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Home , Human placental lactogen, Insulin (medication)

Proc. Nati. Acad. Sci. USA Vol. 82, pp. 3868-3870, June 1985 Medical Sciences -related genes expressed in human from normal and diabetic (RNA homology/fetal growth/) KWANG-SAN LIu*, CHUN-YEH WANG*, NATHANIEL MILLS*, MICHAEL JYVES*, AND JUDITH ILAN*tt Departments of *Reproductive Biology and tDevelopmental Genetics, Case Western Reserve University, Cleveland, Ohio 44106 Communicated by Lester 0. Krampitz, January 30, 1985

ABSTRACT Rapid growth of human fetal tissues requires sponses to acute changes in cord blood (12-14) and insulin or insulin-like growth factors. A high rate of human the factors that control the synthesis and release of insulin in fetal growth occurs between implantation and about 14 weeks the are not known. Human maternal insulin levels can of gestation. Fetal pancreatic insulin secretion begins much be elevated as much as 18-fold without any demonstrable later. Since maternal insulin does not cross the blood/placen- transfer of this across the blood/placental barrier tal barrier, other sources of insulin or insulin-like growth fac- to the fetus (15). Since the fetal has only a limited tors may be provided for fetal development. We report here response to blood glucose levels and since maternal insulin that placental polyadenylylated RNAs from the first and third does not cross the blood/placental barrier, these avenues are trimester of normal as well as from term pregnan- not likely to be the primary source of the insulin or insulin- cies of diabetic mothers hybridize to a 32P-labeled cloned like growth factors necessary for embryonic and fetal devel- cDNA of an insulin-related sequence expressed in fetal pancre- opment. Therefore, the supply of to the develop- as. Moreover, from diabetic women express much ing fetus in normal as well as pathological states must be more of these sequences. These results suggest that insulin- synthesized and secreted by a tissue or tissues that commu- related genes are expressed in placental tissue during fetal de- nicate directly with the fetal circulation. velopment and may be a source of growth-promoting hor- The placenta is the source of hormones, such as mones for the human fetus. developing in diabetic choriogonadotropin and , and steroid hor- women receive a large influx of glucose. This in turn may stim- mones, such as and progestins, and therefore is a ulate the expression of insulin-related sequences, which may likely candidate for the synthesis of growth-promoting hor- result in higher utilization of glucose, thus bringing about the mones, such as insulin and insulin-like growth factors, which macrosomia and high incidence of malformation and still- may be essential for early embryonic development. Since births known to result from pregnancies in diabetics. there are numerous temporal changes in the production of hormones during normal pregnancy, we examined placentas During the first 10 weeks of embryonic and fetal life, which for the temporal appearance, during fetal development, of is a critical period of rapid growth for the human conceptus, RNA sequences related to the insulin gene family. primary and secondary embryonic induction and organogen- esis is taking place (1). Glucose is used as a major source of metabolic energy in the human fetus (2-4) and a glucose im- METHODS balance, in addition to affecting the growth of embryonic tis- sue, may well result in developmental abnormalities such as Tissue. Placentas were obtained primarily by cesarean sec- macrosomia, malformations, and increased rate of still- tion at the MacDonald Hospital for Women, University Hos- births, as is manifested in pregnancies of diabetic women. pitals of Cleveland and were extracted within 30 min of de- Insulin, a regulator of glucose metabolism, is an essential livery. Diabetics were classified according to White (16). growth factor for fast-growing mammalian tissues, including Total placental and pancreatic RNA were prepared as de- human embryonic tissues (2-4); however maternal insulin scribed by Chirgwin et al. (17). Poly(A)+ RNAs were isolat- does not cross the blood/placental barrier (5-8). Although it ed from total cellular RNAs by affinity chromatography on has not been established that insulin or an insulin-like growth oligo(dT)-cellulose (18). The poly(A)+ RNAs were translat- factor is necessary for the early development of the human ed in the cell-free reticulocyte lysate system (New England fetus in utero, such a requirement is not unlikely. Nuclear) under conditions suggested by the supplier. Trans- Explants of multiple tissues from fetal mouse synthesized lation products were resolved by NaDodSO4/12% PAGE insulin-like (9). The production of the insulin- (19). The gel was dried and exposed to Kodak XAR-5 autora- like growth factors IGF-I and IGF-II is developmentally reg- diographic film. ulated, and this pattern of expression is maintained in fibro- For hybridization of poly(A)+ RNA to cDNA probes, pla- blasts derived from fetal as well as older rats (10). Receptors cental and pancreatic poly(A)+ RNAs were electrophoresed for insulin-like growth factors (IGF-I, IGF-II, somatomedin in an agarose minigel (15 ml gel volume) under denaturing A, and multiplication-stimulating activity) and insulin are conditions (20) and transferred to nitrocellulose filter in 3 M present in human fetal tissues at different stages of develop- NaCl/0.3 M sodium citrate, pH 7.1-7.4, by the modified pro- ment (11). This evidence suggests that fetal growth may be cedure of Thomas (20). Blotting of poly(A)+ RNA to nitro- regulated by insulin and/or insulin-like growth factors. cellulose required 16-24 hr; the nitrocellulose filter then was The fetal pancreatic islets of Langerhans are capable of removed from the assembly and baked in a vacuum for 2 hr insulin synthesis only after 14 weeks of gestation, several at 60-80°C. Hybridization was carried out with nick-translat- weeks later than the critical growth period mentioned above. ed (21) cDNA insert under high-stringency conditions (50% Further, the fetal pancreas normally exhibits only limited re- formamide/0.9 M NaCl/0.09 M sodium citrate, pH 7.1-7.4, 42°C). The filter then was autoradiographed. cDNA clones pUC8I-1 and pBM-4 were provided by The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

3868 Downloaded by guest on October 1, 2021 Medical Sciences: Liu et aL Proc. NatL Acad. ScL USA 82 (1985) 3869

L. Villa-Kamaroff (University of Massachusetts Medical A B School). 1 2 3 4 1 2 3 4 RESULTS -28S Poly(A)+ RNA was extracted from placentas at various - 28S normal development as well as from placentas of stages of -.Birt " ^: ;; -18S I - overt diabetic mothers. To examine the quality and integrity 18S 40., 4.b of the poly(A)+ RNA, all samples were assayed in a cell-free 0 reticulocyte lysate translation system. The in vitro transla- tion products ranged in M, from 3000 to well over 100,000, as determined by NaDodSO4/PAGE (Fig. 1). A cDNA clone (pUC8I-1) containing 240 bases encoding from 9 of the prepeptide to amino acid 9 of the A peptide of insulin was used to probe for authentic insulin sequences (22). The DNA sequence of the insulin-related probe (pBM-4) from fetal pancreas contained a 274-base-pair fragment with several regions of substantial homology to the insulin gene (22). Region one has 64% homology to the se- quence encoding the NH2-terminal and insulin B peptide and has an overall homology of 55% to the B-peptide-encoding sequence. Region two with 60% homology, begins at the end FIG. 2. Hybridization of poly(A)+ RNA from different stages of of sequences encoding the insulin B peptide and extends into placental development with human insulin cDNA (pUC8I-1) (A) and sequences encoding the C peptide. A third region, corre- with human fetal pancreatic insulin-related cDNA (pBM-4) (B). Lanes: 1, 0.7 Mg of human adult pancreatic poly(A)+ RNA; 2, 2.0 pg sponding to the COOH-terminal end of the C peptide, has of human first trimester placental poly(A)+ RNA; 3, 2.ff pg of hu- 50% homology to this segment of the insulin sequence. The man full-term placental poly(A)+ RNA; 4, 2.0 pg of overt diabetic last region, with 52% homology, corresponds to part of the human full-term placental poly(A)+ RNA. Markers denote the posi- sequence encoding the leader peptide for insulin. tions of mouse ribosomal RNAs used as standards. To determine whether these sequences were present in placental RNA, poly(A)+ RNAs were separated by electro- tion signals with this probe under the same hybridization phoresis on agarose gels, transferred to nitrocellulose paper, conditions (Fig. 2A, lanes 2-4). and hybridized to the 32P-labeled cDNA insert from the hu- When the same RNA preparations were hybridized with man insulin gene (Fig. 2A) or to the 32P-labeled cDNA of the the human fetal insulin-related probe under the same condi- insulin-related sequence isolated from fetal pancreas (Fig. tions, all placental poly(A)+ RNA samples gave positive sig- 2B). The DNA encoding adult insulin hybridized strongly to nals (Fig. 2B, lanes 2-4), whereas adult pancreatic poly(A)+ adult human pancreatic poly(A)+ RNA (Fig. 2A, lane 1). RNA gave little or no hybridization signal (Fig. 2B, lane 1). However, placental poly(A)+ RNAs showed no hybridiza- With the human fetal insulin-related probe, a major band, corresponding to a molecular size of 8-10 S, appears with placental RNAs from first trimester and term normal preg- 1 3 4 S Mr nancies and from term pregnancies of diabetic mothers. X 10 3 However, larger species which hybridize more weakly are -:...... apparent with all placental poly(A)+ RNA samples. It is also -96 clear (Fig. 2B, lane 4) that poly(A)+ RNA from term diabetic pregnancy contains quantitatively more of the sequences that hybridize strongly to the fetal insulin-related DNA probe. A cDNA probe used as an internal control gave a similar hybridization signal with mRNAs from term placentas of both normal and diabetic _ _, _ _t~~~~~~~3 pregnancies (23). DISCUSSION All of our placental poly(A)+ RNA samples contained se- quences that hybridized to the fetal pancreatic insulin-relat- s ed DNA under conditions where only RNAs with substantial regions of .80% sequence homology would be expected to hybridize. Further, our experiments suggest that although these sequences are present both in first trimester and term placentas, the quantity of insulin-related sequences may vary with different pathophysiological conditions. From comparison with the hybridization results (not shown) of probing placental RNA preparations containing a known percentage of human placental lactogen, RNA, we estimate that the insulin-related sequences represent 0.03%-0.1% of FIG. 1. NaDodSO4/PAGE of cell-free translation products of the total poly(A)+ RNA in placenta. Since this quantity of poly(A)+ RNAs (1 jig) from placentas of first trimester (lane 1) and RNA could code for a substantial amount ofprotein, term normal pregnancy (lane 2) and term overt diabetic pregnancy poly(A)+ (lane 3) and from adult pancreas (lane 4). The 14C-labeled molecular the product may well be a regulatory or growth-type weight standards (lane 5) are phosphorylase b (Mr 96,000), carbonic hormone necessary for early embryonic development as well .X .. ..;sc m~~~~~~~~~~ as other regulatory functions. Some of the known insulin- anhydrase (Mr 30,000), cytochrome (Mr 12,000), and insulin (Mr 3000). related growth factors, which have amino acid sequences Downloaded by guest on October 1, 2021 3870 Medical Sciences: Liu et al. Proc. NatL. Acad. Sci. USA 82 (1985)

similar to that of proinsulin, are the somatomedins (also 9. D'Ercole, A. J., Applewhite, G. T. & Underwood, L. E. known as insulin-like growth factors I and II, or IGF-I and (1980) Dev. Biol. 75, 315-328. these RNAs code for 10. Adams, S. O., Nissley, S. P., Handwerge, S. & Rechler, IGF-II). Alternatively, poly(A)+ may M. M. (1983) Nature (London) 302, 150-153. as yet unrecognized growth-promoting regulatory factors. 11. Sara, V. R., Hall, K., Misaki, M. & Fryklund, L. (1983) J. Whether their possible effects are mediated through their Clin. Invest. 71, 1084-1094. own receptors or by their ability to bind to insulin receptors 12. Shelley, H. J., Bassett, J. M. & Milner, R. D. G. (1975) Br. (24-27) awaits purification and identification of the se- Med. Bull. 31, 37-43. quences for further studies. 13. Bassett, J. M. & Jones, C. T. (1976) in Fetal Physiology and Insulin-related genes expressed in placenta may be analo- Medicine, eds. Beard, R. W. & Nathanielsz, P. W. (Saunders, gous to those of the globin-gene cluster (28, 29), in which y- London), pp. 158-172. globin is expressed in and a- and f3-globins are synthe- 14. Espinosa de Los Monteros, M. A., Driscoll, S. G. & Steinke, in the bone marrow after the J. (1970) Science 168, 1111-1112. sized only parturition. Thus, 15. Wolf, H., Sabata, V., Freriches, H. & Stubbe, P. (1969) Horm. first expression of the insulin multigene family may occur in Metab. Res. 1, 274-275. placenta, and only later, after organogenesis, may the 16. White, P. (1949) Am. J. Med. 7, 609-616. expression of other sequences from the insulin gene family 17. Chirgwin, J. M., Pzybyla, A. E., MacDonald, R. J. & Rutter, occur in the fetal pancreas, brain, and liver. W. J. (1979) Biochemistry 18, 5294-5299. Newborns of diabetic women are usually overweight due 18. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, to macrosomia (30). Pedersen (31) proposed that this charac- 1408-1412. teristic fetal overgrowth was a consequence of maternal hy- 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. perglycemia causing fetal and hyperinsuline- 20. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201- mia. Experiments utilizing fetal sheep preparations have 5205. 21. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) shown that insulin increases fetal glucose uptake as well as J. Mol. Biol. 113, 237-251. fetal glucose utilization (13, 14, 32, 33). Studies of many ani- 22. Liu, K.-S. (1983) Dissertation (Case Western Reserve Univer- mal tissues in culture, including human tissues, have demon- sity, Cleveland, OH), pp. 24, 134, & 135. strated that insulin stimulates glucose uptake and protein 23. Mills, N. C., Gyves, M. & Ilan, J. (1985) Mol. Cell. Endo- synthesis in fetal skin, muscle, brain, adrenal, and (2- crinol. 39, 61-69. 4, 35-41). Since fetal pancreas exhibits none or only a limit- 24. Zapf, J., Schoenle, E. & Froesch, E. R. (1978) Eur. J. Bio- ed response to acute changes in fetal blood glucose levels chem. 87, 285-296. (12-14), the regulation of glucose metabolism in the fetus 25. Zapf, J., Rinderknecht, E., Humbel, R. E. & Froesch, E. R. may be controlled by a hormone of extrapancreatic origin. (1978) Metabolism 27, 1803-1828. 26. Poggi, C., Le Marchand-Brustel, Y., Zapf, J., Froesch, E. R. Further, since glucose utilization by the fetuses of diabetic & Feychet, P. (1978) Endocrinology 105, 723-730. mothers is more rapid than normal, other sources of insulin- 27. Zapf, J., Schoenle, E., Waldvogel, M., Sand, I. & Froesch, related factors may play an important role. E. R. (1981) Eur. J. Biochem. 113, 605-609. 28. Efstratiadis, A., Posakony, J. W., Maniatis, T., Lawn, R. M., We thank Dr. L. Villa-Komaroff for providing the cDNA probe O'Connell, C., Spritz, R. A., DeRiel, J. K., Forget, B. G., for human insulin (pUC8I-1) and the insulin-related probe (pBM-4) Weissman, S. M., Slightom, J. L., Blechl, A. E., Smithies, from fetal pancreas. We are also grateful to Dr. Villa-Komaroff for O., Baralle, F. E., Shoulders, C. C. & Proudfoot, N. J. (1980) the sequence of pBM-4 (in ref. 22). Part of this work was submitted Cell 21, 653-668. to Case Western Reserve University by K.-S.L. in partial fulfillment 29. Proudfoot, N. J. & Maniatis, T. (1980) Cell 21, 537-545. of the requirements for the degree of Doctor of Philosophy. This 30. Hill, D. E. (1976) Prog. Clin. Biol. Res. 10, 127-139. work was supported by National Institutes of Health Grants 31. Pedersen, J. (1975) in Carbohydrate Metabolism in Pregnancy HD14072 and HD11586, the American Association, and and the Newborn, eds. Sutherland, H. W. & Stowers, J. M. Juvenile Diabetes Foundation Research Fellowship 383227. (Churchill-Livingston, London), pp. 247-273. 32. Paxson, C. L., Jr., Morries, F. H., Jr., & Adcock, E. W. 1. 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H. (1973) Pediatr. 7. Chez, R. A., Mintz, D. H. & Epstein, M. F. (1974) Am. J. Ob- Res. 7, 787-793. stet. Gynecol. 120, 690-696. 40. Rabain, F. & Pican, L. (1974) Horm. Metab. Res. 6, 376-380. 8. Adam, P. A. J., King, K. C., Schwartz, R. & Teramo, K. 41. Cooke, P. S. & Nicoll, C. S. (1984) Endocrinology 114, 638- (1972) J. Clin. Endocrinol. Metab. 34, 772-782. 643. Downloaded by guest on October 1, 2021