The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Grampositive bacterium BaciZZus subtiZis. Natural abundance 3C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K+ and proline rose markedly after osmotic upshock. K+ influx from the medium was rapid (< 1 h) but proline synthesis was a slower process (5-9 h). Proline synthesis appeared to be dependent on the prior accumulation of K+ and it is possible that K+ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K+ was also accumulated under these conditions.

PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.The 3-phosphoinositide-dependent protein kinase 1 (PDK1) functions as an upstream activator of a group of AGC family protein kinases that are stimulated by insulin, growth factors, and numerous other agonists (42). These include isoforms of protein kinase B (PKB), also known as Akt (23), p70 ribosomal S6 kinase (S6K) (17), serum and glucocorticoid-induced protein kinase (SGK) (32), and p90 ribosomal S6 kinase (RSK) (27). Activation of PKB and other AGC kinases plays crucial roles in regulating diverse effects of extracellular agonists on cells by phosphorylating regulatory proteins that control metabolism, growth, proliferation, and survival (21). Many if not all of the cellular effects of insulin are mediated through activation of PKB (18, 58). PKB also stimulates the activation of S6K1, which plays an important role in regulating protein synthesis and cell growth (17). Genetic analysis of the PDK1-signaling pathway in Drosophila melanogaster and mice also suggests that this pathway plays an important role in regulating organism size. For example, Drosophila organisms with reduced levels of PDK1 (51), PKB (56), or S6K (40) are all small, possessing cells with reduced volume. Similarly, mice with decreased levels of PDK1 (33) and mice lacking PKB␣ (18) or S6K isoforms (48) also display small-organism and -cell phenotypes.PDK1 activates at least 23 AGC kinases by phosphorylating a specific Thr or Ser residue located within the T-loop of the kinase domain (42). Maximal activation also necessitates phosphorylation of a Ser/Thr residue located C-terminal to the catalytic domain within a region known as the hydrophobic motif. Recent work has established that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 phosphorylate the hydrophobic motif of S6K1 and PKB (52,61). In the case of RSK, a se...

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1985) The osmotic role of mannitol in the Phaeophyta: an appraisal. Phycologia 24: 35-47.Natural abundance l3C nuclear magnetic resonance spectroscopy (NMR) has shown mannitol to be the only major low molecular weight organic compound present in osmotically significant amounts within cells of the fo llowing marine brown algae: Alaria esculenta, Ascophyllum nodosum, Ectocarpus siliculosus, Fucus serratus, F. spiralis, F. vesiculosus, Halidrys siliquosa, Laminaria digitata, L. hyperborea, L. saccharina and Pilayella littoralis. Samples of the top-shore alga Pelvetia canaliculata were fo und to contain volemitol in addition to mannitol. Quantitative gas-liquid chromatographic analyses have confi rmed the presence of mannitol, at concentrations ranging fr om 83.3 to 314.0 mmol kg-1 (expressed in terms of intracellular water content) fo r plants maintained in a fu ll-strength (100%) sea water medium. A study of the changes in intracellular mannitol concentration of six marine brown macroalgae immersed in hyposaline and hypersaline media (20-1 50% EA I sea water medium) showed that mannitol concentration varied as a direct fu nction of salinity in all cases, providing support for the hypothesis that mannitol is intimately involved in osmotic adjustment in response to changes in external water status. Plants of the filamentous form P. littoralis maintained in darkness also showed a marked sensitivity of intracellular mannitol concentration to alterations in the external salt concentration. Overall, the data support an osmotic, rather than a respiratory role fo r the large internal pools of mannitol in such algal cells.