Neurotensin accelerates atherosclerosis and increases circulating levels of short-chain and saturated triglycerides.

BACKGROUND AND AIMS: Obesity and type 2 diabetes are significant risk factors for atherosclerotic cardiovascular disease (CVD) worldwide, but the underlying pathophysiological links are poorly understood. Neurotensin (NT), a 13-amino-acid hormone peptide, facilitates intestinal fat absorption and contributes to obesity in mice fed a high-fat diet. Elevated levels of pro-NT (a stable NT precursor produced in equimolar amounts relative to NT) are associated with obesity, type 2 diabetes, and CVD in humans. Whether NT is a causative factor in CVD is unknown. METHODS: Nt+/+ and Nt-/- mice were either injected with adeno-associated virus encoding PCSK9 mutants or crossed with Ldlr-/- mice and fed a Western diet. Atherosclerotic plaques were analyzed by en face analysis, Oil Red O and CD68 staining. In humans, we evaluated the association between baseline pro-NT and growth of carotid bulb thickness after 16.4 years. Lipidomic profiles were analyzed. RESULTS: Atherosclerotic plaque formation is attenuated in Nt-deficient mice through mechanisms that are independent of reductions in circulating cholesterol and triglycerides but associated with remodeling of the plasma triglyceride pool. An increasing plasma concentration of pro-NT predicts atherosclerotic events in coronary and cerebral arteries independent of all major traditional risk factors, indicating a strong link between NT and atherosclerosis. This plasma lipid profile analysis confirms the association of pro-NT with remodeling of the plasma triglyceride pool in atherosclerotic events. CONCLUSIONS: Our findings are the first to directly link NT to increased atherosclerosis and indicate the potential role for NT in preventive and therapeutic strategies for CVD.

Neurotensin differentially regulates bile acid metabolism and intestinal FXR-bile acid transporter axis in response to nutrient abundance.

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 were 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 the 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.

Absence of neurotensin attenuates intestinal dysbiosis and inflammation by maintaining Mmp7/α-defensin axis in diet-induced obese mice.

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 (Gö6983) 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.

Effects of diet and hyperlipidemia on levels and distribution of circulating lysophosphatidic acid.

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.

ABCG5/ABCG8-independent biliary cholesterol excretion in lactating rats.

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.