Myths and Methodologies: Standardisation in human physiology research-should we control the controllables?

The premise of research in human physiology is to explore a multifaceted system whilst identifying one or a few outcomes of interest. Therefore, the control of potentially confounding variables requires careful thought regarding the extent of control and complexity of standardisation. One common factor to control prior to testing is diet, as food and fluid provision may deviate from participants' habitual diets, yet a self-report and replication method can be flawed by under-reporting. Researchers may also need to consider standardisation of physical activity, whether it be through familiarisation trials, wash-out periods, or guidance on levels of physical activity to be achieved before trials. In terms of pharmacological agents, the ethical implications of standardisation require researchers to carefully consider how medications, caffeine consumption and oral contraceptive prescriptions may affect the study. For research in females, it should be considered whether standardisation between- or within-participants in regards to menstrual cycle phase is most relevant. The timing of measurements relative to various other daily events is relevant to all physiological research and so it can be important to standardise when measurements are made. This review summarises the areas of standardisation which we hope will be considered useful to anyone involved in human physiology research, including when and how one can apply standardisation to various contexts.

Effects of physical form of ß-lactoglobulin and calcium ingestion on GLP-1 secretion, gastric emptying and energy intake in humans: a randomised crossover trial.

The aim of this study was to assess whether adding Ca2+ to aggregate or native forms of ß-lactoglobulin alters gut hormone secretion, gastric emptying rates and energy intake in healthy men and women. Fifteen healthy adults (mean ± sd: 9M/6F, age: 24 ± 5 years) completed four trials in a randomised, double-blind, crossover design. Participants consumed test drinks consisting of 30 g of ß-lactoglobulin in a native form with (NATIVE + MINERALS) and without (NATIVE) a Ca2+-rich mineral supplement and in an aggregated form both with (AGGREG + MINERALS) and without the mineral supplement (AGGREG). Arterialised blood was sampled for 120 min postprandially to determine gut hormone concentrations. Gastric emptying was determined using 13C-acetate and 13C-octanoate, and energy intake was assessed with an ad libitum meal at 120 min. A protein × mineral interaction effect was observed for total glucagon-like peptide-1 (GLP-1TOTAL) incremental AUC (iAUC; P < 0·01), whereby MINERALS + AGGREG increased GLP-1TOTAL iAUC to a greater extent than AGGREG (1882 ± 603 v. 1550 ± 456 pmol·l-1·120 min, P < 0·01), but MINERALS + NATIVE did not meaningfully alter the GLP-1 iAUC compared with NATIVE (1669 ± 547 v. 1844 ± 550 pmol·l-1·120 min, P = 0·09). A protein × minerals interaction effect was also observed for gastric emptying half-life (P < 0·01) whereby MINERALS + NATIVE increased gastric emptying half-life compared with NATIVE (83 ± 14 v. 71 ± 8 min, P < 0·01), whereas no meaningful differences were observed between MINERALS + AGGREG v. AGGREG (P = 0·70). These did not result in any meaningful changes in energy intake (protein × minerals interaction, P = 0·06). These data suggest that the potential for Ca2+ to stimulate GLP-1 secretion at moderate protein doses may depend on protein form. This study was registered at clinicaltrials.gov (NCT04659902).

Are all sugars equal? Role of the food source in physiological responses to sugars with an emphasis on fruit and fruit juice.

High (free) sugar intakes can increase self-reported energy intake and are associated with unfavourable cardiometabolic health. However, sugar source may modulate the effects of sugars due to several mechanisms including the food matrix. The aim of this review was to assess the current state of evidence in relation to food source effects on the physiological responses to dietary sugars in humans relevant to cardiometabolic health. An additional aim was to review potential mechanisms by which food sources may influence such responses. Evidence from meta-analyses of controlled intervention trials was used to establish the balance of evidence relating to the addition of sugars to the diet from sugar-sweetened beverages, fruit juice, honey and whole fruit on cardiometabolic outcomes. Subsequently, studies which have directly compared whole fruit with fruit juices, or variants of fruit juices, were discussed. In summary, the sources of sugars can impact physiological responses, with differences in glycaemic control, blood pressure, inflammation, and acute appetite. Longer-term effects and mechanisms require further work, but initial evidence implicates physical structure, energy density, fibre, potassium and polyphenol content, as explanations for some of the observed responses.

Myths and Methodologies: Standardisation in Human Physiology Research-Should We Control the Controllables?

The premise of research in human physiology is to explore a multifaceted system whilst identifying one or a few outcomes of interest. Therefore, the control of potentially confounding variables requires careful thought regarding the extent of control and complexity of standardisation. One common factor to control prior to testing is diet, as food and fluid provision may deviate from participants' habitual diets, yet a self-report and replication method can be flawed by under-reporting. Researchers may also need to consider standardisation of physical activity, whether it be through familiarisation trials, wash-out periods, or guidance on levels of physical activity to be achieved before trials. In terms of pharmacological agents, the ethical implications of standardisation require researchers to carefully consider how medications, caffeine consumption and oral contraceptive prescriptions may affect the study. For research in females, it should be considered whether standardisation between- or within-participants in regards to menstrual cycle phase is most relevant. The timing of measurements relative to various other daily events is relevant to all physiological research and so it can be important to standardise when measurements are made. This review summarises the areas of standardisation which we hope will be considered useful to anyone involved in human physiology research, including when and how one can apply standardisation to various contexts.

Role of prior feeding status in mediating the effects of exercise on blood glucose kinetics.

Changes in blood glucose concentrations are underpinned by blood glucose kinetics (endogenous and exogenous glucose appearance rates and glucose disappearance rates). Exercise potently alters blood glucose kinetics and can thereby be used as a tool to control blood glucose concentration. However, most studies of exercise-induced changes in glucose kinetics are conducted in a fasted state, and therefore less is known about the effects of exercise on glucose kinetics when exercise is conducted in a postprandial state. Emerging evidence suggests that food intake prior to exercise can increase postprandial blood glucose flux compared with when meals are consumed after exercise, whereby both glucose appearance rates and disappearance rates are increased. The mechanisms underlying the mediating effect of exercise conducted in the fed versus the fasted state are yet to be fully elucidated. Current evidence demonstrates that exercise in the postprandial state increased glucose appearance rates due to both increased exogenous and endogenous appearance and may be due to changes in splanchnic blood flow, intestinal permeability, and/or hepatic glucose extraction. On the other hand, increased glucose disappearance rates after exercise in the fed state have been shown to be associated with increased intramuscular AMPK signaling via a mismatch between carbohydrate utilization and delivery. Due to differences in blood glucose kinetics and other physiological differences, studies conducted in the fasted state cannot be immediately translated to the fed state. Therefore, conducting studies in the fed state could improve the external validity of data pertaining to glucose kinetics and intramuscular signaling in response to nutrition and exercise.

Ketone monoester ingestion increases postexercise serum erythropoietin concentrations in healthy men.

Intravenous ketone body infusion can increase erythropoietin (EPO) concentrations, but responses to ketone monoester ingestion postexercise are currently unknown. The purpose of this study was to assess the effect of ketone monoester ingestion on postexercise erythropoietin (EPO) concentrations. Nine healthy men completed two trials in a randomized, crossover design (1-wk washout). During trials, participants performed 1 h of cycling (initially alternating between 50% and 90% of maximal aerobic capacity for 2 min each interval, and then 50% and 80%, and 50% and 70% when the higher intensity was unsustainable). Participants ingested 0.8 g·kg-1 sucrose with 0.4 g·kg-1 protein immediately after exercise, and at 1, 2, and 3 h postexercise. During the control trial (CONTROL), no further nutrition was provided, whereas on the ketone monoester trial (KETONE), participants also ingested 0.29 g·kg-1 of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate immediately postexercise and at 1 and 2 h postexercise. Blood was sampled immediately postexercise, every 15 min in the first hour and hourly thereafter for 4 h. Serum EPO concentrations increased to a greater extent in KETONE than in CONTROL (time × condition interaction: P = 0.046). Peak serum EPO concentrations were higher with KETONE (means ± SD: 9.0 ± 2.3 IU·L-1) compared with CONTROL (7.5 ± 1.5 IU·L-1, P < 0.01). Serum ß-hydroxybutyrate concentrations were also higher, and glucose concentrations lower, with KETONE versus CONTROL (both P < 0.01). In conclusion, ketone monoester ingestion increases postexercise erythropoietin concentrations, revealing a new avenue for orally ingestible ketone monoesters to potentially alter hemoglobin mass.NEW & NOTEWORTHY To our knowledge, this study was the first to assess the effects of ketone monoester ingestion on erythropoietin concentrations after exercise. We demonstrated that ingestion of a ketone monoester postexercise increased serum erythropoietin concentrations and reduced serum glucose concentrations in healthy men. These data reveal the possibility for ketone monoesters to alter hemoglobin mass.

Whey Protein-Enriched and Carbohydrate-Rich Breakfasts Attenuate Insulinemic Responses to an ad libitum Lunch Relative to Extended Morning Fasting: A Randomized Crossover Trial.

BACKGROUND: Typical breakfast foods are rich in carbohydrate, so they not only elevate blood glucose during the morning, but also elicit a second-meal effect that can attenuate blood glucose responses in the afternoon. OBJECTIVES: To determine whether a reduced-carbohydrate protein-enriched breakfast can elicit similar effects on glucose control later in the day but without hyperglycemia in the morning. METHODS: In a randomized crossover design, 12 healthy men and women (age 22 ± 2 y, BMI 24.1 ± 3.6 kg·m-2; Mean ± SD) completed 3 experimental conditions. In all conditions, participants consumed an ad libitum lunch at 1200 ± 1 h but differed in terms of whether they had fasted all morning (control) or had consumed a standardized porridge breakfast at 0900 ± 1 h (320 ± 50 kcal; prescribed relative to resting metabolic rate) that was either carbohydrate-rich (50 ± 10 g CHO) or protein-enriched (that is, isoenergetic substitution of carbohydrate for 15 g whey protein isolate). RESULTS: The protein-enriched breakfast reduced the morning glycemic response (iAUC 87 ± 36 mmol·L-1·180 min) relative to the carbohydrate-rich breakfast (119 ± 37 mmol·L-1·180 min; P = 0.03). Despite similar energy intake at lunch in all 3 conditions (protein-enriched 769 ± 278 kcal; carbohydrate-rich 753 ± 223 kcal; fasting 790 ± 227 kcal), postlunch insulinemic responses were markedly attenuated when breakfasts had been consumed that were either protein-enriched (18.0 ± 8.0 nmol·L-1·120 min; P = 0.05) or carbohydrate-rich (16.0 ± 7.7 nmol·L-1·120 min; P = 0.005), relative to when lunch was consumed in an overnight fasted state (26.9 ± 13.5 nmol·L-1·120 min). CONCLUSIONS: Breakfast consumption attenuates insulinemic responses to a subsequent meal, achieved with consumption of energy-matched breakfasts typically high in carbohydrates or enriched with whey protein isolate relative to extended morning fasting. TRIAL REGISTRATION NUMBER: NCT03866720 (clinicaltrials.gov).

Restricting sugar or carbohydrate intake does not impact physical activity level or energy intake over 24 h despite changes in substrate use: a randomised crossover study in healthy men and women.

PURPOSE: To determine the effects of dietary sugar or carbohydrate restriction on physical activity energy expenditure, energy intake, and physiological outcomes across 24 h. METHODS: In a randomized, open-label crossover design, twenty-five healthy men (n = 10) and women (n = 15) consumed three diets over a 24-h period: moderate carbohydrate and sugar content (MODSUG = 50% carbohydrate [20% sugars], 15% protein, 35% fat); low sugar content (LOWSUG = 50% carbohydrate [< 5% sugars], 15% protein, 35% fat); and low carbohydrate content (LOWCHO = 8% carbohydrate [< 5% sugars], 15% protein, 77% fat). Postprandial metabolic responses to a prescribed breakfast (20% EI) were monitored under laboratory conditions before an ad libitum test lunch, with subsequent diet and physical activity monitoring under free-living conditions until blood sample collection the following morning. RESULTS: The MODSUG, LOWSUG and LOWCHO diets resulted in similar mean [95%CI] rates of both physical activity energy expenditure (771 [624, 919] vs. 677 [565, 789] vs. 802 [614, 991] kcal·d-1; p = 0.29] and energy intake (2071 [1794, 2347] vs. 2195 [1918, 2473] vs. 2194 [1890, 2498] kcal·d-1; P = 0.34), respectively. The LOWCHO condition elicited the lowest glycaemic and insulinaemic responses to breakfast (P < 0.01) but the highest 24-h increase in LDL-cholesterol concentrations (P < 0.001), with no differences between the MODSUG and LOWSUG treatments. Leptin concentrations decreased over 24-h of consuming LOWCHO relative to LOWSUG (p < 0.01). CONCLUSION: When energy density is controlled for, restricting either sugar or total dietary carbohydrate does not modulate physical activity level or energy intake over a 24-h period (~ 19-h free-living) despite substantial metabolic changes. CLINICAL TRIALS REGISTRATION ID: NCT03509610, https://clinicaltrials.gov/show/NCT03509610.

For Flux Sake: Isotopic Tracer Methods of Monitoring Human Carbohydrate Metabolism During Exercise.

Isotopic tracers can reveal insights into the temporal nature of metabolism and track the fate of ingested substrates. A common use of tracers is to assess aspects of human carbohydrate metabolism during exercise under various established models. The dilution model is used alongside intravenous infusion of tracers to assess carbohydrate appearance and disappearance rates in the circulation, which can be further delineated into exogenous and endogenous sources. The incorporation model can be used to estimate exogenous carbohydrate oxidation rates. Combining methods can provide insight into key factors regulating health and performance, such as muscle and liver glycogen utilization, and the underlying regulation of blood glucose homeostasis before, during, and after exercise. Obtaining accurate, quantifiable data from tracers, however, requires careful consideration of key methodological principles. These include appropriate standardization of pretrial diet, specific tracer choice, whether a background trial is necessary to correct expired breath CO2 enrichments, and if so, what the appropriate background trial should consist of. Researchers must also consider the intensity and pattern of exercise, and the type, amount, and frequency of feeding (if any). The rationale for these considerations is discussed, along with an experimental design checklist and equation list which aims to assist researchers in performing high-quality research on carbohydrate metabolism during exercise using isotopic tracer methods.

Impact of pre-exercise feeding status on metabolic adaptations to endurance-type exercise training.

Nutrition and exercise metabolism are vibrant physiological fields, yet at times it feels as if greater progress could be made by better integrating these disciplines. Exercise is advocated for improving metabolic health, in part by increasing peripheral insulin sensitivity and glycaemic control. However, when a modest-to-high carbohydrate load is consumed before and/or during each exercise bout within a training programme, increases in oral glucose insulin sensitivity can be blunted in both men of a healthy weight and those with overweight/obesity. Exercise training-induced adaptation in the energy sensing AMP-activated protein kinase (AMPK) and the insulin-sensitive glucose transporter GLUT4 protein levels are sensitive to pre-exercise feeding status in both healthy individuals and individuals classified as overweight or obese. Increased lipid oxidation may, in part, explain the enhanced adaptive responses to exercise training performed before (i.e. fasted-state exercise) versus after nutrient ingestion. Evidence in individuals with type 2 diabetes currently shows no effect of altering nutrient-exercise timing for measured markers of metabolic health, or greater reductions in glycated haemoglobin (HbA1c) concentrations with exercise performed after versus before nutrient provision. Since the metabolic inflexibility associated with type 2 diabetes diminishes differences in lipid oxidation between the fasted and fed states, it is plausible that pre-exercise feeding status does not alter adaptations to exercise when metabolic flexibility is already compromised. Current evidence suggests restricting carbohydrate intake before and during exercise can enhance some health benefits of exercise, but in order to establish clinical guidelines, further research is needed with hard outcomes and different populations.

The acute effect of fasted exercise on energy intake, energy expenditure, subjective hunger and gastrointestinal hormone release compared to fed exercise in healthy individuals: a systematic review and network meta-analysis.

OBJECTIVE: To determine the acute effect of fasted and fed exercise on energy intake, energy expenditure, subjective hunger and gastrointestinal hormone release. METHODS: CENTRAL, Embase, MEDLINE, PsycInfo, PubMed, Scopus and Web of Science databases were searched to identify randomised, crossover studies in healthy individuals that compared the following interventions: (i) fasted exercise with a standardised post-exercise meal [FastEx + Meal], (ii) fasted exercise without a standardised post-exercise meal [FastEx + NoMeal], (iii) fed exercise with a standardised post-exercise meal [FedEx + Meal], (iv) fed exercise without a standardised post-exercise meal [FedEx + NoMeal]. Studies must have measured ad libitum meal energy intake, within-lab energy intake, 24-h energy intake, energy expenditure, subjective hunger, acyl-ghrelin, peptide YY, and/or glucagon-like peptide 1. Random-effect network meta-analyses were performed for outcomes containing ≥5 studies. RESULTS: 17 published articles (23 studies) were identified. Ad libitum meal energy intake was significantly lower during FedEx + Meal compared to FedEx + NoMeal (MD: -489 kJ; 95% CI, -898 to -80 kJ; P = 0.019). Within-lab energy intake was significantly lower during FastEx + NoMeal compared to FedEx + NoMeal (MD: -1326 kJ; 95% CI, -2102 to -550 kJ; P = 0.001). Similarly, 24-h energy intake following FastEx + NoMeal was significantly lower than FedEx + NoMeal (MD: -2095 kJ; 95% CI, -3910 kJ to -280 kJ; P = 0.024). Energy expenditure was however significantly lower during FastEx + NoMeal compared to FedEx+NoMeal (MD: -0.67 kJ/min; 95% CI, -1.10 to -0.23 kJ/min; P = 0.003). Subjective hunger was significantly higher during FastEx + Meal (MD: 13 mm; 95% CI, 5-21 mm; P = 0.001) and FastEx + NoMeal (MD: 23 mm; 95% CI, 16-30 mm; P < 0.001) compared to FedEx + NoMeal. CONCLUSION: FastEx + NoMeal appears to be the most effective strategy to produce a short-term decrease in energy intake, but also results in increased hunger and lowered energy expenditure. Concerns regarding experimental design however lower the confidence in these findings, necessitating future research to rectify these issues when investigating exercise meal timing and energy balance. PROSPERO REGISTRATION NUMBER: CRD42020208041. KEY POINTS: Fed exercise with a standardised post-exercise meal resulted in the lowest energy intake at the ad libitum meal served following exercise completion. Fasted exercise without a standardised post-exercise meal resulted in the lowest within-lab and 24-h energy intake, but also produced the lowest energy expenditure and highest hunger. Methodological issues lower the confidence in these findings and necessitate future work to address identified problems.

Addition of Fructose to a Carbohydrate-Rich Breakfast Improves Cycling Endurance Capacity in Trained Cyclists.

It was previously demonstrated that postexercise ingestion of fructose-glucose mixtures can lead to superior liver and equal muscle glycogen synthesis as compared with glucose-based carbohydrates (CHOs) only. After an overnight fast, liver glycogen stores are reduced, and based on this we hypothesized that addition of fructose to a glucose-based breakfast would lead to improved subsequent endurance exercise capacity. In this double-blind cross-over randomized study (eight males, peak oxygen uptake: 62.2 ± 5.4 ml·kg-1·min-1), participants completed two experimental trials consisting of two exercise bouts. In the afternoon of Day 1, they completed a cycling interval training session to normalize glycogen stores after which a standardized high-CHO diet was provided for 4 hr. On Day 2, in the morning, participants received 2 g/kg of CHOs in the form of glucose and rice or fructose and rice, both in a CHO ratio of 1:2. Two hours later they commenced cycling exercise session at the intensity of the first ventilatory threshold until task failure. Exercise capacity was higher in fructose and rice (137.0 ± 22.7 min) as compared with glucose and rice (130.06 ± 19.87 min; p = .046). Blood glucose and blood lactate did not differ between the trials (p > .05) and neither did CHO and fat oxidation rates (p > .05). However, due to the duration of exercise, total CHO oxidation was higher in fructose and rice (326 ± 60 g vs. 298 ± 61 g, p = .009). Present data demonstrate that addition of fructose to a glucose-based CHO source at breakfast improves endurance exercise capacity. Further studies are required to determine the mechanisms and optimal dose and ratio.

Ketone Monoester Ingestion Alters Metabolism and Simulated Rugby Performance in Professional Players.

Ketone ingestion can alter metabolism but effects on exercise performance are unclear, particularly with regard to the impact on intermittent-intensity exercise and team-sport performance. Nine professional male rugby union players each completed two trials in a double-blind, randomized, crossover design. Participants ingested either 90 ± 9 g carbohydrate (CHO; 9% solution) or an energy matched solution containing 20 ± 2 g CHO (3% solution) and 590 mg/kg body mass ß-hydroxybutyrate monoester (CHO + BHB-ME) before and during a simulated rugby union-specific match-play protocol, including repeated high-intensity, sprint and power-based performance tests. Mean time to complete the sustained high-intensity performance tests was reduced by 0.33 ± 0.41 s (2.1%) with CHO + BHB-ME (15.53 ± 0.52 s) compared with CHO (15.86 ± 0.80 s) placebo (p = .04). Mean time to complete the sprint and power-based performance tests were not different between trials. CHO + BHB-ME resulted in blood BHB concentrations that remained >2 mmol/L during exercise (p < .001). Serum lactate and glycerol concentrations were lower after CHO + BHB-ME than CHO (p < .05). Coingestion of a BHB-ME with CHO can alter fuel metabolism (attenuate circulating lactate and glycerol concentrations) and may improve high-intensity running performance during a simulated rugby match-play protocol, without improving shorter duration sprint and power-based efforts.

Carb-conscious: the role of carbohydrate intake in recovery from exercise.

PURPOSE OF REVIEW: The present review summarized evidence on the role of carbohydrates in recovery from exercise within the context of acute and chronic effects on metabolism and performance. RECENT FINDINGS: Recent studies demonstrate that, in contrast to recovery of muscle glycogen stores, the recovery of liver glycogen stores can be accelerated by the co-ingestion of fructose with glucose-based carbohydrates. Three recent studies suggest this can extend time-to-exhaustion during endurance exercise tests. However, periodically restricting carbohydrate intakes during recovery from some training sessions to slow the recovery of liver and muscle glycogen stores may, over time, result in a modest increase in the ability to oxidize fat during exercise in a fasted state. Whether this periodized strategy translates into a performance advantage in the fed state remains to be clearly demonstrated. SUMMARY: To maximize recovery of glycogen stores and the capacity to perform in subsequent endurance exercise, athletes should consider ingesting at least 1.2 g carbohydrate per kilogram body mass per hour - for the first few hours of recovery - as a mixture of fructose and glucose-based carbohydrates. However, if a goal is increased capacity for fat oxidation, athletes should consider restricting carbohydrate intakes during recovery from some key training sessions. VIDEO ABSTRACT: http://links.lww.com/COCN/A15.

Physiological responses to carbohydrate overfeeding.

PURPOSE OF REVIEW: To consider emerging research into the physiological effects of excessive dietary carbohydrate intake, with a particular focus on interactions with physical activity. RECENT FINDINGS: A single episode of massive carbohydrate overload initiates physiological responses to stimulate additional peptide hormone secretion by the gut and the conversion of carbohydrate into lipid by the intestine, liver and adipose tissue. These acute responses maintain glycaemic control both via increased oxidation of carbohydrate (rather than lipid) and via nonoxidative disposal of surplus carbohydrate into endogenous glycogen and lipid storage depots. Sustained carbohydrate overfeeding therefore results in a chronic accumulation of lipid in the liver, skeletal muscle and adipose tissue, which can impair insulin sensitivity and cardiometabolic health in general. Beyond any direct effect of such lipid deposition on body mass/composition, there is not yet clear evidence of physiologically meaningful metabolic or behavioural adaptations to carbohydrate overfeeding in terms of other components of energy balance. However, regular physical exercise can mitigate the negative health effects of carbohydrate overfeeding, independent of any effect on the net carbohydrate surplus. SUMMARY: Research in this area has advanced understanding regarding the mechanisms of weight gain and associated health outcomes within the modern context of an abundant supply of dietary carbohydrate.

Resting skeletal muscle PNPLA2 (ATGL) and CPT1B are associated with peak fat oxidation rates in men and women but do not explain observed sex differences.

NEW FINDINGS: What is the central question of this study? What is the relationship between proteins in skeletal muscle and adipose tissue determined at rest and at peak rates of fat oxidation in men and women? What is the main finding and its importance? The resting contents of proteins in skeletal muscle involved in triglyceride hydrolysis and mitochondrial lipid transport were more strongly associated with peak fat oxidation rates than proteins related to lipid transport or hydrolysis in adipose tissue. Although females displayed higher relative rates of fat oxidation than males, this was not explained by the proteins measured in this study, suggesting that other factors determine sex differences in fat metabolism. ABSTRACT: We explored key proteins involved in fat metabolism that might be associated with peak fat oxidation (PFO) and account for sexual dimorphism in fuel metabolism during exercise. Thirty-six healthy adults [15 women; 40 ± 11 years of age; peak oxygen consumption 42.5 ± 9.5 ml (kg body mass)-1  min-1 ; mean ± SD] completed two exercise tests to determine PFO via indirect calorimetry. Resting adipose tissue and/or skeletal muscle biopsies were obtained to determine the adipose tissue protein content of PLIN1, ABHD5 (CGI-58), LIPE (HSL), PNPLA2 (ATGL), ACSL1, CPT1B and oestrogen receptor α (ERα) and the skeletal muscle protein content of FABP 3 (FABPpm), PNPLA2 (ATGL), ACSL1, CTP1B and ESR1 (ERα). Moderate strength correlations were found between PFO [in milligrams per kilogram of fat-free mass (FFM) per minute] and the protein content of PNPLA2 (ATGL) [rs  = 0.41 (0.03-0.68), P < 0.05] and CPT1B [rs  = 0.45 (0.09-0.71), P < 0.05] in skeletal muscle. No other statistically significant bivariate correlations were found consistently. Females had a greater relative PFO than males [7.1 ± 1.9 vs. 4.5 ± 1.3 and 7.3 ± 1.7 vs. 4.8 ± 1.2 mg (kg FFM)-1  min-1 in the adipose tissue (n = 14) and skeletal muscle (n = 12) subgroups, respectively (P < 0.05)]. No statistically significant sex differences were found in the content of these proteins. The regulation of PFO might involve processes relating to intramyocellular triglyceride hydrolysis and mitochondrial fatty acid transport, and adipose tissue is likely to play a more minor role than muscle. Sex differences in fat metabolism are likely to be attributable to factors other than the resting content of proteins in skeletal muscle and adipose tissue relating to triglyceride hydrolysis and fatty acid transport.

Plasma glucagon-like peptide-1 responses to ingestion of protein with increasing doses of milk minerals rich in calcium.

A high dose of whey protein hydrolysate fed with milk minerals rich in calcium (Capolac®) results in enhanced glucagon-like peptide-1 (GLP-1) concentrations in lean individuals; however, the effect of different calcium doses ingested alongside protein is unknown. The present study assessed the dose response of calcium fed alongside 25 g whey protein hydrolysate on GLP-1 concentrations in individuals with overweight/obesity. Eighteen adults (mean ± sd: 8M/10F, 34 ± 18 years, 28·2 ± 2·9 kgm-2) completed four trials in a randomised, double-blind, crossover design. Participants consumed test solutions consisting of 25 g whey protein hydrolysate (CON), supplemented with 3179 mg (LOW), 6363 mg (MED) or 9547 mg (HIGH) Capolac® on different occasions, separated by at least 48 h. The calcium content of test solutions equated to 65, 892, 1719 and 2547 mg, respectively. Arterialised-venous blood was sampled over 180 min to determine plasma concentrations of GLP-1TOTAL, GLP-17-36amide, insulin, glucose, NEFA, and serum concentrations of calcium and albumin. Ad libitum energy intake was measured at 180 min. Time-averaged incremental AUC (iAUC) for GLP-1TOTAL (pmol·l-1·min-1) did not differ between CON (23 ± 4), LOW (25 ± 6), MED (24 ± 5) and HIGH (24 ± 6). Energy intake (kcal) did not differ between CON (940 ± 387), LOW (884 ± 345), MED (920 ± 334) and HIGH (973 ± 390). Co-ingestion of whey protein hydrolysate with Capolac® does not potentiate GLP-1 release in comparison with whey protein hydrolysate alone. The study was registered at clinical trials (NCT03819972).

Nocturnal whey protein ingestion impairs post-prandial glucose tolerance at breakfast.

Poor post-prandial glucose control is a risk factor for multiple health conditions. The second-meal effect refers to the progressively improved glycaemic control with repeated feedings, an effect which is achievable with protein ingestion at the initial eating occasion. The most pronounced glycaemic response each day therefore typically occurs following breakfast, so the present study investigated whether ingesting protein during the night could improve glucose control at the first meal of the day. In a randomised crossover design, fifteen adults (seven males, eight females; age, 22 (sd 3) years; BMI, 24·0 (sd 2·8) kg/m2; fasting blood glucose, 4·9 (sd 0·5) mmol/l) woke at 04.00 (sd 1) hours to ingest 300 ml water with or without 63 g whey protein. Participants then completed a mixed-macronutrient meal tolerance test (1 g carbohydrate/kg body mass, 2356 (sd 435) kJ), 5 h 39 min following the nocturnal feeding. Nocturnal protein ingestion increased the glycaemic response (incremental AUC) to breakfast by 43·5 (sd 55·5) mmol × 120 min/l (P = 0·009, d = 0·94). Consistent with this effect, individual peak blood glucose concentrations were 0·6 (sd 1·0) mmol/l higher following breakfast when protein had been ingested (P = 0·049, d = 0·50). Immediately prior to breakfast, rates of lipid oxidation were 0·02 (sd 0·03) g/min higher (P = 0·045) in the protein condition, followed by an elevated post-prandial energy expenditure (0·38 (sd 0·50) kJ/min, P = 0·018). Post-prandial appetite and energy intake were similar between conditions. The present study reveals a paradoxical second-meal phenomenon whereby nocturnal whey protein feeding impaired subsequent glucose tolerance, whilst increasing post-prandial energy expenditure.

Determinants of Peak Fat Oxidation Rates During Cycling in Healthy Men and Women.

This study explored lifestyle and biological determinants of peak fat oxidation (PFO) during cycle ergometry, using duplicate measures to account for day-to-day variation. Seventy-three healthy adults (age range: 19-63 years; peak oxygen consumption [V˙O2peak]: 42.4 [10.1] ml·kg BM-1·min-1; n = 32 women]) completed trials 7-28 days apart that assessed resting metabolic rate, a resting venous blood sample, and PFO by indirect calorimetry during an incremental cycling test. Habitual physical activity (combined heart rate accelerometer) and dietary intake (weighed record) were assessed before the first trial. Body composition was assessed 2-7 days after the second identical trial by dual-energy X-ray absorptiometry scan. Multiple linear regressions were performed to identify determinants of PFO (mean of two cycle tests). A total variance of 79% in absolute PFO (g·min-1) was explained with positive coefficients for V˙O2peak (strongest predictor), FATmax (i.e the % of V˙O2peak that PFO occurred at), and resting fat oxidation rate (g·min-1), and negative coefficients for body fat mass (kg) and habitual physical activity level. When expressed relative to fat-free mass, 64% of variance in PFO was explained: positive coefficients for FATmax (strongest predictor), V˙O2peak, and resting fat oxidation rate, and negative coefficients for male sex and fat mass. This duplicate design revealed that biological and lifestyle factors explain a large proportion of variance in PFO during incremental cycling. After accounting for day-to-day variation in PFO, V˙O2peak and FATmax were strong and consistent predictors of PFO.

Galactose Ingested with a High-Fat Beverage Increases Postprandial Lipemia Compared with Glucose but Not Fructose Ingestion in Healthy Men.

BACKGROUND: Fructose ingestion with a high-fat beverage increases postprandial lipemia when compared with glucose. It is unknown whether other sugars, such as galactose, also increase postprandial lipemia. OBJECTIVES: The objective was to assess whether galactose ingestion within a high-fat beverage increases postprandial lipemia relative to glucose or fructose. METHODS: Two experiments were conducted, which contrasted different test drinks under otherwise standardized conditions. In Experiment 1, 10 nonobese men (age: 22 ± 1 y; BMI, 23.5 ± 2.2 kg/2) ingested either galactose or glucose (0.75 g supplemented carbohydrate per⋅kilogram body mass) within a high-fat test drink (0.94 g fat per kilogram body mass). In Experiment 2, a separate group of 9 nonobese men (age: 26 ± 6 y; BMI: 23.5 ± 2.6 kg/m2) ingested either galactose or fructose (identical doses as those in Experiment 1) within the same high-fat test drink. Capillary blood was sampled before and at frequent intervals after ingestion of the test drinks for a 300-min period to determine plasma triacylglycerol, glucose, lactate, nonesterified fatty acid, and insulin concentrations. Paired t tests and 2-way, repeated-measures ANOVA were used to compare conditions within each experiment. RESULTS: The incremental AUC for triacylglycerol was greater following galactose ingestion compared with glucose (127 ± 59 compared with 80 ± 48 mmol⋅L-1 × 300 min, respectively; P = 0.04) but not compared with fructose (136 ± 74 compared with 133 ± 63 mmol⋅L-1 ×300 min, respectively; P = 0.91). Plasma lactate concentrations also increased to a greater extent with galactose compared with glucose ingestion (time-condition interaction: P < 0.001) but not fructose ingestion (time-condition interaction: P = 0.17). CONCLUSIONS: Galactose ingestion within a high-fat beverage exacerbates postprandial lipemia and plasma lactate concentrations compared with glucose but not fructose in nonobese men. These data suggest that galactose metabolism may be more similar to fructose than to glucose, providing a rationale to reassess the metabolic fate of galactose ingestion in humans. This trial was registered at clinicaltrials.gov as NCT03439878.