Adenovirus-mediated transfer of a modified human proinsulin gene reverses hyperglycemia in diabetic mice

Daniel K. Short, Shuichi Okada, Keishi Yamauchi, and Jeffrey E. Pessin

Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242-1109

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

INSULIN IS SYNTHESIZED in the beta -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 beta -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 beta -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 beta -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.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 beta -galactosidase (AdRSVntLacZ), a type 5 adenovirus containing a nuclear-targeted form of beta -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'-CTGCAGGTCCTCT<UNL>CG</UNL>C<UNL>CG</UNL>CCGGCGGGTCT-3' to mutate the B-C junction and the oligo 5'-CACAATGCCACGC<UNL>C</UNL>TC<UNL>C</UNL>GC<UNL>C</UNL>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 <UNL>proins</UNL>ulin with <UNL>t</UNL>etra<UNL>b</UNL>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.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   Amino acid sequence differences between wild-type human proinsulin and the mutant human proinsulin containing the tetrabasic processing sites. Amino acids in bold type were changed by site-directed mutagenesis as described in METHODS. Introduction of these residues converts the endoprotease prohormone convertase (PC) 2 and PC3 cleavage sites to furin processing sites as indicated by the arrows. Amino acids in shaded boxes will be removed by carboxypeptidase E/H after cleavage by the processing enzyme. proins-Tb, human proinsulin with sequence modifications in the processing sites that convert the dibasic sites into tetrabasic sites.

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 beta -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 beta -galactosidase staining was performed by fixing the cryosectioned liver with 1% glutaraldehyde and incubating with 0.2% 5-bromo-4-chloro-3-indolyl beta -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.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   Infection of H4-II-E hepatoma cells with Ad-CMV/InsWT [recombinant adenovirus containing the wild-type human proinsulin gene under control of the cytomegalovirus (CMV) promoter] and Ad-CMV/InsTb (recombinant adenovirus containing the gene for a mutant insulin cDNA in which the dibasic amino acids at the C-A and B-C junctions were changed to tetrabasic sites under the control of the CMV promoter) directs the biosynthesis of proinsulin and insulin, respectively. H4-II-E cells were infected with the wild-type (A) or tetrabasic mutant (B) human proinsulin encoding recombinant adenoviruses, and the cell culture medium was collected over a 24-h period as described in METHODS. Medium (50 ml) was lyophilized, resuspended in 2 ml column buffer, and loaded on Sephadex G-50 Super Fine columns (1.0 × 120 cm). Column fractions (1.5 ml) were collected and assayed for insulin content by radioimmunoassay. Mobility of authentic insulin (I; mol wt = 5,800) and proinsulin (PI; mol wt = 9,400) are indicated by arrows. This is a representative experiment independently performed 2 times.

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).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   Competition of insulin binding and tyrosine kinase activity. H4-II-E cells were infected with AdCMV/InsTb at a multiplicity of infection of 10. One liter of DMEM-0.01% BSA was incubated for 12 h over the cells, concentrated, and passed over 1.0 × 120-cm G-50 superfine columns. Fractions 40-48 (corresponding to the migration point of a human insulin standard) were collected, desalted by dialysis, lyophilized, and resuspended in minimal Eagle's medium containing nucleotides. A: insulin receptor competition assay. Confluent monolayers of Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) were incubated with 50,000 counts/min 125I-monoiodo[A14]insulin and with the indicated concentrations of unlabeled insulin (insulin isolate or human insulin standard). Results are expressed as %bound (B)/unbound (B0). B: antiphosphotyrosine blotting. Confluent monolayers of CHO/IR cells were incubated with the indicated concentrations of insulin isolate or human insulin standard. Cells were solubilized, subjected to SDS-PAGE (7%), and subsequently transferred to a polyvinylidene difluoride membrane and exposed to PY-20-HRP (antiphosphotyrosine) antibody. Detection was by enhanced chemiluminescence (Supersignal Chemiluminescent Substrate, Pierce Chemical, Rockford, IL). Lanes 1-6, cells were exposed to 0, 1, 3, 10, 30, and 100 nM of human insulin standard, respectively. Lanes 7-12, cells were exposed to 0, 1, 3, 10, 30, and 100 nM of insulin isolate, respectively. IR, insulin receptor beta -subunit; IRS-1, insulin receptor substrate-1.

On the basis of these data, we next examined the ability of the isolated material to induce tyrosine phosphorylation of the beta -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 beta -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 beta -galactosidase expression. In situ staining of the liver from these mice demonstrated a higher level of beta -galactosidase expression in >90% of the hepatocytes compared with mock injected animals (Fig. 4, A and B). The expression of beta -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).


View larger version (104K):
[in this window]
[in a new window]
 
Fig. 4.   Injection of mice with Ad-CMV/InsTb results in insulin gene expression in liver cells. Mice were mock injected (A and C) or injected with 109 plaque-forming units (pfu) of either AdRSVntLacZ (B) or Ad-CMV/InsTb (D) as described in METHODS. Four days later, the mice were anesthetized and perfused with 4% paraformaldehyde. Livers were removed, cryosectioned, and either stained for the presence of beta -galactosidase (LacZ, A and B) or incubated with an anti-insulin antibody followed by incubation with a gold-linked secondary antibody and development with silver enhancement (InsTb, C and D) as described in METHODS. Magnification ×160.

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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of Ad-CMV/InsWT and Ad-CMV/InsTb on the production of human insulin and circulating glucose levels in normal mice. Control mice were injected with 109 pfu of the beta -galactosidase expressing recombinant adenovirus (AdRSVntLacZ; black-triangle), the wild-type human proinsulin recombinant adenovirus (Ad-CMV/InsWT; bullet ), and the mutant tetrabasic human proinsulin recombinant adenovirus (Ad-CMV/InsTb; ) as described in METHODS. Serum insulin concentrations (A) were determined by a human insulin specific radioimmunoassay, and blood glucose levels (B) were determined with a One-Touch-II glucometer. Error bars represent means ± SE. This is a representative experiment independently performed 3 times; n = 4 for each group of mice.

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 beta -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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of Ad-CMV/InsWT and Ad-CMV/InsTb on the production of human insulin and circulating glucose levels in diabetic mice. Streptozotocin-induced diabetic mice were injected with 109 pfu of the beta -galactosidase expressing recombinant adenovirus (AdRSVntLacZ; black-triangle), the wild-type human proinsulin recombinant adenovirus (Ad-CMV/InsWT; bullet ), and the mutant tetrabasic human proinsulin recombinant adenovirus (Ad-CMV/InsTb; ) as described in METHODS. Serum insulin concentrations (A) were determined by a human insulin specific radioimmunoassay, and blood glucose levels (B) were determined with with a One-Touch-II glucometer. This is a representative experiment independently performed 2 times. Error bars represent means ± SE. Ad-CMV/InsTb mice: n = 17 for days 0, 4, 8, and 12; n = 16 for day 16; n = 15 for day 20; n = 13 for day 24; n = 5 for day 30. Ad-CMV/InsWT mice: n = 12 for days 0, 4, 8, and 12; n = 11 for days 16 and 20; n = 10 for day 24; n = 5 for day 30.

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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7.   Injection of diabetic mice with Ad-CMV/InsTb results in the production of mature human insulin. A: 8 streptozotocin-induced diabetic mice were injected with 109 pfu each of the tetrabasic mutant human proinsulin recombinant adenovirus (Ad-CMV/InsTb). B: 8 streptozotocin-induced diabetic mice were injected with 109 pfu each of the wild-type human proinsulin recombinant adenovirus (Ad-CMV/InsWT). Five days later the sera were harvested and pooled, and aliquots were separated on a Sephadex G-50 Super Fine column (1.0 × 120 cm). Column fractions (1.5 ml) were collected and assayed for insulin content by a radioimmunoassay specific for human insulin, as described in METHODS. Mobility of authentic insulin (mol wt = 5,800) and proinsulin (mol wt = 9,400) are indicated by arrows.

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).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of adenovirus infection on liver glycogen content in control and diabetic mice. Normal (A) and streptozotocin (STZ)-induced diabetic (B) mice were mock injected (filled bars) or injected with 109 pfu of AdRSVntLacZ (hatched bars), Ad-CMV/InsWT (checkered bars), or Ad-CMV/InsTb (open bars) as described in METHODS. Five days after infection the animals were killed, and the content of liver glycogen was determined. Error bars represent means ± SE; n = 4 mice/group.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The biosynthesis of mature insulin from proinsulin requires disulfide bond formation and several proteolytic processing steps (20). The beta -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 beta -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.

    ACKNOWLEDGEMENTS

We thank Drs. Graeme Bell and Christopher Newgard for providing the human proinsulin and the type 5 adenovirus vector plasmids.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Alarcon, C., B. Cheatham, B. Lincoln, C. R. Kahn, K. Siddle, and C. J. Rhodes. A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor. Biochem. J. 301: 257-265, 1994[Medline].

2.   Barr, P. J. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell 66: 1-3, 1991[Medline].

3.   Becker, T. C., R. J. Noel, W. S. Coats, A. M. Gomez-Foix, T. Alam, R. D. Gerard, and C. B. Newgard. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 43: 161-189, 1994[Medline].

4.   Brakch, N., A. S. Galanopoulou, Y. C. Patel, G. Boileau, and N. G. Seidah. Comparative proteolytic processing of rat prosomatostatin by the convertases PC1, PC2, furin, PACE4 and PC5 in constitutive and regulated secretory pathways. FEBS Lett. 362: 143-146, 1995[Medline].

5.   Chen, S. H., H. D. Shine, J. C. Goodman, R. G. Grossman, and S. L. Woo. Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. Proc. Natl. Acad. Sci. USA 91: 3054-3057, 1994[Abstract].

6.   Duckworth, W. C., D. E. Peavy, F. G. Hamel, J. Liepnieks, M. R. Brunner, R. E. Heiney, and B. H. Frank. Conversion of biosynthetic human proinsulin to partially cleaved intermediates by collagenase proteinases adsorbed to isolated rat adipocytes. Biochem. J. 255: 277-284, 1988[Medline].

7.   Engelhardt, J. F., X. Ye, B. Doranz, and J. M. Wilson. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. USA 91: 6196-6200, 1994[Abstract].

8.   Fang, B., R. C. Eisensmith, H. Wang, M. A. Kay, R. E. Cross, C. N. Landen, G. Gordon, D. A. Bellinger, M. S. Read, P. C. Hu, K. M. Brinkhous, and S. L. C. Woo. Gene therapy for hemophilia B: host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression. Hum. Gene Ther. 6: 1039-1044, 1995[Medline].

9.   Galloway, J. A., S. A. Hooper, C. T. Spradlin, D. C. Howey, B. H. Frank, R. R. Bowsher, and J. H. Anderson. Biosynthetic human proinsulin. Review of chemistry, in vitro and in vivo receptor binding, animal and human pharmacology studies, and clinical trial experience. Diabetes Care 15: 666-692, 1992[Abstract].

10.   Gotoh, B., Y. Ohnishi, N. M. Inocencio, E. Esaki, K. Nakayama, P. J. Barr, G. Thomas, and Y. Nagai. Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3. J. Virol. 66: 6391-6397, 1992[Abstract].

11.   Groskreutz, D. J., M. X. Sliwkowski, and C. M. Gorman. Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin. J. Biol. Chem. 269: 6241-6245, 1994[Abstract/Free Full Text].

12.   Halban, P. A. Proinsulin trafficking and processing in the pancreatic B cell. Trends Endocrinol. Metab. 1: 261-265, 1990.

13.   Halban, P. A. Structural domains and molecular lifestyles of insulin and its precursors in the pancreatic beta cell. Diabetologia 34: 767-778, 1991[Medline].

14.  Halban, P. A. Proinsulin processing in the regulated and the constitutive secretory pathway. Diabetologia 37, Suppl.: S65-S72, 1994.

15.   Hassid, W. Z., and S. Abraham. Chemical procedures for analysis of polysaccharides. Methods Enzymol. 1: 34-38, 1957.

16.   Herz, J., and R. D. Gerard. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc. Natl. Acad. Sci. USA 90: 2812-2816, 1993[Abstract].

17.   Hosaka, M., M. Nagahama, W. S. Kim, T. Watanabe, K. Hatsuzawa, J. Ikemizu, K. Murakami, and K. Nakayama. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J. Biol. Chem. 266: 12127-12130, 1991[Abstract/Free Full Text].

18.   Irminger, J. C., F. M. Vollenweider, M. Neerman-Arbez, and P. A. Halban. Human proinsulin conversion in the regulated and the constitutive pathways of transfected AtT20 cells. J. Biol. Chem. 269: 1756-1762, 1994[Abstract/Free Full Text].

19.   Kakita, K., M. Horino, A. Tenku, S. Nishida, S. Matsumura, M. Matsuki, and S. Kakita. Gel chromatographic separation of human C-peptide and proinsulin. J. Chromatogr. Sci. 222: 33-40, 1981.

20.   Karam, J. K., and P. H. Forsham. Pancreatic hormones and diabetes mellitus. In: Basic and Clinical Endocrinology (4th ed.), edited by F. S. Greenspan, and J. D. Baxter. Norwalk, CT: Appleton & Lange, 1994, p. 571-584.

21.   Kaufmann, J. E., J. C. Irminger, and P. A. Halban. Sequence requirements for proinsulin processing at the B-chain/C-peptide junction. Biochem. J. 310: 869-874, 1995[Medline].

22.   Kiefer, M. C., J. E. Tucker, R. Joh, K. E. Landsberg, D. Saltman, and P. J. Barr. Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15. DNA Cell Biol. 10: 757-769, 1991[Medline].

23.   Kolodka, T. M., M. Finegold, L. Moss, and S. L. Woo. Gene therapy for diabetes mellitus in rats by hepatic expression of insulin. Proc. Natl. Acad. Sci. USA 92: 3293-3297, 1995[Abstract].

24.   Mitanchez, D., R. Chen, J. F. Massias, A. Porteu, A. Mignon, X. Bertagna, and A. Kahn. Regulated expression of mature human insulin in the liver of transgenic mice. FEBS Lett. 421: 285-289, 1998[Medline].

25.   Moore, H. P., M. D. Walker, F. Lee, and R. B. Kelly. Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation. Cell 35: 531-538, 1983[Medline].

26.   Muzzin, P., R. C. Eisensmith, K. C. Copeland, and S. L. Woo. Hepatic insulin gene expression as treatment for type 1 diabetes mellitus in rats. Mol. Endocrinol. 11: 833-837, 1997[Abstract/Free Full Text].

27.   Naggert, J. K., L. D. Fricker, O. Varlamov, P. M. Nishina, Y. Rouille, D. F. Steiner, R. J. Carroll, B. J. Paigen, and E. H. Leiter. Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nat. Genet. 10: 135-142, 1995[Medline].

28.   Nakagawa, T., M. Hosaka, S. Torii, T. Watanabe, K. Murakami, and K. Nakayama. Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4. J. Biochem. (Tokyo) 113: 132-135, 1993[Abstract].

29.   Okada, S., K. Yamauchi, and J. E. Pessin. Shc isoform-specific tyrosine phosphorylation by the insulin and epidermal growth factor receptors. J. Biol. Chem. 270: 20737-20741, 1995[Abstract/Free Full Text].

30.   Orci, L., J. D. Vassalli, and A. Perrelet. The insulin factory. Sci. Am. 259: 85-94, 1988[Medline].

31.   Rehemtulla, A., P. J. Barr, C. J. Rhodes, and R. J. Kaufman. PACE4 is a member of the mammalian propeptidase family that has overlapping but not identical substrate specificity to PACE. Biochemistry 32: 11586-11590, 1993[Medline].

32.   Rehemtulla, A., and R. J. Kaufman. Protein processing within the secretory pathway. Curr. Opin. Biotechnol. 3: 560-565, 1992[Medline].

33.   Smith, T. A., M. G. Mehaffey, D. B. Kayda, J. M. Saunders, S. Yei, B. C. Trapnell, A. McClelland, and M. Kaleko. Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Nat. Genet. 5: 397-402, 1993[Medline].

34.  Steiner, D. F., and D. E. James. Cellular and molecular biology of the beta cell. Diabetologia 35, Suppl.: S41-S48, 1992.

35.   Steiner, D. F., S. P. Smeekens, S. Ohagi, and S. J. Chan. The new enzymology of precursor processing endoproteases. J. Biol. Chem. 267: 23435-23438, 1992[Free Full Text].

36.   Valera, A., C. Fillat, C. Costa, J. Sabater, J. Visa, A. Pujol, and F. Bosch. Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations. FASEB J. 8: 440-447, 1994[Abstract/Free Full Text].

37.   Vollenweider, F., J. C. Irminger, D. J. Gross, L. Villa-Komaroff, and P. A. Halban. Processing of proinsulin by transfected hepatoma (FAO) cells. J. Biol. Chem. 267: 14629-14636, 1992[Abstract/Free Full Text].

38.   Vollenweider, F., J. C. Irminger, and P. A. Halban. Substrate specificity of proinsulin conversion in the constitutive pathway of transfected FAO (hepatoma) cells. Diabetologia 36: 1322-1325, 1993[Medline].

39.   Vollenweider, F., J. Kaufmann, J. C. Irminger, and P. A. Halban. Processing of proinsulin by furin, PC2, and PC3 in (co) transfected COS (monkey kidney) cells. Diabetes 44: 1075-1080, 1995[Abstract].

40.   Wasley, L. C., A. Rehemtulla, J. A. Bristol, and R. J. Kaufman. PACE/furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway. J. Biol. Chem. 268: 8458-8465, 1993[Abstract/Free Full Text].

41.   Yamauchi, K., and J. E. Pessin. Insulin receptor substrate-1 (IRS1) and Shc compete for a limited pool of Grb2 in mediating insulin downstream signaling. J. Biol. Chem. 269: 31107-31114, 1994[Abstract/Free Full Text].

42.   Yanagita, M., H. Hoshino, K. Nakayama, and T. Takeuchi. Processing of mutated proinsulin with tetrabasic cleavage sites to mature insulin reflects the expression of furin in nonendocrine cell lines. Endocrinology 133: 639-644, 1993[Abstract].

43.   Yanagita, M., K. Nakayama, and T. Takeuchi. Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7. FEBS Lett. 311: 55-59, 1992[Medline].

44.   Yang, Y., F. A. Nunes, K. Berencsi, E. Gonczol, J. F. Engelhardt, and J. M. Wilson. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat. Genet. 7: 362-369, 1994[Medline].


Am J Physiol Endocrinol Metab 275(5):E748-E756
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society