Mitochondrial complex I subunit deficiency promotes pancreatic α-cell proliferation

Abstract

Objective: There is strong evidence that mitochondrial DNA mutations and mitochondrial dysfunction play a role in diabetes pathogenesis. The homozygous knock-in mtDNA mutator mouse is a model of premature aging due to the accumulation of mitochondrial DNA mutations. We used this mouse model to investigate the relationship between mitochondrial subunit expression and pancreatic islet cell composition.

Methods: Quadruple immunofluorescence was used to quantify mitochondrial subunit expression (complex I and IV) and cell composition in pancreatic islets from mitochondrial DNA mutator mice (PolgA^mut/mut) and control C57BL/6 mice at 12 and 44 weeks of age.

Results: Mitochondrial complex I subunit expression was decreased in islets from 12 week PolgA^mut/mut mice. This complex I deficiency persisted with age and was associated with decreased insulin staining intensity at 44 weeks. Complex I deficiency was greater in α-cells compared with β-cells in islets from 44 week PolgA^mut/mut mice. Islet cell composition was normal in 12 week PolgA^mut/mut mice, but the β: α cell ratio was decreased in islets from 44 week PolgA^mut/mut mice. This was due to an increase in α-cell number linked to an increase in α-cell proliferation.

Conclusion: Complex I deficiency promotes α-cell proliferation and alters islet cell composition.

Keywords: Mitochondria; Pancreatic islets; mtDNA; mtDNA mutator mice.

Figure 1

Decreased complex I expression is present in PolgA^mut/mut islets from 12 week mice and persists with advancing age. (A): Representative immunofluorescent panel showing labelling of mitochondrial proteins in pancreatic islets from 12 week to 44 week mice. Scale bar, 100 μm. The dashed white line marks the islet perimeter. (B–E): Quantitative analysis of levels of TOMM20 (mitochondrial mass) (B), NDUFB8 (Complex I) (C) MTCO1 (Complex IV) (D), and insulin (E), in islets from 12 week PolgA^mut/mut and PolgA^+/+ mice. Data are presented as Z-scores relative to the PolgA^+/+ mice, n = 4 mice per group. (F–I): Quantitative analysis of levels of TOMM20 (F), NDUFB8 (G) MTCO1 (H), and insulin (I), in islets from 44 week PolgA^mut/mut and PolgA^+/+ mice. Data are presented as Z-scores relative to the PolgA^+/+ mice, n = 5 mice per group. For panels B–I each point represents an individual islet (n = 50 per mouse). Data are presented as mean ± 95% CI. Unpaired t-test. ∗P < 0.05. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Figure 2

Islets from 44 week PolgA^mut/mut mice have increased numbers of α cells resulting in altered islet cell composition (A): Representative immunofluorescent panel showing labelling of nuclei (DAPI), α-cells (Glucagon) and β-cells (insulin) in islets of 44 week PolgA^mut/mut and PolgA^+/+ mice. Scale bar, 100 μm. (B–G): Quantification of the islet size (B), total cell number (C), α-cell percentage (D), β-cell percentage (E), absolute α-cell number (F), and absolute β-cell number (G) in 44 week PolgA^+/+ (n = 4) and PolgA^mut/mut (n = 5) mice. Each point represents one islet (n = 25 for each mouse). Data are presented as mean ± 95% CI. Unpaired t-test. ∗P < 0.05. ∗∗∗P < 0.001. Microscope settings were individually adjusted according to each islet.

Figure 3

Complex I expression is lower in α-cells compared with β-cells in islets from 44 week PolgA^mut/mut mice. A–C: Quantification of levels of TOMM20 (A), NDUFB8 (B) and MTCO1 (C) in α-cells and β-cells in islets from 44 week PolgA^+/+ (n = 5) and PolgA^mut/mut mice (n = 5). Data are presented as Z-scores relative to the PolgA^+/+ β-cells. Each point represents an individual islet (n = 50 for each mouse). Data are presented as mean ± 95% CI. Unpaired t-test. ∗∗P < 0.01. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Figure 4

Islets from 44 week PolgA^mut/mut mice show an increased frequency of proliferating α-cells compared with age matched controls (A): Composite quadruple immunofluorescence panel showing labelling of; nuclei (DAPI, blue); proliferating cells (Ki67, white (highlighted by yellow arrows)); α-cells (Glucagon, red); and β-cells (Insulin, green) in pancreatic islets. (Top panel) PolgA^+/+ mice; (Bottom panel) PolgA^mut/mut mice. Scale bar, 100 μm. (B–D): Quantification of Ki67 positive labelling in PolgA^mut/mut and PolgA^+/+ mice. (B) Mean percentage of islets per mouse with ≥1 Ki67(+) cells. (C) Mean percentage of islets per mouse with ≥1 Ki67(+) α-cells. (D) Mean percentage of islets per mouse with ≥1 Ki67(+) β-cells. PolgA^+/+ mice (n = 4); PolgA^mut/mut mice (n = 4). Each dot represents the mean percentage per mouse (40 islets were quantified per mouse). Data are presented as mean ± 95%CI. Unpaired t-test. ∗P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)