[Epidemiology of the upper respiratory tract diseases in the Cheliabinsk region and socio-hygienic characteristics of the patients].

The study of the prevalence and incidence of upper respiratory tract pathology in the Chelyabinsk region revealed the outstripping growth rate of morbidity of the nasal cavity and accessory sinuses of the nose including their chronic forms. The medico-sociological analysis of characteristics of 450 patients was undertaken to elucidate the social and medical factors responsible for this situation.

Molecular basis for the insulinomimetic effects of C-peptide.

AIMS/HYPOTHESIS: C-peptide, released by the beta-cells of pancreatic islets, elicits salutary responses in Type I (insulin-dependent) diabetes mellitus but the molecular mechanisms behind these effects are not known. We assessed whether synthetic rat C-peptide stimulates insulin-like cellular effects in a classic insulin target tissue. METHODS: To clarify the molecular mechanisms involved in several insulinomimetic actions, we investigated the effect of C-peptide on the insulin signalling pathway in rat skeletal muscle cells. We used L6 myoblasts and myocytes to measure the effects of C-peptide or insulin or both on glycogen synthesis and amino acid uptake. We also studied the effects of C-peptide on insulin receptor autophosphorylation, its tyrosine kinase activity, phosphorylation of IRS-1, PI 3-kinase, Akt, p90Rsk, MAPK, and GSK3 in these cells. RESULTS: In L6 cells, physiological concentrations of C-peptide (0.3-3 nmol/l) significantly activated insulin receptor tyrosine kinase, IRS-1 tyrosine phosphorylation, PI 3-kinase activity, MAPK phosphorylation, p90Rsk, and GSK3 phosphorylation. A scrambled C-peptide sequence - the control - showed no effects. Wortmannin blocked C-peptide-induced glycogen synthesis while pertussis toxin had no effect. Only submaximal insulin concentrations (up to 10 nmol/l) combined with submaximal C-peptide concentrations led to additive effects. CONCLUSION/INTERPRETATION: C-peptide added to the maximal insulin dose (100 nmol/l) did not increase the effect of insulin alone. We thus conclude that the same signalling elements are used by both ligands. However, the lack of Akt activation by C-peptide and the bell-shaped dose response induced by C-peptide indicate that C-peptide has some effects by another distinct mechanism. We speculate that C-peptide could modulate the metabolic effects of insulin by enhancing them at low hormone concentrations and dampening them at high hormone concentrations.

Selective insulin-induced activation of class I(A) phosphoinositide 3-kinase in PIKfyve immune complexes from 3T3-L1 adipocytes.

A diverse range of insulin-regulated cellular processes are dependent on class I(A) phosphatidylinositol 3-kinases (PI 3-Ks) and their association with and activation by up-stream signaling molecules. Here we report on the identification of the phosphoinositide 5'-kinase PIKfyve as a partner of class I(A) PI 3-K. Thus, both p85 and p110 subunits (class I(A)) of PI 3-Ks co-precipitated with anti-PIKfyve antibodies from lysates of resting 3T3-L1 adipocytes and, vice versa, PIKfyve co-precipitated with anti-p85 PI 3-K antibodies. Assignment to class I(A) PI 3-K enzymatic activity was further substantiated by the inhibition of PtdIns 3-P production in PIKfyve immune complexes by low concentrations of wortmannin and Triton X-100, and its preferences for Mg(2+) versus Mn(2+). Insulin but not PDGF or EGF stimulation of 3T3-L1 adipocytes markedly increased the PtdIns 3-P production (4.2-fold) in PIKfyve immune complexes, primarily as a result of increased PI 3-K intrinsic enzymatic activity. Intriguingly, while both insulin and PDGF caused an increase of class I(A) PI 3-K activity co-immunoprecipitated with tyrosine phosphorylated proteins, only insulin treatment yielded an activation of class I(A) PI 3-K in PIKfyve immune complexes. Studies aiming at identifying the underlying mechanism revealed that PIKfyve-class I(A) PI 3-K association and the insulin-induced activation likely operate independently of tyrosine phosphorylated insulin receptor substrate proteins. Together, these results establish PIKfyve as a novel source of activated class I(A) PI 3-K molecules that may be relevant in the insulin-signal transduction pathway.

Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve.

The dual specificity mammalian enzyme PIKfyve phosphorylates in vitro position d-5 in phosphatidylinositol (PtdIns) and PtdIns 3-P, itself or exogenous protein substrates. Here we have addressed the crucial questions for the identity of the lipid products and the role of PIKfyve enzymatic activity in mammalian cells. CHO, HEK293, and COS cells expressing PIKfyve(WT) at high levels and >90% efficiencies increased selectively the intracellular PtdIns 3,5-P(2) production by 30--55%. In these cell types PtdIns 5-P was undetectable. A kinase-deficient point mutant, PIKfyve(K1831E), transiently transfected into these or other cells elicited a dramatic dominant phenotype. Subsequent to a dilation of the PIKfyve-containing vesicles, PIKfyve(K1831E)-expressing cells progressively accumulated multiple swollen lucent vacuoles of endosomal origin, first in the perinuclear cytoplasm and then toward the cell periphery. Despite their drastically altered morphology, the PIKfyve(K1831E)-expressing cells were viable and functionally active, evidenced by several criteria. This phenotype was completely reversed by introducing PIKfyve(WT) into the PIKfyve(K1831E)-transfected cells. Disruptions of the localization signal in the PIKfyve kinase-deficient mutant yielded a PIKfyve(K1831E Delta fyve) protein, incompetent to vacuolate cells, implying that an active PIKfyve enzyme at distinct late endocytic membranes is crucial for normal cell morphology. This was further substantiated by examining the vacuolation-induced potency of several pharmacological stimuli in cells expressing high PIKfyve(WT) levels. Together, the results indicate that PIKfyve enzymatic activity, possibly through the generation of PtdIns 3,5-P(2), and/or yet to be identified endogenous phosphoproteins, is critical for cell morphology and endomembrane homeostasis.

Localization and insulin-regulated relocation of phosphoinositide 5-kinase PIKfyve in 3T3-L1 adipocytes.

The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of phosphatidylinositol 5-P and phosphatidylinositol 3,5-P(2), thought essential in cellular functions, including membrane trafficking. To discern the intracellular loci of PIKfyve products' formation, we have examined the localization of PIKfyve protein versus enzymatic activity and a possible acutely regulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resting cells that were positive for immunoreactive PIKfyve, such as cytosol ( approximately 76%), internal structures (low density microsomal fraction (LDM), composed of recycling endosomes, GLUT4 storage compartment, Golgi, and cytoskeletal elements) ( approximately 20%), and plasma membrane (( approximately )4%), expressed enzymatically active PIKfyve. While the presence of a FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentation, immunoadsorption, and fluorescence microscopy analyses segregated the LDM-associated PIKfyve from the membranes of the recycling endosomes and GLUT4. PIKfyve fluorescence staining largely coincided with trans-Golgi network/multivesicular body markers, indicating PIKfyve's role in the late endocytic/biosynthetic pathways. A subfraction of particulate PIKfyve resisted nonionic detergent treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM, which was coupled with a commensurate increase of PIKfyve lipid kinase activity and an electrophoretic mobility shift. We suggest the recruited PIKfyve specifies the site and timing of phosphoinositide signals that are relevant to the acute insulin action.

PIKfyve lipid kinase is a protein kinase: downregulation of 5'-phosphoinositide product formation by autophosphorylation.

A subset of phosphoinositide 3-kinase family members are dual specificity enzymes; their protein kinase activity is thought to bring about an additional level to their intracellular regulation. Here we have examined whether the 5'-phosphoinositide kinase PIKfyve, reported previously to catalyze the formation of PtdIns 5-P and PtdIns 3,5-P(2) in vitro [Sbrissa et al. (1999) J. Biol. Chem. 274, 21589-21597], displays dual specificity. We now report that PIKfyve possesses an intrinsic protein kinase activity inseparable from its lipid kinase activity and, besides itself, can phosphorylate exogenous proteins in a substrate-specific manner. Both the autophosphorylation and transphosphorylation were demonstrated with PIKfyve immunopurified or affinity-purified from heterologously transfected COS cells, infected Sf9 cells, or native 3T3-L1 adipocytes. Conversely, no protein kinase activity was associated with immunopurified lipid kinase dead point (K1831E) or truncated (Delta1812-2052) PIKfyve mutants. PIKfyve autophosphorylation or transphosphorylation engaged Ser but not Thr or Tyr residues. PIKfyve autophosphorylation was largely abrogated upon pretreatment with PIKfyve lipid substrates or phosphatases. The impact of autophosphorylation on the PIKfyve lipid kinase activity was further examined with purified PIKfyve preparations. A decrease of 70% in the lipid product formation was associated with PIKfyve autophosphorylation, which was reversed upon treatment with phosphatases. In the cellular context, PIKfyve, or a fraction of it, was found in a phosphorylated form. Collectively, these results indicate that PIKfyve is a dual specificity kinase, which can generate and relay protein phosphorylation signals to regulate the formation of its lipid products, and possibly other events, in the context of living cells.

Lithium treatment in ovo: effects on embryonic heart rate, natural death of ciliary ganglion neurons, and brain expression of a highly conserved chicken homolog of human MTG8/ETO.

Understanding the action of the mood stabilizer lithium is dependent on availability of experimental models where lithium treatment at clinically relevant concentrations induces marked phenotypic and genotypic changes. Here we report on such changes in the chicken embryo. Lithium chloride (0.6 mM), applied in ovo 60 h after incubation, markedly delayed the heart rate increase observed from ED2.5 to ED5, and induced the brain expression of a new chicken gene cETO from ED7 to ED15. At the same time the overall developmental dynamics and embryo survival, or the expression of chicken gephyrin were not significantly affected. Furthermore, lithium treatment (0.3 mM, 48 h after incubation) abolished the difference in neuronal number between ED12 ciliary ganglia developing in the presence or absence of postganglionic target muscles. We show that cETO is a close homologue of the human transcription factor MTG8/ETO; named after its location on chromosome 8, and participation in chromosomal translocation 8;21 in myeloid leukemia. The mRNA and protein levels of ETO and gephyrin had a parallel course in chicken brain development suggesting that the expression of both genes is regulated mainly at the level of gene transcription. However, the patterns of expression were markedly different. ETO peaked at ED7 and decreased five-fold at ED15. In contrast, gephyrin levels increased five-fold from ED7 to ED15. We propose that the induction of ETO expression, in concert with lithium-induced upregulation of other genes, such as PEBP2beta and bcl-2, is participating in the neuroprotective effect of chronic lithium treatment.

Complementation analysis in PtdInsP kinase-deficient yeast mutants demonstrates that Schizosaccharomyces pombe and murine Fab1p homologues are phosphatidylinositol 3-phosphate 5-kinases.

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) is widespread in eukaryotic cells. In Saccharomyces cerevisiae, PtdIns(3,5)P(2) synthesis is catalyzed by the PtdIns3P 5-kinase Fab1p, and loss of this activity results in vacuolar morphological defects, indicating that PtdIns(3,5)P(2) is essential for vacuole homeostasis. We have therefore suggested that all Fab1p homologues may be PtdIns3P 5-kinases involved in membrane trafficking. It is unclear which phosphatidylinositol phosphate kinases (PIPkins) are responsible for PtdIns(3,5)P(2) synthesis in higher eukaryotes. To clarify how PtdIns(3,5)P(2) is synthesized in mammalian and other cells, we determined whether yeast and mammalian Fab1p homologues or mammalian Type I PIPkins (PtdIns4P 5-kinases) make PtdIns(3,5)P(2) in vivo. The recently cloned murine (p235) and Schizosaccharomyces pombe FAB1 homologues both restored basal PtdIns(3,5)P(2) synthesis in Deltafab1 cells and made PtdIns(3,5)P(2) in vitro. Only p235 corrected the growth and vacuolar defects of fab1 S. cerevisiae. A mammalian Type I PIPkin supported no PtdIns(3,5)P(2) synthesis. Thus, FAB1 and its homologues constitute a distinct class of Type III PIPkins dedicated to PtdIns(3,5)P(2) synthesis. The differential abilities of p235 and of SpFab1p to complement the phenotypic defects of Deltafab1 cells suggests that interaction(s) with other protein factors may be important for spatial and/or temporal regulation of PtdIns(3,5)P(2) synthesis. These results also suggest that p235 may regulate a step in membrane trafficking in mammalian cells that is analogous to its function in yeast.

General role of GDP dissociation inhibitor 2 in membrane release of Rab proteins: modulations of its functional interactions by in vitro and in vivo structural modifications.

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins in the functional cycle and recycling of Rab GTPases. Previous studies have demonstrated that GDI-1 can operate with multiple Rab proteins. In this study we have addressed a plausible general activity of GDI-2 in supporting Rab membrane release and have analyzed the requirements of sequence-conserved vs variable regions of GDI-2 in these functional interactions. The in vitro function of expressed recombinant GDI-2 wild-type-, point-, or deletion-mutant proteins was investigated toward several Rab family members, divergent in structure, localized and operating on different membranes, including Rab2, Rab4, Rab5, Rab8, Rab9, and Rab11. We demonstrate here a general and nearly invariant ability of GDI-2(WT) to release from membranes this subset of diverse Rabs. Deletion of an 18-residue segment from the C-terminal variable region yielded a fully functional or only slightly defective GDI-2. Conversely, substitution of Met at position 250 of the conserved region markedly abrogated the activity toward all Rabs. Surprisingly, a replacement of an adjacent conserved residue (Y249V) resulted in a selective Rab-dependent response and a profound gain of function toward specific Rabs. To further test whether the endogenous GDI-2 can adopt a gain-of-function conformation, we pharmacologically stimulated intact 3T3-L1 adipocytes to induce GDI-2 tyrosine phosphorylation. We found a pronounced increase of the Rab4 soluble form and its soluble complexes with the tyrosine-phosphorylated GDI-2. Together, these results indicate that (a) GDI-2 displays a general activity to release Rabs from membranes, (b) GDI-2-conserved residues, but not the C-terminal variable region, are essential for this activity, and (c) structural modifications in GDI-2 can enhance its functional activity, directing selective interactions with individual Rabs.

PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin.

One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P(2), have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D., and Ikonomov, O. (1999) Mol. Cell. Biol. 19, 623-634). Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P(2) by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P(2) but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine. By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P(2) generation, implying PIKfyve has a key role in their biosynthesis.

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes.

In insulin-sensitive fat and muscle cells, the major glucose transporter GLUT4 is constitutively sequestered in endosomal tubulovesicular membranes, and moves to the cell surface in response to insulin. While sequence information within GLUT4 appears to be responsible for its constitutive intracellular sequestration, the regulatory elements and mechanisms that enable this protein to achieve its unique sorting pattern under basal and insulin-stimulated conditions are poorly understood. We show here that arrest of endosome acidification in insulin-sensitive 3T3-L1 adipocytes by bafilomycin A1, a specific inhibitor of the vacuolar proton pump, results in the rapid and dose-dependent translocation of GLUT4 from the cell interior to the membrane surface; the effects of maximally stimulatory concentrations of bafilomycin A1 (400-800 nM) were equivalent to 50-65% of the effects of acute insulin treatment. Like insulin, bafilomycin A1 induced the redistribution of GLUT1 and Rab4, but not that of other proteins whose membrane localization has been shown to be insulin-insensitive. Studies to address the mechanism of this effect demonstrated that neither autophosphorylation nor internalization of the insulin receptor was altered by bafilomycin A1 treatment. Bafilomycin-induced GLUT4 translocation was not blocked by cell pretreatment with wortmannin. Taken together, these data indicate that arrest of endosome acidification mimics insulin action on GLUT4 and GLUT1 translocation by a mechanism distal to insulin receptor and phosphatidylinositol 3-kinase activation, and suggest an important role for endosomal pH in the membrane dynamics of the glucose transporters.

Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phosphoinositide kinase in insulin-sensitive cells.

Signaling by phosphorylated species of phosphatidylinositol (PI) appears to regulate diverse responses in eukaryotic cells. A differential display screen for fat- and muscle-specific transcripts led to identification and cloning of the full-length cDNA of a novel mammalian 2,052-amino-acid protein (p235) from a mouse adipocyte cDNA library. Analysis of the deduced amino acid sequence revealed that p235 contains an N-terminal zinc-binding FYVE finger, a chaperonin-like region in the middle of the molecule, and a consensus for phosphoinositide 5-kinases at the C terminus. p235 mRNA appears as a 9-kb transcript, enriched in insulin-sensitive cells and tissues, likely transcribed from a single-copy gene in at least two close-in-size splice variants. Specific antibodies against mouse p235 were raised, and both the endogenously and heterologously expressed proteins were biochemically detected in 3T3-L1 adipocytes and transfected COS cells, respectively. Immunofluorescence microscopy analysis of endogenous p235 localization in 3T3-L1 adipocytes with affinity-purified anti-p235 antibodies documented a punctate peripheral pattern. In COS cells, the expressed p235 N-terminal but not the C-terminal region displayed a vesicular pattern similar to that in 3T3-L1 adipocytes that became diffuse upon Zn2+ chelation or FYVE finger truncation. A recombinant protein comprising the N-terminal but not the C-terminal region of the molecule was found to bind 2.2 mole equivalents of Zn2+. Determination of the lipid kinase activity in the p235 immunoprecipitates derived from 3T3-L1 adipocytes or from COS cells transiently expressing p235 revealed that p235 displayed unique preferences for PI substrate over already phosphorylated PI. In conclusion, the mouse p235 protein determines an important novel class of phosphoinositide kinases that seems to be targeted to specific intracellular loci by a Zn-dependent mechanism.

Circadian clocks and hypertension: genetics and interactions.

Recent advances in molecular genetics of circadian rhythms and hypertension led to the discovery of separate groups of genes implicated in their regulation. Importantly, the identification in both mammals and flies of 6 homologous circadian clock genes strongly indicates that the circadian period is controlled by an evolutionary conserved set of genes. Studies in familial and experimental hypertension reveal that elevated blood pressure is due to mutations in genes implicated in the function of the renin-angiotensin-aldosterone system. A chronobiologic approach to experimental hypertension indicates that hypertension can be associated with selectively inverted circadian rhythm of arterial pressure. Several lines of evidence suggest that the rostral hypothalamus is an area of central integration of the endogenous rhythmic and other regulatory influences that modulate the phase and amplitude of circadian arterial pressure rhythmicity. The combination of advanced molecular genetics and continuous blood pressure monitoring with chronobiologic assessment emerges as a fruitful approach in better understanding the pathogenesis of hypertension.

Innervation and target tissue interactions induce Rab-GDP dissociation inhibitor (GDI) expression during peripheral synapse formation in developing chick ciliary ganglion neurons in situ.

Regulated exocytosis of neurotransmitter from synaptic vesicles involves the function of a small GTP-binding protein, Rab3A. Rab-GDP dissociation inhibitor (GDI) is an important modulator of Rab function and subcellular distribution. We have characterized the respective roles of innervation and target tissue interactions in regulating GDI expression during synapse formation in chick ciliary ganglion (CG) neurons developing in situ. Here we report the first full-length chick GDI cDNA sequence. It is highly homologous to mammalian GDI isoforms and includes all of the sequence-conserved regions critical for Rab3A binding. This chick GDI mRNA is predominantly expressed in neurons as judged by Northern blot analysis of tissue distribution and by in situ hybridization of CG sections. Developmental increases in CG GDI mRNA levels occur in two phases as determined by reverse transcription (RT)-PCR and by Northern analysis of both normal-developing and input- or target tissue-deprived ganglia. The initial phase appears to be independent of cell-cell interactions. In contrast, the second, larger increase is induced by both presynaptic inputs and postganglionic target tissues but does not occur until target tissue innervation. Synaptic interaction with the target seems necessary for the regulatory response to both inputs and target tissues. GDI protein levels show similar changes. The developmentally delayed ability of inputs and targets to influence GDI levels differs from the regulation of neurotransmitter receptor expression in CG neurons. These results suggest that distinct extrinsic regulatory signals influence the expression of synapse-related components at the presynaptic axon terminal versus postsynaptic membrane in an individual neuron.

Modulation of GDP-dissociation inhibitor protein membrane retention by the cellular redox state in adipocytes.

Small GTPases of the Rab family play a key role in the regulation of vesicular transport in eukaryotic cells. As they cycle on and off membranes, Rab proteins rely on the escort services of the GDP-dissociation inhibitor (GDI) proteins. While specific recognition of Rab-GDI complexes by membrane targets is suggested, the mechanisms underlying the subsequent GDI release into the cytosol remain unknown. In this study, we demonstrate that modulations of the cellular redox status in intact rat fat cells, 3T3-L1 adipocytes in culture, and other cultured cell types result in rapid, effective, dose-dependent, and selective membrane dynamics of GDI-1 and -2, membrane retention under reduced redox state, or dissociation under oxidized conditions. GDI retention on adipocyte membranes is associated with a complete arrest of insulin-induced translocation of GLUT4 glucose transporters onto plasma membrane. Together, these data suggest, first, that following Rab delivery to membranes, GDI release is promoted by a shift in the redox state and, second, that arrest of GDIs on membranes inhibits intracellular membrane trafficking events.

Integrative coordination of circadian mammalian diversity: neuronal networks and peripheral clocks.

Diverse circadian rhythms are generated, maintained and/or coordinated by brain structures constituting the circadian timing system. However, the mechanisms underlying the variety in activity types and circadian rhythm phases and amplitudes are currently unknown. We address this problem by comparing rhythms in diurnal and nocturnal mammals, while focusing on alterations not involving the central circadian oscillator. The circadian rhythms are divided into two groups: activity-independent and activity-related. The rhythms in the first group have similar acrophases in all mammals and are anticipated to function as an internal zeitgeber (time giver). Analysis of activity-related circadian rhythms in behavior, blood pressure (BP) and renal excretion suggests separate mechanisms in their regulation in addition to the central suprachiasmatic nuclei-located circadian oscillator. We propose that: (a) a passive hypothalamic oscillator coordinates the phases and underlies the high amplitude of behavioral circadian rhythms; (b) a separate rostral hypothalamic network participates in the regulation of the low-amplitude circadian BP rhythm; and (c) a circadian oscillator in the kidney generates electrolyte excretion rhythms. A model is offered where the overt activity is determined by the phase-relationship between the circadian and the passive hypothalamic oscillator. Specific brain structures or peripheral circadian oscillators integrate circadian and other signals for different activity-related circadian rhythms. The hypothalamic structures implicated in regulation of behavioral and blood pressure rhythms belong to the circadian timing system since they underlie circadian rhythms diversity. The same hypothalamic areas selectively modulate circadian rhythms in response to homeostatic stimuli or stress without engaging the circadian oscillator.

Association of cytosolic Rab4 with GDI isoforms in insulin-sensitive 3T3-L1 adipocytes.

Translocation of an intracellular pool of GLUT4 glucose transporters to the fat and muscle cell surface is thought to involve small GTP-binding proteins such as the Rab4 protein. The cycling of Rab proteins between cytosol and intracellular membranes necessary for their function appears to be regulated by GDP-dissociation inhibitors (GDI), three of which have been cloned thus far. Previous data suggest that Rab4 binds two of these isoforms of GDI (1 and 2) similarly when purified proteins are employed [Shisheva, A., et al. (1994) Mol. Cell. Biol. 14, 3459-3468]. In the present study, we have analyzed the cytosolic Rab4 in complexes with GDI-1 or GDI-2 in intact cells using a coprecipitation technique. We show here that in insulin-sensitive 3T3-L1 adipocytes and other cultured cells, Rab4 simultaneously forms stable cytosolic complexes with both GDI-1 and GDI-2. Acute insulin treatment of the cultured adipocytes significantly increases cytosolic levels of Rab4 which can be quantitatively immunoprecipitated with anti-Rab4 antibodies. Surprisingly, the increased cytosolic Rab4 due to insulin action is predominantly associated with cytosolic GDI-1. The levels of cytosolic Rab4-GDI-2 complexes were virtually unaltered by insulin. Insulin-dependent alterations of Rab4 and GDI-1 phosphorylation were not detected in 32P-labeled 3T3-L1 adipocytes, suggesting another mechanism accounts for the specificity of Rab4 binding to GDI-1. Taken together, these data suggest there is selective formation of Rab4-GDI-1 complexes in response to insulin which plays a role in the action of insulin on membrane trafficking.

Insulin-like actions of vanadate are mediated in an insulin-receptor-independent manner via non-receptor protein tyrosine kinases and protein phosphotyrosine phosphatases.

Most or all mammalian cells contain vanadium at a concentration of 0.1-1.0 microM. The bulk of the vanadium in cells is probably in the reduced vanadyl (IV) form. Although this element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the years 1975-1980 the vanadate ion was shown to act as an efficient inhibitor of Na+,K(+)-ATPase and of other related phosphohydrolyzes as well. In 1980 it was observed that vanadate vanadyl, when added to intact rat adipocytes, mimics the biological actions of insulin in stimulating hexose uptake and glucose oxidation. This initiated a long, currently active, field of research among basic scientists and diabetologists. Several of the aspects studied are reviewed here.