Hepatocyte-Specific Deletion of TIPARP, a Negative Regulator of the Aryl Hydrocarbon Receptor, Is Sufficient to Increase Sensitivity to Dioxin-Induced Wasting Syndrome.

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparpfl/fl mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparpfl/flCreAlb) and whole-body (Tiparpfl/flCreCMV; TiparpEx3-/-) Tiparp null mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice given a single injection of 10 µg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp+/+ mice survived the 30-day treatment. Dioxin-exposed Tiparpfl/flCreAlb and TiparpEx3-/- mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparpfl/flCreAlb mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.

The nuclear phosphatase SCP4 regulates FoxO transcription factors during muscle wasting in chronic kidney disease.

Chronic kidney disease (CKD) and related inflammatory responses stimulate protein-energy wasting, a complication causing loss of muscle mass. Primarily, muscle wasting results from accelerated protein degradation via autophagic/lysosomal and proteasomal pathways, but mechanisms regulating these proteolysis pathways remain unclear. Since dephosphorylation of FoxOs regulates ubiquitin/proteasome protein metabolism, we tested whether a novel nuclear phosphatase, the small C-terminal domain phosphatase (SCP) 4, regulates FoxOs signaling and, in turn, muscle wasting. In cultured mouse myoblast cells, SCP4 overexpression stimulated proteolysis, while knockdown of SCP4 prevented the proteolysis stimulated by inflammatory cytokines. SCP4 overexpression led to nuclear accumulation of FoxO1/3a followed by increased expression of catabolic factors including myostatin, Atrogin-1, and MuRF-1, and induction of lysosomal-mediated proteolysis. Treatment of C2C12 myotubes with proinflammatory cytokines stimulated SCP4 expression in an NF-κB-dependent manner. In skeletal muscle of mice with CKD, SCP4 expression was up-regulated. Similarly, in skeletal muscle of patients with CKD, SCP4 expression was significantly increased. Knockdown of SCP4 significantly suppressed FoxO1/3a-mediated expression of Atrogin-1 and MuRF-1 and prevented muscle wasting in mice with CKD. Thus, SCP4 is a novel regulator of FoxO transcription factors and promotes cellular proteolysis. Hence, targeting SCP4 may prevent muscle wasting in CKD and possibly other catabolic conditions.

Serum matrix metalloproteinase 9 (MMP9) as a biochemical marker for wasting marmoset syndrome.

Use of the common marmoset (Callithrix jacchus) as a non-human primate experimental animal has increased in recent years. Although wasting marmoset syndrome (WMS) is one of the biggest problems in captive marmoset colonies, the molecular mechanisms, biochemical markers for accurate diagnosis and a reliable treatment remain unknown. In this study, as a first step to finding biochemical marker(s) for the accurate diagnosis of WMS, we conducted blood cell counts, including hematocrit, hemoglobin and platelets, and examined serum chemistry values, including albumin, calcium and levels of serum matrix metalloproteinase 9 (MMP9), using a colony of marmosets with and without weight loss. MMP9 is thought to be an enzyme responsible for the degradation of extracellular matrix components and participates in the pathogenesis of inflammatory conditions, such as human and murine inflammatory bowel disease, which, like WMS, are characterized histologically by inflammatory cell infiltrations in the intestines. The values of hematocrit and hemoglobin and levels of serum albumin and calcium in the WMS group were significantly decreased versus the control group. The platelet values and serum MMP9 concentrations were increased significantly in the WMS group compared with the control group. MMP9 could be a new and useful marker for the diagnosis of WMS in addition to hematocrit, hemoglobin, serum albumin and calcium. Our results also indicate that MMP9 could be a useful molecular candidate for treatment.

Gene network of a phosphoglycerate mutase in muscle wasting in mice.

We previously identified the insertion of an intracisternal A-particle retrotransposons (IAPs) sequence in a gene, 9630033F20Rik, that contains domains involved in glycolysis from a mouse model called lethal wasting (lew). However, because both IAP insertion and the muation of vesicle-associated membrane protein 1 (VAMP1) were discovered from lew, the impact of the IAP insertion and Vamp1 on the lew mouse phenotype needs further investigation. In this study, the effect of the 9630033F20Rik and Vamp1 on glycolysis and muscle-wasting genes in heart, muscle, and brain tissues was further investigated using data of gene expression profiles in these tissues. Our data indicated that the expression levels of 9630033F20Rik and Vamp1 are not associated with each other. While 9630033F20Rik affects the expression of several key genes in pathways of glycolysis and muscle wasting, Vamp1 affects a different set of genes, with fewer numbers. In situ hybridization indicated that the expression of 9630033F20Rik is different in musculoskeletal tissues between the muscle-wasting mouse model and the wild-type model. Our data indicated that 9630033F20Rik may play an important role in muscle wasting and that it has a distinguished characterization of gene network. Our data also suggest that both 9630033F20Rik and Vamp1 play functional roles in muscle development and lead to the muscle-wasting phenotype when they are mutated.

Increased extracellular adenosine in Drosophila that are deficient in adenosine deaminase activates a release of energy stores leading to wasting and death.

Extracellular adenosine is an important signaling molecule in neuromodulation, immunomodulation and hypoxia. Adenosine dysregulation can cause various pathologies, exemplified by a deficiency in adenosine deaminase in severe combined immunodeficiency. We have established a Drosophila model to study the effects of increased adenosine in vivo by mutating the main Drosophila adenosine deaminase-related growth factor (ADGF-A). Using a genetic screen, we show here that the increased extracellular adenosine in the adgf-a mutant is associated with hyperglycemia and impairment in energy storage. The adenosine works in this regard through the adenosine receptor as an anti-insulin hormone in parallel to adipokinetic hormone, a glucagon counterpart in flies. If not regulated properly, this action can lead to a loss of energy reserves (wasting) and death of the organism. Because adenosine signaling is associated with the immune response and the response to stress in general, our results mark extracellular adenosine as a good candidate signal involved in the wasting syndrome that accompanies various human pathologies.

Patterned neuroprotection in the Inpp4a(wbl) mutant mouse cerebellum correlates with the expression of Eaat4.

The weeble mutant mouse has a frame shift mutation in inositol polyphosphate 4-phosphatase type I (Inpp4a). The phenotype is characterized by an early onset cerebellar ataxia and neurodegeneration, especially apparent in the Purkinje cells. Purkinje cell loss is a common pathological finding in many human and mouse ataxic disorders. Here we show that in the Inpp4a(wbl) mutant, Purkinje cells are lost in a specific temporal and spatial pattern. Loss occurs early in postnatal development; however, prior to the appearance of climbing fibers in the developing molecular layer, the mutant has a normal complement of Purkinje cells and they are properly positioned. Degeneration and reactive gliosis are present at postnatal day 5 and progress rapidly in a defined pattern of patches; however, Inpp4a is expressed uniformly across Purkinje cells. In late stage mutants, patches of surviving Purkinje cells appear remarkably normal with the exception that the climbing fibers have been excessively eliminated. Surviving Purkinje cells express Eaat4, a glutamate transporter that is differentially expressed in subsets of Purkinje cells during development and into adult stages. Prior to Purkinje cell loss, reactive gliosis and dendritic atrophy can be seen in Eaat4 negative stripes. Our data suggest that Purkinje cell loss in the Inpp4a(wbl) mutant is due to glutamate excitotoxicity initiated by the climbing fiber, and that Eaat4 may exert a protective effect.

NF-kappa B-mediated MyoD decay during muscle wasting requires nitric oxide synthase mRNA stabilization, HuR protein, and nitric oxide release.

Muscle wasting (cachexia) is a consequence of chronic diseases, such as cancer, and is associated with degradation of muscle proteins such as MyoD. The cytokines tumor necrosis factor alpha and gamma interferon induce muscle degeneration by activating the transcription factor NF-kappaB and its target genes. Here, we show that a downstream target of NF-kappaB is the nitric oxide (NO) synthase gene (iNos) and suggest that NO production stimulates MyoD mRNA loss. In fact, although cytokine treatment of iNos(-/-) mice activated NF-kappaB, it did not trigger MyoD mRNA degeneration, demonstrating that NF-kappaB-mediated muscle wasting requires an active iNOS-NO pathway. The induced expression of iNOS by cytokines relies on both transcriptional activation via NF-kappaB and increased mRNA stability via the RNA-binding protein HuR. Moreover, we show that HuR regulates iNOS expression in an AMP-activated protein kinase (AMPK)-dependent manner. Furthermore, AMPK activation results in HuR nuclear sequestration, inhibition of iNOS synthesis, and reduction in cytokine-induced MyoD loss. These results define iNOS and HuR as critical players in cytokine-induced cachexia, establishing them as potential therapeutic targets.

Potential markers of the nutritional status in an experimental model.

The activity of adenine deaminase (ADA) and purine nucleoside phosphorylase (PNP) as potential nutritional markers was analyzed in an experimental model. Weanling Wistar rats were fed a protein-free diet ad libitum to obtain a severe degree of wasting. An age-matched control group received a stock diet. At the end of the experiment, body weight (BW) and thymus weight (TW) were determined. Activity of ADA and PNP was determined on thymocytes of protein-deprived and control rats; the results, expressed as micromoles of uric acid x 10(-1)/W (W = TW/BW(0.75)), were 17.0 +/- 2.6 versus 9.1 +/- 3.0 for ADA and 11.5 +/- 4.2 versus 3.9 +/- 1.0 for PNP (P < 0.01). These results suggest that the nutritional stress provoked by the administration of a protein-free diet from weaning onward affects the development of thymocytes. Moreover, the increase in the activity of ADA and PNP would be an alternative mechanism to avoid the accumulation of high levels of deoxynucleotides, which would be toxic for T lymphocytes. However, some investigators have observed an increase of ADA activity in human serum under some adverse conditions; for this reason and taking into account the present findings, it would be interesting to determine the relation between the activity of ADA and PNP in thymocytes and serum in experimental models to analyze and propose these biochemical parameters as potential and useful markers of nutritional status; it also would be interesting to test this relation in human studies.