ABSTRACT
During cognitive efforts mediated by local neuronal networks, approximately 20% of additional energy is required; this is mediated by chemical messengers such as noradrenaline (NA). NA targets astroglial aerobic glycolysis, the hallmark of which is the end product l-lactate, a fuel for neurons. Biochemical studies have revealed that astrocytes exhibit a prominent glycogen shunt, in which a portion of d-glucose molecules entering the cytoplasm is transiently incorporated into glycogen, a buffer and source of d-glucose during increased energy demand. Here, we studied single astrocytes by measuring cytosolic L-lactate ([lac]i ) with the FRET nanosensor Laconic. We examined whether NA-induced increase in [lac]i is influenced by: (a) 2-deoxy-d-glucose (2-DG, 3 mM), a molecule that enters the cytosol and inhibits the glycolytic pathway; (b) 1,4-dideoxy-1,4-imino-d-arabinitol (DAB, 300 µM), a potent inhibitor of glycogen phosphorylase and glycogen degradation; and (c) 3-nitropropionic acid (3-NPA, 1 mM), an inhibitor of the Krebs cycle. The results of these pharmacological experiments revealed that d-glucose uptake is essential for the NA-induced increase in [lac]i , and that this exclusively arises from glycogen degradation, indicating that most, if not all, d-glucose molecules in NA-stimulated cells transit the glycogen shunt during glycolysis. Moreover, under the defined transmembrane d-glucose gradient, the glycolytic intermediates were not only used to produce l-lactate, but also to significantly support oxidative phosphorylation, as demonstrated by an elevation in [lac]i when Krebs cycle was inhibited. We conclude that l-lactate production via aerobic glycolysis is an essential energy pathway in NA-stimulated astrocytes; however, oxidative metabolism is important at rest.
Subject(s)
Astrocytes/metabolism , Glucose/metabolism , Glycogen/metabolism , Lactic Acid/biosynthesis , Norepinephrine/pharmacology , Animals , Animals, Newborn , Arabinose/pharmacology , Brain/metabolism , Citric Acid Cycle/drug effects , Deoxyglucose/pharmacology , Energy Metabolism , Fluorescence Resonance Energy Transfer , Imino Furanoses/pharmacology , Nitro Compounds/pharmacology , Oxidative Phosphorylation , Primary Cell Culture , Propionates/pharmacology , Rats , Rats, Wistar , Sugar Alcohols/pharmacology , TransfectionABSTRACT
Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolic free glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600µ. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only 56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with the one recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (ΔR) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p < 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose.
Subject(s)
Adipocytes/metabolism , Cell Differentiation/physiology , Cell Membrane Permeability/physiology , Cell Membrane/metabolism , Cytosol/metabolism , Fibroblasts/metabolism , Glucose/metabolism , 3T3-L1 Cells , Adipocytes/cytology , Adrenergic alpha-Agonists/pharmacology , Animals , Cell Differentiation/drug effects , Cell Membrane Permeability/drug effects , Enzyme Inhibitors/pharmacology , Epinephrine/pharmacology , Fibroblasts/cytology , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Iodoacetates/pharmacology , MiceABSTRACT
OBJECTIVES: GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. METHODS: We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. RESULTS: Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. CONCLUSIONS: These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.
Subject(s)
Deoxyglucose/pharmacology , Glycolysis/drug effects , Guanine Nucleotide Dissociation Inhibitors/genetics , Intellectual Disability/genetics , Memory Disorders/prevention & control , Animals , Brain/drug effects , Brain/metabolism , Cells, Cultured , Deoxyglucose/therapeutic use , Down-Regulation/drug effects , Glucose/metabolism , Guanine Nucleotide Dissociation Inhibitors/deficiency , Intellectual Disability/drug therapy , Intellectual Disability/metabolism , Intellectual Disability/pathology , Male , Maze Learning/drug effects , Memory/drug effects , Memory Disorders/genetics , Mice , Mice, KnockoutABSTRACT
Most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons and non-neuronal cells, including astrocytes, which metabolically support neurons with nutrients. Neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline. Activation of astroglial adrenergic receptors with noradrenaline triggers cAMP and Ca2+ signaling and augments aerobic glycolysis with production of lactate, an important neuronal energy fuel. Astrocytes with cytoplasmic TDP-43 inclusions can cause motor neuron death, however, whether astroglial metabolism and metabolic support of neurons is altered in astrocytes with TDP-43 inclusions, is unclear. We measured lipid droplet and glucose metabolisms in astrocytes expressing the inclusion-forming C-terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited a 3-fold increase in the accumulation of lipid droplets versus astrocytes expressing wild-type TDP-43, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increases in intracellular cAMP and Ca2+ levels were reduced by 35% and 31%, respectively, likely due to the downregulation of ß2-adrenergic receptors. Although noradrenaline triggered a similar increase in intracellular lactate levels in astrocytes with and without TDP-43 inclusions, the probability of activating aerobic glycolysis was facilitated by 1.6-fold in astrocytes with TDP-43 inclusions and lactate MCT1 transporters were downregulated. Thus, while in astrocytes with TDP-43 inclusions noradrenergic signaling is reduced, aerobic glycolysis and lipid droplet accumulation are facilitated, suggesting dysregulated astroglial metabolism and metabolic support of neurons in TDP-43-associated ALS and FTD.
Subject(s)
Astrocytes/metabolism , Calcium/metabolism , Cyclic AMP/metabolism , DNA-Binding Proteins/metabolism , Inclusion Bodies/metabolism , Norepinephrine/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Astrocytes/pathology , Cells, Cultured , Glycolysis , Humans , Inclusion Bodies/pathology , Rats, Wistar , Receptors, Adrenergic/metabolism , Signal TransductionABSTRACT
Causal relationship between sodium and hypertension has been proposed and various changes in Na+,K+-ATPase (sodium pump) activity have been described in established primary hypertension. A number of direct vascular effects of estradiol have been reported, including its impact on the regulation of sodium pump activity and vasomotor tone. The effects of estradiol involve the activation of multiple signaling cascades, including phosphatydil inositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44(MAPK)). In addition, some of the effects of estradiol have been linked to activity of cytosolic phospholipase A(2) (cPLA(2)). One possible cardioprotective mechanism of estradiol involves of the interaction between estradiol and the rennin-angiotensin system (RAS). Elevated circulating and tissue levels of angiotensin II (Ang II) have been implicated in the development of hypertension and heart failure. The aim of our investigation was to elucidate the signaling mechanisms employed by estradiol and Ang II in mediating sodium pump, in vascular smooth muscle cells (VSMC). The aim of our investigation was to elucidate the signaling mechanisms employed by estradiol and Ang II in mediating sodium pump activity/expression in VSMC, with particular emphasis on PI3K/cPLA(2)/p42/44(MAPK) signaling pathways. Our primary hypothesis is that estradiol stimulates sodium pump activity/expression in VSMC via PI3K/cPLA(2)/p42/44(MAPK) dependent mechanism and, that impaired estradiol-stimulated sodium pump activity/expression in hypertensive rodent models (i.e. SHR), Ang II-mediated vascular impairment of estradiol is related to a decrease ability of estradiol to stimulate the PI3K/cPLA(2)/p42/44(MAPK) signaling pathways. An important corollary to this hypothesis is that in hypertensive state (i.e. SHR rats) the decreasing in ACE enzyme activity and/or AT1 receptor expression caused by administration of estradiol is accompanying with abrogated ability of Ang II to decrease IRS-1/PI3K association, and consequent PI3K/cPLA(2)/p42/44(MAPK) activity and associated sodium pump activity/expression. A clear characterization of how Ang II attenuates estradiol signaling may lead to a better understanding of the molecular mechanism(s) underlying pathophysiological conditions such as hypertension and to understanding how certain pathophysiological situations affect sodium pump activity/expression in VSMC.
Subject(s)
Estradiol/physiology , Hypertension/metabolism , Models, Cardiovascular , Myocytes, Smooth Muscle/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Angiotensin II/metabolism , Animals , Aorta , Cells, Cultured , Humans , Male , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Rats , Rats, Wistar , Renin-Angiotensin System/physiologyABSTRACT
Dipeptidyl peptidase 4 (DPP4) is a transmembrane glycoprotein involved in protein degradation. Due to its action on incretins, which increase insulin secretion, DPP4 is considered a therapeutic target for type 2 diabetes. Here we have studied the role of single and combined effects of hypoxia and inactivity on the expression of DPP4 in human adipose tissue of 12 adult normal-weight males. Fat biopsies were obtained at baseline and after each of three experimental campaigns. The results revealed that in isolated human preadipocytes the expression of DPP4 was significantly increased by exposure of participants to hypoxia. Physical inactivity per se had no apparent effect on the DPP4 expression. It is concluded that DPP4 may be a marker to monitor indirectly tissue hypoxia, as occurs in obese subjects.
Subject(s)
Adipocytes/metabolism , Dipeptidyl Peptidase 4/metabolism , Hypoxia/metabolism , Adult , Cell Differentiation , Healthy Volunteers , Humans , Male , Young AdultABSTRACT
Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-N-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism.
ABSTRACT
Dipeptidyl peptidase 4 (DPP4), a transmembrane protein, has been identified in human adipose tissue and is considered to be associated with obesity-related type 2 diabetes. Since adipose tissue is relatively hypoxic in obese participants, we investigated the expression of DPP4 in human preadipocytes (hPA) and adipocytes in hypoxia, during differentiation and upon insulin stimulation. The results show that DPP4 is abundantly expressed in hPA but very sparsely in adipocytes. During differentiation in vitro, the expression of DPP4 in hPA is reduced on the addition of differentiation medium, indicating that this protein can be hPA marker. Long term hypoxia altered the expression of DPP4 in hPA. In in vitro hypoxic conditions the protease activity of shed DPP4 is reduced; however, in the presence of insulin, the increase in DPP4 expression is potentiated by hypoxia.
Subject(s)
Adipocytes/cytology , Cell Differentiation , Dipeptidyl Peptidase 4/metabolism , Adipose Tissue/metabolism , Cell Hypoxia , Cells, Cultured , Diabetes Mellitus, Type 2/complications , Gastrointestinal Tract/metabolism , Humans , Insulin/metabolism , Insulin Resistance , Microscopy, Confocal , Obesity/complications , Signal TransductionABSTRACT
Rosiglitazone (Rosi) improves insulin sensitivity and increases the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This involves the fusion of membrane-bound compartments with the plasma membrane, thus increasing the plasma membrane area. However, recent work has shown that in Rosi-pretreated 3T3-L1 adipocytes membrane area did not increase following insulin application, suggesting that the rates of exo- and endocytosis are balanced. Here we examined whether Rosi differentially affects the rates of exo- and endocytosis in 3T3-L1 adipocytes. The immunolabelling of GLUT4 revealed the 3.1-fold increase in PM-resident GLUT4 in Rosi-pretreated, insulin-stimulated cells. By monitoring cumulative exocytosis and endocytosis we found that in Rosi-pretreated cells insulin substantially stimulated the rate of exocytosis and to a similar extent also the rate of endocytosis. We conclude that Rosi-pretreatment balances insulin-stimulated exocytosis and endocytosis, which may prevent insulin-mediated adipocyte cell size increase.
Subject(s)
Adipocytes/metabolism , Endocytosis/drug effects , Exocytosis/drug effects , Insulin/pharmacology , Thiazolidinediones/pharmacology , 3T3-L1 Cells , Adipocytes/drug effects , Animals , Fluorescent Antibody Technique , Fluorometry , Glucose Transporter Type 4/metabolism , Insulin/metabolism , Insulin Resistance , Mice , Microscopy, Confocal , Rosiglitazone , Thiazolidinediones/metabolismABSTRACT
In this study we hypothesized that rosiglitazone, an antidiabetic high-affinity agonist for the peroxisome proliferator-activated receptor gamma, affects the plasma membrane (PM) turnover in single 3T3-L1 adipocytes. To study the PM turnover, the patch-clamp electrophysiological method was used to measure changes in membrane capacitance (Cm), a parameter linearly related to the PM area. Microscopy results show that the presence of rosiglitazone in the differentiating medium significantly increased the differentiation of 3T3-L1 adipocytes in cell culture, based on oil red O-stained area (11.4 +/- 1.2%) vs. controls (3.1 +/- 0.5%). Moreover, rosiglitazone treatment significantly reduced the size of single 3T3-L1 adipocytes; their average radius of 21.1 +/- 1.1 microm in controls was reduced to 17.5 +/- 0.5 microm in rosiglitazone-treated cells. Consistent with this, insulin application increased the rate of Cm increase to 2.34 +/- 0.10%/min, which was significantly different from controls (0.12 +/- 0.08%/min). However, pretreatment of cells with rosiglitazone prior to the treatment with insulin resulted in an attenuated rate of Cm increase. These data support the involvement of insulin in the modulation of membrane area and show that treatment by rosiglitazone reduced the insulin-mediated membrane area increase in 3T3-L1 adipocytes.