Measurement Of Insulin Levels Research Articles

Using an Oral Sugar Test to Biologically Validate the Use of a Commercial Enzyme Immunoassay to Measure Salivary Insulin in Western Lowland Gorillas (Gorilla gorilla gorilla).

Noninvasive evaluations of hormones can contribute to the assessment of health and welfare of animals. Variations in insulin levels and sensitivity, for example, have been linked to health concerns in non-human and human primates including insulin resistance, diabetes, and heart disease, the leading cause of death in zoo-housed gorillas. Few published studies have assessed insulin concentrations in western lowland gorillas (Gorilla gorilla gorilla), and all did so using serum. Anesthesia is typically required to collect blood samples from zoo-housed gorillas, limiting sampling frequency and restricting samples to the fasted state. The ability to measure insulin levels in saliva would eliminate these constraints and provide a minimally invasive means for monitoring this hormone. The purpose of this study was to analytically and biologically validate the measurement of insulin in saliva of western lowland gorillas using a commercially available enzyme immunoassay. For validation, an oral sugar test was conducted on five adult gorillas residing at Cleveland Metroparks Zoo. Such assessments are common practice in both human and equine medicine to evaluate the body's insulin response to ingestion of sugars. The test involved measuring salivary insulin at timed intervals before and after gorillas consumed doses of sugar. Salivary insulin levels increased from 15 min post-sugar ingestion and peaked after 90 to 120 min. One female had a high response and peaked at 990.21 mU/L. The other four gorillas had peaks between 49.82 and 167.04 mU/L. The assessment provided a biological validation for the measurement of salivary insulin in western lowland gorillas.

Development of an In Vivo, Real-Time and Continuous Insulin Sensor Utilizing an Extended Gate Field Effect Transistor-Based Transducer

Introduction In this paper, we aim to utilize an extended gate field-effect transistor (EGFET)-based transducer to measure insulin. The design of EGFETs is characterized by a unique structural arrangement, where a physical barrier exists between the semiconductor component and the analyte or physical medium. Due to this structure, EGFET possesses the advantage of protecting the electrode from intricacies within the measurement solution or medium. The need for a redox probe is also effectively eliminated by incorporating EGFETs, marking a significant breakthrough in electrochemical measurement. The lack of a redox probe allows EGFET-based sensors to conduct measurements with minimal invasiveness and enhanced biocompatibility in accordance with an in vivo monitoring system. As the target analyte is not required to be electrochemically active, EGFET is highly customizable for a wide range of targets, including neurotransmitters, biomarkers, and metabolites. Therefore, we constructed immunosensors immobilized with anti-insulin single-chain antibody (scFv) and measured insulin levels, with the objective of creating an in vivo sensor designed to improve glycemic management. We aimed to implement the inherent semiconductor properties of EGFETs by performing real-time monitoring of changes in electrical properties to quantify various insulin concentrations. Methods Development of an in vivo sensor requires the use of a platform that can be implemented within an in vivo environment. For this purpose, we utilized a gold microwire electrode immobilized with anti-insulin scFv. The gold microwire electrodes were prepared by electrochemically cleaning and further roughening them to increase the active surface area for the purpose of improving immobilization of the antibody. To achieve site-specific immobilization of anti-insulin scFv, we employed a nickel-chelated nitrilotriacetate self-assembled monolayer (Ni-NTA SAM). As the scFv harbors a histidine tag and Ni-NTA has an affinity for histidine residues, we were able to achieve oriented immobilization on the electrode surface. Within this study, we compared the effects of covalent immobilization, which leads to random orientation with the antibody, and non-covalent oriented immobilization by comparing the signal obtained from EGFET measurement. EGFET measurements were conducted using a transistor consisting of source, drain, and gate terminals. In this configuration, a constant potential was maintained between the source and drain terminals, while a sweeping potential was applied between an Ag/AgCl reference electrode and the source terminal. Measurement of insulin was performed within a buffer and artificial interstitial fluid (aISF) to model an in vivo environment. Results and Discussion The interaction between insulin and the immobilized anti-insulin scFv on the electrode surface induces a shift in surface potential, driven by the redistribution of charges across the extended gate. The change in surface potential leads to a subsequent modification of the gate potential, known as Vg. The gate potential, when less than the threshold voltage (Vth), operates in the cutoff region where little to no current is present. However, when Vg is greater than Vth, the system operates in the ohmic region where current can flow. The following relationship allowed us to monitor the drain-source current or Ids within the ohmic region to evaluate insulin concentrations. The signal change due to insulin binding to anti-insulin scFv was observed at a reference voltage of 0.6 volts. Conclusion We measured insulin concentrations using an electrode modified with anti-insulin scFv and demonstrated that EGFET can be used as a working principle for an in vivo monitoring system. Additionally, the measurement principle does not require a redox probe within the measurement solution. Future work will include implanting the anti-insulin scFv immobilized gold microwire electrode within a rat model and measuring insulin.

FACTITIOUSHYPOGLYCEMIA: A DIAGNOSIS NOT TO BE OVERLOOKED

Munchausen syndrome by proxy is a rare psychiatric disorder characterized by inducing symptoms of illness in a child by a close relative, usually the mother, leading to various consultations and more or less invasive medical interventions. Factitious hypoglycemia, a factitious disorder caused by the administration of insulin or other hypoglycemic agents, results in hypoglycemias with severe sequelae. Diagnosis relies on measuring insulin levels, which will be high, and C-peptide levels, which will be low or even undetectable. We report the case of a female infant presenting with factitious hypoglycemia due to an insulin injection, revealed by severe hypoglycemias.

Abstract P188: The Triglyceride-Glucose Index in Africans is an Important Marker of Both an Atherogenic Lipid Profile and Insulin Resistance

Background: Type 2 diabetes (T2D) and cardiovascular disease (CVD) are rapidly increasing in Africa. Both are linked to insulin resistance (IR). Measuring insulin levels and entering the values into formulas such as the Matsuda Sensitivity Index are classic ways to measure IR. But due to cost and technological issues, the insulin assay is unavailable in most clinics in African countries. However, the Triglyceride-Glucose Index (TyG) may be an inexpensive, readily available alternative and could have the added benefit of indicating dyslipidemia. As the value of TyG in Africans is uncertain, we assessed in African-born Blacks living in America, the ability of the TyG index to (a) predict IR, (b) the optimal cut-off for TyG to predict IR, and (c) differences in the strength of the correlations between five lipid parameters which are closely linked to IR, specifically HDL-C, HDL-size, LDL-C, LDL-size and LDL-particle number, and both the TyG and Matsuda Index. Methods: OGTTs and fasting lipid profiles were performed in 589 African-born Blacks living in America (Male: 64%, Age: 39±11y, (mean±SD) (range 20 -69y); BMI 27.9±4.5 kg/m 2 (range 19.5-41.9 kg/m 2 )). The formula for TyG was: ln(fasting glucose * fasting TG)/2. IR was defined by the threshold of the lowest quartile of the population distribution of the Matsuda Index (≤2.89). Area under the receiver operating characteristic (AUC-ROC) curve was used to determine the ability of TyG to predict IR. Youden Index was used to identify the optimal cut-off. Results: T2D, preT2D and normal glucose tolerance occurred in 7% (40/589), 32% (188/589) and 62% (361/589), respectively. AUC-ROC curve for TyG to predict IR was 0.76 (95% CI: 0.71, 0.80). The optimal cut-off was: 4.3. Lipid correlations are provided in the table and were significantly stronger between each lipid parameter and TyG than with the Matstuda Index. Conclusions: TyG is an effective marker of IR in Africans and due to stronger correlations with atherogenic lipids may be a better predictor of CVD than the Matsuda Index.

1762-P: Increased Glucose Sensing and Insulin Secretion Induced by Verapamil Is Associated with Enhanced Oxygen Consumption Rate in MIN6 ß-Cells

It has been shown recently that oral administration of verapamil in persons with type 1 diabetes (T1D) decreased the dependency on exogenous insulin and improved the β cell function. Additionally, it decreased the frequency of hypoglycemic events. This finding encouraged us to test the effect of verapamil on the function of MIN6 mouse β cell-line. Glucose-Stimulated Insulin Secretion (GSIS) assay was performed using static incubation method. MIN6 cells were pre-treated with 50 μM verapamil for 24 hours, then the levels of total insulin content and secreted insulin were measured before and after GSIS assay using highly sensitive mouse insulin ELISA kit. Glucose sensing was estimated by the percentage of secreted insulin over total insulin content for each concentration of glucose. The measured insulin levels were normalized by the level of total protein. Oxygen consumption rate (OCR) was measured using Seahorse system and metabolic stress kit. MIN6 cells were seeded in Agilent Seahorse XF96 cell culture microplate at a cell density of 4×104 cells/well, under normal (5.6 mM) and high (25.2 mM) glucose concentrations for 24 hours, after which, cells were treated with verapamil (50 μM) or left untreated and incubated for 24 hours. Our results show no significant difference between total insulin content of treated vs. untreated cells. GSIS data show that insulin secretion was significantly higher in verapamil-treated cells at 8.4, 11.2, and 16.8 mM, but not at 2.8 and 5.6 mM glucose. The results suggest that β cells were hypersensitized to glucose when treated with verapamil. In metabolic flux analysis for mitochondrial respiration, treatment with verapamil increased basal and maximal respiration. Taken together, our study showed that verapamil increased the level of glucose sensing and oxidative respiration under conditions of high glucose, which may explain its effect on lowering the frequency of hypoglycemic events in clinical studies. Disclosure H.Arefanian: None. S.E.John: None. M.Alhusayan: None. S.J.Kurian: None. N.A.Abukhalaf: None. S.Shenouda: None. H.Alsaeed: None. S.P.Kochumon: None. M.Abu-farha: None. J.Abubaker: None. A.T.Thangavel: None. F.Bahman: None. R.Ahmad: None. F.Almulla: None. A.Al madhoun: None. F.Alzaid: None. S.T.Sindhu: None. M.R.Williams: None. F.Alrashed: None. R.Nizam: None. S.Jacob: None. Funding Kuwait Foundation for the Advancement of Sciences (RACB-2019-001)

BIOSSENSOR PARA NÍVEL DE INSULINA SANGUÍNEO EM ATLETAS

ABSTRACT Introduction: The pancreas releases insulin to assist the human body in utilizing blood glucose. It regulates metabolism by promoting the absorption of glucose into the blood. Objective: This work aimed to create an electrochemical biosensor based on magnetic graphene nanomaterial to measure insulin levels in athletes’ blood. Method: A magnetic graphene nanocomposite created by graphene oxide (GO) and Fe-Ni bimetallic oxides on a glassy carbon electrode was synthesized using the electrochemical deposition method (GCE). Results: The immediate electrical deposition of Fe-Ni bimetallic oxide nanoparticles with the spherical shape on the GO nanosheet without aggregations was validated by structural characterizations of Fe-Ni/GO/GCE using XRD and SEM. The electrochemical results for insulin determination showed good sensitivity and anti-interference capability. The applicability and accuracy of the proposed electrochemical sensor to detect insulin were explored by blood serum samples from sportsmen. Conclusion: The results assigned acceptable RSD values (3.31% to 4.30%) and confirmed the feasibility of the proposed sensor for detecting athletes’ blood insulin. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

Adherence to lifelines diet score and risk factors of metabolic syndrome among overweight and obese adults: A cross-sectional study.

Metabolic syndrome (MetS) is one of the most significant public health issues worldwide, and diet quality is an important controllable environmental factor influencing the incidence of MetS. Numerous dietary scores have been established to assess compliance with dietary recommendations or eating patterns, many of which are not entirely food-based. Hence, Lifelines Diet Score (LLDS) was developed in response to the shortcomings of existing tools. This study aimed to assess any possible links between total food quality and cardiometabolic risk factors among overweight and obese adults. This cross-sectional study included 338 overweight and obese individuals [body mass index (BMI) > 25 kg/m2] aged 20-50 years in Tabriz, Iran. To collect dietary data, we used a validated semi-quantitative Food Frequency Questionnaire (FFQ) for Iranian population. Enzymatic-colorimetric methods were used to assess serum glucose and lipids, and enzyme-linked immunosorbent assay (ELISA) kits were used to measure insulin levels. In addition, the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) and Quantitative Insulin Sensitivity Check Index (QUICKI) were calculated. BMI and hip circumference (HC) were significantly different (P < 0.05) amongst LLDS tertiles. Adherence to the highest tertile of LLDS was associated with lower SBP, and the subjects in higher LLDS tertiles significantly had lower systolic blood pressure (SBP) (P = 0.04). Triglyceride (TG) levels were also lower in the third tertile of LLDS with a near-significant P-value (P = 0.05). According to our results, a higher diet quality score, determined by LLDS, can be associated with a lower risk of MetS. Further experimental and longitudinal studies are needed to better understand this relationship.

Insulinoma: Diagnosis and Management on Two Observations

Insulinoma is an insulin-secreting pancreatic tumor responsible for hypoglycemia. It is a rare tumor that occurs in 1 to 4 persons / million in the population and represents 1 -2% of pancreatic tumors. The diagnosis is made by demonstrating, insulin secretion by measuring insulin levels and C-peptide in the presence of hypoglycemia and by demonstrating a pancreatic tumor by pancreatic CT and/or pancreatic MRI [5]. Surgical resection is the main modality for the treatment of insulinomas [1]. We report 2 observations of an insulinoma. A 47 year old patient, who consulted us for lipothymia associated with cold sweats and hunger, occurring preferably in the morning between 6 and 7 o’clock, The blood sugar level taken at the time of the malaise was 0.35 g/l. Assessment performed : Insulinemia ↑ : 701 pmol/l (18 -173), Peptide C↑: 2,35 pmol/ml ( 0,3- 1,4) in comparison with a hypoglycemia at 0,35 g/l. Abdominal MRI revealed a 16×16 mm nodule in the isthmus of the pancreas with poor vascularity compatible with an insulinoma. Observation 2: patient, 37 years, admitted for investigation and management of hypoglycemia ranging from 0.3 g/l occurring during physical activity. Workup performs: fasting test: peptide C↑: 3 ng/ml (1.1-1.47), insulinemia: 22.6 mUI/l (2.6- 24.9) vs. hypoglycemia 0.50 g/l, insulin/glycemia ratio=0.45 (normal &lt;0.3), turner index:113 (normal &lt;50). Normal abdominal CT scan completed by pancreatic MRI: pancreatic nodule in the isthmic region of 13*8 mm compatible with an insulinoma. Octreoscan: a hyperfixing focus on an isodense nodule of the head-neck junction of the pancreas measuring approximately 12*13 mm compatible with an insulinoma. Surgical management: enucleation. favorable evolution marked by the disappearance of hypoglycemia.

A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin.

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion.

Morphological and biochemical changes in the pancreas associated with acute systemic hypoxia

This study aimed to investigate the changes associated with acute systemic hypoxia in the endocrine system, particularly in pancreatic tissues. The investigation was based on macroscopic, pathohistological, biochemical, and molecular biological findings in cell lines and human cadavers. The results showed that cases of death due to asphyxia more frequently showed severe subcapsular/interstitial hemorrhage versus the other causes of death. Histological examination showed that asphyxia cases were associated with severe morphological changes. Although measured insulin levels in the asphyxia were higher compared to other causes of death, no differences were noted for the glucagon and amylase levels with regard to the cause of death. Increased blood insulin levels were not associated with macro- and micromorphological changes, and did not show any association with glucose or cortisol levels. The experiment conducted under hypoxic conditions in cultured cells demonstrated that insulin mRNA expression and insulin protein levels peaked at 10 min after hypoxia exposure. However, there were no changes in either the amylase mRNA or protein levels. Corticosterone level peaked at 120 min after exposure to hypoxic conditions. Overall, acute systemic hypoxic conditions can directly affect the mechanisms involved in pancreatic insulin secretion.

Application of Sensor Networks for Measuring Insulin Levels

All WBANs are low-speed networks consisting of intelligent sensors and low-power actuators that can be placed on the body or implanted in humans. With the help of these networks, timely on-line data on unusual conditions of the human body can be provided, which leads to significant improvements in the quality of life. The implementation of these networks will save lives, reduce healthcare costs, and eliminate the need for costly monitoring of patients in the hospital. The paper analyzes the possibilities of the Internet of Things technology, communication standards, cloud technologies, and hardware components for developing a system for monitoring insulin levels in diabetic patients. A model and algorithm for design, construction, and management of a sensor network for monitoring insulin levels with simulation software based on the Internet of Things technology proposed. A system for monitoring insulin levels through a cloud structure has designed, which sends a message to a specialized health centre or the attending physician about the condition of diabetic patients. Simulation studies performed, and the operability of the system examined. The results give reason to believe that the considered model and the simulation project of a system for monitoring insulin levels in patients with diabetes is successful, gives realistic and reliable results for early diagnosis and prevention of various complications and can be implemented in practice.

973: Long-term maternal metabolic and cardiovascular phenotypes after pregnancies complicated by mild GDM

To evaluate the association of mild GDM and/or obesity on long-term maternal metabolic and cardiovascular markers. Secondary analysis of 5-10 year follow-up from a mild GDM study that included a treatment trial for mild GDM and an observational cohort of women with abnormal 1-hour GCT only. Women who had maternal 1-hour glucose tolerance tests at the follow-up were included. Maternal serum was analyzed using magnetic bead-based multiplex assays to measure insulin levels and cardiovascular markers VEGF, VCAM-1, CD40L, GDF-15, and ST-2. Insulin levels were used to calculate the insulinogenic index (IGI), a measure of pancreatic ß-cell function, and 1/HOMA, a measure of insulin resistance. These markers were compared by parent study GDM status (treated GDM, untreated GDM, and non-GDM) and by obesity at parent study enrollment. Multivariable linear regression estimated the association of GDM status and obesity with the metabolic and cardiovascular markers, adjusting for age, race/ethnicity and years since index pregnancy. 642 women with serum samples available for testing were included in this analysis (Table). Treated GDM and untreated GDM were associated with lower mean differences in log 1/HOMA, compared with non-GDM (-0.24, 95%CI -0.36–-0.12 and -0.15, 95%CI -0.28–-0.03, respectively, Figure). Treated GDM was also associated with lower mean difference in log VCAM (-0.11, 95%CI -0.19–-0.03). GDM status was not associated with IGI or other cardiovascular markers. Obesity was only associated with lower mean difference in log 1/HOMA (-0.42, 95%CI -0.52–-0.32). Women with mild GDM and obese pregnant women are at increased risk for insulin resistance at long term follow-up. Among this population, neither GDM nor obesity was associated with pancreatic ß-cell dysfunction. Treatment of mild GDM may be associated with less angiogenesis, but neither mild GDM nor obesity was associated with other markers of cardiovascular dysfunction 5-10 years after pregnancy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Soursop (Annona Muricata) Leaf Water Extract (SLWE) Prevent Pancreatic Β-Cell Damage in Male Wistar Rats Induced By High Fat and High Fructose (HFHF) Diet

Background: A high-fructose high fat (HFHF) diet induces dyslipidemia, which can interfere with pancreatic function. Soursop leaf (Annona muricata) water extract (SLWE) has ant hyperlipidemia and antioxidants activity. This research aims to assess the activity of SLWE in preventing pancreatic β-cell damage in male Wistar rats induced by the HFHF diet. Method: This study is a post test group control only research design. Twenty-five wistar rats aged 10-12 weeks, weighing 175- 200 g, were randomly divided into five study groups, namely the normal group (N) laboratory standard diet, the positive group (P) HFHF diet, and the treatment group 1 (T1) HFHF diet+SLWE dose 100mg/kgbw, treatment group 2 (T2) HFHF diet+SLWE dose 200mg/kgbw, and treatment group 3 (T3) HFHF diet+SLWE dose 400mg/kgbw. Diet is given for ten weeks. Measurement of fasting blood glucose levels were taken from the tail using a rapid test conducted at the beginning and end of the study. At the end of the study, the rates were sacrificed under anesthesia. The rats pancreatic organs were surgically removed and blood was taken from the ventricular puncture. Pancreatic organs are used to measure the percentage of pancreatic β cells, which expressed FoxO1 protein, by flow cytometry method, and the average area of pancreatic β cells by immunohistochemistry. Blood is centrifuged to obtain blood plasma, which is used to measure insulin levels. Data were analyzed using One Way Anova and Kruskal Wallis with a significance of p &lt;0.05. Result: SLWE did not cause changes in blood glucose levels, but the administration of SLWE at a doses of 200mg/kgBB tended to increase insulin levels (p&gt; 0.05), decreased percentage number of β-cells expressing FoxO1, and decreased the average area of pancreatic β cells compared to P group (p &lt;0.05). Conclusion: SLWE can prevent pancreatic β-cell damage by increase insulin secretion without causing pancreatic β-cell proliferation.

Isotope-dilution liquid chromatography-tandem mass spectrometry for sensitive quantification of human insulin in serum using derivatization-technique

An isotope-dilution mass spectrometry (IDMS) method for measuring insulin levels in human serum was developed using C-terminal-derivatization method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The carboxyl groups of Glu-C-cleavage products were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity and IDMS quantification, resulting in increases in LC-MS/MS peak areas of derivatized Glu-C-cleavage products of human insulin by ∼23-(A5–17 peptide) to 49-fold(B14–21 peptide), respectively, as compared with results observed in the absence of derivatization. Separation was achieved on a C18 column by gradient elution at 0.3 mL/min, with a mobile phase composed of 0.1% formic acid in acetonitrile and water. Validation studies of target peptides (B1–13 peptide and B14–21 peptide) revealed a linear response in the range of 0.05 ng/mL to 10 ng/mL (regression coefficient, r2 = 0.9987 and 0.9988, respectively), a relative standard deviation within and between days of <8.6%, and spike and recovery test results indicating mean recoveries ranging from 100.2% to 106.6%. Comparison with an established commercial immunoassay showed high correlation (r2 = 0.9943 and 0.9944, B1–13 peptide and B14–21 peptide, respectively) at serum concentrations of between 0.20 ng/mL and 1.51 ng/mL. These findings suggested that this IDMS-based approach was able to quantify human serum insulin with high sensitivity and precision in the reference interval and indicated a potential for determining serum-insulin reference-measurement procedures to allow traceable measurement.

Stimulation of β-adrenergic receptors plays a protective role via increased expression of RAF-1 and PDX-1 in hyperglycemic rat pancreatic islet (RIN-m5F) cells.

IntroductionIt is a widely held view that a progressive reduction of beta-cell mass occurs in the progression of diabetes. RAF-1 kinase and pancreas duodenal homeobox 1 (PDX-1) are major factors that promote survival of cells and maintain normal insulin functions. In this study we investigated the effect of a β-adrenergic receptor agonist and antagonist on RAF-1 and PDX-1, and their respective effects on apoptosis and insulin release in RIN-m5F cells.Material and methodsRIN-m5F cells were cultured in normal (5 mM) and high (25 mM) glucose to mimic diabetic conditions, followed by treatment with 5 µM, 10 µM and 20 µM of isoproterenol and isoproterenol + propranolol for 6, 12 and 24 h. Western blotting and reverse transcription analysis were performed to examine the expression of RAF-1 and PDX-1. Annexin-V-FITC and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were used to investigate apoptosis. ELISA was used to measure insulin levels. Reverse transcription polymerase chain reaction was conducted to investigate the expression of genes.ResultsStimulation of β-adrenergic receptors with isoproterenol significantly induced RAF-1 and PDX-1 genes in a concentration-dependent and time-independent manner. Changes were significant both at protein and mRNA levels. Up-regulation of RAF-1 and PDX-1 was accompanied by improved insulin levels and reduced apoptosis. Concentrations of 10 µM and 20 µM for 12 and 24 h were more effective in achieving significant differences in the experimental and control groups. Propranolol reversed the effect of isoproterenol mostly at maximum concentrations and time periods.ConclusionsA positive effect of a β-adrenergic agonist on RAF-1 and PDX-1, reduction in β-cell apoptosis and improved insulin contents can help to understand the pathogenesis of diabetes and to develop novel approaches for the β-cell dysfunction in diabetes.

Magnetic Optical Microarray Imager for Diagnosing Type of Diabetes in Clinical Blood Serum Samples.

Due to rapidly rising rates of diabetes and prediabetic conditions worldwide and the associated lethal complications, it is imperative to devise new diagnostic tools that reliably and directly measure insulin levels in clinical samples. Herein, we report a simple and sensitive direct imaging of insulin levels in diabetic patient samples using a surface plasmon resonance microarray imager (SPRi). To enhance sensitivity, we utilized magnetic nanoparticles (MNPs) to capture insulin from serum samples either directly or via a capture antibody immobilized on MNPs. The insulin-captured nanoparticles were allowed to bind surface insulin-antibody for detection from pixel intensity increase using a charge coupled device (CCD) built-in with the SPRi. We have compared the analytical figures-of-merit of the SPRi immunoarray on detecting insulin prepared in various percentages of serum solutions. A four parameter logistic model was used to obtain the best fit of microarray responses with insulin concentration and indicated the cooperative binding of insulin-nanoparticle conjugates to surface antibody in both the buffer insulin and the serum insulin conjugates with MNPs. The cooperativity effect is attributed to the greater association of magnetic nanoparticle-bound insulin molecules with increasing concentration of insulin binding to surface antibody. This is the first report of an SPRi immunoarray to accomplish clinical diagnosis of diabetic and prediabetic conditions based on insulin levels with serum matrix effect analysis and comparison between direct and sandwich insulin assay formats.

Magnetic Optical Microarray Imager for Diagnosing Type of Diabetes in Clinical Matrices

Diabetes is a metabolic disorder in which body’s inability to produce enough insulin leads to either low (hypoglycemia) or elevated levels of glucose (hyperglycemia). This imbalance in glucose metabolism can eventually lead to serious health problems such as kidney failure, heart disease, blindness, etc. Insulin (a polypeptide hormone) serves as a valuable predictor in diagnosing the type of diabetes and depending upon its levels in clinical matrices, diabetes can be broadly classified as type 1 (< 50 pM) or type 2 (> 80 pM). In type 1 (insulin dependent), the body does not produce enough insulin, while in type 2 (non-insulin dependent), the excess insulin produced is not utilized by the body, thus elevating the glucose levels. 2012 statistics from American Diabetes Association reported 9.3 % of the population to be diabetic and a predicted 25% of population with pre-diabetic conditions. Diagnosis of diabetes, by ultrasensitive detection methods, has therefore become the need, which can detect and measure insulin levels at clinical sites. We present herein, an ultrasensitive microarray imager, which can detect insulin levels in clinical matrices such as serum and whole blood by an optical tool, surface plasmon resonance. The imager involves an immunoassay utilizing antibody immobilized gold arrays and magnetic particles spiked various insulin concentrations to detect picomolar levels of insulin and diagnose the type of diabetes with high sensitivity. The magnetic microarray insulin imager developed is a promising platform for detecting insulin in serum and whole blood at clinical sites. Acknowledgements. We are grateful for the financial support by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number R15DK103386.

Voltammetric immunosensor assembled on carbon-pyrenyl nanostructures for clinical diagnosis of type of diabetes.

Herein we report the first serum insulin voltammetric immunosensor for diagnosis of type 1 and type 2 diabetic disorders. The sensor is composed of multiwalled carbon nanotube-pyrenebutyric acid frameworks on edge plane pyrolytic graphite electrodes (PGE/MWNT/Py) to which an anti-insulin antibody was covalently attached. The detection of picomolar levels of serum insulin binding to the surface antibody was achieved by monitoring the decrease in voltammetric current signals of a redox probe taken in the electrolyte solution. This method offered a detection limit of 15 pM for free insulin present in serum. This detection limit was further lowered to 5 pM by designing serum insulin conjugates with poly(acrylic acid)-functionalized magnetite nanoparticles (100 nm hydrodynamic diameter) and detecting the binding of MNP-serum insulin conjugate to the surface insulin-antibody on PGE/MWNT/Py electrodes. When tested on real patient serum samples, the sensor accurately measured insulin levels. To our knowledge, this is the first report of a voltammetric immunosensor capable of both diagnosing and distinguishing the type of diabetes based on serum insulin levels in diabetic patients.

Response: The Effect of DPP-4 Inhibitors on Metabolic Parameters in Patients with Type 2 Diabetes (Diabetes Metab J 2014;38:211-9).

We appreciate your interest and comments on our article entitled Effect of DPP-4 Inhibitors on Metabolic Parameters in Patients with Type 2 Diabetes, which was published in Diabetes & Metabolism Journal 2014;38:211-9 [1]. In our study, we showed the different effects of 24-week dipeptidyl peptidase-4 (DPP-4) inhibitor (sitagliptin and vildagliptin) treatments on serum glucose and lipid parameters. The glucose lowering efficacy was not significantly different between both groups, but vildagliptin showed augmented total cholesterol and triglyceride reduction than sitagliptin group. Although it is not clear whether the lipid-lowering effect of DPP-4 inhibitors is the result of direct DPP-4 inhibition or indirect metabolic effects of medication, other studies have also shown the lack of correlation between the reduction of total cholesterol and reduction in blood glucose levels. This suggests that the effects of DPP-4 inhibitors on lipid parameters might be independent of their blood glucose lowering effect. In addition, the direct effect of DPP-4 inhibitors on lipid biosynthesis has been reported in mice model [2]. Regarding body weight change, no significant weight change was observed in both treatment groups (data not shown). Considering that DPP-4 inhibitors have no effect on body weight in general, as reported in many other studies [3,4,5,6], it is unlikely that the change in blood lipid profile is due to weight loss. Unfortunately, we did not measure insulin levels at follow-up visit, and hence we could not calculate homeostatic model assessment-insulin resistance as an index of insulin resistance. In general, all DPP-4 inhibitors are reported to have an effect on postprandial lipid levels [7]. One meta-analysis study reported that vildagliptin and alogliptin could have a greater lipid lowering effect than sitagliptin [8]. A recent observation study showed that a greater reduction in total cholesterol was achieved with vildagliptin (-24 mg/dL) than with sitagliptin (-11 mg/dL) and saxagliptin (-3.6 mg/dL) [9]. These studies are in line with our study and suggest the differential DPP-4 inhibitor effects on blood cholesterol levels. A potential reason for this difference may be due to the pharmacokinetics of vildagliptin versus sitagliptin. Vildagliptin interacts with DPP-4 as a kind of substrate blocker, with vildagliptin continuously binding to DPP-4 as long as vildagliptin levels are adequate. In contrast, sitagliptin, a competitive DPP-4 antagonist, inhibits its activity in a dose-dependent manner [10]. However, further and larger clinical studies and functional studies are needed to better establish the impact of different DPP-4 inhibitors on dyslipidemia in vivo and in vitro. Lastly, although it would be interesting to identify the difference between the patients who showed lipid lowering response to DPP-4 inhibitors and those didn't, we did not assess external factors such as diet or exercise. Instead, we analyzed the effect of statin medication and showed the result in Supplementary Table 1 [1], and found that there was a difference between sitagliptin and vildagliptin on lipid profile change regardless of the use of statin. We would like to thank Dr. Lee for the interest in our study and for your thoughtful comments.

Outcome of Periapical Lesions in a Rat Model of Type 2 Diabetes: Refractoriness to Systemic Antioxidant Therapy

IntroductionThis study evaluated the development of periapical lesions in a rat model of type 2 diabetes and assessed the potential actions of the antioxidant agent tempol in this model. MethodsMale Wistar rats were used; they received tap water (N = 5) or a 20% glucose solution (N = 15) during a period of 9 weeks. At the sixth week, periapical lesions were induced on the first mandibular molars, and the animals were subdivided into 4 groups. The subgroup 1 was composed of nondiabetic rats orally receiving saline solution (10 mL/kg). Chronically glucose-fed rats were divided into the following subgroups: (2) saline-treated animals (10 mL/kg by oral route), and animals treated with tempol by gavage at doses of (3) 50 mg/kg or (4) 100 mg/kg. The body weight was monitored thoroughly. After 21 days of apical periodontitis induction, the animals were killed, and the mandibles were collected and submitted to radiographic and histologic analysis. The livers were collected to determine free radicals, and the blood plasma was used to measure insulin levels. ResultsType 2 diabetic rats displayed a significant decrease of body weight gain and a slight increase of insulin levels, which were allied to reduced levels of the antioxidant components catalase and reduced glutathione; these alterations were reversed by tempol. Concerning the periapical lesions, neither radiographic nor histologic analysis revealed any significant difference between control and type 2 diabetic rats. In diabetic rats, the apical periodontitis was refractory to tempol treatment. ConclusionsThe extent and cellularity of periapical lesions in glucose-fed type 2 diabetic rats were similar to those seen in control rats. Despite affecting other parameters related to diabetes, tempol failed to improve the outcome of endodontic lesions in type 2 diabetic animals.