We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.

There is accumulating evidence that cinnamon extracts contain components that enhance insulin action. However, little is know about the effects of cinnamon on non-insulin stimulated glucose uptake. Therefore, the effects of cinnamaldehyde on the glucose transport activity of GLUT1 in L929 fibroblast cells were examined under both basal conditions and conditions where glucose uptake is activated by glucose deprivation. The data reveal that cinnamaldehyde has a dual action on the glucose transport activity of GLUT1. Under basal conditions it stimulates glucose uptake and reaches a 3.5 fold maximum stimulation at 2.0 mM. However, cinnamaldehyde also inhibits the activation of glucose uptake by glucose deprivation in a dose dependent manner. Experiments with cinnamaldehyde analogs reveal that these activities are dependent on the α,β-unsaturated aldehyde structural motif in cinnamaldehyde. The inhibitory, but not the stimulatory activity of cinnamaldehyde was maintained after a wash-recovery period. Pretreatment of cinnamaldehyde with thiol-containing compounds, such as β-mercaptoethanol or cysteine, blocked the inhibitory activity of cinnamaldehyde. These results suggest that cinnamaldehyde inhibits the activation of GLUT1 by forming a covalent link to target cysteine residue/s. This dual activity of cinnamaldehyde on the transport activity of GLUT1 suggests that cinnamaldehye is not a major contributor to the anti-diabetic properties of cinnamon.

Berberine, which has a long history of use in Chinese medicine, has recently been shown to have efficacy in the treatment of diabetes. While the hypoglycemic effect of berberine has been clearly documented in animal and cell line models, such as 3T3-L1 adipocytes and L6 myotube cells, the mechanism of action appears complex with data implicating activation of the insulin signaling pathway as well as activation of the exercise or AMP kinase-mediated pathway. There have been no reports of the acute affects of berberine on the transport activity of the insulin-insensitive glucose transporter, GLUT1. Therefore, we examined the acute effects of berberine on glucose uptake in L929 fibroblast cells, a cell line that express only GLUT1. Berberine activated glucose uptake reaching maximum stimulation of five fold at >40 μM. Significant activation (P<0.05) was measured within five minutes reaching a maximum by 30 minutes. The berberine effect was not additive to the maximal stimulation by other known stimulants, azide, methylene blue or glucose deprivation, suggesting shared steps between berberine and these stimulants. Berberine significantly reduced the Km of glucose uptake from 6.7 ± 1.9 mM to 0.55 ± 0.08 mM, but had no effect on the Vmax of uptake. Compound C, an inhibitor of AMP kinase, did not affect berberine-stimulated glucose uptake, but inhibitors of downstream kinases partially blocked berberine stimulation. SB203580 (inhibitor of p38 MAP kinase) did not affect submaximal berberine activation, but did lower maximal berberine stimulation by 26%, while PD98059 (inhibitor of ERK kinase) completely blocked submaximal berberine activation and decreased the maximal stimulation by 55%. It appears from this study that a portion of the hypoglycemic effects of berberine can be attributed to its acute activation of the transport activity of GLUT1.

Nitroxyl (HNO) is a molecule of significant interest due to its unique pharmacological properties, particularly within the cardiovascular system. A large portion of HNO biological effects can be attributed to its reactivity with protein thiols, where it can generate disulfide bonds. Evidence from studies in erythrocytes suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond. However, there are no reports that document the effects of HNO on glucose uptake. Therefore, we examined the acute effects of Angeli’s salt (AS), a HNO donor, on glucose uptake activity of GLUT1 in L929 fibroblast cells. We report that AS stimulates glucose uptake with a maximum effective concentration of 5.0 mM. An initial 7.2-fold increase occurs within 2 min, which decreases and plateaus to a 4.0-fold activation after 10 min. About 60% of the 4.0-fold activation recovers within 10 min, and 40% remains after an hour. The activation is blocked by the pretreatment of cells with thiol-reactive compounds, iodoacetamide (0.75 mM), cinnamaldehyde (2.0 mM), and phenylarsine oxide (10 μM). The effects of AS are not additive to the stimulatory effects of other acute activators of glucose uptake in L929 cells, such as azide (5 mM), berberine (50 μM), or glucose deprivation. These data suggest that GLUT1 is acutely activated in L929 cells by the formation of a disulfide bond, likely within GLUT1 itself.

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