The direct effects of pH and osmolarity on cytotoxicity in NIH 3T3 fibroblasts are shown in and , respectively. The ranges for pH and osmolarity that did not induce cytotoxicity, as measured by the WST-1 assay, were approximately 7.0–9.5 and 250–400, respectively.
The effect of (A) pH and (B) osmolarity on cytotoxicity of NIH 3T3 fibroblasts in culture, using the WST-1 cell viability assay.
The results of the LDH and WST-1 assays in our initial experiments were concordant. However, owing to its better reproducibility during repeated measures for 5–7 days, subsequent cytotoxicity assays used the LDH assay. shows the LDH assay results for NIH 3T3 cells exposed to the commercially available lactose-rich glucagon preparation (GlucaGen) at pH 3 after aging for 0, 1, 2, and 7 days. Only the highest concentration (5 mg/ml) led to cytotoxicity, and it was clear that this toxicity was not related to the duration of aging. Indeed, the toxic effect of the highest concentration of glucagon was as prominent at day 0 as it was at day 7. Concentration of the lactose-rich glucagon mixture, not aging duration, was the relevant factor.
Figure 2 LDH assay results in NIH 3T3 fibroblasts exposed to commercially available lactose-rich glucagon preparation (GlucaGen) at pH 3 after aging for 0, 1, 2, and 7 days. Positive and negative controls performed as expected. Only the highest concentration (5 (more ...)
shows the cytotoxicity results for lactose-free glucagon and the lactose-free glucagon analog MAR-D28 at different concentrations with no aging (a) and after aging for 5 days (b). With no aging, there was no significant cytotoxicity at any concentration of the native hormone or the analog at either pH 8.5 or 10. After aging for 5 days, there was some evidence of toxicity at pH 8.5, with similar results for native glucagon and MAR-D28. At 5 days at a pH of 10 in glycine buffer, there was no evidence of cytotoxicity for either preparation.
Figure 3 Cytotoxicity results for lactose-free glucagon and the lactose-free glucagon analog MAR-D28 at different concentrations with (A) no aging and (B) after aging for 5 days. With no aging, there was no significant cytotoxicity at any concentration of the (more ...)
We carried out Congo red studies on GlucaGen reconsti-tuted at 1 mg/ml. The Congo red assay was carried out in triplicate on this lactose-rich preparation immediately after reconstitution and after being aged at 37 °C for various lengths of time. Using a scale of 0–6, with a score of 6 being maximal, we observed Congo red scores of 0 with no aging, 0 after 4 hours of aging, 1.5 after 24 hours of aging, 3.75 after 3 days of aging, and 6 after 5 days of aging (data not shown).
We also carried out a series of Congo red assays on lactose-free preparations aged for longer periods of time, as shown in . This figure shows results of Congo red binding to preparations of lactose-free native glucagon and MAR-D28. There was no Congo red binding at day 0 (freshly prepared, without aging) under any condition. Beginning at day 8, Congo red amyloid intensity was greatest in citrate buffer at pH 3. At a mildly alkaline pH of 8.5, there was substantial Congo red intensity at day 8 with the native glucagon but not with the analog MAR-D28. At a pH of 10, there was no Congo red staining for native glucagon or for the MAR-D28 analog, even after 28 days.
Figure 4 Results of Congo red assay for glucagon preparations aged at 37 °C for varying lengths of time. For fresh glucagon and MAR-D28, there was no Congo red binding under any condition. Beginning at day 8, Congo red amyloid intensity was greatest in (more ...)
In terms of SEC data, the principal metric was the area under the curve (units are absorbance × minutes) for the monomeric glucagon peak, the data for which are shown in . When glucagon was aged in citrate at pH 3, the size of the glucagon monomeric peak declined rapidly and was absent after 7 days. When aged in Tris at pH 8.5, there was also a substantial loss of the glucagon peak. In contrast, when aged in glycine at pH 10, there was very little loss of the monomeric peak at 7 days, although modest loss occurred after 14 and 21 days. There was no obvious difference in the results for the glycine preparation when a mobile phase of PBS (pH 7.4) was used versus a mobile phase of citrate (pH 3).
Figure 5 Results of SEC carried out on fresh and aged preparations of native glucagon. Shown here are the areas under the curve for the main monomeric glucagon peak (molecular weight 3.5 kDa) obtained at different periods of aging at 37 °C up to 21 days (more ...)
Although the monomeric glucagon peak and the peaks from the various protein standards were always well-delineated, there were little if any large molecular weight peaks that would have indicated amyloid aggregates, even in preparations that lost the glucagon peak and were shown by other techniques to be rich in amyloid. We believe that this finding was due to the fact that when the aging solution showed both solids (gels) and liquid, the very narrow gauge pipette tip used to acquire the sample tended to serve as a screen, allowed acquisition only of the nongelled liquid.
shows data for the pig study in which fresh or aged (at 1 mg/ml) GlucaGen was given to anesthetized pigs at two different doses. For the lower dose (1 μg/kg), the hyperglycemic responses were similar for both fresh and aged, although there was a tendency for the aged preparation to have a somewhat lower response. For the high dose (1 mg), hyperglycemic responses were also similar between the fresh and aged preparations, with the aged preparation demonstrating a somewhat higher response. If one compares the low and high doses, the initial rate of glucose rise was similar for both doses, but the maximal response, typically at 45–60 minutes, was somewhat greater for the higher dose.
Figure 6 Data for the pig study in which fresh or aged glucagon (GlucaGen) was given to anesthetized pigs at two different doses. For the lower dose (1 µg/kg), the hyperglycemic responses was similar among the fresh (n = 2) and aged (n = 2), although there (more ...)
shows the plasma levels of glucagon obtained during the high-dose glucagon studies. Although the maximal plasma levels were very similar for fresh vs aged glucagon, there was a tendency for the rise in glucagon to be slightly delayed in the aged preparation.
The plasma levels of glucagon obtained during the high-dose glucagon studies. Although the maximal plasma levels were very similar, there was a tendency for the rise in glucagon to be slightly delayed in the aged preparation.