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Lysophosphatidic acids (LPAs) are bioactive radyl hydrocarbon-substituted derivatives of glycerol 3-phosphate. LPA metabolism and signaling are implicated in heritable risk of coronary artery disease. Genetic and pharmacological inhibition of these processes attenuate experimental atherosclerosis. LPA accumulates in atheromas, which may be a consequence of association with LDLs. The source, regulation, and biological activity of LDL-associated LPA are unknown. We examined the effects of experimental hyperlipidemia on the levels and distribution of circulating LPA in mice. The majority of plasma LPA was associated with albumin in plasma from wild-type mice fed normal chow. LDL-associated LPA was increased in plasma from high-fat Western diet-fed mice that are genetically prone to hyperlipidemia (LDL receptor knockout or activated proprotein convertase subtilisin/kexin type 9-overexpressing C57Bl6). Adipose-specific deficiency of the ENPP2 gene encoding the LPA-generating secreted lysophospholipase D, autotaxin (ATX), attenuated these Western diet-dependent increases in LPA. ATX-dependent increases in LDL-associated LPA were observed in isolated incubated plasma. ATX acted directly on LDL-associated lysophospholipid substrates in vitro. LDL from all human subjects examined contained LPA and was decreased by lipid-lowering drug therapies. Human and mouse plasma therefore contains a diet-sensitive LDL-associated LPA pool that might contribute to the cardiovascular disease-promoting effects of LPA.

Studies demonstrate a role for neurotensin (NT) in obesity and related comorbidities. Bile acid (BA) homeostasis alterations are associated with obesity. We determined the effect of NT on BA metabolism in obese and non-obese conditions. Plasma and fecal BA profiles were analyzed by LC-MS/MS in male and female NT(+/+) and NT(−/−) mice fed low-fat (LFD) or high-fat diet (HFD) for 6 weeks (early stage of obesity) or greater than 20 weeks (late stage of obesity). The nuclear farnesoid X receptor (FXR) and BA transporter mRNA expression was assessed in ileum, mouse enteroids and human cell lines. HFD decreased plasma primary and secondary BAs in NT(+/+) mice; HFD-induced decrease of plasma BAs was improved in NT-deficient mice. In NT(+/+) mice, HFD inhibited ileal FXR and BA transporter expression; HFD-decreased expression of FXR and BA transporters was prevented in NT(−/−) mice. Compared with LFD-fed NT(+/+) mice, LFD-fed NT(−/−) mice had relatively lower levels of ileal FXR and BA transporter expression. Moreover, NT stimulates expression of FXR and BA transporters in Caco-2 cells; however, stimulated expression of BA transporters was attenuated in NT(−/−) enteroids. Therefore, we demonstrate that HFD disrupts the BA metabolism and ileal FXR and BA transporter axis which are improved in the absence of NT, suggesting that NT contributes to HFD-induced disruption of BA metabolism and plays an inhibitory role in the regulation of ileal FXR and BA transporter signaling under obese conditions. Conversely, NT positively regulates the expression of ileal FXR and BA transporters under non-obese conditions. Therefore, NT plays a dual role in obese and non-obese conditions, suggesting possible therapeutic strategies for obesity control.

We previously reported that high levels of plasma neurotensin (NT), a gut hormone released from enteroendocrine cells of the small bowel, contribute to obesity and comorbid conditions. Gut microbiota has been implicated in the obesity development. Paneth cells are critical in maintaining gut microbiota composition and homeostasis by releasing antimicrobial proteins including α-defensins. The purpose of our current study was to determine the possible role of NT in gut microbiota composition and α-defensin gene expression associated with obesity. Here we show that the ratio of Firmicutes/Bacteroidetes (F/B ratio) and intestinal proinflammatory cytokines is significantly increased in NT+/+ mice fed with a high-fat diet (HFD) which were improved in NT-deficient mice. HFD disrupted the intestinal Mmp7/α-defensin axis, which was completely prevented in NT-/- mice. In addition, NT treatment inhibited DEFA5 expression and concurrent NF-κB activity, which was blocked by a pan PKC inhibitor (Go6983) or an inhibitor for atypical PKCs (CRT0066854). More importantly, the shRNA-mediated knockdown of atypical PKCτ reversed NT-attenuated DEFA5 expression and increased NF-κB activity. NT contributes to the HFD-induced disruption of gut microbiota composition and α-defensin expression. PKCτ/λ plays a central role in NT-mediated α-defensin gene expression which might be mediated through the inhibition of NF-κB signaling pathways in Paneth cells.

BackgroundCancer cells are characterized by aberrant phosphatidylcholine (PC) metabolism. PC can be synthesized de novo or absorbed from diet, after digestion, by the intestinal enterocytes. Here, we investigated the association of dietary intake of PC and breast cancer development in mice. MethodsWe used tandem mass spectrometry methods to quantitate PC content of various fat sources used to manufacture rodent diets. Rodent diets were then formulated with either casein or amino acids in place of casein. To test the effects of dietary PC on tumor growth we fed low density lipoprotein receptor-null (LDLR–/–) mice high fat diets formulated with casein (high PC) or amino acids in place of casein (low PC). Endogenous PC biosynthesis and levels of total circulated plasma PC was monitored using stable isotope tracer choline and mass spectrometry analysis. Tumors were induced in mice after 12 weeks of high fat diet feeding. Since PC-derived molecules are important transducers of mitogenic signals, we tested the effects of inhibiting production of lysophosphatidic acid (LPA) using a recently described autotaxin (ATX) inhibitor. Finally, plasma inflammatory cytokine levels were analyzed to determine the effects of diets and ATX inhibition on systemic cytokine milieu. ResultsWe found that casein is the main source of PC when present in rodent diets. Replacing casein with amino acids increased the relative proportion of endogenously biosynthesized PC in mouse plasma. Compared to diets containing casein, amino acid-defined diets decreased primary tumor growth in the hyperlipidemic estrogen-receptor positive E0771 breast cancer mouse model. Inhibition of autotaxin with the potent inhibitor PAT-505 did not attenuate breast cancer development in these hyperlipidemic mice. Further, replacing casein with amino acids or treatment with PAT-505 significantly reduced systemic markers of inflammation. ConclusionOur results show that casein is a significant source of PC when present in rodent diets. Diets formulated with amino acids in place of casein have higher proportion of circulating PC from the endogenous biosynthetic pool. Casein-containing high fat diets promote primary breast tumor development in mice through mechanisms that involve systemic inflammation but is independent of LPA production by autotaxin.

Lysophosphatidic acids (LPA) are bioactive radyl hydrocarbon-substituted derivatives of glycerol 3-phosphate. LPA metabolism and signaling are implicated in heritable risk of coronary artery disease. Genetic and pharmacological inhibition of these processes attenuate experimental atherosclerosis. LPA accumulates in atheromas, which may be a consequence of association with low-density lipoproteins (LDL). The source, regulation, and biological activity of LDL-associated LPA are unknown. We examined effects of experimental hyperlipidemia on levels and distribution of circulating LPA in mice. The majority of plasma LPA was associated with albumin in plasma from wild-type mice fed normal chow. LDL-associated LPA was increased in plasma from high-fat western diet fed mice that are genetically prone to hyperlipidemia (LDL receptor knockout or activated PCSK9 overexpressing C57Bl6). Adipose-specific deficiency of the ENPP2 gene encoding the LPA-generating secreted lysophospholipase D autotaxin (ATX) attenuated these western diet-dependent increases in LPA. ATX-dependent increases in LDL-associated LPA were observed in isolated incubated plasma. ATX acted directly on LDL-associated lysophospholipid substrates in vitro. LDL from all human subjects examined contained LPA and was decreased by lipid lowering drug therapies. Human and mouse plasma therefore contains a diet-sensitive LDL-associated LPA pool that might contribute to the cardiovascular disease promoting effects of LPA.

Lysophosphatidic acid (LPA) is a pleiotropic growth-factor lysophospholipid that promotes cancer cell survival, growth, migration, invasion, metastasis, and resistance to chemotherapy and radiotherapy. The main precursor for LPA, lysophosphatidylcholine (LPC) is derived majorly from the highly abundant phosphatidylcholine (PC). Hydrolysis of LPC to the circulating LPA is catalyzed by the enzyme autotaxin (ATX), a secreted lysophospholipase D. We investigated the effects of altering dietary intake of PC and pharmacological inhibition of ATX on breast cancer development in mice. To formulate diets with defined PC levels we used tandem mass spectrometry to measure the concentration of PC in various components of fat used to manufacture rodent diets. We determined that casein is the main source of PC when present in rodent diets. High fat diets (60% kcal fat) were formulated with casein (high PC) or amino acids in place of casein (low PC). To study the effects of these diets on breast cancer development, six-week old female C57Bl6 low density lipoprotein receptor knockout mice (LDLr-/-) were fed high fat diets for 12 weeks. Subsequently, E0771 syngeneic mammary cells were injected into the inguinal mammary fat pads of mice fed continuously on high fat diets. At the time of tumor cell inoculation, the animals were divided into groups to either receive daily dosage of a novel potent ATX inhibitor or vehicle control. Primary tumor growth was monitored using orthogonal caliper measurements and tumor volumes estimated from width2 × length/2. The number of metastatic nodules in the lungs were counted after staining with India ink. In high fat diet mouse models of breast cancer we observed that feeding mice with diets in which casein is replaced with amino acids decreases primary tumor growth. Contrary to our expectation, treatment with a potent ATX inhibitor resulted in no significant effect on primary tumor growth and an increased number of metastatic nodules compared with vehicle-treated mice. Moreover, treatment with the ATX inhibitor reversed the beneficial effects of casein replacement and instead resulted in enhanced tumor growth and metastasis. These studies suggest that dietary casein promotes primary tumor growth in the hyperlipidemic E0771 models of cancer and that targeting ATX inhibition is not efficacious in these models. Our results show that under certain hyperlipidemic conditions consumption of dietary casein can promote primary tumor growth. We also provide evidence emphasizing the need for further investigation in ascertaining the impact and utility of ATX inhibitors in breast cancer treatment. Citation Format: Fredrick O. Onono, Ebubechi Adindu, Baoxiang Yan, Sony Soman, Courtney Hammill, Andrew J. Morris. Dietary phosphatidylcholine depletion reduces tumor development independent of autotaxin inhibition in hyperlipidemic mouse models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3601.

Lactation is associated with increased expression of bile acid transporters and an increased size and hydrophobicity of the bile acid pool in rats. ATP-binding cassette (ABC) transporters multidrug resistance protein 2 (Mdr2), Abcb11 [bile salt export pump (Bsep)], and Abcg5/Abcg8 heterodimers are essential for the biliary secretion of phospholipids, bile acids, and cholesterol, respectively. We investigated the expression of these transporters and secretion of their substrates in female control and lactating Sprague Dawley rats and C57BL/6 mice. Expression of Abcg5/Abcg8 mRNA was decreased by 97 and 60% by midlactation in rats and mice, respectively; protein levels of Abcg8 were below detection limits in lactating rats. Mdr2 mRNA expression was decreased in lactating rats and mice by 47 and 59%, respectively. Despite these changes in transporter expression, basal concentrations of cholesterol and phospholipid in bile were unchanged in rats and mice, whereas increased Bsep mRNA expression in early lactation coincided with an increased basal biliary bile acid concentration in lactating mice. Following taurocholate infusion, coupling of phospholipid and taurocholate secretion in bile of lactating mice was significantly impaired relative to control mice, with no significant changes in maximal secretion of cholesterol or bile acids. In rats, taurocholate infusion revealed a significantly impaired coupling of cholesterol to taurocholate secretion in bile in lactating vs. control animals. These data reveal marked utilization of an Abcg5/Abcg8-independent mechanism for basal biliary cholesterol secretion in rats during lactation, but a dependence on Abcg5/g8 for maximal biliary cholesterol secretion.