In Vivo Misfolding of Proinsulin Below the Threshold of Frank Diabetes

Article (PDF Available)inDiabetes 60(8):2092-101 · June 2011with44 Reads
DOI: 10.2337/db10-1671 · Source: PubMed
Abstract
Endoplasmic reticulum (ER) stress has been described in pancreatic β-cells after onset of diabetes-a situation in which failing β-cells have exhausted available compensatory mechanisms. Herein we have compared two mouse models expressing equally small amounts of transgenic proinsulin in pancreatic β-cells. In hProCpepGFP mice, human proinsulin (tagged with green fluorescent protein [GFP] within the connecting [C]-peptide) is folded in the ER, exported, converted to human insulin, and secreted. In hProC(A7)Y-CpepGFP mice, misfolding of transgenic mutant proinsulin causes its retention in the ER. Analysis of neonatal pancreas in both transgenic animals shows each β-cell stained positively for endogenous insulin and transgenic protein. At this transgene expression level, most male hProC(A7)Y-CpepGFP mice do not develop frank diabetes, yet the misfolded proinsulin perturbs insulin production from endogenous proinsulin and activates ER stress response. In nondiabetic adult hProC(A7)Y-CpepGFP males, all β-cells continue to abundantly express transgene mRNA. Remarkably, however, a subset of β-cells in each islet becomes largely devoid of endogenous insulin, with some of these cells accumulating large quantities of misfolded mutant proinsulin, whereas another subset of β-cells has much less accumulated misfolded mutant proinsulin, with some of these cells containing abundant endogenous insulin. The results indicate a source of pancreatic compensation before the development of diabetes caused by proinsulin misfolding with ER stress, i.e., the existence of an important subset of β-cells with relatively limited accumulation of misfolded proinsulin protein and maintenance of endogenous insulin production. Generation and maintenance of such a subset of β-cells may have implications in the avoidance of type 2 diabetes.
In Vivo Misfolding of Proinsulin Below the Threshold
of Frank Diabetes
Israel Hodish, Afaf Absood, Leanza Liu, Ming Liu, Leena Haataja, Dennis Larkin, Ahmed Al-Khafaji,
Anthony Zaki, and Peter Arvan
OBJECTIVEEndoplasmic reticulum (ER) stress has been de-
scribed in pancreatic b-cells after onset of diabetesa situation in
which failing b-cells have exhausted available co mpensatory mech -
anisms. Herein we have compared two mouse models expressing
equally small amounts of transgenic proinsulin in panc reatic
b-cells.
RESEARCH DESIGN AND METHODSIn hProCpepGFP
mice, human proinsulin (tagged with green uorescent protein
[GFP] within the connecting [C]-peptide) is folded in the ER, ex-
ported, converted to human insulin, and secreted. In hProC(A7)Y-
CpepGFP mice, misfolding of transgenic mutant proinsulin causes
its retention in the ER. Analysis of neonatal pancreas in both
transgenic animals shows each b-cell stained positively for endog-
enous insulin and transgenic protein.
RESULTSAt this transgene expression level, most male
hProC(A7)Y-CpepGFP mice do not develop frank diabetes, yet
the misfolded proinsulin perturbs insulin production from en-
dogenous proinsulin and activates ER stress response. In non-
diabetic adult hProC(A7)Y-CpepGFP males, all b-cells continue
to abundantly express transgene mRNA. Remarkably, however,
a subset of b-cells in each islet becomes largely devoid of endog-
enous insulin, with some of these cells accumulating large quan-
tities of misfolded mutant proinsulin, whereas another subset of
b-cells has much less accumulated misfolded mutant proinsulin,
with some of these cells containing abundant endogenous insulin.
CONCLUSIONSThe results indicate a source of pancreatic
compensation before the development of diabetes caused by
proinsulin misfolding with ER stress, i.e., the existence of an
important subset of b-cells with relatively limited accumulation
of misfolded proinsulin protein and maintenance of endogenous
insulin production. Generation and maintenance of such a subset
of b-cells may have implications in the avoidance of type 2 di-
abetes. Diabetes 60:20922101, 2011
D
uring early type 2 diabetes, morphological ab-
normalities have been identied within the se-
cretory pathway of pancreatic islet b-cells.
Specically, the endoplasmic reticulum (ER) and
pre-Golgi intermediates become distended (herein called
ER crowding), and some b-cells develop a deciency of
secretory granules (1,2). Similar morphological features
have also been repo rted in various monogenic forms of
diabetes that may develop an impacted-ER phenotype
(3,4). As best we can tell, morphological ER crowding is
correlated with ER stress, as evidenced by activation
of ER stress response signaling pathways. Morphological
ER crowding is not critical in simple overfeeding (3),
suggesting that ER crowding may be a specic link to
b-cell dysfunction. However, most research demonstrating
ER crowding/ER stress in pancreatic b-cells has focused
on models that are already hyperglycemic at the time of
study. Once hyperglycemia commences, additional meta-
bolic insults (a process known as glucotoxicity [57]) may
cause further b-cell injury. Although one can anticipate
that some degree of ER crowding/ER stress may exist even
before deterioration of glycemic control, this process is
less well studied.
Mutant INS geneinduced diabetes of youth (MIDY) (8)
is a syndrome with an established genetic basis (9), caused
by preproinsulin-coding sequence mutations that trigger
misfolding, which leads to autosomal-dominant, insulin
decient diabetes. The same disease occurs also in Akita
(10) and Munich (11) mice. Secondary defects in pro-
insulin folding may also occur as a consequence of alter-
ations in the proinsulin folding environment in the ER
(4,12). Hyperglycemia may exacerbate such an unfavor-
able environment, creating a potential linkage between
proinsulin misfolding in the ER and type 2 diabetes (13
15). Before the onset of overt hyperglycemia, we have our
best chance to identify early pancreatic compensatory
responses that may help to limit diabetes progression.
In this study, we have characterized a mouse model
expressing exclusively in pancreatic b-cells a transgene
containing the same proinsulin-C(A7)Y mutation as that
found in Akita mice (16). It is noteworthy that the folding-
defective proinsulin known as hProC(A7)Y-CpepGFP
(bearing green uorescent protein [GFP] within the
connecting [C]-p eptide) is expressed at subthreshold
levels, such that very f ew mice develop frank diabetes
in the absence of additional metabolic or genetic insult.
These animals can be studied side by side with transgenic
mice that exhibit comparable b-cellspecic expression of
hProCpepGFP lacking any misfolding-inducing mutation
(17). The presence of the GFP tag itself does not prevent
proinsulin folding, trafcking, processing, or secretion (17)
but allows for detection and localization of the protein in
b-cells. The present studies highlight pathways of islet
compensation in the setting of underlying proinsulin mis-
folding, which may have relevance for understanding early
type 2 diabetes.
RESEARCH DESIGN AND METHODS
Materials. Rabbit antisera against GFP was from Immun ology Consultants
(Newberg, OR); antia-tubulin was from Santa Cruz Biotechnology (Santa
Cruz, CA); anti-immunoglobulin heavy chain-binding protein (BiP) was from
Cell Signaling (Danvers, MA); AlexaFluor-488conjugated anti-GFP was from
Invitrogen (Carlsbad, CA); peroxidase-conjugated anti-rabbit and peroxidase-
conjugated antiguinea pig were from Jackson ImmunoResearch Laboratories
From the Division of Metabolism, Endocrinology, and Diabetes, University of
Michigan Medical Center, Ann Arbor, Michigan.
Corresponding authors: Israel Hodish, ihodish@umich.edu; Peter Arvan, parvan@
umich.edu.
Received 1 December 2010 and accepted 17 May 2011.
DOI: 10.2337/db10-1671
Ó 2011 by the American Diabetes Association. Readers may use this article as
long as the work is prope rly cited, the use is educational and not for prot,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
2092 DIABETES, VOL. 60, AUGUST 2011 diabetes.diabetesjournals.org
ORIGINAL ARTICLE
(West Grove, PA); AlexaFluor-555conjugated anti-rabbit was from Invitrogen;
rat insulin radioimmunoassay (RIA) was from Millipore (Billerica, MA); mouse
proinsulin ELISA was from ALPCO (Salem NH); and collagenase-P and pro-
teinase inhibitor mixture were from Roche Applied Science (Indianapolis, IN).
Construction of the hProCpepGFP and hProC(A7)Y-CpepGFP trans-
genes. The emerald GFP cDNA was inserted into the C-peptidecoding se-
quence within the human insulin cDNA to create hProCpepGFP (17). The
hProC(A7)Y-CpepGFP then used PCR mutagenesis to replace Cys(A7) with
Tyr in the coding sequence. XhoI-anking restriction sites were used to insert
these constructs downstream of the 8.3-kb mouse Ins1 promoter and up-
stream of the nontranslated human growth hormone gene (18).
Generation of transgenic mice. The linearized hProC(A7)Y-CpepGFP
transgene was injected into pronuclei of fertilized mouse eggs at the University
of Michigan Transgenic Animal Model Core. PCR genotyping was performed by
PCR with GFP-specic primers (forward primer, 59-AGG TCT ATA TCA CCG
CCG ACA-39; reverse primer, 59-TGC AGT AGT TCT CCA GCT GGT AG-39),
yielding a 400-bp product. One line (of 10) was propagated, housed in a
pathogen-free facility, and fed standard rodent chow in accordance with the
University of Michigan Animal Care Committee. Unless otherwise stated,
all experiments were done with 36-month-old nondiabetic male mice (e.g.,
Figs. 1A,4A, 5, and 8). Intraperitoneal glucose tolerance tests were performed
on fasted animals with 1.0 mg dextrose per gram body weight. For some
experiments, hProCpepGFP and hProC(A7)Y-CpepGFP mice were mated to
Ins2
2/2
mice (from C. Polychronakos, McGill University, Montreal, Quebec,
Canada) to increase the ratio between the products of transgenic and
endogenous Ins1 alleles. In other experiments, compound heterozygotes
bearing both wild-type hProCpepGFP and Akita mutant proinsulin were used
as a control for the hProC(A7)Y-CpepGFP line that carries both elements in
a single allele.
Immunouorescence. Fixed pancreata (formaldehyde plus 10% sucrose) were
snap-frozen; 7-mm sections were permeabilized in acetone, blocked, and
immunolabeled with guinea pig anti-insulin or antiglucagon and secondary
antibodies in PBS containing 0.5% BSA. For BiP and GFP, de-parafnized sec-
tions were heated in citrate (pH 5.0), cooled, blocked, incubated overnight with
anti-BiP and appropriate secondary, blocked again, incubated overnight with
AlexaFluor-488conjugated anti-GFP, and mounted for confocal microscopy.
Pancreatic islet isolation. Pancreata were digested in 2 mg/mL collagenase-P
for 30 min at 37°C, washed, and handpicked and incubated in RPMI 1640 me-
dium containing either 4 or 16.7 mmol/L glucose (plus 10% fetal bovine serum
and 1% penicillinstreptomycin) for 48 h.
Western blotting. Islet proteins were resolved by 412% gradient SDS-PAGE
under reducing conditions; electrotransferred to nitrocellulose; probed with
anti-insulin, anti-GFP, and antia-tubulin (loading control); and probed with
appropriate peroxidase-conjugated secondary antibodies for enhanced chemi-
luminescence.
Real-time PCR. cDNA from template mRNA (RNeasy; QIAGEN, Valencia, CA)
was generated by reverse transcriptase (SuperScript III; Invitrogen) and am-
plied with Taq polymerase in a real-time thermal cycler (denaturation 30 s,
annealing 30 s, and extension 3 min) using appropriate primers, and SYBR
green uorescence followed each cycle. The following primers were used:
FIG. 1. Endogenous proinsulin and transgenic GFP-labeled proinsulin. A: mRNA levels from islets from three to four hProCpepGFP and hProC(A7)
Y-CpepGFP mice. Islet mRNA extraction was followed by synthesis of reverse-transcribed cDNAs; specic primers for endogenous preproinsulin-II and
GFP were then used to measure mRNA abundance relative to b-actin mRNA, as quantied by real-time PCR (2
-DDCT
6 SEM). The ratio of transgene
mRNA to endogenous preproinsulin-II mRNA is similar in the islets of hProCpepGFP (5.5%) and hProC(A7)Y-CpepGFP transgenic mice (4.2%); in
both cases, >95% of insulin mRNA is derived from endogenous genes. B: Distribution of endogenous and GFP-labeled insulin + proinsulin among
the b-cells of neonatal males. Neonatal pancreatic tissue was snap frozen in liquid nitrogen and 7-mm sections were analyzed. GFP epiuorescence
identied transgenic proinsulin and its derived products, while endogenous proinsulin-derived products were immunolabeled with anti-insulin
antibodies. Scale bars are identical for each image shown. Even in hProC(A7)Y-CpepGFP ma les, most neonatal b-cells mutually express both
endogenous insulin and GFP-labeled mutant proinsulin. (A high-quality digital representation of this gure is available in the online issue.)
I. HODISH AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 60, AUGUST 2011 2093
GFP, 59-AGG TCT ATA TCA CCG CCG ACA-39 and 59-TGC AGT AGT TCT CCA
GCT GGT AG-39; preproinsulin-II, 59-CCC TGC TGG CCC TGC TCT T-39 and 59-
AGG TCT GAA GGT CAC CTG CT-39;BiP,59-GGC CAA ATT TGA AGA GCT
GA-39 and 59-GCT CCT TGC CAT TGA AGA AC-39; CHOP, 59 - CAT ACA CCA
CCA CAC CTG AAA G-39 and 59-CCG TTT CCT AGT TCT TCC TTG C-3 9;
XBP-1, 59-TGG CCG GGT CTG CTG AGT CCG-39 and 59-GTC CAT GGG AAG
ATG TTC TGG-39; spliced XBP-1, 59-CTG AGT CCG AAT CAG GTG CAG-39
and 59-GTC CAT GGG AAG ATG TTC TGG-39;andb-actin, 59-TAT TGG CAA
CGA GCG GTT CC-39 and 59-GGC ATA GAG GTC TTT ACG GAT GTC-39.
mRNA levels (normalized to b-actin) were calculated using the comparative
threshold cycle (CT) method (2
2DDCT
).
In situ hybridization. A 175-bp, digoxigeninlabeled oligonucleotide probe
complementary to GFP mRNA was used for in situ hybridization with anti
digoxigenin-phosphatase detection as described elsewhere (19). As a negative
control, pancreatic islets from C57BL/6 mice were negative for detectable
mRNA.
Image analysis. For each genotype, epiuorescence images from twenty
0.5-cm diameter pancreatic (tail) cryosections from three mice, with identical
exposures, were quantied in grayscale using Adobe Photoshop CS3. To
identify the predominant (pro)insulin (GFP-labeled or endogenous) in each
b-cell, a sensitivity threshold was established at midrange of the pixel intensity
histogram for each section. Cellular areas with signals above threshold for
each type of (pro)insulin were demarcated, and DAPI staining identied each
nucleus. Of the total cellular area of uorescence, cells containing .80%
GFP uorescence area were classied as cells expressing primarily GFP-
labeled p roin sul in ; cel ls e xpr ess in g .80% insulin immunouorescence area
were classied as cells expressing primarily en d oge nous insulin; and b-cell s
expressing both in which neither label predominated were classied as ex-
pressing both. Cell boundaries (not shown) were corroborated by immuno-
labeling with antib-catenin.
Quantication was performed using NIH ImageJ 1.37c software. Results
presented are mean 6 SEM. Statistical analyses were performed using one-way
ANOVA with Tukey multiple comparisons; P # 0.05 was considered signicant.
Transmission electron microscopy. Pancreatic tissue from nondiabetic
hProC(A7)Y-CpepGFP mice was xed in buffered glutaraldehyde, postxed in
OsO
4
, dehydrated with graded alcohols and propylene oxide, embedded in
FIG. 2. Blood glucose levels in male mice (median age 3.5 months). A: Random blood glucose was obtained by tail snip from males of the different
genotypes indicated. Mean random glucose of hProC(A7)Y-CpepGFP transgenic males was signicantly higher than that of hProCpepGFP
transgenic males, but less than that of hProCpepGFP transgenic males bearing one endogenous Ins2 Akita allele (bar at right). B: Histograms of
random blood glucose measurements of individual male mice; each animal is shown only once. (Retesting of the small proportion [;10%] of hProC
(A7)Y-CpepGFP animals with random blood glucose exceeding 300 mg/dL reconrmed diabetes in these anima ls.) C: Intraperitoneal glucose
tolerance tests (1 mg dextrose/g body weight) were performed on fasted animals. Compared with hProCpepGFP and C57BL/6 control mice, the
results conrm that the hProC(A7)Y-CpepGFP males have impaired glucose tolerance.
b-CELL HETEROGENEITY IN NONDIABETIC MICE
2094 DIABETES, VOL. 60, AUGUST 2011 diabetes.diabetesjournals.org
Spurrs resin, and sectioned at 70 nm, grids-stained with uranyl acetate and
lead citrate, and examined on a Philips CM-100 electron microscope.
RESULTS
Transgene expression in hProCpepGFP and hProC
(A7)Y-CpepGFP mice. Two transgenic lines were used.
The hProCpepGFP mouse carries a GFP-labeled proinsulin
(17), whereas the hProC(A7)Y-CpepGFP mouse carries
a similar GFP-labeled proinsulin bearing the misfolding-
inducing Akita mutation. In islets isolated from the
two mouse lines, we examined an abundance of mRNAs
encoding these GFP-labeled preproinsulins and the en-
dogenous Ins2 mRNA levels (Ins2 represents the majority
of total insulin transcripts in mouse islets [20]). Using
qPCR (normalized to b-actin mRNA) with concentration
standards of hProCpepGFP plasmid DNA, we conrmed
that the levels of hProCpepGFP and hProC(A7)Y-CpepGFP
mRNA represented only 5.5 and 4.2%, respectively, of that
measured for endogenous Ins2 mRNA (Fig. 1A).
We examined pancreatic cryosections from neonatal
male hProCpe pGFP and hPr oC(A7)Y-CpepGFP mice (sex
determined by Y-chromosome PCR) using intrinsic GFP
uorescence and insulin immunouorescence. At postnatal
day 1, hProCpepGFP and hProC(A7)Y-CpepGFP male mice
demonstrated GFP uorescence (either properly folded
or misfolded mutant proinsulin) in essentially all of the cells
that were immunolabeled for endogenous insulin + pro-
insulin, i.e., in the vast majority of b-cells (Fig. 1B; quanti-
ed in Fig. 7A). Thus, the data in Fig. 1 indicate that the two
animal models demonstrate comparable levels and patterns
of transgene mRNA and protein expression across the b-cell
population in vivo.
Most male hProC(A7)Y-CpepGFP transgenic mice
avoid frank diabetes. We compared body weights and
random glucose levels of hProC(A7)Y-CpepGFP transgenic
mice with those of hProCpepGFP mice that are phenotyp-
ically identical to C57BL/6 littermates (17). Mean body
weight of hProC(A7)Y-CpepGFP males was similar to that
of normal control subjects, and mean random glucose of
hProC(A7)Y-CpepGFP males (between 8:00
A.M. and 12:00
P.M.) was only modestly elevated and signicantly lower
than hProCpepGFP Akita males (Fig. 2A). A histogram of
individual males of each genotype showed that random
blood glucose levels in the vast majority (.85%) of hProC
(A7)Y-CpepGFP males had a distribution largely over-
lapping with that of normal controls (Fig. 2B). Although a
small subset of males (;10% of total) exhibited random
hyperglycemia, the results indicate that the majority of ani-
mals with transgenic expression of hProC(A7)Y-CpepGFP
(driven by the Ins1 promoter) do not have frank diabetes
(Figs. 2A and B), i.e., a considerably milder phenotype
than Akita mice who bear the same mutation in one of the
endogenous Ins2 genes.
To identify subtler defects in islet performance in
hProC(A7)Y-CpepGFP males, we subjected the animals to
intraperitoneal glucose tolerance testing after an over-
night fast. Although fasting glucose values were similar
to those of hProCpepGFP and C57BL/6 control males, the
hProC(A7)Y-CpepGFP males had a signicantly higher
mean blood glucose at 30 min poststimulation than co n-
trol subjects (309 6 32.6 vs. 253.1 6 16.1 mg/dL; P =
0.03). Although a signi cant difference was not sustained
at 120 min postchallenge, the results indicate that hProC
(A7)Y-CpepGFP mice have impaired glucose tolerance
(Fig. 2C ).
Homozygous knockout of the endogenous Ins2 gene
(with homozygous expression of wild-type Ins1 still re-
maining) itself does not cause diabetes (20). Therefore, we
were interested to use homozygous Ins2 knockout to ex-
amine the effect of increasing the relative expression of
mutant hProC(A7)Y-CpepGFP (transgene) to wild-type in-
sulin. In this background, mice with transgenic expression
of wild-type hProCpepGFP were normoglycemic, whereas
mice with transgenic expression of hProC(A7)Y-CpepGFP
developed hyperglycemia that was particularly severe when
both Ins2 alleles were missing, including the cohort of fe-
male animals (Fig. 3, bottom). Thus, although the majority
of mice exhibit sufcient compensation to accommodate
low-level expression of misfolded proinsulin, these animals
are nevertheless predisposed to diabetes, and the relative
expression levels of misfolded and wild-type proinsulin
dictate the phenotype (Fig. 3).
In nondiabetic hProC(A7)Y-CpepGFP mice, the pro-
duction and maturation of endogenous proinsulin is
perturbed by transgenic expression of misfolded mu-
tant proinsulin. To determine whether pancreatic insulin
content was comparable in nondiabetic hProC(A7)Y-
CpepGFP transgenic mice and nonmutant control subjects,
we measured total insulin + proinsulin (endogenous and
transgenic) in isolated pancreatic islets. By RIA, islet in-
sulin + proinsulin content per cell was signicantly less
than that measured for hProCpepGFP transgenic controls
(Fig. 4A), despite that all islets were derived from non-
diabetic animals. In the small subset of diabetic hProC(A7)
FIG. 3. Mean random glucose of transgenic male and female mice par-
tially or completely devoid of endogenous Ins2. Transgenic mice were
rst mated with complete Ins2 knockout animals to generate hProC-
pepGFP or hProC(A7)Y-CpepGFP, Ins2
+/2
animals (rst two sets of
bars) and then back bred to generate transgenic Ins2
2/2
animals
(second two sets of bars). Results are depicted as mean 6 SEM.
I. HODISH AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 60, AUGUST 2011 2095
Y-CpepGFP males, the insulin + proinsulin content was
lower still (,5% of control).
We also used an independent assay to follow proinsulin
(and insulin) content in isolated islets incubated for 48 h in
either 4 or 16.7 mmol/L glucose. Islet lysates were analyzed
by reducing SDS-PAGE, electrotransferred to nitrocellu-
lose, and immunoblotted with either anti-GFP (to follow
the transgene product) or anti-insulin (to follow the en-
dogenous product). In islets from both hProCpepGFP
and hProC( A7)Y-CpepGFP mice, protein encoded by the
transgene was up-regulated in response to incubation at high
glucose. As expected, hProC(A7)Y-CpepGFP did not exhibit
maturation, as measured by its inability to be endoproteo-
lytically converted to CpepGFP, whereas hProCpepGFP was
processed in secretory granules to CpepGFP (Fig. 4B). En-
dogenous proinsulin protein expression also was increased
by high glucose; however, the amount of proinsulin was
markedly less in islets of hProC(A7)Y-CpepGFP transgenic
mice (Fig. 4B). The immunoblotted insulin band in islets
of hProC(A7)Y-CpepGFP transgenic mice did not appear
to be greatly affected by the high-glucose incubation; how-
ever, we found that when loading amounts that linearly
report changes in proinsulin content (Fig. 4B,inset),in-
sulin band-intensity changes were rather insensitive and
may underestimate the insulin recovery defect in hProC(A7)Y-
CpepGFP islets. Indeed by rodent proinsulinspecicRIA,
we found that proinsulin content of the islets was 1.26% of
total hormone content for both hProCpepGFP and C57BL/6
mice and 1.4% for nondiabetic hProC(A7)Y-CpepGFP mice.
Altogether, the data of Fig. 4 su ggest that the presence
of misfolded mutant proinsulin decreases the levels of en-
dogenous bystander proinsulin (Fig. 4B), concomitant with
a decrease in islet insulin that occurs even in nondiabetic
animals (Fig. 4A).
Islets of nondiabetic hProC(A7)Y-CpepGFP trans-
genic mice activate ER stress response. To determine
whether expression of misfolded mutant hProC(A7)Y-
CpepGFP below the diabetogenic threshold triggers ER
stress response, mRNA levels for BiP and spliced XBP-1
were measured in freshly isolated islets from nondiabetic
hProCpepGFP and hProC(A7)Y-CpepGFP transgenic mice.
Indeed, islets of hProC(A7)Y-CpepGFP mice had signif-
icantly increased BiP mRNA, as well as a tendency to
increased splicing of XBP-1 (Fig. 5). Furthermore, hProC(A7)Y-
Cp epGF P islets also exhibited a trend suggesting increased
mRNA for CHOP (Fig. 5), a downstream component of the
unfolded protein response pathway that predisposes to cell
death (2123). The results suggest that proinsulin misfold-
ing by hProC(A7)Y-CpepGFP does induce ER stress, even
in the absence of or before frank diabetes.
In hProC(A7)Y-CpepGFP transgenic mice, a subpopu-
lation of b-cells in each islet exhibits misfolded pro-
insulin accumulation and poor endogenous insulin
production. A recent report of PERK knockout mice
has shown that a subpopulation of islet b-cells exhibits an
impacted-ER phenotype characterized morphologically
FIG. 4. Content of insulin and proinsulin and transgene-encoded products in mouse islets. A: Isolated islets from hProCpepGFP and hProC(A7)Y-
CpepGFP transgenic males were lysed directly, and total insulin + proinsulin content measured by RIA. B: Isolated islets from hProCpepGFP and
nondiabetic hProC(A7)Y-CpepGFP transgenic males were incubated in RPMI 1640 medium containing either 4 or 16.7 mmol/L gluco se for 48 h
before lysis. Islet lysates from three adult males of each genotype (plus C57BL/6 littermate controls) were pooled and analyzed by reducing SDS-
PAGE and Western blotting with anti-insulin (bottom), anti-GFP (middle), and antia-tubulin (top). Note that endogenous proinsulin content is
diminished in islets of nondiabetic hProC(A7)Y-CpepGFP mice. Inset: Different loading volumes of islet lysate from C57BL/6 mice indicate that
Western blotting with anti-insulin appeared more sensitive to changes in proinsulin content than to changes in insulin, which may account for the
relatively small change in appearance of the insulin band in islets of nondiabetic hProC(A7)Y-CpepGFP mice in B despite the larger apparent
change in insulin + proinsulin content noted in A.
b-CELL HETEROGENEITY IN NONDIABETIC MICE
2096 DIABETES, VOL. 60, AUGUST 2011 diabetes.diabetesjournals.org
by an expanded ER with accumulation of proinsulin (4),
consistent with ER crowding (1,2). B ecause of the evi-
dence of ER stress in islets of nondiabetic hProC(A7)Y-
CpepGFP mice (Fig. 5), we looked for morphological
correlates in the b-cells of these animals. In cryosections
of adult male transgenic mice, hProCpepGFP or hProC
(A7)Y-CpepGFP protein expression was detected by in-
trinsic GFP uorescence, whereas anti-insulin immunos-
taining was used to reect endogenous insulin protein.
As in neonatal males (Fig. 1B), islets of hProCpepGFP
adult males (17) expressed the GFP-positive transgene
product in virtually all b-cells, which were simultaneou sly
positive fo r endogenous insulin (Fig. 6A; quantied in
Fig. 7B). Remarkably, in contrast to neonatal animals,
islets of nondiabetic hProC(A7)Y-CpepGFP males repro-
ducibly exhibited heteroge neous b-cell subpopulations
(Fig. 6B). Approximately 40% of the b-cells accumulated the
uorescent, misfolded mutant proinsulin, which, at higher
magnication, exhibited an ER distribution pattern (Fig. 6C).
Within each islet, b-cel ls rich in hProC(A7)Y-CpepGFP
uorescence tended to be largely devoid of insulin immu-
nostaining; another major subpopulation of b-cells exhibited
little GFP uorescence and immunostained clearly for en-
dogenous insulin; and only a small, third subpopulation ac-
cumulated both types of molecules (Fig. 7B).
To begin to understand the origin of the heterogeneity of
hProC(A7)Y-CpepGFP accumulation within the b-cell pop-
ulation, we also examined hProC(A7)Y-CpepGFP mRNA
distribution within the islets by in situ hybridization with
a specic complementary oligonucleotide RNA probe. As
exemplied in Fig. 6D, as much as 60% of transgene mRNA
positive b-cells within the population exhibited little or no
hProC(A7)Y-CpepGFP uorescence. Evidently, the other
;40% subpopulation of b-cells with misfolded proinsulin
accumulation and little or no insulin immunostaining (Figs.
6B and 7B) develops during postnatal life. Even if the larger
subpopulation of b-cells that failed to accumulate or had
not yet accumulated signicant quantities of misfolded
proinsulin within the ER (Fig. 6B
) contained all of the islet
insulin that is recovered (Fig. 4A), the insulin content, even
in this subpopulation of b-cells, must still be decreased
compared with that of hProCpepGFP control islet b-cells.
As the islets of hProC(A7)Y-CpepGFP mice contained
signicantly increased BiP mRNA (Fig. 5), we also looked
at the distribution of BiP protein compared with islets of
hProCpepGFP mice. Although BiP was increased in many
b-cells of hProC(A7)Y-CpepGFP mice, a heterogeneous pat-
tern within the islets was observed (Fig. 8A, left). BiP
tended to be increased in cells that had accumulated
hProC(A7)Y-CpepGFP, but Bi P was also increased in a few
other cells that had not accumulated green uorescence
(Fig. 8A, right), suggesting that these cells also were syn-
thesizing increased amounts of misfolded secretory protein.
To examine b-cell heterogeneity at the ultrastructural
level, we performed transmission electron microscopy of
the islets of nondiabetic hProC(A7)Y-CpepGFP islets. Pop-
ulations of highly granulated b-cells were readily identied
(Fig. 8B, b-cell #1). However, side by side with such cells
were other b-cells with a highly expanded ER and many
fewer (but denite) insulin secretory granules (Fig. 8B,
b-cell #2). Upon close inspection, dilation of the ER was
also seen in the b-cells that retained abundant insulin se-
cretory granules (Fig. 8C and D, b-cell #1). b-Cells with
a dramatically expanded ER compartment tended to have
unusually small insulin secretory granule proles (Fig. 8C,
microgranules). Finally, we were also surprised to discover
the unique morphological appearance of a third type of
b-cell that also had insulin microgranules but lacked an
expanded ER compartment; rather, such cells exhibited a
highly shrunken cytoplasm (Fig. 8D, b-cells #2 and #3). It is
not clear whether this third kind of b-cell has either suf-
cient hProC(A7)Y-CpepGFP protein or endogenous insulin
protein to be detected either by GFP uorescence or insulin
immunouorescence as was measured in Figs. 6 and 7.
Islets of nondiabetic hProC(A7)Y-CpepGFP mice are
hyperplastic. To further clarify whether the decreased is-
let insulin is a consequence of a decrease of insulin within
b-cells or a decrease in islet size and a resultant decrease
of overall b-cell numbers, we examined multiple random
pancreatic tail cryosections of 36-month-old nondiabetic
hProCpepGFP and hProC(A7)Y-CpepGFP male mice. Islets
contained within 0.2-cm
2
pancreatic cross-sections from
three different mice of each genotype were identied,
and, by use of GFP uorescence plus immunostaining with
anti-insulin, the islet boundaries were determined and the
islet cell nuclei counted by DAPI staining. (Antiglucagon
FIG. 5. ER stress response activation in islets of hProC(A7)Y-CpepGFP
transgenic mice. Freshly isolated islets were extracted for RNA, fol-
lowed by synthesis of reverse-transcribed cDNAs. Specic primers for
BiP, spliced XBP1, total XBP1, and CHOP were used to quantify mRNA
by real-time PCR, normalized to b-actin mRNA. Results are depicted as
mean 2
-DDCT
6 SEM. ER stress response is higher in islets of non-
diabetic hProC(A7)Y-CpepGFP mice than in hProCpepGFP mice (which
exhibit BiP, spliced/total XBP1, and CHOP mRNA levels that are not
higher than those of C57BL/6 control islets).
I. HODISH AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 60, AUGUST 2011 2097
immunostaining was also performed in both sets of mice
[not shown], conrming that a-cells did not exceed 10% of
islet cells in either mouse line.)
On average, the random cross-sectional islet area of
nonmutant hProCpepGFP mice (n = 21 islets) was 2,545
mm
2
(6276 SEM), similar to that obtained from C57BL/6
control mice (not shown), whereas random cross- sectional
islet area of hProC(A7)Y-CpepGFP mice (n = 25 islets)
averaged 4,221 mm
2
(6793 SEM), a considerable (65%) in-
crease. On average, each hProCpepGFP islet cross-
section contained 40.0 (66.2 SEM) b-cell proles, whereas
each hProC(A7)Y-CpepGFP islet cross-section contained
62.8 (623.7 SEM) b-cell proles. Dividing average islet
cross-sectional area by average number of b-cell proles
per cross-section, a rough estimate of average b-cell cross-
sectional area was essentially unchanged between the two
mouse lines. Thus, rather than b-cell hypertrophy, the data
indicate an expansion of b-cell number per islet, suggest-
ing b-cell hyperplasia in compensation for expression of
misfolded proinsulin to help these animals avoid diabetes.
Even when considering that many islet b-cells accumu-
lating uorescent misf olded mutant proinsulin have little
or no endogenous insulin (Fig. 7B), there was not an actual
loss of insulin -positive b-cells in nondiabetic hPr oC(A7)Y-
CpepGFP islets, indicating that decreased islet insulin con-
tent in these animals (Fig . 4A) must be cause d by a decrease
of insulin content per b-cell.
DISCUSSION
Studies have suggested b-cell ER crowding/ER stress in
animals in which hyperglycemia was already present (1,3,24).
Glucotoxicity is a potentially confounding variable making
other pathogenic mechanisms more difc ult to analyze.
FIG. 6. Distribution of endogenous and GFP-labeled proinsulin + insulin among the cells within islets of adult males. A: Immunostaining with
anti-insulin and GFP epiuorescence (performed as desc ribed in
RESEARCH DESIGN AND METHODS) in an adult hProCpepGFP male. B: Nond iabetic
hProC(A7)Y-CpepGFP adult male analyzed by identical methodology to that in A. C: A high magnication image showing the relative distribution
of endogenous insulin and misfolded proinsulin in a population of b-cells from a transgenic hProC(A7)Y-CpepGFP adult male. D: Nondiabetic
adult hProC(AY)Y-CpepGFP male analyzed by in situ hybridization to measure transgene-derived mRNA (left), transgene-derived protein assessed
by epiuorescence (middle), and a superimposed image (right). Note that while all b-cells in the islet appear to contain transgene-derived hProC(A7)Y-
CpepGFP mRNA, b-cells accumulating large quantities of GFP-labeled mutant proinsulin appear decient of endogenous insulin + proinsulin.
(A high-quality digital representation of this gure is availabl e in the online issue.)
b-CELL HETEROGENEITY IN NONDIABETIC MICE
2098 DIABETES, VOL. 60, AUGUST 2011 diabetes.diabetesjournals.org
Herein, we have compared nonmutant hProCpepGFP trans-
genic males to nondiabetic hProC(A7)Y-CpepGFP trans-
genic males, with each transgene accounting for only a few
percent of proinsulin mRNA (Fig. 1). Whereas permanent
neonatal diabetes occurs both in humans (9) and Akita
males (10), the hProC(A7)Y-CpepGFP transgene is driven
by the weak Ins1 promoter coexpressed with four wild-type
alleles; thus, diabetes penetrance is more subtle (Fig. 2). A
distinct advantage of this model is that effects of frank di-
abetes are largely excluded, although hProC(A7)Y-CpepGFP
males are rendered severely diabetic upon deletion of en-
dogenous Ins2, i.e., when the relative expression of mis-
folded proinsulin to wild-type proinsulin is increas ed (Fig. 3).
However, even in nondiabetic hProC(A7)Y-CpepGFP males
with a full complement of endogenous Ins genes, islet
b-cells have decreased insulin production (Fig. 4A), inhibi-
ted by coexpressed mutant proinsulin (Fig. 4B) as observed
in b-cells of Akita males (17) (females tend to resist di-
abetes [25,26]).
In hProC(A7)Y-CpepGFP males, misfolded proinsulin
and its dominant inter ference with endogenous proinsulin
causes ER stress that increases BiP (Fig. 5), predating
frank diabetes. Among other ER stress response targets,
CHOP is proapoptotic (2123). Nevertheless, the initial
decrease of insulin production is not accompanied by de-
creased b-cell mass in Akita mice (4) or in prediabetic
hProC(A7)Y-CpepGFP mice (this study). Evidently, either
because of or in spite of ER stress responses, there is
pancreatic compensation for proinsulin misfolding with
decreased insulin production.
Surprisingly, we found that, in islets of nondiabetic adult
hProC(A7)Y-CpepGFP transgenic males, despite that most
or all b-cells express the transgene mRNA (Fig. 6D), mis-
folded proinsulin accumulation appears concentrated in
a subpopulation of islet b-cells (Figs. 6B and 7 B), as has
recently been described in other mouse models (4). The
fact that b-cell subpo pulations are not detected on post-
natal day 1 (Fig. 1B) suggests that b-cell heterogeneity
develops during postnatal life. Preservation of insulin
production includes maintenance of a robust pool of in-
sulin secretory granules in a subpopulation (Figs. 6 and 8)
that is undoubtedly linked to the islet compensation nec-
essary to avoid diabetes. One hypothesis needing to be
tested is that this might be explained by the slowly pro -
gressive appearance of new b-cells that have not yet ac-
cumulated the misfolded proinsul in product. Indeed, islets
of nondiabetic hProC(A7)Y-CpepGFP male mice exhibit
morphological evidence of b-cell hyperplasia with in islets.
Generation of new b-cells may be needed to compensate
for others that have l ost insulin production (generating
only a few microgranules; Fig. 8C and D). Such a hy-
pothesis interdigitates w ell with the fact that there are
already known to be b-cell subpopulations in adult ani-
mals that operate at different rates of proinsulin biosyn-
thesis (2729), and this may include variability in secretory
feedback on proinsulin synthesis (30) and secretion (31)
as well as potentially different susceptibilities to cell
stress (32). Of course, the mutant hProC(A7)Y-CpepGFP
represents only a tiny fraction of total proinsulin mRNA
(Fig. 1A) and an even smaller fraction of total b-cell
protein synthesis. Thus, whether mutant proinsulin protein
is variably synthesized among the different b-cells within
islets (especially under different gluco se conditions) is
unknown.
It seems likely that there is a continuum of proinsulin
misfoldi ng, ER crowding, and ER stres s in a range of
diabetes subtypes, from permanent neonatal diabetes to
type 2 diabetes. Unfortuna tely, not all genetic ally pr e-
disposed individuals exhibit sufcient compensatory
responses to prevent diabetes. Our results are consistent
with the suggestion that b-cell dysfunction exists before
the onset of overt diabetes, and patients with such b-cell
dysfunction may benet from early pharmacological in-
tervention to limit proinsulin misfolding or to preserve
insulin production through suitable compen satory
responses.
FIG. 7. Quantication of the distribution of endogenous and GFP-labeled proinsulin + insulin among the cells within islets of neonatal and adult
transgenic male mice. Islet b-cells were scored for cells in which both endogenous and GFP-labeled proinsulin + insulin were detected (black
bars), versus cells in which either endogenous insulin (light gray) or GFP-labeled proinsulin (dark gray) predominated. Cryosections of three
adult and two neonatal mice of each group were used for quantication; n = number of islets examined. A: Percentage of cells accumulating each
type of insulin + proinsulin in male neonates (sex determined by Y-chromosome PCR). B: Percentage of cells accumulating each type of insulin +
proinsulin in nondiabetic adult males. Note that in hProC(A7)Y-CpepGFP adult males, only 20% of b-cells have accumulated protein derived from
both endogenous and transgene-derived proinsulins.
I. HODISH AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 60, AUGUST 2011 2099
ACKNOWLEDGMENTS
This work was supported primarily by National Institutes of
Health grants R00-DK-077441A (to I.H.) and R01-DK-48280
(to P.A.), a grant from the Michigan Diabetes Research and
Training Center (to M.L.), and Endocrinology T32 grant (to
L.L.).
No potential conicts of interest relevant to this article
were reported.
I.H. researched the data, contributed to the discussion,
and wrote the manuscript. A.A., M.L., and L.H. researched
the data and contributed to the discussion. L.L., D .L.,
A.A.-K., and A.Z. researched the data. P.A. contributed to
the discussion, wrote the manuscript, and reviewed and
edited the m anuscript.
The authors acknowledge assistance from the University
of Michigan Transgenic Mouse Core, Morphology and Image
Analysis Core, Molecular Biology and DNA Sequencing
Cor e, supported in part by P60-DK-20572, the University
of Michigan Gut-Peptide Center, and the University of
Michigan Cancer Center for assistance with electron mi-
croscopy. The authors thank Bill and Dee Brehm for im-
proving diabetes research infrastructure at the University
of Michigan.
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    • "d From control and Akita diabetic mice, the fraction of immunostainable cells that were glucagon-positive (called " Alpha " ) was quantitated and presented as a scatter plot as in panel B, with mean ± SE; *: p < 0.05 Fig. 4Treatment of Akita mice with exendin-4 (4 mice) or sham-treated (physiologic solution; 7 mice) beginning at 3 weeks of age, as noted with arrow one additional wild-type allele); thus, the Akita diabetic mouse reflects a model of individuals with misfolded proinsulin and diminished intra-islet insulin reserve. Indeed, we also examined transgenic hProC(A7)Y-CpepGFP mice in Ins2 −/− and Ins2 +/− genetic backgrounds, to further lower the relative expression of misfolded proinsulin, which is a means to limit the inhibition of intra-islet insulin production [16, 19]. The combined action of insulin and glucagon provide coordinate control of hepatic glucose production, such that even small changes in the circulating glucagon/insulin ratio may affect glycemic control. "
    Article · Dec 2016
    • "The more we learn in this area, the more our studies point out how much we still do not know. For example, it has been established that misfolded proinsulin causes beta cell failure in a dose dependent manner (Hodish et al., 2011; Liu et al., 2012a; Renner et al., 2013), yet it remains unknown whether a specific amount of misfolded proinsulin must first be present before beta cell failure ensues. Given that increased misfolding of wild-type proinsulin occurs under some pathological conditions, further studies are needed to determine the threshold of misfolded proinsulin required to trigger beta cell failure in common forms of diabetes (Fig. 5), as well as to identify genetic modifiers that may render some individuals more susceptible than others. "
    [Show abstract] [Hide abstract] ABSTRACT: To maintain copious insulin granule stores in the face of ongoing metabolic demand, pancreatic beta cells must produce large quantities of proinsulin, the insulin precursor. Proinsulin biosynthesis can account for up to 30-50% of total cellular protein synthesis of beta cells. This puts pressure on the beta cell secretory pathway, especially the endoplasmic reticulum (ER), where proinsulin undergoes its initial folding, including the formation of three evolutionarily conserved disulfide bonds. In normal beta cells, up to 20% of newly synthesized proinsulin may fail to reach its native conformation, suggesting that proinsulin is a misfolding-prone protein. Misfolded proinsulin molecules can either be refolded to their native structure or degraded through ER associated degradation (ERAD) and autophagy. These degraded molecules decrease proinsulin yield but do not otherwise compromise beta cell function. However, under certain pathological conditions, proinsulin misfolding increases, exceeding the genetically determined threshold of beta cells to handle the misfolded protein load. This results in accumulation of misfolded proinsulin in the ER - a causal factor leading to beta cell failure and diabetes. In patients with Mutant INS-gene induced diabetes of Youth (MIDY), increased proinsulin misfolding due to insulin gene mutations is the primary defect operating as a "first hit" to beta cells. Additionally, increased proinsulin misfolding can be secondary to an unfavorable ER folding environment due to genetic and/or environmental factors. Under these conditions, increased wild-type proinsulin misfolding becomes a "second hit" to the ER and beta cells, aggravating beta cell failure and diabetes. In this article, we describe our current understanding of the normal proinsulin folding pathway in the ER, and then review existing links between proinsulin misfolding, ER dysfunction, and beta cell failure in the development and progression of type 2, type 1, and some monogenic forms of diabetes. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Jan 2015
    • "While the male Akita Ins2 heterozygous mice begin to have hyperglycemia shortly after weaning, the male Akita Ins2 homozygous mice develop more severe diabetes shortly after birth (Yoshioka et al., 1997 ). In contrast, when Akita mutant proinsulin is expressed at a lower level from a transgene (not more than 4% of endogenous Ins2), diabetes prevalence is low (less than 10% of males) but the animals display pre-diabetes by glucose tolerance testing (Hodish et al., 2011). Similarly, a diabetic phenotype is not observed in transgenic pig lines that express Akita mutant proinsulin at levels < 15 % of the endogenous INS gene. "
    [Show abstract] [Hide abstract] ABSTRACT: A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS-gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Full-text · Article · Dec 2014
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