© A c t a Endocrinologies (Copenh) 1990, 122,3: 323-328
Erythropoiesis, serum erythropoietin, and serum
I G F - Iin rats during accelerated growth
Armin Kurtz, Robert Matter, Kai-Uwe Eckardt and J ü r g e n Zapf
1Physiologisches Institut der Universität Zürich, and Stoffwechsellabor1, Medizinische Klinik, Universitätsspital, Zürich, Switzerland
Abstract I n this study we have e x a m i n e d the correlation between activity o f erythropoiesis a n d serum concentra- tions o f e r y t h r o p o i e t i n a n d insulin-like growth factor I i n male a n d female rats d u r i n g accelerated growth (day 30-90). We f o u n d that fractional i n c o r p o r a t i o n o f i r o n into newly f o r m e d r e d blood cells was linearily correlated with body weight gain. T o t a l i r o n incorporation into newly f o r m e d r e d blood cells reflecting total daily r e d cell formation increased almost linearily between day 25 a n d 80 after b i r t h i n both sexes. W h i l e serum erythropoietin concentrations decreased i n the time interval investigated (25-120 days), serum IGF-I levels increased i n both sexes between day 25 a n d 55. I n this p e r i o d , i n d i v i d u a l values of total i r o n i n c o r p o r a t i o n into r e d b l o o d cells a n d serum IGF-I concentrations were linearily correlated. O u r ob- servations support the concept that IGF-I rather than erythropoietin modulates erythropoiesis d u r i n g acceler- ated growth a n d thus manages a p r o p o r t i o n a l increase i n body mass a n d oxygen transport capacity.
It is well established that red cell mass increases in strict proportion to the body mass during the growth period of mammals (1). This proportional- ity is physiologically important because it ensures that increased oxygen consumption is matched by an increased oxygen transport capacity. However, the way by which this adaptation is brought about is unknown. Since erythropoiesis in the adult is pri- marily controlled by erythropoietin (2), one could assume that this hormone is of major importance also within this growth process. Another candidate
for the adapation is insulin-like growth factor I.
IGF-I is considered to mediate the growth promot- ing activities of growth hormone (3) and thus to govern growth of the organism. IGF-I has been found to enhance the proliferation of erythroid precursors in vivo (4) and in vitro (5-8). Recent evidence indicates that application of IGF-I to hy- pophysectomized rats leads to a proportional sti- mulation of erythropoiesis and body growth (9).
The relation between the rate of erythropoiesis and serum levels of IGF-I and erythropoietin in intact animals during the growth period is unknown. In order to distinguish possible roles of IGF-I and ery- thropoietin in the regulation of erythropoiesis during growth we have determined
5 9Fe incorpo- ration into red blood cells, serum IGF-I, and serum erythropoietin levels in normal growing rats.
The results obtained are supportive to the con- cept that erythropoiesis during growth is governed by IGF-I.
Materials and Methods
Animals
Sprague-Dawley rats (Ivanovas, Kissleg, F R G ) were b r e d and g r o w n i n the local animal house. T h e animals h a d free access to standard chow (Altromin®) a n d water.
Weights o f animals were controlled daily at n o o n .
59Fe-incorporation into red blood cells
Rates o f erythropoiesis were determined by measuring i r o n i n c o r p o r a t i o n into newly f o r m e d r e d blood cells.
Rats were injected i p with 2uGi/100 g 5 9F e - c h l o r i d e (spe- cific activity 10uGi/ng); 48 h after the injection the rats were anesthesized with ether a n d bled by heart puncture.
T h e 48-h interval was chosen to avoid interference with age-dependency o f i r o n kinetics (10). B l o o d was collected i n n o n - h e p a r i n i z e d plastic tubes. S e r u m was stored at
— 20°C. T h e fractional : , 9F e i n c o r p o r a t i o n into r e d blood cells ( R B C ) was calculated according to the following gen- erally used f o r m u l a : 5 0F e i n c o r p o r a t i o n (%) = (radioac- tivity p e r m l o f blood) X blood volume (ml) X 100/(total radioactivity injected). B l o o d volume was calculated ac- c o r d i n g to published data (I). T o t a l ;'9F e i n c o r p o r a t i o n into R B C was calculated as (total radioactivity injected) x
5 9F e i n c o r p o r a t i o n (%)/100.
Determination of serum erythropoietin concentrations S e r u m e r y t h r o p o i e t i n levels were determined by radio- immunoassay as described previously (11). Erythropoie- tin-enriched rat serum was used as standard which h a d been calibrated i n the e x h y p o x i c mouse assay for erythro- poietin (11). Eadh sample was measured i n duplicate.
Determination of serum IGF-I concentrations
S e r u m I G F I was d e t e r m i n e d by radioimmunoassay (12) using rabbit antiserum batch 6656/251074 (gift from late
D r Reber, H o f f m a n n - L a Roche, Basel, C H ) against h u m a n I G F - I (final d i l u t i o n 1:20 000). I G F - I was sepa- rated f r o m I G F carrier proteins by gel permeation on a Sephadex G-50 c o l u m n i n 1 mol/1 acetic acid. Separated I G F was lyophilized and stored at - 2 0 ° C . After reconsti- tution with 1 m l P B S , p H 7.4, containing 0.1% (w/v) h u m a n serum a l b u m i n , all samples were assayed at 3 dif- ferent dilutions.
Results
The development of body mass of Sprague-Dawley rats with aging is characterized by a sigmoidal curve (Fig. 1). A period of accelerated growth occurs be- tween the 30th and 80th day after birth in both sexes. In this period, body weight gain ranges be- tween 4.4 and 7,4 g/day in male and between 3.4 and 4.7 g/day in female rats. The age dependency of body weight gains for both sexes is shown in Fig.
1 (insert).
As indices for the activity of erythropoiesis both fractional and total : ) 9Fe incorporation into newly formed red blood cells were determined. Frac- tional 5 9F e incorporation was considered to reflect the specific activity of erythropoietic tissues (re- lated to constant body mass), whereas total 5 9Fe in-
10 30 50 70 90 110 130 150 170 age (days)
Fig. 1.
B o d y weight of male and female Sprague-Dawley rats as a function o f age. Weights were d e t e r m i n e d daily. T h e values are shown for different ages as mean ± SEM ( N = 20 to 30 for both sexes). Lines between the means were drawn by inspection (•) male, (o) female.
Insert: body weight gain (b.w.g.) at different ages for male (•) a n d female (o) rats.
10 - 60
c o
« 50 o Q 8 ? 4 0
10 50 100 150 age (days)
F,g. 2.
Fractional (upper panel) and total (lower panel) o 9F e i n - corporation into newly f o r m e d r e d «jlood cells ( R B C ) i n male (•) a n d female (o) rats at different ages. Data are mean ± SEM ( N = 9 to 12 for both sexes).
corporation reflects the absolute amount of red cells formed. The fractional incorporation rate of
59
Fe into red blood cells had a value of around 57%
for both sexes at day 25 after birth (Fig. 2 upper panel) and declined to a value of around 30% with increasing age.
5 9Fe incorporation rates in male an- imals decreased at a slower rate than those of female rats. Fractional
5 9Fe incorporation rates and body weight gain were linearily correlated in both sexes (
5 9Fe incorporation (%) = 28.1 + 3.2 x body weight gain (g/day); r = 0.95). The total incorpo- ration of
5 9F e into red blood cells increased almost linearily between day 30 and 70 (Fig. 3 lower panel) and approached a plateau with increasing age. The plateau in male rats was about 70% higher than that reached in female animals.
Serum-erythropoietin concentrations in both sexes decreased with increasing age in the time in- terval investigated (day 25-120) (Fig. 3, upper panel). Serum IGF-I concentrations on the other
10 -
l I I L _
10 50 100 150 age ( d a y s )
F,g.3.
Immunoreactive serum e r y t h r o p o i e t i n (Epo) (upper panel) a n d serum I G F - I concentrations o f male (•) a n d female (o) rats at different ages. Data are mean ± SEM (N = 7 to 9 for both sexes).
hand increased almost linearily during the phase of growth acceleration (Fig. 3, lower panel). Starting from the same level at day 25, IGF-I increased to around 135 ug/1 in male and to around 95 ug/1 in female rats. After the phase of accelerated growth, IGF-I levels tended to fall slightly in both sexes. In Fig. 4 the correlation between individual serum IGF-I levels and total rates of iron incorporation during the period of accelerated growth is shown.
Both parameters are linearily correlated.
Discussion
In this study we have investigated the correlation between the activity of erythropoiesis, serum ery- thropoiesis, and serum IGF-I levels in growing rats.
The results show that fractional
5 9Fe incorporation,
indicating specific erythropoietic activity, declines
steadily from the neonatal period and onwards
4
CO
o
" 3 E Q
O
2 1 o
1 0 5 0 100 150 IGF I O j g/ I )
/ ^ j . 4.
Relationship of i n d i v i d u a l values for total incorporation
of 5 9F e into newly f o r m e d r e d blood cells to serum I G F - I
levels i n rats d u r i n g accelerated growth. L i n e a r regres- sion as indicated by the dashed line: total r>'*Fe incorpo- ration (cpm 10~6) = - 0 . 2 6 6 + 0.0166 X [IGF-I] (ng/1);
r = 0.81, p < 0 . 0 0 0 1 . (•) male, (o) female animals.
(Fig. 2). This fall of iron incorporation rates is at- tenuated during the phase of accelerated growth, which occurs between 30 and 80 days after birth (Fig. 1). After the neonatal period, fractional
5 9Fe incorporation rates were linearily correlated to the body weight gains. This observation supports the concept that fractional
, 9Fe incorporation rates re- flect the sum of red cell formation necessary to compensate daily red cell degradation and to refill the daily expanding circulatory system. Extrapola- tion of the linear regression curve yielded a frac- tional '"'Fe incorporation of 28% for non-growing animals. This value is similar to the
0 9Fe incorpo- ration rates of growth-arrested, hypophysecto- mized rats (9) determined under comparable ex- perimental conditions.
Total red cell formation is reflected by the total incorporation of iron (Fig. 2, lower panel). The curve obtained fits well with direct calculations of daily red cell formation in rats during growth (1).
Red cell formation increased by a factor of around 10 and 6 in male and female rats, respectively, during the period of accelerated growth. This in- crease in red cell formation was associated with a fall of serum erythropoietin levels (Fig. 3, upper panel). The observed decrease of serum erythro- poietin with age is in accordance with observations by Clemons et al. (13) and Bozzini et al. (14), who also found a difference in serum erythropoietin between neonatal and adult rats. Most likely this decrease of erythropoietin formation is caused by recovery from the anemia which develops during the neonatal period, and which is completely com- pensated 70 days after birth in rats (1).
Considering the inverse relationship of serum erythropoietin to total red cell formation during accelerated growth, it seems unlikely that erythro- poietin only governs the expansion of red cell volume in this period. The conclusion is in accord- ance with previous observations (15, 16) that ery- thropoiesis during rapid growth cannot be blunted by plethora, a condition which is known to suppress erythropoietin formation in the adult (17). Re- cently it has been shown that plethora in fact blunts erythropoietin formation but not the erythropoie- tic activity present in the serum of mice during rapid growth (18).
The temporal relation between IGF-I levels and body weight gain as observed in this study is in accordance with the concept that IGF-I governs the increase in body and organ mass during acceler- ated growth (3). Our findings show that red cell formation and serum IGF-I concentrations are also directly correlated during accelerated growth (Fig.
4). It is tempting to speculate therefore that IGF-I causes proliferation and expansion of hemopoietic tissues in proportion to the increase of body mass.
Although our findings cannot prove a causality, they could provide an essential link in the under- standing of a possible role of IGF-I in the regula- tion of erythropoiesis during growth. Whether this effect is mediated by systemic IGF-I or by locally released IGF-I (19) cannot be answered yet.
The present results show that red cell formation
and body weight gain are linearily correlated, and
that the individual rates of red cell formation are
directly correlated with serum IGF-I levels. Re-
cently it was demonstrated that infusion of IGF-I
into growth-arrested hypophysectomized rats
causes a proportional increase in growth and ery-
thropoiesis (9). IGF-I, moreover, has been shown to
enhance the proliferation of erythroid precursors
in vivo (4) and in vitro (5-8). In addition, the ex- istence of IGF-I receptors on erythroid precusors (20) as well as the existence of IGF-I in the direct environment of erythroid precursors have been demonstrated in vivo (21). The sum of these ob- servations supports the idea that IGF-I governs erythropoiesis during the period of accelerated growth and thus manages a proportional increase in body mass and oxygen transport capacity.
Such a role of IGF-I would not contest an essen- tial involvement of erythropoietin in the mainte- nance of erythropoiesis in growing animals as sug- gested by a recent study (14). In fact, there is some evidence that IGF-I requires the presence of ery- thropoietin for its mitogenic effect on erythroid precusors from adult mammals (6-8). A modular tory role of IGF-I on erythropoiesis maintained by erythropoietin would also be supported by recent findings that serum erythropoietin levels do not change during puberty in humans (22, 23), a situ- ation in which IGF-I levels rise and red cell masses expand.
Acknowledgments
The authors wish to thank C . B a u e r for critical reading o f the manuscript. T h e expert technical assistance p r o v i d e d by W. Gehret a n d I. G r a n d is gratefully acknowledged.
One of us ( K - U E ) acknowledges a fellowship from the Deutsche Forschungsgemeinschaft. T h i s study was sup- ported by a grant o f the Swiss N a t i o n a l Science F o u n d a - tion (grant 3.600-0.86).
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Received September 18th, 1989.
A c c e p t e d N o v e m b e r 16th, 1989.
D r A r m i n K u r t z , Physiologisches Institut, U n i v e r s i t ä t Z ü r i c h , Wintherthurerstrasse 190, C H - 8 0 5 7 Z ü r i c h ,
Switzerland.
