Stimulatory effect of lithium on glucose transport in rat adipocytes is not mediated by elevation of IP1

Xiaoli Chen, Ellen G. McMahon, and Eric A. Gulve

Cardiovascular Disease and Diabetes Research, Monsanto Company, St. Louis, Missouri 63167

    ABSTRACT
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Abstract
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Methods
Results
Discussion
References

Lithium has been shown to increase glucose uptake in skeletal muscle and adipose tissues. The therapeutic effect of lithium on bipolar disease is thought to be mediated by its inhibitory effect on myo-inositol-1-monophosphatase (IMPase). We tested the hypothesis that the stimulatory effect of lithium on glucose uptake results from inhibition of IMPase and the resultant accumulation of inositol monophosphates (IP1) by comparing the effects of lithium and a selective IMPase inhibitor, L-690,488, on isolated rat adipocytes. Insulin produced a concentration-dependent stimulation of 2-deoxy-D-[14C]glucose (2-DG) transport (10 µU/ml caused half-maximal activation). Acute exposure to lithium stimulated basal glucose transport activity in a concentration-dependent manner, with a threefold stimulation at 30 mM lithium. Lithium also potentiated insulin-stimulated 2-DG transport. Lithium produced a concomitant increase in IP1 accumulation. In contrast, L-690,488 increased IP1 to levels comparable to those of lithium without stimulatory effects on 2-DG transport. These results demonstrate that stimulatory effects of lithium on glucose transport are not mediated by the inhibition of IMPase and subsequent accumulation of IP1 in rat adipocytes.

prodrug; inositol monophosphatase inhibitor; insulin; isolated adipocytes

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

LITHIUM is a widely used therapeutic agent for the treatment of manic-depressive disorders. The therapeutic effect of lithium on bipolar disorders is believed to result from an inhibitory action on myo-inositol-1-monophosphatase (IMPase) (6). IMPase plays an important role in phosphatidylinositol metabolism in that this enzyme catalyzes the dephosphorylation of inositol monophosphates (IP1) to produce free inositol. Inositol then combines with cytidine monophosphorylphosphatidate to form phosphatidylinositol (17). Thus exposure to lithium could deplete inositol pools in the brain by inhibition of IMPase, leading to reduced cellular responses to neurotransmitters, the receptors of which are coupled to the phosphatidylinositol signal transduction pathway (6).

Lithium has also been shown to affect various endocrine functions (23). For example, lithium enhances glucose transport and metabolism in insulin-sensitive tissues. In manic-depressive disorder patients, lithium treatment improved glucose tolerance (26). A direct effect of lithium on peripheral glucose disposal in vivo has been demonstrated in rats. Lithium was shown to have antidiabetic effects in a pancreatectomized rat model, where lithium at the plasma concentrations attained in the treatment of bipolar disorder completely restored muscle insulin action to normal (22). The effects of lithium to stimulate glucose metabolism can be demonstrated in isolated tissues. Acute exposure to lithium has been shown to increase glucose uptake in isolated rat diaphragm muscle (14) and adipocytes (8) as well as glycogen synthesis in rat hepatocytes (18), diaphragm muscle (14), and adipocytes (8). Furthermore, the effect of lithium on glucose transport in skeletal muscle mimics the persistent effects of exercise rather than those of insulin, suggesting that a more distal step beyond the insulin receptor is involved in the actions of lithium (25).

Despite this wealth of descriptive information regarding lithium effects on glucose metabolism, the mechanisms underlying this action are poorly understood. One possibility is that these effects depend on the inhibitory action of lithium on IMPase. Recent work has identified potent and selective bisphosphonate inhibitors of IMPase (1). The further development of an ester prodrug (L-690,488) that is cell permeable and converted by the action of esterases to an active inhibitor (2) makes possible the selective inhibition of IMPase in intact cells. The prodrug L-690,488 has been shown to elevate the accumulation of IP1 in isolated cells and brain slices (2).

The present work examined the hypothesis that lithium stimulation of glucose transport activity is mediated by inhibition of IMPase. Isolated adipocytes were used for this purpose because 1) the isolated adipocyte preparation is an excellent model system for studying glucose transport and glucose metabolism, 2) lithium has previously been shown to stimulate glucose transport in this preparation, and 3) the likelihood of inhibitor penetration into isolated cells is greater than in intact skeletal muscle preparations. Therefore, the purpose of the present study was twofold: 1) to characterize the effects of lithium on glucose transport activity and inositol phosphate levels and 2) to determine whether the effects of lithium can be mimicked by inhibition of IMPase by the use of a commercially available prodrug of a bisphosphonate inhibitor of IMPase. Our results suggest that lithium-stimulated glucose transport is not mediated by IMPase inhibition in isolated rat adipocytes.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of Isolated Adipocytes

Male Sprague-Dawley rats weighing 140-200 g were fed with standard Purina Chow and water ad libitum. Epididymal fat pads were isolated under pentobarbital sodium anesthesia (5 mg/100 g body weight ip), and adipocyte suspensions were prepared by treating the fat pads with a crude collagenase, as described by Rodbell (21) with modifications. In most experiments, epididymal fat pads were isolated from three animals and rinsed in warmed saline solution (pH 7.4). Each 25-ml polyethylene Erlenmeyer flask contained two or three fat pads that had been pooled and cut into pieces ~2 mm in diameter. All incubations were carried out in a freshly prepared Krebs Ringer phosphate (KRP) buffer (with the following composition in mM: 125 NaCl, 5 KCl, 1 KH2PO4, 10 HEPES, 1.25 CaCl2 · 2H2O, and 1.25 MgSO4 · 7H2O) containing 1% fraction V BSA and at a pH of 7.4 at 37°C. The fat tissues were dispersed into small fragments by incubating for 1 h with 5 ml of collagenase solution (2.5 mM of glucose and 1 mg/ml of collagenase in KRP buffer) in a shaking incubator (120 cycles/min) at 37°C. Cells were then filtered through nylon mesh. All the cells from different flasks were pooled, washed three times with fresh KRP buffer (40 ml/wash), and centrifuged (1,000 rpm for 30 s). The cells were resuspended in KRP buffer to the desired density. An adipocrit of 25% gave a cell suspension of ~1 million adipocytes/ml.

Glucose Transport Studies

The assay for glucose transport was modified from the method described by Olefsky (19) for adipocyte incubation and uptake of 2-deoxy-D-[14C]glucose (2-DG). 2-DG is transported across the cell membrane and phosphorylated like D-glucose, although it cannot be further metabolized (27). The cell suspension was divided into aliquots (180 µl · sample of 2.5 × 105 cells-1 · ml-1). Triplicate or quadruplicate samples were distributed to 12 × 75-mm polypropylene tubes containing 90 µl KRP buffer with or without the addition of lithium, IMPase inhibitor, or insulin. Because addition of lithium slightly alters the osmotic strength of the incubation medium, the nonpenetrating solute mannitol was added at a level sufficient to match the extra osmolarity resulting from lithium. This ensured that osmolarity was constant across all experimental groups and did not contribute to any observed effects of lithium treatment. After the treatments under the experimental conditions for the designated time periods as described in Figs. 1-5, the transport assay was initiated by adding 30 µl of 2-DG with a final specific activity of 6 µCi/µmol (at a final 2-DG concentration of 0.1 mM).

The transport assay was terminated by transferring a 200-µl aliquot of cell suspension into small polyethylene microcentrifuge tubes containing 100 µl dinonyl phthalate. The cells and the buffer were rapidly separated by centrifugation at 14,000 rpm for 8 s. Tubes were then cut through the oil layer. The cells (the upper layer) were transferred to scintillation vials, scintillation cocktail (EcoLite) was added, and radioactivity was determined by scintillation counting. The residual amount of sugar trapped in the extracellular water space of the cell layers was determined in parallel incubations for each experiment by use of a nonpenetrating marker ([14C]mannitol), as described by Gliemann et al. (13). The extracellular space volume was ~0.07% of total space. All 2-DG uptake data were corrected for this factor. Data were normalized to the dry weight of cells, determined for each cell preparation (with weight of buffer subtracted). The dry weight of cells was routinely 20-30 mg per ml for cell suspensions used for glucose transport assay.

Uptake of 2-DG was markedly stimulated by insulin, with a maximum response of ~20-fold over basal (e.g., see Fig. 2A), and half-maximal activation (EC50) was observed at ~10 µU/ml (data not shown). The transport assay was terminated after a 15-min incubation with 2-DG, conditions under which maximally effective insulin-stimulated 2-DG uptake was linear.

Both basal and insulin-stimulated 2-DG transport was dramatically attenuated by cytochalasin B, an inhibitor of glucose transporters (e.g., 50 µM cytochalasin B reduced maximally effective insulin-stimulated 2-DG uptake by >98%).

Measurement of Inositol Phosphate Production

Labeling and incubation. These procedures were performed according to the methods of Pennington and Martin (20) with modifications. Isolated adipocytes with an adipocrit of 25-30% (160 mg dry weight/ml) were preincubated in triplicate with myo-[3H]inositol (80 µCi/ml) in a total volume of 750 µl in a shaking water bath for 2 h at 37°C. The cell suspensions were centrifuged and washed four times with KRP buffer in 20× cell volume. To prevent cell loss, during washing and centrifugation, stiff tubing was inserted into the centrifuge tube, and buffer was aspirated through a vacuum line connected to the tubing. The cell suspensions were concentrated to 500 µl per sample after the final wash, and the cells were then incubated with agonists at 37°C for the indicated time periods. The reactions were terminated by transferring 500-µl cell suspensions to 2 ml of ice-cold chloroform-methanol (1:2; vol/vol).

Lipid extraction. Lipids were extracted by the method of Bligh and Dyer (7) with modifications. Thus 0.66 ml of chloroform and 0.66 ml of water were added into the mixture of sample and chloroform-methanol. The phases were separated by centrifugation at 2,500 rpm for 20 min at 4°C. An aliquot of the upper aqueous phase was removed for determination of inositol phosphates (IPs).

Separation and assay of IPs. IPs were measured by the method of Berridge et al. (5). Briefly, the 3H-labeled IPs were separated on anion exchange columns of Dowex 1-X8 (100-200 mesh, formate form, 1 ml). After the aqueous samples were loaded (1.8 ml), the columns were washed with distilled water (50 ml), 5 mM disodium tetraborate, and 60 mM sodium formate (30 ml) to remove free [3H]inositol and [3H]glycerophosphoinositol, respectively. IP1, inositol bisphosphate (IP2), and inositol trisphosphate (IP3) were then sequentially eluted with 0.1 M formic acid-0.2 M ammonium formate (30 ml), 0.1 M formic acid-0.4 M ammonium formate (25 ml), and 0.1 M formic acid-1.0 M ammonium formate (25 ml), respectively. These fractions were then counted for radioactivity in a liquid scintillation counter.

Statistical Analysis

For each experiment, data are expressed as means (triplicate or quadruplicate samples for each treatment group) ± SE. The significance of differences in each independent experiment was determined by nonparametric ANOVA and the least squares means procedure for paired groups, or a one-tailed ANOVA followed by Tukey's Studentized range test for multiple comparisons. Experiments were performed multiple times for confirmation (see legends to Figs. 1-5). EC50 was estimated from a four-parameter logistic regression equation.

Materials

Rats were purchased from Charles River Laboratory (Portage, MI). Collagenase (type 1) was obtained from Worthington Biochemical (Freehold, NJ). Porcine insulin (Iletin II) was purchased from Eli Lilly (Indianapolis, IN). Lithium chloride, cytochalasin B, BSA, and 2-DG were purchased from Sigma Chemical (St. Louis, MO). L-690,488 was obtained from Tocris Cookson (St. Louis, MO). 2-[14C]DG and [14C]mannitol were purchased from American Radiolabeled Chemicals (St. Louis, MO). D-myo-[3H]inositol was purchased from Du Pont-NEN (Boston, MA). Dowex 1-X8 was obtained from Bio-Rad Laboratories (Hercules, CA).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of Lithium on Basal Glucose Transport

The basal 2-DG uptake rate was 1.78 nmol · 15 min-1 · 106 adipocytes-1 in the experiment shown in Fig. 1. Lithium increased the basal 2-DG uptake rate in a concentration-dependent manner, with an approximate threefold stimulation at 30 mM. Time-course experiments (2.5-30 min preincubation) demonstrated that lithium (10 mM) stimulated glucose transport activity rapidly, with a maximal effect measured after 5 min of preincubation before the 2-DG transport assay (data not shown). In most of the experiments described below, 30 mM lithium was used to maximize the effects on glucose transport.


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Fig. 1.   Concentration-dependent effect of lithium on basal 2-deoxy-D-[14C]glucose (2-DG) transport in adipocytes. Cells were preincubated with lithium at concentrations indicated for 30 min at 37°C. Uptake of 2-DG was determined over 15 min as described in METHODS. Values are means ± SE of 4 samples. * P < 0.05, significantly different from basal (LiCl 0 mM).

Effect of Lithium on Insulin-Stimulated 2-DG Transport

In addition to the effect of lithium on basal 2-DG transport, insulin-induced 2-DG transport was potentiated by lithium (Fig. 2A). The lithium potentiation of insulin action occurred only at submaximal insulin concentrations. The potentiation of lithium on insulin-stimulated 2-DG transport became less marked at insulin concentrations approaching the maximally effective concentration, where the effects of lithium and insulin were merely additive. When 2-DG uptake was expressed as a percentage of the maximal insulin effect, a substantial shift in the insulin concentration-response curve was apparent (Fig. 2B). The effect of lithium to potentiate glucose transport activity at low insulin concentrations was seen reproducibly in several different experiments. These data suggest that, in addition to stimulating glucose transport in the absence of insulin, lithium also enhances insulin sensitivity in rat adipocytes.


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Fig. 2.   Effect of lithium on insulin-stimulated 2-DG uptake. Cells were incubated with increasing concentrations of insulin in the absence or presence of 30 mM lithium. Cells were preincubated with lithium for 30 min and with insulin for the last 15 min. Uptake of 2-DG was then determined as described in METHODS. Values are means ± SE of triplicate samples. A: data presented as nmol · 15 min-1 · 106 cells-1. B: data expressed as the increment induced by insulin treatment and normalized to response at maximally effective concentration of insulin (10 mU/ml). Similar results were obtained in 3 independent experiments. * P < 0.05, significantly different from paired control group (without LiCl).

Most experiments were performed using 30 mM lithium because that concentration resulted in a very large stimulation of glucose transport activity. Lithium at lower concentrations also potentiated the stimulation of 2-DG uptake by 5 µU/ml of insulin (stimulatory effect of insulin was twofold higher in the presence of 10 mM lithium; data not shown).

Effect of Lithium on IP Production

To determine whether lithium inhibits IMPase in our isolated rat adipocyte preparation, changes in cellular IPs were directly measured after treatment with lithium. IP1, IP2, and IP3 are all present in adipocytes (Fig. 3), with IP1 being the predominant form (90%). The high ratio of IP1 to IP2 plus IP3 is similar to that reported previously in adipocytes by others (11). Treatment with 30 mM lithium for 30 min significantly increased 3H-labeled IP1 accumulation to more than double that under basal conditions (Fig. 3), whereas IP2 and IP3 levels were unaffected (Fig. 3, insert).


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Fig. 3.   Effect of lithium on inositol phosphate production. Cells were labeled with myo-[3H]inositol for 2 h, washed, and then incubated in the absence or presence of lithium (30 mM) for 30 min at 37°C. Values are means ± SE of triplicate samples. Similar results were obtained in 3 independent experiments. IP1, inositol monophosphate. Inset: data expressed with a magnified scale to better show changes in inositol bisphosphates (IP2) and inositol trisphosphates (IP3). * P < 0.05, significantly different from control group.

Effect of a Prodrug of the IMPase Inhibitor on IP Production

The effect of L-690,488, a prodrug of the IMPase inhibitor L-690,330, on IP1 accumulation was also examined. The prodrug at a concentration of 50 µM significantly increased IP1 and IP2 levels in a time-dependent manner (Fig. 4), demonstrating that the prodrug penetrated the adipocyte cell membrane and was cleaved to the active inhibitor. A 30-min exposure to 50 µM of the prodrug increased IP1 levels to about the same extent as exposure to 30 mM lithium, i.e., about a doubling from basal levels (Fig. 4). Longer exposure to the prodrug (45 min) resulted in a further substantial accumulation of IP1 to about three times that seen in the basal state.


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Fig. 4.   Effect of the inositol monophosphatase (IMPase) inhibitor L-690,488 on inositol phosphate production. Prodrug was dissolved in DMSO and diluted into incubation medium. DMSO was added to control cells to maintain the same DMSO concentration in all experimental groups. Cells were incubated in the absence or presence of prodrug (50 µM) for 30 or 45 min at 37°C after 2 h of labeling with myo-[3H]inositol. Values are means ± SE of triplicate samples. Similar results were obtained in 2 independent experiments. Inset: data expressed with a different scale to better show changes in IP2 and IP3. * P < 0.05, significantly different from control group. dagger  P < 0.05, significantly different from prodrug (30-min incubation) group.

Effect of Prodrug on Glucose Transport

We further examined the effects of the prodrug on basal and insulin-stimulated 2-DG transport. Incubation of adipocytes with the prodrug (5 or 50 µM) for 30 min did not enhance either basal or insulin (5 µU/ml)-stimulated 2-DG transport (Fig. 5). In the same experiments, lithium (30 mM) markedly increased both basal and insulin-stimulated 2-DG transport. In fact, the prodrug at 5 and 50 µM reduced basal glucose transport activity by 10 and 28%, respectively (significantly different from control at 50 µM). Incubation of the adipocytes with L-690,488 also produced a significant (22%) decrease in insulin-stimulated 2-DG transport activity when present at 50 µM. Exposure to the prodrug (50 µM) did not alter 2-DG transport after a 90-min incubation (data not shown).


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Fig. 5.   Effects of IMPase inhibitor and lithium on basal and insulin-stimulated 2-DG transport. Cells were incubated at 37°C for 30 min in the absence or presence of the prodrug at indicated concentrations. Insulin (5 µU/ml) was added 15 min after addition of prodrug. Uptake of 2-DG was determined as described in METHODS. Values are means ± SE of triplicate samples. Similar results were obtained in 2 independent experiments. * P < 0.05, significantly different from basal group (without prodrug and insulin). dagger  P < 0.05, significantly different from insulin alone. # P < 0.05, significantly different from LiCl alone.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present study examined the effect of lithium on glucose transport in isolated rat adipocytes and tested the hypothesis that the effect of lithium on glucose transport is mediated by inhibition of IMPase. Our strategy was to examine the effects of a selective inhibitor of IMPase in comparison with those of lithium. The results show that lithium increases basal glucose transport activity in a concentration-dependent manner in adipocytes. In comparison with the previous study by Cheng et al. (8), our adipocytes responded to lower concentrations of lithium, and the responses were much greater than previously reported: 30 mM lithium produced a 198% increase in our study vs. a 50% increase in the previous study (8).

This study provides the first evidence that lithium potentiates the response of adipocytes to other stimuli, such as insulin, as has recently been demonstrated in skeletal muscle (25). Cheng et al. (8) reported that the effects of lithium were not additive to those of insulin, but these investigators examined only a maximally effective concentration of insulin, in which an additive effect is more difficult to distinguish. When a range of insulin concentrations was examined, it became apparent that lithium has multiple effects on glucose transport in adipocytes (Fig. 2): not only does it stimulate basal transport activity, but it also induces a leftward shift in the insulin concentration response (increased insulin sensitivity).

Our results indicate that inhibition of IMPase and subsequent accumulation of IP1 could not mimic the stimulatory effect of lithium on glucose transport activity. Treatment with the prodrug of the IMPase inhibitor, L-690,488, resulted in a significant elevation of IP1 in our adipocyte preparation. Incubation of the adipocytes with 50 µM L-690,488 for 30 min doubled IP1 levels, which is equivalent to the effect of 30 mM lithium. Lithium induced a significant increase in glucose transport activity, whereas the prodrug did not. These results suggest that accumulation of IP1 after inhibition of IMPase is not sufficient to mimic the stimulatory effect of lithium on glucose transport activity. However, we cannot rule out two possibilities. First, the IMPase inhibitor has a slightly different effect from that of lithium, because the IMPase inhibitor elevated IP2 level, but lithium did not. The increase in IP2 may somehow offset the effect of elevated IP1 to stimulate glucose transport. However, we are unaware of any precedent for IP2 to inhibit the activation of glucose transport. The second possibility that we cannot rule out is that lithium treatment increases IP1 in a different cellular compartment from that stimulated by the IMPase inhibitor.

Recent studies by Epps-Fung et al. (10) suggested that phospholipase C (PLC) may play a role in GLUT-4-mediated glucose transport in differentiated 3T3-L1 adipocytes. This hypothesis was supported primarily through experimental interventions that reduced PLC activity. For example, the PLC inhibitor U-73122 reduced epidermal growth factor (EGF)- and insulin-stimulated glucose transport activity and GLUT-4 translocation significantly. The authors speculated that PLC or resultant products, such as IPs, play either a permissive role or mediate the stimulation of GLUT-4 translocation by insulin and EGF. Given that 1) insulin stimulated PLC only slightly (6%), yet the inhibitor reduced insulin-stimulated transport activity extensively, and 2) the inhibitor appeared to reduce GLUT-4 levels in the basal state, PLC activity may only be permissive for GLUT-4 translocation. In that case, increases in PLC products such as IPs above basal levels may not mediate further increases in transport activity. Although Epps-Fung et al. showed that lithium increased glucose transport in the 3T3-L1 adipocytes, they did not report effects of lithium on IP levels. In our studies, we examined only conditions that increase IP levels, so we cannot conclude whether basal levels of these metabolites play a permissive role in maintaining glucose transport activity.

The effects of lithium on glucose metabolism are not limited to glucose transport. Lithium has also been shown to stimulate glycogen synthase activity in skeletal muscle (14), adipocytes (9), and hepatocytes (18). Recently, Klein and Melton (16) showed that lithium regulates cell fate determination in diverse organisms through selective and potent inhibition of glycogen synthase kinase-3 (GSK-3) (16). Therefore, lithium may activate glycogen synthase activity through inhibition of GSK-3, which inactivates glycogen synthase by phosphorylation of the enzyme.

Lithium treatment has also been shown to inhibit adenylate cyclase activation by beta -adrenergic agonists and histamine in neuronal cells (12). Lithium suppresses hormone-induced formation of cAMP in a number of tissues, including the thyroid, kidney, and platelets (12). However, lithium has no effect on basal or epinephrine-induced increases in cAMP levels in muscle, a tissue in which lithium blunts the activation of glycogen phosphorylase by epinephrine and significantly stimulates glucose transport activity (25). Therefore, cAMP does not seem to be a mediator of the lithium effect on glucose transport, at least in skeletal muscle.

Lithium has been shown to modulate GTP-binding proteins. Lithium at therapeutic concentrations completely blocks both adrenergic and cholinergic agonist-induced increases in GTP binding in membranes from rat cerebral cortex (3). These results suggest that G proteins may act as the molecular site of action for both the antimanic and antidepressant effects of lithium in supporting an alternative adrenergic-cholinergic balance hypothesis of bipolar disorders (15). Recently, it has been reported that lithium stimulates rat pancreatic beta -cell replication that is mediated by pertussis toxin-sensitive GTP-binding proteins (24). Interestingly, it has been demonstrated that nonhydrolyzable GTP analogs mimic effects of insulin on glucose transporter recruitment to the cell surface in permeabilized adipocytes (4). There is no direct evidence that lithium alters insulin signaling or enhances translocation of glucose transporters in response to insulin. However, given that lithium potentiates the response to muscle contractions/hypoxia, it seems likely that lithium alters steps common to glucose transport pathways activated by these stimuli (25). A more complete understanding of the molecular basis of lithium action on glucose transport awaits further investigation.

In conclusion, our results demonstrate that lithium stimulates basal glucose transport activity and potentiates insulin-stimulated glucose transport activity in isolated rat adipocytes. However, the stimulatory effect of lithium on glucose transport in adipocytes is not due to an inhibition of IMPase and the resulting accumulation of IP1.

    ACKNOWLEDGEMENTS

We thank Jeanne Sebaugh for the statistical analysis of the data in this study.

    FOOTNOTES

Address for reprint requests: X. Chen, Cardiovascular Disease and Diabetes Research, Monsanto Company, Mail Zone T1G, 800 North Lindbergh Blvd., St. Louis, MO 63167.

Received 2 December 1997; accepted in final form 28 April 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(2):E272-E277
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society