Prevention of vascular and neural dysfunction in diabetic rats by C- peptide

Science

Volumn 277, Issue 5325, 1997, Pages 563-566

  Ido, Y ^a   Vindigni, A ^a   Chang, K ^a   Stramm, L ^b   Chance, R ^b   Heath, W F ^b   DiMarchi, R D ^b   Di Cera, E ^a   Williamson, J R ^a  

^a WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b ELI LILLY AND COMPANY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE TRIPHOSPHATASE (POTASSIUM SODIUM); ALBUMIN; C PEPTIDE; GLUCOSE; GLYCOSYLATED HEMOGLOBIN; INSULIN; ISOPHANE INSULIN;

ANIMAL EXPERIMENT; ANIMAL MODEL; ANIMAL TISSUE; AORTA; ARTICLE; BLOOD FLOW; BLOOD VESSEL PERMEABILITY; CIRCULAR DICHROISM; CONTROLLED STUDY; DIABETES MELLITUS; DIABETIC ANGIOPATHY; DIABETIC NEUROPATHY; ENZYME ACTIVITY; GLUCOSE BLOOD LEVEL; GRANULATION TISSUE; INSULIN SYNTHESIS; MOTOR NERVE CONDUCTION; NONHUMAN; PRIORITY JOURNAL; RAT; RETINA; SCIATIC NERVE; STREPTOZOCIN DIABETES; SUBCUTANEOUS DRUG ADMINISTRATION; UVEA;

AMINO ACID SEQUENCE; ANIMALS; BLOOD CIRCULATION; BLOOD GLUCOSE; C-PEPTIDE; CAPILLARY PERMEABILITY; CIRCULAR DICHROISM; DIABETES MELLITUS, EXPERIMENTAL; DIABETIC ANGIOPATHIES; DIABETIC NEUROPATHIES; HUMANS; MALE; MOLECULAR SEQUENCE DATA; NA(+)-K(+)-EXCHANGING ATPASE; NEURAL CONDUCTION; PEPTIDE FRAGMENTS; PROTEIN STRUCTURE, SECONDARY; RATS; RATS, SPRAGUE-DAWLEY; STEREOISOMERISM;

ANIMALIA;

EID: 0030821262     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5325.563     Document Type: Article

References (46)

1. J. Markussen and H. E. Schiff, Int. J. Pept. Protein Res. 5, 69 (1973); C. R. Snell and D. G. Smyth, J. Biol. Chem. 250, 6291 (1975); D. F. Steiner, Diabetes 27 (suppl. 1), 145 (1978); _, G. I. Bell, H. S. Tager, A. H. Rubenstein, in Endocrinology, L. J. DeGroot et al., Eds. (Saunders, Philadelphia, PA, 1995), vol 2, pp. 1296-1328.
2. J. Markussen and H. E. Schiff, Int. J. Pept. Protein Res. 5, 69 (1973); C. R. Snell and D. G. Smyth, J. Biol. Chem. 250, 6291 (1975); D. F. Steiner, Diabetes 27 (suppl. 1), 145 (1978); _, G. I. Bell, H. S. Tager, A. H. Rubenstein, in Endocrinology, L. J. DeGroot et al., Eds. (Saunders, Philadelphia, PA, 1995), vol 2, pp. 1296-1328.
3. J. Markussen and H. E. Schiff, Int. J. Pept. Protein Res. 5, 69 (1973); C. R. Snell and D. G. Smyth, J. Biol. Chem. 250, 6291 (1975); D. F. Steiner, Diabetes 27 (suppl. 1), 145 (1978); _, G. I. Bell, H. S. Tager, A. H. Rubenstein, in Endocrinology, L. J. DeGroot et al., Eds. (Saunders, Philadelphia, PA, 1995), vol 2, pp. 1296-1328.
4. J. Markussen and H. E. Schiff, Int. J. Pept. Protein Res. 5, 69 (1973); C. R. Snell and D. G. Smyth, J. Biol. Chem. 250, 6291 (1975); D. F. Steiner, Diabetes 27 (suppl. 1), 145 (1978); _, G. I. Bell, H. S. Tager, A. H. Rubenstein, in Endocrinology, L. J. DeGroot et al., Eds. (Saunders, Philadelphia, PA, 1995), vol 2, pp. 1296-1328.
5. A. E. Kitabchi, Metabolism 26, 547 (1977).
6. T. Toyota, K. Abe, M. Kudo, K. Kimura, Y. Goto, Tohoku J. Exp. Med. 117, 79 (1975); J. R. Dryburgh, S. M. Hampton, V. Marks, Diabetologia 19, 397 (1980); C. Wójcikowski, Y. Maier, K. Dominiak, R. Fussganger, E. F. Pfeiffer, ibid. 25, 288 (1983); B.-L. Johansson, S. Sjöberg, J. Wahren, ibid. 35, 121 (1992); B.-L. Johansson, A. Kernell, S. Sjöberg, J. Wahren, J. Clin. Endocrinol. Metab. 77, 976 (1993); J. R. Zierath, A. Handberg, M. Tally, H. Wallberg-Henriksson, Diabetologia 39, 306 (1996).
7. T. Toyota, K. Abe, M. Kudo, K. Kimura, Y. Goto, Tohoku J. Exp. Med. 117, 79 (1975); J. R. Dryburgh, S. M. Hampton, V. Marks, Diabetologia 19, 397 (1980); C. Wójcikowski, Y. Maier, K. Dominiak, R. Fussganger, E. F. Pfeiffer, ibid. 25, 288 (1983); B.-L. Johansson, S. Sjöberg, J. Wahren, ibid. 35, 121 (1992); B.-L. Johansson, A. Kernell, S. Sjöberg, J. Wahren, J. Clin. Endocrinol. Metab. 77, 976 (1993); J. R. Zierath, A. Handberg, M. Tally, H. Wallberg-Henriksson, Diabetologia 39, 306 (1996).
8. T. Toyota, K. Abe, M. Kudo, K. Kimura, Y. Goto, Tohoku J. Exp. Med. 117, 79 (1975); J. R. Dryburgh, S. M. Hampton, V. Marks, Diabetologia 19, 397 (1980); C. Wójcikowski, Y. Maier, K. Dominiak, R. Fussganger, E. F. Pfeiffer, ibid. 25, 288 (1983); B.-L. Johansson, S. Sjöberg, J. Wahren, ibid. 35, 121 (1992); B.-L. Johansson, A. Kernell, S. Sjöberg, J. Wahren, J. Clin. Endocrinol. Metab. 77, 976 (1993); J. R. Zierath, A. Handberg, M. Tally, H. Wallberg-Henriksson, Diabetologia 39, 306 (1996).
9. T. Toyota, K. Abe, M. Kudo, K. Kimura, Y. Goto, Tohoku J. Exp. Med. 117, 79 (1975); J. R. Dryburgh, S. M. Hampton, V. Marks, Diabetologia 19, 397 (1980); C. Wójcikowski, Y. Maier, K. Dominiak, R. Fussganger, E. F. Pfeiffer, ibid. 25, 288 (1983); B.-L. Johansson, S. Sjöberg, J. Wahren, ibid. 35, 121 (1992); B.-L. Johansson, A. Kernell, S. Sjöberg, J. Wahren, J. Clin. Endocrinol. Metab. 77, 976 (1993); J. R. Zierath, A. Handberg, M. Tally, H. Wallberg-Henriksson, Diabetologia 39, 306 (1996).
10. T. Toyota, K. Abe, M. Kudo, K. Kimura, Y. Goto, Tohoku J. Exp. Med. 117, 79 (1975); J. R. Dryburgh, S. M. Hampton, V. Marks, Diabetologia 19, 397 (1980); C. Wójcikowski, Y. Maier, K. Dominiak, R. Fussganger, E. F. Pfeiffer, ibid. 25, 288 (1983); B.-L. Johansson, S. Sjöberg, J. Wahren, ibid. 35, 121 (1992); B.-L. Johansson, A. Kernell, S. Sjöberg, J. Wahren, J. Clin. Endocrinol. Metab. 77, 976 (1993); J. R. Zierath, A. Handberg, M. Tally, H. Wallberg-Henriksson, Diabetologia 39, 306 (1996).
11. T. Toyota, K. Abe, M. Kudo, K. Kimura, Y. Goto, Tohoku J. Exp. Med. 117, 79 (1975); J. R. Dryburgh, S. M. Hampton, V. Marks, Diabetologia 19, 397 (1980); C. Wójcikowski, Y. Maier, K. Dominiak, R. Fussganger, E. F. Pfeiffer, ibid. 25, 288 (1983); B.-L. Johansson, S. Sjöberg, J. Wahren, ibid. 35, 121 (1992); B.-L. Johansson, A. Kernell, S. Sjöberg, J. Wahren, J. Clin. Endocrinol. Metab. 77, 976 (1993); J. R. Zierath, A. Handberg, M. Tally, H. Wallberg-Henriksson, Diabetologia 39, 306 (1996).
12. B. H. Frank, J. M. Pettee, R. E. Zimmerman, P. J. Burck, in PEPTIDES: Synthesis-Structure-Function, D. H. Rich and E. Gross, Eds. (Pierce Chemical Company, Rockford, IL, 1981), pp. 729-738; B. H. Frank and R. E. Chance, Münch Med. Wschr. 125 (Suppl. 1), S14 (1983). Human proinsulin (recombinant DNA origin) from Eli Lilly and Company, Indianapolis, IN, was converted to human insulin and C-peptide by the hydrolytic actions of trypsin and carboxypeptidase B. Human C-peptide was purified with the use of both cation and anion exchange chromatography. The two proinsulin conversion intermediates, des(31,32) split and des(64,65) split human proinsulin, were also derived from biosynthetic human proinsulin by controlled enzymatic digestions with trypsin and carboxypeptidase B and purified by reversed-phase high-performance liquid chromatography. The des(31,32) and des(64,65) split forms of proinsulin are intermediates in the cleavage of C-peptide from proinsulin following formation of disulfide bonds between the A and B chains of insulin. These split forms are produced by cleavage of proinsulin between amino acid residues 32,33 and 65,66 by an endopeptidase and subsequent elimination of the dipeptide residues 31,32 and 64,65 following cleavage by an exopeptidase between residues 30,31 and 63,64. C-peptide remains attached to the A chain of insulin the the des(31,32) split form and is attached to the B chain of insulin in the des(64,64) form.
13. B. H. Frank, J. M. Pettee, R. E. Zimmerman, P. J. Burck, in PEPTIDES: Synthesis-Structure-Function, D. H. Rich and E. Gross, Eds. (Pierce Chemical Company, Rockford, IL, 1981), pp. 729-738; B. H. Frank and R. E. Chance, Münch Med. Wschr. 125 (Suppl. 1), S14 (1983). Human proinsulin (recombinant DNA origin) from Eli Lilly and Company, Indianapolis, IN, was converted to human insulin and C-peptide by the hydrolytic actions of trypsin and carboxypeptidase B. Human C-peptide was purified with the use of both cation and anion exchange chromatography. The two proinsulin conversion intermediates, des(31,32) split and des(64,65) split human proinsulin, were also derived from biosynthetic human proinsulin by controlled enzymatic digestions with trypsin and carboxypeptidase B and purified by reversed-phase high-performance liquid chromatography. The des(31,32) and des(64,65) split forms of proinsulin are intermediates in the cleavage of C-peptide from proinsulin following formation of disulfide bonds between the A and B chains of insulin. These split forms are produced by cleavage of proinsulin between amino acid residues 32,33 and 65,66 by an endopeptidase and subsequent elimination of the dipeptide residues 31,32 and 64,65 following cleavage by an exopeptidase between residues 30,31 and 63,64. C-peptide remains attached to the A chain of insulin the the des(31,32) split form and is attached to the B chain of insulin in the des(64,64) form.
14. Animals were cared for in accordance with National Institutes of Health guidelines on laboratory animal welfare. Diabetes was induced in male Sprague-Dawley rats (body weight, 300 g) by intravenous injection of 50 mg of streptozotocin (Zanosar, Upjohn). Control rats received only buffer.
15. +-ATPase activity was measured in crude homogenates by a modification of the coupled enzyme method (11).
16. J. R. Williamson et al., Diabetes 42, 801 (1993).
17. Diabetes Control and Complications Trial, ibid. 44, 968 (1995); T. Inoguchi et al., Proc. Natl. Acad. Sci. U.S.A. 89, 11059 (1992); G. Pugliese et al., Diabetes 39, 323 (1990); D. Rogers et al., ibid. 37, 1689 (1988).
18. Diabetes Control and Complications Trial, ibid. 44, 968 (1995); T. Inoguchi et al., Proc. Natl. Acad. Sci. U.S.A. 89, 11059 (1992); G. Pugliese et al., Diabetes 39, 323 (1990); D. Rogers et al., ibid. 37, 1689 (1988).
19. Diabetes Control and Complications Trial, ibid. 44, 968 (1995); T. Inoguchi et al., Proc. Natl. Acad. Sci. U.S.A. 89, 11059 (1992); G. Pugliese et al., Diabetes 39, 323 (1990); D. Rogers et al., ibid. 37, 1689 (1988).
20. Diabetes Control and Complications Trial, ibid. 44, 968 (1995); T. Inoguchi et al., Proc. Natl. Acad. Sci. U.S.A. 89, 11059 (1992); G. Pugliese et al., Diabetes 39, 323 (1990); D. Rogers et al., ibid. 37, 1689 (1988).
21. J. R. Williamson et al., J. Clin. Invest. 85, 1167 (1990); B. A. Wolf et al., ibid. 87, 31 (1991). In this model, a circle of skin ∼2 cm in diameter was removed from either side of the lower back/thigh and a plastic chamber was sutured to the surrounding cuff of skin. One week later, 1.5 ml of Hepes buffer solutions (pH 7.4) containing 5 or 30 mM glucose ± C-peptide was added to the chambers twice daily for 7 days, at which time blood flow and vascular albumin permeation were assessed.
22. J. R. Williamson et al., J. Clin. Invest. 85, 1167 (1990); B. A. Wolf et al., ibid. 87, 31 (1991). In this model, a circle of skin ∼2 cm in diameter was removed from either side of the lower back/thigh and a plastic chamber was sutured to the surrounding cuff of skin. One week later, 1.5 ml of Hepes buffer solutions (pH 7.4) containing 5 or 30 mM glucose ± C-peptide was added to the chambers twice daily for 7 days, at which time blood flow and vascular albumin permeation were assessed.
23. With the exception of scrambled human C-peptide, des(13-17) human C-peptide, and all-D-amino acid C-peptide (which are described below), synthetic peptides were from the Lilly Research Laboratories in Indianapolis, IN, and were prepared by solid-phase synthesis on an Applied Biosystems (ABI) Model 430A peptide synthesizer with the use of the Boc protecting strategy. Each residue was double-coupled with the use of either dicyclohexylcarbodi-imide-initiated symmetric anhydride or 1-hydroxybenzotriazole activation. The peptides were cleaved from the resin with the use of 9:1 hydrogen fluoride/ m-cresol at 0°C for 1 hour, followed by extraction with 0.1 M ammonium bicarbonate and lyophilization. The peptides were purified by rpHPLC on a Vydac C18 column using acetonitrile gradient elution with either 0.1 M ammonium acetate or 0.1 M ammonium bicarbonate buffers and analyzed by electrospray ionization mass spectrometry (ESI-MS). Native pig C-peptide (Lilly Research Laboratories) was isolated and purified from a trypsin digestion of des(62,63) split porcine proinsulin [R. E. Chance, In DIABETES: Proceeding of the Seventh Congress of the International Diabetes Federation, R. R. Rodriquez and J. Vallance-Owen, Eds. (Excerpta Medica, Amsterdam, 1971), pp. 292-305]. Scrambled human C-peptide, human C-peptide des(13-17), and all-D-amino acid C-peptide were synthesized by the Protein and Nucleic Acid Chemistry Laboratory at Washington University on an ABI model 431A peptide synthesizer. Amino acid activation was performed with the use of HBTU [2-(1H-benzotriazol-1-yl)1,1,3,3,tetramethyluronium hexafluorophosphate]. The alpha amino group of the amino acids was FMOC (9-fluorenyl-methoxycarbonyl) protected, and the side chain groups were protected by tertiarybutyl (Asp, Ser, and Glu), and trityl (Gln). FMOC deprotection was performed with 20% piperidine in N-methylpyrrolidone. The peptide was simultaneously deprotected and cleaved from the resin with triflouroacetic acid/phenol/thioanisole/water/ ethanedithiol (85:5:4:4:2) for 2 hours. Chromatographic analysis and purification of the peptide were performed as described [J. Gorka, D. W. McCourt, B. D. Schwartz, Pept. Res. 2, 376 (1989)]. Electrospray mass spectrometry of the purified peptide was performed on a Vestec model 201 electrospray mass spectrometer.
24. With the exception of scrambled human C-peptide, des(13-17) human C-peptide, and all-D-amino acid C-peptide (which are described below), synthetic peptides were from the Lilly Research Laboratories in Indianapolis, IN, and were prepared by solid-phase synthesis on an Applied Biosystems (ABI) Model 430A peptide synthesizer with the use of the Boc protecting strategy. Each residue was double-coupled with the use of either dicyclohexylcarbodi-imide-initiated symmetric anhydride or 1-hydroxybenzotriazole activation. The peptides were cleaved from the resin with the use of 9:1 hydrogen fluoride/ m-cresol at 0°C for 1 hour, followed by extraction with 0.1 M ammonium bicarbonate and lyophilization. The peptides were purified by rpHPLC on a Vydac C18 column using acetonitrile gradient elution with either 0.1 M ammonium acetate or 0.1 M ammonium bicarbonate buffers and analyzed by electrospray ionization mass spectrometry (ESI-MS). Native pig C-peptide (Lilly Research Laboratories) was isolated and purified from a trypsin digestion of des(62,63) split porcine proinsulin [R. E. Chance, In DIABETES: Proceeding of the Seventh Congress of the International Diabetes Federation, R. R. Rodriquez and J. Vallance-Owen, Eds. (Excerpta Medica, Amsterdam, 1971), pp. 292-305]. Scrambled human C-peptide, human C-peptide des(13-17), and all-D-amino acid C-peptide were synthesized by the Protein and Nucleic Acid Chemistry Laboratory at Washington University on an ABI model 431A peptide synthesizer. Amino acid activation was performed with the use of HBTU [2-(1H-benzotriazol-1-yl)1,1,3,3,tetramethyluronium hexafluorophosphate]. The alpha amino group of the amino acids was FMOC (9-fluorenyl-methoxycarbonyl) protected, and the side chain groups were protected by tertiarybutyl (Asp, Ser, and Glu), and trityl (Gln). FMOC deprotection was performed with 20% piperidine in N-methylpyrrolidone. The peptide was simultaneously deprotected and cleaved from the resin with triflouroacetic acid/phenol/thioanisole/water/ ethanedithiol (85:5:4:4:2) for 2 hours. Chromatographic analysis and purification of the peptide were performed as described [J. Gorka, D. W. McCourt, B. D. Schwartz, Pept. Res. 2, 376 (1989)]. Electrospray mass spectrometry of the purified peptide was performed on a Vestec model 201 electrospray mass spectrometer.
25. Y. Ido et al., Diabetes 43, 1469 (1994).
26. All results are expressed as mean ± 1 SD. Unless otherwise stated, overall group comparisons for each parameter were performed by the Van der Waerden test [E. L. Lehman, Nonparametrics: Statistical Methods Based on Ranks (Holden-Day, San Francisco, CA, 1975)]. When this test was significant at P < 0.05, individual pairwise group differences were assessed by the general linear model procedure with the use of SAS (SAS Institute, Cary, NC). Schéffe's interval test [H. Schéffe, The Analysis of Variance (Wiley, New York, 1959)] was also performed by SAS.
27. All results are expressed as mean ± 1 SD. Unless otherwise stated, overall group comparisons for each parameter were performed by the Van der Waerden test [E. L. Lehman, Nonparametrics: Statistical Methods Based on Ranks (Holden-Day, San Francisco, CA, 1975)]. When this test was significant at P < 0.05, individual pairwise group differences were assessed by the general linear model procedure with the use of SAS (SAS Institute, Cary, NC). Schéffe's interval test [H. Schéffe, The Analysis of Variance (Wiley, New York, 1959)] was also performed by SAS.
28. Y. Ido et al., data not shown.
29. R. Srinivasan and G. D. Rose, Proteins 22, 81 (1995).
30. R. A. Sayle and E. J. Milner-White, Trends Biochem. Sci. 20, 374 (1995).
31. W. Kabsch and C. Sander, Biopolymers 22, 2577 (1983).
32. P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991).
33. P. Y. Chou and G. D. Fasman, Biochemistry 13, 211 (1974); ibid., p. 222; P. N. Lewis, F. A. Momany, H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 68, 2293 (1971); J. L. Crawford, N. N. Lipscomb, C. G. Schellman, ibid. 70, 538 (1973).
34. P. Y. Chou and G. D. Fasman, Biochemistry 13, 211 (1974); ibid., p. 222; P. N. Lewis, F. A. Momany, H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 68, 2293 (1971); J. L. Crawford, N. N. Lipscomb, C. G. Schellman, ibid. 70, 538 (1973).
35. P. Y. Chou and G. D. Fasman, Biochemistry 13, 211 (1974); ibid., p. 222; P. N. Lewis, F. A. Momany, H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 68, 2293 (1971); J. L. Crawford, N. N. Lipscomb, C. G. Schellman, ibid. 70, 538 (1973).
36. P. Y. Chou and G. D. Fasman, Biochemistry 13, 211 (1974); ibid., p. 222; P. N. Lewis, F. A. Momany, H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 68, 2293 (1971); J. L. Crawford, N. N. Lipscomb, C. G. Schellman, ibid. 70, 538 (1973).
37. M. T. Stubbs et al., Eur. J. Biochem. 206, 187 (1992).
38. R. G. Tilton et al., Diabetes 44, 234 (1995); M. K. Van den Enden, J. R. Nyengaad, E. Ostrow, J. H. Burgan, J. R. Williamson, Invest. Ophthalmol. Vis. Sci. 36, 1675 (1995); H. Ishii et al., Science 272, 728 (1996); R. G. Tilton et al., J. Clin. Invest. 99, 2192 (1997).
39. R. G. Tilton et al., Diabetes 44, 234 (1995); M. K. Van den Enden, J. R. Nyengaad, E. Ostrow, J. H. Burgan, J. R. Williamson, Invest. Ophthalmol. Vis. Sci. 36, 1675 (1995); H. Ishii et al., Science 272, 728 (1996); R. G. Tilton et al., J. Clin. Invest. 99, 2192 (1997).
40. R. G. Tilton et al., Diabetes 44, 234 (1995); M. K. Van den Enden, J. R. Nyengaad, E. Ostrow, J. H. Burgan, J. R. Williamson, Invest. Ophthalmol. Vis. Sci. 36, 1675 (1995); H. Ishii et al., Science 272, 728 (1996); R. G. Tilton et al., J. Clin. Invest. 99, 2192 (1997).
41. R. G. Tilton et al., Diabetes 44, 234 (1995); M. K. Van den Enden, J. R. Nyengaad, E. Ostrow, J. H. Burgan, J. R. Williamson, Invest. Ophthalmol. Vis. Sci. 36, 1675 (1995); H. Ishii et al., Science 272, 728 (1996); R. G. Tilton et al., J. Clin. Invest. 99, 2192 (1997).
42. R. Bessale, A. Kapitkovsky, A. Gorea, I. Shalit, M. Fridkin, FEBS 274, 151 (1990); R. B. Merrifield et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3449 (1995), P. Nicolas and A. Mor, Annu. Rev. Micorobiol. 49, 277 (1995).
43. R. Bessale, A. Kapitkovsky, A. Gorea, I. Shalit, M. Fridkin, FEBS 274, 151 (1990); R. B. Merrifield et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3449 (1995), P. Nicolas and A. Mor, Annu. Rev. Micorobiol. 49, 277 (1995).
44. R. Bessale, A. Kapitkovsky, A. Gorea, I. Shalit, M. Fridkin, FEBS 274, 151 (1990); R. B. Merrifield et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3449 (1995), P. Nicolas and A. Mor, Annu. Rev. Micorobiol. 49, 277 (1995).
45. D. M. Eades, J. R. Williamson, W. R. Sherman, Biomed.Appl. 490, 1 (1989).
46. Supported by NIH grants EY 06600, HL 39934, HL 58141, HL 49413, and DK 20579, the Kilo Diabetes and Vascular Research Foundation, and Eli Lilly, Inc. We thank several colleagues at Washington University, especially E. Groisman and G. R. Marshall, for informative discussions and advice. We acknowledge the excellent assistance of H. B. Long, H. E. Osborne, A. H. Pekar, and J. M. Richardson from the Lilly Research Laboratories; of W. LeJeune, A. Faller, E. Ostrow, and J. Burgan from Washington University in the performance of these experiments; and of M. Himmelmann in preparation of the manuscript.