To investigate the signalling pathways involved in insulin analogues action in colon cancer cells, our initial experiments measured the basal levels of insulin receptor and IGF-IR in the HCT116 cell line. As shown in Fig. 1a, b, HCT116 cells express both receptors at similar levels. These results were confirmed by immunofluorescent microscopy using anti-IGF-IR and anti-insulin receptor antibodies (data not shown). Incubation of the cells for 24 h in serum-free (starvation) medium had no effect on basal receptor levels (Fig. 1a, b). To identify the specific receptor(s) activated by insulin glargine in comparison to regular human insulin or IGF-I, confluent cells were maintained overnight in starvation medium, after which they were treated with a physiological dose of insulin glargine (50 ng/ml) for 10 min. At the end of the incubation, receptor phosphorylation was evaluated by immunoprecipitation assays. Specifically, lysates were immunoprecipitated with anti-insulin receptor (Fig. 1c) or anti-IGF-IR (Fig. 1d) for 24 h, electrophoresed through 10% SDS-PAGE and immunoblotted with anti-phosphotyrosine. Results showed that, at a dose of 50 ng/ml, each receptor was preferentially activated by its cognate ligand, with minimal cross-activation by the heterologous hormone. Similarly to regular insulin, insulin glargine also stimulated insulin receptor phosphorylation; in addition, it also phosphorylated the IGF-IR in an IGF-I-like manner.
Next, we characterised the dose-dependency of insulin receptor and IGF-IR activation by insulin glargine. To this end, starved cells were treated with increasing doses of insulin glargine, regular insulin or IGF-I (5, 10, 25, 50 or 100 ng/ml) for 10 min, while untreated cells served as controls. Cells were then collected and receptor activation assessed by immunoprecipitation assays, as described above. The results revealed that insulin glargine induced insulin receptor phosphorylation in a dose-dependent manner at doses of 5 to 25 ng/ml (Fig. 2a, c). Regular insulin, on the other hand, induced almost maximal insulin receptor phosphorylation at the lowest concentration studied (5 ng/ml). In addition, insulin glargine, similarly to IGF-I, activated the IGF-IR at a dose of 5 ng/ml, whereas regular insulin was unable to stimulate IGF-IR phosphorylation at the doses studied (Fig. 2b, d). Equal loading was assessed by blotting the membranes with antibodies against total insulin receptor or IGF-IR. Together, the results of dose-dependent activation assays indicate that maximal insulin receptor phosphorylation was seen at a dose of 25 ng/ml insulin glargine, whereas maximal IGF-IR phosphorylation was detected at fivefold lower doses of the analogue.
Given that insulin glargine is a long-acting analogue, we evaluated receptor activation over longer periods of time. For this purpose, cells were treated with insulin glargine (50 ng/ml) and receptor activation was assessed by immunoprecipitation after various periods of time (5, 20 and 40 min, and 1, 2, 4 and 6 h). Results indicate that insulin glargine induced sustained insulin receptor phosphorylation (up to 6 h; Fig. 3a, c), whereas its pattern of IGF-IR activation was bi-phasic, with activation peaks at 5 min and 2 h (Fig. 3b, d). In comparison, regular insulin phosphorylated the insulin receptor in a very rapid and transient fashion (Fig. 3e).
Next, we evaluated the ability of insulin detemir to activate the IGF-IR. To this end starved cells were incubated with increasing doses of the analogue (5, 25, 50 and 100 ng/ml) for 20 min (Fig. 4a). In addition, cells were treated with insulin detemir for longer periods of time (1, 2 h) at a dose of 50 ng/ml (Fig. 4b). At the end of the incubation period, cell lysates were prepared and IGF-IR phosphorylation was assessed by immunoprecipitation assays, as described above. The results revealed that insulin detemir induced IGF-IR phosphorylation in a dose-dependent manner. However, at the short incubation period (20 min) insulin detemir was approximately one order of magnitude less potent than IGF-I. After a longer incubation time (1 h), the effect of insulin detemir was comparable to that of IGF-I, although this effect was not sustained, with a marked reduction of insulin detemir-induced IGF-IR activation noted after 2 h.
To evaluate the ability of insulin glargine to stimulate IGF-IR internalisation and redistribution, cells were transfected with an IGF-IR-encoding expression vector fused to a GFP tag. After 48 h, cells were treated with insulin, IGF-I or insulin glargine for 20, 40 or 60 min, and fixed for confocal microscopy. Figure 5 shows the results of IGF-IR internalisation analysis at 40 min. Control cells expressed the IGF-IR primarily on the cell membrane (Fig. 5a). Insulin treatment did not significantly affect the distribution of IGF-IR (Fig. 5b). When treated with IGF-I, a portion of the tagged receptor displayed, in addition to its cell-surface distribution, a perinuclear localisation, implying IGF-IR internalisation (Fig. 5c). Treatment with insulin glargine produced a similar effect to that seen with IGF-I, i.e. reduced membrane localisation and perinuclear accumulation of the IGF-IR (Fig. 5d).
To address the activation of downstream signalling molecules by insulin glargine and insulin detemir in comparison to regular insulin or IGF-I, cells were incubated with the ligands for up to 1 h and Akt phosphorylation was measured by western blot analysis with anti-phospho-Akt antibodies at 15, 30 and 60 min. The results showed that insulin glargine strongly stimulated Akt phosphorylation at 30 min, an induction that paralleled the effect of insulin (Fig. 6a, b). At this time point, IGF-I- and insulin detemir-stimulated Akt phosphorylation were significantly lower. No significant differences between the ligands in terms of Akt phosphorylation were seen at 60 min, whereas at 15 min basal Akt phosphorylation was elevated and no stimulation was seen (not shown). On the other hand, glargine- and detemir-induced ERK1/2 phosphorylation, as measured by western blots with anti-phospho-ERK1/2 antibodies, paralleled the effect of IGF-I (Fig. 6c, d). Under our experimental conditions, regular insulin exhibited the strongest potency at all time points. Notably, the ability of insulin glargine and insulin detemir to activate ERK1/2 was largely reduced at 60 min.
Next, the potential protection from apoptosis afforded by insulin glargine and insulin detemir were evaluated. To this end, cells were starved for 24 h, after which they were treated with the various ligands at a dose of 50 ng/ml for an additional 24 h. Cells were collected and apoptosis was assessed by western blot using anti-PARP. The results show that insulin glargine and insulin detemir treatments were associated with 21% and 50% reductions respectively (vs control cells) in the intensity of the ~85 kDa band, which represents a cleavage product of the full-length PARP (~116 kDa) and serves as a marker of cells undergoing apoptosis (Fig. 7a–c). The intensity of the ~85 kDa band in insulin-treated cells was similar to that in control cells, consistent with a reduced anti-apoptotic effect. As expected, use of IGF-I led to a 58% reduction in PARP cleavage. These results were corroborated using an Annexin V-FITC kit. FACS analysis revealed that IGF-I and insulin glargine reduced the proportion of apoptotic cells from 5.1 ± 0.4% to 3.1 ± 0.7% and 2.8 ± 0.6%, respectively, whereas insulin had no protective effect (Fig. 7d). Finally, we evaluated the ability of insulin glargine to stimulate cell cycle progression. For this purpose cells were treated with 50 ng/ml of insulin glargine, insulin or IGF-I for 10 h, and collected for cell cycle analysis. Untreated cells were used as control. Cells were dyed with propidium iodide and analysed by FACS for DNA content. Quantification of the cell fraction at S-phase revealed that insulin glargine, similarly to IGF-I, enhanced the fraction of cells at S-phase (Fig. 7e). Insulin, on the other hand, had no effect on cell cycle progression.