The characteristics of NKX6.1 defined β-cell dedifferentiation
By immunofluorescence staining with insulin and NKX 6.1 in human pancreatic tissue sections, we identified two types of dedifferentiated β cells with NKX6.1 inactivation: 1) Cells that are insulin-positive but without nucleic NKX6.1 expression are at the early stage of dedifferentiated β cells (NKX6.1Nuc-Ins+, including β cells with cytoplasmic NKX6.1 expression or no NKX6.1 expression, Fig. 1a-b); 2) Cells that are insulin-negative but with cytoplasmic expression of NKX6.1 are at the late stage of dedifferentiated β cells (NKX6.1cytIns-, Fig. 1a-b). The mean absolute number of NKX6.1Nuc-Ins+ cells per islet was 2.69 times that of NKX6.1cytIns- cells. In addition, we also captured the β cells with the expression of NKX6.1 in both nucleus and cytoplasm in the earlier stage of dedifferentiated β cells, but that was rare. NKX6.1 dislocation or lost expression were signs of NKX6.1 inactivation. These results suggest different stages of NKX6.1 inactivation in the dynamic process of β-cell dedifferentiation.
The level of β cell dedifferentiation with NKX6.1 inactivation was elevated and positively correlated with HbA1c in Type 2 diabetic subjects
We next evaluated the level of β-cell dedifferentiation with NKX6.1 inactivation in ND and T2DM subjects (Fig. 2a). The absolute number of NKX6.1Nuc-Ins+ cells per islet in T2DM subjects were increased by 24.41% (Fig. 2b, P < 0.05), and the percentage in β cells increased by 61.56%, compared with ND subjects (Fig. 2b, P < 0.001). The absolute number and percentage of NKX6.1cytIns- cells per islet in T2D subjects increased by 2.41 and 2.31 fold, respectively (Fig. 2c, P < 0.001, P < 0.001). We also found NKX6.1Nuc-Ins+ cells have a significant correlation with HbA1c in T2DM subjects (Fig. 2d, P = 0.03). These results suggest that the level of β-cell dedifferentiation in T2DM islet is elevated and aggravated by hyperglycemia.
Modest hyperglycemia, advanced age, or obesity did not affect the level of β cell dedifferentiation with NKX6.1 inactivation
To evaluate the effect of modest hyperglycemia on β-cell dedifferentiation with NKX6.1 inactivation, the non-diabetic samples were further divided into the non-prediabetic group with normal glycaemia (HbA1c <5.7%) and the prediabetic group with modest hyperglycemia (HbA1c: 5.7-6.4%). The absolute number of NKX6.1Nuc-Ins+ cells per islet was 10.45 ± 0.91 in non-prediabetes and 9.00 ± 1.67 in prediabetes, respectively, and the percentage in β cells was 16.31 ± 1.15% and 16.68 ± 2.40% (Table 3). The absolute number of NKX6.1cytIns- cells per islet was 2.95 ± 0.55 and 2.04 ± 0.91, respectively, and the percentage per islet was 3.57 ± 0.69% and 2.84 ± 1.15%, respectively (Table 3).
To analyze the effects of aging on β-Cell dedifferentiation with NKX6.1 inactivation, we further divided non-diabetic subjects into four groups with ages of 30-39, 40-49, 50-59, and 60-79, respectively. The absolute number of NKX6.1Nuc-Ins+ cells per islet was 9.32 ± 4.20, 11.63 ± 0.92, 8.95 ± 1.29, and 8.57 ± 2.07 in the four groups respectively, the percentages in β cells was 13.19 ± 3.56%, 18.20± 1.16%, 15.72 ± 2.02%, and 14.40 ± 4.03%, respectively (Table 3). The absolute number of NKX6.1cytIns- cells per islet was 3.05 ± 1.16, 2.71 ± 0.82, 2.16 ± 0.58, and 4.67 ± 2.07, the percentage per islet was 3.66 ± 1.38%, 3.24 ± 1.00%, 3.00 ± 0.81%, and 5.34 ± 2.64%, respectively (Table 3).
To analyze the effects of obesity on β-cell dedifferentiation with NKX6.1 inactivation, non-diabetic samples were divided into four groups according to the BMI standards for Asians special BMI: <23 (normal), 23-25 (overweight), 25-30 (obesity), ≥30 (over-obesity). The absolute number of NKX6.1Nuc-Ins+ cells per islet was 12.64 ± 1.64, 9.69 ± 1.71, 9.41 ± 1.24, and 8.18 ± 1.85, respectively, the percentage in β cells was 18.46 ± 1.35%, 15.90 ± 2.21%, 15.92 ± 1.96%, and 14.40 ± 2.73%, respectively (Table 3). The absolute number of NKX6.1cytIns- cells per islet was 3.10 ± 0.87, 3.65 ± 1.81, 2.45 ± 0.69, and 1.98 ± 0.53, respectively, and the percentage per islet was 3.80 ± 1.15%, 4.39 ± 2.21%, 3.05 ± 0.85%, and 2.61 ± 0.67%, respectively (Table 3).
By statistical analysis, we found that there was no association between the number and percentage of NKX6.1Nuc-Ins+ and NKX6.1cytIns- cells and modest hyperglycemia, advanced age, and obesity. Further correlation analysis between NKX6.1Nuc-Ins+ or NKX6.1cytIns- versus age/BMI/HbA1c found no significant correlation (Additional file 1: Supplementary Fig. 1-3). These results suggested that higher risk factors for type 2 diabetes did not affect β-cell dedifferentiation in the non-diabetic subjects.