Biochemical and immunocytochemical localization of the 'GLUT5 glucose transporter' in human skeletal muscle.
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ABSTRACT:
SUBMITTER: Hundal HS
PROVIDER: S-EPMC1132902 | biostudies-other | 1992 Sep
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC1219879 | biostudies-other
Project description:The molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain, MHC) and mechanosensitive signaling (titin) have been well studied in animals but less is known about their roles in humans. The purpose of this study was to perform a comprehensive analysis of titin, collagen and MHC isoform distributions in a large number of human muscles, to search for common themes and trends in the muscular organization of the human body. In this study, 599 biopsies were obtained from six human cadaveric donors (mean age 83 years). Three assays were performed on each biopsy - titin molecular mass determination, hydroxyproline content (a surrogate for collagen content) and MHC isoform distribution. Titin molecular mass was increased in more distal muscles of the upper and lower limbs. This trend was also observed for collagen. Percentage MHC-1 data followed a pattern similar to collagen in muscles of the upper extremity but this trend was reversed in the lower extremity. Titin molecular mass was the best predictor of anatomical region and muscle functional group. On average, human muscles had more slow myosin than other mammals. Also, larger titins were generally associated with faster muscles. These trends suggest that distal muscles should have higher passive tension than proximal ones, and that titin size variability may potentially act to 'tune' the protein's mechanotransduction capability.
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Project description:Intestinal fructose uptake is mainly mediated by glucose transporter 5 (GLUT5/SLC2A5). Its closest relative, GLUT7, is also expressed in the intestine but does not transport fructose. For rat Glut5, a change of glutamine to glutamic acid at codon 166 (p.Q166E) has been reported to alter the substrate-binding specificity by shifting Glut5-mediated transport from fructose to glucose. Using chimeric proteins of GLUT5 and GLUT7, here we identified amino acid residues of GLUT5 that define its substrate specificity. The proteins were expressed in NIH-3T3 fibroblasts, and their activities were determined by fructose radiotracer flux. We divided the human GLUT5 sequence into 26 fragments and then replaced each fragment with the corresponding region in GLUT7. All fragments that yielded reduced fructose uptake were analyzed further by assessing the role of individual amino acid residues. Various positions in the first extracellular loop, in the fifth, seventh, eighth, ninth, and tenth transmembrane domains (TMDs), and in the regions between the ninth and tenth TMDs and tenth and 11th TMDs were identified as being important for proper fructose uptake. Although the p.Q167E change did not render the human protein into a glucose transporter, molecular dynamics simulations revealed a drastic change in the dynamics and a movement of the intracellular loop connecting the sixth and seventh TMDs, which covers the exit of the ligand. Finally, we generated a GLUT7-GLUT5 chimera consisting of the N-terminal part of GLUT7 and the C-terminal part of GLUT5. Although this chimera was inactive, we demonstrate fructose transport after introduction of four amino acids derived from GLUT5.
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Project description:Glucose transporter 1 (GLUT1) is believed to solely mediate basal (insulin-independent) glucose uptake in skeletal muscle; yet recent work has demonstrated that mechanical overload, a model of resistance exercise training, increases muscle GLUT1 levels. The primary objective of this study was to determine if GLUT1 is necessary for basal or overload-stimulated muscle glucose uptake. Muscle-specific GLUT1 knockout (mGLUT1KO) mice were generated and examined for changes in body weight, body composition, metabolism, systemic glucose regulation, muscle glucose transporters, and muscle [3H]-2-deoxyglucose uptake ± the GLUT1 inhibitor BAY-876. [3H]-hexose uptake ± BAY-876 was also examined in HEK293 cells-expressing GLUT1-6 or GLUT10. mGLUT1KO mice exhibited no impairments in body weight, lean mass, whole body metabolism, glucose tolerance, basal or overload-stimulated muscle glucose uptake. There was no compensation by the insulin-responsive GLUT4. In mGLUT1KO mouse muscles, overload stimulated higher expression of mechanosensitive GLUT6, but not GLUT3 or GLUT10. In control and mGLUT1KO mouse muscles, 0.05 µM BAY-876 impaired overload-stimulated, but not basal glucose uptake. In the GLUT-HEK293 cells, BAY-876 inhibited glucose uptake via GLUT1, GLUT3, GLUT4, GLUT6, and GLUT10. Collectively, these findings demonstrate that GLUT1 does not mediate basal muscle glucose uptake and suggest that a novel glucose transport mechanism mediates overload-stimulated glucose uptake.
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Project description:Insulin resistance (IR) in skeletal muscle is a prerequisite for type 2 diabetes and is often associated with obesity. IR also develops alongside muscle atrophy in older individuals in sarcopenic obesity. The molecular defects that underpin this syndrome are not well characterized, and there is no licensed treatment. Deletion of the transforming growth factor-β family member myostatin, or sequestration of the active peptide by overexpression of the myostatin propeptide/latency-associated peptide (ProMyo) results in both muscle hypertrophy and reduced obesity and IR. We aimed to establish whether local myostatin inhibition would have a paracrine/autocrine effect to enhance glucose disposal beyond that simply generated by increased muscle mass, and the mechanisms involved. We directly injected adeno-associated virus expressing ProMyo in right tibialis cranialis/extensor digitorum longus muscles of rats and saline in left muscles and compared the effects after 17 days. Both test muscles were increased in size (by 7 and 11%) and showed increased radiolabeled 2-deoxyglucose uptake (26 and 47%) and glycogen storage (28 and 41%) per unit mass during an intraperitoneal glucose tolerance test. This was likely mediated through increased membrane protein levels of GLUT1 (19% higher) and GLUT4 (63% higher). Interestingly, phosphorylation of phosphoinositol 3-kinase signaling intermediates and AMP-activated kinase was slightly decreased, possibly because of reduced expression of insulin-like growth factor-I in these muscles. Thus, myostatin inhibition has direct effects to enhance glucose disposal in muscle beyond that expected of hypertrophy alone, and this approach may offer potential for the therapy of IR syndromes.
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Project description:BackgroundThis study aim at assessing C. abbreviata aqueous extracts for its potential to exhibit anti-diabetic activity in skeletal muscle cells. In addition to the toxicological and glucose absorption studies, the action of C. abbreviata extracts on some major genes involved in the insulin signaling pathway was established.MethodsThe in vitro cytotoxic effects C. abbreviata was evaluated on muscle cells using the MTT assay and the in vitro glucose uptake assay conducted using a modified glucose oxidase method described by Van de Venter et al. (2008). The amount of GLUT-4 on cell surfaces was estimated quantitatively using the flow cytometry technique. Real time quantitative PCR (RT-qPCR) was used to determine the expression of GLUT-4, IRS-1, PI3 K, Akt1, Akt2, PPAR-γ.ResultsCytotoxicity tests revealed that all extracts tested at various concentrations were non-toxic (LC50 > 5000). Aqueous extracts of leaves, bark and seeds resulted in a dose-dependent increase in glucose absorption by cells, after 1 h, 3 h and 6 h incubation period. Extracts of all three plant parts had the best effect after 3 h incubation, with the leaf extract showing the best activity across time (Glucose uptake of 29%, 56% and 42% higher than untreated control cells after treatment with 1 mg/ml extract at 1 h, 3 h and 6 h, respectively). All extracts, with the exception 500 µg/ml seed extract, induced a two-fold increase in GLUT-4 translocation while marginally inducing GLUT-10 translocation in the muscle cells. The indirect immunofluorescence confirmed that GLUT-4 translocation indeed occurred. There was an increased expression of GLUT-4, IRS1 and PI3 K in cells treated with insulin and bark extract as determined by the RT-qPCR.ConclusionThe study reveals that glucose uptake involves GLUT-4 translocation through a mechanism that is likely to involve the upstream effectors of the PI3-K/Akt pathway.
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Project description:Rat and bovine parotid gland and pancreas contain deoxyribonuclease I (DNAase I) activities in different amounts. The DNAase I activity in tissue homogenates of bovine and rat parotid gland can be inhibited by addition of monomeric actin, as with the enzyme of bovine pancreas. The isolated DNAase I species from bovine and rat parotid gland differ in their molecular weights and also in their affinities for monomeric actin, being lowest for rat parotid DNAase I (5 X 10(6)M(-1). Antibodies raised against rat and bovine parotid and bovine pancreatic DNAase I can be used to study the subcellular localization of DNAase I in these tissues by indirect immunofluorescence. DNAase I was found to be confined solely to the secretory granules of the tissue from which it was isolated.
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Project description:Perilipins (PLINs) coat the surface of lipid droplets and are important for the regulation of lipid turnover. Knowledge about the physiological role of the individual PLINs in skeletal muscle is limited although lipid metabolism is very important for muscle contraction. To determine the effect of long-term exercise on PLINs expression, 26 middle-aged, sedentary men underwent 12 weeks combined endurance and strength training intervention. Muscle biopsies from m. vastus lateralis and subcutaneous adipose tissue were taken before and after the intervention and total gene expression was measured with deep mRNA sequencing. PLIN4 mRNA exhibited the highest expression of all five PLINs in both tissues, and the expression was significantly reduced after long-term exercise in skeletal muscle. Moreover, PLIN4 mRNA expression levels in muscle correlated with the expression of genes involved in de novo phospholipid biosynthesis, with muscular content of phosphatidylethanolamine and phosphatidylcholine, and with the content of subsarcolemmal lipid droplets. The PLIN4 protein was mainly located at the periphery of skeletal muscle fibers, with higher levels in slow-twitch as compared to fast-twitch skeletal muscle fibers. In summary, we report reduced expression of PLIN4 after long-term physical activity, and preferential slow-twitch skeletal muscle fibers and plasma membrane-associated PLIN4 location.
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Project description:BackgroundTissue-engineered muscles ("myobundles") offer a promising platform for developing a human in vitro model of healthy and diseased muscle for drug development and testing. Compared to traditional monolayer cultures, myobundles better model the three-dimensional structure of native skeletal muscle and are amenable to diverse functional measures to monitor the muscle health and drug response. Characterizing the metabolic function of human myobundles is of particular interest to enable their utilization in mechanistic studies of human metabolic diseases, identification of related drug targets, and systematic studies of drug safety and efficacy.MethodsTo this end, we studied glucose uptake and insulin responsiveness in human tissue-engineered skeletal muscle myobundles in the basal state and in response to drug treatments.ResultsIn the human skeletal muscle myobundle system, insulin stimulates a 50% increase in 2-deoxyglucose (2-DG) uptake with a compiled EC50 of 0.27 ± 0.03 nM. Treatment of myobundles with 400 µM metformin increased basal 2-DG uptake 1.7-fold and caused a significant drop in twitch and tetanus contractile force along with decreased fatigue resistance. Treatment with the histone deacetylase inhibitor 4-phenylbutyrate (4-PBA) increased the magnitude of insulin response from a 1.2-fold increase in glucose uptake in the untreated state to a 1.4-fold increase after 4-PBA treatment. 4-PBA treated myobundles also exhibited increased fatigue resistance and increased twitch half-relaxation time.ConclusionAlthough tissue-engineered human myobundles exhibit a modest increase in glucose uptake in response to insulin, they recapitulate key features of in vivo insulin sensitivity and exhibit relevant drug-mediated perturbations in contractile function and glucose metabolism.
| S-EPMC7710786 | biostudies-literature
Project description:The effect of electrically induced muscle contraction, insulin (10 m-units/ml) and electrically-induced muscle contraction in the presence of insulin on insulin-regulatable glucose-transporter (GLUT-4) protein distribution was studied in female Sprague-Dawley rats during hindlimb perfusion. Plasma-membrane cytochalasin B binding increased approximately 2-fold, whereas GLUT-4 protein concentration increased approximately 1.5-fold above control with contractions, insulin, or insulin + contraction. Microsomal-membrane cytochalasin B binding and GLUT-4 protein concentration decreased by approx. 30% with insulin or insulin + contraction, but did not significantly decrease with contraction alone. The rate of muscle glucose uptake was assessed by determining the rate of 2-deoxy[3H]glucose accumulation in the soleus, plantaris, and red and white portions of the gastrocnemius. Both contraction and insulin increased glucose uptake significantly and to the same degree in the muscles examined. Insulin + contraction increased glucose uptake above that of insulin or contraction alone, but this effect was only statistically significant in the soleus, plantaris and white gastrocnemius. The combined effects of insulin + contraction of glucose uptake were not fully additive in any of the muscles investigated. These results suggest that (1) insulin and muscle contraction are mobilizing two separate pools of GLUT-4 protein, and (2) the increase in skeletal-muscle glucose uptake due to insulin + contraction is not due to an increase in plasma-membrane GLUT-4 protein concentration above that observed for insulin or contraction alone.
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