CD36 regulates fatty acid composition and sensitivity to insulin in 3T3-L1 adipocytes.

In the current study, we tested a hypothesis that CD36 fatty acid (FA) transporter might affect insulin sensitivity by indirect effects on FA composition of adipose tissue. We examined the effects of CD36 downregulation by RNA interference in 3T3-L1 adipocytes on FA transport and composition and on sensitivity to insulin action. Transfected 3T3-L1 adipocytes, without detectable CD36 protein, showed reduced neutral lipid levels and significant differences in FA composition when levels of essential FA and their metabolites were lower or could not be detected including gamma linolenic (C18:3 n6), eicosadienic (C20:2 n6), dihomo-gamma linolenic (C20:3 n6), eicosapentaenoic (EPA) (C20:5 n3), docosapentaenoic (DPA) (C22:5 n3), and docosahexaenoic (DHA) (C22:6 n3) FA. Transfected 3T3-L1 adipocytes exhibited a significantly higher n6/n3 FA ratio, reduced 5-desaturase and higher 9-desaturase activities. These lipid profiles were associated with a significantly reduced insulin-stimulated glucose uptake (4.02+/-0.1 vs. 8.42+/-0.26 pmol.10(-3) cells, P=0.001). These findings provide evidence that CD36 regulates FA composition thereby affecting sensitivity to insulin action in 3T3-L1 adipocytes.

Lipopolysaccharide exposure makes allergic airway inflammation and hyper-responsiveness less responsive to dexamethasone and inhibition of iNOS.

Allergic airway disease can be refractory to anti-inflammatory treatment, whose cause is unclarified. Therefore, in the present experiment, we have tested the hypothesis that co-exposure to lipopolysacharide (Lps) and allergen results in glucocorticoid-resistant eosinophil airway inflammation and hyper-responsiveness (AHR). Ovalbumin (Ova)-sensitized BALB/c mice were primed with 10 microg intranasal Lps 24 h before the start of Ova challenges (20 min on 3 consecutive days). Dexamethasone (5 mg/kg/day) was given on the last 2 days of Ova challenges. AHR, cellular build-up, cytokine and nitrite concentrations of bronchoalveolar lavage fluid (BALF) and lung histology were examined. To assess the role of iNOS-derived NO in airway responsiveness, mice were treated with a selective inhibitor of this enzyme (1400W) 2 h before AHR measurements. More severe eosinophil inflammation and higher nitrite formation were found in Lps-primed than in non-primed allergized mice. After Lps priming, AHR and concentrations of T-helper type 2 cytokines in BALF were decreased, but still remained significantly higher than in controls. Eosinophil inflammation was partially, while nitrite production and AHR were observed to be largely dexamethasone resistant in Lps-primed allergized animals. 1400W effectively and rapidly diminished the AHR in Ova-sensitized and challenged mice, but failed to affect it after Lps priming plus allergization. In conclusion, Lps inhalation may exaggerate eosinophil inflammation and reduce responsiveness to anti-inflammatory treatment in allergic airway disease.

HO-1 expression in type II pneumocytes after transpulmonary gene delivery.

Somatic cell gene transfer is a potentially useful strategy to alter lung function. However, achieving efficient transfer to the alveolar epithelium, especially in smaller animals, has not been demonstrated. In this study, the rat heme oxygenase-1 (HO-1) gene was delivered to the lungs of neonatal mice via transpulmonary injection. A bidirectional promoter construct coexpressing both HO-1 and a luciferase reporter gene was used so that in vivo gene expression patterns could be monitored in real time. HO-1 expression levels were also modulated with doxycycline and assessed in vivo with bioluminescent light transmitted through the tissues from the coregulated luciferase reporter. As a model of oxidative stress and HO-1-mediated protection, groups of animals were exposed to hyperoxia. After gene transfer, elevated levels of HO-1 were detected predominantly in alveolar type II cells by immunocytochemistry. With overexpression of HO-1, increased oxidative injury was observed. Furthermore, this model demonstrated a cell-specific effect of lung HO-1 overexpression in oxidative stress. Specific control of expression for therapeutic genes is possible in vivo. The transpulmonary approach may prove useful in targeting gene expression to cells of the alveolar epithelium or to circumscribed areas of the lung.

Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor.

Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'-untranslated region of 2.2 kb. The open reading frame encodes a 1,245-residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells.

Intracellular distribution of photosensitizing porphyrins measured by video-enhanced fluorescence microscopy.

The intracellular distribution of photosensitizing porphyrins with different amounts of aggregates was measured in a fluorescence microscope using video-intensified detection and image processing. Porphyrin fluorescence was localized in the plasma membrane, cytoplasm, nuclear membrane and nucleoli. The fluorescence intensity in the plasma membrane decreased with increasing aggregation of the photosensitizers. In addition, a redistribution of the porphyrin molecules from the plasma membrane to the nuclear membrane and adjacent intracellular sites was observed with increasing incubation time. Photobleaching of porphyrin fluorescence was most pronounced in the plasma membrane and least efficient within the nucleoli.

Primary structure of the mouse laminin B2 chain and comparison with laminin B1.

One of the major components of basement membranes is the glycoprotein laminin, made up of three disulfide-bonded subunits, the A, B1, and B2 chains. We have isolated and sequenced overlapping mouse laminin B2 chain cDNA clones covering 7562 base pairs. The deduced amino acid sequence predicts that the mature B2 chain consists of 1572 residues, has an unglycosylated molecular weight of 173,541, and possesses 14 potential N-linked glycosylation sites. Analysis of the predicted secondary structure shows the presence of six domains, two rich in alpha-helical structure, two composed of homologous cysteine-rich repeat units, and two globular regions. The organization of the molecule is very similar to that of the mouse laminin B1 chain, and significant sequence homology between the B1 and B2 chains was found in their two cysteine-rich domains and in their amino-terminal globular domains.

Carboxyl-terminal sequence of entactin deduced from a cDNA clone.

Entactin is a widely distributed basement membrane sulfated glycoprotein of approximately equal to 150 kDa. The entactin gene is expressed early in mouse embryogenesis. Two cDNA clones complementary to rat entactin mRNA were isolated by antibody screening of an oligo(dT)-primed cDNA library constructed in the lambda gt11 expression vector. One of the clones, lambda 1E, was subcloned into plasmid pBR322 and further characterized. The clone contained sequences complementary to an mRNA species 6 kilobases in length. This mRNA was translated in rabbit reticulocyte lysates to yield a polypeptide of 143 kDa that was precipitated with anti-entactin antiserum. The cDNA insert, 1328 base pairs long, was sequenced and found to contain an open reading frame of 729 base pairs that coded for 243 amino acids at the carboxyl terminus of entactin. Analysis of the peptide revealed no extended alpha-helical or beta-sheet secondary structures. Radiolabeled probes prepared by nicktranslation of p lambda 1E were used to monitor the steady-state levels of entactin mRNA in F9 embryonal carcinoma cells that were induced to differentiate by exposure to retinoic acid and dibutyryl cyclic AMP. The increase in steady-state levels of entactin mRNA lagged behind the increase in mRNA for the B2 chain of laminin, suggesting that laminin and entactin are independently rather than coordinately regulated.