Hypophagia and metabolic adaptations in mice with defective ATGL-mediated lipolysis cause resistance to HFD-induced obesity.

Adipose triglyceride lipase (ATGL) initiates intracellular triglyceride (TG) catabolism. In humans, ATGL deficiency causes neutral lipid storage disease with myopathy (NLSDM) characterized by a systemic TG accumulation. Mice with a genetic deletion of ATGL (AKO) also accumulate TG in many tissues. However, neither NLSDM patients nor AKO mice are exceedingly obese. This phenotype is unexpected considering the importance of the enzyme for TG catabolism in white adipose tissue (WAT). In this study, we identified the counteracting mechanisms that prevent excessive obesity in the absence of ATGL. We used "healthy" AKO mice expressing ATGL exclusively in cardiomyocytes (AKO/cTg) to circumvent the cardiomyopathy and premature lethality observed in AKO mice. AKO/cTg mice were protected from high-fat diet (HFD)-induced obesity despite complete ATGL deficiency in WAT and normal adipocyte differentiation. AKO/cTg mice were highly insulin sensitive under hyperinsulinemic-euglycemic clamp conditions, eliminating insulin insensitivity as a possible protective mechanism. Instead, reduced food intake and altered signaling by peroxisome proliferator-activated receptor-gamma (PPAR-γ) and sterol regulatory element binding protein-1c in WAT accounted for the phenotype. These adaptations led to reduced lipid synthesis and storage in WAT of HFD-fed AKO/cTg mice. Treatment with the PPAR-γ agonist rosiglitazone reversed the phenotype. These results argue for the existence of an adaptive interdependence between lipolysis and lipid synthesis. Pharmacological inhibition of ATGL may prove useful to prevent HFD-induced obesity and insulin resistance.

Deletion of CGI-58 or adipose triglyceride lipase differently affects macrophage function and atherosclerosis.

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58(-/-) mice). CGI-58(-/-) macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL(-/-) macrophages. In contrast to ATGL(-/-) macrophages, however, CGI-58(-/-) macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58(-/-) macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE(-/-) mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor(-/-) mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency.

Mammalian alpha beta hydrolase domain (ABHD) proteins: Lipid metabolizing enzymes at the interface of cell signaling and energy metabolism.

Dysregulation of lipid metabolism underlies many chronic diseases such as obesity, diabetes, cardiovascular disease, and cancer. Therefore, understanding enzymatic mechanisms controlling lipid synthesis and degradation is imperative for successful drug discovery for these human diseases. Genes encoding α/ß hydrolase fold domain (ABHD) proteins are present in virtually all reported genomes, and conserved structural motifs shared by these proteins predict common roles in lipid synthesis and degradation. However, the physiological substrates and products for these lipid metabolizing enzymes and their broader role in metabolic pathways remain largely uncharacterized. Recently, mutations in several members of the ABHD protein family have been implicated in inherited inborn errors of lipid metabolism. Furthermore, studies in cell and animal models have revealed important roles for ABHD proteins in lipid metabolism, lipid signal transduction, and metabolic disease. The purpose of this review is to provide a comprehensive summary surrounding the current state of knowledge regarding mammalian ABHD protein family members. In particular, we will discuss how ABHD proteins are ideally suited to act at the interface of lipid metabolism and signal transduction. Although, the current state of knowledge regarding mammalian ABHD proteins is still in its infancy, this review highlights the potential for the ABHD enzymes as being attractive targets for novel therapies targeting metabolic disease.

Assessing human carcinogenicity risk of agrochemicals without the rodent cancer bioassay.

The rodent cancer bioassays are conducted for agrochemical safety assessment yet they often do not inform regulatory decision-making. As part of a collaborative effort, the Rethinking Carcinogenicity Assessment for Agrochemicals Project (ReCAAP) developed a reporting framework to guide a weight of evidence (WOE)-based carcinogenicity assessment that demonstrates how to fulfill the regulatory requirements for chronic risk estimation without the need to conduct lifetime rodent bioassays. The framework is the result of a multi-stakeholder collaboration that worked through an iterative process of writing case studies (in the form of waivers), technical peer reviews of waivers, and an incorporation of key learnings back into the framework to be tested in subsequent case study development. The example waivers used to develop the framework were written retrospectively for registered agrochemical active substances for which the necessary data and information could be obtained through risk assessment documents or data evaluation records from the US EPA. This exercise was critical to the development of a framework, but it lacked authenticity in that the stakeholders reviewing the waiver already knew the outcome of the rodent cancer bioassay(s). Syngenta expanded the evaluation of the ReCAAP reporting framework by writing waivers for three prospective case studies for new active substances where the data packages had not yet been submitted for registration. The prospective waivers followed the established framework considering ADME, potential exposure, subchronic toxicity, genotoxicity, immunosuppression, hormone perturbation, mode of action (MOA), and all relevant information available for read-across using a WOE assessment. The point of departure was estimated from the available data, excluding the cancer bioassay results, with a proposed use for the chronic dietary risk assessment. The read-across assessments compared data from reliable registered chemical analogues to strengthen the prediction of chronic toxicity and/or tumorigenic potential. The prospective case studies represent a range of scenarios, from a new molecule in a well-established chemical class with a known MOA to a molecule with a new pesticidal MOA (pMOA) and limited read-across to related molecules. This effort represents an important step in establishing criteria for a WOE-based carcinogenicity assessment without the rodent cancer bioassay(s) while ensuring a health protective chronic dietary risk assessment.

Adipose-selective overexpression of ABHD5/CGI-58 does not increase lipolysis or protect against diet-induced obesity.

Adipose triglyceride lipase (ATGL) catalyzes the first step of triacylglycerol hydrolysis in adipocytes. Abhydrolase domain 5 (ABHD5) increases ATGL activity by an unknown mechanism. Prior studies have suggested that the expression of ABHD5 is limiting for lipolysis in adipocytes, as addition of recombinant ABHD5 increases in vitro TAG hydrolase activity of adipocyte lysates. To test this hypothesis in vivo, we generated transgenic mice that express 6-fold higher ABHD5 in adipose tissue relative to wild-type (WT) mice. In vivo lipolysis increased to a similar extent in ABHD5 transgenic and WT mice following an overnight fast or injection of either a ß-adrenergic receptor agonist or lipopolysaccharide. Similarly, basal and ß-adrenergic-stimulated lipolysis was comparable in adipocytes isolated from ABHD5 transgenic and WT mice. Although ABHD5 expression was elevated in thioglycolate-elicited macrophages from ABHD5 transgenic mice, Toll-like receptor 4 (TLR4) signaling was comparable in macrophages isolated from ABHD5 transgenic and WT mice. Overexpression of ABHD5 did not prevent the development of obesity in mice fed a high-fat diet, as shown by comparison of body weight, body fat percentage, and adipocyte hypertrophy of ABHD5 transgenic to WT mice. The expression of ABHD5 in mouse adipose tissue is not limiting for either basal or stimulated lipolysis.

Gαi/o-coupled Htr2c in the paraventricular nucleus of the hypothalamus antagonizes the anorectic effect of serotonin agents.

The anorexigenic effect of serotonergic compounds has largely been attributed to activation of serotonin 2C receptors (Htr2cs). Using mouse genetic models in which Htr2c can be selectively deleted or restored (in Htr2c-null mice), we investigate the role of Htr2c in forebrain Sim1 neurons. Unexpectedly, we find that Htr2c acts in these neurons to promote food intake and counteract the anorectic effect of serotonergic appetite suppressants. Furthermore, Htr2c marks a subset of Sim1 neurons in the paraventricular nucleus of the hypothalamus (PVH). Chemogenetic activation of these neurons in adult mice suppresses hunger, whereas their silencing promotes feeding. In support of an orexigenic role of PVH Htr2c, whole-cell patch-clamp experiments demonstrate that activation of Htr2c inhibits PVH neurons. Intriguingly, this inhibition is due to Gαi/o-dependent activation of ATP-sensitive K+ conductance, a mechanism of action not identified previously in the mammalian nervous system.

Acyl-ghrelin Is Permissive for the Normal Counterregulatory Response to Insulin-Induced Hypoglycemia.

Insulin-induced hypoglycemia leads to far-ranging negative consequences in patients with diabetes. Components of the counterregulatory response (CRR) system that help minimize and reverse hypoglycemia and coordination between those components are well studied but not yet fully characterized. Here, we tested the hypothesis that acyl-ghrelin, a hormone that defends against hypoglycemia in a preclinical starvation model, is permissive for the normal CRR to insulin-induced hypoglycemia. Ghrelin knockout (KO) mice and wild-type (WT) littermates underwent an insulin bolus-induced hypoglycemia test and a low-dose hyperinsulinemic-hypoglycemic clamp procedure. Clamps also were performed in ghrelin-KO mice and C57BL/6N mice administered the growth hormone secretagogue receptor agonist HM01 or vehicle. Results show that hypoglycemia, as induced by an insulin bolus, was more pronounced and prolonged in ghrelin-KO mice, supporting previous studies suggesting increased insulin sensitivity upon ghrelin deletion. Furthermore, during hyperinsulinemic-hypoglycemic clamps, ghrelin-KO mice required a 10-fold higher glucose infusion rate (GIR) and exhibited less robust corticosterone and growth hormone responses. Conversely, HM01 administration, which reduced the GIR required by ghrelin-KO mice during the clamps, increased plasma corticosterone and growth hormone. Thus, our data suggest that endogenously produced acyl-ghrelin not only influences insulin sensitivity but also is permissive for the normal CRR to insulin-induced hypoglycemia.

POMC neurons expressing leptin receptors coordinate metabolic responses to fasting via suppression of leptin levels.

Leptin is critical for energy balance, glucose homeostasis, and for metabolic and neuroendocrine adaptations to starvation. A prevalent model predicts that leptin's actions are mediated through pro-opiomelanocortin (POMC) neurons that express leptin receptors (LEPRs). However, previous studies have used prenatal genetic manipulations, which may be subject to developmental compensation. Here, we tested the direct contribution of POMC neurons expressing LEPRs in regulating energy balance, glucose homeostasis and leptin secretion during fasting using a spatiotemporally controlled Lepr expression mouse model. We report a dissociation between leptin's effects on glucose homeostasis versus energy balance in POMC neurons. We show that these neurons are dispensable for regulating food intake, but are required for coordinating hepatic glucose production and for the fasting-induced fall in leptin levels, independent of changes in fat mass. We also identify a role for sympathetic nervous system regulation of the inhibitory adrenergic receptor (ADRA2A) in regulating leptin production. Collectively, our findings highlight a previously unrecognized role of POMC neurons in regulating leptin levels.

Ghrelin Receptor Agonist Rescues Excess Neonatal Mortality in a Prader-Willi Syndrome Mouse Model.

In the current study, we sought to determine the significance of the ghrelin system in Prader-Willi Syndrome (PWS). PWS is characterized by hypotonia and difficulty feeding in neonates and hyperphagia and obesity beginning later in childhood. Other features include low GH, neonatal hypoglycemia, hypogonadism, and accelerated mortality. Although the hyperphagia and obesity in PWS have been attributed to elevated levels of the orexigenic hormone ghrelin, this link has never been firmly established, nor have ghrelin's potentially protective actions to increase GH secretion, blood glucose, and survival been investigated in a PWS context. In the current study, we show that placing Snord116del mice modeling PWS on ghrelin-deficient or ghrelin receptor [GH secretagogue receptor (GHSR)]-deficient backgrounds does not impact their characteristically reduced body weight, lower plasma IGF-1, delayed sexual maturation, or increased mortality in the period prior to weaning. However, blood glucose was further reduced in male Snord116del pups on a ghrelin-deficient background, and percentage body weight gain and percentage fat mass were further reduced in male Snord116del pups on a GHSR-deficient background. Strikingly, 2 weeks of daily administration of the GHSR agonist HM01 to Snord116del neonates markedly improved survival, resulting in a nearly complete rescue of the excess mortality owing to loss of the paternal Snord116 gene. These data support further exploration of the therapeutic potential of GHSR agonist administration in limiting PWS mortality, especially during the period characterized by failure to thrive.

Hepatocyte toll-like receptor 4 deficiency protects against alcohol-induced fatty liver disease.

OBJECTIVE: Recent studies have suggested a critical role for toll-like receptor 4 (TLR4) in the development of alcoholic liver disease. As TLR4 is widely expressed throughout the body, it is unclear which TLR4-expressing cell types contribute to alcohol-induced liver damage. METHODS: We selectively ablated TLR4 in hepatocytes and myeloid cells. Male mice were fed a liquid diet containing either 5% alcohol or pair-fed a control diet for 4 weeks to examine chronic alcohol intake-induced liver damage and inflammation. In addition, mice were administered a single oral gavage of alcohol to investigate acute alcohol drinking-associated liver injury. RESULTS: We found that selective hepatocyte TLR4 deletion protected mice from chronic alcohol-induced liver injury and fatty liver. This result was in part due to decreased expression of endogenous lipogenic genes and enhanced expression of genes involved in fatty acid oxidation. In addition, mice lacking hepatocyte TLR4 exhibited reduced mRNA expression of inflammatory genes in white adipose tissue. Furthermore, in an acute alcohol binge model, hepatocyte TLR4 deficient mice had significantly decreased plasma alanine transaminase (ALT) levels and attenuated hepatic triglyceride content compared to their alcohol-gavaged control mice. In contrast, deleting TLR4 in myeloid cells did not affect the development of chronic-alcohol induced fatty liver, despite the finding that mice lacking myeloid cell TLR4 had significantly reduced circulating ALT concentrations. CONCLUSIONS: These findings suggest that hepatocyte TLR4 plays an important role in regulating alcohol-induced liver damage and fatty liver disease.

Serotonergic Control of Metabolic Homeostasis.

New treatments are urgently needed to address the current epidemic of obesity and diabetes. Recent studies have highlighted multiple pathways whereby serotonin (5-HT) modulates energy homeostasis, leading to a renewed interest in the identification of 5-HT-based therapies for metabolic disease. This review aims to synthesize pharmacological and genetic studies that have found diverse functions of both central and peripheral 5-HT in the control of food intake, thermogenesis, and glucose and lipid metabolism. We also discuss the potential benefits of targeting the 5-HT system to combat metabolic disease.

The atypical antipsychotic olanzapine causes weight gain by targeting serotonin receptor 2C.

Atypical antipsychotics such as olanzapine often induce excessive weight gain and type 2 diabetes. However, the mechanisms underlying these drug-induced metabolic perturbations remain poorly understood. Here, we used an experimental model that reproduces olanzapine-induced hyperphagia and obesity in female C57BL/6 mice. We found that olanzapine treatment acutely increased food intake, impaired glucose tolerance, and altered physical activity and energy expenditure in mice. Furthermore, olanzapine-induced hyperphagia and weight gain were blunted in mice lacking the serotonin 2C receptor (HTR2C). Finally, we showed that treatment with the HTR2C-specific agonist lorcaserin suppressed olanzapine-induced hyperphagia and weight gain. Lorcaserin treatment also improved glucose tolerance in olanzapine-fed mice. Collectively, our studies suggest that olanzapine exerts some of its untoward metabolic effects via antagonism of HTR2C.

Regulation of Hepatic Triacylglycerol Metabolism by CGI-58 Does Not Require ATGL Co-activation.

Adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) are critical regulators of triacylglycerol (TAG) turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG) and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes.

The serine hydrolase ABHD6 Is a critical regulator of the metabolic syndrome.

The serine hydrolase α/ß hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6's role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

Distinct roles for alpha-beta hydrolase domain 5 (ABHD5/CGI-58) and adipose triglyceride lipase (ATGL/PNPLA2) in lipid metabolism and signaling.

Catabolism of stored triacylglycerol (TAG) from cytoplasmic lipid droplets is critical for providing energy substrates, membrane building blocks, and signaling lipids in most cells of the body. However, the lipolytic machinery dictating TAG hydrolysis varies greatly among different cell types. Within the adipocyte, TAG hydrolysis is dynamically regulated by hormones to ensure appropriate metabolic adaptation to nutritional and physiologic cues. In other cell types such as hepatocytes, myocytes, and macrophages, mobilization of stored TAG is regulated quite differently. Within the last decade, mutations in two key genes involved in TAG hydrolysis, alpha-beta hydrolase domain 5 (ABHD5/CGI-58) and adipose triglyceride lipase (ATGL/PNPLA2), were found to cause two distinct neutral lipid storage diseases (NLSD) in humans. These genetic links, along with supporting evidence in mouse models, have prompted a number of studies surrounding the biochemical function(s) of these proteins. Although both CGI-58 and ATGL have been clearly implicated in TAG hydrolysis in multiple tissues and have even been shown to physically interact with each other, recent evidence suggests that they may also have distinct roles. The purpose of this review is to summarize the most recent insights into how CGI-58 and ATGL regulate lipid metabolism and signaling.

CGI-58/ABHD5-derived signaling lipids regulate systemic inflammation and insulin action.

Mutations of comparative gene identification 58 (CGI-58) in humans cause Chanarin-Dorfman syndrome, a rare autosomal recessive disease in which excess triacylglycerol (TAG) accumulates in multiple tissues. CGI-58 recently has been ascribed two distinct biochemical activities, including coactivation of adipose triglyceride lipase and acylation of lysophosphatidic acid (LPA). It is noteworthy that both the substrate (LPA) and the product (phosphatidic acid) of the LPA acyltransferase reaction are well-known signaling lipids. Therefore, we hypothesized that CGI-58 is involved in generating lipid mediators that regulate TAG metabolism and insulin sensitivity. Here, we show that CGI-58 is required for the generation of signaling lipids in response to inflammatory stimuli and that lipid second messengers generated by CGI-58 play a critical role in maintaining the balance between inflammation and insulin action. Furthermore, we show that CGI-58 is necessary for maximal TH1 cytokine signaling in the liver. This novel role for CGI-58 in cytokine signaling may explain why diminished CGI-58 expression causes severe hepatic lipid accumulation yet paradoxically improves hepatic insulin action. Collectively, these findings establish that CGI-58 provides a novel source of signaling lipids. These findings contribute insight into the basic mechanisms linking TH1 cytokine signaling to nutrient metabolism.