insulin-like growth factor-i and insulin-like growth factor-binding protein in the toad, bufo...

GENERAL AND COMPARATIVE ENDOCRINOLOGY 78, 263-272 (1990)

Insulin-like Growth Factor-l and Insulin-like Growth Factor-Binding Protein in the Toad, Bufo woodhousei

MARTHA K. PANCAK-ROESSLER* AND PHILLIP D. K. LEE*?

*The Children’s Hospital Kempe Research Center, Denver, Colorado 80218, and tThe University of Colorado Medical School, Denver, Colorado 80262

Accepted July 26, 1989

Molecular weight characteristics and plasma concentrations of insulin-like growth factor-I (IGF-I) and its binding protein (IGF-BP) were investigated in the toad, Bufo woodhousei. IGF-I and IGF-BP were measured by radioimmunoassay (RIA, K,, = 0.37 k 0.04 &ml) and charcoal-separated ligand binding assay, respectively, in male toad plasma and adult male human donor plasma using a synthetic human IGF-I standard. Prior to the IGF-I RIA, samples were acid-ethanol extracted. Molecular weight characteristics were determined using size exclusion chromatography. At neutral pH (PH = 7.4), IGF-I immunoreactivity and IGF-BP eluted at molecular weight >66 kDa in both toad and human plasma. Acid chromatography @H -3) resulted in the separation of IGF-I from its binding protein and consequently a shift of IGF-I immunoreactivity to the low molecular weight fractions (-8 kDa) for both toad and human. IGF-BP activity shifted to molecular weight -50 kDa. Toad plasma IGF-I and IGF-BP activity exhibited differences according to season: IGF-I levels were low in the spring (March = 0.48 f 0.11 ng es/ml), increased progressively to reach a peak in July (5.84 2 2.5 ng eqfml), and decreased to low levels again in the fall (October = 0.60 rf: 0.08, November = 0.45 + 0.09 ng eq/ml). Plasma IGF-BP activity demonstrated a similar pattern (March = 17.4 + 2.5, July = 35.0 f 2.4, November = 12.6 ? 3.2% specific binding). IGF-I was produced for at least 72 hr when toad liver explants were cultured in serum-free medium, indicating that the liver is a source of IGF-I in anurans. 0 1990 Academic

Press, Inc.

Insulin-like growth factors (IGF-I and IGF-II) are - 7.5kDa peptides which are structurally similar to insulin and mediate the mitogenic actions of growth hormone (GH) (Van Vliet et al., 1983; Rinderknecht and Humbel, 1978a,b; Blundell et al., 1978; Marquardt et al., 1981; Jansen et al., 1983, 1985; Rotwein, 1986). IGF immunoreactivity and bioactivity have been detected in the sera of representatives from all verte- brate classes (Van den Brande, 1974; Pof- fenbarger et al., 1976; Rothstein et al., 1980; Daughaday et al., 1985; Wilson and Hintz, 1982; Shapiro and Pimstone, 1977) and according to Rinderknecht and Humbel

i To whom correspondence and reprint requests should be addressed at Diabetes Center, Baylor Col- lege of Medicine, 8080 N. Stadium Dr., Houston, TX 77054.

(1978a), the progenitor IGF may have arisen in prevertebrate evolution. IGF-II immunoreactivity has been found in higher vertebrates, but not fish or amphibians, suggesting that a gene duplication giving rise to two separate IGFs occurred at the time of reptilian evolution (Daughaday et al., 1985).

Unlike other peptide hormones, IGFs circulate as macromolecular complexes with specific plasma-binding proteins. Vir- tually no free IGF peptide can be detected in serum. In higher vertebrates, IGFs are found in two primary serum fractions: a 30- kDa fraction containing most of the unsaturated IGF-binding capacity and only a small proportion of endogenous IGF and a 150- to 200-kDa complex which contains most of the IGF and very little unsaturated binding activity (Hintz, 1984; Furlanetto,

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264 PANCAK-ROESSLER AND LEE

1980; Lee et al., 1988). This second class of the IGF-binding complex is GH dependent and predominates in adult plasma (Cohen and Nissley, 1976; Moses et al., 1976).

In mammals, IGFs stimulate skeletal growth and cartilage proteoglycan synthesis and possess insulin-like and mitogenic activity in nonskeletal tissues (Zapf et al., 1984). Furthermore, it is apparent that the IGF-binding proteins regulate IGF action (Ooi and Herington, 1988). Although much is known about IGFs in mammals, very few studies on IGFs or IGF-binding proteins have been performed in lower vertebrates. In the present study, we examined the presence of IGF-BPS and IGF-I in several species of anurans. Since amphibians from temperate climates have predictable seasonal periods of breeding and growth, we measured plasma levels of IGF-I and unsaturated IGF-BP activity in a toad, B&o woodhousei, at different times of the year in order to determine whether circulating levels reflect the changes in the physiology of the animal.

The mammalian liver is a source of IGF-I and can be stimulated to produce IGF-I by GH or PRL (Francis and Hill, 1975; Holder and Wallis, 1977). In order to determine whether the amphibian liver produces IGF- I, we studied in vitro production of IGF-I by cultured toad livers in the presence or absence of bovine growth hormone (bGH) or bovine prolactin (bPRL). Our studies indicate the presence of an IGF-I-like peptide and serum IGF-binding activity in anurans. The characteristics of the IGF-complex are similar to that of humans.

MATERIALS AND METHODS

Materials. Rabbit polyclonal IGF-I antibody (UBK487), kindly provided by Drs. L. E. Underwood and J. J. Van Wyk (Division of Pediatric Endocrinol- ogy, University of North Carolina, Chapel Hill, NC), bGH, and bPRL were distributed by the National Hor- mone and Pituitary Program of the NIDDK, Univer- sity of Maryland School of Medicine. Synthetic re- combinant DNA-derived human IGF-I was purchased from Bachem Inc. (Torrance, CA). (3-[izsI]-

iodotyrosyl) IGF-I,[Th?T was obtained from Amer- sham Corp. (Arlington Heights, IL) and had an initial specific activity of -2000 Ci/mmol.

RIA-grade, insulin-free bovine serum albumin (BSA) was obtained from Miles Laboratories, Inc. (Kankakee, IL). Dulbecco’s phosphate-buffered saline (PBS), pH 7.4, and Tween 20 (Monolaurate) were purchased from Sigma Chemical Co. (St. Louis, MO). Agarose-immobilized goat anti-rabbit IgG was obtained from Bio-Rad Laboratories (Richmond, CA).

Animals. Male toads (B. woodhouse~) were collected near Brighton, Colorado, during the breeding season (May-June) and maintained in an outdoor en- closure located on the campus of the University of Colorado, Boulder. The enclosures contained natural ground cover, pans of water, and insects. The animals’ diets were supplemented with mealworms and crick- ets. At monthly intervals, blood samples were taken by cardiac puncture from 6 to 11 animals using hepa- rinized syringes. The plasma was stored at - 80” until assayed for IGF-I and IGF-BP activity. Samples for the month of March were obtained from hibernating toads in an unheated greenhouse. Aliquots of toad plasma from the months of June and July were pooled and used for the chromatography.

Adult male human donor plasma was obtained from the blood bank of The Children’s Hospital, Denver, Colorado. Citrated phosphate dextrose adenosine (CPDA) was used as an anticoagulant. Aliquots from this donor were used in all of the assays and chromatography procedures.

Size exclusion chromatography. Pooled toad plasma and the human plasma were subjected to the following chromatography procedures. One-milliliter samples were applied to a Pharmacia Sephacryl-200HR column (1.6 X 100 cm) and eluted at 1 ml/mitt with either PBS, pH 7.4, or 1 .O% (v/v) formic acid (pH -3). The column was precalibrated under both neutral and acid elution conditions using dextran blue, bovine serum albumin (66 kDa), soybean trypsin inhibitor (25 kDa), lysozyme (14 kDa), and human insulin (6 kDa). The plasma samples used for acid chromatography were first diluted 1: 1 in 2.0% formic acid and incubated for 1 hr at room temperature before application to the column. Frac- tions (3.25 ml) were collected for the neutral column and 6.0~ml fractions for the acid column. Fractions from the acid column were lyophylized and reconstituted in PBS (pH 7.4) 0.1% Tween 20 and subjected to acid-ethanol extraction prior to assay. Fractions from the neutral column were extracted as described below. For the neutral column, every four fractions (for toad) or two fractions (human) were pooled prior to extraction. For the acid column, every two fractions were pooled for both toad and human before extraction. Protein elution patterns were monitored at A, (neutral column) and A, (acid column).

Sumple extraction method. Since IGF-BPS can in- terfere with the measurement of IGFs, all samples

IGF-I AND IGF-BP IN THE TOAD 265

weresubjected to acid-ethanol extraction prior to the IGF-I RIA (Daughaday et al., 1980). One milliliter of a mixture of 87.5% ethanol and 12.5% 2 N formic acid (v/v) was mixed with 0.25 ml of sample. The mixture was allowed to stand for 30 min at room temperature and then centrifuged at 10,000 rpm for 3 min. One milliliter of the supernatant was transferred to a clean polypropylene tube and lyophylized. ‘Ibe residue was reconstituted with 0.25 ml of assay buffer and assayed for IGF-I. A recovery experiment was also performed in which either known amounts of IGF-I were added to human plasma or a known amount of tracer was added to the plasma prior to extraction.

IGF-I radioimmunoassay procedures. ‘he RIA was performed by preincubation of 0. l-ml sample or standard with 0.05 ml antiserum (UBK 487) in PBS (pH = 7.4) 0.1% Tween 20 for 1 hr at room temperature (final titer 1:16,000). Approximately 10,000 cpm of [‘*‘I]IGF-I (0.05 ml) in assay buffer was added and the mixture was incubated overnight at 4”. Separation of free and bound IGF-I was achieved by incubation for 3 hr with 0.175 ml of an agarose-immobilized aflinity- purified goat anti-rabbit IgG (1 mg/ml) in PBS (PH = 7.4) containing 0.1% BSA at room temperature, followed by centrifugation at 2300 rpm for 10 min at 4”. The pellets were then counted in a gamma spectrometer. All samples were assayed in duplicate. Nonspe- cific binding was determined in the presence of 500 rig/ml pure hIGF-I. RIA data were analyzed using the RIA Data Reduction Program for the IBM-PC (M. L. Jaffe and Assoc., Silver Spring, MD) (Rodbard, 1984) using a four parameter logistic model. IGF-I levels in anuran plasma are expressed as nanogram equivalents of hIGF-I.

ZGF-binding protein activity assay. Unsaturated IGF-binding protein activity was measured by adding 0.05 ml [1251JIGF-I (-10 K cpm) to 0.45 ml of neutral column fractions, 0.1 ml lyophilized and reconstituted acid column fractions, or 0.05 ml of animal plasma in a final assay volume of 0.5 ml (Hintz et al., 1981). After 18 hr at 4”, 1.0 ml of ice-cold 1% (w/v) activated charcoal in assay buffer was added to the mixture. After a 15-min incubation at 4”, the assay tubes were centrifuged at 2300 rpm for 30 min at 4”. The supematant was decanted into another tube and counted in a gamma spectrometer. The corresponding pellets were also counted and the percentage bound calculated, using the following equation: [(supematant cpm x lOO/ (pellet + supematant cpm)] - % nonspecific binding. Nonspecific binding was determined in the presence of buffer only. For the neutral column, all fractions were assayed separately, whereas acid column fractions (every two) were pooled and assayed.

Liver cultures. In order to measure IGF-I production by toad livers, postbreeding animals were anes- thetized terminally in 0.1% MS-222 (3arninobenzoic acid ethyl ester), and the livers were removed surgi- cally and placed in ice-cold medium (60% MEM Ea-

gle’s medium, Sigma Chemical Co.). The tissue was then diced into -l-mm’ pieces, washed several times in medium, and 6-10 pieces were placed in 24 well plates to produce a final concentration of -15-20 mg tissheJml medium. Liver explants were incubated under sterile conditions for 72 hr in air at room temperature. The incubation medium was removed every 24 hr and replaced. The removed media was assayed for IGF-I and IGF-BP activity. In a separate study, liver cultures were exposed to varying concentrations of bovine GH or PRL (0, 50, 100, 250, 500, and 1000 ngknl). The incubation mixture was removed after 24 hr and assayed for IGF-I and IGF-BP activity as described above.

Statistical analysis. Statistical differences were determined by one-way analysis of variance followed by Duncan’s multiple range test. Significance was accepted at the P s 0.05 level. Descriptive data are pre- sented as the arithmetic mean f SEM.

RESULTS

Serial dilutions of toad and human plasma were assayed and compared to the purified synthetic hIGF-I standard curve. Figure 1 illustrates that after acid-ethanol treatment, toad and human plasma and purified human IGF-I standards gave parallel displacement curves. Recovery of IGF-I was approximately 90% after extraction for both toad and human plasma. IGF-I immunoreactivity and IGF-BP activity were detected in all species of anuran tested. Table 1 summarizes these results.

0 20 40 60 80 100

B/B0 X 100

FIG. 1. Displacement curves showing parallelism between hIGF-I and serial dilutions of toad and human plasma. hIGF-I concentrations are plotted against the left axis while the relative concentrations of plasma are plotted against the right axis. Symbols: Standard hIGF-I (W); human plasma (A); toad plasma (0)‘


TABLE 1 LEVELS OF IGF-I (nghl) AND IGF-BP ACTIVITY

(PERCENTAGE BOUND) IN VARIOUS ANURAN SPECIES

Species

Bufo americanus May male Nov. male

B. marinus Oct. female Oct. male

B. woodhousei Male (range)

Rana catesbeana Aug. female

Scaphiopus couchi July male

Xenopus laevis Captive female

a Not determined.

IGF-I IGF-BP (ng es/ml) + SEM (% bound)

4.25 5.4 0.52 1.4

1.09 f 0.47 (n = 2) 20.3 0.29 f 0.06 (n = 3) 10.8

0.45-5.84 5.0-36.0

1.24 10.2

N.D.” 64.0

2.79 3.5

When toad and human plasma were subjected to neutral size-exclusion chromatography, the major peak of IGF-I activity was detected at >66 kDa (Fig. 2). Correspond- ing with this peak was the major portion of IGF-binding protein activity. A small amount of IGF-I also was found in the low molecular weight fractions of toad plasma. Acid chromatography resulted in the separation of IGF-I from its binding protein and consequently+ the major peak of IGF-I activity shifted from the high molecular weight fractions to the low molecular weight fractions (-7.5 kDa) in both human and toad (Fig. 3). Small amounts of IGF-I immunoreactivity , as well as most of the binding protein activity, was detected in the high molecular weight fractions.

Seasonal variations of IGF-I and IGF-BP were studied (Fig. 4). Because of the heter- ogeneity of variances that existed, the data were log transformed before statistical analysis. Plasma IGF-I concentrations were significantly lower in the spring (March = 0.48 rt 0.11 ng eq/ml) and autumn months (October = 0.60 & 0.08, No- vember 0.45 -C 0.09 ng es/ml). Plasma concentrations of IGF-I began to increase during the breeding season (May-June;, 1.04 + 0.13 and 1.89 2’0-34 ng eq/ml, respectively) and significantly peaked in July, 5.85 rig eq/

ml (F = 6.531, P < 0.001). IGF-I decreased slightly in August and September (2.18 + 0.90, 2.86 + 1.63 ng eq/ml, respectively). IGF-BP activity levels showed a similar pattern to IGF-I levels (Fig. 5), with a sig- nificant peak occurring in July, 34.97% binding (F = 3.807, P s 0.004); however, while IGF-I levels remained somewhat elevated in August and September, IGF-BP activity showed a sharp decrease in August to 14.65% binding and remained low during the autumn and spring months, with values ranging from 12.30 to 17.40% binding.

In vitro liver cultures produced progres- sive amounts of IGF-I for the 72-hr period studied (Fig. 6). Neither GH nor PRL stimulated IGF-I production in a dose-de- endent manner over the dose range studied (Fig. 7). However, bPRL was more effec- tive in stimulating IGF-I production than bGH, particularly at doses 3250 rig/ml. No IGF-BP activity was detected in the medium either in the presence or absence of bGH or bPRL.

DISCUSSION

Our studies indicate the presence of IGF- I immunoreactivity in B. woodhousei and other anurans. Plasma levels of IGF-I observed are similar to those reported for anurans by other investigators (Rothstein et al., 1980; Daughaday et al., 1985), and we found parallel displacements in the heterologous RIA. Lack of parallelism in a previous study (Rothstein et al., 1980) may have been due to failure to remove IGF-binding proteins prior to the assays. The observed assay characteristics indicate that we are measuring the anuran homologue of human IGF-I rather than IGF-II. However, the true identity of this peptide and its relation- ship to the human IGFs await amino acid and/or cDNA sequencing.

IGF-binding activity was found in all anuran plasma tested (Table l), in contrast to a previous report in B. marinus (Daugha- day et al.. 1985). This discrepency may be


b 60

24

2s 19

76 ii

52 i3

I

26

90

a0 70

60

50

40

30

20

~ 10

0

8 I- x

8 es I

8 7

0 66 130 195

ELUTION VoLuvlE (ml)

FIG. 2. Fractionation of toad and human plasma by size exclusion chromatography. Samples were loaded onto a 1.6 x lOO-cm Sephacryl- 2MlHR column and eluted at 1 ml/min in PBS, pH 7.4. Fractions (3.25 ml) were collected and assayed for IGF-BP (bars). IGF-I was measured in acid-ethanol extracted aliquots of the fractions using RIA (line). The arrows indicate the molecular weights of calibration proteins. (a) Human plasma. (b) B. wmdkmsei plasma.

due to the time of year of sampling or to differences in assay method. In our study, radioiodinated IGF-I was used as the ligand, whereas Daughaday and colleagues used IGF-II. Furthermore, the previous study used Sephadex G-75 acid-chro- matographed fractions, a procedure which may alter the binding activity in serum (un- published observations).

Neutral and acid chromatography provide evidence that anuran and human IGF- I/IGF-binding activity have similar size dis- tributions. As observed in humans (Rapp et

al., 1988; Ooi and Herington, 1980), most of the IGF immunoreactivity in auuran plasma is associated with an acid-dissociable high molecular weight fraction. Therefore, we postulate that the IGF-binding protein detected in our study may be the anuran homologue of the saturated, GH-dependent complex in humans. The presence of high molecular weight IGF-BPS and the absence of low molecular weight IGF-BPS in the summer toads may be related to high GH levels that are thought to exist during their summer growth period (cited in Snyder and


b 30 1 66hD 2s K, 14kD 6h0 I

130

52

26

0

90

60

70

60

50

40

30

20

10

0 0 60 120 180 240 -

ELUTION VOLUME (ml) FIG. 3. Fractionation of human and toad plasma on a 1.6 X NO-cm SephacryL2OOHR column eluted

at 1 ml/min in 1.0% (v/v) formic acid @H -3). Every two fractions were pooled and assayed for IGF-BP (bars) as described under Materials and Methods. NSF-1 was measured by RIA in acid-ethanol extracted aliquots of the pooled fractions using RIA (line). The arrows indicate the elution patterns for the 66-, 25-, 14-, and 6-kDa standards. (a) Human plasma. (b) B. woodhousei plasma.

Frye, 1972; Jorgensen, 1983, 1986; Wind- Larsen and Jorgensen, 1987). Failure to detect a low molecular weight unsaturated IGF-BP in both human and toad is not sur- prising, since this type of IGF-BP is typi- cally at a minimum in adult humans (Bris- mar et al., 1987; Hintz et al., 1981). We cannot exclude the possibility, however, that a low molecular weight IGF-binding protein does not exist in anurans.

Virtually no free IGF-I circulates in mammalian plasma (Hintz, 1984; Zapf et al., 1979). The small peak of IGF-I activity

found in the low molecular weight fractions of toad plasma under neutral pH may be due to use of heparin during blood collec- tion. Heparin may facilitate IGF-I release from the carrier complex at the tissue level prior to its interaction with the target cell (Clemmons et al., 1983).

When human and toad plasma were subjected to acid chromatography, the IGF-BP activity shifted to the -5O-kDa fractions, suggesting a possible conversion of the Ml- kDa binding protein to a -50 to 60-kDa binding protein as described previously


-2’ ’ ’ j L L n J FEB MAR AR? MAY am Jl AU; SEPT OCT No” OEC

MONTH OF YEAR

FIG. 4. Seasonal variation in circulating levels of IGF-I (ng es/ml) in B. woodhousei. Samples were acid-ethanol extracted prior to RIA. Values represented are means 2 SEM (n = 6-11).

(Hintz and Liu, 1980; Hintz, 1984; Baxter, 1988). The shift of IGF-I immunoreactivity to the low molecular weight fractions (-8 kDa) under acidic conditions (Fig. 3) for both toad and human supports the conclusion that IGF-I is complexed to a binding protein(s) in toads, and that the IGF-I mol- ecule has a molecular weight similar to that of humans. The small peaks of IGF-I activity detected in the higher molecular weight fractions could be due to incomplete separation of IGF-I from its binding proteins during acid chromatography or sample extraction, resulting in the interference of these proteins in the RIA (Daughaday et al., 1980, 1987).

The physiological role of the IGFs in lower vertebrates is not known. Human

FEB MAR Awi MAY .JN AL Ale YPT CCT M” EC

MONTH OF YEAR

FIG. 5. Seasonal variation in circulating IGF-BP activity (percentage bound) in B. woodhousei, measured by charcoal-separated l&and-binding assay. Values represented are means + SEM (n = 6-11).

HOURS OF INCUBATION

FIG. 6. Cumulative production of IGF-I in vitro by liver explants of B. woodhousei over a 72-hr period. This is a plot of the mean values minus the values for medium alone.

GH can stimulate an increase in circulating immunoreactive IGF-I in a teleost, Sparus aurata (Funkenstein et al., 1989). Indirect evidence suggests that IGFs mediate the action of GH in frogs as well (Rothstein, 1982). IGF-I activity in the frog lens is abol- ished by hypophysectomy and can be re- stored by human, bovine, or anuran GH, anuran PRL, TSH, or triiodothyronine (Wainwright et al., 1976; Wainwright et al., 1978; Weinsieder and Roberts, 1980). Moreover, growth in the germinative zone of frog lenses is seasonal and dependent upon the hypothalmo-hypophysial axis

_I F 0 250 500 750 1000 1250

GH or PRL DOSE hQ/“d)

FIG. 7. Dose-response curves for the effect of in- creasing bGH or bPRL concentrations on IGF-I production during in vitro culturing of liver explants of B. woodhousei for 24 hr. Symbols: PRL (0); GH (0).


(Rosenbaum and Rothstein, 1972; Rothstein et al., 1975; Rothstein, 1982). Therefore, the available evidence suggests that IGF and GH in lower vertebrates may be interrelated, as they are in mammals.

We found a seasonal variation in IGF-I immunoreactivity and IGF-BP activity in the plasma of B. woodhousei. This anuran lives in a temperate climate, where growth and reproduction occur on a seasonal basis. IGF-I and its associated binding protein were found to be elevated in the postbreeding toads, a period when toads are known to feed and grow rapidly. It has been sug- gested that GH levels may also be elevated during this period (cited in Synder and Frye, 1972; Jorgensen 1983, 1986) and this may account for the increased IGF-I levels as well as the high molecular weight IGF- BP activity. Other hormones may also be involved, such as PRL, TSH, and thyroidal hormones (Wainwright et al., 1976; Wain- wright, 1978; Weinsieder and Roberts, 1980). Further investigations in this area are clearly needed.

In mammals, a major site for synthesis of IGF-I and the low molecular weight unsaturated IGF-BPS appears to be the liver (Daughaday et al., 1976), although recent studies show that these peptides are syn- thesized at multiple tissue sites (D’Ercole et al., 1984; Underwood et al., 1986; Clem- mons et al., 1981; Adashi et al., 1985). Pre- vious studies provide indirect evidence for anuran liver production of IGF-I (Rothstein et al., 1981a, b). The present study demon- strates the production of IGF-I immunoreactivity but not IGF-binding activity by toad livers in vitro. We also found that incubation with bPRL, but not bGH, stimulated IGF-I production over 24 hr. This finding may reflect interspecies difference in GH and PRL structure since mammalian PRLs have been shown to be similar to amphibian GHs (Hayashida et al., 1973). However, human GH has been reported to bind to frog hepatocytes (Weinsieder and Rothstein, 1980), whereas results from

binding studies with ovine PRL have been variable (Carr and Jaffe, 1980; White, 1981). The lack of a dose response to either GH or PRL is also unexplained, but may relate to the relatively short incubation period of sampling or the doses used. Another possibility is that the livers were from toads collected in the autumn months when the number of receptors for GH and PRL may have been too low.

Failure to detect a liver-produced IGF- binding protein and a low MW unsaturated complex in toad plasma raises the possibility that this protein is not produced in anuran livers. Another explanation may be that the experiment was done in the autumn when circulating IGF-BP activity levels are low and GH levels are presumably low. Perhaps the liver is unresponsive to exoge- nous hormone in terms of IGF-BP production at this time of year. Furthermore, the method of assaying for IGF-BP activity may not have been sensitive enough to detect very low levels of production. Further investigations addressing these issues are underway.

In conclusion, the anuran IGF/IGF- binding protein system bears many similar- ities to the human system, providing a po- tentially valuable model for the study of the physiology and evolution of these proteins in vertebrates.

ACKNOWLEDGMENTS We acknowledge Drs. David 0. Norris and Paul G.

Roessler for their valuable comments and suggestions. This study was supported by BRS Award 922 from the University of Colorado Health Sciences Center (PDKL).

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insulin-like growth factor-i and insulin-like growth factor-binding protein in the toad, bufo...

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