Postnatal β-cell proliferation and mass expansion is dependent on the transcription factor Nkx6.1

Abstract

All forms of diabetes are characterized by a loss of functional β-cell mass, and strategies for expanding β-cell mass could have significant therapeutic benefit. We have recently identified the transcription factor Nkx6.1 as an essential maintenance factor of the functional β-cell state. In addition, Nkx6.1 has been proposed to control β-cell proliferation, but a role for Nkx6.1 in regulating β-cell mass has not been demonstrated. Here, we show that Nkx6.1 is required for postnatal β-cell mass expansion. Genetic inactivation of Nkx6.1 in newly formed β-cells caused a drastic decrease in early postnatal β-cell proliferation, leading to reduced β-cell mass and glucose intolerance. Interestingly, Nkx6.1 was dispensable for prenatal β-cell proliferation. We found that Nkx6.1 regulates the expression of several β-cell maturation markers as well as expression of the nutrient sensors Glut2 and Glp1r. Manifestation of the β-cell mass defect at the transition to postnatal feeding suggests that Nkx6.1 could regulate β-cell growth by enabling β-cells to respond to nutrient-dependent proliferation signals, such as glucose and Glp1. Identification of β-cell-intrinsic regulators that connect nutrient-sensing and proliferation suggests new therapeutic targets for expanding functional β-cell mass.

Figure 1

Nkx6.1 deletion in newly formed β-cells leads to glucose intolerance and reduced β-cell mass. A: Schematic of alleles and transgenes used to inactivate Nkx6.1 in fetal β-cells. Rectangles show coding sequences; triangles show loxP sites; red rectangle shows DsRed coding sequence. B and C: Immunofluorescence staining for Nkx6.1 and insulin reveals loss of Nkx6.1 in most β-cells of Nkx6.1^∆β mice at P0. D: Blood glucose levels in 6-week-old Nkx6.1^∆β mice fed ad libitum compared with control mice (n = 6). E: Intraperitoneal glucose tolerance test shows glucose intolerance in 6-week-old Nkx6.1^∆β mice as compared with control mice (n = 6). F: Quantification of β-cell mass reveals decreased β-cell mass in Nkx6.1^∆β mice at 6 weeks of age (n = 3). Data shown as mean ± SEM. Scale bars = 20 μm. Ins, insulin; YFP, yellow fluorescent protein. *P < 0.05; **P < 0.01.

Figure 2

Nkx6.1 is required for postnatal β-cell mass expansion. A: Quantification of the insulin immunofluorescent area relative to total pancreatic area reveals no difference in β-cell mass between Nkx6.1^∆β and control mice at P0 and a slight but not significant decrease at P4 (n = 3). B–G’’: Immunofluorescence staining for insulin, Nkx6.1, and YFP at P0 (B–C’’), P4 (D–E’’), and 6 weeks of age (F–G’’). H: Quantification of insulin⁺ cells expressing YFP at P0, P4, and 6 weeks shows a progressive decrease of YFP⁺ recombined β-cells in Nkx6.1^∆β mice postnatally (n = 3). I: Quantification of insulin⁺ cells expressing Nkx6.1 reveals a progressive increase of Nkx6.1-expressing unrecombined β-cells in Nkx6.1^∆β mice between P0 and 6 weeks of age (n = 3). Data shown as mean ± SEM. Scale bar = 20 μm. Ins, insulin; YFP, yellow fluorescent protein. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Nkx6.1 is required for postnatal β-cell proliferation. A–C: β-Cells are not apoptotic at P4 in Nkx6.1^∆β or control mice based on TUNEL combined with immunofluorescence staining for insulin and DAPI. TUNEL⁺ cells in the pancreas are shown as a positive control (arrowheads) and TUNEL⁺insulin⁺ cells were quantified. D–G’’’: Immunofluorescence staining for insulin, Ki67, and YFP at P0 and P4. H: Quantification of the percentage of insulin⁺YFP⁺ cells expressing Ki67 shows decreased β-cell proliferation in Nkx6.1^∆β mice at P4, but not at P0 (n = 3). I: Quantification of Ki67-expressing YFP⁺insulin⁺ cells and YFP⁻insulin⁺ cells in Nkx6.1^∆β mice at P4 reveals a selective decrease in proliferation of recombined compared with unrecombined β-cells within the same animal (n = 3). Data shown as mean ± SEM. Scale bar = 20 μm. Ins, insulin; YFP, yellow fluorescent protein. *P < 0.05; **P < 0.01.

Figure 4

Nkx6.1 inactivation leads to a cell autonomous loss of β-cell maturation and nutrient sensing markers. Immunofluorescence staining for insulin, Pdx1, and YFP (A–C’’’) or insulin, MafA, and YFP (D–F’’’) shows Pdx1 but not MafA expression in recombined YFP⁺insulin⁺ cells of Nkx6.1^∆β mice at P4. Unrecombined YFP⁻insulin⁺ cells express Pdx1 and MafA in Nkx6.1^∆β mice. G: qRT-PCR analysis of pancreata from Nkx6.1^∆β and control mice at P2 for genes associated with β-cell maturation (n = 3). Immunofluorescence staining for insulin, Ucn3, and YFP (H–J’’’); insulin, Glut2, and YFP (K–M’’’); or insulin, Glp1r, and YFP (N–P’’’) shows loss of Ucn3, Glut2, and Glp1r expression in recombined YFP⁺insulin⁺ cells but not in unrecombined YFP⁻insulin⁺ cells of Nkx6.1^∆β mice at P4. For each marker, representative areas are shown in lower panels for Nkx6.1^∆β mice, as indicated by a dashed box in the merged middle panel. White arrowheads point to recombined YFP⁺insulin⁺ cells and blue arrowheads to unrecombined YFP⁻insulin⁺ cells. Data shown as mean ± SEM. Scale bar = 20 μm. Ins, insulin; YFP, yellow fluorescent protein. *P < 0.05; **P < 0.01.