Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.     

Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.     

Effects of gender and GH secretory pattern on sterol regulatory element-binding protein-1c and its target genes in rat liver

We investigated whether the sexually dimorphic secretory pattern of growth hormone (GH) in the rat regulates hepatic gene expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its target genes. SREBP-1c, fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (GPAT) mRNA were more abundant in female than in male livers, whereas acetyl-CoA carboxylase-1 (ACC1) and stearoyl-CoA desaturase-1 (SCD-1) were similarly expressed in both sexes. Hypophysectomized female rats were given GH as a continuous infusion or as two daily injections for 7 days to mimic the female- and male-specific GH secretory patterns, respectively. The female pattern of GH administration increased the expression of SREBP-1c, ACC1, FAS, SCD-1, and GPAT mRNA, whereas the male pattern of GH administration increased only SCD-1 mRNA. FAS and SCD-1 protein levels were regulated in a similar manner by GH. Incubation of primary rat hepatocytes with GH increased SCD-1 mRNA levels and decreased FAS and GPAT mRNA levels but had no effect on SREBP-1c mRNA. GH decreased hepatic liver X receptor-alpha (LXRalpha) mRNA levels both in vivo and in vitro. Feminization of the GH plasma pattern in male rats by administration of GH as a continuous infusion decreased insulin sensitivity and increased expression of FAS and GPAT mRNA but had no effect on SREBP-1c, ACC1, SCD-1, or LXRalpha mRNA. In conclusion, FAS and GPAT are specifically upregulated by the female secretory pattern of GH. This regulation is not a direct effect of GH on hepatocytes and does not involve changed expression of SREBP-1c or LXRalpha mRNA but is associated with decreased insulin sensitivity.     

FXR-mediated down-regulation of CYP7A1 dominates LXRalpha in long-term cholesterol-fed NZW rabbits

We investigated how cholesterol feeding regulates cholesterol 7alpha-hydroxylase (CYP7A1) via the nuclear receptors farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha) in New Zealand white rabbits. After 1 day of 2% cholesterol feeding, when the bile acid pool size had not expanded, mRNA levels of the FXR target genes short-heterodimer partner (SHP) and sterol 12alpha-hydroxylase (CYP8B) were unchanged, indicating that FXR activation remained constant. In contrast, the mRNA levels of the LXRalpha target genes ATP binding cassette transporter A1 (ABCA1) and cholesteryl ester transfer protein (CETP) increased 5-fold and 2.3-fold, respectively, associated with significant increases in hepatic concentrations of oxysterols. Activity and mRNA levels of CYP7A1 increased 2.4 times and 2.2 times, respectively. After 10 days of cholesterol feeding, the bile acid pool size increased nearly 2-fold. SHP mRNA levels increased 4.1-fold while CYP8B declined 64%. ABCA1 mRNA rose 8-fold and CETP mRNA remained elevated. Activity and mRNA of CYP7A1 decreased 60% and 90%, respectively. Feeding cholesterol for 1 day did not enlarge the ligand pool size or change FXR activation, while LXRalpha was activated highly secondary to increased hepatic oxysterols. As a result, CYP7A1 was up-regulated. After 10 days of cholesterol feeding, the bile acid (FXR ligand) pool size increased, which activated FXR and inhibited CYP7A1 despite continued activation of LXRalpha. Thus, in rabbits, when FXR and LXRalpha are activated simultaneously, the inhibitory effect of FXR overrides the stimulatory effect of LXRalpha to suppress CYP7A1 mRNA expression.     

On the role of liver X receptors in lipid accumulation in adipocytes

The pivotal role of liver X receptors (LXRs) in the metabolic conversion of cholesterol to bile acids in mice is well established. More recently, the LXRalpha promoter has been shown to be under tight regulation by peroxisome proliferator-activated receptors (PPARs), implying a role for LXRalpha in mediating the interplay between cholesterol and fatty acid metabolism. We have studied the role of LXR in fat cells and demonstrate that LXR is regulated during adipogenesis and augments fat accumulation in mature adipocytes. LXRalpha expression in murine 3T3-L1 adipocytes as well as in human adipocytes was up-regulated in response to PPARgamma agonists. Administration of a PPARgamma agonist to obese Zucker rats also led to increased LXRalpha mRNA expression in adipose tissue in vivo. LXR agonist treatment of differentiating adipocytes led to increased lipid accumulation. An increase of the expression of the LXR target genes, sterol regulatory binding protein-1 and fatty acid synthase, was observed both in vivo and in vitro after treatment with LXR agonists for 24 h. Finally, we demonstrate that fat depots in LXRalpha/beta-deficient mice are smaller than in age-matched wild-type littermates. These findings imply a role for LXR in controlling lipid storage capacity in mature adipocytes and point to an intriguing physiological interplay between LXR and PPARgamma in controlling pathways in lipid handling.     

Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream of beta-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos

In early Ciona embryos, nuclear accumulation of beta-catenin is most probably the first step of endodermal cell specification. If beta-catenin is mis- and/or overexpressed, presumptive notochord cells and epidermal cells change their fates into endodermal cells, whereas if beta-catenin nuclear localization is downregulated by the overexpression of cadherin, the endoderm differentiation is suppressed, accompanied with the differentiation of extra epidermal cells ( Imai, K., Takada, N., Satoh, N. and Satou, Y. (2000) Development 127, 3009-3020). Subtractive hybridization screens of mRNAs between beta-catenin overexpressed embryos and cadherin overexpressed embryos were conducted to identify potential beta-catenin target genes that are responsible for endoderm differentiation in Ciona savignyi embryos. We found that a LIM-homeobox gene (Cs-lhx3), an otx homolog (Cs-otx) and an NK-2 class gene (Cs-ttf1) were among beta-catenin downstream genes. In situ hybridization signals for early zygotic expression of Cs-lhx3 were evident only in the presumptive endodermal cells as early as the 32-cell stage, those of Cs-otx in the mesoendodermal cells at the 32-cell stage and those of Cs-ttf1 in the endodermal cells at the 64-cell stage. Later, Cs-lhx3 was expressed again in a set of neuronal cells in the tailbud embryo, while Cs-otx was expressed in the anterior nervous system of the embryo. Expression of all three genes was upregulated in beta-catenin overexpressed embryos and downregulated in cadherin overexpressed embryos. Injection of morpholino oligonucleotides against Cs-otx did not affect the embryonic endoderm differentiation, although the formation of the central nervous system was suppressed. Injection of Cs-ttf1 morpholino oligonucleotides also failed to suppress the endoderm differentiation, although injection of its synthetic mRNAs resulted in ectopic development of endoderm differentiation marker alkaline phosphatase. By contrast, injection of Cs-lhx3 morpholino oligo suppressed the endodermal cell differentiation and this suppression was rescued by injection of Cs-lhx3 mRNA into eggs. In addition, although injection of delE-Ci-cadherin mRNA into eggs resulted in the suppression of alkaline phosphatase development, injection of delE-Ci-cadherin mRNA with Cs-lhx3 mRNA rescued the alkaline phosphatase development. These results strongly suggest that a LIM-homeobox gene Cs-lhx3 is one of the beta-catenin downstream genes and that its early expression in embryonic endodermal cells is responsible for their differentiation.     

A New Avian Fibroblast Growth Factor Receptor in Myogenic and Chondrogenic Cell Differentiation

We studied the expression of FREK (fibroblast growth factor receptor-like embryonic kinase), a new receptor recently cloned from quail embryo, during the differentiation of skeletal muscle satellite cells and epiphyseal growth-plate chondrocytes. Although FREK mRNA was expressed in both cell types, satellite cells expressed higher levels of this mRNA than chondrocytes. FREK gene expression was found to be modulated by b-FGF in a biphasic manner: low concentrations increased expression, whereas high concentrations attenuated it. In both cell cultures, the levels of FREK mRNA declined during terminal differentiation. Moreover, retinoic acid (RA), which induces skeletal muscle satellite cells to differentiate, also caused a reduction in FREK gene expression in these cells. Induction of chondrocyte differentiation with ascorbic acid was monitored by a decrease in collagen type II gene expression and an increase in alkaline phosphatase activity. Satellite cell differentiation was marked by morphological changes as well as by increased sarcomeric myogenin content and creatine kinase activity and changes in the expression of the regulatory muscle-specific genes, MyoD and myogenin. DNA synthesis in both cell types was stimulated by b-FGF. However, in satellite cells, the response was bell-shaped, peaking at 1 ng/ml b-FGF, whereas in chondrocytes, higher levels of b-FGF were needed. b-FGF-dependent DNA synthesis in satellite cells was decreased by RA at concentrations over 10^-7M . The observed correlation between the level of FREK gene expression and various stages of differentiation, its modulation by b-FGF and RA, as well as the correlation between FREK gene expression and the physiological response to b-FGF, suggest that this specific FGF receptor plays an important role in muscle and cartilage cell differentiation.     

Maternal dietary iron restriction modulates hepatic lipid metabolism in the fetuses

Maternal dietary Fe restriction reduced fasting plasma cholesterol and triglyceride (TG) concentrations in the fetuses, as well as decreased plasma TG levels in the adult offspring. To investigate how maternal Fe restriction was affecting fetal lipid metabolism, we investigated whether there were changes in liver lipid metabolism in the full-term fetuses. There was a approximately 27% (P < 0.05) increase in cholesterol but approximately 29% reduction (P = 0.01) in TG concentrations in the liver of the Fe-restricted fetuses. Hepatic mRNA levels of cholesterol 7alpha hydroxylase and liver X receptor-alpha (LXRalpha) were reduced by approximately 50% (P < 0.01) and approximately 34% (P < 0.01), respectively. As LXRalpha regulates expression of sterol response element binding protein-1c (SREBP-1c) expression, we measured SREBP-1c expression. There was an approximately 43% (P < 0.001) reduction in mRNA levels of SREBP-1c and its response genes, including acetyl-CoA carboxylase by approximately 35% (P = 0.01), fatty acid synthase by approximately 18% (P = 0.05), and diacylglycerol acyltransferase by approximately 19% (P = 0.03). Furthermore, protein levels of CD36 were reduced by approximately 27% (P = 0.02) in Fe-restricted fetuses. In conclusion, changes in liver cholesterol and TG concentrations in Fe-restricted fetuses may be coordinated through reduced expression of heme-containing cholesterol 7alpha hydroxylase and its regulator LXRalpha, mainly via downregulation of expression of genes in bile acid synthesis and fatty acid synthesis pathways.     

22-Hydroxycholesterols regulate lipid metabolism differently than T0901317 in human myotubes

The nuclear liver X receptors (LXRalpha and beta) are regulators of lipid and cholesterol metabolism. Oxysterols are known LXR ligands, but the functional role of hydroxycholesterols is at present unknown. In human myotubes, chronic exposure to the LXR ligand T0901317 promoted formation of diacylglycerol (DAG) and triacylglycerol (TAG), 22-R-hydroxycholesterol (22-R-HC) had no effect, and 22-S-hydroxycholesterol (22-S-HC) reduced the formation. In accordance with this, 22-HC and T0901317 regulated the expression of fatty acid transporter CD36, stearoyl-CoA desaturase-1, acyl-CoA synthetase long chain family member 1 and fatty acid synthase (FAS) differently; all genes were increased by T0901317, 22-R-HC did not change their expression level, while 22-S-HC reduced it. Transfection studies confirmed that the FAS promoter was activated by T0901317 and repressed by 22-S-HC through an LXR response element in the promoter. Both 22-R-HC and T0901317 increased gene expression of LXRalpha, sterol regulatory element-binding protein 1c and ATP-binding cassette transporter A1, while 22-S-HC had little effect. In summary, 22-R-HC regulated lipid metabolism and mRNA expression of some LXR target genes in human myotubes differently than T0901317. Moreover, 22-S-HC did not behave like an inactive ligand; it reduced synthesis of complex lipids and repressed certain genes involved in lipogenesis and lipid handling.     

Potential role of antioxidants during ethanol-induced changes in the fatty acid composition and arachidonic acid metabolites in male Wistar rats

Biochemical assessment of liver damage during ethanol-induced stress was done by measuring the activities of serum enzymes, viz., aspartate transaminase (AST) and alkaline phosphatase (ALP), which were significantly elevated in rats fed ethanol. Ethanol administration for a period of 60 days modifies the fatty acid composition, and the analysis of fatty acids showed that there was a significant increase in the concentrations of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) in liver, kidney, and brain, whereas the concentrations of palmitoleic (16:1) and arachidonic acid (20:4) were significantly decreased. The breakdown products of arachidonic acids (20:4), prostaglandins, were elevated. The antioxidants curcumin and N-acetylcysteine (NAC) decreased the activities of serum AST and ALP. Curcumin and NAC decreased the concentrations of fatty acids, viz., palmitic, stearic, and oleic acid, whereas arachidonic acid and palmitoleic acid were elevated. The prostaglandin concentrations were also decreased after curcumin and N-acetylcysteine treatment. Thus the present investigation shows that curcumin and N-acetylcysteine prevent the fatty acid changes produced by ethanol and also reduce the inflammatory response of ethanol by reducing the level of prostaglandins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improved metabolic control by depletion of Liver X Receptors in mice

Liver X Receptors (LXRs) coordinate the regulation of lipid and carbohydrate metabolism and insulin signaling. LXR-ligands lower plasma glucose in hyperglycemic rodents and have consequently been proposed as anti-diabetic agents. We investigated the metabolic effects induced by high carbohydrate diet in LXRalpha(-/-)beta(-/-) mice. Irrespective of diets, LXRalpha(-/-)beta(-/-) mice had reduced fatty acid, insulin, and C-peptide plasma levels than wild-type controls, suggesting a lower insulin production. High carbohydrate diet decreased the plasma glucose levels and the homeostasis model assessment (HOMA)-index in LXRalpha(-/-)beta(-/-) mice and increased hepatic triglyceride content and mRNA levels of lipogenic genes in wild-type and LXRalpha(-/-)beta(-/-) mice, proportionally. In wild-type mice high carbohydrate diet was associated with induced expression of LXR (1.5-fold), despite unchanged SREBP-1c expression. LXRalpha(-/-)beta(-/-) mice responded to this diet by induction of SREBP-1c. Our study suggests that in LXRalpha(-/-)beta(-/-) mice, glucose utilization seems to be privileged possibly due to reduced circulating free fatty acid levels.