Six Weeks of Morning Fasting Causes Little Adaptation of Metabolic or Appetite Responses to Feeding in Adults with Obesity.

OBJECTIVE: The aim of this study was to determine the effects of sustained morning fasting or breakfast consumption on metabolism, energy intake, and appetite in healthy adults with obesity. METHODS: An independent-measures randomized controlled trial with baseline and follow-up laboratory assessment days separated by a 6-week intervention of either morning fasting (0 kcal until 12:00 pm) or daily breakfast (> 700 kcal by 11:00 am) was performed. Measures included metabolic outcomes (glucose, insulin, nonesterified fatty acids), hormones regulating appetite (total/acylated ghrelin, peptide YY, leptin), and energy expenditure (diet-induced thermogenesis) parameters throughout a laboratory test day and ad libitum intake following a fixed breakfast. RESULTS: Allocation to fasting versus breakfast resulted in minimal adaptation as reflected by the metabolic outcomes or the majority of appetite regulatory outcomes for either area under curve or time-course-based measures (P > 0.05). Ad libitum lunch intake was not different (P = 0.13), nor was diet-induced thermogenesis or a composite appetite score (both P > 0.10). However, there was a reduced total area under the curve for peptide YY (P = 0.05) and increased postprandial hunger ratings (P = 0.05) in the breakfast group. CONCLUSIONS: There was little evidence of metabolic adaptation to acute feeding or negative consequences from sustained morning fasting. This indicates that previously observed differences between breakfast consumers and skippers may be acute effects of feeding or may have resulted from other lifestyle factors.

Postprandial Metabolism and Appetite Do Not Differ between Lean Adults that Eat Breakfast or Morning Fast for 6 Weeks.

Background: It remains unknown whether sustained daily feeding-fasting patterns modify the acute response to specific feedings on a given day. Objective: We conducted a randomized controlled trial to establish if daily breakfast consumption or fasting until noon modifies the acute metabolic and appetitive responses to a fixed breakfast and ad libitum lunch. Methods: With the use of a parallel group design, we randomly assigned 31 healthy, lean men and women (22-56 y) to 6 wk of either consuming ≥700 kcal of self-selected items before 1100 or fasting (0 kcal) until 1200 daily. Following 48 h of diet and physical activity standardization, we examined metabolic and appetite responses to a standardized breakfast and ad libitum lunch before and after the intervention. Data were analyzed using 3- and 2-way ANCOVA. Results: Systemic concentrations of energy balance regulatory hormones total and acylated ghrelin, leptin, and peptide tyrosine-tyrosine) responded similarly to breakfast and lunch before and after 6 wk of either morning fasting or regular breakfast, with the exception of a tendency for increased glucagon-like peptide-1 concentrations from baseline to follow-up in the Breakfast Group compared with a decrease over that period in the Fasting Group [P = 0.06, partial eta squared value (ƞ2) = 0.16]. Subjective appetite sensations also did not differ over the course of the day, and ad libitum energy intake at lunch was not systematically affected by either intervention, decreasing by 27 kcal (95% CI: -203, 149 kcal) with fasting and by 77 kcal (95% CI: -210, 56 kcal) with breakfast. Similarly, glycemic, insulinemic, lipemic, and thermogenic responses to breakfast and lunch were very stable at baseline and follow-up and, thus, did not differ between treatment groups. Conclusions: Our results indicate that a sustained period of either extended morning fasting or eating a daily breakfast has minimal effect upon acute metabolic and appetite responses in lean adults. This trial was registered at www.isrctn.org as ISRCTN31521726.

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.

Is breakfast the most important meal of the day?

The Bath Breakfast Project is a series of randomised controlled trials exploring the effects of extended morning fasting on energy balance and health. These trials were categorically not designed to answer whether or not breakfast is the most important meal of the day. However, this review will philosophise about the meaning of that question and about what questions we should be asking to better understand the effects of breakfast, before summarising how individual components of energy balance and health respond to breakfast v. fasting in lean and obese adults. Current evidence does not support a clear effect of regularly consuming or skipping breakfast on body mass/composition, metabolic rate or diet-induced thermogenesis. Findings regarding energy intake are variable, although the balance of evidence indicates some degree of compensatory feeding later in the day such that overall energy intake is either unaffected or slightly lower when breakfast is omitted from the diet. However, even if net energy intake is reduced, extended morning fasting may not result in expected weight loss due to compensatory adjustments in physical activity thermogenesis. Specifically, we report that both lean and obese adults expended less energy during the morning when remaining in the fasted state than when consuming a prescribed breakfast. Further research is required to examine whether particular health markers may be responsive to breakfast-induced responses of individual components of energy balance irrespective of their net effect on energy balance and therefore body mass.

The causal role of breakfast in energy balance and health: a randomized controlled trial in obese adults.

BACKGROUND: The causal nature of associations between breakfast and health remain unclear in obese individuals. OBJECTIVE: We sought to conduct a randomized controlled trial to examine causal links between breakfast habits and components of energy balance in free-living obese humans. DESIGN: The Bath Breakfast Project is a randomized controlled trial with repeated measures at baseline and follow-up among a cohort in South West England aged 21-60 y with dual-energy X-ray absorptiometry-derived fat mass indexes of ≥13 kg/m(2) for women (n = 15) and ≥9 kg/m(2) for men (n = 8). Components of energy balance (resting metabolic rate, physical activity thermogenesis, diet-induced thermogenesis, and energy intake) were measured under free-living conditions with random allocation to daily breakfast (≥700 kcal before 1100) or extended fasting (0 kcal until 1200) for 6 wk, with baseline and follow-up measures of health markers (e.g., hematology/adipose biopsies). RESULTS: Breakfast resulted in greater physical activity thermogenesis during the morning than when fasting during that period (difference: 188 kcal/d; 95% CI: 40, 335) but without any consistent effect on 24-h physical activity thermogenesis (difference: 272 kcal/d; 95% CI: -254, 798). Energy intake was not significantly greater with breakfast than fasting (difference: 338 kcal/d; 95% CI: -313, 988). Body mass increased across both groups over time but with no treatment effects on body composition or any change in resting metabolic rate (stable within 8 kcal/d). Metabolic/cardiovascular health also did not respond to treatments, except for a reduced insulinemic response to an oral-glucose-tolerance test over time with daily breakfast relative to an increase with daily fasting (P = 0.05). CONCLUSIONS: In obese adults, daily breakfast leads to greater physical activity during the morning, whereas morning fasting results in partial dietary compensation (i.e., greater energy intake) later in the day. There were no differences between groups in weight change and most health outcomes, but insulin sensitivity increased with breakfast relative to fasting. This trial was registered at www.isrctn.org as ISRCTN31521726.

Carbohydrate-rich breakfast attenuates glycaemic, insulinaemic and ghrelin response to ad libitum lunch relative to morning fasting in lean adults.

Breakfast omission is associated with obesity and CVD/diabetes, but the acute effects of extended morning fasting upon subsequent energy intake and metabolic/hormonal responses have received less attention. In a randomised cross-over design, thirty-five lean men (n 14) and women (n 21) extended their overnight fast or ingested a typical carbohydrate-rich breakfast in quantities relative to RMR (i.e. 1963 (sd 238) kJ), before an ad libitum lunch 3 h later. Blood samples were obtained hourly throughout the day until 3 h post-lunch, with subjective appetite measures assessed. Lunch intake was greater following extended fasting (640 (sd 1042) kJ, P< 0.01) but incompletely compensated for the omitted breakfast, with total intake lower than the breakfast trial (3887 (sd 1326) v. 5213 (sd 1590) kJ, P< 0.001). Systemic concentrations of peptide tyrosine-tyrosine and leptin were greater during the afternoon following breakfast (both P< 0.05) but neither acylated/total ghrelin concentrations were suppressed by the ad libitum lunch in the breakfast trial, remaining greater than the morning fasting trial throughout the afternoon (all P< 0.05). Insulin concentrations were greater during the afternoon in the morning fasting trial (all P< 0.01). There were no differences between trials in subjective appetite during the afternoon. In conclusion, morning fasting caused incomplete energy compensation at an ad libitum lunch. Breakfast increased some anorectic hormones during the afternoon but paradoxically abolished ghrelin suppression by the second meal. Extending morning fasting until lunch altered subsequent metabolic and hormonal responses but without greater appetite during the afternoon. The present study clarifies the impact of acute breakfast omission and adds novel insights into second-meal metabolism.

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.

The causal role of breakfast in energy balance and health: a randomized controlled trial in lean adults.

BACKGROUND: Popular beliefs that breakfast is the most important meal of the day are grounded in cross-sectional observations that link breakfast to health, the causal nature of which remains to be explored under real-life conditions. OBJECTIVE: The aim was to conduct a randomized controlled trial examining causal links between breakfast habits and all components of energy balance in free-living humans. DESIGN: The Bath Breakfast Project is a randomized controlled trial with repeated-measures at baseline and follow-up in a cohort in southwest England aged 21-60 y with dual-energy X-ray absorptiometry-derived fat mass indexes ≤11 kg/m² in women (n = 21) and ≤7.5 kg/m² in men (n = 12). Components of energy balance (resting metabolic rate, physical activity thermogenesis, energy intake) and 24-h glycemic responses were measured under free-living conditions with random allocation to daily breakfast (≥700 kcal before 1100) or extended fasting (0 kcal until 1200) for 6 wk, with baseline and follow-up measures of health markers (eg, hematology/biopsies). RESULTS: Contrary to popular belief, there was no metabolic adaptation to breakfast (eg, resting metabolic rate stable within 11 kcal/d), with limited subsequent suppression of appetite (energy intake remained 539 kcal/d greater than after fasting; 95% CI: 157, 920 kcal/d). Rather, physical activity thermogenesis was markedly higher with breakfast than with fasting (442 kcal/d; 95% CI: 34, 851 kcal/d). Body mass and adiposity did not differ between treatments at baseline or follow-up and neither did adipose tissue glucose uptake or systemic indexes of cardiovascular health. Continuously measured glycemia was more variable during the afternoon and evening with fasting than with breakfast by the final week of the intervention (CV: 3.9%; 95% CI: 0.1%, 7.8%). CONCLUSIONS: Daily breakfast is causally linked to higher physical activity thermogenesis in lean adults, with greater overall dietary energy intake but no change in resting metabolism. Cardiovascular health indexes were unaffected by either of the treatments, but breakfast maintained more stable afternoon and evening glycemia than did fasting.

Exercise counteracts the effects of short-term overfeeding and reduced physical activity independent of energy imbalance in healthy young men.

Physical activity can affect many aspects of metabolism but it is unclear to what extent this relies on manipulation of energy balance. Twenty-six active men age 25 ± 7 years (mean ± SD) were randomly assigned either to consume 50% more energy than normal by over-consuming their habitual diet for 7 days whilst simultaneously restricting their physical activity below 4000 steps day(-1) to induce an energy surplus (SUR group; n = 14) or to the same regimen but with 45 min of daily treadmill running at 70% of maximum oxygen uptake (SUR+EX group; n = 12). Critically, the SUR+EX group received additional dietary energy intake to account for the energy expended by exercise, thus maintaining a matched energy surplus. At baseline and follow-up, fasted blood samples and abdominal subcutaneous adipose tissue biopsies were obtained and oral glucose tolerance tests conducted. Insulinaemic responses to a standard glucose load increased 2-fold from baseline to follow-up in the SUR group (17 ± 16 nmol (120 min) l(-1); P = 0.002) whereas there was no change in the SUR+EX group (1 ± 6 nmol (120 min) l(-1)). Seven of 17 genes within adipose tissue were differentially expressed in the SUR group; expression of SREBP-1c, FAS and GLUT4 was significantly up-regulated and expression of PDK4, IRS2, HSL and visfatin was significantly down-regulated (P ≤ 0.05). The pAMPK/AMPK protein ratio in adipose tissue was significantly down-regulated in the SUR group (P = 0.005). Vigorous-intensity exercise counteracted most of the effects of short-term overfeeding and under-activity at the whole-body level and in adipose tissue, even in the face of a standardised energy surplus.

Bath Breakfast Project (BBP)--examining the role of extended daily fasting in human energy balance and associated health outcomes: study protocol for a randomised controlled trial [ISRCTN31521726].

BACKGROUND: Current guidance regarding the role of daily breakfast in human health is largely grounded in cross-sectional observations. However, the causal nature of these relationships has not been fully explored and what limited information is emerging from controlled laboratory-based experiments appears inconsistent with much existing data. Further progress in our understanding therefore requires a direct examination of how daily breakfast impacts human health under free-living conditions. METHODS/DESIGN: The Bath Breakfast Project (BBP) is a randomised controlled trial comparing the effects of daily breakfast consumption relative to extended fasting on energy balance and human health. Approximately 70 men and women will undergo extensive laboratory-based assessments of their acute metabolic responses under fasted and post-prandial conditions, to include: resting metabolic rate, substrate oxidation, dietary-induced thermogenesis and systemic concentrations of key metabolites/hormones. Physiological and psychological indices of appetite will also be monitored both over the first few hours of the day (i.e. whether fed or fasted) and also following a standardised test lunch used to assess voluntary energy intake under controlled conditions. Baseline measurements of participants' anthropometric characteristics (e.g. DEXA) will be recorded prior to intervention, along with an oral glucose tolerance test and acquisition of adipose tissue samples to determine expression of key genes and estimates of tissue-specific insulin action. Participants will then be randomly assigned either to a group prescribed an energy intake of ≥3000 kJ before 1100 each day or a group to extend their overnight fast by abstaining from ingestion of energy-providing nutrients until 1200 each day, with all laboratory-based measurements followed-up 6 weeks later. Free-living assessments of energy intake (via direct weighed food diaries) and energy expenditure (via combined heart-rate/accelerometry) will be made during the first and last week of intervention, with continuous glucose monitors worn both to document chronic glycaemic responses to the intervention and to verify compliance.

Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells.

Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors.

AS160 phosphotyrosine-binding domain constructs inhibit insulin-stimulated GLUT4 vesicle fusion with the plasma membrane.

AS160 (TBC1D4) is a known Akt substrate that is phosphorylated downstream of insulin action and that leads to regulated traffic of GLUT4. As GLUT4 vesicle fusion with the plasma membrane is a highly regulated step in GLUT4 traffic, we investigated whether AS160 and 14-3-3 interactions are involved in this process. Fusion was inhibited by a human truncated AS160 variant that encompasses the first N-terminal phosphotyrosine-binding (PTB) domain, by either of the two N-terminal PTB domains, and by a tandem construct of both PTB domains of rat AS160. We also found that in vitro GLUT4 vesicle fusion was strongly inhibited by the 14-3-3-quenching inhibitors R18 and fusicoccin. To investigate the mode of interaction of AS160 and 14-3-3, we examined insulin-dependent increases in the levels of these proteins on GLUT4 vesicles. 14-3-3γ was enriched on insulin-stimulated vesicles, and its binding to AS160 on GLUT4 vesicles was inhibited by the AS160 tandem PTB domain construct. These data suggest a model for PTB domain action on GLUT4 vesicle fusion in which these constructs inhibit insulin-stimulated 14-3-3γ interaction with AS160 rather than AS160 phosphorylation.

A dominant-negative ESCRT-III protein perturbs cytokinesis and trafficking to lysosomes.

In eukaryotic cells, the completion of cytokinesis is dependent on membrane trafficking events to deliver membrane to the site of abscission. Golgi and recycling endosomal-derived proteins are required for the terminal stages of cytokinesis. Recently, protein subunits of the ESCRT (endosomal sorting complexes required for transport) that are normally involved in late endosome to lysosome trafficking have also been implicated in abscission. Here, we report that a subunit, CHMP3 (charged multivesicular body protein-3), of ESCRT-III localizes at the midbody. Deletion of the C-terminal autoinhibitory domain of CHMP3 inhibits cytokinesis. At the midbody, CHMP3 does not co-localize with Rab11, suggesting that it is not present on recycling endosomes. These results combined provide compelling evidence that proteins involved in late endosomal function are necessary for the end stages of cytokinesis.