Stress-associated conditions such as psychoemotional reactivity and depression have been paradoxically linked to either weight gain or weight loss. This bi-directional effect of stress is not understood at the functional level. Here we tested the hypothesis that pre-stress level of adaptive thermogenesis and brown adipose tissue (BAT) functions explain the vulnerability or resilience to stress-induced obesity.

High-fat diet (HFD) triggers insulin resistance and diabetes mellitus, but their link remains unclear. Characterization of overt hyperglycemia in insulin receptor mutant (Insr(P1195L/+)) mice exposed to HFD (Insr(P1195L/+)/HFD mice) revealed increased glucose-6-phosphatase (G6pc) expression in liver and increased gluconeogenesis from glycerol. Lipolysis in white adipose tissues (WAT) and lipolysis-induced blood glucose rise were increased in Insr(P1195L/+)/HFD mice, while wild-type WAT transplantation ameliorated the hyperglycemia and the increased G6pc expression. We found that the expressions of genes involved in bile acid (BA) metabolism were altered in Insr(P1195L/+)/HFD liver. Among these, the expression of Cyp7a1, a BA synthesis enzyme, was insulin-dependent and was markedly decreased in Insr(P1195L/+)/HFD liver. Reduced Cyp7a1 expression in Insr(P1195L/+)/HFD liver was rescued by WAT transplantation, and the expression of Cyp7a1 was suppressed by glycerol administration in wild-type liver. These findings suggest that unsuppressed lipolysis in adipocytes elicited by HFD feeding is linked with enhanced gluconeogenesis from glycerol and with alterations in BA physiology in Insr(P1195L/+)/HFD liver.

Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity.

Peroxisome proliferator activated receptor (PPARγ) is a nuclear receptor transcription factor that regulates adipogenesis and energy homeostasis. Recent studies suggest PPARγ may mediate some of its metabolic effects through actions in the brain. We used a Cre-recombinase-dependent (using NestinCre) conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ to examine mechanisms by which PPARγ in the nervous system controls energy balance. Inducible expression of PPARγ was evident throughout the brain. Expression of 2 PPARγ target genes, aP2 and CD36, was induced by WT but not P467L PPARγ in the brain. Surprisingly, NesCre/PPARγ-WT mice exhibited severe microcephaly and brain malformation, suggesting that PPARγ can modulate brain development. On the contrary, NesCre/PPARγ-P467L mice exhibited blunted weight gain to high-fat diet, which correlated with a decrease in lean mass and tissue masses, accompanied by elevated plasma GH, and depressed plasma IGF-1, indicative of GH resistance. There was no expression of the transgene in the pancreas but both fasting plasma glucose, and fed and fasted plasma insulin levels were markedly decreased. NesCre/PPARγ-P467L mice fed either control diet or high-fat diet displayed impaired glucose tolerance yet exhibited increased sensitivity to exogenous insulin and increased insulin receptor signaling in white adipose tissue, liver, and skeletal muscle. These observations support the concept that alterations in PPARγ-driven mechanisms in the nervous system play a role in the regulation of growth and glucose metabolic homeostasis.

Melanocortin 4 receptor (MC4R) signaling mediates diverse physiological functions, including energy balance, glucose homeostasis, and autonomic activity. Although the lateral hypothalamic area (LHA) is known to express MC4Rs and to receive input from leptin-responsive arcuate proopiomelanocortin neurons, the physiological functions of MC4Rs in the LHA are incompletely understood. We report that MC4R(LHA) signaling regulates glucose tolerance and sympathetic nerve activity. Restoring expression of MC4Rs specifically in the LHA improves glucose intolerance in obese MC4R-null mice without affecting body weight or circulating insulin levels. Fluorodeoxyglucose-mediated tracing of whole-body glucose uptake identifies the interscapular brown adipose tissue (iBAT) as a primary source where glucose uptake is increased in MC4R(LHA) mice. Direct multifiber sympathetic nerve recording further reveals that sympathetic traffic to iBAT is significantly increased in MC4R(LHA) mice, which accompanies a significant elevation of Glut4 expression in iBAT. Finally, bilateral iBAT denervation prevents the glucoregulatory effect of MC4R(LHA) signaling. These results identify a novel role for MC4R(LHA) signaling in the control of sympathetic nerve activity and glucose tolerance independent of energy balance.

Smokers around the world commonly report increased body weight after smoking cessation as a major factor that interferes with their attempts to quit. Numerous controlled studies in both humans and rodents have reported that nicotine exerts a marked anorectic action. The effects of nicotine on energy homeostasis have been mostly pinpointed in the central nervous system, but the molecular mechanisms controlling its action are still not fully understood. The aim of this study was to investigate the effect of nicotine on hypothalamic AMP-activated protein kinase (AMPK) and its effect on energy balance. Here we demonstrate that nicotine-induced weight loss is associated with inactivation of hypothalamic AMPK, decreased orexigenic signaling in the hypothalamus, increased energy expenditure as a result of increased locomotor activity, increased thermogenesis in brown adipose tissue (BAT), and alterations in fuel substrate utilization. Conversely, nicotine withdrawal or genetic activation of hypothalamic AMPK in the ventromedial nucleus of the hypothalamus reversed nicotine-induced negative energy balance. Overall these data demonstrate that the effects of nicotine on energy balance involve specific modulation of the hypothalamic AMPK-BAT axis. These targets may be relevant for the development of new therapies for human obesity.

We studied interscapular brown adipose tissue (iBAT) activity in wild-type (WT) and glucagon-like peptide 1 receptor (GLP-1R)-deficient mice after the administration of the proglucagon-derived peptides (PGDPs) glucagon-like peptide (GLP-1), glucagon (GCG), and oxyntomodulin (OXM) directly into the brain. Intracerebroventricular injection of PGDPs reduces body weight and increases iBAT thermogenesis. This was independent of changes in feeding and insulin responsiveness but correlated with increased activity of sympathetic fibers innervating brown adipose tissue (BAT). Despite being a GCG receptor agonist, OXM requires GLP-1R activation to induce iBAT thermogenesis. The increase in thermogenesis in WT mice correlates with increased expression of genes upregulated by adrenergic signaling and required for iBAT thermogenesis, including PGC1a and UCP-1. In spite of the increase in iBAT thermogenesis induced by GLP-1R activation in WT mice, Glp1r(-/-) mice exhibit a normal response to cold exposure, demonstrating that endogenous GLP-1R signaling is not essential for appropriate thermogenic response after cold exposure. Our data suggest that the increase in BAT thermogenesis may be an additional mechanism whereby pharmacological GLP-1R activation controls energy balance.

Obesity increases the risk of developing life-threatening metabolic diseases including cardiovascular disease, fatty liver disease, diabetes, and cancer. Efforts to curb the global obesity epidemic and its impact have proven unsuccessful in part by a limited understanding of these chronic progressive diseases. It is clear that low-grade chronic inflammation, or metaflammation, underlies the pathogenesis of obesity-associated type 2 diabetes and atherosclerosis. However, the mechanisms that maintain chronicity and prevent inflammatory resolution are poorly understood. Here, we show that inhibitor of κB kinase epsilon (IKBKE) is a novel regulator that limits chronic inflammation during metabolic disease and atherosclerosis. The pathogenic relevance of IKBKE was indicated by the colocalization with macrophages in human and murine tissues and in atherosclerotic plaques. Genetic ablation of IKBKE resulted in enhanced and prolonged priming of the NLRP3 inflammasome in cultured macrophages, in hypertrophic adipose tissue, and in livers of hypercholesterolemic mice. This altered profile associated with enhanced acute phase response, deregulated cholesterol metabolism, and steatoheptatitis. Restoring IKBKE only in hematopoietic cells was sufficient to reverse elevated inflammasome priming and these metabolic features. In advanced atherosclerotic plaques, loss of IKBKE and hematopoietic cell restoration altered plaque composition. These studies reveal a new role for hematopoietic IKBKE: to limit inflammasome priming and metaflammation.

Activation of brown adipose tissue-mediated thermogenesis is a strategy for tackling obesity and promoting metabolic health. BMP8b is secreted by brown/beige adipocytes and enhances energy dissipation. Here we show that adipocyte-secreted BMP8b contributes to adrenergic-induced remodeling of the neuro-vascular network in adipose tissue (AT). Overexpression of bmp8b in AT enhances browning of the subcutaneous depot and maximal thermogenic capacity. Moreover, BMP8b-induced browning, increased sympathetic innervation and vascularization of AT were maintained at 28 °C, a condition of low adrenergic output. This reinforces the local trophic effect of BMP8b. Innervation and vascular remodeling effects required BMP8b signaling through the adipocytes to 1) secrete neuregulin-4 (NRG4), which promotes sympathetic axon growth and branching in vitro, and 2) induce a pro-angiogenic transcriptional and secretory profile that promotes vascular sprouting. Thus, BMP8b and NRG4 can be considered as interconnected regulators of neuro-vascular remodeling in AT and are potential therapeutic targets in obesity.

One understudied function of white adipose tissue (AT) is its role in postprandial lipid buffering. In this study, we demonstrate that mice lacking the adipose tissue-specific transcription factor peroxisome proliferator-activated receptor γ2 (PPARγ2) exhibit a defect in their rate of adipose tissue lipid storage. Impaired adipose tissue storage rate reduces metabolic flexibility, without compromising fasted glucose tolerance or insulin sensitivity, even following prolonged high-fat feeding. However, acutely overfeeding PPARγ2-KO mice caused a 10-fold increase in insulin levels compared with controls. Although impaired adipose tissue storage rate did not result in insulin resistance in young mice, 1-year-old PPARγ2-KO mice developed skeletal muscle insulin resistance. Our data indicate that failed adipose tissue storage may occur prior to defects in glucose handling and that overfeeding protocols may uncover genes controlling adipose tissue storage rate, as opposed to capacity, and act as a diagnostic test for early-stage human metabolic disease.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous inherited human disorder displaying a pleotropic phenotype. Many of the symptoms characterized in the human disease have been reproduced in animal models carrying deletions or knock-in mutations of genes causal for the disorder. Thinning of the cerebral cortex, enlargement of the lateral and third ventricles, and structural changes in cilia are among the pathologies documented in these animal models. Ciliopathy is of particular interest in light of recent studies that have implicated primary neuronal cilia (PNC) in neuronal signal transduction. In the present investigation, we tested the hypothesis that areas of the brain responsible for learning and memory formation would differentially exhibit PNC abnormalities in animals carrying a deletion of the Bbs4 gene (Bbs4-/-). Immunohistochemical localization of adenylyl cyclase-III (ACIII), a marker restricted to PNC, revealed dramatic alterations in PNC morphology and a statistically significant reduction in number of immunopositive cilia in the hippocampus and amygdala of Bbs4-/- mice compared to wild type (WT) littermates. Western blot analysis confirmed the decrease of ACIII levels in the hippocampus and amygdala of Bbs4-/- mice, and electron microscopy demonstrated pathological alterations of PNC in the hippocampus and amygdala. Importantly, no neuronal loss was found within the subregions of amygdala and hippocampus sampled in Bbs4-/- mice and there were no statistically significant alterations of ACIII immunopositive cilia in other areas of the brain not known to contribute to the BBS phenotype. Considered with data documenting a role of cilia in signal transduction these findings support the conclusion that alterations in cilia structure or neurochemical phenotypes may contribute to the cognitive deficits observed in the Bbs4-/- mouse mode.

Estrogens play a major role in the modulation of energy balance through central and peripheral actions. Here, we demonstrate that central action of estradiol (E2) inhibits AMP-activated protein kinase (AMPK) through estrogen receptor alpha (ERα) selectively in the ventromedial nucleus of the hypothalamus (VMH), leading to activation of thermogenesis in brown adipose tissue (BAT) through the sympathetic nervous system (SNS) in a feeding-independent manner. Genetic activation of AMPK in the VMH prevented E2-induced increase in BAT-mediated thermogenesis and weight loss. Notably, fluctuations in E2 levels during estrous cycle also modulate this integrated physiological network. Together, these findings demonstrate that E2 regulation of the VMH AMPK-SNS-BAT axis is an important determinant of energy balance and suggest that dysregulation in this axis may account for the common changes in energy homeostasis and obesity linked to dysfunction of the female gonadal axis.

We recently reported that mitoquinone (mitoQ, 500 μmol/L) added to drinking water of C57BL/6J mice attenuated weight gain, decreased food intake, increased hypothalamic orexigenic gene expression, and mitigated oxidative stress when administered from the onset of high-fat (HF) feeding. Here, we examined the effects of mitoQ on pre-existing obesity in C57BL/6J mice first made obese by 107 days of HF feeding. In contrast to our preventative study, we found that already obese mice did not tolerate mitoQ at 500 μmol/L. Within 4 days of administration, obese mice markedly decreased food and water intake and lost substantial weight necessitating a dose reduction to 250 μmol/L. Food and water intake then improved. Over the next 4 weeks, body mass of the mitoQ-treated mice increased faster than vehicle-treated controls but did not catch up. Over the subsequent 10 weeks, weights of the mitoQ-treated group remained significantly less than vehicle control, but percent fat and food intake did not differ. Although the mitoQ-treated groups continued to drink less, there was no difference in percent body fluid and no laboratory evidence of dehydration at study end. At the time of killing, hypothalamic NPY gene expression was reduced in the mitoQ-treated mice . Liver fat was markedly increased by HF feeding but did not differ between mitoQ and vehicle groups and, in contrast to our previous preventative study, there was no improvement in plasma alanine amino transferase or liver hydroperoxides. In summary, administration of mitoQ to already obese mice attenuated weight gain, but showed limited overall benefit.

Background: Induction of nonshivering thermogenesis can be used to influence energy balance to prevent or even treat obesity. The pungent component of mustard, allyl-isothiocyanate (AITC), activates the extreme cold receptor transient receptor potential channel, subfamily A, member 1 and may thus induce energy expenditure and metabolic changes.Objective: The objective of our study was to evaluate the potential of mustard AITC to induce thermogenesis (primary outcome) and alter body temperature, cold and hunger sensations, plasma metabolic parameters, and energy intake (secondary outcomes).Design: Energy expenditure in mice was measured after subcutaneous injection with vehicle, 1 mg norepinephrine/kg, or 5 mg AITC/kg. In our human crossover study, 11 healthy subjects were studied under temperature-controlled conditions after an overnight fast. After ingestion of 10 g of capsulated mustard or uncapsulated mustard or a capsulated placebo mixture, measurements of energy expenditure, substrate oxidation, core temperature, cold and hunger scores, and plasma parameters were repeated every 30 min during a 150-min period. Subjects were randomly selected for the placebo and capsulated mustard intervention; 9 of 11 subjects received the uncapsulated mustard as the final intervention because this could not be blinded. After the experiments, energy intake was measured with the universal eating monitor in a test meal.Results: In mice, AITC administration induced a 32% increase in energy expenditure compared with vehicle (17.5 ± 4.9 J · min-1 · mouse-1 compared with 12.5 ± 1.2 J · min-1 · mouse-1, P = 0.03). Of the 11 randomly selected participants, 1 was excluded because of intercurrent illness after the first visit and 1 withdrew after the second visit. Energy expenditure did not increase after ingestion of capsulated or uncapsulated mustard compared with placebo. No differences in substrate oxidation, core temperature, cold and hunger scores, or plasma parameters were found, nor was the energy intake at the end of the experiment different between the 3 conditions.Conclusion: The highest tolerable dose of mustard we were able to use did not elicit a relevant thermogenic response in humans. This trial was registered at www.controlled-trials.com as ISRCTN19147515.

Low concentrations of insulin-like growth factor (IGF) binding protein-1 (IGFBP1) are associated with insulin resistance, diabetes, and cardiovascular disease. We investigated whether increasing IGFBP1 levels can prevent the development of these disorders. Metabolic and vascular phenotype were examined in response to human IGFBP1 overexpression in mice with diet-induced obesity, mice heterozygous for deletion of insulin receptors (IR(+/-)), and ApoE(-/-) mice. Direct effects of human (h)IGFBP1 on nitric oxide (NO) generation and cellular signaling were studied in isolated vessels and in human endothelial cells. IGFBP1 circulating levels were markedly suppressed in dietary-induced obese mice. Overexpression of hIGFBP1 in obese mice reduced blood pressure, improved insulin sensitivity, and increased insulin-stimulated NO generation. In nonobese IR(+/-) mice, overexpression of hIGFBP1 reduced blood pressure and improved insulin-stimulated NO generation. hIGFBP1 induced vasodilatation independently of IGF and increased endothelial NO synthase (eNOS) activity in arterial segments ex vivo, while in endothelial cells, hIGFBP1 increased eNOS Ser(1177) phosphorylation via phosphatidylinositol 3-kinase signaling. Finally, in ApoE(-/-) mice, overexpression of hIGFBP1 reduced atherosclerosis. These favorable effects of hIGFBP1 on insulin sensitivity, blood pressure, NO production, and atherosclerosis suggest that increasing IGFBP1 concentration may be a novel approach to prevent cardiovascular disease in the setting of insulin resistance and diabetes.

Thyroid hormones have widespread cellular effects; however it is unclear whether their effects on the central nervous system (CNS) contribute to global energy balance. Here we demonstrate that either whole-body hyperthyroidism or central administration of triiodothyronine (T3) decreases the activity of hypothalamic AMP-activated protein kinase (AMPK), increases sympathetic nervous system (SNS) activity and upregulates thermogenic markers in brown adipose tissue (BAT). Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus (VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism. This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and increases expression of thermogenic markers in BAT. These effects are reversed by pharmacological blockade of the SNS. Thus, thyroid hormone-induced modulation of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of whole-body energy homeostasis.

Stored glycogen is an important source of energy for skeletal muscle. Human genetic disorders primarily affecting skeletal muscle glycogen turnover are well-recognised, but rare. We previously reported that a frameshift/premature stop mutation in PPP1R3A, the gene encoding RGL, a key regulator of muscle glycogen metabolism, was present in 1.36% of participants from a population of white individuals in the UK. However, the functional implications of the mutation were not known. The objective of this study was to characterise the molecular and physiological consequences of this genetic variant.

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta) has been implicated in important metabolic processes. A mouse lacking PGC-1beta (PGC1betaKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1betaKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1beta ablation was partially compensated by up-regulation of PGC-1alpha in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1betaKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1beta was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1betaKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1betaKO mice have impaired mitochondrial function. Lack of PGC-1beta also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1beta plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.

Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10-20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.

Regulation of body temperature critically depends on thyroid hormone (TH). Recent studies revealed that TH induces browning of white adipose tissue, possibly contributing to the observed hyperthermia in hyperthyroid patients and potentially providing metabolic benefits. Here, we show that browning by TH requires TH-receptor β and occurs independently of the sympathetic nervous system. The beige fat, however, lacks sufficient adrenergic stimulation and is not metabolically activated despite high levels of uncoupling protein 1 (UCP1). Studies at different environmental temperatures reveal that TH instead causes hyperthermia by actions in skeletal muscle combined with a central body temperature set-point elevation. Consequently, the metabolic and thermogenic effects of systemic hyperthyroidism were maintained in UCP1 knockout mice, demonstrating that neither beige nor brown fat contributes to the TH-induced hyperthermia and elevated glucose consumption, and underlining that the mere presence of UCP1 is insufficient to draw conclusions on the therapeutic potential of browning agents.