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).

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.

Thermal Stabilisation of Lysozyme through Ensilication.

Protein therapeutics, vaccines, and other commercial products are often sensitive to environmental factors, such as temperature and long-term storage. In many cases, long-term protein stability is achieved by refrigeration or freezing. One alternative is the encapsulation of the protein cargo within an inert silica matrix (ensilication) and storage or transport at room temperature as a dry powder. In this paper, we test the effect of three commonly used biological buffers on the ensilication, storage, and desilication of the enzyme lysozyme. We show that ensilication protects lysozyme from heat (100 °C for 1 h) and during storage (18 months at room temperature). The choice of ensilication buffer has little effect on the activity of lysozyme after desilication. Our results provide confidence in the continued pursuit of ensilication as a methodology for protein stabilisation and in its compatibility with biological buffers.

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.

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.

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.

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.

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.

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).

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.

Molecular adaptations of adipose tissue to 6 weeks of morning fasting vs. daily breakfast consumption in lean and obese adults.

KEY POINTS: In lean individuals, 6 weeks of extended morning fasting increases the expression of genes involved in lipid turnover (ACADM) and insulin signalling (IRS2) in subcutaneous abdominal adipose tissue. In obese individuals, 6 weeks of extended morning fasting increases IRS2 expression in subcutaneous abdominal adipose tissue. The content and activation status of key proteins involved in insulin signalling and glucose transport (GLUT4, Akt1 and Akt2) were unaffected by extended morning fasting. Therefore, any observations of altered adipose tissue insulin sensitivity with extended morning fasting do not necessarily require changes in insulin signalling proximal to Akt. Insulin-stimulated adipose tissue glucose uptake rates are lower in obese versus lean individuals, but this difference is abolished when values are normalised to whole-body fat mass. This suggests a novel hypothesis which proposes that the reduced adipose glucose uptake in obesity is a physiological down-regulation to prevent excessive de novo lipogenesis. ABSTRACT: This study assessed molecular responses of human subcutaneous abdominal adipose tissue (SCAT) to 6 weeks of morning fasting. Forty-nine healthy lean (n = 29) and obese (n = 20) adults provided SCAT biopsies before and after 6 weeks of morning fasting (FAST; 0 kcal until 12.00 h) or daily breakfast consumption (BFAST; ≥700 kcal before 11.00 h). Biopsies were analysed for mRNA levels of selected genes, and GLUT4 and Akt protein content. Basal and insulin-stimulated Akt activation and tissue glucose uptake rates were also determined. In lean individuals, lipid turnover and insulin signalling genes (ACADM and IRS2) were up-regulated with FAST versus BFAST (ACADM: 1.14 (95% CI: 0.97-1.30) versus 0.80 (95% CI: 0.64-0.96), P = 0.007; IRS2: 1.75 (95% CI: 1.33-2.16) versus 1.09 (95% CI: 0.67-1.51), P = 0.03, respectively). In obese individuals, no differential (FAST versus BFAST) expression was observed in genes involved in lipid turnover (all P > 0.1). GLUT4, Akt protein content and insulin-stimulated Akt phosphorylation were unaffected by FAST versus BFAST in both lean and obese cohorts (all P > 0.1). Lower insulin-stimulated glucose uptake rates in obese versus lean individuals were eradicated when normalised to whole-body fat mass (P = 0.416). We conclude that morning fasting up-regulates lipid turnover genes in SCAT of lean individuals. Secondly, altered SCAT insulin sensitivity with morning fasting is unlikely to be explained by signalling proximal to Akt. Finally, lower insulin-stimulated SCAT glucose uptake rates in obese individuals are proportional to whole-body fat mass, suggesting a compensatory down-regulation, presumably to prevent excessive de novo lipogenesis in adipose tissue. This trial was registered as ISRCTN31521726.

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.

Feeding influences adipose tissue responses to exercise in overweight men.

Feeding profoundly affects metabolic responses to exercise in various tissues, but the effect of feeding status on human adipose tissue responses to exercise has never been studied. Ten healthy overweight men aged 26 ± 5 yr (mean ± SD) with a waist circumference of 105 ± 10 cm walked at 60% of maximum oxygen uptake under either fasted or fed conditions in a randomized, counterbalanced design. Feeding comprised 648 ± 115 kcal 2 h before exercise. Blood samples were collected at regular intervals to examine changes in metabolic parameters and adipokine concentrations. Adipose tissue samples were obtained at baseline and 1 h after exercise to examine changes in adipose tissue mRNA expression and secretion of selected adipokines ex vivo. Adipose tissue mRNA expression of pyruvate dehydrogenase kinase isozyme 4 (PDK4), adipose triglyceride lipase, hormone-sensitive lipase (HSL), fatty acid translocase/CD36, glucose transporter type 4 (GLUT4), and insulin receptor substrate 2 (IRS2) in response to exercise were lower in fed compared with fasted conditions (all P ≤ 0.05). Postexercise adipose IRS2 protein was affected by feeding (P ≤ 0.05), but Akt2, AMPK, IRS1, GLUT4, PDK4, and HSL protein levels were not different. Feeding status did not impact serum and ex vivo adipose secretion of IL-6, leptin, or adiponectin in response to exercise. This is the first study to show that feeding before acute exercise affects postexercise adipose tissue gene expression, and we propose that feeding is likely to blunt long-term adipose tissue adaptation to regular exercise.

Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes.

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.

Insulin regulates Rab3-Noc2 complex dissociation to promote GLUT4 translocation in rat adipocytes.

AIMS/HYPOTHESIS: The glucose transporter GLUT4 is present mainly in insulin-responsive tissues of fat, heart and skeletal muscle and is translocated from intracellular membrane compartments to the plasma membrane (PM) upon insulin stimulation. The transit of GLUT4 to the PM is known to be dependent on a series of Rab proteins. However, the extent to which the activity of these Rabs is regulated by the action of insulin action is still unknown. We sought to identify insulin-activated Rab proteins and Rab effectors that facilitate GLUT4 translocation. METHODS: We developed a new photoaffinity reagent (Bio-ATB-GTP) that allows GTP-binding proteomes to be explored. Using this approach we screened for insulin-responsive GTP loading of Rabs in primary rat adipocytes. RESULTS: We identified Rab3B as a new candidate insulin-stimulated G-protein in adipocytes. Using constitutively active and dominant negative mutants and Rab3 knockdown we provide evidence that Rab3 isoforms are key regulators of GLUT4 translocation in adipocytes. Insulin-stimulated Rab3 GTP binding is associated with disruption of the interaction between Rab3 and its negative effector Noc2. Disruption of the Rab3-Noc2 complex leads to displacement of Noc2 from the PM. This relieves the inhibitory effect of Noc2, facilitating GLUT4 translocation. CONCLUSIONS/INTERPRETATION: The discovery of the involvement of Rab3 and Noc2 in an insulin-regulated step in GLUT4 translocation suggests that the control of this translocation process is unexpectedly similar to regulated secretion and particularly pancreatic insulin-vesicle release.

FGT-1 is the major glucose transporter in C. elegans and is central to aging pathways.

Caenorhabditis elegans is widely used as a model for investigation of the relationships between aging, nutrient restriction and signalling via the DAF-2 (abnormal dauer formation 2) receptor for insulin-like peptides and AGE-1 [ageing alteration 1; orthologue of PI3K (phosphoinositide 3-kinase)], but the identity of the glucose transporters that may link these processes is unknown. We unexpectedly find that of the eight putative GLUT (glucose transporter)-like genes only the two splice variants of one gene have a glucose transport function in an oocyte expression system. We have named this gene fgt-1 (facilitated glucose transporter, isoform 1). We show that knockdown of fgt-1 RNA leads to loss of glucose transport and reduced glucose metabolism in wild-type worms. The FGT-1 glucose transporters of C. elegans thus play a key role in glucose energy supply to C. elegans. Importantly, knockdown of fgt-1 leads to an extension of lifespan equivalent, but not additive, to that observed in daf-2 and age-1 mutant worms. The results of the present study are consistent with DAF-2 and AGE-1 signalling stimulating glucose transport in C. elegans and this process being associated with the longevity phenotype in daf-2 and age-1 mutant worms. We propose that fgt-1 constitutes a common axis for the lifespan extending effects of nutrient restriction and reduced insulin-like peptide signalling.

Ketogenic diet but not free-sugar restriction alters glucose tolerance, lipid metabolism, peripheral tissue phenotype, and gut microbiome: RCT.

Restricted sugar and ketogenic diets can alter energy balance/metabolism, but decreased energy intake may be compensated by reduced expenditure. In healthy adults, randomization to restricting free sugars or overall carbohydrates (ketogenic diet) for 12 weeks reduces fat mass without changing energy expenditure versus control. Free-sugar restriction minimally affects metabolism or gut microbiome but decreases low-density lipoprotein cholesterol (LDL-C). In contrast, a ketogenic diet decreases glucose tolerance, increases skeletal muscle PDK4, and reduces AMPK and GLUT4 levels. By week 4, the ketogenic diet reduces fasting glucose and increases apolipoprotein B, C-reactive protein, and postprandial glycerol concentrations. However, despite sustained ketosis, these effects are no longer apparent by week 12, when gut microbial beta diversity is altered, possibly reflective of longer-term adjustments to the ketogenic diet and/or energy balance. These data demonstrate that restricting free sugars or overall carbohydrates reduces energy intake without altering physical activity, but with divergent effects on glucose tolerance, lipoprotein profiles, and gut microbiome.

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.