The cellular composition of the islet of Langerhans is essential to ensure its physiological function. Morphophysiological islet abnormalities are present in type 2 diabetes but the relationship between fasting plasma glucose (FPG) and islet cell composition, particularly the role of delta cells, is unknown. We explored these questions in pancreases from baboons (Papio hamadryas) with FPG ranging from normal to type 2 diabetic values.
We measured the volumes of alpha, beta and delta cells and amyloid in pancreatic islets of 40 baboons (Group 1 [G1]: FPG < 4.44 mmol/l [n = 10]; G2: FPG = 4.44–5.26 mmol/l [n = 9]; G3: FPG = 5.27–6.94 mmol/l [n = 9]; G4: FPG > 6.94 mmol/l [n = 12]) and correlated islet composition with metabolic and hormonal variables. We also performed confocal microscopy including TUNEL, caspase-3, and anti-caspase cleavage product of cytokeratin 18 (M30) immunostaining, electron microscopy, and immuno-electron microscopy with anti-somatostatin antibodies in baboon pancreases.
Amyloidosis preceded the decrease in beta cell volume. Alpha cell volume increased ∼50% in G3 and G4 (p < 0.05), while delta cell volume decreased in these groups by 31% and 39%, respectively (p < 0.05). In G4, glucagon levels were higher, while insulin and HOMA index of beta cell function were lower than in the other groups. Immunostaining of G4 pancreatic sections with TUNEL, caspase-3 and M30 showed apoptosis of beta and delta cells, which was also confirmed by immuno-electron microscopy with anti-somatostatin antibodies.
In diabetic baboons, changes in islet composition correlate with amyloid deposition, with increased alpha cell and decreased beta and delta cell volume and number due to apoptosis. These data argue for an important role of delta cells in type 2 diabetes.
Plasma glucose levels are tightly regulated by the islets of Langerhans in concert with insulin target tissues. Islets have a complex multi-cellular structure, containing insulin-secreting beta cells, glucagon-secreting alpha cells, somatostatin-secreting delta cells, pancreatic-polypeptide-secreting PP cells and grehlin-secreting epsilon cells. Physiological interactions between these different cell types are essential to ensure appropriate islet function, and changes in islet architecture have functional implications [1–7]. Compared with rodent islets, human and baboon islets contain proportionally fewer beta cells and more alpha cells [8–11]. In rodents, endocrine non-beta cells are confined to the islet periphery (mantle) while beta cells are concentrated in the centre (core) . In sharp contrast, the vast majority of human and baboon islets do not have a core-mantle structure, as the beta cells are intermingled with alpha and delta cells throughout the islet and all the endocrine cells have equivalent access to blood vessels [8–10]. Therefore, human and baboon alpha and delta cells may exert a stronger paracrine control of the adjacent beta cells than those of rodents.
Islet composition undergoes profound changes in humans and non-human primates with type 2 diabetes, with a 50–70% reduction in beta cell mass and a variable absolute or relative increase in alpha cell numbers [13, 14], which account for the defective insulin secretion and the hyperglucagonaemia that are distinctive features of the disease [15–17]. However, studies of islet cell composition in humans have the following limitations: (1) they are necessarily performed on autoptic pancreases obtained 48–72 h post mortem when autolysis causes cell damage; (2) the vast majority of studies have compared non-diabetic vs diabetic subjects and have not examined islet composition in different stages of glucose intolerance; (3) the different glucose-lowering regimens represent an additional confounding factor [18, 19]; and (4) most of the studies do not show correlations between morphometric and biochemical/functional variable parameters.
Delta cell fate in the diabetic pancreas is presently unknown, even though these cells may be more important than commonly thought [2–4]. A heterocellular region in which alpha, delta and beta cells are in close proximity has been described in the islets of all species and is believed to function as a pacemaker for the entire islet under the control of the delta cells [2, 3, 20]. However, information about delta cell number and function in diabetes is limited and whether these cells are also involved in the pathogenesis of diabetes remains unclear. Nevertheless, several lines of evidence support a role of delta cells in the pathogenesis of diabetes. Somatostatin receptors are present on both alpha and beta cells , and somatostatin secreted by delta cells paracrinally controls alpha and beta cell function [22, 23]. Major physiological insulin secretagogues such as glucose, arginine and glucagon-like peptide-1 (GLP-1) also regulate somatostatin release [22–28]. Finally, transgenic mice depleted of delta cells (Sst −/− mice) show enhanced insulin and glucagon release in response to nutrient stimuli , suggesting that delta cells exert a tonic inhibitory influence on insulin and glucagon secretion and are implicated in nutrient-induced suppression of glucagon secretion.
Baboons are an interesting model of spontaneous obesity, insulin resistance and type 2 diabetes in humans [29–33]. In the present study, we analysed the islet cell composition of 40 baboons obtained from a large cohort. The fasting plasma glucose (FPG) concentrations of the baboons were measured in the last year of their lives and the volumes of the islets of Langerhans, beta, alpha and delta cells and amyloid deposits were measured in pancreases. The relationships between changes in islet cell composition and FPG, clinical, biochemical and metabolic variables were investigated.
The study was conducted in accordance with the Principles of Laboratory Care in baboons that died from natural causes, with similar age and weight and different levels of FPG, (Group 1 [G1]: FPG<4.44 mmol/l [n = 10]; G2: FPG = 4.44–5.26 mmol/l [n = 9]; G3: FPG = 5.27–6.94 mmol/l [n = 9]; G4: FPG > 6.94 mmol/l [n = 12]) selected from a large cohort followed during the past 14 years (from 1994 to 2007) at the Texas Biomedical Research Institute. Inclusion criteria were: (1) older than 8 years; and (2) availability of pancreatic tissue and clinical, anthropometric and laboratory measurements. Forty baboons were included in the present work (Table 1). Animals were fed ad libitum with a standard monkey chow (diet 5038, Purina, St Louis, MO, USA) and housed in corrals where they performed unrestrained physical activity.
Complete necropsies were performed on all the baboons, approximately 6–18 h post mortem. Pancreatic tissue from the body-tail region was fixed in 10% neutral-buffered formalin, processed conventionally and embedded in paraffin blocks. Sequential 5 μm sections for each baboon were stained with haematoxylin and eosin and Congo red and immunostained with antibodies for insulin, glucagon and somatostatin for evaluation of islets, amyloid deposits, and beta, alpha and delta cell morphometry, respectively. The detailed description of the automated immunohistochemistry, electron microscopy and immuno-electron microscopy is available in the electronic supplementary material (ESM) Methods section.
The Computer Assisted Stereology Toolbox (CAST) 2.0 system (Olympus, Ballerup, Denmark) was used to perform all the microscopic measurements of beta, alpha and delta cell and amyloid deposit volume using the stereology fundamentals previously described and validated [31, 33–35]. The person who performed the microscopic measurements (RGM) was blinded to the metabolic status of each baboon and the reproducibility of the CAST measurements was estimated twice in five specimens with a coefficient of variation <5%.
To identify apoptotic cells we analysed pancreas sections derived from a total of eight G1 (normal glucose tolerant [NGT]) and eight G4 (type 2 diabetic) baboons, and three different methods were employed: (1) TUNEL; (2) anti-cleaved caspase 3 (Asp175) immunostaining (CC-3); and (3) anti-caspase cleavage product of cytokeratin 18 (M30). The detailed description of immunofluorescence and confocal microscopy methods is available in ESM Methods.
Apoptosis quantification in beta and delta cells
To quantify the percentage of apoptotic cells in each section, the number of M30-positive and TUNEL-positive delta or beta cells was counted in a total of 100 somatostatin-immunoreactive delta cells and 100 insulin-immunoreactive beta cells in three different animals from each group, blind, by two different observers (SL and CP). Cells were counted in sequence, in different islets, until the number of 100 beta and 100 delta cells was reached. Statistical evaluation was performed using the unpaired t test.
Blood glucose was measured by the glucose oxidase method with the SYNCHRON CX System (Beckman Coulter, Brea CA, USA), insulin levels by RIA (Linco Research, St Louis, MO, USA), glucagon by RIA (Euro-Diagnostica AB, Malmö, Sweden) and NEFA concentrations were determined using the fluorometric method.
Insulin resistance (HOMA-IR) and beta cell function (HOMA-B) were measured as previously described .
Data are presented as means ± SEM. ANOVA with Bonferroni correction was used as a post hoc test for comparisons between more than two groups when normal distribution was confirmed and Kruskal–Wallis or log transformed values were used for those with a skewed distribution, confirming a normal distribution after the log transformation. Bivariable correlations were evaluated with Pearson’s correlation coefficient. A p value of <0.05 was considered statistically significant.
Clinical, biochemical and metabolic characteristics
Clinical, anthropometric, biochemical and metabolic data, as well as islet volumes, in the four groups are shown in Table 1. FPG increased linearly from G1 to G4; however, only baboons in the G4 group showed the classic diabetic phenotype characterised by: (1) increased plasma glucagon, NEFA and cholesterol levels; (2) decreased FPI levels; and (3) dramatically impaired beta cell function as calculated by HOMA-B. NEFA, cholesterol and HOMA-IR levels tended to increase from G1 to G3, while HOMA-B tended to decline even though these changes were not statistically significant. In addition, islet volume and size did not vary significantly from G1 to G3, while they showed a significant increase in G4.
Islet cell composition and amyloid deposition
Islet cell composition and architecture in the four groups is shown in Fig. 1. Figure 1a–l are representative islets in pancreatic sections stained for insulin (a–d), glucagon (e–h) and somatostatin (i–l). Figure 1m–p are the volumes per islet of beta (m), alpha (n), delta cells (o) and amyloid deposits (p); the same data expressed as the percentage of the entire pancreatic area are shown in Fig. 1q–t. Amyloid volume showed a striking linear increase from G1 to G4 (Fig. 1p, t). The progressive increases in amyloid deposits were not paralleled by significant changes in beta cell volumes that were in fact similar in G1 and G2, slightly decreased in G3 and dramatically reduced only in G4. Alpha cell volumes increased from G1 to G3 where they reached high statistical significance, but did not increase further in G4 (Fig. 1n, r). The volume of somatostatin-secreting delta cells was similar in G1 and G2 but showed a remarkable decrease (∼41%) in G3 and G4 (Fig. 1o, s).
Correlation between severity of amyloid deposition, FPG and islet cell composition
The analysis of the correlation between the severity of amyloid deposition, FPG levels and volumes of the three islet cell types is shown in Fig. 2. As expected, amyloid severity showed a linear positive correlation with FPG (Fig. 2a, R 2 0.5275, p < 0.001) and an inverse correlation with beta cell volume (Fig. 2b, R 2 0.7679, p < 0.001). By contrast, amyloid deposition and alpha cell volume showed a positive correlation (Fig. 2c, R 2 0.1416, p < 0.05). Finally, the correlation between amyloid deposits and delta cell volume was, similarly to the beta cells, also negative (Fig. 2d, R 2 0.1493, p < 0.05).
Correlation between beta cell volume and biochemical and metabolic variables
The relationship between FPG levels and beta cell volume was negative and hyperbolic (Fig. 3a, R 2 0.5428, p < 0.001). Beta cell volume also correlated inversely with NEFA levels (Fig. 3b, R 2 0.2351, p < 0.001) and positively with FPI levels and beta cell function calculated with HOMA-B (Fig. 3c, R 2 0.2946, p < 0.001; Fig. 3d, R 2 0.6092, p < 0.001). HOMA-B was inversely correlated with NEFA levels (Fig. 3e, R 2 0.2451, p < 0.01).
Correlation between FPG and NEFA levels and volumes of alpha and delta cells
The correlations between FPG and alpha and delta cell volumes were not significant (Fig. 4a, b). Conversely, delta cell volume was inversely correlated with loge NEFA, suggesting a potential toxic effect of increased levels of NEFA, which are observed in G3 and G4 animals in delta cells (Fig. 4c, R 2 0.1926, p < 0.05). Delta cell volume positively correlated with beta cell volume (Fig. 4d, R 2 0.2110, p < 0.01).
Correlation of plasma glucagon levels and beta, alpha and delta cell volumes
Plasma glucagon levels did not correlate significantly with either beta or alpha cell volume (ESM Fig. 1a, b), while glucagon levels showed a significant inverse correlation with delta cell volume (ESM Fig. 1c, R 2 0.2696, p < 0.01).
Pancreatic delta and beta cell apoptosis
To explore the mechanisms involved in the reduction of both delta and beta cell volume we performed a TUNEL assay on pancreatic sections obtained from G1 and G4 baboons. In triple immunofluorescence experiments with antibodies against hormones, ∼5% of delta and ∼3.5% of beta cells were TUNEL positive (apoptotic) in G4 diabetic pancreases (Fig. 5e, f, h, i, k, l), compared with control non-diabetic pancreases (G1 group) where no apoptotic delta cells (p < 0.05) and beta cells (p < 0.005) were observed (Fig. 5d, g, j, m).
To confirm the presence of apoptotic delta cells in diabetic pancreases, in both G1 and G4 pancreases we also performed double label immunohistochemical and triple immunofluorescence staining with antibodies directed against two additional apoptotic cell markers: M30 and CC-3 antibodies.
We observed M30-positive islet cells that were somatostatin negative, likely corresponding to insulin-positive beta cells as previously demonstrated  (ESM Fig. 2c, d, e). Using double label immunohistochemistry we were able to quantify that in diabetic pancreatic islets ∼3% of delta cells (Fig. 5m and ESM Fig. 2f) and ∼2% of insulin-producing beta cells were positive for the M30 antibody (i.e. apoptotic cells). Conversely, in non-diabetic pancreases (G1 group) no apoptotic delta cells (p < 0.001) or beta cells (p < 0.001) were observed. These findings confirm and reinforce the data obtained by TUNEL (Fig. 5m). Finally, consistent with the data obtained by TUNEL confocal microscopy and M30 immunohistochemistry, we could also detect both insulin/CC-3-positive beta as well as somatostatin/CC-3-positive delta cells in G4 (type 2 diabetes) islets of Langerhans (ESM Fig. 3). The number of delta cells per islet area in a subset of baboon pancreases was decreased by ∼30% in baboon pancreases from G3-4 animals (ESM Fig. 4).
While delta cells of G1 baboons were well preserved (Fig. 6a, b and ESM Fig. 5c), in G4 animals, delta cells showed the typical large and uniformly electron-dense secretory granules encircled by a tightly fitting membrane and were immunoreactive for somatostatin, but also presented signs of degeneration, such as pycnotic nuclei and cytoplasmic vacuoles, which are typical features of apoptotic cells (Fig. 6c, d and ESM Fig. 5d).
At the ultrastructural level, the presence of increasing amyloid deposits was associated with signs of cellular injury, including intracellular vacuoles and pycnotic nuclei involving mainly beta cells, but also delta cells in G3 (impaired fasting glucose [IFG]) and G4 (type 2 diabetic) animals, while alpha cell morphology was unaffected by amyloidosis in G4 animals (Figs 7b, c, d and 8b, f). The degenerative/pro-apoptotic ultrastructural characteristics observed in beta and delta cells were also consistent with the decreases in beta and delta cell volume in diabetic animals.
Several studies have shown that the pancreases of subjects with type 2 diabetes display a severe beta cell deficit due to increased beta cell apoptosis [16, 17, 37, 38], and normal or increased alpha cell number [5, 13–15, 19]; interestingly, these alterations are also present in diabetic and insulin resistant/obese baboons [31, 33]. However, little is known regarding islet cell composition in prediabetic conditions such as IFG and impaired glucose tolerance . We believe that another important unresolved question is related to the function and fate of the delta cells in type 2 diabetes. To learn more about these issues, we analysed the islet cell composition in the pancreases of baboons stratified into four quartiles of FPG levels spanning the NGT, IFG and type 2 diabetes ranges (G1–G4; Table 1).
We already reported that FPG levels in baboons are linearly correlated with islet amyloidosis . In the present study, we confirm this finding by showing that as little as 0.83 mmol/l of FPG increase is associated with a significant increase in amyloid deposits that, remarkably, precede the changes in beta cell volumes (Fig. 1p). In fact, beta cell volumes were identical in G1 and G2, decreased in G3 but without reaching statistical significance, and fell dramatically only in G4 (Fig. 1m). These data further strengthen the evidence that amyloid deposition is an early event in islet pathology in baboons and that amyloidosis is an important cause of beta cell death in humans, non-human primates and other species [16, 31, 38, 39].
Similarly to the beta cell, the alpha and delta cell volumes also did not change significantly between G1 and G2. Alpha cell volume increased sharply in G3 (Fig. 1n), while delta cell volume showed a remarkable decrease in this group (Fig. 1o). Thus, alterations in alpha and delta cell composition seemed to occur earlier than changes in beta cell volume. In G4, alpha and delta cell volumes did not change further as compared with G3. This and previous studies indicate that in humans and baboons, alpha cells are resistant to various conditions of beta cell stress, and that delta cells are also involved in the islet remodelling that occurs in type 2 diabetes [20, 21, 40–44].
It has been suggested that metabolic stress and type 2 diabetes in mice can result in beta cell dedifferentiation into a progenitor-like stage followed by the conversion of a subpopulation of dedifferentiated beta cells into alpha cells . Similar results have been shown in primary human beta cells that, when incubated in vitro under conditions of stress, loose insulin granules and transdifferentiate into alpha cells .
There is little information about delta cell fate in diabetes, but previous studies reported increased delta cell volumes in type 1 diabetes  as well as in type 2 diabetes  and in diabetes associated with cystic fibrosis . Delta cell expansion has been interpreted as a compensatory adaptation to hyperglucagonaemia. These studies, however, have the limitation of being performed on a small number of subjects and in the presence of severe beta cell depletion, so that apparent delta cell hyperplasia might be relative to beta cell loss rather than absolute.
Furthermore, together with the evidence of reduced delta cell volumes, we here provide for the first time the direct evidence of ongoing delta cell death in type 2 diabetes. Confocal microscopy examination of control (G1) and G4 pancreatic tissues triple-stained for insulin, somatostatin and TUNEL showed that apoptotic delta cells were absent in pancreases from G1 baboons but present in those from G4 baboons (Fig. 5). Electron microscopy and immune-electron microscopy analysis of G4 baboon pancreases showed somatostatin-positive delta cells with ultrastructural degenerative features and fragmented pycnotic nuclei (Fig. 6 and ESM Fig. 5). Moreover, double immunohistochemical and triple immunofluorescence studies showed co-localisation of somatostatin and the apoptotic markers M30 and CC-3 in islet delta cells of diabetic baboons (ESM Figs 2, 3). The evidence that amyloid severity is inversely correlated to delta cell volumes (Fig. 2d) may suggest that delta cells, like beta cells, are also sensitive to amyloid toxicity. In contrast to beta cell volume (Fig. 3a), delta cell volume did not show a clear negative correlation with FPG (Fig. 4b). Nevertheless, similarly to beta cell volume, delta cell volume correlated inversely with NEFA levels (Figs 3b and 4c). Thus, delta cells appear resistant to glucose-toxicity but sensitive to lipotoxicity . In spite of a slightly different sensitivity to hyperglycaemia, amyloid and NEFA, beta and delta cells share a common fate as their volumes are positively correlated with each other (Fig. 4d). Owing to the known negative paracrine effect of somatostatin on alpha cell secretion, loss of delta cells (in concert with the relative or absolute increase in alpha cell volume) can also play a role in insulin and glucagon secretory changes associated with type 2 diabetes. This possibility is supported by evidence that fasting glucagon concentrations are inversely correlated to delta cell volume (ESM Fig. 1c). Finally, a decrease in delta cell number (considering the negative paracrine effect of somatostatin on insulin secretion) could be viewed as a compensatory adaptation in a situation of beta cell deficiency.
We would like to propose that as glucose and amyloid increase within the islet, dysfunctional islet remodelling occurs that involves not only beta and alpha cells, but also delta cells (Fig. 9).
In conclusion, additional studies should be carried out to confirm the loss of delta cells in human type 2 diabetes and to explore the molecular mechanisms involved, and to investigate novel therapeutic options directed at preserving not only beta cells, but also delta cells.
Computer assisted stereology toolbox
Anti-cleaved caspase 3 (Asp175) immunostaining
Fasting plasma glucose
Fasting plasma insulin
Impaired fasting glucose
Anti-caspase cleavage product of cytokeratin 18
Normal glucose tolerance
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We thank M. Silva for pathology support and expert assistance in tissue processing (Texas Biomedical Research Institute, San Antonio, Texas, TX, USA). We also thank P. Zuo for performing insulin and glucagon RIA and NEFA determinations. Portions of this work were presented in abstract form at the 2014 Endocrine Society Meeting, Chicago, IL, USA. This work is dedicated to the memory of my beloved nephew Matteo Folli (17 April 1995–2 April 2015), who left us in the prime time of his life.
This research was supported in part by NIH-NCRR grant P51 RR013986 to the Southwest National Primate Research Center and conducted in facilities constructed with support from Research Facilities Improvement Program Grant C06 RR014578 and C06 RR015456. FF was partially supported by NIH RO1 DK080148. FF is a visiting professor in the Department of Medicine and the Obesity and Comorbidities Research Center (OCRC), University of Campinas, Campinas, São Paulo State, Brazil, and is supported by FAPESP and CNPq, Brazil. He is on sabbatical leave from the University of Texas Health Science Center at San Antonio, TX, USA. LAV and MJAS are supported by OCRC and FAPESP, Brazil.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
FF is responsible for the integrity of the study and conceived the study. RGM and FF performed experiments and generated, analysed and interpreted the data, wrote the manuscript and reviewed the final version of the manuscript. CP, GF, SL, CC, FS and SM performed experiments, analysed and interpreted the data, wrote the manuscript and contributed to the discussion and the final review of the manuscript. LMJC performed data quantification, analysed and interpreted the data and critically revised the manuscript for important intellectual content. LAV, MJAS and FB analysed and interpreted the data and critically revised the manuscript for important intellectual content. EJD and AD performed experiments, analysed and interpreted the data, wrote the manuscript, contributed to the discussion and reviewed the final version of the manuscript. All authors gave final approval of the version of the manuscript to be published.
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Guardado Mendoza, R., Perego, C., Finzi, G. et al. Delta cell death in the islet of Langerhans and the progression from normal glucose tolerance to type 2 diabetes in non-human primates (baboon, Papio hamadryas). Diabetologia 58, 1814–1826 (2015). https://doi.org/10.1007/s00125-015-3625-5
- Alpha cell
- Beta cell
- Delta cell
- Islet of Langerhans
- Islet remodelling
- Non-human primates
- Papio hamadryas
- Type 2 diabetes mellitus