Melanin-concentrating hormone-producing neurons in the hypothalamus regulate brown adipose tissue and thus contribute to energy expenditure.

KEY POINTS: Melanin-concentrating hormone (MCH) neuron-ablated mice exhibit increased energy expenditure and reduced fat weight. Increased brown adipose tissue (BAT) activity and locomotor activity-independent energy expenditure contributed to body weight reduction in MCH neuron-ablated mice. MCH neurons send inhibitory input to the medullary raphe nucleus to modulate BAT activity. ABSTRACT: Hypothalamic melanin-concentrating hormone (MCH) peptide robustly affects energy homeostasis. However, it is unclear whether and how MCH-producing neurons, which contain and release a variety of neuropeptides/transmitters, regulate energy expenditure in the central nervous system and peripheral tissues. We thus examined the regulation of energy expenditure by MCH neurons, focusing on interscapular brown adipose tissue (BAT) activity. MCH neuron-ablated mice exhibited reduced body weight, increased oxygen consumption, and increased BAT activity, which improved locomotor activity-independent energy expenditure. Trans-neuronal retrograde tracing with the recombinant pseudorabies virus revealed that MCH neurons innervate BAT via the sympathetic premotor region in the medullary raphe nucleus (MRN). MRN neurons were activated by MCH neuron ablation. Therefore, endogenous MCH neuron activity negatively modulates energy expenditure via BAT inhibition. MRN neurons might receive inhibitory input from MCH neurons to suppress BAT activity.

Homeostatic versus hedonic control of carbohydrate selection.

Macronutrient intake is associated with cardiometabolic health, ageing and longevity, but the mechanisms underlying its regulation have remained unclear. Most rodents increase carbohydrate selection under certain physiological and pathological conditions such as fasting. When presented with a choice between a basally preferable high-fat diet (HFD) and a high-carbohydrate diet (HCD) such as a high-sucrose diet, fasted mice first eat the HFD and then switch to the HCD during the first few hours of refeeding and continue to eat the HCD up to 24 h in the two-diet choice approach. Such consumption of an HCD after fasting reverses the fasting-induced increase in the plasma concentration of ketone bodies more rapidly than does refeeding with an HFD alone. 5'-AMP-activated protein kinase (AMPK)-regulated neurons in the paraventricular nucleus of the hypothalamus (PVH) that express corticotropin-releasing hormone (CRH) are necessary and sufficient for the fasting-induced selection of carbohydrate over an HFD in mice. These neurons appear to contribute to a fasting-induced increase in the positive valence of carbohydrate without affecting the preference for more palatable and energy-dense diets such as an HFD. Identification of the neural circuits in which AMPK-regulated CRH neurons in the PVH of mice are embedded should shed new light on the physiological and molecular mechanisms responsible for macronutrient selection.

[Central regulation of appetite and energy metabolism.]

Recent advances of neuroscience shed a light in the neural pathway and mechanisms for food intake regulation. The central nervous system(CNS)has also been recognized as a crucial organ to regulate metabolism in peripheral tissues. This includes the increase in glucose and fatty acid utilization in skeletal muscle as well as in brown adipose tissue and the heart, improving type 1 diabetes without change in insulin secretion. CNS regulation for food intake and energy metabolism is important to understand the mechanism for homeostatic regulation in living body.

[Molecular mechanism for feeding and food preference regulation].

Feeding behavior is regulated by homeostatic and hedonic mechanisms. NPY/AgRP neurons in the arcuate hypothalamus are involved in the homeostatic regulation, and dopaminergic neurons in the ventral tegmental area regulating the nucleus of accumbens are involved in the hedonic regulation, respectively. Food preference also appears to be regulated by both homeostatic and hedonic mechanisms. However, molecular mechanism for food preference regulation remains elusive and further studies are necessary.

Conditional ablation of orexin/hypocretin neurons: a new mouse model for the study of narcolepsy and orexin system function.

The sleep disorder narcolepsy results from loss of hypothalamic orexin/hypocretin neurons. Although narcolepsy onset is usually postpubertal, current mouse models involve loss of either orexin peptides or orexin neurons from birth. To create a model of orexin/hypocretin deficiency with closer fidelity to human narcolepsy, diphtheria toxin A (DTA) was expressed in orexin neurons under control of the Tet-off system. Upon doxycycline removal from the diet of postpubertal orexin-tTA;TetO DTA mice, orexin neurodegeneration was rapid, with 80% cell loss within 7 d, and resulted in disrupted sleep architecture. Cataplexy, the pathognomic symptom of narcolepsy, occurred by 14 d when ∼5% of the orexin neurons remained. Cataplexy frequency increased for at least 11 weeks after doxycycline. Temporary doxycycline removal followed by reintroduction after several days enabled partial lesion of orexin neurons. DTA-induced orexin neurodegeneration caused a body weight increase without a change in food consumption, mimicking metabolic aspects of human narcolepsy. Because the orexin/hypocretin system has been implicated in the control of metabolism and addiction as well as sleep/wake regulation, orexin-tTA; TetO DTA mice are a novel model in which to study these functions, for pharmacological studies of cataplexy, and to study network reorganization as orexin input is lost.

Hypothalamic SIRT1 prevents age-associated weight gain by improving leptin sensitivity in mice.

AIMS/HYPOTHESIS: Obesity is associated with ageing and increased energy intake, while restriction of energy intake improves health and longevity in multiple organisms; the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) is implicated in this process. Pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in the arcuate nucleus (ARC) of the hypothalamus are critical for energy balance regulation, and the level of SIRT1 protein decreases with age in the ARC. In the current study we tested whether conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevents age-associated weight gain and diet-induced obesity. METHODS: We targeted Sirt1 cDNA sequence into the Rosa26 locus and generated conditional Sirt1 knock-in mice. These mice were crossed with mice harbouring either Pomc-Cre or Agrp-Cre and the metabolic variables, food intake, energy expenditure and sympathetic activity in adipose tissue of the resultant mice were analysed. We also used a hypothalamic cell line to investigate the molecular mechanism by which Sirt1 overexpression modulates leptin signalling. RESULTS: Conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevented age-associated weight gain; overexpression in POMC neurons stimulated energy expenditure via increased sympathetic activity in adipose tissue, whereas overexpression in AgRP neurons suppressed food intake. SIRT1 improved leptin sensitivity in hypothalamic neurons in vitro and in vivo by downregulating protein-tyrosine phosphatase 1B, T cell protein-tyrosine phosphatase and suppressor of cytokine signalling 3. However, these phenotypes were absent in mice consuming a high-fat, high-sucrose diet due to decreases in ARC SIRT1 protein and hypothalamic NAD(+) levels. CONCLUSIONS/INTERPRETATION: ARC SIRT1 is a negative regulator of energy balance, and decline in ARC SIRT1 function contributes to disruption of energy homeostasis by ageing and diet-induced obesity.

Alpha-synuclein elicits glucose uptake and utilization in adipocytes through the Gab1/PI3K/Akt transduction pathway.

Insulin is the main glucoregulator that promotes the uptake of glucose by tissues and the subsequent utilization of glucose as an energy source. In this paper, we describe a novel glucoregulator, the alpha-synuclein (SNCA) protein, that has previously been linked to Parkinson's disease. Treatment with recombinant SNCA promotes glucose uptake in vitro in preadipocytes and in vivo in the adipose tissues and skeletal muscles of mice through the LPAR2/Gab1/PI3K/Akt pathway; these effects occur independently of the insulin receptor. This function of SNCA represents a new mechanistic insight that creates novel avenues of research with respect to the process of glucose regulation.

Inhibition of high-fat diet-induced inflammatory responses in adipose tissue by SF1-expressing neurons of the ventromedial hypothalamus.

Inflammation and thermogenesis in white adipose tissue (WAT) at different sites influence the overall effects of obesity on metabolic health. In mice fed a high-fat diet (HFD), inflammatory responses are less pronounced in inguinal WAT (ingWAT) than in epididymal WAT (epiWAT). Here we show that ablation and activation of steroidogenic factor 1 (SF1)-expressing neurons in the ventromedial hypothalamus (VMH) oppositely affect the expression of inflammation-related genes and the formation of crown-like structures by infiltrating macrophages in ingWAT, but not in epiWAT, of HFD-fed mice, with these effects being mediated by sympathetic nerves innervating ingWAT. In contrast, SF1 neurons of the VMH preferentially regulated the expression of thermogenesis-related genes in interscapular brown adipose tissue (BAT) of HFD-fed mice. These results suggest that SF1 neurons of the VMH differentially regulate inflammatory responses and thermogenesis among various adipose tissue depots and restrain inflammation associated with diet-induced obesity specifically in ingWAT.

Role of central leptin signaling in the starvation-induced alteration of B-cell development.

Nutritional deprivation or malnutrition suppresses immune function in humans and animals, thereby conferring higher susceptibility to infectious diseases. Indeed, nutritional deprivation induces atrophy of lymphoid tissues such as thymus and spleen and decreases the number of circulating lymphocytes. Leptin, a major adipocytokine, is exclusively produced in the adipose tissue in response to the nutritional status and acts on the hypothalamus, thereby regulating energy homeostasis. Although leptin plays a critical role in the starvation-induced T-cell-mediated immunosuppression, little is known about its role in B-cell homeostasis under starvation conditions. Here we show the alteration of B-cell development in the bone marrow of fasted mice, characterized by decrease in pro-B, pre-B, and immature B cells and increase in mature B cells. Interestingly, intracerebroventricular leptin injection was sufficient to prevent the alteration of B-cell development of fasted mice. The alteration of B lineage cells in the bone marrow of fasted mice was markedly prevented by oral administration of glucocorticoid receptor antagonist RU486 (11ß-[p-(dimethylamino)phenyl]-17ß-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one). It was also effectively prevented by intracerebroventricular injection of neuropeptide Y Y(1) receptor antagonist BIBP3226 [(2R)-5-(diaminomethylideneamino)-2-[(2,2-diphenylacetyl)amino]-N-[(4-hydroxyphenyl)methyl]pentanamide], along with suppression of the otherwise increased serum corticosterone concentrations. This study provides the first in vivo evidence for the role of central leptin signaling in the starvation-induced alteration of B-cell development. The data of this study suggest that the CNS, which is inherent to integrate information from throughout the organism, is able to control immune function.

Central melanocortin signaling restores skeletal muscle AMP-activated protein kinase phosphorylation in mice fed a high-fat diet.

Little is known about the role of the central melanocortin system in the control of fuel metabolism in peripheral tissues. Skeletal muscle AMP-activated protein kinase (AMPK) is activated by leptin and serves as a master regulator of fatty acid beta-oxidation. To elucidate an unidentified role of the central melanocortin system in muscle AMPK regulation, we treated conscious, unrestrained mice intracerebroventricularly with the melanocortin agonist MT-II or the antagonist SHU9119. MT-II augmented phosphorylation of AMPK and its target acetyl-CoA carboxylase (ACC) independent of caloric intake. Conversely, AMPK/ACC phosphorylation by leptin was abrogated by the coadministration of SHU9119 or in KKA(y) mice, which centrally express endogenous melanocortin antagonist. Importantly, high-fat-diet-induced attenuation of AMPK/ACC phosphorylation in leptin-overexpressing transgenic mice was not reversed by central leptin but was markedly restored by MT-II. Our data provide evidence for the critical role of the central melanocortin system in the leptin-skeletal muscle AMPK axis and highlight the system as a therapeutic target in leptin resistance.

Adiponectin stimulates AMP-activated protein kinase in the hypothalamus and increases food intake.

Adiponectin has been shown to stimulate fatty acid oxidation and enhance insulin sensitivity through the activation of AMP-activated protein kinase (AMPK) in the peripheral tissues. The effects of adiponectin in the central nervous system, however, are still poorly understood. Here, we show that adiponectin enhances AMPK activity in the arcuate hypothalamus (ARH) via its receptor AdipoR1 to stimulate food intake; this stimulation of food intake by adiponectin was attenuated by dominant-negative AMPK expression in the ARH. Moreover, adiponectin also decreased energy expenditure. Adiponectin-deficient mice showed decreased AMPK phosphorylation in the ARH, decreased food intake, and increased energy expenditure, exhibiting resistance to high-fat-diet-induced obesity. Serum and cerebrospinal fluid levels of adiponectin and expression of AdipoR1 in the ARH were increased during fasting and decreased after refeeding. We conclude that adiponectin stimulates food intake and decreases energy expenditure during fasting through its effects in the central nervous system.

Crystal structure of the Ca²âº/calmodulin-dependent protein kinase kinase in complex with the inhibitor STO-609.

Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca(2+) elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV, which directly activate transcription factors. In this study, we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKß isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKKß lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKKß lacks the activation loop phosphorylation site, the activation loop is folded in an active-state conformation, which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKß kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed, mutagenesis demonstrated that the CaMKKß residue Pro(274), which replaces the conserved acidic residue of other protein kinases, is an important determinant for the selective inhibition by STO-609. Therefore, the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKß and for designing novel inhibitors targeting CaMKKß and the related protein kinases.

Brown adipose tissue dysfunction promotes heart failure via a trimethylamine N-oxide-dependent mechanism.

Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.

Structural basis for compound C inhibition of the human AMP-activated protein kinase α2 subunit kinase domain.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor to maintain energy balance at both the cellular and the whole-body levels and is therefore a potential target for drug design against metabolic syndrome, obesity and type 2 diabetes. Here, the crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C. The AMPK α2 kinase domain exhibits a typical bilobal kinase fold and exists as a monomer in the crystal. Like the wild-type apo form, the T172D mutant apo form adopts the autoinhibited structure of the `DFG-out' conformation, with the Phe residue of the DFG motif anchored within the putative ATP-binding pocket. Compound C binding dramatically alters the conformation of the activation loop, which adopts an intermediate conformation between DFG-out and DFG-in. This induced fit forms a compound-C binding pocket composed of the N-lobe, the C-lobe and the hinge of the kinase domain. The pocket partially overlaps with the putative ATP-binding pocket. These three-dimensional structures will be useful to guide drug discovery.

An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes.

An enzymatic assay adapted to photometric analysis with 96-well microplates was evaluated for the measurement of 2-deoxyglucose (2DG) uptake in insulin-responsive tissues and differentiated 3T3-L1 adipocytes. For in vivo measurements, a small amount of nonradiolabeled 2DG was injected into mice without affecting glucose metabolism. For photometric quantification of the small amount of 2-deoxyglucose 6-phosphate (2DG6P) that accumulates in cells, we introduced glucose-6-phosphate dehydrogenase, glutathione reductase, and 5,5'-dithiobis(2-nitrobenzoic acid) to the recycling amplification reaction of NADPH. We optimized the enzyme reaction for complete oxidation of endogenous glucose 6-phosphate (G6P) and glucose in mouse tissues in vivo and serum as well as in 3T3-L1 adipocytes in vitro. All reactions are performed in one 96-well microplate by consecutive addition of reagents, and the assay is able to quantify 2DG and 2DG6P in the range of 5-80 pmol. The results obtained with the assay for 2DG uptake in vitro and in vivo in the absence or presence of insulin stimulation was similar to those obtained with the standard radioisotopic method. Thus, the enzymatic assay should prove to be useful for measurement of 2DG uptake in insulin-responsive tissues in vivo as well as in cultured cells.

Recent Advances in Neural Circuits for Taste Perception in Hunger.

Feeding is essential for survival and taste greatly influences our feeding behaviors. Palatable tastes such as sweet trigger feeding as a symbol of a calorie-rich diet containing sugar or proteins, while unpalatable tastes such as bitter terminate further consumption as a warning against ingestion of harmful substances. Therefore, taste is considered a criterion to distinguish whether food is edible. However, perception of taste is also modulated by physiological changes associated with internal states such as hunger or satiety. Empirically, during hunger state, humans find ordinary food more attractive and feel less aversion to food they usually dislike. Although functional magnetic resonance imaging studies performed in primates and in humans have indicated that some brain areas show state-dependent response to tastes, the mechanisms of how the brain senses tastes during different internal states are poorly understood. Recently, using newly developed molecular and genetic tools as well as in vivo imaging, researchers have identified many specific neuronal populations or neural circuits regulating feeding behaviors and taste perception process in the central nervous system. These studies could help us understand the interplay between homeostatic regulation of energy and taste perception to guide proper feeding behaviors.

Basigin deficiency prevents anaplerosis and ameliorates insulin resistance and hepatosteatosis.

Monocarboxylates, such as lactate and pyruvate, are precursors for biosynthetic pathways, including those for glucose, lipids, and amino acids via the tricarboxylic acid (TCA) cycle and adjacent metabolic networks. The transportation of monocarboxylates across the cellular membrane is performed primarily by monocarboxylate transporters (MCTs), the membrane localization and stabilization of which are facilitated by the transmembrane protein basigin (BSG). Here, we demonstrate that the MCT/BSG axis sits at a crucial intersection of cellular metabolism. Abolishment of MCT1 in the plasma membrane was achieved by Bsg depletion, which led to gluconeogenesis impairment via preventing the influx of lactate and pyruvate into the cell, consequently suppressing the TCA cycle. This net anaplerosis suppression was compensated in part by the increased utilization of glycogenic amino acids (e.g., alanine and glutamine) into the TCA cycle and by activated ketogenesis through fatty acid ß-oxidation. Complementary to these observations, hyperglycemia and hepatic steatosis induced by a high-fat diet were ameliorated in Bsg-deficient mice. Furthermore, Bsg deficiency significantly improved insulin resistance induced by a high-fat diet. Taken together, the plasma membrane-selective modulation of lactate and pyruvate transport through BSG inhibition could potentiate metabolic flexibility to treat metabolic diseases.

Adipocyte/macrophage fatty acid binding proteins control integrated metabolic responses in obesity and diabetes.

Fatty acid binding proteins (FABPs) are cytosolic fatty acid chaperones whose biological role and mechanisms of action are not well understood. Here, we developed mice with targeted mutations in two related adipocyte FABPs, aP2 and mal1, to resolve their role in systemic lipid, glucose, and energy metabolism. Mice lacking aP2 and mal1 exhibited a striking phenotype with strong protection from diet-induced obesity, insulin resistance, type 2 diabetes, and fatty liver disease. These mice have altered cellular and systemic lipid transport and composition, leading to enhanced insulin receptor signaling, enhanced muscle AMP-activated kinase (AMP-K) activity, and dramatically reduced liver stearoyl-CoA desaturase-1 (SCD-1) activity underlying their phenotype. Taken together with the previously reported strong protection against atherosclerosis, these results demonstrate that adipocyte/macrophage FABPs have a robust impact on multiple components of metabolic syndrome, integrating metabolic and inflammatory responses in mice and constituting a powerful target for the treatment of these diseases.

Metabolic adaptation of mice in a cool environment.

Homeothermic animals, including humans, live by adapting to changes in ambient temperature. Numerous studies have demonstrated cold exposure (at approximately 5 degrees C) improves glucose tolerance despite reducing insulin secretion and increasing energy expenditure. To determine the effects of a small reduction in ambient temperature on energy metabolism, we compared two groups of mice; one exposed to a cool environment (20 degrees C) and the other maintained in a near-thermoneutral environment (25 degrees C) for 10 days. Both glucose-induced insulin secretion and glucose response were significantly impaired in mice exposed to a cool environment. In the cool temperature-exposed mice, skin temperatures were reduced, and plasma norepinephrine levels were increased, suggesting that impairment of insulin secretion was facilitated by induction of sympathetic nervous activity due to skin cooling. In addition, expression of GLUT4 mRNA was increased significantly in inguinal subcutaneous adipose tissue (IWAT) but not in epididymal or brown adipose tissue or skeletal muscle in these mice. Moreover, expression of Dok1, a molecule linked to activation of insulin receptors in adipocyte hypertrophy, and Cd36, a molecule related to NEFA uptake, were also increased at mRNA and/or protein levels only in IWAT of the cool temperature-exposed mice. Fatty acid synthesis was also facilitated, and fat weights were increased only in IWAT from mice kept at 20 degrees C. These results suggest that a small reduction in ambient temperature can affect glucose homeostasis through regulation of insulin secretion and preferentially enhances fat storage in IWAT. These adaptations can be interpreted as preparation for a further reduction in ambient temperature.