Rate-limiting Step For Glucose Uptake Research Articles

Photochemically Controlled Release of the Glucose Transporter 1 Inhibitor for Glucose Deprivation Responses and Cancer Suppression Research.

Cancer cells need a greater supply of glucose mainly due to their aerobic glycolysis, known as the Warburg effect. Glucose transport by glucose transporter 1 (GLUT1) is the rate-limiting step for glucose uptake, making it a potential cancer therapeutic target. However, GLUT1 is widely expressed and performs crucial functions in a variety of cells, and its indiscriminate inhibition will cause serious side effects. In this study, we designed and synthesized a photocaged GLUT1 inhibitor WZB117-PPG to suppress the growth of cancer cells in a spatiotemporally controllable manner. WZB117-PPG exhibited remarkable photolysis efficiency and substantial cytotoxicity toward cancer cells under visible light illumination with minimal side effects, ensuring its safety as a potential cancer therapy. Furthermore, our quantitative proteomics data delineated a comprehensive portrait of responses in cancer cells under glucose deprivation, underlining the mechanism of cell death via necrosis rather than apoptosis. We reason that our study provides a potentially reliable cancer treatment strategy and can be used as a spatiotemporally controllable trigger for studying nutrient deprivation-related stress responses.

Glial Growth Factor 2 Regulates Glucose Transport in Healthy Cardiac Myocytes and During Myocardial Infarction via an Akt-Dependent Pathway.

Neuregulin (NRG), a paracrine factor in myocytes, promotes cardiac development via the ErbB receptors. NRG-1β also improves cardiac function and cell survival after myocardial infarction (MI), although the mechanisms underlying these cardioprotective effects are not well elucidated. Increased glucose uptake has been shown to be cardio-protective during MI. We hypothesized that treatment with a recombinant version of NRG-1β, glial growth factor 2 (GGF2), will enhance glucose transport in the healthy myocardium and during MI. Cardiac myocytes were isolated from MI and healthy adult rats, and subsequently incubated with or without insulin or GGF2. Glucose uptake was measured using a fluorescent D-glucose analog. The translocation of glucose transporter (GLUT) 4 to the cell surface, the rate-limiting step in glucose uptake, was measured using a photolabeled biotinylation assay in isolated myocytes. Similar to insulin, acute in vitro GGF2 treatment increased glucose uptake in healthy cardiac myocytes (by 40 and 49%, respectively, P = 0.002). GGF2 treatment also increased GLUT4 translocation in healthy myocytes by 184% (P < 0.01), while ErbB 2/4 receptor blockade (by afatinib) abolished these effects. In addition, GGF2 treatment enhanced Akt phosphorylation (at both threonine and serine sites, by 75 and 139%, respectively, P = 0.029 and P = 0.01), which was blunted by ErbB 2/4 receptor blockade. GGF2 treatment increased the phosphorylation of AS160 (an Akt effector) by 72% (P < 0.05), as well as the phosphorylation of PDK-1 and PKC (by 118 and 92%, respectively, P < 0.05). During MI, cardiac GLUT4 translocation was downregulated by 44% (P = 0.004) and was partially rescued by both in vitro insulin and GGF2 treatment. Our data demonstrate that acute GGF2 treatment increased glucose transport in cardiac myocytes by activating the ErbB 2/4 receptors and subsequent key downstream effectors (i.e., PDK-1, Akt, AS160, and PKC). These findings highlight novel mechanisms of action of GGF2, which warrant further investigation in patients with heart failure.

To perform your best: work hard not long

The overload principle, that improved performance is the result of systematic and progressive training of sufficient frequency, intensity and duration, is the one overriding truth in exercise physiology. The uncertainty that has cost many a coach their job is: what is the optimal frequency, intensity and duration? With the results in this issue of The Journal of Physiology, Gibala et al. (2006) have started to determine how short the duration of exercise can be while still increasing cycling performance. As a result of the short duration, the workload of their subjects is extreme: about 4-times as high as regular endurance training. If you can complete the high-intensity exercise, the improvement in performance, mitochondrial mass, muscle buffering capacity, and glycogen supercompensation is the same following as little as 3 min of cycling as it is in subjects who performed 90–120 min of cycling at 65% of their maximum. While these findings probably won't change the training protocols of elite athletes, they may have important implications for those wishing to maintain fitness and they also provide clues as to the complex nature of the response to endurance exercise. It is becoming increasingly clear that there are at least three molecular signals that can lead to an increase in mitochondrial mass and glucose transport capacity in skeletal muscle following endurance exercise training (Hood et al. 2006). The pulsatile decrease of ATP and concomitant increase in AMP during repeated high-intensity exercise can activate the AMP-activated protein kinase; a long-term rise in intracellular calcium can activate calcium–calmodulin kinases; and the cellular stress associated with intense endurance exercise can activate p38 MAP kinase. The reason that many different types of training have a similar net result on muscle phenotype might be explained by the fact that all three signalling molecules have a similar downstream target. In skeletal muscle, all three of these molecules participate in the phosphorylation of the transcriptional repressor HDAC5 (Al-Khalili et al. 2004; Bassel-Duby & Olson, 2006). Phosphorylation of HDAC5 results in its dissociation from Mef2 and the subsequent activation of transcription. Two of the targets of this transcriptional activation are the GLUT4 and PGC-1α genes (Bassel-Duby & Olson, 2006). GLUT4 catalyses the rate-limiting step in glucose uptake and glycogen synthesis, the transport of glucose into the muscle cell, while PGC-1α coordinates the expression of the nuclear and mitochondrial genes needed for mitochondrial biogenesis. The result is an increase in the capacity to take up and either oxidize or store glucose, the main fuel used during maximal performance, as well as an increased capacity to generate ATP from fat and spare glycogen during lower intensity exercise. In support of this hypothesis, the work of Gibala et al. (2006) draws on studies performed by Shin Terada (Terada et al. 2001, 2005). Dr Terada showed that rats swimming 6 h a day carrying a weight equal to 2% of their body weight had similar increases in GLUT4 and PGC-1α content as rats that swam for 14 intervals lasting 20 s carrying a weight equal to 14% of their body weight. While not all of the cellular signals described above were measured, they did show that the activation of AMP kinase was almost twice as high in the high-intensity group as the low-intensity group (Terada et al. 2005). There are two possible explanations for the fact that the change in GLUT4 and PGC-1α were the same after either 8 days or 6 h. The first possibility is that there is a threshold for the adaptation to endurance training over which no greater increase in PGC-1α or GLUT4 is possible. The second is that the sum total of AMPK, p38 and CamK activation is the same under the two protocols: the greater AMPK activity in high-intensity training is made up for by longer periods of elevated calcium. For those looking to do the least possible work to be fit, the work of Gibala et al. (2006) presents hope. In as little as 3 min (40 min total time including warm up and cool down) we can improve our and performance. However, there are a couple of caveats. First, high-intensity training does not utilize as many calories as long-duration exercise, meaning that it would not be as good if the goal were weight loss. Second, it is unclear whether performance in other sports (i.e. running) are effected in the same way by high-intensity training, and lastly, as a former strength and conditioning coach I can say that while many people are willing to exercise at a low intensity for a long duration, it is VERY hard to get people to exercise at a high intensity for any period of time.

EFFECT OF CARBOHYDRATE SUPPLEMENTATION ON THE TIME-COURSE OF POST-EXERCISE GLYCOGEN RESYNTHESIS IN INSULIN-RESISTANT ZUCKER RATS

Glucose transport is the rate-limiting step for glucose uptake and storage in skeletal muscle. We determined the effect of an acute bout of swimming (8 × 30 min) followed by either carboydrate administration (i.p. 0.5 mg/g weight of glucose and free access to chow; CHO) or fasting (FAST) on the time-course (0, 30 min, 6 h post-exercise) of glycogen resynthesis in skeletal muscle (soleus) and liver from lean (L) and insulin resistant (IR) obese Zucker rats. Muscle [glycogen] at rest was similar between L and IR (29 ± 2 vs. 38 ± 4 μmol/g wet weight; mean ± SEM) and was reduced by ∼65% after exercise (p < 0.05). Liver [glycogen] was greater in IR than L (334 ± 31 vs. 247 ± 16 μmol/g; p < 0.05) and fell by 44% and 94% after exercise (P < 0.05). Muscle and liver [glycogen] remained unchanged after 30 min in L for both FAST and CHO. Liver and muscle [glycogen] was also unaltered by FAST in IR after 30 min, but muscle [glycogen] increased 68% from 14 ± 1 to 24 ± 3 μmol/g (P < 0.05) with CHO. After 6 h FAST, muscle [glycogen] of IR was restored to pre-exercise levels, but liver [glycogen] did not change with FAST or CHO. Muscle [glycogen] for L remained 56% below rest after 6 h, while liver [glycogen] had not increased above post-exercise values. After 6 h CHO, liver [glycogen] in L had been restored to resting levels while muscle [glycogen] was supercompensated by 68% (50 ± 4 μmol/g; p < 0.05). At this time, muscle [glycogen] in IR was 44% higher than rest (55 ± 3 μmol/g; p < 0.05). We conclude that with carbohydrate administration, 1) the rapid (≤ 30 min post-exercise) phase of muscle glycogen resynthesis was greater in insulin-resistant than lean animals, 2) most of the muscle glycogen resynthesis in lean animals occurred in the slow (> 30 min) phase, 3) there was an additive effect on muscle glycogen resynthesis in insulin-resistant animals, and 4) muscle, but not liver, glycogen was supercompensated 6 h following an intense bout of exercise in both lean and insulin-resistant obese Zucker rats.

Insulin does not change the intracellular distribution of hexokinase in rat heart.

Preliminary evidence has suggested that hexokinase in rat heart changes its kinetic properties in response to insulin through translocation to the outer mitochondrial membrane. We reexamined this hypothesis in light of tracer kinetic evidence to the contrary. Our methods were as follows. Working rat hearts were perfused with Krebs-Henseleit buffer containing glucose (5 mmol/l) and sodium oleate (0.4 mmol/l). Dynamic glucose uptake was measured with [2-3H]glucose and with 2-deoxy-2-[18F]fluoroglucose (2-[18F]DG). Hexokinase activity was determined in the cytosolic and mitochondrial fractions. Our results are as follows. Uptake of glucose and uptake of 2-[18F]DG were parallel. Insulin (1 mU/ml) increased glucose uptake threefold but had no effect on 2-[18F]DG uptake. The tracer-to-tracee ratio decreased significantly. The Michaelis-Menten constant of hexokinase for 2-deoxyglucose was up to 10 times higher than for glucose. There was no difference in maximal reaction velocity. Two-thirds of hexokinase was bound to mitochondria. Insulin neither caused translocation nor changed Michaelis-Menten constant or maximal reaction velocity. In conclusion, the insulin-induced changes in the tracer-to-tracee ratio are due to a shift of the rate-limiting step for glucose uptake from transport to phosphorylation by hexokinase. Insulin does not affect the intracellular distribution or the kinetics of hexokinase.

Glucose-transporter (GLUT4) protein content in oxidative and glycolytic skeletal muscles from calf and goat

It is well accepted that skeletal muscle is a major glucose-utilizing tissue and that insulin is able to stimulate in vivo glucose utilization in ruminants as in monogastrics. In order to determine precisely how glucose uptake is controlled in various ruminant muscles, particularly by insulin, this study was designed to investigate in vitro glucose transport and insulin-regulatable glucose-transporter protein (GLUT4) in muscle from calf and goat. Our data demonstrate that glucose transport is the rate-limiting step for glucose uptake in bovine fibre strips, as in rat muscle. Insulin increases the rate of in vitro glucose transport in bovine muscle, but to a lower extent than in rat muscle. A GLUT4-like protein was detected by immunoblot assay in all insulin-responsive tissues from calf and goat (heart, skeletal muscle, adipose tissue) but not in liver, brain, erythrocytes and intestine. Unlike the rat, bovine and goat GLUT4 content is higher in glycolytic and oxido-glycolytic muscles than in oxidative muscles. In conclusion, using both a functional test (insulin stimulation of glucose transport) and an immunological approach, this study demonstrates that ruminant muscles express GLUT4 protein. Our data also suggest that, in ruminants, glucose is the main energy-yielding substrate for glycolytic but not for oxidative muscles, and that insulin responsiveness may be lower in oxidative than in other skeletal muscles.

Glucose transport and NIDDM.

Three major metabolic abnormalities contribute to hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM) including defective glucose-induced insulin secretion, elevated rates of hepatic glucose output, and insulin's impaired ability to stimulate glucose uptake in peripheral target tissues (insulin resistance). These functions involve cellular glucose transport in beta-cells, liver, adipose tissue, and skeletal muscle; and, in some instances, abnormalities in glucose transporter isoforms (GLUT) specifically expressed in these tissues may constitute key biochemical lesions underlying defective glucose homeostasis. In animal models of NIDDM, suppression of GLUT2 in beta-cells is correlated with loss of high-Km glucose transport and glucose-sensitive insulin secretion. Although there are no data on humans with NIDDM, GLUT2 loss would constitute an attractive mechanism for defective glucose sensing in beta-cells if it can be shown that transport then becomes rate limiting for glucose metabolism. In the liver, however, hepatocyte glucose transport via GLUT2 probably plays only a permissive role in sustaining increased glucose efflux. Peripheral insulin resistance is associated with decreased glucose transport activity, the likely rate-limiting step for glucose uptake in fat and muscle. Accordingly, the insulin-responsive GLUT4 isoform expressed exclusively in insulin target tissues has been studied intensively in NIDDM. In these studies, pretranslational suppression of GLUT4 appears to be the key mechanism of insulin resistance in adipocytes. However, levels of GLUT4 protein and mRNA are normal in vastus lateralis and rectus abdominis, inferring that defects in GLUT4 functional activity or insulin-mediated translocation cause insulin resistance in muscle. Thus, the intensified study of glucose transport has provided important new insights into NIDDM pathogenesis over the past 5 yr and has presented investigators with additional intriguing hypotheses.

Glucose transport in primary cultured neurons.

In this study, we have examined glucose uptake and its regulation by insulin in primary cultured neurons. Glucose transport was assessed by measuring the initial rate of uptake of 3H-2-deoxyglucose, a glucose analog that is transported and phosphorylated but not further metabolized. The uptake of 2-deoxyglucose was saturable; measurements of the intracellular concentration of 2-deoxyglucose and 2-deoxyglucose-6-phosphate revealed that hexokinase activity rather than membrane transport is the rate-limiting step for glucose uptake. Insulin had no effect on 2-deoxyglucose uptake at low (0.2 mM) or high (20 mM) concentrations of substrate. The order of potency of other hexoses to competitively inhibit the accumulation of 2-deoxyglucose was D-glucose (0.2 mM) = D-mannose (0.2 mM) greater than 3-0-methylglucose (9 mM) greater than D-galactose (90 mM). Cytochalasin B was a potent inhibitor of 2-deoxyglucose uptake (IC50 = 500 nM) and phloretin was more potent than ploridzin in inhibiting uptake. The structure of glucose transporters was examined by photoaffinity labeling using 3H-cytochalasin B and by immunologic detection using antibodies raised against the human erythrocyte transporter. 3H-cytochalasin B labeled two proteins of 55 kDa and 43 kDa and the antibody recognized primarily a 43 kDa protein. The subcellular distribution of glucose transporters, estimated by measuring the number of specific cytochalasin B binding sites in subfractions of neuronal homogenates, showed 3.62 pmol/mg protein in the 11,000g pellet and 1.34 pmol/mg protein in the 200,000g pellet. 1) Neuronal glucose transport is not acutely regulated by insulin. 2) The kinetics of 2-deoxyglucose uptake into neurons are determined largely by hexokinase activity rather than membrane transport. 3) The apparent molecular weight of neuronal glucose transporters is similar to transporters in other tissues. 4) The number of glucose transporters per milligram of protein is relatively low in neurons compared to other tissues.

2 The Insulin-Like Effect of Muscle Contraction

Muscle contraction results in an increase in membrane permeability to glucose. The actual mechanism by which contractile activity increases membrane permeability is unknown. However, kinetic studies suggest that this increase is due to an increase in the number of glucose transporters associated with the plasma membrane. This is also suggested by the recent finding that cytochalsin B, which competitively inhibits the binding of glucose to the glucose transporter in the plasma membrane, prevents activation of the glucose transport process by muscle contraction. Unlike insulin-stimulated glucose transport, in which permeability is reversed rapidly upon removal of the insulin, the increase in membrane permeability following contractile activity can persist for many hours. It has also been reported that the stimulatory effects of insulin and contraction are additive, and that prostaglandin E2 augments the effect of insulin on glucose transport but has no effect on contraction-facilitated glucose transport. Collectively, these findings suggest that insulin and contractile activity increase membrane permeability to glucose by independent mechanisms. An increase in membrane permeability is only partially responsible for the increase in glucose uptake during exercise in vivo. With an increase in muscle activity, there is an increase in delivery of glucose and insulin to the muscle as a result of an increase in muscle blood flow. Glucose uptake may also be facilitated by an increase in the insulin sensitivity of the muscle. The increases in muscle blood flow and insulin sensitivity may be associated with activation of the kinin-prostaglandin system of the muscle. The increase in muscle insulin sensitivity may also involve an increase in insulin binding to its receptors on the sarcolemma. It should be noted that the increase in insulin binding associated with contractile activity requires the presence of epinephrine. Muscle glycogen may also affect the rate of glucose uptake during exercise. During prolonged, moderately intense exercise, glucose uptake increases as the muscle glycogen level declines. This increase in glucose uptake is inversely related to the glucose-6-phosphate concentration of the cell. During high-intensity exercise, the rate of glycogenolysis is rapid, resulting in the accumulation of glucose-6-phosphate and free glucose. Thus, it appears that the rate-limiting step in glucose uptake during exercise is shifted from transport to glucose phosphorylation, and that this shift is mediated by the intracellular glucose-6-phosphate concentration, which is influenced by the rate of muscle glycogen catabolism.

Insulin: Fundamental Mechanism of Action and the Heart

Insulin accelerates the entry of glucose and amino acids into muscle cells by acting upon the 'carrier-facilitated' transport mechanism. For glucose this process is passive and leads to equilibration of intracellular and extracellular concentrations. In heart muscle, glucose transport is a rate-limiting step for glucose uptake. During hypoxia and ischemia the heart turns to anaerobic glycolysis for energy production and therefore, maximal glucose transport becomes important. Insulin is necessary to insure proper protein synthesis, probably at the level of membrane-bound polyribosomes. However, during myocardial hypoxia, insulin alone cannot restore the associated depression in protein synthesis. Although insulin hyperpolarizes the cell, a change in the ratio of intracellular to extracellular activities of potassium is not its primary mode of action. An insulin-induced configurational change in the plasma membrane could simultaneously account for the effects of insulin on sodium and potassium permeability and the action on facilitated transport. Intracellular levels of cyclic adenylate may be reduced by insulin in adipose tissue because of inhibition of adenyl cyclase or stimulation of phosphodiesterase. However, at this time there is little evidence that insulin alters cyclic AMP levels in the heart. Insulin secretion is depressed in patients with heart disease in proportion to the reduction of cardiac index sustained. Since the ischemic heart is dependent upon glucose as the major fuel, insulin lack may deprive the heart of adequate substrate.

THE UPTAKE OF GLUCOSE INTO CELLS AND THE ROLE OF INSULIN IN GLUCOSE TRANSPORT.

This presentation has been restricted to the role of insulin in glucose transport in muscle cells and deals mainly with experiments using the perfused rat heart. The several possible means for glucose transfer into cells, diffusion, pores, pinocytosis, carriers, and dimerization, have been discussed; and arguments in favor of the carrier theory, namely, specificity, kinetics, inhibition, competition, and counterflow, have been elaborated. Glucose uptake has been considered to consist of three sequential steps: (1) passage of glucose from within the capillary to the cell surface, (2) transport across the cell membrane, and (3) metabolism of glucose within the cell. The first is considered to take place by diffusion and not to be significantly limiting under normal conditions, nor to be influenced by insulin. Transport across the cell membrane is thought to be mainly under the control of insulin and is the major rate-limiting step in glucose uptake when the extracellular glucose levels are in the normal range. Metabolism of glucose within the cell is the major rate-limiting step in glucose uptake when intracellular glucose concentration is so high that its phosphorylation is near saturation.

EFFECT OF PITUITARY LIPID MOBILIZER (ASTWOOD'S PEPTIDE I) UPON GLUCOSE UPTAKE OF THE ISOLATED PERFUSED RABBIT HEART.

EFFECT OF PITUITARY LIPID MOBILIZER (ASTWOOD'S PEPTIDE I) UPON GLUCOSE UPTAKE OF THE ISOLATED PERFUSED RABBIT HEART.