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Journal of Clinical Endocrinology and Metabolism Vol. 55, No. 6
Copyright © 1982 by The Endocrine Society Printed in U.S.A.

Natural and Biosynthetic Insulin Stimulates the Growth

of Human Erythroid Progenitors in Vitro*
NOELLE BERSCH, JEROME E. GROOPMAN, AND DAVID W. GOLDE
Division of Hematology-Oncology, Department of Medicine, UCLA School of Medicine,
Los Angeles, California 90024

ABSTRACT. High concentrations of insulin are known to aug-
ment the growth of various cell types in vitro. We examined the
effect of a purified porcine insulin and biosynthetic human
insulin produced in E. coli on the growth of human erythroid
progenitors in vitro. Both insulins stimulated peripheral blood
erythroid colony formation within the physiological range. An
approximately 2-fold augmentation in colony formation was seen
at insulin concentrations of 8 ng/ml, and as little as 0.1 ng/ml
(0.17 nM) caused detectable stimulation of colony formation.
The effect of subnanomolar concentrations of insulin on eryth-
ropoiesis in vitro suggests that insulin could modulate erythro-
poiesis in vivo. Human responsiveness to insulin's growth-pro-
moting activity can be directly assayed in vitro using peripheral
blood. (JClin Endocrinol Metab 55: 1209, 1982)

T HE GROWTH of erythroid precursors in vitro is

affected by numerous endocrine hormones (1). Hor-
mones that potentiate the growth of large human periph-
ylcellulose was used (3,5,7,8). The complete medium contained
0.8% Methocel E4M premium (Dow Chemical Co., Midland,
MI), Iscove's modification of Dulbecco's medium (Irvine Sci-
eral blood red cell colonies in vitro [known as burst- entific, Santa Ana, CA), 30% fetal calf serum (selected lot), 10~4
forming units-erythroid (BFU-E)] include steroid hor- M a-thioglycerol (Calbiochem, La Jolla, CA), and penicillin-
streptomycin. One-half unit per ml sheep plasma erythropoietin
mones of the androgen and glucocorticoid classes, thyroid
(Connaught, Swiftwater, PA) or human urinary erythropoietin
hormones, GHs, and related growth polypeptides, such (SA, 44 U/mg protein, provided by the NHLBI) was added to
as human chorionic somatomammotropin (1-6). Insulin each culture. Three thousand nucleated buffy coat cells were
is known to bind to various types of hemic cells, and the cultured in each 100-jul well.
binding affinity and number of receptors are believed to Highly purified porcine insulin (lot 615-17J-256) and biosyn-
reflect insulin responsiveness in the organism. We report thetic human insulin (lot 615-70N-174-10) were a gift of Dr.
here that physiological concentrations of highly purified Mary A. Root (Eli Lilly Co., Indianapolis, IN). The porcine
porcine insulin and human insulin produced in E. coli by insulin had a potency of 28.5 U/mg and a water content of 5%.
recombinant technology have a potent growth-promoting It contained less than 10"3% proinsulin and less than 10"4%
activity on circulating human erythroid progenitor cells glucagon. The biosynthetic human insulin was produced in E.
in vitro. coli and was identical to pancreatic human insulin, which differs
from porcine insulin only at the amino acid B-30 (which is
threonine in man and alanine in the pig). The insulin was
Materials and Methods dissolved in 0.01 N HC1 and further diluted with PBS. Appro-
Peripheral blood was obtained from healthy young adult priate concentrations of insulin were added to the cultures in
volunteers after obtaining the appropriate informed consent. 10-jul volumes per microtiter well. Control cultures contained
Blood was collected into preservative-free sterile heparin, and only diluent material. The microtiter plates were incubated at
the buffy coat cells were obtained by centrifugation. The re- 37 C in high humidity with 8% CO2 in air, and the BFU-E (large
maining erythrocytes were lysed with ammonium chloride-Tris hemoglobinized colonies containing at least 50 cells) were enu-
buffer. The buffy coat cells were washed once with Iscove's merated at 10-14 days using a Leitz Diavert microscope (E.
medium and 10% fetal calf serum, and the final cell concentra- Leitz, Inc., Rockleigh, NJ). Duplicate and triplicate cultures
tion was adjusted to 3 x 106/ml. A microtiter modification of were established for each concentration point.
the plate technique for culturing erythroid precursors in meth-
Results
Received April 2, 1982.
Address requests for reprints to: Dr. David W. Golde, Division of When the same healthy volunteer was used in 18
Hematology-Oncology, UCLA School of Medicine, Center for the separate experiments, the mean colony formation was
Health Sciences, Los Angeles, California 90024.
* This work was supported by USPHS Grants CA-30388 and CA- 30/well, with a SE of ±2 and an actual range of 20-58
32737 awarded by the NCI, DHHS, and USPHS Grant RR-00865. colonies/well. Another healthy volunteer studied in 8

1209

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1210 COMMENTS J C E & M < 1982
Vol 55 < No 6

separate experiments showed a mean colony formation active in vivo (15), and we have shown that it has a
of 31, with a SE of ±4 and an actual range of 21-59 potent growth-promoting activity for erythroid progeni-
colonies. A total of 33 control experiments were per- tors in vitro. Half-maximal activity is seen at about 4 ng/
formed on 8 volunteers, resulting in a mean colony for- ml (0.7 nM), and the dose-response curve for the biosyn-
mation of 30, with a SE of ±2 and an actual range of thetic human insulin is almost identical to that of a
19-59. Figure 1 shows the results of 12-20 dose-response highly purified porcine insulin.
experiments performed in duplicate or triplicate on nor- Substantial work has been done on insulin binding to
mal volunteers. As little as 0.1 ng/ml (0.17 nM) produced circulating monocytes and erythrocytes (16-19). The
detectable augmentation in the BFU-E growth assay. number of insulin-binding sites seems to correlate with
Peak activity was seen at about 8 ng/ml, resulting in an metabolism insulin sensitivity measured by other tech-
approximately 2-fold increase in BFU-E. Substantially niques. There is some evidence that the growth-promot-
less stimulating activity was seen at 10 ng/ml, and a ing effects of insulin are modulated through a different
further decrease was observed at 100 ng/ml. Insulin also receptor than that regulating the metabolic effects of
stimulated normal human BFU-E in a serum-substituted insulin (20). The response of peripheral blood BFU-E to
assay system (data not shown) (3, 8). very low concentrations of insulin suggests that insulin is
not acting through an insulin-like growth factor receptor
Discussion in this system. Our studies as well as those of others (21)
suggest that insulin may be important in modulating
Insulin is known to have important growth-promoting erythropoiesis. More importantly, however, the effect of
effects in vitro (9). In most studies, however, very high insulin on erythroid progenitor proliferation in vitro may
concentrations of insulin are used (usually >1 jug/ml) reflect the in vivo level of responsiveness to the growth-
which do not correspond to insulin concentrations occur- promoting effect of insulin.
ring in vivo. We have noted a growth-stimulating effect
of insulin on normal human erythroid progenitors of the
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