Nutrition and somatomedin: Nutritionally regulated release of somatomedins and somatomedin inhibitors from perfused livers in rats

Nutrition

and Somatomedin: and Somatomedin

Nutritionally Regulated Release of Somatomedins Inhibitors from Perfused Livers in Rats S. Goldstein and L.S. Phillips

Circulating somatomedin activity reflects the presence of both somatomedins and somatomedin inhibitors, factors which antagonize the growth-promoting actions of somatomedins. Although both are regulated by nutrition, somatomedin inhibitors respond more rapidly than somatomedins to refeeding in fasted animals. To explore the role of the liver in such responses, release of somatomedin activity and somatomedin inhibitor activity was assessed during perfusion of livers from normal, fasted, and fasted-refed rats. Size-exclusion high-performance liquid chromatography (HPLC) revealed that liver perfusates contain both somatomedin and somatomedin inhibitor activity of apparent molecular weight (mol wt) comparable to that found in the circulation (-7,000 and -30,000, respectively), as well as activity of apparently higher mol wt. In subsequent studies, reponses to nutrition were evaluated as fluctuations in bioactivity only of mol wt comparable to that found in the circulation. Release of both somatomedin and somatomedin inhibitor activity was progressive over at least two hours of recirculating perfusion. Perfusates of livers from normal fed rats had somatomedin activity (stimulation of cartilage SO, uptake) 94 ? 19% above buffer (P < .Ol), which fell to undetectable levels after three days of fasting. With refeeding, perfusate somatomedin activity rose within three hours to -25% of levels in fed rats, but did not become significant until after 12 hours (29 + 7%. P < .02). Perfusates of livers of fed rats also contained somatomedin inhibitor activity (42 & 10% inhibition of cartilage stimulation by normal serum), which rose after three days of fasting to 114 f 22% (P < .02). Perfusate inhibitor activity began to fall after three hours of refeeding, and at both six and 12 hours, was comparable to levels in liver perfusates from fed animals. When the livers of fasted rats were perfused with medium enriched in amino acids as well as glucose, there was a significant increase in release of somatomedin activity and a marginal decrease in release of somatomedin inhibitor activity. Although perfusate somatomedin and somatomedin inhibitor activity were correlated (r = -0.45, P < .04), discrepancy in temporal responses suggested noncoordinate regulation. Thus, although fasting produced both a rise in somatomedin inhibitor and a fall in somatomedin activity, refeeding was associated with complete normalization of perfusate somatomedin inhibitor activity over six to 12 hours, whereas perfusate somatomedin activity did not become statistically significant until after 12 hours, when levels were still -75% below values in normal fed animals. During nutritional repletion of fasted animals, changes in both somatomedin and somatomedin inhibitor activity in liver perfusates appeared to precede comparable changes in the circulation. In combination, these observations indicate that the liver may play a major role in metabolic homeostasis via nutritionregulated delivery of both somatomedins and somatomedin inhibitors to the circulation. o 1989 by Grune & Stratton, Inc.

HE ROLE OF THE LIVER in metabolic regulation appears to include delivery to the circulation not only of metabolic fuels, but also of growth factors which affect use of fuels in the periphery. Thus, the liver is recognized as a source of both somatomedins (insulin-like growth factors [IGFs]) and somatomedin carrier proteins.‘v2 The biological activity of somatomedins is modified by other circulating factors, the somatomedin inhibitor(s).3 Although the somatomedin inhibitors as yet are poorly characterized, circulating somatomedin inhibitor activity is trypsin- and heatlabile, has apparent molecular weight (mol wt) 20,000 to 40,000, and antagonizes both somatomedin and insulin action on target tissues such as muscle, fat, and cartilage.4

T

The

inhibitor(s)

appears

to be metabolically

regulated,

as

in serum of fasted or diabetic animals.’ To evaluate the possibility that nutritionally regulated fluctuations in both somatomedins and somatomedin inhibitors might involve the liver, we measured somatomedin and somatomedin inhibitor activity released by the perfused livers of rats during fasting and early refeeding. shown

by

elevated

levels

MATERIALS AND METHODS

General Experimental

Design

In situ liver perfusion was performed in rats which were either fed ad libitum, fasted three days, or fasted three days followed by three, six, or 12 hours of refeeding. Liver perfusates were fractionated by size exclusion, high-performance liquid chromatography (HPLC). Biologically active somatomedins were assessed according to the Metabolism, Vol 38, No 8 (August), 1989: pp 745-752

ability of samples to stimulate sulfate uptake by costal cartilage from hypophysectomized rats in vitro, and somatomedin inhibitors assessed according to the ability of samples to blunt such stimulation by somatomedins.

Animals and Animal Serum Male Sprague-Dawley rats weighing 100 to 170 g were obtained from Sasco-King Breeding Laboratories (Omaha, NE), housed in wire cages at 24 ? 1°C with lights on from 6 AM to 6 PM, and stabilized for I week with ad libitum intake of Purina chow (Purina, St Louis) and water. Animals were used either in the ad libitum fed state, after fasting for three days, or after fasting for three days followed by ad libitum refeeding for three, six, or 12 hours. For comparison with growth factors released into liver perfusates, somatomedins and somatomedin inhibitors were also evaluated in serum from normal fed rats and from 72-hour fasted rats. From the Division of Endocrinology and Metabolism, Deparimen1 of Medicine. Emory University School of Medicine. Grady Memorial Hospital, Arlanta, GA. This work was supported in part by research Grants DK-3347.5 and DK-34785 from the National Institutes of Health, This work was presented in part in the annual meeting of the American Federation for Clinical Research, I987 (Clin Res 35:504. 1987 [abstr]). Address reprint requests to Lawrence S. Phillips, MD, Department of Medicine. Emory University School of Medicine. 69 Butler Street. SE, Atlanta, GA 30303. CC) I989 by Grune & Stratton, Inc. 0026-0495/89/3808-0008$03.00/0 745

746

GOLDSTEIN AND PHILLIPS

Liver Perfusion Livers from rats with normal and altered nutrition were subjected to recirculating perfusion with a modification of procedures described previously.’ In situ perfusion was performed in pentobarbital-anesthetized animals, with inflow via the portal vein and outflow via the superior vena cava. The recirculating medium was KrebsHenseleit buffer with 20 mmol/L HEPES, pH 7.4, with washed bovine red cells (average hematocrit 22%) and fatty acid-free bovine serum albumin (BSA) 3 g/dL. (This supports oncotic pressure and will prevent adsorption of some, but not all, proteins.) The buffer was supplemented with 100 mg/dL of glucose, with or without minimal essential amino acids (GIBCO) and glutamine (5 mmol/L). Livers were perfused at 37°C at a flow rate of 1.1 mL/min/g liver wet weight (from prior studies relating liver weight to animal weight). Oxygenation was achieved with 95% CO,/5% O2 via a hyperbaric membrane exchanger. The first five minutes of perfusate flow were discarded, followed by an additional 30 minutes of recirculating perfusion to permit hepatic equilibration. Fresh medium was then provided, and recirculated for the following 120 minutes. Samples for pH and p02 were obtained at 30-minute intervals to assure that oxygen consumption was optimal. At selected time points, small volumes of medium were removed, red cells separated via centrifugation, filtered through a 0.22 r,~cellulose membrane, and stored at - 20°C for subsequent determination of contained somatomedins and somatomedin inhibitors.

Size Exclusion HPLC of Liver Perfusates Liver perfusate samples (200 /IL) were chromatographed on TSK-2000 SW (7.5 x 60 cm) in 1.5 mol/L ammonium formate, pH 3.0, at 0.71 mL/min.’ Recovery has been improved by the use of siliconized glass or polypropylene collection tubes. The eluate between V, and V, was divided into 12 equal fractions, lyophilized three times, resuspended in 400 PL of bioassay buffer (vide infra), and stored at -20°C until assay. After such handling, ammonium formate buffer blanks are inactive in the bioassay. Approximate molecular weights of somatomedin and somatomedin inhibitor activity were estimated from the elution of standards of known mol wt: glucagon (mol wt 3.485), insulin (5,840) RNAase (l3,700), LYchymotrypsinogen-A (25,700). ovalbumin (43,000), BSA (68,000), and gamma globulins (I 50,000).

Assay of Somatomedins

and Somatomedin

Inhibitors

Costal cartilage from hypophysectomized rats is very sensitive to specific stimulation by somatomedins, and in these studies was used to estimate the activity of somatomedins and somatomedin inhibitors in fractions of liver perfusates from experimental animals. While these determinations are less accurate and specific than radioimmunoassays, they permit study of factors for which no immunoassay is available (somatomedin inhibitors), and can be used to detect somatomedins in the presence of binding proteins which confound RIA determinations. Although parallel-line analysis is difficult because of the limited number of pieces of rat cartilage that can be included in a single assay, potency is well reflected by cartilage responses to single concentrations lying within the linear portion of the dose-response curve. ‘,’ Thus , single concentrations of serum fractions were tested for somatomedin activity and somatomedin inhibitory activity in the rat cartilage bioassay system.’ Assay conditions for somatomedin and somatomedin inhibitor activities were selected to maximize sensitivity’ and minimize potential contributions from the presence of contamination.‘.’ As described previously? groups of five cartilage segments from a single hypophysectomized rat were incubated at 37OC for 48 hours in 0.5 mL Krebs phosphosaline-amino acid buffer supplemented with 8.9 mmol/L D-glucose, antibiotics, and 1 r.rCi [“S] sulfate, with or

without added whole normal rat serum or column fractions. After washing to remove adsorbed sulfate, cartilage was dried, weighed, hydrolyzed, and [“S] sulfate incorporation estimated by scintillation counting. Somatomedin activity was determined according to the ability of fractions (2.5%, vol/vol) to stimulate sulfate uptake above buffer levels, and expressed relative to sulfate uptake by cartilage incubated in buffer alone (% of buffer). The activity of somatomedin inhibitors in column fractions was determined at 4% (vol/vol) final concentration, according to the ability to blunt cartilage stimulation in the presence of 1% pooled normal rat serum (NRS), added to, provide uniform stimulation by somatomedins. Inhibitory activity was expressed as cartilage stimulation by NRS which was blocked by the addition of test fractions: %NRS inhibited X

= 100

(NRS SO4 uptake)

- ([NRS

(NRS SO4 uptake)

Somatomedin-C/IGF-1

+ sample]

SO1 uptake)

- (buffer SO., uptake)

Radioimmunoassay

Biosynthetic (Th?) somatomedin-C/IGFI, purchased from AMGen Biologicals, Thousand Oaks, CA, was iodinated with lactoperoxidase to specific activities of 100 to 280 rCi/r.rg. SomatomedinC/IGF-1 in aliquots of size-exclusion HPLC fractions was determined by the radioimmunoassay method of Furlanetto et aL9 using the anti-human somatomedin-C/IGF-1 serum provided by Drs Van Wyk and Underwood through the National Hormone and Pituitary Program. Biosynthetic somatomedin-C/IGF-1 was used as a standard to define the dose-reponse curve. Intra-and inter-assay coeflicients of variation were 8% and 12%. respectively.

Somatomedin-C/IGF-1

Binding Activity

Binding protein activity was determined as described previously’0 with minor modifications, Aliquots of HPLC fractions were diluted with phosphate buffered saline containing I % BSA and incubated 16 hours at 4°C with ‘ZSI-somatomedin-C/IGF-l (20,000 cpm) in a total volume of 500 wL. After addition of I-mL assay buffer containing 0.25% charcoal and 0.02% protamine sulfate and incubation for 30 minutes at 4OC, bound Sm-C/IGF-1 was separated from free by centrifugation at 2,000 x g for 40 minutes at 4°C. Normal rat serum (10 l.rL) was used as an assay control, whereas blanks consisted of ‘2SI-somatomedin-C/IGF-l in the presence of buffer alone. Separate studies have demonstrated the specificity and linearity of binding in this system. Results were expressed as percent of added cpm bound.

Liver Glycogen Content Freeze-clamped portions of livers were hydrolyzed in 2 vols of 30% (wt/vol) KOH at IOO’C for 30 minutes. Glycogen was precipitated overnight at room temperature by addition of 0.1 mL 2% (wt/vol) NaTSO, in ethanol to a final concentration of 65% to 70% (vol/vol), and collected by centrifugation. Pellets were heated briefly to remove trace ethanol, and glycogen hydrolyzed to glucose with Aspergillus niger amyloglucosidase and porcine pancreatic aamlyase.” An aliquot of the hydrolysate was diluted in 0.1 mol/L potassium phosphate buffer, pH 6.5, containing glucose oxidase, horseradish peroxidase, and o-dianisidine. Glucose content was estimated from A450 v absorbance of standards.

Statistics Significance of differences between experimental groups was evaluated by two-tailed, unpaired, t tests and by ANOVA. Relationships between variables were examined by linear regression analysis.

HEPATIC RELEASE OF SOMATOMEDINS

AND INHIBITORS

RESULTS

Animal

Liver Perfusates: Fed

Weight and Liver Glycogen Content

Initial animal weight averaged 133 g (Table 1). Over three days of fasting, weight loss averaged 31%. With refeeding, animals regained rapidly. Weight gain over three hours of ad libitum refeeding averaged 13% (P < .Ol), with little further increase over the next nine hours of refeeding. Liver glycogen in fed rats averaged 2.4% (wt/wt), and fell significantly to 0.39% after three days of fasting (P < .Ol). Glycogen content increased rapidly with refeeding, to 2.51% after three hours (comparable to normal fed levels), and 5.92% at six hours. Such alterations in hepatic glycogen demonstrate the conversion by the liver from a catabolic state during fasting to an anabolic state during refeeding. Somatomedin

Activity

in Liver Perfusates

To characterize somatomedins released by the liver, 120minute perfusates were fractionated at pH 3.0, and fractions tested individually for stimulation of sulfate uptake by rat costal cartilage (Fig 1). Somatomedin activity was found both at K,, 0.50 to 0.67 (corresponding to mol wt 12,000 to 42,000). and at K,, 0.67 to 0.92 (corresponding to mol wt 3,000 to 12,000). Fasted rat livers released less somatomedin activity in both regions. With normal livers, there was approximately twice as much activity in the low-mol wt region as in the high-mol wt region, 94 f 19% v 41 i- 23%. The low-mol wt peak is coincident with serum somatomedin activity chromatographed in the same system, and recombinant ‘251-somatomedin-C/IGF-l elutes in identical fractions; thus, the low-mol wt somatomedin activity in liver perfusates appears to be similar in size to somatomedins found in the circulation. Since net stimulation provided by low-mol wt somatomedin activity in 200 WL of normal liver perfusate was comparable to that in 200 PL of normal rat serum (98 +- 23%) it may be inferred that total somatomedin activity in the liver perfusate (60 mL) is approximately ten times that in the circulation of a normal 150-g rat (serum volume approximately 4 to 5 mL). Based on the size similarity between low-mol wt liver perfusate somatomedin in activity and somatomedins in serum, subsequent measurement of liver perfusate somatomedin activity at K,, 0.67 to 0.92 was used to assess the impact of nutrition. Somatomedin

Inhibitor Activity

in Liver Perfusates

Since circulating somatomedin inhibitors are increased fasted rats,’ we first examined somatomedin inhibitors

and Liver Glycogen Content Changes With

RatWeight

Table 1.

in in

Feeding Status Weight Group

n

Initial

Fasted

Normal-fed

8

134t8

3-d

fasted

7

134k8

8957

3-h

refed

7

13228

94k7

6-h

refed

12-h

refed

NOTE:

Values

7

115

6

139+9 are mean

(g)

Hepatic Glycogen

Refed

-

k3

81

z SEM.

2.38

+ .4

0.39

*

k 7

2.51

+ .l

95

+- 3

5.92

k .6

109

+ 9

106 +3

97-c7

% (wt/wt)

.l

K a” Fig 1. Elution profile of somatomedin activity in perfusates of fed and three-day fasted rats, after chromatography on TSK-2000 at pH 3.0. Somatomedin activity was measured according to stimulation of cartilage sulfate uptake in vitro (see Methods). The location of somatomedin activity eluted after chromatography of normal rat serum is shown by hatching. Mean t SEM for five to six perfusate samples at each point.

perfusates from livers of rats fasted for three days. As with somatomedin activity, somatomedin inhibitor activity in liver perfusates was heterogeneous (Fig 2). The major peak of liver perfusate somatomedin inhibitor activity was found at K,, 0.42 to 0.67 (corresponding to mol wt 12,000 to SO,OOO), comparable to the predominant somatomedin inhibitors in the circulation.’ This region was preceded by a higher-m01 wt area of somatomedin inhibitor activity at K,, 0.17 to 0.42 (corresponding to mol wt 50,000 to 80,000). Perfusates of livers from normal rats had less somatomedin inhibitor activity, mostly at K,, 0.42 to 0.67. Since the low-mol wt range of somatomedin inhibitor activity in liver perfusates corresponded to the size of metabolically regulated serum somatomedin inhibitors, only bioactivity eluting in this region was examined to assess the impact of altered nutrition. Time-Course of Hepatic Release of Somatomedins and Somatomedin Inhibitors Following five minutes of wash-out and 30 minutes of equilibration, liver perfusate content of both somatomedin activity and somatomedin inhibitor activity increased progressively during 120 minutes of recirculating perfusion. Somatomedin activity (K,, 0.67 to 0.92) increased from 41%

748

GOLDSTEIN AND PHILLIPS

80

, Liver Perfusates: Fed

60

Liver Perfusates: L 60

I

, FED

FAST 3 DAY

6

3 HR.

REFED

Fig 3. Response of release of somatomedin and refeeding. Somatomedin activity (K,, 0.67 minute perfusates of livers from normal, fasted, rats. Mean + SEM for five to six animals at each

11.

O-.08 -.I7

I

-.25

I,

I

I1

-.50

I

I

-1 .o

K av Fig 2. Size profile of somatomedin inhibitor activity in perfusates of livers from fed and three-day fasted rats, chromatographed as in Fig 1. Somatomedin inhibitor activity was measured by the ability of samples to antagonize stimulation of cartilage sulfate uptake by somatomedins in added normal rat serum (see Methods). The elution profile of somatomedin inhibitor activity in serum from diabetic or fasted rats is shown by hatching. Mean + SEMI for five to six samples at each point.

to 94% (stimulation above buffer). Somatomedin inhibitor activity (K,, 0.42 to 0.67) increased from 52% to 114% (stimulation inhibited). Since significant levels were found only after 120 minutes (both P < .Ol), only this time-point was used to examine the effects of altered nutrition. In separate perfusions, the trend for release of somatomedin activity by livers from normal animals and somatomedin inhibitor activity by livers from fasted animals persisted for six hours (not shown).

12

activity to fasting to 0.92) in 120and fasted/refed point.

six and 12 hours (22 t 18 and 29 + 7%, respectively), significance was reached only after 12 hours of refeeding (P c: .02). Combining the five groups of animals, there was a significant correlation between liver perfusate somatomedin activity and body weight at the time of perfusion (r = SO, P < .OOS). Hepatic Release of Somatomedin Inhibitors During Fasting and Refeeding The perfusates of livers from normal fed rats contained only a low level of somatomedin inhibitor activity, 42 + 10% (Fig 4, stimulation inhibited, P < .05). After animals were fasted for three days, perfusate somatomedin inhibitor activity increased to 114 + 22% (P < .02). After refeeding for three hours, perfusate somatomedin inhibitor activity fell to 96 t 24%. Perfusate somatomedin inhibitor activity fell progressively with continued refeeding, and after six hours

Hepatic Release of Somatomedins During Fasting and Refeeding As shown in Fig 3, somatomedins in perfusates of livers from normal fed rats provided cartilage stimulation 94 f 19% above buffer (P < .Ol). After animals had been fasted for three days, no perfusate somatomedin activity could be detected (P = NS v buffer). The presence of cartilage stimulatory activity apparently below buffer level (nonsignificant) may conceivably be due in part to interference from somatomedin inhibitors, which overlap about 15% with somatomedins at K,, 0.67 to 0.92. After three hours of ad libitum refeeding, perfusate somatomedin activity rose to 25% of levels in fed rats (24 + 22%, P = NS v buffer). Although perfusate somatomedin activity was comparable at

FA’ST 3 DAY

6 HR.

12

REFED

Fig 4. Somatomedin inhibitor activity (K,, 0.42 to 0.67) released by livers of normal, fasted, and fasted/refed rats. Mean + SEM for five to six animals at each point.

HEPATIC

RELEASE

OF SOMATOMEDINS

AND INHIBITORS

749

HEPATIC RELEASE OFSOMATOMEDlNS(O)/INHIBITORS(n)

(44 i 25%) was comparable to levels of activity in perfusates of livers from fed animals. There was no significant further change in inhibitor activity at 12 hours (57 + 11%). Correlations between perfusate somatomedin inhibitor activity and body weight were not significant (r = - .40, P < .06).

I

ESjhct of Altered Liver Perfusion Conditions on Hepatic Release of Somatomedins and Somatomedin Inhibitors To evaluate the impact of altered provision of metabolic fuels in vitro, release of somatomedins and somatomedin inhibitors by livers of fasted rats was assessed in perfusions supplemented with amino acids (as found in “minimal essential medium,” see Methods) as well as glucose as a fuel source. After 120 minutes of perfusion, somatomedin activity (52 + 24% above buffer) was significantly greater than levels from fasted rat livers perfused with glucose alone (P < .02). Somatomedin inhibitor activity appeared to decrease (48 i 41% stimulation inhibited), but changes were not significant. Addition of amino acids did not alter the release of either somatomedin or somatomedin inhibitor activity by the livers of fed animals. Effect of Nutritional Status on Somatomedins and Somatomedin Inhibitors in Liver Perfusates v Serum Although perfusate somatomedin and somatomedin inhibitor activities were correlated (r = -.45, P < .04), additional comparisons suggested noncoordinate regulation. As shown in Fig 5, comparable animals fasted for three days exhibited a rise in both circulating and liver perfusate somatomedin inhibitors, and a fall in both circulating and liver perfusate somatomedins. With refeeding, perfusate somatomedin activity began to rise after three hours, but significant levels could not be detected until I2 hours. Circulating somatomedin activity exhibited no apparent increase after six hours, began to rise after 12 hours, and was restored to fed levels after 24 hours. In contrast, somatomedin inhibitors responded more rapidly to refeeding. Perfusate somatomedin inhibitor activity decreased by 16% after three hours, and reached levels in fed animals after six hours. The fall in perfusate somatomedin inhibitor activity was followed by a comparable fall in circulating somatomedin inhibitor activity, 41% below fasted levels after six hours, returning to levels in fed animals after 24 hours.

-I

A

To determine whether bioassayable somatomedin activity and somatomedin inhibitor activity could be attributed to the presence of somatomedins and/or somatomedin binding proteins. the latter were measured in liver perfusates from both normal and fasted rats (Fig 6). Somatomedin binding activity was comparable in perfusates from normal v fasted animals, maximal at K,, 0.33 to 0.42 and decreased in successive fractions. The dissimilarity between this pattern v that of inhibitory activity (maximal at K,, 0.42 to 0.50), together with the lack of differences between normal v fasted rats, indicate that inhibitory activity cannot be attributed simply to the presence of somatomedin binding proteins.

u HRS.

CIRCULATING

SOMATOMEDINS

Pi. 300

-

:/

‘1

REFED

(.)/INHIBITORS

(A,

‘\ ‘1.

s 2

‘. 200

h-_-q

-

z *

I

Y 0

,00

____&_~_~___

B % 0 FED

B

I 6 a

FAST 3 DAYS

/

HRS.

REFED

Fig 5. (A) Release of somatomedin activity and somatomedin inhibitor activity by perfused livers of normal, fasted, and fasted/ refed rats, from Figs 4 and 5. (B) Somatomedin activity and somatomedin inhibitor activity in serum of comparable animals.” Each point represents determinations from at least five experimental animals. SEM proportional to those in Figs 4 and 5.

Somatomedin immunoreactivity was greater in normal than in fasted liver perfusates and was found largely at K,, 0.67 to 0.92, indicating that bioassayable somatomedin activity in this region can be attributed to somatomedins (dissociated from binding proteins); the region of high-mol wt somatomedin bioactivity had less immunoreactivity, but the profile IGF- 1 BINDING ACTIVITY TSK 2000, pH 3.0 ‘Qrmh,d.,

Somatomedin Immunoreactivity and Somatomedin Binding Activity in Liver Perfusates

FAST 3 DAYS

FED

IMMUNOREACTIVE IGFTSK 2000, pH 3.0 I

LhrPm”..,., *.*

1 I

Fig 6. (A) IGF-1 binding activity in fractionated perfusates from normal and fasted rats. (B) lmmunoreactive IGF-1 in fractionated perfusates. Mean + SEM, n = 3.

750

GOLDSTEIN AND PHILLIPS

appeared comparable to that of Fig 1, consistent with the hypothesis that such activity reflects the presence of a somatomedin precursor. DISCUSSION

The liver plays a major role in maintenance of metabolic homeostasis. Such a role has been envisioned largely in terms of delivery v storage of metabolic fuels, ie, glycogenolysis, gluconeogenesis, and ketogenesis under conditions of catabolism, and storage of glycogen and lipogenesis under conditions of anabolism.‘* However, evidence for participation of the liver in the production of somatomedins and somatomedin inhibitors suggests that the liver may also affect metabolism via delivery of export proteins that have the potential to regulate fuel use in muscle, fat, and other tissues. The somatomedins/IGFs are growth factors with proinsulinlike structure and broad, insulin-like, anabolic and anticatabolic properties on a wide variety of tissues and cultured ce11s.13The hypothesis that somatomedins are critical mediators of both growth hormone and insulin-induced growth is supported by evidence that (a) treatment of hypopituitary animals with GH and diabetic animals with insulin is attended by a rise in somatomedins along with improved growth,“si4 and (b) direct administration of somatomedins to either hypopituitary or diabetic animals can also produce growth in the absence of GH or insulin, respectively.‘43’5 Study of circulating somatomedin immunoreactivity and bioactivity in states of hypoinsulinemia, undernutrition, and GH deficiency suggests that poor growth in these conditions may be mediated not only via decreased somatomedins, but also by an increase in somatomedin inhibitors, factors which can blunt somatomedin action on target tissues. Inhibitor activity rises with induction of diabetes or withdrawal of food, and is restored toward normal with refeeding.‘,16 However, regulation of the inhibitors is not precisely parallel to that of the somatomedins, in that (a) glucocorticoids appear to raise inhibitors without affecting somatomedins (in normal humans” as well as diabetic animals,18 (b) inhibitors rise before somatomedins fall with progressive severity of diabetes,” and (c) inhibitors appear to fall before somatomedins rise with refeeding of fasted animalsI A pivotal role for the liver in contribution of somatomedins to the circulation is hypothesized based on studies of liver perfusion, extraction, and cells in culture. Hepatic release of somatomedin bioactivity and immunoreactivity reflects the nutritional/hormonal status of donor animals as well as enrichment of the perfusing medium with hormones and metabolic fuels. Somatomedins in liver perfusates generally parallel levels in the circulation.‘~*’ Extractable hepatic content of somatomedin activity also reflects donor metabolic status.*’ In fasted and diabetic animals, liver-extract and liver perfusate somatomedin activity fall before a decrease in circulating somatomedin bioactivity can be detected.** Hepatocytes in primary culture23 and liver cell lines” also release somatomedins, again under hormonal control. Schwander et al*’ has estimated from studies of somatomedin production by perfused livers that hepatic generation of somatomedins is sufficient to account for the known turnover of somatomedins in the circulation. That hepatic production of somato-

medins may involve synthesis is indicated by puromycin inhibition of somatomedin release during liver perfusion*’ and cycloheximide inhibition of somatomedin release in liver cell cultures.23 There is less evidence for hepatic production of somatomedin inhibitors. Vassiloupoulou-Sellin et al’ found that perfusates from livers of diabetic or fasted animals had net inhibitory activity in somatomedin bioassays, but did not fractionate perfusates to assess relative contributions of changes in somatomedins v inhibitors. Subsequently, Binoux et a126reported that liver explants released an -45,000 mol wt factor that inhibited somatomedin stimulation of sulfate uptake by embryonic chick cartilage, and which appeared to be under metabolic control. More recently, VassiloupoulouSellin et al have reported that neutral liver extracts have somatomedin inhibitor activity in both rat and chick cartilage assay systems, and can reversibly decrease the size of embryonic cartilage explants during five-day cu1ture.27328To date, there have been no reports of nutrition-regulated changes in either hepatic content or release of somatomedin inhibitors. The present studies demonstrate that the liver releases both somatomedin and somatomedin inhibitor activity of apparent mol wt higher than that found in the circulation, and that hepatic release of somatomedin and somatomedin inhibitor activity (of low mol wt comparable to that found in the circulation) is responsive to nutritional status both in vivo and in vitro. Seventy-two hours of fasting was associated with a marked fall in release of somatomedin activity, together with a marked rise in somatomedin inhibitor activity. With refeeding, somatomedin inhibitor activity began to fall within three hours, and reached normal fed levels after six hours: in contrast, somatomedin activity was restored more slowly, significantly above buffer after 12 hours, but still below levels in normal perfusates. The perturbations in release of somatomedin and somatomedin inhibitor activity associated with fasting appeared to be reversed partially when livers from fasted rats were perfused with medium enriched in amino acids as well as glucose. This may indicate that lack of essential amino acids limits somatomedin production in the fasted state. Although conclusions must be qualified because of quantitation via bioassay methodology, these findings provide additional evidence that the liver may be the locus of nutrition-regulated changes in circulating somatomedins and somatomedin inhibitors, and suggest that amino acids may play a critical regulatory role. The potential impact of added insulin was not investigated. Since release of somatomedins likely reflects both protein synthesis and conversion of high- to low-mol wt forms, and release of somatomedin inhibitors may reflect either protein synthesis26 or protein breakdown as well as conversion, additional studies will be required to ascertain the mechanism by which added amino acids influence release of somatomedins and somatomedin inhibitors. The release of somatomedin activity of mol wt higher than that found in the circulation cannot be attributed simply to the presence of carrier proteins. While perfused livers and cultured liver cells release somatomedin carrier proteinszo”9 that cross-react in somatomedin radioimmunoassays, carrier

HEPATIC RELEASE OF SOMATOMEDINS

751

AND INHIBITORS

proteins should not be stimulatory in cartilage bioassays. Moreover, somatomedins appear to dissociate from serum carrier proteins under our acidic conditions. Thus, high-mol wt somatomedin bioactivity detected in the present studies could reflect either carrier proteins from which somatomedins cannot be dissociated, or putative precursor forms. Although the hypothesis of high-mol wt somatomedins is supported by studies showing that both IGF-1 and IGF-2 genes code for forms of mol wt 15,000 to 22,000,30*3’ to date only in vitro translation of the IGF-2 gene has been shown to result in an immunoprecipitable product of mol wt 2 1,600.32 Release of somatomedins of apparent mol wt 12,000 to 42,000 is consistent with the -20,000 to 40,000 mol wt of somatomedin activity in liver extracts,*’ and with the apparent mol wt of somatomedin immunoreactivity released from cultured cell lines.j3 There is less information regarding high mol wt forms of the somatomedin inhibitors. The majority of circulating somatomedin inhibitor activity has apparent mol wt 20,000 to 40,000,5 consistent with the lower mol wt region of somatomedin inhibitor activity found in the present studies. Serum of normal as well as fasted and diabetic rats contains somatomedin inhibitor activity of higher mol wt5*’ that does not exhibit metabolic regulation. Human serum also contains high-mol wt somatomedin inhibitor activity, which does not increase after glucocorticoid ingestion.16 Such apparent size

is comparable to that of the high-mol wt region of somatomedin inhibitor activity found in the present studies. Our laboratory has previously observed small quantities of highmol wt somatomedin inhibitor activity in serum of diabetic rats fractionated at neutral pH, but not at acid PH.~*’ The somatomedin inhibitor activity found by VassilopoulouSellin et a12’~**in neutral extracts of normal livers has apparent mol wt >50,000, suggesting that extractable inhibitor could be identical to the high-mol wt circulating species. Based on complementary bioassay and immunoassay analyses, the present studies buttress the hypothesis that altered release of both somatomedins and somatomedin inhibitors by the liver may contribute to the fluctuations in circulating somatomedins and somatomedin inhibitors seen in metabolic perturbations such as starvation. Through such mechanisms, the liver may participate in metabolic homeostasis not only via storage and release of critical metabolic fuels, but also via delivery to the circulation of regulatory factors that can modulate fuel use in the periphery.

ACKNOWLEDGMENT We thank P.E. Dawson, D.E. Lamberski, S.S. Welker. L.M. Quinones. and L.M. Amendt for excellent technical assistance, and we thank Aaliyah Salim and Mark S. Goldman. DDS. for assistance in preparation of the mansucript.

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