Heterotrimeric G protein subunits are located on rat liver endosomes

BACKGROUND: Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors. RESULTS: By Western blotting G(sα), G(iα1,2), G(iα3 )and G(β )were enriched in both canalicular (CM) and basolateral (BLM) membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC) > compartment for uncoupling of receptor and ligand (CURL) > multivesicular bodies (MVB) >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC) was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for G(sα), G(iα3 )and G(β )corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers. CONCLUSION: We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s) of internalized insulin receptors

The Effects of Maternal and Postnatal Dietary Methyl Nutrients on Epigenetic Changes that Lead to Non-Communicable Diseases in Adulthood

The risk for non-communicable diseases in adulthood can be programmed by early nutrition. This programming is mediated by changes in expression of key genes in various metabolic pathways during development, which persist into adulthood. These developmental modifications of genes are due to epigenetic alterations in DNA methylation patterns. Recent studies have demonstrated that DNA methylation can be affected by maternal or early postnatal diets. Because methyl groups for methylation reactions come from methionine cycle nutrients (i.e., methionine, choline, betaine, folate), deficiency or supplementation of these methyl nutrients can directly change epigenetic regulation of genes permanently. Although many studies have described the early programming of adult diseases by maternal and infant nutrition, this review discusses studies that have associated early dietary methyl nutrient manipulation with direct effects on epigenetic patterns that could lead to chronic diseases in adulthood. The maternal supply of methyl nutrients during gestation and lactation can alter epigenetics, but programming effects vary depending on the timing of dietary intervention, the type of methyl nutrient manipulated, and the tissue responsible for the phenotype. Moreover, the postnatal manipulation of methyl nutrients can program epigenetics, but more research is needed on whether this approach can rescue maternally programmed offspring

Effects of supplemental creatine and guanidinoacetic acid on spatial memory and the brain of weaned Yucatan miniature pigs

The emergence of creatine as a potential cognitive enhancement supplement for humans prompted an investigation as to whether supplemental creatine could enhance spatial memory in young swine. We assessed memory performance and brain concentrations of creatine and its precursor guanidinoacetic acid (GAA) in 14-16-week-old male Yucatan miniature pigs supplemented for 2 weeks with either 200 mg/kg∙d creatine (+Cr; n = 7) or equimolar GAA (157 mg/kg∙d) (+GAA; n = 8) compared to controls (n = 14). Spatial memory tests had pigs explore distinct sets of objects for 5 min. Objects were spatially controlled, and we assessed exploration times of previously viewed objects relative to novel objects in familiar or novel locations. There was no effect of either supplementation on memory performance, but pigs successfully identified novel objects after 10 (p < 0.01) and 20 min (p < 0.01) retention intervals. Moreover, pigs recognized spatial transfers after 65 min (p < 0.05). Regression analyses identified associations between the ability to identify novel objects in memory tests and concentrations of creatine and GAA in cerebellum, and GAA in prefrontal cortex (p < 0.05). The concentration of creatine in brain regions was not influenced by creatine supplementation, but GAA supplementation increased GAA concentration in cerebellum (p < 0.05), and the prefrontal cortex of +GAA pigs had more creatine/g and less GAA/g compared to +Cr pigs (p < 0.05). Creatine kinase activity and maximal reaction velocity were also higher with GAA supplementation in prefrontal cortex (p < 0.05). In conclusion, there appears to be a relationship between memory performance and guanidino compounds in the cerebellum and prefrontal cortex, but the effects were unrelated to dietary supplementation. The cerebellum is identified as a target site for GAA accretion

Airway tapering: an objective image biomarker for bronchiectasis

Purpose: To estimate airway tapering in control subjects and to assess the usability of tapering as a bronchiectasis biomarker in paediatric populations. Methods: Airway tapering values were semi-automatically quantified in 156 children with control CTs collected in the Normal Chest CT Study Group. Airway tapering as a biomarker for bronchiectasis was assessed on spirometer-guided inspiratory CTs from 12 patients with bronchiectasis and 12 age- and sex-matched controls. Semi-automatic image analysis software was used to quantify intra-branch tapering (reduction in airway diameter along the branch), inter-branch tapering (reduction in airway diameter before and after bifurcation) and airway-artery ratios on chest CTs. Biomarkers were further stratified in small, medium and large airways based on three equal groups of the accompanying vessel size. Results: Control subjects showed intra-branch tapering of 1% and inter-branch tapering of 24–39%. Subjects with bronchiectasis showed significantly reduced intra-branch of 0.8% and inter-branch tapering of 19–32% and increased airway–artery ratios compared with controls (p < 0.01). Tapering measurements were significantly different between diseased and controls across all airway sizes. Difference in airway–artery ratio was only significant in small airways. Conclusion: Paediatric normal values for airway tapering were established in control subjects. Tapering showed to be a promising biomarker for bronchiectasis as subjects with bronchiectasis show significantly less airway tapering across all airway sizes compared with controls. Detecting les

Gut Microbial Gene Expression in Mother-Fed and Formula-Fed Piglets

Effects of diet on the structure and function of gut microbial communities in newborn infants are poorly understood. High-resolution molecular studies are needed to definitively ascertain whether gut microbial communities are distinct in milk-fed and formula-fed infants.Pyrosequencing-based whole transcriptome shotgun sequencing (RNA-seq) was used to evaluate community wide gut microbial gene expression in 21 day old neonatal piglets fed either with sow's milk (mother fed, MF; n = 4) or with artificial formula (formula fed, FF; n = 4). Microbial DNA and RNA were harvested from cecal contents for each animal. cDNA libraries and 16S rDNA amplicons were sequenced on the Roche 454 GS-FLX Titanium system. Communities were similar at the level of phylum but were dissimilar at the level of genus; Prevotella was the dominant genus within MF samples and Bacteroides was most abundant within FF samples. Screened cDNA sequences were assigned functional annotations by the MG-RAST annotation pipeline and based upon best-BLASTX-hits to the NCBI COG database. Patterns of gene expression were very similar in MF and FF animals. All samples were enriched with transcripts encoding enzymes for carbohydrate and protein metabolism, as well as proteins involved in stress response, binding to host epithelium, and lipopolysaccharide metabolism. Carbohydrate utilization transcripts were generally similar in both groups. The abundance of enzymes involved in several pathways related to amino acid metabolism (e.g., arginine metabolism) and oxidative stress response differed in MF and FF animals.Abundant transcripts identified in this study likely contribute to a core microbial metatranscriptome in the distal intestine. Although microbial community gene expression was generally similar in the cecal contents of MF and FF neonatal piglets, several differentially abundant gene clusters were identified. Further investigations of gut microbial gene expression will contribute to a better understanding of normal and abnormal enteric microbiology in animals and humans

Intestinal development of early-weaned piglets receiving diets supplemented with selected amino acids or polyamines

Early-weaned piglets are subjected to various environmental and nutritional stresses that can result in overall poor performance. Several amino acids associated with the urea cycle have been shown to be critical to intestinal development and metabolism. The objective of this research was to examine performance and intestinal development in early-weaned piglets receiving diets supplemented with selected amino acids or polyamines. Forty-two Yorkshire piglets (3.94 ± 0.43 kg) weaned at ~12.5 d were randomly assigned to diets supplemented with either arginine, glutamate, citrulline, ornithine or polyamines, at levels of 0.93, 6.51, 0.94, 0.90 and 0.39%, respectively. Diets were fed for 12 d and various parameters to assess growth and intestinal development were measured. Glutamate supplementation enhanced both total and mucosal growth in several sections of the small intestine (P < 0.05), whereas polyamines were detrimental to intestinal growth. Arginine and glutamate supplementation prevented weaning-induced villus atrophy in the duodenum, compared with both the control and polyamine-fed pigs (P = 0.004). These results indicate that glutamate and arginine supplementation may enhance intestinal development of the early-weaned piglet, whereas polyamine supplementation at the ratios and concentrations used in this experiment is not recommended in typical early-weaned piglet diets

Estimate of the variability of the lysine requirement of growing pigs using the indicator amino acid oxidation technique1

Although AA requirements for the mean in a population of growing pigs are well established, there are no direct estimates of their variability within the population. The indicator AA oxidation method allows repeated measurements in a short period of time so that the AA requirement can be determined for individual pigs. The objective was to determine the Lys requirement in individual pigs to derive a first estimate of the population mean requirement and its variability. Nine individually housed barrows (15 to 18 kg) were surgically implanted with venous catheters for isotope infusion. Pigs were offered, in random order, isonitrogenous and isoenergetic diets with one of seven Lys concentrations (4.8 to 15.5 g of Lys/kg diet, as-fed basis). The pigs were fed twice daily, except for study days when they received one-half of the daily allowance in eight equal hourly meals. After a validated minimum adaptation period, indicator (Phe) oxidation was determined for each dietary Lys level during a 4-h primed, constant infusion of l-[1-14C]Phe at a rate of 464 kBq/h. The Lys requirement was calculated using a two-phase linear regression crossover analysis within individual pigs. For each pig, Phe oxidation decreased linearly (P < 0.02) as the dietary Lys concentration increased until the requirement was reached; thereafter, Phe oxidation was not different. The true ileal digestible Lys requirement ranged from 7.5 to 10.6 g/kg of diet (as-fed basis) for the nine animals. The mean requirement for all pigs was 9.1 g/d (CV, 11.6%) or 93.9% (CV, 9.8%) of the predicted (NRC, 1998) requirement based on each pig’s mean BW and energy intake. The measured and predicted requirements did not differ. The indicator AA oxidation method gave values for Lys requirement similar to conventional methods. The short (<3 wk) experimental period allows, for the first time, the estimate of population variability, which provides for more accurate calculation of the effect of altering Lys intake on herd performance and production economics. This method is suitable to use with all dietary indispensable AA

Effect of common antinutritive factors and fibrous feedstuffs in pig diets on amino acid digestibilities with special emphasis on threonine

Most feedstuffs contain antinutritive factors (ANF) such as insoluble fibers, lignins, tannins, and lectins. Intake of these ANF has the ability to reduce nutrient digestibility and to increase endogenous protein losses, such as through increased intestinal mucus secretion. The objective of this experiment was to determine the apparent ileal digestibilities (AID) of AA of 6 ANF-enriched diets to estimate endogenous protein loss associated with these ingredients in diets for young pigs. Forty-two 10-kg BW pigs fitted with a simple T-cannula at the distal ileum were randomly assigned to 1 of 7 casein-based diets with: no supplement (control), 100 g/kg of canola meal (CM), 100 g/kg of wheat bran (WB), 150 g/kg of barley (BR), 22.5 g/kg of lignin (LG), 15 g/kg of kidney beans [as a lectin (LE) source], and 15 g/kg of tannins (TN). All diets were formulated to be similar in N, indispensable AA, and caloric contents. After a 7-d adaptation to the test diets, N balance was conducted for 5 d, followed by 24 h of collection of digesta for analyses of AA. Pigs fed BR had 17% lower ADG and 15% lower feed conversion ratio (P < 0.05) compared with control and CM pigs. Pigs fed diets containing WB and BR had lower N retention as a percentage of absorbed N compared with all other groups (P = 0.03). The AID for CP was lower in BR, WB, and LE pigs compared with control. Of the AA, AID of Thr was notably lowest in BR, WB, and TN pigs (P < 0.05). The standardized ileal digestibility was lower in WB and BR pigs for most indispensable AA. Altogether, these data suggest that hemicellulose fiber, at concentrations typical in commercial swine diets, reduces AID of AA by increasing endogenous losses. Understanding the differential effects of ANF on endogenous losses of individual dietary AA will improve the accuracy of diet formulation

Guanidinoacetate Is More Effective than Creatine at Enhancing Tissue Creatine Stores While Consequently Limiting Methionine Availability in Yucatan Miniature Pigs

Creatine (Cr) is an important high-energy phosphate buffer in tissues with a high energy demand such as muscle and brain and is consequently a highly consumed nutritional supplement. Creatine is synthesized via the S-adenosylmethionine (SAM) dependent methylation of guanidinoacetate (GAA) which is not regulated by a feedback mechanism. The first objective of this study was to determine the effectiveness of GAA at increasing tissue Cr stores. Because SAM is required for other methylation reactions, we also wanted to determine whether an increased creatine synthesis would lead to a lower availability of methyl groups for other methylated products. Three month-old pigs (n = 18) were fed control, GAAor Cr-supplemented diets twice daily. On day 18 or 19, anesthesia was induced 1–3 hours post feeding and a bolus of [methyl-3H]methionine was intravenously infused. After 30 minutes, the liver was analyzed for methyl-3H incorporation into protein, Cr, phosphatidylcholine (PC) and DNA. Although both Cr and GAA led to higher hepatic Cr concentration, only supplementation with GAA led to higher levels of muscle Cr (P < 0.05). Only GAA supplementation resulted in lower methyl-3H incorporation into PC and protein as well as lower hepatic SAM concentration compared to the controls, suggesting that Cr synthesis resulted in a limited methyl supply for PC and protein synthesis (P < 0.05). Although GAA is more effective than Cr at supporting muscle Cr accretion, further research should be conducted into the long term consequences of a limited methyl supply and its effects on protein and PC homeostasis

Effects of a Diet High in Salt, Fat, and Sugar on Telemetric Blood Pressure Measurements in Conscious, Unrestrained Adult Yucatan Miniature Swine (Sus scrofa)

Radiotelemetry was used to evaluate diet-related elevation of blood pressure in adult Yucatan miniature swine. Systolic arterial blood pressure (SAP), diastolic atrial blood pressure (DAP), heart rate, and locomotor activity were assessed in 9- or 11-mo-old Yucatan miniature pigs fed a standard diet or a North American-type diet high in salt, fat, and sugar (HSFS). Compared with pigs fed standard diet, pigs fed HSFS diet showed markedly elevated SAP (132 ± 3 compared with 156 ± 6 mm Hg), whereas DAP was unchanged (92 ± 2 compared with 99 ± 5 mm Hg). In addition, all pigs were modestly sensitive to short-term changes in dietary salt, as indicated by a 6% to 7% response in blood pressure parameters. According to these data, the increase in SAP for pigs on the HSFS diet was too large to be explained by the NaCl content of the diet alone. We found no evidence of endothelial dysfunction, and the relaxation responses of isolated coronary arteries actually were enhanced in the HSFS group. In conclusion, in a Yucatan miniature pigs model chronically fed a HSFS diet, DAP did not increase, but SAP and pulse pressure appeared to be affected by high dietary levels of fat or sugar (or both)