Figure 3. Insulin signaling drives differentiation of LSKs into lymphoid cells. (A) Sorted WT LSKs were cultured in StemPro-34 medium as described in Materials and methods. For insulin stimulation (With insulin), insulin was added to the medium to a final concentration of 200 nM for 18 h. For insulin depletion (Without insulin), anti-insulin antibody was added to the medium to reach 1 µg/ml for 18 h. Phosphorylation of Insr was detected using antibody against phospho-insulin receptor β (Tyr1150/1151). CM, culture medium. (B) CFC assays of Flk2−LSKs and MPPs pretreated with or without insulin. Sorted cells pretreated with or without 200 nM insulin for 18 h in StemPro-34 medium were seeded in differentiation medium for 10 d and numbers of colonies were calculated. (C) Lymphoid differentiation potential of Flk2−LSKs and MPPs cultured with or without insulin. 1,000 sorted cells, pretreated with or without insulin, were seeded onto OP9 stromal cells (see Materials and methods) and numbers of B220+CD19+ cells were calculated at the indicated intervals. (D) Cells treated with or without insulin were first stained with Hoechst 33342 and then stained with pyronin Y, followed by flow cytometry (left). Percentages of cells in S/G2/M (right). (E) Flk2−LSKs and MPPs treated with or without insulin were permeabilized and stained with antibody against active caspase-3. Cells positive for active caspase-3 were calculated. (D and E) n = 6. (F) 5 IU/kg of insulin or PBS (Vehicle) were injected into mice through a tail vein at the end of the day (8:00 p.m.) six times, and the BM was analyzed for the indicated populations in the morning of the day (8:00 am) after last insulin injection. n = 8 per group. (G and H) Insr reintroduction rescues the phenotype observed in Insr−/− mice. LSKs from Insr+/+ or Insr−/− mice were infected with lentivirus encoding the indicated plasmids (G) and then transplanted to lethally irradiated CD45.1 mice together with 3 × 105 CD45.1 BM cells. 16 wk later, numbers of the indicated CD45.2-positive cells were calculated by flow cytometry (H). n = 6 per group. (I and J) Generation of diabetic mice. Mice were injected with streptozotocin (STZ; 80 mg/kg i.p. for four consecutive days; vehicle, 0.1 M sodium citrate, pH 4.5) to induce diabetes. The indicated mice were examined for the levels of blood glucose (I) and plasma insulin (J) at the indicated intervals. (K) Diabetic mice induced by STZ were examined for the indicated cell populations through flow cytometry 8 wk after STZ stimulation. (L) Sorted cells were first stained with Hoechst 33342, and then stained with pyronin Y. Percentages of cells in S/G2/M were calculated. (M) GMPs and CLPs from control and diabetic mice were permeabilized and stained with antibody against active caspase-3. Cells positive for active caspase-3 were calculated. (K–M) n = 7 per group. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are from experiments repeated three times with similar results.
Figure 3.

Insulin signaling drives differentiation of LSKs into lymphoid cells. (A) Sorted WT LSKs were cultured in StemPro-34 medium as described in Materials and methods. For insulin stimulation (With insulin), insulin was added to the medium to a final concentration of 200 nM for 18 h. For insulin depletion (Without insulin), anti-insulin antibody was added to the medium to reach 1 µg/ml for 18 h. Phosphorylation of Insr was detected using antibody against phospho-insulin receptor β (Tyr1150/1151). CM, culture medium. (B) CFC assays of Flk2−LSKs and MPPs pretreated with or without insulin. Sorted cells pretreated with or without 200 nM insulin for 18 h in StemPro-34 medium were seeded in differentiation medium for 10 d and numbers of colonies were calculated. (C) Lymphoid differentiation potential of Flk2−LSKs and MPPs cultured with or without insulin. 1,000 sorted cells, pretreated with or without insulin, were seeded onto OP9 stromal cells (see Materials and methods) and numbers of B220+CD19+ cells were calculated at the indicated intervals. (D) Cells treated with or without insulin were first stained with Hoechst 33342 and then stained with pyronin Y, followed by flow cytometry (left). Percentages of cells in S/G2/M (right). (E) Flk2−LSKs and MPPs treated with or without insulin were permeabilized and stained with antibody against active caspase-3. Cells positive for active caspase-3 were calculated. (D and E) n = 6. (F) 5 IU/kg of insulin or PBS (Vehicle) were injected into mice through a tail vein at the end of the day (8:00 p.m.) six times, and the BM was analyzed for the indicated populations in the morning of the day (8:00 am) after last insulin injection. n = 8 per group. (G and H) Insr reintroduction rescues the phenotype observed in Insr−/− mice. LSKs from Insr+/+ or Insr−/− mice were infected with lentivirus encoding the indicated plasmids (G) and then transplanted to lethally irradiated CD45.1 mice together with 3 × 105 CD45.1 BM cells. 16 wk later, numbers of the indicated CD45.2-positive cells were calculated by flow cytometry (H). n = 6 per group. (I and J) Generation of diabetic mice. Mice were injected with streptozotocin (STZ; 80 mg/kg i.p. for four consecutive days; vehicle, 0.1 M sodium citrate, pH 4.5) to induce diabetes. The indicated mice were examined for the levels of blood glucose (I) and plasma insulin (J) at the indicated intervals. (K) Diabetic mice induced by STZ were examined for the indicated cell populations through flow cytometry 8 wk after STZ stimulation. (L) Sorted cells were first stained with Hoechst 33342, and then stained with pyronin Y. Percentages of cells in S/G2/M were calculated. (M) GMPs and CLPs from control and diabetic mice were permeabilized and stained with antibody against active caspase-3. Cells positive for active caspase-3 were calculated. (K–M) n = 7 per group. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are from experiments repeated three times with similar results.

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