Reduction In Hexokinase Research Articles

Dexmedetomidine hydrochloride plus sufentanil citrate inhibits glucose metabolism and epithelial‑mesenchymal transition in human esophageal squamous carcinoma KYSE30 cells by modulating the JAK/STAT3/HIF‑1α axis.

Dexmedetomidine hydrochloride (DEX-HCl) and sufentanil citrate (SFC) are commonly used anesthetic drugs for esophageal cancer (EC) surgery. The present study was performed to investigate the effect of DEX-HCl and SFC treatment on glucose metabolism and epithelial-mesenchymal transition in EC. Cell counting kit-8 (CCK8), clonogenic, wound healing and Transwell migration assays were performed to assess the effects of the DEX-HCl and SFC on KYSE30 cell proliferation, invasion and migration. Changes in lactate and glucose levels in KYSE30 cells were also detected. Western blot analysis was used to determine the protein expression levels of the JAK/STAT signaling pathway and glucose metabolism-related proteins. The results of CCK8, clonogenic and wound healing assays demonstrated that DEX-HCl and SFC inhibited KYSE30 cell proliferation, invasion and migration. Similarly, the combined DEX-HCl and SFC treatment significantly reduced lactate production, ATP production and glucose levels in KYSE30 cells. Western blotting indicated that DEX-HCl and SFC could reduce JAK/STAT and metastasis-related protein expression. Demonstrating a reduction in Hexokinase 2, matrix metallopeptidase 2 and 9, N-cadherin and lactate dehydrogenase A protein expression levels. The effects of DEX-HCl and SFC combined treatment were counteracted by the addition of JAK/STAT pathway activator RO8191, which suggested that DEX-HCl and SFC could serve a role in mediating the JAK/STAT signaling pathway in KYSE30 cells.

The role of fenugreek seed extract in alleviating pancreatic toxic effects and altering glucose homeostasis induced by acetamiprid via modulation of oxidative stress, apoptosis, and autophagy

Acetamiprid (ACMP) is a second-generation neonicotinoid that has been extensively used in the last few years. The present study examined the toxic effects of ACMP on the pancreas and glucose homeostasis through the evaluation of histological and biochemical changes and the possible ameliorative role of fenugreek seed extract (FG). Fifty adult albino rats were divided into 5 groups: negative control, positive control, FG-treated, ACMP-treated, and ACMP + FG-treated groups by oral gavage for 12 weeks. The ACMP-treated group highlighted significant elevations in plasma glucose, glycosylated haemoglobin levels (HbA1c), serum amylase, and serum lipase, along with a decrease in plasma insulin levels. In addition, significant increases in tumour necrosis factor- alpha (TNF-α) and malondialdehyde (MDA) were associated with reductions in the levels of interleukin 10 (IL-10), glutathione peroxidase, and catalase. Moreover, glucose-6-phosphatase and glycogen phosphorylase were significantly increased, with a significant reduction in hexokinase and liver glycogen stores. These biochemical changes were associated with histological changes in pancreatic sections stained by haematoxylin and eosin, Masson stain, and Orcein stain. ACMP-treated cells showed a marked reduction in β- cell immune reactivity to insulin, with pronounced p53, and beclin 1 immune expression. The use of FG with ACMP induced partial protection except for hexokinase and glycogen phosphorylase.

Elevated CLOCK and BMAL1 Contribute to the Impairment of Aerobic Glycolysis from Astrocytes in Alzheimer’s Disease

Altered glucose metabolism has been implicated in the pathogenesis of Alzheimer’s disease (AD). Aerobic glycolysis from astrocytes is a critical metabolic pathway for brain energy metabolism. Disturbances of circadian rhythm have been associated with AD. While the role of circadian locomotor output cycles kaput (CLOCK) and brain muscle ARNT-like1 (BMAL1), the major components in the regulation of circadian rhythm, has been identified in the brain, the mechanism by which CLOCK and BMAL1 regulates the dysfunction of astrocytes in AD remains unclear. Here, we show that the protein levels of CLOCK and BMAL1 are significantly elevated in impaired astrocytes of cerebral cortex from patients with AD. We demonstrate that the over-expression of CLOCK and BMAL1 significantly suppresses aerobic glycolysis and lactate production by the reduction in hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) protein levels in human astrocytes. Moreover, the elevation of CLOCK and BMAL1 induces functional impairment by the suppression of glial fibrillary acidic protein (GFAP)-positive filaments in human astrocytes. Furthermore, the elevation of CLOCK and BMAL1 promotes cytotoxicity by the activation of caspase-3-dependent apoptosis in human astrocytes. These results suggest that the elevation of CLOCK and BMAL1 contributes to the impairment of astrocytes by inhibition of aerobic glycolysis in AD.

Effects of cerebral glucose levels in infarct areas on stroke injury mediated by blood glucose changes

Admission hyperglycemia is considered to be related to poor outcomes of ischemic stroke. However, there is controversy regarding effects of attempts to lower blood glucose in stroke patients. This study aimed at determining the effects of blood glucose fluctuation on stroke injury by detection of cerebral glucose levels. A single intraperitoneal injection of glucose (0, 0.5, 1, 1.5 or 2 g kg−1) at 5 min before reperfusion caused blood glucose fluctuation (5–15 mmol L−1) lasting for 2 h after reperfusion. Blood glucose levels of 6–10 mmol L−1 decreased stroke injury after reperfusion for 24 h and 28 days compared with conditions of hyperglycemia (>10 mmol L−1) and hypoglycemia (<6 mmol L−1). High glucose concentration increased neuronal injury and death after oxygen–glucose deprivation. Under hyperglycemia and hypoglycemia, elevations in expression of MMP-2/-9 and decrease of tight junction proteins including occludin, claudin-5 and ZO-1 contributed to blood–brain barrier (BBB) dysfunction in infarct regions after ischemia–reperfusion injury, and reduction of hexokinase, pyruvate kinase and lactate dehydrogenase activities significantly inhibited the glucose metabolism in cortex and striatum after 24 h of reperfusion. BBB damage and reduced glucose metabolism caused accumulation of glucose in infarct areas. An obvious increase in cerebral glucose levels aggravated stroke injury.

Abstract 1130: Unveiling the metabolic response of BRAF mutant melanoma cells to BRAF inhibition

Abstract Background and Purpose: BRAF-MEK1/2 signaling inhibitors have shown remarkable clinical activity in BRAF-mutant melanomas. However, responses are not durable suggesting activation of alternative tumour survival mechanisms that allow resistance to therapy. This work investigates the metabolic consequences of treatment with the BRAF inhibitor vemurafenib in BRAF mutant human melanoma cells that could potentially enable resistance to treatment. Materials and Methods: BRAFV600D WM266.4 human melanoma cells were treated with 2μM vemurafenib (5xGI50) for 24h in standard culture media. Cell extracts were prepared and analyzed by 1H NMR spectroscopy to assess the levels of the aqueous metabolites. The areas of the NMR resonances were analysed using a multivariate method (Partial Least Squares-Discriminant Analysis, PLS-DA) as well as targeted analysis in which metabolite levels were normalized to cell number and an internal standard. Target inhibition was confirmed by western blotting for P-MEK1/2 and P-ERK1/2 protein levels while treatment-induced changes in metabolic enzyme expression were assessed by qRT-PCR and western-blotting. Results: As expected, exposure to vemurafenib led to a fall in P-MEK1/2 and P-ERK1/2 levels, consistent with BRAF inhibition. PLS-DA analyses revealed a good separation of control and treated 1H NMR spectra (correlation coefficient, R2 = 100%, predicted variation, Q2 = 96.5%) mainly based on differences in lactate, myo-inositol, glycine and acetate. This correlated well with the values obtained after targeted analysis of the 1H NMR spectra, which showed a significant reduction in intracellular lactate (to 62±9%) and acetate (to 62±9%), and an increase in glycine (to189±62%,) and myo-inositol (to 251±25%) in vemurafenib-treated cells relative to controls (p≤0.03). These changes are consistent with inhibition of the glycolytic pathway, confirmed by a reduction in hexokinase II and lactate dehydrogenase A expression, both detected by qRT-PCR and western-blotting. qRT-PCR also detected a fall in phosphoglycerate dehydrogenase expression indicating reduced flux to serine-glycine synthesis and suggesting that glycine accumulation may be due to decreased utilization. Reduced pyruvate dehydrogenase kinase 1 expression was also observed indicating enhanced TCA cycle activity. Conclusions: Our data show that BRAF inhibition with vemurafenib affects various metabolic pathways in BRAF mutant human melanoma cells inducing a shift in cellular energetic profile that may enable survival following treatment. Citation Format: Teresa Delgado-Goni, Slawomir Wantuch, Paul Workman, Richard Marais, Martin Leach, Mounia Beloueche-Babari. Unveiling the metabolic response of BRAF mutant melanoma cells to BRAF inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1130. doi:10.1158/1538-7445.AM2015-1130

Abstract 713: Targeting glycolytic pathway with 2-Deoxy-glucose enhances lapatinib sensitivity in HER2-amplified breast cancer

Abstract Background: Aerobic glycolysis, commonly used by cancer cells, converts glucose to lactate regardless of available oxygen. 2-Deoxyglucose (2DG), a glucose metabolism inhibitor, inhibits hexokinase enzyme activity, and can be used in functional tumor imaging, which can enhance efficacy of chemotherapy and radiotherapy in solid tumors. Lapatinib, a HER kinase inhibitor, is used for HER2+ breast cancer treatment, but is limited in efficacy due to emergence of therapeutic resistance. However, data investigating combination of 2DG + lapatinib in HER2+ BC are limited. In this study, we evaluated whether 2DG enhances lapatinib sensitivity in lapatinib-sensitive and resistant HER2+ cells. Materials and methods: SKBR3 and BT474 cells were treated with increasing dose of lapatinib to develop lapatinib-resistant cells: SKBR3-LapR and BT474-LapR. Potential synergism between 2DG and lapatinib was evaluated by viability assays, and calculated via CalcuSyn software or relative risk ratios (RRRs). Glycolysis was monitored in response to drug treatments via western blots and assays for lactate production, glucose uptake, and hexokinase activity. Results: Lapatinib treatment significantly inhibited glycolysis in SKBR3 and BT474 cell lines (P &amp;lt; 0.01). Combination lapatinib + 2DG was synergistic in inhibiting SKBR3 and BT474 proliferation, with average IC50 values of 0.53±0.16 and 0.55±0.01, respectively. SKBR3-LapR cells exhibited higher rates of lactate production, glucose uptake and hexokinase activity compared to lapatinib-sensitive cells (P &amp;lt; 0.05), and BT474-LapR cells exhibited higher hexokinase activity (P &amp;lt; 0.001) and glucose uptake (P &amp;lt; 0.05). RRRs were less than 1 with 2DG + lapatinib combination in SKBR3-LapR (0.88-0.98) and BT474-LapR (0.76-0.96) cells. Moreover, 2DG + lapatinib induced greater apoptosis, especially in lapatinib-resistant cells (P = 0.004 and 0.003 for SKBR3-LapR and BT474-LapR cells, respectively). Finally, 2DG + lapatinib inhibited several steps of the glycolytic pathway, namely: reduction of hexokinase II expression levels, inhibition of lactate production, decreased glucose uptake and impaired hexokinase activity, in both lapatinib-sensitive and resistant cells. Conclusion: 2DG can enhance lapatinib sensitivity by inhibiting aerobic glycolysis in both naïve and lapatinib-resistant HER2+ breast cancer cells. 2DG impinges on multiple steps of glycolysis, and combination with lapatinib may serve to prevent and/ or ameliorate therapeutic resistance in patients treated with lapatinib. Citation Format: Xiaosong Chen, Junjun Liu, Toby Ward, Xiaofei Liu, Yan Mao, Jessica Bockhorn, Kunwei Shen, Mark Pegram. Targeting glycolytic pathway with 2-Deoxy-glucose enhances lapatinib sensitivity in HER2-amplified breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 713. doi:10.1158/1538-7445.AM2015-713

Abstract P214: Reduction of Hexokinase II Exaggerated Cardiac Hypertrophy via Increased ROS Production and Mitochondrial Permeability Transition

Rationale/Objective: Cardiac hypertrophy is associated with a metabolic switch in substrate utilization from fatty acids to glucose. Levels of Hexokinase II (HKII), a key regulator of glucose metabolism, increase in response to hypertrophic stimulation and we hypothesized that a reduction in HKII could attenuate the hypertrophic response. Methods/Results : Contrary to our hypothesis, heterozygous knockout of HKII (HKII+/-) resulted in exaggerated cardiac hypertrophy after the induction of pressure overload by trans-aortic constriction (TAC). HKII+/- mice demonstrated increased heart size, interstitial fibrosis, and pulmonary edema compared to wild-type (WT) 4 weeks after TAC. This was associated with reduced cardiac function and increased mortality in the HKII+/- mice by the 8-week time point, indicating an accelerated transition to heart failure. In primary cultures of neonatal rat cardiomyocytes (NRCM), siRNA knockdown of HKII exacerbated the hypertrophic response to Angiotensin II (AngII) compared to control siRNA. Mechanistically, reduction of HKII resulted in increased ROS levels after hypertrophic stimulation relative to controls. Treatment of NRCM with the antioxidant N -acetylcysteine (NAC) attenuated the hypertrophic response to AngII, and abrogated the increased hypertrophy observed with HKII knockdown. In addition to its enzymatic activity, HKII can bind the outer membrane of the mitochondria, and this interaction increased with hypertrophic agonists. Dissociation of HKII from the mitochondria using a synthetic cell permeable peptide resulted in de novo hypertrophy compared with scrambled peptide control, and this was also attenuated with NAC treatment. Further investigation into the source of ROS revealed that HKII knockdown (+AngII) or mitochondrial dissociation resulted in increased mitochondrial permeability pore (mPTP) opening in the absence of cell death. Conclusions: Overall, the data suggest that HKII and its binding to the mitochondria serve as a negative regulator of hypertrophy by decreasing ROS production, possibly through regulation of the mPTP.

Reduction in Hexokinase II Levels Results in Decreased Cardiac Function and Altered Remodeling After Ischemia/Reperfusion Injury

Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane. We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R. We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII(+/)⁻) mice to I/R by coronary ligation. At baseline, HKII(+/)⁻ mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor-dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O₂ consumption, and contractility. These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes.

Hexokinase II partial knockout impairs exercise-stimulated glucose uptake in oxidative muscles of mice.

Muscle glucose uptake (MGU) is distributively controlled by three serial steps: delivery of glucose to the muscle membrane, transport across the muscle membrane, and intracellular phosphorylation to glucose 6-phosphate by hexokinase (HK). During states of high glucose fluxes such as moderate exercise, the HK activity is of increased importance, since augmented muscle perfusion increases glucose delivery, and increased GLUT4 at the cell membrane increases glucose transport. Because HK II overexpression augments exercise-stimulated MGU, it was hypothesized that a reduction in HK II activity would impair exercise-stimulated MGU and that the magnitude of this impairment would be greatest in tissues with the largest glucose requirement. To this end, mice with a HK II partial knockout (HK+/-) were compared with their wild-type control (WT) littermates during either sedentary or moderate exercise periods. Rg, an index of glucose metabolism, was measured using 2-deoxy-[3H]glucose. No differences in glucose metabolism were detected between sedentary groups. The increase in Rg due to exercise was impaired in the highly oxidative heart and soleus muscles of HK+/- compared with WT mice (7 +/- 10 vs. 29 +/- 9 and 8 +/- 3 vs. 25 +/- 7 micromol. 100 g-1. min-1, respectively). However, the increase in Rg due to exercise was not altered in gastrocnemius and superficial vastus lateralis muscles in HK+/- and WT mice (8 +/- 2 vs. 12 +/- 3 and 5 +/- 2 vs. 8 +/- 2 micromol. 100 g-1. min-1, respectively). In conclusion, MGU is impaired by reductions in HK activity during exercise, a physiological condition characterized by high glucose flux. This impairment is critically dependent on the tissue's glucose metabolic rate and correlates with tissue oxidative capacity.

A Nicotiana tabacum cell culture selected for accelerated growth on mannose has increased expression of phosphomannose isomerase

Phosphomannose isomerase (PMI), a key enzyme in mannose (Man) metabolism, is expressed at very low levels in many plant species. For example, measured PMI activity in Nicotiana tabacum (NT1) suspension cells is relatively low, resulting in slow metabolism of Man. Not surprisingly then, NT1 cultures were observed to grow six times faster on glucose (Glc) than on Man as sole carbon source. We report here the selection of a mutant NT1 cell line that grows four times faster on Man than the parental culture from which it was derived. This cell line had fivefold greater PMI activity than the parental culture, which likely contributes to the increased growth rate on Man. The selected line continued to express elevated PMI activity after transfer to Glc, suggesting a stable genetic change. However, the selected line grew more slowly than the wild type on Glc. This was likely due to a more than 50% reduction in hexokinase (HK) activity, an enzyme that is required for the phosphorylation and subsequent metabolism of Glc. Unlike HK, fructokinase activity was essentially unchanged in the mutant cell lines. However, activities of the carbohydrate metabolic enzymes phosphoglucose isomerase and 6-phosphogluconate dehydrogenase were also higher in mutant cells.

Effect of a moderate intensity exercise training protocol on the metabolism of macrophages and lymphocytes of tumour-bearing rats.

It is commonly accepted that moderate intensity exercise is beneficial to the immune system. We tested the influence of a moderate intensity training protocol (8 weeks) upon immune system function in Wistar tumour-bearing (TB) rats. The metabolism of glucose and glutamine in lymphocytes and macrophages was assessed, together with some functional parameters (hydrogen peroxide production and lymphocyte proliferative response). These substrates were chosen since they represent the most important energetic and synthetic metabolites for these cellular types. The training protocol caused a decrease of 17.4 per cent in the production of H(2)O(2) by macrophages, as well as a decrease in glucose consumption (25 per cent) and lactate production (47.1 per cent), and an increase in the production of labelled CO(2) from the oxidation of [U-(14)C]-glucose, in TB rats. The training protocol was also able to induce changes in the maximal activity of some key enzymes in the metabolism of glucose and glutamine, a reduction of hexokinase (68.8 per cent) activity and an increase in the activity of citrate synthase (10.1 per cent) in TB rats. The training protocol increased the proliferative response of lymphocytes cultivated in the absence of mitogens (75 per cent), of those cultivated in the presence of ConA (38.2 per cent) and in the presence of LPS (45.0 per cent). These cells also showed an increase in the maximal activity of some key enzymes of the glycolytic and glutaminolytic pathways. Our data demonstrated that the training protocol was able to induce an increase in aerobic utilisation of both substrates in lymphocytes and macrophages. The training protocol was also able to prevent several changes in glucose and glutamine metabolism that are normally present in sedentary TB rats. These changes in immune cell metabolism induced by the training protocol were able to increase TB rat survival.

Effect of a moderate intensity exercise training protocol on the metabolism of macrophages and lymphocytes of tumour‐bearing rats

It is commonly accepted that moderate intensity exercise is beneficial to the immune system. We tested the influence of a moderate intensity training protocol (8 weeks) upon immune system function in Wistar tumour-bearing (TB) rats. The metabolism of glucose and glutamine in lymphocytes and macrophages was assessed, together with some functional parameters (hydrogen peroxide production and lymphocyte proliferative response). These substrates were chosen since they represent the most important energetic and synthetic metabolites for these cellular types. The training protocol caused a decrease of 17·4 per cent in the production of H2O2 by macrophages, as well as a decrease in glucose consumption (25 per cent) and lactate production (47·1 per cent), and an increase in the production of labelled CO2 from the oxidation of [U-14C]-glucose, in TB rats. The training protocol was also able to induce changes in the maximal activity of some key enzymes in the metabolism of glucose and glutamine, a reduction of hexokinase (68·8 per cent) activity and an increase in the activity of citrate synthase (10·1 per cent) in TB rats. The training protocol increased the proliferative response of lymphocytes cultivated in the absence of mitogens (75 per cent), of those cultivated in the presence of ConA (38·2 per cent) and in the presence of LPS (45·0 per cent). These cells also showed an increase in the maximal activity of some key enzymes of the glycolytic and glutaminolytic pathways. Our data demonstrated that the training protocol was able to induce an increase in aerobic utilisation of both substrates in lymphocytes and macrophages. The training protocol was also able to prevent several changes in glucose and glutamine metabolism that are normally present in sedentary TB rats. These changes in immune cell metabolism induced by the training protocol were able to increase TB rat survival. Copyright © 2000 John Wiley & Sons, Ltd.

Platelet Functions and Energy Metabolism in a Patient With Hexokinase Deficiency

Abstract We have studied the regeneration of adenosine triphosphate (ATP) in the glycolytic pathway in platelets with a 75% reduction in hexokinase (HK) activity and have investigated aggregation and Ca2+ secretion. HK- deficient platelets had a normal glycolytic flux in the resting state, but responded insufficiently to stimulation with thrombin (5 U/ml). In contrast, glycogen contents and glycogenolysis were normal. When the metabolic adenine nucleotides were labeled with 14C-adenine, the patient's platelets showed a normal adenylate energy charge and a normal level of 14C-ATP. However, the inhibitor of mitochondrial energy generation, CN-, induced a weaker fall in 14C-ATP in the patient's platelets than in the controls. Analysis of secretion markers revealed decreased amounts of granule-bound ATP and secretable Ca2+, whereas granule-bound adenosine diphosphate (ADP), beta-thromboglobulin, N- acetyl-beta-D-glucosaminidase, and beta-glucuronidase were within the normal range. Aggregation and Ca2+ secretion induced by 5 U/ml thrombin were normal and were not changed in the presence of inhibitors of mitochondrial and glycogenolytic energy generation. Aggregation was also normal at 0.1 U/ml thrombin and was independent of these inhibitors, but Ca2+ secretion was greatly impaired when mitochondrial and glycogenolytic ATP resynthesis was abolished. These findings indicate that a severe reduction in HK activity causes insufficient acceleration of the glycolytic flux during stimulation with thrombin. This leads to impaired dense granule secretion in conditions where secretion depends on concurrent ATP resynthesis and glycolysis is rate limiting.

Platelet functions and energy metabolism in a patient with hexokinase deficiency

We have studied the regeneration of adenosine triphosphate (ATP) in the glycolytic pathway in platelets with a 75% reduction in hexokinase (HK) activity and have investigated aggregation and Ca2+ secretion. HK-deficient platelets had a normal glycolytic flux in the resting state, but responded insufficiently to stimulation with thrombin (5 U/ml). In contrast, glycogen contents and glycogenolysis were normal. When the metabolic adenine nucleotides were labeled with 14C-adenine, the patient's platelets showed a normal adenylate energy charge and a normal level of 14C-ATP. However, the inhibitor of mitochondrial energy generation, CN-, induced a weaker fall in 14C-ATP in the patient's platelets than in the controls. Analysis of secretion markers revealed decreased amounts of granule-bound ATP and secretable Ca2+, whereas granule-bound adenosine diphosphate (ADP), beta-thromboglobulin, N-acetyl-beta-D-glucosaminidase, and beta-glucuronidase were within the normal range. Aggregation and Ca2+ secretion induced by 5 U/ml thrombin were normal and were not changed in the presence of inhibitors of mitochondrial and glycogenolytic energy generation. Aggregation was also normal at 0.1 U/ml thrombin and was independent of these inhibitors, but Ca2+ secretion was greatly impaired when mitochondrial and glycogenolytic ATP resynthesis was abolished. These findings indicate that a severe reduction in HK activity causes insufficient acceleration of the glycolytic flux during stimulation with thrombin. This leads to impaired dense granule secretion in conditions where secretion depends on concurrent ATP resynthesis and glycolysis is rate limiting.