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 effect of duration of exercise at the ventilation threshold on subjective appetite and short-term food intake in 9 to 14 year old boys and girls

<p>Abstract</p> <p>Background</p> <p>The effect of exercise on subjective appetite and short-term food intake has received little investigation in children. Despite a lack of reported evaluation of short-duration activity programs, they are currently being implemented in schools as a means to benefit energy balance. The purpose of this study was to determine the effect of duration of exercise at the ventilation threshold (VeT) on subjective appetite and short-term food intake in normal weight boys and girls aged 9 to 14 years.</p> <p>Methods</p> <p>On 4 separate mornings and in random order, boys (n = 14) and girls (n = 15) completed 2 rest or 2 exercise treatments for 15 (short-duration; SD) or 45 min (long-duration; LD) at their previously measured VeT, 2 h after a standardized breakfast. Subjective appetite was measured at regular intervals during the study sessions and food intake from a pizza meal was measured 30 min after rest or exercise.</p> <p>Results</p> <p>An increase in average appetite, desire to eat, and hunger (p < 0.05) was attenuated by SD exercise, but was further increased (p < 0.05) by LD exercise. However, food intake after SD and LD exercise was similar to after rest in both boys and girls (p = 0.55). The energy cost of SD and LD exercise resulted in a lower net energy balance compared to resting during the study measurement period in boys (SD: Δ = -418 ± 301 kJ; LD: Δ = -928 ± 196 kJ) and in girls (SD: Δ = -297 ± 105 kJ; LD: Δ = -432 ± 115 kJ).</p> <p>Conclusion</p> <p>Neither SD nor LD exercise at the VeT increased short-term food intake and SD exercise attenuated increases in appetite. Thus, SD exercise programs in schools may be an effective strategy for maintaining healthier body weights in children.</p

Cystic fibrosis

Advancement in treatment has resulted in a dramatic increase in life expectancy of patients with cystic fibrosis (CF) to well beyond 30 years of age in most centres. What was once a fatal genetic disease in childhood now sees over a third of its CF populations in adult clinics. The improved survival is multifactorial, but most attribute the reasons to more aggressive nutritional care along with better management of the gastrointestinal and pulmonary systems. Many of the nutritional and GI issues of paediatric patients remain similar when they become adults, except that there is the added psychosocial stress associated with this transition. This chapter aims to highlight the gastrointestinal and nutritional issues manifesting at the different stages of life from infancy to adulthood, and the recommended management

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

Variable-Intensity Simulated Team-Sport Exercise Increases Daily Protein Requirements in Active Males

Protein requirements are generally increased in strength and endurance trained athletes relative to their sedentary peers. However, less is known about the daily requirement for this important macronutrient in individuals performing variable intensity, stop-and-go type exercise that is typical for team sport athletes. The objective of the present study was to determine protein requirements in active, trained adult males performing a simulated soccer match using the minimally invasive indicator amino acid oxidation (IAAO) method. After 2 days of controlled diet (1.2 g⋅kg−1⋅day−1 protein), seven trained males (23 ± 1 years; 177.5 ± 6.7 cm; 82.3 ± 6.1 kg; 13.5% ± 4.7% body fat; 52.3 ± 5.9 ml O2⋅kg−1⋅min-1; mean ± SD) performed an acute bout of variable intensity exercise in the form of a modified Loughborough Intermittent Shuttle Test (4 × 15 min of exercise over 75 min). Immediately after exercise, hourly meals were consumed providing a variable amount of protein (0.2–2.6 g⋅kg−1⋅day−1) and sufficient energy and carbohydrate (6 g⋅kg−1⋅day−1). Protein was provided as a crystalline amino acids modeled after egg protein with the exception of phenylalanine and tyrosine, which were provided in excess to ensure the metabolic partitioning of the indicator amino acid (i.e., [1-13C]phenylalanine included within the phenylalanine intake) was directed toward oxidation when protein intake was limiting. Whole body phenylalanine flux and 13CO2 excretion (F13CO2) were determined at metabolic and isotopic steady state from urine and breath samples, respectively. Biphasic linear regression analysis was performed on F13CO2 to determine the estimated average requirement (EAR) for protein with a safe intake defined as the upper 95% confidence interval. Phenylalanine flux was not impacted by protein intake (P = 0.45). Bi-phase linear regression (R2 = 0.64) of F13CO2 resulted in an EAR and safe intake of 1.20 and 1.40 g⋅kg−1⋅day−1, respectively. Variable intensity exercise increases daily protein requirements compared to the safe intake determined by nitrogen balance (0.83 g⋅kg−1⋅day−1) and IAAO (1.24 g⋅kg−1⋅day−1) but is within the range (i.e., 1.2–2.0 g⋅kg−1⋅day−1) of current consensus statements on general recommendations for athletes.Clinical Trial registrationThis trial was registered June 18, 2015 at http://clinicaltrials.gov as NCT02478814

Recent developments in understanding protein needs - How much and what kind should we eat?

A novel method has been developed to determine protein requirements, which is called Indicator amino acid oxidation (IAAO). This technique has been validated by comparison with the “gold standard” nitrogen balance”. Using IAAO we have shown that minimum protein requirements have been under estimated by 30-50%. The National Academy of Sciences have for macro-nutrients proposed “Acceptable Macronutrient Distribution Ranges”, which for protein is 10 to 35% of total energy. In practice, we suggest 1.5-2.2 g/kg/d of a variety of high-quality proteins.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Protein Requirements during Aging

Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR) and recommended dietary allowance (RDA), respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO) method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals.Medicine, Faculty ofOther UBCNon UBCPediatrics, Department ofPopulation and Public Health (SPPH), School ofReviewedFacult