Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8 kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 μM; 0.25μCi) [1-14C]oleate for 6 h increased incorporation of [14C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an ∼89 kDa band in liver and testis from GPAT1 null mice and both 89 and 80 kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis

Modulatory Role for Retinoid-related Orphan Receptor α in Allergen-induced Lung Inflammation

Rationale: Nuclear receptors play a critical role in the regulation of inflammation, thus representing attractive targets for the treatment of asthma

Modulatory Role for Retinoid-related Orphan Receptor α in Allergen-induced Lung Inflammation

Rationale: Nuclear receptors play a critical role in the regulation of inflammation, thus representing attractive targets for the treatment of asthma. Objective: In this study, we assess the potential regulatory function of retinoid-related orphan receptor α (RORα) in the adaptive immune response using ovalbumin (OVA)-induced airway inflammation as a model. Methods: Allergen-induced inflammation was compared between wild-type (WT) and staggerer (RORα(sg/sg)) mice, a natural mutant strain that is deficient in RORα expression. Measurements and Main Results: Despite robust increases in OVA-specific IgE, RORα(sg/sg) mice developed significantly less pulmonary inflammation, mucous cell hyperplasia, and eosinophilia compared with similarly treated WT animals. Induction of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, was also significantly less in RORα(sg/sg) mice. Microarray analysis using lung RNA showed increased expression of many genes, previously implicated in inflammation, in OVA-treated WT mice. These include mucin Muc5b, the chloride channel calcium-activated 3 (Clca3), macrophage inflammatory protein (MIP) 1α and 1β, eotaxin-2, serum amyloid A3 (Saa3), and insulin-like growth factor 1 (Igf1). These genes were induced to a greater extent in OVA-treated WT mice relative to RORα(sg/sg) mice. Conclusions: Our study demonstrates that mice deficient in RORα exhibit an attenuated allergic inflammatory response, indicating that RORα plays a critical role in the development of Th2-driven allergic lung inflammation in mice, and suggests that this nuclear receptor should be further evaluated as a potential asthma target

Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

Lysophosphatidic acid (LPA) is an agonist for peroxisome proliferator activated receptor-γ (PPARγ). Although glycerol-3-phosphate acyltransferase-1 (GPAT1) esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO) cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA) or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression

A comparison of service organisation and guideline compliance between two adjacent European health services

Introduction Outcomes in stroke patients are improved by a co-ordinated organisation of stroke services and provision of evidence-based care. We studied the organisation of care and application of guidelines in two neighbouring health care systems with similar characteristics. Methods Organisational elements of the 2015 National Stroke Audit (NSA) from the Republic of Ireland (ROI) were compared with the Sentinel Stroke National Audit Programme (SSNAP) in Northern Ireland (NI) and the United Kingdom (UK). Compliance was compared with UK and European guidelines. Results Twenty-one of 28 ROI hospitals (78%) reported having a stroke unit (SU) compared with all 10 in NI. Average SU size was smaller in ROI (6 beds vs. 15 beds) and bed availability per head of population was lower (1:30,633 vs. 1:12,037 p &lt; 0.0001 Chi Sq). Fifty-four percent of ROI patients were admitted to SU care compared with 96% of UK patients ( p &lt; 0.0001). Twenty-four–hour physiological monitoring was available in 54% of ROI SUs compared to 91% of UK units ( p &lt; 0.0001). There was no significant difference between ROI and NI in access to senior specialist physicians or nurses or in SU nurse staffing (3.9/10 beds weekday mornings) but there was a higher proportion of trained nurses in ROI units (2.9/10 beds vs. 2.3/10 beds ( p = 0.02 Chi Sq). Conclusion Whilst the majority of hospitals in both jurisdictions met key criteria for organised stroke care the small size and underdevelopment of the ROI units meant a substantial proportion of patients were unable to access this specialised care. </jats:sec

Treating CHO and CHO-GPAT1 cells with LPA, PA, or DiC8∶0 did not enhance PPARγ activity.

<p>CHO and CHO-GPAT1 cells were transfected with 0.1 μg of pRLSV40 (internal LUC control), and equal concentrations (0.4 μg) of a PPARγ expression vector, an RXR expression vector, and a PPRE-CAT reporter vector. Twenty-four hours later, the transfected cells were treated with vehicle (0.1% BSA), 1 μM troglitazone, 5 µM oleoyl-LPA, 5 µM dioleoyl-PA, or 10 µM DiC8∶0. CAT activity was measured 24 h later and normalized to LUC activity. The results (mean +/− SEM; n = 3) are expressed as relative CAT activity. Results are representative of two independent experiments. *p<0.05 and **p≤0.01 when comparing within treatment groups.</p

Adding AGPAT2 and DGKα to CHO-GPAT1 cells decreased PPARγ activity.

<p>(<b>A</b>) CHO and CHO-GPAT1 cells were transfected with 0.1 µg of pRLSV40 (internal LUC control), and equal concentrations (0.4 µg) of expression and reporter vectors, PPARγ RXR, PPRE-CAT, and either empty vector or an AGPAT2 expression vector. (<b>B</b>) Cells were similarly transfected with the vectors described plus either a wild-type (inactive) DGKα expression vector (−) or the constitutively active DGKα Δ196 expression vector (+). Twenty-four hours after transfection, cells were treated with either vehicle or 1 µM troglitazone. CAT activity was measured 24 h later and normalized to LUC activity. The results (mean +/− SEM; n = 3) are expressed as relative CAT activity. Results are representative of three and two independent experiments, respectively. *p<0.05 and **p≤0.01 when comparing within treatment groups.</p