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Introduction
The discovery of novel therapeutical protein or peptide
has become one of the main trends in the development of a
new drug in recent times. However, a natural barrier, the cell
plasma membrane, makes direct intracellular delivery of most
macromolecules impossible to achieve. Until the discovery
of the protein transduction domain
(PTD)[1,2], this problem could not be
addressed[3]. PTD are small cationic peptides
which allow proteins to penetrate the plasma membrane into
nuclei directly and more efficiently[4]. The protein
transduction system also improves the safety of the process by
avoiding the latent risk of gene insertion or mutation. Until recently,
several researchers have applied this novel technology to the
treatment of diseases, for instance,
tumors[5_9], ischemia[10,11],
and inflammation[12]. There is growing focus on applying
protein transduction as a potentially valuable tool to clinics
by transducing the therapeutic protein or peptide into
patients.
Diabetes mellitus is the most prevalent metabolic
disease characterized by hyperglycemia. Type 1 diabetes
results from insulin deficiency caused by autoimmune
destruction of insulin-producing pancreatic β
cells[13_15]. Although islet transplantation has been tested and found to
be effective in restoring normoglycemia, the scarcity of the
tissue supply and the dependency on immunosuppressive
therapy hinder the clinical application of the
treatment[16,17]. Therefore, there is a great medical need to search for
substitute insulin-producing cells.
The intestine has close relations with the pancreas. There
are several developmental similarities between endocrine
cells in the gut and those of the endocrine pancreas. Both of
them are derived from the endoderm, with the embryonic
pancreas originating from a dorsal and ventral protrusion of
the primitive gut epithelium[18]. The plasticity between the
intestine and pancreas has been reported. Although the
expression of the insulin gene is restricted to the
β cell in mature mammals, the intestinal cells have been induced to
express insulin in vivo by transfecting the intestine with
Ad-MafA or Ad-PDX-1[19,20] (recombinant adenovirus
containing MafA or Pdx-1 gene). In vitro, the intestinal cells have
been induced to express insulin by either transfection with
PDX-1 and exposure to the growth factor,
betacellulin[21], or transfection with PDX-1 and the LIM homeodomain
transcription factor, Islet-1[22].
BETA2/neurogenic differentiation (NeuroD), a member
of the basic helix-loop-helix (bHLH) family, plays an
essential role in pancreatic islet morphogenesis at early stages of
development and maintenance of adult β cell function by
positively regulating insulin
expression[23]. The adenovirus-mediated introduction of BETA2/NeuroD could induce
ectopic insulin expression in the
liver[24,25]. Our previous research and others found that BETA2/NeuroD contains its
own protein transduction domain which allows it to cross
the membrane of several kinds of mammalian cells.
Furthermore, the internalized BETA2/NeuroD protein still
preserves insulin transcription
activity[26,27], which opens up the possibility of applying the novel approach of protein
therapy to benefit diabetic patients.
Our present study aims at evaluating whether the
BETA2/NeuroD protein has transduction activity in vivo
and alleviates symptoms of diabetes mellitus by inducing enteric
insulin expression.
Materials and methods
Plasmid construction and protein
purification The method of constructing the plasmids is described in our
previous report[26]. BL21 (DE3) cells containing the expression
plasmids of the NeuroD_EGFP (Enhanced Green
Fluorescent Protein) protein and the EGFP protein were cultured at
37 °C so that the OD600 (Optical Density at 600 nm)
reached 0.8. Isopropyl-β-D-thiogalactopyranoside was added to a
final concentration of 1 mmol/L, and the cells were then
incubated at 24 °C for 12 h. The cells were sonicated, the
supernatants were recovered and applied to a column of
Ni-NTA agarose (medium for affinity chromatography used to bind
recombinant proteins with a 6×His tag) (Novagen, San Diego,
CA, USA), and the target proteins were eluted with an
elution buffer containing 300 mmol/L NaCl, 50 mmol/L
NaH2PO4, and 150 mmol/L imidazole (pH 8.0). The protein purification
procedure was then repeated in order to enhance the purity
of the target proteins. Finally, Amicon Ultra-4 centrifugal
filter devices (Millipore, Billerica, MA,USA) were used to
remove the salt and other contaminating microsolutes. The
fusion proteins were either used immediately after
purification or stored at 4 °C.
Cell culture and protein internalization STC-1
(entero-endocrine cell line) cells were obtained from American Type
Culture Collection (Manassas, VA, USA), and cultured in
high glucose Dulbecco's modified Eagle's medium
containing 10% fetal calf serum. All cells were cultured at 37 °C in a
humidified atmosphere with 5% CO2. The cells were seeded
on a 24-well plate at a density of
1×105 cells/well. After 24 h incubation, the medium was replaced with fresh medium
containing 1 μmol/L EGFP or the NeuroD_EGFP protein. The
cells were incubated with the protein-containing medium for
4 h, washed 3 times with phosphate-buffered solution (PBS),
fixed in 4% paraformaldehyde (PFA), and then observed
using a fluorescence microscope.
Diabetic mice preparation and treatment with
protein ICR male mice(derived from Institute of Cancer Researcch)
(8 weeks of age, weighing 22±2 g, provided by and bred at
the Laboratory Animal Center of the Second Military
Medical University, Shanghai, China; Certificate
No SCXK Jun, 2002-011, China) were made diabetic by an ip(intraperitoneal)
injection of streptozotocin (STZ; 150 mg/kg,
Sigma, St Louis, MO, USA) freshly dissolved in citrate buffer (pH 4.5). The
diabetic mice were injected with either the NeuroD_EGFP
protein (5 mg/kg, n=6) or the EGFP protein (5 mg/kg,
n=5) into the caudal vein 1 week post-STZ injection. After the
protein injection, non-fasting blood glucose levels and body
weights were measured regularly with a portable glucose meter
(Optium, Medisense, Medisense, Bedfold, MA, USA) after
snipping the tail. Sixteen hours after the NeuroD_EGFP and
EGFP protein administration, fasting serum insulin levels were
determined by an insulin radioimmunoassay kit (Linco,
Billerica, MA, USA).
RT-PCR and quantitative PCR analysis Total RNA were
isolated from the small intestine using Trizol agent
(Ambion, Austin, TX, USA). Reverse transcription of
RNA was performed with a M-MLV (Moloney Murine Leukemia
Virus) reverse transcriptase (Promega, Madison, WI).
Two pairs of primers were used for the amplification of
insulin (5'-TGGCTTCTTCTACACCCAAG-3' and 5'-ACAA-TGCCACGCTTCTGCC-3') and GAPDH
(5'-TGGCAAA-GTGGAGATTGTTGCC-3' and
5'-AAGATGGTGATGGG-CTTCCCG-3').
The expression levels of the insulin gene were quantified
by real-time quantitative PCR, using SYBR(Synergy Brands, Inc) green I dye (Invitrogen, Carlsbad, California,
USA) and the ABI (Applied Biosystems) Prism 7900 Real
Time PCR System (ABI, Foster City, CA, USA). The amount
of copy numbers of the target genes in each sample was
calculated and expressed as a ratio to the GAPDH gene.
Primers are described as above.
Immunohistochemistry The mice were killed by
cervical dislocation, and after a midline abdominal incision
was made, the small intestine tissues were removed from
the mice and fixed overnight with 4% PFA. The fixed
tissues were routinely processed for paraffin embedding and
approximately 4 μm sections were prepared and mounted
onto slides. To detect insulin, the Streptavidin-biotin
complex (SABC) method was performed using the SABC Kit
(Boster, Wuhan, Hubei, China). The sections were
incubated overnight with a rabbit anti-insulin antibody (H-86;
Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted at
1:100 in PBS containing 1% BSA (Bovine Serum Albumin),
followed by incubation with biotinylated anti-rabbit IgG
(Immunoglobulin G) (Boster, Wuhan, Hubei, China), SABC
reagent incubation, and 3,3'-diaminobenzidine
tetrahydro-chloride (Boster, Wuhan, Hubei, China) staining.
Statistics The results of the quantitative data were
expressed as mean±SD. Statistical differences between the
groups were analyzed with the t-test, and the significant
level was defined as a P<0.05. The data were analyzed by
SPSS statistical software (SPSS, Chicago, IL, USA).
Results
Transduction of BETA2/NeuroD protein in
intestine To examine whether the BETA2/NeuroD protein can be
transduced into small intestine epithelium cells
in vitro, we purified the NeuroD protein with EGFP fused at the C-terminal
(Figure 1D) and used the fluorescence activity of EGFP to
track the location of the NeuroD protein. Intestine
epithelium-derived STC-1 cells were treated with the purified
NeuroD_EGFP protein. After 4 h incubation, the STC-1 cells
incubated with NeuroD_EGFP were nearly 100% EGFP
positive as determined by a fluorescence microscope (Figure 1A,
left panel). In contrast, hardly any EGFP-positive cells could
be observed when they were treated with EGFP proteins
(Figure 1A, right panel). These results demonstrate that the
BETA2/NeuroD protein can efficiently permeate
intestine epithelium cells in vitro.
We further tested whether the BETA2/NeuroD protein
can preserve its transduction activity and be internalized
into the small intestine epithelium cells in
vivo. The 8-week-old ICR mice were intravenously injected with NeuroD_EGFP
protein (9 mg/kg) while the same dosage of EGFP protein
was used as the negative control. Two hours later, the ICR
mice were killed and tissue samples were isolated from the
liver, pancreas, brain, small intestine, and heart. The mice
were perfused with PBS to avoid possible contamination of
EGFP remnants in body fluid. The tissue samples were then
analyzed for the intensity of fluorescence. The
NeuroD_EGFP protein was found to accumulate at the small intestine
(Figure 1B) and the liver (data not shown). However, no
NeuroD_EGFP protein was clearly detected in the brain,
pancreas, and heart (data not shown). To confirm the
distribution of the NeuroD_EGFP protein in the small
intestine tissue, a technical procedure of frozen section
was then conducted and analyzed. The 5 µm sections of
the small intestine were prepared and treated with
DAPI(4',6-diamidino-2-phenylindole) to stain the nuclei. As
observed by the fluorescent microscope, the small intestine
cells of the NeuroD_EGFP protein-treated mice were almost
100% EGFP positive (Figure 1C). In contrast, no
EGFP-positive cell was detected in the control intestine (Figure 1C).
These results indicate that NeuroD_EGFP can be transduced
into and accumulate at the small intestine epithelium cells
in vivo.
Effect of the internalized BETA2/NeuroD protein on
insulin expression BETA2/NeuroD is known to activate
insulin expression in pancreatic β cells. To test whether
BETA2/NeuroD can stimulate the ectopic expression of the insulin
gene in the small intestine of diabetic mice where the
BETA2/NeuroD protein accumulated, RT-PCR and real-time
quantitative PCR were performed. The 8-week-old ICR mice were
treated with 150 mg/kg STZ. One week later, blood glucose
concentrations were measured and hyperglycemia was
verified. The STZ-induced diabetic mice were intravenously
injected with the NeuroD_EGFP protein (5 mg/kg) or the EGFP
protein (5 mg/kg) as the negative control. After 16 h, the
tissue samples for the jejunum were separated and total
tissue RNA was extracted. Semiquantitative RT-PCR revealed
that the transcription of the insulin gene greatly increased in
the jejunum of NeuroD_EGFP-treated mice (Figure 2A, left
panel). However, no obvious insulin gene transcription was
detected in the jejunum of EGFP-treated mice. Real-time
quantitative PCR further demonstrated that treatment with the
NeuroD_EGFP protein gave a 38-fold increase in the
transcription level of the insulin gene in terms of increase in
mean insulin/GAPDH ratio. For the experiment group,
the ratio reached
(2.109±1.019)×10-2, which was
significantly different from that of the negative control equaling
(5.4±0.9)×10-4 (Figure 2A, right panel,
t-test, n=3, P<0.05). To test
whether the NeuroD_EGFP protein could only stimulate
insulin ectopic expression in the small intestine
in vivo, we examined insulin expression levels in various tissues
(spleen, jejunum, liver, brain, and heart). As shown by
semiquantitative RT-PCR, the increasing expression levels
of insulin was only clearly detected in the jejunum, but not in
any other tissues (Figure 2B).
To examine whether the insulin protein was synthesized
and stored in the jejunum, we used immunohistochemistry to
test the insulin expression after treatment with
NeuroD_EGFP and estimated the number of insulin-producing cells in the
diabetic mice after different treatments. Immunostaining for
insulin 16 h after treatment with the NeuroD_EGFP protein
revealed several insulin-positive cells in the cytoplasm.
These cells were located both in the crypts of Lieberkuhn
(Figure 3B) and the intervillus epithelia (Figure 3A) of the
jejunum. The density of insulin-positive cells 16 h after the
NeuroD_EGFP protein treatment was 1.53±0.28
cell/mm2 (mean±SD, n=5). However, by examining 30 sections
derived from 3 NeuroD_EGFP-treated mice, no insulin-positive
cells were observed 8 d after NeuroD_EGFP treatment (Figure
3C). The insulin-producing cells were also not detectable in
the EGFP-treated jejunum (Figure 3D) or by immunostaining
without a primary antibody (data not shown).
Effect of hyperglycemia alleviation by BETA2/NeuroD
protein treatment To examine whether enteric insulin
production induced by the NeuroD_EGFP protein is capable of
controlling blood glucose levels in diabetic mice, we injected
150 mg/kg STZ into ICR mice, and 1 week later treated the
mice with 5 mg/kg of the NeuroD_EGFP protein or the same
dosage of the EGFP protein as the negative control. Sixteen
hours later, fasting serum insulin levels were determined.
The blood glucose levels and the body weight of the mice
were then measured regularly. The fasting serum insulin
level of the control diabetic mice was 84±23 pg/mL. In
contrast, that of the NeuroD_EGFP-treated diabetic mice was
337±39 pg/mL (t-test, n=5_6,
P<0.01 vs control). The shape of the blood glucose level curve demonstrated that the
administration of the NeuroD_EGFP protein could significantly
reverse hyperglycemia and the effect could last for about
2_3 d (t-test, n=5-6, P<0.01). After that, however, blood
glucose levels gradually increased. We did not detect any
significant decrease in blood glucose levels of the control
diabetic mice (Figure 4A). Moreover, we noticed that,
compared with the control mice, the body weights of the diabetic
mice treated with NeuroD_EGFP gradually increased (Figure
4B).
Discussion
Protein transduction technology has been considered to
be an alternative tool for clinical therapy. Efforts have been
made to alleviate many kinds of diseases in animal models.
In our study, the results revealed that the NeuroD protein
with innate transduction activity could obviously alleviate
diabetic symptoms. NeuroD_EGFP-treated intestinal cells
were as almost as high as 100% EGFP positive, and
displayed rapid biological activity. In addition, since no
extraneous gene has been introduced, the protein
transduction system improves the safety of the process by avoiding
the latent risk of gene insertion or mutation.
In our study, we found that the BETA2/NeuroD protein
could be efficiently transduced into small intestine and liver
(data not shown) tissues in vivo, implying tissue preference
to a certain extent. This result is interesting because both
the small intestine and liver are potential target organs to be
induced to produce insulin. Several researchers have
trans-differentiated liver cells into insulin-expressing
cells by adenovirus-mediated gene
transduction[23,24,26,27]. Never-theless, we did not detect any obvious increase of insulin
gene transcription in the liver by delivery of the BETA2/NeuroD
protein. Therefore, we speculate that some other factors
might be involved in the process of
transdifferentiation from liver cells. For instance, recently, other researchers found that a
host response to an adenovirus was required for the
correction of diabetes using Pdx-1 or neurogenin-3 in the
liver[30]. The transdifferentiation of liver cells may be much more
complicated than we expected.
In our research, the NeuroD_EGFP protein successfully
stimulated intestinal cells to express insulin. This result
further confirms that small intestine can serve as a target organ
in diabetes treatment. Furthermore, we found that most
insulin-positive cells were located at the crypts of
Lieber-kuhn. It is believed that several stem cells lie in the crypt
under a normal, steady-state
condition[31]. Therefore, we propose a hypothesis that the internalized BETA2/NeuroD
protein could bind to an insulin promoter and activate its
expression in the immature intestinal cells. Other researchers
have reported that IEC-6 rat intestinal epithelial crypt cell
line, which have the characteristics of immature intestinal crypt
cells, could be induced to differentiate into insulin-producing
cells in vitro[22]. This result could prove our hypothesis to a
certain extent from another aspect.
In our study, many cells in the small intestine were
permeated by the NeuroD_EGFP protein, but not all cells
expressed substantial amounts of insulin; in fact,
1.53±0.28 cell/mm2 in the jejunum were detected to express substantial
amounts of insulin in the small intestine. Taken into
account the large surface area of the jejunum, we suppose
that the total insulin production in it is sufficient to correct
hyperglycemia.
The BETA2/NeuroD protein could stimulate its own
transcription by binding to its own E
box[32]. Thus, once BETA2/NeuroD is transduced into cells that still have differentiation
potential, it may induce endogenous BETA2/NeuroD
trans-cription, stimulate insulin expression, and facilitate their
differentiation into insulin-producing
cells[3,33]. However, the BETA2/NeuroD protein-treated diabetic mice did not restore
normoglycemia for a long time. Our data suggest that the
effect of reversing hyperglycemia merely lasted 2_3 d. It is
also believed that intestinal epithelial cells turn over every
3_5 d by dissociating from the villi tips into the gut lumen or
by undergoing apoptosis at the tip of the
villi[34]. Therefore, the short-lasting effect may be due to the quick turnover of
intestinal epithelium cells. Moreover, we noticed that
although the NeuroD_EGFP protein could reverse
hyperglycemia in diabetic mice, it could not significantly ameliorate
glucose tolerance (data not shown). We have to admit that
there are still a few disadvantages in our technology, such
as high dosage, immune activity, and unknown long-term
effects. Further research should be conducted to address
these problems, such as aiming at improving target
specificity to reduce the protein dosage. In any case, we have to
overcome a significant obstacle for clinical application of
the protein transduction in diabetes treatment. Nevertheless,
our research provides a new perspective of protein
transduction in handling diabetes and has proven its
effectiveness in correcting hyperglycemia. Put simply, the value of
the research can not be outweighed by its shortcomings.
In conclusion, the protein transduction of the
BETA2/NeuroD protein induces ectopic insulin expression in the
small intestine, which results in improved serum insulin
levels and ameliorates non-fasting glucose levels in STZ-treated
mice. Here we reported an alternative strategy for diabetes
treatment other than insulin injection, which provided
relatively longer maintenance of euglycemia, although further
research is still required for its clinical application.
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