Microphthalmia transcription factor regulates pancreatic β-cell function

Abstract

Precise regulation of β-cell function is crucial for maintaining blood glucose homeostasis. Pax6 is an essential regulator of β-cell-specific factors like insulin and Glut2. Studies in the developing eye suggest that Pax6 interacts with Mitf to regulate pigment cell differentiation. Here, we show that Mitf, like Pax6, is expressed in all pancreatic endocrine cells during mouse postnatal development and in the adult islet. A Mitf loss-of-function mutation results in improved glucose tolerance and enhanced insulin secretion but no increase in β-cell mass in adult mice. Mutant β-cells secrete more insulin in response to glucose than wild-type cells, suggesting that Mitf is involved in regulating β-cell function. In fact, the transcription of genes critical for maintaining glucose homeostasis (insulin and Glut2) and β-cell formation and function (Pax4 and Pax6) is significantly upregulated in Mitf mutant islets. The increased Pax6 expression may cause the improved β-cell function observed in Mitf mutant animals, as it activates insulin and Glut2 transcription. Chromatin immunoprecipitation analysis shows that Mitf binds to Pax4 and Pax6 regulatory regions, suggesting that Mitf represses their transcription in wild-type β-cells. We demonstrate that Mitf directly regulates Pax6 transcription and controls β-cell function.

FIG. 1.

Mitf is found in all five hormone-expressing cell types in the pancreas. A–D: Mitf (green) is detected in the nuclei of hormone-expressing (red) cells at P0. E–H: Mitf expression was observed in insulin-, glucagon-, somatostatin-, and PP-producing cells in islets of 12-week-old mice. Arrows denote Mitf hormone–positive cells.

FIG. 2.

Improved glucose tolerance and increased insulin secretion in Mitf^ce/ce mice. A: Blood glucose levels from random-fed and 6-h-fasted wild-type (wt/het) and Mitf mutant mice, n ≥ 9. B: Plasma glucagon levels from random-fed and 6-h-fasted wild-type and Mitf^ce/ce animals, n ≥ 3. C and D: IPGTT (with overnight, 12-h fasting) of 12-week (C) and 6-month-old (D) Mitf mutant and wild-type mice. Blood glucose measurements were taken at 0, 5, 15, 30, 60, and 120 min after the intraperitoneal glucose injection (2 g of D(+) glucose/kg body wt). E: In vivo glucose-stimulated insulin secretion: intraperitoneal glucose injection with 2 g D(+) glucose/kg body wt. Insulin levels were measured 0, 2, 5, and 15 min after injection; n ≥ 10. t test, *P value <0.05, **P value < 0.01. F: Insulin secretion profile of Mitf^ce/ce mice. Islets isolated from Mitf^ce/ce and control mice treated with different glucose concentrations (2.8, 8.3, and 16.7 mmol/L glucose and 16.7 mmol/L glucose plus 35 mmol/L KCl). Insulin levels were assessed with insulin ELISA; n ≥ 3. **P value < 0.01 with two-way ANOVA.

FIG. 3.

Wild-type (wt/het) and Mitf^ce/ce mutant islets consist of insulin, glucagon, somatostatin, and PP cells (A). B: Average β-cell area is unchanged in Mitf^ce/ce animals; n = 3. C: Quantitative RT-PCR measurement of pancreatic hormone levels in Mitf mutant and wild-type islets (n > 5 per genotype.) The data were normalized to HPRT mRNA levels and are presented as relative to control (set as 1). t test, *P < 0.05, ***P < 0.001.

FIG. 4.

Twelve-week-old Mitf^ce/ce mice show no obvious differences in the expression pattern of markers characteristic for functional β-cells when analyzed by immunohistochemistry. A and B: Pax6 (green) and insulin (red). C and D: Pdx1 (green) and insulin (red). E and F: MafA (green) and insulin (red). G and H: Nkx6.1 (green) and insulin (red). I and J: GLUT2 (green) and insulin (red). K–O: Quantitative RT-PCR analysis of expression of Pax6, Pdx1, MafA, Nkx6.1, and GLUT2 in mutant (Mitf^ce/ce) and wild-type (wt/het) islets; n > 5 per genotype. t test, *P < 0.05, **P < 0.001. INS, insulin.

FIG. 5.

Quantitative gene expression profile of pancreatic transcription factors and glucose metabolism–related (A) and insulin secretory (B) genes in Mitf mutant (Mitf^ce/ce) and wild-type (wt/het) islets (n ≥ 4 per genotype). The data were normalized to HPRT mRNA and are presented relative to control (set to 1). IA-2, insulinoma-associated protein-2; IAPP, islet amyloid polypeptide. ***P < 0.001.

FIG. 6.

Mitf binds to and regulates Pax6 expression in β-TC6 cells. Pancreas-specific Pax6 expression is partially regulated by upstream regulatory elements (summarized in ref. 25). A: The pPax6P0-LUC construct contains the EE and PE elements. B: ChIP analysis was performed on β-TC6 cells transfected with an expression vector containing FLAG-tagged Mitf. The anti-FLAG precipitated chromatin was analyzed by nonquantitative PCR using primers specific for Pax4, Pax6^EE, Pax6^PE, and PEPCK control region sequences. C: ChIP analysis of α-TC6 cells transfected with FLAG-tagged Mitf. PCR analysis with Pax4, Pax6^EE, Pax6^PE, and Runx2 control region sequences. Control PCR reactions were run with input chromatin (1:100 dilution), and DNA was obtained after precipitation with mouse IgG or no DNA. ChIP analysis was repeated from at least three independent chromatin preparations. D: Dual luciferase reporter assays show that pPax6P0-LUC activity is enhanced by cotransfection with Pax6 in HEK293 cells but repressed by cotransfection with Mitf.