Coordinate regulation of collagen and proteoglycan synthesis in costal cartilage of scorbutic and acutely fasted, vitamin C-supplemented guinea pigs

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 246, No. 1, April, pp. 42-51, 1986

Coordinate Regulation of Collagen and Proteoglycan Synthesis in Costal Cartilage of Scorbutic and Acutely Fasted, Vitamin C-Supplemented Guinea Pigs TIMOTHY AND

A. BIRD,l ROBERT G. SPANHEIMER, BEVERLY PETERKOFSKY3

Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 Received

August

19,1985,

and in revised

form

November

13, 1985

The effects of ascorbic acid deficiency and acute fasting (with ascorbate supplementation) on the synthesis of collagen and proteoglycan in costal cartilages from young guinea pigs was determined by in vitro labeling of these components with radioactive proline and sulfate, respectively. Both parameters were coordinately decreased by the second week on a vitamin C-free diet, with a continued decline to 20-30% of control values by the fourth week. These effects were quite specific, since incorporation of proline into noncollagenous protein was reduced by only 30% after 4 weeks on the deficient diet. The time course of the decrease in collagen and proteoglycan synthesis paralleled the loss of body weight induced by ascorbate deficiency. Hydroxylation of proline in collagen synthesized by scorbutic costal cartilage was reduced to about 60% of normal relatively early, and remained at that level thereafter. Neither collagen nor proteoglycan synthesis was returned to normal by the addition of ascorbate (0.2 IIIM) to cartilage in vitro. Administration of a single dose of ascorbate to scorbutic guinea pigs increased liver ascorbate and restored proline hydroxylation to normal levels by 24 h, but failed to increase the synthesis of collagen or proteoglycan. Synthesis of both extracellular matrix components was restored to control levels after four daily doses of ascorbate. A 96-h total fast, with ascorbate supplementation, produced rates of weight loss and decreases in the synthesis of these two components similar to those produced by acute scurvy. There was a linear correlation between changes in collagen and proteoglycan synthesis and changes in body weight during acute fasting, scurvy, and its reversal. These results suggest that it is the fasting state induced by ascorbate deficiency, rather than a direct action of the vitamin in either of these two biosynthetic pathways, which is the primary regulatory factor. 0 1986 Academic PIVSS, IIIC.

crucial role in maintaining the structural integrity of the extracellular matrix. Proteoglycans are most abundant in cartilage, where they constitute approximately 50% of the dry weight. Defective production of these components has been implicated in scurvy, but collagen metabolism has been investigated more extensively. It has been widely accepted that the most characteristic lesion of scurvy, poor wound healing, can be attributed to an underlying impair-

Collagen and proteoglycans are macromolecular components of a wide variety of connective tissues, in which they play a 1 Present address: Strangeways Research Laboratory, Wort’s Causeway, Cambridge CB14RN, England. a Recipient of a Special Emphasis Research Career Award (AM-01435). Present address: Departmentof Medicine, Emory University, 69 Butler St., Atlanta, Ga. 30303. 3 To whom reprint requests should be addressed. 0003-9861/86 Copyright All rights

$3.00

0 1966 by Academic Press, Inc. of reproduction in any form reserved.

42

COLLAGEN

AND

PROTEOGLYCAN

ment of collagen proline hydroxylation (l3), since it is known that the enzymes prolyl (4, 5) and lysyl (6) hydroxylase have a requirement for ascorbate. Ascorbate appears to function by preventing oxidation of free or enzyme-bound iron (7, 8). In a recent study from our laboratory (9) on collagen metabolism in calvarial bones removed from guinea pigs at various stages of vitamin C deficiency, it was found that in addition to a moderate decrease in proline hydroxylation, there was a specific decrease in the rate of collagen synthesis. This decrease was directly correlated with the amount of body weight lost during the course of scurvy, but was temporally dissociated from the decrease in proline hydroxylation. It seems most likely that scurvy-associated inanition is the critical factor in provoking a specific decrease in bone and articular cartilage collagen synthesis, because acutely fasted animals showed similarly decreased synthesis in these tissues even when given saturating doses of ascorbate (10). Defects in cartilage matrix formation and chondrocyte morphology during scurvy have been observed in microscopic studies (11,12). There also have been reports that the incorporation of radiosulfate into the glycosaminoglycan (GAG)4 moiety of proteoglycans is reduced in tissues from scorbutic guinea pigs (13-15), but the question of whether this results from inanition or direct involvement of ascorbate in GAG synthesis in vivo has not been resolved. Friberg (16) showed that fasting lowered the incorporation of 35S04into guinea pig rib cartilage despite the administration of vitamin C. Kofoed and Robertson (17) measured the net replacement of GAG in cartilage following the injection of papain into guinea pigs. Significantly less accumulation was observed in scorbutic animals, but the effect also was observed in pair-fed controls, prompting the hypothesis that decreased GAG synthesis was secondary to inanition and that ascorbate was not required for GAG synthesis. In contrast, a number of studies (H-20) showed * Abbreviations MEM, Eagle’s

used: GAG, minimal essential

glycosaminoglycan; medium.

SYNTHESIS

IN

43

SCURVY

that scurvy selectively decreased the galactosamine content of granulation tissue, and that this decrease did not result from simple inanition (19, 20) although no specific, direct requirements for ascorbate in the various steps leading to proteoglycan synthesis have been reported. In the light of these conflicting data and the relationship between weight loss and collagen synthesis in bone of scorbutic guinea pigs, we undertook this study to examine the temporal relationships between body weight changes and GAG synthesis in costal cartilage, both during the development of scurvy and following ascorbate repletion of scorbutic animals. Since scurvy and fasting would appear to be ideal dynamic systems in which to test the suggestion (21) that collagen and proteoglycan metabolism may be coordinately regulated, we extended our study to compare the effects of scurvy on both parameters. Costa1 cartilage was chosen for this study since the rate of proteoglycan synthesis was much greater than in articular cartilage. The latter tissue was more suitable for isolation of mRNA because more tissue was available, and those studies are described in an accompanying paper (22). MATERIALS

AND

METHODS

Materials. Uniformly labeled L-[“Clproline (273 mCi/mmol) and L-[~-~H] proline (23.8 mCi/pmol) were obtained from New England Nuclear (Boston, Mass.); sodium [%]sulfate (carrier-free) was from Amersham Corporation (Arlington Heights, Ill.). Ultrapure guanidine hydrochloride was from Bethesda Research Laboratories Inc. (Gaithersburg, Md.). Ascorbate oxidase (Cucurbita sp.) was from Boehringer-Mannheim Biochemicals (Indianapolis, Ind.). Bacterial eollagenase was purchased from Worthington Biochemicals Corporation (Freehold, N. J.) and further purified by gel filtration chromatography (23). Chick embryo carrier protein (24) and serum-free Eagle’s minimal essential medium (MEM) containing 50 pg/ml gentamicin sulfate were prepared as described elsewhere (25). Animals. Full details of the routine maintenance of and production of ascorbate deficiency in guinea pigs appear in the accompanying paper (22). Animals to be acutely fasted were transferred to clean cages where they remained, without food, for 96 h while receiving a daily supplement of 50 mg of ascorbic acid as described earlier (10). Although the results pre-

44

BIRD,

SPANHEIMER,

AND

sented here are derived from several batches of animals, the progress of animals fed a scorbutogenic diet followed a predictable course (typical experiment shown in Fig. 1). In this study, the various stages of scurvy have been defined according to the degree of weight loss. Stage I encompasses the first 12-15 days of the experimental period, when animals on the ascorbate-free diet continued to gain weight at the same rate as ascorbate-supplemented controls. Stage II (Days 16-24) was marked by moderate weight loss in many of the scorbutic animals while others maintained their body weight during this period. During stage III (Days 25-28), weight loss became increasingly severe (6-8% per 48 h) and all animals displayed overt signs of scurvy such as lethargy, hind limb paralysis, joint hemorrhage, and bleeding gingiva. In some cases, animals exhibiting typical signs of stage II scurvy (22-24 days) were repleted with ascorbic acid by oral administration of the vitamin as described in the accompanying paper (22). Radiolabeling of cartilage. At appropriate times, animals were sacrificed by decapitation, rib cages were exposed, and cartilage from ribs II through VII was removed, leaving a 2-mm-wide region at each of the costosternal and costochondral junctions. After careful removal of muscle and perichondrium, the cartilage was cut transversly into sections approximately 4 mm long; three to four cartilage segments (about 30 mg wet wt) were used for each incubation. Duplicate or triplicate incubations were carried out, with continual shaking, at 37°C in a total volume of 1 ml under an atmosphere of 95% sir/5% COP in stoppered glass

'12,

I

0

4

8

12 TIME

16

20

24

28

(DAYS1

FIG. 1. Typical growth curves of guinea pigs maintained on ascorbate-supplemented (0) or -free (0) diets. Each point represents the amount of body weight lost or gained during the previous 48 h expressed as a percentage of the earlier weight and is the mean of values from all surviving animals. The initial numbers of animals in each group were 9 controls and 16 scorbutics. Two animals from each group were sacrificed after 1, 2, 3, and 4 weeks on the experimental diets.

PETERKOFSY

culture tubes (15 X 125 mm). To measure the relative rate of collagen synthesis, incubation mixtures consisted of 10 &i of L-[4-aH]proline and 2 &!i of L[14C]proline (0.1 mM) in MEM with or without added sodium ascorbate (0.2 mM). For the measurement of [35S]sulfate incorporation, MEM was replaced by a modified medium (MgC12, 165 mg/ml substituted for MgSO(; after addition of gentamicin sulfate, the total inorganic sulfate content of the medium was about 8 mg/liter) containing 10 pCi pS$ulfate per incubation, with or without ascorbate at 0.2 mM. Incubations were carried out for 15 min prior to the addition of radioactive label and then were continued for 4 h, as extensive pilot studies demonstrated that incorporation of radioactive proline and sulfate into macromolecules was linear for at least that amount of time. Relative rate of collagen synthesis and the degree of proline hydroxylation in newly synthesized collagen After incubation with radioactive proline, the cartilage segments and media were mixed with 1.5 mg chick embryo carrier protein and processed as described previously (9). The amounts of “C radioactivity associated with the collagenase-sensitive and -resistant fractions were used to calculate the relative rates of collagen synthesis. The method of calculation of the degree of proline hydroxylation from the ratio of 3H: l*C in collagenase digests has already been described (26). Values for the percentage of proline hydroxylation obtained by this method are similar to those obtained by other procedures (26). Measurement of radio&fate incorporation After tissues were incubated with [35S]sulfate, the labeling medium was discarded as it contained an insignificant proportion of the total macromolecular %S04 (typically 2-5%). The cartilage was rinsed twice in unlabeled medium and was then suspended in 1.0 ml of 4 M guanidine hydrochloride, buffered to pH 5.8 with 0.1 M sodium acetate and containing the protease inhibitors EDTA (10 ITIM) and benzamidine hydrochloride (5 IXIM). The suspension was homogenized at full speed for 30 s in a Polytron homogenizer (Brinkmann Instruments, Westbury, N. Y.). A further homogenization for 10 s was performed in 0.5 ml of fresh extractant to free retained fragments of tissue. The two homogenates were combined, allowed to stand on ice for 30 min, and clarified by centrifugation at 59008 for 15 min. A portion of each extract was retained for estimation of DNA content, and the remainder was applied to individual Sephadex G-25 PD-10 columns (Pharmacia, Uppsala, Sweden) equilibrated in extractant solution. The V, fraction containing the macromolecular %S was collected in a volume of 3-3.5 ml, and portions were counted to determine the total amount of radioactivity incorporated per microgram of DNA. Complete separation of incorporated from free radioactivity was achieved by this technique. Efficiency of extraction of labeled proteoglycan was identical for control and scorbutic tissues, and averaged 85%.

COLLAGEN

AND

PROTEOGLYCAN

Other deteminatims. The DNA content of solubilized cartilage homogenates was determined by a fluorimetric procedure (27) or, if sufficiently large amounts of DNA were present, by the diphenylamine method (28). For the estimation of the ascorbic acid content of liver, samples were quickly removed at sacrifice, frozen in dry ice, and stored in liquid nitrogen. Ethanol soluble fractions were prepared and assayed as described in a previous paper (lo), using the cy, cY’-dipyridyl-Fe’+ procedure (29).

SYNTHESIS

30 I-

IN

45

SCURVY + 02 mM ASCORBATE

(21T

RESULTS

The efect of ascorbate deficiency in vivo on the relative rate of collagen production and [35Sjsulfate incorporation. The percentage of protein synthesis devoted to collagen production (relative rate) and the uptake of [YS]sulfate into proteoglycan were compared in costal cartilages from control and scorbutic animals which were pulse-labeled in vitro in the presence or absence of ascorbate. Both parameters changed in similar fashion in scorbutic tissue during the experimental period (Fig. 2). By the end of stage I (Days 14-15), there was a reduction in the relative rate of collagen production to 58% (P c 0.001) and in the incorporation of [35S]sulfate into proteoglycan to 67% (P < 0.001) of control values. By stage II, [35S]sulfate incorporation was 62% and collagen production 50% of the control values (both P < 0.001). In the terminal stage of the deficiency (stage III), when the experimental groups were losing between 5 and 8% body weight per day, the relative rate of collagen production averaged only 27% of the control value (P < 0.001) and [35S]sulfate incorporation averaged 19% (P < 0.001). Only at stage III was there a significant difference in incorporation into noncollagen protein in scorbutic cartilage compared to the control values (Fig. 3). It may be concluded that the decrease in the relative rate of collagen synthesis was attributable to a specific effect on collagen and not to an increase in noncollagen protein production. Ascorbate replacement in vitro. Direct addition of ascorbate to the culture system at a final concentration of 0.2 mM did not alter the biosynthetic activities of control or scorbutic cartilage to any significant extent (Figs. 2 and 3). This observation is in

(2)

5

(21

‘2!_

II Ir (4)

I41

14)

cc

cs-

STAGE

I

STAGE

--

s

c

II

STAGE

s

III

FIG. 2. Effects of ascorbate deficiency on the relative rate of collagen production and [?5]sulfate incorporation by guinea pig costal cartilage. Cartilage was pulse-labeled for 4 h at 37”C, and the incorporation of sulfate into macromolecules (upper panel) and the relative rate of collagen production (lower panel; calculated from the ratio of 14C radioactivity in collagenase-sensitive protein to the radioactivity in total proteins) were determined as described under Materials and Methods. For each experimental group, incubations were carried out in the presence of 0.2 mM ascorbate (open bars) or in its absence (solid bars). Each pair of bars is designated C or S to indicate that the cartilage was from control or sorbutic groups, respectively. The number of incubations per data point (mean + SD) is given in parentheses. The minimum number of two incubations represents data from a single animal.

agreement with the earlier findings that the decreased collagen production in scorbutic guinea pig calvaria (9) and articular cartilage (22) was not reversed by the addition of ascorbate to in vitro cultures. This would indicate that scorbutic cartilage requires more than the mere presence of ascorbate for the normalization of collagen and glycosaminoglycan synthesis, at least in the short term.

46

BIRD,

SPANHEIMER,

AND

PETERKOFSY

vidual animals responded: six out of eight continued to lose weight on the first day, but all animals exhibited some degree of weight gain by the second day, and subsequently growth rates were either elevated or normal. A detailed description of weight regain kinetics appears in the accompanying paper (22). Ascorbate depletion proline hydroxylation.

STAGE I

STAGE II

STAGE Ill

FIG. 3. Effects of ascorbate deficiency on the incorporation of [%]proline into collagen and noncollagen proteins by guinea pig costal cartilage. The data are presented as incorporation per microgram of DNA into collagen (top panel) and noncollagen proteins (lower panel). Since no differences were seen in the incorporation of [“Clproline in the absence or presence of ascorbate (see Fig. 2), data from both incubations were used. The open bars represent incubations of control cartilage, and the solid bars show the results obtained with cartilage from ascorbate-deficient animals. Each data point is the mean f SEM; where indicated, results are significantly different from control(***P < 0.001).

Ascorbate replacement in vivo. After 2224 days on the deficient diet, depending on when they reached a rate of weight loss of at least 6% in 48 h, some guinea pigs received ascorbate for 1 or 4 days prior to sacrifice in order to study the kinetics of reversal of the defects in the relative rates of collagen production and [?S]sulfate incorporation in costal cartilage. We had found that in articular cartilage, collagen synthesis returned to near-normal levels after 4 days of ascorbate repletion (22). The kinetics of reversal in costal cartilage differed slightly from those in articular cartilage, in that at 24 h after a single 50-mg dose of ascrobate, collagen synthesis had not increased above the level in severely scorbutic costal cartilage (Fig. 4). The same was true of proteoglycan synthesis. After the guinea pigs had received four daily doses, both activities were restored to the control values. During the recovery period there was some variation in the way indi-

status

and degree of

The degree of hydroxylation of proline in collagen was determined by comparing the 3H:‘4C ratio in the collagenase-digestible material to that in the original mixture of [4-3H]- and [14C]proline. This allowed us to assess the functional activity of endogenous prolyl hydroxylase together with its cofactors (Fig. 5). The extent of proline hydroxylation in scorbutic costal cartilage was about 65% of maximal at stage II of ascorbate deficiency and remained at about the same level throughout the period of the experiment. This is similar to results obtained using calvarial bones (9), but somewhat different from results in articular cartilage, where the changes in hydroxylation appeared later and were less pronounced (22). No significant differences in hydroxylation were seen in the controls at any time point. Ascorbic acid content was measured in pooled samples of liver as this tissue is more abundant than costal cartilage and the ascorbate levels in liver were found to parallel those in bone (9) and artitular cartilage (22). The concentration remained essentially constant in animals maintained on the ascorbate-supplemented diet, but was undetectable in livers of scorbutic animals after the 14th day. One day after administration of a single dose of ascorbate to scorbutic animmals, the tissue concentration of vitamin C increased, but only to 25% of the control values. Nevertheless, proline hydroxylation was indistinguishable from that in controls at that time point, indicating that the vitamin rapidly became available to prolyl hydroxylase and that relatively low levels are saturating. Normal tissue levels of ascorbate were attained by the fourth day of repletion. The severity of the effects of scurvy upon [35Sjsulfate incorporation and the relative rate of collagen production were

COLLAGEN A

T 0

AND

PROTEOGLYCAN

SYNTHESIS

IN

SCURVY

47

0 +0.2mM ASCORBATE m NO ASCORBATE

9 E E

15

g 2 2 8

---------

10

2 5 5 ? F CL

5

0 24 ASCORBATE

REPLACEMENT

(howl

FIG. 4. Effects of ascorbate replacement in viva on the biosynthetic activity of costal cartilage from scorbutic guinea pigs. After receiving the ascorbate-deficient diet for 22-24 days, 10 guinea pigs received an oral dose of 50 mg ascorbate. Six were killed 24 h later and the remaining animals received daily doses of ascorbate until they were sacrificed on the fourth day. Quadruplicate samples of cartilage were taken from each animal for labelling with [%]sulfate (A) of which two were carried out in the presence of ascorbate (open bars) and two in its absence (solid bars). Similar samples were taken from two animals in each group for labeling with radioactive proline (B). For reference, the activities of stage II scorbutic (dashed) or control (solid) cartilages are shown by horizontal lines. All data are means + SEM.

clearly dissociated from the effects upon proline hydroxylation and the level of ascorbate in the liver. The effect of acute fasting on [35Sjsulfate incorporation and collagen production. A major feature of scurvy is the precipitous loss of body weight during the third and fourth weeks and we had previously found that the decrease in collagen synthesis in calvarial bone of scorbutic guinea pigs was correlated with this weight loss (9). Furthermore, this effect could be reproduced by acutely fasting guinea pigs supplemented with vitamin C (10). We carried out a similar analysis of both collagen and proteoglycan synthesis in costal cartilage of acutely fasted animals receiving vitamin C, permitting a comparison between the effects of simple inanition in the presence of high levels of vitamin C and scurvy-associated inanition. The results in cartilage of acutely fasted animals showed that lack of vitamin C is not a prerequisite for the decrease in collagen and proteoglycan synthesis (Table I). Both parameters were

strikingly repressed by fasting, falling within the range observed for scurvy. The accumulated data for control, scorbutic, fasted, and ascorbate-repleted groups were plotted as a function of growth rate (i.e., change in body weight) at the time of sacrifice (Fig. 6). Positive correlations emerged between the extent of weight loss or gain and both [35S]sulfate incorporation (T = 0.694; n = 22) and collagen production (r = 0.894; n = 19). Both correlations were highly significant with P < 0.001. DISCUSSION

In this paper we present evidence for the coregulation of proteoglycan and collagen synthesis in scorbutic costal cartilage. The time course of morphologic changes in guinea pig costal cartilage tissue during scurvy, previously observed by light and electron microscopy (12), closely parallel the changes in collagen and proteoglycan synthesis which we observed. These effects

48

BIRD,

SPANHEIMER, OC~NTROL ~SCORBUTIC q ASCORBATE

AND

REPLACE 0

AGE I STAGE ASCORBATE

PETERKOFSY

Ii ST/ JGE III DEFICIENCY

24h

9Bh

ASCORBATE REPLACEMENT

FIG. 5. Degree of collagen proline hydroxylation and the tissue levels of ascorbate during ascorbate deficiency and replacement. Upper: Cartilage samples were labeled with a mixture of L-[4-‘Hlproline and L-[“Clproline. The extent to which proline became hydroxylated was determined by measuring changes in the ratio of ‘H/“C radioactivity in collagenase digests compared with the initial ratio. Measurements were made for incubations carried out in the presence and absence of added ascorbate (0.2 mM). The percentage of maximal collagen proline hydroxylation was then calculated setting the sample with added ascorbate to 100% in each case. The mean value of samples (n = 18) incubated with ascorbate was 37.9%, which is similar to values found in articular cartilage (22). Solid bars are from incubations with deficient cartilage, open bars represent control incubations, and the hatched bars show the results obtained with cartilage from ascorbate-replaced animals. Lower: The amount of ascorbic acid in the livers of corresponding animals is shown. Levels in scorbutic tissue were barely detectable and are shown as thickened lines.

are more directly linked to the growth rate of the animals than to the availability of ascorbate for proline hydroxylation within the affected tissues. This is clearly shown by the strong correlation between changes in collagen and proteoglycan synthesis with changes in body weight during scurvy and similar reductions in their synthesis in acutely fasted, ascorbate-supplemented animals. These results clarify previous conflicting reports on whether or not proteoglycan synthesis is specifically decreased in scurvy (13-20). Our conclusions also are supported by the lack of response of collagen and proteoglycan synthesis to ascorbate 24 h after its administration in wivo despite the fact that levels of ascorbate were achieved which fully supported pro-

line hydroxylation. The fact that vitamin C deficiency caused a decrease in proteoglycan synthesis, although there is no known requirement for ascorbate in any of the reactions involved, supports the hypothesis that effects on the extracellular matrix components occur indirectly. Most studies agree that weight loss in scorbutic guinea pigs is the result of decreased food intake rather than increased metabolic activity (30). The etiology of the decreased food intake has been attributed to either pain in the joints of the legs preventing movement to the feedbox or pain in the tempero-mandibular joint which would discourage mastication. Our studies (data not shown) confirmed previous observations (30) that there is decreased food

COLLAGEN

AND

PROTEOGLYCAN

SYNTHESIS

TABLE THE EFFECT

Fed Fasted (Fasted/fed)

X 100

49

SCURVY

I

OF ACUTE FASTING ON THE RELATIVE THE INCORPORATION OF [“S]%LFATE Relative rate of collagen production (‘%)

Status

IN

RATE OF COLLAGEN PRODUCTION IN COSTAL CARTILAGES [%]Sulfate (cpm/pg

AND

incorporation DNA/4 h)

Expt 1 (n = 3)

Expt 2 (n = 3)

Expt 3 (n = 2)

Expt 4 (n = 3)

17.3 ? 4.2 5.2 f 2.1

12.3 + 0.6 3.5 f 0.5

17,266 f 4,637 2,705 f 379

13,629 + 1,179 2,711 + 140

30

28

16

20

a Four-week-old guinea pigs were fed ad libitum or fasted for 96 h prior to sacrifice; all animals received 50 mg of ascorbate daily. Portions of costal cartilage were incubated with radiolabeled proline or sulfate and processed as described under Materials and Methods. The results of four separate experiments are shown, each with one animal from each group. Values are the means f SD; n = number of incubations.

intake in scorbutic animals and there also was evidence that the lack of ascorbate itself resulted in decreased appetite drive based on the rapid reversal in the rate of weight loss within 24 h and the subsequent return to a normal rate of weight gain by 48 h after ascorbate repletion. At these early time points, gross examination of the joints revealed the same degree of hemorrhage and distortion as found in scorbutic animals without replacement, making it ulikely that correction of painful joint deformities allowed for increased food intake. Our results provide some basis for speculating that ascorbate may be involved in regulating appetite. The possibility that changes in collagen synthesis or secretion might influence deposition of proteoglycans in the extracellular matrix has been considered by some and it has been reported that in smooth muscle cell cultures, addition of ascorbate resulted in increased deposition of both molecules in the insoluble extracellular matrix (31). In that case, however, total [%]sulfate incorporation was only slightly affected. The kinetics of the effect on each component differed and proteoglycan deposition occurred even when collagen secretion was inhibited by a,a’-dipyridyl, suggesting that the effect of ascorbate on the metabolism of proteoglycans was not related to its role in proline hydroxylation or procollagen secretion.

Comparison of the effects of ascorbate deficiency on collagen synthesis in costal cartilage with those observed previously in calvarial bone (9) and articular cartilage (22) shows great similarities. The effect seems to be on collagen synthesis since degradation of newly synthesized or preexisting collagen was not affected (9). In acutely fasted guinea pigs receiving vitamin C, there also was no degradation of preexissting or newly synthesized collagen (10). Our studies on articular cartilage suggest that the principal mechanism for regulation of collagen synthesis during scurvy and acute fasting with vitamin C supplementation is at the level of mRNA (10,22). There are a number of stages during which the synthesis of proteoglycan might be controlled, including core protein synthesis, elongation of the glycosaminoglycan chain, synthesis of the nucleotide sugar transferases, or alterations in pool sizes of the many precursor sugars. Because the changes occurring in connective tissues during scurvy and fasting are widespread, rapid, and coordinated, it would seem probable that their propagation involves one or more humoral factors. Such factors might include hormones known to be influenced by growth rate and to have an effect upon cartilage metabolism. Serum levels of insulin, somatomedin C, triiodothyronine, and glucocorticoids are known to be altered rapidly during periods

BIRD.

20

SPANHEIMER,

0 0

. .

.

.

10 Q E -15

-10

PERCENT

0%

-5 CHANGE

0 IN WEIGHT/48

+5

+10 HOURS

FIG. 6. Correlation among growth rate, relative rate of collagen production, and uptake of [%]sulfate by costal cartilage in control, fasted, and scorbutic guinea pigs. The data are from the experiments described in Figs. 2-5 and Table I, and are plotted as the incorporation of [?$ulfate (top, n = 22) or the relative rate of collagen production (bottom, n = 19) versus the percentage of gain or loss of body weight during the 48 h preceding sacrifice. Controls (0); scorbutics (0); 96-h acutely fasted, ascorbate-supplemented (0); 24-h ascorbate-repleted (Cl); 96-h ascorbate-repleted (w). The data have been fitted to straight lines by the method of least squares.

of fasting or food restriction (32-3’7), and all have been shown to effect collagen and/ or proteoglycan production in various in vitro or in vivo systems (27, 38-47). These observations and the results we have described here and in previous studies (9,10, 22) suggest that vitamin C deficiency indirectly affects synthesis of extracellular matrix components. We propose that the process is initiated by the anorexia which eventually produces a chronic fasting state during the latter stages of scurvy, concomitant with alterations in circulating levels of one or more of the hormones discussed. REFERENCES 1. PETERKOFSKY, B. (1972) Bioehem. Biophys. Res Commun. 49, X343-1350. 2. BARNES, M. J., AND KODICEK, E. (1972) Warn Harm. 30, l-43.

AND

PETERKOFSY

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