Pyruvate shuttle in muscle cells: high-affinity pyruvate transport sites insensitive to trans-lactate efflux

2003 ◽  
Vol 285 (6) ◽  
pp. E1196-E1204
Author(s):  
Raymond Mengual ◽  
Kaoukib el Abida ◽  
Nassima Mouaffak ◽  
Michel Rieu ◽  
Michele Beaudry
Keyword(s):  
High Capacity ◽  
Muscle Cells ◽  
Type I ◽  
Maximal Velocity ◽  
Type Ii ◽  
Lactate Shuttle ◽  
Transport Efficiency ◽  
High Affinity ◽  
High K ◽  
Transport Kinetic

The specificity of the transport mechanisms for pyruvate and lactate and their sensitivity to inhibitors were studied in L6 skeletal muscle cells. Trans- and cis-lactate effects on pyruvate transport kinetic parameters were examined. Pyruvate and lactate were transported by a multisite carrier system, i.e., by two families of sites, one with low affinity and high capacity (type I sites) and the other with high affinity and low capacity (type II). The multisite character of transport kinetics was not modified by either hydroxycinnamic acid (CIN) or p-chloromercuribenzylsulfonic acid (PCMBS), which exert different types of inhibition. The transport efficiency (TE) ratios of maximal velocity to the trans-activation dissociation constant ( Kt) showed that lactate and pyruvate were preferentially transported by types I and II sites, respectively. The cis-lactate effect was observed with high Ki values for both sites. The trans-lactate effect on pyruvate transport occurred only on type I sites and exhibited an asymmetric interaction pattern ( Kt of inward lactate > Kt of outward lactate). The inability of lactate to trans-stimulate type II sites suggests that intracellular lactate cannot recruit these sites. The high-affinity type II sites act as a specific pyruvate shuttle and constitute an essential relay for the intracellular lactate shuttle.

scholarly journals Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Yong Fu ◽  
Xia Cui ◽  
Sai Fan ◽  
Jing Liu ◽  
Xiao Zhang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Lipid Metabolism ◽  
Toxoplasma Gondii ◽  
Binding Protein ◽  
Ankyrin Repeat ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
High K ◽  
Acyl Coenzyme A

ABSTRACT Acyl coenzyme A (CoA)-binding protein (ACBP) can bind acyl-CoAs with high specificity and affinity, thus playing multiple roles in cellular functions. Mitochondria of the apicomplexan parasite Toxoplasma gondii have emerged as key organelles for lipid metabolism and signaling transduction. However, the rationale for how this parasite utilizes acyl-CoA-binding protein to regulate mitochondrial lipid metabolism remains unclear. Here, we show that an ankyrin repeat-containing protein, TgACBP2, is localized to mitochondria and displays active acyl-CoA-binding activities. Dephosphorylation of TgACBP2 is associated with relocation from the plasma membrane to the mitochondria under conditions of regulation of environmental [K+]. Under high [K+] conditions, loss of ACBP2 induced mitochondrial dysfunction and apoptosis-like cell death. Disruption of ACBP2 caused growth and virulence defects in the type II strain but not in type I parasites. Interestingly, mitochondrial association factor-1 (MAF1)-mediated host mitochondrial association (HMA) restored the growth ability of ACBP2-deficient type II parasites. Lipidomics analysis indicated that ACBP2 plays key roles in the cardiolipin metabolism of type II parasites and that MAF1 expression complemented the lipid metabolism defects of ACBP2-deficient type II parasites. In addition, disruption of ACBP2 caused attenuated virulence of Prugniuad (Pru) parasites for mice. Taking the results collectively, these data indicate that ACBP2 is critical for the growth and virulence of type II parasites and for the growth of type I parasites under high [K+] conditions. IMPORTANCE Toxoplasma gondii is one of the most successful human parasites, infecting nearly one-third of the total world population. T. gondii tachyzoites residing within parasitophorous vacuoles (PVs) can acquire fatty acids both via salvage from host cells and via de novo synthesis pathways for membrane biogenesis. However, although fatty acid fluxes are known to exist in this parasite, how fatty acids flow through Toxoplasma lipid metabolic organelles, especially mitochondria, remains unknown. In this study, we demonstrated that Toxoplasma expresses an active ankyrin repeat containing protein TgACBP2 to coordinate cardiolipin metabolism. Specifically, HMA acquisition resulting from heterologous functional expression of MAF1 rescued growth and lipid metabolism defects in ACBP2-deficient type II parasites, manifesting the complementary role of host mitochondria in parasite cardiolipin metabolism. This work highlights the importance of TgACBP2 in parasite cardiolipin metabolism and provides evidence for metabolic association of host mitochondria with T. gondii.

scholarly journals Relative contributions of Na+-dependent phosphate co-transporters to phosphate transport in mouse kidney: RNase H-mediated hybrid depletion analysis

1997 ◽  
Vol 327 (3) ◽  
pp. 735-739 ◽  
Author(s):  
Ken-ichi MIYAMOTO ◽  
Hiroko SEGAWA ◽  
Kyoko MORITA ◽  
Tomoko NII ◽  
Sawako TATSUMI ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Xenopus Oocytes ◽  
Mouse Kidney ◽  
Rnase H ◽  
Transport Systems ◽  
Kidney Cortex ◽  
Type I ◽  
Type Ii ◽  
High Affinity ◽  
Pi Transport

Reabsorption of Pi in the proximal tubule of the kidney is an important determinant of Pi homoeostasis. At least three types (types I-III) of high-affinity Na+-dependent Pi co-transporters have been identified in mammalian kidneys. The relative roles of these three types of Na+/Pi co-transporters in Pi transport in mouse kidney cortex have now been investigated by RNase H-mediated hybrid depletion. Whereas isolated brush-border membrane vesicles showed the presence of two kinetically distinct Na+/Pi co-transport systems (high Km-low Vmax and low Km-high Vmax), Xenopus oocytes, microinjected with polyadenylated [poly(A)+] RNA from mouse kidney cortex, showed only the high-affinity Pi uptake system. Kidney poly(A)+ RNA was incubated in vitro with antisense oligonucleotides corresponding to Npt-1 (type I), NaPi -7 (type II) or Glvr-1 (type III) Na+/Pi co-transporter mRNAs, and then with RNase H. Injection of such treated RNA preparations into Xenopus oocytes revealed that an NaPi-7 antisense oligonucleotide that resulted in complete degradation of NaPi-7 mRNA (as revealed by Northern blot analysis), also induced complete inhibition of Pi uptake. Degradation of Npt-1 or Glvr-1 mRNAs induced by corresponding antisense oligonucleotides had no effect on Pi transport, which was subsequently measured in oocytes. These results indicate that the type II Na+/Pi co-transporter NaPi-7 mediated most Na+-dependent Pi transport in mouse kidney cortex.

scholarly journals Interferon Regulatory Factor 1 Protects against Chikungunya Virus-Induced Immunopathology by Restricting Infection in Muscle Cells

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Sharmila Nair ◽  
Subhajit Poddar ◽  
Raeann M. Shimak ◽  
Michael S. Diamond
Keyword(s):  
Chikungunya Virus ◽  
Muscle Cells ◽  
Interferon Regulatory Factor ◽  
Ross River Virus ◽  
Type I ◽  
Type Ii ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 1 ◽  
Ifn Γ

ABSTRACT The innate immune system protects cells against viral pathogens in part through the autocrine and paracrine actions of alpha/beta interferon (IFN-α/β) (type I), IFN-γ (type II), and IFN-λ (type III). The transcription factor interferon regulatory factor 1 (IRF-1) has a demonstrated role in shaping innate and adaptive antiviral immunity by inducing the expression of IFN-stimulated genes (ISGs) and mediating signals downstream of IFN-γ. Although ectopic expression experiments have suggested an inhibitory function of IRF-1 against infection of alphaviruses in cell culture, its role in vivo remains unknown. Here, we infected Irf1 −/− mice with two distantly related arthritogenic alphaviruses, chikungunya virus (CHIKV) and Ross River virus (RRV), and assessed the early antiviral functions of IRF-1 prior to induction of adaptive B and T cell responses. IRF-1 expression limited CHIKV-induced foot swelling in joint-associated tissues and prevented dissemination of CHIKV and RRV at early time points. Virological and histological analyses revealed greater infection of muscle tissues in Irf1 −/− mice than in wild-type mice. The antiviral actions of IRF-1 appeared to be independent of the induction of type I IFN or the effects of type II and III IFNs but were associated with altered local proinflammatory cytokine and chemokine responses and differential infiltration of myeloid cell subsets. Collectively, our in vivo experiments suggest that IRF-1 restricts CHIKV and RRV infection in stromal cells, especially muscle cells, and that this controls local inflammation and joint-associated swelling. IMPORTANCE Interferon regulatory factor 1 (IRF-1) is a transcription factor that regulates the expression of a broad range of antiviral host defense genes. In this study, using Irf1 −/− mice, we investigated the role of IRF-1 in modulating pathogenesis of two related arthritogenic alphaviruses, chikungunya virus and Ross River virus. Our studies show that IRF-1 controlled alphavirus replication and swelling in joint-associated tissues within days of infection. Detailed histopathological and virological analyses revealed that IRF-1 preferentially restricted CHIKV infection in cells of nonhematopoietic lineage, including muscle cells. The antiviral actions of IRF-1 resulted in decreased local inflammatory responses in joint-associated tissues, which prevented immunopathology.

Reconstitution of the Functional Mouse Oncostatin M (OSM) Receptor: Molecular Cloning of the Mouse OSM Receptor β Subunit

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 804-815 ◽  
Author(s):  
Minoru Tanaka ◽  
Takahiko Hara ◽  
Neal G. Copeland ◽  
Debra J. Gilbert ◽  
Nancy A. Jenkins ◽  
...  
Keyword(s):  
Amino Acid Level ◽  
Embryonic Stem ◽  
Oncostatin M ◽  
Chromosome 15 ◽  
Type I ◽  
Type Ii ◽  
Β Subunit ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Species Specific

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines that share the gp130 receptor subunit. Of these family members, leukemia inhibitory factor (LIF) is most closely related to OSM, and various overlapping biologic activities have been described between human LIF and OSM (hLIF and hOSM). Two types of functional hOSM receptors are known: the type I OSM receptor is identical to the LIF receptor that consists of gp130 and the LIF receptor β subunit (LIFRβ), and the type II OSM receptor consists of gp130 and the OSM receptor β subunit (OSMRβ). It is thus conceivable that common biologic activities between hLIF and hOSM are mediated by the shared type I receptor and OSM-specific activities are mediated by the type II receptor. However, in contrast to the human receptors, recent studies have demonstrated that mouse OSM (mOSM) does not activate the type I receptor and exhibits unique biologic activity. To elucidate the molecular structure of the functional mOSM receptor, we cloned a cDNA encoding mOSMRβ, which is 55.5% identical to the hOSMRβ at the amino acid level. mOSM-responsive cell lines express high-affinity mOSM receptors, as well as mOSMRβ, whereas embryonic stem cells, which are responsive to LIF but not to mOSM, do not express mOSMRβ. mOSMRβ alone binds mOSM with low affinity (kd = 13.0 nmol/L) and forms a high-affinity receptor (kd = 606 pmol/L) with gp130. Ba/F3 transfectants expressing both mOSMRβ and gp130 proliferated in response to mOSM, but failed to respond to LIF and human OSM. Thus, the cloned mOSMRβ constitutes an essential and species-specific receptor component of the functional mOSM receptor. Reminiscent of the colocalization of the mOSM and mLIF genes, the mOSMRβ gene was found to be located in the vicinity of the LIFRβ locus in the proximal end of chromosome 15.

Reconstitution of the Functional Mouse Oncostatin M (OSM) Receptor: Molecular Cloning of the Mouse OSM Receptor β Subunit

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 804-815 ◽  
Author(s):  
Minoru Tanaka ◽  
Takahiko Hara ◽  
Neal G. Copeland ◽  
Debra J. Gilbert ◽  
Nancy A. Jenkins ◽  
...  
Keyword(s):  
Amino Acid Level ◽  
Embryonic Stem ◽  
Oncostatin M ◽  
Chromosome 15 ◽  
Type I ◽  
Type Ii ◽  
Β Subunit ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Species Specific

Abstract Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines that share the gp130 receptor subunit. Of these family members, leukemia inhibitory factor (LIF) is most closely related to OSM, and various overlapping biologic activities have been described between human LIF and OSM (hLIF and hOSM). Two types of functional hOSM receptors are known: the type I OSM receptor is identical to the LIF receptor that consists of gp130 and the LIF receptor β subunit (LIFRβ), and the type II OSM receptor consists of gp130 and the OSM receptor β subunit (OSMRβ). It is thus conceivable that common biologic activities between hLIF and hOSM are mediated by the shared type I receptor and OSM-specific activities are mediated by the type II receptor. However, in contrast to the human receptors, recent studies have demonstrated that mouse OSM (mOSM) does not activate the type I receptor and exhibits unique biologic activity. To elucidate the molecular structure of the functional mOSM receptor, we cloned a cDNA encoding mOSMRβ, which is 55.5% identical to the hOSMRβ at the amino acid level. mOSM-responsive cell lines express high-affinity mOSM receptors, as well as mOSMRβ, whereas embryonic stem cells, which are responsive to LIF but not to mOSM, do not express mOSMRβ. mOSMRβ alone binds mOSM with low affinity (kd = 13.0 nmol/L) and forms a high-affinity receptor (kd = 606 pmol/L) with gp130. Ba/F3 transfectants expressing both mOSMRβ and gp130 proliferated in response to mOSM, but failed to respond to LIF and human OSM. Thus, the cloned mOSMRβ constitutes an essential and species-specific receptor component of the functional mOSM receptor. Reminiscent of the colocalization of the mOSM and mLIF genes, the mOSMRβ gene was found to be located in the vicinity of the LIFRβ locus in the proximal end of chromosome 15.

scholarly journals Development of muscle fiber specialization in the rat hindlimb.

1981 ◽  
Vol 90 (1) ◽  
pp. 128-144 ◽  
Author(s):  
N A Rubinstein ◽  
A M Kelly
Keyword(s):  
Muscle Fiber ◽  
Muscle Development ◽  
Fiber Type ◽  
Muscle Cells ◽  
Postnatal Period ◽  
Type I ◽  
Fiber Types ◽  
Type Ii ◽  
Slow Myosin ◽  
Fast Myosin

The appearance of fast and slow fiber types in the distal hindlimb of the rat was investigated using affinity-purified antibodies specific to adult fast and slow myosins, two-dimensional electrophoresis of myosin light chains, and electron microscope examination of developing muscle cells. As others have noted, muscle histogenesis is not synchronous; rather, a series of muscle fiber generations occurs, each generation forming along the walls of the previous generation. At the onset of myotube formation on the 15th d of gestation, the antimyosin antibodies do not distinguish among fibers. All fibers react strongly with antibody to fast myosin but not with antibody to slow myosin. The initiation of fiber type differentiation can be detected in the 17-d fetus by a gradual increase in the binding of antibody to slow myosin in the primary, but not the secondary, generation myotubes. Moreover, neuromuscular contacts at this crucial time are infrequent, primitive, and restricted predominantly, but not exclusively, to the primary generation cells, the same cells which begin to bind large amounts of antislow myosin at this time. With maturation, the primary generation cells decrease their binding of antifast myosin and become type I fibers. Secondary generation cells are initially all primitive type II fibers. In future fast muscles the secondary generation cells remain type II, while in future slow muscles most of the secondary generation cells eventually change to type I over a prolonged postnatal period. We conclude that the temporal sequence of muscle development is fundamentally important in determining the genetic expression of individual muscle cells.

scholarly journals Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells

1987 ◽  
Vol 241 (1) ◽  
pp. 249-255 ◽  
Author(s):  
U Vischer ◽  
B Blondel ◽  
C B Wollheim ◽  
W Höppner ◽  
H J Seitz ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Deae Cellulose ◽  
Indirect Evidence ◽  
Exchange Chromatography ◽  
Hill Coefficient ◽  
Type I ◽  
Maximal Velocity ◽  
Type Ii ◽  
Two Dimensional Gel Electrophoresis ◽  
Insulinoma Cells

We have analysed the pattern of expression of the hexokinase isoenzyme group in RIN-m5F insulinoma cells. Three hexokinase forms were resolved by DEAE-cellulose chromatography. The most abundant isoenzyme co-eluted with hexokinase type II from rat adipose tissue and displayed a Km for glucose of 0.15 mM, similar to the adipose-tissue enzyme. Hexokinase type II was in large part associated with a particulate subcellular fraction in RIN-m5F cells. The two other hexokinases separated by ion-exchange chromatography were an enzyme similar to hexokinase type I from brain and glucokinase (or hexokinase type IV). The latter isoenzyme was identified as the liver-type glucokinase by the following properties: co-elution with hepatic glucokinase from DEAE-cellulose and DEAE-Sephadex; sigmoid saturation kinetics with glucose with half-maximal velocity at 5.6 mM and Hill coefficient (h) of 1.54; suppression of enzyme activity by antibodies raised against rat liver glucokinase; apparent Mr of 56,500 and pI of 5.6, as shown by immunoblotting after one- and two-dimensional gel electrophoresis; peptide map identical with that of hepatic glucokinase after proteolysis with chymotrypsin and papain. These data indicate that the gene coding for hepatic glucokinase is expressed in RIN-m5F cells, a finding consistent with indirect evidence for the presence of glucokinase in the beta-cell of the islet of Langerhans. On the other hand, the overall pattern of hexokinases is distinctly different in RIN-m5F cells and islets of Langerhans, since hexokinase type II appears to be lacking in islets. Alteration in hexokinase expression after tumoral transformation has been reported in other systems.

Identification of a common gene signature for type II cytokine–associated myeloid cells elicited in vivo in different pathologic conditions

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 575-583 ◽  
Author(s):  
Gholamreza Hassanzadeh Ghassabeh ◽  
Patrick De Baetselier ◽  
Lea Brys ◽  
Wim Noël ◽  
Jo A. Van Ginderachter ◽  
...  
Keyword(s):  
Expression Profiling ◽  
High Capacity ◽  
Myeloid Cells ◽  
Parasitic Infections ◽  
Type I ◽  
Type Ii ◽  
In Vivo Models ◽  
Signature Genes

Compared with type I cytokine–associated myeloid (M1) cells, the molecular repertoire and mechanisms underlying functional properties of type II cytokine–associated myeloid (M2) cells are poorly characterized. Moreover, most studies have been limited to in vitro–elicited M2 cells. Here, comparative gene expression profiling of M1 and M2 cells, elicited in murine models of parasitic infections and cancer, yielded a common signature for in vivo–induced M2 populations independent of disease model, mouse strain, and organ source of cells. Some of these genes, such as cadherin-1, selenoprotein P, platelet-activating factor acetylhydrolase, and prosaposin, had not been documented as associated with M2. Overall, the common signature genes provide a molecular basis for a number of documented or suggested properties of M2, including immunomodulation, down-regulation of inflammation, protection against oxidative damage, high capacity for phagocytosis, and tissue repair. Interestingly, several common M2 signature genes encode membrane-associated markers that could be useful for the identification and isolation of M2. Some of these genes were not induced by IL-4/IL-13 or IL-10 under various in vitro settings and thus were missed in approaches based on in vitro–activated cells, validating our choice of in vivo models for expression profiling of myeloid cells.

scholarly journals Development of an Immunofluorometric, High-Capacity, Cell-Based Assay for the Measurement of Human Type I and Type II Interferons

1999 ◽  
Vol 4 (1) ◽  
pp. 33-37 ◽  
Author(s):  
Lan Trinh ◽  
Russell Ziegler ◽  
Diane Watling ◽  
R. Michael Snider ◽  
Ed Croze
Keyword(s):  
Limit Of Detection ◽  
High Capacity ◽  
Viable Cell ◽  
Microtiter Plate ◽  
Type I ◽  
Type Ii ◽  
Time Resolved ◽  
Type I Ifns ◽  
Coefficients Of Variation ◽  
High Throughput Assay

We have developed a cell-based 96-well microtiter plate, high throughput assay for measuring both type I and type II interferon (IFN) activity on human cells. This assay makes use of a previously described IFN-specific reporter stably expressed in human HT 1080 cells. The induction of the reporter by IFN is determined by measuring the IFN-dependent expression of CD2 on the cell surface. The cytokine-induced expression of CD2 occurs within 48 h and is measured using a time-resolved fluorometric immunoassay. The limit of detection for type I IFN is >0.4 IU/ml. Interassay and intraassay coefficients of variation were 1.1% and 1.3% for the medium control (31 IU IFNβ1b/ml), respectively. The limit of detection for type II IFN is >8 IU/ml, and the assay coefficients of variation are similar to those determined for type I IFNs. The level of sensitivity for this assay is comparable to other assays commonly used to measure IFN activity on cells. The current assay has an advantage over antiviral and antiproliferative assays, in that there is no requirement for the use of pathogenic virus or for determining viable cell numbers. The current assay is ideally suited for increasing sample screening and high-capacity automation, making it an excellent tool for drug discovery.

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