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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 89-94 |
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doi:10.1111/j.1745-7270.2006.00138.x |
Regulated production of mature
insulin in rat hepatoma
Cells: insulin production is up-regulated by dexamethasone
and down-regulated by insulin
Xin-Yu QIN1*, Kun-Tang
SHEN1,
Lu-Jun SONG1,
Xin ZHANG2,
and Ze-Guang HAN2
1 Department of General Surgery,
2 Chinese National Human Genome Center at
Received:
Accepted: December14, 2005
This work was supported by a grant from the
*Corresponding author: Tel, 86-21-64041990; Fax, 86-21-64038472; E-mail,
[email protected]
Abstract We engineered an artificial b cell line that produces an up-regulation of
insulin in response to dexamethasone, and a down-regulation in response to insulin.
A regulatory secretion system was devised by placing proinsulin cDNA containing
genetically engineered furin endoprotease cleavage sites and a regulatory
promoter for phosphoenolpyruvate carboxykinase (PEPCK), and an insulin
expressing retrovirus vector (pN-PEPCK-mINS) was constructed and transfected
into Hepa1-6 cells. The levels of insulin in culture medium and expression of
insulin gene was estimated by radioimmunoassay and reverse
transcription-polymerase chain reaction (RT-PCR), respectively. The
clone (Hepa1-6/INS21), which secreted the highest level of insulin (10.79 mIU/106 cells per
day), was selected for the regulation experiment. Compared with the non-treated
Hepa1-6/INS21 cells, insulin production was augmented 3.6-fold by the addition
of 10-
Key words insulin; regulation; hepatoma cell;
insulin-dependent diabetes mellitus (IDDM)
The treatment of insulin-dependent
diabetes mellitus (IDDM) by islet transplantation is restricted by several
factors, such as lack of sufficient donors and immune rejection to the
transplanted islets. In order to overcome these problems, it has become a
leading strategy in this field to generate gene-engineered cells secreting
insulin in a glucose-dependent manner in non-b cells in vitro followed by their implantation in
diabetes patients, which may replace insulin therapy and islet transplantation for
IDDM. In our previous studies, mature insulin was successfully secreted from
hepatic cells and several other somatic cells [1], and an artificial b cell line expressing insulin under the control of
doxycycline was also established [2]. However, the regulation of insulin
expression in these cells had not been achieved precisely in a physiological
manner. Phosphoenolpyruvate carboxykinase (PEPCK), a key rate-limiting enzyme
of gluconeogenesis, is specifically expressed in liver. The expression of the PEPCK
gene is regulated under complex hormonal control by insulin, glucagons,
glucocorticoids and cAMP [3]. Insulin inhibits PEPCK gene transcription
whereas glucagon, glucocorticoids and cAMP stimulate its transcription. Some
studies have tried to establish a regulatory production of insulin in
hepatocytes using the PEPCK promoter to drive insulin expression. Mature
or active insulin is also obtained by the introduction of furin-cleavage
sequences into the human proinsulin gene for conversion of proinsulin to insulin
[4,5], but non-virus methods for the transfer of the exogenous genes into
hepatic cells are often not efficient.
In an attempt to explore
an ideal approach for gene therapy for diabetes, this study established an
insulin-regulated artificial b cell line by
generically engineering rat Hepa1-6 cells using an insulin-expressing
retrovirus vector (pN-PEPCK-mINS) which contained a PEPCK gene promoter.
Materials and Methods
Materials
Insulin, dexamethasone,
and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (
Construction of a
retrovirus vector containing PEPCK promoter and mutated human proinsulin gene
Mutated human proinsulin
cDNA was obtained from plasmid pcDNA3.1/mINS by PCR using the forward primer 5'-CATGGATCCTGCCATGGCCCTGTGGATG-3'
and the reverse primer 5'-CAGAAGCTTGCAGGCTGCGTCTAGTTGC-3', which
contained BamHI and HindIII restriction sites. The PCR product
was digested with BamHI and HindIII and purified by agarose gel
electrophoresis. Plasmid pN-PEPCK-INS, containing the PEPCK promoter and mild
proinsulin gene, was digested with BglII and HindIII to remove
the mild proinsulin gene. The resulting fragment was used as a vector, and the
mutated proinsulin gene was cloned to the downstream of the PEPCK promoter with
T4 DNA ligase to construct the pN-PEPCK-mINS vector containing a PEPCK/mutated
proinsulin chimeric gene. After the sequence and the orientation of the
inserted mutated proinsulin gene were confirmed by sequencing, the pN-PEPCK-mINS
plasmid was then used to transfect Hepa1-6 cells.
Cell culture and
transfection
Hepa1-6 cells were
cultured in DMEM supplemented with 10% fetal calf serum. Cells were maintained
at 37 ºC in 5% CO2
until the cells were 80% confluent, and then transfected with
pN-PEPCK-mINS by Lipofectin reagent. Seventy-two hours after transfection,
Hepa1-6 cells were plated into DMEM at a density of 5´104 cells/ml in
the presence of 800 mg/ml G418.
The selection medium was replaced every other day to remove dead cells for a
period of 20 to 25 d until G418-resistant colonies were of sufficient size to
be plated in 24-well culture dishes with clone-disc (Clontech,
Immunofluorescence
staining
Staining for the
presence of insulin in cells was implemented using the mouse anti-human insulin
antibody as a primary antibody. Control and insulin-expressing Hepa1-6 cells
were grown on culture slides for 12 h and then fixed in 2% paraformaldehyde and
0.1% Triton X-100 for 30 min. The non-specific binding sites were saturated
with 5% BSA in phosphate-buffered saline (PBS) (pH 7.4). Cells were then
incubated with the mouse monoclonal anti-human insulin antibody (1:100
dilution) at room temperature for 2 h, and washed and incubated with
fluorescein-conjugated goat anti-mouse antibody (Gibco) (
Treatment of transfected
cells with insulin or dexamethasone
Theoretically, exogenous
insulin and dexamethasone can regulate the activity of artificial cells at both
the transcriptional and protein levels. Hepa1-6/INS21 cells (approximately 1106 cells/well) were grown to a confluent state
(non-growing state) in 6-well plates and then washed twice with insulin-free
medium, followed by the addition of desired concentrations of culture
medium-stimulating chemicals for 24 h, including insulin at 10-
RT-PCR
RT-PCR was performed to test
the effect of these hormones on the expression of the insulin gene as a
preliminary step. Total RNA was extracted from cells with Trizol (Gibco), and
the first chain of cDNA was prepared by RT with SuperscriptII reverse transcriptase (Gibco) using an oligo(dT11)
primer. The proinsulin gene was amplified with 0.5 u of Taq polymerase (Promega) in a total volume of 25 ml. The PCR primers for the proinsulin gene were:
sense primer 5'-catggatcctgccatggccctgtggatg-3'
and antisense primer 5'-cagaagcttgcaggctgcgtctagttgc-3',
resulting in a 366-bp fragment. The primer sequences of b-actin were: sense primer 5'-tcacccacactgtgcccatctacga-3' and
antisense primer 5'-cagcggaaccgctcattgccaatgg-3',
resulting in a 300-bp fragment. The PCR amplification was performed in a reaction
mixture containing
Radioimmunoassay
Insulin in the culture
medium of engineered Hepa1-6 cells was measured using the Human insulin radioimmunoassay
kit according to the manufacturer's instructions.
Statistical analysis
Student's t test
was used for statistical analysis (SPSS10.0) and a P value less than
0.05 was considered statistically significant.
Results
Stable expression of
insulin in transfected Hepa1-6 cells
After transfection with
vector pN-PEPCK-mINS and screening with G418 for more than 2 months,
Regulated expression of
proinsulin gene in transformed Hepa1-6 cells
Hepa1-6/INS21 cells were
seeded in 6-well plates, followed by the addition of different concentrations
of insulin or dexamethasone. Samples of culture medium and total cellular RNA
were collected for estimation of the expression of the proinsulin gene at the
transcriptional and protein levels after incubation for 24 h. All samples were
collected from duplicate wells.
RT-PCR analysis revealed
that the expression level of the proinsulin gene decreased remarkably when
cells were treated with increasing concentrations of exogenous insulin, but
dexamethasone led to an increase in proinsulin mRNA levels (Fig. 2). Fig.
3 shows the abundance ratio of proinsulin to b-actin.
The treatment of
Hepa1-6/INS21 cells with dexamethasone resulted in an increase of 2.4-fold to
3.6-fold for insulin secretion compared with untreated cells, reaching a
maximum at 10-
Discussion
PEPCK is a critical
rate-limiting enzyme of gluconeogenesis and glyceroneogenesis in liver and is induced
during diabetes. It increases the rate of hepatic glucose output and
triglyceride synthesis and makes the diabetic metabolic derangement worse [6].
There are three major factors contributing to the induction of PEPCK in a
diabetic environment. The reduction of insulin concentration in the blood
dramatically weakens its negative regulative effect on PEPCK transcription.
during diabetes, the a-cells of pancreatic islets secrete glucagons in
response to falling insulin levels [7] and subsequently elevate the
concentration of hepatic cAMP, which stimulates PEPCK gene transcription
[8]. The increased concentration of glucocorticoid hormones is known to further
induce PEPCK expression and resultant physiological alterations,
including increased gluconeogenesis [3]. Considering that the PEPCK promoter
is up-regulated under complex hormonal control and induced by dexamethasone,
but inhibited by insulin, we attempted to establish an artificial b cell line secreting mature insulin in a
physiological-regulated manner by genetically engineering liver cells, and
used the PEPCK promoter to direct the expression of human insulin gene.
The mutated human
proinsulin cDNA was added downstream to the PEPCK promoter to construct
an expressing vector, which was used to transfect Hepa1-6 cells by Lipofectin
reagent. However, this method can not achieve persistent expression for the
target gene, therefore, the retrovirus vector was implemented to overcome this
disadvantage. Besides the ability to transfer a gene into various kinds of
target cells with high efficiency, the retrovirus vector is able to integrate a
gene to the cell genomic sequence and make it express stably [9]. In this
study, 20 clones expressing insulin at different levels were obtained after the
transfection of the PEPCK promoter and insulin gene into Hepa1-6 cells and
screening for 2 months. Furthermore, insulin gene expression was up-regulated
at the mRNA and protein levels in cells which were treated with dexamethasone.
The highest level of insulin, which was 3.6-fold high than the basal level, was
noted when the concentration of dexamethasone was 10-
Nevertheless, there are
still several important factors to be determined before using the cells in
vivo. It is necessary to solve the problem that the engineered cells can
grow unlimitedly in case that excessive insulin produced by the cells would
cause hypoglycemia, even death in clinical practice. In order to inhibit the
transplanted cells from proliferating and to maintain a stable release of
insulin, Lu et al. [12] tried to transplant the transfected cells after packaging
by a semi-permeable membrane of 5% gelose-gel. However, this is not yet
reliable, so other approaches like engineering auto-body cells or transferring
the insulin gene directly in vivo will contribute significantly to gene-therapy
for diabetes. Because the kinetics of the transcriptional regulation of insulin
production in engineered cells by the PEPCK promoter is much slower than that
in normal pancreatic b cells, a
slow onset of insulin secretion will probably prolong the exposure to
hyperglycemia when the blood glucose level is high or a relatively low insulin
level occurs. Conversely, a long period of time is required to decrease insulin
production in response to blood glucose decline, which will result in
continuous release of insulin and can potentially lead to hypoglycemia. The
relative stability and high level of insulin mRNA can mainly account for
insulin-induced hypoglycemia, as the half-life of plasma insulin is less than
10 min. Hence, we had tentatively planned to insert instability elements, which
are AU-rich structures residing at the 3'-untranslated region of several
kinds of highly labile mRNA [13], into the downstream of insulin mRNA to
facilitate stable mRNA decay, but this will undoubtedly reduce the level of
insulin production. Recently, an important technical breakthrough was made by
Rivera et al. [14] in the field of regulated insulin secretion in the
liver. In their experiments, insulin was in frame fused with the FK-506-binding
protein (FKBP12) to express a fusion protein, which was retained in the
cytoplasmic reticulum with the aid of an aggregation domain in FKBP12. In
response to a membrane-permeate drug, the aggregate protein dissociates and
exports to the cytoplasm. Then, mature insulin will be released within 20 min
after the addition of the drug as the endoprotease furin can process the
fusion protein by the recognition site between insulin and FKBP12. However,
this regulating system acts through a drug rather than a glucose-responsiveness
pathway. Thus, hypoglycemia may result from a overdosing of the drug or a delay
in the clearance of insulin. So it is imperative to develop an expression
system regulating insulin release in a more perfect manner like that in b cells.
In summary, our results
show that the retrovirus vector efficiently transferred and expressed the
exogenous insulin gene in liver cells in vitro, and the genetically
engineered artificial b cells showed
a response to the concentrations of insulin and dexmethasone in extracellular
fluid. The specific capability of the PEPCK promoter and its
transcriptional activity in diabetes makes it very suitable for regulating
insulin gene expression. Therefore, the transplantation of these
physiological-regulated "b cells" may provide hope for replacing
insulin injections and islet transplantation for the treatment of IDDM.
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