Cloning of an L-3-hydroxyacyl-CoA dehydrogenase that interacts with the GLUT4 C-terminus.

Evidence indicates that the carboxy-terminal cytoplasmic domain of glucose transporter 4 (GLUT4) is important for the regulation of GLUT4 in muscle and adipocytes. We cloned from a human skeletal muscle cDNA library a 34-kDa protein which interacts with GLUT4 C-terminal cytoplasmic domain in a two-hybrid system and also with GLUT4 C-terminus synthetic peptide in an in vitro binding assay. This protein, called YP10, showed a high degree (>90%) of sequence homology with l-3-hydroxyacyl-CoA dehydrogenase (HAD) and had a dehydrogenase activity similar to pig heart HAD, which was inhibited by GLUT4 C-terminus synthetic peptide. An antiserum raised against pig heart HAD also reacted with YP10. Western blot analysis using this antiserum revealed abundant immunoreactivity only in the mitochondria- and plasma membrane-enriched fractions of rat adipocytes. Northern blots revealed that YP10 mRNA is most abundant in skeletal and heart muscle. These findings suggest that YP10, a HAD isoform, interacts with GLUT4 at the plasma membrane and may play a role in cross-talk between glucose transport and fatty acid metabolism.

Hrp12, a novel heat-responsive, tissue-specific, phosphorylated protein isolated from mouse liver.

Previous studies from this laboratory identified a 28-kd nonreducible protein, liver-derived immunoinhibitory factor (LDIF) from the mouse liver. Isolation of this protein resulted in the co-purification of another unique protein called heat responsive protein 12 kd (Hrp12). In contrast to LDIF, Hrp12 was totally reducible to a protein of 12 kd suggesting a dimer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) purification, followed by sequencing of an in situ cyanogen bromide digest of membrane bound Hrp12, yielded an internal 20-amino acid polypeptide. Degenerate oligonucleotides made from this peptide were used to screen a murine liver complementary DNA (cDNA) library. A 1240-bp cDNA clone was obtained with an internal 521-bp open reading frame (ORF). Sequence analysis of the 173-amino acid ORF of mouse Hrp12 showed a high degree of homology with a 99 amino acid rat liver-kidney perchloric acid-soluble protein (LKPS) and a 136-amino acid perchloric acid soluble rat protein (PSP). Transcripts for Hrp12 were mainly restricted to the liver and kidney in mouse and man. The protein was estimated to be approximately 0.8% of the total liver-soluble cytosolic protein. A zoo-blot probed at moderate stringency with labeled cDNA revealed a strong conservation of the gene in all of the mammalian species tested. Analysis of the protein structure of Hrp12 revealed motifs predicted to be targets for protein kinase C (PKC). More importantly, purified mouse Hrp12 could be phosphorylated in vitro with PKC. The protein had significant similarity to DnaK heat shock protein (Hsp)70 and contained a 54-amino acid stretch with sequence similarity to Hsp90. This prompted us to investigate the heat shock response of Hrp12. Isolated hepatocytes and hepatoma cells were exposed to different heat shock temperatures (39.5 degrees C, 42.5 degrees C, and 44.5 degrees C); and then total RNA was extracted and Northern analysis carried out. The message for this novel protein responded atypically to heat shock. Although the steady-state level of the message increased after heat shock, a marked oscillatory pattern was superimposed on it. In contrast, the steady-state levels of Hsp90 and Hsp70 messenger RNA (mRNA) were found to respond to heat shock in the expected manner. Finally, the amount of Hrp12 protein was also found to increase after heat shock in a manner that was consistent with heat-responsive proteins.

Modulation of GLUT1 intrinsic activity in clone 9 cells by inhibition of oxidative phosphorylation.

Brief (1-2 h) exposure of Clone 9 cells to inhibitors of oxidative phosphorylation such as azide is known to markedly increase glucose uptake. Clone 9 cells express GLUT1 but not GLUT2, -3, and -4, and the azide effect was not accompanied by any increase in cellular or plasma membrane GLUT1 level. To identify the molecular event underlying this apparent increase in GLUT1 intrinsic activity, we studied the acute effects of azide on the substrate binding activity of GLUT1 in Clone 9 cells by measuring glucose-sensitive cytochalasin B binding. The glucose-displaceable, cytochalasin B binding activity was barely detectable in membranes isolated from Clone 9 cells under control conditions but was readily detectable after a 60-min incubation of cells in the presence of 5 mM azide showing a 3-fold increase in binding capacity with no change in binding affinity. Furthermore, the cytochalasin B binding activity of purified human erythrocyte GLUT1 reconstituted in liposomes was significantly reduced in the presence of cytosol derived from azide-treated Clone 9 cells but not in the presence of cytosol from control cells; this effect was heat-labile and abolished by the presence of the peptide corresponding to the GLUT1 COOH-terminal sequence. These results suggest that a cytosolic protein in Clone 9 cells binds to GLUT1 at its COOH-terminal domain and inhibits its substrate binding and that azide-induced metabolic alteration releases GLUT1 from this inhibitory interaction. Studying the binding of cytosolic proteins derived from 35S-labeled Clone 9 cells to glutathione S-transferase fusion protein containing glucose transporter COOH-terminal sequences, we identified 28- and 70-kDa proteins that bind specifically to the cytoplasmic domain of GLUT1 and GLUT4 in vitro. We also found a 32P-labeled, 85-kDa protein that binds to GLUT4 but not to GLUT1 and only in cytosol derived from azide-treated cells. The roles, if any, of these glucose transporter-binding proteins in the azide-sensitive modulation of GLUT1 substrate binding activity in Clone 9 cells are yet to be determined.

ATP-sensitive binding of a 70-kDa cytosolic protein to the glucose transporter in rat adipocytes.

We have identified a 70-kDa cytosolic protein (GTBP70) in rat adipocytes that binds to glutathione S-transferase fusion proteins corresponding to the cytoplasmic domains of the facilitative glucose transporter isoforms Glut1, Glut2, and Glut4. GTBP70 did not bind to irrelevant fusion proteins, indicating that the binding is specific to the glucose transporter. GTBP70 binding to the glucose transporter showed little isoform specificity but was significantly subdomain-specific; it bound to the C-terminal domain and the central loop, but not to the N-terminal domain of Glut4. The GTBP70 binding to Glut4 was not affected by the presence of 2 mM EDTA, 2.4 mM Ca2+, or 150 mM K+. The binding was inhibited by ATP in a dose-dependent manner, with 50% inhibition at 10 mM ATP. This inhibition was specific to ATP, as ADP and AMP-PCP (adenosine 5'-(beta, gamma-methylenetriphosphate)) were without effect. GTBP70 did not react with antibodies against phosphotyrosine, phosphothreonine, or phosphoserine, suggesting that it is not a phosphoprotein. The binding of GTBP70 to Glut4 was not affected by the pretreatment of adipocytes with insulin. When these experiments were repeated using rat hepatocyte cytosols, no ATP-sensitive 70-kDa protein binding to the glucose transporter fusion proteins was evident, suggesting that either GTBP70 expression or its function is cell-specific. These findings strongly suggest the possibility that GTBP70 may play a key role in glucose transporter regulation in insulin target cells such as adipocytes.

A cross-country study of commercial contraceptive sales programs: factors that lead to success.

This report presents results from a comparison of subsidized commercial contraceptive marketing programs in 11 developing countries, and makes recommendations for program planning based on findings. The analysis, based on a combined quantitative approach of analysis of variance, factor analysis, and regression analysis, suggests that the successful sale of oral contraceptives is related to level of socioeconomic development, the absence of prescription requirements, and the level of commitment to family planning activities. On the other hand, the successful sale of condoms is related to marketing factors, which include price and advertising. A final discussion of the possible effect of qualitative factors suggests cultural and historical variables that could account for the remaining unexplained variations in sales performances.