Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

Treatment of rats with a self-selected hyperlipidic diet, increases the lipid content of the main adipose tissue sites in a proportion similar to that of the lipids in the rest of organs and tissues

Adipose tissue (AT) is distributed as large differentiated masses, and smaller depots covering vessels, and organs, as well as interspersed within them. The differences between types and size of cells makes AT one of the most disperse and complex organs. Lipid storage is partly shared by other tissues such as muscle and liver. We intended to obtain an approximate estimation of the size of lipid reserves stored outside the main fat depots. Both male and female rats were made overweight by 4-weeks feeding of a cafeteria diet. Total lipid content was analyzed in brain, liver, gastrocnemius muscle, four white AT sites: subcutaneous, perigonadal, retroperitoneal and mesenteric, two brown AT sites (interscapular and perirenal) and in a pool of the rest of organs and tissues (after discarding gut contents). Organ lipid content was estimated and tabulated for each individual rat. Food intake was measured daily. There was a surprisingly high proportion of lipid not accounted for by the main macroscopic AT sites, even when brain, liver and BAT main sites were discounted. Muscle contained about 8% of body lipids, liver 1-1.4%, four white AT sites lipid 28-63% of body lipid, and the rest of the body (including muscle) 38-44%. There was a good correlation between AT lipid and body lipid, but lipid in"other organs" was highly correlated too with body lipid. Brain lipid was not. Irrespective of dietary intake, accumulation of body fat was uniform both for the main lipid storage and handling organs: large masses of AT (but also liver, muscle), as well as in the"rest" of tissues. These storage sites, in specialized (adipose) or not-specialized (liver, muscle) tissues reacted in parallel against a hyperlipidic diet challenge. We postulate that body lipid stores are handled and regulated coordinately, with a more centralized and overall mechanisms than usually assumed.

Effect of feed fat by-products with trans fatty acids and heated oil on cholesterol and oxycholesterols in chicken

Chicken is the most widely consumed meat all over the world due to chickens being easy to rear, their fast growth rate and the meat having good nutritional characteristics. The main objective of this paper was to study the effects of dietary fatty by-products in low, medium and high levels of oxidized lipids and trans fatty acids (TFAs) on the contents of cholesterol and oxycholesterols in meat, liver, and plasma of chickens. A palm fatty acid distillate, before and after hydrogenation, and a sunflower-olive oil blend (70/30, v/v) before and after use in a commercial frying process were used in feeding trials after adding 6% of the fats to the feeds. Highly oxidized lipid and TFA feeds significantly increased the contents of cholesterol and oxycholesterols in all tissues of chicken (0.01 < p <= 0.05). The contents of oxycholesterols in chicken meat, liver and plasma obtained from TFA feeding trials varied between 17 and 48 μg/100 g in meat, 19-42 μg/100 g in liver and 105-126 μg/dL in plasma. In contrast, in the oxidized lipid feeding trials, oxycholesterols varied between 13 and 75 μg/100 g in meat, 30-58 μg/100 g in liver and 66-209 μg/dL in plasma. Meat from chickens fed with feeds containing high levels of TFAs or oxidized lipids may contribute to higher ingestion of cholesterol and oxycholesterols by humans.

Plasma homocysteine and vitamin B12 serum levels, red blood cell folate concentrations, C677T methylenetetrahydrofolate reductase gene mutation and risk of recurrent miscarriage: a case-control study in Spain.

Background: Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) gene mutation have been postulated as a possible cause of recurrent miscarriage (RM). There is a wide variation in the prevalence of MTHFR polymorphisms and homocysteine (Hcy) plasma levels among populations around the world. The present study was undertaken to investigate the possible association between hyperhomocysteinemia and its causative genetic or acquired factors and RM in Catalonia, a Mediterranean region in Spain. Methods: Sixty consecutive patients with ≥ 3 unexplained RM and 30 healthy control women having at least one child but no previous miscarriage were included. Plasma Hcy levels, MTHFR gene mutation, red blood cell (RBC) folate and vitamin B12 serum levels were measured in all subjects. Results: No significant differences were observed neither in plasma Hcy levels, RBC folate and vitamin B12 serum levels nor in the prevalence of homozygous and heterozygous MTHFR gene mutation between the two groups studied. Conclusions: In the present study RM is not associated with hyperhomocysteinemia, and/or the MTHFR gene mutation.

Urinary and plasma catecholamines and metanephrines in dogs with pheochromocytoma, hypercortisolism, nonadrenal disease and in healthy dogs.

BACKGROUND: Diagnosis of pheochromocytoma (PC) is based on a combination of clinical suspicion, finding an adrenal mass, increased plasma, and urine concentrations of catecholamine metabolites and is finally confirmed with histopathology. In human medicine, it is controversial whether biochemically testing plasma is superior to testing urine. OBJECTIVES: To measure urinary and plasma catecholamines and metanephrines in healthy dogs, dogs with PC, hypercortisolism (HC), and nonadrenal diseases (NAD) and to determine the test with the best diagnostic performance for dogs with PC. ANIMALS: Seven PC dogs, 10 dogs with HC, 14 dogs with NAD, 10 healthy dogs. METHODS: Prospective diagnostic clinical study. Urine and heparin plasma samples were collected and stored at -80°C before analysis using high-pressure liquid chromatography (HPLC) coupled to electrochemical detection or tandem mass spectrometry were performed. Urinary variables were expressed as ratios to urinary creatinine concentration. RESULTS: Dogs with PC had significantly higher urinary normetanephrine and metanephrine : creatinine ratios and significantly higher plasma-total and free normetanephrine and plasma-free metanephrine concentrations compared to the 3 other groups. There were no overlapping results of urinary normetanephrine concentrations between PC and all other groups, and only one PC dog with a plasma normetanephrine concentration in the range of the dogs with HC and NAD disease. Performances of total and free plasma variables were similar. Overlap of epinephrine and norepinephrine results between the groups was large with both urine and plasma. CONCLUSION AND CLINICAL IMPORTANCE: Measurement of normetanephrine is the preferred biochemical test for PC and urine was superior to plasma.

4B.05: Plasma Lasma copeptin is associated with insulin resistance in a Swiss population-based study

OBJECTIVE: Previous studies suggest that arginine vasopressin may have a role in metabolic syndrome (MetS) and diabetes by altering liver glycogenolysis, insulin, and glucagon secretion and pituitary ACTH release. We tested whether plasma copeptin, the stable C-terminal fragment of arginine vasopressin prohormone, was associated with insulin resistance and MetS in a Swiss population-based study. DESIGN AND METHOD: We analyzed data from the population-based Swiss Kidney Project on Genes in Hypertension. Copeptin was assessed by an immunoluminometric assay. Insulin resistance was derived from the HOMA model and calculated as follows: (FPI x FPG)/22.5, where FPI is fasting plasma insulin concentration (mU/L) and FPG fasting plasma glucose (mmol/L). Subjects were classified as having the MetS according to the National Cholesterol Education Program Adult Treatment Panel III criteria. Mixed multivariate linear regression models were built to explore the association of insulin resistance with copeptin. In addition, multivariate logistic regression models were built to explore the association between MetS and copeptin. In the two analyses, adjustment was done for age, gender, center, tobacco and alcohol consumption, socioeconomic status, physical activity, intake of fruits and vegetables and 24 h urine flow rate. Copeptin was log-transformed for the analyses. RESULTS: Among the 1,089 subjects included in this analysis, 47% were male. Mean (SD) age and body mass index were 47.4 (17.6) years 25.0 (4.5) kg/m2. The prevalence of MetS was 10.5%. HOMA-IR was higher in men (median 1.3, IQR 0.7-2.1) than in women (median 1.0, IQR 0.5-1.6,P < 0.0001). Plasma copeptin was higher in men (median 5.2, IQR 3.7-7.8 pmol/L) than in women (median 3.0, IQR 2.2-4.3 pmol/L), P < 0.0001. HOMA-IR was positively associated with log-copeptin after full adjustment (β (95% CI) 0.19 (0.09-0.29), P < 0.001). MetS was not associated with copeptin after full adjustment (P = 0.92). CONCLUSIONS: Insulin resistance, but not MetS, was associated with higher copeptin levels. Further studies should examine whether modifying pharmacologically the arginine vasopressin system might improve insulin resistance, thereby providing insight into the causal nature of this association.

Effect of sex and prior exposure to a cafeteria diet on the distribution of sex hormones between plasma and blood cells

It is generally assumed that steroid hormones are carried in the blood free and/or bound to plasma proteins. We investigated whether blood cells were also able to bind/carry sex-related hormones: estrone, estradiol, DHEA and testosterone. Wistar male and female rats were fed a cafeteria diet for 30 days, which induced overweight. The rats were fed the standard rat diet for 15 additional days to minimize the immediate effects of excess ingested energy. Controls were always kept on standard diet. After the rats were killed, their blood was used for 1) measuring plasma hormone levels, 2) determining the binding of labeled hormones to washed red blood cells (RBC), 3) incubating whole blood with labeled hormones and determining the distribution of label between plasma and packed cells, discounting the trapped plasma volume, 4) determining free plasma hormone using labeled hormones, both through membrane ultrafiltration and dextrancharcoal removal. The results were computed individually for each rat. Cells retained up to 32% estrone, and down to 10% of testosterone, with marked differences due to sex and diet (the latter only for estrogens, not for DHEA and testosterone). Sex and diet also affected the concentrations of all hormones, with no significant diet effects for estradiol and DHEA, but with considerable interaction between both factors. Binding to RBC was non-specific for all hormones. Estrogen distribution in plasma compartments was affected by sex and diet. In conclusion: a) there is a large non-specific RBC-carried compartment for estrone, estradiol, DHEA and testosterone deeply affected by sex; b) Prior exposure to a cafeteria (hyperlipidic) diet induced hormone distribution changes, affected by sex, which hint at sex-related structural differences in RBC membranes; c) We postulate that the RBC compartment may contribute to maintain free (i.e., fully active) sex hormone levels in a way similar to plasma proteins non-specific binding.

Obtencion de recubrimientos autolubricados por proyeccion termica de plasma atmosferico (APS) como alternativa a los lubricantes liquidos

La obtención de recubrimientos con bajos coeficientes de fricción, es una innovadora respuesta a la creciente demanda industrial de alternativas para los tradicionales sistemas lubricados con aceites. Para este fin, la introducción de un sólido lubricante, en una matriz con buenas propiedades mecánicas, ha de conferir al conjunto un bajo coeficiente de fricción y mejorar la resistencia al desgaste del recubrimiento, favoreciendo además, la utilización a elevadas temperaturas, en las que los lubricantes líquidos convencionales presentan limitaciones. En el Centro de Proyección Térmica (CPT) de la Universidad de Barcelona se han obtenido mediante proyección de Plasma Atmosférico (APS) recubrimientos autolubricados de Ni-Grafito y Cr2O3-CaF2, estudiando con especial interés las propiedades y el efecto que produce el grafito y el CaF2 en las matrices metálica y cerámica respectivamente. Se ha determinado en los dos casos que el desgaste originado en los recubrimientos es bajo, si bien los coeficientes de fricción varían ostensiblemente en función del sistema indicado. Los resultados obtenidos indican que el recubrimiento de Ni-Grafito posee un comportamiento idóneo capaz de competir con los tradicionales lubricantes líquidos.

Moderately oxidized oils and dietary zinc and alpha-tocopheryl acetate supplementation: effects on the oxidative stability of rabbit plasma, liver, and meat

The aim of this study was to assess the alterations in plasma, liver, and meat oxidative stability and R-tocopherol content when moderately oxidized sunflower oils were added to feeds and when feeds were supplemented with R-tocopheryl acetate (100 mg/kg) and Zn (200 mg/kg). The effects of cooking the meat and its subsequent refrigeration were also studied. When the content of primary oxidation compounds of the oil was high, rabbit plasma, liver, and meat R-tocopherol content was reduced and meat susceptibility to oxidation increased. The addition of oil with a high content of secondary oxidation compounds (oil heated at 140 'C, 31 h) to feed also led to an increase in meat susceptibi- lity to oxidation, although it presented an R-tocopherol content similar to that of nonheated oil. Feed supplementation with R-tocopheryl acetate increased tissue R-tocopherol content and improved the oxidative stability of liver and meat. However, in the latter, it was less effective when oil heated at 55 'C was added.

Oncogenic K-Ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status

Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation - by activation of protein kinase C (PKC) - increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide - for the first time - evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.

Oxidized oils and dietary zinc and alpha-tocopheryl acetate supplementation: effects on rabbit plasma, liver and meat fatty acid composition and meat Zn, Cu, Fe and Se content

The effects of the addition of heated oils to feeds (3%, w/w) and the dietary supplementation with a-tocopheryl acetate (TA; 100 mg/kg) and Zn (200 mg/kg) on rabbit tissue fatty acid (FA) composition and on the Zn, Cu, Fe and Se content in meat were assessed. Heating unrefined sunflower oil (SO) at 558C for 245 h increased its content in primary oxidation products and reduced its a-tocopherol content. However, this did not significantly affect tissue FA composition. Heating SO at 1408C for 31 h increased its content in secondary oxidation products and in some FA isomers asc9,t11-CLA and di-trans CLA. This led to increases in di-trans CLA in liver and in t9,c12-18:2 in meat. The c9,t11-CLA was the most incorporated CLA isomer in tissues. The dietary supplementation with a-TA did not affect the FA composition of plasma, liver or meat. The cooking of vacuum-packed rabbit meat at 788C for 5 min reduced significantly but slightly its polyunsaturated FA content. The dietary supplementation with Zn did not modify the content of Zn, Fe or Se in meat, but it reduced its Cu content. On the other hand, it increased the content of some FAs in meat when SO heated at 1408C for 31 h was added to feeds.

Proteinuria Increases Plasma Phosphate by Altering Its Tubular Handling.

Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesis, we studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF-23) concentration, plasma phosphate concentration, and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross-sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria≥300 mg/24 hours is predictive of higher phosphate levels, independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF-23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations, we induced glomerular proteinuria in two animal models. Rats with puromycin-aminonucleoside-induced nephrotic proteinuria displayed higher renal protein expression of the sodium-phosphate co-transporter NaPi-IIa, lower renal Klotho protein expression, and decreased phosphorylation of FGF receptor substrate 2α, a major FGF-23 receptor substrate. These findings were confirmed in transgenic mice that develop nephrotic-range proteinuria resulting from podocyte depletion. In vitro, albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion, we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubule NaPi-IIa expression secondary to decreased FGF-23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF-23 concentrations, potentially contributing to cardiovascular disease.

Impact of platelet rich plasma and adipose stem cells on lymphangiogenesis in a murine tail lymphedema model.

BACKGROUND: Lymphedema is an underdiagnosed pathology which in industrialized countries mainly affects cancer patients that underwent lymph node dissection and/or radiation. Currently no effective therapy is available so that patients' life quality is compromised by swellings of the concerned body region. This unfortunate condition is associated with body imbalance and subsequent osteochondral deformations and impaired function as well as with an increased risk of potentially life threatening soft tissue infections. METHODS: The effects of PRP and ASC on angiogenesis (anti-CD31 staining), microcirculation (Laser Doppler Imaging), lymphangiogenesis (anti-LYVE1 staining), microvascular architecture (corrosion casting) and wound healing (digital planimetry) are studied in a murine tail lymphedema model. RESULTS: Wounds treated by PRP and ASC healed faster and showed a significantly increased epithelialization mainly from the proximal wound margin. The application of PRP induced a significantly increased lymphangiogenesis while the application of ASC did not induce any significant change in this regard. CONCLUSIONS: PRP and ASC affect lymphangiogenesis and lymphedema development and might represent a promising approach to improve regeneration of lymphatic vessels, restore disrupted lymphatic circulation and treat or prevent lymphedema alone or in combination with currently available lymphedema therapies.