Patients with nonalcoholic fatty liver disease (NAFLD) are reported to have low growth hormone (GH) production and/or hepatic GH resistance. GH replacement can resolve the fatty liver condition in diet-induced obese rodents and in GH-deficient patients. However, it remains to be determined whether this inhibitory action of GH is due to direct regulation of hepatic lipid metabolism. Therefore, an adult-onset, hepatocyte-specific, GH receptor (GHR) knockdown (aLivGHRkd) mouse was developed to model hepatic GH resistance in humans that may occur after sexual maturation. Just 7 days after aLivGHRkd, hepatic de novo lipogenesis (DNL) was increased in male and female chow-fed mice, compared with GHR-intact littermate controls. However, hepatosteatosis developed only in male and ovariectomized female aLivGHRkd mice. The increase in DNL observed in aLivGHRkd mice was not associated with hyperactivation of the pathway by which insulin is classically considered to regulate DNL. However, glucokinase mRNA and protein levels as well as fructose-2,6-bisphosphate levels were increased in aLivGHRkd mice, suggesting that enhanced glycolysis drives DNL in the GH-resistant liver. These results demonstrate that hepatic GH actions normally serve to inhibit DNL, where loss of this inhibitory signal may explain, in part, the inappropriate increase in hepatic DNL observed in NAFLD patients.
Peroxisome proliferator-activated receptor γ (PPARγ) is the target for thiazolidinones (TZDs), drugs that improve insulin sensitivity and fatty liver in humans and rodent models, related to a reduction in hepatic de novo lipogenesis (DNL). The systemic effects of TZDs are in contrast to reports suggesting hepatocyte-specific activation of PPARγ promotes DNL, triacylglycerol (TAG) uptake and fatty acid (FA) esterification. Since these hepatocyte-specific effects of PPARγ could counterbalance the positive therapeutic actions of systemic delivery of TZDs, the current study used a mouse model of adult-onset, liver (hepatocyte)-specific PPARγ knockdown (aLivPPARγkd). This model has advantages over existing congenital knockout models, by avoiding compensatory changes related to embryonic knockdown, thus better modeling the impact of altering PPARγ on adult physiology, where metabolic diseases most frequently develop. The impact of aLivPPARγkd on hepatic gene expression and endpoints in lipid metabolism was examined after 1 or 18wks (Chow-fed) or after 14wks of low- or high-fat [HF] diet. aLivPPARγkd reduced hepatic TAG content but did not impact endpoints in DNL or TAG uptake. However, aLivPPARγkd reduced the expression of the FA translocase (Cd36), in 18wk-Chow and HF-fed mice, associated with increased NEFA after HF-feeding. Also, aLivPPARγkd dramatically reduced Mogat1 expression, that was reflected by an increase in hepatic monoacylglycerol (MAG) levels, indicative of reduced MOGAT activity. These results, coupled with previous reports, suggest that Cd36-mediated FA uptake and MAG pathway-mediated FA esterification are major targets of hepatocyte PPARγ, where loss of this control explains in part the protection against steatosis observed after aLivPPARγkd.
Our group has previously reported de novo lipogenesis (DNL) and hepatic triglyceride content increases in chow-fed male mice within 7 days of hepatocyte-specific GH receptor knockdown (aLivGHRkd). Here, we report that these changes are associated with an increase in hepatic expression of peroxisome proliferator-activated receptor γ (PPARγ), consistent with previous reports showing steatosis is associated with an increase in PPARγ expression in mice with congenital loss of hepatic GH signaling. PPARγ is thought to be an important driver of steatosis by enhancing DNL, as well as increasing the uptake and esterification of extrahepatic fatty acids (FAs). In order to determine whether hepatic PPARγ is critical for the rapid development of steatosis in the aLivGHRkd mouse model, we have generated aLivGHRkd mice, with or without PPARγ (ie, adult-onset, hepatocyte-specific double knockout of GHR and PPARγ). Hepatic PPARγ was not required for the rapid increase in liver triglyceride content or FA indexes of DNL (16:0/18:2 and 16:1/16:0). However, loss of hepatic PPARγ blunted the rise in fatty acid translocase/CD36 and monoacylglycerol acyltransferase 1 expression induced by aLivGHRkd, and this was associated with a reduction in the hepatic content of 18:2. These results suggest that the major role of PPARγ is to enhance pathways critical in uptake and reesterification of extrahepatic FA. Because FAs have been reported to directly increase PPARγ expression, we speculate that in the aLivGHRkd mouse, the FA produced by DNL enhances the expression of PPARγ, which in turn increases extrahepatic FA uptake, thereby further enhancing PPARγ activity and exacerbating steatosis overtime.
Drosophila DIM-7 (encoded by the moleskin gene, msk) is the orthologue of vertebrate Importin-7. Both Importin-7 and Msk/DIM-7 function as nuclear import cofactors, and have been implicated in the control of multiple signal transduction pathways, including the direct nuclear import of the activated (phosphorylated) form of MAP kinase. We performed two genetic deficiency screens to identify deficiencies that similarly modified Msk overexpression phenotypes in both eyes and wings. We identified 11 total deficiencies, one of which removes the Delta locus. In this report, we show that Delta loss-of-function alleles dominantly suppress Msk gain-of-function phenotypes in the developing wing. We find that Msk overexpression increases both Delta protein expression and Delta transcription, though Msk expression alone is not sufficient to activate Delta protein function. We also find that Msk overexpression increases Egfr protein levels, and that msk gene function is required for proper Egfr expression in both developing wings and eyes. These results indicate a novel function for Msk in Egfr expression. We discuss the implications of these data with respect to the integration of Egfr and Delta/Notch signaling, specifically through the control of MAP kinase subcellular localization.
Proper activation of the Ras/MAPK pathway is broadly required during development, and in many cases, signal transduction downstream of the receptor is linear. Thus, different mechanisms exist to properly regulate the large number of specific developmental outputs that are required by the activation of this pathway. Previously, we have reported a regulated cytoplasmic sequestration of phosphorylated MAPK (pMAPK) in developing Drosophila compound eyes and wings “called MAPK Cytoplasmic Hold”. In the developing wing, we have shown that cytoplasmic hold promotes the differentiation of wing vein tissue, while pMAPK nuclear translocation regulates growth and division. We had also suggested that the Ras pathway signals for inducing cell growth and cell division split upstream of the nuclear translocation of MAPK itself. Here, we further refine the role of MAPK in Drosophila. We report evidence that suggests, for the first time, that the phosphorylation of MAPK is itself another step in the regulation of cell growth and division in both Drosophila wing and eye cells. We show that inhibition of MAPK phosphorylation, or pMAPK nuclear translocation, is sufficient to block cell growth, but not cell division. These data suggest that non-phosphorylated MAPK is sufficient to induce cell division, but not cell growth, once inside the nucleus of the cell.
No abstract
Development of multidrug resistance is a major impediment to the successful treatment of cancer. Although many cellular mechanisms are involved in multidrug resistant cancers, overexpression of ATP-binding cassette (ABC) membrane transporters represents one of the main determinants of the multidrug resistance phenotype. We have previously demonstrated a positive correlation of expression of intracellular Notch1 (NIC) and the ABCC1 transporter; we found that NIC contributes to multidrug resistance by upregulation of ABCC1 (S Cho, M Lu et al, PNAS, in press, 2011). In order to determine whether there is a functional relationship between the two proteins, we performed drug efflux experiments. We treated drug-sensitive MCF-7 and multidrug resistant (MDR) MCF-7/VP breast cancer cells with the anticancer drug doxorubicin and measured the amount of drug efflux by flow cytometry over time. The MDR MCF-7/VP cells, which have high levels of activated Notch1 and ABCC1 proteins, exhibited increased doxorubicin efflux, compared to the drug-sensitive MCF-7 cells. Since γ-secretase (GS) is required for the processing of Notch into its active form, NIC, we blocked GS activity using a GS inhibitor, DAPT, in MCF-7/VP cells and assayed drug efflux from them. We found that inhibition of Notch signaling by DAPT blocks ABCC1 mediated drug efflux. To better understand the connection between Notch, ABCC1, and drug efflux, we compared drug efflux in MCF-7 cells stably overexpressing NIC (ICN1-c4, which also overexpress ABCC1), and the “empty vector” control MCF-7 cells (pcDNA3). As expected, we saw increased drug efflux in the ICN1-c4 cells compared to MCF7/pcDNA3 cells. To establish whether the observed drug efflux is ABCC1-specific, we treated ICN1-c4 cells with either the ABCC1-specific inhibitors reversan or probenecid, or with an ABCC1 siRNA to knockdown ABCC1 expression, and then examined these cells for changes in drug efflux. We found that inhibition of ABCC1, by reducing ABCC1 activity or levels in the presence of activated Notch1, blocks drug efflux from ICN1-c4 cells. In addition, we also found that the ABCC1 inhibitor reversan sensitizes ICN1-c4 cells to doxorubicin, and knockdown of ABCC1 reverses the resistance of ICN1-c4 cells to doxorubicin. These results strongly support the idea that Notch1 exerts its effect on ABCC1-mediated drug efflux to a large extent through its induction of ABCC1. Overall, our results reveal a novel functional relationship between expression of Notch and ABCC1. (Supported in part by CA40570 [to WTB] and in part by UIC.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 772. doi:1538-7445.AM2012-772
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