Deficiency of peroxisome proliferator-activated receptor α attenuates apoptosis and promotes migration of vascular smooth muscle cells

Peroxisome proliferator-activated receptor (PPAR) α is widely expressed in the vasculature and has pleiotropic and lipid-lowering independent effects, but its role in the growth and function of vascular smooth muscle cells (VSMCs) during vascular pathophysiology is still unclear. Herein, VSMC-specific PPARα-deficient mice (Ppara^ΔSMC) were generated by Cre-LoxP site-specific recombinase technology and VSMCs were isolated from mice aorta. PPARα deficiency attenuated VSMC apoptosis induced by angiotensin (Ang) II and hydrogen peroxide, and increased the migration of Ang II-challenged cells.     

Rosiglitazone Reverses Inflammation in Epididymal White Adipose Tissue in Hormone-Sensitive Lipase-Knockout Mice

Hormone-sensitive lipase (HSL) plays a crucial role in intracellular lipolysis, and loss of HSL leads to diacylglycerol (DAG) accumulation, reduced FA mobilization, and impaired PPARγ signaling. Hsl knockout mice exhibit adipose tissue inflammation, but the underlying mechanisms are still not clear. Here, we investigated if and to what extent HSL loss contributes to endoplasmic reticulum (ER) stress and adipose tissue inflammation in Hsl knockout mice. Furthermore, we were interested in how impaired PPARγ signaling affects the development of inflammation in epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) of Hsl knockout mice and if DAG and ceramide accumulation contribute to adipose tissue inflammation and ER stress. Ultrastructural analysis showed a markedly dilated ER in both eWAT and iWAT upon loss of HSL. In addition, Hsl knockout mice exhibited macrophage infiltration and increased F4/80 mRNA expression, a marker of macrophage activation, in eWAT, but not in iWAT. We show that treatment with rosiglitazone, a PPARγ agonist, attenuated macrophage infiltration and ameliorated inflammation of eWAT, but expression of ER stress markers remained unchanged, as did DAG and ceramide levels in eWAT. Taken together, we show that HSL loss promoted ER stress in both eWAT and iWAT of Hsl knockout mice, but inflammation and macrophage infiltration occurred mainly in eWAT. Also, PPARγ activation reversed inflammation but not ER stress and DAG accumulation. These data indicate that neither reduction of DAG levels nor ER stress contribute to the reversal of eWAT inflammation in Hsl knockout mice.     

Copper and mercury induced oxidative stresses and antioxidant responses of Spirodela polyrhiza (L.) Schleid

Duckweed is recognized as a phytoremediation aquatic plant due to the production of large biomass and a high level of tolerance in stressed conditions. A laboratory experiment was conducted to investigate antioxidant response and mechanism of copper and mercury tolerance of S. polyrhiza (L.) Schleid. To understand the changes in chlorophyll content, MDA, proline, and activities of ROS-scavenging enzymes (SOD, CAT, GPOD) during the accumulation of Cu⁺² and Hg⁺², S. polyrhiza were exposed to various concentrations of Cu⁺² (0.0–40 μM) and Hg⁺² (0.0–0.4 μM). antioxidant activity initially indicated enhancing trend with application of 10 μM Cu⁺²; 0.2 μM Hg⁺² (SOD), of 20 μM Cu⁺²; 0.2 μM Hg⁺² (CAT) and of 10 μM Cu⁺²;0.2 μM Hg⁺² (GPOD) and then decreased consistently up to 40 μM Cu⁺² and 0.4 μM Hg⁺². In the experiment chlorophyll and frond multiplication initially showed increasing tendency and decreased gradually with the application of increased metal concentration. Application of heavy metal has constantly enhanced proline and MDA content while the maximum increase was observed with the application of 40 μM Cu; 0.4 μM Hg for proline and MDA respectively. The upregulation of antioxidant enzymes and proline reveals that S. polyrhiza has strong biochemical strategies to deal with the heavy metal toxicity induced by the accumulation of Cu⁺² and Hg⁺².     

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.     

Gut microbiome-derived glycine lipids are diet-dependent modulators of hepatic injury and atherosclerosis

Oral and gut Bacteroidetes produce unique classes of serine-glycine lipodipeptides and glycine aminolipids that signal through host Toll-like receptor 2. These glycine lipids have also been detected in human arteries, but their effects on atherosclerosis are unknown. Here, we sought to investigate the bioactivity of bacterial glycine lipids in mouse models of atherosclerosis. Lipid 654 (L654), a serine-glycine lipodipeptide species, was first tested in a high-fat diet (HFD)-fed Ldlr^?/? model of atherosclerosis. Intraperitoneal administration of L654 over 7 weeks to HFD-fed Ldlr^?/? mice resulted in hypocholesterolemic effects and significantly attenuated the progression of atherosclerosis. We found that L654 also reduced liver inflammatory and extracellular matrix gene expression, which may be related to inhibition of macrophage activation as demonstrated in vivo by lower major histocompatibility complex class II gene expression and confirmed in cell experiments. In addition, L654 and other bacterial glycine lipids in feces, liver, and serum were markedly reduced alongside changes in Bacteroidetes relative abundance in HFD-fed mice. Finally, we tested the bioactivities of L654 and related lipid 567 in chow-fed Apoe^?/? mice, which displayed much higher fecal glycine lipids relative to HFD-fed Ldlr^?/? mice. Administration of L654 or lipid 567 for 7 weeks to these mice reduced the liver injury marker alanine aminotransferase, but other effects seen in Ldlr^?/? were not observed. Therefore, we conclude that conditions in which gut microbiome-derived glycine lipids are lost, such as HFD, may exacerbate the development of atherosclerosis and liver injury, whereas correction of such depletion may protect from these disorders.     

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b^?/? mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b^?/? mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.     

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.     

Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation During Hypercholesterolemia

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1^trg/EC^cre) and their control littermates (Smpd1^trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1^trg/EC^cre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1^trg/EC^cre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1^trg/EC^cre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1^trg/EC^cre mice than in their control littermates. Using ECs from Smpd1^trg/EC^cre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1β levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.     

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.     

Optimisation of estrogen receptor subtype-selectivity of a 4-Aryl-4H-chromene scaffold previously identified by virtual screening

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity).     

Sulfatide with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs in renal intercalated cells

Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H⁺-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H⁺-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH₃ and protons into the urine.     

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1^−/− mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND-fed Cyp7b1^−/− mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1^−/− mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1^−/− mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.     

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.     

The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationships in Benzoate Degradation

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades fatty and aromatic acids into acetate, CO₂, formate, and H₂ that are utilized by methanogens and other hydrogen-consuming microbes. S. aciditrophicus benzoate degradation proceeds by a multistep pathway with many intermediate reactive acyl-coenzyme A species (RACS) that can potentially N^ε-acylate lysine residues. Herein, we describe the identification and characterization of acyl-lysine modifications that correspond to RACS in the benzoate degradation pathway. The amounts of modified peptides are sufficient to analyze the post-translational modifications without antibody enrichment, enabling a range of acylations located, presumably, on the most extensively acylated proteins throughout the proteome to be studied. Seven types of acyl modifications were identified, six of which correspond directly to RACS that are intermediates in the benzoate degradation pathway including 3-hydroxypimeloylation, a modification first identified in this system. Indeed, benzoate-degrading enzymes are heavily represented among the acylated proteins. A total of 125 sites were identified in 60 proteins. Functional deacylase enzymes are present in the proteome, indicating a potential regulatory system/mechanism by which S. aciditrophicus modulates acylation. Uniquely, N^ε-acyl-lysine RACS are highly abundant in these syntrophic bacteria, raising the compelling possibility that post-translational modifications modulate benzoate degradation in this and potentially other, syntrophic bacteria. Our results outline candidates for further study of how acylations impact syntrophic consortia.     

Comparative study of the antioxidant activity of the essential oils of five plants against the H2O2 induced stress in Saccharomyces cerevisiae

The purpose of this work was to investigate the protective effect of five essential oils (EOs); Rosmarinus officinalis, Thymus vulgaris, Origanum compactum Benth., Eucalyptus globulus Labill. and Ocimum basilicum L.; against oxidative stress induced by hydrogen peroxide in Saccharomyces cerevisiae. The chemical composition of the EOs was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The in vitro antioxidant activity was evaluated and the protective effect of EOs was investigated. Yeast cells were pretreated with different concentrations of EOs (6.25–25 µg/ml) for an hour then incubated with H₂O₂ (2 mM) for an additional hour. Cell viability, antioxidants (Catalase, Superoxide dismutase and Glutathione reductase) and metabolic (Succinate dehydrogenase) enzymes, as well as the level of lipid peroxidation (LPO) and protein carbonyl content (PCO) were evaluated. The chemical composition of EOs has shown the difference qualitatively and quantitatively. Indeed, O. compactum mainly contained Carvacrol, O. basilicum was mainly composed of Linalool, T. vulgaris was rich in thymol, R. officinalis had high α-Pinene amount and for E. globulus, eucalyptol was the major compound. The EOs of basil, oregano and thyme were found to possess the highest amount of total phenolic compounds. Moreover, they have shown the best protective effect on yeast cells against oxidative stress induced by H₂O₂. In addition, in a dose dependent manner of EOs in yeast medium, treated cells had lower levels of LPO, lower antioxidant and metabolic enzymes activity than cells exposed to H₂O₂ only. The cell viability was also improved. It seems that the studied EOs are efficient natural antioxidants, which can be exploited to protect against damages and serious diseases related to oxidative stress.     

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.     

Ablation of pigment epithelium-derived factor receptor (PEDF-R/Pnpla2) causes photoreceptor degeneration

Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2^−/− mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA₂ enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2^−/− photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2^−/− and Pnpla2^+/− mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.     

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.     

Clinical,Hormonal, and Genetic Characteristics of 5α-Reductase Type 2 Deficiency in 103 Chinese Patients

Objective5α-Reductase type 2 (5α-RD2) deficiency causes variable degrees of undervirilization in patients. The correlation between its genotype and phenotype is unclear.MethodsWe retrospectively evaluated 103 patients with 46,XY disorders of sex development who were diagnosed with 5α-RD2 deficiency.ResultsThe prevalence of female sex assignment (P = .008) and the incidences of cryptorchidism (P = .0003) and bifid scrotum (P = .0002) in the non-p.R227Q variant group were higher, but there were no significant differences in the incidences of hypospadias and isolated microphallus. The external masculinization score in the non-p.R227Q variant group was lower than that in the homozygous p.R227Q variant (P = .019) and compound heterozygous p.R227Q variant groups (P = .013). The level of anti-Mullerian hormone in the non-p.R227Q variant group was lower than that in the homozygous p.R227Q variant (P < .001) and compound heterozygous p.R227Q variant groups (P = .006). The testosterone-to-dihydrotestosterone ratio of the homozygous p.R227Q variant group was higher than that of the non-p.R227Q variant (P = .018) and compound heterozygous p.R227Q variant groups (P = .029). Twenty-three reportedly pathogenic variants and 11 novel steroid 5α-reductase 2 (SRD5A2) variants were identified.ConclusionCompared with patients without p.R227Q, patients with p.R227Q exhibited higher external masculinization scores and anti-Mullerian hormone expression, a lower prevalence of female sex assignment, and lower incidences of cryptorchidism and bifid scrotum. We identified 23 reportedly pathogenic SRD5A2 variants and 11 novel SRD5A2 variants that led to 5α-RD2 deficiency. We established a genotype-phenotype correlation, and patients with p.R227Q showed a relatively mild phenotype.