scholarly journals Biochemical and functional characterization of the GLUT5 fructose transporter in rat skeletal muscle

1998 ◽  
Vol 336 (2) ◽  
pp. 361-366 ◽  
Author(s):  
Froogh DARAKHSHAN ◽  
Eric HAJDUCH ◽  
Søren KRISTIANSEN ◽  
Erik A. RICHTER ◽  
Harinder S. HUNDAL
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Plasma Membranes ◽  
Functional Characterization ◽  
Protein Band ◽  
Subcellular Fractionation ◽  
Rat Skeletal Muscle ◽  
Reverse Transcription Pcr ◽  
Molecular Approaches ◽  
Rat Adipose Tissue

Previous work has demonstrated that human skeletal muscle and adipose tissue both express the GLUT5 fructose transporter, but to date the issue of whether this protein is also expressed in skeletal muscle and adipose tissue of rodents has remained unresolved. In the present study we have used a combination of biochemical and molecular approaches to ascertain whether rat skeletal muscle expresses GLUT5 protein and, if so, whether it possesses the capacity to transport fructose. An isoform-specific antibody against rat GLUT5 reacted positively with crude membranes prepared from rat skeletal muscle. A single immunoreactive band of approx. 50 kDa was visualized on immunoblots which was lost when using anti-(rat GLUT5) serum that had been pre-adsorbed with the antigenic peptide. Subcellular fractionation of skeletal muscle localized this immunoreactivity to a single membrane fraction that was enriched with sarcolemma. Plasma membranes, but not low-density microsomes, from rat adipose tissue also displayed a single protein band of equivalent molecular mass to that seen in muscle. Reverse transcription–PCR analyses, using rat-specific GLUT5 primers, of muscle and jejunal RNA revealed a single PCR fragment of the expected size in jejunum and in four different skeletal muscle types. Sarcolemmal vesicles from rat muscle displayed fructose and glucose uptake. Vesicular uptake of glucose was inhibited by nearly 90% in the presence of cytochalasin B, whereas that of fructose was unaffected. To determine whether fructose could regulate GLUT5 expression in skeletal muscle, rats were maintained on a fructose-enriched diet for 4 days. This procedure increased jejunal and renal GLUT5 protein expression by approx. 4- and 2-fold respectively, but had no detectable effects on the abundance of GLUT5 protein in skeletal muscle or on fructose uptake in rat adipocytes. The present results show that GLUT5 is expressed in the sarcolemma of rat skeletal muscle and that it is likely to mediate fructose uptake in this tissue. Furthermore, unlike the situation in absorptive and re-absorptive epithelia, GLUT5 expression in insulin-sensitive tissues is not regulated by increased substrate supply.

scholarly journals Biochemical and immunocytochemical localization of the ‘GLUT5 glucose transporter’ in human skeletal muscle

1992 ◽  
Vol 286 (2) ◽  
pp. 339-343 ◽  
Author(s):  
H S Hundal ◽  
A Ahmed ◽  
A Gumà ◽  
Y Mitsumoto ◽  
A Marette ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Cellular Localization ◽  
Human Skeletal Muscle ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Protein Band ◽  
Subcellular Fractionation ◽  
Triceps Muscle

Using biochemical and immunocytochemical techniques, we have assessed both the protein expression and the cellular localization of the GLUT5 transporter in human skeletal muscle. Human muscle membranes, prepared by subcellular fractionation, were subjected to SDS/PAGE and Western-blot analyses using antiserum raised against a specific C-terminal amino acid sequence of the human GLUT5 transporter. GLUT5 was detected as a discrete 49 kDa protein band in a plasma-membrane-enriched fraction prepared from either soleus or gracilis muscle. In contrast, GLUT5 protein was not detectable to any significant extent in fractions which were devoid of muscle plasma membranes (mean GLUT5 abundance in intracellular fractions from three muscle preparations amounted to approximately 10% of that in the plasma-membrane-enriched fraction). Immunofluorescence studies using cryostat sections of human triceps muscle supported the biochemical observations and revealed that GLUT5 antibody selectivity labelled the plasma membrane of muscle cells. This immuno-labelling was significantly suppressed after tissue incubation with antiserum in the presence of a 14-amino-acid synthetic peptide corresponding to a specific C-terminus sequence of human GLUT5. These results indicate that human skeletal muscle expresses the GLUT5 transporter and that it is specifically localized to the plasma membrane, where it may participate in regulating hexose transfer across the sarcolemma.

Effect of food restriction on the insulin signalling pathway in rat skeletal muscle and adipose tissue

2005 ◽  
Vol 16 (10) ◽  
pp. 602-609 ◽  
Author(s):  
Ana Alonso ◽  
Yolanda Fernández ◽  
Rebeca Fernández ◽  
Patricia Ordóñez ◽  
María Moreno ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Food Restriction ◽  
Insulin Signalling ◽  
Signalling Pathway ◽  
Rat Skeletal Muscle ◽  
Insulin Signalling Pathway ◽  
Effect Of Food

scholarly journals Properties of phosphatidate phosphohydrolase in rat adipose tissue

1994 ◽  
Vol 301 (3) ◽  
pp. 793-799 ◽  
Author(s):  
S C Jamdar ◽  
W F Cao
Keyword(s):  
Adipose Tissue ◽  
Plasma Membranes ◽  
Lipid A ◽  
Mesangial Cell ◽  
Intraperitoneal Administration ◽  
Subcellular Fractions ◽  
Phosphatidate Phosphohydrolase ◽  
Membrane Bound ◽  
Rat Adipose Tissue ◽  
Triton X

Previously we have identified the presence of two different phosphatidate phosphohydrolase (PPH) activities in rat adipose tissue, based on Mg(2+)-dependency. In the present investigation, we have further characterized these isoenzymes, using both aqueous dispersed and membrane-bound phosphatidate as substrates and differentiated these activities on the basis of both Mg(2+)-dependency and N-ethylmaleimide (NEM)-sensitivity. These two distinguishing criteria gave identical estimates of PPH activities present in the different subcellular fractions. The microsomal and cytosol fractions contained mainly the Mg(2+)-dependent (NEM-sensitive) form, which was inhibited by various thiol reagents, was inactivated by heating at 55 degrees C for 20 min, and was decreased significantly within 2 h after intraperitoneal administration of cystamine (200 mg/kg). Such treatments had no effects on the Mg(2+)-independent (NEM-insensitive) form of PPH, which was mainly located in the plasma membranes, mitochondrial and microsomal fractions. Addition of Lipid A and guanosine 5′-[gamma-thio]triphosphate to the assay mixture had no effect on the PPH activities. The Mg(2+)-independent PPH form, which was thermostable in the intact subcellular fractions, became thermolabile when these fractions were disrupted in the presence of Triton X-100. The present studies demonstrate that: (1) the thermostability is not a satisfactory index to differentiate these isoenzymes; (2) the thiol/disulphide exchange may be involved in the regulation of Mg(2+)-dependent PPH activity; and (3) the PPH isoenzymes do not seem to be under G-protein control in adipose tissue, as reported previously in the mesangial cell line.

Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria

2001 ◽  
Vol 361 (1) ◽  
pp. 49-56 ◽  
Author(s):  
James A. HARPER ◽  
Jeff A. STUART ◽  
Mika B. JEKABSONS ◽  
Damien ROUSSEL ◽  
Kevin M. BRINDLE ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Uncoupling Protein ◽  
Mitochondrial Protein ◽  
Yeast Mitochondria ◽  
Rat Skeletal Muscle ◽  
Muscle Mitochondria ◽  
Skeletal Muscle Mitochondria ◽  
Uncoupling Protein 3 ◽  
Brown Adipose

Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14μg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200–700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24–84μg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.

L(+)-Lactate Binding to a Protein in Rat Skeletal Muscle Plasma Membranes

1995 ◽  
Vol 20 (1) ◽  
pp. 112-124 ◽  
Author(s):  
Karl J. A. McCullagh ◽  
Arend Bonen
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membranes ◽  
Mass Range ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Lactate Transport ◽  
Sds Page ◽  
Lactate And Pyruvate ◽  
Potential Inhibitors ◽  
Dose Dependent

Biochemical studies were conducted to determine the location of a putative lactate transport protein in rat skeletal muscle plasma membranes (PM). PM (50-100 μg protein) were incubated with [U-14C] L(+)-lactate, in the presence or absence of unlabeled monocarboxylates or potential inhibitors, after which proteins were separated by SDS-PAGE. Gel slices (2 mm) were cut and analyzed for14C. [U-14C] L(+)-lactate was bound to plasma membranes in the 30 to 40 kDa molecular mass range. Binding of [U-14C] L(+)-lactate was inhibited by N-ethylmaleimide, unlabeled L-lactate and pyruvate, and in a dose dependent manner by α-cyano-4-hydroxycinnamate (r = 0.995), but not by cytochalasin-B. The inhibition of [U-14C] L(+)-lactate binding was similar to the inhibition of lactate transport. Therefore the transport of L(+)-lactate across skeletal muscle plasma membranes involves a polypeptide of 30 to 40 kDa. Key words: transport, affinity labeling

Effect of phospholipase C on the binding of α-bungarotoxin to isolated plasma membranes of rat skeletal muscle

1986 ◽  
Vol 72 (2) ◽  
pp. 135-140 ◽  
Author(s):  
Nika Adham ◽  
Alan J. Harborne ◽  
Janis K. Shute ◽  
Margaret E. Smith
Keyword(s):  
Skeletal Muscle ◽  
Phospholipase C ◽  
Plasma Membranes ◽  
Rat Skeletal Muscle

The Effects of Hyperinsulinemia and Hyperglycemia on GLUT4 and Hexokinase II mRNA and Protein in Rat Skeletal Muscle and Adipose Tissue

Diabetes ◽  
1993 ◽  
Vol 42 (6) ◽  
pp. 922-929 ◽  
Author(s):  
C. Postic ◽  
A. Leturque ◽  
F. Rencurel ◽  
R. L. Printz ◽  
C. Forest ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Rat Skeletal Muscle ◽  
Hexokinase Ii

scholarly journals METABOLIC AND ULTRASTRUCTURAL CHANGES INDUCED IN ADIPOSE TISSUE BY INSULIN

1960 ◽  
Vol 8 (1) ◽  
pp. 83-101 ◽  
Author(s):  
Russell J. Barrnett ◽  
Eric G. Ball
Keyword(s):  
Adipose Tissue ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Morphological Changes ◽  
Ultrastructural Changes ◽  
Cytoplasmic Matrix ◽  
Smooth Membrane ◽  
Metabolic Action ◽  
Rat Adipose Tissue

The addition in vitro of insulin to rat adipose tissue (epididymal) produces marked metabolic changes which may be followed by measurement of the net gas exchange of the tissue. Using this method to monitor the metabolic action of insulin, concomitant observations with the electron microscope on the tissue have been made. These reveal that pronounced morphological changes are induced by insulin. The plasma membranes of the adipose cells become invaginated at many sites to form minute finger-like indentations. Numerous tiny, membrane-bounded vesicles are also present and arranged in relationship to the plasma membrane in such a way as to suggest that their formation occurred when a recessed fold was pinched off. Deeper in the cytoplasm, especially in specimens that had been incubated a longer time, numerous large, smooth, membrane-limited vesicles are seen. Finally, in these incubated specimens the cytoplasmic matrix has lost much of its granular nature, small lipid droplets are frequently found in the cytoplasm and suggestive changes have occurred in mitochondria. In control specimens, incubated without insulin for identical periods of time, indentations and vesicles in the plasma membrane are sparse at best and no vesicles or membrane-bound spaces appear deeper in the cytoplasm. The metabolic and morphologic changes induced by insulin seem to be interdependent events. Both changes appear to be initiated rapidly and concomitantly in the tissue. Both processes are initiated by insulin at concentrations considered to be physiological, 0.004 µg. (100 µunits) per ml. Insulin treated with alkali fails to initiate either process. It is concluded that insulin initiates pinocytosis in rat adipose tissue and the possible significance of this process in the mode of action of insulin is discussed.

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