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.

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).

The impact of physical inactivity on glucose homeostasis when diet is adjusted to maintain energy balance in healthy, young males.

BACKGROUND & AIMS: It is unclear if dietary adjustments to maintain energy balance during reduced physical activity can offset inactivity-induced reductions in insulin sensitivity and glucose disposal to produce normal daily glucose concentrations and meal responses. Therefore, the aim of the present study was to examine the impact of long-term physical inactivity (60 days of bed rest) on daily glycemia when in energy balance. METHODS: Interstitial glucose concentrations were measured using Continuous Glucose Monitoring Systems (CGMS) for 5 days before and towards the end of bed rest in 20 healthy, young males (Age: 34 ± 8 years; BMI: 23.5 ± 1.8 kg/m2). Energy intake was reduced during bed rest to match energy expenditure, but the types of foods and timing of meals was maintained. Fasting venous glucose and insulin concentrations were determined, as well as the change in whole-body glucose disposal using a hyperinsulinemic-euglycemic clamp (HIEC). RESULTS: Following long-term bed rest, fasting plasma insulin concentration increased 40% (p = 0.004) and glucose disposal during the HIEC decreased 24% (p < 0.001). Interstitial daily glucose total area under the curve (tAUC) from pre-to post-bed rest increased on average by 6% (p = 0.041), despite a 20 and 25% reduction in total caloric and carbohydrate intake, respectively. The nocturnal period (00:00-06:00) showed the greatest change to glycemia with glucose tAUC for this period increasing by 9% (p = 0.005). CGMS measures of daily glycemic variability (SD, J-Index, M-value and MAG) were not changed during bed rest. CONCLUSIONS: Reduced physical activity (bed rest) increases glycemia even when daily energy intake is reduced to maintain energy balance. However, the disturbance to daily glucose homeostasis was much more modest than the reduced capacity to dispose of glucose, and glycemic variability was not negatively affected by bed rest, likely due to positive mitigating effects from the contemporaneous reduction in dietary energy and carbohydrate intake. CLINICAL TRIALS RECORD: NCT03594799 (registered July 20, 2018) (https://clinicaltrials.gov/ct2/show/NCT03594799).

The effect of exercise in a fasted state on plasma low-density lipoprotein cholesterol concentrations in males and females.

Cardiovascular disease (CVD) is the leading cause of death worldwide. Physical activity interventions improve almost all modifiable CVD risk factors, but the effect of physical activity on low density lipoprotein cholesterol (LDL-C) is uncertain. This may be due to lack of research on the feeding status in which the physical activity is performed. The aim of this study is to investigate the effect of fasted versus fed exercise on LDL-C concentrations in males and females. One hundred healthy participants, equal males and females, aged between 25 and 60 years will be recruited and will undergo a home-based 12-week exercise intervention. After baseline testing, participants will be randomized to a fasted exercise (exercise after an 8-h fast) or fed exercise (exercise 90-180 min after ingestion of 1 g kg-1 CHO) group and will perform 50 min of moderate intensity exercise (e.g., 95% heart rate of lactate threshold 1) three times a week either before or after a high carbohydrate (1 g kg-1 ) meal. Participants will visit the laboratory again at week 4 and week 12 and measurements will be taken for body composition, resting blood pressure, fasting blood glucose, lipid profiles and systemic inflammation, lactate threshold, and 14-day blood glucose control.

Influence of traumatic lower-limb amputation on physical activity, body composition, and cardiometabolic risks: A descriptive preliminary study.

BACKGROUND: Following traumatic lower-limb amputation (LLA), humans are predisposed to numerous unfavorable changes in health, including the development of secondary chronic health conditions such as metabolic disorders and cardiovascular disease. OBJECTIVE: To determine within and between group differences in cardiometabolic component risks, body composition, and physical activity (PA) in individuals with traumatic unilateral and bilateral LLA, compared to noninjured controls. DESIGN: Prospective observational cohort study. SETTING: A military complex trauma rehabilitation center. PARTICIPANTS: Sixteen males with traumatic LLA (8 unilateral, mean age 30 ± 5 years and 8 bilateral, mean age 29 ± 3 years). Thirteen active age-matched males with no LLA (28 ± 5 years) acted as controls and performed habitual activities of daily living. INTERVENTION: Participants with LLA attended two 4-week periods of inpatient rehabilitation, separated by two 6-week periods of home-based recovery. MAIN OUTCOME MEASURES: Venous blood samples were taken prior to and following a 75 g oral glucose load, for determination of biomarkers, including insulin and glucose, at baseline and 20 weeks. Body composition (dual X-ray absorptiometry) was measured at baseline, 10 weeks, and 20 weeks. Daily PA was recorded using a triaxial accelerometer for 7 days during inpatient rehabilitation and while at home. Energy expenditure was estimated using population-specific equations. RESULTS: Individuals with bilateral LLA demonstrated more unfavorable mean body composition values, lower PA, and increased cardiometabolic health risk compared to controls. Cardiometabolic syndrome was identified in 63% of individuals with bilateral LLA. No statistically significant differences in cardiometabolic component risk factors, body composition, and estimated daily PA were reported between unilateral LLA and control groups (p > .05). While at home, mean PA counts.day-1 reduced by 17% (p = .018) and 42% (p = .001) in the unilateral and bilateral LLA groups, respectively. CONCLUSIONS: Despite extensive inpatient rehabilitation, cardiometabolic component risks are elevated in individuals with bilateral LLA but are comparable between unilateral LLA and active noninjured control groups. Innovative strategies that improve/support the long-term PA and cardiometabolic health of severely injured individuals with bilateral LLA are warranted.

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.

Muscle-Specific Ablation of Glucose Transporter 1 (GLUT1) Does Not Impair Basal or Overload-Stimulated Skeletal Muscle Glucose Uptake.

Glucose transporter 1 (GLUT1) is believed to solely mediate basal (insulin-independent) glucose uptake in skeletal muscle; yet recent work has demonstrated that mechanical overload, a model of resistance exercise training, increases muscle GLUT1 levels. The primary objective of this study was to determine if GLUT1 is necessary for basal or overload-stimulated muscle glucose uptake. Muscle-specific GLUT1 knockout (mGLUT1KO) mice were generated and examined for changes in body weight, body composition, metabolism, systemic glucose regulation, muscle glucose transporters, and muscle [3H]-2-deoxyglucose uptake ± the GLUT1 inhibitor BAY-876. [3H]-hexose uptake ± BAY-876 was also examined in HEK293 cells-expressing GLUT1-6 or GLUT10. mGLUT1KO mice exhibited no impairments in body weight, lean mass, whole body metabolism, glucose tolerance, basal or overload-stimulated muscle glucose uptake. There was no compensation by the insulin-responsive GLUT4. In mGLUT1KO mouse muscles, overload stimulated higher expression of mechanosensitive GLUT6, but not GLUT3 or GLUT10. In control and mGLUT1KO mouse muscles, 0.05 µM BAY-876 impaired overload-stimulated, but not basal glucose uptake. In the GLUT-HEK293 cells, BAY-876 inhibited glucose uptake via GLUT1, GLUT3, GLUT4, GLUT6, and GLUT10. Collectively, these findings demonstrate that GLUT1 does not mediate basal muscle glucose uptake and suggest that a novel glucose transport mechanism mediates overload-stimulated glucose uptake.

Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand.

Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction.

GLUT4 On the move.

Insulin rapidly stimulates GLUT4 translocation and glucose transport in fat and muscle cells. Signals from the occupied insulin receptor are translated into downstream signalling changes in serine/threonine kinases within timescales of seconds, and this is followed by delivery and accumulation of the glucose transporter GLUT4 at the plasma membrane. Kinetic studies have led to realisation that there are distinct phases of this stimulation by insulin. There is a rapid initial burst of GLUT4 delivered to the cell surface from a subcellular reservoir compartment and this is followed by a steady-state level of continuing stimulation in which GLUT4 recycles through a large itinerary of subcellular locations. Here, we provide an overview of the phases of insulin stimulation of GLUT4 translocation and the molecules that are currently considered to activate these trafficking steps. Furthermore, we suggest how use of new experimental approaches together with phospho-proteomic data may help to further identify mechanisms for activation of these trafficking processes.

The Impact of Long-term Physical Inactivity on Adipose Tissue Immunometabolism.

CONTEXT: Adipose tissue and physical inactivity both influence metabolic health and systemic inflammation, but how adipose tissue responds to chronic physical inactivity is unknown. OBJECTIVE: This work aimed to characterize the impact of chronic physical inactivity on adipose tissue in healthy, young males. METHODS: We collected subcutaneous adipose tissue from 20 healthy, young men before and after 60 days of complete bed rest with energy intake reduced to maintain energy balance and fat mass. We used RNA sequencing, flow cytometry, ex vivo tissue culture, and targeted protein analyses to examine adipose tissue phenotype. RESULTS: Our results indicate that the adipose tissue transcriptome, stromal cellular compartment, and insulin signaling protein abundance are largely unaffected by bed rest when fat mass is kept stable. However, there was an increase in the circulating concentration of several adipokines, including plasma leptin, which was associated with inactivity-induced increases in plasma insulin and absent from adipose tissue cultured ex vivo under standardized culture conditions. CONCLUSION: Physical inactivity-induced disturbances to adipokine concentrations such as leptin, without changes to fat mass, could have profound metabolic implications outside a clinical facility when energy intake is not tightly controlled.

Divergent immunometabolic changes in adipose tissue and skeletal muscle with ageing in healthy humans.

KEY POINTS: Ageing is associated with increased systemic inflammation and metabolic dysfunction that contributes to the development of age-associated diseases. The role of adipose tissue in immunometabolic alterations that take place with ageing is unknown in humans. We show, in healthy, active and lean older adults, that adipose tissue, but not skeletal muscle, displays considerable pro-inflammatory transcriptomic, cellular and secretory changes, as well as a reduction in insulin signalling proteins compared to younger adults. These findings indicate that adipose tissue undergoes substantial immunometabolic alterations with ageing, and that these changes are tissue-specific and more profound than those observed in skeletal muscle or in the circulation. These results identify adipose tissue as an important tissue in the biological ageing process in humans, which may exhibit signs of immunometabolic dysfunction prior to systemic manifestation. ABSTRACT: Ageing and obesity are both characterized by inflammation and a deterioration in metabolic health. It is now clear that adipose tissue plays a major role in inflammation and metabolic control in obesity, although little is known about the role of adipose tissue in human ageing. To understand how ageing impacts adipose tissue, we characterized subcutaneous adipose tissue and skeletal muscle samples from twelve younger (27 ± 4 years [Young]) and twelve older (66 ± 5 years [Old]) active/non-obese males. We performed a wide-range of whole-body and tissue measures, including RNA-sequencing and multicolour flow cytometry. We also measured a range of inflammatory and metabolic proteins in the circulation and their release by adipose tissue, ex vivo. Both adipose tissue and muscle had ∼2-fold more immune cells per gram of tissue with ageing. In adipose tissue, this immune cell infiltration was driven by increased memory/effector T-cells, whereas, in muscle, the accumulation was driven by memory/effector T-cells and macrophages. Transcriptomic analysis revealed that, with ageing, adipose tissue, but not muscle, was enriched for inflammatory transcripts/pathways related to acquired and innate immunity. Ageing also increased the adipose tissue pro-inflammatory secretory profile. Insulin signalling protein content was reduced in adipose tissue, but not muscle. Our findings indicate that adipose tissue undergoes substantial immunometabolic changes with ageing in humans, and that these changes are tissue-specific and more profound than those observed in the circulation and skeletal muscle.

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.

Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells.

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell "SMALPome", membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be "non-specific" than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

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).

Protein- and Calcium-Mediated GLP-1 Secretion: A Narrative Review.

Glucagon-like peptide 1 (GLP-1) is an incretin hormone produced in the intestine that is secreted in response to nutrient exposure. GLP-1 potentiates glucose-dependent insulin secretion from the pancreatic ß cells and promotes satiety. These important actions on glucose metabolism and appetite have led to widespread interest in GLP-1 receptor agonism. Typically, this involves pharmacological GLP-1 mimetics or targeted inhibition of dipeptidyl peptidase-IV, the enzyme responsible for GLP-1 degradation. However, nutritional strategies provide a widely available, cost-effective alternative to pharmacological strategies for enhancing hormone release. Recent advances in nutritional research have implicated the combined ingestion of protein and calcium with enhanced endogenous GLP-1 release, which is likely due to activation of receptors with high affinity and/or sensitivity for amino acids and calcium. Specifically targeting these receptors could enhance gut hormone secretion, thus providing a new therapeutic option. This narrative review provides an overview of the latest research on protein- and calcium-mediated GLP-1 release with an emphasis on human data, and a perspective on potential mechanisms that link potent GLP-1 release to the co-ingestion of protein and calcium. In light of these recent findings, potential future research directions are also presented.

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.

Lipid Metabolism Links Nutrient-Exercise Timing to Insulin Sensitivity in Men Classified as Overweight or Obese.

CONTEXT: Pre-exercise nutrient availability alters acute metabolic responses to exercise, which could modulate training responsiveness. OBJECTIVE: To assess acute and chronic effects of exercise performed before versus after nutrient ingestion on whole-body and intramuscular lipid utilization and postprandial glucose metabolism. DESIGN: (1) Acute, randomized, crossover design (Acute Study); (2) 6-week, randomized, controlled design (Training Study). SETTING: General community. PARTICIPANTS: Men with overweight/obesity (mean ± standard deviation, body mass index: 30.2 ± 3.5 kg⋅m-2 for Acute Study, 30.9 ± 4.5 kg⋅m-2 for Training Study). INTERVENTIONS: Moderate-intensity cycling performed before versus after mixed-macronutrient breakfast (Acute Study) or carbohydrate (Training Study) ingestion. RESULTS: Acute Study-exercise before versus after breakfast consumption increased net intramuscular lipid utilization in type I (net change: -3.44 ± 2.63% versus 1.44 ± 4.18% area lipid staining, P < 0.01) and type II fibers (-1.89 ± 2.48% versus 1.83 ± 1.92% area lipid staining, P < 0.05). Training Study-postprandial glycemia was not differentially affected by 6 weeks of exercise training performed before versus after carbohydrate intake (P > 0.05). However, postprandial insulinemia was reduced with exercise training performed before but not after carbohydrate ingestion (P = 0.03). This resulted in increased oral glucose insulin sensitivity (25 ± 38 vs -21 ± 32 mL⋅min-1⋅m-2; P = 0.01), associated with increased lipid utilization during exercise (r = 0.50, P = 0.02). Regular exercise before nutrient provision also augmented remodeling of skeletal muscle phospholipids and protein content of the glucose transport protein GLUT4 (P < 0.05). CONCLUSIONS: Experiments investigating exercise training and metabolic health should consider nutrient-exercise timing, and exercise performed before versus after nutrient intake (ie, in the fasted state) may exert beneficial effects on lipid utilization and reduce postprandial insulinemia.

Fructose and metabolic health: governed by hepatic glycogen status?

Fructose is a commonly ingested dietary sugar which has been implicated in playing a particularly harmful role in the development of metabolic disease. Fructose is primarily metabolised by the liver in humans, and increases rates of hepatic de novo lipogenesis. Fructose increases hepatic de novo lipogenesis via numerous mechanisms: by altering transcriptional and allosteric regulation, interfering with cellular energy sensing, and disrupting the balance between lipid synthesis and lipid oxidation. Hepatic de novo lipogenesis is also upregulated by the inability to synthesise glycogen, either when storage is inhibited in knock-down animal models or storage is saturated in glycogen storage disease. Considering that fructose has the capacity to upregulate hepatic glycogen storage, and replenish these stores more readily following glycogen depleting exercise, the idea that hepatic glycogen storage and hepatic de novo lipogenesis are linked is an attractive prospect. We propose that hepatic glycogen stores may be a key factor in determining the metabolic responses to fructose ingestion, and saturation of hepatic glycogen stores could exacerbate the negative metabolic effects of excessive fructose intake. Since physical activity potently modulates glycogen metabolism, this provides a rationale for considering nutrient-physical activity interactions in metabolic health.

Preexercise breakfast ingestion versus extended overnight fasting increases postprandial glucose flux after exercise in healthy men.

The aim of this study was to characterize postprandial glucose flux after exercise in the fed versus overnight fasted state and to investigate the potential underlying mechanisms. In a randomized order, twelve men underwent breakfast-rest [(BR) 3 h semirecumbent], breakfast-exercise [(BE) 2 h semirecumbent before 60 min of cycling (50% peak power output)], and overnight fasted exercise [(FE) as per BE omitting breakfast] trials. An oral glucose tolerance test (OGTT) was completed after exercise (after rest on BR). Dual stable isotope tracers ([U-13C] glucose ingestion and [6,6-2H2] glucose infusion) and muscle biopsies were combined to assess postprandial plasma glucose kinetics and intramuscular signaling, respectively. Plasma intestinal fatty acid binding (I-FABP) concentrations were determined as a marker of intestinal damage. Breakfast before exercise increased postexercise plasma glucose disposal rates during the OGTT, from 44 g/120 min in FE {35 to 53 g/120 min [mean (normalized 95% confidence interval)] to 73 g/120 min in BE [55 to 90 g/120 min; P = 0.01]}. This higher plasma glucose disposal rate was, however, offset by increased plasma glucose appearance rates (principally OGTT-derived), resulting in a glycemic response that did not differ between BE and FE ( P = 0.11). Plasma I-FABP concentrations during exercise were 264 pg/ml (196 to 332 pg/ml) lower in BE versus FE ( P = 0.01). Breakfast before exercise increases postexercise postprandial plasma glucose disposal, which is offset (primarily) by increased appearance rates of orally ingested glucose. Therefore, metabolic responses to fed-state exercise cannot be readily inferred from studies conducted in a fasted state.