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.

Structure, function and regulation of mammalian glucose transporters of the SLC2 family.

The SLC2 genes code for a family of GLUT proteins that are part of the major facilitator superfamily (MFS) of membrane transporters. Crystal structures have recently revealed how the unique protein fold of these proteins enables the catalysis of transport. The proteins have 12 transmembrane spans built from a replicated trimer substructure. This enables 4 trimer substructures to move relative to each other, and thereby alternately opening and closing a cleft to either the internal or the external side of the membrane. The physiological substrate for the GLUTs is usually a hexose but substrates for GLUTs can include urate, dehydro-ascorbate and myo-inositol. The GLUT proteins have varied physiological functions that are related to their principal substrates, the cell type in which the GLUTs are expressed and the extent to which the proteins are associated with subcellular compartments. Some of the GLUT proteins translocate between subcellular compartments and this facilitates the control of their function over long- and short-time scales. The control of GLUT function is necessary for a regulated supply of metabolites (mainly glucose) to tissues. Pathophysiological abnormalities in GLUT proteins are responsible for, or associated with, clinical problems including type 2 diabetes and cancer and a range of tissue disorders, related to tissue-specific GLUT protein profiles. The availability of GLUT crystal structures has facilitated the search for inhibitors and substrates and that are specific for each GLUT and that can be used therapeutically. Recent studies are starting to unravel the drug targetable properties of each of the GLUT proteins.

Chemical biology probes of mammalian GLUT structure and function.

The structure and function of glucose transporters of the mammalian GLUT family of proteins has been studied over many decades, and the proteins have fascinated numerous research groups over this time. This interest is related to the importance of the GLUTs as archetypical membrane transport facilitators, as key limiters of the supply of glucose to cell metabolism, as targets of cell insulin and exercise signalling and of regulated membrane traffic, and as potential drug targets to combat cancer and metabolic diseases such as type 2 diabetes and obesity. This review focusses on the use of chemical biology approaches and sugar analogue probes to study these important proteins.

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.

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.

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.

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.

Kinetic evidence for unique regulation of GLUT4 trafficking by insulin and AMP-activated protein kinase activators in L6 myotubes.

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of approximately 20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min(-1)) and was decreased during the steady-state responses to insulin (0.18 min(-1)), AICAR (0.16 min(-1)), and A-769662 (0.24 min(-1)). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.

Translocation of the Na+/H+ exchanger 1 (NHE1) in cardiomyocyte responses to insulin and energy-status signalling.

The Na+/H+ exchanger NHE1 is a highly regulated membrane protein that is required for pH homoeostasis in cardiomyocytes. The activation of NHE1 leads to proton extrusion, which is essential for counteracting cellular acidity that occurs following increased metabolic activity or ischaemia. The activation of NHE1 intrinsic catalytic activity has been well characterized and established experimentally. However, we have examined in the present study whether a net translocation of NHE1 to the sarcolemma of cardiomyocytes may also be involved in the activation process. We have determined the distribution of NHE1 by means of immunofluorescence microscopy and cell-surface biotinylation. We have discovered changes in the distribution of NHE1 that occur when cardiomyocytes are stimulated with insulin that are PI3K (phosphoinositide 3-kinase)-dependent. Translocation of NHE1 also occurs when cardiomyocytes are challenged by hypoxia, or inhibition of mitochondrial oxidative metabolism or electrically induced contraction, but these responses occur through a PI3K-independent process. As the proposed additional level of control of NHE1 through translocation was unexpected, we have compared this process with the well-established translocation of the glucose transporter GLUT4. In immunofluorescence microscopy comparisons, the translocation of NHE1 and GLUT4 to the sarcolemma that occur in response to insulin appear to be very similar. However, in basal unstimulated cells the two proteins are mainly located, with the exception of some co-localization in the perinuclear region, in distinct subcellular compartments. We propose that the mechanisms of translocation of NHE1 and GLUT4 are linked such that they provide spatially and temporally co-ordinated responses to cardiac challenges that necessitate re-adjustments in glucose transport, glucose metabolism and cell pH.

Insulin signaling meets vesicle traffic of GLUT4 at a plasma-membrane-activated fusion step.

A hypothesis that accounts for most of the available literature on insulin-stimulated GLUT4 translocation is that insulin action controls the access of GLUT4 vesicles to a constitutively active plasma-membrane fusion process. However, using an in vitro fusion assay, we show here that fusion is not constitutively active. Instead, the rate of fusion activity is stimulated 8-fold by insulin. Both the magnitude and time course of stimulated in vitro fusion recapitulate the cellular insulin response. Fusion is cell cytoplasm and SNARE dependent but does not require cell cytoskeleton. Furthermore, insulin activation of the plasma-membrane fraction of the fusion reaction is the essential step in regulation. Akt from the cytoplasm fraction is required for fusion. However, the participation of Akt in the stimulation of in vitro fusion is dependent on its in vitro recruitment onto the insulin-activated plasma membrane.

Thrifty Tbc1d1 and Tbc1d4 proteins link signalling and membrane trafficking pathways.

Establishing a complete pathway which links occupancy of the insulin receptor to GLUT4 translocation has been particularly elusive because of the complexities involved in studying both signalling and membrane trafficking processes. However, Lienhard's group has now discovered two related molecules that could function in this linking role. These proteins, Tbc1d4 (also known as AS160) and now Tbc1d1, as reported in this issue of the Biochemical Journal, have been demonstrated to be Rab GAPs (GTPase-activating proteins) that link upstream to Akt (protein kinase B) and phosphoinositide 3-kinase and downstream to Rabs involved in trafficking of GLUT4 vesicles. The data from Leinhard and colleagues suggest that high levels of Rab GAP activity lead to suppression of GLUT4 translocation and this observation has wide significance and is likely to be relevant to the recent discovery that mutations in the Tbc1d1 gene lead to some cases of severe human obesity.

Identification of Insulin-Activated Rab Proteins in Adipose Cells Using Bio-ATB-GTP Photolabeling Technique.

We have recently developed a photolabeling method to identify GTP-loaded Rab proteins. The new biotinylated GTP analogue (Bio-ATB-GTP) binds to GTP-binding proteins and after a UV irradiation a covalent bond is formed between the protein and the photoreactive diazirine group on the photolabel. The tagged protein can then be isolated and detected using the classic biotin-streptavidin interaction. In this chapter, we describe the Bio-ATB-GTP photolabel and discuss the advantages of using this photolabeling approach to detect GTP-loaded Rab proteins compared to other existing methodologies. We also describe a step-by-step procedure for detecting the activated state of a Rab protein in primary rat adipocytes.

Rab28 is a TBC1D1/TBC1D4 substrate involved in GLUT4 trafficking.

The Rab-GTPase-activating proteins (GAPs) TBC1D1 and TBC1D4 play important roles in the insulin-stimulated translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane in muscle cells and adipocytes. We identified Rab28 as a substrate for the GAP domains of both TBC1D1 and TBC1D4 in vitro. Rab28 is expressed in adipose cells and skeletal muscle, and its GTP-binding state is acutely regulated by insulin. We found that in intact isolated mouse skeletal muscle, siRNA-mediated knockdown of Rab28 decreases basal glucose uptake. Conversely, in primary rat adipose cells, overexpression of Rab28-Q72L, a constitutively active mutant, increases basal cell surface levels of an epitope-tagged HA-GLUT4. Our results indicate that Rab28 is a novel GTPase involved in the intracellular retention of GLUT4 in insulin target cells.

Highly Potent and Isoform Selective Dual Site Binding Tankyrase/Wnt Signaling Inhibitors That Increase Cellular Glucose Uptake and Have Antiproliferative Activity.

Compounds 13 and 14 were evaluated against 11 PARP isoforms to reveal that both 13 and 14 were more potent and isoform selective toward inhibiting tankyrases (TNKSs) than the "standard" inhibitor 1 (XAV939)5, i.e., IC50 = 100 pM vs TNKS2 and IC50 = 6.5 µM vs PARP1 for 14. In cellular assays, 13 and 14 inhibited Wnt-signaling, enhanced insulin-stimulated glucose uptake, and inhibited the proliferation of DLD-1 colorectal adenocarcinoma cells to a greater extent than 1.

Thermal stability, storage and release of proteins with tailored fit in silica.

Biological substances based on proteins, including vaccines, antibodies, and enzymes, typically degrade at room temperature over time due to denaturation, as proteins unfold with loss of secondary and tertiary structure. Their storage and distribution therefore relies on a "cold chain" of continuous refrigeration; this is costly and not always effective, as any break in the chain leads to rapid loss of effectiveness and potency. Efforts have been made to make vaccines thermally stable using treatments including freeze-drying (lyophilisation), biomineralisation, and encapsulation in sugar glass and organic polymers. Here for the first time we show that proteins can be enclosed in a deposited silica "cage", rendering them stable against denaturing thermal treatment and long-term ambient-temperature storage, and subsequently released into solution with their structure and function intact. This "ensilication" method produces a storable solid protein-loaded material without the need for desiccation or freeze-drying. Ensilication offers the prospect of a solution to the "cold chain" problem for biological materials, in particular for vaccines.

Long-term metformin treatment stimulates cardiomyocyte glucose transport through an AMP-activated protein kinase-dependent reduction in GLUT4 endocytosis.

Long-term (18 h) metformin treatment of cardiomyocytes increased glucose transport activity 3- to 5-fold, as measured using the phosphorylated sugar 2-deoxy-D-glucose and the nonphosphorylated sugar 3-O-methyl-D-glucose. The affinity for 3-O-methyl-D-glucose transport was not increased by metformin treatment. Total levels of glucose transporter 4 (GLUT4) were not changed by 18-h culture with or without insulin or metformin treatment. GLUT1 levels were elevated after 18 h in culture, but this increase was not altered by insulin or metformin treatment. Metformin-induced stimulation of transport was not inhibited by treatment with wortmannin and was additive with that of insulin. These data suggest that the metformin effect is mediated by a signaling route independent of phosphatidylinositol 3-kinase and Akt. Surprisingly, however, levels of both phospho-AMP-activated protein kinase and phospho-Akt were increased 4- and 3-fold, respectively, after metformin treatment. Chronic treatment with insulin for 18 h led to down-regulation of insulin-stimulated glucose transport. Cotreatment with metformin bypassed this insulin resistance by maintaining high transport levels. These data also indicate an independent point of convergence of metformin and insulin stimuli on GLUT4 regulatory processes. To test the possibility of altered GLUT4 subcellular trafficking, the kinetics of GLUT4 exocytosis and endocytosis were determined. Metformin treatment markedly slowed endocytosis of GLUT4, but exocytosis was not increased. We conclude that metformin treatment leads to a longer residence time of GLUT4 in the plasma membrane due to an AMP-activated protein kinase-dependent reduction in endocytosis. This accounts for metformin's ability to enhance hexose transport activity above insulin-stimulated and Akt-dependent levels.

The human glomerular podocyte is a novel target for insulin action.

Microalbuminuria is significant both as the earliest stage of diabetic nephropathy and as an independent cardiovascular risk factor in nondiabetic subjects, in whom it is associated with insulin resistance. The link between disorders of cellular insulin metabolism and albuminuria has been elusive. Here, we report using novel conditionally immortalized human podocytes in vitro and human glomeruli ex vivo that the podocyte, the principal cell responsible for prevention of urinary protein loss, is insulin responsive and able to approximately double its glucose uptake within 15 min of insulin stimulation. Conditionally immortalized human glomerular endothelial cells do not respond to insulin, suggesting that insulin has a specific effect on the podocyte in the glomerular filtration barrier. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1 and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. Insulin responsiveness in this key structural component of the glomerular filtration barrier may have central relevance for understanding of diabetic nephropathy and for the association of albuminuria with states of insulin resistance.

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.