ABSTRACT
OBJECTIVE: Promoting thermogenesis in adipose tissue has been a promising strategy against obesity and related metabolic complications. We aimed to identify compounds that promote thermogenesis in adipocytes and to elucidate their functions and roles in metabolism. METHODS: To identify compounds that directly promote thermogenesis from a structurally diverse set of 4800 compounds, we utilized a cell-based platform for high-throughput screening that induces uncoupling protein 1 (Ucp1) expression in adipocytes. RESULTS: We identified one candidate compound that activates UCP1. Additional characterization of this compound revealed that it induced cellular thermogenesis in adipocytes with negligible cytotoxicity. In a subsequent diet-induced obesity model, mice treated with this compound exhibited a slower rate of weight gain, improved insulin sensitivity, and increased energy expenditure. Mechanistic studies have revealed that this compound increases mitochondrial biogenesis by elevating maximal respiration, which is partly mediated by the protein kinase A (PKA)-p38 mitogen-activated protein kinase (MAPK) signaling pathway. A further comprehensive genetic analysis of adipocytes treated with these compounds identified two novel UCP1-dependent thermogenic genes, potassium voltage-gated channel subfamily C member 2 (Kcnc2) and predicted gene 5627 (Gm5627). CONCLUSIONS: The identified compound can serve as a potential therapeutic drug for the treatment of obesity and its related metabolic disorders. Furthermore, our newly clarified thermogenic genes play an important role in UCP1-dependent thermogenesis in adipocytes.
Subject(s)
Insulin Resistance , Obesity , Uncoupling Protein 1 , Animals , Mice , Adipocytes/metabolism , Adipose Tissue/metabolism , Adipose Tissue, Brown/metabolism , Energy Metabolism , Obesity/complications , Obesity/drug therapy , Thermogenesis/physiology , Uncoupling Protein 1/antagonists & inhibitorsABSTRACT
Recent research focusing on brown adipose tissue (BAT) function emphasizes its importance in systemic metabolic homeostasis. We show here that genetic and pharmacological inhibition of the mevalonate pathway leads to reduced human and mouse brown adipocyte function in vitro and impaired adipose tissue browning in vivo. A retrospective analysis of a large patient cohort suggests an inverse correlation between statin use and active BAT in humans, while we show in a prospective clinical trial that fluvastatin reduces thermogenic gene expression in human BAT. We identify geranylgeranyl pyrophosphate as the key mevalonate pathway intermediate driving adipocyte browning in vitro and in vivo, whose effects are mediated by geranylgeranyltransferases (GGTases), enzymes catalyzing geranylgeranylation of small GTP-binding proteins, thereby regulating YAP1/TAZ signaling through F-actin modulation. Conversely, adipocyte-specific ablation of GGTase I leads to impaired adipocyte browning, reduced energy expenditure, and glucose intolerance under obesogenic conditions, highlighting the importance of this pathway in modulating brown adipocyte functionality and systemic metabolism.
Subject(s)
Adipocytes, Brown/drug effects , Mevalonic Acid/pharmacology , Protein Prenylation/drug effects , Uncoupling Protein 1/antagonists & inhibitors , Adipocytes, Brown/metabolism , Adolescent , Adult , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Humans , Male , Mice , Mice, Inbred Strains , Middle Aged , Uncoupling Protein 1/metabolism , Young AdultABSTRACT
Glycerol-3-phosphate is an excellent substrate for FAD-linked mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) in brown adipose tissue mitochondria and is regularly used as the primary substrate to measure oxygen consumption and reactive oxygen consumption by these mitochondria. mGPDH converts cytosolic glycerol-3-phosphate to dihydroxyacetone phosphate, feeding electrons directly from the cytosolic side of the mitochondrial inner membrane to the CoQ-pool within the inner membrane. mGPDH activity is allosterically activated by calcium, and when calcium chelators are present in the mitochondrial preparation medium and/or experimental incubation medium, calcium must be added to insure maximal mGPDH activity. It was demonstrated that in isolated brown adipose tissue mitochondria (1) mGPDH enzyme activity is maximal at free calcium ion concentrations in the 350 nM-1 µM range, (2) that ROS production also peaks in the 10-100 nM range in the presence of a UCP1 inhibitory ligand (GDP) but wanes with further increasing calcium concentration, and (3) that oxygen consumption rates peak in the 10-100 nM range with rates being maintained at higher calcium concentrations. This article provides easy-to-follow protocols to facilitate the measurement of mGPDH-dependent UCP1 activity in the presence of calcium for isolated brown adipose tissue mitochondria.
Subject(s)
Adipose Tissue, Brown/cytology , Enzyme Assays/methods , Glycerolphosphate Dehydrogenase/metabolism , Mitochondria/metabolism , Uncoupling Protein 1/analysis , Animals , Calcium/metabolism , Calcium Chelating Agents/pharmacology , Cations, Divalent/metabolism , Enzyme Assays/instrumentation , Female , Guanosine Diphosphate/pharmacology , Male , Mitochondria/drug effects , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Uncoupling Protein 1/antagonists & inhibitors , Uncoupling Protein 1/metabolismABSTRACT
Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.
Subject(s)
Adenine Nucleotides/pharmacology , Phosphates/pharmacology , Uncoupling Protein 1/antagonists & inhibitors , Uncoupling Protein 3/antagonists & inhibitors , Animals , Arginine/chemistry , Binding, Competitive , Fatty Acids/pharmacology , Lipid Bilayers , Liposomes , Mice , Microscopy, Atomic Force , Mutagenesis, Site-Directed , Protons , Recombinant Proteins/drug effects , Uncoupling Protein 1/genetics , Uncoupling Protein 3/geneticsABSTRACT
Apelin is an adipose tissue derived peptidergic hormone. In this study, 40 male Sprague-Dawley rats were used (four groups; n = 10). Apelin-13 at three different dosages (1, 5 and 50 µg/kg) was given intraperitoneally while the control group received vehicle the same route for a period of 14 days. In results, apelin-13 caused significant decreases in serum testosterone, luteinizing hormone and follicle-stimulating hormone levels (p < 0.05). Administration of apelin-13 significantly increased body weights, food intake, serum low-density lipoprotein and total cholesterol levels (p < 0.05), but caused significant decreases in high-density lipoprotein levels (p < 0.05). Serum glucose and triglyceride levels were not significantly altered by apelin-13 administration. Significant decreases in both uncoupling protein (UCP)-1 levels in the white and brown adipose tissues and UCP-3 levels in the biceps muscle (p < 0.05) were noted. The findings of the study suggest that apelin-13 may not only lead to obesity by increasing body weight but also cause infertility by suppressing reproductive hormones.
Subject(s)
Energy Intake/drug effects , Energy Metabolism/drug effects , Feeding Behavior/drug effects , Hypercholesterolemia/chemically induced , Infertility, Male/chemically induced , Intercellular Signaling Peptides and Proteins/toxicity , Overweight/chemically induced , Adipose Tissue, Brown/drug effects , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/drug effects , Adipose Tissue, White/metabolism , Animals , Dose-Response Relationship, Drug , Gonadotropins, Pituitary/antagonists & inhibitors , Gonadotropins, Pituitary/blood , Hypercholesterolemia/blood , Hypercholesterolemia/metabolism , Infertility, Male/blood , Infertility, Male/metabolism , Injections, Intraperitoneal , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Overweight/blood , Overweight/metabolism , Random Allocation , Rats, Sprague-Dawley , Testosterone/antagonists & inhibitors , Testosterone/blood , Toxicity Tests, Chronic , Uncoupling Protein 1/antagonists & inhibitors , Uncoupling Protein 1/genetics , Uncoupling Protein 1/metabolism , Uncoupling Protein 3/antagonists & inhibitors , Uncoupling Protein 3/genetics , Uncoupling Protein 3/metabolism , Weight Gain/drug effectsABSTRACT
Regarding the enormous interest in brown and brite/beige adipose tissue in the context of metabolic disease, reliable quantification of thermogenesis in these adipocytes is a central issue. This requires an assay specific for uncoupling protein 1 (UCP1) mediated thermogenesis in adherent intact cells. In a recent study we identified a major pitfall associated with established procedures generally applied for this purpose. Meaningful respirometry of UCP1-mediated thermogenesis imperatively requires activation of UCP1 and control over free fatty acid levels. By comparison of respiration profiles of wild-type (WT) and UCP1 knock-out (KO) cells we reproducibly quantified the thermogenic capacity enabled by UCP1 in both brown and brite adipocytes. Employing this protocol, we demonstrated that (1) brite adipocytes display a similar thermogenic capacity as classical brown adipocytes, (2) variations in brite adipogenesis known for inbred mouse strains are associated with differential capacities for thermogenesis in these cells, and (3) adipose triglyceride lipase (ATGL) activity is required for UCP1 activation in intact cells. We here further refined our cell-based respirometry assay by implementation of two strategies to inhibit UCP1 in WT cells. First, we employed the purine nucleotide guanosine diphosphate (GDP) to directly quantify the fraction of thermogenesis enabled by UCP1 activity. Second, applying siRNA mediated knockdown of UCP1 and ATGL we demonstrated the feasibility of this technology to study the functional relevance of candidate genes for thermogenesis in brown and brite adipocytes.