Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle

Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.     

Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon

The purpose of this study was to determine the effects of aging and caloric restriction (CR) on insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF-binding protein-3 (IGFBP-3) and IGFBP-4 expression in the stomach and colon of male Fischer 344 rats. Stomach and colonic RNA were prepared from ad libitum (AL) fed or long-term CR rats. Stomach IGF-I, IGFBP-3 and IGFBP-4 mRNA levels increased significantly (P相似文献   

Production of Insulin-like Growth Factors and Their Binding Proteins in Primary Cultures of Rat Liver Parenchymal and Nonparenchymal Cells

Regulation of the production of insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBPs), and their related proteins by various hormones was investigated in primary cultures of rat liver parenchymal and nonparenchymal cells.

Freshly isolated parenchymal cells contained mRNAs of IGF-I, IGF-II, IGFBP-1, IGFBP-4, growth hormone (GH) receptor, and the acid-labile subunit (ALS), which forms a ternary complex with IGF-I and IGFBP-3; however, parenchymal cells did not express the IGFBP-3 gene. In contrast, nonparenchymal cells contained IGFBP-3 mRNA exclusively, as we reported previously [Takenaka et al. Agric. Biol. Chem., 55, 1191–1193 (1991)]. Cultured rat parenchymal cells produced IGF-I, IGFBP-1, and IGFBP-4 prominently. In these cells, secretion of IGF-I and the content of IGF-I mRNA was greatly increased in the presence of GH in the medium. Insulin also increased the production of IGF-I. Secretion of IGFBP-l into the medium was enhanced by treatment with glucagon, dibutyrylcyclic AMP (Bu₂cAMP), and dexamethasone (Dex) and these enhancements with glucagon and Dex reflected the increase in its mRNA content. Insulin depressed the secretion of IGFBP-l. The content of IGFBP-4 in the parenchymal cells was increased by insulin, Bu₂cAMP, and triiodothyronine (T₃), thereby enhancing the production of IGFBP-4 and secretion into the medium. Cultured liver nonparenchymal cells of rats produced IGFBP-1, IGFBP-3, and IGFBP-4. Secretion of IGFBP-l was increased by Bu₂cAMP in the medium, that of IGFBP-3 by IGF-I, and that of IGFBP-4 by both IGF-I and Bu2cAMP. Regulation of the production of IGFBP-3 by IGF-I was demonstrated in these investigations.

These results suggest that GH increases production of IGF-I in the parenchymal cells and this IGF-I, in turn, increases the production of IGFBP-3 in nonparenchymal cells. As we found GH also increases ALS production in parenchymal cells, by these mechanisms, GH increases the formation of the ternary complex of IGF-I, IGFBP-3, and ALS. This study clearly demonstrates the interrelationship between parenchymal and nonparenchymal cells in the production of IGF-I and IGFBPs in the liver.     

Differential Expression and Localization of IGF-I and IGF Binding Proteins in Inflamed Rat Colon

Abstract

Recent studies indicate increased insulin-like growth factor I (IGF-I) expression and altered expression of IGF binding proteins (IGFBP) in the bowel during experimental colitis. This study analyzes the cellular sites of altered IGF-I and IGFBP-expression in large bowel of rats with experimental colitis. Colitis was induced by colonic instillation of 2, 4, 6- trinitrobenzenesulfonic (TNB) acid in ethanol. Animals were sacrificed at 7 days after induction of colitis. Cryostat sections of colon from TNB-treated and control rats were hybridized with ³⁵S-labeled antisense probes for IGF-I, IGFBP-3, IGFBP-4 and IGFBP-5. IGF-I mRNA was up-regulated in lamina propria cells, submucosa and smooth muscle of inflamed colon. IGFBP-3 mRNA was localized to lamina propria and was down-regulated in inflamed colon. IGFBP-4 and IGFBP-5 mRNAs were both up-regulated in inflamed colon. IGFBP-4 mRNA was increased in lamina propria, submucosa and smooth muscle, whereas IGFBP-5 mRNA was increased in smooth muscle. Increased IGF-I expression in mesenchymal layers of colon during experimental colitis supports the hypothesis that IGF-I contributes to hyperplasia and fibrosis in response to inflammation. Altered expression of IGFBP-3, IGFBP-4 and IGFBP-5 in specific bowel layers during colitis suggests that they play a role in modulating IGF-I action.     

Sepsis and inflammatory insults downregulate IGFBP-5, but not IGFBP-4, in skeletal muscle via a TNF-dependent mechanism

The purpose of the present study was to determine whether catabolic stimuli that induce muscle atrophy alter the muscle mRNA abundance of insulin-like growth factor binding protein (IGFBP)-4 and -5, and if so determine the physiological mechanism for such a change. Catabolic insults produced by endotoxin (LPS) and sepsis decreased IGFBP-5 mRNA time- and dose-dependently in gastrocnemius muscle. This reduction did not result from muscle disuse because hindlimb immobilization increased IGFBP-5. Continuous infusion of a nonlethal dose of tumor necrosis factor-alpha (TNF-alpha) decreased IGFBP-5 mRNA 70%, whereas pretreatment of septic rats with a neutralizing TNF binding protein completely prevented the reduction in muscle IGFBP-5. The addition of LPS or TNF-alpha to cultured C(2)C(12) myoblasts also decreased IGFBP-5 expression. Although exogenously administered growth hormone (GH) increased IGFBP-5 mRNA 2-fold in muscle from control rats, muscle from septic animals was GH resistant and no such elevation was detected. In contrast, exogenous administration of IGF-I as part of a binary complex composed of IGF-I/IGFBP-3 produced comparable increases in IGFBP-5 mRNA in both control and septic muscle. Concomitant determinations of IGF-I mRNA content revealed a positive linear relationship between IGF-I and IGFBP-5 mRNA in the same muscle in response to LPS, sepsis, TNF-alpha, and GH treatment. Although dexamethasone decreased muscle IGFBP-5, pretreatment of rats with the glucocorticoid receptor antagonist RU486 did not prevent the sepsis-induced decrease in IGFBP-5 mRNA. In contrast, muscle IGFBP-4 mRNA abundance was not significantly altered by LPS, sepsis, or hindlimb immobilization. In summary, these data demonstrate that various inflammatory insults decrease muscle IGFBP-5 mRNA, without altering IGFBP-4, by a TNF-dependent glucocorticoid-independent mechanism. Finally, IGF-I appears to be a dominant positive regulator of IGFBP-5 gene expression in muscle under both normal and catabolic conditions.     

Differential expression of insulin-like growth factor-I and insulin-like growth factor binding protein-1 in the diabetic rat

Summary Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10–30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p < 0.05) and diaphragm (p < 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p < 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-1 by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals.     

Resection upregulates the IGF-I system of parenterally fed rats with jejunocolic anastomosis

Rats maintained with parenteral nutrition following 60% jejunoileal resection plus cecectomy exhibit minimal adaptive growth in the residual jejunum but a dramatic adaptive growth in the residual colon. Coinfusion of insulin-like growth factor I (IGF-I) with parenteral nutrition induces jejunal growth but has minimal effects in the colon. Our objective was to study the role of the endogenous IGF-I system in the differential responses of jejunum and colon to resection and/or IGF-I during parenteral nutrition. We measured concentrations of immunoreactive IGF-I in plasma, jejunum, and colon, IGF-I receptor binding, and levels of IGF receptor, IGF-I, IGF binding protein (IGFBP)-3 and IGFBP-5 mRNA in residual jejunum and colon 7 days after resection and/or IGF-I treatment. IGF-I receptor number was increased (74-99%) in jejunum and colon due to resection; IGF-I mRNA was increased 5-fold in jejunum and 15-fold in colon due to resection. Resection increased circulating IGFBPs but did not alter plasma IGF-I concentration. Resection induced colonic growth in association with significantly greater colonic IGFBP-5 mRNA and significantly lower colonic immunoreactive IGF-I. IGF-I treatment had no significant effect on IGF-I mRNA or IGF-I receptor number. Concentrations of plasma and jejunal immunoreactive IGF-I were significantly increased in rats given IGF-I in association with jejunal growth. IGF-I treatment significantly increased IGFBP-5 mRNA in the jejunum, which also correlated with jejunal growth. Thus resection upregulated IGF-I receptor number and IGF-I mRNA in residual jejunum and colon, but differential adaptation of these segments correlated with differential regulation of IGFBP-5 mRNA.     

Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.     

Effect of Mild Hypoinsulinemia on Renal Hypertrophy: Growth Hormone/Insulin-Like Growth Factor I System in Mild Streptozotocin Diabetes

The metabolic aberrations associated with diabetes mellitus profoundly alter the growth hormone/insulin-like growth factor I (GH/IGF-I) system. In severe experimental diabetes, serum IGF-I level is reduced, reflecting altered hepatic expression. On the other hand, increased levels of kidney IGF-I have been implicated in the development of diabetic kidney disease. This study aimed to examine the effect of mild experimental diabetes with hypoinsulinemia on both the systemic and renal GH/IGF-I systems in a low-dose streptozotocin (STZ)-induced diabetic rat. Diabetic animals with mild hypoinsulinemia developed renal hyperfiltration within 3 days of diabetes, whereas the renal size increased significantly only between 30 and 48 days of diabetes. Plasma GHlevels were unchanged during the entire course of the study, but a decrease in serum IGF-I, IGF-binding protein 3 (IGFBP-3), and IGF-binding protein 4 (IGFBP-4) occurred after 10, 30, and 48 days. Kidney IGF-I and IGF-binding protein 1 (IGFBP-1) mRNA expression increased after 10 and 30 days of diabetes. A significant increase in kidney IGFBP-1/2, IGFBP-3, and IGFBP-4 proteins was seen after 48 days of diabetes.Apositive correlations was found between renal growth and insulin/glucose ratio (r = .57), kidney IGF-I (r = .57), IGFBP-1 mRNA(r = .43), IGFBP-1/2 (r = .41), and IGFBP-4 levels (r = .40). These results demonstrate hyperfiltration within 3 days of diabetes and a similar response in the IGF-I system in mildly and severely hypoinsulinemic rats; however, renomegaly develops slower in mildly diabetic rats at least partly due to delayed changes in the renal IGF and IGF BPs.     

Experimental arthritis inhibits the insulin-like growth factor-I axis and induces muscle wasting through cyclooxygenase-2 activation

Chronic arthritis induces cachexia associated with an inhibition of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) system and an activation of the E3 ubiquitin-ligating enzymes muscle atrophy F-box (MAFbx) and muscle Ring finger 1 (MuRF1) in the skeletal muscle. The aim of this work was to study the role of cyclooxygenase (COX)-2 in chronic arthritis-induced cachexia. Arthritis was induced in rats by Freund's adjuvant injection, and the effects of two COX inhibitors (indomethacin, a nonspecific inhibitor, and meloxicam, a selective COX-2 inhibitor on pituitary GH and on liver and serum IGF-I levels) were tested. Arthritis decreased body weight gain and GH and liver IGF-I gene expression. In the arthritic rats, both inhibitors, indomethacin and meloxicam, prevented the inhibitory effect of arthritis on body weight gain. Indomethacin and meloxicam administration to arthritic rats increased pituitary GH and liver IGF-I mRNA as well as serum levels of IGF-I. These data suggest that induction of COX-2 during chronic inflammation is involved in the inhibition of the GH-IGF-I axis and in the body weight loss. In the gastrocnemius muscle, arthritis increased the gene expression of tumor necrosis factor (TNF)-alpha, the E3 ubiquitin-ligating enzymes MAFbx and MuRF1, as well as of IGF-I and IGF-binding protein-5 (IGFBP-5). Inhibition of COX-2 by meloxicam administration increased gastrocnemius weight and decreased MAFbx, MuRF1, TNF-alpha, and IGFBP-5 gene expression. In summary, our data indicate that chronic arthritis-induced cachexia and muscle wasting are mediated by the COX-2 pathway resulting in a decreased GH-IGF-I secretion and increased expression of MAFbx and MuRF1 mRNA.     

Adenoviral-mediated expression of human insulin-like growth factor-binding protein-3.

Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary complexes comprising IGF, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGFBP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure-function relationships of the multifunctional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, medium-scale production of functional, glycosylated IGFBP-3. Replication-deficient adenovirus containing human IGFBP-3 cDNA was generated using standard techniques. Secreted, recombinant IGFBP-3 (IGFBP-3(Ad)) was purified from the culture medium of virus-infected cells by IGF-I affinity chromatography followed by reverse-phase HPLC. When analyzed by SDS-PAGE, IGFBP-3(Ad) was similar in size (43- to 45-kDa glycoform doublet) to IGFBP-3(Pl) derived from plasma. In addition, IGFBP-3(Ad) was detected by immunoblot using an antibody specific for human IGFBP-3 and by ligand blot using radiolabeled IGF-I. IGFBP-3(Ad) had similar affinities for IGF-I and ALS and an approximately 25% decreased affinity for IGF-II compared to IGFBP-3(Pl). IGFBP-3(Ad) showed no significant difference in its susceptibility to an IGFBP-3 protease present in medium conditioned by MCF-7 breast cancer cells compared to IGFBP-3(Pl), but appeared more resistant to the IGFBP-3 protease present in pregnancy serum. IGFBP-3(Ad) also exhibited increased binding to T47D cells which may be related to the glycosylation state of the protein.     

Regulation of the components of the 150 kDa IGF binding protein complex in cocultures of rat hepatocytes and Kupffer cells by 3',5'-cyclic adenosine monophosphate

In the circulation, most of IGFs are bound to a high molecular mass complex of 150 kDa that consists of IGF-I (or IGF-II), IGFBP-3 and the acid-labile subunit (ALS). Within rat liver, biosynthesis of these components has been localized to different cell populations with hepatocytes as source of ALS and nonparenchymal cells (endothelial and Kupffer cells (KC)) as source of IGFBP-3. In the present study, the regulatory effects of the cAMP analogs dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP (8-br-cAMP) on IGF-I, ALS, and IGFBP expression were evaluated in primary cultures of rat hepatocytes, KC as well as in cocultures of hepatocytes and KC. In cocultures, biosynthesis of IGFBP-3 and ALS was inhibited dose-dependently by db-cAMP and 8-br-cAMP while that of IGF-I, IGFBP-1, and -4 was stimulated as demonstrated by ligand and Northern blotting. IGFBP-3 expression in primary cultures of pure KC did not respond to cAMP treatment indicating the importance of a cellular interaction between KC and hepatocytes for the decreased IGFBP-3 synthesis. The inhibition of IGFBP-3 in db-cAMP-treated cocultures was due to a decrease of IGFBP-3 mRNA level accompanied by a reduced cellular degradation of IGFBP-3. We conclude that cAMP stimulate the biosynthesis of IGF-I, IGFBP-1, and -4 in cocultures of hepatocytes and KC thereby enabling the formation of binary IGF/IGFBP complexes while the formation of the 150 kDa complex is impaired through downregulation of IGFBP-3 and ALS. This complex regulation may be a prerequisite for the effects of cAMP-dependent hormones on the transfer of IGFs from circulation to peripheral tissues.     

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.     

Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β (TGF-β) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-β stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-β is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF-I and TGF-β differentially regulate IGFBP-3, IGFBP-4, and IGFBP-5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF-I. J. Cell. Physiol. 174:240–250, 1998. © 1998 Wiley-Liss, Inc.     

Burn-induced increase in atrogin-1 and MuRF-1 in skeletal muscle is glucocorticoid independent but downregulated by IGF-I

The present study determined whether thermal injury increases the expression of the ubiquitin (Ub) E3 ligases referred to as muscle ring finger (MuRF)-1 and muscle atrophy F-box (MAFbx; aka atrogin-1), which are muscle specific and responsible for the increased protein breakdown observed in other catabolic conditions. After 48 h of burn injury (40% total body surface area full-thickness scald burn) gastrocnemius weight was reduced, and this change was associated with an increased mRNA abundance for atrogin-1 and MuRF-1 (3.1- to 8-fold, respectively). Similarly, burn increased polyUb mRNA content in the gastrocnemius twofold. In contrast, there was no burn-induced atrophy of the soleus and no significant change in atrogin-1, MuRF-1, or polyUb mRNA. Burns also did not alter E3 ligase expression in heart. Four hours after administration of the anabolic agent insulin-like growth factor (IGF)-I to burned rats, the mRNA content of atrogin-1 and polyUb in gastrocnemius had returned to control values and the elevation in MuRF-1 was reduced 50%. In contrast, leucine did not alter E3 ligase expression. In a separate study, in vivo administration of the proteasome inhibitor Velcade prevented burn-induced loss of muscle mass determined at 48 h. Finally, administration of the glucocorticoid receptor antagonist RU-486 did not prevent burn-induced atrophy of the gastrocnemius or the associated elevation in atrogin-1, MuRF-1, or polyUb. In summary, the acute muscle wasting accompanying thermal injury is associated with a glucocorticoid-independent increase in the expression of several Ub E3 ligases that can be downregulated by IGF-I.     

Effects of dexamethasone on the production of insulin-like growth factor-I and insulin-like growth factor binding proteins in primary cultures of rat hepatocytes.

The effects of dexamethasone (Dex) on insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 production were investigated in primary cultures of rat hepatocytes. Dex enhanced the secretion of IGFBP-1 as measured by ligand blot analysis but did not show any prominent effect on immunoreactive IGF-I secretion. EC50 of Dex on IGFBP-1 secretion was calculated to be 3 x 10(-8) M. The content of IGFBP-1 mRNA in the cells increased greatly in the presence of Dex but the IGF-I mRNA content did not change significantly under the same conditions. Insulin showed the opposite effect of Dex by decreasing the production of IGFBP-1 and the cellular content of IGFBP-1 mRNA. This effect of insulin was observed also with Dex in the medium. These results show that the gene expression of IGF-I and IGFBP-1 is differently regulated by glucocorticoids and insulin in primary cultures of rat hepatocytes. The results most possibly explain the in vivo effects of glucocorticoids and insulin in regulation of IGF-I and IGFBP-1 production by liver.     

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.