Long-term IGF-I exposure decreases autophagy and cell viability

Abstract

A reduction in IGF-I signaling has been found to increase lifespan in multiple organisms despite the fact that IGF-I is a trophic factor for many cell types and has been found to have protective effects against multiple forms of damage in acute settings. The increase in longevity seen in response to reduced IGF-I signaling suggests that there may be differences between the acute and chronic impact of IGF-I signaling. We have examined the possibility that long-term stimulation with IGF-I may have a negative impact at the cellular level using quiescent human fibroblasts. We find that fibroblast cells exposed to IGF-I for 14 days have reduced long-term viability as judged by colony forming assays, which is accompanied by an accumulation of senescent cells. In addition we observe an accumulation of cells with depolarized mitochondria and a reduction in autophagy in the long-term IGF-I treated cultures. An examination of mice with reduced IGF-I levels reveals evidence of enhanced autophagy and fibroblast cells derived from these mice have a larger mitochondrial mass relative to controls indicating that changes in mitochondrial turnover occurs in animals with reduced IGF-I. The results indicate that chronic IGF-I stimulation leads to mitochondrial dysfunction and reduced cell viability.

Figure 1. IGF-I decreases long-term viability of human fibroblasts.

Long-term IGF-I treatment reduces colony formation potential. Colony forming assays were performed on cells that had been maintained in either MCDB 105 medium without additions or with IGF-I (40 ng/ml) for 2 weeks. Cells were seeded in full growth medium to allow colony growth and results are presented in panel A. Bars are number of colonies per 3×10³ cells plated (*, P<0.01) B. Representative micrograph (20X) of senescence-associated β-galactosidase staining of fibroblast colonies. C. Crystal violet stained colonies of plates seeded with 3×10³ cells for colony forming assays.

Figure 2. IGF-I treatment increases mitochondrial depolarization.

WI-38 fibroblasts were maintained for 14 days in MCDB 105 medium, MCDB 105 medium with IGF-I (40 ng/ml), or MCDB 105 medium with EGF (40 ng/ml). Medium with or without growth factors was replenished every 3 days and cells were stained for mitochondrial potential at that time as described in materials and methods. Cells with depolarized mitochondria were visualized by flow cytometry as described in Material and Methods. A. Percentage of cells with depolarized mitochondria as assessed by JC-1 staining (*, P<0.001). B. Representative dot blot of JC-1-stained cultures in MCDB 105 with or without IGF-I or EGF. Y-axis, fluorescence at 590 nm; X-axis, fluorescence at 525 nm. A downward shift on the X-axis is indicative of mitochondrial membrane depolarization.

Figure 3. IGF-I treatment impairs autophagy.

WI-38 fibroblasts stably expressing the GFP-LC3 fusion protein were maintained for 14 days in MCDB 105 medium, or MCDB 105 medium with IGF-I (40 ng/ml). Medium with or without growth factor was replenished every 3 days. A. Number of LC3 puncta per cell in WI-38 GFP-LC3 cells with or without 40 ng/ml IGF-I (**, P<0.01) At least 25 fields and 100 cells per slide were examined. B. Representative fluorescence micrograph (40X) of WI-38 GFP-LC3 cells with or without IGF-I treatment. C. Accumulation of LC3 and p62/SQSTM1 over time in IGF-I-treated cells as assessed by western blot. D. Protein degradation in control and IGF-I-treated cells measured as percentage of the residual ³⁵S-Methionine radioactivity at the indicated time points over time 0 (*, P<0.01).

Figure 4. Rapamycin restores mitochondrial clearance and long-term viability.

WI-38 fibroblasts were maintained for 14 days in MCDB 105 medium, or MCDB 105 medium with IGF-I (40 ng/ml). Medium with or without growth factor was replenished every 3 days. Mitochondrial potential and colony forming assays were performed at the end of the 14 day period. A. Rapamycin reduces the effect of IGF-I on mitochondria depolarization as assessed by JC-1 staining (*, P<0.05). B. Rapamycin restores long-term viability in IGF-I-treated cells as measured by colony forming assay. Bars are number of colonies per 3×10³ cells plated. C. Crystal violet stained colonies of plates seeded with 3×10⁴ cells after 2 weeks in full growth medium.

Figure 5. Rapamycin restores autophagy in IGF-I-treated cells.

WI-38 fibroblasts stably expressing the GFP-LC3 fusion protein were maintained for 14 days in MCDB 105 medium, or MCDB 105 medium with IGF-I (40 ng/ml). Medium with or without growth factor was replenished every 3 days. A. Representative fluorescence micrographs of WI-38 GFP-LC3 cells with or without 40 ng/ml IGF-I and/or 10 nM rapamycin. B. Number of LC3 puncta per cell in WI-38 GFP-LC3 cells (*, P<0.05). At least 25 fields and 100 cells per slide were examined C. Levels of LC3 and p62/SQSTM1 in WI-38 with or without 40 ng/ml IGF-I and/or 10 nM rapamycin as assessed by Western blot analysis.

Figure 6. Impairment of autophagy increases mitochondrial depolarization.

WI-38 fibroblasts and WI-38 cells expressing either an shRNA construct targeting Atg5 (shAtg5) or the same targeting vector expressing a scrambled sequence (Scramble) were maintained for 14 days in MCDB 105 medium. As a control, parallel cultures were maintained in MCBD 105, or MCDB 105 medium with IGF-I (40 ng/ml). Medium with or without growth factor was replenished every 3 days. Cells were stained for mitochondrial potential at that time as described in materials and methods. Cells with depolarized mitochondria were visualized by flow cytometry as described in Material and Methods. Differences in the percent of cells with depolarized mitochondria between cell populations were significant for shAtg5 versus scramble or control cells (*, P<0.05). The difference in the percent of cells with depolarized mitochondria between IGF-I treated and either control or scramble was also significant (*, P<0.05).

Figure 7. IGF-I-depleted mice show markers of increased autophagy.

Tissue sections from IGF-I deficient mice and controls were examined for LC3 containing puncta as described in the Material and Methods section. A. Representative fluorescence micrograph of liver, kidney, and quadriceps tissue slides from wild type and IGF-I-depleted mice stained with anti-LC3 rabbit polyclonal antibody. B. Number of LC3 puncta per nuclei in tissues from wild type and IGF-I-depleted mice. At least 100 nuclei and 25 fields per slide were examined. C. Western blot analysis of LC3 protein levels in liver, kidney, and skeletal muscle tissue lysates. 1,2,3: IGF-I-depleted mice starved for 24 hours. 4,5: IGF-I-depleted mice fed. 6,7,8: wild type mice starved for 24 hour. 9,10: wild type mice fed.

Figure 8. Mouse embryo fibroblasts from IGF-I-depleted mice show increased mitochondrial mass and DNA content.

Mouse embryo fibroblasts (MEFs) were growth from IGF-I deficient mice or control animals as described in materials and methods. The relative mitochondria content in wild type and IGF-I-depleted mice measured by staining with the mitochondrial specific mitotracker green fluorescent dye as described in the Materials and Methods. A. Mean mitochondrial mass of the cell populations at passage 2 and 10 is presented as analyzed by flow cytometery. Differences between the mitochondrial mass in the IGF-I deficient mice and controls was significant (P<0.01 at passage 2 and P<0.05 at passage 10). B. Relative mitochondrial DNA content at passage 2 is presented. The experiment presented is representative of the results of 2 independent measurements on 4 DNA isolates using independent primer sets that amplify mitochondrial and nuclear DNA. The difference in mitochondrial DNA content was significant (P<0.05).