Nkx2.2 regulates beta-cell function in the mature islet

Abstract

Nkx2.2 is a homeodomain transcription factor that is critical for pancreatic endocrine cell specification and differentiation in the developing mouse embryo. The purpose of this study was to determine whether Nkx2.2 is also required for the maintenance and function of the mature beta-cell in the postnatal islet. We have demonstrated previously that a repressor derivative of Nkx2.2 can functionally substitute for endogenous Nkx2.2 to fully restore alpha- and immature beta-cells in the embryonic islet; however, Nkx2.2 activator functions appear to be required to form a functional beta-cell. In this study, we have created transgenic mouse lines to express the Nkx2.2-repressor derivative in the mature beta-cell in the presence of endogenous Nkx2.2. The transgenic mice were assessed for beta-cell function, overall islet structure, and expression of beta-cell-specific markers. Using this transgenic approach, we have determined that the Nkx2.2-repressor derivative disrupts endogenous Nkx2.2 expression in adult mice and causes downregulation of the mature beta-cell factors, MafA and Glut2. Consistently, the Nkx2.2-repressor mice display reduced insulin gene expression and pancreatic insulin content and impaired insulin secretion. At weaning, the male Nkx2.2-repressor mice are overtly diabetic and all Nkx2.2-repressor transgenic mice exhibit glucose intolerance. Furthermore, the loss of beta-cell function in the Nkx2.2-repressor transgenic mice is associated with disrupted islet architecture. These studies indicate a previously undiscovered role for Nkx2.2 in the maintenance of mature beta-cell function and the formation of normal islet structure.

FIG. 1

Expression of Nkx2.2 derivatives in the adult pancreas. Nkx2.2 control (A) and repressor (B) derivatives are expressed in the pancreas under control of the 4.5-kilobase Pdx1 promoter. 2.2HD, Nkx2.2 homeodomain; myc, myc epitope tag; EnR, Engrailed repressor domain. C: Anti-engrailed Western blot to detect expression of the 50-kDa Nkx2.2-repressor protein in lines 7414, 7566, and 7546. D: Anti-myc Western blot to detect expression of a 18-kDa Nkx2.2hdmyc fusion protein in lines 5635 and 5631. In both C and D, anti-lamin B was used as a loading control for nuclear lysates. E: Quantitative real-time PCR for Nkx2.2hdEnR transgene in transgenic mice (TG) and endogenous Nkx2.2 in wild-type (WT) littermates (L7414; n = 3). P < 0.005.

FIG. 2

Nkx2.2-repressor transgenic mice are glucose intolerant with impaired insulin secretion. Two- to 3-month-old mice were fasted overnight and injected with glucose (A, B, and C) or arginine (E and F) at t = 0. Blood glucose levels were measured and tail vein blood collected for insulin enzyme-linked immunosorbent assays. Plasma glucose or plasma insulin concentrations are indicated. A, C, E, and F: Male mice: □, wild-type (WT); ■, Nkx2.2-repressor transgenic (TG) (L7414); ◇, control transgenic (L5635 and L5631); △, Nkx2.2-activator transgenic (L7319 and L7318). B: Female mice: ◇, wild-type; ◆, transgenic (L7414). *P < 0.005; ** P < 0.0001. D: Quantitative real-time PCR on RNA isolated from whole pancreata for insulin mRNA indicated insulin mRNA is decreased in Nkx2.2-repressor transgenic mice.

FIG. 3

Neither male nor female Nkx2.2-repressor transgenic mice are insulin resistant. To determine insulin tolerance, 2- to 3-month-old mice were fasted overnight and injected with insulin at t = 0 and blood glucose levels measured. Plasma glucose concentrations are indicated. A: Male mice: □, wild-type (WT); ■, transgenic (TG) (L7414). B: Female mice ○, wild-type; •, transgenic (L7414).

FIG. 4

Real-time PCR demonstrates that Nkx2.2, MafA and Glut2 are downregulated in Nkx2.2hdEnR islets. Real-time PCR for select islet genes was performed using total RNA from isolated islets of wild-type (■) and Nkx2.2-repressor () mice (n = 4 each; islets were pooled from two mice for each sample). Nkx2.2, MafA, and Glut2 are significantly decreased in Nkx2.2-repressor islets. *P < 0.005. All other genes are not significantly changed. Error bars represent SEM of four individual samples for each genotype.

FIG. 5

Islet architecture is disrupted in Nkx2.2-repressor transgenic mice. Hematoxylin and eosin staining of wild-type (A), Nkx2.2-repressor (L7414) (E) or Nkx2.2 control (L5635) (I) islets demonstrates that transgenic islets are of normal size and morphology. Hormone immunostaining for insulin (red in all panels) and glucagon (green in B, D, F, H, and J) or somatostatin (green in C and G) show that islet organization is disrupted in the Nkx2.2-repressor mice (F, G, and H) in adult islets (B, C, F, G, and J) and in embryonic day 18.5 islets (D and H) compared with wild-type (B–D) or Nkx2.2-control islets (J). Magnification ×20.

FIG. 6

Markers of terminally differentiated β-cells are reduced in Nkx2.2hdEnR mice. A–F: Confocal images of insulin staining (red) with Pdx1 (green) (A–C) or MafA (green) (D–F) demonstrates all insulin-positive cells coexpress Pdx1, but only ~50% of insulin-positive cells coexpress MafA in Nkx2.2-repressor islets (B and E) compared with wild-type islets (A and D) or Nkx2.2hdmyc control islets (C and F) at embryonic day 18.5. E: Arrows indicate MafA-negative cells in Nkx2.2-repressor islets. G–J: Confocal images of glucagon (red) and glut2 (green) staining in neonatal and adult islets indicate that glut2 is significantly reduced at embryonic day 18.5 and lost in adult islets of Nkx2.2-repressor mice. Arrowheads indicate glut2-positive cells (H).