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The Fifth Artificial Pancreas Workshop: Enabling Fully Automation, Access, and Adoption was held at the National Institutes of Health (NIH) Campus in Bethesda, Maryland on May 1 to 2, 2023. The organizing Committee included representatives of NIH, the US Food and Drug Administration (FDA), Diabetes Technology Society, Juvenile Diabetes Research Foundation (JDRF), and the Leona M. and Harry B. Helmsley Charitable Trust. In previous years, the NIH Division of Diabetes, Endocrinology, and Metabolic Diseases along with other diabetes organizations had organized periodic workshops, and it had been seven years since the NIH hosted the Fourth Artificial Pancreas in July 2016. Since then, significant improvements in insulin delivery have occurred. Several automated insulin delivery (AID) systems are now commercially available. The workshop featured sessions on: (1) Lessons Learned from Recent Advanced Clinical Trials and Real-World Data Analysis, (2) Interoperability, Data Management, Integration of Systems, and Cybersecurity, Challenges and Regulatory Considerations, (3) Adaptation of Systems Through the Lifespan and Special Populations: Are Specific Algorithms Needed, (4) Development of Adaptive Algorithms for Insulin Only and for Multihormonal Systems or Combination with Adjuvant Therapies and Drugs: Clinical Expected Outcomes and Public Health Impact, (5) Novel Artificial Intelligence Strategies to Develop Smarter, More Automated, Personalized Diabetes Management Systems, (6) Novel Sensing Strategies, Hormone Formulations and Delivery to Optimize Close-loop Systems, (7) Special Topic: Clinical and Real-world Viability of IP-IP Systems. "Fully automated closed-loop insulin delivery using the IP route," (8) Round-table Panel: Closed-loop performance: What to Expect and What are the Best Metrics to Assess it, and (9) Round-table Discussion: What is Needed for More Adaptable, Accessible, and Usable Future Generation of Systems? How to Promote Equitable Innovation? This article summarizes the discussions of the Workshop., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: DCK is a consultant for Better Therapeutics, EOFlow, Integrity, Lifecare, Nevro, Novo, Sanofi, and Thirdwayv. DK has received research support from Abbott Diabetes Care. AW receives research salary support from UnitedHealth Group. REA, TT, AMY, JH, CEM, WAL have no disclosures.

Diabetes Technology Society hosted its annual Diabetes Technology Meeting from November 3 to November 5, 2022. Meeting topics included (1) the measurement of glucose, insulin, and ketones; (2) virtual diabetes care; (3) metrics for managing diabetes and predicting outcomes; (4) integration of continuous glucose monitor data into the electronic health record; (5) regulation of diabetes technology; (6) digital health to nudge behavior; (7) estimating carbohydrates; (8) fully automated insulin delivery systems; (9) hypoglycemia; (10) novel insulins; (11) insulin delivery; (12) on-body sensors; (13) continuous glucose monitoring; (14) diabetic foot ulcers; (15) the environmental impact of diabetes technology; and (16) spinal cord stimulation for painful diabetic neuropathy. A live demonstration of a device that can allow for the recycling of used insulin pens was also presented.

Diabetes Technology Society hosted its annual Diabetes Technology Meeting on November 4 to November 6, 2021. This meeting brought together speakers to discuss various developments within the field of diabetes technology. Meeting topics included blood glucose monitoring, continuous glucose monitoring, novel sensors, direct-to-consumer telehealth, metrics for glycemia, software for diabetes, regulation of diabetes technology, diabetes data science, artificial pancreas, novel insulins, insulin delivery, skin trauma, metabesity, precision diabetes, diversity in diabetes technology, use of diabetes technology in pregnancy, and green diabetes. A live demonstration on a mobile app to monitor diabetic foot wounds was presented.

Diabetes Technology Society hosted its annual Diabetes Technology Meeting on November 12 to November 14, 2020. This meeting brought together speakers to cover various perspectives about the field of diabetes technology. The meeting topics included artificial intelligence, digital health, telemedicine, glucose monitoring, regulatory trends, metrics for expressing glycemia, pharmaceuticals, automated insulin delivery systems, novel insulins, metrics for diabetes monitoring, and discriminatory aspects of diabetes technology. A live demonstration was presented.

Newly developed patch pumps are starting to occupy a noticeable fraction of the insulin delivery market. New entrants, using novel technologies, promise accurate, flexible insulin delivery at lower costs. In the section, we review the currently available devices, discuss some of the devices on the horizon, and speculate about some fascinating new approaches. In this first article, we provide an overview of the simplified devices-V-Go, PAQ, and One Touch Via-and of the more complex devices-Omnipod, Cellnovo, JewelPump, Solo, SFC Fluidics pump, Libertas, Medtronic pump, and EOPatch. We also discuss controllers, smartphones, and cybersecurity.

Insulin pens are more accurate and easier to teach than other methods of insulin delivery. They also do not suffer from the risk of mismatch of insulin concentration and type of insulin syringe. The ISO standard used to test insulin pens, however, needs to be updated to reflect their clinical use., (© 2015 Diabetes Technology Society.)

With the availability of a smaller mixing pen, mass marketing of less stable medications is possible. Bidureon is one such medication, and the properties of its pen are discussed along with the prospects for future mixing pens., (© 2015 Diabetes Technology Society.)

Self-monitoring of blood glucose provides information about blood glucose control. The data become useful information and knowledge through careful analysis for patterns that are appropriate or can be corrected. Some analyses can be performed on newer blood glucose meters, but most often, this needs to be done on a computer, tablet, or smartphone. There are a few established methods of presenting the data that make analysis easier. In this article, we discuss four types of data presentations and the methods for utilizing them., (© 2013 Diabetes Technology Society.)

The pain associated with lancing can be a significant barrier to self-monitoring of blood glucose (SMBG). The OneTouch® Delica™ lancing device contains features to reduce lancing pain, including improved lancet control and stability, reduced vibration, and a thinner, 33-gauge lancet. This 2-visit, randomized controlled trial assessed perceived pain of lancing with the OneTouch® Delica™ compared with 4 other common lancing devices: OneTouch® Comfort™, ACCU-CHEK® Softclix, ACCU-CHEK® Multiclix, and Ascensia® Microlet™2. Two hundred patients with type 1 or type 2 diabetes mellitus were assigned to the OneTouch® Delica™ and also randomized to 1 of the 4 comparator devices (n=50 per device pair). At visit 1, we determined the minimum depth settings required to produce≥1 μL of fingertip blood for each patient with each device. At visit 2, patients lanced their fingertips with the devices at the predetermined depths and used a 150-mm visual analog scale (VAS) to rate lancing pain relative to their "usual pain" associated with SMBG. The VAS ranged from "much less painful" (0 mm) to "much more painful" (150 mm), with the midpoint (75 mm) labeled as "usual pain." Fingertip pain scores from patients using OneTouch® Delica™ were significantly lower than those obtained using OneTouch® Comfort™, ACCU-CHEK® Multiclix, and Ascensia® Microlet™2. Pain scores for OneTouch® Delica™ and ACCU-CHEK® Softclix were not significantly different. In conclusion, OneTouch® Delica™ was either less painful or no different than the comparator devices when used for fingertip lancing. Innovative lancing devices that cause less pain may improve compliance and persistence with prescribed SMBG.

Regulatory interest has focused on the accuracy of blood glucose monitoring systems. Currently, almost all systems meet the International Organization for Standardization (ISO) 15197 clinical standard (≥95% of the values within 20% of the reference for values above 75 mg/dl and within 15 mg/dl below that level). Should the systems have to meet one of the extended ISO standards of 15%, 10%, or even 5%? There is a wide variety of people with diabetes doing glucose monitoring, and the majority do not need better accuracy. Indeed, when selecting an insulin dose, the inaccuracy of the glucose reading has little effect compared with the inaccuracy in counting carbohydrates and the variability in insulin absorption. It might be far better to evaluate the accuracy in a standard method and provide the accuracy values on a standard label. Patients and health care providers could then select the monitoring system that best meets their needs., (© 2010 Diabetes Technology Society.)

Glucose monitoring has become an integral part of diabetes care but has some limitations in accuracy. Accuracy may be limited due to strip manufacturing variances, strip storage, and aging. They may also be due to limitations on the environment such as temperature or altitude or to patient factors such as improper coding, incorrect hand washing, altered hematocrit, or naturally occurring interfering substances. Finally, exogenous interfering substances may contribute errors to the system evaluation of blood glucose. In this review, I discuss the measurement of error in blood glucose, the sources of error, and their mechanism and potential solutions to improve accuracy in the hands of the patient. I also discuss the clinical measurement of system accuracy and methods of judging the suitability of clinical trials and finally some methods of overcoming the inaccuracies. I have included comments about additional information or education that could be done today by manufacturers in the appropriate sections. Areas that require additional work are discussed in the final section., (Copyright 2009 Diabetes Technology Society.)

Most blood glucose monitoring systems need coding to correct for variation in lots of enzyme, which leads to differences in lots of strips. About 16% of patients miscode the meters, although the magnitude of the miscoding is unstudied. This miscoding has the potential to cause errors as high as 30% and to cause errors in adjusting insulin therapy that could lead to hypoglycemia at least 10% of the time. Studies of these systems suggest that they have accuracy similar to other current meters and have similar physical characteristics. Because they do not require coding, they are often easier to use. No-coding systems have the potential to avoid some errors in blood glucose.

Objective: This study was done to evaluate the accuracy and safety of measuring glucose with the GlucoWatch biographer in children and adolescents with diabetes., Methods: Accuracy was assessed by comparing biographer glucose measurements with hourly blood glucose measurements using the HemoCue (Aktiebolaget Leo, Helsingborg, Sweden) Photometer for up to 12 h of monitoring. Safety was evaluated by examining the biographer application sites immediately upon removal of the devices, and then at regular intervals., Results: Sixty-six subjects each wore three biographers at sites including the forearm, upper arm, leg, and torso. For forearm biographers, the mean absolute relative difference between biographer readings and blood glucose was 21%. Ninety-five per cent of biographer readings fell into the A or B regions of the Clarke error grid, and 97.3% into the A or B regions of the consensus error grid. Data from biographers worn at the alternative sites were similar to data from the forearm biographers. Two strong reactions to the adhesive pad of the biographer AutoSensor were observed. Most skin reactions were mild., Conclusions: The GlucoWatch biographer is well tolerated by children and adolescents with diabetes. Performance is similar when the device is worn at different anatomical sites, and is similar to the performance on the forearm, previously reported in adults.

Objective: The objectives of this study were 1) to construct new error grids (EGs) for blood glucose (BG) self-monitoring by using the expertise of a large panel of clinicians and 2) to use the new EGs to evaluate the accuracy of BG measurements made by patients., Research Design and Methods: To construct new EGs for type 1 and type 2 diabetic patients, a total of 100 experts of diabetes were asked to assign any error in BG measurement to 1 of 5 risk categories. We used these EGs to evaluate the accuracy of self-monitoring of blood glucose (SMBG) levels in 152 diabetic patients. The SMBG data were used to compare the new type 1 diabetes EG with a traditional EG., Results: Both the type 1 and type 2 diabetes EGs divide the risk plane into 8 concentric zones with no discontinuities. The new EGs are similar to each other, but they differ from the traditional EG in several significant ways. When used to evaluate a data set of measurements made by a sample of patients experienced in SMBG, the new type 1 diabetes EG rated 98.6% of their measurements as clinically acceptable, compared with 95% for the traditional EG., Conclusions: The consensus EGs furnish a new tool for evaluating errors in the measurement of BG for patients with type 1 and type 2 diabetes.

Objective: To evaluate the effectiveness, comfort, and ease of use of insulin pen injections with a 31-gauge, 8-mm needle., Methods: In 50 study subjects (24 patients with type 1 insulin-dependent diabetes and 26 insulin-using patients with type 2 diabetes), we assessed the delivery of insulin, residual insulin leakage, glycemic control, plunger depression pressure, and perceived pain associated with the B-D 31-gauge, 8-mm pen needles in comparison with the B-D conventional 30-gauge, 8-mm pen needles, while the patient used their own insulin pens (Novo or B-D). The study subjects injected their usual dose of regular and NPH insulin using the 30-gauge, 8-mm needle during the first 3 weeks of the study. This period was followed by two 3-week crossover segments of the study with either needle assigned in random sequence., Results: No statistically significant differences were noted in glycemic control or perceived pain of injection between the two needles. The interaction between the two needles and the two insulin pen brands on glycemic control was not statistically significant. Plunger depression pressure increased with the increase in the gauge of the needle and with increases in size of dose of injected insulin (P<0.01). B-D pen users reported lower plunger pressure ratings in comparison with Novo pen users (P<0.01), regardless of the needle type and dose range. Both the insulin pen type and the needle type individually had statistically significant (P<0.01) effects on the residual insulin leakage from the needle tip after injection; however, their interaction was not statistically significant. Insulin doses greater than 30 units were associated with increased leakage (P<0.01). As needle retention time decreased, residual insulin leakage from the needle tip after injection increased (P<0.01), regardless of the needle used., Conclusion: The 31-gauge insulin pen needles are safe and effective for the delivery of insulin. With both 30-gauge and 31-gauge needles, attention to injection technique is essential to ensure complete delivery of insulin, particularly with administration of large doses.

Recently, the Diabetes Control and Complication Trial (DCCT) and other similar studies have demonstrated that near-normalization of blood glucose in diabetes will reduce complications up to 75% but translation of these results into practice has been difficult. In an attempt to help provide the best possible control of patients with diabetes, we have produced an attempt to help provide the best possible control of patients with diabetes, we have produced a new disease state management system for diabetes, called "Staged Diabetes Management" (SDM), implemented it in over 100 sites worldwide, and developed a computer program to simplify its use. SDM, designed to change the way we deal with patients with diabetes, is based upon five principles: (1) community involvement in setting care guidelines; (2) negotiation of goals with patients; (3) appropriate timelines for therapeutic success; (4) use of flowcharts for medical decisions; and (5) evaluation of the program. SDM is designed to be altered by a community to meet its needs and resources. It encourages primary care physicians to deliver better diabetes care using a team approach and to refer patients with diabetes to specialists when appropriate. It has a complete set of materials for communities, individual health care providers and patients. SDM has been tested for changes in structure, process and outcomes. A meta-analysis of seven clinical trials with over 500 patients has shown a time-weighted average fall in hemoglobin A1c of 1.7 points (equivalent to a drop in mean blood glucose of about 3.5 mM or 60 mg/dL). Preliminary pharmacoeconomic analysis demonstrates a lifetime cost saving of over $27,000 per patient. A computer program has been developed for the Microsoft Windows environment that contains a client-server database, based upon DiabCare, for the data file structure.

Insulin pens deliver insulin more slowly than syringes because of compressible elements of the insulin cartridges, especially air bubbles. The time required for a pen to deliver 20 Units of insulin increased with increasing air in the cartridge as determined by two independent techniques. The first technique involved no back pressure and was done by injection of [125I]-iodoinsulin onto absorbent paper on a constant angular velocity turntable. The second technique involved the normal back pressure of subcutaneous tissue and was done by robotic, timed injections of [125I]-iodoinsulin into full thickness pigskin. Air dramatically reduced the delivery of insulin in the five seconds that patients normally wait for injection by an insulin pen. Accumulation of more than 50 microliters of air results in a delivery of an unacceptably low percentage of insulin: with 200 microliters of air, a patient would get only 37% of the expected dose. Thus, a patient who injects 20 Units of insulin and withdraws the needle after the recommended 5 seconds would receive only 7.4 Units if there were 200 microliters of air in the cartridge. Since we found that 42 of 50 commercially available insulin cartridges contained air bubbles at purchase (average estimated to be 50 microliters), additional air entering the cartridge could lead to serious underdosing. Previous studies have demonstrated that air accumulates in insulin cartridges when the needle is left on the pen between injections. Therefore, for safety reasons, patients should be strongly advised to remove the needle immediately after each injection as recommended by the manufacturer.

This decade will bring major changes to the therapy of diabetes. New drugs are likely to include monomeric insulins, fatty-acid-oxidation inhibitors, insulin-secretion inducers, and nutrition modifiers. Likely new devices include improved insulin pens, less invasive methods of insulin administration, and noninvasive blood glucose monitoring. The use of computers will integrate this care, and artificial intelligence will provide new approaches to all of health care. An integrated system for using these new technologies, such as staged diabetes management, will ensure an orderly, cost-effective transition in therapy by the entire health-care community.

The insulin hypothesis of hypertension proposes that hyperinsulinemia increases sympathetic nerve activity (SNA) and raises arterial pressure. The goals of this study were 1) to determine if hyperinsulinemia produces regionally uniform or nonuniform increases in SNA and 2) to test the hypothesis that spontaneously hypertensive rats (SHR) have exaggerated sympathoadrenal responses to hyperinsulinemia. We measured plasma insulin, blood glucose, mean arterial pressure, and adrenal, renal, and lumbar SNA in alpha-chloralose-anesthetized SHR and normotensive Wistar-Kyoto (WKY) rats before and during infusion of two doses of insulin for 60 min each while maintaining euglycemia. In WKY rats, graded increases in plasma insulin from 27 +/- 5 (SE) to 200 +/- 29 microU/ml increased lumbar SNA from 100% to 285 +/- 26% but failed to significantly increase adrenal or renal SNA. In SHR rats, similar increases in plasma insulin from 27 +/- 4 to 213 +/- 33 microU/ml caused significant increases in adrenal (100% to 174 +/- 16%) and lumbar (100% to 307 +/- 26%) SNA but not in renal SNA. Despite increases in SNA, mean arterial pressure did not increase significantly in either group of rats. We conclude that 1) hyperinsulinemic euglycemic clamp produces regionally nonuniform increases in sympathetic nerve activity, and 2) there is a potentiated increase in adrenal SNA in SHR compared with WKY rats during hyperinsulinemia, whereas lumbar SNA responses were similar in the two strains, and renal SNA did not increase in either strain.

Self-measurement of blood glucose is an integral part of diabetes mellitus therapy. As many as 65% of diabetic people (4-5 million people) perform some degree of self-monitoring and approximately 20-30% do so frequently. Most patients consider this the most onerous part of their diabetes therapy. It requires obtaining blood, frequently in public, and is usually the most painful part of therapy, being significantly more painful than insulin self-administration. Patients therefore are anxious for a less-invasive method for glucose measurement. Methods exist or are being developed for minimally invasive glucose monitoring, which use body fluids other than blood (e.g., sweat and saliva), subcutaneous tissue, or blood measured less invasively. Sweat and saliva are relatively easily obtained but their glucose concentration lags significantly behind blood glucose. Methods to increase sweating have been developed and seem to increase the timeliness of the sweat glucose measurement. Subcutaneous glucose measurement seems to lag only a few minutes behind blood glucose and may actually be a better measurement of the critical values of glucose concentrations in brain, muscle, and other tissue. Glucose can be measured by noninvasive or minimally invasive methods, such as those making skin or mucous membranes permeable to glucose or those placing a reporter molecule in the subcutaneous tissue. Needle-type sensors have been improved in accuracy, size, and stability and can be placed into the subcutaneous tissue or peripheral veins to monitor blood glucose with miniature instruments.

Increases in membrane lipid unsaturation and drug-induced increases in membrane fluidity have been shown to be associated with increases in insulin receptor concentration in animals, cultured cells, and liposomes. In the current study, we have examined the effect of increased membrane fatty acid unsaturation on insulin action. Friend Erythroleukemia cells were grown with exogenous polyunsaturated fatty acids for three days. After growth in medium supplemented with fatty acids, the unsaturation index of the phospholipids increased from 1.08 to 1.92, and this was associated with a significant decrease in anisotropy, as measured by fluorescence polarization. When measured at 15 degrees C, insulin receptor number rose from 9000 to 22,000 per cell with increased fatty acid unsaturation. The affinity for insulin in the polyunsaturated fatty acid treated cells decreased, however, resulting in similar amounts of insulin binding at low insulin concentrations but more binding at high insulin concentrations when compared to control cells. In contrast, binding of IGF-I was not influenced by increased membrane fatty acid unsaturation. When measured at 37 degrees C there were no changes in binding of insulin or IGF-I. Internalization of insulin was identical in control cells and in cells with increased membrane fatty acid unsaturation. Thymidine incorporation, an insulin-dependent function in these cell, was measured in control and fatty acid treated cells. In control cells, insulin increased thymidine incorporation by 80%, with an ED50 of about 5 nM. In cells treated with polyunsaturated fatty acids, the insulin stimulated thymidine incorporation was slightly higher and the ED50 was about 0.2 nM. In contrast, there was no increase in the sensitivity or responsiveness of fatty acid treated cells to IGF-I. We conclude that increased membrane fatty acid unsaturation greatly influences insulin binding and biological sensitivity, but not that of IGF-I. At low insulin levels, there was a greater insulin bioeffectiveness, despite the same or lower insulin binding, suggesting more efficient coupling of the insulin-effector complex.

The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells (0.62 +/- 0.07 nmol/mg of protein versus 0.27 +/- 0.05 nmol/mg of protein, respectively). Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine (25 microM) decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine. The results show that an extracellular source of ethanolamine significantly influences the phospholipid metabolism of cultured bovine aortic endothelial cells.

We have developed a rapid method for producing homogeneous mono-[125I]A14 iodoinsulin with high specific activity and yield. After iodination by the lactoperoxidase method, the labeled peptides were applied to a C18 Porasil pre-column, washed with aqueous buffer to eliminate the free [125I]-iodide and placed "in-line" with a C-18 HPLC column; mono-[125I]A14 iodoinsulin was then eluted isocratically with 29% acetonitrile in 16 minutes. The labeled hormone was extremely stable, and proved suitable for various biological studies.

Human T-lymphocytes activated by phytohemagglutin acquire insulin receptors in culture. Saturation analysis of insulin-binding activity in the presence of competing ligand revealed curvilinear Scatchard plots. Insulin receptors were not regulated by insulin before mitogen activation and culture of T-lymphocytes. However, insulin-induced downregulation of insulin receptors was: (1) demonstrable in receptor-positive cells, (2) dependent on insulin concentration, (3) temporally unrelated to insulin internalization, and (4) prevented by culture at 4 degrees C but not by cycloheximide at 37 degrees C. Recovery of insulin receptors required further culture of cells in media depleted of insulin for 24 h. Scatchard analysis revealed loss of receptor number without changes in receptor affinity. Insulin-induced increases in glucose transport and oxidation were demonstrable in receptor-positive cells but not in receptor-negative cells. However, these effects were extremely time-dependent. After a 2-h exposure of cells to 10(-8) M insulin, increases in glucose transport were no longer demonstrable. Elution of bound insulin from these cells followed by re-exposure to insulin depressed glucose transport in them. Recovery from this hyporesponsive, desensitized state required a 6-h culture in insulin-depleted media. Glucose oxidation of desensitized cells could be stimulated by spermine but not by insulin. These studies demonstrate the activated human T-lymphocyte is an insulin-sensitive tissue that is capable of limiting its physiologic response to insulin by receptor- and postreceptor-mediated mechanisms.

Mono A14-[125I]-iodoinsulin was incubated with cultured endothelial cells derived from bovine pulmonary arteries at physiologic conditions. The processing of the cell-bound A14-[125I]-iodoinsulin was evaluated by trichloroacetic acid precipitation, gel filtration and high performance liquid chromatography. In contrast to insulin processing in many other cell types, approximately 95% of cell bound insulin was dissociated from the cells in less than 15 minutes, and biologically intact insulin rapidly passed through the endothelial cells. The unique location of endothelial cells coupled with the ability of rapid transport of intact insulin are consistent with an endothelial role for either the transport of insulin out of the bloodstream or as an extra-pancreatic storage area for insulin.

Octyl beta-glucoside (1%), a dialyzable detergent, was used to solubilize the insulin receptor of the turkey erythrocyte membrane. Insulin binding capacity was stable for at least 1 week when the receptor was kept in 1% octyl beta-glucoside at 4 degrees C. The binding properties of the solubilized receptor were examined at detergent concentrations above (1%) and below (0.6%) the critical micelle concentration. A reduction in insulin binding occurred when the detergent concentration was raised above the critical micelle concentration, due to an apparent decrease in the number of binding sites. The specificity of the receptor for insulin analogues was preserved, and the relative affinity of the solubilized receptor, desoctapeptide insulin greater than proinsulin greater than porcine insulin, was similar in 0.6% and 1% detergent. Addition of divalent cations increased insulin binding to a similar extent at both detergent concentrations, but there was a slightly greater stimulation of binding in 0.6% detergent as compared to 1% detergent. The pH optimum for binding was not affected by changes in the detergent concentration. These results indicate that the insulin receptor can be successfully solubilized by octyl beta-glucoside and that the binding activity is quite stable. Therefore, octyl beta-glucoside may be a useful detergent for purification of this receptor. In addition, the data indicate that the binding properties of the insulin receptor can be affected by changes in the physical state of the octyl beta-glucoside.

Previous studies using inhibitors have suggested that protein synthesis is necessary for "down-regulation" of insulin receptors. We have tested this hypothesis without the use of inhibitors by studying the ability of cells of the erythroid series to down-regulate their insulin receptors in vitro. The cells tested include mature erythrocytes and reticulocytes from rabbits and Friend erythroleukemia cells (a model for the basophilic erythroblast, a primitive nucleated erythrocyte). All cells were maintained at 37 degrees C for 18 hr +/- insulin (10(-8)M). Cultures were then incubated with phosphate buffered salines (pH 7.0) at 30 degrees C for 40 min to remove bound insulin. Receptors were quantitated by computerized analysis of Scatchard plots of subsequent insulin binding studies. Cells fully capable of both mRNA synthesis and protein synthesis, such as the undifferentiated and differentiated Friend erythroleukemia cell, had reduction of insulin receptors at 60% and 43%, respectively. Reticulocytes, which were capable of protein synthesis but not mRNA synthesis, had decreases of 25%-30% in 8 separate experiments. Mature erythrocytes, capable of neither RNA nor protein synthesis had no significant changes in receptor concentrations. Since mature erythrocytes do not "down-regulate" their insulin receptor concentration, studies of these receptors in erythrocytes of patients should be interpreted with caution.

CFIs have been implicated in the regulation of several inflammatory mediators; consequently, we have evaluated the effects of CFI on neutrophil chemotactic and lysosomal enzyme release response to C5-derived chemotaxins. Chemotaxis was measured by direct migration under agarose, LER by glucosaminidase release from cytochalasin B-treated neutrophils, and CFI activity by its inhibition of LER. After inactivation by CFI, C5-fr lost their ability to stimulate neutrophils, and acquired a new chemotactic inhibitory activity. On gel chromatography, the stimulatory activity of C5-fr and the inhibitory activity of inactivated C5-fr eluted as separate peaks with different molecular weights. Effects of CFI on neutrophil responses to C5-fr, ZAS, and ZAP were adverse and dose-dependent: maximal neutrophil response to C5-ft decreased in amplitude as CFI levels increased, and a close reciprocal relationship was demonstrated between the endogenous CFI and the chemotaxis activities of ZAS (r = -0.833) and ZAP (r = -0.932) in 10 healthy adults. The data suggest that CFI is a potent regulator of neutrophil response to C5-derived inflammatory mediators.

The detergent-solubilized avian insulin receptor retains negative cooperativity and other binding properties of the membrane bound form. On gel filtration the receptor elutes as a single peak with a Stokes radius of 72 A. Preincubation of the receptor with low levels of insulin leads to the formation of a second, smaller form with a Stokes radius of 40 A. The percent of receptor in this second peak is proportional to the insulin concentration and correlates well with the insulin-induced increase in dissociation rate (negative cooperativity). Both the isolated high molecular weight and the isolated low-molecular-weight forms of the receptor re-equilibrate in the presence of insulin and, upon refiltration of either isolated peak, both forms of the receptor are obtained. These results are compatible with a model of the insulin receptor in which a tetrameric form can dissociate to a monomeric form as a concomitant of negative cooperativity.

We have compared insulin binding, plasma membrane fluidity, and phospholipid composition of three different Friend erythroleukemia clones, a wild type (FLC) a mutant (R3) and the revertant to wild type F+. The R3 clone is a non-differentiating DMSO-resistant clone (R3) and has altered membrane fluidity and dramatically altered insulin-binding properties. The receptor of R3 bound insulin as if it possessed a single class of low affinity receptors that lacks the property of negative cooperativity. The Scatchard plot is linear and there is no ligand-induced acceleration of dissociation. The Hill coefficient for R3 is 1, implying 'no cooperativity', whereas the Hill coefficient for the two DMSO-inducible clones, (FLC and F+) is 0.3, implying 'negative cooperativity'. In addition, the insulin receptor of R3 has a decreased affinity for insulin, manifested as a 40-fold increase in the amount of insulin required to compete for half of the tracer binding (41 nM for R3 vs. 1 nM for FLC and F+). Computer-fitted Scatchard plots analyzed by the negative cooperativity model reveal that R3 has 95 000 receptor sites/cell, with a high affinity constant Ke of 0.016 nM-1, and a low affinity constant, Kf of 0.012 nM-1. Both DMSO-inducible clones have about 40 000 receptor sites/cell with Ke of 0.11 nM-1 and Kf of 0.02 nM-1. Electron spin resonance measurements with the 5-nitroxy stearate spin probe demonstrate that R3 had a more fluid plasma membrane than the FLC and F+ clones. The lipid composition of R3 is different from that of the DMSO-inducible clones. The weight ratio for unsaturated fatty acids to saturated fatty acids for R3 is 2.5, and the FLC clone has a lower ratio of 1.9. These results are consistent with our earlier findings in FLC that very high membrane fluidity is associated with alterations in the binding properties of the insulin receptor.

The Friend erythroleukemia cell has an insulin receptor with all the properties of mammalian insulin receptors: rapid, reversible, and saturable binding of insulin; specific for insulin and insulin analogs; inversely proportional to temperatures; sharply pH dependent (optimum = 8.0); and demonstrated ligand-induced accelerated dissociation consistent with negative cooperativity. There were 17,200 sites per cell. After induction by dimethylsulfoxide, 80% of the cells became benzidine positive (i.e., contained hemoglobin). The receptor concentration dropped to 4300 sites per cell, while the remaining receptors retained all the initial binding characteristics. This loss of receptors could not be attributed directly to either dimethylsulfoxide or changes in cell size. Thus, during the process of differentiation, the concentration of insulin receptors in the Friend erythroleukemia cell decreases.

Primary human T-lymphocytes that have been mitogen activated in chemically defined medium demonstrate cell surface receptor for insulin-like growth factor-II (IGF-II). In contrast resting T-lymphocytes demonstrate little or no IGF-II receptor. Receptors appear within 24 hours of mitogen activation with maximal binding occurring at 72 hours. After this point IGF-II binding declines. Receptor binding of IGF-II to T-lymphocytes does not show a sharp pH dependence but is maximal above pH 7. Insulin does not compete for IGF-II binding sites and proinsulin competes only weakly, suggesting that this is a type 2 IGF receptor and not an insulin receptor. Furthermore, anti-insulin antibodies do not inhibit IGF-II from binding to activated T-lymphocytes indicating divergent binding domains on the two peptide hormones. IGF-II demonstrates stimulating action on T-lymphocyte proliferation probably mediated by binding of IGF-II to this receptor.

Protamine is used widely to reverse the anticoagulant effects of heparin and to delay the absorption of insulin. Although adverse reactions to protamine are reported infrequently and are usually mild, we recently observed the first fatal case of type I anaphylaxis resulting from protamine. This patient had previously been sensitized to protamine during cardiac catheterization and had high levels of protamine-specific immunoglobulin E in the serum. In a prospective study, we found that 10 of 19 diabetic patients (53%) who had received insulin containing insulin also had high levels of antiprotamine immunoglobulin E. In contrast, none of 27 nondiabetic healthy normal controls or 10 diabetics who had never received protamine or protamine-containing insulin had levels of antiprotamine immunoglobulin E over background. This study underscores the risks of routinely administering protamine to susceptible individuals and the need for alternative therapies.

The insulin receptor was solubilized from turkey erythrocyte membranes by extraction with 1% beta-octylglucopyranoside. Insulin binding was enhanced when the solubilized material was reconstituted in phospholipid vesicles. The affinity of the reconstituted vesicles for various insulins was similar to that of the intact membranes: porcine insulin greater than proinsulin greater than desoctapeptide insulin. A curvilinear Scatchard plot was obtained for insulin binding to the reconstituted system at 15 degrees C. A high affinity association constant of 1.4 x 10(9) M-1 was obtained from the Scatchard plot. This is a four-fold increase over the value for the turkey erythrocyte membrane, which contains more highly saturated phospholipids. This suggests that the insulin receptor may be sensitive to the lipid composition of the membranes in which it is embedded.

The presence of a glucocorticoid receptor in human lymphocytes is well established, but factors affecting its regulation have not been described. Using a competitive binding whole cell assay, we have examined the binding of [3H]-dexamethasone at 24 and 37 degrees C in untreated normal subjects and in healthy subjects taking various glucocorticoid preparations. At 24 degrees C normal human lymphocytes had 6000 binding sites/cell and a dissociation constant of 4 x 10(-9) M. The administration of 1 mg of dexamethasone, 5 mg of prednisone, and 37.5 mg of cortisone acetate resulted in a 30% decrease in binding sites after 1 week with no change in binding affinity. No changes in the number of binding sites was noted before 1 week and the diminished number persisted for 1 week after discontinuation of glucocorticoid treatment. Lymphocytes from hospitalized patients taking 40-60 mg of dexamethasone daily demonstrated the same change in number of binding sites that was seen in normal subjects taking 1 mg of dexamethasone. When binding assays were carried out at physiologic temperature there was the same decrease in number of binding sites after dexamethasone administration, and in addition, there was a two-fold increase in binding affinity. Glucocorticoid administration results in a time-dependent decrease in the number of lymphocyte glucocorticoid binding sites that is independent of the type of glucocorticoid administered. This is the first in vivo demonstration that glucocorticoids modulate their own receptors in man.

The binding of insulin and insulin-like growth factor I (IGF-I) and their effect on amino acid and neurotransmitter transport was studied in cultured human Y79 retinoblastoma cells. Y79 cells possess specific receptors for both insulin and IGF-I. Insulin binding to Y79 cells is characterized by a curvilinear Scatchard plot suggesting a two-site or two-affinity binding system. In contrast, IGF-I binding has a linear plot indicative of a one-site, one-affinity binding system. The uptake of glycine, a putative neurotransmitter in the retina occurs by a specific transport system in Y79 cells, independent of the uptake of other neutral amino acids. The uptake of glycine was increased 25-50% by either insulin or IGF-I. The response to insulin or IGF-I on glycine uptake is gradual and concentration dependent. The accumulation of other amino acids and putative retinal neurotransmitters by Y79 cells was not significantly affected by insulin of IGF-I. In addition, the activity of Na+/K+-ATPase was not influenced. The analysis of high affinity glycine uptake indicates that insulin and IGF-I are stimulating glycine transport by increasing the V'max without significantly affecting the K'm. Further analysis suggests that insulin and IGF-I are causing a recruitment of additional glycine transporters at the cell surface or activating otherwise nonfunctional transporters by an unexplained mechanism. Because of the implication that glycine responds as a neuroactive amino acid in Y79 cells these studies suggest that insulin and IGF-I may influence neuroactivity in the human retina by regulating the transport of glycine.

The binding of iodinated cholera and Escherichia coli (LT) enterotoxins to Y1 mouse adrenal cells was studied by using saturation analysis (Scatchard). Each toxin bound to Y1 cells with similar affinity [KA = (1.5--2.0) x 10(9)M-1], but there appeared to be twice as many receptor sites per cell for E. coli toxin (approximately 4 x 10(5). Despite the increased binding of E. coli toxin, Y1 cells respond sooner to, and to smaller concentrations of, cholera toxin. The binding of each toxin was inhibited competitively by both toxins, although twice as much E. coli toxin was required to inhibit 50% of the binding of cholera toxin as was needed for either homologous inhibition or the inhibition of E. coli toxin binding by cholera toxin. The B subunits of both toxins were equally effective in competing for the binding of both iodinated toxins. Whereas the A subunits of both toxins had little or no effect on the binding of E. coli toxin, they consistently inhibited 20--40% of the binding of cholera toxin to cells. These results suggest that there are receptor loci on cells for the A subunit and that conformational differences exist between the two toxins that might explain the greater sensitivity of Y1 cells to cholera toxin. A model is suggested in which cholera toxin exhibits a greater degree of multivalent ligand binding than does the E coli toxin, resulting in a more favorable situation for apposition of the A subunit to its receptor or for its insertion into the membrane.