Current understanding of glucose transporter 4 expression and functional mechanisms

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.