Peroxisome proliferator-activated receptorβ/δ activation is essential for modulating p-Foxo1/Foxo1 status in functional insulin-positive cell differentiation

Abstract

Peroxisome proliferator-activated receptors (PPARs) participate in energy homeostasis and play essential roles in diabetes therapy through their effects on non-pancreas tissues. Pathological microenvironment may influence the metabolic requirements for the maintenance of stem cell differentiation. Accordingly, understanding the mechanisms of PPARs on pancreatic β-cell differentiation may be helpful to find the underlying targets of disrupted energy homeostasis under the pancreatic disease condition. PPARs are involved in stem cell differentiation via mitochondrial oxidative phosphorylation, but the subtype member activation and the downstream regulation in functional insulin-positive (INS+) cell differentiation remain unclear. Here, we show a novel role of PPARβ/δ activation in determining INS+ cell differentiation and functional maturation. We found PPARβ/δ expression selectively upregulated in mouse embryonic pancreases or stem cells-derived INS+ cells at the pancreatic mature stage in vivo and in vitro. Strikingly, given the inefficiency of generating INS+ cells in vitro, PPARβ/δ activation displayed increasing mouse and human ES cell-derived INS+ cell numbers and insulin secretion. This phenomenon was closely associated with the forkhead box protein O1 (Foxo1) nuclear shuttling, which was dependent on PPARβ/δ downstream PI3K/Akt signaling transduction. The present study reveals the essential role of PPARβ/δ activation on p-Foxo1/Foxo1 status, and in turn, determining INS+ cell generation and insulin secretion via affecting pancreatic and duodenal homeobox-1 expression. The results demonstrate the underlying mechanism by which PPARβ/δ activation promotes functional INS+ cell differentiation. It also provides potential targets for anti-diabetes drug discovery and hopeful clinical applications in human cell therapy.

Figure 1

PPARs expressions at pancreatic mature stage in vivo and in vitro. (a) PPAR protein expressions in pancreas at embryonic days E12, 14, 16, 18 of gestation and newborn mouse, n=3. (b) PPARs expressed during INS⁺ cell differentiation stage, n=3. (c) Co-expressions of PPARs with insulin at terminal day (left panel), and insulin expression in mouse isolated islets (right panel) were determined by Immunofluorescence staining. Bar=50 μm. (d) Flow cytometry assay demonstrated the PPARs were co-expressed with insulin at the terminal differentiation, n=4. Values represent mean ±S.D.

Figure 2

PPARβ/δ determined Pdx-1 expression and functional INS⁺ cell generation from mouse ES cells. Cells were treated with PPARs agonists, antagonists from day 5+9, or transfected with sh-PPARβ/δ or sh-NC at day 5+9 and collected at day 5+28 for further detection. PPARα agonist: WY14643; PPARα antagonist: GW6471; PPARβδδ agonist: L165041; PPARβδδ antagonist: GSK0660; PPARγ agonist: GW1929. (a, i) Ratios of INS⁺ cells at day 5+28 were detected by flow cytometry analysis. (a, n=3, i, n=4.) (b) PPARβδδ changed the insulin-staining area and ΔΨm. Bar=100 μm. (c) Released insulin of induced cells and isolated mouse islets were analyzed. Values were normalized with total protein contents, n=3. (d, l) Insulin secretion level of INS⁺ cells was measured at terminal day of differentiation. (d, n=5, l, n=3). (e, k) Pancreatic specific gene expressions were detected by quantitative RT-PCR. n=3. (f, h) Western blot analyzed Pdx-1 protein expression at day 5+28, n=3. (g) PPARβδδ protein expression was receded till day 5+28 after sh-PPARβ/δ transfection, n=3. (j) Insulin staining area was reduced after transfected with sh-PPARβ/δ. Bar=100 μm. Values represent mean ±S.D. Statistical significance was set as *P<0.05, **P<0.01 versus DMSO control, ⁺P<0.05, ⁺⁺P<0.01 versus sh-NC control

Figure 3

Foxo1, Gsk3β and PI3K/Akt signaling were associated with PPARβδδ activation. (a) Protein expressions at day 5+15 and day 5+28 were detected after L165041 or GKS0660 treatment from day 5+9. (b) Protein expressions at day 5+28 were analyzed after PPARβδδ shRNA transfection from day 5+9. Values represent mean ±S.D., n =3. Statistical significance was set as *P<0.05, **P<0.01 versus DMSO control, ⁺P<0.05, ⁺⁺P<0.01 versus sh-NC

Figure 4

Knockdown of Foxo1 improved PPARβ/δ-mediated cell differentiation. Cells were transfected with shRNA against PPARβ/δ, Foxo1 and Gsk3β from day 5+9 and harvested at day 5+28. (a, f) Protein expressions of Foxo1 (a) and Gsk3β (f) were repressed till 5+28 after transfection by targeted shRNA. (b, g) Ratios of INS⁺ cells after transfection were determined by flow cytometry. (c, d and h) Pdx-1 expression was determined by western blot (c, h) and immunofluorescence analysis (d, Bar= 100 μm). (e) Insulin secretion levels of INS⁺ cells detected by ELISA. Values represent mean ±S.D., n=3. Statistical significance was set as ⁺P<0.05, ⁺⁺P<0.01 versus sh-NC, *P<0.05, **P<0.01 versus sh-PPARβ/δ

Figure 5

Inhibition of PI3K disturbed the effects of PPARβδδ on INS⁺ cell differentiation. Cells were treated with L165041 and LY294002 from day 5+9, and harvested at day 5+28 for further analysis. (a) LY294002 inhibited p-Akt expression. (b) The ratios of INS⁺ cells were determined by flow cytometry. (c) Expressions of cytosolic p-Foxo1 and nuclear Foxo1 were changed after treatment with LY294002. (d) Protein expression of Pdx-1 was analyzed by western blot. Values represent mean ±S.D., n =3. Statistical significance was set as *P<0.05, **P<0.01 versus control, ^#P<0.05, ^##P<0.01 versus L165041-treated groups. (e) Schematic diagram of the signaling pathways involved in PPARβδδ-mediated INS⁺ cell differentiation of mouse ES cells

Figure 6

Human ES cell-derived INS⁺ cells share the same function and signaling pathway of PPARβ/δ activation. (a) Co-expressions of PPARβ/δ with insulin in human ES cell-derived INS⁺ cells at the terminal differentiation day. Bar=25 μm. Human ES cells were treated with PPARβ/δ agonist L65041 or antagonist GSK0660 at INS⁺ cell differentiation stage, and the results shown on the terminal day are as follows. (b) The INS⁺ cells were quantified by flow cytometry assay. (c) Expression of insulin mRNA was detected by quantitative RT-PCR. (d) Insulin secretion level of INS⁺ cells was measured. (e, f) The molecular events in the PPARβ/δ signaling pathway demonstrated similar characteristics as those in mouse-ES cell-derived INS⁺ cells, n=3. Values represent mean±S.D. Statistical significance was set as *P<0.05, **P<0.01 versus DMSO control