Larry L. Louters

Effects of cinnamaldehyde on the glucose transport activity of GLUT1

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 2.0mM. 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 cinnamaldehyde is not a major contributor to the anti-diabetic properties of cinnamon.

Acute effects of troglitazone and nitric oxide on glucose uptake in L929 fibroblast cells.

The thiazolidinedione class of antidiabetic drugs, including troglitazone, has an insulin-sensitizing effect for patients with type 2 diabetes. However, in some tissues, studies have shown that troglitazone also has an acute insulin-independent effect on glucose uptake. To determine the extent of this acute action of troglitazone, the effect of troglitazone on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells was measured. Troglitazone stimulated 2DG uptake in a dose dependent manner with a maximum stimulation of >300% at 5-10 microM. In addition, nitric oxide has been shown to stimulate glucose uptake in peripheral muscle tissue. Therefore, the effect of nitric oxide on 2DG uptake in L929 cells was also investigated using the nitric oxide donor, sodium nitroprusside (SNP). SNP stimulated 2DG uptake by >200% with a maximally effective concentration of 5 mM. The combined effect of maximally effective concentrations of both stimulants (10 microM troglitazone + 5 mM SNP) was not additive suggesting a shared pathway for 2DG uptake. However, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 50 microM) had no effect on troglitazone stimulated 2DG uptake, indicating that the troglitazone and nitric oxide pathways converge after nitric oxide production. In addition, 12.5 microM dantrolene was shown to have no effect on either troglitazone or SNP stimulated 2DG uptake suggesting that these stimulatory effects are independent of changes in calcium ion concentrations. These data provide important evidence for the acute regulation of glucose transport through GLUT 1 transporters.

Dual action of phenylarsine oxide on the glucose transport activity of GLUT1

An early event in the toxic effects of organic arsenic compounds, such as phenylarsine oxide (PAO), is an inhibition of glucose uptake. Glucose uptake involving the glucose transporter, GLUT4 is inhibited by PAO indicating an importance of vicinal sulfhydryls in insulin-stimulated glucose uptake. However, the data on effects of PAO on GLUT1 are conflicting. This study investigated the effects of PAO on glucose uptake in L929 fibroblast cells, cells, which express only GLUT1. The data presented here reveal a dual effect of PAO. At low concentrations or short exposure times PAO stimulated glucose uptake reaching a peak activation of about 400% at 3 microM. At higher concentrations (40 microM), PAO clearly inhibited glucose uptake. At intermediate concentrations (10 microM), PAO had no effect under basal conditions but completely inhibited activation of glucose uptake by glucose deprivation and partially inhibited methylene blue-stimulated glucose uptake. PAO increased the specific binding of cytochalasin B to GLUT1 suggesting a direct interaction with the transporter. These data are most consistent with PAO interacting with multiple proteins that regulate the activity of this transporter, one of which may be GLUT1 itself. The identity of these proteins will require further investigation.

Differential regulation of GLUT1 activity in human corneal limbal epithelial cells and fibroblasts

The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. It has been shown that GLUT1 in L929 fibroblast cells and other cell lines can be acutely activated by a variety agents. However, the acute regulation of glucose uptake in corneal cells has not been systematically investigated. Therefore, we examined glucose uptake in an immortalized human corneal-limbal epithelial (HCLE) cell line and compared it to glucose uptake in L929 fibroblast cells, a cell line where glucose uptake has been well characterized. We report that the expression of GLUT1 in HCLE cells is 6.6-fold higher than in L929 fibroblast cells, but the HCLE cells have a 25-fold higher basal rate of glucose uptake. Treatment with agents that interfere with mitochondrial metabolism, such as sodium azide and berberine, activate glucose uptake in L929 cells over 3-fold, but have no effect on glucose uptake HCLE cells. Also, agents known to react with thiols, such cinnamaldehyde, phenylarsine oxide and nitroxyl stimulate glucose uptake in L929 cells 3-4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself.

Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells

The widely expressed mammalian glucose transporter, GLUT1, can be acutely activated in L929 fibroblast cells by a variety of conditions, including glucose deprivation, or treatment with various respiration inhibitors. Known thiol reactive compounds including phenylarsine oxide and nitroxyl are the fastest acting stimulators of glucose uptake, implicating cysteine biochemistry as critical to the acute activation of GLUT1. In this study, we report that in L929 cells glucose uptake increases 6-fold as the pH of the uptake solution is increased from 6 to 9 with the half-maximal activation at pH 7.5; consistent with the pKa of cysteine residues. This pH effect is essentially blocked by the pretreatment of the cells with either iodoacetamide or cinnamaldehyde, compounds that form covalent adducts with reduced cysteine residues. In addition, the activation by alkaline pH is not additive at pH 8 with known thiol reactive activators such as phenylarsine oxide or hydroxylamine. Kinetic analysis in L929 cells at pH 7 and 8 indicate that alkaline conditions both increases the Vmax and decreases the Km of transport. This is consistent with the observation that pH activation is additive to methylene blue, which activates uptake by increasing the Vmax, as well as to berberine, which activates uptake by decreasing the Km. This suggests that cysteine biochemistry is utilized in both methylene blue and berberine activation of glucose uptake. In contrast a pH increase from 7 to 8 in HCLE cells does not further activate glucose uptake. HCLE cells have a 25-fold higher basal glucose uptake rate than L929 cells and the lack of a pH effect suggests that the cysteine biochemistry has already occurred in HCLE cells. The data are consistent with pH having a complex mechanism of action, but one likely mediated by cysteine biochemistry.

Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells.

Nitroxyl (HNO) has a unique, but varied, set of biological properties including beneficial effects on cardiac contractility and stimulation of glucose uptake by GLUT1. These biological effects are largely initiated by HNOs reaction with cysteine residues of key proteins. The intracellular production of HNO has not yet been demonstrated, but the small molecule, hydroxylamine (HA), has been suggested as possible intracellular source. We examined the effects of this molecule on glucose uptake in L929 fibroblast cells. HA activates glucose uptake from 2 to 5-fold within two minutes. Prior treatment with thiol-active compounds, such as iodoacetamide (IA), cinnamaldehyde (CA), or phenylarsine oxide (PAO) blocks HA-activation of glucose uptake. Incubation of HA with the peroxidase inhibitor, sodium azide, also blocks the stimulatory effects of HA. This suggests that HA is oxidized to HNO by L929 fibroblast cells, which then reacts with cysteine residues to exert its stimulatory effects. The data suggest that GLUT1 is acutely activated in L929 cells by modification of cysteine residues, possibly the formation of a disulfide bond within GLUT1 itself.

Vanadates form insoluble complexes with histones

Vanadium oxoanions are known to have a variety of physiological effects including insulin-like activity, inhibition of phosphotyrosine phosphatases, as well as direct interactions with a variety of cellular proteins, such as microtubules. In this study, vanadate was found to form insoluble complexes with histones, as well as other positively charged proteins, in a concentration dependent fashion. This interaction occurred over a 0.5-10 mM range which corresponds to the concentration range required for many of vanadates known physiological effects. Results from precipitation experiments using vanadate solutions with or without the yellow-orange decavanadate indicated that the decamer form is primarily responsible for this precipitation. Vanadate was able to selectively precipitate histones from soluble chromatin as well as from a soluble bacterial protein extract to which a low concentration of histones had been added. Vanadate was also able to effectively precipitate histone from solutions as low as 0.006 mg/mL histone. Thus, the selective precipitation of histones and other positively charged proteins by vanadate can be utilized as a tool for protein purification. In addition, this interaction may provide insight into the mechanisms for the physiological effects of vanadate.

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells, and inhibits uptake in HCLE cells

AIMS Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1. MAIN METHODS We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators. KEY FINDINGS Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 μM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells. SIGNIFICANCE The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration.

Histone H4 stimulates glucose uptake through the insulin receptor

Histone H4 stimulates the uptake of glucose in rat adipocytes and muscle cells. However, the mechanism of this unusual activity is not known. Therefore, we have begun to investigate the mechanism by which histone H4 stimulates the glucose uptake in rat adipocytes. We report that histone H4 requires 15-20 min to achieve its maximum effect and its time course is virtually indistinguishable from the time course of insulin itself. Reduction of the concentration of insulin receptors on the surface of adipocytes, either by trypsin digestion of the receptor, or by insulin-induced down regulation of the receptor, reduced the histone H4 effect as well as the insulin effects. Also, quercetin, a bioflavenoid that inhibits the insulin receptor tyrosine kinase activity, inhibits the actions of both histone H4 and insulin. However, histone H4 activity is somewhat more resistant to these interventions than insulin activity. In contrast to the activity of insulin, histone H4 does not appear to be able to down regulate the insulin receptor, since the pretreatment of adipocytes with histone H4 did not affect the subsequent actions of either insulin or histone H4. Finally, Scatchard analysis of the binding of 125I-insulin in the presence and absence of histone H4 increases the specific binding of insulin in a concentration dependent fashion. Histone H2b, a histone that does not have insulin-like activity, does not affect insulin binding. Taken together, these data suggest that the greatest portion of the insulin-like activity of histone H4 is initiated at the insulin receptor. However, the interaction of histone H4 and the insulin receptor is more complex than a simple binding of H4 to the insulin binding site. These studies may provide additional insight into alternate mechanisms for activation of the insulin receptor.

Determination of GLUT1 Oligomerization Parameters using Bioluminescent Förster Resonance Energy Transfer

The facilitated glucose transporter GLUT1 (SLC2A1) is an important mediator of glucose homeostasis in humans. Though it is found in most cell types to some extent, the level of GLUT1 expression across different cell types can vary dramatically. Prior studies in erythrocytes—which express particularly high levels of GLUT1—have suggested that GLUT1 is able to form tetrameric complexes with enhanced transport activity. Whether dynamic aggregation of GLUT1 also occurs in cell types with more modest expression of GLUT1, however, is unclear. To address this question, we developed a genetically encoded bioluminescent Förster resonance energy transfer (BRET) assay using the luminescent donor Nanoluciferase and fluorescent acceptor mCherry. By tethering these proteins to the N-terminus of GLUT1 and performing saturation BRET analysis, we were able to demonstrate the formation of multimeric complexes in live cells. Parallel use of flow cytometry and immunoblotting further enabled us to estimate the density of GLUT1 proteins required for spontaneous oligomerization. These data provide new insights into the physiological relevance of GLUT1 multimerization as well as a new variant of BRET assay that is useful for measuring the interactions among other cell membrane proteins in live cells.