The Metabolic Requirements for Transcellular Movement and Secretion of Collagen*

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.

From the Departments of Biochemistry and Medicine, University of Washington, Seattle, Washington 98195 SUMMARY Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.
The collagen molecule exists intracellularly as a higher molecular weight biosynthetic precursor, procollagen (1, 2)) and is synthesized primarily, if not exclusively, on membrane-bound ribosomes of the rough endoplasmic reticulum (3)(4)(5). Recent studies employing electron microscope autoradiography (6) and ferritin-labeled antibodies (7,8) have implicated the Golgi complex in the pathway for secretion of procollagen, but the detailed mechanisms and the metabolic requirements for secretion have not been established.
There is evidence for the involvement of microtubules in the transcellular movement of procollagen since agents which disrupt microtubular function also inhibit conversion of procollagen to collagen (9) and secretion of procollagen (10-12). Conversion of procollagen to collagen is thought to occur extracellularly (1, 2, 13), hence lack of conversion would be expected to follow intra-cellular retention of the protein. In the study of Ehrlich and Bornstein (9), an energy requirement for collagen secretion was suggested since incubation of embryonic chick cranial bones with m-Cl-CCP,l an inhibitor of oxidative phosphorylation, resulted in decreased conversion of procollagen to collagen. However, the possibility of nonspecific effects of the drug on the secretory process was not eliminated and metabolic consequences were not analyzed to establish that the production of ATP by oxidative metabolism was inhibited.
An ATl' requirement has been established in pancreatic zymogen and insulin secretion (14-16). In both cases, ATP production by oxidative phosphorylation appeared to be essential for secretion, while inhibition of glycolysis had little effect (15)(16)(17). In the case of zymogen secretion, this apparently reflects the tissue's predominant aerobic metabolism and a limited glycclytic capacity (15). Little is known of the mechanisms by which ATP is utilized in secretory processes, and the question remains whether glycolytic and mitochondrial ATI' are equivalent in meeting the cell's energy needs for this process. This is a pertinent consideration in light of reports suggesting such functional compartmentalization for a number of metabolic processes in animal cells (18)(19)(20)(21).
The present ,work clearly shows an energy requirement for intracellular movement and secretion of collagen. The effect of selective inhibition of ATP-producing pathways on cellular energy metabolism and collagen secretion in cultured chick tendon fibroblasts was examined to determine whether specific metabolic pathways are required for the transcellular movement and secretion of collagen. Co., Inc.) was then placed in the center well and bottles were incubated 90 min at room temperature. Control experiments indicated these conditions accomplished maximal absorption of the 14C02. Three bottles containing confluent cell layers were used for each experiment.
In these experiments it was found to be more accurate to express data on the basis of culture bottles rather than protein content. Protein determinations were unreliable due to the difficulty in quantitatively removing cell protein from the glass surface.

RESULTS
Cell Cuulture-Isolated tendon cells adapted well to culture and exhibited a typical fibroblast-like morphology. Cell division was rapid with a doubling time of 18 hours estimated from the logarithmic growth phase of the curve shown in Fig. 1. Cultures reached a confluent density of 5 to 10 x lo6 cells per flask at 4 to 5 days. Subsequently, cell division was slower as additional layers began to accumulate.

Pulse-Chase
Experiments-It was found necessary to use relatively long incubation periods to obtain sufficient incorporation of proline into collagen for subsequent analyses. Thus, as seen in Fig. 2, radioactive peptidyl proline or hydroxyproline was not released into the culture medium in detectable quantity until 45 min after administration of the isotope. A similar lag in secretion was observed in cultured chick embryo fibroblasts (31), while freshly isolated chick tendon fibroblasts exhibited a shorter lag time (11,22). By 60 min, 9% of the incorporated radioactivity but 20% of the radioactive hydroxyproline was present in the medium ( Fig. 2A), indicating active secretion of radioactive collagen. A 60-min incorporation period was used to obtain maximal labeling of intracellular collagen in subsequent pulsechase experiments.
The data in Fig. 2A indicate that the conditions used during the chase period following a SO-min pulse were effective. Thus, tot,al radioactivity incorporated in the cell layer at the start of the chase period closely approximates that present in the combined cell layer aud medium at all times during the chase. small increase in total radioactive hydroxyproline observed following institution of the chase period ( Fig. 2B) presumably results from hydroxylation of proline present in nascent polypeptide chains at the start of the chase (32, 33).
Since the amount of radioactive collagen appearing in the culture medium was nearly maximal after a chase of 60 min (Fig.  2B), this period was selected for subsequent experiments. However, not all radioactive collagen is released from the cell layer during a chase period. Thus, even after 4 hours, 37% of the radioactive hydroxyproline remains associated with cells (Fig.  2B). In order to determine the nature of this material, collagenous proteins were extracted from cell layers (24) and analyzed by sodium dodecyl sulfate gel electrophoresis (34). Only disulfidebonded procollagen was identified in cultures pulsed for 45 min with radioactive proline. However after a 4-hour chase, a substantial fraction of this material had been converted to collagen (data not shown). Since conversion of procollagen occurs extracellularly (1, 2) the material presumably exists in fibrillar form (35). Apparently a fraction of secreted procollagen in chick cell cultures is converted rapidly and is retained in the cell layer. The remainder of the procollagen in the medium appears to be converted only very slowly to cell-associated collagen (34) evidenced by high rates of glucose utilization and lactate production. More than 90% of the glucose metabolized by such cultures was accounted for by lactate released into the medium (data not shown). This observation is consistent with previous studies of cultured diploid fibroblasts (36, 37). We hoped to utilize the glycolytic inhibitors iodoacetate and 2-deoxyglucose, in conjunction with an alternate energy source such as pyruvate, to examine the effect of selective inhibition of glycolytic ATP production on cellular energy levels and on the ability of the cell to transport and secrete collagen. Experiments with varying concentrations of iodoacetate in the presence of glucose indicated that a concentration of 0.05 mM effected an 85% inhibition of glycolysis as measured by lactate production. This concentration, and a concentration of 6.1 mM 2-deoxyglucose (1 mg/ml) were chosen in experiments to examine the selectivity of these agents.
Incorporation of [3H]tryptophan or [3H]proline into protein was unimpaired in cultures metabolizing pyruvate rather than glucose ( Table I). Secretion of collagen was also normal as judged by appearance of radioactive hydroxyproline in the medium of cultures incubated with [3H]proline (Table II). When iodoacetate was added to pyruvate-containing medium, the reduction in labeled protein was predominantly in the medium fraction, reflecting an inhibition of both protein synthesis and secretion (Table II). Paradoxically, the addition of iodoacetate to cells cultured in the presence of both pyruvate and glucose resulted in a substantially greater reduction in protein synthesis (see below and "Discussion").
2-Deoxyglucose produced a dramatic decrease in proline incorporation into protein, to a level less than 10% of that in control cultures (Table II).
To determine the contribution of a reduced synthesis of high energy compounds to these results, analyses of ATP content as well as lactate production were performed. As shown in Table  4844   TABLE   I   TABLE   III Ability  III, glycolysis, measured by lactate production, was reduced to less than 10% of control values when pyruvate was used as a carbohydrate source, while the ATP content of the cells remained normal. Thus, under these conditions glycolysis should make only a minor contribution to ATP production. Yet, addition of either iodoacetate or 2-deoxyglucose to culture medium, whether supplemented with pyruvate or not, markedly decreased cellular ATP levels (Table III). This should not occur if these drugs act specifi- tally as inhibitors of glycolysis, and suggests that other pathways of energy metabolism may be inhibited as well. Addition of glucose or glucose and pyruvate to cells cultured in the presence of iodoacetate increased lactate production but reduced ATP levels further to almost undetectable levels (see "Discussion").
To obtain additional information on the specificity of these inhibitors, r4C02 production was monitored in cultures incubated with [2-r4C]pyruvate. Production of radioactive COZ was found to be lowered in the presence of either drug (  (Table  V). However, when m-Cl-CCP was added in the absence of glucose and glycolysis was functioning at a low level, ATP levels dropped dramatically. Obviously, a very high rate of glycolysis is required to maintain cellular ATP levels in the presence of m-Cl-CCP. However, in the absence of m-Cl-CCP, high ATP levels were maintained with a very low rate of glycolysis in pyruvat)e-supplemented medium or in the absence of carbohydrate for 1 hour. Apparently, respiratory ATP production maintained cellular ATP levels in the absence of glucose, and was inhibitable by m-Cl-CCP. To corroborate this, we examined CO2 production from pyruvate in the presence of m-Cl-CCP. If m-Cl-CCP is, in fact, effective as an uncoupler of oxidative phosphorylation, an increased oxidation of pyruvate to CO2 resulting from the release of respiratory control should be observed. In fact, a 4-fold stimulation of radioactive CO2 production in the presence of m-Cl-CCP was observed (Table IV), indicating that the drug was effective under the experimental conditions used. Collagen secretion, as monitored by the appearance of radioactive hydroxyproline in the medium, was maintained at control values as long as cellular ATP levels remained high (Table V). Thus, specific inhibition of glycolytic metabolism (by use of pyruvate as an energy source) or of oxidative metabolism (by use of glucose plus m-Cl-021') did not result in appreciably decreased collagen secretion. However, when both glycolysis and oxidative metabolism were interfered with (by use of m-Cl-CCP in the absence of an external energy source), AT'1 levels fell and collagen secretion was inhibited ( Table V). The inhibition of collagen secretion cannot be ascribed to nonspecific effects of m-Cl-CCP since secretion was restored to near normal levels when glucose was present in addition to m-Cl-CCP. Thus, these results show that the secretion of collagen by cultured chick tendon fibroblasts is dependent on an energy source which can be provided by either glycolysis or respiration.
Effects of Inhibition of Protein Synthesis on Collagen Secretion-An inhibition of energy metabolism leading to a reduction in ATP levels is also likely to lead to an inhibition in protein synthesis. Previous studies had suggested that inhibition of protein syn thesis did not interfere with conversion of procollagen to collagen (39) and therefore, presumably, did not interfere with secretion. Nevertheless, it was important to determine whether the inhibition of collagen secretion observed as a result of interference with energy metabolism could be attributed to reduced protein synthesis.
Experiments were therefore performed to determine the extent of inhibition   was included in the chase medium after a pulse with [3H]proline, only a small degree of inhibition of collagen secretion was observed (Table VI). Thus, collagen secretion remained largely unimpaired despite an inhibition of new protein synt.hesis exceeding 90%. The hydroxyprolinc present in the medium of inhibited cultures cannot be attributed to cell lysis since the ratio of radioactive proline to hydroxyproline in the medium of control and inhibited cultures was the same.
Both ribosome-bound polypeptides resulting from cycloheximide administration and prematurely terminated puromycincontaining nascent chains are likely to be retained intracellularly. These chains are at least partially hydroxylated (32,33), and would therefore be detected by the hydroxyproline assay. Assuming a synthesis time of 6 min for a procollagen chain (40), a sufficient percentage of radioactive hydroxyproline could be present in nascent chains at the end of a 60.min pulse period to account for the small degree of inhibition of secretion observed in the presence of puromycin and cycloheximide. DISCUSSION A high rate of glucose utilization and lactate production is characteristic of cultured cells (41). Although we did not attempt to measure cellular respiration in the presence of glucose, the rate of glucose metabolism and respiration observed in other diploid fibroblast cultures indicates that both glycolysis and respiration make significant contributions to ATP production (36, 37, 42). The data in Table V show that chick tendon fibroblasts maintain high ATP levels when either glycolysis or oxidative phosphorylation is inhibited. Thus, these cultures represent a good system to examine the effect of selective inhibition of major energy-producing pathways on intracellular transport and secretion. Our results show that there is an ATP requirement for collagen secretion, and that ATP produced either by glycolysis or by oxidative phosphorylation may be utilized effectively in this process. The slight inhibition of secretion observed in the presence of m-Cl-CCP and glucose may indicate a preferential utilization of mitochondrial ATP. Alternatively, this could represent a minor inhibitory effect of m-Cl-CCP which is unrelated to energy metabolism.
These findings differ from those obtained with embryonic chick cranial bones, in which incubation with m-Cl-CCP, albeit at a higher concentration, in the presence of glucose resulted in a substantial inhibition of collagen secretion (12). The difference may be due to the high glycolytic capacity of cultured cells which allows them to maintain high ATP levels by glycolysis alone, whereas the tissue may not have this capacity. Pancreatic tissue slices apparently are not able to maintain sufficient cellular energy reserves to support zymogen secretion exclusively by glycolysis (15).
Cell fractionation and electron microscope autoradiography have established movement from the transitional elements of the rough endoplasmic reticulum to the condensing vacuoles of the Golgi complex as the first energy-dependent step in the secretory pathway for pancreatic zymogens (15). The situation is less clear with respect to insulin secretion, but there appears to be an energy-dependent step between the Golgi complex and storage vacuoles, and probably an earlier block between the rough endoplasmic reticulum and the Golgi as well (16). Furthermore, the energy dependence of exocytosis of secretory granules from exocrine glands generally is well established (43). Thus, processes in the secretory pathway involving membrane fissionfusion appear to be energy-dependent.
Since thcrc is now good evidence that the secretory pathway for procollagen in chick tendon fibroblasts involves movement through the Golgi complex (668), membrane fission and fusion will almost certainly occur and should be energy-dependent.
If microtubules are involved in intracellular translocation as suggested by several laboratories (g-12), the normal functioning of these subcellular organelles represents another ATP-requiring process. In our experiments the use of iodoacctate resulted in a decrease in cellular ATP which could not be accounted for by glycolytic inhibition alone. In the presence of glucose, iodoacetate produced an even greater reduction in ATP levels (Table III), probably because of utilization of ATP in the production of phosphorylated intermediates in glycolysis. Such compounds accumulate due to the inhibition of glyceraldehyde-3-phosphate dehydrogenase by iodoacetate (18). The concentration of iodoacetate used (0.05 mM) is somewhat less than that commonly used for inhibition of glycolysis (15,17,18) and has been reported to inhibit glycolysis specifically without appreciably affecting respiration in some systems (44). However, a survey of the effects of iodoacetate on respiration in the absence of an added energy source reveals a great deal of variability in specificity of action (44). In a number of cases, including mouse fibroblasts in culture, iodoacetate concentrations in the range of 0.05 mM produced significant inhibition of respiration (44). It is apparent from our results that it is necessary to determine the specificity of inhibition of energy metabolism by iodoacetate when the compound is used as an inhibitor of glycolysis. The decreased COz production from ['"Clpyruvate may indicate direct inhibition of substrate utilization by the Krebs cycle and electron transport system. Alternatively, iodoacetate may inhibit transport of pyruvate into the cell either directly or as a result of selective use of ATP produced by glycolysis in active transport across the plasma membrane. In either case ATP production resulting from respiration would be reduced.
Similar considerations apply to the use of 2-deoxyglucose. 2-Deoxyglucose acts as a competitive inhibitor of glucose transport and is phosphorylated intracellularly, probably by hexokinase (45, 46). The accumulation of 2-deoxyglucose B-phosphate apparently causes competitive inhibition of glucose-6-phosphate isomerase (47). The very low levels of ATP observed in the presence of 2-deoxyglucose (Table III) may result in part from the utilization of ATP in the phosphorylation of 2-deoxyglucose. However, as in the case of iodoacetate, decreased metabolism of pyruvate (Table IV) also appears to play a role. 4847 2-Deoxyglucose has been used as an inhibitor of glycosylation in the synthesis of collagen (48)) immunoglobulins (49)) and yeast glycoproteins (50, 51). The mechanism of inhibition of glycosylation by 2-deoxyglucose may involve interference with uridine nucleotide metabolism with a resultant accumulation of UDP-2deoxyhexose (52-54). Melchers (49) concluded, on the basis of the use of this compound, that glycosylation of immunoglobulins was necessary in order to permit the transcellular movement of these proteins in plasma cells. However, our results indicate that 2-deoxyglucose cannot be regarded as a specific inhibitor of glycosylation or glycolysis. These results are consistent with the observation that the inhibition of incorporation of lysine into protein by embryonic chick tibiae, caused by 2-deoxyglucose, was not relieved by the addition of inosine as an energy source (48). The large decrease in ATP levels caused by 2-deoxyglucose must therefore be considered in experiments purporting to examine the role of glycosylation in intracellular transport and secretion of glycoproteins.