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.

AbstractWe studied whether the use of 8 mm insulin pen needles, shorter than the standard 12.7 mm needles, affects glucose control in obese and non‐obese people with type 1 or type 2 diabetes. A prospective, open‐label, nine‐week, crossover study involved 106 insulin pen‐using patients. During the initial period, all subjects used B‐D brand standard length needles. In the second period, subjects were randomised to either B‐D or NovoFine brand 8 mm pen needles. In the third period, subjects were crossed over to the alternative brand. Fructosamine levels were measured, leakage of insulin from injection sites was quantified, and pain was assessed. Glucose control was not altered in either obese or nonobese subgroups after switching to either brand of 8 mm needle. However, a few individuals, most of whom were obese, experienced a clinically significant increase in fructosamine levels after switching to an 8 mm needle. B‐D 8 mm needles were significantly less painful than either of the other needles tested. Obese subjects were significantly more likely to experience insulin leakage from their injection sites compared to non‐obese people, but the leakage was not significantly correlated with changes in fructosamine levels. Non‐obese people with type 1 or type 2 diabetes can switch to 8 mm insulin pen needles without compromising their glucose control and with less pain. Some obese patients who switch to 8 mm needles may have a higher risk of poorer glucose control, and should therefore consult with their healthcare professional. All patients should carefully monitor their blood glucose when changing to a shorter needle.

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.

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.

Abstract: Neuroblastoma cells were used to determine the effect of sorbinil on myo‐inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo‐inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo‐inositol uptake and in the incorporation of extracellular myo‐inositol into phospholipid. The effect of elevated glucose levels on myo‐inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo‐inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo‐inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.

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 effect of low serum concentration on plasma membrane fluidity and lipid composition, differentiation and insulin binding was investigated in three Friend erythroleukemia clones. Both FLC (clones No. 745) and F(+) (Ostertag F4N) Friend erythroleukemia cells can be induced to differentiate and to produce hemoglobin when exposed to DMSO. Clone R(3) (Ostertag F4-D5-1) is a DMSO-resistant clone when grown under normal conditions (15% serum) but could undergo differentiation with accumulation of protoporphyrin IX upon induction with DMSO when grown in low serum concentration (2.5% serum). Electron spin resonance measurements of the order parameters (S) and S(T parallel) demonstrate that R(3) has a more fluid plasma membrane than the FLC and F(+). The order parameters of the outer hyperfine splittings S(T parallel) at 37 degrees C are 0.60 +/- 0.009, 0.62 +/- 0.008 and 0.64 +/- 0.009 for R(3), F(+) and FLC, respectively. We have used the insulin receptors as a model for a polypeptide hormone receptor associated with the plasma membrane of the Friend clones. Insulin binding studies demonstrated that the receptor of R(3) had a decreased affinity for insulin manifest as a 10-fold increase in the amount of insulin required to compete for half of the tracer binding (18 nM for R(3) vs. 2 nM for FLC and F(+)). Computer-fit Scatchard plot analysis by the negative cooperativity model reveal that R(3) possessed a similar number of sites/cell (about 70,000) as the FLC or F(+) cells, with similar high and low affinities. Growing the DMSO-resistant clone R(3) in low serum concentration caused a decrease in receptor number by 35%, and an increase in receptor affinity to that seen with the differentiable clones. Thus, the abnormal properties of the plasma membrane and insulin receptor of the DMSO-resistant clone in our earlier report (Simon et al. (1984) Biochim. Biophys. Acta 803, 39-47) were partially reversed by growing the cells in a low serum concentration, restoring the cellular response to the differentiation agent.

Abstract: Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo‐inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V1max for inositol uptake was significantly decreased with little change in the K1max. These data suggest that glucose decreases myo‐inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo‐inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo‐inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.