Liver Study Unit

Nebraska City, Iowa, United States

Liver Study Unit

Nebraska City, Iowa, United States
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Czaja M.J.,Yeshiva University | Ding W.-X.,University of Kansas Medical Center | Donohue Jr. T.M.,Liver Study Unit | Kim J.-S.,Florida College | And 10 more authors.
Autophagy | Year: 2013

Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases. © 2013 Landes Bioscience.

Vessey D.A.,Liver Study Unit | Vessey D.A.,University of California at San Francisco | Li L.,Liver Study Unit | Kelley M.,Liver Study Unit
American Journal of Physiology - Heart and Circulatory Physiology | Year: 2011

Protection of the heart from ischemia-reperfusion injury can be achieved by ischemic preconditioning and ischemic postconditioning. Previous studies revealed that a complex of pannexin-1 with the P2X7 receptor forms a channel during ischemic preconditioning and ischemic postconditioning that results in the release of endogenous cardioprotectants. ATP binds to P2X7 receptors, inducing the formation of a channel in association with pannexin-1. We hypothesized that this channel would provide a pathway for the release of these same cardioprotectants. Preconditioning-isolated perfused rat hearts with 0.4 μM ATP preceding 40 min of ischemia minimized infarct size upon subsequent reperfusion (5% of risk area) and resulted in >80% recovery of left ventricular developed pressure. Postconditioning with ATP after ischemia during reperfusion was also protective (6% infarct and 72% recovery of left ventricular developed pressure). Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X7 receptors (brilliant blue G and A438079) blocked ATP pre-and postconditioning, indicating that ATP protection was elicited via the opening of a pannexin-1/P2X7 channel. An antagonist of binding of the endogenous cardioprotectant sphingosine 1-phosphate to its G protein-coupled receptor diminished protection by ATP, which is also consistent with an ATP-dependent release of cardioprotectants. Suramin, an antagonist of binding of ATP (and ADP) to P2Y receptors, was without effect on ATP protection. Benzoyl benzoyl-ATP, a more specific P2X7 agonist, was also a potent pre-and postconditioning agent and sensitive to blockade by pannexin-1/P2X7 channel antagonists. The data point out for the first time the potential of P2X7 agonists as cardioprotectants. © 2011 the American Physiological Society.

Vessey D.A.,Liver Study Unit | Vessey D.A.,University of California at San Francisco | Li L.,Liver Study Unit | Jin Z.-Q.,South Dakota State University | And 5 more authors.
Oxidative Medicine and Cellular Longevity | Year: 2011

Sphingosine kinase (SphK) exhibits two isoforms, SphK1 and SphK2. Both forms catalyze the synthesis of sphingosine 1-phosphate (S1P), a sphingolipid involved in ischemic preconditioning (IPC). Since the ratio of SphK1:SphK2 changes dramatically with aging, it is important to assess the role of SphK2 in IR injury and IPC. Langendorff mouse hearts were subjected to IR (30min equilibration, 50min global ischemia, and 40min reperfusion). IPC consisted of 2min of ischemia and 2min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), ±dP/dtmax, and heart rate between SphK2 null (KO) and wild-type (WT) hearts. In KO hearts, SphK2 activity was undetectable, and SphK1 activity was unchanged compared to WT. Total SphK activity was reduced by 53. SphK2 KO hearts subjected to IR exhibited significantly more cardiac damage (37±1 infarct size) compared with WT (28±1 infarct size); postischemic recovery of LVDP was lower in KO hearts. IPC exerted cardioprotection in WT hearts. The protective effect of IPC against IR was diminished in KO hearts which had much higher infarction sizes (35±2 ) compared to the IPC/IR group in control hearts (12±1 ). Western analysis revealed that KO hearts had substantial levels of phosphorylated p38 which could predispose the heart to IR injury. Thus, deletion of the SphK2 gene sensitizes the myocardium to IR injury and diminishes the protective effect of IPC. Copyright © 2011 Donald A. Vessey et al.

Vessey D.A.,Liver Study Unit | Vessey D.A.,University of California at San Francisco | Li L.,Liver Study Unit | Kelley M.,Liver Study Unit
Molecular and Cellular Biochemistry | Year: 2011

Protection of the ex vivo rat heart from ischemia/reperfusion injury can be provided by ischemic preconditioning (IPC). Previous studies revealed that a complex of pannexin-1 with the P2X7 receptor forms a channel during IPC that results in the release of cardioprotectants such as adenosine and sphingosine 1-phosphate (S1P) that bind to G-protein-coupled cell surface receptors triggering cardioprotective cell signaling pathways. Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X7 receptors (brilliant blue G) are known to block IPC when administered at the time of preconditioning (Vessey et al. J Cardiovasc Pharmacol Ther 15:190, 2010). We now demonstrate that these same antagonists also block the cardioprotective effects of IPC when added after the index ischemia during full reperfusion. Likewise, addition at full reperfusion of binding antagonists to the endogenous cardioprotectants S1P (VPC) or adenosine (8-SPT) reduced the effectiveness of IPC. These data suggest that IPC has a component that requires the release of cardioprotectants via pannexin-1/P2X7 channels not only during preconditioning phase but again during the early stages of reperfusion following the index ischemia. It was found that the level of cardioprotectant release required at reperfusion to achieve cardioprotection was lower when hearts had been preconditioned. Further, pharmacologic preconditioning with S1P or adenosine was also blocked at reperfusion by antagonists of the pannexin-1/P2X7 channels indicating that pharmacologic preconditioning also requires opening of the channel at full reperfusion. In untreated hearts, key components of the PI3 kinase/Akt signaling pathway were revealed by western analysis to be lost during ischemia. This correlates with an inability to generate phospho-Akt at reperfusion. IPC prevents this loss and thereby primes the cell for response to cardioprotectants released at full reperfusion. © 2011 Springer Science+Business Media, LLC.

Waly M.I.,Sultan Qaboos University | Kharbanda K.K.,Liver Study Unit | Deth R.C.,Northeastern University
Alcoholism: Clinical and Experimental Research | Year: 2011

Background: Methionine synthase (MS) is a ubiquitous enzyme that requires vitamin B12 (cobalamin) and 5-methyl-tetrahydrofolate for the methylation of homocysteine to methionine. Previous studies have shown that acute or chronic ethanol (ETOH) administration results in the inhibition of MS and depletion of glutathione (GSH), and it has been proposed that GSH is required for the synthesis of methylcobalamin (MeCbl). Methods: We measured GSH levels and investigated the ability of different cobalamin cofactors [cyano- (CNCbl), glutathionyl- (GSCbl), hydroxo- (OHCbl), and MeCbl] to support MS activity in liver and brain cortex from control and ETOH-treated rats. Results: In control animals, MS activity was higher in liver than in cortex for all cobalamins and MeCbl-based activity was higher than for other cofactors. S-adenosylmethionine (SAM) was required for OHCbl, CNCbl, and GSCbl-based activity, but not for MeCbl. Feeding an ETOH-containing diet for four weeks caused a significant decrease in liver MS activity, in a cobalamin-dependent manner (OHCbl≥CNCbl>GSCbl>MeCbl). In brain cortex, OHCbl, CNCbl, and GSCbl-based activity was reduced by ETOH treatment, but MeCbl-based activity was unaffected. GSH levels were reduced by ETOH treatment in both liver and cortex homogenates, and addition of GSH restored OHCbl-based MS activity to control levels. Betaine administration had no significant effect on GSH levels or MS activity in either control or ETOH-fed groups. Conclusions: The ETOH-induced decrease in OHCbl-based MS activity is secondary to decreased GSH levels and a decreased ability to synthesize MeCbl. The ability of MeCbl to completely offset ETOH inhibition in brain cortex, but not liver, suggests tissue-specific differences in the GSH-dependent regulation of MS activity. © 2010 by the Research Society on Alcoholism.

Rasineni K.,University of Nebraska Medical Center | Casey C.A.,University of Nebraska Medical Center | Casey C.A.,Liver Study Unit
Indian Journal of Pharmacology | Year: 2012

Ethanol abuse and chronic ethanol consumption remains a major public health problem and is responsible for a high rate of morbidity. Alcohol-induced fatty liver generally begins as hepatic steatosis, and if the cause persists, this invariably progresses to steatohepatitis and cirrhosis. The original biochemical explanation for an alcoholic fatty liver centered on the ability of ethanol metabolism to shift the redox state of the liver and inhibit fatty acid oxidation. Subsequent studies found repression of fatty acid oxidation and that the induction of lipogenesis can occur in alcoholic conditions. Ethanol activates sterol regulatory element binding protein 1, inducing a battery of lipogenic enzymes. These effects may be due in part to inhibition of AMP-dependent protein kinase, reduction in plasma adiponectin or increased levels of TNF- the liver. They in turn activate lipogenic pathways and inhibit fatty acid oxidation. Besides the fatty acid synthesis and oxidation, ethanol also alters lipid droplet (LD, the storage form of triglycerides, TG) metabolism in hepatocytes and very low-density lipoprotein (VLDL) secretion from liver. Because steatosis is now regarded as a significant risk factor for advanced liver pathology, an understanding of the molecular mechanisms in its etiology provides new therapeutic targets to reverse the alcoholic fatty liver.

Thomes P.G.,Liver Study Unit | Thomes P.G.,University of Nebraska Medical Center | Ehlers R.A.,Liver Study Unit | Ehlers R.A.,University of Nebraska Medical Center | And 9 more authors.
Autophagy | Year: 2013

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanolmetabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-I (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH+/CYP2E1+) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes. © 2013 Landes Bioscience.

We investigated the hypothesis that postconditioning by FTY720 (FTY) in isolated perfused mouse hearts is independent of the sphingosine 1-phosphate (S1P) pathway. Ex vivo hearts were exposed to postconditioning (POST) by either ischemia or FTY720. Protection against ischemia/reperfusion (IR) injury was measured by recovery of left ventricular developed pressure (LVDP) and infarct size. FTY effectively postconditioned (POST) ex vivo hearts against ischemia/reperfusion (IR) injury as measured by recovery of LVDP and a low infarct size. FTY protection, unlike S1P but like sphingosine (Sph), was insensitive to inhibition of S1P G-Protein Coupled Receptors (GPCRs) or inhibition of PI3 kinase. Protection by FTY and Sph was however blocked by inhibitors of PKA and PKG. Thus, FTY follows the same cardioprotective pathway as Sph. This was further supported by studies of FTY POST in knockout (KO) mice lacking the SphK2 form of Sph kinase that is needed for phosphorylation of FTY to an S1P analog. In the absence of SphK2, FTY (and Sph) POST was still cardioprotective. This differed from the effect of SphK2 KO on protection by ischemic POST (IPOST). IPOST was not effective in KO hearts. To see if the GPCR signaling pathway to protection is normal in KO hearts, we looked at POST by GPCR agonists S1P and adenosine. Both provided effective protection even in KO hearts suggesting that the problem with IPOST in KO hearts is a low level of S1P available for release during IPOST. Thus, pharmacologic POST with FTY or Sph, like adenosine and S1P, is unaffected in the KO. FTY720 administered in vivo might behave in a dual manner showing both S1P-like effects and sphingosine-like effects. It appears that the latter may have been overlooked and may be the more important in aging hearts.

PubMed | Liver Study Unit
Type: | Journal: Current molecular pharmacology | Year: 2015

Here, we describe research on the involvement of the transcription factor, Early Growth Response-1 (Egr-1) in alcohol-induced liver injury, specifically, fatty liver (steatosis), one of the earliest and most frequent signs of liver injury that occurs after heavy drinking. Egr-1 is a ubiquitous transcription factor found in nearly all cell types. However, because the liver is the principal site of ethanol oxidation, it sustains the greatest damage from alcohol abuse. Thus, this review focuses on how alcohol consumption causes changes in the hepatic expression of Egr-1, which, in turn causes downstream alterations in the expression of other genes to cause liver pathology. Ironically, while such changes in Egr-1 expression clearly favor steatosis and even fibrosis development, the absence of Egr-1 expression can actually exacerbate liver injury after excessive alcohol consumption or after exposure to other hepatotoxins. The existing literature on Egr-1 is extensive. Here, we confine our initial description of Egr-1 to its principal molecular characteristics, its biological functions, and its involvement in certain pathologies that are either directly or obliquely related to alcoholic liver disease. We describe experimental data that clearly implicate Egr-1 function in alcohol-induced steatosis and fibrosis, showing that ethanol-elicited regulation of Egr-1 expression depends on the generation of acetaldehyde and that the absence of Egr-1 diminishes alcohol-induced triglyceride accumulation. Overall, the existing evidence for the involvement of Egr-1 as a key link in alcohol-induced liver disease is strong. The evidence underscores the potential role of Egr-1 and several other transcription factors as therapeutic targets in the alleviation of alcoholic liver disease, which, even after decades of treatment options, still remains difficult to manage in the clinic.

PubMed | University of Nebraska Medical Center and Liver Study Unit
Type: Journal Article | Journal: Alcoholism, clinical and experimental research | Year: 2016

Chronic ethanol (EtOH) consumption decelerates the catabolism of long-lived proteins, indicating that it slows hepatic macroautophagy (hereafter called autophagy) a crucial lysosomal catabolic pathway in most eukaryotic cells. Autophagy and lysosome biogenesis are linked. Both are regulated by the transcription factor EB (TFEB). Here, we tested whether TFEB can be used as a singular indicator of autophagic activity, by quantifying its nuclear content in livers of mice subjected to acute and chronic EtOH administration. We correlated nuclear TFEB to specific indices of autophagy.In acute experiments, we gavaged GFP-LC3(tg) mice with a single dose of EtOH or with phosphate buffered saline (PBS). We fed mice chronically by feeding them control or EtOH liquid diets.Compared with PBS-gavaged controls, livers of EtOH-gavaged mice exhibited greater autophagosome (AV) numbers, a higher incidence of AV-lysosome co-localization, and elevated levels of free GFP, all indicating enhanced autophagy, which correlated with a higher nuclear content of TFEB. Compared with pair-fed controls, livers of EtOH-fed mice exhibited higher AV numbers, but had lower lysosome numbers, lower AV-lysosome co-localization, higher P62/SQSTM1 levels, and lower free GFP levels. The latter findings correlated with lower nuclear TFEB levels in EtOH-fed mice. Thus, enhanced autophagy after acute EtOH gavage correlated with a higher nuclear TFEB content. Conversely, chronic EtOH feeding inhibited hepatic autophagy, associated with a lower nuclear TFEB content.Our findings suggest that the effect of acute EtOH gavage on hepatic autophagy differs significantly from that after chronic EtOH feeding. Each regimen distinctly affects TFEB localization, which in turn, regulates hepatic autophagy and lysosome biogenesis.

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