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Glucose regulated production of human insulin in rat hepatocytes

Gene Therapy volume 7, pages 205–214 (2000)Cite this article

Abstract

Insulin gene therapy requires that insulin secretion be coupled to metabolic requirements. To this end, we have developed an insulin transgene whose transcription is stimulated by glucose and inhibited by insulin. Glucose- and insulin-sensitive promoters were constructed by inserting glucose-responsive elements (GlREs) from the rat L-pyruvate kinase (L-PK) gene into the insulin-sensitive, liver-specific, rat insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Glucose (5 to 25 mM) stimulated, and insulin (10−10 to 10−7 M) inhibited, luciferase expression driven by these promoters in primary cultured rat hepatocytes. The capacity of transfected hepatocytes to secrete mature, biologically active insulin was demonstrated using a human proinsulin cDNA (2xfur), modified to allow protein processing by endogenous endopeptidase activity. Medium conditioned by insulin-producing hepatocytes contained greater than 300 μU/ml immunoreactive insulin, while denaturing SDS-PAGE of an anti-insulin immunoprecipitate revealed bands with the mobilities of insulin A, and B chains. Biological activity of hepatocyte-produced insulin was demonstrated in a transfection assay, in which medium conditioned by insulin-producing hepatocytes exerted an effect similar to 10−7 M insulin. We then combined the glucose- and insulin-sensitive promoter with the modified human proinsulin cDNA to create a metabolically sensitive insulin transgene ((GlRE)3BP-1 2xfur). In both H4IIE hepatoma cells stably transfected with this construct, and normal rat hepatocytes (GlRE)3BP-1 2xfur-mediated insulin secretion increased in response to stimulation by glucose. Moreover, a capacity to decrease insulin production in response to diminishing glucose exposure was also demonstrated. We conclude that the transcriptional regulation of insulin production using these glucose- and insulin-sensitive constructs meets the requirements for application in a rodent model of insulin gene therapy.

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References

  1. Kay MA, Rothenberg S, Landen CN, Bellinger DA . In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs Science 1993 262: 117–119

    Article  CAS  Google Scholar 

  2. Applebaum-Bowden D, Kobayashi J, Kashyap VS, Brown DR . Hepatic lipase gene therapy in hepatic lipase-deficient mice. Adenovirus-mediated replacement of a lipolytic enzyme to the vascular endothelium J Clin Invest 1996 97: 799–805

    Article  CAS  Google Scholar 

  3. Alino SF, Bobadilla M, Crespo J, Lejarreta M . Human α1-antitrypsin gene transfer to in vivo mouse hepatocytes Hum Gen Ther 1996 7: 531–536

    Article  CAS  Google Scholar 

  4. Monahan PE, Samulski RJ, Tazelaar J, Xiao X . Direct intramuscular injection with recombinant AAV vectors results in sustained expression in dog model of hemophila Gene Therapy 1998 5: 40–41

    Article  CAS  Google Scholar 

  5. Harding CO, Wild K, Chang D, Messing A . Metabolic engineering as therapy for inborn errors of metabolism – development of mice with phenylalanine hydroxylase expression in muscle Gene Therapy 1998 5: 677–683

    Article  CAS  Google Scholar 

  6. Revers RR, Henry R, Schmeiser L, Kolterman O . The effects of biosynthetic human proinsulin on carbohydrate metabolism Diabetes 1984 33: 762–770

    Article  CAS  Google Scholar 

  7. Simpson AM, Tuch BE, Swan MA, Tu J . Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2) Gene Therapy 1995 2: 223–231

    CAS  PubMed  Google Scholar 

  8. Simpson AM, Marshall GM, Tuch BE, Maxwell L . Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g) Gene Therapy 1997 4: 1202–1215

    Article  CAS  Google Scholar 

  9. Groskreutz DJ, Sliwkowski MX, Gorman CM . Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin J Biol Chem 1994 269: 6241–6245

    CAS  Google Scholar 

  10. Simonson GD, Groskreutz DJ, Gorman CM, MacDonald MJ . Synthesis and processing of genetically modified human proinsulin by rat myoblast primary cultures Hum Gen Ther 1996 7: 71–78

    Article  CAS  Google Scholar 

  11. Lu D, Tamemoto H, Shibata H, Saito I . Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin Gene Therapy 1998 5: 888–895

    Article  CAS  Google Scholar 

  12. Muzzin P, Eisensmith RC, Copeland KC, Woo SLC . Hepatic insulin gene expression as treatment for type 1 diabetes mellitus in rats Mol Endocrinol 1997 11: 833–837

    Article  CAS  Google Scholar 

  13. Luzi L, Hering BJ, Socci C, Raptis G . Metabolic effects of successful intraportal islet transplantation in insulin-dependent diabetes mellitus J Clin Invest 1996 97: 2611–2618

    Article  CAS  Google Scholar 

  14. Fioretto P, Steffes MW, Sutherland DER, Goetz FC . Reversal of lesions of diabetic nephropathy after pancreas transplantation New Engl J Med 1998 339: 69–75

    Article  CAS  Google Scholar 

  15. Sugiyama A, Hattori S, Tanaka S, Isoda F . Defective adenoassociated viral-mediated transfection of insulin gene by direct injection into liver parenchyma decreases blood glucose of diabetic mice Hormone Metabol Res 1997 29: 599–603

    Article  CAS  Google Scholar 

  16. Kolodka TM, Finegold M, Moss L, Woo SLC . Gene therapy for diabetes mellitus in rats by hepatic expression of insulin Proc Natl Acad Sci USA 1995 92: 3293–3297

    Article  CAS  Google Scholar 

  17. Valera A, Fillat C, Costa C, Sabater J . Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations FASEB J 1994 8: 440–447

    Article  CAS  Google Scholar 

  18. Kaneda Y, Iwai K, Uchida T . Introduction and expression of the human insulin gene in adult rat liver J Biol Chem 1989 264: 12126–12129

    CAS  PubMed  Google Scholar 

  19. Selden R, Skoskiewicz MJ, Burke Howie KB, Russell PS . Implantation of genetically engineered fibroblasts into mice: implications for gene therapy Science 1987 236: 714–718

    Article  CAS  Google Scholar 

  20. Robertson DG, Marino EM, Thulé PM, Seneviratne CK . Insulin and glucocorticoids regulate IGFBP-1 expression via a common promoter region Biochem Biophys Res Commun 1994 200: 226–232

    Article  CAS  Google Scholar 

  21. Lefrancois-Martinez A-M, Diaz-Guerra M-J, Vallet V, Kahn A . Glucose-dependent regulation of the L-pyruvate kinase gene in a hepatoma cell line is independent of insulin and cyclic AMP FASEB J 1994 8: 89–96

    Article  CAS  Google Scholar 

  22. Unger R, Foster DW . Diabetes mellitus. In: Wilson JD et al (eds) William's Textbook of Endocrinology WB Saunders Co: Philadelphia 1998 pp 973–1059

    Google Scholar 

  23. Taconic . Hematological Clinical Chemistry Values Sprague Dawley Rats. Tachonic Technical Library, 1–6. 1998, Internet Communication

  24. Smeekens SP, Chan SJ, Steiner DF . The biosynthesis and processing of neuroendocrine peptides: identification of proprotein convertases involved in intravesicular processing Prog Brain Res 1992 92: 235–246

    Article  CAS  Google Scholar 

  25. Steiner DF et al. The new enzymology of precursor processing endoproteases J Biol Chem 1992 267: 23435–23438

    CAS  PubMed  Google Scholar 

  26. Goswami R, Lacson R, Yang E, Sam R . Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter Endocrinol 1994 134: 736–743

    Article  CAS  Google Scholar 

  27. Suh DS, Ooi GT, Rechler MM . Identification of cis-elements mediating the stimulation of rat insulin-like growth factor-binding protein-1 promoter activity by dexamethasone, cyclicadenosine 3′,5′-monophosphate, and phorbol esters, and inhibition by insulin Mol Endocrinol 1994 8: 794–805

    CAS  PubMed  Google Scholar 

  28. Goldstein S, Sertich G, Levan KR, Phillips LS . Nutrition and somatomedin. XIX. Molecular regulation of insulin-like growth factor-I in streptozotocin-diabetic rats Mol Endocrinol 1988 2: 1093–1100

    Article  CAS  Google Scholar 

  29. Pao C-I, Farmer PK, Begovic S, Goldstein S . Expression of hepatic insulin-like growth factor-I and insulin-like growth factor-binding protein-1 genes is transcriptionally regulated in streptozotocin-diabetic rats Mol Endocrinol 1992 6: 969–977

    CAS  PubMed  Google Scholar 

  30. Schnetzler B, Murakawa G, Abalos D, Halban P . Adaptation to supraphysiologic levels of insulin gene expression in transgenic mice: evidence of the importance of posttranscriptional regulation J Clin Invest 1993 92: 272–280

    Article  CAS  Google Scholar 

  31. Vitullo JC, Aron DC, Miller RE . Control of insulin gene expression: implications for insulin gene therapy J Lab Clin Med 1994 124: 328–334

    CAS  PubMed  Google Scholar 

  32. Leibiger B, Moede T, Schwarz T, Brown GR . Short-term regulation of insulin gene transcription by glucose Proc Natl Acad Sci USA 1998 95: 9307–9312

    Article  CAS  Google Scholar 

  33. Gros L, Montoliu L, Riu E, Lebrigand L . Regulated production of mature insulin by non-β-cells Hum Gen Ther 1997 8: 2249–2259

    Article  CAS  Google Scholar 

  34. Regazzi R, Verchere CB, Halban PA, Polonsky KS . Insulin production: from gene to granule Diabetol 1997 40: B33–B38

    Article  Google Scholar 

  35. Tomita N, Higaki J, Oghihara T, Kondo T . A novel gene-transfer technique mediated by HVJ (Sendai virus), nuclear protein, and liposomes Cancer Detect Prevent 1994 18: 485–491

    CAS  PubMed  Google Scholar 

  36. Hartwig R, Danishefsky KJ . Studies on the assembly and secretion of fibrinogen J Biol Chem 1991 266: 6578–6585

    CAS  PubMed  Google Scholar 

  37. Dahn MS, Hsu CJ, Lange MP, Kimball SR . Factors affecting secretory protein production in primary cultures of rat hepatocytes (43570) Proc Soc Exp Biol Med 1993 203: 38–44

    Article  CAS  Google Scholar 

  38. Dixon JL, Ginsberg HN . Hepatic synthesis of lipoproteins and apolipoproteins Semin Liver Dis 1992 12: 364–372

    Article  CAS  Google Scholar 

  39. Vaulont S, Puzenat N, Levrat F, Cognet M . Proteins binding to the liver-specific pyruvate kinase gene promoter. A unique combination of known factors J Mol Biol 1989 209: 205–219

    Article  CAS  Google Scholar 

  40. Cognet M, Lone YC, Vaulont S, Kahn A . Structure of the rat L-type pyruvate kinase gene J Mol Biol 1987 196: 11–25

    Article  CAS  Google Scholar 

  41. Babajko S, Groyer A . Interplay of the liver-enriched trans-acting factors, DBP and HNF1 in the transactivation of human IGFBP-1 promoter Biochem Biophys Res Commun 1993 196: 480–486

    Article  CAS  Google Scholar 

  42. Wanke IE, Wong NC . Specific problems facing gene therapy for insulin-dependent diabetes mellitus: glucose-regulated insulin secretion from hepatocytes Proc Western Pharmacol Soc 1997 40: 131–133

    CAS  Google Scholar 

  43. Tuch BE, Tabiin MT, Casamento FM, Simpson AM . Transplantation of genetically engineered insulin-producing hepatocytes into immunoincompetent mice Transplant Proc 1998 30: 473

    Article  CAS  Google Scholar 

  44. Seglen PO . Preparation of rat liver cells. III. Enzymatic requirements for tissue dispersion Exp Cell Res 1973 82: 391–398

    Article  CAS  Google Scholar 

  45. Ginot F, Decaux J-F, Cognet M, Berbar T . Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression Eur J Biochem 1989 180: 289–294

    Article  CAS  Google Scholar 

  46. Bonifacino J . Analysis of proteins. Current Protocols in Molecular Biology, (10) New York: John Wiley & Sons 1997

    Google Scholar 

  47. Baker A, Saltik M, Lehrmann H, Killisch I . Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery Gene Therapy 1997 4: 773–782

    Article  CAS  Google Scholar 

  48. Marriott D, Gillece-Castro B, Gorman CM . Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones Mol Endocrinol 1992 6: 1441–1450

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was funded in part by National Institutes of Health (NIH) grant R01 DK 33475 to LS Phillips. PM Thulé was supported by a VA Merit Review Award, NIH grant NIDDK/NRSA F32 DK08978, and the Veterans Administration. The authors gratefully acknowledge Dr Timothy Stewart and Genentech, Inc., for supplying the modified human pro-insulin cDNA.

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Authors and Affiliations

  1. Veterans Administration Medical Center, Atlanta, GA, USA

    P M Thulé & J Liu

  2. Division of Diabetes, Endocrinology and Metabolism, Emory University School of Medicine, Atlanta, GA, USA

    L S Phillips

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  1. P M Thulé
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Thulé, P., Liu, J. & Phillips, L. Glucose regulated production of human insulin in rat hepatocytes. Gene Ther 7, 205–214 (2000). https://doi.org/10.1038/sj.gt.3301076

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