Bone matrix studies. Influences of parathyroid extract, calcitonin, and cholecalciferol and of rickets and its treatment.

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by parathyroid extract. Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamin D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.     

Bone matrix studies Influences of parathyroid extract,calcitonin, and cholecalciferol and of rickets and its treatment

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by paratyroid extract.Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamine D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.     

Phytocomponent p-hydroxycinnamic acid stimulates bone formation and inhibits bone resorption in rat femoral tissues in vitro

The objective of this study was to examine alcohol-induced changes of bone in hormone-deficient males using the developed method. In the process of bone resorption, type I collagen crosslinking molecules, pyridinoline (PYD), are released into the circulation and cleared by the kidneys. ²H₂O as a tracer has been applied to measure the synthesis rates of slow-turnover proteins and successfully applied to bone collagen synthesis in our hormone deficiency rats. This study demonstrated for the first time, the early changes of the femur bone degradation in hormone-deficient male individuals, more influenced by alcohol through histopathological study, serum PYD assay, and ²H₂O labeling. We also observed that serum PYD was a sensitive pathological marker of bone degradation in castrated osteoporosis males and the unique features of ²H₂O labeling to measure the bone turnover collagen synthesis rates were excellent markers of bone degradation and aging.     

Gill protein turnover: costs of adaptation

Measurements of gill protein synthesis, and hence turnover, were greatly facilitated over the last decade by the application of "flooding dose" methodology to non-mammalian species. Numerous studies show that in fish and aquatic invertebrates, gills are among the most active tissues with respect to protein turnover, this being true under a variety of environmental and nutritional conditions. The main components being turned over in fish gills are probably collagen, primarily in the gill arches, and epithelial cell proteins in the filaments, both arches and filaments having similar protein synthesis rates. Intriguingly, differences are apparent between protein synthesis rates of adjacent holobranchs, the first (most anterior) being significantly more active than the second or third, perhaps hinting at functional differences between holobranchs. Experimental estimates of energetic costs for protein synthesis, derived from cycloheximide treatment of isolated perfused gills, give a maximum value of 14 mmol O2/g protein synthesized, which is about double theoretical costs. Environmental stressors, such as heavy metals or acid/aluminum, have variable effects on branchial protein turnover. Limited data suggest that zinc or acid exposure depresses protein synthesis, whereas acid/aluminum increases it quite markedly. Calculations indicate that whereas effects within the gills may be substantial, in terms of whole animal energetics, the costs of branchial adaptation are likely to be small.     

Collagen turnover in the adult femoral mid-shaft: modeled from anthropogenic radiocarbon tracer measurements

We have measured the (14)C content of human femoral mid-shaft collagen to determine the dynamics of adult collagen turnover, using the sudden doubling and subsequent slow relaxation of global atmospheric (14)C content due to nuclear bomb testing in the 1960s and 1970s as a tracer. (14)C measurements were made on bone collagen from 67 individuals of both sexes who died in Australia in 1990-1993, spanning a range of ages at death from 40 to 97, and these measurements were compared with values predicted by an age-dependent turnover model. We found that the dataset could constrain models of collagen turnover, with the following outcomes: 1) Collagen turnover rate of females decreases, on average, from 4%/yr to 3%/yr from 20 to 80 years. Male collagen turnover rates average 1.5-3%/yr over the same period. 2) For both sexes the collagen turnover rate during adolescent growth is much higher (5-15%/yr at age 10-15 years), with males having a significantly higher turnover rate than have females, by up to a factor of 2. 3) Much of the variation in residual bomb (14)C in a person's bone can be attributed to individual variation in turnover rate, but of no more than about 30% of the average values for adults. 4) Human femoral bone collagen isotopically reflects an individual's diet over a much longer period of time than 10 years, including a substantial portion of collagen synthesised during adolescence.     

Changes in synthesis of types-I and -III collagen during matrix-induced endochondral bone differentiation in rat.

The changes in rates of hydroxyproline formation and biosynthesis of types-I and -III collagen during bone matrix-induced sequential differentiation of cartilage, bone and bone marrow in rat were investigated. Biosynthesis of types-I and -III collagen at different stages of this sequence was studied by labelling in vivo and in vitro with [2,3-3H]proline. Pepsin-solubilized collagens were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis. The results revealed that maximal amounts of type-III collagen were synthesized on day 3 during mesenchymal-cell proliferation. Thereafter, there was a gradual decline in type-III collagen synthesis. On days 9--20 during bone formation predominantly type-I collagen was synthesized. Similar results were obtained by the use of labelling techniques both in vivo and in vitro.     

Acute IGF-I infusion stimulates protein synthesis in skeletal muscle and other tissues of neonatal pigs

Studies have shown that protein synthesis in skeletal muscle of neonatal pigs is uniquely sensitive to a physiological rise in both insulin and amino acids. Protein synthesis in cardiac muscle, skin, and spleen is responsive to insulin but not amino acid stimulation, whereas in the liver, protein synthesis responds to amino acids but not insulin. To determine the response of protein synthesis to insulin-like growth factor I (IGF-I) in this model, overnight-fasted 7- and 26-day-old pigs were infused with IGF-I (0, 20, or 50 microg. kg(-1). h(-1)) to achieve levels within the physiological range, while amino acids and glucose were clamped at fasting levels. Because IGF-I infusion lowers circulating insulin levels, an additional group of high-dose IGF-I-infused pigs was also provided replacement insulin (10 ng. kg(-0.66). min(-1)). Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, low-dose IGF-I increased protein synthesis by 25-60% in various skeletal muscles as well as in cardiac muscle (+38%), skin (+24%), and spleen (+32%). The higher dose of IGF-I elicited no further increase in protein synthesis above that found with the low IGF-I dose. Insulin replacement did not alter the response of protein synthesis to IGF-I in any tissue. The IGF-I-induced increases in tissue protein synthesis decreased with development. IGF-I infusion, with or without insulin replacement, had no effect on protein synthesis in liver, jejunum, pancreas, or kidney. Thus the magnitude, tissue specificity, and developmental change in the response of protein synthesis to acute physiological increases in plasma IGF-I are similar to those previously observed for insulin. This study provides in vivo data indicating that circulating IGF-I and insulin act on the same signaling components to stimulate protein synthesis and that this response is highly sensitive to stimulation in skeletal muscle of the neonate.     

Increased Collagen Synthesis Rate during Wound Healing in Muscle

Wound healing in muscle involves the deposition of collagen, but it is not known whether this is achieved by changes in the synthesis or the degradation of collagen. We have used a reliable flooding dose method to measure collagen synthesis rate in vivo in rat abdominal muscle following a surgical incision. Collagen synthesis rate was increased by 480% and 860% on days 2 and 7 respectively after surgery in the wounded muscle compared with an undamaged area of the same muscle. Collagen content was increased by approximately 100% at both day 2 and day 7. These results demonstrate that collagen deposition during wound healing in muscle is achieved entirely by an increase in the rate of collagen synthesis.     

Increased bone turnover is associated with protein and energy metabolism in adolescents with sickle cell anemia

Contribution of bone turnover to the hypercatabolic state observed in sickle cell anemia is unknown. We examined the association between markers of bone turnover and basal rates of whole body protein turnover and energy expenditure in 28 adolescents with homozygous sickle cell anemia (HbSS) and in 26 matched controls with normal phenotype (HbAA). Whole body protein breakdown and synthesis were measured using a stable isotope of [15N]glycine, resting energy expenditure was measured by whole room indirect calorimetry, and the rate of pyridinoline cross-link (PYD) excretion in urine and fasting serum levels of the type I procollagen carboxy-terminal propeptide (PICP) were measured with commercial kits. Urinary PYD and serum PICP were significantly elevated in HbSS patients. The increase in procollagen synthesis, indicated by high levels of PICP, was significantly correlated with increased whole body protein synthesis. The increase in type I collagen degradation, indicated by high PYD excretion, was significantly correlated with increased protein breakdown. We conclude that increased rates of bone turnover contribute to the increased rates of protein turnover and energy expenditure observed in adolescents with homozygous sickle cell anemia.     

Type I collagen synthesis by human osteoblasts in response to placental lactogen and chaperonin 10, a homolog of early-pregnancy factor

Recent studies have indicated that maternal skeletal metabolism undergoes significant changes during gestation. The agents that are responsible for eliciting these changes in bone turnover during pregnancy have yet to be defined. We therefore sought to investigate whether chaperonin 10 (Cpn10), a homolog of early-pregnancy factor, or human placental lactogen (PL) were capable of influencing the synthesis of type I collagen by human osteoblasts in vitro. Both Cpn10 and PL are major components of the maternal circulation during pregnancy, but how they might contribute to bone metabolism has not been determined. Type I collagen represents the most abundant component of bone tissue, accounting for approximately 90% of the organic compartment. Both Cpn10 and PL were capable of stimulating the synthesis of type I collagen by human osteoblasts in culture. The inclusion of 17 beta-estradiol or prolactin, however, failed to influence the ability of cells to mobilize type I collagen. These novel findings support a role for PL and Cpn10 in the metabolism of bone tissue during pregnancy. Maternal bone collagen metabolism is clearly an important event during pregnancy, and the identification of the factors responsible will aid our understanding of the regulation of skeletal metabolism during gestation.     

Growth rate correlates negatively with protein turnover in Arabidopsis accessions

Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed ¹³CO₂ pulse‐chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised protein day^?1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade‐off between protein turnover and maximisation of growth rate.     

Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs

Infusion of physiological levels of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates. To determine whether insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonates, insulin secretion was blocked with somatostatin in fasted 7-day-old pigs (n = 8-12/group) while glucose and glucagon were maintained at fasting levels and insulin was infused to simulate either less than fasting, fasting, intermediate, or fed insulin levels. At each dose of insulin, amino acids were clamped at either the fasting or fed level; at the highest insulin dose, amino acids were also reduced to less than fasting levels. Skeletal muscle protein synthesis was measured using a flooding dose of l-[4-(3)H]phenylalanine. Hyperinsulinemia increased protein synthesis in skeletal muscle during hypoaminoacidemia and euaminoacidemia. Hyperaminoacidemia increased muscle protein synthesis during hypoinsulinemia and euinsulinemia. There was a dose-response effect of both insulin and amino acids on muscle protein synthesis. At each insulin dose, hyperaminoacidemia increased muscle protein synthesis. The effects of insulin and amino acids on muscle protein synthesis were largely additive until maximal rates of protein synthesis were achieved. Amino acids enhanced basal protein synthesis rates but did not enhance the sensitivity or responsiveness of muscle protein synthesis to insulin. The results suggest that insulin and amino acids independently stimulate protein synthesis in skeletal muscle of the neonate.     

Gel-filtrated fractions of alveolar bone extract contain factors promoting cell attachment and a mitogenic effect on periodontal ligament fibroblasts

The purpose of this study was to investigate the effect of acetic acid-extracted bone proteins on human periodontal ligament fibroblasts (hPF) with respect to mitogenic and cell attachment promoting activity. Alveolar bone was harvested from healthy donors and subjected to 0.5 M acetic acid extraction, dialysis and lyophilization, and gel filtration. Promotion of cell attachment and stimulation of DNA synthesis by the crude extract and gel-filtrated fractions were studied in cultured hPE Many protein components, varying in molecular weight from 10-14 to 120 kDa, were detectable in 10% SDS-PAGE of the extract. Gel filtration of bone extract disclosed four fractions with molecular weights of 55, 34, 29 and 19-20 kDa. Both the 34 and 55 kDa fractions at a concentration of 5 microg/ml, but not the 29- or 19-20 kDa fractions, were found to promote cell attachment while only the 55 kDa fraction (5 microg/ml) stimulated DNA synthesis of hPF, Both mitogenic activity and the promotion of the cell attachment by gel-filtrated active fractions were resistant to thermal treatment (70 degrees C) and pH (4 to approximately 8) changes. These findings suggest that acetic acid extract of alveolar bone may contain components which are capable of modulating cell attachment and mitogenesis of hPF.     

Accumulation of advanced glycation end products decreases collagen turnover by bovine chondrocytes

The integrity of the collagen network is essential for articular cartilage to fulfill its function in load support and distribution. Damage to the collagen network is one of the first characteristics of osteoarthritis. Since extensive collagen damage is considered irreversible, it is crucial that chondrocytes maintain a functional collagen network. We investigated the effects of advanced glycation end products (AGEs) on the turnover of collagen by articular cartilage chondrocytes. Increased AGE levels (by culturing in the presence of ribose) resulted in decreased collagen synthesis (P < 0.05) and decreased MMP-mediated collagen degradation (P < 0.02). The latter could be attributed to increased resistance of the collagen network to MMPs (P < 0.05) as well as the decreased production of MMPs by chondrocytes (P < 0.02). Turnover of a protein is determined by its synthesis and degradation rates and therefore these data indicate that collagen turnover is decreased at enhanced AGE levels. Since AGE levels in human cartilage increase approximately 50 fold between age 20 and 80, cartilage collagen turnover likely decreases with increasing age. Impaired collagen turnover adversely affects the capacity of chondrocytes to remodel and/or repair its extracellular matrix. Consequently, age-related accumulation of AGE (via decreased collagen turnover) may contribute to the development of cartilage damage in osteoarthritis.     

Effect of administration of oral contraceptives in vivo on collagen synthesis in tendon and muscle connective tissue in young women

Women are at greater risk than men for certain kinds of diseases and injuries, which may at least partly be caused by sex hormonal differences. We aimed to test the influence of estradiol in vivo on collagen synthesis in tendon, bone, and muscle. Two groups of young, healthy women similar in age, body composition, and exercise-training status were included. The two groups were either habitual users of oral contraceptives exposed to a high concentration of synthetic estradiol and progestogens (OC, n = 11), or non-OC-users tested in the follicular phase of the menstrual cycle characterized by low concentrations of estradiol and progesterone (control, n = 12). Subjects performed 1 h of one-legged kicking exercise. The next day collagen fractional synthesis rates (FSR) in tendon and muscle connective tissue were measured after a flooding dose of [(13)C]proline followed by biopsies from the patellar tendon and vastus lateralis in both legs. Simultaneously, microdialysis catheters were inserted in vastus lateralis and in front of the patellar tendon for measurement of insulin-like growth factor I (IGF-I) and its binding proteins. Serum NH(2)-terminal propeptide of type I collagen (PINP) and urine COOH-terminal telopeptides of type-I collagen (CTX-I) were measured as markers for bone synthesis and breakdown, respectively. Tendon FSR and PINP were lower in OC compared with control. An increase in muscle collagen FSR postexercise was only observed in control (P < 0.05). Furthermore, the results indicate a lower bioavailability of IGF-I in OC. In conclusion, synthetic female sex hormones administered as OC had an inhibiting effect on collagen synthesis in tendon, bone, and muscle connective tissue, which may be related to a lower bioavailability of IGF-I.     

Influence of thyroid hormones on collagen content in tissues of guinea pigs.

Collagen fractions content and level of collagen catabolites in body fluids were determined in normal, hypo- and hyperthyroid guinea pigs. An increase of urinary excretion of hydroxyproline and hydroxylysine as well as concentration of these amino acids in blood serum was found in hyperthyroidism, and a decrease was shown in hyperthyroid guinea pigs. Hyperthyroidism stimulated an increase of neutral-salt-soluble and acid-soluble collagen in skin and liver, and a decrease of insoluble collagen in skin as well as increase of all collagen fractions in bone samples. Hypothyroidism induced a decrease of all collagen fractions in skin and liver, and an increase of acid-soluble and insoluble collagen in bone samples.     

Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid

Knowledge of rates of protein turnover is important for a quantitative understanding of tissue synthesis and catabolism. In this work, we have used the racemization of aspartic acid as a marker for the turnover of collagen obtained from healthy and pathological human intervertebral disc matrices. We measured the ratio of the d- and l-isomers in collagen extracted from these tissues as a function of age between 16 and 77 years. For collagen taken from healthy discs, the fractional increase of d-Asp was found to be 6.74 x 10(-4)/year; for degenerate discs, the corresponding rate was 5.18 x 10(-4)/year. Using the racemization rate found previously for the stable population of collagen molecules in dentin, we found that the rate of collagen turnover (k(T)) in discs is not constant but rather a decreasing function of age. The average turnover rate in normal disc between the ages of 20 and 40 is 0.00728 +/- 0.00275/year, and that between the ages of 50 and 80 is 0.00323 +/- 0.000947/year, which correspond to average half-lives of 95 and 215 years, respectively. Turnover of collagen from degenerate discs may be more rapid than that found for normal discs; however, statistical analysis leaves this point uncertain. The finding of a similar correlation between the accumulation of d-Asp and that of pentosidine for three normal collagenous tissues further supports the idea that the accumulation of pentosidine in a particular tissue can, along with the racemization of aspartic acid, be used as a reliable measure of protein turnover.     

Turnover Rates of Hepatic Collagen and Circulating Collagen-Associated Proteins in Humans with Chronic Liver Disease

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of ²H₂O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2–0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.     

Rates of collagen synthesis in lung, skin and muscle obtained in vivo by a simplified method using [3H]proline.

Methods for measurement of rates of collagen synthesis in vivo have thus far been technically difficult and often subject to quite large errors. In this paper a simplified method is described for obtaining synthesis rates of collagen and non-collagen proteins, for tissues of rabbits. This involves an intravenous injection of [3H]proline, administered with a large dose of unlabelled proline, and measurement of the specific radioactivity of proline and hydroxyproline in body tissues up to 3 h later. The specific radioactivity of [3H]proline in plasma and the tissue free pools rises rapidly to a plateau value which is maintained for at least 2 h, when the specific radioactivity of the type I collagen precursors, isolated from the skin, was similar to that of the plasma and tissue-free pool. Furthermore, over this period, the increase in the specific radioactivity of proline in collagen and non-collagen protein was linear with respect to time. These results suggest that the large dose of proline floods the precursor pools for protein synthesis, and that this effect can be maintained for quite long periods of time. Such kinetics greatly simplified the method for obtaining collagen synthesis rates in vivo, which were calculated for lung, heart, skin and skeletal muscle, and shown to be quite rapid, ranging between about 3 and 10%/day. The lung was a particularly metabolically active tissue, with synthesis rates of about 10%/day for collagen and 35%/day for total non-collagen proteins, indicating rapid turnover of both intracellular and extracellular proteins of this tissue.     

Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 1802 labelling.

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.