In Vitro and In Vivo Potency of Insulin Analogues Designed for Clinical Use

Vølund, Aage; Brange, Jens; Drejer, Kirsten; Jensen, I.; Markussen, J.; Ribel, Ulla; Sørensen, Annette; Schlichtkrull, J.

doi:10.1111/j.1464-5491.1991.tb02122.x

Cited by 70 publications

(38 citation statements)

References 24 publications

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“…However these mutations did result in shifts of the concentration dependence of insulin-regulated receptor autophosphorylation, which correlated reasonably well with the changes in affinity for insulin. This correlation is consistent with the results of studies of the biological activities of insulin analogues that bind to this site, in which it was found that changes in potency correlated with changes in affinity for the receptor (35).…”

Section: Resultssupporting

confidence: 80%

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Journal of Biological Chemistry

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Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the ␣ subunit (amino acids [705][706][707][708][709][710][711][712][713][714][715] , and Phe 89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC 50 commensurate with their effect on the affinity of the receptor for insulin.The initiating event in the insulin-signaling cascade is the binding of insulin to a specific plasma membrane receptor. This interaction has been studied extensively and was found to be extremely complex (see Ref. 1 for review). Scatchard plots (2) of equilibrium binding data are concave and curvilinear, suggesting heterogeneity of ligand binding sites, negatively cooperative site-site interactions, or a combination of both (1). These properties and high affinity interactions with insulin are dependent on the dimeric structure of the receptor; insulin binds non-cooperatively to a single population of binding sites in the monomeric receptor (3-5). The stoichiometry of binding to the native receptor appears to be one insulin molecule to one receptor dimer (6). The secreted recombinant extracellular domain of the receptor exhibits properties similar to those of the receptor monomer and has a stoichiometry of two insulin molecules to one receptor dimer (6, 7).A number of hypothetical models of insulin-receptor interactions have been proposed to explain these findings (7-9). The model that best explains the experimental findings is that of De Meyts (1). This proposes that insulin has two topographically distinct receptor binding sites and that the receptor has two topographically distinct insulin binding sites/monomer. In this model, insulin binds asymmetrically to the insulin dimer, with each of its binding sites contacting its cognate receptor site on a separate monomer. Although the detailed structural basis of this model has yet to be elucidated, the structure-function relationships of the insulin molecule and the receptor have been studied extensively and provide support for the essentials of the model.Insulin (1, 10). However, hagfish insulin, in which these residues and the tertiary structure of the molecule are conserved (11), displays anomalous receptor binding behavior (11,12), suggesting that other residues outside this region might also be involved in receptor interactions. This is supported by the finding that two recombinant insulin analogues with substitutions in residues located on the opposite side of the molecule, Leu A13 to Ser and Leu B17 to Gln, exhibited similar receptor binding behavior to hagfish insulin (7,8).The structure and structure-function relationships of the insulin receptor are not as well documented. It is a dimeric transmembrane protein (see Ref. 1 for review). Each monomer consists of disulfide-linked ␣ and ␤ subunits. The ␣ subunits are wholly extracellular and c...

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Section: Resultssupporting

confidence: 80%

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Journal of Biological Chemistry

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“…This is in contrast to the high correlation between insulin receptor binding and glucose transport (29) and lipogenesis (30 -34). This is true for the analogues used here (29,31,(33)(34)(35) and other insulin analogues (29 -35). We also found that glucose incorporation into glycogen in L6 cells and H4 cells correlate with receptor binding.…”

Section: Insulin Analogues and Protein Degradationmentioning

confidence: 84%

Insulin and Analogue Effects on Protein Degradation in Different Cell Types

Fawcett¹,

Hamel²,

Bennett³

et al. 2001

Journal of Biological Chemistry

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In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activity in vitro and in vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (Lys B28 Despite similar binding, LysPro was 11-to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.,The major effect of insulin on whole body protein turnover is inhibition of protein degradation (1). Insulin also stimulates the synthesis of selected proteins and overall protein synthesis under certain conditions such as growth and development (2, 3). In the adult animal, however, total protein synthesis is not increased by insulin, and the protein anabolic effect of the hormone is actually an anti-catabolic property, i.e. inhibition of degradation (1).The mechanisms of cellular protein degradation and the control of these processes are poorly understood. Currently, the major degradative systems are considered to be the lysosome, the proteasome, and various cytoplasmic and cellular proteases such as the calpain family and a recently described giant protease (4 -7), which may functionally overlap proteasome activities. In general, lysosomes appear responsible for degradative processing of most endocytosed proteins and for autophagy under extreme catabolic states (4). The proteasome is a multifunctional organelle with multiple forms, e.g. 20 S and 26 S, which degrades abnormal and targeted proteins (ubiquitin, ATP-dependent pathway) and has various specific functions (antigen processing, etc.) (5). Calpains participate in Ca 2ϩ -mediated degradative activity (6). Various other proteases have specialized or uncertain roles but have a relatively minor contribution to cellular protein balance.Overall, cellular protein degradation can also be divided according to the half-lives of the proteins. This is particularly relevant, because many of ...

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“…These authors could prevent diabetes in NOD mice by treating with a B25Asp insulin analogue, which binds very poorly to the insulin receptor [24], but preserves the sequences targeted by autoimmune T cells. Given that the B25Asp analogue is metabolically inactive, its efficacy was considered a proof of immune protective mechanisms being at play.…”

Section: Discussionmentioning

confidence: 99%

Short‐term subcutaneous insulin treatment delays but does not prevent diabetes in NOD mice

2012

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Despite encouraging results in the NOD mouse, type 1 diabetes prevention trials using subcutaneous insulin have been unsuccessful. To explain these discrepancies, 3-weekold NOD mice were treated for 7 weeks with subcutaneous insulin at two different doses: a high dose (0.5 U/mouse) used in previous mouse studies; and a low dose (0.005 U/mouse) equivalent to that used in human trials. Effects on insulitis and diabetes were monitored along with immune and metabolic modifications. Low-dose insulin did not have any effect on disease incidence. High-dose treatment delayed but did not prevent diabetes, with reduced insulitis reappearing once insulin discontinued. This effect was not associated with significant immune changes in islet infiltrates, either in terms of cell composition or frequency and IFN-γ secretion of islet-reactive CD8 + T cells recognizing the immunodominant epitopes insulin B 15-23 and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) [206][207][208][209][210][211][212][213][214] . Delayed diabetes and insulitis were associated with lower blood glucose and endogenous C-peptide levels, which rapidly returned to normal upon treatment discontinuation. In conclusion, high-but not low-dose prophylactic insulin treatment delays diabetes onset and is associated with metabolic changes suggestive of β-cell "rest" which do not persist beyond treatment. These findings have important implications for designing insulin-based prevention trials.Keywords: Insulin r NOD mouse r Prevention r T cells r Type 1 diabetes IntroductionType 1 diabetes (T1D) is an autoimmune disease in which failures in central and peripheral tolerance mechanisms lead to T-cellmediated destruction of insulin-producing β cells. Several β-cell Ags have been identified, among which insulin and its precursor (pre)proinsulin ((pre)PI) are major targets of islet-reactive T cells, both in humans [1] and in NOD mice [2]. This led to the Correspondence: Dr. Roberto Mallone e-mail: roberto.mallone@inserm.fr hypothesis that subcutaneous insulin administration could prevent disease, the rationale of which is twofold. First, insulin administration could induce a state of "β-cell rest" that would make β cells less vulnerable to metabolic stress, apoptosis [3], and possibly to immune-mediated destruction [4]. Second, repeated subcutaneous insulin injections could act as a vaccination protocol potentially restoring immune tolerance [5]. The relative role of these two putative mechanisms remains unsettled.Studies in the NOD mouse showed that subcutaneously administered insulin, started prior to disease development, is capable of preventing insulitis and diabetes [6]. This observation prompted the Diabetes Prevention Trial-Type 1 (DPT-1) [7] Eur. J. Immunol. 2012. 42: 1553-1561 developing disease [8]. However, both trials were unsuccessful, as no difference in T1D incidence was observed between insulinand placebo-treated individuals [7,8].It is still not clear why this translation from animal studies to human clinical trials fail...

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In Vitro and In Vivo Potency of Insulin Analogues Designed for Clinical Use

Cited by 70 publications

References 24 publications

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Insulin and Analogue Effects on Protein Degradation in Different Cell Types

Short‐term subcutaneous insulin treatment delays but does not prevent diabetes in NOD mice

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