Proliferative effects of insulin analogues on mammary epithelial cells

Structural modification of insulin results in the generation of insulin analogues that show altered binding affinities to the insulin receptor and/or IGF-I receptor. As a consequence these insulin analogues may have increased mitogenic potency. Nine benign or malignant human mammary epithelial cells, which show different insulin receptor and IGF-I receptor expression patterns, were studied regarding mitogenicity of insulin and insulin analogues. Only insulin glargine showed a significantly higher proliferative effect on MCF-7 breast cancer cells compared to regular insulin among a panel of short- or long-acting insulin analogues, that are in clinical use.     

Engineering of insulin receptor isoform-selective insulin analogues

Background

The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues.

Methodology/Principal Findings

Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity.

Conclusions/Significance

We have discovered a new class of IR isoform-selective insulin analogues with 2–4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.     

Photoreactive insulin analogues used to characterise the insulin receptor

Three photoreactive insulin analogues (“photoprobes”) have been prepared in which an aryl azide group was substituted at either the A1, B1 or B29 positions of the insulin molecule. When incubated with rat liver plasma membranes and irradiated all three photoprobes covalently labelled specific insulin binding sites within the membrane. SDS-polyacrylamide gel electrophoresis of plasma membranes covalently tagged with either of the three ¹²⁵I-photoprobes resolved one major specifically labelled polypeptide with an apparent molecular weight of 130,000. The labelled polypeptide migrated anomalously in SDS-polyacrylamide gels and a molecular weight of 90,000 for the polypeptide was determined from a ‘Ferguson’ plot using the combined results from gels of different acrylamide concentrations. Column chromatography of detergent solubilised photoprobe-labelled membranes indicated that the labelled polypeptide may be a subunit of a larger protein complex.     

Experience with insulin analogues in children

Current data on rapid and long-acting insulin analogues in the paediatric age group is limited. While several studies indicate a benefit in reducing hypoglycaemia, particularly at night, with rapid or long-acting insulin analogue treatment, the effect on long-term glycaemic control remains controversial. The continuous glucose monitoring system offers a new option for tailoring treatment with insulin analogues to achieve optimal glycaemia. In 29 adolescents with diabetes this approach confirmed the non-inferiority of postprandial rapid-acting analogue administration compared to preprandial regular insulin, but revealed significant mealtime differences, with increased analogue requirement at breakfast and dinner. Although rapid- and long-acting insulin analogues may offer potential benefits for problems frequently encountered in paediatric diabetology, their value for the individual child still has to be tested in long-term observations in daily clinical practice.     

Insulin, insulin analogues and diabetic retinopathy

Insulin is absolutely vital for living beings. It is not only involved in metabolism, but also in the regulation of growth factors, e.g. IGF-1. In this review we address the role insulin has in the natural evolution of diabetic retinopathy. On the one hand, chronic deficiency of insulin and IGF-1 at the retina is thought to cause capillary degeneration, with subsequent ischaemia. On the other hand, acute abundance of (exogenously administered) insulin and IGF-1 enhances ischaemia-induced VEGF expression. A critical ratio of tissue VEGF-susceptibility: VEGF-availability triggers vascular proliferation (i.e. of micro-aneurysms and/or abnormal vessels). The patent-protected insulin analogues Lispro, Glulisine, Aspart, Glargine and Detemir are artificial insulin derivatives with altered biological responses compared to natural insulin (e.g. divergent insulin and /or IGF-1 receptor-binding characteristics, signalling patterns, and mitogenicity). Their safety profiles concerning diabetic retinopathy remain to be established by randomised controlled trials. Anecdotal reports and circumstantial evidence suggest that Lispro and Glargine might worsen diabetic retinopathy.     

Cost-effectiveness of insulin analogues for diabetes mellitus

Background

Insulin analogues may be associated with fewer episodes of hypoglycemia than conventional insulins. However, they are costly alternatives. We compared the cost-effectiveness of insulin analogues and conventional insulins used to treat type 1 and type 2 diabetes mellitus in adults.

Methods

We conducted a cost-effectiveness evaluation of insulin analogues versus conventional insulins using the Center for Outcomes Research Diabetes Model. We compared rapid-acting analogues (insulin aspart and insulin lispro) with regular human insulin, and long-acting analogues (insulin glargine and insulin detemir) with neutral protamine Hagedorn insulin. We derived clinical information for the comparisons from meta-analyses of randomized controlled trials. We obtained cost and utility estimates from published sources. We performed sensitivity analyses to test the robustness of our results.

Results

For type 1 diabetes, insulin aspart was more effective and less costly than regular human insulin. Insulin lispro was associated with an incremental cost of Can$28 996 per quality-adjusted life-year. The incremental cost per quality-adjusted life-year was Can$87 932 for insulin glargine and Can$387 729 for insulin detemir, compared with neutral protamine Hagedorn insulin. For type 2 diabetes, insulin aspart was associated with an incremental cost of Can$22 488 per quality-adjusted life-year compared with regular human insulin. For insulin lispro, the incremental cost was Can$130 865. Compared with neutral protamine Hagedorn insulin, insulin detemir was less effective and more costly. Insulin glargine was associated with an incremental cost of Can$642 994 per quality-adjusted life-year. The model was sensitive to changes in the effect size of hemoglobin A_1c and to decrements applied to utility scores when fear of hypoglycemia was included as a factor.

Interpretation

The cost-effectiveness of insulin analogues depends on the type of insulin analogue and whether the patient receiving the treatment has type 1 or type 2 diabetes. With the exception of rapid-acting insulin analogues in type 1 diabetes, routine use of insulin analogues, especially long-acting analogues in type 2 diabetes, is unlikely to represent an efficient use of finite health care resources.Insulin agents available for the treatment of diabetes mellitus include conventional insulins and insulin analogues. Insulin analogues were developed to mimic more closely the separate bolus and basal components of insulin secretion.¹ Rapid-acting (bolus or mealtime) and long-acting (basal or background) analogue formulations are available. This new class of drugs has been promoted as providing more flexible treatment schedules and a reduced risk of hypoglycemia relative to conventional insulins.¹The cost of insulin analogues exceeds that of conventional insulins.^2,3 More than US$7.3 billion was spent globally on the purchase of insulin products in 2005 — an increase of 19% over the previous year.⁴ It has been suggested that the increased expenditure was due to both the increasing prevalence of diabetes and the increased use of insulin analogues.⁵We performed an analysis of the cost-effectiveness of insulin analogues compared with conventional insulins in the management of type 1 or type 2 diabetes in adults.     

Studies on the effects of protease substrate analogues on some of the actions of insulin

Added TAME (N alpha-p-tosyl-1-anginine methyl ester) or BAME (benzoyl-anginine methyl ester) inhibited insulin induced activation of glucose oxidation and fat cell PDH activation without affecting spermine action on PDH activation and glucose oxidation in fat cells. BAME inhibited insulin-induced generation of both PDH stimulator and PDH inhibitor from liver particulate fraction. In contrast, insulin-induced internalization of insulin receptors and negative cooperativity of insulin receptors were not affected by protease substrate inhibitors. These results suggest that certain actions of insulin (glucose oxidation, generation of PDH regulators) are mediated by proteolytic events, while insulin-induced down regulation and negative cooperativity of insulin receptors are not mediated by activation of endogenous proteases.     

Genetically engineered insulin and its pharmaceutical analogues

Studies of replacement therapy of diabetes mellitus resulted not only in introduction of series of forms of insulin available at pharmaceutical market but also in new insulin analogues, which exhibit better control of blood glucose level. The present paper deals with basic tendencies in this field.     

Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.     

Superiority of insulin analogues versus human insulin in the treatment of diabetes mellitus

The modern goals of insulin replacement in Type 1 and Type 2 diabetes mellitus (T1, T2DM) are A1C <6.5% long-term, and prevention of hypoglycaemia (blood glucose, BG <70 mg/dl). In addition to appropriate education and motivation of diabetic subjects, the use of rapid- and long-acting insulin analogues, is critical to achieve these goals. The benefits of rapid-acting analogues (lispro, aspart and glulisine have similar pharmacodynamic effects) compared with non-modified human regular insulin, are: (a) lower 1- and 2-h post-prandial blood glucose; (b) lower risk of late post-prandial hypoglycaemia (and therefore lower BG variability); (c) better quality of life (greater flexibility in timing and dosing of insulin). In T1DM, rapid-acting analogues improve A1C only by the extent to which replacement of basal insulin is optimized at the same time, either by multiple daily NPH administrations, or continuous subcutaneous insulin infusion (CSII), or use of the long-acting insulin analogues glargine or detemir. In T2DM, rapid-acting analogues reduce post-prandial hyperglycaemia more than human regular insulin, but systematic studies are needed to examine the effects on A1C. The benefits of long-acting insulin analogues glargine and detemir vs. NPH, are: (1) lower fasting BG combined with lower risk of hypoglycaemia in the interprandial state (night); (2) lower variability of BG. Glargine and detemir differ in terms of potency and duration of action. Detemir should be given twice daily in the large majority of people with T1DM, and in a large percentage of subjects with T2DM as well, usually at doses greater vs those of the once daily glargine. However, when used appropriately for individual pharmacokinetics and pharmacodynamics, glargine and detemir result into similar effects on BG, risk of hypoglycaemia and A1C. Rapid- and long-acting insulin analogues should always be combined in the treatment of T1 and T2DM.     

The role of rapid-acting insulin analogues and inhaled insulin in type 2 diabetes mellitus

sBackground:The availability of rapid-acting insulin analogues and inhaled insulin gives clinicians additional treatmentoptions in the management of patients with diabetes mellitus (DM). Combining rapid-acting insulin analogues with basal insulin can more closely mimic physiologic insulin release to maximize glycemic control.Objective:The objective of this article was to discuss the role of rapid-acting insulin analogues and inhaled insulin inthe treatment of patients with type 2 DM.Methods:Materials for this article were obtained through an online search of MEDLINE/PubMed and Google(1996-2006) using the search terms bolus insulin, postprandial, rapid-acting insulin analogues, titration, hypoglycemia, glycemic control, inhaled insulin, and insulins lispro, aspart, and glulisine.Results:Glycosylated hemoglobin (A1C) levels and number of all hypoglycemic episodes were similar in patients withtype 2 DM taking either mealtime rapid-acting insulin analogues or regular human insulin (RHI). Rapid-acting insulins have been successfully used in basal-bolus regimens with a variety of long- and intermediate-acting insulins, as well as with oral hypoglycemic agents. Injectable rapid-acting insulin analogues markedly decreased postprandial glucose (PPG) levels compared with RHI. Better reduction in PPG levels may be key to achieving target A1C levels in some patients, but long-term outcome studies are needed to assess whether lowering PPG levels decreases cardiovascular risk in patients with type 2 DM. Inhaled insulin may be an option for patients who cannot inject insulin, but route of administration and dosing issues limit its use in many patients. The effect of inhaled insulin on PPG is unclear at this time.Conclusions:Although rapid-acting insulin analogues are effective in the management of patients with type 2 DM, the limited numbers of studies have yet to demonstrate that these agents have any significant long-term advantage compared with RHI. In addition, they cost more than RHI. Further studies are needed to compare the efficacy of the rapid-acting insulin analogues, to compare the different dosing regimens used with mealtime insulin administration, and to ascertain if the decrease in PPG levels seen with the use of rapid-acting insulin analogues translates into improved glycemic control and perhaps even a reduction in cardiovascular risk in patients with type 2 DM. (Insulin. 2007;2:61-67) Copyright 2007 Excerpta Medica, Inc.     

Novel use of fluorescent glucose analogues to identify a new class of triazine-based insulin mimetics possessing useful secondary effects

There is an urgent need to discover new compounds that effectively treat diabetes by mimicking the action of insulin (insulin mimetics). Traditional approaches to studying anti-diabetic agents in cells are inconvenient for screening chemical libraries to identify insulin mimetics. 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 6-NBDG are fluorescent analogues of glucose that could be employed in screening. However, there are no published data about the use of these analogues to identify new insulin mimetics. We have developed a screening system based on 6-NBDG using 3T3-L1 adipocytes in a 96-well culture plate format. 6-NBDG was found to produce a larger signal than 2-NBDG in this screening system. 6-NBDG uptake in 3T3-L1 adipocytes was sensitive to insulin, known insulin mimetics, inhibitors of glucose transport and insulin-sensitizing compounds. To validate our screening system, a chemical library of 576 tagged, triazine-based small molecules was screened. The screening results were identical to that obtained from a commercial enzyme-based glucose assay. Two inducers of glucose uptake were shown to be non-cytotoxic and confirmed as insulin mimetic compounds by their inhibition of epinephrine-stimulated free fatty acid release from adipocytes. These novel insulin mimetics functioned at a markedly lower concentration than two widely studied insulin mimetics, zinc(ii) complexes and vanadium compounds, and also showed novel, beneficial effects on endothelial cell function (a key determinant of secondary complications in diabetes). The discovery of new insulin mimetics using 6-NBDG validates the use of this probe in the development of large-scale, cell-based screening systems based on the uptake of fluorescent-tagged glucose analogues. This research should aid the development of novel strategies to discover new drugs and drug targets for combating the increasing prevalence of diabetes.     

Rapid preparation of human insulin and insulin analogues in high yield by enzyme-assisted semi-synthesis.

Reaction conditions are described that permit the enzyme-assisted semi-synthetic replacement of residue B30 of pig insulin (or of analogue) to proceed in very high yield in 2 h or less. Immobilized trypsin may be used as catalyst, and excess amino acid ester may be recycled after a simple extraction. Alanine-B30 may be replaced by a variety of nucleophiles, including threonine O-t-butyl ether t-butyl ester, in which case the yield of crude product is about 99%. De-protection of the B30 threonyl ester analogue of insulin thus formed then affords human insulin in an overall yield of about 92%, based on pig starting material. The product has full biological potency, as determined by depression of blood glucose concentration in rats, and showed the expected behaviour on radioimmunoassay.     

Synthesis and biological activity of seventeen analogues of human insulin

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.     

Two carbocyclic locked nucleic acid analogues give structural information about the role of hydration in A-type duplexes

Two locked nucleic acid (LNA) analogues with three-carbon 2'-4' linkages, saturated or unsaturated, are synthesized using a ring-closing metathesis based strategy. Strongly stabilized duplexes with complementary RNA and slightly destabilized duplexes with complementary DNA are observed. CD-spectroscopy indicates a less pronounced shift toward A-type duplexes compared to LNA. These results combining a strong N-type conformation with the absence of a 2'-oxygen demonstrate a stronger importance of minor groove hydration in an intermediate duplex type than in an A-type duplex.