Biomedical subjects

A Klip

Publications and source records attributed to A Klip.

At least 19 recordsLinked to original sources

PubMed1992

Biochemical and immunocytochemical localization of the 'GLUT5 glucose transporter' in human skeletal 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.

Blotting, Western↗

PubMed1992

Detection of the GLUT3 facilitative glucose transporter in rat L6 muscle cells: regulation by cellular differentiation, insulin and insulin-like growth factor-I.

The GLUT3 facilitative glucose transporter protein was found to be expressed in rat L6 muscle cells. It was detected at both the myoblast and myotube stage. GLUT3 protein content per mg of total membrane protein increased significantly during L6 cell differentiation. Subcellular fractionation demonstrated that the GLUT3 protein was predominantly localized in plasma membrane-enriched fractions of either myoblasts or myotubes. Short-term exposure of L6 myotubes to IGF-I or insulin caused a redistribution of GLUT3 protein from an intracellular membrane fraction to the plasma membrane, without affecting total membrane GLUT3 protein content. Long-term exposure of L6 myotubes to IGF-I produced an increase of GLUT3 protein in total membranes and all subcellular membrane fractions, especially the plasma membrane. We propose that the GLUT3 glucose transporter may play an important role in glucose metabolism in developing muscle.

Animals↗

PubMed1992

Glycaemia regulates the glucose transporter number in the plasma membrane of rat skeletal muscle.

The number of glucose transporters was measured in isolated membranes from diabetic-rat skeletal muscle to determine the role of circulating blood glucose levels in the control of glucose uptake into skeletal muscle. Three experimental groups of animals were investigated in the post-absorptive state: normoglycaemic/normoinsulinaemic, hyperglycaemic/normoinsulinaemic and hyperglycaemic/normoinsulinaemic made normoglycaemic/normoinsulinaemic by phlorizin treatment. Hyperglycaemia caused a reversible decrease in total transporter number, as measured by cytochalasin B binding, in both plasma membranes and internal membranes of skeletal muscle. Changes in GLUT4 glucose transporter protein mirrored changes in cytochalasin B binding in plasma membranes. However, there was no recovery of GLUT4 levels in intracellular membranes with correction of glycaemia. GLUT4 mRNA levels decreased with hyperglycaemia and recovered only partially with correction of glycaemia. Conversely, GLUT1 glucose transporters were only detectable in the plasma membranes; the levels of this protein varied directly with glycaemia, i.e. in the opposite direction to GLUT4 glucose transporters. This study demonstrates that hyperglycaemia, in the absence of hypoinsulinaemia, is capable of down-regulating the glucose transport system in skeletal muscle, the major site of peripheral resistance to insulin-stimulated glucose transport in diabetes. Furthermore, correction of hyperglycaemia causes a complete restoration of the transport system in the basal state (determined by the transporter number in the plasma membrane), but possibly only an incomplete recovery of the transport system's ability to respond to insulin (since there is no recovery of GLUT4 levels in the intracellular membrane insulin-responsive transporter pool). Finally, the effect of hyperglycaemia is specific for glucose transporter isoforms, with GLUT1 and GLUT4 proteins varying respectively in parallel and opposite directions to levels of glycaemia.

Animals↗

PubMed1992

Stimulation of glucose transport in L6 muscle cells by long-term intermittent stretch-relaxation.

Skeletal muscle stretch increases resting metabolism and causes hypertrophy. We have examined the effect of mechanical stretch in vitro on glucose transport activity and transporter contents in L6 muscle cells. Long-term (24-48 h) stretch-relaxation (25% maximal elongation at 30 cycles per min) of cell monolayers significantly increased glucose uptake by 1.6- to 2-fold in myotubes but not in myoblasts. The presence of serum was required for the stretch-relaxation induced increase in glucose uptake. Cycloheximide inhibited the mechanical stimulation of glucose uptake, and the latter response was not additive to the stimulatory effect of long-term exposure to insulin. GLUT1 and GLUT4 glucose transporter contents were not changed in total cell membranes from mechanically stimulated cells relative to controls. These results indicate that mechanical stimulation through passive stretch may be an important regulation of nutrient uptake in fetal myotubes independent of innervation.

Animals↗

PubMed1992

Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle.

Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.

Animals↗

PubMed1992

Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells.

L6 myoblasts spontaneously undergo differentiation and cell fusion into myotubes. These cells express both GLUT1 and GLUT4 glucose transporters, but their expression varies during myogenesis. We now report that the subcellular distribution and the protein processing by glycosylation of both glucose transporter isoforms also change during myogenesis. Crude plasma membrane and light microsome fractions were isolated from either myoblasts or myotubes and characterized by the presence of two functional proteins, the Na+/K(+)-ATPase and the dihydropyridine receptor (DHPR). Immunoreactive alpha 1 subunit of the Na+/K(+)-ATPase was faint in the crude plasma membrane fraction from myoblasts, but abundant in both membrane fractions from myotubes. In contrast, the alpha 1 subunit of the DHPR, which is expressed only in differentiated muscle, was detected in crude plasma membrane from myotubes but not from myoblasts. Therefore, crude plasma membrane fractions from myoblasts and myotubes contain cell surface markers, and the composition of these membranes appears to be developmentally regulated during myogenesis. GLUT1 protein was more abundant in the crude plasma membrane relative to the light microsome fraction prepared from either myoblasts or myotubes. The molecular size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the GLUT1 transporters in myotubes was smaller than that in myoblasts (Mr 47,000 and 53,000, respectively). GLUT4 protein (Mr 48,000) was barely detectable in the crude plasma membrane fraction and was almost absent in the light microsome fraction prepared from myoblasts. However, GLUT4 protein was abundant in myotubes and was predominantly located in the light microsome fraction. Treatment with endoglycosidase F reduced the molecular size of the transporters in all fractions to Mr 46,000 for GLUT1 and Mr 47,000 for GLUT4 proteins. In myotubes, acute insulin treatment increased the crude plasma membrane content of GLUT1 marginally and of GLUT4 markedly, with a concomitant decrease in the light microsomal fraction. These results indicate that: (a) the subcellular distribution of glucose transporters is regulated during myogenesis, GLUT4 being preferentially sorted to intracellular membranes; (b) both GLUT1 and GLUT4 transporters are processed by N-linked glycosylation to form the mature transporters in the course of myogenesis; and (c) insulin causes modest recruitment of GLUT1 transporters and marked recruitment of GLUT4 transporters, from light microsomes to plasma membranes in L6 myotubes.

Animals↗

PubMed1992

Acute and long-term effects of insulin-like growth factor I on glucose transporters in muscle cells. Translocation and biosynthesis.

Insulin-like growth factor I (IGF-I) rapidly (less than 10 min) stimulated glucose uptake into myotubes of the L6 muscle cell line, at concentrations that act specifically on IGF-I receptors. Uptake remained stimulated at a steady level for 1-2 h, after which a second stimulation occurred. The first phase was insensitive to inhibition of protein synthesis. Subcellular fractionation demonstrated that it was accompanied by translocation of glucose transporters (both GLUT1 and GLUT4) to the plasma membrane from intracellular membranes. Translocation sufficed to explain the first phase increase in glucose transport, and there was no change in the total cellular content of GLUT1 or GLUT4 glucose transporters. The second phase of stimulation was inhibitable by cycloheximide, and involved a net increase in either GLUT1 or GLUT4 transporter content, which was reflected in an increase in transporter number in plasma membranes. These results define a cellular mechanism of metabolic action of IGF-I in muscle cells; furthermore, they suggest that IGF-I has acute metabolic effects that mimic those of insulin, bypassing action on the insulin receptor.

Animals↗

PubMed1992

Acute and chronic signals controlling glucose transport in skeletal muscle.

Glucose transport into muscle cells occurs through facilitated diffusion mediated primarily by the GLUT1 and GLUT4 glucose transporters. These transporter proteins are controlled by acute and chronic exposure to insulin, glucose, muscle contraction, and hypoxia. We propose that acute responses occur through recruitment of pre-formed glucose transporters from an intracellular storage site to the plasma membrane. In contrast, chronic control is achieved by changes in transporter biosynthesis and protein stability. Using subcellular fractionation of rat skeletal muscle, recruitment of GLUT4 glucose transporters to the plasma membrane is demonstrated by acute exposure to insulin in vivo. The intracellular pool appears to arise from a unique organelle depleted of transverse tubule, plasma membrane, or sarcoplasmic reticulum markers. In diabetic rats, GLUT4 content in the plasma membranes and in the intracellular pool is reduced, and incomplete insulin-dependent GLUT4 recruitment is observed, possibly through a defective incorporation of transporters to the plasma membrane. The lower content of GLUT4 transporters in the muscle plasma membranes is reversed by restoration of normoglycemia with phlorizin treatment. In some muscle cells in culture, GLUT1 is the only transporter expressed yet they respond to insulin, suggesting that this transporter can also be regulated by acute mechanisms. In the L6 muscle cell line, GLUT1 transporter content diminishes during myogenesis and GLUT4 appears after cell fusion, reaching a molar ratio of about 1:1 in the plasma membrane. Prolonged exposure to high glucose diminishes the amount of GLUT1 protein in the plasma membrane by both endocytosis and reduced biosynthesis, and lowers GLUT4 protein content in the absence of changes in GLUT4 mRNA possibly through increased protein degradation. These studies suggest that the relative contribution of each transporter to transport activity, and the mechanisms by which glucose exerts control of the glucose transporters, will be key subjects of future investigations.

Animals↗

PubMed1992

Regulation of glucose transport and GLUT1 glucose transporter expression by O2 in muscle cells in culture.

The effect of varying cellular oxygenation on L6 muscle cell 2-deoxy-D-glucose transport, glucose utilization, lactate production, and expression of GLUT1 and GLUT4 transport proteins was investigated. Incubation of L6 myotubes in 3% O2 (mimicking a state of hypoxia) elevated glucose uptake by 6.5-fold over 48 h relative to cells incubated in 21% O2 (normoxia). Incubation of L6 cells in hyperoxic conditions (50% O2) significantly depressed glucose uptake by 0.4-fold. These effects were fully reversible. Incubation in 3% O2 also caused lactate accumulation and enhanced glucose consumption from the medium. Hypoxia elevated 2-deoxy-D-glucose transport even when the concentration of glucose in the medium was kept constant, suggesting that glucose deprivation alone was not responsible for increased cellular glucose uptake. Incubation in 3% O2 also elevated 3-O-methylglucose uptake but not amino acid uptake. Cycloheximide prevented the hypoxia-induced increase in glucose uptake, indicating that de novo synthesis of glucose transport-related proteins was the major means by which cells increased glucose uptake. The content of GLUT1 glucose transporter was significantly elevated in total membranes of cells incubated in 3% O2 and depressed in membranes from cells incubated in hyperoxic conditions, whereas GLUT4 expression was not affected. These results indicate that hypoxia induces an adaptive response of increasing cellular glucose uptake through elevated expression of GLUT1 in an attempt to maintain supply of glucose for utilization by nonoxidative pathways.

3-O-Methylglucose↗

PubMed1992

Abundance, localization, and insulin-induced translocation of glucose transporters in red and white muscle.

D-Glucose protectable cytochalasin B (CB) binding to subcellular membrane fractions was used to determine glucose transporter number in red (quadriceps-gastrocnemius-soleus) and white (quadriceps-gastrocnemius) rat muscle. CB binding was twofold higher in isolated plasma membranes of red than of white muscle. In contrast, the number of transporters in an isolated insulin-sensitive intracellular membrane organelle was similar in the two muscle groups. Immunoblotting and immunofluorescence microscopy with anti-GLUT4 and anti-GLUT1 antibodies indicated that both GLUT1 and GLUT4 transporter isoforms account for the higher abundance of CB binding sites in plasma membranes of red than of white muscle. Immunofluorescence localized GLUT4 to both the surface and the interior of the muscle cell and demonstrated that type I (slow twitch oxidative) and type IIa (fast twitch oxidative-glycolytic) fibers are enriched in GLUT4 protein compared with type IIb (fast twitch glycolytic) fibers. In contrast, GLUT1 reactivity was restricted to the surface of the muscle cell and was also highly enriched in the perineurial sheaths of peripheral nerves and the capsules of muscle spindles present in both red and white muscles. Insulin caused a twofold increase in CB binding in isolated plasma membranes of red or white muscles with a corresponding 40-50% decrease in CB binding in isolated intracellular membranes. These changes in CB binding were paralleled by similar changes in the membrane distribution of the GLUT4 glucose transporter isoform and in glucose transport activity measured after insulin perfusion of hindquarter muscles. In contrast, insulin did not change the distribution of either GLUT1 glucose transporters or Na(+)-K(+)-ATPase alpha 1-subunits. The molar ratio of GLUT4 to GLUT1 in red and white muscle plasma membranes was found to be 4:1 in the basal state and 7:1 in the insulin-stimulated state. These results indicate that red muscle contains a higher amount of GLUT1 and GLUT4 transporters at the plasma membrane than white muscle in the basal and insulin-stimulated states but that GLUT4 translocation does not differ between muscle types. In addition, GLUT4 expression correlates with the metabolic nature (oxidative vs. glycolytic) of skeletal muscle fibers, rather than with their contractile properties (slow twitch vs. fast twitch).

Animals↗

PubMed1992

Glucose transport in human skeletal muscle cells in culture. Stimulation by insulin and metformin.

Primary human muscle cell cultures were established and the regulation of glucose transport was investigated. Primary cultures were allowed to proceed to the stage of myotubes through fusion of myoblasts or were used for clonal selection based on fusion potential. In clonally selected cultures, hexose (2-deoxy-glucose) uptake into myotubes was linear within the time of study and inhibitable by cytochalasin B (IC50 = 400 nM). Cytochalasin B photolabeled a protein(s) of 45,000-50,000 D in a D-glucose-protectable manner, suggesting identity with the glucose transporters. In the myotube stage, the cells expressed both the GLUT1 and GLUT4 glucose transporter protein isoforms at an average molar ratio of 7:1. Preincubation in media of increasing glucose concentrations (range 5-25 mM) progressively decreased the rate of 2-deoxyglucose uptake. Insulin elevated 2-deoxyglucose uptake in a dose-dependent manner, with half maximal stimulation achieved at 3.5 nM. Insulin also stimulated the transport of the nonmetabolizable hexose 3-O-methylglucose, as well as the activity of glycogen synthase, responsible for nonoxidative glucose metabolism. The oral antihyperglycemic drug metformin stimulated the cytochalasin B-sensitive component of both 2-deoxyglucose and 3-O-methylglucose uptake. Maximal stimulation was observed at 8 h of exposure to 50 microM metformin, and this effect was not prevented by incubation with the protein-synthesis inhibitor cycloheximide. The relative effect of metformin was higher in cells incubated in 25 mM glucose than in 5 mM glucose, consistent with its selective action in hyperglycemic conditions in vivo. Metformin (50 microM for 24 h) was more effective than insulin (1 microM for 1 h) in stimulating hexose uptake and the hormone was effective on top of the stimulation caused by the biguanide, suggesting independent mechanisms of action.

Biological Transport↗

PubMed1992

Stimulation of hexose transport by metformin in L6 muscle cells in culture.

L6 muscle cells grown in culture to the stage of fused myotubes were incubated with the oral hypoglycemic drug metformin to test the effects of this drug on glucose transport. Metformin increased the initial rate of uptake of 2-deoxyglucose and 3-O-methylglucose. The effect was time dependent, with half-maximal stimulation at 5-6 h and maximal stimulation by about 16 h. The stimulation of hexose uptake was not prevented by cycloheximide. In 15 mM glucose medium, the basal rate of transport was lower than in 5 mM glucose medium. The stimulation of hexose uptake by metformin was comparable in absolute units in both media; hence, relative to basal uptake, stimulation was greater in the high glucose medium than in the low glucose medium. In 5 mM glucose medium, half-maximal stimulation was obtained with 800 microM metformin when tested for 24 h. The stimulation of hexose transport by metformin was only detectable in fused myotubes and not in perfusion myoblasts. No significant changes were observed in glucose transporter levels in total cell membranes from L6 myotubes (measured as D-glucose-protectable binding sites for cytochalasin-B) or in the total levels of the immunoreactive glucose transporter isoforms GLUT4 or GLUT1. It is concluded that metformin stimulates hexose transport into differentiated muscle cells by acting at a posttranslational level. We speculate that this might also constitute the basis for the ability of the drug to lower glycemia in diabetic individuals.

3-O-Methylglucose↗

PubMed1992

Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells.

The effects of the oral hypoglycemic drug metformin on glucose and amino acid transporter activity and subcellular localization of GLUT1 and GLUT4 glucose transporters were tested in cultured L6 myotubes. In muscle cells preexposed to maximal doses of metformin (2 mM, for 16 h), 2-deoxyglucose uptake was stimulated by over 2-fold from 5.9 +/- 0.3 to 13.3 +/- 0.5 pmol/min.mg protein. Uptake of the nonmetabolizable amino acid analog methylaminoisobutyrate was unaffected by treatment with the drug under identical conditions. Extracellular calcium was required to preserve the full response to the biguanide. Exposure of muscle cells to insulin in the presence of metformin resulted in further activation of 2-deoxyglucose transport. The latter effect was additive to the maximum effect of metformin, suggesting that the biguanide stimulates hexose uptake into muscle cells by an insulin-independent mechanism. Glucose transporter number quantified by performing studies of D-glucose-protectable binding of cytochalasin-B in plasma membranes (PM) and internal membranes (IM) prepared from L6 myotubes revealed that a 16-h treatment with 800 microM metformin significantly elevated glucose transporter number in the PM (by 47%), with an equivalent decrement in glucose transporter number (47%) in the IM. Western blot analysis using antisera reactive with the GLUT1 and GLUT4 isoforms of glucose transporters showed that metformin caused a reduction in GLUT1 content in the IM fraction and a concomitant increase in the PM. Unlike insulin, metformin treatment had no effect on the subcellular distribution of GLUT4. We propose that the molecular basis of metformin action in skeletal muscle involves the subcellular redistribution of GLUT1 proteins from an intracellular compartment to the plasma membrane. Such a recruitment process may form an integral part of the mechanism by which the drug stimulates glucose uptake (and utilization) in skeletal muscle and facilitates lowering of blood glucose in the management of type II diabetes.

Aminoisobutyric Acids↗

PubMed1992

Effects of hyperglycemia on glucose transporters of the muscle: use of the renal glucose reabsorption inhibitor phlorizin to control glycemia.

Individuals with non-insulin dependent or insulin-dependent diabetes mellitus present insulin resistance in peripheral tissues. This is reflected in a subnormal whole body insulin-dependent glucose utilization, largely dependent on skeletal muscle. Glucose transport across the cell membrane of this tissue is rate limiting in the utilization of the hexose. Therefore, it is possible that a defect exists in insulin-dependent glucose transport in skeletal muscle in diabetic states. This review focuses on two questions: is there a defect at the level of glucose transporters in skeletal muscle of diabetic animal models, and is this a consequence of abnormal insulin or glucose levels? The latter question arises from the fact that these parameters usually vary inversely to each other. Glucose transport into skeletal muscle occurs by two membrane proteins, the GLUT1 and GLUT4 gene products. By subcellular fractionation and Western blotting with isoform-specific antibodies, it was determined that isolated plasma membranes (PM) contain GLUT4 and GLUT1 proteins at a molar ratio of 3.5:1 and that an intracellular fraction (internal membranes; IM) different from sarcoplasmic reticulum contains only GLUT4 transporters. The IM furnishes transporters to the PM in response to insulin. Both transporter isoforms bind cytochalasin B in a D-glucose-protectable fashion. In streptozocin-induced diabetes of the rat with normal fasting insulin levels and marked hyperglycemia, the number of cytochalasin B-binding sites and of GLUT4 proteins diminishes in the PM whereas the GLUT1 proteins increase to a new ratio of about 1.5:1 GLUT4:GLUT1. In the IM, the levels of GLUT4 protein drop, as does the cellular GLUT4 mRNA. To investigate if these changes are associated with hyperglycemia, glucose levels were corrected back to normal values for a 24-h period with sc injections of phlorizin to block proximal tubule glucose reabsorption. This treatment restored cytochalasin B binding, restored GLUT4 and GLUT1 values back to normal levels in the PM, and partly restored cytochalasin B binding but not GLUT4 levels in the IM, consistent with only a partial recovery of GLUT4 mRNA. It is concluded that GLUT4 protein in the PM correlates inversely whereas GLUT1 protein correlates directly with glycemia. It is proposed that the decrease in GLUT4 levels is a protective mechanism, sparing skeletal muscle from gaining glucose and experiencing diabetic complications, albeit at the expense of becoming insulin resistant.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle.

Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-DHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca(2+)-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the alpha 1-subunit of the Na(+)-K(+)-ATPase, a surface membrane marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Insulin-stimulated glucose transport in circulating mononuclear cells from nondiabetic and IDDM subjects.

The objectives of this study were 1) to evaluate glucose transport and its regulation by insulin in easily accessible human cells, 2) to investigate the glucose transporter isoforms involved, and 3) to establish whether a defect in glucose transport is associated with peripheral insulin resistance, which is common in insulin-dependent diabetes mellitus (IDDM) patients. We measured 2-deoxyglucose (2-DG) uptake in circulating mononuclear cells from 23 nondiabetic adults, 16 adults with IDDM, and 10 children with IDDM. Circulating mononuclear cells were separated from whole blood by Ficoll gradients and incubated with +/- 1 nM insulin. 2-DG uptake was measured after incubation with [3H]2-DG and cell separation through corn oil-phthalate. Cytochalasin B-inhibitable 2-DG uptake (basal and insulin stimulated) was higher in control than in IDDM subjects (P less than 0.001). Insulin significantly increased 2-DG uptake or 3-O-methylglucose uptake in both groups. Basal and insulin-stimulated 2-DG uptake was similar for adults and children with IDDM and did not correlate with age or body mass index in any group or disease duration, insulin dosage, or HbA1c in IDDM. In separated monocytes and lymphocytes, 2-DG uptake increased in response to insulin only in the monocyte population. Insulin dose-response curves indicated maximal stimulation of hexose uptake at 1-2 nM insulin for both control and diabetic subjects and demonstrated a significant decrease in maximal insulin response in the latter. Immunoblotting with specific antibodies revealed that circulating mononuclear cells and separated monocytes express the GLUT1 but not the GLUT4 isoform of the glucose transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1992

Effect of diabetes on glucoregulation. From glucose transporters to glucose metabolism in vivo.

Peripheral resistance to insulin is a prominent feature of both insulin-dependent and non-insulin-dependent diabetes. Skeletal muscle is the primary site responsible for decreased insulin-induced glucose utilization in diabetic subjects. Glucose transport is the rate-limiting step for glucose utilization in muscle, and that cellular process is defective in human and animal diabetes. The transport of glucose across the muscle cell plasma membrane is mediated by glucose transporter proteins, and two isoforms (GLUT1 and GLUT4) are expressed in muscle. Insulin acutely increases glucose transport in muscle by selectively stimulating the recruitment of the GLUT4 transporter (but not GLUT1) from an intracellular pool to the plasma membrane. In skeletal muscles of streptozocin-induced diabetic rats, there is a decreased GLUT4 protein content in intracellular and plasma membranes. In these rats, insulin induced the mobilization of GLUT4 from the internal pool, but the incorporation of the transporter protein into the plasma membrane is diminished. Conversely, the content of the GLUT1 transporter increases in the plasma membrane of these diabetic rats. Normalization of glycemia with phlorizin fully restores the amount of GLUT1 and GLUT4 proteins to normal levels in the plasma membrane without altering insulin levels. This suggests that glycemia regulates the number of glucose transporters at the cell surface, GLUT1 varying directly and GLUT4 inversely, to glycemia. The regulatory role of glycemia also can be seen in diabetic dogs in vivo, where correction of hyperglycemia with phlorizin restores, at least in part, the defective metabolic clearance rate of glucose seen in these animals. In addition to acutely stimulating glucose transport in muscle, insulin controls exercise- and possibly stress-mediated glucose uptake in vivo, by preventing hyperglycemia and by restraining the effects of catecholamines on lipolysis and/or muscle glycogenolysis. Finally, we postulated a neural pathway that requires the permissive effect of insulin to increase glucose uptake by the muscle. Thus, insulin, glucose, and neural pathways regulate muscle glucose utilization in vivo and are, therefore, important determinants of glucoregulation in diabetes.

Animals↗

PubMed1991

Differential expression of the GLUT1 and GLUT4 glucose transporters during differentiation of L6 muscle cells.

Skeletal muscle is the main tissue responsible for glucose utilization in the fed state, and it expresses the ubiquitous GLUT1 glucose transporter and the muscle/fat specific GLUT4 glucose transporter. Here we investigated the expression of these transporters during muscle cell differentiation in vitro. Rat L6 muscle cells were grown to the stages of myoblasts, alignment and fused myotubes. Glucose (2-deoxy-D-glucose) transport was higher in myoblasts, decreasing with the progression of alignment and cell fusion. Conversely, insulin-stimulated glucose uptake was negligible in myoblasts, and increased with cell alignment and fusion. The cellular content of GLUT1 transporters decreased and that of GLUT4 transporters increased with cell fusion. Insulin rapidly stimulated glucose uptake in fused myotubes maintained in 2% serum but not in 10% serum. In 10% serum, basal glucose uptake increased as did the cellular content of GLUT1 transporters, while GLUT4 transporter content did not change. These results indicate that both transporters are regulated oppositely during muscle cell differentiation, and that high serum concentrations override the capacity of insulin to regulate transport by inducing overexpression of the GLUT1 transporter.

Animals↗