Dietary Fiber Modulates the Release of Gut Bacterial Products Preventing Cognitive Decline in an Alzheimer's Mouse Model.

Fiber intake is associated with a lower risk for Alzheimer´s disease (AD) in older adults. Intake of plant-based diets rich in soluble fiber promotes the production of short-chain fatty acids (SCFAs: butyrate, acetate, propionate) by gut bacteria. Butyrate administration has antiinflammatory actions, but propionate promotes neuroinflammation. In AD patients, gut microbiota dysbiosis is a common feature even in the prodromal stages of the disease. It is unclear whether the neuroprotective effects of fiber intake rely on gut microbiota modifications and specific actions of SCFAs in brain cells. Here, we show that restoration of the gut microbiota dysbiosis through the intake of soluble fiber resulted in lower propionate and higher butyrate production, reduced astrocyte activation and improved cognitive function in 6-month-old male APP/PS1 mice. The neuroprotective effects were lost in antibiotic-treated mice. Moreover, propionate promoted higher glycolysis and mitochondrial respiration in astrocytes, while butyrate induced a more quiescent metabolism. Therefore, fiber intake neuroprotective action depends on the modulation of butyrate/propionate production by gut bacteria. Our data further support and provide a mechanism to explain the beneficial effects of dietary interventions rich in soluble fiber to prevent dementia and AD. Fiber intake restored the concentration of propionate and butyrate by modulating the composition of gut microbiota in male transgenic (Tg) mice with Alzheimer´s disease. Gut dysbiosis was associated with intestinal damage and high propionate levels in control diet fed-Tg mice. Fiber-rich diet restored intestinal integrity and promoted the abundance of butyrate-producing bacteria. Butyrate concentration was associated with better cognitive performance in fiber-fed Tg mice. A fiber-rich diet may prevent the development of a dysbiotic microbiome and the related cognitive dysfunction in people at risk of developing Alzheimer´s disease.

Bidirectional astrocytic GLUT1 activation by elevated extracellular K.

The acute rise in interstitial K+ that accompanies neural activity couples the energy demand of neurons to the metabolism of astrocytes. The effects of elevated K+ on astrocytes include activation of aerobic glycolysis, inhibition of mitochondrial respiration and the release of lactate. Using a genetically encoded FRET glucose sensor and a novel protocol based on 3-O-methylglucose trans-acceleration and numerical simulation of glucose dynamics, we report that extracellular K+ is also a potent and reversible modulator of the astrocytic glucose transporter GLUT1. In cultured mouse astrocytes, the stimulatory effect developed within seconds, engaged both the influx and efflux modes of the transporter, and was detected even at 1 mM incremental K+ . The modulation of GLUT1 explains how astrocytes are able to maintain their glucose pool in the face of strong glycolysis stimulation. We propose that the stimulation of GLUT1 by K+ supports the production of lactate by astrocytes and the timely delivery of glucose to active neurons.

Aerobic Glycolysis in the Brain: Warburg and Crabtree Contra Pasteur.

Information processing is onerous. Curiously, active brain tissue does not fully oxidize glucose and instead generates a local surplus of lactate, a phenomenon termed aerobic glycolysis. Why engage in inefficient ATP production by glycolysis when energy demand is highest and oxygen is plentiful? Aerobic glycolysis is associated to classic biochemical effects known by the names of Pasteur, Warburg and Crabtree. Here we discuss these three interdependent phenomena in brain cells, in light of high-resolution data of neuronal and astrocytic metabolism in culture, tissue slices and in vivo, acquired with genetically-encoded fluorescent sensors. These sensors are synthetic proteins that can be targeted to specific cell types and subcellular compartments, which change their fluorescence in response to variations in metabolite concentration. A major site of acute aerobic glycolysis is the astrocyte. In this cell, a Crabtree effect triggered by K+ coincides with a Warburg effect mediated by NO, superimposed on a slower longer-lasting Warburg effect caused by glutamate and possibly by NH4+. The compounded outcome is that more fuel (lactate) and more oxygen are made available to neurons, on demand. Meanwhile neurons consume both glucose and lactate, maintaining a strict balance between glycolysis and respiration, commanded by the Na+ pump. We conclude that activity-dependent Warburg and Crabtree effects in brain tissue, and the resulting aerobic glycolysis, do not reflect inefficient energy generation but the marshalling of astrocytes for the purpose of neuronal ATP generation. It remains to be seen whether neurons contribute to aerobic glycolysis under physiological conditions.

Neuronal control of astrocytic respiration through a variant of the Crabtree effect.

Aerobic glycolysis is a phenomenon that in the long term contributes to synaptic formation and growth, is reduced by normal aging, and correlates with amyloid beta deposition. Aerobic glycolysis starts within seconds of neural activity and it is not obvious why energetic efficiency should be compromised precisely when energy demand is highest. Using genetically encoded FRET nanosensors and real-time oxygen measurements in culture and in hippocampal slices, we show here that astrocytes respond to physiological extracellular K+ with an acute rise in cytosolic ATP and a parallel inhibition of oxygen consumption, explained by glycolytic stimulation via the Na+-bicarbonate cotransporter NBCe1. This control of mitochondrial respiration via glycolysis modulation is reminiscent of a phenomenon previously described in proliferating cells, known as the Crabtree effect. Fast brain aerobic glycolysis may be interpreted as a strategy whereby neurons manipulate neighboring astrocytes to obtain oxygen, thus maximizing information processing.

Fluid Brain Glycolysis: Limits, Speed, Location, Moonlighting, and the Fates of Glycogen and Lactate.

Glycolysis is the core of intermediate metabolism, an ancient pathway discovered in the heydays of classic biochemistry. A hundred years later, it remains a matter of active research, clinical interest and is not devoid of controversy. This review examines topical aspects of glycolysis in the brain, a tissue characterized by an extreme dependence on glucose. The limits of glycolysis are reviewed in terms of flux control by glucose transporters, intercellular lactate shuttling and activity-dependent glycolysis in astrocytes and neurons. What is the site of glycogen mobilization and aerobic glycolysis in brain tissue? We scrutinize the pervasive notions that glycolysis is fast and that catalysis is channeled through supramolecular assemblies. In brain tissue, most glycolytic enzymes are catalytically silent. What then is their function?

Proton Fall or Bicarbonate Rise: GLYCOLYTIC RATE IN MOUSE ASTROCYTES IS PAVED BY INTRACELLULAR ALKALINIZATION.

Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types.

Near-critical GLUT1 and Neurodegeneration.

Recent articles have drawn renewed attention to the housekeeping glucose transporter GLUT1 and its possible involvement in neurodegenerative diseases. Here we provide an updated analysis of brain glucose transport and the cellular mechanisms involved in its acute modulation during synaptic activity. We discuss how the architecture of the blood-brain barrier and the low concentration of glucose within neurons combine to make endothelial/glial GLUT1 the master controller of neuronal glucose utilization, while the regulatory role of the neuronal glucose transporter GLUT3 emerges as secondary. The near-critical condition of glucose dynamics in the brain suggests that subtle deficits in GLUT1 function or its activity-dependent control by neurons may contribute to neurodegeneration. © 2017 Wiley Periodicals, Inc.

Channel-mediated lactate release by K⁺-stimulated astrocytes.

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

The electrogenic sodium bicarbonate cotransporter NBCe1 is a high-affinity bicarbonate carrier in cortical astrocytes.

The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) is a robust regulator of intracellular H(+) and a significant base carrier in many cell types. Using wild-type (WT) and NBCe1-deficient (NBC-KO) mice, we have studied the role of NBCe1 in cortical astrocytes in culture and in situ by monitoring intracellular H(+) using the H(+)-sensitive dye BCECF [2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein] in wide-field and confocal microscopy. Adding 0.1-3 mm HCO3(-) to an O2-gassed, HEPES-buffered saline solution lowered the intracellular H(+) concentration with a Km of 0.65 mm HCO3(-) in WT astrocytes, but slowly raised [H(+)]i in NBCe1-KO astrocytes. Human NBCe1 heterologously expressed in Xenopus oocytes could be activated by adding 1-3 mm HCO3(-), and even by residual HCO3(-) in a nominally CO2/HCO3(-)-free saline solution. Our results demonstrate a surprisingly high apparent bicarbonate sensitivity mediated by NBCe1 in cortical astrocytes, suggesting that NBCe1 may operate over a wide bicarbonate concentration in these cells.

Higher transport and metabolism of glucose in astrocytes compared with neurons: a multiphoton study of hippocampal and cerebellar tissue slices.

Glucose is the most important energy substrate for the brain, and its cellular distribution is a subject of great current interest. We have employed fluorescent glucose probes, the 2-deoxy-D-glucose derivates 6- and 2-([N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose) (2-NBDG), to measure transport and metabolism of glucose in acute slices of mouse hippocampus and cerebellum. In the hippocampus, 6-NBDG, which is not metabolized and hence indicates glucose transport, was taken up faster in astrocyte-rich layers (Stratum radiatum [S.r.], Stratum oriens [S.o.]) than in pyramidal cells. Metabolizable 2-NBDG showed larger signals in S.r. and S.o. than in Stratum pyramidale, suggesting faster glucose utilization rate in the astrocyte versus the neuronal compartment. Similarly, we found higher uptake and temperature-sensitive metabolism of 2-NBDG in Bergmann glia when compared with adjacent Purkinje neurons of cerebellar slices. A comparison between 6-NBDG transport and glucose transport in cultured cells using a fluorescence resonance energy transfer nanosensor showed that relative to glucose, 6-NBDG is transported better by neurons than by astrocytes. These results indicate that the preferential transport and metabolism of glucose by glial cells versus neurons proposed for the hippocampus and cerebellum by ourselves (in vitro) and for the barrel cortex by Chuquet et al. (in vivo) is more pronounced than anticipated.

NBCe1 mediates the acute stimulation of astrocytic glycolysis by extracellular K+.

Excitatory synaptic transmission stimulates brain tissue glycolysis. This phenomenon is the signal detected in FDG-PET imaging and, through enhanced lactate production, is also thought to contribute to the fMRI signal. Using a method based on Förster resonance energy transfer in mouse astrocytes, we have recently observed that a small rise in extracellular K(+) can stimulate glycolysis by >300% within seconds. The K(+) response was blocked by ouabain, but intracellular engagement of the Na(+)/K(+) ATPase pump with Na(+) was ineffective, suggesting that the canonical feedback regulatory pathway involving the Na(+) pump and ATP depletion is only permissive and that a second mechanism is involved. Because of their predominant K(+) permeability and high expression of the electrogenic Na(+)/HCO(3)(-) cotransporter NBCe1, astrocytes respond to a rise in extracellular K(+) with plasma membrane depolarization and intracellular alkalinization. In the present article, we show that a fast glycolytic response can be elicited independently of K(+) by plasma membrane depolarization or by intracellular alkalinization. The glycolytic response to K(+) was absent in astrocytes from NBCe1 null mice (Slc4a4) and was blocked by functional or pharmacological inhibition of the NBCe1. Hippocampal neurons acquired K(+)-sensitive glycolysis upon heterologous NBCe1 expression. The phenomenon could also be reconstituted in HEK293 cells by coexpression of the NBCe1 and a constitutively open K(+) channel. We conclude that the NBCe1 is a key element in a feedforward mechanism linking excitatory synaptic transmission to fast modulation of glycolysis in astrocytes.

Fast and reversible stimulation of astrocytic glycolysis by K+ and a delayed and persistent effect of glutamate.

Synaptic activity is followed within seconds by a local surge in lactate concentration, a phenomenon that underlies functional magnetic resonance imaging and whose causal mechanisms are unclear, partly because of the limited spatiotemporal resolution of standard measurement techniques. Using a novel Förster resonance energy transfer-based method that allows real-time measurement of the glycolytic rate in single cells, we have studied mouse astrocytes in search for the mechanisms responsible for the lactate surge. Consistent with previous measurements with isotopic 2-deoxyglucose, glutamate was observed to stimulate glycolysis in cultured astrocytes, but the response appeared only after a lag period of several minutes. Na(+) overloads elicited by engagement of the Na(+)-glutamate cotransporter with d-aspartate or application of the Na(+) ionophore gramicidin also failed to stimulate glycolysis in the short term. In marked contrast, K(+) stimulated astrocytic glycolysis by fourfold within seconds, an effect that was observed at low millimolar concentrations and was also present in organotypic hippocampal slices. After removal of the agonists, the stimulation by K(+) ended immediately but the stimulation by glutamate persisted unabated for >20 min. Both stimulations required an active Na(+)/K(+) ATPase pump. By showing that small rises in extracellular K(+) mediate short-term, reversible modulation of astrocytic glycolysis and that glutamate plays a long-term effect and leaves a metabolic trace, these results support the view that astrocytes contribute to the lactate surge that accompanies synaptic activity and underscore the role of these cells in neurometabolic and neurovascular coupling.

Inhibition of the sodium-dependent HCO3- transporter SLC4A4, produces a cystic fibrosis-like airway disease phenotype.

Bicarbonate secretion is a fundamental process involved in maintaining acid-base homeostasis. Disruption of bicarbonate entry into airway lumen, as has been observed in cystic fibrosis, produces several defects in lung function due to thick mucus accumulation. Bicarbonate is critical for correct mucin deployment and there is increasing interest in understanding its role in airway physiology, particularly in the initiation of lung disease in children affected by cystic fibrosis, in the absence of detectable bacterial infection. The current model of anion secretion in mammalian airways consists of CFTR and TMEM16A as apical anion exit channels, with limited capacity for bicarbonate transport compared to chloride. However, both channels can couple to SLC26A4 anion exchanger to maximise bicarbonate secretion. Nevertheless, current models lack any details about the identity of the basolateral protein(s) responsible for bicarbonate uptake into airway epithelial cells. We report herein that the electrogenic, sodium-dependent, bicarbonate cotransporter, SLC4A4, is expressed in the basolateral membrane of human and mouse airways, and that it's pharmacological inhibition or genetic silencing reduces bicarbonate secretion. In fully differentiated primary human airway cells cultures, SLC4A4 inhibition induced an acidification of the airways surface liquid and markedly reduced the capacity of cells to recover from an acid load. Studies in the Slc4a4-null mice revealed a previously unreported lung phenotype, characterized by mucus accumulation and reduced mucociliary clearance. Collectively, our results demonstrate that the reduction of SLC4A4 function induced a CF-like phenotype, even when chloride secretion remained intact, highlighting the important role SLC4A4 plays in bicarbonate secretion and mammalian airway function.

CO2 signaling mediates neurovascular coupling in the cerebral cortex.

Neurovascular coupling is a fundamental brain mechanism that regulates local cerebral blood flow (CBF) in response to changes in neuronal activity. Functional imaging techniques are commonly used to record these changes in CBF as a proxy of neuronal activity to study the human brain. However, the mechanisms of neurovascular coupling remain incompletely understood. Here we show in experimental animal models (laboratory rats and mice) that the neuronal activity-dependent increases in local CBF in the somatosensory cortex are prevented by saturation of the CO2-sensitive vasodilatory brain mechanism with surplus of exogenous CO2 or disruption of brain CO2/HCO3- transport by genetic knockdown of electrogenic sodium-bicarbonate cotransporter 1 (NBCe1) expression in astrocytes. A systematic review of the literature data shows that CO2 and increased neuronal activity recruit the same vasodilatory signaling pathways. These results and analysis suggest that CO2 mediates signaling between neurons and the cerebral vasculature to regulate brain blood flow in accord with changes in the neuronal activity.

Single-Fluorophore Indicator to Explore Cellular and Sub-cellular Lactate Dynamics.

Lactate is an energy substrate and an intercellular signal, which can be monitored in intact cells with the genetically encoded FRET indicator Laconic. However, the structural complexity, need for sophisticated equipment, and relatively small fluorescent change limit the use of FRET indicators for subcellular targeting and development of high-throughput screening methodologies. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed a single-fluorophore indicator for lactate, CanlonicSF. This indicator exhibits a maximal fluorescence change of 200% and a KD of ∼300 µM. The fluorescence is not affected by other monocarboxylates. The lactate indicator was not significantly affected by Ca2+ at the physiological concentrations prevailing in the cytosol, endoplasmic reticulum, and extracellular space, but was affected by Ca2+ in the low micromolar range. Targeting the indicator to the endoplasmic reticulum revealed for the first time sub-cellular lactate dynamics. Its improved lactate-induced fluorescence response permitted the development of a multiwell plate assay to screen for inhibitors of the monocarboxylate transporters MCTs, a pharmaceutical target for cancer and inflammation. The functionality of the indicator in living tissue was demonstrated in the brain of Drosophila melanogaster larvae. CanlonicSF is well suited to explore lactate dynamics with sub-cellular resolution in intact systems.

Astrocytes regulate brain extracellular pH via a neuronal activity-dependent bicarbonate shuttle.

Brain cells continuously produce and release protons into the extracellular space, with the rate of acid production corresponding to the levels of neuronal activity and metabolism. Efficient buffering and removal of excess H+ is essential for brain function, not least because all the electrogenic and biochemical machinery of synaptic transmission is highly sensitive to changes in pH. Here, we describe an astroglial mechanism that contributes to the protection of the brain milieu from acidification. In vivo and in vitro experiments conducted in rodent models show that at least one third of all astrocytes release bicarbonate to buffer extracellular H+ loads associated with increases in neuronal activity. The underlying signalling mechanism involves activity-dependent release of ATP triggering bicarbonate secretion by astrocytes via activation of metabotropic P2Y1 receptors, recruitment of phospholipase C, release of Ca2+ from the internal stores, and facilitated outward HCO3- transport by the electrogenic sodium bicarbonate cotransporter 1, NBCe1. These results show that astrocytes maintain local brain extracellular pH homeostasis via a neuronal activity-dependent release of bicarbonate. The data provide evidence of another important metabolic housekeeping function of these glial cells.

Kinetic validation of 6-NBDG as a probe for the glucose transporter GLUT1 in astrocytes.

In recent years, the use of fluorescent glucose analogs has allowed the study of rapid transport modulation in heterogeneous cell cultures and complex tissues. However, the kinetic behavior of these tracers is not conventional. For instance, the fluorescent glucose analog 6-NBDG permeates the cell 50-100 times slower than glucose but the uptake of 6-NBDG is almost insensitive to glucose, an observation that casts doubts as to the specificity of the uptake pathway. To investigate this apparent anomaly in cultured astrocytes, which are rich in the glucose transporter GLUT1, we first estimated the kinetic parameters of 6-NBDG uptake, which were then incorporated into the kinetic model of GLUT1. The main outcome of the analysis was that 6-NBDG binds to GLUT1 with 300 times higher affinity than glucose, which explains why its uptake is not efficiently displaced by glucose. The high binding affinity of 6-NBDG also explains why cytochalasin B is less effective at inhibiting 6-NBDG uptake than at inhibiting glucose uptake. We conclude that 6-NBDG, used at low concentrations, permeates into astrocytes chiefly through GLUT1, and advise that the exofacial GLUT1 inhibitor 4,6-ethylidine-D-glucose be used, instead of glucose, as the tool of choice to confirm the specificity of 6-NBDG uptake.

Energy Dynamics in the Brain: Contributions of Astrocytes to Metabolism and pH Homeostasis.

Regulation of metabolism is complex and involves enzymes and membrane transporters, which form networks to support energy dynamics. Lactate, as a metabolic intermediate from glucose or glycogen breakdown, appears to play a major role as additional energetic substrate, which is shuttled between glycolytic and oxidative cells, both under hypoxic and normoxic conditions. Transport of lactate across the cell membrane is mediated by monocarboxylate transporters (MCTs) in cotransport with H+, which is a substrate, a signal and a modulator of metabolic processes. MCTs form a "transport metabolon" with carbonic anhydrases (CAs), which not only provide a rapid equilibrium between CO2, HCO3 - and H+, but, in addition, enhances lactate transport, as found in Xenopus oocytes, employed as heterologous expression system, as well as in astrocytes and cancer cells. Functional interactions between different CA isoforms and MCTs have been found to be isoform-specific, independent of the enzyme's catalytic activity, and they require physical interaction between the proteins. CAs mediate between different states of metabolic acidosis, induced by glycolysis and oxidative phosphorylation, and play a relay function in coupling pH regulation and metabolism. In the brain, metabolic processes in astrocytes appear to be linked to bicarbonate transport and to neuronal activity. Here, we focus on physiological processes of energy dynamics in astrocytes as well as on the transfer of energetic substrates to neurons.

Tight coupling of astrocyte energy metabolism to synaptic activity revealed by genetically encoded FRET nanosensors in hippocampal tissue.

The potassium ion, K+, a neuronal signal that is released during excitatory synaptic activity, produces acute activation of glucose consumption in cultured astrocytes, a phenomenon mediated by the sodium bicarbonate cotransporter NBCe1 ( SLC4A4). We have explored here the relevance of this mechanism in brain tissue by imaging the effect of neuronal activity on pH, glucose, pyruvate and lactate dynamics in hippocampal astrocytes using BCECF and FRET nanosensors. Electrical stimulation of Schaffer collaterals produced fast activation of glucose consumption in astrocytes with a parallel increase in intracellular pyruvate and biphasic changes in lactate . These responses were blocked by TTX and were absent in tissue slices prepared from NBCe1-KO mice. Direct depolarization of astrocytes with elevated extracellular K+ or Ba2+ mimicked the metabolic effects of electrical stimulation. We conclude that the glycolytic pathway of astrocytes in situ is acutely sensitive to neuronal activity, and that extracellular K+ and the NBCe1 cotransporter are involved in metabolic crosstalk between neurons and astrocytes. Glycolytic activation of astrocytes in response to neuronal K+ helps to provide an adequate supply of lactate, a metabolite that is released by astrocytes and which acts as neuronal fuel and an intercellular signal.

GLUT1 and GLUT8 support lactose synthesis in Golgi of murine mammary epithelial cells.

The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (p < 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (p < 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (p < 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane-associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in cis-Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.