Whole-body Metabolism Research Articles

Lipid metabolism and food ingredients from the perspective of thermogenic adipocytes.

The high heat-producing capacity of brown and beige adipocytes, collectively known as thermogenic adipocytes, contributes to whole-body energy expenditure and is an attractive target for the management of obesity. It has been revealed that the functions of thermogenic adipocytes are important for the regulation of whole-body carbohydrate and lipid metabolism, and the activation of thermogenic adipocytes seems to have beneficial effects for the management of obesity-related metabolic disorders, such as dyslipidemia. Recent studies have showed that specific food ingredients have the potential to activate thermogenic adipocytes via various mechanisms. Some of these are effective not only in rodents, but also in humans, and effective prevention of obesity using these food ingredients is expected. In this review, we introduce the recent findings on the regulatory mechanisms of lipid metabolism by thermogenic adipocytes and food ingredients, demonstrating the potential to activate thermogenic adipocytes and their underlying mechanisms.

Methods to characterize lactate turnover in aging and Alzheimer's disease; The LEAN study

BackgroundThere is evidence that chronic exercise can benefit the brain, but the effects vary markedly between studies. One potential mechanism for exercise-related benefit is the increase in systemic lactate concentration that is well-characterized to occur during exercise. Lactate is known to cross the blood brain barrier and can be used readily as a fuel for neurons. This may be particularly important in Alzheimer's Disease, which is characterized by cerebral hypometabolism. However, little is known about how whole-body lactate metabolism differs between older adults and individuals with cognitive impairment. This information is critical when considering potential differences in responses to exercise in various cognitive diagnosis groups. MethodsHere we describe the use of a “lactate clamp” procedure to adjust blood lactate levels to approximate those achieved during exercise, but while at rest. This trial will compare lactate oxidation between cognitively healthy older adults and cognitively impaired participants. We will further evaluate the effect of acute lactate infusion on cognitive performance. DiscussionThe findings of the study described here, the Lactate for Energy and Neurocognition trial (clinicaltrials.gov # NCT05207397) will add to our understanding of systemic lactate mechanics in cognitively healthy older adults and individuals with Alzheimer's Disease. These findings will be applicable to ongoing exercise trials and to future studies aimed at modulating systemic bioenergetic function in aging and Alzheimer's Disease.

Nitric oxide donors rescue metabolic and mitochondrial dysfunction in obese Alzheimer’s model

Reduced nitric oxide (NO) bioavailability is a pathological link between obesity and Alzheimer’s disease (AD). Obesity-associated metabolic and mitochondrial bioenergetic dysfunction are key drivers of AD pathology. The hypothalamus is a critical brain region during the development of obesity and dysfunction is an area implicated in the development of AD. NO is an essential mediator of blood flow and mitochondrial bioenergetic function, but the role of NO in obesity-AD is not entirely clear. We investigated diet-induced obesity in female APPswe/PS1dE9 (APP) mouse model of AD, which we treated with two different NO donors (sodium nitrite or L-citrulline). After 26 weeks of a high-fat diet, female APP mice had higher adiposity, insulin resistance, and mitochondrial dysfunction (hypothalamus) than non-transgenic littermate (wild type) controls. Treatment with either sodium nitrite or L-citrulline did not reduce adiposity but improved whole-body energy expenditure, substrate oxidation, and insulin sensitivity. Notably, both NO donors restored hypothalamic mitochondrial respiration in APP mice. Our findings suggest that NO is an essential mediator of whole-body metabolism and hypothalamic mitochondrial function, which are severely impacted by the dual insults of obesity and AD pathology.

Gene Expression Shifts in Emperor Penguin Adaptation to the Extreme Antarctic Environment.

Gene expression can accelerate ecological divergence by rapidly tweaking the response of an organism to novel environments, with more divergent environments exerting stronger selection and supposedly, requiring faster adaptive responses. Organisms adapted to extreme environments provide ideal systems to test this hypothesis, particularly when compared to related species with milder ecological niches. The Emperor penguin (Aptenodytes forsteri) is the only endothermic vertebrate breeding in the harsh Antarctic winter, in stark contrast with the less cold-adapted sister species, the King penguin (A. patagonicus). Assembling the first de novo transcriptomes and analysing multi-tissue (brain, kidney, liver, muscle, skin) RNA-Seq data from natural populations of both species, we quantified the shifts in tissue-enhanced genes, co-expression gene networks, and differentially expressed genes characterising Emperor penguin adaptation to the extreme Antarctic. Our analyses revealed the crucial role played by muscle and liver in temperature homeostasis, fasting, and whole-body energy metabolism (glucose/insulin regulation, lipid metabolism, fatty acid beta-oxidation, and blood coagulation). Repatterning at the regulatory level appears as more important in the brain of the Emperor penguin, showing the lowest signature of differential gene expression, but the largest co-expression gene network shift. Nevertheless, over-expressed genes related to mTOR signalling in the brain and the liver support their central role in cold and fasting responses. Besides contributing to understanding the genetics underlying complex traits, like body energy reservoir management, our results provide a first insight into the role of gene expression in adaptation to one of the most extreme environmental conditions endured by an endotherm.

Metabolic Shifts Induced by Helminth Infections and Their Contribution to Stunting in Vulnerable Populations

Aims: to revisit the condition of chronic metabolic shifting caused by immunomodulatory sequence facilitated by helminth infection together with other deteriorating condition such as persistent exposure to infection which contributes to stunting formation. Discussion: During dynamic interaction between active helminth infection and the host, the helminth and its excretory/secretory products induce and arouse the type 2 immune response which drives host tolerance and plays an important role in promoting tissue repair. Helminths also activates M2 Macrophages and induce a metabolic shift, even metabolic reprogramming towards reliance on oxidative phosphorylation, lipid oxidation and amino acid metabolism. Helminth-induced activation and metabolic reprogramming of macrophages precede the improvement in overall whole-body metabolism, denoted by improved insulin sensitivity, body mass in response to high-fat diet and atherogenic index in mammals. Contributions of altered nutrient uptake, adipose tissue function and/or the intestinal microbiota with the ability of helminths to alter metabolic status play a pivotal role in increased metabolism rate and may lead to wasting and even stunting formation. Conclusion: Helminth drives the type 2 immunity and activates its cell line which is responsible for metabolic shifting and reprogramming.

Metabolic Rate and Oxidative Stress as a Risk Factors in the Development of Colorectal Cancer.

There is growing evidence that the body's energy expenditures constitute a significant risk factor for the development of most deadly diseases, including cancer. Our aim was to investigate the impact of basal metabolic rate (BMR) on the growth and progression of colorectal cancer (CRC). To do so, we used a unique model consisting of three lines of laboratory mice (Mus musculus) artificially selected for high (HBMR) and low (LBMR) basal metabolic rate and randomly bred individuals (non-selected, NSBMR). The experimental individuals were implanted with human colorectal cancer cells DLD-1. The variation in BMR between the lines allowed for testing the impact of whole-body metabolism on oxidative and antioxidant parameters in the liver throughout the cancerogenesis process. We investigated the dependence between metabolic values, reactive oxygen species (ROS) levels, and Kelch-like ECH-associated protein 1-based E3 ligase complexes (Keap1) gene activity in these animals. We found that the HBMR strain had a higher concentration of oxidative enzymes compared to the LBMR and NSBMR. Furthermore, the growth rate of CRC tumors was associated with alterations in the levels of oxidative stress enzymes and Keap1 expression in animals with a high metabolic rate. Our results indicate that a faster growth and development of CRC line DLD-1 is associated with enzymatic redox imbalance in animals with a high BMR.

Emerging roles of osteocytes in regulation of bone and skeletal muscle mass.

Contrary to the popular perception that the bone is merely a structural organ, decades of research have established its functional importance in whole-body metabolism. Osteocytes, which comprise >80% of all bone cells, were also initially thought to be terminally differentiated dormant cells lacking metabolic functions. However, new research is increasingly providing evidence that osteocytes not only play a role in the structural integrity of the bone, but also have secretory functions which regulate other bone cells as well as other organs including skeletal muscle - the structural-mechanical neighbor of the bone - via paracrine, and endocrine pathways. However, the publicly available preclinical and clinical data pertaining to the factors secreted by osteocytes and their functions in the musculoskeletal system largely fail to reach a consensus. This review is thus aimed to objectively collate all information available in the public domain for efficient access by researchers in the field. We strongly believe that this review will help researchers in an unbiased design of therapeutic strategies for musculoskeletal disorders.

Cortical actions of thyroid hormone: An exploration and metabolism crossroad

Classically, the central actions of thyroid hormones (THs) on metabolism occur within the hypothalamus. A recent article published in Cell by Sabatini and colleagues demonstrates that TH modulates cerebral cortical circuits of male mice, which might integrate exploratory behavior and whole-body metabolism.

Wnt7b overexpression in osteoblasts stimulates bone formation and reduces obesity in mice on a high-fat diet.

Previous studies have shown that Wnt7b potently stimulates bone formation by promoting osteoblast differentiation and activity. As high-fat feeding leads to obesity and systemic metabolic dysregulation, here we investigate the potential benefit of Wnt7b overexpression in osteoblasts on both bone and whole-body metabolism in mice fed with a high-fat diet (HFD). Wnt7b overexpression elicited massive overgrowth of trabecular and cortical bone but seemed to ameliorate body fat accumulation in mice with prolonged HFD feeding. In addition, Wnt7b overexpression modestly improved glucose tolerance in male mice on HFD. Collectively, the results indicate that targeted overexpression of Wnt7b in osteoblasts not only stimulates bone formation but also improves certain aspects of global metabolism in overnourished mice.

Inferring the metabolic rate of bone

The bone organ is poorly represented in comparative research on mammalian mass-specific metabolic rates. As a first order attempt to remedy this, from the literature we collected mass-specific metabolic rates for all major organs except for the bone organ, and by subtraction infer the rate for the bone organ. The scaling relationships are given of each whole-organ mass-specific metabolic rate and of the relationship between whole-organ metabolic rate and body mass. Scaling of the lung, adipose depot and bone organ with body mass is higher than would be expected by ¾ power scaling. We interpret the similar scalings of bone and the adipose depot in light of their evolved regulation of whole-body metabolism. We also briefly examine the supra-¾ power scaling of the lung as well as the independence of the mass-specific metabolic rate of the heart from body mass. The bone organ exhibits relatively high energy expenditure with increasing body size. The bone marrow and its medullary adipocyte store may be responsible for engendering the greater share of the bone organ's energetic cost.

Reversing Pdgfrβ signaling restores metabolically active beige adipocytes by alleviating ILC2 suppression in aged and obese mice

ObjectivePlatelet Derived Growth Factor Receptor Beta (Pdgfrβ) suppresses the formation of cold temperature-induced beige adipocytes in aged mammals. We aimed to determine if deleting Pdgfrβ in aged mice could rejuvenate metabolically active beige adipocytes by activating group 2 innate lymphoid cells (ILC2), and whether this effect could counteract diet-induced obesity-associated beige fat decline. MethodsWe employed Pdgfrβ gain-of-function and loss-of-function mouse models targeting beige adipocyte progenitor cells (APCs). Our approach included cold exposure, metabolic cage analysis, and age and diet-induced obesity models to examine beige fat development and metabolic function under varied Pdgfrβ activity. ResultsAcute cold exposure alone enhanced metabolic benefits in aged mice, irrespective of beige fat generation. However, Pdgfrβ deletion in aged mice reestablished the formation of metabolically functional beige adipocytes, enhancing metabolism. Conversely, constitutive Pdgfrβ activation in young mice stymied beige fat development. Mechanistically, Pdgfrβ deletion upregulated IL-33, promoting ILC2 recruitment and activation, whereas Pdgfrβ activation reduced IL-33 levels and suppressed ILC2 activity. Notably, diet-induced obesity markedly increased Pdgfrβ expression and Stat1 signaling, which inhibited IL-33 induction and ILC2 activation. Genetic deletion of Pdgfrβ restored beige fat formation in obese mice, improving whole-body metabolism. ConclusionsThis study reveals that cold temperature exposure alone can trigger metabolic activation in aged mammals. However, reversing Pdgfrβ signaling in aged and obese mice not only restores beige fat formation but also renews metabolic function and enhances the immunological environment of white adipose tissue (WAT). These findings highlight Pdgfrβ as a crucial target for therapeutic strategies aimed at combating age- and obesity-related metabolic decline.

Paraventricular oxytocin neurons impact energy intake and expenditure: projections to the bed nucleus of the stria terminalis reduce sucrose consumption.

The part played by oxytocin and oxytocin neurons in the regulation of food intake is controversial. There is much pharmacological data to support a role for oxytocin notably in regulating sugar consumption, however, several recent experiments have questioned the importance of oxytocin neurons themselves. Here we use a combination of histological and chemogenetic techniques to investigate the selective activation or inhibition of oxytocin neurons in the hypothalamic paraventricular nucleus (OxtPVH). We then identify a pathway from OxtPVH neurons to the bed nucleus of the stria terminalis using the cell-selective expression of channel rhodopsin. OxtPVH neurons increase their expression of cFos after both physiological (fast-induced re-feeding or oral lipid) and pharmacological (systemic administration of cholecystokinin or lithium chloride) anorectic signals. Chemogenetic activation of OxtPVH neurons is sufficient to decrease free-feeding in Oxt Cre:hM3Dq mice, while inhibition in Oxt Cre:hM4Di mice attenuates the response to administration of cholecystokinin. Activation of OxtPVH neurons also increases energy expenditure and core-body temperature, without a significant effect on locomotor activity. Finally, the selective, optogenetic stimulation of a pathway from OxtPVH neurons to the bed nucleus of the stria terminalis reduces the consumption of sucrose. Our results support a role for oxytocin neurons in the regulation of whole-body metabolism, including a modulatory action on food intake and energy expenditure. Furthermore, we demonstrate that the pathway from OxtPVH neurons to the bed nucleus of the stria terminalis can regulate sugar consumption.

GCN2 drives diurnal patterns in the hepatic integrated stress response and maintains circadian rhythms in whole body metabolism during amino acid insufficiency.

Disruptions in circadian rhythms are associated with an increased risk of developing metabolic diseases. General control nonderepressible 2 (GCN2), a primary sensor of amino acid insufficiency and activator of the integrated stress response (ISR), has emerged as a conserved regulator of the circadian clock in multiple organisms. The objective of this study was to examine diurnal patterns in hepatic ISR activation in the liver and whole body rhythms in metabolism. We hypothesized that GCN2 activation cues hepatic ISR signaling over a natural 24-h feeding-fasting cycle. To address our objective, wild-type (WT) and whole body Gcn2 knockout (GCN2 KO) mice were housed in metabolic cages and provided free access to either a control or leucine-devoid diet (LeuD) for 8 days in total darkness. On the last day, blood and livers were collected at CT3 (CT = circadian time) and CT15. In livers of WT mice, GCN2 phosphorylation followed a diurnal pattern that was guided by intracellular branched-chain amino acid concentrations (r2 = 0.93). Feeding LeuD to WT mice increased hepatic ISR activation at CT15 only. Diurnal oscillations in hepatic ISR signaling, the hepatic transcriptome including lipid metabolic genes, and triglyceride concentrations were substantially reduced or absent in GCN2 KO mice. Furthermore, mice lacking GCN2 were unable to maintain circadian rhythms in whole body energy expenditure, respiratory exchange ratio, and physical activity when fed LeuD. In conclusion, GCN2 activation functions to maintain diurnal ISR activation in the liver and has a vital role in the mechanisms by which nutrient stress affects whole body metabolism.NEW & NOTEWORTHY This work reveals that the eIF2 kinase GCN2 functions to support diurnal patterns in the hepatic integrated stress response during natural feeding and is necessary to maintain circadian rhythms in energy expenditure, respiratory exchange ratio, and physical activity during amino acid stress.

Endothelin 3/EDNRB signaling induces thermogenic differentiation of white adipose tissue

Thermogenic adipose tissue, consisting of brown and beige fat, regulates nutrient utilization and energy metabolism. Human brown fat is relatively scarce and decreases with obesity and aging. Hence, inducing thermogenic differentiation of white fat offers an attractive way to enhance whole-body metabolic capacity. Here, we show the role of endothelin 3 (EDN3) and endothelin receptor type B (EDNRB) in promoting the browning of white adipose tissue (WAT). EDNRB overexpression stimulates thermogenic differentiation of human white preadipocytes through cAMP-EPAC1-ERK activation. In mice, cold induces the expression of EDN3 and EDNRB in WAT. Deletion of EDNRB in adipose progenitor cells impairs cold-induced beige adipocyte formation in WAT, leading to excessive weight gain, glucose intolerance, and insulin resistance upon high-fat feeding. Injection of EDN3 into WAT promotes browning and improved whole-body glucose metabolism. The findings shed light on the mechanism of WAT browning and offer potential therapeutics for obesity and metabolic disorders.

Doublecortin-like knockdown in mice attenuates obesity by stimulating energy expenditure in adipose tissue

Crosstalk between peripheral metabolic organs and the central nervous system is essential for body weight control. At the base of the hypothalamus, β-tanycytes surround the portal capillaries and function as gatekeepers to facilitate transfer of substances from the circulation into the cerebrospinal fluid and vice versa. Here, we investigated the role of the neuroplasticity gene doublecortin-like (DCL), highly expressed by β-tanycytes, in body weight control and whole-body energy metabolism. We demonstrated that DCL-knockdown through a doxycycline-inducible shRNA expression system prevents body weight gain by reducing adiposity in mice. DCL-knockdown slightly increased whole-body energy expenditure possibly as a result of elevated circulating thyroid hormones. In white adipose tissue (WAT) triglyceride uptake was increased while the average adipocyte cell size was reduced. At histological level we observed clear signs of browning, and thus increased thermogenesis in WAT. We found no indications for stimulated thermogenesis in brown adipose tissue (BAT). Altogether, we demonstrate an important, though subtle, role of tanycytic DCL in body weight control through regulation of energy expenditure, and specifically WAT browning. Elucidating mechanisms underlying the role of DCL in regulating brain-peripheral crosstalk further might identify new treatment targets for obesity.

The mitochondrial pyruvate carrier regulates adipose glucose partitioning in female mice

ObjectiveThe mitochondrial pyruvate carrier (MPC) occupies a critical node in intermediary metabolism, prompting interest in its utility as a therapeutic target for the treatment of obesity and cardiometabolic disease. Dysregulated nutrient metabolism in adipose tissue is a prominent feature of obesity pathophysiology, yet the functional role of adipose MPC has not been explored. We investigated whether the MPC shapes the adaptation of adipose tissue to dietary stress in female and male mice. MethodsThe impact of pharmacological and genetic disruption of the MPC on mitochondrial pathways of triglyceride assembly (lipogenesis and glyceroneogenesis) was assessed in 3T3L1 adipocytes and murine adipose explants, combined with analyses of adipose MPC expression in metabolically compromised humans. Whole-body and adipose-specific glucose metabolism were subsequently investigated in male and female mice lacking adipocyte MPC1 (Mpc1AD−/−) and fed either standard chow, high-fat western style, or high-sucrose lipid restricted diets for 24 weeks, using a combination of radiolabeled tracers and GC/MS metabolomics. ResultsTreatment with UK5099 or siMPC1 impaired the synthesis of lipids and glycerol-3-phosphate from pyruvate and blunted triglyceride accumulation in 3T3L1 adipocytes, whilst MPC expression in human adipose tissue was negatively correlated with indices of whole-body and adipose tissue metabolic dysfunction. Mature adipose explants from Mpc1AD−/− mice were intrinsically incapable of incorporating pyruvate into triglycerides. In vivo, MPC deletion restricted the incorporation of circulating glucose into adipose triglycerides, but only in female mice fed a zero fat diet, and this associated with sex-specific reductions in tricarboxylic acid cycle pool sizes and compensatory transcriptional changes in lipogenic and glycerol metabolism pathways. However, whole-body adiposity and metabolic health were preserved in Mpc1AD−/− mice regardless of sex, even under conditions of zero dietary fat. ConclusionsThese findings highlight the greater capacity for mitochondrially driven triglyceride assembly in adipose from female versus male mice and expose a reliance upon MPC-gated metabolism for glucose partitioning in female adipose under conditions of dietary lipid restriction.

Genetic Deletion of Skeletal Muscle Inositol Polyphosphate Multikinase Disrupts Glucose Homeostasis and Impairs Exercise Tolerance.

Inositol phosphates are critical signaling messengers involved in a wide range of biological pathways in which inositol polyphosphate multikinase (IPMK) functions as a rate-limiting enzyme for inositol polyphosphate metabolism. IPMK has been implicated in cellular metabolism, but its function at the systemic level is still poorly understood. Since skeletal muscle is a major contributor to energy homeostasis, we have developed a mouse model in which skeletal muscle IPMK is specifically deleted and examined how a loss of IPMK affects whole-body metabolism. Here, we report that mice in which IPMK knockout is deleted, specifically in the skeletal muscle, displayed an increased body weight, disrupted glucose tolerance, and reduced exercise tolerance under the normal diet. Moreover, these changes were associated with an increased accumulation of triglyceride in skeletal muscle. Furthermore, we have confirmed that a loss of IPMK led to reduced beta-oxidation, increased triglyceride accumulation, and impaired insulin response in IPMK-deficient muscle cells. Thus, our results suggest that IPMK mediates the whole-body metabolism via regulating muscle metabolism and may be potentially targeted for the treatment of metabolic syndromes.

Understanding the metabolism of infants using whole-body metabolic models

A whole-body model is a computational representation of sex-specific and organ-resolved whole-body metabolism. In this issue of Cell Metabolism, Zaunseder etal. report whole-body models of infants that represent metabolic, physiological, energetic, and nutritional features, accurately simulating the growth of infants and providing foundations for personalized medicine for infants.

Regulation of mitochondrial metabolism by hibernating bear serum: Insights into seasonal metabolic adaptations

Hibernating animals undergo a unique and reversible decrease in their whole-body metabolism, which is often accompanied by a suppression of mitochondrial respiration. However, the precise mechanisms underlying these seasonal shifts in mitochondrial metabolism remain unclear. In this study, the effect of the serum from active and hibernating Japanese black bears on mitochondrial respiration was assessed. Stromal–vascular cells were obtained from bear white adipose tissue and cultured with or without an adipocyte differentiation cocktail. When the oxygen consumption was measured in the presence of bear serum, the hibernating bear serum reduced maximal respiration by 15.5 % (p < 0.05) and spare respiratory capacity by 46.0 % (p < 0.01) in the differentiated adipocytes in comparison to the active bear serum. Similar reductions of 23.4 % (p = 0.06) and 40.6 % (p < 0.05) respectively were observed in undifferentiated cells, indicating the effect is cell type-independent. Blue native PAGE analysis revealed that hibernating bear serum suppressed cellular metabolism independently of the assembly of mitochondrial respiratory chain complexes. RNA-seq analysis identified 1094 differentially expressed genes (fold change>1.5, FDR<0.05) related to insulin signaling and glucose metabolism pathways. These findings suggest that the rapid alterations in mitochondrial metabolism during hibernation are likely induced by a combination of reduced insulin signaling and suppressed mitochondrial function, rather than changes in respiratory complex assembly.

Mechanisms of spinophilin-dependent pancreas dysregulation in obesity.

Spinophilin is an F-actin binding and protein phosphatase 1 (PP1) targeting protein that acts as a scaffold of PP1 to its substrates. Spinophilin knockout (Spino-/-) mice have decreased fat mass, increased lean mass, and improved glucose tolerance, with no difference in feeding behaviors. Although spinophilin is enriched in neurons, its roles in nonneuronal tissues, such as β cells of the pancreatic islets, are unclear. We have corroborated and expanded upon previous studies to determine that Spino-/- mice have decreased weight gain and improved glucose tolerance in two different models of obesity. We have identified multiple putative spinophilin-interacting proteins isolated from intact pancreas and observed increased interactions of spinophilin with exocrine, ribosomal, and cytoskeletal protein classes that normally act to mediate peptide hormone production, processing, and/or release in Leprdb/db and/or high-fat diet-fed (HFF) models of obesity. In addition, we have found that spinophilin interacts with proteins from similar classes in isolated islets, suggesting a role for spinophilin in the pancreatic islet. Consistent with a pancreatic β cell type-specific role for spinophilin, using our recently described conditional spinophilin knockout mice, we found that loss of spinophilin specifically in pancreatic β cells improved glucose tolerance without impacting body weight in chow-fed mice. Our data further support the role of spinophilin in mediating pathophysiological changes in body weight and whole body metabolism associated with obesity. Our data provide the first evidence that pancreatic spinophilin protein interactions are modulated by obesity and that loss of spinophilin specifically in pancreatic β cells impacts whole body glucose tolerance.NEW & NOTEWORTHY To our knowledge, these data are the first to demonstrate that obesity impacts spinophilin protein interactions in the pancreas and identify spinophilin specifically in pancreatic β cells as a modulator of whole body glucose tolerance.