Expression of conventional and novel glucose transporters, GLUT1, -9, -10, and -12, in vascular smooth muscle cells

Abstract

Intimal hyperplasia is characterized by exaggerated proliferation of vascular smooth muscle cells (VSMCs). Enhanced VSMC growth is dependent on increased glucose uptake and metabolism. Facilitative glucose transporters (GLUTs) are comprised of conventional GLUT isoforms (GLUT1-5) and novel GLUT isoforms (GLUT6-14). Previous studies demonstrate that GLUT1 overexpression or GLUT10 downregulation contribute to phenotypic changes in VSMCs. To date, the expression profile of all 14 GLUT isoforms has not been fully examined in VSMCs. Using the proliferative and differentiated phenotypes of human aortic VSMCs, the present study has determined the relative abundance of GLUT1-14 mRNAs by quantitative real-time PCR analysis. Twelve GLUT mRNAs excluding GLUT7 and GLUT14 were detectable in VSMCs. In the proliferative phenotype, the relative abundance of key GLUT mRNAs was GLUT1 (∼43%)>GLUT10 (∼26%)>GLUT9 (∼13%)>GLUT12 (∼4%), whereas in the differentiated phenotype the relative abundance was GLUT10 (∼28%)>GLUT1 (∼25%)>GLUT12 (∼20%)>GLUT9 (∼14%), together constituting 86-87% of total GLUT transcripts. To confirm the expression of key GLUT proteins, immunoblot and immunocytochemical analyses were performed using GLUT isoform-specific primary antibodies. The protein bands characteristic of GLUT1, -9, -10, and -12 were detected in VSMCs in parallel with respective positive controls. In particular, GLUT1 protein expression showed different molecular forms representative of altered glycosylation. While GLUT1 protein displayed a predominant distribution in the plasma membrane, GLUT9, -10, and -12 proteins were mostly distributed in the intracellular compartments. The present study provides the first direct evidence for GLUT9 and GLUT12 expression in VSMCs in conjunction with the previously identified GLUT1 and GLUT10.

Metformin exaggerates phenylephrine-induced AMPK phosphorylation independent of CaMKKβ and attenuates contractile response in endothelium-denuded rat aorta

“…First, the uptake of 2-deoxyglucose (a key component of metabolic stress) and AICAR (an adenosine analog) in vascular smooth muscle cells is mediated by plasma membrane-localized glucose transporter-1 [28, 46] and presumably equilibrative nucleoside transporter-1 [47, 48], respectively. The present findings from LC-MS/MS MRM analysis show significant amounts of metformin in arterial smooth muscle (~600 pmoles/mg aortic tissue) following ex vivo exposure to a high concentration of metformin (3 mM for 2 hr); however, loss of metformin from the aortic tissue is rapid with a t 1/2 of approximately 1 hr.…”

“…Aortic tissue samples (20 μg protein each) were electrophoresed using pre-cast 4-12% NuPage mini-gels (Life Technologies, Carlsbad, CA), and the resolved proteins were transferred to nitrocellulose membranes (Hybond C, GE Healthcare Life Sciences, Piscataway, NJ) as described [28]. The membranes were blocked in 5% bovine serum albumin, and probed with the primary antibodies specific for phospho-AMPKα (Thr172), AMPKα, CaMKKβ, or β-actin.…”

“…VSMCs (passages 3 to 6) were maintained in culture with vascular cell basal medium containing VSMC growth kit and antibiotic/antimycotic solution in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. After the attainment of confluence, VSMCs were trypsinized, centrifuged, and ~3 million cells were seeded on to 150 mm petri dishes each, as described [28]. Subconfluent VSMCs were deprived of growth supplements for 2 days followed by incubation with KHB buffer for 2 hours.…”

Ras-induced Epigenetic Inactivation of the RRAD (Ras-related Associated with Diabetes) Gene Promotes Glucose Uptake in a Human Ovarian Cancer Model

“…All samples were normalized to ␤-actin. The primer sequences for the glucose transporter have been described previously (29). The sequences of other primers used are listed in Table 3.…”

Effect of stocking density on the expression of glucose transporter protein 1 and other physiological factors in the Lake Victoria Nile tilapia, Oreochromis niloticus (L.)

“…Movement of glucose across the plasma membrane of red blood cells is mediated through facultative glucose transporter protein 1 (Samih et al 2000;Joost and Thorens 2001;Zhao and Keating 2007). Currently, there are 14 members of GLUT protein family (Balmaceda-Aguilera et al 2012;Pyla et al 2013). GLUT 1 protein has been detected in tissues of teleost fish (Wright et al 2000;Moon 2001).…”