Egr1 plays a major role in the transcriptional response of white adipocytes to insulin and environmental cues.

It is believed that insulin regulates metabolic functions of white adipose tissue primarily at the post-translational level via the PI3K-Akt-mediated pathway. Still, changes in transcription also play an important role in the response of white adipocytes to insulin and environmental signals. One transcription factor that is dramatically and rapidly induced in adipocytes by insulin and nutrients is called Early Growth Response 1, or Egr1. Among other functions, it directly binds to promoters of leptin and ATGL stimulating the former and inhibiting the latter. Furthermore, expression of Egr1 in adipocytes demonstrates cell autonomous circadian pattern suggesting that Egr1 not only mediates the effect of insulin and nutrients on lipolysis and leptin production but also, coordinates insulin action with endogenous circadian rhythms of adipose tissue.

Inhibitors of RNA and protein synthesis cause Glut4 translocation and increase glucose uptake in adipocytes.

Insulin stimulates glucose uptake in adipocytes by triggering translocation of glucose transporter 4-containg vesicles to the plasma membrane. Under basal conditions, these vesicles (IRVs for insulin-responsive vesicles) are retained inside the cell via a "static" or "dynamic" mechanism. We have found that inhibitors of RNA and protein synthesis, actinomycin D and emetine, stimulate Glut4 translocation and glucose uptake in adipocytes without engaging conventional signaling proteins, such as Akt, TBC1D4, or TUG. Actinomycin D does not significantly affect endocytosis of Glut4 or recycling of transferrin, suggesting that it specifically increases exocytosis of the IRVs. Thus, the intracellular retention of the IRVs in adipocytes requires continuous RNA and protein biosynthesis de novo. These results point out to the existence of a short-lived inhibitor of IRV translocation thus supporting the "static" model.

Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4.

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin-sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.

The formation of an insulin-responsive vesicular cargo compartment is an early event in 3T3-L1 adipocyte differentiation.

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.

Exploring protein interactions.

Protein Targeting Protocols Edited by Robert A. Clegg. Totowa, Humana, 1998, $79.50 (xii+ 331 pages), ISBN 0-896-03450-X.

RNA-binding protein kinase from amphibian oocytes is a casein kinase II.

RNA-binding protein kinase from amphibian oocytes modifies serine and threonine residues in the molecules of substrates and utilizes both ATP and GTP. Low concentrations of heparin inhibit protein kinase. The foregoing suggests that this enzyme is casein kinase II. It is shown that RNA-binding proteins lack active forms of phosphatases and proteases which may affect the results of phosphorylation of both endogenous and exogenous substrates.

pH-dependent changes in structure and RNA-binding activity of casein kinase 2 from Rana temporaria oocytes.

It is demonstrated by filter-binding assay that casein kinase 2 from Rana temporaria oocytes binds rRNA in vitro with high affinity. Ligand-blotting shows that rRNA-binding activity is inherent to alpha and alpha' subunits of the enzyme. Increase of pH from 6.5 to 7.5 has little effect on casein kinase but completely suppresses rRNA-binding activity of the enzyme. Sedimentation coefficient of casein kinase 2 also depends on pH: at pH 7.5 it is mainly 10 S, and at pH 6.5-18 S. At pH 6.95 the amounts of both forms are equal. The heavy form of casein kinase 2 practically lacks rRNA-binding activity.

Presence of the gypsy (MDG4) retrotransposon in extracellular virus-like particles.

As a first step to investigate the functional activity of gypsy virus-like particles (VLPs) we explored the possibility of preservation of its VLP in extracellular form. The preparations containing extracellular gypsy VLP from Drosophila melanogaster and D. virilis were obtained. Full-length polyA+ RNA and polyA+ RNA-DNA complexes of gypsy were observed in both preparations. The polypeptides with some specificity to gypsy nucleic acids were identified in the obtained VLP preparations. These data accompanied by morphological characteristics of samples testify the presence of intact gypsy VLP in cultured media both from D. melanogaster and D. virilis cultivated cells.

The decreased level of casein kinase 2 in brain cortex of schizophrenic and Alzheimer's disease patients.

The content of casein kinase 2 is considerably decreased in ribosome-free extracts of the frontal cortex of schizophrenic and Alzheimer's disease patients in comparison to normal brains as has been demonstrated by means of immunoblotting. The activity of casein kinase 2 towards endogenous substrates and casein is also diminished in the cases of mental pathologies examined. This phenomenon may explain the well-known aberrations in the phosphorylation of structural proteins of human brain which are intrinsic for the mental diseases.

[Progesterone-induced activation of RNA-binding casein kinase type II during maturation of frog oocytes]. / Indutsiruemaia progesteronom aktivatsiia RNK-sviazyvaiushchei kazeinovoi kinazy tipa II pri sozrevanii ootsitov liagushki.

During progesterone-induced maturation of the Rana temporaria oocytes phosphorylation of RNA(heparin)-binding proteins changes drastically due to the alterations in the activity of RNA-binding casein kinase II. Its activity increases 7 hr later administration of progesterone and correlates with the level of translation in oocytes. Cycloheximide almost completely inhibits the protein biosynthesis but has no effect on the activity of RNA-binding casein kinase and the set of phosphorylated polypeptides. The possibility is discussed that this enzyme participates in the translation control mechanisms.

[Informosomes and translation activity of mRNA in eukaryotic cells]. / Informosomy i transliatsionnaia aktivnost' mRNK v éukarioticheskikh kletkakh.

In this review we summarize recent results which are obtained in the field of structure and functions of cytoplasmic mRNP, or informosomes. These data lead to conclusion, that the informosomal structure of mRNA in eukaryotic cells makes possible the establishment of translational control by masking-demasking of messages.

[Intracellular localization of casein kinase of type II in oocytes of Rana temporaria]. / Vnutrikletochnaia lokalizatsiia kazeinovoi kinazy tipa II v ootsitakh Rana temporaria.

Casein kinase II has been isolated from ribosome-free extracts of Rana temporaria oocytes by chromatography on heparin-sepharose, phosphocellulose and mono Q. It consists of different subunits with molecular weights 43, 41 and 29 Kda. Protein kinase has been labelled in vitro with 125I and injected back into amphibian oocytes. With the help of radioautography it is shown that this enzyme forms in the oocyte cytoplasm a wide concentric ring around the nucleus. Part of the labelled casein kinase II is associated with free cytoplasmic informosomes.

[Protein kinase activity of RNA-binding proteins from amphibian oocytes]. / Proteinkinaznaia aktivnost' RNK-sviazyvaiushchikh belkov ootsitov amfibii.

RNA-binding proteins isolated from amphibian oocytes ribosome-free extract by affinity chromatography on poly (U)-Sepharose possess an endogenous protein kinase activity. Incubation of these proteins with [gamma-32P] ATP leads to the incorporation of labelled phosphate into 6-7 polypeptide chains with molecular masses from 20 000 to 80 000, which are estimated by disk-electrophoresis in the presence of sodium dodecyl sulphate, followed by autoradiography of dried gels. High-voltage paper electrophoresis of acid hydrolysates of labelled proteins showed that mainly [32P]phosphoserine is formed in this reaction. Phosphorylation is not stimulated by cAMP and reaches its maximum by 20 degrees and pH near 8. The presence of poly(U) in the reactional mixture inhibits phosphorylation considerably. The opportunity of partial decrease or loss of RNA-binding activity due to phosphorylation of RNA-binding proteins is discussed.

Insulin regulation of protein traffic in rat adipose cells.

Rat adipocytes were biotinylated with cell-impermeable reagents, sulfo-N-hydroxysuccinimide-biotin and sulfo-N-hydroxysuccinimide-S-S-biotin in the absence and presence of insulin. Biotinylated and nonbiotinylated populations of the insulin-like growth factor-II/mannose 6-phosphate receptor, the transferrin receptor, and insulin-responsive aminopeptidase were separated by adsorption to streptavidin-agarose to determine the percentage of the biotinylated protein molecules versus their total amount in different subcellular compartments. Results indicate that adipose cells possess at least two distinct cell surface recycling pathways for insulin-like growth factor-II/mannose 6-phosphate receptor (MPR) and transferrin receptor (TfR): one which is mediated by glucose transporter isoform 4(Glut4)-vesicles and another that bypasses this compartment. Under basal conditions, the first pathway is not active, and cell surface recycling of TfR and, to a lesser extent, MPR proceeds via the second pathway. Insulin dramatically stimulates recycling through the first pathway and has little effect on the second. Within the Glut4-containing compartment, insulin has profoundly different effects on intracellular trafficking of insulin-responsive aminopeptidase on one hand and MPR and TfR on the other. After insulin administration, insulin-responsive aminopeptidase is redistributed from Glut4-containing vesicles to the plasma membrane and stays there for at least 30 min with minimal detectable internalization and recycling, whereas MPR and TfR rapidly shuttle between Glut4 vesicles and the plasma membrane in such a way that after 30 min of insulin treatment, virtually every receptor molecule in this compartment completes at least one trafficking cycle to the cell surface. Thus, different recycling proteins, which compose Glut4-containing vesicles, are internalized into this compartment at their own distinctive rates.

[Inhibition of casein kinase type 2 activity by formation of complexes with mRNA in vivo]. / Ingibirovanie aktivnosti kazeinovoi kinazy tipa 2 za schet obrazovaniia kompleksov.

It is known that casein kinase 2 possesses, besides the protein kinase, an RNA-binding activity. Using ligand blotting it has been demonstrated that the both activities are localized on the alpha- and alpha'-subunits of the enzyme. Casein kinase 2 is suppressed in vitro by polyuridylic acid. A part of the intracellular pool of casein kinase 2 is found in the informosomes. The informosomes and free proteins were separated by centrifugation in a sucrose density gradient, and each fraction was incubated with casein and [gamma-32P]ATP. The informosome-bound protein kinase is completely inhibited, while the free protein kinases heavily phosphorylate casein. It is concluded that the activity of casein kinase 2 can be regulated by the reversible formation of complexes with RNA.

gp160, a tissue-specific marker for insulin-activated glucose transport.

We have isolated and partially sequenced a M(r) 160,000 glycoprotein whose rate of cycling to and from the adipocyte cell surface is enhanced by insulin in a manner apparently identical to the effect of insulin on GLUT4 cycling. Based on the protein sequence, we have prepared an antipeptide antibody against this protein, gp160. The antibody recognizes a M(r) 160,000 protein whose subcellular distribution is identical to that of GLUT4. This was determined by three separate criteria: (i) Western blotting of fractionated adipocyte membranes from cells exposed to insulin or not, (ii) adsorption of vesicles with anti-GLUT4 antibodies followed by Western blotting, and (iii) separation of microsomal vesicles by sucrose velocity and density gradients. By all three criteria, GLUT4 and gp160 are completely colocalized in rat fat cells. Moreover, gp160 can be detected by Western blot only in fat and cardiac and skeletal muscles and was absent from all other tissues tested. Thus, gp160 is an additional marker for physiologically important, insulin-sensitive glucose transport. Its further study at the protein and DNA level may reveal information about the mechanistic details of insulin-activated GLUT4 translocation as well as information concerning the tissue-specific expression of GLUT4 and gp160.

A Glut4-vesicle marker protein, insulin-responsive aminopeptidase, is localized in a novel vesicular compartment in PC12 cells.

Glut4-containing vesicles represent a regulated recycling compartment in insulin-sensitive fat and skeletal muscle cells, the nature and origin of which are not fully understood. In addition to Glut4 itself, these vesicles compartmentalize a number of proteins, at least one of which, insulin-responsive aminopeptidase, or IRAP, is completely colocalized with Glut4 in insulin-sensitive tissues. However, unlike Glut4, IRAP is expressed in a variety of other tissues and cell lines. Here, we explored the intracellular localization of IRAP in the rat pheochromocytoma cell line PC12. We found that this protein is present in a distinct population of slowly recycling light vesicles. By gradient centrifugations, immunoadsorption and double immunofluorescent staining, these vesicles are different from transferrin-containing endosomes, small synaptic vesicles and secretory granules and may thus represent a novel compartment in PC12 cells. Glut4-GFP chimera transiently expressed in PC12 cells is targeted to IRAP-containing vesicles indicating that cotargeting of Glut4 and IRAP is not specific for adipocytes and myocytes, but is faithful in a foreign cell type. We suggest that PC12 cells may possess a novel type of a vesicular carrier that may represent the homolog of Glut4-vesicles.

Cellugyrin is a marker for a distinct population of intracellular Glut4-containing vesicles.

Although Glut4 traffic is routinely described as translocation from an "intracellular storage pool" to the plasma membrane, it has been long realized that Glut4 travels through at least two functionally distinct intracellular membrane compartments on the way to and from the cell surface. Biochemical separation and systematic studies of the individual Glut4-containing compartments have been limited by the lack of appropriate reagents. We have prepared a monoclonal antibody against a novel component protein of Glut4 vesicles and have identified this protein as cellugyrin, a ubiquitously expressed homologue of a major synaptic vesicle protein, synaptogyrin. By means of sucrose gradient centrifugation, immunoadsorption, and confocal microscopy, we have shown that virtually all cellugyrin is co-localized with Glut4 in the same vesicles. However, unlike Glut4, cellugyrin is not re-distributed to the plasma membrane in response to insulin stimulation, and at least 40-50% of the total population of Glut4 vesicles do not contain this protein. We suggest that cellugyrin represents a specific marker of a functionally distinct population of Glut4 vesicles that permanently maintains its intracellular localization and is not recruited to the plasma membrane by insulin.

The insulin-like growth factor II/mannose 6-phosphate receptor utilizes the same membrane compartments as GLUT4 for insulin-dependent trafficking to and from the rat adipocyte cell surface.

The insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptor recycles in adipose cells between the cell surface and an intracellular storage pool, and the rate of this trafficking is markedly enhanced by insulin. We show here that the IGF-II/Man-6-P receptor is a constituent of the GLUT4-containing compartment ("GLUT4 vesicles") where it represents gp230, a major recycling protein detected earlier by cell surface biotinylation (Kandror, K. V., and Pilch, P. F. (1994) J. Biol. Chem. 269, 138-142). The GLUT4 vesicles include 10-15% of the total and all of the acutely insulin-responsive recycling population of the IGF-II/Man-6-P receptor. The main part of the IGF-II/Man-6-P receptor population is excluded from the pathway of GLUT4 trafficking and either resides permanently in intracellular membranes or has a much slower rate of cycling to the cell surface. Thus, GLUT4 vesicles mediate the insulin-dependent delivery to the cell surface of the IGF-II/Man-6-P receptor as well as the other recyclable proteins with extracellular functional domains (GLUT4 and the aminopeptidase gp160).