Inhibition of polyploidization in Pten-deficient livers reduces steatosis.

The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver-specific deletion of Pten in mice results in the hyper-activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver-specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten-induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten-deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR-gamma signalling. However, we find no evidence that deregulated genes in Pten-deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten-deficient livers.

Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system.

BACKGROUND: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver. OBJECTIVE: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system. ANIMALS: Liver organoids obtained from 6 cats. METHODS: Eight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR. RESULTS: Both T863 (a diacylglycerol O-acyltransferase 1 [DGAT1] inhibitor) and 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P < .0001) and 46% (P = .0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863. CONCLUSIONS AND CLINICAL IMPORTANCE: Two potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid-lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species-specific system and provide the basis for further clinical testing of drugs to treat steatosis.

Depletion of phosphatidylcholine affects endoplasmic reticulum morphology and protein traffic at the Golgi complex.

The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their phosphatidylcholine (PC) level within 24 h when cultured at the nonpermissive temperature (40 degrees C). This is due to a relative rapid breakdown of PC that is not compensated for by the inhibition of de novo PC synthesis. Despite this drastic decrease in cellular PC content, cells are viable and can proliferate by addition of lysophosphatidylcholine. By [(3)H]oleate labeling, we found that the FA moiety of the degraded PC is recovered in triacylglycerol. In accordance with this finding, an accumulation of lipid droplets is seen in MT58 cells. Analysis of PC-depleted MT58 cells by electron and fluorescence microscopy revealed a partial dilation of the rough endoplasmic reticulum, resulting in spherical structures on both sites of the nucleus, whereas the morphology of the plasma membrane, mitochondria, and Golgi complex was unaffected. In contrast to these morphological observations, protein transport from the ER remains intact. Surprisingly, protein transport at the level of the Golgi complex is impaired. Our data suggest that the transport processes at the Golgi complex are regulated by distal changes in lipid metabolism.

Hexadecylphosphocholine causes rapid cell death in canine mammary tumour cells.

Hexadecylphosphocholine (HePC, Miltefosine) is an antitumour phospholipid and known inducer of apoptosis in human breast cancer cells. The mechanism underlying the induction of cell death by HePC, however, is not clear yet. In this study, we have investigated the cytotoxic effects of HePC on canine mammary tumour cells (CMTs) in vitro. Upon addition of HePC, CMTs rapidly exhibited several features that resembled apoptotic cell death. Cells showed externalization of phosphatidylserine, a hallmark of apoptosis, within 5 min after addition of HePC at concentrations as low as 10 microM. Furthermore, rapid swelling of mitochondria was observed. Rounding and detachment of cells followed within 30 min. However, fragmentation of nuclear DNA could not be observed. Overall, HePC was shown to induce a type of cell death in CMTs that in some aspects resembles apoptosis, though the process proceeds much more rapidly than reported for other tumour cell lines.

Inhibition of phosphatidylcholine synthesis is not the primary pathway in hexadecylphosphocholine-induced apoptosis.

The anticancer drug hexadecylphosphocholine (HePC), an alkyl-lysophospholipid analog (ALP), has been shown to induce apoptosis and inhibit the synthesis of phosphatidylcholine (PC) in a number of cell lines. We investigated whether inhibition of PC synthesis plays a major causative role in the induction of apoptosis by HePC. We therefore directly compared the apoptosis caused by HePC in CHO cells to the apoptotic process in CHO-MT58 cells, which contain a genetic defect in PC synthesis. HePC-provoked apoptosis was found to differ substantially from the apoptosis observed in MT58 cells, since it was (i) not accompanied by a large decrease in the amount of PC and diacylglycerol (DAG), (ii) not preceded by induction of the pro-apoptotic protein GADD153/CHOP, and (iii) not dependent on the synthesis of new proteins. Furthermore, lysoPC as well as lysophosphatidylethanolamine (lysoPE) could antagonize the apoptosis induced by HePC, whereas only lysoPC was able to rescue MT58 cells. HePC also induced a rapid externalisation of phosphatidylserine (PS). These observations suggest that inhibition of PC synthesis is not the primary pathway in HePC-induced apoptosis.

Control of the CDPethanolamine pathway in mammalian cells: effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the amount of intracellular diacylglycerol.

For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinese-hamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26- and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [3H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with control-transfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in control-transfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.

Inhibition of phosphatidylcholine synthesis induces expression of the endoplasmic reticulum stress and apoptosis-related protein CCAAT/enhancer-binding protein-homologous protein (CHOP/GADD153).

Inhibition of de novo synthesis of phosphatidylcholine (PC) by some anti-cancer drugs such as hexadecylphosphocholine leads to apoptosis in various cell lines. Likewise, in MT58, a mutant Chinese hamster ovary (CHO) cell line containing a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), an important regulatory enzyme in the CDP-choline pathway, inhibition of PC synthesis causes PC depletion. Cellular perturbations like metabolic insults and unfolded proteins can be registered by the endoplasmic reticulum (ER) and result in ER stress responses, which can lead eventually to apoptosis. In this study we investigated the effect of PC depletion on the ER stress response and ER-related proteins. Shifting MT58 cells to the non-permissive temperature of 40 degrees C resulted in PC depletion via an inhibition of CT within 24 h. Early apoptotic features appeared in several cells around 30 h, and most cells were apoptotic within 48 h. The temperature shift in MT58 led to an increase of pro-apoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP; also known as GADD153) after 16 h, to a maximum at 24 h. Incubation of wild-type CHO-K1 or CT-expressing MT58 cells at 40 degrees C did not induce differences in CHOP protein levels in time. In contrast, expression of the ER chaperone BiP/GRP78, induced by an increase in misfolded/unfolded proteins, and caspase 12, a protease specifically involved in apoptosis that results from stress in the ER, did not differ between MT58 and CHO-K1 cells in time when cultured at 40 degrees C. Furthermore, heat-shock protein 70, a protein that is stimulated by accumulation of abnormal proteins and heat stress, displayed similar expression patterns in MT58 and K1 cells. These results suggest that PC depletion in MT58 induces the ER-stress-related protein CHOP, without raising a general ER stress response.

Phosphatidylcholine and cell death.

Phosphatidylcholine (PC) constitutes a major portion of cellular phospholipids and displays unique molecular species in different cell types and tissues. Inhibition of the CDP-choline pathway in most mammalian cells or overexpression of the hepatic phosphatidylethanolamine methylation pathway in hepatocytes leads to perturbation of PC homeostasis, growth arrest or even cell death. Although many agents that perturb PC homeostasis and induce cell death have been identified, the signaling pathways that mediate this cell death have not been well defined. This review summarizes recent progress in understanding the relationship between PC homeostasis and cell death.