Glucose transporters (GLUTs) are pivotal membrane proteins that facilitate the passive transport of glucose into cells. However, their aberrant overexpression is closely linked to the Warburg effect and chemotherapy resistance of tumors. GLUTs are complex multi-pass transmembrane proteins that require detergents for extraction from the cell membrane during preparation. The persistent presence of detergents in the sample can disrupt lipid-protein interactions, potentially leading to conformational distortion and functional losses of GLUTs, severely hindering the research into their structures and transport mechanisms. To eliminate detergent interference and preserve its authentic conformation, this study employs nanodisc technology and utilizes the self-assembly of the membrane scaffold protein MSP1E3D1 and phospholipids to produce a biomimetic membrane environment, thereby overcoming the limitations of conventional methods. The C-terminal His10-tagged GLUT1 was heterologously expressed in the insect cell Sf9/Bac-to-Bac system, and the GLUT1-nanodisc complex was obtained after detergent solubilization, affinity chromatography purification via anti-His antibody resin, and self-assembly. The successfully reconstituted nanodisc complex was further purified by Ni-NTA affinity chromatography. Nanodisc reconstitution produced monodisperse GLUT1 particles that retained native secondary structure, as confirmed by far-UV circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). Unlike conventional detergent micelles, which lack a true lipid bilayer, distort transmembrane-helix topology, and occlude ligand-binding sites, the nanodisc platform embeds GLUT1 in a phospholipid bilayer that preserves its authentic conformation while eliminating detergent interference. The resulting GLUT1-nanodisc complex is therefore a superior scaffold for high-resolution cryo-EM structural analysis, permitting detailed interrogation of the transporter's conformational cycle, its interactions with partner proteins, and downstream structure-guided, high-throughput drug screening.
Nanostructures/chemistry* ; Glucose Transporter Type 1/biosynthesis* ; Humans ; Animals ; Phospholipids/chemistry* ; Detergents/chemistry*

We constructed inducible and constitutive heterotrophic expression vectors of Dunaliella salina (D. salina) and identified heterotrophic transformants. A gene encoding a glucose transporter (Glut1) was cloned from human placenta tissues by RT-PCR and sequenced. Inducible heterotrophic expression vector pMDDGN-Bar of D. salina, which included a duplicated carbonic anhydrase (DCA) promotor and a Bar selectable marker that could drive expression of the Glut1 gene in D. salina, was constructed by molecular biology methods. In addition, we constructed another vector G5Glut1-Bar that contained a constitutive ubiquitin promotor, Glut1 and Bar box. The two expression vectors were introduced into D. salina by electroporation method, and then screened the transformants with phosphinothricin (PPT). Total RNA of the transformants extracted was used to analyze the integration of the target gene (Glut1) by RT-PCR. The cloned Glut1 sequence was 1479 bp and encoded 493 amino acids. The results of all enzymes digesting showed that two expression vectors were successfully constructed. After screening by PPT for several weeks, the transfomants grew well whereas wild-type cells died completely. The result of RT-PCR indicated that two transformants both had an about 250 bp specific band and the sequence homology was 100% compared with the human Glut1 sequence by Blast analysis. Taken altogether, inducible and constitutive heterotrophic expression vectors of D. salina was constructed successfully and the Glut1 gene was integrated into the genome of D. salina. Expression vectors above-mentioned may be used for the expression of the foreign Glut1 gene in D. salina.
Base Sequence ; Chlorophyta ; genetics ; metabolism ; Cloning, Molecular ; Electroporation ; Genetic Vectors ; genetics ; Glucose Transporter Type 1 ; biosynthesis ; genetics ; Industrial Microbiology ; Molecular Sequence Data ; Transformation, Genetic

It is well known that exercise can have beneficial effects on insulin resistance by activation of glucose transporter. Following up our previous report that caveolin-1 plays an important role in glucose uptake in L6 skeletal muscle cells, we examined whether exercise alters the expression of caveolin-1, and whether exercise-caused changes are muscle fiber and exercise type specific. Fifity week-old Sprague Dawley (SD) rats were trained to climb a ladder and treadmill for 8 weeks and their soleus muscles (SOL) and extensor digitorum longus muscles (EDL) were removed after the last bout of exercise and compared with those from non-exercised animals. We found that the expression of insulin related proteins and caveolins did not change in SOL muscles after exercise. However, in EDL muscles, the expression of insulin receptor beta (IRbeta) and glucose transporter-4 (GLUT-4) as well as phosphorylation of AKT and AMPK increased with resistance exercise but not with aerobic exercise. Also, caveolin-1 and caveolin-3 increased along with insulin related proteins only in EDL muscles by resistance exercise. These results suggest that upregulation of caveolin-1 in the skeletal muscle is fiber specific and exercise type specific, implicating the requirement of the specific mode of exercise to improve insulin sensitivity.
AMP-Activated Protein Kinases ; Animals ; Caveolin 1/*biosynthesis ; Caveolin 3/metabolism ; Female ; Glucose Transporter Type 4/biosynthesis ; Insulin/*physiology ; Multienzyme Complexes/metabolism ; Muscle Fibers, Skeletal/*metabolism ; Muscle, Skeletal/metabolism/*physiology ; Phosphorylation ; *Physical Conditioning, Animal ; Protein-Serine-Threonine Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Insulin/biosynthesis ; Up-Regulation

OBJECTIVETo demonstrate the impact of hypoxia on ER-alpha in both breast cancer tissue and cell line, and its relationship with hypoxia-related parameters.

METHODSExpression of ER-alpha in 51 breast cancer patients with ER positive determined by ligand-binding assay was examined by immunohistochemistry and compared with CA-IX and Glut-1. Impact of hypoxia on breast cancer cell line MCF-7 (ER-alpha positive) was observed by Western Blot and RT-PCR.

RESULTSOf 51 breast cancer patients, 49 were ER-alpha positive. Regional decrease of ER-alpha expression was consistently observed in peri-necrotic regions as compared to distant regions in both in-situ carcinomas (n=29, P <0.0001) and invasive carcinomas (n=20, P=0.0001), which was closely associated with the induction of CA-IX and Glut-1 in hypoxia (P <0.0001). The decreased expression of ER-alpha protein and mRNA in breast cancer cell lines were attributed to hypoxia and not to other stress factors, such as reduced glucose, low pH, and products released from necrotic or hypoxic cells. Chronic intermittent hypoxia could cause persistent down-regulation of ER-alpha in the MCF-7 breast cancer cell line.

CONCLUSIONRegional hypoxia in breast cancer is associated with the reduced ER-alpha expression, and intermittent hypoxia can cause persistent down-regulation. Hypoxia may therefore contribute to the progression of ER-alpha negative status and potentially to the development of resistance to endocrine therapy.

Antigens, Neoplasm ; metabolism ; Breast ; metabolism ; pathology ; Breast Neoplasms ; metabolism ; pathology ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Carcinoma in Situ ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; Cell Hypoxia ; Cell Line, Tumor ; Down-Regulation ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Glucose Transporter Type 1 ; Humans ; Hypoxia ; metabolism ; Monosaccharide Transport Proteins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics