Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242-1109
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ABSTRACT |
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The human proinsulin cDNA was introduced into a replication-defective adenovirus and was found to confer proinsulin expression to a hepatocyte (H4-II-E) cell line upon infection. A second virus was constructed in which the dibasic prohormone convertase recognition sequence was mutated to a tetrabasic furin cleavage site. Cells infected with this virus synthesized both proinsulin and mature insulin. Gel filtration chromatography, competition of insulin binding, and activation of the insulin receptor kinase activity demonstrated that this mature insulin was functionally identical to that of authentic processed insulin. Injection of these viral constructs into the external jugular vein of mice resulted in insulin gene expression in the liver. Expression from the mutated proinsulin virus dramatically improved the glycemic state of diabetic mice. However, the effects of the viral infection were transient, being maximal at ~5-7 days and returning to steady-state levels by 14-21 days. These data demonstrate that somatic cell insulin gene delivery by the use of recombinant adenovirus can be used to transiently reverse the diabetic state in mice.
diabetes; gene therapy; furin
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INTRODUCTION |
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INSULIN IS SYNTHESIZED in the -cells within the
Islets of Langerhans of the pancreas as a preprohormone (30). The
signal sequence is cotranslationally removed in the endoplasmic
reticulum followed by the trafficking of the prohormone to the Golgi
apparatus (12). The proinsulin is then transferred to regulated
secretory vesicles (13) where it is processed into mature insulin by
proteolytic cleavage of the C-peptide; however, a small amount of
proinsulin is maintained and is eventually released into the
circulation (9). The proteolytic processing of proinsulin to insulin
requires specific proteases called prohormone convertases (PC2 and
PC3), which are members of the subtilisin family of endoproteases (35). These convertases cleave peptide bonds carboxyl-terminal to dibasic residues (2) and in combination with carboxypeptidase E/H, which
removes the basic residues from the B chain, generates the mature
functional insulin that is secreted into the circulation upon
stimulation of the
-cells with appropriate agonists (27).
Several studies have demonstrated that heterologous cell types can express either proinsulin or insulin, depending whether they are neuroendocrine cells or not (14, 18, 25, 43). Recently, it has been demonstrated that, if the dibasic processing sequences at the C-A and B-C junctions of proinsulin are changed to tetrabasic sequences and the resulting gene is expressed in cells lacking the regulated secretory pathway, processing to mature insulin can occur (11, 42, 43). This is most likely due to the action of furin, another subtilisin family member endoprotease that is ubiquitously expressed and acts on proteins that are transferred to the cell surface or are secreted from the cell via the constitutive secretory pathway. Furin requires an additional basic residue two positions amino terminal to the dibasic site in order to recognize and process a peptide sequence (17). Thus a tetrabasic processing site should be recognized by furin and cleaved appropriately, with carboxypeptidase E/H subsequently removing all four basic residues at the new carboxyl terminus.
Type I diabetes mellitus results from the specific autoimmune
destruction of the -cells in the Islets of Langerhans, resulting in
insulin deficiency and hence metabolic wasting (20). In a recent study
that demonstrated the feasibility of supplying a diabetic animal with
insulin produced from a heterologous tissue, transgenic mice were
generated carrying the proinsulin cDNA driven by a liver specific
promoter (36). After ablation of the
-cells in these animals, the
production and secretion of proinsulin from the liver were able to
prevent the onset of severe hyperglycemia. Thus these data suggest the
potential use of liver as a gene-targeting site for the expression of
the proinsulin cDNA. In the present study, we have utilized a
replication defective adenovirus vector to direct the transient
expression of both proinsulin and insulin in hepatocyte cell lines and
from the livers of mice. These data demonstrate that expression of an
appropriately mutated proinsulin cDNA results in the secretion of fully
mature and bioactive insulin, which normalizes the hyperglycemic state
of diabetic animals.
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METHODS |
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Materials. The cDNA for human
proinsulin and the type 5 recombinant adenoviral vector construction
plasmids pACCMV.pLpA and pJM17 were kind gifts from Dr.
Graeme Bell (University of Chicago, Chicago, IL) and Dr. Christopher
Newgard (University of Texas Southwestern Medical Center, Dallas, TX),
respectively. Purified recombinant adenovirus containing a nuclear
targeted form of bacterial -galactosidase (AdRSVntLacZ), a type 5 adenovirus containing a nuclear-targeted form of
-galactosidase was
purchased from the University of Iowa Viral Vector Core. The human
proinsulin standard was obtained from Sigma Chemical (St. Louis, MO),
and the human insulin standard (Humulin regular) was purchased from Eli
Lilly (Indianapolis, IN). Bovine serum albumin [A7030, formulated to reduce interference in insulin radioimmunoassays (RIA)] was purchased from Sigma Chemical.
Site-directed mutagenesis of the human proinsulin
gene. The cDNA for human proinsulin was cloned into the
plasmid pSELECT (Promega, Madison, WI). Site-directed mutagenesis of
the human proinsulin gene was performed using the Altered Sites kit
(Promega) with the oligo
5'-CTGCAGGTCCTCCGGCGGGTCT-3'
to mutate the B-C junction and the oligo
5'-CACAATGCCAC
GGGACCCCT-3' to mutate the C-A junction. The oligonucleotides were
used simultaneously in the same mutagenesis reaction. This modification
generated a series of four consecutive arginine residues at both the
C-A and B-C junctions as indicated in Fig.
1. The presence of the mutations was
confirmed by DNA sequence analysis, and the resultant gene was termed
proins-Tb for
ulin with
et
asic
cleavage sites. Both the normal human proinsulin cDNA and
proins-Tb were subcloned into the
adenovirus shuttle plasmid pACCMV.pLpA between the
Kpn I site and the
BamH I site, downstream of the
cytomegalovirus (CMV) early gene promoter.
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Adenovirus construction. Recombinant viruses were generated by homologous recombination between the pACCMV. pLpA plasmids containing the human proinsulin cDNA or proins-Tb and the plasmid pJM17 as previously described (3). The resulting recombinant viruses contain either the human proinsulin cDNA or proins-Tb in place of the E1 region of the adenovirus genome. These viruses were plaque purified, screened by polymerase chain reaction for the presence of inserts, and termed Ad-CMV/InsWT or Ad-CMV/InsTb for the wild-type proinsulin gene or the tetrabasic processing site mutant proinsulin gene under the control of the CMV early gene promoter.
Adenovirus propagation. The
Ad-CMV/InsWT and Ad-CMV/InsTb viruses were propagated as previously
described (3). For large-scale preparations of virus, thirty 15-cm
plates of 293 cells (90% confluence) were infected at a multiplicity
of infection of 10 for 2 h. The media was then aspirated, the plates
were washed with phosphate-buffered saline (PBS: 137 mM NaCl, 10 mM
Na2HPO4,
2.7 mM KCl, and 1.8 mM KH2PO4,
pH 7.4), and new media was placed on the cells. The media was removed
36 h later, and the cells were collected and lysed by three cycles of
freezing and thawing in a dry ice/ethanol bath. The cell debris was
pelleted from the lysate by a brief centrifugation (20 min at 1,500 g), and the supernatant was placed
over a CsCl step gradient (1.28-1.40
g/cm3). This gradient was spun
at 30,000 g in a VAC50 rotor for 3 h. The viral band was removed from the interface of the gradient and
applied over another CsCl step gradient (1.33-1.40
g/cm3) and centrifuged in a
SW41.Ti rotor for 3 h at 30,000 g. The viral band was again removed from the interface, desalted over a PD-10
column (Pharmacia Biotech, Piscataway, NJ), and stored in PBS
containing 3% sucrose at 80°C until use. The viral titers were estimated optically by the absorbance at 260 nm (5).
Cell culture. Human embryonic kidney 293 cells (provided by Dr. Michael Welsh, The University of Iowa, Iowa City, IA) were maintained in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10 mM HEPES, pH 7.6, 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. H4-II-E rat hepatoma cells (provided by Dr. Mark Yorek, The University of Iowa, Iowa City, IA) were maintained in DMEM supplemented with 10% FBS, 10% calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) were isolated and cultured as we have previously described (29).
Insulin RIA. Insulin in culture media and in column fractions derived from culture media was measured using an insulin RIA kit capable of detecting both insulin and proinsulin (ICN Pharmaceuticals, Costa Mesa, CA). Serum insulin concentrations were determined using an ultrasensitive human insulin specific RIA kit (Linco Research, St. Louis, MO), which has <1% cross-reactivity with mouse insulin and 6% cross-reactivity with human proinsulin. Insulin in column fractions derived from serum samples was measured using the ultrasensitive human insulin RIA kit.
Separation of insulin from proinsulin. Insulin was resolved from proinsulin by gel filtration chromatography on 1.0 × 120-cm Sephadex G-50 Super Fine columns (Pharmacia) as previously described (6, 19, 43). Briefly, the columns were equilibrated in 50 mM sodium acetate, pH 5.0, containing 0.01% BSA, after which 2.0-ml samples were loaded onto the columns. Column fractions were collected (1.5 ml) and assayed for immunoreactive insulin. The column fractions in each peak were pooled, dialyzed overnight, lyophilized, and resuspended in a minimal volume of minimal Eagle's medium containing nucleotides. Insulin concentration was determined by insulin RIA (see above).
Insulin receptor binding assay. Confluent monolayers of CHO/IR cells grown in 96-well culture dishes were washed two times with Krebs-Ringer HEPES buffer (KRH; 50 mM HEPES, pH 7.4, 128 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2, and 1.3 mM MgSO4) containing 0.1% BSA and 0.01% bacitracin. The cells were then incubated with 50,000 counts/min of 125I-monoiodo[A14]insulin and the indicated concentrations of cold insulin in a total volume of 50 µl of KRH-BSA buffer for 1 h at room temperature with gentle shaking. The cells were then washed two times with PBS and solubilized in 0.2 ml of 0.1 N NaOH at 37°C. The contents of each well were then transferred to a test tube and counted in a gamma counter (Packard Instrument, Downers Grove, IL) for 1 min.
Stimulation of the insulin receptor tyrosine kinase activity. Confluent monolayers of CHO/IR cells grown in 48-well culture dishes were serum starved for 6 h in minimal Eagle's medium containing nucleotides. The cells were then incubated with the indicated concentrations of either insulin standard or experimentally isolated insulin in 100 µl of minimal Eagle's medium containing nucleotides for 5 min. The media was then removed, and the cells were washed two times with PBS at 4°C and lysed in 100 µl/well of 20 mM HEPES, pH 7.4, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 2 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 0.5 trypsin inhibitory units of aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 10 mM leupeptin for 1 h at 4°C. The resulting lysate was centrifuged (12,000 g for 15 min) to remove insoluble material, combined with an equal volume of 2× Laemmli SDS-sample buffer [120 mM Tris · HCl, pH 6.8, 20% (vol/vol) glycerol, 10% (wt/vol) SDS, 200 mM dithiothreitol, and 0.01% (wt/vol) bromphenol blue], and heated for 5 min at 100°C. The samples were then subjected to phosphotyrosine immunoblotting as previously described (29).
Animal injections. Nine-week-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized with a 30-µl intraperitoneal injection of a 1:10 mixture of ketamine (Ketaject; Phoenix Pharmaceutical, St. Joseph, MO) and xylazine (Rompin; Miles, Shawnee Mission, KS). An incision was made in the skin over the chest and neck, and the external jugular vein was exposed. Each mouse was injected with 0.1 ml of purified virus of the indicated titer into the external jugular vein. The incision was then closed with sutures, and the mice were allowed to recover.
To generate diabetic mice, the mice were injected intraperitoneally with 200 mg/kg of streptozotocin (Zanosar; Upjohn, Kalamazoo, MI). Four days later, the blood glucose was checked using a One-Touch-II glucometer (Lifescan, Milpitas, CA). Those mice with a blood glucose level of 350 mg/dl were used for injections on that day. All of the animal procedures conformed to established guidelines for the appropriate care and use of animals in research and were approved by The Animal Review and Use Committee of The University of Iowa.
Insulin immunohistochemistry and -galactosidase
staining. Nine-week-old C57BL/6 mice were anesthetized
and injected with virus as described above. Four days later, the mice
were anesthetized again and were perfused by injection into the left
ventricle with 5 ml of PBS followed by 5 ml of 4% paraformaldehyde.
The livers were removed, immersed in liquid nitrogen, and
cryosectioned. Insulin staining was performed using a guinea pig
anti-porcine insulin antibody (DAKO, Carpinteria, CA), followed by
incubation with a gold-linked secondary antibody and silver
enhancement. In situ
-galactosidase staining was performed by fixing
the cryosectioned liver with 1% glutaraldehyde and incubating with
0.2% 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside in
10 mM
Na2HPO4,
pH 7.0, 1 mM MgCl2, 150 mM NaCl,
3.3 mM
K3Fe(CN)6,
and 3.3 mM
K4Fe(CN)6
for 1 h at 37°C as previously described (41).
Glycogen determination in liver tissue. Liver glycogen content was determined using the anthrone reagent, after digestion of the tissue by potassium hydroxide as previously described (15). All results were expressed as means ± SE and were compared using the unpaired Student's t-test, with P < 0.05 considered significant.
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RESULTS |
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Infection with Ad-CMV/InsWT induces proinsulin synthesis in hepatoma cells. To determine whether the recombinant adenovirus encoding for wild-type preproinsulin (Ad-CMV/InsWT) could direct synthesis and secretion of insulin, the rat H4-II-E hepatoma cell line was infected with this virus. Sephadex G-50 gel filtration chromatography of the culture medium demonstrated the presence of a single peak of insulin immunoreactive material (Fig. 2A). The mobility of the immunoreactive material on this column was identical to the migration of a proinsulin standard. However, because partially processed insulin (a single cleavage between either the C-A or the B-C junctions) migrates similarly to proinsulin, we cannot rule out the presence of these partially processed insulins. In contrast, infection of the H4-II-E cells with the virus Ad-CMV/InsTb, which encodes for a mutant preproinsulin containing processing sites for ubiquitous constitutive secretory pathway endoproteases, resulted in the production of two immunoreactive species (Fig. 2B). The eluant between fractions 28 and 38 corresponds with the migration of proinsulin or partially processed insulins, whereas the slower eluting protein corresponds with the mobility of fully processed insulin.
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H4-II-E cells infected with Ad-CMV/InsTb produce biologically active insulin. To determine whether the immunoreactive insulin isolated from the infected H4-II-E cells was biologically active, we first determined whether it could bind the insulin receptor. The column fractions corresponding to the migration point of mature insulin were pooled, desalted, and concentrated, and the insulin content was determined by RIA. Insulin receptor binding was then determined using a competition binding assay in which increasing amounts of the isolated material or an insulin standard were used to compete with 125I-monoiodo[A14]insulin for binding to CHO/IR cells, which express the human insulin receptor at high levels (29). Although individual insulin isolates were slightly greater or less effective in competing for 125I-labeled inuslin binding compared with the human insulin standard, in multiple experiments there were no significant differences. Thus these data indicated that the isolated material was able to bind to insulin receptors with a dose-response curve that was statistically indistinguishable from that of the commercial human insulin standard (Fig. 3A).
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On the basis of these data, we next examined the ability of the
isolated material to induce tyrosine phosphorylation of the -subunit
of the insulin receptor. This was accomplished by exposing CHO/IR cells
to increasing concentrations of either the isolated material or the
human insulin standard, solubilizing the cells, and analyzing by
SDS-PAGE followed by antiphosphotyrosine immunoblotting. Although
individual isolates exhibited activity that was somewhat greater or
less than the activity of the insulin standard, the average
dose-response curve of the isolated material was similar to that of an
insulin standard for insulin receptor
-subunit and insulin receptor
substrate-1 tyrosine phosphorylation. A representative blot is shown
(Fig. 3B).
Injection of mice with Ad-CMV/InsWT and with
Ad-CMV/InsTb induces the production of immunoreactive human insulin in
normal mice. Because the recombinant adenoviruses were
able to induce insulin gene expression in cultured hepatocytes, we next
assessed the ability of these viruses to promote insulin production in vivo. Initially, 9-wk-old C57BL/6 mice were injected with
109 plaque-forming units of
AdRSVntLacZ, a recombinant adenovirus encoding for -galactosidase
expression. In situ staining of the liver from these mice demonstrated
a higher level of
-galactosidase expression in >90% of the
hepatocytes compared with mock injected animals (Fig.
4, A and
B). The expression of
-galactosidase was also equally dispersed throughout the liver (data
not shown). Similarly, the livers of mice injected with
109 plaque-forming units of either
AdRSVntLacZ or Ad-CMV/InsTb were examined by immunohistochemistry for
the presence of insulin (Fig. 4, C and
D). The livers from the mice
injected with AdRSVntLacZ had no specific insulin immunoreactivity
(Fig. 4C), whereas the mice injected
with Ad-CMV/InsTb displayed positive immunoreactivity in the majority
of hepatocytes, with some areas having more intense focal staining
(Fig. 4D).
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Consistent with the immunohistochemistry, no human insulin was detected in the sera of the AdRSVntLacZ-injected mice, as determined by an RIA specific for human insulin that has <1% cross-reactivity with mouse insulin (Fig. 5A). In contrast, mice injected Ad-CMV/InsTb displayed significant increases in insulin levels that peaked at ~200 pM 5 days after injection (Fig. 5A). Consistent with these data, RIA indicated an increase in human C-peptide from 5 ± 2.5 to 353 ± 48 pM. Similarly, 5-10 days postinjection with Ad-CMV/InsWT the human insulin levels increased to ~100-125 pM. However, these increases in circulating human insulin levels were transient and returned to baseline levels by 15-20 days for the mice injected with Ad-CMV/InsTb and by 30-35 days for the mice injected with Ad-CMV/InsWT.
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In parallel to the changes in human insulin expression, we observed concomitant alterations in circulating glucose levels (Fig. 5B). The mice injected with Ad-CMV/InsWT had a decrease in blood glucose levels to 93-103 mg/dl between 5 and 15 days postinjection and recovered to baseline values by 20 days. Similarly, glucose levels from the Ad-CMV/InsTb virus-injected animals had a nadir of ~85 mg/dl between 5 and 10 days postinjection, which also recovered to control levels at ~20 days. As expected, mice injected with AdRSVntLacZ had no significant excursions in blood glucose concentration.
Ad-CMV/InsTb can correct the hyperglycemia of diabetic
mice. Because Ad-CMV/InsWT and Ad-CMV/InsTb were able
to induce the synthesis and secretion of immunoreactive insulin in
vivo, we next examined the ability of these recombinant viruses to
improve the metabolic state of diabetic mice (Fig.
6). As observed in the normal mice,
injection of either virus resulted in a transient increase in the
amount of circulating human insulin in the plasma that was maximal at
~8 days and returned to control levels by 25-30 days (Fig.
6A). Mice made diabetic by
destruction of the -cells with streptozotocin had significantly
higher resting glucose levels compared with normal animals (Fig.
6B). However, 8 days postinjection
with Ad-CMV/InsTb the blood glucose levels dramatically decreased from
429 ± 14 to 238 ± 21 mg/dl (Fig.
6B). The decline of glucose levels
persisted for ~12 days at which point it gradually increased back to
the original hyperglycemic state by 30 days. In contrast, mice injected
with Ad-CMV/InsWT experienced only a small improvement in their
glycemic status, despite a nearly identical level of immunoreactive
insulin production, presumably due to the lower bioactivity of
proinsulin versus that of mature fully processed insulin.
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Expression of Ad-CMV/InsTb in vivo results in the processing of the mutant human proinsulin to insulin. To examine the processing of insulin in mice injected with the Ad-CMV/InsTb virus, serum was isolated from diabetic mice and subjected to gel filtration chromatography and insulin RIA (Fig. 7). As observed in the H4-II-E cell line, infection with Ad-CMV/InsTb resulted in the production of two peaks of insulin immunoreactive material that corresponded to the mobility of proinsulin and insulin (Fig. 7A). In contrast, mice infected with Ad-CMV/InsWT were only able to generate the production of proinsulin into the circulation (Fig. 7B). Interestingly, the ability of liver in vivo to process proinsulin to insulin was significantly greater than that observed in the H4-II-E hepatoma cells in vitro. In any case, these data directly demonstrate that, although mouse liver in vivo cannot naturally process human proinsulin into mature human insulin, this can occur upon introduction of two furin cleavage sites into proinsulin.
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Effect of Ad-CMV/InsWT and Ad-CMV/InsTb on liver glycogen content. Under normal steady-state conditions, glycogen accounts for ~10% of the dry weight of liver in mice (Fig. 8A). Surprisingly, injection with the control adenovirus AdRSVntLacZ resulted in a 27-fold decrease in the hepatic glycogen content compared with uninjected mice. Although the livers from diabetic mice had a 6.5-fold decrease in glycogen, adenovirus injection caused a further 3-fold decrease (Fig. 8B). In the nondiabetic animals, injection with Ad-CMV/InsWT or Ad-CMV/InsTb resulted in a partial restoration of liver glycogen (Fig. 8A), whereas only Ad-CMV/InsTb was effective in increasing the glycogen content in the diabetic animals (Fig. 8B).
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DISCUSSION |
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The biosynthesis of mature insulin from proinsulin requires disulfide
bond formation and several proteolytic processing steps (20). The
-cells of the pancreas and several other specialized neuroendocrine
cells express endoproteases (PC2 and PC3) that can cleave peptide bonds
carboxyl to dibasic sequences (34, 35). However, most cell types do not
express these proteases, and several studies have demonstrated that
stable expression of the insulin gene in fibroblasts and in
liver-derived cell lines results in the synthesis of proinsulin (12,
14, 18, 21, 25, 37-39, 42, 43). Consistent with these data, we
have also observed that expression of the human proinsulin cDNA using
recombinant adenovirus (Ad-CMV/InsWT) in the liver cell line H4-II-E
results in the synthesis of proinsulin.
In contrast to PC2 and PC3, most cell types express PACE4 and furin, two endoproteases involved in the constitutive processing of secreted and membrane targeted proteins (1, 4, 22, 28, 31, 32, 40). These enzymes both share the core consensus processing sequence of (R/K)-X-(R/K)-R (10, 37). Many proteins synthesized in the liver contain this processing sequence, which is cleaved before secretion; some examples are human profactors VII (RRRR) and IX (RPKR), factor X (RRKR), protein S (RRRR), complement factors C3 (RRRR) and C4 (RRRR), and the insulin and insulin-like growth factor I receptors (RRKR; see Refs. 8 and 17). We therefore speculated that mutation of the proinsulin processing site at the C-A and B-C junctions (LQKR and KTRR, respectively) to the tetrabasic sequence RRRR would result in the appropriate and more efficient proteolytic cleavage events. In confirmation of this hypothesis, adenovirus expression of the tetrabasic mutant form of proinsulin resulted in the production of both proinsulin and processed insulin as assessed by gel filtration chromatography. Although we did not directly determine whether the tetrabasic RRRR sequence at the B chain carboxyl terminus was removed by carboxypeptidase, this material had identical binding and insulin receptor tyrosine kinase activation properties as authentic mature insulin.
Recently, several studies have also examined the production of insulin from the liver using either retroviral expression in partially hepatectomized rats or by generating transgenic mice carrying the insulin gene under the control of the phosphoenolpyruvate carboxykinase promoter (23, 24, 26, 36). In these studies, expression from either the human or rat insulin 1 cDNA resulted in the predominant production of proinsulin, whereas efficient biosynthesis of insulin was achieved by mutation of the insulin cDNA with furin cleavage sites. Although these expression systems have demonstrated the utility and physiological benefit of low level constitutive insulin production from the liver, neither of these approaches are amenable for use in gene therapy.
On the basis of these data, we explored the potential use of the
recombinant adenovirus expression system as a means for somatic cell
gene transfer of insulin secretion. It has been previously demonstrated
that the major target tissue for adenovirus uptake and expression is
the liver (16, 33). Consistent with these data, in situ
-galactosidase staining and immunohistochemical analysis for insulin
also demonstrated that the primary site of expression was the liver. As
expected, expression of the human proinsulin cDNA in control mice
injected with the Ad-CMV/InsWT virus resulted in the presence of human
proinsulin in the plasma with a small reduction in the circulating
glucose concentration. Similarly, injection with the tetrabasic mutant
proinsulin-carrying virus (Ad-CMV/InsTb) also resulted in a small
decline in glucose levels.
More importantly, injection of diabetic mice with either Ad-CMV/InsWt or Ad-CMV/InsTb not only induced insulin gene expression but also substantially reduced the hyperglycemia of these animals. Although similar amounts of immunoreactive insulin were detected in the serum after infection with either virus, infection with Ad-CMV/InsTb resulted in a larger decline in circulating glucose levels than was seen with Ad-CMV/InsWT infection. Furthermore, insulin expression by Ad-CMV/InsTb also resulted in a greater recovery of liver glycogen than was seen after infection with Ad-CMV/InsWT. These data are consistent with our serum chromatography data showing that mice injected with Ad-CMV/InsTb produce some mature insulin, whereas mice injected with Ad-CMV/InsWT only generate proinsulin, which has a lower biological potency than insulin.
Notably, the ability of these recombinant adenoviruses to induce insulin production in vivo was transient and typically lasted 15-20 days postinfection. The transient nature of adenovirus-mediated gene expression probably results from an immune response directed against viral proteins that are expressed at low levels after infection by the recombinant viruses (7, 44). We are currently testing methods to specifically suppress the immune response to adenovirus and/or to induce the development of tolerance to the adenoviral proteins. Even though the liver does not display regulated exocytosis, the production of a continuous basal level of insulin would be useful by preventing ketoacidic episodes and thereby eliminating the need for long-acting insulin injections.
In conclusion, this study has demonstrated that introduction into the human proinsulin cDNA of tetrabasic processing sites for constitutive endoproteases results in the biosynthesis and constitutive secretion of mature insulin both in vitro and in vivo. In addition, somatic cell gene transfer using the recombinant replication-deficient adenovirus encoding this mutant human insulin cDNA can reduce, at least transiently, the hyperglycemia of diabetic mice to near normal values.
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ACKNOWLEDGEMENTS |
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We thank Drs. Graeme Bell and Christopher Newgard for providing the human proinsulin and the type 5 adenovirus vector plasmids.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Research Grants DK-49012 and DK-25295.
Present address of K. Yamauchi: Dept. of Endocrinology and Geriatrics, Shinshu University School of Medicine, Nagano 390, Japan.
Address for reprint requests: J. E. Pessin, Dept. of Physiology and Biophysics, The University of Iowa, Iowa City, IA 52242-1109.
Received 31 October 1997; accepted in final form 27 July 1998.
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