The hallmark of fibrosis is the excessive accumulation of collagen. The deposited collagen contains increased pyridinoline cross-link levels due to an overhydroxylation of lysine residues within the collagen telopeptides. Lysyl hydroxylase 2b (LH2b) is the only lysyl hydroxylase consistently up-regulated in several forms of fibrosis, suggesting that an enhanced LH2b level is responsible for the overhydroxylation of collagen telopeptides. The present paper reports the effect of profibrotic cytokines on the expression of collagen, lysyl hydroxylases and lysyl oxidase in normal human skin fibroblasts, as well as the effect on pyridinoline formation in the deposited matrix. All three isoforms of TGF-beta induce a substantial increase in LH2b mRNA levels, also when expressed relatively to the mRNA levels of collagen type I alpha2 (COL1A2). The TGF-beta isoforms also clearly influence the collagen cross-linking pathway, since higher levels of pyridinoline cross-links were measured. Similar stimulatory effects on LH2b/COL1A2 mRNA expression and pyridinoline formation were observed for IL-4, activin A, and TNF-alpha. An exception was BMP-2, which has no effect on LH2b/COL1A2 mRNA levels nor on pyridinoline formation. Our data show for the first time that two processes, i.e., up-regulation of LH2b mRNA levels and increased formation of pyridinoline cross-links, previously recognized to be inherent to fibrotic processes, are induced by various profibrotic cytokines.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors are currently in clinical development as potential lipid-lowering and antiatherosclerotic agents. We investigated the effect of avasimibe (Cl-1011), a novel ACAT inhibitor, on bile acid synthesis and cholesterol 7␣-hydroxylase in cultured rat hepatocytes and rats fed different diets. Avasimibe dose-dependently decreased ACAT activity in rat hepatocytes in the presence and absence of ␤-migrating very low-density lipoproteins (␤VLDL) (by 93% and 75% at 10 mol/L) and reduced intracellular storage of cholesteryl esters. Avasimibe (3 mol/L) increased bile acid synthesis (2.9-fold) after preincubation with ␤VLDL and cholesterol 7␣-hydroxylase activity (1.7-and 2.6-fold, with or without ␤VLDL), the latter paralleled by a similar induction of its messenger RNA (mRNA). Hepatocytes treated with avasimibe showed a shift from storage and secretion of cholesteryl esters to conversion of cholesterol into bile acids. In rats fed diets containing different amounts of cholesterol and cholate, avasimibe reduced plasma cholesterol (by 52% to 71%) and triglyceride levels (by 28% to 62%). Avasimibe did not further increase cholesterol 7␣-hydroxylase activity and mRNA in cholesterol-fed rats, but prevented down-regulation by cholate. Avasimibe did not affect sterol 27-hydroxylase and oxysterol 7␣-hydroxylase, 2 enzymes in the alternative pathway in bile acid synthesis. No increase in the ratio of biliary excreted cholesterol to bile acids was found, indicating that ACAT inhibition does not result in a more lithogenic bile. Avasimibe increases bile acid synthesis in cultured hepatocytes by enhancing the supply of free cholesterol both as substrate and inducer of cholesterol 7␣-hydroxylase. These effects may partially explain the potent cholesterol-lowering effects of avasimibe in the rat. (HEPATOLOGY 1999;30:491-500.)

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The influence of different retinoids on apolipoprotein A-I (apoA-I) synthesis and secretion was investigated in primary monolayer cultures of hepatocytes from cynomolgus monkeys. Addition of retinol (vitamin A) and retinoic acid to the culture medium resulted in a time- and dose-dependent increase in the secretion of apoA-I. No effect was observed during the first 24-hour incubation period; however, apoA-I secretion was enhanced 1.5-fold in the following 24-hour period in the presence of 10 mumol/L retinoic acid. Maximal stimulation (2.7-fold) was obtained at 10 mumol/L retinoic acid during a third 24-hour incubation. In these experiments apoB-100 secretion was unaffected. When [35S]methionine incorporation studies were performed de novo synthesis of apoA-I was increased, whereas total protein synthesis remained constant. These observations indicated that the induction of apoA-I synthesis is not part of a general effect of retinoic acid on hepatic protein synthesis. Among different natural and synthetic retinoids, retinoic acid and its 9-cis and 13-cis isomers were equally active and were the most potent inducers of apoA-I synthesis, whereas the maximal stimulation induced by retinol was lower (1.6-fold). ApoA-I mRNA abundance was increased threefold in hepatocytes exposed for 72 hours to 10 mumol/L retinoic acid, which was associated with a twofold increase in the transcriptional rate of the apoA-I gene. In contrast, no changes were found in the apoB-100 mRNA level and transcriptional activity of the apoB-100 gene. We conclude that retinoids enhance apoA-I synthesis in simian hepatocytes by transcriptional regulation.

a b s t r a c tBackground: Muscle atrophy is defined as decreased muscle mass, associated with aging as well as with various chronic diseases and is a fundamental cause of frailty, functional decline and disability. Frailty represents a huge potential public health issue worldwide with high impact on healthcare costs. A major clinical issue is therefore to devise new strategies preventing muscle atrophy. In this study, we tested the efficacy of Vital01, a novel oral nutritional supplement (ONS), on body weight and muscle mass using a caloric restriction-induced mouse model for muscle atrophy. Methods: Mice were calorically restricted for 2 weeks to induce muscle atrophy: one control group received 60% kcal of the normal chow diet and one intervention group received 30% kcal chow and 30 kcal% Vital01. The effects on body weight, lean body mass, muscle histology and transcriptome were assessed. In addition, the effects of Vital01, in mice with established muscle atrophy, were assessed and compared to a standard ONS. To this end, mice were first calorically restricted on a 60% kcal chow diet and then refed with either 100 kcal% chow, a mix of Vital01 (receiving 60% kcal chow and 40 kcal% Vital01) or with a mix of standard, widely prescribed ONS (receiving 60 kcal% chow and 40 kcal% Fortisip Compact). Results: Vital01 attenuated weight loss (−15% weight loss for Vital01 vs. −25% for control group, p b 0.01) and loss of muscle mass (Vital01 with −13%, −12% and −18%, respectively, for gastrocnemius, quadriceps and tibialis vs. 25%, −23% and −28%, respectively, for control group, all p b 0.05) and also restored body weight, fat and muscle mass more efficiently when compared to Fortisip Compact. As assessed by transcriptome analysis and Western blotting of key proteins (e.g. phospoAKT, mTOR and S6K), Vital01 attenuated the catabolic and anabolic signaling pathways induced by caloric restriction and modulated inflammatory and mitochondrial pathways. In addition, Vital01 affected pathways related to matrix proteins/collagens homeostasis and tended to reduce caloric restriction-induced collagen fiber density in the quadriceps (with −27%, p = 0.051). Conclusions:We demonstrate that Vital01 preserves muscle mass in a calorically restricted mouse model for muscle atrophy. Vital01 had preventive effects when administered during development of muscle atrophy. Furthermore, when administered in a therapeutic setting to mice with established muscle atrophy, Vital01 rapidly restored body weight and accelerated the recurrence of fat and lean body mass more efficiently than Fortisip Compact. Bioinformatics analysis of gene expression data identified regulatory pathways that were specifically influenced by Vital01 in muscle.