue. They induced obesity, similar to conventional obesity when the insulin levels increased. These results may serve as additional evidence for importance of the adipogenic insulin function in mechanisms of regulation of general metabolism.

The adipogenic function and other biological effects of insulin

Studies on experimental animals with knockout of the insulin receptor gene (Insr) in the whole body or in certain tissues and/or related genes encoding proteins involved in realization of insulin signal transduction in target cells, have made an important contribution to the elucidation of insulin regulation of metabolism, particularly fat metabolism. Since the whole insulin secreted by β-cells, together with the products of gastrointestinal tract digestion of proteins, fats, and carbohydrates reaches in the liver, the latter is the first organ on which this hormone acts. The liver employs released amino acids for synthesis of proteins, including apo-proteins for various lipoproteins. Glucose is used for synthesis of glycogen, fatty acids, and triglycerides, which enter all the organs in very low density lipoproteins (VLDL). The LIRKO mice with knockout of the insr gene in the liver demonstrated inhibition of synthesis of macromolecular compounds from amino acids, glucose, and fatty acids. Low molecular weight substances demonstrated increased entry to circulation, and together with other disorders induced hyperglycemia. In LIRKO mice blood glucose levels and glucose tolerance demonstrated time-dependent normalization and at later stages the increase in glucose levels was replaced by hypoglycemia. These changes can be well explained if we take into consideration that one of the main functions of insulin consists in stimulation of energy accumulation by means of activation of triglyceride deposition in adipose tissue. FIRKO mice with selective knockout of adipose tissue Insr were characterized by decreased uptake of glucose in adipocytes, and its transformation into lipids. However, the level of body fat in animals remained normal, possibly due to preserved insulin receptor in the liver and insulin-induced activation of triglyceride production which maintained normal levels of body fat stores, the effective functioning of adipose tissue and secretion of leptin by adipocytes during inhibition of glucose transformation into triglyceride in adipose tissue. Knockout of the Insr gene in muscles blocked glucose uptake by myocytes, but it did not induce hyperglycemia, probably due to the increase in glucose uptake by other organs, which retained the insulin receptor, and induced some increase in fat resources in adipose tissue. Similar results were obtained in mice with knockout the glucose transporter 4 GLUT4 in muscle and/or adipose tissue. Insulin microinjections in the brain, in the cerebral ventricle 4 (CVI) and mediobasal hypothalamus (MBH) did not affect the insulin levels in the general circulation, but effectively activate lipogenesis and inhibited lipolysis in adipose tissue. They induced obesity, similar to conventional obesity when the insulin levels increased. These results may serve as an additional confirmation of the importance of the adipogenic insulin function in mechanisms of regulation of general metabolism.

Biological Effects of Insulin and Its Analogs on Cancer Cells With Different Insulin Family Receptor Expression

Hyperinsulinemia is a likely cause of the increased cancer incidence and mortality in diabetic patients, but its role is difficult to define in vivo. Previous in vitro studies testing the mitogenic potential of insulin and its analogs provided incomplete and sometimes contradictory results. To better evaluate cancer cell responsiveness to insulin, to its analogs and to IGF-I, we measured under identical experimental conditions cell proliferation, invasiveness, and foci formation in six cancer cell lines with different insulin receptor family expression levels. The cancer cells studied have a different expression of insulin receptor (IR), its isoforms (IR-A and IR-B), and of the IGF-I receptor. The data indicate that insulin stimulates proliferation in all cancer cell lines, invasiveness in some, and foci formation in none. Cancer cell responses to insulin (and IGF-I) are not related to receptor expression levels; moreover, hormone-stimulated proliferation and invasiveness are not correlated. IGF-I is a more potent stimulator than insulin in most but not all cancer cell lines. Insulin analogs including M1 and M2 Glargine metabolites stimulate cancer cells similar to insulin. However, exceptions occur for specific analogs in particular cancer cells. In conclusion, in vitro insulin is an effective growth factor for all cancer cells but the biological response to insulin cannot be predicted on the basis of receptor expression levels. In the clinical setting, these observations should be taken in account when deciding treatment for diabetic patients who are at risk of undiscovered cancer or survivors of oncological diseases.

IGF-I, IGF-II, and Insulin Stimulate Different Gene Expression Responses through Binding to the IGF-I Receptor

Insulin and the insulin-like growth factors (IGF)-I and -II are closely related peptides important for regulation of metabolism, growth, differentiation, and development. The IGFs exert their main effects through the IGF-I receptor. Although the insulin receptor is the main physiological receptor for insulin, this peptide hormone can also bind at higher concentrations to the IGF-I receptor and exert effects through it. We used microarray gene expression profiling to investigate the gene expression regulated by IGF-I, IGF-II, and insulin after stimulation of the IGF-I receptor. Fibroblasts from mice, knockout for IGF-II and the IGF-II/cation-independent mannose-6-phosphate receptor, and expressing functional IGF-I but no insulin receptors, were stimulated for 4 h with equipotent saturating concentrations of insulin, IGF-I, and IGF-II. Each ligand specifically regulated a group of transcripts that was not regulated by the other two ligands. Many of the functions and pathways these regulated genes were involved in, were consistent with the known biological effects of these ligands. The differences in gene expression might therefore account for some of the different biological effects of insulin, IGF-I, and IGF-II. This work adds to the evidence that not only the affinity of a ligand determines its biological response, but also its nature, even through the same receptor.

Relaxin, Insulin and Diabetes: An Intriguing Connection

Until recently, relaxin and insulin were grouped into the same hormone superfamily because of substantial biochemical homologies. This notion has then changed with the understanding that insulin and relaxin are ligands for different receptors and signal transduction systems, namely, tyrosine kinase and G-protein-coupled receptors respectively. As a matter of fact, relaxin does not mimic the metabolic effects of insulin. The biological effects of insulin are much more clearly delineated than those of relaxin, which is traditionally viewed as a reproductive hormone involved in the maternal adjustments of pregnancy. In the last decade, evidence has been accumulating that relaxin has major effects on the heart, blood vessels and the extracellular matrix within connective tissues. In particular, pertaining to the relationships between relaxin and diabetes, relaxin was shown to promote arterial and microvascular dilation, thereby increasing organ perfusion, counteract ischemic injury, improve adverse cardiac and vascular remodeling, and promote extracellular matrix turn-over, thereby exerting anti-fibrotic effects. Thus, relaxin could blunt or delay the vascular and organ complication of diabetes. Whether relaxin may also synergize with insulin to optimize blood glucose homeostasis remains an unconfirmed issue. However, there are clues in the literature which, if gathered, speak in favor of this perspective. Moreover, preliminary data suggest that exogenous relaxin administration may improve insulin sensitivity in diabetic patients. Considering that human recombinant relaxin is under study as a novel, promising drug for the treatment of heart failure, a broader knowledge of the possible beneficial effects of relaxin in diabetes and its complications can be of interest to the scientific community.

Biological effects of insulin on murine melanoma cells and fish erythrophoroma cells: A comparative study

Insulin is the hormone that plays an essential role in metabolism and mitosis of normal and tumor cells, exerting its pleiotropic effects through binding to specific membrane receptors and promoting the phosphorylation of tyrosine residues of the receptor itself and of other components of the signaling pathway. The aim of this study was to investigate the effects of insulin on melanogenesis and cell growth in three different cell lines: the goldfish GEM-81 erythrophoroma cells (undifferentiated and differentiated with 1.5% dimethylsulfoxide-DMSO), and the murine B16F10 and Cloudman S91 melanoma cells. Undifferentiated GEM-81 and B16F10 cells responded to insulin with a small increase of cell proliferation, whereas S91 cells responded with a decrease of growth. In the two mammalian cell lines, and in DMSO-differentiated GEM-81 cells, the hormone strongly inhibited melanogenesis, by decreasing tyrosinase activity. In undifferentiated GEM-81 cells, insulin had no effect on tyrosinase activity. An increase in the tyrosine phosphorylation status of pp185 (insulin receptor substrate 1 and 2—IRS-1/2) phosphorylation degree was observed in S91 mouse melanoma and in differentiated GEM-81 erythrophoroma cells, suggesting that this specific protein was maintained during transformation process and participates in insulin signaling. Our results imply an ancient and diverse history of the insulin signaling system in vertebrate pigment cells.

Overexpression of membrane glycoprotein PC-1 can influence insulin action at a post-receptor site.

An elevated content of membrane glycoprotein PC-1 has been observed in cells and tissues of insulin resistant patients. In addition, in vitro overexpression of PC-1 in cultured cells induces insulin resistance associated with diminished insulin receptor tyrosine kinase activity. We now find that PC-1 overexpression also influences insulin receptor signaling at a step downstream of insulin receptor tyrosine kinase, independent of insulin receptor tyrosine kinase. In the present studies, we employed Chinese hamster ovary cells that overexpress the human insulin receptor (CHO IR cells; approximately 10(6) receptors per cell), and transfected them with human PC-1 c-DNA (CHO IR PC-1). In CHO IR PC-1 cells, insulin receptor tyrosine kinase activity was unchanged, following insulin treatment of cells. However, several biological effects of insulin, including glucose and amino acid uptake, were decreased. In CHO IR PC-1 cells, insulin stimulation of mitogen-activated protein (MAP) kinase activity was normal, suggesting that PC-1 overexpression did not affect insulin receptor activation of Ras, which is upstream of MAP kinase. Also, insulin-stimulated phosphatidylinositol (PI)-3-kinase activity was normal, suggesting that PC-1 overexpression did not interfere with the activation of this enzyme by insulin receptor substrate-1. In these cells, however, insulin stimulation of p70 ribosomal S6 kinase activity was diminished. These studies suggest, therefore, that, in addition to blocking insulin receptor tyrosine kinase activation, PC-1 can also block insulin receptor signaling at a post-receptor site.

Early Steps in Insulin Action

The biological effects of insulin are initiated by the binding of insulin to the insulin receptor. Insulin binds to the extracellular domain of the insulin receptor and induces conformational changes in the receptor, leading to autophosphorylation of the receptor on intracellular tyrosine residues. These phosphorylated tyrosine residues act as binding sites for proteins which subsequently may be phosphorylated by the insulin receptor. As a result, yet other proteins can be recruited to form larger complexes and, in the case of enzymes, changes in their activity may take place. By a combination of these processes, the activated insulin receptor initiates cascades of biochemical events which are regulated mainly by specific phosphorylation or dephosphorylation reactions. Intermediates which are involved in the normal insulin signalling pathway are subjects of expanding research.

Tissue specific differences in insulin receptor characteristics in the mature bovine animal

Insulin is believed to play a key role in the partitioning of nutrients towards fat and protein deposition. The biological effects of insulin are a function of plasma insulin concentration, insulin-receptor concentration and the affinity of the receptor. The number and affinity of insulin receptors on adipocytes differs between genetically lean and obese pigs (Camara and Mourot, 1996) while receptor number, but not affinity, was shown to differ in a variety of bovine muscles (Boge et al., 1995). The objective of the current work was to determine if insulin receptor affinity and number vary between the principal target tissues (i.e. liver, muscle and adipose tissue) in the bovine animal.Five Charolais-cross steers were offered grass silage ad libitum from 18 months of age until slaughter, at 701± 22.9 kg, at a commercial abattoir. Samples of liver [L], of two skeletal muscles [Ml, M3] and of subcutaneous [S], omental [0]and renal [R] fats were taken as soon as possible after slaughter (typically <30min for L, M3, S and O and <45min for Mland R), wrapped and snap frozen in liquid nitrogen prior to storage at -70 °. Insulin receptors were partially purified by solubilising tissues in 1% Triton X-100 for 24 hours at 4 °C followed by centrifugation (100000 x g, lh, 4°) and affinity chromatography of the supernatants on 0.5 ml wheatgerm agglutinin Sepharose 6MB columns. Bound receptors were eluted from the column with 0.3M N-acetyl-D-glucosamine. Receptor binding was assessed using a tracer amount of A-14 125I-insulin (∼35 pM) and increasing concentrations of unlabelled insulin (0-10 μM) in a total volume of 150 μl of pH 7.4 buffer as described by Magri et al (1990).

Metformin induces an agonist-specific increase in albumin production by primary cultured rat hepatocytes

Metformin (MET) is known to increase several biological effects of insulin (INS), but there is no information concerning its direct effects on protein synthesis. We studied the action of MET on albumin production by primary cultures of freshly isolated rat hepatocytes, alone or in combination with various agonists: INS, IGF-1, EGF, thyroxin, and dexamethasone. While having no effect alone, MET in vitro potentiates the effects of INS, IGF-1, and EGF. When this increasing effect toward INS was studied over a broad concentration range, MET appeared to improve low-acting INS levels and to intensify the maximal INS effects. In contrast, MET did not change the production of albumin stimulated by thyroxin or dexamethasone. Animals chronically pretreated with MET in vivo showed a higher yield of isolated hepatocytes, better attachment, and especially higher viability after liver perfusion and during cell culture. This may largely explain why basal albumin rates were higher than in in vitro-treated cells. The effect of MET in the presence of the agonists exhibited the same agonist-specificity as in vitro. Our data provide new insights into the pharmacology of MET by showing that hepatic protein synthesis is increased by MET and INS. From the specificity of action of MET towards INS, IGF-1, and EGF (but not thyroxin or dexamethasone), we hypothesize that this biguanide may act on intracellular pathways located between membrane receptors and sites of branching in the signaling cascades shared by these agonists.

Insulin resistance in genetically obese (fa/fa) rats: changes in the glycosyl-phosphatidylinositol signaling system in isolated hepatocytes.

In different types of mammalian cells, insulin has been shown to promote the release of an inositol phosphate glycan (InsP-glycan) through the hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns). This InsP-glycan, which has been demonstrated to be taken up by intact cells, may mediate some of the biological effects of insulin. We have investigated how the insulin resistance expressed in genetically obese (fa/fa) rats affects the glycosyl-PtdIns signaling system in isolated hepatocytes compared to what occurs in hepatocytes isolated from lean (Fa/-) rats. The hepatocyte content of glycosyl-PtdIns was reduced by about 30% in obese rats, with respect to that measured in lean rats (2553 +/- 138 vs. 3334 +/- 115 dpm/mg protein; P < 0.01; n = 5). This reduction was accompanied by a marked blockade of the insulin-mediated glycosyl-PtdIns hydrolysis as well as a decrease (approximately 30%) in the rate of InsP-glycan uptake by the isolated liver cells. Obese Zucker rat hepatocytes also showed a significant decrease in the effects of both insulin and InsP-glycan on the stimulation of glycogen synthesis and the activation of glycogen synthase compared to hepatocytes isolated from lean rats. Our results demonstrate that genetic obesity in Zucker (fa/fa) rats is associated with an impairment of the glycosyl-PtdIns-dependent insulin signaling system.

Discordant metabolic actions of insulin in extreme lipodystrophy of childhood.

Congenital lipodystrophy includes a group of disorders characterized by total or partial absence of adipose tissue and insulin resistance. In this study we investigated the nature of insulin resistance in an 11-yr-old girl with one form of congenital lipodystrophy. We examined in vivo insulin and glycemic responses to feeding and iv glucose and in vitro amino acid and thymidine incorporation responses of skin fibroblasts to insulin exposure. In addition, we used stable isotope infusions of glucose, glycerol, and amino acids to investigate the in vivo metabolic actions of insulin on carbohydrate, fat, and protein. At 5 yr of age, she first demonstrated clinical glucose intolerance. Her basal insulin levels were normal (129 and 114 pmol/L), but increased markedly (peak values, 1304 and 5045 pmol/L) after iv glucose and a mixed meal. Insulin antibodies were undetectable, and specific [125I]insulin binding to her skin fibroblasts was normal. Both [3H]aminoisobutyric acid transport and [3H]thymidine incorporation by her fibroblasts were similar to responses obtained using control cells. At 11 5/12 yr of age, while receiving an infusion of stable isotopes, infusions of insulin at doses of 0.1 and 0.3 U/kg BW.h were ineffective in reducing her blood glucose despite elevating her serum insulin level to approximately 2500 pmol/L. Resting metabolic rate, respiratory quotient, VCO2, carbohydrate and lipid oxidation rates, glucose production rate, glycerol appearance rate, and plasma glycerol concentrations were unperturbed by the insulin infusions. By contrast, the insulin infusions reduced plasma leucine concentrations (124.2 to 86.1 to 66.7 mumol/L) and 13CO2 production rates (0.034 to 0.017 to 0.011 mumol/kg/min; baseline, 0.1, and 0.3 U insulin/kg.h, respectively). The leucine appearance rate declined (1.96 to 1.72 mumol/kg.min) in response to the 0.1 U/kg.h dose, but did not decline further in response to the 0.3 U/kg.h dose. The leucine oxidation rate also declined (0.87 to 0.39 to 0.25 mumol/kg.min), and there was a dose-related reduction in most plasma amino acid concentrations. Finally, nonoxidative leucine disposal increased progressively (1.09, 1.34, and 1.48 mumol/kg.min), suggestive of an insulin-induced increase in protein synthesis. These data indicate profound metabolic resistance to the carbohydrate and lipid actions of insulin, with preservation of protein anabolism. These observations suggest that in this patient, the biological effects of insulin on carbohydrate, lipid, and protein are distinct metabolic actions, regulated independently.

Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits.

Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.

Binding and biological effects of insulin, insulin analogues and insulin-like growth factors in rat aortic smooth muscle cells. Comparison of maximal growth promoting activities

Binding and growth promoting effects of insulin, insulin analogues modified in the B chain, proinsulin, insulin-like growth factor-I and -II were studied in cultured rat aortic smooth muscle cells. Specific binding of 125I-insulin was 0.9 +/- 0.2% of total 125I-insulin added, and the IC50-value was estimated to 8.9 pmol/l. The insulin analogue B10 Asp tended to be more potent than insulin in displacing 125I-insulin, B28 Asp was equipotent, B9 Asp/B27 Glu was approximately 100 times less potent and insulin-like growth factor-I more than 1000 times less potent than insulin. Specific binding of 125I-insulin-like growth factor-I after 4 h incubation at 10 degrees C was five times higher than the specific binding of insulin (4.4 +/- 0.4% of total 125I-insulin-like growth factor-I added), and the IC50-value was 0.3 nmol/l. Insulin was approximately 500 times less potent than insulin-like growth factor-I in displacing 125I-insulin-like growth factor-I. The insulin analogue B10 Asp was slightly more potent and analogue B28 Asp was equipotent with insulin. Analogue B9 Asp/B27 Glu was ten times less potent and proinsulin was more than ten times less potent than insulin. The order of potency was similar for 3H-thymidine incorporation into DNA: insulin-like growth factor-I greater than B10 Asp greater than insulin-like growth factor-II greater than insulin greater than or equal to B28 Asp greater than B9 Asp/B27 Glu greater than proinsulin. The maximal effect of insulin-like growth factor-I on 3H-thymidine incorporation was 71 +/- 16% higher than the maximal effect of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulinlike Growth Factor Action and Production in Adipocytes and Endothelial Cells From Human Adipose Tissue

Primary cultures of microvascular endothelial cells and isolated adipocytes were prepared from human omental adipose tissue to study the potentially overlapping roles of insulin and insulinlike growth factors (IGFs) in human adipose tissue. To determine whether adipocytes contain type I IGF receptors, binding experiments were carried out with 125I-labeled IGF-I. At 16 degrees C, saturation of specific binding to adipocytes was reached after 30 min and was 0.7% per 10(6) cells. At 37 degrees C, chloroquine produced an increase in cell-associated 125I-IGF-I, suggesting that IGF-I is internalized and degraded in a manner analogous to insulin. In competition experiments, IGF-I competed for binding more effectively than rat IGF-II or insulin. The concentrations of IGF-I, rat IGF-II, and insulin necessary to displace 50% of 125I-IGF-I binding were 2.5, 15, and 90 nM, respectively. In addition, a monoclonal antibody (alpha-IR3) that has been shown to block the type I IGF receptor was used in competition binding experiments. The antibody also inhibited binding of 125I-IGF-I to adipocytes. The biological effects of insulin and IGF-I were examined by studying adipocyte lipoprotein lipase (LPL). Insulin stimulated [14C]glucose incorporation into cellular lipid in a dose-dependent manner, with 50% effective concentration (EC50) of 0.3 nM. However, an increase in LPL activity was observed only at a high insulin concentration, with an EC50 of approximately 30 nM. In contrast, IGF-I stimulated a progressive increase in LPL, with an EC50 of 3.2 nM. In addition, alpha-IR3 blocked the stimulatory effect of IGF-I on adipocyte LPL.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor binding and biological effects of insulin and insulin-like growth factor I during the cell cycle in a human leukemic cell line

Receptor binding and biological effects of insulin and insulin-like growth factor I during the cell cycle in a human leukemic cell line