Current understanding of glucose transporter 4 expression and functional mechanisms

Table 1 Summary of glucose transporter family members

Protein (gene)	Amino acids	Km (mm)	Expression sites	Function/substrates	Ref.
GLUT1 (SLC2A1)	492	3-7	Ubiquitous distribution in tissues and culture cells	Basal glucose uptake; glucose, galactose, glucosamine, mannose	[24-30]
GLUT2 (SLC2A2)	524	17	Liver, pancreas, brain, kidney, small intestine	High-capacity low-affinity transport; glucose, galactose, fructose, glucosamine, mannose	[25-27,29-34]
GLUT3 (SLC2A3)	496	1.4	Brain and nerves cells	Neuronal transport; glucose, galactose, mannose	[25-27,29,30,33-35]
GLUT4 (SLC2A4)	509	5	Muscle, fat, heart, hippocampal neurons	Insulin-regulated transport in muscle and fat; glucose, glucosamine	[25-27,29,31,36,37]
GLUT5 (SLC2A5)	501	6	Intestine, kidney, testis, brain	Fructose	[25-27,29,30,34,38-42]
GLUT6 (SLC2A6)	507	5	Spleen, leukocytes, brain	Glucose	[25-27,29,30,43]
GLUT7 (SLC2A7)	524	0.3	Small intestine, colon, testis, liver	Fructose and glucose	[25-27,29,30,38]
GLUT8 (SLC2A8)	477	2	Testis, blastocyst, brain, muscle, adipocytes	Insulin-responsive transport in blastocyst; glucose, fructose, galactose	[25-27,29,30,44,45]
GLUT9 (SLC2A9)	Major 540, Minor 512	0.9	Liver, kidney	Glucose, fructose	[25-27,29,30, 46-48]
GLUT10 (SLC2A10)	541	0.3	Heart, lung, brain, skeletal muscle, placenta, liver, pancreas	Glucose and galactose	[25-27,29,30,48,49]
GLUT11 (SLC2A11)	496	0.2	Heart, muscle, adipose tissue, pancreas	Muscle-specific; fructose and glucose transporter	[25-27,29,30,50-54]
GLUT12 (SLC2A12)	617	4-5	Heart, prostate, skeletal muscle, fat, mammary gland	Glucose	[25-27,29,30,53,55]
GLUT13 (SLC2A13)	Rat 618, human 629	0.1	Brain (neurons intracellular vesicles)	H+-myo-inositol transporter	[25-27,29,30,56]
GLUT14 (SLC2A14)	Short 497, Long 520	unknown	Testis	Glucose transport	[25-27,29,30,57]

Table 2 Recent studies of glucose transporter 4 expression and translocation in the skeletal muscle

Methods	Materials	Comparisons	Observations/conclusions	Ref.
Western blot.	Cell fractions of rat L6 myotubes, 3T3-L1, and mouse muscle and adipose tissues. Anti-GLUT4 from Santa Cruz Biotechnology (1:1000).	Cell: Total cell lysate vs membrane fractions. Mouse tissues: Control vs high-fat diets.	Insulin treatments increases GLUT4 levels in membrane fractions without any change in the total cell lysate. GLUT4 levels in adipose tissue and muscle of mice fed a high-fat diet are lower in all fractions than that fed the control diet.	[72]
Western blot.	Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000).	Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR.	GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment.	[73]
Western blot.	Giant sarcolemmal vesicles from soleus muscles of Sprague-Dawley rats. Anti-GLUT4 from Millipore (1:4000).	Tissue samples without or with insulin released in the presence of glucose as a stimulant and lipid as a control.	A glucose-dependent insulinotropic polypeptide increases glucose transport and plasma membrane GLUT4 protein content.	[74]
Real-time PCR for Slc2a4 mRNA levels.	Total RNA of the skeletal muscle from male C57BL/6J and ICR mice fed different diets.	mRNA levels in muscle samples from mice fed the control or CLA supplement diet	Dietary CLA does not affect Slc2a4 mRNA levels in the mouse skeletal muscle	[75]
Western blot.	Preparations of sarcolemmal membrane fractions and crude lysates from male Muscovy ducklings. Anti-GLUT4 from East Acres (1:500).	GLUT4 from a unique crude membrane fraction of rat skeletal muscle was used as an arbitrary unit and from erythrocyte ghost as a negative control	Polyclonal antibodies detect a protein of similar size (approximately 45 kDa) of GLUT4 in the crude membrane preparations from rat (positive control) and duckling skeletal muscle. No signal was obtained for rat erythrocyte ghost membrane preparation.	[76]
ATB-BMPA-labelling of glucose transporters, Immunoprecipitation, liquid-scintillation counting, Western blot.	Tissue samples of isolated and perfused EDL or soleus muscle from GLUT1 transgenic C57BL’KsJ-Leprdbj and control mice. Anti-GLUT4 (R1184; C-terminal) from an unknown source.	Non-transgenic mice vs transgenic mice.	Basal levels of cell-surface GLUT4 in isolated or perfused EDL are similar in transgenic and non- transgenic mice. Insulin induces cell-surface GLUT4 by 2-fold in isolated EDL and by 6-fold in perfused EDL of both transgenic and non-transgenic mice. Western blot results were not shown.	[77]
Preembedding technique (immune reaction occurs prior to resin embedding to label GLUT4), and observations of whole mounts by immunofluorescence microscopy, or after sectioning by immunogold electron microscopy.	Muscle samples from male Wistar rats. Anti-GLUT4 (C-terminal, 1:1000), and anti-GLUT4 (13 N-terminal, 1:500) from unknown species.	Rats were divided in four groups: Control, contraction received saline, insulin and insulin plus contraction groups. They received glucose followed by insulin injection.	Two populations of intracellular GLUT4 vesicles are differentially recruited by insulin and muscle contractions. The increase in glucose transport by insulin and contractions in the skeletal muscle is due to an additive translocation to both the plasma membrane and T tubules. Unmasking of GLUT4 COOH-terminal epitopes and changes in T tubule diameters does not contribute to the increase in glucose transport.	[78]
Immunoprecipitation, and Western blot.	Membrane fractions from skeletal muscle of male Wistar rats treated without or with insulin. Anti-GLUT-4 from Genzyme, Anti-GLUT-4 from Santa Cruz Biotechnology.	Crude membrane preparations and cytosolic fractions in samples of rats treated without or with insulin.	In vitro activation of PLD in crude membranes results in movement of GLUT4 to vesicles/microsomes. This GLUT4 translocation is blocked by the PLD inhibitor, neomycin, which also reduces insulin-stimulated glucose transport in rat soleus muscle.	[79]
Western blot for GLUT4 protein in homogenates of epitrochlearis muscles. Tissue slices labeled with 2-[1,2-3H]-deoxy-d-glucose and counted in a gamma counter.	Muscle homogenate and slices from male Sprague-Dawley rats. Anti- GLUT4 from Dr. Osamu Ezaki.	Sedentary control vs a 5-day swimming training group.	The change of insulin responsiveness after detraining is directly related to muscle GLUT-4 protein content. The greater the increase in GLUT-4 protein content induced by training, the longer an effect on insulin responsiveness lasts after training.	[80]
Immunofluorescence for membrane preparations, and 2-Deoxyglucose uptake in isolated skeletal muscles.	Membrane preparations from L6 cells over-expressing GLUT4myc. Isolated skeletal muscle samples from mice. Anti-GLUT4 from Biogenesis.	L6 cells over-expressing GLUT4myc treated without or with Indinavir.	HIV-1 protease inhibitor indinavir at 100 µmol/l inhibits 80% of basal and insulin-stimulated 2-deoxyglucose uptake in L6 myotubes with stable expression of GLUT4myc.	[81]

AICAR: 5-aminoimidazole-4- carboxamide ribonucleotide; CLA: Conjugated linoleic acid; EDL: Extensor digitorum longus; HIV: Human immunodeficiency virus; ICR: Institute of Cancer Research; PLD: Phospholipase D; GLUT4: Glucose transporter 4.

Table 3 Recent studies of effects of bioactive compounds and chemical drugs on glucose transporter 4 expression and translocation in adipocytes

Methods	Materials	Comparisons	Conclusions	Ref.
Immunoprecipitation, dual fluorescence immunostaining, Western blot.	3T3-L1, anti-GLUT4 from Santa Cruz Biotechnology (1:200).	Treatments without or with kaempferitrin.	Kaempferitrin treatment upregulates total GLUT4 expression and its translocation in 3T3-L1 cells.	[95]
Subcellular fractionations, Western blot.	3T3-L1, anti-GLUT4 from Cell Signaling Technology (1:1000).	Treatments without or with epigallocatechin gallate.	Green tea epigallocatechin gallate suppresses insulin-like growth factor-induced-glucose uptake via inhibition of GLUT4 translocation in 3T3-L1 cells.	[96]
Western blot.	3T3-L1, anti-GLUT4 from Santa Cruz Biotechnology.	Treatments without or with GW9662.	GW9662 increases the expression of GLUT4 protein in 3T3-L1 cells.	[97]
Immunoprecipitation, Western blot.	3T3-L1, anti-GLUT4 from Chemicon.	Treatments without or with p38 inhibition.	Inhibition of p38 enhances glucose uptake through the regulation of GLUT4 expressions in 3T3-L1 cells.	[98]
Western blots, Real-time PCR, Electrophoretic mobility shift assay, Immunofluorescence.	Adipose tissues of Esr1 deletion and wild type female mice, 3T3-L1, anti-GLUT4 from Merck/Millipore for Western blot (1:4000), and for immunofluorescence (1:100).	Tissue and cells without or with gene deletion.	Estradiol stimulates adipocyte differentiation and Slc2a4 mRNA and GLUT4 protein expressions in an ESR1/CEBPA mediated manner in vitro and in vivo.	[99]
Real-time PCR, Solid-phase ELISA.	3T3-L1, anti-GLUT4 antibody from Pierce (1:1000).	Treatments without or with the extract.	The crude extract of stevia leaf can enhance Slc2a4 mRNA and GLUT4 protein levels in 3T3-L1 cells.	[100]
GeXP multiplex for mRNA, Western blot.	3T3-L1, anti-GLUT4 from Millipore (1:20).	Treatments without or with indicated reagents.	Curculigoside and ethyl acetate fractions increase glucose transport activity of 3T3-L1 adipocytes via GLUT4 translocation.	[101]
Real-time PCR, Western blot.	3T3-L1, anti-GLUT4 from Cell Signaling Technology.	Treatments without or with luteolin	Luteolin treatment decreases Slc2a4 mRNA and GLUT4 protein levels in 3T3-L1 cells.	[102]
Western blot.	3T3-L1, anti-GLUT4 from Abcam (ab654-250).	Treatments without or with extract.	Shilianhua extract treatment decreases GLUT4 protein level in 3T3-L1 cells.	[103]
Western blot.	3T3-L1, and male C57BL/6J mice fed a normal-fat or high-fat diet, anti-mouse GLUT4 from AbD SeroTec (1:1000).	Treatments without or with fargesin.	Fargesin treatment increases GLUT4 protein expression in 3T3-L1 cells and adipose tissues of mice.	[104]
Western blot.	3T3-L1, antibody no mentioned.	Treatments without or with phillyrin.	Phillyrin treatment increases the expression levels of GLUT4 protein in 3T3-L1 cells.	[105]
Real-time PCR, Western blot.	3T3-L1, anti-GLUT4 from Santa Cruz Biotechnology.	Treatments without or with 6Hydroxydaidzein.	6Hydroxydaidzein facilitates GLUT4 protein translocation, but did not affect Slc2a4 mRNA level in 3T3-L1 cells.	[106]
Western blot.	3T3-L1, and C57BL/6J mice with SirT1 and Ampkα1 knockdown, anti-GLUT4 from Signalway Antibody.	Treatments without or with indicated reagents.	Seleniumenriched exopolysaccharides produced by Enterobacter cloacae Z0206 increase the expression of GLUT4 protein in mice, but not in 3T3-L1 cells.	[107]
Western blot.	3T3-L1, anti-GLUT4 from Cell Signaling Technology	Treatments without or with extract.	Aspalathin-enriched green rooibos extract increases GLUT4 protein expression in 3T3-L1 cells.	[108]
Transient expression of myc-GLUT4-GFP and fluorescence microscopy.	3T3-L1, fusion protein only.	Treatments without or with indicated reagents.	Gallic acid can increase GLUT4 translocation and glucose uptake in 3T3-L1 cells.	[109]
Real-time PCR, Western blot.	3T3-L1, anti GLUT4 from Santa Cruz Biotechnology (1: 1000).	Treatments without or with Ginsenoside Re.	Ginsenoside Re promotes the translocation of GLUT4 by activating PPAR-γ2. Slc2a4 mRNA is not affected in 3T3-L1 cells.	[110]
Real-time PCR, GLUT4-myc7-GFP from retroviral vector, flow cytometry, fluorescence microscopy.	3T3-L1 with knockdown of PPARγ, fusion protein.	Cells without or with knockdown.	Bone morphogenetic proteins 2 and 6 inhibit PPARγ expression, which increases total GLUT4 levels, but not GLUT4 translocation3T3-L1 cells.	[111]
Western blot, real-time PCR.	3T3-L1, anti-GLUT4 antibody from Santa Cruz Biotechnology (sc-1608).	Treatments without or with pulse or manipulations.	Glucose pulse (25 mM) increases GLUT4 expression. GLUT4 level is partially restored by increasing intracellular NAD/P levels. A liver X receptor element on Slc2a4 promoter is responsible for glucose-dependent transcription.	[112]

GLUT4: Glucose transporter 4; AMPK: AMP-activated protein kinase; CEBPA and C/EBP: CCAAT/enhancer-binding protein alpha; ESR1: Estrogen receptor 1.

Table 4 Recent studies of mechanisms of glucose transporter 4 expression and translocation in adipocytes

Methods	Materials	Comparisons	Observations/conclusions	Ref.
Western blot, real-time PCR, Electrophoretic mobility shift assay.	3T3-L1 pre and differentiated adipocytes. Anti-GLUT4 antibody from Chemicon (1:4,000).	Treatment groups without or with the antagonist.	CB1 receptor antagonist markedly increases Slc2a4 mRNA and GLUT4 protein levels in 3T3-L1 cells via NF-kB and SREBP-1 pathways.	[113]
Immunohistochemistry, Western blot, real-time PCR.	Brown adipose tissue of Arfrp1 flox/flox and Arfrp1 ad-/-mouse embryos (ED 18.5) and 3T3-L1 cells with knockdown of Arfrp1. Anti-GLUT4 without specifying the vendor (1:1000).	Mice without or with deletion, and 3T3-L1 cells without or with knockdown.	In Arfrp1 ad-/- adipocytes, GLUT4 protein accumulates on the cell membrane rather than staying intracellularly without any change of Slc2a4 mRNA. siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes has a similar result and increases basal glucose uptake.	[114]
Real-time PCR, Western blot.	3T3-L1 transfected with Mmu-miR-29a/b/c. Anti-GLUT4 from Santa Cruz Biotechnology (SC-7938).	Cells with or without transfection.	Transfection of miR-29 family members inhibits Slc2a4 mRNA and GLUT4 protein levels in 3T3-L1 cells by inhibiting SPARC expression.	[115]
Northern blot, Western blot, Nuclear run-on assay for the rate of GLUT4 gene transcription.	3T3-L1 pre and differentiated adipocytes. Rabbit polyclonal GLUT4 antibody form Chemicon.	Treatment groups without or with inhibitors.	Inhibitions of proteasome using Lactacystin and MG132 reduce Slc2a4 mRNA and GLUT4 protein levels in 3T3-L1 cells.	[116]
AFFX miRNA expression chips for mRNA, Western blot.	Human Omental adipose tissue, 3T3-L1 pre and differentiated adipocytes with miR-222 silenced by antisense oligonucleotides. Anti-GLUT4 from Abcam.	Groups without or with transfection.	High levels of estrogen reduce the expression and translocation of GLUT4 protein. miR -222 silencing dramatically increases the GLUT4 expression and the insulin-stimulated translocation of GLUT4 in 3T3-L1 adipocytes.	[117]
Northern blot for mRNA, Western blot.	3T3-L1 pre and differentiated adipocytes. Anti-GLUT4 from Chemicon.	Treatment groups without or with oxidative stress.	Oxidative stress mediated by hydrogen peroxide induces expressions of C/EBPα and δ, resulting in altered C/EBP-dimer composition on the GLUT4 promoter, which reduces GLUT4 mRNA and protein levels.	[118]
Real-time PCR, Western blot.	Human Subcutaneous pre and differentiated adipocytes from control and obese subjects, 3T3-L1 pre and differentiated adipocytes transfected with miR-155. Anti-GLUT4 from Abcam.	Primary pre and differentiated adipocytes from normal and obese subjects, and cells without or with transfection.	The level of SLC2A4 is reduced in obese people, and the expression of GLUT4 protein is reduced in 3T3-L1 cells and differentiated human mesenchymal stem cells transfected with miR-155.	[119]
HA-GLUT4-GFP from transfected lentiviral plasmid and analyzed by flow cytometry, and fluorescence microscopy.	3T3-L1 pre and differentiated adipocytes with knockdown of Dennd4C. Fusion protein.	Groups without or with knockdown.	Knockdown of Dennd4C inhibits GLUT4 translocation, and over- expression of DENND4C slightly stimulates it. DENND4C is found in isolated GLUT4 vesicles.	[120]
HA-Glut4-GFP from transfected plasmid, and analyzed by flow cytometry, fluorescence microscopy	3T3-L1 pre and differentiated adipocytes with AS160 knockdown.Fusion protein.	Groups without or with knockdown.	Akt regulates the rate of vesicle tethering/fusion by regulating the concentration of primed, and fusion-competent GSVs with the plasma membrane, but not changing the intrinsic rate constant for tethering/fusion.	[121]
HA-tagged GLUT4 by fluorescence microscopy, Western blots, Immune pulldown.	3T3-L1 pre and differentiated adipocytes without or with GST-ClipR-59 transfection. Rabbit anti-GLUTlut4 from Millipore; Mouse monoclonal anti-GLUT4 from Cell Signaling Technology.	Pull down antibodies.	By interacting with AS160 and enhancing the association of AS160 with Akt, ClipR-59 promotes phosphorylation of AS160 and GLUT4 membrane translocation.	[122]
Transfection of GFP-GLUT4 and indirect immunofluorescence.	3T3-L1 pre and differentiated adipocytes with siRNA knockdown of P-Rex1. Fusion protein.	Without or with knockdown.	P-Rex1 activates Rac1 in adipocytes, which leads to actin rearrangement, GLUT4 trafficking, increase of glucose uptake.	[123]
Transfection of GLUT4-eGFP plasmid and analyzed by fluorescence microscopy.	3T3-L1 pre and differentiated adipocytes. Fusion protein.	Treatment groups without or with activators.	AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin.	[124]

GLUT4: Glucose transporter 4; ARFRP1: ADP-ribosylation factor-related protein 1; AMPK: AMP-activated protein kinase; AS160: Akt substrate of 160 kDa; CB1: Cannabinoid receptor 1; CEBPA and C/EBP: CCAAT/enhancer-binding protein alpha; CLIPR-59: Cytoplasmic linker protein R-59; DENND4C: Differentially expressed in normal and neoplastic cells domain-containing protein 4C; GSV: GLUT4 storage vesicle; NF-Κb: Nuclear factor-κB; PREX1: Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1; SPARC: Secreted protein acidic and rich in cysteine; SREBP-1: Sterol regulatory element-binding protein 1.

Table 5 Recent studies of glucose transporter 4 expression and translocation in the heart

Methods	Materials	Comparisons	Conclusions	Ref.
Western blot.	Cytosol and membrane fractions of left ventricular, heart, and blood from male Sprague-Dawley rats. Anti-GLUT4 from Santa Cruz Biotechnology (1:200).	Groups without or with the indicated treatments. Na+/K+-ATPase and β-actin were loading controls of the membrane and cytosol fractions, respectively. Losartan was used as a positive control.	Ginsenoside Rb1 treatment can increase GLUT4 expression via inhibition of the TGF-β1/Smad and ERK pathways, and activation of the Akt pathway.	[134]
Real-time PCR,Western blot.	Isolated ventricular cells from heart of male adult (aged 6-8 wk) and neonatal (1-3 d old) Wistar rats.Anti GLUT4 from Abcam (unknown dilution).	Groups with or without the ethanol feeding. Gapdh and β-actin were included as loading controls for real-time PCR and Western blot, respectively.	Long-term (22 wk) ethanol consumption increases AMPK and MEF2 expressions, and reduces GLUT4 mRNA and protein expression in rat myocardium	[135]
Western Blot.	Isolated ventricular cells from heart of adult male Wistar rats. Polyclonal rabbit anti-human GLUT4 from AbD Serotec (4670–1704 1:750)	Groups with or without the indicated treatments.	Heart failure and MI reduce glucose uptake and utilization. GGF2 partially rescues GLUT4 translocation during MI.	[136]
Western blot, Immunofluorescence.	Isolated ventricular cells from heart of adult rats. Polyclonal anti-GLUT4 from Thermo Fisher Scientific (1:100).	Treatment groups were compared with that of 100 nM insulin.	Catestatin can induce AKT phosphorylation, stimulate glucose uptake, and increase GLUT4 translocation.	[137]
Western blot, Flow cytometric analysis.	Isolated ventricular cells from heart of adult male Wistar rats. Anti-GLUT4 (H-61) from unknow source (1:1000 for Western) and conjugated to Alexa Fluor 488.	Treatment groups with or without AMPK agonists.	AMPK activation does not affect GLUT4 translocation and glucose uptake in isolated cardiomyocytes.	[138]
Real-time PCRUsing TaqMan® Gene Expression assays.	Blood, heart, frontal cortex cerebellum from male Wistar rats.	Tissues from control and diabetic rats.	Slc2a4’s expression is downregulated in STZ-treated rat’s heart, but unaffected in tissue protected by blood-brain barrier like frontal cortex.	[139]
Western blot, Immunohistochemistry.	Heart from male Sprague-Dawley rats, anti-GLUT4 from Cell Signaling Technology (2213, 1:1000), anti GLUT4 from Abcam (ab654, 1:200 for ICC/IF)	Treatment groups without or with the indicated treatments.	PEDF can increase glucose uptake and GLUT4 translocation in ischemic myocardium.	[140]
Real-time PCR,Western blot.	Heart from male wild type rats and SHRs. Rabbit polyclonal antibody GLUT4 from Millipore	Wild type rats and SHRs without or with the indicated treatments.	Sitagliptin upregulates levels of GLUT4 protein and Slc2a4 mRNA, and its translocation in cardiac muscles of SHRs.	[141]
Real-time PCR,Western blot.	Left ventricles muscle from male Wistar rats. Anti-GLUT4 from Chemicon (1:1000)	Saline as untreated control and reagent treated groups.	Growth hormone stimulates the translocation of GLUT4 to the cell membrane of cardiomyocytes in adult rats.	[142]

GLUT4: Glucose transporter 4; AMPK: Adenosine monophosphate-activated protein kinase; ERK: Extracellular signal-regulated kinase; GGF2: Glial growth factor 2; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; MEF2: Myocyte enhancer factor-2; MI: Myocardial infarction; PEDF: Pigment epithelium-derived factor; SHR: Spontaneously hypertensive rats; STZ: Streptozotocin; TGF: Transforming growth factors.

Table 6 Recent studies of glucose transporter 4 expression and translocation in the brain

Methods	Materials	Comparisons	Conclusions	Ref.
Western blot.	Brain, skeletal muscle, heart, and whiteadipose tissue from mice. Anti-GLUT4 from Chemicon (1:1000).	Samples from wild type and knockout mice.	Deletion of Slc2a4 in the brain leads to insulin resistance, glucose intolerance, and impaired glucose sensing in the VMH.	[154]
Western blot,Real time-PCR,Immunofluorescence.	Cortex, hypothalamus, cerebella samples from CD-1 mice. Monoclonal anti Glut4 (1F8) from Dr. Paul Pilch, Polyclonal anti Glut4 MC2A from Dr. Giulia Baldini, Polyclonal anti Glut4 αG4 from Dr. Samuel Cushman, Polyclonal anti Glut4 (C-20) from Santa Cruz Biotechnology.	Expression profile in the mouse and rat brain samples.	Slc2a4 mRNA is expressed in cultured neurons. GLUT4 protein is highly expressed in the granular layer of the mouse cerebellum. GLUT4 translocation to the plasma membrane can be stimulated by physical activity.	[155]
Western blot.	Brian tissue from STZ-induced diabetic male Sprague-Dawley rats.Anti-GLUT4 from Millipore (1:1000).	Comparing treatment samples using β-actin and NA/K ATPase as loading controls in Western blot.	Chronic infusion of insulin into the VMH in poorly controlled diabetes is sufficient to normalize the sympathoadrenal response to hypoglycemia via restoration of GLUT4 expression.	[156]
Immunocytochemistry.	Cerebellum and hippocampus from male Sprague-Dawley rats.Rabbit anti-GLUT4 antibody from Alomone Labs (AGT-024, RRID: AB_2631197).	Identifying expression profile and translocation.	GLUT4 is expressed in neurons including nerve terminals.Exercising axons rely on translocation of GLUT4 to the cell membrane for metabolic homeostasis.	[157]
Real-time PCR, Immunocytochemistry.	Cerebral cortex, hippocampus, thalamus, cerebellum, medulla oblongata, cervical spinal cord, biceps muscles from male Wistar rats. Unknown source of antibody.	Identifying expression profile using β -actin as loading control in real-time PCR.	Slc2a4 mRNA is detected in many neurons located in brain and spinal cord. GLUT4 protein is detected in different regions of the CNS including certain allocortical regions, temporal lobe, hippocampus, and substantia nigra.	[158]
Immunocytochemistry, Western blot.	Brain, spleen, kidney from Lrrk2 knockout mice.Anti GLUT4 Avivasysbio (ARP43785_P050, 1:100), andanti GLUT4 from R&D Systems (MAB1262, 1:1000).	Samples from wild type and knockout mice, andanti-β-Tubulin as loading control.	Phosphorylation of Rab10 by LRRK2 is necessary for GLUT4 translocation.Lrrk2 deficiency increases GLUT4 expression on the cell surface in “aged” cells.	[159]
Western blot, Immunofluorescence, real-time PCR.	Brain from Cyp27Tg mice. Anti GLUT4 from Cell Signaling Technology (#2213,1:1000 dilution).	Mice with different treatments.	A reduction of GLUT4 protein expression in brain occurs after 27-OH cholesterol treatment.	[160]
Immunohistochemistry.	Brain, hypothalamus, and other tissues from Sprague–Dawley rats.Anti-GLUT4 antibody from Santa Cruz Biochemicals (1:200), Anti GLUT4 from S. Cushman (1:1000).	Identifying the expression profiles. Soleus muscle as GLUT4 positive control. Antibodies after pre-absorption with the corresponding synthetic peptide were used as negative control. for GLUT4 antibody.	GLUT4 is localized to the micro vessels comprising the blood brain barrier of the rat VMH.GLUT4 is co-expressed with both GLUT1 and zonula occludens-1 on the endothelial cells of these capillaries.	[161]
Electrophysiological analyses, fluorescent microscope.	Brain from GLUT4-EYFP transgenic mice. Fusion protein.	Comparing samples from treatments. A scrambled RNA expressed by AAV acted as a negative control.	GLUT4 neurons are responsible for glucose sensing.	[162]
Western blot,Immunohistochemistry.	Brain samples from 7, 11, 15, 21 and 60 d old Balb/c mice. Rabbit anti-rat GLUT4 from an unknow source (1:2500 dilution for Western and 1:2000 for immunohistochemistry).	Determine the expression profiles. Vinculin is used as the loading control in Western blot.	GLUT4 is expressed in neurons of the postnatal mouse brain. GLUT4 and GLUT8 may mediate the effects of insulin, or insulin-like growth factor on regulations of cognition, memory, behavior, motor activity and seizures.	[163]

AAV: Adeno-associated virus; CNS: Central nervous system; CRR: Counterregulatory response; EYFP: Enhanced Yellow Fluorescent Protein; LRRK2: Leucine-rich repeat kinase 2; STZ: Streptozotocin; VMH: Ventromedial hypothalamus.