Beta-cell proliferation, but not neogenesis, following 60% partial pancreatectomy is impaired in the absence of FoxM1

Abstract

Objective: This study was designed to determine whether the transcription factor FoxM1 was required for regeneration of beta-cell mass via proliferation and/or neogenesis in the adult after 60% partial pancreatectomy (PPx).

Research design and methods: Adult mice with a pancreas-wide deletion of Foxm1 (Foxm1(flox/flox);Pdx1-Cre [FoxM1(Deltapanc)]) and their control littermates (Foxm1(flox/flox)) were subjected to PPx or a sham operation, after which islet expression of Foxm1 and several target genes, beta-cell mass, proliferation, beta-cell size, islet size, islet density, and neurogenin-3 expression were analyzed.

Results: In control mice, PPx stimulated beta-cell proliferation and neogenesis and upregulated Foxm1 and several of its known targets (Plk1, Cenp-a, Birc5/Survivin, and Ccnb1) in islets. Within 1 week post-PPx, control mice underwent significant regeneration of beta-cell mass, and average islet size within the regenerating lobe was similar to that after a sham operation. However, FoxM1(Deltapanc) mice exhibited specific impairments in beta-cell mass regeneration and islet growth after PPx, with reduced proliferation of alpha- and beta-cells but no impairments in acinar or ductal cell proliferation. Interestingly, FoxM1 was not required for proliferation of beta-cells within small endocrine cell clusters located in the regenerating portion of the pancreas but was specifically required for proliferation of beta-cells within larger islets. Additionally, FoxM1 was not required for beta-cell neogenesis following PPx.

Conclusions: Our results indicate that FoxM1 is partially required for increased beta-cell proliferation, but not beta-cell neogenesis, stimulated by PPx. Furthermore, FoxM1 seems to be dispensable for proliferation of beta-cells following neogenesis but is required for proliferation of preexisting beta-cells.

FIG. 1.

Experimental timeline for 60% PPx. Either PPx or a sham operation was performed on 8-week-old female mice, after which BrdU was administered in the drinking water for 1 week. IPGTT was performed 1 day before and 7 days following the operation, and the mice were killed on day 7.

FIG. 2.

Expression levels of Foxm1 mRNA and some of its targets were upregulated following 60% PPx. Real-time quantitative RT-PCR was performed on RNA extracted from isolated islets 6 days following either PPx (▪) or sham (□). Error bars represent SE of the mean. Unpaired t tests were used to measure significance. *P < 0.05; **P < 0.01. n = 4–6 per group.

FIG. 3.

Glucose tolerance was maintained following 60% PPx. No significant differences in blood glucose levels were observed in either control or FoxM1^Δpanc mice following PPx compared with sham at any time point during IPGTT. Error bars represent SE. Two-way ANOVA with Bonferroni's posttests was used to measure significance. n = 5–7 per group. □, control sham day 7; ▪, FoxM1^Δpanc sham day 7; ▵, control PPx day 7; ▴, FoxM1^Δpanc PPx day 7.

FIG. 4.

Regeneration of β-cell mass, but not gross pancreatic tissue, was impaired in FoxM1^Δpanc mice. A: Wet weight of the pancreatic splenic lobe was measured immediately following (day 0) or 7 days after PPx or sham. The majority of tissue was removed by PPx, and a moderate amount of regeneration occurred within 7 days in both control (□) and FoxM1^Δpanc (▪) mice. B: β-Cell mass, which takes into account tissue wet weight, was significantly reduced within the splenic lobe of FoxM1^Δpanc pancreata after a sham operation, and regeneration of β-cell mass was not observed in FoxM1^Δpanc mice following PPx. C: β-Cell mass within the duodenal lobe of FoxM1^Δpanc pancreata was only significantly reduced in comparison with control pancreata following PPx. Error bars represent SE. Two-way ANOVA with Bonferroni's posttests, comparing sham day 7 versus PPx day 0 and PPx day 0 versus PPx day 7, was used to measure significance. n = 4–6 per group. ns, not significant.

FIG. 5.

Proliferation of endocrine cells was impaired in FoxM1^Δpanc mice following 60% PPx. BrdU incorporation (red) over 7 days was used as a marker of cell proliferation. Double labeling with insulin (green) revealed low, basal levels of β-cell proliferation within the splenic lobes following a sham operation in both control (A) and FoxM1^Δpanc (C) mice. PPx enhanced BrdU incorporation into all pancreatic cell types within the splenic lobe of control mice (B), but incorporation into endocrine cells was reduced in FoxM1^Δpanc mice (D). β-Cell proliferation was quantified for both the splenic (E) and duodenal (F) pancreatic lobes. β-Cell proliferation within small endocrine cell clusters (eight or less β-cells) after PPx versus sham was enhanced in the splenic lobes of FoxM1^Δpanc and control mice (G) but was not altered in the duodenal lobes (H). β-Cell proliferation within definitive islets (nine or more β-cells) after PPx was enhanced in the splenic lobe of control mice, and this effect was partially inhibited in FoxM1^Δpanc mice (I). PPx also significantly stimulated β-cell proliferation within definitive islets in the duodenal lobe of control, but not FoxM1^Δpanc, mice (J). α-Cell proliferation within the splenic lobe of FoxM1^Δpanc pancreata (K) was also impaired in comparison with control pancreata following PPx. Error bars represent SE. Two-way ANOVA with Bonferroni's posttests was used to measure significance. n = 4–5 per group for β-cell proliferation, n = 3–4 per group for α-cell proliferation. ns, not significant. E–K: □, control; ▪, FoxM1^Δpanc. (Please see http://dx.doi.org/10.2337/db08-0878 for a high-quality digital representation of this figure.)

FIG. 6.

FoxM1^Δpanc mice exhibited increased β-cell and nucleus size. After a sham operation, FoxM1^Δpanc mice exhibited increased β-cell size in both the splenic (A) and duodenal (B) pancreatic lobes. Increased β-cell size was also observed after PPx in the splenic lobe of FoxM1^Δpanc mice versus controls but was significantly reduced compared with after a sham operation. The average β-cell size in control pancreata was unchanged by PPx. FoxM1^Δpanc mice also exhibited increased β-cell nucleus size in the splenic (C) but not duodenal (D) lobe, which was unchanged by PPx. Error bars represent SE. Two-way ANOVA with Bonferroni's posttests was used to measure significance. n = 4–5 per group. □, control; ▪, FoxM1^Δpanc.

FIG. 7.

FoxM1^Δpanc mice exhibited impaired islet growth but no impairment in islet neogenesis after 60% PPx. There was no significant difference in average islet size, measured as the cross-sectional area of each insulin⁺ cell grouping, between control (□) and FoxM1^Δpanc (▪) mice after a sham operation or PPx in either the splenic (A) or duodenal (B) pancreatic lobes. Following PPx, the average islet size in the splenic lobe of control pancreata was similar to that observed in sham-operated mice, but FoxM1^Δpanc islets were significantly reduced in size. Within the duodenal lobe, control mice trended toward increased average islet size after PPx, but no increase was observed in FoxM1^Δpanc mice. FoxM1^Δpanc mice specifically exhibited an increased proportion of small insulin⁺ cell clusters (one to eight β-cells) within the splenic lobe after PPx (C); no differences were observed within the duodenal lobe (D). ▪, more than eight β-cells; □, one to eight β-cells. The number of insulin⁺ cell groupings per section was reduced within the splenic lobe of FoxM1^Δpanc mice compared with control littermates after a sham operation (E), but at day 0 and day 7 following PPx, the number of insulin⁺ cell groupings per section was equivalent between FoxM1^Δpanc and control mice. Seven days after PPx, there was a trend in both groups of mice to show increased numbers of insulin⁺ cell groupings per section within the splenic lobe, but no differences between any groups of mice were observed in the duodenal lobe (F). Ngn3 expression was detected only after PPx within cytokeratin⁺ (CK⁺) ductal epithelial cells located within foci of regeneration in the splenic lobes of both control (G) and FoxM1^Δpanc (H) mice. Error bars represent SE. Two-way ANOVA with Bonferroni's posttests was used to measure significance. n = 4–5 per group. (Please see http://dx.doi.org/10.2337/db08-0878 for a high-quality digital representation of this figure.)