Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target.

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Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) pathway in experimental renal failure Chih-Yuan Lin, MD, Shih-Che Hsu, MS, Herng-Sheng Lee, MD, PhD, Shih-Hua Lin, MD, Chien-Sung Tsai, MD, Shih-Ming Huang, PhD, Chun-Che Shih, MD, PhD, Yu-Juei Hsu, MD, PhD Journal of Vascular Surgery Volume 57, Issue 2, Pages 475-485 (February 2013) DOI: 10.1016/j.jvs.2012.07.037 Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 1 Blood biochemistry values for sham-operated (n = 8) and chronic renal failure (CRF; n = 8) rats are shown for (A) serum blood urea nitrogen (BUN), (B) creatinine, and (C) glucose levels, along with (D) systolic blood pressure (SBP). Data are presented as means ± standard error of the mean. *P < .05 vs sham-operated rats. Journal of Vascular Surgery 2013 57, 475-485DOI: (10.1016/j.jvs.2012.07.037) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 2 Glucose transporter-1 (GLUT1) expression in aortic tissue of chronic renal failure (CRF) rats. A, Representative semiquantitative reverse transcriptase-polymerase chain reaction analysis of GLUT1 messenger RNA in aortic tissue from sham-operated and CRF rats. B, The amount of GLUT1 messenger RNA is expressed as the ratio to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) abundance and depicted as percentages of that in sham-operated rats. C, Representative immunoblots of protein samples from homogenates of aortic tissue of sham-operated and CRF rats were labeled with antibodies against GLUT1 or GAPDH. D, GLUT1 protein expression was quantified by computer-assisted densitometry analysis and presented as the ratio to GAPDH expression levels, in relative percentages compared with those in sham-operated rats. Data are presented as means ± standard error of the mean (n = 8). *P < .05 vs sham-operated rats. Journal of Vascular Surgery 2013 57, 475-485DOI: (10.1016/j.jvs.2012.07.037) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 3 Histology and immunohistochemistry of uremic rat aorta. Representative images of hematoxylin and eosin staining (original magnification ×400) of paraffin-embedded aortas from (A) sham-operated and (B) chronic renal failure (CRF) rats. Representative images of immunohistochemical staining (original magnification ×400) of glucose transporter-1 (GLUT1) protein in the aortae of (C) sham-operated and (D) CRF rats. E, Semiquantification of aortic GLUT1 protein was assessed by immunohistochemical rating score and is depicted as percentage of sham-operated control. Data are presented as means ± standard error of the mean (n = 8). *P < .05 vs sham-operated rats. Journal of Vascular Surgery 2013 57, 475-485DOI: (10.1016/j.jvs.2012.07.037) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 4 Glucose transporter-1 (GLUT1) protein abundance in vascular smooth muscle cells treated with indoxyl sulfate (IS). A, Representative immunoblots of total protein lysates isolated from A7r5 cells exposed to IS (250 μM) with and without rapamycin (Rapa; 25 nM) for 24 hours were labeled with antibodies against GLUT1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, Expression of GLUT1 protein was quantified by computer-assisted densitometry analysis and presented as the ratio to GAPDH expression levels, in relative percentages compared with untreated control. Data are presented as means ± standard error of the mean (n = 4). *P < .05 vs untreated control. Journal of Vascular Surgery 2013 57, 475-485DOI: (10.1016/j.jvs.2012.07.037) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 5 Effect of indoxyl sulfate (IS) on glucose transporter-1 (GLUT1) expression and the Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling pathway in vascular smooth muscle cells (VSMCs). A, Representative immunoblots of GLUT1 expression and the Akt/TSC2/mTOR/S6K signaling pathway in VSMCs incubated with IS (250 μM) with or without rapamycin (Rapa) for 3, 6, or 12 hours. GLUT1 protein expression and Akt, TSC2, and S6K phosphorylation were quantified by computer-assisted densitometry analysis and presented as the ratio to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, Levels of phospho-protein immunoreactivity were normalized to their respective total protein immunoreactivity and are expressed as the percent of control. Data are presented as means ± standard error of the mean (n = 4). *P < .05 vs untreated control (0 hours); #P < .05 vs without treatment with Rapa for the same period. Journal of Vascular Surgery 2013 57, 475-485DOI: (10.1016/j.jvs.2012.07.037) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 6 Effects of glucose transporter-1 (GLUT1) inhibitor on the expression of pro-proliferative and antiapoptotic genes in indoxyl sulfate (IS)-stimulated vascular smooth muscle cells. A, Representative semiquantitative reverse transcriptase-polymerase chain reaction analysis of cyclin D1, p21, and p53 messenger RNA expression in A7r5 cells treated with IS (250 μM) with or without GLUT1 inhibitor phloretin (Phlo; 0.1 mM). B-D, The amount of messenger RNA was expressed as the ratio to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) abundance and depicted as percentages of that in untreated control. Data are presented as means ± standard error of the mean (n = 3). *P < .05 vs untreated control; # P < .05 vs treatment with IS. Journal of Vascular Surgery 2013 57, 475-485DOI: (10.1016/j.jvs.2012.07.037) Copyright © 2013 Society for Vascular Surgery Terms and Conditions