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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go, 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. 1Go). 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. 2Go).



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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 ({square}), or medium ({diamond}). 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 ({square}) after STZ Treatment

For the LX/erINS-treated rats, n = 10; for the LX/ßgeo-treated rats, n = 13.

 
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. 3Go). 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. 3Go), 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 ({blacksquare}) or LX/ßgeo ({square})

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.

 
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. 4Go).



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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 ({square}), or medium ({diamond}). 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.

 
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. 5Go, 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 50–70% 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 48–72 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).

 
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. 6Go). 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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Figure 6. Blood Glucose Levels during 24-h Fasting in LX/erINS-Treated Rats after Induction of Diabetes

Rats were transduced with various doses of LX/erINS. Ten days after STZ treatment, blood glucose levels were determined in the animals at 6-h intervals. For comparison, blood glucose 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 ({circ}); n = 6 for 6 x 106 cfu/rat ({blacktriangleup}); n = 10 for 2 x 106 cfu/rat (•); n = 10 for 6 x 105 cfu/rat ({diamond}); n = 6 for 2 x 105 cfu/rat ({blacksquare}). For the LX/ßgeo-treated group, n = 5 ({square}).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 10–15 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    ACKNOWLEDGMENTS
 
We thank Milton Pyron for his technical assistance and Drs. Swan Thung and Romil Saxena for immunohistochemical analysis of pancreatic tissues.


    FOOTNOTES
 
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. Back

2 Current address: Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, New York 10029. Back

Received for publication February 19, 1997. Accepted for publication March 21, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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