Int J Clin Exp Pathol 2010;3(7):691-705

www.ijcep.com /IJCEP1007010

Original Article
Persistent hyperinsulinemic hypoglycemia of infancy:
constitutive activation of the mTOR pathway with
associated exocrine-islet transdifferentiation and
therapeutic implications
Sanda Alexandrescu1, Nina Tatevian1, Oluyinka Olutoye 2, Robert E Brown1
1Department of Pathology and Laboratory Medicine, University of Texas Health Science Center-Medical School at

Houston, Houston, TX, USA; 2Baylor College of Medicine, Houston, TX, USA.

Received July 26, 2010; accepted August 6, 2010; available online August 8, 2010

Abstract: Background: Amino-acids stimulate the mammalian target of rapamycin complex(mTORC)1; mTORC1 inte-
grates amino-acid and energy-sensing pathways in beta-cells. Rapamycin inhibits mTORC1. We examined the mTOR
pathway and cell cycle data in the exocrine pancreas in diffuse persistent hyperinsulinemic hypoglycemia of infancy
(PHHI). Design: Tissues from two diffuse PHHI cases, one pediatric control and from adult pancreatic tissue microar-
ray were analyzed. The case studies are newborns of non-diabetic mothers, one with SUR1 mutation, and the other
with a family history of PHHI. Immunostaining for (p)-mTOR(Ser2448), phospholipase(PLD)1, cell cycle analytes ( Ki-
67, Skp2, p27Kip1), and insulin were performed. Cell cycle analytes were assessed by automated cellular imaging or
visual quantification. Multispectral imaging of double immunostaining for insulin/p-mTOR and transmission electron
microscopy (TEM) were performed. Results: Hematoxylin-eosin and insulin-staining showed beta-cell hyperplasia in
the exocrine pancreas, without mass effect. Overexpression of (p)-mTOR on the plasmalemmal, but not nuclear com-
partment, consistent with mTORC1, was noted in acinar elements. Residual expression was noted in islets. Double
immunostaining revealed occasional exocrine cells co-expressing mTOR and insulin. No such co-expressions were
seen in the control. TEM showed acinar cells containing zymogen and hormone-secreting granules. No nuclear Skp2
was noted. Obversely, p27Kip1 was expressed. Mitotic index was 1/40 (0.25/10) HPF.Conclusion: Morphoproteomic,
histopathologic and morphometric findings in this study of diffuse PHHI coincide with existing genomic and signal
transduction data in: 1) supporting a role for a constitutively activated and overexpressed mTORC1 pathway in the
acinar pancreas in its pathogenesis; 2) reaffirming transdifferentiation of acinar-to-islet cells; 3) raising the possibility
of rapamycin as a therapeutic option in PHHI.

Keywords: mTOR, PHHI, transdifferentiation, rapamycin

Introduction limited to one or several regions with the rest of

the pancreas showing no pathologic changes. In
Persistent hyperinsulinemic hypoglycemia of the diffuse form of PHHI, the beta-cells are not
infancy (PHHI) has an incidence of 1/50,000 only increased in number but are abnormal with
live births and is considered the most common some having hyperchromatic and hypertrophied
cause of severe hypoglycemia in infants[1]. nuclei that are conventionally accepted to be 3
Clinically, it is manifested by marked hyperinsu- times larger than nuclei of surrounding beta
linemia and severe hypoglycemia with its associ- cells [4]. Furthermore, in the diffuse form, insu-
ated systemic complications and notably, by the lin-producing cells can also be seen within acini,
absence of ketosis (ketonemia and ketonuria). outside any well-defined islet, and ductal to islet
Most authors would classify PHHI morphologi- cell transformation (nesidioblastosis) is also
cally into two forms, focal adenomatous hyper- present. In addition to the histopathologic differ-
plasia and a diffuse abnormality of the islets, ences, the clinical scientific literature provides
respectively [2; 3]. To expand on this, in the fo- genomic and clinical correlates that serve to
cal form the histopathologic abnormalities are characterize the two types. Specifically, the dif-
 PHHI: mTOR activation and transdifferentiation

fuse form is associated with recessive muta- from Texas Children’s Hospital. The infants were
tions in SUR1 or KCNJ11 genes [5]. The focal males, born at term to non-diabetic mothers.
type is associated with a mutation in the pater- One infant had a birth weight of 3760 grams,
nal allele of the SUR1 gene with loss of mater- and he had no genetic anomalies. However, his
nal allele of the KCNJ11 gene [6]. Clinically, the brother had a history of diffuse PHHI. The other
diffuse form of PHHI is managed with continu- infant had a birth weight of 5164.2 grams, and
ous feedings and medical therapies that include the birth was complicated by shoulder dystocia.
a potassium channel activator, glucose infusion He also had SUR1 gene mutation, and both par-
and replacement of pancreatic enzymes. More- ents were heterozygous for the mutation. Both
over, surgery is used in an attempt to physically infants had very low glucose levels immediately
remove the mass of insulin-secreting cells. How- after birth, and they did not respond to conser-
ever, even with surgical removal of 95-98% of vative treatment. Subtotal pancreatectomy was
their pancreas, the children with the diffuse performed, with 95% of the pancreas being re-
form develop hypoglycemic episodes or struggle moved from the first child, and 98% from the
with diabetes mellitus at one point in their lives. second child, after the intraoperative pathology
A better understanding of the pathobiology of consult showed diffuse nesidoblastosis. A de-
the diffuse form is needed so that therapies tailed gestational and perinatal clinical history,
that target and interrupt key pathways in the as well as the initial presentation of the pa-
pathogenetic sequence can be applied in the tients, and their follow up, is presented in Table
hopes of controlling and managing the disease 1.
process.
A paraffin block of pancreas (incidental pancre-
In this context, and because there is a body of atectomy from a 3-year-old trauma child) with
literature that implicates the mammalian target no pathologic changes was used as control
of rapamycin (mTOR) pathway in insulin secre- (case 3). A tissue microarray of pancreas from
tion (vide infra); we studied the mTOR pathway adult patients was also used as control.
in two cases with the diffuse form of PHHI. The
specific objectives and design of this study were We examined tissue cut 4 microns thick and
threefold and sequenced as follows: first, to stained with hematoxylin-eosin (H&E) from the
assess components of the mTOR pathway and two cases of PHHI, and also from the control
their state of activation using a morphoproteo- pediatric case. Sections cut 4 microns in thick-
mic approach [7] and to compare and contrast ness were used for immunohistochemistry
the findings with those in control pancreases staining and transmission electron microscopy.
from adult and pediatric case material; second,
to consider the possibility that exocrine to islet Insulin and p-mTOR Immunohistochemistry
transdifferentiation, in association with the acti-
vation of the mTOR pathway is involved in the Sequential double staining for insulin and p-
histogenesis of the islet cell mass both by look- mTOR was performed on 4 microns sections.
ing for transition forms using dual immunostain- The tissue was deparaffinized and rehydrated
ing, multispectral imaging and transmission before antigen retrieval. Overnight incubation at
electron microscopic techniques, and by em- 4 Celsius degrees was performed, and 1:200
ploying cell cycle analysis; and third, to integrate rabbit monoclonal antibody against phosphory-
our findings regarding the mTOR pathway with lated (p)-mammalian target of rapamycin
the genomic and clinical data into a pathoge- (mTOR) at serine (Ser) 2448, (Cell Signaling
netic sequence that allows for targeted thera- Technology 2971) was applied. A secondary
peutic intervention in the diffuse form of PHHI. antibody (pk 6101 from Vectra kit) was applied.
The tissue was then incubated with anti-insulin
Materials and methods antibody (DAKO rabbit polyclonal 10564) 1:50
for 60 minutes at room temperature, and its
Case and control selection expression was enhanced using red chromogen
(Vulcan kit). The compartmental distribution of
Formalin-fixed, paraffin-embedded blocks of the brown chromogen (mTOR) and red chro-
pancreatic tissue from two cases of diffuse vari- mogen (insulin) were enhanced using a multis-
ant of persistent hyperinsulinemic hypoglycemia pectral imaging device (Nuance Multispectral
of infancy were retrieved (case 1 and case 2) Imaging Systems, CRi).

692 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Table 1. Features of PHHI cases

Gestational age at delivery 36 weeks, SVD 38 4/7 weeks, SVD
Mother’s history G2P2, Caucasian, non-diabetic G3P2, African-American, non-diabetic

Sex Male Male

Weight 5014 grams 3660 grams

Genetic studies SUR1 gene mutation -

Family history No diabetes; healthy brother Brother with PHHI
Grandparent with diabetes
Glucose levels at presentation “in the teens” (by surgeon) <40
Neurologic symptoms Seizures
Preoperative treatment Dextrose infusion Dextrose infusion
Continuous feedings and dextrose infusion
Intra-operative consult Diffuse changes Diffuse changes
Operative procedure 98% pancreatectomy 95% pancreatectomy

Postoperative treatment Cornstarch supplement Insulin drip for two weeks

Gastrostomy tube Gastrostomy tube
Octreotide Octreotide
Diazoxide Pancrealipase

Postoperative glucose level <50; controlled with medication High for the first two weeks
Postoperative course Weaned off diazoxide at 5 months Discharged at 3 months
Weaned off octreotide at 30 Feedings and octreotide
months
G-tube removed at 3 years of age Stable at 4 ½ months

Stable off meds at 4 years of age Increased octreotide at 6 months

Phospholipase D1 Immunohistochemistry p27Kip1 nuclear expression was determined by

counting the number of positive nuclei per 100
IgG mouse monoclonal antibodies were used insulin-expressing cells in each of 10 high
against phospholipase D1 (1:100, Santa Cruz). power fields in the admixed exocrine-endocrine
Then anti-insulin antibody was applied using the pancreas. A mitotic index was derived by count-
same technique described above. ing the number of mitotic figures in 10 high
power fields (4 sets of 10 were counted in each
Cell Cycle Data case and divided by 4).

To analyze the cell cycle progression and associ- Interpretation of the immunohistochemistry
ated rate of proliferation, we applied mono-
clonal antibodies to S phase kinase-associated The slides were screened by three pathologists
protein 2 (Skp2) (1:100, Santa Cruz), p27Kip1 and the expression of protein analytes in the
(1:50, Novacastra), and Ki67 (1:500, DAKO). acinar, ductal and beta-islet cells of the cases
with PHHI and the control case were then ana-
We determined the percentage and intensity of lyzed utilizing a bright field microscope with re-
Ki67 using an automated cellular imaging sys- spect to the following: (1) presence or absence
tem (ACIS, DAKO® Corporation). The nuclear of expression of various analytes, and (2) local-
expression of Skp2 was quantified by counting ization of the protein analytes in the subcellular
the positive nuclei in per 100 cells in each of 10 compartments namely cytoplasmic, plasmalem-
high power fields. Similarly the percentage of mal (cell membrane) and/or nuclear.

693 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Figure 1. H&E examination of the

two cases of diffuse PHHI shows an
increased number of islet-cells,
some organized in well defined is-
lands of Langerhans, but many of
them scattered throughout the tis-
sue, mixed within the exocrine pan-
creas in a random fashion
(1A,x100); despite the apparent
proliferation of these cells, no mass
effect is noted. A closer look (1B,
x400) shows the intimacy between
the exocrine and endocrine compo-
nents in diffuse PHHI. Some of the
islet-cells have karyomegaly with
hyperchromasia (1B, arrow). Islet-
cells budding from the ductal epithe-
lium are also seen (1C, x200, ar-
rows). Insulin immunohistochemistry
highlights the diffuse nature of the
pathologic process in PHHI and con-
firms their beta-cell nature (1D,
x100).

Any degree of antibody expression in various Results

cellular compartments of the acini, ducts, islets
and interstitium was analyzed, and its signifi- Histologic evaluation of the two cases of diffuse
cance was established. Positive and negative PHHI and evaluation of insulin immunohisto-
controls, were run concurrently and the patho- chemistry stain
logic findings of PHHI were compared against
them. Microscopic examination of the H&E and insulin
stains of the two cases of diffuse PHHI showed
Transmission Electron Microscopy (TEM) beta-cells organized in islets, and also in iso-
lated groups scattered in the exocrine compo-
The only available tissue we had was embedded nent throughout the tissue examined (Figure
in paraffin. We deparaffinized the sections and 1A). Some of these islet-cells showed karyo-
treated them on the slide by rehydration with megaly with nuclear hyperchromaticity (Figures
descending alcohols, rinsed the sections with 1B, 1C, arrows). Occasionally, islet-cells were
Millonings Sodium Phosphate Buffer, fixed with noted to be budding from the mature ductal
4% gluteraldehyde. 2% osmium was used for epithelium (Figure 1C, arrow). Despite the in-
post-fixation. The tissue was then re-dehydrated creased number of islet-cells, no mass effect
with a graded series of ethanols and propylene was noted. It seemed that these cells occupy by
oxide, and infiltrated with LX-112. A thin layer of replacement rather than proliferation and divi-
resin (1mm) was left on the slide, and it was sion. Immunohistochemical stain for insulin
baked overnight at 60 Celsius degrees. After confirmed beta-cells scattered throughout the
polymerization, the glass slide and layer of resin entire pancreatic tissue in a disorganized man-
were warmed in a beaker of water that had ner (Figure 1D).
been heated to boiling and removed from the
burner. The tissue was pulled from the slide Constitutive activation and overexpression of
while warm. Different areas of the tissue were mTORC1 pathway in the acinar cells, and acti-
cut using a razor blade and glued onto a blank vation in the ductal cells
polymerized epoxy block for ultrathin sectioning
and analysing under electron microscope [8]. Microscopic examination of PHHI cases showed

694 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Figure 2. mTOR is constitutively activated on the plasmalemmal (cell membrane)aspect of the mature exocrine cells
in diffuse PHHI by virtue of the expression of phosphorylated (p)-mTOR (Ser 2448) ( 2A, 2B, see arrows AC,original
magnification x400).There is immunohistologic variation among the two cases of diffuse PHHI; the case with SUR1
gene mutation (2A) shows a stronger plasmalemmal expression of p-mTOR compared with the case without a con-
firmed gene mutation but with a family history of diffuse PHHI (2B). However, in both cases, p-mTOR was overex-
pressed on the plasmalemma of the acinar cells vis-à-vis the concurrently run pediatric control case (2C, original
magnification x400) and in a subsequent tissue microarray of adult pancreases (2D, original magnificationx400),
where the plasmalemmal expression is confined largely to centroacinar and intercalated duct cells (see arrows DC).
There is no nuclear expression of p-mTOR in the exocrine pancreas and in the context of plasmalemmal expression is
consistent with activation of mTORC1 (p-mTOR, Raptor, mLST8) in PHHI.

plasmalemmal expression of p-mTOR (moderate mTOR (Figure 3A, arrow and inset). Multispec-
intensity) in the acinar cells (Figures 2A, 2B). tral imaging confirmed the co-expression of in-
Immunohistochemic variability was noted in the sulin and plasmalemmal p-mTOR in occasional
expression of plasmalemmal p-mTOR between cells in the acinar pancreas (Figure 3B, arrow
the two cases: case 1 had stronger and more and inset). Mild residual plasmalemmal expres-
uniform expression than case 2 (Figures 2A, sion of p-mTOR was present in the already
2B). p-mTOR was expressed on the plasmalem- formed islands of Langerhans (Figure 3C) but
mal aspect in the ductal and intercalated cells no expression of p-mTOR in the islands of
in the concurrently run pediatric control case Langerhans of the pediatric control case (Figure
and in a subsequently assessed tissue micoar- 3D). No nuclear expression of p-mTOR was pre-
ray of adult exocrine pancreas with minimal sent in the exocrine pancreas. The plasmalem-
plasmalemmal expression of p-mTOR in the mal distribution with nuclear exclusion of p-
acinar cells (Figures 2C and 2D). Insulin- mTOR is consistent with activation of mTOR
secreting granules were present in the islets, complex 1 (mTORC1) [9].
but also were extensively present in the acinar
pancreas that co-expressed plasmalemmal p- For completeness, we also compared our PHHI

695 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Figure 3. Double immunohistochemistry stain for p-mTOR-insulin applied on the cases of diffuse PHHI shows the tran-
sitional cells cells that coexpress p-mTOR(Ser 2448), on the plasmalemmal aspect, and insulin (3A, original magnifi-
cation x200, arrow and inset). These results were enhanced using multispectral pseudofluorescence (CRi, Nuance),
which highlighted the cells that expressed p-mTOR (green with Nuance) and insulin (pink with Nuance) (3B, original
magnification x200, arrow and inset).Microanatomical transition of acinar pancreas into defined islets is also evident
in the form of residual, faint plasmalemmal expression of p-mTOR in islands of Langerhans in our cases of diffuse
PHHI (3C, original magnification x200) supporting the involvement of mTORC1 in the process of transdifferentiation
of exocrine cells into islet cells. Contrastively, no similar pattern of mTORC1 expression was seen in the pediatric
control (3D, original magnification x200).

cases with the pattern of distribution of p-mTOR Transmission electron microscopy evaluation of
in adult pancreatic tissue microarray (Figure pancreatic tissue from the two PHHI cases
2D), and it showed similar results with the pedi- showed transition-type cells with rough endo-
atric control case. plasmic reticulum in which zymogen granules,
Lack of expression of phospholipase D1 in the and endocrine granules are seen intermingled
acinar cells in the cytosol of the same acinar cell (Figure 5).
Such cells have been variously labelled as aci-
Microscopic examination of the PHHI cases nar-islet or intermediate cells [12].
showed moderate plasmalemmal expression of
PLD1 in the ductal cells and lack of expression Expression of cell cycle markers and prolifera-
on the plasmalemmal aspect of acinar cells tion markers
(Figure 4). Only some of the centroacinar/
intercalated duct cells had expression of PLD1. The percentage of Ki67 in the population of
cells with admixed exocrine and endocrine com-
Transmission electron microscopy ponents was determined using the Automate-

696 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Figure 5. To highlight the intermediate forms , we

performed transmission electron microscopy on tis-
Figure 4 . Phospholipase D1 (PLD1) –whose product, sue from the same PHHI paraffinized block, and
phosphatidic acid is a rapamycin inhibitor – is lo- observed zymogen granules in close proximity of
cated only on the plasmalemmal aspect of the ductal endocrine granules, in the same cell (5A, x25K). The
and intercalated duct/centroacinar cells in diffuse latter would be classified as an acinar-alpha interme-
PHHI ( x400). There is no plasmalemmal expression diate cell, as noted in other cases of hyperinsuline-
of PLD1 on the acinar cells. Therefore rapamycin can mia [10].
act to inhibit mTORC1 in diffuse PHHI.

Cdk2-dependent G1 phase. Furthermore, this

inverse relationship of p27Kip1 with Skp2 nu-
Cell Imaging System (ACIS, DAKO® Corporate) clear expression accords with observations in
and a mean positive nuclear score of 30.4% for the literature [12].
case 1, and 28.6% for case 2 were established
(Figure 6A). There was evidence of proliferation The calculated mitotic index for our PHHI cases
only in the interstitium, and focally in the islands was 0.25 mitotic figures /10 HPF, and 0 for the
of Langerhans in the control case (18%) (Figure pediatric control case.
6B).
A summary of the cell cycle data is incorporated
We quantified the nuclear expression of Skp2,
by counting the number of positive nuclei per Table 2. Cell Cycle Parameters in PHHI
100 cells in each of 10 HPF. The percentage of
Skp2 positive nuclei for the control case was Cell Cycle Case 1 Case 2 Control
similar to that in the PHHI cases (0.12% versus Data
0.18%) (Figure 6C). This result suggests that the
Ki67 30.1% 28.6% 18%
insulin-secreting cells that are arising in asso-
ciation with the mature acini and ducts do not p27Kip1 *% *% Only in few
reach S phase of the cell cycle [11]. islet cells

Contrastively, p27Kip1 was diffusely expressed Skp2 0.18% 0.18% 0.12%

in the majority of the nuclei of insulin-expressing
cells within the admixed endocrine and exocrine Mitotic 0.25/10 0.25/10 0/10 HPF
component (Figure 6D, red chromogen- insulin, index HPF HPF
brown chromogen- p27Kip1). Nuclear expres-
sion of p27Kip1 in the pattern described above * Positive in the majority of nuclei in insulin-
suggests that the insulin-secreting cells do not expressing cells within the admixed endocrine and
progress into cell cycle beyond the Cyclin E/ exocrine component (See figure 6D).

697 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Figure 6. Ki67 is expressed in the nuclei of mature acini and ducts (6A, x100, case 1) in a proportion of 30.4% in
case 1 and 28.6% in case 2). The Ki67 expression in the control case is 18%. Furthermore, in the control case, Ki67
is present mainly in the interstitium (6B, x100). Nuclear expression of Skp2 is almost non-existent in the two cases of
diffuse PHHI (0.18% for each case) (6C, x200). Double immunohistochemistry stain for p27Kip1 and insulin shows
that p27Kip1 is present in the majority of the nuclei of the insulin-expressing cells within the admixed exocrine and
endocrine component (6D, original magnification x100). A higher power with the details of the cells that have nuclear
positivity for p27Kip1 is presented in the inset. These findings are consistent with the cell cycle arrest in G0/G1
phase in diffuse PHHI.

in Table 2. and endocrine pancreas and observe that the

acinar cells overexpressing p-mTOR do not ex-
Discussions press PLD1. Finally, we consider the known ef-
fect of rapamycin on mTORC1, and also on beta
The findings of this study on two cases of dif- cells in the context of both our morphoproteo-
fuse PHHI will be discussed in three parts. mic findings and the genomic aspects of diffuse
Firstly, we review the histologic, immunohisto- PHHI and its potential therapeutic application in
chemical, ultrastructural, and morphoproteomic such patients.
evidence that the pathogenesis of diffuse vari-
ant of PHHI involves G0/G1 cell cycle arrest in G0/G1 cell cycle arrest in insulin-producing islet
insulin-producing islet cells, and that these cells cells of persistent hyperinsulinemic hypoglyce-
form through transdifferentiation of mature aci- mia of infancy (PHHI): evidence for islet neofor-
nar and ductal elements. These are considered mation from transdifferentiation of acinar and
in the context of the National Library of Medi- ductal cells
cine’s MEDLINE database. Secondly, we discuss
our novel finding of the constitutive activation Persistent hyperinsulinemic hypoglycemia of
with overexpression of the mTORC1 pathway in infancy is associated with a proliferation of insu-
the acinar cells. In conjunction with this, we lin-producing (beta-type) islet cells of the pan-
analyze the expression of PLD1 in the exocrine creas and increased secretion of insulin into the

698 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Figure 7. p-mTOR triggers

progression of the cells to
G1 phase of the cell cycle.
In diffuse PHHI, the mature
acinar and ductal cells with
admixed endocrine and
exocrine component ex-
press p-mTOR on the plas-
malemmal aspect, and
these cells enter cell cycle,
as it is highlighted by the
expression of Ki67. How-
ever, they do not progress
to S phase (Skp2 of 0.18%).
These cells have nuclear
expression of p27Kip1 –
a Cyclin E/Cdk2 complex
inhibitor; these findings
support the theory that
there is G0/G1 phase arrest
in diffuse PHHI.

system circulation. In general, such a prolifera- gle cells. These cells were diffusely present
tion could involve one or more mechanisms to throughout the pancreas, some of them occupy-
include cell cycle progression in pre-existing ing acini and budding from ducts. Even with a
beta cells, neoformation of beta cells from pluri- very prominent increase in number of insulin-
potential stem/progenitor cells and/or transdif- secreting cells, there was not a “mass effect”
ferentiation of mature acinar and ductal cells of seen in the pancreas. It appears that the beta-
the exocrine pancreas into beta-cells. Similarly, cells were occupying the exocrine pancreas by
the hyperinsulinemia could involve one or more replacement. TEM confirmed the existence of
pathogenetic factors such as a mass action cells with admixed, endocrine and exocrine,
effect consequent to an expanded population of component (transition or intermediate forms).
beta cells, genetic hyper-responsiveness to Moreover, cell cycle analysis revealed a G0/G1
agents such as amino acids, and/or constitutive phase arrest for the cells with admixed endo-
activation of molecular signal transduction path- crine and exocrine components by virtue of the
ways involved in promoting the synthesis and following patterns: moderately elevated Ki-67
release of insulin. (which reflects the G1, S, G2, and M phases), at
30.4 % for case 1 and 28.6% for case 2 respec-
In our study of two cases of diffuse variant of tively, a low S phase kinase-associated protein
PHHI, we observed histologic, immunohisto- (Skp2) percentage coinciding with a high per-
chemical, ultrastructural and molecular/signal centage of islet and exocrine nuclei expressing
transduction pathway evidence that support the p27Kip1, an inhibitor of Cyclin E/Ckd2-
theory of transdifferentiation of mature acinar dependent G1 phase [12]. There was also a low
and ductal elements into insulin-secreting cells. mitotic index (0.25 mitotic figures /10 high
H&E and immunohistochemical staining of the power fields). Such cell cycle data corroborate
tissue for insulin showed endocrine cells organ- the evidence from the literature as summarized
ized in islets, but also in small clusters and sin- below and coincide with the existence of transi-

699 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

tion forms in supporting the histogenetic se- Sprague-Dawlwey rats, if isolated and cultured
quence of acinar and ductal transdifferentiation in suspension, will lose amylase expression, and
into insulin-secreting cells. A schematic of the will convert to cells with a duct-like phenotype.
cell cycle information is included in Figure 7. Insulin-positive cells were also observed at the
periphery of the acini-derived spheroids. There
Our findings regarding cell cycle arrest and aci- were a few insulin-positive cells coexpressing
nar and ductal transdifferentiation into insulin- cytokeratins, suggesting that a spontaneous
secreting cells are complemented and sup- acinar to ductal cell transdifferentiation process
ported by the clinical and preclinical studies of was further going on towards insulin-secreting
others. Sempoux et al. [13], in a study of 18 cells. Moreover, Bouwens L. [17] studied the
cases of PHHI (11 focal and 7 diffuse forms), process of regeneration of insulin producing
observed the proliferation rate of the beta-cells beta-cells after pancreatic injury on rodents. He
by virtue of Ki67 immunohistochemistry. They concluded that these cells regenerate via neo-
observed that these cells, in diffuse PHHI, do genesis from pancreatic exocrine epithelial
not have a significant increase in the prolifera- cells. Using immunohistochemistry for PDX1
tion rate compared with the control cases used (which is first expressed in all cells in pancreas,
(29.4% versus 19.6% in aged-matched con- but it restricts to beta-cells in the adult pan-
trols). They concluded back in 2002 that these creas), glut-2 (first expressed in all cells and
cells do not increase in number through prolif- then only in the insulin secreting cells) and
eration. In a most recent publication, Lovisolo vimentin (which is present in the stem cells)
and co-workers [14] showed that there was an Bouwen concluded that stem cells should ex-
increase in the mean Ki-67 labeling index in the press all the three markers, and he did not ob-
beta cells of the islets in the diffuse form of con- serve cells with such traits in the pancreases
genital hyperinsulinism versus the age-matched examined. There are no dormant stem cells that
controls at 2.41% versus 1.87%; and although would transform into hormone-producing cells
this small difference was statistically significant, in case of pancreatic injury. Finally, Bani and co-
it could simply reflect a G1 phase expression workers [12] described the presence of nesi-
consequent to mTORC1 influence on G1 phase dioblastosis and intermediate cells (acinar-islet
in transdifferentiated cells, as discussed above cells) scattered in the acinar tissue in three pa-
(also see Figures 3, 6 and 7). . Kushner JA. [15] tients with hyperinsulinemic hypoglycemia, two
analyzed the beta-cell replication in mice of dif- adults with insulinoma and one child born to a
ferent ages by the use of 5-bromo-2- diabetic mother. Such intermediate cells in
deoxyuridine (BrdU), a DNA precursor analogue their study were characterized by TEM as acinar
that is faithfully incorporated in the dividing -alpha or acinar-alpha-beta or acinar-beta types.
cells instead of thymidine, and can be detected Neoformation of islets from ductal elements
with the use of specific monoclonal antisera. He similar to our two cases was also noted [10].
concluded that beta-cell proliferation in 3-month Most recently and in a related sense, it has
-old wild type mice was only 0.2% following a 6 been reported by Thorel and associates [18]
hour label. The proliferation rate decreased showed in a preclinical study that alpha islet
even more in older mice. Since he was aware of cells can convert (transform) into beta cells in
a possible toxic effect of BrdU on the beta-cells, response to near-total beta-cell ablation.
he measured the apoptosis with TUNEL stain.
Only very few cells were TUNEL stain positive. In Constitutive activation and overexpression of
the same paper, he studied the dependency of mTORC1 pathway in the acinar cells and consti-
beta-cell growth on cyclinD2/Cdk4 activity, and tutive activation of mTORC1 in the ductal cells
one of his observations is that it is still unknown
how much replication is needed to maintain the Previous extensive research revealed that the
mass of beta-cells, and that these cells could genetic defect in the diffuse variant of PHHI is
conceivably live for the life of the organism. inactivating mutations in SUR1 and KCNJ11
genes [1], which lead to inactivation of ATP-
In further support of transdifferentiation from dependent potassium channel. As a secondary
the exocrine pancreas, Song et al. [16], in their effect, due to accumulation of extracellular po-
research for alternatives for diabetic patients tassium, the cells will depolarize and the cal-
needing beta-cell transplantation, observed that cium channels will become activated. Calcium
pancreatic acini from 7 to 8-weeks-old male will accumulate inside the cells at high concen-

700 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Figure 8. The proposed sequence of events that take place from “genes” to “histopathology” is the following: the
genetic defect in diffuse PHHI is inactivating mutations in SUR-1 and Kir 6.2 genes; the consequence is inactivation
of ATP-sensitive potassium channels, with a secondary effect of increased intracellular influx of calcium. The in-
creased level of intracytoplasmic calcium and the amino acids, especially leucine, activates m-TORC1 (Raptor + p-
mTOR) [35, 36]. (plasmalemmal aspect of acinar and ductal cells, causing them to enter cell cycle, where they arrest
in G0/G1 phase.

trations. The high concentrations of calcium in very interesting pattern; we noticed expression
the cytoplasm, together with nutrients, espe- of p-mTOR on the plasmalemmal aspect of duc-
cially leucine, activate raptor in mammalian tal cells, and its constitutive activation and over-
target of rapamycin complex1 [19-24]. There is expression on the plasmalemmal aspect of aci-
literature evidence that high levels of intracellu- nar cells relative to the controls (Figure 2A, 2B).
lar calcium activates regulatory protein I (reg I) Only residual plasmalemmal p-mTOR was pre-
[25; 26], possible by stimulating the process of sent in the well formed Langerhans islets in
transdifferentiation of insulin-secreting cells diffuse PHHI (Figure 3C). In the control case,
from acinar cells. This physiopathologic pathway plasmalemmal p-mTOR was expressed only in
with genetic implications is represented in Fig- some centroacinar/intercalated duct cells
ures 8 and 9. (Figure 2C). Identical results with the ones ob-
served in the control case were seen in the
Our cases expressed p-mTOR (Ser 2448) in a adult pancreatic tissue microarray examined

701 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Figure 9. Considering the peculiar pattern of distribution of phospholipase D1 –whose product, phosphatidic acid is a
rapamycin inhibitor – in diffuse PHHI (absent from the plasmalemmal aspect of the acinar cells with admixed endo-
crine and exocrine components, residual expression in the beta-islets and expression on the plasmalemmal aspect of
mature ductal cells), we believe that rapamycin would inhibit the expression of m-TORC1 in the acinar, and possibly
to some extent in ductal cells undergoing transdifferentiation, decreasing insulin synthesis in diffuse variant of PHHI.

(Figure 2D). The double immunostaining for p- granules were present only in the Langerhans
mTOR – insulin (Figures 3A and 3B) showed the islets in the control case.
diffuse distribution of insulin-secreting cells
throughout the pancreas, with some acinar and Potential therapeutic application for rapamycin
ductal cells expressing both, p-mTOR and endo- in moderating the acinar-islet transdifferentia-
crine granules. Some beta-cells were observed tion and the release of insulin based on the
budding from mature ductal epithelium. For pathogenesis of hyperinsulinism in diffuse PHHI
completeness, we analyzed the immunohisto-
chemistry results with a multispectral Knowing that phospholipase D1 plays a role in
pseudofluorescence device (Figure 3B), and the activation of mTOR pathway through its
performed transmission electron microscopy product phosphatidic acid [27], but also inhibits
also (Figure 5), to demonstrate the presence of the action of Rapamycin on FKBP-12 (see Fig-
transition forms that contain zymogen granules ure 9), we analyzed its expression in our cases.
and endocrine granules. Insulin secreting To our delight, phospholipase D1 was lacking

702 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

from the plasmalemmal aspect of acinar cells rapamycin. It has been shown that activation of
(Figure 4). It was moderately present in some mammalian target of rapamycin complex 1
centroacinar cells, and also in ductal cells. This (mTORC1) in the pancreas leads to insulin syn-
pattern of distribution of phospholipase D1 thesis by its proliferative and transcriptional
made us believe that rapamycin would act on effects. Bourcier et al. [33] have reported a
the acinar cells undergoing transdifferentiation case of pancreatic insulin-secreting islet cell
to islet cells, and possible on the ducts. Our tumor with metastases, that failed to respond to
correlations show that calcium channel blockers octreotide, diazoxide and continuous glucose
combined with rapamycin [28-31], would be a infusion, but responded to oral dose of 2 mg/dl
great addition to the treatment of this entity, of rapamycin. The effect was due to inhibition of
and would control the concentration of intracy- beta-cell growth and proliferation, as well as
tosolic calcium. They could potentially inhibit the blockade of insulin production. This means that
activation of p-mTORC1, and the process of rapamycin can help the treatment of hypoglyce-
transdifferentiation, by controlling the concen- mic states.
tration of intracytosolic calcium. A normal level
of calcium inside the cells would stop reg I pro- In conclusion, it has been forty years since one
tein from becoming over expressed, and the of us (REB) proposed that leucine-sensitive hy-
process of transdifferentiation of endocrine poglycemia of infancy might be related to the
cells from acinar and ductal cells. With the addi- transformation of acinar and ductal elements
tion of a calcium channel blocker and rapamy- into beta cells by the amino acid, leucine in hy-
cin to the current treatment, the expected ef- perresponsive individuals [34]. Our study on two
fects would be decreased insulin secretion by cases of diffuse variant of PHHI supports this
decreased viability and potency, and also by concept by demonstrating that there is G0/G1
stimulating autophagy of already formed beta- cell cycle arrest of both the mature exocrine
cells. Bas et al. [32] described three cases of cells undergoing transdifferentiation and in the
PHHI that failed to respond to diazoxide and islet cells in the exocrine pancreas and in the
somatostatin, but were successfully controlled islets, and there is constitutive activation and
with nifedipine. The patients had good control of overexpression of p-mTOR on the plasmalem-
hypoglycemia even after 12 months of use of mal aspect of the acinar cells, and activation on
this calcium channel blocker, and there were no the plasmalemmal aspect of the ductal cells.
side effects associated with the treatment. We suggest that the particular distribution of
expression of p-mTOR and PLD1 in PHHI, should
There are recent experimental studies that allow rapamycin to act in an inhibitory fashion
show the possible effect of rapamycin on beta- at the level of acini, where it will prevent the
islets. Bussiere et al. [29] cultured human duc- process of transdifferentiation, and at the level
tal cells (HDC) and neonatal porcine islets (NPI) of already organized clusters and islets of beta-
with Rapamycin, and saw that there is a 50% cells, where it will release the inhibitory effect of
decrease in HDC, and a 28% decrease in NPI p-mTORC1 on autophagy, and will decrease in-
after 24 hours. A negative TUNNEL stain made sulin synthesis by decreasing the survival and
him conclude that the mechanism through potency of these cells. Calcium channel block-
which these cells are disappearing is not apop- ers would assist rapamycin in its action, by de-
tosis. creasing the intracellular level of calcium, which
will lead to decrease activation of p-mTORC1,
Tanemura et al. [28] used pancreatic tissue and inhibition of neoformation of insulin-
from male BL6 mice, to isolate beta-islets. He secreting cells from acinar and ductal elements.
incubated fresh islets for 24 hours in culture
medium, in the presence or absence of Rapa- Despite all the recent progress in elucidating
mycin, either 1 or 10 ng/mL. Western blot the intricacies of this entity there is still not a
analysis showed accumulation of membrane good management to stop the increase in num-
bound LC3-II, which is an early marker of auto- ber of insulin secreting cells. We believe that
phagy. The viability of islets incubated with ra- our findings might revolutionize the medical
pamycin was also analyzed, and there was a approach for infants with diffuse variant of per-
43% decrease in the viability of islets treated sistent hyperinsulinemic hypoglycemia. How-
with 1 ng/mL rapamycin, and a 51% decrease ever, the number of cases we studied is limited
when the islets were incubated with 10ng/mL and further research is needed in this direction.

703 Int J Clin Exp Pathol 2010;3(7):691-705

 PHHI: mTOR activation and transdifferentiation

Aknowledgements [8] Estrada JC, Selim MA, Miller SE. TEM of paraffin-
embedded H&E-stained sections for viral diagno-
We thank Pamela Johnston, and Richard sis (an unusual papovavirus case). Microsc Mi-
Breckenridge from University of Houston at croanal 11(Suppl 2), 2005:964-965
[9] Mori H, Inoki K, Opland D, et al. Critical roles for
Texas – Health Science Center histology lab for the TSC-mTOR pathway in {beta}-cell function.
their technical support and Bheravi Patel for Am. J. Physiol. Endocrinol. Metab., 2009; 8:18
support with the graphics. Patricia Navarro de- [10] Bani D, Bani Sacchi T, Biliotti G. Nesidioblastosis
serves special thanks for her excellent work and and intermediate cells in the pancreas of pa-
for the help she has given us with the electron tients with hyperinsulinemic hypoglycemia. Vir-
microscopy aspects of this project. We are also chows Arch [Cell Pathol] 1995;48:19-32
very grateful to Dr Milton J. Finegold at Texas [11] Chiarle R, Fan Y, Piva R, et al. S-Phase kinase-
Children’s Hospital for providing the two cases associated protein 2 expression in non-
and for his very helpful comments and sugges- Hodgkin’s lymphoma inversely correlated with
p27 expression and defines cells in S phase. Am
tions. J Pathol. 2002 Apr;160(4):1457-66
[12] Chiarle R, Pagano M, Inghirami G. The cyclin
Please address correspondence to: Robert E. Brown, dependent kinase inhibitor p27 and its prognos-
MD, Department of Pathology and Laboratory Medi- tic role in breast cancer. Breast Cancer Res.
cine, University of Texas Health Science Center Medi- 2001; 3(2):91-94
cal School at Houston, 6431 Fannin Street, MSB [13] Sempoux C. Pancreatic beta-cell proliferation in
2.286, Houston, TX 77030, Tel: 713-500-5332, Fax: persistent hyperinsulinemic hypoglycemia of
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