Myo-inositol Influx Research Articles

Hypotonic activation of the myo-inositol transporter SLC5A3 in HEK293 cells probed by cell volumetry, confocal and super-resolution microscopy.

Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P ino [m/s] and expression/localization of SLC5A3. P ino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), P ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

Effect of ammonia and methionine sulfoximine on myo-inositol transport in cultured astrocytes.

Ammonia causes astrocyte swelling which is abrogated by methionine sulfoximine (MSO). Since myo-inositol is an important osmolyte, we investigated the effects of ammonia and MSO on myoinositol flux in cultured astrocytes for periods up to 72 hours. Uptake of myo-inositol was significantly decreased by 26.7 (P < 0.05) and 39.3 (P < 0.006) percent after 48 hours of exposure to 5 or 10 mM ammonia, respectively. The maximum rate of uptake was 14.0+/-0.5 nmol/hour/mg protein which was reduced to 7.45+/-0.27 and 7.02+/-0.57 nmoles/hour/mg protein by 5 or 10 mM ammonia, respectively. The Kms by Michaelis-Menten equation for the control, and in the presence of 5, or 10 mM ammonia were 32.5+/-4.52, 44.4+/-5.82, and 39.3+/-7.0 microM, respectively. Kms by Hanes-Woolf plot for the control, 5, or 10 mM ammonia were 25, 45, and 40 microM, respectively. Treatment of astrocytes with either 5 or 10 mM NH4Cl for 6 hours caused a decrease in myo-inositol content by 66% and 58%, respectively. MSO (3 mM) partially diminished the ammonia-induced inhibition of myo-inositol uptake and decreased myo-inositol content by 31% after 24 hours. Additionally, ammonia increased myo-inositol efflux briefly through the fast efflux component but had little effect on myo-inositol efflux through the slow efflux component of astrocytes exposed to ammonia for up to 72 hours. Predominantly decreased myo-inositol influx coupled with brief efflux through the fast component may represent an adaptive response to diminish the extent of ammonia-induced astrocyte swelling.

Inducible Expression of Na+/Myo-inositol Cotransporter mRNA in Anterior Epithelium of Bovine Lens: Affiliation With Hypertonicity and Cell Proliferation

Bovine lenses were pretreated with physiological (314±6 mosm) or hypertonic medium (>450 mosm) for 20 hours prior to introduction of the lenses into modified Ussing chambers (providing a leakproof seal at the lens equator). The anterior (epithelial-containing) and posterior (cell-free) aspects of the mounted intact lens could thus be independently bathed with myo-[3H]inositol. Myo-inositol uptake as a function of concentration across the anterior aspect was indicative of an active carrier-mediated component and at high concentration, significant myo-inositol influx due to passive diffusion. Myo-inositol uptake was markedly stimulated across the anterior aspect and inhibitable by ouabain but not stimulated across the posterior aspect, when lenses were exposed to hyperosmotic medium by the addition of raffinose or sodium chloride. Myo-inositol rapidly desaturates across both aspects of the lens consistent with its efflux being largely a function of passive diffusional leakout from the extracellular space. In order to corroborate the apparent hypertonic-induced upregulation of gene expression inferred by kinetic analysis, the level of Na+/myo-inositol cotransporter mRNA was determined by reverse transcription and quantitative PCR using harvested anterior epithelial cells from intact lenses exposed to physiologic or hypertonic medium for 20 hours. RNA samples were microextracted from both the central and equatorial regions of the lenticular epithelium of the anterior lens capsule. The abundance of Na+/myo-inositol cotransporter mRNA was similar from epithelium of the central zone irrespective of medium tonicity but was markedly elevated from the equatorial zone of osmotically-stressed lenses. While both the quiescent epithelial cells of the central region and actively dividing epithelial cells of the equatorial zone of the intact lens express Na+/myo-inositol cotransporter mRNA, only the equatorial epithelium responds to hypertonic insult with enhanced uptake of myo-inositol due to increased transcription of the Na+/myo-inositol cotransporter gene, suggesting a possible role for the state of cell proliferation.

Effect of pyruvate on lens myo-inositol transport and polyol formation in diabetic cataract.

In diabetic cataract, sorbitol pathway flux perturbs intracellular metabolism by two putative mechanisms. The osmolyte hypothesis implicates the aldose reductase enzyme, increased rate of reduction of glucose of sorbitol and reciprocal osmoregulatory depletion of organic osmolytes (myo-inositol). Redox hypothesis favors alterations in the ratios (NADP+/NADPH and/or NADH/NAD+ as the primary cause of glucose-induced aldose reductase related defects. Increase in NADH/NAD+ promotes increased oxidation of sorbitol to fructose by polyol dehydrogenase; potential normalization of this ratio by coadministration of pyruvate (which reoxidizes NADH to NAD+ via lactate dehydrogenases reaction) was investigated. Effects of exogenous pyruvate on lens polyol formation and sodium-dependent myo-inositol (MI) cotransporter using two in vitro models of sugar cataract were determined. Rat lenses were incubated for 16 h in either normal (5.5 mM) or high sugar medium, 35.5 mM glucose or 30 mM galactose. Then lens MI influx was compared to polyol, MI and fructose content. Pyruvate did not affect MI influx or sorbitol content in lenses incubated in control medium. In 35.5 mM glucose, coadministration of pyruvate maintained lens MI influx at 76% of control values vs. 43% for lenses without pyruvate. Furthermore, pyruvate treatment diminished lens sorbitol content by 50% and increased lens sugar content (myo-inositol, fructose, lactate) and media lactate levels. Lenses incubated in high galactose medium formed galactitol with a corresponding decreased MI content. Coadministration of pyruvate had no effect on either lens sugar content (galactitol, myo-inositol, fructose) or MI influx, consistent with the fact that galactitol was not metabolized to fructose. In conclusion, pyruvate did not exert a direct effect on the MI co-transporter or prevent galactitol inhibition of MI influx. Coadministration of pyruvate with high glucose altered lens metabolism and promoted reduction of pyruvate to lactate, increased fructose, decreased sorbitol, enhanced MI influx, maintained lens MI content, implicating both osmotic and redox systems.

Kinetics and Stoichiometry of a Proton/myo-Inositol Cotransporter

Voltage clamp recording was used to measure steady-state and presteady-state currents mediated by a myo-inositol transporter cloned from Leishmania donovani and expressed in Xenopus oocytes. Application of myo-inositol resulted in inward currents, which did not require external sodium and which were increased by increasing the extracellular proton concentration and by membrane hyperpolarization. Alkalinization of the extracellular space occurred concomitantly with myo-inositol influx. Correlation of membrane currents with radiolabeled myo-inositol flux revealed that one positive charge is translocated with each molecule of myo-inositol, consistent with cotransport of one proton. The transport concentration dependence on both species suggested ordered binding of a proton followed by a molecule of myo-inositol. In the absence of myo-inositol, a voltage-dependent capacitance was observed that correlated with the transporter expression level. This charge movement obeyed a Boltzmann function, which was used to estimate a turnover of 0.70 +/- 0.06 s-1 at -60 mV. The pH and voltage dependence of the charge movements were simulated with a model involving alternating access of internal and external protons to sites within an occluded pore.

Kinetics of myo-inositol transport in rat ocular lens.

Myo-inositol (MI) influx as a function of concentration in rat lens consisted of a saturable component, fit by a rectangular hyperbola, and a linear component which was more distinct at high myo-inositol concentrations suggesting passive diffusion. The hyperbolic component was half-maximally saturated (Kt) at 61.3 microM and had a maximal transport rate (Jmax) of 44.6 mumol/kg wet wt/h. The linear component had an apparent permeability coefficient of 1.44 x 10(-6) s-1. Sorbitol, which distributed rapidly in the extracellular space (6.83 ml/100 g wet wt), also appeared to enter the intracellular space with a permeability coefficient of 1.37 x 10(-6) s-1, similar to that of myo-inositol. The influx of myo-inositol was critically dependent on the concentration of extracellular sodium consistent with a sodium-myo-inositol cotransport. The kinetics of influx activation by sodium suggested an apparent 2:1 coupling ratio for sodium and myo-inositol. When potassium was used as sodium substitute, a significantly stronger influx inhibition was observed than with nondepolarizing sodium substitutes, indicating that myo-inositol was driven by the electrochemical gradient of sodium rather than the chemical gradient only. Reducing the extracellular Na concentration increased the MI concentration at which transport was half-maximally activated, suggesting an ordered binding sequence of Na followed by MI. Myo-inositol influx was competitively inhibited by phlorizin with an inhibitory coefficient (Ki) of 35 microM. Phloretin also was capable of inhibition but with a much lesser efficacy. Myo-inositol desaturates from the lens at a rate of 0.00862 h-1. Approximately 19% of the efflux can be inhibited with phlorizin, suggesting that it represents carrier-mediated flux. The phlorizin insensitive flux has a rate of 0.00695 h-1 or 1.93 x 10(-6) s-1, similar to the Na-independent passive influx. MI influx is due to a Na-dependent, phlorizin-sensitive active transport while the efflux consists largely of a phlorizin-independent passive leakage.

Mechanisms for D-glucose inhibition of myo-inositol influx into rat lens

Mechanisms for D-glucose inhibition of myo-inositol influx into rat lens

Mechanisms for D-Glucose Inhibition of myo-Inositol Influx Into Rat Lens

Myo-Inositol depletion as a result of hyperglycemia is considered one of the leading contributors to chronic diabetic complications. We investigated the possible mechanisms through which elevated extracellular glucose levels affect the loss of intracellular myo-inositol in rat lens. Short-term incubation (up to 4 h) in solutions with elevated glucose concentrations revealed a concentration-dependent inhibition of myo-inositol influx. This inhibition was caused by both an increase of the transport coefficient and a decrease of maximal flux and thus was a mixed competitive and noncompetitive inhibition. If polyol accumulation was prevented with sorbinil, an aldose reductase inhibitor, the inhibition of myo-inositol influx was partially reduced. The remaining inhibition was the result of an increased transport coefficient without a change in maximal flux and therefore represents a strictly competitive inhibition. A similar competitive inhibition was observed with the nonmetabolizable glucose analogue L-glucose, which cannot be converted to polyol. Longer exposure (16 h) to solutions with high glucose concentrations resulted in an inhibition that correlated with high lens polyol levels. This inhibition persisted after the lenses were returned to solutions with normal glucose concentrations and was the result of a decrease of maximal flux without a significant change in transport coefficient, a strictly noncompetitive inhibition. The noncompetitive inhibition associated with polyol accumulation and the competitive inhibition due to extracellular glucose were additive. Lens myo-inositol depletion after exposure to elevated glucose concentrations thus resulted from a competitive inhibition caused by the interaction of extracellular glucose with the myo-inositol carrier and a noncompetitive inhibition associated with polyol accumulation.

Relationship between myoinositol influx and lipids in diabetic neuropathy

Diabetic neuropathy is associated with abnormalities in lipid metabolism and has been postulated to be associated with abnormal myoinositol metabolism. Leucocyte myoinositol influx was measured using a triple isotope method in long-standing type 1 (insulin-dependent) diabetic patients with and without diabetic neuropathy and in a group of matched controls. No differences in fasting lipid, glucose concentrations or glycated haemoglobin were found in the diabetic groups. Myoinositol influx was significantly but negatively correlated with the serum very low density lipoprotein (VLDL) cholesterol concentration in patients with and without neuropathy but not in the controls. The VLDL cholesterol concentration also correlated negatively with the transporter Km (Rs = -0.87, P < 0.005, neuropathic group only) and Vmax (Rs = -0.93, P < 0.001, neuropathic group; Rs = -0.59, P < 0.05, non-neuropathic group). Myoinositol influx correlated with the glycated haemoglobin only in the diabetic patients without neuropathy. The lipid relationships in diabetic subjects were independent of glucose control, which suggests that myoinositol influx mechanisms represent another transporter affected by intracellular lipid metabolism. The control of VLDL metabolism by fibrates could offer a method for reducing the progression of diabetic neuropathy.

Abnormal myoinositol influx in human leucocytes in diabetes but not specifically in diabetic neuropathy

Abnormal myoinositol influx in human leucocytes in diabetes but not specifically in diabetic neuropathy

Myo-inositol transport in the lens of galactose-maintained rats.

Lens myo-inositol (MI) content is regulated by a pump-leak system consisting of an active Na-dependent MI transport and its passive permeability through the membrane. We measured the active MI uptake and membrane permeability in lenses of rats maintained on a 50% galactose diet for 1, 3 and 7 days. After only 1 day of galactose feeding, active MI uptake in the lens was reduced dramatically by 74% compared to age-matched control lenses; by day 3, active MI transport was decreased by 89% and it was undetectable by day 7. The passive membrane permeability was determined by measuring (a) the passive MI influx and (b) the 3H-sorbitol flux. After 1 day of galactose feeding, the membrane permeability increased such that within 3 days it increased to 5-6 fold. Galactose feeding also led to a rapid increase in lens polyol content. After 1 day, lens polyol increased to 53 mumol/g wet wt compared to a control value of 0.35 mumol/g wet wt and increased further to 65 and 72 mumol/g wet wt after 3 and 7 days of galactose feeding respectively. Lens galactose accumulation was low (3 mumol/g wet wt) up to 7 days; however, it was rapidly increased after 7 days. Our results indicate that galactose feeding rapidly interfered with MI homeostasis by a severe depression of active MI transport and a rapid increase in membrane permeability. These interferences of MI homeostasis correlate with the appearance of high polyol levels.

Myo-Inositol influx into human leucocytes: methods of measurement and the effect of glucose

1. Low intracellular concentrations of myo-inositol in diabetic cells may contribute to the development of tissue damage. The cause of these low levels is unknown, but inhibition of a putative myo-inositol transporter by high concentrations of glucose has been proposed. We have developed a triple-isotope method for estimating myo-inositol influx into human leucocytes and so investigated both the kinetics of this uptake in normal volunteers and the effect of glucose upon it. 2. Uptake was composed of a passive component with a rate constant of 2.4 +/- 0.3 X 10(-2) min-1 and a saturable component with a Km of 61 +/- 23 mumol/l and Vmax of 11.3 +/- 4.5 X 10(-4) mmol min-1 l-1. Ouabain and low extracellular concentrations of sodium partly inhibited influx. Uptake was predominantly into the cytosolic fraction of the cell with 12% entering the membrane-associated fraction at both 5 and 10 min. 3. myo-Inositol influx was significantly inhibited by both D- and L-glucose but not by sucrose. Neither cytochalasin B nor ethyl isopropyl amiloride significantly inhibited uptake. 4. It is concluded that a myo-inositol transporter exists in human leucocytes which is similar to that found in other species and tissues. Our technique allows myo-inositol influx in diabetic subjects to be related to varying glycaemic control and tissue damage.

Myo-inositol transport in the small intestine of the domestic fowl

1. 1. Myo-inositol is transported in chicken small intestine by a mediated route with an apparent K m of 0.1 mM and by a diffusion mechanism. 2. 2. The mediated route is susceptible to inhibition by sugars, though sugars are not transported by this process, nor is myo-inositol transported by the sugar transport system. 3. 3. Myo-inositol influx is inhibitable by phlorizin, sulfhydryl reagents, removal of Na + from the incubation medium, and preloaded sugar; it can be stimulated by theophylline. 4. 4. The ability to absorb this nutrient varies greatly between individual animals.