Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 149-157
doi:10.1111/j.1745-7270.2008.00387.x
Genetically engineered K cells
provide sufficient insulin to correct hyperglycemia in a nude murine model
Yiqun Zhang1,
Liqing Yao1, Kuntang Shen1, Meidong Xu1,
Pinghong Zhou1, Weige Yang1, Xinyuan Liu2, and
Xinyu Qin1*
1 Department of
General Surgery, Zhongshan Hospital of Fudan University, Shanghai 200032, China
2 Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
Received: April 27,
2007
Accepted: October
29, 2007
This work was
supported by grants from the Key Program of the Shanghai Science and
Technology Committee (04DZ19505), the Research Fund for the Doctoral Program of
Higher Education (20040246057) and the Youth Foundation of Fudan University
(EYF152010)
*Corresponding
author: Tel/Fax, 86-21-64037269; E-mail, [email protected]
A gene
therapy-based treatment of type 1 diabetes mellitus requires the development of
a surrogate b cell that can synthesize and secrete functionally active
insulin in response to physiologically relevant changes in ambient glucose
levels. In this study, the murine enteroendocrine cell line STC-1 was
genetically modified by stable transfection. Two clone cells were selected
(STC-1-2 and STC-1-14) that secreted the highest levels of insulin among the 22
clones expressing insulin from 0 to 157.2 mIU/ml/106 cells/d. After glucose concentration in the culture medium
was increased from 1 mM to 10 mM, secreted insulin rose from 40.30.8 to 56.33.2
mIU/ml
(STC-1-2), and from 10.80.8 to 23.62.3 mIU/ml (STC-1-14). After
STC-1-14 cells were implanted into diabetic nude mice, their blood glucose
levels were reduced to normal. Body weight loss was also ameliorated. Our data
suggested that genetically engineered K cells secrete active insulin in a
glucose-regulated manner, and in vivo study showed that hyperglycemia
could be reversed by implantation of the cells, suggesting that the use of
genetically engineered K cells to express human insulin might provide a
glucose-regulated approach to treat diabetic hyperglycemia.
Keywords type 1 diabetes mellitus; insulin; gene therapy;
glucose-dependent insulinotropic polypeptide; STC-1 cell; glucose
Type 1 diabetes mellitus results from autoimmune destruction of the
pancreatic b cells. Due to severe deficiency of insulin, elevated plasma levels
of glucose increase the risk of diabetic complications such as cardiovascular
and kidney diseases or blindness. Although exogenous insulin therapy can
mimic the physiological control of glucose, ideal glucose levels are rarely
attainable. To restore endogenous insulin secretion, pancreas and islet
transplantation have been attempted, but donors of pancreatic tissue are
limited and transplanted islets can be eliminated by autoimmune reactions
[1,2]. Thus, insulin-producing cell-based therapies for treating insulin-dependent
diabetes can provide a more physiological regulation of blood glucose levels
in a less invasive fashion than daily insulin injections. Other than b cells, there
are very few glucose-responsive native endocrine cells in the body. On choosing a target endocrine cell line for expressing and
releasing mature insulin, we found that gut K cells had some advantages. First,
gut K cells are glucose-dependent insulinotropic polypeptide (GIP) positive and
there is a rapid response of GIP secretion on glucose stimulation. Second, GIP
promoter is cell-specific and is likely to be effective in targeting gene
expression specifically to K cells [3,4]. Finally, K cells contain the
necessary enzymes (prohormone convertases 2 and 3) that process proinsulin to
mature insulin [5]. Thus, K cells seem to be very good targets for insulin gene
therapy. Cheung et al engineered a transgenic mouse model for the first
time and showed that K cells genetically engineered to express human insulin
can normalize glucose homeostasis in streptozotocin (STZ)-induced diabetes
[3].In this study, we aimed to generate an insulin-secreting cell line
with glucose responsiveness at millimolar concentrations and explore the
possibility of insulin-dependent diabetes (IDD) treatment by genetically
engineered cells. Here, a vector encoding the human insulin gene linked to
the 3-end of the rat GIP promoter was constructed, and a tumor-derived K cell
line STC-1 [6] was stably transfected with the plasmid. The novel insulin/GIP
co-producing cell line was selected. In vitro results suggested that the
release of human insulin is glucose-dependent. We also attempted to correct
hyperglycemia in vivo in an immunodeficient murine model of diabetes.
Blood glucose levels were efficiently lowered by transplantation of
insulin-producing STC-1 cells.
Materials and Methods
Construction of plasmids
Plasmid pSK-GIP-hIns was kindly provided by Dr. Burton M. Wice [7]
(Washington University School of Medicine, Washington, USA). pcDNA3 was
digested with BglII and HindIII, followed by treatment with
Klenow polymerase to blunt the ends and self-ligate, resulting in
pcDNA3-no-CMV. pSK-GIP-hIns was digested with KpnI and NotI, and
the 3 kb fragment of GIP-hIns was isolated and cloned into KpnI and NotI
sites of pcDNA3-no-CMV. This resulted in the pcDNA3-GIP-hIns construction used
for further studies.
Cells, cell cultures, and
generation of stably-transfected STC-1 cells
The murine enteroendocrine cell line STC-1 was obtained from the
Institute of Genetics, Fudan University (Shanghai, China). Human normal liver
cell line LO2 and normal human lung fibroblast cell line NHLF-1 were purchased
from Shanghai Cell Collection (Chinese Academy of Sciences, Shanghai, China).
The STC-1 and LO2 cells were cultured in Dulbecco’s minimal essential medium
containing 10% fetal bovine serum (Gibco BRL, Gaithersburg, USA), 4 mM
glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 ?C
in a humidified atmosphere with 5% CO2. NHLF-1 cells were
routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine
serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine.
Medium was changed every 34 d.Cells were transfected with pGIP/Ins or pcDNA3 in 60 mm dishes using
Effectene Transfection Reagent (Qiagen, Cologne, Germany). G418 (Sigma,
Deisenhofen, Germany) was added to the cells 72 h after transfection (400 mg/ml). Medium
was changed every 3 d, until individual clones of transfected cells could be
picked. Stable transfected cell clones with pGIP/Ins or pcDNA3 were isolated
for analysis.
RT-PCR analysis
Total cellular RNA was isolated using TRIzol reagent (Invitrogen,
Carlsbad, USA) according to the manufacturer’s protocol. RT-PCR was carried
out with total RNA (2 mg) using the First strand RT-PCR kit (Stratagene, La Jolla, USA). A
cDNA equivalent of 1 ng RNA was amplified by PCR using primers specific for
the target genes. The thermal cycles were 94 ?C for 30 s, 58 ?C for 30 s, and
72 ?C for 30 s for 30 cycles for human insulin cDNA (363 bp). Nucleotide
sequences of human insulin primers were as follows: sense, 5-TAGAACCTGGGAGGGCTAGG-3;
antisense, 5-CTGTGCCGTCTGTGTGTCTT-3. The amplification products were separated
by 1% agarose gel electrophoresis and visualized by ethidium bromide staining.
Fluorescence microscopy of
insulin/pro-insulin staining
Cells were cultured on glass slides. Cells were rinsed once with
phosphate-buffered saline (PBS), treated with sucrose buffer and 0.5% Triton
X-100 for 2 min on ice, then fixed with 4% paraformaldehyde and rinsed once
with 0.5% NP-40 for 5 min. They were incubated with mouse anti-human insulin
monoclonal antibody (1:100) (Zymed, South San Francisco, USA) at 4 ?C
overnight, followed by incubation with horse anti-mouse secondary antibody
(Gibco BRL) labeled with rhodamine for 30 min. Cells were imaged by an Olympus
IX70 fluorescence microscope (Olympus, Tokyo, Japan), and the exposure time
was set as 200 ms. Cells were cultured on glass slides. Cells were rinsed once with
phosphate-buffered saline (PBS), treated with sucrose buffer and 0.5% Triton
X-100 for 2 min on ice, then fixed with 4% paraformaldehyde and rinsed once
with 0.5% NP-40 for 5 min. They were incubated with mouse anti-human insulin
monoclonal antibody (1:100) (Zymed, South San Francisco, USA) at 4 ?C
overnight, followed by incubation with horse anti-mouse secondary antibody
(Gibco BRL) labeled with rhodamine for 30 min. Cells were imaged by an Olympus
IX70 fluorescence microscope (Olympus, Tokyo, Japan), and the exposure time
was set as 200 ms.
Animal care and STC-1 cell
implantation
Male BALB/c nude mice at 4–5 weeks old were obtained from the Shanghai
Slaccas Animal Laboratory (Shanghai, China). The animals were kept under
standard pathogen-free conditions and were handled according to the criteria
outlined in the “Guide for the Care and Use of Laboratory Animals” by
the Chinese Academy of Sciences. Animal welfare and all experiments were carried
out in accordance with the local ethics committee. The animals were allowed to
adapt to the environment 1 week before the start of experiments. Diabetes was
induced by intraperitoneal injection of STZ (Sigma), which is dissolved in 100
mM sodium citrate solution (pH 4.5) containing 150 mM NaCl immediately before
injection, at the dose of 200 mg/kg body weight to nude mice fasted for 24 h.
Mice with blood glucose levels higher than 16.7 mM 48 h after injection were
used as recipients [8–10]. Those mice were divided into three groups: group 1 (cell
therapy diabetic animals, n=7), mice implanted with STC-1-14 cells;
group 2 (cell control diabetic animals, n=6), mice implanted with negative
control cells; group 3 (PBS-treated diabetic animals, n=6), mice
injected subcutaneously with PBS. Four to six days after induction of diabetes,
1?107 STC-1-14 cells, control cells, or 200 ml PBS were
injected subcutaneously into the lower right flank of athymic BALB/c male nude
diabetic mice.
Measurement of glucose,
insulin, and body weight
Blood glucose levels were checked by a portable glucose tester with
a detection limit of 0.6 mM (Accu-Chek Advantage II; Roche Diagnostics,
Mannheim, Germany). Non-fasting blood glucose was measured, except as otherwise
specified under “Results”, between 08:30 and 09:30 am every 34 d.
Body weight was measured at the same time. Immunoreactive insulin in the
culture medium was measured by a sensitive human insulin radioimmunoassay
(RIA) kit (Dongya Immuno-technology Institute, Chinese PLA General Hospital,
Beijing, China) standardized against human insulin, according to the protocol
provided by the company. This kit is specially developed for human insulin and
has no cross-activity with mouse insulin, but can react with proinsulin
partially.The mice were subjected to 5 h of fasting and injected
intraperitoneally with 50% glucose at the dose of 5 g/kg body weight. Blood
glucose was measured at 30 min intervals up to 120 min after glucose
injection. Serum from two or three mice from each group (normal mice, mice that
received PBS, control cells, or the engineered K cells) was mixed to have
enough volume for the determination of serum insulin levels.
Immunohistochemistry
All animals were killed at the end of the study. Tumors were
resected and tissue was fixed and embedded in paraffin. Deparaffinized tumor
section specimens were treated with 3% H2O2, blocked with SuperBlock (ScyTek Laboratories, Logan, USA), and
reacted with mouse anti-human insulin monoclonal antibody (1:100; Zymed), then
reacted with biotinylated goat anti-mouse secondary antibody (Gibco BRL),
followed by 30 min incubation in the presence of avidin-biotin complex reagent
(BioGenex Laboratories, San Ramon, USA) and 5 min exposure to diaminobenzidine.
The slides were counterstained with hematoxylin.
Statistical analysis
Data are expressed as the mean±SD. Statistical analysis was carried
out using Student’s t-test or paired t-test. P<0.05 was considered significant.
Results
Generation of insulin-producing
STC-1 cell lines
The insulin-producing STC-1 cell line would be an important tool
that could be used to investigate whether engineering insulin production by
STC-1 cells is a feasible gene therapy strategy to treat type 1 diabetes mellitus.
STC-1 cells were transfected with pGIP/Ins and subjected to genetic selection
cultured in the presence of G418. Twenty-two clones were isolated. The clones
of cells were analyzed by RIA to determine whether they could secrete human
insulin. As shown in Fig. 1(A), human insulin was not detected in either
wild-type STC-1 cells or cells transfected with pcDNA3 (control cells). In
contrast, 16 out of 22 clones of cells transfected with pGIP/Ins secreted human
insulin. Clone 2 (STC-1-2) secreted the highest level of insulin (157.2 mIU/ml/106 cells/d), and clone 14 (STC-1-14) secreted approximately 50% the
amount of insulin as STC-1-2. Both STC-1-2 and STC-1-14 were selected for the
following studies.
Identification of expression
of human insulin
To examine exogenous human insulin expression, STC-1-2 cells,
control cells, and wild-type STC-1 cells were cultured in 60 mm dishes for 48–72 h, then
harvested and subjected to isolation of RNA for RT-PCR or stained for fluorescence
microscopy imaging. As shown in Fig. 1(B), the human insulin mRNA of the
STC-1-2 group was detectable compared with the control group and the wild-type
STC-1 cell group. Human insulin detection by immunofluorescence confirmed the
results from RT-PCR (Fig. 2).
Cell-specific activity of GIP
promoter
To evaluate activation of the GIP promoter that appears to be
restricted to STC-1 cells, STC-1, NHLF-1, and LO2 cells were transfected with
pGIP/Ins. As shown in Fig. 3, human insulin was detectable only in the
medium of the STC-1 cells, and the other two cell lines scarcely secreted human
insulin. These results showed that the GIP promoter was cell-specific and
likely to be effective in targeting transgene expression specifically to K
cells in vivo.
Glucose sensitivity of engineered
STC-1 cells
To further show that insulin secretion by STC-1 cells is dependent
on glucose concentration, two of the STC-1 cell line clones were assayed for
insulin release. We analyzed insulin secretion from the cells in culture
containing either 1 mM or 10 mM glucose. When STC-1-2 and STC-1-14 cells were
cultured in medium containing 1 mM glucose, the release of insulin was 40.30.8 mIU/ml and
10.80.8 mIU/ml, respectively, whereas 10 mM glucose increased the secretion
of insulin to 56.3±3.2 mIU/ml and 23.6±2.3 mIU/ml, respectively. We observed a 1.4- to 2.2-fold difference after
the increase in glucose concentration. This increase was statistically
significant (P<0.001). This observation suggested that release of human insulin from these cells was glucose-dependent (Fig. 4).
Reversal of hyperglycemia and
body weight in diabetic mice by engineered cells
To assess the feasibility of bioengineered STC-1 cells to correct a
diabetic state, insulin-expressing STC-1 cells (clone 14) were implanted into
diabetic nude mice. Cells were implanted subcutaneously 7 d after successful
induction of a diabetic state (blood glucose concentration >16.7 mM)
through intraperitoneal injection of STZ (200 mg/kg body weight). Animals
treated with control cells or PBS served as negative controls. As shown in Fig.
5(A), control cells or PBS-treated diabetic animals remained highly
hyperglycemic. In contrast, in the cell therapy group implanted with STC-1-14
clone, blood glucose levels were gradually reduced 26 d after implantation and
remained at nearly normal glycemic levels (4–6 mM) until the end of the
study in all mice. In one animal we observed hypoglycemic levels (<2.8 mM). These results suggested that a transplant of insulin-releasing cells could decrease blood glucose levels compatible with long-term survival of the recipients. Significant tumor growth was visible macroscopically during this experiment. Mice were killed 49 d after implantation for removal of STC-1-14 tumors. All animals progressively lost weight following STZ injection. The body weight of the mice implanted with the control cells or PBS decreased during the period of the study. However, the diabetic animals that received cell therapy progressively gained body weight [Fig. 5(B)].
Intraperitoneal glucose
tolerance test
Normal nude mice, control cell implanted diabetic animals,
PBS-treated, or cell therapy diabetic animals were subjected to an
intraperitoneal glucose tolerance test. Control cell or PBS-treated diabetic
animals were severely hyperglycemic both before and after the glucose
ingestion. In contrast, cell therapy diabetic animals showed initial increases
in blood glucose followed by significant decreases between 30 and 60 min after
glucose ingestion, as normal control mice did. At 90 min, the cell therapy
group had blood glucose levels similar to pre-test values [Fig. 6(A)]. Blood samples from each group were also collected for determination
of serum insulin levels. To get enough volume of serum for the measurement,
the blood from two or three mice was mixed and subjected to RIA analysis. There
was no detectable insulin expression in the mice that received PBS or control
cells. In the cell therapy group, serum insulin levels increased from
170.9±22.0 mIU/ml before glucose injection to 828.3±72.3 mIU/ml at 30 min
after glucose infusion [Fig. 6(B)]. The expression level decreased
sharply to 155.419.8 mIU/ml at 120 min after glucose infusion. The kinetics of insulin
expression was consistent with that of blood glucose levels. These results indicated
that human insulin produced from the implanted cells was sufficient to maintain
normal glucose tolerance despite having virtually no pancreatic cells.
Histological examination of pancreas
of normal nude mice and diabetic nude mice, and implanted tumors
The normal architecture of pancreatic islet is shown in Fig. 7(A).
The b cells grew in cords or nests with the acinus surrounding the islet
full of cells. The islets of diabetic immunodeficient nude mice were almost
destroyed 2 months after injection with STZ, and showed thinner cords. The
smaller atrophic cells of destroyed islets showed scant cytoplasm and coarse
chromatin nuclei [Fig. 7(B)]. The implanted tumor cells were small and
spindle-shaped with moderate to little cytoplasm. The cells were closely packed
and intensively stained with round nuclei [Fig. 7(C)].
Expression of insulin protein
in tumors of treated diabetic mice
Approximately 2 months after cell transplantation, STZ-treated mice
were killed and tumors formed by cell implantation were collected and
processed for the detection of human insulin expression. Insulin-positive
cells were observed in the tumors formed by STC-1-14 cell implantation. As
shown in Fig. 8, a brown-colored precipitate indicates the
intracellular presence of insulin and/or proinsulin peptides. In contrast,
there was no signal in the tumors after control cell implantation.
Discussion
In this study, we have shown that K cells could be engineered to
produce human insulin efficiently. STC-1 cells, a tumor-derived K cell line,
are suitable for stable gene transfer and clonal selection, and tumor xenograft
could be established in nude mice. Stable transfection allows both the
selection of clones with high levels of expression and long-term expression of
the human insulin gene. We have shown that significant amounts of human insulin
were secreted to the culture medium of STC-1 cells. We also observed that the
secretion of human insulin from these cells was glucose-dependent. Two studies
[3,11] have previously reported that glucose stimulates insulin secretion
approximately 1.5-fold from GIP-producing cell lines. Ramshur et al [7]
reported that glucose had no effect on insulin secretion by GIP/Ins cells,
although these cells could produce and store human insulin. We are not sure
why Ramshur et al [7] got different results, but the differences could
be due to the followings. Glucose, the major GIP “secretagogue”, does
not act directly on gut K cells. Glucose uptake and metabolism by adjacent
enterocytes is required for glucose-stimulated GIP release by K cells [12].
Another reason might be the heterogeneity between different clones of GIP-producing
cells. Our results showed that the GIP promoter used is cell-specific and
is likely to be effective in targeting transgene expression specifically to K
cells in vivo. The mechanism has been unclear until now. Some
researchers have identified that two transcription factors, GATA-4 and ISL-1,
are involved in the cell-specific transcriptional regulation of GIP [13].
Jepeal et al advocated that cell-specific expression of GIP was
regulated by the transcription factor called pancreas duodenum homeobox-1
[14]. But further studies will be necessary to determine whether these
transcription factors are functional and requisite for cell-specific
expression of GIP.We implanted transduced STC-1-14 cells into diabetic nude mice and
monitored blood glucose and weight gain before and after surgery. These in
vivo data indicated that STC-1-14 cells produced mature insulin that
significantly reduced blood glucose and permitted weight gain toward normal
levels. Control mice that received non-insulin-expressing cells did not show
any decrease in blood glucose. A glucose tolerance test provides a rigorous
assessment of ectopic glucose-regulated insulin delivery. Diabetic mice
implanted with STC-1-14 cells responded to an intraperitoneal glucose tolerance
testing by a marked reduction in blood glucose over the 30–60 min time
period after glucose challenge. Previous attempts to prevent glucosuria and
lethal consequences of diabetes, such as ketoacidosis, were unable to restore
normal glucose tolerance [15,16]. Our data show that insulin production from
STC-1-14 cells might correct blood glucose and restore normal glucose
tolerance (Fig. 6). Cheung et al obtained similar results by
establishing transgenic mice expressing human insulin under control of the GIP
promoter [3]. It is very important to ascertain whether removal of the tumor
results in the return of diabetes. We are carrying out experiments to study
this issue and more research needs to be done to make the transplantation of
the engineered K cells a feasible treatment for diabetes. Based on these results, the use of genetically engineered K cells to
express human insulin might provide a glucose-regulated approach to reduce
diabetic hyperglycemia. However, we must acknowledge the limitation of the
study. STC-1 is a tumor-derived K cell line. Significant tumors were visible
macroscopically 3 weeks after implantation into nude mice. In our study, one
diabetic mouse died of hypoglycemia. An identical problem was found in previous
studies [17–19]. Some studies tried to regulate the secretion of insulin by
controlling cell overgrowth [20–22], but these methods were not ideal. The primary solution is to
develop an effective therapeutic gene delivery system to intestinal cells in
the body. Viral vectors have already been developed that deliver genes to
cells of the intestinal tract, including stem cells [23–26]. Qin et al
revealed that regulated expression of insulin could be achieved by a
retroviral vector [27]. We are fully aware of the limitations of our model because of the
use of a tumor cell line to secrete insulin, but this work provides a starting
point for the treatment of diabetic hyperglycemia through glucose-dependent
expression of insulin. Our results showed that genetically engineered STC-1
cells were able to synthesize, process, and secrete active human insulin.
Furthermore, release of human insulin from these cells was glucose-dependent.
The use of genetically engineered K cells to express human insulin could
provide a glucose-regulated approach to reduce diabetic hyperglycemia. The
present work is a basic scientific model to prove the concept and the next
step is to see whether this concept works after including a long-term
expression vector (Adeno-associated virus or gutless adenovirus) carrying the
GIP promoter and necessary transcription factors to express insulin.
Acknowledgements
We thank Lanying Sun for helping with the cell culture and Lili
Zhao, Aiwen Dong, Yanhong Zhang, Yigang Wang and Ming Zhuo (Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences) for professional technical assistance.
References
1 Hirshberg B, Rother KI, Harlan DM. Islet transplantation:
Where do we stand now? Diabetes Metab Res Rev 2003, 19: 175–178
2 Rother KI, Harlan DM. Challenges facing islet
transplantation for the treatment of type 1 diabetes mellitus. J Clin Invest
2004, 114: 877–883
3 Cheung AT, Dayanandan B, Lewis JT, Korbutt
GS, Rajotte RV, Bryer-Ash M, Boylan MO et al. Glucose-dependent insulin
release from genetically engineered K cells. Science 2000, 290: 1959–1962
4 Jepeal LI, Boylan MO, Wolfe MM. Cell-specific
expression of the glucose-dependent insulinotropic polypeptide gene functions
through a GATA and an ISL-1 motif in a mouse neuroendocrine tumor cell line.
Regul Pept 2003, 113: 139–147
5 Smeekens SP, Montag AG, Thomas G,
Albiges-Rizo C, Carroll R, Benig M, Phillips LA et al. Proinsulin
processing by the subtilisin-related proprotein convertases furin, PC2, and
PC3. Proc Natl Acad Sci USA 1992, 89: 8822–8826
6 Rindi G, Grant SG, Yiangou Y, Ghatei MA,
Bloom SR, Bautch VL, Solcia E et al. Development of neuroendocrine
tumors in the gastrointestinal tract of transgenic mice. Heterogeneity of
hormone expression. Am J Pathol 1990, 136: 1349–1363
7 Ramshur EB, Rull TR, Wice BM. Novel
insulin/GIP co-producing cell lines provide unexpected insights into gut K-cell
function in vivo. J Cell Physiol 2002, 192: 339–350
8 Suzuki A, Nakauchi H, Taniguchi H.
Glucagon-like peptide 1 (1–37) converts intestinal epithelial cells into
insulin-producing cells. Proc Natl Acad Sci USA 2003, 100: 5034–5039
9 Tanaka SI, Yamakawa T, Kimura M, Aoki I,
Kamei J, Okuda K, Mobbs C. Daily nasal inoculation with the insulin gene
ameliorates diabetes in mice. Diabetes Res Clin Pract 2004, 63: 1–9
10 Yasutomi K, Itokawa Y, Asada H, Kishida T, Cui
FD, Ohashi S, Gojo S et al. Intravascular insulin gene delivery as
potential therapeutic intervention in diabetes mellitus. Biochem Biophys Res
Commun 2003, 310: 897–903
11 Kieffer TJ, Huang Z, McIntosh CH, Buchan AM,
Brown JC, Pederson RA. Gastric inhibitory polypeptide release from a
tumor-derived cell line. Am J Physiol 1995, 269: E316–322
12 Wang SY, Chi MM, Li L, Moley KH, Wice BM.
Studies with GIP/Ins cells indicate secretion by gut K cells is KATP channel
independent. Am J Physiol Endocrinol Metab 2003, 284: E988–1000
13 Boylan MO, Jepeal LI, Jarboe LA, Wolfe MM.
Cell-specific expression of the glucose-dependent insulinotropic polypeptide
gene in a mouse neuroendocrine tumor cell line. J Biol Chem 1997, 272: 17438–17443
14 Jepeal LI, Fujitani Y, Boylan MO, Wilson CN,
Wright CV, Wolfe MM. Cell-specific expression of
glucose-dependent-insulinotropic polypeptide is regulated by the transcription
factor PDX-1. Endocrinology 2005, 146: 383–391
15 He CX, Shi D, Wu WJ, Ding YF, Feng DM, Lu B,
Chen HM et al. Insulin expression in livers of diabetic mice mediated by
hydrodynamics-based administration. World J Gastroenterol 2004, 10: 567–572
16 Imai J, Katagiri H, Yamada T, Ishigaki Y,
Ogihara T, Uno K, Hasegawa Y et al. Constitutively active PDX1 induced
efficient insulin production in adult murine liver. Biochem Biophys Res Commun
2005, 326: 402–409
17 Clark SA, Quaade C, Constandy H, Hansen P,
Halban P, Ferber S, Newgard CB et al. Novel insulinoma cell lines
produced by iterative engineering of GLUT2, glucokinase, and human insulin expression.
Diabetes 1997, 46: 958–967
18 Falqui L, Martinenghi S, Severini GM, Corbella
P, Taqlietti MV, Arcelloni C, Sarugeri E et al. Reversal of diabetes in
mice by implantation of human fibroblasts genetically engineered to release
mature human insulin. Hum Gene Ther 1999, 10: 1753–1762
19 Tiedge M, Elsner M, McClenaghan NH, Hedrich
HJ, Grube D, Klempnauer J, Lenzen S. Engineering of a glucose-responsive
surrogate cell for insulin replacement therapy of experimental
insulin-dependent diabetes. Hum Gene Ther 2000, 11: 403–414
20 Tirone TA, Fagan SP, Templeton NS, Wang X,
Brunicardi FC. Insulinoma-induced hypoglycemic death in mice is prevented with
beta cell-specific gene therapy. Ann Surg 2001, 233: 603–611
21 Yoshimoto K, Murakami R, Moritani M, Ohta M,
Iwahana H, Nakauchi H, Itakura M. Loss of ganciclovir sensitivity by exclusion
of thymidine kinase gene from transplanted proinsulin-producing fibroblasts as
a gene therapy model for diabetes. Gene Ther 1996, 3: 230–234
22 Taniguchi H, Fukao K, Nakauchi H. Constant
delivery of proinsulin by encapsulation of transfected cells. J Surg Res 1997,
70: 41–45
23 Lau C, Soriano HE, Ledley FD, Finegold MJ,
Wolfe JH, Birkenmeier EH, Henning SJ. Retroviral gene transfer into the
intestinal epithelium. Hum Gene Ther 1995, 6: 1145–1151
24 Croyle MA, Stone M, Amidon GL, Roessler BJ. In
vitro and in vivo assessment of adenovirus 41 as a vector for gene
delivery to the intestine. Gene Ther 1998, 5: 645–654
25 During MJ, Xu R, Young D, Kaplitt MG, Sherwin
RS, Leone P. Peroral gene therapy of lactose intolerance using an
adeno-associated virus vector. Nat Med 1998, 4: 1131–1135
26 Cheng X, Ming X, Croyle MA. PEGylated
adenoviruses for gene delivery to the intestinal epithelium by the oral route.
Pharm Res 2003, 20: 1444–1451
27 Qin XY, Shen KT, Song LJ, Zhang X, Han ZG.
Regulated production of mature insulin in rat hepatoma cells: Insulin
production is up-regulated by dexamethasone and down-regulated by insulin. Acta
Biochim Biophys Sin 2006, 38: 89–94