Total Hexokinase Activity Research Articles

Biochemical properties and subcellular localization of six members of the HXK family in maize and its metabolic contribution to embryo germination

BackgroundSeed germination is a crucial process in the plant life cycle when a dramatic variation of type and sugar content occurs just as the seed is hydrated. The production of hexose 6 phosphate is a key node in different pathways that are required for a successful germination. Hexokinase (HXK) is the only plant enzyme that phosphorylates glucose (Glc), so it is key to fueling several metabolic pathways depending on their substrate specificity, metabolite regulatory responses and subcellular localization. In maize, the HXK family is composed of nine genes, but only six of them (ZmHXK4–9) putatively encode catalytically active enzymes. Here, we cloned and functionally characterized putative catalytic enzymes to analyze their metabolic contribution during germination process.ResultsFrom the six HXKs analyzed here, only ZmHXK9 has minimal hexose phosphorylating activity even though enzymatic function of all isoforms (ZmHXK4–9) was confirmed using a yeast complementation approach. The kinetic parameters of recombinant proteins showed that ZmHXK4–7 have high catalytic efficiency for Glc, fructose (Fru) and mannose (Man), ZmHXK7 has a lower Km for ATP, and together with ZmHXK8 they have lower sensitivity to inhibition by ADP, G6P and N-acetylglucosamine than ZmHXK4–6 and ZmHXK9. Additionally, we demonstrated that ZmHXK4–6 and ZmHXK9 are located in the mitochondria and their location relies on the first 30 amino acids of the N-terminal domain. Otherwise, ZmHXK7–8 are constitutively located in the cytosol. HXK activity was detected in cytosolic and mitochondrial fractions and high Glc and Fru phosphorylating activities were found in imbibed embryos.ConclusionsConsidering the biochemical characteristics, location and the expression of ZmHXK4 at onset of germination, we suggest that it is the main contributor to mitochondrial activity at early germination times, at 24 h other ZmHXKs also contribute to the total activity. While in the cytosol, ZmHXK7 could be responsible for the activity at the onset of germination, although later, ZmHXK8 also contributes to the total HXK activity. Our observations suggest that the HXKs may be redundant proteins with specific roles depending on carbon and ATP availability, metabolic needs, or sensor requirements. Further investigation is necessary to understand their specific or redundant physiological roles.

Cardioprotective adaptation of rats to intermittent hypobaric hypoxia is accompanied by the increased association of hexokinase with mitochondria.

Chronic hypoxia increases the myocardial resistance to acute ischemia-reperfusion injury by affecting the mitochondrial redox balance. Hexokinase (HK) bears a high potential to suppress the excessive formation of reactive oxygen species because of its increased association with mitochondria, thereby inhibiting the membrane permeability transition pore opening and preventing cell death. The purpose of this study was to determine the effect of severe intermittent hypobaric hypoxia (7,000 m, 8 h/day, 5 wk) on the function and colocalization of HK isoforms with mitochondria in the left (LV) and right ventricles of rat myocardium. The real-time RT-PCR, Western blot, enzyme coupled assay, and quantitative immunofluorescence techniques were used. Our results showed significantly elevated expression of HK isoforms (HK1 and HK2) in the hypoxic LV. In addition, intermittent hypoxia increased the total HK activity and the association of HK isoforms with mitochondria in both ventricles. These findings suggest that HK may contribute to the cardioprotective phenotype induced by adaptation to severe intermittent hypobaric hypoxia.

The effect of standard chow and reduced hexokinase II on growth, cardiac and skeletal muscle hexokinase and low-flow cardiac ischaemia–reperfusion injury

In the present study, we examined whether standard chow (SDS versus Purina 5001; both low fat, high carbohydrate) and reductions in hexokinase (HK) II (wild-type versus HKII(+/-) mice) affect (1) growth parameters, (2) HK levels in cardiac and skeletal muscle and (3) low-flow cardiac ischaemia-reperfusion (IR) injury. Total HK activity and HKI and HKII expressions were determined, and low-flow IR injury was examined in isolated hearts subjected to 40 min 5% low-flow ischaemia and 120 min reperfusion. Standard chow, but not HKII reductions, significantly affected body weight, heart weight and cardiac hypertrophy. Both standard chow and reduced HKII diminished total cardiac and skeletal muscle HK activity. For the heart, the Purina chow-induced decrease in total HK activity was through decreases in HKI expression, whereas for skeletal muscle post-translational mechanisms are suggested. Both standard chow and reduced HKII demonstrated a non-significant trend for affecting cardiac IR damage. However, the low-flow ischaemia model was associated with mild sublethal injury only (∼1% cell death). In conclusion, standard chow affects body weight, heart weight and HK activity and HKI expression in the heart, without altering HKII expression. This implicates standard chow as an important factor in genomic, physiological research models and demonstrates that large differences in fat or carbohydrates in the diet are not necessary to affect growth. In a cardiac low-flow IR model, resulting in only mild injury, standard chow or reduced HKII does not significantly affect IR damage.

Correlation between glucose/fructose discrepancy and hexokinase kinetic properties in different Saccharomyces cerevisiae wine yeast strains

Grape juice contains about equal amounts of glucose and fructose, but wine strains of Saccharomyces cerevisiae ferment glucose slightly faster than fructose, leading to fructose concentrations that exceed glucose concentrations in the fermenting must. A high fructose/glucose ratio may contribute to sluggish and stuck fermentations, a major problem in the global wine industry. We evaluated wine yeast strains with different glucose and fructose consumption rates to show that a lower glucose preference correlates with a higher fructose/glucose phosphorylation ratio in cell extracts and a lower K (m) for both sugars. Hxk1 has a threefold higher V (max) with fructose than with glucose, whereas Hxk2 has only a slightly higher V (max) with glucose than with fructose. Overexpression of HXK1 in a laboratory strain of S. cerevisiae (W303-1A) accelerated fructose consumption more than glucose consumption, but overexpression in a wine yeast strain (VIN13) reduced fructose consumption less than glucose consumption. Results with laboratory strains expressing a single kinase showed that total hexokinase activity is inversely correlated with the glucose/fructose (G/F) discrepancy. The latter has been defined as the difference between the rate of glucose and fructose fermentation. We conclude that the G/F discrepancy in wine yeast strains correlates with the kinetic properties of hexokinase-mediated sugar phosphorylation. A higher fructose/glucose phosphorylation ratio and a lower K (m) might serve as markers in selection and breeding of wine yeast strains with a lower tendency for sluggish fructose fermentation.

Last Word: Point:Counterpoint authors respond to the commentaries on “Glucose phosphorylation is/is not a significant barrier to glucose uptake by the working muscle” that appear in this issue

LETTERS TO THE EDITORLast Word: Point:Counterpoint authors respond to the commentaries on “Glucose phosphorylation is/is not a significant barrier to glucose uptake by the working muscle” that appear in this issueThorkil Ploug, and Jørgen VintenThorkil Ploug, and Jørgen VintenPublished Online:01 Dec 2006https://doi.org/10.1152/japplphysiol.01083.2006This is the final version - click for previous versionMoreSectionsPDF (28 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat The present debate, precipitated by our restatement of E. Lundsgaard’s claim that phosphorylation of glucose is not rate limiting for glucose utilization in muscle, has proved very instructive. It seems presently unchallenged that glucose phosphorylation is hardly rate limiting during insulin stimulation at rest or during moderate exercise stimulation at basal insulin concentrations. In further support of this, muscle glucose uptake increases during moderate intensity contractions in vitro (2) or in vivo (3), when extracellular glucose levels are raised beyond normal physiological levels in the absence of any change in insulin concentrations. This clearly demonstrates that at moderate intensity exercise the hexokinase is not saturated at euglycemic conditions, which means that the free intracellular glucose concentration must be below two to three times the Km value of the hexokinase. In human skeletal muscle, where hexokinase I accounts for three-fourths of total hexokinase activity (4) this translates into a free intracellular glucose concentration below 0.3 mM during moderate intensity exercise. Considering that the extracellular glucose concentration is around 5 mM during moderate intensity exercise, this shows that glucose phosphorylation contributes less than 10% to the overall resistance to glucose uptake by the working muscle.When it comes to strenuous exercise, glucose transport is limiting for uptake for at least the first few minutes, where appreciable GLUT4 translocation has not yet occurred. When maximum GLUT4 translocation has taken place, glucose uptake is limited by glycogen breakdown. This is so, because during strenuous exercise more than 80% of energy production in human muscle is provided by this intracellular process (1), which mainly produces glucose 1-phosphate. This is in turn converted to glucose 6-phosphate by an isomerase, and thus bypasses phosphorylation by the hexokinase. In this situation, glucose 6-phosphate inhibition of hexokinase is increased. However, during such circumstances it does not make much sense to claim that muscle glucose metabolism is limited by phosphorylation by the hexokinase, when the need for carbohydrate fuel is so amply provided by an intracellular fuel store accumulated under control of glucose transport during more quiet periods (e.g., resting periods or alternating recruitment of motor units), where phosphorylation is unlikely to be rate limiting.REFERENCES1 Katz A, Sahlin K, and Broberg S. Regulation of glucose utilization in human skeletal muscle during moderate dynamic exercise. Am J Physiol Endocrinol Metab 260: E411–E415, 1991.Link | ISI | Google Scholar2 Nesher R, Karl IE, and Kipnis DM. Dissociation of effects of insulin and contraction on glucose transport in rat epitrochlearis muscle. Am J Physiol Cell Physiol 249: C226–C232, 1985.Link | ISI | Google Scholar3 Richter EA. Glucose utilization. In: Handbook of Physiology. Exercise: Regulation and Integration of Multiple Systems. Bethesda, MD: Am Physiol Soc, 1996, sect. 12, chapt. 20, p. 912–951.Google Scholar4 Ritov VB and Kelley DE. Hexokinase isozyme distribution in human skeletal muscle. Diabetes 50: 1253–1262, 2001.Crossref | PubMed | ISI | Google Scholar Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 101Issue 6December 2006Pages 1811-1811 Copyright & PermissionsCopyright © 2006 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.01083.2006History Published online 1 December 2006 Published in print 1 December 2006 Metrics

Hexose‐specificity of hexokinase and ADP‐dependence of pyruvate kinase play important roles in the control of monosaccharide utilization in freshly diluted boar spermatozoa

Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L-lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7 +/- 0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3 +/- 0.1 mIU/mg protein to 0.60 +/- 1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15-0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface.

PMA-enhanced neutrophil [ 18F]FDG uptake is independent of integrin occupancy but requires PI3K activity

Objective While respiratory burst enhances neutrophil glucose utilization, many neutrophil functions are critically influenced by extracellular matrix interaction and phosphoinositide-3-OH kinase (PI3K) signaling. We thus evaluated the role of RGD integrin occupancy and PI3K inhibition on respiratory burst and [ 18F]fluorodeoxyglucose ([ 18F]FDG) uptake of stimulated neutrophils. Methods Human neutrophils were stimulated by 100 ng/ml phorbol–myristate–acetate (PMA), and respiratory burst was measured by cumulative luminescence with lucigenin. [ 18F]FDG uptake and total hexokinase activity was measured 20 min after PMA stimulation in the presence or absence of soluble RGD peptides (200 μg/ml) and/or the PI3K inhibitor wortmannin (200 nM). Results Phorbol–myristate–acetate induced a 71.7±0.9 fold increase in neutrophil oxygen intermediate generation. [ 18F]FDG uptake was increased to 194.6±3.7% and hexokinase activity to 145.0±2.0% of basal levels (both P<.0005). RGD peptides attenuated respiratory burst activation to 35.6±0.2% ( P<.005) but did not inhibit stimulated [ 18F]FDG uptake or hexokinase activity. In contrast, without affecting respiratory burst activation, wortmannin inhibited PMA-stimulated [ 18F]FDG uptake to 66.9±1.6% and hexokinase activity to 81.0±4.2% (both P<.0005), demonstrating its dependence on PI3K activity. Neither RGD nor wortmannin reversed the other's inhibitory effect on stimulated [ 18F]FDG uptake and hexokinase activity or respiratory burst, which suggests the involvement of distinct signaling pathways. Conclusion Neutrophil [ 18F]FDG uptake is enhanced by PMA through a mechanism that requires PI3K activity but is independent of integrin receptor occupancy or respiratory burst activation.

Regulation of hexokinase activity by protein kinase C in peritoneal mesothelium cell

To investigate the effect of protein kinase C (PKC) on the activity of hexokinase (HK) in rat peritoneal mesothelium cell (PMC) and the functional consequences in glucose uptake. omentum obtained from albino male rats of the Sprague Dawley strain, PMC were isolated and subculture by enzymatic disaggregation. The antibody to cytokeratin was used for identified by immunofluorescence technique. After treated with PKC activator--phorbol esters (PMA and PDD) and classical PKC (cPKC) inhibitor--Gö6976, total HK activity in PMC were measured by a standard G6PDH-coupled assay, glucose disappearance in the culture medium were tested as glucose net utilization by colorimetric method. The PMC were polygonal when confluent and reacted positively for cytokeratin. PMA induced HK activity in a time and dose-dependent manner and increased net glucose utilization. At concentration of 0.01, 0.1 and 1 micromol/L PMA for 24 h, the increase in HK activity were 30.7%, 59.9% and 99.1%, respectively above the level in the control (P < 0.05). After treated with 1 micromol/L PMA for 6 h, the HK activity of PMC began to up-regulation, the peak reached at 24 h (21.3% vs 100.4%, P < 0.05), and accompanied by increase in net glucose utilization (2 mmol/L, P < 0.05). The expression of HK induced by 0.1 micromol/L PDD were similar to 1 micromol/L PMA, 4 alpha-PDD whose structure resemble to PDD didn't have effect on HK. Gö6976 blocked the activity of HK induced by PMA. After incubated with Gö6976 at concentration of 10, 20, 30, 40 and 50 micromol/L for 30 min, PMC were cultured in 1 micromol/L PMA for 24 h, the decrease in HK activity were 14.6%, 26.8%, 34.2%, 44.2% and 54.9% (P < 0.05). Elevated HK activity by PKC was accompanied with corresponding increase in net glucose utilization, which might take part in the accelerated glucose uptake after long-term peritoneal dialysis. The blocking of HK activity by cPKC inhibitor might be an effective method for increasing ultrafiltration in peritoneal dialysis.

Developmental expression of glucokinase in rat hypothalamus

Neurons in the hypothalamus sense changes in glucose concentration. Glucokinase (GK), a key enzyme for pancreatic (β)-cell glucose sensing, was found in both the embryonic and adult hypothalamus. GK activity accounted for approximately 20% of total hexokinase (HK) activity in both embryonic and adult hypothalamus with no activity measured in cortical samples, indicating that glucose sensing in the hypothalamus initiates early in development and precedes the maturation of glucose signaling in liver.

LPA is a novel lipid regulator of mesangial cell hexokinase activity and HKII isoform expression.

The prototypical extracellular phospholipid mediator, lysophosphatidic acid (LPA), exhibits growth factor-like properties and represents an important survival factor in serum. This potent mesangial cell mitogen is increased in conditions associated with glomerular injury. It is also a known activator of the classic mitogen-activated protein kinase (MAPK) pathway, which plays an important role in the regulation of mesangial cell hexokinase (HK) activity. To better understand the mechanisms coupling metabolism to injury, we examined the ability of LPA to regulate HK activity and expression in cultured murine mesangial cells. LPA increased total HK activity in a concentration- and time-dependent manner, with maximal increases of >50% observed within 12 h of exposure to LPA concentrations > or =25 microM (apparent ED(50) 2 microM). These effects were associated with increased extracellular signal-regulated kinase (ERK) activity and were prevented by the pharmacological inhibition of either MAPK/ERK kinase or protein kinase C (PKC). Increased HK activity was also associated with increased glucose (Glc) utilization and lactate accumulation, as well as selectively increased HKII isoform abundance. The ability of exogenous LPA to increase HK activity was both Ca2+ independent and pertussis toxin insensitive and was mimicked by LPA-generating phospholipase A2. We conclude that LPA constitutes a novel lipid regulator of mesangial cell HK activity and Glc metabolism. This regulation requires sequential activation of both Ca2+-independent PKC and the classic MAPK pathway and culminates in increased HKII abundance. These previously unrecognized metabolic consequences of LPA stimulation have both physiological and pathophysiological implications. They also suggest a novel mechanism whereby metabolism may be coupled to cellular injury via extracellular lipid mediators.

Regulation of Mesangial Cell Hexokinase Activity and Expression by Heparin-binding Epidermal Growth Factor-like Growth Factor: EPIDERMAL GROWTH FACTORS AND PHORBOL ESTERS INCREASE GLUCOSE METABOLISM VIA A COMMON MECHANISM INVOLVING CLASSIC MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY ACTIVATION AND INDUCTION OF HEXOKINASE II EXPRESSION

Heparin-binding epidermal growth factor -like growth factor (HB-EGF) expression and hexokinase (HK) activity are increased in various pathologic renal conditions. Although the mitogenic properties of HB-EGF have been well characterized, its effects on glucose (Glc) metabolism have not. We therefore examined the possibility that HB-EGF might regulate HK activity and expression in glomerular mesangial cells, which constitute the principal renal cell type affected by a variety of pathologic conditions. Protein kinase C (PKC)-dependent classic mitogen-activated protein kinase (MAPK) pathway activation has been associated with increased HK activity in this cell type, so we also examined dependence upon these signaling intermediates. HB-EGF (> or =10 nm) increased total HK activity over 50% within 12-24 h, an effect mimicked by other EGF receptor agonists, but not by IGF-1 or elevated Glc. EGF receptor and classic MAPK pathway antagonists prevented this increase, as did general inhibitors of gene transcription and protein synthesis. Both HB-EGF and phorbol esters activated the classic MAPK pathway, albeit via PKC-independent and PKC-dependent mechanisms, respectively. Both stimuli were associated with increased HK activity, selectively increased HKII isoform expression, and increased Glc metabolism via both the glycolytic-tricarboxylic acid cycle route and the pentose phosphate pathway. HB-EGF thus constitutes a novel regulator of mesangial cell HK activity and Glc metabolism. HKII is the principal regulated isoform in these cells, as it is in insulin-sensitive peripheral tissues, such as muscle. However, the uniform requirement for classic MAPK pathway activation distinguishes HKII regulation in mesangial cells from that observed in muscle. These findings suggest a novel mechanism whereby growth factors may couple metabolism to glomerular injury.

Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa

Incubation of dog spermatozoa with 10 mmol l(-1) glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of L-lactate and CO2. In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l(-1)). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l(-1) to 10 mmol l(-1), fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.

Gamma-linolenic acid alters the composition of mitochondrial membrane subfractions, decreases outer mitochondrial membrane binding of hexokinase and alters carnitine palmitoyltransferase I properties in the Walker 256 rat tumour

Gamma-linolenic acid (GLA) is known to be an inhibitor of Walker 256 tumour growth in vivo and causes changes in both mitochondrial structure and cellular metabolism. The aim of the present study was to investigate in greater detail the changes in energy metabolism and ultrastructure induced by GLA in this tumour model. A diet containing 5.5% GLA, which is sufficient to cause a 45% decrease in tumour growth, was found to almost double the triacylglycerol (TAG) content of the tumour and to increase the quantity of 20:3 n−6, 20:4 n−6, 22:4 n−6 and 22:5 n−6 in the TAG fraction as determined by gas chromatography–mass spectrometry (GCMS) analysis. Morphometric analysis of the tumour by electron microscopy confirmed this increase in TAG content, identifying a doubling of lipid droplet content in the GLA dietary group. The surface density of mitochondrial cristae was reduced, along with a reduction in the number of contact sites (CS) and matrix granules. These three parameters are likely indicators of a reduction in mitochondrial metabolic activity. Measurement of hexokinase activity identified that much of the total hexokinase activity was in the mitochondrially bound form (66.5%) in the control tumour and that GLA caused a decrease in the amount of enzyme in the bound form (39.3%). The fatty acyl chain composition of the tumour mitochondrial subfractions, outer membranes (OM), CSs and inner membranes (IM) was determined by GCMS. All subfractions showed considerable increases in 20:3 n−6 and decreases in 18:1 n−9, 18:2 n−6 and 22:6 n−3, when exposed to GLA diet. These changes were reflected in a large increase in the n−6/ n−3 ratio in the GLA OM vs. the control OM, 21.299 vs. 6.747, respectively. The maximal activity of OM carnitine palmitoyltransferase I (CPT I) was found to be decreased by 61.6% in the GLA diet group. This was accompanied by a decrease in malonyl CoA sensitivity and a decrease in affinity for 16:0 CoA substrate. Such changes in CPT I may be the cause of cytoplasmic acyl CoA accumulation seen in this tumour model. These effects, together with previously reported increases in lipid peroxidation, lead to the conclusion that GLA may cause inhibition of tumour cell growth through separate but interlinked pathways, all of which eventually lead to apoptosis and a decrease in tumour development. The influence of mitochondrial OM fatty acyl chain composition upon two important enzymes of energy metabolism, hexokinase and CPT I, both of which have been linked to apoptosis, is of considerable importance for future studies on fatty acid-induced cell death.

Variability in glucose transporter-1 levels and hexokinase activity in human melanoma

Melanoma exhibits heterogeneous growth patterns and widely varying sensitivities to multiple treatment modalities. This variability may reflect intrinsic genetic differences in factors giving rise to altered metabolism. Glucose is the primary energy source of tumours, including melanoma, and glucose transporter isoform 1 (Glut-1) and hexokinase are key rate-limiting factors in glucose metabolism. The levels of Glut-1 and total hexokinase activity were measured in 31 melanoma biopsies to determine the extent of tumour-to-tumour variability in these parameters. Relative Glut-1 levels were determined by Western immunoblot analysis using human anti-Glut-1 rabbit polyclonal antibody, and hexokinase activity was measured in the same samples by an enzymatic assay monitoring the reduction in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) (in nmol NADP+ reduced/min per mg protein). All melanomas were from patients who had received no therapy prior to surgery. Immediately after excision, tumour biopsies were disaggregated to single cells by collagenase and DNase and frozen in liquid nitrogen. Thirty human melanomas exhibited a 22-fold variation in levels of Glut-1 and 29 exhibited a nine-fold variation in total cellular hexokinase activity. Glut-1 levels and hexokinase activity were not correlated with one another. The broad range in Glut-1 levels and hexokinase activity observed between melanomas suggests that these glycolytic rate-limiting parameters that influence the rate of glucose metabolism may contribute to the variability in melanoma response to treatment modalities.

Hexokinase isozyme distribution in human skeletal muscle.

Two isoforms of hexokinase (type I and type II) are expressed in skeletal muscle; however, the intracellular distribution of these hexokinase isoforms in human skeletal muscle is unclear. The current study was undertaken to assess this issue because binding of hexokinase to subcellular structures is considered to be an important mechanism in the regulation of glucose phosphorylation. Vastus lateralis muscle was obtained from healthy lean individuals. Muscle homogenate was separated at 45,000g into particulate and cytosolic fractions. The activity and subcellular distribution of hexokinase isozymes in human skeletal muscle was determined using ion-exchange chromatography and a highly sensitive high-performance liquid chromatography-based hexokinase assay. This criterion method was used to validate a modified thermal inactivation method for distinguishing type I and type II isoforms. Mean hexokinase activity was 3.88 +/- 0.65 U/g wet wt or 0.64 +/- 0.11 U/mU creatine kinase (CrK) in the particulate fraction and 0.45 +/- 0.22 U/g wet wt or 0.07 +/- 0.03 U/mU CrK in the cytosolic fraction. Hexokinase I and II accounted for 70-75 and 25-30% of total hexokinase activity, respectively. Nearly all (95%) of hexokinase I activity (0.52 +/- 0.09 U/mU CrK) was found in the particulate fraction, consistent with the known high affinity of hexokinase I for mitochondria. Hexokinase II activity was also largely bound to the particulate fraction (72%), but 28% was found within the cytosolic fraction. Thus, within the particulate fraction, the relative contributions of hexokinase I and hexokinase II were 81 and 19%, whereas within the cytosolic fraction, the relative contributions for hexokinase I and hexokinase II were 37 and 63%.

The importance of glucose transport activity as the rate-limiting step of 2-deoxyglucose uptake in tumor cells in vitro

Glucose transporter (GLUT) expression and hexokinase activity are thought to be related to high [ 18F]-fluorodeoxyglucose (FDG) uptake in tumor cells, but their relative importance is still unknown. To determine which is the predominant factor in FDG uptake in tumor cells, cultured tumor cell lines and a normal cell line were studied in vitro with respect to 2-deoxyglucose (DG) uptake, hexokinase activity, and the initial uptake rate of 3- O-methylglucose (3- O-MG) transport, which is generally accepted as indicating the amount of GLUT expressed on the plasma membrane. In 16 types of tumor cells and one fibroblast cell line, DG uptake was assessed for 60 min, the initial uptake rate of 3- O-MG transport was measured for 1 min, and total hexokinase activity, including that in the mitochondrial fraction, was determined. Across all 16 tumor cell lines, there was a significant correlation between DG uptake and 3- O-MG transport ( p = 0.0012, F test), but not between DG uptake and hexokinase activity. Hexokinase activity of the tumor cells was comparable to that of the human fibroblast cells in the exponential growth phase. Most tumor cells showed higher DG uptake and 3- O-MG transport than the human fibroblast cells. The results suggest that DG uptake of cultured tumor cells is governed by GLUT expression, which may be a distinct characteristic of the neoplastic process.

Changes in pulmonary expression of hexokinase and glucose transporter mRNAs in rats adapted to hyperoxia.

Impairment of lung aconitase activity, citric acid cycle, and mitochondrial respiration by hyperoxia necessitates the elevation of glycolysis for energy production and of pentose shunt activity for reducing equivalents. The molecular mechanisms that allow increased glucose utilization are unknown. Adult male and female rats were adapted to sublethal hyperoxia, equivalent to 83% oxygen at sea level, or air for 7 days. Lung RNA and protein increased in hyperoxia (197 and 57%, respectively), whereas total DNA was unchanged. In hyperoxia, lung total hexokinase (HK) activity increased threefold, and mRNAs for HK-II and -III were specifically upregulated. HK-I mRNA was unchanged. mRNAs for HK-II and -III gradually increased during the first 72 h in hyperoxia. HK-II mRNA was significantly elevated at 72 h, preceding changes in lung cell populations. Although virtually absent in air, HK-II activity was highly expressed in hyperoxia. Among lung glucose transporters, specific expression of mRNAs for GLUT-4 (insulin dependent) and sodium-glucose cotransporter-1 was decreased, whereas that for GLUT-1 was minimally changed. Adaptation to hyperoxia involves coordinated changes in gene expression for the proteins regulating pulmonary glucose transport.

Transcriptional Regulation of Hexokinase II in Denervated Rat Skeletal Muscle

Hexokinase II protein is augmented in denervated skeletal muscle; therefore, we determined if hexokinase II gene transcription rates and mRNA levels are increased with denervation. The right hindlimb skeletal muscles of male rats were denervated while the left hindlimbs were sham operated. Seventy-two h following surgery, rats were sacrificed and the gastrocnemius and soleus muscles were harvested for nuclear and RNA isolation. Nuclear run-on and ribonuclease protection analyses indicated that denervation increased hexokinase II transcription rates and mRNA levels 42% and 88%, respectively (p<0.05). Total hexokinase activity rose 23% in denervated gastrocnemius muscle. In conclusion, the increase in hexokinase II gene transcription and mRNA may account for the increase in hexokinase II protein and the subsequent rise in total hexokinase activity in denervated rat skeletal muscle.

Abnormal Regulation of Hexokinase in Insulin-Resistant Skeletal Muscle

The present study has assessed the potential involvement of hexokinase in the control of insulin-mediated glucose metabolism in insulin-sensitive and -resistant skeletal muscle. Soleus muscle strips from lean (insulin-sensitive) and obese (insulin-resistant) Zucker rats were incubated with 10 or 10,000 μU insulin·ml−1and then homogenized using a protocol to maintain the location of hexokinasein situ.Hexokinase is inhibited by glucose 6-phosphate, a metabolic intermediate which may have a central role in the regulation of glycogen synthesis. Two separate measurements of hexokinase activity were made on each muscle homogenate: the total hexokinase activity (glucose 6-phosphate was metabolized immediately by glucose 6-phosphate dehydrogenase) and the fractional hexokinase activity (glucose 6-phosphate accumulated so as to regulate the enzyme asin vivo). The total hexokinase activity was equal in insulin-sensitive and -resistant muscle and was unaffected by the extracellular insulin concentration. The fractional hexokinase activity was significantly increased by insulin (10,000 μU·ml−1) in all muscles (lean, 82%; obese, 52%;P< 0.05) although the stimulated fractional hexokinase activity was lower in the muscle from obese Zucker rats compared to lean (P< 0.05). These results provide evidence that insulin decreases the inhibition of hexokinase by glucose 6-phosphate in insulin-sensitive but not in insulin-resistant muscle. This study has revealed short-term regulation of hexokinase by insulin which is defective in insulin-resistant skeletal muscle. Thus, the study has identified hexokinase as a potential regulatory site of insulin action that is abnormal in insulin resistance. The altered regulation of hexokinase may be a major contributing factor to the reduced insulin-mediated glucose fluxes in insulin-resistant skeletal muscle.

Regulation of hexokinase II and glycogen synthase mRNA, protein, and activity in human muscle.

Insulin regulates the activity of key enzymes of glucose metabolism in skeletal muscle by altering transcription or translation or by producing activity-altering modifications of preexisting enzyme molecules. Because of the small size of percutaneous muscle biopsies, these phenomena have been difficult to study in humans. This study was performed to determine how physiological hyperinsulinemia regulates the activities of hexokinase (HK), glycogen synthase (GS), and GLUT-4 in human skeletal muscle in vivo. We determined mRNA abundance, protein content, and activities for these proteins in muscle biopsies before and after a hyperinsulinemic clamp in normal subjects. HK I, HK II, GS, and GLUT-4 were expressed in muscle. HK II accounted for 80% of total HK activity and was increased by insulin from a basal value of 2.11 +/- 0.26 to 3.35 +/- 0.47 pmol.min-1.mg protein-1 (P < 0.05); HK I activity was unaffected. Insulin increased GS activity from 3.85 +/- 0.82 to 6.06 +/- 0.49 nmol.min-1.mg-1 (P < 0.01). HK II mRNA was increased 3.3 +/- 1.3-fold (P < 0.05) by insulin infusion. HK I, GS, and GLUT-4 mRNA and protein were unaffected. Because insulin infusion increased HK II but not GS mRNA, we conclude that HK II and GS may be regulated by insulin by different mechanisms in human skeletal muscle.