Page 1 of 74 Diabetes
Highly proliferative alpha cell related islet endocrine cells in
human pancreata
Carol J. Lam1, Aaron R. Cox1, Daniel R. Jacobson2, Matthew M. Rankin2, Jake A. Kushner1
Short Running Title: Proliferative Islet Cells in Human Pancreata
1 McNair Medical Institute, Baylor College of Medicine, Houston, TX, 77030
2 Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104
Correspondence: Jake A. Kushner M.D. or Aaron R. Cox PhD., Baylor College of Medicine, Houston, TX, 77030, (832) 822 3780, [email protected] or [email protected]
Keywords: Pancreatic Islet, Endocrine, Cell Differentiation, Morphometry, Cell Proliferation,
β cell, α cell, Sox9, ARX
(Manuscript information: abstract – 197 words, main text – 3990 words; 7 figures, 1 table, 13
supplemental figures, 23 supplemental tables)
Diabetes Publish Ahead of Print, published online January 11, 2018 Diabetes Page 2 of 74
ABSTRACT
The proliferative response of non β islet endocrine cells in response to type 1 diabetes
(T1D) remains undefined. We quantified islet endocrine cell proliferation in a large collection of non diabetic control and T1D human pancreata across a wide range of ages. Surprisingly, islet endocrine cells with abundant proliferation were present in many adolescent and young adult
T1D pancreata. But, the proliferative islet endocrine cells were also present in similar abundance within control samples. We queried the proliferating islet cells with antisera against various islet hormones. Although PP, somatostatin, and ghrelin cells did not exhibit frequent proliferation, glucagon expressing α cells were highly proliferative in many adolescent and young adult samples. Notably, α cells only comprised a fraction (~1/3) of the proliferative islet cells within those samples; most proliferative cells did not express islet hormones. The proliferative hormone negative cells uniformly contained immunoreactivity for ARX (indicating α cell fate) and cytoplasmic Sox9 (Sox9Cyt). These hormone negative cells represented the majority of islet
endocrine Ki67+ nuclei and were conserved from infancy through young adulthood. Our studies
reveal a novel population of highly proliferative ARX+ Sox9Cyt+ hormone negative cells and suggest the possibility of previously unrecognized islet development and/or lineage plasticity within adolescent and adult human pancreata.
Page 3 of 74 Diabetes
INTRODUCTION
Type 1 diabetes (T1D) is characterized by a considerable loss of β cells and subsequent
insulin deficiency (1 7). Although β cells have been reported to persist in T1D pancreata for
several years after diagnosis, we recently found that T1D pancreata do not exhibit evidence of
increased β cell proliferation, nor evidence of β cell neogenesis or transdifferentiation (1).
However, the impact of T1D on non β cells has not been studied. Thus, the regenerative
response of islet endocrine cells to T1D remains poorly understood.
Lineage tracing studies in mice suggest that α cells may have unappreciated plasticity. α
cells appear to transdifferentiate into β cells in mice under some circumstances (2 4). These
results imply that α cells might be a potential source for β cell neogenesis as a novel therapy for
diabetes. Indeed, insulin glucagon co expressing cells have been reported within pancreata of
human patients with acute pancreatitis (5). However, potential compensatory responses from
non β cell sources in human pancreata with longstanding T1D remain poorly understood, as only
a few studies have been performed. Increased Ki67+ islet cells have been observed in both α
and β cells of pancreata from individuals with recent onset T1D (6). Ki67+ ductal cells have also
been described in transplanted pancreas of patients with T1D (7). Increased cell proliferation has
also been reported in pancreatic duct glands of T1D pancreata (8). Taken together, these
observations hint at a role for non β cell sources in T1D pathophysiology or compensation.
Given the lack of consensus, we considered the possibility that other islet endocrine cells
could participate or respond to autoimmunity with attempted regeneration. We surveyed human
islet proliferation in non diabetic control and T1D pancreata from the JDRF nPOD collection,
applying high throughput imaging and analysis using similar techniques as our previous study Diabetes Page 4 of 74
(1). We find that islet proliferation did not increase in response to T1D. But, islet cell proliferation was sharply increased in many adolescent and young adult pancreata of non
diabetic and T1D individuals. We identify a novel population of highly proliferative, α related
cells within many adolescent and young adult pancreata.
Page 5 of 74 Diabetes
RESEARCH DESIGN AND METHODS
Human Pancreatic Samples. Paraffin embedded pancreas tissue sections were obtained
from the JDRF Network for Pancreatic Organ Donors with Diabetes (nPOD) after a waiver from
our institutional review board (IRB). Pancreata were studied based on availability. Tissues were
processed by nPOD by standardized operating procedures (http://www.jdrfnpod.org/for
investigators/standard operating procedures/). Paraffin embedded tissues were fixed in 10%
neutral buffered formalin for 24 h, up to 40 h for pancreata with high fat content (1).
Sample Population: 59 non diabetic controls and 47 T1Ds were studied, selected to
include various ages: infants (0 1.4 yrs), children (1.5 13.9 yrs), adolescents (14 20.9 yrs), young
adults (21 39 yrs), and older adults (≥40yrs), as previously (1). Recent onset T1D was defined as
disease duration ≤10 years. See Table S1 2 for further information.
Immunohistochemistry. Paraffin sections were incubated with primary antisera (Table
S3), followed by the appropriate secondary antisera conjugated to AMCA, Cy2, Cy3, or Cy5
(Jackson ImmunoResearch) and DAPI (Molecular Probes, Eugene, OR), as previously (1).
Primary antisera (1:100) ARX (R&D systems, AF7068), β3 tubulin (Novus NB100 1612), CD3
(ThermoFisher PA1 37282), CD31 (Abcam, ab28364), chromagranin A (Abcam, ab8204),
ghrelin (Phoenix Pharmaceuticals, H 031 77), GLUT1 (Millipore, 07 1401), ISL1&2 (DSHB,
39.4D5), INSM1 (Santa Cruz, sc 271408), NeuN (Millipore, MAB377), Nkx2.2 (Abcam,
ab191077), Nkx6.1 (DSHB, F55A12), pancreatic polypeptide (Invitrogen, 18 0043), PCNA
(Cell Signaling, 2586S), PC1/3 (Millipore, AB10553), Pdx1 (Novus Biologicals, NBP2 38865),
phospho histone H3 (Cell Signaling, 9701S), proinsulin (DSHB, GN ID4), SNAP25 (Millipore,
MAB331), somatostatin (Invitrogen, 18 0078), synaptotagmin 1A (Abcam, ab133856), Sox9
(Millipore, AB5535), Sox9 (pS181) (abcam, ab59252),, and synaptophysin (ThermoFisher, 18 Diabetes Page 6 of 74
0130; Abcam, AB6245); (1:250) glucagon (Abcam, ab8055 & ab10988) and Ki67 (BD
Biosciences, #550609); (1:1500) insulin (Dako, A0564).
Islet morphometry. Islet endocrine and α cell morphometry with Volocity 6.1.1
(PerkinElmer) as previously (9). Zeiss AxioImager M1 (Carl Zeiss Microscopy) with automated
X Y stage and Orca ER camera (Hamamatsu) acquired images of tens of thousands of individual nuclei/sample (Suppl. Table 5).
Proliferation analysis. Ki67+ islet endocrine (synaptophysin), α cell (glucagon), PP, SS,
ghrelin, and Sox9Cyt proliferation were calculated as % total cells. A subset of high proliferators
was quantified as % intraislet Ki67+ cells for insulin and all other markers. High proliferation
was defined as having an islet endocrine cell replication rate >0.71%, corresponding to z score of
+0.5.
TUNEL. Apoptosis analysis was performed in a subset of available control and T1D samples, as previously (1). Total terminal deoxynucleotide transferase mediated dUTP nick end labeling (TUNEL) positive islet endocrine cells were assessed in >85,000 islet cells/condition.
Total TUNEL+ Sox9Cyt cells were assessed in 993 islet cells/condition. In every sample,
TUNEL+ pancreatic ducts were imaged to ensure adequate TUNEL staining. Page 7 of 74 Diabetes
RESULTS
Increased islet endocrine cell proliferation in some adolescent and
young adults, irrespective of T1D status
We previously quantified β cell proliferation in control and T1D nPOD pancreas organ
donor samples and found no evidence for a β cell regenerative response to T1D at any age group
(1). During our studies, we noted some samples had islets with abundant Ki67+ cells, indicating
islet proliferation (Fig. 1a c). Samples with highly proliferative islets represented only a portion
of pancreatic donors, largely from adolescents and young adults. Proliferative islet cells were
present in control and T1D samples; T1D did not influence the proliferative islet cell phenotype
(Fig. 1d f; Table S5). Cell cycle entry within islets, as measured by the percentage of Ki67+ islet
cells amongst total islet nuclei, was equivalent between control and T1D for age matched cohorts
of children and adolescents (0.63 v. 0.81%) or adults (0.38 % v. 0.24%), with proliferation more
variable in controls (Fig. 1g j; Table S5). Most proliferative islet cells did not express insulin,
suggesting they were not β cells (Fig. 1a b). We quantified Ki67+ β cells as a fraction of the
total Ki67+ islet cells amongst the highly proliferative pancreatic samples. The vast majority of
Ki67+ islet cells did not contain insulin; 89.8% of Ki67+ intraislet cells of controls and 98.86%
of Ki67+ intraislet cells of T1Ds did not express insulin (Fig. 1k; Table S6). Moreover, Ki67+
islet cells were not CD3+ or CD31+, thereby excluding lymphocytes or endothelial cells,
respectively (Fig. S1). We therefore considered the possibility of proliferative non β islet
endocrine cells within our samples.
Increased islet endocrine cell proliferation in pancreata from some
adolescent and young adults, irrespective of T1D status Diabetes Page 8 of 74
We imaged and quantified total islet endocrine cell proliferation using Ki67 and
synaptophysin (marker of islet endocrine cells) in 59 control and 47 T1D pancreata to determine
the identity of the proliferative islet cells and to test for a proliferative response to T1D. Similar
to previous findings of β cell proliferation (1; 10; 11), islet endocrine cell proliferation was greatest in pancreatic samples from non diabetic infants and children (up to 8.68%) and declined with age (Fig. 2a h; Table S7 8). Islet endocrine cell proliferation was comparable between control and T1D samples from age matched cohorts of children (0.94±0.59% v. 1.50±1.03%; p =
0.66) (Fig. 2i j; Table S7 8). However, a subset of control and T1D adolescents and young adults exhibited many Ki67+ islet cells (0.48 6.39%; hereby referred to as ‘high proliferators’; Table
S5 8). Islet cell proliferation was not associated with duration of diabetes and was minimal in the other samples from control and T1D adolescents and young adults (Fig. 2k l; Table S7 8).
Pancreatic samples from older control and T1D adults had very few Ki67+ cells and minimal islet endocrine cell proliferation (Fig. 2g h; Table S7 8). Synaptophysin+ Ki67+ cells represented the vast majority of intra islet Ki67+ cells in both control and T1D samples (Fig. 2m;
Table S9). Islet endocrine cell proliferation was verified with other markers of cell cycle entry.
Abundant synaptophysin+ phospho histone H3+ (pHH3) and PCNA+ nuclei were observed, confirming increased cell cycle entry (Fig. S2).
To test for regional variation of the islet cell proliferative phenotype, we studied samples from the opposite region of the pancreas (e.g. head/body when tail had been previously measured) from four adolescent and young adult cases and compared synaptophysin+ Ki67+.
Reassuringly, there was minimal intra sample variation of islet cell proliferation (Fig. S3, Table
S10). Thus, the proliferative islet endocrine cell population did not substantially vary within Page 9 of 74 Diabetes
pancreatic region. Taken together, these studies indicate that proliferative islet endocrine cells
are present within a significant population of pancreata from adolescent and young adult donors.
α cell proliferation declines with age and is greatly increased in some
adolescent and young adult pancreata
We considered the possibility that α cells could represent the proliferating islet endocrine
cells of pancreata from adolescents and young adults. We measured α cell proliferation in
control samples, quantifying glucagon+ Ki67+ nuclei (Fig. 3a f; Table S7). Infants exhibited
very high (up to 8.93%) and older adults very little (0 to 0.42%) α cell proliferation. (Fig. 3g;
Table S7). However, α cell proliferation was quite elevated (up to 3.4% of α cells were Ki67+)
in samples from a fraction (~1/3) of adolescents and young adults. Thus, α cell proliferation
gradually decreased with age, consistent with previous findings in islets (above), α cells (12),
and in β cells (1; 10; 11). Notably, many pancreata from adolescents and young adults with
increased islet endocrine cell proliferation also had abundant α cell proliferation (Table S8).
We tested for a proliferative response of α cells to T1D. Glucagon+ Ki67+ cells were
present in some T1D samples of children, adolescents, and young adults (Fig. 3d f). Abundant α
cell proliferation was present in a fraction of pancreata from children, adolescents, and young
adults (Fig. 3h; Table S8). Similar to controls, α cell proliferation declined as a function of age to
very low levels in older T1D adults (0 0.07%). Notably, α cell proliferation was decreased in
T1D samples from children/adolescents and young/older adults (Fig. 3i j; Table S7 8). Similarly,
Ki67+ α cells per islet was decreased in T1D pancreata from children, adolescents, and young
adults (Table S11). T1D individuals with the most α cell proliferation were mainly children and
adolescents with short disease duration (Fig. 3k l; Suppl. Table S8). α cell proliferation was Diabetes Page 10 of 74
present in the same subset of control and T1D adolescent and young adult samples that exhibited
islet endocrine cell proliferation (Table S7 8).
Increased α cell proliferation represents a fraction of total islet cell proliferation
Given parallel trends of proliferation between islet endocrine cells and α cells, we considered the possibility that α cells could represent the majority of proliferative islet cells.
However, glucagon+ Ki67+ cells only accounted for 24% and 30% of total intra islet cell proliferation in the highly proliferative control and T1D samples, respectively (Fig. 3m; Table
S12). Since β cell proliferation comprised only 10.2% and 1.1% of total intra islet cell proliferation in control and T1D pancreata (Fig. 1g; Table S6), respectively, most islet endocrine proliferation could not be accounted for within α and β cells. Thus, our studies revealed the presence of another population of highly proliferative islet endocrine cells within adolescent and young adults.
PP, somatostatin, and ghrelin cells exhibit minimal proliferation
In a further attempt to identify the highly proliferative islet endocrine cells, we separately quantified Ki67 staining with pancreatic polypeptide (PP), somatostatin, and ghrelin. However, there were very few proliferating PP, somatostatin, and ghrelin cells within a random sampling of adolescent and young adult pancreata (Fig. 4a i; Table S13). No difference was noted between control and T1D for any cell populations. A separate analysis quantified PP, SS, and ghrelin proliferation as a percentage of intraislet proliferation (Fig. 4j l), using the subset of non diabetic and T1D donors identified with high levels of proliferation. PP, somatostatin, and ghrelin cell proliferation accounted for very little of the total intra islet proliferation within control and T1D pancreata (Fig. 4j l; Table S14). Thus, most of the islet cells entering cell cycle within the Page 11 of 74 Diabetes
proliferative samples did not contain any of the five known islet endocrine hormones, although
some Ki67+ cells were glucagon+. Our studies therefore reveal the presence of highly
proliferative islet endocrine cells that do not express any of the known islet hormones.
No association of islet endocrine cell proliferation with pancreas
harvest conditions
Since increased islet endocrine cell proliferation was only present within a fraction of
pancreata from adolescents and young adults in control (9 of 34) and T1D (2 of 31), we
considered the possibility that islet cell cycle entry might be influenced by postmortem state or
conditions of organ harvest. We compared synaptophysin+ Ki67+ cell content of pancreatic
samples versus donor clinical parameters. Reassuringly, there was no association between
duration of intensive care unit (ICU) stay and islet cell proliferation in control or T1D (Fig. S4a
b; Table S15). Similarly, there was no association between α cell proliferation and ICU stay (Fig.
S4c d; Table S15).
Concerns have been raised regarding the potential impact of warm or cold ischemia to
reduce Ki67 immunoreactivity and thus confound measurement of human islet cell proliferation
(13). Although pancreatic transit time varied dramatically, there was no correlation with islet cell
proliferation or α cell proliferation (Fig. S4e h; Table S15). Thus, impaired sample quality did
not seem to explain the variability of islet endocrine proliferation in our population.
Cytoplasmic Sox9 in proliferative islet cells
We considered the possibility that highly proliferative islet endocrine cells could express
markers of islet endocrine progenitors. Ngn3, a key islet endocrine cell progenitor, was not
detected within either control or T1D pancreata (data not shown). We then tested for Sox9
expression, which influences development of pancreatic progenitor cells and adult exocrine cells, Diabetes Page 12 of 74
amongst other organs (14 16). Surprisingly, most Ki67 or PCNA proliferative cells showed Sox9
immunoreactivity in the cytoplasm (Sox9Cyt), in sharp contrast to pancreatic ducts (Fig. 5a b;
Fig. S5a d). To further test Sox9Cyt immunoreactivity, we stained pancreata with phospho specific antisera against serine 181 of Sox9 (p Sox9). Consistent with earlier Sox9 studies (17
23), p Sox9 antisera yielded cytoplasmic immunoreactivity in scattered islet endocrine cells that were uniformly ARX+ (Fig. S6a d). Many p Sox9+ ARX+ cells also expressed glucagon in variable amounts. (Fig. S6c d). In contrast to the above Sox9 studies, p Sox9+ antisera did not stain nuclear antigens in pancreatic acini and only weakly detected nuclei within duct structures
(Fig. S6e f). Collectively, these studies with p Sox9+ antisera support the possibility of Sox9Cyt
immunoreactivity within the highly proliferative ARX+ glucagon variable islet endocrine cells.
Quantification revealed very high amounts of Sox9Cyt Ki67+ cells (up to ~13%) in some
adolescent controls and T1Ds (Fig. 5c; Table S16). Indeed, Sox9Cyt immunoreactive cell proliferation accounted for ~72% of total intra islet proliferation in the subset of controls and
T1Ds with high endocrine cell proliferation (Fig. 5d; Table S16). Thus, Sox9Cyt Ki67+ cells
represented the proliferative hormone negative intra islet cells, which were also detected by p
Sox9 antisera (Fig. S6g h). Notably, Sox9Cyt Ki67+ cells were specific to islets and not primarily associated with pancreatic ducts or pancreatic parenchyma, as detected by ductal structures or cytokeratin 19 (Fig. S5e g; Table S17).
Absence of neuronal markers in cytoplasmic Sox9 immunoreactive
cells
Synaptophysin is a neuroendocrine marker and could also detect neurons in the pancreas or islet. Hence we employed additional neuronal markers to test if Sox9Cyt cells represented de Page 13 of 74 Diabetes
differentiated neurons. Sox9Cyt cells did not express β3 tubulin or NeuN, which detect peripheral
neurons (Fig. S6). Thus, proliferative intra islet cells did not express neuronal related markers.
Absence of β cell specific markers in proliferative islet cells
We employed additional β cell and endocrine differentiation markers to test if
proliferating intra islet cells represented de differentiated β cells. The Ki67+ islet cells did not
express Pdx1 (Fig. S8a), which defines mature β cells and a few somatostatin islet cells. Islet
Sox9Cyt cells also did not express the mature β cell marker, Nkx6.1, which was always found
within insulin+ cells and absent from glucagon+ cells (Fig. S8b) (1). Similarly, Ki67+ cells did
not express MafA, GAD65, PC1/3, and GLUT1 (Fig. S8c f). These studies indicate that the
proliferative intra islet cells did not express β cell related markers.
Proliferative islet endocrine Sox9cyt cells express α cell markers (ARX
and variable glucagon)
Further analysis of islet Sox9Cyt cells demonstrated that virtually all Sox9Cyt co expressed
synaptophysin (Control = 99.9%, T1D = 99.6%; Fig. 6a b; Table S18), which is consistent with
our synaptophysin Ki67 studies. Having excluded β cell related markers, we considered the
possibility that the proliferative islet endocrine Sox9Cyt cells might be related to α cells. We
looked for α cell progenitors after finding increased α cell proliferation, given that proliferative
α cells did not represent the majority of proliferative islet cells. We stained pancreata with
antisera against ARX, a marker of α cell related fate. Indeed, virtually all Sox9Cyt cells were also
ARX+ (Control = 99.3%, T1D = 98.4%; Fig. 6c d; Table S19). ARX+ Sox9Cyt cells variably
expressed glucagon: some ARX+ Sox9Cyt cells contained glucagon while others were glucagon
negative (Fig. 6c d). Sox9Cyt cells also variably expressed GLP1 and many GLP1+ cells co
expressed glucagon (Fig. S9). Notably, many ARX+ glucagon negative cells were Ki67+ (Fig. Diabetes Page 14 of 74
6e f; Table S20). These studies of ARX+ Sox9Cyt Ki67+ glucagon cells reveal that many of the proliferative islet endocrine cells have an α cell related phenotype.
We tested for other islet endocrine markers of ARX+ Sox9Cyt cells. We first looked for
INSMI, a transcription factor present within insulinomas (24) and in murine α , β , somatostatin,
and PP cells (25). INSM1 expression was consistently observed in β and α cells of young adult
control and TID samples and also in ARX+ Sox9Cyt cells (Fig. S10a d). Similarly, Isl1&2 was
readily detected in ARX+ Sox9Cyt cells (Fig. S10e). Additionally, Nkx2.2 marked both α/β cells
and was observed in highly proliferative Ki67+ insulin negative islet cells (Fig. S10f g). Thus,
ARX+ Sox9Cyt Ki67+ cells seem to express many markers common to islet endocrine cells.
To further characterize the endocrine properties of the proliferative cells, we tested for chromogranin A and other vesicular or exocytosis related proteins. ARX+ Sox9Cyt cells
expressed chromagranin A, as well as exocytotic machinery components synaptotagmin 1a and
SNAP25 (Fig. S11a f). Interestingly, secretogranin III, which is typically contained within islet
endocrine cells including α , β cells (26; 27), was absent from Ki67+ cells or some ARX+ cells
(Fig. S11g h). Thus, the ARX+ Sox9Cyt islet endocrine cells exhibit many (but not all) of the
common features of hormone secreting α cells.
ARX+ Sox9Cyt islet endocrine cells are conserved in infant and
children pancreata
To determine if the proliferative islet endocrine cells are unique to adolescents and young
adults, we studied additional pancreata from control infants and young children. Sox9Cyt
synaptophysin+ cells were abundant in islets from infants and young children (Fig. 7a b). ARX+
Sox9Cyt cells were readily detected in islets from infants and young children, with glucagon present in some but not all cells (Fig. 7c d). Similar to adolescents and young adults, most of the
Page 15 of 74 Diabetes
ARX+ Ki67+ cells in younger samples did not express glucagon (Fig. 7e f). ARX+ Sox9Cyt cells
consistently expressed INSM1 (Fig. 7g h). These studies reveal that ARX+ Sox9Cyt islet
endocrine cells are developmentally conserved and readily detected within pancreata across a
wide range of ages.
Cell death within proliferative islet cells
We hypothesized that vast proliferation of ARX+ Sox9Cyt islet endocrine cells might be
counter balanced by increased cell death. We quantified islet endocrine cell death via TUNEL
assay. Some adolescent and young adult control and T1D individuals exhibited intra islet
TUNEL nuclei (Fig. S12a c, f g; Table S21). However, Sox9 cells were not routinely TUNEL+
(Fig. S12d e; Table S22). Thus, the ARX+ Sox9Cyt islet endocrine cells seem to be a stable
population that does not immediately undergo cell death. However, overall islet endocrine cell
death seemed to be dramatically increased within some samples, especially those from
adolescents and young adults.
Increased cell death could limit the net expansion of islet endocrine cells. To directly test
the impact of increased islet endocrine cell proliferation, we quantified islet endocrine cell mass
using synaptophysin as a pan endocrine islet marker. While islet area and mass were decreased
in T1D samples compared to controls, islet endocrine area and mass did not correlate with age in
either cohort (Fig S13; Table S23). Thus, the proliferative islet endocrine cells do not appear to
result in accumulation of islet endocrine mass.
Diabetes Page 16 of 74
DISCUSSION
We analyzed human islet cell proliferation and found high amounts of islet endocrine and
α cell proliferation in some control and T1D pancreata from adolescents and young adults. The proliferative cells appear to be related to α cells and express ARX, Sox9Cyt, and various markers
of islet endocrine cells including INMS1, Isl1&2, and Nkx2.2 (Table 1). Sox9Cyt cells comprised the majority of islet endocrine cell proliferation (up to ~72%). ARX+ Sox9Cyt cells expressed
markers of secretory vesicles and machinery, suggesting some capacity for hormone secretion.
ARX+ Sox9Cyt cells were abundant in non proliferative individuals in a wide age range. Our studies reveal a novel population of highly proliferative α related cells and suggest previously unappreciated replicative capacity and plasticity within islet populations of human pancreata in adolescents and young adults.
Although β cell proliferation decreases dramatically after infancy (1; 10; 11), we found that islet endocrine and α cell proliferation was high in a subset of adolescents and young adults.
Interestingly, α cell proliferation was decreased in T1D compared to controls, suggesting that islet proliferation in adolescents and young adults may be impaired by diabetes. Furthermore, we observed an increase in hormone negative α related cells with substantial proliferation suggesting that adolescent and young adult pancreata may harbor cells with retained plasticity and replicative capacity. These α related cells were relatively abundant and comprised the majority of islet Ki67+ nuclei within donors with highly proliferative islets. They did not exhibit mature β cell markers: Pdx1, Nkx6.1, MafA, and GAD65. Notably, islet cell proliferation in
adolescent T1D pancreata was equivalent to control non diabetic pancreata. Although a
regenerative response to T1D was not observed, few samples were obtained from very recent
T1D onset. In contrast, Willcox et. al demonstrated that individuals with less than 18 months Page 17 of 74 Diabetes
disease duration may have compensatory α and β cell proliferation (6). However, the high rates
of islet and α cell proliferation observed in some T1D adolescents do not appear to be attempted
β cell regeneration from T1D associated β cell deficiency. Rather, these highly proliferative cells
seem to represent previously unrealized developmental plasticity within islet endocrine lineages,
unrelated to T1D. While most islet proliferation occurs during the perinatal period (28), islet cell
proliferation and apoptosis were comparable in our study further suggesting adolescence and
young adulthood as a unique period of high proliferation.
Sox9Cyt cells, which express many islet and α cell markers, might be in a transition state.
However, whether they are transitioning to or from α cells remains unclear. Sox9 is an important
transcription factor in developing pancreas for islet endocrine differentiation and development
(29). In contrast to typical nuclear Sox9 localization (30), we find highly proliferative cells with
Sox9Cyt immunoreactivity. Interestingly, Sox9 activity has been described to change with its
localization in the cytoplasm and nucleus within gonad, chondrocytes, breast, and gastric tissues
(17 23). In cultured cells nuclear translocation of Sox9 has been linked to β catenin degradation
and thus inhibition of Wnt signaling (22). In mature islets, future studies will determine whether
Sox9Cyt plays a role in cell proliferation or differentiation.
Our study has notable strengths but also limitations inherent to autopsy specimens. We
studied a large number of control and T1D samples across a wide age range and quantified vast
numbers of cells within several different cell types. Thus, our findings of highly proliferative
islet endocrine cells seem to represent a genuine phenomenon, and these Ki67+ cells can be
readily detected. However, studies of autopsy tissues are complicated by potential confounding
variables, including the cause and conditions of death of the patient, tissue transport conditions,
and other pre existing medical conditions prior to the patient’s demise. For example, hypoxemia Diabetes Page 18 of 74
or other conditions of ICU stay could alter pancreas physiology and possibly influence islet
endocrine cell proliferation. Thus, it will be difficult to resolve the highly proliferative α related
cells in vivo given the challenges of pancreatic biopsy from T1D individuals, which include pancreatitis (31). Although samples can be obtained from patients who require pancreatic
resection for cancer or tumors (32), such lesions typically occur in the middle aged and elderly.
Consequently, it might be difficult to obtain pancreatic samples from healthy adults. Therefore,
nPOD autopsy specimens represent a unique window into human physiology, albeit with
limitations.
In conclusion, we find no compensatory islet proliferation in T1D. However, we find a highly proliferative population of α related islet endocrine cells in adolescents and young adults.
The presence of proliferative ARX+ Sox9Cyt cells across a range of ages (infants to young adults)
demonstrates developmental conservation of this α related cell population and hints that α cells
may play an essential role in islet function and growth.
Page 19 of 74 Diabetes
ACKNOWLEDGEMENTS
The authors thank the pancreas donors and their families. This research was performed
with the support of nPOD, a collaborative T1D research project sponsored by the Juvenile
Diabetes Research Foundation International (JDRF). Organ Procurement Organizations,
partnering with nPOD to provide research resources, are listed at www.jdrfnpod.org/our
partners.php. We thank M. Yang, I. Kusmartseva, A. Pugliese, C. Wasserfal, M. Campbell
Thompson, and M. Atikinson for their generous and essential contributions to this work.
Additionally, we thank A. Myers, M. Beery, and E. Verney for helping us acquire samples for
this study.
This study was supported by funding from the Robert and Janice McNair Foundation,
NIH (1R01AG040110), and Diabetes Research Center of the Baylor College of Medicine (DRC
P30DK079638).
CJL, ARC, DRJ, MMR, & JAK conceived and designed the experiments. CJL, DRJ,
MMR, ARC performed the experiments. CJL, ARC, DRJ, MMR, & JAK analyzed the data. CJL,
ARC, & JAK wrote the paper. JAK is the guarantor of this work and, as such, had full access to
all the data in the study and takes responsibility for the integrity of the data and the accuracy of
the data analysis.
CJL, ARC, MMR, DRJ, and JAK declare no conflict of interest. JAK serves on the
scientific advisory board for Lexicon and Know Foods. MMR is a current employee of Janssen
Pharmaceuticals of Johnson and Johnson.
Diabetes Page 20 of 74
REFERENCES
1. Lam CJ, Jacobson DR, Rankin MM, Cox AR, Kushner JA: beta Cells Persist in T1D Pancreata Without Evidence of Ongoing beta Cell Turnover or Neogenesis. J Clin Endocrinol Metab 2017;102:2647 2659
2. Thorel F, Nepote V, Avril I, Kohno K, Desgraz R, Chera S, Herrera PL: Conversion of adult pancreatic alpha cells to beta cells after extreme beta cell loss. Nature 2010;464:1149 1154
3. van der Meulen T, Mawla AM, DiGruccio MR, Adams MW, Nies V, Dolleman S, Liu S, Ackermann AM, Caceres E, Hunter AE, Kaestner KH, Donaldson CJ, Huising MO: Virgin Beta Cells Persist throughout Life at a Neogenic Niche within Pancreatic Islets. Cell Metab 2017;25:911 926 e916
4. Ben Othman N, Vieira A, Courtney M, Record F, Gjernes E, Avolio F, Hadzic B, Druelle N, Napolitano T, Navarro Sanz S, Silvano S, Al Hasani K, Pfeifer A, Lacas Gervais S, Leuckx G, Marroqui L, Thevenet J, Madsen OD, Eizirik DL, Heimberg H, Kerr Conte J, Pattou F, Mansouri A, Collombat P: Long Term GABA Administration Induces Alpha Cell Mediated Beta like Cell Neogenesis. Cell 2017;168:73 85 e11
5. Chung CH, Hao E, Piran R, Keinan E, Levine F: Pancreatic beta cell neogenesis by direct conversion from mature alpha cells. Stem Cells 2010;28:1630 1638
6. Willcox A, Richardson SJ, Bone AJ, Foulis AK, Morgan NG: Evidence of increased islet cell proliferation in patients with recent onset type 1 diabetes. Diabetologia 2010;53:2020 2028
7. Martin Pagola A, Sisino G, Allende G, Dominguez Bendala J, Gianani R, Reijonen H, Nepom GT, Ricordi C, Ruiz P, Sageshima J, Ciancio G, Burke GW, Pugliese A: Insulin protein and proliferation in ductal cells in the transplanted pancreas of patients with type 1 diabetes and recurrence of autoimmunity. Diabetologia 2008;51:1803 1813
8. Moin AS, Butler PC, Butler AE: Increased Proliferation of the Pancreatic Duct Gland Compartment in Type 1 Diabetes. J Clin Endocrinol Metab 2017;102:200 209
9. Teta M, Rankin MM, Long SY, Stein GM, Kushner JA: Growth and regeneration of adult beta cells does not involve specialized progenitors. Dev Cell 2007;12:817 826
10. Meier JJ, Butler AE, Saisho Y, Monchamp T, Galasso R, Bhushan A, Rizza RA, Butler PC: Beta cell replication is the primary mechanism subserving the postnatal expansion of beta cell mass in humans. Diabetes 2008;57:1584 1594
11. Cnop M, Hughes SJ, Igoillo Esteve M, Hoppa MB, Sayyed F, van de Laar L, Gunter JH, de Koning EJ, Walls GV, Gray DW, Johnson PR, Hansen BC, Morris JF, Pipeleers Marichal M, Cnop I, Clark A: The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation. Diabetologia 2009;53:321 330 Page 21 of 74 Diabetes
12. Cnop M, Igoillo Esteve M, Hughes SJ, Walker JN, Cnop I, Clark A: Longevity of human islet alpha and beta cells. Diabetes Obes Metab 2011;13 Suppl 1:39 46
13. Sullivan BA, Hollister Lock J, Bonner Weir S, Weir GC: Reduced Ki67 Staining in the Postmortem State Calls Into Question Past Conclusions About the Lack of Turnover of Adult Human beta Cells. Diabetes 2015;64:1698 1702
14. Seymour PA, Freude KK, Dubois CL, Shih HP, Patel NA, Sander M: A dosage dependent requirement for Sox9 in pancreatic endocrine cell formation. Dev Biol 2008;323:19 30
15. McDonald E, Li J, Krishnamurthy M, Fellows GF, Goodyer CG, Wang R: SOX9 regulates endocrine cell differentiation during human fetal pancreas development. Int J Biochem Cell Biol 2012;44:72 83
16. Furuyama K, Kawaguchi Y, Akiyama H, Horiguchi M, Kodama S, Kuhara T, Hosokawa S, Elbahrawy A, Soeda T, Koizumi M, Masui T, Kawaguchi M, Takaori K, Doi R, Nishi E, Kakinoki R, Deng JM, Behringer RR, Nakamura T, Uemoto S: Continuous cell supply from a Sox9 expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat Genet 2011;43:34 41
17. Gasca S, Canizares J, De Santa Barbara P, Mejean C, Poulat F, Berta P, Boizet Bonhoure B: A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination. Proc Natl Acad Sci U S A 2002;99:11199 11204
18. Chakravarty G, Moroz K, Makridakis NM, Lloyd SA, Galvez SE, Canavello PR, Lacey MR, Agrawal K, Mondal D: Prognostic significance of cytoplasmic SOX9 in invasive ductal carcinoma and metastatic breast cancer. Experimental biology and medicine 2011;236:145 155
19. Chakravarty G, Rider B, Mondal D: Cytoplasmic compartmentalization of SOX9 abrogates the growth arrest response of breast cancer cells that can be rescued by trichostatin A treatment. Cancer Biol Ther 2011;11:71 83
20. Malki S, Nef S, Notarnicola C, Thevenet L, Gasca S, Mejean C, Berta P, Poulat F, Boizet Bonhoure B: Prostaglandin D2 induces nuclear import of the sex determining factor SOX9 via its cAMP PKA phosphorylation. EMBO J 2005;24:1798 1809
21. Moniot B, Farhat A, Aritake K, Declosmenil F, Nef S, Eguchi N, Urade Y, Poulat F, Boizet Bonhoure B: Hematopoietic prostaglandin D synthase (H Pgds) is expressed in the early embryonic gonad and participates to the initial nuclear translocation of the SOX9 protein. Dev Dyn 2011;240:2335 2343
22. Topol L, Chen W, Song H, Day TF, Yang Y: Sox9 inhibits Wnt signaling by promoting beta catenin phosphorylation in the nucleus. J Biol Chem 2009;284:3323 3333
23. Leung VY, Gao B, Leung KK, Melhado IG, Wynn SL, Au TY, Dung NW, Lau JY, Mak AC, Chan D, Cheah KS: SOX9 governs differentiation stage specific gene expression in growth plate Diabetes Page 22 of 74
chondrocytes via direct concomitant transactivation and repression. PLoS genetics 2011;7:e1002356
24. Zhang T, Chen C, Breslin MB, Song K, Lan MS: Extra nuclear activity of INSM1 transcription factor enhances insulin receptor signaling pathway and Nkx6.1 expression through RACK1 interaction. Cell Signal 2014;26:740 747
25. Jia S, Ivanov A, Blasevic D, Muller T, Purfurst B, Sun W, Chen W, Poy MN, Rajewsky N, Birchmeier C: Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic beta cell function. EMBO J 2015;34:1417 1433
26. Sakai Y, Hosaka M, Yoshinaga A, Hira Y, Harumi T, Watanabe T: Immunocytochemical localization of secretogranin III in the endocrine pancreas of male rats. Arch Histol Cytol 2004;67:57 64
27. Stridsberg M, Grimelius L, Portela Gomes GM: Immunohistochemical staining of human islet cells with region specific antibodies against secretogranins II and III. J Anat 2008;212:229 234
28. Kassem SA, Ariel I, Thornton PS, Scheimberg I, Glaser B: Beta cell proliferation and apoptosis in the developing normal human pancreas and in hyperinsulinism of infancy. Diabetes 2000;49:1325 1333
29. Seymour PA, Freude KK, Tran MN, Mayes EE, Jensen J, Kist R, Scherer G, Sander M: SOX9 is required for maintenance of the pancreatic progenitor cell pool. Proc Natl Acad Sci U S A 2007;104:1865 1870
30. Kopp JL, Dubois CL, Schaffer AE, Hao E, Shih HP, Seymour PA, Ma J, Sander M: Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas. Development 2011;138:653 665
31. Krogvold L, Edwin B, Buanes T, Ludvigsson J, Korsgren O, Hyoty H, Frisk G, Hanssen KF, Dahl Jorgensen K: Pancreatic biopsy by minimal tail resection in live adult patients at the onset of type 1 diabetes: experiences from the DiViD study. Diabetologia 2014;57:841 843
32. Inaishi J, Saisho Y, Sato S, Kou K, Murakami R, Watanabe Y, Kitago M, Kitagawa Y, Yamada T, Itoh H: Effects of obesity and diabetes on alpha and beta cell mass in surgically resected human pancreas. J Clin Endocrinol Metab 2016:jc20161374
Page 23 of 74 Diabetes
TABLE 1
Table 1. Adolescent and young adult pancreata contain a population of highly proliferative
α related cells that mostly glucagon negative. A summary of markers found to be detected or
absent in α , α’ , β , γ , δ , and ε cells.
α cell α’ cell β cell γ cell δ cell ε cell Glucagon Arx Insulin Pancreatic Somatostatin Ghrelin Polypeptide GLP1 Sox9Cyt Nkx6.1 Arx Nkx2.2 MafA D Sox9Cyt Insm1 GAD65 e t Nkx2.2 Isl1&2 Insm1 e Insm1 Synaptophysin Isl1&2 c Isl1&2 CgA Nkx2.2 t Synaptophysin SCG3 Synaptophysin e SCG3 SNAP25 CgA d SNAP25 SYT1 SCG3 SYT1 Ki67 SNAP25 CgA PCNA SYT1 Ki67 pHH3 Insulin Glucagon Glucagon Ki67 Ki67 Ki67 A Nkx6.1 GLP1 b MafA Insulin Arx Cyt s GAD65 Nkx6.1 Sox9 e MafA Ki67 n GAD65 t Pdx1 SCG3
Diabetes Page 24 of 74
FIGURE LEGENDS
Fig. 1. High levels of intra islet proliferation within some adolescents and young adults
but not within β cells. Islet images for (a) control and (b) T1D pancreata stained for
DAPI (blue), insulin (Ins; green) and Ki67 (red). (c d) Quantification of intra islet
cell proliferation in (c) control and (d) T1D represented by age (years) demonstrate
high intra islet proliferation in both control and T1D islets. Data points represent the
mean value for each of 33 controls and 18 T1D pancreata. (e f) Ki67+ intra islet cells
(% total) in T1D were very similar to control (e) children & adolescent pancreata and
(f) young & older adults pancreata. Results expressed as mean ± SEM for 11 control
and 12 T1D children & adolescents, 17 control and 6 young and older adults. (g)
Ki67+ β cells represent a small fraction of the total intra islet Ki67+ cells in a subset
of highly proliferating control (n=12) and T1D (n=6) pancreata, identified as
described in methods.
Fig. 2. Islet endocrine cell proliferation is greatly increased in some adolescent and
young adult pancreata. Analysis of islet endocrine cells in control and T1D
pancreata. Islet images for (a c) control and (d f) T1D stained for DAPI (blue),
synaptophysin (Syn; green), and Ki67 (red). Scale bar: 100 m. (g h) Quantification
of islet endocrine cell proliferation in (g) control and (h) T1D represented by age
(years). Data points represent the mean value for each of 59 controls and 46 T1D
pancreata. Ki67+ Syn+ (% total) in control and T1D v. age (years) demonstrate that
islet endocrine cell proliferation generally declines with age with notable proliferation
in adolescents and young adults. (i j) Ki67+ Syn+ (% total) in T1D was very similar Page 25 of 74 Diabetes
to control (i) children & adolescent pancreata but reduced compared to control (j)
young & older adults pancreata. Results expressed as mean ± SEM for 23 control and
23 T1D children & adolescents, 28 control and 23 young & older adults. (k l) Islet
endocrine cell proliferation v. T1D duration (years) reveals that most pancreata from
T1D individuals exhibited low rates of islet endocrine cell proliferation. (m) Ki67+
islet endocrine cells represent a small fraction of the total intra islet Ki67+ cells in a
subset of highly proliferating control (n=12) and T1D (n=6) pancreata.
Fig. 3. α cell proliferation is greatly increased in some adolescent and young adult
pancreata; no compensatory β cell proliferation in T1D. Analysis of α cell in
control and T1D pancreata. Islet images for (a c) control and (d f) T1D stained for
DAPI (blue), glucagon (Gcg; green), and Ki67 (red). Scale bar: 100 m. (g h)
Quantification of α cell proliferation in (g) control and (h) T1D represented by age
(years). Data points represent the mean value for each of 59 controls and 47 T1D
pancreata. Ki67+ α cell (% total) in control and T1D v. age (years) demonstrates that
α cell proliferation declines with age with notable proliferation in adolescents and
young adults. (i j) Ki67+ α cell (% total) in T1D was reduced compared to control (i)
children & adolescent pancreata and (j) young & older adults pancreata. Results
expressed as mean ± SEM for 23 control and 24 T1D children & adolescents, 28
control and 23 T1D young & older adults. (k l) α cell proliferation v. T1D duration
(years) demonstrates that most pancreata from T1D individuals exhibited low rates of
α cell proliferation. (m) Ki67+ α cells represent a fraction of the total intra islet
Ki67+ cells in a subset of highly proliferating control (n=12) and T1D (n=6)
pancreata.
Diabetes Page 26 of 74
Fig. 4. PP, somatostatin, and ghrelin cells exhibit minimal proliferation in control and
T1D. (a, d, g) Control and (b, e, h) T1D islets stained for DAPI (blue) and Ki67 (red)
with an islet endocrine hormone (green); (a b) pancreatic polypeptide (PP), (d e)
somatostatin (SS), or (g h) ghrelin. Scale bar: 100 m. (c, f, i) Quantification of PP,
SS, and ghrelin cell proliferation for adolescent control and T1D pancreata. Results
expressed as mean ± SEM for 5 controls (PP/Ghrelin), 6 controls (SS) and 9 T1D. (j)
PP, (k) somatostatin, and (l) ghrelin cells represent a small fraction of the total intra
islet Ki67+ cells in a subset of highly proliferating control (n=3) and T1D (n=3)
pancreata.
Fig. 5. Proliferative Sox9Cyt cells in adolescent and young adult samples represent the
majority of proliferating islet cells. (a b) Islet images for (a) control and (b) T1D
stained for insulin (Ins; blue), Sox9 (green), and Ki67 (red). Insets indicate Ki67+
Sox9Cyt+ cells. Scale bar: 100 m. (c) Quantification of Sox9Cyt cell proliferation in a
random sampling of adolescent control (n=6) and T1D (n=6) pancreata, represented
as % total Sox9Cyt cells. Results expressed as mean ± SEM for controls (n=6) and
T1D pancreata (n=6). (d) Ki67+ Sox9Cyt+ cells comprise the majority of proliferating
intra islet cells measured in a subset of highly proliferative controls (n=3) and T1D
(n=3) pancreata.
Fig. 6. Highly proliferative Sox9Cyt cells are endocrine cells, expressing ARX and
variable glucagon. (a, c, e) Control and (b, d, f) T1D islets stained for endocrine,
Sox9, Ki67, and α cell markers. (a b) Islets stained for synaptophysin (Syn; green)
and Sox9 (red) show synaptophysin, Sox9Cyt copositive cells. (c d) Islets stained for
Sox9 (green), ARX (red), and glucagon (white) indicate some Sox9Cyt, ARX,
Page 27 of 74 Diabetes
glucagon triple positive cells (inset c), and some Sox9Cyt ARX+ cells that are clearly
glucagon negative as visible in the inset of (d). (e f) Islets stained for glucagon
(white), ARX (green), Ki67 (red) indicate ARX and Ki67 copositive cells that do not
express glucagon. Scale bar: 100 m.
Fig. 7. Highly proliferative cytoplasmic Sox9 cells that express ARX, INSM1 and
variable glucagon are also present in infant and children pancreata. Control
islets of (a, c, e, g) infants and (b, d, f, h) children stained for islet endocrine, Sox9,
Ki67, and α cell markers. (a b) Islets stained for synaptophysin (Syn; green) and
Sox9 (red). Insets indicate synaptophysin, Sox9Cyt copositive cells. (c d) Islets stained
for Sox9 (green), ARX (red), and glucagon (Gcg; white). Insets indicate cytoplasmic
Sox9, ARX, glucagon triple positive cells, with a Sox9Cyt ARX+ cell that is clearly
glucagon negative visible in the inset of (d). (e f) Islets stained for glucagon (white),
ARX (green), Ki67 (red). Insets indicate ARX and Ki67 copositive cells that do not
express glucagon. (g h) Islets stained for Sox9 (white), ARX (green), and INSM1
(red). Insets indicate Sox9Cyt ARX+ cells expressing INSMI. Scale bar: 100 m.
a Control DAPI Ins Ki67 b Control DiabetesDAPI Syn pHH3 c Control DAPIPage Syn 28 PCNA of 74 6279 6131 6235 19 yrs 24.2 yrs 30 yrs
d T1D DAPIf Ins Ki67 e T1D DAPI Syn pHH3 f T1D DAPI Syn PCNA 6052 6052 6261 12 yrs 12 yrs 16 yrs
1 yr duration 1 yr duration 14.2 yr duration g 12 Control Intraislet Cell h 12 T1D Intraislet Cell i 8 Children & Adolescents 10 Proliferation 10 Proliferation Infants 6 8 Children 8 Children Adolescents Adolescents 6 6 Young Adults Young Adults 4 4 Older Adults 4 2 2 2 Intraislet Cells (% Total) (% Intraislet Cells Intraislet Cells (% Total) (% Intraislet Cells Intraislet Cells (% Total) (% Intraislet Cells + + + 0 0 0
0 10 20 30 40 50 60 70 Ki67 0 10 20 30 40 50 60 70 Ki67 Ki67 Control T1D Age (Years) Age (Years) j k Control & T1D β-Cell Proliferation 4 Young & Older Adults Control T1D Ki67+ Insulin+ 3 Ki67+ Insulin- 2
1 Intraislet Cells (% Total) (% Intraislet Cells + 0
Ki67 Control T1D
Lam et al Fig. 1 aPage Control 29 of 74 DAPI Syn Ki67 b Control DiabetesDAPI Syn Ki67 c Control DAPI Syn Ki67 6200 6235 6020 Newborn, 2 days 30 yrs 60 yrs
d T1D DAPI Syn Ki67 e T1D DAPI Syn Ki67 f T1D DAPIf Syn Ki67 6052 6261 6138 12 yrs 16 yrs 49.2 yrs
1 yr duration 14.2 yr duration 41 yr duration g Control Islet Endocrine Proliferation h T1D Islet Endocrine Proliferation i 12 12 15 Children & Adolescents 10 10 Infants 8 Children 8 Children 10 Adolescents Adolescents 6 6 cells (% Total) (% cells cells (% Total) (% cells cells (% Total) (% cells Young Adults Young Adults + + + 4 Older Adults 4 Older Adults 5 Syn Syn Syn + + + 2 2 Ki67 Ki67 Ki67 0 0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Control T1D AgeAge (Years) AgeAge (Years) (Years) j k T1D Islet Endocrine Proliferation l T1D Islet Endocrine Proliferation 12 12 4 Young & Older Adults 10 10 3 * 8 Children 8 Children Adolescents Adolescents 2 6 6 cells (% Total) (% cells
Young Adults Total) (% cells cells (% Total) (% cells Young Adults + + + 4 Older Adults 4 Older Adults 1 Syn Syn Syn + + 2 + 2
Ki67 0 Ki67 0 Ki67 0 Control T1D 0 10 20 30 40 50 60 70 0 2 4 6 8 10 Duration of Diabetes (Years) Duration of Diabetes (Years) m Control & T1D Islet Endocrine Cell Proliferation Control T1D Ki67+ Synaptophysin+ Ki67+ Synaptophysin-
Lam et al Fig. 2 a Control DAPI Gcg Ki67 b Control DiabetesDAPI Gcg Ki67 c Control DAPIPage Gcg 30 Ki67 of 74 6092 6279 6009 6 mths 19 yrs 45 yrs
d T1D DAPI Gcg Ki67 e T1D DAPI Gcg Ki67 f T1D DAPIf Gcg Ki67 6228 6261 6150 13 yrs 16 yrs 41.2 yrs
0 yr duration 14.2 yr duration 36 yr duration
10 g Control α-cell Proliferation h 10 T1D α-cell Proliferation i 10 Children & Adolescents
8 Infants 8 8 * Children Children 6 Adolescents 6 Adolescents 6 Young Adults Young Adults
-cells (% Total) (% -cells 4 4 -cells (% Total) (% -cells 4 Older Adults Older Adults Total) (% -cells α
α
α + + 2 + 2 2 Ki67 Ki67 0 Ki67 0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Control T1D Age (Years) AgeAge (Years) (Years) 10 10 j 5 Young & Older Adults k T1D α-cell Proliferation l T1D α-cell Proliferation 8 8 4 * Children Children 3 6 Adolescents 6 Adolescents Young Adults Young Adults 4 4 -cells (% Total) (% -cells 2 Total) (% -cells -cells (% Total) (% -cells Older Adults Older Adults α
α α + + 1 + 2 2 Ki67 Ki67 0 Ki67 0 0 Control T1D 0 10 20 30 40 50 60 70 0 2 4 6 8 10 Duration ofof Diabetes Diabetes (years) (Years) DurationDuration of of Diabetes Diabetes (years)(Years) m Control & T1D Islet α-cell Proliferation Control T1D Ki67+ Glucagon+ Ki67+ Glucagon-
Lam et al Fig. 3 aPage Control 31 of 74 DAPI PP Ki67 b T1D DiabetesDAPI PP Ki67 c T1D 4 PP-cell Proliferation 6153 6087 15.2 yrs 17.5 yrs 3
2
PP Cells (% Total) (% Cells PP 1 +
4 yr duration Ki67 0 Control T1D d Control DAPI SS Ki67 e T1D DAPI SS Ki67 f SS-cell Proliferation 6075 6084 4 16 yrs 14.2 yrs 3
2
SS Cells (% Total) (% Cells SS 1 +
4 yr duration Ki67 0 Control T1D g Control DAPI Ghrelin Ki67 h T1D DAPI Ghrelin Ki67 i Ghrelin-cell Proliferation 6075 6087 4 16 yrs 17.5 yrs * 3
2
1
Total) (% Ghrelin Cells + 4 yr duration 0 Ki67 Control T1D j Control & T1D Islet Pancreatic k Control & T1D Islet Somatostatin l Control & T1D Islet Ghrelin cell Polypeptide (PP) cell Proliferation (SS) cell Proliferation Proliferation Control T1D Control T1D Control T1D
Ki67+ PP+ Ki67+ SS+ Ki67+ Ghrelin+ Ki67+ PP- Ki67+ SS- Ki67+ Ghrelin-
Lam et al Fig. 4 Control Diabetes Page 32 of 74 a ControlControl Ins Sox9 Ki67 b T1D Ins Sox9 Ki67 6057 6052 22 yrs 12 yrs
1 yr duration c Sox9Cyt Proliferation d Control & T1D Islet Sox9Cyt cell proliferation 15 Control T1D
10 Cells (% Total) (% Cells cyt 5 Sox9
+ Cyt Ki67+ Sox + Ki67+ SoxCyt + 0 Ki67+ SoxCyt - Ki67 Control T1D
Lam et al Fig. 5 aPage Control 33Control of 74 Syn Sox9 Diabetesb T1DControl Syn Sox9 6004 6083 33 yrs 15.2 yrs
11 yr duration c Control Sox9 ARX Gcg d T1D Sox9 ARX Gcg 6227 6088 17 yrs 31.2 yrs
5 yr duration e Control Gcg ARX Ki67 f T1D Gcg ARX Ki67 6234 6261 20 yrs 16 yrs
14.2 yr duration
Lam et al Fig. 6 a Control Infant Syn Sox9 Diabetesb Control Child Syn Sox9 Page 34 of 74 6218 6094 5 wks 2.9 yrs
c Control Infant Sox9 ARX Gcg d Control Child Sox9 ARX Gcg 6117 6112 4 mths 6.3 yrs
e Control Infant Gcg ARX Ki67 f Control Child Gcg ARX Ki67 6222 6007 2 mths 9 yrs
g Control Sox9 ARX INSM1 h Control Sox9 ARX INSM1 Infant Child 6214 6106 5 days 2.9 yrs
Lam et al Fig. 7 Page 35 of 74 Diabetes
SUPPLEMENTAL FIGURE LEGENDS
Fig. S1. T-cells or endothelial cells are not proliferating within islets. (a) Lymphocytes or
(b-c) islet images for control pancreata stained for DAPI (blue), CD3 (green), CD31
(green) and Ki67 (red) reveal that proliferating cells are not T-cells or endothelial
cells as indicated by (a-b) CD3 or (c) CD31, respectively; (a) CD3+ lymphocytes
indicate a positive control for CD3 staining. Scale bar: 100 m.
Fig. S2. Proliferating islet endocrine cells verified by multiple proliferative markers. Islet
images for (a, c) control and (b, d) T1D stained for DAPI (blue), synaptophysin (Syn;
green) and proliferative markers (red). Proliferating islet endocrine cells (c-d) Syn+
pHH3+ and (c-d) Syn+ PCNA+ cells found in both control and T1D islets.
Fig. S3. Islet endocrine cell proliferation is consistent across different regions of the
pancreas. (a-d) Representative islet images for control pancreata stained for DAPI
(blue), synaptophysin (Syn; green), and Ki67 (red) demonstrate similar proliferation
rates in head and tail regions of the pancreas. (e) Correlation analysis of islet cell
proliferation measured as % total confirm similar rates of islet cell proliferation in the
differing regions of the pancreas.
Fig. S4. Duration of ICU stay and pancreas transit time are not associated with islet
endocrine cell or α-cell proliferation. (a-d) Duration of ICU stay (days) v. (a-b)
islet cell proliferation (% total) or (c-d) α-cell proliferation (% total) for (a,c) control
and (b,d) T1D pancreata. (e-h) Pancreas transit time (h) v. (e-f) islet cell proliferation
(% total) or (g-h) α-cell proliferation (% total) for (e, g) control and (f, h) T1D
pancreata.
Diabetes Page 36 of 74
Fig. S5. Proliferating islet endocrine Sox9Cyt cells verified by multiple proliferative
markers. Islet images of (a, c, e, g) control and (b, d, f) T1D stained for DAPI (blue),
Sox9 (green), and proliferative markers (PCNA/Ki67; red). Proliferating Sox9Cyt cells
are verified by (a-b) Sox9Cyt+ PCNA+ and (c-f) Sox9Cyt+ Ki67+ cells found in (a, c,
e) control and (b, d, f) T1D islets. (e-f) Ki67+ Sox9Cyt+ cells are not associated with
pancreatic ducts (d, white dashed lines). (g) Nuclear Sox9 in ducts. Sox9Cyt+ cells are
not associated with pancreatic ducts, as detected by CK-19 (green). Scale bar: 100 m.
Fig. S6. Sox9Cyt cells detected by Sox9 (pS181) antibody. Islet and ductal images of (a, c, e,
g) control and (b, d, f, h) T1D samples – stained for DAPI (blue), Sox9 (pS181)s
(green), Ki67 (red) and α-cell markers, ARX (red; blue) and glucagon (Gcg; red). (a-
f) pSox9Cyt intraislet cells express ARX, variable amounts of glucagon, and Ki67. (g-
h) Ductal cells contain occasional pSox9Cyt+ cells. Scale bar: 100 m.
Fig. S7. Sox9Cyt cells do not co-express neuronal markers. (a-b) Control young adult islets
stained for DAPI (blue), Sox9 (green), and neuronal marker, (a) β3 tubulin (red) or
(b) NeuN (red). Scale bar: 100 m.
Fig. S8. Highly proliferative cells do not express β-cell specific markers. Control
adolescent and young adult islets stained for Sox9, Ki67, and various β-cell markers.
Most Ki67+ cells do not express β-cell markers: (a) Pdx1 (green), insulin (blue), Ki67
(red) (b) Nkx6.1 (green), insulin (blue), Sox9 (red) (c) MafA (green), insulin (blue),
Ki67 (red) (d) GAD65 (magenta), insulin (white), Ki67 (green) (e) PC1/3 (magenta),
insulin (white), Ki67 (green). (f) GLUT1 (magenta), insulin (white), Ki67 (green).
Scale bar: 100 m.
Page 37 of 74 Diabetes
Fig. S9. Sox9Cyt cells co-express α-cell marker, GLP1. Islet images for (a-b) control and (c)
T1D stained for GLP1 and various markers. (a) α-cells express ARX (blue), glucagon
(green), and GLP1 (red). (b-c) Sox9Cyt (green) cells express variable GLP1 (red). (c)
DAPI (blue), Sox9 (green), and GLP1 (red). Scale bar: 100 m.
Fig. S10. Sox9Cyt cells co-express INSM1 and Isl1&2, which is common to human α- and
β-cells. (a-b) Islet images for (a) control and (b) T1D stained for insulin (Ins; green),
glucagon (Gcg; white), and INSMI (red) show INSMI expression in both human β-
and α-cells. (c) Control and (d) T1D stained for Sox9 (white), ARX (green), and
INSM1 (red) reveal Sox9Cyt ARX+ cells expressing INSMI. (e) Control islet stained
with Sox9 (white), Isl1&2 (red), and ARX (green) reveal that Sox9Cyt+ cells within
islets contain Isl1&2. (f) Human islets contain Nkx2.2 as shown in control islet
stained with insulin (Ins; green), glucagon (Gcg, white) and Nkx2.2 (red). (g) Control
islet stained with insulin (Ins; white), Nkx2.2 (red), and Ki67 (green) reveals that
Ki67+ insulin negative cells within islets contain Nkx2.2. Scale bar: 100 m.
Fig. S11. Highly proliferative cells express markers of secretory vesicles and exocytotic
machinery. (a-c) Control islets stained for chromagranin A (CgA; green/red), insulin
(blue), glucagon (green), ARX (blue), and pHH3 reveal that pHH3+ ARX+ intra-islet
cells contain CgA which is differentially expressed at higher levels in α-cells than in
β-cells. (c-f) Islet images stained for DAPI (blue), synaptophysin (Syn; green),
Sox9Cyt (green/red), CgA (red), ARX (white), and related secretory vesicular markers
(green). Sox9Cyt cells are islet endocrine cells that express (c) Syn, (d) CgA, (e)
synaptoptagmin 1A (SYT1), and (f) SNAP25. (g-h) SCG3 is absent in (g) some
ARX+ cells or in (h) Ki67+ cells. Scale bar: 100 m.
Diabetes Page 38 of 74
Fig. S12. Adolescent and young adults exhibit increased islet endocrine cell death but no
Sox9Cyt cell death. (a-e) Images stained for DAPI (blue), synaptophysin (Syn; green),
Sox9Cyt (green), and TUNEL (red). (a) Positive control for TUNEL staining within
ductal cells. (b-c) Control islets from (b) low and (c) high proliferating individuals
reveal very low TUNEL rates in islet endocrine cells, with some TUNEL+ islet
endocrine cells increased in highly proliferating adolescent and young adult islets. (d-
e) Control islets images from (d) low and (e) high proliferating individuals are
representative of no TUNEL+ Sox9Cyt cells found in analysis. Scale bar: 100 m. (f-g)
TUNEL+ islet endocrine cells (% total) v. age (years) in (f) control and (g) T1D
pancreata.
Fig. S13. Islet endocrine area and mass do not accumulate with age after adolescence or
with T1D. (a-b) Islet endocrine cell area and mass are reduced in T1D pancreata. (a)
Islet endocrine area (% total) is reduced in T1D pancreata. Results expressed as mean
± SD for (a) controls (n=43) and T1D (n=37). (b) Islet endocrine cell mass (g) is
reduced in T1D pancreata. Results expressed as mean ± SD for controls (n=28) and
T1D pancreata (n=33). (c-d) Correlation analysis of (c) islet endocrine area v. age
(years) and (d) islet endocrine mass v. age (years). No correlation between age and
islet endocrine cell (c) area or (d) mass for adolescents, young, & older adults.
Page 39 of 74 Diabetes
Control Control a Control DAPI CD3 b Control Ins CD3 Ki67 6230 6230 16 yrs 16 yrs
c Control Ins CD31 Ki67 6057 22 yrs
Lam et al Suppl. Fig. 1 Diabetes Page 40 of 74 a Control DAPI Syn pHH3 b T1D DAPI Syn pHH3 6131 6052 24.2 yrs 12 yrs
1 yr duration
c Control DAPI Syn PCNA d T1D DAPI Syn PCNA 6235 6261 30 yrs 16 yrs
14.2 yr duration
Lam et al Suppl. Fig. 2 Page 41 of 74 Diabetes
a ControlControl – Head DAPI Syn Ki67 b ControlControl – Tail DAPI Syn Ki67 6029 6029 24 yrs 24 yrs
c Control – Head DAPI Syn Ki67 d Control – Tail DAPI Syn Ki67 6131 6131 24.2 yrs 24.2 yrs
e 4 r2=0.9775 p < 0.05
3
2 Region1 1
0 Islet Cell Proliferation (% total) (% Proliferation Cell Islet 0 1 2 3 4 Region 2 Islet Cell Proliferation (% Total)
Lam et al Suppl. Fig. 3 Diabetes Page 42 of 74
Infants to Older Adults a 15 Control Islet Cell Proliferation b 15 T1D Islet Cell Proliferation vs. Duration of ICU Stay vs. Duration of ICU Stay
10 r2 = 0.01887 10 r2 = 0.007468 p = 0.3127 p = 0.5770 cells (% Total) (% cells cells (% Total) (% cells + + 5 5 Syn Syn + + Ki67 0 Ki67 0 0 10 20 30 0 5 10 15 20 25 Duration of ICU Stay (days) Duration of ICU Stay (days) c 15 Control !-cell Proliferation d 15 T1D !-cell Proliferation vs. Duration of ICU Stay vs. Duration of ICU Stay
10 10 r2 = 0.00015 r2 = 0.000002 p = 0.9286 p = 0.9758 -cells (% Total) (% -cells -cells (% Total) (% -cells 5 5 ! !
+ + Ki67 Ki67 0 0 0 10 20 30 0 5 10 15 20 25 Duration of ICU Stay (days) Duration of ICU Stay (days) e 15 Control Islet Cell Proliferation f 15 T1D Islet Cell Proliferation vs. Pancreas Transit Time vs. Pancreas Transit Time
10 r2 = 0.05134 10 r2 = 0.008346 p = 0.0994 p = 0.5602 cells (% Total) (% cells cells (% Total) (% cells + + 5 5 Syn Syn + + Ki67 0 Ki67 0 0 10 20 30 40 50 0 10 20 30 40 Pancreas Transit Time (h) Pancreas Transit Time (h) g 15 Control !-cell Proliferation h 15 T1D !-cell Proliferation vs. Pancreas Transit Time vs. Pancreas Transit Time
10 r2 = 0.005854 10 r2 = 0.001809 p = 0.5824 p = 0.7790
-cells (% Total) (% -cells 5 Total) (% -cells 5 ! !
+ +
Ki67 0 Ki67 0 0 10 20 30 40 50 0 10 20 30 40 Pancreas Transit Time (h) Pancreas Transit Time (h)
Lam et al Suppl. Fig. 4 Page 43 of 74 Diabetes Diabetes Page 44 of 74
a Control DAPI p-Sox9 ARX b T1D DAPI p-Sox9 ARX 6057 6084 22 yrs 14.2 yrs
4 yr duration
c Control p-Sox9 ARX Gcg d T1D p-Sox9 ARX Gcg 6075 6089 16 yrs 14.3 yrs
8 yr duration e Control DAPI p-Sox9 f T1D DAPI p-Sox9 6235 6084 30 yrs 14.2 yrs
4 yr duration
g Control DAPI p-Sox9 Ki67 h T1D DAPI p-Sox9 Ki67 6131 6261 24.2 yrs 16 yrs
14.2 yr duration
Lam et al Suppl. Fig. 6 Page 45 of 74 Diabetes
a Control DAPI Sox9 "3 Tubulin b Control DAPI Sox9 NeuN 6057 6030 22 yrs 30.1 yrs
Lam et al Suppl. Fig. 7 Diabetes Page 46 of 74
a Control Ins Pdx1 Ki67 b Control Ins Nkx6.1 Sox9 6232 6235 14 yrs 30 yrs
c Control Ins MafA Ki67 d Control Ins GAD65 Ki67 6232 6279 14 yrs 19 yrs
e Control Ins PC1/3 Ki67 f Control Ins GLUT1 Ki67 6235 6153 30 yrs 15.2 yrs
Lam et al Suppl. Fig. 8 Page 47 of 74 Diabetes
a Control ARX Gcg GLP1 b Control DAPI Sox9 GLP1 6030 6162 30.1 yrs 22.7 yrs
c T1D DAPI Sox9 GLP1 6083 15.2 yrs
11 yrs duration
Lam et al Suppl. Fig. 9 Diabetes Page 48 of 74
Control Control a Control Ins Gcg INSM1 b T1D Ins Gcg INSM1 6235 6245 30 yrs 22 yrs
1 yr duration c Control Sox9 ARX INSM1 d T1D Sox9 ARX INSM1 6174 6039 20.8 yrs 28.7 yrs
12 yr duration e Control Sox9 ARX Isl1&2 f Control Ins Gcg Nkx2.2 6232 6234 14 yrs 20 yrs
g Control Ins Nkx2.2 Ki67 6233 14 yrs
Lam et al Suppl. Fig. 10 Page 49 of 74 Diabetes
a Control ARX CgA pHH3 b Control Ins Gcg CgA 6153 6053 15.2 yrs 25 yrs
c Control ARX Syn CgA d Control DAPI Sox9 CgA 6162 6174 22.7 yrs 20.8 yrs
e f Control SYT1 Sox9 ARX Control SNAP25 Sox9 ARX 6098 6096 17.8 yrs 16 yrs
g ControlControl Ins SCG3 ARX h Control Ins SCG3 Ki67 6233 6235 14 yrs 30 yrs
Lam et al Suppl. Fig. 11 Diabetes Page 50 of 74 a Control – DAPI TUNEL b Control – DAPI Syn TUNEL c Control – DAPI Syn TUNEL Low Cell Low Cell High Cell Turnover Turnover Turnover 6099 6099 6235 14.2 yrs 14.2 yrs 30 yrs
d Control – DAPI Sox9 TUNEL e Control – DAPI Sox9 TUNEL Low Cell High Cell Turnover Turnover 6099 6235 14.2 yrs 30 yrs
1 yr duration 14.2 yr duration f Control Islet Endocrine Cell Death g T1D Islet Endocrine Cell Death 0.5 0.5
0.4 Infants 0.4 Children Children 0.3 Adolescents 0.3 Adolescents Young Adults Young Adults 0.2 0.2 TUNEL TUNEL Older Adults TUNEL 0.1 0.1
(% Total Endocrine Cells) Endocrine Total (% 0.0 Cells) Endocrine Total (% 0.0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Age (Years) Age (Years)
Lam et al Suppl. Fig. 12
Page 51 of 74 Diabetes
a Islet Endocrine Area – b Islet Endocrine Mass– adolescent, young, & older adult adolescent, young, & older adult
5 **** 4 **** 4 3 3 2 2 1 1
0 (g) Mass Endocrine Islet 0
Islet Endocrine Area (% Total) (% Area Endocrine Islet Control T1D Control T1D c Islet Endocrine Area v. Age – d Islet Endocrine Mass v. Age – adolescent, young, & older adult adolescent, young, & older adult
5 Control 4 Control T1D T1D 4 3 3 2 2
1 1
0 (g) Mass Endocrine Islet 0 0 20 40 60 80 0 20 40 60 80 Islet Endocrine Area (% Total) (% Area Endocrine Islet Age (Years) Age (Years)
Lam et al Suppl. Fig. 13
Diabetes Page 52 of 74 9OKKGAHAIN=G :=>GA '$ 5JI#0E=>ANE? 9=HKGA 7JKOG=NEJI% I:94 ?=MA IOH>AL# =CA !SA=LM"# MAR# ANDIE?ENS# ?=OMA JB @A=ND# 3$ KAKNE@A !IC&HG"# 6>1(? ! "# 287# >J@S QAECDN !FC"# K=I?LA=M M=HKGA LACEJI# ?JLLAMKJI@EIC >GJ?F IOH>AL# =I@ HANDJ@ JB NEMMOA LA?JPALS% "I&=" EI@E?=NAM 3$KAKNE@A HA=MOLAHAINM QALA IJN HA=MOLA@ BJL ND=N ?=MA% /JINLJG 2>-'? .J@R 7=I?LA=M I760 -CA /#KAKNE@A 2>-'? .GJ?F :EMMOA 9AQ 1NDIE?ENR /=OMA JB 0A=ND !HHJG% .43 ;AECDN 9=HKGA /=MA # ! 3IB=INM !&#'$*" -((, '%+)' 8 3=O?=ME=I 1IJRE= +%,0 I&= I&= (.%( .%' :=I<=EG ') 9LC=I 4JIJL -'0) '%,'' 5 1BLE?=I 1HALE?=I 1IJRE= '%*, I&= I&= (*%/ 0%' :=I<=EG ') 9LC=I 4JIJL -('* (%, 8 3=O?=ME=I 1IJRE= '%0/ -%( +* (-%/ /%' :=I<=EG '+ 9LC=I 4JIJL -('. )%) 8 1BLE?=I 1HALE?=I 1IJRE= ,%0 ,%) ** (,%0 (,%' :=I<=EG '+ 9LC=I 4JIJL -'0+ )%0 8 1BLE?=I 1HALE?=I 1IJRE= *%,, I&= I&= (,%/ (+%' :=I<=EG '- 9LC=I 4JIJL -('- )%0 8 3=O?=ME=I 1IJRE= .%*- I&= I&= (/%( (-%' :=I<=EG '+ 9LC=I 4JIJL -'', ,%' 5 3=O?=ME=I 3ALA>LJP=M?OG=L&;NLJFA I&= I&= I&= (,%. (-%/ :=I<=EG '+ 9LC=I 4JIJL -(() -%* 5 6EMK=IE? 6A=@ /DEG@LAI !'$+#')$," -)./ ('%' 5 1BLE?=I 1HALE?=I 1IJRE= +%,+ -%* +, )(%* ,)%+ :=I2J@S ') 9LC=I 4JIJL -)*) (+%' 5 3=O?=ME=I 6A=@ -@JGAM?AINM !'*#(&$," -).0 (0%' 8 3=O?=ME=I 6A=@ ! *&" -'(' +.%' 5 3=O?=ME=I 3ALA>LJP=M?OG=L&;NLJFA I&= I&= I&= (0%. ,-%/ :=I<=EG '0 9LC=I 4JIJL -''/ ,'%' 5 3=O?=ME=I 6A=@ 1HIKDPEM "(%,$(*%.# /+-, *,') .') @ 7GZIGXOGT 7KWKHWU[GXIZRGW(CYWUQK )'-+ *,'* *+) & & $ & * +*', .1'* BGT /DSKRQ /*,1 -2'+ -*') : 7GZIGXOGT 5TU]OG 4)'). T(G T(G & & $ & * ,,'0 2.') BGT6UJ^ )- AWMGT 8UTUW /)-) .)') +)') : 7GZIGXOGT 7KWKHWU[GXIZRGW(CYWUQK 4)'). 0', ./ & & $ & * ,*'/ 1.') BGT .B>>:5;5 ,-()"-0 "*/(#+%(&. " 2@D ALIIS (1('' @ 5 AZUVIPU 26.'-/ !* BWFWOMQ 4LMGNIQ (1('' >RXWU 3MRORKMGEOU >3(''$(-() 45* @EFFMV (1('' BLITPR 6MULIT ?2($*.)/) 45*( @EFFMV (1('' 2FGEP EF)/*-+ 4LTRPEKTEQMQ 2 =RWUI (1('' 2FGEP EF/)'+ 7LTIOMQ @EFFMV (1('' ?LRIQMY ?LETPEGIWVMGEOU 8$'*($.. 7OWGEKRQ @EFFMV (1),' 2FGEP EF/',, 7OWGEKRQ =RWUI (1),' 2FGEP EF('0// 7 6KGGC>D>EJ:C 7:;C> '$ 6KDD:HM F? >LG>HBD>EJ:C =>J:BCI$ 186 0IC>J 3K< 1B()" +59" ,MJ 6FL* ,MJ 0IC>J !#<>CC "#<>CC 44 66 /AH>CBE 6FL* ,MJ 6FL* +59" .E=F /2<4?62@ (, (, (, ( ) ( ) $% $% $% & & & ) ) & & & $+ ) .=<=?@ /2<4?62@ (, (, (, ( ) ( ) $% $% $% & & & ) ) & & & $+ ) 064A9=<@ ,FEJHFC -6::@ )%'%&& $,''&$ $,''&$ %&%$ ,,)+ +*% (+%# $&&#)# &)+%( +(#(( $&*( )(&& )#' (+%# (%(+ %$('( $%)), (&)' $(',*' ,,& 9@:6A@ /2<4?62@ '+ '+ '+ , , , ) ) ) ) + * , ) ) & & & $% <"2 .=<=?@ /2<4?62@ 7&- '+ '+ '+ , , , ) ) ) ) + * , ) ) & & & $% <"2 064A9=<@ -6::@ '(+)%) %(,$*( %(,$*( $#++ +,'$ *,+ &%&# *#%,* ')'#& )#+, ),# '##+ %)$ &%&# $'*) $)#** $#$() $$)* +(*)) <"2 Diabetes Page 56 of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xxxxxx & & %$,' +'(- '%$% , .&'+ %$%- +'') '&$%xxxxxx &$* )& &-)+- %$'' +&,) '%$- . &),.' %$%+ +&.- ''$% ($% &' +.(. %$&, *H:D6<: &,$% .' &)(*- %$+( +')* ''$% ,$% +) (+(,, %$&- 3, ($( .' ))+' %$+* +'), ')$% %$+ &' &),-% %$%- 3-0 &$% '- &()* %$'% +'&& ')$% )$% +& &,+*- %$(* +%') '&$% + &)-,+ %$%) +&.+ '+$% &*$% &)' (().- %$)' +&,. '&$- ,' &(%)- %$** *H:D6<: '($' *$' ** '&'-, %$') +&+' ''$, ' ,%'* %$%( 3, &$-( *$( )- &&(+- %$&( +%%( '($% '% &(%*) %$&* 3-0 %$,* '$' '% )+)& %$%* xxxxxx +%'. ')$% + &*)%. %$%) C$H6?G: !H% 8BAFDB?" %$%) xxxxxx & % %$+% +&(& ')$' )&' &,-&, '$(& xxxxxx +%)- (%$% - &%(+* %$%- +'(* (%$% &., &''.( &$+% +%(% (%$& +% -**- %$,% +''. (&$% '% '.(&- %$%, +%&& )+$% * ((++ %$&* +%%- *%$% && *-+- %$&. +&+- *&$% (- &&&)' %$() +%&, *.$% & &&&)+ %$%& +%'% +%$% &, ...* %$&, +%&+ +)$% % (*,, %$%% +%&( +*$% % ..(. %$%% *H:D6<: (-$) *& &&*,+ %$(- 3, &+$* &%* +%), %$+) 3-0 )$% '* &)+, %$&* Page 57 of 74 Diabetes 2FBB>9?9@E5> 356>9 '% -@8=G=8F5> !$79>> BCA>=:9C5E=A@ 9IBC9DD98 5D A: =@EC5=D>9E .='(# 79>>D =@ @A@$8=569E=7 5@8 3&+ B5@7C95E5% 19,-" !$45:: 6D>?I />LDKJ ! 6D>?I /M?I;A? +*%' .*,' '& &%'( )*,- ( &%&. 81 -%/ (-,) '. &%(' ((/, ) &%'+ 825 (%, /(' , &%&- -,+ ' &%&+ Page 59 of 74 Diabetes 9MIIE@F@GL 1CDE?J@G !'%+$')%/" 0N@J xxxxx I$N " *&" .(+. ,1&* +,&( 1**+ ) (&() ).+1 ( (&(( .)+0 ,1&* ,)&( )0*, ( (&(( ),*. ) (&(/ .(,( -(&( *(&( ).(0 ( (&(( )1)0 ( (&(( 0N@J I$N xxxxx Diabetes Page 60 of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iabetes Page 62 of 74 7KGGC>D>EJ:C 8:;C> &&$ .L>H:@> !#<>CC EKD;>H G>H BIC>J 714 &$& ,), '* '( ) %$- /ABC=H>E &$*#&($-! .L>H:@> &%$+ ($, ).%& '( &(+ )% %$&, +'(' &)$% '--' )' &.- &* &$* 70 ($' ($' (%-( (( .% &. %$&, +'(( &)$% &.%- '& &(* &) &$& 714 %$. %$. --' &% '+ * %$%* xxxxx +%.. &)$' )*.* ' +& ,* %$% G#L:CK> L$ +%%& ''$% &&)&) - '.& (. %$& G#L:CK> L$ +%&% ),$% '''( % &%. '% %$% G#L:CK> L$ +%%- *%$% &+-& % *' (' %$% +&*% )&$'xxxxx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xxxxx 714 '$+ ,+* & (% ' %$% G#L:CK> L$ xxxxx Page 63 of 74 Diabetes 3GCC?:@:AF6? 467?: &'% .A9>H>9G6? !$8:?? CDB?>;:D6F>BA :JCD:EE:9 6E B; >AFD6>E?:F />()# 8:??E >A ABA$9>67:F>8 6A9 4&, C6A8D:6F6% 19,-" !$45::@ ,&)& )&%' (/ '&( (.%*) xxxxxx ,&&* ))%& ,/ )/( '-%,& *H:D6<: ('%) *( ',, ()%/- 3, ,%. )* ''* '&%', 3-0 (%& '& )) (%/) Diabetes Page 64 of 74 5IEEA 1@)*# 1@)*# 7<8FG 1@)*# 6DH8A 1@)*# 6DH8A 1@)*# 1@)*# 6DH8A +>< 44# /?F xxxxxx -',) ()%' ( ' )0- '%'' + 0', '%++ ' -+ '%'' -((* (*%( (%- ' (.- '%'' ' ((-. '%'' ' ()0 '%'' -'/+ (+%) + ' )(/ '%'' * (0*- '%(, ' -/ '%'' -'/0 (+%* / ' ,) '%'' ' -.- '%'' ' +* '%'' -'+0 (,%' (' ' ' '%'' ( ()/+ '%'/ ' )') '%'' -'/* (,%) (( ' .( '%'' ( (0., '%', ' (+, '%'' -'/. (.%, + ' (*, '%'' ' ' '%'' ' /, '%'' 6'- -(+, (/%' (( ' ('+ '%'' ( 00/ '%(' ' ). '%'' -'-+ (0%- 0 ' *- '%'' ' ' '%'' ( *, )%/- +J xxxxxx Page 65 of 74 Diabetes 5IEEA 1@)*# 1@)*# 7<8FG 1@)*# 0CHF8@GA .*++ ),&( D'9 ( ,) (&(( )+ */* ,&/0 ( ,- (&(( .)-+ )-&* D'9 ). )+( )*&+) * 0( *&-( ( .) (&(( .(/- ).&( D'9 ) *( -&(( * )* ).&./ ( +0 (&(( xxxxxx +J xxxxxx .(-* )*&( ) ( 11 (&(( , ,*1 (&1+ ( ),, (&(( .))+ )+&) )&. ( ) (&(( ( )0 (&(( ( )1 (&(( .(0, ),&* , ( *) (&(( + ,) /&+* ( +* (&(( +J E%J8AI< !J& :DCHFDA" (&(0,,xxxxxx (&++ (&.+ (&)0 (&,, (&0( (&+- xxxxx (&.1 xxxxx xxxxxx xxxxx xxxxx Diabetes Page 66 of 74 4JFFB=C=DI9B 59:B= &*$ 0D 4/1 &$&-& &$,/ '$+( '$*+ -@AB xxxxx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xxxxxxxxx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xxxxxx +I;E7=; ',$* xxxxxx ** /-& +- )$&' -*$./ 4- '$-+ xxxxxx .) ,&& '&' )$/. (,$'- 4.1 &$-' xxxxxx )* (*+ *' '$,( '&$,. xxxxxx ,&+( '($& '$& '&& --- '(( '($.- .'$/- ,'') ')$' '$, '- *+* (. )$-* ,&$-' ,&.* '*$( *$& ,- +*, /& '($(- -*$** ,&./ '*$) .$& ( .+) ') &$() '+$). ,&*/ '+$& '&$& ( )*+ * &$+. +&$&& 5&- ,'*+ '.$& ''$& & (++ ( &$&& &$&& +I;E7=; '*$* +$/ )' +). *) *$/+ *-$&/ 4- ($&* *$) *( ()- +' ,$&, )($.' 4.1 &$.) '$. '- /- (' ($*- ')$)/ D$I7@H; !I% 9CBGEC@" &$'' xxxxxx &$-+ &$') &$-. &$+) &$'* xxxxxx Diabetes Page 68 of 74 4HDD@;A;BG7@ 578@; &(* 1H9@;7E 4CK) :H9G7@ 9;@@ DEC@?<;E7G?CB /?'(# 4CK)1H9# 6;7EF /?'(# 4CK)1H9# -H9G7@ ,;@@F B32- ,7F; # +=; J?G> -H9G7@ ,;@@F -H9G7@ ,;@@F -?78;G;F ! 5CG7@ -H9G ,;@@F" ,&// '*$( <%4 )'+ & &$&& ,'+) '+$( <%4 (.( & &$&& ,&-+ ',$& <%4 ))' & &$&& ,&/, ',$& <%4 -,' & &$&& ,&/. '-$. <%4 (/-- + &$'- ,CBGEC@ ,(-/ '/$& <%4 +/( ' &$'- +I;E7=; ',$* xxxxxxx .-, ' &$&, xxxxxxx 4- '$-+ xxxxxxx '&*, ( &$&/ xxxxxxx 4.0 &$-' xxxxxxx *(- ' &$&* ,&+( '($&xxxxxxx '$& '-. & &$&& ,'') ')$' '$, .) & &$&& ,&.* '*$( *$& ++/ & &$&& ,&*/ '+$& '&$& (.& & &$&& ,'*+ '.$& ''$& '/- & &$&& 5&- ,'/+ '/$( +$& '-/ & &$&& +I;E7=; '+$) +$* (*, & &$&& 4- ($. *$( ',, & &$&& 4.0 '$' '$- ,. & &$&& xxxxxxx D$I7@H; !I% 9CBGEC@" &$*) xxxxxxx &$'. &$(+ &$'* xxxxxxx Page 69 of 74 Diabetes 0D@@<7=7>C3< 134<7 &'% 0?G(*HC# ;B<7C 7>6?5A;>7 57<?>$6;347C;5 3>6 1&+ B3=@<7B% 5AE6> 4H@# 8:>>D !@F?7:C"$ 4AG13HE# 8:>>D !@F?7:C2 A; DH@# 8:>>D"$ 6@9 4H@# 4AG13HE# 8:>>D !@F?7:C2 A; 4AG13HE# 8:>>D"& "@'6" =@9=86E:D E<: ?:6DFC:?:@E =D @AE 6BB>=867>: ;AC E<: 86D:& 3CADD%<6E8<:9 7AG:D =@9=86E:D E<6E E<: ?:6DFC:?:@E 8AF>9 @AE 7: 86>8F>6E:9& 0H># 0?G(*HC# 273AB 0?G(*HC# *7<# 0?G(*HC# *7</.+ *3B7 # F;C: 0H># *7<# !273AB" *7< )E7A397 ++&( xxxxx /)0* ,// ,/. 11&1( .&/* *?>CA?< 0+ -&* xxxxx *(0. ).1 ).0 (&)/ )&,1 xxxxx 0,- +&( xxxxx )*(, 10 1/ (&)( (&0. .*)* *(&(xxxxx -&( .0/+ ))(. ))(( 11&,. ).&(1 .(*- *+&0 )1&( *.01 *11 *10 11&./ ))&)* .*)) *,&( ,&( .-)- 10/ 10, 11&/( )-&)- )E7A397 **&. 1&+ -+-1 /1/ /1, 11&.) ),&)* 1&+ 0+ *&+ 0&, *+)1 ,+. ,++ (&)+ *&., 0,- )&+ ,&0 )++1 *-* *-( (&(/ )&-* xxxxx @$E3 xxxxx Diabetes Page 70 of 74 0EAA=8>8?D4= 145=8 &'% 0@H')ID (/2 6@$A@C 0@H')ID#(/2# 0@H')ID# )8==C (:8 384BC G (F8B4:8 *- xxxxxxx ,**+ ))+/ ))+( 11&+, *0&.) )@?DB@= 0* -&. xxxxxxx ***- --1 -.) (&00 0&() xxxxxxx 0+, +&* xxxxxxx )*0, +*+ +*, (&-) ,&.+ xxxxxxx .*)* *(&( -&( ,(,- ))(. )(01 10&,. */&+, .(*- *+&0 )1&( )-./ *11 *1. 11&(( )1&(0 .*)) *,&( ,&( ,-,, 10/ 1.. 1/&0/ *)&/* (F8B4:8 **&. 1&+ ++0- /1/ /0, 10&,, **&/) 1&* 0* *&+ 0&, )-1, ,+. ,*/ (&-. ,&** 0+, )&+ ,&0 1*) *-* *,. (&+* *&,, A$F4=E8 !F% 6@?DB@=" (&-+ xxxxxxx (&.* (&,- (&,, (&*) (&+* xxxxxxx Page 71 of 74 Diabetes 5JFFB=C=DI9B 69:B= (&% 3GEBA>=G9IAD? +47# ?BJ;9?ED D=?9IAK= ;=BBH >GEC DED$ xxxxxxxxx +K=G9?= *+&, xxxxxxxxx )/00 ,* -&+/ xxxxxxxxx ,EDIGEB 5- /&( *),) ,/ /&,* xxxxxxxxx 5.1 ,&) xxxxxxxxx )*+. */ ,&*0 xxxxxxxxx .))+ )+&) )&. +/+ )0 ,&0+ .(0, ),&* ,&( +-( +/ )(&-/ .*.) ).&( ),&* ,,, )-1 +-&0) +K=G9?= ),&, .&. +01 /) )/&(/ 6'- 5- )&- .&/ ,1 // ).&,0 5.1 (&0 +&1 *0 ,, 1&-* F$K9BJ= !K% ;EDIGEB" (&)( xxxxxxxxx (&+* (&.( (&++ xxxxxxxxx xxxxxxxxx Diabetes Page 72 of 74 1EAA=9>9?D5= 256=9 '&% (8@=9C79?D CH?5AD@A;HCA 4HA"! 9;?? CDB?><;D7F>BA AG@8;D0 % FBF7?!# FBF7? >E?;F ;A:B9D>A; 9;?? AG@8;D!$ 495BC 23.+,# 1H?# (:9 23.+,# 1H?# ?0/* )5C9 # G 4EAA=:>:?D8= %%$ 561,/# 4@G(*HD# )?8=HC 561,/# ?32+ );: 561,/# .<&'# 4H?# -C=:DC !#" *HD *8C: # !7:8BC" 4@G(# 9:==C 4@G( # 9:==C ! 5@D8= 4H?#" !# A:B -C=:D" +%.. &)$' % &.. %$%%% %$%+% +%.+ &+$% % &() %$%%% %$&*% +%.- &,$- % '+, %$%%% %$%*% +'*( &.$% % &'+ %$%%% %$'-% +'(* (%$% % &-* %$%%% &$)%% +%(% (%$& % -' %$%%% %$*.% )F:B8;: '&$' % &++ %$%%% %$)'' 4+ ,$& % +* %$%%% %$*&. 4,0 '$. % '+$, %$%%% %$'&' Diabetes Page 74 of 74 4HDD@ -)*) (+%' +0%+' )%(. (%'.' -'/+ (+%) +%' >&3 '%0, xxxxxx -)** (+%' -'%0* )%.) (%-,/ -'/0 (+%* /%' ,-%/' '%*.xxxxxx '%)(( -'00 (+%) /,%,- *%,) *%''0 -'+0 (,%' ('%' (,%/' (%0/ '%*() -(,* (,%) -.%., )%*. (%-'. -'/* (,%) ((%' +(%./ (%*0 '%,/' -'., (-%' +.%)0 (%+- '%-0' -)'. (-%' ('%' **%)' '%+) '%(+( -'0- (-%' ,(%,' (%/, '%0,, -)-( (-%' (+%) >&3 *%/( xxxxxx -)*' (-%' --%). )%() (%+'+ -(+/ (.%( .%' )-%*- '%+'xxxxxx '%('+ -)). (.%' -'%+' (%,' '%0'- -'/. (.%, +%' ,/%+' '%+* '%),+ -).( (.%' 0.%00 (%(( (%'0' -(+, (/%' ((%' +)%(0 (%'' '%+)) -'0/ (.%/ 0(%(' *%'* )%.,- -)*. (/%' ()%' *)%,( (%(. '%*/( -),* (0%' /,%)0 (%*+ (%(*0 -(0, (0%) ,%' )0%/' '%,0 '%(., -).0 (0%' /'%(. (%)( '%0.' -(-( (0%) .%' *0%/' '%.' '%).. -)*+ )'%' (0%/- *%'- '%-'. -'-+ (0%- 0%' +-%(, (%(, '%,*) ,;C@ xxxxxx xxxxxx -''( ))%' >&3 )%,' -)+, ))%' .%' *(%,/ )%). '%.(/ xxxxxx -',. ))%' ('+%*- )%*/ )%+/, -')- ))%+ (+%' -.%*' (%*( '%//, -(-) ))%. /(%,' (%'' '%/(/ -'.' ))%- .%' *0%(' '%-) '%)+* -''* )*%' >&3 )%+) xxxxxx -'-0 ))%0 .%' ,,%.' *%(( (%.*+ -')0 )+%' .0%*' (%+) xxxxxx(%(). -'), )*%/ (0%' >&3 '%-, xxxxxx -(*( )+%) ('/%0) (%00 )%(-- -)+. )+%' '%- ,0%.. '%-'xxxxxx '%*,. -',* ),%' >&3 (%)( xxxxxx -)(( )+%' +%' **%'' '%.. '%),* -()- ),%) /'%)' (%(+ xxxxxx'%0(( -(0- )-%' (,%' )/%(0 (%'' '%)/) -',/ ).%' ,)%-/ )%*( (%)(0 -'+( )-%* )*%' *)%,' '%/* '%).' -'+/ *'%' (*0%'' (%)( (%-/' -'*0 )/%. ()%' +)%)' (%-0 '%.(* -)*, *'%' (')%*( (%-+ (%-.. -'// *(%) ,%' *)%'' (%*. '%+*0 -'*' *'%( 0-%*' (%*) (%).( -'/( *(%+ (,%' -)%+* '%,0 '%*-0 -))0 *(%' +,%-' )%*+ (%'-, -'*, *)%( )/%' +)%(' (%-- '%.'( 7CHB= ,;H@GF "('$)+# 7CHB= -'*+ *)%' .,%+' (%/0 (%+)/ ,;H@GF "('$)+# 7CHB= -',+ *,%( *'%' )0%+, (%'0 '%*)( xxxxxx -''+ **%' >&3 (%-' -'*/ *.%) )'%' *0%,' (%(+ '%+,( xxxxxx -'') *0%' >&3 )%'( xxxxxx -'*( *0%' *,%' +'%)' '%0-- '%*// -'(, *0%' >&3 )%') xxxxxx ,I ,I 4/1 (%* ,%/- '%() '%(, D$I8@H< "I% :CBGEC@# '%0**' xxxxx '%'''' '%*'/* '%'''' -''0 +,%' >&3 (%00 xxxxxx -'*- (0%)xxxxx *+%' *(%(* '%0) '%)/, -'(( +-%' >&3 (%,0 xxxxxx -(,' +(%) *,%' ).%(+ '%*. '%('' xxxxxx -'(' +.%' >&3 )%+( ! *&# -(*, +*%, )(%' *(%*0 )%)( '%-0, -''/ ,'%' >&3 '%0- xxxxxx -(*/ +0%) +(%' 00%-- '%+' '%+'* ! *&# xxxxxx -(-/ ,(%' //%.- (%*' (%(,/ -'+' ,'%' )'%' -+%0' '%-( '%*0- xxxxxx -'(. ,0%' >&3 (%./ ,I -'(- -+%' >&3 )%+. xxxxxx 4/1 ,%- +%( (*%0/ '%*+ '%'0. 2@; -'(* -,%' >&3 (%0' xxxxxx D$I8@H< "I% :CBGEC@# '%')/* xxxxxxxxxxxxxxxxxxxxx 2@; 4. .%0 xxxxxxx '%-' xxxxxx 4/1 )%- xxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxx