Correlation of spectral domain optical coherence tomography with histology and electron microscopy in the porcine retina.

Spectral domain optical coherence tomography (SD-OCT) is used as a non-invasive tool for retinal morphological assessment in vivo. Information on the correlation of SD-OCT with retinal histology in the porcine retina, a model resembling the human retina, is limited. Herein, we correlated the hypo- and hyper-reflective bands on SD-OCT with histology of the lamellar architecture and cellular constituents of the porcine retina. SD-OCT images were acquired with the Heidelberg Spectralis HRA + OCT. Histological analysis was performed using epoxy resin embedded tissue and transmission electron microscopy. Photomicrographs from the histologic sections were linearly scaled to correct for tissue shrinkage and correlated with SD-OCT images. SD-OCT images correlated well with histomorphometric data. A hyper-reflective band in the mid-to-outer inner nuclear layer correlated with the presence of abundant mitochondria in horizontal cell processes and adjacent bipolar cells. A concentration of cone nuclei corresponded to a relative hypo-reflective band in the outer portion of the outer nuclear layer. The presence of 3 hyper-reflective bands in the outer retina corresponded to: 1) the external limiting membrane; 2) the cone and rod ellipsoid zones; and 3) the interdigitation zone of photoreceptor outer segments/retinal pigment epithelium (RPE) apical cell processes and the RPE. These correlative and normative SD-OCT data may be employed to characterize and assess the in vivo histologic changes in retinal vascular and degenerative diseases and the responses to novel therapeutic interventions in this large animal model.

Activation of oligodendroglial Stat3 is required for efficient remyelination.

Multiple sclerosis is the most prevalent demyelinating disease of the central nervous system (CNS) and is histologically characterized by perivascular demyelination as well as neurodegeneration. While the degree of axonal damage is correlated with clinical disability, it is believed that remyelination can protect axons from degeneration and slow disease progression. Therefore, understanding the intricacies associated with myelination and remyelination may lead to therapeutics that can enhance the remyelination process and slow axon degeneration and loss of function. Ciliary neurotrophic factor (CNTF) family cytokines such as leukemia inhibitory factor (LIF) and interleukin 11 (IL-11) are known to promote oligodendrocyte maturation and remyelination in experimental models of demyelination. Because CNTF family member binding to the gp130 receptor results in activation of the JAK2/Stat3 pathway we investigated the necessity of oligodendroglial Stat3 in transducing the signal required for myelination and remyelination. We found that Stat3 activation in the CNS coincides with myelination during development. Stimulation of oligodendrocyte precursor cells (OPCs) with CNTF or LIF promoted OPC survival and final differentiation, which was completely abolished by pharmacologic blockade of Stat3 activation with JAK2 inhibitor. Similarly, genetic ablation of Stat3 in oligodendrocyte lineage cells prevented CNTF-induced OPC differentiation in culture. In vivo, while oligodendroglial Stat3 signaling appears to be dispensable for developmental CNS myelination, it is required for oligodendrocyte regeneration and efficient remyelination after toxin-induced focal demyelination in the adult brain. Our data suggest a critical function for oligodendroglial Stat3 signaling in myelin repair.

Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes.

The liver expresses high levels of two proteins with high affinity for long-chain fatty acids (LCFAs): liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2). Real-time confocal microscopy of cultured primary hepatocytes from gene-ablated (L-FABP, SCP-2/SCP-x, and L-FABP/SCP-2/SCP-x null) mice showed that the loss of L-FABP reduced cellular uptake of 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid (a fluorescent-saturated LCFA analog) by ∼50%. Importantly, nuclear targeting of the LCFA was enhanced when L-FABP was upregulated (SCP-2/SCP-x null) but was significantly reduced when L-FABP was ablated (L-FABP null), thus impacting LCFA nuclear targeting. These effects were not associated with a net decrease in expression of key membrane proteins involved in LCFA or glucose transport. Since hepatic LCFA uptake and metabolism are closely linked to glucose uptake, the effect of glucose on L-FABP-mediated LCFA uptake and nuclear targeting was examined. Increasing concentrations of glucose decreased cellular LCFA uptake and even more extensively decreased LCFA nuclear targeting. Loss of L-FABP exacerbated the decrease in LCFA nuclear targeting, while loss of SCP-2 reduced the glucose effect, resulting in enhanced LCFA nuclear targeting compared with control. Simply, ablation of L-FABP decreases LCFA uptake and even more extensively decreases its nuclear targeting.

Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3ß-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together, these results suggest that L-FABP, particularly in the absence of SCP-2, plays a significant role in HDL-mediated cholesterol uptake in cultured primary hepatocytes.

Autophagy regulates functional differentiation of mammary epithelial cells.

Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment. Developmental cues from the environment have been implicated in selective autophagy, or mitophagy, of mitochondria during cell differentiation and tissue development. Mitophagy occurring in this context, termed programmed mitophagy, responds to cell state rather than mitochondrial damage and is often accompanied by a metabolic transition. However, little is known about the mechanisms that engage and execute mitophagy under physiological or developmental conditions. As the mammary gland undergoes post-natal development and lactation challenges mitochondrial homeostasis, we investigated the contribution of mitochondria to differentiation of mammary epithelial cells (MECs). Using lactogenic differentiation of the HC11 mouse MEC line, we demonstrated that HC11 cells transition to a highly energetic state during differentiation by engaging both oxidative phosphorylation and glycolysis. Interestingly, this transition was lost when autophagy was inhibited with bafilomycin A1 or knockdown of Atg7 (autophagy related 7). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover in vitro with the fluorescent probe, pMitoTimer. Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of Prkn/parkin in HC11 cells. We found that MEC differentiation was impaired in shPrkn cells, implying that PRKN is required for MEC differentiation. These studies suggest a novel regulation of MEC differentiation through programmed mitophagy and provide a foundation for future studies of development and disease associated with mitochondrial function in the mammary gland.Abbreviations: AA: antimycin A; ATG5: autophagy related 5; BAF: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; COX8A: cytochrome c oxidase subunit 8A; CQ: chloroquine; CSN2: casein beta; ECAR: extracellular acidification rate; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; FUNDC1: FUN14 domain containing 1; HIF1A: hypoxia inducible factor 1 subunit alpha; L1: lactation day 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEC: mammary epithelial cell; mitoQ: mitoquinol; mROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; P: priming; P16: pregnancy day 16; PARP1: poly(ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PPARGC1A: PPARG coactivator 1 alpha; PRKN: parkin RBR E3 ubiquitin protein ligase; shNT: short hairpin non-targeting control; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TFAM: transcription factor A, mitochondrial; U: undifferentiated.

L-FABP directly interacts with PPARalpha in cultured primary hepatocytes.

Although studies with liver type fatty acid binding protein (L-FABP) gene ablated mice demonstrate a physiological role for L-FABP in hepatic fatty acid metabolism, little is known about the mechanisms whereby L-FABP elicits these effects. Studies indicate that L-FABP may function to shuttle lipids to the nucleus, thereby increasing the availability of ligands of nuclear receptors, such as peroxisome proliferator-activated receptor-alpha (PPARalpha). The data herein suggest that such mechanisms involve direct interaction of L-FABP with PPARalpha. L-FABP was shown to directly interact with PPARalpha in vitro through co-immunoprecipitation (co-IP) of pure proteins, altered circular dichroic (CD) spectra, and altered fluorescence spectra. In vitro fluorescence resonance energy transfer (FRET) between Cy3-labeled PPARalpha and Cy5-labeled L-FABP proteins showed that these proteins bound with high affinity (Kd approximately 156 nM) and in close proximity (intermolecular distance of 52A). This interaction was further substantiated by co-IP of both proteins from liver homogenates of wild-type mice. Moreover, double immunogold electron microscopy and FRET confocal microscopy of cultured primary hepatocytes showed that L-FABP was in close proximity to PPARalpha (intermolecular distance 40-49A) in vivo. Taken together, these studies were consistent with L-FABP regulating PPARalpha transcriptional activity in hepatocytes through direct interaction with PPARalpha. Our in vitro and imaging experiments demonstrate high affinity, structural molecular interaction of L-FABP with PPARalpha and suggest a functional role for L-FABP interaction with PPARalpha in long chain fatty acid (LCFA) metabolism.

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice.

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3alpha-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7alpha-hydroxylase, sterol 27alpha-hydroxylase, Na(+)/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.

Liver fatty acid binding protein gene ablation enhances age-dependent weight gain in male mice.

Although studies performed in vitro and with transfected cells in culture suggest a role for liver fatty acid binding protein (L-FABP) in regulating fatty acid oxidation and fat deposition, the physiological significance of this possibility is not completely clear. To begin to address this question, the effect of L-FABP gene ablation on phenotype of standard rodent chow-fed male mice was examined with increasing age up to 18 months. While young (2-3 months old) L-FABP null mice displayed no visually obvious phenotype, with increasing age >9 months the L-FABP null mice were visibly larger, exhibiting increased body weight due to increased fat and lean tissue mass. Liver lipid concentrations were unaffected by L-FABP gene ablation with the exception of triacylglycerol, which was decreased by 74% in the livers of 3-month-old mice. Likewise, serum lipid levels were not altered in L-FABP null mice with the exception of triacylglycerol, which was increased in the serum of 18-month-old mice. Increased body weight, fat tissue mass, and lean tissue mass in 18-month-old L-FABP null mice were accompanied by increased hepatic levels of low-density lipoprotein (LDL) receptor, peroxisome proliferator-activated receptor (PPAR) alpha, and PPARalpha-regulated proteins such as fatty acid transport protein (FATP), fatty acid translocase (FAT/CD36), carnitine palmitoyl transferase I (CPT I), and lipoprotein lipase (LPL). A key enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase, was down-regulated in L-FABP null mice. These findings were consistent with a proposed role for L-FABP as an important physiological regulator of PPARalpha.

Phytol-induced hepatotoxicity in mice.

Phytanic acid is a branched-chain, saturated fatty acid present in high concentrations in dairy products and ruminant fat. Some other dietary fats contain lower levels of phytol, which is readily converted to phytanic acid after absorption. Phytanic acid is a peroxisome proliferator binding the nuclear transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) to induce expression of genes encoding enzymes of fatty acid oxidation in peroxisomes and mitochondria. Administration of dietary phytol (0.5% or 1%) to normal mice for twelve to eighteen days caused consistent PPARalpha-mediated responses, such as lower body weights, higher liver weights, peroxisome proliferation, increased catalase expression, and hepatocellular hypertrophy and hyperplasia. Female mice fed 0.5% phytol and male and female mice fed 1% phytol exhibited midzonal hepatocellular necrosis, periportal hepatocellular fatty vacuolation, and corresponding increases in liver levels of the phytol metabolites phytanic acid and pristanic acid. Hepatic expression of sterol carrier protein-x (SCP-x) was five- to twelve-fold lower in female mice than in male mice. These results suggest that phytol may cause selective midzonal hepatocellular necrosis in mice, an uncommon pattern of hepatotoxic injury, and that the greater susceptibility of female mice may reflect a lower capacity to oxidize phytanic acid because of their intrinsically lower hepatic expression of SCP-x.

Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that affect lipid and glucose catabolism and storage.

Data on SD-OCT image acquisition, ultrastructural features, and horizontal tissue shrinkage in the porcine retina.

The data presented in this article are related to the research paper entitled "Correlation of Spectral Domain Optical Coherence Tomography with Histology and Electron Microscopy in the Porcine Retina" (Xie et al., 2018) [2]. This research data highlights our technique for retinal fundus image acquisition during spectral domain optical coherence tomography (SD-OCT) in a large animal model. Low and high magnification electron micrographs are included to demonstrate the ultrastructural features of the porcine retina. Data on horizontal tissue shrinkage during processing of the porcine retina are presented.

Spatial segregation of virulence gene expression during acute enteric infection with Salmonella enterica serovar Typhimurium.

UNLABELLED: To establish a replicative niche during its infectious cycle between the intestinal lumen and tissue, the enteric pathogen Salmonella enterica serovar Typhimurium requires numerous virulence genes, including genes for two type III secretion systems (T3SS) and their cognate effectors. To better understand the host-pathogen relationship, including early infection dynamics and induction kinetics of the bacterial virulence program in the context of a natural host, we monitored the subcellular localization and temporal expression of T3SS-1 and T3SS-2 using fluorescent single-cell reporters in a bovine, ligated ileal loop model of infection. We observed that the majority of bacteria at 2 h postinfection are flagellated, express T3SS-1 but not T3SS-2, and are associated with the epithelium or with extruding enterocytes. In epithelial cells, S. Typhimurium cells were surrounded by intact vacuolar membranes or present within membrane-compromised vacuoles that typically contained numerous vesicular structures. By 8 h postinfection, T3SS-2-expressing bacteria were detected in the lamina propria and in the underlying mucosa, while T3SS-1-expressing bacteria were in the lumen. Our work identifies for the first time the temporal and spatial regulation of T3SS-1 and -2 expression during an enteric infection in a natural host and provides further support for the concept of cytosolic S. Typhimurium in extruding epithelium as a mechanism for reseeding the lumen. IMPORTANCE: The pathogenic bacterium Salmonella enterica serovar Typhimurium invades and persists within host cells using distinct sets of virulence genes. Genes from Salmonella pathogenicity island 1 (SPI-1) are used to initiate contact and facilitate uptake into nonphagocytic host cells, while genes within SPI-2 allow the pathogen to colonize host cells. While many studies have identified bacterial virulence determinants in animal models of infection, very few have focused on virulence gene expression at the single-cell level during an in vivo infection. To better understand when and where bacterial virulence factors are expressed during an acute enteric infection of a natural host, we infected bovine jejunal-ileal loops with S. Typhimurium cells harboring fluorescent transcriptional reporters for SPI-1 and -2 (PinvF and PssaG, respectively). After a prescribed time of infection, tissue and luminal fluid were collected and analyzed by microscopy. During early infection (≤2 h), bacteria within both intact and compromised membrane-bound vacuoles were observed within the epithelium, with the majority expressing SPI-1. As the infection progressed, S. Typhimurium displayed differential expression of the SPI-1 and SPI-2 regulons, with the majority of tissue-associated bacteria expressing SPI-2 and the majority of lumen-associated bacteria expressing SPI-1. This underscores the finding that Salmonella virulence gene expression changes as the pathogen transitions from one anatomical location to the next.

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes.

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.

Effect of SCP-x gene ablation on branched-chain fatty acid metabolism.

Despite the importance of peroxisomal oxidation in branched-chain lipid (phytol, cholesterol) detoxification, little is known regarding the factors regulating the peroxisomal uptake, targeting, and metabolism of these lipids. Although in vitro data suggest that sterol carrier protein (SCP)-x plays an important role in branched-chain lipid oxidation, the full physiological significance of this peroxisomal enzyme is not completely clear. To begin to resolve this issue, SCP-x-null mice were generated by gene ablation of SCP-x from the SCP-x/SCP-2 gene and fed a phytol-enriched diet to characterize the effects of lipid overload in a system with minimal 2/3-oxoacyl-CoA thiolytic activity. It was shown that SCP-x gene ablation 1) did not result in reduced expression of SCP-2 (previously thought to be derived in considerable part by posttranslational cleavage of SCP-x); 2) increased expression levels of key enzymes involved in alpha- and beta-oxidation; and 3) altered lipid distributions, leading to decreased hepatic fatty acid and triglyceride levels. In response to dietary phytol, lack of SCP-x resulted in 1) accumulation of phytol metabolites despite substantial upregulation of hepatic peroxisomal and mitochondrial enzymes; 2) reduced body weight gain and fat tissue mass; and 3) hepatic enlargement, increased mottling, and necrosis. In summary, the present work with SCP-x gene-ablated mice demonstrates, for the first time, a direct physiological relationship between lack of SCP-x and decreased ability to metabolize branched-chain lipids.

Gender differences in response of hippocampus to chronic glucocorticoid stress: role of glutamate receptors.

Glucocorticoids (GC) play critical roles in the pathophysiological reactions to environmental stress. In brain, morphological changes were examined in hippocampal CA3 neurons with 2 weeks of chronic elevation of GC in male and female mice. Molecular correlates and underlying mechanisms paralleling these morphologic changes in hippocampus were investigated. Although the hippocampal neurons in the CA3 area in male mice atrophy with chronically elevated GC, female mice show minimal morphological changes with comparable GC regimens. These sexual morphological differences correlate with differences in the postsynaptic dense protein (PSD95) as well as the spectrum of glutamate receptors induced by GC treatment in male and female mice, including NMDA, AMPA, and KA receptors. These findings suggest that synaptic receptor composition is adapted to the unique physiological requirements of males and females and illuminate underlying mechanisms of GC/stress responses in the brain.

Effect of branched-chain fatty acid on lipid dynamics in mice lacking liver fatty acid binding protein gene.

Although a role for liver fatty acid protein (L-FABP) in the metabolism of branched-chain fatty acids has been suggested based on data obtained with cultured cells, the physiological significance of this observation remains to be demonstrated. To address this issue, the lipid phenotype and metabolism of phytanic acid, a branched-chain fatty acid, were determined in L-FABP gene-ablated mice fed a diet with and without 1% phytol (a metabolic precursor to phytanic acid). In response to dietary phytol, L-FABP gene ablation exhibited a gender-dependent lipid phenotype. Livers of phytol-fed female L-FABP-/- mice had significantly more fatty lipid droplets than male L-FABP-/- mice, whereas in phytol-fed wild-type L-FABP+/+ mice differences between males and females were not significant. Thus L-FABP gene ablation exacerbated the accumulation of lipid droplets in phytol-fed female, but not male, mice. These results were reflected in the lipid profile, where hepatic levels of triacylglycerides in phytol-fed female L-FABP-/- mice were significantly higher than in male L-FABP-/- mice. Furthermore, livers of phytol-fed female L-FABP-/- mice exhibited more necrosis than their male counterparts, consistent with the accumulation of higher levels of phytol metabolites (phytanic acid, pristanic acid) in liver and serum, in addition to increased hepatic levels of sterol carrier protein (SCP)-x, the only known peroxisomal enzyme specifically required for branched-chain fatty acid oxidation. In summary, L-FABP gene ablation exerted a significant role, especially in female mice, in branched-chain fatty acid metabolism. These effects were only partially compensated by concomitant upregulation of SCP-x in response to L-FABP gene ablation and dietary phytol.

Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo.

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.

Sexually dimorphic metabolism of branched-chain lipids in C57BL/6J mice.

Despite the importance of branched chain lipid oxidation in detoxification, almost nothing is known regarding factors regulating peroxisomal uptake, targeting, and metabolism. One peroxisomal protein, sterol carrier protein-x (SCP-x), is thought to catalyze a key thiolytic step in branched chain lipid oxidation. When mice with substantially lower hepatic levels of SCP-x were tested for susceptibility to dietary stress with phytol (a phytanic acid precursor and peroxisome proliferator), livers of phytol-fed female but not male mice i). accumulated phytol metabolites (phytanic acid, pristanic acid, and Delta-2,3-pristanic acid); ii). exhibited decreased fat tissue mass and increased liver mass/body mass; iii). displayed signs of histopathological lesions in the liver; and iv). demonstrated significant alterations in hepatic lipid distributions. Moreover, both male and female mice exhibited phytol-induced peroxisomal proliferation, as demonstrated by liver morphology and upregulation of the peroxisomal protein catalase. In addition, levels of liver fatty acid binding protein, along with SCP-2 and SCP-x, increased, suggesting upregulation mediated by phytanic acid, a known ligand agonist of the peroxisomal proliferator-activated receptor alpha. In summary, the present work establishes a role for SCP-x in branched chain lipid catabolism and demonstrates a sexual dimorphic response to phytol, a precursor of phytanic acid, in lipid parameters and hepatotoxicity.