Expression of IGF-binding protein-1 phosphoisoforms in fasted rat skin and its role in regulation of collagen biosynthesis

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen and glycosaminoglycan (GAG) biosynthesis in tissues. IGF-I activity is modulated by a family of IGF-binding proteins (IGFBPs) with different IGF-I binding affinities. At least IGFBP-1 and IGFBP-2 are known as inhibitors of IGF functions. Some IGFBPs (IGFBP-1, IGFBP-3 and IGFBP-5) may undergo phosphorylation that dramatically increase their affinity for IGF. During fasting of animals there is a significant decrease of the collagen and GAG content of the skin, accompanied by a reduction of plasma IGF-I levels. However, in previous studies we showed that in the skin of fasted rats IGF-I as well as IGFBP-1 and IGFBP-2 expressions were not different, compared to control rat skin, although collagen content was significantly decreased. In the present study we show that fasted rat skin contains similar amounts of IGF-I, IGFBP-3 and IGFBP-1, although extract from fasted rat skin induced inhibition of collagen biosynthesis in cultured fibroblasts, compared to control rat skin extract. Western immunoblot analysis of control and fasted rat skin extracts, using anti-phosphoserine antibodies for immunoprecipitated IGFBP-1 and IGFBP-3, revealed that both proteins are present in phosphorylated form. Although no differences were found in the expression of phosphorylated IGFBP-3 between control and fasted rat skins, that of phosphorylated IGFBP-1 in fasted rat skin extract was higher than in control one. We suggest that there is an increased level of IGFBP-1 phosphoisoform in fasted rat skin, associated with increased affinity for IGF-I. The increase of phosphorylated IGFBP-1 in fasted rat skin tissue may augment IGF-I binding affinity for IGF and decrease its bioavailability for receptor interaction. This mechanism may prevent IGF-I dependent stimulation of fibroblasts to produce extracellular matrix components. The specific expression of IGFBPs and their phosphoisoforms in tissues may play an important role in regulation of IGF-I action during physiologic and pathologic responses.     

Inhibition of collagen biosynthesis and increases in low molecular weight IGF-I binding proteins in the skin of fasted rats

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen biosynthesis and prolidase activity in connective tissue cells. The disturbances in skin collagen metabolism (reflected by significant decrease in skin collagen content, collagen biosynthesis and prolidase activity) in fasted rats were accompanied by decrease in serum IGF-I level. Fasted rat serum was found to contain about 58% of IGF-I (101.6 +/- 15.4 ng/ml) as compared to control rat serum (175.7 +/- 19.8 ng/ml), while the skin of control and fasted rats contained similar concentrations of IGF-I (about 77 ng/g tissue). The insulin-like growth factor binding proteins (IGFBPs) of sera and tissue extracts (known to regulate IGF-I activity) were analysed by ligand blotting. In the serum of control rats one IGFBP band of about 46 kDa (corresponding to the acid-dissociated IGFBP-3) was detected. In the serum of fasted rats the 46 kDa IGFBP was not observed, however, an other IGFBP of about 30 kDa (corresponding to low molecular weight IGFBPs, e.g. IGFBP-1 or IGFBP-2) was found. The intensity of IGF-I binding to the 30 kDa IGFBP was much higher than that of IGFBP-3, found in control rat serum. Control and fasted rat skin contained similar IGFBPs, however their IGF-I binding abilities were much lower, compared to their serum counterparts. It was found that 46 kDa and 30 kDa proteins, observed in ligand blotting represent IGFBP-3 and IGFBP-1 or IGFBP-2. respectively as demonstrated by western immunoblot analysis. An increase in IGF-binding to 30 kDa IGFBP-1 and/or IGFBP-2 (known as an inhibitors of IGF-dependent functions) in the skin of fasted rats may explain the mechanism of reduced collagen biosynthesis and deposition in tissues during fasting.     

The effects of fasting and refeeding on serum parathormone and calcitonin concentrations in young and old male rats.

Although fasting and refeeding reveal the existence of age-related changes in carbohydrate and lipid metabolism, the effects of aging on mineral metabolism in refed animals are unknown. We therefore investigated hormonal regulation of calcium metabolism in young (4 months) and old (26 months) male rats fasted for 48 hours and then refed for 4 or 24 hours. Serum concentrations of total and ionized calcium and parathormone were similar in control young and old rats. Serum calcitonin level was higher, and the concentrations of albumin and inorganic phosphate and alkaline phosphatase activity were lower in fed old rats. In young fasted rats, the serum ionized and total calcium was decreased, and phosphate concentration was increased. In old rats, fasting resulted in the increase of serum parathormone level. Fasting reduced serum alkaline phosphatase activity to a similar extent in both age groups. In young rats, refeeding for 24h normalized serum calcium and phosphate levels and alkaline phosphatase activity, and decreased serum concentrations of PTH and calcitonin. In old refed rats, serum calcitonin concentration was raised by 77% compared to fed or fasted animals, whereas parathormone levels were normalized. Our results indicate that old fasted or refed rats maintain normal serum calcium concentration in a different way than young animals, possibly through the increase in serum levels of parathormone and/or calcitonin. Thus, dietary manipulations such as fasting and refeeding constitute an interesting model for the investigation of the effects of aging on the hormonal regulation of serum calcium level.     

Dietary vitamin E supplementation lowers blood pressure in spontaneously hypertensive rats

Pre-eclampsia, is the most common, pregnancy-associated pathological syndrome accompanied by a significant increase in collagen and sulphated glycosaminoglycans (GAGs) contents in the umbilical cord arteries (UCAs). Insulin-like growth factor-I (IGF-I) is expressed in most foetal tissues and it is involved in anabolic effects. It stimulates protein (mainly collagen) and GAG biosynthesis, cell proliferation and differentiation. Previously, we have found that pre-eclampsia is associated with an increase of IGF-I concentration in the umbilical cord blood. A family of IGF-I-binding proteins (BPs) modulates the activity of IGF-I. We demonstrated qualitative differences between BPs of normal and pre-eclamptic human umbilical cord (UC) serum and UC-tissues (UCA-wall and Wharton's jelly) by Western immunoblot analysis. All examined sera and tissues contained BP-1 and BP-5 as well lower molecular weight materials. The BP-2 was recovered from both control and pre-eclamptic sera, while it was not detected in the UC-tissues. Instead, lower molecular weight forms of BP-2 were found as judged by the anti-BP-2 antibody. The BP-3 was detected in sera, UCA and Wharton's jelly. The most distinct expression of BP-3 was found in the UCA. The pre-eclamptic UCA and Wharton's jelly contained additional BP-3-reactive material of lower molecular weight. The BP-4 was strongly expressed in pre-eclamptic UC-serum and the expression was decreased in pre-eclamptic UC-tissues, compared to respective controls. Ligand binding assay revealed that most of IGF-I was bound to 46 kDa region (typical for BP-3) in both control and pre-eclamptic sera and tissues. However, distinctly less IGF-I was bound in pre-eclamptic serum, distinctly more in pre-eclamptic UCA and no differences were found in pre-eclamptic Wharton's jelly, compared to controls. We demonstrated that both normal and pre-eclamptic UC-sera and tissues are able to degrade 46 kDa IGF-I-BP. The degradation may result in a decrease of IGF-I binding, contributing to increase in free IGF-I that may stimulate the cells to produce extracellular matrix (ECM) components. The specific BPs and their proteolytic modification in UC tissues may be important modulators of IGF-I action during foetal development.     

Potential role of pyrroline 5-carboxylate in regulation of collagen biosynthesis in cultured human skin fibroblasts

Although insulin-like growth factor-I (IGF-I) is known as an important stimulator of collagen biosynthesis in collagen-producing cells, the mechanism and endpoints by which it regulate the process remain largely unknown. Serum of acutely fasted rats contained reduced amount of IGF-I (72+/-16 ng/ml) and showed about 75% reduced ability to stimulate collagen and DNA synthesis in confluent human skin fibroblasts in comparison to the effect of control rat serum (IGF-I, 168+/-19 ng/ml). An addition of IGF-I (at least 40 ng/ml) to fasted rat serum restored its mitogenic activity but could not restore its ability to stimulate collagen biosynthesis to control values during 24 h of incubation. However, when the cells were incubated in fasted rat serum supplemented with 40 ng/ml of IGF-I for 48 h, collagen biosynthesis was restored to control values. It suggests that the stimulatory role of IGF-I in collagen biosynthesis undergo indirectly. We considered pyrroline-5-carboxylate (P5C) as a candidate to play a direct role in this process. Since IGF-I and P5C are known to be decreased in serum of fasted rats it seems that the action of IGF-I on collagen biosynthesis may involve participation of P5C. We have found that serum of fasted rats (showing low level of P5C) supplemented with 1 mmol/l P5C induced collagen biosynthesis in confluent human skin fibroblasts during 24 h to control values. Supporting evidence comes from the experiment showing stimulatory action of P5C on collagen biosynthesis in fibroblasts cultured in serum-free medium. Our results postulate potential role of P5C in regulation of collagen biosynthesis and indicate participation of this molecule in the pathway of IGF-I action in this process.     

Effects of short-term fasting in male Sprague-Dawley rats

Fasting is a common procedure for animals in experiments. Although fasting may be necessary for scientific reasons, it should be minimized. In the current study, jugular-catheterized male Sprague-Dawley rats in metabolism cages were fasted for 0 to 24 h before measurement of various physiologic markers (serum chemistry, CBC analysis, serum corticosterone). When controlled for cohort, rats fasted for 6 and 16 h had significantly lower serum glucose than did nonfasted rats. Other values did not differ from controls. Only rats fasted for 24 h had elevated serum corticosterone levels. Therefore, fasting for as long as 16 h has fewer effects on rats that does fasting for 24 h. Fasting for 24 h or more therefore should receive appropriate consideration by both scientists and the IACUC in the experimental design and the animal-use protocol.     

Hepatic reduction of insulin-like growth factor (IGF)-I and IGF binding protein-3 that results from fasting is not attenuated in genetically obese rats

Fasting or caloric restriction causes substantial reductions in serum IGF-I in normal weight humans and animals, and reductions of liver IGF-I and IGFBP-3 mRNAs in animals. Obese humans, however, have attenuated and delayed decrements in IGF-I in serum when subjected to caloric restriction. Obese Zucker rats show a clear tendency to preserve body protein during fasting. To determine whether obesity opposes the effects of fasting on IGF-I and IGFBP-3, and thereby contributes to preservation of lean tissue, we have examined the effect of 72 h of fasting on IGF-I and IGFBP-3 in lean and obese Zucker rats. We observe that between lean and obese animals, fasting for 72 h produces similar decrements in body weight, serum IGF-I, liver IGF-I mRNA, serum IGFBP-3 and liver IGFBP-3 mRNA. Our finding that the reduction of IGF-I and IGFBP-3 in liver that results from 72 h of fasting is not attenuated in obese Zucker rats raises the possibility that conservation of lean tissue in these animals during fasting is not related to the hepatic production of IGF-I and IGFBP-3.     

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.     

Age-related changes in rat adipose tissue in response to fasting: protein, lactate and pyruvate levels

1. The evolution with age of the metabolic response of adipose tissue to fasting has been investigated in two groups of rats, at different ages, fed and fasted. 2. The protein tissue content increases in response to fasting in young rats but not in adult-old ones. This indicates a lower lipomobilizing response to starvation with increasing age. 3. In contrast to young rats, the adult rat lactate is not increased by fasting while pyruvate is increased. 4. As a result of lactate and pyruvate variations, NAD/NADH is also changed: after fasting it decreases in young rats, while it increases in older rats. 5. Absolute NAD level is decreased by fasting both in young and older rats. 6. These data provide experimental support for the decreased sensitivity of fat pads from older animals to stimuli affecting lipolysis.     

Comparative changes with age of the fasting response in circulating ketone bodies, glucose and insulin and oral glucose tolerance test in the rat

1. Body weight loss in 48 hr fasted rats decreased with age. 2. Blood glucose and plasma RIA-insulin levels correlated negatively and positively respectively with body weight in fed rats. Fasting produced a greater fall in blood glucose and a smaller decrease in RIA-insulin in young than in old rats. 3. Blood ketone bodies correlated negatively with body weight after 48 hr fasting. 4. In oral glucose tolerance tests, blood glucose rose more in adult and old rats than in prepuberals when both fed and fasted. RIA-insulin levels rose more in prepuberals than in older rats when fed but not when fasted. 5. Changes in body composition and reduced insulin sensitivity with age are discussed.     

Greater potency of IGF-I than IGF-I/BP-3 complex in catabolic parenterally fed rats

We compared the anabolic effects of recombinant human insulin-like growth factor I (rhIGF-I, 2.5 mg/kg) and equimolar amounts of rhIGF-I prebound to rhIGF binding protein-3 (rhIGF-I/BP-3) coinfused continuously with total parenteral nutrition (TPN) solution in dexamethasone (Dex, 70 microg/day ip)-treated male rats for 6 days. The four TPN groups included control, Dex, Dex + IGF-I, and Dex+IGF-I/BP-3. Pharmacokinetic analysis indicated reduced clearance of IGF-I when infused as IGF-I/BP-3 compared with free IGF-I (0.91 +/- 0.09 vs. 2.01 +/- 0.19 ml serum/min, P < 0.001) and this was associated with significantly greater serum IGF-I concentrations in the Dex+IGF-I/BP-3 group. Despite greater total serum IGF-I levels, infusion of free IGF-I produced greater anabolic responses than IGF-I/BP-3 based on body weight, nitrogen balance, and jejunal cellularity. Treatment with free IGF-I, but not IGF-I/BP-3, significantly reduced serum insulin and glucose levels that were elevated due to Dex. There were no significant differences in liver IGF-I mRNA levels between groups. Serum IGFBP-3 levels were elevated with infusion of IGF-I/BP-3 compared with IGF-I. These results indicate greater anabolic potency of IGF-I compared with IGF-I/BP-3 when administered by continuous parenteral infusion with TPN solution in catabolic rats.     

The influence of fasting on liver sulfhydryl groups, glutathione peroxidase and glutathione-S-transferase activities in the rat

Sulfhydryl groups, glutathione peroxidase (GPx) and glutathione-S-transferase (GST) are important elements of the antioxidant defence in the organism. The efficacy of their antioxidant action is influenced by many factors. In this work, the effect of fasting on total, protein-bound and nonprotein sulfhydryl groups and on the activity of liver and serum GPx and GST in rats were determined. Male Wistar rats were divided into two groups: non-fasted and 18-hour fasted. In fasted animals liver content of nonprotein sulfhydryl groups (represented predominantly by reduced glutathione; GSH) was diminished by 22% in comparison to non-fasted group, whereas total and protein-bound -SH groups were unaffected. The activity of liver and serum GPx was unchanged in food deprived rats. In these animals the activity of GST in serum was reduced by 26%. Fasting had no significant effect on the activity of GST in the liver. Our results demonstrate that in rats deprived of food for 18 hours liver and serum GPx and GST are not involved in protection against action of reactive oxygen species formed during fasting. The observed drop in the content of liver nonprotein sulfhydryl groups without concomitant rise in the activity of GPx and GST indicates that this effect may be due to augmented degradation of GSH, its potentiated efflux from hepatocytes and formation of conjugates with intermediates arising as a result of reactive oxygen species action.     

The fetal rat binding protein for insulin-like growth factors is expressed in the choroid plexus and cerebrospinal fluid of adult rats

The biological effects of the insulin-like growth factors, IGF-I and IGF-II, on their receptors are modulated by IGF-binding proteins. Recently, we isolated a cDNA clone for one member of the family of IGF-binding proteins, BP-3A, a 30 kilodalton (kDa) protein synthesized by the BRL-3A rat liver cell line. BP-3A is related to but distinct from two other cloned IGF-binding proteins, the human amniotic fluid binding protein and the glycosylated binding subunit of the 150 kDa IGF-binding protein complex in serum. It is expressed in multiple nonneural tissues and in serum in the fetal rat and decreases after birth, similar to the developmental pattern of IGF-II expression. IGF-I, IGF-II, and their receptors are expressed in brain. The present study examines the expression of BP-3A in the rat central nervous system. By Northern blot analysis, BP-3A mRNA is present at high levels in brain stem, cerebral cortex, and hypothalamus from 21-day gestation rats and, like IGF-II mRNA, persists in adult rat brain. The site of BP-3A mRNA synthesis was localized by in situ hybridization to coronal sections of adult rat brain using 35S-labeled oligonucleotides, 48 bases in length, complementary and anticomplementary to the coding region of BP-3A. Specific hybridization of the BP-3A probe was observed exclusively to the choroid plexus extending from the level of the medial preoptic nucleus to the arcuate nucleus of the hypothalamus, similar to the previously reported preferential localization of IGF-II mRNA to the choroid plexus. Synthesis of BP-3A mRNA by choroid plexus suggested that BP-3A might be secreted into the cerebrospinal fluid. A 30 kDa IGF-binding protein was demonstrated in rat cerebrospinal fluid that is recognized by antibodies to BP-3A and, like purified BP-3A, has equal affinity for IGF-I and IGF-II. By analogy with other transport proteins synthesized by the choroid plexus, BP-3A may facilitate the secretion of IGF-II to the cerebrospinal fluid and modulate its biological actions at distant sites within the brain.     

Thyroid C cell function during fasting and refeeding of young and old rats

Age-related alterations in the structure and function of many organs often become apparent under stimulation of their function. Although the ageing process affects the regulation of mineral homeostasis, the function of thyroid C-cells that secrete calcitonin (CT) under the conditions of fasting and refeeding, a way of dietary manipulation that reveal the existence of age-related changes of follicular thyroid cells, has not been characterized. Therefore, we investigated the number of C-cells and serum CT concentration in young (4 mo) and old (26 mo) male rats fasted for 48 hours, and then refed for 4 or 24 hours. We found significantly higher number of C-cells in thyroids of old vs young rats both under basal conditions, and after fasting/refeeding. Correspondingly, serum calcitonin level was higher in fed or fasted old rats vs young ones. However, in young rats refeeding decreased, whereas in old animals increased serum concentrations of calcitonin. Thus, the control of serum calcium concentration, that was well preserved in old rats, occurs at the expense of increased serum CT level both under basal conditions, and after refeeding. These observations suggest that C-cell function is altered in ageing.     

Sympatho-adrenal activity and metabolic responses in fasted rats--the role of interscapular brown adipose tissue

1. Sympatho-adrenal (SA) and metabolic responses to fasting were studied in sham-operated (SHAM) rats and in those with interscapular brown adipose tissue (IBAT) removed. 2. Fasting significantly increased adrenaline (A) excretion and serum free fatty acids (FFA), but decreased noradrenaline (NA) excretion and blood glucose level in SHAM rats. 3. IBAT removal did not change metabolic responses while it markedly altered the SA activity. Fasting in animals void of IBAT potentiated the activity of adrenal medulla, inhibited the FFA rise and prevented glucose reduction, which is normally observed in SHAM-fasted rats. 4. Results suggest the significance of IBAT in the regulation of the blood level of energy substrates in fasted rats and in maintaining the basal level of NA excretion.     

Distribution of apolipoprotein A-IV among lipoprotein subclasses in rat serum

The distribution of apolipoproteins (apo) A-I, A-IV, and E in sera of fed and fasted rats was studied using various methods for the isolation of lipoproteins. Serum concentrations of apoA-I and apoA-IV decreased significantly during fasting (16 and 31%, respectively), while apoE concentrations remained essentially the same. Chromatography of sera on 6% agarose columns showed that apoA-IV is present on HDL and as so-called "free" apoA-IV. The concentration of "free" apoA-IV decreased six- to seven-fold during fasting, explaining the decrease in total serum apoA-IV. Serum apoA-I and apoE are almost exclusively associated with HDL-sized particles. When sera are centrifuged at a density of 1.21 g/ml, marked quantities of apoA-I (8-9%) and apoE (11-22%) are recovered in the "lipoprotein-deficient" infranatant, suggesting that ultracentrifugation affects the integrity of serum HDL. The nature of the chromatographically separated carriers of serum apoA-IV was investigated by quantitative immunoprecipitation. From these studies, it is concluded that apoA-IV in rat serum is present in at least three fractions: 1) particles with the size and composition of HDL, containing both apoA-I and apoA-IV and possibly minor quantities of apoE; 2) HDL-sized particles containing apoA-IV, but no apoA-I or apoE; 3) "free" apoA-IV probably containing small amounts of bound cholesterol and phospholipid.     

Absence of effects of short-term fasting on plasma ghrelin and brain expression of ghrelin receptors in the tilapia, Oreochromis mossambicus

Ghrelin is an important endocrine peptide that links the gastrointestinal system and brain in the regulation of food intake and energy expenditure. In human, rat, and goldfish plasma levels of ghrelin and GH are elevated in fasted animals, suggesting that ghrelin is an orexigenic signal and a driving force behind the elevated plasma levels of GH during fasting. Ghrelin's orexigenic action is mediated by the ghrelin receptor (GHS-R1a and GHS-R1b) which is localized on neuropeptide Y (NPY) neurons in the brain. Studies were undertaken to investigate the effect of short-term fasting on plasma ghrelin and brain expression of GHS-R1a, GHS-R1b, and NPY in the tilapia. Fasting for 7 days had no effect on plasma ghrelin concentrations, whereas significant increases in plasma levels of GH were observed on day 3. Fasting significantly reduced plasma levels of IGF-I on days 3 and 7, and of glucose on days 3, 5, and 7. Brain expression of ghrelin and GHS-R1b were significantly elevated in fasted fish on day 3, but were significantly reduced on day 5. This reduction was likely due to a significant increase in the expression in the fed controls on day 5 compared to day 0. No change was detected in the expression of GHS-R1a or NPY in the brain. These results indicate that ghrelin is not acting as a hunger signal in short-term fasted tilapia and is not responsible for the elevated levels of plasma GH.