Evidence of increased islet cell proliferation in patients with recent-onset type 1 diabetes
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- Willcox, A., Richardson, S.J., Bone, A.J. et al. Diabetologia (2010) 53: 2020. doi:10.1007/s00125-010-1817-6
In adults, the rate of beta cell replication is normally very low, but recent evidence suggests that it may increase during insulitis. We therefore studied tissue from donors with recent-onset type 1 diabetes to establish whether islet cell proliferation is increased during the disease process.
Paraffin-embedded pancreatic sections from ten donors with recent-onset type 1 diabetes and a range of relevant controls were stained by immunohistochemical techniques with antibodies against the proliferation markers Ki67 and minichromosome maintenance protein-2 (MCM-2). A combination staining technique involving immunoperoxidase and immunofluorescence methods was developed to quantify the numbers of alpha and beta cells with Ki67-positive nuclei and to investigate the relationship between insulitis and islet cell proliferation.
In non-diabetic control donors, only 1.1 ± 0.3% (mean ± SEM) of islets contained one or more Ki67+ islet cells, whereas this proportion was increased markedly in recent-onset type 1 diabetes (10.88 ± 2.5%; p < 0.005). An equivalent increase in Ki67+ staining occurred in alpha and beta cells and was correlated positively with the presence of insulitis. A significant increase in the labelling of islet cells from type 1 diabetic donors was also seen when MCM-2 staining was employed. Increased islet cell proliferation was not evident in three donors with longer duration type 1 diabetes or in ten type 2 diabetic donors.
Alpha and beta cells undergo a marked increase in proliferation during the progression of type 1 diabetes in humans. The results imply that islet cell proliferation is re-initiated in response to the autoimmune attack associated with type 1 diabetes.
KeywordsAlpha cell Beta cell Human Immunohistochemistry Insulitis Ki67 Minichromosome maintenance protein 2 Proliferation Type 1 diabetes
Gap (in cell cycle)
Minichromosome maintenance protein 2
Type 1 diabetes is a T cell-mediated autoimmune disease in which the insulin-secreting beta cells of the islets of Langerhans are destroyed selectively . Clinical symptoms arise when ∼70% of the total beta cell mass is lost, a process that follows a relatively protracted time course (months to years), during which the net rate of cell loss must exceed the rate of beta cell replacement .
Beta cell proliferation has been studied to only a limited extent in humans, but the available evidence implies that this process occurs at relatively high levels during the first 2 years of life and declines rapidly thereafter [3, 4]. However, it is also clear that, at least in animals, beta cell proliferation can be re-induced in adulthood under conditions when insulin resistance is increased, for example during pregnancy or in obesity [5, 6, 7]. This suggests that beta cells may retain an intrinsic capacity to replicate, but that they become largely quiescent during early childhood.
Recent evidence from a single case report  has revealed that enhanced proliferation of beta cells occurred in an adult human who was characterised as ‘prediabetic’ by virtue of the presence of circulating islet autoantibody positivity and active insulitis on histological examination of the pancreas. This proliferative response was observed prior to the onset of clinical symptoms, thereby raising the possibility that a compensatory increase in beta cell proliferation may occur early in the development of type 1 diabetes in humans. This would be consistent with evidence from animal models, where enhanced beta cell proliferation has also been seen early in the disease process [9, 10, 11], but this evidence differs from the conclusions of Butler et al. , who did not detect enhanced beta cell replication in pancreas sections recovered from a cohort of newly diagnosed type 1 diabetic patients. It is important, therefore, to clarify whether enhanced beta cell proliferation occurs during the course of human type 1 diabetes. If this were to be the case, then it might offer a window of opportunity for therapeutic intervention to replenish beta cell mass, in parallel with attempts to limit the rate of autoimmune-mediated cell loss. One recent study reported the detection of insulin-positive cells in 88% (37/42) of patients with long-standing type 1 diabetes, suggesting a potentially substantial window of opportunity for intervention with potential regenerative therapies .
To address this issue more fully, we analysed pancreatic tissue from a cohort of patients with recent-onset type 1 diabetes. Two independent markers of cell proliferation were studied using both light and fluorescence microscopy to identify and quantify proliferating endocrine cells.
We selected ten human pancreases recovered from deceased patients with recent-onset type 1 diabetes (disease duration ≤ 18 months) who are members of a cohort that has been used previously .
Details of donors with recent-onset type 1 diabetes
Duration of diabetes
Cause of death
Ki67+ islets (n)
ICI with insulitis (n)
IDI with insulitis (n)
Unspecified but recent
Glioma raised intracranial pressure
Details of primary antibodies used
Dako, Ely, UK
Abcam, Cambridge, UK
Combined immunoperoxidase and immunofluorescence immunohistochemistry
A combination technique was used, in which sections were dual-stained using the immunoperoxidase technique described above, followed by immunofluorescence labelling of the same sections. Anti-Ki67 and anti-MCM-2 were detected on separate sections using the immunoperoxidase technique, while endocrine cells and lymphocytes were detected using immunofluorescence. After counterstaining with haematoxylin, Ki67 immunoperoxidase-stained sections from donors with recent-onset type 1 diabetes and from the non-diabetic controls were incubated with primary antibodies against either insulin and glucagon, or insulin and somatostatin, or insulin and CD45, or glucagon and CD45. MCM-2-stained sections were further incubated with antibodies against insulin or CD45. Sections from chronic pancreatitis patients were further incubated in a cocktail of antibodies against insulin and glucagon. The antibodies were detected with goat AlexaFluor 488 or 568 conjugated secondary antibodies (Invitrogen, Paisley, UK). DAPI (1:1,000; Invitrogen) was included in the final secondary incubation to stain cell nuclei. By this method, tissue from nine of the ten deceased type 1 diabetic patients was found to be suitable for analysis; the tissue from the one other patient failed to absorb the DAPI nuclear stain. Sections were mounted in Vectashield hard-set mounting medium (Vector Laboratories, Peterborough, UK) under glass cover slips.
The sections were analysed initially using the brightfield function of a microscope (Eclipse 80i; Nikon, Kingston-upon-Thames, UK) and a monochrome photomicrograph was captured for each islet that contained at least one Ki67+ cell. The fluorescence imaging function of the microscope was then used to capture immunofluorescence staining. The images were precisely overlaid using NIS-Elements BR 3.0 software (Nikon) to determine the endocrine cell sub-type of the Ki67+ or MCM-2+ cells.
Islets were identified morphologically in each pancreas section after counterstaining with haematoxylin. Initially all islets on each section from every donor were assessed for the presence of Ki67+ or MCM-2+ nuclei, and the percentage of islets containing one or more immunopositive cells per section was recorded. Using the combination imaging technique described above, the identity of each of the cells with Ki67+ nuclei was determined. In islets infiltrated with immune cells (which could be readily identified by their unique morphology), any Ki67+ cell that was evidently an immune cell was excluded from the analysis. A Ki67 labelling index was determined for alpha and beta cells in the non-diabetic control and type 1 diabetic donor groups according to the percentage of each islet cell type that was positively stained for Ki67. Cell counting was carried out using Adobe Photoshop CS4 software (Adobe, Uxbridge, UK). Islets were categorised as insulitis-positive if there were five or more CD45+ cells within the islet perimeter .
The percentage of Ki67+ or MCM-2+ islets per section is expressed as the mean ± SEM and statistical significance was calculated by Student’s t test using SPSS 16 software (SPSS, Chicago, IL, USA). The statistical significance of differences in the labelling index of islet cells and also the insulitis correlation were calculated using χ2 analysis.
The percentage of islets with Ki67-positive islet cells was increased in recent-onset type 1 diabetic donors compared with non-diabetic controls
The mean percentage of Ki67+ islets detected in the donor groups
Islets examined (n)
%Ki67+ islets (mean±SEM)
Non-diabetic controls (C1–C14)
1.1 ± 0.3
Recent-onset type 1 diabetes
10.9 ± 2.5a,b
Long duration type 1 diabetes
Type 2 diabetes
1.5 ± 1.6c
Non-diabetic controls (C40–C48)d
0.97 ± 0.37e
Overall, 1,280 and 4,031 islet sections were examined in the type 1 diabetic and control groups, respectively. In the type 1 diabetic group, a mean of 37.1 ± 8.2% (range 6.7–78.4%) of the total islet sections were insulin-positive. Of the islet sections that were Ki67+, 23% were insulin-negative, suggesting that the increased proliferation was not restricted solely to beta cells. The mean area (±SEM) per islet was calculated for the recent-onset type 1 diabetic donors and the non-diabetic controls. These values were 18,227 ± 1,054 µm2 for insulin-positive and 9,017 ± 1,139 µm2 for insulin-negative islets in the type 1 diabetic group, and 12,265 ± 1,095 µm2 for insulin-positive islets in the non-diabetic controls (all islets were insulin positive in this group).
The increase in Ki67+ islet cells in recent-onset type 1 diabetes occurs in alpha and beta cells
The topography of the staining patterns precluded unequivocal identification of 10% to 20% of the Ki67+ islet cells, both in the recent-onset type 1 diabetic pancreatic sections and in the non-diabetic controls.
Increased proliferation of islet cells in type 1 diabetes was also seen using a second marker of cell proliferation
Pancreas sections from three of the ten recent-onset type 1 diabetic donors were double-stained with anti-MCM-2 and anti-insulin in order to assess the percentage of MCM-2+ beta cells. In the 54 MCM-2+ islets examined, 142 MCM-2+ cells were detected. Of these, 60 (42%) were dual-positive for insulin, thereby confirming that they were beta cells (Fig. 4c, d).
To confirm that the majority of MCM-2+ cells were islet cells rather than infiltrating lymphocytes, dual staining was performed with anti-MCM-2 and anti-CD45. We examined in detail 21 MCM-2+ islets that were heavily infiltrated with CD45+ lymphocytes. Very few of the infiltrating CD45+ cells were co-positive for MCM-2 (Fig. 4e). Peripancreatic lymph nodes were often present adjacent to the pancreas sections, and dual staining revealed the presence of MCM-2+ CD45+ cells, demonstrating that immunoperoxidase staining of the nucleus with anti-MCM-2 had not interfered with the ability of the CD45 antibody to stain the lymphocyte cell surface (Fig. 4f).
The presence of Ki67+ islet cells correlates with insulitis in type 1 diabetes
Increased Ki67+ labelling of islet cells was not maintained in donors with longer duration type 1 diabetes
To establish whether the increase in proliferating islet cells is persistent, pancreatic sections from three type 1 diabetic donors with a longer disease duration (one 6, two 12 years) were stained with the anti-Ki67 antibody. One of these donors (disease duration 12 years) still retained some residual beta cells in 53 of 79 of their islet sections; the other donors were entirely beta cell-deficient in the available sections. No Ki67+ islet cells were detected in any of the 561 islet sections examined in these donors (Table 3).
Increased Ki67+ labelling of islet cells was not observed in donors with type 2 diabetes
To determine whether the increase in islet cell proliferation was restricted to patients with (recent-onset) type 1 diabetes, pancreatic sections from ten donors with type 2 diabetes were also stained with the Ki67 antibody. Examination of 1,436 islets revealed that the mean (±SEM) percentage of Ki67+ islets per section (1.5 ± 0.16%; three donors had no Ki67+ cells) was statistically lower than that of the recent-onset type 1 diabetic group (p < 0.005), but not different from the non-diabetic controls (p = 0.55; Table 3). Samples from the type 2 diabetic donors were also compared with those from nine age-matched non-diabetic controls stained for Ki67. In this control group a mean of 0.97 ± 0.37% islets per section contained Ki67+ cells, which was not statistically different from the type 2 diabetic donors (p = 0.5; Table 3).
Increased Ki67+ labelling of islet cells was not observed in donors with pancreatitis
The rate of islet endocrine cell proliferation in humans varies with age. A wave of proliferation occurs early in postnatal life as the endocrine pancreas is released from the intrauterine environment and is remodelled to meet the demands of independent living and changing nutrition . This period, however, is short-lived and is followed by a rapid decline in the proliferation rate; this then stabilises at an extremely low level, which is maintained throughout childhood and into adult life. A similar situation may also pertain in other mammalian species, although, in some species, islet cell proliferation can be re-induced during adulthood by the imposition of increased metabolic demand (e.g. during obesity) and also in response to the hormonal adaptations associated with pregnancy [6, 7]. Importantly, we have now demonstrated that islet cell proliferation is increased in humans during the ongoing process of insulitis and autoimmunity associated with the recent onset of type 1 diabetes.
To study the proliferation of islet cells, we employed immunohistochemical detection of two independent proteins produced uniquely during cell division. Ki67 is widely used as a marker of cell division in fixed tissue, as it is expressed specifically during all stages of the cell cycle, but is degraded before cells re-enter gap (G)0 . MCM-2 plays a role in DNA replication and is produced from early G1 through the remaining phases of the cell cycle, but is degraded prior to entry into G0 . We analysed both proteins and found that they were detected at higher frequency in the nuclei of islet cells from donors with recent-onset type 1 diabetes than in those from non-diabetic controls. Detailed analysis revealed a tenfold increase in the proportion of islets displaying one or more Ki67+ cells in the type 1 diabetic samples vs controls. The increase was somewhat lower when the labelling of MCM-2 was assessed (approximately fivefold), but remained statistically significant. The reduction in the magnitude of the response seen when assessing MCM-2 was principally due to the larger proportion of islet sections from control donors that displayed MCM-2 immunopositivity compared with those that were positive for Ki67. This difference may reflect the different periods of the cell cycle when each protein is produced. Analysis of islet cells from islets of donors with recent-onset type 1 diabetes that displayed increased proliferation revealed that the labelling index of alpha and beta cells for Ki67 was enhanced by approximately tenfold. Thus, unlike the process of immune-mediated islet cell destruction (which is entirely beta cell-specific), the proliferative response appears not to display such cell specificity.
The increase in islet cell proliferation correlated closely with the presence of insulitis suggesting that a signal or signals emanating from infiltrating immune cells might mediate the enhanced proliferative response. Consistent with this hypothesis, an increase in islet cell proliferation has also been reported in the inflamed islets of a patient classified as having ‘pre-diabetes’ . In addition, an 89-year-old man recently diagnosed with type 1 diabetes, had increased Ki67+ islet cells, as well as insulitis in a large percentage of islets . Taken together, these results support the suggestion that human islet cell proliferation can be increased during islet inflammation. Our finding that patients with a long duration of diabetes do not display islet cell proliferation also supports this conclusion, since islet inflammation was minimal in these patients.
Additional support for the possible involvement of immune-derived signals as mediators of islet cell proliferation comes from the NOD mouse and the BioBreeding rat. In the NOD model of diabetes, beta cell proliferation is increased during the prediabetic phase, a finding attributed to the presence of insulitis in the relevant islets [9, 10]. The BioBreeding rat also has a higher than normal islet cell Ki67 labelling index during the prediabetic period when the islets are infiltrated by mononuclear cells . Similar findings have also been reported in a partial pancreatectomy model in the LEW.Han rat , although, unlike our present findings in man, alpha cell proliferation was unchanged in these animals.
Examination of pancreatic sections from patients with pancreatitis, another inflammatory disease of the pancreas, revealed low numbers of Ki67+ alpha or beta cells, which is not consistent with a large-scale proliferative response. However, a significant increase in beta cell replication has been reported previously in sections from patients with severe pancreatitis, suggesting that, under these circumstances, the immune cells may elaborate a stimulus for endocrine cell proliferation . One study has also reported neogenesis of duct epithelial cells into insulin-producing cells in patients with chronic pancreatitis .
We did not characterise the nature of the putative immune cell-derived signal that may promote islet cell proliferation, but very low concentrations of certain pro-inflammatory cytokines (including IL-1β) can elicit beta cell proliferation in isolated human islet preparations . Higher doses of these same cytokines are cytotoxic, suggesting that the gradient of concentration to which islet cells are exposed may determine their ultimate fate. Conceivably, the initial influx of immune cells might promote endocrine cell proliferation, but this response may be ultimately overridden by a net cytotoxicity as the concentration of immune mediators increases further. It should, however, be noted that we did not detect an increase in Ki67+ islet cells in pancreas sections from donors with type 2 diabetes. This form of diabetes has recently been shown to be associated with low-level macrophage infiltration of the islets, but there is no lymphocyte infiltration [14, 23, 24]. This implies that a signal other than IL-1β (which is secreted by macrophages, but not by T or B lymphocytes) may be primarily responsible for mediating the proliferative response. The finding that islet cell proliferation is not increased in patients with type 2 diabetes also implies that hyperglycaemia is unlikely to represent a primary stimulus for the proliferative response, since this is a feature of both forms of the disease.
The present results differ from the recent findings of Butler et al. , who did not find any increase in beta cell proliferation in deceased patients with recent-onset type 1 diabetes. If, as we propose, the proliferative response reflects an early response to insulitis, then this may have been undetectable in several of the samples examined by Butler et al. , since four of the nine persons studied did not have insulitis. However, other undefined factors might also account for the differences.
In summary, the current study reveals that islet cell proliferation is increased in the islets of patients with recent-onset type 1 diabetes. This correlates with inflammation and suggests that the immune cells elaborate a signal that promotes islet cell mitosis. These results suggest that, if mechanisms could be found to facilitate cell proliferation during the prediabetic phase, it might be possible to facilitate the regeneration of beta cells as a therapeutic intervention in patients progressing to type 1 diabetes.
We are grateful to the Juvenile Diabetes Research Foundation for funding this study.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.