Re-expression of IGF-II is important for beta cell regeneration in adult mice

Abstract

Background: The key factors which support re-expansion of beta cell numbers after injury are largely unknown. Insulin-like growth factor II (IGF-II) plays a critical role in supporting cell division and differentiation during ontogeny but its role in the adult is not known. In this study we investigated the effect of IGF-II on beta cell regeneration.

Methodology/principal findings: We employed an in vivo model of 'switchable' c-Myc-induced beta cell ablation, pIns-c-MycER(TAM), in which 90% of beta cells are lost following 11 days of c-Myc (Myc) activation in vivo. Importantly, such ablation is normally followed by beta cell regeneration once Myc is deactivated, enabling functional studies of beta cell regeneration in vivo. IGF-II was shown to be re-expressed in the adult pancreas of pIns-c-MycER(TAM)/IGF-II(+/+) (MIG) mice, following beta cell injury. As expected in the presence of IGF-II beta cell mass and numbers recover rapidly after ablation. In contrast, in pIns-c-MycER(TAM)/IGF-II(+/-) (MIGKO) mice, which express no IGF-II, recovery of beta cell mass and numbers were delayed and impaired. Despite failure of beta cell number increase, MIGKO mice recovered from hyperglycaemia, although this was delayed.

Conclusions/significance: Our results demonstrate that beta cell regeneration in adult mice depends on re-expression of IGF-II, and supports the utility of using such ablation-recovery models for identifying other potential factors critical for underpinning successful beta cell regeneration in vivo. The potential therapeutic benefits of manipulating the IGF-II signaling systems merit further exploration.

Figure 1. IGF-II re-expression in MIG mice after brief Myc activation.

Total pancreas RNA was extracted from pIns-c-MycER^TAM/IGF-II^+/+ (MIG) and pIns-c-MycER^TAM/IGF-II^+/− (MIGKO) mice at 24 hr and 48 hr following Myc activation to induce beta cell ablation (n = 3). RT-PCR data (A) shows the amplified products in MIG mice after 24 hr (1) and 48 hr (3) of Myc activation in the target size of 357 bp. No amplified product was found in MIGKO mice at 24 hr (2) and 48 hr (4) of Myc activation by comparing to the positive control (WT mouse E17.5 placenta RNA) (0). Quantitative RT-PCR was performed and validated the RT-PCR results. IGF-II mRNA was expressed 5-fold higher in MIG mice after brief Myc activation but not in MIGKO mice nor in negative controls (B).

Figure 2. Blood glucose homeostasis.

Myc activation was induced for 11 days in mice following which Myc was deactivated for up to 3 months. Blood glucose levels (Mean ± SEM) were measured in MIG mice n = 3 and MIGKO mice n = 3 during this period (A). Both strains developed hyperglyceamia after Myc activation and MIGKO mice showed a delay of recovery from hyperglycemia at day 4 following Myc deactivation. After 3 months both strains were able to return to normal blood glucose level.

Figure 3. Intraperitoneal glucose tolerance test.

Islet functional study was performed by taking IP glucose tolerance test (IPGTT). Mice were starved overnight before intraperitoneal injection of glucose at 1.5 mg/g of animal body weight. Results of IPGTT showed both stains did not recover 3 weeks after Myc deactivation, comparing to their wildtype litter mates (A). After 3 month recovery both strains were able to maintain normal blood glucose (B).

Figure 4. Loss of IGF-II impedes recovery of beta cell mass following beta cell ablation.

For each individual, beta cell mass was calculated as cross sectional area of insulin immunoreactivity divided by total sectional area for at least 5 levels (n = 3). Results showed in MIG mice beta cell mass increased 2-fold at 4 days Myc deactivation (p = 0.0106), whereas in MIGKO mice the beta cell mass was largely unchanged. After 3 months recovery the beta cell mass increased 6-fold in MIG mice and in MIGKO mice the increase was 5-fold.

Figure 5. Loss of IGF-II affects recovery of beta cell numbers following beta cell ablation.

Beta cell number was counted in up to 200 islets from five pancreas levels per mouse (n = 3) by software co-developed with our collaborators . Results showed in MIG mice beta cell number increased by 60% at 4 days Myc deactivation, whereas in MIGKO mice the beta cell mass was barely changed. Consistently with our previous observation in beta cell mass after 3 months recovery, both MIG and MIGKO mice achieved 3- to 4-fold increase of beta cell numbers.

Figure 6. Glucagon positive cell cluster in MIG mice after 3 months Myc deactivation.

In MIG mice after 3 months of recovery, clusters of glucagon positive cells separate from islets were observed. This might be new islets forming or possible clusters of alpha cells which might then further differentiate into beta cells. Such clusters were not seen in MIGKO mice or control mice pre-ablation.