Hyperosmolarity is a controversial signal for renal cells. It can induce cell stress or differentiation and both require an active lipid metabolism. We showed that hyperosmolarity upregulates phospholipid (PL) de novo synthesis in renal cells. PL synthesis requires fatty acids (FA), usually stored as triglycerides (TAG). PL and TAG de novo synthesis utilize the same initial biosynthetic route: sn-glycerol 3P (G3P) → phosphatidic acid (PA) → diacylglycerol (DAG). In the present work, we evaluate how such pathway contributes to PL and TAG synthesis in renal cells subjected to hyperosmolarity. Our results show an increase in PA and DAG formation under hyperosmotic conditions; augmented DAG production, due to lipin enzyme activity, lead to the increase of both TAG and PL. However, at early stages (24 and 48 h), most of the de novo synthesized DAG was directed to PL synthesis; longer treatments downregulated PL synthesis and the DAG formed was mainly driven to TAG synthesis. Hyperosmolarity induced ACC and FASN transcription which mediated FA de novo synthesis. New FA molecules were stored in TAG. Silencing experiments revealed that hyperosmotic-induction of lipin-1 and -2 was mediated by SREBP1. Interestingly, SREBP1 knockdown also dropped SREBP2, indicating a modulatory action between both isoforms. Impairing SREBP activity leads to a decline in TAG levels but not PL. Membrane homeostasis is maintained through the adequate PL synthesis and renewal and constitute a protective mechanism against hyperosmolarity. The present data reveal the relevance of TAG synthesis and storage for PL synthesis in renal cells.
Background and aims
Non‐alcoholic fatty liver (NAFLD) and its more serious form non‐alcoholic steatohepatitis increase risk of hepatocellular carcinoma (HCC). Lipid metabolic alterations and its role in HCC development remain unclear. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is involved in lipid metabolism, NAFLD and diabetes, but the effects on hepatic lipid metabolism and HCC development is unknown. The aim of this study was to evaluate the role of SPARC in HCC development in the context of NAFLD.
Methods
Primary hepatocyte cultures from knockout (SPARC−/−) or wild‐type (SPARC+/+) mice, and HepG2 cells were used to assess the effects of free fatty acids on lipid accumulation, expression of lipogenic genes and de novo triglyceride (TG) synthesis. A NAFLD‐HCC model was stabilized on SPARC−/− or SPARC+/+ mice. Correlations among SPARC, lipid metabolism‐related gene expression patterns and clinical prognosis were studied using HCC gene expression dataset.
Results
SPARC−/− mice increases hepatic lipid deposits over time. Hepatocytes from SPARC−/− mice or inhibition of SPARC by an antisense adenovirus in HepG2 cells resulted in increased TG deposit, expression of lipid‐related genes and nuclear translocation of SREBP1c. Human HCC database analysis revealed that SPARC negatively correlated with genes involved in lipid metabolism, and with poor survival. In NAFLD‐HCC murine model, the absence of SPARC accelerates HCC development. RNA‐seq study revealed that pathways related to lipid metabolism, cellular detoxification and proliferation were upregulated in SPARC−/− tumour‐bearing mice.
Conclusions
The absence of SPARC is associated with an altered hepatic lipid metabolism, and an accelerated NAFLD‐related HCC development.
NF-κB and TonEBP belong to the Rel-superfamily of transcription factors. Several specific stimuli, including hypertonicity which is a key factor for renal physiology, are able to activate them. It has been reported that, after hypertonic challenge, NF-κB activity can be modulated by TonEBP, considered as the master regulator of transcriptional activity in the presence of changes in environmental tonicity. In the present work we evaluated whether hypertonicity-induced gene transcription mediated by p65/RelA and TonEBP occurs by an independent action of each transcription factor or by acting together. To do this, we evaluated the expression of their specific target genes and cyclooxygenase-2 (COX-2), a common target of both transcription factors, in the renal epithelial cell line Madin-Darby canine kidney (MDCK) subjected to hypertonic environment. The results herein indicate that hypertonicity activates the Rel-family transcription factors p65/RelA and TonEBP in MDCK cells, and that both are required for hypertonic induction of COX-2 and of their specific target genes. In addition, present data show that p65/RelA modulates TonEBP expression and both colocalize in nuclei of hypertonic cultures of MDCK cells. Thus, a sequential and synchronized action p65/RelA → TonEBP would be necessary for the expression of hypertonicity-induced protective genes.
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