Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells

Abstract

Aim/hypothesis: Neonatal beta cells lack glucose-stimulated insulin secretion and are thus functionally immature. We hypothesised that this lack of glucose responsiveness results from a generalised low expression of genes characteristic of mature functional beta cells. Important glucose-responsive transcription factors, Mafa and Pdx1, regulate genes involved in insulin synthesis and secretion, and have been implicated in late beta cell development. The aim of this study was to assess whether Mafa and/or Pdx1 regulates the postnatal functional maturation of beta cells.

Methods: By quantitative PCR we evaluated expression of these and other beta cell genes over the first month compared with adult. After infection with adenovirus expressing MAFA, Pdx1 or green fluorescent protein (Gfp), P2 rat islets were evaluated by RT-PCR and insulin secretion with static incubation and reverse haemolytic plaque assay (RHPA).

Results: At P2 most beta cell genes were expressed at about 10% of adult, but by P7 Pdx1 and Neurod1 no longer differ from adult; by contrast, Mafa expression remained significantly lower than adult through P21. Overexpression of Pdx1 increased Mafa, Neurod1, glucokinase (Gck) mRNA and insulin content but failed to enhance glucose responsiveness. Similar overexpression of MAFA resulted in increased Neurod1, Nkx6-1, Gck and Glp1r mRNAs and no change in insulin content but, importantly, acquisition of glucose-responsive insulin secretion. Both the percentage of secreting beta cells and the amount of insulin secreted per beta cell increased, approaching that of adult beta cells.

Conclusions/interpretation: In the process of functional maturation acquiring glucose-responsive insulin secretion, neonatal beta cells undergo a coordinated gene expression programme in which Mafa plays a crucial role.

Fig. 1

Expression pattern of beta cell transcription factors during the first postnatal month. a Beta cell transcription factors Neurod1 (black circle), Pdx1 (white circle), Mafa (black triangle), Nkx6-1 (white diamond); (b) other key genes: Glp1r (black diamond), Pc (white square), Pcsk1 (white triangle), insulin (black circle), Glut2 (black square) and Gck (white circle) mRNA show different patterns of expression over the neonatal period as measured by quantitative RT-PCR. Data expressed as fold change with respect to adult using S25 as internal control gene. Mean ± SEM, n=4–6 isolated samples per age, each pooled from three to ten animals. The same samples were analysed in Fig. 1a, b and ESM Fig. 1. Quantification of the beta cell proportion showed isolated neonatal islets did not significantly differ from adult in % beta cell [10]. Only P11 had significantly lower % beta cell than adult; P11 also had significantly lower than P1–2 but not P21. Thus the changes in gene expression are likely to be due to changes in phenotype rather than proportion of beta cells. By western blot, MAFA protein (c) and PDX1 (d) protein are low at birth (P2). MAFA increases at P11 but is still low compared with adult. PDX1 increased at P11 and decreased in adult islets in a pattern similar to that of mRNA levels at the same ages. Representative gels of three independent samples. Ad, adult

Fig. 2

PDX1 protein levels change in intensity during the postnatal period. a By immunostaining, PDX1 protein (green) in insulin⁺ cells (red) is low at birth (P2) and increases progressively until most beta cells are PDX1^high in adults. b Quantification of nuclear PDX1 levels shows that already at P2 almost all insulin positive cells produced detectable PDX1 and the intensity of staining increases with age. Undetectable PDX1 (black bars), PDX 1^low (white bars), PDX 1^high (grey bars). Total insulin⁺ cells counted: P2, 463; P11, 2,286; adult, 1,092 (five pancreases per age). Mean ± SEM. *p<0.05 with respect to previous age

Fig. 3

MAFA protein levels change in intensity and localisation during the postnatal period. a By immunostaining, MAFA protein is low at birth (P0) and mainly cytoplasmic at 36 h after birth. By P15 it has increased and has more nuclear localisation. In adult islets MAFA protein is clearly nuclear. b Quantification of nuclear MAFA levels from immunofluorescent images (see ESM Fig. 2) showed at P2 only 30% MAFA^low insulin positive cells (white bar) and less than 5% MAFA^high (grey bar) while the rest had undetectable MAFA levels (black bar). In the adult 95% of beta cells were MAFA⁺ (high+low). Total insulin⁺ cells counted: P2, 654 cells from four pancreases; P11, 1,140 cells from four pancreases; adult, 2,127 cells from five pancreases. Mean ± SEM; *p<0.05 with respect to previous age

Fig. 4

Proteins of selected genes have similar age-dependent increases as mRNA. Insulin (a), GLP1R (b) and PCSK1 (c) had very low levels of immunostaining at P2, increasing until adult. To allow comparison of protein levels at different ages paraffin sections were immunostained in parallel and photographed in confocal mode at same settings. Ad, adult

Fig. 5

Effect of adenoviral-mediated increase of Pdx1 and Mafa on genes important for beta cell function. After 72 h culture adenoviral-mediated overexpression of Pdx1 (a) or MAFA (b) P2 islets had significant increases of total Mafa mRNA (hMAFA; see Methods) and total Pdx1 mRNA. Overexpression of Pdx1 upregulated Mafa, Neurod1 and Gck whereas overexpression of Mafa had upregulated Neurod1, Nkx6-1, Gck and Glp1r. Quantitative RT-PCR; expression compared with Ad-Gfp infected cells (equal to 1, dotted line). Mean ± SEM, n=4–6 independent experiments; *p<0.05

Fig. 6

Insulin secretion in static incubation and insulin content after Ad-Pdx1, Ad-MAFA and Ad-Gfp infection. a Insulin secretion in response to 16.8 mmol/l (white bar) glucose increased in both Ad-Pdx1 and Ad-MAFA infected P2 cells compared with Ad-Gfp infected cells after 5 days culture. However, insulin secretion in 2.6 mmol/l (black bar) glucose from Ad-MAFA infected cells significantly decreased while that from Ad-Pdx1 increased almost as much as with high glucose.*p<0.05 in marked comparisons. b Insulin secretion expressed as fold change in response to glucose stimulation reflects the glucose responsiveness of the cultured cells. As previously shown, neonatal (P2) islets have little glucose responsiveness when compared with adult islets similarly cultured (Ad-GfpP2: 7±2 pg insulin/ng DNA at 2.8 mmol/l and 9±2 at 16.8 mmol/l; Ad-Gfp adult: 11±2 pg insulin/ng DNA at 2.8 mmol/l and 47±13 at 16.8 mmol/l). Only Ad-MAFA increased the glucose responsiveness of the P2 cells; the uninfected, Ad-Gfp and Ad-Pdx1 infected cells had little to no response to the increased glucose concentration. *p<0.05 compared with untreated cultured P2 cells (c). c There was no change in insulin content (pg/ng DNA) in Ad-MAFA infected P2 islets and cultured control islets (untreated or Ad-Gfp infected), but insulin content was significantly higher in Ad-Pdx1 compared with cultured uninfected (c) cells. Mean ± SEM; n=4 independent experiments in duplicate. *p<0.05 compared with untreated (c) cultured P2 cells

Fig. 7

Mafa expression increases percentage of secreting cells and insulin secreted per beta cell as measured by reverse haemolytic plaque assay. a After Ad-MAFA infection, cultured P2 cells have increased insulin secretion index in response to glucose stimulation; 2.6 mmol/l (black bar), 16.8 mmol/l (white bar). Mean ± SEM; n=3 independent experiments. *p<0.03 respect to Ad-Gfp P2. Total number of plaques counted per condition: P2 untreated (c), 223; Ad-Gfp P2, 595; Ad-MAFA P2, 463; untreated (c) cultured adult, 188; cultured adult Ad-Gfp, 256. b The percentage of insulin-secreting beta cells increased after Ad-MAFA infection. *p<0.005 respect to Ad-Gfp P2; 2.6 mmol/l (black bar), 16.8 mmol/l (white bar). c In response to stimulatory 16.8 mmol/l glucose, more Ad-MAFA cells are secreting and with greater individual secretion, as seen in this plot of the subpopulations from a representative experiment. P2 Adv-Gfp (white square), P2 Adv-MAFA (black circle), adult control (white triangle)