Increased collagen metabolism in granulomata induced in rats deficient in endogenous prostaglandin precursors

Collagen metabolism was measured (in terms of various hydroxyproline (HP), DNA and protein ratios) in granulomata obtained after s.c. implantation of carrageenan-impregnated and untreated polyether sponges into normal and essential fatty acid deficient (EFAD) rats for 8 and 15 days. Collagen synthesis (HP/protein) in day 8 and 15 untreated granulomata was the same for both normal and EFAD rats, though collagen breakdown (total HP) appeared to be greater in EFAD granulomata on day 15. With carrageenan-impregnated sponges, collagen synthesis in EFAD granulomata was much greater than in normal granulomata on both day 8 and day 15. Ratios of protein and/or HP to DNA (probably indicative of cellular infiltration) were increased in EFAD rats with both sponge types, though this increase was less pronounced with carrageenan-impregnated sponges. Is is suggested that endogenous prostaglandin (PG) production (markedly reduced during EFA deficiency) may exert a negative feedback effect on collagen metabolism during proliferative inflammation.     

Reduced exudation and increased tissue proliferation during chronic inflammation in rats deprived of endogenous prostaglandin precursors

Two models of chronic inflammation were studied in rats deprived of endogenous precursors of prostaglandins by feeding the animals on essential fatty acid deficient (EFAD) food. During kaolin-induced pouch-granuloma, exudate production was markedly reduced in EFAD rats, when compared with normal animals. The exudates from normal rats contained large amounts of PGE, but in the exudates from EFAD rats the amount of PGE was very markedly reduced. Similarly, with carrageenan-impregnated polyether sponges, the exudative component of inflammation was reduced in EFAD rats. However, the proliferative component was significantly increased, particularly in relation to the stunted growth of EFAD rats. Sponge exudates from EFAD rats contained fewer leucocytes than those from normal animals but the fall in leucocyte count was much smaller than the very marked reduction in PGE activity. EFAD rats also exhibited a significant increase in adrenal weights.The results are discussed in the light of the ambivalent (pro- or anti-inflammatory) role of endogenous PGs. It appears that, in the proliferative phase of inflammation, the anti-inflammatory role of PGs is more dominant.     

The diabetic rat as an impaired wound healing model: stimulatory effects of transforming growth factor-beta and basic fibroblast growth factor

Two models of wound repair compared the effect of defined, recombinant growth factors on the rate of wound repair in both normal and streptozotocin-induced diabetic rats: subcutaneous implantation of polyvinyl alcohol sponges and incisional wounding. Transverse incisional wounds were made on the dorsal surface of rats and closed with steel sutures. Three days postwounding the rats received a single injection of either transforming growth factor-beta or vehicle alone directly into the wound site. Animals were sacrificed 7, 14, and 21 days postwounding, and fresh and formalin-fixed wound tensile strength were measured. Diabetic rats had expected defects in wound repair, including decreased granulation tissue and reduced amounts of collagen, protein, and DNA. Fresh tensile strength of the diabetic incisions was 53% of normal on Day 7 (p < or = .01) and 29% of normal on Day 21. Fixed tensile strength was 41% of normal on Day 7 (p < or = .01) and fell to 78% of normal by Day 21 (p < or = .01), suggesting that collagen concentrations of diabetic wounds increased towards normal but did not undergo maturation. TGF beta produced a moderate increase in tensile strength of fresh and fixed wounds of diabetic rats, but not to the levels of wounds in untreated normal rats. Sponges fill with granulation tissue, their reproducible rate of organization being measured by histological and biochemical methods. A single injection into sponges 3 days postimplantation of basic fibroblast growth factor, transforming growth factor-beta, or vehicle only, was evaluated at 7 and 9 days postimplantation. In the sponge model, bFGF and TGF beta were each able to induce significant increases in the accumulation of granulation tissue in both diabetic and normal rats. TGF beta increased the collagen content of sponges by 136% in sponges from diabetic animals (p < or = .001), thereby raising the collagen content to that of normal control wounds, while stimulating a 49% (p < or = .02) increase in sponges from normal animals on Day 9. By contrast, the response to bFGF was predominantly an increase in the protein and DNA content of the sponges. These results emphasize the differential effects of the two cytokines in accelerating healing under conditions of defective wound repair.     

Synthesis and degradation of collagens in skin of healthy and protein-malnourished rats in vivo, studied by 18O2 labelling.

To explore the effects of growth retardation, caused by restricted protein intake, on collagen turnover in the whole skin, Sprague-Dawley rats (n = 20) were labelled with 18O2 and fed on either an adequate (18%) or a low (3%) lactalbumin diet. Skin biopsies were obtained at intervals during the following 6 months. Independent groups of animals (n = 186) were used to determine the size of the 0.5 M-acetic acid-soluble and -insoluble collagen pools in the entire skin of healthy and malnourished rats. Collagen was estimated by measurement of hydroxyproline. Soluble-collagen synthesis rates were equivalent to 99 +/- 8 mumol of hydroxyproline/day in healthy animals and 11 +/- 2 mumol/day in malnourished rats. Insoluble-collagen synthesis rates were 32 and 5 mumol of hydroxyproline/day in the healthy and protein-depleted rats respectively. The degradation of soluble collagen amounted to 37 +/- 8 and 6 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Efflux of collagen from the soluble collagen, defined as the sum of the rate of soluble collagen that is degraded plus that which matures into insoluble collagen, was 70 +/- 8 and 11 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Insoluble collagen was not degraded in either group. The fraction of soluble collagen leaving the pool that was converted into insoluble collagen was 0.46 in both diet groups. It is concluded that the turnover of soluble collagen is markedly decreased with malnutrition, but degradation and conversion into insoluble collagen account for the same proportions of efflux from the soluble-collagen pool as in rapidly growing rats.     

Hydroxyproline contents and prolyl hydroxylase activities in lungs of rats exposed to low levels of ozone.

Exposure of adult rats to 0.8 ppm ozone enhanced collagen synthesis in the lungs. Collagen synthesis was studied by estimating hydroxyproline (Hyp) content and by following the activity of prolyl hydroxylase (PH), a crucial enzyme in the pathway of collagen biosynthesis. In the early phases (1–2 day) of ozone-induced injury, PH activity was increased twofold over control values and the amount of collagen synthesized (as estimated by Hyp formation) was double the amount of non-collagenous protein synthesized. This resulted, by the third day, in a significant increase (29%) in total lung collagen. In the later stages of the injury (3–7 day), however, increases in PH activity were more gradual, approaching 2.7 times control levels at the end of the 7-day exposure period. The synthesis of non-collagenous protein during this period increased steadily and by the 7th day the ratio of the amounts of collagen to non-collagenous protein synthesized was comparable to that of controls. When the exposed (0.8 ppm O₃/7 days) animals were placed in filtered ambient air, PH activity returned to normal in 13 days whereas Hyp content remained elevated for up to 28 days. These results suggest that environmental ozone exposure could be a contributing factor in pulmonary disorders involving lung collagen synthesis.     

Influence of acetaminophen consumption and exercise on Achilles tendon structural properties in male Wistar rats

Chronic consumption of acetaminophen (APAP) during exercise training leads to a reduction in tendon stiffness and modulus compared with a placebo. We explored whether this effect could be due to a reduction in tendon collagen content or cross-linking. Ten-week-old male Wistar rats (n = 50) were divided into placebo or APAP groups and into sedentary or treadmill-exercised groups. APAP (200 mg/kg) or saline was administered once daily by oral gavage. Rats in the exercise groups ran on a treadmill 5 days per week for 8 wk with progression to 60 min per day, 20 m/min, and 8° incline. After 8 wk, lyophilized Achilles tendon samples were assayed for the collagen-specific amino acid hydroxyproline and cross-linking [hydroxylyslpyridinoline (HP)] content by high-performance liquid chromatrography. Collagen content was not influenced by exercise or APAP (P > 0.05). Compared with placebo, tendon water content was 7% (P = 0.006, main effect) lower in animals consuming APAP (placebo: 54.79 ± 0.8%, APAP: 50.89 ± 1.2%). HP in the Achilles tendon was 36% greater (sedentary: 141 ± 15, exercise: 204 ± 26 mmol/mol collagen) in the exercise-trained rats independent of drug treatment (P = 0.020, main effect). Independent of exercise, HP content was 33% lower (P = 0.032, main effect) in the animals consuming APAP (placebo: 195 ± 21, APAP: 140 ± 19 mmol/mol collagen). Our data suggests that chronic consumption of APAP results in a reduction in collagen cross-linking and a loss of tissue water independent of chronic exercise. This reduction in cross-linking and water content could contribute to the decrease in tendon stiffness noted in humans chronically consuming APAP.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Collagen synthesis in normal BHK cells and temperature-sensitive chemically transformed BHK cells.

Collagen synthesis in normal BHK 21/cl 13 and chemically transformed temperature sensitive BHK 21/cl 13 cells (Me2N4) was assessed by examination of hydroxyproline formation and collagenase-susceptible protein. The Me2N4 cells lost their ability to synthesize collagen at both permissive and nonpermissive temperatures for transformation. These conclusions were confirmed by polyacrylamide-gel eletrophoresis and CM-cellulose chromatography. Prolyl hydroxylase activity was present in both normal and transformed cells even when no collagen could be demonstrated. The production of noncollagen protein, although decreased in the transformed cell, did not change as drastically as the collagen synthesis.     

Turnover of muscle protein in the fowl. Collagen content and turnover in cardiac and skeletal muscles of the adult fowl and the changes during stretch-induced growth

The collagen content and the rate of collagen synthesis were measured in the anterior and posterior latissimus dorsi muscles and in heart from fully grown fowl. This was done by measuring the proline/hydroxyproline ratios in the muscle and by a constant infusion of [¹⁴C]proline. These measurements were also made during the hypertrophy of the anterior muscle in response to the attachment of a weight to one wing of the fowl. In the non-growing muscles the collagen content was higher in the anterior muscle (22.8% of total protein) than in the posterior muscle (9.5% of total protein) and lowest in the heart (3.8% of total protein). In the two skeletal muscles a little over half of the collagen was accounted for by internal collagen (i.e. perimysium and endomysium). Collagen synthesis in these non-growing muscles occurred at 0.59%/day in each of the two skeletal muscles and at 0.88%/day in the cardiac muscle. During hypertrophy the collagen content of the anterior muscle increased, but not as fast as intracellular protein, so that after 58 days the concentration had fallen from 22.8 to 14.4% of total protein. This may have resulted from an incomplete production of the epimysial sheath, since the concentration of internal collagen did not fall and as a result accounted for over 80% of the total in the enlarged muscle. Collagen synthesis increased 8-fold during the first week of the hypertrophy, but never amounted to more than 4% of the total muscle protein synthesis. When the net accumulation of collagen is compared with the increased rate of synthesis it is concluded that between 30 and 70% of the newly synthesized collagen may have been degraded.     

Collagen synthesis in explants from rat intestine

Collagen is the major structural component of the intestinal wall and its metabolism is of special interest for intestinal strength. We describe collagen synthesis in short-term (3 h) incubations of rat intestinal tissue, as measured in terms of incorporation of [3H]proline in collagenase-digestible protein and percentage relative collagen synthesis. In this time span, incorporation of [3H]proline in collagen increases linearly with time and tissue weight. Addition of unlabeled proline during incubation, in excess of the 0.1 microM [3H]proline always present, strongly increases both total protein and collagen synthesis, suggesting that proline transport is rate limiting. Further experiments have been performed in the presence of labeled proline alone and with the addition of 0.35 mM unlabeled proline. Collagen synthesis is significantly higher in colon than in ileum, comprising 0.37 and 0.21%, respectively, of total protein synthesis. Also, collagen synthesis decreases considerably with age, both in ileum and colon. The results presented here demonstrate that rat intestinal explants synthesize measurable amounts of collagen in vitro and that the system used is able to detect changes in in vivo synthetic capability such as those induced by ageing.     

Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E2 (PGE2), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [3H]Thymidine and [3H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h. PGE2 (10(-7) M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10(-7) M) inhibited DNA and collagen synthesis. The addition of PGE2 reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collagen synthesis in central bone to levels above control untreated cultures. We conclude that PGE2 has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase.

Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications.     

Influence of Aloe vera on collagen characteristics in healing dermal wounds in rats

Wound healing is a fundamental response to tissue injury that results in restoration of tissue integrity. This end is achieved mainly by the synthesis of the connective tissue matrix. Collagen is the major protein of the extracellular matrix, and is the component which ultimately contributes to wound strength. In this work, we report the influence of Aloe vera on the collagen content and its characteristics in a healing wound. It was observed that Aloe vera increased the collagen content of the granulation tissue as well as its degree of crosslinking as seen by increased aldehyde content and decreased acid solubility. The type I/type III collagen ratio of treated groups were lower than that of the untreated controls, indicating enhanced levels of type III collagen. Wounds were treated either by topical application or oral administration of Aloe vera to rats and both treatments were found to result in similar effects.     

Dimethyl sulfoxide (DMSO) does not affect epidermal wound healing

We studied the effect of 95% DMSO on dermal/epidermal healing and microbiol flora in partial-thickness wounds. Wounds of 0.3 mm were made in the skin of Yorkshire pigs with a keratome and treated daily with either 95% DMSO, water, or they were left untreated. Wounds were excised on Days 2-7 and the dermis was separated from the epidermis. The dermis was assayed for collagen biosynthesis (by measuring the production of [14C]hydroxyproline (HP) and amount of radioactive peptides released after collagenase digestion) and absolute HP (by spectrophotometric analysis). The epidermis was evaluated macroscopically for resurfacing. Aerobic bacteria from unwounded and wounded skin were identified and quantitated. There were no significant differences between treatment groups in HP incorporation or absolute collagen content from Days 2-6 after wounding. HP incorporation in the total protein fractions and in the collagenase digestible fractions were analogous. Collagen biosynthesis was similar in both unwounded, untreated, and unwounded DMSO-treated skin. Epidermal healing did not differ between treatment groups. There were no differences in the number or types of bacteria in wounds between treatment groups. These results indicate that topical DMSO is neither beneficial nor harmful in the healing of superficial wounds.     

甲状旁腺激素相关蛋白对心肌成纤维细胞增殖的影响

目的：观察外源性甲状旁腺激素相关蛋白（1—40）（PTHrp（1-40））对原代培养新生Wistar鼠心肌成纤维细胞（CFs）增殖及胶原合成的影响。方法：分离、培养Wistar乳鼠心肌成纤维细胞,加入不同浓度的PTHrp（1-40）共培养,用四氮唑盐比色法（MTT法）和^3H—TdR掺入法检测细胞增殖;^3H—Proline掺入法测定胶原合成。结果随着一定浓度PTHrp（1-40）的升高,CFs MTT法A490值及^3H—TdR的掺入量,^3H-脯氨酸掺入率呈明显的递减趋势。结论：PTHrp在一定程度上可抑制成纤维细胞增殖及细胞外基质的沉积。     

Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation, and maturation

Collagen metabolism was studied in the regenerating forelimbs of adult newts (Notophthalmus viridescens) with respect to the pattern of accumulation relative to total protein accretion, maturation, and rate of biosynthesis. Measurements of collagen and noncollagen protein in regenerating limbs at various stages indicate that a preferential enrichment in collagen occurs at two periods correlating with (1) the onset of differentiation and chondrogenesis and (2) the initial period of elongation and outgrowth following morphogenesis. The maturation of collagen was determined by measuring the distribution of collagen into acetic acid soluble and insoluble forms. Soluble collagen increased to 30% during the differentiative period, remained at a high level during digit-formation, and decreased progressively following morphogenesis.Tracer studies were performed to determine whether the net accumulation of collagen resulted from a preferential increase in collagen biosynthesis. Separation of collagen and noncollagen proteins labeled in vivo with [³H]proline was performed enzymatically using purified clostridial collagenase. Rates of incorporation of proline into collagen relative to noncollagen proteins did not vary significantly during regeneration, although a threefold increase in incorporation rates into both species occurs at the onset of differentiation. Collagen synthesis constitutes 7–11% of the total protein synthesis in the regenerate. Estimates of variations in the absolute rates of protein synthesis, based on endogenous levels of proline, indicate that the highest rates of protein synthesis occur during morphogenesis. The relationship between protein content and relative rates of synthesis suggests that the net accumulation is governed by variations in rates of degradation. The relationship between collagen content and solubility also suggests that the rate of insolubilization plays a role in the net accumulation of collagen.     

Existence of malfunctioning pro alpha2(I) collagen genes in a patient with a pro alpha 2(I)-chain-defective variant of Ehlers-Danlos syndrome

Collagen synthesis was examined in skin fibroblasts from a patient with a variant of Ehlers-Danlos syndrome. The relative rate of collagen synthesis to total protein synthesis in the patient's fibroblasts was always one-half of that in fibroblasts from normal controls. Total collagen synthesis, as assessed by quantification of total hydroxyproline, was also significantly lower than that of controls, indicating that the rate of collagen synthesis by the patient's fibroblasts was decreased compared with that by normal fibroblasts. Analysis of procollagen and collagen components showed the absence of the pro alpha 2(I) chain and its derivatives. Dot-blot and Northern-blot analyses showed the patient's fibroblasts to contain less than 10% of the mRNAs for pro alpha 2(I) found in control fibroblasts. In spite of these results, Southern blot analysis of genomic DNA indicated the presence of the same number of genes for the pro alpha 2(I) collagen chain in the patient's fibroblasts as in control fibroblasts, suggesting malfunctioning pro alpha 2(I) collagen genes as the cause for failure of the patient's fibroblasts to synthesize pro alpha 2(I) collagen chains.     

Regulation of cysteine dioxygenase and gamma-glutamylcysteine synthetase is associated with hepatic cysteine level

Two hepatic enzymes, cysteine dioxygenase (CDO) and gamma-glutamylcysteine synthetase (GCS), play important regulatory roles in the response of cysteine metabolism to changes in dietary sulfur amino acid or protein levels. To examine the time-course of changes in CDO and GCS activities, CDO and GCS-catalytic or heavy subunit protein and mRNA levels, and cysteine and glutathione levels, we adapted rats to either a low protein (LP) or high protein (HP) diet, switched them to the opposite diet, and followed these parameters over 6 days. Hepatic CDO activity and amount, but not mRNA level, increased in response to higher protein intake; the t(1/2) of change for CDO activity or protein level was 22 h for rats switched from a LP to a HP diet and 8 h for rats switched from a HP to a LP diet, suggesting that the HP diet decreased turnover of CDO. Hepatic GCS activity, catalytic subunit amount and mRNA level decreased in response to a higher protein intake. GCS catalytic subunit level changed with a similar t(1/2) for both groups, but the change in GCS activity in rats switched from a LP diet to a HP diet was faster (approximately 16h) than for rats switched from a HP to a LP diet (approximately 74h). Hepatic cysteine and glutathione levels reached new steady states within 12 h (LP to HP) or 24 h (HP to LP). CDO activity appeared to be regulated at the level of protein, probably by diminished turnover of CDO in response to higher protein intake or cysteine level, whereas GCS activity appeared to be regulated both at the level of mRNA and activity state in response to the change in cysteine or protein availability. These findings support a role of cysteine concentration as a mediator of its own metabolism, favoring catabolism when cysteine is high and glutathione synthesis when cysteine is low.     

Increasing protein at the expense of carbohydrate in the diet down-regulates glucose utilization as glucose sparing effect in rats

High protein (HP) diet could serve as a good strategy against obesity, provoking the changes in energy metabolic pathways. However, those modifications differ during a dietary adaptation. To better understand the mechanisms involved in effect of high protein diet (HP) on limiting adiposity in rats we studied in parallel the gene expression of enzymes involved in protein and energy metabolism and the profiles of nutrients oxidation. Eighty male Wistar rats were fed a normal protein diet (NP, 14% of protein) for one week, then either maintained on NP diet or assigned to a HP diet (50% of protein) for 1, 3, 6 and 14 days. mRNA levels of genes involved in carbohydrate and lipid metabolism were measured in liver, adipose tissues, kidney and muscles by real time PCR. Energy expenditure (EE) and substrate oxidation were measured by indirect calorimetry. Liver glycogen and plasma glucose and hormones were assayed. In liver, HP feeding 1) decreased mRNA encoding glycolysis enzymes (GK, L-PK) and lipogenesis enzymes(ACC, FAS), 2) increased mRNA encoding gluconeogenesis enzymes (PEPCK), 3) first lowered, then restored mRNA encoding glycogen synthesis enzyme (GS), 4) did not change mRNA encoding β-oxidation enzymes (CPT1, ACOX1, βHAD). Few changes were seen in other organs. In parallel, indirect calorimetry confirmed that following HP feeding, glucose oxidation was reduced and fat oxidation was stable, except during the 1(st) day of adaptation where lipid oxidation was increased. Finally, this study showed that plasma insulin was lowered and hepatic glucose uptake was decreased. Taken together, these results demonstrate that following HP feeding, CHO utilization was increased above the increase in carbohydrate intake while lipogenesis was decreased thus giving a potential explanation for the fat lowering effect of HP diets.