Red blood cell glucose metabolism in trisomy 10p: possible role of hexokinase in the erythrocyte

Red blood cell glucose metabolism was investigated in a male patient with de novo trisomy 10p. According to previous evidence, when assigning hexokinase gene locus in the 10p11 leads to pter region, a triplex dosage effect of hexokinase activity (HK) was found, while all the other erythrocyte glycolytic enzymes were in the normal values range. Red blood cell glucose utilization was 2.87 mumole/hr/ml RBC as compared to 1.43 in normal controls; the rate of glucose metabolized through the hexose monophosphate shunt (HMPS) was unchanged. Glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and dihydroxyacetone phosphate increased with respect to normal controls, while normal levels of 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and ATP were found. The HK activity increased in all the red blood cell fractions obtained by density gradient ultracentrifugation. However, a small difference in the distribution of cells through the gradient was evident. The experiments reported in this article show that in the red blood cells of patients with trisomy 10p, an increased level of HK leads to higher concentrations of glucose-6-phosphate and to a faster glucose utilization in the Embden-Meyerhof pathway, while the HMPS rate is unchanged.     

Identification and Ds-tagged isolation of a new gene at the Cf-4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5

Hexokinase type I (HK I; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the predominant glucose-phosphorylating enzyme in red blood cells, exists in human erythrocytes in multiple molecular forms that differ in isoelectric point and are separable by ion-exchange chromatography. The major forms, designated HK Ia, Ib and Ic, have similar kinetic properties but are characterized by different age-dependent decay and different intracellular distribution in reticulocytes. HK Ib, which elutes between HK I and HK II in the DEAE ion-exchange chromatography, appears to be unique to RBCs and different from any other hexokinase isozyme previously described. Indeed, Murakami and Piomelli recently reported the presence of a specific HK isozyme (named HKr) expressed in K562 cells and in human reticulocytes and, moreover, the resolution of the human HK I gene structure provided the direct evidence of an erythroid-specific exon 1. To further investigate the microheterogeneity of HK I in human RBCs we established a prokaryotic expression system for the HKr isozyme, using the pET plasmid, inducible with IPTG. The recombinant HKr, expressed in bacterial cells as a catalytically active enzyme, was purified to homogeneity by a combination of DEAE ionexchange chromatography followed by hydrophobic interaction chromatography and dye-ligand affinity chromatography. The kinetic and chromatographic properties of the homogeneous recombinant HKr suggest that this erythroid-specific HK isozyme in fact corresponds to the HK isoform previously described in human RBCs and referred to as HK Ib.     

Rifampin, a useful drug for nonmycobacterial infections

Hexokinase I, the pacemaker of glycolysis in brain tissue and red blood cells, is comprised of two similar domains fused into a single polypeptide chain. The C-terminal half of hexokinase I is catalytically active, whereas the N-terminal half is necessary for the relief of product inhibition by phosphate. A crystalline complex of recombinant human hexokinase I with glucose and phosphate (2.8 A resolution) reveals a single binding site for phosphate and glucose at the N-terminal half of the enzyme. Glucose and phosphate stabilize the N-terminal half in a closed conformation. Unexpectedly, glucose binds weakly to the C-terminal half of the enzyme and does not by itself stabilize a closed conformation. Evidently a stable, closed C-terminal half requires either ATP or glucose 6-phosphate along with glucose. The crystal structure here, in conjunction with other studies in crystallography and directed mutation, puts the phosphate regulatory site at the N-terminal half, the site of potent product inhibition at the C-terminal half, and a secondary site for the weak interaction of glucose 6-phosphate at the N-terminal half of the enzyme. The relevance of crystal structures of hexokinase I to the properties of monomeric hexokinase I and oligomers of hexokinase I bound to the surface of mitochondria is discussed.     

Functional analysis of a conditionally transformed pancreatic beta-cell line

Development of beta-cell lines for cell therapy of diabetes is hindered by functional deviations of the replicating cells from the normal beta-cell phenotype. In a recently developed cell line, denoted betaTC-tet, derived from transgenic mice expressing the SV40 T antigen (Tag) under control of the tetracycline (Tc) gene regulatory system, growth arrest can be induced by shutting off Tag expression in the presence of Tc. Here, we compared differentiated cell functions in dividing and growth-arrested betaTC-tet cells, both in culture and in vivo. Proliferating cells stably maintained normal glucose responsiveness for >60 passages in culture. Growth-arrested cells survived for months in culture and in vivo and maintained normal insulin production and secretion. After growth arrest, the cells gradually increased their insulin content three- to fourfold. This occurred without significant changes in insulin biosynthetic rates. At high passage numbers, proliferating betaTC-tet cells exhibited an abnormal increase in hexokinase expression. However, the upregulation of hexokinase was reversible upon growth arrest. Growth-arrested cells transplanted intraperitoneally into syngeneic recipients responded to hyperglycemia by a significant increase in insulin secretion. These findings demonstrate that transformed beta-cells maintain function during long periods of growth arrest, suggesting that conditional transformation of beta-cells may be a useful approach for developing cell therapy for diabetes.     

Hexokinase isoenzymes in the rat placenta

The phosphorylation of glucose to glucose-6-phosphate, the first enzymatic step for glucose utilization is catalysed by a family of four hexokinase isoenzymes (HKI-IV) which display a tissue-specific distribution. The expression of HK isoenzymes was investigated in the rat placenta. High levels of HKI and HKII mRNA were found in the junctional and the labyrinthine zones. HKIII mRNA was present at low levels in the junctional zone and glucokinase (HKIV) mRNA was not detected, indicating that HKI and HKII are the two major placental HK isoenzymes. HKII activity was increased in placenta of insulinopenic diabetic rats. This regulation is likely to support the increase in glucose utilization and storage characteristics of the enlarged placentae of diabetic rats.     

Effect of steroids on DNA synthesis in an in vitro replication system: initial quantitative structure-activity relationship studies and construction of a non-estrogen receptor pharmacophore

Insulin resistance, as is found in skeletal muscle of individuals with obesity and NIDDM, appears to involve a reduced capacity of the hormone to stimulate glucose uptake and/or phosphorylation. The glucose phosphorylation step, as catalyzed by hexokinase II, has been described as rate limiting for glucose disposal in muscle, but overexpression of this enzyme under control of a muscle-specific promoter in transgenic mice has had limited metabolic impact. In the current study, we investigated in a cultured muscle model whether expression of glucokinase, which in contrast to hexokinase II is not inhibited by glucose-6-phosphate (G-6-P), would have a pronounced metabolic impact. We used a recombinant adenovirus containing the cDNA-encoding rat liver glucokinase (AdCMV-GKL) to increase the glucose phosphorylating activity in cultured human muscle cells by fourfold. G-6-P levels increased in AdCMV-GKL-treated cells in a glucose concentration-dependent manner over the range of 1-30 mmol/l, whereas the much smaller increases in G-6-P in control cells were maximal at glucose concentrations <5 mmol/l. Further, cells expressing glucokinase accumulated 17 times more 2-deoxyglucose-6-phosphate than control cells. In AdCMV-GKL-treated cells, the time-dependent rise in G-6-P correlated with an increase in the activity ratio of glycogen synthase. AdCMV-GKL-treated cells also exhibited a 2.5- to 3-fold increase in glycogen content and a four- to fivefold increase in glycolytic flux, proportional to the increase in glucose phosphorylating capacity. All of these observations were made in the absence of insulin. Thus we concluded that expression of glucokinase in cultured human muscle cells results in proportional increases in insulin-independent glucose disposal, and that muscle glucose storage and utilization becomes controlled in a glucose concentration-dependent manner in AdCMV-GKL-treated cells. These results encourage testing whether delivery of glucokinase to muscle in vivo has an impact on glycemic control, which could be a method for circumventing the failure of insulin to stimulate glucose uptake and/or phosphorylation in muscle normally in insulin-resistant subjects.     

Troglitazone prevents the inhibitory effects of inflammatory cytokines on insulin-induced adipocyte differentiation in 3T3-L1 cells

Tumor necrosis factor (TNF) is implicated in wasting syndromes and insulin resistance in chronic infection and obese-linked diabetes. TNF (10 ng/ml) inhibited adipocyte differentiation of 3T3-L1 cells, and in these TNF treated cells little insulin-stimulated glucose uptake was observed. Treatment of 3T3-L1 cells with troglitazone (1-10 microM) partially prevented this inhibitory effect of TNF on adipogenesis, and enhanced expression of C/EBP alpha and GLUT4, even in the presence of TNF. Troglitazone also prevented the inhibitory effects of interleukin-1, interleukin-6, and leukemia inhibitory factor, but not of transforming growth factor beta on adipocyte differentiation of 3T3-L1 cells. These effects might contribute to the antidiabetic effect of troglitazone in obese diabetic animals.     

Overexpression of glycogen phosphorylase increases GLUT4 expression and glucose transport in cultured skeletal human muscle

Skeletal muscle glucose utilization, a major factor in the control of whole-body glucose tolerance, is modulated in accordance with the muscle metabolic demand. For instance, it is increased in chronic contraction or exercise training in association with elevated expression of GLUT4 and hexokinase II (HK-II). In this work, the contribution of increased metabolic flux to the regulation of the glucose transport capacity was analyzed in cultured human skeletal muscle engineered to overexpress glycogen phosphorylase (GP). Myocytes treated with an adenovirus-bearing muscle GP cDNA (AdCMV-MGP) expressed 10 times higher GP activity and exhibited a twofold increase in the Vmax for 2-deoxy-D-[3H]glucose (2-DG) uptake, with no effect on the apparent Km. The stimulatory effect of insulin on 2-DG uptake was also markedly enhanced in AdCMV-MGP-treated cells, which showed maximal insulin stimulation 2.8 times higher than control cells. No changes in HKII total activity or the intracellular compartmentalization were found. GLUT4, protein, and mRNA were raised in AdCMV-MGP-treated cells, suggesting pretranslational activation. GLUT4 was immunodetected intracellularly with a perinuclear predominance. Culture in glucose-free or high-glucose medium did not alter GLUT4 protein content in either control cells or AdCMV-MGP-treated cells. Control and GP-overexpressing cells showed similar autoinhibition of glucose transport, although they appeared to differ in the mechanism(s) involved in this effect. Whereas GLUT1 protein increased in control cells when they were switched from a high-glucose to a glucose-free medium, GLUT1 remained unaltered in GP-expressing cells upon glucose deprivation. Therefore, the increased intracellular metabolic (glycogenolytic-glycolytic) flux that occurs in muscle cells overexpressing GP causes an increase in GLUT4 expression and enhances basal and insulin-stimulated glucose transport, without significant changes in the autoinhibition of glucose transport. This mechanism of regulation may be operative in the postexercise situation in which GLUT4 expression is upregulated in coordination with increased glycolytic flux and energy demand.     

Glucose 6-phosphate produced by glucokinase, but not hexokinase I, promotes the activation of hepatic glycogen synthase

In a previous study (O'Doherty, R. M., Lehman, D. L., Seoane, J., Gómez-Foix, A. M., Guinovart, J. J., and Newgard, C.B. (1996) J. Biol. Chem. 271, 20524-20530), we demonstrated that adenovirus-mediated overexpression of glucokinase but not hexokinase I has a potent enhancing effect on glycogen synthesis in primary hepatocytes. In an effort to understand the underlying mechanism of this differential effect of the two hexokinase isoforms, we have investigated changes in key intracellular metabolites and the activation state of glycogen synthase in cells treated with recombinant adenoviruses expressing the liver isoform of glucokinase (AdCMV-GKL) or hexokinase I (AdCMV-HKI). Glucose 6-phosphate (Glu-6-P) levels are elevated from approximately 1.5 nmol/mg protein to 8-10 nmol/mg protein in both AdCMV-GKL- and AdCMV-HKI-treated hepatocytes as glucose is raised from 1 to 5 mM, levels four times higher than those in untreated cells. In AdCMV-GKL-treated cells, Glu-6-P continues to accumulate at glucose levels greater than 5 mM, reaching a maximum of 120 nmol/mg protein in cells incubated at 25 mM glucose, a value 10 and 50 times greater than the maximal levels achieved in AdCMV-HKI-treated and untreated cells, respectively. In parallel with the changes observed in Glu-6-P levels, increases in UDP-Glc in AdCMV-HKI- and AdCMV-GKL-treated cells were most pronounced at low (1-5 mM) and high (25 mM) glucose levels, respectively. Despite the significant increases in Glu-6-P and UDP-Glc achieved in AdCMV-HKI-treated cells, only AdCMV-GKL-treated cells exhibited increases in glycogen synthase activity ratio and translocation of the enzyme from a soluble to a particulate form relative to untreated control cells. We conclude that Glu-6-P produced by overexpressed glucokinase is glycogenic because it effectively promotes activation of glycogen synthase. Glu-6-P produced by overexpressed hexokinase, in contrast, appears to be unable to exert the same regulatory effects, probably due to the different subcellular distribution of the two glucose-phosphorylating enzymes.     

Sequencing of two alternatively spliced mRNAs corresponding to the extracellular domain of the rat receptor for advanced glycosylation end products (RAGE)

The phosphorylation of glucose to glucose-6-phosphate, catalyzed by hexokinase, is the first committed step in glucose uptake into skeletal muscle. Two isoforms of hexokinase, HKI and HKII, are expressed in human skeletal muscle, but only HKII expression is regulated by insulin. HKII messenger RNA, protein, and activity are increased after 4 h of insulin infusion; however, glucose uptake is stimulated much more rapidly, occurring within minutes. Studies in rat muscle suggest that changes in the subcellular distribution of HKII may be an important regulatory factor for glucose uptake. The present studies were undertaken to determine if insulin causes an acute redistribution of HKII activity in human skeletal muscle in vivo. Muscle biopsies (vastus lateralis muscle) were performed before and at the end of 30 min insulin infusion, performed using the euglycemic clamp technique. Muscle biopsies were subfractionated into soluble and particulate fractions to determine if insulin acutely changes the subcellular distribution of HKII. Insulin decreased HKII activity in the soluble fraction from 2.20 +/- 0.31 to 1.40 +/- 0.18 pmoles/(min[chempt]micrograms) and increased HKII activity in the particulate fraction from 3.02 +/- 0.46 to 3.45 +/- 0.46 pmoles/(min[chempt]micrograms) (P < 0.01 for both). These changes in HKII activity were correlated with changes in HKII protein, as determined by immunoblot analysis (r = 0.53, P = 0.05). Insulin had no effect on the subcellular distribution of HKII activity, which was primarily restricted to the soluble fraction. These studies are consistent with the conclusion that, in vivo in human skeletal muscle, insulin changes the subcellular distribution of HKII within 30 min.     

Lac repressor inducible gene expression in human breast cancer cells in vitro and in a xenograft tumor

As part of an ongoing search for susceptibility loci for NIDDM, we tested 19 genes whose products are implicated in insulin secretion or action for linkage with NIDDM. Loci included the G-protein-coupled inwardly rectifying potassium channels expressed in beta-cells (KCNJ3 and KCNJ7), glucagon (GCG), glucokinase regulatory protein (GCKR), glucagon-like peptide I receptor (GLP1R), LIM/homeodomain islet-1 (ISL1), caudal-type homeodomain 3 (CDX3), proprotein convertase 2 (PCSK2), cholecystokinin B receptor (CCKBR), hexokinase 1 (HK1), hexokinase 2 (HK2), mitochondrial FAD-glycerophosphate dehydrogenase (GPD2), liver and muscle forms of pyruvate kinase (PKL, PKM), fatty acid-binding protein 2 (FABP2), hepatic phosphofructokinase (PFKL), protein serine/threonine phosphatase 1 beta (PPP1CB), and low-density lipoprotein receptor (LDLR). Additionally, we tested the histidine-rich calcium locus (HRC) on chromosome 19q. All regions were tested for linkage with microsatellite markers in 751 individuals from 172 families with at least two patients with overt NIDDM (according to World Health Organization criteria) in the sibship, using nonparametric methods. These 172 families comprise 352 possible affected sib pairs with overt NIDDM or 621 possible affected sib pairs defined as having a fasting plasma glucose value of >6.1 mmol/l or a glucose value of >7.8 mmol/l 2 h after oral glucose load. No evidence for linkage was found with any of the 19 candidate genes and NIDDM in our population by nonparametric methods, suggesting that those genes are not major contributors to the pathogenesis of NIDDM. However, some evidence for suggestive linkage was found between a more severe form of NIDDM, defined as overt NIDDM diagnosed before 45 years of age, and the CCKBR locus (11p15.4; P = 0.004). Analyses of six additional markers spanning 27 cM on chromosome 11p confirmed the suggestive linkage in this region. Whether an NIDDM susceptibility gene lies on chromosome 11p in our population must be determined by further analyses.     

Decreased cerebral glucose utilization in rats during the ebb phase of thermal injury

OBJECTIVE: Thermal injury is associated with the development of encephalopathy. The mechanism(s) for the development of this condition have not been established. In the present study, the effects of thermal injury were determined on rat brain glucose utilization (Rg), using 2-[18F]fluoro-2-deoxy-D-glucose (18FDG). DESIGN: Four types of studies were performed. In one group of rats, the effect of thermal injury on total rat brain glucose utilization (Rg) was determined at 6 hours, 24 hours, and 3 weeks after injury. The brains of thermally injured rats were also assayed for hexokinase and glucose-6-phosphatase activities, since these enzyme activities are responsible for the phosphorylation and dephosphorylation of the 18FDG. We also measured total body oxygen consumption in the thermally injured rats. We wanted to compare the changes produced by thermal injury on rat brain glucose utilization (Rg) with the effects produced by compounds known to modify energy metabolism and/or rat brain glucose utilization (Rg). For that reason, in a second group of rats, an inflammatory state was produced by lipopolysaccharide injection, and rat brain glucose utilization (Rg) was determined. In the third group of rats, overall metabolism in rats was reduced by pentobarbital injection, followed by hypothermia, and rat brain glucose utilization (Rg) was determined. In the fourth group of rats, overall metabolism in rats was stimulated by 2,4-dinitrophenol injection, and rat brain glucose utilization (Rg) was determined. MATERIALS AND METHODS: Glucose utilization (Rg) by the brains of these treated rats was determined using 18FDG. Oxygen consumption in vivo, as well as glucose-6-phosphatase and hexokinase activity in vitro, were measured by standard procedures. MEASUREMENTS AND MAIN RESULTS: Glucose utilization (Rg) by rat brain was significantly reduced (p < 0.01) at 6 and 24 hours after injury, but returned to normal values 3 weeks after injury. These reductions were associated with decreases in rat brain hexokinase activity, increases in rat brain glucose-6-phosphatase activity, and decreased oxygen consumption by rats in vivo. Pentobarbital injection followed by hypothermia reduced rat brain glucose utilization (Rg) (p < 0.01), while 2,4-dinitrophenol treatment elevated rat brain glucose utilization (Rg) (p < 0.01). In contrast, LPS treatment had no effect on rat brain glucose utilization (Rg). CONCLUSIONS: These data indicate that thermal injury decreases glucose utilization (Rg) in rat brain during the hypometabolic phase. This effect can be explained, at least in part, by alterations in hexokinase and glucose-6-phosphatase activities, as well as reductions in oxygen consumption. Thus, the changes in brain glucose utilization (Rg) appear to be associated with the ebb phase of the thermal injury. The present results observed in burned rats may provide evidence to explain the encephalopathy observed in burned patients.     

Laron dwarfism and non-insulin-dependent diabetes mellitus in the Hnf-1alpha knockout mouse

Mice deficient in hepatocyte nuclear factor 1 alpha (HNF-1alpha) were produced by use of the Cre-loxP recombination system. HNF-1alpha-null mice are viable but sterile and exhibit a phenotype reminiscent of both Laron-type dwarfism and non-insulin-dependent diabetes mellitus (NIDDM). In contrast to an earlier HNF-1alpha-null mouse line that had been produced by use of standard gene disruption methodology (M. Pontoglio, J. Barra, M. Hadchouel, A. Doyen, C. Kress, J. P. Bach, C. Babinet, and M. Yaniv, Cell 84:575-585, 1996), these mice exhibited no increased mortality and only minimal renal dysfunction during the first 6 months of development. Both dwarfism and NIDDM are most likely due to the loss of expression of insulin-like growth factor I (IGF-I) and lower levels of insulin, resulting in stunted growth and elevated serum glucose levels, respectively. These results confirm the functional significance of the HNF-1alpha regulatory elements that had previously been shown to reside in the promoter regions of both the IGF-I and the insulin genes.