Obesity has reached pandemic proportions, highlighting the urgent need for continued research to uncover the molecular mechanisms governing lipid homeostasis and ectopic fat deposition in overnutrition. Our recent translational studies demonstrated that STE20-type kinases STK25 and MST3 associate with intracellular lipid droplets and play a pivotal role in regulating the dynamic balance between fat storage and utilization. This study aimed to assess the in vivo effects of the combined inhibition of STK25 and MST3 in obese mice. We performed phenotypic characterization in three cohorts of mice fed a high-fat diet: (1) mice with genetic ablation of Stk25, (2) mice treated with Mst3-targeting antisense oligonucleotide (ASO), and (3) mice depleted of both STK25 and MST3 by injecting Stk25−/− mice with Mst3 ASO. Whole-body metabolic physiology and organ lipotoxicity were examined in the STK25- and/or MST3-deficient mice compared with their respective controls by using histological assessments, immunofluorescence microscopy, molecular profiling, and biochemical assays. We found that the inactivation of STK25 and MST3, either individually or in combination, provided equal protection against ectopic fat accumulation and associated lipotoxic damage in the liver, kidney, and skeletal muscle of obese mice. Strikingly, high-fat diet-fed STK25/MST3-deficient mice, but not mice lacking only one kinase, displayed reduced body and fat mass gain, which was accompanied by markedly increased abundance of thermogenesis markers in the brown adipose tissue (BAT). Dual inhibition of STK25 and MST3 in mice mitigates obesity-triggered lipotoxic injury to metabolic tissues and elevates indicators of BAT thermogenic capacity.

Mice with genetic ablation of PI3Ky are protected from diet-induced obesity. However, the cell type responsible for PI3Ky action in obesity remains unknown. We generated mice with conditional deletion of PI3Ky neurons using the nestin promoter to drive the expression of the Cre recombinase (PI3KyNest mice) and investigated their metabolic phenotype in a model of diet-induced obesity. On a chow diet, lean PI3KyNest mice display reduced linear growth and a normal metabolic phenotype. PI3KyNest mice were largely protected from diet-induced obesity and liver steatosis and showed improved glucose tolerance and insulin sensitivity. This phenotype was associated with increased phosphorylation of hormone-sensitive lipase (HSL) at protein kinase A (PKA) sites in white fat. It is concluded that PI3Ky action in diet-induced obesity depends on its activity in neurons controlling adipose tissue lipolysis. Future clinical studies on PI3Ky inhibitors capable of crossing the brain-blood barrier will reveal the relevance of these findings to humans.

The use of the GLP-1R agonist semaglutide is revolutionizing the treatment of obesity, yet its mechanistic effects on energy balance remain elusive. Here, we demonstrate that reactivation of semaglutide-responsive dorsal vagal complex neurons mimics the drug's effects of reducing food intake and body weight and promoting fat utilization and conditioned taste aversion. We observe that many of the semaglutide-activated area postrema (AP) and nucleus of the solitary tract (NTS) neurons express Adcyap1 mRNA, and ablation of AP/NTS Adcyap1+ neurons largely reverses semaglutide's effects on energy balance acutely in lean mice and in subchronically treated obese mice. Semaglutide-activated AP/NTS Adcyap1+ neurons promote the loss of fat rather than lean mass, with only a modest effect on conditioned taste aversion. Furthermore, NTS Adcyap1+ neurons are engaged by GLP-1R-expressing AP neurons and are necessary for semaglutide-induced activation of several downstream satiety-related structures. Selective targeting of semaglutide-responsive Adcyap1+ neurons holds potential for improved future anti-obesity treatments.

The neurotransmitter acetylcholine has since long been implicated in reward learning and drug addiction. However, the role of specific cholinergic receptor subtypes on different neuronal populations remain elusive. Here, we studied the function of nicotinic acetylcholinergic alpha 7 receptors (α7 nAChRs) in cocaine and food-enforced behaviors. We found that global deletion of α7 nAChRs in mice attenuates cocaine seeking in a Pavlovian conditioned place preference paradigm and decreases operant responding to cocaine in a runway task and in self-administration, without influencing responding to palatable food. This effect can be attributed to alpha 7 receptor signaling in the striatum, as selective deletion of striatal α7 nAChRs using a viral vector approach resulted in a similar decrease in cocaine-preference as that of global deletion. To investigate which type of striatal neurons are responsible for this effect, we selectively targeted Cholinergic (ChAT-expressing) neurons and dopamine D1-receptor (D1R) expressing neurons. Mice with conditional deletion of α7 nAChRs in ChAT-neurons (α7 nAChR-ChATCre) exhibited decreased cocaine place preference and intact place preference for food, while α7 nAChR-D1RCre mice had no changes in reward learning to neither food nor cocaine. Cocaine induction of striatal immediate early gene expression of cFos, FosB, Arc and EGR2 was blocked in α7 nAChR-ChATCre mice, demonstrating the importance of α7 nAChRs on cholinergic neurons for striatal neuronal activity changes. Collectively, our findings show that α7 nAChRs on cholinergic interneurons in the striatum are pivotal for learning processes related to cocaine, but not food reward.

Cancer cachexia is well-recognized in pancreatic cancer, explained by cytokines, prostaglandins and hormones. Recently, neuroendocrine factors as PTHrP and GDF-15 are also proposed as important mediators behind cachexia in animal models. Therefore, our aim was to evaluate how EGFR, PTHrP and GDF-15 may relate to tumour progression and cachexia in patients with PDAC tumours. Blood PTHrP and GDF-15 were quantified by Elisa analyses (n=175). Tumour expression of EGFR, PTHrP and GDF-15 was quantified by Immunohistochemistry (n=33). Blood and tumour levels were compared to clinical variables (standard laboratory tests, food diaries, energy expenditure, body composition, exercise-capacity and Health Related Quality of Life (SF-36, HAD-scales). Simple- and multivariate analyses were used to evaluate statistical inferences. Blood PTHrP and GDF-15 levels were related to altered whole body metabolism, positively correlated to fat oxidation (p<0.001) and negatively to carbohydrate oxidation (p<0-02). Multivariate regressions on tumour tissue proteins imply EGFR as a primary driver of tumour growth (p<0.04), counteracted by PTHrP and GDH-15 in tumour tissue. Statistical inference on blood analyses implies that PTHrP may induce fat browning which promotes energy expenditure, while blood GDF-15 is not a primary statistical factor behind cachexia but may promote anorexia secondarily to systemic inflammation (p<0.001), with insulin resistance. Tumour tissue EGFR is a driver of PDAC growth, while tumour PTHrP- and GDF-15 may rather be counteracting factors. However, blood PTHrP and GDF-15 are produced in several tissues and thereby affect whole body oxidative metabolism and negative energy balance, within networks of systemic inflammation. *Published, submitted and unpublished results.

GFRAL-expressing neurons actuate aversion and nausea, are targets for obesity treatment, and may mediate metformin effects by long-term GDF15-GFRAL agonism. Whether GFRAL+ neurons acutely regulate glucose and energy homeostasis is, however, underexplored. Here, we report that cell-specific activation of GFRAL+ neurons using a variety of techniques causes a torpor-like state, including hypothermia, the release of stress hormones, a shift from glucose to lipid oxidation, and impaired insulin sensitivity, glucose tolerance, and skeletal muscle glucose uptake but augmented glucose uptake in visceral fat. Metabolomic analysis of blood and transcriptomics of muscle and fat indicate alterations in ketogenesis, insulin signaling, adipose tissue differentiation and mitogenesis, and energy fluxes. Our findings indicate that acute GFRAL+ neuron activation induces endocrine and gluco- and thermoregulatory responses associated with nausea and torpor. While chronic activation of GFRAL signaling promotes weight loss in obesity, these results show that acute activation of GFRAL+ neurons causes hypothermia and hyperglycemia.

ObjectiveNon-alcoholic fatty liver disease (NAFLD), the primary hepatic consequence of obesity, is affecting about 25% of the global adult population. The aim of this study was to examine the in vivo role of STE20-type protein kinase TAOK3, which has been previously reported to regulate hepatocellular lipotoxicity in vitro, in the development of NAFLD and systemic insulin resistance in the context of obesity.MethodsTaok3 knockout mice and wild-type littermates were challenged with a high-fat diet. Various in vivo tests were performed to characterize the whole-body metabolism. NAFLD progression in the liver, and lipotoxic damage in adipose tissue, kidney, and skeletal muscle were compared between the genotypes by histological assessment, immunofluorescence microscopy, protein and gene expression profiling, and biochemical assays. Intracellular lipid accumulation and oxidative/ER stress were analyzed in cultured human and mouse hepatocytes where TAOK3 was knocked down by small interfering RNA. The expression of TAOK3-related STE20-type kinases was quantified in different organs from high-fat diet-fed Taok3-/- and wild-type mice.ResultsTAOK3 deficiency had no impact on body weight or composition, food consumption, locomotor activity, or systemic glucose or insulin homeostasis in obese mice. Consistently, Taok3-/- mice and wild-type littermates developed a similar degree of high-fat diet-induced liver steatosis, inflammation, and fibrosis, and we detected no difference in lipotoxic damage of adipose tissue, kidney, or skeletal muscle when comparing the two genotypes. In contrast, the silencing of TAOK3 in vitro markedly suppressed ectopic lipid accumulation and metabolic stress in mouse and human hepatocytes. Interestingly, the hepatic mRNA abundance of several TAOK3-related kinases, which have been previously implicated to increase the risk of NAFLD susceptibility, was significantly elevated in Taok3-/-vs. wild-type mice.ConclusionsIn contrast to the in vitro observations, genetic deficiency of TAOK3 in mice failed to mitigate the detrimental metabolic consequences of chronic exposure to dietary lipids, which may be partly attributable to the activation of liver-specific compensation response for the genetic loss of TAOK3 by related STE20-type kinases.

We previously provided evidence supporting the existence of a novel leptin-independent body weight homeostat ("the gravitostat") that senses body weight and then initiates a homeostatic feed-back regulation of body weight. We, herein, hypothesize that this feed-back regulation involves a CNS mechanism. To identify populations of neurones of importance for the putative feed-back signal induced by increased loading, high-fat diet-fed rats or mice were implanted intraperitoneally or subcutaneously with capsules weighing similar to 15% (Load) or similar to 2.5% (Control) of body weight. At 3-5 days after implantation, neuronal activation was assessed in different parts of the brain/brainstem by immunohistochemical detection of FosB. Implantation of weighted capsules, both subcutaneous and intraperitoneal, induced FosB in specific neurones in the medial nucleus of the solitary tract (mNTS), known to integrate information about the metabolic status of the body. These neurones also expressed tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbH), a pattern typical of norepinephrine neurones. In functional studies, we specifically ablated norepinephrine neurones in mNTS, which attenuated the feed-back regulation of increased load on body weight and food intake. In conclusion, increased load appears to reduce body weight and food intake via activation of norepinephrine neurones in the mNTS.

Variations in the human FTO gene have been linked to obesity and altered connectivity of the dopaminergic neurocircuitry. Here, we report that fat mass and obesity-associated protein (FTO) in dopamine D2 receptor-expressing medium spiny neurons (D2 MSNs) of mice regulate the excitability of these cells and control their striatopallidal globus pallidus external (GPe) projections. Lack of FTO in D2 MSNs translates into increased locomotor activity to novelty, associated with altered timing behavior, without impairing the ability to control actions or affecting reward-driven and conditioned behavior. Pharmacological manipulations of dopamine D1 receptor (D1R)- or D2R-dependent pathways in these animals reveal altered responses to D1- and D2-MSN-mediated control of motor output. These findings reveal a critical role for FTO to control D2 MSN excitability, their projections to the GPe, and behavioral responses to novelty.

The central nervous system (CNS) has an important role in the regulation of peripheral insulin sensitivity and glucose homeostasis. Research in this dynamically developing field has progressed rapidly due to techniques allowing targeted transgenesis and neurocircuitry mapping, which have defined the primary responsive neurons, associated molecular mechanisms and downstream neurocircuitries and processes involved. Here we review the brain regions, neurons and molecular mechanisms by which the CNS controls peripheral glucose metabolism, particularly via regulation of liver, brown adipose tissue and pancreatic function, and highlight the potential implications of these regulatory pathways in type 2 diabetes and obesity.

BACKGROUND:The cancer-anorexia-cachexia syndrome (CACS) negatively affects survival and therapy success in cancer patients. Inflammatory mediators and tumour-derived factors are thought to play an important role in the aetiology of CACS. However, the central and peripheral mechanisms contributing to CACS are insufficiently understood. The area postrema (AP) and the nucleus tractus solitarii are two important brainstem centres for the control of eating during acute sickness conditions. Recently, the tumour-derived macrophage inhibitory cytokine-1 (MIC-1) emerged as a possible mediator of cancer anorexia because lesions of these brainstem areas attenuated the anorectic effect of exogenous MIC-1 in mice.METHODS:Using a rat hepatoma tumour model, we examined the roles of the AP and of vagal afferents in the mediation of CACS. Specifically, we investigated whether a lesion of the AP (APX) or subdiaphragmatic vagal deafferentation (SDA) attenuate anorexia, body weight, muscle, and fat loss. Moreover, we analysed MIC-1 levels in this tumour model and their correlation with tumour size and the severity of the anorectic response.RESULTS:In tumour-bearing sham-operated animals mean daily food intake significantly decreased. The anorectic response was paralleled by a significant loss of body weight and muscle mass. APX rats were protected against anorexia, body weight loss, and muscle atrophy after tumour induction. In contrast, subdiaphragmatic vagal deafferentation did not attenuate cancer-induced anorexia or body weight loss. Tumour-bearing rats had substantially increased MIC-1 levels, which positively correlated with tumour size and cancer progression and negatively correlated with food intake.CONCLUSIONS:These findings demonstrate the importance of the AP in the mediation of cancer-dependent anorexia and body weight loss and support a pathological role of MIC-1 as a tumour-derived factor mediating CACS, possibly via an AP-dependent action.

Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis.

Cutting-edge experiments show a new means to control the activity of specifically genetically targeted neurons in the hypothalamus using electromagnetic force. At the flip of a switch, the system bidirectionally regulates feeding behavior and glucose homeostasis, demonstrating wireless control over deep brain regions and their strong influence over energy balance.

Melanin-concentrating-hormone (MCH)-expressing neurons (MCH neurons) in the lateral hypothalamus (LH) are critical regulators of energy and glucose homeostasis. Here, we demonstrate that insulin increases the excitability of these neurons in control mice. In vivo, insulin promotes phosphatidylinositol 3-kinase (PI3K) signaling in MCH neurons, and cell-type- specific deletion of the insulin receptor (IR) abrogates this response. While lean mice lacking the IR in MCH neurons (IR Delta MCH) exhibit no detectable metabolic phenotype under normal diet feeding, they present with improved locomotor activity and insulin sensitivity under high-fat-diet-fed, obese conditions. Similarly, obesity promotes PI3 kinase signaling in these neurons, and this response is abrogated in IR Delta MCH mice. In turn, acute chemogenetic activation of MCH neurons impairs locomotor activity but not insulin sensitivity. Collectively, our experiments reveal an insulin-dependent activation of MCH neurons in obesity, which contributes via distinct mechanisms to the manifestation of impaired locomotor activity and insulin resistance.

The role of IL-6 in obesity-associated inflammation remains controversial. Bruening and colleagues identify signaling by IL-6 as an important determinant for the alternative activation of macrophages during inflammation.

It is well-established that prostaglandins (PGs) affect tumorigenesis, and evidence indicates that PGs also are important for the reduced food intake and body weight loss, the anorexia-cachexia syndrome, in malignant cancer. However, the identity of the PGs and the PG producing cyclooxygenase (COX) species responsible for cancer anorexia-cachexia is unknown. Here, we addressed this issue by transplanting mice with a tumor that elicits anorexia. Meal pattern analysis revealed that the anorexia in the tumor-bearing mice was due to decreased meal frequency. Treatment with a non-selective COX inhibitor attenuated the anorexia, and also tumor growth. When given at manifest anorexia, non-selective COX-inhibitors restored appetite and prevented body weight loss without affecting tumor size. Despite COX-2 induction in the cerebral blood vessels of tumor-bearing mice, a selective COX-2 inhibitor had no effect on the anorexia, whereas selective COX-1 inhibition delayed its onset. Tumor growth was associated with robust increase of PGE(2) levels in plasma - a response blocked both by non-selective COX-inhibition and by selective COX-1 inhibition, but not by COX-2 inhibition. However, there was no increase in PGE(2)-levels in the cerebrospinal fluid. Neutralization of plasma PGE(2) with specific antibodies did not ameliorate the anorexia, and genetic deletion of microsomal PGE synthase-1 (mPGES-1) affected neither anorexia nor tumor growth. Furthermore, tumor-bearing mice lacking EP4 receptors selectively in the nervous system developed anorexia. These observations suggest that COX-enzymes, most likely COX-1, are involved in cancer-elicited anorexia and weight loss, but that these phenomena occur independently of host mPGES-1, PGE2 and neuronal EP4 signaling. (C) 2013 Elsevier Inc. All rights reserved.

Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

Interleukin-6 (IL-6) is critical for the lipopolysaccharide (LPS)-induced febrile response. However, the exact source(s) of IL-6 involved in regulating the LPS-elicited fever is still to be identified. One known source of IL-6 is hematopoietic cells, such as monocytes. To clarify the contribution of hematopoietically derived IL-6 to fever, we created chimeric mice expressing IL-6 selectively either in cells of hematopoietic or, conversely, in cells of non-hematopoietic origin. This was performed by extinguishing hematopoietic cells in wild-type (WT) or IL-6 knockout (IL-6 KO) mice by whole-body irradiation and transplanting them with new stem cells. Mice on a WT background but lacking IL-6 in hematopoietic cells displayed normal fever to LPS and were found to have similar levels of IL-6 protein in the cerebrospinal fluid (CSF) and in plasma and of IL-6 mRNA in the brain as WT mice. In contrast, mice on an IL-6 KO background, but with intact IL-6 production in cells of hematopoietic origin, only showed a minor elevation of the body temperature after peripheral LPS injection. While they displayed significantly elevated levels of IL-6 both in plasma and CSF compared with control mice, the increase was modest compared with that seen in LPS injected mice on a WT background, the latter being approximately 20 times larger in magnitude. These results suggest that IL-6 of non-hematopoietic origin is the main source of IL-6 in LPS-induced fever, and that IL-6 produced by hematopoietic cells only plays a minor role.