Role of enterocyte Enpp2 and autotaxin in regulating lipopolysaccharide levels,systemic inflammation,and atherosclerosis

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr^−/− mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr^−/−/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr^−/− mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis.     

Hepatic Deletion of X-Box Binding Protein 1 in FXR Null Mice Leads to Enhanced Liver Injury

FXR regulates bile acid metabolism, and FXR null (Fxr^?/?) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1α/X-box binding protein 1 (XBP1) pathway is a protective unfolded protein response pathway activated in response to endoplasmic reticulum stress. Here, we sought to determine the role of the inositol-requiring enzyme 1α/XBP1 pathway in hepatic bile acid toxicity using the Fxr^?/? mouse model. Western blotting and quantitative PCR analysis demonstrated that hepatic XBP1 and other unfolded protein response pathways were activated in 24-week-old Fxr^?/? compared with 10-week-old Fxr^?/? mice but not in WT mice. To further determine the role of the liver XBP1 activation in older Fxr^?/? mice, we generated mice with whole-body FXR and liver-specific XBP1 double KO (DKO, Fxr^?/?Xbp1^LKO) and Fxr^?/?Xbp1^fl/fl single KO (SKO) mice and characterized the role of hepatic XBP1 in cholestatic liver injury. Histologic staining demonstrated increased liver injury and fibrosis in DKO compared with SKO mice. RNA sequencing revealed increased gene expression in apoptosis, inflammation, and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein pathway and cell cycle marker cyclin D1 were also activated in DKO mice. Furthermore, we found that total hepatic bile acid levels were similar between the two genotypes. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. In conclusion, the hepatic XBP1 pathway is activated in older Fxr^?/? mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.     

Dennd5b-Deficient Mice are Resistant to PCSK9-Induced Hypercholesterolemia and Diet-Induced Hepatic Steatosis

Dennd5b plays a pivotal role in intestinal absorption of dietary lipids in mice and is associated with body mass index in humans. This study examined the impact of whole-body Dennd5b deletion on plasma lipid concentrations, atherosclerosis, and hepatic lipid metabolism in mice. Hypercholesterolemia was induced in Dennd5b^?/? mice by infection with an adeno-associated virus expressing the proprotein convertase subtilisin/kexin type 9 serine protease (PCSK9) gain-of-function mutation (PCSK9D377Y) and feeding a Western diet for 12 weeks. Body weight and plasma lipid concentrations were monitored over 12 weeks, and then aortic atherosclerosis and hepatic lipid content were quantified. Compared to Dennd5b^+/+ mice, Dennd5b^?/? mice were resistant to diet-induced weight gain and PCSK9-induced hypercholesterolemia. Atherosclerosis quantified by en face analysis and in aortic root sections, revealed significantly smaller lesions in Dennd5b^?/? compared to Dennd5b^+/+ mice. Additionally, Dennd5b^?/? mice had significantly less hepatic lipid content (triglyceride and cholesterol) compared to Dennd5b^+/+ mice. To gain insight into the basis for reduced hepatic lipids, quantitative PCR was used to measure mRNA abundance of genes involved in hepatic lipid metabolism. Key genes involved in hepatic lipid metabolism and lipid storage were differentially expressed in Dennd5b^?/? liver including Pparg, Cd36, and Pnpla3. These findings demonstrate a significant impact of Dennd5b on plasma and hepatic lipid concentrations and resistance to PCSK9-induced hypercholesterolemia in the absence of Dennd5b.     

Comparison between genetic and pharmaceutical disruption of Ldlr expression for the development of atherosclerosis

Antisense oligonucleotides (ASOs) against Ldl receptor (Ldlr-ASO) represent a promising strategy to promote hypercholesterolemic atherosclerosis in animal models without the need for complex breeding strategies. Here, we sought to characterize and contrast atherosclerosis in mice given Ldlr-ASO with those bearing genetic Ldlr deficiency. To promote atherosclerosis, male and female C57Bl6/J mice were either given weekly injections of Ldlr-ASO (5 mg/kg once per week) or genetically deficient in Ldlr (Ldlr^?/?). Mice consumed either standard rodent chow or a diet high in saturated fat and sucrose with 0.15% added cholesterol for 16 weeks. While both models of Ldlr deficiency promoted hypercholesterolemia, Ldlr^?/? mice exhibited nearly 2-fold higher cholesterol levels than Ldlr-ASO mice, reflected by increased VLDL and LDL levels. Consistent with this, the en face atherosclerotic lesion area was 3-fold and 3.6-fold greater in male and female mice with genetic Ldlr deficiency, respectively, as compared with the modest atherosclerosis observed following Ldlr-ASO treatment. Aortic sinus lesion sizes, fibrosis, smooth muscle actin, and necrotic core areas were also larger in Ldlr^?/? mice, suggesting a more advanced phenotype. Despite a more modest effect on hypercholesterolemia, Ldlr-ASO induced greater hepatic inflammatory gene expression, macrophage accumulation, and histological lobular inflammation than was observed in Ldlr^?/? mice. We conclude Ldlr-ASO is a promising tool for the generation of complex rodent models with which to study atherosclerosis but does not promote comparable levels of hypercholesterolemia or atherosclerosis as Ldlr^?/? mice and increases hepatic inflammation. Thus, genetic Ldlr deficiency may be a superior model, depending on the proposed use.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

LRP1 loss in airway epithelium exacerbates smoke-induced oxidative damage and airway remodeling

The LDL receptor-related protein 1 (LRP1) partakes in metabolic and signaling events regulated in a tissue-specific manner. The function of LRP1 in airways has not been studied. We aimed to study the function of LRP1 in smoke-induced disease. We found that bronchial epithelium of patients with chronic obstructive pulmonary disease and airway epithelium of mice exposed to smoke had increased LRP1 expression. We then knocked out LRP1 in human bronchial epithelial cells in vitro and in airway epithelial club cells in mice. In vitro, LRP1 knockdown decreased cell migration and increased transforming growth factor β activation. Tamoxifen-inducible airway-specific LRP1 knockout mice (club Lrp1^?/?) induced after complete lung development had increased inflammation in the bronchoalveolar space and lung parenchyma at baseline. After 6 months of smoke exposure, club Lrp1^?/? mice showed a combined restrictive and obstructive phenotype, with lower compliance, inspiratory capacity, and forced expiratory volume_0.05/forced vital capacity than WT smoke-exposed mice. This was associated with increased values of Ashcroft fibrotic index. Proteomic analysis of room air exposed-club Lrp1^?/? mice showed significantly decreased levels of proteins involved in cytoskeleton signaling and xenobiotic detoxification as well as decreased levels of glutathione. The proteome fingerprint created by smoke eclipsed many of the original differences, but club Lrp1^?/? mice continued to have decreased lung glutathione levels and increased protein oxidative damage and airway cell proliferation. Therefore, LRP1 deficiency leads to greater lung inflammation and damage and exacerbates smoke-induced lung disease.     

2-Hydroxypropyl-β-cyclodextrin mitigates pathological changes in a mouse model of retinal cholesterol dyshomeostasis

CYP46A1 is a CNS-specific enzyme, which eliminates cholesterol from the brain and retina by metabolism to 24-hydroxycholesterol, thus contributing to cholesterol homeostasis in both organs. 2-Hydroxypropyl-β-cyclodextrin (HPCD), a Food and Drug Administration-approved formulation vehicle, is currently being investigated off-label for treatment of various diseases, including retinal diseases. HPCD was shown to lower retinal cholesterol content in mice but had not yet been evaluated for its therapeutic benefits. Herein, we put Cyp46a1^?/? mice on high fat cholesterol-enriched diet from 1 to 14 months of age (control group) and at 12 months of age, started to treat a group of these animals with HPCD until the age of 14 months. We found that as compared with mature and regular chow-fed Cyp46a1^?/? mice, control group had about 6-fold increase in the retinal total cholesterol content, focal cholesterol and lipid deposition in the photoreceptor-Bruch’s membrane region, and retinal macrophage activation. In addition, aged animals had cholesterol crystals at the photoreceptor-retinal pigment epithelium interface and changes in the Bruch’s membrane ultrastructure. HPCD treatment mitigated all these manifestations of retinal cholesterol dyshomeostasis and altered the abundance of six groups of proteins (genetic information transfer, vesicular transport, and cytoskeletal organization, endocytosis and lysosomal processing, unfolded protein removal, lipid homeostasis, and Wnt signaling). Thus, aged Cyp46a1^?/? mice on high fat cholesterol-enriched diet revealed pathological changes secondary to retinal cholesterol overload and supported further studies of HPCD as a potential therapeutic for age-related macular degeneration and diabetic retinopathy associated with retinal cholesterol dyshomeostasis.     

Curdlan intake changes gut microbial composition,short-chain fatty acid production,and bile acid transformation in mice

Indigestible polysaccharides, such as dietary fibers, benefit the host by improving the intestinal environment. Short-chain fatty acids (SCFAs) produced by gut microbial fermentation from dietary fibers exert various physiological effects. The bacterial polysaccharide curdlan benefits the host intestinal environment, although its effect on energy metabolism and SCFA production remains unclear. Hence, this study aimed to elucidate the effect of curdlan intake on gut microbial profiles, SCFA production, and energy metabolism in a high-fat diet (HFD)-induced obese mouse model. Gut microbial composition of fecal samples from curdlan-supplemented HFD-fed mice indicated an elevated abundance of Bacteroidetes, whereas a reduced abundance of Firmicutes was noted at the phylum level compared with that in cellulose-supplemented HFD-fed mice. Moreover, curdlan supplementation resulted in an abundance of the family Bacteroidales S24-7 and Erysipelotrichaceae, and a reduction in Deferribacteres in the feces. Furthermore, curdlan supplementation elevated fecal SCFA levels, particularly butyrate. Although body weight and fat mass were not affected by curdlan supplementation in HFD-induced obese mice, HFD-induced hyperglycemia was significantly suppressed with an increase in plasma insulin and incretin GLP-1 levels. Curdlan supplementation elevated fecal bile acid and SCFA production, improved host metabolic functions by altering the gut microbial composition in mice.     

Regenerating islet-derived protein (Reg)3β plays a crucial role in attenuation of ileitis and colitis in mice

Regenerating islet-derived protein (Reg)3β belongs to a member of the Reg family of proteins and has pleiotropic functions, including antimicrobial activity and tissue repair. However, whether Reg3β plays a protective role in the development of colitis and ileitis has not been fully investigated. We generated transgenic mice expressing a short form of cellular FLICE-inhibitory protein (cFLIPs) that promotes necroptosis, a regulated form of cell death. cFLIPs transgenic (CFLARs Tg) mice develop severe ileitis in utero. Although Reg3β is undetectable in the small intestine of wild-type embryos, its expression is aberrantly elevated in the small intestine of CFLARs Tg embryos. To test whether elevated Reg3β attenuates or exacerbates ileitis in CFLARs Tg mice, we generated a Reg3b^?/? strain. Reg3b^?/? mice grew to adulthood without apparent abnormalities. Deletion of Reg3b in CFLARs Tg mice exacerbated the embryonic lethality of CFLARs Tg mice. Dextran sulfate sodium-induced colitis, characterized by body weight loss and infiltration of neutrophils, was exacerbated in Reg3b^?/? compared to wild-type mice. Moreover, the expression of Interleukin 6, an inflammatory cytokine and Chitinase-like 3, a marker for tissue repair macrophages was elevated in the colon of Reg3b^?/? mice compared to wild-type mice after DSS treatment. Together, these results suggest that attenuation of colitis and ileitis is a result of Reg3β′s real function.     

Neuronal growth regulator 1 promotes adipocyte lipid trafficking via interaction with CD36

Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol-anchored membrane protein associated with several human pathologies, including obesity, depression, and autism. Recently, significantly enlarged white adipose tissue, hepatic lipid accumulation, and decreased muscle capacity were reported in Negr1-deficient mice. However, the mechanism behind these phenotypes was not clear. In the present study, we found NEGR1 to interact with cluster of differentiation 36 (CD36), the major fatty acid translocase in the plasma membrane. Binding assays with a soluble form of NEGR1 and in situ proximal ligation assays indicated that NEGR1-CD36 interaction occurs at the outer leaflet of the cell membrane. Furthermore, we show that NEGR1 overexpression induced CD36 protein destabilization in vitro. Both mRNA and protein levels of CD36 were significantly elevated in the white adipose tissue and liver tissues of Negr1^?/? mice. Accordingly, fatty acid uptake rate increased in NEGR1-deficient primary adipocytes. Finally, we demonstrated that Negr1^?/? mouse embryonic fibroblasts showed elevated reactive oxygen species levels and decreased adenosine monophosphate-activated protein kinase activation compared with control mouse embryonic fibroblasts. Based on these results, we propose that NEGR1 regulates cellular fat content by controlling the expression of CD36.     

Serum amyloid A augments the atherogenic effects of cholesteryl ester transfer protein

Serum amyloid A (SAA) is predictive of CVD in humans and causes atherosclerosis in mice. SAA has many proatherogenic effects in vitro. However, HDL, the major carrier of SAA in the circulation, masks these effects. The remodeling of HDL by cholesteryl ester transfer protein (CETP) liberates SAA restoring its proinflammatory activity. Here, we investigated whether deficiency of SAA suppresses the previously described proatherogenic effect of CETP. ApoE^−/− mice and apoE^−/− mice deficient in the three acute-phase isoforms of SAA (SAA1.1, SAA2.1, and SAA3; “apoE^−/− SAA-TKO”) with and without adeno-associated virus-mediated expression of CETP were studied. There was no effect of CETP expression or SAA genotype on plasma lipids or inflammatory markers. Atherosclerotic lesion area in the aortic arch of apoE^−/− mice was 5.9 ± 1.2%; CETP expression significantly increased atherosclerosis in apoE^−/− mice (13.1 ± 2.2%). However, atherosclerotic lesion area in the aortic arch of apoE^−/− SAA-TKO mice (5.1 ± 1.1%) was not significantly increased by CETP expression (6.2 ± 0.9%). The increased atherosclerosis in apoE^−/− mice expressing CETP was associated with markedly increased SAA immunostaining in aortic root sections. Thus, SAA augments the atherogenic effects of CETP, which suggests that inhibiting CETP may be of particular benefit in patients with high SAA.     

Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr^?/? mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.     

10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr^?/?) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.     

Elevated granulocyte-colony stimulating factor and hematopoietic stem cell mobilization in Niemann-Pick type C1 disease

Niemann-Pick type C1 (NPC1) disease is a progressive lysosomal storage disorder caused by mutations of the NPC1 gene. While neurodegeneration is the most severe symptom, a large proportion of NPC1 patients also present with splenomegaly, which has been attributed to cholesterol and glycosphingolipid accumulation in late endosomes and lysosomes. However, recent data also reveal an increase in the inflammatory monocyte subset in the Npc1^nih mouse model expressing an Npc1 null allele. We evaluated the contribution of hematopoietic cells to splenomegaly in NPC1 disease under conditions of hypercholesterolemia. We transplanted Npc1^nih (Npc1 null mutation) or Npc1^wt bone marrow (BM) into Ldlr^?/? mice and fed these mice a cholesterol-rich Western-type diet. At 9 weeks after BM transplant, on a chow diet, the Npc1 null mutation increased plasma granulocyte-colony stimulating factor (G-CSF) by 2-fold and caused mild neutrophilia. At 18 weeks after BM transplant, including 9 weeks of Western-type diet feeding, the Npc1 mutation increased G-csf mRNA levels by ～5-fold in splenic monocytes/macrophages accompanied by a ～4-fold increase in splenic neutrophils compared with controls. We also observed ～5-fold increased long-term and short-term hematopoietic stem cells (HSCs) in the spleen, and a ～30–75% decrease of these populations in BM, reflecting HSC mobilization, presumably downstream of elevated G-CSF. In line with these data, four patients with NPC1 disease showed higher plasma G-CSF compared with age-matched and gender-matched healthy controls. In conclusion, we show elevated G-CSF levels and HSC mobilization in the setting of an Npc1 null mutation and propose that this contributes to splenomegaly in patients with NPC1 disease.     

Baat Gene Knockout Alters Post-Natal Development,the Gut Microbiome,and Reveals Unusual Bile Acids in Mice

Bile acids (BAs) are steroid detergents in bile that contribute to fat absorption, cell signaling, and microbiome interactions. The final step in their synthesis is amino acid conjugation with either glycine or taurine in the liver by the enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT). Here, we describe the microbial, chemical, and physiological consequences of Baat gene knockout. Baat^-/- mice were underweight after weaning but quickly exhibited catch-up growth. At three weeks of age, KO animals had increased phospholipid excretion and decreased subcutaneous fat pad mass, liver mass, glycogen staining in hepatocytes, and hepatic vitamin A stores, but these were less marked in adulthood. Additionally, KO mice had an altered microbiome in early life. Their BA pool was highly enriched in cholic acid but not completely devoid of conjugated BAs. KO animals had 27-fold lower taurine-conjugated BAs than wild type in their liver but similar concentrations of glycine-conjugated BAs and higher microbially conjugated BAs. Furthermore, the BA pool in Baat^-/- was enriched in a variety of unusual BAs that were putatively sourced from cysteamine conjugation with subsequent oxidation and methylation of the sulfur group mimicking taurine. Antibiotic treatment of KO mice indicated the microbiome was not the likely source of the unusual conjugations, instead, the unique BAs in KO animals were likely derived from the peroxisomal acyltransferases Acnat1 and Acnat2, which are duplications of Baat in the mouse genome that are inactivated in humans. This study demonstrates that BA conjugation is important for early life development of mice.     

Serum opacity factor rescues fertility among female Scarb1−/− mice by reducing HDL-free cholesterol bioavailability

Human female infertility, 20% of which is idiopathic, is a public health problem for which better diagnostics and therapeutics are needed. A novel cause of infertility emerged from studies of female mice deficient in the HDL receptor gene (Scarb1). These mice are infertile and have high plasma HDL cholesterol (C) concentrations, due to elevated HDL-free cholesterol (FC), which transfers from HDL to all tissues. Previous studies have indicated that oral delivery of probucol, an HDL-lowering drug, to female Scarb1^?/? mice reduces plasma HDL-C concentrations and rescues fertility. Additionally, serum opacity factor (SOF), a bacterial virulence factor, disrupts HDL structure, and bolus SOF injection into mice reduces plasma HDL-C concentrations. Here, we discovered that delivering SOF to female Scarb1^?/? mice with an adeno-associated virus (AAV_SOF) induces constitutive SOF expression, reduces HDL-FC concentrations, and rescues fertility while normalizing ovary morphology. Although AAV_SOF did not alter ovary-FC content, the ovary-mol% FC correlated with plasma HDL-mol% FC in a fertility-dependent way. Therefore, reversing the abnormal plasma microenvironment of high plasma HDL-mol% FC in female Scarb1^-/- mice rescues fertility. These data provide the rationale to search for similar mechanistic links between HDL-mol% FC and infertility and the rescue of fertility in women by reducing plasma HDL-mol% FC.     

Lycopene is superior to moringa in improving fertility markers in diet-induced obesity male rats

Obesity is a condition of chronic tissue inflammation and oxidative stress that poses as a risk factor for male infertility. Moringa oleifera oil extract is known to have cholesterol-lowering properties and a potential to treat obesity, while lycopene is a potent antioxidant. We hypothesize that Moringa or lycopene may improve male fertility markers in an animal model of diet-induced obesity. Male Albino rats (n = 60) were randomized to receive regular chow (RC) or high-fat diet (HFD) for 12 weeks (n = 30 each). Animals in each arm were further randomized to receive gavage treatment with corn oil (vehicle), lycopene (10 mg/kg), or Moringa (400 mg/kg) for four weeks starting on week 9 (n = 10 each). Animals were sacrificed at 12 weeks, and blood was collected to assess lipid profile, serum testosterone, and gonadotropin levels. The testes and epididymides were removed for sperm analysis, oxidative stress and inflammatory markers, and histopathological assessment. In comparison to their RC littermates, animals on HFD showed an increase in body weights, serum lipids, testosterone and gonadotrophin levels, testicular oxidative stress and inflammatory markers, as well as sperm abnormalities and disrupted testicular histology. Moringa or lycopene reduced body weight, improved oxidative stress, and male fertility markers in HFD-fed animals with lycopene exhibiting better anti-antioxidant and anti-lipidemic effects. Lycopene is superior to Moringa in improving male fertility parameters, possibly by attenuating oxidative stress.     

Associations of ApoE4 status and DHA supplementation on plasma and CSF lipid profiles and entorhinal cortex thickness

Apolipoprotein ε allele 4 (APOE4) influences the metabolism of polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The entorhinal cortex (EC) in the brain is affected early in Alzheimer's disease and is rich in DHA. The purpose of this study is to identify the effect of APOE4 and DHA lipid species on the EC. Plasma and cerebrospinal fluid (CSF) lipidomic measurements were obtained from the DHA Brain Delivery Pilot, a randomized clinical trial of DHA supplementation (n = 10) versus placebo (n = 12) for six months in nondemented older adults stratified by APOE4 status. Wild-type C57B6/J mice were fed a high or low DHA diet for 6 months followed by plasma and brain lipidomic analysis. Levels of phosphatidylcholine DHA (PC 38:6) and cholesterol ester DHA (CE 22:6) had the largest increases in CSF following supplementation (P < 0.001). DHA within triglyceride (TG) lipids in CSF strongly correlated with corresponding plasma TG lipids, and differed by APOE4, with carriers having a lower increase than noncarriers. Changes in plasma PC DHA had the strongest association with changes in EC thickness in millimeters, independent of APOE4 status (P = 0.007). In mice, a high DHA diet increased PUFAs within brain lipids. Our findings demonstrate an exchange of DHA at the CSF-blood barrier and into the brain within all lipid species with APOE having the strongest effect on DHA-containing TGs. The correlation of PC DHA with EC suggests a functional consequence of DHA accretion in high density lipoprotein for the brain.     

Novel 13C enrichment technique reveals early turnover of DHA in peripheral tissues

The brain is rich in DHA, which plays important roles in regulating neuronal function. Recently, using compound-specific isotope analysis that takes advantage of natural differences in carbon-13 content (¹³C/¹²C ratio or δ¹³C) of the food supply, we determined the brain DHA half-life. However, because of methodological limitations, we were unable to capture DHA turnover rates in peripheral tissues. In the current study, we applied compound-specific isotope analysis via high-precision GC combustion isotope ratio mass spectrometry to determine half-lives of brain, liver, and plasma DHA in mice following a dietary switch experiment. To model DHA tissue turnover rates in peripheral tissues, we added earlier time points within the diet switch study and took advantage of natural variations in the δ¹³C-DHA of algal and fish DHA sources to maintain DHA pool sizes and used an enriched (uniformly labeled ¹³C) DHA treatment. Mice were fed a fish-DHA diet (control) for 3 months, then switched to an algal-DHA treatment diet, the ¹³C enriched-DHA treatment diet, or they stayed on the control diet for the remainder of the study time course. In mice fed the algal and ¹³C enriched-DHA diets, the brain DHA half-life was 47 and 46 days, the liver half-life was 5.6 and 7.2 days, and the plasma half-life was 4.7 and 6.4 days, respectively. By using improved methodologies, we calculated DHA turnover rates in the liver and plasma, and our study for the first time, by using an enriched DHA source (very high δ¹³C), validated its utility in diet switch studies.