Intracellular monosaccharide and amino acid concentrations and activities and the mechanisms of insulin action.

Abstract

Current amino acid and monosaccharide transport models are based on an assumption which equates the intracellular chemical activity of a solute with its concentration. This assumption was tested for a-aminoisobutyric acid and 3-0-methylglucose in a giant cell, the amphibian oocyte, by using recently developed cryomicrodissection and internal reference phase techniques. We found the following. (i) a-Aminoisobutyric acid and 3-0-methylglucose activities were much greater in cytoplasm than was suggested by concentration data; i.e., activity coefficients were higher than in ordinary water solutions. This is attributable to the inaccessibility of considerable water as solvent (solute exclusion). (ii) Solute concentrations varied regionally as follows: nucleus >> animal cytoplasm > vegetal cytoplasm. Insulin increased the nucleus/cytoplasm concentration asymmetry, apparently by increasing cytoplasmic solute exclusion. (iii) Nuclear activity coefficients more closely resembled those of ordinary saline solutions so that nucleus/ extracellular concentration ratios reflected transmembrane activity gradients better than did cytoplasm (or whole cell)/extracellular ratios. (iv) Mediated passive a-aminoisobutyric acid and 3-O-methylglucose transport were constituent oocyte membrane properties. Membrane active transport was initiated with time (in the presence of substrate) and by insulin. (v) Increased temperature mimicked insulin in enhancing transmembrane a-aminoisobutyric acid activity gradients and increasing the nucleus/cytoplasm concentration asymmetry. These results indicated that concentration data are a misleading measure of cellular amino acid and monosaccharide activity; some consequences of this observation were explored. A model is proposed in which cell water has reduced solvent capacity or is compartmentalized (considered less likely) and is susceptible to physiological modulation. The model accounts for many observations in small cells, suggesting generality of the exclusion phenomenon and a previously unrecognized metabolic control mechanism.Cellular amino acid and monosaccharide concentrations are generally used to describe transport and enzymatic reactions. However, since activities, not concentrations, are the driving force in these reactions (and determine equilibria as well), the practice involves the notion of activity-concentration equivalency which, in effect, assumes that solute activity coefficients within cells are similar to those in the aqueous solutions bathing the cells. Because of the small size of most cells, this assumption (known to lack universality for ions [30]) has never been adequately tested.The To circumvent these difficulties, a giant cell, the amphibian oocyte, has been studied. Oocytes exhibit transport behavior typical of smaller cells: solute size-dependent permeability (19,20), transmembrane Na+-K+ activity gradients (7,23,51), mediated saturable amino acid accumulation (13, 36), and insulin responsiveness (2,9,42,53

Artifacts caused by cell microinjection.

“…Samples were weighed (at -100C), dried over P205 at 60'C, and reweighed to determine water content. Dried samples were extracted in boiling water and extracts were analyzed for [14C]-or [3H]sucrose by liquid scintillation counting and for Na and K by atomic absorption spectroscopy (1,4,5). Na and K data are presented as concentrations (mM) and sucrose data, as RP-normalized concentration ratios-e.g., N/RP equals nuclear dpm per ml of water divided by RP dpm per ml of water.…”

“…The error is conservative, strengthening our conclusions. Data are presented as mean + SEM and the significance of differences between sample means was determined by using Student (5,7). Ooplasm/RP ratios are smaller than unity, in part because of the presence in ooplasm of yolk platelets, which contain water of crystal hydration from which sucrose is excluded (7,8).…”

Diffusive and nondiffusive proteins in vivo.

“…Several controls validate the efficacy of the intracellular RP as a sampler of diffusive proteins: ments of macromoleculer diffusion in cross-linked gels (17) indicate that the gelatin RP will equilibrate with globular diffusive proteins of up to -100,000 daltons within 20 h. (b) In vitro gelatin water has the solvent characteristics of ordinary water for small solutes and proteins ( 1211-myoglobin). Equilibrium dialysis experiments show that neither exclusion from gelatin water nor binding to the gelatin matrix is detectable; and no effects of tracer electrical charge on kinetics or equilibria have been observed (16,18). (c) After 121 1-myoglobin has been introduced into the oocyte dissolved in RP gelatin, it reaches diffusive equilibrium between the RP, cytoplasm, and nucleus within 4 h. For myoglobin, as well as for the dextran tracers discussed immediately below, the nucleus/cytoplasm equilibrium distribution is the same in RP-containing and control (non-RP-injected) oocytes, demonstrating that the intracellular equilibrium distributions of diffusive macromolecules are not affected by the presence ofthe RP.…”

“…Cryomicrodissection (18,21) was used to isolate frozen RP, nucleus, and cytoplasmic samples . The method eliminates artifactual relocations of all diffusive solutes, including macromolecules (18,21,22).…”

“…Cryomicrodissection (18,21) was used to isolate frozen RP, nucleus, and cytoplasmic samples . The method eliminates artifactual relocations of all diffusive solutes, including macromolecules (18,21,22). The frozen oocyte was removed directly from liquid nitrogen to the -45°C dissection stage ofthe cryomicrodissection apparatus, and the nucleus, cytoplasm, and RP were dissected freehand with insulated, stainless steel microtools (Fig.…”