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Original article
Effects of glucocorticoid treatment on beta and alpha cell mass in Japanese adults
with and without diabetes
Seiji Sato1), Yoshifumi Saisho1), Jun Inaishi1), Kinsei Kou1), Rie Murakami1), Taketo
Yamada2), Hiroshi Itoh1)
1) Department of Internal Medicine, Keio University School of Medicine
2) Department of Pathology, Keio University School of Medicine
Running title: Glucocorticoid therapy and β cell mass
Word count: Main text 3,737 words, number of tables: 1, number of figures: 7,
supplementary figures: 1 and supplementary tables: 1
Key words: beta cell mass, alpha cell mass, glucocorticoid, Japanese, diabetes mellitus
Corresponding author:
1
Diabetes Publish Ahead of Print, published online April 16, 2015 Diabetes Page 2 of 50
Yoshifumi Saisho, MD, PhD
Department of Internal Medicine
Keio University School of Medicine
35 Shinanomachi, Shinjuku ku, Tokyo 160 8582, Japan
TEL: +81 3 3353 1211 (x62383)
FAX: +81 3 3359 2745
E mail: [email protected]
Abbreviations
%BCA; fractional beta cell area, %ACA; fractional alpha cell area, BCM; beta cell mass, ACM; alpha cell mass, NGSP; National Glycohemoglobin Standardization
Program, IFCC; International Federation of Clinical Chemistry and Laboratory
Medicine, IQR; interquartile range, ER; endoplasmic reticulum, IGT; impaired glucose tolerance, PP; pancreatic polypeptide
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Abstract The aim of this study was 1) to clarify beta cell regenerative capacity in the face of
glucocorticoid (GC) induced insulin resistance and 2) to clarify the change in beta and
alpha cell mass in GC induced diabetes in humans. We obtained the pancreases from
100 Japanese autopsy cases. The cases were classified according to whether or not they
had received GC therapy prior to death and the presence or absence of diabetes.
Fractional beta cell area (%BCA) and alpha cell area (%ACA) were quantified and the
relationship with GC therapy was evaluated. As a result, in non diabetic cases, there
was no significant difference in %BCA between cases with and without GC therapy
(1.66 ± 1.05% vs. 1.21 ± 0.59%, P = 0.13). %ACA was also not significantly different
between the two groups. In cases with type 2 diabetes, both %BCA and %ACA were
significantly reduced compared with those in non diabetic controls; however,
neither %BCA nor %ACA was significantly decreased in cases with GC induced
diabetes. There was a significant negative correlation between %BCA and HbA1c
measured before death; however, this relationship was attenuated in cases with GC
therapy. In conclusion, the present study suggests that beta and alpha cell mass remain
largely unchanged in the face of GC induced insulin resistance in Japanese individuals,
implying limited capacity of beta cell regeneration in adult humans. The absence of
apparent beta cell deficit in cases with GC induced diabetes suggests that GC induced
diabetes is mainly caused by insulin resistance and/or beta cell dysfunction, but not
necessarily a deficit of beta cell mass.
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Introduction Type 1 (T1DM) and type 2 diabetes (T2DM) are both characterized by a deficit of beta cell mass (BCM)(1; 2). Preservation or recovery of BCM is therefore an important therapeutic strategy for both T1DM and T2DM. However, the regenerative capacity of BCM in humans remains largely unknown.
In rodents, beta cells have been shown to be able to adaptively increase in response to an increased insulin demand such as obesity or pregnancy(3 5). On the other hand, it has been reported that in humans, beta cell proliferation rapidly decreases within five years after birth, and only minimal beta cell proliferation is observed in adult humans(6 8). Estimation of beta cell lifespan by lipofuscin accumulation or radiocarbon dating has also suggested minimal beta cell turnover in adult humans(9; 10). Therefore, clarification of endogenous regenerative capacity in adult humans is critical for interpretation of the results of rodent studies and their application to humans.
It has been reported that BCM is increased by 20 to 50% in obese non diabetic adult humans(8; 11), to a smaller degree than in rodents, which usually show a 2 to 3 fold increase(4; 5), consistent with lower beta cell turnover in adult humans. Recently, we have also reported that in the Japanese population, no significant increase in BCM was observed in obese non diabetic adults(12). These findings further underscore the limited capacity of BCM expansion in adult humans.
Glucocorticoids (GCs) such as prednisolone and dexamethasone, also generally called “steroids”, are potent anti inflammatory agents that are commonly used to treat a broad range of inflammatory and autoimmune conditions(13). GCs are known to increase insulin resistance by facilitating hepatic glucose production and reducing peripheral glucose disposal(14 16). As a result, the use of GCs is associated with a risk
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of development or worsening of glucose intolerance, which is well known as one of the
major adverse effects of GC therapy(17). In rodents, it has been shown that GC induced
insulin resistance increased insulin biosynthesis and secretion, and promoted BCM
expansion through beta cell proliferation(18 20). In humans, GC administration has also
been shown to augment insulin secretion(21 23); however, the effect of GC therapy on
BCM in humans is unknown.
Therefore, in this study, to gain more insight into beta cell regenerative
capacity and the pathophysiological role of BCM in GC induced glucose intolerance in
humans, we sought to address the following questions: 1) Does BCM adaptively
increase to compensate GC induced insulin resistance? 2) Does BCM decrease in
individuals with GC induced diabetes, i.e., steroid diabetes, as well as T1DM and
T2DM? 3) Is there any relationship between glucose intolerance and BCM in
individuals with GC treatment?
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Materials and Methods Subjects
Specimens of pancreas obtained at autopsy were obtained with the permission of the bereaved families. The Keio University School of Medicine Review Board approved this study. Potential cases were first identified by retrospective analysis of the
Keio University autopsy database. To be included, cases were required to have 1) been aged 40 to 79 years, 2) had a full autopsy within 24 h of death, 3) medical information prior to death, 4) no history of pancreatitis, pancreatic tumor or pancreatic surgery, and
5) pancreatic tissue stored that was of adequate size and quality. Cases were excluded if pancreatic tissue had undergone autolysis. We reviewed approximately 1,000 autopsy cases between 2000 and 2013, and found 49 cases that had received GC therapy with either long term (e.g., prednisolone 30 mg/day for 2 years) or short term/intermittent
(e.g., 3 days of pulse methylprednisolone) use prior to death. We only included cases that had received GC administration at least within 3 months prior to death.
Twenty three cases had received long term GC therapy and 26 cases short term/intermittent GC therapy (Supplementary Table 1). The cases were classified into three groups; 1) cases without diabetes (GC NDM), 2) cases with GC induced diabetes (GC DM) and 3) cases diagnosed with T2DM prior to GC therapy (DM2+GC).
Then, we also obtained 51 age and sex matched cases with or without T2DM that had not received GC therapy prior to death as control groups (DM2 and NDM, respectively).
All of them were Japanese. Mean time from death to autopsy was 7.5 ± 5.5 h. Most (N
= 95) specimens were sampled from the body or tail of the pancreas, and 5 specimens were from the head of the pancreas. In addition, we were able to obtain glycosylated hemoglobin (HbA1c) level within one year prior to death in 83 cases (NDM; 18,
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GC NDM; 23, GC DM; 9, DM2; 20, DM2+GC; 13 cases), and HbA1c value was
expressed as the National Glycohemoglobin Standardization Program (NGSP)
value(24).
Pancreatic tissue processing
The pancreas was fixed in formaldehyde at autopsy, and then embedded in
paraffin for subsequent analysis. Then, 5 m sections were stained for light microscopy
as follows: 1) with hematoxylin eosin, 2) for insulin (peroxidase staining) with
hematoxylin, 3) for glucagon with hematoxylin, 4) for insulin and Ki67 for assessment
of beta cell replication, and 5) for insulin and single stranded DNA (ssDNA) for
assessment of beta cell apoptosis, as previously described(12; 25). For
immunohistochemical staining, guinea pig polyclonal antibodies against porcine insulin
and rabbit polyclonal antibodies against human glucagon were used (DAKO Japan,
Kyoto, Japan). Furthermore, murine monoclonal antibodies against human Ki67
(DAKO Japan) and rabbit polyclonal antibodies against ssDNA (IBL, Takasaki, Japan)
were used for the detection of proliferating cells and apoptotic cells, respectively.
Morphometric analysis
To quantify fractional beta cell area (%BCA), the entire pancreatic section was
imaged at 200x magnification (20x objective) using a Mirax Scan and Mirax Viewer
(Carl Zeiss MicroImaging GmbH, Goettingen, Germany). The ratio of beta cell area to
total pancreas area was digitally measured using Image Pro Plus software (Media
Cybernetics, Silver Springs, MD, USA), as previously reported(12; 25). Interlobular
connective tissue, large blood vessels and adipocytes were excluded from total pancreas
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area; thus, total pancreas area consisted to the greatest extent of pancreatic acinar tissue and pancreatic islets. Likewise, the ratio of alpha cell area to total pancreas area
(%ACA) was also digitally measured. All measurements were conducted by a single investigator (S.S.), and the intraobserver coefficient of variance (computed in five cases studied on five occasions) was 7%. All measurements were conducted twice, and the mean of the two measurements was used. At the time of measurement, the investigator was blinded to GC use and the glucose metabolism status of each specimen.
To conduct further morphometric analysis, islet size and density, scattered beta cells and insulin positive duct cells were quantified in randomly selected areas of the pancreas that contained more than 100 islets in each case (112 ± 16 islets per case, total
10,619 islets) using a Mirax Viewer. Scattered beta cells were defined as a cluster of three or fewer beta cells in acinar tissue, and the density of scattered beta cells was determined as the number of scattered beta cells/pancreas area (/mm2). Likewise, the density of islets (/mm2) and islet size ( m2) were also determined in the same area.
Insulin positive duct cells were also counted and expressed as the number of insulin positive duct cells/pancreas area (/mm2). In addition, beta cell replication and apoptosis were quantified in total pancreas sections, and the frequencies of beta cell replication and apoptosis were expressed as percentage of islets. Assuming a difference in %BCA among the groups, these values were further adjusted for %BCA, as previously described(2). A total of 49,450 islets (610 ± 28 islets per case) were assessed for these analyses.
Statistical Analysis
Data are presented as mean ± SD in the text and tables. Data with a non normal
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distribution are expressed as median (interquartile range; IQR). Mann Whitney U test
was used to analyze the difference between the groups, and Spearman’s correlation
coefficient was used to assess the correlation between two parameters. For multivariate
regression analysis, non normally distributed data were log transformed. Statistical
analyses were performed using SPSS version 22 (IBM, Chicago, IL, USA). A P value
<0.05 was considered to be significant for all analyses.
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Results Subjects’ characteristics
The characteristics of the cases are summarized in Table 1, with causes of death in Supplementary Table 1. Mean BMI in each group was 19 to 22, and mean BMI in
GC DM was slightly but significantly lower than that in DM2. Mean HbA1c in GC DM,
DM2 and DM2+GC was 7.0 7.5%, which was significantly higher than that in NDM and GC NDM.
Fractional beta cell area
There was no significant difference in %BCA between NDM and GC NDM
(1.66 ± 1.05% vs. 1.21 ± 0.59%, P = 0.13, Figure 1A). %BCA in DM2 was significantly lower than that in NDM and GC NDM (0.92 ± 0.63%, P = 0.01 and 0.03 vs. NDM and
GC NDM, respectively). On the other hand, there was no significant difference in %BCA in GC DM compared with NDM or GC NDM (1.34 ± 0.53%, P = 0.55 and
0.55, vs. NDM and GC NDM, respectively), and %BCA in GC DM was significantly higher than that in DM2 (P = 0.03). %BCA in DM2+GC was not significantly different from that in DM2 (0.73 ± 0.43%, P = 0.35), but was significantly lower than that in
NDM, GC NDM and GC DM (P = 0.01, P = 0.02, and P = 0.01, respectively).
There was no significant correlation between %BCA and total days or total dose of GC in either GC NDM, GC DM or DM2+GC group (Figure 2A F), although the correlation between %BCA and total days of GC use was almost significant in
GC NDM (R = 0.40, P = 0.06, Figure 2A). When these cases were classified according to short term/intermittent or long term use of GCs, these results were largely unchanged
(Figure 2).
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In line with our prior study(12), there was no significant correlation
between %BCA and BMI in either total or each group of subjects, and the relationships
between %BCA and total days or total dose of GC use were unchanged after adjustment
for BMI (data not shown).
Fractional alpha cell area and alpha to beta cell ratio
Similarly to %BCA, there was no significant difference in %ACA between
NDM and GC NDM (1.07 ± 0.68% vs. 0.88 ± 0.52%, P = 0.36, Figure 1B). There was a
significant decrease in %ACA in DM2 compared with NDM (0.64 ± 0.43%, P = 0.02).
There was no significant difference in %ACA in GC DM compared with NDM or
GC NDM (0.82 ± 0.54%, P = 0.24 and 0.50 vs. NDM and GC NDM,
respectively). %ACA in DM2+GC was also significantly lower than that in NDM or
GC NDM (0.45 ± 0.29%, P = 0.01 and P = 0.01, respectively).
There was a significant positive correlation between %ACA and total dose of
GC in GC NDM (R = 0.52, P = 0.03, Figure 3) and between %ACA and total days of
GC therapy in total subjects (i.e., GC NDM, GC DM and DM2+GC combined, R =
0.34, P = 0.02), although there was no significant correlation between %ACA and total
days or total dose of GC treatment in the GC DM and DM2+GC groups. These
correlations were unchanged after adjustment for BMI (data not shown).
There was no significant difference in alpha cell to beta cell ratio (%ACA
to %BCA ratio) among the groups (Figure 1C), and no significant correlation between
alpha cell to beta cell ratio and total days or dose of GC treatment (Figure 4), which,
again, was unchanged after adjustment for BMI. There was a significant positive
correlation between %BCA and %ACA in cases with and without GC therapy (R = 0.70
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and 0.59, both P = 0.0001, Figure 5).
Islet size and density
Mean islet size and islet density were not significantly different between NDM and GC NDM (6631 ± 2985 vs. 5970 ± 2656 m2, P = 0.48 and 6.11 ± 3.15 vs. 5.53 ±
2.06 /mm2, P = 0.70, respectively) or GC DM (5211 ± 890 m2 and 4.99 ± 2.44 /mm2, P
= 0.66 and 0.29, respectively, Figure 6A and B). Although there was no significant difference in islet density, mean islet size was significantly decreased in DM2 and
DM2+GC compared with NDM, GC NDM and GC DM (all P <0.05), consistent with the reduced %BCA and %ACA in DM2 and DM2+GC.
Beta cell turnover
There was no significant difference in density of scattered beta cells among the groups (Figure 6C). On the other hand, density of insulin positive duct cells was significantly higher in GC NDM and DM2 compared with NDM (0.01 ± 0.03 vs. 0.12 ±
0.16 /mm2 and 0.05 ± 0.06 /mm2, P = 0.001 and 0.002, respectively, Figure 6D).
However, there was no significant correlation between density of insulin positive duct cells and total days or total dose of GC use (Supplementary Figure 1).
There was no significant difference in beta cell replication (i.e., Ki67 positive beta cells) among the groups (Figure 6E). It is of note that beta cell replication was not significantly correlated with BMI in either group (data not shown). Only a few beta cells showing apoptosis (i.e., ssDNA positive beta cells) were found in DM2, but they were not found in the other groups (Figure 6F).
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Association between fractional beta and alpha cell area and HbA1c
There was a significant negative correlation between %BCA and HbA1c in
total cases (R = 0.34, P = 0.001, Figure 7E). When the cases were stratified by the
presence or absence of GC therapy, there was a significant negative correlation
between %BCA and HbA1c in cases without GC therapy (i.e., NDM and DM2 groups
combined, R = 0.45, P = 0.004, Figure 7A). However, the correlation between %BCA
and HbA1c was attenuated in cases that had received GC therapy (i.e., GC NDM,
GC DM and DM2+GC groups combined, R = 0.27, P = 0.08, Figure 7C).
Intriguingly, a significant negative correlation was also observed
between %ACA and HbA1c in total cases (R = 0.36, P = 0.001, Figure 7F). The
negative correlation between %ACA and HbA1c remained significant in both cases with
and without GC therapy (R = 0.44, P = 0.006, R = 0.36, P = 0.02, respectively, Figure
7B and D). These results were unchanged even after excluding one NDM subject with
higher %BCA (4.9%) and %ACA (2.9%).
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Discussion In this study, by examining autopsy pancreas, we report that: 1) There was no significant increase in BCM in non diabetic individuals who received GC therapy. 2)
There was no significant reduction in BCM in individuals with GC induced diabetes, while BCM in individuals with T2DM with or without GC therapy was significantly decreased compared with that in non diabetic individuals. 3) In individuals who received GC therapy, the correlation between BCM and degree of hyperglycemia (i.e.,
HbA1c) was attenuated compared with that in those without GC therapy. 4) There was no significant increase in ACM in individuals who received GC therapy.
GCs such as prednisolone and dexamethasone are well known to have diabetogenic effects by facilitating hepatic glucose production and reducing peripheral glucose disposal(14 16). In healthy humans, it has been reported that insulin secretion is
2 to 4 fold increased to compensate increased insulin resistance after short term GC administration(21 23). In rodents, GC administration has been shown to promote an increase in BCM as well as insulin secretion, through an increase in beta cell replication and beta cell neogenesis(18 20). Based on these previous studies, we hypothesized that
BCM may also be increased in humans who received GC therapy.
In this study, however, we found no significant difference in %BCA in
Japanese non diabetic individuals who had received GC therapy prior to death compared with age , sex and BMI matched non diabetic individuals without GC therapy prior to death. There was also no significant correlation between %BCA and total days or total dose of GC administration, although a higher amount of GC increases the risk of development of diabetes(26). These results were unchanged after adjustment for BMI. We also observed no significant difference in either islet size or islet density
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between non diabetic individuals with and without GC administration. Thus, our
findings suggest that in adult humans, in contrast to rodents, there is little adaptive
increase in BCM in response to GC induced insulin resistance.
Notably, we found no significant decrease in %BCA in individuals with
GC induced diabetes, also called “steroid diabetes”, compared with non diabetic
individuals. Beta cell dysfunction is a hallmark of both T1DM and T2DM(27). It has
been reported that BCM is decreased by >90% and 30 to 65% in patients with T1DM(1)
and T2DM(2; 11; 28 31), respectively, suggesting that deficits of both beta cell function
and mass, collectively called “beta cell functional mass”, are a core pathogenetic feature
of diabetes. Indeed, in this study we confirmed that %BCA was decreased by ~45% in
individuals with T2DM compared with age and BMI matched non diabetic controls,
consistent with other Japanese studies(30; 31). Furthermore, we found a significant
negative correlation between %BCA and HbA1c level in individuals with and without
T2DM, in line with the previous observation(30), whereas this correlation was
attenuated in individuals who received GC therapy. These findings suggest that, unlike
T1DM and T2DM, the development of GC induced diabetes is mainly associated with
insulin resistance and/or beta cell dysfunction, but not necessarily a deficit of BCM.
This may explain why patients with GC induced diabetes often achieve complete
remission after withdrawal of GC administration(21), while patients with T2DM rarely
achieve its remission(27).
Although it has been reported that insulin secretion is increased after a single
dose or short term GC administration in healthy humans, beta cell function assessed by
disposition index has been reported to be unchanged or even impaired in susceptible
populations such as first degree relatives of patients with T2DM and obese
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individuals(21 23; 32), suggesting that GC administration not only induces insulin resistance but also impairs beta cell function. In vitro studies in rodent islets have shown that GCs acutely impair the insulin secretory pathway by reducing the uptake and oxidation of glucose, augmenting outward potassium currents and interfering with the parasympathetic nervous system(17). It has also been reported that GCs augment endoplasmic reticulum (ER) stress and induce beta cell apoptosis(33). However, we did not observe an increase in beta cell apoptosis in individuals who received GC therapy.
We did not observe any significant change in mean islet size and islet density in individuals who received GC therapy, consistent with no significant change in %BCA in those individuals. We also observed no difference in beta cell replication and apoptosis between individuals with and without GC therapy, suggesting little change in beta cell turnover after GC therapy, although it is possible that we were not able to detect a significant difference among the groups because of the limited number of Ki67 and ssDNA positive beta cells under the autopsy condition(34; 35). Scattered beta cells and insulin positive duct cells are considered to be a surrogate marker of beta cell neogenesis(2; 36). Recently, it has been reported that beta cell neogenesis was increased in patients with impaired glucose tolerance (IGT) and new onset T2DM(31), suggesting that beta cell neogenesis is compensatorily increased during the development of diabetes. In this study, we observed a significant increase in insulin positive duct cells in non diabetic individuals who received GC therapy as well as individuals with T2DM compared with non diabetic controls, suggesting that beta cell neogenesis may compensatorily increase under GC induced insulin resistance. However, it should be stressed that this compensatory increase in insulin positive duct cells did not result in an increase in %BCA or islet density, indicating that the compensatory increase in beta cell
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neogenesis is not sufficient for beta cell mass expansion in humans.
Lastly, we found no significant increase in %ACA in individuals who received
GC therapy, whereas it has been reported that GC administration increases plasma
glucagon level(23; 37). However, it is noted that there was a significant positive
correlation between %ACA and total dose of GC in GC NDM, suggesting the
possibility that a high dose of GC may increase alpha cell mass (ACM). Thus, further
study is warranted to clarify this possibility in subjects who received higher dose and/or
longer GC therapy than that in the present study. It will also be of interest to examine
whether alpha cell mass is increased in individuals with endogenous GC overproduction
such as Cushing syndrome.
It is also noted that we did not find a significant increase in %ACA in
individuals with T2DM. Hyperglucagonemia and a paradoxical increase in postprandial
glucagon is a common pathological feature of T2DM(38). However, it remains
controversial whether ACM is increased in patients with T2DM(30; 39). We rather
observed a significant decrease in %ACA in patients with T2DM, inconsistent with
another Japanese study(30). This inconsistency may be derived from the patients’
characteristics and methodological differences, as discussed previously(40). Because the
proportion of alpha cells increases with islet size, this inconsistency may also be derived
from the difference in islet size distribution between individuals. Moreover, since we
did not examine alpha cell turnover in this study, the mechanism by which alpha cell
mass decreased remains unclear. Nonetheless, the significant decrease in islet size with
similar islet density in patients with T2DM observed in this study also indicates that the
total number of islet endocrine cells is indeed reduced in patients with T2DM. Although
recent rodent studies have suggested beta cell to alpha cell transdifferentiation as a
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cause of beta cell loss in patients with T2DM(41), our and previous studies have shown no evidence of alpha cell expansion in patients with T2DM(39). The significant negative correlation between %ACA and HbA1c observed in this study also implies that
ACM expansion is not a main contributor to hyperglycemia in humans with T2DM.
As with other autopsy studies, our study was not free of limitations. First, the underlying diseases such as inflammatory and autoimmune diseases that necessitated
GC therapy might have affected BCM in individuals who received GC therapy, although it is extremely challenging to match underlying diseases between individuals with and without GC therapy. In this study, we tried our best to compare the subjects with control subjects matched for age, sex and BMI. Although the presence of a chronic systemic inflammatory status may decrease insulin sensitivity(42), this effect would tend to increase the difference between subjects with and without GC therapy. However, we are not able to exclude the possibility that other factors such as family history of
T2DM, duration of diabetes and concomitant medications as well as decreased body weight related to the cause of death might also have affected our findings. Second, in this study we assessed BCM and ACM by measuring fractional beta and alpha cell area.
Thus, if there was any difference in pancreas volume between the groups, this might have affected our findings. However, the groups were mostly matched for age and BMI to minimize these effects on pancreas volume(43). It has been reported that the proportions of alpha and beta cells are constant throughout the pancreas except in the pancreatic polypeptide (PP) cell rich ventral portion of the pancreatic head(28; 29). In this study, only five cases were sampled from the pancreatic head and the results were unchanged even after excluding these cases (data not shown). We measured %BCA and %ACA in a single section of the pancreas, which might have resulted in greater
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inter individual variation; however, a wide range of BCM and ACM has been reported
even in the non diabetic population(8; 11; 30; 31; 44; 45). Third, the use of different
kinds of GCs and different regimens of GC therapy might have affected our findings,
although we took account of the total days and total dose of GC use. We also confirmed
that the results did not change when the subjects were classified according to long term
or short term/intermittent administration. Fourth, although the diagnosis and
classification of diabetes were based on medical records before death, undiagnosed or
misclassified cases might have affected our findings. However, we also confirmed the
results based on HbA1c values. It is of note that HbA1c values in non diabetic
individuals were relatively high in this cohort. A recent study has shown that the HbA1c
level was 0.2 0.5% greater in Asians compared with Caucasians with the same plasma
glucose levels(44). Other factors such as anemia also might have affected HbA1c
values(45). Finally, since the subjects of this study were Japanese, who are leaner and
more insulin sensitive than other ethnicities such as Caucasians, Hispanics and
Africans(46), and it has been suggested that there is an ethnic difference in beta cell
change in response to obesity(12; 47), our findings may not be generalizable to other
ethnicities. Because of these limitations, as well as the relatively small sample size of
each group, our results should be confirmed in further studies with different
populations.
In conclusion, GC therapy affected neither BCM nor ACM in adult humans
with and without diabetes. These results suggest that GC induced diabetes is mainly
caused by insulin resistance and/or beta cell dysfunction, but not necessarily a deficit of
BCM, and also underpin the minimal capacity of beta cell expansion in adult humans.
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Author contributions
S.S. and Y.S. researched data and wrote the manuscript. J.I., K.K. and R.M. contributed to discussion and reviewed/edited the manuscript. T.Y. researched data, contributed to discussion and reviewed/edited the manuscript. H.I. contributed to discussion and reviewed/edited the manuscript.
Acknowledgements
We thank Yuko Madokoro, Department of Pathology, Keio University School of Medicine, for technical assistance and Dr. Wendy Gray, self employed, for editing the manuscript.
Disclosure statement
The authors have no conflict of interest. Y.S. 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.
Funding
This study was supported by funding from the Nateglinide Memorial
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Toyoshima Research and Education Fund, the Daiwa Securities Health Foundation, the
Japan Diabetes Foundation and Keio Gijuku Academic Development Funds (Y.S.).
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Figure legends
Figure 1. Fractional beta cell area (%BCA) (A), fractional alpha cell area (%ACA) (B),
and alpha cell to beta cell ratio (%ACA to %BCA ratio) (C) in each group. NDM; cases
without diabetes, GC NDM; cases with GC therapy without diabetes, GC DM; cases
with GC induced diabetes, DM2; cases with type 2 diabetes, DM2+GC; cases with GC
therapy with type 2 diabetes. In cases with GC therapy, circles show cases with
long term GC use and triangles show cases with short term/intermittent GC use. Bars
indicate mean. * P <0.05 vs. NDM. # P <0.05 vs. GC NDM. + P <0.05 vs. GC DM.
Figure 2. Correlation between fractional beta cell area (%BCA) and total days or total
dose of GC therapy in subjects who had received GC therapy; GC NDM (A and B),
GC DM (C and D), DM2+GC (E and F) and total subjects (G and H). GC NDM; cases
with GC therapy without diabetes, GC DM; cases with GC induced diabetes,
DM2+GC; cases with GC therapy with type 2 diabetes. Circles show cases with
long term GC use. Triangles show cases with short term/intermittent GC use.
Figure 3. Correlation between fractional alpha cell area (%ACA) and total days or total
dose of GC therapy in subjects who had received GC therapy; GC NDM (A and B),
GC DM (C and D), DM2+GC (E and F) and total subjects (G and H). GC NDM; cases 31
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with GC therapy without diabetes, GC DM; cases with GC induced diabetes,
DM2+GC; cases with GC therapy with type 2 diabetes. Circles show cases with long term GC use. Triangles show cases with short term/intermittent GC use.
Figure 4. Correlation between alpha cell to beta cell area ratio (%ACA/%BCA ratio) and total days or total dose of GC therapy in subjects who had received GC therapy;
GC NDM (A and B), GC DM (C and D), DM2+GC (E and F) and total subjects (G and
H). GC NDM; cases with GC therapy without diabetes, GC DM; cases with
GC induced diabetes, DM2+GC; cases with GC therapy with type 2 diabetes. Circles show cases with long term GC use. Triangles show cases with short term/intermittent
GC use.
Figure 5. Correlation between fractional beta cell area (%BCA) and alpha cell area
(%ACA) in cases without GC therapy (i.e., NDM; white and DM2; dark gray, A), cases with GC therapy (i.e., GC NDM; light gray, GC DM; gray and DM2+GC; black, B) and total cases (C). In cases with GC therapy, circles show cases with long term GC use and triangles show cases with short term/intermittent GC use.
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Figure 6. Mean islet size (A), islet density (B), scattered beta cells (C), insulin positive
duct cells (D), beta cell replication (E), and beta cell apoptosis (F) in each group. NDM;
cases without diabetes, GC NDM; cases with GC therapy without diabetes, GC DM;
cases with GC induced diabetes, DM2; cases with type 2 diabetes, DM2+GC; cases
with GC therapy with type 2 diabetes. In cases with GC therapy, circles show cases with
long term GC use and triangles show cases with short term/intermittent GC use. Bars
indicate mean. * P <0.05 vs. NDM. # P <0.05 vs. GC NDM. + P <0.05 vs. GC DM.
Figure 7. Correlation between fractional beta cell area (%BCA) or alpha cell area
(%ACA) and HbA1c in cases without GC therapy (i.e., NDM; white, DM2; dark gray, A
and B), cases with GC therapy (i.e., GC NDM; light gray, GC DM; gray, DM2+GC;
black, C and D) and total cases (E and F). In cases with GC therapy, circles show cases
with long term GC use and triangles show cases with short term/intermittent GC use.
HbA1c (NGSP) was converted to HbA1c (International Federation of Clinical
Chemistry and Laboratory Medicine; IFCC) value using the formula: IFCC (mmol/mol)
= [10.93 × NGSP (%)] − 23.50.
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Table 1. Characteristics of subjects. NDM GC NDM GC DM DM2 DM2+GC Total N 26 26 10 25 13 100 Sex 15/11 11/15 4/6 21/4 11/2 62/38 (male/female) Age (years) 63 ± 8 60 ± 12 63 ± 10 66 ± 8 66 ± 7 64 ± 9 BMI (kg/m2) 20.8 ± 2.9 20.4 ± 4.1 18.5 ± 5.1 21.5 ± 4.0+ 20.2 ± 2.1 20.6 ± 3.7 HbA1c (%) 5.5 ± 0.6 5.3 ± 0.6 7.0 ± 1.0*# 7.5 ± 1.2*# 7.2 ± 1.0*# 6.4 ± 1.3 HbA1c 37 ± 6 34 ± 7 53 ± 11*# 59 ± 13*# 55 ± 11*# 46 ± 14 (mmol/mol) Total days of GC 210 180 90 150 － － administration (30 6935) (60 2920) (13.5 195) (30 3102.5) (days) Total dose of 5133 5305 6697.5 5422.5 GC† － － (2325 33397) (1420 13400) (1818 7028) (1941.6 13243.8) (mg) NDM; cases without diabetes, GC NDM; cases with GC therapy without diabetes, GC DM; cases with GC induced diabetes, DM2; cases with type 2 diabetes, DM2+GC; cases with type 2 diabetes and GC therapy. BMI; body mass index, HbA1c; glycosylated hemoglobin, GC; glucocorticoid. Data with normal distribution are expressed as mean ± SD, and data with non normal distribution are expressed as median (interquartile range). † GC dose was expressed as prednisolone equivalent. * P <0.05 vs. NDM, # P <0.05 vs. GC NDM, + P <0.05 vs. GC DM. 34
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130x272mm (300 x 300 DPI)
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165x269mm (300 x 300 DPI)
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168x269mm (300 x 300 DPI)
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170x269mm (300 x 300 DPI)
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114x271mm (300 x 300 DPI)
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184x192mm (300 x 300 DPI)
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174x208mm (300 x 300 DPI)
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Supplementary Table 1. Characteristics of autopsy cases.
Age Sex BMI HbA1c No Group Cause of death Reason for GC use Administration of GC (years) (M/F) (kg/m2) (%) Primary pulmonary 1 NGT 52 M 19.6 5.1 － － hypertension 2 NGT 54 M 20.0 5.1 Chronic kidney disease － － Intracranial aneurysm 3 NGT 54 F 20.7 NA － － rupture 4 NGT 54 F 18.6 5.1 Cerebral hemorrhage － － 5 NGT 56 M 19.8 6.0 Malignant lymphoma － － 6 NGT 56 F 19.4 NA Lung cancer － － 7 NGT 57 F 22.3 NA Ovarian cancer － － 8 NGT 58 F 19.4 NA Ovarian cancer － － Acute myelocytic 9 NGT 58 M 19.4 6.4 － － leukemia 10 NGT 58 M 18.4 6.3 Lung cancer － － Malignant pleural 11 NGT 59 M 19.0 5.8 － － mesothelioma 12 NGT 59 M 19.0 5.9 Lung cancer － － 13 NGT 60 M 19.6 4.2 Alcoholic hepatitis － － 14 NGT 60 F 24.1 NA Ovarian cancer － － 15 NGT 62 M 20.5 NA Pneumonia － － Page 43 of 50 Diabetes
16 NGT 63 M 15.2 5.0 Lung cancer － － 17 NGT 63 F 22.6 5.4 Myocardial infarction － － 18 NGT 65 M 25.5 5.3 Lung cancer － － 19 NGT 69 M 27.5 5.3 Myocardial infarction － － Microscopic 20 NGT 69 M 18.3 6.3 － － polyangiitis 21 NGT 69 M 18.4 NA Malignant lymphoma － － 22 NGT 69 F 22.2 NA Intestinal ischemia － － Hepatocellular 23 NGT 75 F 27.0 4.9 － － carcinoma Aortic aneurysm 24 NGT 75 F 23.1 5.5 － － rupture 25 NGT 76 F NA 5.9 Cerebral infarction － － 26 NGT 77 M 20.8 5.9 Acute heart failure － － Graft versus host 27 GC-NDM 43 F 21.8 4.9 Aplastic anemia Short-term/intermittent disease Myelodysplastic Myelodysplastic 28 GC-NDM 44 M 19.2 5.8 Short-term/intermittent syndrome syndrome 29 GC-NDM 44 F 22.2 5.0 Fistula cancer Crohn's disease Short-term /intermittent Hepatocellular 30 GC-NDM 45 M 17.4 5.6 Crohn's disease Long-term carcinoma 31 GC-NDM 47 M 18.8 6.0 Myelodysplastic Ulcerative colitis Long-term Diabetes Page 44 of 50
syndrome 32 GC-NDM 47 M 28.8 NA Alcoholic hepatitis Alcoholic hepatitis Short-term/intermittent 33 GC-NDM 47 F 15.4 5.6 Interstitial pneumonia Dermatomyositis Short-term/intermittent Primary sclerosing Graft versus host 34 GC-NDM 51 F 23.0 4.4 Short-term/intermittent cholangitis disease Anti-phospholipid 35 GC-NDM 54 F 17.1 5.8 Cerebral hemorrhage Long-term antibody syndrome Acute fulminant Acute fulminant 36 GC-NDM 56 F 26.5 4.9 Short-term/intermittent hepatitis hepatitis Graft versus host 37 GC-NDM 58 F 21.7 3.8 Liver cirrhosis Short-term/intermittent disease 38 GC-NDM 59 M 24.8 5.1 Malignant lymphoma Malignant lymphoma Long-term 39 GC-NDM 60 M 20.9 5.6 Amyloidosis Multiple myeloma Short-term/intermittent 40 GC-NDM 65 M 18.3 NA Lung cancer Lung cancer Short-term/intermittent 41 GC-NDM 66 F 18.4 NA Rheumatoid arthritis Rheumatoid arthritis Long-term 42 GC-NDM 67 F 19.3 5.0 Rheumatoid arthritis Rheumatoid arthritis Long-term 43 GC-NDM 68 M 13.2 5.6 Aspergillus pneumonia Rheumatoid arthritis Short-term/intermittent 44 GC-NDM 68 F 19.0 6.0 Scleroderma Scleroderma Long-term 45 GC-NDM 69 F 22.2 5.1 Rheumatoid arthritis Rheumatoid arthritis Long-term 46 GC-NDM 71 F 26.4 5.7 Rheumatoid arthritis Rheumatoid arthritis Long-term 47 GC-NDM 72 M 14.8 5.6 Interstitial pneumonia Interstitial pneumonia Short-term/intermittent Page 45 of 50 Diabetes
48 GC-NDM 73 F 25.4 5.4 Interstitial pneumonia Interstitial pneumonia Short-term/intermittent 49 GC-NDM 73 F 13.2 4.4 Scleroderma Scleroderma Long-term ANCA associated ANCA associated 50 GC-NDM 73 M 21.4 6.5 Long-term vasculitis vasculitis 51 GC-NDM 75 M 22.4 4.8 Malignant lymphoma Malignant lymphoma Long-term 52 GC-NDM 75 F 17.7 5.0 Ileus Rheumatoid arthritis Long-term 53 GC-DM 41 M 20.4 NA Multiple myeloma Multiple myeloma Long-term 54 GC-DM 59 F 17.3 7.3 Lung cancer Interstitial pneumonia Long-term Graft versus host 55 GC-DM 60 M 13.3 7.0 Malignant lymphoma Short-term/intermittent disease T cell lymphocytic T cell lymphocytic 56 GC-DM 60 M 28.1 6.0 Short-term/intermittent leukemia leukemia Scleroderma, 57 GC-DM 62 F 18.1 5.7 Sepsis Long-term Dermatomyositis 58 GC-DM 65 F 26.4 7.2 Interstitial pneumonia Dermatomyositis Short-term/intermittent 59 GC-DM 67 F 15.4 8.0 Scleroderma Scleroderma Long-term 60 GC-DM 69 M 18.0 6.2 Malignant lymphoma Malignant lymphoma Long-term 61 GC-DM 74 F 13.2 7.1 Ovarian cancer Dermatomyositis Short-term/intermittent 62 GC-DM 75 F 15.1 8.7 Interstitial pneumonia Interstitial pneumonia Long-term 63 DM2 52 F 28.3 7.9 Pituitary carcinoma － － 64 DM2 53 M 22.7 NA Cerebral infarction － － Diabetes Page 46 of 50
65 DM2 55 M 16.2 NA Myocardial infarction － － 66 DM2 57 M 23.2 7.0 Castleman's disease － － 67 DM2 59 M 19.2 8.9 Cerebral hemorrhage － － Nontuberculous 68 DM2 59 F 18.4 6.3 － － mycobacterial infection 69 DM2 59 M 16.9 7.5 Lung cancer － － 70 DM2 60 M 18.4 7.0 Cerebral infarction － － Hepatocellular 71 DM2 62 M 25.2 7.6 － － carcinoma 72 DM2 63 M 26.5 7.3 Cerebral hemorrhage － － 73 DM2 64 M 22.8 NA Myocardial infarction － － 74 DM2 66 F 22.0 NA Chronic kidney disease － － Hepatocellular 75 DM2 66 M 21.7 5.8 － － carcinoma 76 DM2 66 M 27.9 11.7 Liver cirrhosis － － 77 DM2 67 M 25.6 7.0 Sepsis － － Hepatocellular 78 DM2 71 M 22.8 7.8 － － carcinoma 79 DM2 72 M 18.8 7.0 Lung cancer － － 80 DM2 72 M 29.0 6.5 Myocardial infarction － － 81 DM2 74 M 15.5 6.6 Lung cancer － － Page 47 of 50 Diabetes
82 DM2 75 M 18.5 NA Angiosarcoma － － 83 DM2 76 M 20.9 8.5 Sepsis － － 84 DM2 76 M 22.1 7.3 Myocardial infarction － － 85 DM2 76 M 22.4 7.8 Chronic kidney disease － － Acute lymphocytic 86 DM2 77 M 17.8 7.1 － － leukemia Hepatocellular 87 DM2 77 F 14.9 7.6 － － carcinoma 88 DM2+GC 55 M 24.4 5.6 Interstitial pneumonia Interstitial pneumonia Short-term/intermittent 89 DM2+GC 57 F 22.1 8.5 Multiple myeloma Multiple myeloma Short-term/intermittent Myelodysplastic Organizing 90 DM2+GC 57 M 21.2 6.1 Long-term syndromes pneumonia 91 DM2+GC 63 M 19.6 7.7 Myasthenia gravis Myasthenia gravis Long-term 92 DM2+GC 64 M 19.5 6.5 Interstitial pneumonia Interstitial pneumonia Short-term/intermittent 93 DM2+GC 66 M 18.4 8.8 Interstitial pneumonia Interstitial pneumonia Short-term/intermittent Hepatocellular 94 DM2+GC 66 M 18.2 5.7 Optic neuritis Long-term carcinoma 95 DM2+GC 67 M 22.2 7.8 Malignant lymphoma Malignant lymphoma Short-term/intermittent 96 DM2+GC 71 M 19.1 7.2 Interstitial pneumonia Interstitial pneumonia Short-term/intermittent 97 DM2+GC 72 M 21.9 7.0 Intestinal perforation Interstitial pneumonia Short-term/intermittent 98 DM2+GC 72 F 18.6 7.4 Lung cancer Lung cancer Short-term/intermittent Diabetes Page 48 of 50
99 DM2+GC 75 M 16.9 7.9 Malignant lymphoma Malignant lymphoma Short-term/intermittent 100 DM2+GC 79 M 20.1 7.1 Interstitial pneumonia Interstitial pneumonia Long-term GC; glucocorticoid, BMI; body mass index, NA; not available.
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Supplementary Figure 1. Correlation between density of insulin-positive duct cells
and total days or total dose of GC therapy in subjects who had received GC therapy;
GC-NDM (A and B), GC-DM (C and D), DM2+GC (E and F) and total subjects (G
and H). GC-NDM; cases with GC therapy without diabetes, GC-DM; cases with
GC-induced diabetes, DM2+GC; cases with GC therapy with type 2 diabetes. Circles
show cases with long-term GC use. Triangles show cases with short-term/intermittent
GC use. Diabetes Page 50 of 50