ABSTRACT
Cachexia represents a leading cause of morbidity and mortality in various cancers, chronic inflammation and infections. Understanding of the mechanisms that drive cachexia has remained limited, especially for infection-associated cachexia (IAC). In the present paper we describe a model of reversible cachexia in mice with chronic viral infection and identify an essential role for CD8+ T cells in IAC. Cytokines linked to cancer-associated cachexia did not contribute to IAC. Instead, virus-specific CD8+ T cells caused morphologic and molecular changes in the adipose tissue, which led to depletion of lipid stores. These changes occurred at a time point that preceded the peak of the CD8+ T cell response and required T cell-intrinsic type I interferon signaling and antigen-specific priming. Our results link systemic antiviral immune responses to adipose-tissue remodeling and reveal an underappreciated role of CD8+ T cells in IAC.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cachexia/etiology , Virus Diseases/complications , Virus Diseases/immunology , Adipose Tissue/diagnostic imaging , Adipose Tissue/immunology , Adipose Tissue/metabolism , Adipose Tissue/virology , Animals , CD8-Positive T-Lymphocytes/metabolism , Cachexia/diagnostic imaging , Cachexia/metabolism , Cachexia/pathology , Chronic Disease , Cytokines/blood , Cytokines/metabolism , Female , Interferon Type I/metabolism , Lipid Metabolism , Lipolysis , Lymphocyte Activation/immunology , Lymphocytic choriomeningitis virus , Magnetic Resonance Imaging/methods , Male , Mice , Signal Transduction , Virus Diseases/virologyABSTRACT
Cancer-associated cachexia (CAC) is a hypermetabolic syndrome characterized by unintended weight loss due to the atrophy of adipose tissue and skeletal muscle. A phenotypic switch from white to beige adipocytes, a phenomenon called browning, accelerates CAC by increasing the dissipation of energy as heat. Addressing the mechanisms of white adipose tissue (WAT) browning in CAC, we now show that cachexigenic tumors activate type 2 immunity in cachectic WAT, generating a neuroprotective environment that increases peripheral sympathetic activity. Increased sympathetic activation, in turn, results in increased neuronal catecholamine synthesis and secretion, ß-adrenergic activation of adipocytes, and induction of WAT browning. Two genetic mouse models validated this progression of events. 1) Interleukin-4 receptor deficiency impeded the alternative activation of macrophages, reduced sympathetic activity, and restrained WAT browning, and 2) reduced catecholamine synthesis in peripheral dopamine ß-hydroxylase (DBH)-deficient mice prevented cancer-induced WAT browning and adipose atrophy. Targeting the intraadipose macrophage-sympathetic neuron cross-talk represents a promising therapeutic approach to ameliorate cachexia in cancer patients.
Subject(s)
Adipose Tissue, Brown/pathology , Cachexia/pathology , Cell Communication , Neoplasms/complications , Neurons/pathology , Sympathetic Nervous System/pathology , Animals , Cachexia/etiology , Cachexia/metabolism , Gene Expression , Heterografts , Humans , Mice , Neoplasms/metabolism , Receptors, Adrenergic, beta/metabolism , ThermogenesisABSTRACT
Magnetization transfer spectroscopy relies heavily on the robust determination of T 1 relaxation times of nuclei participating in metabolic exchange. Challenges arise due to the use of surface RF coils for transmission (high B 1 + variation) and the broad resonance band of most X nuclei. These challenges are particularly pronounced when fast T 1 mapping methods, such as the dual-angle method, are employed. Consequently, in this work, we develop resonance offset and B 1 + robust excitation RF pulses for 31P magnetization transfer spectroscopy at 7T through ensemble-based time-optimal control. In our approach, we introduce a cost functional for designing robust pulses, incorporating the full Bloch equations as constraints, which are solved using symmetric operator splitting techniques. The optimal control design of the RF pulses developed demonstrates improved accuracy, desired phase properties, and reduced RF power when applied to dual-angle T 1 mapping, thereby improving the precision of exchange-rate measurements, as demonstrated in a preclinical in vivo study quantifying brain creatine kinase activity.
Subject(s)
Magnetic Resonance Spectroscopy , Animals , Magnetic Resonance Spectroscopy/methods , Time Factors , Brain/diagnostic imaging , Brain/metabolism , Radio Waves , AlgorithmsABSTRACT
Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.
Subject(s)
Cryopreservation , Freezing , Lipid Droplets/ultrastructure , Liver/ultrastructure , Mitochondria/ultrastructure , Animals , Mice , Mice, Inbred C57BL , Microscopy, ElectronABSTRACT
Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endolysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and parenchymal and nonparenchymal cells. Tissue samples were obtained from fed, fasted, 2 h refed, and insulin-treated mice, as well as from mice housed at 5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles that were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels, indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue- and cell-type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP plays a role in the functional adaption to nutrient starvation and ambient temperature.
Subject(s)
Lysophospholipids/metabolism , Lysosomes/metabolism , Monoglycerides/metabolism , Animals , Endosomes/metabolism , Macrophages/cytology , MiceABSTRACT
High serum fatty acid (FA) levels are causally linked to the development of insulin resistance, which eventually progresses to type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) generalized in the term metabolic syndrome. Adipose triglyceride lipase (ATGL) is the initial enzyme in the hydrolysis of intracellular triacylglycerol (TG) stores, liberating fatty acids that are released from adipocytes into the circulation. Hence, ATGL-specific inhibitors have the potential to lower circulating FA concentrations, and counteract the development of insulin resistance and NAFLD. In this article, we report about structure-activity relationship (SAR) studies of small molecule inhibitors of murine ATGL which led to the development of Atglistatin. Atglistatin is a specific inhibitor of murine ATGL, which has proven useful for the validation of ATGL as a potential drug target.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Lipase/antagonists & inhibitors , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Animals , Drug Discovery , Lipase/chemistry , Lipase/metabolism , Lipolysis/drug effects , Mice , Structure-Activity Relationship , Triglycerides/bloodABSTRACT
Bis(monoacylglycerol)phosphate (BMP) is a phospholipid that is crucial for lipid degradation and sorting in acidic organelles. Genetic and drug-induced lysosomal storage disorders (LSDs) are associated with increased BMP concentrations in tissues and in the circulation. Data on BMP in disorders other than LSDs, however, are scarce, and key enzymes regulating BMP metabolism remain elusive. Here, we demonstrate that common metabolic disorders and the intracellular BMP hydrolase α/ß-hydrolase domain-containing 6 (ABHD6) affect BMP metabolism in mice and humans. In mice, dietary lipid overload strongly affects BMP concentration and FA composition in the liver and plasma, similar to what has been observed in LSDs. Notably, distinct changes in the BMP FA profile enable a clear distinction between lipid overload and drug-induced LSDs. Global deletion of ABHD6 increases circulating BMP concentrations but does not cause LSDs. In humans, nonalcoholic fatty liver disease and liver cirrhosis affect the serum BMP FA composition and concentration. Furthermore, we identified a patient with a loss-of-function mutation in the ABHD6 gene, leading to an altered circulating BMP profile. In conclusion, our results suggest that common metabolic diseases and ABHD6 affect BMP metabolism in mice and humans.
Subject(s)
Lysophospholipids/metabolism , Metabolic Diseases/metabolism , Monoacylglycerol Lipases/metabolism , Monoglycerides/metabolism , Adult , Aged , Animals , Female , Humans , Lysophospholipids/blood , Male , Metabolic Diseases/blood , Mice , Mice, Knockout , Mice, Transgenic , Middle Aged , Monoacylglycerol Lipases/deficiency , Monoacylglycerol Lipases/genetics , Monoglycerides/blood , PhenotypeABSTRACT
AIMS/HYPOTHESIS: Dysfunction of lipid metabolism in white adipose tissue can substantially interfere with health and quality of life, for example in obesity and associated metabolic diseases. Therefore, it is important to characterise pathways that regulate lipid handling in adipocytes and determine how they affect metabolic homeostasis. Components of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway are involved in adipocyte physiology and pathophysiology. However, the exact physiological importance of the STAT family member STAT5 in white adipose tissue is yet to be determined. Here, we aimed to delineate adipocyte STAT5 functions in the context of lipid metabolism in white adipose tissue. METHODS: We generated an adipocyte specific knockout of Stat5 in mice using the Adipoq-Cre recombinase transgene followed by in vivo and in vitro biochemical and molecular studies. RESULTS: Adipocyte-specific deletion of Stat5 resulted in increased adiposity, while insulin resistance and gluconeogenic capacity was decreased, indicating that glucose metabolism can be improved by interfering with adipose STAT5 function. Basal lipolysis and fasting-induced lipid mobilisation were diminished upon STAT5 deficiency, which coincided with reduced levels of the rate-limiting lipase of triacylglycerol hydrolysis, adipose triglyceride lipase (ATGL, encoded by Pnpla2) and its coactivator comparative gene identification 58 (CGI-58). In a mechanistic analysis, we identified a functional STAT5 response element within the Pnpla2 promoter, indicating that Pnpla2 is transcriptionally regulated by STAT5. CONCLUSIONS/INTERPRETATION: Our findings reveal an essential role for STAT5 in maintaining lipid homeostasis in white adipose tissue and provide a rationale for future studies into the potential of STAT5 manipulation to improve outcomes in metabolic diseases.
Subject(s)
Adipocytes/metabolism , Adiposity/physiology , STAT5 Transcription Factor/metabolism , 3T3-L1 Cells , Adiposity/genetics , Animals , Blotting, Western , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Female , Glucose/metabolism , Insulin Resistance/genetics , Insulin Resistance/physiology , Lipid Metabolism/genetics , Lipid Metabolism/physiology , Lipid Mobilization/genetics , Lipid Mobilization/physiology , Lipolysis/genetics , Lipolysis/physiology , Male , Mice , Quality of Life , Real-Time Polymerase Chain Reaction , STAT5 Transcription Factor/geneticsABSTRACT
Monoglyceride lipase (MGL) is required for efficient hydrolysis of the endocannabinoid 2-arachidonoylglyerol (2-AG) in the brain generating arachidonic acid (AA) and glycerol. This metabolic function makes MGL an interesting target for the treatment of neuroinflammation, since 2-AG exhibits anti-inflammatory properties and AA is a precursor for pro-inflammatory prostaglandins. Astrocytes are an important source of AA and 2-AG, and highly express MGL. In the present study, we dissected the distinct contribution of MGL in astrocytes on brain 2-AG and AA metabolism by generating a mouse model with genetic deletion of MGL specifically in astrocytes (MKO(GFAP)). MKO(GFAP) mice exhibit moderately increased 2-AG and reduced AA levels in brain. Minor accumulation of 2-AG in the brain of MKO(GFAP) mice does not cause cannabinoid receptor desensitization as previously observed in mice globally lacking MGL. Importantly, MKO(GFAP) mice exhibit reduced brain prostaglandin E2 and pro-inflammatory cytokine levels upon peripheral lipopolysaccharide (LPS) administration. These observations indicate that MGL-mediated degradation of 2-AG in astrocytes provides AA for prostaglandin synthesis promoting LPS-induced neuroinflammation. The beneficial effect of astrocyte-specific MGL-deficiency is not fully abrogated by the inverse cannabinoid receptor 1 agonist SR141716 (Rimonabant) suggesting that the anti-inflammatory effects are rather caused by reduced prostaglandin synthesis than by activation of cannabinoid receptors. In conclusion, our data demonstrate that MGL in astrocytes is an important regulator of 2-AG levels, AA availability, and neuroinflammation.
Subject(s)
Astrocytes/enzymology , Gene Deletion , Inflammation/enzymology , Inflammation/pathology , Monoacylglycerol Lipases/metabolism , Nervous System/enzymology , Nervous System/pathology , Animals , Arachidonic Acids/metabolism , Astrocytes/pathology , Behavior, Animal , Brain/enzymology , Cytokines/metabolism , Endocannabinoids/metabolism , Female , Glial Fibrillary Acidic Protein/metabolism , Glycerides/metabolism , Lipopolysaccharides , Mice, Inbred C57BL , Mice, Knockout , Microglia/metabolism , Microglia/pathology , Organ Specificity , Receptor, Cannabinoid, CB1/metabolismABSTRACT
The anabolism and catabolism of myocardial triacylglycerol (TAG) stores are important processes for normal cardiac function. TAG synthesis detoxifies and stockpiles fatty acids to prevent lipotoxicity, whereas TAG hydrolysis (lipolysis) remobilizes fatty acids from endogenous storage pools as energy substrates, signaling molecules, or precursors for complex lipids. This study focused on the role of G0/G1 switch 2 (G0S2) protein, which was previously shown to inhibit the principal TAG hydrolase adipose triglyceride lipase (ATGL), in the regulation of cardiac lipolysis. Using wild-type and mutant mice, we show the following: (i) G0S2 is expressed in the heart and regulated by the nutritional status with highest expression levels after re-feeding. (ii) Cardiac-specific overexpression of G0S2 inhibits cardiac lipolysis by direct protein-protein interaction with ATGL. This leads to severe cardiac steatosis. The steatotic hearts caused by G0S2 overexpression are less prone to fibrotic remodeling or cardiac dysfunction than hearts with a lipolytic defect due to ATGL deficiency. (iii) Conversely to the phenotype of transgenic mice, G0S2 deficiency results in a de-repression of cardiac lipolysis and decreased cardiac TAG content. We conclude that G0S2 acts as a potent ATGL inhibitor in the heart modulating cardiac substrate utilization by regulating cardiac lipolysis.
Subject(s)
Cell Cycle Proteins/genetics , G1 Phase/genetics , Lipolysis/genetics , Myocardium/metabolism , Resting Phase, Cell Cycle/genetics , Triglycerides/metabolism , Animals , Cell Line , Heart Function Tests , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
The coordinated processes of lipid synthesis, degradation, and transport are mediated by enzymes, cofactors, and transport proteins. Accordingly, lipid-metabolizing enzymes represent logical targets for the treatment of dyslipidemia, a major risk factor for typeâ 2 diabetes, atherosclerosis, and other disorders. Small-molecule tool compounds, modulating the functions of such proteins, can substantially facilitate the characterization of target proteins. Such molecules complement genetic studies, and allow time- and dose-dependent control of protein activity in biological systems. This can improve our understanding of physiological processes, give insights into the druggability of target proteins, and might finally result in the development of therapeutic compounds. In this review we summarize the current state of available inhibitors targeting key proteins in neutral lipid metabolism, with a focus on metabolic lipases, acyltransferases, and fatty-acid-binding proteins.
Subject(s)
Lipid Metabolism , Acyltransferases/metabolism , Fatty Acid-Binding Proteins/metabolism , Lipase/metabolismABSTRACT
The protein G0/G1 switch gene 2 (G0S2) is a small basic protein that functions as an endogenous inhibitor of adipose triglyceride lipase (ATGL), a key enzyme in intracellular lipolysis. In this study, we identified a short sequence covering residues Lys-20 to Ala-52 in G0S2 that is still fully capable of inhibiting mouse and human ATGL. We found that a synthetic peptide corresponding to this region inhibits ATGL in a noncompetitive manner in the nanomolar range. This peptide is highly selective for ATGL and does not inhibit other lipases, including hormone-sensitive lipase, monoacylglycerol lipase, lipoprotein lipase, and patatin domain-containing phospholipases 6 and 7. Because increased lipolysis is linked to the development of metabolic disorders, the inhibition of ATGL by G0S2-derived peptides may represent a novel therapeutic tool to modulate lipolysis.
Subject(s)
Cell Cycle Proteins/metabolism , Lipase/antagonists & inhibitors , Peptides/pharmacology , Recombinant Proteins/metabolism , 1-Acylglycerol-3-Phosphate O-Acyltransferase/antagonists & inhibitors , 1-Acylglycerol-3-Phosphate O-Acyltransferase/genetics , 1-Acylglycerol-3-Phosphate O-Acyltransferase/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Chlorocebus aethiops , Dose-Response Relationship, Drug , Humans , Lipase/genetics , Lipase/metabolism , Mice, Knockout , Molecular Sequence Data , Peptides/genetics , Recombinant Proteins/chemistryABSTRACT
Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.
Subject(s)
1-Acylglycerol-3-Phosphate O-Acyltransferase/metabolism , Lipase/metabolism , Lipid Droplets/enzymology , Viral Core Proteins/physiology , Animals , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , HEK293 Cells , Humans , Hydrolysis , Lipid Metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NIH 3T3 Cells , Triglycerides/metabolismABSTRACT
In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120-220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120-220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.
Subject(s)
Adipocytes/enzymology , Insulin Resistance , Lipase/metabolism , Lipolysis/physiology , Proteins/physiology , 3T3-L1 Cells , Adipocytes/drug effects , Adipocytes/metabolism , Animals , Apoptosis Regulatory Proteins , Humans , Insulin/metabolism , Insulin/pharmacology , Mice , Mice, Knockout , Phosphorylation , Protein Binding , Proteins/genetics , Signal Transduction , Triglycerides/metabolismABSTRACT
Systemic knockout of adipose triglyceride lipase (ATGL), the pivotal enzyme of triglyceride lipolysis, results in a murine phenotype that is characterized by progredient cardiac steatosis and severe heart failure. Since cardiac and vascular dysfunction have been closely related in numerous studies we investigated endothelium-dependent and -independent vessel function of ATGL knockout mice. Aortic relaxation studies and Langendorff perfusion experiments of isolated hearts showed that ATGL knockout mice suffer from pronounced micro- and macrovascular endothelial dysfunction. Experiments with agonists directly targeting vascular smooth muscle cells revealed the functional integrity of the smooth muscle cell layer. Loss of vascular reactivity was restored ~50% upon treatment of ATGL knockout mice with the PPARα agonist Wy14,643, indicating that this phenomenon is partly a consequence of impaired cardiac contractility. Biochemical analysis revealed that aortic endothelial NO synthase expression and activity were significantly reduced in ATGL deficiency. Enzyme activity was fully restored in ATGL mice treated with the PPARα agonist. Biochemical analysis of perivascular adipose tissue demonstrated that ATGL knockout mice suffer from perivascular inflammatory oxidative stress which occurs independent of cardiac dysfunction and might contribute to vascular defects. Our results reveal a hitherto unrecognized link between disturbed lipid metabolism, obesity and cardiovascular disease.
Subject(s)
Heart Failure/pathology , Lipase/genetics , Lipid Metabolism/genetics , Obesity/genetics , Triglycerides/metabolism , Adipose Tissue/enzymology , Adipose Tissue/metabolism , Animals , Disease Models, Animal , Gene Expression Regulation, Enzymologic , Heart Failure/enzymology , Humans , Lipase/biosynthesis , Lipase/metabolism , Mice , Mice, Knockout , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Nitric Oxide Synthase/biosynthesis , Obesity/enzymology , Obesity/pathology , Organ Culture Techniques , Oxidative Stress , PPAR alpha/genetics , PPAR alpha/metabolismABSTRACT
BACKGROUND & AIMS: Adipose tissue (AT)-derived fatty acids (FAs) are utilized for hepatic triacylglycerol (TG) generation upon fasting. However, their potential impact as signaling molecules is not established. Herein we examined the role of exogenous AT-derived FAs in the regulation of hepatic gene expression by investigating mice with a defect in AT-derived FA supply to the liver. METHODS: Plasma FA levels, tissue TG hydrolytic activities and lipid content were determined in mice lacking the lipase co-activator comparative gene identification-58 (CGI-58) selectively in AT (CGI-58-ATko) applying standard protocols. Hepatic expression of lipases, FA oxidative genes, transcription factors, ER stress markers, hormones and cytokines were determined by qRT-PCR, Western blotting and ELISA. RESULTS: Impaired AT-derived FA supply upon fasting of CGI-58-ATko mice causes a marked defect in liver PPARα-signaling and nuclear CREBH translocation. This severely reduced the expression of respective target genes such as the ATGL inhibitor G0/G1 switch gene-2 (G0S2) and the endocrine metabolic regulator FGF21. These changes could be reversed by lipid administration and raising plasma FA levels. Impaired AT-lipolysis failed to induce hepatic G0S2 expression in fasted CGI-58-ATko mice leading to enhanced ATGL-mediated TG-breakdown strongly reducing hepatic TG deposition. On high fat diet, impaired AT-lipolysis counteracts hepatic TG accumulation and liver stress linked to improved systemic insulin sensitivity. CONCLUSIONS: AT-derived FAs are a critical regulator of hepatic fasting gene expression required for the induction of G0S2-expression in the liver to control hepatic TG-breakdown. Interfering with AT-lipolysis or hepatic G0S2 expression represents an effective strategy for the treatment of hepatic steatosis.
Subject(s)
Adipose Tissue/metabolism , Fasting/metabolism , Fatty Acids/metabolism , Fatty Liver/genetics , Fibroblast Growth Factors/genetics , Gene Expression Regulation , Liver/metabolism , Animals , Blotting, Western , Diet, High-Fat/adverse effects , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Fatty Liver/metabolism , Fatty Liver/pathology , Fibroblast Growth Factors/biosynthesis , Genes, Switch , Liver/ultrastructure , Mice , Mice, Transgenic , Microscopy, Electron , RNA/genetics , Real-Time Polymerase Chain ReactionABSTRACT
Adipose triglyceride lipase (ATGL) is rate limiting in the mobilization of fatty acids from cellular triglyceride stores. This central role in lipolysis marks ATGL as an interesting pharmacological target as deregulated fatty acid metabolism is closely linked to dyslipidemic and metabolic disorders. Here we report on the development and characterization of a small-molecule inhibitor of ATGL. Atglistatin is selective for ATGL and reduces fatty acid mobilization in vitro and in vivo.
Subject(s)
Lipase/antagonists & inhibitors , Lipase/metabolism , Phenylurea Compounds/pharmacology , Adipose Tissue, White , Animals , Gene Expression Regulation, Enzymologic , Inhibitory Concentration 50 , Lipase/genetics , Mice , Mice, Knockout , Molecular StructureABSTRACT
Adipose triglyceride lipase (ATGL) catalyzes the degradation of cellular triacylglycerol stores and strongly determines the concentration of circulating fatty acids (FAs). High serum FA levels are causally linked to the development of insulin resistance and impaired glucose tolerance, which eventually progresses to overt type 2 diabetes. ATGL-specific inhibitors could be used to lower circulating FAs, which can counteract the development of insulin resistance. In this article, we report about structure-activity relationship (SAR) studies of small molecule inhibitors of ATGL based on a hydrazone chemotype. The SAR indicated that the binding pocket of ATGL requests rather linear compounds without bulky substituents. The best inhibitor showed an IC50=10µM in an assay with COS7-cell lysate overexpressing murine ATGL.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydrazones/chemistry , Hydrazones/pharmacology , Lipase/antagonists & inhibitors , Animals , COS Cells , Chlorocebus aethiops , Enzyme Inhibitors/chemical synthesis , Hydrazones/chemical synthesis , Lipase/metabolism , Lipid Metabolism/drug effects , Mice , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Triglycerides/metabolismABSTRACT
NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.
Subject(s)
Acetyltransferases/metabolism , Adipocytes, Brown/metabolism , Energy Metabolism , Lipid Metabolism , Acetyl Coenzyme A/metabolism , Acetyltransferases/deficiency , Acetyltransferases/genetics , Adipocytes, Brown/cytology , Adipocytes, Brown/enzymology , Adipogenesis , Animals , Cell Cycle Proteins/metabolism , Gene Expression Regulation, Enzymologic , Gene Knockout Techniques , Gene Silencing , Humans , Ion Channels/metabolism , Kinetics , Male , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Size , PPAR alpha/metabolism , Phenotype , Phosphoproteins/metabolism , Protein Kinases/genetics , Protein Transport , Uncoupling Protein 1 , Up-RegulationABSTRACT
BACKGROUND: Cancer-associated cachexia (CAC) is a debilitating syndrome associated with poor quality of life and reduced life expectancy of cancer patients. CAC is characterized by unintended body weight reduction due to muscle and adipose tissue loss. A major hallmark of CAC is systemic inflammation. Several non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested for CAC treatment, yet no single medication has proven reliable. R-ketorolac (RK) is the R-enantiomer of a commonly used NSAID. The effect of RK on CAC has not yet been evaluated. METHODS: Ten- to 11-week-old mice were inoculated with C26 or CHX207 cancer cells or vehicle control (phosphate-buffered saline [PBS]). After cachexia onset, 2 mg/kg RK or PBS was administered daily by oral gavage. Body weight, food intake and tumour size were continuously measured. At study endpoints, blood was drawn, mice were sacrificed and tissues were excised. Immune cell abundance was analysed using a Cytek® Aurora spectral flow cytometer. Cyclooxygenase (COX) activity was determined in lung homogenates using a fluorometric kit. Muscle tissues were analysed for mRNA and protein expression by quantitative real-time PCR and western blotting analysis, respectively. Muscle fibre size was determined on histological slides after haematoxylin/eosin staining. RESULTS: Ten-day survival rate of C26-bearing animals was 10% while RK treatment resulted in a 100% survival rate (P = 0.0009). Chemotherapy resulted in a 10% survival rate 14 days after treatment initiation, but all mice survived upon co-medication with RK and cyclophosphamide (P = 0.0001). Increased survival was associated with a protection from body weight loss in C26 (-0.61 ± 1.82 vs. -4.48 ± 2.0 g, P = 0.0004) and CHX207 (-0.49 ± 0.33 vs. -2.49 ± 0.93 g, P = 0.0003) tumour-bearing mice treated with RK, compared with untreated mice. RK ameliorated musculus quadriceps (-1.7 ± 7.1% vs. -27.8 ± 8.3%, P = 0.0007) and gonadal white adipose tissue (-18.8 ± 49% vs. -69 ± 15.6%, P = 0.094) loss in tumour-bearing mice, compared with untreated mice. Mechanistically, RK reduced circulating interleukin-6 (IL-6) concentrations from 334 ± 151 to 164 ± 123 pg/mL (P = 0.047) in C26 and from 93 ± 39 to 35 ± 6 pg/mL (P = 0.0053) in CHX207 tumour-bearing mice. Moreover, RK protected mice from cancer-induced T-lymphopenia (+1.8 ± 42% vs. -49.2 ± 12.1% in treated vs. untreated mice, respectively). RK was ineffective in ameliorating CAC in thymus-deficient nude mice, indicating that the beneficial effect of RK depends on T-cells. CONCLUSIONS: RK improved T-lymphopenia and decreased systemic IL-6 concentrations, resulting in alleviation of cachexia and increased survival of cachexigenic tumour-bearing mice, even under chemotherapy and independent of COX inhibition. Considering its potential, we propose that the use of RK should be investigated in patients suffering from CAC.