1 Department of Medicine, Section of Endocrinology; 2 Howard Hughes Medical Institute and Departments of Medicine, Molecular Genetics and Cell Biology, 3 Pharmacology and Physiology, University of Chicago, Chicago, Illinois 60637; and 4 Department of Biochemistry, University of California at San Francisco, San Francisco, California 94143
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ABSTRACT |
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To study effects of
Bcl-xL in the pancreatic -cell,
two transgenic lines were produced using different forms of the rat
insulin promoter. Bcl-xL
expression in
-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After
exposure to thapsigargin (10 µM for 48 h), losses of cell viability
in islets of Fd 1 and Fd 2 Bcl-xL
transgenic mice were significantly lower than in islets of wild-type
mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but
not Fd 1 Bcl-xL mice. Pancreatic
insulin content and islet morphology were not different from control in
either transgenic line. However, Fd 2 Bcl-xL islets had impaired insulin
secretory and intracellular free
Ca2+
([Ca2+]i)
responses to glucose and KCl. Furthermore, insulin and
[Ca2+]i
responses to pyruvate methyl ester (PME) were similarly reduced as
glucose in Fd 2 Bcl-xL islets.
Consistent with a mitochondrial defect, glucose oxidation, but not
glycolysis, was significantly lower in Fd 2 Bcl-xL islets than in wild-type
islets. Glucose-, PME-, and
-ketoisocaproate-induced
hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP
production were also significantly reduced in Fd 2 Bcl-xL islets. Thus, although
Bcl-xL promotes
-cell survival,
high levels of expression of
Bcl-xL result in reduced
glucose-induced insulin secretion and hyperglycemia due to a defect in
mitochondrial nutrient metabolism and signaling for insulin secretion.
apoptosis; calcium; islets of Langerhans; mitochondria
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INTRODUCTION |
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APOPTOSIS OR PROGRAMMED cell death is an important
biological process essential for the development and maintenance of
multicellular organisms (36, 42). Changes in the rate of apoptosis may
underlie a variety of common diseases (38). Members of the
bcl-2 protooncogene family encode proteins that
function either to promote or to inhibit apoptosis (3, 12, 43).
Anti-apoptotic products of this gene family, such as Bcl-2 and
Bcl-xL, prevent programmed cell death in response to a wide variety of stimuli (3).
Bcl-xL is present in
mitochondrial, endoplasmic reticulum, and nuclear membranes (38, 29).
Recent studies have demonstrated that Bcl-xL or Bcl-2 inhibits apoptosis
via multiple effects in mitochondria, such as inhibiting release of
mitochondrial cytochrome c (11), increasing mitochondrial Ca2+
uptake (22), facilitating mitochondrial ATP/ADP exchange (40), and
maintaining normal mitochondrial volume (41). Because mitochondria play
an important role in the stimulus-secretion coupling of glucose-induced insulin secretion in pancreatic -cells (19, 24), whether Bcl-xL changes mitochondrial
function in
-cells, thereby affecting insulin secretion, is an
important issue to address.
Apoptosis is known to mediate autoimmune destruction of -cells in
type 1 diabetes (13) and postpartum involution of
-cell mass in the
rat (34). We and others have recently suggested that subtle increases
in the rate of
-cell apoptosis may contribute to loss of
-cell
mass in some animal models of type 2 diabetes, such as the Zucker fatty
diabetic rat (26) and Psammomys obesus (1). In light of these observations, interventions that could reduce
the rate of
-cell apoptosis may have substantial potential in the
prevention and treatment of diabetes. One approach is to overexpress
the anti-apoptotic protein Bcl-xL
in pancreatic
-cells. The present study was undertaken to test the
feasibility of such an approach by producing transgenic mice that
overexpress Bcl-xL in the
pancreatic
-cell. The results demonstrate that
Bcl-xL overexpression does
partially protect
-cells from death induced by thapsigargin, an
agent that induces apoptosis in insulin-secreting
-cells by
inhibiting the sarcoendoplasmic reticulum
Ca2+-ATPase and depleting
intracellular Ca2+ stores (46).
However, marked overexpression of
Bcl-xL resulted in a severe defect
in insulin secretion and hyperglycemia in the Bcl-xL transgenic mice. Further
studies revealed that the impairment in pancreatic
-cell function
can largely be accounted for by alteration in mitochondrial nutrient
metabolism and the consequent generation of ATP or other signaling
molecules for insulin secretion. These unexpected phenotypic and
functional changes associated with the overexpression of
Bcl-xL in
-cells should be
taken into account in efforts that target
Bcl-xL or other proteins to the pancreatic
-cell for therapeutic purposes.
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MATERIALS AND METHODS |
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Generation of the transgenic mice. Two
Bcl-xL rat insulin promoter (RIP)
constructs were employed to express
Bcl-xL in pancreatic -cells
(Fig.
1A).
Both vectors contained the same blunt-ended 0.82-kb human
bcl-xL cDNA (25).
To induce specific expression in
-cells, the
bcl-xL cDNA was
ligated to the 3' ends of either RIP1 or RIP7. RIP1, a 0.50-kb
Sac
I/BstB I fragment of the RIP (9) was
used to generate Fd 1 Bcl-xL
trangenic mice. RIP7, used in the Fd 2 transgenic line, was composed of
a much longer (12.9-kb) promoter/enhancer intron region from the rat
insulin II gene (23). Original founders were screened by PCR analysis using the primers 5'-CCGAATTCTTG GACAATGGAC-TGGTTGA-3' and
3'-CCGAATTCGTAGAG-TGGATGATCAGTG-5' and were confirmed
by Southern blot using a
32P-labeled full-length
bcl-xL cDNA
probe. Once founder lines were established, PCR was used exclusively to
identify transgenic progeny.
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Immunoblotting. Islets from wild-type and Bcl-xL transgenic mice were washed in PBS and lysed by passing them through a 25-gauge needle five times in RIPA buffer (1% Nonidet P-40, 1% deoxycholate, and 0.1% SDS) containing aprotinin, phenylmethylsulfonyl fluoride, and leupeptin. Protein samples (50 µg, quantified by bicinchoninic acid; Pierce, Rockford, IL) were separated in a 15% polyacrylamide-SDS gel. Proteins were transferred to nitrocellulose and blotted with a monoclonal antibody to Bcl-xL (18). Reactive proteins were detected using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL), and the intensities of the bands were quantified with a densitometer (Molecular Dynamics, Sunnyvale, CA).
Immunohistochemistry. Pancreatic tissue was fixed in 10% neutral buffered Formalin and paraffin embedded. Sections were deparaffinized and rehydrated by standard methods before immunohistochemistry. An antigen retrieval protocol (boiling the slides in citrate buffer for 10 min) was employed for Bcl-xL staining in sections of Fd 1 Bcl-xL mice. The primary antibodies used were a polyclonal guinea pig anti-bovine insulin antibody (1:1,000); a cocktail of polyclonal antibodies for glucagon, somatostatin, and pancreatic polypeptide (1:1,000; Linco Research, St. Charles, MO); and a monoclonal anti-Bcl-xL antibody (1:200). All blocking and developing reagents were from a Histomouse-SP Bulk Kit (Zymed Laboratories, San Francisco, CA) and were used according to the manufacturer's instructions. Sections treated with the same protocol in the absence of the primary antibodies served as negative controls.
Isolation of pancreatic islets and tests of in vitro cell viability. Islets of Langerhans were isolated from the pancreata of 8- to 12-wk-old Bcl-xL transgenic mice and their wild-type littermates by collagenase digestion and discontinuous Ficoll gradient separation, a modification of the original method of Lacy and Kostianovsky (14). Islets were cultured in RPMI 1640 medium supplemented with 11 mM glucose, 5% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
Islet cell viability after 48 h exposure to thapsigargin was determined by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay as previously described (20, 46).
Assessment of glucose tolerance. Intraperitoneal glucose tolerance tests (IPGTTs) were performed after a 4-h fast in Bcl-xL transgenic and wild-type control mice. Blood was sampled from the tail vein before and 30, 60, and 120 min after injection of 2 g/kg dextrose intraperitoneally.
Insulin secretion from isolated perifused pancreatic islets. Insulin secretion from isolated islets was measured in a temperature-controlled multichamber perifusion system (ACUSYST-S; Cellex Biosciences, Minneapolis, MN), as previously described (37), after an overnight culture in RPMI 1640 medium (with 11 mM glucose).
Insulin secretion from the in situ-perfused pancreas. The pancreas was perfused in situ in a humidified, temperature-controlled chamber using a modification of a protocol previously described in detail (27).
Intracellular free Ca2+ measurements. Islets attached to glass coverslips were loaded with fura 2-AM (5 µM; Molecular Probes, Eugene, OR) for 30 min at 37°C in the Krebs-Ringer bicarbonate (KRB) medium (no BSA). The coverslip was mounted in a microperifusion chamber on the specimen stage of a microscope equipped for epifluorescence and was perifused with KRB at a rate of 2.5 ml/min at 37°C as described in detail elsewhere (31). Intracellular free Ca2+ concentration ([Ca2+]i) was measured using dual-wavelength fluorescence video microscopy and was expressed as the ratio of the fluorescence intensity (detected at 510 nm) of fura 2 stimulated with light at excitation wavelengths of 340 and 380 nm (ratio 340/380) as described (31).
Glucose utilization and mitochondrial glucose oxidation in islets. These parameters were determined simultaneously by measuring the formation of 3H2O from D-[5-3H]glucose (45) and 14CO2 from [6-14C]glucose (35) in the same test tube. Duplicate batches of 10 islets each were incubated in 100 µl of KRB containing 2 or 24 mM glucose together with 0.35 µCi D-[6-14C]glucose and 1 µCi D-[5-3H]glucose. NAD(P)H autofluorescence measurement. The reduced forms of NAD and NADP, referred to as NAD(P)H, were measured using the same imaging system described for the measurement of [Ca2+]i. Autofluorescence derived from NAD(P)H was excited at 365 nm and was measured at 495 nm in dye-free islets (28). Mitochondrial membrane potential measurement. Rhodamine 123 (Rh123) was used as an indicator of mitochondrial membrane potential ( ![]() |
RESULTS |
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Islet morphology and Bcl-xL expression in pancreatic islets. There were no significant differences in litter size and body weight between the two transgenic founder lines and their wild type littermates throughout their lifetime.
Immunohistochemical staining with anti-insulin antibody (Fig. 2, A-C) or a cocktail of anti-glucagon/somatostatin/pancreatic peptide sera (Fig. 2, D-F) revealed normal islet architecture in both Fd 1 and Fd 2 Bcl-xL mice. As expected, insulin-positive
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Effects of Bcl-xL on thapsigargin-induced
cell death and DNA fragmentation in islets.
In our previous study, we have demonstrated that thapsigargin
consistently induces a sizable degree of apoptosis in islet -cells
(46). Therefore, we used it as a convenient apoptosis inducer to test
whether overexpression of Bcl-xL
confers protection against apoptosis in islet
-cells. As shown in
Fig. 3A,
thapsigargin treatment dramatically reduced the viability of wild-type
islets, whereas Bcl-xL-
overexpressing transgenic islets had significantly higher cell
viability. After exposure to 10 µM thapsigargin for 48 h, 64 ± 4.6% of wild-type islets remained viable compared with 81 ± 1.6%
in Fd 1 and 86 ± 1.0% in Fd 2 Bcl-xL islets
(P < 0.05 vs. wild type for both).
Protection against apoptosis induced by high concentrations of
thapsigargin (20 µM) was greater in Fd 2 than in Fd 1 Bcl-xL transgenic islets, as
indicated by a greater degree of viability (65 ± 4 vs. 27 ± 2%, P < 0.01, Fig. 3A). DNA fragmentation measurement
was used as an additional indicator of thapsigargin-induced apoptosis
in Bcl-xL and wild-type islets. Thapsigargin (10 µM) caused less DNA fragmentation in
Bcl-xL islets (Fig.
3B) than in wild-type islets, which
is consistent with resistance to apoptosis.
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Insulin secretory and
[Ca2+]i
responses to glucose and KCl in isolated perifused islets.
To identify the cause of hyperglycemia in Fd 2 Bcl-xL transgenic mice, insulin
secretory responses to glucose and KCl were measured in perifused
islets. As shown in Table 1, insulin
secretion in response to 16.7 mM glucose was reduced by 60% in islets
from the Fd 2 Bcl-xL (5.5 ± 0.6 vs. 13 ± 1.9 pmol/l, P < 0.001) compared with wild-type islets but was normal in Fd 1 Bcl-xL islets (15 ± 1.9 pmol/l). KCl-stimulated insulin secretion in islets of Fd 2 mice was
also significantly attenuated (by 34%).
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Insulin secretory and [Ca2+]i responses to carbachol. Carbachol, a muscarinic agonist, stimulates insulin secretion by releasing Ca2+ from intracellular stores, a process that is mediated by inositol 1,4,5-trisphosphate (IP3; see Ref. 7). Addition of EGTA (2.5 mM) to the incubation medium, by preventing entry of extracellular Ca2+ into islets, isolated the effects of carbachol on release of Ca2+ from intracellular stores. Addition of 250 µM carbachol in the presence or absence of extracellular Ca2+ triggered similar [Ca2+]i responses in Bcl-xL and wild-type islets (Fig. 5B), suggesting that the intracellular Ca2+ stores and their release mechanisms are intact in Fd 2 Bcl-xL islets. Consistent with this observation, insulin responses to carbachol from perifused Fd 2 Bcl-xL islets in the presence of 2 mM glucose were not impaired (Table 1).
Insulin secretory and
[Ca2+]i
responses to mitochondrial substrates and tolbutamide.
To test mitochondrial function in
Bcl-xL islets, two agents that are
metabolized in mitochondria [PME, a cell-permeable derivative of
pyruvate (44), and -ketoisocaproic acid (KIC)] were used as
secretagogues as well. As shown in Fig. 6,
insulin responses to glucose and PME in pancreata from
Bcl-xL mice were reduced by 79 ± 10 and 76 ± 13%, respectively
(P < 0.01 for both, see Fig.
6B).
[Ca2+]i
responses to 24 mM PME and 10 mM KIC were reduced by 58 ± 3 and 58 ± 5%, respectively (P < 0.01, n = 5) in Fd 2 Bcl-xL islets compared with a 52 ± 7% reduction in the
[Ca2+]i
response to glucose (comparisons were made based on AUC, data not
shown).
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Glucose metabolism in islets from Bcl-xL
transgenic mice.
The rates of glycolytic flux
(3H2O
production from
[5-3H]glucose) and
glucose oxidation within mitochondria
(14CO2
production from
[6-14C]glucose) were
measured simultaneously. As illustrated in Fig. 7A, the
basal glucose metabolic rates at 2 mM glucose were not different in the two groups of islets. At 24 mM glucose, glycolytic flux in Bcl-xL islets was slightly
higher (statistically nonsignificant) than in wild-type islets, whereas
the rate of glucose oxidation in mitochondria was
significantly lower in Fd 2 Bcl-xL
than in wild-type islets (P < 0.01 at 24 mM glucose, Fig. 7A). Hence, the ratio of glucose oxidation to utilization in
Bcl-xL islets was significantly
reduced at 24 mM glucose (Fig. 7B).
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Responses of
m to glucose
and PME.
Nutrient metabolism and NAD(P)H generation in
-cells normally
increases the rate of electron transport and proton efflux across the
innermitochondrial membrane, leading to the hyperpolarization of
m and ATP production. In the
present study, increasing glucose concentration from 2 to 12 mM induced
a two-phase decrease of the Rh123 signal in wild-type islets: a rapid
drop within the first 120 s, followed by a much slower second-phase
decrease lasting for another 120 s before maintaining a hyperpolarized
state (Fig. 9A).
After withdrawal of 12 mM glucose,
m returned almost completely to the prestimulation level. Stimulation of wild-type islets with 24 mM
PME caused a steep drop of
m
that reached the nadir in <1 min. The response of
m to glucose in Fd 2 Bcl-xL islets was markedly
different from that of control islets. The normal biphasic decrease in
m was replaced by a single,
slow decrease of the Rh123 signal in
Bcl-xL islets, and no rapid drop
at the beginning of stimulation was observed. The degree of
hyperpolarization of
m, as
measured by the maximum decrease in Rh123 signal, was decreased by 50%
in Fd 2 Bcl-xL islets relative to
that of wild-type controls (Fig.
9B).
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DISCUSSION |
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In view of the mounting evidence that apoptosis mediates destruction of
-cells in type 1 and possibly type 2 diabetes, interventions that
reduce the rate of
-cell apoptosis could potentially offer therapeutic benefit in the prevention and treatment of these
conditions. The present study was undertaken as a prelude to the
development of such a therapeutic approach. Transgenic mice were
produced in which the anti-apoptotic protein
Bcl-xL was expressed in the pancreatic
-cells. Tissue-specific expression of
Bcl-xL was obtained by using rat
insulin II promoter constructs, and the level of Bcl-xL expression was determined
by the length of the promoters. The longer promoter construct, RIP7,
resulted in levels of Bcl-xL that
were ~10-fold those seen in wild-type islets, whereas a shorter promoter, RIP1 led to an ~2- to 3-fold increase in the level of expression. This experimental design allowed us to study the effects of
two levels of overexpression of
Bcl-xL on the function of
pancreatic islets as well as the ability of islets to resist apoptosis
induced by thapsigargin.
Bcl-xL overexpression in
pancreatic -cells did not alter islet development or morphology in
either founder line of transgenic mice, as evidenced by the normal
islet architecture and insulin staining. Normal pancreatic insulin
content, in conjunction with normal pancreas weight, excludes the
possibility of major changes in
-cell mass in the transgenic
animals. Additionally, increased expression of
Bcl-xL protein in transgenic
islets afforded protection against thapsigargin-induced cell death when
compared with wild-type islets, and the level of protection appeared to
correlate with the quantity of
Bcl-xL expressed in islets. These
results are consistent with previous findings in other cell types and
insulinoma cell lines transfected with Bcl-2 (10, 17). This is the
first demonstration that Bcl-xL
promotes the survival of primary
-cells.
Thapsigargin is known to induce apoptosis in many cell types, including
insulin-secreting -cells, by depleting intracellular Ca2+ stores (46). Cytokines such
as interleukin-1
, interferon-
, and tumor necrosis factor-
have
been widely implicated in autoimmune destruction of
-cells
associated with type 1 diabetes. However, the potency of the cytokines
to induce cell death in intact islets was much lower than thapsigargin
in our hands, and others have shown that cytokines also induce necrosis
in pancreatic
-cells. Because thapsigargin is a more potent inducer
of
-cell death, we would expect that islets overexpressing
Bcl-xL are protected against
cytokine-induced cell death.
The high level of Bcl-xL expression achieved in the Fd 2 animals was associated with significant impairment of glucose tolerance manifested by increases in the blood glucose concentration both in the fasting state and 2 h after the intraperitoneal administration of glucose. The elevation in blood glucose was present at an early age (at least 4 wk) in both male and female animals and was associated with defective insulin secretion in response to glucose and other secretagogues in the isolated perfused pancreas and isolated perifused islets. The reduction in insulin secretion was paralleled by a similar reduction in [Ca2+]i responses to glucose and KCl. However, the pathways that stimulate insulin secretion after release of Ca2+ from intracellular stores appear to be intact, since the insulin secretory responses to carbachol, an agent that activates phospholipase C and generates IP3 (7), were normal. Normal insulin secretory responses to carbachol indicate that the exocytosis process distal to the rise in [Ca2+]i is intact in Bcl-xL transgenic islets.
Taken together with the normal islet morphology and insulin content,
these results indicate that the high level of
Bcl-xL protein expression in the
Fd 2 transgenic mice interferes with the -cell signaling pathways
activated by glucose. The precise mechanisms responsible for the
anti-apoptotic effects of Bcl-xL have not been fully defined. Its subcellular localization to the mitochondrial membrane (12) and the recent demonstration of the effects
of Bcl-xL on mitochondrial
function (11, 22, 29, 40, 41) raised the possibility that the
impairment of
-cell function associated with
Bcl-xL overexpression may be
related to disturbances in mitochondrial signaling mediating
glucose-induced insulin secretion. We therefore tested the hypothesis
that
-cell dysfunction in Fd 2 Bcl-xL transgenic mice may be
related to important mitochondrial effects of
Bcl-xL overexpression.
These studies uncovered considerable evidence for mitochondrial
dysfunction. In Fd 2 Bcl-xL
pancreas or islets, the insulin secretory responses to the
mitochondrial nutrients PME (Fig. 1) and KIC (data not shown) were
reduced to a similar extent as the response to glucose. In addition,
glucose oxidation was significantly reduced at 24 mM glucose in the
face of a normal glycolytic flux. Reduced NAD(P)H responses to PME or
KIC stimulation in Fd 2 Bcl-xL islets provided further evidence for reduced metabolism of these two
nutrients within the mitochondria.
Bcl-xL islets also demonstrated altered regulation of m, as
evidenced by reduced hyperpolarization responses after nutrient
stimulation. The maintenance of a normal
m is crucial to a number of
mitochondrial functions, such as ATP production and homeostasis of
intramitochondrial Ca2+ levels,
which are important for the regulation of metabolism (30). In the
present study, we found that the ATP production in response to glucose
and KIC stimulation was indeed reduced in
Bcl-xL islets compared with
wild-type islets.
The normal NAD(P)H response to glucose in Fd 2 Bcl-xL islets is surprising in light of the alteration in glucose oxidation. The reason for this apparent discrepancy is not known. It may be explained by the difference in the length of time for each experiment. The glucose oxidation is the total amount of oxidation over 120 min, whereas the change in NAD(P)H is the maximal amplitude over 10 min. Over the 120-min period, glucose would continue to be oxidized, and this would occur in all of the cells of the wild-type islets but may not happen in cells overexpressing Bcl-xL. Also, because the change in fluorescence is a relative value, it is difficult to quantitatively compare the two experiments.
ATP plays an important role in the regulation of exocytosis of insulin
(24, 32), and depletion of ATP impairs tolbutamide-induced insulin
secretion (21). The reduction in KCl and tolbutamide-induced [Ca2+]i
and insulin responses seen in islets of Fd 2 Bcl-xL mice are likely due to
mitochondrial dysfunction in the -cell, since similar results have
recently been observed in a pancreatic
-cell line lacking
mitochondrial DNA (39). Because mitochondrial DNA encodes proteins
responsible for ATP production through oxidative phosphorylation, depletion of mitochondrial DNA is expected to abolish mitochondrial ATP
generation. In Fd 2 Bcl-xL islets,
there is indeed a dramatic reduction in nutrient metabolism and ATP
production. These observations indicate that ATP or other factors
generated from mitochondrial nutrient metabolism play an important role
in KCl- and tolbutamide-induced [Ca2+]i
responses and insulin secretion and in glucose-induced insulin secretion.
Increased expression of Bcl-xL seen in islets from Fd 1 and Fd 2 mice was associated with protection from thapsigargin-induced apoptosis. These results are consistent with those of Biroccio et al. (2) and others, who have suggested that a decrease in mitochondrial nutrient metabolism may help to restrain the release of cytochrome c and other death-promoting molecules from the space between the inner and outer mitochondrial membranes to the cytosol. However, although even a modest overexpression of Bcl-xL, as seen in the Fd 1 islets, was sufficient to protect the islets from thapsigargin-induced cell death, only marked overexpression of the protein, as seen in the islets from the Fd 2 animals, resulted in sufficient disturbance of mitochondrial distribution and metabolism to reduce glucose-induced insulin secretion.
Although it is possible that the diabetic phenotype in the Fd 2 line
could also be linked to a very rare insertional disruption of the host
gene, we believe that the chance of this mechanism being responsible
for the observed changes is very small. First, we always performed
wild-type × transgenic breeding to avoid the disruption of both
alleles of a particular gene. Second, the phenotype of the Fd 2 Bcl-xL mice is unique; the animals
were viable, reproductively competent, and had a glucose-signaling
defect consistent with possible effects of
Bcl-xL. It is also unlikely that
we introduced a gene disruption to a position that happened to be more
important for -cell function then elsewhere. However, because we did
not get the chance to study more positive founders generated with the
RIP7 construct, we can not totally rule out insertional disruption of a
host gene as a mechanism for the phenotype of the Fd 2 animals.
In summary, the two- to threefold increase in
Bcl-xL expression seen in islets
from the Fd 1 animals afforded protection against thapsigargin-induced
apoptosis and did not interfere with insulin secretion. The ~10-fold
increase in expression of Bcl-xL
found in the Fd 2 animals afforded greater protection against
thapsigargin-induced apoptosis but was associated with a severe defect
in insulin secretion that caused diabetes. This defect in insulin
secretion is due to a novel mitochondrial defect resulting in reduced
responses to mitochondrial substrates, decreased mitochondrial glucose
oxidation, and abnormal control of
/
m. These results underscore
the complexity of developing therapeutic approaches by transferring
novel proteins into
-cells to modify their underlying function or to
enhance their ability to survive. In designing such therapeutic
strategies, not only must the desired therapeutic effect of the protein
under study be considered but unexpected adverse influences of the
particular protein on islet function need to be considered also. On the
other hand, the Fd 2 Bcl-xL mice
represent a unique model of
-cell-specific mitochondrial impairment
that underscores the critical role of mitochondrial metabolism in
insulin secretion (5, 6).
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ACKNOWLEDGEMENTS |
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We thank Kimberly Fox for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-31842, DK-44840, DK-20595, and DK-49799, the Blum-Kovler Foundation, and the Jack and Dollie Galter Center of Excellence of the Juvenile Diabetes Foundation International. M. Levisetti was supported by a mentor-based postdoctoral fellowship award of American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. S. Polonsky, Section of Endocrinology, Univ. of Chicago, MC 1027, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: polonsky{at}medicine.bsd.uchicago.edu).
Received 10 May 1999; accepted in final form 12 October 1999.
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