Hepatic Insulin Gene Expression as Treatment for Type 1 Diabetes Mellitus in Rats
Patrick Muzzin1,
Randy C. Eisensmith2,
Kenneth C. Copeland and
Savio L. C. Woo2
Department of Cell Biology (P.M., R.C.E., S.L.C.W.), Department
of Pediatrics (K.C.C.), Howard Hughes Medical Institute
(S.L.C.W.), Baylor College of Medicine, Houston Texas 77030
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ABSTRACT
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Type 1 diabetes mellitus is caused by a lack
of insulin that results from the autoimmune destruction of the
pancreatic ß-cells. Severe diabetes, if not controlled by periodic
insulin injections, can lead to ketoacidosis and death. We have
previously shown that sustained low level production of insulin in the
liver of diabetic rats prevented their death from complications of
diabetes. To test the hypothesis that there is a window of serum
insulin concentrations that can prevent ketoacidosis without
significant risk of hypoglycemia secondary to hyperinsulinemia, rats
were infused with various doses of a recombinant retrovirus encoding an
engineered rat preproinsulin-1 gene. The gene was engineered to allow
processing into mature insulin by the protease furin. At the lower
doses tested, fatal ketoacidosis was prevented, but the rats exhibited
nonfasting hyperglycemia. At intermediate doses, which resulted in
serum insulin concentrations of 1.6 mg/ml, the rats achieved
near-normoglycemia and no serum ketones. These rats did not exhibit
hypoglycemia even during a 24-h fast. At high virus doses, the animals
achieved nonfasting normoglycemia but exhibited hypoglycemia during the
fast. In conclusion, we have defined a therapeutic window of hepatic
insulin expression that provides protection against ketoacidosis
without significant risk of hypoglycemia. This window of sustained
hepatic insulin expression might permit its development into a novel
treatment modality for the prevention of ketoacidosis in patients with
severe insulin-dependent diabetes mellitus.
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INTRODUCTION
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Insulin-dependent diabetes mellitus (IDDM) results from the
autoimmune destruction of the insulin-producing ß-cells of the
pancreas. In IDDM, the deficiency of insulin leads to wasting,
hyperglycemia, and death from ketoacidosis (1, 2). The present
treatment for IDDM involves frequent monitoring of blood glucose and
lifelong insulin injection. To minimize hyperglycemia and to ensure the
avoidance of ketoacidosis, intensive diabetes management strategies and
vigorous patient compliance are necessary. The long-term intensive
management has proven to be difficult for some patients, especially
those with very low C peptide levels.
To address these problems, a gene therapy treatment strategy for IDDM
was investigated. As the pancreatic ß-cells have been destroyed,
reconstituted insulin expression was directed into an ectopic organ.
The liver provides an excellent target organ, since it is the principal
effector for glucose homeostasis and ketogenesis. A recombinant
retroviral vector was chosen for transfer of the insulin gene into
hepatocytes. This vector permits stable and persistent transgene
expression in hepatocytes without cytotoxicity. However, as
retrovirus-based vectors can only transduce actively dividing cells,
division of normally quiescent hepatocytes must be stimulated by
surgical partial hepatectomy before retrovirus infusion.
We have previously demonstrated that retrovirus-mediated transfer of
the rat preproinsulin-1 gene into hepatocytes resulted in sustained
levels of insulin expression that were sufficient to prevent
ketoacidosis in diabetic rats (3). To further develop this strategy as
a potential new treatment modality for IDDM, we hypothesize that there
is a therapeutic range of hepatic insulin expression that can prevent
ketoacidosis and death in diabetic rats without significant risk of
hypoglycemia secondary to hyperinsulinemia. This hypothesis was tested
in rats with IDDM induced by administration of high-dose streptozotocin
(STZ).
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RESULTS
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In the first part of this study, we examined the effects of the
infusion of LX/erINS on serum ketone and blood glucose levels in
STZ-treated rats. After a partial hepatectomy, rats were infused with
2 x 106 colony-forming units (cfu) of recombinant
retrovirus encoding either LX/erINS, LX/ß-geo, or medium. Fourteen
days later, LX/ß-geo- and LX/erINS-treated rats were injected with
STZ. As shown in Fig. 1
, at 3 days after STZ treatment,
the LX/ß-geo-treated rats lost 20% of their body weight (120.2
± 6.8 g vs. 149.4 ± 2.4 g, n = 13),
while the weight of the LX/erINS-treated rats remained constant
(142.4 ± 6.5 g vs. 141.4 ± 2.3 g,
n = 10). Ten days after STZ administration, the body weights of
the LX/erINS-treated rats were only 11% less than those of control
rats injected with buffer (Fig. 1
). The body weight gain in
LX/erINS-treated rats is in agreement with previous reports that
insulin expression from the livers of transgenic mice did not cause
abnormal biology (4) and that insulin can act on the liver to promote
body growth (5). Within 17 days of treatment, all 13 LX/ß-geo-treated
rats died, whereas all LX/erINS-treated rats were still alive (Fig. 2
).

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Figure 1. Effects of STZ Treatment on Body Weight in Rats
Twenty-four hours after partial hepatectomy, rats were infused with
2 x 106 cfu of either LX/erINS (), LX/b-geo ( ),
or medium ( ). At day 14, rats infused with the recombinant
retroviruses were treated with STZ. Medium-infused rats were injected
with the STZ buffer. Results are expressed as the mean ±
SEM. For the LX/erINS-treated rats, n = 10; for the
LX/ßgeo-treated rats, n = 13 at day 0 and n = 4 at 25 days;
for the medium-treated rats, n = 5.
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Figure 2. Percent Survival of Rats Infused with 2 x
106 cfu of Either LX/erINS () or LX/ßgeo ( ) after
STZ Treatment
For the LX/erINS-treated rats, n = 10; for the LX/ßgeo-treated
rats, n = 13.
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To confirm that the treatment of the rats with a high dose of STZ
resulted in near total ablation of pancreatic ß-cells,
immunohistological staining of pancreatic sections with antibodies
against insulin was performed in both LX/erINS- and LX/ß-geo-treated
rats. Twelve days after STZ treatment, only one or two insulin-positive
cells per islet were observed in 20% of the islets. The remaining
islets were negative (data not shown). Serum insulin levels in
LX/ß-geo-treated rats were decreased to below 0.1 ng/ml after STZ
treatment. Thus, the administration of a high dose of STZ caused a
near-total destruction of pancreatic ß-cells in all treated rats.
Sera from rats transduced with 2 x 106 cfu of a
recombinant retrovirus encoding either LX/erINS or LX/ß-geo were
assayed for immunoreactive insulin by RIA. Before retrovirus infusion,
nonfasting serum insulin levels were 0.9 ± 0.2 ng/ml and 0.8
± 0.1 ng/ml in LX/erINS- and LX/ß-geo-treated rats, respectively
(Fig. 3
). Ten days after retrovirus infusion, serum
insulin levels increased to 2.1 ± 0.4 ng/ml in the
LX/erINS-treated rats, whereas it did not change significantly in the
control rats. Three and 10 days after STZ injection (at days 18 and 25
in Fig. 3
), insulin concentrations fell to undetectable levels in the
control rats. However, rats treated with LX/erINS maintained serum
insulin at concentrations of 1.6 ± 0.4 ng/ml and 1.5 ± 0.3
ng/ml 3 and 10 days after induction of diabetes, respectively. These
results indicate that near-physiological levels of immunoreactive
insulin produced by the liver can prevent the lethal consequences of
diabetes in rats.

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Figure 3. Serum Insulin Levels in Rats Transduced with 2
x 106 cfu of Either LX/erINS ( ) or LX/ßgeo ( )
Serum insulin concentrations were determined in treated rats after
retrovirus infusion. Diabetes was induced at day 14 after virus
infusion. Eighteen days and 25 days after STZ treatment, serum insulin
levels in the LX/ßgeo-transduced rats were below the limit of
sensitivity of the assay (0.1 ng/ml). The results are expressed as the
mean ± SEM. For the LX/erINS-treated rats, n =
10; for the LX/ßgeo-treated rats, n = 13.
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Because of the slightly higher serum insulin level in the
LX/erINS-treated rats, as compared with normal nonfasting values
(1.5 ± 0.3 ng/ml. vs. 0.9 ± 0.2 ng/ml), a 24-h
blood glucose profile was established under nonfasting conditions.
Before induction of diabetes, blood glucose levels remained constant at
approximately 100 mg/ml throughout the 24-h period. After STZ
treatment, LX/ß-geo-treated rats had blood glucose levels constantly
higher than 250 mg/dl for the entire 24-h period, whereas
LX/erINS-treated rats had reduced blood glucose levels varying from 121
to 178 mg/dl (Fig. 4
).

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Figure 4. Twenty Four-Hour Blood Glucose Profiles
Nonfasting blood glucose levels were determined at 3-h intervals in
rats transduced with 2 x 106 cfu of either LX/erINS
(), LX/ßgeo ( ), or medium ( ). Two weeks after retrovirus
infusion, LX/erINS- and LX/ßgeo-transduced rats were treated with
STZ; the medium-infused rats were injected with the STZ buffer. Seven
days later, blood glucose levels were measured in the experimental
animals. The results are expressed as the mean ± SEM.
For the LX/erINS-treated rats, n = 10; for the LX/ßgeo-treated
rats, n = 9; for the medium-injected rats, n = 5.
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To illustrate that there is a window of insulin expression in the liver
that can prevent ketoacidosis without significant risk of hypoglycemia
secondary to hyperinsulinemia, rats were infused with the concentrated
LX/erINS virus at 2 x 107 cfu, or dilutions
corresponding to 6 x 106 cfu, 2 x
106 cfu, 6 x 105 cfu, and 2 x
105 cfu. The doses used covered 1 order of magnitude both
above and below the initial dose of LX/erINS infused into the animals.
To achieve these higher doses, LX/erINS was concentrated by low-speed
centrifugation. It has been shown that the recovery of virus
concentrated by this technique is greater than 90% and that transgene
expression increases almost linearly with increased virus doses
(6).
As shown in Fig. 5
, 10 days after induction of diabetes,
blood ketones were high in the LX/ß-geo-treated rats (47 ± 6
mg/dl), while no ketones were measured in 5070% of rats transduced
with 2 x 105-2 x 106 cfu of
LX/erINS; the remainder of the rats in these treatment groups had low
levels of ketones (below 15 mg/dl). All rats treated with 6 x
106 or 2 x 107 cfu of LX/erINS had no
serum ketones. However, three of five rats transduced with 2 x
107 cfu of LX/erINS died 4872 h after virus infusion,
presumably from hypoglycemia. The serum insulin levels in the two
surviving rats treated with 2 x 107 cfu LX/erINS were
2.6 ng/ml; serum insulin levels in the six rats treated with 6 x
106 cfu of the virus were 3.7 ± 0.8 ng/ml.

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Figure 5. Serum Ketone Levels of Rats after Induction of
Diabetes
Rats were transduced with various doses of LX/erINS, and ketone levels
were determined 10 days after STZ treatment. For comparison, ketone
levels in LX/ßgeo-treated rats (2 x 106 cfu) are
shown. The results are expressed as the mean ± SEM.
For the LX/erINS-treated groups: n = 2 for 2 x
107 cfu/rat; n = 6 for 6 x 106
cfu/rat; n = 10 for 2 x 106 cfu/rat; n = 10
for 6 x 105 cfu/rat; n = 6 for 2 x
105 cfu/rat. For the LX/ßgeo-treated group, n = 5.
Three days after retrovirus infusion, three rats transduced with 2
x 107 cfu died of hypoglycemia. Serum insulin was measured
in one of these animals and was found to be high (6.7 ng/ml).
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Since near-normoglycemia was achieved in some of the LX/erINS-treated
rats under nonfasting conditions, we next tested whether these animals
developed hypoglycemia upon fasting. Ten days after induction of
diabetes, rats transduced with either LX/ß-geo, 2 x
105 cfu, or 6 x 105 cfu of LX/erINS had
blood glucose levels that were elevated to greater than 250 mg/dl
during the first 12 h of the fast, but then decreased rapidly over
the next 12 h (Fig. 6
). In the rats treated with
6 x 106 or 2 x 107 LX/erINS,
nonfasting blood glucose levels were 128 mg/dl and 106 mg/dl,
respectively. Upon fasting, however, blood glucose levels of both
groups of LX/erINS-treated rats decreased to hypoglycemic levels (below
50 mg/dl) within the first 6 h. Blood glucose levels in the
animals receiving 2 x 106 cfu of the
insulin-expressing vector slowly decreased to normoglycemic levels
within 24 h, with no evidence of hypoglycemia. These results are
consistent with those of our previous study showing that normoglycemia
was achieved within 4 h of the fast and remained in this range for
20 h in rats treated with a recombinant retrovirus encoding the
wild type rat preproinsulin-1 gene (3) and suggest that this dose is
the upper limit for hepatically expressed insulin.
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DISCUSSION
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We have previously shown that sustained low hepatic insulin
expression has potential in the treatment of IDDM, preventing
ketoacidosis and death in diabetic rats (3). The current study has
examined whether a sufficiently wide therapeutic window of hepatic
insulin expression exists that can prevent ketoacidosis and death or
hyperinsulinemia and hypoglycemia at the two extremes. Using a rat
model of STZ-induced IDDM, we have shown that hepatic expression of
insulin after transduction with a recombinant retrovirus encoding an
engineered rat insulin molecule can lead to the production of
sufficient insulin levels to prevent ketoacidosis with no danger of
hypoglycemia under fasting conditions. At the other extreme, we
anticipated that at very low doses of insulin, some diabetic rats would
develop ketoacidosis. In fact, despite the fact that insulin levels
were 0.2 ± 0.2 ng/ml and below 0.1 ng/ml in rats treated with
6 x 105 or 2 x 105 cfu of LX/erINS,
respectively, serum ketone levels were low and ketoacidosis did not
develop. Our results also show that even if rats are transduced with
low doses of LX/erINS, which results in hyperglycemia, a window of
serum insulin concentrations between 0.1 and 1.6 ng/ml can prevent
ketoacidosis in this animal model of diabetes.
Previous studies have shown that hepatically produced native rat
preproinsulin-1 was apparently not fully processed to mature insulin,
as STZ-treated animals exhibited nonfasting mild hyperglycemia (serum
glucose levels of 200 mg/dl) even at serum immunoreactive insulin
concentrations of 1015 mg/ml (3). Hepatocytes have a constitutive
secretory pathway and process secreted proteins by the protease furin
(7). In the present study, mild nonfasting hyperglycemia (serum
glucose = 178 ± 39 mg/dl) was achieved in the
LX/erINS-treated rats with much reduced serum immunoreactive insulin
levels (1.6 ± 0.4 ng/ml in the LX/erINS-treated animals
vs. 0.9 ± 0.2 ng/ml in normal rats). This finding
suggests that the engineered insulin was processed and biologically
active.
An alternative to the present strategy that will constitutively express
higher levels of insulin without risking low glucose levels will
require the regulated synthesis of insulin. The promoter of the
L-pyruvate kinase gene, which is transcriptionally regulated by glucose
(8, 9), might modulate insulin expression in hepatocytes in
vivo. However, since the higher levels of insulin in our animal
model of diabetes caused hypoglycemia within the first 6 h of
fast, rapid and acute regulation of insulin synthesis will be
necessary. Nevertheless, our demonstration of the existence of a window
of hepatic insulin expression that prevents acute ketoacidosis should
permit the future development of a novel treatment for diabetic
patients by insulin gene therapy.
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MATERIALS AND METHODS
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Engineering of the Rat Preproinsulin-1 cDNA Construction
A full-length rat preproinsulin-1 cDNA clone generated by PCR
was described previously by Kolodka et al. (3). PCR
mutagenesis, using the megaprimer method (10), was performed to alter
the B-C junction, from Lys-Ser-Arg-Arg to Arg-Ser-Lys-Arg, which is the
consensus sequence recognized and cleaved by furin, an abundant
protease in the liver (7). Mutant colonies were screened for the
presence of a SacII site (introduced by the mutagenesis),
and positive clones were fully sequenced using a DyeDeoxy Terminator
Cycle Sequencing Kit (Perkin Elmer, Norwalk, CT) to confirm the
presence of the furin recognition sequence. The A-C junction of the rat
pre-proinsulin gene (Arg-Glu-Lys-Arg) already conformed to the furin
recognition sequence and therefore required no engineering.
Construction of a Recombinant Retrovirus Vector Encoding the
Engineered Rat Preproinsulin-1 Gene
A plasmid, pLX/erINS, encoding the 5'-long terminal repeat, the
engineered rat preproinsulin-1 cDNA, and the 3'-long-terminal repeat
was constructed and used to transfect the retrovirus packaging cell
line GPAM-12. Individual colonies were isolated and screened for their
ability to induce insulin production in rat fibroblast 208F cells. A
clone producing 150 ng of immunoreactive rat insulin in the conditioned
medium per 106 cells per day was selected to transduce rat
hepatocytes in vivo. Viral titers of LX/ß-geo were
determined to be 5 x 105 cfu/ml by transduction of
208F fibroblasts followed by X-gal staining. The titer of LX/erINS was
estimated at 1 x 106 cfu/ml.
Concentration of Retrovirus
Recombinant retrovirus was concentrated by low-speed
centrifugation as described (6). Briefly, the supernatant of the virus
producer cell line GPAM-12 was filtered through a 0.45-µm filter and
centrifuged at 6000 x g for 16 h at 4 C. The
virus pellet was gently resuspended in 0.1 volume of culture media to
produce a 10-fold concentration. The concentrated virus suspension was
filtered through a 0.45-µm filter and stored at -70 C. Measurement
of insulin production from fibroblast 208F cells transduced with the
concentrated virus indicated that the recovery was greater than
90%.
Retrovirus Transduction of Rat Hepatocytes in Vivo
and Induction of Diabetes
Various doses of LX/erINS or LX/ß-Geo, an analog virus
encoding a ß-galactosidase-neomycin phosphotransferase fusion
protein, were used to transduce rat hepatocytes in vivo
(11). Briefly, male Lewis rats, 3 weeks old, were subjected to a 70%
partial hepatectomy. Twenty-four hours later, the retrovirus
supernatant was infused into the portal vein. Fourteen days later,
diabetes was induced with an intraperitoneal injection of a high dose
of STZ (250 mg/kg).
Serum Chemistry Analysis
Blood glucose was measured with a One Touch II glucose meter
(Lifescan, Mountain View, CA). Serum insulin was measured with a rat
insulin RIA kit (Linco Research, St. Louis, MO). Serum ketones were
determined by spotting serum on an Ames Ketostix reagent strip
(Lifescan, Mountain View, CA).
Immunocytochemical Examination
For immunohistochemical procedures, the tissues were fixed
in 10% buffered formalin and kept in 70% ethanol. An antibody to
human insulin (Linco Research), which has 50% of cross-reactivity to
rat insulin, was used for the immunostaining.
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ACKNOWLEDGMENTS
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We thank Milton Pyron for his technical assistance and Drs. Swan
Thung and Romil Saxena for immunohistochemical analysis of pancreatic
tissues.
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FOOTNOTES
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Address requests for reprints to: Savio Woo, Institute for Gene Therapy and Molecular Medicine, Box 1496, One Gustave Levy Place, New York, New York 10029-6574.
This work was supported by NIH Grant DK-44080 and by the American
Diabetes Association. S.L.C.W. was an Investigator in the Howard Hughes
Medical Institute.
1 Current address: Department of Medical Biochemistry, University of
Geneva, CH 1211 Geneva 4, Switzerland. 
2 Current address: Institute for Gene Therapy and Molecular Medicine,
Mount Sinai School of Medicine, New York, New York 10029. 
Received for publication February 19, 1997.
Accepted for publication March 21, 1997.
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REFERENCES
|
---|
-
Cahill GF 1971 The physiology of insulin in man. Diabetes 20:785798[Medline]
-
Vignati L, Asmal AC, Black WL, Brink SJ, Hare JW 1985 Coma
and diabetes. In: Marble A, Krall LP, Bradley RF, Christlieb AR,
Soeldner JS (eds): Joslins Diabetes Mellitus. Lea & Febiger,
Philadelphia, p 526 and p 867
-
Kolodka TM, Finegold M, Moss L, Woo SLC 1995 Gene
therapy for diabetes mellitus in rats by hepatic expression of insulin.
Proc Natl Acad Sci USA 92:32933297[Abstract]
-
Valera A, Fillat C, Costa C, Sabater J, Visa J, Pujol A,
Bosch F 1994 Regulated expression of human insulin in the liver of
transgenic mice corrects diabetic alterations. FASEB J 8:440447[Abstract/Free Full Text]
-
Griffen SC, Russel SM, Katz LS, Nicoll CS 1987 Insulin exerts
metabolic and growth-promoting effects by a direct action on the liver
in vivo: clarification of the functional significance of the
portal vascular link between the beta cells of the pancreatic islets
and the liver. Proc Natl Acad Sci USA 84:73007304[Abstract]
-
Bowles NE, Eisensmith RC, Mohuiddin R, Pyron M, Woo SLC 1996 A simple and efficient method for the concentration and purification of
recombinant retrovirus for increased hepatocyte transduction in
vivo. Hum Gene Ther 7:17351742[Medline]
-
Hosaka M, Nagahama M, Kim WS, Watanabe T, Hatsuzawa K,
Ikemizu J, Murakami K, Nakayama K 1991 Arg-X-Lys/Arg-Arg motif as a
signal for precursor cleavage catalyzed by furin within the
constitutive secretory pathway. J Biol Chem 266:1212712130[Abstract/Free Full Text]
-
Thompson KS, Towle HC 1991 Localization of the carbohydrate
response element of the rat L-type pyruvate kinase gene. J Biol
Chem 266:86798682[Abstract/Free Full Text]
-
Chen R, Doiron B, Kahn A 1995 Glucose responsiveness of a
reporter gene transduced into hepatocytic cells using a retroviral
vector. FEBS Lett 365:223226[CrossRef][Medline]
-
Sarkar G, Sommer SS 1990 The "megaprimer" method of
site-directed mutagenesis. BioTechniques 8:404407[Medline]
-
Kolodka TM, Finegold M, Woo SLC 1993 Hepatic gene therapy:
efficient retroviral-mediated gene transfer into rat hepatocytes
in vivo. Somat Cell Mol Genet 19:491497[Medline]