Skeletal muscle mass is determined predominantly by feeding-induced and activity-induced fluctuations in muscle protein synthesis (MPS). Older individuals display a diminished MPS response to protein ingestion, referred to as age-related anabolic resistance, which contributes to the progression of age-related muscle loss known as sarcopenia.
We aimed to determine the impact of consuming higher-quality compared with lower-quality protein supplements above the recommended dietary allowance (RDA) on integrated MPS rates. We hypothesized that increasing total protein intake above the RDA, regardless of the source, would support higher integrated rates of myofibrillar protein synthesis.
Thirty-one healthy older males (72 ± 4 y) consumed a controlled diet with protein intake set at the RDA: control phase (days 1-7). In a double-blind, randomized controlled fashion, participants were assigned to consume an additional 50 g (2 × 25g) of whey (n = 10), pea (n = 11), or collagen (n = 10) protein each day (25 g at breakfast and lunch) during the supplemental phase (days 8-15). Deuterated water ingestion and muscle biopsies assessed integrated MPS and acute anabolic signaling. Postprandial blood samples were collected to determine feeding-induced aminoacidemia.
Integrated MPS was increased during supplemental with whey (1.59 ± 0.11 %/d; P < 0.001) and pea (1.59 ± 0.14 %/d; P < 0.001) when compared with RDA (1.46 ± 0.09 %/d for the whey group; 1.46 ± 0.10 %/d for the pea group); however, it remained unchanged with collagen. Supplemental protein was sufficient to overcome anabolic signaling deficits (mTORC1 and rpS6), corroborating the greater postprandial aminoacidemia.
Our findings demonstrate that supplemental protein provided at breakfast and lunch over the current RDA enhanced anabolic signaling and integrated MPS in older males; however, the source of additional protein may be an important consideration in overcoming age-related anabolic resistance. This trial was registered clinicaltrials.gov as NCT04026607.

UNASSIGNED: \'OMICs encapsulates study of scaled data acquisition, at the levels of DNA, RNA, protein, and metabolite species. The broad objectives of OMICs in biomedical exercise research are multifarious, but commonly relate to biomarker development and understanding features of exercise adaptation in health, ageing and metabolic diseases.
UNASSIGNED: This field is one of exponential technical (i.e., depth of feature coverage) and scientific (i.e., in health, metabolic conditions and ageing, multi-OMICs) progress adopting targeted and untargeted approaches.
UNASSIGNED: Key findings in exercise biomedicine have led to the identification of OMIC features linking to heritability or adaptive responses to exercise e.g., the forging of GWAS/proteome/metabolome links to cardiovascular fitness and metabolic health adaptations. The recent addition of stable isotope tracing to proteomics (\'dynamic proteomics\') and metabolomics (\'fluxomics\') represents the next phase of state-of-the-art in \'OMICS.
UNASSIGNED: These methods overcome limitations associated with point-in-time \'OMICs and can be achieved using substrate-specific tracers or deuterium oxide (D2O), depending on the question; these methods could help identify how individual protein turnover and metabolite flux may explain exercise responses. We contend application of these methods will shed new light in translational exercise biomedicine.

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered 13C3, 15N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.

The key to understanding the mechanisms regulating disease stems from the ability to accurately quantify the dynamic nature of the metabolism underlying the physiological and pathological changes occurring as a result of the disease. Stable isotope tracer technologies have been at the forefront of this for almost 80 years now, and through a combination of both intense theoretical and technological development over these decades, it is now possible to utilise stable isotope tracers to investigate the complexities of in vivo human metabolism from a whole body perspective, down to the regulation of sub-nanometer cellular components (i.e organelles, nucleotides and individual proteins). This review therefore aims to highlight; 1) the advances made in these stable isotope tracer approaches - with special reference given to their role in understanding the nutritional regulation of protein metabolism, 2) some considerations required for the appropriate application of these stable isotope techniques to study protein metabolism, 3) and finally how new stable isotopes approaches and instrument/technical developments will help to deliver greater clinical insight in the near future.

Lipids comprise diverse classes of compounds that are important for the structure and properties of membranes, as high-energy fuel sources and as signaling molecules. Therefore, the turnover rates of these varied classes of lipids are fundamental to cellular function. However, their enormous chemical diversity and dynamic range in cells makes detailed analysis very complex. Furthermore, although stable isotope tracers enable the determination of synthesis and degradation of complex lipids, the numbers of distinguishable molecules increase enormously, which exacerbates the problem. Although LC-MS-MS (Liquid Chromatography-Tandem Mass Spectrometry) is the standard for lipidomics, NMR can add value in global lipid analysis and isotopomer distributions of intact lipids. Here, we describe new developments in NMR analysis for assessing global lipid content and isotopic enrichment of mixtures of complex lipids for two cell lines (PC3 and UMUC3) using both 13C6 glucose and 13C5 glutamine tracers.

The Dietary Guidelines for Americans (DGAs) published an \"ounce equivalents\" recommendation to help consumers meet protein requirements with a variety of protein food sources. However, the metabolic equivalency of these varied protein food sources has not been established.
We have investigated the hypothesis that the anabolic responses to consumption of ounce equivalents of protein food sources would be directly related to the essential amino acid (EAA) content of the protein food source.
Following 3 d of dietary control, a total of 56 healthy young adults underwent an 8.5-h metabolic study using stable isotope tracer methodology. The changes from baseline following consumption of 1 of 7 different protein food sources were compared with the baseline value for that individual (n = 8 per group).
Consumption of ounce equivalents of animal-based protein food sources (beef sirloin, pork loin, eggs) resulted in a greater gain in whole-body net protein balance above baseline than the ounce equivalents of plant-based protein food sources (tofu, kidney beans, peanut butter, mixed nuts; P < 0.01). The improvement in whole-body net protein balance was due to an increase in protein synthesis (P < 0.05) with all the animal protein sources, whereas the egg and pork groups also suppressed protein breakdown compared with the plant protein sources (P < 0.01). The magnitude of the whole-body net balance (anabolic) response was correlated with the EAA content of the protein food source (P < 0.001).
The \"ounce equivalents\" of protein food sources as expressed in the DGAs are not metabolically equivalent in young healthy individuals. The magnitude of anabolic response to dietary proteins should be considered as the DGAs develop approaches to establish healthy eating patterns.

We have recently demonstrated in young adults that an anabolic response with mixed meal protein intake above ~35 g/meal, previously recognized as an \"optimal\" protein dose, was further stimulated. However, it is unknown if this applies to older adults. We therefore examined anabolic response to a mixed meal containing either 35 g (MOD, moderate amount of protein) or 70 g (HIGH, high amount of protein) in a randomized cross-over metabolic study in older adults (n = 8). Primed continuous infusions of L-[2H5] phenylalanine and L-[2H2]tyrosine were performed to determine whole-body protein kinetics and muscle protein fractional synthesis rate (MPS) in basal fasted and fed states. Whole-body protein kinetics (NB, net protein balance; PS, protein synthesis; PB, protein breakdown) and MPS was expressed as changes from the baseline post-absorptive state. Consistent with our previous findings in young adults, both feedings resulted in a positive NB, with HIGH being more positive than MOD. Furthermore, NB (expressed as g protein∙240 min) increased linearly with an increasing amount of protein intake, expressed relative to lean body mass. The positive NB was achieved due mainly to the suppression of PB in both MOD and to a greater extent HIGH, while PS was only increased in HIGH. Consistent with the whole-body data, MPS was significantly higher in HIGH than MOD. Plasma concentrations of essential amino acids and insulin were greater in HIGH vs. MOD. We conclude that in the context of mixed meals, whole-body anabolic response linearly increases with increasing protein intake primarily through the suppression of PB, and MPS was further stimulated with protein intake above the previously considered \"optimal\" protein dose in older adults.

OBJECTIVE: The purpose of the study was to determine if an actinidin protease aids gastric digestion and the protein anabolic response to dietary protein.
METHODS: Hayward green kiwifruit (containing an actinidin protease) and Hort 16A gold kiwifruit (devoid of actinidin protease) were given in conjunction with a beef meal to healthy older subjects. Twelve healthy older males (N = 6) and females (N = 6) were studied with a randomized, double-blinded, crossover design to assess muscle and whole-body protein metabolism before and after ingestion of kiwifruit and 100 g of ground beef. Subjects consumed 2 of each variety of kiwifruit daily for 14 d prior to each metabolic study, and again during each study with beef intake.
RESULTS: Hayward green kiwifruit consumption with beef resulted in a more rapid increase in peripheral plasma essential amino acid concentrations. There were significant time by kiwifruit intake interactions for plasma concentrations of EAAs, branched chain amino acids (BCAAs), and leucine (P < 0.01). However, there was no difference in the total amount of EAAs absorbed. As a result, there were no differences between kiwifruit in any of the measured parameters of protein kinetics.
CONCLUSIONS: Consumption of Hayward green kiwifruit, with a beef meal facilitates protein digestion and absorption of the constituent amino acids as compared to Hort 16A gold kiwifruit.
BACKGROUND: NCT04356573, April 21, 2020 \"retrospectively registered\".

A high fructose intake exacerbates postprandial plasma triacylglycerol (TAG) concentration, an independent risk factor for cardiovascular disease, although it is unclear whether this is due to increased production or impaired clearance of triacylglycerol (TAG)-rich lipoproteins. We determined the in vivo acute effect of fructose on postprandial intestinal and hepatic lipoprotein TAG kinetics and de novo lipogenesis (DNL). Five overweight men were studied twice, 4 weeks apart. They consumed hourly mixed-nutrient drinks that were high-fructose (30% energy) or low-fructose (<2% energy) for 11 h. Oral 2H2O was administered to measure fasting and postprandial DNL. Postprandial chylomicron (CM)-TAG and very low-density lipoprotein (VLDL)-TAG kinetics were measured with an intravenous bolus of [2H5]-glycerol. CM and VLDL were separated by their apolipoprotein B content using antibodies. Plasma TAG (p < 0.005) and VLDL-TAG (p = 0.003) were greater, and CM-TAG production rate (PR, p = 0.046) and CM-TAG fractional catabolic rate (FCR, p = 0.073) lower when high-fructose was consumed, with no differences in VLDL-TAG kinetics. Insulin was lower (p = 0.005) and apoB48 (p = 0.039), apoB100 (p = 0.013) and non-esterified fatty acids (NEFA) (p = 0.013) were higher after high-fructose. Postprandial hepatic fractional DNL was higher than intestinal fractional DNL with high-fructose (p = 0.043) and low-fructose (p = 0.043). Fructose consumption had no effect on the rate of intestinal or hepatic DNL. We provide the first measurement of the rate of intestinal DNL in humans. Lower CM-TAG PR and CM-TAG FCR with high-fructose consumption suggests lower clearance of CM, rather than elevated production, may contribute to elevated plasma TAG, possibly due to lower insulin-mediated stimulation of lipoprotein lipase.