MCF‐7 human mammary adenocarcinoma cells exhibit augmented responses to human insulin on a collagen IV surface

Human mammary cell lines are extensively used for preclinical safety assessment of insulin analogs. However, it is essentially unknown how mitogenic responses can be optimized in mammary cell‐based systems. We developed an insulin mitogenicity assay in MCF‐7 human mammary adenocarcinoma cells, under low serum (0.1% FCS) and phenol red‐free conditions, with 3H thymidine incorporation as endpoint. Based on EC50 values determined from 10‐fold dilution series, β‐estradiol was the most potent mitogen, followed by human IGF‐1, human AspB10 insulin and native human insulin. AspB10 insulin was significantly more mitogenic than native insulin, validating the ability of the assay to identify hypermitogenic human insulin analogs. With MCF‐7 cells on a collagen IV surface, the ranking of mitogens was maintained, but fold mitogenic responses and dynamic range and steepness of dose–response curves were increased. Also, PI3K pathway activation by insulin was enhanced on a collagen IV surface. This study provided the first determination and ranking of the mitogenic potencies of standard reference compounds in an optimized MCF‐7 assay. The optimized MCF‐7 assay described here is of relevance for in vitro toxicological testing and carcinogenicity safety assessment of new insulin compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

Growth receptor binding protein 10 inhibits glucose‐stimulated insulin release from pancreatic β‐cells associated with suppression of the insulin/insulin‐like growth factor‐1 signalling pathway

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Carthamus tinctorius L. extract activates insulin‐like growth factor‐I receptor signaling to inhibit FAS‐death receptor pathway and suppress lipopolysaccharides‐induced H9c2 cardiomyoblast cell apoptosis

Carthamus tinctorius L. (Compositae) is used in Chinese medicine to treat heart disease and inflammation. In our previous study, we found that C. tinctorius L. inhibited lipopolysaccharides (LPS)‐induced tumor necrosis factor‐alpha (TNF‐α) activation, JNK expression, and apoptosis in H9c2 cardiomyoblast cells. The present study was performed to investigate the protective effect of C. tinctorius extract (CTF) on LPS‐challenged H9c2 myocardioblast cell and to explore the possible underlying mechanism. Cell viability assay showed that LPS treatment decreased the cell viability of H9c2 cell, whereas CTF treatment reversed LPS cytotoxicity in a dose‐dependent manner, especially in the LPS + CTF 25 (μg/mL) group. LPS treatment‐induced apoptosis was determined by transferase‐mediated dUTP nick end labeling assay, and by Western blot. LPS‐induced apoptotic bodies were decreased following CTF treatment. Expression of TNF‐α, FAS‐L, FAS, FADD, caspase‐8, BID, and t‐BID was significantly increased in LPS‐treated H9c2 cells. In contrast, it was significantly suppressed by the administration of CTF extract. In addition, CTF treatment activates antiapoptotic proteins, Bcl‐2 and p‐Bad, and downregulates Bax, cytochrome‐c, caspase‐9, caspase‐3, and apoptosis‐inducing factor expression. Furthermore, CTF exerted cytoprotective effects by activating insulin‐like growth factor‐I (IGF‐I) signaling pathway leading to downregulation of the apoptotic proteins involved in FAS death receptor pathway. In addition, AG1024 and IGF‐I receptor (IGF‐IR) inhibitor and siRNA silencing reverses the effect of CTF implying that CTF functions through the IGF‐IR pathway to inhibit LPS‐induced H9c2 apoptosis. These results suggest that treatment with CTF extract prevented the LPS‐induced apoptotic response through IGF‐I pathway.     

Classifying the adverse mitogenic mode of action of insulin analogues using a novel mechanism-based genetically engineered human breast cancer cell panel

Insulin analogues are widely used in clinical practice. Modifications on the insulin molecular structure can affect the affinity and activation towards two closely related receptor tyrosine kinases: the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R). A switch towards higher IGF1R affinity is likely to emphasize mitogenesis rather than glucose metabolism. Relevant well-validated experimental tools to address the insulin analogue activation of either INSR or IGF1R are missing. We have established a panel of human MCF-7 breast cancer cell lines either ectopically expressing the INSR (A or B isoform) in conjunction with a stable knockdown of the IGF1R or ectopically expressing the IGF1R in conjunction with a stable knockdown of the INSR. In these cell lines, we systematically evaluated the INSR and IGF1R receptor activation and downstream mitogenic signalling of all major clinical relevant insulin analogues in comparison with insulin and IGF1R. While most insulin analogues primarily activated the INSR, the mitogenic activation pattern of glargine was highly similar to IGF1 and insulin AspB10, known to bind IGF1R and induce carcinogenesis. Yet, in a long-term proliferation assay, the proliferative effect of glargine was not much different from regular insulin or other insulin analogues. This was caused by the rapid enzymatic conversion into its two metabolic active metabolites M1 and M2, with reduced mitogenic signalling through the IGF1R. In summary, based on our new cell models, we identified a similar mitogenic potency of insulin glargine and AspB10. However, rapid enzymatic conversion of glargine precludes a sustained activation of the IGF1R signalling pathway.     

Novel markers to detect recombinant human insulin‐like growth factor‐I (rhIGF‐I)/rhIGF binding protein‐3 (rhIGFBP‐3) misuse in athletes

Insulin‐like growth factor‐I (IGF‐I) is abused by elite athletes for its metabolic and anabolic effects. We have previously shown that it is possible to detect IGF‐I misuse by measuring serum IGF‐I and procollagen type III amino‐terminal propeptide (P‐III‐NP) but a pilot study suggested measuring IGF‐II, IGF binding protein‐2 (IGFBP‐2) and acid‐labile subunit (ALS) may improve the detection of IGF‐I administration. The aim of the study was to assess this in a randomized controlled trial. Twenty‐six female and 30 male recreational athletes were randomized to 28 days’ treatment with placebo or recombinant human (rh)IGF‐I/rhIGF binding protein‐3 (IGFBP‐3) complex (30 mg/day or 60 mg/day), followed by 56 days’ washout. IGF‐II, IGFBP‐2 and ALS (women only) were measured using commercial immunoassays. IGFBP‐2 increased and IGF‐II decreased in response to both low and high dose rhIGF‐I/rhIGFBP‐3 in both women and men while ALS decreased in women in response to high dose rhIGF‐I/rhIGFBP‐3. Two days after discontinuing treatment, significant differences remained between the three treatment groups in IGFBP‐2 and IGF‐II, but not ALS. Thereafter there were no significant differences between the three treatment groups in any of the markers. Combining IGF‐I with IGF‐II and/or IGFBP‐2 improved the performance of the test to detect rhIGF‐I/rhIGFBP‐3 administration in both women and men. Copyright © 2016 John Wiley & Sons, Ltd.     

LPS‐enhanced IGF‐IIR pathway to induce H9c2 cardiomyoblast cell hypertrophy was attenuated by Carthamus tinctorius extract via IGF‐IR activation

The use of herbs as alternative cardiovascular disease treatment has attracted a great deal of attention owing to their lower toxicity. Whether Carthamus tinctorius extract prevent cardiomyoblast cell hypertrophy remains unclear. The present study was performed to investigate the effect of C tinctorius extract (CTF) on rat cardiomyoblast cell H9c2 and the possible molecular mechanisms. H9c2 cells were treated with lipopolysaccharide (LPS; 2 μg/mL) for 12 hours, subsequently treated with CTF (1‐25 μg/mL) The incubation continued for another 24 hours, and the cells were analyzed with actin staining assay, western blot analysis, and siRNA transfection assays. In the present study, the increased cell size induced by LPS was significantly decreased by pretreating at a concentration of 1‐25 μg/mL CTF. It was found that CTF could inhibit cardiac hypertrophy induced by LPS and decrease hypertrophic proteins calcineurin, p‐GATA‐4, GATA‐4, atrial natriuretic peptide, and B‐type natriuretic peptide levels in H9c2 cells. Additionally, LPS‐induced insulin‐like growth factor‐II receptor (IGF‐IIR) hypertrophy pathway was downregulated by CTF. Moreover, IGF‐IR siRNA or inhibitors both reversed the CTF effects, confirming that CTF activates IGF‐1R to prevent LPS‐induced H9c2 cardiomyoblast cell hypertrophy. The current findings indicate that CTF activates IGF‐IR to inhibit IGF‐IIR signaling pathway which resulted in reducing H9c2 cardiomyoblast cell hypertrophy induced by LPS.     

The development of decision limits for the GH‐2000 detection methodology using additional insulin‐like growth factor‐I and amino‐terminal pro‐peptide of type III collagen assays

The GH‐2000 and GH‐2004 projects have developed a method for detecting GH misuse based on measuring insulin‐like growth factor‐I (IGF‐I) and the amino‐terminal pro‐peptide of type III collagen (P‐III‐NP). The objectives were to analyze more samples from elite athletes to improve the reliability of the decision limit estimates, to evaluate whether the existing decision limits needed revision, and to validate further non‐radioisotopic assays for these markers. The study included 998 male and 931 female elite athletes. Blood samples were collected according to World Anti‐Doping Agency (WADA) guidelines at various sporting events including the 2011 International Association of Athletics Federations (IAAF) World Athletics Championships in Daegu, South Korea. IGF‐I was measured by the Immunotech A15729 IGF‐I IRMA, the Immunodiagnostic Systems iSYS IGF‐I assay and a recently developed mass spectrometry (LC‐MS/MS) method. P‐III‐NP was measured by the Cisbio RIA‐gnost P‐III‐P, Orion UniQ? PIIINP RIA and Siemens ADVIA Centaur P‐III‐NP assays. The GH‐2000 score decision limits were developed using existing statistical techniques. Decision limits were determined using a specificity of 99.99% and an allowance for uncertainty because of the finite sample size. The revised Immunotech IGF‐I – Orion P‐III‐NP assay combination decision limit did not change significantly following the addition of the new samples. The new decision limits are applied to currently available non‐radioisotopic assays to measure IGF‐I and P‐III‐NP in elite athletes, which should allow wider flexibility to implement the GH‐2000 marker test for GH misuse while providing some resilience against manufacturer withdrawal or change of assays. Copyright © 2015 John Wiley & Sons, Ltd.     

Three‐dimensional solution structure of a disulfide bond isomer of the human insulin‐like growth factor‐I

Abstract: The solution structure of a disulfide bond isomer of human insulin‐like growth factor‐I (IGF‐I) was determined using homonuclear NMR methods. A total of 292 interatomic distance constraints, including 12 related to the disulfide bridges, was used in the distance geometry calculations. The determined structures contain two helical rods corresponding to the sequence regions, Ala⁸–Cys¹⁸ and Leu⁵⁴–Cys⁶¹. Comparison with the previously determined structure of native human IGF‐I revealed partial correspondence of the secondary structure (helices I: Ala⁸–Cys¹⁸ and helices III: Leu⁵⁴–Cys⁶¹) and internal packing. Helix II in native human IGF‐I (residues Gly⁴²–Cys⁴⁸) is disrupted in the isomer. A similar relationship has been described between the structure of native insulin and a homologous disulfide isomer, suggesting that these alternative folds represent general features of insulin‐like sequences. In each case the precision of the distance geometry ensemble is low due in part to resonance broadening and a paucity of NOEs relative to other globular proteins of this size. These observations suggest that tertiary structure of the isomer is not highly ordered. Comparison of the biological activities of native and the disulfide bond isomer of human IGF‐I highlight the importance of Tyr²⁴, Phe²⁵, Phe⁴⁹–Cys⁵² and Phe¹⁶ in binding to the IGF‐I receptor or specific IGFBPs. The relationship of this proposed receptor‐binding surface of human IGF‐I to those of insulin is discussed.     

Design,synthesis, and biological evaluation of novel 3‐(thiophen‐2‐ylthio)pyridine derivatives as potential multitarget anticancer agents

A series of novel 3‐(thiophen‐2‐ylthio)pyridine derivatives as insulin‐like growth factor 1 receptor (IGF‐1R) inhibitors was designed and synthesized. IGF‐1R kinase inhibitory activities and cytotoxicities against HepG2 and WSU‐DLCL2 cell lines were tested. For all of these compounds, potent cancer cell proliferation inhibitory activities were observed, but not through the inhibition of IGR‐1R. Selected compounds were further screened against various kinases. Typical compound 22 (50% inhibitory concentration [IC₅₀] values, HepG2: 2.98 ± 1.11 μM and WSU‐DLCL2: 4.34 ± 0.84 μM) exhibited good inhibitory activities against fibroblast growth factor receptor‐2 (FGFR2), FGFR3, epidermal growth factor receptor, Janus kinase, and RON (receptor originated from Nantes), with IC₅₀ values ranging from 2.14 to 12.20 μM. Additionally, the cell‐cycle analysis showed that compound 22 could arrest HepG2 cells in the G1/G0 phase. Taken together, all the experiments confirmed that the compounds in this series were multitarget anticancer agents worth further optimizing.     

A new C‐peptide correction model used to assess bioavailability of regular human insulin

The clinical assessment of new formulations of human insulin is problematic due to the inability to distinguish between endogenous insulin and exogenously administered insulin. The usual methods to surmount the problem of distinguishing between endogenous and exogenous human insulin include evaluation in subjects with no or little endogenous insulin, hyper‐insulinemic clamp studies or the administration of somatostatin to suppress endogenous insulin secretion. All of these methods have significant drawbacks. This paper describes a method for C‐Peptide correction based upon a mixed effects linear regression of multiple time point sampling of C‐Peptide and insulin. This model was able to describe each individual's insulin to C‐Peptide relationship using the data from four different phase I clinical trials involving both subjects with and without type 2 diabetes in which insulin and C‐Peptide were measured. These studies used hyper‐insulinemic euglycemic clamps or meal challenges and subjects received insulin or Glucagon‐like peptide 1 (GLP‐1). It was possible to determine the exogenously administered insulin concentration from the measured total insulin concentration. A simple statistical technique can be used to determine each individual's insulin to C‐Peptide relationship to estimate exogenous and endogenous insulin following the administration of regular human insulin. This technique will simplify the assessment of new formulations of human insulin. Copyright © 2010 John Wiley & Sons, Ltd.     

Insulin‐like growth factor binding protein 7 accelerates hepatic steatosis and insulin resistance in non‐alcoholic fatty liver disease

An association between increased insulin‐like growth factor binding protein‐7 (IGFBP7) expression and insulin resistance in metabolic diseases has been reported. However, the role and molecular mechanism of IGFBP‐7 in non‐alcoholic fatty liver disease (NAFLD) remains largely unknown. Therefore, the potential function of IGFBP7 in the pathological progression of NAFLD was explored in this investigation. For in vivo experiments, an animal model of NAFLD was established in C57BL/6 mice by feeding a high‐fat diet (HFD), and IGFBP7 was knocked down by injecting adeno‐associated adenovirus (AAV)‐mediated short‐hairpin (sh)‐IGFBP7 into the liver. We found that AAV‐sh‐IGFBP7 treatment significantly alleviated hepatocyte injury and inhibited hepatic lipid accumulation by reducing lipogenesis‐associated gene expression. Furthermore, downregulation of IGFBP7 markedly ameliorated IR and restored impaired insulin signalling by elevating the phosphorylation levels of IRS‐1, Akt and GSK3β in HFD‐treated mice. Similar results were also confirmed by an in vitro study in a palmitic acid (PA)‐stimulated HepG2 cell model. In conclusion, our study demonstrates that IGFBP7 contributes to hepatic steatosis and insulin resistance in NAFLD development, which might serve as a novel therapeutic agent for the treatment of NAFLD.     

Investigation of morin‐induced insulin secretion in cultured pancreatic cells

Morin is a flavonoid contained in guava that is known to reduce hyperglycemia in diabetes. Insulin secretion has been demonstrated to increase following the administration of morin. The present study is designed to investigate the potential mechanism(s) of morin‐induced insulin secretion in the MIN6 cell line. First, we identified that morin induced a dose‐dependent increase in insulin secretion and intracellular calcium content in MIN6 cells. Morin potentiated glucose‐stimulated insulin secretion (GSIS). Additionally, we used siRNA for the ablation of imidazoline receptor protein (NISCH) expression in MIN6 cells. Interestingly, the effects of increased insulin secretion by morin and canavanine were markedly reduced in Si‐NISCH cells. Moreover, we used KU14R to block imidazoline I₃ receptor (I‐3R) that is known to enhance insulin release from the pancreatic β‐cells. Without influence on the basal insulin secretion, KU14R dose‐dependently inhibited the increased insulin secretion induced by morin or efaroxan in MIN6 cells. Additionally, effects of increased insulin secretion by morin or efaroxan were reduced by diazoxide at the dose sufficient to open K_ATP channels and attenuated by nifedipine at the dose used to inhibit L‐type calcium channels. Otherwise, phospholipase C (PLC) is introduced to couple with imidazoline receptor (I‐R). The PLC inhibitor dose‐dependently inhibited the effects of morin in MIN6 cells. Similar blockade was also observed in protein kinase C (PKC) inhibitor‐treated cells. Taken together, we found that morin increases insulin secretion via the activation of I‐R in pancreatic cells. Therefore, morin would be useful to develop in the research and treatment of diabetic disorders.     

Activation of imidazoline‐I3 receptors ameliorates pancreatic damage

Agmatine, an endogenous ligand of imidazoline receptors, is reported to exhibit anti‐hyperglycaemic and many other effects. It has been established that the imidazoline I3 receptor is involved in insulin secretion. The current study characterizes the role of the imidazoline I3 receptor in the protection of pancreatic islets. The activity effect of agmatine against on streptozotocin (STZ)‐induced (5 mmol/L) rat β cell apoptosis was examined by using ApoTox‐Glo triplex assay, live/dead cell double staining assay, flow cytometric analysis, and western blot. Imidazoline I3 receptors antagonist KU14R and the phospholipase C inhibitor named U73122 were treated in β cells to investigate the potential signalling pathways. The serum glucose and recovery of insulin secretion were measured in STZ‐treated rats after continuously injected agmatine. The apoptosis in rat β cells was reduced by agmatine in a dose‐dependent manner, cell viability was improved after treatment with agmatine and these effects were suppressed after the blockade of KU14R and U73122. Western blot analysis confirmed that agmatine could decrease caspase‐3 expression and increase the p‐BAD levels. In STZ‐treated rats, injection of agmatine for 4 weeks may significantly lower the serum glucose and recovery of insulin secretion. This improvement of pancreatic islets induced by agmatine was deleted by KU14R in vivo. Agmatine can activate the imidazoline I3 receptor linked with the phospholipase C pathway to induce cell protection against apoptosis induced by a low dose of STZ. This finding provides new insight into the prevention of early stage pancreatic islet damage.     

Titanium dioxide nanoparticles increase plasma glucose via reactive oxygen species‐induced insulin resistance in mice

There have been few reports about the possible toxic effects of titanium dioxide (TiO₂) nanoparticles on the endocrine system. We explored the endocrine effects of oral administration to mice of anatase TiO₂ nanoparticles (0, 64 and 320 mg kg^–1 body weight per day to control, low‐dose and high‐dose groups, respectively, 7 days per week for 14 weeks). TiO₂ nanoparticles were characterized by scanning and transmission electron microscopy (TEM) and dynamic light scattering (DLS), and their physiological distribution was investigated by inductively coupled plasma. Biochemical analyzes included plasma glucose, insulin, heart blood triglycerides (TG), free fatty acid (FFA), low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), total cholesterol (TC), tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐6 and reactive oxygen species (ROS)‐related markers (total SOD, GSH and MDA). Phosphorylation of IRS1, Akt, JNK1, and p38 MAPK were analyzed by western blotting. Increased titanium levels were found in the liver, spleen, small intestine, kidney and pancreas. Biochemical analyzes showed that plasma glucose significantly increased whereas there was no difference in plasma insulin secretion. Increased ROS levels were found in serum and the liver, as evidenced by reduced total SOD activity and GSH level and increased MDA content. Western blotting showed that oral administration of TiO₂ nanoparticles induced insulin resistance (IR) in mouse liver, shown by increased phosphorylation of IRS1 (Ser307) and reduced phosphorylation of Akt (Ser473). The pathway by which TiO₂ nanoparticles increase ROS‐induced IR were included in the inflammatory response and phosphokinase, as shown by increased serum levels of TNF‐α and IL‐6 and increased phosphorylation of JNK1 and p38 MAPK in liver. These results show that oral administration of TiO₂ nanoparticles increases ROS, resulting in IR and increasing plasma glucose in mice. Copyright © 2015 John Wiley & Sons, Ltd.     

Canavanine induces insulin release via activation of imidazoline I3 receptors

The aim of the present study was to identify the effect of canavanine on the imidazoline receptor because canavanine is a guanidinium derivative that has a similar structure to imidazoline receptor ligands. Transfected Chinese hamster ovary‐K1 cells expressing imidazoline receptors (nischarin (NISCH)‐CHO‐K1 cells) were used to elucidate the direct effects of canavanine on imidazoline receptors. In addition, the imidazoline I₃ receptor has been implicated in stimulation of insulin secretion from pancreatic β‐cells. Wistar rats were used to investigate the effects of canavanine (0.1, 1 and 2.5 mg/kg, i.v.) on insulin secretion. In addition the a specific I₃ receptor antagonist KU14R (4 or 8 mg/kg, i.v.) was used to block I₃ receptors. Canavanine decreased blood glucose by increasing plasma insulin in rats. In addition, canavanine increased calcium influx into NISCH‐CHO‐K1 cells in a manner similar to agmatine, the endogenous ligand of imidazoline receptors. Moreover, KU12R dose‐dependently attenuated canavanine‐induced insulin secretion in HIT‐T15 pancreatic β‐cells and in the plasma of rats. The data suggest that canavanine is an agonist of I₃ receptors both in vivo and in vitro. Thus, canavanine would be a useful tool in imidazoline receptor research.     

Insulin aspart: a novel rapid-acting human insulin analogue

In order to improve therapy and increase the quality of life for diabetic patients, it has been of significant interest to develop rapid-acting insulin preparations that mimic the physiological meal-time profile of insulin more closely than soluble human insulin. Insulin aspart (B28Asp human insulin) is a novel rapid-acting insulin analogue that fulfils this criterion. The B28Asp modification weakens the self-association of the insulin molecule and provides a more rapid absorption from the sc. injection site. The preclinical evaluation in vitro and in vivo demonstrates that apart from the more rapid absorption, insulin aspart is equivalent to human insulin. Thus, insulin aspart is equivalent to human insulin on key in vitro parameters such as insulin receptor affinity, insulin receptor dissociation rate, insulin receptor tyrosine kinase activation, IGF-I receptor binding affinity, metabolic and mitogenic potency. In accordance with the equivalent in vitro profiles, the toxico-pharmacological properties of insulin aspart and human insulin are also identical. The available data for insulin aspart and other rapid-acting insulin analogues supports that in vitro assays are sensitive and valuable in the preclinical evaluation of insulin analogues. Clinical studies demonstrate that insulin aspart has a pharmacokinetic and pharmacodynamic profile superior to that of soluble human insulin. In Type 1 diabetic patients on a basal-bolus injection regimen, insulin aspart given immediately before the meals provides an improved postprandial glycaemic control and an improved long-term metabolic control, as compared to soluble human insulin given 30 min before the meals, without increasing the risk of hypoglycaemia. Taken together, the data support the hope that insulin aspart will allow the diabetic patient to combine a more flexible lifestyle with better glycaemic control, without any increased safety risk.     

Antidiabetic agent pioglitazone increases insulin receptors on 3T3-L1 adipocytes

Pioglitazone and other antidiabetic thiazolidinediones appear to overcome insulin resistance by affecting an unidentified early event in insulin action. Because attempts to determine changes in insulin receptors in tissues from thiazolidinedione-treated diabetic animals are complicated by drug-induced reduction of hyperinsulinemia and consequent receptor up-regulation, 3T3-L1 cells were utilized here as an in vitro model system. After the excess insulin required for optimal differentiation of 3T3-L1 adipocytes was removed from the culture medium, there was an increase in specific binding of insulin to the cell surface that was further augmented 25 and 47% in cells treated with pioglitazone for 12 and 24 h, respectively (EC₅₀ about 0.6 μM, maximal at 5–25 μM). Pioglitazone increased the number of insulin receptors without changing binding affinity. Specific IGF-I binding was not affected by pioglitazone in the 3T3-L1 cells. Glycerol 3-phosphate dehydrogenase activity, a marker of adipocyte differentiation, was also increased in a dose-dependent manner by pioglitazone (maximal at 10–25 μM). Tunicamycin, which inhibits the N-linked glycosylation of newly synthesized insulin receptors that is required for their translocation to the cell surface, decreased specific insulin binding by about 50% after 24 h, and this decrease remained unchanged when pioglitazone was simultaneously present. Pioglitazone increased not only cell surface insulin receptors, but also total cellular insulin receptors (quantitated either by specific insulin binding or by immunoblotting) and insulin receptors mRNA. In addition, among several structurally related thiazolidinediones there was a positive correlation between in vivo antihyperglycemic activity and their ability to increase insulin binding in vitro, suggesting a possible common molecular mechanism for these actions. © 1995 Wiley-Liss, Inc.     

The effects of a freeze-thaw cycle and pre-analytical storage temperature on the stability of insulin-like growth factor-I and pro-collagen type III N-terminal propeptide concentrations: Implications for the detection of growth hormone misuse in athletes

A method based on two serum biomarkers – insulin‐like growth factor‐I (IGF‐I) and pro‐collagen type III N‐terminal propeptide (P‐III‐NP) – has been devised to detect growth hormone (GH) misuse. The aims of this study were to determine the stability of IGF‐I and P‐III‐NP concentrations in serum stored at ?20°C and to establish the effects of one freeze‐thaw cycle. Blood was collected from 20 healthy volunteers. Serum aliquots were analyzed after storage for one day at 4°C and one day, one week, five weeks, and three months at ?20°C. IGF‐I and P‐III‐NP results were combined to calculate a GH‐2000 discriminant function score for each volunteer. Inter‐assay precision was determined by analysing one quality control sample at each time‐point. A single freeze‐thaw cycle, storage of serum at 4°C for one day and at ?20°C for up to three months had no significant effect on IGF‐I or P‐III‐NP concentration. Intra‐sample variability for IGF‐I was 6.8% (Immunotech assay) and 12.9% (DSL assay). Intra‐sample variability for P‐III‐NP was 10.9% (Cisbio assay) and 13.7% (Orion assay). When IGF‐I and P‐III‐NP results were combined, intra‐sample variability of the GH‐2000 score expressed as a standard deviation varied between 0.31 and 0.50 depending on the assay combination used. Variability in IGF‐I and P‐‐III‐NP results of stored samples is largely determined by the characteristics of the assays. A single freeze‐thaw cycle, storage of serum at 4°C for one day or at ?20°C for up to 3 months does not result in a significant change in GH‐2000 score. Copyright © 2012 John Wiley & Sons, Ltd.     

Pharmacological evaluation and chemical stability of 2‐benzylthioether‐5′‐O‐(1‐thiotriphosphate)‐adenosine,a new insulin secretagogue acting through P2Y receptors

Activation of P2Y receptors on pancreatic β‐cells by extracellular ATP bring about amplification of glucose‐induced insulin secretion, which has been shown to reduce hyperglycemia in vivo. A new P2 receptor ligand, 2‐benzylthio‐ATP‐α‐S, was synthesized based on a combination of modifications of the ATP skeleton, as a potential insulin secretagogue. The two diastereoisomers of this ligand were separated and are designated A and B. The effects of these compounds on insulin secretion and vascular resistance in the rat isolated and perfused pancreas were evaluated in the presence of a slightly stimulating glucose concentration (8.3 mM) and were compared with ATP‐α‐S and ATP. Both isomers of 2‐benzylthio‐ATP‐α‐S (0.015–1.5 μM) induced a concentration‐dependent increase in glucose‐induced insulin release. The potency of isomer A was not significantly different from that of isomer B, and both were approximately 100‐fold more potent than ATP. ATP‐α‐S induced a similar pattern of insulin response; however, it was only approximately 10‐fold more potent than ATP. These compounds also induced vascular effects: ATP‐α‐S induced a vasodilatation and was transiently vasoconstrictor only at a high concentration, whereas the C2‐substituted derivative constantly induced a vasoconstriction. The chemical stability of these ligands was evaluated under physiological conditions and gastric juice pH. Hydrolysis of 2‐benzylthio‐ATP‐α‐S has been studied both in pH 7.4 and pH 1.4 at 37°C using ³¹P nuclear magnetic resonance (NMR) spectroscopy and high‐performance liquid chromatography. This compound exhibited high chemical stability with respect to hydrolysis of the glycosidic bond and desulfurization of the phosphorothioate moiety. Hydrolysis of the phospho ester bond, which was the only detectable degrading reaction under the investigation conditions (pH 7.4, 37°C), was slow, with a half‐life of 264 h. Moreover, even at gastric juice conditions (pH 1.4, 37°C), hydrolysis of the terminal phosphate was the only detectable reaction, with half‐life of 17.5 h. In conclusion, both isomers of 2‐benzylthio‐ATP‐α‐S are soluble in water and highly chemically stable. These compounds are highly potent and effective insulin secretagogues; however, they increase pancreatic vascular resistance. Drug Dev. Res. 53:33–43, 2001. © 2001 Wiley‐Liss, Inc.