Abstract
Connective tissue activating peptide-III (CTAP-III) is a component of platelet alpha-granules which elicits a series of responses in connective tissue cells referred to as activation, including increased glucose consumption and mitogenesis and increased secretion of hyaluronic acid and glycosaminoglycans. As anticipated by a requirement for glucose or glucose precursors in the activation process, an early event following CTAP-III activation of connective tissue cells is an increase in glucose transport. The present study investigates the molecular basis for this increase in glucose transport. Murine 3T3-F442A fibroblasts were found to respond to CTAP-III in a manner similar to human connective tissue cells (synovial cells, chondrocytes, skin fibroblasts). CTAP-III increases the rate of glucose transport to similar extents at 4 and 24 h, and at physiologic (micrograms/ml) concentrations of CTAP-III. A proteolytic cleavage product of recombinant CTAP-III (rCTAP-III-Leu-21 (des-1-15)), also known as neutrophil-activating peptide-2 (NAP-2), was found to be equally effective as CTAP-III, whereas NAP-1/interleukin-8, another member of the CTAP-III super-family, was ineffective in stimulating glucose transport. This contrasts with neutrophil chemotaxis, in which CTAP-III (des-1-15)/NAP-2 acts similarly to NAP-1/interleukin 8 while CTAP-III is ineffective. CTAP-III appears to elicit a different type of glucose transport response than many other growth factors in that its response is sustained (greater than or equal to 24 h) rather than transient (peak approximately 4 h) in confluent as well as in subconfluent cells. Western blot analysis using antibodies to the GLUT-1 glucose transporter revealed an increased level of GLUT-1 protein in response to CTAP-III isoforms that corresponded in magnitude (on a percentage basis) to the increased level of glucose transport. The increased levels of GLUT-1 protein in response to CTAP-III and rCTAP-III-Leu-21 (des-1-15)/NAP-2 were accompanied by an increase in levels of GLUT-1 mRNA of a magnitude sufficient to account for observed increased levels of GLUT-1. These results are consistent with CTAP-III isoforms stimulating glucose transport in connective tissue cells by increasing levels of GLUT-1 mRNA and is one of the few known instances in which increases in levels of GLUT-1 mRNA and protein are sufficient to account for observed increases in glucose transport. They also provide further evidence that CTAP-III (des-1-15)/NAP-2 binds to more than one type of receptor and that CTAP-III acts in a manner different than other well characterized growth factors (e.g. platelet-derived growth factor, transforming growth factor-beta) in that it causes a sustained (greater than or equal to 24 h) elevation in glucose transport in confluent as well as subconfluent cells.
Highlights
Connective tissue activating peptide-I11 (CTAP-111) served increased levels of GLUT-1
Murine 3T3-F442A fibroblasts were found to transforming growth factor+) in that it causes a susrespond to CTAP-I11 in a manner similar to human tained (224 h) elevation in glucose transport in conconnective tissue cells
Western blot analysis using antibodies to the the actionsof insulin onglucose metabolism and transport have been extensively studied for manyyears, very little effort has been expended to understand how the body regulates glucose metabolism in cells,includingconnective tissue cells, that are relatively insensitive to insulin but occasionally have dramatically increased demands for glucose
Summary
Connective tissue activating peptide-I11 (CTAP-111) served increased levels of GLUT-1. I11 activation of connective tissue cells is an increase more than one type of receptor and thatCTAP-I11acts in glucose transport. Level of GLUT-1 protein in response to CTAP-I11 iso- CTAP-I11 is a major component of plateleta-granules forms thatcorresponded in magnitude (ona percentage (present at 1000 times the quantity of other growth factors basis) to the increased level of glucose transport. Purity of the isoforms was tive tissue cells, since glucose provides the substrate assessed by SDS-PAGEincombination withsilver stainingand for the GAGS of the extracellular matrix which are synthe- Western blotting with anti-CTAP-111 antibody. Aliquots (50 fig of protein) of cell lysates body Bg is a polyclonal anti-glucose transporter antibody obtained from the glucose transport experiments described above were diluted by immunizing arabbit with purifiedband 4.5
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