Changes in selected blood component values of rainbow trout, Salmo gairdneri Richardson, following the blocking of the cortisol stress response with betamethasone and subsequent exposure to phenol or hypoxia

Cortisol was undetectable in rainbow trout plasma 24 h after the fish had been injected with betamethasone. This drug is known to suppress ACTH release and, consequently, cortisol production in teleosts. The blood glucose, chloride, sodium, potassium and total protein concentrations were not significantly affected by betamethasone. However, significantly increased blood packed cell volume values were found.
Betamethasone-injected fish when exposed to hypoxia or phenol had a pattern of blood component changes similar to those found in sham-injected fish also exposed to the pollutants. These changes in turn were similar to those found in exposed but uninjected and unhandled fish. Suppression of rainbow trout plasma cortisol concentrations with betamethasone apparently has no effect on the fish's short-term blood component responses to pollutants under the conditions used in these experiments, but does prevent a stressormediated increase in plasma cortisol concentrations.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Nutrition and Physical Activity Program to Attenuate Obesity and Promote Physical and Metabolic Fitness in Elementary School Children

Obesity and low levels of physical and metabolic fitness are risk factors for cardiovascular disease and diabetes. The purpose of this investigation was to attenuate obesity and improve physical and metabolic fitness in elementary school children. Schools have the opportunity, mechanisms, and personnel in place to deliver nutrition education, fitness activities, and a school food service that is nutritious and healthy. Cohorts from grades 3 to 5 in two school districts in rural Nebraska (Intervention/Control) participated in a 2-year study of physical activity and modified school lunch program. Data collection for aerobic capacity, body composition, blood chemistry, nutrition knowledge, energy intake, and physical activity was at the beginning and end of each year. Int received enhanced physical activity, grade specific nutrition education, and a lower fat and sodium school lunch program. Con continued with a regular school lunch and team sports activity program. At year 2, Int lunches had significantly less energy (9%), fat (25%), sodium (21%), and more fiber (17%). However, measures of 24-hour energy intake for Int and Con showed significant differences for sodium only. Physical activity in the classroom was 6% greater for Int compared to Con (p < 0.05) but physical activity outside of school was ?16 % less for Int compared to Con (p < 0.05). Body weight and body fat were not different between schools for normal weight or obese children. No differences were found for cholesterol, insulin, and glucose; however, HDL cholesterol was significantly greater and cholesterol/HDL was significantly less for Int compared to Con (p < 0.05). It appears that compensation in both energy intake and physical activity outside of school may be responsible for the lack of differences between Int and Con.     

Resynchronization of ovulation and timed insemination in lactating dairy cows III. Administration of GnRH 23 days post AI and ultrasonography for nonpregnancy diagnosis on day 30

The objective was to compare pregnancy rates to resynchronization and timed AI (TAI) protocols in lactating dairy cows that received GnRH at 23 d and were diagnosed not pregnant at 30 d after the pre-enrollment AI. Nonpregnant cows (624) at ultrasonography on day 30 (study day 0) were classified as diestrus (74.8%), metestrus (5.6%) and without a CL (19.5%). Cows in diestrus were assigned either to the GnRH group (PGF2alpha on day 0, GnRH on day 2 and TAI 16 h later, n = 238) or the estradiol cypionate (ECP) group (PGF2alpha on day 0, ECP on day 1, and TAI 36 h later, n = 229). Cows in metestrus were assigned to the Modified Heatsynch Group (GnRH on day 0, PGF(2alpha) on day 7, ECP on day 8 and TAI on day 9, n = 35). Cows without a CL (n = 122) were classified either as proestrus (10.6%), ovarian cysts (7.5%) or anestrus (1.4%), and assigned to factorial treatments (i.e., use of GnRH versus CIDR) to either the GnRH group (GnRH on day 0, PGF2alpha on day 7, GnRH on day 9 and TAI 16 h later, n = 28), the CIDR group (CIDR insert from days 0 to 7, PGF2alpha on day 7, GnRH on day 9 and TAI 16 h later, n = 34), the GnRH + CIDR group (GnRH on day 0, CIDR insert from days 0 to 7, PGF2alpha on day 7, GnRH on day 9 and TAI 16h later, n = 32), and the control group (PGF2alpha on day 7, GnRH on day 9 and TAI 16 h later, n = 28). For cows without a CL, plasma P4 concentrations were determined on days 0, 7, 10 and 17 and ovarian structures determined on days 0, 7 and 17. Pregnancy rates were evaluated at 30, 55 and 90 d after the resynchronized AI. For cows in diestrus, there were no differences in pregnancy rates on days 30, 55 and 90 for cows in the GnRH (27.5, 26.5 and 24.2%) or ECP (29.1, 25.5 and 24.1%) groups. In addition, there were no differences in pregnancy losses between days 30 and 55 and 55 and 90 between the GnRH (7.0 and 8.6%) and ECP (9.8 and 5.4%) groups. For cows without a CL, GnRH on day 0 increased the proportion of cows with a CL on days 7 and 17 and plasma P4 concentration on day 17 in cows with ovarian cysts but not for cows in proestrus. The CIDR insert increased pregnancy rate in cows with ovarian cysts but reduced pregnancy rate for cows in proestrus.     

Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby canine kidney cells

BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.     

Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.     

Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells

ATP-binding cassette (ABC)-type proteins are essential for bile formation in vertebrate liver. BSEP, MDR1, MDR2, and MRP2 ABC transporters are targeted to the apical (canalicular) membrane of hepatocytes where they execute ATP-dependent transport of bile acids, drugs, amphipathic cations, phospholipids, and conjugated organic anions, respectively. Changes in activity and abundance of transporters in the canalicular membrane regulate bile flow; however, little is known regarding cellular proteins that bind ABC transporters and regulate their trafficking. A yeast two-hybrid screen identified HAX-1 as a binding partner for BSEP, MDR1, and MDR2. The interactions were validated biochemically by glutathione S-transferase pull-down and co-immunoprecipitation assays. BSEP and HAX-1 were over-represented in rat liver subcellular fractions enriched for canalicular membrane vesicles, microsomes, and clathrin-coated vesicles. HAX-1 was bound to BSEP, MDR1, and MDR2 in canalicular membrane vesicles and co-localized with BSEP and MDR1 in the apical membrane of Madin-Darby canine kidney (MDCK) cells. RNA interference of HAX-1 increased BSEP levels in the apical membrane of MDCK cells by 71%. Pulse-chase studies indicated that HAX-1 depletion did not affect BSEP translation, post-translational modification, delivery to the plasma membrane, or half-life. HAX-1 depletion resulted in an increased peak of metabolically labeled apical membrane BSEP at 4 h and enhanced retention at 6 and 9 h. HAX-1 also interacts with cortactin. Expression of dominant negative cortactin increased steady state levels of BSEP 2-fold in the apical membrane of MDCK cells, as did expression of dominant negative EPS15. These findings suggest that HAX-1 and cortactin participate in BSEP internalization from the apical membrane.