Phospholipid metabolism during regeneration of rat liver.
ABSTRACT: The concentration and composition of phospholipids and mitotic activity in regenerating rat liver were studied. (1) The total amount of liver phospholipid increased approximately linearly during 48h after operation but without change in the relative concentrations of individual phospholipids. (2) The appearance of mitoses 30h after operation was accompanied by an increased incorporation of (32)P into the liver phospholipids. (3) The regenerating livers incorporated a higher percentage of the label into the phosphatidylserine+phosphatidylinositol fraction than those of control rats. The percentage of the label incorporated into phosphatidylethanolamine in these livers increased but decreased in the phosphatidylcholine.
Project description:Preliminary studies of liver regeneration induced by partial hepatectomy (PHE) identified a substantial depletion of hepatic retinoid stores, by greater than 70%, in regenerating livers of wild-type C57Bl/6J mice. To understand this, we compared responses of wild-type and lecithin:retinol acyltransferase (Lrat)-deficient mice, which totally lack hepatic retinoid stores, to PHE. The Lrat-deficient livers showed delayed regeneration in the first 24 h after PHE. At 12 h after PHE, we observed significantly less mRNA expression for growth factors and cytokines implicated in regulating the priming phase of liver regeneration, specifically for Hgf and Tgf?, but not Tgf?. Compared with wild-type mice, the changes in mRNA levels for p21 and cyclins E1, B1, and A2 mRNAs and for hepatocellular BrdU incorporation and mitoses were delayed (i.e., shifted to later times) in regenerating Lrat(-/-) livers. Concentrations of all-trans-retinoic acid were significantly lower in the livers of Lrat(-/-) mice following PHE, and this was accompanied by diminished expression of known retinoid-responsive genes. At later times after PHE, the rate of liver weight restoration for Lrat(-/-) mice was parallel to that of wild-type mice, although additional biochemical differences were observed. Thus, hepatic retinoid stores are required for maintaining expression of signaling molecules that regulate cell proliferation and differentiation immediately after hepatic injury, accounting for the delayed restoration of liver mass in Lrat(-/-) mice.
Project description:The time course of hepatic zinc-isometallothionein synthesis was studied in the regenerating liver and compared with that produced after the parenteral injection of zinc (6 mg of Zn2+/kg). In the regenerating liver, zinc levels rose rapidly after partial hepatectomy and reached a maximum at approx. 14h before declining to approximately normal levels at 48h post-operation. During this 48h period most of the zinc was incorporated into metallothionein. Purification of the latter into the charge-separable isometallothioneins (i.e. MT1 and MT2) showed that, in the regenerating liver, there was an unequal distribution of zinc between the two isoproteins. Thus at operation the endogenous thionein had an MT2/MT1 ratio of 1; after regeneration this ratio increased, and all times during the time course there was more MT2 than MT1. In contrast, the intraperitoneal injection of zinc produced a biphasic uptake of zinc into the liver with maxima at 10h and 32h. During the first phase of zinc uptake, metallothionein synthesis increased rapidly and, unlike the regenerating liver, the MT2/MT1 ratio of 1 remained constant. Thereafter, this ratio increased in a manner analogous to that exhibited by the regenerating liver. Half-life determinations for thionein disappearance/degradation shows that MT2 and MT1 were degraded with half-lives (t1/2) of 26.18h and 16.44h respectively in the regenerating liver and 14.75h and 9.3h after zinc injection. Thus thionein disappearance/degradation in the regenerating liver was slower than that seen after zinc injection. However, in both situations MT2 was always removed at a slower rate than MT1. Calculation of the rates of thionein synthesis (assuming the above disappearance rates were constant throughout the time course) showed that, in the regenerating liver, the rate of MT2 synthesis was approximately twice that of MT1. This was not the case after zinc injection, where both isometallothioneins were synthesized in equal amounts. These results demonstrate that the rates of synthesis of MT2 and MT1 can be altered according to the metabolic status of the cell and suggest a specific role for MT2 during liver regeneration.
Project description:The release of (14)CO(2) from [7-(14)C]orotic acid was measured in isolated perfused normal and regenerating rat livers. With some limitations, the release of (14)CO(2) from [7-(14)C]orotic acid can be used to estimate UMP synthesis in perfused livers. Isolated perfused livers rapidly pick up labelled orotic acid added to perfusate and convert most of it into UMP. Perfused regenerating livers produce approx. 2.5 times as much UMP/g of liver as do perfused normal livers. However, the absolute amount of orotic acid converted into UMP is higher in perfused normal livers than in perfused regenerating livers. Perfused regenerating livers do not differ in their orotic acid uptake and UMP synthesis from livers of comparable size in which regeneration is not taking place. The total amount of orotic acid taken up by the liver (rather than the rate of uptake) and the size of the liver appear to be the determining factors in UMP production. The results suggest that the decrease in liver size caused by partial hepatectomy may be in itself sufficient to account for an increase in the flow of metabolites in the pyrimidine pathway at the early stages of liver regeneration.
Project description:This study was performed to investigate sequential changes in bile secretion and biliary lipids after taurocholic acid (TCA) loading of regenerating rat liver. TCA was administered intravenously at stepwise-increasing doses to groups of non-operated control and partially hepatectomized rats, 24, 72 and 168 h after surgery. Bile flow, bile-acid output (BAO) and phospholipid output (PLO) (expressed per gram of liver) in partially hepatectomized rats increased more than in the controls. Using an isolated perfusion rat-liver system, TCA infusion was also carried out on groups of non-operated control and hepatectomized rats 72 h after operation. Again bile flow, BAO and PLO (expressed per gram of liver) were significantly higher in the partial hepatectomy case, mirroring the results obtained in vivo. When horseradish peroxidase (HRP) was pulse-loaded in isolated perfusion preparations, the second peak of biliary HRP secretion in hepatectomized rats was significantly higher than in controls. We conclude that increased bile-acid flow in partially hepatectomized rats is dependent upon acceleration of vesicular transport accompanying or following proliferation in regenerating livers.
Project description:In this study, we analyzed global liver gene expression in MCU knock-down (KD) mice. MCUloxp/loxp male mice were treated with an AAV8-Cre under the control of a hepatocyte specific promoter (TBG) to generate liver-specific MCU KD mice. AAV8-TBG-Null treated littermates were used as controls. Knockdown was verified 3 weeks after injection by protein and mRNA (80%). 5 weeks after injection mice were subjected to 70% partial hepatectomy and liver semples were collected 30h after the sugery. Mouse Clariom S (Affymetrix, Santa Clara, CA) arrays were used to obtain global gene expression data from pooled liver samples (pools of 4 biological replicates per array). Overall design: Gene expression was profiled in the regenerating livers of wild type and in liver-specific MCU knockdown mice at t=0 and 30h after 70% partial hepatectomy; pools of 4 biological replicates per genotype.
Project description:Elucidating the mechanism of liver regeneration could lead to life-saving therapy for a large number of patients, especially elderly patients, after segmental liver transplantation or resection of liver tumors. The forkhead box m1b (Foxm1b) transcription factor is required for normal liver regeneration. Here we report that Foxm1b is the first direct farnesoid X receptor (FXR) target gene known to be involved in cell cycle regulation and that aging regenerating livers have delayed activation of FXR, which results in defective induction of Foxm1b and thereby contributes to defective liver regeneration. An inverted repeat 0 (IR-0) FXR response element, acting as an enhancer in intron 3 of the Foxm1b gene, was identified by a combination of transcriptional reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays. Diminished FXR binding to the IR-0 element was found in aging regenerating livers. FXR activation by a novel ligand in aging livers induced Foxm1b expression and elevated hepatocyte DNA replication to about 70% of the levels found in young regenerating livers, which were specifically suppressed by hepatic expression of anti-Foxm1b short hairpin RNA.Our results have revealed Foxm1b as the first known direct FXR target gene involved in cell cycle regulation and have demonstrated that defective activation of FXR could be an intrinsic defect in aging regenerating livers. Activation of FXR alone is largely able to alleviate age-related liver regeneration defects. These findings highlight FXR as a potential target of drug design for promoting liver regeneration in older subjects.
Project description:1. Rats pretreated with Triton WR-1339 to prevent the formation of remnants were injected with [3H]cholesterol-labelled remnants, intact chylomicrons or chylomicrons depleted of most of their surface phospholipids by treatment with phospholipase A2. Within 5 min about 80% of the injected label of remnants and phospholipid-depleted chylomicrons was incorporated into the livers compared with less than 10% of the injected radioactivity of intact chylomicrons. A similar rapid hepatic uptake of radioactivity occurred when rats not pretreated with Triton were injected with [3H]cholesterol-labelled phospholipid-depleted chylomicrons. This rapid hepatic uptake of phospholipid-depleted chylomicrons occurred apparently without any alteration in the apoprotein composition of the particles. 2. The participation of hepatocytes in the uptake of remnants and phospholipid-depleted chylomicrons was examined. Both types of particles were taken up by the hepatocytes. However, small chylomicrons (Sf less than 400) were taken up more efficiently than were large chylomicrons (Sf greater than 400), but neither was taken up as efficiently as the remnants. 3. The results of this study lend support to the hypothesis that phospholipid-depleted chylomicrons and chylomicron remnants are taken up by the liver by a similar mechanism, which depends on the loss of surface phospholipids.
Project description:1. The effect of gamma-irradiation (4000rd) on the synthesis of ribosomal (pre-rRNA) and heterogeneous nuclear RNA (pre-mRNA) in normal and in regenerating rat liver was studied by using 40 min labelling with [6(-14)C]orotic acid. 2. Partial hepatectomy caused a sharp transient increase in the specific radioactivity of the endogenous low-molecular-weight RNA precursors in the livers of both normal and irradiated rats. Irradiation of intact animals did not affect the pool. 3. Irradiation enhanced the synthesis of pre-rRNA for at least 12h. The synthesis of pre-mRNA was also enhanced, but only in the first 3h after irradiation. 4. Partial hepatectomy strongly stimulated the synthesis of both pre-rRNA and pre-mRNA. 5. The synthesis of pre-rRNA was enhanced also in regenerating liver of animals irradiated before or after the operation. The conclusion can be drawn that the early increase in the synthesis of ribosomal RNA is a non-specific cellular response to different injuring factors. 6. The only case where irradiation caused an early inhibition of RNA synthesis was that of pre-mRNA in regenerating liver. This supports the hypothesis that ionizing radiation does not suppress the transcription per se but affects the mechanisms of activation of new genes (cellular programming).
Project description:The involvement of IL-4 in liver regeneration has not yet been recognized. In this article, we show that IL-4, produced by NKT cells that accumulate in regenerating livers after partial hepatectomy, contributes to this process by regulating the activation of complement after liver resection in mice. The mechanism of this regulation was associated with the maintenance of an appropriate level of IgM in mouse blood, because IgM deposited in liver parenchyma most likely initiated complement activation during liver regeneration. By controlling complement activation, IL-4 regulated the induction of IL-6, thereby influencing a key pathway involved in regenerating liver cell proliferation and survival. Furthermore, the secretion of IL-4 was controlled by complement through the recruitment of NKT cells to regenerating livers. Our study thus reveals the existence of a regulatory feedback mechanism involving complement and IL-4 that controls liver regeneration.
Project description:To identify the molecular targets of orosomucoid (Orm1) during liver regeneration, GeneChip analysis was performed at 48 h after partial hepatectomy (PH) in regenerating mouse liver treated with siControl or siOrm. A total of 180 differentially expressed genes in Orm1 konckdown mouse liver by comparing with siControl were identified with a fold change more than 2. Then, pathway analysis performed on the altered gene expression profiles using Ingenuity Pathways Analysis (IPA) program revealed that cell cycle, Toll-like receptor and TGF-beta receptor signaling pathways were under control of Orm1 in regenerating mouse livers. Three days post In vivo knockdown of Orm1 with its siRNA administered to mice using Invivofectamine 3.0 by a single injection, 40% PH was perfomred and gene expression prolifes of regenerating mouse livers at 48 h after PH was measued using Affymetrix GeneChip Mouse Genome 430A 2.0 Array.