Regulation of Glucose Uptake
Our research focuses on glucose transport into cells and how that rate of transport rapidly adjusts as the cells’ environment changes.
Glucose is arguably the most important energy source in biological systems. Proper glucose uptake is critical to the maintenance and health of a wide variety of organisms. For example, in humans, diabetes is a disease in which glucose uptake is compromised either due to the insufficient production of insulin (type 1) or compromised response to insulin at peripheral tissues (type 2). The uptake of glucose across biological membranes is mediated by a family of proteins called GLUTs (GLUcose Transporters). Our lab is particularly interested in the regulation of two of these transporters—GLUT 4, which is responsive to insulin, and GLUT 1, which is found in a variety of tissues and is responsible for basal or background uptake. Acute regulation of GLUT 4 by agents such as insulin and exercise is primarily mediated by a translocation of the transporter from internal stores to the cell surface, thereby increasing glucose uptake. GLUT 1 was initially viewed to be responsible for basal uptake and therefore not acutely regulated by environmental conditions. However, recent data from our lab and others have indicated that cell stressors such as azide, hyperosmolarity, and methylene blue can quickly increase the transport of glucose through GLUT 1. Our current research focus is to understand the acute regulation of glucose uptake with a focus on the activity of GLUT 1. GLUT1 is a membrane glycoprotein of about 55 kD, (see Figure from Lehninger (Freeman)) which transports glucose in and out of the cell. Its proposed structure is shown below. We are currently exploring three plausible mechanisms for the activation of GLUT1.
Our lab works with several cell types—intact mouse muscle (soleus and epitrochlearis), cultured fibroblast cells (L929, which contain only GLUT 1), and cultured myoblast cells and myotubes. Glucose uptake is measure by scintillation spectrometry using the radioactive glucose analog, 2-deoxyglucose or 3-O-methylglucose.
Recent Student Projects
Project: Genetic engineering of GLUT1
We have generated a genetic construct consisting of the GLUT1 gene with the green fluorescent protein attached to the C-terminus. We have transformed L929 fibroblast cells and have shown that the chimeric protein is expressed and locates to the Golgi and cell surface as expected. This fluorescentGLUT1 will be used in FRAP and FRET assays to study changes location or mobility that may result from activation of the transporter. We will also generate a chimeric protein with GFP attached to the N-terminus should the C-terminus GLUT1-GFP prove to be either not able to transport glucose or not able to be activated.
Project: Role of Adjacent Sulfhydrals in the Activation of GluT1
A family of membrane imbedded proteins, known as GluT, transport glucose into cells. Phenylarsine oxide, chemical that binds tightly to the sulfur atoms of neighboring cysteine residues, has a dual action on glucose uptake. At low concentrations (3µM) it activates glucose uptake, and at higher concentrations (10µM) it blocks the activation of glucose transport initiated by either methylene blue treatment or glucose starvation.
Project: Investigating GluT1 Activity in Cardiac and Muscle Cells
We have shown that cell stress, such as glucose starvation can quickly activate the glucose transport activity of GluT1 in fibroblast cells. We are interested in understanding this mode of activation extends to other cell lines. This study investigated the effects of insulin and glucose starvation on a muscle cell line, L6 cells, and on a cardiac cell line, H9c2 cells. In both types of cells, glucose starvation stimulated glucose uptake suggesting activation of GluT1. In L6 muscle cells the effects of insulin, which activates GluT4, was additive to the effects of glucose starvation, again suggesting that insulin and cell stress activated different glucose transporters.
Project: Effects of Cinnamon on the Activity of GluT1 Glucose Transporter
Cinnamon has been recommended as a nutritional aid for patients with type 2 diabetes. In this study, we investigated the effects of a cinnamon extract on the glucose transport activity of GluT1 in fibroblast cells. Surprisingly, we found that the extract potently inhibited glucose transport. The extract was separated into 6 fractions using chromatography. The inhibitor compound was isolated in fraction #4. The purified compound, cinnamaldehyde, mimicked the inhibitory effects of the extract. A systematic study of the cinnamaldehyde and related analogs reveal that low concentration actually active transport and higher concentration inhibit. The activity of cinnamaldehyde can be blocked by prior reaction of cinnamaldehyde with a sulfhydral compound. This provides evidence of the importance of sulfhydrals (cysteine residues) to the activity of GLUT1.
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