Immunogold labeling study of the distribution of GLUT-1 and GLUT-4 in cardiac tissue following stimulation by insulin or ischemia

Abstract

Whereas glucose transporter 1 (GLUT-1) is thought to be responsible for basal glucose uptake in cardiac myocytes, little is known about its relative distribution between the different plasma membranes and cell types in the heart. GLUT-4 translocates to the myocyte surface to increase glucose uptake in response to a number of stimuli. The mechanisms underlying ischemia-and insulin-mediated GLUT-4 translocation are known to be different, raising the possibility that the intracellular destinations of GLUT-4 following these stimuli also differ. Using immunogold labeling, we describe the cellular localization of these two transporters and investigate whether insulin and ischemia induce differential translocation of GLUT-4 to different cardiac membranes. Immunogold labeling of GLUT-1 and GLUT-4 was performed on left ventricular sections from isolated hearts following 30 min of either insulin, ischemia, or control perfusion. In control tissue, GLUT-1 was predominantly (76%) localized in the capillary endothelial cells, with only 24% of total cardiac GLUT-1 present in myocytes. GLUT-4 was found predominantly in myocytes, distributed between sarcolemmal and T tubule membranes (1.84 Ϯ 0.49 and 1.54 Ϯ 0.33 golds/ m, respectively) and intracellular vesicles (127 Ϯ 18 golds/ m 2 ). Insulin increased T tubule membrane GLUT-4 content (2.8 Ϯ 0.4 golds/ m, P Ͻ 0.05) but had less effect on sarcolemmal GLUT-4 (1.72 Ϯ 0.53 golds/ m). Ischemia induced greater GLUT-4 translocation to both membrane types (4.25 Ϯ 0.84 and 4.01 Ϯ 0.27 golds/ m, respectively P Ͻ 0.05). The localization of GLUT-1 suggests a significant role in transporting glucose across the capillary wall before myocyte uptake via GLUT-1 and GLUT-4. We demonstrate independent spatial translocation of GLUT-4 under insulin or ischemic stimulation and propose independent roles for T-tubular and sarcolemmal GLUT-4. glucose transporter; immunogold electron microscopy

Relaxin remodels fibrotic healing following myocardial infarction

“…14 The rationale for Glut-1 staining was based on previous studies using endothelial expression of Glut-1 as a marker of active de novo capillary formation; where 75% of Glut-1 expression in the heart was localized to the capillary endothelium. 23 The morphological assessment of its expression was used as a surrogate marker of relaxin's pro-angiogenic properties, and to supplement the a-SMA staining of larger vessels. In this study, we were able to demonstrate that relaxin's anti-fibrotic, anti-apoptotic and pro-angiogenic actions acted in synergy during post-infarct healing without overt adverse outcomes.…”

“…Serial sections were also used for immunohistochemical staining and morphometric analysis of various selected markers associated with collagen turnover, utilizing monoclonal antibodies to a-smooth muscle actin (a-SMA; a marker for myofibroblast differentiation; 1:250 dilution; Dako) and MMP-13 (collagenase-3; 1:100 dilution, Calbiochem) in addition to a polyclonal antibody to TGF-b1 (1:200 dilution, Santa-Cruz Biotechnology). Macrophage infiltration (inflammation) was assessed utilizing a monoclonal antibody to f4/80 (a transmembrane protein present on the cell surface of mouse macrophages; 1:50 dilution; Serotec); angiogenesis was detected by a-SMA (smooth muscle) and glucose transporter (Glut-1; endothelial 23 ) staining of vessels using a polyclonal antibody to Glut-1 (1:1000 dilution; Chemicon International); while cardiomyocyte apoptosis was detected using the ApopTag (TUNEL based) Plus Peroxidase assay kit (Millipore). Detection of antibody staining was completed using various DAB-based kits: the Vectastain ABC kit for a-SMA, f4/80 and Glut-1, the Dako ARK (mouse on mouse) kit for MMP-13 and the Dako EnVision anti-rabbit kit for TGF-b1.…”

A common trafficking route for GLUT4 in cardiomyocytes in response to insulin, contraction and energy-status signalling

“…Heart muscle glucose transport shares many similarities to that occurring in skeletal muscle and is known to be associated with the translocation of the GLUT4 glucose transporter isoform from intracellular reservoir compartments to the limiting membranes of the sarcolemma and the transverse-tubule system of the cardiomyocytes (Fischer et al, 1997). Using immunogold labelling of GLUT4 in heart cells, Slot et al reported insulin stimulation of GLUT4 translocation to both sarcolemma and the transverse-tubules membranes (Slot et al, 1997), whereas Davey et al reported some selectivity in translocation to the transverse-tubules (Davey et al, 2007).…”

Infusion of a biotinylated bis-glucose photolabel: a new method to quantify cell surface GLUT4 in the intact mouse heart

“…GLUT1 is expressed in cardiomyocytes (13,14,51) and endothelial cells (9). GLUT1 is generally considered to be primarily involved in basal heart glucose transport but also undergoes translocation to plasma membranes in response to insulin (14,51).…”

“…GLUT1 is also expressed in the heart, both in cardiac myocytes, where it has a role in basal glucose uptake (14,41,51), and in endothelial cells (9). GLUT1 undergoes modest translocation to the sarcolemma with insulin and ischemia (14,51), as it does in adipocytes (50).…”