Introduction
Insulin analogues in the treatment of patients with type 1 or type 2 diabetes have been shown to be more efficient, reproducible and convenient than regular insulin [
1]. Due to either sequence or secondary structural modifications, analogues may differ from insulin with respect to metabolic potency, stability, or onset and duration of action. Although these changes were introduced to alter the time–action profile of the respective insulin analogues, they may also lead to an altered activation profile of the insulin receptor (IR) and (or) IGF-1 receptor (IGF1R) signalling pathways, and may change metabolic or mitogenic responses [
2]. A careful investigation of acute and long-term effects of insulin analogues has been a major research focus.
The insulin analogue insulin aspart B10 (B10Asp human insulin) (AspB10) was withdrawn from clinical development due to a higher incidence of breast cancer in rats [
3]. AspB10 differs from human insulin by the substitution of histidine by aspartate in position 10 of the B chain. In vitro, AspB10 displays higher affinity towards the IR and IGF1R, a prolonged occupancy time at the IR and a higher proliferation rate in mammalian cell lines. Although the mechanism by which AspB10 exerts its mitogenic effect is not clear, it is still contended that the analogue’s greater affinity to the IGF1R might be at least in part responsible [
4‐
7]. This has led to the general belief that insulin analogues with increased IGF1R affinity in vitro might per se exert an increased growth-promoting activity in vivo.
However, in vitro studies cannot be directly applied to the in vivo situation. First, the affinity of the endogenous ligand IGF-1 for IGF1R is at least 100-fold greater than that of insulin or insulin analogues. Thus, IGF-1 competes with insulin for IGF1R occupation. Second, in vitro studies use supraphysiological (nanomolar) concentrations vs picomolar concentrations in vivo. Third, the plasma and tissue concentration and distribution of insulin analogues are different in vivo than in vitro. Fourth, insulin analogues undergo biotransformation in vivo, necessitating analysis of the active metabolites.
Insulin glargine (A21Gly,B31Arg,B32Arg human insulin) is a long-acting human insulin analogue with an activity profile very closely mimicking the natural physiological profile of basal endogenous insulin secretion. Glargine differs from human insulin by substitution of asparagine by glycine in position 21 of the A chain and by carboxy-terminal extension of the B chain by two arginine residues. These changes cause a shift in the isoelectric point from pH 5.4 to 6.7. Following s.c. administration as a clear solution of pH 4, insulin glargine precipitates at the injection site because of its low solubility at physiological pH levels. The prolonged blood glucose-lowering activity of insulin glargine may result from the subsequent slow dissolution of the microprecipitate on the basis of a low dissociation rate. The dissolution process is followed by rapid proteolytic degradation of parent glargine, leading to soluble metabolites as demonstrated in metabolism studies in humans, rats and dogs [
8‐
10]. The two main metabolites of insulin glargine, M1 ([GlyA21]insulin) and M2 ([GlyA21,des-ThrB30]insulin) are formed by the sequential removal of the two arginines from the carboxy-terminus of the B chain and additional deamination of threonine in position B30. In plasma, the principal circulating compound is the metabolite M1, the exposure of which increases as the dose of administered insulin glargine increases [
11].
Insulin glargine has an in vitro IR signalling and metabolic profile comparable to that of human insulin, but displays a slightly greater IGF1R affinity in vitro [
4,
5,
7]. However, in 2 year carcinogenicity studies, no difference was observed in the incidence of mammary tumours in mice and rats compared with controls or animals treated with NPH insulin [
12], a finding that can be attributed to the pharmacodynamic effect of M1, which has in vitro metabolic and mitogenic profiles comparable with human insulin [
7].
The aim of this study was to analyse the time–action profile of glargine in responsive tissues of rats with respect to pharmacological and signalling variables and to compare that profile to those of human insulin and AspB10, using therapeutic as well as suprapharmacological doses. We also investigated the effect of human insulin, glargine and AspB10 on the phosphorylation of IGF1R and compared it with the effect of IGF-1.
Discussion
Given that patients with diabetes often require life-long insulin treatment, it is essential to examine all steps in the action of an insulin analogue in vitro and in vivo, to exclude unwanted effects like growth-promoting activities [
15]. Malignant cell growth is often associated with aberrant signalling of both IR isoforms (IR-A and IR-B) and IGF1R. The insulin and IGF receptors trigger a complex range of intracellular signals for metabolism, cell growth and proliferation [
16]. Their relative abundance affects intracellular signalling and has important consequences for tissue-specific responses to insulin, IGFs and insulin analogues [
17,
18]. In addition, it has been demonstrated that overexpression of IR and IGF1R in human breast carcinomas allows insulin and IGF-1 hybrid receptors to form. These hybrid receptors become tyrosine autophosphorylated when breast cancer cells are exposed to IGF-1, but not to insulin, and also mediate growth in response to IGF-1 [
19‐
22].
As cancer cells have aberrant IR and IGF1R signalling patterns, it is important to understand how insulin analogues affect normal and cancerous cells, as this has implications for diabetes, cancer and cancer treatment. It is generally believed that insulin analogues with a higher affinity than human insulin for IGF1R in vitro have greater mitogenic activity in vivo. This belief is based on AspB10, the only insulin analogue with proven carcinogenicity in rats [
3]. Insulin glargine is also thought to have greater mitogenic activity in vivo, based on its slightly higher affinity for IGF1R in vitro, but unlike AspB10, glargine does not have greater affinity for the IR or a prolonged occupancy time at the IR [
4,
5,
7].
It is, however, difficult to predict results in vivo on the basis of in vitro data [
23], and in vitro studies do not conclusively support IGFR activation as the mechanism of increased mitogenic activity. The mitogenic action of insulin glargine is increased in certain cell lines with high IGFR:IR ratios [
6,
7,
24‐
28], but other cell lines with high IGFR:IR ratios do not respond to insulin glargine treatment with increased proliferation [
4,
5,
7,
24,
25,
29‐
34]. Moreover, some cell lines respond to AspB10, but not to other analogues with increased proliferation [
35], while cell lines that do not have a greater affinity for IGFR have also been reported to show increased proliferation upon exposure to other insulin analogues currently used in therapy [
27,
28,
36].
One way of examining mitogenic activity in vivo is to directly determine tumour formation after chronic treatment with insulin and (or) insulin analogues. AspB10 was withdrawn from clinical development due to a higher incidence of breast cancer in rats after 12 months [
3]. In contrast, insulin glargine did not induce a higher incidence of mammary tumours in lifetime carcinogenic studies in female rats and mice [
37], confirming that this basal insulin analogue is unlikely to pose a cancer risk in humans. Moreover, in a mouse model prone to tumour formation, Nagel et al [
38] demonstrated that tumour formation did not increase with insulin glargine vs NPH insulin treatment after 18 months. The balance of evidence indicates that no commercially available insulin analogue has carcinogenic effects in the human clinical setting [
10]. The approach presented here was to examine the time course of phosphorylation of the IR and IGF1R, and the effects on downstream signalling molecules of insulin and insulin analogues in different tissues in rats. Previous studies have been performed in mice [
39,
40]. The one rat study that has been reported to date used suprapharmacological doses and only investigated downstream signalling [
17].
Blood glucose levels dropped immediately after the injection of human insulin or AspB10, with no difference between the two insulins. In contrast, glucose lowering was delayed with insulin glargine, as expected for a long-acting insulin analogue, where the mode of action involves precipitation and subsequent slow release from the tissue depot [
41]. As in humans [
11,
42], the lowering of blood glucose could be correlated to the biotransformation of insulin glargine into the M1 metabolite, which lacks the di-arginine residues [
8]. Glargine parent and M2 were not detectable. Consequently M1, and not glargine itself, is the primary driver of the pharmacodynamic effect and the long-acting time–action profile observed with insulin glargine treatment [
11,
42]. The proteases responsible for this activity appear to be independent of the species investigated [
43]. M1 is the major active metabolite even at a dose of 200 U/kg, suggesting that the protease system involved has a high capacity. However, at this high dose, glargine can be found in the circulation, indicating that saturation of the system can occur.
Peak IR and Akt phosphorylation levels induced by insulin glargine were generally comparable with those achieved with human insulin, although in some tissues the effects of insulin glargine were delayed and (or) prolonged in time. Similar differences have been described by Agouni et al [
39], possibly reflecting differences in pharmacokinetic and/or pharmacodynamic properties across tissues. The comparable peak phosphorylation of insulin glargine vs human insulin reflects the comparable activity of M1 vs human insulin and supports the conclusion that insulin glargine behaves like human insulin in terms of signalling. In contrast, IR and Akt phosphorylation was increased and prolonged with AspB10, most strikingly in muscle and liver. These results are compatible with the greater affinity of the IR for AspB10 and with the prolonged signalling of the IR when exposed to AspB10 in vitro [
6,
7]. Interestingly, at higher doses (12.5 and 200 U/kg), the differences in peak phosphorylation of the IR and Akt observed between AspB10, glargine and insulin were no longer detectable, apparently demonstrating the saturation of peak phosphorylation under these high-dose conditions. Hvid et al have reported that 100 U/kg s.c. resulted in comparable peak phosphorylation of Akt over 150 min [
17,
18]. Consequently, a therapeutic dose of 1 U/kg seems to reflect differences in the affinity of AspB10, insulin and insulin glargine to the IR in vitro [
7].
The presence and activation of the IGF1R in muscle, heart and mammary tissue was demonstrated by intravenous injection of a high dose of IGF-1 (136 nmol/kg), whereas 6 nmol/kg IGF-1 injected s.c. was unable to generate detectable receptor autophosphorylation. A similar result was reported in mouse heart muscle, where the injection of 136 nmol/kg IGF-1 i.v. resulted in strong phosphorylation of the receptor, whereas no signal could be detected after i.v. injection of a therapeutic 4 nmol/kg dose [
44]. The tight control of IGF1R phosphorylation was also demonstrated by Hvid et al [
17], who reported that s.c. injection of a supraphysiological dose (600 nmol/kg) of IGF-1 in rats increased Akt phosphorylation in liver, colon and mammary gland of Sprague–Dawley rats. In agreement with a relatively poor response to IGF-1, Lee et al reported that Akt and ERK phosphorylation occurred in mouse mammary gland tissue only after a large bolus tail vein injection [
45]. Although it has been demonstrated that the large bolus can result in the majority of the IGF-1 being in circulation [
46], it should be noted that part of the dose could be bound to IGF-binding proteins, which would limit the free concentration and explain a reduced response [
45]. In any case, we used des[1-3]IGF-1, which lacks the N-terminal tripeptide Gly-Pro-Glu and has increased potency due to reduced binding to most of the IGF-binding proteins [
13]. Thus i.v. injection of des[1-3]IGF-1 should directly allow characterisation of the tissue IGF1R response.
Neither human insulin nor insulin analogues generated significant IGF1R signals in these tissues at s.c. doses up to 200 U/kg. The same holds true when the analogues were administered i.v. and the phosphorylation pattern was studied after 5 min. Importantly, animals that received an insulin glargine injection of 200 U/kg showed a free serum insulin M1 level of 22 nmol/l, which is 30-fold below the reported affinity of M1 to the IGF1R [
7]. In the same study, the measured concentrations of parent glargine and M2 were 5 nmol/l and 2 nmol/l, respectively, with the total insulin concentration reaching about 30 nmol/l. Assuming similar concentrations were achieved with AspB10 at a dose of 200 U/kg, the lack of IGF1R phosphorylation would be remarkable, since this concentration should be able to increase IGF-1 phosphorylation in vitro [
7]. The tendency for a high dose of AspB10 to show an increase in the tyrosine phosphorylation signal of the immunoprecipitate may be a reflection of phosphorylated IR contamination due to co-precipitation of IR by the antibody used in these studies. It may also reflect IR subunits precipitated as IR and/or IGFR hybrids. Hybrid receptors have been detected in a number of tissues including human skeletal and heart muscle, and adipose tissue [
6,
47]. Glargine itself might have a higher affinity for hybrid receptors in vitro [
6,
47], but M1 is like human insulin and thus no signal was observed in vivo.
In summary, AspB10 treatment resulted in a blood glucose profile comparable to that of insulin. The phosphorylation of signalling molecules was increased and (or) prolonged in most tissues, which resembles in vitro findings. The glycaemic action of insulin glargine was (slightly) retarded compared with insulin. Insulin glargine is rapidly and effectively metabolised to M1 under therapeutic and high-dose conditions, and the phosphorylation of signalling molecules in tissues was generally comparable to that of insulin, but retarded in time in some tissues. IGF1R phosphorylation could not be detected in several tissues upon exposure to insulin glargine or AspB10, even at high dose and different routes of administration. We conclude that in rats AspB10 has a different IR signalling profile to that of insulin and insulin glargine, and that the slightly elevated IGF1R activity of AspB10 in vitro did not translate into IGF1R phosphorylation in vivo. Consequently, we hypothesise that the greater mitogenic effect of AspB10 is most likely to be based on its altered IR profile in vivo. It is therefore tempting to speculate that the greater mitogenic effects of insulin and insulin analogues are solely based on the growth-promoting activity of the IR itself, and that IGF1R activation by insulin analogues may be less relevant under therapeutic conditions than previously discussed.