Leptin facilitates proliferation of hepatic stellate cells through up-regulation of platelet-derived growth factor receptor.

In the present study, we investigated the effect of leptin on proliferation of hepatic stellate cells (HSCs) in vitro. Proliferation of 3-day cultured rat HSCs was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the nuclei. The percentages of BrdU-positive cells were increased in the presence of PDGF-BB (5 ng/ml) for 8h as expected. Co-incubation with leptin (10-100 nM) potentiates this PDGF-dependent increase in BrdU positive cells in a dose-dependent manner. Messenger RNA for PDGF receptor alpha and beta subunits was increased almost 2- to 3-fold by incubation with leptin for 6h. Further, pre-incubation with leptin for 6h enhanced PDGF-induced increases in phospho-p44/42 MAP kinase and phospho-Akt levels in a dose-dependent manner. In the same condition, however, leptin per se did not increase phospho-STAT 3 and phospho-p44/42 MAP kinase levels. Instead, leptin increased phospho-Akt levels in HSCs within 30 min, suggesting that the phosphatidylinositol 3 kinase (PI3K)/Akt pathway is involved in the mechanism by which leptin accelerates the proliferation of HSCs. In conclusion, the present study clearly indicated that leptin potentiates PDGF-dependent proliferative responses of HSCs in vitro.

Expression of leptin receptors in hepatic sinusoidal cells.

Emerging evidence has suggested a critical role of leptin in hepatic inflammation and fibrogenesis, however, the precise mechanisms underlying the profibrogenic action of leptin in the liver has not been well elucidated. Therefore, the present study was designed to investigate the expression and functions of leptin receptors (Ob-R) in hepatic sinusoidal cells. Hepatic stellate cells (HSCs), Kupffer cells and sinusoidal endothelial cells (SECs) were isolated from rat livers by in situ collagenase perfusion followed by differential centrifugation technique, and expression of Ob-Ra and Ob-Rb, short and long Ob-R isoforms, respectively, were analyzed by RT-PCR. Ob-Ra mRNA was detected ubiquitously in HSCs and SECs. In contrast, Ob-Rb was detected clearly only in SECs and Kupffer cells, but not in 7-day cultured HSCs. Indeed, tyrosine-phosphorylation of STAT-3, a downstream event of Ob-Rb signaling, was observed in SECs, but not in HSCs, 1 hr after incubation with leptin. Further, leptin increased AP-1 DNA binding activity and TGF-beta 1 mRNA levels in Kupffer cells and SECs, whereas leptin failed to increase TGF-beta 1 mRNA in HSCs. These findings indicated that SECs and Kupffer cells, but not HSCs, express functional leptin receptors, through which leptin elicits production of TGF-beta 1. It is hypothesized therefore that leptin, produced systemically from adipocytes and locally from HSCs, up-regulates TGF-beta 1 thereby facilitate tissue repairing and fibrogenesis in the sinusoidal microenvironment.

Dietary glycine prevents chemical-induced experimental colitis in the rat.

BACKGROUND & AIMS: In this study, the effect of dietary glycine on experimental colitis induced by 2,4,6-trinitrobenzene sulphonic acid (TNBS) and dextran sulfate sodium (DSS) in the rat was evaluated. METHODS: Male Wistar rats were fed a diet containing 5% glycine or casein as controls starting 3 days before experiments, and were given a single intracolonic injection of TNBS (50 mg/rat, dissolved in 50% ethanol). Similarly, some rats were given 3% DSS orally in drinking water for 5 days to induce colitis as a second model. The severity of colitis was evaluated pathologically, and tissue myeloperoxidase (MPO) activity was measured. Further, mRNA and protein levels for interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, cytokine-induced neutrophil chemoattractant (CINC), and macrophage inflammatory protein (MIP)-2 were detected by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: A diet containing glycine ameliorated diarrhea and body weight loss caused by TNBS, and improved both macroscopic and histologic scores of colitis significantly. TNBS-induced increases in MPO activities in the colonic tissue were blunted significantly in glycine-fed animals. Further, dietary glycine largely prevented increases in IL-1beta and TNF-alpha in the colon 2 days after TNBS, and TNBS induction of CINC and MIP-2 in the colonic tissue also was abrogated by glycine. Importantly, the protective effect of glycine was significant even when TNBS colitis was once established. Moreover, dietary glycine also was preventive in a second, DSS-induced colitis model. CONCLUSIONS: Dietary glycine prevents chemical-induced colitis by inhibiting induction of inflammatory cytokines and chemokines. It is postulated that glycine may be useful for the treatment of inflammatory bowel diseases as an immunomodulating nutrient.

Leptin is required for fibrogenic responses induced by thioacetamide in the murine liver.

In this study, we investigated hepatic fibrogenesis caused by long-term thioacetamide (TAA) administration in ob/ob mice, a naturally occurring leptin deficient animal. In the lean littermates, prominent hepatic fibrosis, as well as positive staining for alpha smooth muscle actin (alpha-SMA), was induced by treatment with TAA (200 microg/g, IP, 3 times per week) for 4 to 8 weeks as expected. In sharp contrast, almost no hepatic fibrosis developed in ob/ob mice given the equivalent doses of TAA, where specific staining for alpha-SMA barely was detected. Induction of alpha1(I) procollagen mRNA caused by TAA also was prevented in ob/ob mice almost completely. Further, transforming growth factor beta (TGF-beta) mRNA was increased in the liver after TAA treatment for 4 weeks in lean littermates, which also was prevented in ob/ob mice. Interestingly, fibrotic septa in the hepatic lobules, as well as increases in alpha1(I) procollagen mRNA, was observed in ob/ob mice, when they were injected with recombinant murine leptin (1 microg/g daily) in combination with TAA treatment. Leptin per se did not cause any fibrotic changes in the liver in ob/ob mice. These findings clearly indicated that leptin deficiency is responsible for the resistance to TAA-induced profibrogenic responses in ob/ob mice. In conclusion, leptin appears to promote profibrogenic responses in the liver, in part, by up-regulation of TGF-beta.

Leptin receptor-mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat.

BACKGROUND & AIMS: In this study, we investigated the role of leptin and its receptors (Ob-R) in profibrogenic responses in the liver using Zucker (fa/fa) rats, a natural occurring Ob-R-deficient animal. METHODS: Male Zucker (fa/fa) rats and their lean (+/?) littermates were given intraperitoneal injections of thioacetamide (TAA) (200 mg/kg body wt, 3 times/wk) for 4-8 weeks, and progression of hepatic fibrosis was evaluated. In vitro transactivation of hepatic stellate cells (HSCs) isolated from Zucker rats was evaluated by Western blotting and immunocytochemistry for alpha-smooth muscle actin and type I collagen. Further, a long-form Ob-R (Ob-Rb) in sinusoidal endothelial cells (SECs) and Kupffer cells was identified by reverse-transcription polymerase chain reaction. Moreover, transforming growth factor (TGF)-beta1 messenger RNA in LSE cells, a human SEC-derived cell line, was measured by Northern blotting. RESULTS: Although the normal liver does not produce leptin, activated HSCs produced leptin in vivo during fibrogenesis caused by TAA. In Zucker rats, TAA-induced hepatic fibrosis was prevented almost completely, whereas induction of TGF-beta1 and activation of HSCs were abolished. It is less likely, however, that leptin plays an essential role in the activation of HSCs as a strong autocrine regulator, because HSCs isolated from Zucker rats undergo normal transactivation process in vitro. In contrast, SECs and Kupffer cells contain Ob-Rb, through which leptin up-regulates the expression of matrix remodeling genes including TGF-beta1. CONCLUSIONS: Collectively, these findings indicated that leptin and its functional receptors (Ob-Rb) play a pivotal role in profibrogenic responses in the liver.

Pioglitazone prevents early-phase hepatic fibrogenesis caused by carbon tetrachloride.

Here we investigated the effect of pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, on early-phase hepatic fibrogenesis in vivo caused by acute carbon tetrachloride (CCl(4)) administration in the rat. Pioglitazone (1 mg/kg BW) prevented pericentral fibrosis and induction of alpha-smooth muscle actin (SMA) 72 h after CCl(4) administration (1 ml/kg BW). CCl(4) induction of alpha1(I)procollagen mRNA in the liver was blunted by pioglitazone to the levels almost 2/3 of CCl(4) alone. Pioglitazone also prevented CCl(4)-induced hepatic inflammation and necrosis, as well as increases in serum tumor necrosis factor-alpha levels. Further, pioglitazone inhibited the induction of alphaSMA and type I collagen in primary cultured hepatic stellate cells in a dose-dependent manner. In conclusion, pioglitazone inhibits both hepatic inflammation and activation of hepatic stellate cells, thereby ameliorating early-phase fibrogenesis in the liver following acute CCl(4).